1. Article
Journal of
Biomedical
Nanotechnology
Copyright © 2013 American Scientific Publishers
All rights reserved
Printed in the United States of America
Vol. 9, 1–8, 2013
www.aspbs.com/jbn
Human Dental Pulp Stem Cell Behavior Using
Natural Nanotolith/Bacterial Cellulose
Scaffolds for Regenerative Medicine
Gabriel Molina Olyveira1 3 ∗ , Gerson Arisoly Xavier Acasigua2 , Ligia Maria Manzine Costa1 ,
Cristiane Regina Scher2 , Lauro Xavier Filho4 , Patricia Pranke5 6 7 , and Pierre Basmaji4
1
Department of Nanoscience and Advanced Materials-UFABC, Rua Santa Adélia, 166, Santo André-SP, Brazil
Dentistry Post-Graduation Programme, Federal University of Rio Grande do Sul, Av. Ipiranga 2752,
Porto Alegre-RS, 90610-000, Brazil
3
Natural Products Laboratory and Biotechnology, UNIT, Aracaju, Brazil
4
Innovatec’s—Biotechnology Research and Development, São Carlos-SP, 13560-042,, Brazil
5
Material Science Post-Graduation Programme, Federal University of Rio Grande do Sul, Av. Ipiranga 2752,
Porto Alegre-RS, 90610-000, Brazil
6
Faculty of Pharmacy, Hematology and Stem Cell Laboratory, Federal University of Rio Grande do Sul,
Av. Ipiranga 2752, Porto Alegre-RS, 90610-000, Brazil
7
Stem Cell Research Institute (SCRI), Federal University of Rio Grande do Sul, Av. Ipiranga 2752, Porto Alegre-RS, 90610-000, Brazil
2
Adhesion and Viability study with human dental pulp stem cell using natural nanotolith/bacterial cellulose scaffolds for
regenerative medicine are presented at first time in this work. Nanotolith are osteoinductors, i.e., they stimulate bone
regeneration, enabling higher cells migration for bone tissue regeneration formation. This is mainly because nanotoliths
are rich minerals present in the internal ear of bony fish. In addition, are part of a system which acts as a depth sensor
and balance, acting as a sound vibrations detector and considered essential for the bone mineralization process, as in
hydroxiapatites. Nanotoliths influence in bacterial cellulose was analyzed using transmission infrared spectroscopy (FTIR).
Results shows that fermentation process and nanotoliths agglomeration decrease initial human dental pulp stem cell
adhesion however tested bionanocomposite behavior has cell viability increase over time.
KEYWORDS: Adhesion and Viability Study, Bacterial Cellulose (Nanoskin), Nanotoliths, Natural Nanocomposites, Regenerative
Medicine, Stem Cells.
INTRODUCTION
Human dental composition is made up of hard tissues
(enamel and dentin) and soft tissues (pulp, periodontal ligament and alveolar bone), which are composed of connective tissues and those from the basement membrane.1 2
Soft tissues are composed of cells and extracellular matrix
(ECM), where there are many types of cells performing different functions, e.g., biosynthesis of the ECM
components, lipid storage, biological protection, maintaining the body homeostasis. The ECM is comprised of many
∗
Author to whom correspondence should be addressed.
Email: gabriel.ufabc@gmail.com
Received: 14 September 2012
Revised/Accepted: 6 January 2013
J. Biomed. Nanotechnol. 2013, Vol. 9, No. xx
different macromolecular structures, which can be grouped
into collagens, glycoprotein’s, and proteolysis, attending to
their major components.3 4
Nanocollagenous proteins present in oral cavity tissues
are involved in several processes, such as the initiation
and formation of apatite crystals (mineralized tissues) and
the regulation of cell proliferation and tissue growth and
development.5–7
The family of collagens accounts for more than 50% of
the total tissular protein. Collagens are well-known proteins whose chemical and tridimensional structures are
clearly elucidated.8
Bacterial cellulose (BC) is produced by microorganism
via biochemical steps and self-assembling of the secreted
cellulose fibrils in the medium. Shaping of BC materials
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doi:10.1166/jbn.2013.1620
1

2. Human Dental Pulp Stem Cell Behavior Using Natural Nanotolith/Bacterial Cellulose Scaffolds
in the culture medium can be controlled by the type of
cultivation that changes chain sizes, origin of strains that
produce different proportion of crystalline phase of BC
and a kind of bioreactor.9
The structural features of microbial cellulose, its properties and compatibility of the biomaterial for regenerative medicine can be changed modifying its culture
medium10–12 or surface modification by physical13 14 and
chemical methods15 16 to obtain a biomaterial with less
rejection when cellular contact and blood cell contact interaction occurs.
