發(fā)布時間：2018-05-07 02:36

  本文選題：熔融紡絲 + 骨組織工程支架��； 參考：《吉林大學(xué)》2014年博士論文

【摘要】：長期以來，生物可降解高分子材料應(yīng)用于骨組織工程一直是組織修復(fù)的熱點(diǎn)，多種高分子材料組織工程支架材料被不斷開發(fā)出來。在組織工程支架材料設(shè)計與制備中，良好的孔隙結(jié)構(gòu)對骨修復(fù)效果十分重要，研究證明：一方面，在多孔材料中可見新生骨形成而在無孔粒子材料中未見骨形成[1]；一方面，當(dāng)孔隙率和孔隙結(jié)構(gòu)在一定范圍內(nèi)增加時對細(xì)胞增殖、粘附、長入以及支架血管化有著顯著地促進(jìn)作用[2-5]。目前，多孔的生物可降解高分子組織工程支架因其具有可控的孔隙率和孔徑、良好的力學(xué)強(qiáng)度和加工性能等優(yōu)點(diǎn)，已經(jīng)成為組織工程支架研究的主流。但目前以傳統(tǒng)粒子瀝濾支架為代表的海綿狀多孔支架仍存在一些缺點(diǎn)，如孔隙之間連通不佳，易于出現(xiàn)閉孔和微孔連接等，在一定程度上限制了組織長入和營養(yǎng)物質(zhì)交換，不利于骨組織修復(fù)。而纖維性多孔支架雖然解決了海綿狀多孔支架的孔隙間連通性問題，但卻存在其他不足，具體來說：以靜電紡絲纖維氈為代表的納米級纖維支架，其具有良好的抗菌性、可控性和利于細(xì)胞增殖和分化等優(yōu)點(diǎn)，但無法提供足夠的厚度以及利于細(xì)胞長入的孔隙直徑，難以滿足治療大段骨缺損的需求；以快速成型法為代表制備的微米級纖維支架，雖然解決了靜電紡絲纖維氈的厚度和孔隙直徑問題，但由于缺乏納米級和亞納米級纖維結(jié)構(gòu)，對細(xì)胞功能的促進(jìn)作用不甚理想。為此，本研究采用一種新型的熔融離心紡絲方法，制備得到棉花樣、直徑分布廣泛（100nm-40μm）的聚乳酸無紡纖維，并采用有機(jī)溶劑氣體表面粘連方法將該種纖維制備成為新型的纖維組織工程支架，，以期克服傳統(tǒng)海綿狀多孔支架和纖維性支架的局限。然后利用明膠表面涂覆的方法對新型纖維支架進(jìn)行修飾，使其從結(jié)構(gòu)和生物活性上均更加接近天然細(xì)胞外基質(zhì)，從而獲得更加智能化的組織工程支架。 首先，明確熔融離心紡絲方法制備聚乳酸纖維所需的適宜條件即溫度和轉(zhuǎn)速，接下來對不同原材料和轉(zhuǎn)速所制備的聚乳酸無紡纖維進(jìn)行掃描電鏡、直徑分布、力學(xué)強(qiáng)度、熱力學(xué)性質(zhì)、結(jié)晶度以及初步的體外生物相容性和功能分析；然后，明確有機(jī)溶劑氣體表面粘連方法制備聚乳酸纖維支架的所需的適宜條件即粘聯(lián)作用時間和所需纖維的最佳密度，然后對不同密度纖維制備的單純聚乳酸纖維組織工程支架進(jìn)行力學(xué)性能、孔隙率、掃描電鏡、初步的體外生物相容性和功能分析，并通過表面明膠涂覆方法對聚乳酸纖維支架進(jìn)行改性修飾，對改性修飾后支架進(jìn)行了掃描電鏡和體外礦化能力研究；最后，將制備所得的聚乳酸纖維復(fù)合明膠支架進(jìn)行兔橈骨缺損修復(fù)實(shí)驗(yàn)，檢驗(yàn)其骨缺損修復(fù)能力。通過上述研究，對新型的熔融離心紡絲方法制備聚乳酸纖維及支架應(yīng)用于臨床提供依據(jù)，期望開發(fā)出可應(yīng)用于臨床的骨組織工程材料。 第一部分：熔融離心紡絲聚乳酸纖維的制備、表征以及生物相容性研究。通過多次試驗(yàn)，得到聚乳酸（PLLA，粘均分子量：90595）進(jìn)行熔融離心紡絲的適宜溫度為：旋碟中心220℃、旋碟邊緣180℃；能夠得到成形纖維的最低轉(zhuǎn)速和最高轉(zhuǎn)速分別為300rpm和500rpm，而制備纖維產(chǎn)量最大的轉(zhuǎn)速為900rpm，得到初次紡絲纖維三種。以制備所得纖維分別作為原材料，按照各自的制備轉(zhuǎn)速進(jìn)行二次紡絲，得到二次紡絲纖維三種。利用掃描電鏡、直徑分布、力學(xué)強(qiáng)度、熱力學(xué)性質(zhì)、結(jié)晶度、熱力學(xué)降解、X線衍射和紅外衍射等對所制備的六種不同聚乳酸纖維進(jìn)行表征。將小鼠胚胎成骨細(xì)胞前體（MC3T3-E1）細(xì)胞種植于六種纖維和靜電紡絲膜上，利用MTT法檢測不同材料在1d、3d和7d對成骨增殖的影響，利用掃描電鏡法觀察不同材料在1d、3d、7d和14d對細(xì)胞形態(tài)和長入能力的影響，并利用六種纖維和靜電紡絲纖維氈的24h材料浸提液，行MTT檢測，檢驗(yàn)材料的急性細(xì)胞毒性。結(jié)果顯示：熔融離心紡絲所獲得聚乳酸無紡纖維擁有棉花樣、直徑分布較寬的三維立體結(jié)構(gòu)；其理化性質(zhì)可根據(jù)不同的原材料和轉(zhuǎn)速進(jìn)行調(diào)整；相對于靜電紡絲纖維氈，熔融離心紡絲纖維其細(xì)胞生物相容性更好。 第二部分：熔融離心紡絲聚乳酸纖維復(fù)合明膠支架的制備、表征以及體外相容性實(shí)驗(yàn)。采用三氯甲烷氣體作為粘連劑，利用溶劑氣體表面粘連法成功制備了聚乳酸纖維支架，其最佳粘連時間為90min，并明確支架成形所需最低的纖維密度為0.1g/cm3，此外還制備了密度為0.15g/cm3以及0.2g/cm3的支架，利用力學(xué)強(qiáng)度、孔隙率、掃描電鏡等對三種不同密度的纖維支架進(jìn)行了表征，并將小鼠胚胎成骨細(xì)胞前體（MC3T3-E1）細(xì)胞種植于0.15g/cm3纖維支架上，利用掃描電鏡法觀察密度為0.15g/cm3纖維支架在7d時對細(xì)胞長入能力的影響。