CN108434520A - 一种引导骨组织再生的支架的制备方法 - Google Patents
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Abstract
本发明公开了一种引导骨组织再生的支架的制备方法:制备聚乳酸‑羟基乙酸的二氯甲烷溶液,将酒精溶液添加到聚乳酸‑羟基乙酸的二氯甲烷溶液中制备W/O型初乳,再制备聚乙烯醇的水溶液,混合聚乙烯醇水溶液和W/O型初乳,制成W/O/W复乳,对复乳进行搅拌固化、冷冻干燥,制成聚乳酸‑羟基乙酸微囊,将微囊在吐温、二氯甲烷和水的混合液中进行融合连接,最终制成聚乳酸‑羟基乙酸微囊支架。本发明制备的微囊支架具有机械强度高,且其上培养细胞的细胞活性值高等优点。
Description
技术领域
本发明属于生物医学材料领域,具体地,涉及一种高分子生物医学材料,更具体地,涉及一种引导骨组织再生的支架的制备方法。
背景技术
组织工程,是一门以细胞生物学和材料科学相结合,进行体外或体内建构组织或器官的新兴学科,其核心在于建立细胞与生物材料的三维空间复合体,用以对病损组织进行形态、结构和功能三个方面的重建,并达到永久性替代。骨组织工程是指将分离的自体高浓度成骨细胞、骨髓基质干细胞或软骨细胞,经体外培养扩增后种植于某种细胞支架或细胞外基质上,然后将这种细胞杂化材料植入骨缺损部位,在生物材料逐步降解的同时,种植的成骨细胞不断增殖,从而达到修复骨组织缺损的目的。
用于骨缺损修复的材料主要有生物无机材料和有机高分子材料两大类。有机高分子材料中,由乳酸和羟基乙酸随机聚合而成的聚乳酸-羟基乙酸(poly(lactic-co-glycolic acid),PLGA)的研究较为广泛,其具有良好的生物相容性、生物可降解性以及降解速率可调节性等优点,而且,PLGA通过酯键水解降解为乳酸和羟基乙酸,最终降解为二氧化碳和水排出体外。
现有的技术主要采用加热工艺使支架成形,这种高温的应用会使一些蛋白质类的药物变性,限制了载药的种类;同时,由于大型加热的设备较少,加热工艺的选择也限制了支架的制作。虽然关于微球载药支架已经有了一些研究,但是微囊支架仍少见报道,微囊支架载药更是如此。
微囊系指固态或液态药物被高分子材料包封形成的微小囊状粒子。目前已形成的微囊制备方法主要分为化学法、物理化学法和物理法三大类。其中,复乳法是在传统的乳化溶剂挥发法的基础上改进形成的,其制备原理是选择一个与水不相混溶的沸点低、蒸气压比水高的溶媒,将囊材溶于其中,然后将包裹物水溶液分散在上述溶液中,形成油包水(W/O)型乳剂,另外配制一个含有稳定剂、保护胶的水溶液作为包囊溶液,在搅拌情况下,将此溶液加到上述乳剂中,形成水包油包水(W/O/W)型复乳,再是溶剂挥发掉,即所谓的W/O/W型复乳水包物干燥法。
中国专利公布CN103550824A一种引导骨组织再生的支架的制备方法公开了一种用于骨组织工程的聚乳酸-羟基乙酸微囊支架的制备方法:将聚乳酸-羟基乙酸溶于二氯甲烷中,向其中加入适量蒸馏水,超声振荡,得到均质初乳,再将其倒入一定量的聚乙烯醇水溶液中,搅拌至完全固化,高速离心、洗涤、冷冻干燥,得到聚乳酸-羟基乙酸微囊;将聚乳酸-羟基乙酸微囊用试验筛筛选出目标粒径的微囊,填充到模具中,采用二氯甲烷蒸气使其融合成型,冷冻干燥后制得聚乳酸-羟基乙酸微囊支架。但是,这种方法制备得到的聚乳酸-羟基乙酸微囊支架的机械强度较低,而且这种支架上生长的细胞的前期细胞活性值较低。
中国专利公布CN103610664A公开了一种制备卡马西平聚乳酸-羟基乙酸共聚物微胶囊的方法:将卡马西平溶解在超临界二氧化碳中,用溶解有卡马西平的超临界二氧化碳对聚乳酸-羟基乙酸共聚物进行塑化溶胀,待卡马西平在超临界二氧化碳和聚乳酸-羟基乙酸共聚物之间达到分配平衡后,逸出二氧化碳,制成卡马西平聚乳酸-羟基乙酸共聚物微胶囊。但是,这种方法主要用于制备载药型聚乳酸-羟基乙酸微胶囊,并没有对聚乳酸-羟基乙酸微囊本身的性能进行改进,而且制备超临界状态的二氧化碳对压力要求较高。
