Feasibility study on the preparation of novel negative pressure materials for constructing new matrix of full-thickness skin defect wounds in rats
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摘要:
目的 探索制备新型负压材料以构建大鼠全层皮肤缺损创面新生基质的可行性。 方法 采用实验研究方法。取负压治疗中常用的聚氨酯泡沫敷料，于扫描电子显微镜下观察其微观结构并测定其孔径（样本数为5）。分别以聚己内酯、聚丁二酸丁二醇酯（PBS）为原材料，采用熔体纺丝技术，以测得的聚氨酯泡沫敷料孔径为纺丝间距，分别以15、25、35 mm/s为纺丝速率制备聚己内酯负压材料和PBS负压材料，于扫描电子显微镜下观察制备的负压材料微观结构并测定其丝径，采用拉力试验机与复合材料试验机分别测定制备的负压材料和聚氨酯泡沫敷料的拉伸强度与拉伸模量（样本数均为5），以筛选后续制备负压材料的纺丝速率。将对数生长期的人皮肤成纤维细胞（Fb）分别与以筛选的纺丝速率制备的聚己内酯负压材料和PBS负压材料共培养，于共培养1、4、7 d，用活/死细胞检测试剂盒检测材料中细胞活性与黏附情况，用细胞计数试剂盒8法检测材料中细胞增殖水平（样本数为5）。于18只5~6周龄雌雄不限的SD大鼠背部各制备1个全层皮肤缺损创面，伤后即刻，将致伤大鼠按随机数字表法分为聚己内酯+聚氨酯组、PBS+聚氨酯组、单纯聚氨酯组（每组6只），用含相应成分材料覆盖创面，以-16.7 kPa负压进行持续负压吸引。分别于负压治疗7、14 d取每组3只大鼠，取创面组织与创面直接接触层材料（以下简称创面取材），行苏木精-伊红染色后观察肉芽组织生长及材料与创面结合情况，行Masson染色后观察胶原纤维沉积情况，采用免疫组织化学法检测并计数CD34、白细胞介素6（IL-6）阳性细胞。对数据行单因素方差分析、析因设计方差分析、LSD-t检验、Kruskal-Wallis H检验、Mann-Whitney U检验及Bonferroni校正。 结果 聚氨酯泡沫敷料微观上呈疏松多孔结构，孔径为（815±182）μm。以800 μm为后续负压材料制备中的纺丝间距。PBS负压材料与聚己内酯负压材料的微观结构均较为规则，层间垂直相通，纺丝连续且粗细均匀，但PBS负压材料纺丝较聚己内酯负压材料平直；不同纺丝速率下由同种原材料制备的负压材料微观结构无明显差别。以3种纺丝速率制备的聚己内酯负压材料的丝径相近（P>0.05），以25、35 mm/s的纺丝速率制备的PBS负压材料的丝径均明显小于以15 mm/s的纺丝速率制备的PBS负压材料（t值分别为4.99、6.40，P<0.01）。以3种纺丝速率制备的聚己内酯负压材料的拉伸强度、拉伸模量均相近（P>0.05）；以15、25 mm/s的纺丝速率制备的PBS负压材料的拉伸强度均明显小于以35 mm/s的纺丝速率制备的PBS负压材料（t值分别为9.20、8.92，P<0.01），拉伸模量均明显小于以35 mm/s纺丝速率制备的PBS负压材料（t值分别为2.58、2.47，P<0.05）。后续选用35 mm/s的纺丝速率制备聚己内酯负压材料，选用15 mm/s的纺丝速率制备PBS负压材料。共培养1、4、7 d，在聚己内酯负压材料和PBS负压材料中黏附生长的人皮肤Fb数随时间延长而增多，2种材料间比较无明显差别。共培养1、7 d，2种负压材料中人皮肤Fb增殖水平均相近（P>0.05）；共培养4 d，PBS负压材料中人皮肤Fb增殖水平显著高于聚己内酯负压材料（t=6.37，P<0.01）。负压治疗7 d，3组大鼠创面取材中材料均清晰可辨且均可见少量胶原纤维，单纯聚氨酯组大鼠创面取材中可见少量肉芽组织；负压治疗14 d，3组大鼠创面取材中均可见大量肉芽组织与大量胶原纤维，聚己内酯+聚氨酯组大鼠创面取材中材料与创面组织分辨不清。负压治疗7、14 d，单纯聚氨酯组大鼠创面取材中胶原纤维均较另外2组致密。负压治疗7 d，每400倍视野下，PBS+聚氨酯组大鼠创面取材中CD34阳性细胞数为（14.8±3.6）个，明显少于单纯聚氨酯组的（27.8±9.1）个（t=3.06，P<0.05）；IL-6阳性细胞数为60（49，72）个，明显多于单纯聚氨酯组的44（38，50）个（Z=2.41，P<0.05）。负压治疗14 d，每400倍视野下，PBS+聚氨酯组大鼠创面取材中IL-6阳性细胞数为19（12，28）个，明显多于聚己内酯+聚氨酯组的3（1，10）个与单纯聚氨酯组的9（2，13）个（Z值分别为2.61、2.40，P<0.05）。 结论 制备的聚己内酯负压材料和PBS负压材料生物相容性好，均可成功构建大鼠全层皮肤缺损创面新生基质，其中聚己内酯负压材料总体上优于PBS负压材料。 Abstract:Objective To explore the feasibility on the preparation of novel negative pressure materials for constructing new matrix of full-thickness skin defect wounds in rats. Methods The experimental research method was applied. The microstructure of polyurethane foam dressing which was commonly used in negative pressure treatment was observed under scanning electron microscope, and its pore diameter was detected (n=5). Polycaprolactone (PCL) and polybutylene succinate (PBS) were used respectively as raw materials for