*3.3. Mechanical Properties of Scaffolds*

The typical stress–strain curves obtained from the compressive stress measurement at 30% strain were used to calculate the compressive modulus of all scaffolds. As presented in Figure 4, the compressive modulus values under the dry condition of the PHB, P(HB-12HV), and P(HB-50HV) scaffolds were found to be 0.75 ± 0.02, 0.39 ± 0.08, and 0.04 ± 0.01 MPa, respectively. The compressive modulus of the PCL scaffold at 0.45 ± 0.01 MPa was not significantly different from the scaffold made with P(HB-12HV) polymers. In addition, the lowest compressive modulus was observed in the P(HB-50HV) scaffold. Our results suggest that increasing the HV content in the P(HB-HV) polymer chain at 50% could lead to a significant decrease in the compressive modulus of a 3D porous scaffold while maintaining the same % porosity. Previous studies have reported that an increase of %HV up to 50–60% could cause a lower melting temperature due to a decrease in the crystallinity of the PHA copolymers, resulting in ductile mechanical properties such as higher elongation to break and greater flexibility with a faster degradation rate under specific physiological conditions [42,43]. Among the P(HB-HV) with various %HV contents produced by *P. denitrificans*, film sheets composed of P(HB-HV) with a HV of 53–60 mol% were found to be more flexible and tougher [19]. Here, the P(HB-50HV) scaffold appeared to be the most soft and flexible scaffold in the dry state.

**Figure 4.** Compressive secant modulus at 30% strain compared between the dry and wet conditions. Data are reported as the average values from six independent scaffolds with standard deviations (*n* = 6). The different letters (a, b, and c) above the bar graph indicate significant differences (*p* < 0.05) between the scaffold materials tested under the same conditions.

The wet-state mechanical properties of 3D porous scaffolds were also investigated in order to determine their compressive behavior in a realistic environment. In Figure 4, the compressive modulus values under wet conditions of the PHB, P(HB-12HV), and PCL scaffolds were found to be approximately in the same levels at 0.33 ± 0.04, 0.25 ± 0.04, and 0.23 ± 0.08 MPa, respectively, which were 62%, 36%, and 49% reduced from the dry condition, respectively. However, there was no change in the compressive modulus of the P(HB-50HV) scaffold under the wet state when compared to the dry state, which still remained at 0.04 ± 0.01 MPa. There was a clear decrease in the mechanical properties from the dry to wet state in all scaffolds, except for P(HB-50HV). Our findings are in line with earlier reports that observed decreased compressive moduli of 3D polymeric scaffolds under wet conditions, which used PBS and cell culture media [27,28]. The water molecules could intersperse and intercalate among the polymer chains that finally spread the polymer chains apart by losing the crystalline network characteristics of the polymer [44]. Notably, there was no discernable difference between the dry and wet conditions on the compressive modulus of the P(HB-50HV) scaffold. One explanation could be that the compressive modulus of P(HB-50HV) at the dry state is already quite low, and that any eventual water plasticizer effect might be too small to be detected.

#### *3.4. Cell Proliferation*

Early cell adhesion and proliferation are necessary in developing scaffolds for periodontal regeneration. The cell adhesion ability and proliferation enhancement of HGFs and PDLSCs on 3D porous scaffolds were studied using the MTT assay. The HGF cells were cultured on different scaffolds for 0, 1, 2, 4, 6, and 8 days. In Figure 5A, the HGF cells grew quite slowly during the first 2 days for all types of scaffolds tested. Interestingly, cell numbers at 8 days were significantly the highest for the P(HB-50HV) scaffold, which showed about a 16-fold increase from the start, followed by the P(HB-12HV) and PCL scaffolds (10-fold), PHB scaffold (8-fold), and 2D control surface (6-fold), respectively.

**Figure 5.** Cell proliferation of (**A**) HGF and (**B**) PDLSC cells grown on different types of 3D porous scaffolds. The values are the means with standard deviation derived from three independent scaffolds (*n* = 3). Bars labeled with different letters (a, b, c, and d) indicate significant differences within the same day (*p* < 0.05).

The proliferation ability of PDLSC cells was investigated at 0, 3, 7, 14, and 21 days, as shown in Figure 5B. Similar cell numbers were found on the 2D control surface and in all scaffolds at day 0. On the last day of the experiment, the highest number of PDLSC cells was significantly increased on the P(HB-50HV) scaffold with a 7-fold increase from the initial cell loaded compared with the P(HB-12HV) and PCL scaffolds (6-fold), and the PHB scaffold and 2D control surface (5-fold).

