CN104740681A - Polylactic acid-glycolic acid/adenovirus composite nano-fiber scaffold material as well as preparation method and application thereof - Google Patents

Polylactic acid-glycolic acid/adenovirus composite nano-fiber scaffold material as well as preparation method and application thereof Download PDF

Info

Publication number
CN104740681A
CN104740681A CN201510092902.7A CN201510092902A CN104740681A CN 104740681 A CN104740681 A CN 104740681A CN 201510092902 A CN201510092902 A CN 201510092902A CN 104740681 A CN104740681 A CN 104740681A
Authority
CN
China
Prior art keywords
adenovirus
polylactic acid
glycolic acid
acid
sucrose
Prior art date
Application number
CN201510092902.7A
Other languages
Chinese (zh)
Inventor
孙宏晨
朱阳
李道伟
方滕姣子
乔春燕
史册
张恺
Original Assignee
吉林大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 吉林大学 filed Critical 吉林大学
Priority to CN201510092902.7A priority Critical patent/CN104740681A/en
Publication of CN104740681A publication Critical patent/CN104740681A/en

Links

Abstract

The invention discloses a polylactic acid-glycolic acid/adenovirus composite nano-fiber scaffold material as well as a preparation method and application thereof in bone repairing, and belongs to the technical field of bone repairing materials. The method comprises the following steps: dissolving PLGA into trichloromethane and N,N-dimethylformamide, and preparing a nano spinning frame material by virtue of a high-pressure electrospinning device at a roomtemeprature; preparing an adenovirus cryoprotectant employing IM sucrose as final concentration; coating the PLGA nano spinning frame surface with sucrose/virus solution; and pre-freezing and carrying out freeze drying to obtain a PLGA/sucrose-adenovirus complex. The polylactic acid-glycolic acid/adenovirus composite nano-fiber scaffold material has good biocompatibility and relatively good biodegradability; the adenovirus can be localized on the PLGA surface, so that the concentration of the virus contacting the cells per unit area is increased; the infection efficiency is increased; the virus activity can be well stored; and once-through operation and long-time preservation can be carried out.

Description

一种聚乳酸-羟基乙酸/腺病毒复合纳米纤维支架材料、制备方法及其应用 A polylactic acid - glycolic acid / adenovirus complex nanofiber scaffolds, preparation method and application

技术领域 FIELD

[0001] 本发明属于骨修复材料技术领域,具体涉及一种聚乳酸-羟基乙酸(PLGA)/腺病毒(Ad)复合纳米纤维支架材料、制备方法及其在骨修复方面的应用。 [0001] The present invention belongs to the technical field of bone repair materials, in particular to a polylactic acid - glycolic acid (PLGA) / adenovirus (Ad) composite nanofiber scaffolds, preparation method and application in terms of bone repair.

背景技术 Background technique

[0002] 目前治疗骨缺损的方法有组织工程科学、骨及骨替代物移植及药物治疗等方法。 [0002] Current methods for treating bone defects organized engineering sciences, bone and bone graft substitutes and drug treatment. 其中,骨及骨替代物移植会产生创伤较大及骨整合不理想等问题,药物治疗中应用辛伐他汀、双磷酸盐等效果并不理想。 Wherein the bone substitute and bone grafting and bone trauma will produce undesirable problems such as integration, simvastatin drug treatment, other bisphosphonates not ideal. 而组织工程科学可以通过支架材料、生长因子及细胞成为一类可期待的方法。 The tissue engineering scaffold material science can, growth factors and cell to become a class method can be expected.

[0003] 纳米纤维支架直径为纳米级,表面积大,因而具有独特的小尺寸效应和表面效应。 [0003] nanofibrous scaffold having a diameter of nanometer level, large surface area, which has a unique small size effect and the surface effect. 另一方面由于其类似于天然细胞外基质,有利于细胞黏附,进而促进细胞增殖及分化,而被广泛应用。 On the other hand because it is similar to natural extracellular matrix, conducive to cell adhesion, promoting cell proliferation and differentiation and thus, is widely used. 但是现阶段研究显示:体内局部细胞不足,单纯的纳米纤维支架促进骨形成的能力有限。 However, studies have shown that stage: lack of local cell in vivo, pure nanofiber scaffolds limited ability to promote bone formation. 因此,需要生长因子或细胞与纳米纤维支架复合。 Therefore, cell growth factors or nanofiber scaffold. 外源细胞引入具有引起机体适应性免疫反应及细胞不能成活等缺点。 Introducing foreign cells having adaptive immune response caused by the body and the cells can not survive other shortcomings. 而促红细胞生成素(EPO)可促进内皮祖细胞(EPCs) 迁移、促进培养的人间充质干细胞(MSCs)趋化。 And erythropoietin (EPO) may facilitate endothelial progenitor cells (EPCs) migration, promote cultured human mesenchymal stem cells (MSCs) chemotaxis. 并能通过增加间充质干细胞的趋性、迁移性,激活基质金属蛋白酶,促进局部血管的形成,为骨再生提供良好的微环境。 And by increasing the inter-mesenchymal stem cell tropism, mobility, activation of matrix metalloproteinases, to promote the formation of local blood vessels, to provide a good environment for micro bone regeneration. 蛋白类生长因子部分造价较高以及具有免疫原性,较难得到广泛应用。 Immunogenic portion higher cost and a more difficult to be widely used protein growth factors. 因此,可以应用负载生长因子基因的载体,通过转染自体细胞原位表达相应生长因子以达到治疗目标。 Thus, the load carrier may be applied growth factor gene, growth factor corresponding to achieve a therapeutic target cell by in situ expression of Transfected. 编码EPO基因的腺病毒Ad-EPO能够在体外促进大鼠骨髓间充质干细胞向成骨细胞分化。 Encoding adenovirus Ad-EPO EPO gene can promote rat bone marrow mesenchymal stem cells into osteoblasts in vitro. 但是,单纯在骨缺损处应用病毒不仅会使病毒随体液扩散,降低病毒浓度,还有可能造成异位感染。 However, the simple application in the bone defect with the body fluids virus will not only make the virus spread, reduce the concentration of the virus, may also cause ectopic infection. 目前的解决方法是利用生物材料将病毒固定于应用部位。 The current workaround is to use the biological material of the virus is fixed to the application site. 例如:将病毒混入静电纺丝溶液中,通过高压静电纺丝装置将病毒与纳米纤维共纺。 For example: the virus is mixed electrostatic spinning solution by electrospinning nanofiber means a virus co-spinning. 其缺点在于高压及有机溶剂会降低病毒活力,而且由于静电纺丝时会损失部分溶液而使病毒不易定量,进而不能控制实际应用时的病毒剂量。 The disadvantage is that the high pressure and an organic solvent can reduce viral activity, and due to some loss of electrostatic spinning solution is not easy to quantify the virus, and thus can not control virus dose when practical use. 这为Ad-EPO在体内应用促进骨缺损修复造成了困难。 This creates difficulties for the Ad-EPO promote bone defect repair in vivo applications.

