Preparation and roles of sliver-loaded viscous hydrogel in healing of full-thickness skin defect wounds with bacterial colonization in mice
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摘要:
目的 制备负载银粒子的改性透明质酸黏性水凝胶，探讨其在小鼠细菌定植全层皮肤缺损创面愈合中的作用及可能的机制。 方法 采用实验研究方法。制备多巴胺修饰的透明质酸（HA-DA）和苯硼酸修饰的透明质酸（HA-PBA），傅里叶变换红外光谱检测其特征峰。在HA-DA和HA-PBA中加入不同质量的丙烯酰胺，制备质量分数为10%、15%、20%丙烯酰胺的黏性水凝胶。观察含质量分数20%丙烯酰胺的黏性水凝胶在37 ℃下倾斜状态和倒立状态的成胶情况，旋转流变仪检测前述3种黏性水凝胶的储存模量和损耗模量。在含质量分数20%丙烯酰胺的黏性水凝胶中加入纳米银离子，制备含银黏性水凝胶，采用电感耦合等离子体质谱仪测量含银黏性水凝胶释放的银离子浓度，并计算累计银离子释放率（样本数为5）。取小鼠成纤维细胞L929，分为磷酸盐缓冲液（PBS）组、黏性水凝胶组及含银黏性水凝胶组并进行相应处理，采用细胞计数试剂盒8法检测培养1、2、3 d细胞存活情况（样本数为5）。取24只6~8周龄雄性C57BL/6小鼠，在其背部建立48个接种大肠埃希菌和金黄色葡萄球菌混合悬液的全层皮肤缺损创面模型，每只小鼠2个创面。将创面分成生理盐水组、黏性水凝胶组、含银黏性水凝胶组并进行相应处理，每组16个创面，且每只小鼠的2个创面纳入不同组。伤后3、7、10、14 d，观察创面愈合情况并计算创面愈合率；伤后3 d，观察并计数创面中大肠埃希菌及金黄色葡萄球菌菌落数；伤后14 d，行苏木精-伊红染色和Masson染色分别观察并分析创面上皮化的表皮厚度和胶原纤维的光密度；伤后3、7、10 d，采用免疫组织化学法检测创面中肿瘤坏死因子α（TNF-α）、转化生长因子β1（TGF-β1）及血管内皮生长因子（VEGF）的表达。 各指标各时间点创面数均为4个。对数据进行析因设计方差分析、单因素方差分析及Bonferroni校正。 结果 HA-PBA在波数为1 369、1 425 cm-1处检测到特征峰，表明苯硼酸成功接枝到透明质酸上；HA-DA在波数为1 516、1 431 cm-1处检测到特征峰，表明多巴胺已成功接枝到透明质酸上。含质量分数20％丙烯酰胺的黏性水凝胶在37 ℃下，无论是倾斜还是倒立时都保持稳定不流动的凝胶状态。随着丙烯酰胺含量的增加，黏性水凝胶储存模量和损耗模量均有所增加，但3种不同丙烯酰胺含量黏性水凝胶的储存模量和损耗模量随振荡频率或时间的增加变化不明显且储存模量均大于损耗模量。含银黏性水凝胶中银离子释放长达7 d，累计银离子释放率最高达65%。培养1、2、3 d，含银黏性水凝胶组细胞存活率明显低于PBS组和黏性水凝胶组（P<0.05或P<0.01）；培养1 d，黏性水凝胶组细胞存活率明显低于PBS组（P<0.01）。随着伤后时间的延长，3组小鼠创面均不断缩小。伤后3、7、10、14 d，含银黏性水凝胶组创面愈合率分别为（53.0±3.6）%、（75.3±6.9）%、（93.3±1.2）%、（96.7±0.8）%，明显高于生理盐水组的（21.8±6.4）%、（53.9±8.2）%、（72.0±7.8）%、（92.5±0.4）%（P<0.01）。伤后3、14 d，含银黏性水凝胶组创面愈合率明显高于黏性水凝胶组的（43.5±2.4）%、（94.1±1.5）%（P<0.05）；伤后3、10 d，黏性水凝胶组创面愈合率明显高于生理盐水组（P<0.01）。伤后3 d，含银黏性水凝胶组创面中2种细菌菌落数明显少于生理盐水组及黏性水凝胶组（P<0.01），黏性水凝胶组创面中2种细菌菌落数明显少于生理盐水组（P<0.05）。伤后14 d，含银黏性水凝胶组创面基本上皮化且表皮厚度更厚，胶原蛋白含量较其他2组明显增多且胶原排列更加有序；含银黏性水凝胶组创面的表皮厚度较其余2组明显增加（P<0.05），胶原纤维光密度较生理盐水组明显增加（P<0.05）。伤后3 d，含银黏性水凝胶组创面TGF-β1和VEGF的表达明显高于生理盐水组（P<0.05或P<0.01），黏性水凝胶组创面VEGF的表达明显高于生理盐水组（P<0.01）。伤后7 d，含银黏性水凝胶组创面TGF-β1的表达明显高于其余2组（P<0.01），VEGF的表达明显高于生理盐水组（P<0.01）。伤后10 d，含银黏性水凝胶组创面TNF-α的表达明显低于生理盐水组（P<0.05），TGF-β1和VEGF的表达明显高于生理盐水组（P<0.05或P<0.01），且VEGF的表达明显高于黏性水凝胶组（P<0.05）。 结论 本研究制备的含银黏性水凝胶具有良好的稳定性和弹性，可以持续释放银离子，有助于加速小鼠细菌定植全层皮肤缺损创面的愈合，生物毒性较低，可以促进创面再上皮化、胶原沉积和血管再生，可能涉及炎症细胞的浸润与消退。 Abstract:Objective To prepare the modified hyaluronic acid viscous hydrogel loaded with sliver particles and to explore the roles and mechanism of the hydrogel in healing of full-thickness skin defect wounds with bacterial colonization in mice. Methods The experimental research method was adopted. Dopamine modified hyaluronic acid (HA-DA) and phenylboric acid modified hyaluronic acid (HA-PBA) were prepared, and their characteristic peaks were detected by Fourier-transform infrared spectroscopy. Different mass of acrylamides was added to HA-DA and HA-PBA to prepare the viscous hydrogel with