## 3 Results
### Physicochemical Evaluation
The XPS spectra of the untreated sample consisted of binding energies for O 1s at 532.3, 530.7, and 528.6 eV; Ca 2p3/2 at 346.7.1 and 344.5 eV; P 2p3/2 at 132.5 eV; and C 1s at 288.3, 286.1, and 284.5 eV (Table 3). The Ca/P ratio was 1.3, typical of a calcium-deficient HA surface. While the chemical treatments decreased the surface Ca and P ratio, as shown in Table 2, they did not modify the binding energies of Ca 2p, P 2p, O 1s, and C 1s (Table 3). Nitrogen atoms belonging to the RGD structure were detected in the RGD-coated samples (p-RGD/HA and c-RGD/HA), \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Samples** & **IP \({}^{\textbf{1}}\)** & **pH \({}^{\textbf{2}}\)** & **ZP (mV)** & **Groups \({}^{\textbf{3}}\)** & **CA (c) \({}^{\textbf{4}}\)** \\ \hline HA & 4.99 (\(\pm\)0.01) & 7.0 (\(\pm\)0.5) & \(-\)20.6 (\(\pm\)1.7) & OH\({}^{-}\) and PO\({}_{4}\)\({}^{-}\) & 61 (\(\pm\)2) \\ p-RGD/HA & 6.76 (\(\pm\)0.06) & 7.7 (\(\pm\)0.3) & 1.0 (\(\pm\)1.9) & COOH\({}^{-}\) and NH\({}_{2}\)\({}^{+}\) & 91 (\(\pm\)1) \\ p-RGD/HA \({}^{\textbf{*}}\) & 5.91 (\(\pm\)0.04) & 7.1 (\(\pm\)0.0) & \(-\)5.5 (\(\pm\)0.6) & COOH\({}^{-}\) & 83 (\(\pm\)2) \\ c-HA1 & 10.05 (\(\pm\)0.03) & 7.9 (\(\pm\)0.4) & 10.6 (\(\pm\)1.3) & NH\({}_{2}\)\({}^{+}\) & 75 (\(\pm\)1) \\ c-HA2 & 8.24 (\(\pm\)0.06) & 7.9 (\(\pm\)0.4) & 2.1 (\(\pm\)0.9) & NH\({}_{2}\)\({}^{+}\) and CH\({}^{3-}\) & 68 (\(\pm\)3) \\ c-RGD/HA & 5.35 (\(\pm\)0.03) & 7.4 (\(\pm\)0.3) & \(-\)5.7 (\(\pm\)0.6) & COOH\({}^{-}\) & 69 (\(\pm\)4) \\ c-RGD/HA \({}^{\textbf{*}}\) & 5.34 (\(\pm\)0.05) & 7.1 (\(\pm\)0.1) & \(-\)4.9 (\(\pm\)0.6) & COOH\({}^{-}\) & 67 (\(\pm\)2) \\ \hline \hline \end{tabular}  
* The isoelectric point (IP) values of each sample demonstrate the modification of the HA surface charge during the immobilization steps; \({}^{\textbf{2}}\) pH values of samples dispersed in potassium chloride (KCl) solutions at the moment of the ZP measurements; the mean value of 3 replicates (\(\pm\)standard deviation); \({}^{\textbf{3}}\) possible functional groups exposed on the HA surface at each stage. However, the overlapping of films does not occur totally and homogeneously; \({}^{\textbf{4}}\) CA values using dripping water; the mean value of 5 replicates (\(\pm\) standard deviation). \({}^{\textbf{*}}\) Sample evaluated after sonication.  
\end{table} 
Table 2: Surface charge analysis of samples before and after RGD physical and chemical immobilization steps using the Zeta Potential (ZP) and Contact Angle (CA) technique. indicating the presence of the peptide. The N/Ca ratio was around 0.06 for the p-RGD/HA and c-RGD/HA samples. This result has suggested that less than one N atom was linked per HA unit cell on the disc surface. The p-RGD/HA and c-RGD/HA discs (Figure 3A,B) displayed higher fluorescent intensity than the auto-fluorescence of HA (Figure 3C) that was present all over the discs (Figure 3A,B). Figure 3D shows that there was no significant difference between the fluorescence of p-RGD/HA and c-RGD/HA samples (\(p\) > 0.05), with the statistical difference with all other groups of \(p\) < 0.0001 (one-way ANOVA) (Figure 3D). Figure 3: Fluorescence emission intensity of physically adsorbed RGD-FITC (p-RGD/HA) and chemically bound RGD-FITC (c-RGD/HA) on HA discs. (**A,B**) Images of the discs after one hour of reaction with RGD-FITC solution and their histograms of fluorescence intensity (a.u. : arbitrary unit) obtained and the fluorescence emission intensity quantified through software Image Zen2-ProPlus version 2 using the peak area of total fluorescence light. (**C**) The auto-fluorescence of HA control sample with an intensity of 1047 a.u. after remaining one hour in milli-Q water. (**D**) A graph of the average fluorescence intensity of the discs measured by a microplate reader; the result shows the average of 3 independent experiments carried out in triplicate. * Statistical difference to all other groups (\(p\) < 0.0001, one-way ANOVA).