天然高分子基多功能止血复合敷料的制备及其性能研究

发布时间：2018-02-11 15:00

本文关键词： 氧化再生纤维素止血胶原蛋白静电纺丝聚己内酯细胞分化　出处：《哈尔滨工业大学》2016年博士论文　论文类型：学位论文

【摘要】：近年来,天然高分子止血材料的研制逐渐成为热点,本文利用天然高分子,如纤维素、胶原蛋白等为原材料制备出一系列具有良好止血效果的复合型止血材料。其中,氧化再生纤维素膜材料具有新颖的物理形态,对该材料的止血机制研究具有重要意义;胶原蛋白/氧化再生纤维素复合止血海绵具有优异的止血效果,同时具有良好的抑菌性能;而聚己内酯/胶原蛋白止血修复膜的制备则开启了止血材料的新思路,使得快速止血、长效抑菌、组织修复等多重功能有机的结合在一起。氧化再生纤维是一种常见的止血材料,然而由于其物理形态的限制,关于其作用机制的研究较少,尚不完全明确。本文利用离子液体(1-烯丙基-3-甲基咪唑高氯酸盐)作为溶剂,在NO2/CCl4体系下制备了不同氧化时间的氧化再生纤维素膜,此种形态非常利于细胞粘附研究。FT-IR和NMR谱图显示此体系在C6位上发生了高选择性氧化反应,随着氧化时间延长,羧基含量有一定程度增加。从接触角分析中明显看到,氧化后的再生纤维素膜表面润湿性明显提高,利于血液铺展。在SEM照片中,可以得出氧化40 h的样品粘附并激活的血小板数量最多,粘附的红细胞数目也较多。本文推测了两种可能的止血机制:一是氧化再生纤维素可吸收血液中液态物质,使血液粘稠,同时表面羧基可激活血小板,进而粘附红细胞、纤维蛋白,形成血栓;二是表面负电荷与血液中的Fe2+发生了络合反应,形成了棕色凝胶从而迅速封闭受伤血管,并保护其不受病毒和细菌的入侵。胶原蛋白是自然界中另一种生物相容性极好的材料,被广泛用于伤口愈合和组织修复。然而,由于胶原蛋白抑菌性能和生物有效性有限,限制了其作为口腔止血和填塞材料的应用。本文所制备的超孔胶原蛋白/氧化再生纤维素复合物(Col/ORC)具有特殊的微观结构及优异的止血、抑菌性能。SEM照片中发现,束状的氧化再生纤维素(ORC)纤维均匀地分布于胶原蛋白(Col)网状结构中。Col/ORC复合物的FT-IR图完全不同于ORC,但是与Col极为相似。物理性能测试结果表明Col-0.25%ORC的润湿性、孔隙率和吸水性最佳。MTT实验表明Col和Col/ORC对活细胞无毒性,在两种体内损伤模型中对止血时间和出血量的评价显示,Col-0.25%ORC生物有效性最佳,植入体内后,3 w内可降解完全,组织炎症反应消失。另外,通过ELISA和理化性能结果分析首次推测了该材料的止血机制:1)具有超孔结构的胶原蛋白海绵可以吸收大量血液,加速血液聚集;2)羧基可聚集并激活血小板;3)内源性凝血途径被激活。Col/ORC复合海绵的止血机制是胶原蛋白和氧化再生纤维素粉末物理吸附与生理止血共同的作用。在术后皮肤创伤恢复过程中,生长因子TGFβ1对于促进肌纤维细胞分化为具有可收缩活性的成肌纤维细胞,发挥了至关重要的作用。本文通过控制纯化I型胶原蛋白(Col I)与聚己内酯(PCL)纺丝原液配比及伤口敷料中的TGFβ1使用量,采用模拟细胞外基质传递过程研究最为合适的参数配比。根据细胞CCK-8活性试验、依托RT-PCR和荧光免疫染色的检测方法,在宏观层面、基因表达层面和蛋白质表达层面加以研究,从而取得可以使得TGFβ1从静电纺丝纳米纤维止血修复膜中自主、持久地释放的关键参数,并优化使得该止血修复膜具有最优的细胞相容性和最适宜的细胞分化速率。同时,本文还研究了添加噬菌体后的止血修复膜的微观形貌及抑菌性能,结果显示,PCL-Col I B止血修复膜(40%/60%,w/w)具有最好的刺激细胞分化功能、较好的止血效果、优异的抑菌性能及适宜的体内降解速率。
[Abstract]:In recent years, the development of natural macromolecule hemostatic materials has become a hot spot, using natural polymers, such as cellulose, collagen and other raw materials for the preparation of a series of good hemostatic effect of compound hemostatic materials. The oxidized regenerated cellulose membrane materials with novel physical form, is of great significance to study the hemostatic mechanism of the material collagen; / oxidized regenerated cellulose composite has excellent hemostatic sponge hemostatic effect, and has good antibacterial properties; and polycaprolactone / collagen hemostatic membrane repair preparation is opening a new idea of hemostatic materials, making rapid hemostasis, long-term antibacterial function, multiple tissue repair combined with organic oxidation regeneration. Fiber is a kind of common hemostatic materials, but because of its physical form, research on the mechanism of the less, this is not entirely clear. Using ionic liquid paper (1- allyl -3- methyl imidazole perchlorate) as solvent in NO2/CCl4 system was prepared under different oxidation time of regenerated cellulose membrane, the shape is very conducive to the study of cell adhesion.FT-IR and NMR spectra showed that this system had high selective oxidation reaction in the C6 site, with the oxidation time, carboxyl the content has increased to a certain extent. From the contact angle analysis showed that the regenerated cellulose film surface wettability after oxidation was improved to blood spreading in SEM. In the photo, the number of platelet adhesion and oxidation of 40 h samples can be drawn and activate the most number of red blood cell adhesion is greater. This paper suggested two possible the hemostatic mechanism: one is the oxidized regenerated cellulose