A cell responds to the environmental cues through the cellular mechanotransduction pathway. The disolveable and insoluble cues regulate/modulate various genes and their own downstream effectors. The physiological outcome of a cell increasing on a scaffold is defined by three factors— natural, biochemical, and biomaterial. [


]. Different techniques with various architectures are used for synthesizing scaffolds for a specific biological or even clinical application. (Figure 


). In the following section we have detailed a few methods that impart architectural uniqueness to scaffold design and their limitations with respect to stem cell programs.

Fig. 1

Cellular response to the particular biophysical microenvironment. Biomaterials with ( the ) fibrous architecture, ( w ) nano grooves/ridges, ( chemical ) surface roughness and varying nanotopographical functions, ( d ) nanodotted surface area, and ( e ) concave and convex curvatures inside a porous scaffold. These microenvironmental mechanical cues have the ability to influence cell adhesion, alignment, expansion, differentiation, and migration

Nanoscale platforms

Among the major challenges in biomaterial science is to generate the substrate topography that mimics the in vivo microenvironment composed of pores, ridges, and channels that provide physical tips to cells at a nanoscale level. Scaffolds are created by the techniques described below.


Electrospinning is one of the most widely used fabrication techniques. Nanofibers associated with sub-microscale diameter are generated by ejecting electrically billed polymer solutions through a needle on to a grounded enthusiast surface [ 7 ]. Usage-dependent customized nanofibers of different architectures and shapes can be fabricated using patterned collectors upon electrospinning. Since the fiber diameter is lesser than the cellular surface area, it is a perfect platform for the cells to organize plus spread around the nanofibers with numerous focal adhesion factors [ 6 ]. Pores of variable sizes can be released during the generation of electrospun scaffolds by incorporating porogens or even sacrificial fibers that get dissolved after fabrication [ 8 , 9 ]. A wide range of polymers of both natural and artificial materials are employed for manufacturing scaffolds [ 10 ]. Although the biological materials promise better clinical applications it is hard to maintain the integral chemical features of the material during the electrospinning process. For example , collagen loses its physicochemical properties whenever fluoroalcohol is used as a solvent to generate nanofibers [ 11 ]. It is much more challenging to precisely control the sizing and morphology of silk fibroin as 3D electrospun scaffolds for specific biomedical and dental applications [ 12 ]. Some drawbacks of this method are the possibility of cellular material squeezing into smaller pores thereby limiting their development potential and the lack of precise control of fiber alignment. Adjustments to the electric charges and the introduction of high-speed rotator mandrels have overcome this limitation [ 13 ]. Tissues seeded on a nanofiber structure tend to maintain the phenotypic form and a guided growth according to nanofiber orientation. Yin ou al. differentiated human tendon stem/progenitor cells to muscles using electrospun poly- l -lactic acid (PLLA) nanofibers [ 14 ]. The mobile morphology was defined by the nano-architecture; cells were a lot more stellate-patterned on random nanofibers and elongated and spindle-shaped fibroblastic phenotype with the aligned nanofibers [ 14 ]. In addition to the differentiation factors, the scaffold architecture is capable of leading lineage specification. For example , human bone marrow stromal cellular material (hBMSCs) cultured on nanofibrous poly-ε -caprolactone (PCL) scaffolds adopted an elongated, highly branched, osteogenic morphology [ 15 ]. Morelli et al. have reported that polylactic acid (PLA) and composite PLA-nanohydroxyapatite electrospun scaffolds had been equally efficient in differentiating human mesenchymal stem cellular material (MSCs) to osteogenic and osteoclastogenic differentiation [ 16 ]. Kai et al. in a recent study demonstrated that will electrospun composite PCL-gelatin scaffolds encapsulated with vascular endothelial growth factor promoted differentiation of MSCs for myocardial regeneration [ 17 ]. Furthermore, Mohtaram et al. discovered that electrospun PCL scaffolds with a smaller diameter cycle mesh induced higher neuronal differentiation compared to thicker spiral from neural progenitors [ 18 ]. Electrospun nanofibrous scaffolds of polylactic-co-glycolic acid (PLGA) and gelatin with inlayed epidermal growth factors have been used for tissue engineered epidermis scaffolds [ 19 ]. Recently, Ortega et al. utilized a combination approach of electrospinning and microstereolithography to generate corneal membranes that mimicked the limbal region of the attention that harbor ocular stem cells [ 20 ]. Sneaking in guidance cues with gradient concentration on electrospun fibers can offer a polarized effect on the cultured cells and tissues. Similarly, strategies such as air brushing are now being used to eliminate the use of natural solvents for the preparation of polymer solutions. One of the prominent feature of electrospinning is controlling the fiber position, which has been achieved for proper control of cellular functions by means of control of cellular morphology and alignment [ 21 , 22 ]. The discharge of encapsulated drugs and biomolecules is being tailored with the use of core-shell fibers [ 23 ].

