Accepted Manuscript Title: Regenerated cellulose scaffolds: Preparation, characterization and toxicological evaluation Author: Adalberto M. de Ara´ujo J´unior Guilherme Braido Sybele Saska Hernane S. Barud Leonardo P. Franchi Rosana M.N. Assunc¸a˜ o Raquel M. Scarel-Caminaga Ticiana S.O. Capote Youn`es Messaddeq Sidney J.L. Ribeiro PII: DOI: Reference:
S0144-8617(15)00931-5 http://dx.doi.org/doi:10.1016/j.carbpol.2015.09.066 CARP 10368
To appear in: Received date: Revised date: Accepted date:
21-5-2015 26-8-2015 21-9-2015
Please cite this article as: J´unior, A. M. A., Braido, G., Saska, S., Barud, H. S., Franchi, L. P., Assunc¸a˜ o, R. M. N., Scarel-Caminaga, R. M., Capote, T. S. O., Messaddeq, Y., and Ribeiro, S. J. L.,Regenerated cellulose scaffolds: preparation, characterization and toxicological evaluation , Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.09.066 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Regenerated cellulose scaffolds: preparation, characterization and toxicological evaluation
2 Adalberto M. de Araújo Júniora*, Guilherme Braidob*, Sybele Saskaa, Hernane S. Baruda,c, Leonardo P.
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Franchid, Rosana M. N. Assunçãoe, Raquel M. Scarel-Caminagab, Ticiana S.O. Capoteb, Younès
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Messaddeqa, Sidney J. L. Ribeiroa**
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Institute of Chemistry, São Paulo State University, UNESP, PO Box 355, Araraquara, SP, 14800-970 Brazil. Emails:
[email protected] (A. M. de Araújo Júnior);
[email protected] (S. Saska);
[email protected] (Y. Messaddeq);
[email protected] (S. J. L. Ribeiro). b Dental School at Araraquara, São Paulo State University, UNESP, Rua Humaitá, 1680, Zip Code 14803-901, Araraquara, SP, Brazil. E-mails:
[email protected] (G. Braido);
[email protected] (R. M. Scarel-Caminaga);
[email protected] (T. S. O. Capote). c Laboratório de Química Medicinal e Medinica Regenerativa (QUIMMERA), Centro Universitário de Araraquara (UNIARA), Araraquara, SP, Brazil. E-mail:
[email protected] (H. S. Barud). d Ribeirão Preto Medical School, University of São Paulo, USP, Av. Bandeirantes, 3900, Ribeirão Preto, SP, Brazil. E-mail:
[email protected] (L. P. Franchi). e Faculdade de Ciências Integradas do Pontal, Universidade Federal de Uberlândia, Campus do Pontal, Rua Vinte, 1600, Zip Code 38304-402, Ituiutaba, MG, Brazil. E-mail:
[email protected] (R. M. N. Assunção).
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These two authors contributed equally to this paper.
**
Corresponding author: Sidney J. L. Ribeiro, e-mail:
[email protected], telephone number +55 16 3301-9631.
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Abstract
41 Regenerated cellulose scaffolds (RCS) may be used as alloplastic materials for tissue repair. In
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this work, the RCS were obtained by viscose process and characterized by Scanning Electron Microscopy
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(SEM), Wide Angle X-ray Diffraction (WAXD), Fourier Transform Infrared Spectroscopy (FTIR) and
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Thermogravimetry Analysis (TG). In vitro enzymatic degradation assay and toxicological assays were
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also evaluated. The physicochemical characterizations revealed the formation of a porous material with
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distinct thermal profile and crystallinity compared to pristine cellulose pulp. Enzymatic degradation assay
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revealed that lysozyme showed a mildest catalytic action when compared to cellulase, Tricoderma reesei
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(Tr). Nevertheless, both enzymes were efficient for degrading the RCS. RCS did not show cytotoxicity,
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mutagenic or genotoxic effects. The systematically characterization of this work suggests that RCS
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presented distinct features that make it a viable material for future studies related to the development of
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scaffolds for biological applications.
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Keywords: regenerated cellulose, scaffold, tissue repair, cytotoxicity, genotoxicity, mutagenicity.
