Experimental Study On Intervention Of Weilingxian On Injectable Chitosan/β-glycerophosphate Gel Encapsulating Allograft Chondrocytes There-dimension Cultured In Vitro

Background and objective Incidence of articular cartilage injuries is high,and repair is quite difficult.The study of repairing articular cartilage injuries has always been the difficulty and research focus of the medical profession.At present,tissue engineering technique has been main research direction and has got advancement.The technique includes of seeds cell, frame and growth factor.Chinese medicine has been manifested satisfactory clinical therapeutic effect on repairing cartilage injuries. We filt Weilingxian as study object. Studies have shown that Weilingxian can maintain and promote the synthesis of proteoglycan and collagenⅡof chondrocyte, and protect articular cartilage by inhibiting the level of interleukin-1 (IL-1) possibly.The topic combine Weilingxian with tissue engineering technique,and observe effect of Weilingxian on developing proliferation of chondrocytes and interfereing in injectable chitosan/p-glycerol phosphate (C/β-GP)encapsulating allograft chondrocytes there-dimension cultured in vitro and Potentiality of cartilaginification. Our study try to provid a foundation to study of repairing cartilage injuries in vivo,and explore methode and value of combineding Chinese medicine with modern medicine.Methods (1) After separating articular hyaline cartilage of four-week-old New Zealand white rabbits with blades, chondrocytes were isolated and cultured by the way of machinery dissociation and enzymatic digestion in vitro. Morphology of chondrocytes was observed under inverted phase contrast microscope. The phenotype was identified by toluidine blue staining.After dispensing Weilingxian medium of 0.01,0.05,0.1,0.5,1.0mg/mL,the third-passage cells in the logarithmic growth phase were randomly divided into groups and given the treatment respectively.Thiazolyl Tetrazolium (MTT) assay method was adopted to observe the influence of Weilingxian with different concentrations on the proliferation of chondrocytes. (2) Chitosan/β-glycerophosphate gel encapsulating allograft chondrocytes was prepared at ratio of 7:1,then was randomly divided into experimental and control groups divided into groups and given corresponding treatment of Weilingxian medium of 0.01,0.05,0.1,0.5,1.0mg/mL and DMEM high glucose mediun respectively.MTT assay method was used to detect the influence of Weilingxian with different concentrations on the proliferation of chondrocytes three-dimensional cultured in gel;scanning electron microscopy was adopted to observe morphological change of chondrocytes in the gel;HE and typeⅡcollagen immunohistochemical staining was used to detect histological changes of chondrocytes at 1,4 week.Result (1) chondrocytes were isolated and cultured by the way of machinery dissociation and enzymatic digestion in vitro. All groups of Weilingxian can promote proliferation of chondrocytes,effect of 0.5mg/mL group was especially obvious,and the peak of proliferation was at the third day. (2) Chondrocytes behave good state of stretch,adhesion,proliferation in C/β-GP gel.All groups of Weilingxian can promote chondrocyte proliferation in the gel,and the effect of 0.5mg/mL group was especially obvious.Weilingxian can also enhance the secretion of collagenⅡ.Conclusions (1)chondrocytes were obtained satisfactory by the way of machinery dissociation and enzymatic digestion in vitro. (2) Weilingxian can promote chondrocyte proliferation. (3) It has good compatibility of chondrocytes in the co-culture with C/-GP gel,Weilingxian can promote chondrocytes proliferation and secretion of collagen II in the C/β-GP gel, and also can induce the development to cartilage.