Mandibular Condylar Cartilage Research Articles

Exploring the genetic expression of Sdf1, Foxc1 and histologic changes following mandibular advancement and recovery phase in Wistar rats.

PurposeThis study evaluated the impact of mandibular advancement on Sdf1 and Foxc1 gene expression in the mandibular condylar cartilage of young Wistar rats. By examining the changes that occur during a unique one-month recovery period, it highlights the critical role of gene expression and condylar adaptation during the recovery phase. The analysis focused on whether, during the recovery period, reversal changes occur when functional appliances are removed and whether genetic expression important for condyle growth and adaptation downregulates. Material and MethodsThe study involved 30 male Wistar rats divided into 2 control groups Appliance Control and Recovery Control groups, and 2 experimental groups, the Appliance group with mandibular advancement bite-jumping appliance for 30 days, and the Recovery group with appliance for 30 days followed by a 30-day recovery. Molecular analysis of condylar cartilage using real-time RT-PCR and histological assessments was conducted. ResultsSignificant genetic expression alterations were noted in both the experimental groups for Sdf1 (p < 0.05) and Foxc1 (p < 0.05). According to histological investigations, significant alterations with an increase in the proliferative and hypertrophic layer in condylar cartilage were seen. ConclusionMandibular advancement bite-jumping appliances induce proliferative and hypertrophic layer changes in mandibular condylar cartilage, shown by elevated Foxc1 levels and decreased Sdf1 levels. Post-appliance removal, persistent gene expression reveals a true joint stimulation.

Phosphotungstic acid-enhanced micro-computed tomography and RNA sequencing provide a new perspective on temporomandibular joint arthritis induced by complete Freund's adjuvant and collagen-induced arthritis in rat models

Background/PurposeTemporomandibular joint (TMJ) arthritis causes inflammation and degradation of the mandibular condylar cartilage and subchondral bone. Complete Freund's adjuvant (CFA) and collagen-induced arthritis (CIA) are models for studying TMJ arthritis. While micro-computed tomography (micro-CT) is crucial for three-dimensional (3D) bone analysis, it has limitations in imaging nonmineralized tissues. Phosphotungstic acid (PTA) enhances soft tissue contrast. However, research on the 3D imaging of mandibular condylar cartilage and the molecular mechanisms of CFA- and CIA-induced arthritis remains unclear. This study aimed to investigate the bone and PTA-stained cartilage in the mandibular condyle using 3D reconstruction and explore the characteristics of enriched gene ontology terms underlying CFA- and CIA-induced TMJ arthritis in rat models. Materials and methodsRat mandibular condyles were collected from control, CFA, and CIA groups. Live micro-CT created 3D bone structures, and PTA-enhanced micro-CT constructed 3D mandibular condylar cartilage. Gene ontology enrichment analysis identified enriched gene ontology terms from differentially expressed genes through RNA sequencing. ResultsMajor deformities in cartilage volume and bone morphology were observed in the arthritis-induced groups. The CIA group exhibited significant correlations between cartilage volume and bone parameters changes. Gene ontology enrichment analysis indicated fewer terms with upregulated differentially expressed genes related to inflammation and immune response in the CIA group than in the CFA group. ConclusionThis study reveals distinct responses between CFA- and CIA-induced TMJ arthritis models. The CIA group exhibited strong correlations between cartilage volume and bone parameter changes and had less pronounced inflammation and immune response than the CFA group.

