Layering Poly (lactic-co-glycolic acid)-based electrospun membranes and co-culture cell sheets for engineering temporomandibular joint disc.

The temporomandibular joint disk (TMJD) lacks blood vessels and is characterized by slow self-repair. Qualitative lesions in TMJD are difficult to repair. In this study, electrospun poly (lactic-co-glycolic acid) (PLGA) scaffolds were used to reconstruct temporomandibular joint discs by tissue engineering. Rabbit temporomandibular joint disc cells (TMJDCs) and rabbit synovium-derived mesenchymal stem cells (SMSCs) were co-cultured in 1:1 ratios. Cell sheets were induced by ascorbic acid incubated with electrospun PLGA scaffolds for 14 days in the presence (10 ng/ml in culture medium) or absence of TGF-ß3. Dimethylmethylene Blue Assay (DMMB) was used to determine the content of glycosaminoglycans in the extracellular matrix. The expression of Col1a1, Col2a1, Sox-9 and Runx-2 was quantified by RT-PCR, and the expression of type II collagen was observed by immunofluorescent staining. After 14 days of cultivation, the electrospun PLGA scaffold-loaded cell sheets could form an articular disc tissue with certain morphological characteristics. The expression of chondrogenic-related genes (Col2a1, Sox-9) and the secretion of extracellular matrix (GAG, type II collagen) in the co-culture group were close to those in the TMJDC group alone. The results suggest that PLGA electrospun scaffold-loaded co-cultured cell membrane could be used in the tissue engineering reconstruction of the temporomandibular joint disc.

Fibro/chondrogenic differentiation of dental stem cells into chitosan/alginate scaffolds towards temporomandibular joint disc regeneration.

Tissue engineering (TE) may provide effective alternative treatment for challenging temporomandibular joint (TMJ) pathologies associated with disc malpositioning or degeneration and leading to severe masticatory dysfunction. Aim of this study was to evaluate the potential of chitosan/alginate (Ch/Alg) scaffolds to promote fibro/chondrogenic differentiation of dental pulp stem cells (DPSCs) and production of fibrocartilage tissue, serving as a replacement of the natural TMJ disc. Ch/Alg scaffolds were fabricated by crosslinking with CaCl2 combined or not with glutaraldehyde, resulting in two scaffold types that were physicochemically characterized, seeded with DPSCs or human nucleus pulposus cells (hNPCs) used as control and evaluated for cell attachment, viability, and proliferation. The DPSCs/scaffold constructs were incubated for up to 8 weeks and assessed for extracellular matrix production by means of histology, immunofluorescence, and thermomechanical analysis. Both Ch/Alg scaffold types with a mass ratio of 1:1 presented a gel-like structure with interconnected pores. Scaffolds supported cell adhesion and long-term viability/proliferation of DPSCs and hNPCs. DPSCs cultured into Ch/Alg scaffolds demonstrated a significant increase of gene expression of fibrocartilaginous markers (COLI, COL X, SOX9, COM, ACAN) after up to 3 weeks in culture. Dynamic thermomechanical analysis revealed that scaffolds loaded with DPSCs significantly increased storage modulus and elastic response compared to cell-free scaffolds, obtaining values similar to those of native TMJ disc. Histological data and immunochemical staining for aggrecan after 4 to 8 weeks indicated that the scaffolds support abundant fibrocartilaginous tissue formation, thus providing a promising strategy for TMJ disc TE-based replacement.

Effect of mechanical loading on the metabolic activity of cells in the temporomandibular joint: a systematic review.

OBJECTIVES: The purpose of this systematic review was to elucidate how different modalities and intensities of mechanical loading affect the metabolic activity of cells within the fibro-cartilage of the temporomandibular joint (TMJ). MATERIALS AND METHODS: A systematic review was conducted according to PRISMA guidelines using PubMed, Embase, and Web of Science databases. The articles were selected following a priori formulated inclusion criteria (viz., in vivo and in vitro studies, mechanical loading experiments on TMJ, and the response of the TMJ). A total of 254 records were identified. After removal of duplicates, 234 records were screened by assessing eligibility criteria for inclusion. Forty-nine articles were selected for full-text assessment. Of those, 23 were excluded because they presented high risk of bias or were reviews. Twenty-six experimental studies were included in this systematic review: 15 in vivo studies and 11 in vitro ones. CONCLUSION: The studies showed that dynamic mechanical loading is an important stimulus for mandibular growth and for the homeostasis of TMJ cartilage. When this loading is applied at a low intensity, it prevents breakdown of inflamed cartilage. Yet, frequent overloading at excessive levels induces accelerated cell death and an increased cartilage degradation. CLINICAL SIGNIFICANCE: Knowledge about the way temporomandibular joint (TMJ) fibrocartilage responds to different types and intensities of mechanical loading is important to improve existing treatment protocols of degenerative joint disease of the TMJ, and also to better understand the regenerative pathway of this particular type of cartilage.

