Cellular and Matrix Response of the Mandibular Condylar Cartilage to Botulinum Toxin

Abstract

Objectives: To evaluate the cellular and matrix effects of botulinum toxin type A (Botox) on mandibular condylar cartilage (MCC) and subchondral bone.

Materials and methods: Botox (0.3 unit) was injected into the right masseter of 5-week-old transgenic mice (Col10a1-RFPcherry) at day 1. Left side masseter was used as intra-animal control. The following bone labels were intraperitoneally injected: calcein at day 7, alizarin red at day 14 and calcein at day 21. In addition, EdU was injected 48 and 24 hours before sacrifice. Mice were sacrificed 30 days after Botox injection. Experimental and control side mandibles were dissected and examined by x-ray imaging and micro-CT. Subsequently, MCC along with the subchondral bone was sectioned and stained with tartrate resistant acid phosphatase (TRAP), EdU, TUNEL, alkaline phosphatase, toluidine blue and safranin O. In addition, we performed immunohistochemistry for pSMAD and VEGF.

Results: Bone volume fraction, tissue density and trabecular thickness were significantly decreased on the right side of the subchondral bone and mineralized cartilage (Botox was injected) when compared to the left side. There was no significant difference in the mandibular length and condylar head length; however, the condylar width was significantly decreased after Botox injection. Our histology showed decreased numbers of Col10a1 expressing cells, decreased cell proliferation and increased cell apoptosis in the subchondral bone and mandibular condylar cartilage, decreased TRAP activity and mineralization of Botox injected side cartilage and subchondral bone. Furthermore, we observed reduced proteoglycan and glycosaminoglycan distribution and decreased expression of pSMAD 1/5/8 and VEGF in the MCC of the Botox injected side in comparison to control side.

Conclusion: Injection of Botox in masseter muscle leads to decreased mineralization and matrix deposition, reduced chondrocyte proliferation and differentiation and increased cell apoptosis in the MCC and subchondral bone.

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.