Elsevier

Biomaterials

Volume 35, Issue 5, February 2014, Pages 1462-1477
Biomaterials

The localisation of inflammatory cells and expression of associated proteoglycans in response to implanted chitosan

https://doi.org/10.1016/j.biomaterials.2013.10.068Get rights and content

Abstract

Implantation of a foreign material almost certainly results in the formation of a fibrous capsule around the implant however, mechanistic events leading to its formation are largely unexplored. Mast cells are an inflammatory cell type known to play a role in the response to material implants, through the release of pro-inflammatory proteases and cytokines from their α-granules following activation. This study examined the in vivo and in vitro response of mast cells to chitosan, through detection of markers known to be produced by mast cells or involved with the inflammatory response. Mast cells, identified as Leder stained positive cells, were shown to be present in response to material implants. Additionally, the mast cell receptor, c-kit, along with collagen, serglycin, perlecan and chondroitin sulphate were detected within the fibrous capsules, where distribution varied between material implants. In conjunction, rat mast cells (RBL-2H3) were shown to be activated following exposure to chitosan as indicated by the release of β-hexosaminidase. Proteoglycan and glycosaminoglycans produced by the cells showed similar expression and localisation when in contact with chitosan to when chemically activated. These data support the role that mast cells play in the inflammatory host response to chitosan implants, where mediators released from their α-granules impact on the formation of a fibrous capsule by supporting the production and organisation of collagen fibres.

Introduction

One of the major problems associated with the implantation of foreign materials is their propensity to induce inflammation and fibrosis, which may result in a loss of implant function. Implantation of materials into the body results in a cascade of events starting with the host's response to injury, inflammation. The most likely long-term host response is fibrosis and formation of a fibrous capsule around the implant [1] and although widely recognised, research into the mechanistic events that occur are still relatively unknown. Strategies to reduce fibrosis generally revolve around alteration of material surfaces to make them anti-fouling or protein resistant, but the role of inflammatory cells in mediating fibrosis capsule formation during the body's inflammatory response to an implanted material remains largely unexplored. Upon implantation of a foreign material, injury to the vasculature occurs, resulting in haemorrhage, infiltration of neutrophils and activation of the complement cascade [2]. A number of products of complement activation function as mediators of inflammation, by recruiting various inflammatory cells. Many different cell types are known to be associated with inflammation and are quite often studied when assessing implant-induced inflammation, including neutrophils, macrophages and foreign-body giant cells, yet the role that mast cells play in response to implanted materials is only now starting to be investigated.

Mast cells, a population of cells associated with inflammation, are derived from hematopoietic stem cells. They are a heterogeneous cell population, and are widely distributed in the connective tissues where they are normally located in close proximity to blood vessels. They are concentrated in tissue at sites directly exposed to the environment including skin, airways and gastrointestinal tract [3]. Mast cells are important for the induction of inflammatory reactions to implanted materials through their release, upon activation, of pro-inflammatory proteases and mediators including cytokines that attract cells to the site of implantation. Mast cells have also been linked with chronic inflammation resulting in fibrosis [4], suggesting that these mediators have a positive effect on the expression of collagen. Mast cell activation can occur through a number of different pathways, and results in the release of the contents of their α-granules, which are bound to the proteoglycan serglycin [3]. Serglycin is an intracellular proteoglycan (PG) produced by hematopoietic and endothelial cells, consisting of a protein core to which chondroitin sulphate (CS), heparan sulphate (HS) and/or heparin chains are attached depending on the origin and species of the producing cell [5]. Mast cells, along with other inflammatory cells, have previously been shown to be present following implantation of foreign materials [6]. Degranulation of mast cells has been correlated with the inflammatory reaction within the surrounding tissue [7]. Mast cells are known to play a role in the acute inflammatory response [8], where they have been shown to regulate the infiltration of neutrophils [9], and macrophages [10], [11]. In addition to their role in the acute inflammatory response, the long term presence of mast cells may be related to the degree of fibrotic encapsulation [12], [13].

