# Osseointegration of Antimicrobial Acrylic Bone Cements Modified with Graphene Oxide and Chitosan
## 3 Results and Discussion
### In Vivo Studies
#### 3.4.2 Bone Implantation of ABCs  
SEM and EDS studies were performed at the interface of bone and cements to evaluate the osseointegration process through the sealing defect and to detect the presence of Ca and P, respectively. According to the SEM micrographs illustrated in Figure 8, Control ABC implantation area in parietal bone of Wistar rats showed that after 3 months of implantation, the cement was not completely integrated with the surrounding bone tissue, since a clear separation between the two was observed (Figure 8a,b). In the area corresponding to the cement, the typical topography of the bone cement was observed with few deposits of extracellular matrix (EM). On the contrary, the formulations modified with GO (Figure 9a,b) or CS (Figure 9c,d) and with both components together (Figure 9e,f) presented the bone-cement interface with an advanced sealing process. In addition, at the interface there were presence of osteoblasts (OB) and abundant deposits of EM that nearly completely seal the periphery of the intraosseous preparation as shown in Figure 9. The better sealing means an enhanced osseointegration or chemical affinity of the modified ABCs  
Figure 7: Micrographics of [PERSON]’s trichrome stain after 1 month of subdermal implantation in Wistar rats (10\(\times\)). (**a**) Collagen control, (**b**) Control ABC, (**c**) ABC with 0.3% GO, (**d**) ABC with 15% CS and (**e**) ABC with 0.3% GO and 15% CS. IZ: Implantation zone. M: Muscle. H: Hypodermis. II: Inflammatory infiltrate. CF: Collagen fibers. with the bone. In accordance, the difference in the EM deposits between the Control ABC and the modified cements (Figures 8 and 9 respectively) is evident. Interestingly, cement with both CS and GO presented a more active interface with a group of osteoblasts growing from the interface towards the bone cement (Figure 9e,f). Figure 8: Micrographs of the implantation zone of Control ABC after 3 months of implantation in parietal bone of Wistar rats. (**a**) 50\(\times\) and (**b**) 200\(\times\). EM: Extracellular matrix and OB: Osteoblasts. Figure 9: SEM micrographs of the implantation zone of modified ABCs after 3 months of implantation in parietal bone of Wistar rats. (**a,b**) ABCs with 0.3% GO, (**c,d**) ABCs with 15% CS and, (**e,f**) ABCs with 0.3% GO and 15% CS. (**a,c,e**) with 50\(\times\) magnification. Areas surrounded by a red line are showed with deeper detail (200\(\times\)) in (**b,d,f**) images, respectively. EM: Extracellular matrix and OB: Osteoblasts. When the cement-bone interface of the ABC 15% CS and ABC0.3% GO-15% CS formulations were analyzed at higher magnification (500\(\times\)) (Figure 10) it was appreciated that the interface of ABCs modified with only CS was not totally sealed, showing cement particles in the process of cellular colonization, while that of the ABC 0.3% GO-15% CS formulation was almost completely sealed. Analysis of EDS spectra at the interface and the bone zones (Figure 11) in all cases demonstrated the presence of bands of calcium (Ca) and phosphorous (P) in their corresponding spectra (Figure 11b,c,e,f,h,i,k,l) supporting the mineralization process and the good biocompatibility of the ABCs studied here. However, analysis of EDS spectra in the cement IZ showed the presence of the Ca signal of low intensity and absence of the that of P (Figure 11a,d,g,j). Interestingly, increasing amounts of Ca and P were found for the formulations with 0.3% GO and/or 15% CS with respect to Control ABC, more marked for the sample containing both components (Figure S2). The deposition of Ca and P elements is a primary evidence of the osteogenesis process and the amount of elemental deposition is associated with a material biocompatibility and osteoconductive behavior [43]. These results agree with those previously obtained by SEM, since the enhanced biomineralization at the interface for the ABC with 0.3% GO and 15% CS sample correlates with a greater integration of this cement with the bone and with an improved biocompatibility compared to the other studied formulations.