所以,目前需要一种能够制备机械强度高,且能够提高其上生长细胞细胞活性值的聚乳酸-羟基乙酸微囊支架的方法。
发明内容
本发明提供的一种引导骨组织再生的支架的制备方法,相比于现有技术,本发明的方法能够制备一种内部为空腔,仅外围是聚乳酸-羟基乙酸囊材的微囊结构,在提高微囊支架孔隙率的同时保持支架较高的机械强度和其上生长细胞的细胞活性值。
说明:本发明中,除另有说明外,所用的水均为去离子水。
本发明公开了一种引导骨组织再生的支架的制备方法,该方法包括以下步骤:
(1)将聚乳酸-羟基乙酸溶解于二氯甲烷中,得到聚乳酸-羟基乙酸的二氯甲烷溶液;
(2)将酒精溶液加入到步骤(1)得到的聚乳酸-羟基乙酸的二氯甲烷溶液中,通过涡旋和超声振荡制备成均匀的初乳;
(3)将聚乙烯醇加入去离子水中,加热到60-90℃,高速搅拌至完全溶解,制得聚乙烯醇溶液;
(4)将步骤(3)得到的聚乙烯醇溶液缓慢添加到步骤(2)得到的初乳中,搅拌至完全固化,离心,去离子水洗涤,冷冻干燥,得到聚乳酸-羟基乙酸微囊;
(5)将步骤(4)得到的聚乳酸-羟基乙酸微囊用试验筛筛出目标粒径的微囊;
(6)先将吐温80和二氯甲烷进行混合,制备吐温80和二氯甲烷的混合溶液,再将该混合溶液和去离子水进行混合,制备微囊融合液;
(7)将步骤(5)制备得到的聚乳酸-羟基乙酸微囊填充到聚四氟乙烯模具中,再将模具放入盛有微囊融合液的容器中1-2min,从模具中取出,去离子水清洗,冷冻干燥,制得聚乳酸-羟基乙酸微囊支架。
进一步地,所述步骤(1)中,聚乳酸-羟基乙酸和二氯甲烷的质量体积比为0.15g/mL-0.3g/mL;优选地,所述步骤(1)中聚乳酸-羟基乙酸和二氯甲烷的质量体积比为0.16-0.24g/mL。
进一步地,所述步骤(2)中,酒精溶液中酒精和水的体积比为1:6-15;优选地,所述步骤(2)中,酒精溶液中酒精和水的体积比为1:8-10。
进一步地,所述步骤(2)中,酒精溶液和聚乳酸-羟基乙酸二氯甲烷溶液的体积比为1:4-15;优选地,所述步骤(2)中,酒精溶液和聚乳酸-羟基乙酸二氯甲烷溶液的体积比为1:8-13。
进一步地,所述步骤(2)中,涡旋转速为2000r/min,振幅为6mm,涡旋时间为10-50s,超声振荡功率为100-500W,超声时间为10s-10min。
进一步地,所述步骤(3)中,聚乙烯醇和去离子水的质量体积比为0.5-4g/L;优选地,所述步骤(3)中,聚乙烯醇和去离子水的质量体积比为1.5-3g/L。
进一步地,所述步骤(4)中,初乳和聚乙烯醇溶液的体积比为15-30:100;优选地,所述步骤(4)中,初乳和聚乙烯醇溶液的体积比为18-25:100。
进一步地,所述步骤(5)中,试验筛目数为50-400目。
进一步地,所述步骤(6)中,每200mL的二氯甲烷中添加1-4滴的吐温80;优选地,每200mL的二氯甲烷中添加1-2滴吐温80。
进一步地,所述步骤(6)中,吐温80和二氯甲烷的混合液与去离子水的体积比为1:30-90;优选地,所述步骤(6)中,吐温80和二氯甲烷的混合液与去离子水的体积比为1:40-70。
与现有技术相比,本发明的有益效果为:
(1)本发明采用复乳法制备W/O/W型结构,通过挥发有机溶剂,得到了内部中空,仅外围是聚乳酸-羟基乙酸材料的聚乳酸-羟基乙酸微囊,能够为细胞提供较大的附着表面。
(2)本发明制备的内部中空的聚乳酸-羟基乙酸微囊,一方面可以加快聚乳酸-羟基乙酸微囊的降解速度,另一方易于装载各类药物以及细胞因子。
(3)本发明通过将聚乳酸-羟基乙酸的二氯甲烷溶液和酒精溶液进行混合制备W/O结构,进而制得最终的微囊支架产品,提高了微囊支架的机械强度。
(4)本发明通过微囊融合液对制得的微囊进行溶解连接,不仅提高了微囊支架上生长细胞的细胞活性值,而且提高了支架的机械强度。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