Among all the calcium phosphates, hydroxyapatite has
lowest rate of biodegradation, or absorption by the animal
organism, where the sequence is the following order at the
rate of resorption. As the main mineral constituent of bone
structure, hydroxyapatite has been extensively studied and
used as material for permanent inclusion in defective or
abnormal bone structure in surgical bone repair depending on his performance as biomaterials (biocompatible,
bioactive and osteoconductive).17–19 However, recent studies reported that hydroxyapatite is a carcinogen material
for bone tissue regeneration.20 In this context, Innovatec’s,
which makes development with natural nanomaterials, discovered a new material with bone tissue regeneration
bone called otolith. This material has same characteristics
related to hydroxyapatite with lower toxicity because is a
natural material.
Based on the results with artificial skin, bacterial cellulose was tested in dental tissue regeneration. Olyveira
et al.21 reported the first otolith/collagen/bacterialcellulose
nanocomposites as a scaffold for bone regeneration. The
success of the subsequent transplantation of the engineered
in vitro construction is due to the properties of the materials but also to the osteoprogenitor cell sources. In addition,
Olyveira et al. reported that otoliths showed biocompatibility with pulp tissues in vivo and the response of pulp
tissue was successful.22 It can be concluded that direct pulp
capping with the preparation of otoliths, similar to calcium
hydroxide, preserves the vitality, stimulates the formation
of mineralized tissue barrier and induces reparative pulp
response. This biomaterial represents a promising biomaterial for use in human dental medicine in the future.
Besides, the success of the scaffold 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.23–25
The interaction of cells with the surfaces of materials
is of extreme importance for the effectiveness of medical implants26 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.27 Cells identify the exposed
2
Olyveira et al.
surface topography and nanofiber features, like porous
matrices and alignment influence the adhesion, spreading, proliferation and gene expression of the cells seeded
onto them. Different cell behavior was found in several
surface topographies obtained from lithography,28 29 phase
separation,30 electrospinning;31 nanoimprinting32 and self–
assembly.33
Stem cells are a non-specialized cell type, which can
self-renew and remain for a long period of time with the
potential to derive in a cell lineage or tissue 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.34 35 Thus, the study of populations
of adult stem cells with plasticity similar to embryonic
stem cells has been the target of numerous researchers. Of
particular interest, stem cells from deciduous teeth have
certain advantages. Miura et al. attributed to these cells
significantly greater potential for proliferation and clonogenisticity when compared to pulp stem cells from permanent teeth and stem cells from bone marrow.36
In order to obtain a bionanocomposite with mechanical properties and an osteoconductive environment that
can facilitate cell attachment37 and to increase in vitro
proliferation and differentiation of osteoblastic cells as
well as the rapid vascularization, it is reported in this
work a novel natural nanotoliths/bacterial cellulose for
future dental materials is reported with adhesion and
viability study using human dental pulp stem cell in
bionanocomposite.
MATERIALS AND METHODS
Materials
Bacterial cellulose membranes (Nanoskin), ∼ 500 mm
thick, and Otoliths were supplied by Innovatec’s—
Biotechnology Research and Development—Brazil.
