Parathyroid hormone ameliorates temporomandibular joint osteoarthritic‐like changes related to age

Abstract Objectives Ageing could be a contributing factor to the progression of temporomandibular joint osteoarthritis (TMJ OA), whereas its pathogenesis and potential therapeutic strategy have not been comprehensively investigated. Materials and methods We generated ageing mouse models (45‐week and 60‐week; 12‐week mice as control) and intermittently injected 45‐week mice with parathyroid hormone (PTH(1‐34)) or vehicle for 4 weeks. Cartilage and subchondral bone of TMJ were analysed by microCT, histological and immunostaining. Western blot, qRT‐PCR, ChIP, ELISA and immunohistochemical analysis were utilized to examination the mechanism of PTH(1‐34)’s function. Results We showed apparent OA‐like phenotypes in ageing mice. PTH treatment could ameliorate the degenerative changes and improve bone microarchitecture in the subchondral bone by activating bone remodelling. Moreover, PTH inhibited phosphorylation level of Smad3, which can combine with p16ink4a gene promoter region, resulting in reduced senescent cells accumulation and increased cellular proliferation of marrow mesenchymal stem cells (MSCs). ELISA also showed relieved levels of specific senescent‐associated secretory phenotype (SASP) in ageing mice after PTH treatment. Conclusions In summary, PTH may reduce the accumulation of senescent cells in subchondral bone by inhibiting p16ink4a and improve bone marrow microenvironment to active bone remodelling process, indicating PTH administration could be a potential preventative and therapeutic treatment for age‐related TMJ OA.

the TMJ articular surfaces increases with advancing age (50% in aged group vs 28% in young group). 7,8 In the mandibular condylar cartilage, ageing causes reduction in chondrocytes number and viability, decrease in matrix synthesis, and the appearance of OA lesions along the articular surface of the joint. 9 In the subchondral bone region, ageing animals exhibited significantly reduced bone volume. 10 Temporomandibular joint osteoarthritis causes changes in cartilage, subchondral bone, synovial membrane and other tissues. 11,12 Recently, an increasing evidence supports the significance of subchondral bone in the pathogenesis of TMJ degenerative disease. 13,14 Subchondral bone locates below the layer of calcified cartilage to provide mechanical support. Its alterations antedate the changes in articular cartilage during OA progression. 15,16 Subchondral bone remodelling is a progression throughout life in TMJ because of occlusion movement. To maintain a physical remodelling process, the activity of osteoclasts, which cause bone resorption, and osteoblasts, which are responsible for bone formation, must be precisely coordinated. During bone remodelling, factors released locally to mediate bone marrow microenvironment, which influences the recruitment and fate of bone marrow mesenchymal stem cells (MSCs) for new bone formation. [17][18][19][20][21] In the early progress of OA, the remodelling of subchondral bone is upregulated, whereas the activity of osteoblasts cannot catch with osteoclasts, resulting in decreased bone mineral density. [22][23][24] Some reasons contribute to this impaired balance, for instance, decreased number of MSCs, and reduced capacity of proliferation or differentiation which occurs with ageing. 25,26 Parathyroid hormone (PTH), an 84-amino-acid polypeptide, is a systemic hormone that regulates calcium homoeostasis by a direct action on bone and kidney. It has been revealed that intermittent injection of PTH (1-34) not only increases apparent bone mass, but also improves microarchitecture of bone such as trabecular number and connectivity by modulating bone marrow microenvironment and increasing the number of osteoblasts in ageing mice. 27,28 Furthermore, studies demonstrated a phenomenon that PTH had an effect on cell cycle progression. 29,30 While intermittent treatment of PTH has been used to prevent the progression of OA in knee or spine osteoarthritis both in animal and humans, 31,32 not much is known about the specific function of PTH administration in TMJ OA. Therefore, in the current study, we set out to address the effect and underlying mechanism of intermittently injection of PTH  in ageing mice with TMJ OA-like changes. We have found that PTH (1-34) treatment ameliorated the degenerative changes in TMJ condyles and improved subchondral bone microarchitecture by increasing the number of osteoblasts in ageing mice. Moreover, PTH inhibited the expression of p16 ink4a , a senescence biomarker as well as relieved the expression of specific senescent-associated secretory phenotype (SASP), indicating PTH administration could ameliorate TMJ OAlike alterations related to age.

