Preliminary investigation on the wound healing effect of three-dimensional bioprinting ink containing human adipose-derived protein complexes
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摘要:
目的 探讨人脂肪来源蛋白复合物（ADPC）对人皮肤成纤维细胞（HSF）和人脐静脉内皮细胞（HUVEC）增殖和迁移能力的影响，以及含ADPC的三维生物打印墨水（Bioink）在裸鼠全层皮肤缺损创面中的修复效应。 方法 采用实验研究方法。收集解放军总医院2020年10月—2021年3月收治的3例行腹部皮瓣转移修补术的女性慢性创面患者（年龄29~34岁）的废弃皮下脂肪组织和同期收治的3名行腹部抽脂术的健康女性（年龄24~36岁）的废弃抽脂术脂肪组织，分别制备正常ADPC（nADPC）及抽脂术ADPC（lADPC）。采用二辛丁酸法测定2种ADPC的蛋白浓度，计算2种ADPC的提取效率，样本数均为3。取对数生长期HSF和HUVEC进行后续实验。取2种细胞均按随机数字表法（分组方法下同）分为磷酸盐缓冲液（PBS）对照组、4 μg/mL nADPC组、20 μg/mL nADPC组、100 μg/mL nADPC组和200 μg/mL nADPC组，每组5孔。PBS对照组细胞采用PBS培养，余4组分别采用含相应终质量浓度的nADPC培养。常规培养24 h，采用细胞计数试剂盒8法检测细胞增殖活力。取HSF和HUVEC，分为PBS对照组、单纯nADPC组、单纯lADPC组、单纯肿瘤坏死因子α（TNF-α）组、TNF-α+nADPC组、TNF-α+lADPC组。PBS对照组与单纯TNF-α组细胞分别加入PBS，单纯nADPC组、单纯lADPC组、TNF-α+nADPC组及TNF-α+lADPC组中分别加入终质量浓度100 μg/mL的nADPC或lADPC，单纯TNF-α组、TNF-α+nADPC组及TNF-α+lADPC组再加入终质量浓度20 ng/mL的TNF-α。进行细胞划痕试验后计算划痕后24 h细胞迁移率（样本数为3），同前检测培养24 h细胞增殖活力（样本数为5）。取明胶-海藻酸钠复合Bioink（Bioink AG），制备含100 μg/mL lADPC的Bioink AG（lADPC-Bioink AG），观察二者在室温下及冷凝后的形态和三维生物打印且交联后的形态，采用流变仪检测流变性能时记录低温成胶时间（样本数为3）。取20只8~10周龄雌性BALB/c-NU裸鼠，建立背部全层皮肤缺损创面模型后，分为常规换药组、单纯lADPC组、单纯Bioink AG组和lADPC-Bioink AG组，每组5只。常规换药组裸鼠创面仅覆盖水胶体敷料和常规换药，其余3组裸鼠创面另予相应lADPC、Bioink AG、lADPC-Bioink AG处理。从治疗0 d起，行大体观察，计算治疗2、6、10 d的创面愈合率。治疗10 d，采用苏木精-伊红染色行创面组织病理学观察。对数据行独立样本t检验、单因素方差分析、重复测量方差分析、SNK-q检验及LSD-t检验。 结果 lADPC的蛋白浓度、提取效率分别为（1.306±0.011）mg/mL、（11.1±1.5）%，明显低于nADPC的（2.039±0.042）mg/mL、（22.2±2.0）%（t=23.83、6.38，P<0.05或P<0.01）。培养24 h，与PBS对照组比较，100 μg/mL nADPC组和200 μg/mL nADPC组HSF（q=6.943、6.375，P<0.01）和HUVEC（q=6.301、6.496，P<0.01）增殖活力均明显下降；与100 μg/mL nADPC组比较，200 μg/mL nADPC组HSF和HUVEC增殖活力无明显变化（P>0.05）。划痕后24 h，与PBS对照组比较，单纯nADPC组、单纯lADPC组、单纯TNF-α组HSF和HUVEC迁移率均明显降低（q=5.642、6.645、11.480，4.772、6.298、10.420，P<0.05或P<0.01）；与单纯nADPC组比较，单纯lADPC组HSF和HUVEC迁移率无明显变化（P>0.05）；与单纯TNF-α组比较，TNF-α+nADPC组、TNF-α+lADPC组HSF迁移率无明显变化（P>0.05），HUVEC迁移率均明显升高（q=8.585、7.253，P<0.01）；与TNF-α+nADPC组比较，TNF-α+lADPC组HSF和HUVEC迁移率无明显变化（P>0.05）。培养24 h，与PBS对照组比较，单纯nADPC组、单纯lADPC组、单纯TNF-α组HSF和HUVEC增殖活力均明显降低（q=5.803、5.371、9.136，11.580、9.493、13.510，P<0.05或P<0.01）；与单纯nADPC组比较，单纯lADPC组HSF和HUVEC增殖活力均无明显变化（P>0.05）；与单纯TNF-α组比较，TNF-α+nADPC组、TNF-α+lADPC组HSF（q=14.990、10.850，P<0.01）和HUVEC（q=7.066、8.942，P<0.01）增殖活力均明显升高；与TNF-α+nADPC组比较，TNF-α+lADPC组HSF和HUVEC增殖活力均无明显变化（P>0.05）。室温及冷凝状态下，lADPC-Bioink AG比Bioink AG外观稍显浑浊；三维生物打印且交联后lADPC-Bioink AG与Bioink AG形态类似。在10 ℃时，lADPC-Bioink AG的凝固时间为（76.6±0.4）s，明显慢于Bioink AG的（74.4±0.6）s（t=4.64，P<0.01）。治疗2 d，常规换药组裸鼠创面渗出较多，其余3组无明显渗出；治疗8 d，lADPC-Bioink AG组裸鼠残余创面面积最小，有明显上皮覆盖。治疗2 d，lADPC-Bioink AG组裸鼠创面愈合率明显高于单纯lADPC组（t=3.59，P<0.05），与常规换药组、单纯Bioink AG组相近（P>0.05）；治疗6 d，lADPC-Bioink AG组裸鼠创面愈合率明显高于常规换药组、单纯lADPC组、单纯Bioink AG组（t=18.70、15.70、3.15，P<0.05或P<0.01）；治疗10 d，lADPC-Bioink AG组裸鼠创面愈合率明显高于常规换药组、单纯lADPC组（t=12.51、4.84，P<0.01），与单纯Bioink AG组相近（P>0.05）。