Evaluating the oxysterol combination of 22(S)-hydroxycholesterol and 20(S)-hydroxycholesterol in periodontal regeneration using periodontal ligament stem cells and alveolar bone healing models

Abstract

Background: Oxysterols, oxygenated by-products of cholesterol biosynthesis, play roles in various physiological and pathological systems. However, the effects of oxysterols on periodontal regeneration are unknown. This study investigated the effects of the specific oxysterol combination of 22(S)-hydroxycholesterol and 20(S)-hydroxycholesterol (SS) on the regeneration of periodontal tissues using in-vitro periodontal ligament stem cells (PDLSCs) and in-vivo models of alveolar bone defect.

Methods: To evaluate the effects of the combined oxysterols on PDLSC biology, we studied the SS-induced osteogenic differentiation of PDLSCs by assessing alkaline phosphatase activity, intracellular calcium levels [Ca²⁺]_i, matrix mineralization, and osteogenic marker mRNA expression and protein levels. To verify the effect of oxysterols on alveolar bone regeneration, we employed tooth extraction bone defect models.

Results: Oxysterols increased the osteogenic activity of PDLSCs compared with the control group. The expression of liver X receptor (LXR) α and β, the nuclear receptors for oxysterols, and their target gene, ATP-binding cassette transporter A1 (ABCA1), increased significantly during osteogenesis. Oxysterols also increased protein levels of the hedgehog (Hh) receptor Smo and the transcription factor Gli1. We further confirmed the reciprocal reaction between the LXRs and Hh signaling. Transfection of both LXRα and LXRβ siRNAs decreased Smo and Gli1 protein levels. In contrast, the inhibition of Hh signaling attenuated the LXRα and LXRβ protein levels. Subsequently, SS-induced osteogenic activity of PDLSCs was suppressed by the inhibition of LXRs or Hh signaling. The application of SS also enhanced bone formation in the defect sites of in-vivo models, showing equivalent efficacy to recombinant human bone morphogenetic protein-2.

Conclusions: These findings suggest that a specific combination of oxysterols promoted periodontal regeneration by regulating PDLSC activity and alveolar bone regeneration.

Keywords: Alveolar bone defect; Bone regeneration; Osteogenic differentiation; Oxysterol; Periodontal ligament stem cells.

Fig. 1

Critical steps in the animal experiment. Timeline for establishment of the alveolar bone defect model and the injection of each agent. β-APN β-aminopropionitrile, BMP‐2 bone morphogenetic protein‐2, SS 22(S)-hydroxycholesterol and 20(S)-hydroxycholesterol

Fig. 2

Effect of oxysterols on PDLSC osteogenesis. Cells treated with different oxysterol concentrations (SS; 0.5, 1, and 5 μM) for 0, 4, 7, or 14 days. a ALP activity, b [Ca²⁺]_i, and c Alizarin red staining. Lower panels (bars) denote Alizarin Red quantification. Values reported as means ± SD of five independent experiments. *P < 0.05 vs control value at each time point. [Ca²⁺]_i intracellular calcium level, SS 22(S)-hydroxycholesterol and 20(S)-hydroxycholesterol

Fig. 3

Effect of oxysterols on osteogenic gene expression. mRNA levels of a OCN, b OSX, c OPN, and d RUNX2 analyzed after 7 days of osteogenic induction. Values reported as means ± SD of five independent experiments. *P < 0.05 vs control value. OCN osteocalcin, OPN osteopontin, OSX osterix, RUNX2 Runt-related transcription factor 2

Fig. 4

Effect of oxysterols on osteogenic-related protein levels. Protein levels of OCN, OSX, and RUNX2 determined by (a–c) immunofluorescence staining and (d) western blotting (OCN 5.5 kDa, OSX 45 kDa, RUNX2 55 kDa). Nuclei stained with DAPI (blue). Representative result from three independent experiments (scale bar, 100 μm). DAPI 4′,6-diamidino-2-phenylindole, OCN osteocalcin, OSX osterix, RUNX2 Runt-related transcription factor 2

Fig. 5

Effect of oxysterols on LXR and Hh signaling. Cells incubated with SS at different concentrations (0.5, 1, and 5 μM). a Protein levels of LXRα (50 kDa) and LXRβ (56 kDa). b mRNA expression of LXRα, LXRβ, and ABCA1. c Protein levels of Smo (85 kDa) and Gli1 (118 kDa). Values reported as means ± SD of four independent experiments. *P < 0.05 vs control value. ABCA1 ATP-binding cassette transporter A1, Hh Hedgehog, LXR liver X receptor, Smo Smoothened

Fig. 6

Evaluation of the cross-reaction between LXR and Hh signaling. Cells were transfected with either LXRα or LXRβ-specific siRNAs or a negative control siRNA for 24 h and further incubated with SS (1 μM) for 48 h, and protein levels of (a) LXRα and (b) LXRβ were assessed. c Cells were transfected with both LXRα and LXRβ-specific siRNAs to a final concentration of 100 nM of each siRNA prior to SS treatment, and protein levels of Smo and Gli1 were analyzed. d Protein levels of LXRα and LXRβ measured after cells were pretreated with 22-NHC before SS treatment. Representative result from five independent experiments. LXR liver X receptor, NC negative control, 22-NHC 22-azacholesterol, siRNA small interfering RNA, SS 22(S)-hydroxycholesterol and 20(S)-hydroxycholesterol

Fig. 7

Effect of LXR and Hh signaling on oxysterol-induced PDLSC osteogenesis. Cells pretreated with 22-NHC or transfected with LXRαβ siRNAs before SS (1 μM) treatment. (a) ALP activity. (b) mRNA expression and (c) protein levels of OCN, OSX, and RUNX2. Data obtained from three independent experiments. *P < 0.05 vs control value; ^# P < 0.05 vs SS treatment alone. LXR liver X receptor, NC negative control, 22-NHC 22-azacholesterol, OCN osteocalcin, OSX osterix, RUNX2 Runt-related transcription factor 2, siRNA small interfering RNA, SS 22(S)-hydroxycholesterol and 20(S)-hydroxycholesterol

Fig. 8

Effect of oxysterols or BMP-2 on alveolar bone regeneration. a Color-coded μCT images of extraction sockets in the control, BMP-2, and SS groups 15 days after tooth extraction. Purple area in the sockets (indicated by white arrows) represents newly formed bone (scale bar, 2 mm). b Newly formed bone volume in sockets (%) measured three-dimensionally (mean ± SD). c Protein levels of COLIA, ALP, RUNX2, and OCN determined to confirm alveolar bone regeneration using western blotting analysis. ALP alkaline phosphatase, BMP‐2 bone morphogenetic protein‐2, Con control, OCN osteocalcin, RUNX2 Runt-related transcription factor 2, SS 22(S)-hydroxycholesterol and 20(S)-hydroxycholesterol