发明内容 SUMMARY

[0004] 本发明的目的在于提供一种促进骨修复的复合纳米纤维支架材料及其制备方法和用途。 [0004] The object of the present invention is to provide a nanofiber scaffolds promoting complex and its preparation method and use of bone repair. 通过制备聚乳酸-羟基乙酸纳米纤维支架与编码促红细胞生成素基因的腺病毒(Ad-EPO)结合,达到局部提高病毒浓度,使病毒原位感染细胞,表达EP0,为骨再生提供一个良好的微环境。 By polylactic acid - glycolic acid nanofiber scaffolds with adenovirus encoding the pro erythropoietin gene (Ad-EPO) in combination, to improve the local concentration of the virus, virus infected cells in situ, EP 0 expression, provide a good bone regeneration Microenvironment. 但是,由于EPO并不可见,所以较难确定处理方法对腺病毒的活性是否有损害,因此,需要一种直观的方法测定病毒活性。 However, the EPO is not visible, it is difficult to determine whether the treatment has activity against adenoviruses damage, therefore, a need for a straightforward method for determination of viral activity. 编码绿色荧光蛋白(EGFP)基因的腺病毒Ad-EGFP感染细胞使细胞表达EGFP,EGFP在荧光显微镜下可发绿光,可以直观的观察病毒的活性,同时,EGFP对细胞的增殖与分化不产生影响。 Ad-EGFP adenovirus encoding green fluorescent protein (EGFP) gene expression activity of cells infected cells EGFP, EGFP fluorescence microscope may emit green light, the virus can be directly observed, at the same time, EGFP cell proliferation and differentiation does not occur influences. 因此,本发明采用Ad-EGFP在体外检测PLGA/腺病毒复合纳米纤维支架上病毒的活力及病毒释放。 Accordingly, the present invention employs Ad-EGFP in vitro detection PLGA / adenovirus virus activity and release of the virus in the fiber composite stent nm. 利用蔗糖作为冷冻保护剂, 米用冷冻干燥法将PLGA与腺病毒复合。 Using sucrose as a cryoprotectant, by freeze-drying the rice PLGA adenovirus complex.

[0005] 本发明所述的聚乳酸-羟基乙酸/腺病毒复合纳米纤维支架材料(PLGA/Ad),其直径为100~200nm,首先由静电纺丝法获得聚乳酸-羟基乙酸纳米纤维,然后利用冷冻干燥法将腺病毒固定于聚乳酸-羟基乙酸纳米纤维表面。 [0005] The polylactic acid according to the present invention - glycolic acid / adenovirus complex nanofiber scaffolds (PLGA / Ad), having a diameter of 100 ~ 200nm, a polylactic acid is first obtained by the electrostatic spinning method - glycolic acid nanofibers, and by freeze drying method adenovirus fixed to a polylactic acid - glycolic acid nano fiber surface.

[0006] 本发明所述的聚乳酸-羟基乙酸/腺病毒复合纳米纤维支架的制备方法,其步骤如下: [0006] The present invention is a polylactic acid - glycolic acid / adenovirus composite nano fiber scaffold preparation methods, the following steps:

[0007] a)制备质量体积分数为0. 2~0. 4g/mL的聚乳酸-羟基乙酸溶液,溶剂为三氯甲烷与N,N-二甲基甲酰胺的混合,混合溶剂中三氯甲烷的体积含量为30~50%,然后在20~30°C条件下磁力搅拌4~8小时直至得到透明溶液; . [0007] a) Preparation of mass volume fraction of 0. 2 ~ 0 4g / mL of polylactic acid - glycolic acid solution, the solvent is chloroform and N, N- dimethylformamide was mixed in, a mixed solvent of Triclosan volume methane content is 30 to 50%, followed by stirring at 20 ~ 30 ° C condition magnetic 4-8 hours until a clear solution;

[0008] b)将步骤a)溶液采用高压静电纺丝装置进行纺丝,然后将纺丝产物在20~30°C 条件下真空干燥,即得到聚乳酸-羟基乙酸纳米纤维支架材料; [0008] b) step a) using a solution electrospinning spinning apparatus, the spinning product was then dried under vacuum at 20 ~ 30 ° C conditions, i.e., to obtain a polylactic acid - glycolic acid nanofiber scaffold;

[0009] c)将聚乳酸-羟基乙酸纳米纤维支架材料裁剪成直径5mm~3cm的圆片,圆片正反面分别进行紫外线照射消毒(功率8~10W、波长250nm~260nm,照射时间20~30分钟); [0009] c) The polylactic acid - glycolic acid nano-fibrous scaffold material was cut into 5mm 3cm diameter wafer, the wafer front and back surfaces are sterilized by ultraviolet irradiation (power 8 ~ 10W, a wavelength of 250nm ~ ~ 260nm, irradiation time of 20 to 30 minute);

[0010]d)取8.55g蔗糖晶体,溶于20mL纯水中,经过带有微孔滤膜的注射滤器(0.22~ 0.45iim微孔滤膜)抽滤除菌,得到I.25M蔗糖溶液;再取浓度为2XIOltl~2X1012VPAiL 的腺病毒原体与I.25M蔗糖溶液以体积比1:4混合,得到蔗糖/腺病毒溶液; [0010] d) sucrose crystals taken 8.55g, was dissolved in 20mL of pure water, through a syringe filter with a microporous membrane (0.22 ~ 0.45iim microporous membrane) filtration sterilization, to give I.25M sucrose solution; then take 2XIOltl ~ 2X1012VPAiL concentration of adenovirus in form from I.25M sucrose solution at a volume ratio of 1: 4 were mixed, to give the sucrose / adenovirus solution;

