Decreased expression of ATP-binding cassette protein G1 promotes abnormal adipogenesis of condylar chondrocytes in temporomandibular joint osteoarthritis.

BACKGROUND: Abnormal lipid metabolism is involved in the development of osteoarthritis (OA). ATP-binding cassette protein G1 (ABCG1) is crucial in mediating the outflow of cholesterol, phosphatidylcholine and sphingomyelin and reducing intracellular lipid accumulation. OBJECTIVE: This study aimed to evaluate whether ABCG1 participates in the abnormal adipogenesis of chondrocytes in osteoarthritic cartilage of temporomandibular joint. METHODS: Eight-week-old female rats were subjected to unilateral anterior crossbite (UAC) to induce OA in the temporomandibular joint (TMJ). Histochemical staining, immunohistochemical (IHC) staining, and qRT-PCR were performed. Primary condylar chondrocytes of rats were transfected with ABCG1 shRNA or overexpression lentivirus and then stimulated with fluid flow shear stress (FFSS). Cells were collected for oil red O staining, immunofluorescence staining, and qRT-PCR analysis. RESULTS: Abnormal adipogenesis, characterized by increased expression of Adiponectin, CCAAT/enhancer-binding protein α (Cebpα), fatty acid binding protein 4 (Fabp4) and Perilipin1, was enhanced in the degenerative cartilage of TMJ OA in rats with UAC, accompanied by decreased expression of ABCG1. After FFSS stimulation, we observed lipid droplets in the cytoplasm of cultured cells with increased expression of Adiponectin, Cebpα, Fabp4 and Perilipin1 and decreased expression of ABCG1. Knockdown of Abcg1 induced abnormal adipogenesis and differentiation of condylar chondrocytes. Overexpression of ABCG1 alleviated the abnormal adipogenesis and differentiation of condylar chondrocytes induced by FFSS. CONCLUSIONS: Abnormal adipogenesis of chondrocytes and decreased ABCG1 expression were observed in degenerative cartilage of TMJ OA. ABCG1 overexpression effectively inhibits the adipogenesis of chondrocytes and thus alleviates TMJ condylar cartilage degeneration.

Occlusal disharmony transiently decrease cognition via cognitive suppressor molecules and partially restores cognitive ability via clearance molecules.

Occlusal disharmony has been reported to be affected not only by cytokine and steroid hormone secretion and sympathetic activation in peripheral organs, but also by neurotransmitter release in the central nervous system. However, little is known about whether occlusal disharmony can decrease cognitive ability. We hypothesized that hyperocclusion decreases cognition via Alzheimer's disease-associated molecule expression in the brain. The present study is aimed to elucidate the relationships among occlusal disharmony, cytokine and cognitive-regulated molecule expression in the brain, and the impairment of learning and memory cognition. We examined the effect of hyperocclusion on the relationships among cytokine expression, cognitive suppressor molecules in the hippocampus, and cognition in behavior using a hyperocclusion mouse model. Hyperocclusion dramatically increased interleukin-1ß expression in the serum and hippocampus 1 week after hyperocclusal loading in 2-month-old mice, but no effects in 12-month-old mice. The social and long-term cognitive abilities of the 2-month-old mice were transiently downregulated close to the level of the 12-month-old mice 1 week after hyperocclusion and recovered to close to basal level via the expression of cognitive suppressor clearing proteins. The expression levels of amyloid-ß and phosphorylated tau were significantly upregulated 1 week after hyperocclusal loading in the hippocampus of 2-month-old mice but were constant in 12-month-old mice. Occlusal disharmony-induced interleukin-1ß expression may contribute to accumulation of cognitive suppressor molecules such as amyloid-ß and phosphorylated tau and activate their clearance proteins, resulting in protection against transient dementia in young but not older individuals.

Indian hedgehog in craniofacial neural crest cells links to skeletal malocclusion by regulating associated cartilage formation and gene expression.

