Articles

2012 Auteur(s) : Boisselier E, Audet ML, Cantin L, Salesse C. Titre : A strategy for purifying glutathione S-transferase in the presence of sodium dodecyl sulfate. Référence : Biotechniques. 2011 Sep;51(3):193-4. Lien : Cliquer ici Résumé : Glutathione S-Transferase (Gst) Is Widely Used To Prepare And Purify Gsttagged Fusion Proteins. Although Gst Improves Protein Solubility, Detergents Must Often Be Used To Achieve Protein Solubilization From Bacterial Lysates. However, Purification Of Gst By Affinity Chromatography Cannot Be Achieved In The Presence Of Even Low Concentrations Of The Detergent Sodium Dodecyl Sulfate (Sds). Here We Show That 2-Methyl-2,4-Pentanediol (Mpd) Can Prevent Sds From Interfering With Purification Of Gst, Thus Enabling Purification Of Proteins That Require Sds To Improve Their Solubility. Auteur(s) : Zaniolo K, Bostan C, Rochette Drouin O, Deschambeault A, Perron MC, Brunette I, Proulx S. Titre : Culture of human corneal endothelial cells isolated from corneas with Fuchs endothelial corneal dystrophy. Référence : Exp Eye Res. 2012 Jan;94(1):22-31. Epub 2011 Nov 19. Lien : Cliquer ici Résumé : The Purpose Of This Study Was To Assess The Feasibility Of Initiating Primary Cultures Of Corneal Endothelial Cells From Patients Suffering From Fuchs Endothelial Corneal Dystrophy (Fecd; Mim# 1036800). We Also Evaluated Which Conditions Yielded The Best Results For Culture. Twenty-Nine Patients Undergoing Descemet Stripping Automated Endothelial Keratoplasty Consented To The Use Of Their Excised Descemet'S Membrane For This Study. Out Of The 29 Specimens, 18 Successfully Initiated A Culture. Cell Morphology Varied Between Endothelial (Rounded, Slightly Elongated Cells, N = 12) And Fibroblastic-Like (Thin And Very Elongated Cells, N = 6). These Differences In Cell Morphology Were Also Observed With The Normal Human Corneal Endothelial Cell Cultures. The Cultures That Initially Presented An Endothelial Morphology Maintained Their Shape In Subcultures. Clusterin Expression Was Similar In Fecd And Normal Endothelial Cells. Transmission Electron Microscopy Of Fecd Descemet'S Membranes Showed A High Degree Of Various Abnormalities Generally Found In This Disease, Such As A Thickened Descemet'S Membrane, Presence Of A Posterior Banded Layer, Presence Of A Fibrillar Layer And Striated Bodies Of Various Sizes And Periodicities. Patient'S Age Was Predictive Of Culture Success, All Younger Fecd Donors Generating Cultures Of Endothelial Morphology. The Absence Of A Fibrillar Layer Was Also A Factor Associated With Greater Success. Culture Success Was Not Dependent On Specimen Size, Specimen Pigmentation, Or Patient'S Preoperative Central Corneal Thickness. In Conclusion, This Paper Shows For The First Time That Central Descemet'S Membranes Of Patients Suffering From Fecd Possess Proliferative Endothelial Cells That Can Be Isolated And Cultured Without Viral Transduction, Opening The Way For New In Vitro Studies Of This Disease. Auteur(s) : Fortier GM, Gauvin R, Proulx M, Vallée M, Fradette J. Titre : Dynamic culture induces a cell type-dependent response impacting on the thickness of engineered connective tissues. Référence : J Tissue Eng Regen Med. 2011 Dec 12. doi: 10.1002/term.522. [Epub ahead of print] Lien : Cliquer ici Résumé : Mesenchymal Cells Are Central To Connective Tissue Homeostasis And Are Widely Used For Tissue-Engineering Applications. Dermal Fibroblasts And Adipose-Derived Stromal Cells (Ascs) Allow Successful Tissue Reconstruction By The Self-Assembly Approach Of Tissue Engineering. This Method Leads To The Production Of Multilayered Tissues, Devoid Of Exogenous Biomaterials, That Can Be Used As Stromal Compartments For Skin Or Vesical Reconstruction. These Tissues Are Formed By Combining Cell Sheets, Generated Through Cell Stimulation With Ascorbic Acid, Which Favours The Cell-Derived Production/Organization Of Matrix Components. Since Media Motion Can Impact On Cell Behaviour, We Investigated The Effect Of Dynamic Culture On Mesenchymal Cells During Tissue Reconstruction, Using The Self-Assembly Method. Tissues Produced Using Ascs In The Presence Of A Wave-Like Movement Were Nearly Twice Thicker Than Under Standard Conditions, While No Difference Was Observed For Tissues Produced From Dermal Fibroblasts. The Increased Matrix Deposition Was Not Correlated With An Increased Proliferation Of Ascs, Or By Higher Transcript Levels Of Fibronectin Or Collagens I And Iii. A 30% Increase Of Type V Collagen Mrna Was Observed. Interestingly, Tissues Engineered From Dermal Fibroblasts Featured A Four-Fold Higher Level Of Mmp-1 Transcripts Under Dynamic Conditions. Mechanical Properties Were Similar For Tissues Reconstructed Using Dynamic Or Static Conditions. Finally, Cell Sheets Produced Using Ascs Under Dynamic Conditions Could Readily Be Manipulated, Resulting In A 2 Week Reduction Of The Production Time (From 5 To 3 Weeks). Our Results Describe A Distinctive Property Of Ascs' Response To Media Motion, Indicating That Their Culture Under Dynamic Conditions Leads To Optimized Tissue Engineering. Copyright © 2011 John Wiley & Sons, Ltd. Auteur(s) : Garcia-Perez ME, Jean J, Pouliot R. Titre : Antipsoriatic Drug Development: Challenges and New Emerging Therapies. Référence : Recent Pat Inflamm Allergy Drug Discov. 2012 Jan 1;6(1):3-21. Lien : Cliquer ici Résumé : Psoriasis Is A Chronic Recurring Skin Disorder Affecting Up To 2% Of The World'S Population. Psoriatic Lesions Are Generally Visible, Leading To Significant Emotional And Social Disabilities For Patients. In The Context Of Psoriasis, The Orchestrated Interplay Between Activated T Cells, Antigen-Presenting Cells And Keratinocytes Leads To The Release Of Proinflammatory Cytokines, Chemokines And Chemical Mediators Responsible For The Perpetuation Of This Disease. Even Though Some Therapies Are Available For Psoriasis Treatment, There Is Still No Cure For This Skin Disorder And Psoriatic Patients Are Significantly Unsatisfied, As Demonstrated By Recent Worldwide Surveys. Unlike Other Diseases, Psoriasis Does Not Have A Generally Accepted Animal Model, Which Complicates The Successful Introduction Of New Antipsoriatic Drugs Into Clinical Phases Of Development. Moreover, Psoriasis Affects Infants, Children And Elderly Patients Which Require Long-Term Therapies. Thus, The Development Of New Therapeutic Approaches Should Consider Multiple Factors Such As Efficacy, Dosing Frequency, Route Of Administration, Toxicity As Well As Co-Morbidities Of Patients. This Article Analyzes Current Challenges For The Antipsoriatic Drug Development And Reviews Recent Patent Applications Gathered From 2000 To 2011 For Psoriasis Treatment. Additionally, Future Perspectives For Antipsoriatic Drug Development Are Summarized. 2011 Auteur(s) : Jean J, Garcia-Perez ME, Pouliot R Titre : Bioengineered Skin: The Self-Assembly Approach Référence : J Tissue Sci Eng S5:001 Lien : Cliquer ici Résumé : Tissue-Engineered Skin Substitutes Represent An Innovative Therapeutic Option For The Treatment Of Burns And Skin Ulcers As Well As A Powerful Tool For Fundamental Research. To Be Efficient, In Vitro Skin Substitutes Must Closely Mimic Human Skin Structures And Exogenous Material Has To Be Reduced As Much As Possible. The Self-Assembly Approach Is Based On The Capacity Of Fibroblasts To Create Their Own Extracellular Matrix In Vitro, Which Allows The Production Of Cell Sheets That Are Easy To Handle. Therefore, A Skin Substitute Devoid Of Exogenous Extracellular Matrix Proteins And Synthetic Material Is Produced, Which Demonstrates Many Histological, Physico-Chemical And Mechanical Characteristics Found In Normal Human Skin In Vivo. A Particularity Of This Approach Is The Possibility To Add Various Other Cell Types (Keratinocytes, Melanocytes, Adipocytes, Endothelial And Immunological Cells, Etc.) According To Needs. Furthermore, Pathological Cells (Hypertrophic Scar, Sclerodermic, Tumoral And Psoriatic Cells) Can Be Used For The Production Of Pathological Skin Substitutes. The Development Of These Models Represents A Key Component In The Fight Against Such Diseases Because They Can Lead To A Better Understanding Of The Pathology And To The Development Of New Pharmaceutical Therapies.This Review Will Present The Need For Tissue-Engineered Skin Substitutes, The Implication Of Tissue Engineering In The Cutaneous Field (Basic And Applied Research), The Self-Assembly Approach And Its Characteristics As Well As The Actual State Of Research On Healthy And Pathological Self-Assembled Skin Models. Auteur(s) : Brunette I, Rosolen SG, Carrier M, Abderrahman M, Nada O, Germain L, Proulx S. Titre : Comparison of the pig and feline models for full thickness corneal transplantation. Référence : Vet Ophthalmol. 2011 Nov;14(6):365-377. Lien : Cliquer ici Auteur(s) : Simon F, Bergeron D, Larochelle S, Lopez-Vallé CA, Genest H, Armour A, Moulin VJ. Titre : Enhanced secretion of TIMP-1 by human hypertrophic scar keratinocytes could contribute to fibrosis. Référence : Burns. 2011 Oct 29. [Epub ahead of print] Lien : Cliquer ici Auteur(s) : Chabaud S, Moulin VJ. Titre : Apoptosis modulation as a promising target for treatment of systemic sclerosis. Référence : Int J Rheumatol. 2011;2011:495792. Epub 2011 Sep 6. Lien : Cliquer ici Auteur(s) : Boisselier E, Audet ML, Cantin L, Salesse C. Titre : A strategy for purifying glutathione S-transferase in the presence of sodium dodecyl sulfate. Référence : Biotechniques. 2011 Sep;51(3):193-4. Lien : Cliquer ici Auteur(s) : Dubé J, Rochette-Drouin O, Lévesque P, Gauvin R, Roberge CJ, Auger FA, Goulet D, Bourdages M, Plante M, Moulin VJ, Germain L. Titre : Human keratinocytes respond to direct current stimulation by increasing intracellular calcium: preferential response of poorly differentiated cells. Référence : J Cell Physiol. 2011 Aug 31. doi: 10.1002/jcp.23008. [Epub ahead of print] Lien : Cliquer ici Auteur(s) : Berthod F, Symes J, Tremblay N, Medin JA, Auger FA. Titre : Spontaneous fibroblast-derived pericyte recruitment in a human tissue-engineered angiogenesis model in vitro. Référence : J Cell Physiol. 2011 Jul 18. doi: 10.1002/jcp.22943. [Epub ahead of print] Lien : Cliquer ici Auteur(s) : Parenteau-Bareil R, Gauvin R, Cliche S, Gariépy C, Germain L, Berthod F. Titre : Comparative study of bovine, porcine and avian collagens for the production of a tissue engineered dermis. Référence : Acta Biomater. 2011 Oct;7(10):3757-65. Lien : Cliquer ici Auteur(s) : Proulx S, Brunette I. Titre : Methods being developed for preparation, delivery and transplantation of a tissue-engineered corneal endothelium. Référence : Exp Eye Res. 2011 Jun 25. [Epub ahead of print] Lien : Cliquer ici Auteur(s) : Imbeault A, Bernard G, Ouellet G, Bouhout S, Carrier S, Bolduc S. Titre : Surgical Option for the Correction of Peyronie's Disease: An Autologous Tissue-Engineered Endothelialized Graft. Référence : J Sex Med. 2011 Nov;8(11):3227-35. Lien : Cliquer ici Auteur(s) : Cattan V, Bernard G, Rousseau A, Bouhout S, Chabaud S, Auger FA, Bolduc S. Titre : Mechanical Stimuli-induced Urothelial Differentiation in a Human Tissue-engineered Tubular Genitourinary Graft. Référence : Eur Urol. 2011 Dec;60(6):1291-8. Lien : Cliquer ici Auteur(s) : Landreville S, Lupien CB, Vigneault F, Gaudreault M, Mathieu M, Rousseau AP, Guérin SL, Salesse C. Titre : Identification of differentially expressed genes in uveal melanoma using suppressive subtractive hybridization. Référence : Mol Vis. 2011;17:1324-33. Lien : Cliquer ici Auteur(s) : Bouhout S, Gauvin R, Gibot L, Aubé D, Bolduc S. Titre : Bladder substitute reconstructed in a physiological pressure environment. Référence : J Pediatr Urol. 2011 Jun;7(3):276-82. Lien : Cliquer ici Auteur(s) : Jean J, Garcia-Perez ME, Guérard S, Pouliot R Titre : Psoriasis: Causes, Treatments and Pathological Models Référence : Dermatology - Laboratory and Clinical Research Series Lien : Cliquer ici Résumé : Psoriasis Is A Common, Inflammatory, Multisystemic Skin Disease Characterized By Hyperproliferation And Abnormal Differentiation Of Epidermis. It Affects Approximately 2 % Of The World’S Population, Both Men And Women, Aged Between 15 And 30 Years. Psoriasis Is Typically Recognized With Sharply Demarcated Red Scaly Dermatological Plaques Affecting Most Body Surfaces, But Especially Knees, Elbows And Scalp. Generally, Psoriasis Is Not A Fatal Disease But The Presence Of Physical And Psychological Pains Can Severely Affect Patients’ Quality Of Life. There Is A Wide Variety Of Therapeutic Options To Treat Psoriasis Including Topical, Phototherapy, Systemic And Biological Therapies. These Treatments Can Control Or Prevent Symptoms; However There Is Still No Cure Available. During The Past Decade, Many Pathological Models Have Been Developed To Better Understand Psoriasis. Among Them, In Vitro Models Offer An Interesting Alternative To Animal Ones. Their Development Represents A Key Component In The Fight Against Psoriasis. This Complete Review Of Psoriasis Presents The Characteristics Of This Pathology, The Suggested Causes, The Treatments (Side Effects And Mechanisms Of Action) And The Current State Of Research On Pathological Models To Better Understand Mechanisms Of Psoriasis. Auteur(s) : Gauvin R, Parenteau-Bareil R, Larouche D, Marcoux H, Bisson F, Bonnet A, Auger FA, Bolduc S, Germain L. Titre : Dynamic mechanical stimulations induce anisotropy and improve the tensile properties of engineered tissues produced without exogenous scaffolding. Référence : Acta Biomater. 2011 May 30. [Epub ahead of print] Lien : Cliquer ici Résumé : Mechanical Strength And The Production Of Extracellular Matrix (Ecm) Are Essential Characteristics For Engineered Tissues Designed To Repair And Replace Connective Tissues That Are Subject To Stress And Strain. In This Study, Dynamic Mechanical Stimulation (Dms) Was Investigated As A Method To Improve The Mechanical Properties Of Engineered Tissues Produced Without The Use Of An Exogenous Scaffold, Referred To As The Self-Assembly Approach. This Method, Based Exclusively On The Use Of Human Cells Without Any Exogenous Scaffolding, Allows For The Production Of A Tissue Sheet Comprised Of Cells And Ecm Components Synthesized By Dermal Fibroblasts In Vitro. A Bioreactor Chamber Was Designed To Apply Cyclic Strain To Engineered Tissues In Order To Determine If Dynamic Culture Had An Impact On Their Mechanical Properties And Ecm Organization. Fibroblasts Were Cultured In The Presence Of Ascorbic Acid For 35Days To Promote Ecm Production And Allow The Formation Of A Tissue Sheet. This Sheet Was Grown On A Custom-Built Anchoring System Allowing For Easy Manipulation And Fixation Of The Tissue In The Bioreactor. Following The 35Day Period, Tissues Were Maintained For 3Days In Static Culture (Sc), Or Subjected Either To A Static Mechanical Stimulation Of 10% Strain, Or A Dynamic Dms With A Duty Cycle Of 10% Uniaxial Cyclic Strain At 1Hz. Ecm Was Characterized By Histology, Immunofluorescence Labeling And Western Blotting. Both Static And Dynamic Mechanical Stimulation Induced The Alignment Of Assessed Cytoskeletal Proteins And Ecm Components Parallel To The Axis Of Applied Strain And Increased The Ecm Content Of The Tissues Compared To Sc. Measurement Of The Tensile Mechanical Properties Revealed That Mechanical Stimulation Significantly Increases Both The Ultimate Tensile Strength And Tensile Modulus Of The Engineered Tissues When Compared To The Non-Stimulated Control. Moreover, We Demonstrated That Cyclic Strain Significantly Increases These Parameters When Compared To A Static-Loading Stimulation And That Mechanical Stimulation Contributes To The Establishment Of Anisotropy In The Structural And Mechanical Properties Of Self-Assembled Tissue Sheets. Auteur(s) : Chabaud S, Corriveau MP, Grodzicky T, Senécal JL, Chartier S, Raymond Y, Moulin VJ. Titre : Decreased secretion of MMP by non-lesional late-stage scleroderma fibroblasts after selection via activation of the apoptotic Fas-pathway. Référence : J Cell Physiol. 2011 Jul;226(7):1907-14. doi: 10.1002/jcp.22520. Lien : Cliquer ici Résumé : Our Hypothesis Is That The Development Of Lesional Areas Of Skin In Patients With Systemic Sclerosis (Ssc) Originates From The Selection Of Profibrotic Cell Subpopulations Within Their Non-Lesional Skin Areas, Due To Their Greater Resistance To Apoptosis. Sensitivity To Apoptosis Of Early-Stage Or Late-Stage Ssc Fibroblasts As Well As Of Healthy Cells Was Compared Using Extrinsic Or Intrinsic Apoptotic Pathway-Inducers. Subpopulations Of Non-Lesional Ssc Cells And Healthy Cells Obtained After Repeated Fas-Induced Apoptosis Were Compared With Respect To Their Fibrotic Parameters Such As Collagen And Mmp Secretion. Only Late-Stage Lesional Ssc Cells Were More Resistant To Fas-Induced Apoptosis Than Their Non-Lesional Counterparts Isolated From The Same Patient. This Result Correlated With An Increase In The Levels Of The Anti-Apoptotic Proteins Cflips And Ciap In Lesional Cells Compared To Non-Lesional Cells. Healthy And Non-Lesional Cell Populations Could Be Selected To Generate A Subpopulation That Was More Resistant To Apoptosis. However, Only The Late-Stage Non-Lesional Ssc Fibroblast Populations Showed A Significant Decrease In Mmp Secretion, One Of Parameters Of The Fibrosis. Our Results Show That Resistance To Apoptosis Is An Important Characteristic Of The Late-Stage Lesional Ssc Fibroblast Phenotype. We Thus Hypothesized That A Selection Of Specific Fibroblast Subpopulations From Late-Stage Non-Lesional Ssc Skin Areas Could Be At The Origin Of Lesional Populations. These Cells Should Become Independent Of Any Exogenous Stimuli And Can Induce Or Maintain Ssc Skin Lesions. Auteur(s) : Landry H, Aminian A, Hoffart L, Nada O, Bensaoula T, Proulx S, Carrier P, Germain L, Brunette I. Titre : Corneal Endothelial Toxicity of Air and SF6. Référence : Invest Ophthalmol Vis Sci. 2011 Apr 8;52(5):2279-86. Print 2011. Lien : Cliquer ici Résumé : Purpose. The Authors Conducted In Vivo Assessment Of Corneal Endothelial Toxicity Of Air And Sf6 In The Feline Model. This Research Was Motivated By The Increased Use Of Air In Anterior Segment Surgery In Human Subjects. Methods. This Was A Prospective Masked Study. The Eyes Of 16 Healthy Adult Cats Were Randomly Assigned For The Injection Of 0.7 Ml Air Into The Anterior Chamber Of One Eye And Sf6 In The Contralateral Eye. Daily Examination Included Slit Lamp Photographs, Pachymetry, And Tonometry. Specular Microscopy Was Performed Before, 7 Days After, And 10 Days After Injection. The Animals Were Euthanatized, And The Corneas Were Processed For Alizarin Red-Trypan Blue Staining And For Light And Electron Microscopy. Results. Sf6 Remained In The Anterior Chamber Significantly Longer Than Air. Both Groups Showed Postinjection Inflammation, Which On Average Was Maximal At Day 2 And More Severe With Sf6. No Difference In Iop Was Observed Between The Two Groups. Specular Microscopy Showed Significant Endothelial Cell Loss In The Sf6 Group (Mean Postinjection Cell Loss, 132 ± 50 Cells/Mm(2)) But Not In The Group Injected With Air. Alizarin Red Staining Revealed Significant Regional Differences In Cell Density Only In The Sf6 Group And More Pronounced Endothelial Cell Loss In The Superior Area. Conclusions. These Results Indicate That Both Air And Sf6 Injected Into The Anterior Chamber Of The Eye Can Induce Intraocular Reaction In The Feline Model And That Sf6 Is More Toxic Than Air In Terms Of Endothelial Cell Loss And Anterior Chamber Inflammation. Auteur(s) : Gauvin R, Guillemette MD, Galbraith T, Bourget JM, Larouche D, Marcoux H, Aubé D, Hayward C, Auger FA, Germain L. Titre : Mechanical Properties of Tissue-Engineered Vascular Constructs Produced Using Arterial or Venous Cells. Référence : Tissue Eng Part A. 2011 Apr 2. [Epub ahead of print] Lien : Cliquer ici Résumé : There Is A Clinical Need For Better Blood Vessel Substitutes Since Current Surgical Procedures Are Limited By The Availability Of Suitable Autologous Vessels And Suboptimal Behavior Of Synthetic Grafts In Small Caliber Arterial Graft (< 5 Mm) Applications. The Aim Of The Present Study Was To Compare The Mechanical Properties Of Arterial And Venous Tissue-Engineered Vascular Constructs Produced By The Self-Assembly Approach Using Cells Extracted From Either The Artery Or Vein Harvested From The Same Human Umbilical Cord. The Production Of A Vascular Construct Comprised Of A Media And An Adventitia (Tevma) Was Achieved By Rolling A Continuous Tissue Sheet Containing Both Smcs And Adventitial Fibroblasts Grown Contiguously In The Same Tissue Culture Plate. Histology And Immunofluorescence Staining Were Used To Evaluate The Structure And Composition Of The Extracellular Matrix (Ecm) Of The Vascular Constructs. The Mechanical Strength Was Assessed By Uniaxial Tensile Testing, While Viscoelastic Behavior Was Evaluated By Stepwise Stress-Relaxation And By Cyclic Loading Hysteresis Analysis. Tensile Testing Showed That The Use Of Arterial Cells Resulted In Stronger And Stiffer Constructs When Compared To Those Produced Using Venous Cells. Moreover, Cyclic Loading Demonstrated That Constructs Produced Using Arterial Cells Were Able To Bear Higher Loads For The Same Amount Of Strain When Compared To Venous Constructs. These Results Indicate That Cells Isolated From Umbilical Cord Can Be Used To Produce Vascular Constructs. Arterial Constructs Possessed Superior Mechanical Properties When Compared To Venous Constructs Produced Using Cells Isolated From The Same Human Donor. This Study Highlights The Fact That Smcs And Fibroblasts Originating From Different Cell Sources Can Potentially Lead To Distinct Tissue Properties When Used In Tissue Engineering Applications. Auteur(s) : Gauvin R, Guillemette MD, Galbraith T, Bourget JM, Larouche D, Marcoux H, Aubé D, Hayward C, Auger FA, Germain L. Titre : Mechanical Properties of Tissue-Engineered Vascular Constructs Produced Using Arterial or Venous Cells. Référence : Tissue Eng Part A. 2011 Lien : Cliquer ici Résumé : There Is A Clinical Need For Better Blood Vessel Substitutes Since Current Surgical Procedures Are Limited By The Availability Of Suitable Autologous Vessels And Suboptimal Behavior Of Synthetic Grafts In Small Caliber Arterial Graft (< 5 Mm) Applications. The Aim Of The Present Study Was To Compare The Mechanical Properties Of Arterial And Venous Tissue-Engineered Vascular Constructs Produced By The Self-Assembly Approach Using Cells Extracted From Either The Artery Or Vein Harvested From The Same Human Umbilical Cord. The Production Of A Vascular Construct Comprised Of A Media And An Adventitia (Tevma) Was Achieved By Rolling A Continuous Tissue Sheet Containing Both Smcs And Adventitial Fibroblasts Grown Contiguously In The Same Tissue Culture Plate. Histology And Immunofluorescence Staining Were Used To Evaluate The Structure And Composition Of The Extracellular Matrix (Ecm) Of The Vascular Constructs. The Mechanical Strength Was Assessed By Uniaxial Tensile Testing, While Viscoelastic Behavior Was Evaluated By Stepwise Stress-Relaxation And By Cyclic Loading Hysteresis Analysis. Tensile Testing Showed That The Use Of Arterial Cells Resulted In Stronger And Stiffer Constructs When Compared To Those Produced Using Venous Cells. Moreover, Cyclic Loading Demonstrated That Constructs Produced Using Arterial Cells Were Able To Bear Higher Loads For The Same Amount Of Strain When Compared To Venous Constructs. These Results Indicate That Cells Isolated From Umbilical Cord Can Be Used To Produce Vascular Constructs. Arterial Constructs Possessed Superior Mechanical Properties When Compared To Venous Constructs Produced Using Cells Isolated From The Same Human Donor. This Study Highlights The Fact That Smcs And Fibroblasts Originating From Different Cell Sources Can Potentially Lead To Distinct Tissue Properties When Used In Tissue Engineering Applications. Auteur(s) : Jean J, Soucy J, Pouliot R. Titre : Effects of Retinoic Acid in Keratinocyte Proliferation and Differentiation in a Psoriatic Skin Model. Référence : Tissue Eng Part A. 2011 Jul;17(13-14):1859-68. Epub 2011 May 25. Lien : Cliquer ici Résumé : Psoriasis Is A Skin Disease Characterized By The Presence Of Red Plaques On The Skin. This Pathology Is Well Known To Be A Retinoid-Sensitive Disease. Previous Investigations Have Shown That Retinoids Can Modulate Epidermal Proliferation With An Anti-Proliferative Potential In Hyperproliferative Skins. The Aim Of This Study Was To Compare The Development Of Psoriatic Substitutes Cultured In A Retinoic Acid Supplemented Medium With Those Cultured In Medium Receiving No Supplement To Define The Effects Of This Growth Factor On Keratinocyte Proliferation And Differentiation. The Self-Assembly Method Was Used To Create Substitutes. Characterization Of The Psoriatic Substitutes Was Performed By Histological And Immunohistochemical Immunolabelling Analyses. Results Showed That Psoriatic Keratinocyte Substitutes Cultured With Retinoic Acid Have A Thinner Epidermis Compared With Psoriatic Keratinocyte Substitutes Cultured Without This Supplement. Furthermore, The Expression Of All Tested Cell Differentiation Markers Was Restored In Psoriatic Keratinocyte Substitutes Cultured In Presence Of Retinoic Acid. No Significant Change In Epidermal Thickness Or In The Expression Of Late Differentiation Markers Were Observed In Healthy Keratinocyte Substitutes Cultured With Or Without Retinoic Acid, However, Some Changes Were Reported For Proliferation And Early Differentiation Markers. Results Suggest That Retinoic Acid Can Modulate Epidermal Differentiation And Proliferation With An Anti-Proliferative Potential In Psoriatic Substitute Such As Observed In Psoriatic Skin In Vivo. Auteur(s) : Guillemette MD, Roy E, Auger FA, Veres T. Titre : Rapid isothermal substrate microfabrication of a biocompatible thermoplastic elastomer for cellular contact guidance. Référence : Acta Biomater. 2011 Feb 15. [Epub ahead of print] Lien : Cliquer ici Résumé : The Use Of Microstructured Substrates To Study And Influence Cell Orientation, Which Plays An Important Role In Tissue Functionality, Has Been Of Great Interest Lately. Silicon And Poly(Dimethylsiloxane) Substrates Have Typically Been Used, But Long Processing Times And Exogenous Protein Surface Coating, Required To Enhance Cell Viability, Limit Their Use As Large-Scale Platforms. There Is Thus A Need For A Non-Biodegradable Biocompatible Substrate That Allows Rapid And Low Cost Microfabrication. In This Paper A Styrene-(Ethylene/Butylene)-Styrene Block Co-Polymer (Sebs) Microstructured By A Rapid Replication Technique Using Low Pressure An Isothermal Hot Embossing Approach Has Been Demonstrated. Sebs Substrates Were Treated With Oxygen Plasma To Enhance Cell Adhesion And Sterilized Using Ethylene Oxide Gas. While Cell Adhesion To And Proliferation On These Substrates Was As Good As On Tissue Culture Polystyrene, Cellular Alignment On Microstructured Sebs Was Also Very High (97.7±0.5%) When Calculated Within A 10° Angle Variation From The Longitudinal Axis. Furthermore, Tissue Sheets On Microstructured Sebs Have Been Produced And Exhibited Cellular Alignment Within The Engineered Tissue. In Addition, These Results Were Obtained Without Coating The Material With Exogenous Proteins. Such Substrates Should Be Helpful In The Culture Of Tissue Engineered Substitutes With An Intrinsic Orientation And To Elucidate Questions In Cell Biology. Auteur(s) : Jean J, Garcia-Perez ME, Guérard S, Pouliot R Titre : Current knowledge in psoriasis: an overview of the skin disease Référence : In: Psoriasis: causes, diagnosis and treatment, Eds: Carrasco JA, Chap 2 Lien : Cliquer ici Résumé : Psoriasis Is A Common, Inflammatory, Multisystemic Skin Disease Characterized By Hyperproliferation And Abnormal Differentiation Of Epidermis. It Affects Approximately 2 % Of The World’S Population, Both Men And Women, Aged Between 15 And 30 Years. Psoriasis Is Typically Recognized With Sharply Demarcated Red Scaly Dermatological Plaques Affecting Most Body Surfaces, But Especially Knees, Elbows And Scalp. Generally, Psoriasis Is Not A Fatal Disease But The Presence Of Physical And Psychological Pains Can Severely Affect Patients’ Quality Of Life. There Is A Wide Variety Of Therapeutic Options To Treat Psoriasis Including Topical, Phototherapy, Systemic And Biological Therapies. These Treatments Can Control Or Prevent Symptoms; However There Is Still No Cure Available. During The Past Decade, Many Pathological Models Have Been Developed To Better Understand Psoriasis. Among Them, In Vitro Models Offer An Interesting Alternative To Animal Ones. Their Development Represents A Key Component In The Fight Against Psoriasis. This Complete Review Of Psoriasis Presents The Characteristics Of This Pathology, The Suggested Causes, The Treatments (Side Effects And Mechanisms Of Action) And The Current State Of Research On Pathological Models To Better Understand Mechanisms Of Psoriasis. Auteur(s) : Ouellet G, Dubé J, Gauvin R, Laterreur V, Bouhout S, Bolduc S. Titre : Production of an optimized tissue-engineered pig connective tissue for the reconstruction of the urinary tract. Référence : Tissue Eng Part A. 2011 Feb 3. [Epub ahead of print] Lien : Cliquer ici Résumé : Non-Urological Autologous Tissues Are Used For Urethral Reconstruction To Correct Urinary Tract Disorders But Are Still Leading To Complications. Other Substitutes Have Been Studied On Small Animal Models Without Great Success. For Preclinical Tests, We Selected The Porcine Model For Its Similarity To The Human Urinary Tract. Up To Now, Porcine Skin Fibroblasts Were Not Able To Synthesize Enough Extracellular Matrix (Ecm) Under Standard Conditions To Sustain The Formation Of An Adequate Tissue For Transplantation Purposes. Therefore, Our Goal Was To Optimize The Harvesting Site And Culture Conditions To Obtain A Thick And Easy To Handle Porcine Fibroblast Tissue. The Oral Mucosa Was Found To Be The Ideal Harvesting Site And A Culture Temperature Of 39°C Enabled The Formation Of A Good Porcine Fibroblast Sheet. We Successfully Superimpose Three Fibroblast Sheets That Merged Into A Thick And Resistant Tissue Where Physiological Ecm Was Produced. Mechanical Resistance Evaluation By Uniaxial Traction On The Three-Layer Fibroblast Constructs Also Demonstrated Its Suitable Properties. The Production Of This Porcine Connective Tissue Offers An Interesting Option In The Field Of Urological Tissue Engineering. Autologous Experiments On A Larger Animal Model Are Now Possible And Accessible, Allowing The Performance Of Long-Term In Vivo Studies. Auteur(s) : Jean J, Bernard G, Duque-Fernandez A, Auger FA, Pouliot R. Titre : Effects of Serum-Free Culture at the Air-Liquid Interface in a Human Tissue-Engineered Skin Substitute. Référence : Tissue Eng Part A. 2011 Apr;17(7-8):877-88. Epub 2011 Jan 10. Lien : Cliquer ici Résumé : Previous Studies Have Reported That Well-Defined Culture Conditions Can Improve Keratinocytes Terminal Differentiation And Reproducibility. The Aim Of Our Study Was To Compare Skin Substitutes Cultured In A Complete Medium With Those Cultured In A Serum-Free Medium At The Air-Liquid Interface To Optimize The Self-Assembly Method. Skin Substitutes, Cultured In A Serum-Free Medium Over 7, 14, And 21 Days, Were Compared With Others Cultured In A Complete Medium (5% Serum) Over The Complete Culture Period. Masson'S Trichrome Staining Showed That The Substitutes Cultured In A Serum-Free Medium Generated A Well-Developed And Differentiated Epidermis. Immunolabeling Analyses Between The Substitutes Cultured Without Serum And Those Cultured In Complete Serum Showed Similar Expression Of Epidermal Differentiation Markers, Dermo-Epidermal Junction, And Dermal Extracellular Matrix Components. On The Basis Of Our Attenuated Total Reflectance-Fourier Transform Infrared (Atr-Ftir) Results, The Skin Substitutes Cultured In Serum-Free Condition Over 21 Days Of Culture At The Air-Liquid Interface Showed Lower Frequencies Of The Ch(2) Symmetric Mode Of Vibrations, Which Means A Better Lipid Organization Of The Stratum Corneum. No Significant Difference In Hydrocortisone Penetration Was Observed Between Serum-Free Medium Substitutes And The Controls. Results Demonstrate That The Absence Of Serum Does Not Compromise The Characteristics Of The Skin Substitutes Observed In This Study. Auteur(s) : Labbé B, Marceau-Fortier G, Fradette J. Titre : Cell sheet technology for tissue engineering: the self-assembly approach using adipose-derived stromal cells. Référence : Methods Mol Biol. 2011;702:429-41. Lien : Cliquer ici Résumé : In The Past Years, Adipose Tissue Has Spurred A Wide Interest, Not Only As A Source Of Adult Multipotent Stem Cells But Also As A Highly Eligible Tissue For Reconstructive Surgery Procedures. Tissue Engineering Is One Field Of Regenerative Medicine Progressing At Great Strides In Part Due To Its Important Use Of Adipose-Derived Stem/Stromal Cells (Ascs). The Development Of Diversified Technologies Combining Ascs With Various Biomaterials Has Lead To The Reconstruction Of Numerous Types Of Tissue-Engineered Substitutes Such As Bone, Cartilage, And Adipose Tissues From Rodent, Porcine, Or Human Ascs. We Have Recently Achieved The Reconstruction Of Connective And Adipose Tissues Composed Entirely Of Cultured Human Ascs And Their Secreted Endogenous Extracellular Matrix Components By A Methodology Known As The Self-Assembly Approach Of Tissue Engineering. The Latter Is Based On The Stimulation Of Ascs To Secrete And Assemble Matrix Components In Culture, Leading To The Production Of Cell Sheets That Can Be Manipulated And Further Assembled Into Thicker Multilayer Tissues. In This Chapter, Protocols To Generate Both Reconstructed Connective And Adipocyte-Containing Tissues Using The Self-Assembly Approach Are Described In Detail. The Methods Include Amplification And Cell Banking Of Human Ascs, As Well As Culture Protocols For The Production Of Individual Stromal And Adipose Sheets, Which Are The Building Blocks For The Reconstruction Of Multilayered Human Connective And Adipose Tissues, Respectively. Auteur(s) : Robayo LM, Moulin VJ, Tremblay P, Cloutier R, Lamontagne J, Larkin AM, Chabaud S, Simon F, Islam N, Goulet F. Titre : New ligament healing model based on tissue-engineered collagen scaffolds. Référence : Wound Repair Regen. 2011 Jan;19(1):38-48. doi: 10.1111/j.1524-475X.2010.00640.x. Epub 2010 Dec 10. Lien : Cliquer ici Résumé : The Anterior Cruciate Ligament (Acl) Is Often The Target Of Knee Trauma. This Ligament Does Not Heal Very Well, Leading To Joint Instability. Long-Term Instability Of The Knee Can Lead To Early Arthritis And Loss Of Function. To Develop Efficient Strategies To Stimulate Posttraumatic Acl Regeneration In Vivo, A Good Healing Model Is Needed In Vitro. Such A Model Must Remain As Simple As Possible, But Should Include Key Features To Provide Relevant Answers To Precise Questions About The Clinical Problem Addressed. Here, We Report Tissue-Engineered Type I Collagen Scaffolds Developed To Establish An Acl Healing Model In Vitro And A Potential Acl Substitute In Vivo. Such Scaffolds Were Used To Evaluate Acl Cell Growth, Migration, And The Capacity To Synthesize And Assemble Collagen Fibers For Up To 40 Days In Vitro And Up To 180 Days In Vivo. They Were Anchored With Two Bone Plugs To Allow Their Static Stretching In Culture And To Facilitate Their Surgical Implantation In Knee Joints. Our Results Have Shown That Living Acl Fibroblasts Can Attach, Migrate, And Colonize This Type Of Scaffold. In Vitro, The Cells Populated The Scaffolds And Expressed Mrnas Coding For The Prolyl-4-Hydroxylase, Involved In Collagen Fibers' Assembly. In Vivo, Acellular Implants Were Vascularized And Populated With Caprine Cells That Migrated From The Osseous Insertions Toward The Center Of The Grafts. This Model Is A Very Good Tool To Study Acl Repair And Identify The Factors That Could Accelerate Its Healing Postsurgery. Auteur(s) : Lévesque P, Gauvin R, Larouche D, Auger FA, Germain L Titre : A computer-controlled apparatus for the characterization of mechanical and viscoelastic properties of tissue-engineered vascular constructs Référence : Cardiovascular engineering and technology. 2011 March; 2:24-34. Lien : Cliquer ici Résumé : Tissue-Engineered Blood Vessels Can Be Partly Characterized By Analyzing Their Mechanical Properties Using Burst Pressure Testing, Compliance Measurement, Creep And Cyclic Testing. Studying These Parameters Provides Information On The Capability Of A Fabrication Method To Produce Tissue-Engineered Blood Vessels (Tebv) And Allow For The Optimization Of Their Resistance And Viscoelastic Properties. This Study Presents The Design And Fabrication Of An Apparatus Allowing Accurate And Reliable Measurements Of The Mechanical Properties Of Tissue-Engineered Vascular Constructs. A Computer-Controlled System Was Designed To Monitor Pressure And Diameter Variations Of Vascular Constructs Submitted To Hydrostatic Loading. The System Was Programmed To Control The Motorized Portion Of The Setup And Allow Simultaneous Data Acquisition, Analysis And Realtime Display. Data Acquisition Cards Allow For Synchronous Monitoring Of Pressure And Diameter Of The Constructs Through A Pressure Transducer And A Ccd Camera. Image Analysis And Pressure Data Computation Resulted In Compliance, Creep And Dynamic Characterization Of The Tested Tissues. This Experimental Setup Succeeded In Measuring The Burst Pressure, Compliance, Creep And Cyclic Behavior Of Tissue-Engineered Vascular Media (Tevm), Adventitia (Teva) And A Combination Of A Media And An Adventitia (Tevma) Reconstructed By The Self-Assembly Method. Our Apparatus Has Proven To Be A Precise And Reliable Tool For The Characterization Of The Mechanical Properties Of Vascular Constructs. Auteur(s) : Larouche D, Cuffley K, Paquet C, Germain L. Titre : Tissue-engineered skin preserving the potential of epithelial cells to differentiate into hair after grafting. Référence : Tissue Eng Part A. 2011 Mar;17(5-6):819-30. Epub 2010 Dec 14. Lien : Cliquer ici Résumé : The Aim Of This Study Was To Evaluate Whether Tissue-Engineered Skin Produced In Vitro Was Able To Sustain Growth Of Hair Follicles In Vitro And After Grafting. Different Tissues Were Designed. Dissociated Newborn Mouse Keratinocytes Or Newborn Mouse Hair Buds (Hbs) Were Added Onto Dermal Constructs Consisting Of A Tissue-Engineered Cell-Derived Matrix Elaborated From Either Newborn Mouse Or Adult Human Fibroblasts Cultured With Ascorbic Acid. After 7-21 Days Of Maturation At The Air-Liquid Interface, No Hair Was Noticed In Vitro. Epidermal Differentiation Was Observed In All Tissue-Engineered Skin. However, Human Fibroblast-Derived Tissue-Engineered Dermis (Hd) Promoted A Thicker Epidermis Than Mouse Fibroblast-Derived Tissue-Engineered Dermis (Md). In Association With Md, Hbs Developed Epithelial Cyst-Like Inclusions Presenting Outer Root Sheath-Like Attributes. In Contrast, Epidermoid Cyst-Like Inclusions Lined By A Stratified Squamous Epithelium Were Present In Tissues Composed Of Hbs And Hd. After Grafting, Pilo-Sebaceous Units Formed And Hair Grew In Skin Elaborated From Hbs Cultured 10-26 Days Submerged In Culture Medium In Association With Md. However, The Number Of Normal Hair Follicles Decreased With Longer Culture Time. This Hair-Forming Capacity After Grafting Was Not Observed In Tissues Composed Of Hd Overlaid With Hbs. These Results Demonstrate That Epithelial Stem Cells Can Be Kept In Vitro In A Permissive Tissue-Engineered Dermal Environment Without Losing Their Potential To Induce Hair Growth After Grafting. Auteur(s) : Lavoie A, Fugère C, Fradette J, Larouche D, Paquet C, Beauparlant A, Gauvin R, Têtu FA, Roy A, Bouchard M, Genest H, Auger FA, Germain L. Titre : Considerations in the choice of a skin donor site for harvesting keratinocytes containing a high proportion of stem cells for culture in vitro. Référence : Burns. 2010 Nov 30. [Epub ahead of print] Lien : Cliquer ici Résumé : The Treatment Of Severely Burned Patients Has Benefited From The Grafting Of Skin Substitutes Obtained By Expansion Of Epithelial Cells In Culture. The Aim Of This Study Was To Evaluate Whether The Anatomic Site Chosen For Harvesting Skin Had An Impact On The Quality Of The Derived Cell Cultures. Considering That Hair Follicles Contain Epithelial Stem Cells, We Compared Hairy Skin Sites Featuring Different Densities And Sizes Of Hair Follicles For Their Capacity To Generate High Quality Keratinocyte Cultures. Three Anatomic Sites From Adult Subjects Were Compared: Scalp, Chest Skin And P-Auricular (Comprising Pre-Auricular And Post-Auricular) Skin. Keratin (K) 19 Was Used As A Marker To Evaluate The Proportion Of Stem Cells. Keratinocytes Were Isolated Using The Two-Step Thermolysin And Trypsin Cell Extraction Method, And Cultured In Vitro. The Proportion Of K19-Positive Cells Harvested From P-Auricular Skin Was About Twice That Of The Scalp. This K19-Positive Cell Content Also Remained Higher During The First Subcultures. In Contrast To These In Vitro Results, The Number Of K19-Positive Cells Estimated In Situ On Skin Sections Was About Double In Scalp As In P-Auricular Skin. Chest Skin Had The Lowest Number Of K19-Positive Cells. These Results Indicate That In Addition To The Choice Of An Adult Anatomic Site Featuring A High Number Of Stem Cells In Situ, The Quality Of The Cultures Greatly Depends On The Ability To Extract Stem Cells From The Skin Biopsy. Auteur(s) : Proulx S, Fradette J, Gauvin R, Larouche D, Germain L. Titre : Stem cells of the skin and cornea: their clinical applications in regenerative medicine. Référence : Curr Opin Organ Transplant. 