Temporomandibular Joint Fibrocartilage Research Articles

Advances in 3D printing techniques for cartilage regeneration of temporomandibular joint disc and mandibular condyle.

Temporomandibular joint (TMJ) osteoarthritis causes fibrocartilage damage to the TMJ disc and mandibular condyle, resulting in local pain and functional impairment that further reduces patients' quality of life. Tissue engineering offers a potential treatment for fibrocartilage regeneration of the TMJ disc and mandibular condyle. However, the heterogeneous structure of TMJ fibrocartilage tissue poses significant challenges for the fabrication of biomimetic scaffolds. Over the past two decades, some researchers have attempted to adopt three-dimensional (3D) printing techniques to fabricate biomimetic scaffolds for TMJ fibrocartilage regeneration, but publications on such attempts are limited and rarely report satisfactory results, indicating an urgent need for further development. This review outlines several popular 3D printing techniques and the significant elements of tissue-engineered scaffolds: seed cells, scaffold materials, and bioactive factors. Current research progress on 3D-printed scaffolds for fibrocartilage regeneration of the TMJ disc and mandibular condyle is reviewed. The current challenges in TMJ tissue engineering are mentioned along with some emerging tissue-engineering strategies, such as machine learning, stimuli-responsive delivery systems, and extracellular vesicles, which are considered as potential approaches to improve the performance of 3D-printed scaffolds for TMJ fibrocartilage regeneration. This review is expected to inspire the further development of 3D printing techniques for TMJ fibrocartilage regeneration.

Detection of the Temporomandibular Joint Fibrocartilage by Magnetic Resonance Imaging

Objective: The aims of this study were to detect the fibrocartilage layer (FC) of the porcine mandible condyle in ex vivo preparations using 3D SPGR (Spoiled GRASS sequence) (T1 WATS) sequences, and, to compare 3D SPGR image results with those of conventional magnetic resonance imaging (MRI) sequences for detecting the fibrocartilage layers of the temporomandibular joint (TMJ) in patients with temporomandibular disorders (TMD). Methods: Four porcine TMJ with and without FC were radiographically and histologically assessed. Additionally, fifty-nine patients were studied using T1 WATS MRI and T2 TSE and PD TSE sequences. Four trained and calibrated observers assessed the MRI images. Results: In porcine, the T1 WATS sequence was able to detect the presence or absence of FC in the mandibular condyle, histologically confirmed. In humans, 98% of fibrocartilages were detected with T1 WATS sequence in temporal fossa, and 91% in mandible condyles. Significantly poorer detection with the T2 TSE and PD TSE sequences was observed. Mean fibrocartilage ROI signal intensity was 74.07 IU (±14.7), without statistical differences between fossa and condyle. Conclusion: T1 WATS is the most reliable and efficient sequence for detecting the TMJ fibrocartilage layer (p <0.05).

A novel approach to establishing a temporomandibular joint fibrocartilage cell line

Background/purposeThe temporomandibular joint (TMJ) is a bi-arthrodial joint that is composed of the temporal bone glenoid fossa and the condylar head of the mandible both having fibrocartilaginous articular surfaces. Functional overloading of the TMJ is the main cause of TMJ osteoarthritis (TMJ OA) disease. The aim of this study was to establish immortalized TMJ fibrocartilage cell clones to provide enough cells to adequately investigate the molecular mechanisms studies of TMJ OA. Materials and methodsWe have isolated temporomandibular condyle chondrocytes from adult Sprague Dawley rat. The cells were cultured and immortalized by treating with Y-27632, a well-characterized inhibitor of Rho-Associated Kinase (ROCK). Clones were characterized on the basis of cell morphology and analyses of marker gene expression through 45 passages. ResultsCells from the condylar fibrocartilage of the TMJ were successfully immortalized by ROCK inhibitor, retaining a consistent cuboidal cell morphology and the expression of several cell markers of polymorphic cell fate. In addition, they retained phenotype features similar to the primary parental TMJ fibrocartilage cells when the cells were challenged with different cytokines and growth factors. ConclusionThese studies establish a novel immortalized cell line through ROCK inhibitor Y-27632, that retains the polymorphic phenotype of primary cell lines from TMJ fibrocartilage chondrocyte cell through a high number of passages, serving as a valuable preclinical resource for mechanistic in vitro assessment of TMJ health, disease, and regeneration.

