Bacterial Cellulose/Chondroitin Sulfate for Dental Materials Scaffolds

1. Copyright © 2014 American Scientific Publishers
All rights reserved
Printed in the United States of America
Journal of
Biomaterials and Tissue Engineering
Vol. 4, 1–5, 2014
Bacterial Cellulose/Chondroitin Sulfate for
Dental Materials Scaffolds
Gabriel Molina de Olyveira1 ∗ , Márcio Luiz dos Santos1 , Paula Braga Daltro1 , Pierre Basmaji2 ,
Gildásio de Cerqueira Daltro3 , and Antônio Carlos Guastaldi1
1
2
Department of Physical Chemistry-UNESP, 14800-900, Brazil
Innovatec’s—Biotechnology Research and Development, São Carlos-SP, 13560-042, Brazil
3
College Hospital Complex Professor Edgard Santos (COM-HUPES)
Bacterial cellulose (BC) has become established as a remarkably versatile biomaterial and can be
used in a wide variety of scientific applications, especially for medical devices. In this work, the
bacterial cellulose fermentation process is modified by the addition of chondroitin sulfate (1% w/w)
to the culture medium before the bacteria are inoculated. Besides, biomimetic precipitation of calcium phosphate of biological interest from simulated body fluid on bacterial cellulose was studied. Chondroitin sulfate influences in bacterial cellulose were analyzed using transmission infrared
spectroscopy (FTIR), XRD (X-ray diffraction) and scanning electron microscopy (SEM). FTIR analysis showed interaction between chondroitin sulfate, bacterial cellulose and calcium phosphate and
XRD demonstrated amorphous calcium phosphate and carbonated apatite on bacterial cellulose
nanocomposites. SEM images confirmed incorporation of calcium phosphate in bacterial cellulose
nanocomposite surface and uniform spherical calcium phosphate particles. Future experiments with
cells adhesion and viability are in course.
Materials.
1. INTRODUCTION
The development of materials for tissue repair or replacement of lost tissues or organs has been encouraged by the
progress of research in the field of biomaterials, contributing to improving the quality and life expectancy of the
population.1
In this way, the advance of research on implants gave
incentive for research of biomaterials, and to study the
reactions that occur in the tissue-implant interface.2
With rapid development technology has enabled
advances in this area, increasing the efficiency of dentistry
implants, and an important functional role in the reconstruction/aesthetics of the patient.3
The commercially pure titanium (cpTi) and its alloys
are the most widely used biomaterials for the manufacture of dental implants due to its excellent biocompatibility
and recognized, low modulus, high strength and corrosion
resistance when compared to other metallic biomaterials.
The main problem in the production of parts of titanium
(Ti) is the high cost of raw materials and their processing.4
∗
Author to whom correspondence should be addressed.
J. Biomater. Tissue Eng. 2014, Vol. 4, No. 2
Nowadays, there is a significant number of metallic biomaterials, ceramics, polymers and composites, as options
for use in various fields of dentistry and medicine. Experience with biomaterials represents a major advance in dentistry implant, but still requires studies of the bioactivity
and biocompatibility in the human body.5 6
As alternative biomaterials, the ceramics-based materials were highlighted because bioceramics has no local or
systemic toxicity, absence of response to a foreign body
or inflammation and apparent ability to integrate with the
host tissue.7–9 Other biomaterials are biopolymers based on
chitosan, polyurethane (PU), poly(lactic-co-glycolic acid)
(PLGA) and among these bacterial cellulose produced by
Acetobacter xylinum.10
Bacterial cellulose (BC), provides an advantage over
the conventional material, as the bacterial culture can be
directed into the production of cellulose chains exclusively, yielding the purest form of the polymer (containing no lignin or hemicelluloses). Experimentally, it was
found that BC also provided the benefit of finer fibrils
of the material, suitable for use in composites. In literature, BC is reported as being produced with high crystallinity, fine microstructure (25–50 nm in diameter) and
2157-9083/2014/4/001/005
doi:10.1166/jbt.2014.1155
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RESEARCH ARTICLE
Keywords: Bacterial Cellulose (Nanoskin), Nanocomposites, Scaffolds, Dental Scaffolds

2. Bacterial Cellulose/Chondroitin Sulfate for Dental Materials Scaffolds
RESEARCH ARTICLE
long fibre length (∼ 5 m).11–16 Bacterial cellulose has also
been found to have a larger degree of polymerization in
comparison to that of wood pulp cellulose.