治疗10 d，lADPC-Bioink AG组裸鼠创面组织血管化程度适中，上皮化充分，愈合效果最好。 结论 抽脂术相关操作会降低ADPC蛋白浓度、提取效率等表征，但lADPC与nADPC具有相同的生物学作用，可在非炎症环境中抑制HSF与HUVEC的增殖与迁移能力，在炎症环境中提高HSF与HUVEC的增殖活力，同时提高HUVEC的迁移能力；将lADPC加入Bioink AG中后，不会明显影响Bioink AG的物理性质及打印性能，并可以提升裸鼠全层皮肤缺损创面的修复效果。 Abstract:Objective To investigate the effects of human adipose-derived protein complex (ADPC) on the proliferation and migration ability of human skin fibroblasts (HSFs) and human umbilical vein endothelial cells (HUVECs), and the repairing effects of ADPC-containing three-dimensional (3D) bioprinting ink (Bioink) in full-thickness skin defect wounds of nude mice. Methods The experimental research method was used. Discarded subcutaneous adipose tissue from 3 female patients with chronic wounds (aged 29-34 years) admitted to PLA General Hospital for abdominal flap transfer from October 2020 to March 2021 and discarded liposuction adipose tissue from 3 healthy female (aged 24-36 years) for abdominal liposuction during the same period were collected to prepare normal ADPC (nADPC) and liposuction-derived ADPC (lADPC), respectively. The protein concentration of the two kinds of ADPC was measured by bicinchoninic acid method, and the extraction efficiency of them was calculated. The sample numbers were 3. HSFs and HUVECs in logarithmic growth phase were taken for the subsequent experiments. HSFs and HUVECs were divided into phosphate buffered saline (PBS) control group, 4 μg/mL nADPC group, 20 μg/mL nADPC group, 100 μg/mL nADPC group, and 200 μg/mL nADPC group according to the random number table (the same grouping method below), with 5 wells in each group. Cells in PBS control group were cultured with PBS, and the cells in the 4 remaining groups were cultured with the corresponding final mass concentration of nADPC. After 24 h of conventional culture, the cell proliferation viability was detected by cell counting kit 8 method. HSFs and HUVECs were taken and divided into PBS control group, nADPC alone group, lADPC alone group, tumor necrosis factor-α (TNF-α) alone group, TNF-α+nADPC group, and TNF-α+lADPC group. Cells in PBS control group and TNF-α alone group were added with PBS. nADPC or lADPC was added to the cells in nADPC alone group, lADPC alone group, TNF-α+nADPC group, and TNF-α+lADPC group with a final mass concentration of 100 μg/mL, respectively. TNF-α with a final mass concentration of 20 ng/mL was added to the cells in TNF-α alone group, TNF-α+nADPC group, and TNF-α+lADPC group. The cell migration rate was calculated after the scratch test at 24 h after scratching (n=3), and the cell proliferation viability was detected after 24 h of culture as above (n=5). Gelatin-alginate composite Bioink (Bioink AG) was taken. Bioink AG containing 100 μg/mL lADPC (lADPC-Bioink AG) was prepared. The morphology of the two at room temperature and after condensation was