Human dental follicle cell-derived conditioned media enhance periodontal regeneration by regulating the osteogenic differentiation and inflammation of periodontal ligament stem cells and macrophage polarization

Dental follicle cells (DFCs) derived from the neural crest are promising seed cells for periodontal tissue engineering. This study aimed to investigate whether conditioned media (CM) from human DFCs could regenerate impaired periodontal tissue and the underlying mechanisms. hDFC-derived CM (hDFC-CM) were obtained via ultracentrifugation. In vitro, human periodontal ligament stem cells (hPDLSCs) were treated with hDFC-CM in normal and inflammatory microenvironments, and the cell proliferation, migration, and osteogenic potential were evaluated. We simulated the inflammatory environment with lipopolysaccharide and detected the expression of osteogenic and Wnt/β-catenin signaling pathway-related proteins. The effect of hDFC-CM on the inhibition of hPDLSC inflammation and macrophage polarization was examined. In vivo, the rat periodontitis model was treated with hDFC-CM. Tissue samples were collected after 4 weeks for micro-computed tomography and histological examination. The results of cell counting kit-8 and scratch experiments showed that hDFC-CM significantly enhanced the proliferation and migration capacities of hPDLSCs. hDFC-CM promoted the osteogenic differentiation of hPDLSCs by showing intense alkaline phosphatase and Alizarin Red staining and upregulated osteogenic protein and gene expression. Western blotting also verified that hDFC-CM promotes the osteogenic differentiation of hPDLSCs by regulating the Wnt/β-catenin pathway in an inflammatory environment. In addition, hDFC-CM inhibited hPDLSC inflammation and polarized macrophages from the M1 to M2 phenotype. In vivo, hDFC-CM effectively promoted periodontal tissue regeneration. hDFC-CM enhances periodontal regeneration by regulating the osteogenic differentiation and inflammation of periodontal ligament stem cells and macrophage polarization, which provided new biochemical cues for the treatment of periodontitis.