Temporomandibular Joint Fibrocartilage Contains CD105 Positive Mouse Mesenchymal Stem/Progenitor Cells with Increased Chondrogenic Potential.

A specific type of mesenchymal stem/progenitor cells (MSPCs), CD105+ is reported to aid in cartilage regeneration through TGF-β/Smad2-signalling. The purpose of this study was to identify and characterize CD105+ MSPCs in temporomandibular joint (TMJ) cartilage. MSPCs were isolated from mouse TMJ condyle explants and evaluated for their clonogenicity and pluripotential abilities. MSPC were examined for CD105 antigen using immunohistochemistry and flow cytometry. Immunohistochemistry revealed presence of CD105+ MSPCs in the proliferative zone of condyle's cartilage. Only 0.2% of isolated MSPCs exhibited CD105, along with the stem cell surface markers CD44 and Sca-1. In CD105+ MSPCs, intracellular immunostaining revealed significantly higher (p < 0.05) protein levels of collagen type 1, 2, proteoglycan 4. Ability for chondrogenic differentiation was found to be significantly higher (p < 0.05) after 4weeks compared to CD105- cells, using alcian blue staining. CD105+ cells were found to resemble an early MSPC subgroup with significantly higher gene expression of biglycan, proteoglycan 4, collagen type 2, Gli2, Sox5 (p < 0.001) and Sox9 (p < 0.05). In contrast, significantly lower levels of Runx2 (p < 0.05), Osterix, Trps1, Col10a1 (p < 0.01), Ihh (p < 0.001) related to chondrocyte senescence and commitment to osteogenic lineage, were observed compared to CD105- cells. The study showed the existence of a CD105+ MSPC subgroup within TMJ fibrocartilage that may be activated to aid in fibrocartilage repair.

Painful temporomandibular joint overloading induces structural remodeling in the pericellular matrix of that joint's chondrocytes.

Mechanical stress to the temporomandibular joint (TMJ) is an important factor in cartilage degeneration, with both clinical and preclinical studies suggesting that repeated TMJ overloading could contribute to pain, inflammation, and/or structural damage in the joint. However, the relationship between pain severity and early signs of cartilage matrix microstructural dysregulation is not understood, limiting the advancement of diagnoses and treatments for temporomandibular joint-osteoarthritis (TMJ-OA). Changes in the pericellular matrix (PCM) surrounding chondrocytes may be early indicators of OA. A rat model of TMJ pain induced by repeated jaw loading (1 h/day for 7 days) was used to compare the extent of PCM modulation for different loading magnitudes with distinct pain profiles (3.5N-persistent pain, 2N-resolving pain, or unloaded controls-no pain) and macrostructural changes previously indicated by Mankin scoring. Expression of PCM structural molecules, collagen VI and aggrecan NITEGE neo-epitope, were evaluated at Day 15 by immunohistochemistry within TMJ fibrocartilage and compared between pain conditions. Pericellular collagen VI levels increased at Day 15 in both the 2N (p = 0.003) and 3.5N (p = 0.042) conditions compared to unloaded controls. PCM width expanded to a similar extent for both loading conditions at Day 15 (2N, p < 0.001; 3.5N, p = 0.002). Neo-epitope expression increased in the 3.5N group over levels in the 2N group (p = 0.041), indicating pericellular changes that were not identified in the same groups by Mankin scoring of the pericellular region. Although remodeling occurs in both pain conditions, the presence of pericellular catabolic neo-epitopes may be involved in the macrostructural changes and behavioral sensitivity observed in persistent TMJ pain.

