Nanofabrication: a cue for cellular differentiation

Abstract Introduction To understand the cellular responses towards its environment regarding mechanical force and topography of its environment, new strategies are being formulated using biomaterials at nanoscale level, providing cellularspecific scaffolding to match differentiation. This review mainly discusses stem cell biology. Conclusion As our understanding of the molecular and whole-cell responses of stem cells to topography increases, there will be enormous scope for the creation of next-generation materials possessing defined features to tailor stem cell fate and functioning to specific laboratory, industrial and therapeutic applications.


Introduction
Cells of all types take cues from the surfaces they encounter in their environment.The cells extract any or all of the mechanical, chemical, spatial and even temporal information from the surface, but which features they read and how they integrate and translate them into specific behavioural responses is still almost a black box.Cellular behaviours, including growth, survival, migration and differentiation, are regulated by the complex interplay between cells and their environment.For the rational design of functional artificial tissue or organ, it is vital to understand the mechanism of biomaterial-cell interaction and the possibility of manipu-Nanofabrication: a cue for cellular differentiation D Nath * lating these implants.Increasing evidence proved that adhesionrelated non-chemical signals such as mechanical force and topography can play an equally important and complementary role like chemical signals.Current efforts are focused on scrutinising the influence of various geometries and densities of ordered nanoparticle arrays on the morphology of pluripotent cells and lineage specification of human mesenchymal stem cells (MSCs).The development of 'smart' materials and their routine application in medical devices will benefit greatly from the identification of topographies that elicit specific cellular responses, understanding how cells interpret these topographic cues and designing new strategies to couple topography with chemical, mechanical and other vital cell stimuli 1,2 .Nanotopographical surface affects cellular behaviour in a wide range of cell types including epithelial cells, fibroblasts, myocytes and osteoblasts 3 .An interesting feature of nanoscale topographic surfaces is the selectivity for cell adhesion.Several investigators have demonstrated the relative diminution of fibroblast adhesion compared withosteoblast adhesion when nano-and microstructured surfaces were evaluated 4,5 .However, until recently, no attempt has been made to utilise a systemic nanoscale environment to investigate the optimum range of nanostructure for cell growth.In this review, the discussion has been focused on the stem cell biology because they play critical roles in the tissues establishment, regeneration and replacement due to their unique capability of both differentiation and self-renewal, and they are the leading candidates for tissue engineering research and regenerative medicine, covering a wide range of therapeutic areas.

Stem cell niche-model for cellular differentiation with variable potency
Isolated from a variety of embryonic, foetal and adult tissues, stem cell populations have the advantage of being significantly proliferative and, therefore, could be extremely efficient in the treatment of presently incurable diseases in the near future.Moreover, their unique characteristics related to the differentiation, regeneration, development, remodelling and replenishment of aged and diseased tissues make them perfect candidates in this area.In a very easy and simple way, stem cells can be conceptually divided into two types: embryonic stem cells (ESCs)derived from a very early embryoand adult stem cells-found in postnatal tissues, of the body (bone marrow (BM), adipose tissue, etc.) and the umbilical cord (UC).Their high self-renewal capacity and pluripotency in differentiating into derivatives of all germ layers in vitro and in vivo have made embryonic stem cells leading candidates for tissue engineering research and regenerative medicine, covering a wide range of therapeutic areas, including the treatment of several neurological and cardiac disorders, diabetes, hematopoietic diseases, liver diseases and lung diseases.Adult somatic cell-derived induced pluripotent stem cell (iPSCs) are increasingly being investigated as a patientspecific alternative to human ESCs with less controversy.Critically, a high degree of similarity exists between iPSCs and ESCs, offering a new hope for the use of pluripotent stem cells for regenerative therapies with fewer ethical concerns and, potentially, enhanced patient specificity 6 .On the other hand, MSCs, one of the many types of adult stem cells, also have a high self-renewal capability and expansive potential ex vivo.Its MSC can differentiate not only into mesodermal cells including osteoblasts, hematopoiesissupporting stromal cells, adipocytes, chondrocytes, myocytes and endothelial cells, but also into nerve ectoderm tissue cell 7 .They play critical roles in the tissues establishment, regeneration and replacement due to their unique capability of both differentiation and selfrenewal.So MSCs can be applied clinically as a new therapy for treating any tissue diseases with mesenchymal origin 8 .

