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Cellulose - Chitosan Nanocomposites

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Submitted: 11 February 2015 , Reviewed: 12 October 2015 , Published: 09 December
2015

DOI: 10.5772/61727
Necessary Preferences Statistics Marketing

W R I T T E N BY
Guillermo H. Riva, Joaquín García-Estrada, Brenda Vega, Fernando
López-Dellamary, María E. Hérnandez and José A. Silva

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FROM THE EDITED VOLUME

IntechOpen

Cellulose
Fundamental Aspects and
Cellulose - Fundamental Aspects and C
Current Trends

Edited by Matheus Poletto urrent Trends

Edited by Matheus Poletto and Heitor Luiz Ornaghi Junior
 Book Details Order Print

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Abstract
This research describes the preparation of membranes with chitosan
(CS) as the polymeric matrix and cellulose nanocrystals (CNC) as
reinforcement. The aim was to evaluate their physical, mechanical and
biological properties, and to determine their potential for biomedical
use. Membranes were prepared via casting CNC suspensions in CS
solution, at CNC concentrations of 0.5%, 1.0% and 2.0% (w/w) with
pure chitosan as a reference. Analysis of membrane properties was
performed using several techniques, such as ATR – FTIR, SEM,
swelling test, maximum water absorption, dynamical mechanical
analysis and in vivo (Winstar rats) biocompatibility and
biodegradability assays for biological evaluation. Experimental results
established that CNC reduced swelling rates and increased the
maximum water absorption when CNC concentration was higher.
Therefore, the presence of CNC in the matrix reduced Young’s modulus
by approximately 50% in comparison with pure chitosan. All
formulations demonstrated biocompatibility and biodegradability
values ranged between 4% and 21% in the 30 days after implantation.
Based on these results, these membranes may be of use for biomedical
applications.

Keywords

Cellulose nanocrystals chitosan biomedical biocompatibility

nanocomposites

Author Information Show +

Chapter sections
1. Introduction

1.1. Nanotechnology in actual context

The advances in nanotechnology for biomedical use are increasing and

are at the forefront of scientific research. In recent years, hot spot areas
such as drug transportation, tissue regeneration or nanomaterial
development for cell-growth scaffolds have been constantly advancing.
Biomaterials that are considered for biomedical applications must
confirm to strict biological, physical and mechanical characteristics.
Some biopolymers in particular, offer advantages in terms of
sustainability and low environment impact compared to ceramics and
metals. These attributes are the biocompatibility (an absence of
inflammatory, cytotoxicity or invasive response in native cells, tissues
or organs in vivo), biodegradability and bioabsorbability (the material
and its by-products will degrade and/or be absorbed or safety
eliminated from the body). More suitable properties may be the
degradation rate (this rate must match the regeneration time of tissue in
damaged zones, as well as transfer the mechanical efforts to new tissue
in a timely manner), porosity (directly linked to mass transport and
efficient tissue regeneration) and surface morphology. As drawbacks,
the physical and mechanical properties of these materials make them
less suitable than petroleum based plastics and other materials (metals,
alloys and clays). As a consequence, reinforcement of the matrix is an
option to counterbalance some of those drawbacks.

1.2. Biopolymers

1.2.1. Cellulose

Cellulose — the most abundant biopolymer on Earth — has an annual

production of 7.5 x 1010 tons. This biopolymer is widely distributed in
higher plants, sea animals (tunicates), and to a lesser degree in algae,
fungi, bacteria, invertebrates, and is even found in protozoans such as
Dictyostelium discoideum. In general, cellulose is a hard, fibrous, and
water insoluble substance that plays an essential function in keeping
the structure of cell walls in plants [1]. Cellulose can be found in its
purest form in plants (i.e. cotton fibers). However, in wood, leaves and
plant stalks, it is found mixed with other materials such as lignin and
hemicelluloses. Cellulose nanofibers have the potential to be used in
multiple ways, notably as a reinforcement material in the development
of nanocomposites [2].

Thus, the preparation of biocompatible nanocomposites employing

cellulose nanocrystals (CNC) as a reinforcement is a natural choice
based on them being inert, biocompatible, biodegradable [3], and non-
cytotoxic. They also contribute to the regeneration of damaged tissues
or organs [4] and have mechanically desirable properties [5].

1.2.1.1. Types of processes used to obtain CNC

Cellulose nanocrystals (CNC) can be obtained by different techniques

and processes:

Mechanical processes [6], i.e. using used bleached pulp of softwoods

and hardwoods as a material raw to obtain nanocrystals from. The
process begins with the soaking and grinding of fibers, followed by
sieving and refining (for hardwoods this process is repeated several
times). Finally, fibers are submitted to high pressure and
homogenization processes (1000 Bar, 180 min), which are repeated
until CNC is obtained. Energy consumption and Young’s modulus
(YM) are higher in hardwoods than softwoods. The tensile resistance
in softwoods is better (75 MPa versus 63 MPa respectively).

Enzymatic processes [7], which have the advantage of simplifying the

mechanical process of obtaining cellulose microfibers, mainly in the
homogenization and disintegration stages. Acid hydrolysis was applied
as a pre-treatment to the mechanical process. The sequence begins with
refining and enzymatic treatment followed by a second refining and
then homogenization. Endoglucanase was used as the enzyme and
microfibers of 10–20 nm diameters were obtained.

Biological processes [8], a new method of obtaining nanocellulose

using the Dutch elm disease fungus was established. The technique
begins with the soaking and disintegrating of pulp in 2 liters of water,
followed by 20 minutes in an autoclave with added sucrose and yeast to
ensure feeding and growth. The fungus was left for 2–4 days at room
temperature with mild stirring. Fibers were added to an autoclave to be
washed again and the process finished with refining high speed
cutting.
Chemical processes [1]. There are two basic methods for chemical
modification: The first changes the surface energy characteristics of
nanocrystals allowing an improvement in compatibility, especially
when they are used with hydrophobic or non-polar matrices in
nanocomposites, thereby obtaining a better dispersion in the matrix.
The second method adds electric charges to the nanofibers (positive
and negative) allowing a good dispersion. Acetylation of nanocrystals
is an example of the first kind of modification [9], making their
surfaces more hydrophobic. Kenaf fibers (Hibiscus cannabius) were
modified using acetic anhydride before cellulose nanofibers were
obtained from acetylated cellulose. This technique includes processes
of disintegration, refining, cryorupture, and high pressure
homogenization.