Estrogen Selectively Enhances TMJ Disc but Not Knee Meniscus Matrix Loss

The preponderance of temporomandibular joint (TMJ) degenerative disorders in women and their early onset during reproductive years have implicated female sex hormones, particularly 17-β estradiol (E2), in the pathogenesis of these disorders. Nevertheless, the mechanisms by which E2 contributes to TMJ degenerative disorders and the reasons for its targeted effects on the TMJ but not other joints remain poorly understood. Here, we developed an ovariectomized mouse model in which systemic E2 concentrations mimicked those in cycling women, and we determined the effect of E2 on the targeted turnover of TMJ fibrocartilage matrix via E2-induced matrix metalloproteinases MMP9 and MMP13. Infusion of E2 and progesterone (P4; hormone control) over 7 d resulted in 5- and 8-fold greater serum E2 and P4 levels relative to controls, respectively, achieving systemic hormone levels similar to high baseline levels in cycling women. Administration of E2 but not P4 caused a significant loss of TMJ collagen and glycosaminoglycans, which was accompanied by amplification of ERα and specific increases in MMP9 and MMP13 expression. This dose of E2 had no effect on knee meniscus fibrocartilage, demonstrating the specificity of the degradative effect of E2. Dose-response experiments showed a greater sensitivity and a higher peak induction of MMP9 and MMP13 in TMJ fibrocartilaginous cells than knee meniscus cells to E2, providing an explanation for the differential responses of these tissues to E2. Using MMP9- and MMP13-null mice, we observed no discernible effects of each proteinase individually to E2-mediated TMJ matrix loss but noted a significant compensatory reciprocal induction of each MMP by E2 in the absence of the other. The redundancy in E2’s induction of MMP9 and MMP13 suggests that the proteinases may together contribute to E2-mediated TMJ fibrocartilage loss. These results advance our understanding of E2-mediated upregulation of MMP9 and MMP13 on fibrocartilage matrix turnover targeted to the TMJ.

17β-estradiol Induces MMP-9 and MMP-13 in TMJ Fibrochondrocytes via Estrogen Receptor α

Temporomandibular joint (TMJ) disorders, including degenerative TMJ disease, occur primarily in women of reproductive age. Previous studies showed elevated estrogen levels in subjects with TMJ disorders relative to controls and the presence of estrogen receptors α and β (ERα and ERβ) in TMJ fibrocartilage. Additionally, estrogen-induced overexpression of specific matrix metalloproteinases (MMPs), including MMP-9 and MMP-13, in TMJ fibrocartilage is accompanied by loss of extracellular matrices. However, the contribution of ERα and ERβ in estrogen-mediated induction of MMP-9 and MMP-13 and the signaling cascade leading to the upregulation of these MMPs have not been elucidated. Here, we show that specific siRNAs and selective ER antagonists effectively block ERα or ERβ expression in primary mouse TMJ fibrochondrocytes, but that only blockage of ERα suppresses MMP-9 and MMP-13 levels induced by 17β-estradiol (E2). Overexpression of ERα but not ERβ enhances E2-induced MMP-9. Using the same loss-of-function and gain-of-function approaches, we demonstrate that E2 stimulates ERK activation through ERα and that inhibition of ERK phosphorylation reduces E2-induced MMP-9. Furthermore, we reveal that E2 promotes NF-κB and ELK-1 activation through ERα/ERK signaling and that knockdown of either one decreases the respective activity of these signaling mediators and MMP-9 expression induced by E2, indicating that both contribute to E2/ERα/ERK-mediated MMP-9 upregulation. This is supported by findings in which mutated binding sites of either NF-κB or ELK-1 in the MMP-9 promoter lead to a significant reduction of E2-stimulated promoter activity. Our findings provide novel molecular mechanisms for the understanding of E2-mediated upregulation of MMPs, having implications to pathophysiologic TMJ cartilage matrix turnover that may yield therapeutic intervention targets for TMJ disorders.

Selective Role of Discoidin Domain Receptor 2 in Murine Temporomandibular Joint Development and Aging

