Carbohydrate Polymers 94 (2013) 713–722

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Chitosan(PEO)/silica hybrid nanofibers as a potential biomaterial for bone

regeneration
Georgios Toskas a,∗ , Chokri Cherif a , Rolf-Dieter Hund a , Ezzeddine Laourine a , Boris Mahltig b ,
Amir Fahmi c , Christiane Heinemann d , Thomas Hanke d
a
Institute of Textile Machinery and High Performance Material Technology (ITM), Technische Universität Dresden, Hohe Str. 6, 01069 Dresden, Germany
b
University of Applied Sciences, Faculty of Textile and Clothing Technology, Webschulstrasse 31, 41065 Mönchengladbach, Germany
c
Rhine-Waal University of Applied Sciences, Faculty of Technology and Bionics, Marie-Curie-Straße 1, 47533 Kleve, Germany
d
Max Bergmann Center of Biomaterials and Institute of Materials Science, Dresden University of Technology, Budapester Str. 27, 01069 Dresden, Germany

a r t i c l e i n f o a b s t r a c t

Article history: New hybrid nanofibers prepared with chitosan (CTS), containing a total amount of polyethyl-
Received 29 November 2012 ene oxide (PEO) down to 3.6 wt.%, and silica precursors were produced by electrospinning. The
Received in revised form 17 January 2013 solution of modified sol–gel particles contained tetraethoxysilane (TEOS) and the organosilane
Accepted 22 January 2013
3-glycidyloxypropyltriethoxysilane (GPTEOS). This is rending stable solution toward gelation and con-
Available online xxx
tributing in covalent bonding with chitosan. The fibers encompass advantages of biocompatible polymer
template silicate components to form self-assembled core–shell structure of the polymer CTS/PEO encap-
Keywords:
sulated by the silica. Potential applicability of this hybrid material to bone tissue engineering was studied
Chitosan(PEO)/silica nanofibers
Sol–gel
examining its cellular compatibility and bioactivity. The nanofiber matrices were proved cytocompat-
Electrospinning ible when seeded with bone-forming 7F2-cells, promoting attachment and proliferation over 7 days.
Cytocompatibility These found to enhance a fast apatite formation by incorporation of Ca2+ ions and subsequent immersion
Bioactivity in modified simulated body fluid (m-SBF). The tunable properties of these hybrid nanofibers can find
applications as active biomaterials in bone repair and regeneration.
© 2013 Elsevier Ltd. All rights reserved.

1. Introduction a silica–gelatin hybrid (Mahony et al., 2010), on silica–collagen

composites (Heinemann et al., 2009) and that of an osteosar-
Sol–gel derived hybrid silica glass nanocomposite materials and coma cell line SaOs-2 on a poly(␥-glutamic acid)/silica hybrid
nanofibers have been recently introduced as scaffolding matrices (Poologasundarampillai et al., 2010).
for bone–tissue regeneration (Heinemann et al., 2009; Heinemann, Chitosan (CTS) a natural cationic polymer offers unique proper-
Coradin, Worch, Wiesmann, & Hanke, 2011; Kim, Kim, & Knowles, ties such as biologically renewable, biodegradable, biocompatible,
2006; Mahony et al., 2010; Poologasundarampillai et al., 2010; Seol, non-antigenic, non-toxic and biofunctional. It is promoting cell
Kim, Kim, & Rhee, 2010). These organic–inorganic nanocomposite adhesion, proliferation and differentiation and evokes a minimal
materials combine both counterparts in one material, advantag- foreign body reaction on implantation (Muzzarelli, 2009). Chitosan
ing the flexibility and good mold ability of the organic part, with silicate hybrids were synthesized with glycidoxypropy-
heat-stability, high strength and chemical resistance of the inor- ltrimethoxy silane (GPTMS), whose epoxy groups are considered
ganic part. Moreover, coating ability of apatite on ceramics has to react with the amino groups of chitosan. The cross-linking den-
been known for a long time (Kokubo, 1991) and has been tested sity was around 80% regardless of the amount of silane (Muzzarelli,
on silica glasses of various alkoxide systems (Kim et al., 2006; 2011). The values of the mechanical parameters indicated that sig-
Poologasundarampillai et al., 2010; Toskas et al., 2011b). The sil- nificant stiffening of the hybrids was obtained upon addition of
ica hybrid materials favor cell attachment and are not cytotoxic the silane while full flexibility was retained. In addition, adhe-
as demonstrated by culture of mesenchymal stem cells (MSCs) on sion and proliferation of the MG63 osteoblast cells cultured on
the hybrid surface were improved compared to those on the
pure chitosan membrane regardless of the silane concentration
(Muzzarelli, 2011; Shirosaki et al., 2005; Shirosaki, Botelho, Lopes,
∗ Corresponding author. Present address: Technological Education Institute of
& Santos, 2009a). MC3T3-E1 cells also showed comparable via-
Piraeus, School of Engineering, Department of Textiles, 250 Thivon & P. Ralli, 12244
Egaleo, Greece. Tel.: +30 210 5381171/5381333; fax: +30 210 5381255. bility on sol–gel derived silica xerogel/chitosan hybrid coatings
E-mail addresses: [email protected], [email protected] (G. Toskas). onto alkali-treated titanium, proving their potential to be used as

