National expert consensus on the diagnosis and surgical treatment of diabetic foot ulcers complicated with lower extremity vasculopathy (2024 version)
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摘要: 糖尿病足溃疡合并下肢血管病变具有发病率高、愈合缓慢、预后差的特点，如不经规范治疗易导致截肢甚至危及生命。针对合并下肢血管病变的治疗对改善糖尿病足溃疡的愈合进程至关重要，在临床实践中逐渐得到重视。近年来，关于糖尿病足溃疡合并下肢血管病变的临床研究已见诸多报道，为了进一步规范临床诊疗，由中国老年医学学会烧创伤分会、中华医学会烧伤外科学分会、中国医师协会创面修复专业委员会牵头的专家组共同审议并编写了《糖尿病足溃疡合并下肢血管病变的外科诊疗全国专家共识（2024版）》。该共识以文献证据为基础，内容涵盖了糖尿病足溃疡合并下肢血管病变的疾病特征、临床诊疗循证证据以及新技术和新治疗方法的应用，旨在为临床工作者提供关于糖尿病足溃疡合并下肢血管病变最佳筛查和诊疗方法的明确指导，希望为从事糖尿病足创面治疗的医务人员提供规范性的临床实践依据。Abstract: Diabetic foot ulcers complicated with lower extremity vasculopathy possess the characteristics of high incidence, slow healing, and poor prognosis, which may eventually lead to amputation or even life-threatening if not treated properly. The treatment of complicated lower extremity vasculopathy is vital to improve the healing process of diabetic foot ulcers, which has gradually received attention in clinical practice. Recently, a number of clinical trials on diabetic foot ulcers complicated with lower extremity vasculopathy were reported. In order to further standardize the clinical diagnosis and treatment of diabetic foot ulcers complicated with lower extremity vasculopathy, an expert group headed by Burns and Trauma Branch of Chinese Geriatrics Society, Chinese Burn Association, and Wound Repair Professional Committee of Chinese Medical Doctor Association deliberated and compiled the National expert consensus on the diagnosis and surgical treatment of diabetic foot ulcers complicated with lower extremity vasculopathy (2024 version) together. This consensus is based on evidences from the literature, covers the disease characteristics, evidence-based evidence of clinical diagnosis and treatment, as well as the application of new technologies and new treatment approaches of diabetic foot ulcers complicated with lower extremity vasculopathy. The goal of this consensus is to provide clear guidance to practitioners on the best approaches for screening, diagnosing, and treating diabetic foot ulcers complicated with lower extremity vasculopathy in individuals, hoping to provide a normative clinical practice basis for medical staff engaged in the treatment of diabetic foot wounds.
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Key words:
- Diabetic foot /
- Diabetic angiopathies /
- Peripheral arterial disease /
- Microcirculation /
- Amputation /
- Wound repair /
- Expert consensus
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(1)证实人脐带间充质干细胞来源的小细胞外囊泡（hUCMSC-sEV）对皮肤创面愈合相关的3种细胞的水平和垂直迁移以及增殖均有促进作用。
(2)证实所构建的负载hUCMSC-sEV的甲基丙烯酸酐化明胶水凝胶能够延长hUCMSC-sEV作用于小鼠全层皮肤缺损创面的时间，从而促进创面愈合。
Highlights:
(1)It was confirmed that the small extracellular vesicles derived from human umbilical cord mesenchymal stem cells (hUCMSCs-sEVs) had promoting effects on horizontal and vertical migration and proliferation of three types of cells related to skin wound healing.
(2)It was confirmed that the constructed gelatin methacrylate anhydride hydrogel loaded with hUCMSCs-sEVs could prolong the time of treatment with hUCMSCs-sEVs on the full-thickness skin defect wounds in mice, thereby promoting wound healing.
