## 3 Results and Discussion  
### Structure of the Nanofibers and 3D Nanofibrous Scaffolds  
Electrospinning resulted in chitosan/RHC nanofibers with smooth structure and relative uniform diameter distribution (Figure 1A). As shown in Figure 1B, the diameter of the chitosan/RHC nanofibers ranged from 80 to 347 nm, and the average diameter was cca. 160 nm. To prepare the 3D nanofibrous scaffolds, the chitosan/RHC nanofibrous were homogenized in a 90% ethanol solution to obtain short nanofibers and tiny nanofibrous. As shown in Figure 1C, the structure of the nanofibers was retained, although the diameter of the nanofibers increased after the treatment. The diameters of the short fibers ranged from 180 to 525 nm, and the average diameter was cca. 300 nm. The increasing on fiber diameters has been reported in previous studies, where they discovered that the EDC mainly concentrated on outer surfaces of collagen fibers, and crosslinking is hampered in the inner part of fibers due to the slow penetrations of EDC [30]. After soaking in water, the uncrosslinked inner part dissolved and expanded until obstructed by the outside part, which lead to increasing on fiber diameters [27].  
As revealed by SEM (Figure 2), the scaffolds showed a noticeable porous architecture. All the scaffolds (1%-0.5 mL, 1%-1 mL, and 2%-1 mL) showed interconnected and hierarchically structured pores with sizes ranging from sub-micrometers to 200 \(\upmu\)m, while the maximum pore size of the electrospun nanofibers was ca. several micrometers. Although the chitosan/RHC scaffolds free of nanofibers showed porous structures, they had very smooth pore walls that might not be suitable for cell adhesion. Meanwhile, all the scaffolds prepared with 1% chitosan/RHC aqueous solution showed porosities higher than 80%, and the porosity of the 2%-1 mL scaffold decreased to 70%; the addition of nanofibers did not affect the porosities significantly (Figure 3A). Both the porosity and pore structure play a critical role in a number of processes such as cellular migration, proliferation, differentiation, and tissue regeneration [17]. In this sense, large pores and high porosities allow effective nutrient supply, gas diffusion, and metabolic waste removal, leading to enhanced cell proliferation and ECM deposition. In contrast, smaller pores are particularly useful for cell attachment and intracellular signaling [2, 31].  
The surface topography of nanostructures could alter the topography and chemistry of the implant surface, providing an apparent affect cell response [32]. As shown in Figure 2A-C, the walls of all the scaffolds were covered by nanofibers, and the nanostructure increased as the number of short nanofibers rose. The walls of the scaffolds became thicker, and the number of nanofibers decreased as the concentration of the chitosan/RHC solution was increased to 2%. Overall, these results indicated that 1%-1 mL scaffolds could provide the most suitable structural environment for cell proliferation and migration.  
Figure 1: Structure of the electrospinning nanofibers and short nanofibers. (**A**) SEM image of the electrospun nanofibers. (**B**) Diameter distribution of the electrospun nanofibers. (**C**) SEM image of the homogenized short nanofibers. (**D**) Diameter distribution of the homogenized short nanofibers.