Carbohydrate Polymers 151 (2016) 313–320

Contents lists available at ScienceDirect

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

Boron nitride nanotubes enhance properties of chitosan-based

scaffolds
Melis Emanet, Emine Kazanç, Zehra Çobandede, Mustafa Çulha ∗
Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Ataşehir, Istanbul 34755, Turkey

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

Article history: With their low toxicity, high mechanical strength and chemical stability, boron nitride nanotubes
Received 1 March 2016 (BNNTs) are good candidates to enhance the properties of polymers, composites and scaffolds. Chitosan-
Received in revised form 13 May 2016 based scaffolds are exhaustively investigated in tissue engineering because of their biocompatibility and
Accepted 20 May 2016
antimicrobial activity. However, their spontaneous degradation prevents their use in a range of tissue
Available online 24 May 2016
engineering applications. In this study, hydroxylated BNNTs (BNNT-OH) were included into a chitosan
scaffold and tested for their mechanical strength, swelling behavior and biodegradability. The results
Keywords:
show that inclusion of BNNTs-OH into the chitosan scaffold increases the mechanical strength and pore
Chitosan
Scaffolds
size at values optimal for high cellular proliferation and adhesion. The chitosan/BNNT-OH scaffold was
Hydroxylated boron nitride nanotubes also found to be non-toxic to Human Dermal Fibroblast (HDF) cells due to its slow degradation rate. HDF
Mechanical strength cell proliferation and adhesion were increased as compared to the chitosan-only scaffold as observed by
Cell adhesion scanning electron microscopy (SEM) and fluorescent microscopy images.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction were embedded in collagen scaffolds, which resulted in increased

viability of smooth muscle cells by delaying gel compaction of the
A search to identify ideal scaffolds for tissue engineering is ongo- collagen scaffolds (MacDonald, Laurenzi, Viswanathan, Ajayan, &
ing. However, there are inadequacies in currently used scaffolds Stegemann, 2005). In addition to the structural reinforcement, the
including weak mechanical strength, uncontrollable degradation, CNTs have been utilized for tissue regeneration as a useful tool for
and inadequate biocompatibility. Therefore, novel scaffolds that cellular growth because of their electrical conductivity For exam-
overcome these drawbacks are sought. The use of nanomaterials ple, the CNTs showed enhanced osteoblast cell proliferation by
is one of the research efforts exploited to achieve this goal. increasing calcium production to 300% (Supronowicz et al., 2002). It
Nanotechnology concept has been increasingly employed in a is found that the CNTs are useful nanomaterials for tissue engineer-
variety of applications including medical and biomedical fields, and ing studies by helping to construct porous surfaces and promote
there are a large number of nanomaterials used to improve prop- cellular proliferation.
erties of scaffolds (Kim, Ahn, Dvir, & Kim, 2014). The main idea is to Boron nitride nanotubes (BNNTs), structural analogues of CNTs,
improve the properties of 3D matrix structure (Shenhar & Rotello, were first synthesized in 1995 (Chopra et al., 1995). The BNNTs were
2003). The role of nanomaterials in scaffold structure is to provide investigated for their potential use in drug delivery, hydrogen stor-
structural support as well as chemical cues to guide cell attach- age, neutron capture therapy, and biomaterial applications such
ment, proliferation and even influencing the cell differentiation. In as orthopaedic implants and composites (Ciofani, Danti, Genchi,
this context, CNTs were used in fabricating scaffolds to provide rein- Mazzolai, & Mattoli, 2013; Wang, Lee, & Yap, 2010). In addition
forcement and mechanical strength to achieve ideal 3D structure to their high mechanical strength and non-degradable structure,
(Harrison & Atala, 2007; Tonelli et al., 2012). For this reason, CNTs the biocompatible nature of the BNNTs made them useful nano-
have been blended with chitosan-based scaffolds resulting in sig- materials for biomedical applications (Emanet, Şen, Çobandede, &
nificant improvements in the mechanical strength of the scaffold Çulha, 2015; Salvetti et al., 2015). Thus, the BNNTs were also used in
structures. (Wang, Shen, Zhang, & Tong, 2005). In one study, CNTs designing scaffolds for tissue engineering and found that they were
promising materials to increase mechanical strength and reinforce-
ment of scaffolds due to their excellent elastic modulus of 1.22
TPa (similar to CNTs) (Li, Chen, & Stachurski, 2013). In our pre-
∗ Corresponding author. vious study, we also showed that addition of BNNTs or BNNT-OH
E-mail addresses: [email protected], [email protected] (M. Çulha).

