CN114732953B - 脱细胞猪真皮基质抗菌导电皮肤支架的制备方法及应用 - Google Patents
脱细胞猪真皮基质抗菌导电皮肤支架的制备方法及应用 Download PDFInfo
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Abstract
本发明公开了脱细胞猪真皮基质抗菌导电皮肤支架的制备方法，具体为：通过水热法制备微米级多孔氧化铜微球，将微米级多孔氧化铜微球与多壁碳纳米管水溶液超声分散均匀并负载到猪脱细胞真皮基质中，再加入聚乙二醇双丙烯酸酯作为覆盖层，将猪脱细胞真皮基质冷冻干燥，得到抗菌导电皮肤支架。本发明制备的猪脱细胞真皮基质/微米级多孔氧化铜微球/碳纳米管抗菌导电皮肤支架兼具抗菌性、导电性、诱导组织再生的能力等多功能特性，可对运动健康实现实时监测，能应用在创口修复和缺失皮肤组织重建，实现“运动健康监测‑组织修复”一体化。
Description
技术领域
本发明属于生物医用材料制备技术领域，具体涉及一种脱细胞猪真皮基质抗菌导电皮肤支架的制备方法，还涉及该支架的应用。
背景技术
皮肤是人体最大的器官，急性创伤如手术切口，慢性创伤如溃疡等都会使皮肤发生损伤甚至缺失，并且创伤部位易感染细菌而对人体造成更大的伤害。在创伤处使用抗生素会伴随耐药性的产生，使用季铵盐、金属银等抑菌物质会带来一定的细胞毒性。由于创伤处细菌代谢会使生理环境变为酸性，根据这一特性开发一种受pH变化而可控释放抗菌物质并加速伤口愈合的医用皮肤支架非常必要。
发明内容
本发明的目的在于提供脱细胞猪真皮基质抗菌导电皮肤支架的制备方法，能够解决现有皮肤支架抑菌效果差、生物相容性低的问题。
本发明的另一目的在于提供上述抗菌导电皮肤支架在创伤修复和缺失皮肤组织重建中的应用。
本发明所采用的技术方案是，脱细胞猪真皮基质抗菌导电皮肤支架的制备方法，具体按照以下步骤实施：
步骤1，通过水热法制备微米级多孔氧化铜微球；
步骤2，将微米级多孔氧化铜微球与多壁碳纳米管水溶液超声分散均匀并负载到猪脱细胞真皮基质中，通过物理吸附、氢键结合等作用力结合在猪脱细胞真皮基质的微纳纤维上，再加入聚乙二醇双丙烯酸酯作为覆盖层，将猪脱细胞真皮基质冷冻干燥，得到抗菌导电皮肤支架。
本发明的特点还在于，
步骤1中，具体为：
将0.01～0.5mol的Cu(NO3)2·3H2O与0.1～5.0mol的乙醇水溶液、0.1～5.0mol的氢氧化铵水溶液以及0.1～5.0mol的氢氧化钠水溶液混合均匀，置于带聚四氟乙烯内衬的反应釜中进行水热反应，待反应结束后，通过离心收集沉淀物并用去离子水和无水乙醇交替洗涤三次，干燥，即可得到微米级多孔氧化铜微球。
水热反应温度为80～160℃，水热反应时间为6～18h；干燥温度为50℃，干燥时间为2～8h。
乙醇水溶液的质量分数为98％；氢氧化铵水溶液的质量分数为35％；氢氧化钠水溶液的质量分数为40％。
步骤2中，具体为：