1.2.1.2. Nanocellulose for biomedical use

Nanocellulose has been called “biomaterials´ eyes” due to its potential

for numerous applications in the biomedical field, including skin grafts
to burn damage and wounds, growing of blood vessels, nerve
reconstruction, brain membranes, and scaffolds in tissue engineering
and bone reconstruction. Tissue engineering (TE) involves searching
for new materials and artefacts to interact in positive ways with
biological tissues. Furthermore, TI is seeking a primal artefact to
cellular development in vitro, rearrangement and development of
tissue when it will be implanted. The main attribute wanted in
biopolymers with a potential biomedical use is a controllable and
specific activity, to be used mainly in cellular scaffolds. Recently, many
of these kinds of materials have been developed, having the required
properties (physical/chemical and mechanical) dependent mostly on
the final application (tissue regeneration, drug releasing, scaffolding,
etc.). The success of scaffolds depends mainly on cellular adhesion and
surface growth. The chemical surface of a biopolymer can cause the
cellular response to interfere with the adhesion, proliferation,
migration and cellular functionalization. The interaction in cell surface
it’s whole important in the graft, including its rejection. For the
regeneration of tissues, three fundamental aspects are important: the
cells, and the bearing and growing factors. The cells synthetize the
matrix to new tissues, the bearing creates a suitable environment for
cell development, and growing factors promote cell regeneration.
Furthermore, regeneration must be promoted and if it is necessary, the
new material must be absorbed or biodegraded. Studies of the
interactions of cell bearing are crucial for the feasibility of grafts.
Different responses of cells can be observed from several materials,
based on the ability of cells to distinguish and/or adapt to the surface of
the material. This last factor is crucial, because it drives different
responses such as cell proliferation, cell migration or feasibility. With
issues regarding the skin, several laboratories have shown an interest in
developing products that offer advantages such as the immediate
mitigation of pain, close adhesion at wound surfaces and the reduction
of infection rates. Nanocellulose has a large surface area that brings a
better capability for water absorption and elasticity, these being the
best characteristics for a recovering bandage, as microbial activity is
stopped. Indeed, nanocellulose is very effective in reducing pain and
promoting the granulation suitable for wound bandages. Another great
advantage of nanocellulose consists in the capability to be built in any
shape and size, making it ideal for covering extensive and difficult
areas of the human body [10].

Hence, cellulose and CNC have been used in biomedicals. Past research
has shown them to be ideal for tissue engineering, producing favorable
results [3, 6]. CS membranes with nanoreinforcement must show a
Young’s modulus of 1,500—2,300 MPa to be suitable for biomedical use
[11].

Furthermore, applications with excellent, proven results have been

reported as follows [10]:

Pharmaceutical. Cellulose has excellent properties of compaction when

it is mixed with other pharmaceutical excipients, forming dense
matrices that make the administration of therapeutic drugs easy.
Nanocellulose offers potential advantages as an excipient in drug
release. Its large surface area and negative charge suggest that higher
quantities of therapeutical drugs can be added to the surface of this
material, showing the potential for a large quantity of charge and the
optimal control of dosification. The proven biocompatibility of
cellulose supports the use of nanocellulose for similar purposes. The
hydroxide groups on the surface offer a site for surface modification to
a broad range of chemical groups, using different methods. The surface
modification can be used to tune the charge and drug release that are
not normally linked with nanocellulose such as hydrophobic and non-
ionized drugs.

Odontology. Nanocellulose can be used as biological barrier due to its

porosity. This makes it ideal for use with infections, loss of fluids and it
has an analgesic effect that allows therapeutic drugs be used easily and
absorb the residual fluids during inflammatory stages, can be rejected
in a controlled and painless way.

Ophthalmology [12]. Researchers explored the potential of

nanocellulose as a scaffold and found it suitable for use in the
development of tissue engineering for the cornea. They studied the
growth of human stem cells in nanocellulose. The growth of corneal
stem cells inside the scaffold was verified with a scanning laser
microscope. The results suggested the potential of this biomaterial as a
scaffold for tissue engineering of artificial corneas.

Vascular surgery [13]. Researchers studied artificial vascular implants

of nanocellulose in two cases: The first was a microsurgery study,
where nanocellulose implants were used as an artificial part of the
carotid artery of rats for a year. These results showed the incorporation
of nanocellulose under the formation of tissues and internal growth of
active fibroblasts over a long period. In a second study, the implants
were used to replace the carotid artery of pigs. After three months, the
implants were retired and analyzed at both macro and microscopic
levels. Seven implants (87.5%) were found in use and just one of them
was blocked. This data showed that the innovative techniques of
nanocellulose engineering have allowed the production of stable
vascular conduits and confirmed the very notable achievement of the
use of tissues for blood vessels in vivo as a part of cardiovascular
programs.

Reconstructive / aesthetic surgery [14] Ideal for nasal reconstruction.

The response of tissue in the presence of nanocellulose in nose bone
was evaluated. In the study, 22 rabbits were used and in 20 of them a
cellulose film was added to the nasal dorsum, with the remaining two
acting as a control. After three and six months, the new bone was
extracted for histopathology studies. Parameters such as blocking of
blood flow, inflammation intensity and inflammation by the presence
of purulent liquids were found to be stable, probably due to the
surgical process itself rather than the presence of cellulose. For the
other parameters, the statistical response was not significant. The
nanocellulose coverage showed good compatibility and remained
unchanged over time, making this material an excellent option for
rebuilding new bone.
1.2.2. Chitosan

Chitosan (CS) is a biomaterial of proven use in the biomedical field due

its biocompatibility, biodegradability and antibacterial activity,
making it ideal for drug transportation, tissue engineering, wound
healing, and antibacterial uses [15]. Furthermore, chitosan is bioactive
and nontoxic. This biopolymer has a wide range of uses such as
substance separation due to its barrier property and sensors, as well as
food packing. Other authors describe the preparation of
bionanocomposites using chitosan and different nanoreinforcements
with the goal of obtaining a better mechanical performance, as well as
the barrier properties and sensing detectors [16]. Chitosan is
commonly amorphous and can be processed in flexible films. It is
bioactive, non-toxic, and suited to biomedical applications such as
pharmaceutical products like films, pearls or spheres, and gels,
powders, etc. Previous works in cellulose-chitosan nanocomposites
showed good results in physical, mechanical and biological tests [17, 18
].

