Local mechanical stimuli shape tissue growth in vertebrate joint morphogenesis

Abstract

The correct formation of synovial joints is essential for proper motion throughout life. Movement-induced forces are critical to creating correctly shaped joints, but it is unclear how cells sense and respond to these mechanical cues. To determine how mechanical stimuli drive joint morphogenesis, we combined experiments on regenerating axolotl forelimbs with a poroelastic model of bone rudiment growth. Animals either regrew forelimbs normally (control) or were injected with a TRPV4 agonist to impair chondrocyte mechanosensitivity during joint morphogenesis. We quantified growth and shape in regrown humeri from whole mount light sheet fluorescence images of the regenerated limbs. Results revealed statistically significant differences in morphology and cell proliferation between the two groups, indicating that mechanical stimuli play a role in the shaping of the joint. Local tissue growth in our finite element model was dictated by a biological contribution, proportional to chondrocyte density, and a mechanical one, driven by fluid pore pressure dynamics. Computational predictions agreed with experimental outcomes, suggesting that interstitial pressure might promote local tissue growth. Predictive computational models informed by experimental findings allow us to explore potential physical mechanisms and regulatory dynamics involved in tissue growth to advance our understanding of the mechanobiology of joint morphogenesis.

In vivoassessment of mechanical properties during axolotl development and regeneration using confocal Brillouin microscopy

“…Whereas atomic force microscopy-based indentation measurements, the current gold standard in mechanobiology [49], have been more extensively used in the field [50–52], albeit with the limitation of measuring processed tissues from ex vivo explants. In urodeles, the only studies on tissue mechanics were performed either in vitro , demonstrating that wound closure of skin explants strongly relies on the substrate biomechanical properties [53], or in silico , predicting that movement-induced interstitial pressure promotes tissue growth during joint morphogenesis [54]. Hence, further in vivo studies need to be carried out to better understand the influence of mechanics on regeneration.…”

Cartilage mechanobiology during limb growth

“…Furthermore, Comellas et al identified differences in joint morphology between control and TRPV4-blocked regenerating limbs in axolotls, emphasizing the need for a comprehensive 3D analysis of the regenerated limb for a more nuanced understanding of morphological distinctions 11 . To enhance our understanding, future studies could incorporate tracking proliferating cells, offering a more comprehensive insight into the effects of TRPV4 impairment on limb regeneration 10 .…”

“…The mechanical loading experienced by growing musculoskeletal tissues generates stimuli that embryonic cells perceive and respond to, translating mechanical signals into molecular responses 5 . Disruption of these mechanical signals during development has been shown to result in significant alterations to joint morphology, tissue composition, and the manifestation of pathology [11][12][13] Methods: This study focused on estimating the viscoelastic material properties during three regeneration stages of axolotl (Ambystoma mexicanum) forelimbs: 27 days post-amputation or 27 DPA (mid-late bud), 41 DPA (palette stage), and fully-grown time points. Material parameters were derived through a stress-relaxation indentation test followed by a two-term Prony series viscoelastic inverse finite element analysis.…”

“…Building upon previous research in our laboratory 11,193 , which leveraged finite element modeling with animal-specific material parameters to discern the impacts of strain rate and fluid flow on tissue metabolism, the results from this thesis could be used to extend this application to study mechanotransduction during limb growth. The derived material properties can now serve as inputs for finite element models, accurately representing the mechanical characteristics of growing limbs at various developmental stages.…”

“…Firstly, the electroporation technique, as demonstrated in this thesis, emerges as a suitable choice, providing a direct and precise means to assess ion channel activity within the local tissue blastema. In contrast, an alternative pharmacological approach involving agonists/antagonists, akin to previous in-vivo studies 11 , could also be considered for characterizing TRPV4, PIEZO1, and PIEZO2 ion channel functionality in the developing axolotl limbs. However, it is crucial to note that pharmacological agents have non-tissue specific targets and may introduce potential systemic health concerns for the animal.…”