Platforms to study
Currently, there are several tissue engineering strategies meant to overcome the incomplete or insufficient bone regeneration conditions offered by autologous bone graft or surgery approaches. In the last decade, attention has been focused toward finding the equilibrium between a suitable scaffold with osteoinductive properties, a cell source with evident potential to develop bone tissue and the appropriate pro-osteogenic factors to condition the differentiation process after cell-scaffold implantation. Consequently, this chapter aims to discuss the benefits that graphene and its derivatives, graphene oxide (GO), bring both to the scaffold biomaterial and to the interaction between the material and the cellular component in order to create a favorable micro-environment for efficient osteogenic differentiation process. Several advantages of including GO in the composition of the materials are shown in relation to cell viability, proliferation, attachment, and osteogenic differentiation.
- graphene oxide
- bone regeneration
- cell-scaffold interaction
- cell adhesion
New materials with outstanding osteoinductive properties and abilities to promote osteogenesis at the implant site are constantly developed for bone tissue engineering applications. One of these new-generation materials with documented pro-osteogenic effects is graphene [1–3]. Graphene and its derivatives are nanomaterials with specific physical and chemical properties compatible with bone regeneration, and therefore, they possess high potential for bone tissue engineering approaches. To date, the information about graphene and its derivatives contribution to bone tissue engineering is relatively limited. In this perspective, superior results were reported after graphene functionalization and immobilization of the derivative on different scaffold biomaterials. This approach was successful probably due to the fact that functional groups can reduce the hydrophobic interactions between graphene and the cellular component , thus enhancing improved biocompatibility of the resulted material. In particular, graphene oxide (GO) have been promoted as one of the most valuable graphene derivatives with excellent results in bone regeneration [5, 6]. Nowadays, the beneficial effects of graphene and its derivatives are tested in various biomedical applications—anti-cancer therapy, biosensors, drug delivery, and tissue engineering [7–9].
2. GO impact on material bioactivity and cytocompatibility
A very strong interconnection exists between the structural, physicochemical properties, and cytotoxic potential of the materials. Characteristics such as the flat shape, surface charges, and uncontrolled nanobiodegradability of graphene and its derivatives condition a relative nanocytotoxicity that has been reported  and currently represents a challenge for the use of graphene-based nanomaterials in clinical applications. Although a lot of positive observations related to the beneficial effects that graphene and GO have on cell growth, expansion, proliferation, and even differentiation of stem cells, caution and safety issues should still be taken into consideration when materials designed with graphene/GO are included in practical tissue engineering.
Most of the
For bone tissue engineering purposes, particularly for orthopedic implants, a composite film based on ultrahigh molecular weight polyethylene (UHMWPE) improved with 0.1–1 wt% graphene nanoplatelets was tested for cytocompatibility with bone cells. The cytotoxicity tests indicated that the increase in graphene nanoplatelets concentration could decrease bone cells viability over 5 days of culture, possibly due to the agglomeration of particles .
Other experiments have shown the contrary—that GO added in certain concentrations in the material has no influence upon cell viability or in some cases even has a positive effect on cell proliferation. In this respect, Sahu et al.  has published a study dedicated to thermosensitive hydrogel with GO content in regard to cytotoxicity and concluded that the addition of GO in the composition had no pro-inflammatory effects and that the hydrogel was biocompatible. Studies performed on titanium substrates coated with GO  also confirmed that graphene derivatives are biocompatible, present low toxicity, and a large dosage loading capacity, thus being able to function as a carrier for delivery of therapeutic proteins.
Conversely, a series of studies highlighted the importance of functionalizing graphene-based materials in order to minimize its potential cytotoxic effects. Graphene is hydrophobic and easily aggregates in solutions with salts, proteins, ions that can produce toxic effects. Covalent or non-covalent modifications can be performed in order to counteract the cytotoxic-susceptible properties of this material . First, it was observed that the addition of polyethylene glycol (PEG) to GO ensures stability in physiological solutions . Another study  emphasized that carboxylated graphene displays higher hydrophilicity and reduced cytotoxicity, due to the fact that carboxylation weakens the hydrophobic interactions between graphene and cellular membranes .
