Rodriguez Navas Marta, Darling Eric M
Institute for Biology, Engineering, and Medicine, Brown University, Providence, Rhode Island, USA.
Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island, USA.
Tissue Eng Part A. 2025 Feb;31(3-4):164-173. doi: 10.1089/ten.tea.2024.0192. Epub 2024 Oct 25.
Mechanical forces are a critical stimulus in both native and engineered tissues. Direct measurement of these microenvironmental forces has been challenging, particularly for cell-dense models. To address this, we previously developed hydrogel-based force sensors that are approximately the size of a cell and can be imaged over time to computationally assess the forces exerted by surrounding cells and matrix. The goal of this project was to identify how the physical characteristics of force sensors impact measurements. Sensors were varied in size, elastic modulus, and surface coating before being included in stem cell suspensions that then spontaneously self-assembled into spheroidal neotissues. Using this model of early mesenchymal condensation, we hypothesized that larger, softer sensors would provide greater sensitivity and precision, whereas protein coatings would influence the directionality of applied forces (tensile vs. compressive). These experiments were conducted using a high-content imaging system that allowed analysis of over a thousand sensors to evaluate the various conditions. Results indicated that measurement fidelity was highest for force sensors that had a diameter >20 µm and modulus ∼0.2 kPa. Extremely soft sensors deformed too much, whereas stiffer sensors deformed too little. Collagen and N-cadherin coatings, which replicated cell-matrix or cell-cell binding, respectively, allowed for tensile forces to be exerted on the sensors, with greater forces being observed for N-cadherin sensors in these highly cellular neotissue constructs. Uncoated sensors were universally compressed due to the lack of cell-sensor adhesion. Disruption of the actin cytoskeleton lessened microenvironmental forces, whereas disruption of microtubules had no measurable effect. Potential future applications of the technology include studies of forces in developing tissues as well as a real-time sensor for monitoring the growth of engineered constructs.
机械力是天然组织和工程组织中的关键刺激因素。直接测量这些微环境力具有挑战性，尤其是对于细胞密集型模型。为了解决这个问题，我们之前开发了基于水凝胶的力传感器，其大小约为一个细胞，可以随时间进行成像，以通过计算评估周围细胞和基质施加的力。该项目的目标是确定力传感器的物理特性如何影响测量结果。在将传感器纳入干细胞悬浮液之前，先改变其大小、弹性模量和表面涂层，然后这些干细胞悬浮液会自发自组装成球形新组织。利用这种早期间充质凝聚模型，我们假设更大、更软的传感器将提供更高的灵敏度和精度，而蛋白质涂层会影响施加力的方向性（拉伸力与压缩力）。这些实验使用了高内涵成像系统进行，该系统能够分析一千多个传感器以评估各种条件。结果表明，直径>20 µm且模量约为0.2 kPa的力传感器测量保真度最高。极其柔软的传感器变形过大，而更硬的传感器变形过小。分别复制细胞-基质或细胞-细胞结合的胶原蛋白和N-钙黏蛋白涂层，使得拉伸力能够作用于传感器，在这些高度细胞化的新组织构建物中，N-钙黏蛋白传感器所受的力更大。未涂层的传感器由于缺乏细胞-传感器粘附而普遍受到压缩。肌动蛋白细胞骨架的破坏会减弱微环境力，而微管的破坏则没有可测量的影响。该技术未来的潜在应用包括对发育中组织中力的研究以及用于监测工程构建物生长的实时传感器。
