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Insight into the Role of the Surface Topography: History
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Contributor: Raluca Tudureanu , Iuliana M. Handrea-Dragan , Sanda Boca , Ioan Botiz

Cell proliferation is an essential process with the key role of maintaining the life of an organism through the healing of wounds, developing the organs and regenerating the tissues. Stem cells are a particular type of cells that have two important properties: self-renewal and developing into different specialized functional cells. According to their potential to differentiate, there are three types of stem cells: totipotent (TSCs), pluripotent (PSCs) and multipotent (MPSCs).  Various cellular behaviors such as adhesion, migration, differentiation, cytoskeleton distortion or even gene expression were shown earlier to clearly depend on the changes in surface topography. 

  • cell differentiation
  • surface topography
  • fibronectin

1. The Impact of the Surface Topography on Cell Proliferation

There are many parameters related to the topography of a substrate (e.g., the shape and dimension of the surface relief patterns, their periodicity or discontinuity, their arrangements, etc.(see Figure 1) that can be controlled in order to induce various, often highly desired effects, including cell directionality or alignment, and to “force” cells to take a specific shape and to adhere more or less prominently to signal various pathways, to migrate and regenerate [1], etc. As will be further described below, the surfaces used in experiments are displaying surface relief patterns that are either coated with biopolymers [2][3][4][5] or are entirely sculptured in biopolymers [6][7][8][9].
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Figure 1. Schematics depicting various parameters that can be controlled within specific topographies and the resulting effects on neural cells: (a) continuous topographies represented by grooves of various dimensions are able to affect the direction of cell outgrowth and polarity; (b) discontinuous topographies such as pillars of different shape, size and arrangements can alter the adhesion, survival and differentiation of neural cells. Adapted with permission from ref. [1].
When learning about cellular behaviors (adhesion, proliferation, orientation, etc.), it seems like a good strategy to consider the report of Nagata et al. from the 1990s which was focused on analyzing the directionality of neuroblasts cultured on artificial microstructures [10]. Specifically, the study outlined that the nervous system’s cells are puzzling when trying to control their behavior by changing the classic properties of culture surfaces (for instance, neurons need good adhesion properties with respect to the substrate to proliferate efficiently [11]). Following this direction, Rangappa et al. Performed one of the first experiments in the nervous system area by culturing dorsal root ganglion (DRG) on laminin-coated poly(L-lactide) (PLLA) filaments. Experiments showed that the grown neurites were longitudinally oriented [2]. Moreover, the laminin coating determined an increase in the longitudinal dimension of neurites from 2 mm on an uncoated surface to 5.8 mm on laminin coated filaments [2]. Changes in orientation were further observed in Schwann cells grown on laminin coated poly (methyl methacrylate) (PMMA) films sculptured with multi-width lines patterns [12]. Here, the laminin coating increased the adhesion properties which contributed to a higher proliferation rate and could even lead to the formation of a monolayer of cells that covered the entire substrate surface, with cells displaying a high order or orientation. Schwann cells along with neurons from the DRG explant were further studied by Miller et al. on laminin coated poly (D,L-lactic acid) (PDLA) substrates displaying a groove-like surface relief pattern, the latter having the role of providing not only the physical guidance but also favoring the growth of axons [13]. The laminin coating was also used to cover the filament membranes of poly (acrylonitrile-co-vinyl chloride) (PAN-PVC) along with fibronectin. Both coatings determined the same cell behavior when different diameter fibers were used as the substrate on which neurons and Schwann cells originated from dorsal root ganglion of mice were deposited. When compared to uncoated control fibers, the outgrowth of neurites was increased, and the increase was more pronounced along the laminin and fibronectin coated fibers of subcellular size (5 µm diameter filament bundles) and along the laminin coated fibers of cellular size (30 µm diameter filament bundles) than along the fibers of supracellular size [14].
