Preparation Of Tissue-engineered Skin Scaffolds And Study Of Full-thickness Skin Construction

Tissue engineered skin has great applications in clinical skin repairing,chemical safety,toxicity and efficacy testing and skin disease research area.In particular,the promotion of animal substitution testing in Europe and American has caused trade barriers and affected Chinese economic development.There is an urgent need to develop effective and simple methods to construct tissue engineered skin with complete tissue structure and function that are more similar to human skin.The technical points that used to construct tissue engineered skin mainly involve the type of cells,materials,preparation of scaffolds and methods for tissue development.Whether fibroblasts can distribute uniform,proliferate and be living in dermis equivalents,keratinocytes can proliferate and differentiate in scaffolds,the structure and function of the skin tissue are complete,all are the key to evaluate the success of full-thickness skin equivalents(FT-SEs)construction.Based on the advantages of great biocompatibility and low cytotoxicity of natural materials,we used sodium alginate,chitosan,collagen,gelatin and fibrin as scaffold materials to construct FT-SEs.Firstly,sodium alginate-chitosan porous scaffolds A1,A2 and A3 whose volume ratio of sodium alginate were 100%,60% and20%,respectively and collagen-chitosan scaffolds B1,B2,and B3 which were cross-linked by 0.25% glutaraldehyde,0.02 M EDC/NHS and lyophilizated twice,respectively and gelatin porous scaffold C1 which were thermal cross-linked,all were obtained by freeze-drying.We compared the water absorption,porosity and structure of scaffolds and three-dimensional spatial distribution and proliferation of fibroblasts seeded on scaffolds among these porous scaffolds.Because fibroblasts can be evenly distributed and proliferated in gelatin scaffold C1 whose water absorption and porosity were higher than 80% and 90%,respectively and pore size were between 70 and 150 ?m.Gelatin porous scaffold C1 has the best performance among these scaffolds so we used it to construct FT-SEs.Finally we successfully obtained FT-SEs which has complete epidermis and dermis structure and can normally express the epidermal marker keratin 10.The full and epidermal thickness of FT-SEs was1000-1100?m and 50-250 ? m,respectively.Since the porous scaffold obtained by freeze-drying requires the fibroblasts generate the extracellular matrix to fill the porous structure and then keratinocytes can be seeded to generate epidermis during the construction of FT-SEs.It will take a long period time to construct FT-SEs.What's more,fibroblasts always difficult to distribute evenly in the thicker porous scaffolds.Based on the proliferation and activity test results of fibroblasts cultured in different concentrations of fibrin hydrogel and its mechanical properties,we finally used 5 mg/mL fibrin hydrogel to construct FT-SEs and obtained the full-thickness skin equivalents with complete epidermal and dermal structure.And keratin 10 expression has increased from day 7to day 14.Finally,we used fibrin gel and the gelatin fiber scaffold that keratinocytes can effectively adhere,proliferate and differentiate in and fabricated by electrospinning to construct FT-SEs in PEGDA tissue culture well.A fluidic environment was constructed using a microfluidic chip and an automated culture system to simulate the dynamic environment of nutrient supply and metabolite removal in vivo.We compared the tissue structure,activity and barrier function of FT-SEs obtained by dynamic culture system and the traditional static culture system.The experimental results showed that after 8 days of cultivation,both culture methods were able to obtain FT-SEs with complete epidermal and dermal structure.And both tissues had great tissue activity and living cells in dermis at day 6.The tissue that obtained by dynamic culture system had less dead cells than static culture system,but the thickness is relatively thin.From the skin barrier test,it can be seen that compared with the first day of FT-SEs,both resistance value significantly increased at day 6,it means that whatever culture methods we use,we all can obtain FT-SEs with certain skin barrier function.In this study,FT-SEs constructed by different types of scaffolds all have complete epidermal and dermal structure and strong applications potential.Further research can be conducted on the application of such directions as skin regeneration and repair,skin disease research,drug screening,and cosmetic toxicology andefficacy evaluation.Using fibrin gel as a scaffold to construct FT-SEs provides favorable conditions for the improvement of the structure and function of FT-SEs.The FT-SEs constructed under dynamic environment can further enhance tissue engineering skin culture environment and also provide an effective technical support for skin-related multi-organ co-culture research.