Sex-Based Differences in the Anterior Cruciate Ligament

While it has long been recognized that anterior cruciate ligament (ACL) injury is more common in females than in males, the underlying cellular and molecular mechanisms are poorly understood. Garcia and coauthors report transcriptional profiling of ACL cells from 4 male and 5 female patients undergoing ACL reconstruction. They identified 5 major cell types (fibroblasts, endothelial cells, pericytes, T cells, and macrophages) in these ACL tissues. Within the fibroblast population, they further identified 5 specific fibroblast subpopulations, and these exhibited significant differences between males and females in the expression of genes related to collagen synthesis and remodeling. They then focused on a fibroblast subpopulation characterized by expression of Tppp3, which marks a progenitor cell population in tendons1. In-depth analysis of gene expression using single-cell RNA sequencing demonstrated that Tppp3+ cells from the ACLs of female patients had upregulated expression of genes involved in extracellular matrix degradation and impaired collagen synthesis, while gene expression patterns in ACL cells from males would support more robust collagen production, collagen organization, and overall matrix integrity. These findings begin to shed some light on the well-established increased prevalence of ACL injury in females. Prior studies have demonstrated the accumulation of microscopic fatigue damage to the ACL due to repetitive submaximal loading2. Such changes may lead to decreased material properties in the ACL, increasing the risk of ligament rupture. The cell-intrinsic transcriptional differences reported here would suggest an impaired ability to repair such accumulated matrix damage in females. Another consideration is the documented increased incidence of ACL injury in females during the ovulatory phase of the menstrual cycle3. Although the current study did not have the ability to identify differences in gene expression activity during different phases of the menstrual cycle, the authors did find upregulation of the gene for matrix metalloproteinase (MMP)-14 in ACL fibroblasts from females. MMP-14 is an estrogen-sensitive protein that cleaves type-I collagen, resulting in decreased tissue stiffness and consequently a possible increase in injury risk. A further clinical implication relates to the recently increased interest in direct repair of the ACL, rather than reconstruction, in select circumstances. The findings related to genes involved in collagen synthesis, degradation, and remodeling would have implications for healing following repair. Prior studies demonstrate that accumulated tissue fatigue damage occurs at the femoral enthesis of the ACL, and it is notable that ACL repair typically involves tissue reattachment at that location, suggesting a risk of impaired healing of ACL repairs in females. An important limitation of the current study is that the authors sampled injured ACL tissue rather than native intact ligament. It is possible that extrinsic cells (for example, cells derived from blood, bone marrow, or synovium) infiltrate the injured ligament. In particular, it would be expected that immune cells may infiltrate the ligament after injury. Nevertheless, the authors compared their data with a publicly available data set from native ACL tissue and found similar cell populations, suggesting that the cells they identified are, in fact, resident in the native ACL. Another consideration is that the molecular profile of ACL cells may be affected by inflammatory mediators and cytokines produced by synovial cells in the injured joint. Although the intrinsic cell populations in the injured ligament may not differ from those in the native ACL, their transcriptional profile may certainly be altered by signaling molecules produced in the injured joint. Further studies are required to examine some of these important issues, but it is clearly a challenge to obtain healthy tissue from clinically relevant populations. Ultimately, an improved understanding of the cellular and molecular mechanisms of soft-tissue injury, degradation, and repair will identify therapeutic targets for innovative treatments. For example, modification of the local microenvironment in ligament tissues using “orthobiologic” agents may represent an opportunity to favorably affect tissue healing and repair. Rapid advancements in the clinical application of gene editing approaches such as CRISPR also hold tremendous promise for biologic augmentation of tissue repair. Finally, the presence of intrinsic progenitor cell populations (the “stem cell niche”) in orthopaedic tissues and the identification of a human skeletal stem cell population suggest the potential to leverage these cells to achieve the as-yet-elusive goal of “regenerative medicine.”4