Bioactivity and Mechanical Properties Characterization of Bioactive Glass Incorporated with Graphene Oxide

In this study, the bioactivity and the mechanical properties (Mechanical compressive strength, Hardness, and density) of bioglass (BG) and bioglass/graphene oxide (BG/GO) were investigated. Bioglass in chemical composition [60SiO2_35CaO_5P2O5] was prepared via the sol–gel method. GO was added to the bioglass (BG) with different contents (0.5, 1, 2, and 3) wt.% named as 0.5%GO, 1%GO, 2%GO, and 3%GO samples respectively. The synthesized specimens were characterized by several techniques Fourier Transform Infrared (FT-IR), X-Ray Diffraction (XRD), and Scanning Electron Microscopy (SEM). Compressive strength, Hardness, and density were studied also by different techniques to obtain the optimum Mechanical samples. The biological activity was studied by an in-vitro test in simulated body fluid (SBF) for 33 days. Results showed that: the 0.5%GO sample exhibited optimum mechanical compressive strength by approximately 82% compared to the BG sample. Hardness was increased from 0.5%GO sample up to 1%GO sample compared to BG sample and gradually decreased in 2%GO Sample and 3%GO. Bioactivity results showed deposition of HA layer on the bioglass surface and there was no significant change in it with the addition of graphene oxide.


Introduction
The second generation of biomaterial like bioactive glass has good bioactivity, biocompatibility, and biodegradability [1][2][3]. They have bone-bonding ability via the formation of a hydroxyapatite layer on their surface and induce angiogenesis [4,5]. To improve their low fracture toughness, which is thought to be the main drawback that restricts the use of BG in load-bearing site applications, polymers, and metal oxides are frequently added to BG [6][7][8][9][10][11][12]. Researchers have attempted to improve the mechanical strength and hardness of burglars, by incorporating different materials into them, including polymers, carbon fibers, and metal oxides [13][14][15]. Graphene oxide (GO) has attracted researchers' attention in the tissue engineering field due to its good mechanical properties and functionalized ease [16]. Graphene oxide (GO) and some types of polymers are used to improve the mechanical properties and cell interaction [17]. Interestingly, recent studies have shown that the mechanical properties and bone formation potential can be enhanced by removing the functional oxygen groups [18]. Given the high strength of GO and excellent biocompatibility of BG, it is expected that the combination of GO and BG could provide a guarantee for improving mechanical strength. Nevertheless, studies have shown that due to significant differences in the physicochemical properties between GO and BG, problems such as poor interfacial bonding [16,19].
The aim of the work, in this study, a bioglass with the chemical composition [60SiO 2 _35CaO_5P 2 O 5 ] was combined with graphene oxide to improve its poor mechanical properties as a bioactive material. BG powder was synthesized by the sol-gel method and mechanically mixed with different contents of graphene oxide (0.5, 1, 2, and 3%) wt. Mechanical properties in terms of compressive strength were studied by compression test. Other mechanical properties like Hardness and density were being examined. The bioactivity of (BG and BG/GO) samples with the optimum mechanical result was studied by in-vitro test in (SBF) for 33 days. Samples were characterized by different techniques (FT-IR, XRD, and SEM) before and after soaking in SBF.

