*4.1. Materials*

The by-product from *P. olivaceus* was provided by EUNHA Marine Co., Ltd. (Busan, Korea). The α-minimum Eagle's medium ( α-MEM), fetal bovine serum (FBS), trypsin (250 U/mg), penicillin/streptomycin, and other materials used in cell culture experiments were purchased from GIBCO ™ (Gaithersburg, MD, USA). Polycaprolactone (PCL), 1-Step p-nitrophenyl phosphate (pNPP), and Alizarin Red S were purchased from Sigma-Aldrich (St. Louis, MO, USA). The other chemical reagents and materials that were used were commercially available and analytical grade.

### *4.2. Extraction and Characterization of Pepsin Soluble MC*

### 4.2.1. Extraction of Pepsin Soluble MC

*P. olivaceus* skin was descaled and desalted by washing with cold water at 4 ◦C for one day and cut into small pieces. Pepsin-soluble MC was extracted from the prepared skin, following the method described by [44] with slight modifications. All steps of the procedure were carried out at 4 ◦C with gentle stirring. Non-collagenous proteins were removed with 0.1 M NaOH at small pieces to a solution ratio of 1:10 (*w/v*) for 2 days. The skins were then washed with ultrapure water until they became a neutral pH. The skins were defatted with acetone with a pieces to solution ratio of 1:10 (*w/v*) for 2 days with a changing to new acetone solution every 12 h, and then thoroughly washed with ultrapure water. Then the skins were suspended in 0.5 M acetic acid with a pieces to solution ratio of 1:20 (*w/v*) for 1 day. After pretreatment, the fish skin was hydrolyzed by pepsin to extract collagen. The skin was dissolved in 0.5 M acetic acid with pepsin (pepsin 1:3000, Sigma, St. Louis, MO, USA) for 24 h at 4 ◦C, then centrifuged at 15,000 rpm for 30 min. The supernatant was salted out by adding NaCl until a final concentration of 0.9 M. The resultant precipitate was collected by centrifugation at 15,000 rpm for 1 h and then dissolved in 0.5 M acetic acid. The solution was then dialyzed against 0.1 M acetic acid for 1 day and ultrapure water for 3 days. The resultant dialysate was lyophilized and was referred to as MC (Figure 7).

**Figure 7.** MC extraction methods from *P. olivaceus* (pepsin hydrolysis).

### 4.2.2. Sodium Dodecyl Sulfate Polyacrylamide-Gel Electrophoresis (SDS-PAGE)

SDS-PAGE was performed, following the method of [45] with slight modifications, using 7.5% separating and 5% stacking gel. The collagen samples were dissolved in the sample buffer and the obtained mixture (1 mg/mL) was heated at 100 ◦C for 5 min. The mixture was centrifuged at 4000 rpm for 5 min using a microcentrifuge at room temperature to remove debris. A total of 20 μg of the sample was loaded onto a polyacrylamide gel and subjected to electrophoresis at a constant voltage (100 V) for 1 h using MiniProtein II unit (Bio-Rad Laboratories, Inc. Richmond, CA, USA). The resultant gel was stained with 0.1% (*w/v*) Coomassie blue R-250 in 50% (*v/v*) methanol and 10% (*v/v*) acetic acid for 2 h and destained with 40% (*v/v*) methanol and 10% (*v/v*) acetic acid. High molecular weight markers were loaded alongside the collagen to estimate the molecular weight of MC, and commercial atelocollagen (Atelocollagen, Dalim Tissen, Korea) was loaded next to the protein marker as standard collagen.

### 4.2.3. UV Absorbance Analysis

The UV absorption spectra of MC from the skin of *P. olivaceus* were studied, following the method reported elsewhere with slight modifications [46]. The MC and commercial atelocollagen samples (1 mg) were dissolved in 1 mL of 0.5 M acetic acid and the collagen solutions were centrifuged at 15,000 rpm for 10 min at 4 ◦C. The collagen solution was placed in a quartz cell with a path length of 1 mm. The collagen solutions were subjected to absorbance at wavelengths between 200 and 500 nm at a scan speed of 2 nm per second with an interval of 1 nm. All spectra were obtained using a UV–visible spectrometer (Epoch 2 Microplate reader, Biotek, Winooski, VT, USA).

### 4.2.4. Amino Acid Contents

Amino acid compositions were analyzed using an automatic analyzer (Hitachi Model 835-50, Tokyo, Japan) with a C18 column (5 μm, 4.6 × 250 nm, Watchers, MA, USA). The reaction was carried out at 38 ◦C, with the detection wavelength at 254 nm and flow rate of 1.0 mL/min. All chemical analyses (from each tank) were carried out in triplicate.

