# Effect of Nanohydroxyapatite on Silk Fibroin-Chitosan Interactions--Molecular Dynamics Study
## 3 Results and Discussion
Similarly, Figure 4 reveals a greater frequency of hydrogen bonds between fibroin and chitosan in the presence of HPO4\({}^{2-}\) and Ca\({}^{2+}\) ions. Notably, hydrogen bonds and electrostatic interactions in fibroin blends containing different polysaccharides including chitosan were analyzed using experimental tools [108]. As  
Figure 2: Visualization of the initial chitosan–silk fibroin structure (**a**) and the optimized complexes containing CH3 COOH (**b**) and HPO4\({}^{2-}\)/Ca\({}^{2+}\) (**c**). Chitosan is presented in a ball model, while fibroin is in ribbon like structure. Fibroin helices are presented as solid turquoise lines, whereas chitosan is represented by a ball–stick model with colors representing the following elements: carbon—turquoise, nitrogen—dark blue, oxygen—red, and hydrogen–white. indicated by these studies, interactions between the protein and polysaccharides can indeed modify the protein structure itself. From the perspective of bone regeneration, it seems to be beneficial to utilize materials that are structurally similar to natural bone tissue. Fibroin emerges as a promising candidate in this context. It is well established that the crystal phase formed during bone mineralization exhibits a characteristic inter-crystalline center spacing of 0.362 nm [109]. Fortunately, this spacing aligns with the \(\beta\)-sheet conformation observed in fibroin [17]. According to the results of infrared spectroscopy measurements, the complexation of fibroin with chitosan favors the \(\beta\)-sheet conformation of the peptide [108; 110; 111]. The \(\beta\)-sheet order was also found in the case of the complexes optimized in this study, as visualized by the red band (Figure 2). As inferred from Figures 3 and 4, the contribution of the ionic contacts and hydrogen bond between chitosan and \(\beta\)-sheet fragments is quite significant compared to the contacts formed by other fragments of the peptide (coil, turn). Furthermore, these interactions involve diverse positions in the primary structure of peptide. The effective arrangement of the chitosan chains with respect to the fibroin fragments characterized by the \(\beta\)-sheet order is shown in Figure 2.  
Figure 4: The distributions of hydrogen bonds in the case of fibroin–chitosan complexes stabilized by \(\text{HPO}_{4}{}^{2-}/\text{Ca}^{2+}\) (**left panel**) and acetic acid aqueous solution (**right panel**). Figure 3: The distributions of ionic contacts in the case of fibroin–chitosan complexes stabilized by \(\text{HPO}_{4}{}^{2-}/\text{Ca}^{2+}\) (**left panel**) and acetic acid aqueous solution (**right panel**). The visualization of all main types of interactions formed in the considered systems (ionic contacts, hydrophobic contacts and hydrogen bonds) was presented in Figures 5 and 6. The optimized structures of fibroin-chitosan complexes are stabilized by strong hydrogen bonds and ionic interactions, which are characterized by short interatomic distances ranging from 1.85 to 2.11 A for hydrogen bonds and from 3.83 to 3.92 A for ionic contacts. The latter type of interactions deserve special attention due to their diversity and the direct involvement of the ions added to the fibroin-chitosan system considered in this study. As established, Ca2+ and HPO42- ions facilitate the formation of ionic bridges. The bridges involving HPO42- were formed through interactions between protonated amino groups in chitosan and lysine (LYS) residues within the fibroin (Figure 6). Interestingly, no analogous interactions were detected in the case of systems containing CH3 COO- ions. In this study, fully deacetylated chitosan was used. It is worth mentioning that previous studies [61; 62] have shown that a degree of chitosan deacetylation enhances the stability of collagen-chitosan complexes. This underscores the importance of the presence of protonated amino groups in stabilizing the system, as can be observed by analyzing key hydrogen bonds (Figure 5) and ionic interactions (Figure 6).