Temporomandibular inflammation regulates the matrix metalloproteinases MMP-2 and MMP-9 in limbic structures.

Temporomandibular disorder (TMD) is characterized by acute or chronic orofacial pain, which can be associated with inflammatory processes in the temporomandibular joint (TMJ) and emotional disorders. Peripheral and central sensitization in painful orofacial processes is common, and it can be triggered by peripheral inflammatory challenge with consequent neuroinflammation phenomena. Such neuroinflammation comes from inflammatory products from supportive cells, blood-brain barrier, and extracellular matrix. Here, we evaluated the possible recruitment of limbic structures for modified matrix metalloproteinases (MMPs) expression and activity during temporomandibular inflammation-induced orofacial persistent pain. The inflammatory process in TMJs of rats was induced by Freund's Complete Adjuvant (CFA) administration. The activity and expression of MMPs-2 and 9 were assessed by in situ zymography and conventional zymography, respectively. A glial colocalization with the MMPs was performed using immunofluorescence. The results evidenced both short- and long-term alterations on MMP-2 and -9 expression in the limbic structures following CFA-induced temporomandibular inflammation. The gelatinolytic activity was increased in the central amygdala, hippocampus, hypothalamus, ventrolateral periaqueductal gray (vlPAG), superior colliculus, and inferior colliculus. Finally, an increase of colocalization of MMP-2/GFAP and MMP-9/GFAP in CFA-induced inflammation groups was observed when compared with saline groups in the central amygdala and vlPAG. It is possible to suggest that glial activation is partly responsible for the production of gelatinases in the persistent orofacial pain, and it is involved in the initiation and maintenance of this process, indicating that inhibition of MMPs might be pursued as a potential new therapeutic target for TMD.

Effect of psychological stress on the structure of the temporomandibular joint and the expression of MMP-3 and TIMP-3 in the cartilage in rats.

Our aim was to observe the effects of psychological stress on the structure of the temporomandibular joint (TMJ), and to evaluate the expression of matrix metallopeptidase-3 (MMP-3) and tissue inhibitor of metalloproteinase-3 (TIMP-3) in condylar chondrocytes in rats. The rats were divided into 3 groups of 12 according to the duration of psychological stress: 3 weeks or 6 weeks, and 6 weeks of recovery. A fourth group of 12 rats was used as controls. Each rat was evaluated by the open-field test and the weight measured. The results confirmed psychological stress in 24 of the 36 rats (67%). The tissues of the TMJ were stained with haematoxylin and eosin and pathological changes were studied under a light microscope. MMP-3 and TIMP-3 expression was investigated using the SP kit. The experimental groups showed thinning of articular cartilage, shedding of collagen fibres, cracks in the articular discs, and other structural changes that were aggravated with time, from three weeks to six weeks. The 6-week recovery group showed an improvement in these changes, which indicated the initiation of joint repair. The MMP-3 expression rate correlated with the degree of joint lesion, while the TIMP-3 rate showed an opposite trend and was highest in the 6-week recovery group. Our findings clearly indicate that psychological stress may play an important part in the development of TMJ diseases in rats; further studies should be made to extrapolate the results to other models before clinical use.

Upregulation of cystathionine-ß-synthetase expression contributes to inflammatory pain in rat temporomandibular joint.

