2 Current Stem Cell Research & Therapy, 2014, Vol. 9, No. 2
port to the extracellular matrix (ECM). Hyaluronan-binding
proteins, called hyaladherins, mediate its interaction with
various extracellular components, including proteoglycans,
collagen and fibrin, which stabilizes both HA and ECM .
However, the success of the scaffolds to be used in tissue
engineering depends, in part, on the adhesion and growth of
cells of interest on its surface. The surface chemistry of the
material may define the cellular material and thus affect the
adhesion, proliferation, migration and cell function [13, 14].
The interaction of cells with the surfaces of materials is
of extreme importance for the effectiveness of medical implants  and may define the degree of acceptance or rejection. Knowledge of basic mechanisms of cell-material interaction and a better understanding of processes at the cellular
level during the accession may contribute to the development
of new biomaterials and the development of new biomedical
products . Cells identify the exposed surface topography
and nanofiber features, such as porous matrices and alignment, influence the adhesion, spreading, proliferation and
gene expression of the cells seeded onto them.
Stem cells are a non-specialized cell type which can selfrenew and remain for a long period of time with the potential
to produce different cell lineages or tissues with specialized
functions. Tissue-specific stem cells, or adult stem cells,
have been considered as an alternative for the use of embryonic stem cells, due to their availability, ease of acquisition
and growth in comparison with mesenchymal stem cells
from bone marrow . ESCs also encounter some problems
related to safety issues such as ESC rejection, the risk of
tumorigenicity and government moratorium, which has restricted research on this type of cell [18, 19]. Mesenchymal
stem cells have thus been studied as a group of cells with
plasticity similar to embryonic stem cells; this has been the
target of numerous researchers.
Several nanocomposites were tested specifically with
stem cell adhesion and differentiation. Cell adherence and
proliferation ability of hBMSCs (human bone mesenchymal
stem cells) on scaffolds were improved by coating with
HA/PLLA nanocomposites . Another study investigated
the effects of nanophase hydroxyapatite (nano-HA), nanoHA/poly(lactide-co-glycolide) (PLGA) composites and a
small peptide derived from bone morphogenetic protein
(BMP-7) on osteogenic differentiation of hMSCs (Human
mesenchymal stem cells). The nanocomposites provided
promising alternatives in controlling the adhesion and differentiation of hMSCs without osteogenic factors from the culture media and,thus, should be further studied for clinical
translation and the development of novel nanocompositeguided stem cell therapies .
The use of poly(L-Lactide) and nanohydroxiapatite was
studied in another work. The culture of bone mesenchymal
stem cells indicated that the composite nanofibrous poly(Llactide) scaffold with 50 wt % nanohydroxyapatite showed
the highest cell viability among various poly(L-lac-tide)based scaffolds  Other nanocomposites and electrospun
nanocomposites succeeded in testing the interaction with
cells [23-26]. Besides this, collagen-based nanocomposites
incorporating nanobioactive glass (Col/nBG) were developed
as a scaffold matrix for dentin–pulp regeneration. The effects
of the novel matrix on the proliferation of human dental pulp
Acasigua et al.
cells (hDPCs) and their differentiation into odontoblastic
lineage were investigated. In conclusion, the nanocomposite
Col/nBG matrix induced the growth and odontogenic differentiation more effectively than Col alone, providing a promising scaffold condition for regeneration of dentin–pulp
complex tissue .
It is of particular interest that stem cells from deciduous
teeth have certain advantages. Miura and colleagues attributed to these cells significantly greater potential for proliferation and clonogenicity when compared to pulp stem cells
from permanent teeth and stem cells from bone marrow .
In this work, novel studies of natural nanocomposites
with bacterial cellulose for functional dental materials is reported. In order to produce scaffolds with drug delivery ability, porous structure and better cell adhesion, fermentation
changes in bacterial cellulose with gelatin and hyaluronic
acid were performed. A link has been established between
fermentation, morphological surface and cell attachment.