2010 Dec 9. [Epub ahead of print] Lien : Cliquer ici Résumé : Purpose Of Review: The Use Of Stem Cells Is Of Great Interest For The Treatment Of Various Pathologies And Ultimately For The Restoration Of Organ Function. Progress Pointing Towards Future Treatments Of Skin And Corneal Epithelial Stem Cell Defects Are Reviewed, Including The Transplantation Of Living Tissue-Engineered Substitutes. Recent Findings: This Article Focuses On Substitutes Optimized For Permanent Replacement Of Skin And Cornea. New Skin Substitutes For Burn Care Are Currently Under Development. More Complex Tissue-Engineered Skin Substitutes In Which Stroma, Adipose Tissue, Capillaries, And Neurons Are Combined With The Epithelium Are Being Developed. Some Dermal/Epidermal Substitutes Have Been Applied To The Treatment Of Patients. Cultured Corneal Epithelial Cells Have Been Characterized And More Complete Corneal Substitutes Are Being Designed. Long-Term Clinical Results On The Transplantation Of Cultured Corneal Stem Cells For The Treatment Of Limbal Stem Cell Deficiency Have Been Reported. Summary: Advances In Tissue Engineering For The Development Of Substitutes That Will Benefit Patients Suffering From Skin Or Corneal Stem Cell Deficiencies Are Reviewed. These Products Are Often A Combination Of Cells, Scaffolds And Other Factors. Key Considerations In The Development Of Corneal And Skin Substitutes For Clinical Applications Are Discussed. Auteur(s) : Gagnon V, Larouche D, Parenteau-Bareil R, Gingras M, Germain L, Berthod F. Titre : Hair Follicles Guide Nerve Migration In Vitro and In Vivo in Tissue-Engineered Skin. Référence : J Invest Dermatol. 2011 Mar 3. [Epub ahead of print] Lien : Cliquer ici 2010 Auteur(s) : Moulin VJ, Mayrand D, Messier H, Martinez MC, Lopez-Vallé CA, Genest H. Titre : Shedding of microparticles by myofibroblasts as mediator of cellular cross-talk during normal wound healing. Référence : J Cell Physiol. 2010 Nov;225(3):734-40. Lien : Cliquer ici Résumé : Interactions Between Cells Are A Crucial Mechanism To Correctly Heal A Wounded Tissue. Myofibroblasts Have A Central Role During Healing But Their Means To Communicate With Other Cells Is Unknown. Microparticles (Mp) Have Demonstrated A Potential Role As Mediators Of Cellular Interactions During Various Diseases. We Have Analyzed The Production Of Mp By Normal (Wmyo) And Pathological (Hypertrophic Scar, Hmyo) Myofibroblasts And Human Dermal Fibroblasts (Fb) When Treated With Serum Or Plasma As Examples Of Body Fluids. We Have Shown That The Presence Of These Body Fluids Induced A Very Significant Increase In Mp Production By Wmyo While No Mp Production Was Denoted For Hmyo And Fb. These Effects Were At Least Due To Thermally Sensitive Protein(S) With A Molecular Mass >30 Kda. Furthermore, The Increase In Mp Production Was Not Linked To An Increase In Apoptotic Wmyo. Mp Characterization Showed That Vegf And Fgf2 Were Present In Mp And That Endothelial And (Myo)Fibroblast Cell Growth Can Be Stimulated By Mp Treatment. We Postulated That Mp Production By Myofibroblasts Could Modulate Mesenchymal Cell Growth And Angiogenesis During Normal Healing. Auteur(s) : Proulx S, d'Arc Uwamaliya J, Carrier P, Deschambeault A, Audet C, Giasson CJ, Guérin SL, Auger FA, Germain L. Titre : Reconstruction of a human cornea by the self-assembly approach of tissue engineering using the three native cell types. Référence : Mol Vis. 2010 Oct 29;16:2192-201. Lien : Cliquer ici Résumé : Purpose: The Purpose Of This Study Was To Produce And Characterize Human Tissue-Engineered Corneas Reconstructed Using All Three Corneal Cell Types (Epithelial, Stromal, And Endothelial Cells) By The Self-Assembly Approach. Methods: Fibroblasts Cultured In Medium Containing Serum And Ascorbic Acid Secreted Their Own Extracellular Matrix And Formed Sheets That Were Superposed To Reconstruct A Stromal Tissue. Endothelial And Epithelial Cells Were Seeded On Each Side Of The Reconstructed Stroma. After Culturing At The Air-Liquid Interface, The Engineered Corneas Were Fixed For Histology And Transmission Electron Microscopy (Tem). Immunofluorescence Labeling Of Epithelial Keratins, Basement Membrane Components, Na+/K+-Atpase α1, And Collagen Type I Was Also Performed. Results: Epithelial And Endothelial Cells Adhered To The Reconstructed Stroma. After 10 Days At The Air-Liquid Interface, The Corneal Epithelial Cells Stratified (4 To 5 Cell Layers) And Differentiated Into Well Defined Basal And Wing Cells That Also Expressed Na+/K+-Atpase α1 Protein, Keratin 3/12, And Basic Keratins. Basal Epithelial Cells From The Reconstructed Epithelium Formed Many Hemidesmosomes And Secreted A Well Defined Basement Membrane Rich In Laminin V And Collagen Vii. Endothelial Cells Formed A Monolayer Of Tightly-Packed Cells And Also Expressed The Function Related Protein Na+/K+-Atpase α1. Conclusions: This Study Demonstrates The Feasibility Of Producing A Complete Tissue-Engineered Human Cornea, Similar To Native Corneas, Using Untransformed Fibroblasts, Epithelial And Endothelial Cells, Without The Need For Exogenous Biomaterial. Auteur(s) : Berthod F, Germain L, Pouliot R, Auger FA Titre : How to Achieve Early Vascularization of Tissue-Engineered Skin Substitutes Référence : In: Advances in Wound Care, Translational Medicine: From Benchtop to Bedside to Community and Back, Eds: Chandan K. SenDirector, Comprehensive Wound Center The Ohio State University Medical Center Columbus, Ohio, Chap 72, pp 445-450 Résumé : The Coverage Of Deep And Extensive Burns With Autologous Tissue-Engineered Skin Is A Promising Strategy To Improve The Cosmetic Aspect And Functionality Of The Skin, Compared To The Transplantation Of Simple Epithelial Sheets. Indeed, A Dermal Compartment Could Markedly Help To Prevent Hypertrophic Scar Formation And To Strengthen The Dermal–Epidermal Junction While Increasing Skin Suppleness And Pliability. The Thickness Of The Dermis Could Be A Limitation To The Survival Of The Tissue After Transplantation, Since Its Vascularization Can Take Up To 2 Weeks To Occur Through Neovascularization. This Delay Could Lead To Graft Necrosis. To Overcome This Problem, The Reconstruction Of A Preformed Network Of Branching Capillaries In The Dermis Before Grafting Has Proven To Be An Efficient Solution In Connecting To The Host’S Vasculature In Less Than 4 Days After Grafting. The Formation Of This Capillary-Like Network Is Achieved By The Coculture Of Human Fibroblasts And Endothelial Cells In A Collagen Sponge For A 1-Month In Vitro Maturation Period. The Successful Inosculation Process Between Human Capillaries And The Host’S Vasculature Was Demonstrated After Grafting Onto Nude Mice. In Addition To Autologous Epithelial Sheets And Splitthickness Autografts, This Endothelialized Reconstructed Skin Made Of The Patient’S Own Cells Could Be A Valuable Additional Strategy To Permanently Cover Deep Wounds. The Reconstruction In Tissue-Engineered Organs Of A Capillary-Like Network Made Of The Patient’S Own Cells Before Grafting Is A Promising Approach To Promote Their Early Vascularization. Auteur(s) : Jean J, Pouliot R Titre : In vivo and in vitro models of psoriasis Référence : In: Tissue engineering, Eds: Eberli D, Chap 18, pp 359-382 Lien : Cliquer ici Résumé : Psoriasis Is A Very Complex Dermatosis Which Is Not Well Understood. No Cure Has Been Found For This Disease Yet. Although In Vitro And In Vivo Models Of Psoriasis Have Been Reported To Replicate Aspects Of The Disease, Research Into Psoriasis And The Subsequent Development Of Therapeutic Strategies, Has Been Hindered By The Absence Of Relevant Models. Each Model Is Based On A Slightly Different Pathogenic Mechanism, And Each Has Its Similarities To Psoriasis As Well As Its Limitations. In This Chapter, Different Sections Are Presented To Update Recent Progress In This Active Field Of Investigative Skin Biology. Highlights Of This Text Include A Complete Review Of In Vivo And In Vitro Models Of Psoriasis. Auteur(s) : Larouche D, Jean J, Berthod F, Germain L, Pouliot R Titre : Markers for an in vitro skin substitute Référence : In: Alternative technologies to animal testing, Methods in Bioengineering Series, Eds: Maguire T and Novik E, Chap 11, pp183-203 Résumé : The Tissue Engineering Self-Assembly Approach Allows The Production Of Skin Substitutes Comprising Both, The Dermis And Epidermis, Using Methods Promoting The Secretion And Organization Of A Dense Extracellular Matrix By Skin Cells. In A Reconstructed Epidermis, All Cellular Layers Of The Native Tissue Are Present. Evaluation Of The Expression And Localization Of A Number Of Specific Protein Markers Revealed That The Self-Assembled Tissue-Engineered Skin Substitute Shares Some Common Features With Normal Human Skin, Such As The Expression Of Ki-67, Keratins 10 And 14, Filaggrin, Involucrin, Transglutaminase, Dlk, α3-Integrin Subunit, Laminin-5, And Collagens I, Ii, Iv And Vii. At The Ultrastructural Level, Many Differentiation Markers Can Be Observed, Including Desmosomes, As Well As An Organized Basement Membrane Presenting Hemidesmosomes, Lamina Densa And Lamina Lucida. In This Chapter, Protocols To Generate Skin Substitutes By The Self-Assembly Approach Will Be Presented And The Methods Including The Labeling Of The Principal Skin Differentiation Markers By Immunofluorescence Will Be Examined. Auteur(s) : Rosa-Fortin, M.-M., Poubelle, P.E., Soucy, J., Pouliot, R. Titre : Cellular interactions in vitro:psoriatic keratinocytes enhance T lymphocyte survival Référence : Psoriasis Forum, 16, no.1, 2010, p.12-15. Lien : Cliquer ici Résumé : The Pathogenesis Of Psoriasis Vulgaris Is Attributed To An Immune Dysregulation Of T Lymphocytes, And Psoriatic Plaques Result From Interactions Between Keratinocytes And T Lymphocytes. To Decipher The Impact Of Keratinocyte-T Lymphocyte Interactions On Cell Survival During Active Cutaneous Psoriasis By Using A New In Vitro Model Of Psoriasis. Cocultures Of Psoriatic Or Healthy Keratinocytes Were Performed With Normal Human Blood T Lymphocytes. Apoptosis Was Detected By Flow Cytometry After Labeling Cells With Annexin V And Propidium Iodide. Psoriatic Keratinocytes Were Significantly More Efficient Than Healthy Keratinocytes In Enhancing T Lymphocyte Survival. Our In Vitro Model Is Pertinent To Understanding The Immunopathogenesis Of Psoriasis And To Discovering New Pharmacological Targets. Auteur(s) : García-Perez ME, Royer M, Duque-Fernandez A, Diouf PN, Stevanovic T, Pouliot R. Titre : Antioxidant, toxicological and antiproliferative properties of Canadian polyphenolic extracts on normal and psoriatic keratinocytes Référence : J Ethnopharmacol. 2010 Aug 18. [Epub ahead of print] Lien : Cliquer ici Résumé : In A First Attempt For Establishing The Possible Utilization Of Polyphenolic Extracts From Barks Of Canadian Wood Species In Psoriasis Treatment, We Aimed To Study (A) Their Antioxidant Capacity, (B) Their Toxicological Properties On Normal Human Keratinocytes (Nhk), And (C) Their Effect On The Growth Of Normal And Psoriatic Keratinocytes (Pk). Polyphenolic Extracts Were Obtained By 90% Ethanolic Maceration And Hot Water Extraction (Hwe). Scavenging Capacity Was Evaluated Towards Hydrogen Peroxide, Hydroxyl, Superoxide, Nitric Oxide And Peroxyl Radicals. Mtt Assay And Trypan Blue Dye Exclusion (Tbde) Method Were Used For Evaluating The Initial Toxicity Of The Most Antioxidant Extracts On Nhk During 24 And 48 H. The Effects Of Extracts On The Growth Of Nhk And Pk At Non-Toxic Concentrations Were Determined After Exposure For 48 H. Yellow Birch Extract Obtained By Maceration (Ybmac) And Black Spruce Extract Obtained By Hwe (Bshwe) Were Determined To Have The Highest Antioxidant Capacity, But Bshwe Was Less Toxic On Nhk. Toxicity Of Extracts On Keratinocyte Plasma Membrane And Mitochondria After 24 H Was Attributed To Their Content Of Hydroxycinnamic Acids And Proanthocyanidins. Bshwe Inhibited The Growth Of Nhk And Non-Lesional Pk, But Was Not Selective For Lesional Pk. Given That Bshwe Presented Elevated Content Of Total Phenols And Flavonoids And Showed A Low Toxicity On Nhk As Well As An Adequate Chemical Reactivity Towards Different Radicals And Some Antiproliferative Properties, It Was Considered As The Most Valuable Extract Obtained From Barks Of Canadian Wood Species. Auteur(s) : Guillemette MD, Gauvin R, Perron C, Labbé R, Germain L, Auger FA. Titre : Tissue-Engineered Vascular Adventitia with Vasa Vasorum Improves Graft Integration and Vascularization Through Inosculation. Référence : Tissue Eng Part A. 2010 May 10. [Epub ahead of print] Lien : Cliquer ici Résumé : Tissue-Engineered Blood Vessel Is One Of The Most Promising Living Substitutes For Coronary And Peripheral Artery Bypass Graft Surgery. However, One Of The Main Limitations In Tissue Engineering Is Vascularization Of The Construct Before Implantation. Such A Vascularization Could Play An Important Role In Graft Perfusion And Host Integration Of Tissue-Engineered Vascular Adventitia. Using Our Self-Assembly Approach, We Developed A Method To Vascularize Tissue-Engineered Blood Vessel Constructs By Coculturing Endothelial Cells In A Fibroblast-Laden Tissue Sheet. After Subcutaneous Implantation, Enhancement Of Graft Integration Within The Surrounding Environment Was Noted After 48 H And An Important Improvement In Blood Circulation Of The Grafted Tissue At 1 Week Postimplantation. The Distinctive Branching Structure Of End Arteries Characterizing The In Vivo Adventitial Vasa Vasorum Has Also Been Observed In Long-Term Postimplantation Follow-Up. After A 90-Day Implantation Period, Hybrid Vessels Containing Human And Mouse Endothelial Cells Were Still Perfused. Characterization Of The Mechanical Properties Of Both Control And Vascularized Adventitia Demonstrated That The Ultimate Tensile Strength, Modulus, And Failure Strain Were In The Same Order Of Magnitude Of A Pig Coronary Artery. The Addition Of A Vasa Vasorum To The Tissue-Engineered Adventitia Did Not Influence The Burst Pressure Of These Constructs. Hence, The Present Results Indicate A Promising Answer To The Many Challenges Associated With The In Vitro Vascularization And In Vivo Integration Of Many Different Tissue-Engineered Substitutes. Auteur(s) : Dube J, Rochette O, Levesque P, Gauvin R, Roberge C, Auger FA, Goulet D, Bourdages M, Plante M, Germain L, Moulin VJ. Titre : Restoration of the transepithelial potential within tissue-engineered human skin in vitro and during the wound healing process in vivo. Référence : Tissue Eng Part A. 2010 May 20. [Epub ahead of print] Lien : Cliquer ici Résumé : Normal Human Epidermis Possesses A Trans-Epithelial Potential (Tep) That Varies In Different Parts Of The Body (10 To 60 Mv). The Role Of Tep In Normal Epidermis Is Not Yet Identified But After Skin Injury, Tep Disruption Induces An Endogenous Direct Current Electric Field (100 To 200 Mv/Mm) Directed Toward The Middle Of The Wound. This Endogenous Electric Field Could Be Implicated In The Wound Healing Process By Attracting Cells Thus Facilitating Reepithelialization. However, Little Is Known On The Restoration Of The Tep During Human Skin Formation And Wound Healing. In The Present Study, The Variations In Tep And Na+/K+ Atpase Pump Expression During The Formation Of The Epithelium Were Investigated In Vitro Using Human Tissue-Engineered Skin (Tes) Reconstituted By Tissue Engineering And In Vivo With A Porcine Wound Healing Model. Results Showed That Tep Undergoes Ascending And Decreasing Phases During Epithelium Formation In Tes As Well As During Wound Repair Within Tes. Similar Results Were Observed During In Vivo Reepithelialization Of Wounds. The Ascending And Decreasing Tep Values Were Correlated With Changes In The Expression Of Na+/K+ Atpase Pump. The Distribution Of Na+/K+ Atpase Pumps Also Varied According To Epidermal Differentiation. Taken Together, These Results Suggest That The Variations In The Expression Of Na+/K+ Atpase Pump Over Time And Across Epidermis Would Be A Determinant Parameter Of The Tep, Dictating A Cationic Transport During The Formation And The Restoration Of The Epidermis. Therefore, This Study Brings A New Perspective To Understand The Formation And Restoration Of Tep During The Cutaneous Wound Healing Process. This Might Have Important Future Medical Applications Regarding The Treatment Of Chronic Wound Healing. Auteur(s) : Zaucha MT, Gauvin R, Auger FA, Germain L, Gleason RL. Titre : Biaxial biomechanical properties of self-assembly tissue-engineered blood vessels. Référence : J R Soc Interface. 2010 Jun 16. [Epub ahead of print] Lien : Cliquer ici Résumé : Along With Insights Into The Potential For Graft Success, Knowledge Of Biomechanical Properties Of Small Diameter Tissue-Engineered Blood Vessel (Tebv) Will Enable Designers To Tailor The Vessels' Mechanical Response To Closer Resemble That Of Native Tissue. Composed Of Two Layers That Closely Mimic The Native Media And Adventitia, A Tissue-Engineered Vascular Adventitia (Teva) Is Wrapped Around A Tissue-Engineered Vascular Media (Tevm) To Produce A Self-Assembled Tissue-Engineered Media/Adventia (Tevma). The Current Study Was Undertaken To Characterize The Biaxial Biomechanical Properties Of Tevm, Teva And Tevma Under Physiological Pressures As Well As Characterize The Stress-Free Reference Configuration. It Was Shown That The Teva Had The Greatest Compliance Over The Physiological Loading Range While The Tevm Had The Lowest Compliance. As Expected, Compliance Of The Sa-Tebv Fell In Between With An Average Compliance Of 2.73 Mpa(-1). Data Were Used To Identify Material Parameters For A Microstructurally Motivated Constitutive Model. Identified Material Parameters For The Teva And Tevm Provided A Good Fit To Experimental Data With An Average Coefficient Of Determination Of 0.918 And 0.868, Respectively. These Material Parameters Were Used To Develop A Two-Layer Predictive Model For The Response Of A Tevma Which Fit Well With Experimental Data. Auteur(s) : Moulin VJ, Mayrand D, Messier H, Martinez MC,Lopez-Vallé CA, Genest H. Titre : Shedding of microparticles by myofibroblasts as mediator of cellular cross-talk during normal wound healing Référence : J Cell Physiol. 2010 Jun 7. [Epub ahead of print] Lien : Cliquer ici Résumé : Interactions Between Cells Are A Crucial Mechanism To Correctly Heal A Wounded Tissue. Myofibroblasts Have A Central Role During Healing But Their Means To Communicate With Other Cells Is Unknown. Microparticles (Mp) Have Demonstrated A Potential Role As Mediators Of Cellular Interactions During Various Diseases. We Have Analyzed The Production Of Mp By Normal (Wmyo) And Pathological (Hypertrophic Scar, Hmyo) Myofibroblasts And Human Dermal Fibroblasts (Fb) When Treated With Serum Or Plasma As Examples Of Body Fluids. We Have Shown That The Presence Of These Body Fluids Induced A Very Significant Increase In Mp Production By Wmyo While No Mp Production Was Denoted For Hmyo And Fb. These Effects Were At Least Due To Thermally Sensitive Protein(S) With A Molecular Mass Greater Than 30 Kda. Furthermore, The Increase In Mp Production Was Not Linked To An Increase In Apoptotic Wmyo. Mp Characterization Showed That Vegf And Fgf2 Were Present In Mp And That Endothelial And (Myo)Fibroblast Cell Growth Can Be Stimulated By Mp Treatment. We Postulated That Mp Production By Myofibroblasts Could Modulate Mesenchymal Cell Growth And Angiogenesis During Normal Healing. (C) 2010 Wiley-Liss, Inc. Auteur(s) : Paquet C, Larouche D, Bisson F, Proulx S, Simard-Bisson C, Gaudreault M, Robitaille H, Carrier P, Martel I, Duranceau L, Auger FA, Fradette J, Guérin SL, Germain L. Titre : Tissue engineering of skin and cornea: Development of new models for in vitro studies. Référence : Ann N Y Acad Sci. 2010 Jun;1197:166-77. Lien : Cliquer ici Résumé : Human Beings Are Greatly Preoccupied With The Unavoidable Nature Of Aging. While The Biological Processes Of Senescence And Aging Are The Subjects Of Intense Investigations, The Molecular Mechanisms Linking Aging With Disease And Death Are Yet To Be Elucidated. Tissue Engineering Offers New Models To Study The Various Processes Associated With Aging. Using Keratin 19 As A Stem Cell Marker, Our Studies Have Revealed That Stem Cells Are Preserved In Human Skin Reconstructed By Tissue Engineering And That The Number Of Epithelial Stem Cells Varies According To The Donor'S Age. As With Skin, Human Corneas Can Also Be Engineered In Vitro. Among The Epithelial Cells Used For Reconstructing Skin And Corneas, Significant Age-Dependent Variations In The Expression Of The Transcription Factor Sp1 Were Observed. Culturing Skin Epithelial Cells With A Feeder Layer Extended Their Life Span In Culture, Likely By Preventing Sp1 Degradation In Epithelial Cells, Therefore Demonstrating The Pivotal Role Played By This Transcription Factor In Cell Proliferation. Finally, Using The Human Tissue-Engineered Skin As A Model, We Linked Hsp27 Activation With Skin Differentiation. Auteur(s) : Gibot L, Galbraith T, Huot J, Auger FA. Titre : A preexisting microvascular network benefits in vivo revascularization of a microvascularized tissue-engineered skin substitute. Référence : Tissue Eng Part A. 2010 Jun 9. [Epub ahead of print] Lien : Cliquer ici Résumé : Delayed Or Absence Of Vascularization Is One Of The Major Reasons For Skin Engraftment Failure In Patients With Extensive Burns. For Such Trauma Victims, The Best Alternative To A Split-Thickness Graft Would Be Wound Coverage With An Autologous In Vitro Reconstructed Skin (Rs) Combining Dermis And Epidermis With An Appropriate Microvascularization. We Have Developed A Microvascularized Reconstructed Skin (Ers) Based On Our Self-Assembly Approach, Which Is Generated From Autologous Cultured Cells Without Any Exogenous Angiogenic Growth Factor Or Scaffold. After Transplantation In Athymic Mice, An Early Inosculation Between The Graft And Host Vasculatures Occurred Within 4 Days. We Also Concurrently Detected An Active Invasion Of The Dermis By Host Capillaries Sprouting From The Wound Bed. Thus, The Microvascular Network Constructed In Vitro Within Our 3D Skin Substitute Did Not Only Develop Functional Anastomoses With The Host'S Blood Vessels But Also Promoted A Rapid, Complete And Optimal Vascularization Of The Implanted Tissues By Exerting An Angiogenic Effect Compared To Control Rs. Our Model May Bring About Interesting Possibilities For Regenerative Medicine By Leading To Faster Vascularization In Clinical Applications. In Addition, The Ers Can Be A Useful In Vitro Angiogenesis Model. Auteur(s) : Bouhout S, Perron E, Gauvin R, Bernard G, Ouellet G, Cattan V, Bolduc S. Titre : In Vitro Reconstruction of an Autologous, Watertight, and Resistant Vesical Equivalent. Référence : Tissue Eng Part A. 2010 Feb 11. [Epub ahead of print] Lien : Cliquer ici Résumé : Purpose: Currently, Bladder Repair Is Performed Using Gastrointestinal Segments; However, This Technique Has A High Morbidity Rate, And New Alternatives Are Thus Needed. The Lack Of Native Or Synthetic Tissue With Similar Properties Of The Bladder Led Us To Develop Autologous Vesical Substitutes Entirely Made By Tissue Engineering And Without Exogenous Matrices. Watertight Function And Mechanical Resistance Are Fundamental For The Model. The Aim Of This Study Was To Determine The Structural And Functional Characteristics Of Our Vesical Equivalent (Ve). Materials And Methods: Porcine Ves Are Produced In 55 Days. The Cellular Types That Make Up The Vesical Wall Are Extracted And Purified Simultaneously From A Small Porcine Bladder Biopsy. Dermal Fibroblasts Are Extracted And Cultured In Vitro To Form Cellular Sheets. Endothelial Cells Were Seeded On The Fibroblast Sheets Before Their Superimposition. Urothelial Cells Are Then Seeded Onto This Cellular Construction. Ves Are Characterized By Histology, Immunostaining, Electron Microscopy, And Cell Viability. Mechanical Properties Of The Reconstructed Substitutes Are Evaluated By Uniaxial Tensile Tests, And Tissue Absorption Is Verified With (14)C-Urea, Which Quantifies The Degree Of Impermeability. Results: This Process Allowed Us To Obtain A Highly Structured Tissue With A Total Fusion Of The Fibroblast Layers. As Expected, Histological Observations Showed A Pseudostratification Of The Urothelium Developing On An Organized Self-Secreted Extracellular Matrix. Positive Markers For Cytokeratin 8/18 In Immunostaining Confirmed The Presence Of A Urinary Epithelium. Electron Microscopy Confirmed The Normal Aspect Of Urothelial Cells. Our Ve'S Permeability To (14)C-Urea Was Significantly Similar To Porcine Bladder, And Characterization Of The Mechanical Properties Indicated That Our Tissue Could Be Suitable For Grafting Since Its Ultimate Tensile Strength Compares Favorably With A Native Porcine Bladder. Conclusion: The Construction Of A Ve Using This Method Seems Very Promising In Meeting The Needs In The Urological Field. Our Substitute Has Proven Its Efficiency As A Barrier To Urea And Has A Sufficient Mechanical Resistance To Support Suturing. Additionally, This Model Is Completely Autologous, And Its Possible Endothelialization Could Promote The Early Vascularization Process After Grafting And Thus Significantly Reducing Inflammation And Possible Rejection. Auteur(s) : Rochon MH, Fradette J, Fortin V, Tomasetig F, Roberge C, Baker K, Berthod F, Auger FA, Germain L. Titre : Normal human epithelial cells regulate the size and morphology of tissue-engineered capillaries. Référence : Tissue Eng Part A. 2009 Nov 25. [Epub ahead of print] Lien : Cliquer ici Résumé : The Survival Of Thick Tissues/Organs Produced By Tissue Engineering Requires Rapid Revascularization After Grafting. Although Capillary-Like Structures Have Been Reconstituted In Some Engineered Tissues, Little Is Known About The Interaction Between Normal Epithelial Cells And Endothelial Cells Involved In The In Vitro Angiogenic Process. In The Present Study, We Used The Self-Assembly Approach Of Tissue Engineering To Examine This Relationship. An Endothelialized Tissue-Engineered Dermal Substitute (Eted) Was Produced By Adding Endothelial Cells To The Tissue-Engineered Dermal Substitute (Ted) Produced By The Self-Assembly Approach. The Latter Consists In Culturing Fibroblasts In Medium Supplemented With Serum And Ascorbic Acid. A Network Of Tissue-Engineered Capillaries (Tecs) Formed Within The Human Extracellular Matrix Produced By Dermal Fibroblasts. To Determine Whether Epithelial Cells Modify Tecs, The Size And Form Of Tecs Were Studied In Eted Cultured In The Presence Or Absence Of Epithelial Cells. In The Presence Of Normal Keratinocytes From Skin, Cornea Or Uterine Cervix, Endothelial Cells Formed Small Tecs (Cross Sectional Area Estimated At Less Than 50 Mum2) Reminiscent Of Capillaries Found In The Skin'S Microcirculation. In Contrast, Tecs Grown In The Absence Of Epithelial Cells Presented Variable Sizes (Larger Than 50 Mum2), But The Addition Of Keratinocyte-Conditioned Media Or Exogenous Vascular Endothelial Growth Factor (Vegf) Induced Their Normalization Towards A Smaller Size. Vegf Neutralization Inhibited The Effect Of Keratinocyte-Conditioned Media. These Results Provide New Direct Evidence That Normal Human Epithelial Cells Play A Role In The Regulation Of The Underlying Tec Network, And Advance Our Knowledge In Tissue Engineering For The Production Of Tissue-Engineered Capillary Networks In Vitro. Auteur(s) : Gauvin R, Ahsan T, Larouche D, Levesque P, Dubé J, Auger FA, Nerem RM, Germain L. Titre : A Novel Single-Step Self-Assembly Approach for the Fabrication of Tissue-Engineered Vascular Constructs. Référence : Tissue Eng Part A. 2009 Dec 28. [Epub ahead of print] Lien : Cliquer ici Auteur(s) : Larouche D, Lavoie A, Paquet C, Simard-Bisson C, Germain L. Titre : Identification of Epithelial Stem Cells In Vivo and In Vitro Using Keratin 19 and BrdU. Référence : Methods Mol Biol. 2010;585:383-400. Lien : Cliquer ici Résumé : Progress In The Identification Of Skin Stem Cells And The Improvement Of Culture Methods Open The Possibility To Use Stem Cells In Regenerative Medicine. Based On Their Quiescent Nature, The Development Of Label Retention Assays Allowed The Localization Of Skin Stem Cells In The Bulge Region Of The Pilosebaceous Units And In The Bottom Of Rete Ridges In Glabrous Skin. The Development Of Markers Such As Keratin 19 Also Permits Their Study In Human Tissues. In This Chapter, Protocols To Identify Skin Stem Cells Based On Their Slow-Cycling Property And Their Expression Of Keratin 19 Will Be Described In Detail. The Methods Include The Labeling Of Skin Stem Cells Within Mouse Or Rat Tissues In Vivo, The Labeling Of Proliferative Human Cells In Vitro Using 5-Bromo-2-Deoxyuridine (Brdu), And The Detection Of Keratin 19 And Brdu By Immunofluorescence Or Auteur(s) : Robitaille H, Simard-Bisson C, Larouche D, Tanguay RM, Blouin R, Germain L. Titre : The Small Heat-Shock Protein Hsp27 Undergoes ERK-Dependent Phosphorylation and Redistribution to the Cytoskeleton in Response to Dual Leucine Zipper-Bearing Kinase Expression. Référence : Journal Invest Dermatol, Jan;130(1):74-85. Lien : Cliquer ici Résumé : Hsp27, A Small Heat-Shock Protein, Has Important Roles In Many Cellular Processes, Including Cytoskeleton Dynamics, Cell Differentiation, And Apoptosis. Its Expression In Normal Epidermis Correlates With Differentiation; However, Little Is Known About The Regulatory Mechanisms Involved. In This Study, We Report That Hsp27 Undergoes Upregulation, Phosphorylation, And Redistribution To The Cytoskeleton During The Late Phase Of Epidermal Keratinocyte Differentiation. Our Results Also Show That The Expression Of The Dual Leucine Zipper-Bearing Kinase (Dlk), An Upstream Activator Of The Map Kinase Pathways, Is Sufficient By Itself To Induce Hsp27 Phosphorylation, Cell Periphery Localization, And Redistribution To The Insoluble Protein Fraction (Cytoskeleton) In Poorly Differentiated Keratinocytes. This Redistribution Correlates With The Insolubilization Of Cornified Envelope-Associated Proteins Such As Involucrin. Interestingly, The Effects Of Dlk On Hsp27 Were Blocked By Pd98059, A Selective Inhibitor Of The Extracellular Signal-Regulated Protein Kinase (Erk) Pathway. Moreover, Downregulation Of Hsp27 By Small Interfering Rna In Epithelial Cells Expressing Dlk Was Accompanied By Attenuated Expression Of Involucrin In The Cytoskeleton. Thus, These Observations Suggest That The Dlk-Erk Signaling Pathway May Act As A Regulator Of The Interaction That Occurs Between Hsp27 And The Cytoskeleton During The Formation Of The Cornified Cell Envelope, A Process Conferring To The Skin Its Crucial Barrier Function.Journal Of Investigative Dermatology Advance Online Publication, 13 August 2009; Doi:10.1038/Jid.2009.185. 2009 Auteur(s) : Snapyan M, Lemasson M, Brill MS, Blais M, Massouh M, Ninkovic J, Gravel C, Berthod F, Götz M, Barker PA, Parent A, Saghatelyan A. Titre : Vasculature guides migrating neuronal precursors in the adult mammalian forebrain via brain-derived neurotrophic factor signaling. Référence : J Neurosci. 2009 Apr 1;29(13):4172-88. Lien : Cliquer ici Résumé : Adult Neuronal Precursors Retain The Remarkable Capacity To Migrate Long Distances From The Posterior (Subventricular Zone) To The Most Anterior [Olfactory Bulb (Ob)] Parts Of The Brain. The Knowledge About The Mechanisms That Keep Neuronal Precursors In The Migratory Stream And Organize This Long-Distance Migration Is Incomplete. Here We Show That Blood Vessels Precisely Outline The Migratory Stream For New Neurons In The Adult Mammalian Forebrain. Real-Time Video Imaging Of Cell Migration In The Acute Slices Demonstrate That Neuronal Precursors Are Retained In The Migratory Stream And Guided Into The Ob By Blood Vessels That Serve As A Physical Substrate For Migrating Neuroblasts. Our Data Suggest That Endothelial Cells Of Blood Vessels Synthesize Brain-Derived Neurotrophic Factor (Bdnf) That Fosters Neuronal Migration Via P75Ntr Expressed On Neuroblasts. Interestingly, Gaba Released From Neuroblasts Induces Ca(2+)-Dependent Insertion Of High-Affinity Trkb Receptors On The Plasma Membrane Of Astrocytes That Trap Extracellular Bdnf. We Hypothesize That This Renders Bdnf Unavailable For P75Ntr-Expressing Migrating Cells And Leads To Their Entrance Into The Stationary Period. Our Findings Provide New Insights Into The Functional Organization Of Substrates That Facilitate The Long-Distance Journey Of Adult Neuronal Precursors. Auteur(s) : Rochon MH, Fradette J, Fortin V, Tomasetig F, Roberge C, Baker K, Berthod F, Auger FA, Germain L. Titre : Normal human epithelial cells regulate the size and morphology of tissue-engineered capillaries. Référence : Tissue Eng Part A. 2009 Nov 25. [Epub ahead of print] Lien : Cliquer ici Résumé : The Survival Of Thick Tissues/Organs Produced By Tissue Engineering Requires Rapid Revascularization After Grafting. Although Capillary-Like Structures Have Been Reconstituted In Some Engineered Tissues, Little Is Known About The Interaction Between Normal Epithelial Cells And Endothelial Cells Involved In The In Vitro Angiogenic Process. In The Present Study, We Used The Self-Assembly Approach Of Tissue Engineering To Examine This Relationship. An Endothelialized Tissue-Engineered Dermal Substitute (Eted) Was Produced By Adding Endothelial Cells To The Tissue-Engineered Dermal Substitute (Ted) Produced By The Self-Assembly Approach. The Latter Consists In Culturing Fibroblasts In Medium Supplemented With Serum And Ascorbic Acid. A Network Of Tissue-Engineered Capillaries (Tecs) Formed Within The Human Extracellular Matrix Produced By Dermal Fibroblasts. To Determine Whether Epithelial Cells Modify Tecs, The Size And Form Of Tecs Were Studied In Eted Cultured In The Presence Or Absence Of Epithelial Cells. In The Presence Of Normal Keratinocytes From Skin, Cornea Or Uterine Cervix, Endothelial Cells Formed Small Tecs (Cross Sectional Area Estimated At Less Than 50 Mum2) Reminiscent Of Capillaries Found In The Skin'S Microcirculation. In Contrast, Tecs Grown In The Absence Of Epithelial Cells Presented Variable Sizes (Larger Than 50 Mum2), But The Addition Of Keratinocyte-Conditioned Media Or Exogenous Vascular Endothelial Growth Factor (Vegf) Induced Their Normalization Towards A Smaller Size. Vegf Neutralization Inhibited The Effect Of Keratinocyte-Conditioned Media. These Results Provide New Direct Evidence That Normal Human Epithelial Cells Play A Role In The Regulation Of The Underlying Tec Network, And Advance Our Knowledge In Tissue Engineering For The Production Of Tissue-Engineered Capillary Networks In Vitro. Auteur(s) : Couture JP, Daviau A, Fradette J, Blouin R. Titre : The mixed-lineage kinase DLK is a key regulator of 3T3-L1 adipocyte differentiation. Référence : PLoS One. 4(3):e4743. Epub 2009 Mar 9. Résumé : Background: The Mixed-Lineage Kinase (Mlk) Family Member Dlk Has Been Proposed To Serve As A Regulator Of Differentiation In Various Cell Types; However, Its Role In Adipogenesis Has Not Been Investigated. In This Study, We Used The 3T3-L1 Preadipocyte Cell Line As A Model To Examine The Function Of Dlk In Adipocyte Differentiation. Methods And Findings: Immunoblot Analyses And Kinase Assays Performed On 3T3-L1 Cells Showed That The Expression And Activity Of Dlk Substantially Increase As Differentiation Occurs. Interestingly, Dlk Appears Crucial For Differentiation Since Its Depletion By Rna Interference Impairs Lipid Accumulation As Well As Expression Of The Master Regulators Of Adipogenesis C/Ebpalpha And Ppargamma2 At Both The Mrna And Protein Levels. In Contrast, Neither The Expression Nor The Dna Binding Activity Of C/Ebpbeta, An Activator For C/Ebpalpha And Ppargamma, Is Affected By Dlk Loss. Conclusions: Taken Together, These Results Suggest That Dlk Is Important For Expression Of Mature Adipocyte Markers And That Its Action Most Likely Takes Place Via Regulation Of C/Ebpbeta Transcriptional Activity And/Or Initiation Of C/Ebpalpha And Ppargamma2 Gene Transcription. Auteur(s) : Guillemette M., Cui B., Roy E., 1Gauvin R., Giasson C., Esch M.B., 1Carrier P., 1Deschambeault A., Dumoulin M., Toner M., Germain L., Veres T., Auger F.A. Titre : Surface topography induces 3D self-orientation of cells and extracellular matrix resulting in improved tissue function. Référence : Integrative Biology, 1: 196-204, 2009. Lien : Cliquer ici Résumé : The Organization Of Cells And Extracellular Matrix (Ecm) In Native Tissues Plays A Crucial Role In Their Functionality. However, In Tissue Engineering, Cells And Ecm Are Randomly Distributed Within A Scaffold. Thus, The Production Of Engineered-Tissue With Complex 3D Organization Remains A Challenge. In The Present Study, We Used Contact Guidance To Control The Interactions Between The Material Topography, The Cells And The Ecm For Three Different Tissues, Namely Vascular Media, Corneal Stroma And Dermal Tissue. Using A Specific Surface Topography On An Elastomeric Material, We Observed The Orientation Of A First Cell Layer Along The Patterns In The Material. Orientation Of The First Cell Layer Translates Into A Physical Cue That Induces The Second Cell Layer To Follow A Physiologically Consistent Orientation Mimicking The Structure Of The Native Tissue. Furthermore, Secreted Ecm Followed Cell Orientation In Every Layer, Resulting In An Oriented Self-Assembled Tissue Sheet. These Self-Assembled Tissue Sheets Were Then Used To Create 3 Different Structured Engineered-Tissue: Cornea, Vascular Media And Dermis. We Showed That Functionality Of Such Structured Engineered-Tissue Was Increased When Compared To The Same Non-Structured Tissue. Dermal Tissues Were Used As A Negative Control In Response To Surface Topography Since Native Dermal Fibroblasts Are Not Preferentially Oriented In Vivo. Non-Structured Surfaces Were Also Used To Produce Randomly Oriented Tissue Sheets To Evaluate The Impact Of Tissue Orientation On Functional Output. This Novel Approach For The Production Of More Complex 3D Tissues Would Be Useful For Clinical Purposes And For In Vitro Physiological Tissue Model To Better Understand Long Standing Questions In Biology. Auteur(s) : Zaucha MT, Raykin J, Wan W, Gauvin R, Auger FA, Germain L, Michaels TE, Gleason R. Titre : A novel cylindrical biaxial computer controlled bioreactor and biomechanical testing device for vascular tissue engineering. Zaucha MT, Raykin J, Wan W, Gauvin R, Auger FA, Germain L, Michaels TE, Gleason R. Référence : Tissue Eng Part A. 2009 Apr 22. [Epub ahead of print] Lien : Cliquer ici Résumé : It Is Becoming Increasingly Obvious That Tissue Engineered Constructs Adapt To Altered Mechanical Loading, And That Specific Combinations Of Multidirectional Loads Appear To Have A Synergistic Effect On The Remodeling. However, Most Studies Of Mechanical Stimulation Of Engineered Vascular Tissue Engineering Employ Only Uniaxial Stimulation. Here We Present A Novel Computer-Controlled Bioreactor And Biomechanical Testing Device Designed To Precisely And Simultaneously Control Mean And Cyclic Values Of Transmural Pressure (At Rates Up To 1 Hz And Ranges Of 40Mmhg), Luminal Flow Rate, And Axial Length (Or Load) Applied To Gel-Derived, Scaffold-Derived, And Self Assembly-Derived Tissue Engineered Blood Vessels (Tebv) Over Long-Term Culture, While Monitoring Vessel Geometry With A Resolution Of 6.6 Microns. Intermittent Monitoring Of The Ecm And Cells Is Accomplished On Live Tissues Using Multi-Photon Confocal Microscopy Under Unloaded And Loaded Conditions At Multiple Time-Points In Culture (On The Same Vessel) To Quantify Changes In Cell And Ecm Content And Organization. This Same Device Is Capable Of Performing Intermittent Cylindrical Biaxial Biomechanical Testing At Multiple Time-Points In Culture (On The Same Vessel) To Quantify Changes In The Mechanical Behavior During Culture. Here We Demonstrate The Capabilities Of This New Device On Self Assembly-Derived And Collagen Gel-Derived Tissue Engineered Blood Vessels. Auteur(s) : Barolet D, Roberge CJ, Auger FA, Boucher A, Germain L. Titre : Regulation of Skin Collagen Metabolism In Vitro Using a Pulsed 660 nm LED Light Source: Clinical Correlation with a Single-Blinded Study. Barolet D, Roberge CJ, Auger FA, Boucher A, Germain L. Référence : J Invest Dermatol. 