BACKGROUND: Hydrogen sulfide (H2S), an endogenous gaseotransmitter/modulator, is becoming appreciated that it may be involved in a wide variety of processes including inflammation and nociception. However, the role for H2S in nociceptive processing in trigeminal ganglion (TG) neuron remains unknown. The aim of this study was designed to investigate whether endogenous H2S synthesizing enzyme cystathionine-ß-synthetase (CBS) plays a role in inflammatory pain in temporomandibular joint (TMJ). METHODS: TMJ inflammatory pain was induced by injection of complete Freund's adjuvant (CFA) into TMJ of adult male rats. Von Frey filaments were used to examine pain behavioral responses in rats following injection of CFA or normal saline (NS). Whole cell patch clamp recordings were employed on acutely isolated TG neurons from rats 2 days after CFA injection. Western blot analysis was carried out to measure protein expression in TGs. RESULTS: Injection of CFA into TMJ produced a time dependent hyperalgesia as evidenced by reduced escape threshold in rats responding to VFF stimulation. The reduced escape threshold was partially reversed by injection of O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA), an inhibitor for CBS, in a dose-dependent manner. CFA injection led to a marked upregulation of CBS expression when compared with age-matched controls. CFA injection enhanced neuronal excitability as evidenced by depolarization of resting membrane potentials, reduction in rheobase, and an increase in number of action potentials evoked by 2 and 3 times rheobase current stimulation and by a ramp current stimulation of TG neurons innervating the TMJ area. CFA injection also led to a reduction of IK but not IA current density of TG neurons. Application of AOAA in TMJ area reduced the production of H2S in TGs and reversed the enhanced neural hyperexcitability and increased the IK currents of TG neurons. CONCLUSION: These data together with our previous report indicate that endogenous H2S generating enzyme CBS plays an important role in TMJ inflammation, which is likely mediated by inhibition of IK currents, thus identifying a specific molecular mechanism underlying pain and sensitization in TMJ inflammation.

Influence of sleep deprivation on expression of MKK4 and c-fos in the mandibular condylar cartilage of rats.

The aim of this study was to investigate the changes in expression of mitogen-activated protein kinase kinase 4 (MKK4) and c-fos in the mandibular condylar cartilage of rats that had been subjected to sleep deprivation. One hundred and twenty female Wistar rats were randomly divided into 6 groups with 20 in each: sleep deprivation for 2 days, 4 days, 6 days, and 8 days, large-platform controls, and cage controls. After sleep deprivation by the modified multiple platform method the sleep-deprived rats were killed. The large-platform and cage control rats were killed at the same time as the rats deprived of sleep for 8 days. Haematoxylin and eosin were used to record the morphological changes in cartilage, and immunohistochemistry and real-time quantitative polymerase chain reaction (PCR) were used to detect the expression of MKK4 and c-fos. Pathological alterations were apparent after 6 and 8 days of sleep deprivation. Compared with control groups, the expression of MKK4 in the sleep-deprived groups was lower, while that of c-fos was higher. As the duration of sleep deprivation increased, the expression of MKK4 decreased. These results indicate that the variation in expression of MKK4 and c-fos may be correlated with pathological changes induced by sleep deprivation in mandibular condylar cartilage in rats.

Sulfotransferase Ndst1 is needed for mandibular and TMJ development.

Heparan sulfate proteoglycans (HS-PGs) regulate several developmental processes, but their possible roles in mandibular and TMJ formation are largely unclear. To uncover such roles, we generated mice lacking Golgi-associated N-sulfotransferase 1 (Ndst1) that catalyzes sulfation of HS-PG glycosaminoglycan chains. Ndst1-null mouse embryos exhibited different degrees of phenotypic penetrance. Severely affected mutants lacked the temporomandibular joint and condyle, but had a mandibular remnant that displayed abnormal tooth germs, substandard angiogenesis, and enhanced apoptosis. In mildly affected mutants, the condylar growth plate was dysfunctional and exhibited thicker superficial and polymorphic cell zones, a much wider distribution of Indian hedgehog signaling activity, and ectopic ossification along its lateral border. Interestingly, mildly affected mutants also exhibited facial asymmetry resembling that seen in individuals with hemifacial microsomia. Our findings indicate that Ndst1-dependent HS sulfation is critical for mandibular and TMJ development and allows HS-PGs to exert their roles via regulation of Ihh signaling topography and action.

Masseter muscular weakness affects temporomandibular synovitis induced by jaw opening in growing rats.

