High-Density Culture and Lipopolysaccharide Cooperatively Activate TAK1–MAPK Signaling to Promote Osteogenic Differentiation in Human Dental Pulp Stem Cells

Human dental pulp stem cells (hDPSCs) are promising for regenerative medicine because of their multipotent differentiation capacity and ease of isolation from extracted teeth. We previously reported that high-density culture enhanced their osteogenic potential; however, the inflammatory environment at the transplantation site may influence osteogenic differentiation. This study investigated the effects of lipopolysaccharide (LPS) on the osteogenic differentiation of hDPSCs cultured at high density. hDPSCs were seeded at either low or high density and stimulated with LPS. High-density culture increased focal adhesion kinase (FAK) phosphorylation, a well-established marker of integrin signaling activation, and enhanced osteogenic differentiation. Under high-density culture conditions, LPS further increased the expression of the osteogenic markers bone morphogenetic protein 2 (BMP2) and runt-related transcription factor 2 (RUNX2) and promoted mineralized nodule formation. Moreover, LPS stimulation induced the phosphorylation of transforming growth factor-β-activated kinase-1 (TAK1), p38 and c-Jun N-terminal kinase (JNK) exclusively under high-density culture conditions. TAK1 inhibition suppressed the phosphorylation of p38 and JNK and reduced BMP2 and RUNX2 expression. TAK1 phosphorylation was attenuated by a FAK inhibitor in LPS-stimulated hDPSCs cultured at high density. These findings suggest that FAK signaling associated with high-density culture facilitates LPS-induced TAK1–MAPK activation, contributing to increased BMP2 and RUNX2 expression and enhanced mineralized nodule formation in hDPSCs.