Construction Of Tissue-engineered Skin Containing Bone Marrow Mesenchymal Stem Cells And Improvement For Skin Wound Healing

Stem cells can renew themselves for long periods through cell division, proliferate rapidly and have the remarkable potential to develop into many different cell types in the body. Bone marrow mesenchymal stem cells(BMMSCs) are able to differentiate into cells of mesenchymal lineage such as osteoblasts, chondrocytes, myoblasts, adipocytes or ligament cells. BMMSCs are self-renewing and are potent in differentiating into multiple cells and tissues. As the development of tissue engineering, BMMSCs are applying in cell therapy, gene therapy and are the cell sources of tissue engineering.In our study, keratinocytes, fibroblasts and bone marrow mesenchymal stem cells were isolated and cultured. An actue skin injury model and a burn wound model were eatablished in pigs. The tissue-engineered skin was used to repair the skin defects and the effects were observed.Part 1 Keratinocytes and fibroblasts were isolated and cultured from the back skin of the new-born pig. Biological conditions of cells were examined by MTT detection and BrdU analysis. We used Percoll separating medium by density gradient centrifugation to isolate pig BMMSCs and amplify in vitro. After several passages, the BMMSCs have the uniform morphology and grow stably in certain conditions, keep undifferentiated and have the potential of differentiation into other tissues. So these cells can be used as the seed cells of tissue-engineered skin.Part 2 TE skin of full thickness was similar with the human skin, it had two layers: dermis and epidermis. 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. BMMSCs skin containg of bone marrow mesenchymal stem cells and bovine I type collagen gel. The two types of skin are in biological condition and can be used to repair the skin defects.Part 3 An actue skin injury model and a burn wound model were eatablished in pigs. The two types of tissue-engineered skins were used to repair the skin defects. When grafted to the skin wounds, the effect of wound repair was observed. The tissue-engineered skin containing BMMSCs showed a better healing condition and keratinization and more vascularization. Grafts proliferated well and contributed to the neo-tissues by fluorescent microscopy and immunohistochemistry. These data suggest that tissue-engineered skin containing BMMSCs in a skin defect can accelerate wound healing and receive satisfactory effects.