Effects and mechanism of water-soluble chitosan hydrogel on infected full-thickness skin defect wounds in diabetic mice
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摘要:
目的 探讨水溶性壳聚糖水凝胶对糖尿病小鼠感染全层皮肤缺损创面的作用及其机制。 方法 采用实验研究方法。采用循环冻融的方法制备由聚乙烯醇和明胶组成的对照水凝胶及由前述2种材料+水溶性壳聚糖组成的水溶性壳聚糖水凝胶。大体观察第1次冻融前后试管中2种敷料流动性，并比较12孔板中2种敷料最终形态的外观差异。取细胞株L929和HaCaT，均分别按照随机数字表法（分组方法下同）分为对照水凝胶组和水溶性壳聚糖水凝胶组，分别加入相应敷料培养24 h，采用细胞计数试剂盒8检测细胞增殖活力。取兔血红细胞悬液，分为生理盐水组、聚乙二醇辛基苯基醚（Triton X-100）组、对照水凝胶组和水溶性壳聚糖水凝胶组，分别作相应处理后孵育1 h，采用酶标仪检测红细胞的溶血程度。取24只11~14周龄雌性db/db小鼠，在其背部制作全层皮肤缺损创面并在创面处滴加耐甲氧西林金黄色葡萄球菌（MRSA）液，72 h后将小鼠分为空白对照组、磺胺嘧啶银水胶组、对照水凝胶组、水溶性壳聚糖水凝胶组，分别作相应处理。伤后0（即刻）、7、14、21 d，大体观察创面愈合情况并计算伤后14、21 d创面愈合率；伤后14 d，检测创面中MRSA浓度；伤后21 d，采用苏木精-伊红染色法对创面进行组织学分析，采用免疫荧光法检测创面中细胞CD31表达并计算其阳性百分率。取Raw264.7细胞，分为进行相应处理的白细胞介素4（IL-4）组、空白对照组、对照水凝胶组、水溶性壳聚糖水凝胶组，培养48 h，采用流式细胞仪检测细胞中CD206阳性细胞百分率。样本数均为3。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、LSD检验及 Dunnett T3检验。 结果 试管中2种敷料在进行冻融前都具有一定的流动性，冻融1次后均形成半固态凝胶。12孔板中2种敷料最终形态均基本呈稳定的半透明片状，透明度差异不大。培养24 h，水溶性壳聚糖水凝胶组L929和HaCaT的细胞增殖活力均明显高于对照水凝胶组（t值分别为6.37、7.50，P<0.01）。孵育1 h，水溶性壳聚糖水凝胶组红细胞溶血程度明显低于Triton X-100组（P<0.01），而与生理盐水组及对照水凝胶组均相近（P>0.05）。伤后0 d，4组小鼠创面情况相似。伤后7 d，空白对照组与对照水凝胶组创面内部淡黄色渗出物较多，磺胺嘧啶银水胶组与水溶性壳聚糖水凝胶组创面观察到少量渗出。伤后14 d，空白对照组与对照水凝胶组创面干燥结痂，无明显上皮覆盖；磺胺嘧啶银水胶组创面痂皮脱落，可见脓性渗出物；水溶性壳聚糖水凝胶组创面基底呈淡红色，创面可观察到明显上皮覆盖。伤后14 d，水溶性壳聚糖水凝胶组创面愈合率显著高于其他3组（P值均<0.01）。伤后21 d，水溶性壳聚糖水凝胶组创面已完全闭合，其他3组创面均未完全愈合；水溶性壳聚糖水凝胶组创面愈合率显著高于其他3组（P值均<0.01）。伤后14 d，水溶性壳聚糖水凝胶组创面的MRSA浓度明显低于空白对照组（P<0.01），但与对照水凝胶组和磺胺嘧啶银水胶组均相近（P>0.05）。伤后21 d，空白对照组创面新生表皮缺损严重；对照水凝胶组创面的表皮亦有大面积缺损；磺胺嘧啶银水胶组创面尚未形成完整新生表皮；水溶性壳聚糖水凝胶组创面不仅被新生表皮完全覆盖，且新生表皮基底细胞排列规整。伤后21 d，水溶性壳聚糖水凝胶组创面中CD31阳性百分率为（2.19±0.35）%，明显高于空白对照组的（0.18±0.05）%、对照水凝胶组的（0.23±0.06）%以及磺胺嘧啶银水胶组的（0.62±0.25）%，P值均<0.01。培养48 h，水溶性壳聚糖水凝胶组Raw264.7中CD206阳性细胞百分率明显低于IL-4组（P<0.01），但明显高于空白对照组与对照水凝胶组（P<0.05或P<0.01）。 结论 水溶性壳聚糖水凝胶生物安全性好，较不含水溶性壳聚糖的对照水凝胶能诱导更高水平的巨噬细胞M2型极化，因此可以提升糖尿病小鼠MRSA感染的全层皮肤缺损创面的修复效果，并促进创面快速愈合。 