Biomimetic recombinant humanized type III collagen peptide-RGD loaded alginate hydrogels enhance proliferation of mesenchymal-lineage cells via Integrin α5β1 activation

Alginate hydrogels (HG) are widely utilized in tissue engineering due to their biocompatibility and tunable physical properties, however, their bio-inert nature limits direct cellular interactions. Moreover, the receptor-level interactions of recombinant humanized type-III collagen-peptide (rCP) and RGD with mesenchymal-lineage cells such as mesenchymal stem cells (MSCs), human osteoblast-like cells (hFOB), and fibroblasts (FB) remain unexplored. To address these limitations, alginate hydrogels were incorporated with RGD (R-HG), rCP (rCP-HG), and both peptides (CPR-HG), and systematically evaluated for their functional, structural, and biological performance. While dual-peptide matrices are widely recognized in biomaterials research, this study specifically elucidates the distinct receptor-level responses and synergistic pathways activated by combining RGD and a specific genetic-engineered recombinant CP within an alginate backbone. Peptide incorporation enhanced HG swelling, with rCP contributing to increased hydration capacity. Drug release studies revealed sustained, media-dependent diffusion of both RGD and rCP. Notably, CPR-HG significantly elevated cell proliferation, particularly in hFOB cells. Among the cells, the incorporationof RGD and rCP significantly accelerated cellular integrin α5 and β1 expression in MSCs than hFOB and FB. SEM analysis demonstrated improved cell morphology and matrix interaction, while immunocytochemistry confirmed upregulated expression of integrin α5 and collagen-I in all cell types. Interestingly, compared to single-peptide and unmodified hydrogels, CPR-HG consistently promoted superior cellular adhesion, spreading, and integrin activation in MSCs, hFOB, and FB cells. For the first time, this study disclosed the synergistic effects of RGD and rCP in alginate hydrogels on MSCs, hFOB, and FB cells. Accordingly, a dual-incorporated CPR-HG hydrogel enhanced bioactivity and cell-specific receptor engagement, underscoring its potential as a versatile platform for advanced tissue regeneration.