Journal of Biomaterials and Nanobiotechnology, 2011, 2, 239-243
doi:10.4236/jbnb.2011.23030 Published Online July 2011 (http://www.SciRP.org/journal/jbnb)
239
First Otoliths/Collagen/Bacterial Cellulose
Nanocomposites as a Potential Scaffold for
Bone Tissue Regeneration
Gabriel Molina de Olyveira1, Daisy Pereira Valido3, Ligia Maria Manzine Costa1,
Plácia Barreto Prata Gois3, Lauro Xavier Filho3, Pierre Basmaji2
1Department of Nanoscience and Advanced Materials, Federal University of ABC, Santo André, Brazil; 2Innovatec’s-Biotechnology
Research and Development, São Carlos, Brazil; 3Natural Products Laboratory and Biotechnology, UNIT, Aracaju, Brazil.
Received March 11th, 2011; revised May 4th 2011; accepted May 15th, 2011.
ABSTRACT
In the present work, we report the first bionanocomposite ma terial formed b y otolith s/ collag en/ bacterial cellu lose (BC)
networks (OCBC). This biomaterial is an osteoinductor or be, stimulates the bone regeneration, enabling b igg er mi gra-
tion of the cells for fo rm ation o f the bon e tissu e regen eration main ly b ecaus e nanoto lith are rich in m inerals considered
essential to the bone mineralization process on a protein matrix (otolin). The objective in this study was to analyze the
regeneration capacity of bone defects treated with this bionanocomposite. Histological experiments shows bone tissue
formation with high regularity, higher osteoblast activity and osteo-reabsorption activities areas. The results suggest
the potential for this new biomaterial as a scaffold for bone tissue regeneration.
Keywords: Bacterial Cellulose, Natural Composites, Bionanocomposit es , Tissue Engineering, Bone T i ss u e Regenerat i on
1. Introduction
Despite many advances in tissue engineering (TE), scien-
tists still face significant challenges in repairing or re-
placing soft tissues such as tendons, ligaments, skin, liver,
nerve and cartilage to improve the quality of people life.
Conventional therapeutic treatments targeted to recon-
struct the injured tissues or organs have some limitations
such as donor limitations and graft rejections [1]. To meet
all the necessary requirements for the success of these
approaches, the choice of polymer, design of the matrices
and knowledge of the factors affecting
cell/tissue-material interactions should be investigated.
The ideal scaffold also requires a desirable mechanical
rigidity and a porous 3D structure which can provide ma-
ximum integration with cells and body fluids, plus have a
nanostructure surface which facilitates the adhesion of
cells [1]. In tissue engineering, matrices are developed to
support cells, promoting their differentiation and prolif-
eration towards the formation of a new tissue. Such
strategies allow for producing hybrid constructs that can
be implanted in patients to induce the regeneration of ti-
ssues or replace failing or malfunctioning organs.
Natural based polymers offer the advantage of being
similar to biological macromolecules, which the biologi-
cal environment is prepared to recognize and deal with
metabolically. Owing to their similarity with the extracel-
lular matrix (ECM), natural polymers may also avoid the
stimulation of chronic inflammation or immunological
reactions and toxicity, often detected with synthetic po-
lymers [2]. Bacterial cellulose (BC) has established to be
a remarkably versatile biomaterial and can be used in
wide variety of applied scientific endeavours. Due to its
unique nanostructure and properties, microbial cellulose
is a natural candidate for numerous medical and tissue-
engineered applications. Moreover, the nanostructure and
morphological similarities with collagen make BC attrac-
tive for cell immobilization and cell support [3-5].
The application of physical barriers in order to regen-
erate bone defects was first described by Dahlin et al. [6]
The involvement of repair by fibrous union occurs in the
bone defect, invasion of fibroblasts in the blood clot. For
this to be avoided, one can resort to surgical techniques
that prevent the blood clot and/or bone lesion are popu-
lated by undesirable cells by application of physical bar-
riers or membranes, thereby facilitating the migration of
cells with osteogenic potential. Thus, it is prevented that

First Otoliths/Collagen/Bacterial Cellulose Nanocomposites as a Potential Scaffold for Bone Tissue Regeneration 240
the fibroblasts to colonize the clo t before the osteoblasts,
which, being more specialized, are slower in its function.