Abstract:Objective To explore the effects and mechanism of water-soluble chitosan hydrogel on infected full-thickness skin defect wounds in diabetic mice. Methods The experimental research method was adopted. The control hydrogel composed of polyvinyl alcohol and gelatin, and the water-soluble chitosan hydrogel composed of the aforementioned two materials and water-soluble chitosan were prepared by the cyclic freeze-thaw method. The fluidity of the two dressings in test tube before and after the first freeze-thawing was generally observed, and the difference in appearance of the final state of two dressings in 12-well plates were compared. According to random number table (the same grouping method below), the cell strains of L929 and HaCaT were both divided into water-soluble chitosan hydrogel group and control hydrogel group, respectively. After adding corresponding dressings and culturing for 24 h, the cell proliferation activity was measured using cell counting kit 8. Rabbit blood erythrocyte suspensions were divided into normal saline group, polyethylene glycol octyl phenyl ether (Triton X-100) group, water-soluble chitosan hydrogel group, and control hydrogel group, which were treated accordingly and incubated for 1 hour, and then the hemolysis degree of erythrocyte was detected by a microplate reader. Twenty-four female db/db mice aged 11-14 weeks were selected, and full-thickness skin defect wounds on their backs were inflicted and inoculated with the methicillin-resistant Staphylococcus aureus (MRSA), 72 h later, the mice were divided into blank control group, sulfadiazine silver hydrogel group, control hydrogel group, and water-soluble chitosan hydrogel group, which were treated accordingly. On post injury day (PID) 0 (immediately), 7, 14, and 21, the healing of the wound was observed. On PID 14 and 21, the wound healing rate was calculated. On PID 14, MRSA concentration in wounds was determined. On PID 21, the wounds were histologically