Tensile force-induced PDGF-BB/PDGFRβ signals in periodontal ligament fibroblasts activate JAK2/STAT3 for orthodontic tooth movement

Abstract

Orthodontic force-induced osteogenic differentiation and bone formation at tension side play a pivotal role in orthodontic tooth movement (OTM). Platelet-derived growth factor-BB (PDGF-BB) is a clinically proven growth factor during bone regeneration process with unclear mechanisms. Fibroblasts in periodontal ligament (PDL) are considered to be mechanosensitive under orthodontic force. Thus, we established OTM model to investigate the correlation between PDGF-BB and fibroblasts during bone regeneration at tension side. We confirmed that tensile force stimulated PDL cells to induce osteogenic differentiation via Runx-2, OCN up-regulation, and to accelerate new bone deposition along the periodontium and the alveolar bone interface. Interestingly, PDGF-BB level was remarkably enhanced at tension side during OTM in parallel with up-regulated PDGFRβ+/α-SMA+ fibroblasts in PDL by immunohistochemistry. Moreover, orthodontic force-treated primary fibroblasts from PDL were isolated and, cultured in vitro, which showed similar morphology and phenotype with control fibroblasts without OTM treatment. PDGFRβ expression was confirmed to be increased in orthodontic force-treated fibroblasts by immunofluorescence and flow cytometry. Bioinformatics analysis identified that PDGF-BB/PDGFRβ signals were relevant to the activation of JAK/STAT3 signals. The protein expression of JAK2 and STAT3 was elevated in PDL of tension side. Importantly, in vivo, the treatment of the inhibitors (imatinib and AG490) for PDGFRβ and JAK-STAT signals were capable of attenuating the tooth movement. The osteogenic differentiation and bone regeneration in tension side were down-regulated upon the treatment of inhibitors during OTM. Meanwhile, the expressions of PDGFRβ, JAK2 and STAT3 were inhibited by imatinib and AG490. Thus, we concluded that tensile force-induced PDGF-BB activated JAK2/STAT3 signals in PDGFRβ⁺ fibroblasts in bone formation during OTM.

Figure 1

Orthodontic tooth movement and representative three-dimensional images of maxillae in SD rats. (A) Orthodontic appliance placement. A coiled-spring was placed between the left maxillary first molar and the maxillary incisors with cured resin on the incisors. (B) A representative image of tooth movement mesially at day 7. Note the amount of space visible between the first and second molar. The distance was indicated by black arrows. (C) Representative three-dimensional images of the maxilla at day 7 and day 14 of OTM. The white arrow indicated the direction of force. Trans transverse, L left, R right. (D) The region of interest (ROI) was defined as the zone of alveolar bone at the distal coronal one-third area of the distal root (the tension area, T) and the mesial coronal one-third area (the pressure area, P) for Micro-CT analysis. The white arrow indicated the direction of force. (E) Analysis of the distance of tooth mesial movement and microstructural parameters of alveolar bone in control group and at day 7 and 14 of OTM. BMD bone mineral density, BV/TV bone volume/total volume, Tb.Sp trabecular separation, Tb.Th trabecular thickness, Tb.N trabecular number. Each column represents the mean value of triplicate experiments. *p < 0.05, **p < 0.01; ^#p < 0.05, ^##p < 0.01 comparison between tension area and pressure area in each group, n = 8/group.

Figure 2

Tension force induced the periodontal ligament change and new bone formation in the tension area during OTM. (A) Schematic graph illustrating the region of interest for histological analysis during OTM. The distal coronal one-third area of the distal root was the tension area (T), and the pressure area (P) was the mesial coronal one-third area. M1 the left maxillary first molar, M2 the left maxillary second molar, AB alveolar bone, DP dental pulp; The black arrow indicated the direction of force. (B) Representative H-E staining images showed the width of periodontal ligament changes in the tension (blue arrows) and compression side (black arrows) in control group and at day 7 of OTM. (C) H-E staining showed the morphology change of periodontal ligament at tension side in Control group, and at day 7 and 14 of OTM. The boxed regions are shown at a higher magnification in their corresponding right figures. (D) Sequential fluorescence labeling observations at day 28 of OTM. Green and red represent labeling by Tetracycline (day 7) and Alizarin Red S (day 14), respectively. The white two-way arrows indicated the newly formed bone. (E) The statistics of mineral apposition rate (MAR) at tension side and pressure side at day 28 of OTM. (F) Van Gieson staining at day 28 of OTM. The black arrow indicated the direction of force. The blue boxed region (tension area) and yellow boxed region (pressure area) were shown at a higher magnification in their corresponding figures, respectively. AB alveolar bone, DP dental pulp, PDL periodontal ligament, NB new bone. Each column represents the mean value of triplicate experiments. **p < 0.01, comparison between Tension group (tension side) and Pressure group (pressure side), n = 8/group.

