Advances in Biomedical and Pharmaceutical Applications of Functional Bacterial Cellulose-Based Nanocomposites

Carbohydrate Polymers 150 (2016) 330352
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Review
Advances in biomedical and pharmaceutical applications of functional

bacterial cellulose-based nanocomposites
Hanif Ullah a,c , Fazli Wahid b , Hlder A. Santos c, , Taous Khan a,
a
b
c
Department of Pharmacy, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan

Biotechnology Program, Department of Environmental Sciences, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland
a r t i c l e
i n f o
Article history:
Received 22 January 2016
Received in revised form 25 April 2016
Accepted 11 May 2016
Available online 14 May 2016
Keywords:
Bacterial cellulose
Bioengineering
Biomedical application
Composites
Nanocellulose
a b s t r a c t
Bacterial cellulose (BC) synthesized by certain species of bacteria, is a fascinating biopolymer with unique
physical and mechanical properties. BCs applications range from traditional dessert, gelling, stabilizing
and thickening agent in the food industry to advanced high-tech applications, such as immobilization
of enzymes, bacteria and fungi, tissue engineering, heart valve prosthesis, articial blood vessels, bone,
cartilage, cornea and skin, and dental root treatment. Various BC-composites have been designed and
investigated in order to enhance its biological applicability. This review focuses on the application of
BC-based composites for microbial control, wound dressing, cardiovascular, ophthalmic, skeletal, and
endodontics systems. Moreover, applications in controlled drug delivery, biosensors/bioanalysis, immobilization of enzymes and cells, stem cell therapy and skin tissue repair are also highlighted. This review
will provide new insights for academia and industry to further assess the BC-based composites in terms
of practical applications and future commercialization for biomedical and pharmaceutical purposes.
2016 Elsevier Ltd. All rights reserved.
Contents
1.
2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
1.1.
Biosynthesis of BC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
1.2.
Effect of bacterial strain on the properties and production of BC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
1.3.
Properties and biocompatibility of BC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332
Applications of BC and BC-based composites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
2.1.
Antimicrobial and antiviral lm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
2.2.
Wound healing, articial skin and skin tissue repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
2.3.
Cardiovascular system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
2.3.1.
Articial blood vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
2.3.2.
Cardiac prosthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
2.4.
Ophthalmic applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340
2.4.1.
Articial cornea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340
2.4.2.
Retinal pigment epithelium substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340
2.4.3.
Contact lenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
2.5.
Application in skeletal systems, cartilage and endodontics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
2.5.1.
Bone regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
2.5.2.
Cartilage, intervertebral discs and articial endocranium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
2.5.3.
Meniscus implant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
2.5.4.
Dental root canal treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
2.5.5.
Ligament and tendon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344
Corresponding authors.
E-mail addresses: helder.santos@helsinki. (H.A. Santos), taouskhan@ciit.net.pk
(T. Khan).
http://dx.doi.org/10.1016/j.carbpol.2016.05.029
0144-8617/ 2016 Elsevier Ltd. All rights reserved.
H. Ullah et al. / Carbohydrate Polymers 150 (2016) 330352
331
2.6.
3.
Applications in drug delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344

2.6.1.
Controlled drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344
2.6.2.
Transdermal drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344
2.7.
Applications in bioengineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
2.7.1.
Biosensors and bioanalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
2.7.2.
Enzymes and cells immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
2.7.3.
Stem cell therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348
Conclusions and overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348
1. Introduction
Cellulose is the most abundant biopolymer on earth that is frequently obtained from the plant sources (Finkenstadt, 2005; Saxena
& Brown, 2012). It is the main structural component of plant cell
wall and has an extraordinary commercial reputation in paper, textile and pulp production units (Gandini, 2008; Huber et al., 2012;
Klemm, Heublein, Fink, & Bohn, 2005). Cellulose is also biosynthesized by some oceanic animals (tunicates), and thus called as
tunicin (Zhao and Li, 2014). Other major pathways for cellulose
production are in vitro enzymatic synthesis and chemosynthesis
from glucose derivatives. The most important pathway of cellulose production is through different microbes, such as algae, fungi
(Klemm et al., 2005) and various aerobic non-pathogenic bacteria
of the genera Agrobacterium, Sarcina, Rhizobium and Glucoacetobacter (formerly Acetobacter) (Khan, Park, & Kwon, 2007; Petersen and
Gatenholm, 2011; Shezad, Khan, Khan, & Park, 2010; Shoda and
Sugano, 2005).
In 1886, Brown reported bacterial cellulose (BC) for the rst
time as a sturdy gelatinous white pellicle on a liquid medium surface during the acetic fermentations (Brown, 1886a, 1886b). The
BC membrane was produced by Bacterium xylinum with a thickness about 25 mm (Brown, 1886a, 1886b). Later on, this bacterium
was renamed as Acetobacter xylinum (A. xylinum), then as Gluconacetobacter xylinus (G. xylinus) and presently it is known as
Komagataeibacter medellinensis (Matsutani et al., 2015; Yamada
et al., 2012; Yamada, 2014).
Although BC is produced in laboratories in small scale for
research, there are some commercial outlets for BC. Besides, traditional nata de coco (Iguchi, Yamanaka, & Budhiono, 2000), a
German company, Fzmb GmbH is considered one of the largest
producers of BC for cosmetics and biomedical applications (Keshk,
2014a). In addition, Xylos Co. in USA is a producer of Prima CelTM ,
a type of BC used for wound dressing. Other brands of BC include
Gengiplex and Bioll (Keshk, 2014a) used as physical barrier for
tissue regeneration. BC is also produced and used by many food
industries in Asian countries (Budhiono, Rosidi, Taher, & Iguchi,
1999; Ng and Shyu, 2004). Sony Corporation, Japan in association
with Ajinomoto, Japan and other rms fabricated the rst BC-based
diaphragm of audio speaker. Ajinomoto, Japan also sells wet BC
(Chawla et al., 2009; Czaja, Krystynowicz, Bielecki, & Brown, 2006).
1.1. Biosynthesis of BC
G. xylinus has been employed as model microbe for basic and
applied studies on BC due to its higher production yields and its
ability to consume variety of sugars and other compounds as carbon
source (Ross, Mayer, & Benziman, 1991; Saxena and Brown, 2012).
In laboratory, glucose is usually added to the fermentation medium
and the biosynthesis of BC takes place in four enzymatic steps: (1)
phosphorylation of glucose by glucokinase to glucose-6-phosphate,
(2) isomerization of glucose-6-phosphate to glucose-1-phosphate
by phosphoglucomutase, (2) conversion of glucose-1-phosphate
to uridine diphosphate glucose (UDP-glucose) by UDP-glucose

pyrophosphorylase, (4) and the synthesis of cellulose from UDPglucose by cellulose synthase (Ross et al., 1991). The activity of
cellulose synthase in G. xylinus is regulated by an allosteric activator c-di-GMP (Ross et al., 1987; Saxena & Brown, 2012), which
is also found in other bacteria (Saxena and Brown, 2005). Cellulose
synthases from different organisms have been found with sequence
similarities (Nobles and Brown, 2004).
BC is biosynthesized in the form of a ribbon that projects from
the pole of bacterial rod. The ribbon elongates at a rate of 2 m/min.
A single G. xylinus cell synthesizes a ribbon composed of 10100
microbrils (Brown, 1985, 1996). There are 50 individual sites for
BC synthesis, organized in a row along the longitudinal axis in close
association with the outer envelope of bacterial cell. The assembly
of BC microbrils is a two-step process: polymerization of glucose
residue to form -1, 4-glucan chain (in the plasma membrane) and
crystallization of glucan chain to cellulose in extracellular medium
(Brown, Willison, & Richardson, 1976). Moreover, crystallization
and polymerization are coupled processes directed by bacterial
cell and the rate of crystallization determines the rate of polymerization (Benziman, Haigler, Brown, White, & Cooper, 1980). In
addition, the assembly of the cellulose crystallite is proposed to
occur in two steps:formation of sheets of glucan chain by van der
Waals forces followed by stacking of the sheets to give crystalline
structure (Cousins & Brown, 1995; Cousins & Brown, 1997a, 1997b).
1.2. Effect of bacterial strain on the properties and production of
BC
Currently, several microorganisms have been reported with the
ability to produce BC having numerous physicochemical properties
for biological applications (Lee, Buldum, Mantalaris, & Bismarck,
2014). For example, Salmonella spp. and Escherichia coli (E. coli)
were reported for the production of BC; however, the amount of
BC produced by these bacteria is lower than that of G. xylinus (Lin,
Calvar et al., 2013). In order to increase the production of BC, E. coli
was transformed with BC producing capability similar to G. xylinus.
The production of gluconic acid during BC biosynthesis decreases
the pH of the medium leading to decreased production of the former. In order to avoid this, De Wulf, Joris, and Vandamme (1996)
genetically engineered, mutant G. xyinus, limiting its ability for gluconic acid production. As a result, the pellicle size was doubled as
compared to the wild type G. xylinus. Similarly, glutamate dehydrogenase decient mutant strain of G. xylinus BPR 2001 (GD-1) does
not produce gluconic acid, but produces twice as much BC than the
parent strain (Shigematsu et al., 2005). Moreover, mutant type G.
xylinus produces cellulose II in which the glucan chains are arranged
antiparallel in comparison to parallel arrangement of glucan chains
(cellulose I) produced by wild type G. xylinus (Kuga, Takagi, &
Brown, 1993). The average length of glucan chain in cellulose II was
about 10 times the width of the strand that suggests the folding of
glucan chain, thus arranged in antiparallel manner. Using sucrose
or glucose as a source of carbon, the main product of G. xylinus is
332
ketogluconate and not cellulose (Masaoka, Ohe, & Sakota, 1993).

Ketogluconate-negative strains have been isolated with increased
BC yield from 1.8 gL1 (the parent strain) to 3.3 gL1 after 10 days of
cultivation, while consumption of glucose decreased from 22.6 gL1
to 7.3 gL1 (Masaoka et al., 1993). The dgc1 gene plays a role
in the biosynthesis of BC through activation of c-di-GMP (Bae,
Sugano, Ohi, & Shoda, 2004). Genetically modied G. xylinus BPR
2001, i.e., dgc1-disrupted mutants were expected to decrease the
BC production. Contrary to this, the dgc1-disrupted mutants gave
approximately the same yield as for the parent strain, in both static
and shaking ask cultures. However, in stirred tank fermenter, the
BC yield of dgc1-disrupted mutants was increased by 36% as compared to the parent strain tank reactor (Bae et al., 2004) with the
assumption that dgc2 and dgc3 complemented or even improved
the production of BC. Conversely, a decrease in BC production with
dgc1-disrupted mutant has also been reported (Tal et al., 1998).
The contradiction could be due to the shorter cultivation time in
the latter case during evaluation of the BC yield (Lee et al., 2014). G.
xylinus secretes the viscous water-soluble polysaccharide acetan
during BC production (Couso, Ielpi, & Dankert, 1987). Acetan is
produced by G. xylinus from UDP-glucose, which is also a precursor for BC. Hence, inhibiting the acetan production is expected to
increase the concentration of UDP-glucose and in turn increasing
the BC yield. However, non-acetan producing mutant strain (EP1),
derived from G. xylinus BPR 2001 decreased the yield of BC in a
shaking ask culture with no signicant difference under static
conditions (Ishida, Sugano, Nakai, & Shoda, 2002). The reduced BC
yield of EP1 strain was attributed to the reduced viscosity of the
culture medium (in absence of acetan) that increased the chance of
coagulation of the cells and BC, and thus decreasing the BC yield.
Likewise, genetically engineered G. xylinus ATCC53582 produced
ve times more BC than ATCC23769 even after all glucose was
consumed (Kawano et al., 2002). The possible reason for this may
be that either gluconic acid was used as a source of carbon or the
strain switched to gluconeogenesis pathway. Similarly, genetically
modied strain with overexpressed endoglucanase gene (CMCax)
exhibited 1.2-fold higher BC yield. Using ultraviolet (UV) radiation and subsequent ethyl methane sulfonate (EMS) treatment,
Hungund and Gupta (2013) reported the strain improvement of
G. xylinus in terms of BC production. The EMS treated mutant has a
50% and 98% more BC yield than parent UV mutant and wild strains,
respectively. Likewise, an improved BC yield was achieved with
a mutant G. xylinus strain produced by high hydrostatic pressure
(250 MPa) at 25 C for 15 min (Wu et al., 2010). Moreover, when
grown on Hestrin-schramm medium with or without ascorbic acid
(vitamin C), the yield of BC from the different strains of G. xylinus
was in the order of 13773 > 13693 > 13772 > 10245 (Keshk, 2014b).
Using Gluconacetobacter europaeus (G. europaeus) and G. xylinus
strains, Zeng et al. (2014) concluded that BC with varying properties can be fabricated by different bacterial strains. BC produced
by G. europaeus exhibited extremely low density, higher porosity
and less lm thickness than that of G. xylinus. Supercritically dried
BC of G. europaeus exhibited good water absorption capacity (110
times of dried weight) as compared to that of G. xylinus (66 times
dried weight). Moreover, BC produced by G. europaeus displayed
robust mechanical properties irrespective of drying method with
higher elastic parameter as compared to BC from G. xylinus (Zeng,
Laromaine, & Roig, 2014). BC of G. europaeus possessed relatively
higher penetration depth, lower hardness and lower Youngs modulus in case of freeze drying and room temperature drying, and the
opposite for supercritical drying (Zeng et al., 2014). Relatively thin
BC brils (510 nm) produced by Asaia bogorensis suggest variation in the biosynthesis or organization of synthesis sites between
bacterial strains (Kumagai et al., 2011).
Furthermore, genetically engineered G. xylinus with genes from
Candida albicans are capable to produce BC with improved in vivo
biodegradability. The cellulose synthase in G. xylinus can consume