In this study, it was clear that all PHA scaffolds could support the attachment and proliferation of HGF and PDLSC cells. This is the first report on the cytotoxicity and biocompatibility of P(HB-50HV) produced by *C. necator* H16 as a candidate scaffold for dental tissue engineering. However, the P(HB-50HV) scaffold appeared to be the most suitable to support both HGF and PDLSC cell growth among the three types of PHA scaffolds, and was even better than the conventional PCL scaffold. Since the chemical properties of PHA and PCL scaffolds are quite similar such as the functional group and hydrophilicity, protein absorption on the material surface is known to be one of the important considerations to promoting cell attachment and the growth of anchorage-dependent mammalian cells on a solid substratum [45]. The amount of protein absorbed on the scaffolds' surface were found to be similar, as shown in Figure 6. Thus, the HGF and PDLSC cell proliferation was related to the mechanical properties of the 3D porous scaffolds. Many previous studies have reported that the stiffness of the material has an effect on cell attachment signaling, leading to a difference in the cell proliferation and differentiation [46,47]. These results indicate that the P(HB-50HV) scaffold had the lowest stiffness, which could promote the greatest adhesion and proliferation of HGF and PDLSC cell adhesion and proliferation, and thus should be considered as a suitable material for the tissue engineering of periodontal cells and other soft tissue-like cells. Regarding the scaffold mechanical properties, our results are in line with the finding reported previously that the fabricated PCL scaffolds with lower modulus values than the PLGA scaffolds showed a 2-fold higher growth rate of stromal cells [48].

**Figure 6.** The adsorption of FBS proteins on 3D porous scaffolds. The values are the means with standard deviation derived from three independent scaffolds (*n* = 3).

#### *3.5. Cell Morphology*

The morphology of healthy HGF and PDLSC cells cultured on the P(HB-50HV) scaffold was further analyzed by fluorescence staining of the nucleus and F-actin filaments. The round-shape of the HGF cells was observed at day 0 (Figure 7A) followed by cell migration and the development of interconnecting network development by day 2 (Figure 7B). After 8 days of culture, the HGF cells were distributed throughout the entire scaffold with a strong presence of F-actin, resulting in a dense interconnecting network of cells (Figure 7C).

**Figure 7.** Confocal micrographs of the (**A**–**C**) HGF and (**D**–**F**) PDLSC cells cultured on the P(HB-50HV) scaffold. Cell nucleus was stained with Hoechst 33342 (blue). F-actin was stained with Alexa Fluor 568 phalloidin (red).

The initial cell adhesion of PDLSC cells at day 0 were observed to be round-shape (Figure 7D). The PDLSC cells proliferated considerably well on the scaffold surface and gradually progressed to high cell density all over the cultured scaffold from day 7 (Figure 7E) to day 21 (Figure 7F). Importantly, both HGF and PDLSC cell morphology on the P(HB-50HV) scaffold showed both spindle and stellate shapes, which are typically good indicators of healthy fibroblasts [49,50]. The results showed that the biocompatibility of the P(HB-50HV) scaffold has promising potential for periodontal tissue engineering.

The scaffold made from bacterially-derived P(HB-50HV) copolymers developed in this study showed a softness property and displayed a capability to promote good proliferation and the interconnection of periodontal cells including HGF and PDLSC cells. The microbial P(HB-50HV) scaffold is not only effective for normal fibroblast cell regeneration, but also demonstrates good potential to promote stem cell proliferation. With these interesting properties, the P(HB-50HV) scaffold is an attractive material for tissue engineering strategies.

#### **4. Conclusions**

In this study, 3D porous scaffolds made from PHB, P(HB-12HV), P(HB-50HV), and PCL polymers were successfully fabricated via the salt leaching method with similar properties in terms of chemical functionality, surface hydrophilicity, surface topography, % porosity, and serum protein absorption. Furthermore, the P(HB-50HV) scaffolds were distinctively different in their compressive modulus by having the lowest stiffness among all of the scaffolds tested. The proliferation of dental cells including HGF and PDLSC cells was investigated with four different types of scaffolds. Interestingly, the P(HB-50HV) scaffold showed the highest proliferation of both HGF and PDLSC cells over all of the PHA scaffolds

and the control PCL scaffold. Cells grown on the P(HB-50HV) scaffold had the characteristic of healthy fibroblasts in forming highly dense interconnecting networks. Taken together with the hydrophilicity, softness property, greater cell proliferation, and morphology of dental cells grown on the P(HB-50HV) scaffold, these results confirm the possibility of using a microbial-derived P(HB-50HV) scaffold as a biomaterial for periodontal tissue engineering and stem cell applications.

**Author Contributions:** Conceptualization, N.N., S.P. (Seubsakul Phuegyod), S.P. (Sasivimon Pramual), J.S. and R.S.; Methodology, S.P. (Seubsakul Phuegyod), S.P. (Sasivimon Pramual), N.W., S.A. and P.S.; Investigation, S.P. (Seubsakul Phuegyod), S.P. (Sasivimon Pramual), N.W., S.A., T.A. and N.N.; Writing—original draft preparation, S.P. (Seubsakul Phuegyod) and S.P. (Sasivimon Pramual); Writing—review and editing, N.N., R.S. and J.S.; Visualization, S.P. (Sasivimon Pramual) and N.N.; Supervision, N.N., R.S. and J.S.; Project administration, N.N.; Funding acquisition, N.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the National Science and Technology Development Agency (NSTDA) through the Coordinating Center for Thai Government Science and Technology Scholarship Students (CSTS): A New Researcher Scholarship of CSTS-MOST, Thailand Science Research and Innovation (TSRI), Chulabhorn Research Institute (grant no. 36821/4274353). The research was also partially supported by the Faculty of Science, Mahidol University.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.