### *4.3. Isolation and Characterization of Carbonated Hydroxyapatite* 4.3.1.IsolationofCarbonated Hydroxyapatitefrom *P.Olivaceus*

*P. olivaceus* bones were cut into small pieces using a bladed cutter. The bone pieces were boiled in 100 ◦C purified water for 1 h to remove unnecessary parts. Then, the bone pieces were boiled in 10 mL of acetone and 2% NaOH for 1 h, and the water was completely removed at 100 ◦C. The dried bone pieces were crushed using a homogenizer. CHA was extracted by boiling the crushed bone pieces for 1 h in 200 ◦C 2 M NaOH to completely remove the organic materials. Collected CHA was washed with purified water to adjust the pH to neutrality and to remove all of the moisture from the dry oven (Figure 8).

**Figure 8.** CHA extraction methods from *P. olivaceus* (Alkaline hydrolysis).

### 4.3.2. Fourier Transform Infrared (FTIR) Spectroscopy

FTIR spectroscopy (Perkin Elmer, Waltham, MA, USA) data were collected from raw fishbones, CHA, HA, MC, and commercial atelocollagen to determine the functional groups of hydroxyapatite and collagen. The IR spectra represent the average of 30 scans between 500 cm<sup>−</sup><sup>1</sup> and 4000 cm<sup>−</sup>1, at a resolution of 4 cm<sup>−</sup>1.

### 4.3.3. X-ray Diffraction (XRD) Analysis

XRD analysis was conducted on the fishbones, HA and CHA using an Ultima IV system (Rigaku Co., Tokyo, Japan) with Cu-K α radiation. The X-ray diffraction intensities were recorded within the range of 5 to 80◦, at a scanning rate of 2◦ min−1.

### *4.4. Fabrication and Characterization of 3D Scaffolds*

In this study, we used a computer-controlled three-axis robot system (EZ-ROBO-5GX ST2520, Iwashita Engineering Inc., Fukuoka, Japan), supplemented with a dispenser to fabricate the PCL, HA/PCL, and CHA/PCL structure. The PCL struts were melted at 100 ◦C in a heating barrel and were extruded through a heated 21G nozzle at a constant pressure (500 ± 25 kPa). Following this condition, the PCL struts were built in a layer-by-layer manner to make a 3D structure with uniform height and porosity. After fabricating the multilayered structure, the fabricated scaffold was sterilized in 70% EtOH. After fabricating the PCL, the HA/PCL and CHA/PCL scaffolds were coated with MC on the surface through 1-ethyl-(3-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) coupling reaction (Figure 9).

**Figure 9.** Schematic diagram of CHA-reinforced PCL scaffold and coated collagen.

### 4.4.1. Scanning Electron Microscope (SEM) Analysis

The structural morphologies of PCL, 2.5% CHA/PCL, 5% CHA/PCL, 10% CHA/PCL, 10% HA/PCL, MC/PCL, 2.5% CHA/MC/PCL, 5% CHA/MC/PCL, 10% CHA/MC/PCL, and 10% HA/MC/PCL scaffolds were examined using a field emission scanning electron microscope (SEM, Tescan, Czech, VEGA II LSU) at 15 kV. The diameter of the 3D scaffolds was measured from the SEM image using image analysis software (Image J, National Institutes of Health, Bethesda, MD, USA).

### 4.4.2. Tensile Properties

The tensile properties of PCL, 2.5% CHA/PCL, 5% CHA/PCL, 10% CHA/PCL, 10% HA/PCL, MC/PCL, 2.5% CHA/MC/PCL, 5% CHA/MC/PCL, 10% CHA/MC/PCL, and 10% HA/MC/PCL scaffold (2.5 × 2.5 mm2) were measured using a universal tensile machine (Top-tech 2000, Chemilab, Kimpo, Korea). The PCL scaffolds were mounted and subjected to a crosshead speed of 0.2 mm/s at room temperature until failure. The elastic modulus was investigated by the average of three runs for each scaffold.

### *4.5. In Vitro Study on Fabricated Scaffolds*

### 4.5.1. Cell Culture and Cell Viability

The MC3T3-E1 subclone 4 cells were purchased from the American Type of Culture Collection (Rockville, MD, USA). The MC3T3-E1 cells were cultured in α-MEM without ascorbic acid, supplemented with 10% fetal bovine serum (FBS), 100 μg/mL streptomycin, and 100 U/mL penicillin. The MC3T3-E1 cells were incubated in 5% CO2 humidified atmosphere and at 37 ◦C and sub-cultured every 2–3 days.

