Pathological mechanism of chondrocytes and the surrounding environment during osteoarthritis of temporomandibular joint

Abstract Temporomandibular joint (TMJ) osteoarthritis is a common chronic degenerative disease of the TMJ. In order to explore its aetiology and pathological mechanism, many animal models and cell models have been constructed to simulate the pathological process of TMJ osteoarthritis. The main pathological features of TMJ osteoarthritis include chondrocyte death, extracellular matrix (ECM) degradation and subchondral bone remodelling. Chondrocyte apoptosis accelerates the destruction of cartilage. However, autophagy has a protective effect on condylar chondrocytes. Degradation of ECM not only changes the properties of cartilage but also affects the phenotype of chondrocytes. The loss of subchondral bone in the early stages of TMJ osteoarthritis plays an aetiological role in the onset of osteoarthritis. In recent years, increasing evidence has suggested that chondrocyte hypertrophy and endochondral angiogenesis promote TMJ osteoarthritis. Hypertrophic chondrocytes secrete many factors that promote cartilage degeneration. These chondrocytes can further differentiate into osteoblasts and osteocytes and accelerate cartilage ossification. Intrachondral angiogenesis and neoneurogenesis are considered to be important triggers of arthralgia in TMJ osteoarthritis. Many molecular signalling pathways in endochondral osteogenesis are responsible for TMJ osteoarthritis. These latest discoveries in TMJ osteoarthritis have further enhanced the understanding of this disease and contributed to the development of molecular therapies. This paper summarizes recent cognition on the pathogenesis of TMJ osteoarthritis, focusing on the role of chondrocyte hypertrophy degeneration and cartilage angiogenesis.


| INTRODUC TI ON
Temporomandibular joint (TMJ) osteoarthritis is a progressive degenerative cartilage disease that affects cartilage and subchondral bone. 1,2 Chondrocyte death, extracellular matrix (ECM) degradation and subchondral bone remodelling are considered to be the main characteristics of TMJ osteoarthritis. 3,4 The characteristic progressive breakdown of cartilage results from the abnormal regulation of chondrocytes and the imbalance between the degradation and formation of tissue.
Chondrocytes maintain balance between synthesis and degradation of the ECM. 5 However, this homeostasis can be disrupted by the uncoupled catabolic and synthetic activities. 6 Moreover, the number of apoptotic chondrocytes increases significantly in the osteoarthritis, which is closely related to the endoplasmic reticulum and death receptor pathways. 7,8 The quality of condylar bone is closely related to the development of TMJ osteoarthritis. When TMJ osteoarthritis occurs, the density of subchondral bone is reduced. 4,9 The mechanism and role of chondrocyte hypertrophy and cartilage angiogenesis in TMJ osteoarthritis have attracted the attention of researchers. Hypertrophic chondrocytes promote ECM degradation and cartilage calcification. This denatured cartilage shows less mechanical adaption to adverse stimuli such as trauma. 10 Apart from hypertrophic chondrocytes, angiogenesis has been reported to participate in TMJ osteoarthritis. 11 Neo-angiogenesis has been shown to exacerbate chronic pain and promote cartilage ossification. 12,13 However, the exact mechanism is unclear. The possible pathogenesis of TMJ osteoarthritis is summarized in Figure 1.
In this review, we summarize the latest studies on the pathogenesis of TMJ osteoarthritis and analyse the pathological changes with an emphasis on chondrocyte hypertrophy and angiogenesis.

| IN VIVO ANIMAL MODEL S AND IN VITRO CELL MODEL S OF TMJ OS TEOARTHRITIS
Animal studies of the development of TMJ osteoarthritis used techniques including modifying the occlusal state, gene editing, injecting inflammatory mediators and surgery (Table 1).
Mechanical modification of the occlusal state is the most commonly used method to induce TMJ osteoarthritis-like changes. The TMJ receives direct occlusal loading, and disruption of occlusal harmony may traumatize the TMJ during functional loading. 14 Intraoral occlusal devices such as unilateral anterior crossbite (UAC) and biteraising plates have been used to induce TMJ osteoarthritis-like lesions. 7,15,16 In addition, an extraoral compression mechanical force device that directly exerts compressive pressure on the TMJ has also been used. 8,17 Similar to other osteoarthritis animal models, gene editing such as overexpression of short stature homeobox 2 (SHOX), transforming growth factor-β1 (TGF-β1), and β-catenin, [18][19][20][21] and inhibiting or knocking out genes such as Discoidin domain receptor 1 (DDR1), small mother against decapentaplegic 3 (SMAD3), and fibroblast growth factor receptor (FGFR3), could lead to the formation of TMJ osteoarthritis. 1,22,23 Intra-articular injection of chemical mediators such as monosodium iodoacetic acid (MIA) and complete Freund's adjuvant (CFA) could mimic the inflammatory response of the TMJ over a short period of time. [24][25][26] Surgical methods have been used to destroy part or all of the disc or cartilage of the condyle. [27][28][29] In addition, since the occurrence of osteoarthritis is closely related to age, 30 aged mice were used in some studies to mimic spontaneous TMJ osteoarthritis. 31 In vitro (Table 1), primary chondrocytes could be directly extracted from TMJ osteoarthritis patients or model animal and used as cell models. Chondrocyte inflammation can also be induced by inflammatory chemical mediators such as IL-1β, 32,33 and mechanical devices such as Flexcell. 34,35 F I G U R E 1 The main pathological changes in TMJ osteoarthritis

