Bioactivities of Chitosan and Its Derivatives - Chitosan Based Hydrogels: Functions and Applications - page 122

microspheres were treated with H 2 O 2 , washed repeatedly and dehydrated successively
with ethanol (30%, 50%, 80%, 95%, and 100%), and finally vacuum dried overnight.

3.2.1.2 Blood Collection and Platelet Isolation

Swine blood was collected from a cannulated femoral artery under nonactivation condi-
tions and anticoagulated with acid citrate dextrose (20 mM citric acid, 110 mM sodium
citrate and 5 mM d-glucose) at a v/v ratio of 9:1 or 1 U/mL heparin, as per the protocol
approved by the DSO Institutional Animal Care and Use Committee. To isolate platelet
solutions, whole blood was centrifuged at 180 g for 20 min, and platelet-rich plasma was
separated from the red blood cell (RBC) fraction and further centrifuged at 1500 g for
15 min to pellet and concentrate the platelets. The platelet pellet was removed and resus-
pended in a buffer (140 mM NaCl, 3 mM KCl, 12 mM NaHCO 3 , 0.4 mM NaH 2 PO 4 and 0.1%
glucose, pH 7.4, adjusted with 4% HEPES). The concentration of the platelet suspension
was measured using the Cell-Dyn 500 hemostasis analyzer (Abbott Laboratories, IL, USA).
Only suspensions with >100,000 platelets/mL were used in experiments.

3.2.1.3 Dynamic Blood Clotting Test

For clotting time measurement, a kinetic method similar to the work described by K. Y. Lee
[84] was used. Microspheres were put into beakers and placed in a thermostat at 37°C for
5 min; then ACD whole blood stay focused on the prepositions (0.25 mL) was dropped onto
the surface of these microspheres, followed by the addition of 0.02 mL CaCl 2 solution
(0.2 mol/L). The blood clotting test was carried out by spectrophotometric measurement at
540 nm. The relative absorbency of 0.25 mL ACD whole blood diluted in 50 mL distilled
water was assumed to be 100. The blood clotting index (BCI) of a biomaterial can be quanti-
fied by using the following equation:

Absorbency of blood in contactwiththe sample
Absorbency of

BCI(%) = × 100

thesolutionofdistilledwater andACD blood

The platelet suspensions (0.5 mL) were spread over the microspheres and incubated at
37°C. After the specified incubation period, the platelet suspensions were counted using a
cytometer.

Totalplatelets Suspension plates
Totalplate

−

Adhesion ratio(%) = × 100

lets

3.2.1.4 Platelet Adhesion and Aggregation

The microspheres after incubation were gently and uniformly washed with 0.1 M PBS
(pH 7.4), fixed with 2.5% glutaraldehyde solution in saline at 4°C for 2 days, washed with
saline and dehydrated with a series of graded ethanol-water solutions (0, 30, 50, 70, 90, and
100%), and dried under vacuum conditions overnight. The dried microspheres after gold
coating were examined with a scanning electron microscope.

3.2.1.5 Hemolysis Test

The hemolytic activity of microspheres was investigated according to Singhal's method
[85]. Fresh rabbit blood was diluted using saline water (1:1.25, v/v). Microspheres with