0.1～5.0mL的多壁碳纳米管水分散液和0.01～0.5mL的微米级多孔氧化铜微球水分散液在10.0～50.0mL的去离子水中超声处理10～30分钟，裁剪直径为3.0cm的猪脱细胞真皮基质浸入混合溶液并在37℃下恒定振荡6～18小时，加入0.1～5.0mL聚乙二醇二丙烯酸酯作为覆盖层封盖微纳纤维，并继续振荡6～18小时，用去离子水清洗猪脱细胞真皮基质并在磷酸盐缓冲溶液浸泡过夜，取出后冷冻干燥，得到pH控释型脱细胞猪真皮基质抗菌导电皮肤支架。
冷冻干燥温度为-60℃，冷冻干燥时间为16～48小时。
多壁碳纳米管在水分散液中的质量分数为0.5％，微米级多孔氧化铜微球在水分散液中的质量分数为0.5％。
本发明所采用的另一技术方案是，脱细胞猪真皮基质抗菌导电皮肤支架能应用在创口修复和缺失皮肤组织重建中。
本发明的有益效果在于：
1)本发明选用猪脱细胞真皮基质作为基底材料，其机械性能良好，微纳纤维间保持着复杂的三维网状立体结构非常利于新生细胞的生长。猪脱细胞真皮基质降解时产生的甘氨酰-组氨酰-赖氨酸三肽能促进胶原、蛋白聚糖和黏多糖合成，激发脯氨酸酶活性从而加速血管生成，其还能黏附血小板从而衍生出生长因子，这些都使得其可成为一种良好的皮肤支架基材；
2)本发明制备的导电皮肤支架，与传统的支架材料相比，最明显的区别是在仿生人体皮肤的基础上具备导电性，仿生微电流的引入能有效促进细胞生长、增殖、分化，体现出其作为支架材料的优势；
3)本发明制备的猪脱细胞真皮基质/微米级多孔氧化铜微球/碳纳米管抗菌导电皮肤支架兼具抗菌性、导电性、诱导组织再生的能力等多功能特性，可对运动健康实现实时监测，能应用在创伤修复和缺失皮肤组织重建，实现“运动健康监测-组织修复一体化”；
4)本发明制备的微米级多孔氧化铜微球大小均一，较大的比表面积能使得铜离子的释放更加高效。铜离子的可控释放能为导电皮肤带来可调节功能的作用：生理环境酸化时释放出较高浓度的铜离子完成杀菌，生理环境中性时释放低浓度的铜离子起到促进细胞和血管的重生，这证明该铜微球具有作为新型智能医用材料的前景。
附图说明
图1为微米级多孔氧化铜微球的扫描电镜图；
图2脱细胞猪真皮基质(pADM)的扫描电镜图；
图3为本发明pH控释型脱细胞猪真皮基质抗菌导电皮肤支架(CNTs-μCuO@pADM)表面的扫描电镜图；
图4为脱细胞猪真皮基质温敏性抗菌导电支架(Ag-pADM@TSDHSiO2)横切面扫描电镜的EDS线扫图；
图5为微米级多孔氧化铜微球水溶液的粒径分布图；
图6为脱细胞猪真皮基质(pADM)以及本发明pH控释型脱细胞猪真皮基质抗菌导电皮肤支架(CNTs-μCuO@pADM)的红外光谱图；
图7为本发明pH控释型脱细胞猪真皮基质抗菌导电皮肤支架(CNTs-μCuO@pADM)在不同pH的磷酸缓冲盐溶液(PBS)中铜离子释放量图；
图8为脱细胞猪真皮基质(pADM)、本发明pH控释型脱细胞猪真皮基质抗菌导电皮肤支架(CNTs-μCuO@pADM)以及对照组(Control)生物相容性测试的OD值柱状图；
图9为生物相容性实验中对照组(Control)的活/死细胞荧光显微镜图；
图10为生物相容性实验中脱细胞猪真皮基质(pADM)的活/死细胞荧光显微镜图；
图11为生物相容性实验中pH控释型脱细胞猪真皮基质抗菌导电皮肤支架(CNTs-μCuO@pADM)的活/死细胞荧光显微镜图。
