Matthew J. Anderson

Musculoskeletal changes following non-invasive knee injury using a novel mouse model of post-traumatic osteoarthritis

OBJECTIVE Post-traumatic osteoarthritis (PTOA) is a common consequence of traumatic joint injury, with 50% of anterior cruciate ligament (ACL) rupture patients developing PTOA within 10-20 years. Currently accepted mouse models of PTOA initiate symptoms using various methods, none of which faithfully mimic clinically-relevant injury conditions. In this study we characterize a novel non-invasive mouse model of PTOA that injures the ACL with a single load of tibial compression overload. We utilize this model to determine the time course of articular cartilage and subchondral bone changes following knee injury. DESIGN Mice were euthanized 1, 3, 7, 14, 28, or 56 days after non-invasive knee injury. Knees were scanned using micro-computed tomography (μCT) in order to quantify subchondral trabecular bone, subchondral bone plate, and non-native bone formation (heterotopic ossification). Development of osteoarthritis (OA) was graded using the osteoarthritis research society international (OARSI) scale on histological sections of injured and uninjured knees. RESULTS Following injury we observed a rapid loss of trabecular bone in injured knees compared to uninjured knees by 7 days post-injury, followed by a partial recovery of trabecular bone to a new steady state by 28 days post-injury. We also observed considerable non-native bone formation by 56 days post-injury. Grading of histological sections revealed deterioration of articular cartilage by 56 days post-injury, consistent with development of mild OA. CONCLUSIONS This study establishes a novel mouse model of PTOA, and describes the time course of musculoskeletal changes following knee injury, helping to establish the window of opportunity for preventative treatment.

Arthroscopic Suture Anchor Repair of the Lateral Ligament Ankle Complex A Cadaveric Study

Background: Operative treatment of mechanical ankle instability is indicated for patients with multiple sprains and continued episodes of instability. Open repair of the lateral ankle ligaments involves exposure of the attenuated ligaments and advancement back to their anatomic insertions on the fibula using bone tunnels or suture implants. Hypothesis: Open and arthroscopic fixation are equal in strength to failure for anatomic Broström repair. Study Design: Controlled laboratory study. Methods: Seven matched pairs of human cadaveric ankle specimens were randomized into 2 groups of anatomic Broström repair: open or arthroscopic. The calcaneofibular ligament and anterior talofibular ligament were excised from their origin on the fibula. In the open repair group, 2 suture anchors were used to reattach the ligaments to their anatomic origins. In the arthroscopic repair group, identical suture anchors were used for repair via an arthroscopic technique. The ligaments were cyclically loaded 20 times and then tested to failure. Torque to failure, degrees to failure, initial stiffness, and working stiffness were measured. A matched-pair analysis was performed. Power analysis of 0.8 demonstrated that 7 pairs needed to show a difference of 30%, with a 15% standard error at a significance level of α = .05. Results: There was no difference in the degrees to failure, torque to failure, or stiffness for the repaired ligament complex. Nine of 14 specimens failed at the suture anchor. Conclusion: There is no statistical difference in strength or stiffness of a traditional open repair as compared with an arthroscopic anatomic repair of the lateral ligaments of the ankle. Clinical Relevance: An arthroscopic technique can be considered for lateral ligament stabilization in patients with mild to moderate mechanical instability.

Long-term administration of AMD3100, an antagonist of SDF-1/CXCR4 signaling, alters fracture repair.

Fracture healing involves rapid stem and progenitor cell migration, homing, and differentiation. SDF‐1 (CXCL12) is considered a master regulator of CXCR4‐positive stem and progenitor cell trafficking to sites of ischemic (hypoxic) injury and regulates their subsequent differentiation into mature reparative cells. In this study, we investigated the role of SDF‐1/CXCR4 signaling in fracture healing where vascular disruption results in hypoxia and SDF‐1 expression. Mice were injected with AMD3100, a CXCR4 antagonist, or vehicle twice daily until euthanasia with the intent to impair stem cell homing to the fracture site and/or their differentiation. Fracture healing was evaluated using micro‐computed tomography, histology, quantitative PCR, and mechanical testing. AMD3100 administration resulted in a significantly reduced hyaline cartilage volume (day 14), callus volume (day 42) and mineralized bone volume (day 42) and reduced expression of genes associated with endochondral ossification including collagen Type 1 alpha 1, collagen Type 2 alpha 1, vascular endothelial growth factor, Annexin A5, nitric oxide synthase 2, and mechanistic target of rapamycin. Our data suggest that the SDF‐1/CXCR4 signaling plays a central role in bone healing possibly by regulating the recruitment and/or differentiation of stem and progenitor cells.