The frontal craniofacial skeleton derived from neural crest cells is vital for facial structure and masticatory functions. The exact role of Indian hedgehog (Ihh) in facial and masticatory development has not been fully explored. In this study, we generated craniofacial neural crest cells-specific Ihh deletion mice (Wnt1-Cre;Ihhfl/fl ;Tomatofl/+ ) and found the gradual dwarfism without perinatal lethality. Morphological and histological analyses revealed unambiguous craniofacial phenotypes in mutants, where we observed skeletal malocclusion accompanied by markedly hypoplastic nasomaxillary complex and reversed incisor occlusion. Both the replacement of nasal concha cartilage by turbinate bones and the endochondral ossification of nasal septum ethmoid bone were substantially delayed. We also observed hypoplastic mandibles in mutants where the mandibular ramus was unexpectedly the most affected. Both the condylar process and mandibular angle cartilages were distorted. However, dental examination showed no significant changes in teeth and dentition. Finally, a comprehensive RNA sequence analysis utilizing condylar cartilage identified Ihh-associated gene network including several cell cycle genes and 16 genes related to the extracellular matrix, sulfate transporters, transcription factors, receptors, a ciliogenesis factor, and an adhesion molecule. Our data provide direct in vivo evidence that Ihh plays crucial roles in midface and masticatory system formation, likely by activating key genes.

Genes and Pathways Associated with Skeletal Sagittal Malocclusions: A Systematic Review.

Skeletal class II and III malocclusions are craniofacial disorders that negatively impact people's quality of life worldwide. Unfortunately, the growth patterns of skeletal malocclusions and their clinical correction prognoses are difficult to predict largely due to lack of knowledge of their precise etiology. Inspired by the strong inheritance pattern of a specific type of skeletal malocclusion, previous genome-wide association studies (GWAS) were reanalyzed, resulting in the identification of 19 skeletal class II malocclusion-associated and 53 skeletal class III malocclusion-associated genes. Functional enrichment of these genes created a signal pathway atlas in which most of the genes were associated with bone and cartilage growth and development, as expected, while some were characterized by functions related to skeletal muscle maturation and construction. Interestingly, several genes and enriched pathways are involved in both skeletal class II and III malocclusions, indicating the key regulatory effects of these genes and pathways in craniofacial development. There is no doubt that further investigation is necessary to validate these recognized genes' and pathways' specific function(s) related to maxillary and mandibular development. In summary, this systematic review provides initial insight on developing novel gene-based treatment strategies for skeletal malocclusions and paves the path for precision medicine where dental care providers can make an accurate prediction of the craniofacial growth of an individual patient based on his/her genetic profile.

Catabolic changes of rat temporomandibular joint discs induced by unilateral anterior crossbite.

BACKGROUND: The temporomandibular joint (TMJ) disc plays a role in joint movement and in load absorbance and distribution. An experimental unilateral anterior crossbite (UAC) prosthesis induces mandibular condylar cartilage degeneration in rats. However, the changes in the articular disc are still unknown. OBJECTIVE: To describe changes in the TMJ discs of UAC rats. METHODS: The discs of fifty-four Sprague-Dawley rats, equally distributed into a UAC group and an age-matched sham-operated control group at 4, 12 and 20 weeks (n = 9), were evaluated by gross and histomorphological observation and by detection at the mRNA or protein expression levels of the markers related to the matrix elements. RESULTS: No macro- or micro-morphological differences were observed between groups. However, there were catabolic degradative changes at the molecular level in the UAC group, showing a significant reduction in the mRNA and/or protein expression levels of many molecules. The reduction became worse with time (P < 0.05). The reduced molecules included: (a) those related to the extracellular matrix, such as type I collagen, decorin and fibromodulin; (b) those related to chondrogenesis, such as type II collagen and aggrecan; and (c) those related to osteogenesis, such as alkaline phosphatase and runt-related transcription factor 2. The mRNA expression of vascular endothelial growth factor did not change. In contrast, fibronectin, which can promote wound healing, and its N-terminal fragment, which can induce cartilage degradation, were accumulated (P < 0.05). CONCLUSION: TMJ discs were stimulated to catabolic changes by the aberrant dental occlusion and seemed to go to inanimate with time.

