*3.3. Biomineralization Activity of the nHA-PBP Hybrid Membranes*

Considering that the biomineralization activity critically influences the biomaterials in bone tissue regeneration, here, the bioactivity of the nHA-PBP hybrid membranes for in vitro apatite forming is assessed by immersion in SBF for 7 days. As shown in Figure 6, the apatite formation capability of the hybrid membranes is significantly affected by the nHA contents. As one can see in Figure 6, the surface of the nHA-PBP hybrid membranes shows new apatite layers relative to the specimens before incubation in SBF (in Figure 3). That is, the mineral is deposited and aggregated in the form of a globular accumulation on the surface of the sample with 0 wt% nHA of the nHA-PBP hybrid membranes as in Figure 6A,B. When the additive of nHA increases to 20 wt% and 30 wt%, the nHA-PBP is covered with densely spherically shaped particles as seen in Figure 6C–F. With the nHA content increasing, the surface morphology of the as-formed hydroxyapatite nanocrystals changes considerably. In addition, it progressively shows needle-like or rodlike characteristics in shape, as shown in Figure 6H,J, showing typical biomineralization characteristics only for bioactive glass materials.

**Figure 6.** Surface morphologies of the nHA-PBP hybrid membranes with different nHA contents after biomineralization in SBF for 7 days. (**A**,**B**) 0 wt% nHA, (**C**,**D**) 20 wt% nHA, (**E**,**F**) 30 wt% nHA, (**G**,**H**) 40 wt% nHA, (**I**,**J**) 50 wt% nHA.

Figure 7 shows the EDS spectra of the nHA-PBP hybrid membranes with various nHA contents after being immersed into SBF for 7 days. It can be seen that, compared to the EDS of the hybrid membrane before being soaked, immersion into SBF leads to the formation of the hydroxyapatite. As the nHA content increases, the formation of hydroxyapatite increases, which is accordant with the published literature. In addition, EDS of the hybrid membrane with the addition of 20 wt% nHA, after being soaked in SBF, shows a significant decrease in the calcium content, indicating a biological apatite formation with a calcium-deficient characteristic [23].

**Figure 7.** Elemental compositions of the nHA-PBP hybrid membranes with different nHA contents after biomineralization in SBF for 7 days. (**A**) 0 wt% (**B**) 20 wt%; (**C**) 30 wt%; (**D**) 40 wt%; (**E**) 50 wt%.

Figure 8 shows the XRD patterns of the hybrid membranes containing different nHA contents and after the 7 days of incubation in SBF, which are employed to investigate the structure of the crystalline phase property of the new forming apatite layer on the hybrid membrane surface. These results indicate that several characteristic peaks are related to crystalline hydroxyapatite. It is also clear to see that the peaks referring to PCL at 2θ = 21.88◦ and 2θ = 23.85◦ are significantly weakened in intensity after 7 days of soaking in SBF, which implies the newly mineralized apatite layer forming on the specimens film. The XRD diffraction peaks at 32◦, 39◦, 46◦, and 49◦ for the hybrids with the addition of 20–50 wt% of nHA correspond to the crystal planes of (211), (310), (222), and (213) of the HA (JCPDS No. 09-0432) [21]. It should be noted that the characteristic peaks of HA are not obvious for the pure PBP hybrid. Clearly, these SEM, EDS, and XRD results demonstrate that the nHA incorporation can remarkably increase the capability for biomineralization in the nHA-PBP hybrid membranes.

**Figure 8.** XRD patterns of the nHA-PBP hybrid membranes with different nHA contents after biomineralization in SBF for 7 days. Representative diffraction peaks of hydroxyapatite were marked in the patterns.

#### *3.4. Osteoblasts Biocompatibility Assessment of the nHA-PBP Hybrid Membranes*

Figure 9 shows the cell attachment and proliferation activity of the osteoblast line (MC3T3-E1) after culturing for 1, 3, and 5 days on the surface of the hybrid membranes. The cells show normal attachment and spreading morphology on the surface of the PBP hybrid membrane, as shown in Figure 9A. While for the nHA-PBP 20 wt% (in Figure 9B) and the nHA-PBP 50 wt% (in Figure 9C) after being cultured for 5 days, there are no significant dead cells observed on the surface of these samples, demonstrating their good cell attachment ability. There are high cell numbers on the surfaces of the hybrid membrane with the incorporation of 20 wt% and 50 wt% nHA compared to the pure PBP hybrid membrane, further suggesting their enhanced cellular biocompatibility. In addition, the cell viability on the PCL and the nHA-PBP hybrid membranes significantly increases as the culture period extends from 1 day to 5 days, which indicates that the as-fabricated hybrid membranes can support the osteoblast proliferation, as seen in Figure 9D. Compared to the PBP control, the osteoblast presents significantly high cell viability after incubating with the nHA-PBP (20% and 50%) for 5 day culture periods. The cell viability is significantly improved as the nHA incorporation increases. These results demonstrate that our nHA-PBP hybrid membranes possess a good osteoblast biocompatibility and the incorporation of nHA can efficiently improve the osteoblast activity of the PBP hybrid membranes.