2009 Jul 9. [Epub ahead of print] Lien : Cliquer ici Résumé : It Has Been Reported That Skin Aging Is Associated With A Downregulation In Collagen Synthesis And An Elevation In Matrix Metalloproteinase (Mmp) Expression. This Study Investigated The Potential Of Light-Emitting Diode (Led) Treatments With A 660 Nm Sequentially Pulsed Illumination Formula In The Photobiomodulation Of These Molecules. Histological And Biochemical Changes Were First Evaluated In A Tissue-Engineered Human Reconstructed Skin (Hrs) Model After 11 Sham Or Led Light Treatments. Led Effects Were Then Assessed In Aged/Photoaged Individuals In A Split-Face Single-Blinded Study. Results Yielded A Mean Percent Difference Between Led-Treated And Non-Led-Treated Hrs Of 31% In Levels Of Type-1 Procollagen And Of -18% In Mmp-1. No Histological Changes Were Observed. Furthermore, Profilometry Quantification Revealed That More Than 90% Of Individuals Showed A Reduction In Rhytid Depth And Surface Roughness, And, Via A Blinded Clinical Assessment, That 87% Experienced A Reduction In The Fitzpatrick Wrinkling Severity Score After 12 Led Treatments. No Adverse Events Or Downtime Were Reported. Our Study Showed That Led Therapy Reversed Collagen Downregulation And Mmp-1 Upregulation. This Could Explain The Improvements In Skin Appearance Observed In Led-Treated Individuals. These Findings Suggest That Led At 660 Nm Is A Safe And Effective Collagen-Enhancement Strategy.Journal Of Investigative Dermatology Advance Online Publication, 9 July 2009; Doi:10.1038/Jid.2009.186. Auteur(s) : Blais M, Grenier M, Berthod F. Titre : Improvement of Nerve Regeneration in Tissue-Engineered Skin Enriched with Schwann Cells. Référence : J Invest Dermatol. 2009 Jul 9. [Epub ahead of print] Lien : Cliquer ici Résumé : The Incorporation Of Schwann Cells In Reconstructed Skin (Rs) Could Have A Major Role In Achieving Functional Recovery Of Cutaneous Sensory Perception. We Showed With A Unique In Vitro Model Of A Tissue-Engineered Innervated Reconstructed Dermis That Schwann Cells Promoted A Twofold Increase In The Number Of Sensory Neurites Migrating In The Three-Dimensional Tissue As Compared With The Control. In Addition, Schwann Cells Spontaneously Colocalized Along Neurites And Achieved The Formation Of Myelin Sheaths In Vitro As Assessed By Transmission Electron Microscopy. We Prepared Rs Samples Enriched Or Not With Schwann Cells And Transplanted Them On Nude Mice For 60-90 Days. We Demonstrated That Schwann Cells Induced A 1.8- And 1.7-Fold Increase In The Number Of Nerve Fibers Migrating In The Graft 60 And 90 Days After Transplantation, Respectively. In Addition, The Rs Sample Enriched With Schwann Cells Had A Current Perception Threshold Similar To That Of Normal Skin For The Large And Myelinated Abeta-Sensory Fibers, In Contrast With The Control. Thus, We Showed That The Addition Of Schwann Cells To Tissue-Engineered Skin Not Only Enhanced Nerve Migration But Also Promoted Myelin Sheath Formation In Vitro And Nerve Function Recovery In Vivo.Journal Of Investigative Dermatology Advance Online Publication, 9 July 2009; Doi:10.1038/Jid.2009.159. Auteur(s) : Beaulieu MM, Tremblay PL, Berthod F. Titre : Tissue-engineered models of the nervous system Référence : Med Sci. 2009 Mar;25(3):288-92. Lien : Cliquer ici Résumé : The Nervous System Is Extraordinarily Complex And Exposed To Various Trauma And Degenerative Diseases That Remain Difficult To Treat. To Facilitate Its Study, In Vitro Models Were Developed By Culturing Neurons And Glial Cells In Monolayer Cultures, Or Through Organotypic Cultures Of Brain Or Spinal Cord Slices. These In Vitro Models Were, And Are Still Very Helpful For The Advancement Of Neurosciences. However, They Are For Some Studies, Either Overly Simplified, Or Too Complex. The Application Of Tissue Engineering To Neurosciences Offers A New And Highly Versatile Approach To Develop Accurate Models Of The Nervous System. These Models Can Be Engineered In Three-Dimensions While Choosing For Each Individual Component, Cellular And Molecular, That Will Compose It. The Level Of Complexity Of The Model Can Be Adjusted From The Simplest To The More Complete As Needed. For Example, Through The Use Of A Three-Dimensional Tissue-Engineered Model Of The Spinal Cord, It Was Possible To Reproduce The Process Of Myelin Sheath Formation Around Motor Neuron Axons For The First Time In Vitro. This Breakthrough Shows The Promising Potential Of Tissue Engineering In The Development Of Powerful In Vitro Models Of The Nervous System. The Combination Of These Models With The Use Of Human Adult Neurons And Glial Cells Obtained From The Differentiation Of Neural Precursor Cells Isolated From Accessible Tissues From Patients (Skin, Fat, Bone Marrow), Opens Promising Perspectives To Better Understand -Neurodegenerative Diseases. Auteur(s) : Auger F.A., Lacroix D., Germain L. Titre : Skin substitutes and wound healing. Référence : Skin Pharmacol Physiol. 2009;22(2):94-102. Lien : Cliquer ici Résumé : Medical Science Has Vastly Improved On The Means And Methods Available For The Treatment Of Wounds In The Clinic. The Production And Use Of Various Types Of Skin Substitutes Has Led To Dramatic Improvements In The Odds Of Survival For Severely Burned Patients, But They Have Also Shown Promise For Many Other Applications, Including Cases Involving Chronic Wounds That Are Not Life Threatening. Nowadays, More Than 20 Products Are Commercially Available, More Are Undergoing Clinical Trials And A Large Number Of New Models Are Being Investigated In Various Research Laboratories Worldwide. Many Of The Current Products Do Not Contain Any Living Cells And Vary In Their Capacity To Harness The Innate Capacity Of The Body To Heal Itself. Others Include Living Cells, Of Allogeneic Or Autologous Origin, And Are Often Referred To As 'Cellular Therapy' Or 'Tissue-Engineered' Products. Modifications And Improvements Are Currently Investigated That Aim At Improving The Healing Potential Of Those Products Through The Use Of Recombinant Growth Factors And Additional Features Such As Microvascularization. Fundamental Research Into Wound Healing And Scar-Free Regeneration Raises The Hope That We Will Eventually Be Able To Restore Almost Completely The Appearance And Function Of Skin After The Healing Of Wounds. Auteur(s) : Carrier P., Deschambeault A., Audet C., Talbot M., Gauvin R., Giasson C.J., Auger F.A., Guérin S.L., Germain L. Titre : Impact of cell source on human cornea reconstructed by tissue engineering Référence : Invest Ophthalmol Vis Sci. 2009 Jun;50(6):2645-52. Lien : Cliquer ici Résumé : Purpose. Investigate The Effect Of The Tissue Origin Of Stromal Fibroblasts And Epithelial Cells On The Reconstructed Corneas In Vitro. Methods. Four Types Of Constructs Were Produced By The Self-Assembly Approach Using The Following Combinations Of Human Cells: Corneal Fibroblasts/Corneal Epithelial Cells, Corneal Fibroblasts/Skin Epithelial Cells, Skin Fibroblasts/Corneal Epithelial Cells, Skin Fibroblasts/Skin Epithelial Cells. Fibroblasts Were Cultured With Ascorbic Acid To Produce Stromal Sheets On Which Epithelial Cells Were Cultured. Following Two Weeks At The Air/Liquid Interface, The Reconstructed Tissues Were Photographed, Absorption Spectra Measured, And Tissues Fixed For Histological Analysis. The Cytokine Expression In Corneal- Or Skin-Fibroblasts-Conditioned Media Was Determined Using Protein Array Membranes. The Effect Of Culturing Reconstructed Tissues With Conditioned Media, Or Media Supplemented With A Cytokine Secreted Mainly By Corneal Fibroblasts Was Determined. Results. The Tissue Source From Which Both Epithelial And Mesenchymal Cells Were Isolated Had A Great Impact On The Macroscopic And Histological Features (Epithelium Thickness And Differentiation) As Well As The Functional Properties (Transparency) Of The Reconstructed Tissues. The Reconstructed Cornea Had Ultraviolet-Absorption Characteristics Resembling Those Of Native Human Cornea. The Regulation Of Epithelial Differentiation And Thickness Was Mesenchyme-Dependent And Mediated By Diffusible Factors. Interleukin-6 (Il-6), Which Is Secreted In Greater Amounts By Corneal Fibroblasts Than Skin Fibroblasts, Decreased The Expression Of The Differentiation Marker Dlk In The Reconstructed Epidermis. Conclusions. The Tissue Origin Of Both Fibroblasts And Epithelial Cells Plays A Significant Role On The Properties Of The Reconstructed Tissues. These Human Models Are Promising Tools To Gain A Thorough Understanding Of Epithelial-Stromal Interactions And Regulation Of Epithelia Homeostasis. Auteur(s) : Proulx S, Bensaoula T, Nada O, Audet C, Uwamaliya JD, Devaux A, Allaire G, Germain L, Brunette I. Titre : Transplantation of a tissue-engineered corneal endothelium reconstructed on a devitalized carrier in the feline model. Référence : Invest Ophthalmol Vis Sci. 2009 Jun;50(6):2686-94. Lien : Cliquer ici Résumé : Purpose. To Evaluate The Functional Outcome Of A Tissue-Engineered Corneal Endothelium Reconstructed On A Devitalized Carrier And Transplanted In The Living Feline Model. Methods. Eighteen Healthy Adult Cats Underwent Full Thickness Corneal Transplantation. In 11 Animals, The Donor Cornea Was Reconstructed From Cultured Allogeneic Feline Corneal Endothelial Cells Seeded On The Denuded Descemet'S Membrane Of A Devitalized Human Cornea. The Reconstructed Corneal Endothelium Was Cultured For Two Weeks Before Transplantation. Five Control Animals Received Either An Autologous (N=1), An Allogeneic (N=3) Or A Human Xenogeneic (N=1) Native Cornea. Two Other Control Animals Were Grafted With The Devitalized Carrier Only (No Cells). Animals Were Observed Daily By Slit-Lamp Until Euthanasia On Day Seven. Postmortem Analysis Included Optical Coherence Tomography (Oct), Alizarin Red Staining, Histology, Fluorescence Microscopy, Scanning (Sem) And Transmission (Tem) Electron Microscopy. Results. Nine Of The 11 Reconstructed Corneal Endothelial Grafts And All 5 Native (Autologous, Allogeneic, And Xenogeneic) Control Grafts Were Clear And Thin Seven Days After Grafting. In Contrast, The Two Control Grafts Consisting In The Carrier Only (Without Endothelium) Remained Thick And Opaque. Alizarin Red Staining, Histology, Sem And Tem Showed That The Transplanted Reconstructed Endothelium Maintained A Normal Morphology And Ultrastructure, And Expressed The Function-Related Proteins Na+/K+-Atpase Alpha1, Na+/Hco3 And Zo-1. Conclusions. This Study Provides Evidence For The Short Term (Seven Days) Anatomical And Functional Success Of Corneal Transplantation With A Tissue-Engineered Corneal Endothelium Reconstructed On A Devitalized Carrier. Auteur(s) : Auxenfans C, Fradette J, Lequeux C, Germain L, Kinikoglu B, Bechetoille N, Braye F, Auger FA, Damour O. Titre : Evolution of three dimensional skin equivalent models reconstructed in vitro by tissue engineering. Référence : 2009 Mar-Apr;19(2):107-13 Lien : Cliquer ici Résumé : Since The Culture Of Keratinocytes On Feeder Layers, Research To Produce Skin Equivalents Has Been Motivated By The Challenge Of Treating Large Burns And Chronic Wounds And By European Regulations Which Both Require Proof Of The Innocuousness And The Effectiveness Of Cosmetic Products, And Which Forbid Animal Testing. The Dynamism In Fundamental Research, Dermocosmetology And The Pharmaceutical Industry Has Led To The Evolution And Complexification Of Reconstructed Skin. The Collagen-Gag-Chitosan Sponge, As Well As The Self-Assembly Model, Allow Dermal Reconstruction In Which The Neosynthesized Extracellular Matrix Contains All Of The Desired Macromolecules. It Is Deposited Forming An Ultrastructurally Organised Architecture. The Quality Of The Dermis Obtained Allows The Development And Regeneration Of A Pluristratified And Differentiated Epidermis Firmly Anchored By An Organised Dermal-Epidermal Junction. Evolution Of Reconstructed Skin Into Models Which Are More And More Similar To The Physiological Skin Results In Higher Graft Take Rates In The Treatment Of Burns And Chronic Wounds, And Brings To Research, To Dermocosmetology And To The Pharmaceutical Industry, A Wide Range Of Products Such As Pigmented, Endothelialized, Immunocompetent, And Now Adipose Reconstructed Skins. The Present Review Will Mainly Concentrate On The Latest Developments In Skin Engineering And Will Mostly Concern The Studies Carried Out By Our Groups. Auteur(s) : Proulx S, Audet C, Uwamaliya JD, Deschambeault A, Carrier P, Giasson CJ, Brunette I, Germain L. Titre : Tissue Engineering of Feline Corneal Endothelium Using a Devitalized Human Cornea as Carrier Référence : Tissue Eng Part A. 2009 Jan 6. [Epub ahead of print] Lien : Cliquer ici Résumé : The Difficulties In Obtaining Good Quality Tissue For The Replacement Of Corneas Of Patients Suffering From Endothelial Dysfunctions Have Prompted Us To Evaluate The Feasibility Of Producing A Tissue-Engineered (Te) Corneal Endothelium Using Devitalized Human Stromal Carriers. Thus, Corneal Substitutes Were Produced By Seeding Cultured Feline Corneal Endothelial Cells On Top Of Previously Frozen Human Corneal Stromas. After Two Weeks Of Culture To Allow Attachment And Spreading Of The Seeded Cells, The Te Corneal Endothelium Was Stained With Alizarin Red For Endothelial Cell Count And Fixed For Histology, Immunofluorescence Labeling, Scanning And Transmission Electron Microscopy. Histology And Hoechst Staining Showed That There Were No Remaining Cells In The Devitalized Stroma. After Seeding, Histology And Transmission Electron Microscopy Showed That The Te Corneal Endothelium Formed A Monolayer Of Tightly Packed Cells That Were Well Adhered To Descemet'S Membrane. Scanning Electron Microscopy Corroborated That The Cells Covered The Entire Posterior Corneal Surface And Had An Endothelial Morphology. Alizarin Staining Showed That Mean Cell Counts Were 2272 +/- 344 Cells/Mm(2), Indicating That The Cell Density Was Appropriate For Grafting. The Te Feline Corneal Endothelium Also Expressed The Function-Related Proteins Na(+)/Hco(3)(-), Zo-1, And Na(+)/K(+)-Atpase Alpha1, And Could Easily Be Marked With A Fluorescent Tracker. This Study Demonstrates The Feasibility Of Reconstructing A Highly Cellular And Healthy Corneal Endothelium On Devitalized Human Corneal Stromas. Auteur(s) : Larouche D, Paquet C, Fradette J, Carrier P, Auger FA, Germain L. Titre : Regeneration of skin and cornea by tissue engineering. Référence : Methods Mol Biol. 2009;482:233-56. Lien : Cliquer ici Résumé : Progress In Tissue Engineering Has Led To The Development Of Technologies Allowing The Reconstruction Of Autologous Tissues From The Patient'S Own Cells. Thus, Tissue-Engineered Epithelial Substitutes Produced From Cultured Skin Epithelial Cells Undergo Long-Term Regeneration After Grafting, Indicating That Functional Stem Cells Were Preserved During Culture And Following Grafting. However, These Cultured Epithelial Sheets Reconstruct Only The Upper Layer Of The Skin And Lack The Mechanical Properties Associated To The Connective Tissue Of The Dermis. We Have Designed A Reconstructed Skin Entirely Made From Human Cutaneous Cells Comprising Both The Dermis And The Epidermis, As Well As A Well-Organized Basement Membrane By A Method Named The Self-Assembly Approach. In This Chapter, Protocols To Generate Reconstructed Skin And Corneal Epithelium Suitable For Grafting Are Described In Details. The Methods Include Extraction And Culture Of Human Skin Keratinocytes, Human Skin Fibroblasts As Well As Rabbit And Human Corneal Epithelial Cells, And A Complete Description Of The Skin Reconstructed By The Self-Assembly Approach And Of Corneal Epithelium Reconstructed Over A Fibrin Gel. Auteur(s) : Pricci M, Bourget JM, Robitaille H, Porro C, Soleti R, Mostefai HA, Auger FA, Martinez MC, Andriantsitohaina R, Germain L. Titre : Applications of Human Tissue-Engineered Blood Vessel Models to Study the Effects of Shed Membrane Microparticles from T-Lymphocytes on Vascular Function. Référence : Tissue Eng Part A. 2009 Jan;15(1):137-145. Lien : Cliquer ici Résumé : Microparticles (Mps) Are Membrane Vesicles Harboring Cell Surface Proteins And Containing Cytoplasmic Components Of The Original Cell. High Levels Of Circulating Mps Have Been Detected In Pathological States Associated With Vascular Dysfunction. We Took Advantage Of The Self-Assembly Method Of Tissue Engineering To Produce In Vitro Three Vascular Constructs From Human Vascular Smooth Muscle Cells And Fibroblasts To Investigate The Role Of The Adventitia In The Modulation Of Vascular Tone By Mps, Comparing The Contractile Response Of Each Of These Constructs To Histamine. The First Two Were Composed Of An Adventitia (Tissue-Engineered Vascular Adventitia (Teva)) Or A Media (Tissue-Engineered Vascular Media (Tevm)) Solely, And The Third One Contained A Media And An Adventitia (Tissue-Engineered Vascular Media And Adventitia (Tevma)). In The Three Constructs, The Results Show That Histamine Induces Contraction Insensitive To Blockade Of Inducible Nitric Oxide (No) Synthase (Inos) And Cyclooxygenase-2 (Cox-2) And Not Affected By Mp Treatment. Mps Decreased No Production And Nuclear Factor (Nf)-Kappab Expression But Did Not Affect Superoxide Anion (O(2)(-)) Release In Teva. Mps Enhanced Nf-Kappab Expression But Did Not Affect Inos And Cox-2 Expression Or No Or O(2)(-) Release In Tevm. In Tevma, Mps Did Not Enhance Nf-Kappab Expression, But Cox-2 Expression Was Higher, And O(2)(-) Release Was Lower. Thus, Mps Affected No, O(2)(-), Nf-Kappab, And Cox-2 In A Subtle Fashion To Maintain The Contractile Response To Histamine. The Use Of Tissue-Engineered Vascular Constructs Results In A Better Understanding Of The Effect Of Mps On Human Adventitia And Media. Auteur(s) : Jean J, Lapointe M, Soucy J, Pouliot R. Titre : Development of an in vitro psoriatic skin model by tissue engineering Référence : J Dermatol Sci. 2009 Jan;53(1):19-25. Epub 2008 Sep 9. Lien : Cliquer ici Résumé : Background: Psoriasis Is A Chronic Skin Disease Characterized By A Thickening And Disorganization Of The Skin'S Protective Barrier. Objectives: This Study Aims To Develop And Characterize A Novel In Vitro Psoriatic Human Skin Model Produced By Tissue Engineering. Methods: The Self-Assembly Method, A Tissue Engineering Approach Based On The Capacity Of Mesenchymal Cells, Such As Fibroblasts, To Create Their Own Extracellular Matrix In Vitro, Was Used To Create Our Substitutes. Manipulatable Sheets Of Fibroblasts Were Superimposed Creating A New Dermis Upon Which Keratinocytes Are Seeded, Leading To A Complete Bilayered Skin Substitute. The Characterization Of The Psoriatic Substitutes Was Performed By Macroscopic, Histological And Immunohistochemical Analyses And Contrasted To Those Constructed From Healthy Cells. Results: Macroscopically, The Psoriatic Substitutes Were More White And Thicker Than The Healthy Substitutes. The Histological Analysis Of Psoriatic Substitutes Stained With Masson'S Trichrome Revealed A Characteristic Thickening Of The Epidermal Layer Seen In Psoriatic Skin In Vivo. Immunohistochemical Analysis Of The Psoriatic Substitutes Showed, Among Other Things, An Overexpression Of Involucrin And An Underexpression Of Filaggrin And Loricrin. Conclusion: These Data Suggest That The Macroscopic, Histological And Immunohistochemical Characteristics Of Psoriasis Are Partially Retained In The Substitutes, Thus Providing A Good Model To Investigate The Mechanisms Of Abnormal Keratinocyte Growth And To Study Cell-Cell Interactions. Auteur(s) : Larouche D, Lavoie A, Proulx S, Paquet C, Carrier P, Beauparlant A, Auger FA, Germain L. Titre : [Regenerative medicine: Stem cells, cellular and matricial interactions in the reconstruction of skin and cornea by tissue engineering.] Référence : Pathologie Biologie Volume 57, Issue 4, June 2009, Pages 299-308 Lien : Cliquer ici Résumé : Le Génie Tissulaire Vise À Produire Des Tissus Ou Organes In Vitro Pour Le Remplacement Permanent Des Tissus Endommagés. À Cette Fin, La Production De Tissus Autologues Possède L’Avantage D’Éviter Tout Risque De Rejet Suite À Leur Transplantation Sur Un Patient. La Maîtrise Des Conditions De Culture Des Cellules Souches Humaines Postnatales Est Essentielle À La Réalisation De Tels Tissus. Il Est Aussi Souhaitable Que Leur Organisation Histologique Et Leur Fonctionnalité Se Rapprochent De Celles Des Tissus Natifs. De Plus, Les Cellules Souches Jouent Un Rôle Essentiel Au Niveau Du Remplacement Des Cellules Épithéliales Différenciées Dans Les Tissus Qui Doivent Constamment Se Renouveler, Tels Que La Peau Et La Cornée. Nous Avons Décrit Une Méthode Qui Permet De Produire Des Organes Vivants In Vitro À Partir De Cellules Humaines Postnatales Sans Ajouter De Biomatériaux. Cette Méthode D’Auto-Assemblage Repose Sur La Capacité Qu’Ont Les Cellules Mésenchymateuses De S’Organiser En Tissu En Présence Des Conditions De Culture Adéquates.Grâce À Différentes Techniques, Ces Tissus Peuvent Ensuite Être Assemblés En Organes Plus Complexes Tels Que Les Vaisseaux Sanguins, Les Valves Cardiaques, La Peau Ou Encore La Cornée. Ces Divers Tissus Pourront Éventuellement Être Utilisés Pour Le Remplacement D’Organes Malades Ou Endommagés Et Fourniront De Nouvelles Alternatives Pour La Médecine Régénératrice De Demain. Cet Article De Revue Sera Concentré Sur La Peau Et La Cornée. L’Importance D’Utiliser Des Conditions D’Isolement Et De Culture Qui Permettent De Conserver Les Cellules Souches Et De Contrôler L’Organisation Des Tissus Afin D’Assurer La Qualité Et La Fonctionnalité Des Organes Reconstitués Par Génie Tissulaire Sera Discutée. 2008 Auteur(s) : Corriveau M.-P., Boufaied I., Lessard J., Chabaud S., Sénécal J.L., Grodzicky T., Chartier S., Raymond Y., Moulin V. Titre : The fibrotic phenotype of systemic sclerosis fibroblasts varies with disese duration and severity of skin involvement: reconstruction of skin fibrosis development using a tissue engineering approach. Référence : J Pathol. 2008: Oct 16. Lien : Cliquer ici Résumé : We Set Out To Examine The Pathophysiologic Mechanisms Of Fibrosis In Diffuse Systemic Sclerosis (Ssc) Using A Tissue Engineering Approach. Skin Fibroblasts Were Isolated From Lesional Skin Of Ssc Patients With A Disease Duration Of Less Than One Year (Early-Stage Ssc) Or More Than 10 Years (Late-Stage Ssc). Fibroblasts Were Also Isolated From Non-Lesional Skin And Compared With Normal Fibroblasts Isolated From Healthy Adults. Cells Were Cultured Using A Tissue-Engineering Method To Reconstruct A Human Dermis, And Histologically Observed. Dermal Thickness Was Measured, As It Reflects The Global And Intrinsic Capacity Of Cells To Reconstitute Matrix. Collagen I, Mmp-1 And Mmp Activity Were Evaluated. Cells Were Treated With Tgf-ß1 Or Ctgf During Dermis Formation To Study Their Fibrogenic Role. Clinical Severity Of Skin Involvement Was Measured By A Modified Rodnan Score. Thickness Of The Dermis Generated With Non-Lesional, Early-Stage Ssc Fibroblasts Was Similar To Normal Cells. In Contrast, Reconstructed Dermis From Lesional, Early-Stage Ssc Fibroblasts And Non-Lesional, Late-Stage Ssc Cells Were Thinner While Lesional, Late-Stage Ssc Fibroblasts Made A Thicker Dermis. Dermis Were Always Thicker When Produced With Tgfß1-Treated Cells, Except When Lesional, Late-Stage Ssc Fibroblasts From Patients With High Rodnan Skin Scores Were Used. Ctgf Did Not Affect Dermal Thickness. Measurements Of Collagen I And Collagenases In The Culture Medium Of The Various Reconstructed Dermis Could Explain Some Of The Changes Observed. We Conclude That The Fibrotic Phenotype Of Ssc Fibroblasts Varies With Disease Duration And With Severity Of Skin Involvement And This Is Clearly Visualized During In Vitro Dermis Reconstruction. Auteur(s) : Cvetkovska B, Islam N, Goulet F, Germain L. Titre : Identification of functional markers in a self-assembled skin substitute in vitro. Référence : In Vitro Cell Dev Biol Anim. 2008 Nov-Dec;44(10):444-50. Lien : Cliquer ici Résumé : Some Functional Parameters Were Identified And Assessed In A Tissue-Engineered Self-Assembled Skin Substitute. This Skin Substitute Was Produced Using Fibroblasts And Keratinocytes Isolated From Adult Human Skin. Keratinocytes Were Seeded On A Dermal Layer, Composed Of Two Fibroblast Sheets Cultured For 35 D. The Epidermal Cells Formed A Stratified And Cornified Epidermis And Expressed Differentiation Markers, Notably Involucrin And Transglutaminase. Interestingly And For The First Time, The Receptor For Vitamin D3 Was Detected In All Of The Epidermal Cell Layers Of The Skin Substitute, As It Is Reported For Normal Human Skin. This Observation Suggests That Keratinocytes Retain Key Receptors During Their Differentiation In The Skin Model. A Network Of Collagen Fibers Was Observed By Electron Microscopy In The Dermal Layer Of The Model. In The Dermis, Collagen Fibers Remodeling And Assembly Is Dependent On Enzymes, Notably Prolyl-4-Hydroxylase. For The First Time In A Skin Construct, The Expression Of Prolyl-4-Hydroxylase Was Detected In Dermal Fibroblasts By In Situ Hybridization. The Secretion Of Collagenases By The Cells Seeded In Our Skin Substitute Was Confirmed By Zymography. We Conclude That The Self-Assembly Approach Allows The Maintenance Of Several Functional Activities Of Human Skin Cells In A Skin Model In Vitro. Auteur(s) : Gingras ME, Masson-Gadais B, Zaniolo K, Leclerc S, Drouin R, Germain L, Guerin SL. Titre : Abstract Binding of the Transcription Factors Sp1, AP-1 and NFI to a Short Regulatory Element from the Human {alpha}5 Integrin Gene Promoter Dictates its Transcriptional Activity. Référence : Invest Ophthalmol Vis Sci. 2008 Sep 4. [Epub ahead of print] Lien : Cliquer ici Résumé : Purpose: Damage To The Corneal Epithelium Results In The Massive Secretion Of Fibronectin (Fn) Shortly After Injury And Induces Expression Of Its Integrin Receptor Alpha5Beta1. We Reported Previously That Fn Induces Alpha5 Expression In Both Human (Hcecs) And Rabbit (Rcecs) Corneal Epithelial Cells By Altering The Binding Of The Transcription Factor (Tf) Sp1 To A Regulatory Element From The Alpha5 Promoter That It Is Also Flanked By Binding Sites For The Tfs Nfi And Ap-1. Here, We Assessed The Function Of Nfi And Ap-1 On Alpha5 Gene Expression And Evaluated The Contribution Of Fn To Their Overall Regulatory Influence. Methods: Tfs Binding To The Alpha5 Promoter Was Evaluated In Vitro By Electrophoretic Mobility Shift Assays (Emsas), And In Vivo By Ligation-Mediated Pcr (Lmpcr) Or Chromatin Immunoprecipitation (Chip). Tfs Expression Was Monitored By Western Blot Whereas Their Influence Was Assessed By Transfection And Rnai Analyses. Results: Co-Expression Of Sp1, Nfi And Ap-1 Was Demonstrated In All Cell Types And Each Tf Was Shown To Bind Efficiently To The Alpha5 Promoter. Whereas Both Ap-1 And Sp1 Activated Expression Directed By The Alpha5 Promoter, Nfi Functioned As A Potent Repressor Of That Gene. Interestingly, Fn Could Either Promote Or Repress Alpha5 Promoter Activity In A Cell Density-Dependent Manner By Differentially Altering The Ratio Of These Tfs. Conclusions: Our Results Suggest That Alpha5 Gene Expression Is Likely Dictated By Subtle Alterations In The Nuclear Ratio Of Tfs That Either Repress (Nfi) Or Activate (Sp1 And Ap-1) Alpha5 Transcription In Corneal Epithelial Cells. Auteur(s) : Magnan M, Lévesque P, Gauvin R, Dubé J, Barrieras D, El-Hakim A, Bolduc S. Titre : Tissue Engineering of a Genitourinary Tubular Tissue Graft Resistant to Suturing and High Internal Pressures. Référence : Tissue Eng Part A. 2008 Aug 31. [Epub ahead of print] Lien : Cliquer ici Résumé : The Aim Of This Study Was To Evaluate The Possibility Of Constructing A Fully Autologous Tissue-Engineered Tubular Genitourinary Graft (Ttgg) And To Determine Its Mechanical And Physiological Properties. Dermal Fibroblasts (Dfs) Were Expanded And Cultured In Vitro With Sodium Ascorbate To Form Fibroblast Sheets. The Sheets Were Then Wrapped Around A Tubular Support To Form A Cylinder. After Maturation, Urothelial Cells (Ucs) Were Seeded Inside The Df Tubes, And The Constructs Were Placed In A Bioreactor. The Ttggs Were Then Characterized According To Histology, Immuno-Histochemistry, Western Blot, Cell Viability, Resistance To Suture, And Burst Pressure. Results Obtained Were Encouraging On All Levels. All Layers Of The Ttggs Had Merged, And A Pluristratified Urothelium Coated The Luminal Surface Of The Tubes. The Burst Pressure Of Non-Sutured Ttggs Was Measured And Found To Be, On Average, Three Times As Resistant As That Of Porcine Urethras. Suturing Was Accomplished Without Difficulty. Results Have Shown That Our Construct Can Sustain An Entire Week Of Pulsatile Stimulation Without Loss Of Mechanical Or Histological Integrity. The Tissue-Engineering Technique Used To Produce This Model Seems Promising For Bioengineering A Urethra Or Ureter Graft And Could Open A Doorway To New Possibilities For Their Reconstruction. Auteur(s) : Tremblay PL, Huot J, Auger FA. Titre : Mechanisms by which E-selectin regulates diapedesis of colon cancer cells under flow conditions. Référence : Cancer Res. 2008 Jul 1;68(13):5167-76. Lien : Cliquer ici Résumé : Diapedesis, The Passage Of Circulating Tumor Cells Across The Endothelium, Is A Critical Determinant In Most Cases Of Metastasis. Using A Laminar Flow Chamber And A Tissue-Engineered Blood Vessel, We Found That E-Selectin Is Required Not Only For The Initial Adhesion And Rolling Of Circulating Ht-29 Colon Cancer Cells On The Endothelium But Also For Their Subsequent Diapedesis. These Processes Require Both The Intracellular And Extracellular Domains Of E-Selectin. We Also Identified Three Distinct Mechanisms By Which Circulating Cancer Cells Interact With E-Selectin To Initiate Their Diapedesis: Formation Of A Mosaic Between Cancer Cells And Endothelial Cells, Paracellular Diapedesis At The Junction Of Three Endothelial Cells, And Transcellular Diapedesis. We Also Obtained Evidence Indicating That E-Selectin-Dependent Paracellular Extravasation Is Independent Of Intercellular Adhesion Molecule And Vascular Cell Adhesion Molecule And That It Requires The Activation Of Extracellular Signal-Regulated Kinase (Erk) Mitogen-Activated Protein Kinase Downstream Of E-Selectin. This Is Supported By The Observation That The Adenoviral-Mediated Expression Of The E-Selectin Mutant Y603F Is Associated With Both An Inhibition Of Erk And Paracellular Extravasation. Our Study Is The First To Clearly Establish, Under Dynamic And Shear Stress Conditions, How E-Selectin Regulates Diapedesis Of Circulating Cancer Cells. These Results Provide New Insights In Understanding The Metastatic Process. Auteur(s) : Trottier V, Marceau-Fortier G, Germain L, Vincent C, Fradette J. Titre : IFATS Series: Using Human Adipose-derived Stem/stromal Cells for the Production of New Skin Substitutes. Référence : Stem Cells. 2008; 10:2713-23. Lien : Cliquer ici Résumé : The Ability To Harvest And Culture Stem Cell Populations From Various Human Postnatal Tissues Is Central To Regenerative Medicine Applications, Including Tissue Engineering. The Discovery Of Multipotent Mesenchymal Stem Cells Within The Stromal Fraction Of Adipose Tissue Prompted Their Use For The Healing And Reconstruction Of Many Tissues. Here, We Examined The Influence Of Adipose-Derived Stem/Stromal Cells (Ascs) On Skin'S Regenerative Processes, From A Tissue Engineering Perspective. Using A Self-Assembly Approach, Human Skin Substitutes Were Produced. They Featured A Stromal Compartment Containing Human Extracellular Matrix Endogenously Produced From Either Dermal Fibroblasts Or Adipose-Derived Stem/Stromal Cells Differentiated Or Not Towards The Adipogenic Lineage. Human Keratinocytes Were Seeded On Each Stroma And Cultured At The Air-Liquid Interface To Reconstruct A Bilayered Skin Substitute. These New Skin Substitutes, Containing Respectively An Epidermis And A Distinctive Stroma Devoid Of Synthetic Biomaterial, Displayed Characteristics Similar To Human Skin. The Influence Of The Type Of Stromal Compartment On Epidermal Morphogenesis Was Assessed By The Evaluation Of Tissue Histology, The Expression Of Key Protein Markers Of The Epidermal Differentiation Program (K14, K10, Transglutaminase), As Well As The Expression Of Dermo-Epidermal Junction Components (Laminins, Collagen Vii) And Presence Of Basement Membrane And Hemidesmosomes. Our Findings Suggest That Adipose-Derived Stem/Stromal Cells Could Usefully Substitute Dermal Fibroblasts For Skin Reconstruction Using The Self-Assembly Method. Finally, By Exploiting The Adipogenic Potential Of Ascs, We Also Describe The Production Of A More Complete Trilayered Skin Substitute Consisting Of The Epidermis, The Dermis As Well As The Adipocyte-Containing Hypodermis, The Skin'S Deepest Layer. Auteur(s) : Gaudreault M, Vigneault F, Gingras ME, Leclerc S, Carrier P, Germain L, Guerin SL. Titre : Transcriptional regulation of the human {alpha}6 integrin gene by the transcription factor NFI during corneal wound healing. Référence : Invest Ophthalmol Vis Sci. 2008; 49(9): 3758-67. Lien : Cliquer ici Résumé : Purpose: Wound Healing Of The Corneal Epithelium Is Highly Influenced By Regulation Of Integrin Genes Expression. Recently, We Demonstrated That Laminin (Lm), A Major Constituent Of The Ecm, Reduces Expression Of The Human Alpha6 Integrin Subunit Gene By Altering The Properties Of The Transcription Factor (Tf) Sp1. In This Work, We Identified A Target Site For The Tf Nuclear Factor I (Nfi) On The Human Alpha6 Gene And Characterized Its Regulatory Influence In Corneal Epithelial Cells. Methods: Plasmids Bearing The Alpha6 Promoter Fused To The Cat Gene Were Transfected Into Human (Hcecs) And Rabbit (Rcecs) Corneal Epithelial Cells Grown On Lm. The Dna Binding Site For Nfi In The Alpha6 Promoter Was Identified By Dnasei Footprinting. Expression And Dna Binding Of Nfi Was Monitored By Western Blot, Rt-Pcr And Electrophoretic Mobility Shift Assays (Emsas) And Its Function Investigated Through Rnai And Nfi Overexpression Assays. Results: All Nfi Isoforms Were Found To Be Expressed In Hcecs And Rcecs. Transfection Analyses Revealed That Nfi Is A Repressor Of Alpha6 Expression In Both Types Of Cells. Lm Increases Expression Of Nfi Whereas Inhibition Of Each Nfi Isoform Increases Promoter Activity Suggesting That Nfi Is A Key Repressor Of Alpha6 Transcription. In Addition, The Negative Influence Of Nfi Appears To Be Potentiated By The Degradation Of Sp1 When Cells Are Grown On Lm. Conclusions: Repression Of Alpha6 Expression Therefore Contributes To The Final Steps Of Corneal Wound Healing By Both Reducing Proliferation And Allowing Attachment Of The Epithelium To The Basal Membrane. Auteur(s) : Vallée M, Côté JF, Fradette J. Titre : Adipose-tissue engineering: Taking advantage of the properties of human adipose-derived stem/stromal cells. Référence : Pathol Biol (Paris). 2008 Jun 3. [Epub ahead of print] Lien : Cliquer ici Résumé : Adipose Tissue Is Now Recognized As An Important Source Of Postnatal Mesenchymal Stem Cells For Regenerative Medicine Applications. For Example, Adipose-Tissue Engineering Is An Emerging Approach That Enables The Development Of Autologous Substitutes That Could Be Used As An Alternative To Fat Transplantation Methods Currently Yielding Variable Outcomes For The Long-Term Repair Of Soft-Tissue Defects. Here, We Describe The Production Of Unique Tissue-Engineered Adipose Tissues Devoid Of Exogenous Biomaterials Produced From Human Adipose-Derived Stem/Stromal Cells. Our Strategy Is Based On The Dual Self-Assembly Of Extracellular Components Secreted And Organized By The Adipose-Derived Stromal Cells After Ascorbic Acid Stimulation, As Well As Their Concomitant Differentiation Into Adipocytes After Adipogenic Induction. When Compared To Stromal Cells Isolated From Resected Fat, Lipoaspirated Fat-Derived Cells Featured An Increased Adipogenic Potential And The Enhanced Ability To Recreate Three-Dimensional Adipose Substitutes In Vitro. These Substitutes Were Histologically Similar To Native Adipose Tissue. They Featured Lipid-Filled Adipocytes Embedded Into An Extracellular Matrix Rich In Fibronectin As Well As Collagens I And V. On A Functional Level, The Reconstructed Adipose Tissues Expressed Adipocyte-Related Transcripts And Secreted Adipokines Typical Of Adipose Tissue, Such As Leptin. Finally, The Successful In Vitro Production Of Human Adipose Substitutes Featuring An Increased Surface Area (>30Cm(2)) Is Described, Reinforcing The Notion That Customized Autologous Reconstructed Adipose Tissues Could Be Produced In The Future To Repair A Wide Range Of Soft-Tissue Defects. Auteur(s) : Caissie R, Beaulieu F, Giroux M, Berthod F, Landry PE. Titre : Cutaneous myiasis: diagnosis, treatment, and prevention. Référence : J Oral Maxillofac Surg. 2008 Mar;66(3):560-8. Lien : Cliquer ici Auteur(s) : Carrier P., Deschambeault A., Talbot M., Giasson C.J., Auger F.A., Guérin S.L., Germain L. Titre : Characterization of Wound Reepithelialization Using a New Human Tissue-Engineered Corneal Wound Healing Model Référence : Investigative Ophthalmology and Visual Science (IOVS), April 2008, 49(4):1376-1385 Lien : Cliquer ici Résumé : Purpose. The Reepithelialization Of The Corneal Surface Is An Important Process For Restoring The Imaging Properties Of This Tissue. The Purpose Of The Present Study Was To Characterize And Validate A New Human In Vitro Three-Dimensional Corneal Wound Healing Model By Studying The Expression Of Basement Membrane Components And Integrin Subunits That Play Important Roles During Epithelial Cell Migration And To Verify Whether The Presence Of Exogenous Factors Could Accelerate The Reepithelialization. Methods. Tissue-Engineered Human Cornea Was Wounded With A 6-Mm Biopsy Punch, And The Reepithelialization From The Surrounding Margins Was Studied. Biopsy Samples Of The Reepithelialized Surface Were Harvested 3 Days After Wounding And Were Processed For Histologic, Electron Microscopic, And Immunofluorescence Analyses. The Effects Of Fibrin And Epithelial Growth Factor (Egf) On Wound Reepithelialization Were Also Studied. Results. Results Demonstrated That This In Vitro Model Allowed The Migration Of Human Corneal Epithelial Cells On A Natural Extracellular Matrix. During Reepithelialization, Epithelial Cell Migration Followed A Consistent Wavelike Pattern Similar To That Reported For Human Corneal Wound Healing In Vivo. This Model Showed A Histologic Appearance Similar To That Of Native Tissue As Well As Expression And Modulation Of Basement Membrane Components And The Integrin Subunits Known To Be Main Actors During The Wound Healing Process. It Also Allowed Quantification Of The Reepithelialization Rate, Which Was Significantly Accelerated In The Presence Of Fibrin Or Egf. The Results Indicated That {Alpha}Vβ6 Integrin Expression Was Increased In The Migrating Epithelial Cells Compared With The Surrounding Corneal Tissue. Conclusions. The Similarity Observed With The In Vivo Wound Healing Process Supports The Use Of This Tissue-Engineered Model For Investigating The Basic Mechanisms Involved In Corneal Reepithelialization. Moreover, This Model May Also Be Used As A Tool To Screen Agents That Affect Reepithelialization Or To Evaluate The Effect Of Growth Factors Before Animal Testing. Auteur(s) : Gingras M, Beaulieu MM, Gagnon V, Durham HD, Berthod F. Titre : In vitro study of axonal migration and myelination of motor neurons in a three-dimensional tissue-engineered model Référence : Glia, 2008 Feb;56(3):354-64 Lien : Cliquer ici Résumé : Primary Motor Neurons Are Difficult To Study In Conventional Culture Systems Because Of Their Short-Term Survival Without Trophic Support From Glia. In Addition, Axonal Migration On A Two-Dimensional Petri Dish Does Not Reflect The Three-Dimensional (3D) Environment In Vivo. A Unique In Vitro 3D Model Of Motor Nerve Regeneration Was Developed To Study Motor Neuron Axonal Migration And Myelination. Mouse Spinal Cord Motor Neurons Were Seeded On A Collagen Sponge Populated With Schwann Cells And Fibroblasts. This Fibroblast-Populated Sponge Was Intended To Mimic The Connective Tissue Through Which Motor Axons Have To Elongate In Vivo. Addition Of Conventional Neurotrophic Supplements Was Not Required For Motor Neuron Survival But Was Necessary To Promote Deep Neurite Outgrowth, As Assessed By Immunostaining Of Neurofilament M. A Vigorous Neurite Elongation Was Detected Inside The Sponge After Only 14 Days Of Neuron Culture, Reaching More Than 850 Microm. The Model Also Allowed The Maturation Of Motor Fibers As One-Third Of Them Were Positive For Neurofilament H. Neurites Growing In The Sponge Were Subject To Myelination When Schwann Cells Were Present, As Shown By Myelin Basic Protein Immunostaining And Electron Microscopy. We Demonstrated In This Model The Spontaneous Formation Of Numerous Thick Myelin Sheaths Surrounding Motor Fibers After Long-Term Culture (28 Days). Thus, This Model Might Be A Valuable Tool To Study The Effect Of Various Cells And/Or Attractive Or Repulsive Molecules On Motor Neurite Outgrowth In Vitro And Also For The Study Of Myelination And Pathogenesis Of Motor Neuron Diseases. Auteur(s) : Larouche D, Tong X, Fradette J, Coulombe PA, Germain L. Titre : Vibrissa hair bulge houses two populations of skin epithelial stem cells distinct by their keratin profile. Référence : FASEB J. 2008; 22(5):1404-15. Lien : Cliquer ici Résumé : Defining The Properties Of Postnatal Stem Cells Is Of Interest Given Their Relevance For Tissue Homeostasis And Therapeutic Applications, Such As Skin Tissue Engineering For Burn Patients. In Hair Follicles, The Bulge Region Of The Outer Root Sheath Houses Stem Cells. We Show That Explants From The Prominent Bulge Area, But Not The Bulb, In Rodent Vibrissa Follicles Can Produce Epidermis In A Skin Model Of Tissue Engineering. Using Morphological Criteria And Keratin Expression, We Typified Epithelial Stem Cells Of Vibrissa Bulge. Two Types Of Slow-Cycling Cells (Bb, Bs1) Featuring A High Colony-Forming Capacity Occur In The Bulge. Bb Cells Are Located In The Outermost Basal Layer, Express K5, K15, K17, And K19, And Feature A Loosely Organized Keratin Network. Bs1 Cells Localize To The Suprabasal Layers Proximal To Bb Cells And Express K5/K17, Correlating With A Network Of Densely Bundled Filaments. These Prominent Bundles Are Missing In K17-Null Mice, Which Lack Vibrissa. Atypically, Both The Bb And Bs1 Keratinocytes Lack K14 Expression. These Findings Show Heterogeneity Within The Hair Follicle Stem Cell Repository, Establish That A Subset Of Slow-Cycling Cells Are Suprabasal In Location, And Point To A Special Role For K5/K17 Filaments In A Newly Defined Subset Of Stem Cells. Our Results Are Discussed In The Context Of Long-Term Survival Of Engineered Tissues After Grafting That Requires The Presence Of Stem Cells.