[0011] e)取10~20UL蔗糖/腺病毒溶液滴加于步骤c)得到的聚乳酸-羟基乙酸纳米纤维支架材料圆片的一侧表面上;在-60~_80°C下预冻20~30h,然后在-60~-80°C 的真空冷冻干燥机中冻干20~30h,获得聚乳酸-羟基乙酸/腺病毒复合纳米纤维支架材料。 [0011] e) takes 10 ~ 20UL sucrose / adenovirus was added dropwise to step c) to give a polylactic acid - glycolic acid on one surface of the nano fiber scaffold wafer; 20 pre-frozen at -60 ~ _80 ° C ~ 30h, and then lyophilized to 20 ~ 30h at -60 ~ -80 ° C in a vacuum freeze dryer, to obtain a polylactic acid - glycolic acid / adenovirus complex nanofiber scaffolds.

[0012] 进一步的,所述的高压静电纺丝装置的纺丝电压为15~25kv,正负电极间的距离为10~20cm,正极是静电纺丝装置的喷口,负极为接地的金属滚轴,正负电压使纺丝液在喷口处的流速为0. 5mL/h~I. 5mL/h;金属滚轴的直径为10~20cm,长度为20~30cm,金属滚轴表面覆盖锡箔纸,锡箔纸用于承接纺丝产物;喷口的直径为〇. 2~0. 4mm,所得纺丝产物的直径范围为100~200nm。 Spinning voltage [0012] Further, the high voltage electrostatic spinning apparatus is 15 ~ 25kv, the distance between the positive and negative electrodes 10 ~ 20cm, for the positive electrode is an electrostatic spinning nozzle apparatus, the negative electrode is a grounded metal roller , positive and negative voltages at the flow rate of the spinning solution at the nozzle is 0. 5mL / h ~ I 5mL / h;. metal rollers of diameter 10 ~ 20cm, a length of 20 ~ 30cm, the surface of the metal roll covered with foil, spinning foil for receiving product; diameter orifice is square 2 ~ 0 4mm, the diameter range of the resulting product was spun 100 ~ 200nm...

[0013] 聚乳酸-羟基乙酸(PLGA)中,聚乳酸的质量百分含量为30~80%,优选为50%。 [0013] The polylactic acid - glycolic acid (PLGA), the mass percentage of the polylactic acid is 30 to 80%, preferably 50%.

[0014] 本发明制备的聚乳酸-羟基乙酸/腺病毒复合纳米纤维支架主要应用于修复受损骨组织的再生。 [0014] The polylactic acid prepared in the present invention - glycolic acid / adenovirus composite nanofibrous scaffolds is mainly used to repair damaged bone tissue regeneration.

[0015] 本发明与现有技术相比,具有以下优点: [0015] Compared with the prior art the present invention has the following advantages:

[0016] (1)本发明制备的聚乳酸-羟基乙酸纳米纤维支架,形貌类似于细胞外基质,利于细胞的粘附和生长;通过控制共混比例,反应温度,时间等因素来控制材料的物理化学性能以及降解速率,这种PLGA/Ad纳米纤维具有良好的生物相容性。 [0016] (1) Preparation of the present invention, the polylactic acid - glycolic acid nano-fibrous scaffold, an extracellular matrix similar morphology, adhesion and conducive to cell growth; blending ratio by controlling the reaction temperature, time and other factors to control material the physicochemical properties and degradation rate, this PLGA / Ad nanofibers have good biocompatibility.

[0017] (2)制备后病毒活性保存良好,能够感染细胞表达相关蛋白。 [0017] (2) Preparation of the preserved viral activity, expression of proteins capable of infecting cells.

[0018] (3)该制备方法简便可行,对设备无特殊要求并具有良好的经济效益。 [0018] (3) The preparation method is simple and requires no special equipment and good economic.

附图说明 BRIEF DESCRIPTION

[0019] 图1 :实施例1制备的PLGA纳米纤维支架材料的扫描电镜图,其中PLGA的质量体积浓度为〇. 3g/mL,图I(a)的放大倍数为10000X,图I(b)的放大倍数为5000X; [0019] Figure 1: Scanning electron micrograph of PLGA nanofibrous scaffold prepared in Example 1, wherein the mass volume concentration of PLGA is square 3g / mL, in FIG I (a) is a magnification of 10000X, FIG I (b). the magnification is 5000X;

[0020] 图2 :实施例3中的PLGA/Ad-EGFP复合纳米纤维支架的病毒存留曲线; [0020] FIG. 2: PLGA in Example 3 / Ad-EGFP virus composite nanofibrous scaffolds retention curve;

[0021] 图3 :实施例3中的PLGA/Ad-EGFP复合纳米纤维支架感染骨髓间充质干细胞的荧光显微镜图像。 [0021] FIG. 3: PLGA in Example 3 / Ad-EGFP between the composite nano-fiber scaffold charge marrow infection fluorescence microscope image of mesenchymal stem cells. 其中,AO、AUA2、A3为Ad-EGFP感染BMMSCs后1,3, 7, 14天时照射的荧光显微镜图像;BO、Bl、B2、B3为在-70°C保存1天后的PLGA/Ad-EGFP感染BMMSCs后1,3, 7, 14天时的荧光显微镜图像;〇)、(:1乂2、03为在-701:保存15天后的?11^/^(146?? 感染BMMSCs后1,3, 7, 14天时的荧光显微镜图像;DO、Dl、D2、D3为在-70°C保存28天后的PLGA/Ad-EGFP感染BMMSCs后1,3, 7, 14天时的荧光显微镜图像; Wherein, AO, AUA2, A3 for the Ad-EGFP infected BMMSCs 1,3, 7, 14 days fluorescence microscopy images illuminated; BO, Bl, B2, B3 is stored in the 1 day -70 ° C PLGA / Ad-EGFP after infection BMMSCs 1,3,. 7, 14 days of a fluorescence microscope image; square), (: 1 as Yi in 2,03 -701: ^ / ^ (146 ?? BMMSCs saved after infection, 3 days 15 11? , 7, 14 days of a fluorescence microscope image; DO, Dl, D2, D3 is stored in the PLGA 28 days -70 ° C / Ad-EGFP 1,3, 7, 14 days after fluorescence microscopic image of BMMSCs infection;

