3D-Morphometric Analysis of Degenerative TMJ Changes in Klotho Mice

Objectives: TMJ functions as loadbearing-absorbants against life-long masticatory stimuli. Reports using klotho (-/-) mice showed multifaceted phenotypic alterations including a short life span, osteoporosis, ectopic calcification so on, but the details of TMJ architecture and age-dependent changes in the mutant remain to be elucidated. We herein aimed to investigate three-dimensionally the after-birth cranio-facial growth and TMJ microarchitecture by combining microfocus X-ray computed tomography (µCT) and serial histologic sectioning. Materials and methods: C57Bl/6 mice, heterozygous with respect to klotho gene, were purchased from a supplier and have been housed at our Institutional animal facility. Homozygous (kl/kl) and wild-type animals at 4-8 weeks old were killed by perfusion with 4% paraformaldehyde fixative. Micro-CT images of the individual specimens were recorded with a high-resolution micro computed tomography system (Nittetsu ELE-SCAN, Japan). After decalcification in EDTA, paraffin-embedded serial sections (4mm thick) were prepared to examine condylar cartilage and cellular phenotypes. A series of histologic sections with Toluidine blue (pH 4.1) staining were also used to reconstruct the entire TMJ regions. All 3D reconstructions were conducted using Ratoc TRI/3D-BON softwares. Results: Klotho-null mice began to display growth retardation at 5-weeks-old and subsequently were severely emaciated at 8 weeks. Morphometry based on µCT 3D-images showed hypoplastic mandible, eruption and occlusion of upper and lower molars with less attrition, and deformation of TMJ inkl/kl mice. In particular, the condylar deformation progressed rapidly, giving rise to a loss of bony core and an erosion of peripheral cortical bone. Conclusion: The present study provides evidence that the kl/kl mice suffer accelerated deformation of the condylar head due to osteoporotic background and continuous occlusion stimuli. This animal model, featured by their short life span, is useful to monitor aging-related adaptation capability of the condylar cartilage against mechanical force. Supported in part by Grants-in-Aid from MEXT/JSPS of Japan (No.1679117).