Objective To investigate the effect of three-dimensional gelatin-chondroitin sulfatehyaluronic acid (Gel-C6S-HA) composite scaffold seeded with genetically modified hair follicle stem cells (HFSCs) on tissue engineering skin angiogenesis.Methods Three-dimensional scaffolds composed of Gel-C6S-HA were fabricated by freeze-lyophilizing.VEGF165 modified HFSCs were seeded on those scaffolds and cellular morphology and adhesion were observed using scanning electron microscope.Eighteen rats were subjected to the full-thickness skin defects with dimensions of 1.2 cm × 1.2 cm on the bilateral sides of the back and covered with VEGF165 transduced HFSCs/Gel-C6S-HA scaffold (Group A),empty-vector transduced HFSCs/Gel-C6S-HA scaffold (Group B),Gel-C6S-HA scaffold (Group C),and vaseline gauze (Group D) respectively according to the random number table.At days 7,14,and 21 after surgery,wound healing was observed,HE staining and immunohistochemistry of CD31 and alpha smooth muscle actin (α-SMA) were performed,and microvessel density (MVD) was used to measure new blood vessel growth.Results Electron microscopy exhibited three-dimensional spongy structure of the scaffold with round or polygon apertures connecting mutually and the scaffold pore size of (133.2 ± 43.4) μm.Cells seeded on the scaffolds spread thoroughly out and anchored firmly after being cultured for 7 days.There were no obvious inflammation reactions on the wounds for all groups at day 7 after operation.Wound healing and scaffold degradation were faster in Group A than in other groups at days 14 and 21 after operation.Histological and immunological detections showed microvasculariztion of the scaffold in Groups A and B with fluffy three-dimensional structure and evenly distributed cells,but scaffolds remained sharp at the edge and there were small cells aggregating at subcutaneous tissue junction area in Group C.At 14 and 21 days after operation,new blood vessels were large and rich in Group A; scaffolds in Groups A and group B were filled with cells,partial of which gathered in the epidermal layer and the scaffold materials were assimilated differentially,whereas a few subcutaneous tissue cells migrated to scaffolds in Group C.MVD was significantly higher in Group A than in Groups B and C at each time point (P ＜ 0.05).Conclusion VEGF165 modified HFSCs compounded with Gel-C6S-HA composite scaffold can facilitate the growth of blood vessels and promote the angiogenesis in wound healing and hence is a promising skin substitute in clinical applications.%目的 探讨基因修饰毛囊干细胞(hair follicle stem cells,HFSCs)复合明胶(gelatin,Gel)-硫酸软骨素(chondroitin sulfate,C6S)-透明质酸(hyaluronic acid,HA)三维支架对组织工程皮肤血管化的影响. 方法 采用冷冻-冻干法制备Gel-C6S-HA三维支架,接种VEGF165基因修饰的HFSCs,电镜观察复合支架内细胞形态、贴壁情况;取18只SD大鼠,每只背部正中线旁开两侧各做2个1.2cm×1.2 cm全层皮肤缺损创面.按随机数字表法分为A组(VEGF165转染HFSCs/Gel-C6S-HA支架)、B组(空载体转染HFSCs/Gel-C6S-HA支架)、C组(Gel-C6S-HA支架)和D组(凡士林纱布).将A、B、C组材料植入创面,D组用无菌敷料覆盖.术后7,14,21 d观察创面愈合情况;采集标本行HE染色;CD31、α-平滑肌肌动蛋白(alpha smooth muscle actin,α-SMA)免疫组化检测,微血管密度(microvessel density,MVD)计数,评价新生血管形成情况. 结果 电镜下支架形成海绵状三维结构,孑孔间存在交通孑孔相连,呈圆形或多边形,孔径(133.2±43.4) μm.复合支架培养7d后,电镜下可见细胞完全铺展开,贴壁牢固.形态学观察术后7d,四组创面无明显红肿反应,术后14,21 d,A组创面愈合速度明显高于其他组,其移植物吸收速度快.组织学和免疫学检测结果表明,术后7d,A、B组移植物内均有微血管生成,支架三维结构形态蓬松,细胞分布均匀,C组支架轮廓清晰,与皮下组织结合处少量细胞聚集;术后14,21 d,A组形成的新生血管最多且大,A、B组支架内充满细胞并有部分向表皮层聚集,支架材料均有不同程度的吸收,C组有皮下组织细胞向支架内迁移.术后各时相点,A组MVD明显高于B、C组(P＜0.05). 结论 VEGF165基因修饰HFSCs复合Gel-C6S-HA支架构建的皮肤替代物可促进创面愈合过程中的血管新生,是一种很有应用前景的组织工程皮肤替代物.
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