Figure 3

Osteogenesis at tension side and osteoclastogenesis at compression side during OTM. (A,B) Immunohistochemistry analysis for Runx-2 and OCN in the pressure area and tension area during OTM (A) and the semi-quantitative analysis (B). The boxed regions are shown at a higher magnification in their corresponding right figures. P pressure area, T tension area. (C,D) TRAP staining for osteoclasts (C) and the number of TRAP-positive osteoclasts (D) at pressure area and tension area in three groups. The boxed regions are shown at a higher magnification in their corresponding right figures. The green arrows indicated osteoclasts. P pressure area, T tension area. Each column represents the mean value of triplicate experiments. ^#p < 0.05, ^##p < 0.01 comparison between tension area and pressure area in each group; *p < 0.05, **p < 0.01, n = 8/group.

Figure 4

PDGF-BB responsive-fibroblasts are up-regulated by orthodontic force. (A) Protein expression of α-SMA, PDGF-BB, PDGFRβ was measured by immunohistochemistry (IHC) in Control group and at day 7 and day 14 of OTM. The boxed regions are shown at a higher magnification in their corresponding right figures. P pressure area, T tension area. (B) The semi-quantitative analysis for IHC. (C) Cell morphology of normal PDL fibroblasts and orthodontic force-induced PDL fibroblasts. (D) Identification of PDL fibroblasts by flow cytometry. Identification of cell surface markers CD11b, CD45, CD29 and CD90. (E) Percentage of PDGRRβ^high PDL fibroblasts in Control group and Force group. **p < 0.01, n = 6/group. (F) Representative immunofluorescence images stained of α-SMA (red), PDGFRβ (green), and DAPI (blue, nuclei) in normal PDL fibroblasts and orthodontic force-induced PDL fibroblasts. The arrows indicated low-expressed PDGFRβ PDL fibroblasts. Each column represents the mean value of triplicate experiments. ^##p < 0.01 comparison between tension area and pressure area in each group, **p < 0.01 comparison with the tension area among three groups, n = 8/group.

Figure 5

Bioinformatics analysis of conceivable factors interacted with PDGFRβ. (A) Biological process (GO). (B) KEGG pathways. (C,D) PDGFRβ predicted functional partners analyzed by STRING. GO Gene Ontology, KEGG Kyoto Encyclopedia of Genes and Genomes.

Figure 6

PDGFRβ/JAK2/STAT3 pathway might regulate bone formation in tension side during OTM. (A) Protein expression of JAK2, STAT3 was measured by immunohistochemistry (IHC) in Control group and at day 7 group and day 14 group. The boxed regions are shown at a higher magnification in their corresponding right figures. P pressure area, T tension area. (B) The semi-quantitative analysis for IHC. (C) Three-dimensional sagittal views of the left maxillae in Vehicle, Imatinib and AG490 groups at day 7 and day 14 of OTM. Note the interdental distance visible between the first and second molar. The red two-way arrows indicated the distance between the first and second molars during OTM. The white arrow indicated the direction of force. (D) Analysis of the distance of tooth mesial movement and microstructural parameters of alveolar bone in tension area of Vehicle, Imatinib and AG490 groups at day 7 and 14 of OTM. BMD bone mineral density, BV/TV bone volume/total volume, Tb.N trabecular number, Tb.Th trabecular thickness, Tb.Sp trabecular separation. Each column represents the mean value of triplicate experiments. *p < 0.05, **p < 0.01 compared with the Vehicle group at day 7 or day 14 of OTM, n = 8/group.

Figure 7

The expressions of PDGFRβ-related pathway during OTM. (A) Protein expression of Runx-2, OCN, JAK2 and STAT3 was measured by immunohistochemistry (IHC) in tension area of Vehicle, Imatinib and AG490 groups at day 7 of OTM. (B) The semi-quantitative analysis for IHC. (C) Schematic graph illustrates the mechanism of the PDGF-BB/PDGFRβ/JAK-2/STAT3 signal axis regulating bone formation under tensile force during OTM. Each column represents the mean value of triplicate experiments. **p < 0.01 compared with the Vehicle group at day 7 of OTM, n = 8/group.