Effects of cerium oxide nanoenzyme-gelatin methacrylate anhydride hydrogel in the repair of infected full-thickness skin defect wounds in mice
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摘要:
目的 探讨氧化铈纳米酶-甲基丙烯酸酐化明胶（GelMA）水凝胶（以下简称复合水凝胶）在小鼠全层皮肤缺损感染创面修复中的作用。 方法 该研究为实验研究。采用水热法制备粒径为（116±9）nm的氧化铈纳米酶，同时制备具有多孔网状结构且成胶性能良好的GelMA水凝胶。筛选出25 μg/mL氧化铈纳米酶可明显促进人皮肤成纤维细胞增殖和具有较高的超氧化物歧化酶活性，将其加入GelMA水凝胶中制备复合水凝胶。计算用磷酸盐缓冲液（PBS）浸泡3、7 d后复合水凝胶中氧化铈纳米酶的释放百分比。将小鼠红细胞悬液分为用相应溶液处理的PBS组、Triton X-100组、氧化铈纳米酶组、GelMA水凝胶组及复合水凝胶组，利用酶标仪检测处理1 h后红细胞的溶血情况。测定用PBS、氧化铈纳米酶、GelMA水凝胶及复合水凝胶培养耐甲氧西林金黄色葡萄球菌（MRSA）和大肠埃希菌2 h后的细菌浓度。以上实验样本数均为3。取24只8周龄雄性BALB/c小鼠，在背部对称位置各制备1个用MRSA感染的全层皮肤缺损创面。将小鼠分为不进行药物干预的对照组及滴加相应溶液的氧化铈纳米酶组、GelMA水凝胶组和复合水凝胶组，每组6只小鼠。观察伤后3、7、14 d创面愈合情况并测量伤后3、7 d剩余创面面积（样本数为5）。取小鼠伤后3 d创面分泌物，检测MRSA的浓度（样本数为3），采用激光散斑血流成像系统观测小鼠伤后5 d创面血流灌注量（样本数为6）。伤后14 d，取小鼠创面组织，行苏木精-伊红染色观察新生上皮情况，行Masson染色观察胶原情况（样本数均为3）。 结果 浸泡3、7 d后，复合水凝胶中氧化铈纳米酶释放百分比分别约为39%、75%。处理1 h后，与Triton X-100组比较，PBS组、GelMA水凝胶组、氧化铈纳米酶组及复合水凝胶组红细胞溶血程度均明显下降（ P<0.05）。与用PBS培养比较，用氧化铈纳米酶、GelMA水凝胶、复合水凝胶培养2 h的MRSA、大肠埃希菌浓度均明显降低（ P<0.05）。伤后3~14 d，4组小鼠创面均逐渐愈合，复合水凝胶组小鼠伤后14 d创面全部愈合。伤后3、7 d，复合水凝胶组小鼠剩余创面面积分别为（29±3）、（13±5）mm 2，明显小于对照组的（56±12）、（46±10）mm 2和氧化铈纳米酶组的（51±7）、（38±8）mm 2（ P值均<0.05），与GelMA水凝胶组的（41±5）、（24±9）mm 2相近（ P值均>0.05）。伤后3 d，复合水凝胶组小鼠创面MRSA浓度明显低于对照组、氧化铈纳米酶组、GelMA水凝胶组（ P值均<0.05）。伤后5 d，复合水凝胶组小鼠创面血液灌注量明显大于对照组、氧化铈纳米酶组、GelMA水凝胶组（ P值均<0.05）。伤后14 d，复合水凝胶组小鼠创面基本完成上皮化，上皮化情况明显优于其他3组；复合水凝胶组小鼠创面胶原含量较其他3组明显增多，排列也更为有序。 结论 复合水凝胶具有良好的生物相容性和体内外抗菌效果，可持续缓释氧化铈纳米酶，改善早期创面血流灌注，促进创面再上皮化及胶原合成，从而促进小鼠全层皮肤缺损感染创面愈合。 Abstract:Objective To investigate the effects of cerium oxide nanoenzyme-gelatin methacrylate anhydride (GelMA) hydrogel (hereinafter referred to as composite hydrogel) in the repair of infected full-thickness skin defect wounds in mice. Methods This study was an experimental study. Cerium oxide nanoenzyme with a particle size of (116±9) nm was prepared by hydrothermal method, and GelMA hydrogel with porous network structure and good gelling performance was also prepared. The 25 μg/mL cerium oxide nanoenzyme which could significantly promote the proliferation of human skin fibroblasts and had high superoxide dismutase activity was screened out. It was added to GelMA hydrogel to prepare composite hydrogel. The percentage of cerium oxide nanoenzyme released from the composite hydrogel was calculated after immersing it in phosphate buffer solution (PBS) for 3 and 7 d. The red blood cell suspension of mice was divided into PBS group, Triton X-100 group, cerium oxide nanoenzyme group, GelMA hydrogel group, and composite hydrogel group, which were treated with corresponding solution. The hemolysis of red blood cells was detected by microplate