2. MATERIALS AND METHODS
The bacterial cellulose raw material (Nanoskin) was provided from Innovatec´s (São Carlos SP, Brazil). Gelatin from
porcine skin and hyaluronic acid sodium salt from
polysaccharide) were purchased from Sigma Aldrich.
The acetic fermentation process was achieved by using
glucose as a carbohydrate source. Results of this process are
vinegar and a nanobiocellulose biomass. The modifying
process is based on the addition of hyaluronic acid and gelatin (1% w/w) to the culture medium before the bacteria is
inoculated. After being added to the culture medium, the
medium is autoclaved at 100 oC degree. Bacterial cellulose
(BC) is produced by Gram-negative bacteria Gluconacetobacter xylinus, which can be obtained from the culture medium in the pure 3-D structure, consisting of an ultra fine
network of cellulose nanofibers .
2.2.2. Bionanocomposite Preparation
In the present study, a novel biomaterial has been explored and different bacterial cellulose nanocomposites have
been prepared; 1) BC/hyaluronic acid, 2) BC/gelatin.
2.3. Samples of Pulp Tissue from Deciduous Teeth
In order to isolate the cells from the pulp tissue and establish their culture, dental pulp was removed from deciduous teeth in the resorption process. After extraction, the teeth
was immersed in 1 mL culture medium DMEM/Hepes
(Sigma Aldrich), 10 % fetal bovine serum (GIBCO),
100U/mL penicillin, 100μg/mL streptomycin (Gibco) and
0.45μg/mL gentamicin (Gibco) at room temperature for
transport to the laminar flow. Those responsible for the patient signed a consent form approved by the Ethics Committee of the Federal University of Rio Grande do Sul, registration number 19273.
Novel Chemically Modified Bacterial Cellulose Nanocomposite
2.3.1. Cell Culture
The handling of the pulp tissue removed was performed
following the protocol established in the laboratory . Cell
suspension in the culture medium was seeded onto a 12 well
culture plate and then incubated at 37˚C in a humidified atmosphere of 5% CO2. The culture medium was changed 24
hours after initial plating and then every 3 days thereafter.
The culture was maintained under these conditions until confluence of approximately 90% when it was then held in its
first passage. The cells in culture were harvested with trypsin-EDTA solution 0.5 % (Sigma-Aldrich) and transferred to
sub-cultures in their culture medium. The sub-culture was
maintained in a monolayer until required for the next raise.
When the cells reached approximately 90% of confluence
between the 5th passage (P5), cell viability was assessed with
trypan blue 4% (Gibco) in a Neubauer chamber and testing,
to verify the interaction between cells and scaffolds, was
perfomed as follows.
2.4.1. Scanning Electron Microscopy (SEM)
Scanning electronic microscopy images were performed
on a PHILIPS XL30 FEG. The samples were covered with
gold and silver paint for electrical contact and to produce the
2.4.2. Transmission Infra-Red Spectroscopy (FTIR, Perkin
Elmer Spectrum 1000)
Influences of hyaluronic acid and gelatin in bacterial cellulose were analyzed in the range between 250 and 4000 cm1
and with 2 cm-1 resolution with samples.
2.4.3. Cell Viability
For the study of cell viability during the 28 days of culture, as performed for the cell adhesion essay, the cells were
seeded onto each type of scaffold in triplicate and then incubated at 37˚C in a humidified atmosphere of 5% CO2. To
collect the initial viability of the seeded cells, the viability of
5 x 104 cells was analyzed, 6 hours after seeding onto the
culture dishes. Analysis was then made 7, 14, 21 and 28 days
after the start of the cultivation of the cells in the biomaterial.
After each trial, cell viability was performed by the salt tre-
Current Stem Cell Research & Therapy, 2014, Vol. 9, No. 2
tazolyum method, a colorimetric assay using bromide 3 (4,5 - dimethylthiazol -2-yl ) -2,5 – diphenyltetrazolium
bromide (MTT). After the experiment time, the culture medium was removed and 200 L MTT solution (0.25 mg/mL)
was added and maintained for 2 hours. The MTT was then
removed and 200μL of dimethyl sulfoxide (DMSO) was
added to dissolve the crystals formed by the reaction. Using
96-well plates, the absorbance of the final solution was analyzed by a spectrophotometer (Wallac EnVision - Perkin
Elmer). The data was calculated using the difference in absorbance between the wavelengths (560 nm – 630 nm). As a
control group, the cells were seeded in a similar way onto
24-well plates (in triplicate) without scaffolds and maintained by the same experimental period and the same procedures for data collection were performed.
