本文選題：動態(tài)液體-靜電紡　切入點：三維支架　出處：《東華大學(xué)》2012年碩士論文

【摘要】：骨是人體重要的支撐結(jié)構(gòu),具有保護(hù)器官和執(zhí)行造血等功能。大塊骨缺損是骨科面臨的一大難題。組織工程為骨缺損的修復(fù)提出了新途徑。支架、種子細(xì)胞、生長因子是組織工程的三要素。骨組織工程支架基本目的是盡可能模仿和接近松質(zhì)骨的機(jī)械性能和結(jié)構(gòu)。骨細(xì)胞外基質(zhì)(extracellular matrix, ECM)由非礦化的有機(jī)組分和礦化的剛性無機(jī)物質(zhì)組成。由于ECM在細(xì)胞的生長過程中起著至關(guān)重要的調(diào)控作用,構(gòu)建具有類似ECM結(jié)構(gòu)與功能的支架是組織工程的關(guān)鍵技術(shù)之一。 本論文采用動態(tài)液體-靜電紡技術(shù)制備了兩類新型可降解的納米纖維三維支架,分別采用磷酸-鈣液交替浸漬法和模擬體液(SBF)浸漬法仿生礦化,模擬天然骨組織的結(jié)構(gòu)和成分。使用MC3T3-E1細(xì)胞研究兩類支架的生物相容性,為骨組織工作支架的構(gòu)建提供新的方法。主要研究內(nèi)容如下： (1)首次采用改良的動態(tài)液體-靜電紡技術(shù)制備了絲素蛋白(silk fibroin,SF)與P(LLA-CL)混紡納米紗和單純P(LLA-CL)的納米紗三維支架,用磷酸-鈣液交替浸漬法仿生礦化。通過掃描電子顯微鏡和紅外光譜、X射線衍射光譜分析礦化前后兩種納米紗的結(jié)構(gòu)和理化性質(zhì)。研究結(jié)果表明,納米紗是由多根納米纖維成股而形成的。在組成單元上,P(LLA-CL)納米紗比SF與P(LLA-CL)混紡的納米紗直徑更大,同時宏觀結(jié)構(gòu)更規(guī)則。SF與P(LLA-CL)的混紡后,通過水的接收,能夠使得SF快速轉(zhuǎn)變?yōu)椴蝗苡谒腂-折疊的silk II結(jié)晶結(jié)構(gòu)。紅外光譜、X射線衍射光譜分析證實通過磷酸-鈣液交替浸漬法能夠使得支架上沉積羥基磷灰石顆粒。細(xì)胞相容性實驗表明,由納米纖維形成的納米紗極大地影響細(xì)胞的粘附和遷移行為。純聚合物材料的P(LLA-CL)由于疏水的表面缺乏細(xì)胞粘附的功能性基團(tuán),不利于細(xì)胞增殖,混合有SF的納米紗支架能夠有效促進(jìn)細(xì)胞增殖。MC3T3-E1細(xì)胞在礦化前后兩種支架上都能夠增殖,其中P(LLA-CL)/SF、P(LLA-CL)-HA和P(LLA-CL)/SF-HA支架上的細(xì)胞數(shù)量出現(xiàn)顯著性增殖(P0.05)。通過HE染色證明,在礦化前后的納米紗支架上都發(fā)現(xiàn)了細(xì)胞的長入,細(xì)胞沿著纖維生長,呈現(xiàn)沿著纖維的走向遷移,遍布了整個支架中。本實驗結(jié)果證明,改良的動態(tài)液體-靜電紡技術(shù)能夠提供一種用于組織工程三維支架材料的制備方法,制備的三維納米紗能夠克服傳統(tǒng)靜電紡制備出的納米纖維膜導(dǎo)致細(xì)胞難以長入支架內(nèi)部的缺點,有望應(yīng)用于骨組織修復(fù)。 (2)為了增強(qiáng)三維結(jié)構(gòu)的納米纖維的力學(xué)性能,本研究利用纖維增強(qiáng)的原理結(jié)合冷凍干燥技術(shù),制備了新型P(LLA-CL)和P(LLA-CL)/SF納米紗增強(qiáng)絲素蛋白的復(fù)合型三維支架,使用75%乙醇蒸汽誘導(dǎo)支架的水不溶性。采用10×SBF浸漬法來進(jìn)行支架的仿生礦化。結(jié)果表明纖維增強(qiáng)這一方式對復(fù)合支架的孔隙率,力學(xué)性能有著巨大的影響。隨著納米紗的加入,相比純SF支架,兩種纖維增強(qiáng)型復(fù)合支架的孔隙率有一定程度的下降,同時力學(xué)性能得到明顯的增加。純SF支架的彈性模量為1.2848±0.1107MPa,孔隙率約為86.28%。PLCL-R-SF支架的彈性模量增加到1.17017±O.1619MPa,孔隙率降低至84.97%,而PLCL/SF-R-SF支架的彈性模量的彈性模量增加到1.7265±0.5042MPa,孔隙率降低至82.82%。礦化沉積的結(jié)晶顆粒的鈣磷比(Ca/P)為1.56,接近羥基磷灰石的Ca/P=1.67。紅外光譜、X射線衍射光譜分析進(jìn)一步證實通過10×SBF浸漬法能夠使得支架上沉積球型的羥基磷灰石顆粒。PLCL-R-SF-HA和PLCL/SF-R-SF-HA支架上的細(xì)胞數(shù)量有了顯著性的增加(P0.05),同時MC3T3-E1在支架上已經(jīng)分泌出了自身的明顯的細(xì)胞外基質(zhì)。說明在納米水平上球狀的羥基磷灰石顆�？赡軙䴗p少MC3T3-E1細(xì)胞在支架上早期的增殖,但是在后期羥基磷灰石對細(xì)胞增殖起了明顯的促進(jìn)作用。本實驗結(jié)果證明,纖維增強(qiáng)這一方式能夠極大的提高三維納米纖維支架的力學(xué)強(qiáng)度,纖維增強(qiáng)型復(fù)合支架在骨組織修復(fù)中具有良好的應(yīng)用潛能。
[Abstract]:Bone is an important supporting structure of human body, can protect organs and hematopoietic function. The execution of large bone defects is a big problem in the Department of orthopedics face. Tissue engineering for the repair of bone defects and puts forward a new way. Scaffolds, seed cells, growth factors are the three elements of tissue engineering. The basic purpose of bone tissue engineering scaffold mechanical properties biomimic the structure and cancellous bone. The bone extracellular matrix (extracellular, matrix, ECM) by non mineralized rigid inorganic substances and mineralization of organic fractions. Because ECM play a vital role in the process of cell growth, construction of stents with similar structure and function of ECM is one of the key technology of tissue engineering.