OBJECTIVE: To evaluate the influence of impaired masseter function during growth on the development of temporomandibular synovitis. MATERIALS AND METHODS: Sixteen 3-week-old male Wistar rats were classified into four groups. The first group served as control; and in the second group, jaw opening was forced for 3 hours when the rats were 9 weeks old. In the third and fourth groups, the masseter muscles were bilaterally resected at 3 weeks of age, and the rats in the fourth group were additionally forced to open their jaw at 9 weeks of age. All rats were sacrificed at 9 weeks. Temporomandibular joint (TMJ) tissue samples were processed for histology, and evaluated for cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expressions by immunohistochemistry to examine the inflammatory changes in the synovial membrane. RESULTS: The control group showed noninflammatory changes. In the jaw-opening group, vascular dilation and weak COX-2 immunoreactivity were induced by jaw opening in the synovium. In the masseter-resection group, the masseter-resected rats exhibited moderate synovial changes while in the resection with opening group, the masseter-resected rats revealed more significant inflammatory changes including synovial hyperplasia, dilated vasculature, fibrin deposits, and intense immunoreactivity for COX-2 and iNOS, all caused by jaw opening. CONCLUSIONS: These results suggest that masseter activity in the growth period is an important factor in the induction of temporomandibular synovitis.

Superoxide dismutase activity in synovial fluids in patients with temporomandibular joint internal derangement.

PURPOSE: To measure the activity of superoxide dismutase (SOD) in the synovial fluid of patients with temporomandibular joint internal derangement and to show the relationship between the activity of SOD and the severity of the disease. MATERIALS AND METHODS: Twenty patients with internal derangement were classified according to Wilkes by clinical radiological examinations. SOD activity was measured by the method based on nitrobluetetrazolium reduction rate. RESULTS: The activity of SOD seemed to be progressively decreased as the stage of the disease increased. CONCLUSION: The reduction of SOD activity observed may result from insufficient scavenging capacity of free radicals. Further investigation and longitudinal studies are required to determine the role of antioxidants that scavenge the free radicals in temporomandibular joint disorders.

Dietary loading and aggrecanase-1/TIMP-3 expression in rat mandibular condylar cartilage.

AIMS: To examine the expression of aggrecanase-1 and a tissue inhibitor of metalloproteinases (TIMP-3) in the condylar cartilage of young rats and to determine their relationship during altered dietary loading at different time points after weaning. METHODS: One hundred Sprague-Dawley rats were randomly assigned to 1 of 2 groups: the soft-diet group, which served as the control group (n=50), or the hard-diet group, which served as the experimental group (n=50). Ten soft- and 10 hard-diet rats were killed at 6 hours, 12 hours, 24 hours, 48 hours, and 9 days after weaning (i.e., after initiation of diet change for hard-diet rats). The right-side temporomandibular joints (TMJs) were prepared for immunohistochemical staining. The cartilage from the left-side mandibular condyles of all 10 animals in each group was combined for Western blot analysis. RESULTS: Immunohistochemical analysis revealed strong staining for aggrecanase-1 localized mainly in the chondrocytes of proliferative and upper hypertrophic cartilage zones at all time points in both groups. The immunohistological expression of aggrecanase-1 was significantly higher in the hard-diet group at 12 and 24 hours than in the soft-diet group. Strong staining for TIMP-3 was mainly localized in the chondrocytes of proliferative and upper hypertrophic zones at all time points in both groups. The expression of TIMP-3 in the hard-diet group was at a significantly lower level compared to the soft-diet group at 6 hours. Western blot analysis also showed time-related differences in aggrecanase-1 and TIMP-3, but there was no significant difference between the 2 groups. CONCLUSION: The temporary change in aggrecanase-1 and TIMP-3 expression reflects the complex interaction of these enzymes in the physiologic range and cartilage response to altered dietary loading.

Influence of estrogen cycle on temporomandibular joint synovial membrane in rat with deviated mandible.

