Fabrication of pillar-like titania nanostructures on titanium and their interactions with human skeletal stem cells

Surface nanotopography is known to influence the interaction of human skeletal (mesenchymal) stem cells (hMSC) with a material surface. While most surface nanopatterning has been performed on polymer-based surfaces there is a need for techniques to produce well-defined topography features with tuneable sizes on relevant load-bearing implant materials such as titanium (Ti). In this study titania nanopillar structures with heights of either 15, 55 or 100 nm were produced on Ti surfaces using anodization through a porous alumina mask. The influence of the surface structure heights on hMSC adhesion, spreading, cytoskeletal formation and differentiation was examined. The 15 nm high topography features resulted in the greatest cell response with bone matrix nodule forming on the Ti surface after 21 days.     

Effect of Focal Adhesion Proteins on Endothelial Cell Adhesion,Motility and Orientation Response to Cyclic Strain

Focal adhesion proteins link cell surface integrins and intracellular actin stress fibers and therefore play an important role in mechanotransduction and cell motility. When endothelial cells are subjected to cyclic mechanical strain, time-lapse imaging revealed that cells underwent significant morphological changes with their resultant long axes aligned away from the strain direction. To explore how this response is regulated by focal adhesion-associated proteins the expression levels of paxillin, focal adhesion kinase (FAK), and zyxin were knocked down using gene silencing techniques. In addition, rescue of endogenous and two mutant zyxins were used to investigate the specific role of zyxin interactions. Cells with decreased zyxin expression levels and rescue with the mutant lacking zyxin/α-actinin binding exhibited lower orientation angles after comparable times of stretching as compared to normal and control cells. However, knockdown of the expression levels of paxillin and FAK and rescue with the mutant lacking zyxin/VASP (vasodilator-stimulated phosphoprotein) binding did not significantly affect the degree of cell orientation. In addition, wound closure speed and cell–substratum adhesive strength were observed to be significantly reduced only for cells with zyxin depletion and the mutation lacking zyxin/α-actinin binding. These results suggest that zyxin and its interaction with α-actinin are important in the regulation of endothelial cell adhesive strength, motility and orientation response to mechanical stretching.     

Expression of Oct4 in human embryonic stem cells is dependent on nanotopographical configuration

The fate of adult stem cells can be influenced by physical cues, including nanotopography. However, the response of human embryonic stem cells (hESCs) to dimensionally well-defined nanotopography is unknown. Using imprint lithography, we prepared well-defined nanotopography of hexagonal (HEX) and honeycomb (HNY) configurations with various spacings between the nanostructures. In serum-free hESC culture medium, basic fibroblast growth factor (bFGF) is required to maintain expression of Oct4, a pluripotent gene. Unexpectedly, hESCs cultured on nanotopography could maintain Oct4 expression without bFGF supplementation. With bFGF supplementation, the HEX nanotopography maintained Oct4 expression whereas the HNY configuration caused down-regulation of Oct4 expression. Thus, we observed that the lattice configurations of the nanotopography cause hESCs to respond to bFGF in different ways. This differential response to a biochemical cue by nanotopography was unforeseen, but its discovery could lead to novel differentiation pathways. Consistent with studies of other cells, we observed that nanotopography affects focal adhesion formation in hESCs. We posit that this can in turn affect cell–matrix tension, focal adhesion kinase signaling and integrin–growth factor receptor crosstalk, which eventually modulates Oct4 expression in hESCs.     

The effect of actin disrupting agents on contact guidance of human embryonic stem cells

Mammalian cells respond to their substrates by complex changes in gene expression profiles, morphology, proliferation and migration. We report that substrate nanotopography alters morpohology and proliferation of human embryonic stem cells (hESCs). Fibronectin-coated poly(di-methyl siloxane) substrates with line-grating (600nm ridges with 600nm spacing and 600+/-150nm feature height) induced hESC alignment and elongation, mediated the organization of cytoskeletal components including actin, vimentin, and alpha-tubulin, and reduced proliferation. Spatial polarization of gamma-tubulin complexes was also observed in response to nanotopography. Furthermore, the addition of actin disrupting agents attenuated the alignment and proliferative effects of nanotopography. These findings further demonstrate the importance of interplay between cytoskeleton and substrate interactions as a key modulator of morphological and proliferative cellular response in hESCs on nanotopography.     

