Zanariah Ujang

In vitro cytotoxicology model of oligo-chitosan and n, o-carboxymethyl chitosan using primary normal human epidermal keratinocyte cultures.

Chitosan (beta-1, 4-D-glucosamine) is a deacetylated form of chitin with excellent biological properties in wound management. The natural properties of chitosan have the physical and chemical limitations to be widely used in biomedical fields. The improvement of the physical and chemical properties of chitosan with some additional chemicals will alter its biocompatibility. Therefore, the biological attribute of the modified chitosan must be evaluated. In this study, the cytotoxicity of oligo-chitosan (OC) and N, O- carboxymethyl-chitosan (NO-CMC) derivatives (O-C 1%, O-C 5%, NO-CMC 1% and NO-CMC 5%) was evaluated using primary normal human epidermal keratinocyte (pNHEK) cultures as an in vitro toxicology model at standardized cell passages (fourth passages). 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl tetrazolium bromide (MTT) was used as a cell viability assay. The O-C 1% is one of the most compatible chitosan derivatives because it steadily sustained >70% of viable cells until 72 hr post-treatment. This was followed by O-C 5%, NO-CMC 5% and NO-CMC 1%. Therefore, oligo-chitosan had the ideal properties of a biocompatible material compared to N, O- carboxymethyl-chitosan in this study.

The Development, Characterization and Application of Water Soluble Chitosan

Chitosan is a linear polysaccharide composed of randomly distributed β-(1-4)-linked Dglucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). Chitosan is produced commercially by the deacetylation of chitin, a long-chain polymer of N-acetylglucosamine which is the structural element in the exoskeleton of crustaceans (crabs, shrimp, etc.) and cell walls of fungi. The degree of deacetylation (%DD) can be determined by NMR spectroscopy, and the %DD in commercial chitosans is in the range 60100 %. A common method for the synthesis of chitosan is the deacetylation of chitin using excess concentrated sodium hydroxide as a reagent. It has the same β(1-4)-Dglucopyranose unit’s backbone as cellulose, except that the 2-hydroxy is replace by an acetamide group. Owing to its specific structure and property, chitosan has attracted significant interest in a broad range of areas such as pharmaceutical (Kato et al., 2003, Kumar et al., 2004), biomedical (Suh and Matthew, 2000, Tucci and Rigotti, 2003, Ng and Swami, 2005) water treatment(Northcott et al., 2005, Crini, 2005), cosmetics (Rinoudo, 2006, Sun et al., 2006 ), agriculture (Boonletniruni et al., 2008,El Hadrami et al., 2010) and food industry (Ham-Pichavant 2005,De Lima et al., 2010). As chitosan is a linear cationic biopolymer, it is only soluble in acidic aqueous solution in which the primary amino groups are protonated and precipitates when neutralized. The presence of rigid crystalline domains, formed by intra-and/or intermolecular hydrogen bonding, is considered to be responsible for the poor solubility of chitosan in high pH solutions (Nishimura et al., 1991). ) The extended applications of chitosan, is therefore frequently limited by its solubility behaviour. The solubility of chitosan can be improved by depolymerization and its chemical modification (Cravotto et al., 2005). Chitosan has reactive amino, primary hydroxyl and secondary hydroxyl groups which can be used for chemical modifications under mild reaction conditions to alter its properties. In particular, chitosan and its derivatives have been considered as biomaterials because of their biocompatibility, biodegradability, low immunogenicity and biological activities. (Hirano 1999, Molinaro et al., 2002) Chitosan has been well known to possess valuable properties for biomedical applications [Li et al, 1997] and being able to accelerate the healing of wound (Kwaeon et al., 2003, Khnor and Lim, 2003). It has also been documented that

Physical properties and biocompatibility of oligochitosan membrane film as wound dressing

Background The physical and biological characteristics of oligochitosan (O-C) film, including its barrier and mechanical properties, in vitro cytotoxicity and in vivo biocompatibility, were studied to assess its potential use as a wound dressing. Methods Membrane films were prepared from water-soluble O-C solution blended with various concentrations of glycerol to modify the physical properties of the films. In vitro and in vivo biocompatibility evaluations were performed using primary human skin fibroblast cultures and subcutaneous implantation in a rat model, respectively. Results Addition of glycerol significantly influenced the barrier and mechanical properties of the films. Water absorption capacity was in the range of 80%-160%, whereas water vapor transmission rate varied from 1,180 to 1,618 g/m2 per day. Both properties increased with increasing glycerol concentration. Tensile strength decreased while elongation at break increased with the addition of glycerol. O-C films were found to be noncytotoxic to human fibroblast cultures and histological examination proved that films are biocompatible. Conclusion These results indicate that the membrane film from O-C has potential application as a wound-dressing material.