Though the technique is widely popular, the process of electrospinning depends upon what polymer solution properties such as viscosity, surface tension, conductivity, and dielectric constant [ 24 ]. The viscosity from the solution maintains the ejecting fiber without breakages [ 25 ]. Voltage applied, flow rate of solution, kind of collector, needle diameter, and the distance between the needle as well as the collector are factors that determine the pattern from the fibers [ 26 ]. Environmental factors such as temperature, dampness, and pressure can have some minor effects on the patterning associated with fibers by electrospinning [ 27 ]. The number of factors influencing the scaffold outcome generated by electrospinning is several, making it difficult to form a standard operating protocol for repeatability of scaffold architecture. The infiltration of cells inside electrospun fibers is rather limited. The fibers are typically not able to serve as scaffolds for load-bearing tissues [ 28 ].

Soft lithography

Soft lithography fabrication uses elastomeric stamps, molds, and conformable photomasks ranging from micrometer to nanometer scale for scaffold era. The synthesizer, based on the application, can customize the spatial distribution of polymer molecules placed on the substrate to help specific outcomes such as nanodots, nanoridges, and grooves within the range from 30  nm to several microns [ 29 , 30 ]. This particular spatial distribution of the polymer molecules also aids in growing and shaping individual as well as groups of cells [ 31 33 ]. Embryonic stem cells are cultured in embryoid body and further differentiated using conditioned culture methods for lineage specificity [ 34 , 35 ]. In an attempt to identify a stem cell shipping system, murine muscle satellite cells were cultured upon 3D polyglycolic acid (PGA) scaffolds fabricated from a mixture of soft lithography and thermal membrane lamination. Cells shipped by scaffold show higher integration to the damaged tibialis anterior muscles in comparison to cells injected intramuscularly [ 36 ]. Grooved patterns of micro- or nanoscale structures advertise cell alignment and differentiation, especially with human wanting stem cells, into neuronal lineage without the need for any dietary supplements [ 37 ]. Hollow spheres are fabricated by treating liquid drops into noncured polydimethylsiloxane (PDMS) mixtures. Additionally, such drops provide a cell culture environment for increasing embryoid bodies [ 38 ]. Micropatterned PDMS scaffolds produced by soft lithography have been used for mimicking musculoskeletal junctions connecting aligned myotubes with acetylcholine receptors [ 39 ]. However , the major limitation with this technique is that it provides a restricted and narrow range of ECM signals for the cells in order to perceive, which can be highly inconsistent in comparison to the vast within vivo microenvironmental cues. This is further substantiated by the within vitro study where PLGA substrates of different groove level promoted human tenocyte alignment with simultaneous upregulation within the expression of chondrogenic and osteogenic genes. On the contrary, inside a rat patellar tendon model, neither of the grooved topographies induced ECM orientation parallel to the substrate. This indicates that will cell phenotype maintenance is well established by two-dimensional (2D) imprinting technologies only in an in vitro condition. Within an in vivo scenario, the neotissue formation and corporation is established by multiple factors [ 40 ]. Another research deciphered the 2D imprinting technique exclusively to evaluate cell function in vitro for phenotype maintenance of human being primary osteoblast phenotype on substrates of different grooves. Moreover, in the in vivo sheep model, none of these topographies promoted osteogenesis [ 41 ].

Soft lithography is limited by distortion in the fabrication associated with single-layer structures [ 42 ]. The defects formed, which usually arise from dust particles, poor adhesion to the base and poor release from the stamp, must be controlled. One more drawback is the formation of a thin film of plastic under the nanometer-sized features. This layer is removed by means of ion etching but leads to damage of small nano-features generated on the fabricated scaffold. Integration of large plus small features in phase-shift lithography is extremely difficult [ 43 ].


