Regulation of stem cell fate by nanomaterial substrates

2015 Stem cells are increasingly studied because of their potential to underpin a range of novel therapies, including regenerative strategies, cell type-specific therapy and tissue repair, among others. Bionanomaterials can mimic the stem cell environment and modulate stem cell differentiation and proliferation. New advances in these fields are presented in this review. This work highlights the importance of topography and elasticity of the nano-/micro-environment, or niche, for the initiation and induction of stem cell differentiation and proliferation.

Over the last few decades, biomaterial-based therapeutic approaches have been successfully employed in regenerative medicine for the repair of tissues [1][2][3][4][5].This novel approach creates new opportunities for stem cell-based regenerative therapies and the advancement of drug delivery and discovery [2,[6][7][8].Inevitably, numerous people lose a part or function of their organs and tissues due to a diverse range of diseases, birth defects or accidental trauma; thus, a tremendous clinical demand exists to promote the regeneration of injured/diseased tissues.Continuous advances in the field of cell and tissue engineering give scientists hope for future developments of implantable tissues, for example, for skin and cartilage, which have already been commercialized or possess high commercialization potential [9].Stem cells, including embryonic (ESCs), adult and induced pluripotent stem cells (iPSCs), are promising cell sources to underpin these novel therapies [10,11].One of the most challenging aspects of regenerative medicine, both in vitro and in vivo, is how to guide stem cell differentiation toward a specific desired lineage [12][13][14][15].In vivo, appropriate differentiation, proliferation and maintenance of potency are regulated by stem cells and their specific microenvironments (niches) [16][17][18].Biomaterials can mimic the niches of stem cells and spe-cifically effect the in vitro differentiation that is necessary for clinical application.Consequently, research efforts have been principally devoted to understanding how a wide range of well-recognized differentiation factors (e.g., growth factors, low molecular weight chemicals, extracellular matrix [ECM] components, cell shape, matrix stiffness and mechanical forces) contribute to the stem cell microenvironment [16,19-23].In this review, the topography that stem cells encounter will be a particular focus.The roles of niche components and architecture in regulating cell behaviors can be elucidated by simplifying the niche structure using well-defined synthetic microenvironments as artificial bioinspired models.It is possible to biomimic the 3D structures that support tissue growth and direct cell behavior through cell-ECM interactions by using natural or artificial polymeric matrices, as will be discussed [24,25].In this review, we highlight new advances in the bionanomaterials field, focusing on stem cell fate together with a perspective on future applications in the next generation of regenerative medicine.

Stem cells & their niches
A biological niche is an instructive multidimensional microenvironment that supplies both chemical and physical guidance to stem future science group Review Mashinchian, Turner, Dalby et al. cells growth and development (Figure 1).This environment must exhibit several anatomic and functional features.The niche should facilitate the differentiation or self-renewal of stem cells as required [26][27][28][29].Specific cues instruct stem cells to preserve or change their fate and to modulate their functions under diverse physiological conditions.Adult stem cell niches are composed of mixtures of extracellular cues that are largely produced by supporting cells and ECM, both of which the stem cells may adhere to [30].Several mammalian niches have been well characterized, such as those of the skin and intestine and to some extent the bone marrow [30][31][32].Close physical contact between supporting cells and stem cells can provide diverse biochemical and mechanical signals via soluble or membrane-bound factors.Similarly biochemical and mechanical signals can be conferred from bound or inherent factors of the protein and sugar rich ECM.In addition, stem cells are able to react with nervous system impulses inside the niche, suggesting circadian regulation [32,33].Stem cell metabolism also appears to be closely controlled by the niche; reactive oxygen species and ions are known to effect stem cell function [34] and metabolomically quiescent mesenchymal stem cells (MSCs) tend to pool unsaturated metabolites to allow redox plasticity upon differentiation [35,36].Our focus is not really on soluble factor regulation, although we acknowledge the important role of this regulation, for example, Wnt glycoproteins, hedgehog proteins and fibroblast growth factors [37][38][39].In addition, while we acknowledge the role of adhesive interactions via adhesion proteins located on the cell surface, which act as an anchor between support and stem cells (i.e., retaining stem cells in close proximity to self-renewal signals), it is not a main focus of our review (see Figure 1) [40,41].
Cell-substrate signaling at the nanoscale Cells communicate with the ECM through cell-adhesion structures, such as integrin proteins that cluster and combine with other proteins to form focal adhesions.Focal adhesions are large macromolecular structures responsible for transmitting mechanical forces to cells.These cell adhesion structures mediate cell signaling and affect cell shape, cell motility and cell-ECM attachment.They also act as trigger points for

Cytoskeleton
• Laminin • Fibronectin • Glycosaminoglycans and... Regulation of stem cell fate by nanomaterial substrates Review cascades of intracellular signaling pathways and affect cellular performance at many levels including cellular/molecular processes, stem cell-matrix interactions and differentiation [42].For example, the pathway of RhoA/Rho-associated kinase regulates focal adhesion formation and the cytoskeletal organization of cells [43].A number of molecular structures are known to be particularly important in stem cell responses to topography, including focal adhesion kinase, paxillin and talin, factors in the ECM (such as surface chemistry, lateral spacing and geometry of nanofeatures) and in the medium (such as the existence of nanoparticles [NPs]) [44].Therefore, bioinspired nanomaterials must be designed to mimic ECM structural complexity including specialized, textured topography (which as described previously are known to direct stem cell behaviors including maintenance of multipotency and differentiation).

