| Deep skin wounds of large area caused by trauma should be treated in clinic with coverage of skin equivalent. Over the last 25 years, tissue engineered skin has developed and progressed at a very rapid rate. Recent evidence indicates that bone marrow contains stem cells with the potential for differentiation into a variety of tissues, including endothelium, liver, muscle, bone, and skin. It may thus be plausible that bone marrow–derived cells can provide progenitor and/or stem cells to wounds during healing. Tissue engineering is restricted by three key agents: seeding cells, scaffold and extracelluar matrix. Among them, the seeding cells, which should have proliferation capacity, long-term biological properts, low antigenicities and strong tissue repair abilities, have always been in the key point. As their characteristics according with those of the seeding cells in tissue engineering, bone marrow-derived mesenchymal stem cells have been used in tissue engineered bone extensively. But despite the great interest in mesenchymal stem cells(MSCs), there is still no well-defined protocol for using of the cells in tissue engineered skin. Can MSCs be used in tissue engineered skin? Can tissue engineered skin made of MSCs as seeding cells promote the healing of deep skin wounds? It is in this vein that our study was conceived. In the repot, we isolated and purified MSCs by the use of a unique method that included a specially designed culture device and transfected MSCs with EGF sequences. For using the MSCs in tissue engineered skin, we also made skin scaffold by using tissue engineering techniques to integrate collagen, chitosan, 6-chondroitin sulfate and other glycosaminoglycans. And the deep skin wound was coved by tissue engineered skin which made by MSCs, scaffold and keratinocytes. The results indicated that the tissue engineered skin could promote the healing of deep skin wound significantly, the healing wound had intact structures of dermis and epidermis, and the tissue engineered skin could repair deep skin wounds much more rapidly by using MSCs transfected by EGF sequences. The main results of the present report are as following:1. MSCs were isolated from human bone marrow aspirates by the use of a unique method that included a specially designed culture device, which was a plastic culture dish comprising a plate with 3-μm pores to sieve out MSCs from bone marrow aspirates. 2. MSCs isolated using this method are a homogeneous population as indicated by morphology and other criteria, such as surface markers. They have the capacity for self-renewal, proliferation, the multilineage potential to differentiate into osteogenic and adipogenic lineages, and satisfy the characteristics of MSCs. 3. MSCs isolated using this method also have the higher ability to differentiate into osteogenic and adipogenic lineages than the cells isolated by the method of gravity gradient centrifugation in the later stage after induction.4. Porous scaffolds for tissue engineered skin were fabricated by freeze-drying the mixture of collagen, glycosaminoglycans and chitosan solutions. And the scaffolds also are coincidence with the biomaterial criteria of collagen porous membrane.5. In vitro culture of MSCs proved that the scaffolds could retain the original good cytocompatibility of collagen to effectively maintain cell infiltration and proliferation.6. The human epidermal growth factor sequences was cloned into vector pcDNA3.1 accurately by the gene manipulation. MSCs transfected with EGF sequences could release EGF up to 1 ng/ml in vitro.7. MSCs transfected with EGF sequences maintained the capacity for self-renewal, proliferation, the multilineage potential to differentiate into osteogenic and adipogenic lineages, and satisfied the characteristics of MSCs. And EGF could promote MSCs proliferation significantly.8. The tissue engineered skin composed of both MSCs and keratinocytes seeding in the scaffold would be ideal. It had capacity for surgery and no influence of immunological function was found in r... |
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