both UDP-glucose and UDP-N-acetylglucosamine (UDP-NAcG) as
substrates (Lee et al., 2001, 2014; Shirai et al., 1994). The presence
of NAcG makes BC vulnerable to the action of lysozyme and also
disturbs the highly ordered crystalline structure of BC. BC produced
by the engineered strain contained NAcG and showed half of crystallinity as compared to that of parent strain. Moreover, BC was
entirely degraded in 10 days (Yadav et al., 2010).
1.3. Properties and biocompatibility of BC
The properties of the BC are fairly diverse from those of plant
cellulose (PC), which allow BC applications in various elds ranging from physics to chemistry and from engineering to biological
sciences (Hu, Chen, Yang, Li, & Wang, 2014). It is produced in an
extremely pure form, which is entirely devoid of pectins, lignin and
hemicelluloses (Chawla, Bajaj, Survase, & Singhal, 2009), leading to
simpler purication process as compared to PC (Shi, Zhang, Phillips,
& Yang, 2014). The as-synthesized BC possesses highly porous
structure with high permeability to uids, favourable for cell adhesion and proliferation, and high water-uptake capability (water
content >90%) that provides moist environment to the wound and
absorbs exudate (Czaja, Young, Kawecki, & Brown, 2007; Klemm,
Schumann, Udhardt, & Marsch, 2001). These properties of BC are
due to its ultrane network structure composed of ribbon-shaped
micro- and nanobrils (100 times thinner than PC bres), which
make it suitable candidate for biomedical applications (Chawla
et al., 2009). BC in never-dried form has exceptional mechanical
characteristics with a stress-strain behaviour that resembles soft
tissue (Svensson et al., 2005; Chawla et al., 2009). The stress at break
of BC pellicles is about 2 106 Pa despite of 99% water content. BC
also possesses exceptional mechanical properties in the dried form
due to its crystalline nanobrillar network. The tensile strength of
a single BC bre is almost comparable to Kevlar and steel (Yano
et al., 2005), making it a suitable candidate for application where
high mechanical performance is desired. BC has been demonstrated
to possess superior properties to conventional dressings in terms
of ease of wound inspection (due to transparency), reduction of
infections and wound pain, improved exudate retention, less healing time, adherence to wound, and ease of removal after enhanced
epithelization (Czaja et al., 2006; Czaja et al., 2007; Keshk, 2014a).
BC mimics extracellular matrix for growth and proliferation of cells
(Petersen and Gatenholm, 2011). BC has many other superior properties (summarized in Fig. 1), which make it an ideal candidate
for various biomedical, pharmaceutical and industrial applications.
Biodegradable scaffold is preferred in some biomedical applications, whereby it is replaced by natural tissue over times, e.g., bone,
skin and wound healing (Petersen and Gatenholm, 2011). BC as an
implant, is almost nonbiodegradable by human body due to lack
of degradation enzymes, i.e., cellulases (Petersen and Gatenholm,
2011). The slow degradation of BC in human body (Czaja et al., 2007)
may be attributed to spontaneous, nonenzymatic hydrolysis of cellulose chains, though this is still a controversial matter. However,
researchers have made some attempts to make BC biodegradable by
modication of cellulose by oxidation (Laurence, Bareille, Baquey,
& Fricain, 2005; Petersen and Gatenholm, 2011; Singh, Ray, Verma,
& Guha, 1979), making composites of BC with cellulases (Hu &
Catchmark, 2011a, 2011b) and in situ incorporation of NAcG to
BC via genetically modied strains (Yadav et al., 2010). The harmless degradation product, i.e., glucose makes BC and its composites
ideal in many wound dressing and tissue engineering applications
for bioresorbable purposes. BC has negligible amount of endotoxin
and has been approved by Food and Drug Administration (FDA)
for use by Xylos Co. as MTATM Surgical Sheet, Xylos Vessel Guard,
Xylos Porous Surgical Mesh and SecurianTM Tissue Reinforcement
Matrix (Petersen and Gatenholm, 2011). BC exhibits higher com-
333
Fig. 1. Various superior properties of bacterial cellulose compared to plant-based cellulose.
plement activation parameters in comparison to conventional graft

materials, such as poly(ethylene terephthalate) (PET) and expanded
polytetrauoroethylene (ePTFE) (Finket al., 2011), but there is a
need for systemic study of the effects of BC on complement activation and immune responses as there is no reported correlation
in these areas of research. Thrombogenicity study in human blood
plasma showed that BC induces a slower coagulation in comparison
to clinically available materials, such as Dacron and Gore-Tex . BC
is also devoid of skin irritation potential (Almeida et al., 2014).
Wounds treated with BC composites have shown proper moisture contents for an extended time, and faster epithelialization
and regeneration in rat models than those treated by Tegaderm
(Lin, Lien, Yeh, Yu, & Hsu, 2013). Skin wounds in Wistar rats
showed faster recovery and less inammatory response when covered with BC in comparison to the conventional gauze (Li et al.,
2015). Similarly, diabetic foot wounds treated with BC have shown
shorter epithelization time in comparison to commercially available XeroformTM petrolatum gauze (Solway, Clark, & Levinson,
2011). BC and its composites possess good hemocompatibility (low
Factor-XII and platelets activation) in comparison to ePTFE (Leitao,
Gupta, Silva, Reviakiftne, & Gama, 2013). Also, BC nanocomposites
have controllable compressive mechanical properties closer to that
of natural tissues, such as articular cartilage, porcine heart valve
(Mohammadi, 2011), rabbit endocranium (Lizhnln, Lixiaozh,
in, & Zhangchozhng,
Zhongm
2011), and pig meniscus
Jiang
(Bodin, Concaro, Brittberg, & Gatenholm, 2007). BC showed better desired properties than paper points for dental applications
(Yoshino et al., 2013). In biosensor applications, BC-based devices
have shown less thrombogenic effects than commercial PVC-based
electrode (Badr, Abdel-Sattar, & Keshk, 2015) and more stability than the commercial Cuprophan membrane (Eisele, Ammon,
Kindervater, Grbe, & Gpel, 1994). Such characteristics are discussed in detail in the respective sections below.
Several studies have conrmed BC and BC-based composites as
biocompatible scaffolds for cell seeding. It has been shown that
different human cells, such as smooth muscle cells (SMC) (Petersen
and Gatenholm, 2011), chondrocytes (Svensson et al., 2005), broblasts, bone forming osteoblasts (Chen et al., 2009), human umbilical
vein endothelial cells (Jeong et al., 2010), Chinese hamster ovary
cells, mouse embryo broblast 3T3 cell (Moreira et al., 2009) and
human embryonic kidney (HEK) cells (Grande, Torres, Gomez, &
2009) can grow and proliferate in the presence of BC. FurBan,
thermore, Helenius et al. (2006) demonstrated the subcutaneous
implantation of BC in rats. Under microscopy, there was no giant
cells or brotic capsule after 12 weeks of implantation, which indicated no foreign body reaction. Furthermore, no swelling, redness
or exudation was found around the implantation site. BASYC prostheses in 5 mice and 7 (out of 8) pigs have shown one year and
90 days patency, respectively, with epithelization and development
of three distinct layers, i.e., the characteristic of natural blood vessel (Schumann et al., 2009). The biocompatibility and nontoxicity
of BC-based biomaterials have been discussed in details in other
sections below.
To incorporate additional properties, such as antibacterial
activity, wound healing capability, improved cell adhesion and proliferation, more even distribution of cells, enhanced mechanical
properties, good biocompatibility and biomimetic capabilities, BCbased composites have been introduced consisting of a matrix and
reinforcement materials (Shah, Ul-Islam, Khattak, & Park, 2013;
Torres, Commeaux, & Troncoso, 2012). In this case, BC provides a
matrix for housing various materials.
Keeping in view the abovementioned exclusive properties of BC,
the objective of this review article is to provide extensive overview
on the current trends of BC-based composites for applications in
biomedical and pharmaceutical elds.
2. Applications of BC and BC-based composites

BC nds extensive applications ranging from conventional paper
and food to advanced drug delivery and tissue engineering. For
example, in food, BC has been traditionally used as a dessert in
Philippines known as nata de coco (Budhiono et al., 1999; Ng &
Shyu, 2004). In this preparation, BC is synthesized from fermentation of coconut water and sliced into small pieces and dipped in
a sugar syrup (Iguchi et al., 2000; Klemm, Heublein, Fink, & Bohn,
2005; Klemm et al., 2006). BC has also been explored as suspending, gelling, stabilizing and thickening agent in food industry (Shi,
Zhang, Phillips, & Yang, 2014). BC has been employed for immobilization of enzymes (Wu and Lia, 2008; Wu, Lia, & Ho, 2013),
bacterial cells (Rezaee, Godini, & Bakhtou, 2008) and fungi (Ton
and Le, 2011). Furthermore, the mechanical properties of BC render this biomaterial as an ideal candidate for scaffold in tissue
engineering including articial cartilage (Nimeskern et al., 2013;
Svensson et al., 2005), bone (Zimmermann, LeBlanc, Sheets, Fox, &
Gatenholm, 2011), heart valve prosthesis (Millon and Wan, 2006),
articial blood vessels (Klemm, Schumann, Udhardt, & Marsch,
2001; Wan et al., 2011), cornea (Wang, Gao, Zhang, & Wan, 2010),
dental root canal treatment (Yoshino et al., 2013), and skin tissue repair (Fu et al., 2012). BC is a non-allergenic biopolymer
and thus has been used in cosmetics as a natural facial scrub
(Hasan, Biak, & Kamarudin, 2012), facial masks to treat dry skin
(Amnuaikit, Chusuit, Raknam, & Boonme, 2011) and as a structuring
agent in cleansing formulations (Heath, Cofndaffer, Kyte, Smith,
& McConaughy, 2012).
The abovementioned studies provide a strong base for use of
BC in various elds; however, to make it more efcient for biological applications, various BC-based nanocomposites have been
designed and validated at the pre-clinical level (Hu et al., 2014). The
334
Fig. 2. Summary of various biomedical and pharmaceutical applications of bacterial cellulose-based nanocomposites.
current biomedical and pharmaceutical applications of BC-based

composites are depicted in Fig. 2, while their usefulness in antimicrobial and antiviral lms, wound dressing systems, cardiovascular
system, ophthalmic and skeletal systems, cartilage and endodontics, drug delivery systems and bioengineering is discussed in detail
in the following sections.
2.1. Antimicrobial and antiviral lm

The growing resistance of pathogenic microbes is a major concern in the pharmacotherapy of injuries and wounds (Alanis,
2005). Hence, new therapeutic candidates should not be exclusively
based on antibiotics, but also on the use of other antimicrobial
approaches. One such strategy can be the use of metallic as well
as non-metallic BC-nanocomposites. In such nanocomposites, BC
offers humid atmosphere to burns and wounds that results in an
enhanced wound healing. However, BC itself does not possess any
antimicrobial activity for the prevention of wound infections (Yang,
Xie, Hong, Cao, & Yang, 2012). In order to inherit antimicrobial properties in BC, various researchers have fabricated BC-composites
with different antimicrobial substances, which will be discussed
in the following paragraphs.
BC nanocomposites with strong antibacterial activity were prepared using in situ biosynthesis by adding partially deacetylated
chitosan nanocrystal (D-ChNC) to the culture medium. Nanocomposites were also synthesized by post-synthetic modication
via mixing aqueous suspension of D-ChNC with BC (Butchosa
et al., 2013). The antibacterial activity of these nanocomposites was increased with increasing D-ChNC concentrations. These
results suggested the potentiality of BC-D-ChNC composites as
a substitute for antimicrobial compounds. In another study, the
in situ modication of BC with chitosan resulted in a BC-chitosan
(BC-Ch) composite material having N-acetylglucosamine and glu-
cosamine units amalgamated in BC sheets (Ciechanska,

