可吸收壳聚糖材料止血性能及其生物相容性研究

发布时间：2018-08-08 10:35

【摘要】：壳聚糖是甲壳素部分脱乙酰的产物,具有止血、抗菌、促进伤口愈合和生物相容性好等优点,广泛应用于血管支架、创伤修复等组织工程领域。目前壳聚糖基止血材料取得巨大成功,其优秀的促凝血效果在军事和民用领域得到验证。然而,FDA批准的现有壳聚糖基止血材料的应用领域仅仅是外用,还不能作为可吸收止血材料长期体内植入。研究表明,现有壳聚糖基止血材料存在以下两个问题：(1)大都呈酸性,在动物体内植入后会导致急性炎症反应和慢性炎症,影响创伤的愈合；(2)降解较缓慢,由此产生组织粘连、纤维囊肿等副作用,并形成严重的疤痕组织。要使壳聚糖材料作为可吸收止血材料体内植入,必须解决这两个存在的缺陷。 (1)通过系统性研究壳聚糖分子量和脱乙酰度对壳聚糖降解速度的调控行为,采用均相氧化降解和非均相氧化降解法制备得到了重均分子量在36537-529652的系列小分子量的壳聚糖,采用甲壳素脱乙酰法和壳聚糖乙酰化法制备脱乙酰度在39.6%～59.6%的系列低脱乙酰度壳聚糖(酸碱滴定)。体外溶菌酶降解壳聚糖实验结果表明,脱乙酰度为39.6%的壳聚糖酶降解速度最快,比明胶海绵快。 (2)创新性的提出了仿手工抄纸工艺,从壳聚糖水凝胶悬浮液出发,利用壳聚糖水凝胶颗粒之间的毛细作用和形成的氢键作用,使用高温烘制或一次冷冻干燥成型,制备了壳聚糖多孔膜和多孔海绵材料。制得的壳聚糖多孔膜形状规整、柔顺性好并能够与创面良好贴敷；壳聚糖多孔海绵具有高孔隙率和高表面粗糙度,这种高孔隙率和高表面粗糙度有利于促进血液凝固。 (3)采用体外动态凝血法测定了不同脱乙酰度纯壳聚糖促凝血性能,其中M0D3(D.D=48.07%,红外)促凝效果最好。通过血小板吸附、红细胞吸附、APTT和TT测试等方法初步研究可壳聚糖的促凝血机理。壳聚糖主要通过对血小板及红细胞的吸附促进血液凝固； (4)将脱乙酰度分别为39.6%,48.07%和93.6%的新型壳聚糖海绵进行大鼠肝脏创伤止血实验,结果显示自制壳聚糖海绵在动物体内也有止血效力,所有壳聚糖海绵止血效果都比明胶海绵好,总出血量都小于明胶海绵组,其中脱乙酰度为48.3%的壳聚糖止血效果最好。 (5)将脱乙酰度分别为39.6%,48.07%和93.6%(酸碱滴定)的新型壳聚糖海绵和明胶海绵植入大鼠肝脏创伤部位,在1周、4周、8周和24周时分别取各组大鼠的肝脏组织标本,做HE.Masson's染色以及免疫组化染色(TGF-β1和IL-1p),检查各种材料的吸收、炎症、创伤愈合情况。结果表明,脱乙酰度39.6%的壳聚糖在第8周时完全被吸收；明胶海绵完全吸收的时间则大于8周,在24周时完全吸收；而脱乙酰度为48.07%和93.6%的壳聚糖在24周还未完全吸收。病理切片和IL-1β染色结果表明,脱乙酰度39.6%的壳聚糖体内炎症反应最弱；,脱乙酰度39.6%的壳聚糖TGF-β1分泌水平低,胶原生成量适中,不形成疤痕性愈合；,脱乙酰度39.6%的壳聚糖,减少其他组织的粘连的几率。
[Abstract]:Chitosan is a product of chitin partial deacetylation. It has many advantages, such as hemostasis, antibacterial, wound healing and good biocompatibility. It is widely used in the tissue engineering fields such as vascular scaffold and wound repair. At present, chitosan based hemostat has been a great success, and its excellent coagulation effect is verified in military and civil fields. However, FD The application of the existing chitosan based hemostat approved by A is only external and can not be implanted in the body for a long time as an absorbable hemostat. Research shows that the existing chitosan based hemostat has two problems: (1) most of them are acidic, and they can lead to acute inflammatory reaction and chronic inflammation after implantation in animals and affect the trauma. Healing; (2) slow degradation, resulting in tissue adhesions, fibrous cysts and other side effects, and forming severe scar tissue. To make the chitosan material as an absorbable hemostatic material to be implanted in the body, these two existing defects must be solved.
(1) by systematic study of the regulation of chitosan molecular weight and deacetylation degree on the degradation rate of chitosan, a series of small molecular weight chitosan with a heavy average molecular weight of 36537-529652 was prepared by means of homogeneous oxidation degradation and heterogeneous oxidation degradation. The deacetylation degree of chitosan was prepared by chitin deacetylation and chitosan acetylation. A series of low deacetylation degree chitosan (acid-base titration) from 39.6% to 59.6%. The results of in vitro lysozyme degradation of chitosan showed that the degradation rate of chitosan was 39.6% faster than that of gelatin sponge.
(2) an innovative handmade papermaking process was proposed. Starting from the chitosan hydrogel suspension, using the capillary action and the hydrogen bond between the chitosan hydrogel particles, the chitosan porous membrane and the porous sponge were prepared by high temperature drying or one freeze drying. The chitosan porous membrane was formed in shape and soft. The chitosan porous sponge has high porosity and high surface roughness. This high porosity and high surface roughness can help to promote blood coagulation.
(3) the procoagulant properties of pure chitosan with different deacetylation degree were measured by dynamic coagulation in vitro, in which the effect of M0D3 (D.D=48.07%, IR) was the best. The coagulation mechanism of chitosan was preliminarily studied by means of platelet adsorption, red cell adsorption, APTT and TT test. Chitosan was mainly used to promote the adsorption of platelets and red blood cells. Blood coagulation;
(4) a new chitosan sponge with a degree of deacetylation of 39.6%, 48.07% and 93.6% was carried out in the rat liver trauma test. The results showed that the self-made chitosan sponge had hemostatic effect in the animal body. All chitosan sponges had better hemostatic effect than gelatin sponge. The total amount of bleeding was less than that of Gelfoam group, and the degree of deacetylation of the chitosan sponge was 48.3%. The hemostatic effect of chitosan is the best.
(5) the deacetylation degree was 39.6%, 48.07% and 93.6% (acid-base titration), the new chitosan sponge and gelatin sponge were implanted in the rat liver trauma site. The liver tissue specimens of rats were collected at 1 weeks, 4 weeks, 8 weeks and 24 weeks respectively. HE.Masson's staining and immunohistochemical staining (TGF- beta 1 and IL-1p) were used to examine the absorption and inflammation of various materials. The results showed that the chitosan was completely absorbed at eighth weeks when the degree of deacetylation was 39.6%; the total absorption time of the gelatin sponge was more than 8 weeks and completely absorbed at 24 weeks. The chitosan with deacetylation of 48.07% and 93.6% was not completely absorbed at 24 weeks. Pathological section and IL-1 beta staining showed that the degree of deacetylation was 39.6% of the shell. The inflammatory reaction was the weakest in the body, and the degree of deacetylation of chitosan TGF- beta 1 was low, the amount of collagen production was moderate, and no scar healing was formed; and the degree of deacetylation of chitosan was 39.6%, reducing the risk of adhesion of other tissues.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：O636.1;R318.08

【共引文献】