| Background and objectiveOnce the articular cartilage defect,the repair is quite difficult,and it 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 defect. We filt Weilingxian as study object. Studies have shown that Weilingxian can maintain and promote the synthesis of proteoglycan and collagen II of 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/β-glycerol PHosPHate(C/β-GP)encapsulating allograft chondrocytes there-dimension cultured in vitro and repairing articular cartilage defects. Our study try to explore methode and value of combineding Chinese medicine with modern medicine.Methods1.chondrocytes were isolated and cultured by the way of enzymatic digestion in vitro,after the PHenotype was identified by toluidine blue staining,the third-passage cells in the logarithmic growth PHase were randomly divided into groups and given the treatment respectively.MorPHology of chondrocytes was observed under inverted PHase contrast microscope,Methyl Thiazolyl Tetrazolium (MTT) assay method was adopted to observe the influence of Weilingxian with different concentrations on the proliferation of chondrocytes, and anti-transcription-polymerase chain-type reaction (RT-PCR) was used to assay the expression changes of transforming growth factor-β1 mRNA.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 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;HE, TB and type II collagen immunohistochemical staining was used to detect histological changes of chondrocytes at 1,4 week.3.After the model of knee articular cartilage defects was established,24 New Zealand rabbits were randomly divided into three groups,that is experimental group (C/β-GP+chondrocytes+Weilingxian medium intra-articular injection group), control group (C/β-GP+chondrocytes+normal medium intra-articular injection group), model group (model group),and given corresponding treatment respectively.The specimens were excised at the 6th and 12th week,examined grossly and analyzed by haematoxylin eosine,toluidine blues staining and typeⅡcollagen immunohistochemistry reaction.Wakitani score method was counted to evaluate the effect of repair.Result1.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.Group of 0.05,0.1,0.5 and 1.0 mg/ml could promote the expression of TGF-β1 mRNA, and there was no significant difference between the four groups (P>0.05), but the peak occurred at 0.5 mg/ml group.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 collagen II and extracellular matrix.3.Defects of articular surface were well filled in experimental group and control group,and hyaline cartilage-like structure were formed,the surface flatness and degree of integration with surrounding tissue of experimental group was better than control group,and model group was not repaired.Formation of cartilage-like and secretion of cartilage matrix and specificity of collagen type II were found in histological slices.Integration of repair tissue with surrounding tissue,histology and amount of type II collagen secretion in experimental group were better than control group. Wakitani scores:scores of experimental group and control group were significantly lower than model group (P<0.01),and scores of experimental group was significantly lower than group control group (P<0.05).Conclusions1.Weilingxian can promote chondrocyte proliferation and TGF-β1mRNA expression,these role like cell growth factor may be one of the possible mechanisms to promote the repair of cartilage defect.2.It has good compatibility of chondrocytes in the co-culture with C/-GP gel,Weilingxian can promote chondrocytes proliferation and secretion of collagenⅡ,proteoglycan cultured in the C/β-GP gel, and also can induce the development to cartilage.3.Weilingxian can promote repair of cartilage defect by the injectable C/β-GP gel composite allogenic chondrocytes,this offer a new idea and method for the combination of Chinese medicine and tissue engineering technologies.
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