Preparation of Bioactive Glass Solution
The chemical composition of the bioactive glass solution prepared in 60% SiO 2 , 35% CaO, and 5% P 2 O 5 ; the chemicals have been used in high purity(Assy) about(97-99%) and were purchased for which tetraethyl orthosilicate (TEOS) Si(OC 2 H 5 ) 4 , Mw (208.3275) g/mol were purchased from Sigma-Aldrich Company, Canada) and triethyl phosphate (TEP; C 6 H 15 O 4 P, Mw (166.15526) g/mol were purchased from the Alfa-Aesar Company, Germany), and distilled water in ethanol (Merck), and Ca (NO 3 ) 2 .4H 2 O, Mw (236.15) g/mol were purchased from (Panreac PRS), were mixed as mentioned below: (22.75) ml of TEOS was added into (5.43) ml of nitric acid (HNO 3 ). The mixture was allowed to react for 60 min to promote the acid hydrolysis of TEOS. Then, (1.11) ml of P 2 O 5 was added and mixed for 60 min. And finally, (14.89) gm of Ca(NO 3 ) 2 .4H 2 O was added to the mixture and kept under stirring for 1.5 h until hydrolysis perfected and polycondensation, following the protocol proposed via sol-gel method steps as shown in Fig. 1 [20]. The sample of bioglass was calcined at 600 °C to eliminate the nitrate. The chemical composition of the bioactive glass solution is presented in Table 1.  Table 2, the flowchart of preparation steps shown in Fig. 2. The graphene oxide was burned at 660 degrees for two hours, at 1 atm, at a rate of 5 degrees per minute, and in a furnace (Fisher Scientific Oven model 818F, U.S.A).

Characterization Techniques:
FT-IR absorption spectra were recorded ranging from 400 cm −1 to 4000 cm −1 at room temperature with a spectrometer of type (FT-IR-400, JASCO, Japan). Identification of the development phases, and amorphous or crystalline nature of the samples using an X-ray diffractometer (model D8 Discover Germany) with a copper target (Cu kα = 1.54060 A°) and Nickel filter) for analysis. Scanning Electron Microscope instrument (EDX; 30 mm 2 Si (Li) R-RSUTW detector). The mechanical compressive strength is tested by a Universal automatic tensile testing machine in the National Research Center (model H001A: 600 kN). The microhardness of the samples was tested by a microhardness tester (model HDNS Kelly MVA-402TS).

Compressive Strength
The mechanical properties of (BG, 0.5%GO, 1%GO, 2%GO, and 3%GO) samples were obtained by compressive strength test as shown in Fig. 3. It was found that, despite of lower mechanical properties of the bioglass (BG), the addition of (GO) up to a certain content enhanced the mechanical performance of [60SiO 2 _35CaO_5P 2 O 5 ] in terms of compressive strength. In detail, it can be seen that the compressive strength (Cs) firstly increased from (55.06 ± 4.49) MPa for the (BG) sample to (100. 23    for (1%GO, 2%GO, and 3%GO) samples respectively, with increasing (GO) content up to 1,2, and 3% wt. It was clear that the best performance of compressive strength was obtained for the 0.5%GO sample, which improve the mechanical properties of BG in terms of compressive strength by (82.03) %. While limited enhancement of (1%GO), (2%GO), and (3%GO) samples, might be because of the degradation in the dispersion of graphene oxide at high concentrations.

Micro-Hardness/Mechanical Properties
To obtain the micro-hardness results, pellets of (BG) and (BG/GO) with a diameter of 10 mm are made using a circular die of 1 cm.
The pellets were made at a load of approximately 2.748 KN (35 MPa) using equal masses (0.7 g) of all powder samples. Also, all pellets were sintered at 660 °C and the dwell time set at 3 h with the heating ramp of 5 °C/min. Pellets were mounted in resin and polished by the silicon-carbide paper of (2000) grift. A 300gf (2.942 N) test force was used for micro-hardness evaluation with a loading time of 5 s. Micro-hardness results of BG and BG/GO samples were shown in Fig. 4.
In detail, there was an increase in micro-hardness from (0.263 ± 0.003) for the BG sample to (0.298 ± 0.031) for the 0.5%GO with an increase of GO weight percentage (0.5 up to 1% wt.). But gradually decreased again to (0.286 ± 0.019) for the 2%GO sample and (0.209 ± 0.056) ( Table 4) for the 3%GO sample. It was cleared that the best performance of micro-hardness is at (BG/GO) 1% wt., or by addition of (GO) content with (0.5 up to 1%) wt.