0144-8617/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbpol.2013.01.068
714 G. Toskas et al. / Carbohydrate Polymers 94 (2013) 713–722

bioactive coating materials in hard tissue engineering (Jun et al., were obtained from Sigma, Germany. Acetic acid was bought
2010). Moreover chitosan was found to improve structural integrity from Normapur (PROLABO), Germany. Deionized water was used
of PEO cross-linked by silicate nanoparticles (Laponite) films and for the preparation of solutions. Dulbecco’s Modified Eagle’s
to enhance the formation of a mineralized extracellular matrix Medium (DMEM—D6046)—low glucose with 1000 mg/L glucose,
and the differentiation of MC3T3-E1 preosteoblast cells (Gaharwar, l-glutamine and sodium bicarbonate, liquid sterile filtered was
Schexnailder, Jin, Wu, & Schmidt, 2010). also purchased from Sigma, Germany.
Glass ceramic silica (SiO2 ) electrospun nanofibers have received
significant attention due to their multifunctional properties in 2.2. Preparation of electrospun nanofibers
biomedical fields (Kim et al., 2006; Seol et al., 2010; Sridhar,
Sundarrajan, Venugopal, Ravichandran, & Ramakrishna, 2012). The modified silica sol was prepared by hydrolyzing
There are two main methods of preparing silica electrospun tetraethoxysilane and 3-glycidyloxypropyltriethoxysilane
nanofibers via solutions (spin dopes): the first is by the direct spin- in a weight ratio of 3:1. The sol was hydrolyzed in 0.01 N
ning from aged sol–gel solutions containing alkoxide precursors; HNO3 and in presence of ethanol, in a volume ratio of:
the second involves carrying polymers and is more advantageous as (TEOS/GPTEOS):HNO3 :C2 H5 OH = 1:0.2:4.2. After stirring for
it can be easily adjusted, generating nanofibers of controllable size 24 h, the solution was ready for the preparation of the spin-dopes.
and uniformity. As alkoxide precursor, tetraethoxysilane (TEOS) A 3 wt.% PEO solution was prepared separately in 0.5 M acetic acid
and tetramethoxysilane (TMOS) are commonly used (Choi, Lee, Im, and stirred for a period of 24 h. Then, 2.5 wt.% of chitosan (CTS)
Kim, & Joo, 2003; Shao et al., 2003). As carrying co-electrospun solution and 3.0% PEO solution in acetic acid were blended with
homopolymer polyvinyl pyrrolidone (PVP), polyvinylidene fluo- variable weight ratios of chitosan to PEO. The optima results as
ride (PVDF), nylon-6 and polyvinyl alcohol (PVA) are common to nanofibers production were obtained at a chitosan/PEO ratio
studied polymers (Gaharwar et al., 2010; Kim, Ahn, & Choi, of 60:40 with 20 wt.% total acetic acid concentration. This ratio
2010; Liu et al., 2008; Shao et al., 2003). Recently, new types of was kept stable for the mixed chitosan(PEO)/silica solutions.
organic–inorganic silica nanofibrous (SiO2 ) composite membranes Afterwards, the CTS(PEO) solutions were mixed in various weight
developed based on one of the most frequently used non-toxic ratios with the alkoxide solution. The resultant mixtures were
and biocompatible polymers, polyethylene oxide (PEO) (Toskas stirred for at least 6 h to ensure adequate mixing.
et al., 2011b). A new organically modified alkoxide sol–gel solu- The electrospinning apparatus was set up horizontally, as it
tion was used containing a mixture of tetraethoxysilane (TEOS) has been previously described (Toskas et al., 2011a). The sil-
and 3-glycidyloxypropyltriethoxysilane (GPTEOS). Organosilanes ica/polymer solution was put into a 1 mL disposable syringe fitted
such as GPTEOS are having the advantage of bearing organic and with 0.60–1.0 mm 23 gauge tip-ground-to-flat needles and fed with
inorganic functionalities in the one molecule which provide the the help of a programmable KD scientific pump. The ambient condi-
ability to bond to organic chains and inorganic moieties accord- tions occurred at temperatures of 20–22 ◦ C and 50–55% RH. Typical
ingly. Moreover, major advantage of this combined alkoxide sol–gel parameters operated in this study were flow rates between 0.4
precursor is to increase the stability of GPTEOS toward gelation up and 1.0 mL/h, voltages between 25 and 28 kV and a tip to collector
to several months (Mahltig, Fiedler, Fischer, & Simon, 2010). It is optimal distance of 11 cm.
also suggested that organosilanes as ␥-glycidoxypropyltrimethoxy
silane (GPTMS) slow condensation reaction rate and enhance the 2.3. Characterization
polymer–silica compatibility (Liu, Su, & Lai, 2004).
Chitosan (CTS) nanofibers via electrospinning were first 2.3.1. Rheology
obtained from neat trifluoroacetic acid (TFA) at 7 wt.% (Ohkawa, A Haake MARS (Haake, Germany) stress-controlled rheometer
Cha, Kim, Nishida, & Yamamoto, 2004). But the more usual method was used for the measurements of the solutions viscosity parame-
involves the use of polyethylene oxide (PEO) as co-blending poly- ters. Cone-plate geometry (35 mm radius, 2◦ , Ti) was used for the
mer, in order to spin chitosan at higher polymer concentrations shear measurements and the oscillatory movement. The gap for all
(Bhattarai, Edmondson, Veiseh, Matsen, & Zhang, 2005; Klossner, solutions was 0.100 mm at a temperature of 20 ± 0.1 ◦ C.
Queen, Coughlin, & Krause, 2008; Toskas, Laourine, Kaeosombon, &
Cherif, 2009). Combining the unique collective properties of both 2.3.2. Microscopy: scanning electron microscopy-EDX,
materials, the fabrication of composite organic–inorganic CTS/SiO2 transmission electron microscopy, fluorescence microscopic
nanofibers via electrospinning is of great interest. Introduction images
of silica into biomaterials was early proved to increase its oxy- A DSM 982 Gemini (Zeiss, Germany) Scanning Electron Micro-
gen permeability, biocompatibility, and biodegradability (Suzuki & scope served for the examination of the morphology of the
Mizushima, 1997; Tian et al., 1997). In this work, a controllable pro- nanofibers. The as-spun nanofibers were dried under vacuum at
cess of creating CTS(PEO)/SiO2 nanofibers is presented; the effect room temperature and sputter-coated with silver/graphite. The
of varying the polymer to silica precursor weight ratio is exam- samples were examined at an accelerating voltage of 1.0 kV and
ined and correlated to the structural properties of the nanofibers. magnifications from 200 to 50,000. The SEM images were then
Subsequently, the ability of these hybrid nanofibers to favor cell used to evaluate the fiber diameter. The average fiber diameter was
attachment of 7F2 osteoblasts is tested and also their capabil- determined using Digital Image Processing System 2.6 (point elec-
ity to modify by incorporating calcium ions resulting in bioactive tronic GmbH) software in conjunction with the SEM image. Ten
hydroxyl carbonate apatite (HCA) crystal formation were in parallel different fiber diameters were determined and averaged to find
examined. the fiber diameter reported for each of the resulting electrospun
mats. The 95% confidence limits of the mean were calculated and
reported with each average fiber diameter.
2. Materials and methods For the transmission electron microscopy (TEM), the samples
were deposited as received in copper TEM grids carbon-coated
2.1. Materials copper grids (400 meshes, AGAR Scientific). Transmission electron
microscope (TEM) from TECNAI Biotwin (FEI Ltd.) at 100 keV was
Chitosan from crab shells with >75% of deacetylation used to observe the samples. Any treatment has been performed
(MW = 200 kD) and polyethylene oxide (PEO) (MW = 900 kD) on the microfibers to avoid any deterioration of the samples.
 G. Toskas et al. / Carbohydrate Polymers 94 (2013) 713–722 715

