Effects and mechanisms of zinc ion-loaded composite hydrogel on infected full-thickness skin defect wounds in diabetic mice
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摘要:
目的 探讨负载锌离子的复合水凝胶（以下简称含锌水凝胶）对糖尿病小鼠全层皮肤缺损感染创面的作用及其机制。 方法 该研究为实验研究。制备聚（甘油癸二酸酯）-共-聚乙二醇-共聚-邻苯二酚/季铵化壳聚糖水凝胶（以下简称单纯水凝胶）和在单纯水凝胶的基础上添加锌离子的疏松多孔且具有良好黏附性的固态含锌水凝胶。计算用磷酸盐缓冲液（PBS）浸泡14 d后含锌水凝胶中锌离子的释放率。检测用单纯水凝胶、含锌水凝胶、PBS培养2 h后耐甲氧西林金黄色葡萄球菌（MRSA）浓度。利用酶标仪检测单纯水凝胶、含锌水凝胶、PBS对1,1-二苯基-2-三硝基苯肼（DPPH）的清除率,反映清除氧自由基能力。测量用单纯水凝胶、含锌水凝胶、PBS培养24 h后人脐静脉内皮细胞（HUVEC）形成的血管长度。采用细胞计数试剂盒8检测用单纯水凝胶、含锌水凝胶、PBS培养24 h后L929细胞的活力。将小鼠红细胞悬液分为用PBS处理的空白对照组和用相应溶液处理的单纯水凝胶组、含锌水凝胶组和Triton X-100组,利用酶标仪检测孵育2 h后红细胞的溶血情况并计算溶血率。以上实验样本数均为3。取21只6~8周龄雄性C57BL/6J小鼠,在背部脊柱对称位置各制备1个用MRSA感染的全层皮肤缺损创面。将小鼠分为滴加PBS的空白对照组和用相应水凝胶处理的单纯水凝胶组和含锌水凝胶组。伤后3 d,检测空白对照组、单纯水凝胶组、含锌水凝胶组小鼠创面中的细菌浓度,样本数为4。于伤后0（即刻）、3、7、14 d,大体观察小鼠创面感染情况并计算伤后3、7、14 d创面愈合率,样本数为5。伤后14 d,行苏木精-伊红染色和Masson染色分别检测小鼠创面中的新生上皮和胶原生成情况,行免疫荧光染色检测小鼠创面中血管新生及M2型巨噬细胞分布情况。 结果 浸泡14 d后,含锌水凝胶中锌离子的释放率为（70.5±4.6）%。与用含锌水凝胶比较,用PBS和单纯水凝胶培养2 h后细菌浓度均明显升高（P<0.05）。含锌水凝胶对DPPH的清除率明显高于PBS、单纯水凝胶（P值均<0.05）。与用PBS比较,用含锌水凝胶培养24 h后HUVEC形成的血管长度明显增长（P<0.05）。与用含锌水凝胶比较,用PBS和单纯水凝胶培养24 h后L929细胞的活力均明显降低（P<0.05）。孵育2 h后,与Triton X-100组相比,空白对照组、单纯水凝胶组、含锌水凝胶组红细胞的溶血率均显著降低（P<0.05）；而后3组的红细胞溶血率相近（P>0.05）。伤后3 d,含锌水凝胶组小鼠创面中的细菌浓度明显低于空白对照组和单纯水凝胶组（P值均<0.05）。伤后3~14 d,3组小鼠创面均逐渐愈合,含锌水凝胶组小鼠伤后14 d创面基本愈合。伤后7 d,含锌水凝胶组小鼠创面愈合率为（72.4±8.4）%,明显高于空白对照组和单纯水凝胶组的（31.6±6.7）%、（44.7±5.4）%（P值均<0.05）。伤后14 d,含锌水凝胶组小鼠创面愈合率为（92.7±4.3）%,明显高于空白对照组的（73.5±7.4）%,P<0.05。伤后14 d,与空白对照组和单纯水凝胶组相比,含锌水凝胶组小鼠创面的新生表皮长度更长、厚度更厚,胶原沉积更多,新生血管和M2型巨噬细胞分布更丰富。 结论 含锌水凝胶具有良好的生物相容性、清除氧自由基能力和体内外抗菌效果、促血管生成能力,可持续缓释锌离子,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 and mechanisms of zinc ion-loaded composite hydrogel (hereinafter referred to as the zinc-containing hydrogel) on infected full-thickness skin defect wounds in diabetic mice. Methods This study was an experimental study. A poly (glycerol sebacate)-co-poly(ethylene glycol)-g-catechol prepolymer/quaternized-chitosan hydrogel (hereinafter referred to as the simple hydrogel) and a solid-state zinc-containing hydrogel with porous and good adhesion by adding zinc ions to the simple hydrogel were prepared. The release rate of zinc ions from the zinc-containing hydrogel after immersion in phosphate buffer solution (PBS) for 14 days was calculated. The concentration of methicillin-resistant Staphylococcus aureus (MRSA) cultured for 2 hours with the simple hydrogel, zinc-containing hydrogel, and PBS was measured. The scavenging ability of the simple hydrogel, zinc-containing hydrogel, and PBS for 1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2, 4, 6-trinitrophenyl) hydrazyl (DPPH) was detected using microplate reader to reflect the ability of oxygen free radical removal. The length of vessels formed by human umbilical vein endothelial cells (HUVECs) cultured for 24 hours with the simple hydrogel, zinc-containing hydrogel, and PBS was measured. The cell viability of L929 cells cultured