Performance of PHMB-surface mesoporous silica composite material and its effect on full-thickness skin defect infected wounds in rats
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摘要:
目的 探讨聚六亚甲基双胍盐酸盐（PHMB）-表面介孔硅石复合材料（以下简称复合材料）的性能及其在大鼠全层皮肤缺损感染创面中的作用。 方法 本研究为包含成组设计、析因设计及重复测量设计的实验研究。通过旋蒸法制备了生物相容性良好的复合材料。采用持续定量检测法检测复合材料体外释放PHMB情况。采用微量营养肉汤法检测PHMB、复合材料对金黄色葡萄球菌和大肠埃希菌的最低抑菌浓度（MIC）和最低杀菌浓度（MBC）。采用持续添加菌悬液法,检测PHMB、复合材料的持续杀菌时长。采用亚致死浓度诱导法检测金黄色葡萄球菌和大肠埃希菌对PHMB和复合材料的抗性出现情况。以上实验样本数均为3。取45只10~12周龄雌性SD大鼠,采用随机数字表法分为常规换药组、PHMB组和复合材料组,每组15只。对所有大鼠制备背部全层皮肤缺损感染创面后,对常规换药组、PHMB组、复合材料组大鼠创面分别采用生理盐水、PHMB终质量分数均为0.100%的PHMB溶液及复合材料分散液冲洗换药。计算伤后6、12、18 d（样本数分别为15、10、5）创面愈合率、感染情况。伤后6、12、18 d,换药前按随机数字表法各组取5只大鼠,采用平板计数法检测创面组织细菌载量。 结果 PHMB和表界网孔硅石质量比1∶1复合材料可持续释放PHMB≥144 h。复合材料无论是对金黄色葡萄球菌还是大肠埃希菌,MIC均较PHMB降低,但2种材料的MBC均一致。PHMB对金黄色葡萄球菌和大肠埃希菌的持续杀菌时长均为3 d,复合材料对金黄色葡萄球菌和大肠埃希菌的持续杀菌时长均为6 d。金黄色葡萄球菌及大肠埃希菌对复合材料在培养60 d后仍未出现抗性,明显晚于对PHMB的抗性。复合材料组大鼠伤后6、12、18 d创面愈合率均明显高于常规换药组和PHMB组（P<0.05）。伤后6、12、18 d,复合材料组大鼠创面均未出现感染,常规换药组大鼠创面均持续出现感染,PHMB组大鼠创面仍有部分创面持续出现感染。复合材料组大鼠伤后6、12、18 d创面组织细菌载量分别（4.6±3.1）、（3.4±3.4）、（1.6±1.5）CFU/mL,明显低于常规换药组[（1 876.0±241.5）、（1 076.0±151.1）、（1 184.0±156.6）CFU/mL]和PHMB组[（824.0±277.6）、（800.0±231.0）、（832.0±350.9）CFU/mL,P<0.05]。PHMB组大鼠伤后6、12 d创面组织细菌载量明显低于常规换药组（P<0.05）。 结论 复合材料具有增效、缓释PHMB活性,并因此延缓细菌抗性产生,可减少创面组织细菌载量,有利于创面愈合。 Abstract:Objective To investigate the performance of polyhexamethylene biguanide hydrochloride (PHMB)-surface mesoporous silica composite material (hereinafter referred to as the composite material) and its effect on full-thickness skin defect infected wounds in rats. Methods This study was an experimental study including group design, factorial design, and repeated measures design. The composite material with good biocompatibility was prepared by rotary evaporation method. The in vitro release of PHMB from the composite material was detected by continuous quantitative release assay. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of PHMB and the composite material against Staphylococcus aureus and Escherichia coli were detected by broth microdilution method. The continuous bactericidal duration of PHMB and the composite material was detected by continuous bacterial challenge assay. The inducing effect of PHMB and the composite material on the resistance of Staphylococcus aureus and Escherichia coli was detected by sub-lethal concentration induction assay. The sample size for the above experiments was 3. Forty-five female SD rats aged 10-12 weeks were divided into routine dressing change group, PHMB group, and composite material group by random number table method, with 15 rats in each group. After preparing full-thickness skin defect infected wounds on the back of all rats, the