Fig. 1. Electrosprayed TEOS/GPTEOS spin dope (a) and electrospinning attempts of 2.5 wt.% CTS/SiO2 ((b) magnification 10,000×, (c) magnification 5000×).

Fluorescence microscopic images have been taken with a Zeiss gentle stirring. After soaking, the samples were carefully rinsed
Axioskop 2 FS mot equipped with both a Zeiss Plan Neofluar® with deionized water, and dried at room temperature.
10×/0.3 and a Zeiss Plan-Apochromat® 20×/0.75. The excitation
was performed by a 50 W-mercury lamp. Filter sets Zeiss Nr 1
(DAPI) as well as Zeiss Nr. 9 (Alexa Fluor 488® ) have been used. 3. Results and discussion

3.1. Synthesis and properties of spin dopes—morphology and

2.3.3. Infrared spectroscopy structure of CTS(PEO)/SiO2 nanofibers
IR spectra were obtained by using the attenuated total reflection
(ATR) method on a FTIR Bruker Tensor 27 spectrophotometer. The organically modified alkoxide sol–gel solution con-
taining the mixture of silanes tetraethoxysilane (TEOS) and
3-glycidyloxypropyltriethoxysilane (GPTEOS) in a weight ratio of
2.3.4. Cell culture and staining
3:1 was prepared for electrospin. No alteration on the gelation of
Nanofibrous membranes for cell culture were prepared on tita-
this combined alkoxide sol–gel precursor over time was observed
nium plates (10 mm diameter) by dip coating. After coating, drying
up to several months due to the increased stability of GPTEOS
at 37 ◦ C and 20% relative humidity was performed over 24 h in
(Mahltig et al., 2010). Electrospinning of the silane solution itself,
a climate chamber. The resulting membranes on titanium plates
designated hereafter as SiO2 , presenting a dynamic viscosity of
were sterilized by gamma-irradiation (25 kGy) before starting the
only 5 mPa s, resulted in nanodroplets through electrospraying
cell culture experiments. Murine osteoblast-like cell line 7F2 was
(see Fig. 1a). Also, electrospinning attempts with pristine chitosan
obtained from the American type Culture Collection (ATCC). Cells
(2.5 wt.% CTS) in mixture with the sol–gel solution in various ratios,
were expanded in minimal essential medium (R-MEM) supple-
were not effective leading to bead formation. Beads possess the
mented with 10% fetal calf serum (FCS), 2 mM l-glutamine, and
same form as these produced by the precursor of silica alone,
the antibiotics penicillin (100 U/mL) and streptomycin (100 U/mL)
but interconnected with nanofibrils (see Fig. 1b and c). Strong
in a humidified atmosphere (37 ◦ C, 7% CO2 ). Medium and all sup-
electrostatic forces are suggested in the origin of this micro-
plements were obtained from Biochrom, Germany.
and nano-particle formation (Coradin & Livage, 2007; Coradin,
The membranes were placed in 48-well plates and soaked in
Durupthy, & Livage, 2002). The attractive force through the elec-
cell culture medium for 24 h. After removing the medium, 40 ␮L of
trostatic interactions between the positively charged ammonium
cell suspension (2500 cells ␮L) was placed onto each sample. Cells
groups of the chitosan backbone and the negatively charged
were allowed to adhere for 30 min in the incubator before filling
poly(silicic acid) [SiOx (OH)4-2x ]n oligomers favors their condensa-
up the wells with additional medium. The medium was changed
tion, inducing silica precipitation (Coradin & Livage, 2007). The
every second day.
addition of polyethylene oxide (PEO) was proved essential to
In order to evaluate cell morphology, spreading, and growth
obtain a successful electrospinning. PEO containing neutral moi-
using fluorescence microscopy actin cytoskeleton as well as the
eties reduces the chitosan solution viscosity relaxing chitosan
nuclei were stained after 1 day and 6 days. To that end, after
chain entanglement. Besides, the possible interaction between PEO
washing and fixing, the cells were permeabilized with 0.2% Triton-
and the sol–gel alkoxide is considered through hydrogen bonds.
X-100 in PBS and blocked with 1% bovine serum albumin (BSA,
According to our hypothesis, PEO could intercalate in between the
Sigma) for 30 min. Cytoskeletal actin was stained with AlexaFluor
positively charged chitosan and the negatively charged silicates. In
488-Phalloidin (Invitrogen), and cell nuclei were stained with 4 ,6-
this model, PEO minimizes the reactivity of silicates and allows dis-
diamidino-2-phenylindole (DAPI, Sigma).
persion of the two phases preventing direct deposition of silicates
as beads during electrospinning.
2.3.5. Bioactivity test Chitosan (CTS) is successfully electrospun with PEO as co-
For the development of apatite nucleation sites on the hybrid blending polymer in various ratios. The polymer system consisting
silicate nanofibers, CaO in the form of calcium chloride dihydrate of 2.5 wt.% of chitosan (CTS) solution and 3.0 wt.% PEO solution in
was added in the initial sol–gel solution, before electrospinning. acetic acid by weight ratio of 60/40, the ultimate aim is to obtain
The molar ratios of CaCl2 • 2H2 O, GPTEOS, TEOS, ethanol, HNO3 and the optima results as to the CTS/PEO nanofiber uniformity and
water were 0.08:0.2:0.6:13.5:0.004:1.9, respectively. The bioactiv- repetitiveness of process (see the Supporting Information, Fig. SF-
ity of the as-obtained nanofibers was assessed for their apatite 1) (Toskas, Hund, Laourine, & Cherif, 2012). Subsequently, varied
forming ability in modified simulated body fluid (m-SBF) accord- the weight ratio between the polymer (CTS/PEO 60/40) to alkox-
ing to Oyane et al. (2002) (see the Supporting Information, Table ide (TEOS/GPTEOS 3/1) solution in order to reveal a composition
1). The specimens of 10 mg were placed in a Petri glass disk and window in which uniform nanofibers could be generated.
immersed in 10 mL of m-SBF solution buffered at pH 7.4 for up to 7 Critical chain entanglement among the macromolecular chains
days without refreshing. Temperature was held at 37 ◦ C through is related to the electrospinning ability of a polymer solution. The
716 G. Toskas et al. / Carbohydrate Polymers 94 (2013) 713–722