Glycoprotein IIb/IIIa and P2Y12 Induction by Oligochitosan Accelerates Platelet Aggregation

Platelet membrane receptor glycoprotein IIb/IIIa (gpiibiiia) is a receptor detected on platelets. Adenosine diphosphate (ADP) activates gpiibiiia and P2Y12, causing platelet aggregation and thrombus stabilization during blood loss. Chitosan biomaterials were found to promote surface induced hemostasis and were capable of activating blood coagulation cascades by enhancing platelet aggregation. Our current findings show that the activation of the gpiibiiia complex and the major ADP receptor P2Y12 is required for platelet aggregation to reach hemostasis following the adherence of various concentrations of chitosan biomaterials [7% N,O-carboxymethylchitosan (NO-CMC) with 0.45 mL collagen, 8% NO-CMC, oligochitosan (O-C), and oligochitosan 53 (O-C 53)]. We studied gpiibiiia and P2Y12 through flow cytometric analysis and western blotting techniques. The highest expression of gpiibiiia was observed with Lyostypt (74.3 ± 7.82%), followed by O-C (65.5 ± 7.17%). Lyostypt and O-C resulted in gpiibiiia expression increases of 29.2% and 13.9%, respectively, compared with blood alone. Western blot analysis revealed that only O-C 53 upregulated the expression of P2Y12 (1.12 ± 0.03-fold) compared with blood alone. Our findings suggest that the regulation of gpiibiiia and P2Y12 levels could be clinically useful to activate platelets to reach hemostasis. Further, we show that the novel oligochitosan is able to induce the increased expression of gpiibiiia and P2Y12, thus accelerating platelet aggregation in vitro.

Effect of the Novel Biodegradable N, O-Carboxymethylchitosan and Oligo-Chitosan on the Platelet Thrombogenicity Cascade in von Willebrand Disease

INTRODUCTION Von Willebrand disease (vWD) is the second least common hemostatic disorder in Malaysia, and it has a low prevalence. This study examined the underlying platelet thrombogenicity cascades in the presence of different formulations of chitosan-derivatives in vWD patients. This paper aimed to determine the significant influence of chitosan biomaterial in stimulating the platelet thrombogenicity cascades that involve the von Willebrand factor, Factor 8, Thromboxane A2, P2Y12 and Glycoprotein IIb/IIIa in vWD. MATERIALS AND METHODS Variable chitosan formulations of N,O-Carboxymethylchitosan (NO-CMC) and Oligo-Chitosan (O-C) were tested. Fourteen vWD subjects voluntarily participated in this study after signing informed consent forms. The patients demographic profiles, family history, type of vWD, clinical symptoms and laboratory profiles were recorded and analyzed. Enzyme-linked immunosorbent assay, flow cytometry and Western blot tests were used to determine the level of the chitosan-adhered-platelet-mechanisms. RESULTS The study revealed that most patients were predominantly affected by vWD type I. The O-C group of chitosans scaffold pores is sufficient to allow for nutrients and cells. The O-C-stimulated-mediators are capable of initiating the platelet actions and were detected to expedite the blood coagulation processes. The oligo-group of chitosans was capable of amplifying and triggering more platelet activators pathways via the studied mediators. The present findings suggest that the ability of each type of chitosan to coagulate blood varies depending on its chemical composition. CONCLUSION The oligo group of chitosans is potentially capable of triggering platelet thrombogenicity cascades by activating platelets in vWD patients to form a platelet plug for hemostasis process.

Cytotoxicity Screening of Modified Chitosan Derivatives Film on Primary Human Dermal Fibroblasts

Chitosan has been proposed for use in biomedical applications because of its biocompatibility and abundance in nature. However, many barriers to the use of chitosan in biomedical applications still exist due to its physical and chemical limitations. Therefore, the biosafety of this chitosan-derived product requires an in-vitro evaluation. The objective of this study is to establish a primary fibroblast culture from human skin tissue and to assess the cyto-compatibility of locally produced chitosan derivatives products. Primary human dermal fibroblasts were isolated by using enzymatic digestion techniques and characterized by immunocytochemical staining. Characterization has been done by using antibodies against heat shock protein 47 (HSP47) and fibroblasts surface protein (FSP) antibody confirmed that the cells were fibroblast. The cytotoxicity of the chitosan derivative products on fibroblast cell culture were compared by measuring cell survival as determined by the tetrazolium salt reduction assay. The results of cytotoxicity testing on fibroblasts using both direct and indirect contact approach showed that oligo chitosan and Ncarboxymethyl chitosan (N-CMC) were the most biocompatible biomaterials in this screening process. Therefore, both locally produced chitosan materials can be used as possible materials for biomedical applications.

Biological Properties of Malaysian Tropical Seaweed for TopicalApplication

The increasing demands on anti-oxidant and skin lightening agents in Asia Pacific market have created vast excitement in the cosmetic and pharmaceutical industry to introduce robust and effective, yet safe invention of topical product. The emergence of various herbal-based products stimulates comprehensive study to evaluate the potential of various tropical plants. Seaweed is a marine tropical alga that has been claimed to possess good anti-aging and anti-pigmentation activity. Nonetheless, these claims are not supported by strong scientific evidences. Three species of Malaysian tropical seaweed, Turbinaria sp. (TR), Gracilaria sp. (G) and Padina sp. (PD) were studied for anti-oxidant and skin lightening properties. Crude aqueous extracts of the selected seaweed species (TRA, GA and PDA) were evaluated using ABTS and mushroom tyrosinase assay, for anti-oxidant and skin lightening activity, respectively. Amongst the extracts, TRA showed the most potent anti-oxidant activity. This extract displayed lower IC50 value of 0.063 ± 0.001 mg/ml, whilst commercial anti-oxidant agent, vitamin C (L-ascorbic acid) demonstrated better IC50 value at 0.008 mg/ml. In addition, TRA showed good skin lightening activity at 2 mg/ml with 50.11 ± 0.04 % inhibition against tyrosinase enzyme activity. Therefore, this study could underpin the dual inhibitory effect of the selected seaweed species as anti-oxidant and skin lightening agent for topical application. The cellular antioxidant activity (CAA) for quantifying the antioxidant activities will then further evaluate to measure the antioxidant potential of seaweed extracts. The cell based bioassay represents a promising analytical tool for potential biological activity.