[0022] 图4 :实施例3中的PLGA/Ad-EPO复合纳米纤维支架体内成骨能力检测图-- Micro-CT。 [0022] FIG. 4: PLGA in Example 3 / Ad-EPO composite nanofibrous scaffolds in vivo osteogenic potential detection map - Micro-CT. 其中,AUA2为对照组在第4周及第8周时的Micro-CT图像;Bl、B2为实验组在第4周及8周时的Micro-CT图像;al、a2、bl和b2为应用IPP图像分析软件检测的Al、 A2、Bl和B2中愈合面积百分比的数据分析。 Wherein, AUA2 control group Micro-CT images at 4 weeks and 8 weeks; Bl, B2 is a Micro-CT image of the experimental group at 4 weeks and 8 weeks; al, a2, bl and b2 for the application IPP image analysis software detected Al, area percent analysis data A2, Bl and B2 healing. *代表P< 0.05。 * Represents P <0.05.

[0023] 图5 :实施例3中的PLGA/Ad-EPO复合纳米纤维支架体内成骨能力检测图--HE 染色。 [0023] FIG. 5: PLGA in Example 3 / Ad-EPO composite nanofibrous scaffolds in vivo osteogenic potential detecting FIG --HE staining. 其中,AUA2为对照组在第4周及第8周时的HE染色图像;Bl、B2为实验组在第4 及8周时的HE染色图像,B3为Bl中箭头处放大10倍的图像;B4为B2中箭头处放大10倍的图像。 Wherein, AUA2 HE staining image of the control group at 4 weeks and 8 weeks; Bl, B2 is a HE staining image in the experimental group at 4 and 8 weeks, B3 arrow Bl at 10 times magnification of the image; B4 is 10 times enlarged image shown by the arrow B2.

具体实施方式 Detailed ways

[0024] 实施例1 : [0024] Example 1:

[0025] 将3g的PLGA(购于sigma公司,相对分子量为3. 6万)溶于IOmL的三氯甲烷与N,N-二甲基甲酰胺的混合溶剂中(体积比为4 :6),磁力搅拌4小时后,得到PLGA的三氯甲烷与二甲基甲酰胺混合溶液;然后利用静电纺丝装置纺丝(电压为18KV,距离为14cm, 纺丝液在喷口处的流速为0. 5mL/h,金属滚轴的直径为10cm,长度为24cm,喷口的直径为0. 3mm),制备得到PLGA的质量体积浓度为0. 3g/mL的聚乳酸-羟基乙酸纳米纤维支架材料,在25°C下真空干燥24h后备用。 [0025] 3g of the PLGA (available from sigma company, a relative molecular weight of 36,000) was dissolved in chloroform and IOmL N, N- mixed solvent of dimethyl formamide (volume ratio 4: 6) after magnetic stirring for 4 hours, chloroform and PLGA dimethylformamide, a mixed solution; and spinning by electrostatic spinning apparatus (voltage of 18KV, a distance of 14cm, the spinning liquid discharge port at a flow rate of 0. 5mL / h, the diameter of the metal roller is 10cm, 24cm in length, orifice diameter of 0. 3mm), prepared mass PLGA volume concentration of 0. 3g / mL of polylactic acid - glycolic acid nano-fibrous scaffold, in 24h after drying in vacuo at alternate 25 ° C. 其纤维直径在IOOnm~200nm,纤维交错,表面平滑,如图1所示。 Fiber diameter 200 nm, fiber interleaved IOOnm ~, smooth surface, as shown in FIG.

[0026] 实施例2 : [0026] Example 2:

[0027] 取8. 55g蔗糖晶体,溶于20mL纯水中,经过注射滤器(0.22 微孔滤膜)抽滤除菌,得到1.25M蔗糖溶液。 [0027] 8. 55g sucrose crystals taken, dissolved in 20mL of purified water through a syringe filter (0.22 microporous membrane) filtration sterilization, to give 1.25M sucrose solution. 取病毒原液Ad-EGFP、Ad-EP0(美国国立卫生研究院,颅颌面研究组,郑长玉研究员馈赠;公众亦可以得到)分别与I. 25M蔗糖溶液以I:4(V/V)混合,方法是将2iiL浓度为2XIO12病毒颗粒(VP) /mL的Ad-EPO溶液,与8iiL浓度为I. 25M的蔗糖溶液混合,吹打混匀后Ad-EPO浓度为4X10nVP/mL,蔗糖的终浓度为1M,此为Ad-EPO/蔗糖溶液;取2iiL浓度为2X101(lVP/mLAd-EGFP溶液,与8iiL浓度为I. 25M的蔗糖溶液混合,吹打混匀后Ad-EGFP浓度为4X109VP/mL,蔗糖的终浓度为1M,此为Ad-EGFP/蔗糖溶液。EGFP 是绿色荧光蛋白,EPO是促红细胞生成素,本专利中的Ad-EGFP对细胞的功能无任何影响, 用于检测病毒活性和病毒释放,而Ad-EPO对细胞的增殖与成骨分化均有一定作用,用于体内成骨能力的检测。 Take stock virus Ad-EGFP, Ad-EP0 (NIH, craniofacial Study Group, researchers gift Zhengzhang Yu; also available public) respectively sucrose solution I and I. 25M: 4 (V / V) mixture, the method is 2iiL 2XIO12 concentration of viral particles (VP) / mL in Ad-EPO solution was mixed with 8iiL concentration of sucrose solution I. 25M, Ad-EPO concentration after mixing of pipetting 4X10nVP / mL, final concentration of sucrose to 1M, this is Ad-EPO / sucrose solution; taken 2iiL concentration of 2X101 (lVP / mLAd-EGFP solution, mixed with a sucrose solution 8iiL I. 25M concentration, Ad-EGFP concentration after mixing of pipetting 4X109VP / mL, sucrose the final concentration of 1M, this is the Ad-EGFP / sucrose solution .EGFP green fluorescent protein, the EPO is erythropoietin, Ad-EGFP in this patent has no effect on cell function, for the detection of viruses and viral activity release, whereas Ad-EPO cell proliferation and differentiation of osteoblasts have a role, for detecting the in vivo osteogenic potential.

