| It is hard to avoid traumatic bleeding that occurs in daily life. Although slight trauma is not fatal, it can be life-threatening if blood loss of the whole body reaches 30% or more in a short time. Excessive bleeding is the important cause of death happened in war, natural disasters, traffic accident and other emergencies. In fact, more than 40% of the casualties can be avoided if the bleeding can be effectively stopped within 30 min when the wounded bleeding occurs. However, the traditional hemostatic materials is often unefficient to stop the massive bleeding, and the rapid hemostasis materials available in the current market are in sufficient to some extent. Therefore, it is many researchers’ goal to develop a new kind of hemostatic materials in biomedical field that can be both fist-aid treatment in war and usual trauma treatment in daily life, with fast hemostatic effect, good biocompatibility and degradation in vivo.Chitosan and gelatin are two biological materials that are biocompatibile, biodegradable, non-toxic, non-immunity, and have a certain blood clotting capability. Pupal shell is the waste when producing silk and rich in chitosan. In this study, the chitosan/gelatin sponge(CG) was prepared via a modified gradual-base extraction and freeze-drying method to produce a rapid absorbable anti-hemorrhagic agent. Chitosan in CG was extracted from silkworm pupae. This paper covers the optimum preparation process of the hemostatic materials, physical and chemical performance, hemostatic effect, hemostatic principle, cell toxicity, biological compatibility and biodegradable properties. Here are the results:(1) The optimum preparation for obtaining chitosan/gelatin composite hemostatic material(CG)Chitosan and gelatin are two biological materials which have good hemostatic effect, and they are often used separately as the main component of hemostatic products. However, there are only a few reports relating the researches of preparing hemostasis products compositing the chitosan extracted from silkworm with gelatin. Regarding the form of the hemostatic material, porous sponge-like structure is considered to be an important feature of a good hemostatic material. Can silkworm pupa chitosan and gelatin be composited to prepare a porous hemostatic material? Actually there are many influencing factors during the composition process. The key of this paper is how to accurately evaluate the impact of various factors on hemostatic effect and obtain the optimum preparation conditions for chitosan/gelatin composite hemostatic material. For this purpose, we conducted the research on the preparation condition of chitosan/gelatin composite hemostatic materials(CG) and got the following results:Composited the chitosan and gelatin, and supplemented a certain concentration of calcium chloride and glycerol with a certain concentration of crosslinking agent. Then we could make a porous spongy hemostatic material via freeze-drying. Glutaraldehyde and tannic acid were proved to have a very good cross-linking effect after the orthogonal experiment and single factor experiment. To test the performance of in vitro coagulation by in vitro coagulation index(BCI), we found out that the clotting effect of tannic acid crosslinked was the best and then got the optimum preparation condition of CG: chitosan mass fraction was 7%, gelatin mass fraction was 8.5%, calcium chloride mass fraction was 1.5%, glycerol mass fraction was 7%, tannic acid mass fraction was 0.05%. Under such condition, the in vitro coagulation index(BCI) of the prepared hemostatic material was 13.3± 0.2, which was a substantial improvement over medical gelatin, the hemostatic effect was increased 3 to 4 times higher than medical gelatin sponge and chitosan sponge respectively.(2) Study on the physical and chemical properties of chitosan/gelatin composite hemostatic material(CG)The physical and chemical properties and internal structure of the hemostatic material had a very important influence on the hemostatic properties. In order to more accurately understand the internal structure and physical and chemical properties of chitosan/gelatin composite hemostatic materials(CG), we conducted the morphology observation, mechanical properties test, chemical structure & characterization and other physical properties research, and got the following results:Chitosan/gelatin composite hemostatic material(CG) was a porous sponge-like structure, uniform aperture size, the porosity interpenetration between the internal porosity. The porosity is 94±1.3%, the aperture size is150~300μ m, and the density is 0.072659 g/cm3. Chitosan and gelatin to each other would form stronger hydrogen bonding force. The formation of three-dimensional interpenetrating network structure was more solid after the CG was crosslinked with tannic acid, and the CG had good mechanical properties: dry strength was 1.57±0.24 MPa, wet strength was 0.79±0.24 MPa, the mechanical properties were better than gelatin sponge(GEL) and chitosan sponge(CS).Chitosan/gelatin composite hemostatic material(CG) has good absorbing properties and hemostatic properties. It absorbed the proportion of different liquids respectively. It could absorb 25.3±1.0 times normal saline(NS), 23.3±1.5 times simulated body fluid(SPF), 14.5±0.7 times fluid absorption rate of whole blood(WB). The absorption amount of liquid was increased 1.8 times, 1.7 times and 1.8 times respectively compared with gelatin sponge(Gel).