Matrix replenishing by BMSCs is beneficial for osteoarthritic temporomandibular joint cartilage.

OBJECTIVES: The present goal was to explore whether matrix replenishment is the primary requirement for osteoarthritic (OA) cartilage. METHODS: Cells isolated from the superficial and deep zone cartilage of a pig temporomandibular joint (TMJ) were exposed to fluid flow shear stress (FFSS). Differences in matrix production and cellular differentiation were detected. Unilateral anterior crossbite (UAC) was applied to C57BL/6J female mice. Green fluorescent protein-labeled exogenous bone marrow stromal cells (GFP-BMSCs) were injected weekly into TMJs, starting from 3 weeks of UAC stimulation and continuing for 4-, 8- and 12-weeks. Another GFP-BMSCs injection UAC group stopped receiving injections for 4-weeks after 8-weeks of injections. Assessments were focused on morphological alterations in UAC mouse TMJ cartilage, the expression levels of DAP3, an anoikis marker, CD163, a scavenger receptor family member, and ki67, a proliferation indicator. RESULTS: FFSS down-regulated type-II collagen expression but stimulated terminal differentiation in cells isolated from deep zone cartilage. It down-regulated aggrecan expression but up-regulated type I collagen in cells isolated from both superficial and deep zones. UAC caused matrix loss and anoikis and enhanced scavenging activity in deep zone chondrocytes without affecting cell proliferation. Superficial fibrillation was obvious in the late stage. Weekly injections of BMSCs largely restored these changes. The implanted BMSCs expressed a high level of CD163 protein but did not show remarkable cell proliferation. Terminating the supply of exogenous BMSCs reversed the restorative effects. CONCLUSIONS: Scavenging the degraded matrix and replenishing the fibrosis-developmental matrix are the primary requirements for the repair of OA cartilage.

The Effect of Functional Mandibular Shift on the Muscle Spindle Systems in Head-Neck Muscles and the Related Neurotransmitter Histamine.

The aim of this study is to explore the effects of abnormal occlusion and functional recovery caused by functional mandible deviation on the head and neck muscles and muscle spindle sensory-motor system by electrophysiological response and endogenous monoamine neurotransmitters' distribution in the nucleus of the spinal tract. Seven-week-old male Wistar rats were randomly divided into 7 groups: normal control group, 2W experimental control group, 2W functional mandible deviation group, 2W functional mandible deviation recovery group, 4W experimental control group, 4W functional mandible deviation group, 4W functional mandible deviation recovery group. Chewing muscles, digastric muscle, splenius, and trapezius muscle spindles electrophysiological response activities at the opening and closing state were recorded. And then the chewing muscles, digastric, splenius, trapezius, and neck trigeminal nucleus were taken for histidine decarboxylase (HDC) detection by high performance liquid chromatography (HPLC), immunofluorescence, and reverse-transcription polymerase chain reaction (RT-PCR). Histamine receptor proteins in the neck nucleus of the spinal tract were also examined by immunofluorescence and RT-PCR. Electromyography activity of chewing muscles, digastric, and splenius muscle was significantly asymmetric; the abnormal muscle electromyography activity was mainly detected at the ipsilateral side. After functional mandibular deviation, muscle sensitivity on the ipsilateral sides of the chewing muscle and splenius decreased, muscle excitement weakened, modulation depth decreased, and the muscle spindle afferent impulses of excitation transmission speed slowed down. Changes for digastric muscle electrical activity were contrary. The functions recovered at different extents after removing the deflector. However, trapezius in all the experimental groups and recovery groups exhibited bilateral symmetry electrophysiological responses, and no significant difference compared with the control group. After functional mandibular deviation, HDC protein and messenger ribonucleic acid (mRNA) levels on the ipsilateral sides of the chewing muscle and splenius increased significantly. HDC level changes for digastric muscle were contrary. After the removal of the mandibular position deflector, HDC protein and mRNA levels decreased on the ipsilateral sides of the chewing muscle and splenius while they increased in the digastric muscle. The difference of histamine decarboxylase content in the bilateral trapezius in each experimental group was small. After functional mandibular deviation, the temporomandibular joint mechanical receptors not only caused the fusimotor fiber hypoallergenic fatigue slow response on the ipsilateral sides of splenius, but also increased the injury neurotransmitter histamine release. The authors' results further support the opinion that the temporomandibular joint receptors may be involved in the mechanical theory of the head and neck muscles nervous system regulation.