[0028] 实施例3: [0028] Example 3:

[0029] 将PLGA纳米纤维支架材料(实施例1)剪裁成直径为5mm和2cm的圆形,正反面分别进行紫外线(功率:8~10W、波长:250nm~260nm)照射消毒30分钟后,取10iiL实施例2制备的Ad-EPO/蔗糖溶液滴加于直径5mm的PLGA纳米纤维支架的一面上;取10iiL 实施例2制备的Ad-EGFP/蔗糖溶液滴加于直径2cm的PLGA纳米纤维支架上。 [0029] The nanofiber PLGA scaffold (Example 1) cut into a circle having a diameter of 5mm and a 2cm, respectively, positive and negative UV (power: 8 ~ 10W, wavelength: 250nm ~ 260nm) 30 minutes after irradiation sterilized, taking Ad-EPO / sucrose solution prepared in Example 2 was added dropwise to the embodiment 10iiL 5mm diameter PLGA nanofiber scaffolds on one side; 10iiL embodiment taken on Ad-EGFP prepared in Example 2 / sucrose solution was added dropwise to a 2cm diameter nanofiber scaffolds of PLGA . 在-70°C下预冻24h使液体充分冰冻,然后在-70°C的真空冷冻干燥机中冻干24h使冰冻的水升华,获得PLGA/Ad-EGFP及PLGA/Ad-EPO两种复合纳米纤维支架材料。 At -70 ° C 24h pre-freezing liquid sufficiently frozen, then lyophilized in vacuo lyophilizer -70 ° C. 24h sublimation of frozen water to obtain PLGA / Ad-EGFP and PLGA / Ad-EPO two complexes nanofiber scaffolds.

[0030] 实施例4: [0030] Example 4:

[0031] 将11个冻干的PLGA/Ad-EGFP复合纳米纤维支架材料置于12孔板中以保证操作条件相同,其他孔空置,每孔加入350iiLTE(IOmMTris-HCl,ImMEDTA,pH7. 5)缓冲液。 [0031] The freeze-dried 11 PLGA / Ad-EGFP composite nanofiber scaffold was placed in a 12 well plate to ensure that the same operating conditions, the other vacant holes, each well was added 350iiLTE (IOmMTris-HCl, ImMEDTA, pH7. 5) buffer. 在第l、2、4、8、15、30min及第l、2、4、8、16h于不同的孔中取出300iiL上清液置于小离心管中。 At l, 2,4,8,15,30min second l, 2,4,8,16h supernatant was removed 300iiL small centrifuge tube to separate wells. 取出的上清液每100UL使用15iiL质量百分浓度为0. 5 %的十二烷基硫酸钠(SDS)-TE 溶液(SDS为溶质,TE缓冲液为溶剂)在室温放置15min,裂解病毒衣壳,使病毒DNA暴露出来。 Each supernatant was removed using 15iiL 100UL mass percent concentration of 0.5% sodium dodecyl sulfate (SDS) -TE solution (SDS for the solute, the TE buffer solution as a solvent) was placed at room temperature for 15min, split virus coat housing the exposed viral DNA. 裂解后的上清液以每孔100UL、每个时间点设3个复孔加于96孔板的33个孔中。 The supernatant after lysis per well 100UL, provided each time point 3 holes 33 added to wells of a 96 well plate. 按照说明书将Quant-iT™PicoGreen®dsDNA定量试剂盒(购买于Invitrogen公司)中的标准品配制成5个梯度浓度(浓度分别为Iyg/ml、100ng/ml、10ng/ml、lng/ml和Ong/ml), 液体分别以IOOu1加于上述96孔板的另5个孔中。 According to the instructions Quant-iT ™ PicoGreen®dsDNA quantification kit (purchased from Invitrogen) standards formulated in a gradient of 5 concentrations (concentrations of Iyg / ml, 100ng / ml, 10ng / ml, lng / ml and Ong / ml), respectively IOOu1 liquid added to the 96 well plates and five other wells. 该试剂盒中的Picogreen试剂以TE 缓冲液稀释200倍,以每孔100yL加于上述样品及标准品所在的孔中。 The Picogreen reagent kit 200-fold dilution in TE buffer, was added per well in 100yL hole of the standards and samples located. 5min避光孵育后, Picogreen试剂可以与双链DNA结合形成DNA-Picogreen复合物,通过Picogreen的突光强度检测DNA的量。 After 5min incubation in the dark, Picogreen reagent may bind to form a double-stranded DNA-DNA complexes Picogreen, the amount of projection light intensity detected by the Picogreen DNA. 待测样品及标准品中的DNA-Picogreen复合物在酶标仪上分别在激发波长485nm、发射波长538nm读取数据。 Test sample and the standard DNA-Picogreen complexes are on a microplate reader at an excitation wavelength 485nm, emission wavelength of the read data 538nm. 对照组为10iiL的Ad-EGFP/蔗糖溶液,对照组与实验组操作相同,以对照组中的双链DNA浓度为100 %。 10iiL control group of Ad-EGFP / sucrose solution, the same operation of the control group and experimental group, double-stranded DNA concentration in the control group as 100%. 如图3所示,病毒在前Ih快速释放,随着时间延长,病毒释放速度减缓,在第16h时在PLGA纳米纺丝支架上存留20%左右。 As shown in FIG. 3, the front Ih virus released rapidly with time, virus release slowed down, during the first 16h in the remaining 20% ​​of the spinning stent PLGA nanoparticles.

