聚乳酸—己内酯乳液静电纺组织工程支架的制备及性能评价

发布时间：2018-06-13 00:02

本文选题：乳液静电纺丝 + 聚乳酸-己内酯　；参考：《东华大学》2014年博士论文

【摘要】：静电纺丝技术利用静电场力来形成超细纤维,是目前制备纳米纤维最为便捷的技术之一。纳米纤维比表面积大,可以形成多孔的纤维支架,并且可以由多种具有良好生物相容性的高聚物制得,这使得纳米纤维在很多方面有得天独厚的应用。在作为组织工程支架方面,静电纺超细纤维也显示出其优势。在形态上,静电纺纤维支架可以极大程度地模拟细胞外间质,为细胞的粘附、增殖及分化提供了必要条件；在功能上,静电纺纤维支架可以作为药物储存和释放的载体。但是,常规的静电纺丝方法在作为药物载体方面具有其局限性。大多数的静电纺丝方法采用将药物和纺丝溶液混溶的方法将药物包覆在超细纤维内,这将导致两种不良后果：(1)如果药物与纺丝溶液的亲疏水性能相反(例如,亲水的蛋白药物与疏水的聚乳酸高聚物溶液)的话,就会形成不均匀的药物溶液,导致药物在纤维内的不均匀分布,不利于药物释放的控制；(2)对于一些具有生物活性的药物(例如生长因子),简单地将其与高聚物溶液混合很容易导致药物失活或者聚集,这样的药物释放至患处,不仅发挥不到积极的治疗作用,而且还有可能被身体识别为异体而发生免疫排斥反应,对患者非常有害。针对上述问题,课题采取了乳液静电纺方法来制备组织工程支架,研究工作分为三个部分： (1)乳液静电纺丝系统的建立将模型蛋白药物牛血清白蛋白(BSA)溶解于水溶液中,有机高聚物聚乳酸-己内酯(PLCL)溶解于有机溶剂氯仿中,将两相溶液制备成为均匀的乳液作为纺丝液。通过共聚焦显微镜(LSCM)对包覆有荧光物质纳米纤维的观察,证实了所制得的纤维为皮芯结构。通过测定纤维支架的水接触角来评定其亲水性能,常规静电纺PLCL纤维与乳液静电纺PLCL BSA纤维的水接触角分别为135.10。和89.30。,这表明乳液静电纺丝纤维的亲水性能得到了明显的提高,这对后期细胞的吸附以及细胞行为都是有利的。药物BSA的体外释放结果表明,在长达28天的释放实验中,只有47.71%的BSA被释放出来,该结果表明,所制备的乳液静电纺载药纤维能有效地抑制药物的突释现象,达到了长期释放的效果。细胞增殖实验表明,在7、14和21天,与PLCL纤维支架相比,PLCL_BSA上的细胞增殖分别高出41.8%、85.0%和49.7%。实验结果证实了乳液静电纺纤维具有较好的生物相容性,与常规静电纺方法所制备的纤维支架相比更加有利于支持细胞的贴附和增殖。 (2)担载单药物生长因子的乳液静电纺纳米纤维支架的制备及性能表征利用血管内皮细胞生长因子(Vascular Endothelial Growth Factor,VEGF)取代第一部分工作中的模型蛋白药物牛血清白蛋白(BSA),并进行纺丝实验,对所制备的纤维支架中纤维的皮芯结构、亲水性能、拉仲性能等进行表征,并且通过细胞增殖MTS实验验证包覆有VEGF的静电纺丝支架的生物相容性。VEGF的释放试验分为两组,一组为利用葡聚糖(Dextran)水溶液作为VEGF的保护剂,记作PLCL-VEGF-DEX;另一组利用BSA水溶液作为保护剂,记作PLCL-VEGF-BSA。两组均收到了良好的释放效果,极大地抑制了初始突释现象。但是保护剂葡聚糖和牛血清白蛋白的选用会明显地影响初始24小时内药物的释放,其释放量分别为1.0%和9.6%；在之后的640小时,二者间的区别不明显,分别为11.6%和11.7%。细胞增值实验MTS结果说明,同纯PLCL支架(常规静电纺纤维支架)相比,含有VEGF的支架极大促进了增殖效果,在细胞培养第10天和第20天时,相比于纯PLCL纤维支架,PLCL-VEGF-BSA纤维支架上的细胞增殖分别高出32.3%和49.9%；PLCL-VEGF-DEX纤维支架上细胞增殖分别高出14.6%和39.8%。实验结果同时也表明,经过静电纺溶液配制和纺丝加工后,VEGF依然保持有明显的活性。为了进一步提高纤维支架的生物相容性,提高对VEGF的保护及担载效果,在皮芯结构的纤维的芯层采用了天然高聚物明胶(Gelatin)来担载VEGF,记作PLCL/GV。为了考察该纤维支架在支持间充质干细胞向心肌细胞方向分化上所起的作用,通过5-氮胞苷(5-AZA)处理的方法在此纤维支架上进行了诱导人间充质干细胞向心肌细胞方向分化的实验。在第10天、第15天与第20天时,PLCL/GV纤维上的细胞增殖情况与纯PLCL支架相比,分别高出35.5%,61.1%和73.4%。从细胞增殖情况来看,5-氮胞苷处理对纤维支架上细胞的增殖情况没有显著影响。实验结果说明：由于VEGF的抗细胞凋零作用,PLCL/GV纤维支架可以有效减少由于5-氮胞苷带来的细胞凋零,实验结果同时也验证了担载在纤维芯层的VEGF具有良好的生物活性。纤维支架上的细胞形态通过5-氯荧光双乙酸钠染色(CMFDA)实验来评价。可以看出,细胞尺寸变大,并且呈现出多边形形态,并且与临近的细胞互相接触。生长在PLCL/GV支架上的细胞与生长在纯PLCL支架上的细胞从数量到形态都有明显区别。PLCL/GV纳米纤维上的细胞更多地呈现出心肌细胞表型,这些细胞尺寸变大,形状有所改变,彼此之间接触并且形成了网络。未分化的间充质干细胞形态细长,彼此平行,类似于成纤维细胞形状。肌动蛋白和重链肌球蛋白两次染色实验均说明PLCL/GV上的细胞表达出了更多的心脏特异性蛋白。 (3)担载双因子的乳液静电纺纳米纤维的制备及性能表征利用乳液静电纺纤维的皮芯结构特性,制备了皮层担载有羟基磷灰石(HA)、芯层担载有层黏连蛋白(Laminin)的乳液静电纺纤维支架,记作PLCL/HA/Lam,并且在纤维支架上进行了成骨细胞的培养。MTS细胞增殖实验显示,从第7天开始,静电纺纤维支架上细胞的增殖情况优于组织培养板(TCP)。从第7天到第21天,PLCL/HA/Lam纤维支架上的细胞增殖情况优于其它支架。譬如21天时,与TCP、PLCL/HA(皮层担载羟基磷灰石的单药物纤维支架)和PLCL/Lam(芯层担载层黏连蛋白的单药物支架)相比,分别高出23.3%、12.0%和10.4%。在第14天和第21天,PLCL/HA/Lam纤维支架上的碱性磷酸酶活性显著高于PLCL/HA和PLCL/Lam纤维支架(P≤0.05),这是由于羟基磷灰石和层粘连蛋白的协同作用效果。EDX给出的数据在TCP,PLCL/HA,PLCL/Lam和PLCL/HA/Lam上的钙含量分别为0%,0.05%,0.09%和0.51%,这与茜素红染色(Alizarin Red S)结果一致。通过细胞的增殖实验,骨蛋白表达,碱性磷酸酶活性以及矿化情况,充分说明了担载有双药物的纳米纤维极大的促进了成骨细胞的增殖、成熟等细胞行为。
