Gingival fibroblasts (GFs) can differentiate into osteoblast-like cells and induce osteoclast precursors to differentiate into osteoclasts. As it is unclear whether these two processes influence each other, we investigated how osteogenic differentiation of GFs affects their osteoclast-inducing capacity. To establish step-wise mineralization, GFs were cultured in four groups for 3 weeks, without or with osteogenic medium for the final 1, 2, or all 3 weeks. The mineralization was assessed by ALP activity, calcium concentration, scanning electron microscopy (SEM), Alizarin Red staining, and quantitative PCR (qPCR). To induce osteoclast differentiation, these cultures were then co-cultured for a further 3 weeks with peripheral blood mononuclear cells (PBMCs) containing osteoclast precursors. Osteoclast formation was assessed at different timepoints with qPCR, enzyme-linked immunosorbent assay (ELISA), TRAcP activity, and staining. ALP activity and calcium concentration increased significantly over time. As confirmed with the Alizarin Red staining, SEM images showed that the mineralization process occurred over time. Osteoclast numbers decreased in the GF cultures that had undergone osteogenesis. TNF-α secretion, a costimulatory molecule for osteoclast differentiation, was highest in the control group. GFs can differentiate into osteoblast-like cells and their degree of differentiation reduces their osteoclast-inducing capacity, indicating that, with appropriate stimulation, GFs could be used in regenerative periodontal treatments.

• 3D fibrin hydrogels promote in vivo-like morphology in gingival fibroblasts. • Gingival fibroblasts remodel fibrin matrices and deposit newly synthesized collagen. • Decreased u-PA/t-PA and increased MMP-2 indicate a shift to collagenolytic remodeling. • Fibrin hydrogel stiffness remains stable despite active matrix remodeling. • 3D fibrin matrices provide a biomimetic model for periodontal soft tissue regeneration.

Bone regeneration requires coordinated interactions between multiple cell types responding to biochemical and biomechanical cues from the extracellular matrix (ECM). Gingival fibroblasts (GFs) and periodontal ligament fibroblasts (PDLFs) contribute to the regeneration of lost alveolar bone, as they can differentiate into osteoblast-like cells and regulate osteoclastogenesis. However, it remains unclear whether these two fibroblast populations contribute similarly to bone regeneration and how ECM properties modulate their osteogenic differentiation and subsequent osteoclast-inducing capacity. Here, we compared the osteogenic potential of GFs and PDLFs from 12 donors, cultured in 2D monolayers or 3D fibrin hydrogels under normal or osteogenic conditions, and assessed how fibroblast-conditioned media regulates osteoclast formation from peripheral blood mononuclear cells (PBMCs). Both fibroblast populations underwent osteogenic differentiation, as evidenced by increased alkaline phosphatase (ALP) activity, calcium deposition, and robust mineral nodule formation in 3D fibrin hydrogels. Osteogenic stimulation reduced macrophage colony-stimulating factor (M-CSF) secretion and increased osteoprotegerin (OPG) release. Functionally, conditioned media from non-osteogenic cultures promoted formation of multinucleated TRAcP-positive osteoclasts and upregulated osteoclast-associated genes, including TRACP, RANK, Cathepsin K, and the fusion marker DC-STAMP. In contrast, osteogenic differentiation attenuated the osteoclast-inducing capacity of both fibroblast populations. Across all parameters, GFs and PDLFs displayed largely comparable osteogenic and osteoclast-modulating phenotypes, irrespective of culture dimensionality. These findings indicate that osteogenic differentiation functions as a regulatory switch that diminishes the osteoclast-inductive potential of oral fibroblasts and support the interchangeable use of GFs and PDLFs in periodontal regenerative strategies.