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Introduction
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Cellulose, the most abundant natural polymer on Earth (Klemm, Heublein, Fink, & Bohn, 2005),
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is a linear homopolysaccharide that represents the largest component of plant biomass. It consists of units
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of β-D-glucopyranose (β-glucose) linked by glycosidic type β-(1→4). Industrially cellulose is widely used in paper production as emulsifying, dispersing agent, gelling
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agent, among other functions, with interesting features such as biodegradability, biocompatibility, no
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toxicity and no allergenicity (Czaja, Krystynowicz, Bielecki, & Brown, 2006). The physicochemical
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properties of cellulose have attracted great interest in the production of new materials in various areas,
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such as electronics (LCD screens), energy (fuel cell membranes), communications (diaphragms for
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microphones and stereo headphones) and medicine (temporary artificial skin for burns and ulcers). In
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recent years, there is an increase in interest for using cellulose for medical applications, especially in
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exploring new porous biomaterials, which might be used for tissue engineering (Hench & Polak, 2002).
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An ideal biomaterial must provide variety of shapes and sizes, and also be tough enough to be
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used in locations where there is impact load. Moreover, it must also be biocompatible, absorbable and
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replaced by new tissue formation, in case of application in tissue engineering (Spector, 2008,
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Karageorgiou & Kaplan, 2005). Currently, natural polymers are important alternatives in obtaining
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scaffolds for tissue repair (Liu & Ma, 2004).
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Concerning regenerated cellulose, it may be obtained by the well known viscose rayon process. In
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short, cellulose pulp is treated with sodium hydroxide and carbon disulfide solutions, leading to cellulose
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xanthate. Regeneration of cellulose occurs by acid hydrolysis of xanthates groups using sulfuric acid
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solution. In this step, the xanthate groups are released from the main chain of cellulose and the cellulose is
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regenerated (Cross, Bevan, & Beadle, 1893a,b).
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The applicability of regenerated cellulose in tissue repair is being evaluated. Martson, Viljanto,
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Hurme, & Saukko (1998) evaluated the biocompatibility of scaffolds based on regenerated cellulose for
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bone repair in rats. They showed that regenerated cellulose was a compatible and osteoconductive matrix,
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promoting new bone formation. Cullen et al. (2002) described a new wound treatment using oxidized
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regenerated cellulose (ORC) and collagen (ORC/collagen). The ORC/collagen composite was able to
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inactivate potentially harmful factors (proteases, oxygen free radicals and excess metal ions) presented in
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chronic wound fluid, and therefore promote healing. Additionally, the effectiveness of pharyngeal mucosal
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healing using ORC was also reported (Liu et al. 2012). Nevertheless, further studies are necessary to
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establish a comparison among different biomaterials and to evaluate the application of regenerated
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cellulose scaffolds in clinical practice. Considering that the human body does not express naturally the
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cellulase enzymes able to degrade cellulose (Hu & Catchmark, 2011a) and in order to obtain in vivo 3
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resorption of the cellulos, this study evaluated the in vitro enzymatic degradation for regenerated cellulose
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scaffolds using two different enzymes, Trichomona reesei cellulase and lysozyme. Once a material undergoes chemical modification, it is necessary to investigate whether these
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chemical changes lead to cytotoxic effects. For this reason, in this study the modified cellulose was
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investigated in respect to their cytotoxic, genotoxic and mutagenic effects. These studies are important to
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evaluate the behavior of the regenerated cellulose for future in vivo applications.
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Therefore, this study aimed to prepare regenerated cellulose scaffold, systematically characterize
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it, and evaluate its enzymatic degradation as well. In addition, the potential cytotoxic, genotoxic and
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mutagenic effects of these materials were evaluated.
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2.1 Preparation of regenerated cellulose scaffolds
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Pristine cellulose pulp (CP) and pristine regenerated cellulose scaffold (RCS), prepared by the
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viscose process, were supplied by Coopercell Ind. de Papel Celofane (São Paulo, Brazil). Cellulose was
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firstly immersed in 18% (w/v) NaOH solution at room temperature; subsequently the alkali cellulose was
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reacted with CS2 solution at 30 °C for 90 min. leading to cellulose xanthate. H2SO4 solution was added
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regenerating cellulose. The formation of regenerated cellulose scaffold with controlled pore size was
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possible using porogenic agent, Na2SO4, with different grain sizes around 100 – 200 μm.
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2.2. SEM characterization of the samples
Scanning electron microscopy images were obtained from a TOPCON 600. Samples were coated with a 20 nm thick gold layer.
