Evaluation of biomarkers in a model of osteoarthritis in ovariectomized rats

MC Carceller1, A Blanco1, MC Terencio1, D Guede2, JR Caeiro3, ML Ferrándiz1, MJ Alcaraz1.

1 University of Valencia, Department of Pharmacology and IDM 46100, Spain, 2Trabeculae, 32900, Spain, 3University of Santiago de Compostela, University Hospital 15706, Spain

Background: Osteoarthritis (OA), one of the most prevalent and disabling conditions, is characterized by synovitis and changes in joint cartilage and subchondral bone. OA and osteoporosis frequently coexist in post-menopausal women because of oestrogen deficiency. Animals models are useful to understand the molecular basis of disease, to investigate biochemical markers and for designing new treatments

Objective: This study was aimed at investigating the pathological characteristics and possible biomarkers of cartilage degradation, bone metabolism and inflammation in an animal model of OA in ovariectomized rats.

Methods: Eight week-old female Wistar rats (150-170 g) were used. Rats were randomly assigned into three groups (n=15): BNO (healthy group), OVX (ovariectomy) and OVX+ACLT (ovariectomy followed by anterior cruciate ligament transection, two weeks later) and were maintained in animal house with water and food ad libitum and the ambient temperature was 21ºC. ACLT was carried out under deep anesthesia with isoflurane (1.5 minimum alveolar concentration) which was followed by the subcutaneous injection of butorphanol (2 mg/kg). Blood samples were collected once a month. The animals were sacrificed at the end of the experiment (third month) and their paws were obtained. Cartilage degradation, bone metabolism and inflammatory mediators were measured in serum and in paw homogenate by ELISA or Multiplex. Joint bone microestructure was analyzed by micro-computed tomography (µCT) and changes in cartilage and synovial membrane were determined by histological procedures. Results were expressed as mean±SEM and analysed by one way ANOVA and Dunnett\'s t-test.

Results: Serum levels of ALP (alkaline phosphatase) increased in OVX (841.7±3.7 U/l, p<0.01) and OVX + ACLT (25.2±2.7 U/l) groups vs BNO (24.0±3.9 U/l) whereas the ratio osteoprotegerin/RANKL (Receptor Activator of Nuclear Factor kappa B Ligand) was reduced (OVX 6.8±0.5, OVX+ACLT 6.9±0.4, p<0.01, vs BNO 14.8±0.9) as well as N-terminal propeptide of type I procollagen (PINP) levels (OVX 12.5±0.8 ng/ml, p<0.05, OVX+ACLT 12.6±0.3 ng/ml, p<0.05, vs BNO 14.6±0.2 ng/ml). On the other hand, levels of inflammatory mediators IL(interleukin)-1β and IL-17 in paw homogenates were significantly increased in OVX (1179.0±114.7 and 141.7±16.1 pg/ml, respectively; p<0.01) and OVX + ACLT (1829.0±100.5 and 143.6±8.9 pg/ml, respectively, p<0.01) groups vs BNO (77.6±96.3 and 77.6±6.1 pg/ml, respectively) while IL-6 levels increased significantly in the OVX + ACLT (9047.0±446.3 pg/ml, p<0.01) group vs BNO (4766.0±404.9 pg/ml,). A decrease in % volume of subchondral bone was observed in OVX (13.4±0.4, p<0.01) and OVX + ACLT (16.1±2.3, p<0.01) groups vs BNO (47.5±9.4) group in the µCT study. Moreover, the histological analysis showed increased synovitis and articular cartilage degradation.

Conclusion: This experimental model mimics the main features of OA and osteoporosis and therefore it could provide insights into pathological components and specific signaling pathways of these conditions leading to future research towards appropriate diagnostic and therapeutic strategies. Our data also confirmed PINP as a very useful biomarker of bone loss.

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