Local injection of abaloparatide promotes mandibular condyle lengthening in adolescent rats via enhancing chondrogenesis and ossification.

BACKGROUND: Mandibular condylar hypoplasia negatively affects patient's facial appearance and dentofacial function. OBJECTIVE: To investigate the effect of local injection of the drug abaloparatide (ABL), an analogue of parathyroid hormone related protein (PTHrP), on promoting lengthening of the mandibular condyle. METHODS: Thirty adolescent male Sprague-Dawley rats were randomly divided into two groups, which received the injection of ABL or normal saline (the control) every 3 days in the temporomandibular joint (TMJ) cavity. Cone-beam computed tomography and immunohistochemistry assays were performed at 2, 4 and 6 weeks since the injection. Mandibular condylar chondrocytes (MCC) and pre-osteoblasts were treated with ABL or PBS, followed by the CCK-8 detection, IC50, real-time PCR assay, Western Blot and immunofluorescence staining. RESULTS: In vivo, compared with the control, the ABL group significantly increased the mandibular condylar process length (by 1.34 ± 0.59 mm at 6 weeks), the thickness of the cartilage layer, and enhanced the matrix synthesis. The ABL group had significant up-regulation of SOX 9, COL II, PTHrP and PTH1R, down-regulation of COL X in the cartilage, up-regulation of RUNX 2, and unchanged osteoclastogenesis in the subchondral bone. In vitro, the intra-TMJ injection of ABL promoted the MCC proliferation, with up-regulated expression of chondrogenic genes, and enhanced osteogenic differentiation of the pre-osteoblasts. CONCLUSIONS: Intra-TMJ injection of abaloparatide promotes mandibular condyle lengthening in the adolescent rats via enhancing chondrogenesis in the mandibular condylar cartilage and ossification in the subchondral bone.

Mechanical stress can regulate temporomandibular joint cavitation via signalling pathways.

The temporomandibular joint (TMJ), composed of temporal fossa, mandibular condyle and a fibrocartilage disc with upper and lower cavities, is the biggest synovial joint and biomechanical hinge of the craniomaxillofacial musculoskeletal system. The initial events that give rise to TMJ cavities across diverse species are not fully understood. Most studies focus on the pivotal role of molecules such as Indian hedgehog (Ihh) and hyaluronic acid (HA) in TMJ cavitation. Although biologists have observed that mechanical stress plays an irreplaceable role in the development of biological tissues and organs, few studies have been concerned with how mechanical stress regulates TMJ cavitation. Based on the evidence from human or other animal embryos today, it is implicated that mechanical stress plays an essential role in TMJ cavitation. In this review, we discuss the relationship between mechanical stress and TMJ cavitation from evo-devo perspectives and review the clinical features and potential pathogenesis of TMJ dysplasia.

Clock gene Per1 regulates rat temporomandibular osteoarthritis through NF-κB pathway: an in vitro and in vivo study.

PURPOSE: Temporomandibular joint osteoarthritis (TMJOA) is a common disease that negatively affects the life quality of human beings. Circadian rhythm acts an important role in life activities. However, whether the clock genes are rhythmic expressed in mandibular condylar chondrocytes, or the clock genes have an effect on the progression of TMJOA remains unknown. In this study, we aim to explore expression of clock genes and regulatory mechanism of TMJOA in rat mandibular condylar chondrocytes. METHODS: After synchronized by dexamethasone, the expression of core clock genes Per1, Per2, Clock, Cry1, Cry2 and Bmal1 and cartilage matrix degrading factor gene Mmp13 were analyzed in mandibular condylar chondrocytes every 4 h with RT-qPCR. The mandibular condylar chondrocytes were stimulated with IL-1ß, and expression of Per1, Mmp13, P65 and p-P65 was assessed by RT-qPCR and Western blot. Sh-Per1 lentivirus was used to assess the effect of clock gene Per1 in IL-1ß-induced chondrocytes, and expression of Mmp13, P65 and p-P65 was measured. After establishing a rat TMJOA model using unilateral anterior crossbite (UAC), micro-CT, H & E, Alcian Blue & Nuclear Fast Red and Safranin O & Fast Green, cartilage thickness was utilized to assess the damage of cartilage and subchondral bone. Immunohistochemistry of PER1, MMP13 and P65 was performed in condylar sections. RESULTS: All core clock genes and Mmp13 were rhythmically expressed. And Mmp13 expression curve was closed in phase and amplitude with Per1. After stimulation with IL-1ß, the expression of MMP13, PER1 and P65 and ratio of p-P65/P65 increased in condylar chondrocytes. After Per1 was down-regulated in condylar chondrocytes, the expression of MMP13 and P65 and ratio of p-P65/P65 decreased. Compared with the condyles of Sham group, the bony parameters of UAC group were significantly worse. The thickness of cartilage in UAC group significantly reduced. The modified Mankin scores and the expression of PER1, MMP13 and P65 in cartilage of UAC group significantly increased compared with Sham group. CONCLUSION: Core clock genes and Mmp13 are rhythmic expressed in rat mandibular condylar chondrocytes. PER1 can regulate the expression of MMP13 through NF-κB pathway in IL-1ß-induced mandibular condylar chondrocytes.

