**3. Fabrication of silk/nHA composite nanofibers**

The regenerated silk fibroin sponge was obtained using the same method as described above. Silk fibroin solutions in the concentration of 18% (w/w) were prepared by dissolv‐ ing the regenerated silk protein sponge into 98% formic acid, and used for electrospin‐ ning [39]. The electrospun silk nanofiber was post-treated by a Calcium–Phosphate (Ca– P) alternate soaking method. That is, mineralization of nHA was achieved by subjecting the nanofibers in a series of calcium and phosphate treatments, deemed as the alternate soaking method [40]. Silk nanofibrous scaffolds were first immersed in 0.5 M of CaCl2 (pH of 7.2) (Aldrich Chemical Company, Inc., St. Louis, State Abbreviation, USA), followed by rinsing with deionized (DI) water. Afterwards, the scaffolds were subsequently immersed in 0.3 M of Na2HPO4 (pH of 8.96) (Merck & Co. Inc., City, NJ, USA) and rinsed with DI water. The above-mentioned step was referred to as 1 cycle of Ca–P treatment. All nanofibers were subjected to 3 cycles of Ca–P treatments, where the first cycle was 10 min (in each chemical solution) and the second and third cycles were 5 min (in each chemical solution). After mineralization, the nanofibers were freeze-dried overnight.

#### **3.1. Morphology of silk/nHA nanofibrous scaffolds**

Mineralization of nHA was successfully deposited on pure silk fibroin nanofibers after 3 cycles [41] of Ca–P treatment as depicted in Figure 7. As shown in Figure 5(b,c), the diameter of obtained silk fibroin nanofibers was around 242 ± 34 nm. It was observed that nHA was homogenously formed on pure silk nanofiber substrates. As evidenced in the high resolution FE-SEM micrograph (Figure 5(d)), nHA particles formed on silk fibroin nanofibrous scaffolds were nanocrystalline in structure and the deposition was occurred predominately on the surfaces of the nanofibrous scaffolds. The size of nHA particles was approximately 30–35 nm in diameter, which was proved by WAXD (see below).

#### **3.2. Crystal Structure of Silk/nHA Nanofibrous Scaffolds**

XRD results as can be seen in Figure 8 clearly demonstrated the presence of nHA in the mineralized silk/nHA nanofibrous scaffolds (Figure 6(b): nHA residues and Figure 6(c): mineralized silk/nHA nanofibers). The broad halo peak at 20.6° in Figure 6(c) was attributed to the silk II form of β-sheet crystalline structure [42, 43]. All the peaks in Figures 6(b) and (c) were consistent with the peaks associated with pure nHA (Figure 6(a)), suggesting that rapid mineralization approach used in our study was effective in producing nHA phases on the silk nanofibrous substrates. But unfortunately, both the EDX and XRD analyses indicate the poor crystallinity of the nHA formed on silk nanofibers. It can be explained that the hydroxyl or amide group, which existed in the silk fibroin macro chains, captured calcium or phosphate ions in the solution by chelation. The supply of calcium or phosphate ions to the apatite nuclei was retarded, and the apatite crystals grew under the condition that the calcium or phosphate ions were not sufficiently supplied. Therefore, the crystal growth of apatite was inhibited along a particular axis and resulted in random orientations of crystals in the miner‐ alized fibroin [44].

**Figure 6.** X-ray diffraction (XRD) patterns of (a) pure HA (control); (b) nHA residues; and (c) mineralized silk/nHA

**Figure 5.** FE-SEM images of pure silk and mineralized silk/nHA nanofibers after 3 cycles of Ca–P treatment. (a) pure silk nanofibers (6000 ×; scale bar, 500 nm), (b–d) mineralized silk/nHA nanofibers after 3 cycles of Ca–P treatment with

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nanofibers.

different magnification.

**3. Fabrication of silk/nHA composite nanofibers**

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The regenerated silk fibroin sponge was obtained using the same method as described above. Silk fibroin solutions in the concentration of 18% (w/w) were prepared by dissolv‐ ing the regenerated silk protein sponge into 98% formic acid, and used for electrospin‐ ning [39]. The electrospun silk nanofiber was post-treated by a Calcium–Phosphate (Ca– P) alternate soaking method. That is, mineralization of nHA was achieved by subjecting the nanofibers in a series of calcium and phosphate treatments, deemed as the alternate soaking method [40]. Silk nanofibrous scaffolds were first immersed in 0.5 M of CaCl2 (pH of 7.2) (Aldrich Chemical Company, Inc., St. Louis, State Abbreviation, USA), followed by rinsing with deionized (DI) water. Afterwards, the scaffolds were subsequently immersed in 0.3 M of Na2HPO4 (pH of 8.96) (Merck & Co. Inc., City, NJ, USA) and rinsed with DI water. The above-mentioned step was referred to as 1 cycle of Ca–P treatment. All nanofibers were subjected to 3 cycles of Ca–P treatments, where the first cycle was 10 min (in each chemical solution) and the second and third cycles were 5 min (in each chemical

solution). After mineralization, the nanofibers were freeze-dried overnight.