Differences of mandibular condylar cartilage thickness of Mus musculus after given different intensities of physical stress

Objective: This study aimed to evaluate the differences in the thickness of Mus musculus mandible cartilage after being given low, moderate, and high-intensity physical stress. Materials and Methods: This research is an experimental laboratory study. A total of 12 Mus musculus were divided into four groups, which are the control group(C), low (LS), moderate(MS), and high-intensity physical stress group(HS). The physical stress group was given treatment with swimming exercises using 3%, 6%, and 9% of body weight with a duration of 40%, 70%, and maximum swimming time calculation. After the experiment was carried out, the animal was decapitated and deparaffinized using xylol to dissolve paraffin in tissue to make histopathology preparations that were stained with hematoxylin-eosin. The thickness of mandibular condylar cartilage was calculated under a light microscope with a magnification of 400x and analyzed using the One Way ANOVA test to determine if the relationship between the sets of data in the treatment group is statistically significant (p&lt;0.05). Results: The thickness of the mandibular cartilage was measured to be thinner in the MS group (56.30 ± 6.11) and the HS group (78.04 ± 19.31) compared to the C group 85,85 ± 7,20. Statistical analysis with One Way ANOVA test showed that all groups had a significant difference in the thickness of mandibular condylar cartilage (p=0.049). Conclusions: There was a difference in the thickness of the mandibular cartilage after giving low, moderate, and high-intensity physical stress.

Formation of the Mandibular Condylar Cartilage in Human Specimens Between 10 and 15 Weeks of Gestation

Background: The study examines some morphological and histological factors that may contribute to condylar cartilage (CC) formation. The specimens used were consecutive sections obtained from 20 human fetuses ranging in age from 10 to 15 weeks. The vascular canals (VC) emerge in the CC and initiate the process of intramembranous ossification. The mandibular condylar cartilage is crucial for the growth and function of the mandible, and it also contributes to the development of the Temporomandibular joint. Understanding the development of mandibular condylar cartilage throughout the early stages of gestation is essential for detecting congenital craniofacial deformities. Objectives: Examine the stages of MCC development in human samples from fetuses at 10-15 weeks of gestation. Study Design: A descriptive Cross-sectional Study. Place and Duration of Study: Department of Anatomy and Obstetric Nowshera Medical College from 15th Nov 2020 to 1st Jun 2021. The changes in the shape and size of the bones and the processes of bone formation should also be determined. Methods: The current study is descriptive, cross-sectional, and involves post-mortem human fetal specimens aged 10-15 weeks. The specimens were obtained from the Department of Obstetrics of QHAMC after miscarriages and intrauterine deaths. The absence of external and congenital malformations was verified. Light microscopic and histological assessment was done with the aid of H&amp;E-stained sections. Results : Twenty fetal specimens were used in the study, with an average gestation period of 12. 5 weeks (±1. 5 weeks). Histological analysis revealed gradual chondrocyte maturation and the beginning of the ossification process. At ten weeks, MCC was comprised of undifferentiated mesenchymal cells. At twelve weeks, it is possible to observe the differentiation of chondrocytes and early ossification. At 15 weeks, the ossification stages of the CRL were at a superior level with the hypertrophic chondrocytes and mineralized cartilage. The p-values for differences in developmental stages between the gestational ages were less than 0. Hence, the differences were statistically significant. Conclusion: The MCC in human specimens at 10–15 weeks' gestation undergoes considerable morphological transformation and commences the ossification process. Knowledge of these stages is essential in identifying and intervening in congenital craniofacial disorders. The authors recommend that future studies focus on investigating molecular processes that may contribute to the development of MCC. Keywords: Mandibular condylar cartilage, Development, Gestation, Ossification

Circadian rhythm disruption upregulating Per1 in mandibular condylar chondrocytes mediating temporomandibular joint osteoarthritis via GSK3β/β-CATENIN pathway