-Larouche, D., Tong, X., Fradette, J., Coulombe, P. A., Germain, L. Vibrissa Hair Bulge Houses Two Populations Of Skin Epithelial Stem Cells Distinct By Their Keratin Profile. 2007 Auteur(s) : Bernard G., Auger M., Soucy J., Pouliot R. Titre : Physical characterization of the stratum corneum of an in vitro psoriatic skin model by ATR-FTIR and Raman spectroscopies. Référence : Biochim Biophys Acta. 2007 Sep;1770(9):1317-23. Epub 2007 Jul 12. Lien : Cliquer ici Résumé : The Stratum Corneum Is An Important Permeability Barrier For The Skin. The Disorganization Of The Skin Protective Barrier Characterizes Some Skin Diseases Such As Psoriasis. Indeed, Psoriatic Skin Is Known To Be More Permeable Than Normal Human Skin. An In Vitro Human Skin Substitute May Be Obtained By The Auto-Assembly Method. This Method Was Adapted To Produce Psoriatic Substitutes. Ftir Spectroscopy Is A Well-Established Method To Evaluate The Order Of Hydrocarbon Chains In Terms Of Population Of Trans And Gauche Conformers. Using Atr-Ftir, We Have Compared The Physicochemical Properties Of The Stratum Corneum In Skin Models Derived From Uninvolved And Involved Psoriatic Cells With Those Derived From Normal Cells. Our Results Suggest That The Stratum Corneum Of Involved Psoriatic Skin Substitutes Is Less Organized Than That Of Normal Skin Substitutes. Also, It Seems That The Properties Of Uninvolved Psoriatic Skin May Vary With Seriousness Of The Disease. The Development Of A New Psoriatic Skin Model Would Be Helpful In The Design Of New Treatments And To Increase The Understanding Of The Mechanisms Of This Pathology. Auteur(s) : Gingras M, Gagnon V, Minotti S, Durham HD, Berthod F. Titre : Optimized protocols for isolation of primary motor neurons, astrocytes and microglia from embryonic mouse spinal cord. Référence : J Neurosci Methods. 2007 Jun 15;163(1):111-8. Lien : Cliquer ici Résumé : Neuron-Glial Interactions Are Important In Development Of The Nervous System And Pathogenesis Of Disease. Primary Cell Cultures Prepared From Nervous Tissue Are Often Used To Study The Properties Of Individual Cell Types And How They Interact With Each Other. Isolation Of Pure Populations Of Cells And Their Culture Is Challenging, Particularly From Murine Spinal Cord. The Purpose Of This Study Was To Optimize Various Protocols To Achieve Efficient, Parallel Isolation And Purification Of Primary Motor Neurons, Microglia And Astrocytes From The Same Mouse Embryonic Spinal Cord Sample. Following Dissociation Of E12 Embryonic Spinal Cords, Motor Neurons Were Isolated At 97% Purity By A Single Step Centrifugation Of The Cell Suspension Through Multiple Discontinuous Density Gradients Of Nycoprep. The Residual Mixed Cell Pellet Was Resuspended And Cultured For 2 Weeks. Mixed Cultures Were Then Shaken To Release Microglia, Which Were Then Harvested From The Medium And Subjected To Another Round Of Differential Adhesion To Achieve 99% Purity. The Astrocytes Remaining In The Mixed Cultures Were Culled To 98% Purity By Treatment With Leucine Methyl Ester And A Subsequent Vigorous Shaking Step To Remove Any Remaining Microglia And Neurons. Furthermore, No Cross Contamination Was Observed In The Glial Cultures. This Technique Provides A Simple, Convenient, And Reliable Method Of Obtaining Highly Purified Preparations Of Motor Neurons, Microglia And Astrocytes From Embryonic Spinal Cord For The Study Of Spinal Cord Cell Biology And Motor Neuron Diseases. Auteur(s) : Caissie R, Landry PE, Paquin R, Champigny MF, Berthod F. Titre : Quantitative Method to Evaluate the Functionality of the Trigeminal Nerve Référence : J Oral Maxillofac Surg. 2007 Nov;65(11):2254-2259.Click here to read Lien : Cliquer ici Résumé : Purpose: The Primary Objective Of This Study Was To Yield Normal Range Values, With A Current Perception Threshold Technique, That Could Be Used To Assess The Functionality Of The Third Division Of The Trigeminal Nerve On A Healthy Population. Moreover, We Wanted To Evaluate The Impact Of Gender And Training On These Values. Patients And Methods: Standardized Current Perception Threshold (Cpt) Measures Using Constant Alternating Current Sinusoid Waveform Stimulus At 5, 250, And 2,000 Hz Were Obtained From 50 Healthy Patients At The Mental Foramen Area Bilaterally Using A Neurometer Current Perception Threshold Device (Neurotron Inc, Baltimore, Md) (2,000 Hz Specifically Stimulates Abeta Fibers, 250 Hz Adelta Fibers, And 5 Hz Stimulates C Fibers). Results: The Mean Cpt Values For The 2,000, 250, And 5 Hz Groups Were Respectively, 157.6 +/- 54.67, 53.10 +/- 27.64, And 33.44 +/- 23.17 Mamp. These Values Were Used To Construct A Cpt Scale To Classify Patients In The Hyperesthetic, Normative, Or Hypoesthetic Range. There Were No Significant Differences When Comparing Cpt Values In Men And Women Except In The 2,000 Hz Group (P < .02; N = 23). In Addition, The Test Was Carried Out A First Time On The Right Side And A Second Time On The Left. This Training Procedure Showed A Significant Decrease In The Cpt Values In Men At 2,000 Hz (P < .01; N = 23) For The Second Measure. Conclusion: The Neurometer Can Be Beneficial For The Evaluation And Intraneural Localization Of Sensory Dysfunctions Associated With The Third Division Of The Trigeminal Nerve. It Also Could Be Used For Initial Diagnosis And Subsequent Evaluation Of The Patient'S Neurologic Status Through The Course Of Their Condition. Auteur(s) : Diebolt M., Laflamme K., Labbe R., Auger F.A., Germain L., Andriantsitohaina R. Titre : Polyphenols modulate calcium-independent mechanisms in human arterial tissue-engineered vascular media. Référence : J Vasc Surg. 2007 Aug 29 Lien : Cliquer ici Résumé : Background: In The Present Study, An Arterial Tissue-Engineered Vascular Media (Tevm) Was Produced From Cultured Human Smooth Muscle Cells Of The Umbilical Artery And We Took Advantage Of This Model To Evaluate The Regulation Of Contraction And The Signalling Pathways Of Polyphenols In Arteries. Methods: Cultured Human Smooth Muscle Cells Of The Umbilical Artery Were Used To Produce Arterial Tevms. Contraction Experiments Were Performed To Determine Intracellular Targets Involved In The Modulation Of Contraction By Polyphenols Extract From Red Wine, Provinols (Seppic Groupe Air Liquide, Paris, France). Results: Smooth Muscle Cells In Arterial Tevm Displayed A Differentiated Phenotype As Demonstrated By The Expression Of Alpha-Smooth Muscle Actin, A Vascular Smooth Muscle-Specific Marker, And Tissue Contraction In Response To Vasoconstrictor And Vasodilator Agents. Contractions Caused By Histamine Were Associated With An Increase In [Ca(2+)](I) And A Ca(2+)-Independent Signalling Pathway. The Latter Pathway Involved Mechanisms Sensitive To Protein Kinase C, Myosin Light Chain Kinase, And Rho-Associated Protein Kinase Inhibitors. The Regulation Of Contraction Induced By Provinols Shows That Treatment Of Arterial Tevm With This Compound Significantly Decreased Histamine-Induced Contraction. This Effect Was Associated With The Inhibition Of The Rho-Associated Protein Kinase Pathway And The Decrease In Alpha-Smooth Muscle Actin Expression. Conclusion: The Use Of Arterial Tevm, Brings New Insights Into The Mechanisms By Which Polyphenols Regulate Vascular Contraction In The Human Artery. Auteur(s) : Auger F.A., D'Orleans-Juste P., Germain L. Titre : Adventitia contribution to vascular contraction: Hints provided by tissue-engineered substitutes. Référence : Cardiovasc Res. 2007 Sep 1;75(4):669-78. Lien : Cliquer ici Résumé : It Is Well Accepted That The Adventitia Is Much More Than A Simple Elastic Membrane Which Surrounds The Media. However, The Extent Of Its Contribution To Vascular Physiology, As Well As The Mechanisms Involved, Remains To Be Clearly Established And Characterised. Investigation Into These Topics Is Hampered By A Few Technical Challenges, Like The Paucity Of Available Healthy Human Vascular Samples And The Variability Such Samples Can Display. Another Challenge Is The Isolation And Preparation Of Intact Adventitia Without Contaminating Cells From The Media. For Those Reasons, Although Other Models Have Proved Useful To Address These Questions, Data From Tissue-Engineered Vascular Substitutes Can Also Provide Quite Valuable Answers. Results From Such Substitutes Indicate That A Reconstructed Adventitial Layer Can Respond To Classic Vasoactive Agents Such As Endothelin And Sodium Nitroprusside. Auteur(s) : Proulx S., Bourget J.M., Gagnon N., Martel S., Deschambeault A., Carrier P., Giasson C.J., Auger F.A., Brunette I., Germain L. Titre : Optimization of culture conditions for porcine corneal endothelial cells. Référence : Mol Vis. 3;13:524-33 Lien : Cliquer ici Résumé : Purpose: To Optimize The Growth Condition Of Porcine Corneal Endothelial Cells (Pcec), We Evaluated The Effect Of Coculturing With A Feeder Layer (Irradiated 3T3 Fibroblasts) With The Addition Of Various Exogenous Factors, Such As Epidermal Growth Factor (Egf), Nerve Growth Factor (Ngf), Bovine Pituitary Extract (Bpe), Ascorbic Acid, And Chondroitin Sulfate, On Cell Proliferation, Size, And Morphology. Methods: Pcec Cultures Were Seeded At An Initial Cell Density Of 400 Cells/Cm(2) In The Presence Or Absence Of 20,000 Murine-Irradiated 3T3 Fibroblast/Cm(2) In The Classic Media Dulbecco'S Modified Eagle'S Medium (Dmem) Supplemented With 20% Fetal Bovine Serum (Fbs). Mean Cell Size And Bromodeoxyuridine Incorporation Was Assessed At Various Passages. Growth-Promoting Factors Were Studies By Seeding Pcec At 8,000 Cells/Cm(2) In Dmem With 20% Fbs Or Opti-Mem I Supplemented With 4% Fbs And One Of The Following Additives: Egf (0.5, 5, 25 Ng/Ml), Ngf (5, 20, 50 Ng/Ml), Bpe (25, 50, 100, 200 Microg/Ml), Ascorbic Acid (10, 20, 40 Microg/Ml) And Chondroitin Sulfate (0.03, 0.08, 1.6%), Alone Or In Combination. Cell Number, Size And Morphology Of Pcec Were Assessed On Different Cell Populations. Each Experiment Was Repeated At Least Twice In Three Sets. In Some Cases, Cell Cultures Were Maintained After Confluence To Observe Post-Confluence Changes In Cell Morphology. Results: Co-Cultures Of Pcec Grown In Dmem 20% Fbs With A 3T3 Feeder Layer Improved The Preservation Of Small Polygonal Cell Shape. Egf, Ngf, And Chondroitin Sulfate Did Not Induce Proliferation Above Basal Level Nor Did These Additives Help Maintain A Small Size. However, Chondroitin Sulfate Did Help Preserve A Good Morphology. Bpe And Ascorbic Acid Had Dose-Dependent Effects On Proliferation. The Combination Of Bpe, Chondroitin Sulfate, And Ascorbic Acid Significantly Increased Cell Numbers Above Those Achieved With Serum Alone. No Noticeable Changes Were Observed When Pcec Were Cocultured With A 3T3 Feeder Layer In The Final Selected Medium. Conclusions: Improvements Have Been Made For The Culture Of Pcec. The Final Selected Medium Consistently Allowed The Growth Of A Contact-Inhibited Cell Monolayer Of Small, Polygonal-Shaped Cells. Auteur(s) : Vermette M., Trottier V., Menard V., Saint-Pierre L., Roy A., Fradette J. Titre : Production of a new tissue-engineered adipose substitute from human adipose-derived stromal cells. Référence : Biomaterials. 2007;28(18):2850-2860. Lien : Cliquer ici Résumé : Adipose Tissue Is An Accessible And Abundant Source Of Mesenchymal Stem Cells For Soft-Tissue Reconstruction. In An Attempt To Create A Novel, Entirely Autologous Tissue-Engineered Adipose Substitute, We Extracted Human Stromal Cells From Either Lipoaspirated Or Resected Fat, And Assessed Their Capacity To Produce A Three-Dimensional Adipose Tissue Using An Adapted "Self-Assembly" Culture Methodology. This Strategy Involved A Concomitant Induction Of Adipogenic Differentiation Whilst Ascorbic Acid Supplementation Stimulated The Stromal Cells To Produce And Organize Their Own "Biomaterial" In The Form Of Extracellular Matrix, Forming Manipulatable Sheets That Are Then Assembled Into Thicker Reconstructed Adipose Tissues. When Compared To Resected Fat, Lipoaspiration-Derived Cells Featured An Increased Adipogenic Potential And The Enhanced Ability To Recreate An Adipose Substitute In Vitro. When Viewed By Scanning Electron Microscopy, The Appearance Of These Reconstructed Adipose Tissues Was Strikingly Similar To Subcutaneous Fat. Furthermore, These Substitutes Secreted Adipokines And Mediated Beta-Adrenergic Receptor-Stimulated Lipolysis, Hence Reproducing Known Major Biological Functions Of White Adipose Tissue. Therefore, Our Cell-Based Tissue Engineering Strategy Led To The Production Of A Functional And Entirely Natural Reconstructed Adipose Tissue, Which Offers The Potential To Be Used For Specific In Vitro Applications As Well As For Autologous Soft-Tissue Reconstruction. Auteur(s) : Gingras, M., Champigny, M.-F., Berthod, F. Titre : Differentiation of human adult skin-derived neuronal precursors into mature neurons Référence : J Cell Physiol., 210(2): 498-506, 2007 Lien : Cliquer ici Résumé : The Isolation Of Autologous Neuronal Precursors From Skin-Derived Precursor Cells Extracted From Adult Human Skin Would Be A Very Efficient Source Of Neurons For The Treatment Of Various Neurodegenerative Diseases. The Purpose Of This Study Was To Demonstrate That These Neuronal Precursors Were Able To Differentiate Into Mature Neurons. We Isolated Neuronal Precursors From Breast Skin And Expanded Them In Vitro For Over Ten Passages. We Showed That 48% Of These Cells Were Proliferating After The First Passage, While This Growth Rate Decreased After The Second Passage. We Demonstrated That 70% Of These Cells Were Nestin-Positive After The Third Passage, While Only 17% Were Neurofilament M-Positive After 7 Days Of Differentiation. These Neuronal Precursors Expressed Betaiii Tubulin, The Dendritic Marker Map2 And The Presynaptic Marker Synaptophysin After 7 Days Of In Vitro Maturation. They Also Expressed The Postsynaptic Marker Psd95 And The Late Neuronal Markers Neun And Neurofilament H After 21 Days Of Differentiation, Demonstrating They Became Terminally Differentiated Neurons. These Markers Were Still Expressed After 50 Days Of Culture. The Generation Of Autologous Neurons From An Accessible Adult Human Source Opens Many Potential Therapeutic Applications And Has A Great Potential For The Development Of Experimental Studies On Normal Human Neurons. Auteur(s) : Morissette G., Germain L., Marceau F. Titre : The antiwrinkle effect of topical concentrated 2-dimethylaminoethanol involves a vacuolar cytopathology Référence : British Journal of Dermatology, 156(3): 433-439, 2007 Lien : Cliquer ici Résumé : Background: The 'Cosmeceutical' Agent 2-Dimethylaminoethanol (Dmae) Is A Tertiary Amine Found In High Concentration In Numerous Topical Antiwrinkle Preparations. Objectives: We Hypothesized That A 337 Mmol L(-1) (3%) Dmae Reservoir Applied To The Skin Could Reproduce The Cytopathology Induced By Other Amines By Maintaining A Millimolar Drug Concentration Within A Certain Depth Of The Skin Layers, And That Vacuolar Cell Expansion Could Account For The Very Rapid Effect On The Apparent Skin Fullness. Methods: Morphological And Functional Assays Were Applied To Cultured Rabbit Dermal Fibroblasts Treated With Tertiary Amines In Vitro. A Morphological Verification Of The Vacuolization Caused By Topical Dmae Was Also Attempted In Vivo Using The Inner Skin Of The Rabbit Ear And In Vitro Using Primary Cultures Of Human Cutaneous Epithelial Cells. Results: Fibroblasts Responded To Dmae (2.5-10 Mmol L(-1)) By Massive Vacuolization (0.5-4 H; Phase Contrast Observations). Triethanolamine, Another Chemical Frequently Used Topically, Was Also Active In This Respect (10 Mmol L(-1)). The Vacuolar Adenosine Triphosphatase Inhibitor Bafilomycin A1 Prevented Dmae- Or Triethanolamine-Induced Vacuolization; Adding Bafilomycin A1 Or Cell Washout Slowly Reversed The Established Vacuolization Induced By Dmae. Further Effects Of Dmae In Cultured Fibroblasts Included A Moderate Cytotoxicity (10 Mmol L(-1)) That Was Abated By Bafilomycin A1 Cotreatment, A Concentration-Dependent Mitotic Arrest (2.5 Mmol L(-1)) And Transient And Mild Effects On Cell Ploidy. The Epidermis Of The Rabbit External Ear Was Significantly Thickened And Exhibited Clear Perinuclear Swelling Indicative Of Vacuolization In Response To 3% Dmae (1 H; Paraffin Tissue Sections). Cultured Human Cutaneous Epithelial Cells Responded To Dmae By Vacuolization (Inhibited By Bafilomycin A1 Cotreatment). Conclusions: The Vacuolar Cytopathology Induced By Concentrated Organic Amines May Be The Cellular Basis Of The Antiwrinkle Effect Of Dmae. 2006 Auteur(s) : Magnan M., Berthod F., Champigny M.-F., Soucy F., Bolduc S. Titre : In vitro reconstruction of a tissue-engineered endothelialized bladder from a single porcine biopsy Référence : Journal of Pediatric Urology: 2(4); 261-270. Lien : Cliquer ici Résumé : Objective: Augmentation Of The Urinary Bladder Using A Tissue-Engineered Approach With Autologous Cells Is A Very Promising Technique. To Prevent Risks Of Necrosis After Transplantation, The Graft Vascularization Process Could Be Markedly Enhanced By Incorporation Of Autologous Endothelial Cells In The Tissue-Engineered Organ. The Purpose Of This Study Was To Develop A Separation Technique To Extract Four Bladder Cell Types From The Same Biopsy, And To Prepare An Endothelialized Reconstructed Bladder. Materials And Methods: Fibroblasts, Smooth Muscle Cells (Smc), Urothelial Cells (Uc) And Endothelial Cells (Ec) Were Extracted From A Small Porcine Bladder Biopsy. The Smc, Fibroblasts And Ec Were Seeded On The Top Of The Sponge And Cultured For 10Days. Then, The Uc Were Seeded On Top Of These Cells For 15 Additional Days To Produce A Three-Dimensional Bladder Wall. Results: The Uc And Ec Extracts From A Single Porcine Biopsy Were 97.2+/-0.6% Keratin 8/18-Positive And 97.7+/-0.3% Pecam-1-Positive Pure Cells, Respectively, As Assessed By Flow Cytometry. The Smc Could Not Be Dissociated From Fibroblasts, And Were Present As 37+/-0.5% Desmin-Positive Cells. Uc Differentiated Into A Urothelium Characterized By Umbrella Cells And A Laminin-Positive Basal Membrane. The Ec Reorganized In The Matrix To Form Pecam-1-Positive Capillary-Like Tubes. Conclusion: This New Model Of Tissue-Engineered Bladder Has The Main Advantages Of Being At Least 2Mm Thick, Autologous, And Able To Promote The Formation Of Capillary-Like Tubes. It Could Be A Promising Alternative To The Use Of Gastrointestinal Segments To Improve Bladder Capacity. Auteur(s) : Tremblay P.L., Auger F.A., Huot J. Titre : Regulation of transendothelial migration of colon cancer cells by E-selectin-mediated activation of p38 and ERK MAP kinases. Référence : Oncogene. 2006 Oct 26;25(50):6563-73. Epub 2006 May 22. Lien : Cliquer ici Résumé : The Invasive Properties Of Cancer Cells Depend On Their Intrinsic Motile Potential And On Their Ability To Breach The Endothelial Barrier. In The Present Work, We Investigated The Mechanisms By Which Adhesion Of Colon Cancer Cells To E-Selectin Expressed By Endothelial Cells Regulates The Barrier Function Of These Cells And Modulates Transmigration Of Cancer Cells. We Found That The Stimulation Of E-Selectin By Activating Antibodies Or The Adhesion Of Ht-29 Cells Results In An Increase In The Activity Of Extracellular Signal-Regulated Kinase (Erk) And P38 Mitogen-Activated Protein Kinases. In Turn, The Activation Of P38 And Erk Enhances Transendothelial Permeability And Migration Of Ht-29 Cells. We Also Obtained Evidence Suggesting That P38-Mediated Increase In Transendothelial Migration Of Cancer Cells Depends On A Myosin Light Chain Phosphorylation-Mediated Formation Of Stress Fibres. On The Other Hand, The Activation Of Erk By E-Selectin Modulates The Opening Of Interendothelial Spaces By Initiating The Activation Of Src Kinase Activities And The Dissociation Of The Ve-Cadherin/Beta-Catenin Complex. We Conclude That Activation Of E-Selectin By Adhering Cancer Cells Is An Important Process That Regulates The Extravasation Of Colon Cancer Cells By Initiating P38- And Erk-Dependent Mechanisms That Both Contribute To Regulate The Integrity Of The Endothelial Layer. Auteur(s) : Auger F.A. Titre : The LOEX perspective on the role of tissue engineering in regenerative medicine. Référence : Biomed Mater Eng. 2006;16(4 Suppl):S19-25. Lien : Cliquer ici Auteur(s) : Grenier G., Remy-Zolghadri M., Bergeron F., Guignard R., Baker K., Labbe R., Auger F.A., Germain L. Titre : Mechanical Loading Modulates the Differentiation State of Vascular Smooth Muscle Cells. Référence : Tissue Eng. 2006 Oct 1; [Epub ahead of print] Lien : Cliquer ici Résumé : The Cause Underlying The Onset Of Stenosis After Vascular Reconstruction Is Not Well Understood. In The Present Study, We Evaluated The Effect Of Mechanical Unloading On The Differentiation State Of Human Vascular Smooth Muscle Cells (Hvsmcs) Using A Tissue-Engineered Vascular Media (Tevm). Hvsmcs Cultured In A Mechanically Loaded Three-Dimensional Environment, Known As A Living Tissue Sheet, Had A Higher Differentiated State Than Cells Grown On Plastic. When The Living Tissue Sheet Was Detached From Its Support, The Release Of The Residual Stress Resulted In A Mechanical Unloading And Cells Within The Extracellular Matrix (Ecm) Dedifferentiated As Shown By Downregulation Of Differentiation Markers. The Relaxed Living Tissue Sheet Can Be Rolled Onto A Tubular Mandrel To Form A Tevm. The Rolling Procedure Resulted In The Reintroduction Of A Mechanical Load Leading To A Cohesive Compacted Tissue. During This Period, Cells Gradually Redifferentiated And Aligned Circumferentially To The Tubular Support. Our Results Suggest That Differentiation Of Hvsmcs Can Be Driven By Mechanical Loading And May Occur Simultaneously In The Absence Of Other Cell Types. The Extrapolation Of Our Results To The Clinical Context Suggests The Hypothesis That Hvsmcs May Adopt A Proliferative Phenotype Resulting From The Mechanical Unloading Of Explanted Blood Vessels During Vascular Reconstruction. Therefore, We Propose That This Mechanical Unloading May Play An Important Role In The Onset Of Vascular Graft Stenosis. Auteur(s) : Giasson C.J., Bouchard C., Boisjoly H., Germain L. Titre : Amnios et problèmes de surface oculaire. Référence : Med Sci (Paris). Jun-Jul;22(6-7):639-44. Lien : Cliquer ici Résumé : The Amniotic Membrane, The Most Internal Placental Membrane, Has Various Properties Useful In Ophthalmology. Collected On Delivery By Elective Caesarean Section, The Amnion Is Prepared Under Sterile Conditions, And, Usually, Cryopreserved Until Its Use As A Biological Bandage Or As A Substrate For Epithelial Growth In The Management Of Various Ocular Surface Conditions. Specifically, The Amnion Is Used To : (1) Limit Formation Of Adhesive Bands Between Eyelids And Eyeball (Symblepharon) Or The Progression Of A Fibrovascular Outgrowth Towards The Cornea (Pterygium) Or To (2) Facilitate The Healing Of Corneal Ulcers, Bullous Keratopathy, And Corneal Stem Cell Deficiency. In This Last Condition, Either Hereditary Or Acquired After A Thermal Or A Chemical Burn, Corneal Stem Cells, Located At A Transitional Zone Between The Cornea And Conjunctiva, Are Lost. These Cells Are Essential For Renewal Of Corneal Epithelium In Normal And In Diseased States. The Loss Of These Cells Leaves The Corneal Surface Free For Invasion By Conjunctival Epithelium. Not Only, Does Conjunctival Epithelium Support The Development Of Vascularisation On The Normally Avascular Cornea, But Some Conjunctival Cells Differentiate Into Mucus Secreting Goblet Cells. Such A Change In Phenotype Leads To Loss Of Corneal Transparency And Visual Disability. The Removal Of This Fibro-Vascular Outgrowth In Combination With Transplantation Of Both Amniotic Membrane And Corneal Stem Cells Are Used To Treat This Condition. The Amnion Stimulates The Proliferation Of Less Differentiated Cells Which Have The Potential To Reconstruct The Cornea. This Potential Is At The Origin Of The Hypothesis That The Amnion May Provide An Alternative Niche For Limbal Stem Cells Of The Corneal Epithelium. It Abounds In Cytokines And Has Antalgic, Anti-Bacterial, Anti-Inflammatory And Anti-Immunogenic Properties, In Addition To Allowing, Like Fetal Skin Does, Wound Healing With Minimal Scar Formation. These Desirable Properties Are Responsible For The Increasing Use Of Amniotic Membrane In Ophthalmology. The Complete Understanding Of The Mechanisms Of Action Of Amniotic Membrane For Ocular Surface Diseases Has Yet To Be Understood. Once Revealed By Research, They May Provide New Pharmacological Avenues To Treat Ocular Surface Diseases. Auteur(s) : Laflamme K, Roberge CJ, Pouliot S, D'Orleans-Juste P, Auger FA, Germain L. Titre : Tissue-engineered human vascular media produced in vitro by the self-assembly approach present functional properties similar to those of their native blood vessels. Référence : Tissue Eng. 2006 Aug;12(8):2275-81. Lien : Cliquer ici Résumé : We Have Developed A Tissue-Engineering Approach For The Production Of A Completely Biological Blood Vessel From Cultured Human Cells. In The Present Study, We Took Advantage Of This Tissue-Engineering Method To Demonstrate That It Can Be Used To Reproduce The Subtle Differences In The Expression Of Receptors Present On The Media Of Native Human Blood Vessels. Indeed, A Small Percentage (3 Of 18) Of Native Human Umbilical Cord Veins (Hucvs) Responded To Endothelin, The Most Powerful Vasopressor Agent Known To Date, Via Both Endothelin A (Et(A)) And Endothelin B (Et(B)) Receptor Activation. In Contrast, Most Hucvs Tested Responded To Et Via Et(A) Receptor Activation Only. Tissue-Engineered Vascular Media (Tevm) Were Next Reconstructed By Using Vascular Smooth Muscle Cells (Vsmcs) Isolated And Cultured From Hucvs Expressing Both Et(A) And Et(B) Receptors To Determine The Functional Integrity Of Our Tevm Model. The Reconstructed Tevm Presents An Endothelin Response Similar To That Of Respective Hucvs From Which Vsmcs Were Isolated. Reverse Transcriptase Polymerase Chain Reaction On Tevm Reconstructed In Vitro Correlated These Vasocontractile Profiles By Showing The Presence Of Messenger Rna For Both Et(A) And Et(B) Receptors. Taken Together With Recently Published Results On Tevm Expressing Only Et(A) Receptor, These Results Show That Our Reconstructed Tevm Present A Similar Et Response Profile As The Blood Vessel From Which The Vsmcs Were Isolated And Cultured. These Findings Indicate That Subtle Differences, Such As Receptor Expression, Are Preserved In The Reconstructed Tissue. Therefore, Our Tevm Offers A Valuable Human In Vitro Model With Which To Study The Functionality Of Human Blood Vessels, Such As Their Vasoactive Response, Or To Perform Pharmacologic Studies. Auteur(s) : Laflamme K, Roberge CJ, Grenier G, Remy-Zolghadri M, Pouliot S, Baker K, Labbe R, D'Orleans-Juste P, Auger FA, Germain L. Titre : Adventitia contribution in vascular tone: insights from adventitia-derived cells in a tissue-engineered human blood vessel Référence : FASEB J. 2006 Jun;20(8):1245-7 Lien : Cliquer ici Résumé : Whether The Adventitia Component Of Blood Vessels Directly Participates In The Regulation Of Vascular Tone Remains To Be Demonstrated. We Have Recently Developed A Human Tissue-Engineered Blood Vessel Comprising The Three Tunicae Of A Native Blood Vessel Using The Self-Assembly Approach. To Investigate The Role Of The Adventitia In The Modulation Of Vascular Tone, This Tissue-Engineering Method Was Used To Produce Three Vascular Constructs From Cells Explanted And Proliferated From Donor Vessel Tunicae 1) An Adventitia + A Media, Or Only 2) An Adventitia, Or 3) A Media. The Vasoconstriction Responses Of These 3 Constructs To Endothelin, The Most Potent Vasopressor Known Up-To-Date, As Well As To Nonselective And Selective Agonists And Antagonists, Were Compared. The Adventitia Contracted To Endothelin-1, -2, Whereas The Media And The Media+Adventitia Contracted To All Three Endothelins. Endothelin-Induced Contraction Of The Adventitia Was Dependent On Et(A) Receptors, Whereas That Of The Media And The Adventitia+Media Was Et(A) And Et(B) Receptor-Dependent. Rt-Pcr Studies Corroborated These Results. Snp Induced A Dose-Dependent Relaxation Of The Three Tissue Constructs. We Also Demonstrated That The Endothelin-Converting Enzyme, Responsible For The Formation Of The Active Endothelin Peptides, Was Present And Functional In The Adventitia. In Conclusion, This Is The First Direct Demonstration That The Adventitia Has The Capacity To Contract And Relax In Response To Vasoactive Factors. The Present Study Suggests That The Adventitia Of A Blood Vessel Could Play A Greater Role Than Expected In The Modulation Of Blood Vessel Tone. Auteur(s) : Solomon, C., Bernier, L., Germain, L., Dufour, R., Davignon, J. Titre : Severe oily ichthyosis in monozygotic twins mimicking Chanarin-Dorfman syndrome but not associated with a mutation of the CGI58 gene Référence : Arch Dermatol, 142(3): 402-3, 2006. Lien : Cliquer ici Auteur(s) : Goulet, F., Napa, I. D., Solomon, L., Morin, O., Islam, N. Titre : Modulated expression of a nuclear-associated glycoprotein during normal rat liver development and in various hepatoma cells. Référence : Prog Neuropsychopharmacol Biol Psychiatry, 30(1): 159-65, 2006. Lien : Cliquer ici Résumé : Liver Plays A Major Role In Systemic Detoxification And Drug Metabolism. Nf-164, A Protein Of 164 Kda Predominantly Localized In Hepatocyte Nuclei, Was Found To Be Present In Increasing Amounts During Liver Maturation. In Addition, Fetal Rat Hepatocytes Had Ten Times, And Neonatal Five Times Less Of This Protein Than Adult Hepatocytes. It Was Also Detected In An Albumin Producing Hepatoma Cell Line, But Not In Three Other Lines That Have Lost Several Differentiated Functions. These Data Suggest That Nf-164 Expression Is Development-Dependent And That It May Be A Marker For Both Normal And Malignant Hepatocyte Differentiation. Nf-164 Seems To Be Liver-Specific, Since It Was Not Detected In Rat Brain, Spleen, Kidney, Lung And Bovine Thymus. It Was Purified From Adult Rat Hepatocyte Nuclei. Its Estimated Pi Is 6.8. Its Total Amino Acid Composition And Partial Amino Acid Sequence Is Also Being Reported. Despite Major Differences Between Their Respective Contents In Amino Acids, Partial Sequences Showed Homologies With Carbamyl Phosphate Synthetase I (Cpsi). These Observations May Suggest That Nf-164 Also Shares Some Functional Features With This Enzyme. Auteur(s) : Talbot M., Carrier P., Giasson C. J., Deschambeault A., Guérin S. L., Auger F. A., Bazin R., Germain L. Titre : Autologous transplantation of rabbit limbal epithelia cultured on fibrin gels for ocular surface reconstruction. Référence : Molecular Vision. 12: 65-75, 2006 Lien : Cliquer ici Résumé : Purpose: Regeneration Of The Corneal Epithelium Could Be Severely Impaired In Patients Suffering From Limbal Stem Cell Deficiency. The Purpose Of This Study Was To Evaluate The Restoration Of The Corneal Epithelium By Grafting Onto Denuded Corneas Autologous Limbal Cells Cultured On Fibrin Gels. The Rabbit Model Was Chosen To Allow The Microscopic Evaluation Over Time After Grafting. Methods: Rabbit Limbal Epithelial Cells (Rlecs) Were Isolated And Cultured From Small Limbal Biopsies (3 Mm2). The Epithelium Was Separated From Stroma After Dispase Digestion And Put In Culture On Lethally Irradiated Fibroblasts Used As A Feeder Layer. At The First Passage, Rlecs Were Cultured On A Fibrin Gel Matrix. At Confluence, The Cultured Epithelia Were Grafted In Vivo On Denuded Autologous Rabbit Corneas. At Different Postoperative Times, Grafted And Control (Without Graft Or Grafted With Fibrin Gels Only) Rabbit Corneas Were Compared In Vivo With A Slit Lamp Microscope, And In Situ By Histological And Immunohistological Microscopy Of Harvested Biopsies. Results: A Small Limbal Biopsy Was Sufficient To Generate Enough Rlecs To Prepare Several Grafts And To Perform Cell Analysis. Only Two Weeks Were Required To Produce A Cultured Epithelium Suitable For Autologous Transplantation. One Month After Grafting, A Normal Corneal Phenotype Was Observed On The Ocular Surface Of Grafted Rabbits In Contrast To The Control Rabbits (Ungrafted Or Grafted With Fibrin Gel Only) Where Histological Signs Of Conjunctivalization Were Found. The Absence Of Goblet Cells And Negative Staining For Keratin 4 Confirmed That The Cultured Cells Persisted And That The Epithelium Regenerated After Grafting Was Not From Conjunctival Origin. Conclusions: Our Results Demonstrate That An Autologous Epithelium Cultured On A Physiologically Biodegradable Matrix Can Be Prepared From A Small Biopsy And Grafted On Denuded Cornea. The Autologous Graft Allows Epithelial Regeneration From Cultured Cells And Promotes Corneal Healing Of Unilateral Total Stem Cell Deficiency. Auteur(s) : Caissie, R., Gingras, M., Champigny, M. F., Berthod, F. Titre : In vivo enhancement of sensory perception recovery in a tissue-engineered skin enriched with laminin. Référence : Biomaterials, 27(15): 2988-2993, 2006. Lien : Cliquer ici Résumé : The Use Of Autologous Reconstructed Skin Appears To Be A Promising Treatment For The Permanent Coverage Of Deep And Extensive Burns. However, The Capability Of Reconstructed Skin Transplanted On Wounds To Promote Recovery Of Sensory Perception Is A Major Concern. Our Aim Was To Assess The Effect Of Laminin On Cutaneous Nerve Regeneration. We Prepared Collagen-Chitosan Sponges Enriched With 0, 1, 10 Or 50 Microg Of Laminin/Sponge To Produce Tissue-Engineered Reconstructed Skins By Culture Of Human Fibroblasts And Keratinocytes, Then Grafted On The Back Of Athymic Mice For 120 Days. Immunohistochemical Studies Demonstrated That There Were 7 Times More Neurofilament 150 Kd-Positive Nerve Fibers Migrating In The Graft In The Samples Enriched With 10 Microg Laminin/Sponge, Compared To Reconstructed Skin Without Laminin, 120 Days After Graft. A Significant Improvement In The Current Perception Threshold Of The Abeta And Adelta Nerve Fibers Was Measured Using A Neurometer In All Grafts Enriched With Laminin. In Addition, The Type C Nerve Fibers Reached An Identical Current Perception Threshold Than Mouse Skin, In All Reconstructed Skins Enriched Or Not With Laminin. We Conclude That The Use Of A Tissue-Engineered Autologous Skin Graft Enriched With Laminin Has The Potential To Efficiently Optimize Cutaneous Sensory Nerve Regeneration In Vivo. Auteur(s) : Germain, L., Larouche, D., Paquet, C. Titre : [Canadian haematopoietic stem cell scientists received the Lasker Award.]. Référence : Med Sci (Paris), 22(2): 212-213, 2006. Lien : Cliquer ici Auteur(s) : Berthod, F., Germain, L., Tremblay, N., Auger, F. A. Titre : Extracellular matrix deposition by fibroblasts is necessary to promote capillary-like tube formation in vitro. Référence : J Cell Physiol, 2006. Lien : Cliquer ici Résumé : The Contribution Of The Cellular And Fibrillar Microenvironment To Angiogenesis Still Remains Unclear. Our Purpose Was To Evaluate The Effect Of The Extracellular Matrix Deposited By Fibroblasts On The Capacity Of Human Endothelial Cells To Form Capillaries In Vitro. We Have Drastically Decreased The Amount Of Extracellular Matrix Surrounding Fibroblasts In Our Model Of Endothelialized-Reconstructed Connective Tissue (Erct) By Culturing It Without Ascorbate. Under These Conditions, The Number Of Capillary-Like Tubes (Clt) Formed By Endothelial Cells Was Reduced By Up To 10-Fold After 31 Days Of Culture Compared To Controls. This Decrease Was Due Neither To A Variation Of Mmp-2 And Mmp-9 Secretion, Nor To A Reduction In The Number Of Fibroblasts And/Or Endothelial Cells, Or A Diminution Of Fibroblast Growth Factor 2 (Fgf2) Synthesis. The Secretion Of Vascular Endothelial Growth Factor (Vegf) By Fibroblasts Accounted For 25-70% Of The Capillary-Like Tube Formation When Tissues Were Cultured In The Presence Or Absence Of Ascorbate, As Demonstrated By Vegf-Blocking Studies. The Culture Of Endothelial Cells On A Similar Extracellular Matrix But In The Absence Of Living Fibroblasts Did Not Promote The Formation Of Clt, Even When Tissues Were Fed With Fibroblast-Conditioned Medium. Thus, The Deposition Of A Rich Extracellular Matrix By Living Fibroblasts Appeared Necessary, But Not Sufficient To Promote Capillary-Like Formation. Fibroblasts Seem To Induce Endothelial Cells To Spontaneously Form Clt By Secreting And Organizing An Abundant Extracellular Matrix, Which Creates A Microenvironment Around Cells That Could In Turn Trap Growth Factors Produced By Fibroblasts And Promote Three-Dimensional Cell Organization. Auteur(s) : Berube, M., Deschambeault, A., Boucher, M., Germain, L., Petitclerc, E., Guerin, S. L. Titre : MMP-2 expression in uveal melanoma: differential activation status dictated by the cellular environment. Référence : Mol Vis, 11(1101-11, 2005. Lien : Cliquer ici Résumé : Purpose: Mmps Are Recognized To Play A Major Role In Tumor Progression And Metastasis Of Many Forms Of Cancers. The Purpose Of This Study Was To Compare The Expression And Activity Of Mmp-2 In Uveal Melanoma Cell Lines Grown Either In Vitro On Plastic Culture Plates Or In Vivo As Tumors Produced In Chick Embryos. Methods: The Chick Chorioallantoic Membrane (Cam) Model Was Used To Evaluate The Tumorigenic Potential Of Uveal Melanoma Cell Lines Derived Either From The Primary Uveal Melanoma Tumor Isolated From Three Different Patients (Cell Lines Sp6.5, Sp8.0, And Tp31) Or From A Metastatic Lesion Derived From The Liver Of A Patient Diagnosed With Uveal Melanoma (Cell Line H79). The Presence Of Mmp-2 In The Vicinity Of The Tumor Cells Was Determined By Immunofluorescence Analyses. Gelatin Zymography Was Used For The Detection Of Latent And Activated Forms Of Mmp-2 In Uveal Melanoma Cell Lines When Grown In Vitro On Plastic, Or In The Solid Tumors These Cell Lines Produced In Vivo On The Cam Of The Chick Embryo. The Gelatinase Activity Was Quantified By Densitometric Analyses And The Active/(Active+Pro-Form) Ratio Was Calculated As The Mmp-2 Activation Ratio. Western Blot Analyses Were Performed To Confirm The Zymographic Profile. Results: Only The Inactive Form Of Mmp-2 Was Expressed And Secreted In Vitro By All Uveal Melanoma Cell Lines, Higher Levels Being Found For The Liver-Derived H79 Cell Line Whereas Sp8.0 Only Expressed Mmp-2 To A Very Low Level. On The Other Hand, All Solid Tumors Produced In The Cam From These Cell Lines Expressed And Secreted, Although To Varying Levels (Sp6.5 And Sp8.0, Tp31 And H79), Primarily The Active Form Of Mmp-2. Gelatinolytic Activities Of Active Mmp-2 Were Significantly Higher In Uveal Melanoma Tissues Than In The Non-Neoplastic Cam, As Revealed By The Measurement Of The Activation Ratio. The Immunolocalization Of Mmp-2 Revealed That All Cell Lines Were Mmp-2-Positive Although A Reduced And More Diffuse Staining Was Observed For H79 And Sp6.5 Than In Sp8.0 And Tp31 Cells. Conclusions: These Results Suggest The Activation Of Prommp-2 As An Important Event In The Process Of Uveal Melanoma Progression. An Elevated Active To Inactive Mmp-2 Ratio In The Tumor Environment Of Uveal Melanoma Suggests That A Potential Mmp-2 Activity Could Be Related To The Progression Of This Type Of Cancer. Auteur(s) : Masson-Gadais, B., Fugere, C., Paquet, C., Leclerc, S., Lefort, N. R., Germain, L., Guerin, S. L. Titre : The Feeder layer-mediated extended lifetime of cultured human skin keratinocytes is associated with altered levels of the transcription factors Sp1 and Sp3. Référence : J Cell Physiol, 206(3): 831-42, 2006. Lien : Cliquer ici Résumé : Primary Cultured Epithelial Cells That Are Used For Basic Research Are Often Cultivated On Plastic Whereas Those Used For Clinical Purposes Are Usually Cultured In The Presence Of A Feeder Layer. Here, We Examined The Influence Of A Feeder Layer On The Expression, Affinity And Dna Binding Ability Of The Transcription Factors, Sp1 And Sp3 In Primary Cultures Of Human Skin Keratinocytes. Co-Culturing Both Newborn And Adult Skin Keratinocytes With Lethally Irradiated 3T3 Cells As A Feeder Layer Contributed To Maintain The Cell'S Morphological And Growth Characteristics And Delayed Terminal Differentiation In Vitro. 