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It is considered that cells can use filopodia, or microspikes, to locate sites suitable for adhesion. This has been investigated using a number of mature cell types, but, to our knowledge, not progenitor cells. Chemical and topographical cues on the underlying substrate are a useful tool for producing defined features for cells to respond to. In this study, arrays of nanopits with different symmetries (square or hexagonal arrays with 120 nm diameters, 300 nm center-centre spacings) and osteoprogenitor cells were considered. The pits were fabricated by ultra-high precision electron-beam lithography and then reproduced in polycarbonate by injection moulding with a nickel stamp. Using scanning electron and fluorescence microscopies, the initial interactions of the cells via filopodia have been observed, as have subsequent adhesion and cytoskeletal formation. The results showed increased filopodia interaction with the surrounding nanoarchitecture leading to a decrease in cell spreading, focal adhesion formation and cytoskeletal organisation.

Current load-bearing orthopaedic implants are produced in 'bio-inert' materials such as titanium alloys. When inserted into the reamed bone during hip or knee replacement surgery the implants interact with mesenchymal populations including the bone marrow. Bio-inert materials are shielded from the body by differentiation of the cells along the fibroblastic lineage producing scar tissue and inferior healing. This is exacerbated by implant micromotion, which can lead to capsule formation. Thus, next-generation implant materials will have to elicit influence over osteoprogenitor differentiation and mesenchymal populations in order to recruit osteoblastic cells and produce direct bone apposition onto the implant. A powerful method of delivering cues to cells is via topography. Micro-scale topography has been shown to affect cell adhesion, migration, cytoskeleton, proliferation and differentiation of a large range of cell types (thus far all cell types tested have been shown to be responsive to topographical cues). More recent research with nanotopography has also shown a broad range of cell response, with fibroblastic cells sensing down to 10 nm in height. Initial studies with human mesenchymal populations and osteoprogenitor populations have again shown strong cell responses to nanofeatures with increased levels of osteocalcin and osteopontin production from the cells on certain topographies. This is indicative of increased osteoblastic activity on the nanotextured materials. Looking at preliminary data, it is tempting to speculate that progenitor cells are, in fact, more responsive to topography than more mature cell types and that they are actively seeking cues from their environment. This review will investigate the range of nanotopographies available to researchers and our present understanding of mechanisms of progenitor cell response. Finally, it will make some speculations of the future of nanomaterials and progenitor cells in tissue engineering.

Nanotopographical material modification represents a possible way of producing surfaces that influence cellular adhesion for biomaterials purposes. Here, two low-adhesion surfaces are studied with human genome microarrays (120nm diameter pits produced by electron beam lithography and 11nm high columns produced by colloidal lithography). In order to present the large numbers of results produced in a succinct and easy to understand manner, two types of recent bioinformatics methods were used; iterative group analysis and Ingenuity pathway analysis. These methods allowed the easy comparison of the nanomaterials and showed large-scale changes in areas of extracellular matrix, cell signalling and inflammation. The results demonstrate that whilst modes of cellular response to low-adhesion materials are similar, the more adhesion is reduced, the further 'shut-down' of critical cellular activities is observed. We also feel that the analysis used could be of interest to biomaterials scientists looking for easy ways to display microarray data efficiently.

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.

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.

We apply a recently developed method for controlling the spreading of cultured cells using electron beam lithography (EBL) to create polymethylmethacrylate (PMMA) substrata with repeating nanostructures. There are indications that the reduced cell spreading on these substrata, compared with planar PMMA, results from a reduced adhesivity since there are fewer adhesive structures and fewer of their associated stress fibres. The reduced cell spreading also results in a reduced nuclear area and a closer spacing of centrosomes within the nucleus, suggesting that the tension applied to the nucleus is reduced as would be expected from the reduction in stress fibres. In order to obtain further evidence for this, we have used specific inhibitors of components of the cytoskeleton and have found effects comparable with those induced by the new substrata. We have also obtained evidence that these subtrata result in downregulation of gene expression which suggests that this may be due to the changed tension on the nucleus: an intriguing possibility that merits further investigation.

Focal adhesions and the cell cytoskeleton (intermediate filaments, microfilaments, microtubules) are involved in mechanotransduction-both direct (transduction of mechanical forces to the nucleus) and indirect (transduction of chemical signaling cascades to the nucleus). Thus, observation of changes in focal adhesion and cytoskeletal organization can be invaluable in research such as drug treatments and medical material testing in vitro. Here we describe how to stain human fibroblasts for vinculin (located to focal adhesions), actin (microfilaments), tubulin (microtubules), and vimentin (intermediate filaments) and how to perform custom microarray experiments. Comparative analysis of the immunofluorescence and array data should allow the researcher to build up a global picture of changes to both direct and indirect mechanotransduction through the cytoskeleton from focal adhesions.