observed. The morphology after 3D bioprinting and cross-linking was observed. The low-temperature gel formation time was recorded when detecting rheological properties using rheometer (n=3). Twenty BALB/c-NU female nude mice of 8-10 weeks old were taken to establish the full-thickness skin defect wounds on the back, and then they were divided into routine dressing change group, lADPC alone group, Bioink AG alone group, and lADPC-Bioink AG group, with 5 nude mice in each group. The wounds of nude mice in routine dressing change group were covered with hydrocolloid dressings and performed with routine dressing changes only, while the wounds of nude mice in the remaining 3 groups were treated with lADPC, Bioink AG, and lADPC-Bioink AG accordingly in addition. General observation was performed from treatment day (TD) 0, and the wound healing rate was calculated on TD 2, 6, and 10. On TD 10, histopathological observation of wounds was performed with hematoxylin eosin staining. Data were statistically analyzed with independent samples t test, one-way analysis of variance, analysis of variance for repeated measurement, Student-Newman-Keuls q test, and least significant difference t test. Results The protein concentration and extraction efficiency of lADPC were respectively (1.306±0.011) mg/mL and (11.1±1.5)%, which were significantly lower than (2.039±0.042) mg/mL and (22.2±2.0)% of nADPC (t=23.83, 6.38, P<0.05 or P<0.01). After 24 h of culture, compared with those in PBS control group, the proliferation viabilities of HSFs (q=6.943, 6.375, P<0.01) and HUVECs (q=6.301, 6.496, P<0.01) were significantly decreased in 100 μg/mL nADPC group and 200 μg/mL nADPC group; compared with those in 100 μg/mL nADPC group, the proliferation viabilities of HSFs and HUVECs in 200 μg/mL nADPC group did not change significantly (P>0.05). At 24 h after scratching, compared with those in PBS control group, the HSF and HUVEC migration rates were significantly lower in nADPC alone group, lADPC alone group, and TNF-α alone group (q=5.642, 6.645, 11.480, 4.772, 6.298, 10.420, P<0.05 or P<0.01); compared with those in nADPC alone group, there were no significant changes in the HSF and HUVEC migration rates in lADPC alone group (P>0.05); compared with those in TNF-α alone group, there were no significant changes in the HSF migration rates in TNF-α+nADPC group or TNF-α+lADPC group (P>0.05), the HUVEC migration rates were significantly higher in TNF-α+nADPC group and TNF-α+lADPC group (q=8.585, 7.253, P<0.01); compared with those in TNF-α+nADPC group, there were no significant changes in the HSF and HUVEC migration rates in TNF-α+lADPC group (P>0.05). After 24 h of culture, compared with those in PBS control group, the HSF and HUVEC proliferation viabilities were significantly lower in nADPC alone group, lADPC alone group, and