3T3 Also Stabilized The Dna Binding Properties Of Sp1 Without Altering Its Transcription. Stimulation Of Sp1/Sp3 Expression Appears To Be Mediated Through Cell-Cell Interactions And By Factors Secreted By 3T3. Thus, We Propose That The Feeder Layer Delay Terminal Differentiation Of Primary Cultured Skin Keratinocytes By Preventing Extinction Of Transcription Factors, Like Sp1 And Sp3, Which Play Pivotal Functions In The Cell Cycle. J.Cell.Physiol. (C) 2005 Wiley-Liss, Inc. 2005 Auteur(s) : Tremblay, P. L., Berthod, F., Germain, L., Auger, F. A. Titre : In vitro evaluation of the angiostatic potential of drugs using an endothelialized tissue-engineered connective tissue. Référence : J Pharmacol Exp Ther, 315(2): 510-6, 2005. Lien : Cliquer ici Résumé : The Development Of A New Pharmacological Strategy, The Angiostatic Therapy, To Inhibit Solid Tumor Progression Has Increased The Need Of Powerful In Vitro Models To Screen The Angiostatic Potential Of New Drug Candidates. We Produced An Endothelialized Reconstructed Connective Tissue (Erct) That Promotes The Spontaneous Formation Of A Human Capillary-Like Network By Coculture Of Human Endothelial Cells Isolated From Umbilical Cord Or From Newborn Foreskin, With Dermal Fibroblasts In A Collagen Sponge. Three Inhibitors Of Angiogenesis, Tamoxifen, Ilomastat, And Echistatin, Were Used To Assess The Efficiency Of Our Erct To Discriminate, In Vitro, An Angiostatic Potential. The Capillary-Like Structures Were Characterized By Their Immunoreactivity To Human Platelet-Endothelial Cellular Adhesion Molecule-1 Antibodies And Were Quantified On Histological Cross-Sections Of Biopsies Taken After 10, 17, 24, And 31 Days Of Culture. A Dose-Response Significant Inhibition Of The Capillary-Like Formation Was Detected When Increasing Concentrations Of Tamoxifen, Ilomastat, Or Echistatin Were Added For 1 Week To The Culture Medium Of The Erct. Tamoxifen Was Found To Be Angiogenic At 10 Microm And To Have A Cytotoxic Effect At 40 Microm 1 Week After Drug Removal. Echistatin Induced A Rapid, Slight, And Reversible Inhibition Of Capillary-Like Formation, Whereas Ilomastat Caused A Very Precocious, Strong, And Reversible Inhibition Of Angiogenesis. In Addition, A 16-H Hypoxia Promoted The Formation Of 10 Times Larger Vessels (>300 Microm(2)), Compared With Normoxic Condition. These Results Suggest That Our Model Could Be Efficiently Used To Study The Long-Term Angiostatic Potential Of Drugs In Vitro In A Very Physiological Environment. Auteur(s) : Tremblay, P. L., Hudon, V., Berthod, F., Germain, L., Auger, F. A. Titre : Inosculation of tissue-engineered capillaries with the host's vasculature in a reconstructed skin transplanted on mice. Référence : Am J Transplant, 5(5): 1002-10, 2005. Lien : Cliquer ici Résumé : The Major Limitation For The Application Of An Autologous In Vitro Tissue-Engineered Reconstructed Skin (Rs) For The Treatment Of Burnt Patients Is The Delayed Vascularization Of Its Relatively Thick Dermal Avascular Component, Which May Lead To Graft Necrosis. We Have Developed A Human Endothelialized Reconstructed Skin (Ers), Combining Keratinocytes, Fibroblasts And Endothelial Cells (Ec) In A Collagen Sponge. This Skin Substitute Then Spontaneously Forms A Network Of Capillary-Like Structures (Cls) In Vitro. After Transplantation To Nude Mice, We Demonstrated That Cls Containing Mouse Blood Were Observed Underneath The Epidermis In The Ers In Less Than 4 Days, A Delay Comparable To Our Human Skin Control. In Comparison, A 14-Day Period Was Necessary To Achieve A Similar Result With The Non-Endothelialized Rs. Furthermore, No Mouse Blood Vessels Were Ever Observed Close To The Epidermis Before 14 Days In The Ers And The Rs. We Thus Concluded That The Early Vascularization Observed In The Ers Was Most Probably The Result Of Inosculation Of The Cls Network With The Host'S Capillaries, Rather Than Neovascularization, Which Is A Slower Process. These Results Open Exciting Possibilities For The Clinical Application Of Many Other Tissue-Engineered Organs Requiring A Rapid Vascularization. Auteur(s) : Hart DA, Shrive NG, Goulet F. Titre : Tissue engineering of ACL replacements. Référence : Sports Med. Arthroscopy Rev. 13: 170-176 , 2005. Auteur(s) : Robitaille H, Proulx R, Robitaille K, Blouin R, Germain L. Titre : The mitogen-activated protein kinase kinase kinase dual leucine zipper-bearing kinase (DLK) acts as a key regulator of keratinocyte terminal differentiation. Référence : J Biol Chem. 2005 Apr 1;280(13):12732-41. Epub 2005 Jan 28. Lien : Cliquer ici Résumé : In The Skin, Epithelial Cells Undergo A Terminal Differentiation Program Leading To The Formation Of The Stratum Corneum. Although It Is Expected That The Last Phases Of This Process Must Be Tightly Regulated Since It Results In Cell Death, The Signaling Pathways Involved In This Induction Remain Ill Defined. We Now Report That A Single Kinase, The Mitogen-Activated Protein Kinase Kinase Kinase Dual Leucine Zipper-Bearing Kinase (Dlk), Acts In The Epidermis To Promote The Terminal Differentiation Of Human Keratinocytes. In Support Of This Notion, We Showed That Dlk Expression Was Restricted To The Granular Layer In Situ. In Addition, Cultured Keratinocytes Infected With A Recombinant Adenovirus Expressing Dlk Exhibited Morphological And Biochemical Changes, Including A Suprabasal Localization, Altered Cell Shape, Compacted Cytoplasm, Dna Fragmentation, And The Up-Regulation Of Filaggrin, That Are Reminiscent Of A Terminally Differentiated Phenotype. Moreover The Expression Of Wild-Type Dlk In Keratinocytes Stimulated Transglutaminase Activity And The Consequent Formation Of The Cornified Cell Envelope, While A Kinase-Inactive Variant Of Dlk Did Not. Together These Results Identify Dlk As A Signaling Molecule Implicated In The Regulation Of Keratinocyte Terminal Differentiation And Cornification. Auteur(s) : Bellemare J., Roberge C.J., Bergeron D., Lopez-Vallé C.A., Roy M., Moulin V.J. Titre : Epidermis promotes dermal fibrosis : role in the pathogenesis of hypertrophic scars Référence : Journal of Pathology 206: 1-8, 2005 Lien : Cliquer ici Résumé : Hypertrophic Scarring Is A Pathological Process Characterized By Fibroblastic Hyperproliferation And By Excessive Deposition Of Extracellular Matrix Components. It Has Been Hypothesized That Abnormalities In Epidermal-Dermal Crosstalk Explain This Pathology. To Test This Hypothesis, A Tissue-Engineered Model Of Self-Assembled Reconstructed Skin Was Used In This Study To Mimic Interactions Between Dermal And Epidermal Cells In Normal Or Pathological Skin. These Skin Equivalents Were Constructed Using Three Dermal Cell Types: Normal Wound (Wmyo) Or Hypertrophic Wound (Hmyo) Myofibroblasts And Normal Skin Fibroblasts (Fb). Epidermis Was Reconstructed With Normal Skin Keratinocytes (Nk) Or Hypertrophic Scar Keratinocytes (Hk). In The Absence Of Keratinocytes, Hmyo Formed A Thicker Dermis Than Wmyo. When Seeded With Nk, The Dermal Thickness Of Hmyo (121.2 +/- 31.4 Microm Vs 196.2 +/- 27.8 Microm) And Fb (43.7 +/- 7.1 Microm Vs 83.6 +/- 16.3 Microm) Dermis Was Significantly (P < 0.05) Reduced, While That Of Wmyo (201.5 +/- 15.7 Microm Vs 160.7 +/- 21.1 Microm) Was Increased. However, The Presence Of Hk Always Induced Significantly Thicker Dermis Formation Than Observed With Nk (Wmyo: 238.8 +/- 25.9 Microm; Hmyo: 145.5 +/- 22.4 Microm; Fb: 74.2 +/- 11.2 Microm). These Results Correlated With Collagen And Mmp-1 Secretion And With Cell Proliferation, Which Were Increased When Keratinocytes Were Added, Except For The Collagen Secretion Of Hmyo And Fb In The Presence Of Nk. The Level Of Dermal Apoptosis Was Not Different When Epidermis Was Added To The Dermis ( Auteur(s) : Grenier G., Rémy-Zolghadri M., Larouche D., Gauvin R., Baker K., Bergeron F., Dupuis D., Langelier È., Rancourt D., Auger F.A., Germain L. Titre : Tissue reorganization in response to mechanical load increases functionality Référence : Tissue Engineering 11(1/2) : 90-100, 2005. Lien : Cliquer ici Résumé : In The Rapidly Growing Field Of Tissue Engineering, The Functional Properties Of Tissue Substitutes Are Recognized As Being Of The Utmost Importance. The Present Study Was Designed To Evaluate The Effects Of Static Mechanical Forces On The Functionality Of The Produced Tissue Constructs. Living Tissue Sheets Reconstructed By The Self-Assembly Approach From Human Cells, Without The Addition Of Synthetic Material Or Extracellular Matrix (Ecm), Were Subjected To Mechanical Load To Induce Cell And Ecm Alignment. In Addition, The Effects Of Alignment On The Function Of Substitutes Reconstructed From These Living Tissue Sheets Were Evaluated. Our Results Show That Tissue Constructs Made From Living Tissue Sheets, In Which Fibroblasts And Ecm Were Aligned, Presented Higher Mechanical Resistance. This Was Assessed By The Modulus Of Elasticity And Ultimate Strength As Compared With Tissue Constructs In Which Components Were Randomly Oriented. Moreover, Tissue-Engineered Vascular Media Made From A Prealigned Living Tissue Sheet, Produced With Smooth Muscle Cells, Possessed Greater Contractile Capacity Compared With Those Produced From Living Tissue Sheets That Were Not Prealigned. These Results Show That The Mechanical Force Generated By Cells During Tissue Organization Is An Asset For Tissue Component Alignment. Therefore, This Work Demonstrates A Means To Improve The Functionality (Mechanical And Vasocontractile Properties) Of Tissues Reconstructed By Tissue Engineering By Taking Advantage Of The Biomechanical Forces Generated By Cells Under Static Strain. Auteur(s) : Laflamme K., Roberge C.J., Labonté J., Pouliot S., D’Orléans-Juste P., Auger F.A., Germain L. Titre : Tissue-engineered human vascular media with a functional endothelin system Référence : Circulation 111 :459-464, 2005. Lien : Cliquer ici Résumé : Background: Cardiovascular Diseases Remain A Major Cause Of Death And Disability In The Western World. Among The Various Approaches Adopted To Counteract The Morbidity Associated With These Diseases, Surgical Procedures And Cardiac And Vascular Xenotransplantations Or Allotransplantations Are Routinely Performed. The Suitable Vascular Graft Would Be As Close As Possible To The Native And Healthy Vessel Composed Exclusively Of Human Components Provided By The Patient And Would Adapt To The Donor'S Hemodynamics. We Have Developed Such A Tissue-Engineered Human Blood Vessel Reconstructed With Human Cells. Because Endothelin Is The Most Potent Vasopressor Known To Date, We Were Interested In Investigating The Functionality Of The Endothelinergic System In Our Reconstructed Human Blood Vessel. Methods And Results: Vasoconstriction Studies Were Performed With Nonselective And Selective Agonists And Antagonists To Demonstrate That Et(A) Receptors Were Present And Functional In Tissue-Engineered Human Vascular Media Constructed With The Self-Assembly Method. Reverse-Transcriptase Polymerase Chain Reaction Studies Demonstrated That Mrna Of The Et(A) But Not The Et(B) Receptor Was Present In These Human Tissue-Engineered Blood Vessels. Furthermore, We Demonstrated That The Endothelin-Converting Enzyme, The Main Enzyme Responsible For The Formation Of The Biologically Active Endothelin Peptides, Was Present And Functional In These Same Bioengineered Vascular Media. Conclusions: Our Results Suggest That The Media Component Of Our Tissue-Engineered Blood Vessel Has The Potential Of Controlling Vascular Resistance Via The Presence Of Functional Endothelin Et(A) Receptors And Endothelin-Converting Enzyme. 2004 Auteur(s) : Landreville S., Coulombe S., Carrier P., Gelb M.H., Guérin S.L., Salesse C. Titre : Expression of phospholipases A2 and C in human corneal epithelial cells Référence : Invest Ophthalmol Vis Sci. 2004 Nov;45(11):3997-4003 Lien : Cliquer ici Résumé : Purpose: To Achieve A Better Understanding Of The Involvement Of Phospholipases In The Inflammation And Wound-Healing Processes In Human Corneal Epithelial Cells (Hcecs), Expression Of Phospholipase A2S (Pla2S) And Phospholipase Cs (Plcs) Was Examined In The Human Corneal Epithelium. Methods: Specific Primers Were Designed For Rt-Pcr Amplification Of The Known Secreted (S)Pla2, Cytosolic (C)Pla2, And Plc Mrnas. Corresponding Pcr Products Were Cloned And The Dna Sequenced. Immunofluorescence Of Flatmounted Corneal Sections And Western Blot Analyses Were Used To Detect The Pla2S And Plcs Expressed By Hcecs. Results: The Mrnas For The Following Phospholipases Were Detected By Rt-Pcr In The Hcecs: Spla2Giii, -Gx, And -Gxiia; Cpla2Alpha And -Gamma; Plcbeta1, -Beta2, -Beta3, -Beta4, -Gamma1, -Gamma2, -Delta1, -Delta3, -Delta4, And -Epsilon. Immunofluorescence Analyses Conducted On Corneal Epithelium Cryosections And Western Blot On Freshly Isolated Hcecs Demonstrated The Presence Of Spla2Giii, -Gx, And -Gxiia; Cpla2Alpha And -Gamma; And Plcbeta2, -Beta3, -Gamma1, -Gamma2, And -Delta3. Conclusions: Many Phospholipase Isoforms Are Expressed By Hcecs And May Play A Major Role In Signal Transduction (Plcs) As Well As In The Release Of Precursors Of Potent Mediators Of Inflammation, Such As Leukotrienes And Prostaglandins (Pla2S). Moreover, The Spla2S Expressed By The Corneal Epithelium Could Be Involved In The Normal Antibacterial Activity In The Tears And In Wound Healing. Auteur(s) : Goulet F, Rancourt D, Cloutier R, Tremblay P, Belzil A-M, Lamontagne J, Bouchard M, Tremblay J, Stevens L-M, Labrosse J, Langelier E, McKee MD. Titre : Torn ACL: a new bioengineered substitute brought from the laboratory to the knee joint. Référence : Applied Bionics Biomech. 1(2): 115-121, 2004. Auteur(s) : Langelier E., Dupuis D., Guillot M., Goulet F., Rancourt D. Titre : Cross-sectional profiles and volume reconstructions of soft tissues using Laser Beam Measurements. Référence : J. Biomechan. Eng., 126:796-802, 2004. Lien : Cliquer ici Résumé : Precise Geometric Reconstruction Is A Valuable Tool In The Study Of Soft Tissues Biomechanics. Optical Methods Have Been Developed To Determine The Tissue Cross Section Without Mechanical Contact With The Specimen. An Adaptation Of The Laser Micrometer Developed By Lee And Woo [Asme J. Biomech. Eng., 110 (2), Pp. 110-114]. Is Proposed In Which The Laser-Collimated Beam Rotates Around And Moves Along A Fixed Specimen To Reconstruct Its Cross Sections And Volume. Beam Motion Is Computer Controlled To Accelerate Data Acquisition And Improve Beam Positioning Accuracy. It Minimizes Time-Dependent Shape Modifications And Increases Global Reconstruction Precision. The Technique Is Also Competent For The Measurement Of Immersed Collagen Matrices. Auteur(s) : Paquette J.-S., Moulin V., Tremblay P., Bernier V., Boutet M., Laviolette M., Auger F.A., Boulet L.-P., Goulet F. Titre : Tissue-engineered human asthmatic bronchial equivalents Référence : European Cells and Materials 7: 1-11, 2004. Lien : Cliquer ici Résumé : The Isolation Of Human Bronchial Epithelial (Hbec) And Fibroblastic Cells (Hbfc) From Biopsies Of Asthmatic And Non-Asthmatic Volunteers Provided Unique Cellular Materials To Be Used For The Production Of Bioengineered Bronchial Equivalents (Be) In Vitro. The Hbec Are Grown On A Mesenchymal Layer Seeded With Hbfc And The Be Can Be Maintained For At Least 15 Days In Culture. Under The Be Culture Conditions Established Previously, Hbec Undergo Differentiation Into Ciliated And Goblet Cells, Within A Pseudostratified Organization Comparable To Human Bronchi. We Published Previously The Results From Histologic And Functional Analyses Of Such Be Produced Exclusively Using Non-Asthmatic Hbec And Hbfc. We Report Here The Comparative Analyses Of Be Produced With Non-Asthmatic And Asthmatic Living Hbec And Hbfc (Nabe And Abe, Respectively). Our Data Indicated That All Asthmatic Hbec Populations Grown On A Mesenchymal Layer, Containing Non-Asthmatic Hbfc, Slowly Reached A Confluent State But Then Detached From The Matrix Upon Culture Time. These Be Appear To Be Very Good Models To Study The Mechanisms Involved In Asthma In Vitro. Auteur(s) : Amiot J., Germain L., Turgeon S., Lemay M., Ory-Salam C., Auger F.A. Titre : Peptides from milk protein hydrolysates to improve the growth of human keratinocytes in culture Référence : International Dairy Journal 14 : 619-626, 2004. Auteur(s) : Auger F.A., Germain L. Titre : Tissue Engineering Référence : Encyclopedia of Biomaterials and Biomedical Engineering, Marcel Dekker : New York : 1477-1483, 2004. Auteur(s) : Germain L., Berthod F., Moulin V., Goulet F., Auger F.A. Titre : Principles of living organ reconstruction by tissue engineering Référence : Tissue Engineering and Novel Delivery Systems, Eds Yaszemski M.J., Trantolo D.J., Lewandrowski K.U., Hasirci V, Altobelli D.E., Wise D.L., ed Marcel Dekker, Inc. : New-York. Chap. 10 : 197-228, 2004. Auteur(s) : Germain L., Giasson C.J., Carrier P., Guérin S.L., Salesse C., Auger F.A. Titre : Tissue Engineering of cornea Référence : Encyclopedia of Biomaterials and Biomedical Engineering, Marcel Dekker : New York : 1534-1544, 2004. Auteur(s) : Auger F.A., Berthod F., Moulin V., Pouliot R., Germain L. Titre : Tissue-engineered skin substitutes : from in vitro constructs to in vivo applications Référence : Biotechnology of Applied Biochemistry 39 : 263-275, 2004. Lien : Cliquer ici Résumé : The Field Of Skin Tissue Engineering Is A Paradigm For The Various Efforts Towards The Reconstruction Of Other Tissues And Organ Substitutes. As Skin Replacement, This Biotechnological Approach Has Evolved From Simple Cultured Autologous Epidermal Sheets To More Complex Bilayered Cutaneous Substitutes. The Various Types Of Such Substitutes Are Herein Presented With Their Intended Use. However, Two Integrative Characteristics Are Analysed More Specifically Because Of Their Critical Role: Neovascularization And Re-Innervation. Furthermore, The In Vitro Use Of These Various Skin Substitutes Has Shed Light On Various Physiological And Pathological Phenomena. Thus, Not Only The In Vivo Application Of These Skin Substitutes As Grafts, But Also Their In Vitro Value As Skin Models, Are Presented. Auteur(s) : Larochelle S, Langlois C, Thibault I, Lopez-Valle CA, Roy M, Moulin V. Titre : Sensitivity of myofibroblasts to H2O2-mediated apoptosis and their antioxidant cell network Référence : Journal of Cellular Physiology 200: 263-271, 2004. Lien : Cliquer ici Résumé : During Wound Healing, The Transition From Granulation To Scar Tissue Shows A Decrease In Myofibroblast Cellularity. Previous Results Have Correlated The Disappearance Of These Cells With The Induction Of Apoptotic Cell Death By Some Unknown Stimuli. In Contrast, Hypertrophic Scar Appearance After Wound Healing Is Thought To Be Linked To A Disorder Of Apoptotic Function Which Induces Myofibroblast Persistence In Granulation Tissue. Oxidative Stress Being An Important Mediator Of Apoptosis, We Have Evaluated The Apoptotic Response Of Normal And Pathological Wound Myofibroblasts (Wmyo And Hmyo Respectively) In Their Interaction With Two Oxidative Stress Inducers: Hydrogen Peroxide, Using A High Concentration As A Single Dose, And Sodium Ascorbate Which Induced A Continuous Release Of H2O2 At A Low Concentration. Our Results Showed That, According To The H2O2 Treatment Type, Hmyo Were More Sensitive (After Ascorbate Treatment) Or Less Sensitive (After H2O2 Treatment) When Compared To Wmyo And Fb. We Next Assessed The Presence Of Several Molecules Known To Be Involved In The Antioxidant Network Protecting Cells Against H2O2 Injury And Found Hmyo To Have A Higher Level Of Activity Of Glutathione Peroxidase And A Lower Level Of Activity Of Catalase Than Wmyo. These Results Can Help Explain The Contradictory Responses Of Myofibroblasts According To The Oxidative Stress Treatment. This Is The First Study Linking Refractory Oxidative Stress Mediated Cell Death To Cellular Phenotype In Hypertrophic Myofibroblasts, And Indicates A Pivotal Role For The Antioxidant Enzyme System In This Type Of Resistance. Auteur(s) : Larouche D., Hayward C., Cuffley K., Germain L. Titre : Keratin 19 as a stem cell marker in vivo and in vitro Référence : Methods in Molecular Biology - Epidermal cells : Methods and protocols, Ed : K. Turksen, Humana Press Inc. : Totowa, NJ. Chap. 12(289) : 103-110, 2004. Lien : Cliquer ici Résumé : The Skin Is A Dynamic Tissue In Which Terminally Differentiated Keratinocytes Are Replaced By The Proliferation Of New Epithelial Cells That Will Undergo Differentiation. The Rapid And Continual Turnover Of Skin Throughout Life Depends On A Cell Population With Unique Characteristics: The Stem Cells. These Cells Are Relatively Undifferentiated, Retain A High Capacity For Self-Renewal Throughout Their Lifetime, Have A Large Proliferative Potential, And Are Normally Slow Cycling. The Long-Term Regeneration Of Grafted Cultured Epidermis Indicates That Epidermal Stem Cells Are Maintained In Cultures. In Animals They Can Be Identified With 3H-Thymidine Or Bromodeoxyuridine Based On Their Property Of Slow Cycling. The Development Of Markers Such As Keratin 19 Also Permits Their Study In Human Tissues. In This Chapter, Protocols To Study Skin Stem Cells Using Their Property Of Slow Cycling And Their Expression Of Keratin 19 Will Be Described In Detail. The Methods Include The Double Labeling Of Tissues For Keratin 19 And Label-Retaining Cells (Auto Radiography Of 3H-Thymidine) In Situ. The Labeling Of Keratin 19 By Immunofluorescence Of By Flow Cytometry Is Described For Cells In Vitro. Auteur(s) : Moulin V., Larochelle S., Langlois C., Thibault I., Lopez-Vallé C.A., Roy M. Titre : Normal skin wound and hypertrophic scar myofibroblasts have differential responses to apoptotic inductors Référence : Journal of Cellular Physiology 198(3): 350-358, 2004 Lien : Cliquer ici Résumé : During Wound Healing, Myofibroblasts Play A Central Role In Matrix Formation And Wound Contraction. At The End Of Healing, There Is Evidence That Myofibroblasts Disappear Via Apoptotic Pathways. Hypertrophic Scars Are A Fibroproliferative Disorder That Leads To Considerable Morbidity. It Has Been Postulated That A Defect In Myofibroblast Apoptosis Could Be Responsible For The Pathological Scar Formation, But No Evidence Exists. We Have Isolated And Cultured Human Normal Wound (Wmyo) And Hypertrophic Scar (Hmyo) Myofibroblasts And Compared Their Basal Apoptotic Rates And Their Sensitivity To Serum Starvation And Fas Antibody-Induced Apoptosis To That Obtained For Dermal Fibroblasts (Fb). A Higher Rate Of Apoptosis As Evidenced By Morphological Criteria And A Propidium Iodide Assay Was Observed For Wmyo In Comparison To Fb And Hmyo. These Results Came Along With A Low Level Of The Anti-Apoptotic Proteins Bcl-2 And Bclx(L) In Wmyo, Whereas There Was An Increase In The Level Of The Pro-Apoptotic Molecule Bax When Compared To The Results Obtained For Fb And Hmyo. Hmyo Showed A Higher Level Of Bcl-2 Compared To Fb But No Difference In The Bax Or Bclx(L) Level. After Serum Starvation, Wmyo Revealed An Increased Apoptotic Rate, Whereas Hmyo And Fb Did Not Show Any Difference. Anti-Fas Treatment Did Not Modify The Levels Of Apoptosis But Strongly Increased The Cell Growth Of Hmyo As Compared To Wmyo. This Is The First Study Presenting A Broad Vision Of The Apoptotic Sensitivity Of Normal And Pathological Myofibroblasts. These Results Confirmed The Hypothesis Of Defects In Apoptosis And Growth During Pathological Scar Formation Impeding Myofibroblast Disappearance At The End Of Healing. Auteur(s) : Rémy-Zolghadri M., Laganière J., Oligny J.-F., Germain L., Auger F.A. Titre : Endothelium properties of a tissue-engineered blood vessel for small-diameter vascular reconstruction Référence : Journal of Vascular Surgery 39(3): 613-620, 2004. Lien : Cliquer ici Résumé : Purpose: A Tissue-Engineered Blood Vessel (Tebv) Produced In Vitro By The Self-Assembly Method Was Developed In Our Laboratory For The Replacement Of Small-Diameter Blood Vessels. The Interior Of This Vessel Is Covered By An Endothelium. The Aim Of The Present Study Was To Evaluate Whether The Endothelial Layer Would Make A Favorable Contribution At The Time Of Implantation Of The Tebv By Investigating In Vitro The Hemocompatible Properties Of The Endothelial Cells Covering Its Interior. Methods: The Secretion Of The Von Willebrand Factor (Vwf) And Expression Of Thrombomodulin By The Endothelium Were Assessed, And The Adhesive Molecules E-Selectin And Intercellular Adhesion Molecule-1 (Icam-1) Were Quantified As A Function Of Maturation Time. To Evaluate The Functional Response Of The Endothelium On Injury, The Cellular Response To Physiological Stimulatory Factors (Thrombin And Lipopolysaccharide [Lps]) Was Analyzed. Results: The Endothelial Cells Formed A Confluent Monolayer Displaying Favorable Hemocompatible Properties (78% +/- 10% Of Cells Expressing Thrombomodulin With Only 12 +/- 3 Mu/10(6) Cells Of Vwf Secreted Over A 2-Hour Period), Which Acquired Their Full Expression After A Culture Period Of 4 Days. Moreover, Pro-Adhesive Properties Toward Inflammatory Cells Were Not Observed. The Cells Were Also Able To Respond To Physiological-Stimulating Agents (Thrombin And Lps) And Demonstrated A Statistically Significant Overexpression Of The Corresponding Molecules Under The Conditions Tested. Conclusions: These Results Indicate That The Endothelium Of The Tissue-Engineered Blood Vessel Produced By The Self-Assembly Approach Displays Advantageous Qualities With Regard To The Vessel'S Future Implantation As A Small-Diameter Vascular Prosthesis. Auteur(s) : Stoclet J.-C., Laflamme K., Auger F.A., Germain L. Titre : Vaisseaux humains reconstitués par génie tissulaire Référence : Médecine/Sciences 20 : 675-678, 2004 Lien : Cliquer ici Résumé : Les Progrès Du Génie Tissulaire Permettent Maintenant De Reconstituer Des Vaisseaux Sanguins Fonctionnels À Partir De Cellules Humaines. Dans Des Conditions Bien Précises, Ces Vaisseaux Possèdent Une Structure, Des Propriétés Mécaniques Et Des Propriétés Fonctionnelles (Notamment En Termes De Vasomotricité) Qui Permettent De Les Utiliser Comme Modèles Pour Contourner Les Difficultés D’Obtention Et D’Utilisation De Vaisseaux Humains Pour La Recherche Expérimentale. En Effet, L’Utilisation De Vaisseaux Humains À Des Fins Expérimentales Est Limitée Par Des Problèmes Éthiques Et Par Les Difficultés D’Interprétation Des Résultats Liées À Leur Hétérogénéité. C’Est Pourquoi Une Grande Partie Des Recherches En Biologie Et En Pharmacologie Vasculaires Est Réalisée Soit Sur Des Modèles Animaux, Soit Sur Des Cellules En Culture, Qui Ne Sont Pas Toujours Représentatifs Des Vaisseaux Humains. Le Génie Tissulaire Peut Apporter Une Source Alternative De Vaisseaux Humains Pour Pallier Ces Inconvénients. 2003 Auteur(s) : Auger F.A., Grenier G., Rémy-Zolghadri M., Germain L. Titre : A full spectrum of functional tissue-engineered blood vessels : from macroscopic to microscopic Référence : Functional Tissue Engineering, Eds Guilak F., Butler D.L., Goldstein S.A., Mooney D.J., ed Springer-Verlag : New-York. Chap. 26: 237-359, 2003. Auteur(s) : Fradette J., Larouche D., Fugère C., Guignard R., Beauparlant A., Couture V., Caouette-Laberge L., Roy A., Germain L. Titre : Dissociation, quantification and culture of normal human merkel cells among epidermal cell populations derived from glabrous and hairy skin sites Référence : Merkell Cells, Eds Baumann K.I., Halata Z., Moll I., ed Springer-Verlag : Berlin, Allemagne. 105-112, 2003. Auteur(s) : Fradette J, Larouche D, Fugere C, Guignard R, Beauparlant A, Couture V, Caouette-Laberge L, Roy A, Germain L. Titre : Normal human merkel cells are present in epidermal cell populations isolated and cultured from glabrous and hairy skin sites Référence : The Journal of Investigative Dermatology 120 (2): 313-317, 2003. Lien : Cliquer ici Résumé : The Merkel Cell Is A Highly Specialized Cell That Primarily Acts As A Slowly Adapting Mechanoreceptor. Merkel Cells Are Scarce In Normal Skin But Can Be Identified By The Expression Of Distinct Keratin Filaments. Merkel Cells Constitute A Very Unique Population And Many Questions Still Remain As To Their Origin, Number, Proliferative Capacity, And Functions In Cutaneous Biology. The Dissociation Of Epidermal Cells From Skin Is A Widely Used Technique To Extract And Culture Keratinocytes. We Took Advantage Of A Two-Step Extraction Method To Quantify Keratin-20-Expressing Merkel Cells Among Total Cutaneous Cells Obtained From Either Hairy Or Glabrous Skin Biopsies. Flow Cytometry Analysis Revealed That Keratin-20-Labeled Merkel Cells Represent Between 3.6% And 5.7% Of Freshly Dissociated Basal Epidermal Cells. No Significant Differences Were Seen Between Samples Derived From Glabrous Palmar And Hairy Anatomic Sites, From Children And Adult, Respectively. We Also Report On The Presence Of Merkel Cells In Primary And First Subcultures Of Epidermal Cells Indicating Their Capacity To Remain Viable After Extraction From Skin Of Various Anatomic Sites. To Our Knowledge, This Is The First Demonstration Of Nontumorigenic Human Merkel Cells In Culture In Vitro. The Persistence Of A Small Number Of Merkel Cells In Culture Suggests That, With The Development Of Appropriate Culture Conditions, These Cells Could Be Amplified And Further Studied To Unravel Long-Standing Questions Relative To Their Paracrine Function Or Epithelial Origin. Auteur(s) : Gaudreault M., Carrier P., Larouche K., Leclerc S., Giasson M., Germain L., Guérin S.L. Titre : Influence of Sp1/Sp3 expression on corneal epithelial cells proliferation and differentiation properties in reconstructed tissues Référence : Investigative Ophtalmology and Visual Science 44 (4): 1447-1457, 2003. Lien : Cliquer ici Résumé : Purpose: Primary Cultured Epithelial Cells Are Widely Used For The Production Of Tissue-Engineered Substitutes And Are Gaining Popularity As A Model For Gene Expression Studies. However, As Such Cells Are Passaged In Culture, They Often Lose Their Ability To Proliferate By Progressing Toward Terminal Cell Differentiation, A Process Likely To Be Determined By Altered Expression Of Transcription Factors That Have Functions Critical For Cell Adhesion And Differentiation. This Study Was Designed To Determine Whether The Variable Life Span Of Primary Cultured Human Corneal Epithelial Cells (Hcecs) Might Be The Consequence Of Varying Expression Levels Of The Well-Known Transcription Factors Sp1 And Sp3 (Sp1/Sp3). Methods: Hcecs Were Obtained From Donor Eyes And Cultured On Irradiated Swiss-3T3. Sp1/Sp3 Expression Was Monitored By Western Blot And Electrophoretic Mobility Shift Assay (Emsa). The Sp1/Sp3 Regulatory Influence Was Evaluated By Transfection Of Hcecs With A Recombinant Plasmid Bearing The Sp1/Sp3-Dependent Poly(Adp-Ribose) Polymerase (Rparp) Promoter Fused To The Cat Reporter Gene. Hcecs That Expressed Various Levels Of Sp1/Sp3 Were Also Used For The Production Of Corneal Substitutes. Results: Expression Of Sp1/Sp3 Was Dramatically Inconsistent Between Hcecs Isolated From The Eyes Of Different Donors. Both Factors Were Highly Expressed During One Passage And Then Totally Disappeared As Cells Terminally Differentiated. Proper Stratification Of Hcecs On Reconstructed Tissue Substitutes Could Be Obtained Only With Cells That Also Had A Delayed Peak Of Sp1/Sp3 Expression When Cultured In Vitro. Conclusions: Expression Of Sp1/Sp3 May Represent A Good Predictor For Selecting Hcecs That Are Most Likely To Proliferate, Stratify, And Differentiate Properly When Used For The Production Of Reconstructed Corneal Substitutes. Auteur(s) : Gingras M., Bergeron J., Déry J., Durham H.D., Berthod F. Titre : In vitro development of a tissue-engineered model of peripheral nerve regeneration to study neurite growth Référence : The FASEB Journal 17: 2124-2126, 2003. Lien : Cliquer ici Résumé : A Unique Tissue-Engineered Model Of Peripheral Nerve Regeneration Was Developed In Vitro To Study Neurite Outgrowth. Mouse Dorsal Root Ganglia Neurons Were Seeded On A Collagen Sponge Populated With Human Endothelial Cells And/Or Human Fibroblasts. Addition Of Nerve Growth Factor (Ngf; 10 Ng/Ml) Was Not Required For Sensory Neurons Survival But Was Necessary To Promote Neurite Outgrowth, As Assessed By Immunostaining Of The 150 Kda Neurofilament. A Vigorous Neurite Elongation Was Detected Inside The Reconstructed Tissue After 14 And 31 Days Of Neurons Culture, Reaching Up To 770 Microm From Day 14. Axons Were Often Observed Closely Associated With The Capillary-Like Tubes Reconstructed In The Model, In A Similar Pattern As In The Human Dermis. The Presence Of Endothelial Cells Induced A Significant Increase Of The Neurite Elongation After 14 Days Of Culture. The Addition Of Human Keratinocytes Totally Avoided The Twofold Decrease In The Amount Of Neurites Observed Between 14 And 31 Days In Controls. Besides The Addition Of Ngf, Axonal Growth Did Not Necessitate B27 Supplement Or Glial Cell Coculture To Be Promoted And Stabilized For Long-Term Culture. Thus, This Model Might Be A Valuable Tool To Study The Effect Of Various Cells And/Or Attractive Or Repulsive Molecules On Neurite Outgrowth In Vitro. Auteur(s) : Gingras M, Paradis I, Berthod F. Titre : Nerve regeneration in a collagen-chitosan tissue-engineered skin transplanted on nude mice Référence : Biomaterials 24: 1653-1661, 2003. Lien : Cliquer ici Résumé : A Reconstructed Skin Made Of A Collagen-Chitosan Sponge Seeded With Human Fibroblasts And Keratinocytes And Grown In Vitro For 31 Days Was Developed For The Treatment Of Deep And Extensive Burns. The Aim Of This Study Was To Assess Whether This Tissue-Engineered Skin Could Promote Nerve Regeneration In Vivo, Since Recovery Of Sensation Is A Major Concern For Burnt Patients. The Human Reconstructed Skin Was Transplanted On The Back Of Nude Mice And The Growth Of Nerve Fibres Within It Was Assessed 40, 60, 90 And 120 Days After Graft. Nerve Growth Was Monitored By Confocal Microscopy Using Immunohistochemical Staining Of Pgp 9.5 And 150 Kd Neurofilament, While Schwann Cell Migration Was Observed Using Protein S100 Expression And Laminin Deposition. Nerve Growth Was First Detected 60 Days After Transplantation And Was More Abundant 90 And 120 Days After Graft. Linear Arrangements Of Schwann Cells Were Observed In The Graft As Early As 40 Days After Graft. Nerve Growth Was Observed Along These Schwann Cell Extensions 60 Days After Transplantation. We Conclude That The Three-Dimensional Architecture Of The Collagen-Chitosan Tissue-Engineered Skin Sponge Encourages Nerve Growth. This Result Provides New Perspectives To Increase Nerve Regeneration Within The Tissue-Engineered Skin By Linkage Of Neurotrophic Factors In The Sponge Before Transplantation. Auteur(s) : Grenier G., Rémy-Zolghadri M., Guignard R., Bergeron F., Labbé R., Auger F.A., Germain L. Titre : Isolation and culture of the three vascular cell types from a small vein biopsy sample Référence : In Vitro Cellular Development Biology – Animal 39 : 131-139, 2003. Lien : Cliquer ici Résumé : The Availability Of Small-Diameter Blood Vessels Remains A Significant Problem In Vascular Reconstruction. In Small-Diameter Blood Vessels, Synthetic Grafts Resulted In Low Patency; The Addition Of Endothelial Cells (Ec) Has Clearly Improved This Parameter, Thereby Proving The Important Contribution Of The Cellular Component To The Functionality Of Any Construct. Because The Optimal Source Of Cells Should Be Autologous, The Adaptation Of Existing Methods For The Isolation Of All The Vascular Cell Types Present In A Single And Small Biopsy Sample, Thus Reducing Patient'S Morbidity, Is A First Step Toward Future Clinical Applications Of Any Newly Developed Tissue-Engineered Blood Vessel. This Study Describes Such A Cell-Harvesting Procedure From Vein Biopsy Samples Of Canine And Human Origin. For This Purpose, We Combined Preexisting Mechanical Methods For The Isolation Of The Three Vascular Cell Types: Ec By Scraping Of The Endothelium Using A Scalpel Blade, Vascular Smooth Muscle Cells (Vsmc), And Perivascular Fibroblasts According To The Explant Method. Once In Culture, Cells Rapidly Grew With The High Level Of Enrichment. The Morphological, Phenotypical, And Functional Expected Criteria Were Maintained: Ec Formed Cobblestone Colonies, Expressed The Von Willebrand Factor, And Incorporated Acetylated Low-Density Lipoprotein (Ldl); Vsmc Were Elongated And Contracted When Challenged By Vasoactive Agents; Perivascular Fibroblasts Formed A Mechanically Resistant Structure. Thus, We Demonstrated That An Appropriate Combination Of Preexisting Harvesting Methods Is Suitable To Isolate Simultaneously The Vascular Cell Types Present In A Single Biopsy Sample. Their Functional Characteristics Indicated That They Were Suitable For The Cellularization Of Synthetic Prosthesis Or The Reconstruction Of Functional Multicellular Autologous Organs By Tissue Engineering. Auteur(s) : Hudon V., Berthod F., Black A.F., D’Amour O., Germain L., Auger F.A. Titre : A tissue-engineered endothelialized dermis to study the modulation of angiogenic and angiostatic molecules on capillary-like tube formation in vitro Référence : British Journal of Dermatology 148 : 1094-1104, 2003. Lien : Cliquer ici Résumé : Background: Because Angiogenesis Is A Major Feature Of Different Physiological And Pathological Situations, The Identification Of Factors That Stimulate Or Inhibit This Process And The Elucidation Of Their Mechanisms Of Action Are Most Certainly Of Clinical Relevance. We Have Produced A New Model Of Endothelialized Reconstructed Dermis That Promotes The Spontaneous Formation Of A Human Capillary-Like Network And Its Stabilization In Vitro For A Period Longer Than 1 Month. Objectives: The Aim Of This Work Was To Describe The Three-Dimensional Structure Of The Capillary-Like Network. Thereafter We Strove To Study, Quantitatively And Qualitatively, The Influence Of Angiogenic And Angiostatic Drugs On Capillary-Like Tube (Clt) Formation In Vitro In The Model. Methods: The Endothelialized Dermis Was Prepared By Coculturing Two Human Cell Types, Dermal Fibroblasts And Umbilical Vein Endothelial Cells, In A Collagen Sponge Biomaterial. Results: The Visualization By Confocal Microscopy Of The Tubes Present In The Model Showed That The Endothelial Structures Were Not Cord-Like But Rather Clts With Well-Defined Lumina. Moreover, These Tubes Were Organized In A Complex Network Of Branching Structures. When Angiogenic Factors (Vascular Endothelial Growth Factor 10 Ng Ml-1 Or Basic Fibroblast Growth Factor 10 Ng Ml-1) Were Added To The Model, 1.8 And 1.4 Times More Capillaries, Respectively, Were Observed, Whereas The Addition Of Progesterone (10 Microg X Ml(-1)) Reduced By 2.4 Times The Number Of Tubes Compared With The Control. Conclusions: These Results Suggest That This Model Is A Highly Efficient Assay For The Screening Of Potentially Angiogenic And Angiostatic Compounds. Auteur(s) : Moulin V, Goulet F, Berthod F, Germain L, Auger FA. Titre : Le génie tissulaire au service de la compréhension du vivant Référence : Médecine/Sciences 19 (10): 1003-1010, 2003. Lien : Cliquer ici Résumé : Le Génie Tissulaire Est Un Nouveau Domaine, Qui Permet L’Étude Des Mécanismes Physiologiques Du Vivant. Il S’Agit D’Une Technologie Fondée Sur La Capacité Des Cellules Vivantes, En Présence Ou Non De Biomatériaux, À S’Assembler En Un Tissu Tridimensionnel. Elle Constitue Une Voie Intéressante Ouvrant Aux Chercheurs La Possibilité De Considérer Les Cellules Dans Un Contexte Proche De Celui Retrouvé In Vivo. Cet Article Résume Les Travaux En Génie Tissulaire Menés Par Le Laboratoire D’Organogenèse Expérimentale (Loex) Au Cours Des Dernières Années, Dans Le But De Comprendre Certains Des Mécanismes Physiologiques Et Pathologiques De L’Organisme Humain. Ainsi, La Cicatrisation Cutanée, Mais Aussi Les Cellules Souches, L’Angiogenèse Et Les Interactions Cellulaires Sont Des Secteurs Ayant Profité De L’Apport Du Génie Tissulaire. Auteur(s) : Paquette J-S., Tremblay P., Bernier V., Auger F.A., Laviolette M., Germain L., Boutet M., Boulet L.P., Goulet F. Titre : Production of tissue-engineered three-dimensional human bronchial models Référence : In Vitro Cellular Development Biology – Animal 39 : 213-220, 2003. Lien : Cliquer ici Résumé : We Have Reported Morphological And Functional Features Of Cells Isolated From Human Bronchial Biopsies. Both Epithelial And Fibroblastic Cells Were Isolated From The Same Biopsies Using Collagenase. A Few Models Have Been Established To Study Normal Bronchial Response To Various Agents And To Understand The Mechanisms Responsible For Some Disorders, Such As Asthma. We Produced Three-Dimensional Bronchial Equivalents In Culture, Using Human Epithelial And Fibroblastic Cells. We Previously Showed That Peripheral Anchorage Can Prevent The Dramatic Collagen Contraction In Gels Seeded With Fibroblasts When Properly Adapted To The Size And Type Of Cultured Tissues. Our Bilayered Bronchial Constructs Were Anchored And Cultured Under Submerged Conditions And At The Air-Liquid Interface. Three Culture Media Were Compared. Serum-Free Medium Supplemented With Retinoic Acid (5 X 10(-8) M) Was Found To Be The Best For Maintenance Of Bronchial Cell Properties In The Reconstructed Bronchial Tissue. Immunohistological And Ultrastructural Analyses Showed That These Equivalents Present Good Structural Organization, Allowing Ciliogenesis To Occur In Culture. Moreover, Human Bronchial Goblet Cells Could Differentiate And Secrete Mucus With Culture Time. Laminin, A Major Constituent Of The Basement Membrane And Basal Cells, Was Also Detected At The Mesenchymoepithelial Interface. Such Models Will Be Useful For Studying Human Bronchial Properties In Vitro. 