| The death of chondrocytes
Several studies have demonstrated that increased turnover of chondrocytes initiates the degeneration of condylar cartilage. 36 Apoptosis, autophagy, and necroptosis, play crucial roles in chondrocyte death.
Apoptosis and necroptosis accelerate the destruction of articular cartilage. 37,38 Chondrocyte apoptosis provides space for neovascularization, and the apoptotic bodies produced by this process are thought to be the source of cartilage mineralization. 10,39 Autophagy is considered a selfprotective mechanism, by which chondrocytes recycle or reuse large biomolecules. 40 In general, abnormal death of chondrocytes not only reduces the number of chondrocytes, but also initiates the degeneration of cartilage and the destruction of subchondral bone. 41 The pathways associated with chondrocyte death in TMJ osteoarthritis are illustrated in Figure 2

| Apoptosis and necroptosis in TMJ osteoarthritis
Calcium plays an essential role in chondrocyte apoptosis and calcium concentration in chondrocytes can be increased under mechanical overload. 42 Endoplasmic reticulum stress (ERS) caused by calcium influx can cause chondrocyte apoptosis, which is known as ERSmediated apoptosis. 17,35,42 A high intracellular calcium concentration can activate inducible nitric oxide synthase (iNOS). Nitric oxide (NO) induced by iNOS inhibits mitochondrial respiration and leads to chondrocyte apoptosis through the release of cytochrome C (Cyt C) and caspase-9. 43 Furthermore, tumour necrosis factor (TNF) and FGFR1 are crucial cytokines that can facilitate chondrocyte apoptosis by acting on the death receptor pathway. 44,45 TA B L E 1 Animal models and cell models used to mimic TMJ osteoarthritis in recent years

| Autophagy in TMJ osteoarthritis
Autophagy is an important mechanism of chondrocyte survival in osteoarthritis. 40 The main process of autophagy involves the formation of autophagosomes, which sequester discarded organelles or macromolecules. Autophagosomes then fuse with lysosomes to form autolysosomes, which eventually degrade the contained materials and release small molecules that can be reused. 47 The markers of autophagy, beclin 1 and light chain 3 beta (LC3B) increase in the early stage of TMJ osteoarthritis but decrease significantly in the late stage. 7 The initial enhancement in autophagy protects chondrocytes from various environmental changes. With the destruction of cartilage, inhibition of autophagy is associated with cell death. 48 In models of abnormal dental occlusion and age-associated spontaneous osteoarthritis, the loss of FGFR1 inhibits the development of osteoarthritis by promoting autophagic activity. 49 In addition, the endoplasmic reticulum-associated proteins endoplasmic reticulum to nucleus signalling 1 (ERN1), mechanistic target of rapamycin kinase complex 1 (MTORC1) and eukaryotic translation initiation factor 2 alpha kinase3 (EIF2AK3) not only induce apoptosis but also act as a regulatory valve to inhibit autophagy. This pathway is known as the ERN1-MTORC1-EIF2AK3 signalling axis. 7 Therefore, regulating autophagy may be an effective mechanism for the treatment of TMJ osteoarthritis.

| Degenerative changes in the ECM
The ECM of cartilage is mainly composed of collagen fibres and large proteoglycans. It not only acts as a protective scaffold against elastic and shear forces for cartilage, but also regulates the behaviour of chondrocytes through matrix-cell interactions. 5,50 In TMJ osteoarthritis, ECM degradation starts with expression of matrix metalloproteinases (MMPs) and a disintegrin and metallopro- In addition, mineralization of cartilage has been found to be involved in the development of TMJ osteoarthritis. 10 The signalling molecules involved in the regulation of ECM degeneration are summarized below.
The role of β-catenin, which regulates MMP13 and ADAMT5, is controversial in TMJ osteoarthritis. Compressive mechanical force reduces endogenous β-catenin and leads to ECM degradation. These pathological changes can be recovered by restoring the β-catenin signalling. 54 However, other authors found that mice with conditional F I G U R E 2 Pathways associated with chondrocyte death in TMJ osteoarthritis. Apoptosis and necroptosis are promoted in TMJ osteoarthritis, but the general trend of autophagy activity is inhibited β-catenin activation showed TMJ osteoarthritis-like phenotypes and up-regulation of MMP13 and ADAMT5 expression. 21,55 The Notch cascade consists of Notch and Notch ligands, which are involved in regulating the proliferation, differentiation, maturation and apoptosis of chondrocytes. 56

