**1. Introduction**

Continuous progress in surgical technologies and biomedical science has allowed tissue or whole-organ transplantation to become potential options to restore native functions such as regeneration of fractured or diseased bones. Unfortunately, the increasing demand for function transplants and the human aspiration for longer living far exceed the available supply of usable donor tissues. Transplantation technology has encountered severe limitations. Therefore, new technologies are needed to reduce this gap in clinical need versus available healthy tissue and organ supplies. In recent years, electrospinning has been employed as a leading technique for generating biomimetic scaffold made of synthetic and natural polymers for tissue engineering. This method can produce electrospun fibers with diameters in the range from several micro‐ meters down to less than 100 nm that have a very high surface area to mass ratio. This kind of thee dimensional, fibrous scaffold with high porosity can closely biomimic that of native extracellular matrix. Thus, facilitate cell attachment, support cell growth and regulate cell differentiation [1, 2].

Silk filament derived from silkworm *Bombyx mori* is a natural protein 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, the silk fibroin. Silk fibroin is a typical fibrous protein that has been studied as a scaffold for tissue engineering because of its excellent biocompatible, bioabsorbability and low level of inflammatory potential [3–5]. In recent years regenerated silk fibroin nanofibers have been developed using electrospinning technique for tissue engineering [4, 5].

In tissue engineering *in vitro*, many researches were directed towards the development of novel hybrid nanofibers scaffold using regenerated silk fibroin by electrospinning technique [6–9] in order to improve cell adhesion, proliferation and differentiation behavior. In current

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research, various electrospun nanofibers have been devised to prepare biomimetic nanocom‐ posites for potential application in tissue engineering. For instance, Mather *et al.* prepared silica from nanofibers by immersion of the PEI/PVP (poly(ethylene imine)/poly(vinyl pyrrolidone)) nanofibers in silica precursor tetramethylorthosilicate (TMOS) and then calcinations [10]. A simple alternative to create silk/silica composites is to coat silk-based material templates with silica precursors (such as tetraethylorthosilicate (TEOS)) and subsequently heat them at 105 °C for several h, as was demonstrated with cocoon fibers of *Bombyx mori* fibroin silkworms [11]. Furthermore, the silk template can subsequently be removed by calcinations, yielding a porous material in which the pore structure is determined by the silk-based material.

activity by accelerating adhesion behavior in the early stages. Secondly, a silk-nHA (nanohydroxyapatite) biocomposite scaffold was also developed by an electrospinning technique and then post-treated by employing a calcium phosphate (Ca–P) alternate soaking method. We hypothesized that well-distributed HA nanoparticles on the silk nanofibrous would improve cell activity by accelerating differentiation in the late stages. Extensive material characterizations and cell culture studies using MC3T3-E1 were conducted to assess the viability and potential application of this material for future bone grafts applications. Fur‐ thermore, we present a novel and effective emulsion electrospinning method in obtaining Fluorescein isothiocyanate-dextran (FITC-dextran)/poly (lactic-co-glycolic acid) (PLGA) and Type I collagen/poly (lactic-co-glycolic acid) (PLGA) fibrous composite scaffolds. Core-sheath structured fibers are successfully fabricated with average diameters of 665 nm and 567 nm for FITC-dextran/PLGA and collagen/PLGA, respectively. *In vitro* release profile shows sustained release of encapsulated FITC-dextran from FITC-dextran/PLGA fibers as long as 7 weeks. The osteoblastic activities of collagen/PLGA nanofibrous scaffold are also investigated by employ‐ ing osteoblastic-like MC3T3-E1 cell line. Lactate dehydrogenase assay results suggest the excellent cytocompatibility. Cell proliferation and alkaline phosphatase (ALP) activity is also ameliorated on this emulsion electrospun collagen/PLGA fibrous scaffold. All the results indicated that this composited scaffold could support the early stages of osteoblast behavior as well as immediate/late stages. The emulsion electrospinning process has good potential for application in drug release device and three-dimensional scaffold in bone regeneration.