http://dx.doi.org/10.1016/j.carbpol.2016.05.074
0144-8617/© 2016 Elsevier Ltd. All rights reserved.
314 M. Emanet et al. / Carbohydrate Polymers 151 (2016) 313–320

into thermally cross-linked gelatin-glucose scaffolds improved the tific, was purchased. BNNTs and HDF cells were obtained from
mechanical strength and cell proliferation (Sen & Culha, 2016).The Nanotechnology Center, Genetic and Bioengineering Department
molecular dynamic approach has shown the tensile strength of of Yeditepe University.
single-walled BNNTs to be about 24 GPa (Lahiri et al., 2010). Since
they are also very flexible, their reinforcement does not adversely 2.2. Design and fabrication
affect the ductility of the scaffolds (Lahiri et al., 2013). There are a
few studies on polymer–BNNT composites with non-biodegradable 2.2.1. BNNT synthesis
polymers like polyaniline (Zhi et al., 2005), polystyrene (Zhou BNNTs were synthesized from colemanite by chemical vapour
et al., 2012) and copolymer of vinylidene chloride and acrylonitride deposition as reported previously (Kalay, Yilmaz, & Culha, 2013). 2 g
(Ravichandran, Manoj, Liu, Manna, & Carroll, 2008) to obtain ideal colemanite and 160 mg Fe2 O3 was dispersed in 2 mL d.i. water at
tensile strength. The fabrication of scaffolds with the BNNTs may 100 ◦ C. The mixture then was put onto an alumina boat, which was
provide suitable molecular dynamics for the regeneration of tissues placed into a tubular furnace (Protherm, Furnaces PTF 14/50/450)
and organs (Lahiri et al., 2010). Previous studies have shown that after the water in the mixture was evaporated with pre-heating on
BNNTs used in composites increase mechanical strength and form a hotplate. The BNNT synthesis was achieved in a NH3 atmosphere
well-designed 3D structures for orthopaedic applications (Lahiri for 3 h at 1280 ◦ C. The white powder, that represents BNNTs at the
et al., 2011; Wang, Liao, Liu, Zhao, & Zhu, 2014). top of alumina boat, was finally collected.
Chitosan, a polysaccharide obtained by deacetylation of chitin
(poly-N-acetyl glucosamine), is a natural biopolymer used for fabri- 2.2.2. Modification of BNNTs with hydroxyl groups
cated scaffolds in tissue engineering. Chitosan is composed of (1–4) The hydroxylation of BNNTs was performed based on a method
linked 2-acetamido-2-deoxy-d-glucose and 2-amino-2-deoxy-d- previously reported (Huang et al., 2013). A 100 mg pure BNNTs and
glucose units. The amino groups in its structure have a pKa of 6.5, 10 mL 30% H2 O2 solution were mixed and sonicated (>20 kHz) to
thus it is soluble when the amino groups are protonated in acidic disperse BNNTs in H2 O2 at room temperature for 1 h. Then, the mix-
solutions (Dutta, Dutta, & Tripathi, 2004). Its pH-dependent solu- ture was refluxed during 48 h at 110 ◦ C while stirring. The obtained
bility provides a convenient mechanism for processing under mild hydroxylated BNNTs (BNNT-OH) were precipitated by centrifuga-
conditions. The enzymatic degradation and biocompatibility also tion (15 min, 10,000 rpm) and washed with deionized water five
makes chitosan a material of choice in tissue engineering studies. times and dried at 60 ◦ C. The BNNT and BNNT-OH were analysed
Chitosan-based scaffolds are used as wound covering biomaterials with fourier transform infrared spectroscopy (FT-IR) and visualized
to heal skin defects originating from burns (Dai, Tanaka, Huang, with transmission electron microscopy (TEM).
& Hamblin, 2011), skin ulcers (Hirose et al., 2007) and traumas
(Cho, Shi, & Borgens, 2010; Mohd Hilmi et al., 2013). In addition, 2.2.3. Preparation of BNNT-OH solution
chitosan has been shown to be useful in other tissue engineering 10 mg of BNNT-OH was dissolved in 10 mL of distilled water.
