Experimental study on the effect of three-dimensional porous structures on the vascularization rate of artificial dermis
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摘要:
目的 探索定向排列的三维多孔网状（A型）结构和蜂窝煤状垂直贯穿的三维多孔网状（B型）结构对人工真皮血管化速率的影响。 方法 采用实验研究方法。本研究中的人工真皮为硅胶层和支架层双层结构，根据支架层结构不同，分为含A型结构和B型结构的人工真皮（以下分别简称A型真皮、B型真皮），其中的A型结构和B型结构分别采用梯度冷冻干燥技术和物理制孔技术制得。采用扫描电镜观测2种真皮支架的微观形貌。采用比重瓶法测定2种真皮支架的孔隙率。参照国家医药行业标准中的方法，于降解4、8、13、24 h测定2种真皮降解液及残留物中羟脯氨酸的含量，反映2种真皮降解率。取L929细胞，按照随机数字表法分为A型真皮组、B型真皮组、阴性对照组、阳性对照组，阳性对照组加入含体积分数5%二甲基亚砜的MEM培养基，阴性对照组加入高密度聚乙烯浸提液，其余2组加入相应的浸提液培养24 h，采用噻唑蓝试剂测定细胞增殖率，并对细胞毒性进行定级。取L929细胞和人脐静脉内皮细胞（HUVEC），接种于预先置有2种真皮的孔板。接种后1、4、7、14 d，采用免疫荧光法检测L929细胞在2种真皮支架表面的黏附生长状况。接种后7 d，采用免疫荧光法和苏木精-伊红（HE）染色法检测前述2种细胞长入2种真皮支架的情况。在3只6个月龄雄性巴马小型猪背部两侧各制作3个5.0 cm×5.0 cm的全层皮肤缺损创面，6列创面按照随机数字表法分为A型真皮两步法组、B型真皮两步法组和B型支架一步法组。A型真皮两步法组和B型真皮两步法组的创面分别先行A型真皮或B型真皮移植后，再行自体刃厚皮片的移植，B型支架一步法组的创面行B型真皮（揭除硅胶层）+自体刃厚皮片一步法移植。大体观察Ⅱ期术后7 d A型真皮两步法组和B型真皮两步法组及Ⅰ期术后14 d B型支架一步法组的猪背创面出血、渗液和感染情况。同时，采用透明胶片网格法测定自体皮移植面积并计算其存活率。Ⅰ期术后4、7、14 d，HE染色法检测3组猪背创面中支架的炎症细胞、成纤维细胞（Fb）和毛细血管浸润情况。Ⅰ期术后7 d，免疫组织化学法进一步检测3组猪背创面中支架的血管化情况。Ⅰ期术后28 d、3个月，HE染色法检测A型真皮两步法组和B型支架一步法组猪背创面中支架的降解情况。对数据行单因素方差分析、独立样本t检验、Bonferroni校正。 结果 A型真皮支架表面均匀分布着大量圆形和椭圆形的微孔，纵切面可观察到柱状孔壁大体呈平行定向排列；B型真皮支架表面的蜂窝煤状贯穿大孔呈矩阵有序排列，纵切面蜂窝煤状贯穿孔的孔壁由微孔相互连通成网络结构。A型真皮支架的孔隙率为（93.2±0.7）%，与B型的（95.9±1.0）%相近（t=4.653，P>0.05）。A型真皮在4、8、13、24 h的降解率与B型真皮对应时间点的降解率相近（t=0.232、0.856、0.258、7.716，P>0.05）。培养24 h，A型真皮组、B型真皮组、阴性对照组L929细胞的增殖率显著高于阳性对照组（t=2 393.460、2 538.270、1 077.770，P<0.01）；阳性对照组细胞毒性评级为4级，其余3组为0级。接种后1、4、7、14 d，L929细胞和HUVEC在2种真皮支架中均呈时间依赖性增殖；且2种细胞在B型真皮上的黏附生长、增殖速率高于A型真皮。接种后7 d，L929细胞和HUVEC均已长满B型真皮支架层且至硅胶层一侧；而前述2种细胞向A型真皮内部迁移速度较慢，硅胶层一侧仅见少量细胞。Ⅱ期术后7 d A型真皮两步法组和B型真皮两步法组及Ⅰ期术后14 d B型支架一步法组创面均未出现出血、渗液、感染等情况；3组各6个创面的自体皮植皮存活率均为100%。Ⅰ期术后4、7、14 d，炎症细胞、Fb、毛细血管等逐渐向创面的支架层浸润，且细胞浸润速率从高到低依次为B型支架一步法组、B型真皮两步法组、A型真皮两步法组。Ⅰ期术后7 d 3组创面中支架的血管化速率从高到低依次为B型支架一步法组、B型真皮两步法组、A型真皮两步法组。B型支架一步法组猪背创面中的支架在术后28 d逐渐溃散，术后3个月完全降解；A型真皮两步法组猪背创面中的支架降解情况与前述相似。 结论 与A型结构相比，B型结构可加速人工真皮支架血管化进程，利于联合自体刃厚皮一步法移植修复猪全层皮肤缺损创面。一步法移植的效果与分次移植人工真皮与自体刃厚皮的两步法一致，可为创面治疗提供更优选择。 Abstract:Objective To explore the effects of orienting three-dimensional porous network (type A) and honeycomb briquette-shaped vertically penetrating three-dimensional porous network (type B) on the vascularization rate of artificial dermis. Methods The experimental research method was used. The artificial dermis was composed of a double layer of silicone layer and scaffold layer. Based on the difference of scaffold layer, they