Nanotopography in directing osteogenic differentiation of mesenchymal stem cells: potency and future perspective

Severe bone injuries can result in disabilities and thus affect a person's quality of life. Mesenchymal stem cells (MSCs) can be an alternative for bone healing by growing them on nanopatterned substrates that provide mechanical signals for differentiation. This review aims to highlight the role of nanopatterns in directing or inducing MSC osteogenic differentiation, especially in bone tissue engineering. Nanopatterns can upregulate the expression of osteogenic markers, which indicates a faster differentiation process. Combined with growth factors, nanopatterns can further upregulate osteogenic markers, but with fewer growth factors needed, thereby reducing the risks and costs involved. Nanopatterns can be applied in scaffolds for tissue engineering for their lasting effects, even in vivo, thus having great potential for future bone treatment.

Besides the microenvironment, another aspect that influences stem cell development is the epigenetic aspect. Epigenetic factors are intracellular processes that do not involve genetic factors. Epigenetic mechanisms usually cause changes in gene expression without changing the DNA structure or sequence. Some examples of epigenetic mechanisms include cytosine DNA methylation, histone modification by histone acetylation and methylation, and gene expression regulatory activity of small noncoding RNA [20]. Mechanical forces can be transduced into the cell through the cytoskeleton to the chromatin in the nucleus, thereby influencing epigenetic regulation. Nuclear lamins can interact with DNA, chromatin, and histones in the lamina-associated domains (LADs). LADs play a role in the regulation of histone modifications and also changes in chromatin structure through nucleoskeletal reorganization. In LADs, there are heterochromatin markers such as H3K9me3 and H3K27me3 that play a role in stem cell differentiation to prevent cell reprogramming and silencing certain line-specific genes with the outcome that differentiated cells can maintain their identity. However, during cell reprogramming, H3K9me3 functions to maintain the undifferentiated state by suppressing the activation of transcription factors and arranging chromatin into dense heterochromatin, while H3K27me3 plays a role in facultative heterochromatin silencing. Thus, cytoskeleton and lamins rearrangements can regulate histone modifications and thereby influence MSC differentiation. This rearrangement of the cytoskeleton and lamins can occur as a result of mechanical signals by topography [21]. Therefore, substrate topography, in which the cells grow, plays an important role in the onset, which then determines the next step of cell fate.
Stem cells can be administered directly through injection or by transplantation through a scaffold medium [22]. Scaffolds can serve as a place for cells to grow and differentiate in vitro in such a way that afterward they can be transplanted into the body. In vitro differentiation in tissue culture is usually regulated by adding growth factors to the growth medium. However, the use of growth factors has some limitations, especially in vivo. Proteolytic activity or protein degradation by enzymes in the body causes growth factors to become unstable and degrade; as a result, they have to be administered several times or in larger doses than normally found in the body to maintain an effective concentration. Therefore, costs are higher and unwanted effects may also occur [23]. Developments in topographic engineering methods to control cell behavior and differentiation can be a solution to these problems and can increase our understanding of the interactions and signaling processes that occur during MSCs' regulation of differentiation and regeneration [3]. Wang et al. were one of the first who showed that topography can affect MSC's differentiation. In their work, they found that elongated topographies can result in neural differentiation rather than osteogenic differentiation [24]. Besides neural proliferation, elongated topographies such as nanogrids was also suitable for diffraction [25].
Micropatterning is a method to engineer substrate topographies that resemble the microenvironment suitable for cells. The substrate is made with micron-sized patterns that define the adhesion of cells to the substrate [19]. Since its discovery, microtopography of substrates has been extensively studied to see how these features interact with cells. However, microenvironments also have nanoscale features. Nanopatterning, or the manufacture of nano motifs on substrates, can now be accomplished due to ever-developing nanotechnology [16]. How far or big the role of nanotechnology in creating microenvironments will greatly affect substrate-dependent osteogenic differentiation.