for 24 hours with the simple hydrogel, zinc-containing hydrogel, and PBS was detected using the cell counting kit-8. The mouse red blood cell suspension was divided into blank control group treated with PBS, simple hydrogel group, zinc-containing hydrogel group, and Triton X-100 group treated with corresponding solution. Hemolysis was detected using microplate reader after 2 hours of treatment, and the hemolysis rate was calculated. All experiments had a sample size of 3. Twenty-one C57BL/6J mice aged 6-8 weeks were taken, and a full-thickness skin defect wound was prepared in the symmetrical position on the back spine and infected with MRSA. Mice were divided into blank control group treated with PBS, simple hydrogel group, and zinc-containing hydrogel group treated with the corresponding hydrogel. Three days after injury, bacterial concentration in the wounds were measured in all groups of mice (n=4). On day 0 (immediately), 3, 7, and 14 after injury, the wound infection status of mice was generally observed and the wound healing rate was calculated (n=5). Hematoxylin-eosin staining and Masson staining were used to detect new epithelium and collagen formation in the wounds of mice on day 14 after injury. Immunofluorescence staining was used to detect neovascularization and distribution of M2 macrophages in the wounds of mice. Results After immersion for 14 days, the release rate of zinc ions of the zinc-containing hydrogel was (70.5±4.6)%. Compared with the zinc-containing hydrogel, the bacterial concentration was significantly increased after 2 hours of culture with PBS and the simple hydrogel (P<0.05). The DPPH scavenging rate of the zinc-containing hydrogel was significantly higher than that of PBS and the simple hydrogel (with P values all <0.05). The length of vessels formed by HUVECs cultured for 24 hours with the zinc-containing hydrogel was significantly longer than that cultured with PBS (P<0.05). Compared with PBS and the simple hydrogel, the cell viability of L929 cells cultured for 24 hours with the zinc-containing hydrogel was significantly higher (P<0.05). After 2 hours of incubation, compared with that in Triton X-100 group, the hemolysis rate of red blood cells in blank control, simple hydrogel, and zinc-containing hydrogel groups was significantly reduced (P<0.05); and the hemolysis rate of red blood cells in the latter three groups was similar (P>0.05). On day 3 after injury, the bacterial concentration in the wounds of mice in zinc-containing hydrogel group was significantly lower than that in blank control and simple hydrogel groups (with P values all <0.05). From day 3 to day 14 after injury, the wounds of mice in all the three groups were gradually healing, and on day 14 after injury, the wounds of mice in the zinc-containing hydrogel group were basically healed. On day 7 after injury, the wound healing rate of mice in zinc-containing hydrogel group was (72.4±8.4)%, which was