A tough act to follow: collagen hydrogel modifications to improve mechanical and growth factor loading capabilities.

Collagen hydrogels are among â€‹the most well-studied platforms for drug delivery and in situ tissue engineering, thanks to their low cost, low immunogenicity, versatility, biocompatibility, and similarity to the natural extracellular matrix (ECM). Despite collagen being largely responsible for the tensile properties of native connective tissues, collagen hydrogels have relatively low mechanical properties in the absence of covalent cross-linking. This is particularly problematic when attempting to regenerate stiffer and stronger native tissues such as bone. Furthermore, in contrast to hydrogels based on ECM proteins such as fibronectin, collagen hydrogels do not have any growth factor (GF)-specific binding sites and often cannot sequester physiological (small) amounts of the protein. GF binding and in situ presentation are properties that can aid significantly in the tissue regeneration process by dictating cell fate without causing adverse effects such as malignant tumorigenic tissue growth. To alleviate these issues, researchers have developed several strategies to increase the mechanical properties of collagen hydrogels using physical or chemical modifications. This can expand the applicability of collagen hydrogels to tissues subject to a continuous load. GF delivery has also been explored, mathematically and experimentally, through the development of direct loading, chemical cross-linking, electrostatic interaction, and other carrier systems. This comprehensive article explores the ways in which these parameters, mechanical properties and GF delivery, have been optimized in collagen hydrogel systems â€‹and examines their in vitro or in vivo biological effect. This article can, therefore, be a useful tool to streamline future studies in the field, by pointing researchers into the appropriate direction according to their collagen hydrogel design requirements.

Cell migration on material-driven fibronectin microenvironments.

Cell migration is a fundamental process involved in a wide range of biological phenomena. However, how the underlying mechanisms that control migration are orchestrated is not fully understood. In this work, we explore the migratory characteristics of human fibroblasts using different organisations of fibronectin (FN) triggered by two chemically similar surfaces, poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA); cell migration is mediated via an intermediate layer of fibronectin (FN). FN is organised into nanonetworks upon simple adsorption on PEA whereas a globular conformation is observed on PMA. We studied cell speed over the course of 24 h and the morphology of focal adhesions in terms of area and length. Additionally, we analysed the amount of cell-secreted FN as well as FN remodelling. Velocity of human fibroblasts was found to exhibit a biphasic behaviour on PEA, whereas it remained fairly constant on PMA. FA analysis revealed more mature focal adhesions on PEA over time contrary to smaller FAs found on PMA. Finally, human fibroblasts seemed to remodel adsorbed FN more on PMA than on PEA. Overall, these results indicate that the cell-protein-material interface affects cell migratory behaviour. Analysis of FAs together with FN secretion and remodelling were associated with differences in cell velocity providing insights into the factors that can modulate cell motility.

Osteogenic and bactericidal surfaces from hydrothermal titania nanowires on titanium substrates.

Nanotopographical cues on Ti have been shown to elicit different cell responses such as cell differentiation and selective growth. Bone remodelling is a constant process requiring specific cues for optimal bone growth and implant fixation. Moreover, biofilm formation and the resulting infection on surgical implants is a major issue. Our aim is to identify nanopatterns on Ti surfaces that would be optimal for both bone remodelling and for reducing risk of bacterial infection. Primary human osteoblast/osteoclast co-cultures were seeded onto Ti substrates with TiO2 nanowires grown under alkaline conditions at 240 °C for different times (2, 2.5 or 3 h). Cell growth and behaviour was assessed by scanning electron microscopy (SEM), immunofluorescence microscopy, histochemistry and quantitative RT-PCR methods. Bacterial colonisation of the nanowire surfaces was also assessed by confocal microscopy and SEM. From the three surfaces tested the 2 h nanowire surface supported osteoblast and to a lesser extent osteoclast growth and differentiation. At the same time bacterial viability was reduced. Hence the 2 h surface provided optimal bone remodeling in vitro conditions while reducing infection risk, making it a favourable candidate for future implant surfaces.

Nanotopography and plasma treatment: redesigning the surface for vascular graft endothelialisation.