Nanopattern technology also has many challenges, such as difficulties in manufacturing and designing nanoscale geometries, as well as high costs with relatively low yields [26]. The effect of nanopatterns on osteogenic differentiation is evident through the upregulation of osteogenic markers on nanopattern surfaces compared with controls. Research by Kim et al. showed that the expression of Col I, RUNX2, and OPN was higher on nanopattern surfaces. In addition, their research also shows that nanopatterns can be combined with biochemical compounds to further improve results [27]. Further, Amaral et al. proved that nanopatterns can be embedded into scaffolds and can induce osteogenic differentiation even without osteogenic-inducing medium [28]. The role of nanopatterns in directing osteogenic differentiation is intriguing and also important to understand in the endeavor to develop bone tissue engineering applications.
Nanopatterns have great potential in tissue engineering because they can induce MSC differentiation in scaffolds through biophysical signals, which have several advantages over biochemical compounds such as growth factors. However, fabricating nanopatterns is quite difficult. Moreover, there are many types of nanopatterns, and pattern selection can affect cell contractility, which ultimately affects cell differentiation. Many aspects need to be considered, such as the required technology, the use of materials and the production costs [29]. These factors can be a challenge in the development of nanopattern fabrication methods. This concise review highlights several uses of nanopattern methods in inducing osteogenic differentiation, as well as the potential for their development in the future.

Resist
Resist as mask during etching process Etching process on surfaces without the resist Removal of resist to reveal the patterns formed Adapted with permission from [81] using Biorender.
the cytosol to the nucleus when activated by mechanical signals. A stiffer surface will increase integrin clustering and FA formation such that F-actin polymerization and stress fiber formation increase. Cells will spread out more and cover a larger area which will support YAP/TAZ translocation to the nucleus [37]. It is this balance between the ration of YAP and TAZ in the cytoplasm and nucleus that plays a role in the regulation of cell differentiation [37][38][39]. The role of YAP protein itself in osteogenesis has yet to be determined because contradictory results are often obtained. However, TAZ plays a role in osteogenesis by coactivating transcription by RUNX2 for osteogenesis as well as inhibiting transcription by PPARγ for adipogenesis [37]. Another signaling pathway that plays a particular role in osteoblast maturation is the Wnt/β-catenin pathway. Wnt proteins interact with frizzled (FZD) surface receptors such that the effector β-catenin is translocated to the nucleus while at the same time inhibiting the complex that degrades β-catenin, namely AXIN1 [40,41]. The Wnt/βcatenin signaling pathway is associated with calcification and osteogenic marker expression. It is also involved in a mutually reinforcing crosstalk with integrins during differentiation. Integrins regulate differentiation via the Wnt pathway, and Wnt increases integrin expression, resulting in further regulation of osteogenic differentiation [40,42]. The signaling pathways mentioned are involved in promoting osteogenic differentiation. Hence, the activity of proteins involved in these pathways, such as YAP and Wnt, can become an indicator or marker of osteogenic differentiation activity in cells [39,42]. Likewise, in nanotopographic engineering, markers from these signaling pathways are used to determine the role of nanopatterns in osteogenesis.

Development of nanopattern fabrication method
Nanopattern is a substrate engineering method to produce nanoscale structures. The first cultured cells utilizing topographic signals were reported in 1914 [43]. Developments in nanoscale fabrication technology made it possible to create nanotopographic structures. Nanopatterns themselves are used not only in the biological field but also in fields, for instance in electronics for electrochemical cells [44]. Nanopattern developments in the study of cell behavior started about three decades ago [45]. Even now, research on nanopatterns to find the optimal method or combination to direct cells according to needs is ongoing. Developments in this area are, of course, very dependent on advancements in the field of nanotechnology.
Different methods have been used to produce nanopatterns, with the research is still underway because many challenges in nanofabrication remain, such as difficulties in designing and producing nanoscale geometries as well as high costs with low yields [26]. Nanofabrication methods continue to shift over time. Various methods have been carried out through research to produce nanopatterns, such as sonication [46], sandblasting [47], solvothermal method [48], plasma oxidation [49] and other methods. However, several methods that are found quite often are lithography, etching and anodization. Lithography and its variations were used quite widely in the early 2010s [27,50,51], were continued with etching [52,53], and then were shifted to anodization during the end of the decade [54][55][56][57][58][59][60][61][62][63][64][65]. Developments of various nanopattern fabrication methods, the resulting patterns and the materials used can be seen in Table 1.