Cell Adhesion - 5 x 10 4 cells in 50 μL of concentrated
culture medium were seeded on each type of scaffold (in
triplicate) and incubated at 37 ˚C in a humidified atmosphere
of 5 % CO2. After 6 hours of culture, the culture medium
was removed and the samples were washed three times with
phosphate-buffered saline to remove non-adherent cells on
the scaffolds. The cells attached to the scaffolds were then
fixed with 4 % paraformaldehyde for 20 minutes. Following
this, staining was performed with 0.5 mg/mL 4,6-diamidino2-phenylindole (DAPI), a fluorescent marker which binds
strongly to DNA. Confocal microscopy images were then
obtained, corresponding to different randomly distributed
3. RESULTS AND DISCUSSION
3.1. Bacterial Cellulose Nanocomposite Mats
Bacterial cellulose nanocomposite mats were characterized by SEM. Fig. (1a and 1b) and Fig. (2a and 2b) show
SEM images of bacterial cellulose/hyaluronic acid and bacterial cellulose/gelatin nanocomposites, respectively. Different
morphological surface with gelatin and hyaluronic acid addition to fermentation medium can be observed. A connection
has been established between fermentation changes and bacterial cellulose biosynthesis to explain such behavior.
Bacterial cellulose contains a thin peptidoglycan layer
adjacent to the cytoplasmic membrane. In addition, it contains an outer membrane composed of phospholipids and
lipopolysaccharides with invaginations of the cell membrane,
Fig. (1). (a, b) Scanning electron microscopy (SEM) of bacterial cellulose/hyaluronic acid.
4 Current Stem Cell Research & Therapy, 2014, Vol. 9, No. 2
Acasigua et al.
Fig. (2). (a, b) Scanning electron microscopy (SEM) of bacterial cellulose/gelatin.
which can be either simple folds, such as vesicular or tubular
structures. Several functions have been observed, such as the
role of bacterial cellulose in cell division and respiration of
the bacteria .In general, aerobic respiration uses glucose
more efficiently to produce energy (i.e. electrons), when
compared to the fermentation process for energy production
It is believed that the tubular fibrils become positioned
within tunneling distance of the cofactors with little consequence to denaturation. The combination of symbioses with
redox active enzymes would appear to offer an excellent and
convenient platform for a fundamental understanding of biological redox reactions .
The biosynthesis of the sugar in cell structures begins by
the synthesis of the units of sugar nucleotides. The supply of
the sugars for the biosynthesis is dependent on the intracellular sugar nucleotide levels that are influenced by the activities of the intracellular enzymes involved in their biosynthesis .
The size of the cellulose molecules is normally expressed
in terms of their polymerization degree (PD), this is the
number of anhydroglucose units present in a chain. However, the conformational analysis of cellulose indicates that
cellobiose (4-O- -D-glucopyranosyl- -D-glucopyranose) is
its basic structural unit . The conformation of the repeating unit of cellulose can be explained if the model proposed
for the biosynthesis of glucose is considered .
The active site of the enzyme cellulose synthase, responsible for the synthesis of cellulose, contains two consecutive
sites for binding to the uridine-diphosphoglucose precursor
(UDP-glucose), positioned at 180°C from each other and a
binding site at the non-reducing end of the -glucan. The
hydroxyl at C-5 of glucose residues linked to these sites is
activated by a mechanism of general base catalysis by promoting the dephosphorylation of UDP-glucose units and
establishing new links (1-4). The resulting -glucan, which
has little affinity with the binding sites for UDP-glucose,
moves to the binding site for -glucan, which is a better location. The synthesis can then be continued with the addition
of two new units of UDP-glucose .
