Poster 203: Mechanical Loading Effects on OA Progression Following ACL Injury and Reconstruction in a Murine Model: Radiographic Assessment of Synovial Fibroblast Activity

Abstract

Objectives:

Anterior cruciate ligament (ACL) injury is one of the leading orthopedic injuries in young individuals and even with surgical repair it is reported that around 50% of patients develop post-traumatic osteoarthritis (PTOA). Although ACL reconstruction improves anterior-posterior knee joint stability and cartilage contact biomechanics, some studies have shown persistent abnormalities in the tibiofemoral kinematics. Extensive rehabilitation work is often required following both ACL tear and ACL reconstruction surgery. New bone formation (osteophyte) and synovial inflammation (synovitis) are often associated with the progression and severity of OA post-injury and surgery, however the effect of mechanical loading stimulus in musculoskeletal repair remains relatively unexplored. The role played by resident synovial stromal cells in the activation of their local inflammatory and regenerative programming is poorly understood. Work in our laboratory has shown that in vivo imaging of activated fibroblasts using ⁶⁸Ga-FAP radiotracer may be a valuable tool to follow the progression of OA and the bone-tendon interface healing. This study aims to explore the therapeutic effect of exercise following ACL reconstruction on the progression or mitigation of PTOA following an ACL injury, evaluating the mechanism and contribution of synovial fibroblast activation using microPET/CT imaging.

Methods:

All study procedures were approved by Weill Cornell IACUC (#2019-0034). Thirty, 12 week old male C57BL/6 mice underwent a non-invasive ACL rupture induced by a single axial compressive load of the tibia at a rate of 1mm/s on the right knee (Fig. 1). One week following injury, the animals were allocated into 4 treatment groups (n=6 per group): non-treated (G1); non-treated with sham surgery (G2); treated with ACL reconstruction (G3); treated with ACL reconstruction and treadmill exercise starting 1 week post-operatively (G4). Six mice were used to harvest the ACL tendon graft (allograft) and their knees scanned in the microCT as healthy controls.Treadmill exercise was performed 5 days/week, at 10m/min for 40min. PET/CT (Inveon scanner - Siemens Medical Solutions) imaging was performed pre-injury (baseline) and at 1, 2, 3, 4 and 8 weeks post-injury to measure fibroblast activation in the joint. One hundred μl of a 7% v/v EtOH/saline solution containing our ⁶⁸Gallium (Ga)68-labeled fibroblast activation protein alpha (FAP-α) inhibitor (3.7-18.5MBq) was administered by tail vein injection and the mice were scanned for 60 minutes under anesthesia. The knee joint was identified as the region of interest (ROI) and the radioactivity concentration (Bq/ml x %ID/g) within the ROI was calculated using the Siemens imaging software. All animals were euthanized at 8 week post-injury. ACL graft healing and OA development were evaluated by micro-CT, X-Ray and routine histology. Statistical analyses were performed by Two-way ANOVA and Tukey’s post hoc test analysis was used to compare means ± SD. Significance level was defined as p ≤ 0.05.

Results:

PET/CT scans demonstrated accumulation of ⁶⁸Ga-FAP in the injured and surgical knees compared to the contralateral healthy control knees (Fig. 2). FAP signal intensity peaked at 1 week after injury and continued increasing at 2 weeks in the surgical groups, with significant differences demonstrated in the ACL reconstructed groups (G3 and G4, p<0.0001) compared to the non-treated group (G1). Group 3 showed significantly higher signal intensity at 3 weeks compared to the non-treated groups (G1 and G2, p<0.0001). There was significantly lower FAP activity in Group 4 (ACLr with exercise) compared to the surgical non exercised group (G3, p=0.043) at 4 weeks. All groups were significantly higher than the baseline at 1, 2, 3 and 4 weeks. No significant differences were found at 8 weeks between the groups or when compared to the pre-operative time point. Micro-CT analysis revealed marked anatomical changes in the meniscus volume, femoral and tibial bones, as well as ectopic ossification and osteophyte formation mainly in the medial compartment (Fig. 3). These changes were more evident in the non-treated groups (G1 and G2), demonstrating a prominent increase in the contact stress in the posterior medial compartment leading to an erosion of the tibial plateau. In contrast, the ACL reconstruction groups (G3 and G4) demonstrated less or no erosion of the posterior medial tibial plateau. Additionally, the amount of new bone formed at the tendon-bone interface was greater and more consistent in the tibia tunnel of Group 4; however, the differences were not statistically significant.

Conclusions:

The intimal layer of the native synovium is composed of 2 main cell types: macrophages and fibroblast-like cells. Recent studies have shown a correlation between activated macrophages and their association with OA severity and joint symptoms; however, the cross-talk between these 2 types of cells and their contribution to PTOA following ACL injury and reconstruction surgery is poorly understood. Our study identified time-dependent changes in FAP activity following ACL reconstruction and mechanical loading stimulus that could be correlated to PTOA development. Our data demonstrates that ACL reconstruction followed by a low intensity exercise regimen leads to a decrease of FAP activity in the ACL injured knee. We also found that the ACL reconstruction groups had less degenerative changes in the posterior medial tibial plateau, which may be due to an improvement of anterior-posterior joint stability, and the exercise group showed a greater amount of bone formed at the tendon-bone interface of the tibia suggesting a superior graft healing process. Immunochemistry for FAP-α is currently being analyzed to confirm the spatial localization of FAP expression.

Figure 1.

Study design, All animals underwent a non-invasive ACL rupture and were allocated into 4 treatment groups: non-treated (Gl); non-treated with sham surgery (G2); treated with ACL reconstruction (G3); treated with. ACL reconstruction and treadmill exercise.

Figure 2.

PET/CT representative coronal images of the 4 different groups and quantitative results for 68Ga-FAPI uptake over time.

Figure 3.

microCT analysis demonstrating the surface morphology of the mouse knee joint showing the marked structural changes in the posterior medial compartment leading to erosion of the tibial plateau (A, yellow circle) and the amount of new bone formed al the tendon-bone interface of the tibia in groups 3 and 4 (B).