Fabrication of Nanofibrous Scaffolds by Electrospinning

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

105

**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

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,

rate for spinning.

Bioactive ceramics, such as hydroxyapatite (HA) has also been used in many medical appli‐ cations in orthopedic and dental surgery owing to its osteoconductivity and osteophilicity [12– 14]. In the past few years, various electrospun nanocomposite fibers, such as PCL/CaCO3 [15], HA/gelatin [16], silk/HA [17], PLA/HA [18], and triphasic HA/collagen/PCL [19] had been devised and explored for potential bone regeneration applications. However, since most of these electrospun composite fibers were prepared by electrospinning of blends made by simply mixing the prior obtained inorganic nanoparticles with viscous spinning solutions of polymers, it usually results in nanocomposites with very limited or lacking of specific interactions between the organic and inorganic phases [20].

Besides the widely recognized merits of electrospun fibers, Core-sheath structural nanofibrous scaffold incorporated with bioactive agents is supposed to promote cell migration, prolifera‐ tion, and gene expressions because the controllable and sustainable release of bioactive agents from the fibers and the preservation of bioactivity. A functional nanofibrous scaffolds incor‐ porated with bioactive agents depends on two factors: the controllable and sustainable release of bioactive agents from the fibers and the preservation of bioactivity. A majority of the reported works on drug release scaffolds tends to adopt the route of simple mixing of bioactive agents and the carrier polymers for blend-electrospinning. The resultant blend formulation would usually lead to initial burst release of drug, which is undesirable for an effective and controllable device. [11] Moreover, simple blending of electrospinning dopes leads to the direct exposure of bioactive agent to aggressive solvent environment that potentially denatures the biomolecules and loses the bioactivity. Therefore, developing novel processes capable of providing controllable system for the release of biomolecules from the electrospun fibers while preserving the bioactivity is of great importance.

In this study, three kinds of nanocomposite scaffold were prepared by electrospinning for improving cell cultivation. Firstly, we describe the formation of regenerated silk fibroin/ tetramethoxysilane (TMOS) nanofibers hybrid nanocomposites obtained by electrospinning of their blends. Hydrolysis and condensation of alkoxy silicon monomer (TMOS) shows that the Si–O–Si bonds join together in order to form a network made of porosities. Moreover, the amine groups catalyze the hydrolysis process due to the alternating presence of protonated and non-protonated amine groups in the fibroin molecular chains, which allows hydrogen bond formation with the oxygen adjacent to silicon in the precursor and thus facilitate –Si–O– Si– bond formation [21]. Here we hypothesize that the hybrid of silk fibroin and TMOS could improve hydrophilicity of the fibrous nanocomposites, furthermore, it would improve cell activity by accelerating adhesion behavior in the early stages. Secondly, a silk-nHA (nanohydroxyapatite) biocomposite scaffold was also developed by an electrospinning technique and then post-treated by employing a calcium phosphate (Ca–P) alternate soaking method. We hypothesized that well-distributed HA nanoparticles on the silk nanofibrous would improve cell activity by accelerating differentiation in the late stages. Extensive material characterizations and cell culture studies using MC3T3-E1 were conducted to assess the viability and potential application of this material for future bone grafts applications. Fur‐ thermore, we present a novel and effective emulsion electrospinning method in obtaining Fluorescein isothiocyanate-dextran (FITC-dextran)/poly (lactic-co-glycolic acid) (PLGA) and Type I collagen/poly (lactic-co-glycolic acid) (PLGA) fibrous composite scaffolds. Core-sheath structured fibers are successfully fabricated with average diameters of 665 nm and 567 nm for FITC-dextran/PLGA and collagen/PLGA, respectively. *In vitro* release profile shows sustained release of encapsulated FITC-dextran from FITC-dextran/PLGA fibers as long as 7 weeks. The osteoblastic activities of collagen/PLGA nanofibrous scaffold are also investigated by employ‐ ing osteoblastic-like MC3T3-E1 cell line. Lactate dehydrogenase assay results suggest the excellent cytocompatibility. Cell proliferation and alkaline phosphatase (ALP) activity is also ameliorated on this emulsion electrospun collagen/PLGA fibrous scaffold. All the results indicated that this composited scaffold could support the early stages of osteoblast behavior as well as immediate/late stages. The emulsion electrospinning process has good potential for application in drug release device and three-dimensional scaffold in bone regeneration.