INTRODUCTION: Vascular graft materials in clinical use, such as polytetrafluoroethylene (PTFE) and Dacron, do not endothelialise and have low patency rates. The importance of an endothelial cell layer on the luminal surface of a vascular graft is well-known with surface topography and chemistry playing an important role. The aim of this study was to investigate the potential of plasma treatment and topographical structures on the luminal graft surface to enhance the self-endothelialisation potential of a nanocomposite vascular graft. METHODS: POSS-PCU is a polycarbonate urea urethane (PCU) with a nanoparticle, polyhedral oligomeric silsesquioxane (POSS) incorporated within it. Planar, microgrooved, and nanopit patterned polymer films were fabricated using photolithography, electron beam lithography, reactive ion etching, and replication by solvent casting. Films were then exposed to oxygen plasma treatment at different powers for a fixed time (40 W, 60 W, 80 W/60 seconds). Effects of plasma treatment were assessed using scanning electron microscopy, atomic force microscopy and water contact angle analysis. Human umbilical vein endothelial cell (HUVEC) proliferation and morphology were characterised using immunostaining, live/dead staining, and Coomassie blue staining. RESULTS: Successful embossing of the micro- and nanostructures was confirmed. Oxygen plasma treatment of the different samples showed that increasing power significantly increased the hydrophilicity of the samples (p < .0001). Improved HUVEC adhesion was seen on plasma modified compared with untreated samples (p < .0001). Coomassie blue staining showed that after 5 days, cells started to form monolayers and live/dead staining showed the cells were viable. Immunostaining showed that HUVECs expressed nitric oxide synthase on all topographies with focal adhesions appearing more pronounced on nanopit surfaces, showing retention of morphology and function. CONCLUSION: These encouraging results indicate a future important role for plasma treatment and nanotopography in the development of endothelialised vascular grafts.

Luminal surface engineering, 'micro and nanopatterning': potential for self endothelialising vascular grafts?

OBJECTIVE: New technologies are being explored to meet the clinical need for an 'off-the-shelf' small diameter vascular graft with superior or at least equivalent properties to autologous vessel. The field of nanotechnology and fabrication promises major advances in biomaterial design and wall structure to deliver biomimetic grafts. This review brings together recent work on this topic. METHODS: A literature search was conducted of PubMed and ISI Web of Knowledge using relevant keywords. Articles published after January 2005 were given preference. Personal communications and PhD theses were also used as sources. RESULTS: An evolving focus on surface patterning of biomaterials has been found to carry great potential. Influencing cellular behaviour on prosthetic grafts using graft luminal surface modulation at the micro- and nano-levels is the basis of this recent concept in vascular graft development. CONCLUSION: This technology may deliver small diameter grafts with the potential for spontaneous in situ endothelialisation without the need for prior 'seeding', with the potential to open a new chapter in vascular graft development.

Biocompatible, smooth, plasma-treated nickel-titanium surface--an adequate platform for cell growth.

High nickel content is believed to reduce the number of biomedical applications of nickel-titanium alloy due to the reported toxicity of nickel. The reduction in nickel release and minimized exposure of the cell to nickel can optimize the biocompatibility of the alloy and increase its use in the application where its shape memory effects and pseudoelasticity are particularly useful, e.g., spinal implants. Many treatments have been tried to improve the biocompatibility of Ni-Ti, and results suggest that a native, smooth surface could provide sufficient tolerance, biologically. We hypothesized that the native surface of nickel-titanium supports cell differentiation and insures good biocompatibility. Three types of surface modifications were investigated: thermal oxidation, alkali treatment, and plasma sputtering, and compared with smooth, ground surface. Thermal oxidation caused a drop in surface nickel content, while negligible chemistry changes were observed for plasma-modified samples when compared with control ground samples. In contrast, alkali treatment caused significant increase in surface nickel concentration and accelerated nickel release. Nickel release was also accelerated in thermally oxidized samples at 600 °C, while in other samples it remained at low level. Both thermal oxidation and alkali treatment increased the roughness of the surface, but mean roughness R(a) was significantly greater for the alkali-treated ones. Ground and plasma-modified samples had 'smooth' surfaces with R(a)=4 nm. Deformability tests showed that the adhesion of the surface layers on samples oxidized at 600 °C and alkali treatment samples was not sufficient; the layer delaminated upon deformation. It was observed that the cell cytoskeletons on the samples with a high nickel content or release were less developed, suggesting some negative effects of nickel on cell growth. These effects were observed primarily during initial cell contact with the surface. The most favorable cell responses were observed for ground and plasma-sputtered surfaces. These studies indicated that smooth, plasma-modified surfaces provide sufficient properties for cells to grow.

