Two-dimensional material-based bionano platforms to control mesenchymal stem cell differentiation
In the past decade, stem cells, with their ability to differentiate into various types of cells, have been proven to be resourceful in regenerative medicine and tissue engineering. Despite the ability to repair damaged parts of organs and tissues, the use of stem cells still entails several limitations, such as low differentiation efficiency and difficulties in guiding differentiation. To address these limitations, nanotechnology approaches have been recently implemented in stem cell research. It has been discovered that stem cells, in combination with carbon-based functional materials, show enhanced regenerative performances in varying biophysical conditions. In particular, several studies have reported solutions to the conventional quandaries in biomedical engineering, using synergetic effects of nanohybrid materials, as well as further development of technologies to recover from diverse health conditions such as bone fracture and strokes.
In this review, we discuss several prior studies regarding the application of various nanomaterials in controlling the behavior of stem cells. We focus on the potential of different types of nanomaterials, such as two-dimensional materials, gold nanoparticles, and three-dimensional nanohybrid composites, to control the differentiation of human mesenchymal stem cells (hMSCs). These materials have been found to affect stem cell functions via the adsorption of growth/differentiation factors on the surfaces of nanomaterials and the activation of signaling pathways that are mostly related to cell adhesion and differentiation (e.g., FAK, Smad, Erk, and Wnt).
Controlling stem cell differentiation using biophysical factors, especially the use of nanohybrid materials to functionalize underlying substrates wherein the cells attach and grow, is a promising strategy to achieve cells of interest in a highly efficient manner. We hope that this review will facilitate the use of other types of newly discovered and/or synthesized nanomaterials (e.g., metal transition dichalcogenides, non-toxic quantum dots, and metal oxide frameworks) for stem cell-based regenerative therapies.
KeywordsGraphene Two-dimensional materials Gold nanoparticles Three-dimensional graphene composites Human mesenchymal stem cell Differentiation
Alizarin red S
Chemical vapor deposition
Human mesenchymal stem cell
Recently, a wide variety of stem cells have been investigated for their extensive utility in biomedical applications, owing to their abilities to differentiate into specific cell lineages, and to generate more stem cells. Mesenchymal stem cells (MSCs), which are multipotent stromal stem cells, have been extensively investigated for their accessibility, versatility, and low risk of teratoma formation. Their multipotency allows them to differentiate into several specific cell types (e.g. adipocytes, osteoblasts, chondrocytes), to form fat , bone , and cartilage tissues . Traditionally, the process of stem cell differentiation has been controlled using media containing specific regulator proteins and biomolecules (e.g., dexamethasone, ascorbic acid, and β-glycerophosphate) . However, since cells actively interact with the underlying substrates/surfaces wherein they attach and grow, a method for controlling their functions including proliferation, migration, and differentiation, via biophysical factors, instead of induction media or the combination of two, has been recently proposed [5, 6, 7]. Such biophysical stimuli are induced by modifying the substrate/surface with cell-matrix interactions, which ultimately influence both cytoskeletal mechanics and cellular gene/protein expression [8, 9].
Until now, a variety of nanomaterials, including carbon nanotube (CNT) , fullerenes, and graphene , have been reported to guide stem cell differentiation with or without the presence of soluble differentiation factors. Among such materials, graphene and its derivative, graphene oxide (GO), have gained attention as unique materials to induce the physical stimulation required for stem cell differentiation. It has been reported that these features of amphiphilicity, surface chemistry, and honeycomb structures of GO [12, 13] affect cytoskeletal dynamics of cells adhered to the GO surface, which ultimately result in the changes of cell spreading, morphology and proliferation [14, 15, 16].
Several studies have reported the application of two-dimensional materials including graphene and its derivatives for productive differentiation of stem cells into desired lineages. Particularly, graphene oxide micropatterns, graphene nanopatterns, graphene, and nanomaterials hybrid platforms have been reported to promote the differentiation of hMSCs into osteocytes, adipocytes, and chondrocytes [17, 18, 19, 20, 21, 22]. This is also attributed to unique surface properties such as absorption/repulsion of specific differentiation factors, and the enhancement of cell adhesion through interactions between the cell membrane and the surface of the carbon materials [23, 24].
Mesenchymal stem cells behaviors on two-dimensional materials
hMSCs have been shown as a promising source for stem cell therapies and regenerative medicine due to their ability to self-renew and differentiate toward various types of cells, such as osteocytes, adipocytes, and chondrocytes [37, 38, 39, 40]. In addition, they can be easily isolated from the bone marrow, fat, and umbilical cord, and successfully expanded in vitro [41, 42]. However, several carbon-based materials have been lauded as versatile tools for establishing the future generation of biomaterials [43, 44, 45]. Although each carbon-based material, such as fullerene, carbon nanotubes, and graphene, presents its own advantages and disadvantages, graphene and its derivatives in particular have been used to guide the behavior of hMSCs [21, 46, 47, 48]. Graphene has several features that are advantageous for biomedical applications, owing to unique physiochemical properties, from its surface chemistry, amphiphilicity, and specific carbon structures . Subsequently, graphene and GO, once fully exploited, would drastically influence the spreading, morphology, and proliferation of stem cells, and become prospects for osteogenic differentiation of hMSCs [50, 51].