Based on positive results reported on grapheme derivates, we have recently tested for cytocompatibility nanomaterials based on polysulfone (PS) and different concentrations of carboxylated graphene (PS/G-COOH). Preliminary observations indicated that cells displayed a very good viability and adhesion in contact with these materials and that proliferation rates were improved as compared with control materials (pure polymer materials) (manuscript under revision).
In the same context, our group published a series of studies highlighting the importance of GO present in either bidimensional (2D) or tridimensional (3D) biomaterials for cell viability and proliferation.
When testing the cytocompatibility of chitosan/GO composite films , with 0.5, 1, 2.5, and 6 wt% GO content, MC3T3-E1 murine preosteoblasts adapted faster and proliferated more in contact with the chitosan/GO biocomposites with a higher content of GO. The biocomposite chitosan/GO 6 wt% proved to be biocompatible and displayed the most equilibrated ratio between the pro-proliferative and cytotoxic potential. In this case, viability and proliferation potential was assessed at 2, 4, and 7 days both quantitatively by MTT assay and qualitatively by LiveDead assay and by means of fluorescence microscopy. Fluorescence microscopy images revealed that cells progressively proliferated and reached confluent monolayers on all chitosan/GO biocomposite films, but the cellular density was found to be higher on the composite materials with 2.5 and 6 wt% GO content than that on the chitosan/GO composite films with lower GO content or 2D control. Additionally, a particular cell distribution was noticed for 2.5 and 6 wt% GO biomaterials, suggesting that GO could have an influence on cell behavior and distribution. The composites with 2.5 and 6 wt% GO content registered increased cell proliferation than the films with low GO loading and controls, particularly after 7 days of culture, as shown by MTT. Conversely, LDH quantification showed a significantly lower profile for chitosan/GO 6 wt% biocomposite than for control chitosan, thus supporting the hypothesis that increase in GO content in material’s composition positively influences cell proliferation.
Further on, similar studies were carried out for graphene oxide/chitosan–polyvinyl alcohol films (CS–PVA/GO) in order to determine the cytocompatibility of these materials and the possible interference of GO with cell viability and proliferation . Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray diffraction (XRD) were first employed to assess CS–PVA/GO nanocomposites structural and surface properties. Good GO nanosheets dispersion within the polymer matrix and excellent thermal stability and mechanical strength were shown for these composites, while the highest tensile modulus was obtained for CS–PVA/GO 6 wt%. During biocompatibility tests, an interesting cell distribution was highlighted when the GO concentration increased in the composition of the nanomaterials. Cell alignment and behavior were correlated with the observed GO nanosheets small aggregations within the polymer matrix. Simultaneously, no significant cytotoxic potential was reported for the composites even when increasing the GO concentration to 2.5 or 6 wt% and a general increasing profile of cell viability and proliferation was described during 7 days of
Similar results were obtained for nanofibrous biocomposite scaffolds of PVA/GO  using the same MC3T3-E1 preosteoblasts. In this case, cells were able to grow and attach to the surface of the materials and not change in cell viability was indicated when increasing GO concentration up to 5 wt% in the composition.
A composite with particular good results, holding promises for future biomedical application as a filtration membrane, nanocarrier, or support for bone regeneration, is a bidimensional film based on polysulfone (PS) and GO nanosheets . In this case, PS composites with 0.25, 0.5, and 1 wt% GO were compared in terms of cytocompatibility with PS controls. Based on special conditions of synthesis, the GO nanosheets were uniformly distributed within the PS matrix, thus ensuring a more ordered structure, as revealed by XRD analysis. Clear improvement of thermal and mechanical properties of the composites was revealed when GO was added in the matrix. These changes in the structure were correlated with the bioactivity tested for PS/GO nanomaterials. Very low levels of cytotoxicity were detected during 1 week of culture for all compositions, and no relevant increase in LDH levels was found when 0.25–1 wt% GO was added, suggesting that the low cytotoxic potential of the composite was due to the basal cytotoxicity of the PS substrate. Conversely, quantitative data showed a slight increase in cell viability during 7 days of
Similarly, membranes based on poly(ε-caprolactone) (PCL) reinforced with GO nanoplatelets revealed good results toward use in bone regeneration due to the improvements in bioactivity . PCL/GO nanocomposites showed better mechanical properties than PCL films due to the fiber organization and strengthening offered by GO, reflected also in better bioactivity due to the anionic functional groups on GO surface.