Topographical cues by nanoscale biomaterials in cellular differentiation
Manipulation of cell fate on biomaterials is a fundamental goal of regenerative medicine 9,10 .Next-generation solutions include implantable materials that will actively participate in tissue formation.This can be achieved by programming multiple cellinstructive cues into the biomaterial itself [11][12][13][14][15][16] .For example, biomaterials can release growth factors, present ligands and deliver physical cues in order to sequentially recruit, organise and differentiate stem cell populations.These smart biomaterials can also provide transformative tools for probing the mechanisms that instruct cell behaviour and biosensing applications.The architecture of the biomaterial surface is a critical element in controlling cell behaviour through contact guidance 17 and is the foundation from which physical and chemical extracellular signals that are essential to defining phenotype are presented to cells (i.e.adhesion ligands and material elasticity).

Nanofabrication controlling physical characteristics of cellular differentiation Adhesion dynamism
The ability to control stem cell differentiation using topography alone has focused attention on elucidating the mechanism by which a cell perceives these topographical cues and relays this information into the nucleus to initiate cellular responses.Focal adhesions, the sites of cell attachment to the underlying substrate, play a pivotal role in all subsequent cell actions in response to nanotopography.These dynamic adhesions are subject to complex regulation involving integrin binding to extracellular matrix (ECM) components, and the reinforcement of the adhesion plaque by recruitment of additional proteins.In addition to their adhesive functions, integrins mediate bidirectional signalling between the cell and the ECM, activating both direct signalling and indirect molecular cascades that regulate transcription factor activity, gene and protein expression and ultimately growth and differentiation.Studies to date indicate that integrin clustering and the formation of focal adhesions are modulated by nanofeatures in vitro 18 , and that subsequent changes in both focal adhesion density and length are linked to changes in stem cell function and differentiation 19 .
Topographic features, such as pillars, islands or pits, with an inter-feature or z-scale dimension greater than 50-60 nm impair focal adhesion formation and the cell response 20 .Conversely, decreasing the inter-feature distance or z-dimension beyond 50 nm or increasing to the microscale facilitates stem cell adhesion and functional differentiation.The integrindependent signalling pathway is mediated by non-receptor tyrosine kinases, most notably focal adhesion kinase (FAK), which is constitutively associated with the β-integrin subunit.FAK localises at focal adhesions or focal contacts and can influence cellular transcriptional events through adhesion-dependent phosphorylation of downstream signalling molecules.In particular, the extracellular signal-regulated kinase (ERK) signalling cascade 21 , a member of the mitogen-activated protein kinase (MAPK) family of pathways, is activated by focal adhesion elongation and acts as a mediator of cellular differentiation.Primitive cells of the embryonic inner cell mass undergo integrin-dependent activation of ERK during early gastrulation, inducing cellular differentiation and the formation of the primitive endoderm.Functional differentiation in skeletal stem cell populations is highly dependent on focal adhesion formation and cellular spreading processes which are, to a degree, dependent on nanotopographical cues.Indeed, it has been reported that adipogenic differentiation of skeletal stem cells versus osteospecific differentiation is directly related to cellular spreading.It can be inferred that nanoscale features influence differential pathways in adherent stem cell populations by modulating integrin clustering and adhesion formation, and that subsequent activation of FAK acts to regulate the ERK signalling pathway and influence stem cell differentiation and tissue neogenesis.