(3) Study on hemostatic performance of chitosan/gelatin composite hemostatic materialIt is a very important content for developing biomedical materials to establish a set of accurate rating hemostatic properties of biological materials. Hemostatic performance of chitosan/gelatin composite hemostatic material(CG) would be evaluated by coagulation in vitro performance testing and animal models in this paper. The coagulation test contained the whole blood clotting time in vitro and dynamic clotting time; animal hemorrhage was evaluated by ear arterial hemorrhage, femoral artery hemorrhage, liver bleeding model respectively. The following results were obtained.Chitosan/gelatin composite hemostatic material(CG) has a significant role in promoting blood coagulation. The whole blood clotting time in vitro was 138.2±17.3s, better than chitosan(CS) and gelatin(GEL) sponge sponge. Dynamic blood clotting test showed that the blood clot formed by CG and blood was stronger and better in coagulation performance. Chitosan/gelatin composite hemostatic material(CG) had good hemostatic effect on uncontrol bleeding which was caused by rabbit artery, femoral artery, hepatic hemorrhage model; the hemostasis time is 46.8±3.1s, 39.3±2.5s, 38.5±5.2s respectively. The hemostatic effect was better than that of control group pure chitosan(CS) and gelfoam sponge(GEL).(4) Study on hemostatic mechanism of chitosan/gelatin composite hemostatic materialHemostatic properties of biological materials usually act together in a variety of factors. The specific hemostatic mechanism of chitosan and gelatin is not yet a very clear conclusion. In order to better understand the concrete hemostatic principle of composite hemostatic material of chitosan/gelatin, the prothrombin time of CG(PT), activated partial thrombin time(APTT), platelet aggregation, erythrocyte aggregation, thrombin production, thromboxane B2 content and other indicators would be discussed respectively in this paper. The following conclusion was obtained:Chitosan/gelatin composite hemostatic material(CG) mainly improved blood coagulation by intrinsic coagulation pathway and intercommunity clotting pathway. Its primary hemostatic mechanism was that CG could accelerate the blood factor Ⅻ activation, and thereby induce the inducible factor Ⅺ Ⅸ, Ⅷ sequencially activated, and finally activate factor X into factor Xa; and in the presence of factor Ⅳ and PF3, then form prothrombin complex. In the presence of Ca2+, prothrombin complex further activated the blood prothrombin into thrombin. CG could accelerate platelet aggregation to its surface, make platelet activation and release the clotting factors. Meanwhile, its high water absorption capacity would quickly elevate plasma concentrations of thrombin and other coagulation factors, and promote the role in vitro rapid hemostatic under a variety of factors working together.(5) Study on biocompatibility of chitosan/gelatin composite hemostatic materialNew rapid hemostatic materials should not only meet the physical and chemical properties and biological mechanics performance when used, also have no cytotoxicity, without doing any harm to the body, and have good biological compatibility with body tissue, to ensure its safety in clinical application. In this paper, the biocompatibility test for chitosan/gelatin composite hemostatic material(CG) was carried out, including cytotoxicity test, primary skin irritation test, intradermal reaction test, heat test and acute systemic toxicity test, and the antibacterial properties were discussed in this paper. The following conclusions were obtained:Chitosan/gelatin composite hemostatic material(CG) had strong antibacterial properties and good biocompatibility. CG had good antibacterial properties foe against staphylococcus aureus and escherichia coli, and it has better bacteriostasis to staphylococcus aureus in e. coli. Determined by MTT method, the test result showed that the CG had no obvious cytotoxicity. Its internal porous structure was very beneficial for cell attachment, growth. L929 cells grew well within the CG and proliferated rapidly. CG had no stimulation, excluding pyrogens, and no acute systemic toxicity. In the tests of rabbit subcutaneous implants, muscle implants and liver implants, there were no obvious lumps, severe inflammation and other obvious immune response and complementarity-mutual exclusiveness at the site of the CG implants. Tissue cells could grow rapidly in the implanted CG, and gradually break down the material, which showed that CG had good biocompatibility and could be used as an implant material present in a biological organism for a long time.(6) Study on in vivo degradation performance of chitosan/gelatin composite hemostatic material(CG)A portion of hemostatic biomaterial in surgery process would inevitably be left in the wounds. Therefore, it is necessary to study the biocompatibility and degradability of hemostatic material. In this paper, there are two systems based on the in vivo and in vitro to study its degradation performance. The with Physiological saline, simulated body fluid(SPF) and lysozyme solution were used for degradation experiment in vitro; while for in vivo degradation, the material was implanted respectively to the rabbit back under the skin, muscles and in the liver. The materials were taken on a regular basis in oder to observe the degradation of implant materials. The following conclusion was obtained:The degradation situation of Chitosan/gelatin composite hemostatic material(CG) was different betwwen in vivo and in vitro. With the increase of time, CG can slow degradation in these three simulation degradation liquid, physiological saline, simulated body fluid(SPF) and lysozyme in solution, and the corresponding degradation rate within eight weeks reached 61.8±3.9%, 46.8±2.4% and 58.6±1.9%. CG could complete substantial degradation after 6 weeks when it was planted into rabbit subcutaneous, and the tissue cells of the implanted site grew well. Part of the CG could be degradated when it was implanted into rabbit muscles after 4 months, and the tissue cells of the implanted site grew well. Portion of the implanted material still could be observed after six months when it was implanted into rabbit liver, observated by tissue section HE dyeing, the materials begun degradation, the surrounding of the implanted material was full of new blood vesssels and the growth of the tissue cells was normal.(7) chitosan/gelatin composite hemostatic material(CG) can be considered to be a rapid and biodegradable hemostatic material with good effect. |
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