[7] Some in vitro studies have been conducted to verify
the performance of membranes in the cartilage tissue as
flattering to the migration of chondrogenic cells, as oc-
curs in bone tissue [8,9]. Biosynthetic membranes show
good acceptance by the body, protecting and aiding the
repair of damaged areas by selective permeability, and
prevent contamination by microorganisms [10,11]. The
biosynthetic membrane based on cellulose was used in
experiments in dogs trochleoplasty, not interfering in
biomechanics and even in the intra-articular [12]. More-
over, it accelerated the initial repair of the trochleoplasty
area, showing good integration of newly formed tissue
with the adjacent cartilage. However, as a single part, is
not effective to promote complete regeneration of articu-
lar cartilage [12]. According to Helenius et al. [13], the
cellulose membrane obtained from bacteria has good
compatibility and has promising potential for use in tis-
sue engineering.
The mimicking of ECM by using natural origin mate-
rials has been further attempted using complementary
approaches too, in order to improve the performance of
these materials. A nano- and microfiber combined
starch-based scaffold [14] showed that its unique archi-
tecture, being able to support and guide cells, can also
provide an ideal structure for cell deposition and organi-
zation to be used to bone tissue engineering. Grande et al.
[15] fabricated BC-Hap nanocomposites to bone tissue
regeneration by the formation of cellulose nanofibrils in
the presence of a mineral phase in a static culture. In or-
der to suspend Hap nanoparticles, bacteria culture me-
dium were modified with carboxymethylcellulose (CMC).
In vitro biocompatibility and v iability was assessed using
HEK cells. The pore size and fibre diameter of BC net-
works are influenced by the water extraction method.
Studies reported by Bhattarai et al. [16] and Yang et al.
[17] indicate that the pore size and fibre diameter of
scaffolds influence the cell growth.
In this work, we report a new bionanocomposite mate-
rial formed by otoliths/collagen/bacterial cellulose (BC)
networks (OCBC). The biomaterial (OCBC) is consti-
tuted of some elements constituents of the bones, as col-
lagen (protein) and nano-otoliths, beyond the membrane
of bacterial cellulose. Collagen is regarded by many as
an ideal scaffold or matrix for tissue engineering as it is
the major protein component of the extracellular matrix,
providing support to connective tissues such as skin,
tendons, bones, cartilage, blood vessels, and ligaments
[18,19]. In its native environment, co llagen interacts with
cells in connective tissues and transduces essential sig-
nals for the regulation of cell anchorage, migration, pro-
liferation, differentiation, and survival [19,20]. Collagen
scaffolds, due to their fast degradation , do not allow iso-
morphous replacement with a newly formed bone. Pre-
sumably, due to the stable macro porous structure and
slow degradation, the progression and extent of osteo-
genesis were markedly and significantly higher for silk
and RGD–silk scaffolds when compared with collagen
scaffolds [20]. Then, BC is our option to be a substrate
with collagen and nano-otholits.
Nano-otholits is an osteoinductor or be, stimulates the
bone regeneration, enabling bigger migration of the cells
for formation of the bone tissue. Otoliths of Cynoscion
acoupa are small particles, composed of a combination of
a gelatinous matrix and calcium carbonate, present in the
ear internal bony fishes and are part of a system which
acts as a sensor of depth and balance, so as a detector of
sound vibrations. The Cynoscion acoupa is commercial-
ized in all the coast of Brazil. The Cynoscion acoupa of
the fish demonstrated to be an important source of colla-
gen too; the membranes of collagens can be gotten from
the acid extraction and posterior saline precipitation.
New advances in bone tissue engineering have moti-
vated the search for new materials that are biocompatib le
with the different bioactive functions which actually oc-
cur in live, growing tissues. [21] This b iomaterial has po-
tential also as help in others kinds of bone regeneration,
of threesomes or small fractures or same cases associated
to the osteoporosis. The goal of this study is to produce
highly efficacious scaffolds to engineer functional bone
tissue with natural bone histological structure and prop-
erties for the cure of bone loss in clinical settings.
2. Experimental Details
2.1. Materials
Bacterial cellulose membranes, ~500 mm thick, were
supplied from Innovatecs-Produtos Biotecnológicos Ltda,
Brazil; Otoliths were supplied by VIAFARMA LTDA,
Brazil and Collagen were supplied by Sigma Aldrich.
2.2. Synthesis of Bacterial Cellulose
Bacterial Cellulose (BC) produced by Gram-negative
acetic acid bacteria Gluconacetobacter xylinus can be
obtained from the culture medium in the pure 3-D struc-
ture consisting of an ultra fine network of cellulose nano-
fibres (3 - 8 nm), highly hydrated (99% in weight), and
displaying higher molecular weight, higher cellulose
crystallinity (60% - 90%), enormous mechanical strength
and full biocompatibility [2 2].