Due to the tridimensional structure of the bone, in certain bone reconstruction applications, a tridimensional porous scaffold is required to mimic bone and to resemble the appropriate conditions for regeneration. Thus, tridimensional materials with mechanical and physical–structural properties close to bone were investigated for biocompatibility and potential for bone tissue engineering. In this respect, the cytocompatibility of chitosan/GO scaffolds improved with 0.5 and 3 wt% GO has been tested both by means of indirect and direct studies . Previous reports have shown that chitosan is particularly attractive for bone reconstruction medical applications due to its good biocompatibility, biodegradability, and ability to support osteoblast attachment and proliferation [28, 29]. Remarkably, the addition of GO to the composition of the scaffolds did not affect cell viability, but even resulted in a lower cytotoxicity of the extract collected from chitosan/GO 3 wt% after 24 h of contact with cells. These observations were correlated with the increasing proliferation profile obtained by MTT assay after 7 days of direct contact between murine preosteoblasts from MC-3T3 line and the materials. The data showed that the addition of 3 wt% GO to the chitosan matrix greatly improved the composite properties and bioactivity, suggesting that GO could have positive effects on cell behavior and metabolic activity .
Another combination of chitosan (CS) and GO was used as a template to fabricate hydroxyapatite (HA) nanocomposites resembling bone structure . CS–GO–HA and GO–HA matrices displayed good properties to support murine fibroblast and human osteoblast
Preliminary positive results for tridimensional GO-containing scaffolds designed specifically for bone tissue repair were also recently reported for gelatin–poly(vinyl alcohol) biocomposites reinforced with GO . In this case, the combination between a naturally occurring compound (gelatin), a synthetically derived one (polyvinyl alcohol) and GO resulted in a biocomposite with equilibrated physical–chemical properties and low cytotoxic profile that allowed murine preosteoblasts viability.
Further tests are required to select the most appropriate biocomposites to serve as platforms to study osteogenic differentiation and thus to validate the most promising biomaterials with application in bone regeneration therapies.
3. GO effects on cell adhesion
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.  suggested that the initiation of focal adhesions is in direct correlation with the nanotopography conditioned by GO.
From our experience, GO also induced a positive effect on murine preosteoblasts adhesion to polysulfone/GO biofilms . 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 observed for pure polysulfone or plysulfone with 0.5–1 wt% GO addition.
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 . An obvious dependency of F-actin fiber distribution with the GO content in the biomaterial was reported in this case, confirming our observations.
Other studies  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 osteocytes capable to produce bone-specific extracellular matrix.
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.  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.  have also shown that GO-covered materials accelerated osteogenic differentiation of human mesenchymal stem cells, as compared to the non-GO-treated-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.
Great emphasis has been placed on the development of biomaterials that mimic the structure, composition, and properties of endogenous tissue using the biomimetic method . 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
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.  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. Bone-specific 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 .
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 . 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 . 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
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  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
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 , 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  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 .
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 . 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.
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
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 engineering. 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 the idea of creating an experimental platform for the evaluation of graphene properties for bone regeneration, Lu et al.  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.