Temporomandibular disorders (TMD) are known to be more prevalent and severe in women than in men, especially in those who are in their reproductive age. In those patients reproductive hormones may play a vital role in the host adaptive capacity of the temporomandibular joint (TMJ). In order to clarify the relationship between TMD prevalence and estrogen cycle, a mandible deviated animal model was carried out, and the expression of inducible nitric oxide synthase (iNOS), an essential enzyme in the pathogenesis of inflammatory arthritis, was investigated in the rat's synovial tissue. An appliance was attached to the rat's incisors to produce a lateral deviation of the mandible during the metestrus phase, and the animals were sacrificed in the proestrus and estrus phase, when the estrogen was at the highest and lowest level, respectively. Immunostaining was then performed for 2 consecutive estrous cycles to demonstrate iNOS expression in the synovial membrane of the TMJ. The immunoreactivity for iNOS was more intense in the synovial membrane on the contralateral side in the proestrus phase (estrogen peak phase). These observations suggest that iNOS expression in the synovial membrane with mandibular deviation may be exacerbated in the presence of estrogen.

The effects of age and sex on the expression of aromatase in the rat temporomandibular joint.

AIMS: To investigate the expression of aromatase in temporomandibular joints(TMJs) of male and female rats of different ages. METHODS: The expression of aromatase in terms of protein and mRNA was examined by immunohistochemistry, Western blot, and in situ hybridization in the TMJs of Sprague-Dawley rats. The expression level of aromatase protein was also quantified by the density of aromatase-positive chondrocytes in TMJ condylar cartilage. A tritiated water assay was used to analyze the activity of aromatase in cartilage chondrocytes. RESULTS: Strong aromatase protein and mRNA hybridization signals were detected in hypertrophic and mature chondrocytes in all the cases examined. The density of aromatase-positive chondrocytes was relatively stable at higher levels before 8 weeks of age. The density decreased gradually in females after 8 weeks of age but not in males. CONCLUSION: These results demonstrate that aromatase is expressed intensely in condylar cartilage and suggest that it may play an important role in pathophysiological mechanisms in condylar cartilage.

Regulation of matrix metalloproteinase expression by dynamic tensile strain in rat fibrochondrocytes.

OBJECTIVE: We sought to determine the molecular basis for the anticatabolic effects of mechanical signals on fibrocartilage cells by studying the expression of a variety of matrix metalloproteinases (MMPs). Furthermore, we examined whether the effects of biomechanical strain on MMP gene expression are sustained. METHODS: Fibrochondrocytes from temporomandibular joint (TMJ) discs were exposed to dynamic tensile strain for various time intervals in the presence of interleukin (IL)-1beta. The regulation of the messenger RNA (mRNA) expression and synthesis of MMPs and tissue inhibitors of MMPs (TIMPs) were examined by end-point and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) as well as Western blot analysis. RESULTS: Fibrochondrocytes expressed mRNA for MMP-2, -3, -7, -8, -9, -11, -13, -14, -16, -17, and -19 as well as TIMP-1, -2, and -3, IL-1beta induced a significant (P<0.05) upregulation of mRNA for MMP-3, -7, -8, -9, -13, -16, -17, and -19. The IL-1beta-stimulated upregulation of these MMPs was significantly (P<0.05) abrogated by dynamic tensile strain. However, MMP-2, -11, -14, and TIMPs were not affected by either IL-1beta or tensile strain. Biomechanical strain also inhibited the IL-1beta-stimulated protein synthesis of MMP-3, -7, -8, -9, -13, -16, and -17. Application of mechanical strain for various time intervals during a 24-h incubation with IL-1beta showed that the suppressive effects of mechanical signals are sustained. CONCLUSIONS: The data provide evidence that biomechanical signals can downregulate the catabolic activity of fibrocartilage cells in an inflammatory environment by inhibiting the expression of a variety of MMPs. Furthermore, the matrix-protective effects of biomechanical signals are sustained even in an inflammatory environment.

