Nanotopographical Control of Stem Cell Differentiation

Stem cells have the capacity to differentiate into various lineages, and the ability to reliably direct stem cell fate determination would have tremendous potential for basic research and clinical therapy. Nanotopography provides a useful tool for guiding differentiation, as the features are more durable than surface chemistry and can be modified in size and shape to suit the desired application. In this paper, nanotopography is examined as a means to guide differentiation, and its application is described in the context of different subsets of stem cells, with a particular focus on skeletal (mesenchymal) stem cells. To address the mechanistic basis underlying the topographical effects on stem cells, the likely contributions of indirect (biochemical signal-mediated) and direct (force-mediated) mechanotransduction are discussed. Data from proteomic research is also outlined in relation to topography-mediated fate determination, as this approach provides insight into the global molecular changes at the level of the functional effectors.

Related papers:

title: Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment.
abstract: Commitment of stem cells to different lineages is regulated by many cues in the local tissue microenvironment. Here we demonstrate that cell shape regulates commitment of human mesenchymal stem cells (hMSCs) to adipocyte or osteoblast fate. hMSCs allowed to adhere, flatten, and spread underwent osteogenesis, while unspread, round cells became adipocytes. Cell shape regulated the switch in lineage commitment by modulating endogenous RhoA activity. Expressing dominant-negative RhoA committed hMSCs to become adipocytes, while constitutively active RhoA caused osteogenesis. However, the RhoA-mediated adipogenesis or osteogenesis was conditional on a round or spread shape, respectively, while constitutive activation of the RhoA effector, ROCK, induced osteogenesis independent of cell shape. This RhoA-ROCK commitment signal required actin-myosin-generated tension. These studies demonstrate that mechanical cues experienced in developmental and adult contexts, embodied by cell shape, cytoskeletal tension, and RhoA signaling, are integral to the commitment of stem cell fate.

title: Matrix Elasticity Directs Stem Cell Lineage Specification
abstract: Microenvironments appear important in stem cell lineage specification but can be difficult to adequately characterize or control with soft tissues. Naive mesenchymal stem cells (MSCs) are shown here to specify lineage and commit to phenotypes with extreme sensitivity to tissue-level elasticity. Soft matrices that mimic brain are neurogenic, stiffer matrices that mimic muscle are myogenic, and comparatively rigid matrices that mimic collagenous bone prove osteogenic. During the initial week in culture, reprogramming of these lineages is possible with addition of soluble induction factors, but after several weeks in culture, the cells commit to the lineage specified by matrix elasticity, consistent with the elasticity-insensitive commitment of differentiated cell types. Inhibition of nonmuscle myosin II blocks all elasticity-directed lineage specification-without strongly perturbing many other aspects of cell function and shape. The results have significant implications for understanding physical effects of the in vivo microenvironment and also for therapeutic uses of stem cells.

title: Harnessing Traction-Mediated Manipulation of the Cell-Matrix Interface to Control Stem Cell Fate
abstract: Stem cells sense and respond to the mechanical properties of the extracellular matrix. However, both the extent to which extracellular matrix mechanics affect stem cell fate in 3D micro-environments and the underlying biophysical mechanisms are unclear. We demonstrate that the commitment of mesenchymal stem cell (MSC) populations changes in response to the rigidity of 3D micro-environments, with osteogenesis occurring predominantly at 11–30 kPa. In contrast to previous 2D work, however, cell fate was not correlated with morphology. Instead, matrix stiffness regulated integrin binding as well as reorganization of adhesion ligands on the nanoscale, both of which were traction-dependent and correlated with osteogenic commitment of MSC populations. These findings suggest that cells interpret changes in the physical properties of adhesion substrates as changes in adhesion ligand presentation, and that cells themselves can be harnessed as tools to mechanically process materials into structures that feedback to manipulate their fate.

title: The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder.
abstract: A key tenet of bone tissue engineering is the development of scaffold materials that can stimulate stem cell differentiation in the absence of chemical treatment to become osteoblasts without compromising material properties. At present, conventional implant materials fail owing to encapsulation by soft tissue, rather than direct bone bonding. Here, we demonstrate the use of nanoscale disorder to stimulate human mesenchymal stem cells (MSCs) to produce bone mineral in vitro, in the absence of osteogenic supplements. This approach has similar efficiency to that of cells cultured with osteogenic media. In addition, the current studies show that topographically treated MSCs have a distinct differentiation profile compared with those treated with osteogenic media, which has implications for cell therapies.

title: Stem cell fate dictated solely by altered nanotube dimension
abstract: Two important goals in stem cell research are to control the cell proliferation without differentiation and to direct the differentiation into a specific cell lineage when desired. Here, we demonstrate such paths by controlling only the nanotopography of culture substrates. Altering the dimensions of nanotubular-shaped titanium oxide surface structures independently allowed either augmented human mesenchymal stem cell (hMSC) adhesion or a specific differentiation of hMSCs into osteoblasts by using only the geometric cues, absent of osteogenic inducing media. hMSC behavior in response to defined nanotube sizes revealed a very dramatic change in hMSC behavior in a relatively narrow range of nanotube dimensions. Small (≈30-nm diameter) nanotubes promoted adhesion without noticeable differentiation, whereas larger (≈70- to 100-nm diameter) nanotubes elicited a dramatic stem cell elongation (≈10-fold increased), which induced cytoskeletal stress and selective differentiation into osteoblast-like cells, offering a promising nanotechnology-based route for uniqueorthopedics-related hMSC treatments.

title: Nantotechniques and approaches in biotechnology.
abstract: Nanotechnology has enabled the development of an amazing variety of methods for fabricating nanotopography and nanopatterned chemistry in recent years. Some of these techniques are directed towards producing single component particles, as well as multi-component assembly or self-assembly. Other methods are aimed at nanofeaturing and patterning surfaces that have a specific chemistry or topography. This article concentrates mainly on surface-directed nanobiotechnologies because they are nearer to commercial realisation, such as use in tissue engineering, control of biofouling and cell culture, than those directed at producing nanoparticles.

title: Tissue engineering: the biophysical background.
abstract: Tissue engineering is the construction, repair or replacement of damaged or missing tissue in humans and other animals. This engineering may take place within the animal body or as tissue constructs to be made in a bioreactor for later grafting into the animal. The minimal set of materials for this are the appropriate types of cell. Usually, however, non-living substrata are used as well. These substrata may be nothing more than materials that bulk up any voids in the damaged tissue and provide the mechanical strength that has been lost when the tissue is damaged or removed. They may serve a similar pair of functions in the bioreactor. They can do much more in terms of pattern formation. The orientations and morphology of the cells, the arrangement of intercellular material as it is laid down and the relationships between different cell types in the repairing or construct tissue are all of importance, for these should resemble the correct normal tissue as closely as possible. Most of these requirements are ones involving pattern formation. This review discusses the various ways in which tissue pattern can be engineered chiefly from a biophysical standpoint. Unpatterned cells are effectively not tissue. This engineering includes the use of topography on the substrata, chemical patterning of adhesive and other cues for the cells, mechanical force application to cause cell orientation and appropriate synthetic responses and electrical fields. The review also discusses the methods used to impart the appropriate cues to and through the materials which are often biodegradable polymers. The article gives particular attention to regions of research and practice where the involvement of the physicist or biophysicist is of importance.

title: Topographical control of cell behaviour. I. Simple step cues.
abstract: The photolithographic techniques of the microelectronics industry have allowed us to fabricate patterned plastic substrata to investigate contact guidance of animal tissue cells. The reactions of cells to single steps on a substratum were examined using time-lapse videorecording and scanning electron microscopy. BHK cells and chick embryonic neural cell processes exhibited gradual inhibition of crossing steps with a concomitant increase in alignment at steps dependent on increasing step height. Comparison of these cells' reactions, with those of chick heart fibroblasts and rabbit neutrophils, at a 5 micron step revealed that the influence of topography is also dependent on cell type, the neutrophils being relatively unaffected. The presence of an adhesive difference at a series of steps altered BHK cells' reactions such that the frequency of crossing was dependent on the direction of approach to a step. Although our data are consistent with Dunn & Heath's proposal (1976) that the inflexibility of the cytoskeleton of a moving cell's protrusion is the cellular property determining such reactions to topography, we have found that, on encountering a topographical feature, the response of a cell may be predictable on a probabilistic basis, i.e. the topographical feature reduces the probability of a cell making a successful protrusion and contact in a given direction, that even the largest features tested did not act as absolute barriers to cell locomotion since a small proportion of a population of cells were able to overcome them, and that other guidance cues could significantly alter a cell's response. Even in situations where it is not the primary cue in directing cell locomotion, topographical control may be an important factor during morphogenesis since it must, at the very least, influence the efficiency of other cues.

title: Geometric control of cell life and death.
abstract: Human and bovine capillary endothelial cells were switched from growth to apoptosis by using micropatterned substrates that contained extracellular matrix-coated adhesive islands of decreasing size to progressively restrict cell extension. Cell spreading also was varied while maintaining the total cell-matrix contact area constant by changing the spacing between multiple focal adhesion-sized islands. Cell shape was found to govern whether individual cells grow or die, regardless of the type of matrix protein or antibody to integrin used to mediate adhesion. Local geometric control of cell growth and viability may therefore represent a fundamental mechanism for developmental regulation within the tissue microenvironment.

title: Nucleus alignment and cell signaling in fibroblasts: response to a micro-grooved topography.
abstract: 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.

title: Synergistic and hierarchical adhesive and topographic guidance of BHK cells.
abstract: Guided cell movement is a fundamental process in development and regeneration. We have used microengineered culture substrates to study the interaction between model topographic and adhesive guidance cues in steering BHK cell orientation. Grooves 0.1, 0.5, 1.0, 3.0, and 6.0 microm deep together with pitch-matched aminosilane tracks 5, 12, 25, 50, and 100 microm wide were fabricated on fused silica substrates using photolithographic and dry-etching techniques. The cues were presented to the cells individually, simultaneously in parallel and orthogonally opposed. Cells aligned most strongly to 25-microm-wide adhesive tracks and to 5-microm-wide, 6-microm-deep grooves. Stress fibers and vinculin were found to align with the adhesive tracks and to the grooves and ridges. Cell alignment was profoundly enhanced on all surfaces that presented both cues in parallel. Cells were able to switch alignment from ridges to grooves, and vice versa, depending on the location of superimposed adhesive tracks. Cells aligned preferentially to adhesive tracks superimposed orthogonally over grooves of matched pitch, traversing numerous grooves and ridges. The strength of the cues was more closely matched on narrower 3- and 6-microm-deep gratings with cells showing evidence of alignment to both cues. Confocal fluorescence microscopy revealed two groups of mutually opposed f-actin stress fibers within the same cell, one oriented with the topographic cues and the other with the adhesive cues. However, the adhesive response was consistently dominant. We conclude that cells are able to detect and respond to multiple guidance cues simultaneously. The adhesive and topographic guidance cues modeled here were capable of interacting both synergistically and hierarchically to guide cell orientation.

title: Activation of macrophage‐like cells by multiple grooved substrata. Topographical control of cell behaviour.
abstract: We studied the influence of substrata topography on the behaviour of murine P388D1 macrophage cell line. Cells were plated on plain fused silica substrata or substrata with microfabricated grooves of varying depth and width. Cell spread area, elongation, orientation and F‐actin content were measured on plain substratum and 6 sets of gratings. The speed and persistence of cell movement were also studied.

title: Differential in-gel electrophoresis (DIGE) analysis of human bone marrow osteoprogenitor cell contact guidance.
abstract: We have used a recent comparative proteomics technique, differential in-gel electrophoresis (DIGE), to study osteoprogenitor cell response to contact guidance in grooves. In order to increase protein output from small sample sizes, we used bioreactor culture before protein extraction and gel electrophoresis. Mass spectroscopy was used for protein identification. A number of distinct proteins were observed to exhibit significant changes in expression. These changes in protein expression suggest that the cells respond to tailored grooved topographies, with alterations in their proteome concurrent with changes in osteoprogenitor phenotype.

title: In vitro reaction of endothelial cells to polymer demixed nanotopography.
abstract: 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.

title: Interaction of animal cells with ordered nanotopography.
abstract: Animal cells live in a complex and diverse environment where they encounter a vast amount of information, a considerable amount of which is in the nanometer range. The surface topography that a cell encounters has a role to play in influencing cell behavior. It has been demonstrated widely that surface shape can directly influence the behavior of cells. In this paper, we discuss the interactions of animal cells with engineered nanotopography, fabricated in quartz and reverse embossed into polycaprolactone, fibroblast cells show reduced adhesion to the ordered nano pits. We show that the area of cells spreading on a structured nanotopography is reduced compared with that on a planar substrate. Furthermore, cytoskeletal organization is disrupted as indicated by a marked decrease in number and size of focal contacts.

title: The influence of microscale topography on fibroblast attachment and motility.
abstract: The ability of a cell to attach and migrate on a substrate or scaffold is important in the field of tissue engineering and biomaterials, and is thus extensively studied. When considering tissue-engineering applications, a highly porous scaffold is required to guide cell growth and proliferation in three dimensions. However existing scaffolds are less than ideal for actual applications, not only as they lack mechanical strength due to pore size and have regular distribution, but also they do not ensure cell attachment, in-growth and organisation. In this study, microfabrication technology was used to create regular arrays of pits on a two-dimensional quartz surface (7, 15 and 25 microm diameter, 20 and 40 microm spacing). The patterned surface thus exhibited spatially separated mechanical edges akin to the basic structural element of a three-dimensional network, and was used as a model system for studying the effects of substrate microgeometry on fibroblast attachment and motility. Results clearly showed that fibroblast interaction with the pit edges depended on both diameter, and therefore angle of circumference, and inter pit spacing, with the largest diameter permitting cells to enter the pits. Interestingly, the highest cell proliferation rates were recorded on the smaller pits. Such information may provide details on possible pore sizes for use in synthetic tissue engineering scaffolds that aim to support fibroblast in-growth and subsequent proliferation.

title: Polymer-demixed nanotopography: control of fibroblast spreading and proliferation.
abstract: 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.

title: Attempted endocytosis of nano-environment produced by colloidal lithography by human fibroblasts.
abstract: 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.

title: Proteomic analysis of human osteoprogenitor response to disordered nanotopography
abstract: Previous studies have shown that microgroove-initiated contact guidance can induce bone formation in osteoprogenitor cells (OPGs) and produce changes in the cell proteome. For proteomic analysis, differential in-gel electrophoresis (DIGE) can be used as a powerful diagnostic method to provide comparable data between the proteomic profiles of cells cultured in different conditions. This study focuses on the response of OPGs to a novel nanoscale pit topography with osteoinductive properties compared with planar controls. Disordered near-square nanopits with 120 nm diameter and 100 nm depth with an average 300 nm centre-to-centre spacing (300 nm spaced pits in square pattern, but with ±50 nm disorder) were fabricated on 1×1 cm2 polycaprolactone sheets. Human OPGs were seeded onto the test materials. DIGE analysis revealed changes in the expression of a number of distinct proteins, including upregulation of actin isoforms, beta-galectin1, vimentin and procollagen-proline, 2-oxoglutarate 4-dioxygenase and prolyl 4-hydroxylase. Downregulation of enolase, caldesmon, zyxin, GRASP55, Hsp70 (BiP/GRP78), RNH1, cathepsin D and Hsp27 was also observed. The differences in cell morphology and mineralization are also reported using histochemical techniques.

