Summary

This study demonstrates the effects of short-term semi-rigid casting after ACL reconstruction, showing that casting improves early new bone formation within the bone tunnel and tendon-to-bone healing. Short-term semi-rigid immobilization may be a key factor in accelerating early healing and facilitating an early return to sport after ACL reconstruction.

Abstract

Introduction

Functional recovery following anterior cruciate ligament reconstruction (ACL-R) requires secure healing of the tendon graft in the bone tunnel, and early post-operative immobilization may accelerate this process. However, there is a lack of consensus amongst clinicians with regard to use and duration of knee immobilizers post-operatively. Many animal studies focusing on post-operative immobilization have utilized external fixators and reported positive effects after ACL-R. However, the external fixator method is rigid, invasive, and not used following ACL-R in the clinical setting. A three-dimensional printed, semi-rigid knee cast has been utilized previously for the mouse knee and represents a non-invasive, more clinically relevant method of knee immobilization. The purpose of the current study is to elucidate the effects of short-term immobilization using semi-rigid casting following ACL-R in mice. We hypothesized that short-term casting would improve early tendon-to-bone healing and functional outcomes after ACL-R.

Methods

All experiments were approved by the Institutional Animal Care and Use Committee. Forty-five 12-week-old male C57BL/6J mice (average weight: 26 g) were used in this study and underwent open ACL transection followed by immediate ACL-R surgery in the right knee using ipsilateral flexor digitorum longus tendon autograft. The mice were randomly assigned to one of the three groups post-operatively: 1) no casting (free cage) group, 2) 5-day casting group followed by free cage activity, and 3) 14-day casting group. All mice were euthanized at two weeks post-surgery. The primary outcome measures were new bone formation and tendon-to bone interface healing within the bone tunnel, assessed by micro-CT and histology (tendon-to-bone tunnel healing [TBTH] score). Secondary outcomes included biomechanical properties, knee range of motion (ROM), and gene expression related to pain (assessed in the lumbar dorsal root ganglia) and muscle atrophy markers (measured in the tibialis anterior muscle). Gait analysis was also performed using DigiGait. Histological and biomechanical assessments were conducted on 5 and 10 mice per group, respectively. qPCR was performed on 6 mice per group, while the other measurements were conducted on 15 mice per group. Statistical analysis was carried out using one-way ANOVA followed by multiple comparisons using the Bonferroni test, with statistical significance set at p < 0.05.

Results

In both casting groups, bone volume formation (BV/TV) and bone mineral density (BMD) in the tibial tunnel were higher than in the free cage group. The TBTH score in the tibial tunnel was also higher in both casting groups compared to the free cage group. No significant differences were observed in biomechanical ultimate load to failure. All groups exhibited restricted ROM compared to the contralateral knee, with the 14-day casting group showing significantly less ROM than the other groups. However, the 5-day casting group had significantly greater ROM than the free cage group. Additionally, the variability in paw area and angle of the affected limb measured with gait analysis was significantly decreased in the 5-day casting group. There were no significant differences in pain marker gene expression among groups, but genes related to muscle atrophy (CAPN1 and CTSB) were significantly upregulated in both casting groups.

Discussion

These findings suggest that postoperative casting improves new bone formation within the bone tunnel and tendon-to-bone healing in the tibial tunnel at 2 weeks after ACL-R. The 5-day casting group demonstrated better ROM and improvements in some gait parameters, while the 14-day casting group showed significantly decreased knee ROM. We hypothesize that the improvement in knee motion at 2 weeks following an initial period of casting is due to resolution of the postsurgical inflammatory process. However, genes related to muscle atrophy were upregulated in both casting groups, and muscle atrophy after ACL-R can be deleterious to recovery and return to sport. This study suggests that non-invasive short-term immobilization followed by free cage activity may improve tendon-to-bone healing while also improving ROM and some gait parameters. Further research is needed to determine the optimal regimen and to elucidate the molecular mechanisms underlying this short-term casting effect.