reader after 1 h of treatment. The bacterial concentrations of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli were determined after being cultured with PBS, cerium oxide nanoenzyme, GelMA hydrogel, and composite hydrogel for 2 h. The sample size in all above experiments was 3. Twenty-four 8-week-old male BALB/c mice were taken, and a full-thickness skin defect wound was prepared in the symmetrical position on the back and infected with MRSA. The mice were divided into control group without any drug intervention, and cerium oxide nanoenzyme group, GelMA hydrogel group, and composite hydrogel group applied with corresponding solution, with 6 mice in each group. The wound healing was observed on 3, 7, and 14 d after injury, and the remaining wound areas on 3 and 7 d after injury were measured (the sample size was 5). The concentration of MRSA in the wound exudation of mice on 3 d after injury was measured (the sample size was 3), and the blood flow perfusion in the wound of mice on 5 d after injury was observed using a laser speckle flow imaging system (the sample size was 6). On 14 d after injury, the wound tissue of mice was collected for hematoxylin-eosin staining to observe the newly formed epithelium and for Masson staining to observe the collagen situation (the sample size was both 3). Results After immersion for 3 and 7 d, the release percentages of cerium oxide nanoenzyme in the composite hydrogel were about 39% and 75%, respectively. After 1 h of treatment, compared with that in Triton X-100 group, the hemolysis of red blood cells in PBS group, GelMA hydrogel group, cerium oxide nanoenzyme group, and composite hydrogel group was significantly decreased ( P<0.05). Compared with that cultured with PBS, the concentrations of MRSA and Escherichia colicultured with cerium oxide nanoenzyme, GelMA hydrogel, and composite hydrogel for 2 h were significantly decreased ( P<0.05). The wounds of mice in the four groups were gradually healed from 3 to 14 d after injury, and the wounds of mice in composite hydrogel group were all healed on 14 d after injury. On 3 and 7 d after injury, the remaining wound areas of mice in composite hydrogel group were (29±3) and (13±5) mm 2, respectively, which were significantly smaller than (56±12) and (46±10) mm 2 in control group and (51±7) and (38±8) mm 2 in cerium oxide nanoenzyme group (with P values all <0.05), but was similar to (41±5) and (24±9) mm 2 in GelMA hydrogel group (with P values both >0.05). On 3 d after injury, the concentration of MRSA on the wound of mice in composite hydrogel group was significantly lower than that in control group, cerium oxide nanoenzyme group, and