Several techniques of surface modification of surgical implants were developed in order to optimize the
adhesion of bone tissue and therefore the implant to the
bone. Among these, we highlight the process of deposition of coatings on implants that combine features of
specific surface coating with structural properties of the
substrate. To increase biocompatibility and bioactivity of
titanium, the use of hydroxyapatite phase (HA) has been
recommended for coating which accelerates the amount of
bone fixation and increases the longevity of the surgical
implant.17 18
Based on these properties, therefore, a biomaterial
should provide consistency with the mechanical properties
of the tissue to be regenerated and provide interface stability tissue/implant, as well as being biodegradable during
bone regeneration. Several works reported bacterial cellulose nanocomposites with chemical changes in bacterial
surface for hydroxyapatite adhesion.19 20
In this work, it is reported chondroitin sulfate/bacterial
cellulose for dental materials scaffolds without membrane chemical modification, this proposal aims to obtain
nanocomposite biomaterial for implant and bone regeneration with functionality. Future experiments with cells
adhesion and viability are in course.
2. MATERIALS AND METHODS
2.1. Materials
The bacterial cellulose raw material (Nanoskin) was provided from Innovatec’s (São Carlos SP, Brazil). Chondroitin sulfate was provided by MAPRIC (Brazil).
2.2. Methods
2.2.1. Synthesis of Bacterial
Cellulose/Chondroitin Sulfate
The acetic fermentation process is achieved by using glucose as a carbohydrate source and green tea as nitrogen source. Results of this process are vinegar and a
nanobiocellulose biomass. The modified process is based
on the addition of chondroitin sulfate (1% w/w) to the
culture medium (green tea) before the bacteria are inoculated. Bacterial cellulose (BC) is produced by Gramnegative bacteria Gluconacetobacter xylinus, which can
be obtained from the culture medium in a pure 3-D
structure, consisting of an ultra fine network of cellulose
nanofibers.21
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 and (2) BC/chondroitin sulfate. After,
BC pieces were immersed in 1.5 SBF solution at 37 C
for 7 days. The 1.5SBF solution was prepared from a
2
Olyveira et al.
Fig. 1. Flow Diagram of bacterial cellulose synthesis and bionanocomposite preparation.
protocol developed by Aparecida.22 Finally, the obtained
BC nanocomposites were oven dried. In Figure 1 is ilustred flow diagram demonstrating the synthesis of bacterial
cellulose as well as bionanocomposite preparation.
2.3. Characterization
Scanning Electron Microscopy (SEM) images were performed on a PHILIPS XL30 FEG. The samples were covered with gold and silver paint for electrical contact and
to perform the necessary images.
Transmission infrared spectroscopy (FTIR, Perkin
Elmer Spectrum 1000) Influences of chondroitin sulfate in
bacterial cellulose were analyzed in a range between 250
and 4000 cm−1 and with 2 cm−1 resolution with samples.
XRD (X-ray diffraction) were performed on a diffractometer Rigaku-DMax/2500PC, Japan ) with Cu–Ka radiation ( = 1 5406 Å), scan speed 0.02 /min in a range of
10–70 . Crystallographica search match software was used
to identify the crystal structure of samples.
3. RESULTS AND DISCUSSION
3.1. SEM
Pure Bacterial cellulose mats and chondroitin sulfate/bacterial cellulose were characterized by scanning
electron microscopy (SEM). Figures 2(a) and (b) shows,
as an example, SEM images of both bacterial cellulose
mats. It can be observed similar morphology with chondroitin sulfate/(1% w/w) bacterial cellulose sample and
pure bacterial cellulose mats which proved the addition
of the appropriate amount chondroitin sulfate causes no
major morphological changes.