Comparison of loading rate-dependent injury modes in a murine model of post-traumatic osteoarthritis.

Post‐traumatic osteoarthritis (PTOA) is a common long‐term consequence of joint injuries such as anterior cruciate ligament (ACL) rupture. In this study we used a tibial compression overload mouse model to compare knee injury induced at low speed (1 mm/s), which creates an avulsion fracture, to injury induced at high speed (500 mm/s), which induces midsubstance tear of the ACL. Mice were sacrificed at 0 days, 10 days, 12 weeks, or 16 weeks post‐injury, and joints were analyzed with micro‐computed tomography, whole joint histology, and biomechanical laxity testing. Knee injury with both injury modes caused considerable trabecular bone loss by 10 days post‐injury, with the Low Speed Injury group (avulsion) exhibiting a greater amount of bone loss than the High Speed Injury group (midsubstance tear). Immediately after injury, both injury modes resulted in greater than twofold increases in total AP joint laxity relative to control knees. By 12 and 16 weeks post‐injury, total AP laxity was restored to uninjured control values, possibly due to knee stabilization via osteophyte formation. This model presents an opportunity to explore fundamental questions regarding the role of bone turnover in PTOA, and the findings of this study support a biomechanical mechanism of osteophyte formation following injury.

Strength of Bone Tunnel Versus Suture Anchor and Push-Lock Construct in Broström Repair

Background: Operative treatment of mechanical ankle instability is indicated for patients who have had multiple sprains and have continued episodes of instability despite bracing and rehabilitation. Anatomic reconstruction has been shown to have improved outcomes and return to sport as compared with nonanatomic reconstruction. Hypothesis: The use of 2 suture anchors and a push-lock anchor is equal to 2 bone tunnels in strength to failure for anatomic Broström repair. Study Design: Controlled laboratory study. Methods: In 7 matched pairs of human cadaver ankles, the calcaneofibular ligament (CFL) and anterior talofibular ligament (ATFL) were incised from their origin on the fibula. A No. 2 Fiberwire suture was placed into the CFL and a separate suture into the ATFL in a running Krackow fashion with a total of 4 locking loops. In 1 ankle of the matched pair, the ligaments were repaired to their anatomic insertion with bone tunnels. In the other, 2 suture anchors were used to reattach the ligaments to their anatomic origins, and a push-lock was used proximally to reinforce these suture anchors. The ligaments were cyclically loaded 20 times and then tested to failure. Torque to failure, degrees to failure, and stiffness were measured. The authors performed a matched pair analysis. An a priori power analysis of 0.8 demonstrated 6 pairs were needed to show a difference of 30% with a 15% standard error at a significance level of .05. Results: There was no difference in the degrees to failure, torque to failure, and stiffness. A post hoc power analysis of torque to failure showed a power of .89 with 7 samples. Power for initial stiffness was .97 with 7 samples. Eleven of 14 specimens failed at either the suture anchor or the bone tunnel. Conclusion: There is no statistical difference in strength or stiffness for a suture anchor and push-lock construct as compared with a bone tunnel construct for an anatomic repair of the lateral ligaments of the ankle. Clinical Relevance: The use of suture anchors in lateral ligament stabilization allows for a smaller incision, less surgical dissection, and improved surgical efficiency. It is up to the discretion of the performing surgeon based on preference, ease of use, operative time, and cost profile to choose either of these constructs for anatomic repair of the lateral ligaments of the ankle. The suture repair at the ligament was significantly strong enough such that the majority of ankles failed at the bone interface.