Chitosan can form flexible, clean and hard films [19] with a good
oxygen barrier [20]. Furthermore, it can be used as packing material,
mainly as a covering and edible film [21] extending the average life of
foods [22, 23]. Chitosan can also form a semipermeable covering to
modify the inner atmosphere, thereby reducing the transpiration rate
of the product in the packaging [24]. Despite good results with respect
to their mechanical properties, chitosan films can be brittle, making it
necessary to use plasticizers to increase their flexibility [25].
Plasticizers such as glycerol can improve the processability, as well as
the mechanical properties of chitosan [26]. In another study,
researchers reported a concentration of 20% (w/w) of glycerol as the
appropriate concentration to improve the flexibility of chitosan films
[27]. To prevent this drawback of rigidity and brittleness of chitosan,
the addition of reinforcement has showed to be useful in enhancing its
mechanical, thermal and barrier properties. When the particles are
smaller, the interaction with the matrix is better [28], with the low cost
a sign of efficiency [29]. Fillers on a nanometric scale (called
nanoparticles or nanoreinforcements) with good dispersion drive an
interface matrix / filler, changing the molecular mobility, relaxation
behavior and thermal and mechanical properties of the material [30].

A positive result in the elaboration of chitosan – CNC nanocomposites

was obtained, using electrospinning for fibers of a derivative of
chitosan / cellulose in an ionic liquid (IL). The chitosan / cellulose
composite were electrospun in the ethanol co-solvent, using the IL to
dissolve the chitosan and cellulose at the same time. Furthermore, the
IL was capable of building fibers of pure chitosan / cellulose composite
after the IL was removed by the ethanol. The fibers of this composite
were manufactured as a three-dimensional shape, offering
antibacterial activity to treat burns, bedsores and skin ulcers [31].

Nanofibers were obtained from chitosan and cellulose, with chitin used
as a reinforcement material at different concentrations (from 1.25% to
5.0% w/w). This allowed the optimizing of the process conditions to
obtain homogeneous and porous nanofibers. This material has a
potential for use in wound bandages and skin burns [32].

Layer-by-Layer technique (LbL) is a technique to elaborate

nanocomposites of chitosan and cellulose whiskers. The interactions
between amine groups (chitosan) and sulfate groups (cellulose
whiskers) ensure the linkages between matrix and nanoreinforcement
to elaborate the films. The average thickness of each bi-layer (whiskers
/ chitosan) was 7 nm and each film was formed by 30 bi-layers. These
materials have a wide range of uses such as packaging and biomedicals
[15].

Chitosan films used for cell scaffolding in the regeneration of the

tympanic membrane (type I experimental tympanoplasty) were
elaborated. These films were grafted in New Zealand rabbits with
successful results of tympanic tissue regeneration [33].

A dv e rt i s e m e n t

2. Objective

Demonstrate that CNC can improve the mechanical and physical

properties of a chitosan matrix. Furthermore, determine the
biocompatibility and biodegradability of CS and CNC nanocomposites
via biological tests and then based on the results obtained, determine a
potential use of these nanocomposites in the biomedical area.

A dv e rt i s e m e n t

3. Experimental

3.1. Materials

Materials used for this study were Biomedical Grade chitosan from
Sigma Aldrich (Deacetylation grade 75–85%), acetic acid, alpha
cellulose (Neucel Cellulose Ltd.), male laboratory rats (Wistar),
cellulose acetate membranes for dialysis, vacuum oven, high resolution
microscopy, dynamical-mechanical and chemical analysis. The
formulations employed in this research are presented in Table 1.

Material Reinforcement Formulation (w/w)

Pure CS

CS + CNC (0.5%)
CS CNC
CS + CNC (1.0%)

CS + CNC (2.0%)

Table 1.
Formulations of CS + CNC films

3.2. Methodology

Preparation of CNC

Alpha cellulose was ground and mixed with sulfuric acid (64%
concentration) for one hour under constant stirring and at a controlled
temperature (approximately 50° C in a warm bath). After that, the
liquor was added to deionized water, cooled at 8° C 1:10 (v/v) to stop
the reaction. The liquor was centrifuged at 4000 RPM for 5 minutes,
separating the liquor into two phases: solid (cellulose gel in the bottom
of recipient) and liquid (with acid remainders). The liquid phase was
disposed of and deionized water was added to the recipient to remove
excess acid from the gel (containing CNC), prior to centrifugations
(three in total). The washed gel was put in dialysis membranes in
deionized water under stirring until it reached a pH of 5. After that, the
CNC were submitted to ultrasound treatment for 2 minutes and finally
vacuum filtered using 0.45 micron Wharton paper, and kept cooled.

Chitosan – CNC films elaboration

Chitosan (4 g) was dissolved in acetic acid at 2% (v/v) per each

formulation, under constant stirring for 2 hours. Next, CNC in
different concentrations were added (0.5%, 1.0% and 2.0%) and then
stirred for two more hours. The substance was cast on Petri plates
affording a concentration of 0.4 mL/cm2 to obtain the same quantity of
nanocomposite on the plates. The plates were then put into a vacuum
oven (28°C–70 MPa) for 96 hours. After drying, NaOH (1.0 N) was
added to the Petri dishes to precipitate the films and then the CS + CNC
films were washed with deionized water until they reached a pH of 7.
Finally, films were dried at room temperature for 48 hours. The
thickness of films was measured with a micrometer and dimensioned
for physical and mechanical testing.