Temporomandibular joint (TMJ) disorders are often associated with development of osteoarthritis-like changes in the mandibular condyle. Discoidin domain receptor 2 (DDR2), a collagen receptor preferentially activated by type I and III collagen found in the TMJ and other fibrocartilages, has been associated with TMJ degeneration, but its role in normal joint development has not been previously examined. Using Ddr2 LacZ-tagged mice and immunohistochemistry, we found that DDR2 is preferentially expressed and activated in the articular zone of TMJs but not knee joints. To assess the requirement for Ddr2 in TMJ development, studies were undertaken to compare wild-type and smallie (slie) mice, which contain a spontaneous deletion in Ddr2 to produce an effective null allele. Analysis of TMJs from newborn Ddr2slie/slie mice revealed a developmental delay in condyle mineralization, as measured by micro–computed tomography and histologic analysis. In marked contrast, knee joints of Ddr2slie/slie mice were normal. Analysis of older Ddr2slie/slie mice (3 and 10 mo) revealed that the early developmental delay led to a dramatic and progressive loss of TMJ articular integrity and osteoarthritis-like changes. Mutant condyles had a rough and flattened bone surface, accompanied by a dramatic loss of bone mineral density. Mankin scores showed significantly greater degenerative changes in the TMJs of 3- and 10-mo-old Ddr2slie/slie mice as compared with wild-type controls. No DDR2-dependent degenerative changes were seen in knees. Analysis of primary cultures of TMJ articular chondrocytes from wild-type and Ddr2slie/slie mice showed defects in chondrocyte maturation and mineralization in the absence of Ddr2. These studies demonstrate that DDR2 is necessary for normal TMJ condyle development and homeostasis and that these DDR2 functions are restricted to TMJ fibrocartilage and not seen in the hyaline cartilage of the knee.

Unilateral Impact of Altered Loading by Changing Teeth Height on the TMJ Fibrocartilage: Disc and Condyle of Wistar Rats

Temporomandibular joint (TMJ) is sensitive to loading and mechanical stress that provokes morphological changes produced by the impact in the occlusal plane. Here, this impact is evaluated in TMJ articular disc and articular cartilage using an in vivo model of unilateral occlusal plane impact and by analysis of serial tissue sections stained with Hematoxylin-Eosin (H-E) or with Masson trichrome technique. Thus, six groups of 5 Wistar rats (200 - 250 g) are subjected to biomechanical dental stimulation by placing unilateral resin occlusal interference, or unilateral tooth wear made by upper left molars artificial mechanical devastation (1 control and 2 experimental groups for each treatment). Each treatment is evaluated two times at 1 and 15 days post-treatment. By H-E staining, control groups show chondrocytes arrangement as several cord cell groups in comparison with the experimental groups, which show an arrangement in one cord cell along of articular disc. However, this yields no significant difference (p < 0.05) in cell number between control and experimental groups. In contrast, in articular cartilage chondrocytes are random distributed along the superficial zone in control groups, while in experimental groups cell-free regions are observed in superficial zone. An image Blue hue analysis for trichrome stain is performed to quantify collagen; this shows a significant collagen decrease (p < 0.05) in almost all experimental groups compared with the controls. A degenerative process biomechanically induced by unilateral occlusal plane modification, causes cell and tissue changes on the TMJ structures that remain the degenerative changes observed in early osteoarthritis.

Temporomandibular joint fibrocartilage degeneration from unilateral dental splints

ObjectiveThe objective of this study was to determine the extent to which altered loading in the temporomandibular joint (TMJ), as might be associated with a malocclusion, drives degeneration of articulating surfaces in the TMJ. We therefore sought to quantify the effects of altered joint loading on the mechanical properties and biochemical content and distribution of TMJ fibrocartilage in the rabbit. DesignAltered TMJ loading was induced with a 1mm splint placed unilaterally over the maxillary and mandibular molars for 6 weeks. At that time, TMJ fibrocartilage was assessed by compression testing, biochemical content (collagen, glycosaminoglycan (GAG), DNA) and distribution (histology), for both the TMJ disc and the condylar fibrocartilage. ResultsThere were no changes in the TMJ disc for any of the parameters tested. The condylar fibrocartilage from the splinted animals was significantly stiffer and the DNA content was significantly lower than that in control animals. There was significant remodeling in the condylar fibrocartilage layers as manifested by a change in GAG and collagen II distribution and a loss of defined cell layers. ConclusionsA connection between the compressive properties of TMJ condylar fibrocartilage after 6 weeks of splinting and the changes in histology was observed. These results suggest a change in joint loading leads to condylar damage, which may contribute to pain associated with at least some forms of TMJ disease.

