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INTRODUCTION
Although a complete understanding of the mechanisms by which ECM stiffness impacts cellular development has not been fully achieved, the biomechanical properties of ECM have been shown to play a significant role in regulating cell proliferation (Mih et al. 2012), migration (Ehrbar et al. 2011), and differentiation (Han et al. 2020) among other phenomena. Furthermore, the ever-changing needs of cells and tissues during development and maturation are reflected in the dynamic nature of their ECM. An ongoing challenge in tissue engineering is that the pared-down complexity present in engineered tissues is generally insufficient to recapitulate these complex, spatiotemporal cell-ECM dynamics. Recently though, we have demonstrated the use of oxidized sucrose (SOx) as a cytocompatible small molecule that can be deployed at different time points during tissue maturation to dynamically modulate the ECM (Silberman et al. 2023).
Here, we show that tissue development and maturation can be controlled by introducing SOx at different time points according to tissues’ developmental needs. In particular, we have shown that deploying SOx too early impaired cells’ ability to remodel their own microenvironment and to establish essential cell-cell and cell-ECM interactions. By encapsulating cells in a soft hydrogel matrix and dynamically modulating the biomechanical microenvironment to match the cells’ evolving needs, on the other hand, we enhanced cell and tissue maturation.
METHODS
Oxidized sucrose (SOx) was synthesized as previously reported (Silberman et al. 2023). Endothelial cells (ECs) and cardiomyocytes (CMs) were encapsulated within an ECM-based hydrogel (Shevach et al. 2015, Edri et al. 2019) and cultured under static conditions. Either on Day 0 or at specified time points during tissue maturation, SOx was added to the culture, incubated overnight, and removed the following day. Tissue morphology, maturation, and functionality were assessed at the end of the culture period.
RESULTS & DISCUSSION
Most significantly, we observed that deploying precisely the same concentration of SOx to manipulate the ECM at different stages of a tissue’s maturation led to markedly different outcomes. When exposed to SOx prematurely, ECs failed to organize into discernible blood vessels. ECs cultured in microenvironments dynamically modulated to regulate their ever-changing needs, on the other hand, generated clear capillary networks. Moreover, the timely addition of Sox induced the formation of thicker, more mature blood vessels than was possible to achieve without dynamic tissue modulation. These same observations, that timely SOx deployment is associated with a more mature phenotype, were also evident when working with CMs. Premature deployment led to a lack of synchronization of cellular contractions, while timely deployment led to more powerful cardiac contractions. This work thus demonstrates both why the ability to dynamically control the biomechanical properties of the tissue during maturation is an essential requirement for advancing tissue maturation, and it represents a significant step to achieving more accurate in vitro recapitulation of these complex processes vital for tissue engineering.