This review focuses on how cells respond to low-adhesion nanotopographies. In order to do this, fabrication techniques, how cells may locate nanofeatures through the use of filopodia and possible mechanotransductive mechanisms are discussed. From this, examples of low-adhesion topographies and sizes and arrangements that may lead to low-adhesion are discussed. Finally, it is hypothesized as to how specifically low-adhesion materials may fit into the outlined mechanotransductive mechanisms.

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.

Growing cells on surfaces bearing nanotopography signals makes many changes in cell gene expression and downstream changes in phenotype but the mechanisms for this have, so far, been obscure. We consider the question of whether the topography directly nanoimprints onto the cell as a component of the signal transduction system. Evidence we present from SEM, TEM and fluorescence detection of the arrangements of cytoskeletal components is consistent with the possibility that cells are nanoimprinted by the substrate. The nanoprinting does not interfere with integrin-mediated adhesion processes and may perhaps work through them. Time-lapse video studies of cells moving from areas bearing nanotopography to flat areas and vice versa suggests that the nanoimprinting takes 1-6h to appear on the cell and a similar time to disappear when the cell moves from a flat surface to a nanotopographic one and back. This nanoprinting of cells would appear to be a novel type of cell signalling.

In bone tissue engineering, it is desirable to use materials to control the differentiation of mesenchymal stem cell populations in order to gain direct bone apposition to implant materials. It has been known for a number of years that microtopography can alter cell adhesion, proliferation and gene expression. More recently, the literature reveals that nanotopography is also of importance. Here, the reaction of primary human osteoprogenitor cell populations to nanotopographies down to 10 nm in size is considered. The topographies were originally produced by colloidal lithography and polymer demixing on silicon and then embossed (through an intermediate nickel shim) into polymethylmethacrylate. The biological testing considered cell morphology (image analysis of cell spreading and scanning electron microscopy), cell cytoskleton and adhesion formation (fluorescent staining of actin, tubulin, vimentin and vinculin) and then subsequent cell growth and differentiation (fluorescent staining of osteocalcin and osteopontin). The results demonstrated that the nanotopographies stimulated the osteoprogenitor cell differentiation towards an osteoblastic phenotype.

In the development of the next generation of orthopaedic implants for load-bearing applications, an ability to influence osteoprogenitor population activity and function will be highly desirable. This will allow the formation of hard-tissue directly onto the implant, i.e. osseointegration, rather than the formation of fibrous capsules which form around many of the current, non-bioactive, prosthesis. The formation of capsules leads to micromotion due to modulus mismatch and ultimately to fracture and the need for revision surgery. Microtopography and latterly nanotopography have been shown to elicit influence over adhesion, proliferation and gene expression of a wide number of cell types. This study has examined the possibility of modulating cell adhesion using a range of nanometric scale shallow pits and grooves. The topographies were manufactured using photolithography followed by the production of nickel shims and finally embossing into polymethylmethacrylate. Cell testing with human osteoprogenitor populations showed that the nanotopographies allowed control of cell adhesion, cytoskeleton, growth and production of the osteoblastic markers osteocalcin and osteopontin. It is concluded that the human bone marrow stromal cells are highly responsive to nanoscale features.

This review is designed to introduce the cytoskeleton and then discuss how mechanical forces may be transduced to the cell nucleus. In addition to this, it also tries to explain current thinking as to how the nucleus turns these mechanical cues into gene changes and is especially interested in mechanotransduction arising from topographically induced morphological changes, specifically nanotopography. Thus, this review also describes cell responses to topography.

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.

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.

In order for cells to react to topography, they must be able to sense shape. When considering nano-topography, these shapes are much smaller than the cell, but still strong responses to nano-topography have been seen. Filopodia, or microspikes, presented by cells at their leading edges are thought to be involved in gathering of special information. In order to investigate this, and to develop an understanding of what size of feature can be sensed by cells, morphological observation (electron and fluorescent microscopy) of fibroblasts reacting to nano-pits with 35, 75 and 120 nm diameters has been used in this study. The nano-pits are especially interesting because unlike many of the nanofeatures cited in the literature, they have no height for the cells to react to. The results showed that cell filopodia, and retraction fibres, interacted with all pit sizes, although direct interaction was hard to image on the 35 nm pits. This suggests that cells are extremely sensitive to their nanoevironment and that should be taken in to consideration when designing next-generation tissue engineering materials. We suggest that this may occur through nanocontact guidance as filopodia are moved over the pits.