Another type of coating frequently used in the cell proliferation research is the collagen type I. Hsu et al. used silicone as a substrate for a Schwann cells culture that, prior to coating with collagen type I, was micropatterned with different models. The results showed that the grooves with larger width/spacing (20/20 µm) and depth led to a more evident increase of the percentage of aligned cells than any other grooves of smaller width/spacing (10/10 µm) and depth [15] (the different behavior of cells depending on the dimensions and types of grooves is schematically represented in Figure 1 [1]). Nonetheless, the alignment of cells on the laminin coated grooves was shown to increase to 60%, as compared to a 51% increase on the collagen type I coated grooves and to only a 41% increase on the uncoated surface [15]. These results indicated that laminin was better favoring the proliferation process than collagen type I. Other studies showed that there is a difference in the adhesion rate of Schwann cells on materials coated with laminin, collagen type I and fibronectin [16], hence the proliferation on collagen type I seems indeed to be weaker than on laminin [15][16] or fibronectin [16]. Even so, collagen type I is over excelling when compared to polymers such as poly (lactic acid-co-glycolic acid) (PLGA). For example, in the bone-area research, osteosarcoma cells (human Saos-2 cell line) grown on collagen and PLGA, both on pillar-like patterns and on flat surfaces, have shown that cells spread more on a collagen surface than on a PLGA surface, demonstrating the superior cell adhesive property of collagen [5]. Moreover, confocal laser scanning microscopy studies further showed that Saos-2 cell proliferation was sensitive to the type of collagen coated micropillars (Figure 2a–d), with the actin fibers being stretched along the cell axis and fine filopodia, and with the abundance of filopodia specifically observed on the patterned surfaces (Figure 2e–g).
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Figure 2. (a–d) Confocal laser scanning micrographs depicting the behavior of Saos-2 cells on collagen on 8 µm high pillars of different lateral dimensions: 8 × 8 µm2 and 4 µm spacing (a), 16 × 16 µm2 and 4 µm spacing (b), 8 × 8 µm2 and 8 µm spacing (c), 16 × 16 µm2 and 8 µm spacing (d). (e) SEM and confocal (inset) micrographs of the Saos-2 cells cultured on the surfaces patterned with 8 × 8 µm2 and 16 × 16 µm2 collagen pillars (all spaced at 8 µm), respectively. Images showed that cells had coarse fibers stretching along the cell axis, and distinct, fine filopodia (indicated by the yellow chevrons point to filopodia). (f,g) SEM (f) and confocal (g) micrographs of the Saos-2 cells cultured on the plain collagen. Adapted with permission from ref. [5].
Other nervous cells include hippocampal neurons. These can be cultured on polymer substrates previously coated with two biopolymeric systems such as polylysine and laminin (this double coating is being used for enhancing the adhesion of the cells to the substrates) [17]. Various isotropic patterns defined by their symmetry along both axes (dots, squares, grids) and anisotropic patterns such as gratings, triangles and others were designed through soft lithography. These patterns displayed diverse dimensions (with their width, diameter or space between pattern units ranging between 2 and 20 µm) and were used to analyze their specific effects on cells when in contact. For example, the axons grew the longest on the gratings having a width of 5 or 15 µm, compared to the samples exhibiting the narrowest widths with constant interspacing of 2 µm. Compared with axonal growth on planar surfaces, the grating patterns showed an average of 60% greater growth. This percentage was of almost 48% for circular patterns. Moreover, the strongest axon guided growth was seen on gratings and circles compared to any other type of patterns, although the axon branching was very reduced [17].
Bacterial cellulose (BC), a biopolymer used in biomedical, food or chemical products industries, can be produced, purified [18] and further coated with gelatin in order to provide suitable surfaces for human dermal fibroblasts (HDF) cultures (note here that the uncoated BC already determines a higher proliferation rate due to its high tensile strength and a degree of polymerization higher than that of usual cellulose; coating BC with gelatin is further favoring cell interactions [19]). Moreover, such gelatin coated surfaces can be further patterned with 1 µm deep grooves exhibiting different widths (2, 10 and 100 µm). Systematic cell studies revealed that cell migration velocity on substrates with narrower patterns was significantly reduced as compared to flat surfaces, while the cell alignment on grooves exhibiting sizes comparable to the size of the cells was more prominent [19]. Moreover, an in vivo experiment showed that the groove patterns on a BC based skin wound dressing were able to favor the infiltration of fibroblasts and deposition of collagen necessary for wound healing.
