CN104307039A - 一种同时固定rgd和ha纤维膜的制备方法 - Google Patents
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Abstract
本发明涉及一种同时固定两种生物活性物质精氨酸-甘氨酸-天冬氨酸多肽(RGD)和羟基磷灰石(HA)的纤维膜的制备方法。聚乙交酯-丙交酯(PLGA)是一种重要的制备生物可吸收的引导骨再生膜材料,但其生物性能较差;RGD是一种重要的细胞粘附序列,细胞对RGD三肽序列的亲和力极强,使黏附面积增加,增强聚酯纤维膜的生物性能;HA是人和动物骨的主要无机成分,有良好的生物相容性和骨传导作用,与骨组织结合能力强,是优良的骨组织缺损填充材料。对RGD和HA进行修饰后,引入到PLGA纤维膜,纤维膜由PLGA、HA-g-PLLA和RGD-PEG-PCL三种成分组成。本发明的优点是加强纤维膜的生物性能,促进细胞黏附,使细胞获得较好的生长环境,有利于细胞增殖。
Description
技术领域
本发明涉及一种同时固定RGD和HA纤维膜的制备方法。
背景技术
引导骨再生(Guide bone regeneration, GBR)是一种手术治疗骨缺损的有效手段,利用多孔膜作为机械屏障,在骨缺损处营造一个独立的空间,屏蔽其他组织的竞争,完成新骨重建。生物可降解聚合物,如聚乳酸(PLA),聚己内酯(PCL),聚丙交酯-乙交酯(PLGA),具有柔韧性,良好的生物相容性和可调谐的生物降解性已广泛用于膜材料,然而因为没有促进细胞黏附和增殖的生物学效应,生物可降解的GBR膜只拥有有限的临床疗效。
固定细胞识别位到生物可降解聚合物,增强细胞和聚合物之间的生物作用,对于成功应用GBR膜修复受损骨是至关重要的。RGD三肽序列是细胞基质中的许多黏附蛋白共有的细胞识别位点,研究发现许多细胞外基质蛋白,如胶原蛋白、层粘连蛋白、纤维蛋白原和玻连蛋白与细胞外基质(Extracellular matrix, ECM)通过整联蛋白的结合刺激特定的细胞的行为。羟基磷灰石(HA)具有与人体骨中无机成分相似的结构和组成,优良的骨传导性能,植入体内后,钙和磷会游离出材料表面被身体组织吸收,并促进新组织生成,但是HA与生物可降解聚酯的界面结合力较差,当复合材料植入体内,HA与聚酯基体的界面首先遭到破坏,HA纳米粒子很快就从基体中分离出来。
电纺丝是一种简单的制备合成和天然聚合物纳米纤维膜的方法。纳米纤维膜有高的比表面积和孔隙率,结构类似于天然EMC,适用于伤口敷料,药物传递,组织工程和GBR膜。本专利分别对RGD、HA进行修饰获得RGD-PEG-PCL和HA-g-PLLA,然后选用PLGA为主体聚合物,将HA-g-PLLA、RGD-PEG-PCL与其均匀共混,电纺成同时固定两种生物功能物质的纤维膜。
发明内容
本发明提供一种同时固定RGD和HA的纤维膜的制备方法。所述纤维膜是将接枝聚合物RGD-PEG-PCL和HA-g-PLLA通过电纺丝引入到PLGA纤维基体,因为固定了RGD和HA,刺激细胞在合成材料表面粘附,增强了纤维膜与细胞间的相互作用。
本发明中同时固定RGD和HA的电纺丝膜的制备方法包括如下步骤:
所述的一种接枝HA-g-PLLA的制备方法和步骤,请参阅论文,纳米羟基磷灰石表面接枝聚合左旋丙交酯,高分子学报,(1)2013。
所述的一种接枝RGD-PEG-PCL的步骤和条件如下:
