CN114272439A - 一种具有改良纳米结构的皮肤移植物 - Google Patents
一种具有改良纳米结构的皮肤移植物 Download PDFInfo
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Abstract
本发明提供一种具有改良纳米结构的皮肤移植物,以聚乳酸羟基乙酸、鱼胶原为原料溶于六氟异丙醇中得到电纺液,电纺液中聚乳酸羟基乙酸和鱼胶原的浓度分别为20%W/V、2%W/V;将电纺液置于静电纺丝设备中,纺制得到取向型的电纺膜,制得电纺膜的纺丝直径为319±100nm,拉伸强度达到11.95MPa,具有生物相容性与降解性;将其应用于作为皮肤移植物时,在皮肤缺损愈合14天时仍可见电纺膜材料存留,保证电纺膜持续作用在皮肤缺损愈合的凝血期、炎症期、增殖修复期。
Description
技术领域
本发明属于生物材料技术领域,具体涉及一种具有改良纳米结构的皮肤移植物。
背景技术
当前医疗行业常用的皮肤移植物包括天然的移植物,以及人工合成的移植物,技术敏感因素多。静电纺丝技术因为其可以模拟细胞外基质的拓扑结构、便于负载生长因子等优势,近年来越来越多被用于组织工程化皮肤移植物的构建。
在电纺移植物的材料选择上,聚乳酸-羟基乙酸共聚物(PLGA)作具有良好的生物相容性、良好的成膜的性能,被广泛应用于医用工程材料。PLGA通过FDA认证,被正式作为药用辅料收录进美国药典。鱼胶原蛋白(FC)提取简便,还具有很多修复因子,具有良好生物相容性、生物降解性、低抗原性和低动物疾病传染性的特点,在生物材料制备上有潜力代替传统哺乳动物来源的胶原。FC的加入可以与PLGA分子链相互作用、形成分子网络结构,有助于增加材料的力学性能。
在以FC/PLGA为成分的电纺膜制备上,如何制备有更高机械强度、更佳生物相容性的膜材料是需要进一步探讨的问题。在移植物结构设计上,由于材料的表面微观结构显著影响材料的力学性能以及细胞的粘附、生长和功能,多种组织工程修复材料展示出对特定尺寸的倾向性。比如:人成纤维细胞生长于直径在350-1100nm聚乳酸羟基乙酸(PLGA)的电纺膜时增殖比较好。与微纤维支架相比,纳米电纺支架的炎症反应最小。还有研究表明:相比直径约1μm的支架,约250-300nm组成的电纺支架对人真皮成纤维细胞增殖的支持作用更强。对于构建皮肤移植物,利用基于聚乳酸羟基乙酸、鱼胶原和六氟异丙醇的静电纺丝膜,制备哪种纤维直径尺寸的静电纺丝膜更有利于皮肤组织再生尚无明确结论。
发明内容
基于以上问题,本发明的目的在于提供一种具有改良纳米结构的皮肤移植物,其纤维。
为了实现以上目的,本发明采用的技术方案为:一种具有改良纳米结构的皮肤移植物,制作方法包括:
步骤1:以聚乳酸羟基乙酸、鱼胶原为原料溶于六氟异丙醇中得到电纺液,电纺液中聚乳酸羟基乙酸和鱼胶的浓度分别为20%W/V、2%W/V;
步骤2:将电纺液置于静电纺丝设备中,纺制得到取向型的电纺膜,静电纺丝设备参数如下:
①施加电压-2Kv,5kv;
②使用21G金属针头(内径0.5mm),针头尖端距离收集器11cm;
③电纺液推注速率0.018mm/min;
④收集器为2800rpm滚筒;
⑤电纺时间4~8小时。
制得电纺膜的纺丝直径为319±100nm,拉伸强度达到11.95MPa,具有生物相容性与降解性;将其应用于作为皮肤移植物时,在皮肤缺损愈合14天时仍可见电纺膜材料存留,保证电纺膜持续作用在皮肤缺损愈合的凝血期、炎症期、增殖修复期。
具体的,上述方法还包括步骤3:将步骤2中得到的电纺膜进行真空干燥72h后,4℃浸泡于NHS/EDC溶液中交联24h;然后用75%乙醇浸泡材料10min进行预收缩,再次真空干燥24h,辐照灭菌备用。
本发明的有益效果为:
(1)、本发明提供的具有改良纳米结构的皮肤移植物,具有促进皮肤缺损愈合、促角化、促血管化、降低纤维化程度等优势,为构建组织工程化皮肤提供了新选择。
(2)、本发明提供的具有改良纳米结构的皮肤移植物,纺丝直径达319±100nm,其纳米取向型的电纺纤维使皮肤细胞的细胞骨架沿纤维走向发生改变,对于细胞具有优良的“地形调控”作用,因而可提高细胞迁移效率,从而加快皮肤伤口关闭。
