Increased joint loading induces subchondral bone loss of the temporomandibular joint via the RANTES-CCRs-Akt2 axis

Abstract

Early-stage temporomandibular joint osteoarthritis (TMJOA) is characterized by excessive subchondral bone loss. Emerging evidence suggests that TMJ disc displacement is involved, but the pathogenic mechanism remains unclear. Here, we established a rat model of TMJOA that simulated disc displacement with a capacitance-based force-sensing system to directly measure articular surface pressure in vivo. Micro-CT, histological staining, immunofluorescence staining, IHC staining, and Western blot were used to assess pathological changes and underlying mechanisms of TMJOA in the rat model in vivo as well as in RAW264.7 cells in vitro. We found that disc displacement led to significantly higher pressure on the articular surface, which caused rapid subchondral bone loss via activation of the RANTES-chemokine receptors-Akt2 (RANTES-CCRs-Akt2) axis. Inhibition of RANTES or Akt2 attenuated subchondral bone loss and resulted in improved subchondral bone microstructure. Cytological studies substantiated that RANTES regulated osteoclast formation by binding to its receptor CCRs and activating the Akt2 pathway. The clinical evidence further supported that RANTES was a potential biomarker for predicting subchondral bone loss in early-stage TMJOA. Taken together, this study demonstrates important functions of the RANTES-CCRs-Akt2 axis in the regulation of subchondral bone remodeling and provides further knowledge of how disc displacement causes TMJOA.

Keywords: Bone Biology; Chemokines; Osteoarthritis; Osteoclast/osteoblast biology.

Figure 1. Disc displacement causes subchondral bone loss in early-stage TMJOA.

Male Sprague-Dawley rats underwent sham or right-sided unilateral DDw/oR surgery. Rats were euthanized at baseline (0 days), 3 days, and 1, 2, 4, and 8 weeks after surgery. (A) Micro-CT images of TMJ condyles. Lines 1 and 4: sagittal view in 2-dimensional images; lines 2 and 5: sagittal view in 3-dimensional images; lines 3 and 6: horizontal view in 3-dimensional images. The yellow arrows show condylar surface erosion. The red arrows indicate condylar surface flattening and local sclerosis. Scale bar: 1 mm. (B–G) Quantitative analysis of (B) BMD, (C) BV/TV, (D) BS/BV, (E) Tb.Th, (F) trabecular number (Tb.N), and (G) Tb.Sp in subchondral bone of TMJ condylar heads determined by micro-CT measurements. (H–J) Quantitative analysis of (H) length, (I) width, and (J) height of TMJ condylar heads determined by micro-CT measurements. Data are presented as mean ± 95% CI, and 1 representative image of 8 independent samples per group is shown. Statistical analyses were determined by 2-way ANOVA with Bonferroni’s multiple comparison test. *P < 0.05, **P < 0.01.

Figure 2. Histopathological changes of TMJs in a rat TMJOA model induced by DDw/oR.

Male Sprague-Dawley rats underwent right-sided unilateral DDw/oR surgery. Rats were euthanized at baseline (0 days), 1, 2, 4, and 8 weeks (w) after surgery. (A) Representative images of TRAP staining in TMJ sagittal sections. The black dotted line represents the demarcation between articular cartilage and subchondral bone. The black arrows indicate TRAP⁺ osteoclasts with 3 or more nuclei in subchondral bone. Scale bar: (top row) 200 μm, (bottom row) 50 μm. (B) Quantitative analysis of the number of TRAP⁺ osteoclasts in subchondral bone of condyles. (C) Representative images of Masson’s trichrome staining in TMJ sagittal sections. The black arrows indicate unmineralized bone (osteoid) in subchondral bone. Scale bar: 100 μm. (D) Quantitative analysis of the percentage of unmineralized bone (osteoid) area. (E) Representative images of Safranin O–fast green staining in TMJ sagittal sections. The black dotted line represents the demarcation between articular cartilage and subchondral bone. Scale bar: 200 μm. (F) Quantitative analysis of the OARSI score of articular cartilage. (G) Representative images of H&E staining in TMJ sagittal sections. The black arrows indicate synovial lining hyperplasia. Scale bar: 100 μm. (H) Quantitative analysis of the synovitis score of TMJ synovium. Data are presented as mean ± 95% CI, and 1 representative image of 6 independent samples per group is shown. Statistical analyses were determined by 1-way ANOVA with Bonferroni’s multiple comparison test. *P < 0.05, **P < 0.01. Abbreviations: Oc, osteoclast; OARSI, Osteoarthritis Research Society International.

