Distinct hyaluronan content and hierarchical organization of collagen-elastic fiber assemblies in subcutaneous fascia

Previous studies have demonstrated that the subcutaneous fascia is a continuous connective tissue layer consisting of morphologically distinct "loose regions" and "dense regions". However, the molecular basis of this regional heterogeneity remains unclear. This study aimed to analyze the biochemical and compositional differences between these two regions to elucidate the factors responsible for their functional and structural distinctions. Loose and dense fascia were isolated from chicken subcutaneous fascia and subjected to collagen composition analysis using SDS-PAGE, hyaluronan (HA) quantification, and fluorescence and electron microscopy combined with various biochemical treatments. The results showed no significant differences in the composition ratios of major fibrillar collagens (types I, III, and V) or in total collagen content per dry weight between the two regions. In contrast, significant differences were found in non-collagenous matrix components; specifically, loose fascia contained approximately twice as much HA as dense fascia (loose: 0.31%, dense: 0.15%). Collagen hybridizing peptide (CHP) staining, which detects denatured/disrupted collagen chains, showed strong signals at the bundle boundaries in dense fascia, suggesting region-specific differences in supramolecular organization and locally increased collagen triple-helix dynamics/instability at bundle peripheries. In addition, elastase treatment led to loosening/disruption of collagen bundles, an observation consistent with a contribution of elastic-fiber components to maintaining higher-order bundle integrity, although enzyme purity and secondary mechanical effects cannot be fully excluded. These results suggest that the regional mechanical behaviors are not fully explained by differences in major collagen composition and are more strongly associated with differences in hyaluronan abundance and supramolecular organization. These findings provide a molecular and ultrastructural basis that may underlie the distinct mechanical behaviors previously reported for loose and dense regions of the subcutaneous fascia.