可吸收壳聚糖材料止血性能及其生物相容性研究
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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H&E.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。
Chitosan is partially deacetylated from chitin and it has many fascinating biological properties such as hemostatic, anti-bacterial, and wound healing ability and good biocompatibility. Chitosan has been used widely in the tissue engineering field such as vascular stents reapir and wound repair. Chitosan-based hemostatic material achieved a great successand its excellent procoagulant effect has been proved in both military and civilian situations. However, the existing chitosan-based hemostatic materials have got FDA approval of applications only for external use, and are not recommended as absorbable hemostatic for long-term implantation. Studies showed that existing chitosan-based hemostatic materials have disadvantages:(1) Because of their acidic characteristic, they caused acute inflammation and chronic inflammation after in vivo implantation and hindered the wound healing process;(2) Slow degradation of them resulted in tissue adhesions, cystic fibrosis and other side effects, and formed a serious scar tissue. These shortcomings of chitosan should be overcome before it can be used as an absorbable hemostatic.
(1) Chitosans with low molecular weight ranging from36537to529652were fabricated through oxydative degradation both in homogeneous and heterogeneous solution. And chitosans with low D.D (from39.6%to59.6%, acid base titration) were fabricated through deacetylation of chitin and acetylation of chitosan. Results of in vitro degradation by lysozyme showed that the chitosan with degree of deacetylation of40.07%was degraded fastest, even faster than the gelatin sponge.
(2) By mimicing papermaking method, porous chitosan films and sponges were fabricated form chitosan hydrogel suspension after heating or freeze-drying. Hydrogel were crosslinked by capillary force and the formation of hydrogen bonds between the chitosan hydrogel particles. The resultant chitosan porous films are regular in shape, flexible and compliable to tissue surface; chitosan sponges have a high porosity and surface roughness in micrometer which will contribute to hemostatsis.
(3) In vitro dynamic blood clotting test was carried out to study effect of o degree of deacetylation on procoagulant performance of chitosans. M0D3(DD=48.07%, acid base titration) showed maximum procoagulant effect on blood clotting among all chitosans. Besides, chitosan was found to activate the adhesion of platelet, red blood cells and white blood cell. Chitosan did not affect the blood parameters such as APTT and TT.
(4) Chitosans sponges with degree of deacetylation of39.6%,48.07%and93.6%(acid base titration) achieved hemostasis in a rat liver trauma model.All chitosan sponge resulted in less total blood loss than gelatin sponges,and chitosan with degree of deacetylation of48.07%chitosan showed the best hemostatic effect.
(5) Chitosan sponges with degree of deacetylation of39.6%,48.07%and93.6%and gelatin sponge were implanted in rat liver defects.1week,4weeks,8weeks and24weeks later, rats were sacrificed and implantation with surrounding liver tissues were collected and stained with H&E, Masson's trichrome staining and immunohistochemical staining (TGF-β1and IL-1β). The absorption rate, inflammatory response and wound healing effects of various materials were compared. The results showed that chitosan with D.D of39.6%were completely absorbed after eight weeks of implantation; it took more than eight weeks for gelatin sponge to be completely absorbed and after24weeks of implantation, gelatin sponges were completely absorbed; chitosans with D.D of48.07%and93.6%lasted for24weeks. Biopsy and IL-1βstaining results showed that chitosan with D.D of39.6%showed weakest inflammatory response. Chitosan (D.D=39.6%) resulted in low level of of TGF-β1, moderate collagen depositon, and scarless wound healing. Moreover, chitosan(D.D=39.6%) reduced the chance of the adhesion to other organizations.
(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H&E.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。
Chitosan is partially deacetylated from chitin and it has many fascinating biological properties such as hemostatic, anti-bacterial, and wound healing ability and good biocompatibility. Chitosan has been used widely in the tissue engineering field such as vascular stents reapir and wound repair. Chitosan-based hemostatic material achieved a great successand its excellent procoagulant effect has been proved in both military and civilian situations. However, the existing chitosan-based hemostatic materials have got FDA approval of applications only for external use, and are not recommended as absorbable hemostatic for long-term implantation. Studies showed that existing chitosan-based hemostatic materials have disadvantages:(1) Because of their acidic characteristic, they caused acute inflammation and chronic inflammation after in vivo implantation and hindered the wound healing process;(2) Slow degradation of them resulted in tissue adhesions, cystic fibrosis and other side effects, and formed a serious scar tissue. These shortcomings of chitosan should be overcome before it can be used as an absorbable hemostatic.
(1) Chitosans with low molecular weight ranging from36537to529652were fabricated through oxydative degradation both in homogeneous and heterogeneous solution. And chitosans with low D.D (from39.6%to59.6%, acid base titration) were fabricated through deacetylation of chitin and acetylation of chitosan. Results of in vitro degradation by lysozyme showed that the chitosan with degree of deacetylation of40.07%was degraded fastest, even faster than the gelatin sponge.
(2) By mimicing papermaking method, porous chitosan films and sponges were fabricated form chitosan hydrogel suspension after heating or freeze-drying. Hydrogel were crosslinked by capillary force and the formation of hydrogen bonds between the chitosan hydrogel particles. The resultant chitosan porous films are regular in shape, flexible and compliable to tissue surface; chitosan sponges have a high porosity and surface roughness in micrometer which will contribute to hemostatsis.
(3) In vitro dynamic blood clotting test was carried out to study effect of o degree of deacetylation on procoagulant performance of chitosans. M0D3(DD=48.07%, acid base titration) showed maximum procoagulant effect on blood clotting among all chitosans. Besides, chitosan was found to activate the adhesion of platelet, red blood cells and white blood cell. Chitosan did not affect the blood parameters such as APTT and TT.
(4) Chitosans sponges with degree of deacetylation of39.6%,48.07%and93.6%(acid base titration) achieved hemostasis in a rat liver trauma model.All chitosan sponge resulted in less total blood loss than gelatin sponges,and chitosan with degree of deacetylation of48.07%chitosan showed the best hemostatic effect.
(5) Chitosan sponges with degree of deacetylation of39.6%,48.07%and93.6%and gelatin sponge were implanted in rat liver defects.1week,4weeks,8weeks and24weeks later, rats were sacrificed and implantation with surrounding liver tissues were collected and stained with H&E, Masson's trichrome staining and immunohistochemical staining (TGF-β1and IL-1β). The absorption rate, inflammatory response and wound healing effects of various materials were compared. The results showed that chitosan with D.D of39.6%were completely absorbed after eight weeks of implantation; it took more than eight weeks for gelatin sponge to be completely absorbed and after24weeks of implantation, gelatin sponges were completely absorbed; chitosans with D.D of48.07%and93.6%lasted for24weeks. Biopsy and IL-1βstaining results showed that chitosan with D.D of39.6%showed weakest inflammatory response. Chitosan (D.D=39.6%) resulted in low level of of TGF-β1, moderate collagen depositon, and scarless wound healing. Moreover, chitosan(D.D=39.6%) reduced the chance of the adhesion to other organizations.
引文
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