Mineralization of nHA was successfully deposited on pure silk fibroin nanofibers after 3 cycles [41] of Ca–P treatment as depicted in Figure 7. As shown in Figure 5(b,c), the diameter of obtained silk fibroin nanofibers was around 242 ± 34 nm. It was observed that nHA was homogenously formed on pure silk nanofiber substrates. As evidenced in the high resolution FE-SEM micrograph (Figure 5(d)), nHA particles formed on silk fibroin nanofibrous scaffolds were nanocrystalline in structure and the deposition was occurred predominately on the surfaces of the nanofibrous scaffolds. The size of nHA particles was approximately 30–35 nm

XRD results as can be seen in Figure 8 clearly demonstrated the presence of nHA in the mineralized silk/nHA nanofibrous scaffolds (Figure 6(b): nHA residues and Figure 6(c): mineralized silk/nHA nanofibers). The broad halo peak at 20.6° in Figure 6(c) was attributed to the silk II form of β-sheet crystalline structure [42, 43]. All the peaks in Figures 6(b) and (c) were consistent with the peaks associated with pure nHA (Figure 6(a)), suggesting that rapid mineralization approach used in our study was effective in producing nHA phases on the silk nanofibrous substrates. But unfortunately, both the EDX and XRD analyses indicate the poor crystallinity of the nHA formed on silk nanofibers. It can be explained that the hydroxyl or amide group, which existed in the silk fibroin macro chains, captured calcium or phosphate ions in the solution by chelation. The supply of calcium or phosphate ions to the apatite nuclei was retarded, and the apatite crystals grew under the condition that the calcium or phosphate ions were not sufficiently supplied. Therefore, the crystal growth of apatite was inhibited along a particular axis and resulted in random orientations of crystals in the miner‐

**3.1. Morphology of silk/nHA nanofibrous scaffolds**

in diameter, which was proved by WAXD (see below).

alized fibroin [44].

**3.2. Crystal Structure of Silk/nHA Nanofibrous Scaffolds**

**Figure 5.** FE-SEM images of pure silk and mineralized silk/nHA nanofibers after 3 cycles of Ca–P treatment. (a) pure silk nanofibers (6000 ×; scale bar, 500 nm), (b–d) mineralized silk/nHA nanofibers after 3 cycles of Ca–P treatment with different magnification.

**Figure 6.** X-ray diffraction (XRD) patterns of (a) pure HA (control); (b) nHA residues; and (c) mineralized silk/nHA nanofibers.

## **3.3. Proliferation behavior of silk/nHA nanofibrous scaffolds**

In Figure 7, immunofluorescence of actin filaments demonstrates the cytoskeletal organization (green). Since the high surface area to volume of nanofibers which is used to mimic the extracellular matrix environment of cells, the MC3T3-E1 cells in Figure 7(I) is investigated to spread in spindle or polygonal morphology after 24 h cultivation. Moreover, intensive vinculin signals can be found along the stretching cellular axis. The MC3T3-E1 cell's adhesion activity in Figure 7(I) suggested that the mineralization of nHA on silk fibrous mats didn't outweigh the benefit of silk nanofibrous scaffold. 3D network culturing morphology of MC3T3-E1 in Figure 7(II) was determined by laser depth-of-focus scanning about 20µm of the silk-nHA scaffold. Together with the cross-section image of Figure 7(III) where a portion of cells are penetrated into fabricated channels, silk-nHA fibrous mats were proved to be suitable for supporting the MC3T3-E1's 3D cultivation.

preferential attachment of the pseudopodia to nHA regions. In addition, a greater cell spreading on silk/nHA nanofibers was observed after 2 days of cell culture (Figure 8(d)), compared to that after 1 day (Figure 8(b)). When the culture period is prolonged in our study, full cell coverage was found on the nanofibers, and eventually osteoblasts covered most of the nanofiber surfaces after 7 days of cell culture with extended lamellipodia, creating a cell multi-

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**Figure 8.** Osteoblasts on pure and mineralized silk/nHA nanofibers. (a) pure silk (day 1); (b) silk/nHA (day 1); (c) pure

