| The repair of soft tissue (skin and fat) defects after trauma, infection, diabetic ulcers, and tumor resection, has long been a problem for clinicians. Current methods of clinical treatment have a variety of shortcomings. With the development of tissue engineering and stem cell technology, the application of tissue-engineered skin and tissue-engineered fat by biological materials and stem cells is the most promising approach. Adipose tissue-derived stem cells is adult mesenchymal stem cells, which has self-renewal and multilineage differentiation capacity. Adipose tissue-derived stem cells sufficient for its origin, drawing convenience, less trauma, no rejection, proliferative ability, and can be amplified by a large number of training, etc., is recognized as an ideal source of seed cells for tissue engineering. Firstly, we isolated adipose tissue-derived stem cells from SD rat, and demonstrated that they promote wound healing in rat skin defect model. To explore the role of adipose tissue-derived stem cells in the preparation of tissue-engineered skin, we isolated human adipose tissue-derived stem cells, and mixed them with dermal fibroblast cells to mimic the microenvironment of natural dermis. Furthermore, tissue-engineered fat was reconstructed by human adipose tissue-derived stem cells with injectable gelatin microspheres. In our study, tissue-engineered skin and tissue-engineered fat were reconstructed by adipose tissue-derived stem cells and biomaterials, which can be used in soft tissue defects repair. The contents of the present study are as follows:1 The role of adipose tissue-derived stem cells in the wound healing of skinObjective: To culture the rat adipose tissue-derived stem cells in vitro and explore biological characteristics, then seed them into the collagen gel to make tissue engineered dermis and evaluate the effect on skin defect repair and to investigate the feasibility of ADSCs as the seed cells for tissue-engineered skin. Methods: Adipose tissues were isolated from a SD rat groin. The fat was digested in a collagenase solution. The ADSCs were detected for cell growth characteristics by MTT and identified by flow cytometry and analyzed for their multilineage differentiation. The amplified cells marked with fluorescence were inoculated in collagen gel to form tissue engineered dermis. Then the tissue engineered dermis was used to repair the skin defect. The wound repair was randomly divided into three groups: adipose tissue-derived stem cells with collagen transplantation group (ADSC+Col), pure collagen transplantation group (Col), the blank control group (Cont.). The effect of wound repair was observed by histological methods and time of wound healing. Results: The cultivated cells have the ability to proliferate continuously and were expressed of mesenchymal stem cell surface markers. They can differentiate into several cell lineages such as fat, bone and nerves. In in vivo experiment, the time of wound healing in the ADSCs with collagen transplantation group, pure collagen transplantation group and the blank control group were (14.3±1.7) days, (16.9±2.5) days and (21.2±4.2) days respectively, which have significant differences (P<0.05). Histological observation showed that dermal fibroblasts and collagen matrix grew into the bed of wounds in adipose tissue-derived stem cells with collagen transplantation group. Furthermore, epidermis covered the wound and spikes-like structure formed. However there were no hair follicles, sweat glands and other skin appendages in that group. In pure collagen transplantation group, epidermis did not cover the wound completely. In blank control group, there were enrich microvasculars in the dermis but the epithelial crawling was slow. Two weeks after transplantation, adipose tissue-derived stem cells marked with Hoechst33342 were detected in the ADSCs with collagen transplantation group. Conclusions: The method of isolation and cultivation was successfully established and can be used for further research. Tissue engineered dermis reconstructed by adipose tissue-derived stem cells with collagen, can repair skin defects and promote wound healing. It is suggested that ADSCs is an ideal seed cell for tissue-engineered skin reconstruction.2 The role of human adipose tissue-derived stem cells in tissue- engineered skin preparationObjective: To isolate human adipose tissue-derived stem cells in vitro and explore their biological characteristics. Many studies demonstrate that single mesenchymal cell type can affect the epidermal morphogenesis of bilayered tissue-engineered skin. However, whether the mixture of different mesenchymal cell types can improve the epidermal morphogenesis of bioengineered skin remains unknown. To explore the role of mixed adipose