analyzed by hematoxylin and eosin staining; the expression of CD31 in the wounds was detected by immunofluorescence method, and its positive percentage was calculated. Raw264.7 cells were taken and divided into interleukin-4 (IL-4) group, blank control group, control hydrogel group, and water-soluble chitosan hydrogel group, which were treated accordingly. At 48 h of culture, the percentages of CD206 positive cells were detected by flow cytometry. The number of samples was all 3. Data were statistically analyzed with independent sample t test, one-way analysis of variance, analysis of variance for repeated measurement, least significant difference test, and Dunnett T3 test. Results Two dressings in test tube had certain fluidity before freeze-thawing and formed semi-solid gels after freeze-thawing for once. The final forms of two dressings in 12-well plates were basically stable and translucent sheets, with little difference in transparency. At 24 h of culture, the cell proliferation activities of L929 and HaCaT in water-soluble chitosan hydrogel group were significantly higher than those in control hydrogel group (with t values of 6.37 and 7.50, respectively, P<0.01). At 1 h of incubation, the hemolysis degree of erythrocyte in water-soluble chitosan hydrogel group was significantly lower than that in Triton X-100 group (P<0.01), but similar to that in normal saline group and control hydrogel group (P>0.05). On PID 0, the traumatic conditions of mice in the 4 groups were similar. On PID 7, more yellowish exudates were observed inside the wound in blank control group and control hydrogel group, while a small amount of exudates were observed in the wound in sulfadiazine silver hydrogel group and water-soluble chitosan hydrogel group. On PID 14, the wounds in blank control group and control hydrogel group were dry and crusted without obvious epithelial coverage; in sulfadiazine silver hydrogel group, the