Pub Date : 2024-01-20DOI: 10.3760/cma.j.cn501225-20231020-00126
Z Z Yan, Y X Wang, T L Zhang, J N Xun, Y C Ma, C Ji, J Gao, S C Xiao
<p><p><b>Objective:</b> To explore the properties of gelatin-polyethylene glycol hydrogel loaded with silver nanoparticle (AgNP) <i>Chlorella</i> (hereinafter referred to as the composite hydrogel) and its effects on healing of infected full-thickness skin defect wounds in mice. <b>Methods:</b> The research was an experimental research. The simple gelatin-polyethylene glycol hydrogel (hereinafter referred to as the simple hydrogel) and the composite hydrogel were prepared, and the appearance and injectability of the two hydrogels were observed at 55 and 37 ℃, and under the irradiation of 808 nm near-infrared light, respectively. An electronic universal testing machine was employed to assess the tensile and compressive stress-strain properties of both types of hydrogels at room temperature. Additionally, the cyclic compressive stress-strain properties of the composite hydrogel were examined at 80% of the maximum compressive stress. <i>Staphylococcus aureus</i> or <i>Escherichia coli</i> solution was added to phosphate buffer solution (PBS), simple hydrogel, and composite hydrogel, respectively. The part of composite hydrogel containing <i>Staphylococcus aureus</i> or <i>Escherichia coli</i> solution was irradiated with near-infrared light for 5 minutes. After each sample was incubated for 6 h, the dilution plating method was used to detect and calculate the mortality rates of the two bacteria at 24 h of culture (<i>n</i>=5). The discarded foreskin tissue was taken from a 6-year-old healthy boy admitted to the Department of Urology of the First Affiliated Hospital of Naval Medical University for circumcision. Primary human fibroblasts (HFbs) were isolated using the enzyme extraction method, routinely cultured to the 3<sup>rd</sup> to 6<sup>th</sup> passages for subsequent cellular experiments. Composite hydrogel extracts with final mass concentrations of 100.0, 50.0, 25.0, 12.5, and 0 mg/mL were respectively prepared and used to culture HFbs, and the cell proliferation after 24 h of culture was detected using a cell counting kit 8 (<i>n</i>=3). A total of twenty 6-8 weeks old C57BL/6J female mice were utilized, and a full-thickness skin defect was surgically created on the back of each mouse. The wounds were infected with <i>Staphylococcus aureus</i> solution. The infected mice were divided into blank control group, simple hydrogel group, composite hydrogel group, and combined treatment group according to the random number table, and the wounds were treated with PBS, simple hydrogel, composite hydrogel, and composite hydrogel+light irradiation (under the irradiation of 808 nm near-infrared light for 5 min), respectively, with 5 mice in each group. On post injury day (PID) 0 (immediately after the first wound treatment), 3, 7, and 14, an overall assessment of wound exudation and healing were conducted, and the wound healing rates on PID 7 and 14 were calculated (<i>n</i>=5). On PID 14, hematoxylin-eosin staining was performed to observe histopatholo
目的探索明胶-聚乙二醇水凝胶负载银纳米粒子（AgNP）小球藻（以下简称 "复合水凝胶"）的特性及其对小鼠感染性全厚皮肤缺损伤口愈合的影响。研究方法本研究为实验研究。制备了单纯明胶-聚乙二醇水凝胶（以下简称单纯水凝胶）和复合水凝胶，并分别在 55 ℃ 和 37 ℃ 及 808 nm 近红外线照射下观察了两种水凝胶的外观和可注射性。使用电子万能试验机评估了两种水凝胶在室温下的拉伸和压缩应力应变特性。此外，还检测了复合水凝胶在最大压缩应力的 80% 时的循环压缩应力-应变特性。将金黄色葡萄球菌或大肠杆菌溶液分别加入磷酸盐缓冲溶液（PBS）、简单水凝胶和复合水凝胶中。将含有金黄色葡萄球菌或大肠杆菌溶液的复合水凝胶部分用近红外光照射 5 分钟。每个样本培养 6 小时后，采用稀释平板法检测并计算两种细菌在培养 24 小时后的死亡率（n=5）。废弃的包皮组织取自海军军医大学第一附属医院泌尿外科一名接受包皮环切术的 6 岁健康男孩。用酶提取法分离出原代人成纤维细胞（HFbs），常规培养至第 3-6 个传代，用于随后的细胞实验。分别制备终质量浓度为 100.0、50.0、25.0、12.5 和 0 mg/mL 的复合水凝胶提取物用于培养 HFbs，并使用细胞计数试剂盒 8 检测培养 24 小时后的细胞增殖情况（n=3）。利用 20 只 6-8 周大的 C57BL/6J 雌性小鼠，在每只小鼠背部用手术造成全厚皮肤缺损。伤口感染金黄色葡萄球菌溶液。按随机数字表将感染小鼠分为空白对照组、单纯水凝胶组、复合水凝胶组和联合治疗组，分别用 PBS、单纯水凝胶、复合水凝胶和复合水凝胶+光照射（在 808 nm 近红外线灯下照射 5 分钟）处理伤口，每组 5 只。在损伤后第 0 天（即第一次伤口处理后）、第 3 天、第 7 天和第 14 天，对伤口渗出和愈合情况进行全面评估，并计算第 7 天和第 14 天的伤口愈合率（n=5）。PID 14时，进行苏木精-伊红染色，观察小鼠伤口的组织病理学变化。结果简单水凝胶和复合水凝胶在 55 ℃ 时都处于溶液状态，冷却到 37 ℃ 时过渡到凝胶状态。两种水凝胶经近红外光照射后，只有复合水凝胶重新升温并恢复到溶液状态，具有可注射性。复合水凝胶的最大拉伸应力为 301.42 kPa，相应应变为 87.19%；最大压缩应力为 413.79 kPa，相应应变为 91.67%，与简单水凝胶的拉伸和压缩性能相似。经过 10 次压缩循环后，复合水凝胶的最大压缩应力仍达到第一次压缩应力的 84.1%。培养 24 小时后，用单纯水凝胶处理的金黄色葡萄球菌的死亡率明显高于用 PBS 处理的金黄色葡萄球菌的死亡率（PEscherichia coli and Staphylococcus aureus treated with composite hydrogel alone were significantly higher than those with simple hydrogel）（PEscherichia coli and Staphylococcus aureus treated with composite hydrogel+light irradiation were significantly higher than those with composite hydrogel alone（PPPPPPConclusions）：制备的复合水凝胶具有优异的热敏性、光热性和注射性，以及良好的机械性能、抗菌性能和生物相容性，可促进小鼠感染性全厚皮肤缺损伤口的愈合。
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