Lithography is a fabrication method that prints a pattern onto a target surface through a thin layer called resist. After the resist pattern has been printed, a subtractive transfer is carried out, which etches the target surface but only on the parts without the resist so that it forms a pattern ( Figure 1). Resist acts as a barrier during etching. Afterward, an additive transfer can also be carried out, which adds a layer of material to the opening that has 10 Shielded plasma oxidation and imprinting lithography   been formed. There are many types of lithography, such as photolithography, x-ray lithography, electron beam lithography, nano-imprint lithography and others [81][82][83].
Etching is indeed part of the processes in lithography. It is the process of engraving on a surface and does not require printing patterns on the resist. For example, Han et al. used the electrochemical etching method to form nanopores on GaN films. The film was immersed in an electrolyte solution and then electrified with a certain voltage such that pores formed on the film randomly [52]. Several etching methods are wet etching, dry etching and reactive ion etching (RIE). Wet etching is a chemical etching process that uses chemical solutions; dry etching is an etching method using high-energy ions fired onto surfaces under airtight conditions; and RIE is a combination of chemical and physical etching via radiofrequency plasma [81].
The anodization method has been widely used in recent years. Patterns formed by this method are usually nanotubes using titanium and its alloys as the substrate material. Titanium has advantages as a biomaterial, specifically, that is light, strong, biocompatible and rust-resistant. However, titanium's Young's modulus is often not enough to be used as a biomaterial, and therefore titanium is usually replaced with its alloy Ti6Al4V [84,85]. Anodization is the most popular method used for titanium material. This method is also relatively easy to do and does not require sophisticated tools. As the diameter of the resulting nanotubes can be customized by adjusting the applied voltage, the cost is not excessive. Tong et al. proved that western blot results in several osteogenic marker genes have higher relative mRNA expression on surfaces with nanotubes than those in flat surface controls [55].
Based on the three types of methods previously discussed, the most widely used type of nanopattern in the last 2 years is nanotubes produced through anodization. This method is commonly used for titanium material, which does have good performance in bone tissue engineering both in vitro and in vivo [86]. Among the three methods discussed, anodization is a relatively simple and easy to perform and regulate because it uses the principle of electrochemical oxidation with voltage variations. Although this method generally forms nanotubes, it can also be combined with other methods to form other types of nanopatterns. One example is nanopores by electrochemical nanopattern formation (ENF) which consists of anodization, sonication and chemical etching [64]. Considering anodization's simplicity, flexibility regarding customization, suitability of the material, and osteogenic differentiation ability, it has the potential for further developments in bone tissue engineering.

Role of nanopatterns in osteogenic differentiation induction
Nanopatterns were developed in the field of stem cell culture mainly to direct cell differentiation to the cell line that suits specific needs. Generally, cell differentiation is induced by chemical compounds such as growth factors. However, the use of chemical compounds for induction has its limitations. Growth factors need to be used in large doses to achieve the appropriate phenotype. Consequently, the costs can be great. In addition, maintenance of hMSCs in vivo after they are administered to patients is also very difficult [ Research on nanopatterns is driven by the knowledge that small changes in topography can have a significant influence in directing stem cell development [69]. For example, Kim et al. used several types of nanopatterns functionalized by BMP-2 (bone morphogenetic protein-2). They investigated how stem cells respond when grown on functionalized (compared with nonfunctionalized) nanopatterns, and with the addition (compared with lack) of osteogenic induction. qRT-PCR results of osteogenic markers expression (Col I, RUNX2, OPN) showed that surfaces with nanopatterns could upregulate osteogenic markers expression when compared with flat surfaces and further increased with BMP-2 functionalization [27].
Besides the presence of nanopatterns, the shapes and sizes of nanopatterns can also affect differentiation. Kim et al. showed that different patterns further increase different osteogenic markers expression. Col I and RUNX2 were expressed mainly in hexagonal dot patterns in substrates without functionalization and without induction medium. On substrates with induction medium, Col I expression was enhanced on substrates with BMP-2 functionalization and was more visible on groove-patterned substrates than dot-patterned substrates [27]. Li et al. demonstrated that differences in nanotubes diameter can affect differentiation. Fluorescence staining results showed that the levels of osteocalcin (OC), osteopontin (OP), and xylenol orange (calcification) were higher on substrates with 39 nm (20 V) diameter than 83 nm (40 V) [60].
