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Toxicology Letters 189 (2009) 235–
Contents lists available at ScienceDirect
Toxicology Letters
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t o x l e t
BC nanofibres: In vitro study of genotoxicity and cell proliferation
Susana Moreira
a, 1
, Naisandra Bezerra Silva
b, 1
, Jailma Almeida-Lima
b
, Hugo Alexandre Oliveira Rocha
b
Silvia Regina Batistuzzo Medeiros c^ , Clodomiro Alves Jr. d^ , Francisco Miguel Gama a,∗
a (^) IBB, Institute for Biotechnology and Bioengineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal b (^) Departamento de Bioquímica, Universidade Federal do Rio Grande do Norte – UFRN, Brazil c (^) Departamento de Biologia Celular e Genética, Universidade Federal do Rio Grande do Norte – UFRN, Brazil d (^) Departamento de Física, Universidade Federal do Rio Grande do Norte – UFRN, Brazil
a r t i c l e i n f o
Article history: Received 2 April 2009 Received in revised form 31 May 2009 Accepted 4 June 2009 Available online 12 June 2009
Keywords: Nanofibres Nonotoxicology Bacterial cellulose Genotoxicity
a b s t r a c t
Nanomaterials have unusual properties not found in the bulk materials, which can be exploited in numer-
ous applications such as biosensing, electronics, scaffolds for tissue engineering, diagnostics and drug
delivery. However, research in the past few years has turned up a range of potential health hazards,
which has given birth to the new discipline of nanotoxicology. Bacterial cellulose (BC) is a promising
material for biomedical applications, namely due its biocompatibility. Although BC has been shown not
to be cytotoxic or genotoxic, the properties of isolated BC nanofibres (NFs) on cells and tissues has never
been analysed. Considering the toxicity associated to other fibre-shaped nanoparticles, it seems crucial
to evaluate the toxicity associated to the BC-NFs.
In this work, nanofibres were produced from bacterial cellulose by a combination of acid and ultrasonic
treatment. The genotoxicity of nanofibres from bacterial cellulose was analysed in vitro , using techniques
previously demonstrated to detect the genotoxicity of fibrous nanoparticles. The results from single cell
gel electrophoresis (also known as comet assay) and the Salmonella reversion assays showed that NFs are
not genotoxicity under the conditions tested. A proliferation assay using fibroblasts and CHO cells reveals
a slight reduction in the proliferation rate, although no modification in the cell morphology is observed.
© 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
The development of artificial materials with biomimetic
behaviour is essential for tissue engineering purposes. Scaffolds
based on nanofibres (NFs) mimic the natural extracellular matrix
and its nanoscale fibrous structure. Several approaches have been
described in order to achieve materials based on nanofibres from
synthetic or natural polymers (Ma et al., 2005; Ashammakhi et al.,
Bacterial cellulose (BC), secreted by Gluconacetobacter xylinus ,
has been presented as a biocompatible scaffold for the engineering
of cartilage and blood vessels, wound dressing, guided tissue regen-
eration, among other applications (Astley et al., 2003; Entcheva et
al., 2004; Svensson et al., 2005; Tabuchi and Baba, 2005; Czaja et al.,
2007; Teeri et al., 2007; Andrade et al., 2008; Backdahl et al., 2008;
Maneerung et al., 2008; Rambo et al., 2008). BC has unique char-
acteristics including high purity, high crystallinity and remarkable
mechanical properties, due to the uniform ultrafine-fibre network
structure, the high planar orientation of the ribbon-like fibres when
∗ (^) Corresponding author. Tel.: +351 253 604 400; fax: +351 253 678 986. E-mail address: [email protected] (F.M. Gama). (^1) These authors gave the same contribution for this work.
compressed into sheets, the good chemical stability, and the high
water holding capacity (Svensson et al., 2005). Several materials
based on bacterial cellulose, recognized as non-genotoxic and non-
cytotoxic, have been commercialized (Schmitt et al., 1991; Jonas
and Farah, 1998).
Since nanomaterials have unusual properties, not found in
the bulk material, such as high surface reactivity and ability to
cross-cell membranes, concerns about their safety and toxicology
emerged. The impact of nanostructural features in the interac-
tion of a material with cells and tissues is dependent on the size,
chemical composition, surface structure, solubility, shape, and on
the supramolecular structural organization (Barnes et al., 2008).
