Temporomandibular Joint Disc Cells Research Articles

A bioinspired and high-strengthed hydrogel for regeneration of perforated temporomandibular joint disc: Construction and pleiotropic immunomodulatory effects.

Due to the lack of an ideal material for TMJ (temporomandibular joint) disc perforation and local inflammation interfering with tissue regeneration, a functional TGI/HA-CS (tilapia type I gelatin/hyaluronic acid-chondroitin sulfate) double network hydrogel was constructed in this paper. It was not only multiply bionic in its composition, structure and mechanical strength, but also endowed with the ability to immunomodulate microenvironment and simultaneously induce in situ repair of defected TMJ discs. On the one hand, it inhibited inflammatory effects of inflammasome in macrophages, reduced the extracellular matrix (ECM)-degrading enzymes secreted by chondrocytes, reversed the local inflammatory state, promoted the proliferation of TMJ disc cells and induced fibrochondrogenic differentiation of synovium-derived mesenchymal stem cells (SMSCs). On the other hand, it gave an impetus to repairing a relatively-large (6mm-sized) defect in mini pigs' TMJ discs in a rapid and high-quality manner, which suggested a promising clinical application.

Effects of glucose concentration and oxygen partial pressure on the respiratory metabolism of sheep temporomandibular joint disc cells.

Temporomandibular joint (TMJ) disc degeneration is a common disease characterized by a decrease in metabolic function. The present study aimed to investigate the pathogenesis of TMJ disc degeneration by analyzing the effects of oxygen and glucose concentrations on metabolism in a simulated TMJ disc cell growth environment. Cell samples were divided into 10 groups and cultured in different nutritional environments, including 21 and 2% O2 partial pressures and various glucose concentrations (0, 0.5, 3, 5.5 and 22.5 mmol/l). Cell proliferation, extracellular matrix content, mitochondrial function, and cell metabolism were subsequently analyzed. The results demonstrated that hypoxia and a low glucose concentration inhibited cell growth, and low glucose concentration inhibited extracellular matrix synthesis and adenosine 5'-monophosphate-activated protein kinase expression. Hypoxic conditions also induced a compensatory increase in the number of mitochondria, whereas mitochondrial deformation and swelling were observed in the absence of glucose. According to this study, the primary metabolic pathway of TMJ disc cells is glycolysis. It was concluded that hypoxic conditions and normal glucose concentrations are needed for the growth of TMJ disc cells. Glucose is necessary to ensure cell survival, extracellular matrix synthesis and mitochondrial function. Glucose deficiency may be related to disc degeneration, aging and disease mechanisms.

Mechanical overload induces TMJ disc degeneration via TRPV4 activation.

The temporomandibular joint (TMJ) disc cushions intraarticular stress during mandibular movements. While mechanical overloading is related to cartilage degeneration, the pathogenesis of TMJ disc degeneration is unclear. Here, we determined the regulatory role of mechanoinductive transient receptor potential vanilloid 4 (TRPV4) in mechanical overload-induced TMJ disc degeneration. We explored the effect of mechanical overload on the TMJ discs in a rat occlusal interference model in vivo, and by applying sustained compressive force in vitro. TRPV4 inhibition was delivered by small interfering RNA or GSK2193874; TRPV4 activation was delivered by GSK1016790A. The protective effect of TRPV4 inhibition was validated in the rat occlusal interference model. Occlusal interference induced TMJ disc degeneration with enhanced extracellular matrix degradation in vivo and mechanical overload promoted inflammatory responses in the TMJ disc cells via Ca2+ influx with significantly upregulated TRPV4. TRPV4 inhibition reversed mechanical overload-induced inflammatory responses; TRPV4 activation simulated mechanical overload-induced inflammatory responses. Moreover, TRPV4 inhibition alleviated TMJ disc degeneration in the rat occlusal interference model. Our findings suggest TRPV4 plays a pivotal role in the pathogenesis of mechanical overload-induced TMJ disc degeneration and may be a promising target for the treatment of degenerative changes of the TMJ disc.