Synthesis of Bacterial Cellulose
Bacterial Cellulose (BC) produced by Gram-negative
acetic acid bacteria Gluconacetobacter xylinus can be
obtained from a culture medium in the pure 3-D structure consisting of an ultra fine gel network of cellulose
nanofibres (3–8 nm), highly hydrated (99% in weight), and
displaying higher molecular weight, higher cellulose crystallinity (60–90%), enormous mechanical strength and full
biocompatibility.38
Nanotoliths Gels
The material in this study was prepared with 1 g powder
of otolith of Cynoscion acoupa with particle size 60 mesh
and addiction 0.25 g of hydrolyzed collagen, diluted in
distilled water. The final product was packaged in petri
dishes and sterilized in UV rays (25 min). Subsequently,
1.0 g of the otoliths were diluted in 100 mL of distillated
water and the pH of the compound was assessed using
J. Biomed. Nanotechnol. 9, 1–8, 2013

3. Olyveira et al.
Human Dental Pulp Stem Cell Behavior Using Natural Nanotolith/Bacterial Cellulose Scaffolds
phmeter Digimed® . Stable gel is formulated with a otolith
calcium salt concentration solution.
Scaffolds Preparation
In the present study, different natural scaffold materials
were prepared; (a) Pure BC, (b) BC with nanotoliths. Bacterial cellulose nanocomposite was obtained by immersion
of dried bacterial cellulose into nanotolith gels and posterior soft drying at 50 C for 12 hours.
Samples of Pulp Tissue from a Deciduous Tooth
In order to isolate the cells from the pulp tissue and
establish their culture, dental pulp was removed from a
deciduous tooth in the process of rhizolysis. After extraction, the tooth was immersed in 1 ml of culture medium
DMEM/Hepes (Sigma Aldrich), 10% fetal bovine serum
(Laborclin), 100 U/ml penicillin, 100 g/mL streptomycin
(Gibco) and 0.45 g/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 Universidade Tiradentes, it being
passed on 10.2.2006, under protocol 171 205, in accordance with ethical for the use of laboratory animals, legally
stipulated 1153-B 1995.
Cell Culture
The handling of the pulp tissue removed was performed
following the protocol established in the laboratory.39 Cell
suspension in the culture medium was seeded onto a culture plate of 12 wells 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. In sub-cultures, 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
performed.
Characterization
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 necessary images.
Transmission
Electron
Microscopy
(TEM)—
Transmission electron micrographs of bacterial cellulose
were taken with a Hitachi-600 transmission electron
microscope with an acceleration voltage of 5 kV.
J. Biomed. Nanotechnol. 9, 1–8, 2013
Transmission infrared spectroscopy (FTIR, Perkin
Elmer Spectrum 1000)—Influences of nanotoliths in bacterial cellulose were analyzed in the range between 250
and 4000 cm−1 and with of 2 cm−1 resolution with
samples.
Cell Adhesion—5 × 104 cells in 50 L 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 nonadherent cells on the scaffolds. The cells attached to
the scaffolds were then fixed with 4% paraformaldehyde
for 20 min. Following this, staining was performed with
0.5 mg/ml 4,6-diamidino-2-phenylindole (DAPI), a fluorescent marker that binds strongly to DNA. From each
sample nine images were obtained (Olympus CX50, 400×
magnification) corresponding to nine different randomly
distributed microscopic fields. Thus, it was possible to
quantify the number of cells attached by means of the
number of cells/field. As a control group, the cells was
seeded in a similar way onto cell culture plates of 24 wells
(in triplicate) without scaffolds, and the same procedures
for data collection were carried out.
Viability cell—For the study of cell viability during
the 28 days of culture, similar to that performed for the
cell adhesion essay, cells were seeded onto each type of
scaffold (in triplicate) and then incubated at 37 C in a
humidified atmosphere of 5% of CO2 . To collect the initial
viability of the seeded cells, the viability of 5 × 104 cells
was analysed, 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 tretazolyum method, a colorimetric assay using the bromide
3-(4,5-dimethylthiazol-2-yl)-2,5-difeniltetrazolio (MTT).
Elapsed times set were removed and the culture medium
added to 200 L of MTT (0.25 mg/mL) for 2 hours. The
MTT was then removed and 200 L 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 analysed 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 periods and the same procedures for data collection
were performed.
RESULTS AND DISCUSSION
Pure Bacterial Cellulose and Bacterial
Cellulose Nanocomposites Mats
Pure Bacterial cellulose mats were characterized by SEM.
Figures 1(a) and (b) show, as an example, SEM images and
3

4. Human Dental Pulp Stem Cell Behavior Using Natural Nanotolith/Bacterial Cellulose Scaffolds
(a)
Olyveira et al.