title: Whole proteome analysis of osteoprogenitor differentiation induced by disordered nanotopography and mediated by ERK signalling.
abstract: Topographic features can modulate cell behaviours such as proliferation, migration, differentiation and apoptosis. Biochemical mechanotransduction implies the conversion of mechanical forces (e.g. changes in cell spreading and morphology from changing surface topography) into biochemical signal via biomolecules. Still, little is known concerning which pathways may be directly involved in cell response to changes in the material surface. A number of pathways have been implicated using focused studies of 'selected' biomolecules rather than a global analysis of signal pathways. This study used a controlled disorder nanopit topography (NSQ50, fabricated by electron beam lithography) to direct osteoblast differentiation of progenitor cells. This topography is unique as it represents a middle route (from absolute order or random roughness) that allows osteoconversion with similar efficiency as dexamethasone and ascorbate treatment. Two direct-comparison proteomics techniques, firstly gel-based and then chromatography-based, were used to analyse progenitor proteome changes in response to the nanotopography. Many of the changed proteins form part of the Extracellular Signal-regulated Kinase (ERK1/2) pathway.

title: Increasing fibroblast response to materials using nanotopography: morphological and genetic measurements of cell response to 13-nm-high polymer demixed islands.
abstract: 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.

title: Enhanced functions of osteoblasts on nanostructured surfaces of carbon and alumina
abstract: It is of the utmost importance to increase the activity of bone cells on the surface of materials used in the design of orthopaedic implants. Increased activity of such cells can promote either integration of these materials into surrounding bone or complete replacement with naturally produced bone if biodegradable materials are used. Osteoblasts are bone-producing cells and, for that reason, are the cells of interest in initial studies of new orthopaedic implants. If these cells are functioning normally, they lay down bone matrix onto both existing bone and prosthetic materials implanted into the body. It is generally accepted that a successful material should enhance osteoblast function, leading to more bone deposition and, consequently, increased strength of the interface between the material and juxtaposed bone. The present study provided the first evidence of greater osteoblast function on carbon and alumina formulations that mimic the nano-dimensional crystal geometry of hydroxyapatite found in bone.

title: Enhanced osteoclast-like cell functions on nanophase ceramics.
abstract: Synthesis of tartrate-resistant acid phosphatase (TRAP) and formation of resorption pits by osteoclast-like cells, the bone-resorbing cells, on nanophase (that is, material formulations with grain sizes less than 100nm) alumina and hydroxyapatite (HA) were investigated in the present in vitro study. Compared to conventional (that is, grain sizes larger than 100 nm) ceramics, synthesis of TRAP was significantly greater in osteoclast-like cells cultured on nanophase alumina and on nanophase HA after 10 and 13 days, respectively. In addition, compared to conventional ceramics, formation of resorption pits was significantly greater by osteoclast-like cells cultured on nanophase alumina and on nanophase HA after 7, 10, and 13 days, respectively. The present study, therefore, demonstrated, for the first time, enhanced osteoclast-like cell function on ceramic surfaces with nanometer-size surface topography.

title: Nano-topography sensing by osteoclasts
abstract: Bone resorption by osteoclasts depends on the assembly of a specialized, actin-rich adhesive ‘sealing zone’ that delimits the area designed for degradation. In this study, we show that the level of roughness of the underlying adhesive surface has a profound effect on the formation and stability of the sealing zone and the associated F-actin. As our primary model substrate, we use ‘smooth’ and ‘rough’ calcite crystals with average topography values of 12 nm and 530 nm, respectively. We show that the smooth surfaces induce the formation of small and unstable actin rings with a typical lifespan of ~8 minutes, whereas the sealing zones formed on the rough calcite surfaces are considerably larger, and remain stable for more than 6 hours. It was further observed that steps or sub-micrometer cracks on the smooth surface stimulate local ring formation, raising the possibility that similar imperfections on bone surfaces may stimulate local osteoclast resorptive activity. The mechanisms whereby the physical properties of the substrate influence osteoclast behavior and their involvement in osteoclast function are discussed.

title: Polymers with nano-dimensional surface features enhance bladder smooth muscle cell adhesion.
abstract: Previous studies investigating the design of synthetic bladder wall substitutes have involved polymers with micro dimensional structures. Since the body is made up of nano-structured components (e.g., extracellular matrix proteins), the focus of the present in vitro study was to design nano-structured polymers for use as synthetic bladder constructs that mimic the topography of natural bladder tissue. In order to complete this task, novel nano-structured biodegradable polymeric films of poly-lactic-co-glycolic-acid (PLGA), poly-ether-urethane (PU), and poly-caprolactone (PCL) were fabricated and separately treated with various concentrations of NaOH (for PLGA and PCL) and HNO(3) (for PU) for select time periods. These treatments reduced the polymer surface feature dimensions from conventional micron dimensions to biologically inspired nanometer dimensions. Select cytocompatibility properties of these biomaterials were tested in vitro. Results provide the first evidence that adhesion of bladder smooth muscle cells is enhanced as polymer surface feature dimensions are reduced into the nanometer range. In addition, surface analysis results reveal that the polymer nanometer surface roughness is the primary design parameter that increases bladder smooth muscle cell adhesion. For this reason, the "next generation" of tissue-engineered bladder constructs with increased efficacy should contain surfaces with nanometer (as opposed to micron) surface features.

title: Nanoscale features influence epithelial cell morphology and cytokine production.
abstract: Available, easy and fast fabrication methods of nanostructured surfaces, and the knowledge that cells in vivo interacts with nanometer-sized structures/objects, led us to study the impact of nanotopography on cell morphology and cytokine production. Uroepithelial cells were seeded on three different substrate types: two with defined nanometer topographies and a flat control, all three having identical surface chemistry. The nanostructured substrates contained hemispherical pillars or step edges, the latter in the form of parallel grooves and ridges. Qualitative and quantitative analysis of cell morphology and cytokine production were studied. Both quantities were significantly different between cells cultured on hemispherically structured surfaces compared to flat control surfaces. Cells cultured on hemispherically structured surfaces showed a decrease in IL-6 and IL-8 production and were less spread, less round and more stellate (larger dispersion). Only cell morphology differed between cells cultured on grooved surfaces and flat control surfaces. These findings suggest that epithelial cell morphology and cytokine production are dependent on the underlying nanotopography.

title: Influence of systematically varied nanoscale topography on the morphology of epithelial cells
abstract: With the knowledge that cells can react to lithographically manufactured nanometer-sized surface objects, our interest concerned whether cells would respond to surface structures of systematically increasing size. Our approach to answer this question was to fabricate surfaces with the same surface chemistry and similar surface roughness but increasing size of structural features. To fabricate large areas of patterned surfaces, required for cell culture studies, we used colloidal lithography utilizing colloidal particles as a template for surface nanostructuring. The fabricated surfaces contained hemispherical nanopillars with diameters ranging from 60 to 170 nm. Changes in cell morphology of a pancreatic epithelial cell line (AR4-2J) were studied by evaluating cell area and cell shape. The latter was studied by applying the cell shape classification method using three shape descriptors. The pancreatic cells responded in a systematic way to the surface nanostructures. The cells spread more and became more nonround when cultured on surfaces with increasing size of the topographic features.

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

title: Electron beam lithography: resolution limits and applications
abstract: We report on the resolution limits of Electron Beam Lithography (EBL) in the conventional polymethylmethacrylate (PMMA) organic resist. We show that resolution can be pushed below 10 nm for isolated features and how dense arrays of periodic structures can be fabricated at a pitch of 30 nm, leading to a density close to 700 Gbit/in2. We show that intrinsic resolution of the writing in the resist is as small as 3 to 5 nm at high incident electron energy, and that practical resolution is limited by the development of the resist after exposure and by pattern transfer. We present the results of our optimized process for reproducible fabrication of sub-10 nm lines by lift-off and 30-nm pitch pillar arrays by lift-off and reactive ion etching (RIE). We also present some applications of these nanostructures for the fabrication of very high density molds for nano-imprint lithography (NIL) and for the fabrication of Multiple Tunnel Junction devices that can be used for single electron device applications or for the connection of small molecules.

title: Arrays of nano-dots for cellular engineering
abstract: Efficient patterning of large areas with nanometre features is required for cellular engineering applications. The final product must be made at an economic cost. 100 nm diameter pits on a 300 nm pitch have shown to be non-adhesive to a range of different mammalian cells. A strategy is described which uses an electron beam lithographic tool with focussed Gaussian beam to define shapes directly. Features as small as 20 nm in diameter on 100 nm pitch were fabricated. It was found that focus of 20 nm spot size is critical. The effect of deflection aberrations across the writing field was investigated. Various resists have been examined. Patterns were successfully embossed in poly(caprolactone). The overall cost of a die in terms of machine time has been significantly reduced.

title: The use of materials patterned on a nano- and micro-metric scale in cellular engineering
abstract: Biological cells form distinct groupings in tissue depending on their function. This is as essential in the regrowth of damaged tissue as it is in the development of the mature animal from its egg. 
 
For a number of years we have been exploring the effect of surfaces patterned with a topographic relief pattern and with patterns of bound active bio-molecules. 
 
The response of cells to micron-sized features, the response of cells to nano-metric features and the effects of soft materials are discussed.

title: Surface Topography and Composition of Deuterated Polystyrene-Poly(bromostyrene) Blends
abstract: Surface structure, obtained from atomic force microscopy and X-ray reflectivity, and surface chemical analysis data, obtained from X-ray photoelectron and static secondary ion mass spectroscopy, are reported for blends of poly(p-bromostyrene) with poly(deuteriostyrene). When high speeds are used in the spin-coating process, the atomic force microscopy measurements reveal that the surface structure consists of islands, the distribution and number changing with the poly(bromostyrene) content. A ribbon structure is observed at just above 50% (w/w) poly(bromostyrene) in the mixture. These ribbons merge to form more continuous structures, leaving voids at higher concentrations. X-ray reflectivity data from the films were consistent with the topographical features observed with the AFM. At low spinning speeds, continuous films with little or no topographical structure are formed. The islands observed at high spinning speeds are predominately poly(bromostyrene) and reflect the importance of thermodynamic and kin...

title: Topography and Surface Composition of Thin Films of Blends of Polystyrene with Brominated Polystyrenes: Effects of Varying the Degree of Bromination and Annealing
abstract: A series of thin films of blends of poly(styrene-d8) and poly(styrene-co-p-bromox-styrene), where 1 ≥ x ≥ 0, cast on to silicon wafers are examined by atomic force microscopy, X-ray photoelectron spectroscopy, and static secondary ion mass spectrometry. Films deposited on wafers stripped of the native oxide are smooth. The poly(styrene-d8) component segregates to the polymer−air interface, and the extent of segregation increases with the degree of bromination. An inverse linear correlation is obtained between the extent of segregation and the polymer compatibility, the latter measured by the interfacial width of bilayer films. For films deposited on wafers retaining the oxide, topographical features are observed with dimensions depending on the blend composition and degree of bromination of the polymer. The bromopolymer forms islands that are raised. The most pronounced topography is found with blends containing the fully brominated styrene. Reducing the degree of bromination increases the polymer compati...

title: Fabrication of nanostructured polymer surfaces using colloidal lithography and spin-coating
abstract: We present an original method for fabricating nanostructured polymer surfaces exhibiting homogeneous chemistry using colloidal lithography combined with polymer spin-coating. The first step of the strategy consists of creating gold surfaces with hemisphere-like protrusions, by adhesion of negatively charged polystyrene colloidal particles onto smooth positively charged surfaces, followed by coating with a thin layer of gold. In the second step, gold substrata are spin-coated with ultrathin polystyrene films. Using X-ray photoelectron spectroscopy and atomic force microscopy, we show that the nanotopography of the obtained polymer surfaces can be modulated by changing three parameters, i.e., the particle diameter, the particle concentration, and the polystyrene film thickness, while keeping the surface chemistry unmodified.

title: Tissue-engineered autologous bladders for patients needing cystoplasty
abstract: Patients with end-stage bladder disease can be treated with cystoplasty using gastrointestinal segments. The presence of such segments in the urinary tract has been associated with many complications. We explored an alternative approach using autologous engineered bladder tissues for reconstruction.



Seven patients with myelomeningocele, aged 4-19 years, with high-pressure or poorly compliant bladders, were identified as candidates for cystoplasty. A bladder biopsy was obtained from each patient. Urothelial and muscle cells were grown in culture, and seeded on a biodegradable bladder-shaped scaffold made of collagen, or a composite of collagen and polyglycolic acid. About 7 weeks after the biopsy, the autologous engineered bladder constructs were used for reconstruction and implanted either with or without an omental wrap. Serial urodynamics, cystograms, ultrasounds, bladder biopsies, and serum analyses were done.



Follow-up range was 22-61 months (mean 46 months). Post-operatively, the mean bladder leak point pressure decrease at capacity, and the volume and compliance increase was greatest in the composite engineered bladders with an omental wrap (56%, 1.58-fold, and 2.79-fold, respectively). Bowel function returned promptly after surgery. No metabolic consequences were noted, urinary calculi did not form, mucus production was normal, and renal function was preserved. The engineered bladder biopsies showed an adequate structural architecture and phenotype.