Physical evaluation

When performing swelling tests, modifications in the dimensions of

specimens were made to evaluate swelling changes over time. The dry
weight (Wi) of each specimen was taken and then films of each
formulation were put in deionized water to control the weight each
minute until a constant value was achieved [34]. For maximum water
retention (MWR), the dry weight (Wi) of each specimen (8 per
formulation) was taken and it was then added to deionized water.
Weights were controlled at 30, 60, and 120 minutes, and then 24 hours,
before the final weight was obtained (Wf) [34]. Finally, the MWR was
determined by the following formula:

MWR (%) = (Wf – Wi ) / (Wi ) x 100

 BB1

Mechanical evaluation

Dynamical mechanical analysis was used in static mode to evaluate

Young’s modulus for all formulations, with the stress-strain test
operating in the controlled force mode. Typical testing conditions were
0.1 N preload, 1 N/min ramp and a gauge length of ca. 10 mm. Strips of
0.075 mm x 5 mm x 20 mm were used. The temperature range was 37.05
± 0.05°C and the moisture content was 98%, determined by a TA
Instruments DMA 800 used in wet conditions.

Biological evaluation

The best way to test our material was in living specimens.

Biocompatibility and biodegradability tests were carried out. For these,
the dry weight (Wi) of films was controlled. The biological subjects for
testing were 16 Lab rats (of the Wistar breed). The animals were
submitted to a surgical procedure involving grafting two portions of
films for each formulation (4 rats per formulation). Each animal had
two sub-cutaneous cuts (in the middle of back, on the right side for
biocompatibility and on the left side for biodegradability). Every three
days, the rats were controlled to prevent any infection or adverse
reaction to the nanomaterial. Specimens were euthanized 30 days after
surgery and the two portions of membrane were retired, lyophilized
and controlled for dry weight (Wf) in order to obtain the
biodegradability value:

Biodegradability (%) = (Wi – Wf) / (Wi) x 100

Biocompatibility was proved by SEM images at the moment the grafts

were retired, the mortality rate of specimens and the non-presence of
encapsulation, fibrillation or any rotten portion of membrane after 30
days.

Characterization of nanocomposites

To characterize the morphology of films, the response and the different

levels of biocompatibility and biodegradability, a JEOL JM 6300 with a
double gold layer to avoid the electrical charge of the sample was used.
The magnification was from around 500x up to 35,000x and the voltage
employed was between 7kV and 20kV.

IR spectroscopy

This technique was used to determine changes in functional groups of

nanocomposite as a consequence of the presence of CNC in the
chitosan matrix, or previous chemical treatment. Portions of thin films
of all formulations were analyzed in an IP Spectrometer in
transmittance mode (4,000 to 500 cm-1). Other parameters were: 16
scans per spectrum, ATR mode and a resolution of 0.4 cm-1. A Perkin
Elmer Spectrum GX FT-IR System was used.

A dv e rt i s e m e n t

4. Results

4.1. CNC + chitosan films

The films obtained from the combination of chitosan and CNC showed
a transparent aspect with a slight yellow color. Transparence in the
films suggests a good distribution of CNC in the CS matrix. At the
same time, the slight yellow color is due to the natural presence of
impurities in this biopolymer [35]. The average thickness of films was
30 microns and the surface was smooth. Visible CNC agglomerates and
dark points were absent from these films as can be seen in Figure 1.

These characteristics in all formulations of pure CS and CS + CNC

were the same, and accorded with findings of other authors [19, 21, 35,
36]. Even with CNC concentrations over 20% (w/w), transparent films
were reported, although with CNC concentrations over 60% the films
showed a translucent appearance (a greyish color) and a rigid but
brittle consistency [35]. Chitosan films and polyethylene oxide (PEO)
employing electrospinning produced good results in transparence and
homogeneity [32].
Figure 1.
a. Film of CS + CNC (0.5%). b. Film of CS + CNC (1.0%) and c. Film of CS +
CNC (2.0%)

4.2. Morphology of CS + CNC films

SEM images showed a good dispersion of CNC in the nanocomposites

due to the affinity between the nanoreinforcement and the matrix
(having similar chemical structure and hydrophilic nature), as well as a
good interaction of negative charges of the sulfate groups of CNC with
the amine groups of CS [35]. The presence of nanoparticles was
observed in different formulations of nanocomposites, as can be seen
in Figure 2. These bundles formed by several tens of CNC, with
dimensions of between 150–200 nm up to 500 nm on average, and in
some cases up to 1 or 2 microns, mainly in CS + CNC (2.0%). The
phenomenon was common when the CNC concentration was higher
(over 5% and 10%) and even when working with concentrations of 5%
and 10% it was possible to see white punctuations in the matrix surface
via SEM images, seen in the cross section of CNC [36]. Another
possible phenomenon linked to a higher concentration of CNC in the
matrix, is the formation of a polyelectrolyte macroion complex
between CS and CNC. The size of these complexes could reach several
microns in length. These particles are formed by CNC and surrounded
by CS chains. The shape of these complexes depends on the quantity of
NH2 in CS. When the concentration of NH2 is high, the shape of the
complex tends to be spherical [37]. These particles could have potential
in biomedicals for drug transportation and controlled release due to
the charges on cellulose and the linkage that can be achieved with other
substances and active composites.
Figure 2.
a SEM image of a cross section of CS + CNC (1.0%) film at 950x. b. SEM image
of CS film at 4,300x. c. SEM image of clusters of CS + CNC (0.5%) film at 8,500x
(circled in red), d. SEM image of clusters of CS + CNC (0.5%) at 35,000x
(circled in red). e CS – CNC (2.0%) macroion at 5,500x and 2f. CS + CNC
(2.0%) at 15,000x.