| Animal
Seven-week-old C57BL/6J male mice were purchased from Chengdu Dossy Biological Technology Co., Ltd and housed in animal centre of West China Hospital, and were randomly selected at the age of 12, 45 or 60 weeks (n = 5 per group) to sacrifice. For the PTH treatment experiment, 45-week-old mice were assigned into two groups (n = 10 per group), subcutaneous injected with human PTH (1-34) (40 μg/kg per day, Bachem, Inc) or an equivalent volume of vehicle (1 mmol/L acetic acid in phosphate buffered saline (PBS)), 5 days per week, for 4 weeks. 33

| MicroCT analysis
Temporomandibular joint samples were harvested at indicated times and scanned using a microCT scanner (μCT50; SCANO, Switzerland) at a voltage of 50 kVp, a current of 200 µA and a resolution of 3.0 µm per pixel. The images were reconstructed and performed three-dimensional histomorphometry based on a previous report. 10 Briefly, two cubic regions of interest (each 100 × 100 × 100 µm 3 ) were defined from the middle of the centre and posterior third of the condylar subchondral cancellous bone. The selected regions were analysed to determine the trabecular bone volume fraction (BV/TV), trabecular thickness (Tb. Th), trabecular number (Tb. N) and trabecular separation (Tb. Sp).

| Histologic and immunologic staining
Samples were fixed in 4% paraformaldehyde for 3 days. After decalcification in 20% EDTA (pH 7.5), samples were processed, embedded in paraffin and cut into 4-μm sections using a microtome (Leica, RM2235, Germany). Haematoxylin/eosin (HE) and Safranin-O staining were performed to assess condyle histomorphology. The Mankin and Osteoarthritis Research Society International (OARSI) scores were used to assess histologic grading of cartilage degeneration. 34 Tartrate-resistant acid phosphatase (TRAP) staining, using a standard protocol (Sigma-Aldrich), was used to detect osteoclasts. For quantitative analysis, condylar subchondral cancellous bone was regarded as regions of interest, and cell counting in bone marrow was conducted by a blinded observer with ImageJ software. OSX + , TRAP + and TUNEL + cells were normalized by the bone surface, and p16 + , pSMAD3 + and pCREB + were normalized by the number of total cells. Three different sections were used from each sample, and three different samples were used for each group.

| Isolation of MSCs from mouse mandibular and cell culture
Mesenchymal stem cells from mouse mandible were isolated as described by previously. 35 Briefly, we collected mandible from 12-or 45-week-old mice and removed the attached soft tissues and teeth.
Digest with 3 mg/mL collagenase type I (Worthington Biochem) and 4 mg/mL dispase II (Roche Diagnostic) for 60 minutes at 37°C.

| Chromatin immunoprecipitation assay (ChIP)
Cells were fixed with 1% PFA, and ChIP assay was performed according to manufacturer's guidelines (Cell Signaling Technology). DNA that co-precipitated with Smad3 (Cell Signaling Technology) was ana-     Figure 1A,B). Furthermore, immunologic analysis showed that the expression of matrix metalloproteinase 13 (MMP13) and collagenase X (Col X) was significantly higher in 45-and 60-weeks relative to 12-weeks mice in cartilage layer ( Figure 1C).

| Statistics
In subchondral bone region, immunofluorescent staining of collagenase I (Col I) and Runx2 showed downregulated expression levels in ageing mice compared with 12-weeks controls. The number of Osx + osteoprogenitors was statistically decreased in 45-and 60-weeks mice, with the minimum number in 60-weeks group ( Figure 1D,F). TRAP staining and cathepsin K (CtsK) expression suggested that osteoclasts number was significantly increased with ageing ( Figure 1E,F). MicroCT analysis further revealed that 60-weeks mice had significantly decreased bone volume (%, BV/TV) and increased trabecular space (mm, Tb.Sp) ( Figure 1G,H). In accordance with this phenotype, the phosphorylation level of Smad3 was significantly increased in 45-and 60-weeks mice when compared to 12-weeks controls ( Figure 1I,J).

| PTH directly induces phosphorylation of CREB (pCREB) in mandibular condyle
We next asked whether subcutaneous injection of PTH (1-34) activates its downstream signalling in mandibular condyle subchondral bone. It has been found that PTH can stimulate downstream intracellular signalling, including cAMP/PKA signalling by virtue of phosphorylation of CREB. 39 Therefore, we performed immunostaining of phosphorylated CREB (pCREB) and visualized that pCREB expression was significantly induced in subchondral bone region at 30 minutes and peaked