基础实施例
一种引导骨组织再生的支架的制备方法,该方法包括以下步骤:
(1)将聚乳酸-羟基乙酸溶解于二氯甲烷中,得到聚乳酸-羟基乙酸的二氯甲烷溶液,其中聚乳酸-羟基乙酸和二氯甲烷的质量体积比为0.15g/mL-0.3g/mL;
(2)将酒精和水的体积比为1:6-15的酒精溶液加入到步骤(1)得到的聚乳酸-羟基乙酸的二氯甲烷溶液中,其中酒精溶液和聚乳酸-羟基乙酸二氯甲烷溶液的体积比为1:10,先以2000r/min的涡旋速度涡旋40s,再以振幅为6mm、功率为400W的强度超声5min,制备成均匀的初乳;
(3)将聚乙烯醇加入去离子水中,加热到60-90℃,高速搅拌至完全溶解,制得质量体积比为3g/L的聚乙烯醇溶液;
(4)将步骤(3)得到的聚乙烯醇溶液缓慢添加到步骤(2)得到的初乳中,其中,初乳和聚乙烯醇溶液的体积比为15-30:100,搅拌至完全固化,离心,去离子水洗涤,冷冻干燥,得到聚乳酸-羟基乙酸微囊;
(5)将步骤(4)得到的聚乳酸-羟基乙酸微囊用试验筛筛出目标粒径的微囊;
(6)先将1-4滴吐温80和200mL的二氯甲烷进行混合,制备吐温80和二氯甲烷的混合溶液,再将1体积份的该混合溶液和30-90体积份的去离子水进行混合,制备微囊融合液;
(7)将步骤(5)制备得到的聚乳酸-羟基乙酸微囊填充到聚四氟乙烯模具中,再将模具放入盛有微囊融合液的容器中1-2min,从模具中取出,去离子水清洗,冷冻干燥,制得聚乳酸-羟基乙酸微囊支架。
通过调整基础实施例中的具体实验参数,得到具体实施例1-9,如表1-3所示。为了突出本发明的有益效果,进一步设置了对比例1-2,其中对比例1如表2所示。
通过调整聚乳酸-羟基乙酸和二氯甲烷的质量体积比、酒精和水的体积比、初乳和聚乙烯醇溶液的体积比、200mL二氯甲烷中吐温的添加量以及二氯甲烷吐温混合液和水的体积比,得到了表1中的
实施例1-4。实验数据显示,采用本发明的方法制备的聚乳酸-羟基乙酸微囊支架的机械强度为0.07MPa左右,其上生长的细胞,第1d的细胞活性值为2.72左右,第9d的细胞活性值为3.31左右。
表1
为了探究酒精添加量对聚乳酸-羟基乙酸微囊支架机械强度的影响,根据酒精添加量不同,本发明设置了表2中的实施例3、实施例5-7和对比例1。实施例6、实施例5、实施例3和实施例7中酒精和水的体积比依次减小,即酒精的添加量依次减少,对应的聚乳酸-羟基乙酸微囊支架的机械强度呈现依次减小的趋势。将实施例3和实施例5-7与对比例1进行比较,可以发现,酒精和水的体积比为0:10也就是制备过程中没有添加酒精的对比例1中制备的支架的机械强度明显低于实施例中的机械强度。所以,表2中的实验数据表明,在聚乳酸-羟基乙酸微囊支架的制备过程中,添加一定量的酒精,可以提高微囊支架的机械强度。
表2
对比例2采用以下步骤制备聚乳酸-羟基乙酸微囊支架:
(1)将聚乳酸-羟基乙酸溶解于二氯甲烷中,得到聚乳酸-羟基乙酸的二氯甲烷溶液,其中聚乳酸-羟基乙酸和二氯甲烷的质量体积比为0.25g/mL;
(2)将酒精和水的体积比为1:10的酒精溶液加入到步骤(1)得到的聚乳酸-羟基乙酸的二氯甲烷溶液中,其中酒精溶液和聚乳酸-羟基乙酸二氯甲烷溶液的体积比为1:10,先以2000r/min的涡旋速度涡旋40s,再以振幅为6mm、功率为400W的强度超声5min,制备成均匀的初乳;
(3)将聚乙烯醇加入去离子水中,加热到60-90℃,高速搅拌至完全溶解,制得质量体积比为3g/L的聚乙烯醇溶液;
(4)将步骤(3)得到的聚乙烯醇溶液缓慢添加到步骤(2)得到的初乳中,其中,初乳和聚乙烯醇溶液的体积比为15:100,搅拌至完全固化,离心,去离子水洗涤,冷冻干燥,得到聚乳酸-羟基乙酸微囊;
(5)将步骤(4)得到的聚乳酸-羟基乙酸微囊用试验筛筛出目标粒径的微囊;