2002 Auteur(s) : Auger F.A., Rémy-Zolghadri M., Grenier G., Germain L. Titre : A truly new approach for tissue engineering : The LOEX self-assembly technique Référence : Stem cell transplantation and tissue engineering, A. Haverich, H. Graf, eds, Springer-Verlag, Berlin. Chap. 6 : 73-88, 2002. Lien : Cliquer ici Auteur(s) : Germain L., Goulet F., Moulin V., Berthod F., Auger F.A. Titre : Engineering human tissues for in vivo applications Référence : Engineering human tissues for in vivo applications" Annals of the New York Academy of Sciences 961: 268-270, 2002. Lien : Cliquer ici Auteur(s) : Germain L., Rémy-Zolghadri M., Auger F.A. Titre : Mesenchymal cell culture: blood vessels Référence : Methods of Tissue Engineering Chap. 28: 359-370, 2002. Auteur(s) : Pouliot R., Larouche D., Auger F.A., Juhasz J., Xu W., Li H., Germain L. Titre : Reconstructed human skin produced in vitro and grafted on athymic mice Référence : Transplantation 73 (11): 1751-1757, 2002. Lien : Cliquer ici Résumé : Background: The Best Alternative To A Split-Thickness Graft For The Wound Coverage Of Patients With Extensive Burns Should Be In Vitro Reconstructed Autologous Skin Made Of Both Dermis And Epidermis And Devoid Of Exogenous Extracellular Matrix Proteins And Synthetic Material. We Have Designed Such A Reconstructed Human Skin (Rhs) And Present Here Its First In Vivo Grafting On Athymic Mice. Methods: The Rhs Was Made By Culturing Newborn Or Adult Keratinocytes On Superimposed Fibrous Sheets Obtained After Culturing Human Fibroblasts With Ascorbic Acid. Ten Days After Keratinocyte Seeding, Reconstructed Skins Were Either Cultured At The Air-Liquid Interface Or Grafted On Athymic Mice. We Present The Macroscopic, Histologic, And Phenotypic Properties Of Such Tissues In Vitro And In Vivo After Grafting On Nude Mice. Results: After Maturation In Vitro, The Reconstructed Skin Exhibited A Well-Developed Human Epidermis That Expressed Differentiated Markers And Basement Membrane Proteins. Four Days After Grafting, A Complete Take Of All Grafts Was Obtained. Histological Analysis Revealed That The Newly Generated Epidermis Of Newborn Rhs Was Thicker Than That Of Adult Rhs After 4 Days But Similar 21 Days After Grafting. The Basement Membrane Components (Bullous Pemphigoid Antigens, Laminin, And Type Iv And Vii Collagens) Were Detected At The Dermo-Epidermal Junction, Showing A Continuous Line 4 Days After Grafting. Ultrastructural Studies Revealed That The Basement Membrane Was Continuous And Well Organized 21 Days After Transplantation. The Macroscopic Aspect Of The Reconstructed Skin Revealed A Resistant, Supple, And Elastic Tissue. Elastin Staining And Elastic Fibers Were Detected As A Complex Network In The Rhs That Contributes To The Good Elasticity Of This New Reconstructed Tissue. Conclusions: This New Rhs Model Gives Supple And Easy To Handle Skins While Demonstrating An Adequate Wound Healing On Mice. These Results Are Promising For The Development Of This Skin Substitute For Permanent Coverage Of Burn Wounds. 2001 Auteur(s) : Auger F.A., Hayward C.J., Lòpez Valle C.A., Guignard R., Germain L. Titre : The effect of the tissue glue Tisseel® on the grafting of cultured human epidermal sheets and their evolution in vivo Référence : Cultured Human Keratinocytes and Tissue Engineered Skin Substitutes, Eds : Horch R.E., Munster A.M., Achauer B.M., ed Georg Thieme Verlag : Allemagne. Section III : 287-297, 2001. Auteur(s) : Berthod F., Germain L., Li H., Xu W., Damour O., Auger F.A. Titre : Collagen fibril network and elastic system remodeling in a reconstructed skin transplanted on nude mice Référence : Matrix Biology 20: 463-473, 2001. Lien : Cliquer ici Résumé : Wound Healing Of Deep And Extensive Burns Can Induce Hypertrophic Scar Formation, Which Is A Detrimental Outcome For Skin Functionality. These Scars Are Characterized By An Impaired Collagen Fibril Organization With Fibril Bundles Oriented Parallel To Each Other, In Contrast With A Basket Weave Pattern Arrangement In Normal Skin. We Prepared A Reconstructed Skin Made Of A Collagen Sponge Seeded With Human Fibroblasts And Keratinocytes And Grown In Vitro For 20 Days. We Transplanted It On The Back Of Nude Mice To Assess Whether This Reconstructed Skin Could Prevent Scar Formation. After Transplantation, Murine Blood Vessels Had Revascularized One-Third Of The Sponge Thickness On The Fifth Day And Were Observed Underneath The Epidermis At Day 15. The Reconstructed Skin Extracellular Matrix Was Mostly Made Of Human Collagen I, Organized In Loosely Packed Fibrils 5 Days After Transplantation, With A Mean Diameter Of 45 Nm. After 40-90 Days, Fibril Bundles Were Arranged In A Basket Weave Pattern While Their Mean Diameter Increased To 56 Nm, Therefore Exactly Matching Mouse Skin Papillary Dermis Organization. Interestingly, We Showed That An Elastic System Remodeling Was Started Off In This Model. Indeed, Human Elastin Deposits Were Organized In Thin Fibrils Oriented Perpendicular To Epidermis At Day 90 Whereas Elastic System Usually Took Years To Be Re-Established In Human Scars. Our Reconstructed Skin Model Promoted In Only 90 Days The Remodeling Of An Extracellular Matrix Nearly Similar To Normal Dermis (I.E. Collagen Fibril Diameter And Arrangement, And The Partial Reconstruction Of The Elastic System). Auteur(s) : Laplante A.F., Germain L., Auger F.A., Moulin V. Titre : Mechanisms of wound reepithelialization: hints from a tissue-engineered reconstructed skin to long standing questions Référence : The FASEB Journal 15: 2377-2389, 2001. Lien : Cliquer ici Résumé : Wound Closure Of Epithelial Tissues Must Occur Efficiently To Restore Rapidly Their Barrier Function. We Have Developed A Tissue-Engineered Wound-Healing Model Composed Of Human Skin Keratinocytes And Fibroblasts To Better Understand The Mechanisms Of Reepithelialization. It Allowed Us To Quantify The Reepithelialization Rate, Which Was Significantly Accelerated In The Presence Of Fibrin Or Platelet-Rich Plasma. The Reepithelialization Of These 6 Mm Excisional Wounds Required The Contribution Of Keratinocyte Proliferation, Migration, Stratification, And Differentiation. The Epidermis Regenerated Progressively From The Surrounding Wound Margins. After 3 Days, The Neoepidermis Showed A Complete Spectrum Of Changes. Near The Wound Margin, The Differentiation Of The Neoepidermis (Keratins 1/10, Filaggrin, And Loricrin) And Regeneration Of The Dermoepidermal Junction (Laminin 5 And Collagen Iv) Were More Advanced Than Toward The Wound Center, Where The Proliferative Index Was Significantly Increased. The Spatial Distribution Of Keratinocytes Distinguished By Particular Features Suggests Two Complementary Mechanisms Of Reepithelialization: 1) The Passive Displacement Of The Superficial Layers Near The Wound Margin That Would Rapidly Regenerate A Barrier Function And 2) The Crawling Of Keratinocytes Over Each Other At The Tip Of The Progressing Neoepidermis. Therefore, This Study Brings A New Perspective To Long-Standing Questions Concerning Wound Reepithelialization. Auteur(s) : L'Heureux N., Stoclet J.-C., Auger F.A., Lagaud G.J.-L., Germain L., Andriantsitohaina R. Titre : A human tissue-engineered vascular media: a new model for pharmacological sudies of contractiles responses Référence : The FASEB Journal 15: 515-524, 2001. Lien : Cliquer ici Résumé : Our Method For Producing Tissue-Engineered Blood Vessels Based Exclusively On The Use Of Human Cells, I.E., Without Artificial Scaffolding, Has Previously Been Described (1). In This Report, A Tissue-Engineered Vascular Media (Tevm) Was Specifically Produced For Pharmacological Studies From Cultured Human Vascular Smooth Muscle Cells (Vsmc). The Vsmc Displayed A Differentiated Phenotype As Demonstrated By The Re-Expression Of Vsmc-Specific Markers And Actual Tissue Contraction In Response To Physiological Stimuli. Because Of Their Physiological Shape And Mechanical Strength, Rings Of Human Tevm Could Be Mounted On Force Transducers In Organ Baths To Perform Standard Pharmacological Experiments. Concentration-Response Curves To Vasoconstrictor Agonists (Histamine, Bradykinin, Atp, And Utp) Were Established, With Or Without Selective Antagonists, Allowing Pharmacological Characterization Of Receptors (H1, B2, And P2Y1, And Pyrimidinoceptors). Sustained Agonist-Induced Contractions Were Associated With Transient Increases In Cytosolic Ca2+ Concentration, Suggesting Sensitization Of The Contractile Machinery To Ca2+. Atp Caused Both Ca2+ Entry And Ca2+ Release From A Ryanodine- And Caffeine-Sensitive Store. Increased Cyclic Amp Or Cyclic Gmp Levels Caused Relaxation. This Human Tevm Displays Many Of Functional Characters Of The Normal Vessel From Which The Cells Were Originally Isolated, Including Contractile/Relaxation Responses, Cyclic Nucleotide Sensitivity, And Ca2+ Handling Mechanisms Comparable To Those Of The Normal Vessel From Which The Cells Were Originally Isolated. These Results Demonstrate The Potential Of This Human Model As A Versatile New Tool For Pharmacological Research. Auteur(s) : Méthot S., Moulin V., Rancourt D., Bourdages M., Goulet D., Plante M., Auger F.A., Germain L. Titre : Morphological changes of human skin cells exposed to a DC electric field in vitro using a new exposure system Référence : The Canadian Journal of Chemical Engineering 79: 668-677, 2001. Auteur(s) : Moulin V., Tam B.Y.Y., Castilloux G., Auger F.A., O'Connor-McCourt M.D., Philip A., Germain L. Titre : Fetal and adult human skin fibroblasts display intrinsic differences in contractile capacity Référence : Journal of Cellular Physiology 188: 211-222, 2001. Lien : Cliquer ici Résumé : One Of The Differences Between Fetal And Adult Skin Healing Is The Unique Ability Of Fetal Wounds To Heal Without Contracture And Scar Formation. Studies Have Shown That The Ratio Between The Three Isoforms Of Tgfbeta Is Different In Adult And Fetal Wounds. Thus, We Analyzed The Capacity Of Adult And Fetal Human Skin Fibroblasts To Contract Collagen Gels After Stimulation With Tgfbeta Isoforms. In Control Medium, Fetal Fibroblasts Had A Contractile Capacity Similar To That Of Adult Fibroblasts. However, The Growth Capacity Of Fetal Fibroblasts Was Completely Inhibited, In Contrast To Adult Fibroblasts. When Cells Were Treated With Tgfbeta, Fetal Fibroblasts Showed An Inhibition Of Their Contractile Capacity Whereas Adult Fibroblasts Further Contracted Gels. The Contractile Response Was Similar For All Isoforms Of Tgfbeta Although Tgfbeta3 Always Had The Strongest Effect. We Considered That The Regulation Of Cell Contractile Capacity By Tgfbeta May Be Dependent On Receptor Expression For This Cytokine, On Myofibroblast Differentiation Of The Cells, Or In Cell Links With Matrix. Since Tgfbeta Receptor Analysis Did Not Show Differences In Receptor Affinity, We Studied The Expression Of Alpha-Smooth Muscle (Sm) Actin, A Fibroblast Contractile Marker And Of Three Integrins, The Cell Surface Receptors Specific Of The Attachment Of The Fibroblasts With Collagen Matrix. We Observed That The Expression Of Alpha-Sm Actin And Alpha3 And Beta1 Integrin Subunits Was Increased When Tgfbeta Was Added To The Medium Of Adult Fibroblasts Whereas The Levels Of The Alpha1 And Alpha2 Subunits Were Unchanged. In Contrast, Fetal Fibroblasts Treated With Tgfbeta Showed A Decrease Of Alpha1, Alpha2, And Beta1 Integrin Expression But No Change In Alpha3 Integrin And In Alpha-Sm Actin Expression. These Results Indicate That Intrinsic Differences Between Fetal And Adult Fibroblasts Might Explain Their Opposite Responses To Tgfbeta Stimuli. The Variations In Their Alpha-Sm Actin And Integrin Expression Patterns Represent Potentially Important Mechanisms Used By Fetal Fibroblasts To Regulate Their Response To Cytokines, And Likely Contribute To The Resultant Differences In The Quality Of Wound Repair. Auteur(s) : Rochon M.-H., Auger F.A., Gauthier M.-J., Germain L. Titre : Simultaneous isolation of keratinocytes and fibroblasts from a human cutaneous biopsy for the production of autologous reconstructed skin Référence : The Canadian Journal of Chemical Engineering 79: 663-667, 2001. Auteur(s) : Tam B.Y.Y., Larouche D., Germain L., Hooper N.M., Philip A. Titre : Characterization of a 150 kDa accessory receptor for TGF-ƒÒ1 on keratinocytes: direct evidence for a GPI anchor and ligand binding of the released form Référence : Journal of Cellular Biochemistry 83: 494-507, 2001 Lien : Cliquer ici Résumé : Transforming Growth Factor-Beta (Tgf-Beta) Is A Key Modulator Of Epidermal Development And Homeostasis, And Has Been Shown To Potently Regulate Keratinocyte Migration And Function During Wound Repair. There Are Three Cloned Tgf-Beta Receptors Termed Type I, Type Ii, And Type Iii That Are Found On Most Cell Types. The Types I And Ii Are The Signaling Receptors, While The Type Iii Is Believed To Facilitate Tgf-Beta Binding To The Types I And Ii Receptors. Recently, We Reported That In Addition To These Receptors, Human Keratinocytes Express A 150 Kda Tgf-Beta 1 Binding Protein (R150) Which Forms A Heteromeric Complex With The Tgf-Beta Signaling Receptors. This Accessory Receptor Was Described As Glycosyl Phosphatidylinositol-Specific Anchored Based On Its Sensitivity To Phosphatidylinositol Phospholipase C (Piplc). In The Present Study, We Demonstrate That The Gpi-Anchor Is Contained In R150 Itself And Not On A Tightly Associated Protein And That It Binds Tgf-Beta 1 With An Affinity Similar To Those Of The Types I And Ii Tgf-Beta Signaling Receptors. Furthermore, The Piplc Released (Soluble) Form Of This Protein Is Capable Of Binding Tgf-Beta 1 Independently From The Signaling Receptors. In Addition, We Provide Evidence That R150 Is Released From The Cell Surface By An Endogenous Phospholipase C. Our Observation That R150 Interacts With The Tgf-Beta Signaling Receptors, Together With The Finding That The Soluble R150 Binds Tgf-Beta 1 Suggest That R150 In Either Its Membrane Anchored Or Soluble Form May Potentiate Or Antagonize Tgf-Beta Signaling. Elucidating The Mechanism By Which R150 Functions As An Accessory Molecule In Tgf-Beta Signaling May Be Critical To Understanding The Molecular Mechanisms Underlying The Regulation Of Tgf-Beta Action In Keratinocytes. Auteur(s) : Wang C.S., Goulet F., Auger F.A., Tremblay N., Germain L., Têtu B. Titre : Production of bioengineered cancer tissue constructs in vitro: epithelium-mesenchyme heterotypic interactions Référence : In Vitro Cellular and Developmental Biology - Animal 37: 434-439, 2001. Lien : Cliquer ici Résumé : A Few Models Have Been Established To Study Cancer Cells In Vitro. However, The Cellular Interactions Have Rarely Been Studied Specifically Using Bioengineered Cancer Constructs Combining Human Carcinoma Cells And Tumor-Associated Fibroblasts. We Developed An In Vitro Model Of Tridimensional Bioengineered Cancer Tissue Constructs (Bctc) By Seeding Mammary Epithelial Cancer Cells Or Normal Keratinocytes Over A Mesenchymal Layer Containing Tumor-Derived Fibroblastic Cells Or Normal Skin Fibroblasts. After The Introduction Of Epithelial Cells, Each Construct Was Cultured For Another 10 D. Histologic Analyses Showed That Carcinoma Cell Lines Could Invade The Subjacent Mesenchymal Layer And That The Capacity To Migrate Was Related To The Invasive Potential Of Cancer Cells And The Type Of Fibroblasts Used, While Noninvasive Populations Did Not. Of The Tested Epithelial Cells, Mda-Mb-231 And, To A Lesser Degree, Hdq-P1 Cell Lines Were Invasive, And The Invasion Was Deeper Into The Mesenchymal Component Containing Tumor-Derived Fibroblasts. However, With Normal Skin Fibroblasts, The Mesenchymal Layer Was Degraded Twice Faster Than With Tumor-Derived Fibroblastic Cells. Mda-Mb-231 Cells And Normal Keratinocytes Induced The Highest Level Of Gelatinase B, And The Level Was Lowest With The Mcf-7 Cell Line. The Activated Form Of Gelatinase B Was, However, Induced To The Highest Levels In The Keratinocyte-Seeded Bctc Containing Tumor-Derived But Not Normal Fibroblasts. Mda-Mb-231 Was The Only Epithelial Cancer Cell Line Whose Activity Of Gelatinase A Was Reduced When Cocultured With Tumor-Derived Fibroblasts But Not Under Normal Fibroblast Stimulation. Finally, A 50/48-Kda Gelatinase Band Has Been Observed In Bctcs With Noninvasive Epithelial Cells Only. Our Study Demonstrates The Selective Secretion Of Gelatinases According To The Phenotype Of The Cells Seeded In The Various Bctcs. Auteur(s) : Wang C.S., Goulet F., Tremblay N., Germain L., Auger F.A., Têtu B. Titre : Selective culture of epithelial cells from primary breast carcinomas using irradiated 3T3 cells as feeder layer Référence : Pathology Research and Practice 197: 175-181, 2001. Lien : Cliquer ici Résumé : The Main Drawback Of The Selective Culture Of Human Mammary Epithelial Cells From Primary Breast Cancer Is The Overgrowth Of Tumor-Associated Stromal Fibroblasts. This Drawback May Be Overcome By Using, In Primary Culture, Lethally Irradiated 3T3 Cells Which Act As A Feeder Layer To Maintain Tumor-Derived Epithelial Cell Proliferation. These 3T3 Cells, Exposed To 60 Gy At Confluence, Form A Specific Cellular Substrate Which Constitutes An Obstacle To Fibroblast Attachment. Enzyme-Disaggregated Breast Cells From Six Primary Breast Carcinomas Were Cocultured Over Lethally Irradiated But Living 3T3 Cells. The Method Led To The Purification Of Tumor-Derived Epithelial Cells From All Six Cancer Samples, And Long-Term Culture Was Obtained In One. The Epithelial Nature Of These Purified Tumor-Derived Epithelial Cells Was Demonstrated By Their General Morphology And By The Expression Of Cytokeratins And Epithelial Membrane Antigen. These Results Confirm The Stimulatory Effect Of A This Stromal Feeder Layer On Breast Epithelial Cell Growth And Show That This Stromal Feeder Layer Can Also Control The Fibroblast Overgrowth. Our Results Provide An Alternative Approach In The Selective Culture Of Epithelial Cells From Primary Breast Carcinoma. 2000 Auteur(s) : Germain L., Damour O., Herbage D., Auger F.A. Titre : From the Tissue Engineering and Biomaterials Symposium of the XIe Entretiens du Centre Jacques Cartier held in Lyon in December 1998 Référence : Med Biol Eng Comput. 2000 Mar;38(2):204. Lien : Cliquer ici Auteur(s) : Auger F.A. Titre : Le génie tissulaire: du rêve à la réalité Référence : Médecine/Sciences 16 (12): 1324-1331, 2000. Auteur(s) : Auger F.A., Pouliot R., Tremblay N., Guignard R., Noël P., Juhasz J., Germain L., Goulet F. Titre : Multistep production of bioengineered skin substitutes: sequential modulation of culture conditions Référence : In Vitro Cellular and Developmental Biology - Animal 36(2): 96-103, 2000. Lien : Cliquer ici Résumé : Many Studies Are Being Conducted To Define The Role Of Growth Factors In Cutaneous Physiology In Order To Add Cytokines In A Timely Fashion For Optimal Tissue Engineering Of Skin. This Study Is Aimed At Developing A Multistep Approach For The Production Of Bioengineered Skin Substitutes, Taking Into Account The Effects Of Various Growth Factors According To The Culture Time. The Use Of A Serum-Supplemented Medium Throughout The Whole Culture Period Of Skin Substitutes Was Compared To The Sequential Use Of Specific Additives At Defined Culture Steps. Histological Analysis Revealed That Serum Was Necessary For Keratinocyte Proliferation And Migration On Dermal Substitutes During The First 2 D After Their Seeding. However, The Serum-Free Medium Presented Some Advantages When Supplemented With Different Additives At Specific Culture Steps. Interestingly, Ascorbic Acid Added To The Dermal Substitutes Before And After Keratinocyte Seeding Maintained Their Cuboidal Morphology In The Basal Epidermal Layer. In The Absence Of Serum, Collagen Matrix Degradation Slowed Down, And A Better Multilayered Epidermal Organization Was Obtained, Notably With Retinoic Acid. Stratum Corneum Formation Was Also Enhanced By Fatty Acids. Thus, Sequential Addition Of Exogenous Factors To The Medium Used To Produce Skin Substitutes Can Improve Their Structural Features And Functional Properties In Vitro. Auteur(s) : Auger F.A., Rémy-Zolghadri M., Grenier G., Germain L Titre : The self-assembly approach for organ reconstruction by tissue engineering Référence : e-biomed 1: 75-86, 2000 REVIEW. Auteur(s) : Auger F.A., Rémy-Zolghadri M., Grenier G., Germain L. Titre : Where to now... That we have tissue engineered blood vessels? Tissue engineered blood vessels! Heavens, what will they think of next! Référence : International Society Applied Cardiovascular Biology Circulator (ISACB) Nov. 2000. Auteur(s) : Chakir J., Dubé J., Laviolette M., Goulet F., Germain L., Auger F.A., Boulet L.-P. Titre : Isolation and characterization of human airway fibroblasts in culture Référence : Methods in molecular medicine 44: 53-65, 2000. Auteur(s) : Germain L., Carrier P., Auger F.A., Salesse C., Guérin S.L. Titre : Can we produce a human corneal equivalent by tissue engineering? Référence : Progress in Retinal and Eye Research 19(5): 497-527, 2000. Lien : Cliquer ici Résumé : Tissue Engineering Is Progressing Rapidly. Bioengineered Substitutes Are Already Available For Experimental Applications And Some Clinical Purposes Such As Skin Replacement. This Review Focuses On The Development Of Reconstructed Human Cornea In Vitro By Tissue Engineering. Key Elements To Consider In The Corneal Reconstruction, Such As The Source For Epithelial Cells And Keratocytes, Are Discussed And The Various Steps Of Production Are Presented. Since One Application Of This Human Model Is To Obtain A Better Understanding Of Corneal Wound Healing, The Mechanisms Of This Phenomenon As Well As The Function Played Both By Membrane-Bound Integrins And Components From The Extracellular Matrix Have Also Been Addressed. The Analysis Of Integrins By Immunohistofluorescence Labelling Of Our Reconstructed Human Cornea Revealed That Beta(1), Alpha(3), Alpha(5), And Alpha(6) Integrin Subunits Were Expressed But Alpha(4) Was Not. Laminin, Type Vii Collagen And Fibronectin Were Also Detected. Finally, The Future Challenges Of Corneal Reconstruction By Tissue Engineering Are Discussed And The Tremendous Applications Of Such Tissue Produced In Vitro For Experimental As Well As Clinical Purposes Are Considered. Auteur(s) : Germain L., Fradette J., Robitaille H., Guignard R., Grondin G., Nadeau A., Blouin R. Titre : The mixed lineage kinase leucine-zipper protein kinase exhibits a differentiation-associated localization in normal human skin and induces keratinocyte differentiation upon overexpression Référence : The Journal of Investigative Dermatology 115 (5): 860-867, 2000. Lien : Cliquer ici Résumé : Leucine-Zipper Protein Kinase/Dual Leucine Zipper Bearing Kinase/Mitogen-Activated Protein Kinase-Upstream Kinase Is A Recently Described Protein Serine/Threonine Kinase Which Belongs To The Mixed Lineage Kinase Family. The Overall Pattern Of Expression Of The Leucine-Zipper Protein Kinase/Dual Leucine Zipper Bearing Kinase/Mitogen-Activated Protein Kinase-Upstream Kinase Gene In Embryonic And Adult Mouse Tissues Suggested That This Kinase Could Be Involved In The Regulation Of Epithelial Cell Proliferation And Differentiation. In Order To Get More Insights Into The Potential Role Of Leucine-Zipper Protein Kinase In These Cellular Processes, We Characterized Its Expression In Normal Human Skin, Both At The Mrna And Protein Levels. In Situ Hybridization, Western Blotting, And Indirect Immunofluorescence Studies Were Conducted To Localize Leucine-Zipper Protein Kinase On Various Human Skin Tissues. This Is One Of The First Reports That Leucine-Zipper Protein Kinase Has A Very Precise Pattern Of Expression In Human Skin Epithelia, As Both Mrna And Protein Are Restricted To The Granular Layer Of The Epidermis And Inner Root Sheath Of Hair Follicles. Detection Of Leucine-Zipper Protein Kinase Protein On Skin From Various Body Sites, Donors Of Different Ages As Well As On Reconstructed Human Skin Always Reveals That Leucine-Zipper Protein Kinase Is Present Only In The Very Differentiated Keratinocytes Of Epidermis And Hair Follicles. To Determine Directly Whether Leucine-Zipper Protein Kinase Exhibits Any Effect On Cell Growth And Differentiation, Keratinocytes Were Transfected With An Expression Vector Harboring The Leucine-Zipper Protein Kinase Cdna. The Presence Of This Construct In Keratinocytes Results In Growth Arrest Together With A Concomitant Increase In Filaggrin Expression. Collectively, Our Results Indicate That Leucine-Zipper Protein Kinase Plays An Active Part In Cellular Processes Related To Terminal Differentiation Of Epidermal Keratinocytes. Auteur(s) : Germain L., Rémy-Zolghadri M., Auger F.A Titre : Tissue engineering of the vascular system: from capillaries to larger blood vessels Référence : Medical & Biological Engineering & Computing 38: 232-240, 2000 REVIEW. Auteur(s) : Goulet F., Rancourt D., Cloutier R., Germain L., Poole R.A., Auger F.A. Titre : Tendons and ligaments Référence : Principles of Tissue Engineering Chap.50: 711-722, 2000. Auteur(s) : Moulin V., Auger F.A., Garrel D., Germain L. Titre : Role of wound healing myofibroblasts on re-epithelialization of human skin Référence : Burns 26: 3-12, 2000. Lien : Cliquer ici Résumé : In Human Skin, Large Burned Surfaces Heal Using Two Concomitant Phenomena: Re-Epithelialization And Dermal Neoformation. Numerous Studies Report The Role Of Interactions Between Keratinocytes And Fibroblasts, But The Relationship Between Wound Healing Myofibroblasts And Keratinocytes Is Not Clear, Even Though These Two Cell Types Coexist During Healing. We Investigated The Influence Of Myofibroblasts On Keratinocyte Growth And Differentiation Using An In Vitro Skin Model. A Histological Study Was Performed To Determine The Speed And Quality Of Epithelialization. When The Dermis Was Populated With Fibroblasts, A Continuous Epidermis Was Formed In 7-10 Days. In Contrast, With Wound Healing Myofibroblasts Or Without Cell In Dermis, The Complete Reepithelialization Never Occurred Over The 10-Day Period Studied. After 7 Further Days Of Epidermal Differentiation, Histology Showed An Epidermis More Disorganized And Expression Of Basement Membrane Constituents Was Reduced When Wound Healing Myofibroblasts Or No Cells Were Added In The Dermis Instead Of Fibroblasts. These Results Suggest That Wound Healing Myofibroblasts Are Not Efficient To Stimulate Keratinocyte Growth And Differentiation. Treatment Of Fibroblasts With Tgfbeta1 Induced An Increase Of Epidermal Cell Differentiation As Seen When Myofibroblasts Were Present. However, This Cytokine Did Not Change Re-Epithelialization Rate And Induced An Increase Of Basement Membrane Matrix Deposition In Opposition To Myofibroblasts. Thus, Tgfbeta1 Action Is Not Sufficient To Explain All The Different Keratinocyte Reactions Towards Fibroblasts And Wound Healing Myofibroblasts. Our Conclusion Is That Myofibroblasts Seem To Have A Limited Role In The Re-Epithelialization Process And Might Be More Associated With The Increased Extracellular Matrix Secretion. Auteur(s) : Wang C.S., Goulet F., Lavoie J., Drouin R., Auger F.A., Champetier S., Germain L., Têtu B. Titre : Establishment and characterization of a new cell line derived from a human primary breast carcinoma Référence : Cancer Genetic Cytogenetic 120: 58-72, 2000. Lien : Cliquer ici Résumé : A New Cell Line, Designated Hdq-P1, Was Successfully Established From A Primary Ductal Infiltrating Mammary Carcinoma By Using A 3T3 Feeder Layer Lethally Irradiated To 60 Gy. The Hdq-P1 Cells Have Been Grown In Culture For Over 115 Passages And Have A Doubling Time Of 60 Hours. Characterization Of The Cell Line Was Performed. This Included Morphology By Light And Transmission Electron Microscopy, Karyotype, Growth Rate, Telomerase Expression, Tumor Antigen Expression, Xenograft Implantation Into Nude Mice, Colony Formation In Soft Agar, Tp53 Sequencing, And Gene Copy Number Of C-Myc, C-Erbb-2, And C-H-Ras Oncogenes. The Epithelial Nature Of This Cell Line Was Confirmed By Ultrastructural Analysis, Expression Of Cytokeratins, And Epithelial Membrane Antigen. The Hdq-P1 Cells Possess An Extensively Rearranged And Polyploid Karyotype, With An Average Of 20 Recurrent Marker Chromosomes. Scatchard Analysis Demonstrated That Both Primary Tumor And Hdq-P1 Cells Were Estrogen- And Progesterone-Receptor Negative. The Hdq-P1 Cells Had The Same Expression Of Human Telomerase Reverse Transcriptase As Other Established Breast Cancer Cell Lines Such As Mda-Mb-231, Sk-Br-3, And Mcf-7. Direct Dna Sequencing Showed A Point Mutation Which Yielded To A Stop Codon At The Amino Acid 213 In Exon 6 Of The Tp53 Gene. A Five-Fold Amplification Of C-Myc Was Observed In Hdq-P1 Cells. No Amplification Of C-Erbb-2 And C-H-Ras Genes Were Observed. This Cell Line Presents Unique Characteristics And May Prove To Be A Good Experimental Model For Investigating Breast Cancer Biology. 1999 Auteur(s) : Auger F.A., Berthod F., Goulet F., Germain L. Titre : What is new in mechanical properties of tissue-engineered organs Référence : Current Topics in Pathology 93: 87-93, 1999. Lien : Cliquer ici Auteur(s) : Black A.F., Hudon V., Damour O., Germain L., Auger F.A. Titre : A novel approach for studying angiogenesis: a human skin equivalent with a capillarylike network Référence : Cell Biology and Toxicology 15, 1999. Lien : Cliquer ici Résumé : Angiogenesis Results From An Ordered Set Of Events That Can Be Modulated In Vivo By A Variety Of Angiogenesis-Enhancing Or Inhibiting Agents. We Review In Vitro Angiogenesis Models And The Agents That Enhance Or Inhibit Angiogenesis. We Also Discuss A New In Vitro Angiogenesis Model Created Within A Skin Equivalent. Briefly, Endothelial Cells Were Combined With The Cutaneous Cells Of A Standard Skin Equivalent And Cultured In A Chitosan Cross-Linked Collagen-Glycosaminoglycan Scaffold Of This Endothelialized Skin. This Model Enables The Formation Of Capillary-Like Structures In A Coculture Environment Containing Newly Synthesized Extracellular Matrix By Fibroblasts And Keratinocytes. Several Morphological Characteristics Associated With The Microvasculature In Vivo Were Observed In The Endothelialized Skin Equivalent Such As Histotypic Organization Of Tubular Structures, Basement Membrane Deposition, And Intercellular Junction Formation. Auteur(s) : Germain L., Auger F.A., Grandbois E., Guignard R., Giasson M., Boisjoly H., Guérin S.L. Titre : Reconstructed human cornea produced in vitro by tissue engineering Référence : Pathobiology 67: 140-147, 1999 Lien : Cliquer ici Résumé : The Aim Of The Present Study Was To Produce A Reconstructed Human Cornea In Vitro By Tissue Engineering And To Characterize The Expression Of Integrins And Basement Membrane Proteins In This Reconstructed Cornea. Epithelial Cells And Fibroblasts Were Isolated From Human Corneas (Limbus Or Centre) And Cultured On Plastic Substrates In Vitro. Reconstructed Human Corneas Were Obtained By Culturing Epithelial Cells On Collagen Gels Containing Fibroblasts. Histological (Masson'S Trichrome Staining) And Immunohistological (Laminin, Type Vii Collagen, Fibronectin As Well As Beta1, Alpha3, Alpha4, Alpha5, And Alpha6 Integrin Subunits) Studies Were Performed. Human Corneal Epithelial Cells From The Limbus Yielded Colonies Of Small Fast-Growing Cells When Cultured On Plastic Substrates. They Could Be Subcultured For Several Passages In Contrast To Central Corneal Cells. In Reconstructed Cornea, The Epithelium Had 4-5 Cell Layers By The Third Day Of Culture; Basal Cells Were Cuboidal. The Basement Membrane Components Were Already Detected After 3 Days Of Culture. Integrin Stainings, Except For The Alpha4 Integrin, Were Also Positive After 3 Days. They Were Mostly Detected At The Epithelium-Stroma Junction. Such In Vitro Tissue-Engineered Human Cornea, Which Shows Appropriate Histology And Expression Of Basement Membrane Components And Integrins, Provides Tools For Further Physiological, Toxicological And Pharmacological Studies As Well As Being An Attractive Model For Gene Expression Studies. Auteur(s) : Langelier E., Rancourt D., Bouchard S., Lord C., Stevens P.P., Germain L., Auger F.A. Titre : Cyclic traction machine for long-term culture of fibroblast-populated collagen gels Référence : Annals of Biomedical Engineering 27: 67-72, 1999. Lien : Cliquer ici Résumé : Our Research Group Has Been Investigating The Effect Of Cyclic Deformations On The Evolution Of Fibroblast Populated Collagen Gels (Fpcg). Since Existing Traction Machines Are Not Designed For Such An Application, We Had To Design A Cyclic Traction Machine Adapted To Tissue Culture Inside An Incubator Over An Extended Period Of Time. Biocompatible Materials Were Used For Fabrication To Allow For Easy Sterilization And To Prevent Any Adverse Reaction From The Tissue. The Traction Machine Is Based On A Computer-Controlled Stepping Motor System For Easy Adjustment Of The Deformation Amplitude And Frequency. The Maximum Stretching Speed Achieved Is Around 1 Mm/S. The Traction Machine Can Measure Fpcg Mechanical Properties And Perform Rupture Tests To Determine Its Ultimate Strength. Several Fpcgs Have Been Successfully Cultured With The Machine For Up To Four Weeks Without Any Adverse Reaction. Auteur(s) : Michel M., L'Heureux N., Pouliot R., Xu W., Auger F.A., Germain L. Titre : Characterization of a new tissue-engineered human skin equivalent with hair Référence : In Vitro Cellular and Developmental Biology - Animal 35: 318-326, 1999. Lien : Cliquer ici Résumé : We Designed A New Tissue-Engineered Skin Equivalent In Which Complete Pilosebaceous Units Were Integrated. This Model Was Produced Exclusively From Human Fibroblasts And Keratinocytes And Did Not Contain Any Synthetic Material. Fibroblasts Were Cultured For 35 D With Ascorbic Acid And Formed A Thick Fibrous Sheet In The Culture Dish. The Dermal Equivalent Was Composed Of Stacked Fibroblast Sheets And Exhibited Some Ultrastructural Organization Found In Normal Connective Tissues. Keratinocytes Seeded On This Tissue Formed A Stratified And Cornified Epidermis And Expressed Typical Markers Of Differentiation (Keratin 10, Filaggrin, And Transglutaminase). After 4 Wk Of Culture, A Continuous And Ultrastructurally Organized Basement Membrane Was Observed And Associated With The Expression Of Laminin And Collagen Iv And Vii. Complete Pilosebaceous Units Were Obtained By Thermolysin Digestion And Inserted In This Skin Equivalent In Order To Assess The Role Of The Transfollicular Route In Percutaneous Absorption. The Presence Of Hair Follicles Abolished The Lag-Time Observed During Hydrocortisone Diffusion And Increased Significantly Its Rate Of Penetration In Comparison To The Control (Skin Equivalent With Sham Hair Insertion). Therefore, This New Hairy Human Skin Equivalent Model Allowed An Experimental Design In Which The Only Variable Was The Presence Of Pilosebaceous Units And Provided New Data Confirming The Importance Of Hair Follicles In Percutaneous Absorption. Auteur(s) : Moulin V., Garrel D., Auger F.A., O'Connor-McCourt M., Castilloux G., Germain L. Titre : What's new in human wound-healing myofibroblasts?" Référence : Current Topics in Pathology 93: 123-133, 1999. Lien : Cliquer ici Auteur(s) : Pouliot R., Germain L., Auger F.A., Tremblay N., Juhasz J. Titre : Physical characterization of the stratum corneum of an in vitro human skin equivalent produced by tissue engineering and its comparison with normal human skin by ATR-FTIR spectroscopy and thermal analysis (DSC) Référence : Biochimica et Biophysica Acta 1439: 341-352, 1999. Lien : Cliquer ici Résumé : An In Vitro Human Skin Equivalent May Be Obtained By Culturing Human Keratinocytes On A Collagen Gel Containing Fibroblasts. The Anchored Skin Equivalent Cultured At The Air-Liquid Interface Closely Resembles Human Skin And Is Acceptable For In Vitro Percutaneous Absorption. However, It Is Still More Permeable Than Human Skin. Since Intercellular Lipids Have Been Recognized To Play An Important Role In Skin Permeability, Infrared Spectroscopy And Differential Scanning Calorimetry Were Performed On The Stratum Corneum Of Bovine Or Human Skin Equivalents Grown At Different Days Of Air-Liquid Culture. The Symmetric And Asymmetric Ch(2) Stretching Vibrations Suggested That For All Days Observed, The Intercellular Lipids Were Less Organized Than Those In Human Skin, Irrespective Of Whether Bovine Or Human Collagen Was Used. Different Culture Conditions Were Also Tested And The Medium Without Serum And No Epidermal Growth Factor At The Air-Liquid Culture Showed Results Significantly More Comparable To Human Skin. Actually, The Thermal Behavior Of In Vitro Stratum Corneum Showed Transitions At Lower Temperatures Than Human Skin. The Transition Around 80 Degrees C, In The Form Of A Lipid-Protein Complex, Was Absent. These Results Showed That The Structural Arrangement Of Intercellular Lipids And Their Thermodynamic Properties Hold A Crucial Role In The Barrier Function Of The Stratum Corneum. 1998 Auteur(s) : Auger F.A., Rouabhia M., Goulet F., Berthod F., Moulin V., Germain L. Titre : Tissue-engineered human skin substitutes developed from collagen-populated hydrated gels: clinical and fundamental applications Référence : Medical and Biological Engineering and Computing 36: 801-812, 1998. Lien : Cliquer ici Résumé : The Field Of Tissue Engineering Has Opened Several Avenues In Biomedical Sciences, Through Ongoing Progress. Skin Substitutes Are Currently Optimised For Clinical As Well As Fundamental Applications. The Paper Reviews The Development Of Collagen-Populated Hydrated Gels For Their Eventual Use As A Therapeutic Option For The Treatment Of Burn Patients Or Chronic Wounds: Tools For Pharmacological And Toxicological Studies, And Cutaneous Models For In Vitro Studies. These Skin Substitutes Are Produced By Culturing Keratinocytes On A Matured Dermal Equivalent Composed Of Fibroblasts Included In A Collagen Gel. New Biotechnological Approaches Have Been Developed To Prevent Contraction (Anchoring Devices) And Promote Epithelial Cell Differentiation. The Impact Of Dermo-Epidermal Interactions On The Differentiation And Organisation Of Bio-Engineered Skin Tissues Has Been Demonstrated With Human Skin Cells. Human Skin Substitutes Have Been Adapted For Percutaneous Absorption Studies And Toxicity Assessment. The Evolution Of These Human Skin Substitutes Has Been Monitored In Vivo In Preclinical Studies Showing Promising Results. These Substitutes Could Also Serve As In Vitro Models For Better Understanding Of The Immunological Response And Healing Mechanism In Human Skin. Thus, Such Human Skin Substitutes Present Various Advantages And Are Leading To The Development Of Other Bio-Engineered Tissues, Such As Blood Vessels, Ligaments And Bronchi. Auteur(s) : Beaulieu M.