中华创伤杂志
中華創傷雜誌
중화창상잡지
Chinese Journal of Traumatology
2014年
6期
598-604
,共7页
软组织损伤%毛囊%干细胞%血管化
軟組織損傷%毛囊%榦細胞%血管化
연조직손상%모낭%간세포%혈관화
Soft tissue injuries%Hair follicle%Stem cells%Vascularization
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.
目的 探討基因脩飾毛囊榦細胞(hair follicle stem cells,HFSCs)複閤明膠(gelatin,Gel)-硫痠軟骨素(chondroitin sulfate,C6S)-透明質痠(hyaluronic acid,HA)三維支架對組織工程皮膚血管化的影響. 方法 採用冷凍-凍榦法製備Gel-C6S-HA三維支架,接種VEGF165基因脩飾的HFSCs,電鏡觀察複閤支架內細胞形態、貼壁情況;取18隻SD大鼠,每隻揹部正中線徬開兩側各做2箇1.2cm×1.2 cm全層皮膚缺損創麵.按隨機數字錶法分為A組(VEGF165轉染HFSCs/Gel-C6S-HA支架)、B組(空載體轉染HFSCs/Gel-C6S-HA支架)、C組(Gel-C6S-HA支架)和D組(凡士林紗佈).將A、B、C組材料植入創麵,D組用無菌敷料覆蓋.術後7,14,21 d觀察創麵愈閤情況;採集標本行HE染色;CD31、α-平滑肌肌動蛋白(alpha smooth muscle actin,α-SMA)免疫組化檢測,微血管密度(microvessel density,MVD)計數,評價新生血管形成情況. 結果 電鏡下支架形成海綿狀三維結構,孑孔間存在交通孑孔相連,呈圓形或多邊形,孔徑(133.2±43.4) μm.複閤支架培養7d後,電鏡下可見細胞完全鋪展開,貼壁牢固.形態學觀察術後7d,四組創麵無明顯紅腫反應,術後14,21 d,A組創麵愈閤速度明顯高于其他組,其移植物吸收速度快.組織學和免疫學檢測結果錶明,術後7d,A、B組移植物內均有微血管生成,支架三維結構形態蓬鬆,細胞分佈均勻,C組支架輪廓清晰,與皮下組織結閤處少量細胞聚集;術後14,21 d,A組形成的新生血管最多且大,A、B組支架內充滿細胞併有部分嚮錶皮層聚集,支架材料均有不同程度的吸收,C組有皮下組織細胞嚮支架內遷移.術後各時相點,A組MVD明顯高于B、C組(P＜0.05). 結論 VEGF165基因脩飾HFSCs複閤Gel-C6S-HA支架構建的皮膚替代物可促進創麵愈閤過程中的血管新生,是一種很有應用前景的組織工程皮膚替代物.
목적 탐토기인수식모낭간세포(hair follicle stem cells,HFSCs)복합명효(gelatin,Gel)-류산연골소(chondroitin sulfate,C6S)-투명질산(hyaluronic acid,HA)삼유지가대조직공정피부혈관화적영향. 방법 채용냉동-동간법제비Gel-C6S-HA삼유지가,접충VEGF165기인수식적HFSCs,전경관찰복합지가내세포형태、첩벽정황;취18지SD대서,매지배부정중선방개량측각주2개1.2cm×1.2 cm전층피부결손창면.안수궤수자표법분위A조(VEGF165전염HFSCs/Gel-C6S-HA지가)、B조(공재체전염HFSCs/Gel-C6S-HA지가)、C조(Gel-C6S-HA지가)화D조(범사림사포).장A、B、C조재료식입창면,D조용무균부료복개.술후7,14,21 d관찰창면유합정황;채집표본행HE염색;CD31、α-평활기기동단백(alpha smooth muscle actin,α-SMA)면역조화검측,미혈관밀도(microvessel density,MVD)계수,평개신생혈관형성정황. 결과 전경하지가형성해면상삼유결구,혈공간존재교통혈공상련,정원형혹다변형,공경(133.2±43.4) μm.복합지가배양7d후,전경하가견세포완전포전개,첩벽뢰고.형태학관찰술후7d,사조창면무명현홍종반응,술후14,21 d,A조창면유합속도명현고우기타조,기이식물흡수속도쾌.조직학화면역학검측결과표명,술후7d,A、B조이식물내균유미혈관생성,지가삼유결구형태봉송,세포분포균균,C조지가륜곽청석,여피하조직결합처소량세포취집;술후14,21 d,A조형성적신생혈관최다차대,A、B조지가내충만세포병유부분향표피층취집,지가재료균유불동정도적흡수,C조유피하조직세포향지가내천이.술후각시상점,A조MVD명현고우B、C조(P＜0.05). 결론 VEGF165기인수식HFSCs복합Gel-C6S-HA지가구건적피부체대물가촉진창면유합과정중적혈관신생,시일충흔유응용전경적조직공정피부체대물.
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.
