本文選題：靜電紡絲 + 組織工程��； 參考：《東華大學(xué)》2013年博士論文

【摘要】：心血管疾病被認為是引起人類死亡的重要原因,其比例已經(jīng)占全球死亡總數(shù)的30%,據(jù)統(tǒng)計在美國每年有14萬例以上的外科血管植入手術(shù),且多數(shù)患者的年齡大于50歲。由于世界上還沒有一套理想的制備小口徑血管組織的技術(shù)產(chǎn)品,醫(yī)生只有取病人身體其他部位的不重要的靜脈來替代硬化的冠狀動脈,此類“二次手術(shù)”無疑是給病人引入了另一種痛苦。與血管疾病情況類似的是,外周神經(jīng)損傷是臨床上的常見疾患,主要由事故造成的創(chuàng)傷引起。僅美國每年就有約1萬1千人因為神經(jīng)受損而癱瘓,造成經(jīng)濟損失愈70億美金,而外周神經(jīng)手術(shù)則超過了5萬例/年。這些修復(fù)神經(jīng)斷裂和缺損的手術(shù)也使用了病人其他部位相對不重要的神經(jīng)來替代,實為權(quán)宜之計。 有鑒于此,科研工作者們一直致力于研究可替代自體血管或神經(jīng)的人造支架。20世紀80年代,組織工程學(xué)科的建立為人造小口徑血管和神經(jīng)導(dǎo)管的開發(fā)提供了新的思路。通過在可降解的人造組織工程支架上種植相應(yīng)細胞,有可能獲得功能化的組織或器官,并最終替代受損組織。而支架的最終降解,也避免了取出支架時的二次手術(shù)對病人造成的額外傷害。 組織工程支架的眾多成型方法中,靜電紡絲法有著最廣泛的使用。該方法獲得的超細纖維能夠有效模擬生命體中的細胞外基質(zhì),有利于細胞的黏附和增殖。在血管組織工程與神經(jīng)組織工程中,使用靜電紡絲法制備的管狀支架仍有待改進。其中,血管支架難以兼具良好的力學(xué)與生物學(xué)性能,而神經(jīng)導(dǎo)管支架的成型方法始終存有缺陷。為了解決這些問題,本文通過材料選擇、纖維制備、支架成型、性能表征等方法,在制備出不同結(jié)構(gòu)的管狀支架并對這些支架進行系統(tǒng)研究的基礎(chǔ)上,探討其在血管與神經(jīng)修復(fù)中的應(yīng)用潛力。論文取得了一些具有重要學(xué)術(shù)價值和應(yīng)用前景的研究成果。 1.血管支架的研究中,以獲得天然蛋白-多糖為主要組成材料的血管支架為主要目的。蛋白-多糖靜電紡纖維能夠從材料組成和結(jié)構(gòu)兩方面模擬人體細胞外基質(zhì),以達到更好的組織修復(fù)效果。在制備管狀纖維支架之前,先通過制備纖維膜進行性能表征,其中膠原蛋白與殼聚糖占比為75%(膠原蛋白／殼聚糖=4:1),并添加了25%的熱塑性聚氨酯提高支架的力學(xué)性能。為使所獲的共混纖維不溶于水溶液,使用戊二醛對其進行了交聯(lián),掃描電鏡照片顯示當(dāng)交聯(lián)時間為48小時的時候,纖維的形貌得到最佳保持。紅外圖譜證明,交聯(lián)后的殘留戊二醛可通過將樣品長時間置于真空干燥箱中除去。生物相容性測試表明,內(nèi)皮細胞和雪旺細胞在該類支架均能較快粘附和增殖。進一步對細胞生長形態(tài)進行觀察,發(fā)現(xiàn)細胞的生長方向受纖維排列方向的影響較大。當(dāng)纖維取向排列時,兩種細胞均能夠沿纖維排列的方向定向生長�；诖祟惞不炖w維制備的管狀支架具有較好的彈性,可滿足體內(nèi)血管對于力學(xué)性能的要求。 2.在獲得了共混管狀支架的基礎(chǔ)上,對該成型方法加以改進,采用復(fù)合法制備出了蛋白-多糖-合成材料的管狀支架。膠原蛋白和殼聚糖以4:1的比例共混作為支架的內(nèi)、外層,而聚乳酸聚己內(nèi)酯則被用作熱塑性聚氦酯的替換材料,以期賦予支架更好的降解性能。該類復(fù)合支架的特點在于內(nèi)外壁表面均為天然蛋白-多糖材料,避免了合成材料與細胞的直接接觸,從而提高了支架的生物相容性。而中間的合成材料可用作加筋層,為支架提供了優(yōu)良的力學(xué)性能。通過自制的體外動態(tài)細胞培養(yǎng)裝置,培養(yǎng)內(nèi)皮細胞7天后,將支架置于共聚焦顯微鏡下觀察,可發(fā)現(xiàn)其表面被細胞鋪滿,形成了內(nèi)皮細胞單層。考慮到血管結(jié)構(gòu)的復(fù)雜性,除復(fù)合法外,又以管狀支架中的纖維聚集形式為出發(fā)點,成功設(shè)計并制備出軸向取向、徑向取向、層疊、梯度結(jié)構(gòu)的管狀纖維支架。這些支架各具特點,有望在不同方面滿足血管支架的要求,其結(jié)構(gòu)與成型方法可為下一代靜電紡血管支架的發(fā)展提供重要借鑒。 3.神經(jīng)導(dǎo)管的研究中,考慮到導(dǎo)管上的纖維若能沿導(dǎo)管的軸向方向排列,將有利于再生神經(jīng)的生長,因此重點研究了軸向取向纖維導(dǎo)管支架的制備技術(shù)。從起初使用的卷繞-縫合法,到經(jīng)過改進的滾輪法和轉(zhuǎn)盤法,再到之后的旋轉(zhuǎn)磁性導(dǎo)體棒-絕緣棒接收裝置,成功制備出纖維沿支架軸向取向的管狀支架。纖維受到電場、磁場以及拓撲結(jié)構(gòu)的共同作用,定向沉積在絕緣棒上,令支架呈現(xiàn)出軸向取向的結(jié)構(gòu),而接收裝置的旋轉(zhuǎn)保證了管狀支架壁厚的均勻性。此外,在靜電紡絲開始前,對絕緣棒表面進行覆糖,待到紡絲結(jié)束后用水將糖溶去,可方便支架的取出。為驗證該方法的普適性,使用多種聚合物進行靜電紡絲,并通過掃描電鏡和快速傅里葉轉(zhuǎn)變法表征所獲管狀支架上纖維的取向度,發(fā)現(xiàn)不同材料的纖維均能夠沿著支架的軸向方向取向排列,且在支架內(nèi)壁表面的纖維具有最高的取向度。 4.獲得軸向取向管狀支架后,又對纖維自身的結(jié)構(gòu)進行優(yōu)化,目的在于使纖維本身就能夠具有軸向取向的平行溝槽,將“軸向取向”這一特點增強。選擇醋酸丁酸纖維素作為聚合物溶質(zhì),溶于二甲基乙酰胺和丙酮的混合溶劑后靜電紡絲,成功制備出帶有“軸向取向溝槽”結(jié)構(gòu)的纖維。纖維制備完畢后,著重研究了該結(jié)構(gòu)的成型機理。通過改變紡絲參數(shù)、使用不同分子量的聚合物材料、比較溶劑溶質(zhì)性能、研究紡絲液揮發(fā)過程等方法,發(fā)現(xiàn)該軸向取向溝槽結(jié)構(gòu)主要是由靜電紡絲過程中,具有高揮發(fā)速度的溶劑迅速揮發(fā)后,所引起的相分離現(xiàn)象形成。聚合物流體暴露在空氣中,在溶劑富集區(qū)形成了孔洞,而這些孔洞在電場力作用下被進一步拉伸、細化成溝槽結(jié)構(gòu)。整個成型過程中,聚合物流體的粘度對于纖維形貌的影響起到了舉足輕重的作用,而粘度又受到溶液濃度、聚合物分子量的直接影響�；谶@些認識,使用相同方法對多種聚合物進行靜電紡絲,并觀察所獲纖維的形貌,使這一成型機理得到了進一步的驗證。 5.分別使用偏振紅外、X射線衍射和力學(xué)拉伸等手段對纖維的理化性能進行了表征,并分別在光滑醋酸丁酸纖維素纖維和取向溝槽纖維上種植大鼠雪旺細胞,評價其體外生物相容性。