Density Test
The density of BG and BG/GO was measured by the Archimedes method Eq. (1) Theoretical densities of BG and GO were assumed to be (2.7 and 1.84) g/cm3 respectively [21][22][23][24]. It was noted, that the density of (BG) was decreased after the addition of different contents (0.5, 1, 2, and 3) wt.% of (GO). In detail, the obtained results showed that the densities are (2.02) g/cm 3 for the (BG) sample, but (1.86, 1.836, 1.837, 1.82) g/cm 3 for   5%GO, 1%GO, 2%GO, and 3%GO) wt. From the above results (as expected) it can be seen that the bioglass density is higher than (BG/GO) density as shown in Fig. 5. This significant decrease in the density of BG after addition, different content of GO might correspond to the density of GO being lower than the density of BG. The density results agreed with the (SEM) results, which confirmed that the addition of (GO) increases the porosity of the surface, which is necessary for bioactivity.

Fourier Transform Infrared (FTIR)
FTIR spectroscopy is a widely used analytical technique applied to the characterization of the organic and mineral components of bone.
Calcium-phosphate bioglass is the mineral component of bone. So, the chemical composition of these minerals was investigated by FTIR spectroscopy.
The spectrum of the typical bands of graphene oxide GO. It reveals six absorption bands. The first band observed at 654 cm −1 is attributed to the bending mode of C-Cl. The second band appeared around 976 cm −1 corresponding to C-C bending vibrations. Characteristic 3rd band at 1474 cm −1 attributed to stretching vibrations of C-O. 4th absorption band at 2039 cm −1 corresponds to the asymmetric stretching mode of -NH3. The 5th band at 2754 cm −1 is associated with symmetric stretching vibration C-H. The characteristic 6th band that is seen at 2876 cm −1 , can be related to the -CH2 stretching vibration [25][26][27][28][29][30] as shown in Fig. 6.
The spectrum is the typical bands of the silica network in sample BG. It reveals two bands, absorption bands. The 1st band observed at 455 cm −1 is attributed to the deformation mode of Si-O-Si [31]. The 2nd band appeared around 1042 cm −1 corresponding to asymmetric Si-O-Si stretch [32]. Characteristic phosphate bands were appearing at 567 and 602 cm −1 [31].
FTIR spectrum of BG/GO sample exhibits the following bands, the 1st band observed at 469 cm −1 is attributed to the deformation mode of Si-O-Si [31]. The 2nd band appeared around 1095 cm −1 corresponding to asymmetric Si-OH stretch [33].
The 3rd band at 1423 cm −1 is associated with symmetric stretching vibration -CO 3 −2 in the graphene oxide [34]. Characteristic 4th band at 1493 cm −1 attributed to stretching vibrations of -OH [35]. The 5th band observed at 1620 cm −1 is attributed to the bending mode of C = C in graphene oxide [36]. The final band appears around 3413 cm −1 and is associated with hydroxyl groups -OH [37].
The FTIR spectra of BG and BG/GO after immersion in SBF for 33 days as shown in Fig. 7.  The spectrum exhibits the typical bands of the silica network of bioglass in two samples BG and BG/GO. It reveals four broad absorption bands. The first band observed at 470 cm −1 is attributed to the bending mode of Si-O-Si that shifted to the wave number of 471 cm −1 in the spectra of (GO) containing sample [38,39]. The second band appeared around 1092 cm −1 corresponding to Si-OH bending vibrations that shifted (blue shift) to a wave number of 1110 cm −1 in BG/GO sample [33,35].
Characteristic bands at 1500 cm −1 attributed to stretching vibrations of C = O are detected for graphene oxide [40]. 1630 cm −1 band is assigned to the stretching modes of C = C is detected for graphene oxide [36] third Broad absorption band at 3440 cm −1 corresponding to the asymmetric stretching mode of-OH (asym) can be related to hydroxyl groups [41][42][43].
The characteristic bands of hydroxyapatite at (969, 858, and 801 cm −1 ) were attributed to stretching vibration of (OH-hydroxyapatite, P-O, and Si-O-NBO) (non-bridging oxygen) deformational vibrations of the Ca-OH groups.