2.3. WAXD characterization
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WAXD patterns were obtained in a Siemens Kristalloflex diffractometer using nickel filtered Cu
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Kα radiation, step pass of 0.02° and a step time of 3 s, from 4 to 70° (2θ angle). The separation of the
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crystalline peaks and the amorphous halo was performed by deconvolution using a Pseudo-Voigt function.
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WL and WG are widths at half height for the Gaussian and the Lorentz components of the equation, A is
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the area and mu is the sum factor.
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(1) 4
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The crystallinity index (CrI) can be calculated from Equation 2. Where Ac and Aa are areas of the crystalline peaks and amorphous halo, respectively.
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2.4. FTIR characterization
FTIR spectra were obtained with dried powdered samples on a PerkinElmer Spectrum 1000
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Fourier Transform Infrared Spectrophotometer. Pellets were prepared from mixtures of the samples and
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KBr (1:100 in weight). Twenty-eight scans were accumulated at a resolution of 4 cm−1.
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2.5. Thermal behavior
TG curves were obtained with a SDT equipment from TA Instruments. The conditions used in the
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experiments were the following: nitrogen atmosphere at a flow rate of 50 mL min−1, heating rate of 10 °C
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min−1 from 10° to 600 °C and alumina pans.
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2.6. Enzymatic degradation
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In vitro enzymatic degradation was evaluated using two enzymes, Trichoderma reesei cellulase
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(Sigma®) and Lysozyme (Sigma®). Two different concentrations of these enzymes (0.5 U mL-1 and 1 U
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mL-1) were used and solutions were prepared in Na2HPO4-Citric acid buffer solution (pH 5.0), containing
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0.1 mol L-1 citric acid and 0.2 mol L-1 sodium hydrogen phosphate in a ratio 1:1. The lyophilized RCS
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samples were cut 1 cm3 in size, and 1 mL of each respective enzymatic solution was dripped over the
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samples; then, these samples were lyophilized for 24 h. Degradation of the samples was performed in
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simulated body fluid (SBF) solution (Kokubo, Kushitani, Sakka, Kitsugi, & Yamamuro, 1990), pH 7.2 at
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37 °C for 56 days. Samples were placed into 24-wells plate for cell culture (Corning®) and 2.5 mL of SBF
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solution was added to each well. Control samples, without enzyme, were also prepared for comparison.
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The degree of degradation was evaluated by weighing method using semi-analytical balance at 3, 7, 14,
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21, 28, 42 and 56 days. After each analysis period, the samples were removed from the SBF solution and
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lyophilized for 48 h. The lyophilized samples were weighed to calculate the percentage of mass loss. The
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experiment was performed in triplicate.
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2.7 Cell culture experiments
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Eluates of RCS were made according to ISO 10993-12:2008, considering the weight (0.2 g mL).
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The RCS were immersed in 1:1 Ham-F10 + D-MEM medium (Sigma®, St. Louis, MO) without fetal 5
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bovine serum (FBS) at 37 ºC for 72 h, shaking at 133 rpm in an incubator (New Brunswick Scientific –
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Excella E24 Incubator Shaker Series). Four different concentrations were used: RCS100% (100% eluate),
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RCS75% (75% eluate + 25% 1:1 Ham-F10 + D-MEM medium), RCS 50% (50% eluate + 50% 1:1 Ham-
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F10 + D-MEM medium) and RCS25% (25% eluate + 75% 1:1 Ham-F10 + D-MEM medium).
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CHO-K1 cells were cultured in 1:1 Ham-F10 + D-MEM medium (Sigma®, St. Louis, MO)
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supplemented with 10% FBS (Cultilab, Campinas, Brazil) and antibiotics [(0.06 g L-1 penicillin (Sigma®),
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0.10 g L-1 streptomycin (Sigma®), 1% kanamycin (Gibco, Carlsbad, CA) and 1% ciprofloxacin
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(Hifloxan®, Halexistar)] in 25 cm2 culture flasks at 37 °C, 5% CO2. Cells were used between the third and
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eighth passages.
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2.7.2 Cytotoxicity tests
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2.7.2.1 XTT assay
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After 24 h of seeding, CHO-K1 cells (2×104 cells seeded) were treated with RCS100%, RCS75%,
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RCS50% and RCS25% eluates (in duplicate) for 24 h in 24-well plates. Each well containing eluate was
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supplemented with 10% FBS. Negative controls (NC) were wells with culture medium supplemented with
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10% FBS in the absence of any eluate (untreated controls), while positive controls (PC), well containing
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CHO-K1 cells, were treated with doxorubicin (3 μg mL-1) for 24 h (all treatments were carried out in
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duplicate). After treatment, the cultures were washed with PBS solution and fresh medium was added.