Submicron poly(L-lactic acid) pillars affect fibroblast adhesion and proliferation

Controlling cell adhesion and proliferation on synthetic polymers is key to tissue engineering scaffold development. It is accepted that surface topography influences cell response but the mechanisms behind this remain unclear. In this work, cell response is assessed to topographies larger than focal complexes (FXs) but smaller than focal adhesions (FAs). Poly(L-lactic acid) was patterned with 400- and 700-nm pillars via replication molding. Human fibroblast adhesion and proliferation were assessed. The development of focal contacts and actin microfilaments were evaluated via immunofluorescence. Cell interactions with surface topography were observed via scanning electron microscopy. Initial fibroblast adhesion (<1 day) increased with texture as 400 nm > 700 nm > smooth, but proliferation (>1 day) decreased with texture. Increased FX formation was observed on textured surfaces. However, FAs were narrower on textured surfaces compared with smooth materials and confined to interpillar regions. SEM showed that fibroblasts deformed the 400-nm pillars. It is hypothesized that surface texture mediated FX formation and increased cell adhesion, possibly via increased material surface area. Texture geometry limited maturation of FXs to FAs, decreasing proliferation. We conclude that surface texture can alter cell adhesion and proliferation and propose geometric constraint as a mechanism for this process.     

The regulation of integrin-mediated osteoblast focal adhesion and focal adhesion kinase expression by nanoscale topography

An important consideration in developing physical biomimetic cell-stimulating cues is that the in vivo extracellular milieu includes nanoscale topographic interfaces. We investigated nanoscale topography regulation of cell functions using human fetal osteoblastic (hFOB) cell culture on poly(l-lactic acid) and polystyrene (50/50 w/w) demixed nanoscale pit textures (14, 29, and 45nm deep pits). Secondary ion mass spectroscopy revealed that these nanotopographic surfaces had similar surface chemistries to that of pure PLLA because of PLLA component surface segregation during spin casting. We observed that 14 and 29nm deep pit surfaces increased hFOB cell attachment, spreading, selective integrin subunit expression (e.g., alphav relative to alpha5, beta1, or beta3), focal adhesive paxillin protein synthesis and paxillin colocalization with cytoskeletal actin stress fibers, and focal adhesion kinase (FAK) and phosphorylated FAK (pY397) expression to a greater degree than did 45nm deep pits or flat PLLA surfaces. Considering the important role of integrin-mediated focal adhesion and intracellular signaling in anchorage-dependent cell function, our results suggest a mechanism by which nanostructured physical signals regulate cell function. Modulation of integrin-mediated focal adhesion and related cell signaling by altering nanoscale substrate topography will have powerful applications in biomaterials science and tissue engineering.     

Chemical and topographical influence of hydroxyapatite and beta-tricalcium phosphate surfaces on human osteoblastic cell behavior

The objective of this work was to evaluate the relative role of the calcium phosphate surface chemistry and surface topography on human osteoblast behavior. Highly dense phosphate ceramics (single-phase hydroxyapatite HA and beta-tricalcium phosphates TCP) presenting two distinct nano roughnesses were produced. Some samples were gold-sputter coated in order to conveniently mask the surface chemical effects (without modification of the original roughness) and to study the isolated effect of surface topography on cellular behavior. Our results indicated that the nanotopography of the studied ceramics had no effect on the cellular adhesion (cell spreading, focal contacts and stress fibers formation). On the contrary, strong topographical effects were verified on cell proliferation and differentiation. Moreover, the phosphate chemistry was responsible for changes in adhesion, proliferation and cell differentiation. On TCP, it was shown that the main influent parameter was surface chemistry, which negatively affected the initial cell adhesion but positively affected the subsequent stage of proliferation and differentiation. On HA, the main influent parameter was surface topography, which increased cell differentiation but lowered proliferation.     

