Histological Characterization of Epiphyseal Bone and Articular Cartilage in a Rat Model of Post-traumatic Knee Osteoarthritis

Purpose: Several experimental animal models have been developed for human osteoarthritis (OA) and used to study the preclinical efficacy of disease modifying OA drug candidates in various species. These animal models have been characterized and the preclinical efficacy of OA drug candidates have been studied using numerous microscopic scoring systems. The histopathology initiative of Osteoarthritis Research Society International (OARSI) has presented recommendations for histological OA assessment in different species in order to standardize the histopathological evaluation of OA in animal models. Recommendations for the histopathological evaluation of knee joints in rat OA models focus mainly on articular cartilage. As OA drug candidates may affect also bone tissue, the histological OA assessment could be completed with the analysis of epiphyseal bone. In this study, we performed a detailed characterization of epiphyseal bone by bone histomorphometry in a rat model of post-traumatic knee OA used frequently in preclinical efficacy studies. Methods: This study was conducted using male Lewis rats. Unilateral OA was induced in their knee joints at the age of 3 months by surgical medial meniscal tear (MMT) and medial collateral ligament transection (MCLT). Knee joint samples were harvested for histological analyses both from ipsilateral and contralateral hind limbs before the surgical operations and at 18 and 25 days after the operations. The samples were fixed and dehydrated in ethanol, defatted in xylene, and embedded in plastic for sectioning in a coronal plane. Prior to the dehydration, representative knee images were obtained by micro-computed tomography (SkyScan 1072; Bruker microCT, Kontich, Belgium). The effects of the MMT+MCLT operation on articular cartilage were analyzed in medial tibial plateau using Toluidine Blue stained sections and following the recommendations of OARSI histopathology initiative. The effects of the MMT+MCLT operation on epiphyseal bone were characterized by bone histomorphometry in medial tibial epiphysis using Masson-Goldner Trichrome stained sections for static parameters and unstained sections for dynamic parameters. For the determination of dynamic parameters, bone labelling was performed with oxytetracycline at 11 days and with calcein green at 2 days before sacrifice. All histological analyses were performed using OsteoMeasure7 histomorphometry system (OsteoMetrics, Atlanta, GA, USA). This experimental protocol was approved by National Animal Experiment Board, Regional State Administrative Agency for Southern Finland, Hämeenlinna, Finland. Results: The histological assessment of OA changes demonstrated that the surgical MMT+MCLT operation induced apparent degenerative changes in the articular cartilage of medial tibial plateau already at 18 days after the operations. These changes were most pronounced on the outer 1/3 of the plateau followed by the middle 1/3. The mildest degenerative changes were observed on the inner 1/3 of the plateau. The degenerative changes included the loss of chondrocytes, proteoglycans and collagen matrix from superficial layer down to tidemark. Bone histomorphometry demonstrated that the MMT+MCLT operation increased trabecular bone area fraction in medial tibial epiphysis. This increase was associated with an increase in the number and surface of osteoclasts on trabecular bone surface and with an increase in dynamic histomorphometry parameters. This indicated enhanced bone resorption and increased trabecular bone turnover and formation in medial tibial epiphysis. Bone histomorphometry demonstrated also that the total amount of trabecular bone remained unchanged, whereas the size of medial tibial epiphysis and its marrow cavity decreased in MMT+MCLT-operated knee. This suggested a compression deformation of the medial tibial epiphysis. Dynamic histomorphometry parameters revealed also that the growth of articular cartilage was even ceased in the medial side of MMT+MCLT-operated knee. Conclusions: This study characterized the effects of surgical MMT+MCLT operation on epiphyseal bone in the rat model of post-traumatic knee OA. The MMT+MCLT operation resulted in increased trabecular bone resorption, turnover and formation without affecting the total amount of trabecular bone. As the operation led to the decreased size of medial tibial epiphysis, trabecular bone area fraction became increased in MMT+MCLT-operated knee. All these findings should be taken into account when selecting animal models for studying the preclinical efficacy of disease modifying OA drug candidates.