Periodic Reporting for period 1 - ProxDistReg (Implications of tissue stiffness in growth control during limb regeneration in salamanders (Ambystoma mexicanum))
Période du rapport: 2021-05-01 au 2023-04-30
Differential adhesion strength and extracellular matrix (ECM) were reported along the proximodistal (PD) axis during axolotl limb regeneration. Therefore, considering that cell–cell interactions and cell-ECM interactions play key roles in force transmission to and between cells, controlling signalling pathways that regulate stem cell self-renewal and differentiation, we hypothesize that tissue mechanical properties are regulated in gradient along the PD axis and are thus majorly responsible for the differential growth rates observed during regeneration between proximally and distally-amputated limbs.
Therefore, the central aim of this project is understanding how biomechanical properties of tissues affect regeneration, which may have important implications for the design of biomaterials to be used in regenerative medicine.
Limb growth: tissue growth during regeneration was evaluated in differently sized animals and data was used to generate a mathematical model describing growth dynamics and differences. From such model we were able to demonstrate that there is a linear correlation between the amputation site and maximum growth rate, as well as the mean cell cycle length during early regeneration phases. We also demonstrated that animal’s development is not affected by limb amputations.
Cell proliferation: we demonstrated that cell cycle is regulated differentially along the P/D axis during regeneration. Such differences were already detected during the early blastema stage.
Cell differentiation: we show that limb patterning occurs faster after distal amputations, as opposed to proliferation, suggesting a differential regulation of cell proliferation/differentiation decisions along the PD axis.
Tissue mechanics: we optimized 2 different techniques for the measurement of mechanical properties in the regenerating axolotl limbs: Brillouin confocal and atomic force microscopy. Our results indicate that distally amputated limbs present stiffer tissues during regeneration than proximally amputated ones.
Mechanical dependence: we proved that cultured blastema cells derived from proximal and distal blastemas don’t present any differences in proliferation, revealing the importance of the in vivo context for the regulation of growth. Furthermore, we developed a technique to culture axolotl cells in three-dimensional gels of differing degrees of stiffness, and proved a mechanical dependence in which cells decrease their proliferation when cultured in stiffer extracellular contexts.
In summary, our results go in line with the hypothesis proposed in the application, i.e. faster growth rate correlates with softer tissue during early phases of regeneration, whereas slower growth rate with stiffer tissues. We are currently characterizing the molecular mechanism behind our observations.
Thus far, the state-of-the-art was lacking studies associating growth regulation with mechanical properties during regeneration. Therefore, our results will importantly contribute to the field of regenerative biology.