具体实施方式
下面结合附图和具体实施方式对本发明进行详细说明。
本发明脱细胞猪真皮基质抗菌导电皮肤支架的制备方法，具体按照以下步骤实施：
步骤1，通过水热法制备微米级多孔氧化铜微球；
具体为：将0.01～0.5mol的固体硝酸铜水合物Cu(NO3)2·3H2O与0.1～5.0mol的乙醇水溶液、0.1～5.0mol的氢氧化铵水溶液以及0.1～5.0mol的氢氧化钠水溶液混合均匀，置于带聚四氟乙烯内衬的反应釜中进行水热反应，待反应结束后，通过离心收集沉淀物并用去离子水和无水乙醇交替洗涤三次，干燥，即可得到微米级多孔氧化铜微球；
乙醇水溶液的质量分数为98％；氢氧化铵水溶液的质量分数为35％；氢氧化钠水溶液的质量分数为40％；
水热反应温度为80～160℃，水热反应时间为6～18h；干燥温度为50℃，干燥时间为2～8h；
步骤2，将微米级多孔氧化铜微球与多壁碳纳米管水溶液超声分散均匀并负载到猪脱细胞真皮基质中，再加入聚乙二醇双丙烯酸酯作为覆盖层，将猪脱细胞真皮基质冷冻干燥，得到兼具抗菌和促进伤口修复的抗菌导电皮肤支架；
具体为：0.1～5.0mL的多壁碳纳米管水分散液和0.01～0.5mL的微米级多孔氧化铜微球水分散液在10.0～50.0mL的去离子水中超声处理10～30分钟，裁剪直径为3.0cm的猪脱细胞真皮基质浸入混合溶液并在37℃下恒定振荡6～18小时，加入0.1～5.0mL聚乙二醇二丙烯酸酯作为覆盖层，并继续振荡6～18小时，用去离子水清洗猪脱细胞真皮基质并在磷酸盐缓冲溶液浸泡过夜，得到pH控释型脱细胞猪真皮基质抗菌导电皮肤支架。
多壁碳纳米管在水分散液中的质量分数为0.5％，微米级多孔氧化铜微球在水分散液中的质量分数为0.5％。
冷冻干燥温度为-60℃，冷冻干燥时间为16～48小时。
本发明制备的微米级多孔氧化铜微球大小均一，具有很大的比表面积，能有利于铜离子的释放。
本发明所制备的pH控释型脱细胞猪真皮基质抗菌导电皮肤支架具有抗菌和导电两个主要性能，能应用在创伤修复和缺失皮肤组织重建。
皮肤在日常生活中能起到保护、排泄、感受外界刺激等一系列关键作用，而创伤修复的原理就是通过构建皮肤的临时屏障，一方面抵挡住细菌侵害而导致的炎症，另一方面诱导皮肤细胞的再生从而完成皮肤的重建。从这两个方面切入，首先本发明中引入的铜是一种高效的杀菌剂，铜会通过接触和破坏细菌膜使得细菌内蛋白类物质泄漏而凋亡，但长时间的铜离子释放必将带来细胞毒性，在此根据细菌代谢会酸化生理环境这一现象构建出pH控制释放体系，在细菌存在时铜离子被释放出来杀死细菌，在没有细菌时释放极低浓度的铜离子起到促进细胞生长的作用。其次，猪脱细胞真皮基质、低浓度的铜离子以及仿生微电流的引入能从三个方面协同促进新细胞的生长，加速皮肤的重建，最终制备出一种抗菌、促愈合、导电的仿生电子皮肤支架。该发明旨在实现缺失皮肤组织修复过程的保护以及修复的加速。
实施例1
1)微米级多孔氧化铜微球的制备：准确称取2.5g硝酸铜水合物Cu(NO3)2·3H2O与20mL乙醇、20mL氢氧化铵水溶液和10mL氢氧化钠水溶液混合，将混合液转移到水热反应器中140℃下加热14小时，通过离心收集沉淀物并用去离子水和无水乙醇交替洗涤三次，在50℃的烘箱中干燥8h，得到微米级多孔氧化铜微球。
