In vitro biocompatibility of chitosan porous skin regenerating templates (PSRTs) using primary human skin keratinocytes

Abstract

Biopolymer chitosan (β-1,4-d-glucosamine) comprises the copolymer mixture of N-acetylglucosamine and glucosamine. The natural biocompatibility and biodegradability of chitosan have recently highlighted its potential use for applications in wound management. Chemical and physical modifications of chitosan influence its biocompatibility and biodegradability, but it is unknown as to what degree. Hence, the biocompatibility of the chitosan porous skin regenerating templates (PSRT 82, 87 and 108) was determined using an in vitro toxicology model at the cellular and molecular level on primary normal human epidermal keratinocytes (pNHEK). Cytocompatibility was accessed by using a 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl tetrazolium bromide (MTT) assay from 24 to 72 h. To assess the genotoxicity of the PSRTs, DNA damage to the pNHEK was evaluated by using the Comet assay following direct contact with the various PSRTs. Furthermore, the skin pro-inflammatory cytokines TNF-α and IL-8 were examined to evaluate the tendency of the PSRTs to provoke inflammatory responses. All PSRTs were found to be cytocompatible, but only PSRT 108 was capable of stimulating cell proliferation. While all of the PSRTs showed some DNA damage, PSRT 108 showed the least DNA damage followed by PSRT 87 and 82. PSRT 87 and 82 induced a higher secretion of TNF-α and IL-8 in the pNHEK cultures than did PSRT 108. Hence, based on our experiments, PSRT 108 is the most biocompatible wound dressing of the three tested.

Introduction

Chitin is a β-(1,4)-d-linked polymer of N-acetylglucosamine that is usually extracted from shellfish waste or the cell walls from crustaceans and arthropods. When the monomers of chitin are deacetylated to form a mixture of N-acetylglucosamine and glucosamine, it is called chitosan (Illum, 1998). Wound dressing in porous structures, such as Integra™ has been typically used as the wound dressing for partial and full-thickness wounds. The biocompatibility and biodegradability of chitosan have made it a potentially useful pharmaceutical material especially for wound management (Diegelmann et al., 1996, Halim et al., 1998). In this study, chitosan was porous-structured using different acidic solvents and neutralized either by ethanol serial-hydration or sodium bicarbonate (NaHCO₃) methods before lyophilization. However, various chemical modifications aimed at further developing the chitosan into various forms might adversely influence the biocompatibility and biodegradability of chitosan. Therefore, an evaluation of the biocompatibility of chemically and physically modified chitosan must be performed to ensure that the final product is biocompatible as a wound management product.

In vitro, cells respond to the location and manner in which the biomaterials are applied. Fibroblasts or keratinocytes, which are the main cell constituents of the dermal and epidermal layers respectively, can be cultured to investigate the biocompatibility of chitosan derivatives as wound dressings. The in vitro toxicology model simplifies the system due to its ability to assess the cellular and molecular reactions outside an organism (Schreier et al., 1993, Cha et al., 1996). Additionally, an in vitro system often has higher sensitivity than an in vivo assessment (Ulreich and Chavapilin, 1983).

At the cellular level, the effect of chitosan formulations was evaluated with a 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl tetrazolium bromide (MTT) assay, using primary human keratinocytes (Lim et al., 2007). Traditional assays have measured cytotoxicity in vitro by means of either an end-stage event such as permeability of cytoplasmic membranes of dead and dying cells or metabolic parameters such as cell division or an enzymatic reaction (Hanks et al., 1996). The MTT assay is a simple, metabolic activity based assay which is used to determine detrimental intracellular effects on mitochondria. The MTT assay utilized the selective ability of viable cells to reduce tetrazolium bromide into purple formazan crystals which are only soluble in organic solvents. Previous reports have shown that MTT assays adequately assess cytocompatibility, give results that are easily interpreted and are cost effective (Janvikul et al., 2005, Mei et al., 2005, Lim et al., 2007, Neamnark et al., 2007).

There are numerous assays that have been used to detect deoxyribonucleic acid (DNA) damage, including the Ames, micronuclei, mutations and structural chromosomal aberrations assays. However, a more recent and useful approach for assessing DNA damage is the comet assay (Singh et al., 1988). A general guideline provided by Tice et al. (2000) is that a cytotoxicity assay should be performed prior to the comet assay, and a cell viability of above 70% is required in order to exclude false positive results, due to apoptosis. There are many excellent reviews on the use of the comet assay on aquatic animal cells to assess the DNA damage caused by genotoxicants (Nacci et al., 1996, Mitchelmore and Chipman, 1998, Cotelle and Ferard, 1999).

In addition to DNA damage assays, expression analysis of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) is another advantage of the in vitro assay that ensures the biocompatibility of the biomaterials with human skin cells (Allen et al., 2001).

In this study, the newly developed chitosan derivatives in the form of porous skin regenerating templates (PSRTs) were tested for biocompatibility in a direct-contact method with primary normal human epidermal keratinocyte (pNHEK) cultures. Cytotoxicity, genotoxicity and inflammation were assessed using the MTT assay, the comet assay and analysis of the pro-inflammatory cytokines (TNF-α and IL-8), respectively.