[Abstract]:Electrospun technology is one of the most convenient technologies for preparing nanofibers by using electrostatic field force. It is one of the most convenient technologies to prepare nanofibers. The nanofibers can form porous fiber scaffolds with a large surface area, and can be made of a variety of polymers with good biocompatibility. This makes the nanofibers unique in many ways. ESD superfine fiber also shows its advantages in the aspect of tissue engineering scaffolding. In form, ESD fiber scaffolds can greatly simulate extracellular matrix, and provide necessary conditions for cell adhesion, proliferation and differentiation; in function, ESD fiber scaffold can be used as a carrier for drug storage and release. The conventional electrospinning method has its limitations as a drug carrier. Most of the electrospun methods use the method of mixing the drug and spinning solution into the superfine fiber, which will lead to two adverse consequences: (1) if the drug is opposite to the hydrophobic property of the spinning solution (for example, hydrophilic protein drugs) With the hydrophobic polylactic acid polymer solution), the inhomogeneous drug solution will be formed, which leads to the uneven distribution of the drug in the fiber and is not conducive to the control of the drug release; (2) for some bioactive drugs (such as growth factors), it is easy to mix it with the polymer solution and easily lead to drug inactivation or aggregation. The release of such drugs to the affected area not only does not play an active therapeutic role, but it may also be recognized by the body as a variant of the immune rejection, which is very harmful to the patient.
In view of the above problems, the emulsion electrospinning method is used to prepare tissue engineering scaffolds. The research work is divided into three parts.
(1) establishment of emulsion electrostatic spinning system
The model protein drug, bovine serum albumin (BSA), was dissolved in aqueous solution, and organic polymer Polylactic Acid Hexyl Ester (PLCL) was dissolved in the organic solvent chloroform. The two phase solution was prepared into a homogeneous emulsion as a spinning solution. The fiber coated with fluorescent material was observed by confocal microscopy (LSCM), and the obtained fibers were confirmed. By measuring the water contact angle of the fiber support, the hydrophilic properties of the PLCL fiber and the emulsion electrospun PLCL BSA