Figure 9 shows cell proliferation on pure and mineralized silk nanofibers onward 3 days of cultivation. It was observed that when compared with the pure silk nanofibers, the cell numbers were smaller for mineralized silk/nHA nanofiber scaffold and TCD until 7 days cultivation. This is different from what was observed in other studies where osteoblast proliferation was improved on nanophase HA materials [48, 49]. Probably, the difference was due to the size effect of hydroxyapatite nanoparticles on proliferation as well as the density or bulk distribution. Moreover, previous studies showed that curved nHA surface at a nanometer level could decrease osteoblast-like cells on early period of proliferation [50]. The previously reported results [51–53] were also coincided with those observed in our study: surface topography had a crucial influence on cell behavior, which was accompanied by attenuated proliferation rates on rougher surfaces. Nevertheless, after 14 days of cultivation, cell number on mineralized silk is of no significant differences between pure silk and TCD controls (*p* ≥ 0.05), suggesting that the addition of nHA had no negative effect on later period of cell

silk (day 2); (d) silk/nHA (day 2); (e) pure silk (day 7); and (f) silk/nHA (day 7).

proliferation.

layers on the fibers (Figure 8(f)).

**Figure 7.** Fluorescent staining of F-actin (green), vinculin (red), and cell nuclei (blue) for MC3T3-E1 cells after 24 h cul‐ tivation on silk/nHA fibrous scaffold. (I) 2D morphology of cultivation; (II) 3D morphology by laser scanning of fibrous scaffold; and (III) cross section of II.

As evidenced in the FE-SEM micrographs, osteoblasts were successfully seeded on both pure and mineralized silk nanofibers where the cells were partly adhered to nHA regions in the silk/nHA nanofibers (Figure 8(b)). The deposition of nHA did not affected the MC3T3-E1 attachment compared to those on those grown on pure silk nanofibers after 1 day cultivation (Figures 8(a) and 8(b)) [45, 46], Likewise, cell spreading in a spindle-like shape was also observed on HA-based composites after 2 days of cell culture due to the physical contacts between cells which is maintained *via* the formation of filopodia or lamellipodia [47]. As seen in Figure 8(d), the cells were strongly anchored on the silk/nHA nanofibrous scaffolds, with preferential attachment of the pseudopodia to nHA regions. In addition, a greater cell spreading on silk/nHA nanofibers was observed after 2 days of cell culture (Figure 8(d)), compared to that after 1 day (Figure 8(b)). When the culture period is prolonged in our study, full cell coverage was found on the nanofibers, and eventually osteoblasts covered most of the nanofiber surfaces after 7 days of cell culture with extended lamellipodia, creating a cell multilayers on the fibers (Figure 8(f)).

**3.3. Proliferation behavior of silk/nHA nanofibrous scaffolds**

supporting the MC3T3-E1's 3D cultivation.

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scaffold; and (III) cross section of II.

In Figure 7, immunofluorescence of actin filaments demonstrates the cytoskeletal organization (green). Since the high surface area to volume of nanofibers which is used to mimic the extracellular matrix environment of cells, the MC3T3-E1 cells in Figure 7(I) is investigated to spread in spindle or polygonal morphology after 24 h cultivation. Moreover, intensive vinculin signals can be found along the stretching cellular axis. The MC3T3-E1 cell's adhesion activity in Figure 7(I) suggested that the mineralization of nHA on silk fibrous mats didn't outweigh the benefit of silk nanofibrous scaffold. 3D network culturing morphology of MC3T3-E1 in Figure 7(II) was determined by laser depth-of-focus scanning about 20µm of the silk-nHA scaffold. Together with the cross-section image of Figure 7(III) where a portion of cells are penetrated into fabricated channels, silk-nHA fibrous mats were proved to be suitable for

**Figure 7.** Fluorescent staining of F-actin (green), vinculin (red), and cell nuclei (blue) for MC3T3-E1 cells after 24 h cul‐ tivation on silk/nHA fibrous scaffold. (I) 2D morphology of cultivation; (II) 3D morphology by laser scanning of fibrous

As evidenced in the FE-SEM micrographs, osteoblasts were successfully seeded on both pure and mineralized silk nanofibers where the cells were partly adhered to nHA regions in the silk/nHA nanofibers (Figure 8(b)). The deposition of nHA did not affected the MC3T3-E1 attachment compared to those on those grown on pure silk nanofibers after 1 day cultivation (Figures 8(a) and 8(b)) [45, 46], Likewise, cell spreading in a spindle-like shape was also observed on HA-based composites after 2 days of cell culture due to the physical contacts between cells which is maintained *via* the formation of filopodia or lamellipodia [47]. As seen in Figure 8(d), the cells were strongly anchored on the silk/nHA nanofibrous scaffolds, with

**Figure 8.** Osteoblasts on pure and mineralized silk/nHA nanofibers. (a) pure silk (day 1); (b) silk/nHA (day 1); (c) pure silk (day 2); (d) silk/nHA (day 2); (e) pure silk (day 7); and (f) silk/nHA (day 7).

