CN111825859A - 一种具有自修复功能的仿生电子皮肤医用支架材料及其制备方法 - Google Patents
一种具有自修复功能的仿生电子皮肤医用支架材料及其制备方法 Download PDFInfo
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Abstract
本发明涉及明胶基生物医用材料领域,具体为一种具有自修复功能的仿生电子皮肤医用支架的制备方法和应用,其特征在于以从生物安全性较好的脱细胞胎牛皮真皮基质提取的明胶为原料,再与生物相容性良好的导电高分子聚3,4‑乙烯二氧噻吩:聚苯乙烯磺酸盐(PEDOT:PSS)溶液和自制的酸化碳纳米管(H‑MWCNTs)复合,通过1‑(3‑二甲氨基丙基)‑3‑乙基碳二亚胺盐酸盐(EDC)交联得到一种具有多功能性的三维网络空间结构的水凝胶支架,该水凝胶生物相容性好,力学强度高,且兼具自修复性能和诱导组织再生修复性能,可广泛应用于皮肤创伤修复治疗。
Description
技术领域
本发明公开了一种具有自修复功能的仿生电子皮肤支架的制备方法和应用,属于生物医用材料领域。
背景技术
皮肤是人体最大的器官,是人体与外界环境相接触的屏障,皮肤屏障是指皮肤表层由皮脂膜和角质层组成的天然防线,既可对外来刺激进行有效防御也可锁住皮肤内水分。在现实生活中,会因为烧伤、创伤、糖尿病、慢性溃疡等原因造成皮肤的缺损,而人体无法通过自身完全愈合任何直径大于4 cm的全层皮肤缺损。
组织工程皮肤以三维支架为载体,通过将细胞种植在支架上而获得,可以从根本上解决重建或修复皮肤屏障的问题,具有良好的发展前景。支架材料作为组织工程的三要素之一,按化学性质可将其分为合成类和生物类。合成类支架材料大多为聚乳酸、聚L乳酸、聚羟基乙酸等聚酯类,其最大的问题是缺乏细胞信号识别,不利于细胞粘附及特异基因的激活,需要在材料表面接枝特异识别位点(RGD序列)以增加支架的细胞生物活性,从而增加支架材料的成本和制作难度。甲壳素、壳聚糖、海藻酸盐、胶原蛋白、透明质酸、明胶、琼脂等天然高分子因为其本身具有相同或类似于细胞外基质的结构,可以促进细胞的黏附,增殖和分化,所以常用作于生物类支架材料。其来源较为广泛,制作简单,且价格低廉。但它也存在力学性能较差,抗原性消除不确定,降解速率不宜控制等问题。
另一方面,活体组织中存在电场,跨表皮电压在20~50 mV之间,皮肤表皮电压较低。通常情况下,当损伤发生时,在深部组织和皮肤表面之间会产生“损伤电流”。研究表明,“损伤电流”可能通过吸引参与修复的细胞、改变细胞膜的通透性、增加细胞的分泌产物和指导细胞结构的重新塑性。将微电流应用于各种皮肤创伤治疗的研究表明,微电流能激活心肌及其他组织的再生过程。其调控机制为:(1)增加BMP6的分泌;(2)下调核因子rd3活性;(3)上调血管内皮生长因子mRNA的表达。
发明内容
综上所述,首先,理想的组织支架应具备以下特点:(1)良好的生物相容性,无明显的排斥反应和炎症反应,无疾病传播的风险;(2)允许细胞再起表面粘附,促进细胞增殖、分化;(3)合适的降解速度,且降解产物无毒,可吸收或可及时排除体外;(4)具有高孔隙率的三维立体结构从而为细胞的粘附、细胞外基质的再生和细胞扩散提供足够的空间;(5)具有一定的机械强度,能够抵抗一定的组织应力,起到支撑和模板作用。
其次,考虑到微电流对刺激组织再生的作用,进一步提出仿生电子皮肤医用支架材料。该具有导电性的支架材料可以通过定期加载外置电源增强微电流的作用,促进细胞的增殖。导电支架材料主要通过传递微电流实现促进细胞再生的目的。
进一步地,在细胞粘附增殖过程中,支架材料难免组织应力的作用,将不可避免地产生损伤和微裂纹,并由此引发宏观裂缝而发生断裂。如果我们能对这种早期损伤和裂纹进行修复,那么对于消除安全隐患、增强材料强度和使用寿命具有重大意义,赋予水凝胶支架材料自修复能力可以大大提高其使用寿命和功能的可靠性。故对该支架材料进一步提出自修复功能的要求。
