## 4 Discussion
In the c-RGD/HA sample, the chemical binding of RGD with the HA surface led to a decrease in FBS protein adsorption. However, the amount of proteins still binding to the disc surface (5.7 micrograms/mL) was enough to stimulate cell adhesion, as also observed on the HA and p-RGD surfaces. This result revealed that the simple presence of FBS proteins on the surface might be sufficient to start the effective adhesion process. Previous studies have reported similar findings, where serum proteins, rich in fibronectin (FN) and vitronectin (VN), preferentially mediate cell adhesion over RGD-functionalized surfaces. By being rich in FN and VN, FBS may activate cell adhesion equally or better than the peptide sequences containing RGD when adsorbed on the HA surfaces [10; 17; 18], even when compared to the specific sequences of the proteins FN-RGD and VN-RGD [27]. [PERSON] et al. [29] compared the coated CaP with FBS, VN, and FN separately and observed that human mesenchymal stem cells (hMSCs) still preferred HA when covered by FBS, while Saos-2 cells preferred HA with FN or FBS to surfaces with immobilized FN-RGD and VN-RGD segments. RGD segments bind to specific groups of integrins, such as cRGDK to \(\alpha\
u\beta\)3 and \(\alpha\
u\beta\)5, highly expressed by the angiogenic endothelial cells [7]. The MC3T3-E1 pre-osteoblastic cells bind preferentially to the FN and VN proteins through the expressed integrins of \(\alpha\)5\(\beta\)1 and \(\alpha\
u\beta\)3, which are directly involved in the initial adhesion of the bone cells [27]. Such specificity may answer the preference of MC3T3-E1 for FBS-coated HA surfaces compared to HA that is associated with the RGD tripeptide, as previously described by [PERSON] et al. [13]. There are still some important considerations about the functionality of RGD that may impair the establishment of comparisons between studies, such as the type of peptides, as well as the concentrations used and the methodology employed, both in the production and characterization of the functionalized surfaces and in the biological evaluation, making it more difficult to reach a more precise conclusion on the subject. In this regard, one of the inherent challenges in this study was determining the precise amount of RGD modification or adsorption and ensuring identical levels across the physical and chemical immobilization methods. The distinct mechanisms underlying physical adsorption and chemical bonding of RGD to the HA surface create intrinsic differences in the way RGD interacts with the material. While fluorescence assays using RGD-FITC were employed to quantify the relative amounts of RGD on the surfaces, and our results indicated similar levels of RGD incorporation by both methods, achieving exact quantitative equivalence is inherently complex. This complexity arises from the different surface chemistries and interaction dynamics involved in each immobilization method. Despite this limitation, our approach provides valuable insights into the functional implications of these differences, particularly in the context of cell adhesion and surface interactions. By focusing on the biological outcomes rather than absolute quantitative equivalence, we highlight the relevance of each method in creating functional biomaterials. The present findings indicate that the physical adsorption of RGD on HA promotes cell adhesion, but the low stability of the peptide on the surface reduces its efficacy. Stabilizing RGD on the HA surface through chemical association may be a good solution if the HA active surface sites remain partially available after functionalization to interact with proteins and growth factors that participate in cell adhesion and proliferation. The results of this study highlight the differential effects of RGD immobilization on the surface morphology and biological performance of hydroxyapatite (HA).