本文亮点：

(1) 制备了含氧化石墨烯（GO）的甲基丙烯酸酐化明胶（GelMA）水凝胶，进一步观察到原位光聚合含0.1 μg/mL GO的GelMA水凝胶能促进小鼠全层皮肤缺损创面血管新生，且新生血管富集于GO附近。

(2) 证实 GO 的促血管新生作用与创面细胞分泌的 VEGF 增加相关。

梁莉婷 1   宋薇 2    张超 2    李曌 2    姚斌 2    张孟德 2    袁星宇 2     恩和吉日嘎拉 2    付小兵 2    黄沙 2    朱平 3   

1 华南理工大学医学院，广州 510006； 2 解放军总医院医学创新研究部创伤修复与组织 再生研究中心，北京 100048；3 广东省医学科学院 广东省人民医院心外科 广东省心血管病研究所，广州 510080

通信作者：黄沙，Email：该Email地址已收到反垃圾邮件插件保护。要显示它您需要在浏览器中启用JavaScript。；朱平，Email：该Email地址已收到反垃圾邮件插件保护。要显示它您需要在浏览器中启用JavaScript。

【摘要】

      目的 制备含氧化石墨烯（GO）的甲基丙烯酸酐化明胶（GelMA）水凝胶并探讨原位光 聚合 GO-GelMA 复合水凝胶对小鼠全层皮肤缺损创面血管化的影响。 方法 采用实验研究方法。 将 0.2 mg/mL 的 GO 溶液 50 μL 均匀涂抹于导电胶上，烘干后于场发射扫描电子显微镜下观察 GO 的结构和大小。将人皮肤成纤维细胞（HSF）分为采用相应终质量浓度 GO 处理的 0 μg/mL GO（不加 GO 溶液，下同）组、0.1 μg/mL GO 组、1.0 μg/mL GO 组、5.0 μg/mL GO 组、10.0 μg/mL GO 组，用酶标仪检测细胞培养 48 h 的吸光度值，以此表示细胞增殖活性（样本数为 6）。将 HSF 和人脐静脉血管内皮细胞（HUVEC）分别分为采用相应终质量浓度 GO 处理的 0 μg/mL GO 组、0.1 μg/mL GO 组、1.0 μg/mL GO组、5.0 μg/mL GO 组，采用划痕试验检测划痕后 24、36 h HSF 的迁移率（样本数为 5）及划痕后 12 h HUVEC 的迁移率（样本数为 3），采用酶联免疫吸附测定法检测培养 4、6、8 h 后 HSF 分泌的血管内皮生长因子（VEGF）水平（样本数为 3）。将配制的含相应终质量浓度 GO 的 GO-GelMA 复合水凝胶设为0 μg/mL GO 复合水凝胶组、 0.1 μg/mL GO 复合水凝胶组、1.0 μg/mL GO 复合水凝胶组、5.0 μg/mL GO 复合水凝胶组，观察其交联前后的性状，检测用磷酸盐缓冲液浸泡 3、7 d 后 GO 的释放情况（样本数为 3）。在 16 只 6 周龄雌性 C57BL/6 小鼠背部制作全层皮肤缺损创面，将采用原位交联的含相应终质量浓 度 GO 的 GO-GelMA 复 合 水 凝 胶 处 理 的 小 鼠 按 随 机 数 字 表 法 分 为 0 μg/mL GO 复 合 水 凝 胶 组 、0.1 μg/mL GO 复合水凝胶组、1.0 μg/mL GO 复合水凝胶组、5.0 μg/mL GO 复合水凝胶组，每组 4 只，观察治疗 3、7、14 d 创面大体情况并计算创面愈合率，采用激光多普勒血流仪检测治疗 3、7、14 d 创面血流灌注并计算平均灌注单位（MPU）比值，采用苏木精-伊红染色观察治疗 7 d 创面血管新生情况并计算血管密度（样本数均为 3）。取 0 μg/mL GO 复合水凝胶组和 0.1 μg/mL GO 复合水凝胶组治疗 7 d 的创面组织，采用苏木精-伊红染色观察 GO 分布与血管新生的关系（样本数为 3），行免疫组织化学染色后观察 VEGF 的表达。对数据行重复测量方差分析、单因素方差分析、Tukey 法。 结果 GO 为多层片状结构，宽度约为 20 μm、长度约为 50 μm。培养 48 h，10.0 μg/mL GO 组 HSF 的吸光度值明显低于0 μg/mL GO 组（q=7.64，P<0.01）。划痕后 24 h，4 组 HSF 迁移率相近（P>0.05）；划痕后 36 h，0.1 μg/mLGO 组 HSF 迁移率明显高于 0 μg/mL GO 组、1.0 μg/mL GO 组、5.0 μg/mL GO 组（q 值分别为 7.48、10.81、10.20，P 值 均 <0.01）。 