Cytokines and VEGF induction in orthodontic movement in animal models.

Orthodontics is a branch of dentistry that aims at the resolution of dental malocclusions. The specialist carries out the treatment using intraoral or extraoral orthodontic appliances that require forces of a given load level to obtain a tooth movement in a certain direction in dental arches. Orthodontic tooth movement is dependent on efficient remodeling of periodontal ligament and alveolar bone, correlated with several biological and mechanical responses of the tissues surrounding the teeth. A periodontal ligament placed under pressure will result in bone resorption whereas a periodontal ligament under tension results in bone formation. In the primary stage of the application of orthodontic forces, an acute inflammation occurs in periodontium. Several proinflammatory cytokines are produced by immune-competent cells migrating by means of dilated capillaries. In this paper we summarize, also through the utilization of animal models, the role of some of these molecules, namely, interleukin-1ß and vascular endothelial growth factor, that are some proliferation markers of osteoclasts and osteoblasts, and the macrophage colony stimulating factor.

Changes in the expression of aromatase, estrogen receptor α and ß in mandibular condylar cartilage of rats induced by disordered occlusion.

BACKGROUND: Estrogens play an important role in modulating the morphology and function of temporomandibular joints (TMJs), which is suggested to act via estrogen receptors (ERs). The present study was to investigate the expression of aggrecan, collagen type II (Col II), Col X, aromatase, ERα and ERß in degenerative changes of mandibular condylar cartilage. METHODS: Forty male and 40 female 8-week-old rats were enrolled in this study. In experimental groups, the disordered occlusion was created by moving the first molars mesially and the third ones distally. Immunohistochemistry and real-time PCR were performed at the end of the second or fourth week. RESULTS: Degenerative changes, characterized by interrupted continuity of hypertrophic layer, pyknotic and eosinophilic lesion with few nuclei, areas filled with eosinophilic nuclei, were observed in more joints from female experimental groups than male ones. However, thickening changes in hypertrophic layer were only found in male experimental groups. The gene expression of Col II, Col X and aggrecan increased in 4-wk male experimental subgroup (both P < 0.01), but decreased in 2-wk and 4-wk female subgroups (P < 0.05). The gene expression of ERα decreased in 2-wk male and female experimental subgroups (both P < 0.01), however, that of ERß increased except the 2-wk female experimental subgroup (all P < 0.01). The expression of aromatase decreased in both male and female experimental subgroups (all P<0.01). CONCLUSIONS: Mandibular condylar cartilage responses differently to the disordered occlusion in male and female rats. The levels of locally synthesized estrogen, ERα and ERß may have limited attribution, if any, to the sex-specific cartilage response.

Differential expression of IGF1, IGFR1 and IGFBP3 in mandibular condylar cartilage between male and female rats applied with malocclusion.