2004).
These composites had a number of valuable features including high
moisture-keeping properties, good wet-state mechanical properties, mono- and oligosaccharides release under the action of
lysozyme, bacteriostatic activity against Gram-positive and Gramnegative bacteria, and bactericidal activity against Gram-positive
bacteria. Such characteristics can make BC-Ch an exceptional
antimicrobial material for the treatment of various kinds of ulcers,
burns and wounds.
Wanling et al. (2012) reported that the air permeability of BC
can be improved for biomedical applications by adding poly(vinyl
alcohol) (PVA) in as low as 0.5% concentration. The effective pore
size of the resulting composite lm was increased and the mechanical properties were improved as compared to the parent BC. In this
patent work, BC-based protective material having effective pore
size of 5070 nm with anti-viral function was also prepared by compounding it with chitosan (Wanling et al., 2012). Most of the viruses
had an average size of more than 50 nm; therefore, these BC composites (5070 nm) could lter out most of the viruses, and thus
preventing the viral infection. Furthermore, BC-PVA composites
lms were also developed with sorbic acid (SA) for antimicrobial
purposes (Dobre et al., 2012). In this preparation, the crushed BC
worked as reinforcing bres, PVA as polymer matrix and SA as
an antibacterial agent. This BC composite lm showed antibacterial activity against E. coli (K12-MG1655). This indicates that these
novel BC-PVA-SA composites could be used as promising antimicrobial lm.
Silver (Ag0 ) nanoparticles were also incorporated into BC-PVA
membrane as an antimicrobial agent (Dobre and Stoica-Guzun,
2013). The resultant BC-PVA-Ag0 lms showed antibacterial activity against E. coli (K12-MG1655). Similarly, Ag0 nanoparticles were
incorporated in BC membranes through reducing silver nitrate
(AgNO3 ) with the help of sodium borohydride (NaBH4 ) (as a
reducing agent). The interaction between Ag0 nanoparticles and

oxygen in cellulose caused a stable nanoparticles adsorption on BC
nanobers, and showed antimicrobial activity greater than 99.99%
against Staphylococcus aureus (S. aureus) and E. coli (Jung, Kim, Kim,
& Jin, 2009). These observations were also supported by another
study where lyophilized BC-Ag0 exhibited strong antibacterial
action against Gram-negative (E. coli) as well as Gram-positive (S.
aureus) bacteria (Maneerung, Tokura, & Rujiravanit, 2008). Likewise, the antimicrobial activity of BC-Ag0 nanocomposites was
tested against Klebsiella pneumoniae (K. pneumoniae), S. aureus and
Bacillus subtilis (B. subtilis). These nanocomposites were effective
against the tested pathogens due to the presence of Ag0 nanoparticles in the cellulosic bres in concentration as low as 5.0 104 %
(w/w) (Pinto et al., 2009). These properties of BC-Ag0 lm make
it a strong candidate for burns and wounds dressing systems with
antimicrobial properties.
A simple strategy was also reported for the development of magnetic Ag0 nanocomposite, whereby the 3D nanobrous structure
of BC was subjected to homogenization with a mixture of ferrous
and ferric salts (Sureshkumar, Siswanto, & Lee, 2010). Nanoparticles of magnetite were precipitated and impregnated into BC
nanostructure by ne-tuning the pH of the blend in alkaline range.
The magnetic BC nanobers thus formed were coated with a selfpolymerized polydopamine (PDA) layer. Due to the reducing action
of PDA surface for Ag+ , the nanoparticles of Ag0 were incorporated into the PDA coated magnetic BC by soaking it in AgNO3
solution. These magnetic BC-Ag0 nanocomposites possessed significant antibacterial activity against B. subtilis and E. coli. In addition,
these magnetic BC-Ag0 nanocomposites showed potential for mild
sterilization.
In a recent study, a facile and environmentally friendly
approach was proposed for the preparation of Ag0 nanoparticles
by hydrothermal synthesis using BC as reducing as well as stabilizing agent without the use of any chemicals (Barud et al., 2011).
Under optimum conditions, small Ag0 nanoparticles with a narrow
size distribution (17.1 5.9 nm) were obtained on the BC matrix
having Ag0 content of 1.78% (w/w). The resultant product showed
sustained Ag0 release and extended antibacterial activity against S.
aureus (Yang, Xie, Deng, Bian, & Hong, 2012). Antimicrobial BC-Ag0
composite membranes were also fabricated by an in situ preparation of Ag0 nanoparticles from hydrolytic reduction of AgNO3
solution using triethanolamine (TEA) as complexing and reducing
agent. The BC-Ag0 composite membranes exhibited strong antimicrobial activity against both Gram-negative and Gram-positive
bacteria (Barud et al., 2011). Furthermore, BC-Ag0 nanocomposite
membranes were fabricated by an in situ method with an excellent antibacterial activity by introducing carboxyl groups into BC
with 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated oxidation reaction followed by an ion-exchange reaction to change the
sodium ions (Na+ ) into Ag+ . Finally, the Ag+ was reduced to Ag0 by
distinct reducing agents, sodium citrate and NaBH4 . The prepared
BC-Ag0 exhibited strong bactericidal effects against both Grampositive (S. aureus) and Gram-negative (E. coli) pathogens, with
higher antimicrobial activity of NaBH4 -reduced BC-Ag0 nanocomposites (Feng et al., 2014). Several other studies on BC-Ag0
nanocomposites for anti-microbial purposes are also summarized
in Table 1.
Inspite of efcient antibacterial effects, metallic nanoparticles
are well-known for their potential adverse effects on human health
and the environment (Ray, Yu, & Fu, 2009; Wang and Wang,
2014). For example, Trop et al. (2006) evaluated the reversible
hepatotoxicity and argyria-like symptomsin a patient with 30%
burns who was treated with Ag0 nanoparticles dressing. Moreover,
the photo, geno and cytotoxicity of Ag0 nanoparticles on human
skin HaCaT keratinocytes has also been reported (Lu et al., 2010).
335
However, this toxicity was preventable by coating Ag0 nanoparticles with a biodegradable polymer. Likewise, Koohi et al. (2011)
reported the dermal toxicity (corrosion/irritation) being more terrible with smaller sizes of Ag0 nanoparticles than with the larger
ones. Samberg et al. (2010) demonstrated the porcine dermal biocompatibility along with the dose-dependent oedema formation
and hyperplasia with Ag0 nanoparticle deposition in supercial
layers of the stratum corneum (SC). Furthermore, dose-dependent
histopathological changes in Guinea pigs bone, heart and kidney
were also reported. Moreover, in vitro size-dependent cutaneous
penetration of gold (Au0 ) nanoparticles has been reported through
the Franz diffusion cell method using rat skin model (Sonavane
et al., 2008). The smaller Au0 particles penetrated deeper while the
larger ones accumulated mostly in the supercial epidermis and
dermis (Sonavane et al., 2008). No acute toxicity was found with
Au0 nanoparticles (Connor, Mwamuka, Gole, Murphy, & Wyatt,
2005), however, the lack of toxic effects on human health associated
with long-term use of such nanoparticles has not been yet established (Korani, Rezayat, & Bidgoli, 2013). On the basis of the toxicity
concerns of these nanoparticles, the BC-based antimicrobial lms
are still far away from practical biomedical applications.
Ex situ and in situ strategies have been employed to fabricate
stable nanocomposites containing copper (Cu0 ) nanollers, i.e.,
nanospheres and nanowires in cellulose matrices (Pinto, Daina,
Sadocco, Pascoal Neto & Trindade, 2013; Pinto, Neves, Neto &
Trindade, 2012). The antimicrobial activity of these nanocomposites was tested against K. pneumonia, and S. aureus, which revealed
that the morphology and chemical stability of Cu0 has a great inuence on the antimicrobial activity. The nanowires exhibited lower
antimicrobial activity due to the smaller reactive surface for oxidation. Similarly, the antimicrobial activity of nanollers with BC
was lower than that with PC. Although both BC and PC have the
same chemical structure, the difference in antimicrobial activity
may be attributed to a lower chemical stability of Cu0 nanostructures grown on PC bres than oxidation of those present in BC
bres. Despite having good antimicrobial activity, the Cu0 nanoparticle associated toxicity on somatosensory neurons of rat (Prabhu,
Ali, Murdock, Hussain, & Srivatsan, 2010) and liver of juvenile sh
(Wang, Long, Cheng, Liu, & Yan, 2014) is still unknown. Further
studies are needed to assess the release and toxicity of metallic
nanoparticles in the BC-based nanocomposites
The combination of BC with silver sulfadiazine (SSD) formed
an antimicrobial composite (BC-SSD), which was investigated for
antimicrobial activity (Luan et al., 2012). The BC-SSD membrane
showed signicant antibacterial activity against S. aureus, E. coli
and Pseudomonas aeruginosa (P. aeruginosa) (Luan et al., 2012; Wen
et al., 2015).
Recently, BC-montmorillonite (MMT) nanocomposite lms (BCMMT) were developed with potent antimicrobial activity (Ul-Islam,
Khan, Khattak, & Park, 2013). These nanocomposites were fabricated by impregnating BC sheets with 2 and 4% suspensions of
MMT, Cu-MMT, Ca-MMT and Na-MMT. The antimicrobial activities of the composites were evaluated against S. aureus and E. coli.
BC-Cu-MMT (2%) composites showed inhibition against the tested
bacteria. The antimicrobial activity of BC-MMTs (4%) was in the
order of BC-Cu-MMT > BC-Ca-MMT > BC-Na-MMT > BC-MMT.
2.2. Wound healing, articial skin and skin tissue repair

Wound healing is a complex biological process consisting of four
basic phases including haemostasis, inammation, proliferation
and maturation (Broughton, Janis, & Attinger, 2006). Wound secrets
exudates containing elevated levels of reactive oxygen species
(ROS), cytokines and proteolytic enzymes. These exudates lead
to a decreased concentration of proteinase inhibitors and growth
336
Table 1
Various applications of bacterial cellulose-based nanocomposites as antimicrobial, antiviral lm and wound dressing system.
Co-former
Method
Finding
Reference
Deacetylated chitin
Loading during synthesis + post-synthesis
Antimicrobial activity increased with chitin amount
Butchosa et al. (2013)
Chitosan
Loading during synthesis
Bacteriostatic against Gram-positive and Gram-negative,

bactericidal against Gram-positive
Increased cell adhesion, proliferation and biocompatibility
Proper moisture maintenance and faster wound healing in
comparison to Tegaderm
(2004)
Ciechanska
Effective pore size against virus (2070 nm), antibacterial

activity against Gram-positive and Gram-negative bacteria
Antibacterial activity against E. coli
Antibacterial activity against E. coli
Wanling et al. (2012)
Antibacterial activity against S. aureus and E. coli

Antibacterial activity against K. pneumonia, S. aureus and B.
subtilis
Antibacterial activity against B. subtilis and E. coli, sterilization
of fermentation medium upon removal by external magnet
Effective against S. aureus with MIC = 1.30 104 g/CFU,
sustained release of Ag0 nanoparticles
Antibacterial activity against Gram-positive and
Gram-negative bacteria
Antibacterial activity against S. aureus and E. coli
Jung et al. (2009), Maneerung et al. (2008)

Pinto et al. (2009)
Post-synthesis loading
PVA + SA
PVA + Ag0
Post-synthesis, in situ by Tollens reaction
Ag0
Post-synthesis, in situ reduction of AgNO3 through NaBH4

Post-synthesis, in situ reduction of AgNO3 through NaBH4 and
treatment with UV radiation
Post-synthesis, in situ loading of BC with iron salt, coating with
ploydopamine and treatment with AgNO3 solution
Hydrothermal synthesis using BC both as reducing and
stabilizing agent
Post-synthesis, in situ hydrolytic reduction of AgNO3 using
triethanolamine (TEA)
Post-synthesis, TEMPO oxidized BC was immersed in aqueous
solution of AgNO3 , followed by reduction with sodium citrate
and NaBH4
Microwave-assisted reduction of AgNO3 in ethylene glycol (EG)
Dobre et al. (2012)

Dobre and Stoica-Guzun (2013)
Sureshkumar et al. (2010)

Yang, Xie, Deng et al. (2012), Yang, Xie, Hong et al.
(2012)
Barud et al. (2011)
Feng et al. (2014)
Antimicrobial activity against Gram-positive and