Focal adhesions in osteoneogenesis.

As materials technology and the field of tissue engineering advance, the role of cellular adhesive mechanisms, in particular, interactions with implantable devices, becomes more relevant in both research and clinical practice. A key tenet of medical device technology is to use the exquisite ability of biological systems to respond to the material surface or chemical stimuli in order to help to develop next-generation biomaterials. The focus of this review is on recent studies and developments concerning focal adhesion formation in osteoneogenesis, with an emphasis on the influence of synthetic constructs on integrin-mediated cellular adhesion and function.

Focal adhesion interactions with topographical structures: a novel method for immuno-SEM labelling of focal adhesions in S-phase cells.

Current understanding of the mechanisms involved in osseointegration following implantation of a biomaterial has led to adhesion quantification being implemented as an assay of cytocompatibility. Such measurement can be hindered by intra-sample variation owing to morphological changes associated with the cell cycle. Here we report on a new scanning electron microscopical method for the simultaneous immunogold labelling of cellular focal adhesions and S-phase nuclei identified by BrdU incorporation. Prior to labelling, cellular membranes are removed by tritonization and antigens of non-interest blocked by serum incubation. Adhesion plaque-associated vinculin and S-phase nuclei were both separately labelled with a 1.4 nm gold colloid and visualized by subsequent colloid enhancement via silver deposition. This study is specifically concerned with the effects microgroove topographies have on adhesion formation in S-phase osteoblasts. By combining backscattered electron (BSE) imaging with secondary electron (SE) imaging it was possible to visualize S-phase nuclei and the immunogold-labelled adhesion sites in one energy 'plane' and the underlying nanotopography in another. Osteoblast adhesion to these nanotopographies was ascertained by quantification of adhesion complex formation.

Adhesion formation of primary human osteoblasts and the functional response of mesenchymal stem cells to 330nm deep microgrooves.

The surface microtexture of an orthopaedic device can regulate cellular adhesion, a process fundamental in the initiation of osteoinduction and osteogenesis. Advances in fabrication techniques have evolved to include the field of surface modification; in particular, nanotechnology has allowed for the development of experimental nanoscale substrates for investigation into cell nanofeature interactions. Here primary human osteoblasts (HOBs) were cultured on ordered nanoscale groove/ridge arrays fabricated by photolithography. Grooves were 330nm deep and either 10, 25 or 100microm in width. Adhesion subtypes in HOBs were quantified by immunofluorescent microscopy and cell-substrate interactions were investigated via immunocytochemistry with scanning electron microscopy. To further investigate the effects of these substrates on cellular function, 1.7K gene microarray analysis was used to establish gene regulation profiles of mesenchymal stem cells cultured on these nanotopographies. Nanotopographies significantly affected the formation of focal complexes (FXs), focal adhesions (FAs) and supermature adhesions (SMAs). Planar control substrates induced widespread adhesion formation; 100microm wide groove/ridge arrays did not significantly affect adhesion formation yet induced upregulation of genes involved in skeletal development and increased osteospecific function; 25microm wide groove/ridge arrays were associated with a reduction in SMA and an increase in FX formation; and 10microm wide groove/ridge arrays significantly reduced osteoblast adhesion and induced an interplay of up- and downregulation of gene expression. This study indicates that groove/ridge topographies are important modulators of both cellular adhesion and osteospecific function and, critically, that groove/ridge width is important in determining cellular response.

The effects of nanoscale pits on primary human osteoblast adhesion formation and cellular spreading.

