CN110124086B - 一种复合纳米纤维垫、水凝胶/海绵敷料及制备方法和应用 - Google Patents
一种复合纳米纤维垫、水凝胶/海绵敷料及制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种复合纳米纤维垫、水凝胶/海绵敷料及制备方法和应用,涉及医学材料技术领域。一种复合纳米纤维垫,包括接收板,接收板上复合有去细胞的基质和高分子材料,去细胞的基质与高分子材料的添加比为:1:1~1:6,复合纳米纤维垫为微纳米纤维结构。本发明提供了一种水凝胶/海绵敷料,该水凝胶/海绵敷料可以用来负载营养因子、药物等促进创面再生和修复的成分,水凝胶/海绵具有很好的生物相容性,无明显细胞毒性;水凝胶/海绵敷料材料柔软,可紧密覆盖所有的创面伤口,抵抗细菌入侵,避免创面感染。
Description
技术领域
本发明涉及医学材料技术领域,具体而言,涉及一种复合纳米纤维垫、水凝胶/海绵敷料及制备方法和应用。
背景技术
伤口指受伤破裂的地方,多指人或其他动物的皮肤、肌肉、黏膜等而言。伤口一般可以分为急性伤口和慢性难愈合伤口,伤口的愈合一般可以分为四个独特但重叠的阶段,分别为凝血/止血,炎症反应/迁移,增殖/血管化/再生,组织重构。正常的伤口愈合一般需要三周左右的时间,但是慢性难愈合伤口由于容易受到细菌感染会延长其伤口愈合的时间。人类伤口外露往往容易造成细菌感染,同时为了满足局部用药、保湿和吸收引流液等需求,往往需要使用伤口敷料改善伤口的局部化学和物理环境以促进伤口的愈合。
去细胞基质是一种保留天然组织的细胞外基质成分的材料,此类材料移植到体内后极有可能带来免疫原性、疾病传染等问题。去细胞技术通过物理或化学方法将细胞破坏,使其与组织的细胞外基质相分离,将DNA/RNA这些会引起免疫反应和传染疾病的因素剔除后,得到单纯的细胞外基质材料。
理想的伤口敷料应该具有以下特征,即1.防止伤口再次感染;2.保护伤口以免再次创伤;3.提供药物;4.保持一定湿度;5.具有良好的通透性;6.吸收引流物及清理伤口;7.具有一定力学强度;8.移除时不会损伤皮肤。目前的医用伤口敷料包括天然纱布、合成纤维类敷料、多聚膜类敷料、发泡多聚类敷料、水胶体类敷料、藻酸盐敷料等。
传统的伤口敷料如纱布敷料和合成纤维敷料,通透性太高,容易使创面脱水且外界环境微生物容易通过,交叉感染的机会较高,容易粘着创面,更换时会造成再次性机械性损伤。
新型合成材料制备的其他伤口敷料如多聚膜类敷料吸收渗液能力差,创面周围皮肤浸渍机会大;发泡多聚体类敷料由于太强的吸收性能,对于低度渗出创面可能会影响到自身清创过程,因不透明,不方便观察创面;水胶体类敷料吸收能力不是很强,因此对于高渗出性创面,常需要使用其他辅助敷料来加强吸收性能,个别患者可能存在对成分过敏。藻酸盐敷料大多数产品不具备自粘性,需要辅助敷料加以固定。
与上述材料相比,去细胞基质具有与天然组织相近的成分与结构,但去细胞基质材料由于力学强度较低,不能单独地用作敷料使用。
鉴于此,特提出本发明。
发明内容
本发明的目的在于提供一种复合纳米纤维垫,具有微纳米纤维的致密结构,能起到隔离外部细菌感染的作用。
本发明的另一目的在于提供一种复合纳米纤维垫的制备方法,该制备方法为去细胞的基质纳米纤维起到力学支撑作用,克服了单纯去细胞的基质纳米纤维强度弱的问题,同时又能保持内部微纳米的仿生结构,有利于细胞的黏附,增值和迁移,为药物提供了很好的载体。
