Chitosan inclusions in the subcutaneous space of rats: clinic, histologic and morphometric evaluation

Abstracts

BACKGROUND: Chitosan is a polymer derivative from chitin applied in many medical specialties. OBJECTIVE:To evaluate the biocompatibility of chitosan membranes used as inclusion material into subcutaneous of rats. METHODS: Twenty male Wistar rats received chitosan membranes into the subcutaneous area of the dorsal medial region. The animals were randomly divided in four groups with 5 animals each, sacrificed on 7th (G1), 15th (G2), 30th (G3) and 60th (G4) postoperative day (PO). All animals were clinically evaluated daily and by photo-documentation at the sacrifice moment. The animals and the material were assessed for evidence of host response by histological and morphometric evaluation of the implants and the surrounding soft tissues. The material was fixed in 10% formalin and then the sections were stained with hematoxylin and eosin. RESULTS: None of animals presented side effects attributable to the implants. The histological evaluation showed smooth and homogeneous inclusions with no host cells inside and were encircled by a pseudocapsule of fibroblasts and inflammatory cells. The morphometric evaluation showed no statistical difference between different groups (P>0.05).

Biocompatible materials; Chitosan; Materials testing; Rats, Wistar

FUNDAMENTOS: A quitosana é polímero derivado da quitina, com vários tipos de aplicação na área médica. OBJETIVO: Avaliar a biocompatibilidade de membranas de quitosana no subcutâneo de ratos. MÉTODOS: Foram utilizados 20 ratos "Wistar" machos, nos quais foram implantadas membranas de quitosana, na região mediana dorsal. Os animais foram sacrificados: sete, 15, 30 e 60 dias após a cirurgia, tendo sido avaliados clinicamente durante o período experimental e com fotodocumentação no momento do sacrifício. Após o sacrifício, as membranas e tecidos adjacentes foram removidos e preparados para exame histológico e morfométrico. RESULTADOS: Nenhum animal apresentou efeitos adversos que pudessem ser atribuídos à implantação das membranas. O exame histológico mostrou que as inclusões são lisas e homogêneas e não são colonizadas por células do hospedeiro, sendo circundadas por pseudocápsula composta por fibroblastos e células inflamatórias. A morfometria da pseudocápsula revelou espessura semelhante durante todo o período experimental (P>0,05). CONCLUSÃO: A quitosana pode ser opção para uso como implante não integrado. Novos estudos devem ser realizados para comprovar a biocompatibilidade a longo prazo.

Materiais biocompatíveis; Quitosana; Ratos Wistar; Teste de materiais

INVESTIGATION

Chitosan inclusions in the subcutaneous space of rats: clinic, histologic and morphometric evaluation

Maila Karina Mattos de Brito^I; Silvana Artioli Schellini^II; Carlos Roberto Padovani^III; Claudia Helena Pellizzon^IV; Cypriano Galvão da T. Neto^V

^I

^IIFull Professor, Department of Ophthalmology, Faculdade de Medicina de Botucatu - Universidade Estadual Paulista (Unesp) - Botucatu (SP), Brazil

^IIIFull Professor, Department of Biostatistics, Institute of Biosciences - Universidade Estadual Paulista (Unesp) - Rio Claro (SP), Brazil

^IVPh.D., Professor, Department of Morphology, Institute of Biosciences - Universidade Estadual Paulista (Unesp) - Rio Claro (SP), Brazil

^VPh.D. studies under course. Department of Polymers, Universidade Federal do Rio Grande do Norte (UFRN) - Natal (RN), Brazil

Mailing Address

ABSTRACT

BACKGROUND: Chitosan is a polymer derivative from chitin applied in many medical specialties.

OBJECTIVE: To evaluate the biocompatibility of chitosan membranes used as inclusion material into subcutaneous of rats.

METHODS: Twenty male Wistar rats received chitosan membranes into the subcutaneous area of the dorsal medial region. The animals were randomly divided in four groups with 5 animals each, sacrificed on 7th (G1), 15th (G2), 30th (G3) and 60th (G4) postoperative day (PO). All animals were clinically evaluated daily and by photo-documentation at the sacrifice moment. The animals and the material were assessed for evidence of host response by histological and morphometric evaluation of the implants and the surrounding soft tissues. The material was fixed in 10% formalin and then the sections were stained with hematoxylin and eosin.

CONCLUSIONS: None of animals presented side effects attributable to the implants. The histological evaluation showed smooth and homogeneous inclusions with no host cells inside and were encircled by a pseudocapsule of fibroblasts and inflammatory cells. The morphometric evaluation showed no statistical difference between different groups (P>0.05).

Keywords:Biocompatible materials; Chitosan; Materials testing; Rats, Wistar

INTRODUCTION

Chitosan is a natural polymer, derived from chitin, material that participates in the composition of exoskeleton of shellfish, such as shrimps, lobsters and crabs and insects, such as ants and beetles ¹.

