## 5 Conclusions  
This study demonstrated that both physical adsorption and chemical immobilization methods effectively bound RGD peptides to HA surfaces at similar concentrations. However, the immobilization method significantly influenced the surface characteristics and biological performance of the material. In serum-free conditions, chemically immobilized RGD showed enhanced cell adhesion compared to physically adsorbed RGD and untreated surfaces. This improvement is likely due to the more stable and bioactive surface created by chemical bonding, which also resulted in a smoother topography and modified surface charge. However, in serum-supplemented conditions, the impact of RGD functionalization on cell adhesion was less pronounced, suggesting that serum proteins may play a more crucial role in mediating cell adhesion on HA surfaces than RGD functionalization alone. These findings underscore the importance of carefully selecting the immobilization method for RGD when designing biomaterials for bone regeneration. While chemical immobilization provides greater stability and potentially enhances cell adhesion in environments devoid of serum proteins, it also reduces the material's ability to interact with key serum proteins, which could limit its effectiveness in vivo. On the other hand, physical adsorption maintains more of the native HA surface properties but may lead to weaker peptide attachment, particularly in the presence of serum. The choice of immobilization method should thus be tailored to the specific clinical application, balancing stability, bioactivity, and interaction with serum proteins to optimize the scaffold's performance in promoting bone regeneration.  
The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app14177479/s1](https://www.mdpi.com/article/10.3390/app14177479/s1)]([https://www.mdpi.com/article/10.3390/app14177479/s1](https://www.mdpi.com/article/10.3390/app14177479/s1)), Figure S1: Fourier-transform infrared spectroscopy (FTIR) of hydroxyapatite (HA) with thermal treatment at 1000 \({}^{\circ}\)C, with OH- at 3570 and (PO\({}_{4}\))\({}^{3-}\) bands at 1091, 1044, 959, 632, 601, and 569 cm\({}^{-1}\); Figure S2: The X-ray photoelectron spectroscopy (XPS) spectra of the uncoated sample consisted of binding energies of O 1s (532.3, 530.7, and 528.6 eV), Ca 2p3/2 (346.7.1 and 344.5 eV), P 2p3/2 (132.5 eV), and C 1s (288.3, 286.1, and 284.5 eV); Table S1: Chemical analyses by X-ray fluorescence (XRF).  
**Author Contributions:** Conceptualization, G.G.A. and A.M.R.; methodology, M.L., E.M., G.N.F., M.S.S., A.C., E.L. and J.M.G.; software, M.L., E.M., M.S.S., A.C., G.N.F., E.L. and J.M.G.; validation, A.M.R. and C.F.M.; formal analysis, A.M.R., T.R., A.P. and C.F.M.; investigation, A.M.R., T.R. and C.F.M.; resources, G.G.A. and A.M.R.; data curation, M.L., E.M., G.N.F., M.S.S., A.C., E.L. and J.M.G.; writing--original draft preparation, M.L., G.G.A. and A.M.R.; writing--review and editing, A.M.R., N.D.P. and T.R.; visualization, T.R., N.D.P. and A.P.; supervision, A.M.R. and G.G.A.; project administration, A.M.R.; funding acquisition, A.M.R. All authors have read and agreed to the published version of the manuscript.  
This research received no external funding.  
**Institutional Review Board Statement:** Not applicable.  
**Informed Consent Statement:** Not applicable.  
**Data Availability Statement:** The original contributions presented in the study are included in the article/supplementary material, and further inquiries can be directed to the corresponding author.  
**Acknowledgments:** Authors would like to acknowledge FAPERJ, CAPES, and CNPq.  
**Conflicts of Interest:** The authors declare no conflicts of interest.