Co-cultured spheroids of human periodontal ligament mesenchymal stem cells and vascular endothelial cells enhance periodontal tissue regeneration.

INTRODUCTION: Human periodontal ligament mesenchymal stem cells (hPDLMSCs) have been known that they play important roles in homeostasis and regeneration of periodontal tissues. Additionally, spheroids are superior to monolayer-cultured cells. We investigated the characteristics and potential of periodontal tissue regeneration in co-cultured spheroids of hPDLMSCs and human umbilical vein endothelial cells (HUVECs) in vitro and in vivo. METHODS: Co-cultured spheroids were prepared with cell ratios of hPDLMSCs: HUVECs = 1:1, 1:2, and 2:1, using microwell chips. Real-time polymerase chain reaction (PCR) analysis, Enzyme-Linked Immuno Sorbent Assay (ELISA), and nodule formation assay were performed to examine the properties of co-cultured spheroids. Periodontal tissue defects were prepared in the maxillary first molars of rats and subjected to transplantation assay. RESULTS: The expression levels of stemness markers, vascular endothelial growth factor (VEGF), osteogenesis-related genes were up-regulated in co-cultured spheroids, compared with monolayer and spheroid-cultured hPDLMSCs. The nodule formation was also increased in co-cultured spheroids, compared with monolayer and spheroid cultures of hPDLMSCs. Treatment with co-cultured spheroids enhanced new cementum formation after 4 or 8 weeks of transplantation, although there was no significant difference in the new bone formation between co-cultured spheroids and hPDLMSC spheroids. CONCLUSIONS: We found that co-cultured spheroids enhance the periodontal tissue regeneration. Co-cultured spheroids of hPDLMSCs and HUVECs may be a useful therapy that can induce periodontal tissue regeneration.

Thymus-expressed chemokine enhances Porphyromonas gingivalis LPS-induced osteoclast formation via NFATc1 activation.

OBJECTIVE: P. gingivalis is a gram-negative anaerobic bacterium and a major periodontal pathogen. LPS produced by P. gingivalis promotes osteoclast formation. TECK is a CC chemokine whose expression is increased in gingival epithelial cells exposed to P. gingivalis LPS. In this study, we investigated the effect of TECK in osteoclastogenesis induced by P. gingivalis LPS. DESIGNS: Real time reverse transcriptase polymerase chain reaction (RTPCR) analysis and western blotting were performed to confirm TECK in MG63, human osteoblast cell line and primary murine osteoblasts and CCR9 in RAW 264.7 cells and murine bone marrow macrophages (BMMs) as osteoclast precursors. P. gingivalis LPS-treated BMMs and Raw 264.7 cells were cultured with or without TECK or TECK antibody to examine the effect of TECK on osteoclast formation. Cocultures with murine osteoblasts and bone marrow cells were also treated with or without TECK or TECK antibody. Luciferase assay and western blotting were used to determine whether TECK-CCR9 induced osteoclastogenesis was mediated through NFATc1 or NF-kB signaling. RESULTS: TECK was shown to be expressed by osteoblasts, and its receptor, CCR9, by osteoclast precursors. TECK increased P. gingivalis LPS-induced osteoclast numbers in an in vitro osteoclast formation assay using osteoclast precursors. The enhanced osteoclast formation by TECK was mediated by NFATc1, but not by NF-kB signaling. CONCLUSION: TECK may be a novel regulator of osteoclast formation induced by P. gingivalis LPS in periodontitis.