| Remodelling of subchondral bone in TMJ osteoarthritis
Abnormal subchondral bone remodelling is one of the first patho- The role of sex hormones, such as oestrogen and progesterone, in TMJ osteoarthritis is still unclear. 66,67 oestrogen is involved in the formation of condylar fibrocartilage and subsequently maintains cartilage stability by inhibiting the Wnt pathway through oestrogen receptorα. 68 The level of oestrogen is associated with the severity of TMJ osteoarthritis. In the early stage of TMJ osteoarthritis, a high level of oestrogen exerts protective effects by inhibiting osteoclast activity and reversing the abnormal absorption of subchondral bone. 66 In contrast, a low level of oestrogen may increase the severity of TMJ osteoarthritis. 69 However, the results of different studies are inconsistent. Some studies have suggested that oestrogen promotes MIA-induced subchondral bone erosion by activating oestrogen receptorβ. 70 Interestingly, a high level of progesterone exerts a protective effect by reducing the degeneration of subchondral bone by inhibiting NF-κB activity. 67

| THE PER S PEC TIVE S ON HYPERTROPHY AND ANG IOG ENE S IS IN TMJ OS TEOARTHRITIS
Endochondral osteogenesis-like changes have been reported in both human osteoarthritis and experimental models of the osteoarthritic process. 71 Chondrocyte hypertrophy and cartilage angiogenesis are important features of endochondral osteogenesis. 71,72 Interestingly, these two processes have also been shown to play an important role in TMJ osteoarthritis. Cytokines secreted by hypertrophic chondrocytes can direct and attract endothelial cells into cartilage. 39 Increasing evidence has suggested that most hypertrophic chondrocytes have the potential to transform into osteoblasts and osteocytes, which are then involved in bone formation. 73

| Angiogenesis promotes the development of TMJ osteoarthritis
Studies have revealed that angiogenesis can promote the development of osteoarthritis. 87, 88 Wang et al found that the number of newly formed blood vessels at the osteochondral junction was increased in mice with TMJ osteoarthritis-like changes. 89 These new blood vessels can transport inflammatory mediators and sustain inflammation in TMJ osteoarthritis. 90 When new blood vessels invade the cartilage, they promote both chondrocyte hypertrophy and mineral deposition in the matrix. 88 Osteophytes can then incorporate with the newly formed vessels on the surface of the joint to facilitate hard tissue formation through the process of endochondral osteogenesis. 88 Neurogenesis follows angiogenesis. An increase in the formation of blood vessels and nerves between the subchondral bone and articular cartilage may mediate the pathological process of osteoarthritis and contribute to the pain associated with osteoarthritis. 12 Vascular endothelial growth factor (VEGF) is crucial for angiogenesis and is an important mediator of TMJ osteoarthritis. Injection

F I G U R E 3 Molecular mechanism of chondrocyte hypertrophy in TMJ osteoarthritis
of VEGF led to TMJ osteoarthritis-like changes in mice by stimulating endothelial cell proliferation and migration and stabilizing newly formed blood vessels. 91 The expression of VEGF is up-regulated by several transcription factors, such as hypoxia-inducible factor-1 (HIF-1). 11,92 High levels of the dickkopf-related protein-1 (DKK-1) protein were found in the synovial fluid of TMJ osteoarthritis patients. It has been suggested that DKK-1 and high-mobility group box 1 (HMGB1) direct the nuclear localization of HIF-1 and thus promote the synthesis of VEGF. 92 The growth of nerve endings also plays a role in angiogenesis. Nerve growth factor (NGF), which regulates nerve growth, survival and repair, can stimulate angiogenesis in TMJ osteoarthritis. 99 The regulatory mechanism and role of angiogenesis in TMJ osteoarthritis are shown in Figure 4.

| CON CLUS ION
The TMJ in animals has been successfully constructed in a variety of ways to stimulate the structural and organizational changes in osteoarthritis. Chondrocyte death, ECM degradation and subchondral bone remodelling play essential roles in TMJ osteoarthritis.
Moreover, the endochondral osteogenesis-like changes such as chondrocyte hypertrophy and endochondral angiogenesis suggest that the pathological changes of TMJ osteoarthritis are closely related to the process of osteogenesis. The molecular pathways that regulate TMJ osteoarthritis help us to better understand the pathological mechanisms of TMJ osteoarthritis. More importantly, these signalling molecules may serve as potential therapeutic targets to help find effective disease-modifying strategies for TMJ osteoarthritis.

ACK N OWLED G EM ENTS
The work was supported by the National Natural Science Foundation of China (grants 81470712 and 81670960), the Natural Science

DATA AVA I L A B I L I T Y S TAT E M E N T
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