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After 24 h of incubation, the cultures were washed with PBS solution and immediately 500 µL of DMEM
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without phenol red were added, followed by the addition of 60 µL of the XTT/electron solution (50:1)
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(Cell Proliferation Kit II - Roche Applied Science). After 3 h reaction, the supernatant was transferred to a
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96-well
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(VersaMax, Molecular Devices, Sunnyvale, CA) at 492 and 690 nm. The absorbance is directly
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proportional to the number of viable cells in each treatment after 24 h of exposure. Three independent
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experiments were conducted.
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2.7.2.2 Clonogenic assay
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After 24 h of seeding, CHO-K1 cells (5×104 cells seeded) were exposed for 24 h to RCS100%,
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RCS75%, RCS50% and RCS25% eluates (in duplicate) for 24 h in 24-well plates. Each eluate well was
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supplemented with 10% FBS. Negative controls (NC) were wells with culture medium supplemented with
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10% FBS in the absence of any eluate, while positive controls (PC), well containing CHO-K1 cells, were
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treated with hydrogen peroxide (80 µmol L-1 for 10 min) (all treatments were carried out in duplicate).
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After exposure, the cultures were washed with PBS solution and fresh medium was added. Exponentially
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growing cells were seeded after treatment at a number of 150 cells per 25 cm2 flasks, in duplicate for each
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treatment. The flasks were incubated at 37 °C, 5% CO2, for 7 days without medium exchange. The
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colonies that formed were fixed with methanol:acetic acid:water (1:1:8 v/v/v) and stained with 5%
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Giemsa. The colonies were counted, and the cell surviving fraction was calculated as percent colonies in
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treated flasks relative to untreated controls (NC). Three independent experiments were conducted.
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2.7.3 Genotoxicity and Mutagenicity assays
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2.7.3.1 Comet assay
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The alkaline version of the comet assay was used according to the method described by Singh,
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McCoy, Tice, & Schneider (1988). CHO-K1 cells were seeded (5×104 cells seeded) and after 24 h they
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were exposed to RCS100%, RCS75%, RCS50% and RCS25% eluates (in duplicate) for 24 h (in duplicate)
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in 24-well plates. Each eluate well was supplemented with 10% FBS. Negative controls (NC) were wells
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with culture medium supplemented with 10% FBS in the absence of any eluate, while positive controls
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(PC) were treated with hydrogen peroxide (80 µmol L-1 for 10 min) (all treatments were carried out in
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duplicate). After exposure, the cultures were washed with PBS solution and harvested with trypsin. Five
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hundred microliters of cells in suspension were obtained, kept on ice and protected from light. After
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centrifugation, the pellet was re-suspended in 200 μL of 0.5% (w/v) low melting point agarose and the
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mixture was spread onto two microscope slides (Knittel, Germany) pre-coated with 1.5% (w/v) normal
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melting point agarose (Gibco). Coverslips were placed over the gel. When the gels had solidified, the
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coverslips were gently removed and the slides were immersed in cold (4 °C) lysis solution (1% Triton X-
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100, 10% DMSO, 2.5 mmol L-1 NaCl, 100 mmol L-1 Na2EDTA, 100 mmol L-1 Tris, pH 10) for 24 h.
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Immediately after this step, slides were placed in a horizontal electrophoresis unit containing freshly
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prepared electrophoresis buffer (1 mmol L-1 Na2EDTA, 300 mmol L-1 NaOH, pH > 13). The DNA was
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allowed to unwind for 20 min and subsequently electrophoresis was performed at 25 V, 300 mA for 20
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min. Afterward, the slides were gently immersed in neutralization buffer (0.4 mol L-1 Tris–HCl, pH 7.5)
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for 15 min and then fixed with ethanol. All the steps of comet assay were conducted under subdued light.
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Three independent experiments were conducted.
