The Primary Study Of Keratinocyte Influence On Contraction Of Composite Chitosan Tissue Engineered Skin Equivalent

Objective:1. To investigate the relationship between myofibroblast and contraction of composite chitosan tissue engineered skin equivalent in vitro. 2. To investigate the influence of the cross-reaction between keratinocyte and composite chitosan tissue engineered dermal equivalent on the contraction of composite chitosan tissue engineered skin equivalent in vitro, and the possible way.Methods:1. Establish a co-culture equipment with millipore filter and two chambers, and measure area changes of three kind of skin equivalents during the culture in vitro. The skin equivalents include composite chitosan tissue engineered skin equivalent, composite chitosan tissue engineered dermal equivalent and the composite chitosan tissue engineered dermal equivalent co-cultured with keratinocyte without direct contact. And then detect the area chang of those three skin equivalents when lost the cells in them. 2. When those skin equivalents have been contracted completely,to observe the micro morphology and Alpha-smooth muscle actin(α-SMA) immunostaining in order to find the difference among these three skin equivalents. 3. Collect culture solution at different time point of these three kind of skin equivalents and monolayer keratinocyte before and after culture, and detect basic fibroblast growth factor(bFGF), epidermal growth factor(EGF), platelet-derived growth factor-BB(PDGF-BB),transform growth factor-β1(TGF-β1) by double antibody method enzyme-linked immunosorbent assay(ELISA).Results:1. The co-culture equipment allows the protein with molecular weight below 80KD pass through freely. 2. When cultured in vitro, composite chitosan tissue engineered skin equivalent and the composite chitosan tissue engineered dermal equivalent co-cultured with it reach the same area in the end, and the composite chitosan tissue engineered dermal equivalent cultured alone doesn't contract as severe as former two. All of their areas won't chang when cells are lost. 3. A few fibroblasts(Fb) adjacent to epidermis are stained byα-SMA immunostaining in composite chitosan tissue engineered skin equivalent, and negative in the other. 4. Growth factors have been shown to decrease as a whole during culture,and there are differences among composite chitosan tissue engineered skin equivalent, composite chitosan tissue engineered dermal equivalent and the composite chitosan tissue engineered dermal equivalent co-cultured with keratinocyte without direct contact. Besides, concentrations of the growth factors are not at equal pace with skin equivalents'contraction completely.Conclusion: Myofibroblast have nothing with the contraction of composite chitosan tissue engineered skin equivalent being in culture in vitro. When composite chitosan tissue engineered skin equivalent has contracted completely, the cell of it behaves no tensile strength for contraction. Keratinocyte can enhance composite chitosan tissue engineered dermal equivalent's contraction by diffusion factor and direct contact.