基于此,针对上述目的,本发明以从生物安全性较好的脱细胞胎牛皮真皮基质提取的明胶为基材,复合无细胞毒性的导电高分子聚3,4-乙烯二氧噻吩:聚苯乙烯磺酸盐(PEDOT:PSS)溶液和自制的酸化碳纳米管(H-MWCNTs),再通过1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)交联制备了一种具有自修复功能的仿生电子皮肤医用支架。明胶含有大量RDG序列及官能团,可促进细胞的粘附和生长;PEDOT:PSS溶液具有高导电性,可提高材料的导电率;酸化碳纳米管不仅可以进一步提高材料的导电性,还可作为水凝胶支架的增强填料,提高水凝胶支架的力学性能;EDC交联后水凝胶支架的机械强度得到大幅度提升,足以应对组织应力。该水凝胶生物相容性好,力学强度高,且兼具自修复性能和诱导组织再生修复性,可广泛应用于皮肤创伤修复治疗。
其中明胶作为胶原的水解产物,具有以下优势:(1)较于胶原的免疫原性大大降低;(2)具有高生物相容性和可降解性,并且含有大量的RGD序列,有利于细胞的粘附和生长;(3)具有温度可逆性,可通过调节温度制备抗张强度高,具有类似真皮形态结构的水凝胶材料;(4)分子中含有大量不同种类的官能团,可以通过化学修饰的方法对其进行调控或负载药物等以增强其使用价值;(5)另外,明胶来源广泛、价格便宜,更具有实际应用价值。
因此,本发明的目的是结合明胶和微电流对于促进细胞再生的优势构建一种具有自修复功能的水凝胶材料,作为新一代仿生电子皮肤医用支架。
为了实现上述目的,本发明采用的技术方案是:选用以从清除抗原后的胎牛皮脱细胞真皮基质提取的明胶为原料,再与无细胞毒性的导电高分子聚3,4-乙烯二氧噻吩:聚苯乙烯磺酸盐(PEDOT:PSS)溶液和自制的酸化碳纳米管(H-MWCNTs)复合,通过1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)交联得到具有多功能性的三维网络状水凝胶,制备了一种具有自修复功能的仿生电子皮肤医用支架,可应用于医学临床。
具体地,由包括如下步骤的方法获得:
步骤一:(1)酸化碳纳米管分散液的制备:准确称取1.0~5.0 g 多壁碳纳米管(MWCNTs)、50.0~250.0 g浓H2SO4 (98%)、20.0~100.0 g HNO3 (65~68%)于三口烧瓶中,升温至50℃~100℃,反应时间为5~15小时;反应完全后采用梯度转速离心,梯度转速离心条件为:①1000~5000 rpm的转速离心10~60 min,过滤;②加入100.0~500.0 mL超纯水,以5000~8000 rpm的转速离心10~60 min,过滤;③加入100.0~500.0 mL超纯水,以8000~10000 rpm的转速离心10~60 min,每个离心步骤重复操作3~5次,冷冻干燥储存;称取0.1~1.0 g酸化碳纳米管粉末,于50.0~200 mL超纯水中超声0.5~2.0 h,制备得到0.05~2.0% (m/v)的酸化多壁碳纳米管分散液,储存备用;
步骤二:酸化碳纳米管/PEDOT:PSS复合分散液的制备:量取10.0~50.0 mL超纯水于单口烧瓶中,并移取1.0~5.0 mL PEDOT:PSS溶液以及1.0~5.0 mL 0.05~1.0%酸化碳纳米管分散液于烧瓶中,以50~100 W,40 kHz超声30~120 min;
步骤三:明胶基复合导电凝胶溶液的制备:准确称取1.0~10.0g明胶于上述混合溶液中,升温至30~60℃,恒温搅拌30~120 min,使明胶完全溶解,体系分散均匀;
步骤四:1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)交联明胶基复合导电凝胶的制备:准确称取0.1~0.5 g EDC粉末溶于1.0~5.0 mL H2O中得到2~50% (m/v)的EDC溶液;准确移取0.1~2.0 mL 2~50%的EDC溶液缓慢滴入步骤(3)中的复合溶液体系中,继续恒温搅拌30~120 min,得到EDC交联改性的明胶基复合导电凝胶溶液;