Gram-negative bacteria
PVP and gelatine inhibited coalescence of Ag0 nanoparticles,
Excellent antimicrobial activity against E. coli
Li et al. (2010)
No toxicity to human adipose-derived mesenchymal stem cells
Figueiredo et al. (2013)
Signicant antibacterial activities, favourable biocompatibility,

fast epithelialization and higher wound healing activity in rat
models with burns
Luan et al. (2012), Wen et al. (2015)
Post-synthesis, ex situ (cellulose was dipped in dispersion of

nanowires previously formed by reduction of CuII nitrate with
hydrazine)
In situ (CuSO4 in BC was reduced with sodium NaBH4 )
strategies
Antimicrobial activity against K. pneumoniae and S. aureus,

copper together with vegetable cellulose bers results in
better antibacterial materials against both species
BC has an inhibitory effect on the oxidation of Cu nanoparticles
incorporated in the matrix and has rmly held the Cu
nanoparticles
Pinto et al. (2013)
MMT
PEG
Kaolin
Col1
During synthesis loading
Ul-Islam et al. (2013)

Cai and Kim (2009)
Wanna et al. (2013)
Wiegand et al. (2006)
Col
RGDC and gentamicin
Vaccarin
Post-synthesis chemical modication with RGDC followed by
covalent attachment of gentamicin
Cellulases
NAcG
In situ by genetically modied strains
Inhibition of S. aureus and E. coli

More biocompatible
Efcient clotting with increased amount of Kaolin
Reduction of interleukins and proteases, showed antioxidant
capability
Better biocompatibility than pure BC
Bactericidal against Streptococcus mutans, but non-toxic to
human dermal broblasts.
Better cell viability and cell attachment, Better wound healing
rat models
Biodegradable wound dressing
Biodegradable wound dressing
Post synthesis, direct in situ metallization by treating Ag+

containing BC membranes with different reducing agents in
aqueous solutions containing 0.2% w/v of PVP or gelatine
PHEMA
SSD
Cu0
Post-synthesis, in situ radical polymerization of

2-hydroxyethyl methacrylate (HEMA) using variable amounts
of poly(ethylene glycol) diacrylate (PEGDA) as cross-linker.
Maria et al. (2010)
Pinto et al. (2012)
Zhijiang and Guang (2011)

Rouabhia et al. (2014)
Qiu et al. (2016)
Hu and Catchmark (2011a, 2011b)
Yadav et al. (2010)
PVA and Chitosan
Cai and Kim (2009)

Lin, Calvar et al. (2013)
factors resulting in degradation of tissue and severe damage. The

exudates also prolong the inammatory phase, which disturbs the
healing process and the wounds fail to heal in a proper manner
(White and Cutting, 2006). Wet or moist treatment environment
is promising for optimal wound healing in terms of enhanced reepithelialization and reduced scar formation, as compared to dry
healing environment (Junker, Kamel, Caterson, & Eriksson, 2013).
Hence, there is a dire need for wound dressing system equipped
with dual-functionality in terms of providing moist environment
and absorbing exudates during the inammatory phase (Chawla
et al., 2009). Such dressing system should be with barrier properties
against infection and uid loss, easy and close wound coverage, and
enable drug delivery to the wound. It should also exhibit high elasticity, mechanical strength and transparency for wound inspection
(Chawla et al., 2009).
Being a highly porous and excellent absorbent (Gayathrya and
Gopalaswamy, 2014), BC alone or in composite form can be an ideal
candidate for lowering or complete removal of the exudates from
wound tissues, for delivery of antibiotics or other medicines into
the wound while at the same time providing an efcient physical barrier against any external infection (Muangman, Opasanon,
Suwanchot, & Thangthed, 2011); thus satisfying the characteristics
of modern wound dressings (Czaja et al., 2006). As an additional
advantage, due to their biocompatibility, and cell adhesion and
proliferation capability, BC and its composites act as articial skin
not only to cover the wounded area, but can also promote skin
tissue regeneration. The transparency of BC can further help in
wound inspection (Keshk 2014a; Qiu & Netravali, 2014). In this
regard, Xylos Co. (USA) claims that its BC based product, XCell ,
possesses dual-functionality of absorption and hydration, which
provides an ideal wet environment for the efcient healing process
(Hoon, Oster, Damien, Wang, & Seraca, 2004; Czaja et al., 2006).
According to the manufacturer, this wound dressing can provide
uid to dry wounds and thus is especially useful for the chronic dry
wounds with dry necrotic tissue on the surface. The same dressing system also possesses the ability to absorb uid away from the
exuding wound. Additionally, XCell does not degrade to leave any
harmful residue at the wound site. It does not adhere to the surface
of wound and thus has no damaging effect on tissue upon removal
(Hoon et al., 2004).
Other commercial BC-based wound dressing products include
Bioll and Bioprocesss , Produtos Bioetecnologicos, Brazil.
Bioll is used as a temporary articial skin in the treatment
of burns and ulcers, while Gengiex is applied in the treatment of periodontal diseases (Czaja et al., 2006; Jonas & Farah,
1998). According to the manufacturers, Bioll is produced within
shorter duration of time (2 days), whereas being thicker, Gengiex
requires relatively longer duration (8 days) of fermentation (Czaja
et al., 2006; Jonas & Farah, 1998). In a case study, 28% of the total
body surface area with deep partial-thickness (ame) burn was
treated with Nanocell , a BC based product, applied once to the face
wound without further dressing change. During observation of the
healing progress until complete epithelialization for 2 weeks, the
patient did not show any allergic reaction or irritation to the dressing. No evidence for presence of bacteria was found in wound swab
culture (Muangman et al., 2011). Thus, BC can be used as articial
or temporary skin for deep wound healing with barrier properties
to microbes.
By preparing different BC-based composites, the desired properties of BC lm can further be tailored to design the most suitable
material for articial skin and skin tissue repair. In this context, the
histological examinations revealed that wounds treated with BC-Ch
epithelialized and regenerated earlier than those treated with BC or
Tegaderm . There was no cytotoxicity in L929 broblasts incubated
with BC-Ch lms, while the cells showed higher viability, adhesion
and proliferation in rat models (Lin, Calvar et al., 2013). Therefore,
337
BC-Ch composites have good potentiality for biomedical applications as a wound dressing material and tissue engineering scaffold
because it is biologically active, less toxic, and appropriate for cell
adhesion, epithelialization and regeneration with good mechanical
properties.
Recently, SSD particles were impregnated with BC to produce
BC-SSD composite membrane. In addition to in vitro antimicrobial activity against S. aureus, P. aeruginosa and E. coli, BC-SSD
demonstrated good wound healing activity in burnt rat models. It
also exhibited good in vitro biocompatibility for epidermal cells.
Upon histological examination, epithelialization was developed
in BC-SSD treated wound (Luan et al., 2012; Wen et al., 2015).
Likewise, Rouabhia et al. (2014) developed a BC-based wound
dressing system with enhanced surface characteristics in terms of
better physiologic interaction with human cells, and the presence
of antimicrobial agents. BC was chemically modied with RGDC
(arginine, glycine, aspartic acid, cysteine) peptide and then gentamicin was covalently attached to the surface of the BC membrane.
BC-RGDC-gentamicin membranes were bactericidal against Streptococcus mutans, but non-toxic to human dermal brobalsts.. Thus,
BC-RGDC-gentamicin may be useful for multipurpose applications,
such as wound healing along with drug delivery.
BC nanocomposite lms in combination with poly(2hydroxyethyl methacrylate) was also prepared by an in situ radical
polymerization of 2-hydroxyethyl methacrylate (HEMA), utilizing
variable amounts of poly(ethylene glycol) diacrylate (PEGDA)
as cross-linker (Figueiredo et al., 2013). The thin lms obtained
showed superior properties to both BC (improved translucency)
and poly(2-hydroxyethyl methacrylate) (PHEMA) (enhanced
mechanical performance and thermal stability). BC-PHEMA
nanocomposites demonstrated no toxicity, good biocompatibility,
and cell adhesion and proliferation for human adipose-derived
mesenchymal stem cells. Therefore, these nanocomposites were
considered promising wound dressings with stem cell-mediated
tissue regenerative capability in biomedicine.
In order to achieve biocompatible wound healing material, BCpoly(ethylene glycol) (PEG) composite (BC-PEG) were fabricated
by submersing wet BC pellicle in aqueous solution of PEG (Cai and
Kim, 2009). After freeze-drying the product acquired a foam-like
structure, in which the PEG molecules were not only layered on
the surface of BC brils but also got penetrated into the BC bre
networks. It was noted that the presence of PEG decreased the
crystallinity of BC due to a change in the preferential orientation
of the plane during the process of drying. The thermal stability was
improved from 263 to 293 C due to the strong interaction between
PEG and BC. The biocompatibility of composite was assessed by
adhesion studies using 3T3 broblast cells, which showed cell adhesion and proliferation. The above study showed that the BC-PEG
composites were more biocompatible than BC.
Similarly, a single BC-kaolin (BC-K) composite system was
developed for wound healing purposes, whereby short-term
(kaolin) and longer-term (BC) wound healing mechanisms were
combined without any adhesives to bind the BC and kaolin (Wanna,
Alam, Toivola, & Alam, 2013). Ultrane kaolin particles with a size
distribution in the range of 50800 nm were added to the BC gel in
a specic weight percentage in order to produce slurries containing 5, 15 and 30% of BC (w/w). These slurries were then pressure
ltrated into BC sheets followed by drying at 80 C. The resultant
product is a combination of short and long term biomedical wound
dressing/healing materials. However, there is still a need for further
research in order to optimize the properties of BC-K with respect to
both blood clotting potential and uid/moisture ingress for better
wound healing.
BC as wound dressing system was also established by in situ
incorporating collagen type-1 (Col1 ) into the BC pellicle. This novel
biomaterial possessed antioxidant properties and was capable to
338
Fig. 3. SEM images of the (a) BC surface, (b) BC-Col composite surface, (c) cross section of BC and (d) cross section of BC-Col.
Reproduced with permission (Zhijiang and Guang, 2011), Copyright, John Wiley and Sons.
signicantly reduce the selected inammatory interleukins and

proteases (Wiegand, Elsner, Hipler, & Klemm, 2006). In addition,
novel composites of BC with collagen (BC-Col) were prepared by
dipping wet BC sheets in solution of collagen followed by the process of lyophilisation (Zhijiang and Guang, 2011). The product thus
formed has sponge-like structure as shown in Fig. 3 (Zhijiang and
Guang, 2011). The collagen molecules were not only coated on the
surface of BC nanobrils, but were also impregnated inside the
BC. The biocompatibility of BC-Col composite was assessed by cell
adhesion studies using 3T3 broblast cells. The biocompatibility of
BC-Col was much better than BC. Vaccarin is a major avonoid glycoside that can attenuate oxidative stress injury of endothelial cells
(Qiu, Qiu, Cui, & Wei, 2016). Recently, Qiu et al. (2016) fabricated
BC composites with vaccarin (BC-Vac) by immersing BC in vaccarin
solution. The prepared BC-Vac composite showed enhanced cell
viability and cell attachment demonstrating the absence of cytotoxicity and its potential for cell growth. Using in vivo rat skin models,
the histological examination revealed that wounds treated with
BC-Vac underwent epithelialization and regeneration faster than
those treated with BC alone (Qiu et al., 2016). The results suggested
the potential of BC-Vac composite as wound healing materials.
Moreover, full-thickness skin wounds in Wistar rats showed faster
recovery and less inammatory response when covered with bottom side of the BC in comparison to the gauze and the top side of
the BC membrane (Li et al., 2015).
The development of bioabsorbable materials is very important in the biomedical eld for wound healing and other tissues
regeneration applications, where natural tissue may replace the
biomaterial (Petersen and Gatenholm, 2011). In this regard,
improvement of bioresorption of oxidized cellulose has been
described in the literature (Laurence, Bareille, Baquey, & Fricain,
2005; Petersen and Gatenholm, 2011; Singh, Ray, Verma, & Guha,
1979). Bioresorbable BC composites were also prepared by incorporating cellulase enzymes into BC (Hu & Catchmark, 2011a, 2011b).
However, it was shown that some of the enzymes may lose up
to 90% of their activities in a suboptimal pH condition, which is a
challenge in the practical applications of this approach due to variation in the pH of wound or tissue. To address this issue, Hu and
Catchmark (2011a,b) incorporated buffer ingredients into the BC in
order to create a more optimal pH microenvironment for the preferred acid cellulases. It was demonstrated that with this approach,
the activity of the cellulases was retained, with an increase in glucose release from 30% (without buffers) to 97% from degraded
materials. An improved material degradation was observed using
simulated tissue padding and body uid (SBF) that mimics real
wound environment. Moreover, the tensile strength and extensibility of the materials was similar to human skin, particularly when
hydrated with saline water (Hu and Catchmark, 2011a). Yadav et al.
(2010) also biosynthesized biodegradable BC in vivo by incorporating NAcG through genetically engineered bacterial strains. Yielding
the harmless glucose as the degradation product, these composites may be ideal for many wound dressing and tissue engineering
applications for the bioresorbable purpose.
The studies described above deal with the possible application
of BC-based composites to control bacterial, viral infections and its
potential for wound dressing system, articial skin and skin tissue
repair, which are further summarized in Table 1.
2.3. Cardiovascular system