本发明还提供了一种水凝胶/海绵敷料的制备方法,通过该方法制备出的敷料上层为复合纳米纤维垫,下层为去细胞的基质水凝胶或海绵,内部存在微纳米结构多孔的疏松结构,敷料上下层能同时修复损伤皮肤的表皮层和真皮层,同时由于敷料上下层均含有去细胞的基质,保留了较多的生物成分,比传统敷料更有利于细胞的黏附,增值和迁移。
本发明是这样实现的:
一种复合纳米纤维垫,复合有去细胞的基质和高分子材料,去细胞的基质与高分子材料的添加比为:1:1~1:6,复合纳米纤维垫为微纳米纤维结构,优选的,去细胞的基质与高分子材料的添加比为:1:6。
在本发明应用较佳的实施例中,上述去细胞的基质的材料来源可以是猪,牛和人中的任意一种,去细胞的基质可以是皮肤细胞外基质,小肠黏膜下层细胞外基质和羊膜细胞外基质中的任意一种或多种的组合。
在本发明应用较佳的实施例中,上述高分子材料可以是聚己内酯、聚乳酸、左旋聚乳酸、透明质酸及明胶中的任意一种。
一种复合纳米纤维垫的制备方法,分别制备去细胞的基质和高分子材料的电纺丝溶液,将去细胞的基质的电纺丝溶液转移到第一注射器中,将高分子材料的电纺丝溶液转移到第二注射器中,采用电纺丝法将去细胞的基质和高分子材料的电纺丝溶液共纺或混纺于接收板上制得复合纳米纤维垫。
在本发明应用较佳的实施例中,上述第一注射器的注射速度为1mL/h~4mL/h,第二注射器的注射速度为1mL/h~4mL/h,第一注射器和第二注射器的针头均为国际标准8~9号双针头,同轴针头和Y型国际标准9号针头中的任意一种,针头与接收板之间的距离为6-10cm;
优选的,第一注射器的注射速度为3mL/h,第二注射器的注射速度为3mL/h,第一注射器和第二注射器的针头均为Y型国际标准9号针头,针头与接收板之间的距离为7cm。
在本发明应用较佳的实施例中,上述针头的外加正电压为8-25kV,接收板的外加负电压为0.5-1.0kV;
优选的,针头的外加正电压为13kV,接收板的外加负电压为1.0kV。
在本发明应用较佳的实施例中,上述接收板由导电的材料制成,优选的,接收板为锡纸包封的不锈钢板。
一种水凝胶/海绵敷料,水凝胶/海绵敷料包括复合纳米纤维垫和去细胞的基质水凝胶/海绵,复合纳米纤维垫设置于去细胞的基质水凝胶/海绵的下层。
一种水凝胶/海绵敷料的制备方法,用消化液对去细胞的基质进行消化后,离心,调节pH,制得裂解溶液,将裂解溶液涂敷在的复合纳米纤维垫中,保温制得水凝胶/海绵敷料。
在本发明应用较佳的实施例中,上述消化液为胃蛋白酶、胰蛋白酶和木瓜蛋白酶中的任意一种或组合,优选的,消化液为胃蛋白酶,调节pH步骤包括先用NaOH调节pH值大于8,再用HCl调节pH=7~7.5,制得裂解溶液;保温步骤包括将涂敷有裂解溶液的复合纳米纤维垫置于35~40℃保温箱中静置2~10分钟制得水凝胶敷料,将涂敷有裂解溶液的复合纳米纤维垫真空冻干制得海绵敷料;真空冻干的条件为-50℃,0.1Pa。
在本发明应用较佳的实施例中,使用10%的胃蛋白酶的盐酸消化液消化去细胞的基质12~48小时,得到的酶解液置于高速离心机中在30000rpm、4℃下高速离心45分钟,用NaOH调节调节pH值大于8,用HCl调回pH=7~7.5,得到预凝胶溶液;在4℃下,将10xPBS加入到已调至中性的预凝胶溶液中,涂敷在复合纳米纤维垫中,置于37℃保温箱中静置2~10分钟制得水凝胶敷料。
在本发明应用较佳的实施例中,使用10%的胃蛋白酶的盐酸消化液消化去细胞的基质12~48小时,得到的裂解液置于高速离心机中在30000rpm、4℃下高速离心45分钟2次,用NaOH调节pH值=7,得到的酶解液涂敷在复合纳米纤维垫中,在-50℃,0.1Pa下真空冻干,得到海绵敷料。
