Poster 02. Protein Response to ACL Injury in Humans Show Early Cartilage Remodeling and Subtle Differences by Sex
Year: 2026
Type: article
Study design: Observational Study
Abstract: Objective: Females have a higher risk to sustain anterior cruciate ligament (ACL) tears and a higher prevalence and incidence of developing knee osteoarthritis (OA) later in life, compared to age-matched males. Anatomical, biomechanical, and hormonal factors are known to be behind these disparities. However, studies defining a sex-specific response at the tissue level in human cartilage after ACL injury are lacking. Furthermore, mouse models of post-traumatic OA (PTOA) do not recapitulate the same sex differences observed in humans, as male mice develop cartilage degeneration faster than female mice, limiting efforts in the advancement of women's health research. Therefore, the aim of this study was to unveil the molecular response elicited in human knee articular cartilage after ACL injury based on biological sex. Our goal is to understand changes occurring after injury but before any development of PTOA. We hypothesize that there are sex differences in the molecular pathways triggered by cartilage injury. Methods: We compared the proteomic profiles of normal cartilage with injured cartilage. Normal cartilage from post-mortem donors was obtained from AlloSource (5 males, 5 females, ages 37-39). Injured cartilage samples were cartilage biopsies taken from the lateral notch during ACL-repair surgery performed up to 6 months post injury, with IRB approval and patient consent (5♀, 5♂, 22-39 y.o.). Importantly, this tissue was not exposed to direct damaging physical impact, but rather to the injurious environment triggered by an ACL tear. Specimens were snap frozen and processed for proteomic analysis using Tandem Mass Tag (TMT) for quantification of relative abundance. Differentially expressed proteins between normal and injured cartilage per sex were considered for pathway analysis when log2 fold change was <-2 and >2 and with adjusted p value <0.05. Pathway enrichment analysis was done using gene ontology (GO) and gene set enrichment analysis (GSEA) compared against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Extracellular matrix (ECM) proteins were further analyzed using the MatrisomeDB2.0 database. A second pathway analysis was done considering only those differentially expressed proteins that were not shared between male and female cartilage (sex-specific analysis). These targets had a log2 fold change of <-1 and >1, and an adjusted p value <0.05. Results: Principal component analysis indicates that samples separate mostly by condition (injured vs normal) rather than by biological sex suggesting that male and female cartilage share similar responses to injury (Fig.1A-B). This was confirmed by clustering analysis of differentially expressed genes by sex, indicating that only a small number of responsive proteins are sex specific (Fig.1C). Indeed, from the 1,660 total proteins identified in the analysis, male and female shared 1,121 upregulated and 23 downregulated proteins. Enriched pathway analysis further revealed similarities. Nevertheless, sex differences were found in some pathways. Males were enriched in ' ECM-receptor interaction ', ' complement and coagulation cascades ', and ' Focal adhesion ' (Fig2A), while females were enriched in ' Citrate cycle (TCA cycle) ', ' Fatty acid degradation ', ' Ribosome ', and ' Fatty acid metabolism ' (Fig.2B). Further analysis using the MatrisomeDB2.0 database revealed a larger number of core matrisome proteins and associated to matrisome targets in male compared to female cartilage. Although, changes in expression of some of the major ECM components and metalloproteases were shared between sexes (Table 1). From the sex-specific group of proteins, 252 were upregulated and 19 were downregulated only in males, while 242 were upregulated and 3 were downregulated only in females. Among those, male-specific upregulated proteins included ADAM9 (metallopeptidase domain 9), FBLN1 (fibulin 1), MMP2 (matrix metalloprotease 2), NID2 (nidogen 2), and CRTAC1 (cartilage acidic protein 1), while male-specific downregulated proteins included COL9A3 (collagen IX alpha 3), CILP2 (cartilage intermediate layer protein 2), and TIMP3 (tissue inhibitor of metallopeptidase 3). Among the female-specific targets, CRTAP (cartilage associated protein), DSE (dermatan sulfate epimerase), SDC2 (syndecan 2), and SLIT3 (slit guidance ligand 3) were upregulated, while only LTBP1 (latent transforming growth factor beta binding protein 1) was downregulated. Analysis of molecular functions of the sex-specific targets shows an enrichment of ECM binding in males and ribosome-related functions in females (Fig.3). Conclusions: The most striking finding from this study is that cartilage taken from ACL surgeries, which has only been exposed to an injurious environment likely including pro-inflammatory conditions, shows signs of cartilage remodeling in both sexes even in the absence of PTOA. These changes are occurring shortly after injury, as samples were taken between 0.5- and 6-months post injury. Further investigation is needed to determine whether this acute matrix remodeling is permanent or resolves over time. In terms of sex-specific analysis, male and female cartilage share mostly similar responses to ACL injury, with similar changes at the ECM level and only subtle sex-specific signatures. Nevertheless, within this small group of targets, we observed that females were enriched in proteins and pathways related to metabolism and protein synthesis, while males were enriched in proteins and pathways related to focal adhesions and cell-ECM interaction. Additional research efforts are needed to define the impact of these subtle sex differences over time; for example, whether they amplify at a later time point and lead to differential processes for cartilage degeneration. A limitation of our study is the small sample size per sex.
Language: English
Subfield: Rheumatology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $1,500
OpenAlex ID: W7204790809
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