A major concern with fibres is their carcinogenic potential. There
is sufficient evidence that all forms of asbestos (generic term for
a group of six naturally occurring fibrous silicate minerals) are
carcinogenic and co-carcinogen to man (Speit, 2002; Dopp et al.,
2005). Moreover, recent studies described the toxicity of mate-
rials associated to size or shape; namely, the toxicity of carbon
nanotubes (Donaldson et al., 2006; Poland et al., 2008) and the
size-dependence toxicity of gold or ferric oxide nanoparticles was
reported (Pan et al., 2007; Backdahl et al., 2008; Wang et al., 2009).
The toxicity associated with inhaled fibres such as asbestos has
been described. Inhaled fibres may be toxic, particularly when they
are “long, thin and durable” (Donaldson et al., 2006). Asbestos
0378-4274/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2009.06.
fibres are dangerous because the fibres split lengthwise, produc-
ing thin fibres that can enter the lungs, being “moderately durable”
once there (Speit, 2002). Although cellulose fibres, from wood
pulp and textile fabric, are used without “significant concern”, cel-
lulose fibres share similar features with asbestos, including the
needle-like shape and biopersistence. Moreover, the inflammatory
responses of respirable cellulose fibres (wood pulp) using animal
models were already reported (Cullen et al., 2000). In light of these
results, it seems crucial to evaluate the toxicity of the BC nanofibres.
It must be remarked that, although BC cannot be enzymatically
degraded in the human body, the inflammatory processes may
actually degrade cellulose to some extent. Given the current focus of
BC as a promising biomaterial with a variety of applications, it is rel-
evant to evaluate not only the toxicity of BC membranes or scaffolds,
but also of its degradation products, including BC nanofibres.
Indeed, although in vivo studies demonstrate the BC biocom-
patibility (Helenius et al., 2006), and lack of mutagenicity (Schmitt
et al., 1991), no reports are available on the BC nanofibres toxicity.
Although BC is not expected to be degraded in vivo , safety concerns
makes this study mandatory. It is well accepted that in vitro studies
using cell systems are valuable tools to clarify the cellular mecha-
nisms involved in genototoxic effects, including DNA damage (Speit,
2002; Dusinská et al., 2004). Therefore, the aim of this study is to
evaluate the genotoxicity of cellulose nanofibres at cellular level
using the single cell gel electrophoresis and the Salmonella rever-
sion assays. The cell proliferation in the presence of nanofibres was
also evaluated. These tests are useful as a screening tool for set-
ting priorities because they are an inexpensive and a quick way to
help single out substances that should be targeted for further test-
ing. Furthermore, these assays were already used to demonstrate
the genotoxic effect of asbestos fibres in mammalian cells in vitro
(Speit, 2002; Dusinská et al., 2004).
2. Materials and methods
2.1. Bacterial strain, cells and culture medium
The cellulose was produced by G. xylinus (ATCC 53582), purchased from the American Type Culture Collection, grown statically in Hestrin and Schramm (1954) medium, pH 5 at 30 ◦^ C, 5 days. In the Salmonella reversion assay, four strains of Salmonella tryphimurium (Dr. B.N. Ames, Biochemistry Department, University of California, Berkeley, USA) were utilized, namely, TA97a [ his D6610, rfa , uvrB^ , bio −^ , pKM101 (ApR^ )], TA98 [ his D3052, rfa , uvrB^ , bio −^ , pKM101 (ApR^ )], TA100 [ his G46, rfa , uvrB^ , bio −^ , pKM101 (ApR^ )], and TA102 [ his D428, rfa , pKM101 (ApR^ ), pQA1 (TtR^ )] (Levin et al., 1982; Maron and Ames, 1983). The proliferation assays were performed using mouse embryo fibroblasts 3T (ATCC CCL-164), grown in Dulbecco’s modified Eagle’s media (DMEM) supplemented with 10% newborn calf serum (Invitrogen), and Chinese Hamster Ovary (CHO), grown in DMEM media supplemented with 10% fetal bovine serum (Invitrogen), both culture medium were supplemented with penicillin/streptomycin (1 g/ml) (Sigma–Aldrich, St. Louis, USA) and the incubation was at 37 ◦^ C, in a fully modified air containing 5% CO 2. The same conditions were used to grow CHO cells in Comet assay. The cell viability was assessed using the MTT (3-[4,5-dimethylthiazol-2-yl]- 2,5-diphenyl-tetrazolium bromide) assay, obtained from Invitrogen.
