**2. Fabrication of silk/TMOS composite nanofibers by co-electrospinning**

In the electrospinning system [22, 23], a high-voltage power supply (Har-100\*12, Matsusada Co., Tokyo, Japan), capable of generating voltages up to 100 kV, was used as the source of the electric field. The regenerated silk protein solution was contained in a plastic tube connected with a capillary tip with an inner diameter of 0.6 mm. The copper wire connected to a positive electrode (anode) was inserted into the polymer solution, and a negative electrode (cathode) was attached to a metallic collector. The solution volume was controlled to keep proper flow rate for spinning.

Silkworm Bombyx mori is a natural protein that is mainly made of sericin and fibroin proteins, *i.e.*, sericin (the outer coating) and fibroin (the inner brins). The sericin protein is removed by a process called degumming in industry, so that the term silk is commonly improperly used to define only one of its two components. In this work, the cocoons of Bombyx mori were degummed three times in an aqueous Na2CO3 (0.02 M) at 100 °C for 30 min and washed with distilled water in order to remove sericin from the surface of silk fibers and then the silk fibroin was obtained. The silk fibroin was then dissolved in a ternary solvent system of CaCl2/ CH3CH2OH/H2O in 1:2:8 molar ratio at 70 °C with vigorous stirring. After dialysis against distilled water with cellulose tubular membrane with molecular weight cutoffs (MWCO) ranging from 12,000 to 16,000 Daltons for 4 days at 25 °C, the regenerated silk fibroin sponge was obtained by lyophilization (−20 °C, 24 h). the solutions were prepared by dissolving 8% (w/w) regenerated silk protein in 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP), after 24 h stirring, 5% and 15% (on the weight of silk fibroin) of TMOS was added to the fibroin solution within 30mins under stirring [24–26].

O–Si– linkages as proved by TG result. Furthermore, the water contact of silk/TMOS hybrid nanocomposites was closed to that of TCD (Tissue culture Dish) template (75.6°), which indicated that it may be more suitable for cell attachment than pure silk nanofibers because the optimum water contact of the surface for fibroblast adhesion is reported in the range between 55° and 75° [28]. The adhesion behavior will be discussed in the following text.

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Lactate dehydrogenase (LDH) leakage assay results in Figure 3(a) suggest that cell culturing on silk/TMOS fibrous scaffolds cause LDH release near 8% while that on silk is near 7%. Both of them are of no significant difference with the LDH release from TCD as a control. The results showed that the incorporation of TMOS in the fibrous material didn't affect the excellent biocompatibility of silk fibroin. From the live/dead fluorescence micrographs in Figure 3(b) and (c), the majority of cells incubated for 12 h on silk/TMOS and silk scaffolds were alive and parts of them revealed spindle shaped morphology. Cytotoxicity assays indicate that L929 cells

The adhesion ratio of L929 cells on pure silk, silk/TMOS nanofibers and TCD controls were shown in Figure 4(a). The cell adhesion ratio of silk/TMOS nanofibers was significantly higher than pure silk nanofibers and TCD controls in all the culture times, it reached as high as 95% after 90 mins' cultivation while that on pure silk was near 85%. Although an increase in adhesion ratio on both pure silk nanofibers and TCD controls after 30 to 90 mins of cell culture were observed, results of adhesion ratio of silk/TMOS nanofibers showed excellent attachment behavior to L929 cells which could be attributed to the melioration of hydrophilicity. The incorporation of TMOS on silk fibroin nanofibers had enhanced the adhesion behavior of L929

Immunofluorescence microscopy of L929 cells grown on pure silk, silk/TMOS fibrous scaffold and TCD after 6 h cultivation are shown in Figure 4(b-d). Blue fluorescence of cell nuclei

on silk/TMOS fibrous scaffold have comparable viability on silk fibrous scaffold.

**2.4. Adhesion behavior of electrospun regenerated silk/TMOS nanofibers**

**Figure 2.** Water contact angle of (a) pure silk nanofibers; and (b) silk/TMOS nanofibers.

**2.3. Cytotoxicity assay**

cells as expected.

#### **2.1. Morphology of silk/TMOS nanocomposites scaffolds**

Electrospun nanofibers of regenerated silk fibroin and its blends with TMOS from their HFIP solutions were obtained using conditions specified above. Figure 1(a) shows SEM image of pure regenerated silk fibroin nanofibers electrospun from a regenerated silk solution dissolved in HFIP at a concentration of 8 wt %. SEM analysis indicates a broad diameter distribution, with an average diameter of 1252 nm and standard deviation (SD) of 410 nm.

