A Wet-and-Dry Research Model: Unveiling Biomechanics in Odontoblast Polarization

Aim: Odontoblast polarization is key to the formation and regeneration of tubular dentine. However, the molecular mechanisms, particularly the mechanobiological cues that guide odontoblast polarization, remain unrecognized, hindering the advancement of regenerative endodontics. To address this, we implemented a Wet-and-Dry platform that integrates photolithographic micropatterning and mathematical modelling, aiming to characterize the mechanobiological features underlying odontoblast polarization at the single cell level.

Methodology: A combined wet-and-dry platform was designed. In the wet experiments, PEGDA-based photolithography created adhesive micropatterns with defined areas and aspect ratios, onto which human dental pulp stem cells (hDPSCs) were seeded. Cell morphology, polarity metrics, odontogenic marker expression and mechano-inductive protein levels were quantified by immunofluorescence and image analysis. Mechanisms were probed by disrupting the Cytoskeleton, inhibiting Rho-ROCK signalling and blocking YAP1 activity. Using an ex vivo mouse tooth germ model, the role of RhoA/ROCK in odontoblast polarization was confirmed under inhibitor treatment. In the dry experiments, a vertex-based computational model simulated single hDPSCs adhesion and spreading on the micropatterns, and predicted the spatiotemporal force distribution during polarization, including deformation energy, adhesion strength and traction forces. The simulation outputs were validated against experimental measurements.

Results: Micropatterned substrates mimicking odontoblast-like cell shapes provide an ideal platform for single-cell mechanobiology. Patterns with 2700 μm² area and 1:4 aspect ratio most strongly promoted hDPSCs polarization and odontogenic differentiation. Polarized cells exhibited increased Myosin II and increased nuclear YAP1. A vertex-based model predicted that high aspect ratio increases traction and adhesion forces. Loss-of-function experiments confirmed that Rho-dependent cytoskeletal remodelling and increased tension are required for geometry-driven polarization and differentiation. Ex vivo tooth germ culture showed that inhibiting RhoA/ROCK disrupts odontoblast polarization and tissue organization, underscoring the importance of this signalling axis.

Conclusion: By integrating micropatterned substrates with in silico modelling and ex vivo tissue validation, we demonstrate that microenvironmental geometry directs hDPSCs' polarization and odontogenic differentiation via a Vinculin-RhoA-YAP signalling axis, which clarifies a core mechanobiological mechanism underlying odontoblast polarization.