research, various electrospun nanofibers have been devised to prepare biomimetic nanocom‐ posites for potential application in tissue engineering. For instance, Mather *et al.* prepared silica from nanofibers by immersion of the PEI/PVP (poly(ethylene imine)/poly(vinyl pyrrolidone)) nanofibers in silica precursor tetramethylorthosilicate (TMOS) and then calcinations [10]. A simple alternative to create silk/silica composites is to coat silk-based material templates with silica precursors (such as tetraethylorthosilicate (TEOS)) and subsequently heat them at 105 °C for several h, as was demonstrated with cocoon fibers of *Bombyx mori* fibroin silkworms [11]. Furthermore, the silk template can subsequently be removed by calcinations, yielding a porous

Bioactive ceramics, such as hydroxyapatite (HA) has also been used in many medical appli‐ cations in orthopedic and dental surgery owing to its osteoconductivity and osteophilicity [12– 14]. In the past few years, various electrospun nanocomposite fibers, such as PCL/CaCO3 [15], HA/gelatin [16], silk/HA [17], PLA/HA [18], and triphasic HA/collagen/PCL [19] had been devised and explored for potential bone regeneration applications. However, since most of these electrospun composite fibers were prepared by electrospinning of blends made by simply mixing the prior obtained inorganic nanoparticles with viscous spinning solutions of polymers, it usually results in nanocomposites with very limited or lacking of specific

Besides the widely recognized merits of electrospun fibers, Core-sheath structural nanofibrous scaffold incorporated with bioactive agents is supposed to promote cell migration, prolifera‐ tion, and gene expressions because the controllable and sustainable release of bioactive agents from the fibers and the preservation of bioactivity. A functional nanofibrous scaffolds incor‐ porated with bioactive agents depends on two factors: the controllable and sustainable release of bioactive agents from the fibers and the preservation of bioactivity. A majority of the reported works on drug release scaffolds tends to adopt the route of simple mixing of bioactive agents and the carrier polymers for blend-electrospinning. The resultant blend formulation would usually lead to initial burst release of drug, which is undesirable for an effective and controllable device. [11] Moreover, simple blending of electrospinning dopes leads to the direct exposure of bioactive agent to aggressive solvent environment that potentially denatures the biomolecules and loses the bioactivity. Therefore, developing novel processes capable of providing controllable system for the release of biomolecules from the electrospun fibers while

In this study, three kinds of nanocomposite scaffold were prepared by electrospinning for improving cell cultivation. Firstly, we describe the formation of regenerated silk fibroin/ tetramethoxysilane (TMOS) nanofibers hybrid nanocomposites obtained by electrospinning of their blends. Hydrolysis and condensation of alkoxy silicon monomer (TMOS) shows that the Si–O–Si bonds join together in order to form a network made of porosities. Moreover, the amine groups catalyze the hydrolysis process due to the alternating presence of protonated and non-protonated amine groups in the fibroin molecular chains, which allows hydrogen bond formation with the oxygen adjacent to silicon in the precursor and thus facilitate –Si–O– Si– bond formation [21]. Here we hypothesize that the hybrid of silk fibroin and TMOS could improve hydrophilicity of the fibrous nanocomposites, furthermore, it would improve cell

material in which the pore structure is determined by the silk-based material.

interactions between the organic and inorganic phases [20].

104 Advances in Nanofibers

preserving the bioactivity is of great importance.