Although nanopatterns can induce osteogenic differentiation, studies have shown that nanopatterns alone are not effective enough compared with using a combination of osteogenic induction medium with nanopattern. You et al. showed that ALP and Cbfa1 were expressed both in nanopattern with osteogenic medium and with growth medium only. However, OP and OC were significantly lower in nanopatterns with growth medium compared with those using the osteogenic medium. These results show that early-stage differentiation can be initiated using nanopatterns and growth medium only because there are no significant differences with when an osteogenic medium is used. However, for later-stage osteogenic differentiation, stronger induction from the osteogenic medium is required [50].
Further, Watari et al. conducted a study that was slightly similar to You et al. They used nanopatterns without osteogenic medium and nanopatterns with an added osteogenic medium. Nanopatterns with pitch sizes of 400 nm can significantly increase the expression of RUNX2, BGLAP and calcification compared with controls with or without an added osteogenic medium. The combination of nanopatterns with the osteogenic medium can provide additional effects that may be beneficial for people with serious conditions. Its effects as a biophysical signal on osteogenic differentiation can also last up to 2 weeks, whereas those from the biochemical medium usually last only 8 days or fewer [88]. The increase in expression on nanopatterns compared with control was higher in the nonosteogenic medium compared with the osteogenic medium. This result was observed on RUNX2 on day 3, BGLAP on day 10, and calcification on day 14. Thus, although nanopatterns cannot completely replace osteogenic medium, nanopatterns themselves can increase the expression of osteogenic markers compared with the control. Additional osteogenic medium is then beneficial to further increase the expression, if more is needed. Further research is needed to confirm whether using nanopattern alone for early differentiation and using induction medium for late differentiation generates consistent results, how this approach compares to using nanopatterns alone or using osteogenic medium from the beginning, and also what is the underlying mechanism.
Although nanopatterns can enhance osteogenic differentiation, as shown through osteogenic markers expression, the process still requires the addition of osteogenic mediums. Therefore, when using nanopatterns to achieve a certain level of differentiation, the required biochemical compounds for the osteogenic medium is reduced. Qian et al. used substrates with a roughness level of 1 and 200 nm with five types of mediums, namely control medium, complete osteogenic induction medium, induction medium without dexamethasone, induction medium without ascorbic acid, and induction medium without β-glycerophosphate. His research showed that the level of ALP expression was higher in the induction medium without dexamethasone compared with control, medium without ascorbic acid, and medium without β-glycerophosphate. Expression of RUNX2, ALP, and OPN on nanorough along with induction medium without dexamethasone was higher compared with the flat surface with complete osteogenic induction medium [53]. Another study, by Thiagarajan et al., stated that dexamethasone, which is commonly used for osteogenic differentiation, can trigger pleiotropic effects, which means it can cause adverse effects on the body. Two other compounds commonly used in inducing osteogenic differentiation, ascorbic acid and β-glycerophosphate, function to promote collagen production and supply inorganic phosphate for mineralization in vitro. Because these two compounds are naturally available in vivo, inducing agents aren't necessarily needed [89]. If nanopatterns can indeed replace dexamethasone's role, the number of induction compounds needed and also the risk of unwanted effects are also reduced. Nanopatterns with the addition of ascorbic acid and β-glycerophosphate alone may be sufficient during in vitro conditions, and during in vivo, induction effects can be maintained by the presence of nanopatterns.
These studies prove that nanopatterns can enhance osteogenic differentiation either alone or in combination with osteogenic media. The findings indicate that nanopatterns can also reduce the costs required for biochemical compounds, either by reducing their use or by increasing the expression of osteogenic markers, and hence make their use more efficient. Higher marker expressions on surfaces with nanopatterns indicate that stem cells' development into bone cells occurs faster than on surfaces without nanopatterns. Further, mechanical signals from nanotopography have the advantage of being more stable, controlled, and more quickly transmitted to the nucleus than chemical signals [90]. With the right size, nanopatterns can also form stable focal adhesion [91]. Focal adhesion stability can keep cytoskeletal tension stable in order for signals from nanopatterns to reach the nucleus properly. Initial cell attachment to the surface can affect osteogenic differentiation and the long-term stability of scaffolds after transplantation. Rapid and stable osseointegration, which is recognized through the cell's osteogenic differentiation rate, can reduce the risks of failure after implantation [65]. Thus, nanopatterns can be a very important first step in directing osteogenic differentiation and maintaining the stability of the differentiation process.