In vivo GO effects during bone regeneration therapies
Regeneration of large bone defects requires development of bioactive scaffolds with distinct properties of promoting stem cells osteogenic differentiation and inducing the
|Material||Post-implant analysis||Biological effects||References|
oxide (rGO) and
|GO-coated titanium implants||Mouse
|2, 4, and
flakes suspended in
fibrin gels (GO/F) for
|Subcutaneous sites of rats||
|Calcium silicate (CS) ceramic reinforced with 1.5 wt% graphene plates (GPs)||Rabbit femur condyle defect||1–3 months||
Up to date, there is a small number of
The authors acknowledge the sources of funding that supported their own studies in the field of graphene-based composites for bone tissue engineering: a grant of the Romanian National Authority for Scientific Research, Executive Agency for Higher Education, Research, Development and Innovation, Project Number PN-II-PCCA-140/2012. Additionally, the studies were financed by the Institute for Research of the University of Bucharest (ICUB), through “Scholarships for Excellence in Research for young researchers, 2015 competition” project.
Nayak T.R., Andersen H., Makam V.S., Khaw C., Bae S., Xu X., Ee P.L., Ahn J.H., Hong B.H., Pastorin G., Özyilmaz B. Graphene for controlled and accelerated osteogenic differentiation of human mesenchymal stem cells. ACS Nano. 2011; 5:4670–4678.
Depan D., Misra R.D. The interplay between nanostructured carbon-grafted chitosan scaffolds and protein adsorption on the cellular response of osteoblasts: structure function property relationship. Acta Biomater. 2013; 9:6084.
Gu M., Liu Y., Chen T., Du F., Zhao X., Xiong C., Zhou Y. Is graphene a promising nano-material for promoting surface modification of implants or scaffold materials in bone tissue engineering? Tissue Eng Part B Rev. 2014; 20:477–491.
Salavagione H.J., Martinez G., Ellis G. Recent advances in the covalent modification of graphene with polymers. Macromol Rapid Commun. 2011; 32:1771.
Depan D., Girase B., Shah J.S., Misra R.D. Structure–process–property relationship of the polar graphene oxide-mediated cellular response and stimulated growth of osteoblasts on hybrid chitosan network structure nanocomposite scaffolds. Acta Biomater. 2011; 7:3432.
Misra R.D., Chaudhari P.M. Cellular interactions and stimulated biological functions mediated by nanostructured carbon for tissue reconstruction and tracheal tubes and sutures. J Biomed Mater Res A. 2013; 101:528
Fiorillo M., Verre A.F., Iliut M. Graphene oxide selectively targets cancer stem cells, across multiple tumor types: implications for non-toxic cancer treatment, via “differentiation-based nano-therapy”. Oncotarget. 2015; 6(6):3553–3562.
Pumera M. Graphene in biosensing. Mater Today. 2011; 14(7–8):308–315. doi:10.1016/S1369-7021(11)70160-2
Goenka S., Sant V., Sant S. Graphene-based nanomaterials for drug delivery and tissue engineering. J Control Release. 2014; 173:75–88.
Shadjou N., Hasanzadeh M. Graphene and its nanostructure derivatives for use in bone tissue engineering: recent advances. 2016. doi:10.1002/jbm.a.35645
Cai X., Tan S., Yu A., Zhang J., Liu J., Mai W. Sodium 1-naphthalenesulfonate-functionalized reduced graphene oxide stabilizes silver nanoparticles with lower cytotoxicity and long-term antibacterial activity. Chem Asian J. 2012; 7:1664.
Hong B.J., Compton O.C., An Z., Eryazici I., Nguyen S.T. Successful stabilization of graphene oxide in electrolyte solutions: enhancement of biofunctionalization and cellular uptake. ACS Nano. 2012; 6:63.
Chng E.L., Pumera M. The toxicity of graphene oxides: dependence on the oxidative methods used. Chemistry. 2013; 19:8227.
Liao K.H., Lin Y.S., Macosko C.W., Haynes C.L. Cytotoxicity of graphene oxide and graphene in human erythrocytes and skin fibroblasts. ACS Appl Mater Interfaces. 2011; 3:2607.
Yang K., Li Y., Tan X., Peng R., Liu Z. Behavior and toxicity of graphene and its functionalized derivatives in biological systems. Small. 2013; 9:9–10.