Limitations for chemical factors
Although potent regulators of cell behavior (discussed further in the section 'Effects of chemical properties'), chemical factors are limited in that they are thought to be only one component of the 'triangle' of cell control, whereby in addition to chemical factors inducing stem cell differentiation inside the niche, topography and matrix elasticity are other chief factors influencing cell fate [45-50].Other important aspects that significantly influence the attachment of cells to biomaterials in vivo are surface moisture retention [51], material structure [52] and preparation methodologies [53].Feeder cells, which are obtained primarily from animal sources, are routinely used for the differentiation and maintenance of ESCs as they provide suitable chemical factors for stem cell maintenance.In addition to the risk of immune recognition, there is another prominent hazard due to the different sources of stem cells and feeder cells that can spread viruses and other unsafe infectious agents [54].In this context, accurate/safe controlling of the stem cells fate in the laboratory/clinical setting is a crucial issue and also alternative strategies besides a conventional method (e.g., growth factors) should be considered in order to completely induce the mature cells.

Synthetic substrates & artificial niches: the three generations of biomaterials
Hench and Polak define the evolution for biomaterials as first generation (structural, e.g., titanium orthopedic implants), second generation (bioactive, e.g., hydroxyapatite and bioglass) and third generation (reproducible molecular control of cells, e.g., nanotopography) [55].A third generation biomaterial designed for use in tissue engineering requires incorporation of multiple types of signals, which increases the complexity of the system and further complicates the interpretation of results.In this regard, cells are capable of complex tissue engineering, able, ultimately to form organs given the right cues.However, once the body is formed, the capacity of cells for self-renewal and regeneration is limited; many factors are responsible for healing, including supporting nutrition and environmentally optimized conditions.Thus, tissue engineering can be used to assist the body by various biomimetic strategies.A combination of cells, material(s) and physico-chemical factors have emerged as important in the tissue engineering field to assist the body in this healing process [56,57].Early studies focused on the nontoxicity of materials and the preservation of cell viability, whereas later studies have focused on cell functions, including proliferation, migration and expression.The physical and chemical properties of materials play an important role in regulating cell function and thus chemical characteristics and physical structures must be mimicked [58].
Both specialized (fully differentiated) and nonspecialized stem cells can be isolated from tissues in vitro using mechanical or enzymatic techniques.For tissue engineering to be a success, cells should be incorporated into an appropriate scaffold as soon as possible in order to retain their native features.The scaffold and culture medium must be precisely selected; in this way, chemical and physico-mechanical properties are mimetically correlated with the natural environment [59].For example, cartilage tissue (largely an avascular hydrogel) contains specialized cells, chondrocytes that are nourished by a synovial fluid flow of nutrients through it.Hydrogels from different materials, such as alginate, chitosan, polyvinyl alcohol and PEG, have been used to mimic this structure [60,61].Although preliminary observations showed that chondrocytes cultured in 3D hydrogels can keep some of their functional characteristics, chemical induction by TGF-β or physical stimulation by hydrostatic pressure enhance cellular responses [62].Thus, MSCs exposed to similar stimulation (chemical and/or physical) might be differentiated to exhibit the chondrocyte phenotype [63].In general, cells can lose their original natural phenotype after transportation and culture in vitro.The most prevalent containers for in vitro cell cultures are fabricated from hydrophobic transparent polystyrene, which has been used for many years for a range of cell types and is not designed specifically for stem cells.Because adherent cells attach loosely to the uncharged polystyrene plate, the modification of its surface by introducing carboxyl and amine functional groups is normally performed using, for example, plasma.The rigidity of this substrate is not ideal for stem cell growth, often resulting in random and spontaneous differentiation (e.g., MSCs differentiating into fibroblasts).In some cases, especially in ESC or keratinocyte cultures, supporting feeder layer cells must be used to provide biochemical signal cues.For example, a skin substitute developed by Genzyme (MA, USA; Epicel ® ) employs mouse fibroblasts as supporting cells for keratinocyte cultures.However, the separation of these two different cell types requires additional equipment and, from an immunological stand point, this strategy does not appear to be safe in clinical applications.Matrigel, a basal lamina ECM mix from murine origin, is now routinely used in stem cell culture; however, it is poorly defined and from an animal source.Although several protocols have been assessed for feeder-free culturing of diverse types of stem cells [64], the coculture procedure is sometimes utilized because it significantly improves the identification of what actually organizes a stem cell native niche [65].In this context, chemically crosslinked feeder cells (i.e., chemical fixation technique) have been fabricated as culture substrates for stem cells which could reduce significant time and effort in feeder cell preparation [66].Based on research carried out in 1995 [67], chemically cross-linked donor cells were employed in order to investigate the biological effects of membrane-anchored EGF on the acceptor cells.Further studies have revealed keratinocyte growthpromoting activity on glutaraldehyde-fixed fibroblast feeder cells [68].Accordingly, chemically cross-linked feeder cells can be utilized to maintain the undifferentiated state of stem cells.In this respect, chemically cross-linked human stromal cells have been applied as ex vivo supporter for expansion of the human cord blood hematopoietic progenitor cells [69].Moreover, the process of decellularizing tissues is another important consideration for tissue engineering, because it retains the physical complexity of the ECM thus presenting essential information to diverse cells.With regard to efficiency of decellularized ECMs on cell commitment, a specific decellularized ECM derived from mouse embryonic fibroblasts have been shown to maintain human ES cells in serum-free medium [70].Although many studies have been conducted recently, we are still far from suggesting an appropriate cell culture substrate for in vitro use.These results suggest that, biomimicked surfaces such as cell-formed ECM-derived substrates or decellularized ECMs, can be recognized as potential alternatives to maintain the undifferentiated state and/or enhance the differentiation induction of stem cells with a normal karyotype.

