3D打印皮肤成体干细胞来源类器官人工皮肤修复小鼠皮肤缺损  

Repair of skin defect with 3D-bioprinted organoid artificial skin derived from adult stem cells in mice

作　　者：张涛 崔进 刘媛媛[2] 苏佳灿 Zhang Tao;Cui Jin;Liu Yuanyuan;Su Jiacan(Department of Orthopedics,First Affiliated Hospital,Naval Medical University,Shanghai 200433,China;Institute of Translational Medicine,Shanghai University,Shanghai 200444,China;Department of Orthopedics,Xinhua Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200092,China)

机构地区：[1]海军军医大学第一附属医院创伤骨科,上海200433 [2]上海大学转化医学研究院,上海200444 [3]上海交通大学医学院附属新华医院骨科,上海200092

出　　处：《中华创伤杂志》2024年第1期40-47,共8页Chinese Journal of Trauma

基　　金：国家自然科学基金(82172098)。

摘　　要：目的构建3D打印皮肤成体干细胞来源类器官人工皮肤并探讨其修复小鼠皮肤缺损的效果。方法将人皮肤角质形成细胞、成纤维细胞、血管内皮细胞以2∶1∶1制备细胞混合悬液,在超低吸附培养板中培养,倒置相差显微镜观察细胞球的形态变化。培养7 d后收集细胞球,进行免疫荧光染色,检测其真皮、表皮及血管标志物的表达情况及结构分布情况。采用3D打印技术打印类器官人工皮肤,观察打印出的人工皮肤的形态。将10只免疫缺陷balb/c雌性小鼠按随机数字表法分为水凝胶组和类器官组,每组5只。所有小鼠建立直径1 cm的全层皮肤缺损模型,水凝胶组创面覆盖水凝胶敷料,类器官组创面覆盖相同大小类器官人工皮肤。建模后0、4、8、12及16 d观察两组创面愈合大体情况及创面愈合率。建模后16 d时创面皮肤取材,HE染色观察创面表皮角化情况及表皮真皮连接情况,Masson染色观察创面胶原纤维的疏松致密程度及真皮层纤维厚度。结果(1)角质形成细胞、成纤维细胞、血管内皮细胞的细胞混合悬液在超低吸附培养板中可自聚集形成细胞球;倒置显微镜观察显示,随着培养时间延长,细胞球体积逐渐增大。(2)细胞球免疫荧光染色结果显示,细胞球外层表达表皮标志物如角蛋白(K)1、K10及K14,核心表达真皮标志物如波形蛋白(VIM)及血管标志物CD31,即表皮位于球体的外层,真皮和血管位于球体的中央,符合皮肤类器官的结构特点。(3)3D打印出的类器官人工皮肤呈圆形透明状,直径10 mm,厚度1 mm。(4)小鼠创面大体观察结果显示,两组小鼠创面面积随治疗时间延长均有缩小,类器官组创面愈合速度更快,建模后4 d时即出现明显上皮化,16 d时创面基本愈合。建模后0 d时两组创面外观无差异;建模后4、8 d时类器官组创面愈合率分别为(31.7±1.0)%、(52.4±5.4)%,水凝胶组分别为(24.3±6.8)%、(45.4±7.0)%(P>0.Objective To construct 3D-bioprinted organoid artificial skin derived from adult stem cells and investigate their effects on repair of skin defect in mice.Methods The cell suspension mixture was prepared with human skin keratinocytes,fibroblasts and vascular endothelial cells with a ratio of 2∶1∶1 and cultured in ultra-low attachment plates,and morphological changes of cell spheres were observed with an inverted phase contrast microscope.After 7 days of culture,cell spheres were collected and immunofluorescence staining was performed to characterize the expression and structural distribution of the epidermis,dermis and blood vessels.The artificial skin composed of skin organoids were printed through 3D printing and morphology of printed artificial skin and dressing was observed.Ten immunodeficient balb/c female mice were divided into hydrogel group and organoid group,with 5 mice in each group with the method of random number table.The full-thickness skin defect model with a diameter of 1 cm was established in all mice,and the wound was covered with the hydrogel dressings in hydrogel group and with 3D-printed skin organoids of the same size in organoid group.Wound healing and healing rate of the two groups were observed at 0,4,8,12 and 16 days after modeling.At 16 days after modeling,HE staining was performed on wound skin samples to observe the epidermal keratosis and dermal epidermal junction of the wound surface and Masson staining to observe the density of collagen fibers and dermal fiber thickness of the wound surface.Results(1)The cell suspension mixture of keratinocytes,fibroblasts and vascular endothelial cells could self-aggregate into cell spheres in the ultra-low attachment plates,and it was observed with the inverted phase contrast microscope that the volume of cell spheres gradually increased with the extension of culture time.(2)Immunofluorescence staining of the cell spheres showed that epidermal markers such as keratin(K)1,K10,and K14 were expressed in the outer layer of the cell spheres,and de

关 键 词：类器官 成体干细胞 软组织损伤 伤口愈合 

分 类 号：R622[医药卫生—整形外科]