940 nm diode laser induced differentiation of human adipose derived stem cells to temporomandibular joint disc cells

BackgroundTemporomandibular disorder (TMD) refers to a group of disorders that affect temporomandibular joint (TMJ) and its associated muscles with very limited treatment options. Stem cell research is emerging as one of the promising fields in the treatment of degenerative diseases. The ability of human adipose derived stem cells to differentiate into many cell types is driving special interest in several disease management strategies. Photobiomodulation has enhanced the role of these stem cells through their ability to promote cell proliferation and differentiation. Hence, this study examined the differentiation potential of human adipose derived stem cells (ADSCs) into fibroblasts and chondrocytes using a 940 nm diode laser for possible TMD therapy.Materials and methodsADSCs were cultured at different seeding densities and for different time intervals. After irradiation at 24, 48, 72 h, 1, 2 and 3 weeks, ADSC viability and morphological changes were assessed in groups with and without basic fibroblast growth factor. Additionally, the level of adenosine triphosphate (ATP) in the cells was also recorded. The differentiated fibroblasts and chondrocytes were characterized with flow cytometry and immunofluorescence techniques, at 1- and 2-weeks post-irradiation.ResultsIncreased ATP proliferation and cell viability above 90% were observed in all post-irradiation experimental groups. Post irradiation results from flow cytometry and immunofluorescence at 1- and 2‐weeks confirmed the expression of chondrogenic and fibroblastic cell surface markers.ConclusionThis study describes stimulatory techniques utilized to differentiate ADSCs into fibroblastic and chondrogenic phenotypes using diode lasers at 940 nm. The study proposes a new treatment model for patients with degenerative disc diseases of the TMJ. The study will offer new possibilities in tissue engineering and TMJ disc management through photobiomodulation of ADSCs using a 940 nm diode laser.

Laser-Induced Differentiation of Human Adipose-Derived Stem Cells to Temporomandibular Joint Disc Cells.

Temporomandibular disorder (TMD) is an incapacitating disease with temporomandibular joint (TMJ) disc degenerative changes in patients. Despite several research attempts to find a definitive treatment, there is no evidence of a permanent solution. The objective of the current study was to observe the role of 660 nm diode laser in the differentiation of human adipose-derived stem cells (ADSCs) to fibroblasts and chondrocytes. After irradiation, the morphology, viability, and adenosine triphosphate (ATP) proliferation of the ADSCs were analyzed at different time intervals. The differentiation of ADSCs toward fibroblastic and chondrogenic phenotypes was supported using flow cytometry and immunofluorescence at 1- and 2-week post-irradiation. More than 90% of viable cells were observed in all experimental groups, with an increase in ATP proliferation. Flow cytometry analyses and immunofluorescence demonstrated the presence of chondrogenic and fibroblastic cell surface markers at 1- and 2-week post-irradiation. This study has demonstrated methods to induce the differentiation of ADSCs toward fibroblastic and chondrogenic phenotypes with a 660 nm diode laser. The study also proposes a future alternative method of treatment for patients with degenerative TMJ disc disorders and presents a positive prospect in the application of photobiomodulation and ADSCs in the treatment of degenerative TMJ disc. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

Effects of dynamic radial tensile stress on fibrocartilage differentiation of bone marrow mesenchymal stem cells

BackgroundUniaxial/biaxial tensile stress has been employed to induce chondrocyte differentiation of mesenchymal stem cells. However, the effects of radial tensile stimuli on differentiation of MSCs into fibrocartilage remain unclear.ResultsIt was found that induced bone marrow mesenchymal stem cells (BMSCs) were not only similar to TMJ disc cells in morphology, but also could synthesize type I collagen (Col I), a small amount of type II collagen (Col II) and glycosaminoglycans (GAGs). The synthesis of Col I significantly increased while that of Col II gradually decreased with increasing tensile strength. The ratio of Col I to Col II was 1.8 to 1 and 2 to 1 in the 10% and 15% stretching groups, respectively. The gene expression of Col I and GAGs was significantly upregulated, whereas that of Col II was downregulated. However, the higher tensile stimulation (15%) promoted the synthesis of α-smooth muscle actin (α-SMA). Too much α-SMA is not conducive to constructing engineered tissue.ConclusionTherefore, the 10% radial tensile stimulus was the optimal strength for inducing the BMSCs to differentiate into fibrochondrocytes of the temporomandibular joint (TMJ) disc. This work provided a novel approach for inducing BMSCs to differentiate into fibrochondrocytes.