Effects of chemical properties
Chemically inductive molecules have been shown to control cell behavior, reprograming cells and directing their differentiation both in vitro and in vivo [71][72][73].These molecules can be incorporated into the scaffold structure or delivered to the substrate for retaining or reprograming cell fate [74].Other signals that are important in controlling cell behavior include metal ions, inorganic substances, organic molecules and other physicochemical factors such as temperature and pH.For example, hydroxyapatite is a well-known calcium phosphate derivative that naturally contains variable amounts of impurities that affect its physicochemical properties [75,76].Elements such as magnesium, fluoride or strontium can be introduced into hydroxyapatite, and their presence shown to influence cell behavior [77][78][79], for example, fluoride increases osteoblast proliferation and alkaline phosphatase secretion (which is an osteoblastic bone formation indicator) [80].It is thought that these ions may affect the protein adsorption profile and cell attachment properties of the hydroxyapatite [81].
Cell-matrix adhesions and interfacial interactions between cells and substrates are important for cell functions [82,83].Some of the most important molecules in natural ECMs are polypeptides (such as collagen, elastin, laminin and fibronectin) and polysaccharides, (including chondroitin sulfate, heparan sulfate and hyaluronic acid).Using these components in the fabrication of artificial ECMs is a very promising method to mimic the biochemical properties of the natural ECM.Although these strategies may not preserve the biochemical activity of pure ECM components, promising results in different cases have been reported [10,[84][85][86].Other natural or artificial polymers, such as silk, spider silk [87], chitosan or polyurethanes, may be used to regulate stem cell behavior.For instance, a blend composed of silk and cellulose (25:75) can induce chondrogenesis in MSCs [88].Polymer networks composed of silk fibroin for wound dressing applications showed noncytotoxic effects when evaluated by cell proliferation methods [89] and specifically, they promoted the maintenance of soft tissue in vivo by providing longer-term structural integrity [90].Several peptide sequences, such as RGD, IKVAV and YIGSR, found in ECM proteins are recognized as cell mediators; surfaces can, for example, be chemically modified with RGD to enhance cell attachment or alter cell morphology in addition to promoting osteogenesis or chondrogenesis [14,91].These peptides can directly regulate stem cell functions through interactions between adhesions molecules such as integrins on the cell surface and ligands on ECM molecules [92].
Surface modification of cell culture substrates can alter cell attachment, morphology, proliferation and gene expression.For example, differences in cell morphology and spatial distribution of fibronectin or vitrofuture science group Regulation of stem cell fate by nanomaterial substrates Review nectin were reported on glass substrates modified with carboxyl and amine functional groups [93].Curran et al. [94] developed silane-modified surfaces with different functional groups, including methyl (-CH 3 ), amino (-NH 2 ), silane (-SH), hydroxyl (-OH) and carboxyl (-COOH), to evaluate MSC differentiation.Osteogenicity on amine and silane-modified surfaces and chondrogenicity on hydroxyl and carboxyl-modified surfaces were reported, whereas the MSC phenotype was maintained on methyl-modified surfaces.Carboxylic acid functional groups abundant in cartilage glycosaminoglycan, phosphate groups prevalent in mineralized bone and hydrophobic groups such as t-butyl that are found in lipids were, respectively, chosen to induce chondrogenesis, osteogenesis and adipogenesis in functionalized PEG encapsulated MSCs, respectively [95].The differentiation of bone marrow derived MSCs was investigated on a variety of silane modified surfaces in vitro [96].
Additionally, the surface hydrophobic/hydrophilic properties of substrates play an important role on the kinetics of protein adsorption and their folded conformation, which in turn could influence cell activities [97,98].Interestingly, the hydrophilic properties of the substrate can also have an impact on apoptosis, while a reduced expression of proinflammatory cytokines have been demonstrated on hydrophilic surfaces [99].
A recent study has demonstrated that small changes in matrix hydrophobicity, a result of adding or removing of CH 2 groups, can modify cell-matrix interactions and subsequently have a profound influence on various cellular behaviors (e.g., adhesion, motility, shape, cytoskeletal organization and differentiation fate) [100].As a consequence, it can be concluded that the chemical aspect of cell/stem cell-material interactions is a crucial direction for biological studies and applications.Additionally, surface chemistry analysis and chemical modification processes should be considered in order to design 3D functional biointerfaces that appropriately control different biological phenomena (e.g., cell adhesion, migration, recognition and uptake, among others).