步骤五:将完成反应后的混合溶液,倒入定制的聚四氟乙烯模具成型,并放入-4~4℃的冰箱陈化12~24 h,取出后用pH为7.0~7.4的PBS溶液浸泡12~24 h,最终得到一种具有自修复功能的仿生电子皮肤医用支架。
本发明的进一步改进在于,步骤(1)中所制备的酸化碳纳米管在水中具有良好的分散性、稳定性及导电性。
本发明的进一步改进在于,步骤(2)中提前将酸化碳纳米管分散液以及PEDOT:PSS溶液混合溶液进行超声分散,使其能够在水凝胶均匀分散,有利于后续得到均相凝胶溶液,从而提高水凝胶支架的导电性。
本发明的进一步改进在于,步骤(3)中所用的明胶具有高生物相容性以及大量RGD序列,并且具有诱导细胞粘附、生长,是优良的电子皮肤支架基材。
本发明的进一步改进在于,步骤(4)中的EDC能够活化明胶分子链上的氨基和羧基,使其产生酰胺键,从而达到交联明胶的目的。
本发明的进一步改进在于,步骤(5)所制备的水凝胶溶胶适用于各种形状的模具,得到任意形状的支架材料。
本发明的进一步改进在于,步骤(5)所制备的支架材料,通过低温陈化进一步提升去机械性能,并结合 PBS浸泡后去除反应副产物尿素,从而较少其细胞毒性,使其可作为医用支架材料应用于伤口临床治疗。
本发明的进一步改进在于,步骤(5)所制备了具有自修复功能的电子皮肤支架,可以应对组织应力的损伤,及时修复裂纹,减少其对细胞粘附、增殖的不良影响。
本发明的进一步改进在于,步骤(5)所制备的具有导电功能的医用支架材料可结合生物微电流,给予定期定量的微电流刺激,可加强微电流的刺激作用,从而促进细胞的生长和分化。
本技术与现有技术相比,具有如下优点:
(1)本发明以从清除抗原后的胎牛皮脱细胞真皮基质提取的明胶为原料,明胶分子含有大量RGD序列,能够促进细胞的粘附和生长并诱导细胞的增殖和分化,是优异的仿生皮肤支架基材;
(2)本发明制备的明胶基水凝胶支架具有高孔隙率的三维立体结构,从而为细胞的粘附、细胞外基质的再生和细胞扩散提供足够的空间,且具有一定的机械强度,能够抵抗一定的组织应力,起到支撑和模板作用;
(3)本发明制备的电子皮肤医用支架,与传统的支架材料相比,最明显的区别是具有导电性,微电流促进细胞生长、增殖、分化,增加其作为支架材料的优势;
(4)本发明所制备的水凝胶支架具有良好的拉伸性能及自修复性能,可应对组织应力的变形和微裂纹进行自我修复,更有利于保护细胞形态,防止细胞从支架上脱落;
(5)本发明制备的电子皮肤医用支架材料含有大量的活性官能团,可接枝负载不同类型的生长因子及药物,得到缓释、温敏的多功能支架材料,具有应用于术后修复及创伤愈合的新型智能医用材料的前景。
附图说明
图1为仿生电子皮肤医用支架的实物图:该支架材料胶具有良好的可塑性、导电性、拉伸及压缩性能;
图2为仿生电子皮肤医用支架的扫描电镜图(SEM):该支架材料具有孔径约为200 μm的多孔结构,能为皮肤组织细胞的生长提供3D空间;
图3仿生电子皮肤医用支架的体外溶胀性能图:该水凝胶具有良好的溶胀性能,可以充分吸收创面渗出液,减少细菌感染风险;
图4仿生电子皮肤医用支架自愈合性能宏观照片:将圆片水凝胶某一条半径切断,37℃进行自愈合,10 min后水凝胶几乎完全愈合,具有快速愈合性能;
图5仿生电子皮肤医用支架生物相容性(MTT)测试结果:该支架材料具有较高的生物相容性。
具体实施方式
下面结合实施方式对本发明进行具体的描述,有必要在此指出的是本实施例只用于对本发明进行进一步说明,而不能理解为对本发明保护范围的限制,该领域的技术熟练人员可以根据上述发明的内容作出非本质的改进和调整。
实施例1
(1)酸化碳纳米管分散液的制备:准确称取1.0 g多壁碳纳米管(MWCNTs)、50.0 g浓H2SO4(98%)、100 g HNO3(65~68%)于三口烧瓶中,升温至50℃,反应时间为15小时;反应完全后采用梯度转速离心,梯度转速离心条件为:①5000 rpm的转速离心60 min,过滤;②加入100.0 mL超纯水,以8000 rpm的转速离心60 min,过滤;③加入100.0 mL超纯水,以10000rpm的转速离心60 min,每个离心步骤重复操作3次,冷冻干燥储存;称取0.1 g酸化碳纳米管粉末,于200 mL H2O中超声0.5 h,制备得到0.05%(m/v)的酸化多壁碳纳米管分散液,储存备用;