In vivo, macrophages and fibroblasts have the tendency to adhere to implanted materials and to widely spread, leading to complications [20]. Considering this, there is a need to find a way to develop antiadhesive topographies for improving their utility in implant engineering. Antiadhesive properties of surfaces exhibiting relief patterns are based on the physical interference of the topographic features with the determination and maturation of focal adhesion. In order to study the behaviors of the HDF and macrophages on antiadhesive substrates covered with various patterns, the former were cultured on biocellulose and on polydimethylsiloxane (PDMS) substrates, both coated with fibronectin. These substrates were then imprinted with patterns such as hexagonal pits with different diameters (6–20 µm), lateral spacings (6–23 µm distance between pits) and shapes (perfectly isotropic centered-hexagonal and quasi-isotropic squares) [21]. The adhesion rate-based results showed that, when compared with flat surfaces, the spread of HDF cultured for 72 h on fibronectin/PDMS and fibronectin/biocellulose decreased with almost 60% on almost all patterned surfaces, the only exceptions being represented by the hexagonal or square arrays with a diameter of 10 µm and a lateral spacing of 13 µm. The highest reduction of adhesion rate (65%) was seen on hexagonal and square pits having a diameter of 5 µm and a lateral spacing of 10 µm. Interestingly, after one week of cell culture, the adhesion decreases further, finally reaching 75% [21]. Moreover, the long-term interaction between HDF and the antiadhesion biocellulose-based topographies decreased the proliferation rate to only 10%. Furthermore, a detailed analysis of the circularity of the cells revealed that the biocellulose pits did not sustain the elongation of the cells seen on PDMS substrates, indicating that biocellulose-based geometries reduced the interactions between the substrate and the cells [21].
A slightly different approach to study the process of cell proliferation is to replace the surface relief patterns coated with biopolymers with surface relief patterns entirely sculptured within biopolymers. For instance, collagen can be used to fabricate films that can further undergo patterning [22]. Specific patterns can also be obtained from chitosan [6], fibronectin, cellulose, various proteins, enzymes, etc. (see Table 1). Additionally, collagen may be combined with other materials such as glycosaminoglycan to get scaffolds for tissue engineering [23]. This variety of surfaces has its own use as there are always advantages and disadvantages to using them in cell studies. For example, in films prepared from collagen extracted from rat′s tails, the adhesion of cells can be weak and, therefore, osteoblasts may not grow as expected [22]. For adhesion, osteoblasts prefer fibronectin to the detriment of type I and IV collagen. Furthermore, while the adhesion is weaker on laminin and type V collagen, osteoblasts do not adhere to type III collagen [24]. These observations are highly important when dealing with tissue engineering applications based on Schwann cells and neurons [14][15][25][26][27], osteoblasts [22], fibroblasts [28], or human corneal keratocytes and retinal pigment epithelial cells [29].
Table 1. Summary of various biopolymeric surface relief patterns that can be created using top-down and bottom-up lithographic methodologies.
Lithography Patterned Material Resulting Pattern Pattern Dimension Ref.
DLW chitosan, starch pores μm size [30]
UV light silk protein non-spherical particles several μm [31]
UV light wool keratin protein lines
circular patterns
crosses
triangles		 2 μm/width
3 μm/diameter
3 μm/width
tens of μm		 [32]
EBL sugar-based polymer moth-eye patterns 120 nm/period [33]
EBL biotinylated PEG pads ~10 μm [34]
IBL DNA oligonucleotides neutravidin
anti-mouse IgG		 line assays
complex stripes-based patterns		 1–2 μm/width
down to 100 nm/width		 [35]
NIL chitosan lines
circular pillars		 10 μm/width
500 nm/diameter		 [6]
[36]
NIL proteins lines 700 nm/period [37]
NIL gelatins/genipin grooves
holes
pillars		 500 nm/width
500 nm/diameter
100 nm/diameter		 [38]
NIL cellulose holes
lines
square pillars
rhombus pillars
holes		 400 nm/diameter
140 nm/width
1 μm/diameter
600 nm/width
600 nm/diameter		 [39]
[40]
μCP protein/Sylgard 527 arrays of nanodots 200 nm × 200 nm [41]
μCP biomolecules/poly(4-aminostyrene) stripes
pads		 ~2 μm/width
~7 μm/diameter		 [42]
μCP silk lines hundreds of μm/width [43]
μCP neutravidin/biotin arrays of nanodots ~62 nm/diameter [44]
μCP amyloid spider web arrays hundreds of μm/width [6]
TCSPL enzyme rectangles
squares
lines
dots		 4.5 μm × 1.5 μm
100 nm ×100 nm
8–9 nm/width
8 nm/diameter		 [45]
PL streptavidin patches 15 nm/diameter [46]
DNSA DNA squares
disks
five-point stars
rectangles
triangles		 ~100 nm/diameter
~100 nm/diameter
~100 nm/diameter
~100 nm/diameter
~100 nm/diameter		 [47]