were divided into type A and type B artificial dermis (type A dermis and type B dermis, for short) containing type A and type B structure, respectively. The type A and type B structures were prepared by gradient freeze-drying technique and physical pore-making technique, respectively. The micro-morphology of two kinds of dermis scaffold was observed by scanning electron microscopy. The porosity of two kinds of dermis scaffold was measured by the Pyrex method. According to the method of national medical industry standard, the hydroxyproline content in degradation liquids and their residues in two kind of dermis were determined after degradation at 4, 8, 13, and 24 h, reflecting the degradation rates of two kinds of dermis. According to the random number table, L929 cells were divided into type A dermis group, type B dermis group, negative control group, and positive control group. The positive control group was added with minimum essential medium (MEM) containing 5% dimethyl sulfoxide, The negative control group was added with high-density polyethylene extract, and the other two groups were added with the corresponding extract. At 24 hours after culture, the growth rate of L929 cells was detected by methyl thiazolyl tetrazolium, and the cytotoxicity was graded. L929 cells and human umbilical vein endothelial cells (HUVECs) were inoculated into pore plates with two kinds of dermis preinstalled. On 1, 4, 7, and 14 d after inoculating, the adhesion and growth of L929 cells on the surfaces of the two kinds of scaffolds were detected by immunofluorescence method. On 7 d after inoculating, the migration of the above two kinds of cells into the two kinds of dermal scaffolds was detected by immunofluorescence and hematoxylin-eosin (HE) staining. Three full-thickness skin defect wounds of 5.0 cm×5.0 cm were created on both sides of the back of three 6-month-old healthy male Ba-Ma mini pigs. According to the random number table, six columns of wounds were divided into type A dermis two-step method group, type B dermis two-step method group, and type B dermis one-step method group. The wounds in type A dermis two-step method group and type B dermis two-step method group were transplanted with type A or type B dermis respectively before, and with autologous split-thickness skin grafting later. The wounds in type B dermis one-step method group were transplanted in a synchronous procedure including type B dermis (without silicone layer) and autologous skin grafting simultaneously. The bleeding, exudation, and infection of the wounds on the back in type A dermis two-step method group and type B dermis two-step method group on the 7th day after the second transplantation and in type B dermis one-step method group on the 14th day after the first transplantation were generally observed. The area of autologous skin graft was measured by the transparent film grid method, and the survival rate of autologous skin was calculated. On 4, 7, and 14 d after the first transplantation, the inflammatory cells, fibroblasts (Fbs), and capillary infiltration into the scaffolds of the three groups were detected by HE staining. On 7, 14 d after the first transplantation, the vascularization of the scaffolds was further observed by immunohistochemistry. On 28, 90 d after the first operation, the degradation of the scaffolds of type A dermis and type B dermis was observed by HE staining. Data were statistically analyzed with one-way analysis of variance, independent sample t test, and Bonferroni correction. Results A large number of round and oval micropores were evenly distributed on the surface of type A scaffold, and the cylindrical hole walls could be observed arranging in a parallel direction in the longitudinal section. The honeycomb briquette-shaped penetrating macropores on the surface of type B scaffold were arranged in an orderly matrix. The pore walls of the honeycomb briquette-shaped penetrating macropores were connected by micropores to form a network structure. The porosity of type A dermis was (93.21±0.72)%, which was similar to (95.88±1.00)% of type B dermis (t=4.653, P>0.05). The degradation rates of type A dermis at 4, 8, 13, and 24 h were similar to those of type B dermis at the corresponding time point (t=0.232, 0.856, 0.258, 7.716, P>0.05). At 24 h after culture, the proliferation rates of L929 cells in the type A dermis group, type B dermis group, and negative control group were significantly higher than those of the positive control group (t=2 393.46, 2 538.27, 1 077.77, P<0.01). The cytotoxicity rating of cells in positive control group was grade 4, while that of the other three groups was grade zero. On 1, 4, 7, and 14 d after inoculation, both L929 cells and HUVECs proliferated in a time-dependent manner in two kinds of dermal scaffolds. The adhesion growth and proliferation rate of the two kinds of cells