X-Ray Diffraction (XRD)
The XRD diagrams of GO, BG, and BG/GO before the in-vitro assay are shown in Fig. 8. The XRD diagram characterized by a broad hump centered at 2θ ~ 23, confirmed the amorphous nature of the prepared samples [47].
The characteristic sharp (0 0 1) peak of graphene oxide appeared at 2Ө of 11.8º [48]. The weakened peak at 2Ө of 26.4º is attributed to the (0 0 2) planes of graphene [49]. An additional, the (1 0 0) peak that appeared in 42.2º is visible [50]. Figure 9 shows the XRD patterns of BG and BG/GO biocomposite after immersion in SBF for 33 days. Three welldefined hydroxyl apatite (HA) peaks developed at 2θ = (25.8°, 32.9°, and 46.7°) according to the standard (JCPDS file no. 09-0432) correspond to the HA layer formation on the surface of the two samples [51]. In addition to another peak at 2θ = 25.8° shows with the hydroxyapatite reference pattern (Ref. Code 00-024-0033) confirming the formation of HA [52] On the surfaces of the two samples, a layer of tetra-calcium phosphate (TeCP) has distinct peaks at 2θ = 29.3° [53]. The peak at 2θ = 29.54° was attributed to the presence of the calcium silicate phase [47].
The trace of graphene oxide remained in the sample BG/ GO a residual weak (0 0 2) peak (2ϴ = 26.4°) belonging to graphene oxide (hexagonal) was identified in the pattern, and additional diffraction lines originating (1 0 1) weak peak (2θ = 44.5°) for GO [54]. There was no noticeable change in the percentage of apatite formed on the surfaces of both  samples, which makes the sample BG/GO mixture whose mechanical properties have been improved without affecting the vital activity and formation of apatite. Figure 10 presents the SEM micrographs illustrating the surface morphology of the BG/GO Sample before immersion in SBF at three different (150 × , 300 × , and 600 ×) magnifications. In general, the result indicated that BG/GO composite displays a porous membrane structure with interconnecting open pores.

Morphological Analysis of Samples (SEM)
In the magnification Fig. 10 one can see that there is a smooth surface spreading of particles in various areas due to the incorporation of bioactive glass particles with graphene oxide. The porosity of the obtained composite increases the loading capacity of the physiological fluid into biomaterials which favors the interfacial reactions between biomaterials and physiological solution. The porosity of composite is necessary for bone tissue engineering for the easy migration of fluid, organic matter, biological cells, and then the proliferation of osteoblasts. Figure 11 presents the SEM micrographs illustrating the surface morphology of the BG/GO Sample after immersion in SBF for 33 days at three different (1000 × , 3000 × , and 6000 ×) magnifications. In general, the result indicated that the surface was almost partially covered with apatite crystals.

Conclusion
A composite of BG incorporated with different contents GO (0.5, 1, 2, and 3 wt.) was prepared via the sol-gel method. All prepared samples were characterized by various techniques (FTIR and XRD). Compressive stress, micro-hardness, and density were measured to determine the optimum mechanical properties. The biological activity was studied by an in-vitro test in simulated body fluid (SBF) for 33 days. Results showed that 0.5% of GO increases the compressive strength of BG by approximately 82% compared to pure BG. Hardness was increased from 0.5%GO sample up to 1%GO sample compared to BG sample and gradually decreased in 2%GO Sample and 3%GO. HA layer was deposited on the bioglass surface and there was no significant change in it with the addition of graphene oxide.

Declarations
Consent for publication Not applicable.

Conflict of interest
The authors declare that they have no conflict of interest.
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