Vrana et al. studied the alteration of properties of collagen-based micropatterned films by growing keratocytes and epithelial cells with the goal to eventually design a functional artificial cornea [7]. Here, while the unseeded collagen films suffered a reduction of strength, the growth of keratocytes improved the mechanical behavior of the films. On the other hand, the pigment epithelial cell line D407 seeded on the same films deteriorated the mechanical properties of the latter. Instead, collagen-patterned ridges oriented keratocytes and gave them an elongated shape. Moreover, after a period of three weeks this behavior changed, as the keratocytes had the ability to adhere to the inclined walls of the ridges once they occupied the base of the patterns [7]. Furthermore, relief patterns changed the cytoskeletal arrangement of keratocytes, the f-actin filaments being aligned with the groove direction after a period of seven days. Nonetheless, when considering the proliferative rates, the authors have shown that D407 cells grew better on flat surfaces, as patterns prevented the formation of cell-to-cell contacts. Instead, keratocytes better conformed to relief patterns and led to an oriented layer of cells once the relief patterns were degraded by the enzymes produced by keratocytes. The resulting films seeded with keratocytes presented better mechanical properties as the proliferation of cells compensated for the loss of the substrate integrity [7]. A relation between the cell proliferation and the presence of relief patterns was further demonstrated when investigating the role played by the size of collagen-glycosaminoglycans pores on the growing of osteoblasts. The results showed that after a period of seven days, osteoblasts proliferated with a higher rate in larger-sized pores made in collagen-based scaffolds, as such a size of the pores better favored the migration of the cells [23].
More recent studies placed neurons or Schwann cells on patterned chitosan surfaces [6] and on gelatin electro spun fibrous substrates [27]. On chitosan ridge/groove patterns, Schwann cells exhibited an orientational growth, as such patterns controlled the alignment of cell growth (Figure 3a). This was not the case for the flat chitosan surfaces where cells grew isotropically (Figure 3b) [6]. On the other hand, analyzing the effect of the orientation of gelatin fibers on primary Schwann cells and the RT4-D6P2T Schwann cells line, Gnavi et al. have demonstrated that although the alignment of fibers actually reduced the adhesion and proliferation rates as compared to random fibers (Figure 3c,d), it still favored the alignment of actin filaments of Schwann cells [27]. The reported data showed that Schwann cells were elongated with their longitudinal body along the aligned gelatin fibers. Similarly, when cultured on gelatin aligned fibers, the B5011 neuron cells were aligned and exhibited parallel axon growth. The authors concluded that the orientation of fibers could be used to modulate Schwann cells and axon organization in vitro [27]. .
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Figure 3. (a,b) Optical micrographs revealing the behavior of Schwann cells on a substrate patterned with 30 µm wide chitosan grooves (a) and on a flat chitosan control substrate (b). (c,d) Proliferation of RT4-D6P2T (c) and primary Schwann cells (d) on substrates covered with aligned and random gelatin fibers, as well as on control poly-L-lysine coated coverslips. Asterisks ** and *** indicated significant statistical differences with p ≤ 0.01 and p ≤ 0.001, respectively. Adapted with permission from ref. [6] (a,b) and ref. [27] (c,d).

2. The Impact of the Surface Topography on Cell Differentiation

As researchers mentioned above, the culture medium may contain different chemical stimuli for guiding cells to differentiate into a specific type of cell. Moreover, the process of cell differentiation can be also initiated by different biomaterials having specific topographies [48]. For example, the most common stem cells used in the differentiation studies on substrates with various topographical features are MSCs [49][50][51][52][53] and iPSCs [54]. Therefore, hereafter researchers analyze the cell differentiation behavior with respect to specific surface relief patterns that were either coated with biopolymers or directly sculptured in biopolymer films [48][51].
Following the idea that many tissues have an anisotropic architecture, Lanfer et al. created aligned structures out of collagen type I using a microfluidic set-up. Furthermore, they incorporated glycosaminoglycan heparin for studying multiple extracellular matrix (ECM) components at once, and researched multiple differentiation lineages of MSCs (osteogenic, adipogenic, chondrogenic) [49]. The most important impact of the substrate was observed in the osteogenic differentiation, where the osteogenic medium induced the formation of mineralized nodules on substrates of aligned collagen structures. In comparison, these structures were missing on flat glass substrates [49]. Moreover, the substrate comprised of aligned collagen fibers influenced the osteogenesis of human MSCs by directing the ordered matrix mineralization. The ability of undifferentiated stem cells to sense the mechanical properties of their growing environment and to differentiate accordingly was further demonstrated by Park et al. [55]. They have shown that bone marrow MSCs, which have the ability to differentiate into smooth muscle cells or chondrogenic cells, can become either one of these types of cells, depending on the stiffness of the matrix there are growing on. MSCs on soft collagen substrates spread less and showed lower proliferation rate than MSCs cultured on stiff collagen substrates. Also, the stiff matrix promoted the differentiation into smooth muscle cells while the soft matrix promoted the differentiation into chondrogenic and adipogenic cell lines [56].