of the chains (Fig. 2). The CTS(PEO)/SiO2 70/30 wt.% electrospun

nanofibers present a “sticky” surface and are interconnected as the
CTS/PEO 60/40 wt.% electrospun alone (see Supporting Information,
Fig. SF-1a and b). When introducing over 80% in wt.% of the silica
component fibers become less uniform with a large diameter dis-
tribution ranging from 866 to 249 nm. Then, over a CTS(PEO)/SiO2
10/90 wt.% ratio beads appear within the non-uniform nanofiber
network. The minimum amount of carrying polymers required for
the continuous fiber production via electrospinning is found to
4.5 wt.% CTS and 3.6 wt.% PEO correspondingly. Compared with the
average nanofiber diameter of CTS/PEO 60/40 wt.% of 88 ± 15 nm,
the nanofiber diameters measured with the silica blend systems are
about 2–5 times enlarged by increasing the silica content. These are
found also higher in average diameter compared with pristine PEO
nanofibers measured 151 ± 23 nm. An enhanced compact struc-
Fig. 2. Shear viscosity as a function of shear rate of various polymer/silica blends of
CTS(PEO)/SiO2 compared to 3.0 wt.% PEO alone and CTS/PEO 60/40. ture is resulted by chitosan due to a stronger interaction among
silicate and CTS(PEO) polymer chains. The investigated formula-
tion showed also an excellent electrospinnability when a rotating
viscosity of these solutions is an expression of the intermolecu- collector with a 4 mm diameter was used. At a rotational speed
lar interactions among the polymer chains. First, the effect of the of 200 rpm, a solution of CTS(PEO)/SiO2 30/70 wt.% produced a
interactions of polymer CTS(PEO) content in the mixed sol–gel thick nanofiber mat. The nanofibers produced with this method
solution was examined. This would affect the viscosity and thus presented also smooth surfaces and possessed a smaller average
the spinnability of solutions. The shear viscosity as a function of diameter of 339 ± 65 nm (see in Supporting Information, Fig. SF-3).
shear rate was measured for solutions of different CTS(PEO)/SiO2 It is worth to mention that silicon is detected by EDX on the fiber
ratios as shown in Fig. 2. The initial dynamic viscosity of 3.0 wt.% surface of all samples (Supporting Information, Fig. SF-3, insert).
PEO was measured 0.407 Pa s, whereas weight ratio of CTS/PEO Transmission electron microscopy (TEM) permitted to identify
60/40 giving uniform nanofibers with diameters down to 40 nm the inner morphology of the nanofibers. The micrographs demon-
was found 1.44 Pa s. Dynamic viscosities of mixtures CTS(PEO)/SiO2 strate that the nanofibers are composed of compact aligned dots
ranged in between the above mentioned values: 0.86 Pa s for the encapsulated by the other material in a core–shell structure (Fig. 5).
CTS(PEO)/SiO2 30/70 wt. and 1.25 Pa s for the ratio 50/50 wt.% (see The differences in contrast along the nanofibers show dark dots
Fig. 2). The images of the corresponding as-spun CTS(PEO)/SiO2 and having the form of elongated capsules indicated by red circles
CTS/PEO nanofibers are shown in Fig. 4 and Fig. SF-1. in Fig. 5a and b and in more detail in Supporting Information,
The diagram in Fig. 2 shows that the viscosity of the Fig. SF-4a and b. This effect is observed to CTS(PEO)/SiO2 ratios
CTS(PEO)/SiO2 blends decreased monotonically with decreasing of 50/50 and 30/70 wt.% whereas increasing the amount of sil-
polymer content. Nevertheless, the curve of the CTS(PEO)/SiO2 ica in the system stretched the capsules within the fibers. With
50/50 wt.% ratio superposes that of 70/30 wt.% ratio. This is sug- the ratio CTS(PEO)/SiO2 of 10/90 wt.% where the relative polymer
gested due to stronger intermolecular interactions among the mass is much inferior to the silicate component, these capsules
polymer chains and the silicon alkoxide network. In fact, other rhe- are stretched to form a uniform core fiber (indicated by a red
ological factors such as elastic modulus (G ) and loss modulus (G ) arrow in Fig. 5c and in more detail in Supporting Information,
are playing significant role in enhancing chain entanglement. In the Fig. SF-4c). As confirmed by EDX and mainly by ATR–FTIR analysis
spin experimental dopes, it was found that the loss modulus G is (Supporting Information, Fig. SF-3 and Fig. 6), silicon was always
dominating the elastic modulus G (G > G ) at low frequencies (see detected on the fiber surface. This finding suggests that the exter-
in Supporting Information, Fig. SF-2). This is a typical behavior for nal coating is consisted of the silicate component, chitosan/PEO
an entangled solution. Moreover, for the CTS(PEO)/SiO2 70/30 wt.% being incorporated as a core material in self assembled hybrid
ratio, a crossover occurs (G = G = 70.9 Pa) at 9.4 Hz and from that nanofibers’ structures. A complex phase separation occur leading
point on at higher frequencies, the elastic modulus (G ) dominates to organic polymer inclusion. The formation of core–shell structure
G (Fig. SF-2c). On the contrary for the CTS(PEO)/SiO2 50/50 wt.% in various polymer–polymer–solvent systems has been attributed
ratio, the crossover observed at G = G = 16.13 Pa has shifted to a to factors that include the different solubility of the two compo-
lower frequency of 5.45 Hz (Fig. SF-2d). This shift of the crossover nents, variations in rheological parameters, diverse in the blending
point has been correlated with the effective cross-linking change ratio molecular weight of the polymers and temperature (Wei, Lee,
or chain entanglement (Chronakis & Ramzi, 2002). Strong hydro- Kang, & Mead, 2005; Zhang et al., 2009). The binodal phase separa-
gen bonding between the silicate and CTS(PEO) polymer chains are tion of the ternary system by nucleation and growth, assisted by the
formed. Furthermore, covalent bonds involving the epoxy group of stronger mobility of chitosan polymer chains induced by acetic acid,
the silane GPTEOS are considered to be at the origin of this enhanced seems more likely ascribe to the core–shell structure of our sys-
entanglement. tem. This hypothesis is based on the consideration that CTS/PEO is
Uniform nanofibers with smooth surface were obtained within a homogenously behaving polymer system. Actually, no phase sep-
a composition window of CTS(PEO)/SiO2 ranging from 70/30 to aration is observed in nanofibers obtained by CTS/PEO 60/40 wt.%
20/80 wt.% ratios (see Fig. 3). Spinning conditions as voltage, tem- electrospun alone (see the Supporting Information, Fig. SF-1c). For
perature and relative humidity were kept fixed so that only the CTS(PEO)/SiO2 hybrids, solvent surface tension and removal of the
polymer/solvent and silica oxide properties could influence the solvent during electrospinning process plays an important role in
fiber morphology. The average diameters of the as-spun nanofibers the core–shell formation. In fact, electrospinnability of this hybrid
are increasing from 239 ± 13 nm for the CTS(PEO)/SiO2 70/30 wt.% system is influenced by the solution surface tension. It is lowered
ratio to 508 ± 62 nm for the 30/70. The CTS(PEO)/SiO2 50/50 wt.% by the addition of the alkoxide solution containing about 87% w/v
ratio presents the smaller average diameter rating 182 ± 16 nm ethanol (surface tension  = 22.3 mN/m) to the aqueous CTS/PEO
(see Fig. 4). This finding is in good agreement with the rheolo- solution (∼80% (w/v) H2 O  = 72 mN/m; (∼20% (w/v) CH3 COOH
gical results discussed above, provoking a tighter entanglement  = 27.6 mN/m). Hence, ethanol is more rapidly evaporated
 G. Toskas et al. / Carbohydrate Polymers 94 (2013) 713–722 717