Fibroblast response to a controlled nanoenvironment produced by colloidal lithography

It is thought that by understanding how cells respond to topography, that better tissue engineering may be achievable. An important consideration in the cellular environment is topography. The effects of microtopography have been well documented, but the effects of nanotopography are less well known. Previously, methods of nanofabrication have been costly and time-consuming, but research by engineers, physicists, and chemists is starting to allow the production of nanostructures using low-cost techniques. In this report, nanotopography is specifically considered. Controlled patterns of 160 nm high nanocolumns were produced for in vitro cell culture using colloidal lithography. By studying cell adhesion with time and cytoskeletal (actin, tubulin, and vimentin) maturity, insight has been gained as to how fibroblasts adhere to these nanofeatures.     

The interaction of cells and bacteria with surfaces structured at the nanometre scale

The current development of nanobiotechnologies requires a better understanding of cell–surface interactions on the nanometre scale. Recently, advances in nanoscale patterning and detection have allowed the fabrication of appropriate substrates and the study of cell–substrate interactions. In this review we discuss the methods currently available for nanoscale patterning and their merits, as well as techniques for controlling the surface chemistry of materials at the nanoscale without changing the nanotopography and the possibility of truly characterizing the surface chemistry at the nanoscale. We then discuss the current knowledge of how a cell can interact with a substrate at the nanoscale and the effect of size, morphology, organization and separation of nanofeatures on cell response. Moreover, cell–substrate interactions are mediated by the presence of proteins adsorbed from biological fluids on the substrate. Many questions remain on the effect of nanotopography on protein adsorption. We review papers related to this point. As all these parameters have an influence on cell response, it is important to develop specific studies to point out their relative influence, as well as the biological mechanisms underlying cell responses to nanotopography. This will be the basis for future research in this field. An important topic in tissue engineering is the effect of nanoscale topography on bacteria, since cells have to compete with bacteria in many environments. The limited current knowledge of this topic is also discussed in the light of using topography to encourage cell adhesion while limiting bacterial adhesion. We also discuss current and prospective applications of cell–surface interactions on the nanoscale. Finally, based on questions raised previously that remain to be solved in the field, we propose future directions of research in materials science to help elucidate the relative influence of the physical and chemical aspects of nanotopography on bacteria and cell response with the aim of contributing to the development of nanobiotechnologies.     

Multiscale patterned transplantable stem cell patches for bone tissue regeneration

Stem cell-based therapy has been proposed as an enabling alternative not only for the treatment of diseases but also for the regeneration of tissues beyond complex surgical treatments or tissue transplantation. In this study, we approached a conceptual platform that can integrate stem cells into a multiscale patterned substrate for bone regeneration. Inspired by human bone tissue, we developed hierarchically micro- and nanopatterned transplantable patches as synthetic extracellular matrices by employing capillary force lithography in combination with a surface micro-wrinkling method using a poly(lactic-co-glycolic acid) (PLGA) polymer. The multiscale patterned PLGA patches were highly flexible and showed higher tissue adhesion to the underlying tissue than did the single nanopatterned patches. In response to the anisotropically multiscale patterned topography, the adhesion and differentiation of human mesenchymal stem cells (hMSCs) were sensitively controlled. Furthermore, the stem cell patch composed of hMSCs and transplantable PLGA substrate promoted bone regeneration in vivo when both the micro- and nanotopography of the substrate surfaces were synergistically combined. Thus, our study concludes that multiscale patterned transplantable stem cell patches may have a great potential for bone regeneration as well as for various regenerative medicine approaches.     

Quantitative assessment of the response of primary derived human osteoblasts and macrophages to a range of nanotopography surfaces in a single culture model in vitro

The effect of nanotopography on a range of Ti oxide surfaces was determined. Flat Ti, 3%, 19%, 30% and 43% topography densities of 110 nm high hemispherical protrusions were cultured in contact with primary derived human macrophages and osteoblasts in single culture models. Prior to introduction of the test substrate the phenotype and optimum conditions for in vitro cell culture were established. The cellular response was investigated and quantified by assessments of cytoskeletal development and orientation, viable cell adhesion, cytokine production and release and RT-PCR analysis of osteogenic markers. The tested nanotopographies did not have a statistically significant effect on viable cell adhesion and subsequent cytoskeletal formation. Surface chemistry was the dominant factor as established via incorporation of a tissue culture polystyrene, TCPS, control. The topography surfaces induced a release of chemotactic macrophage activation agents at 1 day in conjunction with stress fibre formation and a subsequent fibronectin network formation. Osteoblasts migrated away from the topography surfaces to the exposed TCPS within the wells during the 7-day period.     