mass fraction of acrylamide in 10%, 15%, and 20%. The gelation of the viscous hydrogel with mass fraction of acrylamide in 20% was observed in the state of tilt and inversion at 37 ℃, and the storage modulus and loss modulus of the above 3 kinds of viscous hydrogels were detected by rotational rheometer. The sliver-loaded viscous hydrogel was prepared by adding nano silver ions to the viscous hydrogel with mass fraction of acrylamide in 20%. The concentration of silver ions released by sliver-loaded viscous hydrogel was measured by inductively coupled plasma mass spectrometer, and the cumulative release rate of silver ion was calculated (n=5). The mouse fibroblasts L929 were divided into phosphate buffered saline (PBS) group, viscous hydrogel group, and sliver-loaded viscous hydrogel group, which were dealt correspondingly, and the cell survival was detected by cell counting kit 8 method after 1, 2, and 3 d of culture (n=5). Twenty-four male C57BL/6 mice aged 6-8 weeks were selected, and forty-eight full-thickness skin defect wounds were inflicted and inoculated with the mixture of Escherichia coli and Staphylococcus aureus in the back of the mice, with two wounds in each mouse. The wounds were divided into normal saline group, viscous hydrogel group, and sliver-loaded viscous hydrogel group, which were dealt correspondingly, with 16 wounds in each group, and two wounds in each mouse were divided into different groups. On post injury day (PID) 3, 7, 10, and 14, the wound healing was observed and the wound healing rate was calculated. On PID 3, the colony forming units of Escherichia coli and Staphylococcus aureus in wounds were observed and counted. On PID 14, the epithelized epidermal thickness and the optical density of collagen fiber in wounds were observed and analyzed after hematoxylin eosin staining and Masson staining, respectively. On PID 3, 7, and 10, the expressions of tumor necrosis factor α (TNF-α), transforming growth factor β1 (TGF-β1), and vascular endothelial growth factor (VEGF) were detected by immunohistochemistry. The number of wounds in each index detecting at each time point was four. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, and Bonferroni correction. Results The characteristic peaks of HA-PBA were detected at the wave numbers of 1 369 and 1 425 cm-1, indicating that phenylboric acid had been successfully grafted on hyaluronic acid, and the characteristic peaks of HA-DA were detected at the wave numbers of 1 516 and 1 431 cm-1, indicating that dopamine had been successfully grafted on hyaluronic acid. The viscous hydrogel with mass fraction of acrylamide in 20% maintained the stable and no-flow condition of gelation in the state of tilt and inversion at 37 ℃. The storage modulus and loss modulus