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

1Department of Zootechnics, Federal University of Mato Grosso, Cuiab (MT), Brasil 2Veterinary Medicine, Federal University of Gois, Campus Jata, Jata (GO), Brasil

3Chemistry 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 (0.5-

1 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 Minerlization

M.G.C.* Osteoid tissue

Fibroblasts Osteoblasts

Macrophages Osteoclasts

Neovascularizazion Primary bone

Polymorphonuclears

Mononuclears

Other findings** 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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