Fig. 3. CTS(PEO)/SiO2 electrospun nanofibers from 50/50 wt.% ratio ((a) magnification 5000×, (b) magnification of 20,000×; inset: magnification 50,000×), 30/70 weight
ratio ((c) magnification 5000×, (d) magnification 20,000×; inset: magnification 50,000×), 20/80 ((e) magnification 5000×, (f) magnification 20,000×) and 70/30 wt.% ratios
((g) magnification 10,000×, (f) magnification 20,000×).
718 G. Toskas et al. / Carbohydrate Polymers 94 (2013) 713–722

␯(Si O) bands in the 1110–1000 cm−1 region. The Si O Si stretch-

ing assignment of silica at 1078 cm−1 is superposed with the
strong peak of PEO assigned to C O C stretching at 1104 cm−1
and has shifted to lower wavelengths with a maximum of
around 1065 cm−1 . The medium secondary shoulder band at about
1192 cm−1 indicates the presence of an epoxy group from the 3-
glycidyloxypropyltriethoxysilane. The silane assignments at about
940 cm−1 and 793 cm−1 ascribed to the Si O Si and Si OH stretch-
ing vibrations related to the surface of SiO2 , are also present. The
amide I and II bands of chitosan at 1650 and 1565 cm−1 respec-
tively are much weaker but still present, except when the SiO2
content is higher than 80 wt.% (Fig. 6d). Besides, the weak band at
Fig. 4. Effect of polymer (CTS(PEO)) weight ratio on fiber diameter.
2875 cm−1 probably results from the ethoxy groups of GPTEOS, the
broad band at 3275–3244 cm−1 indicating that most of the ethoxy
groups are hydrolyzed to silanol groups (Si OH). These hydroxyl
during electrospinning, decreasing temperature at the fiber surface. groups are considered on the electrospun silicate component sur-
This reduction of temperature varying for the two components, face. These findings corroborate our hypothesis that new compact
can lead to a thermally induced phase separation of silicate and self-assembled hybrid nanofibers are formed.
CTS/PEO, inducing diverse free energy surfaces and resulting in self Moreover, it is likely that a certain fraction of GPTEOS in
assembled core–shell structures. the hybrid is linked via the epoxy group to chitosan. First, the
hydrolyzation and condensation of GPTEOS with TEOS molecules
3.2. Composition and integrity of the hybrid nanofibers give rise to Si O Si bridging oxygen and yield silanol groups
(Si OH). The organosilane formed, conduct organic and inorganic
The interactions of the hybrid fibers compositions were con- domains in the same molecule (see 1st step in Scheme 1). Simul-
firmed by ATR–FTIR spectra. Fig. SF-5a in Supporting Information taneously, GPTEOS could be functionalized through nucleophilic
is presenting the FTIR spectrum of the TEOS/GPTEOS (designated attack to the most substituted carbon of the oxirane, which forms
as SiO2 ) nanoparticles electrosprayed from the initial alkoxide the most stable carbocation. This reaction can catalyzed by the
solution. The characteristic bands of silica xerogel are observed: acetic acid and lead to ring opening of the epoxy group. As to
at 1078 cm−1 and 950 cm−1 , 795 cm−1 are assigned to Si O Si the possible interactions between chitosan and the epoxy group of
and Si OH stretching vibrations, respectively (Allo, Rizkalla, & alkoxide, different approaches have been yet proposed. According
Mequanint, 2010). The wide peak in the range 3300–3700 cm−1 to Liu et al. (2004), a direct acid-catalyzed amino-oxirane addition
is assigned to the hydroxyl stretching of the silanol groups. In reaction, cross-linking NH2 to CH3 end unit of the ring, is taking
the stronger peak of 1078 cm−1 a shoulder at about 1192 cm−1 place. Unlike to this mechanism, Spirk et al. (2012) have recently
is attributed to the presence of epoxy group from the 3- considered that covalent bonds between silanes and chitosan can
glycidyloxypropyltriethoxysilane while a smaller peak at about only proceed via the hydroxyl groups of chitosan (see 2nd step
2840 cm−1 can be attributed to the ethoxy group ( OCH2 CH3 ). The (a) in Scheme 1), due to the formation of strong Si O bonds (av.
ATR–FTIR spectrum of CTS/PEO electrospun nanofibers is demon- 440 kcal mol−1 ) rather than weaker Si N bonds (av. 0 kcal mol−1 ).