In this paper, two kinds of novel biodegradable nanofiber scaffolds prepared by liquid electrospinning technique, respectively using phosphoric acid and calcium liquid alternate immersion method and simulated body fluid (SBF) immersion method of biomimetic mineralization, structure and composition of the simulated natural bone tissue compatibility. The use of MC3T3-E1 cell research two kinds of biological scaffold and provide a new method for constructing bone tissue scaffold work. The main contents are as follows:
(1) for the first time in the preparation of silk fibroin by dynamic modified liquid electrospinning technology (silk fibroin SF) and P (LLA-CL) blended yarn and pure nano P (LLA-CL) nano yarn scaffold, phosphate - calcium liquid alternate immersion biomimetic mineralization by scanning electron microscopy and infrared spectroscopy. X ray diffraction spectrum analysis of mineralization structure before and after the two nanometer yarn and physicochemical properties. The results show that the nano yarn is formed by a plurality of nano fiber strands. In the unit, P (LLA-CL) than SF and P nano yarn (LLA-CL) nano blended yarn diameter is larger, while the macro structure more rules.SF and P (LLA-CL) of the blend, through the water receiver, can make SF silk II a rapid change of crystal structure of folding as insoluble B-. Infrared spectroscopy, X ray diffraction spectrum analysis confirmed by calcium phosphate liquid alternate immersion method can make the deposition of hydroxyapatite scaffold on Particles. The cell compatibility experiment showed that nano fiber yarn formed by nanoparticles greatly affects cell adhesion and migration behavior of pure polymer materials. P (LLA-CL) due to the lack of hydrophobic surface functional groups is not conducive to cell adhesion, cell proliferation, mixed with nano SF yarn stent can effectively promote the cell proliferation of.MC3T3-E1 cells can the proliferation in mineralization before and after the two support, including P (LLA-CL) /SF, P (LLA-CL) -HA and P (LLA-CL) the number of cells on /SF-HA scaffolds induced significant proliferation (P0.05). Proved by HE staining in the nano yarn before and after stent mineralization were found on the growth of cells, cell along the fiber growth, showing migration along the fiber direction throughout the scaffold. The experimental results show that the dynamic modified liquid electrospinning technology can provide a method for preparing three-dimensional scaffold for tissue engineering, system The three dimensional nano yarn prepared by this technology can overcome the shortcomings of the traditional nanofiber membrane produced by electrospinning and cause cells to not grow into the inner part of the scaffold, which is expected to be applied to bone tissue repair.