on the surface of type B dermis was higher than that of type A dermis. On 7 d after inoculation, both L929 cells and HUVECs covered the surface of type B dermis and migrated into one side of the silicone layer. However, the above two kinds of cells migrated slowly into type A dermis, and only a few cells were found on one side of the silicone layer. There was no bleeding, exudation, or infection in the wounds repaired by type A and type B dermis. The survival rate of autologous skin grafting of 6 wounds in each group was 100%. On 4, 7, and 14 d after the first operation, inflammatory cells, Fbs, and capillaries gradually infiltrated into the scaffold layer, and the cell infiltration rate from high to low was type B dermis one-step method group, type B dermis two-step method group, and type A dermis two-step method group. The scaffold in wound in the type B dermis one-step method group gradually collapsed on 28 d after the first operation, and completely degraded in 3 months after the first operation. The scaffold degradation rate of type A dermis two-step method group was similar to that mentioned above. Conclusions The honeycomb briquette-shaped vertically penetrating three-dimensional porous network structure of type B scaffold can accelerate its vascularization process, which is beneficial to autogenous split-thickness skin in one-step procedure to repair full-thickness skin defects wound in Ba-Ma mini pigs. Compared with the "two-step method" of staged transplantation of type A scaffold and autologous split-thickness skin, and one-step transplantation has equal efficacy and can provide a better choice for wound treatment. -
Key words:
- Skin, artificial /
- Wound healing /
- Three-dimensional /
- Pore structure /
- Full-thickness skin defect /
- Vascularization
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参考文献
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1 2种人工真皮试样支架表面和纵切面形貌观察。1A.A型真皮表观形貌，表面均匀分布着大量圆形和椭圆形的微孔 扫描电镜×30，图中标尺为1 mm；1B.B型真皮表观形貌，可见蜂窝煤状贯穿大孔呈矩阵有序排列 扫描电镜×30，图中标尺为1 mm；1C.A型真皮纵切面形貌，可观察到柱状孔壁大体呈平行纵向排列 扫描电镜×200，图中标尺为200 μm；1D.B型真皮纵切面形貌，清晰可见蜂窝煤状贯穿大孔断面（白色虚线标记） 扫描电镜×100，图中标尺为300 μm
注：A型真皮指含定向排列三维多孔网状结构的人工真皮，B型真皮指含蜂窝煤状垂直贯穿三维多孔网状结构的人工真皮
2 不同时间点L929细胞在2种支架上的黏附生长情况 鬼笔环肽-罗丹明-4',6-二脒基-2-苯基吲哚×400，图中标尺为100 μm。2A、2B、2C、2D.分别为细胞在A型真皮接种1、4、7、14 d黏附生长情况，接种后的细胞均呈时间依赖性增殖；2E、2F、2G、2H.分别为细胞在B型真皮分别接种1、4、7、14 d黏附生长情况，与图2A、2B、2C、2D相比，细胞在B型真皮上的黏附生长、增殖速率更高
注：细胞骨架阳性染色为红色，细胞核阳性染色为蓝紫色；A型真皮指含定向排列三维多孔网状结构的人工真皮，B型真皮指含蜂窝煤状垂直贯穿三维多孔网状结构的人工真皮
