*2.6. Cell Proliferation and Viability of the Hybrid Membranes*

The cellular biocompatibility of the hybrid membrane was evaluated by using the osteoblast cell line (MC3T3-E1). Cells were cultured in a standard Dulbecco's modified essential medium (DMEM, Invitrogen) in a humidified atmosphere with 5% CO2 and supplemented with 10% fetal calf serum (FCS). All samples with a size of 10 mm × 10 mm were sterilized by ultraviolet (UV) irradiation for 30 min on each side before cell seeding. MC3T3-E1 cells were seeded on the surface of the hybrid materials at a density of 5000 cells per well. The cell attachment and morphology were then evaluated with a LIVE/DEAD viability kit (Molecular Probes) after the 3-day culture. The staining procedure was according to the manufacture instruction. The cell morphology was observed with a fluorescence microscope (IX53, Olympus, Tokyo, Japan).

After the incubation for 1, 3, and 5 days, the cell viability and proliferation were determined by using a commercial Alamar Blue™ assay kit (Life Technologies). A tissue culture plate (TCP) was used as a control. The cell metabolic activity after incubation with an Alamar Blue kit was performed by a microplate reader (Molecular Devices) according to the instruction book. At least 5 species per sample were analyzed to obtain mean value and standard deviation (SD).

#### *2.7. Statistics Analysis*

Mean ± standard deviation (SD) indicated all data. The student's test analysis of Social Science Statistical Program Software (SPSS 19.0, Inc., Chicago, IL, USA) used to detect the statistical differences between the groups of measurements. Statistically significant difference was represented as \* *p* less than 0.05 and \*\* *p* less than 0.01.

#### **3. Results and Discussion**

#### *3.1. Morphological Measurement*

Through the direct hybridization of PDMS-BG sol, PCL solution, and nHA, the nHA-PBP hybrids were successfully obtained as shown in Figure 1. After thermal casting and incubation, the crack-free hybrid membrane formed. In the hybrid structure, PDMS may have a strong interaction with BG sol through the Si-O-Si bonds. Furthermore, the hydrophobic alkyl chains may have high affinity with the PCL phase. Therefore, the molecular-level inorganic–organic phase structure of the as-fabricated hybrid membranes can be facilely formed. In addition, the nanoscale HA particles are efficiently incorporated into the PBP matrix, which may enhance their surface nanostructure and bioactivity, as well as the osteoblasts biocompatibility.

**Figure 1.** Process diagram and optical images of crack-free nHA-PBP hybrid monoliths fabricated by the representative sol-gel route.

Figure 2 reveals the crystalline phase composition and structure of the as-fabricated nHA-PBP hybrids with various amounts of nHA (0, 20, 30, 40, and 50 wt%) by XRD characterization. In spite of the variations observed in crystallization, one can clearly observe the XRD peaks at 2θ = 21.88◦ and 2θ = 23.85◦, which are ascribed to the representative characteristic peaks of the PCL (semi-crystalline polymer). It is also observed that the PCL peaks significantly decrease in intensity with the increase in nHA content (20–50 wt%). Furthermore, the appearance and significant enhancement in intensity of the peaks at 32◦, 46◦, and 49◦ demonstrates the presence of nHA in the nHA-PBP hybrids.

The surface microstructures and morphologies of the nHA-PBP hybrid membranes containing different nHA contents are shown in Figure 3. It can be observed that the surface roughness of the hybrids increases significantly with the addition of nHA. There are some joints and protuberances on the surface of these composites, which indicates that HA nanoparticles are attached to PCL surfaces. The SEM images also show that the HA particles (particle areas) density increases when the loading concentration increases (Figure 3C–E). Figure 4 shows EDS spectra of the nHA-PBP hybrid membranes. The results confirm that calcium (Ca), phosphorous (P), carbon (C), and oxygen (O) are present in the matrix. The diagram demonstrates that the chemical composition changes with the addition of different nHA contents. The Ca and P peaks in intensity significantly rise with the increase in nHA content. These results reveal that nHA can be effectively crosslinked and hybridized with the PBP matrix.

**Figure 2.** XRD patterns of the nHA-PBP hybrid membranes with different nHA contents.

**Figure 3.** Surface microstructures and morphologies of the nHA-PBP hybrid membranes. (**A**) 0 wt% nHA, (**B**) 20 wt% nHA, (**C**) 30 wt% nHA, (**D**) 40 wt% nHA, (**E**) 50 wt% nHA.

**Figure 4.** EDS analysis spectra of the nHA-PBP hybrid membranes. (**A**) 0 wt% nHA, (**B**) 20 wt% nHA, (**C**) 30 wt% nHA, (**D**) 40 wt% nHA, (**E**) 50 wt% nHA.

#### *3.2. Mechanical Properties Assessment of the nHA-PBP Hybrid Membranes*

The tensile tests are used to assess the mechanical properties of nHA-PBP hybrid membranes, as shown in Figure 5. Figure 5A shows the tensile stress–strain behavior of nHA-PBP hybrids with varying nHA contents (20, 30, 40 wt%). All samples show representative stress–strain behaviors in the initial 10% stain range. The ultimate tensile strength of hybrid membranes decreased from 4.77 ± 0.30 to 2.77 ± 0.25 MPa with increasing nHA content from 20 to 40 wt% (Figure 5B). The Young's modulus of nHA-PBP 20 wt% hybrids indicated a high value of 87.94 ± 1.32 MPa as compared to the 59.58 ± 2.54 of 40 wt% (Figure 5C). The failure stress showed a similar tendency to change with ultimate tensile strength for nHA-PBP from 20 to 40 wt% (Figure 5D). The results show that increasing the amount of nHA in the nHA-PBP hybrids reduced flexural strength. When the nHA content is high, the nHA may not be hybridized well with the polymer phase, and the uniform structure may induce the decrease in flexural strength. Since nHA has poor mechanical properties, its utilization is limited to clinical load bearing applications. To make nHA-PBP hybrid materials play an important role in bone regeneration, some weaknesses of each component need to be improved in order to provide excellent quality and interfacial attachment of new bone tissue.

**Figure 5.** Mechanical properties assessment of nHA-PBP hybrid membranes with different nHA contents. (**A**) Stress–strain behavior; (**B**) Ultimate tensile strength; (**C**) Young's modulus; (**D**) Elongation at break.