[0032] 实施例5: [0032] Example 5:

[0033] 以股骨全骨髓提取法提取大鼠骨髓间充质干细胞BMMSCs(吉林大学基础医学部动物实验中心,Wistar雄性大鼠,4~5周,体重80~100g),在培养瓶中传代至第3 代(JinH,ZhangK,QiaoC,etal.Efficientlyengineeredcellsheetusinga complexofpolyethylenimine-alginatenanocompositesplusbonemorphogenetic protein2genetopromotenewboneformation[J].Internationaljournalof nanomedicine,2014,9:2179.),以每孔1.5X105个细胞,每孔2ml培养基铺于六孔板上。 [0033] extracted in the femoral whole bone marrow extraction rat bone marrow mesenchymal stem cells of BMMSCs (Experimental Animal Center of Basic Medical Jilin University, Wistar male rats, 4 to 5 weeks weighing 80 ~ 100g), the flasks were passaged through 3 generations (JinH, ZhangK, QiaoC, etal.Efficientlyengineeredcellsheetusinga complexofpolyethylenimine-alginatenanocompositesplusbonemorphogenetic protein2genetopromotenewboneformation [J] .Internationaljournalof nanomedicine, 2014,9:. 2179), 1.5X105 cells per well, 2ml culture medium per well were plated on six-well plates . 24h细胞贴壁后将培养基更换为无血清的培养基。 After 24h cells were adherent the medium was replaced with serum-free medium. 对照组为未与材料复合的病毒液体,病毒量与实施例3制备的PLGA/Ad-EGFP中的病毒量相同,实验组为在-70°C下保存1、15、30天时取出的实施例3中制备的PLGA/Ad-EGFP,将PLGA/Ad-EGFP覆盖于细胞上,每个时间点设三个复孔。 Example control group was not complexed with the viral material liquid, the same amount of viral load viral PLGA prepared in Example 3 / Ad-EGFP in the experimental group was removed at days 1,15,30 stored -70 ° C. preparation of 3 PLGA / Ad-EGFP, the PLGA / Ad-EGFP covered on the cell, provided each time point triplicate wells. 然后置于含体积分数5% 0)2的371:恒温细胞培养箱(细胞培养箱中原有为空气,人为输入CO2,使其体积分数达到5% )中培养,12h后将培养基更换为有血清的培养基。 Then placed in the volume fraction% 0 3715) 2: The thermostat cell incubator (incubator original air cells, human input CO2, so that the volume fraction of 5%) in culture medium was replaced with a after 12h serum medium. 然后,每两天换一次体积为2mL的细胞培养基,细胞培养基中包含体积分数为85%-90%低葡萄糖-杜尔伯科改良伊格尔培养基L-DMEM(购买于Gibco公司),及胎牛血清(购买于BI 公司),胎牛血清体积分数为10%~15%。 Then, every two days for a volume of 2mL of the cell culture medium, cell culture medium comprising a volume fraction of 85% -90% low glucose - Dulbecco's Modified Eagle's Medium L-DMEM (purchased from Gibco) and fetal bovine serum (purchased from companies BI), the volume fraction of 10% fetal calf serum to 15%. 在第1、3、7、14天于倒置荧光显微镜下观察并拍照。 Observed and photographed on day 1,3,7,14 an inverted fluorescence microscope. 如图4所示,-70°C下保存30天后病毒仍能感染大鼠BMMSCs,使细胞表达绿色荧光蛋白(图4中明亮部分)。 30 days after the viral infection can still save BMMSCs rats at 4, -70 ° C, the cells were expressing GFP (bright portion in FIG. 4). 说明经过低温保存后Ad-EGFP仍具有活性。 Description After cryopreservation Ad-EGFP still active. 另外,与对照组相比, 实验组的荧光面积更大,说明病毒与材料复合,可以使病毒的局部浓度提高,并缩减病毒在培养基中的沉降时间,增加单位时间病毒进入细胞的数目,从而达到提高转染效率的效果。 Further, compared with the control group, the experimental group of a larger area of ​​fluorescence, indicating that the virus with a composite material, can improve the local concentration of the virus, the virus and reduce the settling time in the medium, increasing the number of viral entry unit of time, so as to improve the effect of transfection efficiency.

[0034] 实施例6: [0034] Example 6:

[0035] 在Wistar雄性大鼠(吉林大学基础医学部动物实验中心,8~10周,体重280~ 300g)的头部耳缘后做长度为Icm的横行切口,分离骨膜,用慢速环形去骨钻在头盖骨人字缝区域制备直径为5mm的圆形缺损,实验组为将实施例3制备的PLGA/Ad-EPO复合纳米纤维支架以1片/只植入缺损处,以只进行手术的大鼠作为对照组,每组5只。 [0035] In the ear head Wistar male rats (Basic Medical Experimental Animal Center of Jilin University, 8 to 10 weeks, weighing 280 ~ 300g) made of transverse incision length Icm, isolated periosteum, with a slow loop boneless cranial drill with a diameter of 5mm lambdoid suture circular defect region, the experimental group of PLGA was prepared in Example 3 / Ad-EPO composite nanofiber scaffold at 1 / implant only defect, for only the big surgery as a control group of mice, 5 per group. 操作过程中不断用生理盐水冲洗,最后用3/0手术缝合线缝合。 During operation continuously rinsed with saline, and finally 3/0 surgical sutures. 术后八周将大鼠心脏灌流固定(质量体积分数为40mg/mL的多聚甲醛)处死后,将头盖骨取出,用多聚甲醛固定处理48小时后用于Micro-CT测量。 Eight weeks postoperatively (paraformaldehyde mass volume fraction of 40mg / mL) is perfused rat heart after sacrifice, the skull removed, fixed with paraformaldehyde treated after 48 hours for Micro-CT measurement. 如图4所示,通过面积计算,可以发现在第4周实验组促进骨缺损明显缩小,缺损边缘不光滑,在第8周,实验组促进骨缺损进一步缩小。 4, by calculating the area can be found at week 4 facilitate the experimental group significantly reduced bone defects, defects in the edge is not smooth, at 8 weeks, the experimental group further refine Bone Defects. 经过HE染色证实,如图5所示,在第4周实验组由于Ad-EPO的作用,使缺损局部产生大量红细胞,并在第8周骨缺损边缘出现骨再生。 After HE staining confirmed, as shown in FIG. 5, in the experimental group 4 weeks due to the effect of Ad-EPO, a large amount of red blood cells so that the local defects, and regeneration of bone at 8 weeks bone defect edges. 说明本发明产物具有促进骨再生的功效。 DESCRIPTION products of the invention promote bone regeneration effect.

