Senolytics alleviate the degenerative disorders of temporomandibular joint in old age

Abstract Aging is one of the major risk factors for degenerative joint disorders, including those involving the temporomandibular joint (TMJ). TMJ degeneration occurs primarily in the population over 65, significantly increasing the risk of joint discomfort, restricted joint mobility, and reduced quality of life. Unfortunately, there is currently no effective mechanism‐based treatment available in the clinic to alleviate TMJ degeneration with aging. We now demonstrate that intermittent administration of senolytics, drugs which can selectively clear senescent cells, preserved mandibular condylar cartilage thickness, improved subchondral bone volume and turnover, and reduced Osteoarthritis Research Society International (OARSI) histopathological score in both 23‐ to 24‐month‐old male and female mice. Senolytics had little effect on 4 months old young mice, indicating age‐specific benefits. Our study provides proof‐of‐concept evidence that age‐related TMJ degeneration can be alleviated by pharmaceutical intervention targeting cellular senescence. Since the senolytics used in this study have been proven relatively safe in recent human studies, our findings may help justify future clinical trials addressing TMJ degeneration in old age.

Micro-CT. The MCC and subchondral bone were analyzed using micro-computerized tomography (micro-CT) (SCANCO Medical AG, Bruttisellen, Switzerland) as previously published . The samples were scanned in 70% alcohol and 55 kV and 145 μA was used to acquire serial tomographic projections, with a voxel size of 6μm and 1000 projections per rotation were collected at 300,000 μs. An automated algorithm using local threshold segmented the reconstructed grey scale images to distinguish calcified tissue from non-calcified tissue. The mushroom shaped head of the condyle was our region of interest.
Bone volume over total volume (BV/TV%) and bone tissue density (mg/ccm HA) were assessed.
Histological staining and quantification. Tissue sections were stained for Tartrate Resistant Acid Phosphatase (TRAP) using ELF97 (Life Tech, Waltham, MA, USA), which generates a yellow fluorescent signal. After imaging for TRAP, the same slide was stained for alkaline phosphatase activity using a fluorescent fast red substrate (Sigma) and for cell nuclei using DAPI (Thermo Fisher Scientific, Waltham, MA, USA) then reimaged. After that, the slide was rinsed in distilled water and stained with TB (IHC WORLD, LLC; Woodstock, MD, USA)) to examine proteoglycans, and reimaged using bright field microscopy. TB stains the cartilage matrix/proteoglycans in the matrix intense blue to light blue. Finally sections were stained for safranin O (IHC WORLD, LLC; Woodstock, MD, USA). The cartilage will stain orange to red and nuclei will be stained black. The background (usually bone) will be stained green. We examined TRAP and AP activity in the MCC and subchondral bone by counting the number of yellow and red pixels dividing by the total number of pixels in the whole region. AP distance was measured by assessing the distance from superficial layer of the cartilage to mineralized cartilage layer (AP positive layer) in 10 different locations throughout the MCC.
Cartilage thickness was measured in TB-stained sections. The distance from the outer cellular layer of the MCC to the tidemark was measured and normalized to the scale bar in 10 different locations throughout the entire MCC, and averaged absolute thickness (µm) was shown. Immunostaining for MMP13 (#39012, targeting both latent and active form, Abcam, Cambridge, UK), Rela (#231481, Abcam), Bmp2 (#14933, Abcam) and p21 (sc-6246, Santa Cruz Biotechnology, Dallas, TX) were performed as previously published Wang et al., 2020). For all staining images, 3-4 images were taken per mouse (covering most of the MCC region), and total of 300-800 cells per mouse were counted for quantification.
OARSI score assessment. OARSI score was assessed according to the OARSI cartilage histopathology assessment system (Pritzker et al., 2006) in safranin O-stained sections. Single examiner did the measurement of the OARSI scores. The examiner was blinded to the groups and did the measurement twice (3 days apart) on the randomly generated samples provided to him. Cohen's kappa statistic for the intra-examiner reliability was 0.95.

Statistical analysis.
Both Two-Way ANOVA with Fisher's LSD test and unpaired Student's t-test were used to compare 2 groups. contributed to the data analysis and the manuscript preparation. Y.Z., and M.X., wrote the manuscript with input from all coauthors. M.X. and S.Y. oversaw all experimental design,