Vincent P. Willard

An Interspecies Comparison of the Temporomandibular Joint Disc

The temporomandibular joint (TMJ) disc plays a critical role in normal function of the joint, and many disorders of the TMJ are a result of disc dysfunction. Previous quantitative TMJ characterization studies examined either the human or a specific animal model, but no single study has compared different species, in the belief that differences in joint morphology, function, and diet would be reflected in the material properties of the disc. In this study, we examined topographical biochemical (collagen, glycosaminoglycan, and DNA content) and biomechanical (tensile and compressive) properties of the human TMJ disc, and also discs from the cow, goat, pig, and rabbit. Regional and interspecies variations were identified in all parameters measured, and certain disc characteristics were observed across all species, such as a weak intermediate zone under mediolateral tension. While human discs possessed properties distinct from those of the other species, pig discs were most similar to the human, suggesting that the pig may be a suitable animal model for TMJ bioengineering efforts.

Fibrochondrogenesis of hESCs: Growth Factor Combinations and Cocultures

The successful differentiation of human embryonic stem cells (hESCs) to fibrochondrocyte-like cells and characterization of these differentiated cells is a critical step toward tissue engineering of musculoskeletal fibrocartilages (e.g., knee meniscus, temporomandibular joint disc, and intervertebral disc). In this study, growth factors and primary cell cocultures were applied to hESC embryoid bodies (EBs) for 3 weeks and evaluated for their effect on the synthesis of critical fibrocartilage matrix components: glycosaminoglycans (GAG) and collagens (types I, II, and VI). Changes in surface markers (CD105, CD44, SSEA, PDGFR alpha) after the differentiation treatments were also analyzed. The study was conducted in three phases: (1) examination of growth factors (TGF-beta 3, BMP-2, BMP-4, BMP-6, PDGF-BB, sonic hedgehog protein); (2) comparison of two cocultures (primary chondrocytes or fibrochondrocytes); and (3) the combination of the most effective growth factor and coculture regimen. TGF-beta 3 with BMP-4 yielded EBs positive for collagens I, II, and VI, with up to 6.7- and 4.8-fold increases in GAG and collagen, respectively. Analysis of cell surface markers showed a significant increase in CD44 with the TGF-beta 3 + BMP-4 treatment compared to the controls. Coculture with fibrochondrocytes resulted in up to a 9.8-fold increase in collagen II production. The combination of the growth factors BMP-4 + TGF-beta 3 with the fibrochondrocyte coculture led to an increase in cell proliferation and GAG production compared to either treatment alone. This study determined two powerful treatments for inducing fibrocartilaginous differentiation of hESCs and provides a foundation for using flow cytometry to purify these differentiated cells.

Mechanical characterization of differentiated human embryonic stem cells

Human embryonic stem cells (hESCs) possess an immense potential in a variety of regenerative applications. A firm understanding of hESC mechanics, on the single cell level, may provide great insight into the role of biophysical forces in the maintenance of cellular phenotype and elucidate mechanical cues promoting differentiation along various mesenchymal lineages. Moreover, cellular biomechanics can provide an additional tool for characterizing stem cells as they follow certain differentiation lineages, and thus may aid in identifying differentiated hESCs, which are most suitable for tissue engineering. This study examined the viscoelastic properties of single undifferentiated hESCs, chondrogenically differentiated hESC subpopulations, mesenchymal stem cells (MSCs), and articular chondrocytes (ACs). hESC chondrogenesis was induced using either transforming growth factor-beta1 (TGF-beta1) or knock out serum replacer as differentiation agents, and the resulting cell populations were separated based on density. All cell groups were mechanically tested using unconfined creep cytocompression. Analyses of subpopulations from all differentiation regimens resulted in a spectrum of mechanical and morphological properties spanning the range of hESCs to MSCs to ACs. Density separation was further successful in isolating cellular subpopulations with distinct mechanical properties. The instantaneous and relaxed moduli of subpopulations from TGF-beta1 differentiation regimen were statistically greater than those of undifferentiated hESCs. In addition, two subpopulations from the TGF-beta1 group were identified, which were not statistically different from native articular chondrocytes in their instantaneous and relaxed moduli, as well as their apparent viscosity. Identification of a differentiated hESC subpopulation with similar mechanical properties as native chondrocytes may provide an excellent cell source for tissue engineering applications. These cells will need to withstand any mechanical stimulation regimen employed to augment the mechanical and biochemical characteristics of the neotissue. Density separation was effective at purifying distinct populations of cells. A differentiated hESC subpopulation was identified with both similar mechanical and morphological characteristics as ACs. Future research may utilize this cell source in cartilage regeneration efforts.

