## 4 Discussion
It is also necessary to ensure that the RGD carboxylic sites can interact with the cellular integrins. These chemical immobilization methods may lead to positive results regarding cell adhesion and spreading, as shown by [PERSON] et al. [22] and [PERSON] et al. [21]. However, in both studies, the adhesion tests occurred under a supplemented medium (10% FBS and 5% bovine serum albumin, BSA, respectively). Furthermore, no work has compared the in vitro performance of these different RGD immobilization methods in the presence or absence of serum proteins. Our results extend this understanding by showing that while chemical immobilization stabilizes RGD on the HA surface, it also modifies the surface properties in ways that reduce serum protein adsorption, leading to a complex interplay between the surface's chemical stability and its biological activity in different culture conditions. The present work used a nanocrystalline powder with Ca/P = 1.68 to analyze the changes in the HA surface after the RGD immobilization and test the efficiency of the physical and chemical methods used to immobilize RGD. XPS analyses showed that sample treatments to associate RGD physically or chemically to the HA surface changed the chemical composition and stoichiometry of the HA surface significantly, even though the Ca/P ratio was not affected by the treatments. The detection of nitrogen binding energy confirmed the RGD association with the HA surface in p-RGD/HA and c-RGD/HA samples. The N/Ca ratio was around 0.06 for both the p-RGD/HA and c-RGD/HA samples, suggesting that less than one N atom from the RGD molecule was linked per HA unit cell in both samples. The fluorescence assays using RGD-FITC confirmed similar amounts of RGD on p-RGD/HA, and c-RGD/HA also supported the XPS analysis that showed the expected Ca and P binding energies of the uncoated-HA [23], identifying the NHx and N-C bonds that were associated with RGD [20]. Altogether, the presented results indicate that similar RGD amounts were immobilized onto the HA surface by the physical and chemical methods using free -COOH sites, suggesting a similar efficiency for both methods, at least in the present experimental conditions. However, the choice of immobilization method significantly influenced the surface topography and chemistry of the HA samples. Atomic force microscopy (AFM) revealed that chemically immobilized RGD (c-RGD/HA) produced a smoother surface compared to physically adsorbed RGD (p-RGD/HA), which displayed a higher average surface roughness. This difference in topography likely results from the more uniform coating achieved through chemical immobilization, which may reduce the exposure of HA's underlying structure. These surface topography and chemical composition modifications caused by the surface treatment on p-RGD/HA and c-RGD/HA samples also led to changes in surface charge and surface wettability. ZP analysis assigned an IP of 4.99 to HA powder, assuring a high negative surface charge in the in vitro assays' conditions. After physical adsorption, the p-RGD/HA sample's IP increased to a value of 6.76, indicating that RGD is bound to the HA surface with the -N terminals that were likely exposed on the surface, thereby modifying the surface charge by the presence of RGD. In the first immobilization step, the APTES coating introduced amine (NH2+) radicals on the silanized HA c-HA1 sample surface [11], leading to an IP of 8.24 and a positive surface charge at neutral pH. After treatment with carbodiimide and RGD, the surface IP was markedly reduced to 5.35 due to the new layers rich in COO\({}^{-}\) when introduced on the HA surfaces [11; 10]. These results confirmed the significant difference in HA surface charge when RGD molecules are associated with the surface through physical or chemical bonds. It is important to highlight the relevance of removing the weakly bound peptides to obtain the functionalized surfaces, predominantly exposing the RGD -C terminus, which is directly involved in binding cellular integrins [24].