临床上对创面的治疗方法通常为换药、负压吸引及手术治疗 ［ 1］ ，随着患者对创面修复要求的提高，现有的治疗方法逐渐无法满足临床需求。既往研究表明，KC和Fb是皮肤创面愈合中的关键细胞 ［ 2, 3］ 。如何增强皮肤细胞的活性，增强其再生功能，进而促进创面愈合，是目前创面修复领域的研究热点。随着再生医学的发展，干细胞疗法为创面的修复和再生性愈合带来了新的思路 ［ 4, 5］ 。
间充质干细胞（mesenchymal stem cell，MSC）是一种多能干细胞，近期研究显示，MSC主要依靠旁分泌产生的细胞外囊泡（extracellular vesicle，EV）发挥效应，小EV（small extracellular vesicle，sEV）是直径<200 nm的EV ［ 6］ 。研究表明人MSC来源的sEV可以作用于靶细胞，发挥促进组织修复的作用 ［ 7, 8］ 。但直接外用或浅表注射至创面的sEV在创面的有效存留时间较短，无法达到预期疗效，且提高其剂量并不能显著改善上述情况 ［ 9］ 。因此，如何延长sEV在创面的存留时间，使其更好地发挥疗效是目前亟须解决的问题。
水凝胶是一种有良好生物相容性的材料。甲基丙烯酸酐化明胶（gelatin methacrylate anhydride，GelMA）水凝胶含有的多肽序列有利于细胞及组织黏附 ［ 10］ ，且可滴加GelMA溶液至创面部位，使其在紫外光照射下原位成胶 ［ 11］ 。
本研究着眼于人脐带MSC（human umbilical cord mesenchymal stem cell，hUCMSC）来源的sEV（small extracellular vesicle derived from human umbilical cord mesenchymal stem cell，hUCMSC-sEV）对创面的全层修复，详尽表征了其对创面愈合的重要细胞：人表皮KC（human epidermal keratinocyte，HEK）、人真皮Fb（human dermal fibroblast，HDF）及人脐静脉内皮细胞（human umbilical vein endothelial cell，HUVEC）的作用，并进一步开发了一种负载hUCMSC-sEV的GelMA（以下简称hUCMSC-sEV/GelMA）水凝胶，探究该水凝胶能否改善伤处皮肤细胞的功能从而促进皮肤创面愈合，以期为临床全层皮肤缺损创面治疗提供新思路。
1. 材料与方法
本实验研究遵循解放军总医院实验动物伦理审查委员会和国家有关实验动物管理使用的规定。
1.1 动物、细胞及主要试剂与仪器来源
24只健康清洁级6周龄体重23~25 g雄性C57BL/6J小鼠购自北京科宇动物养殖中心，许可证号：SCXK（京）2018-0010。HUVEC细胞系、原代hUCMSC、原代HEK、原代HDF购自武汉普诺赛生命科技有限公司。
DMEM培养基、EpiLife培养基、人KC生长添加剂、胎牛血清、青霉素-链霉素溶液购自美国Gibco公司，无EV血清购自美国SBI公司，小鼠抗人CD63、CD9、肿瘤易感基因101（tumor susceptibility gene 101，TSG101）及钙联蛋白单克隆抗体购自美国Abcam公司，辣根过氧化物酶标记的山羊抗小鼠IgG多克隆抗体购自北京中杉金桥生物技术有限公司，BeyoClick™ 5-乙炔基-2'-脱氧尿嘧啶核苷（5-ethynyl-2'-deoxyuridine，EdU）-488细胞增殖检测试剂盒及结晶紫染色液购自上海碧云天生物技术有限公司，Transwell培养板购自美国Corning公司，GelMA购自苏州永沁泉智能设备有限公司。311型二氧化碳培养箱购自美国Thermo Fisher公司，GelDoc XR+凝胶成像分析系统购自美国Bio-Rad公司，CKX53型倒置荧光显微镜购自日本Olympus公司，DMI1型倒置相差显微镜、TCS SPE型激光扫描共聚焦显微镜购自德国Leica公司，LVEM 25E型透射电子显微镜购自加拿大 Delong Instrument公司，Sigma 300型扫描电子显微镜购自德国ZEISS公司。