Choosing sheep (Ovis aries) as animal model for temporomandibular joint research: Morphological, histological and biomechanical characterization of the joint disc.

Preclinical trials are essential to the development of scientific technologies. Remarkable molecular and cellular research has been done using small animal models. However, significant differences exist regarding the articular behavior between these models and humans. Thus, large animal models may be more appropriate to perform trials involving the temporomandibular joint (TMJ). The aim of this work was to make a morphological (anatomic dissection and white light 3D scanning system), histological (TMJ in bloc was removed for histologic analysis) and biomechanical characterization (tension and compression tests) of sheep TMJ comparing the obtained results with human data. Results showed that sheep processus condylaris and fossa mandibularis are anatomically similar to the same human structures. TMJ disc has an elliptical perimeter, thinner in the center than in periphery. Peripheral area acts as a ring structure supporting the central zone. The disc cells display both fibroblast and chondrocyte-like morphology. Marginal area is formed by loose connective tissue, with some chondrocyte-like cells and collagen fibers in diverse orientations. Discs obtained a tensile modulus of 3.97±0.73MPa and 9.39±1.67MPa, for anteroposterior and mediolateral assessment. The TMJ discs presented a compressive modulus (E) of 446.41±5.16MPa and their maximum stress value (σmax) was 18.87±1.33MPa. Obtained results suggest that these animals should be considered as a prime model for TMJ research and procedural training. Further investigations in the field of oromaxillofacial surgery involving TMJ should consider sheep as a good animal model due to its resemblance of the same joint in humans.

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

OBJECTIVE: 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. DESIGN: 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. RESULTS: 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). CONCLUSIONS: 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.

OBJECTIVE: 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. DESIGN: 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. RESULTS: 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. CONCLUSIONS: 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 17ß-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.

Alterations in intermediate filaments expression in disc cells from the rat temporomandibular joint following exposure to continuous compressive force.

The articular disc in the temporomandibular joint (TMJ) that serves in load relief and stabilizing in jaw movements is a dense collagenous tissue consisting of extracellular matrices and disc cells. The various morphological configurations of the disc cells have given us diverse names, such as fibroblasts, chondrocyte-like cells and fibrochondrocytes; however, the characteristics of these cells have remained to be elucidated in detail. The disc cells have been reported to exhibit heterogeneous immunoreaction patterns for intermediate filaments including glial fibrillary acidic protein (GFAP), nestin and vimentin in the adult rat TMJ. Because these intermediate filaments accumulate in the disc cells as tooth eruption proceeds during postnatal development, it might be surmised that the expression of these intermediate filaments in the disc cells closely relates to mechanical stress. The present study was therefore undertaken to examine the effect of a continuous compressive force on the immunoexpression of these intermediate filaments and an additional intermediate filament - muscle-specific desmin - in the disc cells of the TMJ disc using a rat experimental model. The rats wore an appliance that exerts a continuous compressive load on the TMJ. The experimental period with the appliance was 5 days as determined by previous studies, after which some experimental animals were allowed to survive another 5 days after removal of the appliance. Histological observations demonstrated that the compressive force provoked a remarkable acellular region and a decrease in the thickness of the condylar cartilage of the mandible, and a sparse collagen fiber distribution in the articular disc. The articular disc showed a significant increase in the number of desmin-positive cells as compared with the controls. In contrast, immunopositive cells for GFAP, nestin and vimentin remained unchanged in number as well as intensity. At 5 days after removal of the appliance, both the disc and cartilage exhibited immunohistological and histological features in a recovery process. These findings indicate that the mature articular cells are capable of producing desmin instead of the other intermediate filaments against mechanical stress. The desmin-positive disc cells lacked α-smooth muscle actin (α-SMA) in this study, even though desmin usually co-exists with α-SMA in the vascular smooth muscle cells or pericytes. Because the precursor of a pericyte has such an immunoexpression pattern during angiogenesis, there is a further possibility that the formation of new vessels commenced in response to the extraordinary compressive force.

[Effects of basic fibroblast growth factor on bone marrow mesenchymal stem cell differentiation into temporomandibular joint disc cells].