2.2. Production of BC nanofibres
The production of bacterial cellulose was performed by growing G. xylinus in Hestrin–Schramm medium, pH 5. After inoculation, the culture (100 ml) was incu- bated, first with agitation during 8 h, and then statically at 30 ◦^ C, for 5–7 days. BC pellicles were purified in a 4% NaOH solution at 70 ◦^ C, for 90 min. BC was then neu- tralised by thoroughly washing with water. Finally, BC pellicles were lyophilised prior to use. The nanofibres production, by acidic and/or ultrasonic treatment, was based on previous works (Roman and Winter, 2004; Zhao et al., 2007). The acid hydrolysis was performed as follows: 20 mg of dry BC was sliced in small pieces and 2 ml of H 2 SO 4 (50%, v/v) was added. The mixture was kept at 40 ◦^ C, for 2 h with vigorous stirring. To stop the hydrolysis, 10 ml of cold water was added and the cellulose was recovered by filtration, using a membrane with a 0.45 m pore size. Then, the cellulose was washed out with 20 ml of water and the recovered pellet was resuspended in 10 ml of water. This suspension was treated by sonication at 40 W (Branson Ultrasonic Disruptor, Sonifier II/W450) for 10 min (samples were maintained on ice during
sonication). Then, the NFs suspension was centrifuged (1 h, 15,000 rpm), and the pellet resuspended in water and sonicated again, in the same condition, for another 10 min. The yield of the process was evaluated by quantifying the total sugar in the samples, using the phenol-sulphuric method (Dubois et al., 1956).
2.3. TEM analysis
The NFs obtained were stained with uranyl acetate and analysed by transmission electronic microscopy (TEM, Zeiss 902A Orius SC 1000; 50 kV).
2.4. Evaluation of cellulose nanofibres mutagenicity by Salmonella reversion assay
Four S. tryphimurium strains were used to study the potential mutagenicity effect of the cellulosic NFs. The procedure was to some extent modified from the original description by Kado et al. (1986). This assay was performed in miscrosus- pension with or without S9 mixture (MoltoxTM^ , North Carolina, USA), using 0.1, 0. or 1.0 mg/ml of NFs suspension. The negative control (NC) was distilled water, and the positive controls (PC) employed were: 0.1 g/plate 4NQO (4-nitroquinoline 1- oxide) for the TA97a and TA98 strains; 5.0 g/plate sodium azide for the TA strain; and 0.5 g/plate mytomicyn C for the TA102 strain. Briefly, 105 l of a mixture containing the NFs suspension and cell suspension (10^9 cells/ml) were incubated at 37 ◦^ C for 90 min. Then, 2.5 ml of molten Top agar (0.6% bacto-agar and 0.5% NaCl) was added, before plating in a Petri dish containing minimal agar (1.5% agar, Vogel- Bonner E medium). The His+^ revertant colonies were counted after 72 h of incubation at 37 ◦^ C. All experiments were repeated at least three times with three replicas. The mutagenicity of cellulose NFs was evaluated according to the following parameters: the maximum number of revertants in the presence of the NFs should be 2-fold or more relative to the negative control; a dose-dependent increase in the number of revertants should be observed (Mortelmans and Zeiger, 2000).
2.5. Proliferation assays
The proliferation assays were performed in vitro as follows: 1 ml of the CHO or mouse embryo fibroblast 3T3 cell suspension (10^4 cells/ml) was seeded in a 24-well polystyrene plate (TPP, Switzerland). The cells were allowed to adhere for 4 h. Before the addition of cellulose NFs, the medium with non-adherent cells was removed and the NFs containing medium (to a final concentration of 1, 0.5 or 0.1 mg/ml) was added. A control without NFs was carried out. The cellular growth at 0, 24, 48 and 72 h of incubation was evaluated by MTT assay, a colorimetric test that gives a measure of the mitochondrial activity. The effect of NFs on the cell morphology was evaluated by microscopic observation using a Nikon Eclipse TE300 Inverted Microscope.