PCL端羧基活化(PCL-COOH)。PCL 5000的合成,以异丙醇为引发剂,甲苯为溶剂,合成分子量为5000的PCL。0.2g异丙醇为引发剂,16.7g ε-己内酯以辛酸亚锡为催化剂,50mL甲苯为溶剂,115℃,N2环境下反应24h,乙醇沉降,真空干燥。PCL的端基羧化,5g PCL与20mg丁二酸酐、24.4g DMAP(4-二甲氨基吡啶)、20.2mL TEA(三乙胺)加入到的30mL二氯甲烷中,在氮气环境下室温反应24h,乙醇沉降,真空干燥。得PCL-COOH。PCL-COOH端羧基活化,4g端羧基的PCL溶解在30mL二氯甲烷中,加入0.306g EDC·HCl [1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐]和0.184g NHS(N-羟基琥珀酰亚胺)搅拌,在室温下反应12h。反应结束后乙醇沉降,真空干燥,得PCL-NHS。
PEG端基氨化。PEG400 使用甲苯共沸除水后,真空干燥后待用。15g PEG以30mL二氯甲烷为溶剂,加入三乙胺(物质的量为甲基磺酰氯物质的量的1.1倍)和15mL的甲基磺酰氯(物质的量为PEG上羟基物质的量的20倍以上,须缓慢滴加,过快易产生副反应),在室温下反应3天。G4砂芯漏斗过滤,去除三乙胺的盐酸盐。饱和食盐水洗五次后,加入无水硫酸镁除水后过滤,使用旋转蒸发仪浓缩,乙醚沉降,真空干燥。18g 酰氯处理过的样品放入到500mL的烧瓶中,加入22g 氯化铵和400mL的浓氨水在40℃条件下氨解三天,加入氯化钠使溶液饱和,用三氯甲烷萃取五次,再用饱和食盐水洗两次,浓缩后乙醚沉降,真空干燥,获得端氨基的PEG。
制备RGD-PEG-PCL。PCL-PEG-NH2的合成,2.5g PCL-NHS和3g双端氨基的PEG(Mn=4000)加入到30mL二氯甲烷中,在常温下反应24h,乙醇沉降,真空干燥,得PCL-PEG-NH2。RGD-PEG-PCL的合成,将材料3g PCL-PEG-NH2溶于二氯甲烷,加入的0.067g丁二酸酐,在常温下搅拌反应24h,乙醇沉降三次,真空干燥,得PCL-PEG-COOH。将2g PCL-PEG-COOH和0.085g EDC·HCl(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)和0.051g NHS(N-羟基琥珀酰亚胺)搅拌,在室温下反应12h。反应结束后乙醇沉降,真空干燥,得PCL-PEG-NHS。0.3g PCL-PEG-NHS和12mg RGD加入到10mL DMF中在室温下反应24h,放入Mw=3500的透析袋中透析48h,冻干,得RGD-PEG-PCL。
所述的固定RGD和HA的纤维膜的步骤和条件如下:
纺丝溶液的配制,将PLGA、HA-g-PLLA、PCL-PEG-RGD按质量比1:0:0, 95:5:0,90:5:5以氯仿为溶剂配置成浓度为7%、8%、8%的混合溶液,超声分散30min后,搅拌10h。在室温下,在注射器中装入纺丝溶液后将其固定在微量注射泵上,使注射器活塞与泵的推进部分紧密接触,将静电发生器的输出电缆接到注射器前端的9号金属针头上并将接收装置(铝箔)接地,调节针头与接收装置的距离为15cm。启动微量注射泵,流速设为0.2mL/h,使纺丝溶液在毛细管口形成半球形液滴。电源电压为20kV。纺丝完成后将纤维膜取下,真空干燥,获得同时固定RGD和HA的纤维膜。
纤维膜生物性能的测量。各种纤维膜(纯PLGA,HA-g-PLLA/PLGA 和PCL-PEG-RGD/HA-g-PLLA/PLGA的)被分别涂到15mm的圆玻片上,圆玻片已用2%的二甲基二氯硅烷预处理并在180℃加热4h后使用。制备好的样品,在室温下真空干燥48h,然后用紫外辐射灭菌30min。