將密度為0.15g/cm3的纖維支架進(jìn)行明膠表面涂覆修飾，制備得到0.1%、0.5%、1%和10%濃度明膠表面涂覆修飾的復(fù)合支架。利用掃描電鏡法觀察改性后支架的表面形態(tài)和體外礦化后沉積物表面形態(tài)，利用礦化前后質(zhì)量改變、沉積物X線衍射以及等離子體發(fā)射光譜等方法對復(fù)合支架進(jìn)行表征。結(jié)果顯示：采用三氯甲烷氣體作為粘連劑，粘連作用60-90min后可成功制備出聚乳酸纖維支架；其力學(xué)強(qiáng)度及孔隙率等性質(zhì)可根據(jù)粘連時間和纖維密度等條件進(jìn)行人為調(diào)節(jié)；密度為0.15g/cm3的單純聚乳酸纖維支架具有良好的力學(xué)強(qiáng)度和孔隙結(jié)構(gòu)，并且利于細(xì)胞的長入；利用0.5%濃度明膠對支架進(jìn)行表面修飾后可獲得孔隙率和體外礦化能力良好的類細(xì)胞外基質(zhì)支架。 第三部分：熔融離心紡絲聚乳酸纖維復(fù)合明膠支架的兔橈骨缺損修復(fù)實(shí)驗(yàn)。根據(jù)前期工作，選擇力學(xué)強(qiáng)度、孔隙結(jié)構(gòu)和礦化能力性能較好的聚乳酸/明膠復(fù)合支架進(jìn)行兔橈骨缺損修復(fù)試驗(yàn)，分別于2week、4week、6week、9week和12week進(jìn)行大體觀察和X線觀察。結(jié)果顯示：兔橈骨缺損修復(fù)效果：聚乳酸纖維/明膠復(fù)合支架10%羥基磷灰石/聚乳酸粒子粒濾支架單純聚乳酸纖維支架單純聚乳酸粒子瀝濾支架商品化納米羥基磷灰石/聚酰胺復(fù)合支架空白對照組。
[Abstract]:For a long time, biodegradable polymer materials used in bone tissue engineering have always been the hot spots of tissue repair. A variety of polymer scaffold materials have been developed. In the design and preparation of tissue engineering scaffold materials, good pore structure is very important to the effect of bone repair. New bone is found in the material and no [1] is found in the porous material. On the one hand, when the porosity and pore structure increase in a certain range, the cell proliferation, adhesion, long entry and stent vascularization have a significant effect on [2-5].. The advantages of controlled porosity and pore size, good mechanical strength and processing performance have become the mainstream in the research of tissue engineering scaffolds. However, there are still some shortcomings in the spongy porous scaffolds represented by the traditional particle leaching support, such as the poor connectivity between the pores, the easy out of the obturator and the microporous connection and so on, to a certain extent. The tissue long entry and the exchange of nutrients are not conducive to the repair of bone tissue. While the fibrous porous scaffold solves the problem of the connectivity between the pores of the spongy porous scaffold, but there are other deficiencies. Specifically, the nanoscale fiber scaffold, represented by the electrospun fiber felt, has good antibacterial, controllability and benefit. Cell proliferation and differentiation, but can not provide enough thickness and the pore diameter