Claims (7)

1. 一种聚乳酸-羟基乙酸/腺病毒复合纳米纤维支架材料的制备方法,其步骤如下: a) 制备质量体积分数为0. 2~0. 4g/mL的聚乳酸-羟基乙酸溶液,溶剂为三氯甲烷与N,N-二甲基甲酰胺的混合,混合溶剂中三氯甲烷的体积含量为30~50%,然后在20~ 30°C条件下磁力搅拌4~8小时直至得到透明溶液; b) 将步骤a)溶液采用高压静电纺丝装置进行纺丝,然后将纺丝产物在20~30°C条件下真空干燥,即得到聚乳酸-羟基乙酸纳米纤维支架材料; c) 将聚乳酸-羟基乙酸纳米纤维支架材料裁剪成直径5mm~3cm的圆片,圆片正反面分别进行紫外线照射消毒; d) 取8. 55g蔗糖晶体,溶于20mL纯水中,经过带有微孔滤膜的注射滤器抽滤除菌,得到I. 25M蔗糖溶液;再取浓度为2 X IOltl~2 X 10 12VPAiL的腺病毒原体与I. 25M蔗糖溶液以体积比1 :4混合,得到蔗糖/腺病毒溶液; e) 取10~20 y L蔗糖/腺病毒溶液滴 1. A polylactic acid - glycolic acid / adenovirus composite nano fiber scaffold preparation methods, the following steps:. A) Preparation of mass volume fraction of 0. 2 ~ 0 4g / mL of polylactic acid - glycolic acid solution, the solvent trichloromethane and N, N- dimethylformamide was mixed in a mixed solvent of chloroform in a volume content of 30 to 50%, followed by stirring at 20 ~ 30 ° C condition magnetic 4-8 hours until a clear solution; b) step a) using a solution electrospinning spinning apparatus, the spinning product was then dried under vacuum at 20 ~ 30 ° C conditions, i.e., to obtain a polylactic acid - glycolic acid nano-fibrous scaffold; c) the the polylactic acid - glycolic acid nanofiber scaffold material was cut into 5mm 3cm diameter wafer, the wafer front and back surfaces, respectively, ultraviolet light disinfection ~; d) sucrose crystals take 8. 55g, was dissolved in 20mL of purified water, after having micropores membrane syringe filter sterilized suction, to give I. 25M sucrose solution; then take a concentration of 2 X IOltl ~ adenovirus protomer I. 25M with 2 X 10 12VPAiL sucrose solution to a volume ratio of 1: 4 were mixed, to give sucrose / adenovirus solution; E) takes 10 ~ 20 y L sucrose / adenovirus added dropwise 于步骤c)得到的聚乳酸-羟基乙酸纳米纤维支架材料圆片的一侧表面上;在-60~_80°C下预冻20~30h,然后在-60~-80°C的真空冷冻干燥机中冻干20~30h,获得聚乳酸-羟基乙酸/腺病毒复合纳米纤维支架材料。 In step c) to obtain a polylactic acid - glycolic acid side surface of the nanofiber material wafer holder; at -60 ~ _80 ° C pre-freezing 20 ~ 30h, and then frozen in vacuo to -60 ~ -80 ° C dry lyophilized machine 20 ~ 30h, to obtain a polylactic acid - glycolic acid / adenovirus complex nanofiber scaffolds.
2. 如权利要求1所述的一种聚乳酸-羟基乙酸/腺病毒复合纳米纤维支架材料的制备方法,其特征在于:高压静电纺丝装置的纺丝电压为15~25kv,正负电极间的距离为10~ 20cm,正极是静电纺丝装置的喷口,负极为接地的金属滚轴,正负电压使纺丝液在喷口处的流速为〇. 5mL/h~I. 5mL/h ;金属滚轴的直径为10~20cm,长度为20~30cm,金属滚轴表面覆盖锡箔纸,锡箔纸用于承接纺丝产物;喷口的直径为〇. 2~0. 4mm,所得纺丝产物的直径范围为100~200nm。 2. A polylactic acid according to claim 1 - glycolic acid / adenovirus fiber scaffold preparation of composite nano material, wherein: a voltage of electrospinning the spinning device is 15 ~ 25kv, between positive and negative electrodes 10 ~ 20cm of distance, the positive electrode is a nozzle of an electrospinning apparatus, the negative electrode is a grounded metal roller, positive and negative voltages at the flow rate of the spinning solution at the nozzle was square 5mL / h ~ I 5mL / h;.. metal roller diameter 10 ~ 20cm, a length of 20 ~ 30cm, the metal roller surface covering foil, aluminum foil and spun for receiving product;. diameter orifice is square 2 ~ 0 4mm, the diameter of the resultant spun product in the range of 100 ~ 200nm.
3. 如权利要求1所述的一种聚乳酸-羟基乙酸/腺病毒复合纳米纤维支架材料的制备方法,其特征在于:聚乳酸-羟基乙酸中,聚乳酸的质量百分含量为30~80%。 3. A polylactic acid according to claim 1 - glycolic acid / adenovirus fiber scaffold preparation of composite nano material, wherein: the polylactic acid - glycolic acid, the mass percentage of the polylactic acid is 30 to 80 %.
4. 如权利要求3所述的一种聚乳酸-羟基乙酸/腺病毒复合纳米纤维支架的制备方法,其特征在于:聚乳酸-羟基乙酸中,聚乳酸的质量百分含量为50%。 4. The polylactic acid according to claim 3 - glycolic acid / adenovirus composite nano fiber scaffold preparation method, wherein: the polylactic acid - glycolic acid, the polylactic acid has a mass percentage of 50%.
5. 如权利要求1所述的一种聚乳酸-羟基乙酸/腺病毒复合纳米纤维支架材料的制备方法,其特征在于:紫外线照射消毒的功率为8~10W、波长为250nm~260nm、照射时间20~30分钟。 Irradiation time of ultraviolet light disinfection power of 8 ~ 10W, having a wavelength of 250nm ~ 260nm,: glycolic acid / adenovirus fiber scaffold preparation of composite nano material, characterized in that - the polylactic acid as claimed in claim 1 20 to 30 minutes.
6. -种聚乳酸-羟基乙酸/腺病毒复合纳米纤维支架材料,其特征在于:由权利要求1~5任何一项所述的方法制备得到。 6. - Species polylactic - glycolic acid / adenovirus complex nanofiber scaffolds, characterized in that: prepared by any one of claims 1 to 5, obtained by the claims.
7. 权利要求6所述的一种聚乳酸-羟基乙酸/腺病毒复合纳米纤维支架材料在骨修复方面的应用。 Application of glycolic acid / adenovirus complex nanofiber scaffold in bone repair area - polylactic acid according to claim 6.
CN201510092902.7A 2015-03-02 2015-03-02 Polylactic acid-glycolic acid/adenovirus composite nano-fiber scaffold material as well as preparation method and application thereof CN104740681A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201510092902.7A CN104740681A (en) 2015-03-02 2015-03-02 Polylactic acid-glycolic acid/adenovirus composite nano-fiber scaffold material as well as preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201510092902.7A CN104740681A (en) 2015-03-02 2015-03-02 Polylactic acid-glycolic acid/adenovirus composite nano-fiber scaffold material as well as preparation method and application thereof

