Matrix Stiffness-Induced Mechanical Memory of Periodontal Ligament Cell Promotes Osteogenesis in Periodontal Bone Repair

Objective Human periodontal ligament cells (hPDLCs) can sense and respond to local mechanical stimuli, which further influence the cellular behaviors, including cell proliferation, apoptosis, differentiation. However, when the microenvironment changes, instead of responding to the change instantly, cells harbor a memory of their past microenvironment, namely mechanical memory, which could regulate the cellular fates and outcomes. This study aimed to explore how mechanical memory, induced through matrix stiffness, affects the osteogenic potential of hPDLCs and their role in periodontal bone defect repair. Methods We built different substrate stiffness models by adjusting the concentration of GelMA hydrogel (soft matrix: 12 kPa; stiff matrix: 159 kPa), thereby endowing hPDLCs with different mechanical memories. Live/dead staining and CCK8 assays were performed to investigate the biocompatibility of GelMA hydrogels and cell proliferation. ALP staining and real-time PCR were performed to determine the osteogenic differentiation ability. Furthermore, we transplanted hPDLC sheets preconditioned on different substrates into a rat alveolar bone defect model, in which standardized defects (5 × 2 × 1 mm³) were surgically created on the buccal side of the mandibular incisor. Micro-CT, HE, and Masson staining confirmed that stiff-matrix groups promoted superior alveolar bone regeneration compared to soft-matrix and control groups. Results Both types of GelMA hydrogels exhibited favorable biocompatibility, and the stiff matrix significantly enhanced the osteogenic differentiation of hPDLCs compared to the soft matrix, as evidenced by increased ALP activity, upregulation of osteogenic markers (ALP, COL-1, OPN, RUNX2), and improved mineral deposition. In vivo, stiff matrix-induced mechanical memory drove persistent osteogenesis even after environmental withdrawal via cell sheet transplantation. Conclusion Stiff matrix-induced mechanical memory in hPDLCs promotes osteogenic differentiation in vitro and enhances alveolar bone regeneration in vivo. Clinical Significance These findings suggest that mechanical memory can regulate the osteogenic behavior of hPDLCs, offering a promising strategy for periodontal bone regeneration.