2.3.1. Articial blood vessels
In normal physiological conditions, blood circulates through
vessels all over the body. However, these blood vessels are blocked
or severely injured in pathological conditions (Adams, Xiao, & Xu,
2007), which in some cases must be replaced by another blood
vessel harvested from patients own body or donor, or articial
blood vessels. There are serious concerns with patients own vessel as the double surgery may create other health issues including
multiple surgeries, blood loss, prolonged anaesthesia, and pain and
surgical complications at the donor site. The donor strategies also
have limitations in form of high chance of tissue rejections and
development of arteriosclerosis (Parveen, Krishnakumar, & Sahoo,
2006). Some research groups are developing articial blood vessels
in order to solve these issues (Parveen et al., 2006; Shors, 1990).
Similarly, non-compliance between the native tissue and synthetic
graft has been a major concern for the ultimate failure of the currently available graft replacement (Campbell, Efendy, & Campbell,
1999; Edelman, 1999). Synthetic grafts, such as ePTFE and Dacron
(polyester) induce thrombosis and therefore are not suitable substitutes for smaller blood vessels with diameter less than 6 mm
(Saxena and Brown, 2012).
BC and its composites are excellent candidates for articial
blood vessel formation due to its non-toxic nature, purity, greater
tensile strength, ultrane brous network, foldability and moldability (Zang et al., 2015). In this context, the development of
BASYC represents a landmark in biomedical applications of BC.
The carotid artery (small diameter blood vessel) in ve mice was
replaced with BASYC , which demonstrated one year patency
(being open). Likewise, BASYC has proved (90 days) patency in 7
out of 8 pig prostheses. The prosthesis has shown epithelialization
and developed three distinct layered structure, the characteristic of a natural blood vessel (Schumann et al., 2009). It is worthy
to mention that besides articial blood vessels, BASYC alone or
loaded with a neuroregenerative drug has also exhibited promising
resultsin terms of innervation of tissues in animal models after neurosurgery (Klemm et al., 2001, 2005). Moreover, Brown et al. (2011)
fabricated BC-brin composites treated with glutaraldehyde (GLA)
(cross-linker) that well-matched the mechanical properties with
the natural small-diameter blood vessels. The time-dependent viscoelastic behaviour of BC-brin composites was similar to that of
the natural blood vessel (bovine coronary artery). These composites also showed tensile strength, modulus, stress-strain response
and mechanical properties similar to a small-diameter blood vessel.
However, the strain at breakwas much lower than that of natural
blood vessel. Furthermore, no literature has been reported for the
assessment of cell viability, particularly cytotoxicity.
A novel approach was also used through introduction of starch
of various particle sizes and parafn wax in a culture of G. xylinus for
the preparation of scaffolds having 3D network with controllable
microporosity (Bckdahl, Esguerra, Delbro, Risberg, & Gatenholm,
2008). The BC scaffolds obtained had different morphologies and
interconnectivity. In order to achieve microporous BC scaffold,
porogens were effectively removed and no residue was detected
in physicochemical analysis. The prepared scaffolds were seeded
with SMC, which proliferated on the surface and partly into the
scaffolds. Moreover, graphene oxide (GO) was added to BC forming bacterial culture media and novel BC-GO composite material
was formulated (Zhu, Li, He, & Duan, 2015). The GO nanosheets
were entrenched in the nanobers network of BC with hydrogen
bond interaction. BC-GO composite displayed a better biocompatibility than the individual corresponding items and stimulated the
cell proliferation. Due to these properties, BC-GO has the potential
applications in articial blood vessels. In the future, these scaffolds can be investigated for the proliferation, differentiation and
migration of endothelial cells, which may lead to the preparation of semi-synthetic or articial blood vessels. Likewise, Andrade
et al. (2010) demonstrated the potential of BC modied with cellulose binding module and adhesion peptide for articial blood
vessel. The modied BC was able to enhance endothelial cell adhesion and stimulate angiogenesis. In a recent report, Leito et al.
(2016) demonstrated good tensile and suture retention strength
of BC as small-caliber blood vessel. The BC grafts were surgically
incorporated to the hind limb femoral artery of domestic limb.
All BC grafts revealed good integration into the surrounding tissue
without any signicant brosis or external evidence of inammation. Upon histological analysis, a distinct three-layered structure
was found having cellular adhesion and inltration on both the
adventitial and luminal sides of the graft, and a larger BC central region without cell adhesion. After one month of grafting,
CD31 positive cells were observed on the luminal surface and were
believed to be endothelial or progenitor endothelial cells, which
drifted from the grafted femoral artery. A one month patency was
achieved with neo-vascularization and endothelialisation. However, the graft occluded due to thrombosis after 2 months of grafting
339
(Leito et al., 2016). The current research regarding the application

of BC nanocomposites for the preparation of blood vessels has been
summarized in Table 2.
2.3.2. Cardiac prosthesis

Cardiac prosthesis is the replacement of diseased heart valves
with suitable articial valves. This is an advanced surgical therapy, which reduces the morbidity and mortality in cardiac patients
(Takatani, 2000). Different biomedical materials have been tested
and some of them are already in medical practice; however, the
available materials have limitations in long term use, such as calcication, oxidative reactions with blood and severe side effects
(Mohammadi, 2011). Therefore, research efforts are continuing in
order to nd alternative materials for making articial heart valves.
In the last decade, BC and its nanocomposites have also been evaluated for the preparation of the articial heart valve (Andrade et al.,
2011; Fink et al., 2010; Millon and Wan, 2006; Mohammadi, 2011).
BC in combination with PVA has been comprehensively studied by various researchers for biomedical applications as blood
contacting materials. The initial attempts were made in 2013
(Leito, Silva, Dourado & Gama, 2013), in which BC-PVA composite was prepared with lower values of Youngs modulus and
tensile strength. By incorporating PVA, the Youngs modulus of
dry pristine BC decreased from 3.5 GPa to 1 GPa, while the tensile strength decreased ve times. The hemocompatibility of the
BC-PVA nanocomposites was investigated by assessing the FactorXII activation, plasma recalcication, whole blood clotting time,
complement activation, hemolytic index, and platelet adhesion and
activation (Leitao et al., 2013). The results showed that these materials have a good hemocompatibility, owing to both low Factor-XII
and platelets activation. It was also found that platelet adhesion
and the activation proles of both BC and BC-PVA were superior
in comparison to ePTFE. The BC-PVA composites with properties
similar to heart valve (Mohammadi, 2011) were hemocompatible
and thus have a potential for applications in cardiac prosthesis.
Similar attempt was also made by Castro et al. (2014) in which
they biofabricated PVA-based nanocomposites reinforced with BC,
which was produced in a water-soluble PVA modied culture
medium. These PVA-BC composites exhibited synergistic property
contributions from both PVA and BC. The PVA was chemically
cross-linked with glyoxal in order to avoid its loss during the
purication process and to improve the functional characteristics
of composites. BC reinforcement resulted in nanocomposites with
outstanding dimensional, thermal and mechanical properties, as
well as stability in moisture. The incorporation of BC increased the
strength at break and Youngs modulus of nanocomposites. Similarly, Mohammadi (2011) also used BC-PVA composites for the
fabrication of heart valve leaets. The aim of the work was to mimic
the non-linear mechanical properties exhibited by heart valve of
porcine origin and its anisotropic behaviour by application of a controlled strain to the composites while undergoing thermal cycling
(low temperature) for the induction of oriented mechanical properties. These composites possessed mechanical properties almost
similar to porcine heart valve.
Similarly, hydroxyapatite (HAp) was also incorporated into BC
using hot (55 C) alkaline solution of calcium carbonate (CaCO3 ) and
dicalcium phosphate dehydrates (Chen and Lai, 2013). The resistance to compression of the experimental sample reached about
141.36 MPa, and the level of endotoxin decreased to 0.3 EU mL1
upon sintering the raw biocomposite using alginate as a hardener at a temperature of 1200 C for 1 h. Due to its higher level of
resistance to compression and low endotoxin level, the prepared
biocomposite possesses a great potential for employing a lling
material for future applications in articial heart valve and vascular
340
Table 2
Applications of bacterial cellulose-based nanocomposites in articial blood vessels.
Co-former
Method
Model for assessment
Finding
Reference
Fibrin
Viscoelastic behaviour, tensile strength and modulus
Brown et al. (2011)
Starch and
parafn wax
GO
During synthesis
loading
During synthesis
loading
Post-synthesis
modication
Seeded with SMC
All characteristics were similar to a

small-diameter blood vessel
Proliferated on the BC composites
scaffold
Better biocompatibility and stimulated
the cell proliferation
Enhanced endothelial cell adhesion
and angiogenesis
Cellulose
binding
domain
Cytotoxicity assays
Cell adhesion and cell viability assay
Bckdahl et al. (2008)

Zhu et al. (2015)
Andrade et al. (2010)
Table 3
Applications of BC-PVA composites in cardiac prosthesis.
Method
Finding
Reference
Thermal processing
method
Stress-strain tensile properties
Stress-strain tensile properties were matched

to porcine aorta
Youngs modulus and tensile strength
decreased
Thermal, mechanical properties and stability in
moisture improved
Millon et al. (2009)
Exhibited better hemocompatibility
Leito et al. (2013)
Possessed mechanical properties almost

similar to porcine heart valve
Mohammadi 2011
Resistance increased and endotoxin level

decreased
Chen and Lai (2013)
During synthesis
loading, cross-linked
by glyoxal
During synthesis
loadinga
a
Youngs modulus, tensile strength

Thermal and mechanical properties as well as
stability in moisture
Factor-XII activation, plasma recalcication,
whole blood clotting time, complement
activation, hemolytic index, and platelet
adhesion and activation
Comparison of mechanical response in the
circumferential and the radial directions with
porcine heart valve leaet
Resistance to compression, endotoxin level
Leito et al. (2013)

Castro et al. (2014)
HAp instead of PVA.
stents. The aforementioned studies on BC-composites for potential

applications in cardiac prosthesis are listed in Table 3.
2.4. Ophthalmic applications
2.4.1. Articial cornea
Cornea is the outmost part of the eye, which is avascular in
nature. The corneal diseases are the major cause of vision loss
around the world as in pathological conditions the corneal transparency is lost (Foster, 2003; Whitcher, Srinivasan, & Upadhyay,
2001). In order to reverse the vision loss, keratoplasty is a common
technique to replace damaged cornea; however, xerophthalmia,
corneal neovascularisation, donor shortage and graft rejection are
the associated adverse effects (Wang, Wei, & Zhao, 2013). Recently,
tissue and bio-engineering techniques have been applied to design
corneal replacement. These include the synthesis of articial cornea
and tissue engineering for cornea production. In this context, BC has
also been evaluated for articial cornea and scaffolds for corneal tissue engineering. BC possesses light transmittance, unusual material
properties and biocompatibility and thus has been investigated as
an innovative scaffold (Hui et al., 2009). It was shown that corneal
stromal cells can be maintained, grown and proliferated on the BC
scaffolds. It was further demonstrated that BC scaffold supported
the ingrowths of corneal stromal cells, suggesting its potentials in
tissue engineering for cornea production. Likewise, the modied
BC-porous PVA-nano hydroxy apatite (nHA) (BC-PVA-nHA) composite (Kharaghani, Meskinfam, Rezaeikanavi, Balagholi, & Fazili,
2015) demonstrated that the water content of the BC-PVA-nHA
composites (8284%) was almost similar to the natural human
cornea (78%).
2.4.2. Retinal pigment epithelium substrate
Age-related macular degeneration (AMD) is one of the leading causes of irreversible vision loss (Lim, Mitchell, Seddon, Holz,
& Wong, 2012). The retinal pigment epithelium (RPE) is one of