One of the important aspects to consider in tissue engineering implementation, besides biology and security, is the cost. As discussed, nanopatterns are generally able to show higher expression of osteogenic markers compared with controls. This expression is even higher when nanopatterns are combined with an osteogenic induction medium. Therefore, nanopatterns can accelerate the process of osteogenic differentiation and also reduce the use of growth factors. Nanopatterns can also provide more stable signaling compared with biochemical compounds. With these considerations, nanopatterns are economical and worth further development. Research in this area is still very wide open, including further comprehensive research on the financial aspect to see whether nanopatterns can reduce the use of growth factors, and thus be not only safer but also even more economical.

Nanopatterns potential in bone tissue engineering
The use of nanopatterns is currently still in the research stage. However, the ultimate goal of nanopattern research is for application, specifically in bone treatment by either injecting differentiated stem cells or transplanting them together with the scaffolds into the body. If transplanted, the nanopatterns must already be embedded in the scaffold, and the scaffold must be biocompatible in order to be transplanted into the body and to facilitate bone cells growth. In general, scaffolds for bone tissue engineering must meet several criteria. Biological criteria of scaffolds include biocompatibility, nontoxicity and biodegradability. In addition to biological criteria, there are also mechanical criteria. Scaffolds must be mechanically designed according to natural bone tissues to prevent complications. Finally, there are structural criteria, which include having certain levels of porosity to improve osseointegration and also being harnessed with nanotopography [92].
Nanotopographic structures of the scaffold play an important role in osteoinduction and also osteointegration (ability to integrate with bone). Nanopatterns provide biophysical signals and are transmitted through mechanotransduction into the nucleus with the help of integrins and protein complexes in the cell. Integrin will bind to proteins such as FAK, talin, vinculin and paxillin which will indirectly be connected to actin cytoskeletal components and subsequently will affect gene expression via signaling cascades. However, under conditions with nanopatterns, the FA formed is larger so that F-actin will undergo crosslinking to form stress fibers. Cell shape becomes tenser so that it can facilitate osteogenesis better [34]. Thus, biophysical signals from nanopatterns can regulate the expression of genes related to proliferation as well as differentiation of MSCs into osteoblasts.
Scaffolds can be transplanted into damaged bones when cells have reached the osteoblast stage. Osteoblasts can interact with other osteoblasts as well as with osteocytes. Osteoblasts can secrete matrixes that are beneficial for the mineralization process of osteoblasts themselves. Then, osteocytes present in bones can provide paracrine stimulation of osteoblasts to form more osteocytes and also inhibit osteoclast formation [93]. A functional scaffold that has a good degree of osseointegration will have a density similar to the natural bones with which it is integrated. Nanopatterns can affect the conformation of adsorbed proteins and increase attachment by integrins and ultimately promote osseointegration (Figure 2) [93,94]. Of course, the entire process requires support from plenty of research because the mechanisms involved are very complex. Nanopatterns on scaffolds will be able to maintain osteogenic induction effects after being transplanted because nanopatterns are already part of the scaffold, in contrast to biochemical compounds whose effects can decrease if not continuously supplemented. Notably, after transplantation, administrations of induction compounds pose some risks to the person. Negative effects that may be caused by biochemical compounds, such as the pleiotropic effect of dexamethasone, can be avoided by utilizing nanopatterns [89]. In this case, nanopatterns have benefits compared with adding growth factors. Materials for scaffolds, as previously discussed, are very important and have certain criteria. Different types of materials have been used for nanopattern research in inducing osteogenic differentiation, such as barium titanate ceramics [28], PDMS [74] and PS [75] polymers, graphene [76], silicones [78], carbon nanotubes [79], chitosan [95] and many others. Akhavan et al. showed one of the first works relating to the effect of surface nanotopography from graphene (surface coated by graphene nanoribbons in the form of nanogrids) on MSC's proliferation and differentiation [96]. Recently, graphene has been used in combination with nanostructures to create substrates that can enhance osteogenesis. Therefore, graphene are also highly promising because of its biocompatibility and cost-effectiveness [97,98]. However, titanium and its alloys are the most