2)pH控释型脱细胞猪真皮基质抗菌导电皮肤支架的制备：取0.5mL多壁碳纳米管水溶液和0.05mL微米级多孔氧化铜微球水溶液在5.0mL去离子水中超声处理10分钟，裁剪直径为3.0cm的猪脱细胞真皮基质浸入混合溶液并在37℃下恒定振荡14小时，加入0.5mL聚乙二醇二丙烯酸酯作为覆盖层，并继续振荡14小时，用去离子水清洗猪脱细胞真皮基质并在磷酸盐缓冲溶液浸泡过夜，并冷冻干燥，得到pH控释型脱细胞猪真皮基质抗菌导电皮肤支架。
实施例2
1)微米级多孔氧化铜微球的制备：准确称取3.0g硝酸铜水合物Cu(NO3)2·3H2O与30mL乙醇、30mL氢氧化铵水溶液和20mL氢氧化钠混合，将混合液转移到水热反应器中120℃下加热12小时，通过离心收集沉淀物并用去离子水和无水乙醇交替洗涤三次，在50℃的烘箱中干燥8h，得到微米级多孔氧化铜微球。
2)pH控释型脱细胞猪真皮基质抗菌导电皮肤支架的制备：取0.1mL多壁碳纳米管水溶液和0.1mL微米级多孔氧化铜微球水溶液在7.5mL去离子水中超声处理20分钟，裁剪直径为3.0cm的pADM浸入混合溶液并在37℃下恒定振荡12小时，加入1.5mL聚乙二醇双丙烯酸酯作为覆盖层，并继续振荡12小时，用去离子水清洗猪脱细胞真皮基质并在磷酸盐缓冲溶液浸泡过夜，并冷冻干燥，得到pH控释型脱细胞猪真皮基质抗菌导电皮肤支架。
实施例3
1)微米级多孔氧化铜微球的制备：准确称取5.0g硝酸铜水合物Cu(NO3)2·3H2O与40mL乙醇、40mL氢氧化铵水溶液和30mL氢氧化钠混合，将混合液转移到水热反应器中100℃下加热6小时，通过离心收集沉淀物并用去离子水和无水乙醇交替洗涤三次，在50℃的烘箱中干燥6h，得到微米级多孔氧化铜微球。
2)pH控释型脱细胞猪真皮基质抗菌导电皮肤支架的制备：取2.0mL多壁碳纳米管水溶液和0.2mL微米级多孔氧化铜微球水溶液在10.0mL去离子水中超声处理10分钟，裁剪直径为3.0cm的pADM浸入混合溶液并在37℃下恒定振荡10小时，加入2.5mL聚乙二醇双丙烯酸酯作为覆盖层，并继续振荡10小时，，用去离子水清洗猪脱细胞真皮基质并在磷酸盐缓冲溶液浸泡过夜，并冷冻干燥，得到pH控释型脱细胞猪真皮基质抗菌导电皮肤支架。
实施例4
一种pH控释型脱细胞猪真皮基质抗菌导电皮肤支架的制备方法，包括如下步骤：
步骤1，制备微米级多孔氧化铜微球；
步骤2，将氧化铜微球与多壁碳纳米管水溶液超声分散均匀并负载到猪脱细胞真皮基质中，再加入聚乙二醇双丙烯酸酯作为覆盖层，将猪脱细胞真皮基质冷冻干燥得到兼具抗菌和促进伤口修复的医用导电皮肤支架。
步骤1具体又包括以下步骤：
微米级多孔氧化铜微球的制备：通过水热法制备微米级多孔氧化铜微球，将0.01mol固体硝酸铜水合物Cu(NO3)2·3H2O与0.1mol乙醇水溶液(98％)、0.1mol氢氧化铵水溶液(35％)以及0.05mol氢氧化钠水溶液(40％)混合倒入聚四氟乙烯内衬中，将内衬到水热反应釜中150℃下加热14小时，通过离心收集沉淀物并用去离子水和无水乙醇交替洗涤三次，在50℃的烘箱中干燥8h得到微米级多孔氧化铜微球。
