Regional Articular Cartilage Deterioration Following Knee Injury in an Osteoarthritis In Vivo Rat Model

Purpose (the aim of the study): Osteoarthritis (OA) in vivo models are essential for studying disease progression and the efficacy of novel treatments. This study utilizes a mechanical non-invasive anterior cruciate ligament (ACL) tear method to mimic the natural progression of post-traumatic OA in an in vivo rat model. Key timepoints over a span of 10-weeks post-injury were examined to assess cartilage deterioration and the efficacy of an intra-articular (IA) treatment in slowing OA progression. Region-specific mechanical testing and histology provided complementary insights into functional and structural degradation.

Methods: Following Canadian guidelines for animal care and experimentation, forty-four 14-week-old female Lewis rats were assigned to 3 experimental groups: Control (N=12), ACL Rupture Non-Treated (N=24), and ACL Rupture Treated (N=8). ACL ruptures were induced by anterior subluxation of the tibia over the femur using a uniaxial mechanical tester and custom fixtures for rapid tibial compression. Complete ACL tear was verified through manual drawer tests and confirmed by force-displacement data analysis and microscopic imaging. The ACL Rupture Treated group received weekly IA hyaluronan injections from weeks 4 to 6 post-injury. Necropsies took place at week 4, 7, and 10, at which time femoral condyles and tibial plateaus were harvested for mechanical testing. Mechanical properties, including cartilage thickness and instantaneous modulus (IM), were averaged by region from measurements at ~30 positions per articular surface using a triaxial mechanical tester. Samples were then formalin-fixed, decalcified in 0.5N HCl/1% glutaraldehyde, and paraffin-embedded. Paraffin sections (5 µm thick) were stained with Safranin O/Fast Green and H&E for histological assessment. Statistical analysis was performed using a linear mixed model.

Results: Macroscopic inspection of joints at necropsy confirmed complete ACL ruptures with intact surrounding ligaments. In the Control group, cartilage thickness and IM remained stable across timepoints for both femoral condyles and tibial plateaus (p = 0.39 and p = 0.19, respectively), thus all Control group data were pooled. Non-Treated ACL rupture joints exhibited significant cartilage thinning in the anterior medial femoral condyle starting at 4 weeks post-injury (p < 0.001 vs. Control), persisting through 10 weeks (Fig.1A). In contrast, the lateral condyle showed cartilage swelling from 4 to 7 weeks, followed by thinning at 10 weeks (Fig.1B). The IM showed similar regional differences as it increased at 4 and 7 weeks in the anterior medial condyle before decreasing at 10 weeks (Fig.1C), while remaining stable in the anterior lateral condyle until a final increase at 10 weeks (Fig.1D). Mechanical data suggests that IA injections were able to slow the deterioration of cartilage mechanical properties. Histology also revealed severe stages of OA in the medial anterior condyle of Non-Treated samples with complete cartilage denudation by 7 weeks and bone remodeling on the margins of the denuded articular surface evident by 10 weeks (Fig.2). In the lateral anterior condyle, Non-Treated joints exhibited normal cartilage structure and morphology at 4 weeks with some focal defects and thinning at 10 weeks. Despite preserved mechanical properties, Treated femoral condyles showed similar histological erosion in the anterior regions.

Conclusions: This non-invasive method effectively induced consistent ACL ruptures for this in vivo rat model, leading to typical OA knee pathology. Region-specific alterations were identified in the condyles, with the medial anterior region showing pronounced cartilage erosion and mechanical weakening while the lateral anterior region showed milder signs of OA. While effects were most pronounced in the anterior condyle, changes were also observed in the posterior regions and the tibial plateaus, indicating broader impacts of the ACL injury. The IA treatment partially mitigated cartilage degradation by preserving mechanical properties closer to Control levels but showed no signs of reversing cartilage erosion. These findings validate the utility of this OA model for studying disease progression and evaluating therapeutic interventions.