On the other hand, Lee et al.  discovered that the binding ability of graphene with several osteogenic differentiation factors could enhance the differentiation of hMSCs into the osteogenic lineage. They conducted an experiment wherein they cultured hMSCs on the CVD graphene. Several osteogenic differentiation factors, such as dexamethasone, ascorbic acid, and β-glycerophosphate, were used in the culturing process. The result showed that graphene had the ability to promote osteogenesis of hMSCs within 12 days, which was 9 days shorter than the prior studies. It indicates that osteogenesis in the presence of graphene could be achieved earlier than with conventional substrates.
Figure 3 (b) and (e), represent the oil red O (ORO) staining, which stains lipids when hMSCs are differentiated into adipocytes, demonstrating the adipogenetic capacity of hMSCs. Upon quantification, it was confirmed that the rate of adipogenesis in other substances was much higher than that in GO. Moreover, Fig. 3 (f) shows that the gene expression level of the hMSCs grown on BN substrates was the highest among the test substrates.
In addition, it is also known that graphene oxide nanoribbon (GONR) and reduced graphene oxide nanoribbon (rGONR) grids influence the osteogenic differentiation and proliferation of hMSCs, regardless of the presence or absence of differentiation inducing factors .
When osteogenic factors were present, the fastest osteogenic differentiation of hMSCs in rGONR grids was found to occur in about 7 days. The rapid osteogenic differentiation in rGONR was thought to be due to the high adsorption of differentiation inducing substances by rGONR and the physical properties induced by the surface characteristics of the nanogrids.
Moreover, many studies have confirmed the effects of graphene on the differentiation of other stem cells as well as hMSCs. For example, Chen et al.  reported the biocompatibility of G and GO toward long-term culture of induced pluripotent stem cells (iPSCs). Interestingly, iPSCs cultured on G and GO showed imbalance in differentiation tendencies. Especially, in the endodermal lineages, G interrupted spontaneous differentiation. On the other hand, GO promotes the differentiation of iPSCs most prominently along the ectodermal pathway, but differentiation into ectoderm and mesodermal is similar to iPSC incubated in both G and GO.
Consequently, it was found that graphene, GO, and other two-dimensional materials with their unique chemical and physical characteristics, enhance and guide the osteogenic or adipogenic differentiation of hADMSCs. In addition, we could confirm that 2D materials have various effects on the differentiation of hMSCs as well as other types of stem cells. This demonstrated that carbon-based materials were potential materials not only for regenerative medicine but also for the biomedical fields.
The effect of gold nanoparticles (AuNPs) on hMSCs growth and differentiation
Another type of nanomaterial with broad potential in biomedical application is gold nanoparticles (AuNPs). As mentioned already, AuNPs have been proposed as an attractive material for regenerative medicine, owing to their favorable physical properties, including biocompatibility arising from their low cytotoxicity, and abundant control over the particle size [56, 57, 58]. Numerous studies have investigated their application in biomedical fields such as biological imaging, chemical sensing, drug carriers, and disease treatments [59, 60, 61, 62, 63]. Most importantly, the negative charge on the surface of AuNPs makes gold nanoparticles more easily modifiable than the other NPs, such that the AuNPs can be functionalized by a wide range of biomolecules, drugs, DNA, antibodies, and functional peptides/polymers for favorable biomedical research and therapy .
Previously, AuNPs, functionalized with polymers such as chitosan-conjugated AuNPs, were developed to achieve advanced differentiation of human mesenchymal Stem Cells (hMSCs) . Chitosan, a type of aminated polysaccharide that has been utilized in bone tissue engineering, shows similarity to glycosaminoglycan, which plays an important role in extracellular matrix (ECM) interaction during cell adhesion. Moreover, further investigation discovered that chitosan polymers can promote osteogenic differentiation through Wnt/β-catenin signaling pathway [66, 67]. However, AuNPs themselves have been found to promote osteogenic differentiation of hMSCs by their stimulation through protein kinase 38 (p38) mitogen-activated protein kinase (MAPK) pathway. The difference in charge and the moiety of AuNPs have been shown to induce a series of cell responses towards osteogenesis . Hence, Yi et al. studied the use of AuNPs as a novel biomaterial to enhance the osteogenic differentiation of hMSCs and the associated molecular mechanisms .
Therefore, it can be concluded that AuNP surface functionalization with biomolecules is an effective strategy to enhance stem cell growth and differentiation. Although the use of AuNPs is highly promising in the field of stem cells for regenerative and therapy, further studies are needed to examine and develop the compatibility of various molecules in terms of nanoparticles conjugation for biological research.
