Dual Adhesion Pathways and Mechanotransduction of Mesenchymal Stem Cells on Glycated Collagen Substrates

Glycation-induced modifications of extracellular matrix (ECM) proteins, including collagen, are increasingly recognised as critical modulators of cellular behaviour, particularly in pathophysiological contexts such as ageing 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 signalling 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 minutes following substrate engagement, and a later-stage adhesion process predominantly governed by integrins. Immunofluorescence analysis demonstrated maximal nuclear localisation 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 mechano-transductive signalling. 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 minutes, 2 hours, and 5 hours), 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-organised 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 organisation. These matrix alterations were closely associated with changes in adhesion kinetics and mechanotransduction profiles. Taken together, our findings demonstrate that collagen glycation modulates both MSC adhesion dynamics and mechanosensitive signalling 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.