The aim of this paper was to investigate the effects of cross-linked reaction on physicochemical properties of chitosan film by using genipin as cross-linker agent. Series of chitosan film samples with different amount concentration of genipin loaded (0-3 wt %) were prepared and characterized. The physicochemical properties of films were evaluated by Fourier Transform Infra-red (FTIR), UV-vis spectroscopy, Oxygen Transmission Rate (OTR), Scanning Electron Microscopy (SEM), water vapour and tensile test. The cross-linking reaction had affected on colour changing of chitosan film samples from light yellow to dark blue in line with the increasing of genipin concentration. Thus, UV-vis spectroscopy on the cross-linked samples showed the absorbance value at 600 nm wavelength due to genipin content. FTIR observation on cross-linked film samples showed no characteristic of –OCH3 peak from genipin at 1444 cm-1 which resulted by new covalent bonding occurred between chitosan and genipin. Cross-linking also had increased the oxygen barrier and reduced the water vapor rate through the film. Chitosan film sample with addition of 1 wt% genipin achieved the highest tensile stress average at 49.46 MPa compared to other samples while percent of elongation at break reduced with the increasing of genipin concentration loaded
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.
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