Large animal models are necessary to appropriately evaluate regenerative medicine strategies towards TMJ therapies . It is of critical importance to characterize and elucidate the intrinsic similarities and differences between models, such as the pig and the sheep, to facilitate clinical translation to humans. Toward advancing these tissue engineering strategies for the TMJ, this study undertook the characterization and comparison of morphology and bone phenotype and disc ECM of sheep TMJ and that of often cited gold standard porcine model. One human sample was also used for gross morphologic and μCT data that together with historical information [16, 17] provides comparative insights of the relevance of each of these species for human applications.
Because joint and disc anatomy, biochemical composition, and biomechanical properties are determined by the mechanical function, chewing patterns and diet, the morphology and tissue phenotype of the TMJ reflects this structure–function relationship. Thus, despite the TMJ being a characteristic structure found in mammals, there is remarkable variation in morphology and function among species , such that no one animal model serves as an optimal analog for humans. Also, the large spectrum of TMJ disorders makes it impossible for any single model to recapitulate every type of TMJ dysfunction. Nevertheless, pig TMJs are similar in morphology to humans and other higher primates, and are also close to human in function, as the pig TMJ allows for a large range of motion including translational and rotational movements during chewing. For these reasons, the pig is considered the gold standard TMJ model . Unlike the pig, the sheep TMJ joint anatomy permits a functional pattern specialized in translational movements in the transverse plane. Correspondingly, the sheep condyle is more concave than humans, although both sheep and pigs have similar TMJ disc size and shape . Furthermore, our data, along with others [20,21,22], show that quantitative morphometry parameters of the trabecular condyle of the human also fall within the range found in pig and sheep TMJs. These findings suggest that while the overall shape of the sheep TMJ is more distinct from humans than that of pigs, both species serve as good models to assess bone mineral densities and other bone morphometry parameters.
Porcine and sheep TMJs have anatomical similarities in their discal attachments, in that both species, as well as humans, have a posterior attachment superiorly to the temporal bone and inferiorly to the posterior condyle, and an anterior attachment superiorly to the eminence and inferiorly to the anterior condyle [23, 24]. The histological and immunohistology evaluations of the disc and attachments demonstrate several similar characteristics in content and regional organization between pig and sheep, with both species showing the presence of collagen I and elastin and much of the matrix is a continuous blended network. However, we and others [25, 26] have shown complex regional variation in the matrix of the TMJ disc that may differ between species. In this work, while both species show low collagen I staining in the anterior region, porcine discs have significantly greater relative areas of the intermediate/posterior and intermediate/anterior zones staining for collagen than those of sheep. These differences and those of other ECM findings [25, 26] likely reflect disparities in functional and biomechanical demands placed on the disc in the two species.
Cross-linked elastin fibers are found in much lower quantities than collagen fibers which are 90% tissue mass [27, 28] with elastin fibers comprising only 1–2% of the tissue distributed throughout the disc and discal attachments. Yet, elastin fibers are critical in function since their compliant characteristics are responsible for restoring the original shape of the disc following loading [29,30,31]. The disc itself is highly fibrous and shows circumferential alignment of collagen fibers throughout the periphery and antero-posterior alignment through the central region (Fig. 4). This alignment of collagen fibers corresponds with the zonal structure–function relationship of the disc, with an antero-posterior alignment supporting the tensile forces imposed on the disc during functional movement. As shown here, the differences between elastin and collagen content of the attachments and the disc are relatively small between sheep and pig. However, even minor variations in ECM content and organization likely have a significant influence on the mechanical and functional properties of the tissue [32,33,34].
While absent from porcine discs, GAGs are localized in the intermediate and posterior/intermediate zones of sheep discal tissues (Fig. 3). These results corroborate those of others  and support the concept that GAG content is often high in joint soft tissues that sustain large compressive forces. In contrast to the lack of GAG localization in the porcine disc, humans TMJ discs demonstrate positive GAG staining , which corresponds with our findings in sheep in the intermediate and immediate posterior disc (Fig. 2), suggesting that sheep TMJ disc may be a more suitable human-analog for regenerating discal tissues with appropriate compressive tissue phenotype. A thorough overview of species and region-dependent biochemical properties suggest that in several species, including goats and humans, the intermediate zone has a higher GAG content than the rest of disc [25, 26], and concurs with our findings on the sheep. Furthermore, the posterior of the disc also has more collagen content than the other regions for all species. Besides these characterizations, it is notable that this current work is the first to also compare the bone morphology between human TMJs and pig and sheep.
Despite the utilization of one human TMJ for morphological and µCT data, the abundant historical findings on humans [16, 17] provide the necessary comparative information for our studies. While our findings from skeletally mature-aged sheep and pig add to the literature, it is likely that functional and maturational adaptations throughout the lifespan are additional important considerations in selection of animal models for TMJ regenerative therapeutics. Thus, age-related characterization of the TMJ in these large animal models would also be worthy of deeper analyses for further determining their applicability to human disorders and therapies. Lastly, quantitative biochemical measures need to be performed in the future to validate and provide further detail of our histological findings. Overall, our results suggest that while there are differences in the porcine and sheep TMJ morphologies, and bone and disc phenotypes, the sheep is as appropriate a model for TMJ regenerative therapies as the pig and each should be used for defined purposes to address specific concepts that closely mimic those found in humans.