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DNA damage was determined in a blind test in 100 nucleoids per slide. Duplicate slides were
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prepared, stained with ethidium bromide, and screened with a fluorescent microscope (ZEISS®, Jena,
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Thuringia, DEU) equipped with an excitation filter of 515–560 nm, a barrier filter of 590 nm and a 40×
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objective. The level of DNA damage was assessed by an image analysis system (TriTek CometScore®
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1.5, 2006, Sumerduck, VA, USA), and the DNA percentage in the tail and Tail Moment were obtained for 7
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each treatment. A visual score was also used to compute DNA damage from comets. Five classes were
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used, from 0 (no tail) to 4 (almost all DNA in tail) (Azqueta et al. 2011; Collins, 2004). The DNA Damage
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Index (DDI) was calculated using the following formula (Teixeira et al. 2009):
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DDI = 0(n level 0) + 1(n level 1) + 2(n level 2) + 3(n level 3) + 4(n level 4) Total of comets analyzed
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2.7.3.2 Cytokinesis-blocked micronucleus (CBMN) assay for mutagenicity evaluation
CBMN assay was performed according to Fenech (2000) with minor modifications. CHO-K1 cells
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(37×104 cells/culture flask) were seeded in 25 cm2 culture flasks at 37 °C, 5% CO2. After 24 h of seeding,
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cells were exposed for 24 h to RCS100%, RCS75%, RCS50% and RCS25% eluates (in duplicate). Each
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eluate culture flask was supplemented with 10% FBS. Negative controls (NC) were culture flasks with
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culture medium supplemented with 10% FBS in the absence of any eluate (untreated controls), while
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positive controls (PC) were treated with doxorubicin (0.15 μg mL-1) for 4 h. Cytochalasin-B (CytB) was
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added to the CHO-K1 cultures at a final concentration of 5 μg mL-1 and left for 20 h. After treatments, the
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cultures were washed with PBS solution, trypsinized and centrifuged for 5 min at 1500 rpm. The pellet
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was then resuspended in cold hypotonic solution (0.3% KCl w/v) for 3 min. Cells were fixed twice with
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methanol:glacial
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homogenized carefully with a Pasteur pipette. The cell suspensions were dripped on a slide with a film of
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distilled water at 4 °C. The slides were stained with 5% Giemsa solution diluted in phosphate buffer
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(Na2HPO4 0.06 mol L-1, KH2PO4 0.06 mol L-1 - pH 6.8) for 7 min, washed with distilled water, air dried
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and examined by light microscopy (400× magnification). Three independent experiments were conducted.
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Five hundred (500) viable cells were scored to determine the frequency of cells with 1, 2, 3 or 4
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nuclei and calculated the nuclear division index (NDI) using the formula: [NDI = M1 + 2(M2) + 3(M3) +
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4 (M4)/N], where M1–M4 represents the number of cells with 1 to 4 nuclei, respectively, and N is the
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total number of viable cells scored (Fenech, 2000; Eastmond & Tucker, 1989). The frequency of
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binucleated cells with micronuclei (MNBCF) and the total frequency of micronuclei (MF) were scored in
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1000 binucleated cells. The criteria used for identifying micronuclei were based on Fenech (2000).
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2.7.4 Statistical analysis
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The results were expressed as mean and standard error. One-way analysis of variance (ANOVA)
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followed by Tukey’s test were applied to the data. Data from treated groups were compared to the
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negative control using Dunnett’s test. BioEstat statistical package v.5 was used (UFPA, Belém, Brazil) to
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perform the tests. Differences were considered statistically significant when p0.05; Dunnett’s test).
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Clonogenic assays, or colony formation assays, measure clonogenic potential, i.e., the
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proliferative ability of single cells to form a colony (Yalkinoglu, Schlehofer, & zur Hausen, 1990).
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Besides colony formation, this assay measures cell survival and is routinely used as a sensitive model for
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assessing long-term cytotoxicity.
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The cell surviving fraction results obtained by clonogenic survival assay show that RCS100%,
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RCS75% and RCS50% exhibit surviving fraction statistically similar to NC (p>0.05). RCS25% presented
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a significant increase of the proliferative capacity of the CHO-K1 cells compared to NC (p0.05, Dunnett’s test).
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Numeric results are shown in supplementary material.
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Observing the results of the regenerated cellulose scaffolds in different assays, it was verified that
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the RCS eluates did not show cytotoxic, mutagenic and genotoxic potential when in contact with CHO-K1
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cells for 24 hours.