of the viscous hydrogel increased with the increase of acrylamide content, the storage modulus and loss modulus of the 3 kinds of viscous hydrogels had no obvious changes with the increase of the oscillation frequency or time, and the storage modulus of the 3 kinds of acrylamide hydrogels were greater than the loss modulus. The release of silver ion in the sliver-loaded viscous hydrogel lasted for 7 days, and the cumulative release rate of silver ion was up to 65%. After 1, 2, and 3 d of culture, the cell survival rates in sliver-loaded viscous hydrogel group were significantly lower than those in PBS group and viscous hydrogel group (P<0.05 or P<0.01), while after 1 d of culture, the cell survival rate in viscous hydrogel group was significantly lower than that in PBS group (P<0.01). With extension of time after injury, the wounds of mice in the 3 groups shrank gradually. On PID 3, 7, 10, and 14, the wound healing rates in sliver-loaded viscous hydrogel group were (53.0±3.6)%, (75.3±6.9)%, (93.3±1.2)%, and (96.7±0.8)%, which were significantly higher than (21.8±6.4)%, (53.9±8.2)%, (72.0±7.8)%, and (92.5±0.4)% in normal saline group (P<0.01). On PID 3 and 14, the wound healing rates in sliver-loaded viscous hydrogel group were significantly higher than (43.5±2.4)% and (94.1±1.5)% in viscous hydrogel group (P<0.05). On PID 3 and 10, the wound healing rates in viscous hydrogel group were significantly higher than those in normal saline group (P<0.01). On PID 3, the colony forming units of two bacteria in wound of sliver-loaded viscous hydrogel group were significantly less than those in normal saline group and viscous hydrogel group (P<0.01), while the colony forming units of two bacteria in wound of viscous hydrogel group were significantly less than those in normal saline group (P<0.05). On PID 14, the wounds were basically epithelialized and the epidermis was thicker, with collagen protein content being increased significantly and more orderly arranged collagen in sliver-loaded viscous hydrogel group compared with those in the other 2 groups. On PID 14, the epidermal thickness in wounds of sliver-loaded viscous hydrogel group was significantly increased compared with that in the other two groups (P<0.05), and the optical density of collagen fiber was significantly increased compared with those in normal saline group (P<0.05). On PID 3, the expressions of TGF-β1 and VEGF in wounds of sliver-loaded viscous hydrogel group were significantly higher than those in normal saline group (P<0.05 or P<0.01), while the expression of VEGF in wounds of viscous hydrogel group was significantly higher than that in normal saline group (P<0.01). On PID 7, the expression of TGF-β1 in wounds of sliver-loaded viscous hydrogel group was significantly