GelMA hydrogel group, respectively (with Pvalues all <0.05). On 5 d after injury, the volume of blood perfusion in the wound of mice in composite hydrogel group was significantly higher than that in control group, cerium oxide nanoenzyme group, and GelMA hydrogel group, respectively ( P<0.05). On 14 d after injury, the wound of mice in composite hydrogel group basically completed epithelization, and the epithelization was significantly better than that in the other three groups. Compared with that in the other three groups, the content of collagen in the wound of mice in composite hydrogel group was significantly increased, and the arrangement was also more orderly. Conclusions The composite hydrogel has good biocompatibility and antibacterial effect in vivo and in vitro. It can continuously sustained release cerium oxide nanoenzyme, improve wound blood perfusion in the early stage, and promote wound re-epithelialization and collagen synthesis, therefore promoting the healing of infected full-thickness skin defect wounds in mice. -
1 氧化铈纳米酶和GelMA水凝胶的微观形貌。1A.氧化铈纳米酶颗粒均匀 扫描电子显微镜×60 000；1B.GelMA水凝胶呈多孔网状结构 扫描电子显微镜×100
注：GelMA为甲基丙烯酸酐化明胶
3 4组小鼠伤后各个时间点全层皮肤缺损感染创面愈合情况。3A、3B、3C、3D.分别为对照组、氧化铈纳米酶组、GelMA水凝胶组、复合水凝胶组伤后3 d的创面，图3D剩余创面面积明显小于图3A、3B；3E、3F、3G、3H.分别为对照组、氧化铈纳米酶组、GelMA水凝胶组、复合水凝胶组伤后7 d的创面，图3H剩余创面面积明显小于图3E、3F、3G
注：对照组小鼠创面仅用无菌手术敷贴封闭；氧化铈纳米酶组、甲基丙烯酸酐化明胶（GelMA）水凝胶组、复合水凝胶组小鼠创面先分别滴加氧化铈纳米酶、GelMA水凝胶及氧化铈纳米酶-GelMA水凝胶，再用无菌手术敷贴封闭
4 4组小鼠伤后5 d全层皮肤缺损感染创面血流灌注情况。4A、4B、4C、4D.分别为对照组、氧化铈纳米酶组、GelMA水凝胶组、复合水凝胶组创面血流灌注情况，图4D创面血流灌注量明显多于图4A、4B、4C
注：对照组小鼠创面仅用无菌手术敷贴封闭，氧化铈纳米酶组、甲基丙烯酸酐化明胶（GelMA）水凝胶组、复合水凝胶组小鼠创面先分别滴加氧化铈纳米酶、GelMA水凝胶及氧化铈纳米酶-GelMA水凝胶，再用无菌手术敷贴封闭；黑色代表无血流灌注，绿色代表血流灌注少，红色代表血流灌注充足
5 4组小鼠伤后14 d全层皮肤缺损感染创面新生上皮形成和胶原生成情况。5A、5B、5C、5D.分别为对照组、氧化铈纳米酶组、GelMA水凝胶组、复合水凝胶组创面新生上皮情况，图5D上皮化程度明显高于图5A、5B、5C 苏木精-伊红×5；5E、5F、5G、5H.分别为对照组、氧化铈纳米酶组、GelMA水凝胶组、复合水凝胶组创面胶原生成情况，图5H创面新生胶原含量明显多于图5E、5F、5G Masson×5
注：对照组小鼠创面仅用无菌手术敷贴封闭，氧化铈纳米酶组、甲基丙烯酸酐化明胶（GelMA）水凝胶组、复合水凝胶组小鼠创面先分别滴加氧化铈纳米酶、GelMA水凝胶及氧化铈纳米酶-GelMA水凝胶，再用无菌手术敷贴封闭
表1 4组小鼠伤后各时间点全层皮肤缺损感染创面剩余面积比较（mm 2，
表1. Comparison of the remaining areas of infected wounds with full-thickness skin defects in four groups of mice at various time points after injury
组别 样本数 3 d 7 d 对照组 5 56±12 46±10 氧化铈纳米酶组 5 51±7 38±8 GelMA水凝胶组 5 41±5 24±9 复合水凝胶组 5 29±3 13±5 F值 13.38 15.95 P值 <0.001 <0.001 P 1值 <0.001 <0.001 P 2值 <0.001 <0.001 P 3值 0.076 0.145 注：对照组小鼠创面仅用无菌手术敷贴封闭；氧化铈纳米酶组、甲基丙烯酸酐化明胶（GelMA）水凝胶组、复合水凝胶组小鼠创面上先分别滴加氧化铈纳米酶、GelMA水凝胶及氧化铈纳米酶-GelMA水凝胶，再用无菌手术敷贴封闭；处理因素主效应， F=31.15， P<0.001；时间因素主效应， F=29.22， P<0.001；两者交互作用， F=0.34， P=0.797； F值、 P值为组间各时间点总体比较所得； P 1值、 P 2值、 P 3值分别为对照组、氧化铈纳米酶组、GelMA水凝胶组与复合水凝胶组比较所得 
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