strated in Fig. SF-5b. The wide peak at 3300–3700 cm−1 is assigned We consider that an ionic bond between the under acidic condi-
to the stretching of the CTS hydroxyl groups. The medium band tions (pH 3.0) protonated amine groups ( NH3 ) of chitosan and the
at 2889 cm−1 and the strong triplet band at 1147 cm−1 , 1104 cm−1 negatively charged esterified oxygen, resulting of the epoxy group,
and 1064 cm−1 with a maximum at 1104 cm−1 are attributed to the is possible. Besides, an addition copolymerization, implicating the
C H stretch and C O C accordingly of the PEO. The bands assigned chitosan amine groups and GPTEOS/TEOS silane carbonyls of the
to the C H wagging at 1343 cm−1 , C H twist at 1280 cm−1 and esterified ends and resulting in amide synthesis with methanol as
C H rocking at 961 cm−1 and 848 cm−1 are also observed. In the by-product, is much feasible (see 2nd step (b) in Scheme 1). In any
same spectrum (Fig. SF-5b), the carbonyl, C O NHR absorption case, covalent bonds are most likely to be present into the matrix
band was observed at 1653 cm−1 and the amine NH2 absorption together with hydrogen bonds (Poologasundarampillai et al., 2010).
band at 1560 cm−1 , respectively. These are characteristic of chi- Further quantification is under way in order to unveil the bridging
tosan and are denoted as amide I and II, respectively (Shirosaki configurations of this complex system.
et al., 2009b). For applications such as tissue repair and regeneration, mechan-
ATR–FTIR spectra of CTS(PEO)/SiO2 nanofibers of various ratios ical integrity of the scaffolds is critical in order to support cell
are shown in Fig. 6. All of these spectra resemble that of alkox- attachment, differentiation and growth. Chitosan does not dissolve
ide alone (Fig. SF-5a). The GPTEOS-containing hybrids showed in water under near neutral conditions. On the contrary, PEO has

Fig. 5. TEM micrographs of CTS(PEO)/SiO2 in various ratios revealing the core–shell structure: (a) 50/50, (b) 30/70 and (c) 10/90.
 G. Toskas et al. / Carbohydrate Polymers 94 (2013) 713–722 719

a high solubility in water, and electrospun pure PEO fibrous mem-

branes dissolve readily in water at 37 ◦ C. We have examined the
integrity of the hybrid CTS(PEO)/SiO2 nanofibers in deionized water
at 37 ◦ C after immersing 1 day and 6 days. After 1 day of immer-
sion, the nanofibers remain intact. The CTS(PEO)/SiO2 nanofibers
with ratio 30/70 wt.% present rougher surfaces (see in Supporting
Information, Fig. SF-6a and b, upper panel). A contraction of 27%
is measured for the 30/70 wt.% ratio nanofibers while only a small
decrease of 1.2% is observed for the 50/50 wt.% ratio nanofibers.
After 7 days immersion, the nanofibers of CTS(PEO)/SiO2 30/70 wt.%
ratio retain their integrity in water and morphology, although they
show a slight swelling (Fig. SF-6d, lower panel). The nanofibers
of CTS(PEO)/SiO2 50/50 wt.% ratio although they retain their ini-
tial fibrous structure, present a first stage of polymer (probably
PEO) release, eventually caused by acetic acid remaining traces (Fig.
SF-6c).
Pyrolysis is usually applied in polymer/silica systems in order to
eliminate the carrying polymer from the nanofibers. Chitosan does
not melt but degrade at a temperature higher of 313 ◦ C, according
to the degree of deacetylation (Agboh and Qin, 1996). Polyethylene
oxide used in this study, has been provided by the manufac-
turer (Aldrich) a melt point of 61.7–66.6 ◦ C and a flash point of
>113.0 ◦ C. Therefore, a pyrolysis temperature of 500–600 ◦ C was
attained at 10 ◦ C/min and maintained for 2 h in order to decompose
CTS/PEO. The nanofibers preserve their morphology and a decrease
of 8–40% in size of nanofiber diameters is observed (Supporting
Information, Fig. SF-7). Although the regression on the nanofibers of
CTS(PEO)/SiO2 30/70 wt.% ratio corresponds roughly to the organic
polymer content, that of 50/50 wt.% ratio represents only a small
amount of CTS/PEO showing a little effect of pyrolysis. In this last
case, an effective coating of the organic part by the silicate com-
ponent and an efficient compact structure could protect the core
material from decomposing.