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In literature, studies focusing toxicity of regenerated cellulose are very scarce. Song et al. (2014)
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investigated cellulose film regenerated from Styela clava tunics (SCT-CF), which were implanted in
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Sprague–Dawley rats for various lengths of time. The authors did not find differences in the number of
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white blood cells or metabolic enzymes representing liver and kidney toxicity in the serum of SCT-CF
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implanted rats in comparison with vehicle implanted rats. Moreover, no significant alteration of the
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epidermal hyperplasia, inflammatory cell infiltration, redness, and edema were observed in rats implanted
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with SCT-CF (Song et al., 2014). No in vivo toxicity test was performed in the present study but from the
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in-vitro test it can be said that similar non-toxicity results were observed.
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4. Conclusions
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Regenerated cellulose scaffolds were prepared by viscose process. The samples exhibited
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structure compatible with the cellulose II, low crystallinity and a lower thermal stability. These
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requirements are important to ensure biodegradability of the scaffold. RCS also showed a slower in vitro
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degradation profile for samples containing lysozyme compared to samples containing Tr cellulase. In
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addition, RCS did not show cytotoxicity, mutagenic and genotoxic potential. Thus, we can conclude that
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the regenerated cellulose scaffold is a viable biomaterial with promising features, which allows us to 15
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follow up the investigations regarding the development of scaffolds for medical and biological
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applications. Future in vivo studies will be conducted in order to confirm the potential of regenerated
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cellulose for such applicability.
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Acknowledgements
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The authors thank Brazilian agencies FAPESP, CAPES and CNPq for financial support. In addition, the
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company Coopercell Ind. de Papel Celofane (São Paulo, Brazil) for supplying the pristine regenerated
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cellulose and the LMA-IQ for FEG-SEM facilities.
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Regenerated cellulose scaffolds: preparation, characterization and toxicological evaluation
549 Adalberto M. de Araújo Júniora*, Guilherme Braidob*, Sybele Saskaa, Hernane S. Baruda,c,
551
Leonardo P. Franchid, Rosana M. N. Assunçãoe, Raquel M. Scarel-Caminagab, Ticiana S.O.
552
Capoteb, Younès Messaddeqa, Sidney J. L. Ribeiroa**
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550
553 554
a
555
14800-970 Brazil. E-mails:
[email protected] (A. M. de Araújo Júnior);
556
[email protected] (S. Saska);
[email protected] (Y. Messaddeq);
557
[email protected] (S. J. L. Ribeiro).
558
b
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Code 14803-901, Araraquara, SP, Brazil. E-mails:
[email protected] (G.
560
Braido);
[email protected] (R. M. Scarel-Caminaga);
[email protected] (T. S. O.
561
Capote).
562
c
563
Universitário
564
[email protected] (H. S. Barud).
565
d
566
Preto, SP, Brazil. E-mail:
[email protected] (L. P. Franchi).
567
e
568
Pontal,
569
[email protected] (R. M. N. Assunção).
us
cr
Institute of Chemistry, São Paulo State University, UNESP, PO Box 355, Araraquara, SP,
M
an
Dental School at Araraquara, São Paulo State University, UNESP, Rua Humaitá, 1680, Zip
Laboratório de Química Medicinal e Medinica Regenerativa (QUIMMERA), Centro Araraquara
(UNIARA),
Araraquara,
SP,
Brazil.
E-mail:
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d
de
Ribeirão Preto Medical School, University of São Paulo, USP, Av. Bandeirantes, 3900, Ribeirão
Faculdade de Ciências Integradas do Pontal, Universidade Federal de Uberlândia, Campus do
570 571
Rua
572
*
573
**
574
16 3301-9631.
Vinte,
1600,
Zip
Code
38304-402,
Ituiutaba,
MG,
Brazil.
E-mail:
These two authors contributed equally to this paper. Corresponding author: Sidney J. L. Ribeiro, e-mail:
[email protected], telephone number +55
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578 579 580 581
SUPLEMENTARY MATERIAL
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582 FTIR spectra obtained for CP and RCP are observed in Fig. SM-1. The profile of the
584
presented spectra is typical of cellulosic materials. Moreover, the main observed bands are those
585
at 3430 cm-1, assigned to the stretching of the bond O-H; 2900 cm-1, assigned to stretch group C –
586
H; 1645 cm-1, attributed to the angular deformation water molecules that are absorbed in the
587
structure of pulp; 1169 cm-1, attributed to asymmetric stretching of the connection (C1 - O - C5)
588
and 898 cm-1, assigned to a stretching of the glycosidic bond (C1 - O - C4) (Colom, Carrilho,
589
Nogués, & Garriga, 2003).