Engineered bladder tissues, created with autologous cells seeded on collagen-polyglycolic acid scaffolds, and wrapped in omentum after implantation, can be used in patients who need cystoplasty.

title: Adaptation of human embryonic stem cells to feeder-free and matrix-free culture conditions directly on plastic surfaces.
abstract: Previous studies have shown that cultivation of undifferentiated human embryonic stem (hES) cells requires human fibroblasts (hF) or mouse embryonic fibroblast (mEF) feeders or a coating matrix such as laminin, fibronectin or Matrigel in combination with mEF or hF conditioned medium. We here demonstrate a successful feeder-free and matrix-free culture system in which undifferentiated hES cells can be cultured directly on plastic surfaces without any supportive coating, in a hF conditioned medium. The hES cells cultured directly on plastic surfaces grow as colonies with morphology very similar to cells cultured on Matrigel(TM). Two hES cell lines SA167 and AS034.1 were adapted to matrix-free growth (MFG) and have so far been cultured up to 43 passages and cryopreserved successfully. The lines maintained a normal karyotype and expressed the expected marker profile of undifferentiated hES cells for Oct-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and SSEA-1. The hES cells formed teratomas in SCID mice and differentiated in vitro into derivates of all three germ layers. Thus, the MFG-adapted hES cells appear to retain pluripotency and to remain undifferentiated. The present culture system has a clear potential to be scaleable up to a manufacturing level and become the preferred culture system for various applications such as cell therapy and toxicity testing.

title: Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors
abstract: Differentiated cells can be reprogrammed to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with embryonic stem (ES) cells. Little is known about factors that induce this reprogramming. Here, we demonstrate induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture conditions. Unexpectedly, Nanog was dispensable. These cells, which we designated iPS (induced pluripotent stem) cells, exhibit the morphology and growth properties of ES cells and express ES cell marker genes. Subcutaneous transplantation of iPS cells into nude mice resulted in tumors containing a variety of tissues from all three germ layers. Following injection into blastocysts, iPS cells contributed to mouse embryonic development. These data demonstrate that pluripotent stem cells can be directly generated from fibroblast cultures by the addition of only a few defined factors.

title: piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells
abstract: Transgenic expression of just four defined transcription factors (c-Myc, Klf4, Oct4 and Sox2) is sufficient to reprogram somatic cells to a pluripotent state. The resulting induced pluripotent stem (iPS) cells resemble embryonic stem cells in their properties and potential to differentiate into a spectrum of adult cell types. Current reprogramming strategies involve retroviral, lentiviral, adenoviral and plasmid transfection to deliver reprogramming factor transgenes. Although the latter two methods are transient and minimize the potential for insertion mutagenesis, they are currently limited by diminished reprogramming efficiencies. piggyBac (PB) transposition is host-factor independent, and has recently been demonstrated to be functional in various human and mouse cell lines. The PB transposon/transposase system requires only the inverted terminal repeats flanking a transgene and transient expression of the transposase enzyme to catalyse insertion or excision events. Here we demonstrate successful and efficient reprogramming of murine and human embryonic fibroblasts using doxycycline-inducible transcription factors delivered by PB transposition. Stable iPS cells thus generated express characteristic pluripotency markers and succeed in a series of rigorous differentiation assays. By taking advantage of the natural propensity of the PB system for seamless excision, we show that the individual PB insertions can be removed from established iPS cell lines, providing an invaluable tool for discovery. In addition, we have demonstrated the traceless removal of reprogramming factors joined with viral 2A sequences delivered by a single transposon from murine iPS lines. We anticipate that the unique properties of this virus-independent simplification of iPS cell production will accelerate this field further towards full exploration of the reprogramming process and future cell-based therapies.

title: Differentiation of human embryonic stem cells on three-dimensional polymer scaffolds
abstract: Human embryonic stem (hES) cells hold promise as an unlimited source of cells for transplantation therapies. However, control of their proliferation and differentiation into complex, viable 3D tissues is challenging. Here we examine the use of biodegradable polymer scaffolds for promoting hES cell growth and differentiation and formation of 3D structures. We show that complex structures with features of various committed embryonic tissues can be generated, in vitro, by using early differentiating hES cells and further inducing their differentiation in a supportive 3D environment such as poly(lactic-co-glycolic acid)/poly(l-lactic acid) polymer scaffolds. We found that hES cell differentiation and organization can be influenced by the scaffold and directed by growth factors such as retinoic acid, transforming growth factor β, activin-A, or insulin-like growth factor. These growth factors induced differentiation into 3D structures with characteristics of developing neural tissues, cartilage, or liver, respectively. In addition, formation of a 3D vessel-like network was observed. When transplanted into severe combined immunodeficient mice, the constructs continue to express specific human proteins in defined differentiated structures and appear to recruit and anastamose with the host vasculature. This approach provides a unique culture system for addressing questions in cell and developmental biology, and provides a potential mechanism for creating viable human tissue structures for therapeutic applications.

title: The influence of three-dimensional nanofibrous scaffolds on the osteogenic differentiation of embryonic stem cells.
abstract: Embryonic stem cells represent a potentially unlimited cell source for tissue engineering applications. However, in order to be used for such applications, embryonic stem cells' differentiation must be controlled to only the desired lineages. In this study, we examine the effects of nanofibrous architecture and biochemical cues on the osteogenic differentiation of embryonic stem cells compared to the more traditional architecture without the nanofibrous features in two dimensions (thin matrix or flat films) and three dimensions (scaffolds) in vitro. After three weeks of culture the nanofibrous thin matrices were capable of supporting mRNA expression of osteogenic differentiation markers in embryonic stem cells without osteogenic supplements, while solid films required osteogenic supplements and growth factors to achieve mRNA expression of osteogenic differentiation markers. Nanofibrous scaffolds substantially enhanced mRNA expression of osteogenic differentiation markers compared to solid-walled scaffolds, nanofibrous thin matrices or solid films. After 4 weeks of culture, nanofibrous scaffolds were found to contain 3 times more calcium and stronger osteocalcin stain throughout the scaffolds than the solid-walled scaffolds. Overall, the nanofibrous architecture enhanced the osteogenic differentiation and mineralization of embryonic stem cells compared to the solid-walled architecture in both two and three-dimensional cultures.

title: Three‐Dimensional Nanofibrillar Surfaces Promote Self‐Renewal in Mouse Embryonic Stem Cells
abstract: The regulation of mouse embryonic stem cell (mESC) fate is controlled by the interplay of signaling networks that either promote self‐renewal or induce differentiation. Leukemia inhibitory factor (LIF) is a cytokine that is required for stem cell renewal in mouse but not in human embryonic stem cells. However, feeder layers of embryonic fibroblasts are capable of inducing stem cell renewal in both cell types, suggesting that the self‐renewal signaling pathways may also be promoted by other triggers, such as alternative cytokines and/or chemical or physical properties of the extracellular matrix (ECM) secreted by feeder fibroblasts. We have recently used a synthetic polyamide matrix (Ultra‐Web) whose three‐dimensional (3D) nanofibrillar organization resembles the ECM/basement membrane. Growth of mESCs on this nanofibrillar surface greatly enhanced proliferation and self‐renewal in comparison with growth on tissue culture surfaces without nanofibers, despite the presence of LIF in both systems. Enhanced proliferation and self‐renewal of the stem cells on nanofibrillar surfaces were correlated with the activation of the small GTPase Rac, the activation of phosphoinositide 3‐kinase (PI3K) pathway, and the enhanced expression of Nanog, a homeoprotein required for maintenance of pluripotency. Inhibitors of PI3K reduced the expression level of Nanog in mESCs cultured on 3D nanofibrillar surfaces. These results provide support for the view that the three‐dimensionality of the culture surface may function as a cue for the activation of Rac and PI3K signaling pathways, resulting in stem cell proliferation and self‐renewal.

title: Skeletal stem cells: Phenotype, biology and environmental niches informing tissue regeneration
abstract: Advances in our knowledge of the biology of skeletal stem cells, together with an increased understanding of the regeneration of normal tissue offer exciting new therapeutic approaches in musculoskeletal repair. Skeletal stem cells from various adult tissues such as bone marrow can be identified and isolated based on their expression of a panel of markers associated with smooth muscle cells, pericytes and endothelial cells. Thus, skeletal stem cell-like populations within bone marrow may share a common perivascular stem cell niche within the microvascular network. To date, the environmental niche that nurtures and maintains the stromal stem cell at different anatomical sites remains poorly understood. However, an understanding of the osteogenic and perivascular niches will inform identification of the key growth factors, matrix constituents and physiological conditions that will enhance the ex vivo amplification and differentiation of osteogenic stem cells to mimic native tissue critical for tissue repair. This review will examine skeletal stem cell biology, the advances in our understanding of the skeletal and perivascular niche and interactions therein and the opportunities to harness that knowledge for musculoskeletal regeneration.

title: Multilineage potential of adult human mesenchymal stem cells.
abstract: Human mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro. These adult stem cells could be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic lineages. Individual stem cells were identified that, when expanded to colonies, retained their multilineage potential.

title: Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model.
abstract: Bone marrow stromal cells can give rise to several mesenchymal lineages. The existence of a common stem/progenitor cell, the mesenchymal stem cell, has been proposed, but which developmental stages follow this mesenchymal multipotent progenitor is not known. Based on experimental evidence, a model of mesenchymal stem cell differentiation has been proposed in which individual lineages branch directly from the same progenitor. We have verified this model by using clonal cultures of bone marrow derived stromal fibroblasts. We have analyzed the ability of 185 non-immortalized human bone marrow stromal cell clones to differentiate into the three main lineages: osteo-, chondro- and adipogenic. All clones but one differentiated into the osteogenic lineage. About one third of the clones differentiated into all three lineages analyzed. Most clones (60-80%) displayed an osteo-chondrogenic potential. We have never observed clones with a differentiation potential limited to the osteo-adipo- or to the chondro-adipogenic phenotype, nor pure chondrogenic and adipogenic clones. How long the differentiation potential of a number of clones was maintained was assessed throughout their life span. Clones progressively lost their adipogenic and chondrogenic differentiation potential at increasing cell doublings. Our data suggest a possible model of predetermined bone marrow stromal cells differentiation where the tripotent cells can be considered as early mesenchymal progenitors that display a sequential loss of lineage potentials, generating osteochondrogenic progenitors which, in turn, give rise to osteogenic precursors.

title: Effects of transforming growth factor-beta 1 and ascorbic acid on differentiation of human bone-marrow-derived mesenchymal stem cells into smooth muscle cell lineage
abstract: Bone-marrow-derived mesenchymal stem cells (MSCs) can differentiate into a variety of cell types including smooth muscle cells (SMCs). We have attempted to demonstrate that, following treatment with transforming growth factor-beta 1 (TGF-β1) and ascorbic acid (AA), human bone-marrow-derived MSCs differentiate into the SMC lineage for use in tissue engineering. Quantitative polymerase chain reaction for SMC-specific gene (α smooth muscle actin, h1-calponin, and SM22α) expression was performed on MSCs, which were cultured with various concentrations of TGF-β1 or AA. TGF-β1 had a tendency to up-regulate the expression of SMC-specific genes in a dose-dependent manner. The expression of SM22α was significantly up-regulated by 30 μM AA. We also investigated the additive effect of TGF-β1 and AA for differentiation into SMCs and compared this effect with that of other factors including platelet-derived growth factor BB (PDGF-BB). In addition to SMC-specific gene expression, SMC-specific proteins increased by two to four times when TGF-β1 and AA were used together compared with their administration alone. PDGF did not increase the expression of SMC-specific markers. MSCs cultured with TGF-β1 and AA did not differentiate into osteoblasts and adipocytes. These results suggest that a combination of TGF-β1 and AA is useful for the differentiation of MSCs into SMCs for use in tissue engineering.

title: Neurogenic potential of human mesenchymal stem cells revisited: analysis by immunostaining, time-lapse video and microarray
abstract: The possibility of generating neural cells from human bone-marrow-derived mesenchymal stem cells (hMSCs) by simple in vitro treatments is appealing both conceptually and practically. However, whether phenotypic modulations observed after chemical manipulation of such stem cells truly represent a genuine trans-lineage differentiation remains to be established. We have re-evaluated the effects of a frequently reported biochemical approach, based on treatment with butylated hydroxyanisole and dimethylsulphoxide, to bring about such phenotypic conversion by monitoring the morphological changes induced by the treatment in real time, by analysing the expression of phenotype-specific protein markers and by assessing the modulation of transcriptome. Video time-lapse microscopy showed that conversion of mesenchymal stem cells to a neuron-like morphology could be reproduced in normal primary fibroblasts as well as mimicked by addition of drugs eliciting cytoskeletal collapse and disruption of focal adhesion contacts. Analysis of markers revealed that mesenchymal stem cells constitutively expressed multi-lineage traits, including several pertaining to the neural one. However, the applied `neural induction' protocol neither significantly modulated the expression of such markers, nor induced de novo translation of other neural-specific proteins. Similarly, global expression profiling of over 21,000 genes demonstrated that gene transcription was poorly affected. Most strikingly, we found that the set of genes whose expression was altered by the inductive treatment did not match those sets of genes differentially expressed when comparing untreated mesenchymal stem cells and immature neural tissues. Conversely, by comparing these gene expression profiles with that obtained from comparisons between the same cells and an unrelated non-neural organ, such as liver, we found that the adopted neural induction protocol was no more effective in redirecting human mesenchymal stem cells toward a neural phenotype than toward an endodermal hepatic pathway.

title: Synthetic nanostructures inducing differentiation of human mesenchymal stem cells into neuronal lineage.
abstract: Human mesenchymal stem cells (hMSCs) have been shown to trans-differentiate into neuronal-like cells by culture in neuronal induction media, although the mechanism is not well understood. Topography can also influence cellular responses including enhanced differentiation of progenitor cells. As extracellular matrix (ECM) in vivo comprises topography in the nanoscale, we hypothesize that nanotopography could influence stem cell differentiation into specific non-default pathways, such as transdifferentiation of hMSCs. Differentiation and proliferation of hMSCs were studied on nanogratings of 350 nm width. Cytoskeleton and nuclei of hMSCs were aligned and elongated along the nanogratings. Gene profiling and immunostaining showed significant up-regulation of neuronal markers such as microtubule-associated protein 2 (MAP2) compared to unpatterned and micropatterned controls. The combination of nanotopography and biochemical cues such as retinoic acid further enhanced the up-regulation of neuronal marker expressions, but nanotopography showed a stronger effect compared to retinoic acid alone on unpatterned surface. This study demonstrated the significance of nanotopography in directing differentiation of adult stem cells.

title: Molecular mechanisms mediating proliferation/differentiation interrelationships during progressive development of the osteoblast phenotype.
abstract: I.  A FUNCTIONAL relationship between cell growth and the initiation and progression of events associated with differentiation has been a fundamental question challenging developmental biologists for more than a century. In the case of bone, as observed with other cells and tissue, the relationship of growth and differentiation must be maintained and stringently regulated, both during development and throughout the life of the organism, to support tissue remodeling. For many years, bone was defined anatomically and examined largely in a descriptive manner by ultrastructural analysis and by biochemical and histochemical methods. These studies provided the basis for our understanding of bone tissue organization and orchestration of the progressive recruitment, proliferation, and differentiation of the various cellular components of bone tissue. Now, complemented by an increased knowledge of molecular mechanisms that are associated with and regulate expression of genes encoding phenotypic compone...

title: Direct differentiation of human embryonic stem cells into selective neurons on nanoscale ridge/groove pattern arrays.
abstract: Human embryonic stem cells (hESCs) are pluripotent cells that have the potential to be used for tissue engineering and regenerative medicine. Repairing nerve injury by differentiating hESCs into a neuronal lineage is one important application of hESCs. Biochemical and biological agents are widely used to induce hESC differentiation. However, it would be better if we could induce differentiation of hESCs without such agents because these factors are expensive and it is difficult to control the optimal concentrations for efficient differentiation with reduced side effects. Moreover, the mechanism of differentiation induced by these factors is still not fully understood. In this study, we present evidence that nanoscale ridge/groove pattern arrays alone can effectively and rapidly induce the differentiation of hESCs into a neuronal lineage without the use any differentiation-inducing agents. Using UV-assisted capillary force lithography, we constructed nanoscale ridge/groove pattern arrays with a dimension and alignment that were finely controlled over a large area. Human embryonic stem cells seeded onto the 350-nm ridge/groove pattern arrays differentiated into neuronal lineage after five days, in the absence differentiation-inducing agents. This nanoscale technique could be used for a new neuronal differentiation protocol of hESCs and may also be useful for nanostructured scaffolding for nerve injury repair.