Microfluidic devices make an excellent platform to study cells under different microenvironmental conditions such as stress capillary flow, chemical gradient, and the effects of single/low cell numbers on the temporal plus spatial resolution. In microfluidics, the capillary flow keeps a constant soluble microenvironment and has a large surface area to quantity ratio similar to biological systems [ 44 ]. This has already been used extensively to study cell biological aspects such as mobile adhesion forces, the cytoskeleton, and for in vitro lifestyle techniques. Microfluidics are used for high-throughput screening because of the capacity in order to culture a limited number of cells in a controlled manner. This type of system can thereby standardize culture conditions for difference without altering the cell number. Major limitations of microfluidics in long-term stem cell cultures are because of water evaporation, protein adsorption, leaching of non-reactive compounds, plus hydrophobic recovery. Despite the aforementioned limitations, microfluidics provide a prospect of simultaneous multi-parametric analysis with respect to the differentiation paradigm [ 45 ]. Mouse embryonic stem cell (mESC) differentiation studies making use of microfluidic systems have elucidated the decisive roles of fibroblast growth factor (FGF)4 and notch signaling during neuroectodermal lineage [ 46 ]. Three-dimensional microfluidics mimic the within vivo situation more closely, hence this microenvironment will be ideal for studying organogenesis and differentiation.

Much like soft lithography, the field of microfluidics uses strategies wherein the cells are grown on a substrate within 2D format and subjected to fluid flow. Designing microfluidic systems for 3D scaffolds remains a challenge and is simply starting to be investigated. This fluidic strategy has to be utilized a lot more for clinical application [ 47 ].

Nanoparticles in nanotechnology

Nanoparticles have contributed immensely in changing the physicochemical properties of the scaffold because of their variable sizes and shapes. Most of the properties attributed to nanoparticles are driven by their higher surface to volume ratio, improved solubility, electrical plus heat conductivity, and improved catalytic activity on the surface [ 48 ]. These nanoparticles can be used for altering the scaffold architecture either by decorating the scaffold surface in order to impart surface features and varying surface chemistry along with being incorporated in the matrix during scaffold synthesis to alter mechanical properties, electrical conductivity, and so forth. The most widely used nanoparticles in this field can be classified into five groups depending on their nature: carbon based, inorganic base, metal centered, nanostructured hydrogels and quantum dots based [ 49 , 50 ].

Carbon nanotubes

Carbon nanotubes (CNTs) derived from graphene sheets are prepared with precise power over orientation, alignment, nanotube length, diameter, purity, and denseness. They are constructed as single-walled (SWNTs) or multi-walled co2 nanotubes (MWNTs). CNTs have tunable chemical and mechanised properties, like conductivity, biocompatibility, and nanoscale dimensions, that will serve as topographical cues and to generate electrophysiological properties [ 51 , 52 ]. Composites with polycarbonate membrane and collagen sponges promote the osteogenic potential of stem cells. Connections with fibroblasts were noted to be enhanced in polyurethane material composite scaffolds. Better adherence and enhanced proliferation might be observed in endothelial cells cultured on composite polyurethane scaffolds. Polyacrylic acid composites aided in neuronal differentiation through embryonic stem cells [ 52 56 ]. The major drawback of CNTs is the presence of impurities of carbonaceous particles like nanocrystalline graphite, amorphous carbon, fullerenes, and different metals (typically Fe, Co, Mo or Ni) used as catalysts during the synthesis phase, and also concerns with toxicity because they are resistant to degradation in vivo [ 51 , 57 ]. Recently, other forms of carbonaceous nanoparticles such as graphene and nanodiamonds are also being investigated [ 58 , 59 ].