TNF-α alone group (q=5.803, 5.371, 9.136, 11.580, 9.493, 13.510, P<0.05 or P<0.01); compared with those in nADPC alone group, the HSF and HUVEC proliferation viabilities in lADPC alone group did not change significantly (P>0.05); compared with those in TNF-α alone group, the HSF (q=14.990, 10.850, P<0.01) and HUVEC (q=7.066, 8.942, P<0.01) proliferation viabilities were significantly higher in TNF-α+nADPC group and TNF-α+lADPC group; compared with those in TNF-α+nADPC group, the HSF and HUVEC proliferation viabilities in TNF-α+lADPC group did not change significantly (P>0.05). At room temperature and in the condensed state, lADPC-Bioink AG had a more slightly turbid appearance than Bioink AG. lADPC-Bioink AG had a similar morphology to Bioink AG after 3D bioprinting and cross-linking. At 10 ℃, the coagulation time of lADPC-Bioink AG was (76.6±0.4) s, which was significantly slower than (74.4±0.6) s of Bioink AG (t=4.64, P<0.01). On TD 2, the nude mice in routine dressing change group had more wound exudation, while the nude mice in the remaining 3 groups had no significant exudation. On TD 8, the nude mice in lADPC-Bioink AG group had the smallest residual wound area and obvious epithelial coverage. On TD 2, the wound healing rate of nude mice in lADPC-Bioink AG group was significantly higher than that in lADPC alone group (t=3.59, P<0.05) and similar to the rates in routine dressing change group and Bioink AG alone group (P>0.05). On TD 6, the wound healing rate of nude mice in lADPC-Bioink AG group was significantly higher than the rates in routine dressing change group, lADPC alone group, and Bioink AG alone group (t=18.70, 15.70, 3.15, P<0.05 or P<0.01). On TD 10, the wound healing rate of nude mice in lADPC-Bioink AG group was significantly higher than the rates in routine dressing change group and lADPC alone group (t=12.51, 4.84, P<0.01) but similar to that in Bioink AG alone group (P>0.05). On TD 10, the wounds of nude mice in lADPC-Bioink AG group had moderate vascularization of the traumatic tissue, adequate epithelialization, and the best healing effect. Conclusions Liposuction-related operations have little effect on the characterization of ADPC protein concentration and extraction efficiency. LADPC and nADPC have the same biological effects, which can inhibit the proliferation and migration ability of HSFs and HUVECs in non-inflammatory environment and improve the proliferation viabilities of HSFs and HUVECs in inflammatory environment, while improving the migration ability of HUVECs. Adding lADPC to Bioink AG does not significantly affect the physical properties or printing performance of Bioink AG, but can enhance the wound repair effect of full-thickness skin defect wounds in nude mice. -
(1)应用足部相应位置的微型皮瓣游离移植修复手指Ⅳ度电烧伤创面，组织结构相似度高，功能相近，患指愈后外观自然。
(2)足部作为皮瓣供区，位置相对隐蔽，可供切取皮瓣面积相对较小，愈后无功能障碍。
