Inhibition Of Osteoarthritis In Rats By Electroporation With Interleukin-1 Receptor Antagonist

ObjectiveA gene therapeutic approach to treat osteoarthritis (OA) appears to be the horizon for people who suffer from this disease. To development of safe and efficient gene deliveries is the key to the clinical success of gene therapy. The present study was designed to evaluate the electroporation (EP) as non-viral gene vectors for gene therapy of OA, and compared its efficiency with adeno-associated virus (AAV) vector.Materials and MethodsSelection of 8-week-old healthy male SD rats were established osteoarthritis model by the right knee anterior cruciate ligament transaction and medial meniscus forefoot resection. Under different electroporation parameters, EP with the Luciferase reporter gene into rat knee, using a small animal in vivo imaging system measure the fluorescence intensity, the intensity indicate that the leve of Luciferase gene expression at different electroporation parameters. Clearly preferred electroporation parameter by comparing the fluorescence intensity, and carry out the in vivo treatment experiment of rat according to this parameter. One week after OA induced operation, selecting the interleukin-1 receptor antagonist (IL-IRa) gene suppress the IL-1 induced inflammation. Depending on the treatment methods, experiment divided into five groups:Simple osteoarthritis untreated group (OA group, n= 24); Osteoarthritis sole target plasmid treatment group (NP group, n= 24); Osteoarthritis electroporation target gene therapy group (EP group, n= 24); Osteoarthritis adeno-associated virus gene transfection therapy group (AAV group, n= 24); Normal control group (Normal, n= 24). One week after the treatment begin to evaluate, drawn 1 week, a total of 4 times. Local pain was evaluated by monitoring right hind paw intensity through CatWalk test, whereas cartilage and synovium IL-1Ra, IL-1?. TNF-a. MMP-13 and ADAMTS-4 mRNA levels were analyzed using Real-time PCR. ELISA testing the changes of synovial fluid protein content. The structural analysis of subchondral bone change at medial femoral condyle by Micro-CT. In addition, histological evaluations were performed using sections of knee joint specimens stained with hematoxylin and eosin (H-E) and Toluidine blue and Saffron-0 and immunohistochemistry of IL-lRa and collagen type II.ResultsWhen pulse width was 30ms, plasmid dose was 50?g, the fluorescence intensity of the right knee joint was significantly higher than other groups (p<0.05). In the EP group and AAV group, level of pain caused by the inflammation was significantly lower than the OA group and NP group (p<0.05), in the cartilage defect site selected region of interest, subchondral bone mineral density and trabecular thickness increased significantly less than the OA group and NP group (p<0.05). PCR results showed that: EP group and AAV group upregulate IL-1Ra gene expression was significantly higher than other groups (p<0.05), when the first two weeks, IL-1 Ra expression of AAV group was significantly higher than the EP group, the opposite results after two weeks. In the OA group and NP group, IL-1? and TNF-a expression was significantly upregulated. EP group and AAV group, IL-1? and TNF-a expression was higher than normal but there was no statistically significant difference (p>0.05). MMP-13 and ADAMTS-4 is similar trends to IL-1? and TNF-a. ELISA and PCR results showed the same trends. Pathology results showed that:Four weeks, the group EP and group AAV no significant cartilage damage and extracellular matrix degredation, The positive expression of IL-1Ra in the regional distribution of cartilage around the nucleus, the positive expression of type ? collagen in cartilage mainly accumulated in surface and intermediate layer, no difference between the two groups. OA group and NP group cartilage severely damaged, remain few matrix, local area weakly positive expression of IL-1Ra and collagen type ?.ConclusionThese results indicated that EP with transfection efficiency comparable to AAV and longer gene expression potential might be an efficient non-viral deliver system for OA gene therapy.