-J., Li H., Bergeron J., Ross G., Auger F.A., Rouabhia M. Titre : Involvement of male-specific minor histocompatibility antigen H-Y in epidermal equivalent allograft rejection Référence : Cell Transplantation 7(1): 11-23, 1998. Lien : Cliquer ici Résumé : This Study Describes The Involvement Of Male-Specific Minor Histocompatibility Antigen H-Y In Vitro Cultured Epidermal Equivalent (Ee) Rejection. Male And Female Balb/C Or C3H/Hen Keratinocytes Were Isolated And Cultured Separately. Male Ee Were Grafted Onto Adult Male (Isografts) And Adult Female (H-Y Allografts) Mice. As Controls, Balb/C Ee Were Grafted Onto Adult C3H/Hen (Complete Allografts) Mice. Fourteen, 21, And 30 Days Postgrafting, Histological Studies Showed Well-Organized Cutaneous Tissues With Complete Basement Membranes (Laminin And Type Iv Collagen Deposition) In H-Y Allografts Compared To The Isografts. This Cutaneous Organization Was Altered 150 Days Postgrafting, Which Is A Sign Of The H-Y Ee Allograft Rejection. Complete Allografts Were Totally Rejected 21 Days Postgrafting. Immunological Studies Revealed Leucocyte Infiltration Of H-Y Allografts. Significant Infiltration Was Detected Even 150 Days Postgrafting. Leucocyte Phenotyping Revealed The Presence Of Mac-I+, Cd8+ And Cd4+ Cells In The H-Y Allografts. Humoral Immune Analysis Revealed The Presence Of Circulating Anti-H-Y Allogeneic Keratinocyte Cytotoxic Antibodies In Female Recipient Sera. Our Data Suggest That Male-Specific Minor Histocompatibility Antigen H-Y Induces Cellular And Humoral Activation Of The Recipient Immune System Even After Grafting Ee Free Of Cutaneous Active Immune Cells Such As T Lymphocytes And Langerhans Cells. Auteur(s) : Black A.F., Berthod F., L'Heureux N., Germain L., Auger F.A. Titre : In vitro reconstruction of a human capillary-like network in a tissue-engineered skin equivalent Référence : The FASEB Journal 12: 1331-1340,1998. Lien : Cliquer ici Résumé : For Patients With Extensive Burns, Wound Coverage With An Autologous In Vitro Reconstructed Skin Made Of Both Dermis And Epidermis Should Be The Best Alternative To Split-Thickness Graft. Unfortunately, Various Obstacles Have Delayed The Widespread Use Of Composite Skin Substitutes. Insufficient Vascularization Has Been Proposed As The Most Likely Reason For Their Unreliable Survival. Our Purpose Was To Develop A Vascular-Like Network Inside Tissue-Engineered Skin In Order To Improve Graft Vascularization. To Reach This Aim, We Fabricated A Collagen Biopolymer In Which Three Human Cell Types Keratinocytes, Dermal Fibroblasts, And Umbilical Vein Endothelial Cells Were Cocultured. We Demonstrated That The Endothelialized Skin Equivalent (Ese) Promoted Spontaneous Formation Of Capillary-Like Structures In A Highly Differentiated Extracellular Matrix. Immunohistochemical Analysis And Transmission Electron Microscopy Of The Ese Showed Characteristics Associated With The Microvasculature In Vivo (Von Willebrand Factor, Weibel-Palade Bodies, Basement Membrane Material, And Intercellular Junctions). We Have Developed The First Endothelialized Human Tissue-Engineered Skin In Which A Network Of Capillary-Like Tubes Is Formed. The Transplantation Of This Ese On Human Should Accelerate Graft Revascularization By Inosculation Of Its Preexisting Capillary-Like Network With The Patient'S Own Blood Vessels, As It Is Observed With Autografts. In Addition, The Ese Turns Out To Be A Promising In Vitro Angiogenesis Model. Auteur(s) : Dubé J., Chakir J., Laviolette M., Saint-Martin S., Boutet M., Desrochers C., Auger F.A., Boulet L.-P. Titre : In vitro procollagen synthesis and proliferative phenotype of bronchial fibroblasts from normal and asthmatic subjects Référence : Laboratory Investigation 78(3): 297-307, 1998. Lien : Cliquer ici Résumé : Asthma Is Characterized Histologically By A Bronchial Subepithelial Fibrosis. Cytokines And Other Mediators Released In The Asthmatic Chronic Inflammatory Microenvironment Can Activate The Repair Process That Leads To Fibroblast Proliferation And Collagen Synthesis. To Our Knowledge, There Are No Data Regarding The Effect Of A Chronic Inflammatory Microenvironment On The Phenotype Of Human Bronchial Fibroblasts. In The Present Study, We Address This Issue By Comparing Bronchial Fibroblasts Isolated From Normal And Asthmatic Subjects In Terms Of: (A) Proliferation Over Cell Passage; (B) In Vitro Lifespan; (C) Proliferative Response To Transforming Growth Factor-Beta 1, Platelet-Derived Growth Factor-Bb, Dexamethasone, And Retinoic Acid; And (D) Base-Line Synthesis Of Procollagens I And Iii. Bronchial Fibroblasts From Asthmatic Subjects Demonstrated Lower Dna Synthesis With Cell Passage Than Bronchial Fibroblasts From Normals. The In Vitro Lifespan Of Asthmatic Bronchial Fibroblasts Was Lower Than In Those From Normal Subjects And Was Significantly Correlated With Airway Responsiveness. Platelet-Derived Growth Factor-Bb And Dexamethasone Increased 3H-Thymidine Incorporation In Asthmatic Bronchial Fibroblasts Without Having Any Significant Effect On Normal Fibroblast Proliferation. Transforming Growth Factor-Beta 1 And Retinoic Acid Had No Significant Effect On Bronchial Fibroblast Proliferation. Base-Line Procollagens I And Iii Synthesis Measurements Showed No Differences Between Normal And Asthmatic Fibroblasts. Taken Together, These Results Indicate That The Chronic Inflammatory Microenvironment Found In Asthma Can Modulate Some Aspects Of Bronchial Fibroblast Phenotype. Auteur(s) : Fradette J., Germain L., Seshaiah P., Coulombe P.A. Titre : The type 1 keratin 19 possesses distinct and context-dependent assembly properties Référence : The Journal of Biological Chemistry 273(52): 35176-35184, 1998. Lien : Cliquer ici Résumé : Keratins (K), The Cytoplasmic Intermediate Filament (If) Proteins Of Epithelial Cells, Are Encoded By A Multigene Family And Expressed In A Tissue- And Differentiation-Specific Manner. In Human Skin, Keratinocytes Of The Basal Layer Of Epidermis And The Outer Root Sheath Of Hair Follicles Express K5 And K14 As Their Main Keratins. A Small Subpopulation Of Basal Cells Exhibiting Stem-Cell Like Characteristics Express, In Addition, K19. At 40 Kda, This Keratin Is The Smallest If Protein Due To An Exceptionally Short Carboxyl-Terminal Domain. We Examined The Assembly Properties Of K19 And Contrasted Them To K14 In Vitro And In Vivo. Relative To K5-K14, We Find That K5-K19 Form Less Stable Tetramers That Polymerize Into Shorter And Narrower Ifs In Vitro. When Transiently Co-Expressed In Cultured Baby Hamster Kidney Cells, The K5 And K19 Combination Fails To Form A Filamentous Array, Whereas The K5-K14 And K8-K19 Ones Readily Do So. Transient Expression Of K19 In The Epithelial Cell Lines T51B-Ni And A431 Results In Its Integration Into The Endogenous Keratin Network With Minimal If Any Perturbation. Collectively, These Results Indicate That K19 Possesses Assembly Properties That Are Distinct From Those Of K14 And Suggest That It May Impart Unique Properties To The Basal Cells Expressing It In Skin Epithelia. Auteur(s) : Lafrance H., Yahia L'H., Germain L., Auger F.A Titre : Mechanical properties of human skin equivalents submitted to cyclic tensile forces Référence : Skin Research and Technology 4: 228-236, 1998. Auteur(s) : Laplante A.F., Moulin V., Auger F.A., Landry J., Li H., Morrow G., Tanguay R.M., Germain L. Titre : Expression of heat shock proteins in mouse skin during wound healing Référence : The Journal of Histochemistry and Cytochemistry 46(11): 1291-1301, 1998. Lien : Cliquer ici Résumé : Wound Healing Conditions Generate A Stressful Environment For The Cells Involved In The Regeneration Process And Are Therefore Postulated To Influence The Expression Of Heat Shock Proteins (Hsps). We Have Examined The Expression Of Four Hsps (Hsp27, Hsp60, Hsp70 And Hsp90) And A Keratin (Keratin 6) By Immunohistochemistry During Cutaneous Wound Repair From Day 1 To Day 21 After Wounding In The Mouse. Hsps Were Constitutively Expressed In Normal Mouse Epidermis And Their Patterns Of Expression Were Modified During The Healing Process. The Changes Were Not Directly Linked To The Time Course Of The Healing Process But Rather Were Dependent On The Location Of Cells In The Regenerating Epidermis. In The Thickened Epidermis, Hsp60 Was Induced In Basal And Low Suprabasal Cells, Hsp70 Showed A Reduced Expression, And Hsp90 And Hsp27 Preserved A Suprabasal Pattern With An Induction In Basal And Low Suprabasal Cells. All Hsps Had A Uniform Pattern Of Expression In The Migrating Epithelial Tongue. These Observations Suggest That The Expression Of Hsps In The Neoepidermis Is Related To The Proliferation, The Migration, And The Differentiation States Of Keratinocytes Within The Wound. Auteur(s) : L'Heureux N., Pâquet S., Labbé R., Germain L., Auger F.A. Titre : A completely biological tissue-engineered human blood vessel Référence : The FASEB Journal 12: 47-56, 1998. Lien : Cliquer ici Résumé : Mechanically Challenged Tissue-Engineered Organs, Such As Blood Vessels, Traditionally Relied On Synthetic Or Modified Biological Materials For Structural Support. In This Report, We Present A Novel Approach To Tissue-Engineered Blood Vessel (Tebv) Production That Is Based Exclusively On The Use Of Cultured Human Cells, I.E., Without Any Synthetic Or Exogenous Biomaterials. Human Vascular Smooth Muscle Cells (Smc) Cultured With Ascorbic Acid Produced A Cohesive Cellular Sheet. This Sheet Was Placed Around A Tubular Support To Produce The Media Of The Vessel. A Similar Sheet Of Human Fibroblasts Was Wrapped Around The Media To Provide The Adventitia. After Maturation, The Tubular Support Was Removed And Endothelial Cells Were Seeded In The Lumen. This Tebv Featured A Well-Defined, Three-Layered Organization And Numerous Extracellular Matrix Proteins, Including Elastin. In This Environment, Smc Reexpressed Desmin, A Differentiation Marker Known To Be Lost Under Standard Culture Conditions. The Endothelium Expressed Von Willebrand Factor, Incorporated Acetylated Ldl, Produced Pgi2, And Strongly Inhibited Platelet Adhesion In Vitro. The Complete Vessel Had A Burst Strength Over 2000 Mmhg. This Is The First Completely Biological Tebv To Display A Burst Strength Comparable To That Of Human Vessels. Short-Term Grafting Experiment In A Canine Model Demonstrated Good Handling And Suturability Characteristics. Taken Together, These Results Suggest That This Novel Technique Can Produce Completely Biological Vessels Fulfilling The Fundamental Requirements For Grafting: High Burst Strength, Positive Surgical Handling, And A Functional Endothelium. Auteur(s) : Moulin V., Castilloux G., Auger F.A., Garrel D., O'Connor-McCourt M.D., Germain L. Titre : Modulated response to cytokines of human wound healing myofibroblasts compared to dermal fibroblasts Référence : Experimental Cell Research 238: 283-293, 1998. Lien : Cliquer ici Résumé : Myofibroblasts Play An Important Role In Normal Wound Healing. They Are Present Transiently During Tissue Repair. Their Differentiation From Fibroblasts And Their Role In Granulation Tissues Are Most Likely To Be Modulated By Cytokines. As These Cells Are Derived From Normal Fibroblasts, Their Responses To Cytokines Are Assumed To Be Similar. Until Now, However, The Difficulties In Obtaining And Maintaining Normal Human Wound Healing Myofibroblasts In Vitro Have Hampered Comparison. The Present Study Was Designed To Determine The Effect Of Tgf-Beta 1 And Ifn-Gamma, Two Cytokines Known To Modulate Fibroblast Morphology, On Wound Healing Myofibroblasts And To Compare It To Fibroblasts. Morphological And Phenotypic Changes Were Followed By Light And Electron Microscopy (Stress Fibers) And Immunofluorescence Cytochemistry (Alpha-Sm Actin). Functional Parameters Such As The Capacity To Synthesize Collagen And Collagen Gel Contraction Were Studied. Both Cytokines Induced A Strong Modification Of Growth Rate And Phenotypic And Morphological Parameters In Fibroblasts Whereas Collagen Synthesis Was Slightly Changed. Furthermore, Tgf-Beta 1 Increased Contractile Capacity Of Fibroblasts Whereas Ifn-Gamma Greatly Decreased It. In Myofibroblasts, Tgf-Beta 1 And Ifn-Gamma Did Not Induce Any Variation Of Morphology Or Growth Rate. Interestingly, A Strong Modulation Of Functional Parameters Was Observed: Collagen Synthesis Was Highly Modified And, As For Fibroblasts, The Contractile Capacity Was Altered. However, Inhibition Of Contraction By Ifn-Gamma Was Irreversible In Myofibroblasts But Not In Fibroblasts. These Results Suggest That Fibroblastic Cells Show Modulated Responses To Cytokines According To Their Stage Of Differentiation During Wound Healing. Auteur(s) : Paquette J.S., Goulet F., Boulet, L-P., Laviolette M., Tremblay N., Chakir J., Germain L., Auger F.A. Titre : Three-dimensional production of bronchi in vitro Référence : Canadian Respiratory Journal 5(1): 43, 1998. Lien : Cliquer ici Auteur(s) : Pouliot R., Robert M., Germain L., Noël-Hudson M.-S., Lindenbaum A., Juhasz J., Auger F.A., Wepierre J. Titre : Influence of endothelial cells on structure, biochemistry and functionality of epidermis reconstructed on synthetic porous membrane Référence : Skin Pharmacology and Applied Skin Physiology 11: 193-206, 1998. Lien : Cliquer ici Résumé : The Model Of Keratinocytes Cultured On A Synthetic Porous Membrane At The Air-Liquid Interface Leads To The Formation Of A Pluristratified And Cornified Epidermis With Histological And Biochemical Characteristics Near Those Observed In Vivo. In The Present Study, We Evaluated The Effect Of Proliferative Endothelial Cells On Epidermalization. Keratinocytes Were Grown In Three Culture Conditions: In Defined Medium (Dm; Control), In Medium Previously Conditioned By Proliferative Endothelial Cells (Cm) And In Medium With Proliferative Endothelial Cells (Pec). The Structures Of Reconstructed Epidermis Were Analyzed By Electron Microscopy, Their Biochemistry By Dna, Protein And Cytokine Analyses And Finally Their Functionality Was Evaluated By Estradiol And Water Absorption Testing. Ultrastructural Analysis Showed A Well-Developed And Cornified Epidermis For Each Culture Condition. In Addition, Living Epidermis Was Thinner In The Presence Of Endothelial Cells, Revealing Faster Epidermal Differentiation. Dna And Protein Analyses Were In Accordance With These Results. Secreted Soluble Factors Varied According To Culture Conditions. At 37 Degreesc, The Permeability Of Reconstructed Epidermis In Dm, In Cm Or With Pec Was 5- To 10-Fold Higher Than That Of Native Human Epidermis With Both Tracers. Laminin Coating Of The Inserts Led To Similar Absorption Results Except For The Dm Where The Barrier Function To Estradiol Was Decreased 2-Fold. At 32 Degreesc, Reconstructed And Native Epidermis Were, Respectively, 1.5- And 2-Fold Less Permeable To Estradiol Compared To 37 Degreesc. In Conclusion, This Model Is Adequate For Fundamental And Pharmacological Studies Since It Allows The Study Of Interactions Between Two Cell Types Without Their Direct Contact As Well As Percutaneous Absorption Tests Directly Performed In The Modified Culture Chamber. Auteur(s) : Tam B.Y.Y., Germain L., Philip A Titre : TGF-beta receptor expression on human keratinocytes: a 150kDa GPI-anchored TGF-beta1 binding protein forms a heteromeric complex with type I and type II receptors Référence : Journal of Cellular Biochemistry 70: 573-586, 1998. Lien : Cliquer ici Résumé : Keratinocytes Play A Critical Role In Re-Epithelialization During Wound Healing, And Alterations In Keratinocyte Proliferation And Function Are Associated With The Development Of Various Skin Diseases. Although It Is Well Documented That Tgf-Beta Has Profound Effects On Keratinocyte Growth And Function, There Is A Paucity Of Information On The Types, Isoform Specificity And Complex Formation Of Tgf-Beta Receptors On Keratinocytes. Here, We Report That In Addition To The Types I, Ii, And Iii Tgf-Beta Receptors, Early Passage Adult And Neonatal Human Keratinocytes Display A Cell Surface Glycosylphosphatidylinositol (Gpi)-Anchored 150 Kda Tgf-Beta1 Binding Protein. The Identities Of The Four Proteins Were Confirmed On The Basis Of Their Affinity For Tgf-Beta Isoforms, Immunoprecipitation With Specific Anti-Receptor Antibodies, Sensitivity To Phosphatidylinositol Specific Phospholipase C And Dithiothreitol, And 2-Dimensional Electrophoresis. Interestingly, The Antitype I Tgf-Beta Receptor Antibody Immunoprecipitated Not Only The Type I Receptor, But Also The Type Ii Receptor And The 150 Kda Component, Suggesting That The 150 Kda Component Form Heteromeric Complexes With The Signalling Receptors. In Addition, Two-Dimensional (Nonreducing/Reducing) Electrophoresis Confirmed The Occurrence Of A Heterotrimeric Complex Consisting Of The 150 Kda Tgf-Beta1 Binding Protein, The Type Ii Receptor, And The Type I Receptor. This Technique Also Demonstrated The Occurrence Of Types I And Ii Heterodimers And Type I Homodimers Of Tgf-Beta Receptors On Keratinocytes, Supporting The Heterotetrameric Model Of Tgf-Beta Signalling Proposed Using Mutant Cells And Cells Transfected To Overexpress These Receptors. The Keratinocytes Responded To Tgf-Beta By Markedly Downregulating All Four Tgf-Beta Binding Proteins And By Potently Inhibiting Dna Synthesis. The Demonstration That The 150 Kda Gpi-Anchored Tgf-Beta1 Binding Protein Forms A Heteromeric Complex With The Tgf-Beta Signalling Receptors Suggests That This Gpi-Anchored Protein May Modify Tgf-Beta Signalling In Human Keratinocytes. 1997 Auteur(s) : Berthod F., Auger F.A. Titre : In vitro applications of skin substitutes for dermatological purposes Référence : : Skin substitute production by tissue engineering: clinical and fundamental applications. Chap. 9: 211-237, 1997. Auteur(s) : Berthod F., Damour O. Titre : In vitro reconstructed skin models for wound coverage in deep burns Référence : British Journal of Dermatology 136: 809-816, 1997. Lien : Cliquer ici Auteur(s) : Berthod F., Germain L., Guignard R., Lethias C., Garrone R., Damour O., van der Rest M., Auger F.A. Titre : Differential expression of collagens XII and XIV in human skin and in reconstructed skin Référence : The Journal of Investigative Dermatology 108(5): 737-742, 1997. Lien : Cliquer ici Résumé : Collagens Xii And Xiv Localize Near The Surface Of Collagen Fibrils And May Be Involved In Epithelial-Mesenchymal Interactions As Well As In The Modulation Of Tissue Biomechanical Properties. Moreover, Human Skin Fibroblasts Cultured In Monolayer Are Known To Lose Their Ability To Produce Collagen Xiv And To Switch The Transcription Of Collagen Xii From The Small Splice Variant (220 Kda) To The Large (320 Kda), Whereas The Small Form Is The Main Form Found In Human Skin. We Have Investigated The Expression Patterns Of These Two Molecules In Human Skin As A Function Of Donor Age And Anatomic Site, By Using Immunohistology With Specific Monoclonal Antibodies. We Demonstrated Changes In The Expression Patterns Of Collagens Xii And Xiv In Human Skin After Birth. Moreover, In Adult Scalp Skin, Very Strong Staining Of Collagen Xii Fibril Bundles Was Observed Around Hair Follicles, In Association With Very Low Expression Of Collagen Xiv. We Also Investigated The Expression Of Collagens Xii And Xiv By Fibroblasts And Keratinocytes Cultured In A Reconstructed Skin. In These Culture Conditions, Fibroblasts Recovered Their Ability To Produce Collagen Xiv And Re-Expressed The Small Splice Variant Of Collagen Xii. These Results Could Be Explained By The Deposition Of Large Amounts Of Collagen Fibrils By Fibroblasts In This Culture System. Thus, The Re-Expression Of These Collagens Suggests That The Deposition Of Banded Collagen Fibrils Is A Pre-Requisite For The Expression Of Collagen Xiv And Small Variant Of Collagen Xii. Auteur(s) : Berthod F., Rouabhia M. Titre : Exhaustive review of clinical alternatives for damaged skin replacement Référence : Skin substitute production by tissue engineering: clinical and fundamental applications. Rouabhia M., ed. R.G. Landes Bioscience: Austin TX, USA. Chap. 2: 23-45, 1997. Auteur(s) : Chouinard N., Leblanc R., Rouabhia M. Titre : In vitro engineered human skin substitutes for UVB harmful effect assessment Référence : Skin substitute production by tissue engineering: clinical and fundamental applications. Rouabhia M., ed. R.G. Landes Bioscience: Austin TX, USA. Chap. 7: 153-175, 1997. Auteur(s) : Germain L., Michel M., Fradette J., Xu W., Godbout M.-J., Li H. Titre : Skin stem cell identification and culture: a potential tool for rapid epidermal sheet production and grafting Référence : Skin substitute production by tissue engineering: clinical and fundamental applications. Rouabhia M., ed. R.G. Landes Bioscience: Austin TX, USA. Chap. 8: 177-210, 1997. Auteur(s) : Goulet F., Germain L., Rancourt D., Caron C., Normand A., Auger F.A. Titre : Tendons and Ligaments Référence : Lanza R., Langer R., Chick W.L. eds., R.G. Landes Bioscience: Austin TX and Academic Press, Inc.: San Diego. Chap. 39: 633-644, 1997. Auteur(s) : Li H., Berthod F., Xu W., Damour O., Germain L., Auger F.A. Titre : Use of in vitro reconstructed skin to cover skin flap donor site Référence : Journal of Surgical Research 73: 143-148, 1997. Lien : Cliquer ici Résumé : Background: The Skin Flap Technique Is Widely Used In Reconstructive Surgery For The Coverage Of Deep Burns Of The Face, Neck, And Joints. Facial Deformities And Joint Contractures Are Avoided By Transplanting Vascularized Full-Thickness Skin On Wounds. The Major Drawback Of This Technique Is The Injury Inflicted Upon The Donor Site, Which Corresponds To A Third Degree Burn. The Usual Technique To Cover The Flap Donor Site Is The Transplantation Of Split-Thickness Autografts. In The Case Of Patients With Deep And Extensive Burns, The Harvesting Of Good Quality Autografts Is Often Difficult Because Of Multiple Scars. In Order To Avoid Additional Trauma To The Patient By Split-Thickness Skin Harvesting, We Have Experimented The Use Of A New Model Of In Vitro Reconstructed Skin Graft For Flap Donor Site Coverage In A Mouse Model. Materials And Methods: The Reconstructed Skin Was Grafted On The Back Of Nude Mice At The Skin Flap Donor Site, While Flap Was Used To Cover A Wound Generated On Joint Of The Posterior Leg. Results: A 100% Graft Take Was Achieved (16 Mice Were Used) And A Limited Contraction Of The Reconstructed Skin Was Observed 30 Days Posttransplantation (78% Of The Initial Surface Area Of The Graft Remained). Histological Analysis Of The Graft Demonstrated Healing Of A Well Differentiated Epidermis Laying On A Dense Dermis. Conclusions: Since This Technique Would Prevent Additional Trauma To The Patient While Achieving A Good Healing Of The Wound, It May Be A Useful Approach In The Coverage Of Skin Flap Donor Site In Humans. Auteur(s) : Michel M., L’Heureux N., Auger F.A., Germain L. Titre : From newborn to adult: phenotypic and functional properties of skin equivalent and human skin as a function of donor age Référence : Journal of Cellular Physiology 171: 179-189, 1997. Lien : Cliquer ici Résumé : The Skin'S Most Important Function Is To Act As A Barrier Against Fluid Loss, Microorganism Infections, And Percutaneous Absorption. To Fulfill This Role, Keratinocytes Proliferate And Differentiate To Produce A Protective Layer: The Stratum Corneum. Because Stem Cells Are Responsible For The Production Of Differentiated Progeny And Stem Cells (K19-Expressing Cells) Are Less Abundant In Skin From Older Donors, The Purpose Of This Study Was To Establish Whether Histological And Functional Properties Of Differentiating Skin Is Influenced By Donor Age. The In Vitro Model Developed For The Evaluation Of Skin Properties (Michel Et Al., 1995) Was Used To Produce Skin Equivalents From Newborn, Child, And Adult Keratinocytes. Throughout Maturation, Skin Equivalents Were Compared With Corresponding Skin Biopsies For Keratin, Filaggrin, And Transglutaminase Expression. Percutaneous Absorptions Of Hydrocortisone Also Were Measured And Correlated With Lipid Content. After 1 Wk Of Immersed Culture, The Epidermal Layer Of Newborn Skin Equivalents Was Thicker Than Child And Adult Epidermis. As Expected, A Greater Proportion Of Cutaneous Stem Cells Was Present In Newborn Compared With Children And Adult Skin Equivalents. No Age-Related Difference Was Observed For Differentiation Markers. When Skin Equivalents Were Cultured At The Air-Liquid Interface, Cell Differentiation And Stratum Corneum Formation Were Induced, And The Age-Related Variation In The Thickness Of The Epidermal Layer Disappeared. Percutaneous Absorption Through These Matured Skin Equivalents Did Not Vary With Age. Their Lipid Density And Profile Were Similar. Accordingly, Skin Biopsies Exhibited Comparable Percutaneous Absorption Profiles Independently Of Donor Age. In Conclusion, Although Newborn Skin Equivalents, Or Skin Biopsies, Contained More Stem Cells Than Child And Adult Counterparts, No Age-Related Histological Difference Was Observed In The Differentiated Tissues. Moreover, The Functional Barrier Property Of Skins And Matured Skin Equivalents Did Not Vary With Age. Therefore, Both Newborn And Adult Keratinocytes Produce Useful In Vitro Models To Study Epidermal Differentiation Processes Involved In Both Normal And Pathological States. Auteur(s) : Moulin V., Auger F.A., O’Connor-McCourt M., Germain L. Titre : Fetal and postnatal sera differentially modulate human dermal fibroblast phenotypic and functional features in vitro Référence : Journal of Cellular Physiology 171: 1-10, 1997. Lien : Cliquer ici Résumé : Fetal Wounds Heal Without Scar Formation, Fibrosis, Or Contracture. Compared With Adult Wounds, They Are Characterized By Major Differences In The Extracellular Matrix And The Absence Of Myofibroblastic Cells. The Reasons For These Differences Are Not Well Known And Determination Of Factors Affecting The Absence Of Scarring In The Fetus May Lead To Strategies For Controlling Adult Pathological Scarring. In The Present Study, We Have Assessed The Effects Of Serum On The Behavior Of Normal Human Dermal Fibroblasts. Using An In Vitro Approach, We Investigated The Effects Of Fetal And Adult Serum On Cell Properties Such As Growth Rate, Collagen Synthesis, Gelatinase Activities, And Differentiation To Myofibroblasts Using Biochemical, Morphological, And Ultrastructural Parameters. We Studied The Induction Of Alpha-Smooth Muscle (Alpha-Sm) Actin In Fibroblasts, And Its Correlation With Increased Collagen Gel Contraction By The Cells. Our Results Showed That, Compared With Fbs (Fetal Bovine Serum), Postnatal Calf Serum (Pcs) Decreased Mitogenic Activity And Collagenase Synthesis But Not Collagen Synthesis. Furthermore, Cells Cultured With Pcs Differentiated To Myofibroblasts With An Increase In Cell Diameter, Number Of Stress Fibers, Alpha-Sm Actin Expression, And Collagen Gel Contraction. To Characterize The Molecules Involved In This Differentiation Process, The Amount Of Transforming Growth Factor Beta (Tgfbeta) In Fbs And Pcs Was Determined And The Effect Of Neutralizing Anti-Tgfbeta Antibody Was Evaluated. It Was Determined That Fbs Contained More Tgfbeta Than Pcs, But That Essentially All The Tgfbeta Was Latent In Both Sera. However, Results Obtained With Anti-Tgfbeta Antibody Show That Active Tgfbeta Is Present When Human Dermal Fibroblasts Are Cultured With Medium Containing Pcs. These Results Suggest That, In The Presence Of Pcs But Not Fbs, The Cells Either Produce Active Tgfbeta Or An Enzyme That Is Able To Activate Latent Serum Tgfbeta. Alternatively, Sera May Contain Two Different Forms Of Latent Tgfbeta, The Pcs Form Being Activated By The Dermal Fibroblast Cells. A Similar Mechanism May Be Involved, At Least In Part, In Skin Wound Healing And May Underlie The Appearance Of Myofibroblasts In Postnatal Wounds. Auteur(s) : Rouabhia M. Titre : Structural and functional complexity of the skin Référence : Skin substitute production by tissue engineering: clinical and fundamental applications. Rouabhia M., ed. R.G. Landes Bioscience: Austin TX, USA. Chap. 1: 3-22, 1997. Auteur(s) : Rouabhia M. Titre : Skin regeneration after heterologous epidermal substitute grafting Référence : Annals of Burns and Fire Disasters 10 (2): 98-104, 1997. 1996 Auteur(s) : Goulet F., Boulet L.-P., Chakir J., Tremblay N., Dubé J., Laviolette M., Boutet M., Xu W., Germain L., Auger F.A. Titre : Morphologic and functional properties of bronchial cells isolated from normal and asthmatic subjects Référence : American Journal of Respiratory Cell and Molecular Biology 15: 312-318, 1996. Lien : Cliquer ici Résumé : Recent Advances In Biomedical Sciences Have Led To The Development Of Various Methods For The Evaluation Of The Physiopathology Of Respiratory Diseases. This Study Reports Morphologic And Functional Features Of Cells Isolated By A New Method From Bronchial Biopsies Of Normal And Asthmatic Subjects. Both Epithelial And Fibroblastic Cells Were Isolated From The Same Biopsies Using Collagenase. The Cells Were Cultured For Several Passages And Stored Frozen. Two Selective Culture Media Were Used In Order To Obtain Pure Epithelial And Fibroblastic Cell Populations. Immunofluorescence Analysis Of Intermediate Filaments, Keratins, And Vimentin Confirmed The Type Of The Isolated Cells. The Proportions Of Alpha-Actin-Expressing Cells Varied Among The Fibroblastic Cell Populations Isolated From Normal And Asthmatic Subjects. Interestingly, The Population Containing High Numbers Of Alpha-Actin-Expressing Cells And Presenting The Fastest Collagen Contraction Kinetic Was Isolated From Bronchial Biopsies Of An Asthmatic Subject. Moreover, The Fibroblastic Cells That Showed The Best Contractile Properties 24 H After Their Seeding In Floating Collagen Gels Were Isolated From Bronchial Biopsies Of Asthmatic Patients Having Pc20 Values Below 1 Mg/Ml. On The Basis Of These Data, We Propose A New Approach To Isolate, Culture And Characterize Human Bronchial Cells In Vitro. Auteur(s) : Goulet F., Germain L., Caron C., Rancourt D., Normand A., Auger F.A. Titre : Tissue Engineered Ligament Référence : Ligaments and Ligamentoplasties Yahia L'H., ed. Springler-Verlag Publishers: Germany. 367-377, 1996. Auteur(s) : Goulet F., Poitras A., Rouabhia M., Cusson D., Germain L., Auger F.A. Titre : Stimulation of human keratinocyte proliferation through growth factor exchanges with dermal fibroblasts in vitro Référence : Burns 22(2): 107-112, 1996. Lien : Cliquer ici Résumé : Progress In Biotechnology Has Led To New Therapeutic Approaches In Various Fields Of Human Health Care, Such As The Autologous Grafting Of Cultured Epidermal Cell Sheets On Burned Patients. These Cultures Depend On Various Parameters But Growth Factors Are Of Paramount Importance. Cutaneous Cells Are Known To Secrete Various Growth Factors In Vivo, Although Only A Few Have Been Identified. The Aim Of This Study Was To Determine If Such Factors Are Secreted From Human Cutaneous Cells In Culture, To Evaluate Their Effects On Epidermal Cell Proliferation In Vitro And To Analyse Them On Sds-Page. Human Skin Fibroblasts And Keratinocytes Were Co-Cultured For 8-10 Days Using A Costar Trans-Filter System. Dermo-Epidermal Cooperation Was Observed In Such A Co-Culture System Through The Exchange Of Secretion Products In The Culture Medium. Epidermal Cell Growth And Metabolic Activities Were Highly Stimulated In Co-Culture (2-Fold And 1.5-Fold, Respectively, P < 0.02) Compared To The Control. The De Novo Synthesis Of Secretion Products, Notably Of A Protein Of About 40 Kda, Was Specifically Induced In Co-Culture. The Identification Of New Keratinocyte Growth Factors Could Accelerate Graftable Epidermal Sheet Production In Vitro For Human Wound Coverage And Possibly Enhance Wound Healing In Vivo. Auteur(s) : López Valle C.A., Germain L., Rouabhia M., Xu W., Guignard R., Goulet F., Auger F.A. Titre : Grafting on nude mice of living skin equivalents produced using human collagens Référence : Transplantation 62(3): 317-323, 1996. Lien : Cliquer ici Résumé : Autologous Epidermal Transplantation For Human Burn Management Is An Example Of A Significant Breakthrough In Tissue Engineering. However, The Main Drawback With This Treatment Remains The Fragility Of These Grafts During And After Surgery. A New Human Bilayered Skin Equivalent (Hse) Was Produced In Our Laboratory To Overcome This Problem. The Aim Of The Present Work Was To Study Skin Regeneration After Hse Grafting Onto Nude Mice. A Comparative Study Was Carried Out Over A Period Of 90 Days, Between Anchored Bovine Skin Equivalent, Hse And Hse+, The Latter Containing Additional Matrix Components Included At Concentrations Similar To Those In Human Skin In Vivo. The Addition Of A Dermal Layer To The Epidermal Sheet Led To Successful Graft Take, Enhanced Healing, And Provided Mechanical Resistance To The Grafts After Transplantation. In Situ Analysis Of The Grafts Showed Good Ultrastructural Organization, Including The Deposition Of A Continuous Basement Membrane 1 Week After Surgery. Auteur(s) : Michel M., Török N., Godbout M.-J., Lussier M., Gaudreau P., Royal A., Germain, L. Titre : Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage Référence : Journal of Cell Science 109: 1017-1028, 1996. Lien : Cliquer ici Résumé : This Study Was Undertaken To Evaluate Keratin 19 (K19) As A Biochemical Marker For Skin Stem Cells In Order To Address Some Long Standing Questions Concerning These Cells In The Field Of Cutaneous Biology. We First Used The Well-Established Mouse Model Enabling Us To Identify Skin Stem Cells As [3H]Thymidine-Label-Retaining Cells. A Site Directed Antibody Was Raised Against A Synthetic Peptide Of K19. It Reacted Specifically With A 40 Kda Protein (K19) On Immunoblotting. It Labelled The Bulge Area Of The Outer Root Sheath On Mouse Skin By Immunohistochemistry. Double-Labelling Revealed That K19-Positive-Cells Were Also [3H]Thymidine-Label-Retaining Cells, Suggesting That K19 Is A Marker For Skin Stem Cells Of Hair Follicles. K19-Expression Was Then Used To Investigate The Variation In Mouse And Human Skin Stem Cells As A Function Of Body Site, Donor Age And Culture Time. K19 Was Expressed In The Hair Follicle And Absent From The Interfollicular Epidermis At Hairy Sites (Except For Some K18 Coexpressing Merkel Cells). In Contrast, At Glabrous Sites, K19-Positive-Cells Were In Deep Epidermal Rete Ridges. K19 Expressing Cells Also Contained High Levels Of Alpha 3 Beta 1 Integrin. The Proportion Of K19-Positive-Cells Was Greater In Newborn Than Older Foreskins. This Correlated With Keratinocyte Culture Lifespan Variation With Donor Age. Moreover, It Could Explain Clinical Observations That Children Heal Faster Than Adults. In Conclusion, K19 Expression In Skin Provides An Additional Tool To Allow Further Characterization Of Skin Stem Cells Under Normal And Pathological Conditions In Situ And In Vitro. Auteur(s) : Moulin V., Castilloux G., Jean A., Garrel D.R., Auger F.A., Germain L. Titre : In vitro models to study wound healing fibroblasts Référence : Burns 22(5): 359-362, 1996. Lien : Cliquer ici Résumé : Phenotypic And Contractile Properties Of Human Fibroblasts From Dermis And From An Experimental Wound Model Were Studied In Vitro. When Cultured In Monolayer, Dermal Fibroblasts Had An Elongated Spindle Shape, Were Small In Diameter And Grew At A High Rate. Wound Fibroblasts Grew Slowly And Were Large, Star Shaped And Had Cytoplasmic Stress Fibres. Smooth Muscle Alpha Actin Was Detected In 10 Percent Of Dermal Cells, Whereas 20-80 Per Cent Of Wound Fibroblasts Contained This Protein In Their Cytoplasm. The Contractile Property Of Cells Was Evaluated Using A Three-Dimensional Cell Culture Model. Our Results Show That Wound Fibroblasts Contract Collagen Gels During The First Days More Strongly Than Dermal Fibroblasts. These Results Show That, In Vitro, Wound Fibroblasts Have Greater Contractile Capacity Than Dermal Cells. The Significant Proportion Of Wound Fibroblasts Containing Alpha-Smooth Muscle Actin Suggests That Alpha-Smooth Muscle Actin Ratio May Be Related To Wound Contraction. Auteur(s) : Pâquet I., Chouinard N., Rouabhia M. Titre : Cutaneous cell and extracellular matrix responses to ultraviolet-B irradiation Référence : Journal of Cellular Physiology 166: 296-304, 1996. Lien : Cliquer ici Résumé : The Present Study Examined Fibroblasts And Keratinocytes In Monolayers And Cultured Within Dermal And Skin Substitutes And Their Use In Assessing The Effect Of Uvb Irradiation On Cutaneous Cells And Extracellular Matrix Organization. Dermal Substitutes (Ds) Were Produced By Incorporating Normal Fibroblasts Into A Collagen Lattice And Skin Substitutes (Ss) Were Obtained By Seeding Normal Keratinocytes Onto The Ds. Keratinocyte Monolayers, Fibroblast Monolayers, Ds, And Ss Were Exposed Once A Day To A Uvb Source (10 Mj/Cm2). The Irradiation Protocol Was Stopped When The Keratinocytes Of The Non-Irradiated Cultures (Control Groups) Had Reached Confluence. Microscopic Observations Revealed That Uvb Radiation Decreased Both Fibroblast And Keratinocyte Growth And Enhanced Their Differentiation Resulting In (1) Less Fibroblasts In The Ds And Ss, And (2) Incomplete Coverage Of The Ds By Keratinocytes. Microscopic Observations And Histological Analyses Revealed Major Morphological Changes. Both Cell Types Became Bigger And Presented Wide Nuclei And Vacuoles In The Cytoplasm. No Organized Deep Epidermal Layer Was Observed In Irradiated Compared To Non-Irradiated Ss. Irradiated Ds And Ss Extracellular Matrices Showed An Irregular Aggregating Collagen Fiber Organization With Serious Discrepancies Suggesting Large Defects In The Structural Properties Of The Extracellular Matrix. The Present Study Demonstrated That Exposure To A Uvb Source Led To Profound Morphological And Functional Disturbances In Both Cutaneous Cells And In The Extracellular Matrices Of The Ds And Ss. The Present Technology Would Be Of Great Interest For Step-By-Step Studies Of Uvr Effects On Cutaneous Cell Morphology And Functional Properties, And Could Be An Alternative To Using Animals For Pharmacological And Toxicological Evaluations. Auteur(s) : Rouabhia M. Titre : Permanent skin replacement using chimeric epithelial cultured sheets comprising xenogeneic and syngeneic keratinocytes Référence : Transplantation 61(9): 1290-1300, 1996. Lien : Cliquer ici Résumé : The Present Study Was Undertaken To Evaluate The Possibility Of Permanent Skin Replacement Using Chimeric Xenogeneic-Syngeneic Graftable Sheets Previously Obtained In Vitro. Newborn ( Auteur(s) : Rouabhia M. Titre : In vitro production and transplantation of immunologically active skin equivalents Référence : Laboratory Investigation 75 (4): 503-517, 1996. Lien : Cliquer ici Résumé : In This Study, We Produced In Vitro Epidermal Equivalents (Ee) And Skin Equivalents (Se) With And Without Spleen Lymphocytes. These Skin Substitutes Were Used For In Vitro And In Vivo (After Isograft) Histologic Studies Of Cell And Extracellular Matrix Organization And For Protein Synthesis. Then, Using Spleen Lymphocytes In Syngeneic And Allogeneic Se, We Assessed The Immunogenicity Of These Skin Substitutes After Transplantation. In Vitro Histologic Analyses Showed That The Epidermal Organization Of Ee Was Comparable To That Of Se. Fibroblasts And Spleen Lymphocytes Were Present In The Extracellular Matrix, As Is The Case In Normal Skin. Comparative Immunohistologic Studies After Ee And Se Isografting Showed That The Newly Generated Cutaneous Tissues Were Well Structured And Vascularized. There Were Indications Of Physiologically Active Skin. The Dermal Component In These Regenerated Skins Was, However, More Organized After Se Than After Ee Isografting, Which Indicates The Importance Of The Dermis. Lastly, Allografting Of Se With And Without Spleen Lymphocytes Showed Interesting Results. Indeed, 10 Days After Allografting, All Se Allowed Skin Regeneration Comparable To Isografts. Moreover, Leukocyte Infiltration In Allografts Was Observed As Early As 10 Days And Increased During The Postgrafting Period. Also, The Presence Of Allogeneic Spleen Lymphocytes Alone In Syngeneic Se Initiated Recipient Immune Activation And Induced Leukocyte Infiltration And Graft Rejection. The Density Of Infiltrating Leukocytes Was Higher In The Complete Allograft (Allogeneic Keratinocytes, Fibroblasts, And Spleen Lymphocytes) Compared With The Partial Allograft (Only Spleen Lymphocytes Were Allogeneic), With The Allograft (Allogeneic Keratinocytes And Fibroblasts), And With The Partial Isograft (Presence Of Syngeneic Lymphocytes With Allogeneic Keratinocytes And Fibroblasts). Mac-1+ And Cd8+ Cells Were Present In These Leukocyte Infiltrations, Which Indicates Recipient Immune System Activation And Allograft Rejection. Cd4-Positive Cells Were Not, However, Seen In These Leukocyte Infiltrations. These Results Suggest That The Incorporation Of Spleen Lymphocytes In Se Enhanced Their Immunogenicity As Judged By Leukocyte Infiltration And The Presence Of Cd8+ Cells In The Implants. Auteur(s) : Rouabhia M. Titre : Une nouvelle méthode de culture cellulaire pour le remplacement permanent du revêtement cutané endommagé Référence : Médecine/Sciences 12(12): 1370-1377, 1996. Auteur(s) : Stoclet J.