Ahmad Hazri Abdul Rashid

In vitro capacity of different grades of chitosan derivatives to induce platelet adhesion and aggregation

Chitosan-derived hemostatic agents with various formulations may have distinct potential in hemostasis. This study assessed the ability of different grades and forms of chitosan derivatives as hemostatic agents to enhance platelet adhesion and aggregation in vitro. The chitosan derivatives utilized were 2% NO-CMC, 7% NO-CMC (with 0.45 mL collagen), 8% NO-CMC, O-C 52, 5% O-CMC-47, NO-CMC-35, and O-C 53. Samples of chitosan derivatives weighing 5mg were incubated at 37°C with 50 μL of phosphate buffer saline (PBS) (pH 7.4) for 60 min. The morphological features of the platelets upon adherence to the chitosan were viewed using scanning electron microscope (SEM), and the platelet count was analyzed with an Automated Hematology Analyzer. For platelet aggregation, we added an adenosine diphosphate (ADP) agonist to induce the chitosan-adhered platelets. O-C 52 bound with platelets exhibited platelet aggregates and clumps on the surface of the membrane layer with approximately 70-80% coverage. A statistically significant correlation (p<0.01) for the platelet count was identified between the baseline value and the values at 10 min and 20 min. The results indicate that O-C 53 and O-C 52 were able to promote clotting have the potential to induce the release of platelets engaged in the process of hemostasis.

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.

CTCF, YB-1, c-myc, and p53 expressions of primary human hypertrophic scar and normal fibroblast skin cells in response to novel chitosan derivatives sheet

The primary human hypertrophic scar and normal skin fibroblasts were successfully established and identified by heat shock protein 47 and fibroblast surface protein markers. However, different patterns of protein expressions were found in cultured fibroblasts from hypertrophic scar and in normal fibroblast skin cells after treatment with the chitosan derivatives sheet. The CTCF protein was up-regulated in fibroblast hypertrophic scar on treatment with chitosan derivatives. In contrast, the amount of CTCF protein was found unchanged in normal skin fibroblasts both treated and untreated. The YB-1 protein was expressed almost similarly in normal and hypertrophic scar when treated with chitosan but the expression differed when untreated. The c-myc and p53 proteins were expressed in fibroblast hypertrophic scar followed by up-regulation after treatment with chitosan derivatives. The c-myc and p53 expressions were not detected in normal fibroblasts neither untreated nor treated. The CTCF, YB-1, c-myc, and p53 proteins acted in different manners in human hypertrophic scar and normal fibroblast skin cells. The novel chitosan derivatives sheet in this study may play roles in the control of cell growth and proliferation of human hypertrophic scar and normal fibroblast skin cells. The mechanisms underlying expression of these protein factors remain unclear, and further studies are still undergoing in our laboratory.

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.

Study of Covalent/Ionic Cross-Linked Modification on Physical and Mechanical Properties of Chitosan Film as Potential Material in Medical Application

The aim of this study was to investigate the effects of covalent and ionic cross-linked reactions which were respectively done by using genipin and tripolyphosphate (tpp), on the structure and mechanical properties of chitosan film. Both cross-linked and uncross-linked films were prepared by solution casting method and characterized. FTIR spectra showed no characteristic of –OCH3 peak from genipin at 1444 cm-1 which resulted by a new covalent bonding in chitosan film. Reduction in absorption intensity at 1560 cm-1 wave number in chitosan cross-linked tpp films were due to the presence of ionic interaction between the positive charged of amino group in chitosan and negatively charged of phosphate group by tpp. The pattern area from the XRD results showed that the covalent cross-linked had significantly changed on the chitosan`s degree of crystallinity. The water contact angle on the surface of covalent/ionic cross-linked chitosan film reached the highest θ at 82.72° which indicated more hydrophobic properties was formed. Covalent/ionic cross-linked chitosan also showed the higher mechanical strength with average tensile stress value at 71.25 MPa. All finding results demonstrated that cross-linked modification on the chitosan film had successfully reduced the film’s hydrophilicity and increased the mechanical properties of the film.