This study was designed to investigate the expression differences of insulin-like growth factor-1 (IGF1), IGF type 1 receptor (IGFR1) and IGF-binding protein-3 (IGFBP3) in mandibular condylar cartilage between male and female rats with experimentally created malocclusion. A total of 40 male and 40 female rats were used, and malocclusion was created by moving the first molars mesially and the third molars distally in the experimental group. Animals were killed at the end of the second and fourth weeks. Haematoxylin and eosin (HE) staining was performed to monitor the changes in cartilage morphology and thickness. Immunohistochemistry and real-time PCR were used to detect the expression of IGF1, IGFR1 and IGFBP3. Osteoarthritis (OA)-like changes were observed in the experimental groups, with 2-week females showing larger OA-like regions than 2-week males (P < 0·05). Compared to their age- and sex-matched controls, both 2- and 4-week males in the experimental groups displayed increased cartilage thickness in the posterior regions (P < 0·05). Compared to their age- and sex-matched controls, the expression of IGF1 was lower in 2-week female group (P < 0·05), but higher in 4-week female, 2- and 4-week male experimental groups (P < 0.05). Similarly, the expression of IGFR1 was lower in 2-week female experimental group (P < 0.05), but higher in 2-week male experimental group (P < 0.05). The higher expression of IGFBP3 was observed in 2-week female, 2- and 4-week male experimental groups (P < 0·05). These results indicate that condylar cartilage from male and female rats respond differently to the malocclusion in early stage of OA, with more serious degeneration in females.

Alterations in the masseter muscle and plasma IL-6 level following experimentally induced occlusal interference and chronic stress--a study in rats.

This study was undertaken to examine the alteration of masseter and plasma interleukin-6 after inducing occlusal interference and chronic stress. Male Wistar rats were submitted to chronic stress procedure, exposed to occlusal interference, or exposed to both mentioned procedures. Whole blood and masseter tissue were collected to determine interleukin-6 level, measured by means of ELISA. Masseter pain was evaluated using the orofacial formalin test. Masseter interleukin-6 level was significantly higher in animals submitted to combination of occlusal interference and chronic stress than in the control group (p<0.05). There was positive and significant correlation between pain response and masseter interleukin-6 level (r=0.5741; p<0.0003). No significant differences in plasma interleukin-6 level were found between groups (p>0.05), as well as no correlation with pain (p>0.05). Combination of occlusal interference and chronic stress leads to strong local reaction characterized by high levels of masseter interleukine-6. High concentrations of muscle interleukin-6 and its correlation with pain point to inflammatory background of masticatory muscle pain.

Spinal CCK1 Receptors Contribute to Somatic Pain Hypersensitivity Induced by Malocclusion via a Reciprocal Neuron-Glial Signaling Cascade.

Recent studies have shown that the incidence of chronic primary pain including temporomandibular disorders (TMD) and fibromyalgia syndrome (FMS) often exhibit comorbidities. We recently reported that central sensitization and descending facilitation system contributed to the development of somatic pain hypersensitivity induced by orofacial inflammation combined with stress. The purpose of this study was to explore whether TMD caused by unilateral anterior crossbite (UAC) can induce somatic pain hypersensitivity, and whether the cholecystokinin (CCK) receptor-mediated descending facilitation system promotes hypersensitivity through neuron-glia cell signaling cascade. UAC evoked thermal and mechanical pain hypersensitivity of the hind paws from day 5 to 70 that peaked at week 4 post UAC. The expression levels of CCK1 receptors, interleukin-18 (IL-18) and IL-18 receptors (IL-18R) were significantly up-regulated in the L4 to L5 spinal dorsal horn at 4 weeks post UAC. Intrathecal injection of CCK1 and IL-18 receptor antagonists blocked somatic pain hypersensitivity. IL-18 mainly co-localized with microglia, while IL-18R mainly co-localized with astrocytes and to a lesser extent with neurons. These findings indicate that the signaling transduction between neurons and glia at the spinal cord level contributes to the descending pain facilitation through CCK1 receptors during the development of the comorbidity of TMD and FMS. PERSPECTIVE: CCK1 receptor-dependent descending facilitation may mediate central mechanisms underlying the development of widespread somatic pain via a reciprocal neuron-glial signaling cascade, providing novel therapeutic targets for the clinical treatment of TMD and FMS comorbidities.

Expression of integrinalpha5beta1, focal adhesion kinase and integrin-linked kinase in rat condylar cartilage during mandibular lateral displacement.

