## 3 Results and Discussions  
### Structure and Composition of Chitosan/CaCO\({}_{3}\) Composites  
The microstructures of the 3D printed chitosan film and chitosan/CaCO\({}_{3}\) composite hydrogel prepared via the in situ growth of CaCO\({}_{3}\) were investigated via scanning electron microscopy (SEM). The 3D-printed chitosan films changes from transparent to white color  
Figure 2: Microstructures of chitosan hydrogel before and after mineralization. (**a**) Images of 3D printed pure chitosan hydrogel samples at their unmineralized state. (**b**) Images of chitosan/CaCO\({}_{3}\) composite hydrogel samples after 12 h of mineralization. (**c**) SEM images of the surface of pure chitosan hydrogel at its unmineralized state. (**d**) SEM image of the chitosan/CaCO\({}_{3}\) composite hydrogel surface after 12 h of mineralization. (**e**) SEM images of the cross-section of unmineralized chitosan hydrogel. (**f**) SEM image of the cross-section of the chitosan/CaCO\({}_{3}\) composite hydrogel after 12 h of mineralization. Nanoparticles are noticed. after the reaction (Figure 2a,b). Micrographs of the surfaces and cross-sections of the hydrogel samples before and after 12 h of mineralization are shown in Figure 2c-f. The average thicknesses of the 3D-printed films are around 50-60 \(\upmu\)m. Figure 2c,d show the surfaces of the chitosan hydrogel before and after mineralization. Large microscale crystals with an average size of 5-6 \(\upmu\)m are clearly observed on the samples after CaCO\({}_{3}\) growth. While in the cross-sections of the samples, microscale crystals are no longer existing even after 12 h mineralization. Nanoparticles with an average size of \(\sim\)100 nm are noticed (Figure 2f). The results clearly indicate that the crystals grown on the surfaces and cross-sections of the chitosan hydrogel exhibit different morphologies and sizes. Thus, the chitosan molecule network inside the films can indeed affect the crystal growth of CaCO\({}_{3}\). The presence of nanoparticles inside the organic network might be caused by the heterogeneous nucleation and confinement of the chitosan molecules. The detailed mechanisms will be discussed later. Since SEM alone could not fully determine the crystal phases grown in the sample, XRD, Raman spectroscopy, and FTIR were employed in the subsequent analysis. The XRD results corresponded to the calcite standard pattern, aside from the characteristic peak of the chitosan polymer at approximately 20\({}^{\circ}\) (Figure 3a). Additionally, the Raman spectrum displayed a carbonate characteristic peak at around 1084 cm\({}^{-1}\) (Figure 3c). The FTIR spectra of pure chitosan showed a broad, strong absorption peak near 3353 cm\({}^{-1}\), which is attributed to the telescopic vibration peak resulting from the overlap of O-H and N-H stretching vibrations (Figure 3d). Absorption peaks near 2869 cm\({}^{-1}\) represent the -CH\({}_{2}\)-and -CH\({}_{3}\) structures, and characteristic absorption peaks near 1656 cm\({}^{-1}\) and 1579 cm\({}^{-1}\) correspond to the stretching vibration of C=O in the amide I band and the bending vibration of N-H in the amide II band. Peaks near 1022 cm\({}^{-1}\) are attributed to the stretching vibrations of C-O-C (ether bond) or C-OH (hydroxyl group), which represent the glycan ring structure of chitosan. In addition to these characteristic chitosan peaks, the FTIR spectrum of chitosan/CaCO\({}_{3}\) revealed a strong, asymmetric C=O stretching vibration peak near 1373 cm\({}^{-1}\), a C-O bending vibration peak near 870 cm\({}^{-1}\), and a smaller absorption peak near 710 cm\({}^{-1}\), corresponding to the O-C-O bending vibration, indicating the CO\({}_{3}\)\({}^{2-}\) structure of CaCO\({}_{3}\). The results indicate that CaCO\({}_{3}\) nanoparticles were successfully encapsulated within the cross-linked chitosan layer, forming a chitosan/CaCO\({}_{3}\) composite hydrogel structure.