1.2 细胞培养
用含有体积分数10%的无sEV血清和常规浓度的青霉素-链霉素溶液的DMEM培养基培养hUCMSC，用含有体积分数1%的人KC生长添加剂与常规浓度的青霉素-链霉素溶液的EpiLife完全培养基培养HEK，用含有体积分数10%的胎牛血清和常规浓度的青霉素-链霉素溶液的DMEM完全培养基培养HDF与HUVEC。将细胞置于37 ℃、含体积分数5%二氧化碳培养箱中培养，每2天更换1次培养液。于细胞生长至70%融合时进行传代。
1.3 hUCMSC-sEV的提取与鉴定
取第3~5代hUCMSC培养48 h至80%融合时，收集上清液，采用超速离心法提取hUCMSC-sEV。用无菌PBS重新悬浮沉淀后于-80 ℃冻存。取适量hUCMSC-sEV于透射电子显微镜100 000倍放大倍数下观察形态。取第5代hUCMSC及提取的hUCMSC-sEV，采用蛋白质印迹法检测CD9、CD63、TSG101及钙联蛋白的表达，其中一抗为小鼠抗人CD9、CD63、TSG101及钙联蛋白单克隆抗体（稀释比均为1∶1 000），二抗为辣根过氧化物酶标记的山羊抗小鼠IgG多克隆抗体（稀释比为1∶5 000），增强化学发光法显影，采用凝胶成像分析系统获取条带。
1.4 细胞迁移情况
1.4.1 细胞划痕试验
取HUVEC、第3代HEK和HDF，按每孔5×10 6个接种于6孔板中，将细胞分为空白对照组与hUCMSC-sEV组，空白对照组细胞加入2 mL 1.2中的相应完全培养基培养，hUCMSC-sEV组细胞加入2 mL含100 μg/mL hUCMSC-sEV的相应完全培养基培养。待细胞生长至90%融合时用容积为1 mL移液器吸头竖直向下沿孔直径划一条直线，PBS清洗3次后加入前述培养基继续培养。于划痕后0（即刻）、6、12、24 h，于倒置相差显微镜50倍放大倍数下观察划痕情况并拍照，采用Image J 2.1.0图像分析软件（美国国立卫生研究院）测量不同时间点的划痕面积并计算划痕后6、12、24 h的细胞迁移率，细胞迁移率=（划痕后0 h划痕面积-划痕后6、12、24 h划痕面积）÷划痕后0 h划痕面积×100%。该实验样本数为3。
1.4.2 细胞Transwell试验
取HUVEC、第4代HEK和HDF，同1.4.1分组后用相应完全培养基悬浮细胞，调整细胞浓度为4×10 7个/mL，取125 μL细胞悬液加入Transwell培养板上室中。向各Transwell培养板下室中加入与其上室细胞培养基相同的培养基600 μL。培养12 h后进行结晶紫染色（阳性染色为紫色），于倒置相差显微镜40倍放大倍数下观察细胞迁移情况，每组取3个视野，统计迁移至下室的细胞数。该实验样本数为3。
1.5 细胞增殖情况
取HUVEC、第4代HEK和HDF，同1.4.1分组后将2组细胞均按每孔1×10 4个接种于24孔板中，同1.4.1培养24 h后采用EdU-488细胞增殖检测试剂盒行EdU、Hoechst染色。封片后，于倒置荧光显微镜200倍放大倍数下观察、拍照，细胞EdU染色阳性为绿色，细胞核Hoechst 33342染色阳性为蓝色，并计算增殖细胞比例。增殖细胞比例=（EdU染色阳性细胞数÷细胞核数）×100%。该实验样本数为3。
1.6 GelMA水凝胶的制备与表征
1.6.1 GelMA水凝胶的形貌
称取适量GelMA固体溶解于含2.5 g/L光引发剂的PBS中，分别添加PBS或200 μg/mL的hUCMSC-sEV悬液，制成终质量分数15%的GelMA（以下简称单纯GelMA）溶液及含终质量分数15% GelMA和终质量浓度0.2 g/L hUCMSC-sEV的hUCMSC-sEV/GelMA溶液，用波长405 nm的紫外光照射30 s制成水凝胶（光照处理下同）。