3.3. Cell culture and bioactivity

The as-spun nanofibrous membranes were subsequently exam-

ined in respect to their cellular compatibility. The fluorescence
microscopic images (Fig. 7) show an excellent adherence and
spreading of the bone forming 7F2-cells on the nanofiber mats even
after a short culture time of 1 day. Despite the different situation
on CTS(PEO)/SiO2 50/50 wt.% mats (cf. left panel Fig. 7a) compared
with 30/70 wt.% mats (cf. Fig. 7e), in both cases the 7F2-cells are
well attached to the substrate and spread. Stretched cells with
well shaped actin fibers and filopodia are visible. This points an
immediate initial adherence, a good and fast attachment as well
as the absence of cytotoxic substances. After 6 days on both types
of nanofibrous membranes fully confluent layers have been grown
(right panel Fig. 7b, d and f). It stands for an undisturbed prolif-
eration and a good cytocompatibility, as it is commonly known
for titanium, our reference material (cf. Fig. 7c). From this point
of view, the chitosan/polyethylene oxide/silica nanofibers are a
suitable substrate for growing bone forming cells and might find
application in constructing scaffolds for bone tissue engineering.
Bioactive materials establish key biological interactions through
surface chemistry at the interface of this material and a tissue
(Saravanapavan & Hench, 2001). The living bone-bonding ability of
bioactive glasses depends greatly on the formation of biologically
active hydroxyapatite (HAP, [Ca10 (PO4 )6 (OH)2 ]) or hydroxycarbon-
Fig. 6. ATR–FTIR spectra of CTS(PEO)/SiO2 nanofibers of various ratios (a) 70/30, (b) ate apatite (HCA) as to their chemical resemblance to human bones.
50/50, (c) 30/70 and (d) 20/80. This mineralization process is taking place in contact with physi-
ological fluids or with simulated body fluids (SBF). Among various
forms of bioactive materials, nanofibers derived from silicate pre-
cursors via sol–gel exhibit the net advantage of large surface area
resulting in fast HAP crystal nucleation (Kim et al., 2006). Moreover,
the core–shell nanofibers by the use of the biocompatible polymers
720 G. Toskas et al. / Carbohydrate Polymers 94 (2013) 713–722

Scheme 1. Potential interactions of the epoxy silica GPTEOS group with chitosan.

Fig. 7. Fluorescence microscopic images of 7F2-cells on CTS(PEO)/SiO2 nanofiber mats (a) upper panel left: in a ratio of 50/50 wt.% after 1 day culture time; (b) upper panel
right: after 6 days and (d) middle panel right: after 6 days, more detailed image, (c) middle panel left: pure titanium after 3 days as a reference; (e) lower panel left: in a ratio
of 30/70 wt.% after 1 day culture time and (f) lower panel right: after 6 days.
 G. Toskas et al. / Carbohydrate Polymers 94 (2013) 713–722 721

Fig. 8. (a) Effect of CaO as nucleating agent: weak apatite nuclei formation in absence of initially added CaO. (b) Hexagonal 6/m hydroxycarbonate apatite/hydroxyapatite
formation on CaO-CTS(PEO)/SiO2 nanofibers after immersion in m-SBF for 7 days and in (c) EDX spectrum of the hydroxyapatite formation on respective area (b).

