**4. GO benefits for cell differentiation processes**

Scaffolds with different GO content have been previously reported as good substrates for osteogenic differentiation and consequently, for bone tissue regeneration therapies. The ability of graphene and GO to improve the characteristics of scaffold materials and to promote mesenchymal stem cells adhesion, proliferation, and differentiation toward osteogenic lineages has been intensely studied and demonstrated [3, 38, 1, 2, 39]. Lee et al. [33] have reported a proportional correlation between GO presence in the substrate material and the degree of cell osteogenic differentiation. Particularly, this study has highlighted the possibility that graphene-based substrates behave like concentration platforms for pro-osteogenic induction factors. Nayak et al. [1] have also shown that GO-covered materials accelerated osteogenic differentiation of human mesenchymal stem cells, as compared to the non-GOtreated-substrates. They concluded that the rate of differentiation conditioned by the GO scaffold is comparable to the osteogenic differentiation induced by specific growth factors and inducers in a conditional media.

**3. GO effects on cell adhesion**

156 Advanced Techniques in Bone Regeneration

direct correlation with the nanotopography conditioned by GO.

osteocytes capable to produce bone-specific extracellular matrix.

**4. GO benefits for cell differentiation processes**

observed for pure polysulfone or plysulfone with 0.5–1 wt% GO addition.

In general, it has been shown that the addition of GO favors the interaction between a cellular component and a material substrate, thus ensuring a positive effect on cell adhesion. Several studies [32, 33, 1] have demonstrated that bone marrow mesenchymal stem cells (BM-MSCs) developed a fusiform phenotype with multiple elongations and focal adhesion points in contact with graphene derivatives. These observations support the idea that GO favors cytoskeleton development and enhances cell adhesion to the material that contains GO. Experimental conditions used for 3D scaffolds based on chitosan ± GO or nylon ± GO [34, 2, 6] also concluded that osteoblasts or preosteoblasts adhered better in the presence of GO to the substrate materials. The mechanism underlying GO enhancement of cell adhesion has not been elucidated yet, but Kim et al. [35] suggested that the initiation of focal adhesions is in

From our experience, GO also induced a positive effect on murine preosteoblasts adhesion to polysulfone/GO biofilms [25]. A more developed F-actin cytoskeleton has been identified in the presence of 3 wt% GO by confocal microscopy, as compared to the cell cytoskeleton

To support this hypothesis, a substrate based on collagen and GO was developed and tested together with rat BM-MSCs for bioactivity in terms of cell viability, cell adhesion, and cell differentiation to bone cells [36]. An obvious dependency of F-actin fiber distribution with the

Other studies [37] described an increased cell adhesion when using GO in conjunction with fibronectin and titanium substrates. In this case, adhesion was evaluated by looking at focal adhesion molecules expression and localization. Vinculin was found to be highly active in the central and peripheral contact area of the cells cultivated in contact with fibronectin and GO. Good adhesion of cells to their substrate is crucial for cellular processes such as survival, growth, and activation of molecular pathways involved in proliferation. In particular, it has been shown several times that adhesion to the material is essential to induce the molecular program underlying osteogenic differentiation and maturation to functional osteoblasts and

Scaffolds with different GO content have been previously reported as good substrates for osteogenic differentiation and consequently, for bone tissue regeneration therapies. The ability of graphene and GO to improve the characteristics of scaffold materials and to promote mesenchymal stem cells adhesion, proliferation, and differentiation toward osteogenic lineages has been intensely studied and demonstrated [3, 38, 1, 2, 39]. Lee et al. [33] have reported a proportional correlation between GO presence in the substrate material and the degree of cell osteogenic differentiation. Particularly, this study has highlighted the possibility

GO content in the biomaterial was reported in this case, confirming our observations.

Great emphasis has been placed on the development of biomaterials that mimic the structure, composition, and properties of endogenous tissue using the biomimetic method [10]. Since the osteogenic process is based on a combination of signals that will promote the nucleation of hydroxyapatite [40–42], it is essential that the bioengineered scaffold has properties that will induce the assembly of bone*-like* apatite, resembling the natural bone [10]. Considering that charged groups can resemble extracellular matrix proteins and induce the mineralization process, functionalization of GO by bioactive molecules such as dopamine and carrageenan [43] or creation of an interface by modification of GO by gelatin [42] resulted in biomimetic mineralization of hydroxyapatite. Correlated to this enhancement in mineralization, higher cell proliferation, adhesion, and osteogenic potential as shown by alkaline phosphatase activity were reported for MC3T3-E1 preosteoblasts cultured in contact with GO–gelatin surface, as compared to the negative controls [42]. Consequently, these observations can further contrib‐ ute to the development of more efficient cell–scaffold interfaces based on GO properties for successful application in bone surgery.

