Regulation of implant surface cell adhesion: characterization and quantification of S‐phase primary osteoblast adhesions on biomimetic nanoscale substrates

Abstract

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 AE50 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) immunofluorescent 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. ß

Cartilage Tissue Engineering: the Application of Nanomaterials and Stem Cell Technology

“…Interestingly enough, research has also shown the MSC osteoblastic differentiation can be hampered by regularly arranged hexagonal nanopits arrays compared with arrays with a slight irregularity (39). Similar results were found by Biggs et al 2007, where highly ordered nanopits resulted in decreased formation and length of focal adhesions, compared with controlled disorder increasing focal adhesion formation and size (13). With more research being conducted into the in vitro effects of surface nanopatterning on cell behaviour, there are implications for cartilage tissue engineering research.…”

Selective and uncoupled role of substrate elasticity in the regulation of replication and transcription in epithelial cells

“…3E). These spots were assigned to focal complexes and originated as dot-like structures (Biggs et al, 2007). During this time period, DNA replication was still inactive (Fig.…”

“…Mature FAs are typically dash-shaped with areas of 2-5 m 2 . Mature FAs were identified as structures containing vinculin, paxillin, and talin (Biggs et al, 2007). At this time, cells still remained in S phase (Fig.…”

Machine grinding as an alternative method for creating functional surfaces for controlling cell behaviour

“…These results suggest that cells are able to attach to the machine ground surface much more quickly than is the case for the unprocessed polymer surface, as the MTT test was performed after a duration of only 2 h. If this attachment time was increased, a similar attachment level may be seen. The effect of surface topography on cell behaviour has been shown previously, with cell alignment shown when cells are seeded onto grooved surfaces [10,19] as well as when being seeded onto highly ordered nanopits that effect cytoskeleton organisation [2]. Fibroblasts, in particular, have been shown to adhere with more affinity, when exhibiting a well-spread, flattened morphology (i.e.…”