Mechanical loading and autophagy: A study on the BoNT-A injection-induced condylar cartilage degeneration.

Botulinum toxin A (BoNT-A) has emerged as a treatment option for temporomandibular disorder (TMD). By injecting BoNT-A into the masseter muscle, it is possible to reduce mechanical loading on the temporomandibular joint (TMJ). However, numerous prior studies have indicated excessive reduction in mechanical loading can have detrimental effects on TMJ cartilage. This study proposes that autophagy, a process influenced by mechanical loading, could play a role in BoNT-A-induced mandibular condyle cartilage degeneration. To explore this hypothesis, we employed both BoNT-A injection and an excessive biting model to induce variations in mechanical loading on the condyle cartilage of C57BL/6 mice, thereby simulating an increase and decrease in mechanical loading, respectively. Results showed a significant reduction in cartilage thickness and downregulation of Runt-related transcription factor 2 (Runx2) expression in chondrocytes following BoNT-A injection. In vitro experiments demonstrated that the reduction of Runx2 expression in chondrocytes is associated with autophagy, possibly dependent on decreased YAP expression induced by low mechanical loading. This study reveals the potential involvement of the YAP/LC3/Runx2 signaling pathway in BoNT-A mediated mandibular condylar cartilage degeneration.

Longitudinal effects of estrogen on mandibular growth and changes in cartilage during the growth period in rats.

Estrogen is a steroid hormone that induces skeletal growth and affects endochondral ossification of the long tubular bone growth plate during the growth period. However, the effects of estrogen on endochondral ossification of the mandibular condylar cartilage are unclear. In this study, ovariectomized Wistar/ST rats were used to investigate the longitudinal effects of estrogen on mandibular growth. The rats were administered different doses of estrogen. Longitudinal micro-computed tomographic scanning, histological staining and ELISA on plasma growth hormone were performed to examine the effects of estrogen on mandibular growth. The results showed that mandibular growth was suppressed throughout the growth period by estrogen in a dose-dependent manner. In addition, long-term administration of a high dose of estrogen to the rats resulted in significant increase in growth hormone throughout the growth period, significant circularization of cell nuclei in the proliferative layer, intensely staining cartilage matrix in the subchondral bone, and significant suppression of estrogen receptor (ER) alpha and beta expression in the mandibular cartilage. However, regardless of estrogen concentration, in the posterior part of the mandibular cartilage, ER expression extended to both the hypertrophic and proliferative layers. These results indicate that estrogen suppresses mandibular growth throughout the growth period. Additionally, it influences endochondral ossification via its effect on ERs.

[Effect of chronic sleep deprivation on condylar cartilage in rats].