wounds of rats in routine dressing change group, PHMB group, and composite material group were rinsed and dressed with normal saline, PHMB solution and composite material dispersion with a final mass fraction of 0.100% PHMB, respectively. The wound healing rate and infection status were calculated at post injury day (PID) 6, 12, and 18 with sample sizes of 15, 10, and 5, respectively. At PID 6, 12, and 18, 5 rats from each group were selected by random number table method before dressing change, and the wound tissue bacterial load was detected by viable plate count method. Results The composite material with a mass ratio of PHMB to surface mesoporous silica of 1:1 could continuously release PHMB for ≥ 144 h. For both Staphylococcus aureus and Escherichia coli, the MICs of the composite material were lower than those of PHMB, but the MBCs of both materials were identical. The continuous bactericidal duration of PHMB against Staphylococcus aureus and Escherichia coli was both 3 d, and the continuous bactericidal duration of the composite material against Staphylococcus aureus and Escherichia coli was both 6 d. Staphylococcus aureus and Escherichia coli did not show resistance to the composite material after 60 d of culture, which was significantly later than the resistance to PHMB. The wound healing rates of rats in composite material group at PID 6, 12, and 18 were significantly higher than those in routine dressing change group and PHMB group (P<0.05). At PID 6, 12, and 18, no infection occurred in the wounds of rats in composite material group, whereas all wounds in routine dressing change group continuously showed infection, and some wounds in PHMB group still continuously showed infection. The wound tissue bacterial loads of rats in composite material group at PID 6, 12, and 18 were (4.6±3.1), (3.4±3.4), and (1.6±1.5) CFU/mL, respectively, which were significantly lower than those in routine dressing change group (( 1876.0±241.5), (1 076.0±151.1), and (1 184.0±156.6) CFU/mL) and PHMB group ((824.0±277.6), (800.0±231.0), and (832.0±350.9) CFU/mL, P<0.05). The wound tissue bacterial load of rats in PHMB group at PID 6 and 12 was significantly lower than that in routine dressing change group (P<0.05). Conclusions The composite material has the activities of synergistic effect and sustained release of PHMB, thereby delaying the generation of bacterial resistance, reducing the bacterial load of wound tissue, and facilitating wound healing. -
参考文献
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图 3 3种材料的致敏检测和皮内刺激检测 皮肤镜 ×4。3A、3C、3E.分别为PHMB溶液、表面介孔硅石分散液、复合材料分散液致敏实验中持续涂抹3周后大鼠皮肤表现,未表现出致敏性；3B、3D、3F.分别为PHMB溶液、表面介孔硅石分散液、复合材料分散液皮内刺激实验中皮内注射72 h后兔皮肤表现,未表现出皮内刺激性
