Use of protein-engineered fabrics to identify design rules for integrin ligand clustering in biomaterials
Use of protein-engineered fabrics to identify design rules for integrin ligand clustering in biomaterials
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DOI：
10.1039/c5ib00258c
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发表时间：
2016-01-01
影响因子：
2.5
通讯作者：
Heilshorn, Sarah C.
中科院分区：
文献类型：
--
作者：
Benitez, Patrick L.;Mascharak, Shamik;Heilshorn, Sarah C.
虽然已知配体聚集可以增强整合素的激活，但这一见解很难应用于可植入生物材料的设计，因为使聚集增强整合素信号的局部和全局配体密度是不可预测的。在这里，阐明了生物材料配体聚类的两个一般设计原则。首先，当整体配体密度（即整个细胞长度尺度上的配体密度）接近配体的有效解离常数（K-D,K-eff）时，聚类配体增强了整合素依赖信号。其次，当局部配体密度（即单个焦点黏附的长度尺度上的配体密度）小于过度拥挤阈值时，簇状配体增强了整合素的激活。为了确定这些原理，我们制作了一系列类似弹性蛋白的静电纺丝织物，这些织物可以独立控制精氨酸-甘氨酸-天冬氨酸（RGD）配体的局部（0到122000个配体μ m(-2)）和全局（0到71000个配体μ m(-2)）密度。抗体阻断研究证实，人脐静脉内皮细胞与这些蛋白质工程生物材料的粘附主要是由于α (V) β(3)整合素结合。在配体K-D、K-eff为12 000 RGD μ m（-2）时，聚类配体增强了细胞增殖、局灶黏附数和局灶黏附激酶在配体附近的表达。接近这个整体配体密度时，配体簇化织物上的细胞表现类似于在明显更大的整体配体密度但没有簇化的织物上生长的细胞。然而，这种增强的配体聚类效应在阈值截止浓度以上未被观察到。当局部配体密度为122000 RGD μ m（-2）时，细胞分裂、局灶黏附数和局灶黏附激酶的表达量明显低于具有相同全局配体密度和较低局部配体密度的织物。因此，当聚类导致配体过度拥挤时，整合素受体不再能够有效地与其靶配体结合。总之，这两个关于细胞对配体在细胞-基质界面聚集的反应的见解可以作为开发未来几代植入式生物材料的设计原则。
While ligand clustering is known to enhance integrin activation, this insight has been difficult to apply to the design of implantable biomaterials because the local and global ligand densities that enable clustering-enhanced integrin signaling were unpredictable. Here, two general design principles for biomaterial ligand clustering are elucidated. First, clustering ligands enhances integrin-dependent signals when the global ligand density, i.e., the ligand density across the cellular length scale, is near the ligand's effective dissociation constant (K-D,K-eff). Second, clustering ligands enhances integrin activation when the local ligand density, i.e., the ligand density across the length scale of individual focal adhesions, is less than an overcrowding threshold. To identify these principles, we fabricated a series of elastin-like, electrospun fabrics with independent control over the local (0 to 122 000 ligands mu m(-2)) and global (0 to 71 000 ligand mu m(-2)) densities of an arginine-glycine-aspartate (RGD) ligand. Antibody blocking studies confirmed that human umbilical vein endothelial cell adhesion to these protein-engineered biomaterials was primarily due to alpha(V)beta(3) integrin binding. Clustering ligands enhanced cell proliferation, focal adhesion number, and focal adhesion kinase expression near the ligand's K-D,K-eff of 12 000 RGD mu m(-2). Near this global ligand density, cells on ligand-clustered fabrics behaved similarly to cells grown on fabrics with significantly larger global ligand densities but without clustering. However, this enhanced ligand-clustering effect was not observed above a threshold cut-off concentration. At a local ligand density of 122 000 RGD mu m(-2), cell division, focal adhesion number, and focal adhesion kinase expression were significantly reduced relative to fabrics with identical global ligand density and lesser local ligand densities. Thus, when clustering results in overcrowding of ligands, integrin receptors are no longer able to effectively engage with their target ligands. Together, these two insights into the cellular responses to ligand clustering at the cell-matrix interface may serve as design principles when developing future generations of implantable biomaterials.