Surface Modification of 3D-Poly(glycerol-sebacate)/ Poly(L-lactic-acid) Porous Scaffolds by Oxygen Plasma Enhances Proliferation and Differentiation of Goat Temporomandibular Joint Disc Cells

3D poly(glycerol-sebacate)/poly(L-lactic-acid) (PGS/PLLA) scaffolds, produced by thermally induced phase separation (TIPS) method were treated with oxygen plasma in order to reduce the hydrophobicity of the PGS/PLLA membranes. After the oxygen plasma treatment, the pore size of scaffolds became uniform as well as an increasing in the roughness of the surface. No influence on chemical characteristics such as composition and bond types of PLLA was detected after the treatment. However, the change in the ratio of the scaffold elements before and after the oxygen plasma treatment was detected. Oxygen plasma treatment reduced the average of water contact angle of the TIPS film from 103.7± 10.1 to 64.7± 12.6 at the maximum exposure time (970 s) and power (100 W). The results showed that the goat temporomandibular joint (TMJ) cells attached well to the surface of the oxygen plasma modified scaffold. Furthermore, this modification significantly enhanced cell viability, increased cell proliferation and expression of related genes (Collagen type I mRNA and Collagen type II mRNA). The results demonstrated that it is a promising modification method to promote applications of PGS/PLLA based scaffolds to the regeneration of TMJ discs.

Effects of staurosporine on the contraction of self-assembled constructs of goat temporomandibular joint disc cells

The effects of the staurosporine on contraction of self-assembled constructs and extracellular matrix syntheses of goat temporomandibular joint discs were investigated. Goat temporomandibular joint disc cells were isolated and cultured to P3, and 5.5×10⁶ cells were combined with different concentrations of staurosporine (0, 0.1, 1, 10, 100 nmol·L⁻¹) in agarose wells and cultured for one week. The samples were frozen and sectioned. Safranin-O, Picro-sirius red and immunohistochemical staining were performed to observe the distributions of the extracellular matrix and the expression of alpha-smooth muscle actin (α-SMA). Enzyme linked immunosorbent assay (ELISA) and Blyscan kits were utilized to quan--titatively detect the contents of type Ⅰ collagen (ColⅠ) and glycosaminoglycans (GAGs). Each group of goat temporo-mandibular joint disc cells in the agarose wells were gathered to self-assemble into a disc-shaped base for 4 hours and then to gradually contract into a round shape. The Picro-sirius red staining was strong and indicated collagen distribution. The Safranin-O staining observed GAGs throughout the entire construct. The expression of ColⅠ was strongly posi-tive in the staurosporine groups; however, the expression of α-SMA was weak. ColⅠ and GAGs contents in the stau-rosporine groups were greater than that of the control group, especially in the 10 nmol·L⁻¹ group (P<0.01). Staurosporine has a certain effect on the shrinkage of self-assembled constructs; however, such effect is not prominent. Staurosporine contributes to the construction synthesis of extracellular matrix.

Effect of different oxygen tension on the cytoskeleton remodeling of goat temporomandibular joint disc cells

Objective The effect of different oxygen tensions on the cytoskeleton remodeling of goat temporomandibular joint (TMJ) disc cells were investigated. Methods Goat TMJ disc cells were cultured under normoxia (21% O₂) and hypoxia (2%, 4%, and 8% O₂). Toluidine blue, picrosirius red, and type Ⅰ collagen immunocytochemical staining were performed to observe the changes in cell phenotype under different oxygen levels. Immunofluorescent staining and real-time reverse transcription-polymerase chain reaction analysis were then performed to identify actin, tubulin, and vimentin in the cultured disc cells. Results TMJ disc cells still displayed fibroblast characteristics under different oxygen levels and their cytoskeletons had regular arrangement. The fluorescence intensities of actin and vimentin were lowest at 4% O₂(P<0.05), whereas that of tubulin was highest at 2% O₂ (P<0.05). No significant difference among the other groups was observed (P>0.05). Actin mRNA levels were considerably decreased at 2% O₂ and 4% O₂ in hypoxic conditions, while actin mRNA expression was highest in 21% O₂. Tubulin mRNA levels considerably increased at 2% O₂, while tubulin mRNA expression was lowest in 8% O₂ (P<0.05). Vimentin mRNA expression was lowest at 4% O₂ and highest at 21% O₂, and significant differences were observed between vimentin mRNA expression levels among these oxygen levels (P<0.05). Conclusion Cytoskeletons were reconstructed in different oxygen tensions, and 2% O₂ may be the optimal oxygen level required to proliferate TMJ disc cells.

