The Experimental Study Of Co-culture Technology For The Construction Of Engineered Cartilage In Vitro

Part I:Isolation and culture of human articular chondrocytes and human bonemarrow mesenchymal stem cells in vitroObjective To investigate the culture and identification of isolated human bonemarrow mesenchymal stem cells(hBMSCs) and human articular chontrocytes(hACs) invitro. To observe the chondrogenesis of hBMSCs by TGF-β1induction.Methods Human bone marrow mesenchymal stem cells were harvested from childrensuffering from limbs long bone fractures which need fixation of intramedullary pin. Wesucked bone marrow by injector. Human bone marrow mesenchymal stem cells wereobtained after centrifugalization and digestion.Than we put them into L-DMEM medium.Flow cytometer was used to indentify hBMSCs.Check up biological characteristics of thecells by applying the ways of microscope. Human bone marrow mesenchymal stem cellswere induced in chondrogenic medium which include TGF-β1,FBS, ITS+et al. Identifyingthe cells by applying the ways of Alcian blue staining, safrarine “o” staining，Immunohistochemical staining and depicts the growth curve.Human joint chondrocytes were obtained from interphalangeal joint which wereexcised from Multi-fingered children.Results Human bone marrow mesenchymal stem cells grew along the walls.About10days to15days later the primary cells had grew and covered one hole of Six-well cultureplates. The results of flow cytometer and immuncytochemistry showed that the expresionsof stem cell-related antigens(CD29)were positive,and those of hematopoiesis-relatedantigens(CD34,CD45)andother were negative.These passaged cells grew well in first fivegenerations and began to apoptosis in tweenty generations.The test of Alcian blue stainingand safrarine “o” staining had showed positve which mean the expression ofproteoglycan.The test of Immunohistochemical staining had showed positive which wasstandard of the expression of II collagen. The primary hACs grew slowly,10days to15days later covered one hole of Six-well culture plates. Conclusion1. We had successfully established a way to isolate and culture hACs andhBMSCs.2. Human bone marrow mesenchymal stem cells could be harvested from limps longbone of5to8years old children.3. Transforming growth factor-1plays a positive role in promoting chondrogenicdifferentiation of MSCs.Part II: Coculture of human bone marrow mesenchymal stem cells and humanarticular chondrocytes to construct tissue engineering cartilage.Objective To study the best cell ratio of the co-culture system of the hBMSCs andhuman articular chondrocytes(hACs). To compare the effect of the induction of BMSCs byco-culture BMSCs and chondrocytes induction and transforming growth factor (TGF)-β1induction.Methods Co-culture cells were divided into3groups by the ratio(hBMSCs:hAC) of2:1,1:1and1:2(Group A B C). Simultaneously, TGF-β1induction group and humanserved as a control D.Human chondrocytes group served as a control E.All of the groupsabove were implanted into silk fibroin scaffolds.Cells were cultured in scaffolds for threeweeks and their cellularity,cartilage-like matrix formation and chondrogenic geneexpression levels (collagen II,aggrecan) were measured.The content of DNA wasquantified using the Alcian blue binding assay.The content of DNA was quantified by theHoechst33258assay.Results No difference in size had been seen in all of the groups.The engineeredcartilages of co-cultured group A and control group D were rough and gloomy.Weobserved lower levels of cell proliferation and excellular matrix secreted.The engineeredcartilages of group C were smooth and glossy which was iomproved to be best in tissuestaining and quantitive analysis of GAG and DNA.We invesgated almost the same qualityof engineered cartilages in group B and E.Conclusion1.Results displayed that3D coculture of hACs and hBMSCs keptchondrocyte morphology and protein expression property. Equally robust Co-culturechondrogenesis was also observed in co-cultures when the ratio of hACs and hBMSCs was1:2compare to that of TGF-β1induction.2.When chondrocytes come to rate1:1,the better chondrogenisis was obtained whichwas nearly the same as the group of soly chondrocytes. Co-culture of hACs and hBMSCs can improve the quality of engineered cartilages, and the best ratio of coculture was2:1.