Use of a rotating bioreactor toward tissue engineering the temporomandibular joint disc

This objective of this study was to determine the effects of a rotating bioreactor in temporomandibular joint (TMJ) disc tissue engineering. Porcine TMJ disc cells were seeded at a density of 20 million cells/mL onto nonwoven poly(glycolic acid) (PGA) scaffolds in spinner flasks for 1 week and then cultured either under static conditions or in a rotating bioreactor for a period of 6 weeks. A series of analyses was performed, including mechanical testing, measurement of cellularity, quantification of matrix biosynthesis with a hydroxyproline assay and enzyme-linked immunosorbent assays, and observation of matrix distribution with immunohistochemistry. Between the bioreactor and static cultures, there were marked differences in gross appearance, histological structure, and distribution of collagen types I and II. Engineered constructs from the bioreactor contracted earlier and to a greater extent, resulting in a denser matrix and cell composition. In addition, immunostaining intensity was generally uniform in static constructs, in contrast to higher intensity around the periphery of bioreactor constructs. Moreover, bioreactor constructs had higher amounts of collagen II than did static constructs. However, differences in total matrix content and compressive stiffness were generally not significant. On the basis of the results of this study there is no clear benefit from use of the rotating bioreactor, although a sequence of static culture followed by rotating bioreactor culture may prove in the future to be more beneficial than either alone.     

The regional difference of viscoelastic property of bovine temporomandibular joint disc in compressive stress-relaxation

An in vitro experimental technique was performed to measure the viscoelastic properties of the bovine disc. Thirteen TMJ discs from young cattle (3-year-old) were used. Each disc was divided into five specimens of anterior, central, posterior, lateral and medial regions, and they were used for compression tests. A series of stress-relaxation tests was conducted for each specimen from 5% strain up to 20% strain with 5% intervals. The stress-relaxation was monitored over a period of 5 min. Each region exhibited a different biomechanical behavior, which is presumably related to the organization and distribution of proteoglycans that indirectly modulate the stiffness of the collagen network. It is suggested that an improved understanding of the viscoelastic properties of the disc under function may guide consideration for design and selection of biomaterials for TMJ reconstruction.     

The proteoglycan contents of the temporomandibular joint disc influence its dynamic viscoelastic properties

The collagen fibers and proteoglycans in the disc of temporomandibular joint provide resistance to various loadings. Thus far, however, the role of the proteoglycans in determining the viscoelastic properties of the disc has not been investigated. In the present study the hypothesis was tested that the viscoelastic behavior of the disc decreases by the removal of proteoglycans. In 32 bovine discs, dynamic tensile tests with a wide range of frequencies were performed. Before testing, specimens were treated with different concentrations of alpha-amylase to remove proteoglycans. As the frequency increased from 0.1 to 100 Hz, the disc became more viscoelastic. Increasing the concentration of alpha-amylase significantly decreased its viscoelasticity. It was concluded that proteoglycans play an important role in determining the viscoelastic properties of the disc and, therefore, give the disc a greater capacity for distributing and reducing stresses.     

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.     

Seeding techniques and scaffolding choice for tissue engineering of the temporomandibular joint disk

Temporomandibular joint (TMJ) disk removal, or diskectomy, is a detrimental yet necessary surgery for patients with extremely displaced disks. Tissue engineering is an enticing methodology for improvement of the postoperative outcome of diskectomy. Unfortunately, the field of tissue engineering of the TMJ disk is only in the early stages of development. The initial objective of this investigation was to study the cellular response of TMJ disk cells in alginate culture. However, a marked decrease in cell population and the lack of detection of extracellular matrix (ECM) products did not support the use of alginate culture. The second objective was then to attempt TMJ disk cell culture in polyglycolic acid (PGA) nonwoven meshes. However, as suitable seeding methods for TMJ disk cells on PGA had not been determined, three techniques were selected for study: spinner flask, orbital shaker, and a novel pelleting technique. PGA constructs maintained cellularity throughout the culture period, and scaffolds seeded with the spinner flask produced about 35 microg of collagen per construct. Thus, as evidenced by the production of a major extracellular component, PGA nonwoven meshes seeded with TMJ disk cells, using a spinner flask, may be a first positive culturing step in tissue engineering the TMJ disk.     

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.     

