Structural analysis of a collagen-fibrin scaffold which promotes osteogenic differentiation and templated biomineralization of dental pulp stem cells

Objective: Clinical regenerative endodontic treatment (RET) studies report enhanced intracanal mineralization when collagen-based scaffolds are combined with blood, but the underlying mechanisms remain unclear. This study used a simplified in vitro collagen-fibrin model, incorporating fibrin, a key blood component, into collagen. The structural, chemical, and mechanical properties of collagen-fibrin scaffolds were compared with those of the individual components and correlated with osteogenic differentiation and biomineralization by dental pulp stem cells (DPSCs). Methods: Collagen, fibrin, and collagen-fibrin hydrogels were fabricated and characterized by Raman spectroscopy, cryo-scanning electron microscopy (cryo-SEM), atomic force microscopy (AFM), and oscillatory shear rheology. DPSCs were cultured on the scaffolds for up to 28 days without dexamethasone to isolate scaffold-driven effects. Osteogenic differentiation and mineralization were evaluated by qRT-PCR, Raman spectroscopy, Alizarin Red S (ARS) staining, and SEM–EDX. Results: Collagen-fibrin scaffolds formed composite networks with increased modulus and distinct microstructure relative to the individual hydrogels. DPSCs cultured on collagen-fibrin scaffolds exhibited enhanced osteogenic differentiation, including increased expression of osteocalcin and bone sialoprotein. Although calcium phosphate formed on all scaffolds, collagen-fibrin composites uniquely supported templated, fiber-associated hydroxyapatite with crystallinity comparable to native bone. Significance: Collagen-fibrin hydrogels provide a mechanically reinforced, extracellular matrix-mimicking scaffold that promotes biologically regulated mineralization by DPSCs. This model offers mechanistic insight into clinically observed hard tissue formation in RET and highlights fibrin as a key contributor to mineralized tissue formation, with translational relevance when bleeding induction is limited.