(3)、本发明提供的具有改良纳米结构的皮肤移植物,拉伸强度达到11.95MPa,力学性能好,同时具有良好生物相容性和生物降解性,在皮肤缺损愈合14天时仍可见部分材料存留,保证材料在皮肤缺损愈合的凝血期、炎症期、增殖修复期持续发挥作用。
附图说明
图1为微观形貌测试结果图;
图2为纤维行直径分布统计结果图;
图3为拉伸试验的SEM图像;
图4为典型应力-应变曲线;
图5为拉伸强度测试结果图;
图6为应变率;
图7为WCA图像和相应分析;
图8为水性图像和相应分析;
如图9为降解试验结果;
图10为L929增殖活性结果;
图11为HOK增殖活性结果;
图12、13为HOK的PI荧光染色结果;
图14为HOK细胞的SEM图像;
图15和16为大鼠皮肤缺损的宏观图像和相应分析;
图17和18为大鼠皮肤缺损的H&E剩余间隙图像和相应分析;
图19为大鼠皮肤缺损H&E角化层图像分析;
图20、21和22为角化标志物K5和K10的RNA表达结果;
图23为皮肤毛囊再生结果;
图24和25为HE染色结果-血管化结果;
图26和27为血管标志物CD31的RNA表达;
图28和29为异物反应(FBR)-纤维化结果;
图30和31为炎性细胞浸润结果。
具体实施方式
实施例
本实施例提供一种具有改良纳米结构的皮肤移植物,制作方法包括:
步骤1:以聚乳酸羟基乙酸、鱼胶原为原料溶于六氟异丙醇中得到电纺液,电纺液中聚乳酸羟基乙酸和鱼胶的浓度分别为20%W/V、2%W/V;
步骤2:将电纺液置于静电纺丝设备中,纺制得到取向型的电纺膜,静电纺丝设备参数如下:
①施加电压-2Kv,5kv;
②使用21G金属针头(内径0.5mm),针头尖端距离收集器11cm;
③电纺液推注速率0.018mm/min;
④收集器为2800rpm滚筒;
⑤电纺时间8小时;
步骤3:将步骤2中得到的电纺膜进行真空干燥72h后,4℃浸泡于NHS/EDC溶液中交联24h;然后用75%乙醇浸泡材料10min进行预收缩,再次真空干燥24h,辐照灭菌备用。
制得电纺膜的纺丝直径为319±100nm,以下命名为A300。
对照例1
本实施例作为实施例的对照例,其基本操作与实施例相同,区别之处在于以下静电纺丝设备参数:针头尖端距离收集器16cm;电纺液推注速率0.068mm/min;电纺时间4小时。制得电纺膜的纺丝直径为687±138nm,以下命名为A600。
对照例2
本实施例作为实施例的对照例,其基本操作与对照例1相同,区别之处在于:电纺液中聚乳酸羟基乙酸和鱼胶的浓度分别为25%W/V、2%W/V,制得电纺膜的纺丝直径为1048±130nm,以下命名为A100。
对上述实施例、对照例1、对照例2分别制得的A300、A600、A1000进行测试,其中有些测试必要时引入Ctr对照组(生理盐水组),具体测试如下:
一、微观形貌及机械性能
1、对三种不同直径的电纺膜A300、A600和A1000进行扫描电镜(SEM)检测,并进行纤维行直径分布统计,如图1所示SEM图像和图2所示不同直径取向静电纺丝膜的相应直径分布,所有组别均呈现出近似正态的均匀分布,具有一定的取向性,直径分别为A300:319±100nm、A600:687±138nm、A1000:1048±130nm,对比A300、A600可知,同样浓度的静电纺丝液采用不同的静电纺参数,即使参数的差别很小,得到的电纺膜的尺寸差别具大;对比A600、A1000可知,不同浓度的静电纺丝液采用相同的静电纺参数,即使浓度的差别很小,得到的电纺膜的尺寸差别具大;整体对比A300、A600、A1000可知,静电纺丝液的浓度、静电纺的各种参数对得到的电纺膜的影响很大且没有具体的、常规的规律,本发明相较于对照例1、2,微小的改变了静电纺丝液的浓度、静电纺的各种参数,得到的电纺膜的纤维直径尺寸就产生了40%以上的缩小。
2、电纺膜机械性能检测,如图3所示拉伸试验的SEM图像、图4所示各种膜的典型应力-应变曲线、图5所示拉伸强度和图6所示应变率,A300组拉伸强度(11.95MPa)强于A600(6.80MPa)和A1000(8.86MPa)组,可见,本发明相较于对照例1、2,微小的改变了静电纺丝液的浓度、静电纺的各种参数,得到的电纺膜的拉伸强度大幅度增加。
二、亲水性及降解性测试
