Inhibition of Fibrous Adhesion Formation in the Temporomandibular Joint of Tenascin-C Knockout Mice

Tenascin-C (TNC) is a large hexameric extracellular matrix glycoprotein that is expressed in developing organs and tumors. It has been reported that TNC is expressed in inflamed synovial membranes and deformed discs of temporomandibular joint (TMJ) disorder. However, the role of TNC in TMJ is not fully known. In this study, the role of TNC in fibrous adhesion formation of TMJ was examined using TNC knockout (TNCKO) mice. Hypermobility was produced by excessive mouth opening method on the TMJ of both wild-type (WT) and TNCKO mice. TMJ wound healing was compared histologically, and the expression of TNC, fibronectin (FN), and α-smooth muscle actin (α-SMA) in the wounded TMJ was examined by immunohistochemical and immunoblot analyses. Based on histologic analysis, fibrous adhesions were observed in the TMJ of both TNCKO and wild-type (WT) mice after excessive mouth opening. However, fibrous adhesion formation in TNCKO mice occurred later than in WT mice. TNC was expressed in the wounded TMJ disc and mandibular fossa. Although FN and α-SMA expression in the TMJ of TNCKO and WT mice was up-regulated after excessive mouth opening, FN and α-SMA protein levels were higher in WT mice at the same time points. In the wounded TMJ, TNC appears to enhance the expression of FN and α-SMA, and a lack of TNC may reduce fibrous adhesion formation in the TMJ. TNC plays an important role in TMJ wound healing, especially for wounds generated by mechanical stress.Key words: Temporomandibular joint, excessive mouth opening, fibrous adhesion, tenascin-C, fibronectin, α-smooth muscle actin     

Tumor necrosis factor-alpha in temporomandibular joint synovial fluid predicts treatment effects on pain by intra-articular glucocorticoid treatment

The aim of this study was to investigate the influence of tumor necrosis factor-alpha (TNF-alpha) in temporomandibular joint (TMJ) synovial fluid and blood on the treatment effect on TMJ pain by intra-articular injection of glucocorticoid in patients with chronic inflammatory TMJ disorders. High pretreatment level of TNF-alpha in the synovial fluid was associated with a decrease of TNF-alpha and elimination of pain upon maximal mouth opening. Elimination of this TMJ pain was accordingly associated with decrease in synovial fluid level of TNF-alpha. There was also a significant decrease of C-reactive protein and TMJ resting pain after treatment. In conclusion, this study indicates that presence of TNF-alpha in the synovial fluid predicts a treatment effect of intra-articular injection of glucocorticoid on TMJ movement pain in patients with chronic TMJ inflammatory disorders.     

Prevalence of TMJ Signs and Symptoms in the Elderly

Investigations of temporomandibular joint (TMJ) disorders have included assessment of the prevalence of signs such as joint clicking, limitation of jaw opening, and deviation of the mandible on function, and symptoms such as pain on function in patient and non-patient populations. The former (signs) might be viewed as the prevalence of TMJ “disease”, and the latter (symptom presentation) might be viewed as TMJ “illness behavior.” The majority of the reports profiling TMJ patients suggest that most are females (80%) in the 20–40 year age group. Reports of the relationship of TMJ signs and symptoms to age have provided inconsistent findings, i.e. signs and symptoms have been found to increase with age, to decrease with age, or to be unrelated to age. Many of the inconsistencies reflect differences in the populations studied and lack of standardized criteria for diagnosing mandibular function/dysfunction. This study assessed the prevalence of the signs and symptoms of TMJ disorders in community-dwelling, non-health care seeking participants in the Baltimore Longitudinal Study of Aging, the intramural longitudinal aging study of the National Institute on Aging. The only age difference noted was in limitation of jaw opening among the older subjects.     