scabs fell off and purulent exudate was visible on the wound; in water-soluble chitosan hydrogel group, the base of wound was light red and obvious epithelial coverage could be observed on the wound. On PID 14, the wound healing rate in water-soluble chitosan hydrogel group was significantly higher than that in the other 3 groups (all P<0.01). On PID 21, the wound in water-soluble chitosan hydrogel group was completely closed, while the wounds in the other 3 groups were not completely healed; the wound healing rate in water-soluble chitosan hydrogel group was significantly higher than that in the other 3 groups (all P<0.01). On PID 14, the concentration of MRSA in the wound in water-soluble chitosan hydrogel group was significantly lower than that in blank control group (P<0.01), but similar to that in control hydrogel group and sulfadiazine silver hydrogel group (P>0.05). On PID 21, the new epidermis was severely damaged in blank control group; the epidermis on the wound in control hydrogel group also had a large area of defect; complete new epidermis had not yet being formed on the wound in sulfadiazine silver hydrogel group; the wound in water-soluble chitosan hydrogel group was not only completely covered by the new epidermis, the basal cells of the new epidermis were also regularly aligned. On PID 21, the percentage of CD31 positivity in the wound in water-soluble chitosan hydrogel group was (2.19±0.35)%, which was significantly higher than (0.18±0.05)% in blank control group, (0.23±0.06)% in control hydrogel group, and (0.62±0.25)% in sulfadiazine silver hydrogel group, all P<0.01. At 48 h of culture, the percentage of CD206 positive Raw264.7 cells in water-soluble chitosan hydrogel group was lower than that in IL-4 group (P>0.01) but significantly higher than that in blank control group and control hydrogel group (P<0.05 or P<0.01). Conclusions The water-soluble chitosan hydrogel has good biosafety and can induce higher level of macrophage M2 polarization than control hydrogel without water-soluble chitosan, so it can enhance the repair effect of MRSA-infected full-thickness skin defect wounds in diabetic mice and promote rapid wound healing. -