Effects of physical & mechanical properties
Considered to be a supporting structure, in addition to chemical cues the ECM provides physical and mechanical signals for cell functions, including adhesion, migration and differentiation [21,101,102].ECM physical cues can be mimicked by using, for example, porous and/or fibrillar structures with dimensions similar to the natural ECM.For example, the potential of bioinspired nanofibers based on polyvinyl alcohol-chondroitin sulfate for the differentiation of MSCs has been recently investigated [103].Proteins are adsorbed to these fibrous structures in a different profile compared with nonfibrous substrates [104].As a result, the attachment, spreading and migration of cells were altered, demonstrating how the physical architecture of fibrous meshes, such as their diameter or porosity (or bead formation), affect protein adsorption and cell fate [105].Studies show that different distributions of fiber diameters in tissues containing fibrils are related to the ages of those tissues or the types of mechanical loading [106,107].For example, characterization of the fibrous structure of aortic heart valve leaflets showed regional differences in the hinge (rectilinear patterns) and belly (radially oriented striped pattern) parts [108] and Abrams et al. [109] showed that fibrous features on the human corneal basement membrane varied from 22 to 191 nm in diameter.A technique that can be used to fabricate fibers is electrospinning; however, its application is limited due to several drawbacks, including: denaturation of natural proteins (e.g., collagen) by organic solvents (e.g., hexafluoro-2-propanol) during electrospinning [110] and the formation of fibers with larger diameters than native tissues, which also limit porosity [111].Scaffold porosity is considered to be another important physical cue that can be adjusted to resemble the natural ECM.For example, accordion honeycomb scaffolds were fabricated with varying anisotropy for myocardial repair applications [112].
It is desirable to produce substrates with mechanical properties that mimic those of the ECM.However, due to the heterogeneity of mechanical properties between species, tissues and even tissue ages in the same person, it is extremely difficult to predict the viscoelasticity of the cellular environment.Preliminary studies (in the 1920s) confirmed that cells cultured in clots with different stiffnesses showed dissimilar shapes [113].It is now commonly accepted that cells sense and respond to the stiffness of their environment [114,115].Substrate mechanical properties are important for the regulation of cell shape, growth and even cell survival [116,117].Reduced cell spreading and higher motility rates have been observed in cells cultured on flexible substrates compared with rigid ones [118].Stroka et al. [119] showed that the motility of neutrophils decreased with increasing surface rigidity due to the stronger attachments formed.It can be concluded that matrix elasticity can initially trigger the induction of differentiation in stem cells but is likely to be insufficient to complete terminal differentiation [120].Trapmann et al. [12] cultured stem cells on polydimethylsiloxane and polyacrylamide hydrogel surfaces with different stiffnesses (0.1 kPa-2.3MPa).They found that cell spreading and differentiation was modulated by the elastic modulus of polyacrylamide.Reprinted with permission from [130] © American Chemical Society (2009).

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Although the outcome of recent studies clarifies the ability of elasticity to affect cell differentiation, more studies on chemically similar structures with different elasticities must be performed.Other research has shown that myogenic markers of MSCs were upregulated when cells were grown on softer gels coated with type I collagen that simulated muscle elasticity; conversely, stem cells showed osteogenic differentiation when grown on more rigid gels [19].It should be mentioned that polydimethylsiloxane is broadly known as a suitable substrate for cell growth and proliferation, due to its biocompatibility, mechanical stability and also nontoxicity [121][122][123].This influence of the hardness or softness of matrices has also been shown to affect the differentiation of neuronal stem cells [124].
Blau et al. fabricated hydrogels with different rigidities (2, 12 and 42 kPa) by altering the amount of PEG in the hydrogel structure.The self-renewal potential of muscle stem cells in vitro depended on the elasticity of the substrate, and these cells retained their regenerative potential after transplantation [125].In addition to MSCs, ESC differentiation can be influenced depending on how tight or loose adhesions are, which in turn dependent on surface stiffness.ESCs were found to respond to stiffness on polyelectrolyte films; poly(l-lysine) and hyaluronan multilayered native films reduced mouse ESC (mESC) proliferation in comparison to highly crosslinked nanofilms; however, the surface stiffness of these nanomaterials did not affect the expression of mESC markers, such as Nanog, Sox2 and Oct-4.A low proliferation rate of mESCs has been demonstrated on less stiff polydimethylsiloxane substrates.In addition to the varying expression levels of self-renewal markers, the expression levels of mesodermal markers, such as brachyury and goosecoid, were found to be enhanced on nanofilm substrates [126].
The mechanical profile of bone is well known as an important factor in bone/cartilage tissue engineering.Because of the exceptional mechanical strength of carbon nanotubes and carbon nanofibers, they can be utilized as reinforcing agents in composite materials/scaffolds [127,128].In one study, biodegradable nanocomposites reinforced by single-wall carbon nanotubes were reported to be lighter, less dense and much stronger than metallic-based bone substitutes, for example, the titanium and stainless steel that are typically used in orthopedics [129].In another study, laminin-single-wall carbon nanotubes films were shown to have a potential effect on the growth/proliferation of neural stem cells.These thin composite films can serve as biological substrates for promoting cellular adhesion and differentiation (Figure 2) [130].A recent article by Pulskamp et al. [131] demonstrated that human cells treated with commercial carbon nanotube (CNT) products revealed no acute toxicity on viability, which was performed with different assays (WST-1, PI-staining) and also, none of them induced inflammatory mediators.On the other side, some reports showed inconsistency between research findings on the effects of both refined and raw CNTs on mice lungs [132,133].These findings illustrate the difficulty of assessing toxicity of CNTs due to their agglomerative features in aqueous solutions and consequently, mechanical blockage of airways is the main result of mortality [134].
From another point of view, mechanical properties are thought to be important in cancer research; studies have been reported that tumors are stiffer than normal tissue and may be detected by physical palpation [135].This rigidity is reported to have an inductive effect on the invasiveness of tumor cells [136].Although the physical mechanisms employed by metastatic cancer cells in their functions (migration) and gene expression are poorly understood, studies in this field can be helpful for the development of new therapies that target tumor ECM and for understanding how normal tissue differentiation is achieved [135,137,138].
The lack of appropriate 3D models is a hindrance in tissue engineering, largely a result of 2D in vitro culture methods providing different conditions for cells in comparison to in vivo conditions.An increased number of actin-myosin fibers as a result of cell culture on tissue culture polystyrene has been reported by Discher et al. [139].Alternatives to 2D culture systems, which more closely resemble the nat- Regulation of stem cell fate by nanomaterial substrates Review ural 3D architecture of tissues are being sought.For example, a simple protocol was proposed by Fischer et al. [140] to produce 3D structures with stiffness similar to the ECM, in which the cells are sandwiched between a 3D fibrillar ECM and a polyacrylamide gel [141].In summary, physico-mechanical properties such as stiffness, elastic and viscous moduli should be precisely tuned to entirely mimic biological tissues.
In particular, in native structures (i.e., surrounding matrix) some important biological variables could be affected by the complexities of weak or strong cues such as: confinement/nonconfinement of the environment, softness and/or rigidity of the ECM and also continuity/discontinuity of the targeted ECM.

