applied sciences  
Effects of Physically Adsorbed and Chemically Immobilized RGD on Cell Adhesion to a Hydroxapatite Surface  
[PERSON] 1, [PERSON] 1, [PERSON] 2, [PERSON] 1, [PERSON] 1, [PERSON] 3, [PERSON] 3, [PERSON] 4,5,*, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,  
its surface chemistry does not favor chemical binding to specific biological terminals in proteins/enzymes [2]. In general, interactions between proteins and HA surfaces occur according to electrostatic interactions [3]. In some cases, a surface interaction may induce inactivation by conformational changes, leading to protein denaturation or non-exposure of the site of interest at the time of adsorption [4]. Different approaches seek a more controlled interaction between cells and biomaterials, associating material surfaces with essential proteins and adhesion peptides, such as tripeptide RGD (arginylglycylaspartic acid, ArgGly-Asp), aiming at the obtention of innovative biomaterials for osteointegration [5; 6]. RGD was discovered in the early 1980s as one of the cell adhesion motifs common to several matrix proteins, such as fibronectin (FN), vitronectin (VN), and bone sialoprotein (BSP) [7]. Since then, the idea of associating RGD as an isolated peptide segment into several biomaterials to increase cell adhesion and tissue growth has increased. Other derivative peptide segments were also developed, presenting linear or cyclic longer chains that still have the RGD sequence in their structure, such as VNRGD, FNRGD, GRGDSP, cycloRGD, and others, which are often called \"RGD\" or \"segments containing RGD\" [8; 9]. The most used methods for RGD immobilization onto calcium phosphate surfaces involve physical adsorption or covalent bonds [10]. The latter occurs by introducing functional groups on the biomaterial surface, such as the alkyl, amino, and carboxyl groups, conducted using activated plasma, sputtering, or chemical reactions using additives, such as the silanizing agent (3-aminopropyl)triethoxysilane (APTES) [11]. Although the specific interaction of RGD segments with cell integrins is well known, there is controversy regarding the efficiency of RGDs in promoting cell adhesion when the peptide is associated with the HA surface. In vitro studies have indicated that the RGD/HA association may increase the number of adhered cells, even suggesting osteogenesis [10; 12], while other authors have not observed such effects [13; 14; 15]. Previous studies have observed the increased adhesion of cells cultured in a serum-supplemented medium onto HA surfaces covalently bound to the peptide [16; 17]. Furthermore, [PERSON] et al. [18], who physically adsorbed the peptide onto an HA surface, observed increased cell adhesion and spreading, although smaller than the surface covered with fetal bovine serum (FBS). [PERSON] et al. [14], on the other hand, observed the inhibition of cell adhesion, suggesting that the differences in the biological response to HA/RGD associations may depend on the peptide's physicochemical interaction with the biomaterial surface, which, in turn, may be affected by the choice of immobilization method. Both studies claimed that serum proteins could compete for the HA binding sites, thus displacing the weakly bound peptides onto the surface, possibly triggering an anoikis apoptotic pathway. These results have led to the hypothesis that the differences in the biological response to RGD/HA associations may depend on the peptide's physicochemical interaction with the biomaterial surface, which, in turn, may be affected by the choice of immobilization method. However, this hypothesis remained untested since most cell/surface interaction studies employed RGD immobilized through physical adsorption, and there is no direct evidence in the literature comparing the two methods on cell adhesion effects in the presence or absence of serum proteins.