fiber are 135.10. and 89.30. respectively, which indicates that the hydrophilic properties of the electrospun fiber are obviously improved, which is the adsorption of the later cells and the cell behavior. The results of the drug BSA release in vitro showed that only 47.71% of BSA was released in the 28 day release experiment. The results showed that the prepared emulsion electrostatically spun fiber could effectively inhibit the release of the drug, and reached the effect of long-term release. The cell proliferation experiment showed that in 7,14 and 21 days, with PLCL fiber Compared with the scaffolds, the cell proliferation on PLCL_BSA was higher than 41.8%, 85% and 49.7%. showed that the emulsion electrostun fiber had better biocompatibility and was more conducive to supporting cell attachment and proliferation compared with the fiber scaffolds prepared by conventional electrospun methods.
(2) preparation and characterization of emulsion electrospun nanofiber scaffolds supported by single drug growth factor
Vascular Endothelial Growth Factor (VEGF) was used to replace the model protein drug bovine serum albumin (BSA) in the first part of the work, and the spinning experiment was carried out to characterize the skin core structure, hydrophilicity, and secondary properties of the fibers in the prepared fibrous scaffold, and the MTS experiment of cell proliferation was carried out by cell proliferation. The test of biocompatibility.VEGF of the electrospun scaffold coated with VEGF is divided into two groups, one is to use the aqueous solution of dextran (Dextran) as a protective agent for VEGF, to be recorded as PLCL-VEGF-DEX, and the other group of BSA water solution as a protectant, which is recorded as a PLCL-VEGF-BSA. two group and has received a good release effect, greatly inhibited. Initial release phenomenon. However, the selection of protective agent glucan and bovine serum albumin could significantly affect the release of drugs within the initial 24 hours, and the release amount was 1% and 9.6%, respectively, and the difference between the two groups was not obvious at the end of the 640 hours, and the 11.6% and 11.7%. cells added value, respectively, with the pure PLCL stent (conventional electrostenting). Fibrous stents compared with VEGF scaffolds that greatly enhanced the proliferation effect. Cell proliferation on PLCL-VEGF-BSA fiber scaffolds was 32.3% and 49.9% higher than that on pure PLCL scaffolds at tenth and twentieth days in cell culture, and 14.6% and 39.8%. results on PLCL-VEGF-DEX fiber scaffolds, respectively, showed that After spinning and spinning, the VEGF still has obvious activity.
