Mechanoregulation of Jaw Tendon Development

Introduction: Little is known about the development of the tendons that aid in jaw function. This lack of knowledge has limited advancements in treatment of pathologies and injuries that involve jaw tendons, such as temporalis tendinitis and temporomandibular joint disorders[1]. Mechanical stimulation through physical therapy has shown promise as an approach to stimulate healing of jaw tissue structures[2], but the specific effects of mechanical stimulation on jaw tendons during tissue formation (e.g., healing) are unknown. Here, we establish the chick embryo as a model to study jaw tendon formation and to analyze the role of mechanical loading in jaw tendon formation. We focused on the tendon attaching to the musculus adductor mandibulae externus (TmAM) as a jaw closing tendon that is comparable in function to human jaw closing tendons[1] (Fig. 1a).

Materials and

Methods: White leghorn chicken embryos were sacrificed at different developmental stages from embryonic day (D)9 to D19. The head was fixed and sectioned for histology, immunostaining, and histomorphometry to characterize cell and extracellular matrix (ECM) morphology and spatiotemporal distribution. The TmAM was harvested for Brillouin imaging to characterize mechanical properties. We injected developing embryos with pharmacological agents to induce either paralysis or hypermotility, or with saline as a control condition for 48h and then performed Brillouin imaging on the TmAM. Because the TmAM is too small for tensile testing, we used Brillouin imaging to measure longitudinal modulus[3], which was then converted to elastic modulus using a calibration curve generated based on Brillouin imaging and tensile testing of chick embryo calcaneal tendons[4]. One-way ANOVA with Tukey’s multiple comparisons was used to analyze the differences between developmental stages. Unpaired Student t-test was used to analyze the difference between two treatment groups.

Results, Conclusions, and Discussions: The TmAM was detectable by D9 based on histological staining and tenascin-C localization. Collagen content, maturity, and alignment increased, cell density decreased, and cell nuclei elongated during development. Collagen type III, tenascin-C, lysyl oxidase, and matrix metalloproteinases exhibited dynamic spatiotemporal distributions, suggesting stage-specific roles for these proteins in TmAM formation. Elastic modulus was constant from D15 to D17 and then increased from D17 to D20 (Fig. 1b). Decreased movement frequency (paralysis) resulted in a lower elastic modulus, whereas increased movement frequency (hypermotility) resulted in a higher elastic modulus (Fig. 1c, d).. Our data reveal that jaw tendons develop biologically, structurally, and mechanically during embryonic development. Based on ECM characterizations, developing jaw tendons may share overlapping mechanisms and biology as limb tendons[1,5-7]. It was exciting to discover that mechanical stimulation via jaw movement is necessary for jaw tendon mechanical development, and that increased movement, via hypermotility induction, leads to a significantly higher elastic modulus compared to the control. Ongoing work is using this model to elucidate mechanisms of mechanoregulation of jaw tendon development. This study establishes the chick embryo as an exciting new model to study the role of mechanics in jaw tendon formation. We anticipate mechanistic studies with this model will inform the development of physical therapy and regenerative medicine approaches to treat adult jaw tendon disorders and injuries.

Acknowledgements (Optional):

References: [1] Korntner et al., Front. Cell. Dev. Biol., 2022; [2] Michelotti et al., J. Orofac. Pain, 2004; [3] Scarcelli et al., Nat. Photonics, 2008; [4] Jana et al., ORS abstract, 2022; [5] Marturano et al., PNAS, 2013; [6] Pan et al., Philos Trans R Soc Lond B Biol Sci, 2018; [7] Nguyen et al., Connec Tissue Res, 2008