Abstract
Objectives:
Recent studies have been using the anterior cruciate ligament reconstruction (ACLR) mouse model to study the tendon-bone interface (TBI) healing process and the development of post-traumatic osteoarthritis. Our prior research indicated that moderate treadmill exercise following ACLR had a positive impact on TBI healing in both the femoral and tibial tunnels. However, no study has yet examined the impact of treadmill exercise following an initial period of post-operative immobilization following an ACLR in mice. The purpose of this study is to explore the effects of immobilization followed by delayed onset of mechanical loading on TBI healing following ACLR in mice. We hypothesized that moderate loading following an initial regimen of short-term immobilization would have a beneficial effect on TBI healing, and would ameliorate the adverse effects of prolonged immobilization following ACLR.
Methods:
A total of 108 male C57BL/6 mice, aged 12 weeks, underwent an ACL reconstruction in the right knee, as previously described. The speed and frequency of treadmill exercise were based on our prior research. The mice were divided into 7 groups (Figure 1): (1) control, (2) No immobilization (fix) + free cage (FC), (3) No fix + moderate treadmill (10m/min for 50min), (4) 5-day fix + FC, (5) 28-day fix + FC, (6) 5-day fix + moderate treadmill, and (7) 5-day fix + intense treadmill (20m/min for 50min). A three-dimensional printed knee brace was utilized for immobilization (Figure 2) to avoid inflammation associated with pin-dependent external fixation. All mice underwent a 5-day treadmill acclimation protocol prior to surgery.
Starting 7 days post-surgery, the treadmill exercise was performed 5 days/week for a total of 3 weeks. All animals were euthanized at 4 weeks post-surgery. Outcome measures included knee range of motion (ROM), gait, trabecular bone microstructure (microCT), histology (tendon-to-bone tunnel healing [TBTH] score), biomechanical properties, and gene expression. ROM was measured using a handmade three-dimensional goniometer, where the knee holder was subjected to a force equivalent to half the body weight. Gait patterns were analyzed using the DigiGait Imaging system (Mouse Specifics, Inc.). Micro-CT imaging was performed using a Scanco μCT 45 system (Scanco Medical). The bone tunnel was identified, a region of interest (ROI) was defined as the bone tunnel diameter (0.64mm) for evaluation of each value. The TBTH score was assessed using an established histological scoring system. The tunnel was segmented into two subregions: extra-articular and intra-articular part. Biomechanical testing was performed using a materials-testing machine (EnduraTEC ELF 3200). The specimens were subjected to tensile loading at a rate of 5.0 mm/min and the load-to-failure (N) data and displacement (mm) were recorded. qPCR analysis of the tibialis anterior (TA) and lumbar dorsal root ganglia from L2 to L6 was performed with mouse-specific primers for genes relevant to muscle atrophy and pain evaluation, as previously described.
Results:
Except for group 6 (5-day fix + moderate treadmill), ROM was significantly restricted compared to the control group. Gait was significantly impaired in group 5 (28-day fix + FC) compared to all other groups. Micro-CT evaluation revealed that group 4 and 6 (5-day fix and/or 10m/min) exhibited significantly higher values compared to group 2 (No fix + FC) in certain categories, both in the femur and tibia. Histological evaluation demonstrated significantly higher TBTH scores in group 6 (5-day fix + moderate treadmill) compared to the free cage activity group in all groups, except for the extra-articular part of the tibia which exhibited substantial healing in all groups (Figure 3). No significant difference was observed in failure load among all groups while stiffness was significantly lower in group 5 (28-d fix + FC). Genes associated with nociception (Cgrp and Ngf) were significantly upregulated in group 2 and 3 (no fix and/or moderate treadmill), while genes associated with muscle atrophy (FOXO 1 and FOXO32) were significantly upregulated in group 5 (28-day fix + FC).
Conclusions:
The most important finding of this study is superior healing in group 6 (5-day fix+ moderate treadmill) compared to group 2 (no fix + FC), based on ROM that was comparable to group 1 (control), and higher trabecular thickness/bone mineral density, as well as TBI healing score. Additionally, prolonged immobilization (group 5, 28-day fix + FC) demonstrated a significantly impaired gait pattern, lower stiffness, and upregulated expression of genes related to muscle atrophy. These findings suggest that long-term immobilization has a detrimental effect on physical recovery, while moderate treadmill exercise following an initial period of short-term immobilization following ACLR has a positive impact on TBI healing, confirming our hypothesis. We believe that the combination of both approaches represents the optimal strategy to optimize TBI healing throughout the bone tunnel. Further studies are warranted to investigate alternative post-operative loading regimens and follow-up timepoints to identify the optimal protocol to improve TBI healing following ACLR. A moderate load rehabilitation protocol following an initial period of post-operative immobilization might result in improved healing of the tendon graft-to-bone interface following ACLR.
