Fig 1.
Reduced bone volume and density and reduced condyle width at the Botox injected side.
Coronal micro-CT images of condyles of control (A) and Botox (B) injected side masseter 4 weeks after unilateral Botox injection. Quantification of bone parameters: C) BVF—bone volume fraction, D) Trabecular Thickness, E) Trabecular Spacing, F) Tissue Density. Morphometric measurements (G-H) performed in Faxitron xray images of control and Botox injected side mandibles: I) Mandibular Lengh, J) Condyle Head Length, L) Condyle Width. Histograms (C-F, I-L) represent means ± SD for n = 13 per group. *Significant difference between control and Botox injected side (p < 0.05). Scale bar = 500μm.
Fig 2.
Decrease in osteoclastic activity and mineralization at the Botox injected side condyle.
Sagittal sections of control (left side) (A) and Botox (right side) (B) injected side condyles stained for TRAP. C) Quantification of TRAP positive pixels (yellow) over subchondral bone area. Sagittal sections stained for alkaline phosphatase, control (D) and Botox injected side (E). F) Quantification of distance mapping. Histograms (C,F) represents means ± SD for n = 7 per group. * Significant difference between control and Botox injected side (p < 0.05). No significant difference between control and Botox injected side for alkaline phosphatase distance mapping (F). Scale bar = 500μm.
Fig 3.
Reduced Col10a1 positive cells, decreased cell proliferation and increased cell apoptosis at the MCC of Botox injected side.
Representative sagittal sections of condyles of transgenic mice (Col10a1-RFPcherry), control (A) and Botox (B) injected side. C) Quantification of Col10a1 positive pixels (red) over MCC area. Sagittal sections of control (D) and Botox (E) injected side condyles stained for EdU. F) Quantification of EdU positive pixels (yellow) over DAPI positive pixels (blue) at the proliferative zone. TUNEL staining in sections of control (G) and Botox (H) injected side condyles. Histograms (C-I) represents means ± SD for n = 7 (C-F) and n = 5 (I) per group. * Significant difference between control and Botox injected side (p < 0.05). Scale bar = 500μm.
Fig 4.
Reduced proteoglycan secretion at the Botox injected side MCC.
Sagittal sections of control (A) and Botox (B) injected side condyles stained for toluidine blue. C-F) Quantification of toluidine blue stained area and distance mapping at the MCC. Safranin O staining for control (E) and Botox injected side condyles (F). Histograms (C-D) represent means ± SD for n = 7 per group. * Significant difference between control and Botox injected side (p < 0.05). Scale bar = 500μm.
Fig 5.
Decreased expression of SMAD1/5/8 and VEFG in the Botox injected side MCC and subchondral bone.
Immunohistochemistry for SMAD1/5/8 in sagittal sections of control (A) and Botox (B) injected side condyles. VEGF Immunohistochemistry in sections of control (C) and Botox (D) injected side condyles. Scale bar = 500μm.