The present paper is aimed to observe the effects of basic fibroblast growth factor (bFGF) on bone marrow mesenchymal stem cell (BMSCs) differentiation. The bFGF was used to stimulate BMSCs and histology, immunohistochemistry and enzyme linked immunosorbent assay (ELISA) were used to examine the extracellular matrix produced by induced BMSCs, evaluated the feasibility of BMSCs being the seeding cells of temporomandibular joint (TMJ) disc tissue engineering. The results showed that having been induced with bFGF, the BMSCs could differentiate into fibroblast-like cells, which could synthesize GAG and collagen type I matrix. So it is feasible for BMSCs as seeding cells for engineered TMJ disc.

Regional cell density distribution and oxygen consumption rates in porcine TMJ discs: an explant study.

OBJECTIVE: To determine the regional cell density distribution and basal oxygen consumption rates (based on tissue volume and cell number) of temporomandibular joint (TMJ) discs and further examine the impact of oxygen tension on these rates. DESIGN: TMJ discs from pigs aged 6-8 months were divided into five regions: anterior, intermediate, posterior, lateral and medial. The cell density was determined using confocal laser scanning microscopy. The change in oxygen tension was recorded while TMJ disc explants were cultured in sealed metabolism chambers. The volume based oxygen consumption rate of explants was determined by theoretical curve-fitting of the recorded oxygen tension data with the Michaelis-Menten equation. The rate on a per-cell basis was calculated based on the cell density measurements and volume based rate measured in another group of discs. RESULTS: The overall cell density [mean, 95% confidence interval (CI)] was 51.3 (21.3-81.3) × 10(6) cells/mL wet tissue. Along the anteroposterior axis, the anterior band had 25.5% higher cell density than the intermediate zone (P<0.02) and 29.1% higher than the posterior band (P<0.008). Along the mediolateral axes, the medial region had 26.2% higher cell density than the intermediate zone (P<0.04) and 25.4% higher than the lateral region (P<0.045). The overall volume and cell based maximum oxygen consumption rates were 1.44 (0.44-2.44) µmol/mL wet tissue/h and 28.7 (12.2-45.2)nmol/10(6)cells/h, respectively. The central regions (intermediate, lateral, and medial) had significantly higher volume based (P<0.02) and cell based (P<0.005) oxygen consumption rates than the anterior and posterior bands. At high oxygen tension, the oxygen consumption rate remained constant, but dropped as oxygen tension fell below 5%. CONCLUSIONS: The TMJ disc had higher cell density and oxygen consumption rates than articular cartilage reported in the literature. These results suggest that a steeper oxygen gradient may exist in the TMJ disc and may be vulnerable to pathological events that impede nutrient supply.

[Cell sources for engineered temporomandibular joint disc tissue: present and future].

The purpose of this review is to provide a reference for researchers in investigating the tissue engineering of the temporomandibular joint (TMJ) disc. Currently tissue engineering of the TMJ disc is in its infancy, and cell source is one of the key factors that define the development of the tissue engineering of TMJ disc. In this paper, 6 kinds of cells: the TMJ disc native cells, chondrocytes, dermal fibroblasts, bone marrow-derived mesenchymal stem cells, adipose-derived stem cells, and embryonic stem cells are introduced. In addition, the possibility that these cells can be used as cell sources for TMJ disc tissue engineering is described.

Alteration of structural and mechanical properties of the temporomandibular joint disc following elastase digestion.

The temporomandibular joint disc is a fibrocartilaginous structure, composed of collagen fibers, elastin fibers, and proteoglycans. Despite the crucial role of elastin fibers in load-bearing properties of connective tissues, its contribution in temporomandibular joint disc biomechanics has been disregarded. This study attempts to characterize the structural-functional contribution of elastin in the temporomandibular joint disc. Using elastase, we selectively perturbed the elastin fiber network in porcine temporomandibular joint discs and investigated the structural, compositional, and mechanical regional changes through: (a) analysis of collagen and elastin fibers by immunolabeling and transmission electron microscopy; (b) quantitative analysis of collagen tortuosity, cell shape, and disc volume; (c) biochemical quantification of collagen, glycosaminoglycan and elastin content; and (d) cyclic compression test. Following elastase treatment, microscopic examination revealed fragmentation of elastin fibers across the temporomandibular joint disc, with a more pronounced effect in the intermediate regions. Also, biochemical analyses of the intermediate regions showed significant depletion of elastin (50%), and substantial decrease in collagen (20%) and glycosaminoglycan (49%) content, likely due to non-specific activity of elastase. Degradation of elastin fibers affected the homeostatic configuration of the disc, reflected in its significant volume enlargement accompanied by remarkable reduction of collagen tortuosity and cell elongation. Mechanically, elastase treatment nearly doubled the maximal energy dissipation across the intermediate regions while the instantaneous modulus was not significantly affected. We conclude that elastin fibers contribute to the restoration and maintenance of the disc resting shape and actively interact with collagen fibers to provide mechanical resilience to the temporomandibular joint disc.