that is beneficial to cell growth. It is difficult to meet the need for the treatment of large bone defects. Micrometer fiber scaffolds, represented by rapid prototyping, have solved the problem of the thickness and pore diameter of the electrospun fiber felt, but due to the lack of nanoscale and subgrade. Nano fiber structure is not ideal for promoting the function of cell. Therefore, a new type of melt centrifugal spinning method was used to prepare a cotton like fiber with a wide diameter (100nm-40 mu m) in diameter, and a new fiber tissue was prepared by the method of organic solvent gas surface bonding. In order to overcome the limitations of the traditional spongy porous scaffold and fibrous scaffold, the scaffold is used to modify the new type of scaffold with gelatin surface coating to make it closer to the natural extracellular matrix from the structure and biological activity, so as to obtain a more intelligent tissue engineering scaffold.
First, the suitable conditions for the preparation of polylactic acid fibers were defined as temperature and rotational speed. Then, the scanning electron microscopy, diameter distribution, mechanical strength, thermodynamic properties, crystallinity, and preliminary biocompatibility and functional analysis of polylactic acid non woven fibers prepared by different raw materials and rotational speeds were followed. The optimum conditions required for the preparation of polylactic acid fiber scaffolds by the organic solvent gas surface adhesion method are the optimal density of the adhesion time and the required fiber, and the mechanical properties, porosity, scanning electron microscopy, and primary biocompatibility and work in vitro are then carried out for the simple polylactic tissue engineering scaffolds prepared by different density fibers. The modified polylactic acid fiber scaffold was modified by surface gelatin coating method, and the modified scaffold was studied by scanning electron microscope and in vitro mineralization ability. Finally, the prepared poly (lactic acid) composite gelatin scaffold was used to repair the radius defect of rabbit and to test the repair ability of bone defect. The application of the new method of melting centrifugal spinning to the preparation of polylactic acid fibers and scaffolds is provided for clinical application of bone tissue engineering materials.