A new technique to demonstrate flow limitation in partial expiratory flow-volume curves in infants

Partial expiratory flow-volume (PEFV) curves in infants are generated by applying a compressive pressure over the chest wall with an inflatable jacket. This study addresses two issues: pressure transmission to and across the chest wall and whether flow limitation can be identified. Eleven infants sedated with chloral hydrate were studied. Pressure transmission to the chest wall, measured with neonatal blood pressure cuffs placed on the infant's body surface, was 72 +/- 4% of jacket pressure during compression maneuvers. The pressure transmission to the air spaces, determined by measuring airway pressure during a compression maneuver against an occluded airway, was 56 +/- 6% of jacket pressure. A significant amount of the applied pressure is therefore lost across both the jacket and chest wall. Rapid pressure oscillations (RPO) were superimposed on static jacket pressures while expiratory flow was measured. Absence of associated oscillations of flow measured at the mouth was taken to indicate that flow was independent of driving pressure and therefore limited. Flow limitation was demonstrable with the RPO technique in all infants for jacket pressures greater than 50 cmH2O; however, it was evident at jacket pressures less than 30 cmH2O jacket pressure in four infants with obstructive airway disease. The RPO technique is a useful adjunct to the compression maneuver utilized to generate PEFV curves in infants because it facilitates the recognition of expiratory flow limitation.     

Proteomic signature of Temporomandibular Joint Disorders (TMD): Toward diagnostically predictive biomarkers

The temporomandibular joint (TMJ) articulates the mandible with the maxilla. Temporomandibular joint disorders (TMD) are dysfunctions of this joint, which range from acute to chronic inflammation, trauma and dislocations, developmental anomalies and neoplasia. TMD manifest as signs and symptoms that involve the surrounding muscles, ligaments, bones, synovial capsule, connective tissue, teeth and innervations proximal and distal to this joint. TMD induce proximal and distal, chronic and acute, dull or intense pain and discomfort, muscle spasm, clicking/popping sounds upon opening and closing of the mouth, and chewing or speaking difficulties. The trigeminal cranial nerve V, and its branches provide the primary sensory innervation to the TMJ. Our clinical work suggests that the auriculotemporal (AT) nerve, a branch of the mandibular nerve, the largest of the three divisions of the trigeminal nerve, plays a critical role in TMD sequelae. The AT nerve provides the somatosensory fibers that supply the joint, the middle ear, and the temporal region. By projecting fibers toward the otic ganglion, the AT nerve establishes an important bridge to the sympathetic system. As it courses posteriorly to the condylar head of the TMJ, compression, injury or irritation of the AT nerve can lead to significant neurologic and neuro-muscular disorders, including Tourette's syndrome,Torticolli, gait or balance disorders and Parkinson's disease. Here, we propose that a proteomic signature of TMD can be obtained by assessing certain biomarkers in local (e.g., synovial fluid at the joint) and distal body fluids (e.g., saliva, cerebrospinal fluid), which can aid TMD diagnosis and prognosis.     

Immediate effects of the serotonin antagonist granisetron on temporomandibular joint pain in patients with systemic inflammatory disorders

The aim of this study was to investigate if the 5-HT3 antagonist granisetron reduces temporomandibular joint (TMJ) pain in patients with systemic inflammatory joint disorders. Sixteen patients with systemic inflammatory joint disease with pain localized over the TMJ region and tenderness to digital palpation of the TMJ were included. The current resting pain (VASRest) and the pain during maximum mouth opening (VAS(MVM)) of the TMJs were assessed with a 100 mm visual analogue scale. An electronic pressure algometer was used to estimate the pressure pain threshold (PPT) over the lateral aspect of the TMJ. Venous blood was collected for measurement of the plasma and serum levels of 5-HT, erythrocyte sedimentation rate, rheumatoid factor and C-reactive protein. The selective 5-HT3 receptor antagonist granisetron or saline were injected into the posterior part of the upper TMJ compartment in a randomized double-blind manner. The patients in the granisetron group had lower VASRest than the patients in the saline group after 10 min. In the granisetron group, VASRest was decreased after 10 min, while VAS(MVM) was decreased and PPT increased after 20 min. In the saline group, VAS(MVM) was decreased after 20 min. In conclusion, granisetron has an immediate, short-lasting and specific pain reducing effect in TMJ inflammatory arthritis. The 5-HT3 receptor may therefore be involved in the mediation of TMJ pain in systemic inflammatory joint disorders.     

