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. 2024 Jul-Aug;38(4):1594-1600.
doi: 10.21873/invivo.13609.

Effect of Cells Derived from Periodontal Ligament Tissue on Bone Formation

Norika Kobayashi  1 , Hiroshi Kadokura  2 , Eisuke Iso  2 , Takako Tsuchiya  2 , Satoshi Yokose  2
Affiliations

Effect of Cells Derived from Periodontal Ligament Tissue on Bone Formation

Norika Kobayashi et al. In Vivo. 2024 Jul-Aug.

Abstract

Background/aim: Recent reports indicate that sclerostin is secreted by periodontal ligament tissue-derived (PDL) cells during orthodontic force loading and that the secreted sclerostin contributes to bone metabolism. However, the detailed mechanism is poorly understood. The aim of this study was to determine how PDL cells affect bone formation.

Materials and methods: Rat periodontal ligament tissue was immunohistochemically stained for sclerostin. Cultured primary PDL cells, osteoblasts, and skin fibroblasts (Sfbs) isolated from rat periodontal ligament tissue, calvaria, and skin, respectively, were examined. Osteoblasts were cultured with control conditioned medium (Cont-CDM) and PDL cell culture conditioned medium (PDL-CDM) for up to 21 days. Cultured osteoblasts were then stained with alkaline phosphatase and von Kossa stain. Osteoblasts cultured in each conditioned medium were analyzed by real-time quantitative PCR for bone Gla protein (Bgp), Axin2, and Ki67 expression. PDL cells used to obtain conditioned medium were analyzed for Sost, Ectodin and Wnt1 expression and compared with expression in Sfbs.

Results: Expression of sclerostin was observed in periodontal ligament tissue by immunohistochemical staining. The formation of mineralization nodules was inhibited in PDL-CDM compared with Cont-CDM in osteoblast culture. In PDL-CDM, the expression levels of Bgp and Axin2 in osteoblasts were decreased compared with Cont-CDM. In PDL cells, expression levels of Sost and Ectodin were much higher than in Sfbs; however, expression of Wnt1 was lower in PDL cells compared with Sfbs.

Conclusion: PDL cells secrete various proteins, including sclerostin and suppress osteogenesis in osteoblasts through the canonical Wnt pathway.

Keywords: Periodontal ligament; bone formation; sclerostin.

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Conflict of interest statement

All Authors state that they have no conflicts of interest to disclose in relation to this study.

Figures

Figure 1
Figure 1. Immunohistochemical staining for sclerostin in periodontal tissues of 8-week-old rat mandible. H-E staining of periodontal ligament (PDL) tissues (A, B, and C). Expression of sclerostin was observed in PDL cells, osteocytes, and odontoblasts (D, E, and F). AB: Alveolar bone; D: dentin. Bars (A, D)=100.0 μm; (B, C, E, and F)=20.0 μm.
Figure 2
Figure 2. Alkaline phosphatase (ALP) staining and von Kossa staining on day 21 in rat osteoblasts cultured in conditioned medium. Osteoblasts were cultured with conditioned medium prepared from cell-free culture conditioned medium (Cont-CDM) (A, B) or periodontal ligament (PDL) cell culture conditioned medium (CDM) (C, D). Higher-magnification views of each culture well (B, D). ALP-positive cells were observed in Cont-CDM and PDL-CDM (A, C). In PDL-CDM, the size of von Kossa staining–positive mineralized nodules was smaller than that of nodules in Cont-CDM (D). Bars (B, D, and F)=200 μm.
Figure 3
Figure 3. Number of mineralization nodules on day 21 in rat osteoblasts cultured in conditioned medium. Few mineralized nodules were observed in periodontal ligament-cell culture conditioned medium (PDL-CDM) compared with Cont-CDM. Each bar represents the mean of the measured value±standard deviation of three cultures. *p<0.05 compared with Cont-CDM.
Figure 4
Figure 4. Real-time quantitative PCR analysis of gene expression on day 21 in osteoblasts cultured in conditioned medium (Cont-CDM, PDL-CDM). Expression levels of Bgp (A), Axin2 (B), and Ki67 (C). Each bar represents the mean of the measured value±standard deviation of three cultures. *p<0.05 compared with Cont-CDM. CDM: Cell culture conditioned medium; PDL: periodontal ligament.
Figure 5
Figure 5. Real-time quantitative PCR analysis of gene expression in periodontal ligament (PDL) cells and Sfbs used to obtain conditioned media for osteoblast culture. Expression levels of Sost (A), Ectodin (B), and Wnt1 (C) on day 21. Each bar represents the mean of the measured value±standard deviation of three cultures. *p<0.05 compared with PDL cells.
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