applications such as liver healing (Shilpa, Naijil, Nandhu, & Paulose, This solution was placed into a sonicator to disperse BNNT-OH for
2012) and cornea regeneration (Chen et al., 2005). Chitosan shows 2 h at room temperature.
antimicrobial effects against bacteria, viruses and fungi (Badawy &
Rabea, 2011). Furthermore, chitosan is an important material rou- 2.2.4. Chitosan/BNNT-OH and chitosan scaffold preparation
tinely used in gene (Koping-Hoggard et al., 2001) and drug delivery The chitosan scaffold was prepared by adding 2 g of chitosan
studies (Dini, Alexandridou, & Kiparissides, 2003). into 100 mL of distilled water, 10 mL of glacial acetic acid into this
Although chitosan has most of the desired properties from a solution and mixing this solution with a vortex briefly (Saravanan
scaffold such as biodegradability, biocompatibility and antimicro- et al., 2011). 1 mL of pure glutaraldehyde was added after the vor-
bial nature, some of its properties such as uncontrolled degradation, texing step. The solution was kept in the water bath at 75 ◦ C for
high swelling ratio and low mechanical strength need improve- 10 min. Finally, 2 mL of chitosan solution and 1 mL of d.i. H2 O were
ments. In this report, we continue to extend our effort to use BNNTs mixed and this suspension was used for the chitosan scaffold prepa-
in scaffolds for tissue engineering studies. The BNNTs were dis- ration. For the chitosan/BNNT-OH composite scaffold preparation,
persed in chitosan after hydroxylation of BNNTs (BNNT-OH) and 2 mL of chitosan and 1 mL BNNT-OH suspension in H2 O was used.
the scaffolds from chitosan and chitosan/BNNT-OH composite were This process was performed in a 24-well-culture plate. The pre-
prepared with freeze-drying technique. The prepared scaffolds pared scaffolds were placed in a freezer at −80 ◦ C for 4 h, and then
were comparatively evaluated for pore size, swelling, mechanical placed into a freeze dryer to remove water without destroying the
strength and in vitro bio-degradation. In addition, the biocompat- 3D structure of scaffold by sublimation under vacuum at −40 ◦ C for
ibility and tissue regeneration capacity were evaluated with cell two days.
culturing studies using human dermal fibroblast (HDF) cells to anal-
yse the cytotoxicity, cell attachment, proliferation and adhesion 2.3. Analysis and testing
capacity of cells on the scaffolds. The cell behaviours on the scaf-
folds were monitored with scanning electron microscopy (SEM) 2.3.1. Characterization of chitosan/BNNT-OH and chitosan
and fluorescent microscopy. scaffolds
The surface morphology of the chitosan and chitosan/BNNT-OH
scaffolds was characterized with a SEM (Carl Zeiss Evo 40). The
2. Materials and methods average pore size of each scaffold was determined by measuring a
minimum of randomly chosen 10 pores on the SEM images.
2.1. Materials
2.3.2. Swelling studies
Chitosan powder (low molecular weight, 75–85% deacetylated), The dried chitosan and composite of chitosan/BNNT-OH scaf-
acetic acid (100%), and Dulbecco’s modified eagle medium (DMEM) folds were weighed and the weight was recorded as Wo . The
were purchased from Sigma–Aldrich. Fetal bovine serum (FBS) scaffolds were immersed in 2 mL of PBS buffer solution for 24 h at
from GIBCO, glutaraldehyde (25%) from MERCK, lysozyme Fluka 37 ◦ C. After the incubation, the scaffolds were removed and washed
(70,000 U/mg), WST-1 colorimetric cell proliferation assay reagent with distilled water. Then, they were blotted by using filter paper
from Roche, PBS (phosphate buffered saline) from Thermo Scien- to remove the water that was entered into scaffold pores, were
 M. Emanet et al. / Carbohydrate Polymers 151 (2016) 313–320 315