划 痕 后 12 h，0.1 μg/mL GO 组 HUVEC 迁 移 率 明 显 高 于 0 μg/mL GO 组 、1.0 μg/mL GO 组、5.0 μg/mL GO 组（q 值分别为 7.11、8.99、14.92，P 值均<0.01），5.0 μg/mL GO 组 HUVEC迁移率明显低于 0 μg/mL GO 组和 1.0 μg/mL GO 组（q 值分别为 7.81、5.33，P<0.05 或 P<0.01）。培养 4、6 h，4 组 HSF 的 VEGF 表达均相近（P>0.05）；培养 8 h，0.1 μg/mL GO 组 HSF 的 VEGF 表达明显高于0 μg/mL GO 组和 5.0 μg/mL GO 组（q 值分别为 4.75、4.48，P 值均<0.05）。4 组 GO-GelMA 复合水凝胶在交联前均呈红色液体状，交联后呈微黄色凝胶状且流动性无明显差异。0 μg/mL GO 复合水凝胶组复合水凝胶各时间点均无 GO 释放，其余 3 组 GO-GelMA 复合水凝胶中的 GO 于浸泡 3 d 部分释放，至浸泡 7 d 全部释放。治疗 3~14 d，4 组小鼠创面可见水凝胶敷料覆盖在位并保持湿润，创面逐渐愈合。治疗 3、7、14 d，4 组小鼠创面愈合率均相近（P>0.05）。治疗 3 d，0.1 μg/mL GO 复合水凝胶组小鼠创面MPU 比值明显高于 0 μg/mL GO 复合水凝胶组、1.0 μg/mL GO 复合水凝胶组、5.0 μg/mL GO 复合水凝胶组（q 值分别为 10.70、11.83、10.65，P<0.05 或 P<0.01）。治疗 7、14 d，4 组小鼠创面 MPU 比值均相近（P>0.05）。0.1 μg/mL GO 复合水凝胶组小鼠创面治疗 7 d 的 MPU 比值明显低于治疗 3 d（q=14.38，P<0.05），治疗 14 d 的 MPU 比值明显低于治疗 7 d（q=27.78，P<0.01）。治疗 7 d，0.1 μg/mL GO 复合水凝胶组小鼠创面新生血管密度为每 200 倍视野下（120.7±4.1）根，明显高于 0 μg/mL GO 复合水凝胶组、1.0 μg/mL GO 复合水凝胶组、5.0 μg/mL GO 复合水凝胶组的每 200 倍视野下（61.7±1.3）、（77.7±10.2）、（99.0±7.9）根（q 值分别为 12.88、7.79、6.70，P 值均<0.01）；1.0 μg/mL GO 复合水凝胶组和 5.0 μg/mL GO复合水凝胶组小鼠创面新生血管密度均明显高于 0 μg/mL GO 复合水凝胶组（q 值分别为 5.10、6.19，P<0.05）。治疗 7 d，相较于 0 μg/mL GO 复合水凝胶组，0.1 μg/mL GO 复合水凝胶组小鼠创面中成簇新生血管更多，且聚集于 GO 附近；0.1 μg/mL GO 复合水凝胶组小鼠创面中 GO 和新生血管分布区域有大量 VEGF 表达。 结论 GO 质量浓度低于 10.0 μg/mL 对 HSF 增殖活性无明显影响，0.1 μg/mL 的 GO能够促进 HSF 和 HUVEC 迁移，能促进 HSF 分泌 VEGF。原位光聚合 GO-GelMA 复合水凝胶敷料能够通过促进小鼠全层皮肤缺损创面血管新生，增加创面早期血流灌注，且 GO 对新生血管有富集作用，其机制可能与 GO 促进创面细胞分泌 VEGF 相关。

【关键词】 伤口愈合； 水凝胶； 血管内皮生长因子 A； 血管新生； 甲基丙烯酸酐化明胶； 氧化石墨烯

基金项目：国家自然科学基金青年科学基金项目（32000969、82002056）；中国医学科学院医学与健康科技创新工程项目（2019-I2M-5-059）；解放军总医院军事医学创新研究项目（CX19026）；王正国创 伤 医 学 发 展 基 金 会 生 长 因 子 复 兴 计 划（SZYZ-TR-03）；广 州 市 科 学 研 究 计 划 重 点 项 目（201904020047）；广东省人民医院登峰计划专项（DFJH201812、KJ012019119、KJ012019423）