an
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cr
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The main bands related to structural changes in cellulose appear in the region between
591
1700-600 cm-1, as shown in Fig. SM-1b. For CP, a band observed at 1430 cm-1 is assigned to
592
angular deformation of CH2 group. For RCP this band shifted to lower wavelength, 1416 cm-1
593
indicating a modification of the hydrogen bond pattern to a new pattern formation in the cellulose
594
II structure. Moreover, the region between 1200-1300 cm-1 showed a set of bands for RCP as a
595
result of the modification suffered during the mercerization process (Colom et al., 2003).
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590
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Bands at 1430 cm-1, 895 cm-1 and those between 1372 cm-1 (attributed to deformation of
597
the C – H) and 2900 cm-1 are directly proportional to the crystallinity degree of the samples
598
(Uesu, Pineda, & Hechenleitner, 2000). The ratio between the absorbance of bands 1372 and
599
2900 cm-1 (A1372/A2900) (Ciolacu, Ciolacu, & Popa, 2011) was applied in order to assess the
600
degree of order of samples, being equal to 1.21 and 0.90 for CP and RCP respectively. This
601
present result is in accordance to the patterns observed for the X - ray, where the RCP had lower
602
degree of crystallization than CP.
603
Bands at 1430, 1162, 1111 and 893 cm-1 can also be used to study the different samples
604
(Nelson & O’connor, 1964a, b). The band at 1420 cm-1 observed for RCP, is characteristic of
605
cellulose II and amorphous cellulose. If the content of cellulose I is quite significant the band can
606
be shifted to 1430 cm-1. An increase in bandwidth could represent a mixture of cellulose I and II
607
structures. Furthermore, the band clearly defining cellulose II is the one appearing at 893 cm-1. It
20
Page 20 of 28
is not observed for cellulose I. Fig. SM-1b leads to the conclusion that no cellulose I is present
609
(Nelson & O’connor, 1964a, b).
610 611
Fig. SM-1. Fourier Transform Infrared Spectroscopy of CP and RCP in the range (a) 4000-500
612
cm-1 and range (b) 1700 and 600 cm-1.
613
Ac ce pt e
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608
614
Fig. SM-2 shows SEM images obtained for samples submitted to the enzymatic
615
degradation (samples RCS-control, RCS 0.5 U lysozyme and RCS 0.5 U Tr) in the period of 7
616
days. Samples containing enzymes exhibit more porous surface morphology compared to the
617
control sample, whose sample demonstrated a greater integrity of the cellulose fibers in relation
21
Page 21 of 28
618
to the degraded samples. Therefore, these images confirm the efficiency of the degradation of
619
RCS by both enzymes.
620
B
C
D
E
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A
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621
F
622 623
22
Page 22 of 28
624
Fig. SM-2. SEM images at day 7 for enzymatic degradation of the samples: (a) RCS control
625
(without enzyme)- magnification 500× and (b) magnification 1,000×; (c) RCS containing
626
lysozyme- magnification 500× and (d) magnification 1,000×; € RCS containing Tr cellulose-
627
magnification 500× and (f) magnification 1,000×.
ip t
628 The results of cytotoxicity (XTT cell viability assay) are shown in Fig. SM-3. The cell
630
viability is related to the absorbance measure. Negative control was considered 100% of cell
631
viability. The technique principle is based on the cleavage of the yellow tetrazolium salt XTT by
632
metabolically active cells, forming an orange formazan dye. Thus, this conversion occurs only in
633
viable cells. The tested eluates presented high cell viability and they did not present statistically
634
significant difference compared to the NC (p>0.05; Dunnett’s test).
us
cr
629
an
635 120
M
80
d
60
40
Ac ce pt e
Cell Viability (%)
100
20
a
0
636 637
NC
PC
RCS100%
RCS75%
RCS50%
RCS25%
638
Fig. SM-3. Cell viability evaluated by XTT. NC represents 100% of cell viability. Columns
639
indicate the mean value of cell viability (%). Bars indicate standard error. Lowercase letter
640
represents statistically significant difference (a=p0.05). RCS25% presented a significant
654
increase of the proliferative capacity of the CHO-K1 cells compared to NC (p