significantly higher than that of blank control and simple hydrogel groups, being (31.6±6.7)% and (44.7±5.4)%, respectively(with P values all< 0.05). On day 14 after injury, the wound healing rate of mice in zinc-containing hydrogel group was (92.7±4.3)%, which was significantly higher than (73.5±7.4)% in blank control group (P<0.05). On day 14 after injury, compared with that in blank control and simple hydrogel groups, the newly formed epidermis in mice wound of zinc-containing hydrogel group was longer and thicker, with more collagen deposition, and a more abundant distribution of new vessels and M2 macrophages. Conclusions The zinc-containing hydrogel exhibits good biocompatibility, oxygen free radical scavenging capacity, and antimicrobial effects both in vitro and in vivo, as well as angiogenic promotion capability. It can provide sustained release of zinc ions to promote re-epithelialization and collagen synthesis, thus enhancing the healing of infected full-thickness skin defect wounds in diabetic mice. -
Key words:
- Biocompatible materials /
- Hydrogel /
- Infection /
- Diabetes mellitus, experimental /
- Zinc ions /
- Bacterial resistance /
- Wound repair
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参考文献
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图 1 含锌水凝胶的表征及微观结构。1A.成胶前呈流动状态；1B.成胶后呈暗红色,倒立时可保持稳定不流动；1C.水凝胶可牢固黏附在手指上；1D.水凝胶呈多孔结构 扫描电子显微镜×500
注：含锌水凝胶指负载锌离子的聚(甘油癸二酸酯)-共-聚乙二醇-共聚-邻苯二酚/季铵化壳聚糖水凝胶
图 2 3组糖尿病小鼠伤后各时间点全层皮肤缺损感染创面的愈合情况。2A、2B、2C、2D.分别为空白对照组伤后0（即刻）、3、7、14 d创面情况；2E、2F、2G、2H.分别为单纯水凝胶组伤后0、3、7、14 d创面情况；2I、2J、2K、2L.分别为含锌水凝胶组伤后0、3、7、14 d创面情况,图2B、2F、2J的创面均被感染且情况相似,图2K剩余创面面积明显小于图2C、2G,图2L的创面基本愈合且剩余创面面积明显小于图2D、2H
注：空白对照组、单纯水凝胶组、含锌水凝胶组小鼠创面感染耐甲氧西林金黄色葡萄球菌后分别用磷酸盐缓冲液、聚(甘油癸二酸酯)-共-聚乙二醇-共聚-邻苯二酚/季铵化壳聚糖水凝胶、负载锌离子的聚(甘油癸二酸酯)-共-聚乙二醇-共聚-邻苯二酚/季铵化壳聚糖水凝胶处理；每张分图底部为刻度尺刻度
图 3 3组糖尿病小鼠伤后14 d全层皮肤缺损感染创面组织学分析。3A、3B、3C.分别为空白对照组、单纯水凝胶组、含锌水凝胶组小鼠创面,图3C中的新生上皮较3A、3B更加完整 苏木精-伊红×100；3D、3E、3F.分别为空白对照组、单纯水凝胶组、含锌水凝胶组小鼠创面,图3F胶原沉积（蓝色区域）较图3D和图3E明显增多 Masson×100
注：空白对照组、单纯水凝胶组、含锌水凝胶组小鼠创面感染耐甲氧西林金黄色葡萄球菌后分别用磷酸盐缓冲液、聚(甘油癸二酸酯)-共-聚乙二醇-共聚-邻苯二酚/季铵化壳聚糖水凝胶、负载锌离子的聚(甘油癸二酸酯)-共-聚乙二醇-共聚-邻苯二酚/季铵化壳聚糖水凝胶处理
图 4 3组糖尿病小鼠伤后14 d全层皮肤缺损感染创面中新生血管及M2型巨噬细胞分布情况 Alexa Fluor 594-4',6-二脒基-2-苯基吲哚×200。4A、4B、4C.分别为空白对照组、单纯水凝胶组、含锌水凝胶组小鼠创面,图4C中的新生血管明显较图4A、4B多；4D、4E、4F.分别为空白对照组、单纯水凝胶组、含锌水凝胶组小鼠创面,图4F中的M2型巨噬细胞明显较图4D、4E多
注：空白对照组、单纯水凝胶组、含锌水凝胶组小鼠创面感染耐甲氧西林金黄色葡萄球菌后分别用磷酸盐缓冲液、聚(甘油癸二酸酯)-共-聚乙二醇-共聚-邻苯二酚/季铵化壳聚糖水凝胶、负载锌离子的聚(甘油癸二酸酯)-共-聚乙二醇-共聚-邻苯二酚/季铵化壳聚糖水凝胶处理；CD31和CD206（分别表示新生血管和M2型巨噬细胞）阳性染色为红色,细胞核阳性染色为蓝色
Table 1. 3组糖尿病小鼠伤后各时间点全层皮肤缺损感染创面的愈合率比较（%,
组别 样本数 3 d 7 d 14 d 空白对照组 5 8.8±2.9 31.6±6.7a 73.5±7.4a 单纯水凝胶组 5 8.2±2.0 44.7±5.4a 85.4±5.7 含锌水凝胶组 5 8.7±1.3 72.4±8.4 92.7±4.3 F值 0.11 168.00 13.28 P值 0.900 <0.001 <0.001 注：空白对照组、单纯水凝胶组、含锌水凝胶组小鼠创面感染耐甲氧西林金黄色葡萄球菌后分别用磷酸盐缓冲液、聚(甘油癸二酸酯)-共-聚乙二醇-共聚-邻苯二酚/季铵化壳聚糖水凝胶、负载锌离子的聚(甘油癸二酸酯)-共-聚乙二醇-共聚-邻苯二酚/季铵化壳聚糖水凝胶处理；处理因素主效应,F=39.35,P<0.001；时间因素主效应,F=856.67,P<0.001；二者交互作用,F=22.64,P<0.001；与含锌水凝胶组相比,aP<0.05 
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