the preparation of PCL and PBS negative pressure materials by melt spinning technology, with the measured pore diameter of polyurethane foam dressing as the spinning spacing at the spinning rates of 15, 25, and 35 mm/s, respectively. The microstructures of the prepared negative pressure materials were observed under scanning electron microscope, and their fiber diameters were measured. The tensile strength and tensile modulus of the prepared negative pressure materials and polyurethane foam dressing were measured by tensile testing machine and composite testing machine, respectively (n=5), to screen the spinning rate for subsequent preparation of negative pressure materials. Human skin fibroblasts (Fbs) in logarithmic growth phase were co-cultured with PCL negative pressure material and PBS negative pressure material prepared at the selected spinning rate, respectively. After 1, 4, and 7 day (s) of co-culture, the cell activity and adhesion in the materials was detected by living/dead cells detection kit, and the cell proliferation level in the materials was detected by cell counting kit 8 method (n=5). A full-thickness skin defect wound was prepared on the back of 18 5-6 weeks old Sprague-Dawley rats (gender unlimited). Immediately after injury, the injured rats were divided into PCL+polyurethane group, PBS+polyurethane group, and polyurethane alone group according to the random number table (with 6 rats in each group). The wounds were covered with materials containing corresponding component and performed with continuous negative pressure suction at the negative pressure of -16.7 kPa. The wound tissue along with materials directly contacted to the wound (hereinafter referred to as wound specimens) were collected from 3 rats in each group after 7 and 14 days of negative pressure treatment (NPT), respectively. The growth of granulation tissue and the attachment of material to wound surface were observed after hematoxylin-eosin staining, the collagen fiber deposition was observed after Masson staining, and CD34 and interleukin-6 (IL-6) positive cells were detected and counted by immunohistochemical staining. Data were statistically analyzed with one-way analysis of variance, analysis of variance for factorial design, least significant difference-t test, Kruskal-Wallis H test, Mann-Whitney U test, and Bonferroni correction. Results The microstructure of polyurethane foam dressing was loose and porous, with the pore diameter of (815±182) μm. The spinning spacing for the subsequent negative pressure material was set as 800 μm. The microstructures of PBS negative pressure