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

PLoS One. 2016 Oct 10;11(10):e0164599. doi: 10.1371/journal.pone.0164599. eCollection 2016.


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.

MeSH terms

  • Animals
  • Apoptosis / drug effects*
  • Botulinum Toxins, Type A / adverse effects*
  • Botulinum Toxins, Type A / pharmacology
  • Calcification, Physiologic / drug effects
  • Cartilage* / diagnostic imaging
  • Cartilage* / metabolism
  • Cartilage* / pathology
  • Cell Proliferation / drug effects*
  • Chondrocytes* / metabolism
  • Chondrocytes* / pathology
  • Collagen Type X / biosynthesis
  • Collagen Type X / genetics
  • Extracellular Matrix* / metabolism
  • Extracellular Matrix* / pathology
  • Gene Expression Regulation
  • Mandibular Condyle* / diagnostic imaging
  • Mandibular Condyle* / metabolism
  • Mandibular Condyle* / pathology
  • Masseter Muscle / diagnostic imaging
  • Masseter Muscle / metabolism
  • Masseter Muscle / pathology
  • Mice
  • Mice, Transgenic
  • Smad Proteins / genetics
  • Smad Proteins / metabolism
  • Vascular Endothelial Growth Factor A / biosynthesis
  • Vascular Endothelial Growth Factor A / genetics
  • X-Ray Microtomography*


  • Col10a1 protein, mouse
  • Collagen Type X
  • Smad Proteins
  • Vascular Endothelial Growth Factor A
  • vascular endothelial growth factor A, mouse
  • Botulinum Toxins, Type A

Grant support

The study was funded partially by American Association of Orthodontic Foundation and partially by startup funds of Sumit Yadav.