higher than that in the other 2 groups (P<0.01), and the expression of VEGF was significantly higher than that in normal saline group (P<0.01). On PID 10, the expression of TNF-α in wounds of sliver-loaded viscous hydrogel group was significantly lower than that in normal saline group (P<0.05), the expressions of TGF-β1 and VEGF in wounds of sliver-loaded viscous hydrogel group were significantly higher than those in normal saline group (P<0.05 or P<0.01), and the expression of VEGF in wounds of sliver-loaded viscous hydrogel group was significantly higher than that in viscous hydrogel group (P<0.05). Conclusions The sliver-loaded viscous hydrogel prepared in this study has good stability and elasticity, which can continuously release silver ions and help to accelerate the healing of full-thickness defect wounds with bacterial colonization in mice. Besides, the sliver-loaded viscous hydrogel has low biological toxicity and can promote re-epithelialization, collagen deposition as well as angiogenesis of wounds, which may be related to the infiltration and regression of inflammatory cells. -
Key words:
- Wound healing /
- Hydrogel /
- Hyaluronic acid /
- Nano silver /
- Re-epithelialization /
- Collagen deposition /
- Angiogenesis
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参考文献
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1 HA-DA和HA-PBA的红外光谱图显示苯硼酸、多巴胺已成功接到透明质酸上
注：黑色线为透明质酸，红色线为多巴胺，黄色线为苯硼酸，蓝色线为多巴胺修饰的透明质酸（HA-DA），绿色线为苯硼酸修饰的透明质酸（HA-PBA）；HA-PBA光谱图上2个箭头分别表示在波数为1 369、1 425 cm-1附近出现归因于B-O键和芳香化合物C-C键的特征峰，HA-DA光谱图上2个箭头分别表示在波数为1 516、1 431 cm-1附近出现归因于芳香化合物C-C键的特征峰
3 3种不同丙烯酰胺含量黏性水凝胶的储存模量和损耗模量。3A.储存模量和损耗模量随振荡频率变化不明显；3B.储存模量和损耗模量随时间变化不明显
注：该图为经过lg处理的数据形成的描记图;坐标轴数据为未经lg处理的原始数据；黑色、绿色、浅蓝色分别为含质量分数10％、15％、20％丙烯酰胺黏性水凝胶的储存模量；红色、深蓝色、紫色分别为含质量分数10％、15％、20％丙烯酰胺黏性水凝胶的损耗模量
5 3组小鼠伤后各时间点全层皮肤缺损创面愈合情况。5A、5B、5C、5D.分别为生理盐水组伤后3、7、10、14 d创面情况，创面逐渐缩小；5E、5F、5G、5H.分别为黏性水凝胶组伤后3、7、10、14 d创面情况，图5E、5F、5G、5H中创面分别较图5A、5B、5C、5D有所缩小；5I、5J、5K、5L.分别为含银黏性水凝胶组伤后3、7、10、14 d创面情况，图5I、5J、5K、5L中创面分别较图5E、5F、5G、5H有所缩小，较图5A、5B、5C、5D明显缩小
6 3组小鼠伤后3 d全层皮肤缺损创面中金黄色葡萄球菌及大肠埃希菌菌落分布情况。6A、6B、6C.分别为生理盐水组、黏性水凝胶组、含银黏性水凝胶组金黄色葡萄球菌菌落情况，图6B中细菌菌落数较图6A中明显减少，图6C中细菌菌落数较图6A、6B明显减少；6D、6E、6F.分别为生理盐水组、黏性水凝胶组、含银黏性水凝胶组大肠埃希菌菌落情况，图6E中细菌菌落数较图6D明显减少，图6F中细菌菌落数较图6D、6E明显减少
7 3组小鼠伤后14 d全层皮肤缺损创面表皮厚度和胶原生成情况观察。7A、7B、7C.分别为生理盐水组、黏性水凝胶组、含银黏性水凝胶组表皮厚度，图7A部分上皮化且上皮化后表皮较薄，图7B、7C基本上皮化，但图7C表皮较图7B厚 苏木精-伊红×100；7D、7E、7F.分别为生理盐水组、黏性水凝胶组、含银黏性水凝胶组胶原生成情况，图7D胶原蛋白含量较少且胶原排列紊乱，图7E中胶原蛋白含量较图7D增多但排列仍较紊乱，图7F胶原蛋白含量较图7E增多且排列有序 Masson×200
8 3组小鼠伤后各时间点全层皮肤缺损创面肿瘤坏死因子α（TNF-α）的阳性（棕色）表达 二氨基联苯胺-苏木精×400。8A、8B、8C.分别为生理盐水组伤后3、7、10 d TNF-α表达；8D、8E、8F.分别为黏性水凝胶组伤后3、7、10 d TNF-α表达，分别与图8A、8B、8C相近；8G、8H、8I.分别为含银黏性水凝胶组伤后3、7、10 d TNF-α表达，仅图8I中TNF-α表达明显低于图8C