A new approach to test the relationship between substrate topography and cell differentiation process was developed in 2014 by Younesi et al., who have built a 3D scaffold of anisotropically oriented collagen fibers (collagen was used for its properties that support the attachment and growth of some primary cells). This structure mimicked the properties of a native tendon presenting a porosity of 80%. MSCs cultured on the scaffold faced tenogenic differentiation without the presence of the growth factors. The differentiation was seen by the presence of tenomodulin, cartilage oligomeric matrix protein (COMP) and collagen type I in the synthetized matrix (specific tenogenic markers that were up-regulated), and represents a promising alternative for repairing tendons and ligaments [51].
Another biopolymer that can be used in bio-coating to improve seeding surfaces is fibronectin [50][52][57]. For instance, PLGA coated with fibronectin can be patterned with spatially defined geometries and further used for establishing control over the morphology of bone marrow derived human MSCs and eventually for altering the cell differentiation [50]. The results showed that while cells that have been grown on 20 µm wide strips were highly elongated and exhibited an area coverage of ~2000 µm2, cells grown on flat, unpatterned surfaces displayed a much larger area coverage of ~ µm2. Moreover, an ulterior analysis on gene expression indicated that while the elongated cells exhibited up-regulation of several markers associated with neurogenesis and myogenesis, the markers associated with osteogenesis were down-regulated or remained at its nominal level. This demonstrated that the mechanical deformation of cells can be translated into biochemical response and suggested the idea that cell differentiation can be altered by the substrate, in absence of other differentiation factors [50].
As technology is advancing, new ways of improving the materials’ properties are discovered. Substrates coated with rod-shaped turnip mosaic virus (TMV) and spherical turnip yellow mosaic virus (TYMV) were used for culturing bone marrow derived MSCs. This represents a new approach in studying osteogenic differentiation. By analyzing osteogenic markers such as bone morphogenetic protein 2 (BMP-2), an acceleration of osteogenic differentiation process by seven days in both cases was shown [58]. An exact explanation for these mechanisms is not yet available, but some studies showed that cells may use some molecular mechanisms for sensing different topographies which are able to coordinate the focal adhesion of cells [59].
Also, a small guanosine triphosphatase (GTPase) named RhoA and Rho-associated kinase (ROCK) are known to have effects on the control of cell fate by cell spreading through their action on actomyosin contractility [60]. Other studies suggest that some sort of cell membrane and cytoskeleton stress induce cytoskeletal tension mediated by the RhoA/ROCK complex, resulting in the beginning of osteogenesis. In this way, it can be supposed that the virus nanoparticles supply some topographical features that induce mechanical stress to the cell membrane which enables it to trigger an earlier osteogenic process [61].
Furthermore, for the onset of fibrosis, the cells not only have to adhere and to proliferate, but they also have to differentiate for the deposition and contraction of the fibrotic matrix on the surface of implants. To study the differentiation of fibroblasts into contractile myofibroblasts, the expression of α-Smooth Muscle Actin (α-SMA) of cells on different dimension pits, on substrates covered with both hexagonal and square patterns, was analyzed [21]. The most important reduction of α-SMA expression was encountered on the hexagonal disposition of pits with a diameter of 5 µm and a lateral spacing of 10 µm. When compared to the cells cultured on TCP (tissue culture plastic) or MED6015 (medical grade silicone), results have shown that the biomaterial properties did not support the activation of the required signals for differentiation, and that the upgrade of the biocellulose surface with pits-patterns introduced a significant additional inhibition of differentiation, preventing the fibrosis [21].