The silk/TMOS nanofibers, shown in Figure 1(b, c), were obtained by adding 5 wt % TMOS in 8 wt % regenerated silk fibroin solution within 30 mins under stirring, electrospinning at a voltage of 16 kV and a TCD of 10 cm, and finally drying at 25 °C for 24 h under humidity of 20%. Interestingly, the adjacent fibers in silk/TMOS hybrid electrospun nanofibers caused to 'weld' at fiber contact points [27], as evident in the SEM images (Figure 1(b)). Compared to welded hybrid fibers, the pure silk nanofibers shown in Figure 1(a) are intact and do not show flash welding. Additionally, the fiber diameters showed almost same to 1,287 nm (SD = 367 nm) (Figure 1(b)), to fibers spun from the pure silk solution (Figure 1(a)). The observed 'weld' at contact points may be due to the equilibrium water content, as was verified by TGA analysis shown later. Moreover, as the TMOS concentration increased to 15%, the fibers became belts and the juncture extended like a sheet which could not be identified as nanofiber mats at all (Figure 1(c)). So in this study we just investigated the hybrid nanofibers with the TMOS concentration of 5%.

**Figure 1.** SEM images of the nanofibers (a) regenerated silk fibroin nanofibers; (b) silk/TMOS hybrid nanofibers with TMOS concentration of 5 wt %; and (c) silk/TMOS hybrid nanofibers with TMOS concentration of 15 wt %.

#### **2.2. Hydrophilicity properties of electrospun regenerated silk/TMOS nanofibers**

The hydrophilicity of electrospun nanofibers composites can be seen from Figure 2. Water contact angle showed a sharp decrease of electrospun silk nanofibers incorporated with TMOS than pure regenerated silk fibroin nanofiber from 116.2° to 84.8°. Although silk fibroin has many hydrophilic groups such as –OH and –COOH, the incorporation of TMOS result in higher water capacity in the fibers due to the formation of spatial net structure formed *via* Si– O–Si– linkages as proved by TG result. Furthermore, the water contact of silk/TMOS hybrid nanocomposites was closed to that of TCD (Tissue culture Dish) template (75.6°), which indicated that it may be more suitable for cell attachment than pure silk nanofibers because the optimum water contact of the surface for fibroblast adhesion is reported in the range between 55° and 75° [28]. The adhesion behavior will be discussed in the following text.

**Figure 2.** Water contact angle of (a) pure silk nanofibers; and (b) silk/TMOS nanofibers.

#### **2.3. Cytotoxicity assay**

5% and 15% (on the weight of silk fibroin) of TMOS was added to the fibroin solution within

Electrospun nanofibers of regenerated silk fibroin and its blends with TMOS from their HFIP solutions were obtained using conditions specified above. Figure 1(a) shows SEM image of pure regenerated silk fibroin nanofibers electrospun from a regenerated silk solution dissolved in HFIP at a concentration of 8 wt %. SEM analysis indicates a broad diameter distribution,

The silk/TMOS nanofibers, shown in Figure 1(b, c), were obtained by adding 5 wt % TMOS in 8 wt % regenerated silk fibroin solution within 30 mins under stirring, electrospinning at a voltage of 16 kV and a TCD of 10 cm, and finally drying at 25 °C for 24 h under humidity of 20%. Interestingly, the adjacent fibers in silk/TMOS hybrid electrospun nanofibers caused to 'weld' at fiber contact points [27], as evident in the SEM images (Figure 1(b)). Compared to welded hybrid fibers, the pure silk nanofibers shown in Figure 1(a) are intact and do not show flash welding. Additionally, the fiber diameters showed almost same to 1,287 nm (SD = 367 nm) (Figure 1(b)), to fibers spun from the pure silk solution (Figure 1(a)). The observed 'weld' at contact points may be due to the equilibrium water content, as was verified by TGA analysis shown later. Moreover, as the TMOS concentration increased to 15%, the fibers became belts and the juncture extended like a sheet which could not be identified as nanofiber mats at all (Figure 1(c)). So in this study we just investigated the hybrid nanofibers with the TMOS

**Figure 1.** SEM images of the nanofibers (a) regenerated silk fibroin nanofibers; (b) silk/TMOS hybrid nanofibers with

The hydrophilicity of electrospun nanofibers composites can be seen from Figure 2. Water contact angle showed a sharp decrease of electrospun silk nanofibers incorporated with TMOS than pure regenerated silk fibroin nanofiber from 116.2° to 84.8°. Although silk fibroin has many hydrophilic groups such as –OH and –COOH, the incorporation of TMOS result in higher water capacity in the fibers due to the formation of spatial net structure formed *via* Si–

TMOS concentration of 5 wt %; and (c) silk/TMOS hybrid nanofibers with TMOS concentration of 15 wt %.

**2.2. Hydrophilicity properties of electrospun regenerated silk/TMOS nanofibers**

with an average diameter of 1252 nm and standard deviation (SD) of 410 nm.

