异体表皮干细胞对裸鼠全层皮肤缺损创面 移植异体全厚皮成活的影响及其机制

黄韶斌 1 胡志成 1 张逸 2 唐冰 1 王鹏 1 徐海琳 1 王志勇 1 董云先 1 程璞 1 荣燕超 1 吴军 3 朱家源 1

1 中山大学附属第一医院烧伤科，广州 510080；

2 南通大学附属医院烧伤整形外科226001；

3 深圳大学第一附属医院烧伤整形科 518037

通信作者：朱家源，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 探讨异体表皮干细胞（ESC）对裸鼠全层皮肤缺损创面移植异体全厚皮成活的影响及其机制。方法 采用实验研究方法。采用酶消化法从 1 只 4 周龄雄性 BALB/c-NU 裸鼠（品系、鼠龄与性别下同）中获取培养 7 d 呈铺路石状原代 ESC，其第 3 代细胞经流式细胞仪鉴定阳性表达ESC 标志物 CD44 且阴性表达 CD45，经免疫荧光法鉴定阳性表达 ESC 标志物 p63 与整合素 6α 且阴性表达 CD71。取对数生长期的第 3 代 ESC 进行后续实验。取 26 只裸鼠，采用随机数字表法平均分为磷酸盐缓冲液（PBS）组和 ESC 组，在每只裸鼠背部制备全层皮肤缺损创面后，2 组创面分别喷涂等体积的 PBS、ESC，创面上均移植从另外 4 只裸鼠背部切取制备的全厚皮。分别取 2 组 10 只裸鼠，观察术后0（即刻）、3、7、14、21 d 创面愈合与皮片存活情况并计算术后 3、7、14、21 d 皮片存活比和皮片收缩率（样本数为各时间点存活皮片数），采用激光散斑血流成像仪检测术后 3、7、14 d 皮片的血流灌注情况并计算各时间点 ESC 组与 PBS 组裸鼠皮片血流灌注比（样本数为各时间点 2 组配对均存活皮片对数）；分别取 2 组剩余 3 只裸鼠，取术后 7 d 皮片组织，分别采用实时荧光定量反转录 PCR 法和蛋白质印迹法检测组织中肿瘤坏死因子 α（TNF-α）、白细胞介素 8（IL-8）、IL-10、Ⅰ型胶原、Ⅲ型胶原与基质金属蛋白酶 9（MMP-9）的 mRNA 和蛋白表达。对数据行 Log-rank 检验、重复测量方差分析、单因素方差分析、独立样本 t 检验与 Bonferroni 校正。

结果 以术后 0 d 情况为参照，2 组裸鼠术后 3、7、14、21 d 创面逐渐愈合，皮片收缩情况逐渐明显，其中 PBS 组裸鼠创面收缩愈合情况较 ESC 组明显。术后3 d，ESC 组 1 只裸鼠皮片移植失败，PBS 组 3 只裸鼠皮片移植失败；术后 7 d，PBS 组又有 1 只裸鼠皮片移植失败。2 组裸鼠术后 3、7、14、21 d 皮片存活比相近（P>0.05）。术后 3、7、14、21 d，ESC 组裸鼠皮片收缩率分别为（9.2±0.4）%、（19.7±1.2）%、（53.6±3.5）%、（62.2±5.1）%，显著低于 PBS 组的（11.0±0.9）%、（47.8±2.8）%、（86.1±7.1）%、（89.7±9.0）%（t=5.719、26.650、11.940、7.617，P<0.01）。术后 3、7、14 d，2 组裸鼠皮片均有血流灌注信号；ESC 组与 PBS 组裸鼠皮片血流灌注比均值均大于 1，3 个时间点总体比较，差异无统计学意义（P>0.05）。术后 7 d，与 PBS 组比较，ESC 组裸鼠皮片组织中 TNF-α、IL-8、Ⅰ型胶 原 和 Ⅲ 型 胶 原 的 mRNA（t=2.823、2.934、2.845、2.860，P<0.05）和 蛋 白 表 达 均 明 显 降 低 ，IL-10 和MMP-9 的 mRNA（t=3.877、2.916，P<0.05）和蛋白表达均明显升高。

结论 异体 ESC 可减轻裸鼠全层皮肤缺损创面移植异体全厚皮片收缩，促进移植皮片与创面之间新生血管的形成，减轻炎症反应，降低胶原蛋白表达，促进 MMP-9 的表达，从而提高移植皮片的成活质量。

【关键词】 干细胞移植； 皮肤移植； 伤口愈合； 表皮干细胞

基金项目：国家自然科学基金面上项目（81871565）；广东省科技计划（2016B090916001）；广东省基 础 与 应 用 基 础 研 究 基 金（2019A1515012208）；中 山 大 学 临 床 医 学 研 究 5010 计 划（2013001、2018003）；中山大学高校基本科研业务费（19ykpy66）