BackgroundTemporomandibular joint osteoarthritis (TMJOA) has a high incidence rate, but its pathogenesis remains unclear. Circadian rhythm is an important oscillation in the human body and influences various biological activities. However, it is still unclear whether circadian rhythm affects the onset and development of TMJOA.MethodsWe disrupted the normal rhythm of rats and examined the expression of core clock genes in the mandibular condylar cartilage of the jaw and histological changes in condyles. After isolating rat mandibular condylar chondrocytes, we upregulated or downregulated the clock gene Per1, examined the expression of cartilage matrix-degrading enzymes, tested the activation of the GSK3β/β-CATENIN pathway and verified it using agonists and inhibitors. Finally, after downregulating the expression of Per1 in the mandibular condylar cartilage of rats with jet lag, we examined the expression of cartilage matrix-degrading enzymes and histological changes in condyles.ResultsJet lag led to TMJOA-like lesions in the rat mandibular condyles, and the expression of the clock gene Per1 and cartilage matrix-degrading enzymes increased in the condylar cartilage of rats. When Per1 was downregulated or upregulated in mandibular condylar chondrocytes, the GSK3β/β-CATENIN pathway was inhibited or activated, and the expression of cartilage matrix-degrading enzymes decreased or increased, which can be rescued by activator and inhibitor of the GSK3β/β-CATENIN pathway. Moreover, after down-regulation of Per1 in mandibular condylar cartilage in vivo, significant alleviation of cartilage degradation, cartilage loss, subchondral bone loss induced by jet lag, and inhibition of the GSK3β/β-CATENIN signaling pathway were observed. Circadian rhythm disruption can lead to TMJOA. The clock gene Per1 can promote the occurrence of TMJOA by activating the GSK3β/β-CATENIN pathway and promoting the expression of cartilage matrix-degrading enzymes. The clock gene Per1 is a target for the prevention and treatment of TMJOA.

Chondrogenic potential of superficial versus cartilage layer cells of the temporomandibular joint condyle in photopolymerizable gelatin-based hydrogels.

The objectives of this study were to compare the chondrogenic potential of cells derived from different layers of Mandibular condyle cartilage and to gain further understanding of the impact of chondrogenic cues when embedded into a novel hydrogel scaffold (PGH, a polymer blend of poly (ethylene glycol), gelatin, and heparin) compared to a gelatin hydrogel scaffold (GEL). Cartilage layer cells (CLCs) and fibroblastic superficial layer cells (SLCs) were harvested from the mandibular condyle of boer goats obtained from a local abattoir. After expansion, cells were seeded into PGH and GEL hydrogels and cultured in chondrogenic media for 3 weeks. Scaffolds were harvested at 0, 1, and 3 week(s) and processed for gross appearance, histochemical, biochemical, and mechanical assays. In terms of chondrogenesis, major differences were observed between scaffold materials, but not cell types. Glycosaminoglycan (GAG) staining showed GEL scaffolds deposited GAG during the 3 week period, which was also confirmed with the biochemical testing. Moreover, GEL scaffolds had significantly higher compressive modulus and peak stress than PGH scaffolds at all time points with the largest difference seen in week 3. It can be concluded that GEL outperformed PGH in chondrogenesis. It can also be concluded that materials play a more important role in the process of chondrogenesis than the tested cell populations. Fibroblastic SLCs were shown to have similar chondrogenic potential as CLCs cells, suggesting a rich pool of progenitor cells in the superficial fibroblastic layer capable of undergoing chondrogenesis given appropriate physical and chemical cues.

Gestational intermittent hypoxia reduces mandibular growth with decreased Sox9 expression and increased Hif1a expression in male offspring rats.

Maternal obstructive sleep apnea (OSA) during pregnancy is the risk factor for impaired fetal growth with low birth weight in the offspring. However, it is unclear whether gestational intermittent hypoxia (IH, a hallmark of maternal OSA) has long-term detrimental consequences on the skeletal development of offspring. This study aimed to investigate postnatal maxillofacial bone growth and cartilage metabolism in male and female offspring that were exposed to gestational IH. Mother rats underwent IH at 20 cycles/h (nadir, 4% O2; peak, 21% O2; 0% CO2) for 8h per day during gestational days (GD) 7-20, and their male and female offspring were analyzed postnatally at 5 and 10weeks of age. All male and female offspring were born and raised under normoxic conditions. There was no significant difference in whole-body weight and tibial length between the IH male/female offspring and their control counterparts. In contrast, the mandibular condylar length was significantly shorter in the IH male offspring than in the control male offspring at 5 and 10weeks of age, while there was no significant difference in the female offspring. Real-time polymerase chain reaction (PCR) showed that gestational IH significantly downregulated the mRNA level of SOX9 (a chondrogenesis marker) and upregulated the mRNA level of HIF-1α (a hypoxia-inducible factor marker) in the mandibular condylar cartilage of male offspring, but not in female offspring. Gestational IH induced underdeveloped mandibular ramus/condyles and reduced mRNA expression of SOX9, while enhancing mRNA expression of HIF-1α in a sex-dependent manner.

