| Backgroud :Low back pain is one of common and frequently occurring diseases in modern life with increasingly large heath-care and socioeconomic costs.it also the labor force lose.Disc degeneration diseases(DDD),such as lumbar disc herniation,are the major etiological factors. Surgical therapeutical approaches such as discectomy or spinal arthrodesis with fusion sacrifice IVD function and mobility and predispose the adjacent vertebral levels to faster degeneration.The ideal resolvement for the treatment degenerated disc diseases maybe is to develop the transplants to re-establish the structure and functions of degenerated discs.Tissue engineering is an emerging field in modern medicine. Therapies involve the combination of cells and scaffold materials that can be loaded with bioactive factors, ideally resulting in the regeneration or replacement of lost or damaged tissues and organs. The quick development and breakthrough of critical techniques are also displayed in nucleus pulposus tissue engineering. It is essential to select appropriate seed cells and scaffolds in tissue engineering. Multiple cell sources have been investigated for their possible applicability in nucleus pulposus tissue engineering. Although the lineage of the different cell types in the intervertebral disc is not known with certainty,it is agreed that chondrocytes and cells of the nucleus pulposus share many common features. MSCs are considered as ideal seed cells due to their multi-directional differentiation potency and the ability to be induced to chondrocyte-like cells.Embryonal stem cells are the most potent stem cells; however, their use is controversial and has mayor ethical considerations. Mesenchymal stem cells (MSCs) can be obtained from the adult and are widely used because of their differentiation potential. In addition to bone marrow, adipose tissue also appear to be sources of MSCs. Another crucial point in nucleus pulposus tissue engineering is to creat optimal scaffolding, which can satisfy all the goals required, including strength, toughness, controlled degradation, inflammatory response, and formability. The goal of our research is to identify new composite materials (chitosan-alginate gel) and develop novel synthetic processes for fabricating these scaffolds. Porosity, size and communication of pores, and cellular biocompatibility with ADSCs were detected.OBJECTIVES:1. To identify Chondrogenic capability of rabbit ADSCs(rADSCs) and biological characteristics in vitro.2. To fabricate a new type of chitosan-alginate gels scaffold and to detect the pore properties ,and evaluate the proliferation and differentiation of rabbit ADSCs on chitosan-alginate gels.METHOD:1. ADSCs were isolated from rabbit adipose tissue and cultured with DMEM and Chondrogenic Medium(CM). The morphology of ADSCs was observed by inverted phase-contrast microscope,and light microscope with HE staining, Growth curves of ADSCs were evaluated by trypan blue. collagen II and GAG were detected by AB and Van Gieson staining,and the expression of collagen typeⅡwas analyzed with immunohistochemical study.2. Create chitosan-alginate scaffolds, The porosity of processed samples was calculated by liquid displacement method; the pore microstructure was analysed using light microscope and scanning electron microscope.3.The rMSCs were seeded on the chitosan-alginate scaffolds and induced into chondrocyte-like cells by Chondrogenic Medium(CM). Cell morphous was observed by inverted phase-contrast microscope and SEM,At the end of the every culture period( 7d, 14d, 21d) , the compound of scaffolds and rMSCs were processed for histological study, the contents of GAG and collagen typeⅡin the compound were calculated as well.Results:1.Rabbit primary generation ADSCs survived well.ADSCs attachment cutured in monolayer is greatly enhanced and cell clones are abundant while the cells attachment is rather difficult and cell clones are less when cutured in CM, After inducing with CM, cells exhibited a round morphology,the cell growth became slowly, and the doubling time prolonged from 34.90h to 43.73h. The results of staining with Van Gieson and AB was positive.2.The chitosan-alginate 3-D compound scaffolds with porous exterior surfaces and the porosity were 80.57% and the pore sizes ranged from 50μm to 200μm.3.The cells grew well on the chitosan/alginate scaffolds.At the end of the every culture period ( 7d, 14d, 21d) , the GAG contents(ug/ml) in the ECM were 18.69±3.56,53.92±3.22,73.97±7.31 in induced culture group vs. 21.31±2.1,36.63±4.14,38.02±9.34 in the control group.At each time point, there was significant difference in the GAG content between control group and induced culture group( P<0.05) . The content of collagen typeⅡwas higher in the induced group than the control group. The results of staining with Safranin-O and Alcian Blue was positive.Conclusion:1. The method is effective to isolate ADSCs from adipose tissue.ADSCs showed chondrogenesis properties when cultured with CM. ADSCs may be appropriate seed cells for nucleus pulposus tissue engineering2. Chitosan-alginate gel is a suitable substrate for rADSCs proliferation and differentiation and is a candidate for 3-D scaffolds.This compound scaffolds, which possesed a morphological structure of 3-D form, showed a good biocompatibility with ADSCs in vitro. It could be used as a biomaterial for nucleus-pulposus tissue engineering.3. The cells grew well on the chitosan-alginate scaffolds,and under certain conditions,the rADSCs may differentiate towards the Chondrocyte-like cells...
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