Proteomic signature of Temporomandibular Joint Disorders (TMD): Toward diagnostically predictive biomarkers

The temporomandibular joint (TMJ) articulates the mandible with the maxilla. Temporomandibular joint disorders (TMD) are dysfunctions of this joint, which range from acute to chronic inflammation, trauma and dislocations, developmental anomalies and neoplasia. TMD manifest as signs and symptoms that involve the surrounding muscles, ligaments, bones, synovial capsule, connective tissue, teeth and innervations proximal and distal to this joint. TMD induce proximal and distal, chronic and acute, dull or intense pain and discomfort, muscle spasm, clicking/popping sounds upon opening and closing of the mouth, and chewing or speaking difficulties. The trigeminal cranial nerve V, and its branches provide the primary sensory innervation to the TMJ. Our clinical work suggests that the auriculotemporal (AT) nerve, a branch of the mandibular nerve, the largest of the three divisions of the trigeminal nerve, plays a critical role in TMD sequelae. The AT nerve provides the somatosensory fibers that supply the joint, the middle ear, and the temporal region. By projecting fibers toward the otic ganglion, the AT nerve establishes an important bridge to the sympathetic system. As it courses posteriorly to the condylar head of the TMJ, compression, injury or irritation of the AT nerve can lead to significant neurologic and neuro-muscular disorders, including Tourette''s syndrome,Torticolli, gait or balance disorders and Parkinson’s disease. Here, we propose that a proteomic signature of TMD can be obtained by assessing certain biomarkers in local (e.g., synovial fluid at the joint) and distal body fluids (e.g., saliva, cerebrospinal fluid), which can aid TMD diagnosis and prognosis.     

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

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

Expression and localization of versican during postnatal development of rat temporomandibular joint disc

To analyze the growth-related changes in extracellular matrix components in temporomandibular joint (TMJ) discs, the expression and localization of the core protein of a large chondroitin sulphate proteoglycan, versican, in rat TMJ discs during postnatal development (2–32 weeks) were examined using Western blot analysis, real-time quantitative PCR and immunohistochemistry. Western blot analysis showed that rat TMJ discs predominantly expressed one isoform (V1) and the core protein sharply increased after birth, reached a peak at 8 weeks, and then gradually decreased up to 32 weeks. Real-time quantitative PCR with TaqMan probes indicated that mRNA expression of versican was highest at 2 weeks and gradually decreased with growth. An immunohistochemical study showed that staining for versican was weak and evenly distributed in TMJ discs at 2 weeks. Regional differences in staining for versican became prominent after 8 weeks; staining was intense in the anterior and posterior peripheral attachments, and weak in the central part of the discs. These results demonstrate that growth-related changes and regional differences exist in the expression of versican in the TMJ discs of growing rats, and these probably reflect the changes in the biomechanical environment caused by the development of orofacial functions.     

Testosterone and estrogen have opposing actions on inflammation-induced plasma extravasation in the rat temporomandibular joint

The present study was designed to test the hypothesis that estrogen exacerbates inflammation of the temporomandibular joint (TMJ). Evans blue dye was used to quantify plasma extravasation (PE) around the rat TMJ. In an initial set of experiments, TMJ PE was compared in na?ve intact male and female rats, as well as in both groups after complete Freund's adjuvant (CFA)-induced inflammation of the TMJ. In contrast to our hypothesis, TMJ PE was significantly greater in both na?ve and CFA-inflamed male rats than in females. To determine whether these differences were due to gonadal hormones, four additional groups of rats were studied: gonadectomized (Gx) males and females, Gx males with chronic testosterone (T) replacement, and Gx females with chronic estrogen (E) replacement. The sex difference in baseline TMJ PE appeared to reflect the actions of T. However, in the presence of TMJ inflammation, T augmented TMJ PE in males, while E attenuated TMJ PE in females. Changes in PE were also assessed in the contralateral TMJ. Results from this analysis indicated that there is a transient contralateral increase in TMJ PE in females but not males. Given that there is an inverse relationship between PE and joint damage, our results suggest that testosterone may mitigate, but estrogen may exacerbate, TMJ damage, particularly in the presence of overt inflammation. Importantly, our results may help explain both the higher prevalence and severity of temporomandibular disorder pain in females than males.     

mu-Opioid receptor mRNA expression and immunohistochemical localization in the rat temporomandibular joint

This study was undertaken to examine the presence and distribution of the mu-opioid receptor (MOR) in the non-inflamed rat temporomandibular joint (TMJ) using non-radiographic in situ hybridization at the mRNA level and immunohistochemistry at the protein level. MOR mRNA and MOR-like immunoreactivity (MOR-LI) were found around the small blood vessels in the anterior part of the synovial membrane. The number of MOR mRNA signals in the anterior synovial membrane was significantly higher than that in the posterior part. Morphologically, MOR mRNA and MOR-LI were localized in amorphous materials considered to be nervous tissue, as well as some cell types considered to be macrophages, mast cells and endothelial cells. The present study showed the distribution of MOR in the rat TMJ synovial membrane and suggests that the opiate system plays an important role in endogenous analgesia in the TMJ.     