Mechanical signals
Mechanical signals can regulate cellular functions in two modes, transmission and transduction.The former is mainly based on the intracellular cytoskeleton system, with which external signals can be passed to the remote part of the cell 21 ; the latter can convert the mechanical signals into biochemical signals.Mechanotransduction from extracellular stimuli to intracellular signals can be based on force-sensitive ion channels, such as stretch-activated ion channels, or on the ECM-integrin complex.In particular, integrins are a family of adhesion molecules on the cell surface that play key roles in mechanotransduction.Under mechanical stimulation, integrin can adjust its conformation by binding to ECM proteins to form a high-affinity and activation condition, which will promote the connection between its cytoplasmic tail and cytoskeleton as well as the formation of stress fibres and focal adhesions, thereby triggering the intracellular signalling transduction pathway and ultimately affecting physiological functions 22 .Indeed, a large number of signalling molecules have been reported to be activated and involved in mechanotransduction, including Src and small Rho GTPases such as RhoA, Rac1 and Cdc42.
Recent studies show that mechanical forces, including gravity, tension, stiffness, compression, pressure and shear stress, play a vital role in regulating gene and protein expression of stem cells.For example, in MSCs derived from BM, cyclic stretching can have a profound influence on cell lineage commitment 23 and promote the differentiation of MSCs into smooth muscle cells (SMCs).Similarly, in adipose-derived stem cells, cyclic strain alone or in combination with transforming growth factor beta 1 induced the differentiation towards the SMC lineage 24 .Static mechanical compression, on the other hand, seems to promote the chondrogenesis of MSCs.In fact, cyclic compression enhanced the gene and protein expressions, including chondrogenic markers type II collagen and aggrecan 25 .Hydrostatic pressure also enhanced the chondrogenic differentiation of human MSCs (HMSCs).Stem cells were also shown to be sensitive to the stiffness of the ECM.In fact, the stiffness of extracellular elastic matrices can guide the differentiation of HMSCs, committing towards the neuronal cell lineage on soft matrices and towards the osteogenic cell lineage on hard matrices 26 .

Nanofabrication and ECM mimicking
The spatial presentation of ECM has profound effects on cell adhesion and function.In the early 1960s, Curtis and Varde 27 investigated the effect that the topology of a surface has on cell behaviour.They used silica fibres with diameters between 8 and 40 μm in between two chicken embryo heart explants.Fibroblasts migrated out of the explants onto the fibres to form sheets of cells between them.The explants further exhibited a topology preference by predominantly forming sheets at the acute angles of two intersecting fibres and in the concave bends of curved fibres.They also examined cells migrating onto substrates with grooves and ridges of microscale dimensions and observed that the migration was more extensive on the ridges than in the grooves.These early experiments indicated that topography of the substrate was relevant to the cell-substrate interactions.Improved fabrication methods have since made it possible to produce nanoscale features on substrates, which resemble the fibres, pores, peaks and depressions found in the ECM.In mimicking the random structure of the ECM, multiwalled carbon nanofibres have been made by chemical vapour deposition to identify the range of cell functions that nanotopology can affect.Moreover, the predominated mechanosensors of the ECM environment are the cytoskeleton and focal adhesions and are likely to be involved in sensing topology.To understand the topology sensitivity, changes in gene expression of cells can be monitored using microarray analysis.Combining this technique with nanotopology surfaces will help to determine which gene targets may be involved in the topographical-related responses.Nanoscale grooves have been created in substrates as a means of studying the effects of spatial guidance on cellular shape and function.Nanogrooves present surfaces that resemble commonly encountered ECM structures such as topographical length of collagen-fibre bundles.Nanofeatures may also cooperate with existing signalling pathways initiated by soluble factors in order to guide cellular function.

Conclusion
While significant advances have been made since Curtis and Vardehypothesised that topology was important in determining cell behaviour, we are only beginning to understand their effects on cell function.Until recently, research in this area had been limited mainly to quantifying morphological parameters such as alignment, elongation, and area, perhaps because of the engineering background of the investigators.A major limitation has been a lack of control of the surfaces with defined chemistries, difficulties in characterisation of the protein adsorption process and understanding how cells bind to such nanotopographic surfaces.With the barrier between biologists and engineers disappearing, however, these limitations will quickly be addressed, and future investigations will shift towards understanding changes in signalling pathways and gene expression.As our understanding of the molecular and whole-cell responses of stem cells to topography increases, there will be enormous scope for the creation of next-generation materials possessing defined features to tailor stem cell fate and functioning to specific laboratory, industrial and therapeutic applications.