This type of material has been successfully used in the medical and pharmaceutical area, employed for the production of cosmetic products, in the development of artificial skin to treat burn cases, to simulate the permeability of active substances through human skin ^1,2, in the production of dressings that can hold hemorrhages ^3,4, to prevent infections ⁵, in the development of films with antimicrobial action to preserve meat, fruit and cereals ^6,7, membranes for hemodialysis, preventing the adsorption of proteins, platelet adhesion and formation of thrombus by electrical repulsion mechanism, given that the interactions that lead to thrombosis occur in the blood-biomaterial interface ^3,4,7.

Despite the great variability of suggested uses, very little is known about the biocompatibility of the material.

A polymer to be used in the biomedical area should be biocompatible, defined as a material that does not cause adverse reaction and should have ability to trigger an appropriate response to a specific application in the body ^8,9.

However, biocompatibility of the material depends on different intrinsic body factors, such as species, genetic heritage and implantation site or action and the material itself; and on inherent material characteristics which are its shape, size, rugosity and surface chemistry, composition, morphology, duration of contact with the body and degradation. Natural polymers are normally biodegradable and have excellent biocompatibility when compared to other synthetic polymers ^10-12.

The present study was developed to assess the biocompatibility of chitosan membranes, implanted into the subcutaneous space of rats.

MATERIAL AND METHODS

This was an experimental randomized study of interventional character, whose protocol was approved for execution by the Ethics and Animal Experiment Committee at Faculdade de Medicina de Botucatu (2005/494).

The study was carried out at Faculdade de Medicina de Botucatu and we used 20 male rats from Wistar lineage, from the Central Animal Lab of UNESP. Rats were randomly divided into four experimental moments and each one had five animals, sacrificed after seven (G1), 15 (G2), 30 (G3) and 60 (G4) days from inclusion in the study.

We performed the following assessments: 1) clinical: daily assessments, observing inflammatory or infectious signs at the implantation site and also possible abnormalities of behavior that could suggest systemic effects; 2) photodocumentation: performed at the sacrifice, by the same observer, maintaining the same distance from the object to be photographed; 3) histological assessment: after the sacrifice, the implantation region was removed and prepared for analysis under light microscopy, observing the interface between the host and the inclusion and the implanted material itself; 4) morphometric assessment: for quantification of pseudocapsule formed around the implant, using Tukey test.

Characterization of the chitosan used: Chitosan was manufactured by Polymar Ltda, Brasil, with deacetylation grade of about 80% with molar mass determined by viscometric method, using the equation by Mark-Howink-Sakurada. ^13,14 Chitosan was dissolved in aqueous solution of acetic acid at 2% (v/v) under 24h agitation to obtain a solution of polymer at 1.5% (m/v) ¹⁵. After this period, the solution was filtered to eliminate solid residues, placed 24h in the hot oven at 50o C to evaporate the solvent, followed by addition of water solution of NaOH 5% for 2h to neutralize it, followed by rinsing with distilled water. After stretching and drying at room temperature, we obtained membranes with average thickness of 40 µm, of 2 X 1 cm in size (layer measurer DIGIDERM by Mitutoyo). ^13,15

Surgical procedure for membrane implantation: procedures were made under general anesthesia, using intra-peritoneal injection of sodium pentobarbital 3% (ml/Kg weight) and in aseptic and antiseptic conditions. We performed trichotomy on the dorsal region of the rats and longitudinal incision of approximately 1.5cm on the dorsal region using knife and 15 lamina, followed by creation of a subcutaneous pouch with blunt scissors that received the material. Chitosan membrane was introduced below the subcutaneous and fixed on the site with separated stitches, using absorbable thread Vicryl 5-0 ("ETHICON") (Figure 1). At the end of the procedure, animals received a dose of antibiotic enrofloxacin at 10% intraperitoneal application and mupirocin ointment 2%.

Animals were maintained in individual boxes, with constant temperature of 36º C and artificial light cycle of 12/12 h, receiving ad libitum filtered water and feed Purina, specific for the species.

Sacrifice was made using overdose of sodium pentobarbital at 3% intraperitoneal application. After sacrifice, chitosan membrane was removed (Figure 2), and the material was fixed in formal 10%, dehydrated in increasing series of alcohols, diaphanized, included in paraffin, cut in 4µm thick sections using a rotation microtome RM-2155 (Leica) and stained with hematoxylin-eosin (HE). Histological analysis tried to observe the integrity of the membrane, intensity of the inflammatory reaction on the surrounded tissue and the level of integration of the biomaterial with the recipient tissue.