Effects of topography
More recently the effects of surface topography on cell behaviors have been investigated [142-148].With reference to a comprehensive review by Langer et al., [149] topographies can be used as important signaling modalities to control cell fate and can be designed to mimic the structure of natural ECM (see Figure 3).
Substrates with microscale patterning of greater than 100 μm can be utilized for colony shape-induced differentiation [150].Khademhosseini et al. [151] manufactured concave microwell arrays with widths between 200 and 1000 μm, with greater neuronal and cardiomyocyte differentiation observed in ESC colonies grown on larger microwells.Other reports also suggest that the differentiation of embryonic stem cells can be regulated by the size of the suspended aggregates (i.e., embryoid bodies) [152].For example, the gene and protein expression levels of ectodermal markers were increased in aggregates with an initial microwell size of 100 μm in comparison to 500 μm, in which higher levels of mesodermal and endodermal markers were observed [152].When the aggregate size was constrained to 200 μm, cardiac differentiation was enhanced [153].Tissue culture plates could be produced with these well-like topographies to control the proliferation or differentiation of ESCs.Features with dimensions greater than 100 μm are well suited for aggregations, whereas single cells can sense smaller micro-or nanoscale topographies and respond to the shape (ridge, groove and pillar) of surface features.
Surface roughness is known to impact cell behaviors.Studies on silicon substrates with different roughness and similar surface energies demonstrated that moderately rough substrates could boost cell proliferation [154].Vandrovcova et al. [155] reported a larger spreading area and a reduced rate of proliferation for osteosarcoma cells cultured on TiO 2 films with increased surface roughness.In addition, collagen accumulation and/or related gene expres-sion were positively modulated in MSCs cultured on a titanium rough surface [156].In another study, enhancement in the activation of Wnt/β-catenin signaling in cells seeded on titanium surfaces (i.e., rough surface topography) was observed [157].Human ESCs (hESCs) have been observed to exhibit spontaneous differentiation on vitronectin-coated nanorough glass surfaces.In this experiment, hESCs that adhered selectively to smooth surface fragments showed high expression levels of OCT-4 (a selfrenewal marker) with enhanced proliferation rates, whereas the nanorough surfaces tended to induce hESCs to spontaneously differentiate.A mouse embryonic fibroblast-conditioned media supplemented with a specific synthetic polymer coating of poly(2-(methacryloyloxy)-ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide) (PMEDSAH) could maintain the proliferation/pluripotency of hESCs when cultured on roughened surfaces (Figure 4) [20].
Cells can be embedded in structures formed by micron scaled columns, pits, grooves or pillars, and their function changes according to these topographic configurations.Migration is also effected by micron scale topographies, for example the migration rate of fibroblasts was increased when cultured on a microscale pillar patterned structure compared with a flat surface [158].It was proposed that the strength of local adhesion and contraction was regulated by increasing the sur- Reprinted with permission from [20] © American Chemical Society (2012).
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face contact area [158].Nanoscaled topographies have also been shown to regulate cell behaviors, for example, a wide range of cell types, including nerve cells and MSCs, respond to both nano-and micro-scaled grooved substrates by elongating in the direction of the groove [159].Alterations in focal adhesion molecules and cytoskeletal organization of cells cultured on different nanotopographies has been observed by Yim et al. [160].Bucaro et al. [161] reported altered mouse ESC morphology when cultured on different patterned silicon nanopillars (diameter of 400 nm, length of 5 μm).The cells showed spindle-like morphology on surfaces with lower interpillar spacing (0.8-1.25 μm) but appeared spherical on surfaces with higher spacing (1.5-2.5 μm).Improved alignment and elongation of hESCs when cultured on poly(dimethyl siloxane) substrates with nanoscale line-gratings further confirm the effects (i.e., geometry and dimensions) of surface topography on ESC behaviors [162].Additionally, vertically aligned TiO 2 nanotubes with diameters of 15 nm are able to increase MSC differentiation