Metal plus metal oxide nanoparticles

Metallic oxide nanoparticles provide structural variabilities by exhibiting conductor or insulator characters. Oxide nanoparticles display unique chemical substance and physical properties with differential charge on the middle and corner of the nanoparticle [ 60 ]. They have mainly been used in tracking stem cells post-transplantation [ 61 63 ]. MSCs incubated with magnetized iron oxide nanoparticles advertised calcium nodule formation in the presence of osteogenic lifestyle medium [ 64 ]. Superparamagnetic iron oxide nanoparticles chill H 2 O 2 and thereby promoted growth of MSCs [ 65 ]. Delcroix et al. showed that rat MSCs, when loaded with superparamagnetic iron oxide nanoparticles coated along with 1-hydroxyethylidene-1. 1-bisphosphonic acid and injected, showed migratory conduct only on creating a lesion [ 66 ]. Copper oxide nanoparticles did not show any effect on the differentiation possible of rat MSCs to osteogenic and chondrogenic family tree. Enhanced genotoxicity could be observed in the MSCs with maximizing dosage of copper oxide nanoparticles [ 67 ].

Inorganic based

These are ceramic-based nanoparticles synthesized by a combination of a metal and a non-metal component. These are formed under higher temperature and stress [ 68 , 69 ]. These materials have high mechanical power and low biodegradability. Hydroxyapatite and tricalcium phosphate nanoparticles have been shown to promote bone formation [ 70 ]. Silica nanoparticles enhance actin polymerization and promoted osteogenesis through MSCs [ 71 ]. Furthermore, these nanoparticles coated upon scaffolds promote cellular growth of adipose-derived stem tissue in culture through Erk kinase activation [ 72 ]. Fibrin-poly(lactide-caprolactone) nanoparticle-based scaffolds enhance the human adipose-derived stem cellular seeding efficacy and promote cell growth and chondrogenic differentiation [ 73 ]. Embryonic stem cells cultured upon polystyrene nanoparticles differ in their morphology based on their tradition density. At lower density, the embryonic stem tissue transform to embryoid bodies, whereas at higher denseness they became fibroblastic when cultured on polystyrene nanoparticles [ 74 ].

Quantum dots based

These are nano-sized semiconductors that can emit gentle in different colors. These comprise atoms for releasing bad particals and cadmium as one of the chemicals. Most of their usage is restricted to tracking stem cells undergoing differentiation and immigration [ 75 , 76 ]. These are photostable and have longer longevity. Up to now there have been no reports on the effect of quantum dots within altering stem cell proliferation or differentiation [ 77 ].

A major concern with the use of nanoparticles is their toxicity and environmental effects. The environmental impact during production of nanoparticles itself is a global problem [ 78 ]. Moreover, when used in scaffolds, the extensive effect in vivo is not well understood.

Nanostructured hydrogels

Hydrogels are THREE DIMENSIONAL polymeric materials of a hydrophilic nature capable of holding huge amounts of water. Co-polymerization/crosslinking free-radical polymerizations are commonly used to create hydrogels by causing hydrophilic monomers to react along with multifunctional crosslinkers to form a network. Hydrogels can be further categorized into nanogels and micellar gels [ 79 ]. Nanogels are hydrophobic in nature and hence can be used to deliver items to cells. Nanostructured hydrogels are self-assembled injectable service providers of cells and proteins [ 80 ]. Chemically or even physically crosslinked nanostructure scaffolds are fabricated by photo-irradiation of vinyl monomer conjugated to polyethlyne glycol, pluronic copolymers, and hyaluronic acid [ 80 ]. The degree associated with crosslinking determines the mechanical strength, durability, and inflammation properties on the nanostructured hydrogels [ 81 ]. Most of the nanostructured hydrogels are primarily used for carrying genes and protein to be delivered [ 82 ]. The environmental conditions are crucial for crosslinking the monomers in a temperature- or pH-responsive crosslinking strategy [ 83 ]. This ability of the nanostructured hydrogels to transform from sol to gel type makes them the smart hydrogels. Mesenchymal stem cells cultured upon nanostructured PEG-based hydrogels with nano-sized micelles showed an increased gene expression of mesenchymal stem cell marker when compared with those cultured without micelles [ 82 ]. Neural come cells of human origin showed enhanced adhesion plus proliferation when cultured on self-assembling peptide-based nanostructured hydrogels [ 84 ]. Nanostructured hydroxyapatite along with demineralized bone matrix were used for generating nanostructured hydrogels for growing mesenchymal stem cells. These cells showed increased osteocalcin manufacturing with alkaline phosphatase indicating higher osteogenic specific difference [ 85 ]. Nanostructured hydrogel with porous baghdadite displays sustained release of dexamethasone disodium phosphate, promoting osteogenic regeneration [ 86 ]. The in situ forming clever hydrogel can be functionalized by bioactive molecules to enhance development and other functionality of stem cells. The sol-gel changeover of the nanostructured hydrogels serve as carriers for drug plus protein delivery in supporting regenerative medicine.