Regional variation in the mechanical role of knee meniscus glycosaminoglycans

High compressive properties of cartilaginous tissues are commonly attributed to the sulfated glycosaminoglycan (GAG) fraction of the extracellular matrix (ECM), but this relationship has not been directly measured in the knee meniscus, which shows regional variation in GAG content. In this study, biopsies from each meniscus region (outer, middle, and inner) were either subjected to chondroitinase ABC (CABC) to remove all sulfated GAGs or not. Compressive testing revealed that GAG depletion in the inner and middle meniscus regions caused a significant decrease in modulus of relaxation (58% and 41% decreases, respectively, at 20% strain), and all regions exhibited a significant decrease in viscosity (outer: 29%; middle: 58%; inner: 62% decrease). Tensile properties following CABC treatment were unaffected for outer and middle meniscus specimens, but the inner meniscus displayed significant increases in Youngs modulus (41% increase) and ultimate tensile stress (40% increase) following GAG depletion. These findings suggest that, in the outer meniscus, GAGs contribute to increasing tissue viscosity, whereas in the middle and inner meniscus, where GAGs are most abundant, these molecules also enhance the tissues ability to withstand compressive loads. GAGs in the inner meniscus also contribute to reducing the circumferential tensile properties of the tissue, perhaps due to the pre-stress on the collagen network from increased hydration of the ECM. Understanding the mechanical role of GAGs in each region of the knee meniscus is important for understanding meniscus structure-function relationships and creating design criteria for functional meniscus tissue engineering efforts.

The regional contribution of glycosaminoglycans to temporomandibular joint disc compressive properties.

Understanding structure-function relationships in the temporomandibular joint (TMJ) disc is a critical first step toward creating functional tissue replacements for the large population of patients suffering from TMJ disc disorders. While many of these relationships have been identified for the collagenous fraction of the disc, this same understanding is lacking for the next most abundant extracellular matrix component, sulfated glycosaminoglycans (GAGs). Though GAGs are known to play a major role in maintaining compressive integrity in GAG-rich tissues such as articular cartilage, their role in fibrocartilaginous tissues in which GAGs are much less abundant is not clearly defined. Therefore, this study investigates the contribution of GAGs to the regional viscoelastic compressive properties of the temporomandibular joint (TMJ) disc. Chondroitinase ABC (C-ABC) was used to deplete GAGs in five different disc regions, and the time course for >95% GAG removal was defined. The compressive properties of GAG depleted regional specimens were then compared to non-treated controls using an unconfined compression stress-relaxation test. Additionally, treated and non-treated specimens were assayed biochemically and histologically to confirm GAG removal. Compared to untreated controls, the only regions affected by GAG removal in terms of biomechanical properties were in the intermediate zone, the most GAG-rich portion of the disc. Without GAGs, all intermediate zone regions showed decreased tissue viscosity, and the intermediate zone lateral region also showed a 12.5% decrease in modulus of relaxation. However, in the anterior and posterior band regions, no change in compressive properties was observed following GAG depletion, though these regions showed the highest compressive properties overall. Although GAGs are not the major extracellular matrix molecule of the TMJ disc, they are responsible for some of the viscoelastic compressive properties of the tissue. Furthermore, the mechanical role of sulfated GAGs in the disc varies regionally in the tissue, and GAG abundance does not always correlate with higher compressive properties. Overall, this study found that sulfated GAGs are important to TMJ disc mechanics in the intermediate zone, an important finding for establishing design characteristics for future tissue engineering efforts.