It is believed that the primary hydroxyl group of cellulose does not have enough reactivity to grow hydroxyapatite. Therefore, surface modification is needed to stimulate
apatite formation on cellulose. The pre-incubation in the
J. Biomater. Tissue Eng. 4, 1–5, 2014

3. Olyveira et al.
Bacterial Cellulose/Chondroitin Sulfate for Dental Materials Scaffolds
CaCl2 solution is believed to provide the supersaturation
of Ca2+ ions around BC through ionic interaction between
calcium ions and the negatively charged OH groups available on BC and/or physical entrapment due to the 3-D network structure of the BC with tiny hollow spaces. Then the
incorporated calcium ions can bind phosphate ions to form
the initial nuclei. Once the apatite nuclei are formed, they
grow by uptake of calcium and phosphate ions from the
surrounding SBF fluid.17–20
However, biomimetic precipitation of calcium phosphate
from simulated body fluid (SBF) on bacterial cellulose
(BC) was perfomed and it can be observed by SEM images
in Figures 3(a), (b) that calcium phosphate are deposited
on bacterial cellulose nanocomposites, which shows that
the deposition process do not need surface chemistry for
the incorporation of calcium phosphate.
3.2. FTIR
The main features of the bacterial cellulose in
infrared spectroscopy is: 3500 cm−1 : OH stretching,
J. Biomater. Tissue Eng. 4, 1–5, 2014
Fig. 3. SEM aimages of Bacterial cellulose nanocomposites with superficial calcium phosphate.
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.21 23
It can be observed from Figure 4(a), the transmittance intensity is different of bacterial cellulose and bacterial cellulose nanocomposites, which means the exposed
groups are interacting with bacterial cellulose components.
Changes in symmetrical stretching CH2 bonds of bacterial
cellulose structures in 1640 cm−1 and another absorption
peak was obtained in a range of 1490 cm−1 , which shows
the presence of a carbonyl group in the bacterial cellulose together with bonds corresponding to those of glycoside, including C O C at 1162 cm−1 (as in case of
natural cellulose). These results, clearly, shows one possible interaction between bacterial cellulose and chondroitin
sulfate mainly by hydrogen interactions between hydroxyl
and carbonyl groups.21 23
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Fig. 2. (a) SEM image of bacterial cellulose; (b) SEM image of bacterial cellulose/chondroitin sulfate.

4. Bacterial Cellulose/Chondroitin Sulfate for Dental Materials Scaffolds
Olyveira et al.
(a)
BC
BC/SBF
Intensity
80
60
40
CH2
Transmittance (u.a)
100
0
4000
C=O
C–O–C
20
Bacterial cellulose
Bacterial cellulose/chondroitin sulfate
3500
3000
2500
2000
1500
1000
500
10
20
30
Wavenumber (cm–1)
3500
3000
2500
2000
Wavenumber
1500
PO4
P-OH
PO4–CO3
Transmittance (u.a)
RESEARCH ARTICLE
60
70
4. CONCLUSION
BC/SBF
BC/chondroitin sulfate SBF
1000
(cm–1)
Fig. 4. (a) FTIR spectra from bacterial cellulose and bacterial cellulose nanocomposites; (b) bacterial cellulose and nanocomposites
with SBF.
In Figure 4(b), sample was subsequently soaked in 1.5
SBF solution in order to promote absorption of calcium
phosphate on nanocomposites surface. The bands displayed by IR spectroscopy were characteristic of the
groups (PO4 in 454, 544 and 595 cm−1 P OH (879 and
944 to 1095 cm−1 ) and carbonated apatite types A and B
(respectively, 1547 and 1311, 1468 cm−1 ).
3.3. XRD
It can be seen in Figure 5 behavior with respect to the
coating SBF. The peaks observed at 14.4 , 16.9 and 22.5
are attributed to bacterial cellulose, BC was identified as
native cellulose (PDF #50-2241) and the characteristic
peaks are indexed. Besides, amorphous calcium phosphate
(PDF #18-303) and carbonated apatite (PDF #19-272)
peaks are indexed in sample with calcium phosphate.22
Then, this indicates the amorphous calcium phosphate and
carbonated apatite are formed on the surface of bacterial
cellulose.
4
50
Fig. 5. XRD patterns of bacterial cellulose and bacterial cellulose/chondroitin sulfate.
(b)
4000
40
2θ
Bacterial cellulose was successfully modified by changing
the fermentation medium as shown with SEM and FTIR,
which produced scaffolds with different surface morphology because of calcium phosphate deposition. Natural
scaffolds with bacterial cellulose and bacterial cellulose
nanocomposites have good calcium phosphate incorporation over time between tested samples, being an extremely
effective material for dental scaffolds application, such
nanocomposites present similar behavior without membrane chemical modification with what is seen in the literature. Future experiments with cells adhesion and viability
are in course.
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