Effect of temporomandibular joint disc perforation on expression of type Ⅰ collagen in temporomandibular joint disc cells

Objective: To investigate the effect of temporomandibular joint (TMJ) disc perforation on expression of type Ⅰ collagen in TMJ disc cells. Methods: The fibroblastic-like cells from the surgical removed TMJ disc tissue (disc perforation or TMJ condyle hyperplasia) were cultured. The cultured cells were identified as fibroblastic-like cells by toluidine blue and immunofluorescence staining. The expression of type Ⅰ collagen was detected with Western blotting and the content of type Ⅰ collagen was examined by enzyme linked immunosorbent assay (ELISA). Results: Fibroblastic-like cells were cultured from TMJ disc cells and the controls. The collagen-Ⅰ and collagen-Ⅱ were positive in both toluidine blue and immunofluorescence staining. In Western blotting, the expression of typeⅠcollagen in cells from joints with disc perforation was lower than that from normal joints. The content of collagen-Ⅰ was (1.62±0.52) μg/L from controls, and (0.85±0.33) μg/L from disc perforation respectively (P=0.0134). Conclusions: The disc cells from TMJ with disc perforation expressed lower type Ⅰ collagen than that from controls, which may be related to the lower content of collagen-Ⅰ in TMJ disc and the formation of TMJ disc perforation.

Isolation and characterization of mesenchymal stromal progenitors from the temporomandibular joint disc.

Disorders of the temporomandibular joint (TMJ) complex affect 6-12% of the population; the joint's disc is usually involved. Tissue engineering and regenerative medicine may constitute a promising therapeutic approach, with resident stromal progenitor cells a key factor in the process. We hypothesized that the TMJ disc (TMJD) contains multipotent stromal progenitors that may play an important role in regeneration of the disc. TMJD cells were cultured and evaluated for growth kinetics and colony-forming units (CFUs). Single cell-derived clones were isolated and induced to differentiate toward the osteogenic, adipogenic and chondrogenic lineages by culturing in various induction media. Flow cytometry was used to identify multipotent stromal cell surface markers in additional cell samples, and reverse transcription-polymerase chain reaction (RT-PCR) was used to determine gene expression patterns within isolated cells. High numbers of CFUs were observed, indicating cell self-renewal. Biochemical assays showed significantly higher alkaline phosphatase (ALP) activity, lipid droplet concentration and glycosaminoglycan levels in cells cultured in osteogenic, adipogenic and chondrogenic induction medium, respectively. Approximately 1% of the total cell population demonstrated the capability to differentiate into all three mesenchymal lineages. Chondrogenic gene levels within TMJD-derived cells were significantly reduced in passaged culture. Our results support the hypothesis that multipotent stromal progenitor cells populate the TMJD and possess proliferation and differentiation capabilities. These cells may contribute to the regeneration potential of dysfunctional tissue and become the primary component in future attempts at tissue engineering or regeneration of this complex. Copyright © 2015 John Wiley & Sons, Ltd.

