Bioactivities of Chitosan and Its Derivatives - Chitosan Based Hydrogels: Functions and Applications - page 126

adhesion without forming any dimensional network structure or adsorbing any plasma pro-
teins at first. The reason for the promotion of erythrocyte aggregation may be due to its cat-
ionic nature. Furthermore, it can also adsorb various plasma proteins, which may enhance
procoagulation.
Hemolysis of blood is the problem associated with bioincompatibility [94]. Previous
studies indicated that chitosan promoted surface-induced hemolysis, which can be attrib-
uted in part to electrostatic interactions [90,95]. In the literature [93], when the concentra-
tion of microspheres adds up to 100 mg/mL, chitosan showed a little hemolysis. However,
the largest hemolytic activity was lower than 10%, which indicated a wide safety margin
in blood contacting applications and suitability for i.v. administration [96,97]. Furthermore,
by comparing the low hemolytic activity with the high erythrocyte agglutination, it was
shown that chitosan only induces the adhesion of erythrocytes but does not seriously
damage the cell membrane.
The procoagulation properties of chitosan microspheres give them the potential for use
as thrombospheres. Most thrombospheres (Hemosphere, Irvine, CA) are composed of
cross-linked human albumin with human fibrinogen bound to the surface. A similar prod-
uct, Synthocytes (Andaris Group Ltd, Nottingham, UK), has just entered clinical trials in
Europe [98].
3.2.2 Hemostatic Potential
3.2.2.1 Characterization
Whole-blood clotting : The blood clotting test was adapted from Shih et al. [99]. Dressings
were placed in polypropylene (PP) tubes and prewarmed to 37°C. Citrated whole blood
(0.2 mL) was then dispensed onto the dressings, and 20 mL of 0.2 M CaCl 2 solution was
added to start coagulation. The tubes were incubated at 37°C and shaken at 30 rpm. After
10 min, RBCs that were not trapped in the clot were hemolyzed with 25 mL of water, and
the absorbance of the resulting hemoglobin solution was measured at 540 nm.
Platelet adhesion : The platelet adhesion assay was adapted from Vanickova et al. [100].
Before the start of the test, platelets were reconstituted to 2.5 mM CaCl 2 and 1.0 mM
MgCl 2 . Then 1.5 mL of the platelet suspension was added to each dressing. After incuba-
tion at 37°C for 1 h, the dressings were removed and dip rinsed twice in PBS to remove
platelets that were not attached. Samples were then placed in PBS containing 0.9%
Triton-X100 for 1 h at 37°C to lyse the adhered platelets. The lactate dehydrogenase (LDH)
enzyme that was released was measured using a kit (Promega, USA) as per the manufac-
turer's instructions. A platelet calibration curve was generated by serially diluting a
known number of platelets, followed by lysis with 0.9% Triton-X100 and measurement of
LDH. Samples were also fixed in 4% paraformaldehyde and 0.5% glutaraldehyde for
electron microscopy studies.
Thrombin generation : Thrombin-antithrombin complex (TAT), a marker of thrombin neu-
tralization, is an indicator of how much thrombin was formed over a period of time [101].
Dressings were incubated with 1 mL of heparinized whole blood for 30 min at 37°C and
then 20 mL of sodium citrate (0.633 M) was added to stop thrombin generation. TAT levels
in blood samples were measured by an ELISA kit (DadeBehring, Germany) as per the
manufacturer's instructions.
Blood and simulated body fluid ( SBF ) absorption : The absorption efficiency of dressings was
determined in citrated whole blood and SBF, a solution with ion concentrations similar to
that of human plasma. SBF consists of 142 mM Na + , 5.0 mM K + , 2.5 mM Ca 2+ , 148 mM Cl − ,