**Figure 9.** Osteoblasts biocompatibility investigation of the nHA-PBP hybrid membranes with different nHA contents (20 wt% and 50 wt% nHA). MC3T3-E1 cell attachment morphology at 3 days ((**A**), 0 wt%, (**B**), 20 wt% and (**C**), 50 wt% nHA) and proliferation activity after 1–5 days of culture (**D**). \* *p* < 0.05 and \*\* *p* < 0.01 represent the significance differences between groups (n = 5).

In our previous work, the crack-free PBP hybrid membrane was successfully prepared by a conventional sol-gel method, which developed the functional hybrid membranes by incorporating HA particles into PBP sol. The relation between the hybrid properties and apatite-forming bioactivity was investigated, as well as attachment and proliferation in vitro. As one knows, PDMS is well compatible with silicon-based sol phase because it has a typical Si-O-Si skeleton chain and side chain, which induces a strong interaction with the hydrophobic PCL polymer. However, the biomineralization capability and biocompatibility of osteoblasts with the materials still need further improvement. Due to its highly biomimetic chemical structure and composition, HA is a typical bioactive ceramic and was successfully used in bone regeneration. The SEM results show that the HA particles can be uniformly dispersed into the PCL matrix. As a result, in this material system, it is easy to form a homogeneous inorganic–organic hybrid structure. The additive of nHA significantly enhances the biomineralization activity (apatite-forming ability) of the PBP hybrid membranes, as previously reported [24–26]. It is known that MC3T3-E1 cells have different reactions to changes in hybrid surface properties. The surface roughness of these two samples (i.e., 0 wt% and 20 wt% of the nHA) was not significantly different (Figure 3A,B), the number of attached cells on nHA 20 wt% was slightly higher than that of the nHA 0 wt%. This suggests that MC3T3-E1 cells prefer HA-containing samples to adhesion and proliferation. One possible explanation is that HA exists on a composite surface, resulting in more permanent interaction with adsorbed protein. It is absorbed by serums and proteins in the culture medium, or the protein is absorbed by the cell itself. It is also apparent that the cells are distributed more evenly on the nHA-PBP hybrid membrane surface (Figure 9B,C), which further suggests that HA favors the uniform distribution of adsorbed proteins. The addition of nHA also greatly enhances the osteoblasts biocompatibility of the as-fabricated PBP hybrid membranes. In addition, these results match earlier studies that reported the important role of nHA in polymer nanocomposites [27–29].

In bone tissue regeneration applications, the ideal biomaterials should be facilely synthesized and have high bioactivities, including biomineralization activity, for bone-bonding and osteoblast biocompatibility for regeneration. However, the PBP hybrid membrane needs a long processing time (more than 72 h), which is unfavorable for large-scale production and, thereby, limits applications. Based on the requirement for reducing the processing time and enhancing biomineralization activity and osteoblast biocompatibility, the present new developed nHA-PBP hybrid membranes may have promising applications in future bone tissue regeneration.

## **4. Conclusions**

To sum up, highly bioactive and crack-free nHA-PBP hybrid membrane ingredients were successfully prepared via the conventional sol-gel method. Results indicate that adding HA can significantly improve the surface roughness and biomineralization activity of hybrid membranes. The nHA-PBP hybrid membranes after being soaked in SBF can easily induce a crystalline apatite layer on the surface, indicating their excellent biomineralization activity. The optimized nHA-PBP hybrids also show significantly enhanced osteoblast biocompatibility. The hybrids containing 20 wt% nHA show an optimized elastic modulus and toughness. The crack-free structure, short processing time, and high bioactivity of the production of hydroxyapatite formation and biomimetic hybrid composition make the as-fabricated nHA-PBP hybrid membrane a desired candidate as a guidance membrane for future applications in biomedical materials.

**Author Contributions:** Methodology, J.C. and B.L. (Bo Lei).; software, J.C. and B.L. (Beibei Li); investigation, J.C.; writing—original draft preparation, J.C.; writing—review and editing, J.C., B.L. (Bo Lei) and W.Q.; funding acquisition, J.C. and B.L. (Beibei Li). All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Natural Science Basic Research Plan in Shaanxi Province of China (No. 2019JM-520 and 2020JQ-890), the 3-year action plan of Xi'an University (2021xdjh34).

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All the data supporting the results of the study are included in the paper.

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

#### **References**