Integrins are cell-surface mechanochemical sensors and transducers involved in various cellular processes in combination with extracellular ligands. The aim of this study was to investigate the effect of mechanical stress on the expression of integrinalpha5beta1 and its downstream kinases, focal adhesion kinase (FAK) and integrin-linked kinase (ILK), in condylar cartilage during mandible lateral shift in young rats. Sixty 4-week-old male Wistar rats were divided at random into five control groups and five experimental groups. All rats in the experimental groups were fitted with a resin plate to functionally displace the mandible 2mm to the left (ipsilateral side). The rats were killed 1, 3, 7, 14 and 28 days after attachment of the appliance. Serial 6-mum sagittal sections were cut through the condylar head and processed for immunostaining of integrinalpha5beta1, FAK and ILK. The results were quantified using an image analysing system. Integrinalpha5beta1 expression in the superior-posterior region of the condylar cartilage on the ipsilateral side increased from 3 to 14 days compared with the contralateral side, with an intermediate level of expression in the control groups. Expression of FAK and ILK was similar to integrinalpha5beta1 expression, and they were also upregulated on the ipsilateral side compared with the contralateral side at the early stages of the experiment. The different mechanical loading on the two sides of the condylar cartilage led to different expression patterns of integrinalpha5beta1, FAK and ILK, which may correlate with the different morphological and histological changes seen between sides during mandibular lateral shift.

Malocclusion induces chronic stress.

We examined the effect of occlusal disharmony in senescence-accelerated (SAMP8) mice on plasma corticosterone levels, spatial learning in the water maze, fos induction, hippocampal neuron number, expression of glucocorticoid receptors (GR) and glucocorticoid receptor messenger ribonucleic acid (GRmRNA) in hippocampus and inhibitor of glucocorticoid (metyrapone). Bite-raised aged mice had significantly greater plasma corticosterone levels than age-matched control mice as well as impaired spatial memory and decreased Fos induction and a number of neurons in hippocampus. GR and GRmRNA expressions were significantly decreased in aged bite-raised mice compared with age-matched control mice. Pretreatment with metyrapone inhibited not only the bite-raised induced increase in plasma corticosterone levels, but also the reduction in the number of hippocampal neurons and impaired spatial learning. These datas suggest that the bite-raised condition may enhance the aging process in hippocampus, thereby leading to impairment of spatial memory by stress.

[Mitochondrial calcium overload in the masseter muscle of rats with occlusal interference: ionic changes and regulation by calmodulin kinase II].

OBJECTIVE: To investigate the changes in mitochondrial calcium and extracellular sodium concentrations in the masseter muscle of rats with occlusal interference and the regulatory mechanism of mitochondrial Ca2+ overload by calmodulin kinase II (CaMK II). METHODS: SD rat models of occlusal interference were established by placing a stainless steel segments (0.8 mm in diameter) to raise the occlusal surface of the upper right first molar. At 3, 7, 14, and 21 days after occlusal interference and at 3 days after removal of occlusal interference, HE staining was used to observe the histomorphological changes of the masseter muscle. Mitochondrial calcium concentration in the masseter muscle was detected using fluorescence spectrophotometry, and direct turbidimetry with potassium pyroantimonate was used to detect the extracellular sodium concentration; the expression levels of masseter muscle p-CaMK II (Thr287) and CaMK II were detected using Western blotting. RESULTS: Compared with those in the corresponding control groups, mitochondrial Ca2+ concentration in the masseter muscle on occlusal interference side increased significantly at 3, 7, 14 and 21 days after occlusal interference (P<0.05), but was significantly lowered at 3 days after removal of the interference (P<0.05). The concentration of extracellular Na+ increased progressively with time at 3, 7, 14 and 21 days after occlusal interference (P<0.05), and was significantly decreased at 3 days after interference removal (P<0.05). Occlusal interference for 3, 7 and 14 days resulted in significantly increased expressions of p-CaMK II (Thr287) and CaMK II (P<0.05), which was significantly decreased at 21 days compared with those in the control groups (P<0.05) and further decreased after removal of occlusal interference (P<0.05). Similar changes were also observed on the side without interference, but the changes on the interference side were more obvious (P<0.05). CONCLUSION: Occlusal interference causes elevated mitochondrial Ca2+ and extracellular Na+ concentrations in the masseter muscle of rats to lead to calcium overload; the increase in mitochondrial Ca2+ concentration is correlated with the phosphorylation level of CaMK II signaling pathway, suggesting a negative feedback regulation mechanism by the CaMK II signal pathway.