title: Low oxygen tension and synthetic nanogratings improve the uniformity and stemness of human mesenchymal stem cell layer.
abstract: A free-standing, robust cell sheet comprising aligned human mesenchymal stem cells (hMSCs) offers many interesting opportunities for tissue reconstruction. As a first step toward this goal, a confluent, uniform hMSC layer with a high degree of alignment and stemness maintenance needs to be created. Hypothesizing that topographical cue and a physiologically relevant low-oxygen condition could promote the formation of such an hMSC layer, we studied the culture of hMSCs on synthetic nanogratings (350 nm width and 700 nm pitch) and either under 2 or 20% O(2). Culturing hMSCs on the nanogratings highly aligned the cells, but it tended to create patchy layers and accentuate the hMSC differentiation. The 2% O(2) improved the alignment and uniformity of hMSCs, and reduced their differentiation. Over a 14-day culture period, hMSCs in 2% O(2) showed uniform connexon distribution, secreted abundant extracellular matrix (ECM) proteins, and displayed a high progenicity. After 21-day culture on nanogratings, hMSCs exposed to 2% O(2) maintained a higher viability and differentiation capacity. This study established that a 2% O(2) culture condition could restrict the differentiation of hMSCs cultured on nanopatterns, thereby setting the foundation to fabricate a uniformly aligned hMSC sheet for different regenerative medicine applications.

title: Control of neural stem cell differentiation on honeycomb films
abstract: Control of neural stem cell (NSC) differentiation is ongoing interest in neural tissue engineering. Formation of neural networks on various patterned substrates was reported in previous studies. In this study, we cultured NSCs derived from the cerebral cortex of embryonic day-14 mice on honeycomb (HC) films with highly regular pores prepared by casting a polymer solution of water-immiscible solvent under high humidity. The differentiation of NSCs was analyzed by immunostaining for Nestin and MAP2. The differentiation of NSC was controlled for the first time by manipulating the pore size on HC films. The highest suppression of NSC differentiation was observed on HC film with 3 μm pore specifically.

title: Directed growth and selective differentiation of neural progenitor cells on micropatterned polymer substrates.
abstract: Directional growth and differentiation of adult rat hippocampal progenitor cells (AHPCs) were investigated on micropatterned polymer substrates in vitro. Astrocytes or AHPCs cultured on micropatterned polystyrene substrates chemically modified with laminin exhibited over 75% alignment in the groove direction. AHPCs co-cultured with astrocytes preferentially acquired neuronal morphology, with nearly double the percentage of cells expressing class III beta-tubulin on the micropatterned half of the substrate, as opposed to the planar half of the substrate, or compared to those growing in the absence of astrocytes. This indicates that substrate three-dimensional topography, in synergy with chemical (laminin) and biological (astrocytes) guidance cues, facilitates neuronal differentiation of the AHPCs. Through multi-dimensional cell-cell interactions, this environment provides spatial control selectively enhancing neuronal differentiation and neurite alignment on topographically different regions of the same substrate. Integrating these cues is important in understanding and controlling neural stem cell differentiation and designing scaffolds for guided nerve regeneration.

title: The influence of fiber diameter of electrospun substrates on neural stem cell differentiation and proliferation.
abstract: Neural stem/progenitor cells (NSCs) are capable of self-renewal and differentiation into all types of neural lineage under different biochemical and topographical cues. In this study, we cultured rat hippocampus-derived adult NSCs (rNSCs) on laminin-coated electrospun Polyethersulfone (PES) fiber meshes with average fiber diameters of 283+/-45 nm, 749+/-153 nm and 1452+/-312 nm; and demonstrated that fiber diameter of PES mesh significantly influences rNSC differentiation and proliferation. Under the differentiation condition (in the presence of 1 microM retinoic acid and 1% fetal bovine serum), rNSCs showed a 40% increase in oligodendrocyte differentiation on 283-nm fibers and 20% increase in neuronal differentiation on 749-nm fibers, in comparison to tissue culture polystyrene surface. SEM imaging revealed that cells stretched multi-directionally to follow underlying 283-nm fibers, but extended along a single fiber axis on larger fibers. When cultured on fiber meshes in serum free medium in the presence of 20 ng/mL of FGF-2, rNSCs showed lower proliferation and more rounded morphology compared to that cultured on laminin-coated 2D surface. As the fiber diameter decreased, higher degree of proliferation and cell spreading and lower degree of cell aggregation were observed. This collective evidence indicates fiber topography can play a vital role in regulating differentiation and proliferation of rNSCs in culture.

title: Relationship between adsorbed fibronectin and cell adhesion on a honeycomb-patterned film
abstract: Substratum surface morphology plays a vital roles in cellular behavior. Here, we characterized adsorption of fibronectin (Fn) as a typical cell adhesion protein onto honeycomb-patterned films made of poly(e-caprolactone) (PCL) by using atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). In order to determine how cells adhere to a honeycomb-patterned film, focal adhesion of cardiac myocytes (CMYs) and endothelial cells (ECs) on the films were studied by using fluorescence labeling of vinculin. Fn adsorbs around the pore edges to form ring-shaped structures. CMYs and ECs adhere onto the honeycomb-patterned films at focal contact points localized around pore edges distributed over the entire cellular surface. The focal contact points on the honeycomb-patterned films correspond well with the adsorption sites of Fn. We suggest that the cell response to honeycomb-patterned films is associated with the adsorption pattern of Fn on the film.

title: Activation of integrin function by nanopatterned adhesive interfaces.
abstract: To study the function behind the molecular arrangement of single integrins in cell adhesion, we designed a hexagonally close-packed rigid template of cell-adhesive gold nanodots coated with cyclic RGDfK peptide by using block-copolymer micelle nanolithography. The diameter of the adhesive dots is < 8 nm, which allows the binding of one integrin per dot. These dots are positioned with high precision at 28, 58, 73, and 85 nm spacing at interfaces. A separation of > or = 73 nm between the adhesive dots results in limited cell attachment and spreading, and dramatically reduces the formation of focal adhesion and actin stress fibers. We attribute these cellular responses to restricted integrin clustering rather than insufficient number of ligand molecules in the cell-matrix interface since "micro-nanopatterned" substrates consisting of alternating fields with dense and no nanodots do support cell adhesion. We propose that the range between 58-73 nm is a universal length scale for integrin clustering and activation, since these properties are shared by a variety of cultured cells.

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

title: Nanotopographical stimulation of mechanotransduction and changes in interphase centromere positioning
abstract: 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.  2007.

title: Nanosize and vitality: TiO2 nanotube diameter directs cell fate.
abstract: We generated, on titanium surfaces, self-assembled layers of vertically oriented TiO2 nanotubes with defined diameters between 15 and 100 nm and show that adhesion, spreading, growth, and differentiation of mesenchymal stem cells are critically dependent on the tube diameter. A spacing less than 30 nm with a maximum at 15 nm provided an effective length scale for accelerated integrin clustering/focal contact formation and strongly enhances cellular activities compared to smooth TiO2 surfaces. Cell adhesion and spreading were severely impaired on nanotube layers with a tube diameter larger than 50 nm, resulting in dramatically reduced cellular activity and a high extent of programmed cell death. Thus, on a TiO2 nanotube surface, a lateral spacing geometry with openings of 30-50 nm represents a critical borderline for cell fate.

title: Adhesion formation of primary human osteoblasts and the functional response of mesenchymal stem cells to 330 nm deep microgrooves
abstract: 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 330 nm deep and either 10, 25 or 100 μm 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, 1.7 K 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; 100 μm wide groove/ridge arrays did not significantly affect adhesion formation yet induced upregulation of genes involved in skeletal development and increased osteospecific function; 25 μm wide groove/ridge arrays were associated with a reduction in SMA and an increase in FX formation; and 10 μm 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.

title: Cell spreading and focal adhesion dynamics are regulated by spacing of integrin ligands.
abstract: Integrin-mediated adhesion is regulated by multiple features of the adhesive surface, including its chemical composition, topography, and physical properties. In this study we investigated integrin lateral clustering, as a mechanism to control integrin functions, by characterizing the effect of nanoscale variations in the spacing between adhesive RGD ligands on cell spreading, migration, and focal adhesion dynamics. For this purpose, we used nanopatterned surfaces, containing RGD-biofunctionalized gold dots, surrounded by passivated gaps. By varying the spacing between the dots, we modulated the clustering of the associated integrins. We show that cell-surface attachment is not sensitive to pattern density, whereas the formation of stable focal adhesions and persistent spreading is. Thus cells plated on a 108-nm-spaced pattern exhibit delayed spreading with repeated protrusion-retraction cycles compared to cells growing on a 58-nm pattern. Cell motility on these surfaces is erratic and nonpersistent, leaving thin membrane tethers bound to the RGD pattern. Dynamic molecular profiling indicated that the adhesion sites formed with the 108-nm pattern undergo rapid turnover and contain reduced levels of zyxin. These findings indicate that a critical RGD density is essential for the establishment of mature and stable integrin adhesions, which, in turn, induce efficient cell spreading and formation of focal adhesions.

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

title: Lateral spacing of integrin ligands influences cell spreading and focal adhesion assembly.
abstract: Cell-extracellular matrix (cell-ECM) interactions mediated by integrin receptors are essential for providing positional and environmental information necessary for many cell functions, such as proliferation, differentiation and survival. In vitro studies on cell adhesion to randomly adsorbed molecules on substrates have been limited to sub-micrometer patches, thus preventing the detailed study of structural arrangement of integrins and their ligands. In this article, we illustrate the role of the distance between integrin ligands, namely the RGD (arginine-glycine-aspartate) sequence present in ECM proteins, in the control of cell adhesion. By using substrates, which carry cyclic RGD peptides arranged in highly defined nanopatterns, we investigated the dynamics of cell spreading and the molecular composition of adhesion sites in relation to a fixed spacing between the peptides on the surface. Our novel approach for in vitro studies on cell adhesion indicates that not only the composition, but also the spatial organization of the extracellular environment is important in regulating cell-ECM interactions.

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

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

title: pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions.
abstract: Expression of the Rous sarcoma virus-encoded oncoprotein, pp60v-src, subverts the normal regulation of cell growth, which results in oncogenic transformation. This process requires the intrinsic protein-tyrosine kinase activity of pp60v-src and is associated with an increase in tyrosine phosphorylation of a number of cellular proteins, candidate substrates for pp60v-src. We report here the isolation of a cDNA encoding a protein, pp125, that is a major phosphotyrosine-containing protein in untransformed chicken embryo cells and exhibits an increase in phosphotyrosine in pp60v-src-transformed chicken embryo cells. This cDNA encodes a cytoplasmic protein-tyrosine kinase which, based upon its predicted amino acid sequence and structure, is the prototype for an additional family of protein-tyrosine kinases. Immunofluorescence localization experiments show that pp125 is localized to focal adhesions; hence, we suggest the name focal adhesion kinase.

title: Adult Human Mesenchymal Stem Cell Differentiation to the Osteogenic or Adipogenic Lineage Is Regulated by Mitogen-activated Protein Kinase*
abstract: Adult human mesenchymal stem cells are primary, multipotent cells capable of differentiating to osteocytic, chondrocytic, and adipocytic lineages when stimulated under appropriate conditions. To characterize the molecular mechanisms that regulate osteogenic differentiation, we examined the contribution of mitogen-activated protein kinase family members, ERK, JNK, and p38. Treatment of these stem cells with osteogenic supplements resulted in a sustained phase of ERK activation from day 7 to day 11 that coincided with differentiation, before decreasing to basal levels. Activation of JNK occurred much later (day 13 to day 17) in the osteogenic differentiation process. This JNK activation was associated with extracellular matrix synthesis and increased calcium deposition, the two hallmarks of bone formation. Inhibition of ERK activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked the osteogenic differentiation in a dose-dependent manner, as did transfection with a dominant negative form of MAP kinase kinase (MEK-1). Significantly, the blockage of osteogenic differentiation resulted in the adipogenic differentiation of the stem cells and the expression of adipose-specific mRNAs peroxisome proliferator-activated receptor γ2, aP2, and lipoprotein lipase. These observations provide a potential mechanism involving MAP kinase activation in osteogenic differentiation of adult stem cells and suggest that commitment of hMSCs into osteogenic or adipogenic lineages is governed by activation or inhibition of ERK, respectively.

title: Laminin-5 induces osteogenic gene expression in human mesenchymal stem cells through an ERK-dependent pathway.
abstract: The laminin family of proteins is critical for managing a variety of cellular activities including migration, adhesion, and differentiation. In bone, the roles of laminins in controlling osteogenic differentiation of human mesenchymal stem cells (hMSC) are unknown. We report here that laminin-5 is found in bone and expressed by hMSC. hMSC isolated from bone synthesize laminin-5 and adhere to exogenous laminin-5 through alpha3beta1 integrin. Adhesion to laminin-5 activates extracellular signal-related kinase (ERK) within 30 min and leads to phosphorylation of the osteogenic transcription factor Runx2/CBFA-1 within 8 d. Cells plated on laminin-5 for 16 d express increased levels of osteogenic marker genes, and those plated for 21 d deposit a mineralized matrix, indicative of osteogenic differentiation. Addition of the ERK inhibitor PD98059 mitigates these effects. We conclude that contact with laminin-5 is sufficient to activate ERK and to stimulate osteogenic differentiation in hMSC.

title: Critical role of the extracellular signal–regulated kinase–MAPK pathway in osteoblast differentiation and skeletal development
abstract: Ge et al. 2007.  Text][2]

 : {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1083%252Fjcb.200610046%26rft_id%253Dinfo%253Apmid%252F17325210%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%

title: Modulation of human gingival fibroblast adhesion, morphology, tyrosine phosphorylation, and ERK 1/2 localization on polished, grooved and SLA substratum topographies.
abstract: Attachment of connective tissue to dental implants, which is influenced by surface topography, is an important determinant of implant success. Approaches employed to alter topography include acid etching or blasting to produce roughened surfaces, and production of precisely defined topographies using microfabrication techniques. The aim of this study was to assess the influence of polished, microgrooved, and sand-blasted, large grit, acid-etched (SLA) topographies on fibroblast adhesion, morphology, activation, and ERK 1/2 phosphorylation and localization. Human gingival fibroblasts (HGFs) spread on all tested surfaces within 2 h, and topography influenced the pattern of phosphotyrosine localization. Fibrillar adhesion formation was prominent in HGFs cultured on microgrooves and SLA at 24 h compared with smooth. No significant difference in ERK 1/2 phosphorylation was observed at 2 or 24 h, but nuclear localization depended on culture time and substratum topography. Nuclear localization of ERK 1/2 occurred at 2 h on polished surfaces, but was not evident at 1 week. In contrast, cells on SLA and grooved surfaces did not exhibit nuclear localization of ERK 1/2 at early times, but did at 1 week. The results of this study suggest that rough and microfabricated topographies influence fibroblast adhesion and intracellular signaling through focal adhesion/integrin-dependent mechanisms in a time-dependent manner.