Aquaporin 1 expression in human normal temporomandibular disc

The aquaporins (AQPs) are a family of hydrophobic membrane channel proteins that are expressed in many epithelial, endothelial and other tissues and that participate in many physiological and pathological processes. In particular aquaporin-1 (AQP1), a member of this family proteins, is expressed by the masseter and infrahyoid muscles, meckel’s cartilage and submandibular gland. Literature data on the role and the localization of AQP1 in articular joints are very poor, and none providing data on the normal temporomandibular joint (TMJ) disc. Accordantly, the present paper prompted this immunohistochemical and western blotting investigation, on the presence and distribution of AQP1 in human TMJ discs without any degenerative changes, in order to elucidate further aspects of the TMJ homeostasis system and disc tribology. Twelve TMJ discs, were obtained bilaterally from six cadavers and they were processed for immunohistochemical and Western Blot analysis. The results showed an AQP1 immunoexpression in few fibroblasts-like cells and fibrochondrocytes of normal human TMJ discs demonstrating a constitutive expression of this protein. No difference between the disc regions was seen in normal specimens. Western blot analysis of disc samples confirm the physiological AQP1 expression. In this view it appears reasonable a physiological expression of AQP1 by TMJ fibrocartilage cells and interpretable as a physiological mechanism of tissue homeostasis. Further future interesting studies could be conducted to elucidate the full AQPs profile in TMJ pathological disc investigating their change of the expression patterns during TMJ disc diseases.

Assessment of growth factor treatment on fibrochondrocyte and chondrocyte co-cultures for TMJ fibrocartilage engineering

Treatments for patients suffering from severe temporomandibular joint (TMJ) dysfunction are limited, motivating the development of strategies for tissue regeneration. In this study, co-cultures of fibrochondrocytes (FCs) and articular chondrocytes (ACs) were seeded in agarose wells, and supplemented with growth factors, to engineer tissue with biomechanical properties and extracellular matrix composition similar to native TMJ fibrocartilage. In the first phase, growth factors were applied alone and in combination, in the presence or absence of serum, while in the second phase, the best overall treatment was applied at intermittent dosing. Continuous treatment of AC/FC co-cultures with TGF-β1 in serum-free medium resulted in constructs with glycosaminoglycan/wet weight ratios (12.2%), instantaneous compressive moduli (790kPa), relaxed compressive moduli (120kPa) and Young’s moduli (1.87MPa) that overlap with native TMJ disc values. Among co-culture groups, TGF-β1 treatment increased collagen deposition ∼20%, compressive stiffness ∼130% and Young’s modulus ∼170% relative to controls without growth factor. Serum supplementation, though generally detrimental to functional properties, was identified as a powerful mediator of FC construct morphology. Finally, both intermittent and continuous TGF-β1 treatment showed positive effects, though continuous treatment resulted in greater enhancement of construct functional properties. This work proposes a strategy for regeneration of TMJ fibrocartilage and its future application will be realized through translation of these findings to clinically viable cell sources.

Matrix Metalloproteinase Induction by Relaxin Causes Cartilage Matrix Degradation in Target Synovial Joints

Our long-term goal is to understand the mechanisms by which relaxin and estrogen potentially contribute to joint diseases, particularly those afflicting the fibrocartilaginous temporomandibular joint (TMJ). Previously, we showed that relaxin produces a dose-dependent induction of tissue-degrading enzymes of the matrix metalloproteinase (MMP) family, specifically MMP-1 (collagenase-1), MMP-3 (stromelysin-1), MMP-9 (92-kDa gelatinase), and MMP-13 (collagenase-3) in cell isolates and tissue explants from TMJ fibrocartilage. The induction of these MMPs is accompanied by loss of collagen and glycosaminoglycans (GAGs), which was blocked by a pan-MMP inhibitor. We also found the targeted in vivo loss of collagen and GAGs in TMJ discs of ovariectomized rabbits treated with beta-estradiol, relaxin, or both hormones together. Progesterone attenuated the induction of MMPs and matrix loss by relaxin and estrogen. The modulation of matrix composition in TMJ fibrocartilage by these hormones was similar to that observed in the pubic symphysis and differed from that of the knee meniscus. The two target tissues showing the greatest modulation of MMPs and matrix loss, namely, the TMJ disc and pubic symphysis, had similar expression profiles of the estrogen receptors alpha and beta, relaxin-1 receptor (RXFP1, LGR7), and insulin-like peptide 3 receptor (RXFP2, LGR8) and these profiles differed from those in cells from the knee meniscus. These findings suggest a novel model for targeted tissue turnover of cartilage of specific joints through hormone-mediated induction of select MMPs.