细胞黏附实验:将培养好的成骨细胞接种在24孔细胞培养板上,每孔为2×104个细胞。在37℃,5%CO2细胞培养箱中分别培养12、24和48 h。在不同的时间间隔取出样品,吸弃培养基,用PBS缓冲液洗涤三次。然后将样品分别用4%多聚甲醛溶液固定10 min,并用PBS洗涤三次。加入浓度为0.2mg/mL-1的异硫氰酸荧光素,染色10min,用PBS缓冲液洗涤5次,细胞成功着色。用荧光倒置显微镜对每个样品的细胞黏附的形态进行观察。使用数码相机对每个样品9个位置拍照然后用NIHJ图像分析软件得到每个样品上的成骨细胞的平均黏附率。
增殖实验:上述材料中的细胞存活率评价使用噻唑蓝(MTT)法。将样品放置在24孔培养板中,在紫外线辐射下灭菌30 min。每个样品做6个平行样。在37℃,5%CO2的细胞培养箱中分别培养为1、3和7天,分别在24孔板中培养成骨细胞(每孔2×104个细胞)。培养基每2天更换一次。每孔加入50μL的MTT溶液并孵育4h。吸弃培养基每孔加入500μL的盐酸异丙醇溶液,充分震荡各孔中的溶液。每孔取出200μL溶液并转移至另一个96孔板中,然后在540nm的波长上使用酶标仪测定溶液的吸光值。使用6个样品的读数的平均值作为最终结果。
有益效果:本发明通过固定接枝修饰的RGD和HA调整纤维膜的生物性能,增强细胞黏附,使细胞获得较好的生长环境,有利于细胞增殖。
附图说明:图1:实施例2 RGD的400M核磁氢谱。
图2:实施例2所制得RGD-PEG-PCL的1H NMR图谱。
图3:实施例3所制得PLGA纤维膜、HA-g-PLLA/PLGA纤维膜、HA-g-PLLA/RGD-PEG-RGD/PLGA纤维膜的SEM图片。
图4:实施例3所制得PLGA纤维膜、HA-g-PLLA/增加PLGA纤维膜、HA-g-PLLA/ RGD-PEG-PCL/PLGA纤维膜的水接触角数据图。
图5:实施例3所制得PLGA、HA-g-PLLA/PLGA 和HA-g-PLLA/PCL-PEG-RGD/PLGA表面MC3T3细胞黏附面积图。
图6:实施例3所制得PLGA、PLGA/HA-g-PLLA and PLGA/HA-g-PLLA/PCL-PEG-RGD表面细胞在不同时间的MTT实验数据。
如图1所示,实施例2 RGD的400M核磁氢谱。
如图2所示,实施例2所制得RGD-PEG-PCL的1H NMR图谱,图中b=1.78处为RGD中-CH2CH(NH2)CO-中亚甲基的氢振动峰,e =2.55处为RGD中-CHCH2CO-中亚甲基的氢振动峰,f=3.88处为RGD中-NHCH2CO-中亚甲基的氢振动峰,说明RGD成功接枝。
如图3所示,实施例3所制得PLGA纤维膜、HA-g-PLLA/PLGA纤维膜、HA-g-PLLA/RGD-PEG-RGD/PLGA纤维膜的SEM图片,三种纤维膜中的纤维分布比较均匀,纤维表面较光滑,没有明显的结点出现,纤维的直径在1μm左右。
如图4所示,实施例3所制得PLGA纤维膜、HA-g-PLLA/增加PLGA纤维膜、HA-g-PLLA/RGD-PEG-PCL/PLGA纤维膜的水接触角数据图,固定RGD-PEG-PCL的纤维膜比HA-g-PLLA/PLGA和PLGA纤维膜呈现出了亲水性增加的趋势。