将2种GelMA水凝胶样本经真空冻干12 h，从样本中间切开，暴露水凝胶内部表面，真空喷金后分别在扫描电子显微镜500、1 000倍放大倍数下观察水凝胶的微观形貌，并采集图像。
1.6.2 hUCMSC-sEV在hUCMSC-sEV/GelMA水凝胶中的分布
将200 μg/mL的hUCMSC-sEV悬液（1 mL）与1 μmol/L的1，1'-二十八烷基-3，3，3'，3'-四甲基氨基氰高氯酸盐染液（0.1 mL）在37 ℃下避光共孵育3 h得到红色荧光染色的hUCMSC-sEV。按照1.6.1中方法制备单纯GelMA水凝胶及hUCMSC-sEV/GelMA水凝胶，其中hUCMSC-sEV/GelMA水凝胶使用荧光染色的hUCMSC-sEV制备，于激光扫描共聚焦显微镜400倍放大倍数下观察2种水凝胶中hUCMSC-sEV的分布情况。
1.6.3 hUCMSC-sEV/GelMA水凝胶中hUCMSC-sEV的释放
同1.6.1分别制备3份100 μL的hUCMSC-sEV/GelMA水凝胶及单纯GelMA水凝胶样本，并放入48孔板中，每孔加入200 μL PBS。每2天吸出上清液，另加入200 μL PBS。分别于浸泡0（即刻）、2、4、6、8、10、12 d通过蛋白比色定量法测定释放到上清液中的hUCMSC-sEV蛋白量，进而计算hUCMSC-sEV的累积释放率，各时间点hUCMSC-sEV累积释放率=浸泡各时间点内hUCMSC-sEV的蛋白释放总量÷加入的hUCMSC-sEV蛋白总量×100%。该实验样本数为3。
1.7 动物实验
1.7.1 创面模型建立与分组处理
将24只小鼠常规麻醉备皮，按随机数字表法分为PBS组、单纯hUCMSC-sEV组、单纯GelMA水凝胶组及hUCMSC-sEV/GelMA水凝胶组，每组6只，并使用打孔器在每只小鼠背部造1个直径为12 mm的全层皮肤缺损创面。伤后即刻采用微量注射器在PBS组小鼠创缘周围3、6、9、12点方向均匀注射PBS（共100 μL），对单纯hUCMSC-sEV组小鼠采用同样方式注射200 μg/mL的hUCMSC-sEV悬液（100 μL），对单纯GelMA水凝胶组小鼠在其创面表面滴加100 μL单纯GelMA溶液后行光照处理，对hUCMSC-sEV/GelMA水凝胶组小鼠在其创面表面滴加100 μL hUCMSC-sEV/GelMA溶液后行光照处理。
1.7.2 创面愈合情况
于伤后0（即刻）、4、8、12 d观察所有小鼠创面愈合情况并拍照，采用Image J 2.1.0图像分析软件测量创面面积并计算伤后4、8、12 d的创面愈合率，创面愈合率=（伤后0 d创面面积-伤后4、8、12 d创面面积）÷伤后0 d创面面积×100%。
1.7.3 创面组织病理学情况
伤后12 d，于观察创面愈合情况后每组取6只小鼠麻醉后断颈处死并取背部创面及距创缘0.5 cm内的皮肤组织常规制作切片后行HE染色，于倒置相差显微镜20倍放大倍数下观察创面新生组织结构。
1.8 统计学处理
采用SPSS 20.0统计软件进行数据分析，计量资料数据均符合正态分布，以
2. 结果
2.1 hUCMSC-sEV的鉴定
提取的hUCMSC-sEV呈杯状结构；hUCMSC-sEV表达CD9、CD63和TSG101，几乎不表达钙联蛋白。hUCMSC不表达CD9、CD63和TSG101，表达钙联蛋白。综上，hUCMSC-sEV鉴定成功。见 图1。
2.2 细胞迁移情况
2.2.1 细胞划痕试验
划痕后6、12、24 h，hUCMSC-sEV组HEK、HDF、HUVEC的迁移率均明显高于空白对照组（ P<0.05）。见 图2、 表1。