(2)酸化碳纳米管/PEDOT:PSS复合分散液的制备:准确量取10.0 mL超纯水于单口烧瓶中,并准确移取1.0 mL PEDOT:PSS溶液以及5.0 mL 1.0 %酸化碳纳米管分散液于烧瓶中,以50 W,40 kHz超声30 min;
(3)明胶基复合导电凝胶溶液的制备:准确称取2.0 g明胶于上述混合溶液中,升温至30℃,恒温搅拌30 min,使明胶完全溶解,体系分散均匀;
(4)1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)交联明胶基复合导电凝胶的制备:准确称取0.1 g EDC粉末溶于5.0 mL H2O中得到2%(m/v)的EDC溶液;准确移取2.0 mL2%的EDC溶液缓慢滴入步骤三中的复合溶液体系中,继续恒温搅拌30 min,得到EDC交联改性的明胶基复合导电凝胶溶液;
(5)将完成反应后的混合溶液,倒入定制的聚四氟乙烯模具成型,并放入-4℃的冰箱陈化24 h,取出后用pH为7.0的PBS溶液浸泡12 h,最终得到一种具有自修复功能的仿生电子皮肤医用支架。
实施例2
(1)酸化碳纳米管分散液的制备:准确称取3.0 g多壁碳纳米管(MWCNTs)、250.0 g浓H2SO4 (98%)、20 g HNO3 (65~68%)于三口烧瓶中,升温至80℃,反应时间为10小时;反应完全后采用梯度转速离心,条件为:①3000 rpm的转速离心30 min,过滤;②加入300.0 mL超纯水,以6500 rpm的转速离心30 min,过滤;③加入300.0 mL超纯水,以9000 rpm的转速离心30 min,每个离心步骤重复操作4次,冷冻干燥储存;称取0.5 g酸化碳纳米管粉末,于100mL H2O中超声1.0 h,制备得到0.5% (m/v)的酸化多壁碳纳米管分散液,储存备用;
(2)酸化碳纳米管/PEDOT:PSS复合分散液的制备:准确量取50.0 mL超纯水于单口烧瓶中,并准确移取3.0 mL PEDOT:PSS溶液以及2.5 mL 0.5%酸化碳纳米管分散液于烧瓶中,以80 W,40 kHz超声80 min;
(3)明胶基复合导电凝胶溶液的制备:准确称取5.0 g明胶于上述混合溶液中,升温至45℃,恒温搅拌60 min,使明胶完全溶解,体系分散均匀;
(4)1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)交联明胶基复合导电凝胶的制备:准确称取0.25 g EDC粉末溶于2.5 mL H2O中得到10% (m/v)的EDC溶液;准确移取1.0mL 25%的EDC溶液缓慢滴入步骤三中的复合溶液体系中,继续恒温搅拌60 min,得到EDC交联改性的明胶基复合导电凝胶溶液;
(5)将完成反应后的混合溶液,倒入定制的聚四氟乙烯模具成型,并放入0℃的冰箱陈化18 h,取出后用pH为7.2的PBS溶液浸泡18 h,最终得到一种具有自修复功能的仿生电子皮肤医用支架。
实施例3
(1)酸化碳纳米管分散液的制备:准确称取5.0 g多壁碳纳米管(MWCNTs)、150.0 g浓H2SO4 (98%)、50 g HNO3 (65~68%)于三口烧瓶中,升温至100℃,反应时间为5小时;反应完全后采用梯度转速离心,条件为:①1000 rpm的转速离心10 min,过滤;②加入100.0 mL超纯水,以5000 rpm的转速离心10 min,过滤;③加入500.0 mL超纯水,以8000 rpm的转速离心10 min,每个离心步骤重复操作3次,冷冻干燥储存;称取1.0 g酸化碳纳米管粉末,于50mL H2O中超声2.0 h,制备得到2.0% (m/v)的酸化多壁碳纳米管分散液,储存备用;
(2)酸化碳纳米管/PEDOT:PSS复合分散液的制备:准确量取25.0 mL超纯水于单口烧瓶中,并准确移取5.0 mL PEDOT:PSS溶液以及1.0 mL 2.0%酸化碳纳米管分散液于烧瓶中,以100 W,40 kHz超声120 min;
