Substrate stiffness modulates human gingival fibroblast paracrine signaling to promote osteogenic differentiation of human periodontal ligament cells.

Human gingival fibroblasts (HGFs) are stromal cells that maintain periodontal tissue structure and extracellular matrix (ECM) dynamics. ECM stiffness serves as a physical cue that regulates HGF behavior and secretory profiles. This study investigated how substrate stiffness modulates the secretome of HGFs and observed the subsequent effects of this secretome on the osteogenic differentiation of human periodontal ligament cells (HPDLCs). HGFs isolated from healthy donors were cultured on polydimethylsiloxane substrates, representing soft or hard periodontal tissue under normal and lipopolysaccharide (LPS)-induced inflammatory conditions. The expression of cytokines and chemokines was analyzed using qRT-PCR and ELISA, with p38 MAPK inhibitors used to identify stiffness-associated signaling involvement. HPDLCs were treated with conditioned medium from HGFs (HGF-CM) under osteogenic induction, osteogenic marker expression was examined using qRT-PCR and immunofluorescence, with mineralization assessed by Alizarin red S staining. To establish mechanistic causality, functional blocking was conducted using a C-X-C motif chemokine receptor 4 (CXCR4) inhibitor. Hard substrates significantly increased the expression of anti-inflammatory cytokines and the C-X-C motif chemokine ligand 12 (CXCL12) in HGFs, whereas inhibition of p38 mitogen-activated protein kinase (MAPK) activity attenuated stiffness-associated CXCL12 expression. Under LPS-induced inflammatory conditions, hard substrates-maintained matrix metalloproteinase-9 suppression and tissue inhibitor of metalloproteinases 1 upregulation, although CXCL12 protein levels were decreased. Furthermore, HPDLCs treated with HGF-CM derived from hard substrates in osteogenic induction media exhibited elevated CXCR4 expression, increased osteogenic marker levels at days 14 and 21, and enhanced mineral deposition compared to those treated with HGF-CM from soft substrates. In addition, functional blocking with a CXCR4 inhibitor significantly reduced the expression of osteogenic markers (ALP, RUNX2, COL1A1, and OSX) and confirmed a subsequent decrease in matrix mineralization. Substrate stiffness modulated the paracrine behavior of HGFs, with CXCL12 serving as a representative example of a stiffness-responsive factor. These alterations in the HGF-derived secretome were associated with altered osteogenic and inflammatory responses in HPDLCs. These findings support the influence of the physical microenvironment on fibroblast-periodontal ligament cell interactions on anti-inflammatory response and periodontal tissue stabilization.