In designing new biomaterials, specific chemical and topographical cues will be important in guiding cell response. Filopodia are actin-driven structures produced by cells and speculated to be involved in cell sensing of the three-dimensional environment. This report quantifies filopodia response to cylindrical nano-columns (100 nm diameter, 160 nm high) produced by colloidal lithography. Also observed were actin cytoskeleton morphology by fluorescence microscopy and filopodia morphology by electron microscopy (scanning and transmission). The results showed that the fibroblasts used produced more filopodia per microm of cell perimeter and that filopodia could often be seen to interact with the cells' nano-environment. By understanding as to which features evoke spatial reactions in cells, it may be possible to design better biomaterials., (Copyright 2003 Elsevier Ltd.)

Control of the cells' nanoenvironment is likely to be important in the future of cell and tissue engineering. Microtopography has been shown to provide cues to cells that elicit a large range of cell responses, including control of adhesion, morphology, apoptosis and gene regulations. Now, researchers are focusing on nanotopography as techniques such as colloidal and electron beam lithography and polymer demixing have become available. In this study, human fibroblast response to nanocolumns (160-nm high, 100-nm diameter, 230-nm centre-centre spacing) produced by colloidal lithography are considered. Using electron microscopy and immunofluorescence to image the cytoskeleton, clathrin and dynamin, it was observed that the cells try to endocytose the nanocolumns. It also appeared that a small population of the cells changed to unusual morphologies with macrophage-like processes and highly disrupted cytoskeleton. These observations could have implications for nanomaterials science in areas such as cell transfection and drug delivery.

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 perspective article, we attempt to illustrate that a possible mechanism is direct transmittal of forces encountered by cells during spreading to the nucleus via the cytoskeleton. We further try to illustrate that this 'self-induced' mechanotransduction may alter gene expression by changing interphase chromosome positioning. Whilst the observations described here to show how we think nanotopography can be developed as a tool to look at mechanotransduction are preliminary, we feel they indicate that topography may give cell biologists a non-invasive tool with which to investigate in vitro cellular mechanisms.

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., (Copyright 2004 Wiley Periodicals, Inc. J Biomed Mater Res 69A: 314-322, 2004)

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.

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.

Magnetic nanoparticles have been used for bio-medical purposes including drug delivery, cell destruction and as MRI contrast agents for several years. A more recent biological application has focused on targeted drug delivery. To this end, a wide variety of iron oxide nanoparticles have been synthesised. This study involves the use of magnetic nanoparticles synthesised and derivatised with human transferrin, compared to identical underivatised particles. Human fibroblasts were used, representative of a tissue cell-type. The influence in vitro was determined using light and fluorescence microscopy, scanning and transmission electron microscopy, and 1718 gene microarray. The results indicate that the transferrin derivatised particles appear to localise to the cell membrane without instigating receptor-mediated endocytosis, and also induce up-regulation in the cells for many genes, particularly in the area of cytoskeleton and cell signalling. The microscopy results further support these findings.

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.

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.

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., (Copyright 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 1025-1032, 2003)

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.

The in vitro response of primary human osteoblast-like (HOB) cells to a novel hydroxyapatite (HA) coated titanium substrate, produced by a low temperature electrochemical method, was compared to three different titanium surfaces: as-machined, Al(2)O(3)-blasted, plasma-sprayed with titanium particles. HOB cells were cultured on different surfaces for 3, 7 and 14 days at 37 degrees C. The cell morphology was assessed using scanning electron microscopy (SEM). Cell growth and proliferation were assessed by the measurement of total cellular DNA and tritiated thymidine incorporation. Measurement of alkaline phosphatase (ALP) production was used as an indicator of the phenotype of the cultured HOB cells. After three days incubation, the electrochemically coated HA surface produced the highest level of cell proliferation, and the Al(2)O(3)-blasted surface the lowest. Interestingly, as the incubation time was increased to 7 days all surfaces produced a large drop in tritiated thymidine incorporation apart from the Al(2)O(3)-blasted surface, which showed a small increase. Cells cultured on all four surfaces showed an increased expression of ALP with increased incubation time, although there was not a statistically significant difference between surfaces at each time point. Typical osteoblast morphology was observed for cells cultured on all samples. The HA coated sample showed evidence of a deposited phase after three days of incubation, which was not observed on any other surface. Cells incubated on the HA coated substrate appeared to exhibit the highest number of cell processes attaching to the surface, which was indicative of optimal cell attachment. The crystalline HA coating, produced by a low temperature route, appeared to result in a more bioactive surface on the c.p. Ti substrate than was observed for the other three different Ti surfaces.