PURPOSE: The aim of this study was to investigate the morphological changes of condylar cartilage of temporomandibular joint (TMJ) and the expression changes of IL-1ß,TNF-α,IGF-1 and VEGF in condylar cartilage of TMJ by establishing a chronic sleep deprivation model in rats. METHODS: Sixty rats were randomly divided into experimental group, control group and recovery group. Modified multiple platforms method (MMPM) was used to build chronic sleep deprivation models in experimental and recovery groups. Rats in the recovery group received 1 week of cage feeding after sleep deprivation. H-E staining was used to observe morphological change of the condyle. Immunohistochemical method was performed to detect the changes of IL-1ß, TNF-α, IGF-1 and VEGF. The data was processed by using SPSS 23.0 software package. RESULTS: MMPM can establish chronic sleep deprivation model effectively. H-E staining showed condylar cartilage of the experimental group was split stripped, and the boundaries of cartilage cell layer became blurred. Compared with the control group, the recovery group had less cracks in the fibrous layer or some of the cracks were occupied by fibrous tissue. Immunohistochemistry showed that the positive expression intensity of IL-1ß and TNF-α in the experimental group was significantly higher than in the control group (P＜0.05), the positive expression intensity in the recovery group was significantly lower than in the experimental group(P＜0.05). The positive expression intensity of IGF-1 and VEGF in the experimental group was significantly higher than in the control group(P＜0.05). The expression of IGF-1 and VEGF decreased significantly in the recovery group which received sleep deprivation no more than 3 weeks(P＜0.05). CONCLUSIONS: Chronic sleep deprivation can increase the expression of IL-1ß, TNF-α and VEGF in condylar cartilage and aggravate osteoarthritis. Chronic sleep deprivation can lead to increase of IGF-1 in condylar cartilage tissue, which plays a crucial role in protecting and promoting the reconstruction of condylar cartilage. After chronic sleep deprivation, the expressions of IL-1ß, TNF-α, IGF-1 and VEGF in the condylar cartilage of rats were decreased after 1 week of recovery, and the condylar cartilage underwent restorative reconstruction.

Modificación de la actividad metabólica por medio de un dispositivo intraoral en pacientes con hiperplasia condilar confirmada por SPECT / Metabolic activity modification by means of an intraoral device in patients with condylar hyperplasia confirmed by SPECT

Objetivo: Evaluar los efectos de la aplicación de un dispositivo intraoral de uso permanente en el comportamien- to de los cóndilos con hiperplasia condilar (HC) confirmada por tomografía computarizada de emisión por fotón único (SPECT), estableciendo una comparación con un grupo de pacientes con HC que no utilizó el dispositivo. Materiales y métodos: 30 pacientes con una edad promedio de 21,7 años (+/-5,56) con HC confirmada con SPECT fueron asignados al azar a dos grupos: a los del grupo I (n=18) se les colocó un dispositivo intraoral de uso perma- nente para modificar la posición de la mandíbula, mientras que a los del grupo II (n=12) no se les colocó ningún dispo- sitivo. Se realizaron evaluaciones de dolor, del desvío de la línea media, de la apertura máxima y del disconfort al inicio del estudio y a los 2, 4, 6, 10, 12 y 14 meses. A los 19 meses promedio, la actividad osteoblástica (AO) fue reevaluada me- diante SPECT. Resultados: En el grupo I, la AO en los cortes coro- nales y transversales cesó o disminuyó (p<0,001) respecto a la condición inicial, mientras que en el grupo II la AO au- mentó (p<0,001). Los datos fueron analizados utilizando el test de Wilcoxon de rangos signados. Al ajustar un modelo de ANCOVA robusto utilizando el valor inicial como covariable también se observa que el efecto del grupo fue estadística- mente significativo en ambos cortes (p<0,001). Conclusiones: La aplicación de un dispositivo intrao- ral de uso permanente mejora la evolución de la hiperplasia condilar, lo que lo puede convertir en un tratamiento de uti- lidad para el tiempo que se aguarda para realizar una condi- lectomía alta de cuello de cóndilo, o incluso para evitar este procedimiento (AU)

Objective: To evaluate the effects of the application of an intraoral device for permanent use on the behavior of con- dyles with condylar hyperplasia (CH) confirmed by single photon emission computed tomography (SPECT), establish- ing a comparison with a group of patients with CH that did not use the device. Materials and methods: Thirty patients with an aver- age age of 21.7 years (+/-5.56) with CH confirmed by SPECT were randomly divided into two groups: the ones in group I (n=18) received an intraoral device for permanent use to align the mandible, while those in group II (n=12) did not get any device. Pain, midline shift, maximum opening, and discomfort were evaluated at the beginning of the study and at 2, 4, 6, 10, 12, and 14 months. At an average of 19 months, osteoblastic activity (AO) was reassessed by SPECT. Results: In group I, the AO in the coronal and trans- verse sections ceased or decreased (p<0.001) in comparison to the initial condition, while in group II the AO increased (p<0.001). The data was analyzed by the Wilcoxon signed rank test. Adjusting a robust ANCOVA model using the ini-tial value as a covariate made it possible to observe that the effect of the group was statistically significant in both cuts (p<0.001). Conclusions: The application of an intraoral device for permanent use improves the evolution of condylar hyperpla- sia, which can make it a useful treatment until a high condylectomy of the neck of the condyle is performed, or even to avoid this procedure (AU)

Changes of the condylar cartilage and subchondral bone in the temporomandibular joints of rats under unilateral mastication and expression of Insulin-like Growth Factor-1.

