浓缩血小板复合功能纱布的研制

发布时间：2018-04-28 20:39

本文选题：创面愈合 + 富血小板血浆　；参考：《南方医科大学》2017年硕士论文

【摘要】：近年来,国内外学者研究发现血小板除具有止血作用外,在创伤修复和再生领域也发挥着重要的作用。富血小板血浆(Platelet-rich Plasma,PRP)作为血小板的常规应用形式,因其富含高浓度的复合生长因子和细胞因子等生物活性分子,广泛用于组织工程和再生医学领域。但是PRP的主要形式是以液体存在,单独运用有一定局限性,通过将其负载在生物材料上,可以更好的应用在创面发挥止血愈创作用。羧甲基壳聚糖是壳聚糖经羧甲基化后的衍生物,因为其良好的水溶性,规避了壳聚糖用稀酸作为溶剂的弊端,此外,其分子中含有羟基和氨基,可与多种功能活性物质结合,充当其载体,从而使其得到广泛的应用和深入的研究。PRP是否能与其联合应用,国内尚未见报道。本研究将从以下几个方面研究负载浓缩血小板纱布的制备工艺并对其进行初步功能评价。本研究中,我们以临床上常用的可吸收性止血纱布为基础,筛选出合适的原料为壳聚糖类,出于溶解性和降解周期的考虑,我们不用壳聚糖而选择将羧甲基壳聚糖作为本课题制备功能性纱布的原材料,然后将其与富血小板血浆复合,设计不同梯度的浓度、温度及混合比例等条件,对该负载浓缩血小板的可吸收性纱布的制备条件进行探究。为证明其有效性,本研究进一步从促细胞增殖、动物急性创伤修复和外伤止血等几个层面对其功能进行评价。一.复合纱布制备材料的筛选以临床上常用的可吸收性止血纱布为基础,选取临床上常用的几种代表性的纱布。制备浓缩血小板,并对其进行冻干前的预处理。然后在各组纱布的表面滴加制备好的浓缩血小板,并经过预冻、冻干等程序。然后对冻干后的材料进行复溶,测量其pH值并加入相应的激活剂,观察凝胶的的形成情况。结果发现,各组复合冻干品质地均呈现硬而脆,且只有壳聚糖类的复合冻干品能够形成血小板凝胶,其余材料均呈现不同程度的酸性,未见凝胶形成。二.羧甲基壳聚糖/浓缩血小板复合冻干品制备条件的探究设计不同壳聚糖浓度、预冻温度和CC/PRP共混比例等条件,制备CC/PRP复合冻干品,并对其进行肉眼观和扫描电镜观察,抗压缩性能及所负载生长因子含量等进行测试,结果发现,2-3%的壳聚糖溶液浓度,预冻温度为-80℃,CC/PRP按1:1共混比例混合时,所制备出的复合冻干品具有较好的抗压缩性能,且负载的生长因子含量较高。三.CC/PRP可吸收复合材料对HUVEC促增殖功能的研究将不同CC、PRP共混比例制备的新型可吸收性复合血小板功能材料用基础培养基浸提48h后,用凝血酶-葡萄酸钙溶液激活,用其浸提上清作用于HUVEC,检测复合功能材料对HUVEC的促增值效果。研究发现,不同共混比例的复合功能材料均具有促进细胞增殖效果,且相同条件下,2%(wt/vol)浓度CC共混的复合材料,促增殖效果更好。四.CC/PRP可吸收复合材料促进小鼠皮肤全层缺损模型的研究前面的研究,我们已经发现复合止血纱布上已经成功负载了较高浓度的生长因子,并且具有促进细胞增殖的作用。为进一步证明其促进创面修复的能力,通过建立小鼠皮肤全层缺损模型,将48只小鼠随机分为4组(空白组、CC/PRP组、CC组和FDP组),观察其促创面修复效果,结果发现,实验早期,CC/PRP组、CC组、FDP组均具有较高的促进创面愈合率,均明显高于空白对照组。病理切片检查提示,制备的复合材料具有缩短创伤炎症期,加速伤口上皮化和创面愈合率、促进血管新生和胶原纤维的沉积与成熟等。由此可见,其具有明显的促进创面愈合的作用。五.CC/PRP可吸收复合材料对新西兰白兔耳动脉出血的止血作用研究32只新西兰白兔随机分为4组(空白组、CC/PRP组、CC组和FDP组),建立了新西兰白兔耳动脉出血模型,观察所制备的复合CC/PRP对外伤性动脉出血的止血作用,并与FDP、CC进行比较。结果表明,CC/PRP、CC、FDP均具有一定的止血作用,且与单独使用CC、FDP相比,两者联合制备的CC/PRP具有更显著的止血效果。综上所述,本研究制备的CC/PRP可吸收复合功能性纱布,成功负载了生物活性分子,具有促进止血和创伤愈合作用,有望作为一种新型复合功能敷料应用于临床。
[Abstract]:In recent years, scholars at home and abroad have found that platelets play an important role in the field of trauma repair and regeneration. Platelet-rich Plasma (PRP), as a conventional form of platelet, is widely used because of its high concentration of compound growth factor and cytokine, and other biological active molecules. It is in the field of tissue engineering and regenerative medicine. But the main form of PRP is the existence of liquid, which has some limitations. By loading it on biological materials, it can be better applied to the wound to play a hemostatic effect. Carboxymethyl chitosan is a derivative of chitosan after carboxymethylation, because of its good solubility in water. In addition, the chitosan with dilute acid as a solvent, in addition, its molecules contain hydroxyl and amino groups, can be combined with a variety of functional active substances and act as its carrier, so that it can be widely used and deeply study whether.PRP can be combined with its application, and it has not been reported in China. This study will study the small load concentration from the following aspects. In this study, we screened the suitable raw materials for chitosan on the basis of the commonly used absorbable hemostatic gauze. In consideration of solubility and degradation cycle, we chose carboxymethyl chitosan as a functional gauze to prepare carboxymethyl chitosan without the use of chitosan. The preparation conditions of the absorbable gauze of the concentrated platelets were investigated by combining with the platelet rich plasma, the concentration of different gradients, the temperature and the mixing ratio, etc., the preparation conditions of the absorbable gauze of the concentrated platelets were explored. Firstly, the selection of composite gauze preparation materials is based on the