## 4 Discussion  
The association of RGD with a hydroxyapatite surface has been proposed as a tool to improve the biological efficiency of osteeconductive scaffolds in a bone regeneration process [6]. Despite the numerous studies on the subject, there is still controversy regarding the biological effects of RGD/HA association. Non-convergent results from different studies may be attributed to differences between HA samples, the methods for immobilizing RGD on the HA surface, association with various types of RGD, cell culture using different cell types, and the use or not of a supplemented culture medium. This study aimed to address these inconsistencies by comparing the effects of physical adsorption and chemical immobilization of RGD on HA surfaces, focusing on their impacts on surface characteristics and cell adhesion in the presence or absence of serum proteins. It is well-established that the physicochemical properties of HA, such as stoichiometry, crystallinity, surface chemistry, topography, wettability, and surface energy, play critical roles in mediating the interaction between RGD and the biomaterial surface, which in turn influences cellular responses [13]. The present study demonstrated that both immobilization methods alter the HA surface's physicochemical characteristics, with each method presenting distinct advantages and challenges. The chemical immobilization method, in particular, led to a more stable RGD attachment, but it also modified the surface properties in ways that could influence protein adsorption and cell interactions differently compared to physical adsorption. The control of binding RGD to the surface is one of the main challenges for a reliable assessment of the role of RGD in cell adhesion. Many works use physical adsorption, which  
Figure 7: Quantitative analysis of the total FBS proteins adsorbed on the sample surfaces after 3 h of incubation in a 10% FBS solution. Afterward, the samples were washed gently in ultrapure water and reacted for 3 h in a 1.5% sodium dodecyl sulfate (SDS) solution. The collected supernatant was read on a Thermo Scientific(tm) NanoDrop 2000c Spectrophotometer at a wavelength of 280 nm. Statistical analyses were considered (n = 5) for each sample. The statistical analyses consisted of One-way ANOVA, with [PERSON]’s multiple comparisons test (\({}^{\star}\)\(p\) < 0.0001). links the peptide terminals of NH3+ and COO- to the HA surface's negative and positive sites through electrostatic interactions. Interactions with proteins from biological fluids may break these weak bonds, releasing the surface's peptide [11; 13]. In addition, there is no certainty that the RGD active site (-COO-) would be available to interact with the cell ligands. Our findings suggest that while physical adsorption is simpler and maintains the HA surface's native properties to some extent, the weak interaction between RGD and HA limits the effectiveness of this method in promoting cell adhesion, particularly in the presence of competing serum proteins. Despite some studies showing that RGD adsorption promotes cell adhesion and spreading [16; 17], [PERSON] et al. [18] found that HA discs adsorbed with RGD promoted lower interactions with the mesenchymal cells than the same system after an overcoating with serum. The authors concluded that the interaction of serum proteins with the HA surface could neutralize the effect of adsorbed RGD on cell adhesion. This study corroborates these findings, demonstrating that the presence of serum proteins significantly influences the effectiveness of RGD-functionalized surfaces, with the chemically immobilized RGD showing a reduced protein adsorption capability, which could explain the lower cell adhesion observed in serum-supplemented conditions. Some studies have used the chemical immobilization of RGD on the surface of HA [21; 22]. This method ensures the peptide's stability on the surface, but it requires surface functionalization with chemical agents, such as APTES and carbodiimide.