Mechanical force modulates periodontal ligament stem cell characteristics during bone remodelling via TRPV4

Abstract

Objectives: Mechanical force plays an important role in modulating stem cell fate and behaviours. However, how periodontal ligament stem cells (PDLSCs) perceive mechanical stimulus and transfer it into biological signals, and thereby promote alveolar bone remodelling, is unclear.

Materials and methods: An animal model of force-induced tooth movement and a compressive force in vitro was used. After force application, tooth movement distance, mesenchymal stem cell and osteoclast number, and proinflammatory cytokine expression were detected in periodontal tissues. Then, rat primary PDLSCs with or without force loading were isolated, and their stem cell characteristics including clonogenicity, proliferation, multipotent differentiation and immunoregulatory properties were evaluated. Under compressive force in vitro, the effects of the ERK signalling pathway on PDLSC characteristics were evaluated by Western blotting.

Results: Mechanical force in vivo induced PDLSC proliferation, which was accompanied with inflammatory cytokine accumulation, osteoclast differentiation and TRPV4 activation; the force-stimulated PDLSCs showed greater clonogenicity and proliferation, reduced differentiation ability, improved induction of macrophage migration, osteoclast differentiation and proinflammatory factor expression. The biological changes induced by mechanical force could be partially suppressed by TRPV4 inhibition. Mechanistically, force-induced activation of TRPV4 in PDLSCs regulated osteoclast differentiation by affecting the RANKL/OPG system via ERK signalling.

Conclusions: Taken together, we show here that TRPV4 activation in PDLSCs under mechanical force contributes to changing their stem cell characteristics and modulates bone remodelling during tooth movement.

Figure 1

Periodontal ligament stem cells (PDLSCs) and osteoclasts accumulate on the compression side of periodontal tissues following application of mechanical force in vivo. A, Representative occlusal view and micro‐CT images of tooth movement for 3 and 7 d. Semi‐quantitative analysis showed that the distance of tooth movement gradually increased after force was applied for 3 and 7 d (F 3 d and F 7 d, n = 6). The arrow shows the direction of mechanical force. ***P < .001 vs control. B, Representative immunofluorescence images of the compression side of distobuccal roots. The number of CD146⁺ (green) and Ki67⁺ (red) PDLSCs was increased in F 3 d and F 7 d. N = 6, *P < .05, **P < .01, vs control, ^# P < .05 vs F 3 d. Scale bar: 100 μm. C, Representative H&E and TRAP staining of the compression side of distobuccal roots. The number of TRAP‐positive osteoclasts was increased in the periodontal tissues after force application. The arrow shows the direction of mechanical force. Scale bar: 100 μm. N = 6, **P < .01 vs control. D, Representative immunofluorescence staining and semi‐quantitative analysis of IL‐6 and IL‐1β in the periodontal tissues after force was applied. The number of cells positive for IL‐6 and IL‐1β increased around the periodontal tissues after force was applied. Scale bar: 100 μm. N = 6, *P < .05 vs control. ^# P < .05 vs F 3 d

Figure 2

Biological characteristics of force‐induced rPDLSCs ex vivo. A, Growth curves of force‐induced PDLSCs (F‐PDLSCs) and normal PDLSCs (N‐PDLSCs) as determined by CCK‐8 assay. F‐PDLSCs and N‐PDLSCs isolated ex vivo proliferated at a similar rate for 1‐2 d, but F‐PDLSCs showed faster proliferation after 3 d. N = 6, *P < .05, **P < .01 vs N‐PDLSCs. B, Representative images and quantitative comparison of colony‐forming units‐fibroblastic (CFU‐F) of two different rPDLSCs. N = 6, *P < .05 vs N‐PDLSCs. C, Compared to N‐PDLSCs, the F‐PDLSCs showed a decreased capacity to form mineralized nodules, assessed by ARS staining and quantification. N = 5, ***P < .001 vs N‐PDLSCs. D, Oil Red O staining and quantification of two different rPDLSCs. F‐PDLSCs showed less accumulation of lipid‐rich vacuoles. N = 5, *P < .05 vs N‐PDLSCs. Scale bar: 400 μm. E, Representative images of crystal violet staining of RAW264.7 macrophages in Transwell assays. Conditional medium from F‐PDLSCs enhanced the migration of macrophages compared with the control. N = 6, **P < .01 vs N‐PDLSCs. Scale bar: 400 μm. F, Representative images of TRAP staining of osteoclasts among RAW264.7 macrophages co‐cultured with PDLSCs. Osteoclastic differentiation of RAW264.7 macrophages was significantly enhanced by force loading. N = 6, **P < .01 vs N‐PDLSCs. Scale bar: 200 μm. G, Relative mRNA levels of inflammation‐related genes. The mRNA levels of IL‐1β, TNF‐α, IL‐6 and MCP‐1 were upregulated in the F‐PDLSC group. ***P < .001 vs N‐PDLSCs. H, Western blot of Ki67. The protein level of Ki67 was upregulated in the force‐treated PDLSC group. *P < .05, **P < .01 vs N‐PDLSCs. Three independent assays were performed for each cell population

