## 3 Results and Discussions
### Crystal Growth Mechanisms of CaCO\({}_{3}\) Nanoparticles in Chitosan Hydrogels  
In this study, chitosan/CaCO\({}_{3}\) composite hydrogels were successfully prepared using a simple in situ mineralization method. To better understand the growth mechanism of CaCO\({}_{3}\) nanocrystals within the polymer network, samples at various stages of mineralization were further studied via SEM (Figures 4 and 5). Micrographs of samples mineralized for 5 min, 1 h, 3 h, and 6 h were acquired. The results revealed that in the cross-section of samples mineralized for 5 min, the CaCO\({}_{3}\) nanocrystals inside the chitosan matrix, as well as microparticles on the surfaces, already start forming. Figure 4a shows the overall distribution of calcite nanoparticles. In areas near the surfaces, nanoparticles with a size around \(\sim\)100 nm are observed, while in the center, almost no calcite nanoparticles are noticed. After 1 h of mineralization, the distribution of CaCO\({}_{3}\) nanocrystals became more uniform. Nanoparticles start forming in the center areas (Figure 4d-f). As the mineralization time increases to 3 h (Figure 5), the CaCO\({}_{3}\) crystals within the sample became more uniform and stable in both size and distribution. Most of the nanoparticles are in the range of 80-100 nm, even in the center areas. The result of mineralization for 6 h is similar to that of 3 h, indicating that the overall crystallization process is no longer limited by the diffusion process. Figure 3: Testing and analysis of chitosan/CaCO\({}_{3}\) composite hydrogels. (**a**) XRD test curve of the chitosan/CaCO\({}_{3}\) composite hydrogel. (**b**) TGA of chitosan/CaCO\({}_{3}\) composite hydrogel. (**c**) Raman spectral profile of chitosan/CaCO\({}_{3}\) composite hydrogels. (**d**) FTIR analysis of hydrogel before and after mineralization. (**e**) WAXD diffractogram of un mineralized pure chitosan hydrogel. (**f**) WAXD diffractogram of chitosan/CaCO\({}_{3}\) composite hydrogel. Unlike the direct blending method of ceramics and polymers, the in situ growth of CaCO\({}_{3}\) nanoparticles using chitosan hydrogel as a template more closely replicates the microenvironment of biomineralization found in nature. To verify whether the ceramic materials and polymers in the chitosan/CaCO\({}_{3}\) composite hydrogel produced through this biomimetic mineralization process resemble natural mineralized tissues, TEM was conducted to further analyze the nanostructure and interfaces of chitosan molecules and calcite crystals (Figure 6). The calcite nanoparticles are embedded in the chitosan matrix during the mineralization process, forming a composite nanostructure (Figure 6a-d). It is clearly shown in Figure 6a that the large nanoparticle is composed of many small particles of ~100 nm, which is very much like the particle attachment crystallization process. Figure 6c-e show the crystal lattice of calcite nanoparticles, indicating a single crystal-like diffraction pattern. Due to the existence of chitosan molecules, the CaCO\({}_{3}\) nanoparticles in the mineralized hydrogel are observed bounding to the chitosan molecules (Figure 6f). Amorphous areas are noticed both inside the particle and at the edges. This combination of  
Figure 4: SEM images of chitosan/CaCO\({}_{3}\) composite hydrogels after 5 min and 1 h mineralization. (**a–c**) show SEM images of the chitosan/CaCO\({}_{3}\) composite hydrogel cross-sections after 5 min of mineralization, with (**b**) representing the area near the surface and (**c**) representing the area near the center. (**d–f**) show SEM images of the chitosan/CaCO\({}_{3}\) composite hydrogel cross-sections after 1 h of mineralization, with (**e**) representing the area near the surface and (**f**) representing the area near the center. amorphous and crystalline regions is very similar to the chitin/hydroxyapatite composite nanocoating of mantis shrimp dactyl clubs (Figure 6g,h).