In Vivo Fluorescence Reflectance Imaging of Protease Activity in a Mouse Model of Post-Traumatic Osteoarthritis

OBJECTIVE Joint injuries initiate a surge of inflammatory cytokines and proteases that contribute to cartilage and subchondral bone degeneration. Detecting these early processes in animal models of post-traumatic osteoarthritis (PTOA) typically involves ex vivo analysis of blood serum or synovial fluid biomarkers, or histological analysis of the joint. In this study, we used in vivo fluorescence reflectance imaging (FRI) to quantify protease, matrix metalloproteinase (MMP), and Cathepsin K activity in mice following anterior cruciate ligament (ACL) rupture. We hypothesized that these processes would be elevated at early time points following joint injury, but would return to control levels at later time points. DESIGN Mice were injured via tibial compression overload, and FRI was performed at time points from 1 to 56 days after injury using commercially available activatable fluorescent tracers to quantify protease, MMP, and cathepsin K activity in injured vs uninjured knees. PTOA was assessed at 56 days post-injury using micro-computed tomography and whole-joint histology. RESULTS Protease activity, MMP activity, and cathepsin K activity were all significantly increased in injured knees relative to uninjured knees at all time points, peaking at 1-7 days post-injury, then decreasing at later time points while still remaining elevated relative to controls. CONCLUSIONS This study establishes FRI as a reliable method for in vivo quantification of early biological processes in a translatable mouse model of PTOA, and provides crucial information about the time course of inflammation and biological activity following joint injury. These data may inform future studies aimed at targeting these early processes to inhibit PTOA development.

Contribution of mechanical unloading to trabecular bone loss following non-invasive knee injury in mice.

Development of osteoarthritis commonly involves degeneration of epiphyseal trabecular bone. In previous studies, we observed 30–44% loss of epiphyseal trabecular bone (BV/TV) from the distal femur within 1 week following non‐invasive knee injury in mice. Mechanical unloading (disuse) may contribute to this bone loss; however, it is unclear to what extent the injured limb is unloaded following injury, and whether disuse can fully account for the observed magnitude of bone loss. In this study, we investigated the contribution of mechanical unloading to trabecular bone changes observed following non‐invasive knee injury in mice (female C57BL/6N). We investigated changes in gait during treadmill walking, and changes in voluntary activity level using Open Field analysis at 4, 14, 28, and 42 days post‐injury. We also quantified epiphyseal trabecular bone using μCT and weighed lower‐limb muscles to quantify atrophy following knee injury in both ground control and hindlimb unloaded (HLU) mice. Gait analysis revealed a slightly altered stride pattern in the injured limb, with a decreased stance phase and increased swing phase. However, Open Field analysis revealed no differences in voluntary movement between injured and sham mice at any time point. Both knee injury and HLU resulted in comparable magnitudes of trabecular bone loss; however, HLU resulted in considerably more muscle loss than knee injury, suggesting another mechanism contributing to bone loss following injury. Altogether, these data suggest that mechanical unloading likely contributes to trabecular bone loss following non‐invasive knee injury, but the magnitude of this bone loss cannot be fully explained by disuse.

Osteophyte formation after ACL rupture in mice is associated with joint restabilization and loss of range of motion.

Osteophytes are a typical radiographic finding during osteoarthritis (OA). Osteophytes are thought to form in response to joint instability; however, the time course of osteophyte formation and joint stabilization following joint injury is not well understood. In this study, we investigated the time course of osteophyte formation and joint function following non‐invasive knee injury in mice. We hypothesized that initial joint instability following knee injury would initiate osteophyte formation, which would in turn restabilize the joint and reduce range of motion (ROM). Mice were subjected to non‐invasive anterior cruciate ligament (ACL) rupture. Anterior–posterior (AP) joint laxity, ROM, and chondro/osteophyte formation were measured immediately after injury, and 2, 4, 6, and 8 weeks post‐injury. Chondrophyte areas at each time point were measured with histology, while mineralized osteophyte volume was determined using micro‐computed tomography. Immediately after ACL rupture, AP joint laxity was increased twofold, while ROM was increased 11.7%. Chondrophytes appeared by 2 weeks post‐injury, corresponding with a decrease in AP joint laxity and ROM. By 8 weeks post‐injury, considerable osteophyte formation was observed around the joint, AP joint laxity returned to control levels, and joint ROM decreased to 61% of control values. These data support a role for chondro/osteophytes in joint restabilization after injury, and provide crucial insight into the time course and pathology of joint degeneration during OA development in the mouse. Statement of Clinical Significance: Results from this study increase understanding of conditions leading to osteophyte formation.© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:466–473, 2017.