Long-term haplodeficency of DSPP causes temporomandibular joint osteoarthritis in mice

BackgroundExtracellular matrix (ECM) protein malfunction or defect may lead to temporomandibular joint osteoarthritis (TMJ OA). Dentin sialophophoprotein (DSPP) is a mandibular condylar cartilage ECM protein, and its deletion impacted cell proliferation and other extracellular matrix alterations of postnatal condylar cartilage. However, it remains unclear if long-term loss of function of DSPP leads to TMJ OA. The study aimed to test the hypothesis that long-term haploinsufficiency of DSPP causes TMJ OA.Materials and methodsTo determine whether Dspp+/– mice exhibit TMJ OA but no severe tooth defects, mandibles of wild-type (WT), Dspp+/–, and Dspp homozygous (Dspp−/−) mice were analyzed by Micro-computed tomography (micro-CT). To characterize the progression and possible mechanisms of osteoarthritic degeneration over time in Dspp+/– mice over time, condyles of Dspp+/– and WT mice were analyzed radiologically, histologically, and immunohistochemically.ResultsMicro-CT and histomorphometric analyses revealed that Dspp+/– and Dspp−/− mice had significantly lower subchondral bone mass, bone volume fraction, bone mineral density, and trabecular thickness compared to WT mice at 12 months. Interestingly, in contrast to Dspp−/− mice which exhibited tooth loss, Dspp+/– mice had minor tooth defects. RNA sequencing data showed that haplodeficency of DSPP affects the biological process of ossification and osteoclast differentiation. Additionally, histological analysis showed that Dspp+/– mice had condylar cartilage fissures, reduced cartilage thickness, decreased articular cell numbers and severe subchondral bone cavities, and with signs that were exaggerated with age. Radiographic data showed an increase in subchondral osteoporosis up to 18 months and osteophyte formation at 21 months. Moreover, Dspp+/– mice showed increased distribution of osteoclasts in the subchondral bone and increased expression of MMP2, IL-6, FN-1, and TLR4 in the mandibular condylar cartilage.ConclusionsDspp+/– mice exhibit TMJ OA in a time-dependent manner, with lesions in the mandibular condyle attributed to hypomineralization of subchondral bone and breakdown of the mandibular condylar cartilage, accompanied by upregulation of inflammatory markers.

Mandibular Endochondral Growth Is Specifically Augmented by Nutritional Supplementation with Myo-Inositol Even in Rabbits.

Mandibular retrognathism occurs by insufficient mandibular growth and causes several issues, such as respiratory difficulty and diminished masticatory function. At present, functional orthodontic appliances are used for stimulating mandibular growth in pediatric cases. However, the effectiveness of functional appliances is not always stable in daily practices. A more effective, reliable, and safer therapeutic method for mandibular growth promotion would be helpful for growing mandibular retrognathism patients. As we previously discovered that nutritional supplementation of myo-inositol in growing mice specifically increases mandibular endochondral growth, we performed preclinical animal experiments in rabbits in this study. Briefly, six-week-old male Japanese white rabbits were fed with or without myo-inositol supplementation in laboratory chow until 25 weeks old, and 3D image analysis using micro CT data and histological examinations was done. Myo-inositol had no systemic effect, such as femur length, though myo-inositol specifically augmented the mandibular growth. Myo-inositol increased the thickness of mandibular condylar cartilage. We discovered that the nutritional supplementation of myo-inositol during the growth period specifically augmented mandibular growth without any systemic influence, even in rabbits. Our results suggest the possibility of clinical use of myo-inositol for augmentation of the mandibular growth in growing mandibular retrognathism patients in the future.