正常及关节盘移位后颞下颌关节滑膜OPG/OPGL的表达及意义

目的： 观察正常及关节盘移位后颞下颌关节滑膜内护骨素OPG及其配体OPGL的表达及意义。方法： 32只日本大耳白兔，25只建立颞下颌关节盘前移位的动物模型，术后1、2、4、8、12周处死，5只行模拟手术作为假手术对照，另2只不行手术作为空白对照。取颞下颌关节标本，HE染色观察镜下结构， S-P免疫组化法检测滑膜中OPG和OPGL的表达与分布。结果： 各实验组动物术后体重无减轻，伤口愈合良好，大体及显微镜下观察关节盘明显前移位。对照组OPG主要表达于滑膜下层血管内皮细胞及部分滑膜衬里细胞，滑膜内 OPGL无表达或鲜有表达。关节盘移位后，仅1、2周各1个样本滑膜下层少量OPGL表达，其它样本未见明显OPGL阳性细胞；OPG表达强度无减弱。结论： 正常颞下颌关节滑膜内OPG表达明显强于OPGL，对正常状态骨软骨代谢平衡的维持有重要意义；关节盘移位后OPG和OPGL无明显变化，保证了关节盘移位后骨软骨代谢能维持平衡。     

A comparison of human umbilical cord matrix stem cells and temporomandibular joint condylar chondrocytes for tissue engineering temporomandibular joint condylar cartilage

The temporomandibular joint (TMJ) presents many problems in modern musculoskeletal medicine. Patients who suffer from TMJ disorders often experience a major loss in quality of life due to the debilitating effects that TMJ disorders can have on everyday activities. Cartilage tissue engineering can lead to replacement tissues that could be used to treat TMJ disorders. In this study, a spinner flask was used for a period of 6 days to seed polyglycolic acid (PGA) scaffolds with either TMJ condylar chondrocytes or mesenchymal-like stem cells derived from human umbilical cord matrix (HUCM). Samples were then statically cultured for 4 weeks either in growth medium containing chondrogenic factors or in control medium. Immunohistochemical staining of HUCM constructs after 4 weeks revealed a strong presence of collagen I and minute amounts of collagen II, whereas TMJ constructs revealed little collagen I and no collagen II. The HUCM constructs were shown to contain more GAGs than the TMJ constructs quantitatively at week 0 and histologically at week 4. Moreover, the cellularity of HUCM constructs was 55% higher at week 0 and nearly twice as high after 4 weeks, despite being seeded at the same density. The increased level of biosynthesis and higher cellularity of HUCM constructs clearly demonstrates that the HUCM stem cells outperformed the TMJ condylar cartilage cells under the prescribed conditions. HUCM stem cells may therefore be an attractive alternative to condylar cartilage cells for TMJ tissue engineering applications. Further, given the availability and ease of obtaining HUCM stem cells, these findings may have far-reaching implications, leading to novel developments in both craniofacial and orthopaedic tissue replacement therapies.     

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.     

Soft tissue mechanics of the temporomandibular joint

Direct measurement of temporomandibular joint (TMJ) tissue deformation requires animal experimentation. Most of the available data pertain to the mechanical strain on the bone surfaces around the joint. However, bone is rarely the first joint tissue to show injury, being affected after damage to collagenous tissues such as the disc or capsule. Capsular ligaments guide or limit movement, while the intra-articular disc may also distribute joint loads. However, these tissues are difficult to visualize dynamically and not suitable for strain gage attachment, so in vivo deformations are poorly understood. Using pigs as the best nonprimate model for human TMJ function, we implanted differential variable reluctance transducers to measure antero-posterior strain in the lateral aspect of the intra-articular disc. The results were compared to previously published data on the TMJ capsule. Passive manipulation in anesthetized animals indicated that opening, protrusion, and contralateral movements caused the disc to elongate. On the contrary, closing, retrusion and ipsilateral movements caused disc shortening. These strains are opposite to those observed in the capsule and are expected on anatomical grounds. Surprisingly, disc strain during mastication differed from that during manipulation. The disc elongated during jaw closure, more on the retruding balancing side (16% +/- 1) than on the working side (8% +/- 2). This anomalous behavior may reflect compressive loading, such that the disc elongates as a result of the Poisson effect rather than condylar movement. Because the capsule also elongates during the power stroke, especially on the balancing side, both disc and capsule are maximally loaded on the same side at the same moment.     