OBJECTIVES: This study aimed to define changes in the rat condylar cartilage and subchondral bone using the unilateral mastication model. MATERIALS AND METHODS: In this study, forty 4-week-old Wistar rats were randomly divided into experimental (n = 20) and control group (n = 20). In the experimental group, unilateral dental splints were placed on the occlusal surface of left maxillary molars. The rats were sacrificed at 1, 2, 3, and 4 weeks after placement of the splint. Micro-CT scanning and histological staining were performed to observe the changes in the mandibular condylar cartilage and subchondral bone. Levels of insulin-like growth factor-1 (IGF-1) were determined via immunohistochemistry to analyse the occurrence of osteogenic changes. RESULTS: Micro-CT scanning findings demonstrated the occurrence of asymmetric growth of condyle in the experimental group. The condylar cartilage and subchondral bone exhibited degradation on the chewing side of the experimental group and showed decreased bone mineral density, thinner cartilage thickness, and increased degree of degeneration and osteoclast activity. Compared with the control group, the expression of IGF-1 was remarkably higher on the non-chewing side. CONCLUSION: Long-term unilateral mastication can lead to the occurrence of degenerative changes in the condylar cartilage and subchondral bone during growth and development. IGF-1 may play a role in promoting the process of osteogenesis.

ADAMTS5 is required for normal trabeculated bone development in the mandibular condyle.

OBJECTIVE: Determine the role of the extracellular matrix protease ADAMTS5 in development of the trabeculated bone of the mandibular condyle. METHODS: The mandibular condyles of wild type and mice deficient in the protease ADAMTS5 were examined for histopathology with Safranin O staining. Microcomputed tomography was performed to analyze the developing bone of the mandibular condyle. RNAscope and immunohistochemistry were utilized to investigate cell type and extracellular matrix expression. RESULTS: Mice deficient in Adamts5, (Adamts5tm1Dgen/J) exhibit an increase in trabecular separation (n = 37 wild type; n = 27: P < 0.0001) and reduction of trabecular thickness P = 0.0116 and bone volume fraction P = 0.0869 in the mandibular condylar head compared to wild type littermates. The altered bone parameters were more pronounced in male Adamts5-/- mice compared to female Adamts5-/- mice (TbSp; P = 0.03). Adamts5 was co-expressed with versican and Gli1 in mesenchymal, stem-like cells in the transition zone where the trabeculated bone is adjacent to mature hypertrophic chondrocytes. Loss of Adamts5 caused a reduction of Bglap expressing osteoblasts throughout mandibular condylar development and in young adult mice. The protease Mmp13, that is involved in mineralization and is expressed by hypertrophic chondrocytes and osteoblasts, was reduced in the mandibular condyle of Adamts5 deficient mice. CONCLUSION: This is the first report of a novel and critical role for Adamts5 in bone formation within the mandibular condyle of the temporomandibular joint. These data indicate Adamts5 may be required in the transdifferentiation of hypertrophic chondrocytes to osteoblasts during trabecular bone formation in development of the mandibular condyle.

Assessment of bone marrow fat fractions in the mandibular condyle head using the iterative decomposition of water and fat with echo asymmetry and least-squares estimation (IDEAL-IQ) method.

The prevalence of temporomandibular joint disorder (TMD) is gradually increasing, and magnetic resonance imaging (MRI) is becoming increasingly common as a modality used to diagnose TMD. Edema and osteonecrosis in the bone marrow of the mandibular condyle have been considered to be precursors of osteoarthritis, but these changes are not evaluated accurately and quantitatively on routine MRI. The iterative decomposition of water and fat with echo asymmetry and least-squares estimation (IDEAL-IQ) method, as a cutting-edge MRI technique, can separate fat and water using three asymmetric echo times and the three-point Dixon method. The purpose of this study was to analyze the quantitative fat fraction (FF) in the mandibular condyle head using the IDEAL-IQ method. Seventy-nine people who underwent MRI using IDEAL-IQ were investigated and divided into 1) the control group, without TMD symptoms, and 2) the TMD group, with unilateral temporomandibular joint (TMJ) pain. In both groups, the FF of the condyle head in the TMJ was analyzed by two oral and maxillofacial radiologists. In the TMD group, 29 people underwent cone-beam computed tomography (CBCT) and the presence or absence of bony changes in the condylar head was evaluated. The FF measurements of the condyle head using IDEAL-IQ showed excellent inter-observer and intra-observer agreement. The average FF of the TMD group was significantly lower than that of the control group (p < 0.05). In the TMD group, the average FF values of joints with pain and joints with bony changes were significantly lower than those of joints without pain or bony changes, respectively (p < 0.05). The FF using IDEAL-IQ in the TMJ can be helpful for the quantitative diagnosis of TMD.