Induction of MMP-3 by hyaluronan oligosaccharides in temporomandibular joint chondrocytes.

Low-molecular-weight hyaluronan (LMW-HA) is often increased in osteoarthritic joints; however, its biological function in cartilage has not been clarified. We hypothesize that LMW-HA causes the catabolic activation of chondrocytes through its interaction with CD44. Cartilage explants and chondrocytes, derived from bovine temporomandibular joints (TMJ), were examined for matrix loss and the expression of matrix metalloproteinase-3 (MMP-3) following treatment with hyaluronan oligosaccharides (HA(oligos)). Hyaluronan and CD44 were uniformly distributed throughout the fibrous and cartilaginous zones of the TMJ condyle. Treatment of cartilage explants with HA(oligos) resulted in cartilage matrix loss with increased secreted caseinolytic activity. HA(oligos) treatment of TMJ chondrocytes resulted in enhanced MMP-3 expression, whereas wash-out of the HA(oligos) in the middle of the experimental period reduced this induction. These results suggest that HA(oligos) activate chondrocytes, resulting in a substantial enhancement of proteinase expression, and the removal of HA(oligos) by wash-out reverses this catabolic activation.

The prospective use of COX-2 inhibitors for the treatment of temporomandibular joint inflammatory disorders.

Development of a new class of drugs designed to selectively inhibit the inducible cyclooxygenase isoenzyme, COX-2, was initially prescribed for individuals diagnosed with osteoarthritis or rheumatoid arthritis. Although these inflammatory disorders are more typically related to the joints of the knee, ankle, or hand, the temporomandibular joint (TMJ) plays a special role due to its involvement in our normal day-to-day activities of eating and communicating. The TMJ, unlike most of the other joints, contains some unique morphological characteristics that support various inflammatory disorders. An overview of these characteristics and the prospective use of the COX-2 inhibitors for temporomandibular joint inflammation are presented.

The effects of mechanical strain on synovial fibroblasts.

PURPOSE: Arthritic diseases of the temporomandibular joint, such as rheumatoid arthritis and osteoarthritis, suggest that inflammatory mediators and metalloproteinases may play a role in their pathogenesis. Recent clinical evidence from physical therapy and other modalities has shown a significant decrease in temporomandibular joint symptoms in patients with early disease. This project examines the effect of mechanical strain on synovial fibroblasts' production of inflammatory mediators including prostaglandin E(2) (PGE(2)) and proteinases. MATERIALS AND METHODS: An established synovial fibroblast cell line (HIG-82) was grown to confluency in modified Eagle's medium supplemented with 10% fetal calf serum. The monolayer of fibroblasts was then subjected to mechanical strain using the Flexercell Strain Unit (Flexcell International Corporation, McKeesport, PA) at 3 cycles per minute, with 10 seconds' elongation of up to 24% and 10 seconds of relaxation. Levels of PGE(2) were determined by radioimmunoassay using commercially available product and measured in nanograms per milliliter of supernatant. Proteinases collagenase, gelatinase, and stromelysin were measured by H(3) radioactive labeling of acidic anhydride to the specific substrate. Enzymatic proteolysis of the radiolabeled substrate was then measured in supernate as units per milliliter. Statistical analysis of all results was performed using Student's t test in triplicate. RESULTS: PGE(2) levels of mechanically activated cells was 18.1 +/- 13.4 ng/mL, with control levels being 58.0 +/- 9.2 ng/mL. This is a statistically significant decrease, between strained and unstrained cells with P <.05. In control cells, proteinase activity that degrades collagen, gelatin, or casein was 4.27 +/- 1.5, 4.62 +/- 0.11, or 0.11 +/- 0.01 U/mL, respectively. Levels for mechanically strained cells were 3.99 +/- 1.90, 4.02 +/- 0.90, and 0.12 +/- 0.01 U/mL, respectively. These results show that there is a significant decrease in PGE(2) levels of synovial fibroblasts undergoing mechanical strain. Proteinases examined show no difference in levels between mechanically activated fibroblasts and their controls. CONCLUSION: This decrease in PGE(2) production in synovial fibroblasts could help elucidate the mechanism by which physical therapy, and in particular continuous passive motion, may decrease inflammatory mediators of the temporomandibular joint.