The spatial shape plus alignment in stem cell function

All techniques used for generating scaffolds provide a geometrical control of the morphology of cells. The effects of geometrical energies on cells are explained using theoretical models of system and continuum mechanical models. While a continuum mechanised model describes the distribution of adherent cells, the network model is based on the contractile cytoskeleton deciphering the connection between the force distribution and shape of adherent cells.

Correctly revealed modulation of cellular functions such as proliferation plus differentiation based on cell-specific locations in tissues with specific geometry. Stem cells in a 3D matrix respond to THREE DIMENSIONAL architectural features at different scales from nanometers in order to micrometers and further to millimeters with differing functions within apoptosis, proliferation, and differentiation. The dominating effect of matrix geometrical force on cell fate incitement is critical in tissue-specific regeneration (Fig.  



Fig. 2

Schematic representation defining the importance of various scaffold architectures in determining the specific lineage of stem tissues. Stem cells cultured on various nanostructured scaffolds yeild different differentiated cell types, such as a bone marrow stem cells cultivated on nanofibrous PCL scaffold promotes osteogenic fate, b embryonic stem cellular cultured on annosclae ridge or groove promote neuronal fate, c tendons stem cells culutred on aligned and random PLLA directed tendon and stellate lineage, respectively, d mesenchymal stem cells upon PDMS promote osteogenic as well as adipogenic fate.

Since cell shape plus function are tightly linked together, scaffolds that regulate cell shape can dictate cell functions, for example the lengthy body of neurons for effective delivery of indicators, and the spherical shape of adipocytes for lipid storage. Individual MSCs grown on microcontact-printed PDMS show osteogenic features at the edges of the matrix and adipogenic nature to the inner region of the scaffold [ 87 ].

Microenvironmental cues including mechanical forces are very important for the formation of “ stem cell niches”. Certainly, mechanical forces appear to either promote or block difference signals induced by growth factors and cytokines and they also supersede the influence of soluble factors. To investigate the consequence of mechanical forces on MSC differentiation, Kurpinski et ‘s. used a micropatterned strip to align the cells across the direction of the uniaxial strain. They found an increased manifestation of calponin 1 (a smooth muscle marker) and also a decrease in the expression of cartilage matrix marker. Nevertheless , when the cells were aligned perpendicularly to the direction from the strain, the changes in gene expression were reduced [ 88 ]. This experiment suggests that mechanical strain posseses an important role in gene expression and fate of come cells.

An amalgamation of methods with a microengineered system comprising of a soft hydrogel can be used for inducing difference of stem cells (Fig.  


). The outcome of come cell lineage specification has been tabulated with specific biomaterials in Table 



Fig. 3

Various nanoscale platforms for directing stem cell fate. Scaffolds along with ( a ) nanofibrous structures, ( b ) soft lithography, ( c ) hydrogel, plus ( d ) carbon nanotubes. These microenvironmental cues direct stem cell differentiation to some specific lineage

Table 1

Strategic mode of directing the fate of originate cells through nanotopography of the synthetic scaffolds


Rat hair follicle stem cells (HFSCs)

Aligned poly-ε -caprolactone (PCL) nanofiber

Neuronal lineage


Mouse embryonic stem cells (ESCs)

Poly- l -lactic acid (PLLA) nanofiber

Osteogenic lineage


Retinal progenitor cells (RPCs)

Microfabricated PCL

Differentiated RPC


Mouse ESCs

Aligned PCL nanofibers

Neuronal family tree


Human mesenchymal stem cellular material (MSCs)

Type 1 collagen nanofiber

Osteogenic differentiation


Human MSCs

Poly(lactide-co-glycolide) nanofiber

Osteogenic lineage


Rat mesenchymal stem cells (MSCs)

PCL nanofiber



Human MSCs

Polydimethylsiloxane (PDMS) nanogroove

Neuronal lineage


Human MSCs

Polyacrylamide hydrogel

Neuronal family tree


Rat hippocampal progenitor tissue

Micropatterned polystyrene with laminin

Neuronal lineage