Overloaded Orthopedic Force Induces Condylar Subchondral Bone Absorption by Stimulating Rat Mesenchymal Stem Cells Differentiating into Osteoclasts via mTOR-Regulated RANKL/OPG Secretion in Osteoblasts.

Appropriate orthopedic force led to bone remodeling of mandibular condyle, while overloaded orthopedic force (OOF) induced condylar bone absorption. Bone absorption is ascribed to the imbalanced activities between osteoclasts (OCs) and osteoblasts (OBs), mechanism of which remains unclear. This study aimed to observe the condylar changes induced by OOF by mandible advancement appliance and to further investigate the role of mammalian target of Rapamycin (mTOR) and RANKL/OPG in osteoclastic differentiation of stem cells in vivo and in vitro. In vivo, the results of micro-CT analysis indicated that condylar bone resorption was induced by OOF through mandibular advancement appliance for 2 weeks and worsened time dependently. Morphologically, cartilage thickness was reduced, subchondral cortical bone line appeared not continuous, and subchondral bone exhibited irregular-shaped and owned uneven surface. The bone mineral density (BMD), bone volume/tissue volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) were decreased accomplished with the increased trabecular separation (Tb.Sp) determined by micro-CT. In addition, based on immunofluorescent labeling, OOF activated both OCs and OBs, but osteoclastogenesis prevailed over osteogenesis. The mTOR activation and ratio of RANKL/OPG in OBs were elevated by OOF. In vitro, the results of western blot and polymerase chain reaction (PCR) consistently suggested that the mTOR and RANKL/OPG ratio were upregulated by overloaded mechanical stretch. Pretreatment with mTOR inhibitor, rapamycin, could attenuate the activation of mTOR and the secretion of RANKL in OBs. Interestingly, based on the Trap staining, the supernatant of OBs exposed to OOF could promote osteoclastic differentiation of mesenchymal stem cells (MSCs), while its role was weakened by inhibition of mTOR in OBs. Collectively, OOF induced condylar bone absorption; in the process, osteoclastogenesis was prominent than osteogenesis. The activation of mTOR and secretion of RANKL/OPG were enhanced by OOF and were involved in promoting MSCs differentiating into OCs.

Mandibular lateral deviation induces alteration in vascular endothelial growth factor expression and oxidative stress/nitric oxide generation in rat condyle, synovial membrane and masseter muscle.

OBJECTIVE: We aimed to investigate alteration in cellular signaling mediated by vascular endothelial growth factor (VEGF) and parameters of oxidative stress/nitric oxide generation, superoxide dismutase (SOD) and neuronal nitric oxide synthase (nNOS), underlying altered functional mechanical loading of TMJ (temporomandibular joint) during lateral mandibular deviation. DESIGN: Thirty-eight 5-week-old male Wistar rats were divided into experimental group, which received acrylic resin appliance that shifted mandible to the left during closure, and control group. Computed tomography and histomorphometry were used for condyle analyses, while samples of condyle, synovial membrane and m. masseter were analyzed with enzyme-linked immunosorbent assay and spectrophotometry to determine VEGF and nNOS protein concentrations, and SOD activity. RESULTS: Experimental group of rats developed smaller and asymmetrical mandibles. Less of new bone and cartilage formation and larger bone marrow cavities area were found in the experimental group. Higher VEGF expression in condyle and m. masseter as well as higher nNOS expression in m. masseter and synovial membrane were found in the experimental compared to the control group. Alteration of SOD activity was found in m. masseter and synovial membrane in the experimental group. CONCLUSIONS: Lateral mandibular deviation induces mandibular and condylar morphological changes as well as significant cellular signaling alterations in condyle, synovial membrane and masticatory muscle. Cellular VEGF protein overexpression and oxidative stress/nitric oxide disbalance could be the mechanisms underlying unbalanced functional TMJ loading due to mandibular deviation.