The expression of cyclooxygenase-2 in human temporomandibular joint samples: an immunohistochemical study.

The aim of this investigation was to evaluate the immunohistochemical expression of cyclooxygenase-2 (COX-2) in the temporomandibular joint (TMJ) and to compare it with that control specimens. Expression of COX-2 in the TMJ disc and the synovial membrane in 26 human TMJ samples (internal derangement of TMJ; n=16, and control; n=10) was measured by an immunohistological technique using paraffin-embedded tissue and specific antihuman COX-2 polyclonal antibody. There were obvious distinction of COX-2 immunoreactivity between the control specimens and internal derangement cases, in the region of posterior and/or anterior loose connective tissues. In particular, intensive COX-2 expression was detected in the synovial membrane of internal derangement cases. The findings of the present study suggest that COX-2 might be an important mechanism regulating inflammation in the synovial membrane with internal derangement of TMJ.

Expression of matrix metalloproteinases in articular cartilage of temporomandibular and knee joints of mice during growth, maturation, and aging.

OBJECTIVE: This study examined the involvement of different matrix metalloproteinases (MMPs) in articular cartilage in the process of growth, maturation, and aging of mice, and compared the temporal changes in the expression of MMPs between temporomandibular joints (TMJ) and knee joints. METHODS: Homogenates of intact tibial plateau, femoral condyle, and TMJ condyle cartilages from animals of different ages were assessed for gelatinase (MMP-2 and MMP-9) activity by zymography. The messenger RNA (mRNA) expression of MMPs 1, 2, 3, 9, and 13 in tibial plateau cartilage was determined by semiquantitative reverse transcription-polymerase chain reaction, and immunohistochemistry was used to localize MMPs 2, 3, 9, and 13 in the knee joints and TMJ from mice of different ages. RESULTS: The pattern of gelatinase (MMP-2 and MMP-9) activity and their protein expression as well as that of MMPs 3 and 13 varied with the age of the mouse, and differences in expression were observed between the knee and TMJ cartilage. The expression of mRNA for the MMPs in the tibial plateau was also age related. CONCLUSION: This study demonstrated changes in the protein and mRNA expression of MMPs 2, 9, 3, and 13 during growth, maturation, and aging in mice. The temporal changes were characteristic of the joint, and distinct differences were observed between the TMJ and knee cartilage. The differences in temporospatial expression of MMPs between the knee joint and TMJ may be the result of differences in load and function of these joints. The information provided in this study contributes to a better understanding of the role of these MMPs in the maintenance and integrity of cartilage tissue.

The distribution of cyclooxygenase-1 in human temporomandibular joint samples: an immunohistochemical study.

Cyclooxygenase-1,2 (COX-1,2) or prostaglandin (PG) H synthase, is the first enzyme of the pathway in which arachidonic acid is oxidized to PGs. Thus, we examined the expression of COX-1 in 16 human temporomandibular joint (TMJ) samples with internal derangement and in 10 control specimens by an immunohistological technique using paraffin-embedded tissue and specific antihuman COX-1 polyclonal antibody. There was obvious distinction of COX-1 immunoreactivity between the control specimens and internal derangement cases, at the endothelial cells and fibroblasts, in the region of posterior and/or anterior loose connective tissues and synovial membrane. The findings of the present study suggest that COX-1 might be an important mechanism for maintaining normal homeostasis at the endothelial cells and fibroblasts with internal derangement of TMJ.

Role of interleukin-1 in induction of matrix metalloproteinases synthesized by rat temporomandibular joint chondrocytes and disc cells.

