Multiscale analysis reveals distinct temporal trajectories of morphological reconstruction and mechanical maturation during mouse digit tip regeneration

Mouse digit tip regeneration offers a model for mammalian epimorphic regeneration, yet the spatiotemporal evolution between emerging tissue architecture and its mechanical environments remain poorly understood. In this study, we employed a multiscale approach, integrating in vivo micro-CT imaging, atomic force microscopy (AFM) nanomechanical mapping, and finite element modeling (FEM), to quantitatively characterize the temporal evolution of structural organization, local nanomechanical properties, and tissue-scale mechanical environments throughout mouse digit tip regeneration following amputation. AFM measurements revealed distinct patterns of structural reconstruction during regeneration: while the proximal stump underwent rapid repair-associated ossification, the distal tip underwent blastema-driven regeneration characterized by the formation and progressive coalescence of discrete mineralized regions within a highly compliant extracellular matrix. Comparison of the multiscale measurements showed that structural reconstruction and local mechanical maturation progressed along distinct temporal trajectories, with gross anatomical continuity restored by 21 days post-amputation (DPA), whereas native-like local nanomechanical properties continued to mature through 28-31 DPA. FEM analyses further showed progressive restoration of dorsal-ventral stress symmetry during regeneration, with the ventral-to-dorsal mechanical stress and strain ratio approaching the near-unity value observed in unamputated digits, while evolving distal geometry progressively altered stress distributions within the regenerated bone. Together, these findings establish a quantitative multiscale dataset for comparing structural reconstruction, local mechanical maturation, and the evolution of tissue-scale mechanical environments throughout digit regeneration. This multiscale perspective provides a quantitative reference for evaluating functional recovery and may inform the development of bio-inspired strategies for enhancing musculoskeletal regeneration in non-regenerative contexts. STATEMENT OF SIGNIFICANCE: Mammalian digit tip regeneration is a model of complex tissue regrowth, yet how structural organization, local mechanical properties, and tissue-scale mechanical environments evolve during regeneration remains poorly understood. Here, we integrate in vivo micro-CT imaging, atomic force microscopy, and finite element modeling to quantitatively characterize the multiscale structural and mechanical evolution of regenerating mouse digits. Our findings show that structural reconstruction, local matrix maturation, and tissue-scale mechanical environments evolve over distinct spatial and temporal patterns throughout regeneration, providing quantitative insight into the progression of matrix organization and mechanical function. This multiscale framework establishes a quantitative basis for investigating mechanobiological regulation during mammalian tissue regeneration and may facilitate evaluation of regenerative strategies for musculoskeletal tissues.