Histopathological features of condylar hyperplasia and condylar Osteochondroma: a comparison study.

BACKGROUND: Both mandibular condylar hyperplasia and condylar osteochondroma can lead to maxillofacial skeletal asymmetry and malocclusion, although they exhibit different biological behavior. This study attempted to compare the histological features of mandibular condylar hyperplasia and condylar osteochondroma using hematoxylin-and-eosin (H&E) staining, and immunohistochemistry staining of PCNA and EXT1 with quantitative analysis method. RESULTS: The H&E staining showed that condylar hyperplasia and condylar osteochondroma could be divided into four histological types and exhibited features of different endochondral ossification stages. There was evidence of a thicker cartilage cap in condylar osteochondroma as compared condylar hyperplasia (P = 0.018). The percentage of bone formation in condylar osteochondroma was larger than was found in condylar hyperplasia (P = 0.04). Immunohistochemical staining showed that PCNA was mainly located in the undifferentiated mesenchymal layer and the hypertrophic cartilage layer, and there were more PCNA positive cells in the condylar osteochondroma (P = 0.007). EXT1 was mainly expressed in the cartilage layer, and there was also a higher positive rate of EXT1 in condylar osteochondroma (P = 0.0366). The thicker cartilage cap, higher bone formation rate and higher PCNA positive rate indicated a higher rate of proliferative activity in condylar osteochondroma. The more significant positive rate of EXT1 in condylar osteochondroma implied differential biological characteristic as compared to condylar hyperplasia. CONCLUSIONS: These features might be useful in histopathologically distinguishing condylar hyperplasia and osteochondroma.

Effects of Twin Inclined Plane Device on Adaptation and Ultrastructure Variations in Condyle of Growing Rats.

OBJECTIVE: This study investigates the effects of using a twin inclined plane device (TIPD) on the remolding and ultrastructure variation of mandibular condyle in growing rats. MATERIALS AND METHODS: Forty-eight male Wistar rats (six weeks old, body weight of approximately 190-210 g) were divided into experimental group (wearing appliance, n = 32) and control group (no appliance, n = 16). Samples were collected on days 3, 14, 30, and 60. The immunohistochemical analysis for vascular endothelial growth factor (VEGF) and type II collagen was carried out. Tartrate-resistant acid phosphatase (TRAP) reaction was performed to evaluate the osteoclastic activity. Three-dimensional morphometric images were reconstructed for morphometric analysis by microcomputed tomography (micro-CT). The ultrastructure of the condylar surface was observed by scanning electron microscopy (SEM). RESULTS: The expression of VEGF significantly increased, while the expression of type II collagen decreased in the experimental group at days 30 and 60. Furthermore, the enhanced osteoclast activity was observed under the subchondral bone, which was highest at day 30, and decreased to the lowest at day 60 in the experimental group. In addition, adaptive subchondral bone remolding in the posterior part of the condyle was observed at day 60 in the experimental group, and the SEM revealed the ultrastructure variations after installation of the TIPD. However, these changes began to reverse after 30 days. CONCLUSION: Condylar tissue changes point to the osteoclastic activity in the posterior region of the condyle. These adaptive changes point to bone resorption in the posterior condyle. Type II collagen and VEGF contribute to the MCC remolding induced by the TIPD. The ultrastructural changes in the posterior condylar area in response to mechanical stresses are recoverable at the initial stage.

OPG is Required for the Postnatal Maintenance of Condylar Cartilage.

Osteoprotegerin (OPG) is one of the protective factors of bony tissue. However, the function of OPG in cartilage tissues remains elusive. The aim of this study is to explore the function of OPG in the postnatal maintenance and the occurring of osteoarthritis (OA) of temporomandibular joint (TMJ) in the rodent models. We found that OPG expressed in the hypertrophic layer of the condylar cartilage and upregulated in the hyperocclusion-induced-TMJ-trauma rat. In the absence of OPG, the cartilage degradation occurred prior to that in WT mice, and the 3-month-old OPG-Knockout (OPG-KO) condyle showed decreased chondrocyte proliferation and increased chondrocyte apoptosis, whereas the number of chondroclasts was comparable to WT condyle. The isolated chondrocytes from the OPG-KO mice also showed impaired survival and promoted chondrogenic differentiation. Furthermore, the hyperocclusion model deteriorated TMJ degradation in the OPG-KO mice. OPG plays a protective role in the condylar chondrocytes' survival, and it is required for the postnatal maintenance of TMJ.