30mins under stirring [24–26].

106 Advances in Nanofibers

concentration of 5%.

**2.1. Morphology of silk/TMOS nanocomposites scaffolds**

Lactate dehydrogenase (LDH) leakage assay results in Figure 3(a) suggest that cell culturing on silk/TMOS fibrous scaffolds cause LDH release near 8% while that on silk is near 7%. Both of them are of no significant difference with the LDH release from TCD as a control. The results showed that the incorporation of TMOS in the fibrous material didn't affect the excellent biocompatibility of silk fibroin. From the live/dead fluorescence micrographs in Figure 3(b) and (c), the majority of cells incubated for 12 h on silk/TMOS and silk scaffolds were alive and parts of them revealed spindle shaped morphology. Cytotoxicity assays indicate that L929 cells on silk/TMOS fibrous scaffold have comparable viability on silk fibrous scaffold.

#### **2.4. Adhesion behavior of electrospun regenerated silk/TMOS nanofibers**

The adhesion ratio of L929 cells on pure silk, silk/TMOS nanofibers and TCD controls were shown in Figure 4(a). The cell adhesion ratio of silk/TMOS nanofibers was significantly higher than pure silk nanofibers and TCD controls in all the culture times, it reached as high as 95% after 90 mins' cultivation while that on pure silk was near 85%. Although an increase in adhesion ratio on both pure silk nanofibers and TCD controls after 30 to 90 mins of cell culture were observed, results of adhesion ratio of silk/TMOS nanofibers showed excellent attachment behavior to L929 cells which could be attributed to the melioration of hydrophilicity. The incorporation of TMOS on silk fibroin nanofibers had enhanced the adhesion behavior of L929 cells as expected.

Immunofluorescence microscopy of L929 cells grown on pure silk, silk/TMOS fibrous scaffold and TCD after 6 h cultivation are shown in Figure 4(b-d). Blue fluorescence of cell nuclei

**Figure 3.** LDH release (a) and fluorescence micrographs of Calcein AM/PI-stained L929 cells with live cells fluorescing green and dead cells fluorescing red after 12 h culture on the Silk/TMOS; (b), silk; (c) nanofibrous scaffold; and TCD (d).

revealed round, well-spaced, and regularly distributed nuclei across the surface of both fibrous scaffolds. Compared to the L929 cells on pure silk that showed round shaped, the cytoskeletal organization (green fluorescence) of most cells on silk/TMOS scaffold showed obvious spindleshaped morphology, while both round and spindle-shaped L929 cells have been investigated on TCD as a control. Moreover, only L929 cells on silk/TMOS showed vinculin signals (red fluorescence) at the extremities of cellular extensions. Consisting with the adhesion ration in Figure 4 (a), these results mean a better adhesion and stretching behavior of L929 cells on silk/ TMOS nanofibrous scaffold than that on pure silk scaffold.

neat silk because of the formation of spatial net structure formed *via* Si–O–Si– linkages. Studies about the wettability, initial protein adsorption, and the cell adhesion showed that one of the fibronectin state has more active conformation (secondary structure rearrangements) being on a hydrophilic surface [32, 33]. This will consequently lead to more spreading of fibroblasts and ultimately the sufficient cell adhesion and spreading. It has been reported that the optimum wettability of the surface for fibroblast adhesion is in the range between 55° and 75° [28]. The TCD used in this study as control has a water contact of 75.6° (data not shown) and the incorporation of TMOS has change the hydrophobic silk surface of 116.2° to hydrophilic 84.8°. Secondly, SEM images in Figure 1(b, c) showed the interesting adjacent fibers in silk/TMOS hybrid electrospun nanofibers caused to 'weld' at contact points. It has been known that the substrate's topography has a great influence on the behavior of cells at interface. Studies showed that contact guidance happened to cells of different types on different materials with different sizes and shapes of patterns [34–36]. Probably, this kind of 'weld' in silk/TMOS nanofibrous mats influence the surface microstructure of the fiber that might has positive effect to the L929 cell adhesion, though more intensive study is necessary for the conclusion. Nevertheless, considering the complexity of cell surface interaction, which involves protein absorption and specific binding, the function groups that existed in TMOS and net charges presented on the silk/TMOS hybrid scaffold might also influence the protein adsorption and

**Figure 4.** The adhesion ratio (a) for L929 cells after 90 min culture on pure silk, silk/TMOS nanofibers and TCD con‐ trols. Significant difference between different materials groups were denoted as \* (p < 0.05). and Fluorescent staining of F-actin (green), vinculin (red), and cell nuclei (blue) for L929 cells after 6 hs culture on silk; (b) fibrous scaffold, silk/

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therefore cell adhesion in some degree [37, 38].

TMOS; (c); and TCD (d).

