**3. Results and Discussion**

#### *3.1. Characterization of Polymer Films*

The FTIR spectra shown in Figure 1 were used to assess the functional groups present in the polymers. The FTIR spectra of the PHB, P(HB-12HV), P(HB-50HV), and PCL polymers are also shown for comparison. Since both PHAs and PCL contain ester bonds, peaks of C=O stretching were observed around 1730 and 1625 cm−<sup>1</sup> for the PHAs and PCL, respectively. Both the PHA and PCL spectra also showed slightly different C–H stretching and bending, located from 3000 to 2800 cm−<sup>1</sup> and from 1500 to 1000 cm−<sup>1</sup> [32]. Although the PHB, P(HB-12HV), and P(HB-50HV) polymers are chemically similar, the differences in the HV composition of the polymers could be distinguished by FTIR spectra. The PHB homopolymer showed characteristic peaks at 1724 cm−<sup>1</sup> for C=O stretching and 1281 cm−<sup>1</sup> for C–O stretching [33–35]. Apart from additional peaks at 797 cm<sup>−</sup>1, responsible for C–H bending, the presence of HV in the P(HB-HV) copolymers could be identified by observing the FTIR peak shifts. A major shift occurred at the C=O stretching region, in which the peak shifted from 1724 cm−<sup>1</sup> in PHB to 1735 cm−<sup>1</sup> in P(HB-HV). The greater the change to the higher wavenumber, the higher the %HV monomer in the polymer chain. This phenomenon was also observed in other peaks such as C–O stretching at 1281 cm−<sup>1</sup> and the C–H stretching region around 3000 cm−1. In addition, several peaks from FTIR can be used to denote the crystallinity state of different PHA polymers. The peaks at 1453, 1380, 1281, 1057, and 826 cm−<sup>1</sup> shifted to a higher wavenumber when the crystallinity was low [36,37]. Our results showed that there were around five to 10 wavenumber shifts in the mentioned peaks among PHB, P(HB-12HV), and P(HB-50HV). Therefore, the PHAs used in this study were confirmed as having differences in %HV as well as their crystallinity.

The hydrophilicity of a polymer surface is the key parameter affecting cell–material interaction and the adsorption of protein on the polymer surface, which subsequently influence cell behaviors [38]. The results of the water contact angle measurements are summarized in Table 1. All samples showed contact angles of below 90◦ considering hydrophilic behavior. The highest contact angle value of the PCL film indicated the greater hydrophobicity of PCL than the other PHAs. The contact angle value of P(HB-50HV) was significantly higher than the other PHA films tested (*p* < 0.05). This might be due to more ethyl groups of the HV monomer present in the side chain of the copolymers [39]. Kim et al. reported a water contact angle of 79.5◦ of the P(HB-60HV) film produced by *Haloferax mediterranei* ES1 [20].

**Table 1.** Surface hydrophilicity of the polymer films.


(Mean ± SD, *n* = 3, different superscript letters indicate a significant difference at *p* < 0.05).

**Figure 1.** FTIR spectra of the (A) PHB, (B) P(HB-12HV), (C) P(HB-50HV) and (D) PCL polymers.

#### *3.2. Characterization of Scaffolds*

The PHB, P(HB-12HV), P(HB-50HV), and PCL scaffolds were fabricated via a particulate salt leaching technique. All fabricated porous scaffolds exhibited a high porosity of 90% with a sponge-like appearance (Figure 2). The structure of the pores as well as the surface and cross-sectional topologies of the 3D porous scaffolds were examined using SEM, as shown in Figure 3. All polymeric scaffolds were similar in terms of the surface and cross-sectional topographies that comprised of interconnected open pores throughout the scaffolds. The well-tailored pore sizes ranged between 425 and 500 μm on both the surface and inside the scaffolds, suggesting sufficient surface areas for cell attachment. Furthermore, the pore shape observed was similar to the shape of the imprinted salt crystals. Our results agree with earlier findings for scaffolds with pore sizes of around 400 μm, which are considered suitable for the growth and proliferation of bone cells [40]. In general, the scaffolds were highly porous with interconnected pore networks that facilitate nutrient and oxygen diffusion and waste removal during tissue formation. The interconnected networks between open pores are also important for cellular attachment, proliferation, and migration for tissue vascularization [26,41].

**Figure 2.** Photographic images of 3D porous scaffolds via the particulate salt leaching technique.

**Figure 3.** SEM micrographs of representative 3D porous scaffold samples with pore sizes ranging from 425 to 500 μm: PHB scaffold (**A**,**E**), P(HB-12HV) scaffold (**B**,**F**), P(HB-50HV) scaffold (**C**,**G**), and PCL scaffold (**D**,**H**).