Chitosan, is a naturally occurring polysaccharide polymer, consisting of glucosamine and N-acetyl glucosamine linked by β(1–4) glycosidic bonds and is commercially produced by the deacetylation of chitin, which is most frequently derived from crustaceans. Typically, deacetylation of chitosan ranges from around 65–95%, though lower and higher degrees are possible depending on the method of processing. Due to the broad degree of deacetylation and molecular weight range (5–500 kDa) available, the material properties of chitosan have a wide range of variability. The applications of chitosan span numerous fields, including agriculture, water treatment, cosmetics and the biomedical sciences [14]. It has been investigated for a variety of biomedical applications [15] including wound healing [16], bone regeneration as well as being developed as a drug and growth factor delivery vehicle [17]. All of these applications make use of the antibacterial, haemostatic and non-toxic properties of this naturally derived polymer.

The aim of this study was to investigate mast cell interactions, including activation, with chitosan in vivo and in vitro. This study investigated early infiltration of mast cells in vivo following implantation of chitosan, with or without acid pre-treatment, subcutaneously into rats and as well as Surgicel, a commercially available haemostatic cellulose dressing. Histological assessment was carried out following 7 days implantation to investigate mast cell infiltration in response to the implanted material, along with expression of markers known to be produced by mast cells or involved in the inflammatory response. In vitro experiments on the rat mast cell line, RBL-2H3, were also carried out to investigate activation and the expression of PGs and glycosaminoglycans (GAGs) produced by these cells upon contact with chitosan.

Section snippets

Materials

Chemicals were purchased from Sigma–Aldrich (Castle Hill, Australia) unless stated otherwise. Materials used for implantation included Surgicel (Johnson and Johnson, USA) and chitosan (Ultrapure FMC-NovaMatrix, Norway, Batch no# 1148013, 460 kDa, 91% degree of deacetylation) were used as received and prepared for implantation as described below.

Rat implantation model

Preparation of chitosan for implantation involved either an acid or non-acid process. Chitosan gels were prepared, 2% (w/v) chitosan in 2% (v/v) acetic

In vivo response to implanted materials

The inflammatory response to chitosan with different pre-treatment process was evaluated following 7 days implantation. The level of response due the implanted chitosans as well as Surgicel, a commercially haemostatic agent derived from cellulose was assessed. Following dissection of the implanted materials, the behaviour of the chitosan was observed to differ due to the difference in preparation prior to implantation. Chitosan without acid (CWOA) pre-treatment maintained its original

Discussion

The inflammatory response to material implants is known to involve the infiltration of inflammatory cells, such as neutrophils, monocytes and macrophages, which lead to fibrous capsule formation and remodelling of tissue. Histological analyses presented in this study showed that an inflammatory response was evoked following implantation of all material types. The response varied between the materials, particularly evident through the amount of collagen present within the fibrous capsule. This

Conclusions

This study has presented data analysing the inflammatory response to implanted materials, going beyond the traditional methods of analysis in order to gain insight into the mechanisms behind host response and tissue remodelling. H&E staining showed varying levels of inflammatory response between the different material implants, primarily indicated by thickness of the fibrous capsule. Additionally picrosirius red staining showed the presence of collagen within the fibrous capsules, although its

Acknowledgements

This work was supported by funding from the Australian Research Council under the Linkage Project (LP0776293) scheme. The authors acknowledge Prof. Bruce Caterson, Cardiff University, UK, for the supply of the CS stub antibody (clone 2B6) and Prof. Achilleas Theocharis, University of Patras, Greece, for the supply of the polyclonal rabbit anti-serglycin antibody. The authors also acknowledge Dr Christine Chung Heart Research Institute, for assistance in histological staining.

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    Present address: Children's Cancer Institute Australia for Medical Research, Lowy Cancer Research Centre, University of New South Wales, Sydney NSW 2052, Australia.

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