100
BC
BC/nanotoliths
90
Transmitance u.a.
80
70
60
50
40
30
20
10
0
–10
4000
3500
3000
2500
2000
1500
1000
500
Wavelenght (cm–1)
(b)
Figure 2. FTIR spectra of bacterial cellulose and bacterial cellulose/otoliths nancomposites.
(c)
features of the bacterial cellulose in infrared spectroscopy
is: 3500 cm−1 : OH stretching, 2900 cm−1 : CH stretching of alkane and asymmetric CH2 stretching, 2700 cm−1 :
CH2 symmetric stretching, 1640 cm−1 : OH deformation,
1400 cm−1 : CH2 deformation, 1370 cm−1 : CH3 deformation, 1340 cm−1 : OH deformation and 1320–1030 cm−1 :
CO deformation.40
It can be observed in Figures 2 and 3 that that nanotoliths
addiction changes symmetrical stretching CH2 bonds in
bacterial cellulose structures in 1640 cm−1 41 Besides in
Figure 3 nanotoliths bands at 712 and 874 cm−1 can be
observed; characteristic absorption bands of CaCO3 .42 43
The band of 874 cm−1 was just overlapped with the absorption band of Bacterial cellulose from 892 cm−1 .
Cell Adhesion and Viability
The use of matrices associated with the cells is the target of interest for researchers dedicated to this area.44–48
Thus, the characterization of scaffolds is needed to verify
Figure 1. (a), (b) Scanning electron microscopy (SEM) and
TEM of Pure bacterial cellulose. (c) Cellulose bacterial/otoliths
surface morphology.
80
712
BC
BC/otoliths
TEM images of pure Bacterial cellulose mats. Figure 1(c)
shows SEM images from bacterial cellulose/otoliths surface morphology. It can be observed some changes in bacterial cellulose surface morphology with otolith. A high
roughness changes opacity of the film and has influences
in superficial properties mainly in adhesion and viability
cells as proved by other characterizations in this work.
Interaction Between Bacterial
Cellulose with Nanotoliths
Influences of nanotoliths in bacterial cellulose were analysed in the range between 250 and 4000 cm−1 and
with resolution of 2 cm−1 with FTIR analysis. The main
4
Transmittance (a.u.)
70
60
50
40
30
20
874
10
0
–10
1400
1200
1000
800
600
400
Wave Number (cm–1 )
Figure 3. FTIR spectra of the samples BC, BC/otoliths.
J. Biomed. Nanotechnol. 9, 1–8, 2013

5. Olyveira et al.
Human Dental Pulp Stem Cell Behavior Using Natural Nanotolith/Bacterial Cellulose Scaffolds
their structure and determine whether they have the necessary requirements for their application to stem cells from
deciduous teeth used in this study. As described by Pham
et al.49 the morphology of the nanofibers is the result of
the combination of different factors. Normally, the primary
teeth will go through the process of resorption of their
roots during childhood.50
For the successful application of scaffolds in tissue engineering, a crucial feature is that the matrices promote
cell adhesion. According to Andrews,51 cell adhesion is
mediated by the adsorption of extracellular matrix proteins produced by cells on the surface of the scaffold. The
signaling pathways are then activated and cell adhesion
occurs in the mould by means of receptors. Therefore,
accommodation and cell behaviour is strongly affected
by the structure of the scaffolds and cell adhesion assay
becomes important in order to determine whether the scaffolds have a good structure for the initial interaction with
cells.
The next step was staining with 0.5 mg/ml 4,6diamidino-2-phenylindole (DAPI), a fluorescent marker
that binds strongly with DNA. By staining cell nucleus
with DAPI, it is possible to verify the capacity of adhesion to SCDT (stem cells from deciduous cells) in different
scaffolds. Bacterial cellulose (AP) and bacterial cellulose/nanotholits (A1) was statistically lower than the control condition on the culture plates—25.03, 25.73 and
62.69 cells per field in groups: bacterial cellulose (AP),
bacterial cellulose/nanotholits (A1) and control respectively (C), with no statistical difference between the bacterial cellulose groups and the bacterial cellulose/nanotholits
(Fig. 5). It can be observed in Figure 4 images obtained
from DAPI treatment to verify cell adhesion in the control group (a), bacterial cellulose sample (b), and the
bacterial cellulose/nanotholits sample (c). This fact demonstrates the quality of the nanofibers in enabling cells to
Figure 4. Images obtained from DAPI treatment to verify cell
adhesion: control group (a), bacterial cellulose (b), bacterial
cellulose/nanotoliths (c).