Antinociceptive and anti-inflammatory effects of electroacupuncture on experimental arthritis of the rat temporomandibular joint

This study investigated the antinociceptive and anti-inflammatory effects of electroacupuncture (EA) on zymosan-induced acute arthritis of the rat temporomandibular joint (TMJ). Male Wistar rats were injected with saline or zymosan (control group; 2?mg) into the left TMJ. Low frequency EA (10?Hz, 30?min) was performed at acupoints (LI4, LI11, ST36, ST44) or sham points 2?h after or 1?h before zymosan administration. Mechanical hypernociception was accessed by the electronic Von Frey method after zymosan administration. Rats were sacrificed 6?h after zymosan administration and the joint was removed for histopathological analysis, myeloperoxidase activity assessment, vascular permeability observations, and immunohistochemical verification of inflammatory mediators. The results showed that EA inhibited zymosan-induced hypernociception, compared with the control group and with the sham group (p?< 0.05). The results showed that EA inhibited inflammatory parameters such as neutrophil migration, vascular permeability, and tumour necrosis factor α (TNF-α), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) expression in the TMJ compared with the sham group (p < 0.05). Histopathological analysis showed that EA significantly inhibited edema and periarticular infiltration (p?< 0.05) compared with the control and sham groups. EA at acupoints produced antinociceptive and anti-inflammatory effects on zymosan-induced arthritis in the rat TMJ.     

Phenotypic and genetic trends of patellar luxation in Dutch Flat‐Coated Retrievers

Canine patellar luxation has been described in various dog breeds, with high prevalence especially in smaller dogs. Most dogs suffer from medial displacement of the patella, although in larger dogs lateral displacement is also seen. A sex predisposition has been described for females. Patellar luxation is considered a polygenic, multifactorial disorder. From 1990 to 2007, in total 3834 Flat‐Coated Retrievers were screened; 23.6% of those animals were affected with patellar luxation. Lateral displacement of the patella was most common in this breed (61% of cases), whereas medial (31% of cases) and lateral and medial (8% of cases) were less common. Unilateral involvement (51% of cases) was just as often observed as was bilateral involvement (49% of cases). Females were more often affected with patellar luxation (30% of all tested females) than were males (17% of all tested males). The heritability of patellar luxation was 0.17 ± 0.03 in this population, and breeding with one affected parent increased the prevalence of patellar luxation in offspring by 45% compared to that with two unaffected parents. Since the start of the screening program, there was an initial decrease from 28% to 18% in incidence, but this stagnated thereafter. The annual average estimated breeding values followed the same pattern. With approximately one quarter of the Dutch Flat‐Coated Retrievers being affected with patellar luxation, this population shows unusually high prevalence compared with reports in other large‐breed dogs. The heritability for patellar luxation in this population was moderate (0.17), indicating that environmental factors play a large role in the manifestation of the disorder. A screening program reduced the prevalence of patellar luxation in this breed, but improvement has recently stagnated. Inclusion of breeding values in the screening program could improve its effectiveness.     

Immunohistochemical localization of phosphatidylcholine in rat mandibular condylar surface and lower joint cavity by cryotechniques

The immunolocalization of phospholipids has not yet been clearly demonstrated in temporomandibular joints (TMJs). We have examined the distribution of one of phospholipids, phosphatidyl-choline (PC), in the rat mandibular condylar surface and lower joint cavity. Some fresh resected TMJs with their disks attached were immediately plunged into isopentane-propane cryogen (-193 degrees C). Cryostat sections were cut, mounted on NH3+-coated slides, and fixed with paraformaldehyde (PF). Cryosections were first immunostained with anti-mouse PC antibody (JE-1). Subsequently, they were labeled with immunogold particles following silver enhancing for light microscopic analyses. Some cryosections were subjected to double immunofluoresecence labeling with anti-fibronectin antibody or hyaluronic acid-binding protein in combination with the anti-PC antibody. As an immunocontrol, other cryosections were pretreated with phospholipase A2 before such immunofluorescence labeling. We have confirmed the presence of PC in the lower joint cavity of rat TMJs as well as on the mandibular condylar surface layer, which was colocalized with hyaluronic acid and fibronectin respectively. However, by treatment with phospholipase A2, such immunolabeling for PC was clearly decreased, showing that the PC is a component in the rat in vivo TMJ. These findings suggest that PC, hyaluronic acid and fibronectin may interact each other in the TMJ articular surface areas to play a functional role for lubrication in TMJ.     