注：HEK为人表皮角质形成细胞，HDF为人真皮成纤维细胞，HUVEC为人脐静脉内皮细胞，hUCMSC-sEV为人脐带间充质干细胞来源的小细胞外囊泡；空白对照组细胞仅常规培养，hUCMSC-sEV组细胞培养液中另加入hUCMSC-sEV表1 2组HEK、HDF、HUVEC划痕后各时间点迁移率比较（%，组别 样本数 HEK HDF HUVEC 6 h 12 h 24 h 6 h 12 h 24 h 6 h 12 h 24 h 空白对照组 3 0.9 14.0 58.1 9.3 19.5 47.8 7.2 10.1 39.1 hUCMSC-sEV组 3 51.4 88.2 95.7 19.8 35.4 62.2 15.1 38.9 56.7 t值 25.94 20.98 20.04 3.18 5.68 4.28 4.32 19.33 4.00 P值 <0.001 <0.001 <0.001 0.033 0.004 0.013 0.012 <0.001 0.016 注：HEK为人表皮角质形成细胞，HDF为人真皮成纤维细胞，HUVEC为人脐静脉内皮细胞，hUCMSC-sEV为人脐带间充质干细胞来源的小细胞外囊泡；空白对照组细胞仅常规培养，hUCMSC-sEV组细胞培养液中另加入hUCMSC-sEV；HEK、HDF、HUVEC处理因素主效应， F值分别为1 387.01、40.65、66.89， P值分别为<0.001、0.003、0.001；时间因素主效应， F值分别为381.30、209.70、200.13， P值均<0.001；两者交互作用， F值分别为51.20、0.93、5.90， P值分别为<0.001、0.430、0.027 2.2.2 细胞Transwell试验
培养12 h，hUCMSC-sEV组HEK、HDF及HUVEC迁移数量分别为（550±23）、（235
注：HEK为人表皮角质形成细胞，HDF为人真皮成纤维细胞，HUVEC为人脐静脉内皮细胞，hUCMSC-sEV为人脐带间充质干细胞来源的小细胞外囊泡；空白对照组细胞仅常规培养，hUCMSC-sEV组细胞培养液中另加入hUCMSC-sEV；细胞结晶紫阳性染色为紫色2.3 细胞增殖情况
培养24 h，hUCMSC-sEV组HEK、HDF及HUVEC增殖细胞比例分别为（48.7
注：HEK为人表皮角质形成细胞，HDF为人真皮成纤维细胞，HUVEC为人脐静脉内皮细胞，hUCMSC-sEV为人脐带间充质干细胞来源的小细胞外囊泡，EdU为5-乙炔基-2'-脱氧尿嘧啶核苷；空白对照组细胞仅常规培养，hUCMSC-sEV组细胞培养液中另加入hUCMSC-sEV；各图中右上小图为EdU阳性染色（绿色）图，右下小图为Hoechst 33342阳性染色（蓝色）图，各图中左侧大图为右侧2张小图的叠加图像，绿色+蓝色双荧光染色的细胞为增殖细胞2.4 GelMA水凝胶的表征
2.4.1 GelMA水凝胶的形貌
2种GelMA水凝胶内部呈疏松多孔的海绵状结构，凝胶成分均匀光滑。其中在放大后的hUCMSC-sEV/GelMA水凝胶微观结构图像中可见负载在水凝胶中的hUCMSC-sEV，这些sEV呈团块状分散在水凝胶的孔隙和骨架中。见 图5。
2.4.2 hUCMSC-sEV在hUCMSC-sEV/GelMA水凝胶中的分布
单纯GelMA水凝胶中未见hUCMSC-sEV，hUCMSC-sEV/GelMA水凝胶中可见hUCMSC-sEV均匀分布。见 图6。
2.4.3 hUCMSC-sEV/GelMA水凝胶中hUCMSC-sEV的释放
hUCMSC-sEV/GelMA水凝胶浸泡2 d后hUCMSC-sEV累积释放率曲线趋于平缓，浸泡12 d时hUCMSC-sEV累积释放率为（59.2
2.5 hUCMSC-sEV/GelMA水凝胶对小鼠全层皮肤缺损创面愈合的影响
2.5.1 创面愈合情况