4.3. Physical properties

Swelling capability is presented in Figure 3. Swelling capability showed

an evident change with the presence of CNC in the matrix. For water
swelling, pure CS films showed saturation times of 3–4 minutes. With
formulations of CS + CNC (0.5%) saturation times reduced to 1–2
minutes, while for formulations with CNC of 1% and 2%, the
saturation time was 1 minute. These results show that CNC allow water
entrance between CS chains as an unstructuring element, making the
material more hydrophilic. This behavior was opposite to that observed
in another study, which used higher concentrations of CNC (from 5%
up to 60%) making the CS films less permeable, thereby reducing the
swelling capability significantly [35, 36, 38]. The maximum water
retention (MWR) showed a similar response with the presence of
CNC, as presented in Table 2.

Figure 3.
Swelling capability for CS + CNC nanocomposites

Variation in time
Formulation
W30min W120min W24h

Pure CS 189.72 198.86 187.49

CS + CNC (0.5%) 237.68 222.67 226.64

CS + CNC (1.0%) 346.52 277.79 258.18

CS + CNC (2.0%) 234.68 236.70 214.65

Table 2.
Values of MWR for CS + CNC nanocomposites

For pure CS films, the MWR was 187% of its own weight in water,
while CS + CNC (0.5%) films obtained values of 226%, CS + CNC
(1.0%) values of 258% and CS + CNC (2.0%) values of 214%, as can be
seen in Figure 4. In these cases, the low concentrations of CNC
improve the water retention properties of films in comparison with
results of other authors [15, 35] which showed a remarkable reduction
in the physical properties of CS + CNC films, using concentrations
from 5% up to 60%. This phenomenon can be explained by the affinity
of CNC to the water, due their hydrophilic nature (and O–H linkages).
Another reason is the plasticizing effect of CNC at low concentrations
of CS matrix, contributing to the unstructuring effect in CS chains
allowing the passage of water inside the polymer. This effect makes the
film more flexible, facilitating the entry of water into the polymer
chains. A similar effect was reported when adding different
concentrations of glycerol (from 2% to 6%), used as a plasticizer in
starch films with CS covering. Even at the highest CS concentration,
the presence of glycerol increased this parameter, with values from
71.8% to 125.4% in comparison with pure CS (35.7% to 74.8%) [21].

Figure 4.
Values (%) of maximum water retention for pure CS and CS + CNC films

4.4. Mechanical properties

The values obtained from formulations with CNC suggest that these
modified the mechanical properties in comparison with pure CS.
Young’s modulus (YM) reached by pure CS was 2,916 ± 933 MPa,
similar to that observed in another study [35], while for formulations
with CNC the values were CS + CNC (0.5%) 1,575 ± 82 MPa, CS + CNC
(1.0%) 1690 ± 433 MPa and for CS + CNC (2.0%) the value was 1657 ± 4
MPa, as can be seen in Figure 5. There was a large variation in the
results for YM in pure CS, with values from 1,080 MPa up to 2,852 MPa.
The average for pure CS ranged between 2,300 and 2,500 MPa. This
variation is because the matrix polymer has crystalline and amorphous
zones, which will exhibit different resistances (high and low values).
Furthermore, residual efforts stored in films as a consequence of some
conditions in their elaboration process (casting solution), appears in
the first stages of assay, when the machine break these efforts first
before apply all the force in the film in a homogeneous way. The graphs
showed an inflexion point in effort–strain curves. This phenomenon
was present in some films of CS + CNC (1.0%) and CS + CNC (2.0%).

Some explanations for the decrease of Young’s Modulus in the films

with CNC suggest the cause as the formation of aggregates and CS–
CNC complexes due to the agglomeration of nanoparticles in one single
place, generating stresses around them and becoming a potential weak
point in the film. It could be deduced that CNC acted as a plasticizer of
the matrix, making it less rigid. However, CS + CNC formulations
showed values lower than those obtained for pure CS. However, these
values were less variable than pure CS values and a further detail was
observed: CNC aggregation didn’t contribute to the deformation or
rupture of films.

Another possible cause of the observed decrease in YM could be the

low concentrations of CNC employed in this study (0.5%, 1.0% and
2.0%) in comparison with other studies that used concentrations above
5%, 10% until 60% [15, 35] that saw improvements in YM of 78% to
150% and 230% to 320% respectively. The decrease in YM must be
understood as a loss of stiffness, making a more flexible material, but
with low strain values (1–2%). This last can be improved if plasticizers
(i.e. glycerol) are included in the formulations. The values obtained are
similar with CS films with chitin concentrations of 2.7% to 37.5%,
having values from 1,622 ± 377 MPa to 2,318 ±619 MPa respectively [11].
Based on these values, a potential use of these films could be in healing
and wound recovery, as well as scar prevention.
Figure 5.
Young’s Modulus values of pure CS and CS + CNC films

4.5. IR Spectroscopy of CS + CNC films

As can be seen in Figure 6, for pure CS the IR spectrum showed bands

at 3,360 and 3,265 cm-1 typical of O–H and N–H bonds respectively. The
band at 2,870 cm-1 corresponds to C–H bonds and the signal at 1,650
cm-1 is commonly seen with type I amides and C=O bonds linked to
acetyl and amine groups, and 1,560 cm-1 corresponds to amide groups
(N–H). Bands of 1,420 and 1,375 cm-1 correspond to residual CH3N–
acetyl glucosamine and –CH2 groups respectively. Finally, bands of
1,060 and 1,030 cm-1 confirm the presence of C–O bonds.

Regarding nanocomposites, formulations of CS + CNC of 0.5%, 1.0%

and 2.0% didn’t show variations in their spectrums when compared
with a pure CS spectrum, as shown in Figure 7. This is due to the
similarity between cellulose and chitosan in terms of their chemical
structure. For that reason, the bands for both polysaccharides are the
same, except for the presence of an amine group with chitosan [15].
Another cause is the low concentration of CNC, which could be
undetected by FTIR. A similar phenomenon has been observed in
chitin, chitosan and glycerol nanocomposites [38].
Figure 6.
IR spectrum of pure CS film

Figure 7.
IR spectrum of CS + CNC (2.0%) film

4.6. Biological testing

Biological results showed 100% biocompatibility for all formulations in

16 rats. After 30 days, the specimens had not shown an adverse
reaction, septicemia or death in the presence of CNC. At the point the
grafts were retired, no negative influence was observed over the
surface of muscles or sub-cutaneous tissues (no evidence of
coagulation, fibrosis, and/or rotten or dead tissues), as can be seen in
Figure 8. A further typical sign of CS in living tissues is the slight
yellow color over the muscle or tissue. In a few cases rolled grafts were
found, due mainly to movement of the specimen during the 30-day
period. A further possible cause could be that the graft was not fixed
with sutures or staples in the biological test, in order to prevent a
different reaction or influence over the grafts.