Figure 3. Expression levels of osteoclasts and osteoblasts in a rat TMJOA model induced by DDw/oR.

Male Sprague-Dawley rats underwent sham or right-sided unilateral DDw/oR surgery. Rats were euthanized at 1 and 8 weeks (w) after surgery. (A and B) Representative images of (A) RANKL and (B) OPG IHC staining in TMJ sagittal sections. The black dotted line represents the demarcation between articular cartilage and subchondral bone. The black arrows indicate positive cells in subchondral bone, and the asterisks indicate negative signals in subchondral bone. Scale bar: 50 μm. (C and D) Quantitative analysis of the percentage of (C) RANKL⁺ cells and (D) OPG⁺ cells in subchondral bone of condyles. (E) Quantitative analysis of the ratio of RANKL⁺ cells to OPG⁺ cells in subchondral bone of condyles. (F and G) Representative images of (F) CTSK and (G) OCN IHC staining in TMJ sagittal sections. The black dotted line represents the demarcation between articular cartilage and subchondral bone. The black arrows indicate positive cells in subchondral bone. Scale bar: 50 μm. (H) Quantitative analysis of the number of CTSK⁺ cells in subchondral bone of condyles. (I) Quantitative analysis of the percentage of OCN⁺ cells in subchondral bone of condyles. Data are presented as mean ± 95% CI, and 1 representative image of 4 to 6 independent samples per group is shown. Statistical analyses were determined by 2-way ANOVA with Bonferroni’s multiple comparison test. *P < 0.05, **P < 0.01. Abbreviations: Oc, osteoclast; OCN, osteocalcin.

Figure 4. The RANTES-CCRs-Akt2 axis is activated in DDw/oR–induced early-stage TMJOA.

Male Sprague-Dawley rats underwent right-sided unilateral DDw/oR surgery. Rats were euthanized at baseline (0 weeks), early (1 week), and late (8 weeks) time points after surgery. (A) Representative images of RANTES and CD68 immunofluorescence costaining in TMJ sagittal sections. RANTES⁺ cells appear in green and CD68⁺ cells appear in red. The white dotted line represents the demarcation between articular cartilage and subchondral bone. The white arrows indicate RANTES⁺ cells in articular cartilage. Scale bar: 50 μm. (B) Representative images of p-Akt2 and CD68 immunofluorescence costaining in TMJ sagittal sections. p-Akt2⁺ cells appear in green and CD68⁺ cells appear in red. The white dotted line represents the demarcation between articular cartilage and subchondral bone. The white arrows indicate p-Akt2 and CD68 double-positive cells in subchondral bone, and the asterisks indicate CD68 single-positive cells. Scale bar: 50 μm. (C) Quantitative analysis of the number of RANTES⁺ cells in articular cartilage of condyles. (D) Quantitative analysis of the number of CD68⁺ cells in subchondral bone of condyles. (E) Quantitative analysis of the ratio of p-Akt2⁺ and CD68 double-positive cells to CD68-positive cells in subchondral bone of condyles. Data are presented as mean ± 95% CI, and 1 representative image of 4 independent samples per group is shown. Statistical analyses were determined by 1-way ANOVA with Bonferroni’s multiple comparison test. **P < 0.01. Abbreviation: w, week.

Figure 5. Expression levels of p-Akt1, p-Akt2, and CTSK in subchondral bone of a rat TMJOA model induced by DDw/oR.