3 接种后7 d 2种细胞在2种真皮支架中的长入情况 鬼笔环肽-罗丹明-4',6-二脒基-2-苯基吲哚×400，图中标尺为200 μm。3A、3B.分别为人脐静脉内皮细胞分别向A、B型真皮支架纵向长入情况，细胞长入B型真皮支架的数量明显多于A型真皮支架；3C、3D.分别为L929细胞分别向A、B型真皮支架纵向长入情况，细胞长入B型真皮支架的数量明显多于A型真皮支架
注：细胞骨架阳性染色为红色，细胞核阳性染色为蓝紫色；A型真皮指含定向排列三维多孔网状结构的人工真皮，B型真皮指含蜂窝煤状垂直贯穿三维多孔网状结构的人工真皮
4 接种后7 d 2种细胞在2种真皮支架中的长入情况 苏木精-伊红×400，图中标尺为100 μm。4A、4B.分别为人脐静脉内皮细胞分别向A、B型真皮支架长入情况，细胞长入B型真皮支架的数量明显多于A型真皮支架；4C、4D.分别为L929细胞分别向A、B型真皮支架长入情况，细胞长入B型真皮支架的数量明显多于A型真皮支架
注：细胞核阳性染色为蓝紫色（↑）；A型真皮指含定向排列三维多孔网状结构的人工真皮，B型真皮指含蜂窝煤状垂直贯穿三维多孔网状结构的人工真皮
5 3组猪背全层皮肤缺损创面移植自体皮存活情况。5A.A型真皮两步法组Ⅱ期术后7 d创面情况，创面移植的自体皮完全存活；5B.B型真皮两步法组Ⅱ期术后7 d的创面情况，创面移植的自体皮完全存活；5C.B型真皮支架一步法组术后14 d创面，创面移植的自体皮完全存活
注：图中小孔为柱状活检样取出后遗留的痕迹；A型真皮两步法组Ⅰ期术后14 d行Ⅱ期自体皮移植，B型真皮两步法组Ⅰ期术后7 d行Ⅱ期自体皮移植；A型真皮指含定向排列三维多孔网状结构的人工真皮，B型真皮指含蜂窝煤状垂直贯穿三维多孔网状结构的人工真皮
6 3组猪背全层皮肤缺损创面移植支架中的细胞浸润情况 苏木精-伊红×400，图中标尺为100 μm。6A.Ⅰ期术后4 d，A型真皮两步法组创面中支架内有炎症细胞浸润，未见成纤维细胞（Fb）浸润；6B.Ⅰ期术后7 d，A型真皮两步法组创面中支架内炎症细胞较图6A略有增加，支架创基侧有较多Fb浸润；6C.Ⅰ期术后14 d，A型真皮两步法组创面中支架内炎症细胞较图6B增多，整层支架完全被Fb和毛细血管浸润；6D.Ⅰ期术后4 d，B型真皮两步法组创面中的支架与创基交界处已有少量Fb浸润；6E.Ⅰ期术后7 d，B型真皮支架一步法组创面中的支架整层已被大量Fb浸润；6F.Ⅰ期术后14 d，B型真皮支架一步法组创面中的支架内炎症反应明显消退，支架孔隙间已生成大量致密的新生胶原
注：A型真皮指含定向排列三维多孔网状结构的人工真皮，B型真皮指含蜂窝煤状垂直贯穿三维多孔网状结构的人工真皮
7 3组猪背全层皮肤缺损创面中移植支架的血管化情况 二氨基联苯胺-苏木精×100，图中标尺为500 μm。7A.Ⅰ期术后7 d，A型真皮两步法组创面支架中仅基底侧局部可见血管腔；7B. Ⅰ期术后14 d，A型真皮两步法组创面中支架内有较多细小血管样结构均匀长入；7C.Ⅰ期术后7 d，B型真皮两步法组创面中支架的蜂窝煤状贯穿大孔中有大量CD31阳性信号表达；7D.Ⅰ期术后7 d，B型支架一步法组创面中支架完全血管化
注：AD为支架层，AS为移植的自体皮；红色虚线标记处为蜂窝煤状贯穿大孔；棕色为CD31阳性表达，反映细胞血管化情况；A型真皮指含定向排列三维多孔网状结构的人工真皮，B型真皮指含蜂窝煤状垂直贯穿三维多孔网状结构的人工真皮
8 B型真皮支架一步法组和A型真皮两步法组猪背创面中的移植支架降解情况。8A.A型真皮两步法组术后28 d创面中支架的降解情况，支架溃散，仅可见大块碎片（↑） 苏木精-伊红×400，图中标尺为100 μm；8B.A型真皮两步法组术后90 d创面中支架降解情况，支架完全降解，所移植自体皮表皮突清晰可见，未见明显瘢痕增生，炎症反应完全消失 苏木精-伊红×100，图中标尺为500 μm；8C.B型真皮支架一步法组术后28 d创面中支架降解情况与8A相似 苏木精-伊红×400，图中标尺为100 μm；8D.B型真皮支架一步法组术后90 d创面中支架降解情况与8B相似 苏木精-伊红×100，图中标尺为500 μm
注：A型真皮指含定向排列三维多孔网状结构的人工真皮，B型真皮指含蜂窝煤状垂直贯穿三维多孔网状结构的人工真皮
《中华烧伤杂志》第六届编辑委员会通讯编委名单按姓氏拼音排序
贲道锋 卞徽宁 曹永倩 晁生武 陈辉 陈婧 陈朗 陈铭锐 陈鹏 陈晓东 陈忠勇 程君涛 迟云飞 储国平 党永明 邓呈亮 狄海萍 丁国兵 丁若虹 董茂龙 段红杰 段鹏 樊东力 房贺 冯光 付忠华 郭毅斌 韩兆峰 侯春胜 胡德林 胡炯宇 胡骁骅 胡晓燕 黄红军 纪世召 江华 姜丽萍 姜玉峰 雷娜 黎宁 李东杰 李峰 李靖 李晓东 李晓鲁 梁钢 梁鹏飞 林才 林国安 林源 刘德伍 刘健 刘军 刘淑华 龙奕 卢长虹 鲁峰 吕开阳 吕强 马思远 牛轶雯 欧阳军 乔亮 覃凤均 邱学文 曲滨 任超 沈江涌 石继红 宋慧锋 苏海涛 苏永涛 孙勇 孙瑜 谭江琳 唐修俊 滕苗 田社民 涂家金 汪虹 汪洋 王爱萍 王德怀 王洪涛 王会军 王良喜 王爽 王献珍 王志永 温冰 邬佳敏 吴红 吴继炎 吴巍巍 吴祖煌 向飞 向军 谢举临 谢松涛 辛海明 许喜生 许学文 薛斌 杨建民 杨敏烈 杨薛康 姚明 姚兴伟 叶祥柏 易成刚 易南 于东宁 岳丽青 翟红军 詹日兴 张博 张东霞 张红艳 张菊芳 张玲娟 张庆红 张彦琦 张寅 张元海 张志 赵全 赵冉 赵雄 郑德义 郑东风 郑军 周国富 周俊峄 周琴 周万芳 朱峰 朱宇刚 祝筱梅 邹立津 邹晓防 
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