2.6. Evaluation of cellulose nanofibres genotoxicity by single cell gel assay (comet assay)
The DNA integrity was evaluated by alkaline single cell gel assay (also kwon as comet assay) using CHO cells grown in the presence of different NFs concentration. In this assay, 2 ml of CHO cell suspension (10^5 cells/ml) were seeded on a 6- well polystyrene plate (TPP, Switzerland). After 16 h, the medium was refreshed with medium containing the NFs (0.1, 0.5 or 1 mg/ml). Cells were incubated with NFs suspension during 48 h. Hydrogen peroxide (100 mM) and water were used as positive and negative controls, respectively. The alkaline comet assay was per- formed as described by Singh et al. (1988). Briefly, cells were trypsinized from 6-well polystyrene plate, and resuspended in 50 l of medium. The cell viability was deter- mined in a Neubauer counting chamber using the trypan blue exclusion test. A volume of 10 l of the cellular suspension were embedded in 0.5% low-melting- point agarose and plated on agarose-coated microscope slide. Then, the slides with cells were treated with lysis solution (2.5 M NaHO, 0.1 M EDTA, 0.010 M Tris, 1% Triton X-100, 10% DMSO, adjusted to pH 10) for 12 h at 4 ◦^ C, rinsed with distilled water, and placed in the electrophoresis buffer (0.3 M NaOH, pH 13 and 0.001 M EDTA), for 20 min to allow DNA unwinding. Following electrophoresis (30 min, at 25 V and 300 mA), the slides were neutralised with 0.4 M Tris buffer (pH 7.5) and stained with ethidium bromide (20 mg/ml). The slides were analysed through flu- orescence microscopy (Nikon Eclipse TE300 microscope equipped with a Nikon E600 camera, 0.488 m/pixel). At least 300 cells per condition tested were anal- ysed. The DNA damages were evaluated by image analysis using the “Comet Assay IV version 4.2” image analysis system. Data collected from each cell included tail length (TL), tail migration (TMi), percent tail DNA (TI), and tail moment (TM), which correspond the product of the comet length and the amount of DNA in the tail (Olive and Durand, 1992).
2.7. Statistic analysis
The one-way analysis of variance (ANOVA) was applied to statistics evaluation of the comet scores and to the proliferation assays results. The post-test Tukey–Kramer Multiple Comparisons test was used to compare the scores of the samples and positive control, the analysis were performed using GraphPad Prisma v 3.05.
Table 1 Results obtained in Salmonella reversion assay.
Revertant colonies/plate ± SD (without S9) Revertant colonies/plate ± SD (with S9)
TA97a a^ TA98a^ TA100a^ TA102a^ TA97a a^ TA98a^ TA100a^ TA102a
PC 540 ± 54 389 ± 17 1531 ± 183 1026 ± 36 191 ± 21 195 ± 76 485 ± 14 2356 ± 196 NC 143 ± 17 36 ± 6 228 ± 18 350 ± 27 93 ± 8 16 ± 4 82 ± 44 958 ± 20 0.1 124 ± 6 31 ± 6 235 ± 9 327 ± 12 93 ± 4 20 ± 1 133 ± 7 691 ± 61 0.5 132 ± 14 43 ± 2 220 ± 2 327 ± 13 91 ± 10 20 ± 1 112 ± 14 656 ± 35 1.0 147 ± 12 42 ± 4 225 ± 7 333 ± 18 108 ± 7 26 ± 4 112 ± 33 859 ± 109
PC: positive control: 0.1 g/plate of 4NQO to TA97a and TA98, 5.0 g/plate sodium azide to TA100 and 0.5 g/plate mytomicyn C to TA102; NC: negative control: H 2 O; SD: standard deviation. a (^) Strain.