Occlusal disharmony attenuates glucocorticoid negative feedback in aged SAMP8 mice.

To evaluate the mechanism underlying impaired cognitive function due to occlusal disharmony, we examined the effect of the bite-raised condition on spatial performance and hippocampal expression of glucocorticoid receptors (GR) and glucocorticoid receptor messenger ribonucleic acid (GRmRNA) using behavioral, immunohistochemical, and in situ hybridization techniques. Learning ability in the water maze test was significantly impaired in aged bite-raised mice compared with age-matched control mice. There was no difference between control and bite-raised young and middle-aged mice. Also, immunohistochemical and in situ hybridization analysis showed that the bite-raised condition enhanced the age-related decrease in GR and GRmRNA expression in the hippocampus. In particular, GR and GRmRNA expressions were significantly decreased in aged bite-raised mice compared to age-matched control mice. These findings suggest that the bite-raised condition in aged SAMP8 mice decreases GR and GRmRNA, which impairs the hypothalamic-pituitary-adrenal feedback inhibition, thereby leading to memory deficits.

ROCK/actin/MRTF signaling promotes the fibrogenic phenotype of fibroblast-like synoviocytes derived from the temporomandibular joint.

Malocclusion caused by abnormal jaw development or muscle overuse during mastication results in abnormal mechanical stress to the tissues surrounding the temporomandibular joint (TMJ). Excessive mechanical stress against soft and hard tissues around the TMJ is involved in the pathogenesis of inflammatory diseases, including osteoarthritis (OA). OA-related fibrosis is a possible cause of joint stiffness in OA. However, cellular and molecular mechanisms underlying fibrosis around the TMJ remain to be clarified. Here, we established a cell line of fibroblast­like synoviocytes (FLSs) derived from the mouse TMJ. Then, we examined whether the Rho­associated coiled­coil forming kinase (ROCK)/actin/myocardin-related transcription factor (MRTF) gene regulatory axis positively regulates the myofibroblast (MF) differentiation status of FLSs. We found that i) FLSs extensively expressed the MF markers α­smooth muscle actin (α­SMA) and type I collagen; and ii) an inhibitor against the actin­polymerizing agent ROCK, Y­27632; iii) an actin-depolymerizing agent cytochalasin B; iv) an inhibitor of the MRTF/serum response factor­regulated transcription, CCG­100602, clearly suppressed the mRNA levels of α­SMA and type I collagen in FLSs; and v) an MF differentiation attenuator fibroblast growth factor­1 suppressed filamentous actin formation and clearly suppressed the mRNA levels of α-SMA and type I collagen in FLSs. These results strongly suggest that the ROCK/actin/MRTF axis promotes the fibrogenic activity of synoviocytes around the TMJ. Our findings partially clarify the molecular mechanisms underlying the emergence of TMJ­OA and may aid in identifying drug targets for treating this condition at the molecular level.

Morphofunctional compensation of masseter muscles in unilateral posterior crossbite patients.