Figure 8.
Surgical stage of retiring the portions of CS + CNC (0.5 %) (circled in blue), to
evaluate biocompatibility and biodegradability in the specimen.

The biodegradability values were partial but positive for all

formulations, as presented in Table 3. In pure CS, the values reached
between 6.21% and 8.55% in parallel. One sample had 100%
biodegradability and another sample had more weight than the initial
weight, suggesting the presence of new tissue as fascia and/or loose
connective tissue over the graft (as presented in Figure 9), or cells or
blood inside the membrane (biodegradability value: -12.89%). The CS
+ CNC (0.5%) had the most weight gain in their films in comparison
with other formulations (-13.63%, -12.64% and -55.83% respectively).
Just one sample of this group had a low biodegradability value (7.82%).
For CS + CNC (1.0%) the values reached 0.71% to 21.02%. The highest
values of biodegradability were shown by CS + CNC (2.0%) being
19.59% to 60.37% respectively) with just one specimen that gained
weight.
Figure 9.
Presence of loose connective tissue and blood vessels (circled in blue) formed
over the portion of CS + CNC (1.0%)

Biodegradability values (%)

Pure CS CS+CNC (0.5%) CS+CNC (1.0%) CS+CNC (2.0%)

Specimen 1 -12.89 -13.48 8.33 19.59

Specimen 2 8.55 -12.64 0.71 60.37

Specimen 3 100.00 7.82 21.02 -4.92

Specimen 4 6.21 -55.83 5.52 40.52

Table 3.
Biodegradability values for CS + CNC nanocomposites

Indeed, a relationship between the concentration of CNC and higher

values of biodegradability can be deduced. The unstructuring effect of
CNC in CS chains allows the inflow of water, cells and living fluids in
the membrane. This relationship is important because it informs
predictions and design of future research into different presentations
of nanocomposites with higher concentrations of CNC, in order to
study control of the rate of biodegradation for specific biomedical uses
(depending on the part or organ in the body to be used).

The presence of loose connective tissue and fascia were evident for all
formulations, as well as the formation of small blood vessels
surrounding the graft. Based on the principle of time of
biodegradability of new tissue, these films presented different values
after 30 days. For example, CS + CNC (0.5%) showed the best results
for new scaffold tissue in three formulations. In the other cases, we can
estimate the total time for biodegradation to be 3–6 months (less time
with higher concentrations of CNC based on these results). This could
be useful in the future development of possible biomedical uses of
these films.

Another aspect to consider with respect to biodegradation values is the

type of films. In this study, non porous films were elaborated. The
porosity rate and the size of pores directly affect the biodegradability
of films, because they allow the easy passage of water, blood, cells and
other fluids. From their condition, these films could be useful for tissue
scaffolding as can be seen in Figures 10 and 11 (SEM images). A typical
pattern of degradation of chitosan shown as hexagonal borders was
observed in all formulations, at different stages. The formation of
fascia in different stages was evident.

Figure 10.
a. Presence of loose connective tissue in CS + CNC (0.5%) films at 100x and Fig.
b. Hexagonal pattern of chitosan degradation in CS + CNC (0.5%) at 500x.
Figure 11.
a. Presence of loose connective tissue (circled in red) in CS + CNC (1.0%) films
at 100x and b. 300x. Fig. 11c First steps of degradation of chitosan d. Presence of
fascia (circled in red) in CS + CNC (1.0%) films at 500x

A pattern of biodegradation of CS films is supported by the fact that

the biodegradation rate of CS has a linear relationship with the Degree
of Deacetylation (DD). When the DD value (from 0 to 100) is closer to
100, the material shows a slow rate of biodegradation [39]. Other
aspects to consider are that CS is degraded by proteases (mainly
lysozymes) that attack N-acetyl glucosamine linkages making the
process faster, but these linkages have less presence in CS chains when
DD values are higher. Also, in this state, lysozyme activity is low and
the biodegradation rate is slow. Furthermore, CS is a semi crystalline
polymer. In amorphous zones, lysozyme activity is intense and when
the positive charges increase, the interactions between cells with CS are
better, thereby improving biocompatibility [39].

CS scaffolds with a low Degree of Acetylation (DA) and low molecular

weight have a higher rate of biodegradation. Indeed, CS scaffolds with
a low DA present lower biodegradation times, smaller pores, better
mechanical properties, moderate water absorption and more intense
cell activity than CS scaffolds with a higher DA [40]. A pore size of
between 60 and 90 microns is suitable to allow lysozymes inside the
structure and to act in polymer chains.

The CS type used in this research has a DD between 75–85% and a

medium molecular weight related to low biodegradation values. In CS
+ CNC films, the CNC had an unstructuring effect, creating spaces
between CS chains, thereby allowing water (and lysozymes) access
into the scaffold structure, and showing larger values of
biodegradation when CNC concentration was higher.

The attachment, morphology and proliferation of cells on CS scaffolds

is highly dependent on the type of cell lines, the source and
characteristics of CS, the methods of CS scaffold preparation, and the
characteristics of the chitosan scaffolds. In addition, chitosan with a
low molecular weight and low DA has excellent potential as a
scaffolding material for a variety of tissue regeneration systems [40].

A dv e rt i s e m e n t

5. Conclusions

The addition of CNC improves physical properties, increasing their

capability of swelling and water capitation. Mechanically, the main
factor was the unstructuring effects over the chitosan chains. In
biological tests, CNC show positive results for biocompatibility and
biodegradability.

CS + CNC nanocomposites showed physical, mechanical and

biological properties suitable for biomedical use in recovering /
wound healing, tissue scaffolding and scar prevention.