The effects of oxygen level and glucose concentration on the metabolism of porcine TMJ disc cells

To determine the combined effect of oxygen level and glucose concentration on cell viability, ATP production, and matrix synthesis of temporomandibular joint (TMJ) disc cells. TMJ disc cells were isolated from pigs aged 6-8 months and cultured in a monolayer. Cell cultures were preconditioned for 48 h with 0, 1.5, 5, or 25 mM glucose DMEM under 1%, 5%, 10%, or 21% O2 level, respectively. The cell viability was measured using the WST-1 assay. ATP production was determined using the Luciferin-Luciferase assay. Collagen and proteoglycan synthesis were determined by measuring the incorporation of [2, 3-(3)H] proline and [(35)S] sulfate into the cells, respectively. TMJ disc cell viability significantly decreased (P < 0.0001) without glucose. With glucose present, decreased oxygen levels significantly increased viability (P < 0.0001), while a decrease in glucose concentration significantly decreased viability (P < 0.0001). With glucose present, decreasing oxygen levels significantly reduced ATP production (P < 0.0001) and matrix synthesis (P < 0.0001). A decreased glucose concentration significantly decreased collagen synthesis (P < 0.0001). The interaction between glucose and oxygen was significant in regards to cell viability (P < 0.0001), ATP production (P = 0.00015), and collagen (P = 0.0002) and proteoglycan synthesis (P < 0.0001). Although both glucose and oxygen are important, glucose is the limiting nutrient for TMJ disc cell survival. At low oxygen levels, the production of ATP, collagen, and proteoglycan are severely inhibited. These results suggest that steeper nutrient gradients may exist in the TMJ disc and it may be vulnerable to pathological events that impede nutrient supply.

Immortalization and characterization of mouse temporomandibular joint disc cell clones with capacity for multi-lineage differentiation

Despite the importance of temporomandibular joint (TMJ) disc in normal function and disease, studying the responses of its cells has been complicated by the lack of adequate characterization of the cell subtypes. The purpose of our investigation was to immortalize, clone, characterize and determine the multi-lineage potential of mouse TMJ disc cells. Cells from 12-week-old female mice were cultured and immortalized by stable transfection with human telomerase reverse transcriptase (hTERT). The immortalized cell clones were phenotyped for fibroblast- or chondrocyte-like characteristics and ability to undergo adipocytic, osteoblastic and chondrocytic differentiation. Of 36 isolated clones, four demonstrated successful immortalization and maintenance of stable protein expression for up to 50 passages. Two clones each were initially characterized as fibroblast-like and chondrocyte-like on the basis of cell morphology and growth rate. Further the chondrocyte-like clones had higher mRNA expression levels of cartilage oligomeric matrix protein (COMP) (>3.5-fold), collagen X (>11-fold), collagen II expression (2-fold) and collagen II:I ratio than the fibroblast-like clones. In contrast, the fibroblast-like clones had higher mRNA expression level of vimentin (>1.5-fold), and fibroblastic specific protein 1 (>2.5-fold) than the chondrocyte-like clones. Both cell types retained multi-lineage potential as demonstrated by their capacity to undergo robust adipogenic, osteogenic and chondrogenic differentiation. These studies are the first to immortalize TMJ disc cells and characterize chondrocyte-like and fibroblast-like clones with retained multi-differentiation potential that would be a valuable resource in studies to dissect the behavior of specific cell types in health and disease and for tissue engineering.

Transcriptional regulation of proteoglycan 4 by 17β‐estradiol in immortalized baboon temporomandibular joint disc cells

Temporomandibular joint disorders (TMDs) affect a significant portion of the population of the USA, with the majority of those seeking treatment being women of childbearing age. Owing to this striking sexual dimorphism it has been postulated that sex hormones play a role in the maintenance of normal temporomandibular joint (TMJ) function. Proteoglycan 4 (PRG4) is a secreted lubricating molecule required for maintaining low frictional levels within articular joints; however, its role in the TMJ is not well characterized. In this study we describe the development of immortalized baboon cells isolated from specific regions of the TMJ disc and their use in the investigation of PRG4 expression and localization patterns in the TMJ. We identified conserved estrogen response elements within the 5' flanking region of the PRG4 gene of several species, and found that treatment of baboon TMJ disc cells with estrogen led to reduced PRG4 promoter activity and reduced expression of PRG4 mRNA in vitro. The observed negative regulation of PRG4 by estrogen could lead to increased friction and degradation of joint components over time. This study, for the first time, provides evidence of the regulatory potential of estrogen on PRG4 gene expression and suggests a novel etiology for the gender disparity observed among TMD patients.