Cellular response to scaffold materials is of great importance in cellular and tissue engineering, and it is perhaps the initial cell contact with the scaffold that determines development of new tissue. Material surface morphology has strong effects on cell cytoskeleton and morphology, and it is thought that cells may react to the topography of collagen and surrounding cells during tissue embryology. A poorly understood area is, however, gene-level responses to topography. Thus, this paper used microarray to probe for consistent gene changes in response to lithographically produced topography (12.5 x 2-microm grooves) with time. The results showed many initial gene changes and also down-regulation of gene response with time. Cell and nucleus morphology were also considered, with nuclear deformation linked to cell signaling.

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.

Poly(ethylmethacrylate) (PEMA) bone cement has been developed, and the cements mechanical properties are improved by the incorporation of particulate fillers, such as hydroxyapatite (HA). In this in vitro study, human osteoblast-like (HOB) cells were used to examine the effect on cellular behavior of the addition of HA to PEMA using a plain PEMA control. Thymidine uptake ((3)H-TdR) and total DNA were used to assess cell growth and proliferation. Confocal laser scanning microscopy (CLSM) was used to study focal contacts and actin cytoskeletal organisation. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to assess cell morphology and cellular ultrastucture. The early time points showed preferential anchorage to the HA exposed on the cement surface, but no difference in adhesion or proliferation. These results have been attributed to increases in residual monomer with HA incorporation, as shown by proton nuclear magnetic resonance (H(1)-NMR) spectra.

Cell response to nanometric scale topography is a growing field. Nanometric topography production has traditionally relied on expensive and time-consuming techniques such as electron beam lithography. This presents disadvantages to the cell biologist in regard to material availability. New research is focusing on less expensive methods of nanotopography production for in vitro cell engineering. One such method is the spontaneous demixing of polymers (in this case polystyrene and polybromostyrene) to produce nanometrically high islands. This article observes fibroblast response to nanometric islands (13, 35, and 95 nm in height) produced by polymer demixing. Changes in cell morphology, cytoskeleton, and proliferation are observed by light, fluorescence, and scanning electron microscopy. Morphological features produced by cells in response to the materials were selected, and cell shape parameters were measured with shape-recognition software. The results showed that island height could either increase or reduce cell spreading and proliferation in relation to control, with 13-nm islands producing cells with the greatest area and 95 nm islands producing cells with the lowest areas. Interaction of filopodia with the islands could been seen to increase as island size was increased.

HAPEX (hydroxyapatite-reinforced polyethylene composite) is a second-generation orthopedic biomaterial designed as a bone analog material, which has found clinical success. The use of topography in cell engineering has been shown to affect cell attachment and subsequent response. Thus, by combining bioactivity and enhancing osteoblast response to the implant surface, improved tissue repair and implant life span may be achieved. In this study a primary human osteoblast-like cell model has been used to study the influence of surface topography and chemistry produced by three different production methods. Scanning electron microscopy, fluorescence microscopy, and confocal scanning laser microscopy have been used to study cell adhesion; tritiated thymidine uptake has been used to observe cell proliferation; and the reverse transcriptase-polymerase chain reaction and biochemical methods have been used to study phenotypic expression. Transmission electron microscopy has also been used to look at more long-term morphology. The results show that topography significantly influences cell response, and may be a means of enhancing bone apposition on HAPEX.

The introduction of topography to material surfaces has been shown to strongly affect cell behaviour, and the effects of micrometric surface morphologies have been extensively characterised. Research is now starting to investigate the reaction of cells to nanometric topography. This study used polymer demixing of polystyrene and poly(4-bromostyrene) producing nanometrically high islands, and observed endothelial cell response to the islands. Three island heights were investigated; these were 13, 35 and 95 nm. The cells were seen to be more spread on the manufactured topographies than that on flat surfaces of similar chemistry. Other morphological differences were also noted by histology, fluorescence and scanning electron microscopy, with many arcuate cells noted on the test surfaces, and cytoskeletal alignment along the arcuate features. Of the nanotopographies, the 13 nm islands were seen to give the largest response, with highly spread cell morphologies containing well-defined cytoskeleton.