the areas affected by AMD that leads to the damage of the photoreceptors (Ambati and Fowler, 2012; Binder, Stanzel, Krebs, &
Glittenberg, 2007). Transplanted RPE cells have the potential to
save photoreceptors (da Cruz, Chen, Ahmado, Greenwood, & Coffey,
2007; Lim et al., 2012). Hence, the delivery of RPE cells as an
epithelial sheet on a Bruchs membrane (BM) prosthetic substrate
is an important therapeutic approach for treating AMD (Binder
et al., 2007; Goncalves et al., 2015). In addition to biocompatibility, a BM prosthetic substrate must have the ability to perform
the BMs primary functions, i.e., regulation of the passive diffusion
of biomolecules between RPE and choroid, providing support for
RPE cells adhesion, migration, and possibly differentiation (Booij
et al., 2010). The unique properties of BC make it a potential candidate for the successful BM substrate (Booij et al., 2010). Likewise,
BC-PVA-nHA composite (Kharaghani et al., 2015) has shown signicant growth of RPE cells, demonstrating its biocompatibility
and potential for RPE substrate. Recently, Goncalves et al. (2015)
demonstrated the feasibility of modied BC as BM substrate for
RPE cells. Among all the modied BC substrates, the acetylated
BC (ABC) substrate was found to be the most fascinating one in
terms of the mechanical properties, cell adhesion and proliferation,
and the amount of endotoxins. All the substrate possessed similar
bulk properties, such as porosity, permeability up to 300 kDa, and
dimensional stability (especially the ABC ones). Moreover, surface
modications demonstrated better results in terms of cell adhesion
and proliferation in comparison to unmodied BC with ABC showing enhanced cell adhesion and similar proliferation in comparison
to the other modied BC substrates (Goncalves et al., 2015). To further study the RPE morphology and function, Goncalves et al. (2016)
demonstrated ABC substrates coated with urinary bladder matrix
(UBM) for the adhesion and proliferation of RPE cells. Interestingly,
these substrates supported the development of RPE cells monolayer with its desired phenotype, that is, polygonal shaped cells
having microvilli. This phenotype expressed cytoskeletal (ZO-1)

and metabolic (RPE65) proteins. The UBM coated substrates exhibited proper mechanical properties and fascinating physicochemical
features, such as no residual content in endotoxins, easy manipulation, no signs of hydrolytic degradation, low swelling and high
mechanical strength (Goncalves et al., 2016). Hence, UBM coated
ABC substrates exhibit nearly all the desired characteristic of RPE
substrate for representing a very promising advancement in the
eld of ophthalmology.
2.4.3. Contact lenses
More than 2.3 billion people across the globe suffer from visionary defects due to refractive error (RE) (Naidoo and Jaggernath,
2012). RE is the most common vision defect, which results in poor
vision, such as presbyopia, astigmatism, hyperopia and myopia
with severe economic and social implications, if not corrected
(Bourne, Dineen, Huq, Ali, & Johnson, 2004). RE is very simple to be
diagnosed, measured, and corrected with the help of refractive surgical procedures or devices, such as spectacles and contact lenses
(Kempen et al., 2004)
Contact lenses are extensively used for the correction of visionary faults in human. Contact lenses are available in hard and soft
varieties. Classically, the hard type contact lenses are fabricated
from glass or a rigid polymer, such as poly(methyl methacrylate)
(PMMA), poly(hydroxyethyl methacrylate) (PHEMA) and polycarbonates, while the soft lenses are usually made from a soft
polymeric material (Levinson and Glonek, 2010).
BC can be used to fabricate contact lenses due to its transparency and high degree of light transmittance. BC-based contact
lens was fabricated from viscous solution of BC in 1-butyl-3methylimidazolium chloride. The BC solution was poured into a
mold and a clear material was precipitated when the solution was
treated rst with isopropanol and then with water. The synthetic
contact lens naturally detached from the mold surface. The residual solvent 1-butyl-3-methylimidazolium chloride diffused from
the contact lens to water. The hydrated BC contact lens was stable
for more than eight weeks, retaining its transparency and shape
(Levinson and Glonek, 2010). A transparent polymeric nanocomposite based hydrogel was also fabricated by combining water
insoluble nanober of BC and water soluble PHEMA polymer. The
prepared material possessed about 40% (w/w) water content, good
strength and mechanical integrity (Li, Wan, & Panchal, 2010). The
designed BC-PHEMA nanocomposites have the ability to be used in
hydrogel applications, particularly contact lenses.
Keeping in view the above ndings, BC alone or in composites
can thus be used as articial cornea, scaffold for corneal cells and
RPE cells, and as contact lenses.
2.5. Application in skeletal systems, cartilage and endodontics
2.5.1. Bone regeneration
Synthetic implantable biomaterials, such as polymers, ceramics
and metals provide many benets over allografts and autografts
for the treatment of bone defects (Shu, Liu, Palumbo, Luo, &
Prestwich, 2004). However, the practical application of natural
materials as tissue engineering scaffolds is a major concern because
these materials have to exhibit high biocompatibility, biodegradability, chemical functionality, and have to be rather inexpensive
(Markovic, Karadzic, Jokanovic, Vukovic, & Vucic, 2016). A biomaterial is a pharmacologically and systematically inert substance
designed for incorporation or implantation within a living tissue or
system (Rogel, Qiu & Ameer, 2008). It works as scaffolds for maintenance and growth of cells or help in tissue regeneration (Kroeze,
Helder, Govaert, & Smit, 2009).
In depth studies have been conducted for biomaterials as an
alternative to grafts in bone tissue engineering (BTE) due to their
341
non-toxic and biocompatible properties (Ramirez, 2010). Collagen, mineralized by hydroxyapatite (HAp), constitutes a major
part of bone matrix (Petersen and Gatenholm, 2011). Guided bone
regeneration (GBR) is a medical practice, which prompts in vivo
re-growth of bone tissue by the use of osteopromoting llers and
membranes. Being collagen-like material, BC bres were used as
a basis for biomimetic HAp growth with the ultimate aim of fabricating ller materials for GBR (DeMello, 2012). BC bres were
phosphorylated with phosphoric acid and preconditioned with calcium (Ca+2 ) for nucleation of HAp. SBF enhanced the growth of
HAp. Over a period of 14 days, the BC-HAp maintained a Ca-to-P
ratio as high as 1.45 0.92, covering the standard of 1.67 for HAp.
Higher Ca-to-P ratios were detected on the pellicle surface suggesting the deposition of HAp crystals. The study indicated the potential
for formation of 3D-samples and a basis for further optimization
of BC-HAp scaffold for GBR. One of the biomimetic mineralization
pathways of BC has been depicted in Fig. 4 (Na et al., 2011).
Due to its osteoconductivity, HAp was used for the fabrication of
3D network-based nanocomposites with BC via biomimetic route.
This was achieved when phosphorylated BC was soaked in SBF,
which led to the formation of uniform HAp crystals (Wan et al.,
2007) with structural similarities comparable to biological apatite.
Furthermore, Shi et al. (2009) also synthesized BC-based calcium
decient carbonate containing HAp nanocomposites (BC-CaDHAp)
using a biomimetic mineralization technique. The CaDHAp in the
nanocomposites partially contained carbonate that resembled with
apatite (Ap) of natural bone. The study demonstrated that a 3D
network of BC offered a good template for the well-organized
deposition of BC-CaDHAp spherical particles (0.5 m), which were
composed of nanosized squama-shape Ap crystals. The BC-CaDHAp
nanocomposites therefore possess a great potential for BTE.
In another study, it was shown that tubular-shaped (T-shaped)
BC was a natural nanoscale brous hydrogel scaffold for BTE. The
freeze-dried T-shaped BC and its composites demonstrated aligned
nanobrous morphological properties (Favi, 2014). Moreover,
these were cytocompatible with equine-derived bone marrow
mesenchymal stem cells (EqMSCs). Both the T-shaped BC scaffolds
and its composites supported the EqMSCs adhesion, proliferation
and osteogenic differentiation. From this study, it was concluded
that the mineralized porous and oxidized T-shaped BC scaffolds
seem to have potentiality as a scaffold for bone regeneration. Similarly, BC-HAp nanocomposites scaffold were prepared using the
biomimetic approach and investigated the differentiation and proliferation of human bone marrow derived stromal cells (hBMSC)
on these nanocomposites (Fang, Wan, Tang, Gao, & Dai, 2009). The
hBMSC seeded on the BC-HAp nanocomposites showed improved
attachment, proliferation and differentiation as compared to BC.
Real-time reverse transcription polymerase chain reaction data
showed that the expression of molecular players for bone formation and regeneration was better in the nanocomposites group than
inBC. The alkaline phosphatase (ALP) activity and the expression of
ALP, bone sialoprotein, osteocalcin and osteopontin were all higher
for up to 7 days in hBMSC cultured on the nanocomposites as compared to BC in the absence and presence of osteogenic supplements
(dexamethasone, glycerophosphate and l-ascorbic acid). BC-HAp
nanocomposites scaffold displayed in vitro biocompatibility and
had the potential to be use in BTE. Thus, all the aforementioned
studies have demonstrated that BC based biodegradable scaffolds
have an excellent potential for GBR.
2.5.2. Cartilage, intervertebral discs and articial endocranium
Regardless of the recognized usage of the total joint replacement
to treat advanced articular cartilage degeneration, the component
loosening due to osteolysis and wearing limits the durability of
these particular prostheses (Dattani, 2007). BC and its composities
have been investigated as a substitute for the cartilage due to their
342
Fig. 4. FE-SEM images of (a) BC, (b) BC-PVP, (c) 5-PVP-HAp-BC, (d) 7-PVP-HAp-BC and (e) schematic synthesis of PVP-BC-HAp by biomimetic mineralization.
Reproduced with permission (Na et al., 2011), Copyright, Elsevier.
similarities in properties (Petersen and Gatenholm, 2011). In this

context, nanocomposites consisting of PVA and BC nanobers were
synthesized by adding less than 1% of BC to PVA and exposing the
blend to thermal cycling (Millon, Oates, & Wan, 2009). The prepared
BC-PVA composites were studied as possible improved replacement materials for articular cartilage. The BC-PVA nanocomposites
showed controllable compressive mechanical properties with elastic modulus values closer to that of natural articular cartilage. The
nanocomposites also showed improved dependence on strain-rate
and adequate viscoelastic properties. The BC-PVA nanocomposites showed promising properties for the replacement of localized
articular cartilage injuries and other orthopaedic problems such
as injured intervertebral discs. Likewise, BC-based composites
with poly(dimethylacrylamide) fabricated by Azuma et al. (2007)
showed potential for applications a cartilage substitute. Moreover,
Svensson et al. (2005) demonstrated that BC exceeds plastic and

alginates in terms of chondrocytes proliferation and migration and
thus has potentials to be used as cartilage scaffold. These studies
are summarized in Table 4.
On the basis of mechanical properties, foldability, nocytotoxicity and low price, the preparation of BC-based articial
endocranium (AE) was patented (Lizhnln et al., 2011). In this
preparation, the BC wet sheet, after harvesting from fermentation
of G. xylinus, was crushed. Then PVA, sodium alginate and carboxymethylcellulose (CMC) were added to the BC homogenate to
get a liquid with moldability. The mixture was then poured into
a mold followed by freeze-drying to get the AE. The strength at
break point of the fabricated AE was reported to be 45 N mm2 ,
the breaking elongation was in the range of 4598%, and the hook
strength was between 5 to 8 N mm2 , while the water permeability
343
Table 4
Applications of BC-based composites in bone regeneration.
Co-former
Method
Finding
Reference
HAp
Post-synthesis
phosphorylation
Loading of HAp to BC
Ca-to-P ratio
High Ca-to-P ratio
DeMello (2012)
Assessment of HAp loading
Wan et al. (2007)
Biomimetic approach
hBMSC
Biomimetic
mineralization
Crystallinity
More HAp loading in

phosphorylated BC
Improved cell adhesion and
activity
Lower crystallinity
2
2+
Fang et al. (2009)

Shi et al. (2009)
XPS analysis, ALP activity,

osteoblast growth and
formation of bone nodule
Ca and PO4 present,

signicant improvement of
osteoblast growth, adhesion
and osteoconductivity on
BC-HAp membranes
Tazi et al. (2012)
Post-synthesis
chemical reaction
In vitro EqMSCs
Supported the EqMSCs

adhesion, proliferation and
osteogenic differentiation.
Favi (2014)
Ca-to-P ratio
Better mineralization
Na et al. (2011)
HAp
Biomimetic
mineralization
Ca-to-P ratio, in vivo

inammatory tests
Saska et al. (2011)
Otoliths + collagen
Histological examination
HAp or Col with/without OGP
Col1
Post-synthesis
cross-linking
CHO-K1 cells, CBMN assay,

comet assay, XTT assay and
clonogenic assay
Tensile strength, elastic
modulus, strain at break,
osteogenic cells, morphology
and cell proliferation
Ca-to-P ratio similar to natural

bone, no inammation
detected
Formation of bone tissue with
higher osteoblast activity, high
degree of regularity and
osteo-reabsorption activities
No mutagenic, genotoxic or
cytotoxic effects
Saska et al. (2012)
Bone morphogenic protein