CTS/PEO have additional advantages: (i) the inorganic silica com- to the large amount (17.892 g L−1 ) of 2-(4-(2-hydroxyethyl)-1-
ponent being on the shell/surface of the fiber, thus encapsulating piperazinyl)ethanesulfonic acid (HEPES), introduced in m-SBF
the organic polymer, reacts directly with the SBF solutes, and (ii) (Oyane et al., 2002).
they do not require thermal treatment/sintering for the elimination Hydroxyapatite/silica nanofibers have been found promoting
of the polymer, avoiding thus carcinogenesis issues. Certainly, the new bone formation when implanted in vivo (Seol et al., 2010).
ability of the silanol groups on silicate ions to act as a nucleation site Thereby, the CTS(PEO)/SiO2 mineralized nanofibrous scaffold could
to form apatite is well known (Cho et al., 1996; Kokubo & Takadama, promote new bone formation when implanted in defected sites i.e.
2006; Li et al., 1992). From the various types of SBF solutes used for the periodontal tissue reconstruction.
in vitro investigations, the modified simulated body fluid, m-SBF
has the advantages of containing ion concentrations equal to those
4. Conclusions
of blood plasma and being stable for a long time stored at 5 ◦ C. In our
study, the concentration of HCO3 − is decreased to the level of satu-
Sol–gel derived hybrid silica glass nanofibers containing chi-
ration with respect to the calcite (CaCO3 ) phase (Oyane et al., 2002).
tosan (PEO) were fabricated and their structural properties were
When the as-spun CTS(PEO)/SiO2 nanofibers were incubated for 7
comprehensively studied. The nanofibers present an enhanced
days in absence of a nucleation initiator, they showed only a small
compact structure as confirmed by SEM and TEM measurements.
capability in producing spherical apatite (see Fig. 8a). Nonetheless,
The chitosan fraction in wt.% ratio has been found to influence
the nanofibrous non-woven fabric displays an interesting compact
the length scale of the structure formation, as the CTS(PEO)/SiO2
3D structure with a thickness of 5.8 ␮m and small pores of around
50/50 wt.% ratio generating nanofibers with small average diame-
1 ␮m (see in more detail the Supporting Information, Fig. SF-8). The
ter of rating 182 ± 16 nm. This tighter chain entanglement might
formation of apatite was greatly favored on nanofibers in which
be explained by the strong interaction between the epoxy group
a small amount down to 1.2 wt.% of CaO, in the form of calcium
of the organosilane GPTEOS used and the amine groups of chi-
chloride dehydrate, was initially introduced in the sol–gel solu-
tosan chains, triggering covalent bonds. The TEM micrographs
tion. After immersion and incubation for 7 days at 37 ◦ C, the fibers
revealed nanofibers based on self-assembled core–shell structure.
are covered with a dense layer of sheet-like apatite (see Fig. 8b). In
These results have been confirmed by ATR–FTIR analysis, whereas
some parts of the coated fibers, one can also observe the initiation
CTS/PEO is encapsulated to form the fibers ‘core’ coated by the sil-
of hexagonal 6/m structures characteristic for the apatite crystal
icate component as shell. The consequence is a fast apatite crystal
nucleation system. Thereby, the crystals observed in Fig. 8b can be
nucleation when immersed in m-SBF. The as-spun nanofibers were
attributed to the calcium precipitated as nanocrystalline phase.
in parallel proved cytocompatible. Cell fluorescence microscopy
The EDX spectroscopy analysis, on a single fiber (Fig. 8c) unveils
has shown that nanofiber membranes promote the attachment
a high concentration of P on the surface and reveals a Ca/P coating
and proliferation of murine osteoblast-like 7F2-cells. The combi-
ratio of approximately 1.3, lower to the stoichiometry of hydroxy-
nation of these hybrid nanofibers made of biocompatible polymers
apatite (Ca/P = 1.67) but closer to that of hydroxycarbonate apatite
CTS/PEO and silicate facilitate advantages of both materials and
(Ca/P = 1.5) (Kim et al., 2006; Saravanapavan & Hench, 2001). This
offer novel tunable properties for creating bioactive scaffolds
ratio determined by semiquantitative EDX analysis is compara-
for bone repair. Further future investigations will comprise the
ble to that of bonelike minerals (Mann, 1988). The silanol groups
mechanical properties, biodegradation and inner chemical config-
(Si OH), present through the sol–gel system induce Ca3 (PO4 )2
uration of various polymer/silica ratios.
nucleation via precipitation of Ca2+ and PO4 3− and also CO3− . In
addition, carbonate ions have shown to replace the hydroxyl ions
in the apatite crystal lattice (Lee, Lu, Baer, Markel, & Murphy, 2010). Acknowledgements
Furthermore, the presence of the positively charged amine groups
of chitosan could reduce the apatite formation ability and lower First author cordially thanks his early Professors at the Uni-
the Ca/P ratio, through interaction with the negatively charged versité Pierre et Marie Curie, Paris, France: Messrs. Pierre Sigwalt,
silica species. This phenomenon was also observed in the case of Jean-Pierre Vairon, Patrick Hemery and Michel Moreau, for teach-
silica–collagen xerogel (Heinemann et al., 2009). ing him Polymer Science and Ethics. He is also indebted to Emeritus
The major elements detected confirmed the presence of the Professors Messrs. Hartmut Worch (Technische Universität Dres-
ions used for the m-SBF composition. Increased carbon (C) con- den) and Horst Böttcher (Gesellschaft zur Förderung von Medizin-,
tent could also result from the sputtering of the pellets to provide Bio- und Umwelttechnologien e.V. (GMBU), Dresden, Germany)
the surface conductivity for SEM observation. Silicon (Si) is still for their precious encouragement. Authors are also grateful to
present due to the large amount of silanol groups on the nanofiber Dr. Sascha Heinemann of the Max Bergmann Center of Bioma-
surface. The observed strong peak of sulfur (S) can be attributed terials Dresden (TUD) for preparation of the substrates for cell
722 G. Toskas et al. / Carbohydrate Polymers 94 (2013) 713–722

experiments and kind supply of the m-SBF solution. They also thank Liu, Y., Sagi, S., Chandrasekar, R., Zhang, L., Hedin, N. E., & Fong, H. (2008). Preparation
Mr. Axel Mensch responsible for the SEM laboratory of the Institute and characterization of electrospun SiO2 nanofibers. Journal of Nanoscience and
Nanotechnology, 8, 1528–1536.
of Materials Science of the Technical University of Dresden (TUD) Mahltig, B., Fiedler, D., Fischer, A., & Simon, P. (2010). Antimicrobial coatings on
and his collaborators Mrs. Silvia Muehle and Mrs. Ortrud Trom- textiles-modification of sol–gel layers with organic and inorganic biocides. Jour-
mer for their invaluable help; also thank Mrs. Martina Dziewiencki nal of Sol–Gel Science and Technology, 55, 269–277.
Mahony, O., Tsigkou, O., Ionescu, C., Minelli, C., Ling, L., Hanly, R., et al.
(ITM/TUD) for the FTIR and Mr. Nicolas Cheval (University of Not- (2010). Silica–gelatin hybrids with tailorable degradation and mechani-
tingham) for the TEM measurements. cal properties for tissue regeneration. Advanced Functional Materials, 20,
3835–3845.
Mann, S. (1988). Molecular recognition in biomineralization. Nature, 332, 119–124.
Appendix A. Supplementary data Muzzarelli, R. A. A. (2009). Chitins and chitosans for the repair of wounded skin,
nerve, cartilage and bone. Carbohydrate Polymers, 76, 167–182.
Supplementary data associated with this article can be found, Muzzarelli, R. A. A. (2011). Chitosan composites with inorganics, morphogenetic
proteins and stem cells, for bone regeneration. Carbohydrate Polymers, 83,
in the online version, at http://dx.doi.org/10.1016/j.carbpol.2013.
1433–1445.
01.068. Ohkawa, K., Cha, D., Kim, H., Nishida, A., & Yamamoto, H. (2004). Electrospinning of
chitosan. Macromolecular Rapid Communications, 25(18), 1600–1605.
Oyane, A., Kim, H.-M., Furuya, T., Kokubo, T., Miyazaki, T., & Nakamura, T. (2002).
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