There is a direct relationship between higher concentrations of

CNC and higher values of biodegradability.

The formation of the CS-CNC macroion complex in this study

could have potential for controlled drug release taking into account
other presentations of product (microspheres, nanopowders or
nanoparticles).

A dv e rt i s e m e n t

Acknowledgments
Guillermo H. Riva, wishes to express his appreciation to the following
institutions: German Exchange Students Agency (DAAD) for the
scholarship and kind support during his M.Sc. Studies in Mexico; to
the University of Guadalajara (UdG) for his formation as a M.Sc., for
sponsorship and the financial support of this study, and Neucel
Specialty Cellulose Limited (BC, Canada) for donating the alpha
cellulose used in this study.

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References

1. Habibi, Y., L. A. Lucia, Rojas, O. J. Cellulose Nanocrystals:

Chemistry, Self-Assembly, and Applications. Chemical Reviews.
2010; 110(6): 3479-3500 DOI 10.1021/cr900339w

2. Alemdar, A. and M. Sain. Biocomposites from wheat straw

nanofibers: Morphology, thermal and mechanical properties.
Composites Science and Technology. 2008; 68(2): 557-565.
DOI:10.1016/j.compscitech.2007.05.044

3. Sannino, A., Demitri, C., Madaghiele, M. Biodegradable Cellulose-

based Hydrogels: Design and Applications. Materials. 2009; 2: 353-
 373. DOI:10.3390/ma2020353

4. Shanmuganathan, K., J. R. Capadona, et al. Biomimetic

mechanically adaptive nanocomposites. Progress in Polymer
Science. 2010; 35(1–2): 212-222.
DOI:10.1016/j.progpolymsci.2009.10.005

5. Jonoobi, M., J. Harun, et al. Mechanical properties of cellulose

nanofiber (CNF) reinforced polylactic acid (PLA) prepared by twin
screw extrusion. Composites Science and Technology. 2010; 70(12):
1742-1747. DOI: 10.1016/j.compscitech.2010.07.005

6. Oksman, K., A. P. Mathew, Bondeson, D., Kvien, I. Manufacturing

process of cellulose whiskers/polylactic acid nanocomposites.
Composites Science and Technology. 2006; 66(15): 2776-2784.
DOI:10.1016/j.compscitech.2006.03.002

7. Siddiqui, N. Characterization of Mechanically and Enzymatically

Produced Cellulose Nanofibers from Wood Pulp. [thesis]. The
Graduate School: Maine, University of Maine. 2008.

8. Janardhnan, S., Sain, M. Targeted disruption of hydroxyl chemistry

and crystallinity in natural fibers for the isolation of cellulose nano-
fibers via enzymatic treatment. BioResources. 2011; 6(2): 1242-1250.
DOI: 10.15376/biores.6.2.1242-1250

9. Jonoobi, M., Harun, J., Mathew, A.P., Hussein, M.Z., Oksman, K.

Preparation of cellulose nanofibers with hydrophobic surface
characteristics. Cellulose. 2010; 17: 299-307. DOI 10.1007/s10570-
009-9387-9

10. Kalia, S., A. Dufresne, Cherian, B., Keith, B. S., Avérous, L.,
Njuguna, J. Cellulose-Based Bio- and Nanocomposites: A Review.
International Journal of Polymer Science. 2011, (Article ID 837875):
35. DOI:10.1155/2011/837875

11. Shelma R., Willi Paul and Sharma C.P. Chitin Nanofibre reinforced
Chitosan Films for Wound Healing Application. Trends Biomater.
Artif. Organs. 2008; 22(2): 111-115

12. Hui, J., Yuanyuan, J., Jiao, W., Yuan, H., Yuan, Z., Shiru, J.
Potentiality of Bacterial Cellulose as the Scaffold of Tissue
Engineering of Cornea. 2nd International Conference on
Biomedical Engineering and Informatics (BMEI 2009). DOI
10.1109/BMEI.2009.5305657
13. Schumann, D., J. Wippermann, et al. Artificial vascular implants
from bacterial cellulose: preliminary results of small arterial
substitutes. Cellulose. 2009; 16(5): 877-885. DOI 10.1007/s10570-
008-9264-y

14. Amorim, W. L., Costa, H. O., Coehlo de S., F., Germanos de C.,
M., da Silva, L. Experimental study of the tissue reaction caused
by the presence of cellulose produced by Acetobacter xylinum.
Brazilian Journal of Otorhinolaringology. 2009; 75(2): 200-207.
DOI 10.1590/S0034-72992009000200008

15. de Mesquita, J. P., Donnici, C. L., Pereira, F. V. Biobased

Nanocomposites from Layer-by-Layer Assembly of Cellulose
Nanowhiskers with Chitosan. Biomacromolecules. 2010; 11(2): 473-
480. DOI 10.1021/bm9011985

16. Darder, M., López-Blanco, M., Aranda, P., Aznar, A.J., Bravo, J.,
Ruiz-Hitzky, E. Microfibrous chitosan-sepiolite nanocomposites.
Chemical Materials. 2006; 18: 1602-1610. DOI 10.1021/cm0523642

17. Gupta, K. C. and M. N. V. R. Kumar. Structural changes and release

characteristics of crosslinked chitosan blends in response to
solution pH. Journal of Macromolecular Science, Part A. 1999;
36(5-6):827-841.DOI:10.1016/S1381-5148(00)00038-9

18. [18} Madihally, S. V. and H. W. T. Matthew. Porous chitosan

scaffolds for tissue engineering. Biomaterials. 1999; 20(12): 1133-
1142. DOI:10.1016/S0142-9612(99)00011-3

19. Azeredo, H. M., L. H. Mattoso, Avena-Bustillos, R. Ceotto, G.,

Munford, M., Wood, D., Mc Hugh, T. Nanocellulose reinforced
chitosan composite films as affected by nanofiller loading and
plasticizer content. Journal of Food Science. 2010; 75(1): N1-7. DOI:
10.1111/j.1750-3841.2009.01386.x