-C., Andriantsitohaina R., L'Heureux N., Martinez C., Germain L., Auger F.A. Titre : Use of human vessels and human vascular smooth muscle cells in pharmacology Référence : Cell Biology and Toxicology 12: 223-225, 1996. Lien : Cliquer ici Résumé : Relatively Limited Information Is Available Regarding The Mechanisms Controlling Vasomotricity In Human Vessels. Isolated Vessels Obtained From Patients Undergoing Surgery Were Used To Characterize The Role Of Endothelial Factors And To Study Coupling Mechanisms Between Receptors, Intracellular Calcium, And Contraction. However, These Investigations Are Limited By The Availability Of Tissues And Many Uncontrolled Factors. Cultured Human Vascular Cells Were Also Used, But These Cells Rapidly Lose At Least Some Of Their Differentiated Characters. Recently, A Human Blood Vessel Equivalent Was Constructed In Vitro From Cultured Cells, Using Tissue Engineering. This Technique Allowed Us To Obtain Vessel Equivalents Containing Intima, Media, And Adventitia Layers Or Tubular Media Layer Only. Contraction And Rises In Intracellular Calcium Produced By Agonists Were Studied, Indicating That Such Human Vessel Equivalents May Provide Valuable Models For Pharmacological Studies. Auteur(s) : Xu W., Germain L., Goulet F., Auger F.A. Titre : Permanent grafting of living skin substitutes: surgical parameters to control for successful results Référence : Journal of Burn Care and Rehabilitation 17(1): 7-13, 1996. Lien : Cliquer ici Résumé : Autologous Mesh Grafting, Widely Used In The Treatment Of Severe Burns, Remains The Most Conventional Approach For Permanent Skin Replacement. However, During The Last Decade Several Types Of Skin Substitutes Were Reported As Suitable Alternatives For Full-Thickness Burn Wound Coverage. The Clinical Use Of Such Dressings Requires New Surgical Skills To Maintain The Integrity Of The Grafts And Favor Their Permanent Implantation In Vivo. This Article Reports Observations Made On Nude Mice Grafted With Cultured Human Skin Equivalents. Some Parameters Such As The Quality Of Adhesion Between The Implant And The Graft Bed, The Size, The Stability And The Thickness Of The Graft, The Humidity Of The Chamber, And The Protocol Of Antibiotic Administration Were Identified As Crucial For The Success Of The Surgery. The Grafting Procedures Are Described In This Paper. These Results Should Be Taken Into Consideration In All Transplantations Of Skin Grafts In Vivo. Auteur(s) : Xu W., Li H., Brodniewicz T., Auger F.A., Germain L. Titre : Cultured epidermal sheet grafting with HemaseelTM HMN fibrin sealant on nude mice Référence : Burns 22(3): 191-196, 1996. Lien : Cliquer ici Résumé : Grafting Of Cultured Epidermal Sheets Is A Promising Technique For Skin Restoration In Extensive Burns, But The Technique Has Some Limitations, Resulting In Variable Graft Takes. These Experiments Were Designed To Evaluate The Innocuity Of Hemaseel Hmn Fibrin Sealant In The Grafting Process And In Vivo Evolution Of Cultured Epidermis. A Total Of 30 Mice Were Grafted, 15 Were Controls, 15 Received Tissue Sealant Application Before The Deposition Of The Cultured Human Epidermal Sheets. Seven Days After Transplantation, Compared To Controls, The Percentage Of Graft Take Over The Total Surface Area Grafted Was Greater In Animals That Had Received The Tissue Sealant Application. No Difference Was Found 14 And 21 Days Postgrafting. In Contrast, The Percentage Of Graft Take Over The Bony Area (Spinal) Was Significantly Increased In Animals Grafted With Previous Application Of Sealant Compared To Controls At 7, 14 And 21 Days Postgrafting. Immunohistological And Ultrastructural Analysis Showed That The Evolution Of The Cultured Human Epidermis After Transplantation Was Similar In Both Groups. The Basement Membrane Was Well Structured 21 Days After Transplantation. The Sealant Was Present At 4 Days But Not At 21 Days Postgrafting. Therefore, We Conclude That The Application Of Fibrin Sealant Before Cultured Epidermal Sheet Deposition On Nude Mouse Graft Bed Is Innocuous And Enhances Their Mechanical Stability. Since In This Nude Mouse System Hemaseel Hmn Fibrin Sealant Increased The Percentage Of Graft Take Over Areas Difficult To Engraft, We Think That It May Be Advantageous In Cultured Epidermal Sheet Grafting On Burn Patients. 1995 Auteur(s) : Auger F.A., López Valle C.A., Guignard R., Tremblay N., Noël B., Goulet F., Germain L. Titre : Skin equivalent produced with human collagen Référence : In Vitro Cellular and Developmental Biology - Animal 31: 432-439, 1995. Lien : Cliquer ici Résumé : Several Studies Have Recently Been Conducted On Cultured Skin Equivalent (Se), Prepared Using Human Keratinocytes Seeded On Various Types Of Dermal Equivalents (De). We Previously Showed The Advantages Of Our Anchorage Method In Preventing The Severe Surface Reduction Of De Due To Fibroblast Contractile Properties In Vitro. A New Anchored Human Se Was Established In Our Laboratory In Order To Obtain A Bioengineered Tissue That Would Possess The Appropriate Histological And Biological Properties. In Order To Compare The Effects Of Different Collagen Origins On The Evolution Of Se In Vitro, Human Keratinocytes Were Seeded On Three Types Of Anchored De. A Comparative Study Was Carried Out Between Bovine Se (Bse), Human Se (Hse), And Human Skin Equivalent Containing Additional Dermal Matrix Components (Hse+). Immunohistological Analysis Showed That Hse And Hse+ Presented Good Structural Organization, Including The Deposition Of Several Basement Membrane Constituents. Higher Amounts Of Transglutaminase, Ceramides, And Keratin 1 Were Detected In The Epidermal Layers Of All Se When Cultured At The Air-Liquid Interface. However, A 92 Kda Gelatinase Activity Was Higher In Bovine Skin Equivalent (Bse) Compared To Hse Cultures. The Use Of Human Collagens Comparatively To Bovine Collagen As Se Matricial Component Delayed The Degradation Of The Dermal Layer In Culture. Auteur(s) : Fradette J., Godbout M.-J., Michel M., Germain L. Titre : Localization of Merkel cells at hairless and hairy human skin sites using keratin 18 Référence : Biochemistry and Cell Biology 73: 635-639, 1995. Lien : Cliquer ici Résumé : Merkel Cells Are Neurosecretory Cells Of The Skin With Epithelial Features Such As Desmosomes And Expression Of Keratins 8, 18, 19, And 20. Merkel Cells Are Scarcely Distributed In Adult Human Skin. Although They Are Present In Hair Follicles, Their Density Is Higher At Hairless Anatomic Sites Such As Palms And Soles. These Cells Are Often Innervated By Sensory Nerve Fibers And Are Thought To Be Specialized Mechanosensory Skin Receptor Cells. However, Their Precise Origin And Function Are Not Clearly Established. The Aim Of This Study Was To Localize Merkel Cells In Human Hairless And Hairy Skin By Immunohistochemistry With Antibodies Ks18.174 And Ks19.1 Directed Against Keratins 18 And 19, Respectively. In Glabrous Skin Of Palm And Sole, Merkel Cells Have Been Localized At The Bottom Of The Rete Ridges, In The Epidermal Basal Layer. To Study Merkel Cell Distribution At Hairy Anatomic Sites, We Have Chosen Breast Skin, A Tissue Containing Small Hair Follicles Typical Of Those Covering Most Of The Body'S Surface. Merkel Cells Were Present In The Interfollicular Epidermis. In Hair Follicles, They Have Been Identified In The Isthmus Region. Auteur(s) : Germain L., Auger F.A. Titre : Tissue engineered biomaterials: biological and mechanical characteristics. Référence : In Encyclopedic Handbook of Biomaterials and Bioengineering. Part B: Applications, Eds.: Wise D.L., Trantolo D.J., Altobelli D.E., Yaszemski M.J., Gresser J.D., Schwartz E.R. Marcel Dekker, Inc. Publishers: New York, USA. Vol. 1, chap. 25: 699-734, 1995. Auteur(s) : Germain L., Guignard R., Rouabhia M., Auger F.A. Titre : Early basement membrane formation following the grafting of cultured epidermal sheets detached with thermolysin or dispase Référence : Burns 21(3): 175-180, 1995. Lien : Cliquer ici Résumé : The Basement Membrane Zone Is Important For Graft Adhesion And Stability. The Aim Of The Present Study Was To Visualize The Regeneration Of The Basement Membrane And Determine The Sequential Appearance Of Its Constituents In The Early Postgrafting Period Of Cultured Human Epidermal Sheets. A Keratinocyte Single Cell Suspension, Devoid Of Dermal Fibroblast Contamination, Was Obtained From Human Skin By A Two-Step Tissue Digestion Method With Thermolysin And Trypsin. After Culturing, Epidermal Sheets Were Generated, Detached Enzymatically By Incubating With Thermolysin (For 20-30 Min) Or Dispase (For 45-60 Min), And Deposited On A Muscular Graft Bed Of Athymic Mice. Immunohistochemistry And Ultrastructural Analyses Were Performed On Biopsies Harvested 2, 4 And 21 Days Postgrafting. Bullous Pemphigoid Antigens And Laminin Were Detected At The Dermo-Epidermal Junction, Showing An Almost Continuous Line 2 Days Postgrafting. Type Iv Collagen Was Generally Absent At This Time, But It Was Detected 4 Days Postgrafting. Type Vii Collagen Was Labelled As A Discontinuous Line Of Increasing Intensity From 2 To 21 Days Postgrafting. Ultrastructural Analysis Revealed Hemidesmosomes And A Discontinuous Lamina Densa 2 Days Postgrafting, And A Complete Basement Membrane With A Continuous Lamina Densa, Hemidesmosomes And Anchoring Fibrils 21 Days Postgrafting. The Sequence Of Appearance Of Major Basement Membrane Components Was Similar For Cultured Sheets Detached With Thermolysin Or Dispase. However, It Differed From That Of Other Wound Healing Models. Results Are Discussed In Terms Of The Variable Keratinocyte Migration Requirement Between Various Wound Healing Models. Auteur(s) : Lafrance H., Guillot M., Germain L., Auger F.A. Titre : A method for the evaluation of tensile properties of skin equivalents Référence : Medical Engineering & Physics 17(7): 537-543, 1995. Lien : Cliquer ici Résumé : In Vitro Production Of Anchored Skin Equivalent Is A New Therapeutical Option For Burn Patients. A Skin Equivalent Is A Combined Culture Of Dermal And Epidermal Layers. The Dermal Layer Provides Important Mechanical Properties, Such As Tensile Resistance And Nonlinear Elasticity, To The Skin Equivalent During Its Development. Prior To Any In Vivo Human Transplantation, The Tensile Properties Of Cutaneous Equivalents Have To Be Evaluated As A Function Of Its Structural Components, In View Of Establishing The Culture Conditions Leading To The Best Mechanical Resistance And Stretchability Characteristics. However, The Handling And Clamping Of Skin Equivalents Are Frequent Causes Of Tearing And Lack Of Repeatability In The Measuring Of Tensile Properties. A New Indentation Method Involving A Specially Designed Culture Dish Has Been Developed To Minimize The Risk Of Damage. Using This New Culture Dish, Cutaneous Equivalents Were Installed On An Indentation Apparatus. The Central Loading Of A Spherical Tip Was Transmitted To The Central Area Of A Circular Anchored Cutaneous Equivalent And Was Recorded With Tip Position. The Tests Were Achieved At A Constant Low Deflection Rate Of The Tip. This New And Accurate Method Gave Repeatability In Three Central Load-Deflection Characteristics Of Anchored Dermal Equivalent: The High-Modulus (0.15 G Mm-1), The Central Load Of Rupture (1.49 G), The Rupture Deflection (0.470 Mm). This Indentation Test Is Expected To Be An Efficient Tool In The Evaluation Of Various Skin Equivalent Models Tensile Properties. Auteur(s) : Lafrance H., Yahia L'H., Germain L., Guillot M., Auger F.A. Titre : Study of the tensile properties of living skin equivalents Référence : Bio-Medical Materials and Engineering 5(4): 195-208, 1995. Lien : Cliquer ici Résumé : The Living Skin Equivalent Is One Of The More Advanced Clinical Applications In The Field Of Tissue Engineering. It Is A Promising Therapeutic Option For Burn Victims And A Strong Potential For Manifold In Vitro Experiments. However, Researchers Have Encountered Major Drawbacks In The Reconstruction Of The Dermal Layer. Peripheral Anchorage Of The Dermal Equivalent Component Has Been A Valuable Solution To Many Of These Problems. In This Work, We Have Carried Out The Mechanical Analysis Of Skin Equivalent Models, Based On This Dermal Anchoring Technique, With A Study Of Their Biaxial Tensile Properties. Differences Between Models Were Related To The Origin Of Collagen, Either Bovine Or Human, And On The Culture Techniques: Immersion Or At The Air-Liquid Interface. The Study Was Accomplished In Vitro Using 25.4-Mm-Diameter Disk-Shaped Specimens With An Indentation Test. In Appropriate Wet Condition, The Specimens Were Punctured With A Spherical Tip At A Quasi-Static Rate. We Measured The Load Applied Against The Tip Vs Deflection Up To The Breaking Point. Our Results Show That Skin Equivalents Presented A Typical Exponential Load-Deflection Relationship. All Skin Equivalents Presented Large Extensibility Up To 1.41 Expressed In A Ratio Of Deflection Vs Specimen'S Radius. The Maximum Tensile Strength (0.871-1.169 Newton) And Energy Calculations (3.75-6.432 N.Mm) Was Offered By Living Skin Equivalent, Made With Human Types I And Iii Collagens, Cultured At The Air-Liquid Interface. In These Conditions, Our Results Suggest The Tensile Properties Of Living Skin Equivalents Were Enhanced Due To The Development Of Well Stratified Stratum Corneum. Auteur(s) : Michel M., Germain L., Bélanger P.M., Auger F.A. Titre : Functional evaluation of anchored skin equivalent cultured in vitro: percutaneous absorption studies and lipid analysis Référence : Pharmaceutical Research 12 (3): 455-458, 1995. Lien : Cliquer ici Auteur(s) : Moulin V. Titre : Growth factors in skin wound healing Référence : European Journal of Cell Biology 68: 1-7, 1995. Lien : Cliquer ici Résumé : The Healing Of Skin Involves A Wide Range Of Cellular, Molecular, Physiological And Biochemical Events. During The Healing Process, Cells Migrate To Wound Sites Where They Proliferate And Synthesize Extracellular Matrix Components In Order To Reconstitute A Tissue Closely Similar To The Original One. This Activity Is Regulated By Mediators Secreted From The Wound Border Cells Such As Pdgf, Egf, Tgf Beta And Many Other Cytokines. Their Effects On Cells Has Been Demonstrated In Vivo And In Vitro. The Aim Of This Article Is To Summarize The Sequential Events That Occur During Wound Healing Notably Cell Migration, Proliferation And Phenotypic Differentiation And To Describe The Cellular Interactions Involving Growth Factors At The Molecular Level. Auteur(s) : Rouabhia M., Germain L., Bergeron J., Auger F.A. Titre : Allogeneic-syngeneic cultured epithelia: A successful therapeutic option for skin regeneration Référence : Transplantation 59(9): 1229-1235, 1995. Lien : Cliquer ici Résumé : Organ Transplantation Has Progressed Rapidly During The Last Decades. Furthermore, Tissue Engineering Has And Will Continue To Enlarge The Scope Of Organ Grafting. Thus, Severe Skin Wounds, As Observed In Large Burn Trauma Patients, Can Now Be Permanently Treated With Cultured Autologous Epithelial Sheets. However, The Time Required For Autologous Cell Growth Is A Major Limitation. We Propose To Alleviate This Pitfall Through A Novel Chimeric (Allogeneic-Syngeneic) Epithelial Cell Culture Technique. These Chimeric Epidermal Grafts Implanted In An Animal Model Have Been Shown To Allow The Reappearance Of A Histologically Normal Epidermal Coverage Similar To Simultaneously Performed Isografts. The Regenerated Epidermis Contained Only Syngeneic Keratinocytes. Thus, Allogeneic Cells Were Eliminated Passively. This New Culture Technology Could Find Multiple Applications, Notably In Various Skin Disease Therapies. 1994 Auteur(s) : Choinière M., Auger F.A., Latarjet J. Titre : Visual analogue thermometer: a valid and useful instrument for measuring pain in burned patients Référence : Burns 20(3): 229-235, 1994. Lien : Cliquer ici Résumé : This Study Assessed The Psychometric Qualities Of A New Pain Rating Instrument--The Visual Analogue Thermometer (Vat)--Which Was Developed To Measure Pain In Burned Patients. The Validity And Utility Of The Vat Was Assessed And Compared With A Conventional Numeric (Num) And Adjective Pain Scale (Adj) With A Group Of 103 Burned Patients And 51 Nurses. Analyses Of The Results Support The Concurrent And Construct Validity Of The Vat As A Pain Measure. Furthermore, The Vat Gave More Sensitive And Precise Pain Measures Than The Adj And/Or Num Scales. No Major Difference Between The Three Scales Emerged In The Patients' Preference. The Same Was True For The Nurses' Evaluation Except For Those Who Had More Clinical Experience With The Vat And Who Tended To Prefer This Scale For Its Accuracy And Ease Of Utilization. The Vat Appears To Be A Valid, Sensitive And Clinically Useful Tool To Measure Pain In Burned Patients. A Systematic Pain Assessment Procedure Which Can Be Easily Implemented In Burn Care Facilities Is Presented. Auteur(s) : Germain L., Jean A., Auger F.A., Garrel D.R. Titre : Human wound healing fibroblasts have greater contractile properties than dermal fibroblast Référence : Journal of Surgical Research 57: 267-273, 1994. Lien : Cliquer ici Résumé : Contractile And Phenotypic Properties Of Human Fibroblasts From Healing Wounds Were Compared To Those Of Dermal Fibroblasts Using In Vitro Models. Wound Fibroblasts Were Recovered From Implants, Made Of A Polyvinyl Alcohol Sponge Threaded Into A Perforated Silicone Tube, 12 Days After Their Subcutaneous Implantation In Human Volunteers. Dermal Fibroblasts Were Isolated From The Skin Of Healthy Subjects. Two Morphologically Different Fibroblast Populations Were Observed In Cells Cultured From Implants. In Order To Characterize These Fibroblast Populations, Intracellular Alpha-Actin Expression Was Studied By Immunofluorescence Labeling Of Cells Cultured In Monolayer. This Protein Was Detected In Less Than 1% Of The Dermal Fibroblasts. By Contrast, 30 To 40% Of Wound Fibroblasts Were Labeled And Contained Fiber Networks Of Alpha-Actin. These Results Confirm The Presence Of Myofibroblasts In Human Wound Healing Tissues. The Contractile Property Of Fibroblasts And Myofibroblasts Was Evaluated Using A Three-Dimensional Cell Culture Model (Fibroblast Populated Collagen Gels). Cells Were Incorporated In A Collagen Matrix And Cultured For 14 Days. The Surface Area Of Collagen Gels Was Measured Every Day. Our Results Show That Wound Fibroblasts Strongly Contract Collagen Gels During The First 24 Hr (Surface Area At 24 Hr = 20-55% Of Initial Surface Area) In Comparison To Dermal Fibroblasts (Surface Area At 24 Hr = 70-75% Of Initial Surface Area). This Superior Level Of Contraction Was Observed Until The Fifth Day Of Culture. Auteur(s) : Rouabhia M., Germain L., Bergeron J., Auger F.A. Titre : Successful transplantation of chimeric allogeneic-autologous cultured epithelium Référence : Transplantation Proceedings 26(6): 3361-3362, 1994. Lien : Cliquer ici Auteur(s) : Rouabhia M., Jobin N., Doucet R. Jr., Bergeron J., Auger F.A. Titre : CD36+ dendritic epidermal cells: a putative actor in the cutaneous immune system Référence : Cell Transplantation 3(6): 529-536, 1994. Lien : Cliquer ici Résumé : In The Present Study We Have Investigated By Indirect Immunofluorescence Staining And Mixed Lymphocyte Reaction Methods, The Localization, Distribution, Percentage, And The Immunological Involvement Of Cd36(+)-Dendritic Epidermal Cells (Cd36(+)-Decs) In Normal Human Skin. Human Epidermal Cell Suspensions Were Obtained From Skin Specimen Of Healthy Persons. First, An Indirect Immunofluorescent Staining Method Was Performed On Frozen Skin Sections, Freshly Isolated Cells, Nonadherent And Adherent Cells And Second, The Allogeneic Mixed Epidermal Cell-Lymphocyte Reaction (Elr) Method Was Performed With Human Peripheral Blood Mononuclear Cells And Irradiated Cd36(+)-Decs Plus Cd-1A+ (Langerhans Cells) And/Cona (At 10 Micrograms/Ml). We Found That Cd36(+)-Decs Were Localized In The Epidermis Mainly In The Basal Layer. They Were Non Adherent Cells. The Percentage Of These Cd36(+)-Decs Was Of About 2%. These Cd36(+)-Decs Were Ae3 (Which Recognizes Keratin Normally Expressed By Keratinocytes) Positive Cells. Our Immunoreactivity Study Using Allogeneic Mixed Elr, Showed That Cd36(+)-Decs Stimulated Allogeneic Lymphocyte Proliferation. Their Stimulatory Effects Were Important When Langerhans Cells And Cona Were Added Separately Or Together To The Pbmcs Culture. The Above Results Suggest That Cd36(+)-Decs May Contribute To The Immunological Role Of Skin And Could Be Involved In Cutaneous Allograft Recognition And Rejection. Abbreviations: Decs: Dendritic Epidermal Cells; Cona: Concanavalin A; Dpm: Disintegrations Per Minute; Elr: Epidermal Cell-Lymphocyte Reaction; Lc: Langerhans Cells; Pbmcs: Peripheral Blood Mononuclear Cells. 1993 Auteur(s) : Auger F.A., Guignard R., López Valle C.A., Germain L. Titre : Role and innocuity of Tisseel®, a tissue glue, in the grafting process and in vivo evolution of human cultured epidermis Référence : British Journal of Plastic Surgery 46: 136-142, 1993. Lien : Cliquer ici Résumé : Cultured Epidermal Sheets Are Currently Used For Burn Wound Treatment But Reported Results On Graft Take Are Variable. This Study Was Designed To Evaluate The Role And Influence Of Tisseel, A Fibrin Glue, In The Take Of Cultured Human Epidermal Sheets In An Athymic Mouse Model. On Days 4, 10 And 21 Post-Grafting, Histology, Electron Microscopy And Immunofluorescence Staining Confirmed The Presence Of A Human Epithelium And The Development Of A Basement Membrane. Tisseel Was Detectable On Day 4 Only, But Overall Treated And Untreated Grafts Were Similar. The Use Of Tisseel Enhanced The Mechanical Stability Of These Fragile Grafts, Increased The Percentage Of Graft Take, And Its Innocuity On The In Vivo Evolution Of Cultured Epidermal Sheets Was Demonstrated. For These Reasons, We Think That Tisseel May Be Advantageous In A Clinical Setting. Auteur(s) : Germain L., Guignard R., Jacques P., Garceau S., Gilbert M., Auger F.A. Titre : Human epidermal culture model to evaluate the effect of topical antibiotics on keratinocyte growth Référence : British Journal of Dermatology 129 (S 42): 22, 1993. Auteur(s) : Germain L., Rouabhia M., Guignard R., Carrier L., Bouvard V., Auger F.A. Titre : Improvement of human keratinocyte isolation and culture using thermolysin Référence : Burns 19(2): 99-104, 1993. Lien : Cliquer ici Résumé : We Propose A Modification Of The Conventional Keratinocyte Isolation Method Which Has Shown A Significant Improvement In The Purity, Colony Forming Efficiency (C.F.E.) And Growth Capacity Of The Isolated Epidermal Cell Population. This Method Utilized Thermolysin Since It Selectively Digests The Dermo-Epidermal Junction. Following Separation From The Dermis, The Epidermis Was Digested With Trypsin To Obtain A Single Cell Suspension. Compared With The Conventional Procedure, This Isolation Method Was Shorter And Resulted In (I) Cells Displaying A Higher Colony Forming Efficiency, (Ii) Cells Reaching Confluence 1-3 Days Earlier, (Iii) Cells Not Contaminated By Fibroblasts, (Iv) A Cell Population Containing All The Basal Layer Keratinocytes. These Cells Were Suitable For The Establishment Of Primary Cultures And Could Be Subcultured. Such Cell Populations Should Be Advantageous In Studies Of Epithelial-Mesenchymal Interactions In Which Keratinocyte Populations, Free Of Fibroblasts, Are Desirable. In The Treatment Of Extensively Burned Patients Using Cultured Epidermal Sheets, The Main Problem Remains The Time Required For Their Production. Thus, The Absence Of Fibroblast Overgrowth Of The Keratinocyte Cultures And The Significantly Reduced Time To Obtain Confluent Cultures And Epidermal Sheets With Our Method Have Very Important Implications For The Treatment Of Large Burn Wounds. Auteur(s) : L'Heureux N., Germain L., Labbé R., Auger F.A. Titre : In vitro construction of a human blood vessel from cultured vascular cells: a morphologic study Référence : Journal of Vascular Surgery 17(3): 499-509, 1993. Lien : Cliquer ici Résumé : Urpose: The Purpose Of This Study Was To Create A Tubular Vascular Model Exclusively Made Of Human Cells And Collagen. Methods: The Blood Vessel Equivalent Was Constructed With The Three Following Human Cell Types: Vascular Smooth Muscle Cells, Endothelial Cells, And Fibroblasts. A Tissuelike Structure Was Obtained From The Contraction Of A Tubular Collagen Gel (Human Origin) By Vascular Smooth Muscle Cells, Which Created A Media-Like Structure. An Adventitia-Like Tissue Was Added Around The Media-Like Structure By Embedding Fibroblasts Into A Collagen Gel. An Endothelium Was Established Within The Tubular Structure After Intraluminal Cell Seeding. Results: Cell Orientation And Gel Contraction Were Followed Up Over Time. Vascular Smooth Muscle Cells Developed A Complex Tridimensional Network And Were Oriented In A Circular Fashion Around The Tube'S Axis. In Contrast, Fibroblasts Were Randomly Oriented. A Viable, Homogeneous, And Well-Characterized Endothelium Was Observed. These Endothelial Cells Showed A Slightly Elongated Structure And Were Oriented Parallel To This Vascular Equivalent Axis. Conclusion: An In Vitro Tridimensional Vascular Model That Exhibits Some Phenotypic Characteristics Of In Vivo Vascular Cells Could Be Useful In The Study Of Events That Lead To Atherosclerotic Plaque Formations. Auteur(s) : Michel M., Auger F.A., Germain L. Titre : Anchored skin equivalent cultured in vitro: a new tool for percutaneous absorption studies Référence : In Vitro Cellular and Developmental Biology 29A: 834-837, 1993. Lien : Cliquer ici Auteur(s) : Rouabhia M., Germain L., Bélanger F., Auger F.A. Titre : Cultured epithelium allografts: Langerhans cell and thy-1+ dendritic epidermal cell depletion effects on allograft rejection Référence : Transplantation 56(2): 259-264, 1993. Lien : Cliquer ici Résumé : The Effects Of In Vitro Dendritic Cell (Dc) Depletion On The Survival Of Epidermal Sheet Allografts Were Studied In A Murine Model. Newborn (1-3 Days Old) Mouse Skin Was Used. Langerhans Cell (Lc) And Thy-1+ Dendritic Epidermal Cell (Thy-1+ Dec) Depletion Was Achieved Using: (1) A Prolonged Culture Period (7 Days), Or (2) The Anti-Ia And Anti-Thy-1.2 Mabs Followed By Complement Treatment. Dc (Lc And Thy-1+ Dec) Depletion Was Assessed On Sheets And Cultured Cell Suspensions By An Indirect Immunofluorescence Procedure. They Showed That, After 7 Days Of Culture Or After The Antibody-Complement Treatment, Epidermal Cultures Were Depleted Of Lc And Thy-1+ Dec. Cultured Sheets Were Grafted Onto The Muscle Of H-2-Incompatible Recipients. The Control Experiments Were: (1) Full Epidermis And Dc Undepleted Sheet Allografts, And (2) Dc Depleted And Undepleted Sheet Isografts. The Full Epidermis Was Totally Rejected After 9 Days. However, No Rejection Sign Was Ever Seen In Any Of The Isografts. The In Vitro Produced And Allografted Epithelia Did Not Show Any Necrotic Sign Until The 11Th Day Postgrafting. However, On Days 12-13, The Dc Depleted Allograft'S Color Changed From Pink To Brown. On Days 14-16, Degradation Of The Allografts Resulted In A Complete Denudation Of The Underlying Muscle. Immunohistological Analysis Of The Allografts Revealed The Presence Of A Monocyte And Lymphocyte Infiltration Starting From The 11Th Day Postgrafting, With The Presence Of Polymorphonuclear Leukocytes On Day 14. These Results Suggest That Lc And Thy-1+ Dec Depletions Were Not Sufficient To Prevent Allograft Rejection. 1992 Auteur(s) : Bouvard V., Germain L., Rompré P., Roy B., Auger F.A. Titre : Influence of dermal equivalent maturation on the development of a cultured skin equivalent Référence : Biochemistry and Cell Biology 70: 34-42, 1992. Lien : Cliquer ici Résumé : Histologic And Immunofluorescence Methods Were Used To Analyse The Presence Of Fibronectin, Chondroitin-4-Sulphate And Chondroitin-6-Sulphate, Type Iii And Iv Collagens, Laminin, And Keratins To Assess The Maturation Level Of Cultured Dermal And Skin Equivalents. In A First Phase, Fibroblasts In Monolayer Culture Were Compared With Dermal Equivalents In Which Fibroblasts Are Embedded In A Type I Collagen Gel. Different Fluorescent Patterns Were Observed Depending On The Culture System Used. A Sequential Appearance Of Macromolecules Was Noticed In Dermal Equivalents. Fibronectin Was First Detected After 4 Days Of Culture, Whereas Chondroitin-4-Sulphate And Chondroitin-6-Sulphate And Type Iii Collagen Were Present After 7 Days. In Contrast, All Three Macromolecules Were Detected At 24 H Of Culture In Fibroblastic Monolayer Cultures. In A Second Phase, The Quality Of Our Skin Equivalents Was Evaluated According To The Seeding Time Of Epidermal Cells Upon Dermal Equivalents (1, 4, Or 7 Days). A Satisfactory Stratification Was Obtained When Keratinocytes Were Seeded After 4 And 7 Days Of Dermal Equivalent Culture. Laminin And Fibronectin Were Detected At The Dermo-Epidermal Junction, But Type Iv Collagen Was Absent. Various Keratins, As Detected By The Ae1, Ae2, And Ae3 Antibodies, Were Present In The Epidermal Layer. Following Keratinocyte Confluence, A Change In The Organization Pattern Of Type Iii Collagen In The Dermal Fraction Of The Skin Equivalent Was Also Noticed. Our Comparative Results Show That Seeding Of Epidermal Cells On A More Mature Dermal Equivalent Leads To Improved Differentiation Status Of The Epidermal Layer. Auteur(s) : López Valle C.A., Auger F.A., Rompré P., Bouvard V., Germain L. Titre : Peripheral anchorage of dermal equivalents Référence : British Journal of Dermatology 127: 365-371, 1992. Lien : Cliquer ici Résumé : Human Fibroblasts Can Induce Collagen Gel Contraction With Different Kinetics Depending On The Number Of Cells And On The Collagen Concentration Within This Lattice, Which Has Been Considered As A Dermal Equivalent. Skin Equivalent Is A Combined Culture Of Dermo-Epidermal Layers Which May Be Of Therapeutic Value In The Treatment Of Burn Patients. However, The Current Production Of The Dermal Equivalent Component Gives Results That Present Many Drawbacks For Their Eventual Clinical Use As A First Step In Obtaining A Skin Equivalent. These Include: (I) Final Surfaces Which Are Very Small; Less Than 20% Of The Initial Size (Ii) Excessive Thickness Which May Hamper Successful Graft Take (Iii) Fibroblasts That Do Not Have An Arrangement Comparable With Normal Dermal Tissue. We Propose, As A Solution To These Problems, The Utilization Of A 5-Mm-Wide Fibre-Glass Filter Ring Peripherally Attached To The Surface Of The Petri Dishes To Prevent Inordinate Contraction While The Fibroblasts Reorganize The Collagen Gel. Using This Technique The Initial Surface Was Preserved And The Dermal Equivalent Contracted Only In Thickness. Histological Analysis Of These Anchored Equivalents Confirmed An Alignment Of Fibroblasts And Collagen Fibres Resembling Normal Dermal Tissue. We Consider This Method Useful In The Development Of Dermo-Epidermal Sheets For Clinical Purposes. Auteur(s) : López Valle C.A., Germain L., Auger F.A. Titre : Modèle chirurgical murin pour l'étude des greffons cultivés Référence : Annales de Chirurgie 46(9): 845-850, 1992. Lien : Cliquer ici Résumé : The Objective Of The Study Was To Establish An Animal Model For In Vivo Studies Of Cultured Cutaneous Equivalents. The Model On Athymic Mice That We Already Described (López-Valle C.A. Et Al., Plast Reconstr Surg, 1992, 89, 139-143) Satisfied The Criteria Of Immobilization Of The Recipient Site And Physical Stability Of The Graft, But Still Allowing Complete Movement Freedom Of The Animal Used. Nevertheless, This Technique Encountered Two Long Term Weaknesses; A) A Significant Grafted Surface Reduction Caused By The Wound Contraction And B) The Absence Of A Physical Barrier Between Human And Murin Keratinocytes. We Propose Some Modifications To This Technique To Correct Both Problems. Moreover, The Implantation Of Polypropylene, Instead Of Glass Pellets, To Generate Granulation Tissue On The Recipient Bed When Needed, Constitutes An Easier Method For Both The Surgeon And The Animal. Finally, In Order To Minimize Wound Care, Some Modifications Were Made To The Rodent Cages. Immunohistological Analyses Of The Biopsies, 21 Days Post-Grafting, Revealed A Continuous Basal Membrane. This Animal Model Allows In Vivo Studies On The Behavior, Cicatrization And Immunology Of Human Cultured Skin Equivalents. Auteur(s) : López Valle C.A., Glaude P., Auger F.A. Titre : Surgical model for in vivo evaluation of cultured epidermal sheets in mice Référence : Plastic and Reconstructive Surgery 89(1): 139-143, 1992. Lien : Cliquer ici Résumé : He Objective Of This Study Was To Establish A Novel Surgical Model For The Grafting Of Cultured Epidermal Sheets In Mice In Order To Optimize Studies On The Behavior Of These Grafts. Graft-Related Skin Immunology And Wound-Healing Studies Also Would Benefit From This Adapted Surgical Approach. Adapted Tie-Over Surgical Procedures Were Established For Mice And Promptly Applied. Early-Stage Observation Of Grafts Was Made Possible By Replacing The Cotton Dressing With A Saddle-Like Plastic Tube Dressing With A Screw Cap. We Grafted Normal Balb/C Mice And Athymic Nude (Nu/Nu) Mice With Cultured Human Epidermis. Evaluation Of Graft Rejection Was Carried Out With The First Group, Whereas The Second Provided Information On Epidermal Stratification And Terminal Differentiation. This Innovation Permitted Direct Evaluation Of The Grafted Tissue At Any Time. Advances In Applied Transplantation Research Will Certainly Provide Additional Tools For Human Applications. Auteur(s) : Paradis D., Vallée F., Allard S., Bisson C., Daviau N., Drapeau C., Auger F.A., Lebel M. Titre : Comparative study of pharmacokinetics and serum bactericidal activities of cefpirome, ceftazidime, ceftriaxone, imipenem and ciprofloxacin Référence : Antimicrobial Agents and Chemotherapy 36(10): 2085-2092, 1992. Lien : Cliquer ici Résumé : We Compared The Pharmacokinetics And The Serum Bactericidal Activities Of Cefpirome, Ceftazidime, Ceftriaxone, Imipenem, And Ciprofloxacin. Fifteen Healthy Volunteers Received 1 G Of Cefpirome, Ceftazidime, And Ceftriaxone Intravenously, 500 Mg Of Imipenem-Cilastatin Intravenously, And 500 Mg Of Ciprofloxacin Orally. High-Performance Liquid Chromatographic Assays Were Used To Quantitate Unchanged Antibiotic In Plasma And Urine. Serum Bactericidal Activities Were Determined Against Six Clinical Isolates Each Of Staphylococcus Aureus, Enterobacter Cloacae, And Pseudomonas Aeruginosa By Using A Modified Microdilution Method Of Reller And Stratton (L. B. Reller And C. W. Stratton, J. Infect. Dis. 136:196-204, 1977). Overall, Cefpirome Exhibited Pharmacokinetics Similar To Those Of Ceftazidime: Half-Life (T1/2), 1.95 H; Concentration At 1 H (C1H), 47 To 49 Micrograms/Ml For Both Antibiotics. Ceftriaxone Displayed The Longest T1/2 (7.65 H) And The Highest C1H (137.8 Micrograms/Ml), While We Observed The Shortest T1/2 (1.05 H) And The Lowest C1H (19.85 Micrograms/Ml) With Imipenem. At 1 H, Cefpirome And, Even More So, Imipenem Showed Significantly Better Serum Bactericidal Activities Against S. Aureus (1:273 And 1:80) Than Did The Other Antibiotics (P Less Than 0.0005; Analysis Of Variance With Randomized Block Design And Bonferroni Correction). Against E. Cloacae, We Observed The Highest Serum Bactericidal Titers At 1 H With Cefpirome, And This Superiority Vis-À-Vis The Other Antibiotics Tested Was Maintained For Up To 8 H After Dosing. Ceftazidime Remained The Most Active Agent Tested Against P. Aeruginosa (Serum Bactericidal Activity Titers, 1:43 At 1 H) Up To 8 H. In Summary, The Study Showed That Cefpirome And Imipenem Provide More Potent Serum Bactericidal Activities Than Do Broad-Spectrum Cephalosporins Against S. Aureus; Thus, Both Of These Antibiotics Should Be Adequate Against Serious S. Aureus Infections. In Addition, Cefpirome Appears To Be A Promising Alternative For Treatment Of Infections Caused By E. Cloacae And P. Aeruginosa. Auteur(s) : Rouabhia M., Germain L., Bélanger F., Guignard R., Auger F.A. Titre : Optimization of murine keratinocyte culture for the production of graftable epidermal sheets Référence : The Journal of Dermatology 19(6): 325-334, 1992. Lien : Cliquer ici Résumé : The Aim Of The Present Study Was To Optimize Murine Epidermal Cell Cultures In Order To Obtain Graftable Sheets. Newborn (1-3 Days Old) Balb/C Mice Skin Were Used To Optimize Culture Media And Plating Cell Concentration, Then Epidermal Sheet Production, And Grafting. Epidermal Cells Were Plated At Various Concentrations In Different Culture Media Containing Low (0.1 Mm) Or High (Greater Than 1 Mm) Ca2+ Levels. After A 3 Day Culture At The 10(4) Cells/Cm2 Plating Cell Concentration, The Percentage Of Differentiated Cells Was More Than 80% In The High Ca2+ Culture Medium And Less Than 50% With Bulky Cells In The Low Ca2+ Culture Medium. Under These Conditions Confluence Was Not Obtained. At The 10(5) Cells/Cm2 Seeding Inoculum, The Percentage Of Confluence Increased To 95-100% During The First 72 H Of Culture In Both High And Low Ca2+ Culture Media. Three-Day-Old Culture Showed Stratified Multilayer Epidermal Sheets In The High Calcium Medium, And Monolayer Epidermal Sheets Were Present In The Low Calcium Medium After Seeding Keratinocytes In Fibronectin Precoated Flasks. Seven Days After Plating, Post Confluent Cultures Were Composed Of A High Percentage Of Differentiated Cells (90%) With An Increase In Shedding Cells In The Medium. Considering The Above Morphological Observations, Sheets Obtained With 10(5) Cells/Cm2 In Mcdb-153 (A), Dme-Ham (B) Or Gmem (C) Media After 3 Days In Culture Were Grafted. Twenty Days After Grafting, Histological Analysis Of Biopsies Showed An Epidermal Structure And Organization Comparable To Normal Murine Epidermis Without Hair Follicles. Epidermal Transplants Showed A Complete Basement Membrane, Hemidesmosomes, And Tonofilament Bundles. Sheets Obtained After Seven Day Culture In All Media Showed Lower Coverage Of The Wound Bed. These Studies Point Out The Importance Of The Plating Cell And Ca2+ Concentrations, And The Culture Time For Murine Keratinocyte Confluence And Differentiation To Obtain Graftable Epidermal Sheets. Auteur(s) : Rowden G., Colp P., Dean S., Auger F.A., López Valle C.A. Titre : Comparative epidermal Langerhans cell migration studies in epidermal and epidermal/dermal equivalent grafts Référence : The Journal of Investigative Dermatology 99(5)S: 59-61, 1992. Lien : Cliquer ici Résumé : Immigration Of Langerhans Cell Precursors From The Peripheral Blood To The Skin Was Studied In Human Grafts Placed On Severe Combined Immunodeficient (Scid) Mice. Monocyte Fractions Of Human Blood Were Injected Intraperitoneally To Scid Bearing Either Reconstituted (Langerhans Cell Free) Epidermal Sheets (E) Or Living Skin Equivalents (E/D) Consisting Of Both Epidermis And Dermis. A Range Of Immunocytochemical And Ultrastructural Markers Was Employed To Monitor The Colonization Of The Grafts, I.E., Cd1A/C, Birbeck Granules. In Situ Hybridization With Probes Against Alu Sequences Of Human Dna Were Employed Together With Immunostaining For Mhc Class I Mouse And Human Antigens To Document Graft Survival. Although Unequivocal Lc Were Detected Within E Grafts, Including Both Human (Cd1A Positive) And Murine (Nldc-145 Positive), No Migration Was Achieved In The E/D Situations. 1990 Auteur(s) : Rompré P., Auger F.A., Germain L., Bouvard V., López Valle C.A., Thibault J., LeDuy A. Titre : Influence of initial collagen and cellular concentrations on the final surface area of dermal and skin equivalents: A Box-Behnken analysis Référence : In Vitro Cellular and Developmental Biololgy 26: 983-990, 1990. Lien : Cliquer ici Résumé : Our Laboratory Has Been Involved In Finding Optimal Conditions For Producing Dermal And Skin Equivalents. As An Original Approach, A Box-Behnken Experimental Design Was Used To Study The Effects Of The Initial Collagen And Fibroblast Concentrations And The Initial Gel Thickness On The Contraction Of Dermal And Skin Equivalents. The Final Surface Area Of Dermal Equivalent Varied Significantly With The Initial Concentration Of Collagen And Fibroblast, Whereas The Initial Thickness Of Gel Had No Appreciable Effect On The Contraction Of The Dermal Equivalent. When Keratinocytes Were Grown On These Dermal Equivalents They Produced A Very Severe Contraction, To An Extent That All Skin Equivalents Had A Similar Final Surface Area. This Severe Contraction Was Independent Of Collagen And Fibroblast Concentrations. Models For The Prediction Of The Final Percentage Contraction Of Dermal And Skin Equivalents As A Function Of The Initial Concentration Of Collagen, The Logarithm Of Fibroblast Concentration, And The Initial Gel Thickness Were Obtained And Analyzed. Keratinocytes Grown At The Lowest Seeding Density Did Not Contract The Equivalents. However, Histologic Analysis Has Shown An Incomplete Coverage By These Cells Of The Equivalents. The Extensive Contraction Of The Skin Equivalent Presenting Adequate Morphology Is A Major Drawback Toward Its Clinical Utilization For Burn Wound Coverage. 1988 Auteur(s) : Auger F.A. Titre : The role of cultured autologous human epithelium in large burn wound treatment Référence : Transplantation/Implantation Today 5: 21-24

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