It is becoming clear that cells can respond not only to micometric scale topography, but may also to nanometric scale topography. The production of reproducibly sized nanometric features has relied heavily on expensive and time-consuming methods of manufacture, such as electron beam lithography. Polymer demixing of polystyrene and polybromostyrene has been found to produce nanoscale islands of reproducible height, and the islands have been previously shown to effect cell spreading compared to planar surfaces. This study observes morphological, cytoskeletal, and molecular changes in fibroblast reaction to 13-nm-high islands. The methods employed include scanning electron microscopy, fluorescent microscopy, and 1718 gene microarray. The results show that the cells respond to the islands by broad gene up-regulation, notably in the areas of cell signaling, proliferation, cytoskeleton, and production of extracellular matrix proteins. Microscopical results provide confirmation of the microarray findings.

A bone cement, poly(ethylmethacrylate)/n-butylmethacrylate (PEMA/nBMA) has been developed with lower exotherm and monomer leaching compared to the traditional poly(methylmethacrylate)/methylmethacrylate (PMMA/MMA) cement. This study compares the in vitro biological response to the cements using primary human osteoblast-like cells (HOB). Cell attachment was qualified by immunolocalization of vinculin and actin cytoskeleton, showing more organization on PEMA/nBMA compared to PMMA/MMA. Proliferation was assessed using tritiated thymidine incorporation, and phenotype expression determined by measuring alkaline phosphatase (ALP) activity. An increase in proliferation and ALP activity was observed on PEMA/nBMA compared to PMMA/MMA. The results confirm the biocompatability of PEMA/nBMA, and an enhanced cell attachment and expression of differentiated cell phenotype.

Two of the major concerns for tissue engineering materials are inflammatory responses from blood cells and fibrous encapsulation by the body in order to shield the implant from blood reaction. A further hurdle is that of vascularization. In order to develop new tissues, or to repair parts of the vascular system, nutrients need to be carried to the basal cell layers. If a material promotes tissue formation, but not vascularization, necrosis will be observed as multilayered cells develop. In this paper, polymer demixed island topography with a 95-nm Z axis was tested using human mononuclear blood cells, platelets, fibroblasts, and endothelial cells. The results showed no difference in blood response between the islands and the flat controls, suggesting that in vivo there would be negligible immunological difference. Fibroblasts reacted by changing morphology into a rounded shape with thick processes and poorly developed cytoskeleton. Retardation of fibroblast growth may be an advantageous, as it is this cell type that forms the fibrous capsule, preventing growth of the required tissue type. Finally, endothelial cells were seen to form arcuate, or curved, morphologies in response to the islands. This is the normal, in vivo, morphology for vascular endothelium. This result suggests that the nano-features are promoting a more phenotypically correct morphology.

When considering the complicated nature of cell/tissue interactions with biomaterials, especially materials with nanometric surface features, observation of changes in one or two selected genes or proteins may not be sufficient. To get a fuller understanding of the scope of responses effected by nanotopography on cells, many genes need to be surveyed. Recent developments in molecular biology have lead to the commercial production of microarrays. Microarray presents a powerful tool by which many genes (up to many thousands) can be probed simultaneously. In this study, 1718 gene arrays have been used to measure human fibroblast response to 13-nm-high polymer demixed islands. The results have shown many changes in genes involved in signaling, cytoskeleton, extracellular matrix, gene transcription, and protein translation; these results have been used to build a more complete overview of fibroblast response to the islands. The use of microarray has expanded the range of observations possible using established microscopical and biochemical techniques.

The interactions between an implant material and the surrounding tissue are of a complicated nature, and the initial attachment of cells to the surface is important in determining the implant success. HAPEX has been developed as a second-generation orthopaedic biomaterial, with both mechanical and biological characteristics that make it suitable for bone augmentation. Further optimisation of the material is being continued to increase the attachment of osteoblasts coupled with improving mechanical characteristics, so it may be used in load bearing applications. It has been previously observed that polishing followed by roughening the surface of HAPEX enhances osteoblast proliferation and phenotype. This article discusses the recruitment of primary human osteoblast cells onto the optimised surface, by examining morphology and cytoskeletal changes using scanning electron microscopy and confocal laser scanning microscopy. The results show that the cells attach in greater numbers to the optimised surface, and develop notably faster, than cells on machined HAPEX.