20. Jeon, Y. J., Kamil, J.Y.V.A., Shahidi, F. Chitosan as an edible

invisible film for quality preservation of herring and Atlantic cod.
Journal of Agriculture and Food Chemistry. 2002; 50: 5167–5178.
DOI: 10.1021/jf011693l

21. Bangyekan, C., D. Aht-Ong, Aht-Ong, D., Srikulkit, K.

Preparation and properties evaluation of chitosan-coated cassava
starch films. Carbohydrate Polymers. 2006; 63(1): 61-71.
DOI:10.1016/j.carbpol.2005.07.032
22. Durango, A. M., Soares, N.F.F., Andrade, N.J. Microbiological
evaluation of an edible antimicrobial coating on minimally
processed carrots. Food Control. 2006; 17: 336-341.
DOI:10.1016/j.foodcont.2004.10.024

23. Campaniello, D., Bevilacqua, A., Sinigaglia, M., Corbo, M. R.

Chitosan: antimicrobial activity and potential applications for
preserving minimally processed strawberries. Food Microbiology.
2008; 25(8): 992-1000. DOI:10.1016/j.fm.2008.06.009

24. Nisperos-Carriedo. Edible coatings and films based on

polysaccharides. Edible coatings and films to improve food quality.
1994. J. M. Krochta, Baldwin, Nisperos-Carriedo. Lancaster, U. K.,
Technomic. 305-335.

25. Suyatma, N. E., Copinet, A., Coma, V., Tighzert, L. Mechanical

and barrier properties of biodegradable films made from chitosan
and poly(lactic acid) blends. Journal of Polymer Environment.
2004; 12: 1-6. DOI: 10.1023/B:JOOE.0000003121.12800.4e

26. Suyatma, N. E., Tighzert, L., Copinet, A. Effects of hydrophilic

plasticizers on mechanical, thermal, and surface properties of
chitosan films. Journal of Agriculture and Food Chemistry. 2005;
53(10): 3950-3957. DOI 10.1021/jf048790+

27. Di Gioia, L., Guilbert, S. Corn Protein-based Thermoplastic

Resins: Effect of some Polar and Amphiphilic Plasticizers. Journal
of Agriculture and Food Chemistry. 1999; 47: 1254-1261. DOI:
10.1021/jf980976j

28. Ludueña, L. N., Alvarez, V.A., Vasquez, A. Prossesing and

microstructure of PCL/clay nanocomposites. Materials Science
and Engineering A. 2007; 460-461: 121-129.
DOI:10.1016/j.msea.2007.01.104

29. Sorrentino, A., Gorrasi, G., Vittoria, V. Potential perspectives of

bionanocomposites for food packaging applications. Trends in
Food Science & Technology. 2007; 18(2): 84-95.
DOI:10.1016/j.tifs.2006.09.004

30. Vaia, R. A., Wagner, H.D. Framework for nanocomposites.

Materials Today. 2004; 7: 32-37. DOI:10.1016/S1369-
7021(04)00506-1

31. Park, T.-J., Y. Jung, Choi, S. W., Park, H., Kim, H., Kim, E., Lee, S.
 H., Kim, J. H. Native chitosan/cellulose composite fibers from an
ionic liquid via electrospinning. Macromolecular Research. 2011;
19(3): 213-215. DOI:10.1007/s13233-011-0315-0

32. Jacobs, V., Patanaik, A and Anandjiwala, R.D. Electrospun

chitosan nanofibre membranes for antimicrobial application: role
of electrospinning processing parameters. European Cells and
Materials. 2010; Vol. 19. Suppl. 1, pp 4. ISSN 1473-2262.

33. Macías, H. Quitosana como andamiaje celular en regeneración de

membrana timpánica en conejo Nueva Zelanda (timpanoplastía
experimental tipo I) [thesis]. Departamento de Neurociencias.
Guadalajara, Universidad de Guadalajara 2010.

34. ASTM D 587 - 98 Standard Test Method for Swelling and Water
Retention (1998)

35. Fernandes, S., Freire, C., Silvestre, A., Neto, C. P., Gandini, A.,
Berglund, L., Salmén, L. Transparent chitosan films reinforced
with a high content of nanofibrillated cellulose. Carbohydrate
Polymers. 2010; 81: 394-401. DOI:10.1016/j.carbpol.2010.02.037

36. Khan, A., Khan, R. A., Salmieri, S., Le Tien, C., Riedl, B.,
Bouchard, J., Chauve, G., Tan, V., Kamal, M. R., Lacroix, M.
Mechanical and barrier properties of nanocrystalline cellulose
reinforced chitosan based nanocomposite films. Carbohydrate
Polymers. 2012; 90: 1601– 1608. DOI:10.1016/j.carbpol.2012.07.037

37. Wang, H., Roman, M. Formation and properties of chitosan-

cellulose nanocrystal polyelectrolite-macroion complexes for drug
delivery applications. Biomacromolecules. 2011; 12: 1585-1593. DOI:
10.1021/bm101584c

38. Mathew, A. P., Laborie, M.P., Oksman, K. Cross-linked

chitosan/chitin crystal nanocomposites with improved permeation
selectivity and pH stability. Biomacromolecules. 2009; 10(6): 1627-
1632. DOI: 10.1021/bm9002199

39. Croisier, F., Jerome, C. Chitosan based biomaterials for Tissue

Engineering. European Polymer Journal. 2013; (49) 780 – 792. DOI:
10.1016/j.eurpolymj.2012.12.009.

40. Han, T., Nwe, N., Furuike, T., Tokura, S., Tamura, H. Methods of
N – acetylated chitosan scaffolds and it’s in vitro biodegradation by
lysozyme. J. Biomedical Science and Engineering, 2012; (5): 15-23.
 DOI: 10.4236/jbise.2012.51003.

W R I T T E N BY
Guillermo H. Riva, Joaquín García-Estrada, Brenda Vega, Fernando
López-Dellamary, María E. Hérnandez and José A. Silva

Submitted: 11 February 2015 , Reviewed: 12 October 2015 , Published: 09

December 2015

R E G I ST E R TO D O W N LOA D F O R F R E E

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