Current understanding of the mechanisms involved in ossesoinegration following implantation of a biomaterial has led to an emphasis being placed on the modification of material topography to control interface reactions. Recent studies have inferred nanoscale topography as an important mediator of cell adhesion and differentiation. Biomimetic strategies in orthopaedic research aim to exploit these influences to regulate cellular adhesion and subsequent bony tissue formation. Here experimental topographies of nanoscale pits demonstrating varying order have been fabricated by electron-beam lithography in (poly)carbonate. Osteoblast adhesion to these nanotopographies was ascertained by quantification of the relation between adhesion complex formation and total cell area. This study is specifically concerned with the effects these nanotopographies have on adhesion formation in S-phase osteoblasts as identified by BrdU incorporation. Nanopits were found to reduce cellular spreading and adhesion formation.

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

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) 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.

The interaction of human bone marrow cells with nanotopographical features in three dimensional constructs.

Until now, nanotopography has been considered in 2D construct designs. This has been due to fabrication limitations with traditional lithographic processes relying on the ability to focus radiation that will expose a radiation sensitive resist (e.g. photolithography and electron beam lithography). More recently, alternative methods that offer rapid and cheap nanofabrication have been developed; such methods include polymer demixing and colloidal lithography. Polymer demixing in 2D has relied on spin casting of polymer blends-such as polystyrene and polybromostyrene in a solvent such as toluene. As the solvent evaporates, the polymers phase separate and form nanoislands. In this study, the polymer blend solution has been blown through fine tubes and allowed to demix, thus providing 3D constructs for cell biology. The ability to fabricate in tubes may be useful in many applications, for example stents, conduits, and bone repair (when considering structures such as Haversian tubes and Volkmann's canals). As proof of concept, human osteoprogenitor cells have been used to test the cell response to the nanopatterned tubes. The results show that nanofeatures of size X, diameter Y, and spacing Z decrease cell spreading, reduce cytoskeletal organization, and increase endocytotic activity within the cells.

Morphological and microarray analysis of human fibroblasts cultured on nanocolumns produced by colloidal lithography.

The environment around a cell during in vitro culture is unlikely to mimic those in vivo. Preliminary experiments with nanotopography have shown that nanoscale features can strongly influence cell morphology, adhesion, proliferation and gene regulation, but the mechanisms mediating this cell response remain unclear. In this study a well defined nanotopography, consisting of 100 nm wide and 160 nm high cylindrical columns, was used in fibroblast culture. In order to build on previously published morphological data that showed changes in cell spreading on the nanocolumns, in this study gene regulation was monitored using a 1718 gene microarray. Transmission electron microscopy, fluorescent observation of actin and Rac and area quantification have been used to re-affirm the microarray observations. The results indicate that changes in cell spreading correlate with a number of gene up- and down-regulations as will be described within the manuscript.

Cells react to nanoscale order and symmetry in their surroundings.

Mammalian cells react to microstructured surfaces, but there is little information on the reactions to nanostructured surfaces, and such as have been tested are poorly ordered or random in their structure. We now report that ordered surface arrays (orthogonal or hexagonal) of nanopits in polycaprolactone or polymethylmethacrylate have marked effects in reducing cell adhesion compared with less regular arrays or planar surfaces. The pits had diameters of 35, 75, and 120 nm, respectively, with pitch between the pits of 100, 200, and 300 nm, respectively. The cells appear to be able to distinguish between different symmetries of array. We suggest that interfacial forces may be organized by the nanostructures to affect the cells in the same way as they affect liquid crystal orientations.

Investigating the limits of filopodial sensing: a brief report using SEM to image the interaction between 10 nm high nano-topography and fibroblast filopodia.

Having the ability to control cell behaviour would be of great advantage in tissue engineering. One method of gaining control over cell adhesion, proliferation, guidance and differentiation is use of topography. Whilst it has be known for some time that cells can be guided by micro-topography, it is only recently becoming clear that cells will respond strongly to nano-scale topography. The fact that cells will take cues from their micro- and nano-environment suggests that the cells are in some way 'spatially aware'. It is likely that cells probe the shape of their surroundings using filopodia, and that this initial filopodia/topography interaction may be critical to down-stream cell reactions to biomaterials, or indeed, the extracellular matrix. One intriguing question is how small a feature can cells sense? In order to investigate the limits of cell sensing, high-resolution scanning electron microscopy has been used to simultaneously view cell filopodia and 10 nm high nano-islands. Fluorescence microscopy has also been used to look at adhesion formation. The results showed distinct filopodial/nano-island interaction and changes in adhesion morphology.

