The Research Of The Methods Of Culturing The Epiphyseal Plate Chondrocytes In Vitro, Identification And Its Cellular Phenotype In Rabbits

Background:The growth plate injuries in children account for all long bonefractures in15%-30%,10%of the epiphyseal plate injury will lead todevelopment of bone and joint deformities, not only have seriouspsychological effects on children, and the impact of physical function, oreven loss of the Capacity[1]. Tissue engineering for the treatment ofepiphyseal plate injury provides a new opportunity. Seed cells of tissueengineering has been the core issue, the ideal seed cells should have easilyavailable, easy to proliferation in vitro, long-term passage does not changethe biological characteristics, antigenicity, and tissue repair of smallcapacity, and other features.Objective:To cultured rabbit growth plate chondrocytes, and explore simple,efficient method of cell culture. Preliminary studies on cultured cells,microscopic observation of cell morphology, cell ultrastructure underelectron microscope to identify whether the cells secreted proteoglycan andits content, identify whether the cells can synthesize type II collagen andwhether the expression of the corresponding mRNA. The epiphyseal platetissue engineering to find good seed cells.Methods:Select6New Zealand white rabbits aged4weeks, male or female,healthy, young rabbits weighing about200-300g. Anatomical cut proximal tibia, distal femur epiphyseal plate, the three-step enzymatic digestion toobtain cells. Will receive10%of chondrocytes in fetal calf serumcontaining DMEM high glucose medium on behalf of the Central Plains,passage in vitro. By inverted microscope and transmission electronmicroscopy morphology and ultrastructure of cultured cells, cell growthstate. MTT tetrazolium salt colorimetric assay with different passages ofcell growth activity of epiphyseal plate cartilage. Alcian blue stainingidentified cells are generated and quantitative detection of proteoglycan,immunohistochemical staining of collagen type II production, PCRconfirmed cell type II collagen mRNA production.ResultsIn this study, rabbits epiphyseal plate chondrocytes were cultured invitro, collection process is simple and efficient. Culture of epiphyseal plateobserved in adherent cell growth and the growth of the whole process ofpassage, after multiple passages decreased cell proliferation, cellmorphology gradually to the "fibrosis"cell change. The first3generationcells have a high biology and functional characteristics of expression. After4generations from the gradual loss of cell shape changes characteristicstructure of epiphyseal cartilage cells, cartilage cells and collagen typeII-specific proteoglycan gradually disappear, appear to diversity. PCRdetection of mRNA with the expression of collagen type II, Alcian bluestaining showed that the3rd generation cellular proteoglycan content, MTTshows the growth curve of cells2,3,4inverted "S" type, the2th and3thgeneration of cell activity Significantly better than the4th generation,cultured cells appeared at day10were inhibitory.Conclusions:1. rabbit epiphyseal plate cartilage cells for two-enzyme digestionmethod is simple, to obtain a large number of epiphyseal platechondrocytes.2. rabbits in vitro epiphyseal plate before the3represents the type of stable cells, the expression of biology and functional characteristicsof a good school, in vitro2nd generation,3rd generation cellular growthplate is more suitable as sources of seed cells for tissue engineering.3.Successfully established epiphyseal cartilage cells cultuer in vitro method,and confirmed that the cultured cells are epiphyseal chondrocytes.