Biocompatibility of nanostructured chitosan/PEO membranes: in vitro and in vivo

evaluation
Vanessa S.F.1, Valcinir A.S.V.2, Janice R.P.3, Rogrio E.R.2, Diego, P.A.2
1

Department of Zootechnics, Federal University of Mato Grosso, Cuiab (MT), Brasil

2
Veterinary Medicine, Federal University of Gois, Campus Jata, Jata (GO), Brasil
3
Chemistry Institute of So Carlos, University of So Paulo, So Carlos (SP), Brasil
E-mail: vsfranzo@hotmail.com

Abstract. Electrospinning is used to produce fibers in the nanometer range by stretching a polymeric jet
using electric fields of high magnitude. Chitosan is an abundant natural polymer that can be used to
obtain biocompatible nanostructured membranes. The objectives of this work were to obtain
nanostructured membranes based on blends of chitosan and polyoxyethylene (PEO), and evaluate
their in vivo biocompatibility by histomorphometric studies of rats implants to complemente the
preliminar results of in vitro biocompatibility. The results of the cytotoxicity tests evidenced that the
chitosan/PEO membranes are non-toxic to the cells studied in this work. The histomorphometric studies
showed that at 7 and 15 days a considerable number of neutrophils and macrophages,
which declined with time. At 30 and 45 days after implantation, the material was completely degraded,
verifying the presence of fibroblasts and connective tissue fill. Then, was observed the Intense
neovascularization around all implants in very little variable depending on the time of explant. Explants
in 15 days there were discrete formation of peri-implant fibrosis and no differences in thickness.
The occurrence of adhesion in the interface of the material and surrounding tissue were minimal,
demonstrating that the material does not stimulate acute organic reactions of rejection.

Key-words: Nanotechnology, Electrospinning, Chitosan, Polymer, Biocompatibility.

1.

INTRODUCTION

Electrospinning is the only technique that allows dimensional control, fiber

formation in different arrangements, use of several polymers and production in great
quantities compared to the other methods (YARIN et al., 2001). This technique is based
on the application of high magnitude electric potentials (5-50 kV) and low current (0.51 A) in which a jet of fluid material is accelerated and stretched, producing fibers of
reduced diameter (LI D and XIA Y, 2004; LANNUTTI et al., 2007).
Chitosan is derived from chitin deacetylation and is a natural polymer that is
suited for electrospinning in order to produce nanostructured fibrous mats. Usually, the
electrospinning of chitosan requires blending this polymer with other biocompatible
synthetic polymers such as PEO. Chitosan has been used for many years in tissue
regeneration, in some drug delivery controlled systems, for covering wounds and for
other biomedical devices (HAMMAN, 2010; NAIR et al., 2009). Several studies have
shown the chitosan biocompatibility in different forms (membranes, gel, composites,
films and matrices). Therefore, there is an increasing interest in the production of
chitosan-based nanostructured biomaterials (COSTA SILVA et al., 2006; BERGER et
al., 2004).

A biomaterial should be biocompatible and present an appropriate response for a

specific situation, causing minimum allergic, inflammatory and toxic reactions when in
contact with live tissues or organic fluids. The in vitro evaluation can supply fast and
financially accessible results about biological interactions and decrease the use of
animals in research (ISSO, 1992). Cytotoxicity tests are done in vitro and evaluate the
toxic effect for the cells of some materials, which can be cell death, changes in
membrane permeability, enzymatic inhibition, etc. This technique uses cell culture and
the toxicity can be measured quantitatively by cell lysis (cell death), inhibition of cell
growth and other effects caused by the devices, materials and extracts.
The histomorphometric testes are conducted through implant in animals. The
recommendation is to be used initially implants into the subcutaneous tissue of rats, to
understand the inflammatory process involved. Samples of tissue and implant are
removed and studied under the microscope, describing the cellular events.
The objective of this study was to obtain nanostructured membranes of
Chitosan/Peo using the electrospinning technique and evaluate their in vivo
biocompatibility to complement the biocompatibility in vitro cytotoxicity assays
performed before (VULCANI et al. 2012).
2.