Deletion of Runx2 in condylar chondrocytes disrupts TMJ tissue homeostasis.

Runt-related transcription factor-2 (Runx2) is essential for chondrocyte maturation during cartilage development and embryonic mandibular condylar development. The process that chondrocytes, especially a subgroup of hypertrophic chondrocytes (HC), could transform into bone cells in mandibular condyle growth makes chondrocytes crucially important for normal endochondral bone formation. To determine whether Runx2 regulates postnatal condylar cartilage growth and tissue homeostasis, we deleted Runx2 in chondrocytes in postnatal mice and assessed the consequences on temporomandibular joint (TMJ) cartilage growth and remodeling. The cell lineage tracing data provide information demonstrating the role of chondrocytes in subchondral bone remodeling. The histologic and immunohistochemical data showed that Runx2 deficiency caused condylar tissue disorganization, including loss of HC and reduced hypertrophic zone, reduced proliferative chondrocytes, and decreased cartilage matrix production. Expression of Col10a1, Mmp13, Col2a1, Aggrecan, and Ihh was significantly reduced in Runx2 knockout mice. The findings of this study demonstrate that Runx2 is required for chondrocyte proliferation and hypertrophy in TMJ cartilage and postnatal TMJ cartilage growth and homeostasis, and that Runx2 may play an important role in regulation of chondrocyte-derived subchondral bone remodeling.

Insulin-like growth factor-1 engaged in the mandibular condylar cartilage degeneration induced by experimental unilateral anterior crossbite.

OBJECTIVE: To investigate the changes in insulin-like growth factor-1 (IGF-1) expression levels in the degenerative mandibular condylar cartilage. DESIGN: Thirty-six rats were divided into the unilateral anterior crossbite and control groups. The expression levels of IGF-1; IGF-1 receptor (IGF-1R); IGF-binding protein-3 and -5 (IGFBP-3 and -5); proliferating cell nuclear antigen (PCNA); aggrecan; type-I, -II, -VI, and -X collagen; tissue inhibitor of metalloproteinases-1 and -3 (TIMP-1 and -3); metalloproteinases of matrix metalloproteinases-3 and-13 (MMP-3 and -13); a disintegrin and metalloproteinase thrombospondin-4 and -5 (ADAMTS-4 and -5); alkaline phosphatase (ALP); ß-glucuronidase; and N-acetyl-ß-glucosaminidase in the mandibular condylar cartilage were assessed. RESULTS: The protein expression levels of IGF-1and IGF-1R were increased from week 4 in the unilateral anterior crossbite group. The mRNA expression level of IGFBP-3 and -5 was upregulated from week 4 and week 2, respectively; that of IGFBP-3 was downregulated at week 8; and that of PCNA, type-II collagen, type-X collagen, aggrecan, TIMP-1, and TIMP-3 was downregulated, whereas that of MMP-3, MMP-13, ADAMTS-4, ADAMTS-5, ß-glucuronidase, and N-acetyl-ß-glucosaminidase were upregulated from week 2. The positive area size of type-I collagen was increased and that of type VI collagen was decreased from week 2. The positive area size of type X collagen was increased at week 2 but decreased at week 8. The percentage of ALP-positive cells was increased from week 4. CONCLUSIONS: Unilateral anterior crossbite stimulated the multifarious expression of IGF-1 and IGFBP, which may be linked to chondrocyte proliferation and differentiation in the mandibular condylar cartilage that showed progressive degeneration.

MiR-15b is a key regulator of proliferation and apoptosis of chondrocytes from patients with condylar hyperplasia by targeting IGF1, IGF1R and BCL2.