      Effects and mechanisms of allogeneic epidermal stem cells on the survival of allogeneic full-thickness skin grafts in nude mice with full-thickness skin defect wounds Huang Shaobin1, Hu Zhicheng1, Zhang Yi2, Tang Bing1, Wang Peng1, Xu Hailin1, Wang Zhiyong1, Dong Yunxian1, Cheng Pu1, Rong Yanchao1, Wu Jun3, Zhu Jiayuan1

1 Department of Burns, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China

2 Department of Burns and Plastic Surgery, Affiliated Hospital of Nantong University, Nantong 226001, China ;;

3 Department of Burns and Plastic Surgery, the First Affiliated Hospital of Shenzhen University, Shenzhen 518037, China Corresponding author: Zhu Jiayuan,

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【Abstract】

      Objective To investigate the effects and mechanisms of allogeneic epidermal stem cells (ESCs) on the survival of allogeneic full-thickness skin grafts in nude mice with full-thickness skin defect wounds. Methods Experimental research methods were applied. Primary ESCs that appeared paving stone-like after being cultured for 7 d were obtained by enzymatic digestion method from one 4-week-old male BALB/c-NU nude mouse (the same strain, age, and sex below). The cells of third passage were identified by flow cytometry to positively express ESC marker CD44 and negatively express CD45, meanwhile, the positive expression of ESC markers of p63 and integrin 6α, and negative expression of CD71 were identified by immunofluorescence method. The ESCs of third passage in the logarithmic growth phase were used for the following experiments. Twenty-six nude mice were equally divided into phosphate buffered saline (PBS) group and ESCs group according to the random number table. A full -thickness skin defect wound was made on the back of each nude mouse, and then the wounds of the two groups were sprayed with equal volumes of PBS and ESCs, respectively. The wounds were transplanted with full -thickness skin grafts cut from the backs of 4 other nude mice. Each ten nude mice from the two groups were selected, the wound healing and skin survival on post surgery day (PSD) 0 (immediately), 3, 7, 14, and 21 were observed, and the survival ratio and shrinkage rate of skin grafts on PSD 3, 7, 14, and 21 were calculated (the number of sample was the number of surviving skin grafts at each time point); the blood perfusion in the skin grafts on PSD 3, 7, and 14 was detected by the laser speckle blood flow imager, and the blood flow ratio of nude mice skin grafts in ESCs group to PBS group at each time point was calculated (the number of sample was the pair number of surviving skin grafts in group pairing at each time point). The skin graft tissue of each 3 nude mice remained in the two groups were collected on PSD 7, and the mRNA expressions and protein expressions of tumor necrosis factor α (TNF-α), interleukin 8 (IL-8), IL-10, type Ⅰ collagen, type Ⅲ collagen, and matrix metalloproteinase 9 (MMP-9) in the tissue were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction and Western blotting, respectively. Data were statistically analyzed with Log-rank test, analysis of variance for repeated measurement , one-way analysis of variance , independent sample t test, and Bonferroni correction. Results Taking the condition on PSD 0 as a reference, the wounds of nude mice in the two groups healed gradually on PSD 3, 7, 14, and 21, and the shrinkage of skin grafts was gradually obvious. Among them, the shrinkage healing of wound of nude mice in PBS group was more significant than that in ESCs group. On PSD 3, the skin graft of 1 nude mouse failed in ESCs group, while the skin graft of 3 nude mice failed in PBS group. On PSD 7, the skin graft of another nude mouse failed in PBS group. The survival ratio of skin grafts of nude mice in the two groups was similar on PSD 3, 7, 14, and 21 (P>0.05).On PSD 3, 7, 14, and 21, the shrinkage rates of skin grafts of nude mice in ESCs group were (9.2±0.4)% , (19.7±1.2)% , (53.6±3.5)% , and (62.2±5.1)% , respectively, which was significantly lower than (11.0±0.9)% , (47.8±2.8)% , (86.1±7.1)% , and (89.7±9.0)% in PBS group (t=5.719, 26.650, 11.940, 7.617, P<0.01). On PSD 3, 7, and 14, blood perfusion signals were observed in the skin grafts of nude mice in the two groups. The average blood perfusion ratios of the skin grafts of nude mice in ESCs group to PBS group were greater than 1, and there was no statistically significant difference in the overall comparison of 3 time points (P>0.05). On PSD 7, compared with those of PBS group, the mRNA and protein expressions of TNF-α, IL-8, type Ⅰ collagen, and type Ⅲ collagen in the skin graft tissue of nude mice in ESCs group were significantly reduced, while the mRNA and protein expressions of IL-10 and MMP-9 in the skin graft tissue of nude mice in ESCs group were significantly increased (in mRNA comparison, t=2.823, 2.934, 2.845, 2.860, 3.877, 2.916, P<0.05). Conclusions Allogeneic ESCs can reduce the shrinkage of allogeneic full-thickness skin grafts transplanted on full-thickness skin defect wounds in nude mice, promote the formation of new blood vessels between the skin graft and the wound , reduce inflammation and collagen protein expression, and promote the expression of MMP -9, thus improving the survival quality of skin grafts.