title: The effect of substratum topography on osteoblast adhesion mediated signal transduction and phosphorylation.
abstract: Substratum surface topography is a powerful modulator of cell behaviour, but how it influences intracellular signaling is largely unknown. We investigated the influence of microfabricated topographies on the activation of nonreceptor tyrosine kinases Src, FAK and ERK 1/2, as well as the transcription factor, Runx2, in rat osteoblasts, cultured on substrata that varied in their ability to promote bone-like tissue formation. Total tyrosine phosphorylation increased on grooves, tapered pits, and gap cornered boxes, relative to the levels found on smooth surfaces, with the greatest activity at 1 week. Src levels was higher on smooth than on any other surface, but FAK and ERK 1/2 phosphorylation were highest on groove and gap-cornered boxes up to 6 weeks. Inhibition of Src phosphorylation with PP2 inhibited FAK and ERK 1/2 phosphorylation on grooves, but had no detectable effect on either FAK or ERK 1/2 on smooth substratum. We suggest that osteoblast response to substrata with specific topographical features requires FAK-Y397-Src-Y416 complexes for ERK 1/2 phosphorylation, but on smooth surfaces, Src independent methods of ERK 1/2 activation are present.

title: Integrins are required for the differentiation of visceral endoderm
abstract: Integrins of the β1 subfamily are highly expressed in the early mouse embryo and are essential for the formation of primitive germ layers from the inner cell mass (ICM). We investigated the mechanisms by which αβ1 integrins regulate ICM morphogenesis by using the embryonic-stem-cell-derived embryoid body (EB), a model for peri-implantation development. Ablation of integrin β1 in EBs resulted in endoderm detachment and in maturation defects, which were manifested by the mislocalization of GATA4 in the cytoplasm and the markedly reduced synthesis of basement membrane (BM) components and the lineage marker disabled homolog 2. The mutant endoderm cells failed to spread on BM substrates, but could spread on vitronectin, which induced upregulation of αvβ3 integrin and integrin-dependent GATA4 nuclear translocation. Forced expression of integrin β3 in the mutant EBs completely rescued endoderm morphogenesis, suggesting that integrin β3 can substitute for integrin β1 in the endoderm. Furthermore, the mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 (ERK1/2) and p38 were activated in endoderm in an integrin-dependent fashion. Pharmacological inhibition of ERK1/2 or p38 MAPK blocked vitronectin-induced GATA4 nuclear translocation and endoderm maturation, whereas expression of a constitutively active ERK kinase (MEK1) or p38 MAPK in the mutant cells rescued endoderm maturation in integrin-β1-null endoderm cells. Collectively, these results suggest that integrins are required for both the stable adhesion and maturation of visceral endoderm, the latter being mediated through the activation of ERK1/2 and p38 MAPK.

title: Osteoprogenitor response to low-adhesion nanotopographies originally fabricated by electron beam lithography
abstract: 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.

title: Regulation of implant surface cell adhesion: characterization and quantification of S‐phase primary osteoblast adhesions on biomimetic nanoscale substrates
abstract: Integration of an orthopedic prosthesis for bone repair must be associated with osseointegration and implant fixation, an ideal that can be approached via topographical modification of the implant/bone interface. It is thought that osteoblasts use cellular extensions to gather spatial information of the topographical surroundings prior to adhesion formation and cellular flattening. Focal adhesions (FAs) are dynamic structures associated with the actin cytoskeleton that form adhesion plaques of clustered integrin receptors that function in coupling the cell cytoskeleton to the extracellular matrix (ECM). FAs contain structural and signalling molecules crucial to cell adhesion and survival. To investigate the effects of ordered nanotopographies on osteoblast adhesion formation, primary human osteoblasts (HOBs) were cultured on experimental substrates possessing a defined array of nanoscale pits. Nickel shims of controlled nanopit dimension and configuration were fabricated by electron beam lithography and transferred to polycarbonate (PC) discs via injection molding. Nanopits measuring 120 nm diameter and 100 nm in depth with 300 nm center–center spacing were fabricated in three unique geometric conformations: square, hexagonal, and near‐square (300 nm spaced pits in square pattern, but with ±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.

title: Vinculin modulation of paxillin–FAK interactions regulates ERK to control survival and motility
abstract: Cells lacking vinculin are highly metastatic and motile. The reasons for this finding have remained unclear. Both enhanced survival and motility are critical to metastasis. Here, we show that vinculin null (vin−/−) cells and cells expressing a vinculin Y822F mutant have increased survival due to up-regulated activity of extracellular signal–regulated kinase (ERK). This increase is shown to result from vinculin's modulation of paxillin–FAK interactions. A vinculin fragment (amino acids 811–1066) containing the paxillin binding site restored apoptosis and suppressed ERK activity in vin−/− cells. Both vinY822F and vin−/− cells exhibit increased interaction between paxillin and focal adhesion kinase (FAK) and increased paxillin and FAK phosphorylation. Transfection with paxillin Y31FY118F dominant-negative mutant in these cells inhibits ERK activation and restores apoptosis. The enhanced motility of vin−/− and vinY822F cells is also shown to be due to a similar mechanism. Thus, vinculin regulates survival and motility via ERK by controlling the accessibility of paxillin for FAK interaction.

title: Activation of FAK is necessary for the osteogenic differentiation of human mesenchymal stem cells on laminin‐5
abstract: Human mesenchymal stem cell (hMSC) differentiation into osteoblasts and the signaling events involved are poorly understood. We recently established that contact with specific extracellular matrix (ECM) proteins, in particular laminin‐5, is sufficient to induce an osteogenic phenotype in hMSC through an extracellular signal‐related kinase (ERK)‐dependent pathway. Activation of ERK 1/2 by laminin‐5 induces phosphorylation of the runx2/cbfa‐1 transcription factor that controls osteogenic gene expression. We hypothesized that focal adhesion kinase (FAK) mediated signaling pathways supply a link between cell surface integrin‐ECM binding and activation of ERK 1/2, and that laminin‐5 promotes its osteogenic effects through this pathway. To test this hypothesis, we plated hMSC on a laminin‐5 matrix in the presence or absence of FAK‐specific small inhibitory RNAs (siRNA), and assayed for phosphorylation of runx2/cbfa‐1 as well as expression of established osteogenic differentiation markers (bone sialoprotein, osteocalcin, alkaline phosphatase, calcium deposition, and mineral:matrix ratio). We found that siRNA treatment reduced total endogenous FAK protein by ∼40%, and reduced FAK phosphorylation on Y397 by ∼33% in cells plated on laminin‐5 for 30 min. SiRNA treated cells exhibited a decrease in ERK 1/2 phosphorylation after 1 h, and reduced serine/threonine phosphorylation of Runx2/Cbfa‐1 after 8 days. Finally, FAK inhibition blocked osteogenic differentiation of hMSC, as assessed by lowered expression of osteogenic genes (RT‐PCR), decreased alkaline phosphatase activity, greatly reduced calcium deposition, and a lower mineral:matrix ratio after 28 days in culture. These results establish FAK as an important mediator of laminin‐5‐induced osteogenic differentiation of hMSC.  2007.

title: Regulation of fibronectin, integrin and cytoskeleton expression in differentiating adipocytes: inhibition by extracellular matrix and polylysine.
abstract: The differentiation of 3T3 preadipocytes into adipocytes is characterized by major changes in cell morphology from a fibroblastic to a rounded shape and by the induction of gene expression related to lipid metabolism. We have studied the synthesis and mRNA levels of proteins involved in the formation of cell-matrix contacts and in defining cell shape to determine the role and molecular basis of these morphological changes during adipose conversion. When confluent preadipocyte cultures were stimulated with adipogenic medium there was a gradual decrease in the expression of fibronectin, beta-integrin, actin and in the microfilament-associated proteins vinculin, alpha-actinin and tropomyosin. The changes in extracellular matrix and cytoskeletal mRNA levels were apparent before the accumulation of glycerophosphate dehydrogenase (GPD) mRNA and continued during the massive increase in GPD mRNA level. The culturing of preadipocytes on an extracellular matrix deposited on the dish by corneal endothelial cells, or on substrata coated with polylysine, prevented the morphological changes, the decrease in the level of assembled actin, the accumulation of lipid and the shifts in the expression of integrin, cytoskeletal proteins and GPD. In cells cultured on malleable hydrated collagen gels, adipocyte differentiation proceeded at normal rates. The results suggest that the regulated expression of proteins involved in the formation of the transmembrane linkage between the extracellular matrix and the microfilaments are programmed regulatory events that affect cell adhesion and thereby cell shape during adipocyte differentiation.

title: Cleavage of focal adhesion kinase (FAK) is essential in adipocyte differentiation.
abstract: During adipocyte differentiation, the cells experience dramatic alterations in morphology, motility and cell-ECM contact. Focal adhesion kinase (pp125FAK), a widely expressed non-receptor tyrosine kinase in integrin signaling, has been reported to participate in these events in various cells. Utilizing 3T3-L1 cells and primary rat preadipocytes, we explored the role of FAK in adipocyte differentiation. Gradual cleavage of FAK was demonstrated during adipcoyte differentiation, both in vitro and in vivo. This cleavage of FAK was mediated by calpain. Inhibition of calpain activity resulted in the rescue of FAK degradation, accompanied with the disturbance of final maturation of adipocyte. Our study revealed that FAK participated in adipocyte differentiation, and its cleavage by calpain was required to fulfill the final maturation of adipocytes.

title: Disruption of cell-matrix interactions by heparin enhances mesenchymal progenitor adipocyte differentiation.
abstract: Differentiation of marrow-derived mesenchymal progenitors to either the osteoblast or adipocyte lineage is reciprocally regulated. Factors that promote osteoblastogenesis inhibit adipogenesis, while adipogenic factors are inhibitory to osteoblast differentiation. Heparin, a soluble glycosaminoglycan, inhibits bone formation in vivo and osteoblast cell differentiation and function in vitro, and has been shown to promote adipocyte differentiation. To elucidate the role that heparin plays in the adipogenic induction of murine mesenchymal progenitors, we studied immortalized marrow stromal cells (IM-MSC), the MSC cell line, ST2, and 3T3L1 pre-adipocytes. Heparin alone was not sufficient to induce adipogenesis, but enhanced the induction under a variety of adipogenic cocktails. This effect was both dose- and time-dependent. Heparin showed a positive effect at concentrations > 0.1 microg/ml when applied before day 3 during the induction course. Heparin's effect on adipogenesis was independent of cell proliferation, cell density, and extracellular lipid. This effect is likely related to the unique structure of heparin because another polyanionic glycosaminoglycan, dextran sulfate, did not promote adipogenic differentiation. Heparin treatment altered morphology and adhesion characteristics of progenitor cells, resulting in cell rounding and aggregation. As well, heparin counteracted the known inhibitory effect of fibronectin on adipogenesis and decreased basal focal adhesion kinase and paxillin phosphorylation. We conclude that heparin-mediated disruption of cell-matrix adhesion enhances adipogenic potential.

title: Interactions with nanoscale topography: adhesion quantification and signal transduction in cells of osteogenic and multipotent lineage.
abstract: Polymeric medical devices widely used in orthopedic surgery play key roles in fracture fixation and orthopedic implant design. Topographical modification and surface micro-roughness of these devices regulate cellular adhesion, a process fundamental in the initiation of osteoinduction and osteogenesis. Advances in fabrication techniques have evolved the field of surface modification; in particular, nanotechnology has allowed the development of nanoscale substrates for the investigation into cell-nanofeature interactions. In this study human osteoblasts (HOBs) were cultured on ordered nanoscale pits and random nano "craters" and "islands". Adhesion subtypes were quantified by immunofluorescent microscopy and cell-substrate interactions investigated via immuno-scanning electron microscopy. To investigate the effects of these substrates on cellular function 1.7 k microarray analysis was used to establish gene profiles of enriched STRO-1+ progenitor cell populations cultured on these nanotopographies. Nanotopographies affected the formation of adhesions on experimental substrates. Adhesion formation was prominent on planar control substrates and reduced on nanocrater and nanoisland topographies; nanopits, however, were shown to inhibit directly the formation of large adhesions. STRO-1+ progenitor cells cultured on experimental substrates revealed significant changes in genetic expression. This study implicates nanotopographical modification as a significant modulator of osteoblast adhesion and cellular function in mesenchymal populations.

title: The response of fibroblasts to hexagonal nanotopography fabricated by electron beam lithography.
abstract: It has been known for many years that cells will react to the shape of their microenvironment. It is more recently becoming clear that cells can alter their morphology, adhesions, and cytoskeleton in response to their nanoenvironment. A few studies have gone further and measured cellular response to high-adhesion nanomaterials. There have, however, been practical difficulties associated with genomic studies focusing on low-adhesion nanotopographies. Because of advancement in fabrication techniques allowing the production of large area of structure and the ability to amplify mRNA prior to microarray hybridization, these difficulties can be overcome. Here, electron beam lithography has been used to fabricate arrays of pits with 120 nm diameters, 100 nm depth and 300 nm center to center spacing in hexagonal arrangement. Electron and fluorescent microscopies have been used to observe morphological changes in fibroblasts cultured on the pits. 1.7k gene microarray was used to gauge genomic response to the pits. The results show reduction in cellular adhesion, decrease in spreading, and a broad genomic down-regulation. Also noted was an increase in endocytotic activity in cells on the pits.

title: Focal Contacts as Mechanosensors Externally Applied Local Mechanical Force Induces Growth of Focal Contacts by an Mdia1-Dependent and Rock-Independent Mechanism
abstract: The transition of cell–matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II–driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein–tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. ), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force.

title: The role of the dynamics of focal adhesion kinase in the mechanotaxis of endothelial cells
abstract: The migration of vascular endothelial cells (ECs) is critical in vascular remodeling. We showed that fluid shear stress enhanced EC migration in flow direction and called this “mechanotaxis.” To visualize the molecular dynamics of focal adhesion kinase (FAK) at focal adhesions (FAs), FAK tagged with green fluorescence protein (GFP) was expressed in ECs. Within 10 min of shear stress application, lamellipodial protrusion was induced at cell periphery in the flow direction, with the recruitment of FAK at FAs. ECs under flow migrated with polarized formation of new FAs in flow direction, and these newly formed FAs subsequently disassembled after the rear of the cell moved over them. The cells migrating under flow had a decreased number of FAs. In contrast to shear stress, serum did not significantly affect the speed of cell migration. Serum induced lamellipodia and FAK recruitment at FAs without directional preference. FAK(Y397) phosphorylation colocalized with GFP-FAK at FAs in both shear stress and serum experiments. The total level of FAK(Y397) phosphorylation after shear stress was lower than that after serum treatment, suggesting that the polarized change at cell periphery rather than the total level of FAK(Y397) phosphorylation is important for directional migration. Our results demonstrate the dynamics of FAK at FAs during the directional migration of EC in response to mechanical force, and suggest that mechanotaxis is an important mechanism controlling EC migration.

title: Force transduction by Triton cytoskeletons
abstract: Force-initiated signal transduction can occur either via membrane-based ionic mechanisms or through changes in cytoskeletal–matrix linkages. We report here the stretch-dependent binding of cytoplasmic proteins to Triton X-100 cytoskeletons of L-929 cells grown on collagen-coated silicone. Triton X-100–insoluble cytoskeletons were stretched by 10% and incubated with biotinylated cytoplasmic proteins. Analysis with two-dimensional gel electrophoresis showed stretch-dependent binding of more than 10 cytoplasmic protein spots. Bound cytoplasmic proteins were purified by a photocleavable biotin tag and stretch-dependent binding of paxillin, focal adhesion kinase, and p130Cas was found, whereas the binding of vinculin was unchanged and actin binding decreased with stretch. Paxillin binding upon stretch was morphologically and biochemically similar in vitro and in vivo, that is, enhanced in the periphery and inhibited by the tyrosine phosphatase inhibitor, phenylarsine oxide. Thus, we suggest that transduction of matrix forces occurs through force-dependent conformation changes in the integrated cytoskeleton.