在本发明应用较佳的实施例中,使用10%的胃蛋白酶的盐酸消化液消化去细胞的基质12~48小时,得到的裂解液置于高速离心机中在30000rpm、4℃下高速离心45分钟,用NaOH调节调节pH值大于8,用HCl调回pH=7~7.5,得到预凝胶溶液;在4℃下,将10xPBS加入到已调至中性的预凝胶溶液中,涂敷在复合纳米纤维垫中,置于37℃保温箱中静置2~10分钟成胶,成胶后置于-50℃,0.1Pa的真空冻干机中真空冻干,得到海绵敷料。
一种复合纳米纤维垫在制备水凝胶/海绵敷料中的用途。
本发明具有以下有益效果:
本发明提供了一种复合纳米纤维垫,该复合纳米纤维垫具有微纳米纤维的致密结构,能起到隔离外部细菌感染的作用,该复合纳米纤维垫通过加入高分子材料,提升了去细胞的基质纳米纤维强度。
本发明还提供了一种复合纳米纤维垫的制备方法,该制备方法为去细胞的基质纳米纤维起到力学支撑作用,克服了单纯去细胞的基质纳米纤维强度弱的问题,同时又能保持内部微纳米的仿生结构,有利于细胞的黏附,增值和迁移,为药物提供了很好的载体。
本发明还提供了一种水凝胶/海绵敷料的制备方法,通过该方法制备出的敷料上层为复合纳米纤维垫,下层为去细胞的基质水凝胶或海绵,内部存在微纳米结构多孔的疏松结构,敷料上下层能同时修复损伤皮肤的表皮层和真皮层,同时由于敷料上下层均含有去细胞的基质,保留了较多的生物成分,比传统敷料更有利于细胞的黏附,增值和迁移。
一种复合纳米纤维垫在制备水凝胶/海绵敷料中的用途。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1为本发明实例1提供的去细胞的基质纳米纤维垫的扫描电镜图;
图2为本发明实例2提供的去细胞的基质水凝胶敷料侧面的扫描电镜图;
图3为本发明实例2提供的去细胞的基质水凝胶敷料上层水凝胶与下层纳米纤维垫交界面的扫描电镜图;
图4为本发明实例2提供的去细胞的基质水凝胶敷料上层水凝胶的扫描电镜图;
图5为本发明实例3提供的去细胞的基质海绵敷料侧面的扫描电镜图;
图6为本发明实例3提供的去细胞的基质海绵敷料上层海绵与下层纳米纤维垫交界面的扫描电镜图;
图7是本发明实例3提供的去细胞的基质海绵敷料上层海绵的扫描电镜图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
制备复合纳米纤维垫
(1)制备去细胞的基质。取猪来源皮肤组织20m3,切成小块,经过温水洗净,使用3%的tritonX-100浸泡12小时,再用去离子水洗净15分钟3次,用4%脱氧胆酸钠溶液处理24小时,去离子水洗净15分钟3次,真空冻干2天,冻干机温度为-50℃,压强为0.1MPa。取相当于去细胞的组织器官基质10倍体积的乙醇/二氯甲烷(体积比为2:1)对上述真空冻干的基质进行脱脂处理2次,每次1天,去离子水洗净3次,每次15分钟,真空抽滤过夜,真空冻干2天,打粉,过40目筛后在-40℃保存,即得去细胞的基质粉末。
去细胞的基质的材料来源可以是猪,牛和人中的任意一种,去细胞的基质可以是皮肤细胞外基质,小肠黏膜下层细胞外基质和羊膜细胞外基质中的任意一种或多种的组合。需要说明的是,在本发明中,其他的细胞外基质作为主要原料同样具有相同效果。
(2)准备电纺丝溶液。取(1)的去细胞基质粉末0.25g,溶于10ml六氟异丙醇中搅拌4~6天,搅拌结束后,分成5份加入到2ml的EP管中,每个EP管加入2颗直径为5mm,1颗直径为1mm的钢珠或氧化锆小球,并将5支EP管放置于实验条件为-10℃,60Hz的球磨机中球磨10分钟2次,球磨结束后转移到注射器1中;取聚己内酯1.5g,溶于10ml六氟异丙醇或三氟乙醇中搅拌1天,搅拌结束后转移到注射器2中。