into the osteogenic lineage (see Figure 5) [163].In human MSCs, 100 nm diameter TiO 2 nanotubes were seen to be more osteoinductive [164].In the presence of osteogenic supplements in the medium, mouse ESCs cultured on randomly oriented fibrous scaffolds showed higher degrees of osteogenic differentiation compared with flat films [165].Electrospun nanofibers (both random and aligned) displayed an inductive potential for stem cells to differentiate into specific neural lineages and initiate neurite outgrowth [166,167].For instance, multiwalled carbon nanofibers can induce osteoblast proliferation compared with flat glass surfaces [168].Additionally, these nanofibers can increase alkaline phosphatase activity, indicating osteoblastic bone formation [169].
As a concluding point, the biological response of stem cells to nanotopographical stimuli is the result of complex mechanotransduction pathways that accommodate new loading settings from the environment.Mechanotransduction is a specific process whereby physical cues, such as topography and rigidity, exert forces on cells that can alter biochemical signaling and also induce adaptive cellular functional changes.In this respect, some cues that originated from cell-substrate interactions can directly transduce to the nucleus which in turn may effect the cell phenotype.For instance, it has been demonstrated that β-catenin signaling is involved in topography induced mechanotransduction [170].These findings further define 'complicated topography' as a new benchmark for optimizing the growth of stem cell-based engineered tissues [171].
Nanoscale engineering: toward niche manipulation ECM elements of the niche have diverse but specific properties, for example, surface chemistry and  nanopits can maintain MSC multipotency [36,172].However, add in just a small amount of disorder to the pattern and MSCs are stimulated to form osteoblasts [173].ESCs have been seen to respond to similar disordered nanopits by differentiating along the mesodermal lineage [174]; they have also been shown to have enhanced self-renewal on highly ordered surfaces [175] and other reports illustrate similar findings [20,176].Artificial nanoscale banding patterns have been shown as stimulatory; MSCs cultured on nanohelices with a band pattern similar to collagen (∼67 nm) form osteoblasts; however, if banding periodicity is increased to a less physiologically relevant approximately 100 nm, this is not observed [177].
These examples illustrate how both embryonic and adult stem cells show unique differentiation behavior in response to nanoscale features.We note that proteins adsorb to materials, both in vivo and in vitro, and that this is important as cells do not interact directly with materials [178].Materials can be used to organize the nanonetworks of proteins, for example, to enhance cell binding through opening of fibronectin binding sites [179], and this can cause changes in cell response [180].
In a different kind of study of this phenomenon, the aggregation of embryoid bodies was observed with no adverse effects after the penetration and accumulation of poly(lactic-co-glycolic acid) NPs in human ESC colonies [181].In another report, the proteins fibronectin and E-cadherin-Fc were embedded in carbonate apatite NPs to accelerate transgene expression in ESCs upon nonviral transgene delivery [182,183].Biodegradable NPs were used to increase transfection efficiency of plasmid DNA incorporated in poly(β-amino esters) and were shown to maintain hESC viability and pluripotency following transfection [184].More recently, polyamidoamine dendrimermodified magnetic NPs have been demonstrated to increase the delivery of lentivirus expression plasmids for reprograming of human dermal fibroblasts to iPSCs [185].
A commercial 3D nanofibrillar scaffold (applied on coverslips) called Ultra-Web has been shown to produce larger mESC colonies with enhanced proliferation characteristics.This is important because developing artificial proliferation-promoting scaffolds is one of the key strategies to providing nanomaterialniche composites.The Ultra-web nanofibers were prepared to reconstruct the fibrous network of the ECM, which improves stem cell attachment and transplantation efficiency [186].Numerous features make nanofibrillar constructs distinctive, such as large surface area, tailored chemistry and the high density of epitopes achievable [187].Consequently, the chemical, mechanical and 3D properties of these fibrous scaffolds may be able to influence the activation of different signaling pathways and control cell fate and differentiation.

