高亲水性静电纺丝短纤维海绵促创面愈合的作用

Effect of high hydrophilic electrospun short fibrous sponge on wound repair

目的·构建一种含有氧化石墨烯(graphene oxide,GO)的高亲水性静电纺丝短纤维海绵(Sponge@GO),探究其对慢性创面的促愈合作用。方法·分别将不含及含有GO的纺丝液经过纺丝成膜、打碎、塑形并用戊二醛交联后,制备出2种短纤维海绵(Sponge和Sponge@GO)。采用扫描电子显微镜(scanning electron microscope,SEM)观察2种海绵的内部结构,通过接触角及吸水率观察两者的亲水性能。经CCK-8法和活/死细胞染色验证海绵的生物相容性。将12只6周龄SD雄性大鼠分为对照组、Sponge组和Sponge@GO组,每组各4只;通过腹腔注射1%的链脲佐菌素溶液建立糖尿病模型,建模后每只大鼠背部制备3块直径1.0 cm的全层皮肤缺损;将材料覆盖于创面,使用医用纱布覆盖创面并固定(对照组仅覆盖无菌纱布包扎);分别于术后第7、14日测量并计算创面愈合率,同时取创周0.5 cm内组织行苏木精-伊红染色(hematoxylin and eosin staining,H-E染色)、Masson染色观察病理学改变;并在第14日通过α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)免疫荧光染色,观察血管形成情况。结果·SEM观察显示,与Sponge海绵比较,Sponge@GO海绵的纤维直径较细,孔隙率偏大。2种短纤维海绵在10 min内均基本达到最大吸水率,Sponge@GO海绵的吸水率较高。Sponge@GO海绵的接触角小于Sponge海绵,差异有统计学意义(P=0.000)。CCK-8法检测结果显示,术后第3、5日Sponge组与对照组相比,尚呈现出细胞增殖的状态(均P<0.05),而Sponge@GO组与对照组的差异无统计学意义。活/死细胞染色结果显示,3组细胞均呈现良好的增长态势。SEM和荧光染色观察显示,Sponge@GO海绵内部的细胞数量明显较Sponge海绵多。3组大鼠的创面均未见感染。Sponge@GO组和Sponge组术后第7日的创面愈合率均显著高于对照组(均P<0.05);第14日,Sponge@GO组的创面愈合率仍显著高于对照组(P=0.009),而Sponge组与对照组之间的差异无统计学意义。术后第14日,H-E染色显示Sponge@GO组创面肉芽组织更加成熟,结构更为均匀致密;Masson染色显示Sponge@GO组胶原更加致密,上皮化显著;α-SMA免疫荧光染色显示Sponge@GO组新生血管数量更多,密度更高。结论·Sponge@GO海绵在吸收渗液后可维持创面微湿润环境,有利于促进伤口愈合。

Objective·To construct an electrospun short fibrous sponge(Sponge@GO)laden with graphene oxide(GO)for chronic wound healing.Methods·Two types of short fibrous sponges(Sponge and Sponge@GO)without and with GO were prepared by means of electrospinning,homogenizing,shaping and crosslinking with glutaraldehyde,respectively.The internal structures of the two sponges were observed with a scanning electron microscope(SEM),and their hydrophilic properties were observed via contact angle and water absorption rate.The biocompatibility of the sponge was verified by CCK-8 and live/dead staining.Twelve 6-weekold SD male rats were divided into control group,Sponge group and Sponge@GO group,with 4 rats in each group.The diabetes models were established by intraperitoneal injection of 1%streptozotocin solution,and three full-layer skin defects with a diameter of 1.0 cm were prepared on the back of each rat after modelling.Covering on the wound,the material was fixed with medical gauze.The control group was only covered with sterile gauze dressing.The wound healing rate was measured and calculated on Day 7 and 14,respectively,while hematoxylin-eosin(H-E)staining and Masson staining were performed on tissues within 0.5 cm around the wound to observe pathological changes.The angiogenesis was observed byα-smooth muscle actin(α-SMA)immunofluorescence staining on Day 14.Results·SEM observation showed that the fiber diameter of Sponge@GO was significantly thinner and the porosity increased.The two types of short fiber scaffolds basically reached the maximum water uptake within 10 min,but the Sponge@GO scaffold showed better water absorption performance.The water contact angle of Sponge@GO scaffold was significantly smaller than that of Sponge,and the difference was statistically significant(P=0.000).The results of CCK-8 method showed that on Day 3 and 5,the Sponge group had better cell proliferation compared with the control group(both P<0.05),while there was no statistical significance between Sponge@GO group and control group.The results of live/dead staining showed that all the three groups of cells showed good cell growth trend.SEM and fluorescence staining showed that there were more cells in the Sponge@GO scaffold.In vivo experiment,no infection was found on the wound surface of the three groups of rats.The wound healing rate of Sponge@GO and Sponge groups was significantly higher than that of control group on Day 7(both P<0.05).On Day 14,the wound healing rate of the Sponge@GO group was still significantly higher than that of the control group(P=0.009),while the difference between the Sponge group and the control group was not statistically significant.On Day 14,H-E staining showed more mature granulation tissue and more uniform and dense structure in the Sponge@GO group;Masson staining showed more dense collagen and significant epithelialization in the Sponge@GO group;α-SMA immunofluorescence staining showed more neovascularization and higher density in the Sponge@GO group.Conclusion·Sponge@GO sponge can ensure micro-moist environment on the wound surface after absorbing exudate and has shown promising results in promoting wound healing.