ABS 015: Relaxin-2–Mediated Modulation of Wnt and MMP2 Signaling in ACL Tissue In Vivo

Peter Ko ¹, Li Yue ², Brett D. Owens ² ³

¹ Department of Biology at Brown University, Providence, RI
² Department of Orthopaedics at the Warren Alpert Medical School of Brown University and Rhode Island Hospital, Providence, RI
³ University Orthopedics, Providence, RI

The Van Wickle Journal (2026) Volume 2, ABS015

Introduction: Female athletes experience anterior cruciate ligament (ACL) injuries at substantially higher rates than males, suggesting that sex-specific biological factors may contribute to ligament vulnerability. One hormone of interest is relaxin-2, a peptide hormone known to influence connective tissue remodeling. Prior studies have shown that relaxin-2 can increase matrix metalloproteinase activity and reduce collagen expression in ligament cells, potentially weakening the extracellular matrix that provides the ACL with structural integrity.

This project investigates how relaxin-2 affects ACL tissue in female rats, with a particular focus on matrix metalloproteinase-2 (MMP2) and Wnt/β-catenin signaling. MMP2 is associated with collagen degradation and extracellular matrix remodeling, while Wnt/β-catenin signaling is typically linked to collagen synthesis, tissue repair, and ligament homeostasis. Because relaxin-2 may alter both degradation and repair pathways, characterizing its effect on these markers may clarify how hormonal signaling contributes to ACL weakening. However, the in vivo relationship between relaxin-2 exposure, MMP2-mediated matrix remodeling, and Wnt/β-catenin signaling in ACL tissue remains largely incompletely characterized.

The objective of this study is to determine whether relaxin-2 treatment is associated with increased MMP2 expression and altered Wnt signaling in female rat ACL tissue. We hypothesize that relaxin-2 promotes ligament remodeling by increasing MMP2-mediated extracellular matrix degradation and disrupting the balance of Wnt/β-catenin–mediated repair. By identifying molecular changes associated with relaxin-2 exposure, this study may help define mechanisms underlying sex-specific ACL injury risk and inform future development of targeted preventative or therapeutic strategies.

Methods: Female Sprague-Dawley rats aged 10–12 weeks were treated under a broader in vivo protocol examining relaxin-2 and relaxin-2 antagonist effects on ACL integrity. For the present analysis, untreated control rats were compared with rats receiving intravenous relaxin-2 for 10 days. Following CO₂ euthanasia, femur–ACL–tibia complexes were isolated for tissue analysis and biomechanical testing. Blood serum was collected to measure circulating relaxin levels using a competitive ELISA assay. ACL tissues were fixed in 10% formalin, embedded in paraffin, sectioned at 6 μm, and stained with hematoxylin and eosin, MMP2, Wnt, and β-catenin antibodies. Immunofluorescent images were analyzed by counting DAPI-positive nuclei and marker-positive cells. Wnt/DAPI and MMP2/DAPI ratios were compared between untreated and relaxin-treated groups using GraphPad Prism 8, with statistical significance set at p<0.05.

Results: Relaxin-2 treated ACL tissue showed increased MMP2 expression compared with untreated controls, suggesting enhanced extracellular matrix degradation. Wnt expression also appeared elevated in the relaxin-treated group. Total DAPI counts were not markedly different between groups, indicating that relaxin-2 primarily affected marker expression rather than overall cellularity. Visual assessment also suggested greater collagen fiber disorganization in relaxin-treated ACL tissue, with fibers appearing less uniform and more irregular than controls.

Discussion: These preliminary findings suggest that relaxin-2 may weaken ACL integrity by increasing MMP2-mediated matrix remodeling while also altering Wnt/β-catenin signaling. Although Wnt signaling is typically associated with tissue repair, its elevation alongside MMP2 may represent a compensatory but insufficient response to collagen degradation. Future studies should test whether relaxin-2 antagonists, such as folic acid or NADH, can reduce MMP2 activity, normalize Wnt signaling, and preserve ACL structure. This work may contribute to sex-specific ACL injury prevention strategies.