commonly used according to literatures [57][58][59][60][62][63][64][65][66]68]. Titanium has been mentioned as an advantageous material and can be fabricated into nanotubes by anodizing. However, the main disadvantage of using titanium as a material for scaffolds is that titanium is a non-biodegradable metal. Therefore, during the process of bone healing, a further operation is needed to remove the scaffold because there is the possibility of problems emerging in the long term. To address this issue, research on biodegradable metals has been carried out, for example magnesium alloys. However, the degradation of magnesium implant material in the body occurs through corrosion, raising concerns about its effects on the body [92]. Other biodegradable materials have also been widely used as scaffolds in nanopattern research. Some examples are polymers such as PCL [70][71][72][73], bioceramics such as calcium phosphate (CP) and β-TCP [69,77], and natural polymers such as chitosan [80]. Cellulose nanofibers with specific morphologies have also been used and may serve as a greener option [99]. It is known as a very effective, useful and biocompatible material for osteogenic differentiation. Composite nanofiber scaffolds from nano-sized demineralized bone powders (DBP) and biodegradable poly(Llactide) (PLA) have also been used for in vivo bone formation. The osteoconductive effect of PLA/DBP scaffolds showed greater results compared with PLA scaffolds and the composite scaffolds in vitro [100]. Further research on suitable scaffold materials is still needed, especially materials that can facilitate the differentiation process toward osteogenicity.

Conclusion & future perspective
Nanopattern as a method to induce osteogenic differentiation of MSCs has been widely studied. Various methods to produce nanopatterns have been and are being developed, and their development is highly dependent on the advancement of nanotechnology. Anodization has been researched widely in the last 2 years and has the potential to be further developed because it is quite simple and flexible regarding being varied. This method is usually used to create nanotube patterns that have been shown to increase osteogenic markers expression, which also indicates a faster differentiation process when compared with a flat surface.
However, the use of nanopatterns does not mean that growth factors do not need to be used. Combinations of nanopattern and growth factors can increase the effectiveness of osteogenic differentiation compared with nanopatterns alone. Nanopatterns can reduce the use of growth factors, which is beneficial for reducing costs and risks of side effects that can be caused by growth factors. In addition, mechanical signals provided by nanopatterns can provide a more stable, controlled, and faster signal that is transmitted to the nucleus.
In tissue engineering, nanopatterns can be applied in scaffolds such that their induced effects can persist even in vivo. The material used for scaffolds is also important. Titanium is one of the widely materials for bone implants because of its suitable characteristics. However, the use of metal has drawbacks because it is not biodegradable. Therefore, the development of biomaterials for nanopatterned scaffolds is wide open for exploration in the future for bone tissue engineering applications.
This short review has discussed a little about the nanopattern fabrication method and its role in inducing osteogenic differentiation, especially for tissue engineering. It provides a perspective regarding the potential use of nanopattern and recent advances in nanopattern function toward osteogenic differentiation of MSC for bone tissue engineering. Nanopatterns have great potential, especially in bone treatment, that further research and development may shed light on. It provides a perspective regarding the potential use of nanopattern and recent advances in nanopattern function toward osteogenic differentiation of MSC for bone tissue engineering.

Executive summary
• Nanopatterns have great potential in tissue engineering because they can induce mesenchymal stem cell differentiation in scaffolds via biophysical signals. • Regulation of mesenchymal stem cell behavior by biophysical signals occurs through mechanotransduction.
• Anodization can be a method with great potential for further development in bone tissue engineering.
• Nanopatterns can increase osteogenic markers expression and reduce costs of production and maintenance.
• A combination of nanopatterns and growth factors can further increase the effectivity of osteogenic differentiation induction; hence biochemical compounds are not required in large quantities. • Nanopatterned scaffolds made of suitable materials can improve osseointegration in bone tissue treatment.

Author contributions
A Barlian was responsible for the idea, analysis and synthesization. K Vanya was responsible for drafting and creating the manuscript. No writing assistance was utilized in the production of this manuscript.

Open access
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