material and PCL negative pressure material were regular, with vertically interconnected layers and continuous fibers in even thickness, but the fibers of PBS negative pressure material were straighter than those of PCL negative pressure material. There was no obvious difference in the microstructure of negative pressure materials prepared from the same raw material at different spinning rates. The fiber diameters of PCL negative pressure materials prepared at three spinning rates were similar (P>0.05). The fiber diameters of PBS negative pressure materials prepared at spinning rates of 25 mm/s and 35 mm/s were significantly smaller than the fiber diameter of PBS negative pressure material prepared at the spinning rate of 15 mm/s (with t values of 4.99 and 6.40, respectively, P<0.01). Both the tensile strength and tensile modulus of PCL negative pressure materials prepared at three spinning rates were similar (P>0.05). The tensile strength of PBS negative pressure materials prepared at spinning rates of 15 mm/s and 25 mm/s was significantly lower than that of PBS negative pressure materials prepared at the spinning rate of 35 mm/s (with t values of 9.20 and 8.92, respectively, P<0.01), and the tensile modulus was significantly lower than that of PBS negative pressure materials prepared at the spinning rate of 35 mm/s (with t values of 2.58 and 2.47, respectively, P<0.05). Subsequently, PCL negative pressure material was prepared at the spinning rate of 35 mm/s, and PBS negative pressure material was prepared at the spinning rate of 15 mm/s. After 1, 4, and 7 day (s) of co-culture, the number of human skin Fbs that adhered to PCL negative pressure material and PBS negative pressure material increased with time, and there was no significant difference between the two materials. After 1 and 7 day (s) of co-culture, the proliferation levels of human skin Fbs between the two negative pressure materials were similar (P>0.05). After being co-cultured for 4 days, the proliferation level of human skin Fbs in PBS negative pressure material was significantly higher than that in PCL negative pressure material (t=6.37, P<0.01). After 7 days of NPT, the materials were clearly identifiable and a small amount of collagen fibers were also observed in the wound specimens of rats in the three groups; a small amount of granulation tissue was observed in the wound specimens of rats in polyurethane alone group. After 14 days of NPT, a large number of granulation tissue and collagen fibers were observed in the wound specimens of rats in the three groups; the materials and wound tissue