**3. Results**

To overcome these problems, two crucial issues should be addressed. First, the PGA-based scaffold should be prefabricated into the exact shape of human nose. Second, the mechanical strength of the above-mentioned scaffold should be further enhanced so that it can retain the

In order to meet these requirements, in the current study, a computer aided design and manufacturing (CAD/CAM) technique was employed to fabricate a set of negative molds, which was then used to press the PGA fibers into the pre-designed nose structure. Further‐ more, the mechanical strength of the scaffold was enhanced by coating the PGA fibers with an

10 mg of unwoven PGA fibers (provided by Dong Hua University, Shanghai, China) were compressed into a cylinder shape of 5mm in diameter and 2mm in thickness. A solution of 0.3 % PLA (Sigma, St. Louis, MO, USA) in dichloromethane was evenly dropped onto the PGA scaffold, dried in a 65 ºC oven, and weighed. The PLA mass ratio was calculated according to the formula: PLA%= (final mass-original mass)/final mass×100%. The above procedures were repeated until the predetermined PLA mass ratios of 0%, 10%, 20%, 30%, 40% and 50% were achieved. The scaffolds were examined by SEM (Philips XL-30, Amsterdam, Netherlands) [18].

**Cell seeding:** Chondrocytes were isolated from the articular cartilage of newborn swine (2-3weeks old) as previously described [19]. The harvested chondrocytes were adjusted to a

scaffold. The cell-scaffold constructs were then incubated for 4h at 37ºC with 95% humidity

**Cell adhesion:** After 4 hours of incubation, the cell-scaffold constructs were gently transferred into a new 6-well plate. The remaining cells were collected and counted. The cell seeding efficiencies of the scaffolds with different PLA contents were calculated based on the formula:

A patient's normal nose was scanned by CT to obtain the geometric data (Figure 3). These data were further processed by a CAD system to generate both positive and negative of the normal nose, and the resultant data were input into a CAM system (Spectrum 510, Z Corporation) for the fabrication of the resin models by 3D printing. The negative mold was composed of two

and 5% CO2 to allow for complete adhesion of the cells to the scaffolds.

(total cell number- remaining cell number)/ total cell number×100% [14].

parts: the anterior part and the posterior part. (Figure 4A)

cells/mL, and a 100uL cell suspension was pippeted onto each

pre-designed shape.

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optimized amount of PLA.

**2. Materials and methods**

final concentration of 50×106

**2.3. Mold fabrication by CAD/CAM**

**2.1. Preparation of scaffolds with different PLA contents**

**2.2. Biocompatibility evaluation of the scaffolds**

#### **3.1. SEM observation of the scaffolds with different PLA contents**

PLA/PGA scaffold compositions were visualized under SEM. (Figure 1) The pure PGA scaf‐ fold (0% PLA added) appeared as a smooth fiber mesh. In PGA scaffolds supplemented with 10% PLA, the PLA coating can be seen connecting some fibers, particularly at nodes where PGA fibers cross. In the 20% PLA embedded scaffold, most mesh nodes visualized were covered with PLA. 30% PLA scaffold had not only most mesh nodes embedded in PLA, but also the PLA coating was seen covering small portions of the mesh itself, minimally obstructing the porosity of the fiber network. In the 40% PLA embedded scaffold, most of the mesh porosity is obscured by a PLA. In the 50% PLA scaffold, the mesh is almost completely obscured by a PLA sheet.

**Figure 1.** SEM examination. Scaffolds with different PLA contents (0%, 10%, 20%, 30%, 40% and 50%) show differ‐ ent pore structures. The white arrows indicate the coated PLA.

#### **3.2. Evaluation of the biocompatibility of the scaffolds with different PLA contents**

Cell seeding efficiencies were performed to evaluate the influence of PLA contents on cell compatibility of the scaffolds. The results showed that the increase in PLA content could lead to the reduction in the ability of the scaffolds to absorb the cell suspensions (Figure 2A). Quantitative analysis (Figure 2B) demonstrated that all the groups with PLA presented significantly lower cell seeding efficiencies compared to the group without PLA (p<0.05). If the acceptable cell adhesion rate is defined over 80%, these results indicate that 20% but not 30% is an acceptable PLA amount for preparing the scaffolds in terms of cell seeding efficiency.

**Figure 2.** The influences of PLA contents on cell seeding efficiency. (A): Scaffolds with different PLA contents absorb different volumes of the cell suspension. (B): Cell seeding efficiencies decrease with increasing PLA contents in the scaffolds with significant decreases (p<0.05).

#### **3.3. Mold preparation and fabrication of the nose-shaped scaffold**

Because good biocompatibility could be achieved in the scaffold with 20% PLA, this formu‐ lation was further used for the fabrication of the human nose shaped scaffold. In order to prepare the scaffold into a shape of normal nose, a set of negative molds was produced according to image of the normal nose (Figure 3). The resulting nose-shaped scaffold (Figure 4C) achieved a precise shape compared to its positive mold (Figure 4B). These results indicate that the mold produced by CAD/CAM technology is allowed to accurately fabricate a scaffold into a nose shape.

**Figure 3.** image of a patient's normal nose.

**3.2. Evaluation of the biocompatibility of the scaffolds with different PLA contents**

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Cell seeding efficiencies were performed to evaluate the influence of PLA contents on cell compatibility of the scaffolds. The results showed that the increase in PLA content could lead to the reduction in the ability of the scaffolds to absorb the cell suspensions (Figure 2A). Quantitative analysis (Figure 2B) demonstrated that all the groups with PLA presented significantly lower cell seeding efficiencies compared to the group without PLA (p<0.05). If the acceptable cell adhesion rate is defined over 80%, these results indicate that 20% but not 30% is an acceptable PLA amount for preparing the scaffolds in terms of cell seeding efficiency.

**Figure 2.** The influences of PLA contents on cell seeding efficiency. (A): Scaffolds with different PLA contents absorb different volumes of the cell suspension. (B): Cell seeding efficiencies decrease with increasing PLA contents in the

scaffolds with significant decreases (p<0.05).

**Figure 4.** Mold preparation and the fabrication of the nose-shaped scaffolds. (A): The resin negative mold: anterior part and posterior part; (B): The resin positive mold; (C): the nose-shaped PLA/PGA scaffold.