步骤2中，具体为：
0.1mL多壁碳纳米管水溶液和0.1mL微米级多孔氧化铜微球水溶液在10.0mL去离子水中超声处理10分钟，裁剪直径为3.0cm的pADM浸入混合溶液并在37℃下恒定振荡12小时，加入0.1mL聚乙二醇双丙烯酸酯作为覆盖层，并继续振荡12小时，用去离子水清洗猪脱细胞真皮基质并在磷酸盐缓冲溶液浸泡过夜，并冷冻干燥得到pH控释型脱细胞猪真皮基质抗菌导电皮肤支架。
实施例5
一种pH控释型脱细胞猪真皮基质抗菌导电皮肤支架的制备方法，包括如下步骤：
步骤1，制备微米级多孔氧化铜微球；
步骤2，将氧化铜微球与多壁碳纳米管水溶液超声分散均匀并负载到猪脱细胞真皮基质中，再加入聚乙二醇双丙烯酸酯作为覆盖层，将猪脱细胞真皮基质冷冻干燥得到兼具抗菌和促进伤口修复的医用导电皮肤支架。
步骤1具体包括以下步骤：
微米级多孔氧化铜微球的制备：通过水热法制备微米级多孔氧化铜微球，将0.2mol固体硝酸铜水合物Cu(NO3)2·3H2O与2.5mol乙醇水溶液(98％)、2.5mol氢氧化铵水溶液(35％)以及1.0mol氢氧化钠水溶液(40％)混合倒入聚四氟乙烯内衬中，将内衬到水热反应釜中130℃下加热10小时，通过离心收集沉淀物并用去离子水和无水乙醇交替洗涤三次，在50℃的烘箱中干燥6h，得到微米级多孔氧化铜微球；
步骤2具体为：
2.5mL多壁碳纳米管水溶液和0.25mL微米级多孔氧化铜微球水溶液在25.0mL去离子水中超声处理15分钟，裁剪直径为3.0cm的pADM浸入混合溶液并在37℃下恒定振荡14小时，加入0.1～5.0mL聚乙二醇双丙烯酸酯作为覆盖层，并继续振荡14小时，用去离子水清洗猪脱细胞真皮基质并在磷酸盐缓冲溶液浸泡过夜，并冷冻干燥，得到pH控释型脱细胞猪真皮基质抗菌导电皮肤支架。
图1为实例5制得的微米级多孔氧化铜微球的扫描电镜照片。通过观察可知，微球表面由花瓣状颗粒组成，粒径大小均一。
图2为猪脱细胞真皮基质(pADM)的扫描电镜照片。通过观察可知，纤维分散性良好，内部呈现出复杂的三维结构。脱细胞猪真皮基质由江苏省江阴奔翔生物科技有限公司提供。
图5为实例5制得的微米级多孔氧化铜微球水溶液粒径分布图。由图可知微球尺寸在4～5微米分布最多，少量的微球团聚造成10微米以上的粒径分布。
图3为实例5制得的pH控释型脱细胞猪真皮基质抗菌导电皮肤支架(CNTs-μCuO@pADM)表面的扫描电镜照片。通过观察可知，皮纤维表面形成连续的导电层，氧化铜微球嵌在纤维缝隙间。
图4为实例5得的pH控释型脱细胞猪真皮基质抗菌导电皮肤支架(CNTs-μCuO@pADM)横断面EDS线扫结果图。由图可知在纤维断面线扫处铜元素均匀分布。
图6为猪脱细胞真皮基质(pADM)以及实例5制得的pH控释型脱细胞猪真皮基质抗菌导电皮肤支架(CNTs-μCuO@pADM)表面红外光谱图。由图可知碳纳米管和铜微球被成功加载到脱细胞真皮基质上，目标皮肤支架被成功制备。
图7为实例5制得的pH控释型脱细胞猪真皮基质抗菌导电皮肤支架(CNTs-μCuO@pADM)在不同pH的磷酸缓冲盐溶液(PBS)中铜离子释放量图。由图可知在pH分别为5.0和7.4时铜离子释放量相差较大，有效完成了杀菌铜离子的可控释放。