2.3. Nano-Otholits 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, di-
luted in distilled water. The final product was packaged

First Otoliths/Collagen/Bacterial Cellulose Nanocomposites as a Potential Scaffold for Bone Tissue Regeneration 241
in dishes Petri and sterilized in UV rays (25 min). Sub-
sequently, 1.0 g of the otoliths was diluted in 100 mL of
distillated water, and the pH of the compound was as-
sessed by using phmetro Digimed® (São Paulo, SP, Bra-
zil) according to the manufacturer instructions. Stable gel
is formulated with a otoliths calcium salt concentration
solution. The use of poorly water-soluble salts (CaCO3)
influences gelatin rate and, consequently, mechanical
properties.
2.4. Bionanocomposite Preparation
In the present study, we have explored a novel biomate-
rial, and prepared different bacterial cellulose nanocom-
posites(BC); 1) Pure BC, 2) BC with collagen and 3)
BC/otoliths/collagen. Bacterial cellulose nanocomposite
was obtained by immersion of dried bacterial cellulose
into collagen and otolith/collagen gels and posterior soft
drying at 50˚C by 12 hours.
Bionanocomposites Characterization
Scanning Electron Microscopy (SEM)-Scanning elec-
tronic microscopy images were performed on a PHILIPS
XL30 FEG. The samples were covered with gold and
silver paint for electrical contact and to perform the nec-
essary images.
Histological Experiment—The experiment was per-
formed with 20 Wistar rats randomized into two groups,
in which a bone defect was inflicted in th e tibia. In group
1 (experimental), bone cavities were filled with otoliths
nanocomposites [23].
3. Results and Discussion
3.1. Scanning Electronic Microscopy (SEM)
Bacterial cellulose mats were characterized by SEM.
Figure 1 shows, as an example, SEM image of Bacterial
cellulose mats.
In order to obtain a bionanocomposite with mechanical
properties and an osteoconductive environment that can
facilitate cell attachment [24] and to increase in vitro pro-
liferation and differentiation of osteoblastic cells [25] as
well as the rapid vascularization and deposition of con-
nective tissue and calcified matrix in vivo, BC/col- la-
gen/nano-otholits were obtained.
In Figures 2 and 3, scanning electron microscopy
(SEM) image of the bacterial cellulose/collagen surface
morphology and bacterial cellulose/collagen/otholits na-
nocomposites is ilustred.
3.2. Cellulose Bacterial/Otoliths/Collagen
Membrane in Vivo Biological Performance
The Histological sections revealed that the newly formed
trabecular bone was denser and the periosteal reabsorp-
tive activity was less conspicuous in the experimental
Figure 1. Scanning elec tron microscopy (SEM) of pure bac-
terial cellulose.
Figure 2. Cellulose bacterial/collagen surface morphology.
Figure 3. Cellulose bacterial with otoliths/collagen mem-
brane.

First Otoliths/Collagen/Bacterial Cellulose Nanocomposites as a Potential Scaffold for Bone Tissue Regeneration 242
(a)
(b)
Figure 4. (a) Osteotomized area showing irregular trabecu-
lar bone, intertwined aspect and thin thickness; (b) The
trabecular were stretching out, in the general form, from a
side to other of the bone defect induced artificially, showing
osteoblast activity and the periosteum demonstrated mod-
erate degree of fibrosis. Besides, it can be observed areas of
intense infiltration of polymorphonuclear neutrophils
(PMN) and formation of microabscesses (MAB) in Figure
4(a) and (b).
group (group 1) reported in Figures 4(a) and (b). Bio-
chemical (calcium (Ca2+) and alkaline phosphatase (Alp)
parameters were within normal range. In all the cases the
neo formations of trabecular bone was observed, the ma-
jority with irregular appearance, intensely intertwined
aspect and thin thickness. Besides, it can be observed
areas of intense infiltration of polymorphonuclear neu-
trophils (PMN) and formation of microabscesses (MAB)
in Figures 4(a) and (b).
In the experimental group (group1), we observed in
the Figures 4(a) and (b), that the bone surface tissue
present high regularity and higher osteoblast activity in
addition, osteo-reabsorption activities areas is can be
observed cleary in Figures 4( a) and (b).
4. Conclusions
We report the first otoliths/collagen/cellulose bacterial na-
nocomposites as a potential scaffold for bone regenera-
tion. The success of subsequent transplantation of the in
vitro engineered construct is due to the properties of the
materials but also on the osteoprogenitor cell sources. It
is expected that th e seeded cells will secrete specific ECM
components in vitro, to induce proliferation and differen-
tiation into osteoblasts and result in the formation of a
new bone in vivo. Otoliths/collagen/CB new scaffolds
should be designed for different applications, such as: to
induce vascularization; facilitate the deposition of oto-
liths in predefined regions; guide the regeneration of tis-
sue in certain directions permit the development of dif-
ferent tissues; or inhibit calcification and cell adhesion.
The field is widely open for new creative research- ers in
real clinical applications.
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