A Study On Inhibition Of The Formation Of Joint Adhesions By ERK2Small Interfering RNAs

Objective To observe the therapeutic effect of siRNA, which could disturb the expression of ERK2, by setting the rabbit knee synovectomy model of acute local period, we injected a lentivirus-mediated siRNA argeting ERK2, then by siRNA interference ERK2, blocking SMAD and MAPK pathways to inhibit fibroblast proliferation and collagen expression, thereby inhibiting the formation of joint adhesions, and further to explore its repair mechanisms.Methods30healthy adult Japanese white rabbits were randomly divided into three groups:blank control group,Ms siRNA group and ERK2siRNA group. After a skin incision, the knee was opened through a lateral parapatellar approach and the medial and lateral sides of the femoral condyle were exposed. A partial capsulotomy and synovectomy were performed using an osteotome and an osteochondral portion of the condyle was removed until the underlying cancellous bone was exposed. Each operated limb underwent knee joint immobilization at140°of flexion for30days, and the left knee without any treatment. In the ERK2siRNA group,0.1ml of culture medium containing the lentivirus-mediated ERK2siRNA was injected into the articular cavity after incision closure at days3and7, respectively. Similarly,0.1ml of culture medium containing the lentivirus-mediated MS siRNA was applied in the MS siRNA group, and the same volume of medium without a viral vector was applied in the control group. In vivo bioluminescence assay.At28days after transfection, the luciferase expression and distribution in the individual rats in the ERK2siRNA group were measured. Measurement of the contracture angle.Animals were euthanized at30days, and immediately subjected to biomechanical evaluations. The angle of flexion contracture was determined on a lateral view radiograph of the right knee taken under an extension torque of5N, and the angle "a" was defined as the contracture angle. Following measurement of the contracture angle, each joint was exposed by a parapatellar skin incision and held at140°of flexion. Histological evaluation of adhesion tissues. After macroscopic evaluation of the adhesions, the knee joints were excised while preserving all the connective tissues involved in fibrotic adhesive scar formation. Each biopsy was fixed in10%buffered formalin for1week and decalcified for2weeks. The tissue was embedded in paraffin, and6-lm sections were prepared in the vertical plane to the femoral axis. The sections were stained with hematoxylin and eosin and examined microscopically.Results Among the30rats used in this study, three died on days14and20, respectively. These3animals were excluded from the study. The other animals gained weight and appeared healthy, with no signs of impaired wound healing. The luciferase fluorescence detected at28days after transfection demonstrated that local delivery of the lentivirus-mediated ERK2siRNA caused a localized silencing effect in the articular cavity.The flexion contracture angles in the control and MS siRNA groups ranged from71°to91°ith a mean value of82°. Although no significant differences were observed between the MS siRNA and control groups, the contracture angles in these two groups were significantly larger than those in the ERK2siRNA group (range:25-38°; mean value:32°). At30days after the surgery, thick fibrous adhesions developed in the knees of the rats in the control and MS siRNA groups. In contrast, the adhesions in the ERK2siRNA group were soft and weak, and were easily stretched. The adhesion scores for the ERK2siRNA group were significantly lower than those in the control and MS siRNA groups (data not shown).Histologically, the adhesion tissues in the control and MS siRNA groups were dense, thick and fibrous, while those in the ERK2siRNA group were loose and thin with sparse fiber formation. In all three groups, the predominant cells were considered to be fibroblasts, and few inflammatory cells were observed. There was no apparent difference in the cell densities between the control and MS siRNA groups. On the contrary, the cell density in the ERK2siRNA group was low.Conclusion Local delivery of a lentivirus-mediated siRNA targeting ERK2ameliorated joint adhesion formation effectively and safely in a rabbit model. This study provides a novel and promising strategy that will serve as an alternative for the prevention of joint adhesion formation. Further studies involving larger animals are required to support the hypothesis.