【Abstract】 Objective The seed cells source is a research focus in tissue engineered cartilage. To observe whether the post-RNA interference (RNAi) chondrocytes could be used as the seed cells of tissue engineered cartilage. Methods Chondrocytes were separated from Sprague Dawley rats. The first passage chondrocytes were used and divided into 2 groups: normal chondrocytes (control group) and post-RNAi (experimental group). Normal and post-RNAi chondrocytes were seeded into chitosan/gelatin material and cultured in vitro to prepare tissue engineered cartilage. The contents of Aggrecan and Aggrecanase-1, 2 were measured by HE and Masson staining, scanning electron microscope (SEM), and RT-PCR. Results The histological results: no obvious difference was observed in cell number and extracellular matrix (ECM) between 2 groups at 2 weeks; when compared with control group, the secretion of ECM and the cell number increased in experimental group with time. The RT-PCR results: the expression of Aggrecan mRNA in experimental group was significantly higher than that in control group (P lt; 0.05); but the expressions of Aggrecanase-1, 2 mRNA in experimental group were significantly lower than those in control group (P lt; 0.05). The SEM results: the cell number in experimental group was obviously more than that in control group, and the cells in experimental group were conjugated closely. Conclusion The post-RNAi chondrocytes can be used as the seed cells for tissue engineered cartilage, which can secrete more Aggrecan than normal chondrocytes. But their biological activities need studying further.
Objective To review the progress of a disintegrin and metalloproteinase with thrombospondin motif 4 (ADAMTS-4) and ADAMTS-5 in osteoarthritis. Methods Recent literature about the ADAMTS-4 and -5 in osteoarthritis was analyzed; the structure, function, inhibitors of the ADAMTS-4 and -5, and the relationship between the proteases and osteoarthritis were analyzed and summarized. Results ADAMTS-4 and -5 can reduce chondrocyte and extracellular matrix by degrading aggrecan and cartilage oligomeric matrix protein, which induced the pathogenesis of osteoarthritis. Conclusion ADAMTS-4 and -5 have been demonstrated to play important roles in osteoarthritis. It can better guide treatment and prevention of osteoarthritis to further study related mechanism of ADAMTS-4 and -5, and to promote the establishment of a clinical drug targets.
ObjectiveTo observe the effect of lentivirus-mediated cyclooxygenase 2 (COX-2) and Aggrecanase-1 silencing and insulin-like growth factor 1 (IGF-1) in BMSCs after injecting into the knee joint cavity in cynomolgus monkeys with knee osteoarthritis (OA). MethodsBMSCs were isolated from the bone marrow of 10 donors. The lentivirus vector expressing genes of COX-2, Aggrecanase-1, and IGF-1 were constructed, and transfected into the third generation human BMSCs at 40 multiplicity of infection (virus group); BMSCs transfected with lentivirus-empty vector served as blank-virus group. The growth status and number of BMSCs were observed under inverted phase contrast microscope, and normal BMSCs were used as normal control group. At 1 week after transfected, the mRNA expressions of COX-2, Aggrecanase-1, and IGF-1 were detected with RT-PCR. Nine 3-year-old cynomolgus monkeys were selected to establish the OA model according to Hulth modeling method, and were randomly divided into 3 groups (n=3). At 6 weeks after remodeling, the right knee joint cavity was injected accordingly with 1 mL BMSCs (about 1×107 cells) in virus group and blank-virus group, with 1 mL of normal saline in the blank control group; the left knee served as normal controls. The general condition was observed after injection; at 1, 4, and 6 weeks, the concentrations of prostaglandin E2 (PGE2), IL-1, Aggrecanase-1, and IGF-1 of double knee liquid were detected with ELISA; at 6 weeks, MRI, general observation, histology method, and immunohistochemistry method were used to detect the knee cartilage changes and the expressions of COX-2, Aggrecanase-1, and IGF-1 were measured with RT-PCR. ResultsNo significant difference was found in cell morphology and growth curve between 2 groups after transfection. By RT-PCR, COX-2, and Aggrecanase-1 expressions were significantly reduced, IGF-1 expression was significantly increased in virus group when compared with normal control group and the blank-virus group (P < 0.05). All monkeys survived to the end of the experiment after injection. When compared with blank-virus group and blank control group, the concentrations of PGE2, Aggrecanase-1, and IL-1 significantly decreased and the concentration of IGF-1 significantly increased in the virus group (P < 0.05), but the indicators in 3 groups were significantly higher than those in the normal control group (P < 0.05). MRI showed that abnormal articular surface with high density could be found in virus group, blank-virus group, and blank control group, while the virus group had the minimum area. Gross observation and histological observation showed that the cartilage morphology of virus group, blank-virus group, and blank control group was accordance with early OA articular cartilage changes, but virus group was better than blank-virus group and blank control group in repair degree, whose improved Pineda score was significantly lower (P < 0.05). Immunohistochemical staining showed that the virus group had deeper dyeing with occasional brown particles and more chondrocytes than blank-virus group and blank control group. By RT-PCR, COX-2 and Aggrecanase-1 mRNA expressions of cartilage in virus group were significantly decreased, and IGF-1 expression was significantly increased when compared with blank control group and the blank-virus group (P < 0.05). ConclusionLentivirus-mediated multi-genes co-transfection in BMSCs can inhibit the expressions of COX-2 mRNA and Aggrecanase-1 mRNA, and enhance the IGF-1 mRNA expression, which decreases the concentration of inflammatory factors, and protects the joint cartilage effectively.