To identify cartilage-degrading enzymes and cell types that can be specifically induced by interleukin-1 (IL-1)alpha, we studied matrix metalloproteinase (MMP) activities of cultured rat temporomandibular joint (TMJ) chondrocytes and disc cells. The cells were isolated from TMJs pre-injected with normal physiological saline (CR) or recombinant human IL-1alpha (AR). MMP activities in the conditioned media were assayed by gelatin enzymography, and they were identified by Western blot analyses. MMP mRNAs in these cells were also detected by RT-PCR. IL-1alpha significantly induced an increase of active MMP9 as well as pro- and active MMP3, but had no effect on the MMP2 activity in both types of cells. MMP3 and MMP9 mRNAs were also inducible in these cells by IL-1alpha stimulation. Furthermore, disc cells were more susceptible to IL-1alpha than chondrocytes. AR cells spontaneously produced the same MMPs in vitro as the CR cells synthesized under IL-1alpha stimulation. The results indicate that MMP9 and MMP3 were predominantly produced by disc cells, and these may be considered to play a pivotal role in ECM degradation during pathological conditions of the TMJ, such as IL-1-induced TMJ arthritis.

Identification of matrix metalloproteinases and their inhibitor in the articular disc of the craniomandibular joint of the rabbit.

Connective tissue cells synthesize and secrete matrix metalloproteinases (MMPs), a family of matrix-degrading enzymes (comprising collagenases, gelatinases and stromelysins), which are capable of degrading all the constituent molecules of connective tissues at physiological pH. This investigation documents the synthesis and distribution of MMPs and their inhibitor TIMP-1 (tissue inhibitor of metalloproteinases-1) in the developing articular disc of the craniomandibular joint of the rabbit using indirect immunofluorescence microscopy. Cells of the disc synthesised all three classes of MMPs as well as TIMP-1 in all regions of the disc at all stages examined. MMPs and TIMP-1 were detected as bright intracellular accumulations probably within Golgi vesicles and as occasional diffuse, matrix-bound deposits. These results suggest that MMP-mediated matrix remodelling is a prominent feature of growth in craniomandibular joint disc.

Characterization and identification of proteinases and proteinase inhibitors synthesized by temporomandibular joint disc cells.

The adult mammalian temporomandibular joint (TM) disc is a fibrocartilaginous tissue that undergoes normal developmental remodeling, requiring removal of the existing extracellular matrix and its replacement by new matrix macromolecules. This remodeling is probably mediated by matrix-degrading enzymes, but to date none has been demonstrated in association with the TMJ disc. We characterized, identified, and determined the regulation of proteinases and proteinase inhibitor (PIs) synthesized by TMJ disc cells in organ and cell cultures. TMJ discs were retrieved from 14-week-old male NZW rabbits and both tissue- and disc-derived cells were cultured in serum-free medium. The conditioned media were retrieved at 12-hour intervals and assayed for proteinases and PIs in gelatin- and casein-impregnated polyacrylamide gels. Three proteinases with gelatinolytic activities at 92 kDa, 72 kDa, and 42/57 kDa and one caseinolytic activity at 51/54 kDa were detected. All were inhibited by 1,10-1 phenanthroline, thus characterizing these enzymes as matrix metalloproteinases (MMPs), most likely 92-kDa gelatinase (proMMP-9), 72-kDa gelatinase (proMMP-2), procollagenase (proMMP-1), and prostromelysin (proMMP-3). The identity of the latter two MMPs was confirmed by Western blots. Two PIs and 30 kDa and 20 kDa, probably tissue inhibitors of metalloproteinase (TIMP) and TIMP-2, were observed on reverse zymograms. TPA, a protein kinase-C agonist, increased the expression of 92-kDa gelatinase and 30-kDa PI by both explanted discs and isolated disc cells. The profile of MMPs constitutively expressed by disc cells is similar to that of synovial fibroblasts but different from that of chondrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)