Accordingly, intensive researches have been carried out in order to manipulate cellular behavior by modifying the relative properties of materials. Y. Sasai *et al.* induce durable hydrophilicity on the hydrophobic of polystyrene surface and further modified it by RGD sequence which can be recognized by the receptor protein on the cellular membrane to enhance the adhesion and proliferation of PC12 cell [29]. Vera A. *et al.* induced stable cell adhesion by manipulating the surface topography to the hydrogel poly (ethylene glycol) although fibro‐ blast is intrinsically non-adhesive to the smooth surface [30]. Mohammad *et al.* found that the positive surface of the titanium cylinder results in favorable NCTC clone 929 fibroblast cell adhesion [31]. The results in our study suggested that the cell adhesion ratio and spreading on silk/TMOS has been enhanced comparing to the pure silk. This can be explained by the change of fibrous surface properties in the terms of hydrophilicity and surface morphology change. First of all, water contact angle showed that silk/TMOS have better hydrophilicity than

**Figure 4.** The adhesion ratio (a) for L929 cells after 90 min culture on pure silk, silk/TMOS nanofibers and TCD con‐ trols. Significant difference between different materials groups were denoted as \* (p < 0.05). and Fluorescent staining of F-actin (green), vinculin (red), and cell nuclei (blue) for L929 cells after 6 hs culture on silk; (b) fibrous scaffold, silk/ TMOS; (c); and TCD (d).

revealed round, well-spaced, and regularly distributed nuclei across the surface of both fibrous scaffolds. Compared to the L929 cells on pure silk that showed round shaped, the cytoskeletal organization (green fluorescence) of most cells on silk/TMOS scaffold showed obvious spindleshaped morphology, while both round and spindle-shaped L929 cells have been investigated on TCD as a control. Moreover, only L929 cells on silk/TMOS showed vinculin signals (red fluorescence) at the extremities of cellular extensions. Consisting with the adhesion ration in Figure 4 (a), these results mean a better adhesion and stretching behavior of L929 cells on silk/

**Figure 3.** LDH release (a) and fluorescence micrographs of Calcein AM/PI-stained L929 cells with live cells fluorescing green and dead cells fluorescing red after 12 h culture on the Silk/TMOS; (b), silk; (c) nanofibrous scaffold; and TCD (d).

Accordingly, intensive researches have been carried out in order to manipulate cellular behavior by modifying the relative properties of materials. Y. Sasai *et al.* induce durable hydrophilicity on the hydrophobic of polystyrene surface and further modified it by RGD sequence which can be recognized by the receptor protein on the cellular membrane to enhance the adhesion and proliferation of PC12 cell [29]. Vera A. *et al.* induced stable cell adhesion by manipulating the surface topography to the hydrogel poly (ethylene glycol) although fibro‐ blast is intrinsically non-adhesive to the smooth surface [30]. Mohammad *et al.* found that the positive surface of the titanium cylinder results in favorable NCTC clone 929 fibroblast cell adhesion [31]. The results in our study suggested that the cell adhesion ratio and spreading on silk/TMOS has been enhanced comparing to the pure silk. This can be explained by the change of fibrous surface properties in the terms of hydrophilicity and surface morphology change. First of all, water contact angle showed that silk/TMOS have better hydrophilicity than

TMOS nanofibrous scaffold than that on pure silk scaffold.

108 Advances in Nanofibers

neat silk because of the formation of spatial net structure formed *via* Si–O–Si– linkages. Studies about the wettability, initial protein adsorption, and the cell adhesion showed that one of the fibronectin state has more active conformation (secondary structure rearrangements) being on a hydrophilic surface [32, 33]. This will consequently lead to more spreading of fibroblasts and ultimately the sufficient cell adhesion and spreading. It has been reported that the optimum wettability of the surface for fibroblast adhesion is in the range between 55° and 75° [28]. The TCD used in this study as control has a water contact of 75.6° (data not shown) and the incorporation of TMOS has change the hydrophobic silk surface of 116.2° to hydrophilic 84.8°. Secondly, SEM images in Figure 1(b, c) showed the interesting adjacent fibers in silk/TMOS hybrid electrospun nanofibers caused to 'weld' at contact points. It has been known that the substrate's topography has a great influence on the behavior of cells at interface. Studies showed that contact guidance happened to cells of different types on different materials with different sizes and shapes of patterns [34–36]. Probably, this kind of 'weld' in silk/TMOS nanofibrous mats influence the surface microstructure of the fiber that might has positive effect to the L929 cell adhesion, though more intensive study is necessary for the conclusion. Nevertheless, considering the complexity of cell surface interaction, which involves protein absorption and specific binding, the function groups that existed in TMOS and net charges presented on the silk/TMOS hybrid scaffold might also influence the protein adsorption and therefore cell adhesion in some degree [37, 38].