title: Stretching Single Talin Rod Molecules Activates Vinculin Binding
abstract: The molecular mechanism by which a mechanical stimulus is translated into a chemical response in biological systems is still unclear. We show that mechanical stretching of single cytoplasmic proteins can activate binding of other molecules. We used magnetic tweezers, total internal reflection fluorescence, and atomic force microscopy to investigate the effect of force on the interaction between talin, a protein that links liganded membrane integrins to the cytoskeleton, and vinculin, a focal adhesion protein that is activated by talin binding, leading to reorganization of the cytoskeleton. Application of physiologically relevant forces caused stretching of single talin rods that exposed cryptic binding sites for vinculin. Thus in the talin-vinculin system, molecular mechanotransduction can occur by protein binding after exposure of buried binding sites in the talin-vinculin system. Such protein stretching may be a more general mechanism for force transduction.

title: Force Sensing by Mechanical Extension of the Src Family Kinase Substrate p130Cas
abstract: How physical force is sensed by cells and transduced into cellular signaling pathways is poorly understood. Previously, we showed that tyrosine phosphorylation of p130Cas (Cas) in a cytoskeletal complex is involved in force-dependent activation of the small GTPase Rap1. Here, we mechanically extended bacterially expressed Cas substrate domain protein (CasSD) in vitro and found a remarkable enhancement of phosphorylation by Src family kinases with no apparent change in kinase activity. Using an antibody that recognized extended CasSD in vitro, we observed Cas extension in intact cells in the peripheral regions of spreading cells, where higher traction forces are expected and where phosphorylated Cas was detected, suggesting that the in vitro extension and phosphorylation of CasSD are relevant to physiological force transduction. Thus, we propose that Cas acts as a primary force sensor, transducing force into mechanical extension and thereby priming phosphorylation and activation of downstream signaling.

title: Force activates smooth muscle α-actin promoter activity through the Rho signaling pathway
abstract: In pressure or volume overload, hypertrophic growth of the myocardium is associated with myofibroblast differentiation, a process in which cardiac fibroblasts express smooth muscle α-actin (SMA). The signaling mechanisms that mediate force-induced myofibroblast differentiation and SMA expression are not defined. We examined the role of the Rho–Rho-kinase pathway in force-induced SMA expression in fibroblasts using an in vitro model system that applies static tensile forces (0.65 pN/μm2) to integrins via collagen-coated magnetite beads. Force maximally induced RhoA activation at 10 minutes that was localized to force application sites and required intact actin filaments. Force application induced phosphorylation of LIM kinase (5-10 minutes) and an early dephosphorylation of cofilin (5 minutes) that was followed by prolonged cofilin phosphorylation. These responses were blocked by Y27632, an inhibitor of Rho kinase. Force promoted actin filament assembly at force application sites (10-20 minutes), a process that required Rho kinase and cofilin. Force application induced nuclear translocation of the transcriptional co-activator MRTF-A but not MRTF-B. Nuclear translocation of MRTF-A required Rho kinase and intact actin filaments. Force caused 3.5-fold increases of SMA promoter activity that were completely blocked by transfection of cells with dominant-negative MRTF-A or by inhibition of Rho kinase or by actin filament disassembly. These data indicate that mechanical forces mediate actin assembly through the Rho–Rho-kinase–LIMK cofilin pathway. Force-mediated actin filament assembly promotes nuclear translocation of MRTF and subsequent activation of the SMA promoter to enhance SMA expression.

title: Local force and geometry sensing regulate cell functions
abstract: The shapes of eukaryotic cells and ultimately the organisms that they form are defined by cycles of mechanosensing, mechanotransduction and mechanoresponse. Local sensing of force or geometry is transduced into biochemical signals that result in cell responses even for complex mechanical parameters such as substrate rigidity and cell-level form. These responses regulate cell growth, differentiation, shape changes and cell death. Recent tissue scaffolds that have been engineered at the micro- and nanoscale level now enable better dissection of the mechanosensing, transduction and response mechanisms.

title: Mechanical behavior in living cells consistent with the tensegrity model
abstract: Alternative models of cell mechanics depict the living cell as a simple mechanical continuum, porous filament gel, tensed cortical membrane, or tensegrity network that maintains a stabilizing prestress through incorporation of discrete structural elements that bear compression. Real-time microscopic analysis of cells containing GFP-labeled microtubules and associated mitochondria revealed that living cells behave like discrete structures composed of an interconnected network of actin microfilaments and microtubules when mechanical stresses are applied to cell surface integrin receptors. Quantitation of cell tractional forces and cellular prestress by using traction force microscopy confirmed that microtubules bear compression and are responsible for a significant portion of the cytoskeletal prestress that determines cell shape stability under conditions in which myosin light chain phosphorylation and intracellular calcium remained unchanged. Quantitative measurements of both static and dynamic mechanical behaviors in cells also were consistent with specific a priori predictions of the tensegrity model. These findings suggest that tensegrity represents a unified model of cell mechanics that may help to explain how mechanical behaviors emerge through collective interactions among different cytoskeletal filaments and extracellular adhesions in living cells.

title: SUN1 Interacts with Nuclear Lamin A and Cytoplasmic Nesprins To Provide a Physical Connection between the Nuclear Lamina and the Cytoskeleton
abstract: Nuclear migration and positioning within cells are critical for many developmental processes and are governed by the cytoskeletal network. Although mechanisms of nuclear-cytoskeletal attachment are unclear, growing evidence links a novel family of nuclear envelope (NE) proteins that share a conserved C-terminal SUN (Sad1/UNC-84 homology) domain. Analysis of Caenorhabditis elegans mutants has implicated UNC-84 in actin-mediated nuclear positioning by regulating NE anchoring of a giant actin-binding protein, ANC-1. Here, we report the identification of SUN1 as a lamin A-binding protein in a yeast two-hybrid screen. We demonstrate that SUN1 is an integral membrane protein located at the inner nuclear membrane. While the N-terminal domain of SUN1 is responsible for detergent-resistant association with the nuclear lamina and lamin A binding, lamin A/C expression is not required for SUN1 NE localization. Furthermore, SUN1 does not interact with type B lamins, suggesting that NE localization is ensured by binding to an additional nuclear component(s), most likely chromatin. Importantly, we find that the luminal C-terminal domain of SUN1 interacts with the mammalian ANC-1 homologs nesprins 1 and 2 via their conserved KASH domain. Our data provide evidence of a physical nuclear-cytoskeletal connection that is likely to be a key mechanism in nuclear-cytoplasmic communication and regulation of nuclear position.

title: Geometric cues for directing the differentiation of mesenchymal stem cells
abstract: Significant efforts have been directed to understanding the factors that influence the lineage commitment of stem cells. This paper demonstrates that cell shape, independent of soluble factors, has a strong influence on the differentiation of human mesenchymal stem cells (MSCs) from bone marrow. When exposed to competing soluble differentiation signals, cells cultured in rectangles with increasing aspect ratio and in shapes with pentagonal symmetry but with different subcellular curvature—and with each occupying the same area—display different adipogenesis and osteogenesis profiles. The results reveal that geometric features that increase actomyosin contractility promote osteogenesis and are consistent with in vivo characteristics of the microenvironment of the differentiated cells. Cytoskeletal-disrupting pharmacological agents modulate shape-based trends in lineage commitment verifying the critical role of focal adhesion and myosin-generated contractility during differentiation. Microarray analysis and pathway inhibition studies suggest that contractile cells promote osteogenesis by enhancing c-Jun N-terminal kinase (JNK) and extracellular related kinase (ERK1/2) activation in conjunction with elevated wingless-type (Wnt) signaling. Taken together, this work points to the role that geometric shape cues can play in orchestrating the mechanochemical signals and paracrine/autocrine factors that can direct MSCs to appropriate fates.

title: Gold-nanoparticle-assisted laser perturbation of chromatin assembly reveals unusual aspects of nuclear architecture within living cells.
abstract: Chromatin organization within the nucleus is a vital regulator of genome function, yet its mechanical coupling to the nuclear architecture has remained elusive. To directly investigate this coupling, we locally modulated chromatin structure in living cells using nanoparticle-based laser perturbation. Unusual differences in the response of the cell nucleus were observed depending on the nuclear region that was perturbed--the heterochromatin, the euchromatin, and the nuclear envelope. This response varied under different conditions of cellular perturbations such as ATP depletion, apoptosis, and inhibition of histone deacetylases. Our studies implicate heterochromatin organization in imparting mechanical stability to the cell nucleus and suggest that nuclear size and shape are the result of interplay between nuclear and cytoplasmic anchors.

title: Mechanical continuity and reversible chromosome disassembly within intact genomes removed from living cells
abstract: Chromatin is thought to be structurally discontinuous because it is packaged into morphologically distinct chromosomes that appear physically isolated from one another in metaphase preparations used for cytogenetic studies. However, analysis of chromosome positioning and movement suggest that different chromosomes often behave as if they were physically connected in interphase as well as mitosis. To address this paradox directly, we used a microsurgical technique to physically remove nucleoplasm or chromosomes from living cells under isotonic conditions. Using this approach, we found that pulling a single nucleolus or chromosome out from interphase or mitotic cells resulted in sequential removal of the remaining nucleoli and chromosomes, interconnected by a continuous elastic thread. Enzymatic treatments of interphase nucleoplasm and chromosome chains held under tension revealed that mechanical continuity within the chromatin was mediated by elements sensitive to DNase or micrococcal nuclease, but not RNases, formamide at high temperature, or proteases. In contrast, mechanical coupling between mitotic chromosomes and the surrounding cytoplasm appeared to be mediated by gelsolin‐sensitive microfilaments. Furthermore, when ion concentations were raised and lowered, both the chromosomes and the interconnecting strands underwent multiple rounds of decondensation and recondensation. As a result of these dynamic structural alterations, the mitotic chains also became sensitive to disruption by restriction enzymes. Ion‐induced chromosome decondensation could be blocked by treatment with DNA binding dyes, agents that reduce protein disulfide linkages within nuclear matrix, or an antibody directed against histones. Fully decondensed chromatin strands also could be induced to recondense into chromosomes with pre‐existing size, shape, number, and position by adding anti‐histone antibodies. Conversely, removal of histones by proteolysis or heparin treatment produced chromosome decondensation which could be reversed by addition of histone H1, but not histones H2b or H3. These data suggest that DNA, its associated protein scaffolds, and surrounding cytoskeletal networks function as a structurally‐unified system. Mechanical coupling within the nucleoplasm may coordinate dynamic alterations in chromatin structure, guide chromosome movement, and ensure fidelity of mitosis.

title: Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure.
abstract: We report here that living cells and nuclei are hard-wired such that a mechanical tug on cell surface receptors can immediately change the organization of molecular assemblies in the cytoplasm and nucleus. When integrins were pulled by micromanipulating bound microbeads or micropipettes, cytoskeletal filaments reoriented, nuclei distorted, and nucleoli redistributed along the axis of the applied tension field. These effects were specific for integrins, independent of cortical membrane distortion, and were mediated by direct linkages between the cytoskeleton and nucleus. Actin microfilaments mediated force transfer to the nucleus at low strain; however, tearing of the actin gel resulted with greater distortion. In contrast, intermediate filaments effectively mediated force transfer to the nucleus under both conditions. These filament systems also acted as molecular guy wires to mechanically stiffen the nucleus and anchor it in place, whereas microtubules acted to hold open the intermediate filament lattice and to stabilize the nucleus against lateral compression. Molecular connections between integrins, cytoskeletal filaments, and nuclear scaffolds may therefore provide a discrete path for mechanical signal transfer through cells as well as a mechanism for producing integrated changes in cell and nuclear structure in response to changes in extracellular matrix adhesivity or mechanics.

title: The complex language of chromatin regulation during transcription
abstract: An important development in understanding the influence of chromatin on gene regulation has been the finding that DNA methylation and histone post-translational modifications lead to the recruitment of protein complexes that regulate transcription. Early interpretations of this phenomenon involved gene regulation reflecting predictive activating or repressing types of modification. However, further exploration reveals that transcription occurs against a backdrop of mixtures of complex modifications, which probably have several roles. Although such modifications were initially thought to be a simple code, a more likely model is of a sophisticated, nuanced chromatin 'language' in which different combinations of basic building blocks yield dynamic functional outcomes.

title: Mechanotransduction at a distance: mechanically coupling the extracellular matrix with the nucleus
abstract: Research in cellular mechanotransduction often focuses on how extracellular physical forces are converted into chemical signals at the cell surface. However, mechanical forces that are exerted on surface-adhesion receptors, such as integrins and cadherins, are also channelled along cytoskeletal filaments and concentrated at distant sites in the cytoplasm and nucleus. Here, we explore the molecular mechanisms by which forces might act at a distance to induce mechanochemical conversion in the nucleus and alter gene activities.

title: Mechanotransduction gone awry
abstract: Cells sense their physical surroundings through mechanotransduction — that is, by translating mechanical forces and deformations into biochemical signals such as changes in intracellular calcium concentration or by activating diverse signalling pathways. In turn, these signals can adjust cellular and extracellular structure. This mechanosensitive feedback modulates cellular functions as diverse as migration, proliferation, differentiation and apoptosis, and is crucial for organ development and homeostasis. Consequently, defects in mechanotransduction — often caused by mutations or misregulation of proteins that disturb cellular or extracellular mechanics — are implicated in the development of various diseases, ranging from muscular dystrophies and cardiomyopathies to cancer progression and metastasis.

title: Fluorescence two-dimensional difference gel electrophoresis for biomaterial applications
abstract: Fluorescence two-dimensional difference gel electrophoresis (DiGE) is rapidly becoming established as a powerful technique for the characterization of differences in protein expression levels between two or more conditions. In this review, we consider the application of DiGE—both minimal and saturation labelling—to biomaterials research, considering the challenges and rewards of this approach.

title: Differential in-gel electrophoresis (DIGE) analysis of human bone marrow osteoprogenitor cell contact guidance.
abstract: We have used a recent comparative proteomics technique, differential in-gel electrophoresis (DIGE), to study osteoprogenitor cell response to contact guidance in grooves. In order to increase protein output from small sample sizes, we used bioreactor culture before protein extraction and gel electrophoresis. Mass spectroscopy was used for protein identification. A number of distinct proteins were observed to exhibit significant changes in expression. These changes in protein expression suggest that the cells respond to tailored grooved topographies, with alterations in their proteome concurrent with changes in osteoprogenitor phenotype.

