Regulation of implant surface cell adhesion: Characterization and quantification of S-phase primary osteoblast adhesions on biomimetic nanoscale substrates
Regulation of implant surface cell adhesion: Characterization and quantification of S-phase primary osteoblast adhesions on biomimetic nanoscale substrates
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DOI：
10.1002/jor.20319
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发表时间：
2007-02-01
影响因子：
2.8
通讯作者：
Dalby, M. J.
中科院分区：
文献类型：
--
作者：
Biggs, Manus J. P.;Richards, R. G.;Dalby, M. J.
用于骨修复的骨科假体的整合必须与骨整合和植入物固定相关联，这是可以通过植入物/骨界面的地形修改来实现的理想。据认为，成骨细胞使用细胞的延伸，以收集空间信息的地形周围的粘附形成和细胞扁平化之前。粘着斑（Focal adhesions，FA）是与肌动蛋白细胞骨架相关的动态结构，其形成聚集的整合素受体的粘着斑，所述整合素受体在将细胞细胞骨架偶联至细胞外基质（ECM）中起作用。脂肪酸含有结构和信号分子至关重要的细胞粘附和生存。为了研究有序纳米形貌对成骨细胞粘附形成的影响，将原代人成骨细胞（HOB）培养在具有确定的纳米级凹坑阵列的实验基底上。通过电子束光刻法制备了可控纳米孔尺寸和结构的镍纳米片，并通过注射成型将其转移到聚碳酸酯（PC）盘上。测量120 nm直径和100 nm深度的纳米孔与300 nm的中心-中心间距被制造成三种独特的几何构造：正方形、六边形和近正方形（正方形图案中的300 nm间距的凹坑，但具有+/-50 nm的无序）。纽蛋白的免疫荧光标记允许HOB粘附复合物通过图像软件可视化和量化。观察到周围粘连以及核周区域内的粘连，并且观察到粘连长度和数量相对于光滑PC在纳米孔基底上变化。用溴脱氧尿苷（BrdU）免疫荧光检测鉴定实验基质上的S期细胞，从而在细胞周期的S期内对均匀扁平的细胞群体进行粘附定量。这项研究的结果表明，有序的纳米孔粘附形成的破坏性影响和纳米特征的构象在调节这些影响中发挥的作用。高度有序的纳米孔阵列导致粘附形成减少和粘附长度减少，同时引入存在于近正方形阵列中的一定程度的受控无序，显示出增加焦点粘附形成和尺寸。HOB的形态也受到纳米孔的存在和构象的影响。有序阵列影响细胞扩散，并诱导伸长的细胞表型，指示增加的运动性，而近正方形纳米孔对称性诱导HOB扩散。据推测，纳米孔通过直接或间接影响粘附复合物的形成来影响成骨细胞-基质粘附，这是一种依赖于纳米孔尺寸和构象的现象。(c)2006骨科研究学会。由威利期刊公司出版
Integration of an orthopedic prosthesis for bone repair must be associated with osseointegration and implant fixation, an ideal that can be approached via topographical modification of the implant/bone interface. It is thought that osteoblasts use cellular extensions to gather spatial information of the topographical surroundings prior to adhesion formation and cellular flattening. Focal adhesions (FAs) are dynamic structures associated with the actin cytoskeleton that form adhesion plaques of clustered integrin receptors that function in coupling the cell cytoskeleton to the extracellular matrix (ECM). FAs contain structural and signalling molecules crucial to cell adhesion and survival. To investigate the effects of ordered nanotopographies on osteoblast adhesion formation, primary human osteoblasts (HOBs) were cultured on experimental substrates possessing a defined array of nanoscale pits. Nickel shims of controlled nanopit dimension and configuration were fabricated by electron beam lithography and transferred to polycarbonate (PC) discs via injection molding. Nanopits measuring 120 nm diameter and 100 nm in depth with 300 nm center-center spacing were fabricated in three unique geometric conformations: square, hexagonal, and near-square (300 nm spaced pits in square pattern, but with +/- 50 nm disorder). Immunofluorescent labeling of vinculin allowed HOB adhesion complexes to be visualized and quantified by image software. Perhipheral adhesions as well as those within the perinuclear region were observed, and adhesion length and number were seen to vary on nanopit substrates relative to smooth PC. S-phase cells on experimental substrates were identified with bromodeoxyuridine (BrdU) immunofluoreseent detection, allowing adhesion quantification to be conducted on a uniform flattened population of cells within the S-phase of the cell cycle. Findings of this study demonstrate the disruptive effects of ordered nanopits on adhesion formation and the role the conformation of nanofeatures plays in modulating these effects. Highly ordered arrays of nanopits resulted in decreased adhesion formation and a reduction in adhesion length, while introducing a degree of controlled disorder present in near-square arrays, was shown to increase focal adhesion formation and size. HOBs were also shown to be affected morphologicaly by the presence and conformation of nanopits. Ordered arrays affected cellular spreading, and induced an elongated cellular phenotype, indicative of increased motility, while near-square nanopit symmetries induced HOB spreading. It is postulated that nanopits affect osteoblast-substrate adhesion by directly or indirectly affecting adhesion complex formation, a phenomenon dependent on nanopit dimension and conformation. (c) 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.