研究發(fā)現(xiàn),纖維表面帶有的取向二級結(jié)構(gòu)有利于雪旺細胞的黏附和增殖,尤其是早期黏附,細胞在培養(yǎng)8小時后呈現(xiàn)出典型的雪旺細胞“雙極”形態(tài)。7天之后再觀察,發(fā)現(xiàn)在取向溝槽纖維支架上,細胞的取向生長情況與增殖數(shù)量均優(yōu)于光滑纖維。比較體外生物性能后,以這兩種纖維構(gòu)建神經(jīng)導(dǎo)管支架,管外層使用了靜電紡聚乳酸聚羥基乙酸纖維作為力學(xué)增強。將導(dǎo)管支架植入大鼠坐骨神經(jīng)處,修復(fù)一段15毫米的神經(jīng)缺損。術(shù)后12周剖開導(dǎo)管,觀察神經(jīng)再生情況,可發(fā)現(xiàn)再生神經(jīng)的形成。電生理檢查、髓鞘檢查、腓腸肌檢查和大鼠行走足印等結(jié)果顯示,取向溝槽纖維構(gòu)成的軸向取向管狀支架可以進一步促進再生神經(jīng)的生長。雖然其修復(fù)效果與自體神經(jīng)相比仍有一定距離,但比光滑纖維支架有了明顯提高。這些結(jié)果說明,具有“軸向取向溝槽結(jié)構(gòu)”的纖維有利于神經(jīng)愈合,在構(gòu)建神經(jīng)導(dǎo)管支架領(lǐng)域具有較好的應(yīng)用潛力。
[Abstract]:Cardiovascular disease is considered to be an important cause of human death, which accounts for 30% of the total number of deaths in the world. According to statistics, there are more than 140 thousand cases of surgical vascular implantation in the United States each year, and most patients are older than 50. Only an unimportant vein of other parts of the patient's body is taken to replace the sclerotic coronary artery. This "two operation" is undoubtedly another pain to the patient. Similar to the vascular disease, peripheral nerve injury is a common clinical disease, mainly caused by the trauma caused by the accident. Only about 10 thousand and 1 in the United States. Thousands of people were paralyzed by nerve damage, resulting in more than $7 billion in economic loss and more than 50 thousand cases of peripheral nerve surgery. These repair of nerve breaks and defects have also used a relatively unimportant nerve to replace the other parts of the patient. It is an expedient measure.
In view of this, researchers have been working on the study of artificial scaffolds that can replace autologous blood vessels or nerves in the 80s.20 century. The establishment of the tissue engineering discipline provided a new idea for the development of artificial small caliber vessels and nerve conduits. It is possible to gain work by planting the corresponding cells on a biodegradable scaffold. An energetic tissue or organ, which eventually substitutes for the damaged tissue, and the final degradation of the scaffold avoids the additional damage to the patient from the two operation when the stent is removed.
The electrostatic spinning method is the most widely used method in many molding methods for the tissue engineering scaffold. The ultrafine fiber obtained by this method can effectively simulate the extracellular matrix in the life body, which is beneficial to the cell adhesion and proliferation. In the vascular tissue engineering and the neural tissue engineering, the tubular scaffold prepared by the electrostatic spinning method still needs to be improved. Among them, vascular stents are difficult to have good mechanical and biological properties, and the forming methods of the nerve conduit stents have always been defective. In order to solve these problems, this paper makes a systematic study of these scaffolds by means of material selection, fiber preparation, scaffolding molding and performance characterization. On the basis of this, we discussed its potential application in vascular and nerve repair. Some research results with important academic value and application prospect were obtained.
