To study the method of isolating and culturing synovium-derived MSCs (SMSCs), and to investigate its multiple differentiation potential in vitro. Methods Three 2-month-old Changfeng hybrid swines weighing 8-10 kg (male and female) were used. SMSCs were harvested from the synovium of swine knee joints and cultured in vitro. When the SMSCs at passage 3 reached confluence, basic culture medium was removed, and the multi ple differentiationpotential of SMSCs was demonstrated in specific induction media (experimental group). The cells at passage 3 cultured with basic culture medium served as control group. After 21 days of chondrogenic differentiation, the cells underwent toluidine blue staining, immunohistochemistry staining and real-time fluorescence quantitative PCR detection. After 10 and 21 days of osteogenic differentiation, the cells underwent ALP staining and Al izarin red staining, respectively. After 21 days of adipogenic differentiation, the cells underwent Oil red O staining. Results SMSCs displayed long and thin or polygonal morphology 24 hours after culture. They prol iferated fast 48 hours after culture and presented large number of spindle-shaped cells with few globular cells 72 hours after culture. For the experimental group 21 days after chondrogenic induction, the cells were positive for toluidine blue staining with the formation of Aggrecan outside the cells; the immunohistochemistry staining revealed the expression of Col II; the real-time fluorescence quantitative PCR detection showed that the expressions of Col II A1, Aggrecan and SOX9 mRNA of the experimental group were greater than that of control group (P lt; 0.05). The cells were positive for ALP staining 10 days after osteogenic induction, and positive for Al izarin red staining 21 days after osteogenic induction, with the formation of calcium nodules. Oil red O staining displayed the formation of l i pid droplets inside the cells 21 days after adi pogenic induction. For the control group, the results of all the staining assays were negative except the ALP staining presenting with sl ight positive result. Conclusion SMSCs can be isolated from knee joint of swine and proliferate and differentiate into osteogenic, adi pogenic and chondrogenic cells in vitro. SMSCs may be a promising source of seed cells for tissue engineering.
To construct the retroviral vector containing human interleukin 1 receptor antagonist (IL-1Ra)and to investigate the property of the transfected articular chondrocytes from osteoarthritic patients in vitro. Methods Retroviral vector PLXRN carrying IL-1Ra (PLXRN-IL-1Ra) gene was constructed by inserting IL-1Ra gene at the sites of Sal I and BamH I. The recombinant retroviral plasmid was homologously recombinated in bacterial cells. After screening and ampl ification, the recombinant retroviral plasmid was obtained and transfected into PT67 cells. The repl ication-defective retrovirus PLXRN-IL- 1Ra was packed and ampl ified in the PT67 cells. Viral titer was determined by infecting NIH/3T3 cells with serially diluted viral supernatants produced with a control vector. Experiments were divided into 3 groups: non-transducted group (group A), PLXRN transduction group (group B), PLXRN-IL-1Ra transduction group (group C). Primary articular chondrocytes from osteoarthritic patients were transduced with PLXRN and PLXRN-IL-1Ra.The positive chondrocytes clones, which were G418- resistant, were cultured for 3-4 weeks after being selected by G418. The expression of IL-1Ra mRNA in the chondrocytes was determined by RT-PCR. Levels of IL-1Ra protein synthesis in the supernatants were measured by ELISA. Results Restric tive endonuclease identification and gene sequencing confirmed that the recombinant contained IL-1Ra cDNA．Virus titer could reach 3 × 104 CFU/mL. Primary chondrocytes cultured in vitro were polygonal or spindle and were stained with purple particles by toluidine blue staining. After stable transduction into the chondrocytes the 311 bp fragment of IL-1Ra was detected in group C by semi-quantitative RT-PCR. ELISA showed that IL-1Ra in supernatants of the group A and group B were below the level of detection. The concentrations were（60.47 ± 15.13）ng/L in group C .There were significant differences between gene transduction group and control groups (P lt; 0.05). Conclusion The construction of recombinant retrovirus vector by homologous recombination in bacterial cells can be quickly and easily performed. Stable and effective expression of IL-1Ra can be achieved by transduction with retroviral vectors in osteoarthritic articular chondrocytes, indicating potential util ity in gene therapy for osteoarthritis．