9 3组小鼠伤后各时间点全层皮肤缺损创面转化生长因子β1（TGF-β1）的阳性（棕色）表达 二氨基联苯胺-苏木精×400。9A、9B、9C.分别为生理盐水组伤后3、7、10 d TGF-β1表达；9D、9E、9F.分别为黏性水凝胶组伤后3、7、10 d TGF-β1表达；9G、9H、9I.分别为含银黏性水凝胶组伤后3、7、10 d TGF-β1表达，明显高于图9A、9B、9C，图9H中TGF-β1表达明显高于图9E
10 3组小鼠伤后各时间点全层皮肤缺损创面血管内皮生长因子（VEGF）的阳性（棕色）表达 二氨基联苯胺-苏木精×400。10A、10B、10C.分别为生理盐水组伤后3、7、10 d VEGF表达；10D、10E、10F.分别为黏性水凝胶组伤后3、7、10 d VEGF表达，仅图10D中VEGF表达较图10A明显增加；10G、10H、10I.分别为含银黏性水凝胶组伤后3、7、10 d VEGF表达，分别较图10A、10B、10C明显增加，且图10I较图10F中VEGF表达明显增加
表1 3组小鼠成纤维细胞L929培养各时间点存活率比较（％，
组别 样本数 1 d 2 d 3 d PBS组 15 97.2±1.1 96.3±0.8 96.0±2.4 黏性水凝胶组 15 93.5±1.3a 93.7±2.3 93.7±2.1 含银黏性水凝胶组 15 84.5±1.4ab 87.3±2.7ab 89.0±2.4ac F值 125.553 24.807 12.084 P值 <0.01 <0.01 <0.01 注：各组各时间点样本数为5；PBS为磷酸盐缓冲液；处理因素主效应，F=95.869，P<0.01；时间因素主效应，F=1.310，P>0.05；两者交互作用，F=2.971，P>0.05；与PBS组比较，aP<0.01；与黏性水凝胶组比较，bP<0.01,cP<0.05 
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表2 3组小鼠伤后各时间点全层皮肤缺损创面愈合率比较（%，
组别 创面数（个） 3 d 7 d 10 d 14 d 生理盐水组 4 21.8±6.4 53.9±8.2 72.0±7.8 92.5±0.4 黏性水凝胶组 4 43.5±2.4a 66.9±3.6 88.3±2.2a 94.1±1.5 含银黏性水凝胶组 4 53.0±3.6ab 75.3±6.9a 93.3±1.2a 96.7±0.8ab F值 51.107 10.841 22.303 17.740 P值 <0.01 <0.01 <0.01 <0.01 注：处理因素主效应，F=73.626，P<0.01；时间因素主效应，F=328.066，P<0.01；两者交互作用，F=6.461，P<0.01；与生理盐水组比较，aP<0.01；与黏性水凝胶组比较，bP<0.05
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表3 3组小鼠伤后3 d全层皮肤缺损创面大肠埃希菌和金黄色葡萄球菌菌落数比较（CFU/mL，
组别 创面数（个） 金黄色葡萄球菌 大肠埃希菌 生理盐水组 4 （3.1±0.7）×108 （7.3±0.7）×105 黏性水凝胶组 4 （2.0±0.4）×108 a （6.1±0.5）×105 a 含银黏性水凝胶组 4 （3.0±0.4）×104 bc （9.0±1.3）×102 bc F值 39.881 255.464 P值 <0.01 <0.01 注：CFU为集落形成单位；与生理盐水组比较，aP<0.05，bP<0.01；与黏性水凝胶组比较，cP<0.01
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表4 3组小鼠伤后14 d全层皮肤缺损创面表皮厚度和胶原纤维光密度比较（
组别 创面数（个） 表皮厚度（μm） 胶原纤维光密度 生理盐水组 4 38±5 0.261±0.011 黏性水凝胶组 4 41±13 0.281±0.012 含银黏性水凝胶组 4 73±18ab 0.289±0.008a F值 8.875 7.923 P值 <0.01 <0.05 注：与生理盐水组比较，aP<0.05；与黏性水凝胶组比较，bP<0.05
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表5 3组小鼠全层皮肤缺损创面伤后各时间点3种细胞因子的表达（
组别与时间点 创面数（个） TNF-α TGF-β1 VEGF 生理盐水组 12 伤后3 d 0.271±0.017 0.215±0.010 0.191±0.009 伤后7 d 0.254±0.006 0.235±0.011 0.221±0.007 伤后10 d 0.239±0.012 0.281±0.016 0.260±0.025 黏性水凝胶组 12 伤后3 d 0.275±0.017 0.229±0.007 0.225±0.005b 伤后7 d 0.245±0.014 0.253±0.005 0.243±0.016 伤后10 d 0.223±0.015 0.291±0.012 0.290±0.014 含银黏性水凝胶组 12 伤后3 d 0.302±0.021 0.234±0.010a 0.238±0.009b 伤后7 d 0.237±0.015 0.287±0.013bc 0.263±0.014b 伤后10 d 0.206±0.014a 0.316±0.013a 0.341±0.016bd F1值 3.581 4.680 37.686 P1值 >0.05 <0.05 <0.01 F2值 2.018 25.251 10.693 P2值 >0.05 <0.01 <0.01 F3值 5.884 6.551 18.302 P3值 <0.05 <0.05 <0.01 注：各组各时间点样本数均为4；肿瘤坏死因子α（TNF-α）、转化生长因子β1（TGF-β1）及血管内皮生长因子（VEGF）处理因素主效应，F=0.843、29.080、48.415，P>0.05、P<0.01、P<0.01；时间因素主效应，F=48.664、114.758、98.790，P<0.01、P<0.01、P<0.01；两者交互作用，F=5.381、2.550、3.057，P>0.05、P<0.01、P<0.05；F1值、P1值，F2值、P2值，F3值、P3值分别为3组伤后3、7、10 d各指标总体比较所得；与生理盐水组相同时间点比较，aP<0.05，bP<0.01；与黏性水凝胶组相同时间点比较，cP<0.01，dP<0.05
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