Key words:
- Hydrogel /
- Chitosan /
- Infection /
- Skin /
- Chronic wound /
- Wound repair
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(1)自体刃厚皮具有容易切取、取材范围广、成活率高等特点，此前鲜见关于将其作为移植物预制缺损尿道的报道。
阴茎作为男性主要生殖器官，具有性交、排尿和射精等功能。阴茎位置较隐蔽，一般情况下不易受伤，一旦损伤则会给患者造成巨大的心理和生理障碍 ［ 1］ 。手术是目前唯一能有效治疗因外伤致阴茎缺损伴尿道损伤的手段。文献报道，尿道中段缺损的手术方法多种多样 ［ 2］ ，但尚无一种公认的令人满意的术式。尤其是外伤致严重尿道中段缺损伴重度畸形，手术难度大、术后并发症多 ［ 3, 4, 5］ ，术者往往需要综合考虑致伤原因、缺损长度、局部组织条件等多方面因素，才能确定最适合患者的手术方式 ［ 6, 7］ 。
临床上，尿道成形术和端端吻合术 ［ 8］ 是尿道损伤的典型干预措施。然而，对于缺损长度≥3 cm、存在局部并发症的复杂尿道缺损患者，往往需要行局部或游离组织移植 ［ 9］ ，临床中常用的自体移植物包括皮瓣或口腔黏膜（颊黏膜、舌黏膜和唇黏膜） ［ 10, 11, 12］ 。研究表明，皮瓣的获取和移植技术要求高，较皮片移植更为复杂，且皮瓣移植术后并发症多，患者并不倾向于采用这种治疗方法 ［ 13］ 。有些患者可能存在口腔疾病或损伤，导致供体组织（如颊黏膜或其他黏膜移植物）不充足或者不可用。临床研究显示，将口腔黏膜组织作为移植物，取材范围不能超过8 cm×6 cm ［ 14］ ，且需要受区条件适宜，如新鲜的创基或良好的血运等，且口腔黏膜组织移植术后2周内都影响患者进食 ［ 15］ 。本课题组实践表明，对于一些复杂且损伤程度较重的尿道中段缺损伴阴茎缺损，自体刃厚皮移植预制尿道联合阴囊皮瓣修复是一种较佳的选择。
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。根据空军军医大学第一附属医院伦理委员会政策，可以在不泄露患者身份的前提下对其临床资料进行使用及分析。患者或其家属签署知情同意书。
1.1 入选标准
纳入标准：（1）年龄14~60岁，男性；（2）热力烧伤、电击伤、交通伤等外伤导致尿道中段缺损伴阴茎缺损形成尿瘘的患者；（3）尿道缺损长3~5 cm者；（4）接受尿道及阴茎重建手术治疗及长期随访者。排除标准：临床资料不全者。
1.2 临床资料
2015年1月—2022年1月，空军军医大学第一附属医院收治8例符合入选标准的外伤致尿道中段缺损伴阴茎缺损形成尿瘘男性患者。患者年龄14~58岁，其中2例未成年患者年龄分别为14、16岁，尿道缺损长度为3~5 cm。致伤原因包括热力烧伤（4例）、电击伤（2例）、交通伤（2例），部分患者伤后3个月因瘢痕挛缩伴有阴茎偏曲。患者于伤后48~96 h入院。
1.3 治疗方法
1.3.1 术前准备
术前充分与患者或其家属沟通，告知治疗方法、治疗过程及术后注意事项等。根据致伤原因，予抗感染、控制血糖、纠正电解质紊乱、营养支持治疗，改善全身一般状况。行尿道创面分泌物标本微生物培养，根据药物敏感试验结果选用敏感抗生素并静脉给药。术前1 d或手术当天进手术室前对术区行常规剃毛。
1.3.2 手术方法
所有患者手术由同一位经验丰富的主任医师主刀完成。手术分2期进行：Ⅰ期行自体皮移植预制缺损段尿道，Ⅱ期行尿道吻接及单侧阴囊皮瓣转移重建尿道及阴茎。
Ⅰ期手术于全身麻醉下进行，术区常规碘伏消毒、铺巾，留置导尿管。用电动取皮刀切取大腿外侧与缺损尿道长度相当的自体刃厚皮，其宽度为导尿管的周径，大腿继发创面用油纱覆盖包扎。将所取的刃厚皮修剪后，包裹在由2个末端拼接的导尿管形成的尿道模具外面，以6-0可吸收缝线缝合，形成自体刃厚皮卷管。将卷管植入一侧阴囊肉膜内，预制成缺损段尿道，以3-0缝线间断缝合。术后24 ｈ内应用头孢类、喹诺酮类抗生素或根据术前尿道创面分泌物标本微生物培养结果及药物敏感试验结果选用抗生素抗感染，术后10 ｄ拆除缝线。Ⅰ期术后1个月行Ⅱ期手术，Ⅰ、Ⅱ期手术期间每隔2周更换1次导尿管。