关节盘移位对颞下颌关节内应力分布的影响

目的 通过对不同关节盘移位的数值模拟,探究各种移位情况下颞下颌关节（temporomandibular joint,TMJ）内各结构的应力分布规律。方法 依据CT图像,建立包含下颌骨、全牙列、关节盘和关节软骨的正常TMJ三维有限元模型;参考关节盘前、后、外、内移位的临床特征,建立对应的4个模型。关节盘与关节软骨间考虑接触,用缆索元模拟下颌韧带和关节盘附着,施加正中咬合荷载。结果 前移位将导致关节盘中带产生过高的压应力,达到3.23 MPa;后、内、外移位时关节盘的整体应力水平比前移位和正常TMJ高;各种移位都使关节结节后斜面的应力值大幅度增加,但对髁突关节面的影响却不大。结论 各种移位都将导致关节盘和关节结节后斜面产生过高的应力,且后、内、外移位更为危险,更容易造成关节结构和功能的损伤。     

Tensile stress patterns predicted in the articular disc of the human temporomandibular joint

The direction of the first principal stress in the articular disc of the temporomandibular joint was predicted with a biomechanical model of the human masticatory system. The results were compared with the orientation of its collagen fibers. Furthermore, the effect of an active pull of the superior lateral pterygoid muscle, which is directly attached to the articular disc, was studied. It was hypothesized that the markedly antero-posterior direction of the collagen fibers would be reflected in the direction of the tensile stresses in the disc and that active pull of the superior lateral pterygoid muscle would augment these tensions. It was found that the tensile patterns were extremely dependent on the stage of movement and on the mandibular position. They differed between the superior and inferior layers of the disc. The hypothesis could only be confirmed for the anterior and middle portions of the disc. The predicted tensile principal stresses in the posterior part of the disc alternated between antero-posterior and medio-lateral directions.     

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.     

Regional variations in canine descending aortic tissue mechanical properties change with formalin fixation

Background/introductionDiseases of the aorta can alter the local mechanical properties of the tissue, leading to aneurysms and plaque instability. Local tissue properties are best evaluated from surgical samples or autopsy tissue using mechanical testing ex vivo. We examined whether formalin-fixed tissues preserve regional and local variations in tissue properties when compared to fresh tissues in order to determine if fixed tissue can be used to infer mechanical changes associated with tissue remodeling.MethodsEquibiaxial mechanical tests were performed on canine descending thoracic aorta to quantify the regional and local tissue stiffness. Samples were taken from four locations along the aorta and from the lateral and medial side at each location. Half of the samples were randomly formalin fixed and used to measure the effect of fixation on local thickness, stiffness, and anisotropy.ResultsIn fresh tissue, regional differences in tissue stiffness and thickness are present. Aortic tissue stiffens and thins along the aorta. Fixation did not alter thickness, significantly increased tissue stiffness, and altered the directional dependency of the mechanical properties (anisotropy) at low strain. Formalin fixation altered local stiffness of the aorta near the aortic arch.ConclusionThe changes in mechanical properties along the aorta were preserved in formalin-fixed samples. However, our results show that formalin fixation can change the variation in tissue stiffness and significantly affects the anisotropic properties of vascular tissues. Formalin fixation introduces spurious changes in mechanical properties, and we therefore strongly recommend the use of fresh aortic tissues for biomechanical analysis.     

The pathology of joint replacement and tissue engineering

Joint replacement is very common and undertaken in most hospitals in one form or another. Tissue engineering of connective tissues generally, and joints in particular, is becoming more common. With increased usage, these techniques generate iatrogenic morbidity. The diagnosis and exclusion of iatrogenic disease is an increasingly important area of pathologists' working lives. This article discusses the disorders that can arise in association with joint replacement and tissue engineering of joints and describes a relatively new disease (Adverse Reaction to Metal Debris [ARMD]) the first of what may become many new disorders associated with the new therapeutics covered in this article.     

Regional variations in the cellular matrix of the annulus fibrosus of the intervertebral disc

The three-dimensional architecture of cells in the annulus fibrosus was studied by a systematic, histological examination using antibodies to cytoskeletal components, in conjunction with confocal microscopy. Variations in cell shape, arrangement of cellular processes and cytoskeletal architecture were found both within and between the defined zones of the outer and inner annulus. The morphology of three, novel annulus fibrosus cells is described: extended cordlike cells that form an interconnected network at the periphery of the disc; cells with extensive, sinuous processes in the inner region of the annulus fibrosus; and cells with broad, branching processes specific to the interlamellar septae of the outer annulus. The complex, yet seemingly deliberate arrangement of various cell shapes and their processes suggests multiple functional roles. Regional variations in the organization of the actin and vimentin cytoskeletal networks is reported across all regions of the annulus. Most notable is the continuous, strand arrangement of the actin label at the disc's periphery in contrast to its punctate appearance in all other regions. The gap junction protein connexin 43 was found within cells from all regions of the annulus, including those which did not form physical connections with surrounding cells. These observations of the cellular matrix in the healthy intervertebral disc should contribute to a better understanding of site-specific changes in tissue architecture, biochemistry and mechanical properties during degeneration, injury and healing.