Although it was confirmed by an increasing number of studies, the molecular mechanism underlying the ability of graphene or GO to induce by itself the osteogenic differentiation process has not yet been elucidated. Xie et al. [44] designed bidimensional and tridimensional graphene-based substrates to comparatively evaluate the crucial molecular events taking place during periodontal ligament stem cells differentiation to bone cells in these substrates. Bonespecific markers such as RUNX2, collagen type I, osteocalcin were found to be upregulated at gene and protein levels of expression in GO substrates, as a proof of differentiation. A combination of physical and chemical properties of graphene act synergistically to control the osteoinductive effect of graphene [44].

Since they did not show significant cytotoxicity during the biocompatibility studies, graphitic nanomaterials based on carbon nanotubes and carboxylated graphenes were evaluated for capacity to stimulate osteogenesis in the perspective of bone regeneration nanomedicine [45]. The study showed that the activation of the osteogenic differentiation program, synthesis of specific bone markers, and mineral deposition was possible for murine preosteoblasts in MC3T3-E1 cells cultivated in contact with these materials.

An interesting approach in order to evaluate the positive effects of GO on cell differentiation to bone was to incorporate GO nanoparticles in the structure of a scaffold designed for bone tissue reconstruction. Hybrid nanoparticles resulted from reduced GO nanosheets and strontium metallic nanoparticles were then incorporated in poly(ε-caprolactone) matrix with the purpose to test the composite for osteoinductive properties [46]. Increased rates of osteoblast proliferation and differentiation were detected for the scaffold containing GO nanoparticles, as compared to the control, and this bioactivity was associated with the release of strontium ions from the system.

Apart from its positive influence on cell viability and proliferation, functionalized graphene or GO proved also to favor efficient osteogenesis. By coating fibrin on the surface of GO, a novel nanocomposite (FGO) resulted as a potential solution for bone tissue engineering applications. Based on the analysis of bone markers' profile, release of calcium ions and alkaline phosphatase activity registered in osteoblast*-like* cells MG-63 cultivated in contact with this material, FGO was confirmed to have osteoinductive properties and to be a good candidate for medical applications [47]. Following the same trend of functionalized GO, another group of researchers [48] developed a gelatin functionalized GO composite with the purpose to use the surface charged proteins to mimic mineralization of hydroxyapatite and to obtain func‐ tional bone tissue and matrix. The gelatin–GO surface allowed bioactivity as cell adhesion and proliferation, and additionally, it promoted the formation of osteoid mineral matrix during murine cells osteogenic differentiation when compared to control glass surfaces.

The success and efficiency in bone regenerative medicine applications greatly depend on the structure and properties of the implantable biomaterials, but also on the source and type of cells used to condition regeneration. In the past few years, attention was focused on the use of adult stem cells that display the capacity to differentiate toward bone lineage. In this respect, mesenchymal stem cells became most widely used for bone replacement therapies since it was observed their preferential tendency to differentiate to osteogenic lineage when exposed to mechanically stiff scaffolds resembling bone tissue structure. One study [49] showed that when including GO flakes in the composition of soft collagen scaffolds, the resulted composite acquired the necessary stiffness and properties to support MSCs differentiation to bone*-like* cells. Moreover, enhanced osteogenesis was found in cells exposed to GO composite conditions as a result of good MSCs adhesion to the substrate.

An enhanced cell adhesion to the scaffold appears to be crucial for an efficient osteogenic differentiation process. Preosteoblasts, which were previously shown to strongly adhere to fibronectin/GO surface (Fn-Tigra) developed on titanium materials by electrodropping [37], were also shown to differentiate to mature osteoblasts able to produce osteocalcin, type I collagen, and calcium during 2 weeks of culture in contact with this substrate.

Bioceramics became very important in the context of bone tissue engineering. A group of researchers [50] designed a β-tricalcium phosphate covered in modified GO (β-TCP-GRA) and studied the interaction between this bioceramics, GO and stem cells, for bone reconstruction. This combination was found favorable for bone production, since the bioceramics significantly enhanced human BM-MSCs proliferation and osteogenic differentiation, as shown by alkaline phosphatase gene expression levels. Successful osteogenesis was also reported in the case of graphene nanogrids, which promoted the differentiation of human mesenchymal stem cells isolated from umbilical cord toward bone cells [51].

