# Osseointegration of Antimicrobial Acrylic Bone Cements Modified with Graphene Oxide and Chitosan
## 3 Results and Discussion
### In Vivo Studies
#### 3.4.1 Subdermal Implantation  
The SEM micrographs shown in Figure 3 correspond to representative images of the four cements response after 1 month of subdermal implantation in Wistar rat tissues. In Figure 3b, a structure compatible with one cell (marked with triangle in Figure 3) is observed, while Figure 3c,d showed better surface texture and abundant deposits of extracellular matrix on their surface (marked with arrows in Figure 3), compared with the Control ABC (Figure 3a). Since cells of mesenchymal origin, including fibroblasts, are rugophilic, it is valid to think that the surface of the loaded cements, being more irregular than the Control ABC, is more encouraging for the cells. Several pieces of research had demonstrated that a higher roughness of the surface of the biomaterials favors the adhesion and  
Figure 3: SEM micrographics (200\(\times\)) of ABC surfaces after subdermal implantation in rats for 1 month. (**a**) Control ABC, (**b**) ABC with 0.3% GO, (**c**) ABC with 15% CS and (**d**) ABC with 0.3% GO and 15% CS samples (Triangles indicate cells and arrows indicate extracellular matrix).  
proliferation of the cells [61; 62; 63; 64; 65]. The presence of cells and deposits of extracellular matrix on the cements confirmed their biocompatibility because according to [PERSON] [61], the adhesion of cells to surfaces is one of the vital aspects in biocompatibility.  
As far as histological analysis, the samples corresponding to the collagen control at 1 month of implantation are showed in Figure 4 which revealed the presence of remaining material (Col) in the implantation zone (IZ) in process of degradation/absorption and abundant inflammatory infiltration (II).  
Figure 5 corresponds to the Control ABC formulation, showing a capsule (C) surrounding the zone of implantation (IZ) and including an inflammatory infiltrate (II).  
The three formulations of implanted cements including GO and/or CS (Figure 6) showed similar response to that of Control ABC (Figure 5), characterized by a localized inflammatory response, surrounded by a capsule (C) with the presence of inflammatory infiltrate (II). However, compared to collagen samples (Figure 4), the latter had a more diffuse inflammatory response with abundant inflammatory infiltrate.  
The tissues stained with the [PERSON]'s trichrome stain (Figure 7) showed a fibrous capsule at the IZ composed of collagen type I fibers for collagen control and the studied cements.  
Figure 4: H&E staining of collagen membrane after 1 month of subdermal implantation in Wistar rats. (**a**) 4\(\times\) and (**b**) 10\(\times\). IZ: Implantation zone. Col: Collagen. M: Muscle. H: Hypodermis. D: Dermis. E: Epidermis. II: Inflammatory infiltrate.  
Figure 5: H&E staining of Control ABC after 1 month of subdermal implantation in Wistar rats. (**a**) 4\(\times\) and (**b**) 10\(\times\). IZ: Implantation zone. Col: Collagen. M: Muscle. H: Hypodermis. D: Dermis. E: Epidermis. II: Inflammatory infiltrate and C: capsule.  
The presence of the capsule and the inflammatory infiltrate surrounding the cement samples can be considered a normal in vivo response to materials that have been implanted in soft tissue and promote healthy wound healing [66]. For example, [PERSON] et al. [67] reported the formation of a transparent capsule surrounding samples of polyhydroxyalkanoates implanted in rats. Fibrous encapsulation has also been reported for various biomaterials, such as nano-crystalline hydroxyapatite [68] and ABCs with CS [4].  
Figure 6: H&E staining after 1 month of subdermal implantation in Wistar rats. (**a**,**b**) ABC with 0.3% GO, (**c**,**d**) ABC with 15% CS and (**e**,**f**), ABC with 0.3% GO and 15% CS. (**a**–**c**,**e**) micrographs to 4\(\times\) and 4\(\times\), and (**b**–**d**,**f**), micrographs at 10\(\times\). IZ: Implantation zone. M: Muscle. H: Hypodermis. D: Dermis. E: Epidermis. II: Inflammatory infiltrate and C: capsule.