Collagen Formulation in Xenogeneic Bone Substitutes Influences Cellular Responses in Periodontal Regeneration: An In Vitro Study

Abstract

BACKGROUND: Bone regeneration is a key therapeutic objective in periodontology, particularly in the treatment of alveolar defects caused by periodontal disease, dentoalveolar trauma, or surgical interventions. Among current regenerative strategies, collagen-enriched biomaterials have demonstrated an active role in modulating cellular behavior during bone repair. However, the specific effects of different collagen formulations on human dental pulp stem cells (hDPSCs) have not yet been fully characterized. OBJECTIVE: To evaluate the impact of xenogeneic bone grafts with and without collagen-OsteoBiol(®) Gen-Os(®) (GO), OsteoBiol(®) GTO(®) (GTO), and Geistlich Bio-Oss(®) (BO)-on cell viability, adhesion, migration, osteogenic differentiation, and mineralization potential of hDPSCs, and to explore the molecular mechanisms underlying their effects. METHODS: In vitro assays were conducted to assess viability (MTT and fluorescence staining), adhesion (SEM), migration (wound healing assay), and mineralization (Alizarin Red S staining). Gene expression analyses (RT-qPCR) were performed for adhesion/migration markers (FN, SDF-1, COL1A1), angiogenic/proliferation markers (VEGF, FGF2), and osteogenic differentiation markers (RUNX2, ALP, COL1A1). RESULTS: GO showed a higher early expression of genes associated with adhesion, migration, angiogenesis (FN, SDF-1, VEGF and FGF2: p < 0.05; COL1A1: p < 0.01), and osteogenic differentiation (7 days: COL1A1 and ALP (p < 0.001)); (14 days: RUNX2, ALP: p < 0.001; COL1A1: p < 0.05), indicating a sequential activation of molecular pathways and mineralization capacity comparable to the control group. GTO demonstrated the best biocompatibility, with significantly higher cell viability (p < 0.05), strong adhesion, and markedly increased mineralization at 21 days (p < 0.001), despite moderate early gene expression. BO showed reduced cell viability at 10 mg/mL (p < 0.05) and 20 mg/mL (p < 0.001), with mineralization levels similar to the control group. CONCLUSION: Collagen-based xenografts demonstrate favorable interactions with hDPSCs, enhancing viability and promoting osteogenic differentiation. Our findings suggest that beyond the presence of collagen, the specific formulation of these biomaterials may modulate their biological performance, highlighting the importance of material design in optimizing regenerative outcomes. CLINICAL SIGNIFICANCE: The formulation of collagen in xenogeneic bone substitutes may be a determining factor in enhancing periodontal regenerative outcomes by modulating the early cellular response and osteogenic activity in stem cell-based tissue engineering.