本文選題：乳液靜電紡絲 + 聚乳酸-己內(nèi)酯　； 參考：《東華大學(xué)》2014年博士論文

【摘要】：靜電紡絲技術(shù)利用靜電場力來形成超細纖維,是目前制備納米纖維最為便捷的技術(shù)之一。納米纖維比表面積大,可以形成多孔的纖維支架,并且可以由多種具有良好生物相容性的高聚物制得,這使得納米纖維在很多方面有得天獨厚的應(yīng)用。在作為組織工程支架方面,靜電紡超細纖維也顯示出其優(yōu)勢。在形態(tài)上,靜電紡纖維支架可以極大程度地模擬細胞外間質(zhì),為細胞的粘附、增殖及分化提供了必要條件；在功能上,靜電紡纖維支架可以作為藥物儲存和釋放的載體。但是,常規(guī)的靜電紡絲方法在作為藥物載體方面具有其局限性。大多數(shù)的靜電紡絲方法采用將藥物和紡絲溶液混溶的方法將藥物包覆在超細纖維內(nèi),這將導(dǎo)致兩種不良后果：(1)如果藥物與紡絲溶液的親疏水性能相反(例如,親水的蛋白藥物與疏水的聚乳酸高聚物溶液)的話,就會形成不均勻的藥物溶液,導(dǎo)致藥物在纖維內(nèi)的不均勻分布,不利于藥物釋放的控制；(2)對于一些具有生物活性的藥物(例如生長因子),簡單地將其與高聚物溶液混合很容易導(dǎo)致藥物失活或者聚集,這樣的藥物釋放至患處,不僅發(fā)揮不到積極的治療作用,而且還有可能被身體識別為異體而發(fā)生免疫排斥反應(yīng),對患者非常有害。 針對上述問題,課題采取了乳液靜電紡方法來制備組織工程支架,研究工作分為三個部分： (1)乳液靜電紡絲系統(tǒng)的建立 將模型蛋白藥物牛血清白蛋白(BSA)溶解于水溶液中,有機高聚物聚乳酸-己內(nèi)酯(PLCL)溶解于有機溶劑氯仿中,將兩相溶液制備成為均勻的乳液作為紡絲液。通過共聚焦顯微鏡(LSCM)對包覆有熒光物質(zhì)納米纖維的觀察,證實了所制得的纖維為皮芯結(jié)構(gòu)。通過測定纖維支架的水接觸角來評定其親水性能,常規(guī)靜電紡PLCL纖維與乳液靜電紡PLCL BSA纖維的水接觸角分別為135.10。和89.30。,這表明乳液靜電紡絲纖維的親水性能得到了明顯的提高,這對后期細胞的吸附以及細胞行為都是有利的。藥物BSA的體外釋放結(jié)果表明,在長達28天的釋放實驗中,只有47.71%的BSA被釋放出來,該結(jié)果表明,所制備的乳液靜電紡載藥纖維能有效地抑制藥物的突釋現(xiàn)象,達到了長期釋放的效果。細胞增殖實驗表明,在7、14和21天,與PLCL纖維支架相比,PLCL_BSA上的細胞增殖分別高出41.8%、85.0%和49.7%。實驗結(jié)果證實了乳液靜電紡纖維具有較好的生物相容性,與常規(guī)靜電紡方法所制備的纖維支架相比更加有利于支持細胞的貼附和增殖。 (2)擔載單藥物生長因子的乳液靜電紡納米纖維支架的制備及性能表征 利用血管內(nèi)皮細胞生長因子(Vascular Endothelial Growth Factor,VEGF)取代第一部分工作中的模型蛋白藥物牛血清白蛋白(BSA),并進行紡絲實驗,對所制備的纖維支架中纖維的皮芯結(jié)構(gòu)、親水性能、拉仲性能等進行表征,并且通過細胞增殖MTS實驗驗證包覆有VEGF的靜電紡絲支架的生物相容性。VEGF的釋放試驗分為兩組,一組為利用葡聚糖(Dextran)水溶液作為VEGF的保護劑,記作PLCL-VEGF-DEX;另一組利用BSA水溶液作為保護劑,記作PLCL-VEGF-BSA。兩組均收到了良好的釋放效果,極大地抑制了初始突釋現(xiàn)象。但是保護劑葡聚糖和牛血清白蛋白的選用會明顯地影響初始24小時內(nèi)藥物的釋放,其釋放量分別為1.0%和9.6%；在之后的640小時,二者間的區(qū)別不明顯,分別為11.6%和11.7%。