Lahiri D., Dua R., Zhang C., de Socarraz-Novoa I., Bhat A., Ramaswamy S., Agarwal A. Graphene nanoplatelet-induced strengthening of ultrahigh molecular weight polyethylene and biocompatibility in vitro. ACS Appl Mater Interfaces. 2012; 4(4):2234–2241. doi:10.1021/am300244s
Sahu A., Choi W.I., Tae G. A stimuli-sensitive injectable graphene oxide composite hydrogel. Chem Commun. 2012; 48:5280.
La W.G., Park S., Yoon H.H., Jeong G.J., Lee T.J., Bhang S.H., Han J.Y., Char K., Kim B.S. Delivery of a therapeutic protein for bone regeneration from a substrate coated with graphene oxide. Small. 2013; 9:4051–4060.
Zhang B., Wang Y., Zhai G. Biomedical applications of the graphene-based materials. Mater Sci Eng C. 2016; 61:953–964. doi:10.1016/j.msec.2015.12.073
Liu Z., Robinson J.T., Sun X., Dai H. PEGylated nano-graphene oxide for delivery of water insoluble cancer drugs. J Am Chem Soc. 2008; 130(33):10876–10877.
Sasidharan A., Panchakarla L.S., Chandran P., Menon D., Nair S., Rao C.N., Koyakutty M. Differential nano-bio interactions and toxicity effects of pristine versus functionalized graphene. Nanoscale. 2011; 3(6):2461–4.
Pandele A.M., Dinescu S., Costache M., Vasile E., Obreja C., Iovu H., Ionita M. Preparation and in vitro, bulk, and surface investigation of chitosan/graphene oxide composite films. Polym Compos. 2013; 34(12):2116–2124.
Pandele A.M., Ionita M., Crica L., Dinescu S., Costache M., Iovu H. Synthesis, characterization, and in vitrostudies of graphene oxide/chitosan–polyvinyl alcohol films. Carbohydr Polym. 2014; 102:813–820.
Qi Y.Y., Tai Z.X., Sun D.F., Chen J.T., Ma H.B., Yan X.B., Liu B., Xue Q.J. Fabrication and characterization of poly(vinyl alcohol)/graphene oxide nanofibrous biocomposite scaffolds. J Appl Polym Sci. 2013; 127:1885–1894.
Ionita M., Vasile E., Crica L.E., Voicu S.I., Pandele A.M., Dinescu S., Predoiu L., Galateanu B., Hermenean A., Costache M. Synthesis, characterization and in vitrostudies of polysulfone/graphene oxide composite membranes. Compos Part B. 2015; 72:108–115.
Wan C.Y., Chen B.Q. Poly(epsilon-caprolactone)/graphene oxide biocomposites: mechanical properties and bioactivity. Biomed Mater. 2011; 6(5):055010. doi:10.1088/1748-6041/6/5/055010
Dinescu S., Ionita M., Pandele A.M., Galateanu B., Iovu H., Ardelean A., Costache M., Hermenean. In vitro cytocompatibility evaluation of chitosan/graphene oxide 3D scaffold composites designed for bone tissue engineering. Biomed Mater Eng. 2014; 24(6):2249–2256.
Guo B., Glavas L., Albertsson A.C. Biodegradable and electrically conducting polymers for biomedical applications. Prog Polym Sci. 2013; 38:1263–1286.
Lan Levengooda S.K., Zhang M. Chitosan-based scaffolds for bone tissue engineering. J Mater Chem B. 2014; 2:3161–3184.
Li M., Wang Y.B., Liu Q., Li Q.H., Cheng Y., Zheng Y.F., Xi T.F., Wei S.C. In situ synthesis and biocompatibility of nano hydroxyapatite on pristine and chitosan functionalized graphene oxide. J Mater Chem B. 2013; 1(4):475–484 doi:10.1039/c2tb00053a
Ionita M., Crica L.E., Tiainen H., Haugen H.J., Vasile E., Dinescu S., Costache M., Iovu H. Gelatin–poly(vinyl alcohol) porous biocomposites reinforced with graphene oxide as biomaterials. J Mater Chem B. 2016; 4:282–291. doi:10.1039/C5TB02132D
Kalbacova M., Broz A., Kong J., Kalbac M. Graphene substrates promote adherence of human osteoblasts and mesenchymal stromal cells. Carbon. 2010; 48:4323–4329.