title: Anisomycin superinduces annexin V mRNA expression through the ERK1/2 but not the p38 MAP kinase pathway.
abstract: Annexin V is a Ca(2+)-dependent phospholipid-binding protein belonging to the annexin family whose regulation is currently not well understood. In this study, we utilized anisomycin, a protein synthesis inhibitor that activates MAP kinases (MAPKs), to examine the role of MAPKs in annexin V expression in the MCAS ovarian carcinoma cell line. A one-step real-time TaqMan-based reverse transcriptase-PCR method was developed to quantify annexin V mRNA expression. We found that annexin V was induced 13.3-fold by anisomycin and that this superinduction was attenuated by pretreatment with the MEK inhibitors, U0126 and PD98059, but not with the p38 MAPK inhibitor, SB203580. In addition, immunoblotting showed that anisomycin stimulated the phosphorylation of ERK1/2 as well as p38 MAPK and that the phosphorylations were blocked by the three kinase inhibitors. Taken together, these results suggest that anisomycin superinduces annexin V mRNA expression through the ERK1/2 MAPK pathway, but not through the p38 MAPK pathway.

title: 14-3-3ζ interacts with human thromboxane receptors and is involved in the agonist-induced activation of the extracellular-signal-regulated kinase
abstract: Thromboxane receptor (TP) signaling results in a broad range of cellular responses including kinase activation and subsequent nuclear signaling events involved in cell transformation, proliferation, and cell survival. Proteins that may participate in the early signaling following receptor activation remain to be identified. We found that 14-3-3ζ is a novel protein interacting with TP intracellular loop 3 (i3) by yeast two-hybrid system. This interaction was further confirmed by GST pull-down and co-immunoprecipitation methods. Site-directed mutagenesis studies indicated that Pro-236 of the TP-i3 was involved in the binding to the 14-3-3ζ. Co-immunoprecipitation studies in the same cell lysate by TP antibody showed that TP binds not only with the 14-3-3ζ but also with the Raf-1. Our data also demonstrated that TP receptor activation induced by agonist rapidly recruited 14-3-3ζ and Raf-1 to form a complex with the TP on the plasma membrane. The significance of assembling this protein complex was examined by TP agonist-induced extracellular-signal-regulated kinase (ERK) phosphorylation in intact cells. TP agonist, I-BOP, induced ERK phosphorylation in HEK 293 cells expressing wild type TPα but significantly lower in those expressing TPα-P236V mutant. Attenuation of the expression of 14-3-3ζ by 14-3-3ζ siRNA decreased I-BOP-induced ERK phosphorylation indicating the involvement of the 14-3-3ζ in the signal transduction process. These results suggest that 14-3-3ζ may serve as a scaffold protein to form a protein complex consisting of TP, 14-3-3ζ, and Raf-1, and that this protein complex may be involved in the activation of ERK pathway following TP receptor activation.

title: Cellular functions of 14-3-3ζ in apoptosis and cell adhesion emphasize its oncogenic character
abstract: 14-3-3 proteins are relevant to cancer biology as they are key regulators of major cellular processes such as proliferation, differentiation, senescence and apoptosis. So far, the sigma isoform (14-3-3σ) has most directly been implicated in carcinogenesis and was recognized as a tumour-suppressor gene. The other six members of the mammalian 14-3-3 gene family likely behave as oncogenes, although direct evidence supporting this view is largely circumstantial. In this report, we show that knockdown of 14-3-3ζ induces at least two isoform-specific phenotypes that are consistent with a potential oncogenic activity during tumorigenesis. Firstly, downregulation of 14-3-3ζ sensitized cells to stress-induced apoptosis and JNK/p38 signalling and secondly, it enforced cell–cell contacts and expression of adhesion proteins. Apparently, the zeta isoform restrains both cell adhesion and the cellular propensity for apoptosis, two activities that are also restrained during carcinogenesis. The assumption that 14-3-3ζ has oncogenic properties was substantiated with a web-based meta-analysis (Oncomine), revealing that 14-3-3ζ is overexpressed in various types of carcinomas. As the highly conserved human 14-3-3 gene family encodes proteins with either tumour-promoting or tumour-suppressing activities, we infer that the cellular balance between the various 14-3-3 isoforms is crucial for the proper functioning of cells.

title: Inhibition of human angiogenin by DNA aptamers: nuclear colocalization of an angiogenin-inhibitor complex.
abstract: Specific ligands (aptamers) for angiogenin were selected from a 72-mer oligodeoxynucleotide library consisting of 28 randomized positions flanked by two constant regions of 22 residues each. From a starting pool of approximately 10(14) molecules, 19 angiogenin-binding ligands were obtained. Among them, two oligonucleotides showed significant inhibition of the ribonucleolytic activity of angiogenin with apparent Kis of 0.65 and 0.60 micro M, respectively. One of them was shortened on the basis of its secondary structure to provide a 45-mer oligonucleotide that retained much of the inhibitory properties of the parent molecule. It inhibits both the angiogenic and cell proliferative activities of angiogenin but does not interfere with its nuclear translocation in human endothelial cells. Importantly, the inhibitor is cotranslocated to the nucleus with angiogenin in a approximately 1:1 stoichiometric ratio. These results demonstrate that the inhibition of angiogenin-induced cell proliferation and angiogenesis by the oligonucleotide is due to suppression of the ribonucleolytic activity of angiogenin, an event that occurs most likely within the cell nucleus.

title: Ribonuclease inhibitor: structure and function.
abstract: Publisher Summary The mammalian ribonuclease inhibitor (RI) is a 50‐kDa cytosolic protein that binds to pancreatic‐type ribonucleases with femtomolar affinity and renders them inactive. The biological role of RI is not known in its entirety. The ribonucleases recognized by RI are secreted proteins, whereas RI resides exclusively in the cytosol. Nevertheless, RI affinity has been shown to be the primary determinant of ribonuclease cytotoxicity: Only ribonucleases that evade RI can kill a cell. In addition, the complex of RI with human angiogenin (ANG), which stimulates neovascularization by activating transcription in the nucleus, is the tightest of known RI.ribonuclease complexes. The chapter hypothesizes multiple biological roles for RI: (1) To protect cells from invading ribonucleases, (2) to regulate or terminate the activity of ribonuc leases with known intracellular functions, and ( 3) to monitor the oxidation state of the cell in response to factors such as aging and oxidative stress. RI possesses remarkable affinity for pancreatic‐type ribonucleases, despite their limited sequence identity. The resulting noncovalent complexes are some of the tightest known in biology. Details of the molecular interactions within RI.ribonuclease complexes have been elucidated from structural and biochemical investigations. Moreover, RI is known to be a sentry, protecting mammalian cells against invading ribonucleases, which abound in extracellular fluids. Still, many questions remain regarding the biological activity of RI.

title: Heat stress-induced heat shock protein 70 expression is dependent on ERK activation in zebrafish (Danio rerio) cells.
abstract: Heat shock response is a common event that occurs in many species. Despite its evolutionary conservation, comparative studies of heat shock response have been largely unexplored. In mammals, heat shock response decreases with age through unclear mechanisms. Understanding how the age-related decline in heat shock response occurs may provide information to understanding the biology of aging. We have previously shown that heat shock response similarly declines with age in zebrafish. However, signaling pathways that regulate the heat shock response in zebrafish are unknown. In mammals there is evidence that mitogen-activated protein kinases (MAPKs) of the ERK family alter Hsp70 transcription, serving as a potential regulator of the heat shock response. We explored if heat stress-induced Hsp70 expression is altered by activation of ERK in the zebrafish Pac2 fibroblast cell line as occurs in mammalian cells. Heat stress induced both Hsp70 mRNA expression and phosphorylation of both ERK1 and ERK2 (ERK1/2) in Pac2 cells. ERK inhibitors PD98059 and U0126 blocked both heat stress-induced and plated-derived growth factor (PDGF)-induced ERK1/2 phosphorylation, and also diminished heat-induced Hsp70 expression. Pac2 cell viability was not affected by either the ERK inhibitors or heat stress. These results demonstrate that induction of Hsp70 in response to heat stress is dependent on ERK activation in Pac2 cells. This suggests that the heat shock response in zebrafish utilizes a similar signaling pathway to that of mammals and that zebrafish are a good model for comparative studies of heat shock response.

title: Ras-Dependent Recruitment of c-Myc for Transcriptional Activation of Nucleophosmin/B23 in Highly Malignant U1 Bladder Cancer Cells
abstract: U1 bladder cancer cells of high malignancy exhibited higher proliferation capacity than U4 premalignant cells. Higher expression of Ras, c-Myc, and nucleophosmin/B23 and greater c-Myc transactivation and nucleophosmin/B23 promoter activities were detected in U1 cells compared with U4 cells. Moreover, c-Myc and nucleophosmin/B23 were increased in U1 but not in U4 cells upon serum stimulation from quiescence. Likewise, only in U1 cells could serum stimulate transcriptional activity of nucleophosmin/B23 promoter and c-Myc response element. The increase of nucleophosmin/B23 promoter activity could be abrogated by mitogen-activated protein kinase/extracellular signal-regulated kinase activating kinase inhibitor and was associated with recruitment of c-Myc to the promoter. U1 cells constitutively expressing dominant-negative Ras reduced the levels of Ras, nucleophosmin/B23, and p-ERK, and consequently abolished the serum-induced up-regulation of nucleophosmin/B23 promoter activity and c-Myc promoter recruitment. Our results indicate that Ras and c-Myc play important roles in the up-regulation of nucleophosmin/B23 during proliferation of cells associated with a high degree of malignancy, thus outlining a signaling cascade involving these factors in the cancer cells.

title: Induction of SPARC by VEGF in human vascular endothelial cells.
abstract: SPARC/osteonectin/BM-40 is a matricellular protein that is thought to be involved in angiogenesis and endothelial barrier function. Previously, we have detected high levels of SPARC expression in endothelial cells (ECs) adjacent to carcinomas of kidney and tongue. Although SPARC-derived peptide showed an angiogenic effect, intact SPARC itself inhibited the mitogenic activity of vascular endothelial growth factor (VEGF) for ECs by the inhibiting phosphorylation of flt-1 (VEGF receptor 1) and subsequent ERK activation. Thus, the role of SPARC in tumor angiogenesis, stimulation or inhibition, is still unclear. To clarify the role of SPARC in tumor growth and progression, we determined the effect of VEGF on the expression of SPARC in human microvascular EC line, HMEC-1, and human umbilical vein ECs. VEGF increased the levels of SPARC protein and steady-state levels of SPARC mRNA in serum-starved HMEC-1 cells. Inhibitors (SB202190 and SB203580) of p38, a mitogen-activated protein (MAP) kinase, attenuated VEGF-stimulated SPARC production in ECs. Since intact SPARC inhibits phosphorylation ERK MAP kinase in VEGF signaling, it was suggested that SPARC plays a dual role in the VEGF functions, tumor angiogenesis, and extravasation of tumors mediated by the increased permeability of endothelial barrier function.

title: Involvements of mitochondrial thioredoxin reductase (TrxR2) in cell proliferation.
abstract: Mammalian cells contain two forms of thioredoxin reductase (TrxR), cytosolic TrxR1 and mitochondrial TrxR2. To investigate the biological roles of TrxR2, we generated stable HeLa cell lines expressing a dominant negative form of TrxR2 (TrxR2DN) under the control of the tetracycline-off system. We observed that TrxR2DN-induced cells, following stimulation with EGF, produced more hydrogen peroxide than uninduced cells. The extent of protein tyrosine phosphorylation of many proteins including ERK was higher in TrxR2DN-induced cells than in uninduced cells when stimulated with fetal bovine serum or EGF. Induction of TrxR2DN also resulted in the increased rate of progression of G1 to S phase in cell cycle and cell proliferation and affected the expression of many proteins involved in cell cycle. These results suggest that TrxR2 participates in the regulation of protein tyrosine phosphorylation and cell growth as a component of the mitochondria specific H2O2-eliminating system that includes peroxiredoxin III and thioredoxin 2.

title: Regulated expression of galectin-1 during T-cell activation involves Lck and Fyn kinases and signaling through MEK1/ERK, p38 MAP kinase and p70S6 kinase
abstract: Recent evidence has implicated galectins and their carbohydrate ligands as novel regulators of T-cell homeostasis. Galectin-1 (Gal-1), a member of this family, inhibits clonal expansion, induces apoptosis of antigen-primed T lymphocytes and suppresses the development of T-cell-mediated autoimmune diseases in vivo. Because the β-galactoside-binding protein is expressed in activated but not resting T cells, it has been hypothesized that Gal-1-induced apoptosis may constitute an autocrine suicide mechanism to eliminate activated T cells contributing to the termination of an effector immune response. We undertook this study to investigate the signals and intracellular pathways leading to Gal-1 expression during T-cell activation. When T cells were stimulated either with anti-CD3 or anti-CD28 monoclonal antibody plus PMA in the presence of accessory cells, a sustained up-regulation of Gal-1 was observed, reaching a plateau between days 3 and 5 following CD3 engagement or costimulation through CD28. Investigation of the signal transduction events involved in this process revealed a role for Lck and Fyn kinases, since the Src kinase inhibitor PP1 inhibited the up-regulated expression of Gal-1 following T-cell activation. Downstream signaling routes involve mitogen-activated protein kinase (MAPK) kinase (MEK)1/extracellular signal-regulated kinase (ERK) and p38 MAPK, as Gal-1 expression was prevented by U0126 and SB202190. In addition, expression of Gal-1 involves interleukin (IL)-2-dependent signaling routes triggered by p70S6 kinase, as it could be inhibited by rapamycin. This is the first demonstration of the intracellular pathways that control activation-induced expression of Gal-1, which may reveal potential targets for immune intervention to modulate expression of this β-galactoside-binding protein in pathological disorders. ( 2004)

title: The ERK1/2 mitogen-activated protein kinase pathway as a master regulator of the G1- to S-phase transition
abstract: The Ras-dependent extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein (MAP) kinase pathway plays a central role in cell proliferation control. In normal cells, sustained activation of ERK1/ERK2 is necessary for G1- to S-phase progression and is associated with induction of positive regulators of the cell cycle and inactivation of antiproliferative genes. In cells expressing activated Ras or Raf mutants, hyperactivation of the ERK1/2 pathway elicits cell cycle arrest by inducing the accumulation of cyclin-dependent kinase inhibitors. In this review, we discuss the mechanisms by which activated ERK1/ERK2 regulate growth and cell cycle progression of mammalian somatic cells. We also highlight the findings obtained from gene disruption studies.

title: Mechanism of Suppression of the Raf/MEK/Extracellular Signal-Regulated Kinase Pathway by the Raf Kinase Inhibitor Protein
abstract: We have recently identified the Raf kinase inhibitor protein (RKIP) as a physiological endogenous inhibitor of the Raf-1/MEK/extracellular signal-regulated kinase (ERK) pathway. RKIP interfered with MEK phosphorylation and activation by Raf-1, resulting in the suppression of both Raf-1-induced transformation and AP-1-dependent transcription. Here we report the molecular mechanism of RKIP's inhibitory function. RKIP can form ternary complexes with Raf-1, MEK, and ERK. However, whereas MEK and ERK can simultaneously associate with RKIP, Raf-1 binding to RKIP and that of MEK are mutually exclusive. RKIP is able to dissociate a Raf-1–MEK complex and behaves as a competitive inhibitor of MEK phosphorylation. Mapping of the binding domains showed that MEK and Raf-1 bind to overlapping sites in RKIP, whereas MEK and RKIP associate with different domains in Raf-1, and Raf-1 and RKIP bind to different sites in MEK. Both the Raf-1 and the MEK binding sites in RKIP need to be destroyed in order to relieve RKIP-mediated suppression of the Raf-1/MEK/ERK pathway, indicating that binding of either Raf-1 or MEK is sufficient for inhibition. The properties of RKIP reveal the specific sequestration of interacting components as a novel motif in the cell's repertoire for the regulation of signaling pathways.