Fig. 1. TEM images of BNNTs (a) and BNNT-OHs (b) and inlet images show their magnified size, comparison of FT-IR spectra (c), suspension of BNNTs and BNNT-OHs in water
(d). Yellow arrow shows the BNNT aggregates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

weighed and recorded as wet weight, Ww . The ratio of swelling number of cells was 5 × 103 in each well of the 96-well-plate). The
was calculated using the formula, 100 × (Ww − Wo )/Wo . following day, the cell culture medium was totally collected and
the cells incubated for 1 day in the scaffold degradation product-
2.3.3. Mechanical strength studies containing medium. Then, the cell viability was evaluated by the
The mechanical strength of the chitosan only and WST-1 colorimetric assay.
chitosan/BNNT-OH scaffolds were measured by compression
studies with an Instron 4502 mechanical tester (Instron Corpo- 2.3.6. Cell proliferation and adhesion studies
ration, Canton, MA). The scaffolds were prepared in 5 cm length In order to perform cell culture on the scaffolds, these were
and 10 mm diameter for mechanical strength measurements. firstly sterilized by incubation in 70% ethanol solution for 24 h.
The crosshead speed was set at 4.8 mm/min (initial strain rate After the sterilization, the ethanol was removed from the scaffold
of 0.01 s−1 ). Eight specimens were tested for each sample. The by incubation in PBS for two hours and the procedure was repeated
averages and standard deviations were calculated. three times. In the same time, HDF cells were prepared in DMEM
medium, which contains 10% FBS and 1% Penicillin and Strepta-
2.3.4. In vitro bio-degradation studies vidin. The sterilized scaffolds, placed in 24-well plates, were seeded
A solution containing 2 mL of DMEM and 1 mg of lysozyme with 1 mL medium containing 7 × 104 cells and incubated for 1, 3
enzymes for each scaffold was prepared and thereafter poured onto and 7 days to observe the cell proliferation and adhesion.
the scaffolds. Then, the scaffolds were incubated with these solu-
tions for 1, 3 and 7 days at 37 ◦ C. At the end of the incubation periods,
the scaffolds were washed with distilled water and dried. This step 2.3.7. Characterization of cell proliferation and adhesion studies
was repeated for each incubation time. The dry weights of scaffolds 2.3.7.1. SEM imaging. The cells on the scaffolds were prepared for
were recorded as Wt . The degradation of the scaffold was calculated the SEM imaging by fixating the cells on the scaffolds. The cells
with the formula Wo − Wt . were fixed in 2.5% glutaraldehyde for 30 min at 4 ◦ C and they were
washed in 50%, 70%, 90% and 100% ethanol respectively. Then, the
2.3.5. Cytotoxicity studies scaffolds were incubated at 37 ◦ C for 4 h as a drying step and visu-
In order to determine the cytotoxicity of degradation products, alized at SEM.
2 mL of DMEM was poured onto each scaffold, and incubated for
1, 3 and 7 days at 37 ◦ C. After the incubations, the media contain- 2.3.7.2. Fluorescent microscopy imaging. The cells were visualized
ing the scaffold degradation products were collected and applied to on the scaffolds with fluorescent microscopy using calcein and
HDF cells for the analysis of the cytotoxicity of the scaffolds. Mean- diamidino-2-phenylindole (DAPI) fluorescent dyes. The scaffolds
while, for preparing the cytotoxicity measurements, the HDF cells were incubated with 2 ␮L/mL calcein and 4 ␮L/mL DAPI contain-
were seeded in a 96-well plate and incubated for 1 day at 37 ◦ C (the ing PBS solutions for 15 min at 37 ◦ C. After the staining process, the
316 M. Emanet et al. / Carbohydrate Polymers 151 (2016) 313–320