(2) in order to enhance the mechanical properties of nano fiber three-dimensional structure, this study utilizes the principle of fiber reinforced with freeze drying technology to prepare P model (LLA-CL) and P (LLA-CL) /SF nano yarn reinforced composite scaffold of silk protein scaffolds, induced by using 75% ethanol steam water insoluble. To support the biomimetic mineralization with 10 x SBF impregnation method. The results show that the porosity of the fiber reinforced composite scaffolds, have great effects on mechanical properties. With the addition of nano yarn, compared with pure SF stent, two kinds of fiber reinforced composite scaffold porosity decreased to a certain extent, and mechanical properties are increased obviously. The elastic modulus of pure SF scaffold was 1.2848 + 0.1107MPa, the porosity is about 86.28%.PLCL-R-SF elastic modulus of the scaffolds increased to 1.17017 + O.1619MPa, the porosity decreased to 84.97%, while the elastic modulus of PLCL/SF-R-SF support The elastic modulus increased to 1.7265 + 0.5042MPa, the porosity crystalline particles to 82.82%. mineralization of calcium and phosphorus ratio (Ca/P) for 1.56 Ca/P=1.67., close to the infrared spectrum of hydroxyapatite, X ray diffraction spectrum analysis further confirmed by 10 * SBF impregnation method can make the number of.PLCL-R-SF-HA PLCL/SF-R-SF-HA stent and hydroxyapatite particles deposited on the ball type the cells are significantly increased (P0.05), and MC3T3-E1 in the scaffold has produced a distinct extracellular matrix. Its hydroxyl apatite particles in spherical nanometer level may reduce the proliferation of MC3T3-E1 cells in the early support, but in the late stage of hydroxyapatite on cell proliferation was significantly promoted. The experimental result shows that the fiber reinforced this way can greatly improve the three-dimensional nanofiber scaffold mechanical strength, fiber The reinforced composite scaffold has a good potential in the repair of bone tissue.

【學(xué)位授予單位】：東華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R318.08

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 曹現(xiàn)林;劉玲蓉;張其清;;制備工藝條件對膠原多孔支架材料結(jié)構(gòu)和性質(zhì)的影響[J];國際生物醫(yī)學(xué)工程雜志;2006年05期

2 王麗雪;尹志娟;劉海鷗;;玻璃纖維增強(qiáng)環(huán)氧樹脂單向復(fù)合材料力學(xué)性能分析[J];黑龍江工程學(xué)院學(xué)報(自然科學(xué)版);2009年03期

3 陳艷雄;陳敏;朱譜新;杜宗良;;絲素蛋白的研究和應(yīng)用進(jìn)展[J];紡織科技進(jìn)展;2007年02期

4 何創(chuàng)龍,王遠(yuǎn)亮,楊立華,張軍,夏烈文;骨組織工程天然衍生細(xì)胞外基質(zhì)材料[J];中國生物工程雜志;2003年08期

5 張金鵬;湯遜;;納米羥基磷灰石復(fù)合支架材料的研究與應(yīng)用[J];中國組織工程研究與臨床康復(fù);2011年42期

，

本文編號：1661398