图8为脱细胞猪真皮基质(pADM)，实例5制得的pH控释型脱细胞猪真皮基质抗菌导电皮肤支架(CNTs-μCuO@pADM)以及对照组(Control)生物相容性测试的OD值柱状图。由图可知制备出的皮肤支架有着良好的生物相容性，碳纳米管和铜微球的加入对脱细胞真皮基质的生物相容性影响较小。
图9-11为对照组(Control)、脱细胞猪真皮基质(pADM)以及实例5制得的pH控释型脱细胞猪真皮基质抗菌导电皮肤支架(CNTs-μCuO@pADM)的活/死细胞荧光显微镜图。由图可知制得的皮肤支架具有良好的生物相容性。
本发明制备的pH控释型脱细胞猪真皮基质抗菌导电皮肤支架具有pH控释下的抗菌性能，导电性能，具有加速皮肤组织修复的性能。
Claims (5)
1.脱细胞猪真皮基质抗菌导电皮肤支架的制备方法，其特征在于，具体按照以下步骤实施：
步骤1，通过水热法制备微米级多孔氧化铜微球；具体为：
将0.01～0.5mol的Cu(NO3)2·3H2O与0.1～5.0mol的乙醇水溶液、0.1～5.0mol的氢氧化铵水溶液以及0.1～5.0mol的氢氧化钠水溶液混合均匀，置于带聚四氟乙烯内衬的反应釜中进行水热反应，待反应结束后，通过离心收集沉淀物并用去离子水和无水乙醇交替洗涤三次，干燥，即可得到微米级多孔氧化铜微球；
步骤2，将微米级多孔氧化铜微球与多壁碳纳米管水溶液超声分散均匀并负载到猪脱细胞真皮基质中，再加入聚乙二醇双丙烯酸酯作为覆盖层，将猪脱细胞真皮基质冷冻干燥，得到抗菌导电皮肤支架；
具体为：0.1～5.0mL的多壁碳纳米管水分散液和0.01～0.5mL的微米级多孔氧化铜微球水分散液在10.0～50.0mL的去离子水中超声处理10～30分钟，裁剪直径为3.0cm的猪脱细胞真皮基质浸入混合溶液并在37℃下恒定振荡6～18小时，加入0.1～5.0mL聚乙二醇二丙烯酸酯作为覆盖层，并继续振荡6～18小时，用去离子水清洗猪脱细胞真皮基质并在磷酸盐缓冲溶液浸泡过夜，冷冻干燥，得到pH控释型脱细胞猪真皮基质抗菌导电皮肤支架；多壁碳纳米管在水分散液中的质量分数为0.5％，微米级多孔氧化铜微球在水分散液中的质量分数为0.5％。
2.根据权利要求1所述的脱细胞猪真皮基质抗菌导电皮肤支架的制备方法，其特征在于，水热反应温度为80～160℃，水热反应时间为6～18h；干燥温度为50℃，干燥时间为2～8h。
3.根据权利要求1所述的脱细胞猪真皮基质抗菌导电皮肤支架的制备方法，其特征在于，乙醇水溶液的质量分数为98％；氢氧化铵水溶液的质量分数为35％；氢氧化钠水溶液的质量分数为40％。
4.根据权利要求1所述的脱细胞猪真皮基质抗菌导电皮肤支架的制备方法，其特征在于，冷冻干燥温度为-60℃，冷冻干燥时间为16～48小时。
5.如权利要求1-4任一项所述方法制备的脱细胞猪真皮基质抗菌导电皮肤支架，其特征在于，所述脱细胞猪真皮基质抗菌导电皮肤支架能应用在创口修复和缺失皮肤组织重建中。
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