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

**Figure 9.** Cell proliferation on pure and mineralized silk nanofibers after 3 to 14 days of cell culture. Significant differ‐

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

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

superior to that of TCD as a control from 7 to 14 days of cell culture.

ence between different materials groups were denoted as \* (p ≥ 0.05).

114 Advances in Nanofibers

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 of the encapsulated biomolecules during the emulsifying process.

In this study, poly (lactic-co-glycolic acid) (PLGA), a hydrophilic polymer with excellent biocompatibility and biodegradability which has been widely used in drug delivery and scaffold application, [61-63] was dissolved in chloroform/toluene (C/T) mixed solvent to form the oil phase of the emulsion. SPAN80 (Sorbitan Monooleate) was selected as a non-ionic surfactant widely used in pharmaceuticals and presumed to be non-toxic for biomedical use. The Fol-8Col dissolved in aqueous solution was emulsified with the PLGA oil phase to prepare the emulsion electrospinning dope. (Figure.11) The past work in emulsion electrospinning has been limited to relatively low water content of 4 vol.% (volume percent). [64] For some biomacromolecules that have comparatively low solubility in water, higher water content in the emulsions may be advantageous for their desirable encapsulation in fibers. The concept of using emulsion as a modulator in electrospinning was reported by J.C. Sy *et al*. [65] Here, we propose to introduce emulsions with high water content of 10 wt.% (weight percent). The distribution and inner layer structure of the encapsulated Fol-8Col was investigated. More‐ over, release profiles of encapsulated Fol-8Col from the fibrous mats and its short-term cell cytocompatibility to fibroblasts cell line L929 were tested for its potential application as a drug release device as well as tissue engineering scaffold.

fluorescence micrographs of NHS-Fluorescein labeled Fol-8Col/PLGA showed fluorescence emitting fibers (Figure 12), suggesting the homogeneous presence of Fol-8Col in the emulsion electrospun fibers. Consisting with the SEM images in Figure 1, bead defects of fibrous

Fabrication of Nanofibrous Scaffolds by Electrospinning

http://dx.doi.org/10.5772/57093

117

TEM observation was further conducted to identify the layer structure of emulsion electro‐ spun fibers in this study. The TEM image of Figure 13 suggested that the inner component Fol-8Col ofW/Oemulsion was properly wrapped in the centre ofresultant composite fiber. The boundary in the TEMimages reflects the difference of electron transmission ability between the core (Fol-8Col) and sheath (PLGA). However, a slanted portion of boundary can be observed

**Figure 12.** Fluorescence microphotograph of Fol-8Col/PLGA fibers electrospun with Fol-8Col (5 wt% aqueous con‐

**Figure 13.** TEM images of Fol-8Col/PLGA fibers electrospun from Fol-8Col [5 (I) and 10wt%(II) aqueous content], with

which is associated with the miscibility of amphiphilic surfactant (SPAN80) molecule.

morphology were not observed.

tent), a C/T weight ratio of 75/25, and 10wt% PLGA.

a C/T weight ratio of 75/25 and a PLGA concentration of10 wt%.

**Figure 11.** Schematic of emulsion electrospinning.