Publications (1)

Publication Number Publication Date
CN104740681A true CN104740681A (en) 2015-07-01

Family

ID=53581290

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201510092902.7A CN104740681A (en) 2015-03-02 2015-03-02 Polylactic acid-glycolic acid/adenovirus composite nano-fiber scaffold material as well as preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN104740681A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106521646A (en) * 2016-10-05 2017-03-22 桂林理工大学 Preparing method of polylactide-co-glycolide electrostatic spinning solution

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5631236A (en) * 1993-08-26 1997-05-20 Baylor College Of Medicine Gene therapy for solid tumors, using a DNA sequence encoding HSV-Tk or VZV-Tk
CN1378445A (en) * 1999-08-06 2002-11-06 得克萨斯系统大学评议会 Drug releasing biodegradable fiber implant
CN1994476A (en) * 2006-08-29 2007-07-11 北京华世本全科技有限公司 Degradable compound biomaterial membrane for medical purpose
US20120029653A1 (en) * 2002-06-13 2012-02-02 Evans Douglas G Devices and methods for treating defects in the tissue of a living being
CN102965849A (en) * 2012-11-22 2013-03-13 天津大学 Method for preparing medical barrier membrane by electrostatic spinning

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5631236A (en) * 1993-08-26 1997-05-20 Baylor College Of Medicine Gene therapy for solid tumors, using a DNA sequence encoding HSV-Tk or VZV-Tk
CN1378445A (en) * 1999-08-06 2002-11-06 得克萨斯系统大学评议会 Drug releasing biodegradable fiber implant
US20120029653A1 (en) * 2002-06-13 2012-02-02 Evans Douglas G Devices and methods for treating defects in the tissue of a living being
CN1994476A (en) * 2006-08-29 2007-07-11 北京华世本全科技有限公司 Degradable compound biomaterial membrane for medical purpose
CN102965849A (en) * 2012-11-22 2013-03-13 天津大学 Method for preparing medical barrier membrane by electrostatic spinning

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106521646A (en) * 2016-10-05 2017-03-22 桂林理工大学 Preparing method of polylactide-co-glycolide electrostatic spinning solution
CN106521646B (en) * 2016-10-05 2019-06-07 桂林理工大学 A kind of preparation method of poly lactide-glycolide acid electrostatic spinning solution

Similar Documents

Publication Publication Date Title
Gupta et al. Aligned and random nanofibrous substrate for the in vitro culture of Schwann cells for neural tissue engineering
Nie et al. Three‐dimensional fibrous PLGA/HAp composite scaffold for BMP‐2 delivery
Yu et al. Tissue-engineered scaffolds are effective alternatives to autografts for bridging peripheral nerve gaps
Li et al. Biological response of chondrocytes cultured in three‐dimensional nanofibrous poly (ϵ‐caprolactone) scaffolds
Lawrence et al. Silk film biomaterials for cornea tissue engineering
Bisson et al. Acrylic acid grafting and collagen immobilization on poly (ethylene terephthalate) surfaces for adherence and growth of human bladder smooth muscle cells
US9101693B2 (en) Cell-independent fabrication of tissue equivalents
Huang et al. Electrospun collagen–chitosan–TPU nanofibrous scaffolds for tissue engineered tubular grafts
Cai et al. Permeable guidance channels containing microfilament scaffolds enhance axon growth and maturation
Wu et al. Preparation and assessment of glutaraldehyde‐crosslinked collagen–chitosan hydrogels for adipose tissue engineering
Shapira-Schweitzer et al. Matrix stiffness affects spontaneous contraction of cardiomyocytes cultured within a PEGylated fibrinogen biomaterial
Meinel et al. Silk implants for the healing of critical size bone defects
Lu et al. Nanofibrous architecture of silk fibroin scaffolds prepared with a mild self-assembly process
Andrade et al. Improving bacterial cellulose for blood vessel replacement: Functionalization with a chimeric protein containing a cellulose-binding module and an adhesion peptide
Li et al. Effect of carbon nanotubes on cellular functions in vitro
US20060153815A1 (en) Tissue engineering devices for the repair and regeneration of tissue
Jeong et al. Tissue-engineered vascular grafts composed of marine collagen and PLGA fibers using pulsatile perfusion bioreactors
Miyagi et al. Biodegradable collagen patch with covalently immobilized VEGF for myocardial repair
Mosahebi et al. Addition of fibronectin to alginate matrix improves peripheral nerve regeneration in tissue-engineered conduits
EP2129772B1 (en) Tissue-engineered silk organs
US9522218B2 (en) Method for preparing porous scaffold for tissue engineering, cell culture and cell delivery
Manning et al. Controlled delivery of mesenchymal stem cells and growth factors using a nanofiber scaffold for tendon repair
Hamada et al. Spatial distribution of mineralized bone matrix produced by marrow mesenchymal stem cells in self‐assembling peptide hydrogel scaffold
Oliveira et al. The osteogenic differentiation of rat bone marrow stromal cells cultured with dexamethasone-loaded carboxymethylchitosan/poly (amidoamine) dendrimer nanoparticles
Baiguera et al. Electrospun gelatin scaffolds incorporating rat decellularized brain extracellular matrix for neural tissue engineering

Legal Events

Date Code Title Description
C06 Publication
C10 Entry into substantive examination
WD01