Unilateral posterior crossbite is a widespread, asymmetric malocclusion characterized by an inverse relationship of the upper and lower buccal dental cusps, in the molar and premolar regions, on one side only of the dental arch. Patients with unilateral posterior crossbite exhibit an altered chewing cycles and the crossbite side masseter results to be less active with respect to the contralateral one. Few studies about morphological features of masticatory muscle in malocclusion disorders exist and most of these have been performed on animal models. The aim of the present study was to evaluate morphological and protein expression characteristics of masseter muscles in patients affected by unilateral posterior crossbite, by histological and immunofluorescence techniques. We have used antibody against PAX-7, marker of satellite cells, and against α-, ß-, γ-, δ-, ε- and ζ-sarcoglycans which are transmembrane glycoproteins involved in sarcolemma stabilization. By statistical analysis we have evaluated differences in amount of myonucley between contralateral and ipsilateral side. Results have shown: i) altered fibers morphology and atrophy of ipsilateral muscle if compared to the contralateral one; ii) higher number of myonuclei and PAX-7 positive cells in contralateral side than ipsilateral one; iii) higher pattern of fluorescence for all tested sarcoglycans in contralateral side than ipsilateral one. Results show that in unilateral posterior crossbite hypertrophic response of contralateral masseter and atrophic events in ipsilateral masseter take place; by that, in unilateral posterior crossbite malocclusion masticatory muscles modify their morphology depending on the function. That could be relevant in understanding and healing of malocclusion disorders; in fact, the altered balance about structure and function between ipsilateral and contralateral muscles could, long-term, lead and/ or worsen skeletal asymmetries.

Effects of occlusal disharmony on the hippocampal dentate gyrus in aged senescence-accelerated mouse prone 8 (SAMP8).

BACKGROUND AND OBJECTIVE: Malocclusion induced by raising the bite causes chronic stress. Chronic stress leads to increased plasma corticosterone levels and impaired hippocampal function due to impaired neurogenesis or increased apoptosis in the hippocampus. The present study aimed to clarify the mechanisms underlying the impaired hippocampal function induced by the bite-raised condition in aged senescence-accelerated mouse prone 8 (SAMP8). DESIGN: Nine-month-old aged SAMP8 mice were randomly divided into control and bite-raised groups. The vertical dimension of the bite was raised by applying resin to the molars. We evaluated newborn cell proliferation, survival, differentiation, and apoptosis in the hippocampal dentate gyrus (DG). Hippocampal brain-derived neurotrophic factor (BDNF) levels were also measured. RESULTS: The bite-raised mice exhibited a significant decrease in proliferation, survival, and differentiation of newborn cells into neurons in the hippocampal DG compared with controls. The number of apoptotic cells in the hippocampal DG was increased at 7 and 14 days after the bite-raising procedure. Expression of BDNF protein and mRNA in the hippocampus was also decreased in the bite-raised mice. CONCLUSION: Bite-raised aged SAMP8 mice exhibited decreased neurogenesis, increased apoptosis in the hippocampal DG, and decreased hippocampal BDNF expression, in association with hippocampus-dependent learning and memory deficits.

Occlusal disharmonies modulate central catecholaminergic activity in the rat.

Occlusal disharmonies have classically been thought to be involved in the etiopathogenesis of bruxism, as have, more recently, alterations in central neurotransmission, particularly dopaminergic neurotransmission. However, the connection between these two factors has still not been established. In this study, we assessed the effects of diverse occlusal disharmonies, maintained for either 1 day or 14 days, on neurochemical indices of dopaminergic and noradrenergic activity in the striatum, frontal cortex, and hypothalamus of the rat. The in vivo activity of tyrosine hydroxylase, determined as the accumulation of 3,4-dihydroxyphenylalanine (DOPA), 30 min after the administration of 3-hydroxybenzylhydrazine, a DOPA decarboxylase inhibitor, and dopamine and noradrenaline contents were quantified by high-performance liquid chromatography with electrochemical detection. The wearing of an acrylic cap on both lower incisors for 1 day induced a significant increase in DOPA accumulation in the regions analyzed, with parallel increases in dopamine levels in the hypothalamus and dopamine and noradrenaline in the frontal cortex. After the cap was maintained for 14 days, DOPA accumulation tended to return to control values, except in the left striatum, thereby causing an imbalance between hemispheres. In contrast, 1 or 14 days after the lower left and the upper right incisors were cut, less pronounced changes in catecholaminergic neurotransmission were found in the brain areas studied. Moreover, the cutting of one lower incisor did not modify either DOPA accumulation or dopamine and noradrenaline contents in the striatum or hypothalamus. These results provide experimental evidence of a modulation of central catecholaminergic neurotransmission by occlusal disharmonies, being dependent on the nature of the incisal alteration and on the time during which it was maintained.