Reconstruction Of A SD Rat Skin Equivalent By Tissue Engineering Method

Postgraduate student for Master degree: Wang Junlin1 Supervisors: Jin Yan11. Department of Oral Histology & Pathology. College of Stomatology, the Fourth Military Medical University, Xi'an, 710032Tissue engineering is the product of co-development and combination of modern cell biology, biomaterial science, and engineering. The aim of tissue engineering is to investigate and restore tissue and organ substitutes. In this study, a bi-layer SD rats skin was reconstructed in vitro by employing the method of tissue engineering.1. Isolation of keratinocytes, and dermal fibroblasts from new-born SD Rat skin: To develop a rapid and reproducible method for the isolation of the two populations of cells (fibroblasts and keratinocytes) from a SD rat skin biopsy, the keratinocytes, and dermal fibroblasts were isolated and cultured in six different methods. The biological conditions of the cells were examined by attaching efficiency, colony-forming efficiency, MTT detection and BrdU analysis. The ability of the cells to construct a skin replacement was also investigated. The results showed that thegreatest number of keratinocytes and dermal fibroblasts in good conditions can be attained rapidly by Dispase treating. Our results also demonstrated the importance of the incubation time and temperature in the recuperation of skin cells. They also showed the feasibility of isolating two cell types from a single skin biopsy, and culturing those cells to recreate an autologous reconstructed skin.2. Culture of SD rats keratinocytes in three different media: The keratinocytes were cultured in three different media, FAD, MCDB153(-), MCDB153(+). The proliferation ability was investigated by cell growth curve, MTT detection and FCM analysis. Results showed that the keratinocytes growed best in serum-free medium supplemented with EOF and BPE.3. Effects of EOF on the cell cycle of SD rats dermal fibroblasts: The dermal fibroblasts were cultured in DMEM containing 50mg ?L"1 EOF. The proliferation ability was investigated by MTT detection and FCM analysis. The expression of cyclinDl and CDK-4 was investigated by immunohistochemisty method. Results demonstrated that the dermal fibroblasts cultured in DMEM containing 50mg ?L"1 EOF were in better conditions than those cultured in DMEM without EOF. And EOF shortened the Gl phase of the cell cycle.4. The effect of different concentration collagens on rat fibroblasts using three-dimensional culture models: Fibroblasts of 1 ?2 day new born SD rat from back skin were isolated and seeded into 6.4mg/ml, 5.3mg/ml, 4mg/ml collagen, respectively. The status and numbers of fibroblasts were evaluated under light microscope. The number of fibroblasts was also analysized using t test. Results-7-showed that the appropriate concentration of collagen for the growth of fibroblasts was 6.4mg/ml.5. Construction of SD rats tissue-engineering skin of full thickness: keratinocytes and fibroblasts were isolated from the back skin of new-born SD rats. Fibroblasts were seeded into bovine I type collagen gel and cultured for 3 days. Keratinocytes were seeded on the surface of collagen gel and cultured for another 2 days, then expose the equivalent skin to air-liquid interface to generate a protective cornified layer. Results showed that tissue-engineering skin of full thickness was a fine biological living skin equivalent and can be used to repair the defects of full thickness skin.6. Study of bFGF and FN in tissue engineering skin allograft to rapair rat skin defection: To culture tissue engineering skin which draws the materials from neonatal SD rats, and graft it to adult Wistar rats and detected the expression of bFGF and FN of wound healing with immunohistochemistry staining. Results showed the changes of bFGF and FN maybe play a important rol...