伤后0~12 d，4组小鼠创面均未见明显感染迹象，且随时间延长，创面均不断缩小。伤后12 d，hUCMSC-sEV/GelMA水凝胶组小鼠创面已完全愈合，其余3组小鼠均可见残余创面。伤后4、8、12 d，hUCMSC-sEV/GelMA水凝胶组小鼠创面愈合率均明显高于其余3组（ P<0.05），单纯GelMA水凝胶组、单纯hUCMSC-sEV组小鼠创面愈合率均明显高于PBS组（ P<0.05）；伤后8、12 d，单纯hUCMSC-sEV组创面愈合率均明显高于单纯GelMA水凝胶组（ P<0.05）。见 图8、 表2。
注：PBS为磷酸盐缓冲液，GelMA为甲基丙烯酸酐化明胶，hUCMSC-sEV为人脐带间充质干细胞来源的小细胞外囊泡；对PBS组、单纯hUCMSC-sEV组、单纯GelMA水凝胶组和hUCMSC-sEV/GelMA水凝胶组小鼠创面分别行PBS注射、hUCMSC-sEV悬液注射、单纯GelMA水凝胶覆盖、hUCMSC-sEV/GelMA水凝胶覆盖；直尺刻度为1 mm表2 4组小鼠伤后各时间点全层皮肤缺损创面愈合率比较（%，组别 样本量 4 d 8 d 12 d PBS组 6 58.3±1.3 63.7±0.4 77.8±1.3 单纯GelMA水凝胶组 6 65.4±4.4 69.2±1.8 92.8±0.5 单纯hUCMSC-sEV组 6 67.8±0.9 75.7±0.4 97.2±0.6 负载hUCMSC-sEV的GelMA水凝胶组 6 80.2±2.6 97.9±0.6 100 F值 70.00 1 332.52 1 001.81 P值 <0.001 <0.001 <0.001 P 1值 <0.001 <0.001 <0.001 P 2值 <0.001 <0.001 <0.001 P 3值 <0.001 <0.001 <0.001 P 4值 0.076 <0.001 <0.001 P 5值 <0.001 <0.001 <0.001 P 6值 <0.001 <0.001 0.031 注：PBS为磷酸盐缓冲液，GelMA为甲基丙烯酸酐化明胶，hUCMSC-sEV为人脐带间充质干细胞来源的小细胞外囊泡；对PBS组、单纯GelMA水凝胶组、单纯hUCMSC-sEV组和hUCMSC-sEV/GelMA水凝胶组小鼠创面分别行PBS注射、单纯GelMA水凝胶覆盖、hUCMSC-sEV悬液注射、hUCMSC-sEV/GelMA水凝胶覆盖；处理因素主效应， F=461.49， P=0.001；时间因素主效应， F=1 714.92， P<0.001；两者交互作用， F=76.83， P=0.001； P 1值、 P 2值、 P 3值分别为单纯GelMA水凝胶组、单纯hUCMSC-sEV组、hUCMSC-sEV/GelMA水凝胶组与PBS组各时间点比较所得； P 4值、 P 5值分别为单纯hUCMSC-sEV组、hUCMSC-sEV/GelMA水凝胶组与单纯GelMA水凝胶组各时间点比较所得； P 6值为单纯hUCMSC-sEV组与hUCMSC-sEV/GelMA水凝胶组各时间点比较所得 2.5.2 创面组织病理学情况
伤后12 d，hUCMSC-sEV/GelMA水凝胶组小鼠创面上皮化程度最佳，真皮胶原排列松散有序，未见明显炎症细胞浸润；其余3组小鼠创面可见真皮胶原排列致密且存在不同程度的炎症细胞浸润。
3. 讨论
创面愈合是一个复杂的过程，局部感染、氧化应激、炎症反应和血管生成受损是导致创面愈合困难的因素 ［ 12］ 。MSC是目前再生医学研究的焦点之一，常被用于修复包括皮肤创面在内的各种类型的组织损伤 ［ 13, 14］ ，其旁分泌产生的纳米级囊泡被认为是其发挥修复功能的关键。
为探究hUCMSC-sEV对创面的全层修复效果，本研究通过对提取物形貌以及表面标志物进行测定后证实成功提取hUCMSC-sEV并进行体外实验，详尽探究了其对于HDF、HUVEC以及HEK的迁移与增殖的作用，结果显示hUCMSC-sEV在细胞水平和垂直迁移以及增殖方面均显示出了促进作用，与以往相关文献中MSC来源的sEV能够加速创面愈合的结论 ［ 15, 16, 17, 18］ 一致。然而，单纯的sEV难以在需修复的组织中持续缓慢释放，因此其促进创面愈合的作用在一定程度上受到限制。