Male Sprague-Dawley rats underwent right-sided unilateral DDw/oR surgery. Rats were euthanized at baseline (0 days), 3 days, and 1, 2, 4, and 8 weeks (w) after surgery. (A) Representative Western blot bands showing the expression of p-Akt1 and t-Akt1 in the condylar subchondral bone. (B) Quantitative analysis of the relative intensity of p-Akt1. The level of p-Akt1 is normalized to t-Akt1. (C) Representative Western blot bands showing the expression of p-Akt2 and t-Akt2 in the condylar subchondral bone. (D) Quantitative analysis of the relative intensity of p-Akt2. The level of p-Akt2 was normalized to t-Akt2. (E) Representative Western blot bands showing the expression of CTSK and β-actin in the condylar subchondral bone. (F) Quantitative analysis of the relative intensity of CTSK. β-actin was used as an internal control. Data were presented as mean ± 95% CI, and 1 representative image of 5 to 6 independent samples per group is shown. Statistical analyses were determined by 1-way ANOVA with Bonferroni’s multiple comparison test. *P < 0.05, **P < 0.01.

Figure 6. Specific inhibition of RANTES alleviates subchondral bone loss in early-stage TMJOA.

Male Sprague-Dawley rats were treated with shCtrl or shRantes and underwent sham or right-sided unilateral DDw/oR surgery. Rats were euthanized at 1 week after surgery. (A) Micro-CT images of TMJ condyles (sagittal view). The yellow arrows show condylar surface erosion. Scale bar: 1 mm. (B) Representative images of TRAP staining in TMJ sagittal sections. The black dotted line represents the demarcation between articular cartilage and subchondral bone. The black arrows indicate TRAP⁺ osteoclasts with 3 or more nuclei in subchondral bone. Scale bar: 100 μm. (C–G) Quantitative analysis of (C) BMD, (D) BV/TV, (E) BS/BV, (F) Tb.Th, and (G) Tb.Sp in subchondral bone of TMJ condylar heads determined by micro-CT measurements. (H) Quantitative analysis of the number of TRAP⁺ osteoclasts in subchondral bone. Data are presented as mean ± 95% CI, and 1 representative image of 5 independent samples per group is shown. Statistical analyses were determined by 2-way ANOVA with Bonferroni’s multiple comparison test. *P < 0.05, **P < 0.01. Abbreviation: Oc, osteoclast.

Figure 7. Specific inhibition of Akt2 alleviates subchondral bone loss in early-stage TMJOA.

Male Sprague-Dawley rats were treated with shCtrl or shAkt2 and underwent sham or right-sided unilateral DDw/oR surgery. Rats were euthanized at 1 week after surgery. (A) Micro-CT images of TMJ condyles (sagittal view). The yellow arrows show condylar surface erosion. Scale bar: 1 mm. (B) Representative images of TRAP staining in TMJ sagittal sections. The black dotted line represents the demarcation between articular cartilage and subchondral bone. The black arrows indicate TRAP⁺ osteoclasts with 3 or more nuclei in subchondral bone. Scale bar: 100 μm. (C–G) Quantitative analysis of (C) BMD, (D) BV/TV, (E) BS/BV, (F) Tb.Th, and (G) Tb.Sp in subchondral bone of TMJ condylar heads determined by micro-CT measurements. (H) Quantitative analysis of the number of TRAP⁺ osteoclasts in subchondral bone. Data were presented as mean ± 95% CI, and 1 representative image of 5 independent samples per group is shown. Statistical analyses were determined by 2-way ANOVA with Bonferroni’s multiple comparison test. *P < 0.05, **P < 0.01. Abbreviation: Oc, osteoclast.

Figure 8. Increased pressure on the anterior surface of condyles is yielded by TMJ disc displacement.

Male Sprague-Dawley rats underwent right-sided unilateral DDw/oR surgery. (A) The graphic illustration of capacitance-based force-sensing system fastened to the anterior surface of condyles. (B) Capacitance variations (ΔC) during the cycles from mouth closure to mouth opening in a rat before and after DDw/oR surgery. C₀, initial capacitance. (C) The pressures on the anterior surface of condyles at the maximum passive mouth-opening position before and after DDw/oR surgery. Data are presented as mean ± 95% CI, and 1 representative image of 5 independent samples per group is shown. Statistical analysis was determined by Student’s t-test. **P < 0.01.