Effect of Interleukin-1beta and Dehydroepiandrosterone on the Expression of Lumican and Fibromodulin in Fibroblast-Like Synovial Cells of the Human Temporomandibular Joint

Several epidemiological studies have reported that temporomandibular disorders (TMDs) are more prevalent in women than in men. It has recently been proposed that sex hormones such as estrogen, testosterone and dehydroepiandrosterone (DHEA) are involved with the pathogenesis of TMDs. Although studies have investigated the relationship between estrogen and testosterone and the restoration of TMDs, the relationship between DHEA and TMDs is unknown. The synovial tissue of the temporomandibular joint (TMJ) is made up of connective tissue with an extracellular matrix (ECM) composed of collagen and proteoglycan. One proteoglycan family, comprised of small leucine-rich repeat proteoglycans (SLRPs), was found to be involved in collagen fibril formation and interaction. In recent years, the participation of SLRPs such as lumican and fibromodulin in the internal derangement of TMJ has been suggested. Although these SLRPs may contribute to the restoration of the synovium, their effect is still unclear. The purpose of this study was to investigate the effect of DHEA, a sex hormone, on the expression of lumican and fibromodulin in human temporomandibular specimens and in cultured human TMJ fibroblast-like synovial cells in the presence or absence of the pro-inflammatory cytokine interleukin-1beta (IL-1beta). In the in vivo study, both normal and osteoarthritic (OA) human temporomandibular synovial tissues were immunohistochemically examined. In the in vitro study, five fibroblast-like synoviocyte (FLS) cell lines were established from human TMJ synovial tissue of patients with osteoarthritis. The subcultured cells were then incubated for 3, 6, 12 or 24 h with/without IL-1beta (1 ng/mL) in the presence or absence of DHEA (10 μM). The gene expression of lumican and fibromodulin was examined using the real-time polymerase chain reaction (PCR) and their protein expression was examined using immunofluorescent staining. We demonstrated that the expression of lumican differs from that of fibromodulin in synovial tissue and furthermore, that IL-1beta induced a significant increase in lumican mRNA and immunofluorescent staining in FLS compared to cells without IL-1beta. DHEA plus IL-1beta induced a significant increase in fibromodulin, but not in lumican mRNA, compared to DHEA alone, IL-1beta alone and in the absence of DHEA and IL-1beta. In immunofluorescent staining, weaker fibromodulin staining of FLS cells was observed in cells cultured in the absence of both DHEA and IL-1beta compared to fibromodulin staining of cells cultured with DHEA alone, with DHEA plus IL-1beta, or with IL-1beta alone. These results indicate that DHEA may have a protective effect on synovial tissue in TMJ by enhancing fibromodulin formation after IL-1beta induced inflammation. DHEA enhancement of fibromodulin expression may also exert a protective effect against the hyperplasia of fibrous tissue that TGF-beta1 induces. In addition lumican and fibromodulin are differentially expressed under different cell stimulation conditions and lumican and fibromodulin may promote regeneration of the TMJ after degeneration and deformation induced by IL-1beta.Key words: Temporomandibular joint, dehydroepiandrosterone, lumican, fibromodulin, small leucine rich repeat proteoglycan     

Blood serotonin and joint pain in seropositive versus seronegative rheumatoid arthritis

AIM: To investigate whether blood serotonin (5-hydroxytryptamine) (5-HT) modulates musculoskeletal pain differently in seropositive and seronegative rheumatoid arthritis (RA). METHODS: Patients with temporomandibular joint (TMJ) involvement of seropositive RA (33 patients) or seronegative RA (28 patients) and 26 healthy individuals were included. TMJ pain, general musculoskeletal pain, plasma and serum 5-HT, acute phase reactants and thrombocyte count were investigated. RESULTS: The patients with seropositive RA had higher serum (median = 1130 nmol/l) and plasma (55 nmol/l) levels of 5-HT than the healthy individuals (704 nmol/l, p = 0.044 and 23 nmol/l, p < 0.001, respectively), and higher plasma levels of 5-HT than the seronegative patients (14 nmol/l, p < 0.001). There was no significant correlation between serum and plasma levels of 5-HT in any group. In the seropositive RA patients, positive correlations were found between serum levels of 5-HT and the number of painful mandibular movements (r(s) = 0.36, n = 33, p = 0.042), as well as pain on maximum mouth opening (r(s) = 0.41, n = 24, p = 0.047) and tenderness to digital palpation (r(s) = 0.49, n = 33, p = 0.003). In the healthy individuals, there was a negative correlation between plasma level of 5-HT and the TMJ pressure pain threshold (r(s) = -0.47, n = 20, p = 0.037). CONCLUSION: Peripheral serotonergic pain mechanisms seem to be activated by blood 5-HT in patients with seropositive RA, in contrast to seronegative patients.     