OBJECTIVE: This study aimed to explore potential microRNAs (miRNAs), which participate in the pathological process of condylar hyperplasia (CH) through targeting specific proliferation- and apoptosis- related genes of chondrocytes. METHODS: Insulin-like growth factor 1 (IGF1), IGF1 receptor (IGF1R) and B-cell CLL/lymphoma 2 (BCL2) in CH cartilage were detected by real-time polymerase chain reaction (PCR), Western blot, immunohistochemistry and immunofluorescence. MiRanda and TargetScanS algorithms were used to predict certain miRNAs in CH chondrocytes concurrently modulating the above three genes. MiR-15b was screened and identified using real-time PCR. After transfection of miR-15b mimics or inhibitor into CH chondrocytes, expression of the above three genes was detected by real-time PCR and western blot, meanwhile, cell proliferation and apoptosis was examined by CCK8, cell cycle assays, flow cytometry and Hoechst staining. Dual luciferase activity was performed to identify the direct regulation of miR-15b on IGF1, IGF1R and BCL2. RESULTS: Expression of IGF1, IGF1R and BCL2 increased in CH cartilage. Seven microRNAs concurrently correlated with IGF1, IGF1R and BCL2. Among them, only miR-15b significantly changed in CH chondrocytes. Overexpression of miR-15b in CH chondrocytes suppressed the expression of IGF1, IGF1R and BCL2, while it increased when miR-15b was knockdown. Furthermore, miR-15b suppressed their expression by directly binding to its 3'-UTR in these cells. Besides, miR-15b hampered chondrocytes proliferation through targeting IGF1 and IGF1R and accelerated chondrocytes apoptosis through targeting BCL2. CONCLUSION: Suppressed miR-15b contributed to enhanced proliferation capacity and weakened apoptosis of chondrocytes through augmentation of IGF1, IGF1R and BCL2, thereby resulting in development of CH.

Biomechanically stimulated chondrocytes promote osteoclastic bone resorption in the mandibular condyle.

OBJECTIVE: Chondrocyte signaling is important in osteoclastic bone resorption in mice tibiae. The present study aimed to test whether biomechanically stimulated chondrocytes promote osteoclastic bone resorption in the mandibular condyle. METHODS: Primary chondrocytes isolated from rat condylar cartilage were stimulated by fluid flow shear stress (FSS) for 30, 60, 120 min at intensities of 10, 20, or 30 dynes/cm2. The levels of pro-osteoclastic factors and pro-osteoclastic function of FSS-stimulated chondrocytes were tested. Abnormal molar occlusion was established in rats, and the relationship between cartilage degeneration and osteoclastogenesis in the subchondral bone of the mandibular condyle, and the expression of pro-osteoclastic factors in condylar cartilage, were evaluated. RESULTS: The mRNA and protein levels of SDF-1 and TGFß-1 increased significantly in all FSS-treated groups; the levels of RANKL and RANKL:OPG increased in all intensities and in 60 and 120 min of FSS; and those of Wnt5 A increased in all time-points and in 20 and 30 dynes/cm2 of FSS-treated groups (all compared with their levels the controls; P < 0.05). The percent area of degenerative cartilage changes correlated positively with osteoclast number and osteoclast surface/bone surface in the mandibular condyles of abnormal occlusion rats (P < 0.05). Abnormal occlusion increased the immune-positive area and the mRNA expression levels of Sdf1, Tgfb1, Rankl, Wnt5a and the RANKL:OPG ratio in rat condylar cartilage compared with those in the controls (all P < 0.05). CONCLUSION: Chondrocytes under mechanical stimulation could express higher levels of pro-osteoclastic factors and induced condylar subchondral bone resorption by promoting osteoclastogenesis.

Estrogen Promotes Mandibular Condylar Fibrocartilage Chondrogenesis and Inhibits Degeneration via Estrogen Receptor Alpha in Female Mice.

Temporomandibular joint degenerative disease (TMJ-DD) is a chronic form of TMJ disorder that specifically afflicts people over the age of 40 and targets women at a higher rate than men. Prevalence of TMJ-DD in this population suggests that estrogen loss plays a role in the disease pathogenesis. Thus, the goal of the present study was to determine the role of estrogen on chondrogenesis and homeostasis via estrogen receptor alpha (ERα) during growth and maturity of the joint. Young and mature WT and ERαKO female mice were subjected to ovariectomy procedures and then given placebo or estradiol treatment. The effect of estrogen via ERα on fibrocartilage morphology, matrix production, and protease activity was assessed. In the young mice, estrogen via ERα promoted mandibular condylar fibrocartilage chondrogenesis partly by inhibiting the canonical Wnt signaling pathway through upregulation of sclerostin (Sost). In the mature mice, protease activity was partly inhibited with estrogen treatment via the upregulation and activity of protease inhibitor 15 (Pi15) and alpha-2-macroglobulin (A2m). The results from this work provide a mechanistic understanding of estradiol on TMJ growth and homeostasis and can be utilized for development of therapeutic targets to promote regeneration and inhibit degeneration of the mandibular condylar fibrocartilage.