Tissue Engineering of the Temporomandibular Joint

Temporomandibular joint (TMJ) morbidities affect around a quarter of the US population, yet there are no consistently successful treatment solutions. The TMJ comprises the articulating tissues between the mandibular condyle and the temporal fossa. Its fibrocartilaginous components form the articulating surfaces, with the biconcave TMJ disc allowing for smooth movement between the condyle and fossa during normal mastication. Each tissue within the joint displays unique cellular, biochemical, and biomechanical characteristics. These characteristics are important for the tissue engineer to understand, given the joints limited ability for self-repair following injury. The TMJ is susceptible to pathologies such as osteoarthritis and internal derangement of the disc, which are extremely painful and often require clinical intervention. Current therapies include anti-inflammatory measures, occlusal splints, and in extreme cases, total joint replacement. These therapies, however, are only semipermanent and fail to restore full functionality to the joint. Tissue engineering may provide functional biological replacements for TMJ tissues, resulting in a long-term solution to TMJ pathologies. Research using alternate cell sources, scaffolds, bioactive factors, and mechanical stimulation has shown promise, but more research must be done to determine optimal combinations of these factors.

The attachments of the temporomandibular joint disc: A biochemical and histological investigation

OBJECTIVE The complex movement of the temporomandibular joint (TMJ) disc during mastication is controlled in large part by the discs attachments to the surrounding tissues. This study seeks to address the lack of available quantitative data characterizing the extracellular matrix composition of the discal attachments and how these properties compare to the disc. DESIGN Porcine TMJ disc-attachment complexes were carefully dissected into six discal attachments and five TMJ disc regions. All samples were assayed biochemically for total collagen, glycosaminoglycan (GAG), DNA, and hydration. Additionally, histology was performed on the whole joint to investigate the anatomy of the disc-attachment complex, and to verify the regional distribution of matrix components. RESULTS Quantitative biochemical assays showed that overall water content was fairly constant in all disc and attachment regions. Disc regions generally showed higher sulfated GAG and collagen content than the attachments. In contrast, the attachments contained greater DNA content than the disc. Histological staining supported the quantitative results and also indicated more elastic fibres to be present in the attachments than the disc. CONCLUSIONS Although macroscopically the TMJ disc and its attachments form a seamless complex within the joint, a closer look at regional biochemical constituents reveals that these two components are distinct. Whilst the disc and attachments both contain the same major constituents, the relative amounts of these components vary based on the functional requirements of the tissue. These results can further understanding of both TMJ biology and pathology.

The temporomandibular joint disc of Asian elephant (Elephas maximus) and African elephant (Loxodonta africana)

The temporomandibular joint (TMJ) is a synovial articulation between the mandibular head of the condylar process of the mandible and the mandibular fossa of the squamous temporal bone. Extensions of the fibrous capsule into the joint space form a biconcave disc that functions as an articulating surface and divides the joint into dorsal and ventral compartments. The TMJ disc plays a crucial role in normal functioning of the joint, and differences in cranial morphology, mastication patterns, and diet are reflected in the material and biochemical properties of the disc. The purpose of the present case study was to compare the regional histologic differences between two elephant genera and quantify the biochemical and biomechanical properties of the African elephant disc. This study provides a unique insight into the elephant TMJ disc and also provides a comparison between the African and the Asian elephant genera. The results demonstrate several remarkable findings. First, structure–function relationships exist within the elephant TMJ disc. Second, regional variations exist in the elephant TMJ disc, and these are likely to correlate with its functional requirement. Additionally, it is apparent that some properties of the disc vary with the specific anatomy, diet requirement, and jaw motion. Finally, in comparison with the TMJ disc of other species, it is clear that, although the elephant disc is unique, it has properties that transcend and are preserved among the species.

6.9 Tissue Engineering of the Temporomandibular Joint

Temporomandibular joint (TMJ) morbidities affect around a quarter of the US population, yet there are no consistently successful treatment solutions. The TMJ comprises the articulating tissues between the mandibular condyle and the temporal fossa. Its fibrocartilaginous components form the articulating surfaces, with the biconcave TMJ disc allowing for smooth movement between the condyle and fossa during normal mastication. Each tissue within the joint displays unique cellular, biochemical, and biomechanical characteristics. These characteristics are important for the tissue engineer to understand, given the joint׳s limited ability for self-repair following injury. The TMJ is susceptible to pathologies such as osteoarthritis and internal derangement of the disc, which are extremely painful and often require clinical intervention. Current therapies include anti-inflammatory measures, occlusal splints, and in extreme cases, total joint replacement. These therapies, however, are only semipermanent and fail to restore full functionality to the joint. Tissue engineering may provide functional biological replacements for TMJ tissues, resulting in a long-term solution to TMJ pathologies. Research using alternate cell sources, scaffolds, bioactive factors, and mechanical stimulation has shown promise, but more research must be done to determine optimal combinations of these factors.