Figure 9. The RANTES-CCRs-Akt2 axis is activated by increased pressure.

Isolated rat condylar heads were stimulated under 0, 100, and 600 kPa in vitro. (A) Representative images of RANTES immunofluorescence staining in condylar sagittal sections. The white dotted line represents the demarcation between articular cartilage and subchondral bone. The white arrows indicate RANTES⁺ cells in articular cartilage. Scale bar: 50 μm. (B) Representative images of p-Akt2 and CD68 immunofluorescence costaining in condylar sagittal sections. p-Akt2⁺ cells appear in green and CD68⁺ cells appear in red. The white dotted line represents the demarcation between articular cartilage and subchondral bone. The white arrows indicate p-Akt2 and CD68 double-positive cells in subchondral bone. Scale bar: 50 μm. (C) Representative images of CTSK immunofluorescence staining in condylar sagittal sections. The white dotted line represents the demarcation between articular cartilage and subchondral bone. The white arrows indicate CTSK⁺ cells in subchondral bone. Scale bar: 50 μm. (D) Quantitative analysis of the number of RANTES⁺ cells in articular cartilage. (E) Quantitative analysis of the ratio of p-Akt2⁺ and CD68 double-positive cells to CD68-positive cells in subchondral bone. (F) Quantitative analysis of the number of CTSK⁺ cells in subchondral bone. Data are presented as mean ± 95% CI, and 1 representative image of 4 independent samples per group is shown. Statistical analyses were determined by 1-way ANOVA with Bonferroni’s multiple comparison test. *P < 0.05, **P < 0.01.

Figure 10. The RANTES-CCRs-Akt2 axis facilitates osteoclast formation.

RAW264.7 cells were treated with different osteoclast-inducing conditions in vitro. (A) Representative Western blot bands of the expression of p-Akt2, t-Akt2, CTSK, and β-actin. (B and C) Quantitative analysis of the relative intensity of (B) p-Akt2 and (C) CTSK. The level of p-Akt2 was normalized to t-Akt2. The level of CTSK was normalized to β-actin. (D) Representative Western blot bands of the expression of p-Akt2, t-Akt2, CTSK, and β-actin. (E and F) Quantitative analysis of the relative intensity of (E) p-Akt2 and (F) CTSK. The level of p-Akt2 was normalized to t-Akt2. The level of CTSK was normalized to β-actin. (G) Representative images of TRAP staining. The black arrows indicate TRAP⁺ osteoclasts, and the asterisks indicate negative signals. Scale bar: 100 μm. (H) Quantitative analysis of the number of TRAP⁺ osteoclasts. (I) Quantitative analysis of the number of TRAP⁺ osteoclasts. Data are presented as mean ± 95% CI, and 1 representative image of 3 to 5 independent experiments with biological replicates is shown. Statistical analyses were determined by 2-way ANOVA with Bonferroni’s multiple comparison test. *P < 0.05, **P < 0.01. Abbreviation: Oc, osteoclast.

Figure 11. The relationship between the concentration of RANTES and different stages of TMJOA in humans.

Synovial fluid samples were collected from patients diagnosed with TMJ DDw/oR. (A–C) Radiographic changes of TMJ condylar bone remodeling (coronal view) in the natural course of a patient with DDw/oR. (A) Normal condylar bone with no sign of OA 2 weeks (w) after disc displacement. (B) Early-stage OA after 5 months (m). (C) Late-stage OA after 3 years (y). The yellow arrow shows condylar bone erosion. The red arrow indicates condylar bone flattening and sclerosis. (D) Comparison of RANTES levels in synovial fluid of patients with different stages of DDw/oR. (E) ROC curve of the logistic regression model of RANTES drawn by the back-substitution method. The AUC of RANTES was 0.798. Data are presented as mean ± 95% CI, and 1 representative of 13 to 15 independent samples per group is shown. Statistical analysis was determined by 1-way ANOVA with Bonferroni’s multiple comparison test. *P < 0.05, **P < 0.01.