## 2 Materials and Methods
### Materials
#### 2.1.1 Preparation of CNC/PCL Composite Materials  
The CNC was isolated from Humulus Japonicus stem using sulphuric acid hydrolysis method which has been reported in our previous study [30]. Briefly, the HJS powder was treated with 4% sodium hydroxide solution at 80 \({}^{\circ}\)C for 2 h and treated 3 times with 5% acidified sodium chlorite at 80 \({}^{\circ}\)C for 6 h (2 h each time) to remove hemi-cellulose and lignin. Next, the bleached HJS powder was hydrolyzed using 60% sulfuric acid at 45 \({}^{\circ}\)C for 1 h with strong agitation using an electric mixer (OS-Pro, Kexing, Shanghai, China) at 2000 rpm. The reaction was terminated by the addition of a volume of water 10 times that of the reaction solution. The suspension was filtered using a filter paper (GF/B1442-150, [PERSON], England, UK) with a pore size of 2.5 um and subsequently centrifuged using a centrifuge (80-2B, JTLIANGYOU, Changzhou, China) at 10 \({}^{\circ}\)C and 10,000 rpm and dialyzed using a pH Sensor (51343050, Mettler Toledo, Shanghai, China) until its pH value was to 7. This process is known to produce finely divided CNC (See the supplementary material Figure S1 for the TEM images of CNC). Then the CNC after freeze-drying spray drying treatment using a vacuum dryer (DZF-6020, Jingfu, Shanghai China) were added to the dichloromethane solution and dispersed using an ultrasonic (WZB-DC, Weizhibo, Beijing, China) for 48 h to ensure the formation of a homogeneous solution. The CNC extracted by sulfuric acid hydrolysis method was used as biocompatible material. We dialyzed it repeatedly until its pH value reached 7 to ensure the removal of residual sulfuric acid. In addition, the materials were disinfected before cell culture, soaked in alcohol for 24 h, and treated with ultraviolet light for 24 h. The whole printing process is aseptic. Four equal sets of PCL particles were dissolved in the mixed solution and stirred until a homogeneous solution was formed. As shown in Table 1, The filling amount of the CNC was 0%, 1%, 3%, and 5% of the PCL particle mass. The samples were dried by vacuum dryer and were labeled as CNC-X/PCL, and X was the filling amount of the CNC. For the filled pattern, a new filled pattern is designed with the idea that each layer of the scaffold is divided into several concave hexagons that are separated from each other. As shown in Figure 1a, three sides of each concave hexagon are stacked on the filaments below to improve the vertical compression resistance of the bracket. The other three sides are suspended and attached to the filaments below to form a simply supported beam structure to increase the porosity of the bracket. This is shown in Figure 1b. In order to evaluate the porosity and mechanical properties of the scaffolds printed through different filled patterns, a 3D model (20 mm \(\times\) 20 mm \(\times\) 1 mm) was created using Solidworks 2018 software. For this material, we designed a study using the common orthogonal, triangular, and designed concave hexagon filled patterns. The models of the three different filled patterns are shown in Figure 2. \begin{table}
\begin{tabular}{c c c} \hline
**Sample Name** & **Specific Gravity of Dry CNC (\%)** & **Specific Gravity of Dry PCL (\%)** \\ \hline PCL & 0 & 100 \\ CNC-1/PCL & 1 & 99 \\ CNC-3/PCL & 3 & 97 \\ CNC-5/PCL & 5 & 95 \\ \hline \end{tabular}
\end{table} 
Table 1: Chemical compositions of samples2.2.2 Simulation Modeling of a different scaffold pattern. Figure 1: Layer of concave hexagons (**a**) and a stack of concave hexagons (**b**). Solving the above theoretical model, the scaffold material is PCL/CNC, and to calculate the porosity of the scaffold printed using different filling methods, several physical parameters were evaluated by Solidworks. The theoretical volume (V) was calculated by measuring the length (L), width (W), and height (H) of the scaffold. After entering the theoretical density (\(\rho\)), the actual weight (m) was evaluated by Solidworks.