Network of telocytes in the temporomandibular joint disc of rats

The phenotypes of the temporomandibular joint (TMJ) disc cells range from fibroblasts to chondrocytes. There are relatively few reported studies using transmission electron microscopy (TEM) to determine the ultrastructural features of these cells. It was hypothesized that at least a subpopulation of TMJ stromal cells could be represented by the telocytes, cells with telopodes. In this regard a TEM study was performed on rat TMJ samples. Collagen-embedded networks were found built-up by cells with telopodes with subplasmalemmal caveolae, moderate content in matrix secretory organelles and well-represented intermediate filaments. Appositions of cell bodies were found. Prolongations of such cells were closely related to nerves and microvessels. Our study indicates that the TMJ disc attachment seems equipped with telocytes capable of stromal signaling. However, further studies are needed to assess whether the telocytes belong to a renewed cell population derived from circulating precursors.

Lubricin in human temporomandibular joint disc: An immunohistochemical study

AimsTo evaluate, immunohistochemically, the presence and distribution of lubricin in human temporomandibular joint (TMJ) discs without any degenerative changes, obtained from autopsies, in order to elucidate the TMJ lubrication system and disc tribology. MethodsImmunohistochemistry for lubricin detection was carried out on 34 TMJ discs. Any disc had signs of degenerative or inflammatory joint disease nor disc were displaced. Sections were incubated with diluted rabbit polyclonal anti-lubricin antibody and scored according to the percentage of lubricin immunopositive cells. Three different TMJ disc tissue compartments taken from the intermediate zone were analysed, namely: the central region as well as the temporal (superior) and condylar (inferior) disc surfaces. The Friedman test, was used to compare lubricin at a protein level expression, amongst the regions of disc specimens. ResultsStaining was noted within the TMJ disc cell populations in every disc tissue sample, however, the number of disc cells immunolabelled varied according to disc tissue regions. The percentage of immunostained cells, was statistically significant lower in the central region than in each disc surface (p<0.0001), whilst any statistically significant difference was found when comparing the two surfaces one another. ConclusionsLubricin is present in several location of TMJ disc being significantly more expressed at disc surfaces than in the central part.

Immunolocalization and expression of lubricin in the bilaminar zone of the human temporomandibular joint disc

Lubricin, which is a boundary joint lubricant, was investigated immunohistochemically in the bilaminar zone (BZ) of the human temporomandibular joint (TMJ), without any degenerative changes. Immunohistochemistry for lubricin detection was carried out on 33 TMJ discs obtained from 17 cadavers. Sections were incubated with diluted rabbit polyclonal anti-lubricin antibody and scored according to the percentage of lubricin immunopositive cells. Three different TMJ disc tissue compartments were analyzed, namely: the upper lamina, the inferior lamina and the loose connective tissue in the space between the laminae. The Mann–Whitney U test was used to compare protein expression (lubricin) among disc specimens’ regions. Staining was noted within the TMJ disc cell populations in every disc tissue sample, with almost every cell immunolabeled by the lubricin antibody. The number of disc cells immunolabeled was almost the same in the 3 bilaminar zone regions. Positive extracellular matrix staining was also seen. The results of the present study suggest that lubricin is expressed in the TMJ disc bilaminar zone. Lubricin may have a role in normal disc posterior attachment physiology through the prevention of cellular adhesion as well as providing lubrication during normal bilaminar zone function.

Expression of lumican and fibromodulin following interleukin-1 beta stimulation of disc cells of the human temporomandibular joint