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

OBJECTIVE: The effects of the staurosporine on contraction of self-assembled constructs and extracellular matrix syntheses of goat temporomandibular joint discs were investigated. METHODS: 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). RESULTS: 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). CONCLUSIONS: 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.

Tissue Engineering for the Temporomandibular Joint.

Tissue engineering potentially offers new treatments for disorders of the temporomandibular joint which frequently afflict patients. Damage or disease in this area adversely affects masticatory function and speaking, reducing patients' quality of life. Effective treatment options for patients suffering from severe temporomandibular joint disorders are in high demand because surgical options are restricted to removal of damaged tissue or complete replacement of the joint with prosthetics. Tissue engineering approaches for the temporomandibular joint are a promising alternative to the limited clinical treatment options. However, tissue engineering is still a developing field and only in its formative years for the temporomandibular joint. This review outlines the anatomical and physiological characteristics of the temporomandibular joint, clinical management of temporomandibular joint disorder, and current perspectives in the tissue engineering approach for the temporomandibular joint disorder. The tissue engineering perspectives have been categorized according to the primary structures of the temporomandibular joint: the disc, the mandibular condyle, and the glenoid fossa. In each section, contemporary approaches in cellularization, growth factor selection, and scaffold fabrication strategies are reviewed in detail along with their achievements and challenges.

Clinically relevant cell sources for TMJ disc engineering.

Tissue-engineering of the temporomandibular joint (TMJ) disc aims to provide patients with TMJ disorders an option to replace diseased tissue with autologous, functional tissue. This study examined clinically relevant cell sources by comparing costal chondrocytes, dermal fibroblasts, a mixture of the two, and TMJ disc cells in a scaffoldless tissue-engineering approach. It was hypothesized that all constructs would produce matrix relevant to the TMJ disc, but the mixture constructs were expected to appear most like the TMJ disc constructs. Costal chondrocyte and mixture constructs were morphologically and biochemically superior to the TMJ disc and dermal fibroblast constructs, and their compressive properties were not significantly different. Costal chondrocyte constructs produced almost 40 times more collagen and 800 times more glycosaminoglycans than did TMJ constructs. This study demonstrates the ability of costal chondrocytes to produce extracellular matrix that may function in a TMJ disc replacement.

Scaffold and growth factor selection in temporomandibular joint disc engineering.

Temporomandibular joint disc tissue-engineering studies commonly fail to produce significant matrix before construct contraction. We hypothesized that poly-L-lactic acid (PLLA) non-woven meshes would limit contraction, allow for comprehensive mechanical evaluation, and maintain viability relative to polyglycolic acid (PGA) non-woven mesh controls. Additionally, we proposed that growth factor stimulation, while limiting contraction, would increase construct properties relative to previous reports. After 4 wks, cell proliferation and matrix deposition were similar between the two meshes, but PGA constructs had contracted significantly. Furthermore, only PLLA constructs could be tested in tension and compression. Additional PLLA constructs were formed, then treated with insulin-like growth factor-1 (10 ng/mL), transforming growth factor-beta 1 (5 ng/mL), or transforming growth factor-beta 3 (5 ng/mL). Transforming growth factor-beta 1 yielded the most cells, collagen, and glycosaminoglycans at 6 wks; these constructs also demonstrated improved mechanics. Analysis of these data demonstrated significant temporomandibular joint disc-engineering potential for PLLA and transforming growth factor-beta 1.

A mechanical evaluation of three decellularization methods in the design of a xenogeneic scaffold for tissue engineering the temporomandibular joint disc.

Tissue-engineered temporomandibular joint (TMJ) discs offer a viable treatment option for patients with severe joint internal derangement. To date, only a handful of TMJ tissue engineering studies have been carried out and all have incorporated the use of synthetic scaffold materials. These current scaffolds have shown limited success in recapitulating morphological and functional aspects of the native disc tissue. The present study is the first to investigate the potential of a xenogeneic scaffold for use in tissue engineering the TMJ disc. The effects of decellularization agents on the disc's mechanical properties were assessed using three common decellularization protocols: Triton X-100, sodium dodecyl sulfate (SDS) and an acetone/ethanol solution. Decellularized scaffolds were subsequently characterized through cyclic mechanical testing at physiologically relevant frequencies to determine which chemical agent most accurately preserved the native tissue properties. Results have shown that porcine discs treated with SDS most closely matched the energy dissipation capabilities and resistance to deformation of the native tissue. Treatments using Triton X-100 caused the resultant tissue to become relatively softer with inferior energy dissipation capabilities, while treatment using acetone/ethanol led to a significantly stiffer and dehydrated material. These findings support the potential of a porcine-derived scaffold decellularized by SDS as a xenograft for TMJ disc reconstruction.