(6)将筛选出的聚乳酸-羟基乙酸微囊填充到聚四氟乙烯模具中,再将模具放入含有以液体体积计为10mL的二氯甲烷蒸汽的容积为385mL的玻璃容器中,3min后将处理过的聚乳酸-羟基乙酸为囊取出,-45℃冷冻干燥48h,制得聚乳酸-羟基乙酸微囊支架。
该实施例微囊支架的机械强度为0.04MPa,培养1d的细胞活性值为2.45,培养9d的细胞活性值为2.67。
和实施例3相比,对比例2的不同之处在于对比例2将目标粒径的聚乳酸-羟基乙酸微球置于二氯甲烷蒸汽环境中,对其进行融合连接。对比例2制备的微囊支架的机械强度小于对实施例3中微囊支架的机械强度,对比例2制备的微囊支架上培养细胞的活性值也明显低于实施例3中微囊支架上培养细胞的活性值,这说明,采用本发明的方法,对聚乳酸-羟基乙酸微囊进行融合连接,有利于提高微囊支架的机械强度和其上生长细胞的活性值。
表3中的实验数据表明,在微囊支架的制备过程中,吐温的添加量会对微囊支架的机械强度产生影响,具体的表现为,在本发明的添加量范围内,吐温的添加量越低,微囊支架的机械强度越高。
表3
Claims (10)
1.一种引导骨组织再生的支架的制备方法,其特征在于,该方法包括以下步骤:
(1)将聚乳酸-羟基乙酸溶解于二氯甲烷中,得到聚乳酸-羟基乙酸的二氯甲烷溶液;
(2)将酒精溶液加入到步骤(1)得到的聚乳酸-羟基乙酸的二氯甲烷溶液中,通过涡旋和超声振荡制备成均匀的初乳;
(3)将聚乙烯醇加入去离子水中,加热到60-90℃,高速搅拌至完全溶解,制得聚乙烯醇溶液;
(4)将步骤(3)得到的聚乙烯醇溶液缓慢添加到步骤(2)得到的初乳中,搅拌至完全固化,离心,去离子水洗涤,冷冻干燥,得到聚乳酸-羟基乙酸微囊;
(5)将步骤(4)得到的聚乳酸-羟基乙酸微囊用试验筛筛出目标粒径的微囊;
(6)先将吐温80和二氯甲烷进行混合,制备吐温80和二氯甲烷的混合溶液,再将该混合溶液和去离子水进行混合,制备微囊融合液;
(7)将步骤(5)制备得到的聚乳酸-羟基乙酸微囊填充到聚四氟乙烯模具中,再将模具放入盛有微囊融合液的容器中1-2min,从模具中取出,去离子水清洗,冷冻干燥,制得聚乳酸-羟基乙酸微囊支架。
2.根据权利要求1所述的一种引导骨组织再生的支架的制备方法,其特征在于,所述步骤(1)中,聚乳酸-羟基乙酸和二氯甲烷的质量体积比为0.15g/mL-0.3g/mL。
3.根据权利要求1所述的一种引导骨组织再生的支架的制备方法,其特征在于,所述步骤(2)中,酒精溶液中酒精和水的体积比为1:6-15。
4.根据权利要求1所述的一种引导骨组织再生的支架的制备方法,其特征在于,所述步骤(2)中,酒精溶液和聚乳酸-羟基乙酸二氯甲烷溶液的体积比为1:4-15。
5.根据权利要求1所述的一种引导骨组织再生的支架的制备方法,其特征在于,所述步骤(2)中,涡旋转速为2000r/min,振幅为6mm,涡旋时间为10-50s,超声振荡功率为100-500W,超声时间为10s-10min。
6.根据权利要求1所述的一种引导骨组织再生的支架的制备方法,其特征在于,所述步骤(3)中,聚乙烯醇和去离子水的质量体积比为0.5-4g/L。
7.根据权利要求1所述的一种引导骨组织再生的支架的制备方法,其特征在于,所述步骤(4)中,初乳和聚乙烯醇溶液的体积比为15-30:100。
8.根据权利要求1所述的一种引导骨组织再生的支架的制备方法,其特征在于,所述步骤(5)中,试验筛目数为50-400目。
9.根据权利要求1所述的一种引导骨组织再生的支架的制备方法,其特征在于,所述步骤(6)中,每200mL的二氯甲烷中添加1-4滴的吐温80。
10.根据权利要求1所述的一种引导骨组织再生的支架的制备方法,其特征在于,所述步骤(6)中,吐温80和二氯甲烷的混合液与去离子水的体积比为1:30-90。
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