Bacterial cellulose has cell division and DNA synthesis
within the cells. DNA is organized into long structures called
chromosomes. During cell division these chromosomes are
duplicated in the process of DNA replication, providing each
cell with its own complete set of chromosomes, it will then
subsequently assemble the -1,4-glucan chains outside the
cell in a precise, hierarchical process. The production of cellulose characterizes this bacteria both in liquid medium,
where it produces a thick pellicle on the air–liquid interface,
and isolated cells where a cellulose ribbon can be clearly
seen attached to the long side of the cell .
In this scope, hyaluronic acid structure is similar to bacterial cellulose and may derive from Streptococcus bacterium.
Biosynthesis with bacterial cellulose and hyaluronic acid was
then produced more successfully in fibers structure than bacterial cellulose/gelatin, as observed in Figs. (1 and 2).
3.2. Interaction Between Bacterial
Hyaluronic Acid and Gelatin
Influences of gelatin and hyaluronic acid in bacterial cellulose were analyzed in the range between 250 and 4,000 cm1
and with resolution of 2 cm-1 with FTIR analysis. The main
features of the bacterial cellulose in infrared spectroscopy is:
3,500 cm-1:OH stretching, 2,900 cm-1:CH stretching of alkane and asymmetric CH2 stretching, 2,700 cm-1:CH2 symmetric stretching, 1,640 cm-1:OH deformation, 1,400 cm1
:CH2 deformation, 1,370 cm-1:CH3 deformation, 1,340 cm1
:OH deformation and 1,320-1,030 cm-1:CO deformation
It can be observed in Figs. (3 and 4) that the intensity of
transmittance of the bio nanocomposites is smaller than that
of bacterial cellulose, which means that exposed groups on
the bacterial cellulose molecules are interacting with other
components and in comparison, hyaluronic acid is interacting more than gelatin with bacterial cellulose (Fig. 5). More
OH stretching (at 2,900 cm-1) can be observed, in bacterial
cellulose/hyaluronic nanocomposites than with gelatin composites, mainly because of NH2 interaction with hydroxyl
groups (Fig. 5). Besides this, changes can be observed in the
symmetrical stretching of CH2 bonds of bacterial cellulose
structures in the absorption peak of 1,640cm-1. Another absorption peak was obtained in the range of 1,490 cm 1 in
both samples, which shows the presence of a carbonyl group
in the bacterial cellulose together with bonds corresponding
Novel Chemically Modified Bacterial Cellulose Nanocomposite
to those of glycoside, including C–O–C at 1,162 cm 1 (as in
the case of natural cellulose) . These results clearly show
one possible interaction between bacterial cellulose and gelatin/hyaluronic acid, mainly by hydrogen interactions between
hydroxyl and carbonyl groups.
Current Stem Cell Research & Therapy, 2014, Vol. 9, No. 2
3.3. Cell Adhesion and Viability
The cells used in this study were characterized as mesenchymal stem cells, keeping the profile positive for CD29,
CD44 and CD90 (>95%) and with a low expression of
CD34, CD45, CD146, STRO-1 and HLA-DR (<2%), showing ability to differentiate into adipocytes, osteoblasts and
For the successful application of scaffolds in tissue engineering, a crucial feature is that the matrices promote cell
adhesion. According to Andrews and colleagues , cell
adhesion is mediated by the adsorption of extracellular matrix proteins produced by cells on the surface of the scaffold.
The signalling pathways are then activated and cell adhesion
occurs in the mould by means of receptors. Therefore, accommodation and cell behavior is strongly affected by the
structure of the scaffolds and cell adhesion assay becoming
important in order to determine whether the scaffolds have a
good structure for the initial interaction with cells.
Fig. (3). FTIR spectra of bacterial cellulose/gelatin nanocomposites.
Fig. (4). FTIR spectra of bacterial cellulose/hyaluronic acid.