对三种不同直径的电纺膜A300、A600和A1000进行湿接触角(WCA)和亲水性检测,如图7的WCA图像和相应分析、图8的水性图像和相应分析所示:A300组和A600组差异无统计学意义,但均优于A1000组;对三种不同直径的电纺膜A300、A600和A1000进行降解性检测,如图9的降解试验结果所示:A300组降解性强于A600和A1000组。
三、生物相容性测试
将电纺膜A300、A600和A1000裁剪成5mm*5mm正方形贴于96孔板底部,小鼠皮肤成纤维细胞系(L929细胞)和人口腔黏膜角质形成细胞(HOK细胞)接种于孔板中(细胞密度:4*10^4个/ml),CCK-8试剂盒及PI染色检测细胞活力。L929在电纺膜材料上的增殖活性如图10所示:A300组增殖活性强于A600和A1000组,略低于Ctr对照组(生理盐水组)。HOK在电纺膜材料上的增殖活性如图11所示:差异无统计学意义。HOK在电纺膜材料上的死细胞数量,如图12和13的HOK的PI荧光染色结果所示:差异无统计学意义。
将HOK细胞接种于材料上(3*10^4cells/cm2),培养24h结果显示如图14所示:A300组细胞伸展明显强于其他组。
四、皮肤缺损修复的动物试验
1.缺损愈合面积评价
将三种不同直径的电纺膜——A300、A600和A1000植入大鼠背部皮肤缺损(6mm圆形缺损),缺损上部覆盖Tegaderm(3M),并用硅胶圈(内径8mm,外径10mm,边宽2mm,厚度1mm)缝合固定,避免背部肌肉收缩关闭创口,以达到客观评价皮肤伤口爬行愈合的能力。根据缺损愈合面积统计,如图15和16所示的大鼠皮肤缺损的宏观图像和相应分析表明:A300组皮肤缺损愈合快于其他组。HE染色结果-剩余间隙,如图17和18所示的大鼠皮肤缺损的H&E剩余间隙图像和相应分析表明:A300组剩余缺损间隙小于其他组。
2.组织切片评价角化
大鼠皮肤缺损H&E角化层图像分析如图19所示:A300组在第7天先于其他组出现角化层。角化标志物K5和K10的RNA表达结果如图20、21和22所示:第7天K5和K10的RNA和蛋白表达,A300组优于其他组。皮肤毛囊再生结果如图23所示:第14天,A300组毛囊再生数优于其他组。
3.组织切片评价血管化
HE染色结果-血管化结果如图24和25所示:A300组在第7天血管化面积高于其他组。血管标志物CD31的RNA表达如图26和27所示:第7天CD31的RNA和蛋白表达,A300组强于其他组。
4.组织切片评价纤维化
将材料植入于大鼠皮下,异物反应(FBR)-纤维化如图28和29所示:A300组在第7天纤维包囊厚度低于其他组。炎性细胞浸润结果如图30和31所示:A300组在第7天炎性细胞浸润量低于其他组。
本发明的上述实施例仅仅是为说明本发明所作的举例,而并非是对本发明的实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其他不同形式的变化和变动。这里无法对所有的实施方式予以穷举。凡是属于本发明的技术方案所引申出的显而易见的变化或变动仍处于本发明的保护范围之列。
Claims (2)
1.一种具有改良纳米结构的皮肤移植物,其特征在于,制作方法包括:
步骤1:以聚乳酸羟基乙酸、鱼胶原为原料溶于六氟异丙醇中得到电纺液,电纺液中聚乳酸羟基乙酸和鱼胶的浓度分别为20%W/V、2%W/V;
步骤2:将电纺液置于静电纺丝设备中,纺制得到取向型的电纺膜,静电纺丝设备参数如下:
①施加电压-2Kv,5kv;
②使用21G金属针头(内径0.5mm),针头尖端距离收集器11cm;
③电纺液推注速率0.018mm/min;
④收集器为2800rpm滚筒;
⑤电纺时间4~8小时;
制得电纺膜的纺丝直径为319±100nm,拉伸强度达到11.95MPa,具有生物相容性与降解性;将其应用于作为皮肤移植物时,在皮肤缺损愈合14天时仍可见电纺膜材料存留,保证电纺膜持续作用在皮肤缺损愈合的凝血期、炎症期、增殖修复期。
2.根据权利要求1所述的具有改良纳米结构的皮肤移植物,其特征在于,还包括以下步骤3:将步骤2中得到的电纺膜进行真空干燥72h后,4℃浸泡于NHS/EDC溶液中交联24h;然后用75%乙醇浸泡材料10min进行预收缩,再次真空干燥24h,辐照灭菌备用。
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