纳米拓扑结构的构建以及对细胞行为的影响

生物材料的拓扑形貌是影响细胞行为的重要因素之一,近年来随着纳米生物和医学技术的进展,研究发现细胞能够通过“接触诱导”感应基底材料的纳米拓扑结构,产生强烈的响应。我们对纳米结构的不同构建方法进行了综述,分析不同的纳米拓扑结构对不同种类细胞及其功能的作用,包括对细胞黏附、增殖、分化、凋亡以及细胞外基质的重建等行为的影响和可能的发生机制,探讨了纳米拓扑结构的构建对于从分子和细胞水平上控制生物材料与细胞间的相互作用,从而引发特异性细胞反应的重要作用,以及对于组织再生与修复的潜在应用前景和意义。     

Fabrication and characterization of a recombinant fibronectin/cadherin bio-inspired ceramic surface and its influence on adhesion and ossification in vitro

This study has investigated the effects of a bio-inspired ceramic surface modified with a novel recombinant protein on surface parameters and cell behavior. The surface of a biphasic calcium phosphate ceramic was functionalized with a recombinant protein spanning the fragments of fibronectin module III7–10 and extracellular domains 1 and 2 of cadherin 11 (rFN/CDH) using a dimethyl-3,3′-dithiobispropionimidate cross-linking method. The surface was characterized by scanning electron microscopy, X-ray photoelectron spectroscopy and protein adsorption and surface density measurements. The material exhibited desirable properties for cell adhesion and proliferation. The effects of the surface on the adhesion and proliferation of human mesenchymal stem cells (hMSC) were investigated using a cell adhesion centrifugal assay and the 3-(4,5-dmethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. The data demonstrated that the adhesive capacity and proliferation rate were significantly improved as compared with fibronectin and cadherin positive controls. Moreover, the rFN/CDH bio-inspired ceramic surface also induced osteoblastic differentiation, as evidenced by the higher alkaline phosphatase activity and osteocalcin mRNA expression level of hMSC cultured in osteogenic media for 7–10 days. Furthermore, a functional blocking assay with a site-specific antibody against phosphotyrosine 397 (pY397) of focal adhesion kinase revealed that pY397 is involved in adhesion and ossification. These results suggest that the rFN/CDH bio-inspired BCP surface possesses enhanced functionality in adhesion, proliferation and ossification and may be a promising scaffold for tissue engineering.     

Zyxin and vinculin distribution at the cell-extracellular matrix attachment complex (CMAX) in corneal epithelial tissue are actin dependent

Avian embryonic corneal epithelia are two cell layers thick. If isolated without (-) basal lamina, the basal cells have unorganized actin and project cytoplasmic protrusions termed blebs. The actin-based cytoskeleton at the cell-extracellular matrix junction (termed the actin cortical mat) is disrupted. These epithelia respond to soluble extracellular matrix molecules by reorganizing the actin cortical mat. Sheets of epithelia were isolated + or -basal lamina. Epithelia isolated -basal lamina were cultured +/- laminin-1 and/or +/- cytochalasin D (CD). The intracellular localization of zyxin, vinculin, paxillin, focal adhesion kinase, and tensin was determined using indirect immunohistochemistry. Protein levels were determined by Western blot analysis. Zyxin and vinculin were concentrated in two areas of the tissue. The interface between the upper cell layer (periderm) and the basal cells. The second area of concentration was at the inferior 1-4 microns of the basal cells in an area with multiple actin bundles termed the actin cortical mat. The actin bundles align toward zyxin and vinculin that were located near basal lateral membranes. Zyxin was displaced from the basal compartment of blebbing basal cells. In contrast tensin, vinculin and focal adhesion kinase were found diffusely throughout the blebs. Zyxin and vinculin redistributed to the basal-lateral membranes as actin bundles reorganized in laminin-stimulated epithelia. In contrast to the altered protein distribution, extractable protein levels were similar in blebbing and laminin-stimulated epithelia. Zyxin, vinculin, and other associated proteins were disrupted in the CD-treated tissues and do not colocalize with each other or CD-induced actin aggregates. The intracellular localization of zyxin and vinculin were concentrated in distinct areas along the inferior basolateral membranes of basal cells termed the cell-extracellular matrix attachment complex (CMAX). The distribution of CMAX proteins was dependent upon actin bundle organization.     