In order to further improve the biocompatibility of the fibrous scaffold and to improve the protection and loading effect of VEGF, the core layer of the core structure is loaded with natural polymer gelatin (Gelatin) to load VEGF. It is recorded as PLCL/GV. to investigate the role of the fiber scaffold in supporting the differentiation of mesenchymal stem cells into the direction of cardiac myocyte. Through 5- 5-AZA treatment was used on this scaffold to induce human mesenchymal stem cells to differentiate into cardiomyocytes. On the tenth day, fifteenth days and twentieth days, the proliferation of PLCL/GV fibers was 35.5%, 61.1%, and 73.4%., respectively, compared with the pure PLCL scaffold. From the cell proliferation, 5- nitrocytidine treatment There was no significant effect on cell proliferation on the fiber scaffold. The experimental results showed that the PLCL/GV fiber scaffold could effectively reduce the cell withering caused by 5- azytidine due to the anti cell withering effect of VEGF. The experimental results also verified the good biological activity of the VEGF supported on the fiber core. The cell morphology is evaluated by the 5- chlorine fluorescent diacetate staining (CMFDA) experiment. It can be seen that the cell size becomes larger and presents polygonal shape and contact with adjacent cells. Cells growing on the PLCL/GV scaffold and the cells growing on the pure PLCL scaffold are distincently different from the.PLCL/GV nanofibers from the number to the form. The cells showed more phenotype of the cardiomyocyte, the size of the cells, the shape change, the contact between each other and the formation of the network. The undifferentiated mesenchymal stem cells were elongated, parallel to each other, similar to the shape of fibroblasts. Actin and heavy chain muscle globulin two staining experiments showed the cell surface on PLCL/GV More heart specific proteins are available.
(3) preparation and characterization of double factor emulsion electrospun nanofibers
Using the skin core structure characteristics of the emulsion electrostatic spun fiber, the emulsion electrostatic spun scaffolds containing hydroxyapatite (HA) loaded with hydroxyapatite (HA) and core layer loaded with laminin (Laminin) were prepared and recorded as PLCL/HA/Lam. The proliferation test of.MTS cells in the culture of osteoblasts on the fiber scaffold showed that the electrostatic spun fiber branch started from seventh days. The proliferation of the cells on the shelf was superior to that of the tissue culture plate (TCP). From seventh to twenty-first days, the proliferation of cells on the PLCL/HA/Lam fiber scaffold was better than that of other scaffolds. For example, compared with TCP, PLCL/HA (the single drug scaffold loaded with hydroxyapatite) and PLCL/Lam (single drug scaffold for the core layer of laminin), respectively. The alkaline phosphatase activity on the PLCL/HA/Lam fiber scaffold was significantly higher than that of PLCL/HA and PLCL/Lam scaffolds on fourteenth and twenty-first days after 23.3%, 12% and 10.4%. (P < 0.05). This was due to the synergistic effect of hydroxyapatite and laminin. The.EDX data were 0%, 0, on TCP, PLCL/HA, PLCL/Lam and PLCL/HA/Lam respectively. .05%, 0.09% and 0.51%, which coincide with the results of alizarin red staining (Alizarin Red S). Through cell proliferation experiments, bone protein expression, alkaline phosphatase activity and mineralization, it is suggested that the nanofibers carrying double drugs have greatly promoted the proliferation and maturation of osteoblasts.
【学位授予单位】：东华大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R318.08;TQ340.64

【共引文献】