Mesenchymal stem cells isolated from goat cultivated on graphene-coated plates were also used as a potential platform for testing osteogenic differentiation in the view of bone tissue engineering [52]. This study emphasized the ability of oxidized graphene alone to induce osteogenesis process in goat MSCs in the absence of osteogenic inducers, thus proving the osteoinducing character of graphenes.

nanoparticles, as compared to the control, and this bioactivity was associated with the release

Apart from its positive influence on cell viability and proliferation, functionalized graphene or GO proved also to favor efficient osteogenesis. By coating fibrin on the surface of GO, a novel nanocomposite (FGO) resulted as a potential solution for bone tissue engineering applications. Based on the analysis of bone markers' profile, release of calcium ions and alkaline phosphatase activity registered in osteoblast*-like* cells MG-63 cultivated in contact with this material, FGO was confirmed to have osteoinductive properties and to be a good candidate for medical applications [47]. Following the same trend of functionalized GO, another group of researchers [48] developed a gelatin functionalized GO composite with the purpose to use the surface charged proteins to mimic mineralization of hydroxyapatite and to obtain func‐ tional bone tissue and matrix. The gelatin–GO surface allowed bioactivity as cell adhesion and proliferation, and additionally, it promoted the formation of osteoid mineral matrix during

murine cells osteogenic differentiation when compared to control glass surfaces.

as a result of good MSCs adhesion to the substrate.

isolated from umbilical cord toward bone cells [51].

The success and efficiency in bone regenerative medicine applications greatly depend on the structure and properties of the implantable biomaterials, but also on the source and type of cells used to condition regeneration. In the past few years, attention was focused on the use of adult stem cells that display the capacity to differentiate toward bone lineage. In this respect, mesenchymal stem cells became most widely used for bone replacement therapies since it was observed their preferential tendency to differentiate to osteogenic lineage when exposed to mechanically stiff scaffolds resembling bone tissue structure. One study [49] showed that when including GO flakes in the composition of soft collagen scaffolds, the resulted composite acquired the necessary stiffness and properties to support MSCs differentiation to bone*-like* cells. Moreover, enhanced osteogenesis was found in cells exposed to GO composite conditions

An enhanced cell adhesion to the scaffold appears to be crucial for an efficient osteogenic differentiation process. Preosteoblasts, which were previously shown to strongly adhere to fibronectin/GO surface (Fn-Tigra) developed on titanium materials by electrodropping [37], were also shown to differentiate to mature osteoblasts able to produce osteocalcin, type I

Bioceramics became very important in the context of bone tissue engineering. A group of researchers [50] designed a β-tricalcium phosphate covered in modified GO (β-TCP-GRA) and studied the interaction between this bioceramics, GO and stem cells, for bone reconstruction. This combination was found favorable for bone production, since the bioceramics significantly enhanced human BM-MSCs proliferation and osteogenic differentiation, as shown by alkaline phosphatase gene expression levels. Successful osteogenesis was also reported in the case of graphene nanogrids, which promoted the differentiation of human mesenchymal stem cells

Mesenchymal stem cells isolated from goat cultivated on graphene-coated plates were also used as a potential platform for testing osteogenic differentiation in the view of bone tissue engineering [52]. This study emphasized the ability of oxidized graphene alone to induce

collagen, and calcium during 2 weeks of culture in contact with this substrate.

of strontium ions from the system.

158 Advanced Techniques in Bone Regeneration

However, a small number of studies have focused until present on the effect of GO on human adipose derived stem cells (hASCs) osteogenic differentiation in 3D biomaterials designed for bone tissue engineering [53, 35]. hASCs have revealed encouraging results for adipose and cartilage tissue engineering and proved to be a valuable and more accessible source of adult stem cells than MSCs isolated from bone marrow. Thus, we have developed a strategy for *in vitro* differentiating hASCs inside chitosan-based biomaterials improved with 0.5–3 wt% GO for 28 days in order to study (i) the correlation between GO concentration and the degree of osteogenic differentiation; (ii) osteogenic markers gene expression evolution by qPCR; (iii) osteogenic markers protein expression by confocal microscopy; and (iv) accumulation of bonespecific extracellular matrix by histological staining in our experimental conditions (manu‐ script in preparation). Our results suggested that the degree of differentiation is strongly influenced by the content of GO in the material and that these materials are suitable for bone regeneration therapies.

Another hybrid scaffold between chitosan and GO was used as a template material for biomineralization of hydroxyapatite and tested as a possible material for bone tissue engi‐ neering. This combination proved to be beneficial for cellular activity including proliferation and attachment to the HAP–CS–GO system. Additionally, the scaffold allowed osteoblast growth and an increasing rate of mineralization during *in vitro* cell differentiation, confirming our results and the potential of chitosan/GO nanomaterials for bone regenerative therapies [54].

In the idea of creating an experimental platform for the evaluation of graphene properties for bone regeneration, Lu et al. [55] developed a self-supporting graphene hydrogel film (SGH), which proved to be cytocompatible and to allow cell adhesion and proliferation.

Nevertheless, the great potential of graphene and its derivatives for biomedical applications and their positive effects on cell viability, proliferation, adhesion, and osteogenic differentiation process have been already well documented. At this point, the challenge remains to elucidate the molecular pathways, which are active in the interaction between graphene and the cellular component and to explore and maximize the potential of graphene/ GO-based biomaterials as platforms for bone repair therapies and tissue engineering.