A PTHrP Gradient Drives Mandibular Condylar Chondrogenesis via Runx2

The mandibular condylar cartilage (MCC) is an essential component of the temporomandibular joint, which orchestrates the vertical growth of the mandibular ramus through endochondral ossification with distinctive modes of cell differentiation. Parathyroid hormone–related protein (PTHrP) is a master regulator of chondrogenesis; in the long bone epiphyseal growth plate, PTHrP expressed by resting zone chondrocytes promotes chondrocyte proliferation in the adjacent layer. However, how PTHrP regulates chondrogenesis in the MCC remains largely unclear. In this study, we used a Pthrp-mCherry knock-in reporter strain to map the localization of PTHrP+ cells in the MCC and define the function of PTHrP in the growing mandibular condyle. In the postnatal MCC of Pthrp mCherry/+ mice, PTHrP-mCherry was specifically expressed by cells in the superficial layer immediately adjacent to RUNX2-expressing cells in the polymorphic layer. PTHrP ligands diffused across the polymorphic and chondrocyte layers where its cognate receptor PTH1R was abundantly expressed. We further analyzed the mandibular condyle of Pthrp mCherry/mCherry mice lacking functional PTHrP protein (PTHrP-KO). At embryonic day (E) 18.5, the condylar process and MCC were significantly truncated in the PTHrP-KO mandible, which was associated with a significant reduction in cell proliferation across the polymorphic layer and a loss of SOX9+ cells in the chondrocyte layers. The PTHrP-KO MCC showed a transient increase in the number of Col10a1+ hypertrophic chondrocytes at E15.5, followed by a significant loss of these cells at E18.5, indicating that superficial layer–derived PTHrP prevents premature chondrocyte exhaustion in the MCC. The expression of Runx2, but not Sp7, was significantly reduced in the polymorphic layer of the PTHrP-KO MCC. Therefore, PTHrP released from cells in the superficial layer directly acts on cells in the polymorphic layer to promote proliferation of chondrocyte precursor cells and prevent their premature differentiation by maintaining Runx2 expression, revealing a unique PTHrP gradient-directed mechanism that regulates MCC chondrogenesis.

The Nrf2 Pathway Alleviates Overloading Force-Induced TMJ Degeneration by Downregulating Oxidative Stress Reactions.

Oxidative stress is involved in the mechanisms associated with temporomandibular joint (TMJ) diseases. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial oxidative stress marker, but the specific mechanisms of its regulation in the early stages of mandibular condylar cartilage (MCC) degeneration remain unclear. This study aimed to explore the regulatory role of Nrf2 and its related oxidative stress signaling pathway in the early stage of MCC degeneration. Overloading force-induced MCC degeneration was performed in wild-type and Nrf2 knockout mice, as well as in mice after treatment with the Nrf2 activator cardamonin. Changes in MCC degeneration and the expression of oxidative stress markers in the corresponding situations were observed. Nrf2 and NADPH oxidase 2 (NOX2) expression were elevated during early MCC degeneration induced by an overloading force. MCC degeneration was aggravated when Nrf2 was knocked out, accompanied by increased NOX2 and superoxide dismutase 2 (SOD2) expression. The MCC degeneration process was alleviated after cardamonin treatment, with activation of the Nrf2 pathway and decreased NOX2 and SOD2 expression. Early MCC degeneration is accompanied by mild oxidative stress progression. Activated Nrf2 and related pathways could alleviate the degeneration of MCC.

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.