Lee W.C., Lim C.H., Shi H., Tang L.A., Wang Y., Lim C.T., Loh K.P. Origin of enhanced stem cell growth and differentiation on graphene and graphene oxide. ACS Nano. 2011; 5:7334–7341.
Shi X.T., Chang H.X., Chen S., Lai C., Khademhosseini A., Wu H.K. Regulating cellular behavior on few-layer reduced graphene oxide films with well-controlled reduction states. Adv Funct Mater. 2012; 22:751.
Kim J., Kim Y.R., Kim Y., Lim K.T., Seonwoo H., Park S. Graphene-incorporated chitosan substrata for adhesion and differentiation of human mesenchymal stem cells. J Mater Chem B. 2013; 1:933.
Zhang W.H., Sun H.Y., Mou Y.C., Jiang X.X., Wei L.J., Wang Q.Y., Wang C.Y., Zhou J., Cao J.K. Graphene oxide-collagen matrix accelerated osteogenic differentiation of mesenchymal stem cells. J Biomater Tissue Eng. 2015; 5(4):257–266. doi:10.1166/jbt.2015.1314
Subbiah R., Du P., Van S.Y., Suhaeri M., Hwang M.P., Lee K., Park K. Fibronectin-tethered graphene oxide as an artificial matrix for osteogenesis. Biomed Mater. 2014; 9(6):065003. doi:10.1088/1748-6041/9/6/065003
Mooney E., Dockery P., Greiser U., Murphy M., Barron V. Carbon nanotubes and mesenchymal stem cells: biocompatibility, proliferation and differentiation. Nano Lett. 2008; 8(8):2137–2143.
Bressan E., Ferroni L., Gardin C., Sbricoli L., Gobbato L., Ludovichetti F., Tocco I., Carraro A., Piattelli A., Zavan B. Graphene based scaffolds effects on stem cells commitment. J Transl Med. 2014;c 12(1):296.
Hunter G.K., Goldberg H.A. Modulation of crystal formation by bone phosphoproteins: role of glutamic acid-rich sequences in the nucleation of hydroxyapatite by bone sialoprotein. Biochem J. 1994; 302:175–179.
Liu H., Xi P., Xie G., Shi Y., Hou F., Huang L., Chen F., Zeng Z., Shao C., Wang J. Simultaneous reduction and surface functionalization of Graphene oxide for hydroxyapatite mineralization. J Phys Chem C. 2012; 116:3334–3341.
Liu H., Cheng J., Chen F., Bai D., Shao C., Wang J., Xi P., Zeng Z. Gelatin functionalized graphene oxide for mineralization of hydroxyapatite: biomimetic and in vitro evaluation. Nanoscale. 2014; 6:5315–5322.
Cheng J., Liu H., Zhao B., Shen R., Liu D., Hong J., Wei H., Xi P., Chen F., Bai D. MC3T3-E1 preosteoblast cell-mediated mineralization of hydroxyapatite by polydopamine-functionalized graphene oxide. J Bioact Compat Polym. 2015; 10:1–13.
