| Objective:A kind of porous chitosan microspheres(CSM)with controllable particle size was created and characterized,and its biosafety was investigated by cytological experiments,and the correlation between the particle size of chitosan microspheres and the hemostatic effect in vivo and in vitro was explored,and the particle size range of chitosan microspheres with the best hemostatic effect was initially obtained.Methods:1.Microspheres of different particle size ranges were prepared by precipitation method and coaxial airflow shear method using airflow intensities of 1 L/min,3 L/min,and 5 L/min,and the microspheres prepared by 1 L/min,3 L/min,and 5 L/min airflow parameters were divided into 1.0,3.0,and 5.0 groups in subsequent experiments;2.The structure on the surface and cross section of microspheres were observed by scanning electron microscopy.The diameter range of chitosan microspheres in each group was obtained by measuring the diameter of 100 microspheres in each group through optical microscope observation.The specific surface area,average pore size and porosity of the microspheres were measured by BET specific surface area and N2 adsorption-desorption isotherm,and the water absorption capacity of the microspheres was measured by water absorption test.3.The in vitro hemolysis rate and coculture cell viability of the prepared microspheres were measured by in vitro hemolysis test and CCK-8 method;the biological safety of the material was evaluated by live-dead cell staining.4.Blood was drawn from the ear margin of New Zealand rabbits and the coagulation time of the microspheres of different particle sizes was measured in vitro.Blood Clotting Time(BCT)and Blood Clotting Index(BCI)were measured in vitro for different particle sizes of microspheres;5.An SD rat arterial/venous hemorrhage model was established,and gauze hemostasis groups,1.0,3.0 and 5.0 chitosan groups were set up to investigate the hemostatic effect of chitosan microspheres of different particle sizes in terms of hemostatic time and blood loss.In vivo experiments were conducted to verify the hemostatic effect of chitosan microspheres in different particle size ranges,and to compare the difference of in vivo hemostatic effect with two groups of powdered chitosan currently on the market.Results:The nanopore structure was observed on the smooth microsphere surface under electron microscope.The outer surface of the microspheres will form a denser surface structure.In the sphere section,larger pores can be seen in the 5.0 group microspheres,followed by the 3.0 group microspheres,and the internal pore structure of the 1.0 group microspheres is the smallest and denser of the three groups.The diameters of 913.3±32.26 μm,505.8±42.41μm,and 308.9±23.03μm were obtained for groups 1.0,3.0,and 5.0,respectively,under light microscopy.hysteresis lines were observed for groups 1.0 and 3.0 between a relative pressure of 0.6<P/P0<1.0 and a hysteresis return line can be observed in the 5.0 group between relative pressure 0.9<P/P0<1.0,indicating the presence of mesopores in the microspheres.The BET specific surface area was 97.62,91.83,and 11.34 m2/g for groups 1.0,3.0,and 5.0,respectively.the corresponding mean pore sizes were 10.94,15.30,and 17.38 nm.the results of the water absorption experiments were 97.62,91.83,and 11.34 m2/g for groups 1.0,3.0,and 5.0,respectively.the corresponding mean pore sizes were 10.94,15.30,and 17.38 nm for groups 1.0,3.0,and 5.0,respectively.The corresponding pore volumes are 0.25,0.59,and 0.03 cm3/g.Groups 1.0,3.0,and 5.0 corresponding to 17.5,9.5 and 5.8 times of the initial state of water absorption,respectively.The hemolysis rate of the material was 0.42%,and the cell viability of the microsphere groups in each group was greater than 90%at 1 d,3 d,and 7 d.Many green/live cells and only a few red/dead cells were visible in the photographs taken by fluorescence,and the number of cells all increased with time.The BCT of the 1.0 group was smaller than that of the 5.0 group and had statistical differences(p<.001);There were statistical differences(p<.0001)between the 1.0,3.0,5.0,and Celox groups and the control group;There was no statistical difference between the other groups.The BCI of the 1.0 group was 8.80±1.147%,which was lower than that of the 3.0 group(22.56 ± 4.098%)(p<.001);The BCI of the 1.0 group was 8.80±1.147%,which was significantly lower than that of the 5.0 group(34.45±4.279%)(p<0001);There was a statistical difference(p<.05)between the 3.0 group BCI and the 5.0 group.The results of the rat femoral artery/vein bleeding experiment showed that the BCT of the gauze group was 394.0±37.82 seconds,and the other five experimental groups showed a decrease in BCT,with a statistical difference(p<.001)compared to the gauze group.The BCT of the 1.0 group was as low as 72.00 ± 13.04 seconds,the 3.0 group was 146.0±41.59 seconds,and the 5.0 group was 240.00 ± 94.04 seconds,both of which were significantly shorter than the hemostatic time of the gauze control group(394.00±37.82)(p<.001).The 1.0 and 3.0 groups were superior to the 5.0 group(p<.05).The blood loss in the 1.0 group was as low as 1.96 ±1.743 g,while the blood loss in the 3.0 and 5.0 groups was 3.04± 0.53 g and 5.02± 2.46 g,both significantly lower than that in the gauze control group(6.72 ± 2.41 g)(p<.001),and showed an increasing trend with the decrease of microsphere size.In the measurement of hemostasis time,compared with the domestic product group(158.0 ± 27.75 s)and the Celox imported product group(136.0±15.17 s),the hemostasis time of the 1.0 group was significantly shorter than that of the domestic product group(p<.05);In the experiment of blood loss,the Celox group(4.34 ± 1.64 g)and the domestic commodity group(5.82 ± 2.20 g)both had a greater blood loss than the 1.0 group,but only the 1.0 group and the domestic commodity group showed a statistically significant difference in blood loss(p<.05),indicating that the hemostatic effect of the 1.0 group was better than that of mainstream powder hemostatic products on the market.Conclusion:Through the preparation method of this study,CSM of different pore sizes with better uniformity of particle size could be prepared.1.0 group(particle size:913.3 ± 32.26 μm)CSM showed better hemostatic performance in water absorption experiments,in vitro BCT,BCI and rat femoral arterial/veins bleeding experiments,which tentatively verified that the hemostatic effect of chitosan microspheres prepared by this method tended to increase with increasing diameter and was better than that of domestic and imported commercial Celox.The hemostatic effect of the chitosan microspheres prepared by this method tends to increase with the increase of diameter,and the hemostatic effect of the 1.0 group is more excellent than that of the domestic and imported Celox products.Therefore,the porous CSM prepared by this method can be used as a material with excellent hemostatic effect and has a broad clinical application potential,but further experimental verification is necessary. |
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