水凝胶作为一种传统的生物材料，常作为封闭创面的普通敷料或作为支架材料被用于组织工程研究。本研究中制备的hUCMSC-sEV/GelMA水凝胶可均匀负载hUCMSC-sEV并使其缓慢释放。研究表明，sEV的抗氧化能力随浓度的升高而提高，当sEV浓度达到100 μg/mL时，抗氧化效率约为50% ［ 19］ 。在本实验中，课题组测得在浸泡12 d时hUCMSC-sEV/GelMA水凝胶中的hUCMSC-sEV累积释放率约为60%。为了保证创面处sEV浓度能够达到促进创面修复以及抗氧化的作用，本研究选择在水凝胶溶液中加入200 μg/mL的hUCMSC-sEV。
单纯GelMA水凝胶组与hUCMSC-sEV/GelMA水凝胶组小鼠创面愈合过程中炎症反应不明显，这是由于具有良好组织相容性的GelMA水凝胶能够覆盖并保护创面，减轻炎症程度。但单纯的GelMA水凝胶缺乏生物活性，故而单纯GelMA水凝胶组小鼠创面愈合速度慢于hUCMSC-sEV/GelMA水凝胶组和单纯hUCMSC-sEV组。hUCMSC-sEV/GelMA水凝胶组小鼠创面愈合速度最快，这进一步证明了水凝胶良好的生物相容性以及在水凝胶中载入sEV可以增强sEV的稳定性，并有助于sEV在所需部位的持续释放。
组织病理学结果显示，在伤后12 d，单纯GelMA水凝胶组和单纯hUCMSC-sEV组小鼠创面处仍有少量炎症细胞浸润，而hUCMSC-sEV/GelMA水凝胶组小鼠创面处未见明显炎症细胞浸润。这表明在创伤初期，hUCMSC-sEV可以在一定程度上抑制创面炎症，但随着创面处免疫系统的激活以及小鼠的活动，单纯的hUCMSC-sEV很快被分解、失活，而hUCMSC-sEV/GelMA水凝胶在小鼠创面上牢固附着、保护创面、缓释hUCMSC-sEV，最终起到持续抗炎、促进创面快速愈合的作用。
将水凝胶作为载体促进组织修复是近年来的研究热点。近期研究显示hUCMSC-sEV/GelMA水凝胶可有效改善皮肤激光损伤小鼠的早期炎症反应，从而促进创面愈合，但该研究并未分析hUCMSC-sEV对皮肤创面愈合相关细胞的影响 ［ 20］ 。目前国内外对于负载sEV的GelMA水凝胶的研究大多仅关注sEV对皮肤或血管的某种细胞的作用。本研究团队拟在后续的研究中进一步完善hUCMSC-sEV/GelMA水凝胶对糖尿病或大面积烧伤创面的作用及其机制的相关研究，完善Masson染色、免疫组织化学染色等实验结果。
综上，本研究成功构建了hUCMSC-sEV/GelMA水凝胶，以GelMA水凝胶作为支架，在创面处缓慢释放hUCMSC-sEV，延长了hUCMSC-sEV作用于创面的时间，促进了创面愈合相关的3种细胞的增殖与迁移，从而促进全层皮肤缺损创面愈合。本研究结果为皮肤创面的临床治疗提供了一定的理论依据，为进一步采用新型生物材料治疗临床创面提供了新思路。
所有编写组成员均声明不存在利益冲突，无商业组织向本共识编写组支付费用。本共识制订过程中需要的资金均来自本文著录的基金项目，且推荐意见未受资助影响 -
参考文献
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1 糖尿病足溃疡合并下肢血管病变的评估和分级方法
注：CTA为CT血管成像，MRA为磁共振血管成像，DSA为数字减影血管造影，SINBAD为部位、缺血、神经病变、细菌感染、面积、深度，TcPO2为经皮氧分压，WIfI为创面、缺血和足感染；1 mmHg=0.133 kPa
表1 2009版牛津大学循证医学中心证据分级标准
表1. Oxford Centre for Evidence-Based Medicine: levels of evidence (2009)