The effects of protein-coated surfaces on passaged porcine TMJ disc cells.

OBJECTIVE: Implantation of synthetic temporomandibular joint (TMJ) disc replacements aimed to alleviate pain and restore functional losses caused by TMJ disorders. Unfortunately, these synthetic replacements have been largely unsuccessful and in some instances have incited severe immune responses. Tissue engineering, however, may provide viable TMJ disc replacements. Towards this end, we have studied TMJ disc gene expression as a measure of protein production potential. With passage, collagen type I and aggrecan gene expression decrease in TMJ disc cell cultures. We hypothesize that surfaces coated with TMJ disc proteins may rapidly recover the lost gene expression in passaged TMJ disc cells. DESIGN: To study these effects, passages 0, 1, and 2 TMJ disc cells were plated in wells coated with aggrecan, collagen type I, collagen type II, or decorin. Safranin O staining was conducted to visualize cell aggregation. RESULTS: At passage 0, cultures appeared similar on each surface; however, by passages 1 and 2, aggrecan-coated and decorin-coated surfaces appeared to have more cell aggregates. Gene expression data did not correspond to these visual changes. No treated surface offered a significant change in aggrecan, collagen type I, or decorin expression relative to untreated controls. Furthermore, aggrecan and collagen type I gene expression dropped relative to samples taken prior to plating. CONCLUSIONS: These results indicate that, despite visual changes described by cell aggregates, protein coatings have limited effects for recovering TMJ disc gene expression in monolayer cultures.

Effect of passage and topography on gene expression of temporomandibular joint disc cells.

The temporomandibular joint (TMJ) disc is maintained by a population of fibrochondrocytes. Although articular chondrocytes exhibit zonal differences and de-differentiate in monolayer culture, such variations are unknown for fibrochondrocytic populations. This study's objective was to define topographical cellular variations in the porcine TMJ disc and investigate changes in the disc's gene expression levels over multiple passages using quantitative reverse transcriptase polymerase chain reaction. For topographical characterization, samples were acquired from posterior, anterior, lateral, medial, and intermediate zone sections and subdivided into inferior and superior halves. For passage characterization, cells were plated and passaged for 35 days, with samples acquired at every passage. The medial region had the lowest expression of genes indicative of fibroblastic activity, but in general, topographical variations were limited. Passage effects were evident; gene expression levels of aggrecan, collagen type I, and collagen type II dropped 20%, 23%, and 73% per passage, respectively. In contrast, decorin and glyseraldehyde-3-phosphate dehydrogenase gene expression increased 33% and 27% per passage, respectively. These data indicate that TMJ disc cells undergo significant changes due to monolayer expansion, experiencing losses in major chondrocytic markers (aggrecan and collagen type II) and fibroblastic markers (collagen type I) and posing a serious impediment to studies in which cell passaging is required.

Improving culture conditions for temporomandibular joint disc tissue engineering.

BACKGROUND: The temporomandibular joint (TMJ) is extremely important for activities like eating and talking, which can become painful and difficult for patients with TMJ dysfunction. Tissue engineering is a potential alternative to current surgical interventions through replacement of diseased or injured tissue with a functional construct. Since research with TMJ disc cells began relatively recently, optimal culturing conditions must be determined. METHODS: Metabolic additives, L-glutamine, L-alanyl-L-glutamine, sodium pyruvate, and insulin, were examined for their effects on TMJ disc cells in monolayer. Effects of L-proline were examined in three-dimensional (3-D) culture at concentrations of 0, 25 and 100 mg/l. RESULTS: The combination of L-glutamine, sodium pyruvate, and insulin improved cell proliferation rates without affecting collagen production or gene expression. No differences were observed in mechanical properties of the engineered constructs; however, collagen and glycosaminoglycan quantities normalized to cell number decreased at the highest concentration of L-proline. CONCLUSION: This work identified supplements for 2-D monolayer expansion. Other supplements or culture conditions still need to be investigated for 3-D tissue production. This work improves upon porcine TMJ disc cell culturing conditions, taking us closer to being able to engineer the TMJ disc.