Rapid fibroblast adhesion to 27nm high polymer demixed nano-topography.

It is well known that many cell types react strongly to micro-topography. It is rapidly becoming clear than cells will also react to nano-topography. Polymer demixing is a rapid and low-cost chemical method of producing nano-topography. This manuscript investigates human fibroblast response to 27nm high nano-islands produced by polymer demixing. Cell spreading, cytoskeleton, focal adhesion and Rac localisation were studied. The results showed that an initial rapid adhesion and cytoskeletal formation on the islands at 4 days of culture gave way to poorly formed contacts and vimentin cytoskeleton at 30 days of culture.

Cell response to nano-islands produced by polymer demixing: a brief review.

This review looks at the present literature available regarding cell response to nano-islands produced by nanotopography. Polymer demixing is a chemical method of fabricating large areas of nanotopography quickly and cheaply, making it ideal for cell testing and thus allowing it to be one of the first well-researched methods in cell engineering. The review shows that cells respond strongly to the islands (cell types observed include endothelial cells, fibroblasts, osteoblasts, leucocytes and platelets). Such changes include differences in adhesion, growth, gene expression and morphology.

Nonadhesive nanotopography: fibroblast response to poly(n-butyl methacrylate)-poly(styrene) demixed surface features.

It is becoming clear that cells do not only respond to micrometric scale topography, but may also respond to topography at the nanometric scale. Nano-fabrication methods such as electron beam lithography are, however, expensive and time consuming. Polymer demixing of poly(styrene) and poly(4-bromostyrene) has been found to produce nano-scale islands of reproducible height, and the islands have been previously shown to effect cell events such as adhesion, spreading, proliferation, and differentiation. This study uses demixed poly(styrene) and poly(n-butyl methacrylate) to produce nano-islands with closer packing and narrower widths compared with those previously studied. Observations have been made of morphological and cytoskeletal changes in human fibroblasts interacting with 10- and 50-nm-high islands. The methods used included scanning electron microscopy, fluorescent microscopy, and optical microscopy. The results indicated that the cells do not respond differently to the 10-nm islands compared with planar samples but, in contrast, the 50-nm islands are nonadhesive.

Fibroblast reaction to island topography: changes in cytoskeleton and morphology with time.

In order to develop next-generation tissue engineering materials, the understanding of cell responses to novel material surfaces needs to be better understood. Topography presents powerful cues for cells, and it is becoming clear that cells will react to nanometric, as well as micrometric, scale surface features. Polymer-demixing of polystyrene and polybromostyrene has been found to produce nanoscale islands of reproducible height, and is very cheap and fast compared to techniques such as electron beam lithography. This study observed temporal changes in cell morphology and actin and tubulin cytoskeleton using scanning electron and fluorescence microscopy. The results show large differences in cell response to 95 nm high islands from 5 min to 3 weeks of culture. The results also show a change in cell response from initial fast organisation of cytoskeleton in reaction to the islands, through to lack of cell spreading and low recruitment of cell numbers on the islands.

Enhanced HAPEX topography: comparison of osteoblast response to established cement.

The use of poly(methylmethacrylate) PMMA cement by Charnley in the 1960s revolutionized orthopaedic medicine. Since this time, however, little has changed. The development of bioactive composites, such as HAPEX (a composite of 40% vol hydroxyapatite (HA) in a polyethylene matrix) have potential in orthopaedic applications. The composite has been shown to allow direct bone bonding in vivo, and in vitro studies have shown preferential attachment to HA exposed on the composite surface. In vitro study has also shown that altering the topography HAPEX can enhance osteoblast response. This study uses microscopical investigation of osteoblast cytoskeleton, and biochemical measurement of proliferation (by thymidine incorporation) and phenotype (by alkaline phosphatase activity) to compare primary human osteoblast (HOB) activity on HAPEX and PMMA cement. The study shows large increases in HOB response to the new generation material compared to PMMA, the current implant standard.