(3)证实将足部游离微型皮瓣携带的感觉神经与患指损伤处的近端神经吻合之后，手指感觉恢复接近正常。
手指Ⅳ度电烧伤创面深达深筋膜以下，往往伴有肌腱和骨外露等，修复困难，通常需要应用皮瓣进行修复。随着显微外科技术及皮瓣解剖技术的不断发展，人们对创面修复有了更高的要求，更加注重功能的重建和外观的修复 ［ 1］ 。因此如何选择合适的皮瓣修复手指Ⅳ度电烧伤创面仍值得探讨。本研究中应用足部微型皮瓣游离移植Ⅰ期修复手指Ⅳ度电烧伤创面，取得了较好的临床效果。
1. 对象与方法
本回顾性观察性研究符合《赫尔辛基宣言》的基本原则。根据郑州市第一人民医院（以下简称本院）伦理委员会的相关规定，可以在不泄露患者信息的情况下对其有关临床资料进行分析研究。
1.1 入选标准
纳入标准：（1）性别、年龄不限；（2）确诊为手指Ⅳ度电烧伤但不伴指坏死者；（3）采用足部游离微型皮瓣修复者。排除标准：（1）术中或随访资料不完整者；（2）随访时间不足10个月者。
1.2 临床资料
2017年7月—2022年2月，本院收治20例符合入选标准的手指Ⅳ度电烧伤患者，其中男19例、女1例，年龄18~64岁，致伤电压为380 V~10 kV，均为单指受累。共20处创面，其中15处创面位于掌侧，包括拇指8处、示指5处、中指2处；5处创面位于背侧，包括示指1处、中指4处；原始创面的面积为3.0 cm×2.0 cm~6.0 cm×2.5 cm；其中肌腱、血管和/或神经外露创面15处，骨或关节外露创面5处。手术时间为伤后7~20 d。
1.3 手术方法
1.3.1 受区创面床准备
根据患者意愿，选择全身麻醉或者臂丛神经阻滞+腰硬联合麻醉。根据烧伤部位，对指掌侧创面清创后，探查指掌侧固有神经、血管及指深屈肌腱的损伤程度，保留轻度变性的肌腱，对缺损的指深屈肌腱应用患指近端指浅屈肌腱或掌长肌腱重建。解剖损伤严重的指固有血管至正常喷血，或者解剖至指总动脉处备用，解剖近端指掌侧固有神经至组织正常处备用。对指背侧创面清创后，探查伸指肌腱的损伤程度及关节完好程度，彻底清除坏死组织，保留轻度变性的肌腱和关节周围腱性组织，应用掌长肌腱修复患指伸指肌腱缺损部分。在鼻烟窝处解剖桡动脉末端及头静脉分支备用。最后测量创面面积，应用蓝布拓模，用以设计切取皮瓣。本组患者清创后创面面积为4.5 cm×2.0 cm~7.0 cm×3.0 cm。
1.3.2 皮瓣设计与切取
根据手指创面位置，遵循组织结构相似性原则，采用足底内侧皮瓣修复10处创面、采用拇趾腓侧皮瓣修复5处创面、采用足背动脉皮瓣修复5处创面。应用拓模分别在足底内侧、拇趾腓侧、足背设计皮瓣，切口边缘均在拓模外侧0.5 cm左右。所有皮瓣设计完成后，在血管蒂处设计三角瓣，用以覆盖吻合处血管，减轻吻合口处压力，避免卡压血管吻合口。
1.3.2 .1 足底内侧皮瓣
沿设计线切开皮瓣内外侧缘，在深筋膜深层锐性向皮瓣中心分离，注意保护进入皮瓣的血管穿支。在拇展肌与屈趾肌间隙内探寻足底内侧动脉浅支主干，由远及近游离皮瓣。在皮瓣近端沿胫后动脉走行向内踝下方做“Z”字形切口，于拇展肌起点以远1 cm处切断该肌，在拇展肌深面分离足底内侧动静脉及足底内侧神经，切断并结扎远端血管，并切取足够长度的血管、神经蒂，游离皮瓣准备移植。本组患者足底内侧皮瓣切取面积为7.0 cm×3.0 cm~8.0 cm×3.5 cm。
1.3.2 .2 拇趾腓侧皮瓣
以健侧拇趾趾掌侧固有动脉为皮瓣轴线，首先沿设计线切开皮瓣内侧缘，并沿拇趾腓侧向足背做“Z”字形切口，切开皮肤，在皮下筋膜层寻找合适的包含在皮瓣内的浅静脉。然后切开皮瓣外缘皮肤，向内侧分离至血管蒂处，分离出足够长度的拇趾趾掌侧固有动脉及其伴行神经，必要时在第1和第2跖骨间探查拇趾腓侧血管的来源。最后，切开皮瓣远端，在第1趾蹼处解剖并结扎通向第2趾的分支，保留足够长度的血管、神经，游离皮瓣准备移植。本组患者拇趾腓侧皮瓣切取面积为5.0 cm×2.5 cm~5.5 cm×3.0 cm。
1.3.2 .3 足背动脉皮瓣
在健侧足背按足背动脉走行对称设计皮瓣，切取平面为深筋膜深面。首先，切开皮瓣近侧缘皮肤，在浅筋膜层分离穿入皮瓣的大隐静脉，于皮瓣外侧在深筋膜层面锐性向皮瓣中心分离，探查足背动脉及其伴行静脉，同时注意保护趾长伸肌腱腱膜。然后，切断并结扎与足底深支动静脉血管交通支，由远及近分离皮瓣。最后，在留取合适长度的足背动静脉后，于血管近端离断血管蒂，游离皮瓣准备移植。本组患者足背动脉皮瓣切取面积为7.0 cm×3.5 cm~7.5 cm×3.5 cm。
1.3.3 皮瓣移植和供区处理
本组患者的皮瓣切取面积为5.0 cm×2.5 cm~8.0 cm×3.5 cm。将上述皮瓣断蒂后分别移植于相应手指受区。采用足底内侧皮瓣或者拇趾腓侧皮瓣修复指掌侧创面时，将皮瓣血管蒂动脉与患手指掌侧固有动脉或指掌侧总动脉近端端端吻合，将血管蒂静脉与患手指背静脉端端吻合，将皮瓣携带的神经与指掌侧固有神经近端外膜端端吻合，不桥接缺损的血管、神经。本组患者共15处创面与皮瓣行神经吻合。采用足背动脉皮瓣修复指背侧创面时，将皮瓣血管蒂动脉与患侧手背鼻烟窝处桡动脉末端端端吻合，血管蒂静脉与患侧手背鼻烟窝处头静脉分支端端吻合。最后，检查皮瓣确定血运良好后，将皮瓣与创缘间断缝合，采用无菌敷料包扎术区并留观察窗。皮瓣供区创面移植大腿中厚皮覆盖并加压固定。
1.4 术后处理
嘱患者绝对卧床10~14 d，将患肢抬高。采用红外线烤灯照射保温，观察皮瓣血运情况，常规行抗感染、抗痉挛、抗凝治疗。术后12~14 d视创面愈合情况拆线，并开始功能锻炼；行肌腱、神经修复者采用高分子夹板外固定3~4周后再开始功能锻炼。
1.5 观测指标
术后观察皮瓣和皮片成活情况。随访观察皮瓣外观以及患指末梢温度、颜色。于末次随访时，采用中华医学会手外科学会上肢部分功能评定试用标准 ［ 2］ 评估患指关节功能及皮瓣感觉恢复情况，测量吻合神经皮瓣处皮肤两点辨别觉距离；采用张浩等 ［ 3］ 制订的疗效满意度评分表（总分5~10分为非常满意、0~4分为一般满意、-5~-1分为不满意）从创面愈合、皮瓣形态、皮瓣感觉、皮瓣温度及日常功能方面调查患者的疗效满意度，并计算非常满意率；采用综合评价量表（优：皮瓣存活好，指功能恢复正常，感觉基本正常；良：皮瓣存活好，指功能基本恢复，感觉略有异常；中：皮瓣存活，指功能尚可，感觉未明显恢复；差：皮瓣未成活）评价皮瓣修复效果，并计算优良率。
2. 结果
2.1 总体情况