commonly used absorbable hemostat as a basis to select several typical representative gauze used clinically. To prepare concentrated platelets and pretreat them before freeze drying. After the procedure of prefreezing and freeze drying, the material was redissolved, the pH value was measured and the corresponding activator was added to observe the formation of the gel. The results showed that the quality of the freeze-dried compound was hard and crisp, and only the composite lyophilized freeze-dried products of chitosan could form the platelets gel, and the rest of the materials showed different degrees. Two. The preparation conditions of carboxymethyl chitosan / concentrated platelets were prepared by the preparation of different chitosan concentration, prefreezing temperature and CC/PRP blending ratio, and the preparation of CC/PRP composite freeze dried products, and its naked eye view and scanning electron microscope observation, anti compression performance and the load growth factor content and so on. The results showed that the concentration of chitosan solution in 2-3%, the prefreezing temperature of -80 C, and the mixture of CC/PRP in 1:1 blending ratio, the prepared freeze-dried products had better compression resistance, and the load growth factor content was higher. Three.CC/PRP absorbable composite materials for HUVEC proliferation promoting function will be different CC, PRP blending ratio system The new absorbable composite platelet functional material was extracted with 48h, activated by thrombin - calcium acid calcium solution, and used to extract the supernatant on HUVEC. The effect of compound functional material on HUVEC was detected. Under conditions, 2% (wt/vol) concentration of CC blend composites, the effect of promoting proliferation is better. Four.CC/PRP absorbable composites promote the study of the model of full layer skin defect in mice. We have found that the composite hemostatic gauze has successfully loaded the higher concentration of growth factor and has the effect of promoting cell proliferation. The 48 mice were randomly divided into 4 groups (blank group, CC/PRP group, CC group and FDP group) and the effect of wound healing was observed. The results showed that early experiment, group CC/PRP, CC group and FDP group had higher healing rate of wound healing, which were significantly higher than that of blank control. Pathological section examination suggested that the prepared composite could shorten the period of wound inflammation, accelerate wound epithelialization and wound healing, promote the deposition and maturation of angiogenesis and collagen fibers. This shows that it has a significant effect on promoting wound healing. Five.CC/PRP absorbable composite material for New Zealand white rabbit ear bleeding 32 New Zealand white rabbits were randomly divided into 4 groups (blank group, group CC/PRP, group CC and group FDP), and a New Zealand white rabbit ear artery hemorrhage model was established. The hemostatic effect of the compound CC/PRP was observed and compared with FDP and CC. The results showed that CC/PRP, CC and FDP all had a certain hemostatic effect, and with the single one. Compared with CC alone, the combination of FDP and CC/PRP has more significant hemostatic effect. To sum up, the CC/PRP can absorb the compound functional gauze, which has successfully loaded the bioactive molecules, and has the effect of promoting hemostasis and wound healing. It is expected to be used as a new type of compound functional dressing for clinical application.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.08

【参考文献】