In the study of 1. vascular stents, the main purpose of the vascular scaffold is to obtain the natural protein polysaccharide as the main material. The protein polysaccharide electrostatic spun fiber can simulate the human extracellular matrix from two aspects of material composition and structure, so as to achieve better tissue repair effect. The fibrous membrane is prepared first by the preparation of the tubular fibrous scaffold. The ratio of collagen to chitosan was 75% (collagen / chitosan =4:1), and 25% of the thermoplastic polyurethane was added to improve the mechanical properties of the scaffold. The infrared map showed that the residual glutaraldehyde after cross linking could be removed in a vacuum drying box for a long time. Biocompatibility test showed that endothelial cells and Schwann cells were able to adhere and proliferate faster in the scaffold. The influence of the rectangle on the direction of fiber arrangement is great. When the fiber orientation is arranged, the two kinds of cells can grow along the direction of fiber arrangement. The tubular scaffold based on this kind of blend fiber has good elasticity, which can meet the requirements of the mechanical properties of the blood vessels in the body.
2. on the basis of obtaining the blend tubular scaffold, the molding method was improved. The tubular scaffold of protein polysaccharide synthetic material was prepared by the compound method. The collagen and chitosan were blended with 4:1 as the inner and outer layer of the scaffold. And polylactic acid polyhexyl ester was used as a replacement material for thermoplastic polyhelium. The scaffold has better degradation performance. The characteristic of this kind of composite scaffold is that the surface of the internal and external wall is natural protein - polysaccharide, which avoids the direct contact between the synthetic material and the cell, thus improving the biocompatibility of the scaffold. The intermediate synthetic material can be used as the stiffened layer and provides the excellent mechanical properties. Through the self-made body In the dynamic cell culture device, the endothelial cells were cultured for 7 days, and the scaffolds were observed under confocal microscopy. The surface of the cells was found to be covered with cells and formed a monolayer of endothelial cells. Considering the complexity of the vascular structure, the fiber aggregation in the tubular scaffold was the starting point except for the complex method. Tubular fiber scaffolds with radial orientation, cascading and gradient structure. These scaffolds have various characteristics and are expected to meet the requirements of vascular scaffolds in different aspects. The structure and molding methods can provide important reference for the development of the next generation of electrostatic spun stent.