本组患者术后皮瓣和皮片均成活。术后随访10~18个月，皮瓣外观自然、不臃肿，存活良好，患指末梢温度、颜色与正常手指皮肤基本一致。末次随访时，患指关节功能恢复情况：11个患指关节活动度在正常范围，6个患指总主动活动度恢复到健侧的85%，3个患指总主动活动度恢复到健侧的75%。皮瓣感觉恢复情况：吻合神经的15个皮瓣感觉均恢复到S3 +级，皮瓣处皮肤两点辨别觉距离为7.0~9.0 mm（平均8.2 mm）；未吻合神经的有1个皮瓣感觉恢复到S2级，4个皮瓣感觉恢复到S1级。20例患者的疗效满意度：非常满意者16例、一般满意者4例，非常满意率为80%；20个皮瓣修复效果：优16个、良2个、中2个，优良率为90%。
2.2 典型病例
例1
男，47岁，工作时左手示指接触1 kV高压电，导致指掌侧Ⅳ度电烧伤。伤后先在当地医院行保守治疗，伤后10 d转入本院。入院时体格检查见创面位于左手示指掌侧偏桡侧，面积约3.0 cm×2.0 cm，基底可见黑色腐肉样组织。伤后14 d，手术清除创面内坏死组织，可见示指桡侧固有血管和神经、指深屈肌腱缺损，近指间关节少许开放，中节指骨部分外露，清创后创面面积为4.5 cm×2.0 cm。应用患侧掌长肌腱修复指深屈肌腱缺损，采用面积为5.0 cm×2.5 cm健侧拇趾腓侧皮瓣携带拇趾背侧皮神经移植于左手示指掌侧创面，同前行血管、神经吻合。皮瓣供区移植大腿中厚皮覆盖。术后14 d拆线，术后4周皮瓣及皮片均存活良好。术后12个月末次随访时，皮瓣存活良好、外观自然、不臃肿，患指屈伸功能基本正常，皮瓣感觉达S3 +级，皮瓣处皮肤两点辨别觉距离为7.4 mm，患者对疗效表示非常满意，皮瓣修复效果为优。见 图1。
例2
男，22岁，工作时右手拇指接触1 kV高压电，导致指掌侧Ⅳ度电烧伤。伤后先在当地医院行保守治疗，伤后17 d转入本院。入院时体格检查见创面位于右手拇指掌侧，面积约6.0 cm×2.5 cm，基底可见黑色腐肉样组织。伤后20 d，手术清除创面内坏死组织，可见拇指指间关节开放，清创后创面面积为7.0 cm×3.0 cm。在健侧足部设计面积为8.0 cm×3.5 cm的足底内侧皮瓣，移植于右手拇指掌侧创面，同前行血管、神经吻合。皮瓣供区移植大腿中厚皮覆盖。术后14 d拆线，术后4周皮瓣及皮片均存活良好。术后14个月末次随访时，皮瓣外观自然、不臃肿，存活良好，患指屈伸功能基本正常，皮瓣感觉达S3 +级，皮瓣处皮肤两点辨别觉距离为8.2 mm，患者对疗效表示非常满意，皮瓣修复效果为优。见 图2。
3. 讨论
在手外伤中，手指电烧伤是比较严重的一种创伤。电烧伤对组织的损伤具有选择性，电阻较低的血管、神经、肌肉等组织损伤重，因此电烧伤常常伴有组织缺损、肌腱骨质外露，创面边界不清 ［ 4, 5］ ，创面基底受皮能力差，修复难度较大，严重时可能发生指坏死而导致截指，造成手部残疾。
对于手指的小面积电烧伤创面，黄书润等 ［ 6］ 尝试应用人工真皮+VSD联合自体皮移植修复，手术操作简单，术后手指外形好。但由于人工真皮移植对创面基底和创面面积要求较高，容易并发组织继发性坏死或感染等，该方法应用时受到基底血运的限制，且效果不如薄皮瓣好。也有学者应用传统或者改良的腹部皮瓣移植修复手指的电烧伤创面取得了较好的效果 ［ 6, 7, 8, 9］ 。但该方法需要制动上肢3周，术后患肢关节僵硬、皮瓣臃肿，需要多次手术修薄，且患者住院时间长 ［ 10］ 。还有报道应用指动脉逆行或顺行岛状皮瓣、掌背动脉皮瓣 ［ 11, 12］ 、邻指皮瓣 ［ 13］ 、鱼际皮瓣、V-Y推进皮瓣 ［ 14, 15］ 等修复手指电烧伤或外伤导致的皮肤缺损，修复后外形美观，但是局部损伤较大，皮瓣面积有限，术后也可能因瘢痕挛缩、关节僵直、创伤性关节炎等问题而导致各手指掌指关节、近指间关节和远指间关节的屈曲失能、背伸失能及关节强直，遗留手部功能障碍 ［ 16, 17］ 。
随着显微外科技术的不断普及，穿支皮瓣更加受到推崇，皮瓣切取面积越来越小，皮瓣供区也更加丰富，采用游离皮瓣修复手指电烧伤创面成为最有效和可靠的方法 ［ 18］ 。修复水平的提高促使修复方式向功能修复和外观修复发展。更多的游离微型皮瓣被应用到手指电烧伤创面修复中，如游离腕部或前臂穿支皮瓣 ［ 19, 20］ 、游离股前外侧穿支皮瓣 ［ 21］ 、动脉化血流桥接静脉皮瓣 ［ 22, 23］ 等。该类皮瓣的优点是皮瓣营养血管与受区血管口径匹配，同时由于皮瓣面积小，循环建立快，供区创面多可直接缝合。但手指结构复杂，指掌侧皮肤偏厚且皮纹深，皮下脂肪致密，防滑且耐磨、神经末梢丰富；而指背皮肤相对较薄，皮下组织少，皮肤褶皱丰富，弹性及延展性好，有利于手指的屈曲。所以应根据手指不同的部位选择合适的皮瓣进行修复，才能更好地恢复手指的功能及外观。
本研究遵从相似部位且具有相似功能的组织具有相似结构的原则 ［ 24］ ，应用足部微型皮瓣游离移植修复手指Ⅳ度电烧伤创面。修复时，针对指掌侧创面，应用足底内侧皮瓣或拇趾腓侧皮瓣进行修复，皮瓣组织与受区组织结构近似，同时吻合动静脉血管及神经，皮瓣血运有保障，感觉恢复快，能够达到功能修复的目的。而针对指背侧创面，选择应用足背动脉皮瓣修复，供区皮瓣组织与指背皮肤组织相似，皮下组织少、伸展性好，更加有利于手功能恢复，尽可能避免术后皮瓣修薄处理。本研究共纳入20处手指创面，均应用足部游离微型皮瓣来修复，大部分皮瓣达到较为满意的修复效果。吻合神经的皮瓣感觉恢复较快，术后12个月感觉即可达S3 +级；未吻合神经的皮瓣感觉恢复慢，术后18个月仅达S1、S2级。因此选择合适的皮瓣同时吻合神经对提高后期修复效果有不可忽视的作用。
手部功能是由5个手指协调作用的结果。根据手部功能评价标准将其功能系数分配为拇指40%，示指及中指各20%，环指及小指各10% ［ 2］ 。本研究选择拇指、示指及中指单一创面的修复进行讨论，是因为拇指、示指及中指的功能占全手功能的80%，这3个手指的修复对手功能的恢复意义更大。本组病例应用上述方案修复后，手指功能恢复至伤前或健侧手指功能的75%及以上，大部分患者对修复效果非常满意。
为了尽可能提高修复效果，本课题组认为有2点需要特别注意：（1）术中创面清创后，应首先探查受区血管射血情况，一般修复指掌侧创面需要在创面近端的指总血管处吻合，而修复指背侧创面则在鼻烟窝以远探查桡动脉终末支进行吻合，血流量方能满足移植皮瓣需要；（2）在皮瓣断蒂前要充分测量需要的血管蒂长度，避免皮瓣转移时血管蒂长度不足。
本研究表明，采用足部微型皮瓣游离移植修复手指Ⅳ度电烧伤创面具有较好的疗效，其优点在于：足部作为皮瓣供区位置隐蔽，皮瓣组织结构和功能与手指相似度高；术后皮瓣感染率、坏死率低；可切取多种类型皮瓣，可满足不同面积和形状的创面修复需求；术后患指功能恢复良好，疗效满意度高。