Effects of topography on protein attachment
During in vitro culture, cells are suspended in a liquid medium containing serum proteins.Most cells require a few hours for attachment to the substrate and this attachment is affected by serum protein adsorption to the substrate.It seems that surface topography indirectly influences the composition, orientation or conformation of adsorbed serum proteins and thus can also influence cell adhesion.The exact mechanisms and general trends of protein activity and adsorption on such surface features are not well understood [188].Probably because of the larger number of possible adsorption-promoting interactions, surfaces with a hydrophobic nature are more frequently used in protein adsorption studies compared with hydrophilic surfaces [189,190].For instance, albumin protein strongly adsorbs on hydrophobic self-assembled monolayers [191].Most in vitro studies determined a favorable cellular response to charged and hydrophilic surfaces [178].It should be noted that some studies report no alteration in protein adsorption on different surface topographies [192].Table 1 provides a brief summary of reported alterations in protein adsorption according to topographical features [188].

Synthetic nanoenvironments & artificial niches
Advanced nanomaterials with nanospecific features, such as nanofilms, nanotubes, nanofibers and bioscaffolds, can be produced by a range of techniques, for example, chemical/physical/electrochemical vapor deposition, electrospinning, electron-beam lithography, dip-pen nanolithography and electrohydrodynamic lithography [42,[201][202][203].As previously described, nanotechnological approaches can provide surfaces that mimic natural ECM features, which are also on the nanometer scale.This approach has specific potential for the manufacture of an environment for stem cells and adult cells.For example, graphenecoated glass surfaces can restrict the loss of Nanog and Oct4 expression in mouse iPSCs without the need for any chemical reagents such as leukemia inhibitory factor (LIF) [204].Recently, Mahmoudi et al. [22] fabricated smart nanoenvironments using soft lithography, whereby in vitro cell cultures were used as templates for the transfer of cell-imprinted patterns into artificial polymer substrates.The imprinted substrates were used to induce stem cell differentiation toward future science group Regulation of stem cell fate by nanomaterial substrates Review desired matured cell types, which had themselves been used as templates for the cell-imprinting.These results suggest that the dynamic plasma membrane of cultivated stem cells is capable of adopting the shape of matured cells imprinted in artificial polymer substrates [205].

Conclusion & future perspective
Despite extensive ongoing studies to design biomimetic materials for regenerative medicine, few biofunctionalized biomaterials have been successfully translated into the clinical setting.The complete mechanisms governing the regulation of stem cell differentiation into specific desired lineages remain unclear due to the difficulty of reproducing a complicated biological microenvironment and the regulatory factors of tissue repair.In this regard, stem cell culture materials need to be equipped with physicochemical inductive cues to completely control the diverse phases of healing.Many innovative efforts have been made to enhance the efficacy of products for tissue regeneration.However, the lack of success thus far emphasizes the importance of engineered biomaterials to provide numerous signals in concert.These 'bioinspired' platforms can be effectively tuned to direct stem cell fate according to a defined application.Continued advances in the design of sophisticated devices will lead to improvements in patient care and quality of life.Therefore, in the future, highly integrated nano/microdevices will find increasing use in biomedical and pharmaceutical activities.

Protein effect
Ref.

7-11
Less fibrinogen adsorption on 11-nm rough surfaces compared with other surfaces Fibronectin conformation changed on nanorough surfaces compared with flat [194] Polymer de-mixing, alumina 32 Increased unfolding of vitronectin compared with conventional alumina (mean roughness 16 nm) [195] Particles Silica Diameter less than 20 Lysozyme adsorbed in a native conformation [196] Grooves Electron beam lithography, silicon Spacing 90 Fibronectin conformation altered on grooved substrates as determined by reduced osteoblast adhesion compared with planar controls [197] Pits Colloidal lithography, titanium Diameter 40, depth 10 Fibrinogen conformation altered on nanopits as determined by platelet adhesion [198] Nanoporous surface β-type Ti-25Nb-25Zr alloy Pore size <15 Surface nanotopography did not alter the surface roughness or hydrophilicity of the Ti25Nb25Zr alloy but was capable of inducing biological responses, such as, protein adsorption, cell adhesion, cell migration, cell proliferation and cell mineralization [199] Average surface roughness Solution casting, β-phase PVDF 50-300 Increased fibronectin adsorption toward the cell-material interface was demonstrated on β-phase PVDF films [200] PVDF: Poly(vinylidene fluoride).Reprinted with permission from [188] © Elsevier (2010).
future science group Review Mashinchian, Turner, Dalby et al.

Stem cells & their niches
• The native biological stem cell niche is a multidimensional nano-to micro-scale environment, which supplies both chemical and physical guidance to control stem cell quiescence, proliferation and development.• Different cues instruct stem cells under diverse physiological environments.
Cell-substrate signaling at the nanoscale • Cell adhesions transmit external mechanical forces intracellularly and potentially mediate cell signaling effecting behaviors such as cell shape, motility and differentiation.• One type of cell-substrate adhesion is the focal adhesion; FAK, paxillin and talin are important components and play a role in stem cell responses to substrate topography.

Limitations for chemical factors
• In vivo translation of chemically controlled cell behaviors may present problems, for example, feeder cells can spread viruses and other unsafe infectious agents to the culture.• Topography and elasticity are other principal factors influencing stem cell fate, making a 'triangle' of control.

Synthetic substrates & artificial niches: the three generations of biomaterials
• The evolution of biomaterials has been described as falling into three different generations: first generation (structural), second generation (bioactive) and third generation (reproducible molecular control of cells).• A combination of cells, material(s) and physico-chemical factors have emerged in the tissue engineering field as crucial in the bodys healing process.Thus the chemical and/or physico-mechanical properties of synthetic substrates should be mimetically correlated with the native environment of the cell.