(BMP2 )
In vitro mouse broblast-like

C2C12 cells, in vivo bone
formation
Gelatine
Parafn wax
Loading during
synthesis
Crystallinity index, tensile

strength, strain at break,
thermal stability, Youngs
modulus and 3T3 broblast
adhesion
MC3T3-E1 osteoprogenitor
cells, confocal microscopy and
histology
Tensile strength and elastic

modulus for BC-Col1 decreased,
slight increase in strain at
break, similar cell morphology
and cell proliferation/viability
Mouse broblast-like C2C12
differentiated to cells into
osteoblasts, formation of bone
with high calcium content
in vivo
Crystallinity index, tensile
strength, strain at break,
decreased, thermal stability,
Youngs modulus and cell
adhesion increased
More MC3T3-E1
osteoprogenitor cells clustered
in microporous composite
2-chloroN,Ndimethylethylamine
hydrochloride, glycidyl
trimethyl ammonium chloride,
and monochloro acetic acid
sodium salt
HAp + PVP
Olyveira et al. (2011)
Scarel-Caminaga et al. (2014)
Shi, Li et al. (2012), Shi, Zang

et al. (2012)
Cai and Kim (2010)
Zaborowska et al. (2010)
was 0. All these values were closer to that for a rabbit endocranium.
Moreover, the AE was not amenable to easy tear and off-clip during
the stitching via sutures.
and mechanical performance of BC can be further improved provided that the cell migration and control on the shape is not
affected.
2.5.3. Meniscus implant

Meniscal lesions due to trauma or degenerative diseases are the
most common problems (Tienen et al., 2004). Extended or total
meniscectomy can bring out degenerative changes in the affected
area. Such lesions frequently proceed to osteoarthritis. Allogenic
meniscus implantation is carried out for the symptomatic treatment of middle-aged patients after they have gone through partial
or total mensicectomy (Tienen et al., 2004). The mechanical properties of BC gel were compared with traditional collagen meniscal
implant material and real pig meniscus (Bodin et al., 2007). It
was observed that the Youngs modulus of BC gel was similar
to the one of pig meniscus, and 5-fold higher than the collagen
material. BC is biocompatible and showed promising cell migration along with controlled meniscus shape, which indicated that
BC could be an attractive material as meniscus implant. However, by forming suitable nanocomposites, the tensile strength
2.5.4. Dental root canal treatment

Promotion of dental caries to dental pulp infection needs dental
root canal treatment (DRCT). The major goal of DRCT is to provide a complete decontamination to the dental root canal system
(Trowbridge, 1981; Yoshino et al., 2013). Dental root canal morphology is complicated and dental roots of many humans have
inaccessible areas (Tikku, Pragya, & Ivy, 2012). Furthermore, recurrence of dental infection is equally common (Yoshino et al., 2013).
In conventional treatment, a PC made paper point and cotton
pellet is utilized for drying and sterilization of dental root canal
(Taneja, Kumari, & Parkash, 2010). Such sterilization procedure
requires a treatment material having high absorbency for removal
of any residue, high biocompatibility and capability to increase the
efcacy of intracanal medication (Yoshino et al., 2013). BC was
fabricated in a pointed form and its usability was assessed as a
novel material for dental root canal treatment in respect of tensile
344
strength, expansion, absorption of solutions, biocompatibility and

drug release (Yoshino et al., 2013). BC showed higher expansion
and absorption than paper points and maintained a higher tensile
strength even in wet form. The cumulative drug release was signicantly greater for BC as compared to paper points. In addition,
BC was more compatible than paper points. Considering the properties of BC and abovementioned results, BC-drug composite has
great potentiality for applications in DRCT.
2.5.5. Ligament and tendon
Bio-based brous PC-Col nanocomposites possessed mechanical performance (in sterilized form) closer to that of natural tendon
and ligament in SBF. These nanocomposites endorsed human
endothelial cell and human ligament cell growth, adhesion and
differentiation that is extremely encouraging for ligament tissue engineering without compromising the moisture stability or
mechanical performance (Mathew, Oksman, Pierron, & Harmand,
2013). Although the cellulose used here was of vegetable origin, it
paved the way for BC based composites to be used as substitute for
ligament and tendon.
2.6. Applications in drug delivery systems
2.6.1. Controlled drug delivery
Drugs with shorter half-lives usually need to be administered
in a controlled manner, which incorporates some advantages to
the dosage form including reduction in dose frequency, decreased
uctuation in drug plasma concentration, improved patient compliance and therapeutic efcacy (Deshpande, Rhodes, Shah &
Malick, 1996). BC and other polymeric matrices have been extensively investigated for controlled drug delivery. Fabrication of
BC-based nanocomposites to optimize the control drug delivery
is the most important among the strategies that are employed in
order to enhance the drug release retardant effects of BC. In this
context, the combination of BC and polyacrylic acid (PAA) (BC-PAA)
has been studied in detail. BC-PAA was synthesized by polymerization initiated by electron beam irradiation using different doses
of radiation (Amin, Ahmad, Halib, & Ahmad, 2012; Halib, Amin, &
Ahmad, 2010). It was observed that the degree of swelling of the
composites increased with increasing radiation dose and decreasing ionic strength. These composites also showed sensitivity to pH
with maximum swelling at pH 7. These BC-PAA nanocomposite
hydrogels were tested as pH-responsive materials for controlled
in vitro drug delivery using different loadings of bovine serum albumin (BSA) as model compound (Amin et al., 2012). The drug-release
proles were also controlled using sequentially simulated gastric
uid (SGF) for 2 h and a simulated intestinal uid (SIF) without
enzymes until the maximum drug release. It was observed that the
release of drug was much slower (about 15%) in SGF at the end of
2 h. On the other hand, the release rate was considerably higher in
SIF, but decreased with the increasing radiation dose. For the lowest radiation doses a complete release was achieved at the end of
8 h, whereas at highest radiation doses it took 1314 h for maximum drug release (Fig. 5). The different release rates in SGF and SIF
were due to the effect of pH over the swelling rate of the materials.
It was reported that the pH-responsive behaviour of the material
toward drug delivery was similar to native BC membranes (Amin
et al., 2012).
A similar study was conducted by Ahmad, Amin, Mahali, Ismail,
and Chuang (2014) who fabricated stimuli-responsive hydrogel BCPAA nanocomposites for oral delivery of proteins. When assessed
for BSA release, the cumulative release was less than 10% in SGF.
This demonstrated the potential of BC-PAA for protecting BSA from
the harsh acidic environment of the stomach. Upon conformational
stability analyses by an esterase activity assay, circular dichroism,
and sodium dodecyl sulphate-polyacrylamide gel electrophoresis;
the structural integrity and bioactivity of BSA was preserved and

maintained by the hydrogels as shown in Fig. 6. Furthermore, ex vivo
permeation experiments demonstrated an increase in BSA penetration across the intestinal mucosa. The BC-PAA showed outstanding
cytocompatibility with no signs of toxicity (Ahmad et al., 2014).
Moreover, biodegradable monolayer and multilayer lms of BC
and sorbic acid (SA) (BC-SA) were designed for antibacterial purposes against E. coli K12-MG1655 (Jipa, Stoica-Guzun, & Stroescu,
2012). The authors reported that both BC and SA concentration
affected water absorption, drug release rate and antimicrobial
activity. The release of SA was faster when the BC concentration
was higher, but became signicantly slower by increasing SA content due to slower dissolution of formed crystals. Multilayer lms
showed faster SA release rate as compared to monolayer lms.
These studies provide a strong base for the application of BC-based
composites in drug delivery applications. Further details of these
studies are summarized in Table 5.
2.6.2. Transdermal drug delivery
BC is a biomaterial having unique physical and mechanical
properties, which prompted substantial interest for biomedical applications including transdermal drug delivery. In a study,
freeze-dried BC was treated with gamma irradiation to obtain BC
nanocomposites (Olyveira, Costa, & Basmaji, 2013). The prepared
BC nanocomposites were used as an alternative mean for transdermal drug delivery. Drug diffusion across BC nanocomposites
was studied through diffusion cell method. Lower density of pores
was produced in non-irradiated samples than in irradiated, which
resulted in faster diffusion of the drug than that of the irradiated
membrane. To assess the therapeutic feasibility of BC membrane,
in vitro permeation study of two model drugs (ibuprofen and lidocaine hydrochloride) was carried out through in vitro diffusion
studies. The drug release from BC membrane was studied through
human epidermis and compared with other formulation systems,
such as lidocaine hydrochloride gel, aqueous solution, ibuprofen
gel and solution in PEG400. Lidocaine hydrochloride in BC membrane provided lower permeation rate than that of conventional
formulations. However, the permeation rate was higher (3-fold) for
lipophilic drug (ibuprofen) as compared to gel or PEG400 solution
of ibuprofen as depicted in Fig. 7 (Trovatti et al., 2012). In all cases,
water content the BC membranes were partially removed followed
by absorption of a solution of the selected drug having glycerol
that imparts a plasticizing effect, which turns the BC membrane
more exible and suitable for transdermal application, facilitating
rehydration and swelling of BC. Glycerol is also a humectant and
plasticizer of the SC with a favourable effect on epidermal permeation of the drugs (Trovatti et al., 2012, 2011).
BC membranes have also been explored as novel nanostructured
transdermal delivery systems for diclofenac sodium (Silva et al.,
2014). Diclofenac sodium was loaded into BC membrane and glycerol was used as plasticizer. The drug containing BC membrane was
quite exible, homogeneous and presented a considerable swelling
behaviour. In vitro diffusion studies using human epidermal membranes exhibited that diclofenac incorporated BC membrane has
comparable permeation rates to commercial patches and lower
than that of a commercial gel.
Bodhibukkana et al. (2006) studied BC composites with molecularly imprinted polymeric (MIP) matrices for the enantioselective
transdermal delivery of racemic propranolol. MIP matrices having
specic binding sites for drug were obtained by in situ copolymerization of methacrylic acid, using ethylene glycol dimethacrylate
as a cross-linker, in the presence of S- or R-propranolol as the template molecules. The selective transport of S-propranolol through
the MIP composite membrane was obtained, which was mostly due
to the cellulose membrane with some ancillary contributory effect
from the MIP layer. Simple methods for drug-loading, comparable
345
Fig. 5. In vitro drug release proles (a) 1% (w/v) BSA and (b) 0.5% (w/v) BSA.
Reproduced with permission (Amin et al., 2012), Copyright, Elsevier.
Fig. 6. Graphical presentation of stimuli-responsive BC-PAA composites.

Reproduced with permission (Ahmad et al., 2014), Copyright, American Chemical Society.
Table 5
Applications BC-based composites in drug delivery.
Method
Drug
Finding
Reference
BSA
In vitro dissolution
Amin et al. (2012)
BSA
followed by irradiation
followed by irradiation
SA
In vitro dissolution, ex vivo

permeation, stability of BSA,
bioocompatability
Antibacterial activity against
E. coli
Diffusion cell
Sustained drug release for

1314 h directly proportional
to irradiation dose
Potentials for oral protein
delivery, stable BSA and
biocompatible hydrogel
Possessed antibacterial activity
Slower release from irradiated
composites
Lower permeation rate for
lidocaine hydrochloride and
higher for ibuprofen
Comparable permeation rates
to commercial patches and
signicantly lower than that of
a commercial gel
, Olyveira, Costa et al.