Ⅱ期手术于气管插管全身麻醉及仰卧位下进行。切除损伤尿道周围瘢痕，充分暴露新鲜尿道组织，尽量保留尿道海绵体及阴茎海绵体，避免损伤阴茎背侧血管束，于海绵体内注入生理盐水使阴茎勃起。清创后，阴茎缺损创面的面积为5.0 cm×2.5 cm~7.0 cm×5.5 cm。按创面面积切取皮瓣，依次切开阴囊皮肤、皮下组织及肉膜，在肉膜下潜行分离，且保留足够长的肉膜作为血管蒂，形成单侧阴囊皮瓣。阴囊皮瓣的面积为6.0 cm×3.0 cm~8.0 cm×6.0 cm。分离预制尿道，勿伤及成型的卷管，彻底止血，确保所预制的尿道与单侧阴囊皮瓣为一体转移至阴茎腹侧创面上。取出卷管内的尿道模具，更换导尿管，并将导尿管由阴茎头端穿过预置尿道至膀胱内。将自体刃厚皮卷管与原尿道缺损断端桥接吻合，将吻合口两端剪成斜面，用4-0可吸收缝线内翻缝合，恢复尿道的连续性，检查无张力即可。旋转阴囊皮瓣后，用阴囊肉膜修复尿道海绵体，同时包裹外露的阴茎海绵体，彻底止血，依次分层缝合，恢复阴茎的连续性，完成阴茎重建。将供瓣区创面直接缝合，受区放置引流片。将阴茎部依次用油纱、湿纱布、干纱布覆盖并打包包扎。术后24~48 h拔除阴囊部引流片，术后3周拔除导尿管。自术后72 h起，嘱患者每日至少自主排尿1次。术后10 d拆除术区缝线、局部敷料。
1.4 观测指标
观察并记录Ⅱ期术后7 d皮瓣成活情况。Ⅱ期术后3周，采用尿流率检测仪测定患者尿流率（尿流率>15 mL/s为排尿通畅） ［ 16］ ，观察有无尿瘘、勃起功能，采用本研究团队自行设计的疗效满意度调查表 ［ 17］ 对患者进行疗效满意度调查，调查表包括身体约束度、舒适度及认可度3个指标，总分为0~15分，分数越高表示对疗效越满意。Ⅱ期术后，每3个月门诊复查1次，1年后每年复查1次，随访时观察皮瓣受区外观，采用温哥华瘢痕量表（VSS） ［ 18］ 评估供皮区及供瓣区瘢痕情况，同前检测尿流率，观察有无尿道狭窄、尿瘘及勃起功能，调查患者对疗效的满意度。
1.5 数据处理
采用SPSS 23.0 统计软件进行数据分析。计量资料数据均不符合正态分布，以 M（ Q 1， Q 3）表示。
2. 结果
2.1 术后及随访情况
8例患者Ⅱ期术后7 d皮瓣完全成活。Ⅱ期术后3周，患者尿流率为25.3（18.0，38.5）mL/s，排尿通畅，无尿瘘，有勃起功能，对疗效的满意度评分为14.3（14.0，15.0）分。随访1~7年，8例患者皮瓣受区外形饱满、不臃肿，质地柔软，颜色与周围组织相近，供皮区VSS评分为11.5（10.0，13.0）分，供瓣区VSS评分为10.5（9.3，12.0）分，尿流率为24.6（17.7，34.1）mL/s，无尿道狭窄、尿瘘及勃起功能受限，对疗效的满意度评分为13.5（13.3，14.8）分。
2.2 典型病例
患者男，14岁，因火焰烧伤全身多处伴疼痛于伤后48 h被收入空军军医大学第一附属医院。入院时体格检查显示：全身除腹部、左下肢及背部部分皮肤外，其余皮肤均被烧伤，烧伤总面积为85%TBSA，大部分烧伤创面表皮脱落，创基多红白相间，部分呈苍白色，头皮部分烧焦，四肢末梢血运尚可。入院后予抗感染、抗休克、营养支持治疗，纠正电解质紊乱，改善全身一般状况后，分次有计划地植皮封闭创面。全身创面基本封闭后，同前行2期手术。尿道缺损长度为4 cm，清创后阴茎缺损创面面积为7.0 cm×5.0 cm。伤后2个月，Ⅰ期行自体皮移植预制缺损段尿道；伤后3个月，Ⅱ期行尿道吻接及单侧阴囊皮瓣（面积为8.0 cm×5.5 cm）转移重建尿道及阴茎。Ⅱ期术后24 h，拔除阴囊部引流片。Ⅱ期术后7 d，阴囊皮瓣存活良好；Ⅱ期术后3周，患者站立式排尿，尿流率为29.0 mL/s，排尿通畅，无尿瘘，有勃起功能，对疗效的满意度评分为14.0分。随访7年，患者未出现尿瘘、尿道狭窄，尿流率为32.0 mL/s，排尿功能及勃起功能正常，供皮区瘢痕VSS评分为10.0分，供瓣区VSS评分为8.0分，阴茎、阴囊发育正常，对疗效的满意度评分为13.0分。见 图1。