尽管足部微型皮瓣游离移植在手指Ⅳ度电烧伤创面修复中表现出较好的疗效，但仍存在一些挑战和限制。首先，该术式对操作者的技术水平要求较高，需要操作者具备丰富显微外科经验。手术过程中须精确吻合血管，以保证皮瓣的存活和创面的有效修复；其次，术后抗感染、抗凝等治疗同样重要，需严格遵守医嘱，进行系统治疗；再者，由于电烧伤创面的特殊性，可能存在深部组织损伤和神经损伤，因此术后功能恢复可能需要较长时间；最后，在临床实践中，应根据患者的具体情况选择最合适的治疗方法。因此，未来的研究应进一步探讨足部游离微型皮瓣在手指电烧伤创面修复中的应用范围和限制。同时，应研究更先进的技术和治疗方法，以提高皮瓣的成活率和功能恢复效果。此外，应加强临床护理和康复治疗，促进患者的手指功能恢复，改善患者生活质量。
综上所述，采用足部微型皮瓣游离移植修复手指Ⅳ度电烧伤创面是一种有效的治疗方法，值得临床推广。但仍需在临床实践中不断探索和改进，以进一步提高治疗效果，为患者提供更好的治疗体验和生活质量。
所有作者均声明不存在利益冲突与细菌相关的创面感染及抗生素耐药给患者和医疗保健体系带来了沉重的负担。因此，迫切需要开发一种既能有效预防创面感染，又能促进创面愈合且无抗生素的多功能创面敷料来解决这一难题。该研究基于细菌纤维素(BC)、明胶和硒纳米颗粒(SeNP)等材料，构建了一系列具有显著抗菌、抗氧化和抗炎能力的多功能纳米复合水凝胶用于修复创面。该BC/明胶/SeNP纳米复合水凝胶具有优越的机械性能，良好的溶胀性、柔韧性、生物可降解性及生物相容性，并且可以缓慢、持久地释放SeNP。SeNP的加入赋予了该水凝胶优越的抗氧化和抗炎能力，并对常见的细菌(大肠埃希菌和金黄色葡萄球菌)及多重耐药菌具有优异的抗菌活性。此外，BC/明胶/SeNP纳米复合水凝胶在大鼠全层皮肤缺损模型中表现出较好的促进创面愈合能力，创面具体表现为明显的炎症减轻、创面闭合、肉芽组织形成、胶原沉积、血管生成以及Fb的活化和分化。该研究表明，该多功能BC/明胶/SeNP纳米复合水凝胶作为创面敷料在预防创面感染和促进皮肤再生方面具有巨大的临床应用前景。张清荣，编译自《Adv Healthcare Mater》，2021，10(14):e2100402；于云龙，审校 -
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1 划痕试验观察6组人皮肤成纤维细胞划痕后各时间点迁移情况 倒置荧光显微镜×50，图中标尺为500 μm。1A、1B、1C、1D、1E、1F.分别为磷酸盐缓冲液（PBS）对照组、单纯正常脂肪来源蛋白复合物（nADPC）组、单纯抽脂术脂肪来源蛋白复合物（lADPC）组、单纯肿瘤坏死因子α（TNF-α）组、TNF-α+nADPC组、TNF-α+lADPC组划痕后即刻；1G、1H、1I、1J、1K、1L.分别为PBS对照组、单纯nADPC组、单纯lADPC组、单纯TNF-α组、TNF-α+nADPC组、TNF-α+lADPC组划痕后24 h，图1G剩余划痕面积最小，图1H、1I剩余划痕面积相近，图1J、1K、1L剩余划痕面积相近且均大于图1H、1I
2 划痕试验观察6组人脐静脉内皮细胞划痕后各时间点迁移情况 倒置荧光显微镜×50，图中标尺为500 μm。2A、2B、2C、2D、2E、2F.分别为磷酸盐缓冲液（PBS）对照组、单纯正常脂肪来源蛋白复合物（nADPC）组、单纯抽脂术脂肪来源蛋白复合物（lADPC）组、单纯肿瘤坏死因子α（TNF-α）组、TNF-α+nADPC组、TNF-α+lADPC组划痕后即刻；2G、2 H、2I、2J、2K、2L.分别为PBS对照组、单纯nADPC组、单纯lADPC组、单纯TNF-α组、TNF-α+nADPC组、TNF-α+lADPC组划痕后24 h，图2G剩余划痕面积最小，图2H、2I剩余划痕面积相近，图2J剩余划痕面积最大，图2K、2L细胞剩余划痕面积相近
3 明胶-海藻酸钠复合三维生物打印墨水（Bioink AG）与含抽脂术脂肪来源蛋白复合物的Bioink AG（lADPC-Bioink AG）室温、冷凝下、三维生物打印且交联后形态。3A、3B.分别为Bioink AG与lADPC-Bioink AG室温下形态，图3B较图3A浑浊；3C、3D.分别为Bioink AG与lADPC-Bioink AG冷凝下形态，图3D较图3C浑浊；3E、3F.分别为Bioink AG与lADPC-Bioink AG三维生物打印且交联后形态，二者外观无明显区别
4 4组全层皮肤缺损裸鼠治疗各时间点创面大体情况。4A.常规换药组治疗2 d可见大量淡黄色渗出物覆盖创面；4B.单纯抽脂术脂肪来源蛋白复合物（lADPC）组治疗2 d创面湿润，无明显渗出；4C.单纯明胶-海藻酸钠复合三维生物打印墨水（Bioink AG）组治疗2 d创面打印组织有降解，无明显渗出；4D.lADPC-Bioink AG组治疗2 d打印组织覆盖创面，有降解，创面无明显渗出；4E.常规换药组治疗8 d创面干燥结痂，面积较图4A缩小，创面基底呈淡红色，无明显上皮覆盖；4F.单纯lADPC组治疗8 d创面面积较图4E缩小，创面基底呈鲜红色，无明显上皮覆盖；4G.单纯Bioink AG组治疗8 d创面面积较图4F缩小，创面基底呈淡粉色，有明显上皮覆盖；4H.lADPC-Bioink AG组治疗8 d创面面积最小，创面基底呈淡红色，有明显上皮覆盖
5 4组全层皮肤缺损裸鼠治疗10 d组织形态 苏木精-伊红，图中标尺为1 mm。5A.常规换药组创面表面有痂覆盖，痂下见较多新生血管和炎症细胞浸润；5B.单纯抽脂术脂肪来源蛋白复合物（lADPC）组创面可见少量上皮细胞增殖，创面基底中有较多新生血管和炎症细胞浸润；5C.单纯明胶-海藻酸钠复合三维生物打印墨水（Bioink AG）组创面可见明显上皮细胞增殖，但创面基底中的新生血管较少；5D.lADPC-Bioink AG组创面可见明显上皮细胞增殖，创面基底新生血管较图5C多，较图5A、5B少
表1 5组HSF和HUVEC常规培养24 h细胞增殖活力比较（
组别 样本数 HSF HUVEC PBS对照组 5 1.013±0.034 0.821±0.027 4 μg/mL nADPC组 5 1.109±0.065 0.822±0.083 20 μg/mL nADPC组 5 0.886±0.019 0.648±0.101 100 μg/mL nADPC组 5 0.792±0.064 0.516±0.049 200 μg/mL nADPC组 5 0.810±0.017 0.506±0.063 F值 18.26 10.32 P值 <0.01 <0.01 q1值 3.015 0.030 P1值 >0.05 >0.05 q2值 4.007 3.560 P2值 >0.05 >0.05 q3值 6.943 6.301 P3值 <0.01 <0.01 q4值 6.375 6.496 P4值 <0.01 <0.01 q5值 0.568 0.195 P5值 >0.05 >0.05 注：HSF为人皮肤成纤维细胞，HUVEC为人脐静脉内皮细胞，PBS为磷酸盐缓冲液，nADPC为正常脂肪来源蛋白复合物；F值、P值为2种细胞5组间总体比较所得；q1值、P1值，q2值、P2值，q3值、P3值，q4值、P4值分别为PBS对照组与4 μg/mL nADPC组、20 μg/mL nADPC组、100 μg/mL nADPC组、200 μg/mL nADPC组比较所得；q5值、P5值为100 μg/mL nADPC组与200 μg/mL nADPC组比较所得 
