Mechanically induced experimental knee osteoarthritis benefits from anti-inflammatory and immunomodulatory properties of simvastatin via inhibition of matrix metalloproteinase-3

Twenty-seven male albino Wistar rats weighing approximately 300 g each were obtained from Gazi University Experimental Animal Research Laboratory and were randomly divided into three groups of equal numbers. All surgical procedures were accomplished after intramuscular administration of 40 mg/kg ketamine hydrochloride (Ketalar, Pfizer Inc, USA) and 5 mg/kg xylazine hydrochloride. After being shaved and disinfected, right knees of animals in group 1 and group 2 underwent anterior cruciate ligament transection (ACLT) via a medial parapatellar arthrotomy to simulate knee instability. After surgery, the joint surface was washed with sterile saline solution, and both capsule and skin were sutured using Vicryl 4-0 (Ethicon, Edinburgh, UK) absorbable suture. Animals in group 1 were left untreated, whereas each animal in group 2 was treated with orally administered simvastatin (Merck Sharp & Dohme, Middlesex, UK) 20/mg per kilogram per day by gavage for 8 weeks starting from the first day of surgery. Animals in group 3 were sham operated with an arthrotomy only using the same approach. All animals were housed in different cages according to their groups, were allowed unlimited activity, and were sacrificed 8 weeks after the operation using an intravenous overdose of anesthesia. Twenty-seven knee joints in all groups were subsequently divided into femur (F) and tibia (T) subgroups. After disarticulation of the right knee joint, both femur and tibia were dissected from muscle, and cartilage was harvested from both medial and lateral tibial plateaus and femoral condyles.

Excisional biopsy specimens of proximal tibia and distal femur were fixed in 10% formalin for 1 week and then processed in 10% formic acid solution for decalcification. Formic acid solution was renewed in every 24 h, and the consistency of the bone was checked every day. The bone specimens could be cut at 72 h. Proximal tibia specimens were cut into two pieces in the sagittal plane, proximal femoral specimens were cut into two pieces in the coronal plane, and specimens were embedded in paraffin blocks to be sectioned on their cut surfaces. Three-micron-thick sections were cut on two slides: one to be stained by hematoxylin and eosin and the other by MMP-3 antibody. Immunohistochemical assays were performed on formalin-fixed, paraffin-embedded sections. After deparaffinization, sections were rehydrated with distilled water. Endogen peroxidase activity was blocked by 3% hydrogen peroxide for 10 min and the specimens were then washed in distilled water. Antigen retrieval was performed in citrate buffer pH 6.0 in a microwave oven at high temperature for 20 min for MMP-3. After standing for 20 min at room temperature, slides were washed in phosphate-buffered saline (PBS) pH 7.6 for 5 min. After protein blockage (Lab Vision Ultra V block, Fremont, CA, USA) for 10 min, tissue sections were incubated in primary antibodies against MMP-3 dilution 1/100 (Abcam, Cambridge, CB4, 0FW, UK) for 1 h at room temperature. After washing by PBS for 5 min, tissue sections were incubated in biotinylated secondary antibodies (Lab vision Fremont, CA, USA) for 20 min and then washed with PBS for 5 min. Slides were incubated in streptavidin/peroxidase complex (Lab vision Fremont, CA, USA) for 20 min and washed by PBS for 5 min. Sections were developed with the 3,3’ diaminobenzidine (DAB) as the chromogen and counterstained with hematoxylin. Sections were incubated overnight in primary antibodies against MMP-3 at 4°C. Placenta sections were used as positive controls for MMP-3.

The slides were screened by one pathologist. Positive staining was determined as cytoplasmic staining in chondrocytes. Total number of chondrocytes and positive-staining chondrocytes were counted in the femur and tibia, respectively, in three or more sections on 3–6 high-power fields (×400) according to the size of articular sections, and mean count of these sections was used to determine a proportion of cells expressing MMP-3. The pathologist was masked to the experimental procedures.

Histopathological assessment was made using the OARSI cartilage OA histopathology assessment system by the same pathologist. This system is based on six grades and four stages, reflecting the severity and extent of OA over the joint surface. Grade is defined as OA depth progression into cartilage and is an index of severity or biologic progression of the osteoarthritic process. Grade is assessed by noting the most advanced grade present within cartilage, irrespective of its horizontal extent (grades 1–4: articular cartilage changes only; grades 5–6: cartilage and subchondral bone). Stage is defined as the horizontal extent of cartilage involvement irrespective of underlying grade. Score is defined as OA grade and OA stage (grade × stage). Therefore, scores represents a combined assessment of OA severity and extent [18, 19].

Statistical comparisons were generated using Statistical Package for Social Sciences-11 for Windows program (SPSS, Chicago, IL, USA). All data are expressed as means ± standard deviation (SD). Histopathology and immunohistochemistry data were analyzed using a nonparametric Mann–Whitney U test. P values <0.05 were considered statistically significant.