# Osseointegration of Antimicrobial Acrylic Bone Cements Modified with Graphene Oxide and Chitosan
## 3 Results and Discussion
### In Vitro Studies  
#### 3.3.1 Antibacterial Activity against Escherichia coli  
_E. coli_ has been used in this research only as a model to evaluate the antimicrobial activity of different modifications of bone cements and prove that the biological activity of the GO and CS is maintaining after curing reaction of ABC. Previous results [31] shows that the CFU of _E. coli_ for Control ABC, ABC 0.3% GO, ABC 15% CS and ABC 0.3 GO-15 CS were 96 \(\pm\) 3, 17 \(\pm\) 2, 69 \(\pm\) 2 and 12 \(\pm\) 6, respectively. The former indicate than the effect of GO incorporation on the antibacterial activity of ABCs was much more significant compared to the introduction of only CS. ABCs loaded with GO showed a reduction in CFU by more than 80%, while those containing only CS reduced the number of colonies by 28%, as compared to the Control ABC. The result obtained for ABCs with both loadings (CS and GO) did not show statistical significance comparative with ABC loaded with 0.3% of GO.  
SEM micrographs of cements with bacteria (Figure 1) confirmed a higher bacterial population with the formulations that did not contain GO. These results suggested that GO introduction is the main caused for antibacterial activity of ABCs and is promising for clinical applications devoted to avoiding bacterial infections, a common health risk during surgeries and implantations.  
Figure 1: SEM micrographs (1000\(\times\)) of ABC surfaces after incubation of _E. coli_ cultures for 24 h of (**a**) Control ABC, (**b**) ABC with 0.3% GO, (**c**) ABC with 15% CS and (**d**) ABC with 0.3% GO and 15% CS.  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Formulation** & **C (MPa) [31]** & **B (MPa)** & **E (MPa)** \\ \hline Control ABC & \(93.3\pm 2.2\) & \(48.7\pm 4.2\) & \(2593\pm 178\) \\ ABC 0.3\% GO & \(94.0\pm 0.5\) & \(51.8\pm 0.4\) & \(2599\pm 154\) \\ ABC 15\% CS & \(62.6\pm 1.1\)** & \(32.8\pm 2.9\)** & \(2141\pm 212\)** \\ ABC 0.3 GO-15 CS & \(77.2\pm 2.6\)** & \(44.6\pm 4.1\) & \(2379\pm 132\)* \\ \hline \hline \end{tabular} Asterisks denote significant differences of modified ABC samples with respect to Control ABC at significance levels of * \(p\) \(<\) 0.05; **_p_ \(<\) 0.01.  
\end{table}
Table 3: Compressive strength (C), bending strength (B) and bending modulus (E) in ABCs formulations. Data are reported as a mean \(\pm\) SD.  
[PERSON] et al. [9] reported low antibacterial activity of CS in ABC formulations, suggesting the need to add other synergistic antibacterial properties. The mechanisms of the antimicrobial activity of CS involve electrostatic interactions between the positively charged chitosan chains and bacterial cell membrane, affecting cell permeability and leading to bacterial death [12]. Membrane damage, interaction with DNA/RNA, metal chelation and deposition on the microbial surface are also other possible chitosan antimicrobial mechanisms reported [52]. In this work, the antibacterial activity exhibited by the studied ABCs is enhanced by the presence of GO which presents antibacterial mechanisms due to its extremely sharp edges that cause physical damage to the bacterial membrane [49; 50]. In addition, oxidative stress with production of reactive oxygen species (ROS) disrupts the balance in redox processes within the cell leading to a disruptive membrane cell mechanism [53; 54; 55; 56; 57] and cell lysis [58; 53].