Fig. (5). FTIR spectra of bacterial cellulose/hyaluronic acid and
The metabolic activity was assessed by measuring the activity of the enzyme mitochondrial succinate dehydrogenase
(MTT assay), which is widely used in in vitro evaluation of
cell viability [40, 41]. It was tested for analysis of cell performance over time, allowing the monitoring of cell viability
during the experimental period.
Through the MTT assay, it was found that after 1 day of
experiment, the three groups (samples I and II and control)
showed similar behavior, with no statistical difference. At
seven days, similar behavior was observed between the
groups, with no statistical difference. It was noted that the
absorbance increased over the first day in three groups, indicating that the proliferation was similar between the two
nanocomposites and the control group. On the 15th day, a
statistically significant difference in the absorbance was observed between the test groups and the control group. The
control group had an absorbance greater than the two test
groups, indicating greater proliferation of cells in the control
group. The test groups maintained with similar absorbance
until the seventh day, indicating that the number of cells remained constant from the seventh to the fifteenth day of culture - which is nonetheless a good result (Fig. 6a). This
shows that the fibers prepared for the study provide an initial
adhesion and increase of viability in the initial stage and that
they have ability to promote maintenance of viability in the
It can, therefore, be concluded that gelatin nanocomposites have lower cell adhesion over time because of the fiber
agglomeration during bacterial cellulose biosynthesis Based
on recent literature, there are other variables which have influence on adhesion and cell viability, such as cell dispersion
 and different types of stem cell cultured on poly-Dlysine, poly-L-lysine and collagen, in order to improve cell
differentiation . Besides this, factors such as the deposition of apatite using SBF (simulated body fluid) will be
tested in future experiments [44, 45].
Through images obtained by confocal microscope, it can
be observed that 1 day after the cells had been seeded onto
the scaffolds, they were distributed in a homogenous manner
over the entire scaffold structure. By applying digital zoom
on the images, it was possible to observe that the cells adhered on the nanocomposite surface and exhibited a spread
6 Current Stem Cell Research & Therapy, 2014, Vol. 9, No. 2
morphology in the red colored area in Figs. (6b and 6c).
Confocal images in Fig. (6b), bacterial cellulose/hyaluronic
acid and Fig. (6c), bacterial cellulose/gelatin, confirmed that
after 1 day in culture the cells assumed a fusiform aspect,
showing that the surface of nanocomposites promote and
favor cell adhesion and development. These results demonstrate the quality of the nanocomposites and how they allow
cell adhesion and maintenance in a similar way to the culture
Acasigua et al.
Furthermore, it can be observed that fermentation produced different surface morphologies with the addition of
hyaluronic acid/gelatin and there was more agglomeration of
the fiber formation process in the bacterial cellulose/gelatine
than the bacterial cellulose/hyaluronic acid, which has a detrimental effect on cellular adhesion, as illustrated in Fig.
Bacterial cellulose was successfully modified by changing the fermentation medium as shown with SEM and FTIR,
which produced scaffolds with different surface morphology
but similar cell adhesion and attachment. Natural scaffolds
with bacterial cellulose and bacterial cellulose nanocomposites had good cell adhesion over time between tested samples, being an extremely effective material for tissue regeneration. Such nanocomposites present adhesion behavior
similar with that which can be seen in the literature. However, a better controlled development in methods for production, purification and surface morphology is essential for
widespread use of these scaffolds.
Fig. (6a). Cell viability assay over a time period of 1, 7 and 15days
in bacterial cellulose/hyaluronic acid (sample I), bacterial cellulose/gelatin (sample II) and control group.
CONFLICT OF INTEREST
The authors confirm that this article content has no conflicts of interest.
Nanoskin – Bacterial cellulose produced by Innovatec's Biotechnology Research and Development- Brazil.
LIST OF ABBREVIATIONS
Dulbecco´s Modified Eagle Medium
Ethylenediamine Tetraacetic acid
Embryonic stem cell
Figs. (6). Confocal microscopy images obtained to check cell adhesion and morphology:(b) bacterial cellulose/hyaluronic acid; (c)
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Accepted: November 14, 2013