Anterior cruciate ligament injury as a driver of post-traumatic osteoarthritis: mechanisms and outcomes elucidated from preclinical models

Abstract

Anterior cruciate ligament (ACL) injuries are among the most prevalent orthopedic injuries in active populations and are a leading cause of knee post-traumatic osteoarthritis (PTOA), a debilitating joint condition. PTOA remains a challenging clinical condition, particularly due to its multifactorial pathogenesis and lack of disease-modifying therapies. Emerging research indicates that biological sex significantly influences both the susceptibility to ACL injuries and the progression of PTOA, yet the underlying mechanisms remain poorly understood. Additionally, limitations in current surgical approaches to ACL reconstruction continue to impede long-term joint preservation. The overarching goal of this dissertation was to investigate both biological and surgical determinants of PTOA with the intent of informing improved therapeutic strategies for individuals following ACL rupture. This work is structured around three aims: 1) investigating the role of sex in PTOA progression, 2) probing estrogen signaling as a modulator of PTOA development, and 3) evaluating novel surgical reconstruction strategies in a large animal model. In Aim 1, we used a non-surgical, mechanical mouse model of ACL rupture to explore sex-specific differences in the development of PTOA following joint trauma. Male and female C57BL/6J mice were subjected to unilateral ACL rupture and were monitored over time using functional and structural assessments to comprehensively characterize PTOA progression. These included weekly mobility analyses such as voluntary mobility, static weight-bearing, and gait analyses to track symptomatic development. In parallel, joint degeneration was evaluated longitudinally through radiographic imaging and at terminal timepoints via histological scoring. ACL tissues from injured and control limbs were collected at multiple early timepoints for untargeted, label-free proteomic profiling, enabling characterization of dynamic molecular responses to injury. Our findings revealed that male mice demonstrated greater impairment in mobility, including reduced overall activity and altered hindlimb stance, in comparison to females. Histological analysis further confirmed more severe joint pathology in males. Interestingly, radiographic measures showed limited sex-based differences, suggesting that early structural joint changes may be more sensitively captured by histology and behavior than by imaging alone. Proteomic analysis revealed a markedly different injury response between sexes. Female mice exhibited an early, robust molecular response peaking at three days post-injury, with upregulation of pathways associated with inflammation, coagulation, and estrogen signaling. Males showed a more attenuated and prolonged proteomic response, suggesting sex-specific differences in the temporal dynamics of molecular signaling post-injury. Collectively, these findings identify significant sex-based variation in both the symptomatic and molecular trajectory of PTOA following ACL rupture, reinforcing the need for sex-specific approaches to therapeutic development and clinical management. Aim 2 built upon these findings by exploring the role of estrogen signaling in modulating PTOA progression, with a focus on its contributions in the male mouse joint. Given the absence of early estrogenic signaling in male mice observed in Aim 1, we hypothesized that localized manipulation of estrogen pathways could influence PTOA disease trajectory. Using our established non-surgical ACL rupture model, we administered a single intra-articular (IA) injection of either 17β-estradiol to enhance estrogen signaling or Fulvestrant, an estrogen receptor antagonist, to suppress it. Mice were evaluated over a 35-day period for symptomatic and structural indicators of PTOA development, including voluntary mobility, gait, weight-bearing, and histopathologic scoring of the injured joint. 17β-estradiol delivery led to measurable improvements in multiple outcome parameters, including enhanced mobility and smaller enthesophyte formation compared to saline controls. These findings suggest that estrogen may exert protective effects on PTOA development. However, interpretation of the therapeutic potential of 17β-estradiol was complicated by the biological activity of the vehicle control, β-cyclodextrin, which independently influenced mobility outcomes, osteophyte maturity, and estrogen receptor beta (ERβ) expression. Fulvestrant treatment produced an opposing phenotype characterized by decreased voluntary mobility, increased osteophyte maturity, and altered estrogen receptor dynamics. Notably, Fulvestrant led to a transient increase in estrogen receptor alpha (ERα)) expression in ACL remnants at 3 days post-injury, potentially suggesting a compensatory feedback loop following initial receptor antagonism. These receptor-specific findings, combined with the distinct treatment effects on joint morphology and function, underscore the complex role of estrogen signaling in PTOA. Collectively, these data highlight both the promise and the challenge of developing hormone-based therapies for PTOA and emphasize the need for refined delivery methods and better understanding of sex-specific receptor profiles. In Aim 3, we transitioned to a preclinical ovine model to investigate novel surgical strategies aimed at improving ACL graft healing and minimizing downstream joint degeneration. One reason for the inability of the ACL to spontaneously heal is the presence of plasmin in synovial fluid, which prevents a fibrin-platelet clot from forming between the torn ends of the ACL. Thus, we sought to determine if delivering vascular supply to ACL remnants via a graft could improve overall healing. In this pilot study, we evaluated two innovative reconstruction techniques designed to preserve native blood supply to the graft: one utilizing the lateral digital extensor tendon (LaDET) and the other employing the infrapatellar fat pad (IFP). Post-operative outcomes were evaluated through static weight-bearing, MRI, arthroscopy, macroscopic joint scoring, and histology over six months. The LaDET graft showed successful graft incorporation, histologic continuity with native ACL remnants, minimal synovial inflammation, and restored limb function. In contrast, the IFP graft demonstrated poor structural integration, persistent inflammation, and inferior functional recovery. These results suggest that vascularized tendon grafts, such as the LaDET, may provide a biologically informed surgical strategy for enhancing graft healing in ACL reconstruction. Overall, this dissertation advances our understanding of how biological sex, hormonal signaling, and surgical technique influence joint outcomes following ACL injury. The findings emphasize that PTOA development is driven by a combination of molecular and mechanical factors, and that effective treatment must address both. Sex-based differences in disease progression and the complex role of estrogen signaling suggest that personalized, biology-informed approaches may be potential therapeutic avenues to prevent or slow joint degeneration. Additionally, the exploration of vascularized grafts presents a promising direction for improving ACL reconstruction outcomes. Together, this work provides insight for future research aimed at integrating biological sex, molecular pathways, and surgical strategies to develop more effective, personalized interventions that improve outcomes and reduce the long-term impact of ACL injury.