title: Reciprocal regulation of extracellular signal regulated kinase 1/2 and mitogen activated protein kinase phosphatase-3.
abstract: Mitogen activated protein kinase phosphatase-3 (MKP-3) is a putative tumor suppressor. When transiently overexpressed, MKP-3 dephosphorylates and inactivates extracellular signal regulated kinase (ERK) 1/2. Little is known about the roles of endogenous MKP-3, however. We previously showed that MKP-3 is upregulated in cell lines that express oncogenic Ras. Here we tested the roles of endogenous MKP-3 in modulating ERK1/2 under conditions of chronic stimulation of the Ras/Raf/MEK1/2/ERK1/2 pathway by expression of oncogenic Ras. We used two cell lines: H-ras MCF10A, breast epithelial cells engineered to express H-Ras, and DLD-1, colon cancer cells that express endogenous Ki-Ras. First, we found that MKP-3 acts in a negative feedback loop to suppress basal ERK1/2 when oncogenic Ras stimulates the Ras/Raf/MEK1/2/ERK1/2 cascade. ERK1/2 was required to maintain elevated MKP-3, indicative of a negative feedback loop. Accordingly, knockdown of MKP-3, via siRNA, increased ERK1/2 phosphorylation. Second, by using siRNA, we found that MKP-3 helps establish the sensitivity of ERK1/2 to extracellular activators by limiting the duration of ERK1/2 phosphorylation. Third, we found that the regulation of ERK1/2 by MKP-3 is countered by the complex regulation of MKP-3 by ERK1/2. Potent ERK1/2 activators stimulated the loss of MKP-3 within 30 min due to an ERK1/2-dependent decrease in MKP-3 protein stability. MKP-3 levels recovered within 120 min due to ERK1/2-dependent resynthesis. Preventing MKP-3 resynthesis, via siRNA, prolonged ERK1/2 phosphorylation. Altogether, these results suggest that under the pressure of oncogenic Ras expression, MKP-3 reins in ERK1/2 by serving in ERK1/2-dependent negative feedback pathways.

title: Regulation of the Raf–MEK–ERK pathway by protein phosphatase 5
abstract: The Raf–MEK–ERK pathway couples growth factor, mitogenic and extracellular matrix signals to cell fate decisions such as growth, proliferation, migration, differentiation and survival. Raf-1 is a direct effector of the Ras GTPase and is the initiating kinase in this signalling cascade. Although Raf-1 activation is well studied, little is known about how Raf-1 is inactivated. Here, we used a proteomic approach to identify molecules that may inactivate Raf-1 signalling. Protein phosphatase 5 (PP5) was identified as an inactivator that associates with Raf-1 on growth factor stimulation and selectively dephosphorylates an essential activating site, Ser 338. The PP5-mediated dephosphorylation of Ser 338 inhibited Raf-1 activity and downstream signalling to MEK, an effect that was prevented by phosphomimetic substitution of Ser 338, or by ablation of PP5 catalytic function. Furthermore, depletion of endogenous PP5 increased cellular phospho-Ser 338 levels. Our results suggest that PP5 is a physiological regulator of Raf-1 signalling pathways.

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

title: Bone responses to titanium implants surface-roughened by sandblasted and double etched treatments in a rabbit model.
abstract: The objective of this study was to investigate bone responses to titanium implants surface-roughened by sandblasted and double-etched treatments in a rabbit model.



Sixty implants of 10 mm in length (30 machined and 30 roughened) were inserted into femurs of 30 rabbits and 30 implants of 8 mm in length (15 machined and 15 roughened) were inserted into tibias of 15 rabbits. At 2, 4, and 8 weeks postimplantation, femurs and tibias were retrieved and prepared for removal torque tests (RTQ) and histomorphometric evaluation, respectively.



The roughened implants showed 66.21%, 89.06%, and 115.00% greater RTQ values than did the machined implants at 2, 4, and 8 weeks. Histomorphometric evaluation demonstrated the roughened implants significantly increased bone-implant contact and peri-implant bone formation during all observation periods.



These results suggest this surface-roughened approach provides the implant surface with a considerable osteoconductive potential promoting a high level of bone integration with bone.

title: Discrete deposition of hydroxyapatite nanoparticles on a titanium implant with predisposing substrate microtopography accelerated osseointegration
abstract: We report here a new versatile method to deposit discrete hydroxyapatite (HA) nanoparticles on a titanium (Ti) implant with predisposing substrate microtopography, which exhibited an unexpectedly robust biological effect. Commercially pure Ti substrates were treated with 3-aminopropyltriethoxysilane, on which HA nanoparticles (20 nm) were deposited and chemically bonded to TiO2. The HA deposition rate was linearly related to the treatment time and HA nanoparticles were deposited on up to 50% of the substrate surface. As a result, the discrete deposition of HA nanoparticles generated novel 20–40 nm nanotopography on the Ti substrate with microtopography that was smooth (turned) or roughened by double acid etching (DAE). The experimental implants with or without HA nanoparticles were surgically placed in rat femur and an implant push-in test was performed after two weeks of healing. The deposition of HA nanoparticles on the DAE surface increased the mechanical withstanding load by 129% and 782% as compared to the control DAE and turned implants, respectively. Micro-computed tomography-based 3D bone morphometry revealed equivalent bone volumes around the DAE implant with or without HA nanoparticles. These data suggest that the discrete deposition of HA nanoparticles accelerates the early osseointegration process, likely through increased shear bonding strengths.

title: Acid-etched microtexture for enhancement of bone growth into porous-coated implants.
abstract: We designed an in vivo study to determine if the superimposition of a microtexture on the surface of sintered titanium beads affected the extent of bone ingrowth. Cylindrical titanium intramedullary implants were coated with titanium beads to form a porous finish using commercial sintering techniques. A control group of implants was left in the as-sintered condition. The test group was etched in a boiling acidic solution to create an irregular surface over the entire porous coating. Six experimental dogs underwent simultaneous bilateral femoral intramedullary implantation of a control implant and an acid etched implant. At 12 weeks, the implants were harvested in situ and the femora processed for undecalcified, histological examination. Eight transverse serial sections for each implant were analysed by backscattered electron microscopy and the extent of bone ingrowth was quantified by computer-aided image analysis. The extent of bone ingrowth into the control implants was 15.8% while the extent of bone ingrowth into the etched implants was 25.3%, a difference of 60% that was statistically significant. These results are consistent with other research that documents the positive effect of microtextured surfaces on bone formation at an implant surface. The acid etching process developed for this study represents a simple method for enhancing the potential of commonly available porous coatings for biological fixation.

title: The removal torque of titanium screw inserted in rabbit tibia treated by dual acid etching.
abstract: Chemical acid etching alone of the titanium implant surface have the potential to greatly enhance osseointegration without adding particulate matter (e.g. TPS or hydroxyapatite) or embedding surface contaminants (e.g. grit particles). The aims of the present study were to evaluate any differences between the machined and dual acid etching implants with the removal torque as well as topographic analysis. A total of 40 custom-made, screw-shaped, commercially pure titanium implants with length of 5 mm and an outer diameter of 3.75 mm were divided into 4 groups, 10 screws in each, and chemical modification of the titanium implant surfaces were achieved using HF and HCl/H(2)SO(4) dual acid etching. The first exposure was to hydrofluoric acid and the second was to a combination of hydrochloric acid and sulfuric acid. The tibia metaphysics was exposed by incisions through the skin, fascia, and periosteum. One implant of each group was inserted in every rabbit, 2 in each proximal tibia metaphysics. Every rabbit received 3 implants with acid etched surfaces and 1 implant with a machined surface. Twelve weeks post-surgically, 7 rabbits were sacrificed, Subsequently, the leg was stabilized and the implant was removed under reverse torque rotation with a digital torque gauge (Mark-10 Corporation, USA) . Twelve weeks after implant placement, the removal torque mean values were the dual acid etched implants (24%HF+HCl/H(2)SO(4), group C) required a higher average force (34.7 Ncm), than the machined surface implants (group A) (p=0.045) (Mann-Whiteney test). Scanning electron micrographs of acid etching of the titanium surface created an even distribution of very small (1-2 microm) peaks and valleys, while machining of the titanium surface created typical microscopically grooved surface characteristics. Nonetheless, there was no difference in surface topography between each acid etched implant groups. Therefore, chemically acid etching implant surfaces have higher strengths of osseointegration than machined implant surfaces. There is less correlation between removal torque and the difference in HF volume%.

title: Investigation on plasma immersion ion implantation treated medical implants.
abstract: In this work the biocompatibility of osteosynsthesis plates treated with plasma immersion ion implantation (PIII) was tested using a rat model. Small rods (Ø 0.9 mm, and length 10 mm) prepared from different materials-pure Ti, anodised Ti, and two NiTi alloys (SE 508, and SM 495)-were implanted with oxygen by PIII to form a rutile surface layer and subsequently inserted into rat femurs, together with a control group of untreated samples. The results of the biomechanical tests correlate with the histological results, and show that plasma immersion ion implantation leads to an increase of biocompatibility and osseointegration of titanium and NiTi, albeit no improvement of the (bad) biocompatibility of the anodised Ti. Despite the layer thickness of up to 0.5 microm a strong influence of the base material is still present.

title: The impact of diamond nanocrystallinity on osteoblast functions.
abstract: Nanocrystalline diamond has been proposed as an anti-abrasive film on orthopedic implants. In this study, osteoblast (bone forming cells) functions including adhesion (up to 4h), proliferation (up to 5 days) and differentiation (up to 21 days) on different diamond film topographies were systematically investigated. In order to exclude interferences from changes in surface chemistry and wettability (energy), diamond films with nanometer and micron scale topographies were fabricated through microwave plasma enhanced chemical-vapor-deposition and hydrogen plasma treatment. Scanning electron microscopy (SEM), atomic force microscopy (AFM), Raman spectroscopy and water contact angle measurements verified the similar surface chemistry and wettability but varied topographies for all of the diamond films prepared on silicon in this study. Cytocompatibility assays demonstrated enhanced osteoblast functions (including adhesion, proliferation, intracellular protein synthesis, alkaline phosphatase activity and extracellular calcium deposition) on nanocrystalline diamond compared to submicron diamond grain size films for all time periods tested up to 21 days. An SEM study of osteoblast attachment helped to explain the topographical impact diamond had on osteoblast functions by showing altered filopodia extensions on the different diamond topographies. In summary, these results provided insights into understanding the role diamond nanotopography had on osteoblast interactions and more importantly, the application of diamond films to improve orthopedic implant lifetimes.

title: Induction and regulation of differentiation in neural stem cells on ultra-nanocrystalline diamond films.
abstract: The interaction of ultra-nanocrystalline diamond (UNCD) with neural stem cells (NSCs) has been studied in order to evaluate its potential as a biomaterial. Hydrogen-terminated UNCD (H-UNCD) films were compared with standard grade polystyrene in terms of their impact on the differentiation of NSCs. When NSCs were cultured on these substrates in medium supplemented with low concentration of serum and without any differentiating factors, H-UNCD films spontaneously induced neuronal differentiation on NSCs. By direct suppression of mitogen-activated protein kinase/extracellular signaling-regulated kinase1/2 (MAPK/Erk1/2) signaling pathway in NSCs using U0126, known to inhibit the activation of Erk1/2, we demonstrated that the enhancement of Erk1/2 pathway is one of the effects of H-UNCD-induced NSCs differentiation. Moreover, functional-blocking antibody directed against integrin beta1 subunit inhibited neuronal differentiation on H-UNCD films. This result demonstrated the involvement of integrin beta1 in H-UNCD-mediated neuronal differentiation. Mechanistic studies revealed the cell adhesion to H-UNCD films associated with focal adhesion kinase (Fak) and initiated MAPK/Erk1/2 signaling. Our study demonstrated that H-UNCD films-mediated NSCs differentiation involves fibronectin-integrin beta1 and Fak-MAPK/Erk signaling pathways in the absence of differentiation factors. These observations raise the potential for the use of UNCD as a biomaterial for central nervous system transplantation and tissue engineering.

title: Additive enhancement of implant fixation following combined treatment with rhTGF-beta2 and rhBMP-2 in a canine model.
abstract: Gaps at the interface between implant and bone increase the risk of diminished implant fixation and eventual loosening. The purpose of the present study was to determine if combined use of recombinant human transforming growth factor-beta 2 (rhTGF-beta2) and bone morphogenetic protein 2 (rhBMP-2) led to greater implant fixation strength in the presence of interface gaps than the use of either growth factor alone.



Twenty-eight skeletally mature adult male dogs received one porous-coated titanium implant in the proximal part of each humerus, for a total of fifty-six implantation sites. Spacers were used to establish an initial 3-mm gap between the implant and the host bone at all fifty-six sites. Forty-two implants were coated with hydroxyapatite-tricalcium phosphate and were used in three growth-factor-treatment groups in which the implants placed in the left humerus were loaded with 12 microg of rhTGF-beta2 (Group 1, seven animals), 25 microg of rhBMP-2 (Group 2, seven animals), or 12 microg of rhTGF-beta2 combined with 25 microg of rhBMP-2 (Group 3, seven animals). In these animals, the twenty-one implants that were placed in the right humerus were loaded with buffer only to serve as contralateral controls. In Group 4 (seven animals), the implants were not coated with hydroxyapatite-tricalcium phosphate, the gap in the left humerus was lightly packed with autogenous bone graft, and the gap in the right humerus was left empty to serve as a contralateral control. All animals were killed at twenty-eight days. The primary end points included three mechanical variables: fixation strength, interface stiffness, and energy to failure. Secondary end points included bone ingrowth and bone volume and trabecular architecture in the gap and in a region located 2 mm medial to the implantation site.



The hydroxyapatite-tricalcium phosphate coating had no effect on implant fixation, bone ingrowth, or bone formation in the 3-mm gap. Individual growth factor treatments led to 2.3 to 3.2-fold increases in fixation strength and stiffness as compared with the values for the contralateral controls (p < 0.05). The combined growth factor treatment led to 5.7-fold increases in fixation strength and stiffness compared with the values for the contralateral controls (p < 0.01). Autogenous bone graft treatment was associated with 4.5 to 6.4-fold increases in implant fixation strength and stiffness as compared with the values for the contralateral controls (p < 0.01). Compared with the relevant contralateral controls, energy to failure was increased 3.5-fold in association with TGF-beta2 alone (p < 0.05), 4.5-fold in association with TGF-beta2 combined with BMP-2 (p < 0.01), and 2.5-fold in association with autogenous bone-grafting. As much as 63% of the variance in the mechanical end points was associated with variance in bone volume and architecture in the 3-mm gap and in the region of interest located 2 mm medial to the implantation site (p < 0.01).



In this animal model, the combined use of TGF-beta2 and BMP-2 led to more secure mechanical fixation of the implant than did the use of either growth factor alone and demonstrated results that were similar to those associated with the use of autogenous bone graft.