The Hemostatic Activity And Biological Safety Evaluation Of Chitosan

Chitosan is a natural biodegradability polysaccharide with hemostasis and wound healingactivities. It has been a research highlight as a kind of hemostatic biomaterials in recent years,and there are several kinds of topical chitosan-based dressing approved for listing. However,the development and application of chitosan as an implanted hemostatic material are still intheir infancy, which is limited by its unclear hemostatic effect and biological safety in vivo.The purposes of this thesis are to evaluate the hemostatic capacity and biological safety ofchitosan in animals, to lay research foundations for its development and offer academic basesfor its application as an in vivo hemostatic dressing, and this has important theoretical andpractical values in the developments of chitosan industry.In order to screen a sample with good hemostatic activity and evaluate its hemostaticcapability in vivo, the effects of a series of chitosan powder and solution with different DDand Mw to the coagulation time of rabbit blood in vitro were studied by Lee-White method,and the effects of chitosan powder samples with different properties to the time and theamount of bleeding of mouse tail were investigated by tail cutting method. The samplescreened according to these was applied to the organ wounds of rabbits in vivo, and the timeand amount of bleeding were measured. The results showed that the Mw and DD had greateffects on the hemostatic activity of chitosan, the coagulation time was shorter while the Mwand DD were higher, and the activity of powder was better than the solution at the same dose.All chitosan powder samples could shorten the bleeding time of mouse tail more than53.4%and CS powder, whose DD was85.32%and Mw was1.22×106, could reduce the bleedingtime by74.7%and the amount of bleeding by86.4%. CS powder showed a good hemostaticeffect on the bleeding wounds of substantive organs with rich blood （such as liver and spleen）,while the bleeding time could be shortened63.5%and the amount reduced72.9%at most.To explore the possible way of CS to play its hemostatic activity in vivo, it wasco-cultured with endothelial cells, which were widely distributed in the vessel lumen surface,for24h. The changes of procoagulant factors （such as vWF, TF, PAI, and ET-1） andanti-coagulant factors （such as ENOs, TM, PS, tPA, and TFPI） synthesized and secreted byendothelial cells were measured by RT-PCR and fluorescence quantitative analysis, whichcould denote the changes of coagulation capability of endothelial cells. The results showedthat CS could inhibited the mRNA expressions of TFPI, tPA, and ENOs, and their expressionamounts in the highest CS group were34.40%,0.77%, and9.24%of control group,respectively. Therefore, CS could promote blood clotting by inhibiting the synthesis andsecretion of anti-coagulant factors, and this might be one way of chitosan to stop the bleedingin vivo.The key researches about the biological safety of CS were as follow.（1） Acute and chronic toxicity: CS suspension was intraperitoneally injected into ICRmice at2000mg/kg and5000mg/kg, respectively. The survival condition and symptoms ofadverse effects were observed for14days, and hematological and histological tests wereperformed. Then CS suspension was singly injected intraperitoneally into ICR mice and the observation lasted for90days. The survival condition, symptoms of adverse effects,hematological and histological changes were observed to evaluate the chronic toxicity. Theresults showed that the LD₅₀of CS was over5000mg/kg in mice, and it was a less-toxicmaterial. Some adverse effects, such as decreasing of body weight and food consumption,erected hair, serious diarrhea, and sharply reduced movements, were observed. Acuteinflammations of body to strange materials were noted, inflammatory cells were increased inlivers and kidneys, and spleens were enlarged notably for the disseminated hyperplasis ofwhite pulp area. The long-term existence of CS in vivo could induce chronic inflammation ofbody, persistive enlargement of spleen, and continued increasement of platelet.（2） Genotoxicity and cytotoxicity: the genotoxicity and mutagenicity of CS were studiedby Ames test, mouse micronucleus test and sperm abnormality test, and the effect of CS to theproliferation of HUVEC was tested by MTT method to denote its cytotoxicity. The resultsproved that CS had no genotoxicity in the test dose range because there were no abnormalalterations of test bacteria or cells. CS could promote the proliferation and have nocytotoxicity in the test dose range （0.0625¹mg/mL）.（3） Reproductive, developmental and teratogenic toxicity: CS were injected into micebefore mating or on GD6, and the alterations in reproductive parameters, mating, fertilization,gestation, delivery, and lactation of F0mice were observed. The embryogenesis anddevelopment, growth and teratogenic conditions of offspring were recorded. The resultsshowed that there were no influences of CS in reproductive parameters of F0male mice,however, the corpora lutea counts and implantation sites of F0female mice were reducedsignificantly even at125mg/kg, which could lead to the reducing of the numbers of livefetuses. Therefore, the NOAEL of CS in the reproductive toxicity was considered to be lessthan125mg/kg. CS could induce emaciation and threatened abortion in F0mice at500mg/kg,and the numbers of live fetuses were decreased notably. However, the developments of itsoffspring were not affected. Therefore, the NOAEL of CS in the developmental toxicity wasconsidered to be125mg/kg. In addition, there was no difference in the teratogenic rate amongall groups, so the NOAEL of teratogenic toxicity was considered to be more than2000mg/kg.In summary, CS, whose DD was85.32%and Mw was1.22×106, screened by in vitro andbody surface hemostatic tests and displayed good hemostatic activity in vivo, which couldsignificantly shorten the bleeding time and reduce the amount of bleeding. So it has a gooddevelopment and research prospect. The biological safety evaluation tests of CS as an internalhemostatic material were performed for the first time, and the results showed that the LD₅₀ ofCS in mice was over5000mg/kg, and it had not shown genotoxicity, cytotoxicity orteratogenic toxicity. However, CS could induce some clinical adverse effects, especially in thereproductive process of female mouse and pregnant mouse. These results have importanttheoretical values and practical meaning for the development and application ofchitosan-based implantable hemostatic dressing.