can absorb liquid substances in the blood, the blood viscosity, and the surface carboxyl groups can activate platelets, red blood cells and adhesion, fibrin, The formation of thrombus; two is the negatively charged surface complexation reaction with Fe2+ in blood, forming a brown gel which quickly closed the injured vessel, and to protect against viruses and bacteria invasion. Collagen is another excellent biocompatible materials in nature, is widely used in wound healing and tissue repair. However, the antibacterial properties and bioavailability of collagen Co., limited its application as oral hemostatic and packing materials. The preparation of compound collagen / oxidized regenerated cellulose super hole preparation (Col/ORC) with micro structure and excellent hemostatic special antibacterial properties, found.SEM photo, oxidized regenerated cellulose bundle (ORC) fiber evenly distributed in collagen (Col).Col/ORC complexes in the network structure of FT-IR is different from ORC and Col, but are very similar. Test results show that the physical properties of Col-0 The wettability of.25%ORC, porosity and water absorption of the best.MTT experiments showed that Col and Col/ORC in living cells is non-toxic, display on the bleeding time and bleeding volume in the evaluation of two kinds of internal damage model, the bioavailability of Col-0.25%ORC best, after implantation can be completely degraded within 3 W, disappearance of inflammation. In addition, through ELISA and physicochemical properties analysis results for the first time that the hemostatic mechanism of the material: 1) with collagen sponge pore structure can absorb large amounts of blood, accelerate blood aggregation; 2) can activate platelet aggregation and carboxyl group; 3) endogenous coagulation pathway is activated the hemostatic mechanism of.Col/ORC composite sponge is the role of collagen and oxidized regenerated cellulose powder physical adsorption and hemostasis. Common in skin trauma and postoperative recovery process, growth factor beta 1 TGF to promote the differentiation of muscle cells with contractile activity into The muscle cells, play a crucial role in this paper. By controlling the purification of type I collagen (Col I) and polycaprolactone (PCL) spinning dope ratio and wound dressing in beta 1 TGF usage, the simulation of extracellular matrix transfer process is the most suitable parameter ratio. According to the CCK-8 cell activity test, detection methods based on the RT-PCR and immunofluorescence staining, at the macro level, gene expression level and protein expression level to study, so as to achieve to make TGF beta 1 from electrospun fibrous membrane in hemostasis and repair are key parameters, sustained release, and the optimization of hemostasis and repair membrane has the optimal cell compatibility and the most the suitable cell differentiation rate. At the same time, this paper also studies the microstructure and antibacterial properties, add hemostasis and repair after the phage membrane showed that PCL-Col I B (40%/60%, w/w, hemostasis and repair membrane) out It has the best function of stimulating cell differentiation, better hemostatic effect, excellent bacteriostasis and suitable degradation rate in the body.

【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08;TQ317

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