J. Biomed. Nanotechnol. 9, 1–8, 2013
Figure 5. Statistical analysis of stem cells from a deciduos
tooth in the control group (C), bacterial cellulose (AP), bacterial cellulose/nanotoliths (A1).
perform adhesion, though even this adhesion was statistically lower than in the control group mainly because
of production method of biocomposite using fermentation
roughness surface morphology were obtained, illustrated
in Figure 4(b). With the addition of nanotoliths there was
an agglomeration of nanotoliths on the surface of the scaffolds plus specific cell adhesion and localized punctually, which showed satisfactory results, as illustrated in
Figure 4(c).
The metabolic activity was assessed by measuring the
activity of the mitochondrial enzyme succinate dehydrogenase (MTT assay), which is widely used in in vitro evaluation of cell viability.52–54 It was tested for analysis of cell
performance over time, allowing for the monitoring of cell
viability during the experimental periods.
As can be seen in Figure 6, on the seventh day of culture, cell viability in the control group was statistically
higher than in the bacterial cellulose nanocomposite and
bacterial cellulose. The test groups did not differ among
themselves. At 14 days, there was a statistical difference
between the control and test groups, and cell viability of
bacterial cellulose nanocomposites showed a lower value
than the bacterial cellulose. At 21 and 28 days of culture,
there was no difference in the viability of the three groups,
with the performing groups testing similarly to the control
group.
This shows that, although the fibers prepared for the
study do not provide an initial adhesion and maintenance
of viability in the initial stage, they have the ability to promote a gradual increase in the long-term (21 days) since,
after 21 days of culture, the test groups were presented
in a manner similar to the control group and the standard
for cell culture. Based in recent literature, there is another
variables which has influence in adhesion and cells viability like cells dispersion.55 56
In this scope, natural scaffolds with bacterial cellulose and bacterial cellulose nanocomposites had lower cell
adhesion at the beginning of the test; however, during
the test their response was very positive, being a material
extremely effective for bone and tissue regeneration in the
body.
5

6. Human Dental Pulp Stem Cell Behavior Using Natural Nanotolith/Bacterial Cellulose Scaffolds
Olyveira et al.
Figure 6. Viability cell essay over a time period of 7 days, 14 days, 21 days and 28 days in control group (C), bacterial cellulose (AP), and bacterial cellulose/nanotoliths (A1).
CONCLUSION
Pure Bacterial cellulose and bacterial cellulose nanocomposites showed great response with cell essays over time
and these results show BC/BC nanocomposite as potential
biomaterial for cell delivery applications, mainly because
their natural properties and constitution are like the extracellular matrix. Their degradability, biocompatibility, low
cost and intrinsic cellular interaction make them very
attractive candidates for biomedical applications. However,
a better controlled development in methods for production,
fermentation and filler agglomeration control is essential
for better surface morphology with higher adhesion and
viability cells to widespread use of these scaffolds. However, another variables like adhesion and cells viability will
be study in future work. Thus, undoubtedly, natural-origin
polymers or nature-inspired materials appear as the natural
and desired choice for medical applications.
LIST OF ABBREVIATIONS
Nanoskin—Bacterial cellulose produced by Innovatec’s—
Biotechnology Research and Development—Brazil.
BC—Bacterial cellulose.
DMEM—Dulbecco’s Modified Eagle Medium.
EDTA—Ethylenediamine Tetraacetic acid.
ECM—Extracellular matrix.
OTL—Otoliths supplied by Innovatec’s—Biotechnology
Research and Development-Brazil.
6
Acknowledgments: This work was supported by grants
from the National Council for Scientific and Technological
Development (CNPq) and FAPERGS (Fundação de Apoio
à pesquisa do Estado do Rio Grande do Sul) and Stem
Cell Research Institute (SCRI).
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