Effects and mechanism of water-soluble chitosan hydrogel on infected full-thickness skin defect wounds in diabetic mice
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摘要:
目的 探讨水溶性壳聚糖水凝胶对糖尿病小鼠感染全层皮肤缺损创面的作用及其机制。 方法 采用实验研究方法。采用循环冻融的方法制备由聚乙烯醇和明胶组成的对照水凝胶及由前述2种材料+水溶性壳聚糖组成的水溶性壳聚糖水凝胶。大体观察第1次冻融前后试管中2种敷料流动性，并比较12孔板中2种敷料最终形态的外观差异。取细胞株L929和HaCaT，均分别按照随机数字表法（分组方法下同）分为对照水凝胶组和水溶性壳聚糖水凝胶组，分别加入相应敷料培养24 h，采用细胞计数试剂盒8检测细胞增殖活力。取兔血红细胞悬液，分为生理盐水组、聚乙二醇辛基苯基醚（Triton X-100）组、对照水凝胶组和水溶性壳聚糖水凝胶组，分别作相应处理后孵育1 h，采用酶标仪检测红细胞的溶血程度。取24只11~14周龄雌性db/db小鼠，在其背部制作全层皮肤缺损创面并在创面处滴加耐甲氧西林金黄色葡萄球菌（MRSA）液，72 h后将小鼠分为空白对照组、磺胺嘧啶银水胶组、对照水凝胶组、水溶性壳聚糖水凝胶组，分别作相应处理。伤后0（即刻）、7、14、21 d，大体观察创面愈合情况并计算伤后14、21 d创面愈合率；伤后14 d，检测创面中MRSA浓度；伤后21 d，采用苏木精-伊红染色法对创面进行组织学分析，采用免疫荧光法检测创面中细胞CD31表达并计算其阳性百分率。取Raw264.7细胞，分为进行相应处理的白细胞介素4（IL-4）组、空白对照组、对照水凝胶组、水溶性壳聚糖水凝胶组，培养48 h，采用流式细胞仪检测细胞中CD206阳性细胞百分率。样本数均为3。对数据行独立样本t检验、单因素方差分析、重复测量方差分析、LSD检验及 Dunnett T3检验。 结果 试管中2种敷料在进行冻融前都具有一定的流动性，冻融1次后均形成半固态凝胶。12孔板中2种敷料最终形态均基本呈稳定的半透明片状，透明度差异不大。培养24 h，水溶性壳聚糖水凝胶组L929和HaCaT的细胞增殖活力均明显高于对照水凝胶组（t值分别为6.37、7.50，P<0.01）。孵育1 h，水溶性壳聚糖水凝胶组红细胞溶血程度明显低于Triton X-100组（P<0.01），而与生理盐水组及对照水凝胶组均相近（P>0.05）。伤后0 d，4组小鼠创面情况相似。伤后7 d，空白对照组与对照水凝胶组创面内部淡黄色渗出物较多，磺胺嘧啶银水胶组与水溶性壳聚糖水凝胶组创面观察到少量渗出。伤后14 d，空白对照组与对照水凝胶组创面干燥结痂，无明显上皮覆盖；磺胺嘧啶银水胶组创面痂皮脱落，可见脓性渗出物；水溶性壳聚糖水凝胶组创面基底呈淡红色，创面可观察到明显上皮覆盖。伤后14 d，水溶性壳聚糖水凝胶组创面愈合率显著高于其他3组（P值均<0.01）。伤后21 d，水溶性壳聚糖水凝胶组创面已完全闭合，其他3组创面均未完全愈合；水溶性壳聚糖水凝胶组创面愈合率显著高于其他3组（P值均<0.01）。伤后14 d，水溶性壳聚糖水凝胶组创面的MRSA浓度明显低于空白对照组（P<0.01），但与对照水凝胶组和磺胺嘧啶银水胶组均相近（P>0.05）。伤后21 d，空白对照组创面新生表皮缺损严重；对照水凝胶组创面的表皮亦有大面积缺损；磺胺嘧啶银水胶组创面尚未形成完整新生表皮；水溶性壳聚糖水凝胶组创面不仅被新生表皮完全覆盖，且新生表皮基底细胞排列规整。伤后21 d，水溶性壳聚糖水凝胶组创面中CD31阳性百分率为（2.19±0.35）%，明显高于空白对照组的（0.18±0.05）%、对照水凝胶组的（0.23±0.06）%以及磺胺嘧啶银水胶组的（0.62±0.25）%，P值均<0.01。培养48 h，水溶性壳聚糖水凝胶组Raw264.7中CD206阳性细胞百分率明显低于IL-4组（P<0.01），但明显高于空白对照组与对照水凝胶组（P<0.05或P<0.01）。 结论 水溶性壳聚糖水凝胶生物安全性好，较不含水溶性壳聚糖的对照水凝胶能诱导更高水平的巨噬细胞M2型极化，因此可以提升糖尿病小鼠MRSA感染的全层皮肤缺损创面的修复效果，并促进创面快速愈合。 Abstract:Objective To explore the effects and mechanism of water-soluble chitosan hydrogel on infected full-thickness skin defect wounds in diabetic mice. Methods The experimental research method was adopted. The control hydrogel composed of polyvinyl alcohol and gelatin, and the water-soluble chitosan hydrogel composed of the aforementioned two materials and water-soluble chitosan were prepared by the cyclic freeze-thaw method. The fluidity of the two dressings in test tube before and after the first freeze-thawing was generally observed, and the difference in appearance of the final state of two dressings in 12-well