Age-Related Gene and Protein Expression in Mouse Mandibular Condyle Analyzed by Cap Analysis of Gene Expression and Immunohistochemistry

Introduction: Aging, an inevitable physiological process, leads to morphological and histological degenerative changes in the mandibular condylar cartilage (MCC); however, the molecular mechanism has not yet been elucidated, and little information is available on age-related factors. Therefore, this study was designed to identify age-related factors by investigating the age-related differentially expressed genes (DEGs) and localization of their translated protein expression in the mandibular condyle. Methods: Mandibular condyles were collected from 10- and 50-week-old mice. Total RNA was extracted from the samples and then analyzed using cap analysis of gene expression (CAGE) to identify age-related DEGs. Gene ontology (GO) enrichment analysis was performed to determine which biological processes were most affected by aging in terms of gene expression using Metascape. The mandibular condyle samples were processed for histology to investigate morphological changes caused by aging and for immunohistochemistry to localize the protein expression encoded by age-related genes identified with CAGE. Semi-quantitative immunohistochemistry was performed to assess age-related extracellular matrix (ECM) protein levels in the MCC. The histological sections were also used for Alcian blue histochemistry to detect glycosaminoglycans (GAGs). Results: GO enrichment analysis revealed that the genes related to “extracellular matrix organization,” including Acan, Col1a1, Col1a2, Col2a1, Mmp3, Mmp9, and Mmp13, were most differentially expressed in the aged mandibular condyle. Among these seven genes, Mmp3 was upregulated, and the others were downregulated with aging. Histological examination showed the age-related morphological and histological changes in the MCC. Immunohistochemical investigation showed the localization of matrix metalloproteinases (MMPs)-3, -9, and -13 and their substrate proteins, aggrecan, type I collagen, and type II collagen, in the mandibular condyle at 10 and 50 weeks, indicating different localizations between the young and the aged. In the aged MCC, semi-quantitative immunohistochemistry showed a significant decrease in the aggrecan protein level, and Alcian blue histochemistry showed a decrease in GAGs. Conclusion: MMP-3, MMP-9, and MMP-13 contribute to the remodeling of the ECM of the MCC and subchondral bone during aging by degrading ECM proteins at specific times and sites under the regulation of their production and secretion.

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

Mandibular condylar hypoplasia negatively affects patient's facial appearance and dentofacial function. 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. 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. 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. 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.

Fourier-transform infrared study on effects of ageing, oestrogen level and altered dietary loading on rat mandibular condylar cartilage.

Mandibular condylar cartilage (MCC) of the rat was examined with the Fourier-transform infrared (FITR) spectroscopic imaging to study the effects of ageing, oestrogen level and altered dietary loading on the structure of MCC. The Sprague-Dawley rats (n = 96) aged 5 and 14 months were divided into 12 subgroups according to age, oestrogen status (ovariectomized [OVX], non-ovariectomized [non-OVX)]) and diet (hard, normal, soft). Specimens of the MCC were examined with FTIR spectroscopic imaging to quantify the distribution of collagens and proteoglycans. MCC was divided sagittally into three segments: anterior, most superior and posterior. From each segment, the collagen and proteoglycan contents at different depths of cartilage were statistically compared between the groups using an N-way analysis of variance (ANOVA). The amount of collagen content was significantly associated with old age in the deep layer of the anterior segment and in the middle layer of the posterior segment of MCC. In the deep layer of the most superior segment, the collagen content also increased with ageing. The amount of proteoglycan content increased significantly when dietary loading increased, and the oestrogen level decreased in the deep layer of the most superior segment of MCC. Ageing, oestrogen level and altered dietary loading have a significant effect on the location and content of collagens and proteoglycans of rat MCC. Ageing significantly increased the amount of collagen content in the superior and posterior segments, being highest in the older soft-diet rats. Decreased oestrogen levels and increased dietary loading increased the amount of proteoglycan content.

CaSR modulates proliferation of the superficial zone cells in temporomandibular joint cartilage via the PTHrP nuclear localization sequence.