in the wound specimens of rats in PCL+polyurethane group could not be clearly distinguished. After 7 and 14 days of NPT, the collagen fibers in the wound specimens of rats in polyurethane alone group were denser than those in the other two groups. After 7 days of NPT, the number of CD34 positive cells in the wound specimens of rats in PBS+polyurethane group was 14.8±3.6 per 400 times visual field, which was significantly less than 27.8±9.1 in polyurethane alone group (t=3.06, P<0.05); the number of IL-6 positive cells was 60 (49, 72), which was significantly more than 44 (38, 50) in polyurethane alone group (Z=2.41, P<0.05). After 14 days of NPT, the number of IL-6 positive cells in the wound specimens of rats in PBS+polyurethane group was 19 (12, 28) per 400 times visual field, which was significantly more than 3 (1, 10) in PCL+polyurethane group and 9 (2, 13) in polyurethane alone group (with Z values of 2.61 and 2.40, respectively, P<0.05). Conclusions The prepared PCL negative pressure material and PBS negative pressure material have good biocompatibility, and can successfully construct the new matrix of full-thickness skin defect wounds in rats. PCL negative pressure material is better than PBS negative pressure material in general. -
Key words:
- Negative-pressure wound therapy /
- Wound healing /
- Tissue engineering /
- Skin /
- Fibroblasts /
- Melt spinning technology
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1 聚己内酯+聚氨酯组大鼠全层皮肤缺损创面的负压治疗模型制备过程。1A.制备大鼠背部全层皮肤缺损创面；1B.将聚己内酯负压材料作为创面直接接触层置于创面上；1C.在聚己内酯负压材料上放置聚氨酯泡沫敷料；1D.用自黏性薄膜封闭创面并连接负压导管装置
3 2种新型负压材料微观结构 扫描电子显微镜×100，图中标尺为1 mm。3A.以纺丝速率35 mm/s制备的聚己内酯负压材料呈规则网格结构，纺丝连续且粗细均匀；3B.以纺丝速率25 mm/s制备的聚丁二酸丁二醇酯负压材料微观结构大体与图3A相近，纺丝较图3A平直
4 以不同纺丝速率制备的2种新型负压材料的拉伸强度与拉伸模量比较（样本数为5，
注：与纺丝速率为35 mm/s相比，aP<0.01，bP<0.05
5 于激光扫描共聚焦显微镜下观察人皮肤成纤维细胞分别与2种新型负压材料共培养各时间点细胞活性与黏附情况 钙黄绿素×100，图中标尺为500 μm。5A、5B、5C.分别为共培养1、4、7 d时聚己内酯负压材料中细胞生长情况，图5A中细胞黏附数较少，图5B中细胞黏附数较图5A增加，图5C中细胞黏附数最多且部分材料交叉处细胞融合成片；5D、5E、5F.分别为共培养1、4、7 d时聚丁二酸丁二醇酯负压材料中细胞生长情况，图5D、5E、5F中细胞活性与黏附情况分别与图5A、5B、5C相近
注：活细胞阳性染色为绿色
6 3组全层皮肤缺损大鼠负压治疗各时间点创面组织和创面直接接触层材料中肉芽组织生长和材料与创面组织结合情况及胶原纤维排列情况 图中标尺为100 μm。6A、6B、6C与6D、6E、6F.分别为负压治疗7 d与14 d时聚己内酯+聚氨酯组、PBS+聚氨酯组、单纯聚氨酯组中肉芽组织生长和材料与创面组织结合情况，图6A、6B、6C中材料均清晰可辨，图6C可见少量肉芽组织（粉色），图6D中肉芽组织较图6A增多且材料与创面组织分辨不清，图6E、6F中可见大量新生肉芽组织且材料均可辨认 苏木精-伊红×200；6G、6H、6I与6J、6K、6L.分别为负压治疗7 d与14 d时聚己内酯+聚氨酯组、PBS+聚氨酯组、单纯聚氨酯组中胶原纤维排列情况，图6G与6H中可见少量胶原纤维（蓝色），图6I中胶原纤维较图6G、6H致密，图6J、6K、6L中胶原纤维分别较图6G、6H、6I致密，且图6L中胶原纤维较图6J、6K致密 Masson×200
注：聚己内酯+聚氨酯组、聚丁二酸丁二醇酯（PBS）+聚氨酯组、单纯聚氨酯组大鼠创面上分别放置含相应成分的材料作为创面直接接触层，各材料上均另放置聚氨酯泡沫敷料；图中箭头指示残余材料