证据级别 定义 A 1a 基于同质RCT的系统评价 1b 单个RCT研究 1c 全或无病案研究 B 2a 基于同质队列研究的系统评价 2b 单个队列研究（包括低质量RCT，如随访率<80%） 2c 结果研究或生态学研究 3a 基于同质病例对照研究的系统评价 3b 病例对照研究 C 4 单个病例系列研究（包括低质量队列研究和病例对照研究） D 5 基于未经严格论证的专家意见 注：RCT为随机对照试验 
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表2 针对糖尿病足溃疡评估的SINBAD分级
表2. SINBAD classification for diabetic foot ulcer assessment
类型 定义 评分（分） 部位 前足掌 0 中、后足掌 1 缺血 足部血运正常，至少可触及1处动脉搏动 0 有足部血流减少的临床证据 1 神经病变 保护性感觉存在 0 保护性感觉缺失 1 细菌感染 无 0 有 1 面积 溃疡面积<1 cm 2 0 溃疡面积≥1 cm 2 1 深度 溃疡局限在皮肤和皮下组织 0 溃疡深达肌肉、肌腱或更深 1 注：表格引自文献［ 7］；SINBAD为部位、缺血、神经病变、细菌感染、面积、深度
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表3 针对糖尿病足溃疡患者的WIfI分级各组合的1年内截肢风险等级
表3. The risk grade of amputation within one year for each combination of WIfI classification for patients with diabetic foot ulcers
创面等级 0级缺血 1级缺血 2级缺血 3级缺血 0级足感染 1级足感染 2级足感染 3级足感染 0级足感染 1级足感染 2级足感染 3级足感染 0级足感染 1级足感染 2级足感染 3级足感染 0级足感染 1级足感染 2级足感染 3级足感染 0 极低 极低 低 中等 极低 低 中等 高 低 低 中等 高 低 中等 中等 高 1 极低 极低 低 中等 极低 低 中等 高 低 中等 高 高 中等 中等 高 高 2 低 低 中等 中等 中等 中等 高 高 中等 高 高 高 高 高 高 高 3 中等 中等 高 高 高 高 高 高 高 高 高 高 高 高 高 高 注：WIfI为创面、缺血和足感染
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表4 针对糖尿病足溃疡患者的WIfI分级各组合的血运重建获益等级
表4. The benefit grade of revascularization for each combination of WIfI classification for patients with diabetic foot ulcers
创面等级 0级缺血 1级缺血 2级缺血 3级缺血 0级足感染 1级足感染 2级足感染 3级足感染 0级足感染 1级足感染 2级足感染 3级足感染 0级足感染 1级足感染 2级足感染 3级足感染 0级足感染 1级足感染 2级足感染 3级足感染 0 极低 极低 极低 极低 极低 低 低 中等 低 低 中等 中等 中等 高 高 高 1 极低 极低 极低 极低 低 中等 中等 高 中等 高 高 高 高 高 高 高 2 极低 极低 极低 极低 中等 中等 高 高 高 高 高 高 高 高 高 高 3 极低 极低 极低 极低 中等 中等 中等 高 高 高 高 高 高 高 高 高 注：WIfI为创面、缺血和足感染
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糖尿病足溃疡合并下肢血管病变的外科诊疗全国专家共识（2024版）-附件表格.docx
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