Preparation And Efficacy Of Novel Antibacterial Composite Absorbable Hemostatic Microspheres

Effective hemostasis is an essential step to prevent major blood loss or death during the accident,war or surgical operations.However,our body’s self-coagulation process is hard to accomplish hemostasis without the assistance of hemostatic agents.Recently,some new biomaterails have been developed and applicated in hemostasis.Nonetheless,they still have a lot of defects,such as low hemostasis efficacity,or poor biocompatibility.In particular with large wound surface or a gash,most hemostatic agents are not able to prevent excessive bleeding accompany with high probability of wound infection.Therefore,the development of a safe,reliable hemostatic material with good hemostatic effect and antibacterial properties has theoretical research interests and biomedical application requirements.This research is based on the following biomass polymer materials:sodium alginate(SA),carboxymethyl chitosan(CMC),collagen(Collagen).Calcium chloride is added as a cross-linking agent,using emulsion cross-linking-spray drying method to prepare Absorbable Hemostatic Microspheres(CSCM).We have added berberine hydrochloride as the antibacterial component to prepare a novel antibacterial Absorbable Hemostatic Microsphere(CSCM-B).Scanning electron microscopy(SEM),laser particle size analyzer,and Fourier transform infrared spectroscopy(FTIR)were uesd to characterize the physical and chemical properties of the material.In vitro evaluation experiments,such as swelling capacity analysis,degradation performance analysis,platelet effect analysis,hemolysis rate,and bacteriostatic test were carried out.In vivo safety testing,such as intradermal stimulation test,elimination rat,cytotoxicity,wound repairation were performed to study the hemostatic properties,antibacterial properties,biocompatibility and safety of CSCM and CSCM-B as hemostatic agents.Firstly,we studied the preparation process of the CSCM by optimizing the compounds ratio of sodium alginate,cardiomyopathy chitosan and collagen.SEM analysis showed that CSCM exhibited a hollow spherical structure.The particle size analysis showed an average particle size of 39.33 μm,and 90%of the microspheres have the particle size below 79.60 μm.In the tail clipping experiment test with rats,the average time of hemostasis with CSCM was(249.2±44.7 s).However,the average time of hemostasis with the commercial hemostatic microspheres was 370.0±43.1 s and the average time of hemostasis with the gauze was 428.0±83.3 s.In the hemolysis experiment,CSCM showed a hemolysis rate of 0.83%,much lower than 5%(Pharmacopoeia standard),which indicate that CSCM has no hemolysis reaction and has a good biocompatibility.In the intradermal stimulation test,the stimulation index was analysed 24 hours after injection of CSCM or olive oil(control).The stimulation index of the PBS infusion group was 0.05,and the olive oil infusion group was 0-0.05.No significant difference between the control group and the blank control group were observed,indicating that the CSCM has very slight intradermal irritation.During the in vivo safety test,the CSCM was implanted in SD(Sprague-Dawley)rats and analysed within 7 days,the volume was significantly decreased,and completely degraded within 42 days.Various biochemical and immunological indicators of the rats detected during this period and showed no abnormal effects,which indicated that the material has good biosafety and biocompatibility.However,this material has no significant antibacterial effect,so we stated to modify the hemostatic materialOn the basis of the ancient process,we added berberine hydrochloride with a certain proportion(1%,5%,10%of the total mass of SA/CMC/C)to further prepared sodium alginate/chitosan/collagen/hydrochloric acid,in order to obtain the modified hemostatic material(CSCM-1B,CSCM-5B,CSCM-10B).SEM analysis showed that the CSCM-B series microspheres also had a hollow microsphere-like structure with a relatively rough outer surface and an average particle size of 81.69 μm,slightly higher than the CSCM group.In the in vitro swelling degradation experiment,CSCM-10B has the highest swelling ratio(5500%)and the fastest degradation rate(15～30 min).In the hemostasis experiment,the mean time of coagulation of CSCM-10B was(180.0±6.0 s),which was better than CSCM(249.2±44.7 s)and CSCM-1B(209.0±42.0 s)and CSCM-5B(217.0±26.0 s)..At the same time,we also analyzed its effect on platelets.In the cytotoxicity(according to MTT assay experiments)the positive control with commercially available hemostatic agents and CSCM-10B showed no significant cytotoxicity at either low concentrations(10 μg/mL)or high concentrations(100μg/mL).In the bacteriostatic experiment,CSCM,CMPHP,CSCM-1B,CSCM-5B and ampicillin(Amp)and CSCM-10B showed excellent antibacterial effects at the same concentrationIn conclusion,we have prepared and studied structure,the capacity of hemostasis,the toxicity and antibacterial effects of the the absorbable hemostatic microspheres(CSCM)and the new antibacterial absorbable hemostatic microspheres(CSCM-B),we obtained the optimal preparation process of CSCM-B with antibacterial effects,hemostasis capacity and low toxicity This series of experiments indicate that CSCM-B is a promising new hemostatic agent and provides several evidences for further research on this new type of biomaterial.