An interesting and innovative approach proposed the use of fibronectin coating on a different substrate. Starting from the fact that cellular functions such as proliferation and differentiation can be upregulated by enhancing the focal adhesion (FA) between cells, ECM and intracellular actin polymerization (AP), Seo et al. improved the culture substrate by patterning it with tailor-made micrometer-sized pits (tMP) coated with fibronectin [52]. Mouse MSCs (C3H10T1/2) were cultured on tMPs substrates and the obtained results showed that although the cell spreading area was not affected by this topography, the cells FAs were increased together with AP and traction forces. Therefore, the osteogenic differentiation increased as well. This was observed by reverse transcription polymerase chain reaction (RT-PCR) techniques and western blotting that showed upregulation of specific osteogenic markers such as alkaline phosphatase (ALP), collagen type I, osteocalcin (OCN) and runt-related transcription factor 2 (Run × 2)/core-binding factor 1 (Cbfa1). OCN is a late dominant marker of osteogenic differentiation and, as it is emphasized in Figure 4, its intensity was significantly higher in cells grown on the tMP surfaces than those on the flat surfaces [52].
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Figure 4. Fluorescence micrographs depicting the staining of OCN cells cultured on substrates patterned with 4 µm sized tMPs (a), on substrates patterned with 2 µm sized tMPs (b) and on flat surfaces (c), respectively. Scale bars represent 15 μm. Adapted with permission from ref. [52].
Fibronectin was further employed by Shukla et al., who fabricated micropatterned fibronectin arrays of biomimetic geometries that replicated the morphology aspect of mature cells. Adipocytes cultured in 2D were imaged and used to create biomimetic virtual masks which were then employed to pattern the fibronectin surfaces via the laser scanning lithography. Reported results pointed out a clear influence of the pattern geometry on human MSC differentiation (Figure 5). While human MSCs seeded on nonpatterned fibronectin surfaces in differentiation medium showed positive staining for both lipids and ALP (~10% of the cells stained positive for lipids, ~20% of the cells stained positive for ALP and the remaining cells were not positive for either marker), the MSCs cultured on mimetic patterns in the same differentiation medium showed positive staining for lipids (45% of the cells) and ALP staining was not present in any of the MSCs [57]. In conclusion, the mimetic geometry determined the human MSCs to differentiate into adipocytes.
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Figure 5. Actin cytoskeleton of human stem cells on adipocyte mimetic fibronectin patterns, modified adipocyte fibronectin patterns as well as square and circle fibronectin patterns. Here gold color stands for F-actin while blue color stands for the nucleus. The scale bar is 25 μm. Reproduced with permission from ref. [57].
For culturing of human MSCs cells, fibronectin can be also used in combination with gelatin to coat the PDMS substrates. The latter can be patterned with 10 μm or 20 μm wide and 3 μm deep grooves (the depth of grooves was chosen to match the few micrometers sized heart muscle matrix fibers) [62]. After the visualization of the arranged cytoskeleton, the changed morphology of the cells, as well as the enhanced expression of GATA4, troponin I and troponin T on the surface covered with relief patterns, it was concluded that the 20 μm wide grooves promoted better the cardiomyogenic differentiation of human MSCs [62]. These results were of significant impact, knowing that the arrangement of the cytoskeleton, especially of actin filaments, is important in a lot of signaling pathways [61].
Another interesting study was implemented using scaffolds based on loosely packed silk nanofibers on which the MC3T3-E1 pre-osteoblast mice cell line was cultured. The physicochemical properties of the cells grown on the aligned fibers were compared to the properties of the cells cultured on random-structured fibers. Results showed that cells on the aligned structures not only exhibited an elongated morphology and a more ordered arrangement, but also presented faster and deeper infiltration. The latter promoted proliferation and osteogenic differentiation of the pre-osteoblasts, as indicated by the expression of ALP activity and the presence of the macroscopic mineral nodes after 14 days [63]. Note that the importance of physicochemical properties of the substrates on cell differentiation is highly important, especially when a controlled differentiation of cells is desired. This was exemplified inclusively in a 3D extracellular matrix, when growing human amniotic mesenchymal stem cells (AMSCs) on fibrin hydrogels loaded or not with gold nanowire, and exhibiting different elasticity of the substrate. The latter was shown to clearly affect the osteogenic or chondrogenic differentiation [53].
Besides MSCs, iPSCs are also used for studying differentiation properties on surface-modified substrates. For instance, aligned chitosan fibers can mimic the native tendon’s microstructure and its mechanical properties. Therefore, while human iPSCs could differentiate into MSCs on a smooth surface (with the differentiation process being confirmed by the presence of characteristic MSC surface markers), iPSCs subsequently cultured on well aligned fibers differentiated into tenocyte-like cells through the activation of the mechanic-signal pathway [54].

This entry is adapted from the peer-reviewed paper 10.3390/ijms23147731

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