細胞增值實驗MTS結(jié)果說明,同純PLCL支架(常規(guī)靜電紡纖維支架)相比,含有VEGF的支架極大促進了增殖效果,在細胞培養(yǎng)第10天和第20天時,相比于純PLCL纖維支架,PLCL-VEGF-BSA纖維支架上的細胞增殖分別高出32.3%和49.9%；PLCL-VEGF-DEX纖維支架上細胞增殖分別高出14.6%和39.8%。實驗結(jié)果同時也表明,經(jīng)過靜電紡溶液配制和紡絲加工后,VEGF依然保持有明顯的活性。 為了進一步提高纖維支架的生物相容性,提高對VEGF的保護及擔載效果,在皮芯結(jié)構(gòu)的纖維的芯層采用了天然高聚物明膠(Gelatin)來擔載VEGF,記作PLCL/GV。為了考察該纖維支架在支持間充質(zhì)干細胞向心肌細胞方向分化上所起的作用,通過5-氮胞苷(5-AZA)處理的方法在此纖維支架上進行了誘導(dǎo)人間充質(zhì)干細胞向心肌細胞方向分化的實驗。在第10天、第15天與第20天時,PLCL/GV纖維上的細胞增殖情況與純PLCL支架相比,分別高出35.5%,61.1%和73.4%。從細胞增殖情況來看,5-氮胞苷處理對纖維支架上細胞的增殖情況沒有顯著影響。實驗結(jié)果說明：由于VEGF的抗細胞凋零作用,PLCL/GV纖維支架可以有效減少由于5-氮胞苷帶來的細胞凋零,實驗結(jié)果同時也驗證了擔載在纖維芯層的VEGF具有良好的生物活性。纖維支架上的細胞形態(tài)通過5-氯熒光雙乙酸鈉染色(CMFDA)實驗來評價。可以看出,細胞尺寸變大,并且呈現(xiàn)出多邊形形態(tài),并且與臨近的細胞互相接觸。生長在PLCL/GV支架上的細胞與生長在純PLCL支架上的細胞從數(shù)量到形態(tài)都有明顯區(qū)別。PLCL/GV納米纖維上的細胞更多地呈現(xiàn)出心肌細胞表型,這些細胞尺寸變大,形狀有所改變,彼此之間接觸并且形成了網(wǎng)絡(luò)。未分化的間充質(zhì)干細胞形態(tài)細長,彼此平行,類似于成纖維細胞形狀。肌動蛋白和重鏈肌球蛋白兩次染色實驗均說明PLCL/GV上的細胞表達出了更多的心臟特異性蛋白。 (3)擔載雙因子的乳液靜電紡納米纖維的制備及性能表征 利用乳液靜電紡纖維的皮芯結(jié)構(gòu)特性,制備了皮層擔載有羥基磷灰石(HA)、芯層擔載有層黏連蛋白(Laminin)的乳液靜電紡纖維支架,記作PLCL/HA/Lam,并且在纖維支架上進行了成骨細胞的培養(yǎng)。MTS細胞增殖實驗顯示,從第7天開始,靜電紡纖維支架上細胞的增殖情況優(yōu)于組織培養(yǎng)板(TCP)。從第7天到第21天,PLCL/HA/Lam纖維支架上的細胞增殖情況優(yōu)于其它支架。譬如21天時,與TCP、PLCL/HA(皮層擔載羥基磷灰石的單藥物纖維支架)和PLCL/Lam(芯層擔載層黏連蛋白的單藥物支架)相比,分別高出23.3%、12.0%和10.4%。在第14天和第21天,PLCL/HA/Lam纖維支架上的堿性磷酸酶活性顯著高于PLCL/HA和PLCL/Lam纖維支架(P≤0.05),這是由于羥基磷灰石和層粘連蛋白的協(xié)同作用效果。EDX給出的數(shù)據(jù)在TCP,PLCL/HA,PLCL/Lam和PLCL/HA/Lam上的鈣含量分別為0%,0.05%,0.09%和0.51%,這與茜素紅染色(Alizarin Red S)結(jié)果一致。通過細胞的增殖實驗,骨蛋白表達,堿性磷酸酶活性以及礦化情況,充分說明了擔載有雙藥物的納米纖維極大的促進了成骨細胞的增殖、成熟等細胞行為。
[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.
【學(xué)位授予單位】：東華大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R318.08;TQ340.64

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