Fig. 2. Upper SEM images show chitosan scaffold surfaces (a–c), lower SEM images show chitosan/BNNT-OH scaffold surfaces (d–f). Pore size histogram of the chitosan and
chitosan/BNNT-OH scaffolds (g), the average compressive modulus of chitosan and chitosan/BNNT-OH scaffolds before (h) and after (i) bio-degradation process.

scaffolds were washed with PBS three times thoroughly to remove safer approach including non-covalent coating with a biocompat-
residual calcein and DAPI. Finally, the fluorescent images of the cells ible molecule such as a protein, or chemical modification such as
on scaffolds were acquired at maximum absorption and emission OH groups can be pursued. In this study, the functionalization of
wavelengths for calcein (495–515 nm) and DAPI (355–460 nm). the BNNTs with OH was pursued to improve the dispersion of
Samples were examined with a Zeiss LSM 700 confocal laser scan- BNNTs in aqueous phase and polymer. Fig. 1a and b show in TEM
ning microscope. images of the BNNTs before and after hydroxylation process. As
seen, some of the BNNTs are damaged and perhaps opened to form
hexagonal boron nitrides (hBNs).
3. Results and discussion Fig. 1c shows the comparison of the FT-IR spectra of the BNNTs
before and after the hydroxylation process. A broad band in the
3.1. BNNT modification 3000–3600 cm−1 region indicates the presence of OH groups at
the BNNT defects and ends. Fig. 1d shows the picture of BNNTs
BNNTs have a high hydrophobic character and cannot be dis- and BNNT-OHs dispersed suspensions in water. Before the hydrox-
persed in aqueous media. Their dispersion can be achieved through ylation process, the BNNTs are not well dispersed in water. The
several approaches including treatment with a surfactant or chem- yellow arrow on Fig. 1d shows BNNT aggregates on the inner walls
ical functionalization (Ciofani, Raffa, Menciassi, & Dario, 2008). In of the quartz cuvette while a cloudy suspension is obtained after
medical and biomedical applications, the use of a surfactant is the hydroxylation process indicating improved dispersion.
not preferred since it may cause additional toxicity problems. A
 M. Emanet et al. / Carbohydrate Polymers 151 (2016) 313–320 317

Fig. 3. Swelling ratio (a) and bio-degradation (b) of chitosan and chitosan/BNNT-OH scaffolds. Cytotoxicity of biodegradation products of chitosan and chitosan/BNNT-OH
scaffolds (c). The swelling, degradation and cytotoxicity significance of groups were assessed with Student’s t-tests (*p < 0.05). Fluorescence microscopy images of DAPI
stained cells incubated 7 days on chitosan (d) and chitosan/BNNT-OH (e) scaffolds with the inset images, which show the cells incubated 1 day on scaffolds.