plates were compared. According to random number table (the same grouping method below), the cell strains of L929 and HaCaT were both divided into water-soluble chitosan hydrogel group and control hydrogel group, respectively. After adding corresponding dressings and culturing for 24 h, the cell proliferation activity was measured using cell counting kit 8. Rabbit blood erythrocyte suspensions were divided into normal saline group, polyethylene glycol octyl phenyl ether (Triton X-100) group, water-soluble chitosan hydrogel group, and control hydrogel group, which were treated accordingly and incubated for 1 hour, and then the hemolysis degree of erythrocyte was detected by a microplate reader. Twenty-four female db/db mice aged 11-14 weeks were selected, and full-thickness skin defect wounds on their backs were inflicted and inoculated with the methicillin-resistant Staphylococcus aureus (MRSA), 72 h later, the mice were divided into blank control group, sulfadiazine silver hydrogel group, control hydrogel group, and water-soluble chitosan hydrogel group, which were treated accordingly. On post injury day (PID) 0 (immediately), 7, 14, and 21, the healing of the wound was observed. On PID 14 and 21, the wound healing rate was calculated. On PID 14, MRSA concentration in wounds was determined. On PID 21, the wounds were histologically analyzed by hematoxylin and eosin staining; the expression of CD31 in the wounds was detected by immunofluorescence method, and its positive percentage was calculated. Raw264.7 cells were taken and divided into interleukin-4 (IL-4) group, blank control group, control hydrogel group, and water-soluble chitosan hydrogel group, which were treated accordingly. At 48 h of culture, the percentages of CD206 positive cells were detected by flow cytometry. The number of samples was all 3. Data were statistically analyzed with independent sample t test, one-way analysis of variance, analysis of variance for repeated measurement, least significant difference test, and Dunnett T3 test. Results Two dressings in test tube had certain fluidity before freeze-thawing and formed semi-solid gels after freeze-thawing for once. The final forms of two dressings in 12-well plates were basically stable and translucent sheets, with little difference in transparency. At 24 h of culture, the cell proliferation activities of L929 and HaCaT in water-soluble chitosan hydrogel group were significantly higher than those in control hydrogel group (with t values of 6.37 and 7.50, respectively, P<0.01). At 1 h of incubation, the hemolysis degree of erythrocyte in water-soluble chitosan hydrogel group was significantly lower than that in Triton X-100 group (P<0.01), but similar to that in normal saline group and control hydrogel group (P>0.05). On PID 0, the traumatic conditions of mice in the 4 groups were similar. On PID 7, more yellowish exudates were observed inside the wound in blank control group and control hydrogel group, while a small amount of exudates were observed in the wound in sulfadiazine silver hydrogel group and water-soluble chitosan hydrogel group. On PID 14, the wounds in blank control group and control hydrogel group were dry and crusted without obvious epithelial coverage; in sulfadiazine silver hydrogel group, the scabs fell off and purulent exudate was visible on the wound; in water-soluble chitosan hydrogel group, the base of wound was light red and obvious epithelial coverage could be observed on the wound. On PID 14, the wound healing rate in water-soluble chitosan hydrogel group was significantly higher than that in the other 3 groups (all P<0.01). On PID 21, the wound in water-soluble chitosan hydrogel group was completely closed, while the wounds in the other 3 groups were not completely healed; the wound healing rate in water-soluble chitosan hydrogel group was significantly higher than that in the other 3 groups (all P<0.01). On PID 14, the concentration of MRSA in the wound in water-soluble chitosan hydrogel group was significantly lower than that in blank control group (P<0.01), but similar to that in control hydrogel group and sulfadiazine silver hydrogel group (P>0.05). On PID 21, the new epidermis was severely damaged in blank control group; the epidermis on the wound in control hydrogel group also had a large area of defect; complete new epidermis had not yet being formed on the wound in sulfadiazine silver hydrogel group; the wound in water-soluble chitosan hydrogel group was not only completely covered by the new epidermis, the basal cells of the new epidermis were also regularly aligned. On PID 21, the percentage of CD31 positivity in the wound in water-soluble chitosan hydrogel group was (2.19±0.35)%, which was significantly higher than (0.18±0.05)% in blank control group, (0.23±0.06)% in control hydrogel group, and (0.62±0.25)% in sulfadiazine silver hydrogel group, all P<0.01. At 48 h of culture, the percentage of CD206 positive Raw264.7 cells in