The superficial zone cells in mandibular condylar cartilage are proliferative. The present purpose was to delineate the relation of calcium-sensing receptor (CaSR) and parathyroid hormone-related peptide nuclear localization sequence (PTHrP87-139 ), and their role in the proliferation behaviors of the superficial zone cells. A gain- and loss-of-function strategy were used in an in vitro fluid flow shear stress (FFSS) model and an in vivo bilateral elevation bite model which showed mandibular condylar cartilage thickening. CaSR and PTHrP87-139 were modulated through treating the isolated superficial zone cells with activator/SiRNA and via deleting CaSR or parathyroid hormone-related peptide (PTHrP) gene in mice with the promoter gene of proteoglycan 4 (Prg4-CreERT2 ) in the tamoxifen-inducible pattern with or without additional injection of Cinacalcet, the CaSR agonist, or PTHrP87-139 peptide. FFSS stimulated CaSR and PTHrP expression, and accelerated proliferation of the Prg4-expressing superficial zone cells, in which process CaSR acted as an up-streamer of PTHrP. Proteoglycan 4 specific knockout of CaSR or PTHrP reduced the cartilage thickness, suppressed the proliferation and early differentiation of the superficial zone cells, and inhibited cartilage thickening and matrix production promoted by bilateral elevation bite. Injections of CaSR agonist Cinacalcet could not improve the phenotype caused by PTHrP mutation. Injections of PTHrP87-139 peptide rescued the cartilage from knockout of CaSR gene. CaSR modulates proliferation of the superficial zone cells in mandibular condylar cartilage through activation of PTHrP nuclear localization sequence. Our data support the therapeutic target of CaSR in promoting PTHrP production in superficial zone cartilage.

Disrupting mechanical homeostasis promotes matrix metalloproteinase-13 mediated processing of neuron glial antigen 2 in mandibular condylar cartilage.

Post-traumatic osteoarthritis in the temporomandibular joint (TMJ OA) is associated dysfunctional cellmatrix mediated signalling resulting from changes in the pericellular microenvironment after injury. Matrix metalloproteinase (MMP)-13 is a critical enzyme in biomineralisation and the progression of OA that can both degrade the extracellular matrix and modify extracellular receptors. This study focused on MMP-13 mediated changes in a transmembrane proteoglycan, Neuron Glial antigen 2 (NG2/CSPG4). NG2/CSPG4 is a receptor for type VI collagen and a known substrate for MMP-13. In healthy articular layer chondrocytes, NG2/CSPG4 is membrane bound but becomes internalised during TMJ OA. The objective of this study was to determine if MMP-13 contributed to the cleavage and internalisation of NG2/CSPG4 during mechanical loading and OA progression. Using preclinical and clinical samples, it was shown that MMP-13 was present in a spatiotemporally consistent pattern with NG2/CSPG4 internalisation during TMJ OA. In vitro, it was illustrated that inhibiting MMP-13 prevented retention of the NG2/CSPG4 ectodomain in the extracellular matrix. Inhibiting MMP-13 promoted the accumulation of membrane-associated NG2/CSPG4 but did not affect the formation of mechanical-loading dependent variant specific fragments of the ectodomain. MMP- 13 mediated cleavage of NG2/CSPG4 is necessary to initiate clathrin-mediated internalisation of the NG2/ CSPG4 intracellular domain following mechanical loading. This mechanically sensitive MMP-13-NG2/CSPG4 axis affected the expression of key mineralisation and OA genes including bone morphogenetic protein 2, and parathyroid hormone-related protein. Together, these findings implicated MMP-13 mediated cleavage of NG2/CSPG4 in the mechanical homeostasis of mandibular condylar cartilage during the progression of degenerative arthropathies such as OA.

FGF Ligands and Receptors in Osteochondral Tissues of the Temporomandibular Joint in Young and Aging Mice.