Influence of polymer membrane porosity on C3A hepatoblastoma cell adhesive interaction and function

The effect of the porosity of acrylonitrile-N-vinylpyrrolidone copolymer membranes on human C3A hepatoblastoma cell adhesive interaction and functioning is investigated on four membranes with an average pore size ranging between 6 and 12 nm. Adhesion of C3A cells was quantified and characterized by studying overall cell morphology and focal adhesion formation. Cell-cell interactions were characterized by E-cadherin expression and organization. Cell growth, fibronectin synthesis and cytochrome P450 activity were estimated as criteria of functional cell activity. The results suggest that membrane porosity influences the initial cell-surface interactions since an increasing pore size augmented cell adhesion and aggregate formation. Cell growth after 7 d was diminished on membranes with an average pore size of 12 nm. The activity of P450 measured by 7-ethoxycoumarin conversion at day 7 was influenced by membrane topography representing a clear optimum in the range of 7-10 nm pore size. These results indicate that membrane porosity is a determinant for the function of hepatocytes in extracorporal liver assist devices.     

The threshold at which substrate nanogroove dimensions may influence fibroblast alignment and adhesion

The differences in morphological behaviour between fibroblasts cultured on smooth and nanogrooved substrata (groove depth: 5-350 nm, width: 20-1000 nm) have been evaluated in vitro. The aim of the study was to clarify to what extent cell guidance occurs on increasingly smaller topographies. Pattern templates were made using electron beam lithography, and were subsequently replicated in polystyrene cell culture material using solvent casting. The replicates were investigated with atomic force microscopy (AFM). After seeding with fibroblasts, morphological characteristics were investigated using scanning electron microscopy (SEM) and light microscopy, in order to obtain qualitative and quantitative information on cell alignment. AFM revealed that the nanogroove/ridge widths were replicated perfectly, although at deeper levels the grooves became more concave. The smooth substrata had no distinguishable pattern other than a roughness amplitude of 1 nm. Interestingly, microscopy and image analysis showed that fibroblast after 4 h had adjusted their shape according to nanotopographical features down to cut-off values of 100 nm width and 75 nm depth. After 24 h culturing time, fibroblasts would even align themselves on groove depths as shallow as 35 nm. It appears depth is the most essential parameter in cellular alignment on groove patterns with a pitch ratio of 1:1. On the smooth substrata, cells always spread out in a random fashion. Analysis of variance (ANOVA) demonstrated that both main parameters, topography and culturing time, were significant. We conclude that fibroblast cells cultured on nanotopography experience a threshold feature size of 35 nm, below this value contact guidance does no longer exist.     

The fibroblast response to tubes exhibiting internal nanotopography

The use of three-dimensional scaffolds in cell and tissue engineering is widespread; however, the use of such scaffolds, which bear additional cellular cues such as nanotopography, is as yet in its infancy. This paper details the novel fabrication of nylon tubes bearing nanotopography via polymer demixing, and reports that the topography greatly influenced fibroblast adhesion, spreading, morphology and cytoskeletal organisation. The use of such frameworks that convey both the correct mechanical support for tissue formation and stimulate cells through topographical cues may pave the way for future production of intelligent materials and scaffolds.     