(3)制备上层去细胞的基质/高分子材料复合纳米纤维垫。电纺丝制备去细胞的基质/高分子材料复合纳米纤维垫的条件是:注射器1注射速度为3mL/h,注射器2注射速度为3mL/h,注射器1和注射器2均采用Y型国际标准9号针头,针头与接收平板之间距离为7cm,接收板是以锡纸包封的不锈钢板,注射器1和注射器2的针头均施加正电压13kV,接收平板施加负电压1.0kV。
实施例2
制备去细胞的基质全层皮肤修复水凝胶敷料
(1)称取0.01g胃蛋白酶混入10ml,0.01%的盐酸溶液中搅拌30分钟制得搅拌液,搅拌结束后,在搅拌液中添加实施例1中的去细胞的基质粉末0.1g,制得消化液,继续搅拌消化24小时,得到的酶解液加入到离心管中,将离心管置于高速离心机中在30000rpm、4℃下高速离心45分钟,离心2次,取上清液用0.1%的NaOH调节酶解液的pH值大于8,再用HCl调回pH=7~7.5,得到预凝胶溶液;
(2)在4℃下,将10xPBS加入到已调至中性的预凝胶溶液中,涂敷在实施例1的复合纳米纤维垫中,置于37℃保温箱中静置10分钟成胶,得到去细胞的基质全层皮肤修复水凝胶敷料。
实施例3
制备去细胞的基质全层皮肤修复海绵敷料
称取0.01g胃蛋白酶,将胃蛋白酶混入10ml,0.01%的盐酸溶液中搅拌30分钟,搅拌结束后加入实施例1的去细胞的基质粉末0.1g,制得消化液,继续搅拌消化24小时,将得到的裂解液加入到离心管中,将离心管置于高速离心机中,在30000rpm、4℃下高速离心45分钟,离心2次,用0.1%的NaOH调节裂解液的pH值=7,得到的裂解液涂敷在实施例1的复合纳米纤维垫中,在-50℃,0.1Pa下真空冻干3天,得到去细胞的基质全层皮肤修复海绵敷料。
在本发明的其他实施例中,制备去细胞的基质全层皮肤修复海绵敷料也可以采用如下方法制备得到:
称取0.01g胃蛋白酶混入10ml,0.01%的盐酸溶液中搅拌30分钟,搅拌结束后加入实施例1中去细胞的基质粉末0.1g,制得消化液。继续搅拌消化24小时,将得到的裂解液加入到离心管中,将离心管置于高速离心机中在30000rpm、4℃下高速离心45分钟,离心2次,去上清液用0.1%的NaOH调节酶解液的pH值大于8,再用HCl调回pH=7~7.5,得到预凝胶溶液;在4℃下,将10xPBS加入到已调至中性的预凝胶溶液中,涂敷在实施例1中的复合纳米纤维垫中,置于37℃保温箱中静置10分钟成胶,成胶后置于-50℃,0.1Pa下的真空冻干机中真空冻干3天,得到去细胞的基质全层皮肤修复海绵敷料。
实施例4
将实施例1、实施例2和实施例3得到的材料分别在扫描电镜(日立高新技术S-4800)下观察其结构。
实施例1得到的纳米纤维垫的扫描电镜放大图参照图1所示。
实施例2得到的去细胞的基质水凝胶敷料侧面的扫描电镜图参照图2所示,去细胞的基质水凝胶敷料上层水凝胶与下层纳米纤维垫交界面的扫描电镜图参照图3所示,上层水凝胶的扫描放大电镜图参照图4所示。实施例3得到去细胞的基质海绵敷料侧面的扫描电镜图参照图5所示,去细胞的基质海绵敷料上层海绵与下层纳米纤维垫交界面的扫描电镜图参照图6所示,海绵敷料上层海绵的扫描放大电镜图参照图7所示。
由图1可得,实施例1中的复合纳米纤维垫的纤维直径较小,纤维致密,具有微纳米结构,有利于隔绝外部细菌的入侵,有利于内部细胞的黏附和分化。
由图2-图4可得,实施例2制得的去细胞的基质水凝胶敷料具有微-纳米多级纤维的特性,纤维孔径较大,有利于细胞附着、分化和迁移,下层为复合纳米纤维垫,上层为水凝胶。
