Preparation Of Chitosan Based Porous Microspheres And Their Hemostatic Properties

Chitosan (CS) is the second most abundant natural renewable resource next to cellulose. It has many attractive properties such as non-toxic, pollution-free, biocompatible and biodegradable. The CS contains a large amount of -NH2 groups, which can interact with blood cells, platelets and proteins in blood, so it has been developed to hemostatic materials. This research focused on improving the hemostatic efficiency of chitosan by preparing chitosan particles with special microstructure. Combining emulsion technique and thermally induced phase separation (TIPS), a series of chitosan based microspheres with high porosity and water absorption ratio were prepared by regulating the emulsion and quenching conditions. The main results are listed below.(1) The CS microspheres (CSMS) were porous. Their average diameter was successfully controlled by adjusting the emulsion conditions such as emulsification temperature and the blending ratio of emulsifiers. An array of microspheres with uniform size of 167±38.5 ?m was synthesized under conditions of Tween 60/Span 80= 0.2/4.8 and emulsification temperature= 40 ?. The surface average pore diameter decreased with decreasing quenching temperature. It also decreased when CS was blended with polyvinyl alcohol (PVA). The surface average pore diameter was reduced to 0.49±0.07 ?m, which was the smallest among all the microspheres, when the blending ratio of CS/PVA was 6:4. The density, porosity and water absorption ratio of CSMS-7 were 0.0609g/cm3,97.55% and 1552%, respectively. Meanwhile, the density increased with reducing surface pore size, but the water absorption ratio decreased. The CSMS could be enzymatically degradaded by 60% in 4 weeks, better than CS/PVA microspheres. FTIR and thermogravimetic analysis (TGA) tests illustrated that the composite microspheres with a blending ratio of 6 (CS)/4 (PVA) had a better compatibility.(2) The hemostatic properties of CSMSs were closely related to their average surface pore diameter. Compared with commercial hemostatic gauze Celox, CSMSs with surface pore diameter larger than the size of erythrocyte and platelet, showed no significant improvement in hemostatic properties. However, CSMSs with surface pore diameter smaller than the size of erythrocytes and platelets displayed notable improved hemostatic properties. The hemostatic time of CSMS-7 in rat tail amputation model and liver puncture model was respectively 189 s and 80 s, and the in vitro blood coagulation time was 105 s. They are much shorter than the corresponding values of Celox (229 s,117s and 155 s, respectively). The rate of whole blood clotting also increased. The histology photos of rats' livers indicated that CSMS did not cause exothermic reaction or stimulation to tissues. The porous structure would not affect the interaction between chitosan and blood cells and the rough surface porous structure might benefit the adhesion of erythrocytes and platelets. When CSMSs were loaded with tranexamic acid (TA), TA could be released quickly in blood, which also improved its hemostatic properties. The hemostatic time of CSMS-7TA in rat tail amputation model and liver puncture model were respectively 138 s and 61s, and the in vitro blood coagulation time was 70 s.(3) The porous CSMSs demonstrated non-cytotoxic. Hepatocytes could proliferate regularly on both surface and internal of CSMSs at 12h,24h and 72h. Compared with commercial cell carrier Cytodex, CSMS even had a better cell adhesion rate. The big pores of CSMS-5 were beneficial to cell proliferation significantly. Although CSMS-5 was not a better hemostatic agent than CSMS-7, it might be a new high density cell carrier.