Effects of in situ cross-linked graphene oxide-containing gelatin methacrylate anhydride hydrogel on wound vascularization of full-thickness skin defect in mice

Liang Liting1, Song Wei2, Zhang Chao2, Li Zhao2, Yao Bin2, Zhang Mengde2, Yuan Xingyu2, Enhejirigala 2, Fu Xiaobing2, Huang Sha2, Zhu Ping3

1 School of Medicine, South China University of Technology, Guangzhou 510006, China; 2 Research Center for Wound Repair and Tissue Regeneration, Medical Innovation Research Department, the PLA General Hospital, Beijing 100048, China; 3 Guangdong Cardiovascular Institute, Department of Cardiac Surgery of Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, China

Corresponding authors:Huang Sha, Email : 该Email地址已收到反垃圾邮件插件保护。要显示它您需要在浏览器中启用JavaScript。; Zhu Ping , Email: 该Email地址已收到反垃圾邮件插件保护。要显示它您需要在浏览器中启用JavaScript。

【Abstract】

      Objective To prepare graphene oxide (GO) -containing gelatin methacrylate anhydride (GelMA) hydrogel and to investigate the effects of in situ photopolymerized GO-GelMA composite hydrogel in wound vascularization of full-thickness skin defect in mice.

      Methods The experimental study method was used. The 50 μL of 0.2 mg/mL GO solution was evenly applied onto the conductive gel, and the structure and size of GO were observed under field emission scanning electron microscope after drying. Human skin fibroblasts (HSFs) were divided into 0 μg/mL GO (without GO solution, the same as below) group, 0.1 μg/mL GO group, 1.0 μg/mL GO group, 5.0 μg/mL GO group, and 10.0 μg/mL GO group treated with GO of the corresponding final mass concentration, and the absorbance value was detected using a microplate analyzer after 48 h of culture to reflect the proliferation activity of cells (n=6). HSFs and human umbilical vein vascular endothelial cells (HUVECs) were divided into 0 μg/mL GO group, 0.1 μg/mL GO group, 1.0 μg/mL GO group, and 5.0 μg/mL GO group treated with GO of the corresponding final mass concentration, and the migration rates of HSFs at 24 and 36 h after scratching ( n=5) and HUVECs at 12 h after scratching ( n=3) were detected by scratch test , and the level of vascular endothelial growth factor (VEGF) secreted by HSFs after 4, 6, and 8 h of culture was detected by enzyme-linked immunosorbent assay method ( n=3). The prepared GO-GelMA composite hydrogels containing GO of the corresponding final mass concentration were set as 0 μg/mL GO composite hydrogel group, 0.1 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group to observe their properties before and after cross -linking , and to detect the release of GO after soaking with phosphate buffer solution for 3 and 7 d (n=3). The full -thickness skin defect wounds were made on the back of 16 6-week-old female C57BL/6 mice. The mice treated with in situ cross-linked GO-GelMA composite hydrogel containing GO of the corresponding final mass concentration were divided into 0 μg/mL GO composite hydrogel group, 0.1 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group according to the random number table, with 4 mice in each group. The general condition of wound was observed and the wound healing rate was calculated on 3, 7, and 14 d of treatment, the wound blood perfusion was detected by laser Doppler flowmetry on 3, 7, and 14 d of treatment and the mean perfusion unit (MPU) ratio was calculated, and the wound vascularization on 7 d of treatment was observed after hematoxylin-eosin staining and the vascular density was calculated (n=3). The wound tissue of mice in 0 μg/mL GO composite hydrogel group and 0.1 μg/mL GO composite hydrogel group on 7 d of treatment was collected to observe the relationship between the distribution of GO and neovascularization by hematoxylin -eosin staining (n=3) and the expression of VEGF by immunohistochemical staining . Data were statistically analyzed with analysis of variance for repeated measurement, one -way analysis of variance , and Tukey's method.