Nanopattern-induced changes in morphology and motility of smooth muscle cells

Cells are known to be surrounded by nanoscale topography in their natural extracellular environment. The cell behavior, including morphology, proliferation, and motility of bovine pulmonary artery smooth muscle cells (SMC) were studied on poly(methyl methacrylate) (PMMA) and poly(dimethylsiloxane) (PDMS) surfaces comprising nanopatterned gratings with 350 nm linewidth, 700 nm pitch, and 350 nm depth. More than 90% of the cells aligned to the gratings, and were significantly elongated compared to the SMC cultured on non-patterned surfaces. The nuclei were also elongated and aligned. Proliferation of the cells was significantly reduced on the nanopatterned surfaces. The polarization of microtubule organizing centers (MTOC), which are associated with cell migration, of SMC cultured on nanopatterned surfaces showed a preference towards the axis of cell alignment in an in vitro wound healing assay. In contrast, the MTOC of SMC on non-patterned surfaces preferentially polarized towards the wound edge. It is proposed that this nanoimprinting technology will provide a valuable platform for studies in cell-substrate interactions and for development of medical devices with nanoscale features.     

Zyxin and vinculin distribution at the Cell‐extracellular Matrix Attachment Complex (CMAX) in corneal epithelial tissue are actin dependent

Avian embryonic corneal epithelia are two cell layers thick. If isolated without (−) basal lamina, the basal cells have unorganized actin and project cytoplasmic protrusions termed blebs. The actin‐based cytoskeleton at the cell‐extracellular matrix junction (termed the actin cortical mat) is disrupted. These epithelia respond to soluble extracellular matrix molecules by reorganizing the actin cortical mat. Sheets of epithelia were isolated + or ‐basal lamina. Epithelia isolated ‐basal lamina were cultured ± laminin‐1 and/or ± cytochalasin D (CD). The intracellular localization of zyxin, vinculin, paxillin, focal adhesion kinase, and tensin was determined using indirect immunohistochemistry. Protein levels were determined by Western blot analysis. Zyxin and vinculin were concentrated in two areas of the tissue. The interface between the upper cell layer (periderm) and the basal cells. The second area of concentration was at the inferior 1–4 microns of the basal cells in an area with multiple actin bundles termed the actin cortical mat. The actin bundles align toward zyxin and vinculin that were located near basal lateral membranes. Zyxin was displaced from the basal compartment of blebbing basal cells. In contrast tensin, vinculin and focal adhesion kinase were found diffusely throughout the blebs. Zyxin and vinculin redistributed to the basal‐lateral membranes as actin bundles reorganized in laminin‐stimulated epithelia. In contrast to the altered protein distribution, extractable protein levels were similar in blebbing and laminin‐stimulated epithelia. Zyxin, vinculin, and other associated proteins were disrupted in the CD‐treated tissues and do not colocalize with each other or CD‐induced actin aggregates. The intracellular localization of zyxin and vinculin were concentrated in distinct areas along the inferior basolateral membranes of basal cells termed the cell‐extracellular matrix attachment complex (CMAX). The distribution of CMAX proteins was dependent upon actin bundle organization. Anat Rec 254:336–347, 1999. © 1999 Wiley‐Liss, Inc.     

Modulation of alignment, elongation and contraction of cardiomyocytes through a combination of nanotopography and rigidity of substrates

The topographic and mechanical characteristics of engineered tissue constructs, simulating native tissues, should benefit tissue engineering. Previous studies reported that surface topography and substrate rigidity provide biomechanical cues to modulate cellular responses such as alignment, migration and differentiation. To fully address this issue, the present study aimed to examine the influence of nanogrooved substrates with different stiffnesses on the responses of rat cardiomyocytes. Nanogrooved substrates (450nm in groove/ridge width; 100 or 350nm in depth) made of polystyrene and polyurethane were prepared by imprinting from polydimethylsiloxane molds. The morphology and orientation of cardiomyocytes attached to the substrates were found to be influenced mainly by the nanogrooved structures, while the contractile function of the cells was regulated by the coupled effect of surface topography and substrate stiffness. The distribution of intracellular structural proteins such as vinculin and F-actin showed that the surface topography and substrate stiffness regulated the organization of the actin cytoskeleton and focal adhesion complexes, and consequently the contractile behavior of the cardiomyocytes. The beating rates of the cultured cardiomyocytes were dependent on both the surface topography and the substrate stiffness. The study provides insights into the interaction between cardiomyocytes and biomaterials, and benefits cardiac tissue engineering.