Small leucine-rich repeat proteoglycans (SLRP) are present in the extracellular matrix of the temporomandibular joint (TMJ) disc. Lumican and fibromodulin, classified as class 2 SLRPs, play important roles in TMJ assembly, proliferation and inflammation. Degenerative change in the TMJ disc gives rise to the process of internal derangement (ID). In this study, we immunohistochemically examined the expression of lumican and fibromodulin in nine human TMJ specimens and examined the gene expression of both proteoglycans in cultured human TMJ disc cells under interleukin-1 beta (IL-1 β)-stimulated conditions. An articular disc cell line was established by collagenase treatment of a TMJ disc. The subcultured cells were then incubated for 1, 3, 6, 12, 24 or 48 h under both normal and IL-1 β (1 ng/mL) conditions. The gene expression of lumican and fibromodulin was examined using the reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR. We demonstrated that the expression of lumican significantly differs from that of fibromodulin in the deformed disc and that IL-1 β induces a significant increase in lumican mRNA, but not in fibromodulin mRNA, after 24∼48 h culture compared to cells cultured in the absence of IL-1 β (P<0.05). These results indicate that lumican and fibromodulin display different behaviors and that lumican may promote regeneration of the TMJ after degeneration and deformation induced by IL-1 β.

Expression of hyaluronan synthase 3 in deformed human temporomandibular joint discs: in vivo and in vitro studies

The present study aimed at investigating the expression of a hyaluronan synthase (HAS) 3 in tissue samples of deformed human temporomandibular joint (TMJ) discs and cells obtained from the discs. Fifteen adult human TMJ discs (twelve diseased discs and three normal discs) were used in this study. The twelve diseased discs were obtained from twelve patients with internal derangement (ID) of TMJ. These patients all had anteriorly displaced discs and deformed discs. The tissues were immunohistochemically stained using HAS3 antibodies. In addition, the subcultured TMJ disc cells under both normal and hypoxic conditions (O2: 2%) were incubated for 3, 6, 12, and 24 h after addition of interleukin-1β (IL-1β) (1 ng/mL). Subsequently, the expression of HAS3 was examined using real-time reverse transcription-polymerase chain reaction (RT-PCR). The control group showed from negative to weak positive reactions for HAS3 on immunohistochemical staining. The discs extracted from twelve cases with ID presented from moderate to strong positive reactions for HAS3. The quantity of HAS3 mRNA was compared with a control group, and showed a 204-fold increase at 3 h, a 26-fold increase at 6 h, a 2.5-fold increase at 12 h and a 32-fold increase at 24 h under hypoxia with the addition of IL-1β. The expression of HAS3 mRNA was significantly enhanced at 3 h and 24 h. The results obtained suggest that HAS3 is related to the pathological changes of human TMJ discs affected by ID.

Female hormone receptors are differentially expressed in mouse fibrocartilages

Despite the female predilection for joint diseases, and the known effects of female hormones in regulating chondrocyte function, the various female hormone receptor subtypes in joints are not well characterized, and comparisons in receptor profiles between joints and genders are lacking. This investigation characterized and compared the relative levels of estrogen receptors (ER)-alpha and -beta, relaxin receptors LGR7 and LGR8, and progesterone receptor (PR) in the temporomandibular joint (TMJ) disc, knee meniscus (KM) and pubic symphysis fibrocartilages. Fibrocartilaginous cells from 12-week-old mice were maintained in serum-containing alpha-modified Eagle's medium (MEM) until confluence. Total RNA and cell lysates were assayed by RT-PCR, qRT-PCR, immunocytochemistry and Western blots, and joint sections subjected to immunohistochemistry. All hormone receptors assayed were present in the three joints, but showed substantial differences in expression levels between joints. TMJ cells had higher ER-alpha (>2.8-fold), ER-beta (>2.2-fold), LGR7 (>3-fold) and PR (>1.8-fold), and lower LGR8 (0.5-fold) gene expression levels than KM cells. The ratio of ER-alpha:ER-beta and LGR7:LGR8 was 1.8- and 7.5-fold higher, respectively, in TMJ than in KM cells. The profile of hormone receptors in the TMJ disc was similar to those in the pubic symphysis. Immunochemistry confirmed the differential expression patterns of these receptors in the three tissues. The TMJ cells demonstrated sexual dimorphism in the levels of both ER isoforms, but not of LGR7, LGR8 or PR. The findings suggest that these fibrocartilages are putative target tissues for actions of female hormones. The differential expression profiles of the hormone receptors in the three joint fibrocartilages and the sexual dimorphism in ERs in TMJ disc cells are likely to result in varied downstream effects in response to hormones within these fibrocartilaginous tissues.