MATERIAL AND METHODS

The membranes were prepared by chitosan of medium molecular weight with

deacetylation degree of 80% and viscosity of 284 cps obtained from Aldrich, glacial
acetic acid PA from Synth and poly ethylene oxide (PEO), 900,000 g.mol1, also from
Aldrich. All materials were used as received and the deionized water was used to
compose the solvent system.
The electrospinning equipment used to obtain chitosan/PEO membranes was
built at the Escola de Engenharia Qumica do Departamento de Tecnologia de
Polmeros da Universidade de Campinas* (UNICAMP), Campinas, SP, Brazil with the
following components: high voltage power supply, a 10ml glass syringe (Luer lock
inlet) containing a stainless steel needle with internal diameter of 1 mm, an electrode, a
sustaining claw for the syringe (connected to a sustaining support for the claw), a
grounded-aluminum-collector and a rotary-device (80 rpm) constituted by a 3.2 cm
diameter stainless steel tube between the capillary and the collect table16-19.
Membranes were made by the electrospinning technique according to the
methodology described by BIZARRIA et al. (2010) using the setup described in the
previous paragraph and the blend composed by three parts of a 4% (w.w1) chitosan
solution and one part of a 3% (w.v1) PEO solution, resulting in a final concentration of
approximately 80% chitosan and 20% PEO. This blend composition was selected to
make the membranes because, among the solution blends that showed good processing
conditions, in the referred previous study, it was the one with greatest chitosan
concentration.
Preparing the 4% (w.w1) chitosan solution, an aqueous solution with high
concentration of acetic acid 90% (v.v1) was used as solvent. The chitosan solution was
left under magnetic stirring (not being removed) until its complete homogenization,
which took a few days. The 3% (w.v1) PEO solution was prepared under magnetic
stirring using deionized water as solvent. To prepare the blend solution to be
electrospun, a part of the 3% PEO solution (by volume) was added to three parts (by
weight) of the 4% chitosan solution. The resulting blend was left under magnetic
stirring for two hours before being electrospun. The preparation of the solutions, as well
as the preparation of the blend solution and its electrospinning process were run at room

temperature. The flow rate was determined by the viscosity of the solution being
approx. 1 mL/h. and the distance between the needle tip to collector cylinder 7.5 cm at
an applied voltage of 20 kV.
Cytotoxicity evaluation using the agar diffusion method
Fragments of the membrane measuring about 0.25 cm2 of superficial area were
placed on agar before their complete solidification. The Petri dishes were kept in the
cell incubator with 5% CO2 at 37 C during 24 hours. Latex fragments were used as
positive control and confirmedly non-toxic filter paper discs as negative control,
respecting the dimension of the superficial area. The plates were analyzed
macroscopically and microscopically using the halo presence and the cell integrity
around the sample as parameters, respectively (ISSO, 1992).
In vivo biocompatibility
An experimental, randomized and interventional study was fulfilled using 30 rats
of Wistar strain divided into five experimental moments, each containing six animals
were euthanized seven (G1), 15 (G2), 30 (G3), 45 (G4) and 60 (G5) days after the
implantation of material. After implantation, the animals were clinically evaluated,
observing for signs of inflammation or infection at the implantation site for possible
changes in behavior that might suggest a systemic effect.
The procedures were performed under general anesthesia by intraperitoneal
injection of sodium pentobarbital 3% (1ml/kg) and under sterile conditions and
antiseptic. After shaving the dorsal region of the mouse and made a longitudinal
incision, approximately 1.5 cm in the dorsal region, followed by creation of the
subcutaneous pocket with blunt scissors. The chitosan membrane and inserted below the
subcutaneous tissue and fixed in place with sutures using 5-0 Vicryl absorbable sutures
("Ethicon") (Figure 4). At the end of procedure, the animals received a dose of
antibiotic intraperitoneal enrofloxacin and mupirocin cream.
The animals were kept in boxes with constant temperature of 25C and artificial
light cycle of 12/12 hours, receiving filtered water and Purina supply suitable for the
species ad libitum. The animals were sacrificed using an overdose of sodium
pentobarbital intraperitoneally.
The fragments are surgically removed and fixed in formalin (10%), phosphate
buffered (pH 7.2) for at least 24 hours. After this period, the material was processed for
paraffin embedding. Serial sections are made of 5 mm thick and stained
with hematoxylin-eosin
(HE), Masson's trichrome
and Von Kossa, the
cuts being evaluated under light microscopy and graded by a pathologist blinded to their
identities, with
degrees of
histological
changes described
in
Table
1,
and possibly other observed by the pathologist.
The Microscopic analysis was increased by objective data obtained by
histometric analysis. For this purpose, we used binocular microscope (Olympus BX
60 sr) with equipment for microphotography, camera image capture and image analyzer
(Image Pro-plus. Cybernetics. California, USA). Of each histological section were 20
randomly chosen for cell counting. We quantified the inflammation and
neovascularization following the classification: 0 + / 5 as a minimum and 5/5 as
maximum.

Table 1. Variables examined on slides prepared from samples taken from the implants
of nanoestructured chitosan interface. Distribution in alphabetical order.
Findings at histologic exam
Evaluation of the inflammatory process

Evaluation of metaplasia

Fibrous capsule
M.G.C.*
Fibroblasts
Macrophages
Neovascularizazion
Polymorphonuclears
Mononuclears
Other findings**

Minerlization
Osteoid tissue
Osteoblasts
Osteoclasts
Primary bone

Other findings**

* Multinucleated giant cell

** Possible variables not provided

For the cell count data were analyzed the normality of errors studentized
(Cramer-Von Mises test) and homogeneity of variances (Brown-Forsythe test). After
verifying compliance with these assumptions, the data were subjected to analysis of
variance employing the General Linear Model of SAS and in case of difference (P
0.05), means were compared Tukey's test, considering the 5% level of probability.
3.