注：PHMB为聚六亚甲基双胍盐酸盐；复合材料为PHMB-表面介孔硅石复合材料
图 4 2种材料针对大肠埃希菌的持续杀菌时长。4A、4B、4C.分别为聚六亚甲基双胍盐酸盐（PHMB）与大肠埃希菌共培养第1、2、3天培养基表现,其中第3天开始可见菌落生长；4D、4E、4F.分别为PHMB-表面介孔硅石复合材料与大肠埃希菌共培养第1、2、3天,直到第6天仍无菌落生长
图 5 3组大鼠伤后各时间点全层皮肤缺损感染创面愈合情况。5A、5B、5C.分别为常规换药组伤后6、12、18 d创面愈合情况；5D、5E、5F.分别为PHMB组伤后6、12、18 d创面愈合情况；5G、5H、5I.分别为复合材料组伤后6、12、18 d创面愈合情况,其中图5H创面面积明显小于图5B、5E,图5I创面面积明显小于图5C、5F
注：对常规换药组、聚六亚甲基双胍盐酸盐（PHMB）组、PHMB-表面介孔硅石复合材料（复合材料）组大鼠创面分别采用生理盐水、PHMB终质量分数均为0.100%的PHMB溶液及复合材料分散液冲洗换药
Table 1. 培养48 h后不同终质量分数2种材料对2种细菌的杀菌率（%,
材料与PHMB终质量分数 样本数 金黄色葡萄球菌 大肠埃希菌 PHMB 0.100% 3 100.0±0.0 100.0±0.0 0.010% 3 95.0±2.3 98.2±0.8 0.001% 3 74.2±4.0 85.1±1.9 PHMB-表面介孔硅石复合材料 0.100% 3 100.0±0.0 100.0±0.0 0.010% 3 99.2±0.9 100.0±0.0 0.001% 3 89.6±1.8 93.0±2.9 t1值 0 0 P1值 0 0 t2值 -3.01 -3.95 P2值 0.040 0.017 t3值 -6.11 -3.96 P3值 0.004 0.017 注：PHMB为聚六亚甲基双胍盐酸；t1值、P1值为PHMB终质量分数为0.100%的PHMB与PHMB-表面介孔硅石复合材料各指标比较所得；t2值、P2值为PHMB终质量分数为0.010%的PHMB与PHMB-表面介孔硅石复合材料各指标比较所得；t3值、P3值为PHMB终质量分数为0.001%的PHMB与PHMB-表面介孔硅石复合材料各指标比较所得；金黄色葡萄球菌材料类型因素主效应,F=46.23,P<0.001；质量分数因素主效应,F=136.27,P<0.001；两者交互作用,F=22.59,P<0.001；大肠埃希菌材料类型因素主效应,F=22.33,P<0.001；质量分数因素主效应,F=104.71,P<0.001；两者交互作用,F=12.30,P=0.001 
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Table 2. 2种细菌在培养各时间点针对2种材料的抗性出现情况
细菌种类与材料 样本数 10 d 20 d 30 d 40 d 50 d 60 d 金黄色葡萄球菌 PHMB 3 - + + ++ ++ ++ PHMB-表面介孔硅石复合材料 3 - - - - + + 大肠埃希菌 PHMB 3 - - + ++ ++ ++ PHMB-表面介孔硅石复合材料 3 - - - - - - 注：PHMB为聚六亚甲基双胍盐酸盐；-表示全部菌株未出现抗性,+表示部分菌株出现抗性,++表示全部菌株出现抗性
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Table 3. 3组大鼠伤后各时间点全层皮肤缺损感染创面愈合率比较（%,
组别 样本数 6 d 样本数 12 d 样本数 18 d 常规换药组 15 16.6±2.4 10 33.8±3.4 5 67.6±3.8 PHMB组 15 17.1±2.7 10 35.3±3.0 5 69.0±3.2 复合材料组 15 28.7±2.9 10 56.6±2.6 5 86.3±3.5 F值 97.62 183.94 43.25 P值 <0.001 <0.001 <0.001 P1值 >0.999 0.804 >0.999 P2值 <0.001 <0.001 <0.001 P3值 <0.001 <0.001 <0.001 注：对常规换药组、聚六亚甲基双胍盐酸盐（PHMB）组、PHMB-表面介孔硅石复合材料（复合材料）组大鼠创面分别采用生理盐水、PHMB终质量分数均为0.100%的PHMB溶液及复合材料分散液冲洗换药；组别因素主效应,F=242.59,P <0.001；时间因素主效应,F=2 126.72,P<0.001；两者交互作用,F=16.42,P<0.001；F值、P值为3组间各时间点总体比较所得；P1值为常规换药组与PHMB组比较所得；P2值为常规换药组与复合材料组比较所得；P3值为PHMB组与复合材料组比较所得
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Table 4. 3组大鼠伤后各时间点全层皮肤缺损感染创面组织细菌载量比较（CFU/g,
组别 样本数 6 d 12 d 18 d 常规换药组 5 1 876.0±241.5 1 076.0±151.1 1 184.0±156.6 PHMB组 5 824.0±277.6 800.0±231.0 832.0±350.9 复合材料组 5 4.6±3.1 3.4±3.4 1.6±1.5 F值 97.55 61.07 37.45 P值 <0.001 <0.001 <0.001 P1值 <0.001 0.045 0.082 P2值 <0.001 <0.001 <0.001 P3值 <0.001 <0.001 <0.001 注：对常规换药组、聚六亚甲基双胍盐酸盐（PHMB）组、PHMB-表面介孔硅石复合材料（复合材料）组大鼠创面分别采用生理盐水、PHMB终质量分数均为0.100%的PHMB溶液及复合材料分散液冲洗换药；组别因素主效应,F=178.73,P<0.001；时间主效应,F=8.66,P=0.002；两者交互作用,F=8.07,P<0.001；F值、P值为3组间各时间点总体比较所得；P1值为常规换药组与PHMB组比较所得；P2值为常规换药组与复合材料组比较所得；P3值为PHMB组与复合材料组比较所得
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