【Key words】 Stem cell transplantation; Skin transplantation; Wound healing; Epidermal stem cells

Fund program : General Program of National Natural Science Foundation of China (81871565); Science and Technology Planning Project of Guangdong Province of China (2016B090916001); Foundation for Basic and Applied Basic Research of Guangdong Province of China (2019A1515012208); Sun Yat-sen University Clinical Research 5010 Program (2013001, 2018003); Fundamental Scientific Research of Sun Yat-sen University (19ykpy66)

Rat epidermal stem cells promote the angiogenesis of full-thickness wounds

Shaobin Huang1,2† , Zhicheng Hu1† , Peng Wang1 , Yi Zhang3 , Xiaoling Cao1 , Yunxian Dong1 , Pu Cheng1 , Hailin Xu1, Wenkai Zhu4 , Bing Tang1* and Jiayuan Zhu1*

* Correspondence: 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。; 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。 † Shaobin Huang and Zhicheng Hu contributed equally to this work.

1 Department of Burn, First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, Guangdong Province, People’s Republic of China Full list of author information is available at the end of the article

Abstract

Background: Full-thickness wounds severely affect patients’ life quality and become challenging problems for clinicians. Stem cells have great prospects in the treatment of wounds. Our previous study confirmed that autologous basal cell suspension could promote wound healing, and epidermal stem cells (ESCs) were detected in the basal cell suspension. Herein, this study aimed to explore the effect of ESCs on full-thickness wounds.

Methods: Rat ESCs were isolated and expanded and then were transfected with lentivirus to stably express enhanced green fluorescent protein. The experimental rats were randomly divided into 2 groups: in the ESC group, the rat ESCs were sprayed on the full-thickness wounds of rats; in the control group, phosphate-buffered saline was sprayed the on the wounds. Next, wound healing and neovascularization were evaluated. Colonization, division, and differentiation of ESCs on the wound were analyzed by immunofluorescence.

Results: The rat ESCs colonized, divided, and proliferated in the wound. Additionally, rat ESCs around blood vessels differentiated into vascular endothelial cells and formed a lumen-like structure. Compared with the control group, the ESC group showed enhanced angiogenesis and accelerated wound healing.

Conclusions: Our study confirmed that rat ESCs are safe and effective for treating full-thickness wounds. Additionally, under certain conditions, ESCs can differentiate into vascular endothelial cells to promote angiogenesis and wound healing.

Keywords: ESCs, Cell differentiation, Angiogenesis, Full-thickness wounds

Randomized clinical trial of autologous skin cell suspension combined with skin grafting for chronic wounds

Z .-C. Hu1, D. Chen2, D. Guo3, Y.-Y. Liang1, J. Zhang1, J.-Y. Zhu1 and B. Tang1

Departments of 1Burn Surgery and 2Hepatobiliary Surgery, The First Affiliated Hospital of Sun Yat-sen University, and 3Department of Plastic Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China

Correspondence to: Dr B. Tang, Department of Burn Surgery, The First Affiliated Hospital of Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou 510080, China (e-mail: 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。)

Background: Treatment of chronic wounds using traditional surgical procedures is challenging because of the low graft take rates. This study investigated the combination approach of split-thickness autografts with harvested skin cell suspension for chronic wound treatment.

Methods: This randomized clinical trial enrolled patients with chronic wounds between March 2012 and December 2013. Patients who were assigned randomly to the active treatment received a split-thickness autograft combined with harvested skin cell suspension. Control patients received the split-thickness autograft alone. The primary outcome was the rate of complete wound closure by postoperative day 28. Analysis was by intention to treat. Patients who achieved wound closure were followed up for a minimum of 6 months to evaluate the quality of healing.

Results: A total of 88 patients were included, 44 in each group. More patients achieved complete wound closure in the skin cell group than in the control group (41 versus 34 patients; P = 0⋅035). Complete wound closure was observed at a median of 14 (95 per cent c.i. 12⋅0 to 16⋅0) days in the skin cell group and 20 (15⋅7 to 24⋅3) days in the control group (P = 0⋅001). The skin cell group had significantly fewer complications (4 versus 11 patients; P = 0⋅047). The autografted sites displayed better physical attributes and a reduced tendency for wound recurrence in the skin cell group.