3.3. In vitro proliferation assay
The cellular morphology and proliferation may be affected
by the presence of nanostructural patterns. Several studies anal-
ysed the proliferation of different cell lines on BC membranes,
confirming its non-toxicity and applicability as scaffold for cell
proliferation. However, depending on the cells used, the effect of
the biomaterial on the proliferation rate and the cell morphology
may be quite different (Sanchavanakit et al., 2006). Several stud-
ies showed that the cytotoxicity of a nanomaterial is many times
cell-specific (Cullen et al., 2002). Recently, De Nicola et al. (2007)
reported that, although carbon nanotubes do not present cyto-
toxic effect on human leukemic U937 cells, the proliferation rate is
deeply altered. Moreover, Bottini et al. (2006) showed that the same
nanotubes refereed above induce apoptosis in T lymphocytic cells,
suggesting that cytotoxicity may be cell-specific. In addition, it has
been reported that asbestos fibres inhibits the growth of CHO cells
(Speit, 2002), and yet the same fibres stimulate the proliferation of
different kinds of cells, in vitro , including fibroblasts (Bernstein et
al., 2005). Taking in consideration the evidence of contradictory,
cell-specific effects arising from the interaction cell-biomaterial,
the evaluation of the NFs effect on proliferative rate was performed
both with CHO cells and fibroblasts. In both cases, the proliferation
was about 15–20% lower in the presence of NFs, after 72 h of cell
culture, irrespective of the concentration used (Fig. 2). The lower
proliferation rate may stem from the insolubility of NFs and their
slow deposition on the polystyrene plate. It is known that cell pro-
liferation is dependent on characteristics of material surface, such
as it roughness. In addition, it was also described that cell prolif-
eration on BC membrane is slower than on the cell culture plate
Fig. 2. MTT results from proliferation assays using mouse embryonic fibroblast 3T3 and CHO (mean ± SD; ** P < 0.05; *** P < 0.005). Image obtained by optical microscopy of fibroblasts grown in the presence of cellulose NFs during 72 h. Scale bar = 20 m.
(Backdahl et al., 2006). However, the microscopic observations did
not reveal differences in the cellular morphology.
3.4. Evaluation of cellulose nanofibres genotoxicity by comet
assay
The genotoxicity of a material may be measured by analysing the
damages caused on DNA. The comet assay is based on the ability
of negatively charged loops/fragments of DNA to be drawn through
an agarose gel, in response to an electric field. The extent of DNA
migration depends directly on the DNA damage present in the cells
(Collins et al., 2008). The advantages of the comet assay, relative
to other genotoxicity tests, include its high sensitivity for detecting
low levels of both single and double stranded breaks in damaged
DNA, the requirement for small numbers of cells per sample, flex-
ibility, low cost, and ease of application (Collins et al., 1997, 2008).
Moreover, the comet assay is arguably one of the most widely used
tests for genotoxicity available, being already described as a repro-
ducible assay to evaluate nanoparticles genotoxicity (Collins et al.,
1997), and suggested as a diagnostic tool for clinical management
of cancer (Collins et al., 2008). The nanomaterial’s genotoxicity
may result from a direct interaction with DNA, or from an indirect
response caused by several factors, including surface stress through
direct particle influences on DNA, the release of toxic ions from sol-
uble nanoparticles, or generation of oxidative stress (Donaldson et
al., 2006). It has been proposed that (oxidative) DNA damage plus
structural and numerical chromosome aberrations are the most
sensitive genetic endpoints for detection of asbestos-induced geno-
toxicity detectable by in vitro assay. The comet assay has indeed
proven to be a sensitive test to detect genotoxic effect of asbestos
fibres in mammalian cell in vitro (Speit, 2002; Dusinská et al., 2004).
Therefore, cells grown in NFs-containing medium were analysed by
the comet assay, in order to evaluate their genotoxicity. Cells grown
on bacterial cellulose membrane were also tested as a control.
The DNA damages were evaluated by visual scoring and image
analysis. Fig. 3 shows a representative image obtained for each NFs
Fig. 3. Fluorescent microscopy images of ethidium bromide stained DNA and results from visual scoring in the comet assay. PC: positive control (H 2 O 2 ); NC: negative control (H 2 O); 0.1–1.0 NFs concentration in mg/ml. The images were scored and classified into five classes and given a value according to tail intensity, from 0 (no tail) to 4 (almost all DNA in the tail). Scale bar = 50 m.
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