(2013)
Trovatti et al. (2012)
Insulin
Ibuprofen and
lidocaine
hydrochloride
Diclofenac sodium
Human epidermis
Human epidermis
Ahmad et al. (2014)
Jipa et al. (2012)
Silva et al. (2014)
346
Fig. 7. Drug loaded BC membrane (a) lidocaine hydrochloride, (b) ibuprofen (SEM), and drug permeation study from (c) lidocaine hydrochloride, (d) ibuprofen.
Reproduced with permission (Trovatti et al., 2012), Copyright, Elsevier.
drug release prole and ease of application indicated the potentialities of BC membrane for transdermal administration of different
type of drugs. These studies are summarized in Table 5.
2.7. Applications in bioengineering
2.7.1. Biosensors and bioanalysis
Recently, biosensors have attained an ample attention in the
eld of nanotechnology and medicines (Hasan et al., 2014). Such
biosensors have been developed to be used in diverse elds, such
as for applications in biotechnology, tissue engineering and regenerative medicine (Malhotra, Singhal, Chaubey, Sharma, & Kumar,
2005). Receptors, antibodies and enzymes have been extensively
used in biosensors and bioanalysis as biosensing elements (Kim
et al., 2011). These devices can check real-time biological signals in
vivo, such as the release of antibodies or proteins in response damaged tissue, infections, inammatory events, cardiac infarction or
muscular dystrophy. Thus, biosensors have the capability to inform
about the health-care complications in time and thus help in earlier
detection of disease and treatment in clinical settings (Giepmans,
Adams, Ellisman, & Tsien, 2006). Synthetic BC-Ag0 nanocomposites
have been reported to be used in bioanalysis as surface enhanced
Raman scattering (SERS) substrates using 2,2-dithiodipyridine and
thiosalicylic acid as analytes (Marques, Nogueira, Pinto, Neto, &
Trindade, 2008). The detection limits for the abovementioned analytes were much lower (104 moldm3 ) than the conventional
plant derived cellulose analogues. The BC-Ag0 nanocomposites
were used in bioanalysis of amino acids (l-histidine, l-glutamine
and l-phenylalanine). Moreover, one of the most frequent biosensor in use is for blood glucose monitoring (Khatun, Nurunnabi, Cho,
& Lee, 2012). Furthermore, Wang, Gao, Zhang, and Wan (2010)
and Wang, Li et al. (2010) fabricated nanocomposite between Au0
nanoparticles and BC nanobers (BC-Au0 ) as a source for amper-
ometric glucose determination. The prepared enzymatic glucose

biosensor was highly sensitive for the detection of glucose and was
able to detect glucose concentration as low as 2.3 M with a linear
range of 10400 M. This BC-Au0 nanocomposites based biosensor
was effectively used to determine glucose in human blood samples. Furthermore, Zhang et al. (2010) used BC nanobers as robust
biotemplate for the facile fabrication of novel (BC-Au0 ) nanocomposites. For this purpose, BC nanobers were immersed in aqueous
suspension of Au0 using poly(ethyleneimine) both as the reducing
and the linking agent. The obtained BC-Au0 nanocomposites (Fig. 8)
were used for immobilization of horseradish peroxidase (HRP),
which ultimately worked as an innovative H2 O2 biosensor with
detection sensitivity of lower than 1 M. In another study, polyaniline (PAni) was used for in situ preparation of nano-assembly
with BC nanobers to enhance the electric conductivity (Shi, Li
et al., 2012; Shi, Zang et al., 2012). The electric conductivity of
BC-PAni composite was improved from 108 to 102 S cm1 . The
BC-PAni composite works as an electro-conductive hydrogel, which
may nd applications in biosensors and bioanalysis. Similarly,
Chen et al. (2011) developed a novel Au0 nanoparticles/self-doped
PAni nanobers (Au-SPAN) biosensor having grooves for the HRP
immobilization on glassy carbon electrode surface. The developed
biosensor possessed electro-activity in high pH medium with good
linear response (102000 M) and a detection limit of 1.6 M
under optimum conditions. The biosensor exhibited acceptable
accuracy for the quantication of H2 O2 in real sample. In addition, the Au-SPAN biosensor was selective, reproducible and stable
(Chen et al., 2011), which suggests its potential applications for
other enzyme-based biosensors. In another study, Eisele et al.
(1994) used BC as outer membrane in glucose biosensors for amperometric determination of glucose oxidase reaction products. A
long-term stability (200 h) was achieved with diluted blood, (1:10)
as compared to commercial Cuprophan membrane (stability 30 h)
347
Fig. 8. SEM images of BC-Au0 nanocomposites prepared at pH 2.5 for 1 h (a and b) and pH 11.5 (c and d). [PEI] = 1 gL1 , [HAuCl4 ] = 1 mM. The arrows in (a) and (d) indicates
Au0 nanoparticles and naked BC nanobers, respectively. The inset in (c) shows the high magnied image of dispersed Au0 nanoparticles.
Reproduced with permission (Zhang et al., 2010), Copyright, John Wiley and Sons.
under the same conditions. Similar patterns in terms of stability

were observed with undiluted human blood; BC-based biosensor
was more stable (more than 24 h) in comparison to Cuprophan
(only 34 h). The measuring range of biosensor for glucose could be
extended up to 170 mM by treating BC membrane with polyamide
(Eisele et al., 1994). PVC-based membrane electrodes used for direct
in vivo detection of electrolytes are associated with thrombogenic
concerns. In a study, Badr et al. (2015) modied the surface of
PVC-based electrode by sandwiching it in BC (BC-PVC) or heparin
modied BC (HBC-PVC). The response characteristics of modied
PVC-based electrodes (BC-PVC and HBC-PVC) including response
slope, linear range, limit of detection and selectivity coefcients,
and overall response were comparable to those of control PVCbased membrane electrodes (Badr et al., 2015). Moreover, BC-PVC
and HBC-PVC were more biocompatible than PVC, which suggest
their potential in designing biocompatible PVC-based potentiometric membrane electrodes. Similarly, uorogenic biosensors for
detection of esterase have been developed using chemically modied BC, whereby uorescein-tetraethylene glycol-azide (FTA) was
attached to the celluloses (including BC). This biosensor has the
advantage that the uorophore remains attached to the substrate
by covalent bond, preventing its diffusion and subsequent harmful
effects during in vivo bioanalysis (Derikvand, Yin, Barrett, & Brumer,
2016). Furthermore, functionalized BC, inorganic-organic hybrid
nanopaper has been fabricated by using nanoparticulate oxides of
titanium, vanadium and a mixture of both (Gutierrez, Fernandes,
Mondragon, & Tercjak, 2013). One of the future potential applications may be its use in biosensors and bioanalysis. However, further
studies are needed to investigate such potentials of these nanocomposites.
2.7.2. Enzymes and cells immobilization

BC in pellet form has potential applications in enzyme immobilization (Wu et al., 2013). Glucoamylase, extensively used in food
industry, was immobilized on BC beads (Wu and Lia, 2008; Wu
et al., 2013). The wet BC beads of the smallest size (0.51.5 mm)
were found the best support for glucoamylase immobilization
(Wu and Lia, 2008). The immobilization enhanced the stability of
enzyme against changes in the temperature (particularly in the
lower temperature region) and pH value. The relative activity of the
immobilized glucoamylase was still above 77% at pH 2.0, being the
highest value reported in the literature. The relative activities were
more than 68% in the lower temperature region even at 20 C (Wu
and Lia, 2008). Moreover, when immobilized via periodate oxidation method, the activity of enzyme was increased about 40% than
the previous study (Wu et al., 2013). Thus, BC beads act as promising
support for the preparation of immobilized enzymes in industrial
applications.
Other studies revealed that immobilized yeast on BC for
repeated batch fermentation in the production of wine improves
the cost effectiveness due to reduction of inoculum preparation
and simple separation for recovery of yeast at the end of the fermentation process (Nguyen, Ton, & Le, 2009; Ton and Le, 2011).
In 10 cycles of the repeated batch fermentation, the immobilized yeast in BC always showed higher metabolic activities than
the free/mobilized yeast. Similarly, BC-alginate (BC-Al) composites
have also been successfully designed for immobilization of yeast
by the process of freeze-drying (Kirdponpattara and Phisalaphong,
2013). The BC-Al composites exhibited several advantages, such
as strong hydrophilicity, appropriate pore size, high porosity, and
high thermal, chemical and mechanical stabilities. After 48 h of the
fermentation process, the maximum ethanol content biosynthesized by the yeast immobilized culture (IC) in the BC-Al carriers
348
was approximately 100 gL1 , which was about 13% and 45% greater
than that of suspended culture (SC) and IC in Ca-alginate matrix,
respectively (Kirdponpattara and Phisalaphong, 2013). IC in BC-Al
composites gave more stable repeated-batch ethanol productions
than those using IC in Ca-alginate matrix or SC. The improved
ethanol production was attributed mainly to the properly interconnected pore structure and water uptake ability that help in
substantial mass transfer during the production process. Using the
abovementioned methods, BC can be used for ethanol production,
which is a product of pharmaceutical and medical importance.
Similarly, BC-based composites can be used for immobilization of
various types of important enzymes and cells.
It can be concluded from the abovementioned literature that BC
nanocomposites can be employed for the immobilization of numerous enzymes, and may nd potentialities for applications in the
eld of biosensors, bioelectroanalysis and bioelectrocatalysis.
Acknowledgments
Dr H. A. Santos acknowledges nancial support from the
Academy of Finland (grant nos. 252215 and 281300), the University
of Helsinki Research Funds, the Biocentrum Helsinki, and the European Research Council (FP/20072013, grant no. 310892). Hanif
Ullah is thankful to Higher Education Commission of Pakistan for
granting the scholarship under HEC Indigenous Fellowship Program and for 6 months nancial support to be a visiting researcher
at University of Helsinki, under International Research Support
Initiative Program. The manuscript was reviewed for English
language/grammar by Tamas Kriska, PhD, Department of Pharmacology and Toxicology, Medical College of Wisconsin, USA. The
authors wish to thank Tamas for his assistance.
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2.7.3. Stem cell therapy
In last decade, extensive research has been carried out on stem
cell therapy (Zahra, Muzavir, Ashraf, & Ahmad, 2015). Various biomaterials including BC nd applications in stem cell research. In a
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Recently, there has been a growing interest in the use of cellulose
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Carbohydrate Polymers 150 (2016) 330352

Contents lists available at ScienceDirect

Advances in biomedical and pharmaceutical applications of functional

Department of Pharmacy, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan

H. Ullah et al. / Carbohydrate Polymers 150 (2016) 330352

Applications in drug delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344

to uridine diphosphate glucose (UDP-glucose) by UDP-glucose

H. Ullah et al. / Carbohydrate Polymers 150 (2016) 330352

ketogluconate and not cellulose (Masaoka, Ohe, & Sakota, 1993).

biodegradability. The cellulose synthase in G. xylinus can consume

H. Ullah et al. / Carbohydrate Polymers 150 (2016) 330352

Fig. 1. Various superior properties of bacterial cellulose compared to plant-based cellulose.

plement activation parameters in comparison to conventional graft

2. Applications of BC and BC-based composites

H. Ullah et al. / Carbohydrate Polymers 150 (2016) 330352

current biomedical and pharmaceutical applications of BC-based

2.1. Antimicrobial and antiviral lm

cosamine units amalgamated in BC sheets (Ciechanska,

H. Ullah et al. / Carbohydrate Polymers 150 (2016) 330352

reducing agent). The interaction between Ag0 nanoparticles and

2.2. Wound healing, articial skin and skin tissue repair

Loading during synthesis + post-synthesis

Antimicrobial activity increased with chitin amount

Butchosa et al. (2013)

Loading during synthesis

Bacteriostatic against Gram-positive and Gram-negative,

Effective pore size against virus (2070 nm), antibacterial

Wanling et al. (2012)

Antibacterial activity against S. aureus and E. coli

Jung et al. (2009), Maneerung et al. (2008)

Post-synthesis, in situ reduction of AgNO3 through NaBH4

Dobre et al. (2012)

Sureshkumar et al. (2010)

Antimicrobial activity against Gram-positive and

No toxicity to human adipose-derived mesenchymal stem cells

Figueiredo et al. (2013)

Signicant antibacterial activities, favourable biocompatibility,

Luan et al. (2012), Wen et al. (2015)

Post-synthesis, ex situ (cellulose was dipped in dispersion of

Antimicrobial activity against K. pneumoniae and S. aureus,

Pinto et al. (2013)

Ul-Islam et al. (2013)

Inhibition of S. aureus and E. coli

Post synthesis, direct in situ metallization by treating Ag+

Post-synthesis, in situ radical polymerization of

Maria et al. (2010)

Pinto et al. (2012)

Zhijiang and Guang (2011)

H. Ullah et al. / Carbohydrate Polymers 150 (2016) 330352

PVA and Chitosan

Cai and Kim (2009)

H. Ullah et al. / Carbohydrate Polymers 150 (2016) 330352

factors resulting in degradation of tissue and severe damage. The

H. Ullah et al. / Carbohydrate Polymers 150 (2016) 330352

signicantly reduce the selected inammatory interleukins and

2.3. Cardiovascular system

H. Ullah et al. / Carbohydrate Polymers 150 (2016) 330352

(Leito et al., 2016). The current research regarding the application

2.3.2. Cardiac prosthesis

H. Ullah et al. / Carbohydrate Polymers 150 (2016) 330352

Model for assessment

Viscoelastic behaviour, tensile strength and modulus

Brown et al. (2011)