3.2. Effects of BNNT-OH on the structure of the chitosan scaffolds ture. Further, the BNNT-OH including scaffolds also show higher
strength after bio-degradation processes desired for tissue engi-
The scaffold porosity and pore size plays important roles for neering applications.
cellular adhesion and proliferation. Fig. 2 shows the SEM images Fig. 3a shows the swelling ratio of the two types of scaffolds.
of the chitosan (see Fig. 2a–c) and of the chitosan/BNNT-OH (see The swelling ratio was calculated as 100 and 60 for the chitosan
Fig. 2d–f) scaffolds at three different magnifications. Fig. 2g shows scaffold and chitosan/BNNT-OH scaffold, respectively. The smaller
the comparison pore sizes of both types of scaffolds. The aver- swelling ratio for the chitosan/BNNT-OH scaffolds can be associated
age pore sizes were determined from the randomly chosen pores with the cross-linking between the chitosan and BNNT-OH. The
on the SEM images of a scaffold. As seen, the pore sizes of the OH groups of BNNTs and free OH groups of chitosan caused the
chitosan/BNNT-OH scaffold were larger than the chitosan only. stronger incorporation of BNNT-OH into the scaffold. Since BNNT-
Although the BNNTs become hydrophilic with the introduction of OH is still relatively hydrophobic, their presence helps to exclude
hydroxyl groups into their structures, they are not truly hydrophilic the water molecules from the composite structure.
as they still show some hydrophobicity. When they are added into Lysozyme enzyme was used for in vitro bio-degradation stud-
chitosan, their hydrophobicity becomes less problematic since this ies, and the concentration of lysozyme in solution was adjusted
degree of hydrophobicity allows dispersion in fairly hydrophobic to the equal level to that used for lysozyme digestions as consis-
chitosan. The presence of relatively hydrophobic BNNT-OH in the tent with a cellular digestion (Braun & Rehn, 1969). Fig. 3b shows
scaffold structure causes water molecules to form larger droplets the comparative biodegradation of both types of scaffolds. As seen,
since water molecules try to avoid the hydrophobic structures. the degradation ratio of chitosan/BNNT-OH scaffold was approxi-
When these droplets sublimate, large pores are formed (Mallick mately stabilized at 4,6% up to 3 day incubation, then it increased to
& Cox, 2013). 5,2% up to 7 day incubation while the degradation ratio of the chi-
tosan scaffold irregularly increased to 6,2% during 7 day incubation.
From these results, it can be concluded that the degradation of the
3.3. Mechanical properties of the scaffolds chitosan/BNNT-OH scaffold is slower than that of the chitosan only
scaffold, and the biodegradation process increases with increas-
The effects of the BNNT-OH inclusion into chitosan scaffolds ing incubation time. In theory, scaffolds should be bio-degradable,
on mechanical strength and swelling rate were evaluated. The but this degradation should be slow and controlled. As seen, the
compressive mechanical strength of the samples was investi- use of BNNT-OH into scaffold provides a slower degradation rate,
gated before and after degradation by lysozyme exposure as which allows cells to tolerate toxic effect of degradation prod-
seen in Fig. 2h and i. In the mechanical strength experiments, ucts. It is known that decreased swelling and increased mechanical
chitosan/BNNT-OH scaffolds need a compressive stress of 55 kPa strength may positively affect the bio-degradation and toxicity
while the chitosan scaffolds need 35 kPa for obtaining a 70% because rapid biodegradation may cause toxic effect for cell prolif-
compressive deformation of scaffold structures. After the bio- eration and also prevent integration of the scaffold into the desired
degradation experiments, the BNNT-OH included scaffolds need cells and tissue.
35 kPa while the only chitosan scaffold needs 18 kPa. This clearly The cytotoxicity was tested on HDF cells using WST-1 cell via-
indicates that the BNNT-OH increased the strength of the chitosan bility assay. Fig. 3c shows the comparative evaluation of the cell
scaffold against mechanical forces due to the requirement for high viability using the biodegradation products of both chitosan and
mechanical stress (∼100 and 260 MPa) to destroy BNNT struc-
318 M. Emanet et al. / Carbohydrate Polymers 151 (2016) 313–320

Fig. 4. Fluorescence microscopy (a, b, e, f, i and j) and SEM images (c, d, g, h, k and l) of cells on the chitosan only and chitosan/BNNT-OH scaffolds.

chitosan/BNNT-OH scaffolds. As shown in Fig. 3c, the HDF cell via- improvement for the chitosan/BNNT-OH scaffold can be attributed
bility is decreased to 80% on chitosan only scaffold while the cell to the slower degradation and higher mechanical strength of the
viability increased to 115% on the chitosan/BNNT-OH scaffold. This chitosan/BNNT-OH scaffold.
 M. Emanet et al. / Carbohydrate Polymers 151 (2016) 313–320 319

3.4. Effects of BNNT-OH on cell proliferation and adhesion on Yeditepe University. The authors also would like to acknowledge
scaffolds Asst. Prof. Onur C. Namli for his help in mechanical strength exper-
iments.
In order to evaluate cell proliferation on the chitosan or
chitosan/BNNT-OH scaffolds, HDF cells were seeded on these struc-
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