Regional Expression of Vimentin, S100, and Epithelial Membrane Antigen in the Human Medial Collateral Ligament: A Robust Two-Way Analysis of Variance

Background: The epiligament (EL) of the medial collateral ligament (MCL) has recently attracted increasing attention as a biologically active structure. Emerging evidence suggests that it may contribute to ligament healing by providing progenitor cells, vascular components, and signaling mediators. However, its cellular composition and possible regional variability remain insufficiently characterized. Aim: This study evaluated the expression of vimentin, S100 protein, and epithelial membrane antigen (EMA) to better characterize the EL compared with the ligament proper (LP). Methods: Twelve human MCLs obtained from twelve deceased donors were divided into proximal, middle, and distal segments. Thirty-six paraffin blocks were prepared, from which 180 sections were obtained and equally assigned for immunohistochemical staining of vimentin, S100 protein, and EMA (60 slides for each marker). Systematic quantification of seven to eight non-overlapping microscopic fields per slide generated 900 standardized observations for each investigated marker. This sampling strategy provided 150 measurements for each sub-region (EL and LP across the three anatomical segments). Immunoreactivity was quantified using ImageJ software. Statistical differences were analyzed using a robust two-way analysis of variance (ANOVA), while biological associations between markers were assessed using Spearman’s rank correlation analysis. Results: Vimentin and S100 expression were consistently higher in the EL than in the LP across all anatomical regions (p < 0.0001). The highest vimentin values were observed in the proximal region (median 17.34 vs. 10.14) and distal region (median 19.34 vs. 11.23), whereas S100 showed the greatest expression in the proximal (median 16.9 vs. 7.2) and distal regions (median 14.1 vs. 8.9). EMA expression was generally lower overall; however, it remained significantly higher in the EL than in the LP within the proximal (median 6.87 vs. 5.77) and middle regions (median 4.80 vs. 3.26). No significant difference was identified in the distal region. Spearman rank correlation analysis demonstrated significant positive associations among all investigated markers (p < 0.001), with the strongest relationship observed between vimentin and S100 protein (Spearman correlation coefficient = 0.430). Conclusions: The EL of the MCL is a structurally and biologically distinct component, characterized by significantly higher expressions of vimentin, S100, and EMA than the LP. The significant positive correlations observed among these markers support the concept that the EL functions as an integrated biological microenvironment with clear regional heterogeneity, particularly within the proximal and distal segments. Further studies are warranted to clarify the functional relevance of these findings and their potential implications for clinical management and ligament healing strategies.