Morphometric study of the mandibular condyle of the rat during postnatal development

A morphometric technique for the use of the temporomandibular joint (TMJ) of the rat as an experimental model was developed to study TMJ growth from birth to 120 days of age. Experimental conditions for the reduction and embedding of the specimen, as well as for data collection were standardized. Morphometric data were obtained from projected drawings and the areas occupied by the various structures were determined by counting points with a specially constructed integration grid. The areas occupied by the layers making up the mandibular condyle remained relatively constant, forming an architectural pattern from the 30th postnatal day on. Between the 10th and 30th day they underwent modifications interpreted to be functional adaptations conditioned by changes in the animal's feeding habits.     

Expression of Pituitary Adenylate Cyclase-Activating Peptide (PACAP) and PAC1 in the Periodontal Ligament After Tooth Luxation

Pituitary adenylate cyclase-activating peptide (PACAP) is widely distributed throughout the nervous system. PACAP not only acts as a neurotransmitter but also elicits a broad spectrum of biological action via the PACAP-specific receptor, PAC1. However, no studies have investigated PACAP and PAC1 in the periodontal ligament (PDL), so we aimed to perform this investigation in rats after tooth luxation. In the PDL of an intact first molar, there are few osteoclasts and osteoblasts. However, at days 3 and 5 after luxation, large PAC1-positive cells, thought to be osteoclasts because of their expression of the osteoclast marker, tartrate-resistant acid phosphatase, were detected in appreciable numbers. Osteoblast numbers increased dramatically on day 7 after luxation, and PAC1-positive mononuclear small cells were increased at day 14, many of which expressed the osteoblast marker, alkaline phosphatase. PACAP-positive nerve fibers were rarely detected in the PDL of intact first molars, but were increasingly evident at this site on days 5 and 7 after luxation. Double-immunofluorescence analysis demonstrated the relationship between PACAP-positive nerve fibers and PAC1-positive osteoclasts/-blasts in the PDL. At 5 days after luxation, PACAP-positive nerve fibers appeared in close proximity to PAC1-positive osteoclasts. At 7 days after luxation, PACAP-positive nerve fibers appeared in close proximity to PAC1-positive osteoblasts. These results suggest that PACAP may have effects on osteoclasts and osteoblasts in the PDL after tooth luxation and thus regulate bone remodeling after these types of injury.     

Enhancement of chondrocyte autophagy is an early response in the degenerative cartilage of the temporomandibular joint to biomechanical dental stimulation

Autophagy is a cell protective mechanism for maintaining cellular homeostasis. The present study aimed to investigate whether autophagy is enhanced in the biomechanically induced degenerative cartilage of the temporomandibular joint (TMJ) and the potential role of mitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3) and mammalian Target of rapamycin (mTOR) in this observation. To induce degenerative changes in the TMJs, rats were subjected to biomechanical dental stimulation by moving 4 molars away from their original position as we previously reported. The ultrastructure of autophagosome was observed by transmission electron microscopy. The number of lysosomes was analyzed by flow cytometry. The expression levels of Beclin1 and LC3 and the involvement of MAP4K3 activity were detected by immunohistochemistry, real-time PCR and western blot. The activity of the mTOR pathway indicated by p-mTOR and p-p70S6 K was assayed by western blot. TMJ degeneration, characterized by irregular cell arrangement and cell-free area, was induced in the experimental groups. Under transmission electron microscopy, we observed the presence of autophagosomes, small patches of condensed chromatin, abundant rough endoplasmic reticulum and Golgi apparatus. The number of lysosomes and the expression levels of Beclin1 and LC3 increased, while the activity of mTOR and the expression level of MAP4K3 decreased in the experimental groups. Cartilage in TMJ which was induced to be degenerative biomechanically exhibited autophagy accompanied by reduced mTOR and MAP4K3 activity.