(L-452) Anterior cruciate ligament rupture via a non-invasive knee injury causes structural cartilage degeneration, increased pain sensitivity, and impaired limb function in a preclinical rat model.

Introduction:: Post-traumatic osteoarthritis (PTOA) is an onerous joint disease that develops following injury and is associated with greater years lived with disability than primary osteoarthritis. Injuries, such as anterior cruciate ligament rupture (ACLR), dramatically increase the risk of developing PTOA. Current treatments largely focus on reducing symptomatic pain and no available drugs prophylactically target or reverse disease progression. The PTOA research community remains challenged by a limited understanding of joint disease etiology and a lack of animal models that effectively recapitulate the human PTOA disease state. The medial meniscal transection (MMT) surgery is the most common preclinical model that results in a consistent disease pathology primarily impacting the medial 1/3 of the medial tibial plateau^1–3. However, surgical destabilization fails to capture how PTOA develops in humans following a knee injury (e.g., ACLR). Therefore, this study used a noninvasive knee injury device (NIKI)⁴ to produce ACLR in a rat PTOA model. Limb function and pain sensitivity were longitudinally assessed, and PTOA structural pathology was examined in the knee joint via contrast enhanced micro-computed tomography (µCT). We hypothesized that NIKI-induced ACLR would result in structural changes representative of a mild PTOA pathology after 4 weeks and more severe degeneration by 8 weeks, with both timepoints accompanied by impaired limb function and increased pain sensitivity.

Materials and Methods:: A non-invasive compressive load was applied to the left hindlimb of male Lewis rats to cause either ACLR-induced PTOA (n=20) or sham injury, consisting of an atraumatic static preload (n=7). All procedures were conducted under local IACUC approved protocols. Spontaneous pain behavior as a measure of limb function (Bioseb Dynamic Weight Bearing System) and pain sensitivity (tactile allodynia) were assessed using von Frey testing at 4, 6, and 8 weeks post-procedure with measurements normalized to individual baseline values. Rats with ACLR were euthanized after 4 weeks (n=10) or 8 weeks (n=10) and sham animals (n=7) after 8 weeks. Limbs were subsequently harvested and fixed. Knee joint tissues were carefully dissected to visually confirm ACLR and isolate both tibiae and femora. Contrast-enhanced-µCT (Scanco Medical, Switzerland) and custom evaluation scripts previously validated in the MMT disease model^1–3 were used to compute structural morphometrics in the medial 1/3 and full medial tibial plateau (cartilage volume, cartilage thickness, cartilage X-ray attenuation, osteophyte volumes, etc.) (n=6/group). The remaining limbs were prepared for whole joint histology for verifying ACLR and qualitative joint tissue changes (n=4/group).

Longitudinal outcomes were statistically analyzed using a mixed-model analysis of variance (ANOVA) with post-hoc Bonferroni analyses for multiple comparisons (Graphpad Prism, Boston, MA). Statistical significance is reported as p< 0.05.

Results, Conclusions, and Discussions::

Results: Significantly reduced hind limb weight bearing was recorded in the injured hindlimb at all timepoints versus sham controls, with the greatest differences at 4 weeks (Fig 1A). Notably, this was accompanied by a corresponding increase in weight bearing on the contralateral hind limb and further corroborated by reduction in the ratio of weight bearing on the injured versus contralateral limbs compared to baseline measurements (Fig 1B). Pain sensitivity measurements showed injury resulted in a significantly lower force to induce a paw withdrawal response to the von Frey probe at 4 and 6 weeks versus baseline sensitivity but recovered by 8 weeks (Fig 1C). Conversely, a decrease in pain sensitivity was observed in contralateral uninjured limbs at 6 weeks, followed by a trend towards greater sensitivity at 8 weeks.

Thickening of the medial tibial plateau cartilage, osteophyte formation, subchondral bone remodeling effects, and severe degradation of the posterior aspects of the medial tibial plateau were qualitatively observed in µCT sagittal and axial slices of injured hind limb tibias (Fig 2). Quantitative morphometric analyses for cartilage, osteophytes, and subchondral bone remain ongoing. Preliminary evaluations suggest average medial 1/3 cartilage thickness measurements may provide a more sensitive metric of degenerative changes compared to the average cartilage thickness over the entire medial tibial plateau (Fig 3), although samples sizes are not yet fully powered.

Discussion and Conclusion: Changes in limb function and pain sensitivity suggest animals offload to the contralateral hind limb and ipsilateral forelimb following ACLR. Moreover, this is accompanied by dramatic increases in injured hind limb pain sensitivity for 6 weeks after injury, which recovers by 8 weeks. Interestingly, reduced sensitivity in the contralateral hind limb at 6 weeks, followed by increased sensitivity at 8 weeks, may capture the detrimental effects of limb offloading. While µCT evaluations remain ongoing to fully power these metrics, current results suggest quantitative differences in cartilage thickness. Continuing work will more fully evaluate ACLR-induced cartilage swelling as well as quantify aberrant bone morphology (bone remodeling, osteophytes) with revised evaluation scripts. Future studies will seek to both elucidate and target mechanisms of PTOA progression following ACLR.

Acknowledgements (Optional): :

Funding for this work was provided by the Wu Tsai Human Performance Alliance at Oregon.

References (Optional): :

1.           Willett NJ, Thote T, Hart M, Moran S, Guldberg RE, Kamath RV. Quantitative pre-clinical screening of therapeutics for joint diseases using contrast enhanced micro-computed tomography. Osteoarthritis and Cartilage. 2016;24(9):1604-1612. doi:10.1016/j.joca.2016.04.021

2.           Willett NJ, Thote T, Lin AS, et al. Intra-articular injection of micronized dehydrated human amnion/chorion membrane attenuates osteoarthritis development. Arthritis Res Ther. 2014;16(1):R47. doi:10.1186/ar4476

3.           D S Reece, T  Thote, A S P Lin, N J Willett, R E Guldberg. Contrast enhanced μCT imaging of early articular changes in a pre-clinical model of osteoarthritis. Osteoarthritis and cartilage. 26 1. doi:10.1016/j.joca.2017.10.017

4.           Brown SB, Hornyak JA, Jungels RR, et al. Characterization of Post-Traumatic Osteoarthritis in Rats Following Anterior Cruciate Ligament Rupture by Non-Invasive Knee Injury (NIKI). J Orthop Res. 2020;38(2):356-367. doi:10.1002/jor.24470