F I G U R E 2 (Continued)
at 1 hour after PTH (1-34) single injection. Its expression was returned to normal after 6 hours post-injection. These data revealed the direct signalling of PTH in subchondral bone region (Figure 2A).

| Mouse orofacial bone marrow-derived MSCs (OMSCs) have self-renew and multi-lineage differentiation potential
We have previously demonstrated PTH functions to direct long bone MSCs cell fate. 40 Recent studies discovered OMSCs resided in mandibular bone region, which exhibited several distinct traits when compared to long bone MSCs. 41 To further examine the function of PTH on mandibular bone in vitro, we isolated mouse mandibu-

| PTH (1-34) induces osteogenic differentiation in young and ageing OMSCs
OMSCs of young and ageing mice were utilized to determine the ef-

| PTH regulates p16 ink4a expression in OMSCs through TGF-β signalling
We have previously shown that pSMAD3 expression was activated along with ageing while PTH (1-34) treatment reduced its activity in vivo ( Figures 1I and 2K). Indeed, Western blot analysis demonstrated that pSmad3 protein expression was dramatically increased in aged-OMSCs, while PTH (1-34) treatment significantly inhibited pSmad3 expression ( Figure 5A). Furthermore, qRT-PCR and Western blot analysis indicated that aged-OMSCs had higher p16 ink4a expression than young cells and PTH (1-34) treatment significantly downregulated its expression at both transcript and protein levels ( Figure 5A,B).
In addition, it has been proved that Smad3 can assemble in the promoter region of p16 ink4a to regulate its expression in muscle satellite cells, 42 and p16 ink4a regulates cell cycle progression by preventing S phase entry. 36,37 Thus, we sought to analyse the relationship between Smad3 and p16 ink4a in OMSCs using ChIP assay.
Using primers specific for 5′ promoter regulatory regions of p16 ink4a by qRT-PCR and PCR, the results showed Smad3 can combine with the promoter region of p16 ink4a in OMSCs ( Figure 5C). The percentage distribution of cell cycle phases (G0/G1, S and G2/M) of

| PTH regulates senescence of OMSCs and subchondral bone marrow microenvironment
It has previously suggested that p16 ink4a is one of the biomarkers in cellular senescence. Senescent cells express SASP leading to alterations in bone marrow microenvironment. 43 Therefore, we analysed the number of senescent cells and the expression of SASP factors.
SA-β gal staining appeared increased tendency in aged-OMSCs; however, the number of senescent cells reduced after 7-day PTH

| D ISCUSS I ON
The present study provides evidence that intermittent PTH (1-34) administration could ameliorate the degenerative alterations F I G U R E 4 OMSCs derived from ageing mice have an intense potential of osteogenesis with PTH treatment. A,B, Western blot analysis showing pCREB and CREB levels in OMSC after treated with PTH at different time points, and quantitative analysis. C,D, ALP staining and osteogenic-related gene expression analysis after osteogenic induction for 7 days with PTH or vehicle. E,F, ARS and osteogenic-related gene expression analysis after osteogenic induction for 14 d with PTH or vehicle. N = 3 per group. *P < .05, ***P < .005. All data were expressed as the mean ± SD associated with TMJ OA in ageing mice. It could improve subchondral bone microarchitecture by accelerating bone remodelling and modulate senescence phenotype through inhibiting p16 ink4a and SASP expression. While age has always been regarded as a very important aetiologic agent for OA, 5 the pathogenesis of TMJ OA development is more complex than we thought because of age-related changes in the musculoskeletal system. 7,44,45 We first gener- Continuous PTH exposure results in catabolic effects, while intermittent exposure is associated with anabolic effects. 52 Thus, inter- promoter, suggesting PTH treatment may function to suppress pSmad3, leading to the subsequent reduction in p16 ink4a .
Some studies found that senescent cells depend on pro-survival pathways to defend themselves against their pro-apoptotic microenvironment, 58 and the accumulation of aged cells not only losses proliferation potential, but also creates their own microenvironment, a low-grade inflammation, to affect adjacent cells by SASP. 59  It has been found that senescent cells can defend themselves against apoptosis, 58 as well as affect adjacent cells by senescent-associated secretory phenotype. 59 Therefore, it would be viable to promote senescent cells undergoing apoptosis process to reduce their burden. 60,61,63 Our study showed increased apoptotic

ACK N OWLED G EM ENTS
This study was supported by grants from National Natural Science China Association for Science and Technology (2018QNRC001).

CO N FLI C T O F I NTE R E S T
The authors declare no competing financial interest.