Xie H., Cao T., Gomes JV., Neto A.H.C., Rosa V. Two and three-dimensional graphene substrates to magnify osteogenic differentiation of periodontal ligament stem cells. Carbon. 2015;93:266–275. doi:10.1016/j.carbon.2015.05.071
Mahmood M., Villagarcia H., Dervishi E., Mustafa T., Alimohammadi M., Casciano D., Khodakovskaya M., Biris A.S. Role of carbonaceous nanomaterials in stimulating osteogenesis in mammalian bone cells. J Mater Chem B. 2013; 1(25):3220–3230. doi:10.1039/c3tb20248h
Kumar S., Chatterjee K. Strontium eluting graphene hybrid nanoparticles augment osteogenesis in a 3D tissue scaffold. Nanoscale. 2015; 7(5):2023–2033. doi:10.1039/c4nr05060f
Deepachitra R., Chamundeeswari M., Kumar B.S., Krithiga G., Prabu P., Devi M.P., Sastry T.P. Osteo mineralization of fibrin-decorated graphene oxide. Carbon. 2013; 56:64–76. doi:10.1016/j.carbon.2012.12.070
Liu H.Y., Cheng J., Chen F.J., Bai D.C., Shao C.W., Wang J., Xi P.X., Zeng Z.Z. Gelatin functionalized graphene oxide for mineralization of hydroxyapatite: biomimetic and in vitro evaluation. Nanoscale. 2014; 6(10):5315–5322. doi:10.1039/c4nr00355a
Kang S., Park J.B., Lee T.J., Ryu S., Bhang S.H., La W.G., Noh M.K., Hong B.H., Kim B.S. Covalent conjugation of mechanically stiff graphene oxide flakes to three-dimensional collagen scaffolds for osteogenic differentiation of human mesenchymal stem cells. Carbon. 2015; 83:162–172. doi:10.1016/j.carbon.2014.11.029
Wu C., Xian L., Han P., Xu M., Fang B., Wang J., Chang J., Xiao Y. Graphene oxide modified beta-tricalcium phosphate bioceramics stimulate in vitro and in vivo osteogenesis. Carbon. 2015; 93:116–129.
Akhavan O., Ghaderi E., Shahsavar M. Graphene nanogrids for selective and fast osteogenic differentiation of human mesenchymal stem cells. Carbon. 2013; 59:200–211.
Elkhenany H., Amelse L., Lafont A., Bourdo S., Caldwell M., Neilsen N., Dervishi E., Derek O., Biris A.S., Anderson D. Graphene supports in vitro proliferation and osteogenic differentiation of goat adult mesenchymal stem cells: potential for bone tissue engineering. J Appl Toxicol. 2015; 35(4):367–374. doi:10.1002/jat.3024
Lyu J.Y., Cao C.H., Luo D., Fu Y.X., He Y.S., Zou D.R. Induction of osteogenic differentiation of human adipose-derived stem cells by a novel self-supporting graphene hydrogel film and the possible underlying mechanism. ACS Appl Mater Interfaces. 2015; 7(36):20245–20254. doi:10.1021/acsami.5b05802
Depan D., Pesacreta T.C., Misra R.D.K. The synergistic effect of a hybrid graphene oxide-chitosan system and biomimetic mineralization on osteoblast functions. Biomater Sci. 2014; 2(2):264–274. doi:10.1039/c3bm60192g
Lu J.Y., He Y.S., Cheng C., Wang Y., Qiu L., Li D., Zou D.R. Self-Supporting graphene hydrogel film as an experimental platform to evaluate the potential of graphene for bone regeneration. Adv Funct Mater. 2013; 23(28):3494–3502. doi:10.1002/adfm.201203637.
Lee J.H., Shin Y.C., Lee S.M., Jin O.S., Kang S.H., Hong S.W., Jeong C.M., Huh J.B., Han D.W. Enhanced osteogenesis by reduced graphene oxide/hydroxyapatite nanocomposites. Sci Rep. 5:18833. doi:10.1038/srep18833
La W.G. et al. Delivery of bone morphogenetic protein-2 and substance p using graphene oxide for bone regeneration. Int J Nanomed. 2014; 9:107–116.
La W.G., Jung M.J., Yoon J.K., Bhang S.H., Jang H.K., Lee T.J., Yoon H.H., Shin J.Y., Kim B.S. Bone morphogenetic protein-2 for bone regeneration—dose reduction through graphene oxide-based delivery. Carbon. 2014; 78:428–438.
Xie Y., Li H., Ding C., Zheng X., Li K. Effects of graphene plates’ adoption on the microstructure, mechanical properties, and in vivo biocompatibility of calcium silicate coating. Int J Nanomed. 2015; 10:3855–3863.