Figure 9 shows cell proliferation on pure and mineralized silk nanofibers onward 3 days of cultivation. It was observed that when compared with the pure silk nanofibers, the cell numbers were smaller for mineralized silk/nHA nanofiber scaffold and TCD until 7 days cultivation. This is different from what was observed in other studies where osteoblast proliferation was improved on nanophase HA materials [48, 49]. Probably, the difference was due to the size effect of hydroxyapatite nanoparticles on proliferation as well as the density or bulk distribution. Moreover, previous studies showed that curved nHA surface at a nanometer level could decrease osteoblast-like cells on early period of proliferation [50]. The previously reported results [51–53] were also coincided with those observed in our study: surface topography had a crucial influence on cell behavior, which was accompanied by attenuated proliferation rates on rougher surfaces. Nevertheless, after 14 days of cultivation, cell number on mineralized silk is of no significant differences between pure silk and TCD controls (*p* ≥ 0.05), suggesting that the addition of nHA had no negative effect on later period of cell proliferation.

**Figure 9.** Cell proliferation on pure and mineralized silk nanofibers after 3 to 14 days of cell culture. Significant differ‐ ence between different materials groups were denoted as \* (p ≥ 0.05).

**Figure 10.** ALP activity on pure and mineralized silk/nHA nanofibers after 5, 7, 10 and 14 days of cell culture. Signifi‐

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There are two possible approaches of incorporating biomolecules in the fibers during electro‐ spinning. These approaches include coaxial electrospinning and emulsion electrospinning. The coaxial electrospinning utilizes two feeding capillary channels for different solutions thus maintaining the functional activity of bimolecular. [57] However, special apparatus is required for coaxial electrospinning and it demands careful adjustment of the operational conditions in order to obtain desirable results. On the other hand, emulsion electrospinning has attracted growing interests, [58-60] due to its relative simplicity and capability of preparing core-sheath type nanofibers using normal solution electrospinning process. In emulsion electrospinning, an aqueous drug solution is prepared and dispersed into a polymer solution in an organic solvent to form a water-in-oil emulsion electrospinning dope. However, the emulsifying process by ultra-sonication might cause conformational changes of biomolecules that affect its bioactivity. Thus, it is necessary not only to prepare emulsion electrospun fibrous scaffold where encapsulated proteins can be controllably released but also to preserve the bioactivities

**4. Fabrication of core-sheath structured nanofibers by emulsion**

cant difference between different materials groups were denoted as \* (p < 0.05).

of the encapsulated biomolecules during the emulsifying process.

**electrospinning**

#### **3.4. Alkaline phosphatase (ALP) activity of silk/nHA nanofibrous scaffolds**

One of the properties of nHA is its bioactive nature which promotes osteoblastic differentiation *in vitro* [54–56]. ALP-hydrolyzed phosphate esters play an essential role in the initiation of the cell differentiation process. Thus, ALP activity, as a marker of osteoblastic activity and a standard to evaluate the differentiation of MC3TC-E1 cells, were measured and shown in Figure 10. There was a slight reduction in ALP activity on the pure silk and mineralized silk/nHA nanofibers than TCD after 5 days of cell culture, while a significant increase in ALP activity on both pure silk and mineralized silk/nHA nanofibers after 7 to 14 days of cell culture, compared to TCD counterparts. Results of ALP activity of pure silk and mineralized silk/nHA nanofibers were comparable on an early stage after 5 days of cell culture, but after 7 days of cell culture ALP activity was meliorated in mineralized silk/nHA than pure silk substrates. The incorporation of nHA on silk fibroin nanofibers had enhanced the differentiation activity of MC3T3-E1 from day 7 to 14. After 14 days of cell culture, ALP activity on mineralized silk/ nHA nanofibers was nearly 1.6 times higher than that of pure silk nanofibers. One noteworthy observation was that ALP activity in pure silk and mineralized silk/nHA nanofibers was superior to that of TCD as a control from 7 to 14 days of cell culture.

**Figure 10.** ALP activity on pure and mineralized silk/nHA nanofibers after 5, 7, 10 and 14 days of cell culture. Signifi‐ cant difference between different materials groups were denoted as \* (p < 0.05).