如图5所示,随着培养时间的增长,实施例3所制得三种纤维膜表面的细胞逐渐伸展,细胞面积不断增大。培养24h时,RGD-PEG-PCL/HA-g-PLLA/PLGA纤维膜和HA-g-PLLA/PLGA纤维膜的细胞面积比(Area fraction)都明显高于PLGA纤维膜并有显著性差异(P<0.05)。在培养48h时,RGD-PEG-PCL/HA-g-PLLA/PLGA纤维膜的细胞面积比高于HA-g-PLLA/PLGA 纤维膜和PLGA纤维膜。表明同时固定RGD和HA的纤维膜更有利于细胞黏附。
如图6所示,实施例3所制得PLGA、HA-g-PLLA/PLGA和 RGD-PEG-PCL/HA-g-PLLA/PLGA表面细胞在不同时间的MTT实验数据,随着培养时间的增长,各种纤维膜上的细胞活力和细胞个数在增加,RGD-PEG-PCL/HA-g-PLLA/PLGA 和HA-g-PLLA/PLGA纤维膜的增殖情况优于PLGA纤维膜。培养3天时,HA-g-PLLA/PLGA与RGD-PEG-PCL/HA-g-PLLA/PLGA有显著性差异。培养7天时,RGD-PEG-PCL/HA-g-PLLA/PLGA纤维膜的细胞数量明显多于其他两种纤维膜。这表明成骨细胞在固定RGD和HA纤维膜上的增殖能力和细胞活性优于HA-g-PLLA/PLGA和PLGA纤维膜。
具体实施方式:实施例1:HA-g-PLLA纳米粒子的制备
在HA表面接枝PLLA的制备方法和结构参阅专利发明人论文,纳米羟基磷灰石表面接枝聚合左旋丙交酯,高分子学报,(1)2013。
实施例2:RGD-PEG-PCL的制备
2.5g PCL端羧基活化后,与3g含双端氨基PEG反应,获得PCL-PEG-NH2。3g PCL-PEG-NH2溶于二氯甲烷,加入的0.067g丁二酸酐反应,得PCL-PEG-COOH。2g PCL-PEG-COOH和0.085g EDC·HCl(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)和0.051g NHS(N-羟基琥珀酰亚胺)搅拌,得PCL-PEG-NHS。0.3g PCL-PEG-NHS和12mg RGD加入到10mL DMF中在室温下反应24h,放入Mw=3500的透析袋中透析48h,冻干,得RGD-PEG-PCL。
实施例3:固定RGD-PEG-PCL和HA-g-PLLA的PLGA纤维膜的制备
PLGA、HA-g-PLLA和RGD-PEG-PCL按质量比1:0:0, 95:5:0,90:5:5,以氯仿为溶剂配制成浓度为7%,8%,8%的混合纺丝溶液,超声分散30min后,搅拌10h。在室温下,在注射器中装入纺丝溶液后将其固定在微量注射泵上,使注射器活塞与泵的推进部分紧密接触,将静电发生器的输出电缆接到注射器前端的9号金属针头上并将接收装置(铝箔)接地,调节针头与接收装置的距离为15cm。启动微量注射泵,流速设为0.2mL/h,使纺丝溶液在毛细管口形成半球形液滴。电源电压为20kV。纺丝完成后将纤维膜取下,真空干燥,获得RGD体相改性纤维膜。
Claims (2)
1.同时固定RGD和HA纤维膜的特征在于分别对RGD、HA进行修饰制备RGD-PEG-PCL和HA-g-PLLA,然后将PLGA、HA-g-PLLA和RGD-PEG-PCL均匀共混,电纺成纤维膜,由于PLLA、PCL与PLGA具有良好的相容性,因而使HA和RGD固定于PLGA纤维上,促进细胞粘附于合成材料表面,增强纤维膜的生物性能,所述PLGA由80%丙交酯和20%乙酸乙交酯组成,数均分子量120,000。
2.如权利要求1所述的纤维膜,其特征在于用于组织工程,特别是引导骨再生膜。
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