      Results GO had a multilayered lamellar structure with the width of about 20 μm and the length of about 50 μm. The absorbance value of HSFs in 10. 0 μg/mL GO group was significantly lower than that in 0 μg/mL GO group after 48 h of culture (q=7.64, P<0.01). At 24 h after scratching, the migration rates of HSFs were similar in the four groups ( P>0.05); at 36 h after scratching, the migration rate of HSFs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group, 1 .0 μg/mL GO group , and 5.0 μg/mL GO group (with q values of 7.48, 10.81, and 10 .20, respectively , P<0.01). At 12 h after scratching, the migration rate of HUVECs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group, 1.0 μg/mL GO group, and 5.0 μg/mL GO group (with q values of 7.11, 8.99, and 14.92, respectively, P<0.01), and the migration rate of HUVECs in 5.0 μg/mL GO group was significantly lower than that in 0μg/mL GO group and 1.0 μg/mL GO group (with q values of 7.81 and 5.33, respectively, P<0.05 or P<0.01).At 4 and 6 h of culture,the VEGF expressions of HSFs in the four groups were similar(P>0.05); at 8 h of culture, the VEGF expression of HSFs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group and 5.0 μg/mL GO group (with q values of 4.75 and 4.48, respectively, P<0.05). The GO-GelMA composite hydrogels in the four groups were all red liquid before cross -linking, which turned to light yellow gel after cross -linking, with no significant difference in fluidity. The GO in the GO -GelMA composite hydrogel of 0 μg/mL GO composite hydrogel group had no release of GO at all time points; the GO in the GO -GelMA composite hydrogels of the other 3 groups was partially released on 3 d of soaking, and all the GO was released on 7 d of soaking. From 3 to 14 d of treatment, the wounds of mice in the 4 groups were covered with hydrogel dressings, kept moist, and gradually healed. On 3, 7, and 14 d of treatment, the wound healing rates of mice in the four groups were similar (P>0.05). On 3 d of treatment, the MPU ratio of wound of mice in 0.1 μg/mL GO composite hydrogel group was significantly higher than that in 0 μg/mL GO composite hydrogel group , 1.0 μg/mL GO composite hydrogel group , and 5 .0 μg/mL GO composite hydrogel group (with q values of 10.70 , 11.83, and 10.65, respectively, P<0.05 or P<0.01). On 7and 14 d of treatment, the MPU ratios of wound of mice in the four groups were similar (P>0.05).The MPU ratio of wound of mice in 0.1 μg/mL GO composite hydrogel group on 7 d of treatment was significantly lower than that on 3 d of treatment (q=14.38 ,P<0.05), and that on 14 d of treatment was significantly lower than that on 7 d of treatment ( q=27.78, P<01). On 7 d of treatment , the neovascular density of wound of mice on 7 d of treatment was 120.7±4.1 per 200 times of visual field, which was significantly higher than 61.7±1.3, 77.7±10.2, and 99.0±7.9 per 200 times of visual field in 0 μg/mL GO composite hydrogel group , 1. 0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group (with q values of 12 . 88 , 7.79, and 6.70, respectively, P<0.01), and the neovascular density of wound of mice in 1.0 μg/mL GO composite hydrogel group and 5.0 μg/mL GO composite hydrogel group was significantly higher than that in 0 μg/mL GO composite hydrogel group (with q values of 5.10 and 6.19, respectively, P<0.05). On 7 d of treatment, cluster of new blood vessels in wound of mice in 0.1 μg/mL GO composite hydrogel group was significantly more than that in 0 μg/mL GO composite hydrogel group, and the new blood vessels were clustered near the GO; a large amount of VEGF was expressed in wound of mice in 0.1 μg/mL GO composite hydrogel group in the distribution area of GO and new blood vessels.

      Conclusions GO with mass concentration lower than 10.0 μg/mL had no adverse effect on proliferation activity of HSFs, and GO of 0.1 μg/mL can promote the migration of HSFs and HUVECs , and can promote the secretion of VEGF in HSFs. In situ photopolymerized of GO -GelMA composite hydrogel dressing can promote the wound neovascularization of full -thickness skin defect in mice and increase wound blood perfusion in the early stage, with GO showing an enrichment effect on angiogenesis, and the mechanism may be related to the role of GO in promoting the secretion of VEGF by wound cells.

    【Key words】 Wound healing; Hydrogel; Vascular endothelial growth factor A; Neovascularization ; Gelatin methacrylate anhydride ; Graphene oxide

      Fund program: Youth Science Foundation of National Natural Science Foundation of China (32000969 , 82002056); Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2019 -I2M-5-059); Military Medical Innovation Research Project of PLA General Hospital (CX 19026 ); Wang Zhengguo Foundation for Traumatic Medicine Growth Factor Rejuvenation Plan (SZYZ -TR-03); Key Program of Guangzhou Science Research Plan (201904020047); Special Project of Dengfeng Program of Guangdong Provincial People's Hospital (DFJH201812, KJ012019119, KJ012019423)