In the study of 3. nerve conduits, considering that the fibers on the catheter were aligned along the axial direction of the catheter, it would be beneficial to the growth of the regenerative nerve. Therefore, the preparation techniques of the axially oriented fibrous stent were focused on. A body rod insulation rod receiving device has successfully prepared a tubular support with the axial orientation of the fiber support. The fiber is subjected to the joint action of electric field, magnetic field and topological structure. The fiber is directed to the insulation rod, making the bracket present an axial orientation structure, and the rotation of the receiving device ensures the uniformity of the wall thickness of the tubular support. In addition, the electrostatic spinning is used. In order to verify the universality of the method, a variety of polymers were used for electrostatic spinning to verify the universality of the method, and the orientation degree of the fibers on the tubular scaffold was characterized by scanning electron microscopy and rapid Fu Liye transformation, and the fiber of different materials was found. All dimensions are aligned along the axial direction of the support, and the fibers on the inner surface of the support have the highest orientation.
4. after the axial orientation tubular scaffold is obtained, the structure of the fiber is optimized. The purpose is to make the fiber itself have a parallel groove in the axial direction, and enhance the characteristic of "axial orientation". The fiber with "axial orientation groove" structure was successfully prepared. After the preparation of the fiber, the forming mechanism of the structure was emphatically studied. By changing the spinning parameters, using the polymer materials with different molecular weights, comparing the solute properties of the solvent and studying the volatilization process of the spinning solution, it was found that the axial orientation groove structure was mainly composed of static state. In the process of electrospinning, the phase separation phenomenon is caused by the rapid volatilization of the solvent with high volatilization. The polymer fluid is exposed to the air and formed holes in the solvent enrichment area. These holes are further stretched under the electric field force to refine the groove structure. In the whole process, the viscosity of the polymer fluid is to the fiber. The influence of the dimensional morphology plays an important role, and the viscosity is directly influenced by the concentration of the solution and the molecular weight of the polymer. Based on these knowledge, the same method is used to electrospun a variety of polymers, and the morphology of the obtained fibers is observed, so that the forming mechanism is further verified.
5. the physical and chemical properties of the fiber were characterized by polarizing infrared, X ray diffraction and mechanical stretching respectively. The rat Schwann cells were planted on the smooth cellulose acetate cellulose fiber and the orientation grooves fiber respectively, and the biocompatibility in vitro was evaluated. The study found that the orientation two structure with the surface of the fibrous surface was beneficial to Schwann fine. Cell adhesion and proliferation, especially early adhesion, showed a typical Schwann cell "bipolar" form.7 days after 8 hours of culture. It was found that the orientation growth and proliferation of the cells were superior to smooth fibers on the orientation grooved fiber scaffold. After comparing the biological properties of the cells in vitro, the two fibers were used to construct the nerve. The catheter stent was used as a mechanical enhancement by electrostatically spun poly (glycolic glycolic acid) fibers. The catheter stent was implanted into the rat sciatic nerve to repair a segment of the nerve defect of 15 millimeters. After 12 weeks, the catheter was opened to observe the regeneration of the nerve. Electrophysiological examination, myelin examination, gastrocnemius examination, and large amount of nerve were found. The results showed that the axially oriented tubular scaffold made of orientation grooves could further promote the growth of the regenerative nerve. Although the repair effect was still a certain distance compared with the autologous nerve, it was significantly better than the smooth fiber scaffold. These results suggest that the fiber with the "axial orientation groove structure" is beneficial. In nerve healing, it has a good potential in the field of constructing nerve conduit scaffolds.
【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】：2013
【分類號】：R318.11

【引證文獻】

相關(guān)碩士學(xué)位論文 前2條

1 何南霏;膠原針刺非織造材料及其在軟骨/骨組織工程中的應(yīng)用研究[D];東華大學(xué);2014年

2 劉欣;絲素基納米纖維膜的可控制備及在皮膚燙傷修復(fù)中的應(yīng)用[D];浙江理工大學(xué);2014年

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本文編號：1920804