Conclusion: Complementary split-thickness autologous skin grafting with autologous skin cells harvested using ReCell® (Avita Medical, Cambridge, UK) technology improved the healing rate of chronic wounds. Registration number: UMIN000011966 (http://www.umin.ac.jp/ctr).

Randomized clinical trial of autologous skin cell suspension for accelerating re-epithelialization of split-thickness donor sites

Z. Hu1, D. Guo3, P. Liu1, X. Cao1, S. Li2, J. Zhu1 and B. Tang1

Departments of 1 Burn Surgery and 2Plastic Surgery, First Affiliated Hospital of Sun Yat-sen University, and 3Department of Plastic Surgery, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China

Correspondence to: Dr B. Tang, Department of Burn Surgery, First Affiliated Hospital, Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou 510080, China (e-mail: 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。)">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。)

Background: Split-thickness skin graft (STSG) is used frequently, but may result in complications at the donor site. Rapid healing of donor-site wounds is critical to relieving morbidity. This study investigated whether autologous skin cell suspension could improve healing of STSG donor-site wounds.

Methods: Between September 2014 and February 2016, patients requiring STSGs were randomized to receive autologous skin cell suspension plus hydrocolloid dressings (experimental group) or hydrocolloid dressings alone (control group) for the donor site. The primary outcome was time to complete re-epithelialization. Secondary outcomes included pain and itching scores measured on a visual analogue scale, and adverse events. Patients were followed for 12 weeks to evaluate quality of healing. Analysis was by intention to treat.

Results: Some 106 patients were included, 53 in each group. Median time to complete re-epithelialization was 9⋅0 (95 per cent c.i. 8⋅3 to 9⋅7) days in the experimental group, compared with 13⋅0 (12⋅4 to 13⋅6) days in the control group (P < 0⋅001). Overall postoperative pain and itching scores were similar in both groups. No between-group differences in treatment-related complications were observed. Both patients and observers were more satisfied with healing quality after autologous skin cell suspension had been used.

Conclusion: The use of autologous skin cell suspension with hydrocolloid dressings accelerated epithelialization and improved healing quality of the donor site compared with hydrocolloid dressings alone. Registration number: UMIN000015000 (http://www.umin.ac.jp/ctr).

Paper accepted 11 January 2017

Published online 5 April 2017 in Wiley Online Library (www.bjs.co.uk). DOI: 10.1002/bjs.10508

Parafricta bootees compared with standard care to prevent heel pressure ulcers: a multicentre pragmatic randomised controlled trial

Background: Parafricta bootees are made of low friction material intended to prevent heel pressure ulcers (PU).

Aims: To compare, in hospitalised patients, whether the bootees, added to standard care (SC), prevent heel PU compared with SC alone.

Methods: Patients with Waterlow score ≥20 and no heel PUs at baseline were randomly allocated to either bootees plus SC, or SC alone. Target sample size was 450 patients. Patients’ heels were clinically assessed for heel PUs at day 3 and day 14. 

Results: Slow recruitment stopped the study early. In 31 recruited patients there were zero incident heel PUs (intervention group, 0%) versus 1 (SC group, 6%) at day 3 and no new heel pressure ulcers at Day 14.

Conclusion: This study failed to reach sufficient statistical power to assess the efficacy of the bootees in preventing heel PUs. No adverse events were related to the bootees. Only 1 patient in the SC group developed a heel PU.

KEY WORDS Pressure ulcer Bootees Friction Medical device-related pressure ulcers Shear

ANDREW CLEVES, Researcher, Cedar, Cardiff and Vale University Health Board, Cardiff Medicentre, University Hospital of Wales, Cardiff

NICOLA IVINS, Clinical Research Director, Welsh Wound Innovation Centre, Rhodfa Marics, Ynysmaerdy, Pontyclun

MICHAEL CLARK, Commercial Director, Welsh Wound Innovation Centre, Rhodfa Marics, Ynysmaerdy, Pontyclun

GRACE CAROLAN-REES, Cedar Director (Retired), Cedar, Cardiff and Vale University Health Board, Cardiff Medicentre, University Hospital of Wales, Cardiff

NIA JONES, Advanced Clinical Podiatrist, seconded to the Welsh Would Innovation Centre, Rhodfa Marics, Ynysmaerdy, Pontyclun.

JUDITH WHITE, Researcher, Cedar, Cardiff and Vale University Health Board, Cardiff Medicentre, University Hospital of Wales, Cardiff.

RHYS MORRIS,Cedar Director, Cedar, Cardiff and Vale University Health Board, Cardiff Medicentre, University Hospital of Wales, Cardiff

原位交联含氧化石墨烯的甲基丙烯酸酐化 明胶水凝胶对小鼠全层皮肤缺损创面 血管化的影响

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)