Summary

Hindlimb unloading prior to restabilization surgery is an effective biomechanical intervention for slowing PTOA progression following non-invasive ACL injury in mice, while restabilization surgery after one week of normal weight bearing post-injury accelerates OA progression compared to injury alone.

Abstract

Introduction

Post-traumatic osteoarthritis (PTOA) occurs following injury such as anterior cruciate ligament (ACL) rupture. We previously showed that restabilizing the knee immediately after ACL injury effectively slows PTOA progression in mice. It is currently unclear if unloading the injured joint following injury prior to surgical stabilization alters PTOA progression. The goal of this study was to determine if one week of hindlimb unloading (HLU) following ACL injury prior to surgical restabilization would affect PTOA progression in the mouse. We hypothesized that HLU would slow the progress of PTOA by diminishing inflammation and tissue damage in the joint, whereas restabilizing knee joints after normal weight bearing would accelerate PTOA progression.

Methods

53 female skeletally mature C57BL/6J mice were used in four experimental groups: control (Uninjured), injured-unoperated (Injured), injured with restabilization surgery after one week of normal cage activity (Restabilized), and injured with restabilization surgery after one week of hindlimb unloading (HLU-Restabilized). Mice were subjected to non-invasive ACL injury using a single tibial compression overload. HLU mice were tail-suspended following injury in a 30-degree head-down angle and were provided with food, water, and environmental enrichment.
Surgical stabilization was achieved extra-articularly by passing a suture through a transverse bone tunnel in the anterior proximal tibia around the posterior aspect of the femoral condyle under the fabellofemoral ligaments and tied on the lateral side of the joint.
At 2, 4, and 6-weeks post-injury, mice were imaged with in vivo near-infrared fluorescence reflectance imaging (FRI) to determine protease activity in the joint using a commercially-available fluorescence activatable probe. At 3 and 6-weeks post-injury, whole knees were analyzed with microcomputed tomography to measure epiphyseal trabecular bone microstructure and osteophyte volume.

Results

At week 3, Injured mice experienced epiphyseal trabecular bone volume loss (-15% BV/TV); Restabilized mice had ~30% loss of trabecular bone volume regardless of HLU. MicroCT analysis revealed considerable osteophyte formation in Injured, Restabilized, and HLU-Restabilized mice, although osteophyte volume in HLU-Restabilized mice was significantly less than in Restabilized. FRI analysis showed inflammatory protease activity was significantly increased in Restabilized, but not in the other groups.

Discussion

HLU after ACL injury prior to restabilization can effectively slow PTOA progression and osteophyte formation but does not mitigate loss of epiphyseal trabecular bone. Restabilization surgery results in more trabecular bone loss than ACL injury alone, suggesting that bone loss may be driven by biological factors such as inflammation.
In mice, loading before restabilization accelerates protease activity and OA progression. Unloading injured limbs prior to restabilization may slow PTOA progression, especially during the early inflammatory phase. However, further studies are needed to identify the specific underlying mechanisms.

SIGNIFICANCE/CLINICAL RELEVANCE: Hindlimb unloading of mice after non-invasive ACL injury prior to restabilization is an effective biomechanical intervention for slowing PTOA progression, while restabilization surgery after one week of normal weight bearing post-injury will accelerate OA progression compared to injury alone. The results of this study may affect “prehab” strategies for human subjects after ACL injuries prior to reconstruction, highlighting the efficacy of early unloading for slowing or preventing the initiation of PTOA.