Slides were analyzed and photodocumented using microscope Leica DM, coupled to software Leica QWin Version 3.1 (Leica - UK). We have morphometrically analyzed the thickness of the pseudocapsule formed around the implantation of chitosan, using the semi-automatic linear method module of the software. The result was transferred to an Excel spreadsheet and submitted to statistical analysis, using the variance analysis technique to experiment with factors in independent groups considering the test of multiple comparison of Tukey ¹⁶, considering P > 0.05.

RESULTS

Animals remained apparently healthy and well fed up to the sacrifice, and there were no spontaneous deaths.

Clinical Examination

The surgical wound progressed well, without signs of infection, dehiscence or extrusion for all animals in the four groups. Healing was complete and it was difficult to detect the site where the procedure had been made when the animals were sacrificed 30 to 60 days later owing to regrowth of hairs that prevented visualization of the scar. The membrane did not cause any salience, nor any other visible skin affection over the site where it was implanted, meaning that it would have been impossible to know it were in the subcutaneous if it were not for the surgical scar (Figure 3).

Histological Analysis

G1: the inclusion was identified in histological sections as smooth, thick and homogenous material, formed by similar blocks attached one to the other. Around the inclusion, we observed formation of a pseudocapsule, comprising young fibroblasts. Active neovessels, red blood cells, collagen fibers and few inflammatory cells, most of which were neutrophils and lymphocytes. Fibrosis was narrowed to the lower portion of the inclusion and over its whole surface we observed cells distributed in one single layer (Figure 4A).

   

G2: after 15 days from surgical procedure, there was a great number of fibroblasts with characteristics of young cells. There were also many inflammatory cells around the inclusion (Figure 4B).

G3: fibrosis was thicker, with large fibroblasts. Inflammatory cells were around the inclusion and were predominantly lymphocytes, monocytes, neutrophils and foreign body giant cells (Figure 5A).

   

G4: the implant was surrounded by cell figure, with mature fibroblasts and there was considerable reduction of inflammatory reaction (Figure 5B).

Morphometric Analysis

Even though at macroscopic and histological examination there were suggestions of thick fibrosis (pseudocapsule), numerically there was no significant difference between the groups (Table 1).

DISCUSSION

The present paper was performed to validate the biocompatibility of chitosan membrane using a rat experimental model, given that rats are easy to handle and maintain . ^8,17 Clinical assessment of animals during the studied period showed absence of evident inflammatory signs or extrusion of the implants. It was possible to observe the growth of hairs over the surgical region, which is an indication that the implanted membrane does not cause damage to the implanted region, as observed in previous studies. ^9,18,19 Histological analysis basically revealed proliferation of fibroblast (pseudocapsule) and inflammatory cells around the chitosan membrane, response compatible with tissue regeneration by connective tissue.

Even though inflammatory cells were abundant at the beginning of experiment, with time, there was reduction of tissue reaction. However, in the 30 day assessment after implantation, we detected giant cells, a response of the body to the introduction of foreign material, given that the cells could envelope and even phagocyte small particles that do not belong to the recipient body .^20,21

Thus, we considered that chitosan membrane induces little inflammatory chronic reaction. There were no signs of rejection, which may be a favorable indicator to the use of this material for implantations. This was also the conclusion that other authors have reached, but using the material to other ends .^1,19,22,23

Another aspect to point out was the fact that there was no colonization of the implant by host cells - the material has no pores with completely smooth surface. Therefore, chitosan implants may be considered as implants that do not suffer integration with the host, maintaining the characteristics they had before the contact with the receptor.

Morphometric examination of the pseudocapsule that is formed around the implant intended to define indications of increase in tissue reaction against the implanted material, which was not confirmed, given that there was no increase in it throughout the whole experiment. This type of measurement had already been used to analyze muscle fibers 24, to assess cancer cells and quality of meat ^25,26.

Similarly to other polymers such as polyesters, polyaminoacids, lecithin, etc., chitosan has been used for medical purposes, owing to the need of employing biocompatible and low cost materials to be used in dermatology, orthopedics and ophthalmology .^6,7,19

The results observed here lead us to suggesting that chitosan membrane may be applied also in implants, in the form of membranes to be used for tissue repair. New studies should be performed to define the outcomes or the long-term repercussions of the use of material.

CONCLUSION

The insertion of chitosan membranes in the subcutaneous layer of rats generates little inflammatory reaction, with formation of fibrosis (pseudocapsule) around it. The observed characteristics let us conclude that this type of material does not get integrated and may be used as an implant in tissue repairs.

ACKNOWLEDGEMENT:

We would like to thank Dr Alexandre Paratela Gama, ophthalmologist, who supported the performance of surgical procedures to place the implants.

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*

Trabalho foi desenvolvido na Faculdade de Medicina de otucatu e Instituto de Biociências da Universidade Estadual Paulista (Unesp) - São Paulo (SP), Brasil.

Publication Dates

• Publication in this collection
  23 June 2009
• Date of issue
  Feb 2009

History

• Accepted
  26 Dec 2008
• Received
  25 Mar 2008