*4.8. Solubility*

### 4.8.1. Effect of pH

The effect of pH on collagen solubility was determined using the method described by Chen et al. (2016) [18], with bovine serum albumin (BSA) as the protein standard. The samples were dissolved in 0.5 M acetic acid at the final concentration of 0.2 mg/mL. The pH of the sample solution (5 mL) was adjusted from 2 to 10, with 6 M HCl or 6 M NaOH. Then, the sample solutions were mixed with distilled water of the same pH until the solution volume reached 10 mL. The relative solubility was calculated through comparison with the solubility obtained at the pH that exhibited the highest solubility.

Collagen solubility was determined at various pH levels using the method described by Chen et al. (2016) [18] with slight modifications. The samples were dissolved in 0.5 M acetic acid at a concentration of 0.3% ( *w*/*v*) with gentle stirring at 4 ◦C for 12 h. The collagen solution (8 mL) was placed in a centrifuge tube. Then, the pH was adjusted to different levels, ranging from 2 to 10, using 6 M HCl or 6 M NaOH. The final volume was brought to 10 mL by distilled water previously adjusted to the same pH as the collagen solution tested. The solutions were gently stirred at 4 ◦C for 30 min and left overnight. Next, the supernatants were collected after centrifugation for 30 min at 10,000× *g*. Protein content in the supernatant was calculated using the Lowry method (1951) [52], with BSA as the protein standard. The relative solubility was determined in comparison with that obtained at the pH level that provided the highest solubility.

### 4.8.2. Effect of NaCl

The effect of NaCl on collagen solutions was measured in accordance with the method described by Chen et al. [18], BSA was used as standard. The samples were dissolved in 0.5 M acetic acid at a concentration of 0.2 mg/mL. The sample solution (5 mL) was mixed with 5 mL of a series of NaCl concentrations containing 0.5 M acetic acid to obtain the final solutions with NaCl concentrations of 0%, 2%, 4%, 6%, 8%, 10%, 12%, and 14%, *<sup>w</sup>*/*<sup>v</sup>*. The protein content was measured as described in Section 4.8.1, and the relative solubility was calculated using the solution with final NaCl concentrations of 0% ( *w*/*v*) as a control.

### *4.9. Rheological Properties*

The rheological properties of collagen were measured by a rheometer (MCR 302, Anton Paar, Graz, Austria) using a stainless-steel cone/plate geometry (0.5◦ cone angle, 60 mm cone diameter, gap of 57 μm). The sample (20 mg/mL) was dissolved in 0.5 M acetic acid and then assessed by dynamic frequency sweeps with a constant strain of 30%. The elastic modulus (G) and viscous modulus (G) of the sample were measured as functions of the frequency range of 0.01 to 10 Hz, at 25 ◦C [41]. Each sample was equilibrated for 10 min before measurement.

### *4.10. Cell Compatibility and Cell Morphology*

The cytotoxicity of collagen to the HaCaT and MC3T3-E1 cells was evaluated using a CCK-8 assay with some modifications as described by Sripriya et al. (2015) [53]. The collagen samples were dissolved in distilled water at a concentration of 5 mg/mL. The bottom of the 96-well plates was coated with the collagen solutions (5 mg/mL) and dried under a laminar airflow hood followed by UV disinfection. The cells were seeded with a density of 1 × 10<sup>4</sup> cells per well and then incubated at 37 ◦C in a humidified atmosphere with 5% CO2 for 24 h and 48 h. The CCK-8 solution was added to each well, and incubation was continued for 1.5 h. The absorbance values were measured at 450 nm (Mithras<sup>2</sup> LB 943, Berthold, Germany), and the uncoated wells were used as controls. The cell viability was

calculated using Equation (2). Subsequently, the morphology of each group was observed under an inverted microscope (ECLIPSE Ti, Nikon, Japan).

$$\text{Cell viability } (\%) = \left( 1 - \frac{\text{absorbrane of treatment}}{\text{absorbrane of control}} \right) \times 100\% \tag{3}$$

### *4.11. Statistical Analyses*

The analysis of variance (ANOVA) was performed using SPSS Version 17.0 software (IBM SPSS Statistics, Ehningen, Germany), and a value of *p* < 0.05 was used to indicate a significant deviation. The different letters indicate significant differences between the samples.
