Dual Adhesion Pathways and Mechanotransduction of Adipose-Derived Mesenchymal Stem Cells on Glycated Collagen Substrates-Morphological Evidence.

Glycation-induced modifications of extracellular matrix (ECM) proteins, including collagen, are increasingly recognized as critical modulators of cellular behavior, particularly in pathophysiological contexts such as aging and diabetes. While their impact on general cell adhesion has been explored, the specific consequences for mesenchymal stem cell (MSC) mechanotransduction remain poorly defined. In this study, we investigated the temporal and mechanistic aspects of adhesion and mechanosensitive signaling in adipose-derived MSCs (ADMSCs) cultured on native versus glycated collagen substrates. Our findings identify two temporally distinct adhesion mechanisms: an initial pathway mediated by the receptor for advanced glycation end-products (RAGE), which is activated within the first 30 min following substrate engagement, and a later-stage adhesion process predominantly governed by integrins. Immunofluorescence analysis demonstrated maximal nuclear localization of YAP/TAZ transcriptional regulators during the initial adhesion phase, coinciding with RAGE engagement. This nuclear enrichment was progressively attenuated as integrin-mediated focal adhesions matured, suggesting a dynamic shift in receptor usage and mechanotransductive signaling. Interestingly, glycated collagen substrates accelerated early cell attachment but impaired focal adhesion maturation, suggesting a disruption in integrin engagement. Endogenous collagen synthesis was consistently detected at all examined time points (30 min, 2 h, and 5 h), suggesting a constitutive biosynthetic activity that remains sensitive to the glycation state of the substrate. Atomic force microscopy (AFM) demonstrated that glycation disrupts collagen fibrillogenesis: while native collagen forms a well-organized network of long, interconnected fibrils, GL-1 substrates (glycated for 1 day) displayed sparse and disordered fibrillary structures, whereas GL-5 substrates (5-day glycation) exhibited partial restoration of fibrillar organization. These matrix alterations were closely associated with changes in adhesion kinetics and mechanotransduction profiles. Taken together, our findings demonstrate that collagen glycation modulates both adhesion dynamics and mechanosensitive signaling of MSCs through a dual-receptor mechanism. These insights have significant implications for the design of regenerative therapies targeting aged or metabolically compromised tissues, where ECM glycation is prevalent.