1 自体皮移植预制尿道联合阴囊皮瓣修复烧伤患者尿道中段缺损伴阴茎缺损。1A.伤后1个月，下腹部至双大腿仅有少量正常皮肤，会阴周围均为Ⅲ度烧伤创面；1B.Ⅰ期术前（伤后2个月）会阴周围瘢痕形成，尿道中段缺损伴阴茎部分缺损；1C.用自体刃厚皮制备卷管，植入单侧阴囊前；1D.预制尿道植入阴囊并缝合后；1E.Ⅱ期术前（伤后3个月），会阴周围瘢痕增生明显，阴囊术区愈合良好；1F.Ⅱ期术中寻找并游离预制自体刃厚皮卷管；1G.Ⅱ期术中将自体刃厚皮卷管两端与原尿道缺损断端吻合；1H.Ⅱ期术后7 d，皮瓣存活良好，创面愈合良好；1I.术后1年随访，阴囊皮瓣存活良好，站立式排尿，排尿通畅，无尿道狭窄；1J.术后7年随访，勃起功能正常，阴茎、阴囊发育正常3. 讨论
外伤导致的尿道损伤的修复困难重重，随着尿道修复技术的发展，重建尿道的方式多种多样，但手术失败率仍较高 ［ 19］ 。目前国内外均认为自体组织游离移植是尿道中段损伤修复最可靠的方法 ［ 20, 21, 22］ ，包括皮肤游离移植、黏膜组织游离移植。为确保移植后口腔黏膜成活 ［ 23, 24］ ，用口腔黏膜修复的缺损长度不宜超过5 cm。Mundy ［ 25］ 提出将颊黏膜作为移植物修复尿道，但术后仍存在移植物收缩问题，针对该情况他改用薄层自体刃厚皮修复尿道，防止了因术后移植物收缩而导致的尿道狭窄。因此对于外伤导致的尿道损伤患者，自体皮移植成为较佳的选择。本研究选用自体刃厚皮移植进行尿道重建，具有皮片容易切取、取材范围广、成活率高等特点。外伤后缺损的尿道有较多的定植菌，分泌物较多，感染概率高，不仅可导致移植物存活不良而且可引起再造尿道狭窄。本研究中8例患者随访未出现尿道狭窄，这可能与自体皮移植后组织血管化较好有关。黏膜抗感染能力差，而将无血运的刃厚皮Ⅰ期埋植于阴囊肉膜内寄养，不损伤包裹睾丸和精索的被膜，Ⅱ期随阴囊皮瓣修复重建尿道，降低感染概率。有研究者采用邻近尿道的包皮、睾丸鞘膜、阴囊皮肤组织制作皮管，Ⅱ期转移修复缺损的尿道，尿瘘、尿道狭窄等并发症少 ［ 26］ 。然而对于热力烧伤、电击伤、交通伤等外伤导致尿道中段缺损形成尿瘘的复合伤患者，原有的包皮损伤且通常伴有瘢痕，导致会阴区无可利用的组织。采用大腿自体刃厚皮移植预制尿道联合阴囊皮瓣修复缺损尿道，可避免利用因外伤导致受区周围条件差的组织及再造尿道因移植组织量不足影响修复效果。本研究中8例患者采用自体皮移植预制缺损尿道联合阴囊皮瓣修复尿道后，无尿瘘及尿道狭窄，其中2例未成年患者随年龄的增长，阴茎及阴囊正常发育，勃起功能正常，排尿通畅。
阴囊皮瓣血供丰富、抗感染能力强，移植该皮瓣后，创面可以在相对较短的时间内愈合，且该皮瓣可受神经支配对外界刺激做出反应，是修复尿道及阴茎缺损的较佳选择之一。预制尿道在阴囊皮瓣内寄养1个月后，与周围结构一体化，更易存活。阴囊皮瓣取材面积大，旋转角度较灵活，既可以为受区提供良好的血供，又相当于在尿道外增加了一层组织，可预防尿瘘的发生。阴囊皮肤在质地、颜色和纹理上与阴茎皮肤高度相似，且供区位置隐蔽，不会遗留明显的手术瘢痕。本研究中8例患者术区阴囊未见明显瘢痕，受区颜色、质地与周围正常皮肤相近。移植阴囊皮瓣后，不仅填补了筋膜结构的缺失，而且恢复了阴茎悬韧带的连续性，保留了重建后的阴茎勃起功能。阴囊是男性阴茎根部下方容纳和保护睾丸及附睾的多层结构囊袋，其皮肤有聚成小皱襞的能力，用阴囊皮瓣转移重建阴茎，不仅可使阴茎恢复收缩功能，还保留了其深层感觉。术后及随访观察到，本研究中8例患者阴囊皮瓣完全成活，排尿通畅，均有勃起功能，对疗效的满意度高。
本术式注意事项：（1）将吻合口两端剪成斜面，在无张力下精细严格地内翻对位缝合，避免术后出现吻合口瘘；（2）术中需彻底止血，以减少血肿的形成和感染的发生；（3）充分游离、切除纤维化或形成了瘢痕的尿道及周围组织，改善重建后尿道狭窄及阴茎偏曲；（4）若同期行阴茎背侧瘢痕松解植皮，植皮区加压包扎敷料的首次更换时间不宜过早，避免影响新生血管重建的数量和速度，以术后10 d为宜 ［ 27］ ；（5）尽量避开瘢痕纤维化明显的部位取阴囊皮瓣，减少切口愈合过程中瘘的发生，保留一侧肉膜作为血管蒂，减小皮瓣旋转牵拉的角度，便于移植物覆盖；（6）由于此类患者致病因素的特殊性，手术需分期进行，临床上也可根据患者阴茎发育情况、阴茎瘢痕挛缩程度、尿道缺损长短来综合判断是否需行分期手术。
本研究为单中心回顾性研究，采用自体刃厚皮游离移植重建复杂性尿道中段缺损，手术方法简便实用，具有临床可行性，手术成功率高，术后效果好；阴囊皮瓣血运可靠、组织层次多、厚度佳、有一定收缩功能、转移灵活。自体皮游离移植联合阴囊皮瓣修复缺损尿道及阴茎，不仅重建了尿道结构及阴茎外形，而且恢复了阴茎的感觉及勃起功能，术后并发症少、瘢痕增生不明显、患者满意度高，值得临床推广。