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表2 6组HSF和HUVEC划痕后24 h细胞迁移率比较（%，
组别 样本数 HSF HUVEC PBS对照组 3 68.1±2.7 56.8±3.3 单纯nADPC组 3 55.3±2.4 44.3±4.6 单纯lADPC组 3 53.0±5.5 40.3±2.8 单纯TNF-α组 3 42.0±2.6 29.6±5.1 TNF-α+nADPC组 3 46.0±2.7 52.0±2.9 TNF-α+lADPC组 3 47.6±2.2 48.5±2.5 F值 16.48 13.48 P值 <0.01 <0.01 q1值 5.642 4.772 P1值 <0.05 <0.05 q2值 6.645 6.298 P2值 <0.01 <0.01 q3值 11.480 10.420 P3值 <0.01 <0.01 q4值 1.003 1.526 P4值 >0.05 >0.05 q5值 1.760 8.585 P5值 >0.05 <0.01 q6值 2.486 7.253 P6值 >0.05 <0.01 q7值 0.726 1.331 P7值 >0.05 >0.05 注：HSF为人皮肤成纤维细胞，HUVEC为人脐静脉内皮细胞，PBS为磷酸盐缓冲液，nADPC为正常脂肪来源蛋白复合物，lADPC为抽脂术脂肪来源蛋白复合物，TNF-α为肿瘤坏死因子α；F值、P值为2种细胞6组间总体比较所得；q1值、P1值，q2值、P2值，q3值、P3值分别为PBS对照组与单纯nADPC组、单纯lADPC组、单纯TNF-α组比较所得；q4值、P4值为单纯nADPC组与单纯lADPC组比较所得；q5值、P5值，q6值、P6值分别为单纯TNF-α组与TNF-α+nADPC组、TNF-α+lADPC组比较所得；q7值、P7值为TNF-α+nADPC组与TNF-α+lADPC组比较所得
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表3 6组HSF和HUVEC常规培养24 h细胞增殖活力比较（
组别 样本数 HSF HUVEC PBS对照组 5 1.01±0.03 0.821±0.027 单纯nADPC组 5 0.79±0.06 0.516±0.049 单纯lADPC组 5 0.81±0.04 0.571±0.028 单纯TNF-α组 5 0.66±0.06 0.465±0.046 TNF-α+nADPC组 5 1.24±0.05 0.651±0.031 TNF-α+lADPC组 5 1.08±0.08 0.700±0.037 F值 31.24 24.53 P值 <0.01 <0.01 q1值 5.803 11.580 P1值 <0.05 <0.01 q2值 5.371 9.493 P2值 <0.05 <0.01 q3值 9.136 13.510 P3值 <0.01 <0.01 q4值 0.432 2.088 P4值 >0.05 >0.05 q5值 14.990 7.066 P5值 <0.01 <0.01 q6值 10.850 8.942 P6值 <0.01 <0.01 q7值 4.138 1.876 P7值 >0.05 >0.05 注：HSF为人皮肤成纤维细胞，HUVEC为人脐静脉内皮细胞，PBS为磷酸盐缓冲液，nADPC为正常脂肪来源蛋白复合物，lADPC为抽脂术脂肪来源蛋白复合物，TNF-α为肿瘤坏死因子α；F值、P值为2种细胞6组间总体比较所得；q1值、P1值，q2值、P2值，q3值、P3值分别为PBS对照组与单纯nADPC组、单纯lADPC组、单纯TNF-α组比较所得；q4值、P4值为单纯nADPC组与单纯lADPC组比较所得；q5值、P5值，q6值、P6值分别为单纯TNF-α组与TNF-α+nADPC组、TNF-α+lADPC组比较所得；q7值、P7值为TNF-α+nADPC组与TNF-α+lADPC组比较所得
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表4 4组全层皮肤缺损裸鼠治疗各时间点创面愈合率比较（%，
组别 鼠数（只） 2 d 6 d 10 d 常规换药组 3 11.4±2.0 45.2±2.2 77.3±1.9 单纯lADPC组 3 4.2±1.6 51.0±1.9 90.5±1.6 单纯Bioink AG组 3 7.4±0.7 75.5±2.1 98.1±2.4 lADPC-Bioink AG组 3 9.0±0.7 81.6±1.4 99.4±0.9 F值 10.01 169.10 65.83 P值 <0.01 <0.01 <0.01 t1值 1.74 18.70 12.51 P1值 >0.05 <0.01 <0.01 t2值 3.59 15.70 4.84 P2值 <0.05 <0.01 <0.01 t3值 1.19 3.15 0.72 P3值 >0.05 <0.05 >0.05 注：lADPC为抽脂术脂肪来源蛋白复合物，Bioink AG为明胶-海藻酸钠复合三维生物打印墨水；处理因素主效应，F=109.30，P<0.01；时间因素主效应，F=7 273.00，P<0.01；两者交互作用，F=97.48，P<0.01；F值、P值为各时间点组间总体比较所得；t1值、P1值，t2值、P2值，t3值、P3值分别为常规换药组、单纯lADPC组、单纯Bioink AG组与1ADPC-Bioink AG组各时间点比较所得
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1. 宋薇，李曌，朱世钧，张超，姚斌，孔毅，梁莉婷，恩和吉日嘎拉，付小兵，黄沙. 含人脐血来源富血小板血浆的三维生物打印墨水在裸鼠全层皮肤缺损治疗中的应用. 中华烧伤与创面修复杂志. 2022(10): 905-913 . 
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