Fibroblast growth factors (FGFs) are a family of 22 proteins and 4 FGF receptors (FGFRs) that are crucial elements for normal development. The contribution of different FGFs and FGFRs for the homeostasis or disease of the cartilage from the mandibular condyle is unknown. Therefore, our goal was to characterize age-related alterations in the protein expression of FGF ligands and FGFRs in the mandibular condyle of mice. Mandibular condyles of 1-, 6-, 12-, 18-, and 24-month-old C57BL/6J male mice (5 per group) were collected and histologically sectioned. Immunofluorescence for FGFs that have been reported to be relevant for chondrogenesis (FGF2, FGF8, FGF9, FGF18) as well as the activated/phosphorylated FGFRs (pFGFR1, pFGFR3) was carried out. FGF2 and FGF8 were strongly expressed in the cartilage and subchondral bone of 1-month-old mice, but the expression shifted mainly to the subchondral bone as mice aged. FGF18 and pFGFR3 expression was limited to the cartilage of 1-month-old mice only. Meanwhile, pFGFR1 and FGF9 were mostly limited to the cartilage with a significant increase in expression as mice aged. Our results indicate FGF2 and FGF8 are important growth factors for mandibular condylar cartilage growth in young mice but with limited role in the cartilage of older mice. In addition, the increased expression of pFGFR1 and FGF9 and the decreased expression of pFGFR3 and FGF18 as mice aged suggest the association of these factors with aging and osteoarthritis of the cartilage of the mandibular condyle.

Immunohistochemical characterization of the mandibular condyle for type I and II collagen, aggrecan, MMP-9, and MMP-13 in MMP-2-deficient mice.

Mice devoid of matrix metalloproteinase (MMP)-2 due to gene targeting have been reported to show articular cartilage destruction in the knee joint; however, the phenotype of the mandibular condylar cartilage remains unknown. Thus, in the present study, we investigated the mandibular condyle in Mmp2-/- mice. We obtained and bred Mmp2-/- mice from the same source as the previous study, and performed genotyping using genomic DNA extracted from finger snips. The mandibular condyle of Mmp2-/- mice and wild-type (WT) mice was immunohistochemically examined for the localization of extracellular matrix (ECM) proteins (type I and II collagen, and aggrecan), and MMP-9 and MMP-13. No cartilage destruction was observed in the mandibular condyle of Mmp2-/- mice, and no difference was found in the localization of the ECM proteins between the Mmp2-/- mice and WT mice. However, the bone marrow cavity in the subchondral bone of the mandibular condyle was more distinct in Mmp2-/- mice than in WT mice at the age of 50 weeks. Of note, MMP-9 characteristically localized in multinucleated cells in the mandibular condyle in 50-week-old Mmp2-/- mice. MMP-2 may be involved in the regulation of osteoclast differentiation and the formation of the bone marrow cavity in aged mice.

Bulk RNA-seq analyses of mandibular condylar cartilage in a post-traumatic TMJ osteoarthritis rabbit model.

The temporomandibular joint (TMJ) is anatomically comprised of the mandibular condylar cartilage (CC) lined with fibrocartilaginous superficial zone and is crucial for eating and dental occlusion. TMJ osteoarthritis (OA) leads to pain, joint dysfunction and permanent loss of cartilage tissue. However, there are no drugs clinically available that ameliorate OA and little is known about global profiles of genes that contribute to TMJ OA. Furthermore, animal models that recapitulate the complexity of signalling pathways contributing to OA pathogenesis are crucial for designing novel biologics that thwart OA progression. We have previously developed a New Zealand white rabbit TMJ injury model that demonstrates CC degeneration. Here, we performed genome-wide profiling to identify new signalling pathways critical for cellular functions during OA pathology. Temporomandibular joint OA was surgically induced in New Zealand white rabbits. Three months following injury, we performed global gene expression profiling of the TMJ condyle. RNA samples from TMJ condyles were subjected to sequencing. After raw RNA-seq data were mapped to relevant genomes, differential expression was analysed with DESeq2. Gene ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis were conducted. Our study revealed multiple pathways altered during TMJ OA induction including the Wnt, Notch and PI3K-Akt signalling pathways. We demonstrate an animal model that recapitulates the complexity of the cues and signals underlying TMJ OA pathogenesis, which is essential for developing and testing novel pharmacologic agents to treat OA.