**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 [10] and currently represents a challenge for the use of graphene-based nanomaterials in clinical applications. Although a lot of positive observa‐ tions 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 *in vitro* studies, which have aimed to evaluate different material compositions with GO content for biocompatibility, have reported a slight decrease in cell viability after contact with GO [11, 12]. However, cell response in contact with biomaterials can vary depending on the GO concentration and the material form of synthesis. Chng and Pumera study from 2013 [13] revealed that GO degree of cytotoxicity was related to the carbon/oxygen (C/O) ratio and the number and distribution of carbonyl residues on the surface of the material. Additionally, the particular conformation adopted by the GO sheets inside a material structure can have an impact on cell behavior in contact to the material [14]. Particularly, a higher degree of com‐ paction in GO sheets determined a lower viability in dermal fibroblasts. This decrease in viability was also associated with the increase in the levels of reactive oxygen species (ROS) in human dermal fibroblasts [14, 15]. Related to this, the activation of caspase-3 pro-apoptotic marker, as well as the release of lactate dehydrogenase (LDH) by PC12 cells, was also reported when the cells were cultured in highly condensed GO sheets materials. These observations lead to the hypothesis that added in very high concentrations to the scaffold or distributed as a very dense network to support material's structure, GO could actually determine a negative influence upon cell viability and response.

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 [16].

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 compo‐ nent [4], 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,

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 [10] and currently represents a challenge for the use of graphene-based nanomaterials in clinical applications. Although a lot of positive observa‐ tions 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

Most of the *in vitro* studies, which have aimed to evaluate different material compositions with GO content for biocompatibility, have reported a slight decrease in cell viability after contact with GO [11, 12]. However, cell response in contact with biomaterials can vary depending on the GO concentration and the material form of synthesis. Chng and Pumera study from 2013 [13] revealed that GO degree of cytotoxicity was related to the carbon/oxygen (C/O) ratio and the number and distribution of carbonyl residues on the surface of the material. Additionally, the particular conformation adopted by the GO sheets inside a material structure can have an impact on cell behavior in contact to the material [14]. Particularly, a higher degree of com‐ paction in GO sheets determined a lower viability in dermal fibroblasts. This decrease in viability was also associated with the increase in the levels of reactive oxygen species (ROS) in human dermal fibroblasts [14, 15]. Related to this, the activation of caspase-3 pro-apoptotic marker, as well as the release of lactate dehydrogenase (LDH) by PC12 cells, was also reported when the cells were cultured in highly condensed GO sheets materials. These observations lead to the hypothesis that added in very high concentrations to the scaffold or distributed as a very dense network to support material's structure, GO could actually determine a negative

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

biosensors, drug delivery, and tissue engineering [7–9].

152 Advanced Techniques in Bone Regeneration

tissue engineering.

influence upon cell viability and response.

**2. GO impact on material bioactivity and cytocompatibility**

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. [17] 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 [18] 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-suscep‐ tible properties of this material [19]. First, it was observed that the addition of polyethylene glycol (PEG) to GO ensures stability in physiological solutions [20]. Another study [21] emphasized that carboxylated graphene displays higher hydrophilicity and reduced cytotox‐ icity, due to the fact that carboxylation weakens the hydrophobic interactions between graphene and cellular membranes [19].

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 [22], 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 [23]. 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 aggre‐ gations 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 *in vitro* culture. Particularly, the composite material with 6 wt% GO proved to display the lowest cytotoxic potential by levels of lactate dehydrogenase released in the cell culture media and to favor most efficiently the proliferation of murine preosteoblasts during 1 week of culture in standard conditions. Statistical significant differences were observed in terms of viability and proliferation between nanomaterials with low GO content (0.5 and 1 wt%) and high GO content (2.5 and 6 wt%).

Similar results were obtained for nanofibrous biocomposite scaffolds of PVA/GO [24] 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 concentra‐ tion 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 [25]. 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 improve‐ ment 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 *in vitro* culture, but statistically significant values were obtained only for the composite with 1 wt% GO, when comparing cell viabilities at 7 and 4 days of culture. Additionally, the tendency of cell grouping was emphasized by fluorescence microscopy only for PS/GO 1 wt%, as compared to the other composites and to the PS membrane [25].

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

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 [23]. 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 aggre‐ gations 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 *in vitro* culture. Particularly, the composite material with 6 wt% GO proved to display the lowest cytotoxic potential by levels of lactate dehydrogenase released in the cell culture media and to favor most efficiently the proliferation of murine preosteoblasts during 1 week of culture in standard conditions. Statistical significant differences were observed in terms of viability and proliferation between nanomaterials with low GO content (0.5 and 1 wt%) and high GO content

Similar results were obtained for nanofibrous biocomposite scaffolds of PVA/GO [24] 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 concentra‐

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 [25]. 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 improve‐ ment 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 *in vitro* culture, but statistically significant values were obtained only for the composite with 1 wt% GO, when comparing cell viabilities at 7 and

proliferation.

154 Advanced Techniques in Bone Regeneration

(2.5 and 6 wt%).

tion up to 5 wt% in the composition.

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 [26]. 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 [27]. Previous reports have shown that chitosan is particularly attractive for bone reconstruc‐ tion 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 [27].

Another combination of chitosan (CS) and GO was used as a template to fabricate hydroxya‐ patite (HA) nanocomposites resembling bone structure [30]. CS–GO–HA and GO–HA matrices displayed good properties to support murine fibroblast and human osteoblast*-like* cells proliferation, but when compared in terms of viability and bioactivity toward minerali‐ zation, chitosan functionalized GO matrix provided better conditions for bone repair.

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 [31]. 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.