The MC3T3-E1 cells were seeded onto the scaffolds by dropping them onto scaffolds at a density of 1 × 10<sup>5</sup> per scaffold. Before the cells were seeded, the scaffolds were sterilized with 70% ethyl alcohol (EtOH) and UV light.

The cell viability and distribution of the MC3T3-E1 pre-osteoblasts cultured on the CHA-reinforced scaffolds were determined by Cell Counting Kit-8 (CCK-8) assay (Dojindo, Kumamoto, Japan) and live/dead fluorescence staining. Cell viability at 3, 5, and 7 days via CCK-8 assay was evaluated, according to the manufacturer's protocol. A total of 10 μL of CCK-8 solution along with 100 μL of α-MEM were added to each well; the mixture was incubated at 37 ◦C. for 2 h. The cell viability was evaluated at 450 nm by using a microplate reader (Biotek, Winooski, VT, USA). The cell viability was evaluated using a fluorescence microscope through the Live/Dead assay. After 7 days, the MC3T3-E1 seeded on the CHA-reinforced scaffolds were stained with fluorescein diacetate (8 μg/mL) and propidium iodide (20 μg/mL) for 15 min at room temperature after washing three times with PBS. The stained MC3T3-E1 on the scaffolds were qualitatively examined under a fluorescence microscope (Axio Observer A1, Zeiss, Jena, Germany).

### 4.5.2. ALP Activity and Mineralization Analysis on 3D Scaffolds

MC3T3-E1 cells were cultured in α-MEM containing 50 μg/mL ascorbic acid and 10 mM β-glycerophosphate for the osteogenic differentiation. For the incubation, the osteogenic differentiation media was changed every 2 days.

After 7 days, the 1-StepTM PNPP reagen<sup>t</sup> (100 μL) was added to wells and incubated for 30 min. The 2N NaOH solution was added to stop reactions. The absorbance was measured at 405 nm with a microplate reader. Mineralization in the scaffolds was calculated by subtracting the Alizarin Red values, which were stained by the HA and CHA contained in the cell-free scaffolds.

After 14 and 21 days, the MC3T3-E1 cells were fixed with 10% formalin and stained with Alizarin Red S (40 mM). After staining with Alizarin Red S, the Alizarin Red S was removed and washed three times with D.W. The staining was dissolved through cetylpyridinium chloride and the absorbance of the dissolved stain was measured at 550 nm using a microplate reader.

### *4.6. In Vivo Study in Mouse Calvarial Defect Model*

The experimental protocol was approved by the Animal Care and Experiment Committee of Pukyong National University and performed following relevant guidelines and regulations for the care and use of laboratory animals. To determine the bone regeneration ability of the containing MHA and MC, Male CrljOri:CD1 (ICR) mice (approximate weight of 35 g) were used as an in vivo bone defect model. The experiment was conducted according to the protocols approved by the Institutional Animal Care and Use Committee. The ICR mice were maintained on a 12 h light/dark cycle in a controlled environment (relative humidity: 40–70%; temperature: 20–24 ◦C).

### 4.6.1. Mouse Calvarial Defect Model and Implantation of 3D Scaffolds

Anesthesia of ICR mice was performed by intraperitoneal injection of Zoletil 50. After brightening and disinfecting the upper part of the head, the surgical site was vertically incised and the soft tissue was removed to expose the calvarium. Two 3 mm diameter defects created on both side in the exposed calvarium and PCL, 10% HA/MC/PCL, and

10% CHA/MC/PCL scaffolds were implanted into the bone defect site. The non-treated group was used as a control group.

### 4.6.2. Micro-Computed Tomography Analysis

After the calvarial defect was created, it was implanted with PCL, 10% HA/MC/PCL, and 10% CHA/MC/PCL scaffolds. After 20 weeks of operation, ICR mice were euthanatized and the calvarial specimens were harvested and fixed in 10% formalin for further characterization and analysis. Micro-CT (NFR Polaris-G90, NanoFocusRay Co., Ltd., Korea) was firstly used to detect the defect area with the settings (80 kV, 0.06 mA). The 3D structures of calvarium were reconstructed through Mimic software (Radiant, Poznan, Poland).

### 4.6.3. Histological Analysis

Calvarial specimens were fixed in 10% formalin for 5 days at room temperature and decalcified in 8% formic acid and 8% hydrochloric acid, frozen in mounting media. The frozen blocks were cut into 5 μm-thick sections across the center of the defect area and stained with HE, picrosirius red, and MT staining to evaluate the bone regeneration.

### *4.7. Statistical Analysis*

All quantitative data are presented as means ± standard deviation (SD) with at least three individual experiments that were conducted using fresh reagents. Significant differences among the groups were determined using the unpaired Student's *t*-test. The differences were considered statistically significant at *p* < 0.05.
