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

https://doi.org/10.1016/j.ijbiomac.2012.10.001Get rights and content

Abstract

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 5 mg 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.

Highlights

► Chitosan. ► Platelet adhesion. ► Platelet count. ► Morphology. ► Platelet aggregation.

Introduction

Since the middle of World War II, half of recorded combat deaths have occurred due to exsanguinating hemorrhage. A military post-mortem study of casualties in Operation Iraqi Freedom (OIF) suggested that up to 24% of all battlefield mortality could be reduced with improved anti-hemorrhaging methods and that 85% of deaths were caused by uncontrolled hemorrhage [1]. The development of new methods or devices for hemorrhage control may contribute to a future reduction in hemorrhage morbidity and mortality [2]. Recently, chitosan-derived antihemorrhage biomaterial, which contains N-acetyl glucosamine (found abundantly as a major component in shells of arthropods such as crabs, shrimps, lobsters and insects) [3] was identified as having potential clinical utility.

Chitosan has become one of the most promising local hemostatic agents. It is of particular importance as it functions independently on platelets and normal clotting mechanisms. Chitosan derivatives also possesses amino and hydroxyl groups that permit them to be chemically adjusted by processes such as acylation, N-phthaloylation, alkylation, Schiff base formation, reductive alkylation, tosylation, O-carboxymethylation, N-carboxyalkylation, and graft copolymerization [4], [5]. Although there are numerous studies on the hemostatic capacity of chitosan, to the best of our knowledge there are a limited numbers of in vitro studies on the capacity of chitosan derivatives acting on platelets as an absorbable surgical hemostatic agent. Driven by the significant role of adherence in platelet response during the hemostasis process, in our present study we conducted platelet adhesion and aggregation tests to characterize platelet capacity in the presence of chitosan.

The major functions of platelets in hemostasis involve their adherence at the sites of vessel injury, activation of internal signaling pathways, and formation of plugs by aggregation and clumping [6]. For platelet aggregation, we have added an ADP agonist to induce chitosan-adhered platelets. We used N,O-carboxymethylchitosan (NO-CMC), O-carboxymethylchitosan (O-CMC) and Oligo-chitosan (O-C), produced by Standard and Industrial Research Institute of Malaysia (SIRIM Berhad), with a degree of deacetylation of 75–98%. Lyostypt®, a commercial hemostatic agent was used as a positive control. Our results show that platelets respond differently to the presence of chitosan derivatives with differing molecular weights and degrees of deacetylation.

Section snippets

Different grades and forms of chitosan

In this study, different percentage levels of chitosan sponges and two distinct powdered types of chitosan were used. The following sponge forms were used: 2% NO-CMC, 7% NO-CMC (with 0.45 mL collagen), 8% NO-CMC, O-C 52, and 5% O-CMC-47. The powdered forms of chitosan were NO-CMC-35 and O-C 53.

Subjects

Blood was withdrawn from antecubital veins and collected in vials containing 3.8% sodium citrate. We recruited 30 healthy donors aged 18–40 who had not consumed drugs in the previous two weeks. Informed

Platelet count

Fig. 1 showing the most significant increase from the baseline 297.3 ± 47.19 × 103/μL was observed for O-C 52: 248.6 ± 42.20 × 103/μL after 10 min and 221 ± 41.07 × 103/μL after 20 min. The percentage of platelet counts decreased slightly from the baseline (25.66%) over the 20 min (16.38%) time interval. The next largest increase was observed for O-C 53. The percentage change in the platelet count increased from 11.86% to 17.82%. Table 1 pointing the analysis of correlation indicated that the relationships

Conclusion

Chitosan derivatives exert a combined effect on thrombogenesis by causing platelets to aggregate and form pseudopodal shapes. Various formulations of chitosan exhibited different capabilities in vitro based on the chemical composition of the material. We conclude that O-C 52 and O-C 53 were superior to other types of chitosan in achieving hemostasis. Further studies are needed to elucidate the precise mechanism of action of chitosan derivatives on platelets.

Acknowledgements

This project is funded by USM Short term grant 304/PPSP/61310037. We thank all the donors who participated in this study.

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