Hyaline cartilage cells outperform mandibular condylar cartilage cells in a TMJ fibrocartilage tissue engineering application

To compare temporomandibular joint (TMJ) condylar cartilage cells in vitro to hyaline cartilage cells cultured in a three-dimensional (3D) environment for tissue engineering of mandibular condylar cartilage. Mandibular condylar cartilage and hyaline cartilage cells were harvested from pigs and cultured for 6 weeks in polyglycolic acid (PGA) scaffolds. Both types of cells were treated with glucosamine sulfate (0.4 mM), insulin-like growth factor-I (IGF-I) (100 ng/ml) and their combination. At weeks 0 and 6, cell number, glycosaminoglycan (GAG) and collagen content were determined, types I and II collagen were visualized by immunohistochemistry and GAGs were visualized by histology. Hyaline cartilage cells produced from half an order to a full order of magnitude more GAGs and collagen than mandibular condylar cartilage cells in 3D culture. IGF-I was a highly effective signal for biosynthesis with hyaline cartilage cells, while glucosamine sulfate decreased cell proliferation and biosynthesis with both types of cells. In vitro culture of TMJ condylar cartilage cells produced a fibrous tissue with predominantly type I collagen, while hyaline cartilage cells formed a fibrocartilage-like tissue with types I and II collagen. The combination of IGF and glucosamine had a synergistic effect on maintaining the phenotype of TMJ condylar cells to generate both types I and II collagen. Given the superior biosynthetic activity by hyaline cartilage cells and the practical surgical limitations of harvesting cells from the TMJ of a patient requiring TMJ reconstruction, cartilage cells from elsewhere in the body may be a potentially better alternative to cells harvested from the TMJ for TMJ tissue engineering. This finding may also apply to other fibrocartilages such as the intervertebral disc and knee meniscus in applications where a mature cartilage cell source is desired.

Accelerated osteoarthritis in the temporomandibular joint of biglycan/fibromodulin double-deficient mice

To investigate whether the absence of biglycan and fibromodulin, two proteoglycans expressed in cartilage, bone and tendon, resulted in accelerated osteoarthritis in the temporomandibular joint (TMJ). Histological sections of TMJ from 3-, 6-, 9- and 18-month-old wild-type (WT) and biglycan/fibromodulin double-deficient (DKO) mice were compared. Immuno-stainings for biglycan, fibromodulin and proliferating cell nuclear antigen (PCNA) were performed. Biglycan and fibromodulin were highly expressed in the disc and articular cartilage of the TMJ. At 3 months of age, both WT and DKO presented early signs of cartilage degeneration visible as small acellular areas under the articular surfaces and superficial waving. From 6 months of age, DKOs developed accelerated osteoarthritis compared to WT. At 6 months, small vertical clefts in the condylar cartilage and partial disruption of the disk were visible in the DKO. In addition, chondrocytes had lost their regular columnar organization to form clusters. At 9 months, these differences were even more pronounced. At 18 months, extended cartilage erosion was visible in DKOs when by comparison the thickness of the articular cartilage in WT controls was basically intact. PCNA staining was stronger in 3-month-old WT TMJ fibrocartilage than in 3-month-old DKO TMJ fibrocartilage suggesting that chondrocyte proliferation might be impaired in DKOs. The biglycan/fibromodulin double knock-out mouse constitutes a useful animal model to decipher the pathobiology of osteoarthritis in the TMJ.

Mandibular exercises to control bruxism and deviation problems.

A discussion of bruxism, mandibular deviation and hypermobility problems producing temporomandibular joint (TMJ) and masticatory muscle pathology is presented. The pathophysiology of painful masticatory muscle hyperactivity, TMJ dysfunction and chondromalacia are presented. The role that pain mediators play in the degradation of TMJ fibrocartilage, acute synovitis and joint and muscle pain is analyzed. Isokinetic and stretching exercises are described to assist in correcting or preventing pathology and dysfunction of these structures.