The 'triangle' of control: effects of chemical properties
• Chemically inductive molecules have been shown to regulate cell behavior, inducing their differentiation and could affect the reprogramming process.• Other important signals in controlling the cell behavior include: metal ions, inorganic substances, organic molecules, temperature and pH.• Modified surfaces on cell culture substrates can affect cell attachment, morphology and proliferation (e.g., substrates modified with carboxyl and amine functional groups have altered the spatial distribution of fibronectin or vitronectin).• Cell-matrix adhesions and interfacial interactions between cells and natural/synthetic substrates are important factors in cell function.

The 'triangle' of control: effects of physical & mechanical properties
• Extracellular matrix (ECM) physical and mechanical cues can be imitated using porous and/or fibrillar structures with dimensions similar to the natural ECM.• Physical architecture of fibrous meshes (e.g., diameter or porosity) can effect protein adsorption and cellular fate.• Scaffold porosity is considered to be an important physical cue which can be adjusted to mimic the natural ECM.• One well-known example where the mechanical profile of the tissue is known to be important is in bone/cartilage tissue engineering.

The 'triangle' of control: effects of topography
• Topography can be used as a signaling modality in order to control cell fate.
• It has been demonstrated that moderately rough silicon substrates can boost cell proliferation.
• Migration can be stimulated by micron scale topographies.
• A wide range of cell types, such as nerve cells and mesenchymal stem cells, respond to nano-to-micro-scaled grooved substrates by extending in the direction of the groove.

Nanoscale engineering: toward niche manipulation
• Biomaterials can be manipulated in order to organize the nanonetworks of diverse proteins, for example, to improve cell binding through opening of fibronectin binding sites thus altering cell responses to the substrate.

Effects of topography on protein attachment
• Surface topography can influence cell adhesion by indirectly affecting the composition, orientation and conformation of adsorbed serum or plasma proteins.

Synthetic nanoenvironments & artificial niches
• Nanomaterials with nanospecific features (e.g., nanofilms, nanotubes, nanofibers and bioscaffolds) can be fabricated by a range of methodologies such as lithography-based approaches, chemical/physical/ electrochemical vapor deposition and electrospinning.
future science group Regulation of stem cell fate by nanomaterial substrates Review

Figure 1 .
Figure 1.Schematic illustration of the stem cell niche.Adult stem cells are contained within a 3D microenvironment comprised of complex mixtures of extracellular cues (biochemical, physical and mechanical)(A-C).The niche includes, amongst other components, cell-cell adhesion proteins, soluble factors and extracellular matrix.For color figures, please see online at www.futuremedicine.com/doi/full/10.2217/NNM.14.225 future science group Review Mashinchian, Turner, Dalby et al.

Figure 2 .
Figure 2. Spontaneously differentiated neural stem cells analyzed by immunostaining on heat-treated single-walled carbon nanotube/laminin thin films.Blue color highlights nuclei.(A) The presence of neurons (GFAP staining, red; MAP-2 staining, green) in this image provides clues as to which successful neuronal connections could be formed between cells grown on the carbon nanotube substrates and the surrounding tissue.(B) GFAP staining, red; nestin staining, green.(C) Synapsin staining, red.Synapsin is present in differentiated cells; therefore, its presence confirms that neuronal cell growth and differentiation on the SWNT/laminin thin films results in functional neuronal networks.Scale bars: 2 μm.Reprinted with permission from[130] © American Chemical Society (2009).

Figure 5 .
Figure 5. Focal contact formation, apoptosis, and differentiation of mesenchymal stem cells on 15-and 100-nm nanotubes.(A) Focal contact formation and stress fiber assembly was apparent 1 and 3 days after plating on 15-nm nanotubes (A & C) but was reduced on 100-nm nanotubes (B & D).Antipaxillin staining in red: (A-D); antiactin staining in green, and DAPI nuclear staining in blue.(B) Scanning electron microscope micrographs (immunogold staining with paxillin and β1-integrin antibodies) were used for analyzing the focal contacts, which revealed dense packing of paxillin in focal contacts on 15-nm tubes.(C) Analysis of FAK and ERK phosphorylation confirmed the high extent of focal contact signaling on nanotubes smaller than 30-nm compared with 100-nm nanotubes.(D) Osteogenic differentiation occurred on 15-nm nanotubes as observed by osteocalcin staining (red, upper panels) but was infrequently detectable on 100-nm nanotubes.(E) Hypothetical model demonstrating the lateral spacing of focal contacts on nanotubes with different diameters.A 15-nm spacing was optimal for integrin assembly into focal contacts, inducing the assembly of actin filaments and signaling to the nucleus, but diameters greater than 70 nm could not support focal contact formation, resulting in apoptosis.Reprinted with permission from [163] © American Chemical Society (2007).
Regulation of stem cell fate by nanomaterial substrates Review topography with nanoscale features that present cues to guide cell behavior/fate[8,28].Nanotopographical features inside the niche (e.g., pores, whorls, pits, ridges and grooves) and their symmetry are important factors affecting the differentiation of stem cells toward specific lineages.For instance, highly ordered future science group Review Mashinchian, Turner, Dalby et al.

Financial & competing interests
disclosure L-A Turner and MJ Dalby are funded by the BBSRC and BBSRC, MRC and EPSRC, respectively.The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.

Table 1 .
A brief list of reports describing the effects of different topographical features on protein adsorption.