Résumé
BACKGROUND:Articular chondrocytes with the ability of autocrine and paracrine can provide the growth factors and microenvironment for synovial mesenchymal stem cels differentiating into the chondrocyte. The three-dimensional scaffold could provide space for cels adhesion, proliferation and differentiation. OBJECTIVE: To study the ability of chondrogenesis by co-culturing synovial mesenchymal stem cels and chondrocytes under the three-dimensional condition. METHODS:The synovial membrane and articular cartilage were harvested from rat knee joint. The synovial mesenchymal stem cels and chondrocytes were obtained through the method of enzyme digestion. The passage 3 synovial mesenchymal stem cels and passage 2 chondrocytes were co-cultured in the chitosan/I colagen composite scaffolds at the ratio of 1:2. Then, the cels/scaffold composite was harvested to be examined morphologicaly, histologicaly and immunohistochemicaly after being cultured 21 days. The confocal laser was also employed to detect the cels distribution in the scaffold. RESULTS AND CONCLUSION: After being cultured 72 hours, it could be observed from the cels/scaffold composite examined through the scanning electron microscope that the cels adhered on the surface of the scaffold and extracelular matrix surrounding the cels was seen on the scaffold. After being cultured 21 days, it could be found through the confocal laser scanning that the cels were wel-distributed on the scaffold, and cels decreased gradualy. Type II colagen was positive in the extracelular matrix immunohistochamicaly. It suggested from this study that the synovial mesenchymal stem cels could be co-cultured with chondrocytes in the chitosan/I colagen composite scaffolds and have the ability of chondrogenesis differentiation.
Résumé
BACKGROUND:Compared with other sources of mesenchymal stem cells, synovial-derived mesenchymal stem cells have significant characteristics of chondrogenesis and cloning. Therefore, synovial-derived mesenchymal stem cells are one of the most promising seed cells in cartilage tissue engineering. OBJECTIVE:To isolate and culture synovial-derived mesenchymal stem cells of Sprague-Dawley rats, identify the multipotential differentiation and the potential ability of chondrogenic differentiation in three-dimensional culture condition. METHODS:The synovium tissue was harvested from Sprague-Dawley rats. The synovial-derived mesenchymal stem cells were isolated with typeⅠcol agen enzyme digestion method and cultured in vitro. The passage 3 cells were detected with giemsa staining, the cellcycle, adipogenic and osteogenic differentiation were determined. The passage 3 cells were centrifuged as pel ets and cultured in the chondriogenic medium for 21 days. And the pel ets were examined by toluidine blue staining, typeⅡcol agen immunohistochemical staining and RT-PCR. RESULTS AND CONCLUSION:The mesenchymal stem cells isolated from the synovium tissue of rats have the characteristics of mesenchymal stem cells, and exhibit fibroblast-like morphology after cultured in vitro. The multilineage differentiation potentials were also revealed. After the cellwere cultured in chondrogenic medium for 21 days, chondroid tissue was found, type II col agen and aggrecan could be detected positively by toluidine blue staining, typeⅡcol agen immunohistochemical staining, and expressed by RT-PCR examination. Therefore, synovial mesenchymal stem cells have a chondrogenic differentiation potential.
Résumé
The objective of this study is to explore the application possibility of chitosan/pcDNA-EGFP-TGFPbeta1 nanoparticles in the transfection of synovial-derived mesenchymal stem cells (SDMSCs). Chitosan/pcDNA-EGFP-TGFbeta1 nanoparticles were fabricated through method of ionic crosslinking. The SDMSCs were harvested from rabbit joints and cultured to passage 3. The SDMSCs were then transfected with chitosan/pcDNA-EGFP-TGFbeta1 nanoparticles. Scanning electronic microscope (SEM) was employed to detect the shape and diameter of the nanoparticles. The transfected SDMSCs were examined under the fluorescence microscope and detected through the flow cytometry (FCM). The SEM examination showed that the contour of the fabricated chitosan/pcDNA-EGFP-TGFbeta1 nanoparticles was round and its average diameter was 50 nm. After being cultured for 48 h, the SDMSCs transfected by chitosan/pcDNA-EGFP-TGFbeta1 nanoparticles could be detected under the fluorescence microscope, and the live SDMSCs could also be examined through FCM. The transfection rate was 8% - 10%. Therefore, it suggested that the chitosan/pcDNA-EGFP-TGFbeta1 nanoparticles fabricated through the method of ionic crosslinking could transfect the SDMSCs, but the transfection rate should be improved.