7 3组全层皮肤缺损大鼠负压治疗各时间点创面组织和创面直接接触层材料中CD34和IL-6阳性细胞分布 辣根过氧化物酶-二氨基联苯胺-苏木精×400，图中标尺为50 μm。7A、7B、7C与7D、7E、7F.分别为负压治疗7 d与14 d时聚己内酯+聚氨酯组、PBS+聚氨酯组、单纯聚氨酯组中CD34阳性细胞分布，图7A中CD34阳性细胞数较多，图7B中CD34阳性细胞数与图7A相近，图7C中CD34阳性细胞数多于图7B，图7D、7E、7F中CD34阳性细胞数分别较图7A、7B、7C增多；7G、7H、7I与7J、7K、7L.分别为负压治疗7 d与14 d时聚己内酯+聚氨酯组、PBS+聚氨酯组、单纯聚氨酯组中IL-6阳性细胞分布，图7G中IL-6阳性细胞数较多，图7H中IL-6阳性细胞数与图7G相近但明显多于图7I，图7J中IL-6阳性细胞数较图7G减少，图7K中IL-6阳性细胞数较图7H减少且明显多于图7J，图7L中IL-6阳性细胞数较图7I减少且明显少于图7K
注：聚己内酯+聚氨酯组、聚丁二酸丁二醇酯（PBS）+聚氨酯组、单纯聚氨酯组大鼠创面上分别放置含相应成分的材料作为创面直接接触层，各材料上均另放置聚氨酯泡沫敷料；细胞阳性染色为棕黄色；IL-6为白细胞介素6
表1 制备2种新型负压材料的相关参数
材料名称 电压（kV） 熔融温度（℃） 聚焦高度（mm） 针头内径（mm） 材料厚度（mm） 聚己内酯 5~6 70~75 140 0.41 1 聚丁二酸丁二醇酯 3~4 175~190 140 0.41 1 
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表2 人皮肤成纤维细胞分别与2种新型负压材料共培养各时间点细胞增殖水平比较（
材料名称 样本数 1 d 4 d 7 d 聚己内酯负压材料 5 0.341±0.025 0.676±0.020 1.370±0.175 聚丁二酸丁二醇酯负压材料 5 0.333±0.030 0.816±0.045 1.262±0.109 t值 0.46 6.37 1.18 P值 0.660 <0.001 0.274 注：表中数据为吸光度值，表示细胞增殖水平；处理因素主效应，F=0.06，P=0.808；时间因素主效应，F=313.86，P<0.001；两者交互作用，F=5.04，P=0.015
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表3 3组全层皮肤缺损大鼠负压治疗各时间点创面组织和创面直接接触层材料中CD34和IL-6阳性细胞数比较（个）
组别 样本数 CD34阳性细胞数（ IL-6阳性细胞数［M（min，max）］ 7 d 14 d 7 d 14 d 聚己内酯+聚氨酯组 5 18.2±6.2 32.2±5.9 53（42，62） 3（1，10） PBS+聚氨酯组 5 14.8±3.6 29.6±2.3 60（49，72） 19（12，28） 单纯聚氨酯组 5 27.8±9.1 38.8±7.7 44（38，50） 9（2，13） 统计量值1 t=0.80 t=0.71 Z=1.26 Z=2.61 P1值 >0.999 >0.999 0.666 0.024 统计量值2 t=2.26 t=1.81 Z=1.47 Z=1.26 P2值 0.129 0.286 0.453 0.666 统计量值3 t=3.06 t=2.52 Z=2.41 Z=2.40 P3值 0.030 0.080 0.048 0.048 注：于400倍视野下计数；IL-6为白细胞介素6；CD34阳性细胞数的处理因素主效应，F=8.43，P=0.002；时间因素主效应，F=33.73，P<0.001；两者交互作用，F=0.26，P=0.776；统计量值1、P1值，统计量值2、P2值，统计量值3、P3值分别为聚己内酯+聚氨酯组和聚丁二酸丁二醇酯（PBS）+聚氨酯组、聚己内酯+聚氨酯组和单纯聚氨酯组、PBS+聚氨酯组和单纯聚氨酯组各指标各时间点两两比较所得
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《中华烧伤与创面修复杂志》第六届编辑委员会编辑委员名单
终身顾问 盛志勇 程天民 王正国 樊代明 付小兵 夏照帆 卞修武 顾晓松 李校堃 顾 问 肖光夏 杨宗城 汪仕良 孙永华 柴家科 黄跃生 岑瑛 王旭 名誉总编辑 彭毅志 总编辑 罗高兴 以下按姓氏拼音排序 副总编辑 郭光华 韩春茂 胡大海 郇京宁 梁光萍 刘毅 吕国忠 吴军 谢卫国 姚咏明 常务编辑委员 官浩 贺伟峰 李孝建 李宗瑜 刘琰 陆树良 马显杰 申传安 沈余明 孙炳伟 谭 谦 王达利 王一兵 夏成德 肖仕初 徐庆连 于家傲 袁志强 张丕红 张庆富 张逸 章一新 编辑委员 巴特 陈国贤 陈炯 陈俊杰 陈欣 陈旭 陈旭林 陈昭宏 程飚 崔正军 邓 君 范锟铻 方勇 冯世海 冯正直 官浩 郭光华 韩春茂 韩军涛 郝岱峰 贺伟峰 胡大海 郇京宁 黄沙 霍然 姜笃银 金培生 赖文 雷晋 李德绘 李小兵 李晓亮 李孝建 李学拥 李 毅 李智 李宗瑜 梁光萍 刘文军 刘小龙 刘旭盛 刘琰 刘毅 陆树良 罗高兴 吕大伦 吕国忠 马朋林 马显杰 潘云川 彭曦 齐鸿燕 邱林 荣新洲 申传安 沈余明 沈运彪 史春梦 宋保强 宋国栋 宋华培 孙炳伟 孙天骏 谭谦 唐洪泰 陶克 童亚林 王达利 王德运 王光毅 王凌峰 王新刚 王杨 王一兵 魏在荣 吴健 吴军 吴银生 夏成德 肖厚安 肖健 肖仕初 谢挺 谢卫国 徐庆连 颜洪 杨磊 姚咏明 于家傲 袁志强 曾元临 詹剑华 张恒术 张家平 张建祥 张明华 张丕红 张 勤 张庆富 张逸 章一新 赵耀华 赵永健 朱世辉 以下按英文首字母排序 Chong Si Jack（新加坡） David N. Herndon（美国） Fiona Wood（澳大利亚） Malcolm Xing（邢孟秋，加拿大） Naiem S. Moiemen（英国） Ronald G. Tompkins（美国） Steven E. Wolf（美国） Tina L. Palmieri（美国） Yong-Ming Yu（尤永明，美国）
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