由图5-图7可得,实施例3制得的去细胞的基质海绵敷料具有微-纳米多级孔状的特性,海绵孔径较大,有利于细胞附着、分化和迁移,下层为复合纳米纤维垫,上层为海绵。
本发明通过提供一种复合纳米纤维垫,隔离了外部细菌感染的作用,该复合纳米纤维垫通过加入高分子材料,提升了去细胞的基质纳米纤维强度。本发明通过提供一种复合纳米纤维垫的制备方法,该制备方法为去细胞的基质纳米纤维起到力学支撑作用,克服了单纯去细胞的基质纳米纤维强度弱的问题,同时又能保持内部微纳米的仿生结构,有利于细胞的黏附,增值和迁移,为药物提供了很好的载体。
本发明还提供了一种水凝胶/海绵敷料的制备方法,通过该方法制备出的敷料上层为复合纳米纤维垫,下层为去细胞的基质水凝胶或海绵,内部存在微纳米结构多孔的疏松结构,敷料上下层能同时修复损伤皮肤的表皮层和真皮层,同时由于敷料上下层均含有去细胞的基质,保留了较多的生物成分,比传统敷料更有利于细胞的黏附,增值和迁移。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (9)
1.一种复合纳米纤维垫的制备方法,其特征在于,分别制备去细胞的基质和高分子材料的电纺丝溶液,将所述去细胞的基质的电纺丝溶液转移到第一注射器中,将所述高分子材料的电纺丝溶液转移到第二注射器中,采用电纺丝法将所述去细胞的基质和高分子材料的电纺丝溶液共纺或混纺于接收板上制得复合纳米纤维垫;
所述去细胞的基质与所述高分子材料的添加比为1:6;所述第一注射器的注射速度为3mL/h,所述第二注射器的注射速度为3mL/h;
所述高分子材料可以是聚己内酯、聚乳酸、左旋聚乳酸中的任意一种。
2.根据权利要求1所述的复合纳米纤维垫的制备方法,其特征在于,所述第一注射器和所述第二注射器的针头均为国际标准8~9号双针头,同轴针头和Y型国际标准9号针头中的任意一种,所述针头与所述接收板之间的距离为6-10cm。
3.根据权利要求2所述的复合纳米纤维垫的制备方法,其特征在于,所述第一注射器和所述第二注射器的针头均为Y型国际标准9号针头,所述针头与所述接收板之间的距离为7cm。
4.根据权利要求2所述的复合纳米纤维垫的制备方法,其特征在于,所述针头的外加正电压为13kV,所述接收板的外加负电压为1.0kV。
5.一种由权利要求1-4任一项所述的制备方法制得的复合纳米纤维垫,其特征在于,复合有去细胞的基质和高分子材料,所述去细胞的基质与所述高分子材料的添加比为1:6,所述复合纳米纤维垫为微纳米纤维结构。
6.一种水凝胶/海绵敷料的制备方法,其特征在于,用消化液对去细胞的基质进行消化后,离心,调节pH,制得裂解溶液,将所述裂解溶液涂敷在权利要求5的复合纳米纤维垫中,保温制得水凝胶/海绵敷料。
7.根据权利要求6所述的水凝胶/海绵敷料的制备方法,其特征在于,所述消化液为胃蛋白酶、胰蛋白酶和木瓜蛋白酶中的任意一种或组合,所述调节pH步骤包括先用NaOH调节pH值大于8,再用HCl调节pH=7~7.5,制得裂解溶液;所述保温步骤包括将涂敷有所述裂解溶液的复合纳米纤维垫置于35~40℃保温箱中静置2~10分钟制得水凝胶敷料;将涂敷有所述裂解溶液的复合纳米纤维垫真空冻干制得海绵敷料,所述真空冻干的条件为-50℃,0.1Pa。
8.一种水凝胶/海绵敷料,其特征在于,所述水凝胶/海绵敷料包括权利要求5所述的复合纳米纤维垫和去细胞的基质水凝胶/海绵,所述复合纳米纤维垫设置于所述去细胞的基质水凝胶/海绵的下层。
9.一种如权利要求5所述的复合纳米纤维垫在制备水凝胶/海绵敷料中的用途。
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