Figure 3

TRPV4 is present in F‐PDLSCs ex vivo. A, Relative mRNA levels of TRPV1‐4. TRPV1‐4 mRNAs were detected in rPDLSCs, while that of TRPV4 was increased in the F‐PDLSCs. **P < .01 vs N‐PDLSCs. B, Western blot of TRPV4 in rPDLSCs. The TRPV4 protein level in rPDLSCs was upregulated after force loading. ***P < .001 vs N‐PDLSCs. C, Representative immunofluorescence images and semi‐quantitative analysis of the compression side of distobuccal roots. The number of CD146⁺ (red) and TRPV4⁺ (green) PDLSCs was increased in F 3 d and F 7 d. N = 6, *P < .05 vs control, ^# P < .05 vs F 3 d. Scale bar: 100 μm. Data are means ± SD of three independent experiments

Figure 4

Inhibition of TRPV4 represses biological characteristics of F‐PDLSCs ex vivo. A, Growth curves of F‐PDLSCs and GSK219‐pre‐treated PDLSCs (GSK219). CCK‐8 assays showed that the promotion of proliferation after force loading was inhibited by the TRPV4 antagonist GSK219. N = 6, *P < .05, **P < .01 vs F‐PDLSCs. B, Representative images and quantitative comparison of CFU‐F of two different rPDLSCs. N = 6, *P < .05 vs F‐PDLSCs. C, Representative images of TRAP staining of osteoclasts among RAW264.7 macrophages co‐cultured with rPDLSCs. TRAP staining showed a significant decline in the number of TRAP⁺ osteoclasts among GSK219‐pre‐treated PDLSCs. N = 6, *P < .05 vs F‐PDLSCs. Scale bar: 200 μm. D, Representative immunofluorescence images of F‐PDLSCs and GSK219‐pre‐treated PDLSCs. The number of CD146 (green) and IL‐6 (red) double‐stained PDLSCs decreased after GSK219 treatment. N = 5, ***P < .001 vs F‐PDLSCs. Scale bar: 50 μm. E, Relative mRNA levels of inflammation‐related genes. The mRNA levels of IL‐1β, TNF‐α, IL‐6 and MCP‐1 were decreased in GSK219‐pre‐treated PDLSCs. Three independent assays were performed for each cell population

Figure 5

TRPV4 regulates force‐induced inflammation‐related gene expression and the receptor activator of nuclear factor‐κB ligand (RANKL)/osteoprotegerin (OPG) system in hPDLSCs via the ERK signalling pathway. A, Relative mRNA levels of inflammation‐related genes. The mRNA levels of IL‐6 and TNF‐α were upregulated in the force group and downregulated in the force + GSK219 group compared with the force group. B, Western blot and semi‐quantifications of Ki67 in hPDLSCs. The protein level of Ki67 was upregulated after mechanical force loading, which was mostly reversed by TRPV4 treatment. GAPDH served as an internal control for equal loading. C, GSK219 inhibition of TRPV4 decreased the force‐induced upregulation of the RANKL/OPG ratio. The protein levels of RANKL and OPG were determined in control PDLSCs or cells subjected to mechanical force with or without GSK219 treatment. D, Western blot and semi‐quantifications of TRPV4, phosphorylated ERK (P‐ERK), and total ERK (ERK) levels in hPDLSCs. The TRPV4 level and the proportion of P‐ERK/ERK were upregulated after mechanical force application and attenuated by the inhibition of TRPV4. E, Western blot and semi‐quantifications of TRPV4, P‐ERK and total ERK levels in hPDLSCs. The TRPV4 level and the proportion of P‐ERK/ERK were upregulated after mechanical force stimulation and further enhanced by a simultaneous treatment with GSK101. Data are means ± SD of three independent experiments. *P < .05, **P < .01, ***P < .001 vs control. ^# P < .05, ^## P < .01, ^### P < .001 vs force

Figure 6

The activation of TRPV4 in PDLSCs under mechanical force contributes to the changes in their biological properties and modulates bone remodelling during tooth movement