朱萌：酝酿和设计实验、实施研究、采集数据、数据整理、统计分析、撰写论文；陈禹州：酝酿和设计实验、实施研究、采集数据、撰写论文；区锦钊：数据整理、统计分析；李曌、鞠晓燕：文献调研、分析/解释数据；黄沙、胡骁骅、田野、牛忠伟：研究指导、论文修改、经费支持所有作者均声明不存在利益冲突 -
参考文献
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1 2种水凝胶在试管中冻融1次前后流动性以及在12孔板中循环冻融后最终形态。1A、1B.分别为对照水凝胶和水溶性壳聚糖水凝胶，冻融1次后，2种溶液均由可流动状态变成不流动的半固态；1C、1D.分别为在12孔板中最终形成的对照水凝胶和水溶性壳聚糖水凝胶（培养皿中展示），二者均为稳定规则的圆形半透明片状
注：对照水凝胶由聚乙烯醇和明胶制得，水溶性壳聚糖水凝胶由聚乙烯醇、明胶和水溶性壳聚糖制得
2 4 组db/db小鼠耐甲氧西林金黄色葡萄球菌感染全层皮肤缺损创面伤后各时间点愈合情况。2A、2B、2C、2D.分别为空白对照组伤后0、7、14、21 d创面，图2E显示空白对照组14 d后创面缩小不明显；2F、2G、2H、2I.分别为对照水凝胶组伤后0、7、14、21 d 创面，图2J显示对照水凝胶组各时间点创面面积均分别较图2E有所缩小；2K、2L、2M、2N.分别为磺胺嘧啶银水胶组伤后0、7、14、21 d创面，图2O显示磺胺嘧啶银水胶组各时间点创面面积分别较图2J有所缩小；2P、2Q、2R、2S.分别为水溶性壳聚糖水凝胶组伤后0、7、14、21 d创面，图2T显示水溶性壳聚糖水凝胶组各时间点创面面积均分别较图2E、2J、2O明显缩小
注：对照水凝胶由聚乙烯醇和明胶制得，水溶性壳聚糖水凝胶由聚乙烯醇、明胶和水溶性壳聚糖制得；图中圆形参照物直径为2 cm；图2E、2J、2O、2T为ImageJ图像软件处理后量化图，图中红色、橙色、黄色、绿色边界内范围均分别表示伤后0（即刻）、7、14、21 d创面面积大小
3 4组db/db小鼠耐甲氧西林金黄色葡萄球菌感染的全层皮肤缺损创面伤后21 d组织学变化 苏木精-伊红。3A、3B、3C、3D.分别为空白对照组、对照水凝胶组、磺胺嘧啶银水胶组、水溶性壳聚糖水凝胶组创面，图3D中的新生表皮长度明显长于图3A、3B、3C，图片放大倍数为1.25；3E、3F、3G、3H.分别为图3A、3B、3C、3D局部创面放大图，从左至右显示未封闭的创缘宽度依次缩减，其中图3H所示创面已完全愈合，图片放大倍数为2.50
注：对照水凝胶由聚乙烯醇和明胶制得，水溶性壳聚糖水凝胶由聚乙烯醇、明胶和水溶性壳聚糖制得；图3A、3B、3C、3D中的红色双向箭头指示新生上皮长度，图3E、3F、3G、3H中的红色单向箭头之间区域指示尚未封闭的创缘宽度
4 4组db/db小鼠耐甲氧西林金黄色葡萄球菌感染的全层皮肤缺损创面伤后21 d CD31阳性表达情况 Alexa Fluor 594-4', 6-二脒基-2-苯基吲哚×40。4A、4B、4C、4D.分别为空白对照组、对照水凝胶组、磺胺嘧啶银水胶组、水溶性壳聚糖水凝胶组，图4D中的CD31阳性表达明显高于图4A、4B、4C
注：对照水凝胶由聚乙烯醇和明胶制得，水溶性壳聚糖水凝胶由聚乙烯醇、明胶和水溶性壳聚糖制得；细胞核阳性染色为蓝色，CD31阳性细胞染色为红色（指示新生血管）
表1 4组db/db小鼠耐甲氧西林金黄色葡萄球菌感染的全层皮肤缺损创面伤后各时间点愈合率比较（%，
组别 样本数 14 d 21 d 空白对照组 3 36.94±3.00 58.93±4.69 对照水凝胶组 3 18.92±4.42 84.33±4.04 磺胺嘧啶银水胶组 3 39.71±5.03 88.10±2.01 水溶性壳聚糖胶组 3 83.74±3.30 99.76±0.24 F值 140.70 83.74 P值 <0.001 <0.001 P1值 <0.001 <0.001 P2值 <0.001 <0.001 P3值 <0.001 0.002 注：对照水凝胶由聚乙烯醇和明胶制得，水溶性壳聚糖水凝胶由聚乙烯醇、明胶和水溶性壳聚糖制得；处理因素主效应，F=422.86，P<0.001；时间因素主效应，F=1 788.78，P<0.001；两者交互作用，F=68.88，P<0.001；F值、P值为4组间各时间点总体比较所得；P1值为空白对照组与水溶性壳聚糖水凝胶组比较所得；P2值为对照水凝胶组与水溶性壳聚糖水凝胶组比较所得；P3值为磺胺嘧啶银水胶组与水溶性壳聚糖水凝胶组比较所得 
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