RESULTS E DISCUSSION

The results of the cytotoxicity tests with the membrane prepared with cell lines
showed that the three cell lines did not present halo formation around the fragments of
chitosan nanostructured membrane similarly to the negative controls, suggesting that the
material is not cytotoxic to fibroblasts and epithelial cells. The use of three lines
originated from human, monkey and mouse cells reinforce the large applicability of the
membrane prepared since it demonstrated to be biocompatible in all cases.
This non-toxic characteristic of the chitosan was observed by AMARAL (2006),
who submitted blends of collagen and chitosan to the same in vitro cytotoxicity tests,
i.e., the agar diffusion method. Despite the fact that the chitosan membrane used by that
author was not nanostructured, their result corroborates with the present material
biocompatibility findings. Similarly, Asiah et al. (2006) verified the in vitro
biocompatibility of chitosan films by submitting them to human fibroblast cultures and
In relation to the inflammatory process, it was found in the fragments at seven
days the predominance of polymorphonuclear (on average 2 + / 5) as well as moderate
amount of lymphocytes (an average of 1 + / 5) (Fig. 1A and 1B). Both classes of cells
were in higher concentration in the interface material and adjacent tissue and not within
the implant. There were significant differences in the amount of polymorphonuclear
cells and lymphocytes in different experimental moments. The number of neutrophils
reduced (on average a + / 5), while the number of lymphocytes increased (on average 2
+ / 5).
The lymphocytosis increased significantly at 15 days post-implantation, however
decreased over time because the material degradation was intense. At 30, 45 and 60
days post implantation, the material had been completely degrade, making evident the

interface biomaterial / adjacent tissue, formed by a small capsule of connective tissue

(Fig. 1C and 1D). For macrophages, it was observed seven days to moderate
concentration (mean + 0/5), which tended to increase to 15 days (mean + 1/5), however
no significant difference. From 30 days decreased significantly the amount of
macrophages, however the material had undergone total degradation.
The count of fibroblasts showed that at seven days had a slight presence around
the implant (on average 0 + / 5). However, this amount increased significantly from
15 days (mean + 2/5) having a high amount at 30 days post-implantation (on
average 3 + / 5). At 45and 60 days the healing process was resolved, with no difference
in fibroblasts.
There
was intense
neovascularization around all
the
implants in
quantities varying very little with respect to time of explant. In relation to
the degradation of the material, there was intense decrease in mass both the interior
and the periphery of the sample, the first seven days post-implantation, which is
accentuated, until the complete degradation of the material over time of implantation
(Fig. 1D). The verification of metaplasia in the region of implantation showed
no significant result of cellular.

Figure 1. In A and B sample taken at seven days post-implant. It

is evident the inflammatory process, the invasion
of lymphocytes and neutrophils predominantly around
the implant (200, 400X and Von Kossa staining). In
C can be observed the interface formed by fibrosis,
separating the adjacent tissue of the implant. The
material was degraded, leaving only the fibrous tissue
(arrow) (200X and H.E. staining). In D the material at
seven days post-implantation, suffering severe
degradation. The arrows shows the membrane
fragments (400X and H.E. staining).
The occurrence of adhesion at the interface material and surrounding tissue was
minimal, demonstrating that the material does not stimulate organic reactions of acute

rejection. Furthermore, the inflammatory process proved decreasing in function of time,

which can also be attributed to degradation of material and consequent reduction of
mass and surface exposure to the surrounding tissue.
This pattern of decreasing inflammatory process was observed in studies with
chitosan and collagen, as well as the prevalence of lymphocytes and fibroblasts. When
comparing materials that last longer, such as collagen the lymphocytosis persists to a
considerable degradation of the material and then starts increasing fibroblasts24. This
occurred in implants of chitosan nanostructured at 15 days post implantation, as much
of the material had been degraded. That is, the inflammatory process appears to be
regulated by the quantity of material.
However, chitosan nanostructured can influence cell proliferation as mentioned
by DUAN et al., 2009, who used as composites and check the growth of human
fibroblasts in the material. The authors stated that the presence of chitosan
nanostructured promoted the proliferation of cells, mimicking more suitable matrix
extracelular25 These results are similar to those obtained in test nanostructured
membrane collagen and chitosan, which were used as matrices for growth of human
fibroblasts and found that the nature of the composite non-toxic to cells. The authors
also composite used in animals to verify the efficacy of skin healing as compared to
commercially available collagen membranes and concluded that the composite obtained
better results in the healing (JYH-PING et al., 2008)
The formation of new blood vessels also occurs as a function of time resolution
of the inflammatory process and the degradation (ASIAH et al., 2006; JYH-PING et al.,
2008). The studies showed that materials that are degraded more slowly and therefore
promote a more pronounced inflammatory process tends to delay the onset of blood
vessels.
4.

CONCLUSION

Concluded that the membranes of chitosan / PEO nanostructured shown to be

biocompatible by in vitro tests and in contact with living organisms did not cause acute
rejection reactions.
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