Restriction of Postoperative Joint Loading in a Murine Model of Anterior Cruciate Ligament Reconstruction: Botulinum Toxin Paralysis and External Fixation

Abstract

Control of knee motion in small animal models is necessary to study the effect of mechanical load on the healing process. This can be especially challenging in mice, which are being increasingly used for various orthopedic reconstruction models. We explored the feasibility of botulinum toxin (Botox; Allergan, Dublin, Ireland) paralysis and a newly designed external fixator to restrict motion of the knee in mice undergoing anterior cruciate ligament (ACL) reconstruction. Nineteen C57BL/6 mice were allocated to two groups: (1) Botox group ( = 9) and (2) external fixator group ( = 10). Mice in Botox group received two different doses of Botox: 0.25 unit ( = 3) and 0.5 unit ( = 6). Injection was performed 72 hours prior to ACL reconstruction into the quadriceps, hamstring, and calf muscles of the right hind leg. Mice in external fixator group received an external fixator following ACL reconstruction. Mice were monitored for survival, tolerance, and achievement of complete knee immobilization. All mice were meant for sacrifice on day 14 postoperatively. No perceptible change in gait was observed with 0.25 unit of Botox. All mice that received 0.5 unit of Botox had complete hind limb paralysis documented by footprint analysis 2 days after injection but failed to tolerate anesthesia and were euthanized 24 hours after operation due to their critical condition. In contrast, the external fixator was well tolerated and effectively immobilized the limb. There was a single occurrence of intraoperative technical error in the external fixator group that led to euthanasia. No mechanical failure or complication was observed. Botox paralysis was not a viable option for postoperative restriction of motion and joint loading in mice. However, external fixation was an effective method for complete knee immobilization and can be used in murine models requiring postoperative control of knee loading. This study introduces a robust research tool to allow control of postoperative joint loading in animal models such as ACL reconstruction, permitting study of the effects of mechanical load on the biologic aspects of tendon-to-bone healing.

Postoperative Tendon Loading With Treadmill Running Delays Tendon‐to‐Bone Healing: Immunohistochemical Evaluation in a Murine Rotator Cuff Repair Model

“…The animals were randomly allocated to either the unloaded group or the treadmill group. Mice in the unloaded group were injected with botulinum toxin in the supraspinatus muscle at the time of surgery, with the goal of producing paralysis of the muscle . The contralateral shoulder in the unloaded group at each time point was used as an unoperated control.…”

“…Minimum score is 7 meaning poor maturation and maximum score is 28 meaning excellent maturation. This score is modified from previously reported literature …”

Amplifying Bone Marrow Progenitors Expressing α‐Smooth Muscle Actin Produce Zonal Insertion Sites During Tendon‐to‐Bone Repair

“…The anterior cruciate ligament (ACL) is one of the most common reconstructed ligaments. In this study, a mouse model of ACL reconstruction, similar to a previous work, is used. Because of the vast array of transgenic mouse models, specific cell populations can be genetically modified to better understand the mechanisms of the repair process.…”

Bone marrow stromal cells from a GDF5 origin produce zonal tendon-to-bone attachments following anterior cruciate ligament reconstruction

“…Two events are crucial to successful biological repair following ACL reconstruction: 1) cells need to revitalize the graft, as resident cells within the graft frequently die following reconstruction, and 2) the soft tissue graft needs to integrate with the adjacent bone in the tunnels through the formation of zonal attachments 6,21 . We developed this mouse ACL reconstruction model, which is similar to previous murine ACLR models [10][11][12] , to monitor graft revitalization following reconstruction and the formation of mineralized attachments within the bone tunnel ( Figs. 4-6).…”

“…In addition, a key signaling pathway in enthesis embryogeneis, hedgehog signaling, was shown to be active in tunnel integration in a rat ACL reconstruction model 9 . ACL reconstruction in mice has been established [10][11][12] , which opens up the possibility of using powerful genetic tools found in various mouse strains to better define the molecular regulation of tendonto-bone repair.…”