(3)明胶基复合导电凝胶溶液的制备:准确称取10.0 g明胶于上述混合溶液中,升温至60℃,恒温搅拌120 min,使明胶完全溶解,体系分散均匀;
(4)1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)交联明胶基复合导电凝胶的制备:准确称取0.5 g EDC粉末溶于1.0 mL H2O中得到50% (m/v)的EDC溶液;准确移取0.1 mL50%的EDC溶液缓慢滴入步骤三中的复合溶液体系中,继续恒温搅拌120 min,得到EDC交联改性的明胶基复合导电凝胶溶液;
(5)将完成反应后的混合溶液,倒入定制的聚四氟乙烯模具成型,并放入4℃的冰箱陈化12 h,取出后用pH为7.4的PBS溶液浸泡24 h,最终得到一种具有自修复功能的仿生电子皮肤医用支架。
Claims (9)
1.一种复合导电凝胶的制备方法,其特征在于,包括如下步骤:
在含有酸化碳纳米管、PEDOT:PSS、明胶的均匀分散体中,加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐反应交联,得复合导电凝胶。
2.如权利要求1所述的方法,其特征在于,包括如下步骤:
取1.0~5.0 mL PEDOT:PSS溶液以及1.0~5.0 mL 0.05~1.0 g/ mL的酸化碳纳米管分散于10.0~50.0 mL水中,均匀后加入1.0~10.0g明胶,令体系分散均匀;向体系加入1.0~5.0mL 0.02~0.5 g/ mL的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的水溶液,30-60℃搅拌反应,得复合导电凝胶。
3.如权利要求1所述的方法,其特征在于,包括如下步骤:
1)量取10.0~50.0 mL超纯水于反应器中,加入1.0~5.0 mL PEDOT:PSS溶液以及1.0~5.0 mL 0.05~1.0 g/ mL的酸化碳纳米管分散液,以50~100 W,40 kHz超声30~120 min;
2)称取5.0~20.0 g明胶加入反应体系,升温至30~60℃,恒温搅拌30~120 min,使明胶完全溶解,体系分散均匀;
3)称取0.1~0.5 g EDC粉末溶于1.0~5.0 mL H2O中得到0.02~0.5 g/ mL的EDC溶液;取0.1~2.0 mL的EDC溶液缓慢滴入反应体系,继续恒温搅拌30~120 min,得到复合导电凝胶。
4.如权利要求1所述的方法,其特征在于,所述明胶来源于脱细胞胎牛皮真皮基质。
5.权利要求1-4任一项所述方法得到的复合导电凝胶。
6.一种具有自修复功能的仿生电子皮肤医用支架材料的制备方法,其特征在于,包括如下步骤:
将权利要求5所述的复合导电凝胶注入模具,在-4~4℃陈化12~24 h,然后用pH为7.0~7.4的PBS溶液浸泡12~24 h,得到具有自修复功能的仿生电子皮肤医用支架。
7.如权利要求6所述的方法,其特征在于,所述酸化碳纳米管由包括如下步骤的方法得到:
令1.0~5.0 g的多壁碳纳米管、50.0~250.0 g的98%浓H2SO4、20.0~100.0 g 的65~68%的HNO3 在50℃~100℃反应5~15小时,得到酸化碳纳米管。
8.如权利要求7所述的方法,其特征在于,制备酸化碳纳米管的反应完成后,采用梯度转速离心、冷冻干燥;所述梯度转速离心包括如下步骤:1)1000~5000 rpm的转速离心10~60min,过滤;2)加入100.0~500.0 mL超纯水,以5000~8000 rpm的转速离心10~60 min,过滤;3)加入100.0~500.0 mL超纯水,以8000~10000 rpm的转速离心10~60 min;前述离心步骤1)-3)各重复操作3~5次。
9.权利要求6-8任一项所述方法得到的医用支架材料。
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