Controlling differentiation of hMSCs using modified 3D graphene-based platform
Summary of studies using 3D graphene-based substrate for differentiation of MSCs
Type of carbon-based material
Type of cells
GFs promoted osteogenic differentiation without the use of chemical inducers.
rGO-Collagen hybrid (PADM-rGO)
Reduced Graphene Oxide
PADM-rGO promoted the differentiation of MSCs into neural cells after 7 days under neural differentiation condition.
The graphene / calcium silicate (GC) composite coating promoted adhesion and osteogenic differentiation of hMSCs and greatly increased the expression of genes involved in differentiation
Reduced Graphene Oxide
rGO - coated HA not only significantly enhanced osteogenic differentiation capacity of hMSC in osteogenic medium, but also significantly increased differentiation ability in basal medium.
Reduced Graphene Oxide
GO-PEDOT controlled osteogenic differentiation of hMSCs through electrical stimulation.
Among numerous candidates, it has been previously studied that composite coating with HA/CNTs presented higher durability and longer maintenance period than the conventional HA coating [89, 90, 91]. In this regard, graphene has received substantial attention, which is composed of the same material as CNTs , but has a higher surface area, thermal conductivity, and flexibility. It is also well known for its high biocompatibility and harmlessness, which is considered to be important for grafting . In this regards, Xie et al. studied graphene-reinforced calcium silicate coating (GC) technique, which was found be effective to generate a hierarchical nano−/microstructured surface . The hMSCs were cultured on the GC. As a result, the wear resistance was increased compared with the conventional CS coating, and the adhesion and proliferation of hMSCs in vitro were enhanced when the GC coating was applied. In addition, it was confirmed that gene expression related to osteogenesis, alkaline phosphatase (ALP), osteocalcin (OC), and osteopontin (OPN), was increased. In addition, rGO exhibits exceptional properties, similar to properties of graphene, as mentioned above. In addition, it has been recently shown by many researchers that graphene has the potential to guide osteogenesis of hMSCs [4, 11]. Hydroxylapatite (HA), in the form of microparticles, forms a three-dimensional environment to enhance cell adhesion and proliferation . By employing the advantages of these two materials, the authors developed rGO coated HA. Moreover, they demonstrated the enhancement of osteogenic differentiation of hMSCs when incubated in basal medium without any osteo-inductive molecules . In addition, the osteogenic activity of cells was further improved in osteogenic medium. The researchers assumed that the initial exposure of rGO-coated HA to cells facilitated intracellular signaling via a more intricate pathway. However, further research is required to explore the actual mechanism.
Conclusively, we have highlighted several 3D graphene-based platforms as a substrate for differentiation of hMSCs in this Review. The biocompatibility of these modified 3D scaffolds could be widely utilized for tissue engineering applications such as bone regeneration therapy.
In this review, we focused on several studies that used various nanohybrid materials for biomedical applications, with a particular focus on the use of two-dimensional materials, gold nanoparticles, and three-dimensional graphene composites .
Some of the prior reports have confirmed that two-dimensional materials and nanomaterials in combination with biological materials (e.g., growth factors, peptide, and proteins) enhance a number of cellular behaviors including cell adhesion, proliferation, migration and differentiation. Interestingly, these materials were especially excellent in performing as an attracting signal, not just for the osteogenesis of hMSCs but also for the enhancement of bone regeneration process.
In addition, three-dimensional carbon nanomaterials also have been utilized as the platform to support stem cell growth and differentiation. Unlike the two-dimensional platforms, which turned out to be suitable for controlling stem cells functions/behaviors in vitro, the three-dimensional carbon nanomaterials were found to be excellent in constructing 3D in vivo-like conditions ex vivo. Such approaches were useful to mimic structures of human tissues/organs, which is critical for the development of a new types of in vitro drug-screening tool such as organ-on-a-chip, as well as to develop tissue-biomaterial composites for the transplantation purpose. Although the use of bionano platforms for tissue regeneration is still in its early stages of development, certain biochemical and physical properties of those platforms, which includes tunable physical sizes, shapes, surface hydrophilicity, functional groups, entail a promising future for its development in biomedical fields, especially for the stem cell-based regenerative therapies.
This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare (grant number: HI15C3051); by a grant from the National Research Foundation of Korea (NRF) (grant number: 2016R1C1B1016088); and by the Nano Material Technology Development Program through the National Research Foundation of Korea (NRF) funded by the Korea Government (MSIP) (NRF-2014M3A7B4051907).
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ESK, DSK, IRS, WHL and IS searched references and collected information. THK established the structure of whole manuscript. ESK, DSK, IRS, WHL, IS and THK managed the figures and figure captions. All authors wrote and reviewed the manuscript. All authors read and approved the final manuscript.
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