tissue-derived stem cells and dermal fibroblasts on epidermal morphogenesis. Methods: Adipose tissues were isolated from human and digested in a collagenase solution, human adipose tissue-derived stem cells were obtained by low-density seeding. The cells were identified by flow cytometry and analyzed for their multilineage differentiation. keratinocytes, dermal fibroblasts and adipose tissue-derived stem cells (ADSCs) were respectively isolated and purified from human skin and subcutaneous fat. The effect of conditioned medium (CM) generated from the mixture of dermal fibroblasts and ADSCs at the ratio of 1:1 on keratinocyte proliferation was analyzed by MTT assay. Furthermore, cytokine levels of human hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF, also known as FGF7) in mixed fibroblasts/ADSCs group were detected by enzyme-linked immunosorbent assay (ELISA). To examine the potential roles of mixed fibroblasts and ADSCs on epidermal morphogenesis, a three-dimensional tissue-engineered skin system was applied. Epidermal morphogenesis was analyzed by histological, histomorphometric and transmission electron microscopy methods. Results: The isolated human adipose tissue-derived stem cells have self-renewal capacity, and expressed mesenchymal stem cell surface markers. They can differentiate into several cell lineages such as fat, bone and cartilage. Conditioned medium (CM) generated from the mixture of dermal fibroblasts and ADSCs at the ratio of 1:1 was superior to that from fibroblasts or ADSCs alone in promoting the keratinocyte proliferation, as indicated by MTT assay. Furthermore, ELISA results showed that the cytokine levels of human hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF, also known as FGF7) in mixed fibroblasts/ADSCs group was higher than those in ADSCs or dermal fibroblasts group. Histological analyses demonstrated that keratinocytes proliferated extensively over the mixture of fibroblasts and ADSCs, and formed a thick epidermal layer with well-differentiated structures. Keratin 10 (epidermal differentiation marker) was expressed in suprabasal layer of bilayered TE skin in the mixed fibroblasts and ADSCs group. Desmosomes and hemidesmosomes were detected in the newly formed epidermis by Transmission electron microscopy (TEM) analysis. Conclusion: These findings revealed for the first time that the mixture of fibroblasts and ADSCs in the bilayered TE-skin can improve the epidermal morphogenesis.3 Tissue-engineered fat reconstructed by human adipose tissue- derived stem cells and biodegradable gelatin microspheresObjective: To evaluate the effect of transplantation of human adipose tissue-derived stem cells (hADSCs) and EDC cross-linked gelatin microspheres cultured in three-dimensional conditions by bioreactor for adipose tissue regeneration. Methods: Gelatin microspheres were prepared and detected the cell and tissue compatibility. PKH26 labeled human adipose tissue-derived stem cells combined with gelatin microspheres were cultured in rotating culture system (RCCS). Adipogenesis was examined in nude mice injected subcutaneously with hADSCs and gelatin microspheres cultured in adipogenic medium for 7 days. After 4 weeks, survival cells and newly formed adipose tissue were observed by fluorescent microscope and Oil red O staining. Results: EDC cross-linked gelatin microspheres were spherical and uniform size, the particle size was 75μm-150μm. Adipose tissue-derived stem cells grew well on the microspheres. There was no significant inflammatory reaction on the site of microspheres implanted. Adipose tissue-derived stem cells grew well on the surface of gelatin microspheres in rotating culture system, after adipogenic seven days in vitro, fat cell related genes PPAR-γand C/EBPαwere detected in the mRNA levels. Adipogenesis was examined in nude mice injected subcutaneously with hADSCs attached gelatin microspheres cultured in adipogenic medium for 7 days and control (without induction). After 4 weeks, newly formed adipose tissue was observed in hADSCs attached gelatin microspheres cultured in adipogenic medium for 7 days group. Oil red O staining of newly formed tissue showed that there was substantially more fat tissue regeneration. PKH26 labeled adipose tissue-derived stem cells can be detected in both groups with or without adipogenic induction. Conclusion: Gelatin is a good biocompatibility, biocompatible and biodegradable natural polymer materials. Tissue-engineered fat reconstructed by human adipose tissue-derived stem cells and biodegradable gelatin microspheres provides significant evidence that hADSCs and gelatin microspheres can be applied as a promising soft tissue filler to generate adipose tissue for clinical use.
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