A hydroxyapatite (HA) reinforced polyethylene (PE) composite (designated HAPEX), with high mechanical specification and a bioactive HA phase, has been optimised as a bone analogue material. Manufacturing conditions and machining of the materials were carefully controlled to give a reproducible material surface roughness with minimal batch variation. The effect of surface composition was examined in vitro using primary human osteoblasts (HOB). HOBs were cultured in direct contact with the test materials containing 20% and 40% vol. HA. The results showed that 40% HA/PE enhanced cellular activity by increasing proliferation rate and differentiation compared to the 20% vol. HA composite. The cytoskeletal organisation of the cells was also examined and HOBs cultured on 40% HA/PE were flatter and had an enhanced rate of cytoskeletal organisation and an increase in focal contact points compared to the 20% HA/PE.

Poly(methylmethacrylate) (PMMA) is the current standard for cement held prostheses. It forms a strong bond with the implant, but the bond between the cement and the bone is considered to be weak, with fibroblastic cells observed at the implant site, rather than direct bone contact, a contributing factor leading to implant failure. Incorporation of hydroxyapatite (HA) increases the biological response to the cement from tissue around the implant site, thus giving increased bone apposition. In this study, PMMA discs with 0, 4.6 and 8.8 vol%. HA were examined. Primary human osteoblast-like cells (HOBs) were used for the biological evaluation of the response to the cements in vitro. Morphology was observed using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Measurement of tritiated thymidine (3H-TdR) incorporation and alkaline phosphatase (ALP) activity were used to assess proliferation and differentiation. A synergy between increasing focal contact formation, cytoskeletal organisation, cell proliferation and expression of phenotype was observed with increasing HA volume. Preferential anchorage of HOBs to HA rather than PMMA was a prominent observation.

Failure of the bone/cement interface in cemented joint prostheses is a contributor to implant loosening. The introduction of a bioactive phase, such as hydroxyapatite (HA), to cement may enhance fixation by encouraging direct bone apposition rather than encapsulation of the implant by fibrous tissue. The effect of poly(methylmethacrylate) (PMMA) bone cement (incorporating 17.5% HA wt.) on bioactivity has been investigated using primary human osteoblast-like cells (HOB). A significantly higher cell proliferation and differentiation was seen on the PMMA/HA cement compared to the PMMA cement alone, with retention of phenotype up to 21 days of culture on both materials.

HAPEX, a bone analog material, with similar properties to cortical bone, has been studied in vitro with particular reference to the effect of surface topography. The stimulation of a favorable bone response by this composite depends on optimization of the hydroxyapatite (HA) content in relation to the material bioactivity without compromising the mechanical characteristics. In this study we have started to investigate the effects of surface topography on cell attachment and subsequent cellular behavior in relation to proliferation. Different volumes of HA (20% and 40%) were added to a high density polyethylene (HDPE) matrix to produce the test materials. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were used to examine cell morphology on HAPEX, and the surface characteristics produced by different machining protocols. The measurement of cellular DNA and tritiated thymidine ([3H]-TdR) incorporation has been used to asses cell proliferation upon the materials. The results show that the material surface topography has a large influence on cell proliferation and attachment, and with a controlled material topography the 40% vol HA/HDPE composite gives the greater biological response compared to the 20% vol HA/HDPE composite., (Copyright 2000 Kluwer Academic Publishers)

The nature of the orthopedic implant surface affects the interaction between cells and subsequent bone formation. The bone/cement interface in cement-held prostheses is considered to be the main cause of fracture leading to implant revision. It is thought that the introduction of a bioactive phase, such as hydroxyapatite (HA), to cement may permit a stronger implant by encouraging direct bone apposition rather than encapsulation of the implant by fibrous tissue. Thus, a poly(methylmethacrylate) (PMMA) cement incorporating 17.5% HA by weight has been investigated. In this study, in order to analyze the interaction at the cellular level, the in vitro biological response of the HA/PMMA to a similar PMMA without HA incorporation has been studied. Primary human osteoblast-like cells (HOB) were used as they are a model of the cell type the cements might encounter in vivo. Cell proliferation and growth were assessed by measurement of total cellular DNA and tritiated thymidine ([3H]-TdR) incorporation. Alkaline phosphatase (ALP) production was measured as an indicator of HOB phenotype upon the cements. The results showed that HA/PMMA was a better substrate for HOB cells, resulting in increased proliferation and ALP activity. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that HOB cells cultured on the HA-filled PMMA preferentially anchored to HA particles exposed at the cement surface, with a close intimacy observed between HA and HOB cells., (Copyright 1999 Kluwer Academic Publishers)