Electronic Supplementary Material
Characterization and in vitro evaluation of bacterial cellulose membranes functionalized with
osteogenic growth peptide for bone tissue engineering
Sybele Saska1a*, Raquel Mantuaneli Scarel-Caminaga2a, Lucas Novaes Teixeira3a, Leonardo Pereira
Franchi4a, Raquel Alves dos Santos4b, Ana Maria Minarelli Gaspar2b, Paulo Tambasco de Oliveira3b,
Adalberto Luiz Rosa3c, Catarina Satie Takahashi4c,5, Younès Messaddeq1b, Sidney José Lima Ribeiro1c,
Reinaldo Marchetto1d
1
Univ. Estadual Paulista, UNESP, Institute of Chemistry, Rua Francisco Degni 55, Zip Code: 14.800-900 -
Araraquara-SP,
Brazil.
E-mail:
1asybele_saska@yahoo.com.br,
1byounes.messaddeq@copl.ulaval.ca,
1csidney@iq.unesp.br, 1dmarcheto@iq.unesp.br
2
Univ. Estadual Paulista, UNESP, Dental School at Araraquara, Department of Morphology, Rua Humaitá, 1680,
Zip Code: 14.801-903 – Araraquara - SP, Brazil. E-mail: 2araquel@foar.unesp.br, 2banamaria@foar.unesp.br
3
University of São Paulo, USP, Cell Culture Laboratory, Faculty of Dentistry of Ribeirão Preto, Av. do Café s/n,
CEP 14040-904, Ribeirão Preto, São Paulo, Brazil. E-mail: 3anovaesrp@yahoo.com.br, 3btambasco@usp.br,
3cadalrosa@forp.usp.br
4
University of São Paulo, USP, Department of Genetics, Faculty of Medicine of Ribeirão Preto, Bloco G, Av.
Bandeirantes, 3900, Ribeirão Preto, SP, Brazil. E-mail:
4aleonardofranchi@yahoo.com.br,
4brasantosgen@yahoo.com.br, 4ccstakaha@usp.br
5
University of São Paulo, USP, Department of Biology, Faculty of Philosophy Sciences and Letters of Ribeirão
Preto, Av. Bandeirantes, 3900, Ribeirão Preto, SP, Brazil.
*
Corresponding author: E-mail: sybele_saska@yahoo.com.br. Univ. Estadual Paulista, UNESP,
Institute of Chemistry. Address: Rua Francisco Degni, 55, Zip Code: 14.800-900 - AraraquaraSP, Brazil. Telephone: 55 16 3301-9631 Fax: 55 16 3301-9632
Introduction
Bacterial cellulose (BC) has a three-dimensional structure consisting of an ultrafine network of
cellulose nanofibers. Thus, considering the genotoxicity associated to other fiber-shaped nanoparticles, as
asbestos [1] and carbon nanotubes [2] it seems crucial to evaluate this type of toxicity before any kind of
use of BC. Moreover, BC induced a proliferative rate less than negative control as observed by Bäckdahl
[3] and by Moreira [4] and also observed by us with XTT experiments (Fig. 7), DNA damage can induce
cell cycle arrest to repair the injuries which could induce a slow cell growth [5].
Furthermore, DNA damaged by peptides was observed by Xue [6] and also by Santiard-Baron
[7]. The osteogenic growth peptide (OGP) has structure identical to the C-terminal sequence of histone
H4. Midorikawa [8] described that histone H4 peptide with specific sequence AKRHRK can cause DNA
damage under metal-overloaded condition. Thus the peptides represent a second worry.
In another point of view, LPS is one of the major constituents of the outer membrane of Gramnegative bacteria (such as Gluconacetobacter xylinus) and this molecule can induce DNA damage [9].
Humans are extremely sensitive to endotoxins, its removal is essential. Therefore, the genotoxicity studies
are relatively inexpensive and sensitive to detect impurities, it seems an appropriate assay to use and to
confirm the absence of genotoxic contaminants produced during synthesis, or remaining after purification
of this biological molecules.
Experimental
Comet assay
Prior to the comet assay, CHO-K1 cells were assayed for viability using trypan blue dye
exclusion [10] and the cultures showing cell viability above 70 % were considered suitable for the comet
assay. Comet assays were performed as described previously by Singh [11]. Treatment and cell culture
were performed similar to that of the clonogenic assay. DNA damage was determined in 100 nucleoids
(50 on each slide) in a blind test using a fluorescent microscope (40x objective, Zeiss Axiovision,
Germany). To quantify the extent of DNA damage, each nucleus was classified visually according to the
migration of the fragments in: class 0 (no damage, or < 5 % of migrated DNA); class 1 (little damage
with a short tail length smaller than the diameter of the nucleus, or 5 to 20 % of migrated DNA); class 2
(medium damage with a tail length one or two times the diameter of the nucleus, or 20 to 40 % of
migrated DNA); class 3 (significant damage with a tail length between two and a half to three times the
diameter of the nucleus, or 40 to 95 % of migrated DNA); and class 4 (significant damage with a long tail
of damage greater than three times the diameter of the nucleus, or < 95 % of migrated DNA) [12, 13]. To
facilitate management of the data, an average of DNA migration (DNA damage index) was calculated as
follows: (number of cells with score 1) × 1 + (number of cells with score 2) × 2 + (number of cells with
score 3) × 3 + (number of cells with score 4) × 4/100 [14].