The first part: the preparation, characterization and biocompatibility study of the melt centrifuged poly (lactic acid) fiber. Through several experiments, the optimum temperature of poly lactic acid (PLLA, viscosity average molecular weight: 90595) for melting centrifuge spinning is: 220 centigrade and 180 centigrade at the edge of rotating disc; the minimum speed and maximum speed of the forming fiber can be obtained. Not 300rpm and 500rpm, and the maximum speed of fiber production is 900rpm, and the first spinning fiber three kinds of fibers are obtained. The fibers are prepared as raw materials and spinning two times according to their respective rotational speed, and three kinds of spinning fibers are obtained. Scanning electron microscope, diameter distribution, mechanical strength, thermodynamic properties, crystallinity and thermodynamics are used. Six kinds of poly (lactic acid) fibers were characterized by degradation, X-ray diffraction and infrared diffraction. The mouse embryonic osteoblast precursor (MC3T3-E1) cells were planted on six kinds of fibers and electrospun membranes. The effects of different materials on the osteogenesis of 1D, 3D and 7d were detected by MTT, and the different materials were observed by scanning electron microscopy in 1D, 3D, 7. The effects of D and 14d on the cell morphology and the ability to grow, and using the 24h extracts of six kinds of fibers and electrospun fiber felt, were used to test the acute cytotoxicity of the material by MTT test. The results showed that the poly lactic acid non spun fiber obtained by molten centrifugal spinning has a three dimensional structure with a wide diameter distribution of cotton and its physical and chemical properties. It can be adjusted according to different raw materials and rotational speed. Compared with the electrospun fiber mat, the melt centrifugal spinning fiber has better cell biocompatibility.
The second part: the preparation, characterization and in vitro compatibility of the melt centrifuged poly (lactic acid) fiber composite gelatin scaffold. Using trichloromethane gas as an adhesion agent, the polylactic acid fiber scaffold was successfully prepared by the solvent gas surface adhesion method. The optimum adhesion time was 90min, and the minimum fiber density needed for the stent formation was clearly defined. For 0.1g/cm3, the scaffolds with density of 0.15g/cm3 and 0.2g/cm3 were also prepared. Three kinds of fiber scaffolds with different densities were characterized by mechanical strength, porosity and scanning electron microscope, and the mouse embryonic osteoblast precursor (MC3T3-E1) cells were planted on the 0.15g/cm3 fibrous scaffold. The density was observed by scanning electron microscope (0.15g/cm3). The effect of fiber scaffold on the cell growth ability at 7d. The surface coating of 0.1%, 0.5%, 1% and 10% gelatin surfaces was prepared by coating gelatin surface with the density of 0.15g/cm3 fiber scaffold. The surface morphology of the modified stent and the surface morphology after the mineralization were observed by scanning electron microscope. The composite scaffolds were characterized by quality changes before and after mineralization, X-ray diffraction of sediments and plasma emission spectroscopy. The results showed that chloroform gas was used as adhesion agent, after adhesion of 60-90min, the polylactic acid fiber scaffold could be successfully prepared. The mechanical strength and porosity could be based on the adhesion time and fiber. Density and other conditions are adjusted artificially; the simple polylactic acid fiber scaffold with a density of 0.15g/cm3 has good mechanical strength and pore structure, and is beneficial to the growth of the cell. After the surface modification of the scaffold with 0.5% concentration gelatin, the porosity and good extracorporeal mineralization ability of the extracellular matrix scaffold can be obtained.
The third part: a rabbit radial defect repair experiment with a melt centrifuged poly (lactic acid) fiber composite gelatin scaffold. According to the earlier work, a polylactic acid / gelatin composite scaffold with better mechanical strength, pore structure and mineralizing ability was selected to repair the radius defect of rabbit, and the general observation was made in 2week, 4week, 6week, 9week and 12week, respectively. The results showed that the repair effect of rabbit radial defect: polylactic fiber / gelatin composite scaffold 10% hydroxyapatite / polylactic acid particle filter scaffold simple polylactic acid scaffold for pure polylactic acid leaching stent commercial nano hydroxyapatite / polyamide composite scaffold in empty white control group.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R318.08

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Lung tissue flap repairs esophagus defection with an inner chitosan tube stent[J];World Journal of Gastroenterology;2009年12期

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