water-soluble chitosan hydrogel group was lower than that in IL-4 group (P>0.01) but significantly higher than that in blank control group and control hydrogel group (P<0.05 or P<0.01). Conclusions The water-soluble chitosan hydrogel has good biosafety and can induce higher level of macrophage M2 polarization than control hydrogel without water-soluble chitosan, so it can enhance the repair effect of MRSA-infected full-thickness skin defect wounds in diabetic mice and promote rapid wound healing. -
Key words:
- Hydrogel /
- Chitosan /
- Infection /
- Skin /
- Chronic wound /
- Wound repair
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参考文献
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1 2种水凝胶在试管中冻融1次前后流动性以及在12孔板中循环冻融后最终形态。1A、1B.分别为对照水凝胶和水溶性壳聚糖水凝胶，冻融1次后，2种溶液均由可流动状态变成不流动的半固态；1C、1D.分别为在12孔板中最终形成的对照水凝胶和水溶性壳聚糖水凝胶（培养皿中展示），二者均为稳定规则的圆形半透明片状
注：对照水凝胶由聚乙烯醇和明胶制得，水溶性壳聚糖水凝胶由聚乙烯醇、明胶和水溶性壳聚糖制得
2 4 组db/db小鼠耐甲氧西林金黄色葡萄球菌感染全层皮肤缺损创面伤后各时间点愈合情况。2A、2B、2C、2D.分别为空白对照组伤后0、7、14、21 d创面，图2E显示空白对照组14 d后创面缩小不明显；2F、2G、2H、2I.分别为对照水凝胶组伤后0、7、14、21 d 创面，图2J显示对照水凝胶组各时间点创面面积均分别较图2E有所缩小；2K、2L、2M、2N.分别为磺胺嘧啶银水胶组伤后0、7、14、21 d创面，图2O显示磺胺嘧啶银水胶组各时间点创面面积分别较图2J有所缩小；2P、2Q、2R、2S.分别为水溶性壳聚糖水凝胶组伤后0、7、14、21 d创面，图2T显示水溶性壳聚糖水凝胶组各时间点创面面积均分别较图2E、2J、2O明显缩小
注：对照水凝胶由聚乙烯醇和明胶制得，水溶性壳聚糖水凝胶由聚乙烯醇、明胶和水溶性壳聚糖制得；图中圆形参照物直径为2 cm；图2E、2J、2O、2T为ImageJ图像软件处理后量化图，图中红色、橙色、黄色、绿色边界内范围均分别表示伤后0（即刻）、7、14、21 d创面面积大小
3 4组db/db小鼠耐甲氧西林金黄色葡萄球菌感染的全层皮肤缺损创面伤后21 d组织学变化 苏木精-伊红。3A、3B、3C、3D.分别为空白对照组、对照水凝胶组、磺胺嘧啶银水胶组、水溶性壳聚糖水凝胶组创面，图3D中的新生表皮长度明显长于图3A、3B、3C，图片放大倍数为1.25；3E、3F、3G、3H.分别为图3A、3B、3C、3D局部创面放大图，从左至右显示未封闭的创缘宽度依次缩减，其中图3H所示创面已完全愈合，图片放大倍数为2.50
注：对照水凝胶由聚乙烯醇和明胶制得，水溶性壳聚糖水凝胶由聚乙烯醇、明胶和水溶性壳聚糖制得；图3A、3B、3C、3D中的红色双向箭头指示新生上皮长度，图3E、3F、3G、3H中的红色单向箭头之间区域指示尚未封闭的创缘宽度
4 4组db/db小鼠耐甲氧西林金黄色葡萄球菌感染的全层皮肤缺损创面伤后21 d CD31阳性表达情况 Alexa Fluor 594-4', 6-二脒基-2-苯基吲哚×40。4A、4B、4C、4D.分别为空白对照组、对照水凝胶组、磺胺嘧啶银水胶组、水溶性壳聚糖水凝胶组，图4D中的CD31阳性表达明显高于图4A、4B、4C
注：对照水凝胶由聚乙烯醇和明胶制得，水溶性壳聚糖水凝胶由聚乙烯醇、明胶和水溶性壳聚糖制得；细胞核阳性染色为蓝色，CD31阳性细胞染色为红色（指示新生血管）
表1 4组db/db小鼠耐甲氧西林金黄色葡萄球菌感染的全层皮肤缺损创面伤后各时间点愈合率比较（%，
组别 样本数 14 d 21 d 空白对照组 3 36.94±3.00 58.93±4.69 对照水凝胶组 3 18.92±4.42 84.33±4.04 磺胺嘧啶银水胶组 3 39.71±5.03 88.10±2.01 水溶性壳聚糖胶组 3 83.74±3.30 99.76±0.24 F值 140.70 83.74 P值 <0.001 <0.001 P1值 <0.001 <0.001 P2值 <0.001 <0.001 P3值 <0.001 0.002 注：对照水凝胶由聚乙烯醇和明胶制得，水溶性壳聚糖水凝胶由聚乙烯醇、明胶和水溶性壳聚糖制得；处理因素主效应，F=422.86，P<0.001；时间因素主效应，F=1 788.78，P<0.001；两者交互作用，F=68.88，P<0.001；F值、P值为4组间各时间点总体比较所得；P1值为空白对照组与水溶性壳聚糖水凝胶组比较所得；P2值为对照水凝胶组与水溶性壳聚糖水凝胶组比较所得；P3值为磺胺嘧啶银水胶组与水溶性壳聚糖水凝胶组比较所得 
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