Cytokinesis-blocked micronucleus (CBMN) assay
The CBMN assay was performed according to published procedures by Fenech [15] with minor
modifications. CHO-K1 cells were seeded in 24-well plates at a density of 5×104 cells/well. After 24 h of
seeding, similar to the clonogenic assay, cells were exposed for 24 h to the BC, BC-OGP and BC-OGP
[10-14] membranes. Negative controls (NC) were wells without any BC membranes and positive controls
(PC) were treated with doxorubicin (0.3 g.mL-1) for 4 h (all experiments were carried out in duplicate).
Cytochalasin-B (CytB) was added to the CHO-K1 cultures at a final concentration of 5 µg.mL-1 and
maintained for 20 h. One thousand (1,000) cells were scored to evaluate the percentage of mono-, bi-, tri-,
and tetra-nucleated cells. The nuclear division index (NDI) was calculated according to the formula: [NDI
= M1 + 2(M2) + 3(M3) + 4 (M4)/N], where M1–M4 represents the number of cells with 1 – 4 nuclei,
respectively, and N is the total number of scored cells. Micronuclei (MN) were scored in 1000
binucleated cells. MN is a biomarker of DNA damage and instability. The criteria for identifying MN
were based on Fenech [15].
Statistical analysis
At least 3 experiments were conducted for each parameter analyzed. The experimental results are
expressed as mean and standard error. The Shapiro-Wilk test was used to assess the normality of the data
and Levene’s test for homogeneity. In view of the results, parametric tests were utilized. One-way
analysis of variance (ANOVA) followed by Tukey’s test was applied to the data. Data from treated
groups were compared to the negative control. BioEstat statistical package v.5 was used (UFPA, Belém,
Brazil) to perform the tests. Differences were considered statistically significant when p<0.05
Results and Discussion
Evaluation of genotoxicity and mutagenicity
Genotoxicity was evaluated by the comet assay and the results of the DNA damage index (DDI)
of CHO-K1 cells treated with all tested materials are shown in Table 1. The DDI of the NC and the PC
were statistically different (p<0.05), but no statistical differences were obtained for any materials based
on BC in comparison with the NC. Mutagenicity was assessed by the CBMN test, demonstrating the
results of both the nuclear division index (NDI) and the micronuclei in binucleated cell frequency
(MNBCF) in Table 1. The NDI was similar for all the studied groups (p=0.68), indicating that any
treatment that the CHO-K1 were subjected to could influence cell division. Similar to the DDI, the
MNBCF of the NC was statistically different to that of the PC (p<0.05). In comparison to the NC, no
statistical differences were obtained for any tested materials, indicating no genotoxic or mutagenic
effects. Non-genotoxic effects of BC have been described previously by Schmitt [16], as well as those of
BC nanofibers, which were evaluated by visual scoring analysis of comet assays [4]. To our knowledge,
this is the first study to investigate the mutagenic potentiality of BC membranes. It is worth bearing in
mind that the comet assay is not used to detect mutations, but to detect genomic lesions that could render
a mutation [17]. CBMN assays detect chromosome breaks and aneuploidy, drastic lesions that cannot be
repaired by the cell DNA repair machinery [18]. Besides demonstrating the absence of mutagenic
potential of all the tested materials, the methodologies employed in this study also showed that the
developed osteogenic growth peptides can be safely used because they were not genotoxic or mutagenic.
Regarding the OGP and OGP [10-14] peptides, no previous studies have been found to focus on
genotoxicity and mutagenicity effects.
In fact, we didn’t observe a significant induction of DNA damage, so another mechanism is
involved in that acute reductions in XTT viability assay (Fig. 7). The results show that the BC and
peptides modified-BC is free of Gluconacetobacter xylinus endotoxins genotoxicity.
Table 1. DNA damage after exposure to the different BC membranes evaluated by micronuclei and comet
assays in CHO-K1 cells.
NDI
MNBCF
DDI
Mean ± SE
Mean ± SE
Mean ± SE
NC
1.81 ± 0.02
6.3 ± 0.3
0.82 ± 0.21
PC
1.83 ± 0.03
350.3 a ± 42.0
1.81 a ± 0.17
BC
1.79 ± 0.01
12.0 ± 1.2
1.08 ± 0.20
BC-OGP
1.77 ± 0.01
14.7 ± 3.7
0.93 ± 0.17
BC-OGP [10-14]
1.83 ± 0.05
13.3 ± 4.3
1.36 ± 0.10
Treatment
p = 0.68
NDI = nuclear division index; MNBCF = micronucleated binucleated cells frequency; DDI = DNA
damage index; SE = Standard Error. Different letters in collums mean statistically significant difference
between groups (ANOVA, Tukey test).
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