Biomechanical, histologic, and molecular characteristics of graft-tunnel healing in a murine modified ACL reconstruction model

PURPOSE: The purpose of our study was to introduce and validate a metal-free, reproducible and reliable mouse model of anterior cruciate ligament (ACL) reconstruction (ACLR) surgery as an effective tool for a better understanding of molecular mechanisms of graft-tunnel healing after ACLR. METHODS: A total of 150 C57BL/6 mice were randomly allocated into five Groups: Group 1 (mice with intact ACL), Group 2–4 (mice underwent modified ACLR surgery and sacrificed 1-, 2-, and 4-weeks after surgery), and Group 5 (mice underwent unmodified ACLR surgery and sacrificed 4 weeks after surgery). Micro-computed tomography (CT), biomechanical histological as well as immunohistochemical (IHC) analyses were performed to characterize the modified ACLR. RESULTS: Micro-CT analysis demonstrated there is a non-significant increase in BV/TV and BMD of the bone tunnel during the tendon-to-bone healing following ACLR. Biomechanical tests showed that the mean load-to-failure forces of Group 3 and 4 are equal to 31.7% and 46.0% of that in Group 1, while the stiffness was 33.1% and 57.2% of that of Group 1, respectively. And no obvious difference in biomechanical parameters was found between Group 4 and 5. Histological analysis demonstrated that formation of fibrovascular tissue in the tibial tunnel and aperture in Groups 4 and 5 and direct junction appeared between tendon graft and tunnel both in Groups 4 and 5. IHC results showed that there are gradually enhanced expression of Patched1, Smoothened and Gli2 concomitant with decreased Gli3 protein in the tendon-bone interface during the tendon-bone healing process. CONCLUSION: We introduced a metal-free, reproducible and reliable mouse model of ACLR compared to the unmodified ACLR procedure, and characterized the expression pattern of key molecules in Ihh signaling during the graft healing process. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: In the present study we introduced and validated, for the first time, a metal-free, reproducible and reliable ACLR mouse model, which could be used to investigate the detailed molecular mechanisms of graft-tunnel healing after ACLR. We also explored new strategies to promote the healing of tendon-to-bone integration.