## 4 Discussion
Moreover, the wettability of HA surfaces, as indicated by contact angle (CA) measurements, was altered by RGD immobilization. The surface hydrophilicity is an indicator of the potential of the calcium phosphate surface to interact with molecules and cells [25]. Functional groups, such as -OH, -CH3, COOH, and NH2, influenced the adsorption of the proteins on the calcium phosphate surfaces and, consequently, the protein-cell interactions. Moreover, the coatings of N+, O+, and Si+ ions reduce the surface's free energy. In general, it is well established that a hydrophilic character reduces the adsorption of the proteins and cell adhesion by providing low free interfacial energy [26]. However, adhesive proteins containing RGD, such as FN and VN, prefer hydrophilic surfaces, favoring the adhesion of cells such as osteoblasts [27]. CA measurements of the water droplets on the HA disc surface showed a significant decrease in HA surface hydrophilicity after RGD physical adsorption. In contrast, the complete chemical immobilization of RGD on the HA surface led to a slight increase in the contact angle, from 61\({}^{\circ}\) to 69\({}^{\circ}\), suggesting that hydrophilicity was slightly affected in the RGD bond functionalized HA surface. The low hydrophilicity of the c-RGDHA sample was attributed to -NH2+ terminals, which was in agreement with the results from the Zeta potential assessments [23]. These topographical changes, in combination with surface charge modifications detected by zeta potential (ZP) analysis, suggest that chemical immobilization may create a more stable environment for RGD, potentially enhancing its bioactivity. Despite these modifications, our cell adhesion assays revealed that the number of pre-osteoblast cells (MC3T3-E1) adhered to RGD-functionalized surfaces did not significantly differ from the untreated HA when cultivated in a serum-free medium. This result is in convergence with previous reports [10; 18], where treatment with serum was necessary to identify the effects of RGD in cell adhesion over HA, indicating that serum proteins play a crucial role in mediating cell adhesion on HA surfaces, potentially outcompeting RGD for binding sites and facilitating cell attachment. In contrast, when cultivated in a serum-supplemented medium, chemically immobilized RGD surfaces showed a slight but statistically significant increase in cell adhesion compared to physically adsorbed RGD, although the overall difference was modest. [PERSON] et al. [9] demonstrated that cyclic RGD was physically adsorbed using a branched-chain polypeptide, resulting in a more significant electrostatic interaction that accelerated the adipocyte adhesion (AdMSC) in the serum-free medium. The weak binding of RGD to the HA surface may explain the different results of cell adhesion when RGD is physically immobilized on the HA surface. In this case, RGD may be released from the surface because adsorption occurs through the electrostatic interaction mediated by the water molecules chemically bound to the HA surface. On the other hand, the stabilization of RGD on the HA surface by chemical binding aimed to increase the number of adhered cells by offering higher attachment points for adhesion, accelerating the interaction process. The higher cell adhesion on c-RGD/HA confirmed the role of RGD in cell adhesion in the serum-free medium and the need for stabilized RGD onto the biomaterial surface to improve cell-surface interactions. Uncoated HA, p-RGD/HA, and c-RGD/HA adsorbed the proteins present in the FBS via electrostatic interaction, thus promoting similar cell adhesion in all tested groups. Hydrophilic surfaces, such as HA, favor the adsorption of the adhesive proteins present in FBS by electrostatic interactions, thus promoting cell adhesion [28]. According to the FBS adsorption assays, the p-RGD/HA surface adsorbed the same amount of FBS as HA did, suggesting an RGD displacement by the FBS proteins, as proposed by [PERSON] et al. [10] and [PERSON] et al. [14].