Keating M, Kurup A, Alvarez-Elizondo M, Levine A J, Botvinick E
University of California, Irvine, Department of Biomedical Engineering, Irvine 92697-2730, USA.
Technion, Israel Institute of Technology, Department of Biomedical Engineering, Technion City, 32000, Israel.
Acta Biomater. 2017 Jul 15;57:304-312. doi: 10.1016/j.actbio.2017.05.008. Epub 2017 May 5.
Bulk tissue stiffness has been correlated with regulation of cellular processes and conversely cells have been shown to remodel their pericellular tissue according to a complex feedback mechanism critical to development, homeostasis, and disease. However, bulk rheological methods mask the dynamics within a heterogeneous fibrous extracellular matrix (ECM) in the region proximal to a cell (pericellular region). Here, we use optical tweezers active microrheology (AMR) to probe the distribution of the complex material response function (α=α'+α″, in units of µm/nN) within a type I collagen ECM, a biomaterial commonly used in tissue engineering. We discovered cells both elastically and plastically deformed the pericellular material. α' is wildly heterogeneous, with 1/α' values spanning three orders of magnitude around a single cell. This was observed in gels having a cell-free 1/α' of approximately 0.5nN/µm. We also found that inhibition of cell contractility instantaneously softens the pericellular space and reduces stiffness heterogeneity, suggesting the system was strain hardened and not only plastically remodeled. The remaining regions of high stiffness suggest cellular remodeling of the surrounding matrix. To test this hypothesis, cells were incubated within the type I collagen gel for 24-h in a media containing a broad-spectrum matrix metalloproteinase (MMP) inhibitor. While pericellular material maintained stiffness asymmetry, stiffness magnitudes were reduced. Dual inhibition demonstrates that the combination of MMP activity and contractility is necessary to establish the pericellular stiffness landscape. This heterogeneity in stiffness suggests the distribution of pericellular stiffness, and not bulk stiffness alone, must be considered in the study of cell-ECM interactions and design of complex biomaterial scaffolds.
Collagen is a fibrous extracellular matrix (ECM) protein widely used to study cell-ECM interactions. Stiffness of ECM has been shown to instruct cells, which can in turn modify their ECM, as has been shown in the study of cancer and regenerative medicine. Here we measure the stiffness of the collagen microenvironment surrounding cells and quantitatively measure the dependence of pericellular stiffness on MMP activity and cytoskeletal contractility. Competent cell-mediated stiffening results in a wildly heterogeneous micromechanical topography, with values spanning orders of magnitude around a single cell. We speculate studies must consider this notable heterogeneity generated by cells when testing theories regarding the role of ECM mechanics in health and disease.
大块组织的硬度与细胞过程的调节相关，反之，细胞已被证明会根据对发育、体内平衡和疾病至关重要的复杂反馈机制重塑其细胞周围组织。然而，大块流变学方法掩盖了细胞近端区域（细胞周围区域）内异质纤维细胞外基质（ECM）中的动力学。在这里，我们使用光镊主动微流变学（AMR）来探测I型胶原ECM（一种组织工程中常用的生物材料）内复材响应函数（α=α'+α″，单位为µm/nN）的分布。我们发现细胞使细胞周围材料发生弹性和塑性变形。α'高度异质，单个细胞周围的1/α'值跨越三个数量级。在无细胞的1/α'约为0.5nN/µm的凝胶中观察到了这一现象。我们还发现，抑制细胞收缩性会立即软化细胞周围空间并降低硬度异质性，这表明该系统发生了应变硬化，且不仅是塑性重塑。其余高硬度区域表明细胞对周围基质进行了重塑。为了验证这一假设，将细胞在含有广谱基质金属蛋白酶（MMP）抑制剂的培养基中于I型胶原凝胶中孵育24小时。虽然细胞周围材料保持硬度不对称，但硬度大小降低了。双重抑制表明，MMP活性和收缩性的结合对于建立细胞周围硬度格局是必要的。这种硬度异质性表明，在研究细胞与ECM相互作用以及设计复杂生物材料支架时，必须考虑细胞周围硬度的分布，而不仅仅是大块硬度。
胶原蛋白是一种纤维细胞外基质（ECM）蛋白，广泛用于研究细胞与ECM的相互作用。ECM的硬度已被证明可指导细胞，而细胞反过来又可修饰其ECM，这在癌症和再生医学研究中已有体现。在这里，我们测量了细胞周围胶原微环境的硬度，并定量测量了细胞周围硬度对MMP活性和细胞骨架收缩性的依赖性。有能力的细胞介导的硬化导致了高度异质的微观力学地形，单个细胞周围的值跨越多个数量级。我们推测，在测试关于ECM力学在健康和疾病中的作用的理论时，研究必须考虑细胞产生的这种显著异质性。
