Nie Jiaying, Yi Yangyan, Zhu Yuanzheng
Department of Plastic Surgery, the Second Affiliated Hospital of Nanchang University, Nanchang Jiangxi, 330006, P.R.China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2020 Feb 15;34(2):226-233. doi: 10.7507/1002-1892.201903064.
To explore the possibility of constructing tissue engineered adipose by adipose tissue derived extracellular vesicles (hAT-EV) combined with decellularized adipose tissue (DAT) scaffolds, and to provide a new therapy for soft tissue defects.
The adipose tissue voluntarily donated by the liposuction patient was divided into two parts, one of them was decellularized and observed by HE and Masson staining and scanning electron microscope (SEM). Immunohistochemical staining and Western blot detection for collagen type Ⅰ and Ⅳ and laminin were also employed. Another one was incubated with exosome-removed complete medium for 48 hours, then centrifuged to collect the medium and to obtain hAT-EV via ultracentrifugation. The morphology of hAT-EV was observed by transmission electron microscopy; the nanoparticle tracking analyzer (NanoSight) was used to analyze the size distribution; Western blot was used to analyse membrane surface protein of hAT-EV. Adipose derived stem cells (ADSCs) were co-cultured with PKH26 fluorescently labeled hAT-EV, confocal fluorescence microscopy was used to observe the uptake of hAT-EV by ADSCs. Oil red O staining was used to evaluate adipogenic differentiation after hAT-EV and ADSCs co-cultured for 15 days. The DAT was scissored and then injected into the bilateral backs of 8 C57 mice (6-week-old). In experimental group, 0.2 mL hAT-EV was injected weekly, and 0.2 mL PBS was injected weekly in control group. After 12 weeks, the mice were sacrificed, and the new fat organisms on both sides were weighed. The amount of new fat was evaluated by HE and peri-lipoprotein immunofluorescence staining to evaluate the ability of hAT-EV to induce adipogenesis .
After acellularization of adipose tissue, HE and Masson staining showed that DAT was mainly composed of loosely arranged collagen with no nucleus; SEM showed that no cells and cell fragments were found in DAT, and thick fibrous collagen bundles could be seen; immunohistochemical staining and Western blot detection showed that collagen type Ⅰ and Ⅳ and laminin were retained in DAT. It was found that hAT-EV exhibited a spherical shape of double-layer envelope, with high expressions of CD63, apoptosis-inducible factor 6 interacting protein antibody, tumor susceptibility gene 101, and the particle size of 97.9% hAT-EV ranged from 32.67 nmto 220.20 nm with a peak at 91.28 nm. Confocal fluorescence microscopy and oil red O staining showed that hAT-EV was absorbed by ADSCs and induced adipogenic differentiation. experiments showed that the wet weight of fat new organisms in the experimental group was significantly higher than that in the control group ( =2.278, =0.048). HE staining showed that the structure of lipid droplets in the experimental group was more than that in the control group, and the collagen content in the control group was higher than that in the experimental group. The proportion of new fat in the experimental group was significantly higher than that in the control group ( =4.648, =0.017).
DAT carrying hAT-EV can be used as a new method to induce adipose tissue regeneration and has a potential application prospect in the repair of soft tissue defects.
探讨脂肪组织来源的细胞外囊泡（hAT-EV）与去细胞脂肪组织（DAT）支架构建组织工程脂肪的可能性，为软组织缺损提供新的治疗方法。
将抽脂患者自愿捐献的脂肪组织分为两部分，一部分进行去细胞处理，通过苏木精-伊红（HE）染色、Masson染色及扫描电子显微镜（SEM）观察。同时采用免疫组织化学染色及蛋白质免疫印迹法检测Ⅰ型和Ⅳ型胶原蛋白及层粘连蛋白。另一部分与去除外泌体的完全培养基孵育48小时，然后离心收集培养基，通过超速离心获得hAT-EV。采用透射电子显微镜观察hAT-EV的形态；用纳米颗粒跟踪分析仪（NanoSight）分析其大小分布；用蛋白质免疫印迹法分析hAT-EV的膜表面蛋白。将脂肪干细胞（ADSCs）与PKH26荧光标记的hAT-EV共培养，用共聚焦荧光显微镜观察ADSCs对hAT-EV的摄取情况。hAT-EV与ADSCs共培养15天后，用油红O染色评估成脂分化情况。将DAT剪碎后注射到8只6周龄C57小鼠的双侧背部。实验组每周注射0.2 mL hAT-EV，对照组每周注射0.2 mL PBS。12周后处死小鼠，称取双侧新生成脂肪组织的重量。通过HE染色及周围脂蛋白免疫荧光染色评估新脂肪生成量，以评价hAT-EV诱导脂肪生成的能力。
脂肪组织去细胞处理后，HE染色和Masson染色显示DAT主要由排列疏松的无细胞核胶原蛋白组成；SEM显示DAT中未见细胞及细胞碎片，可见粗大的纤维状胶原束；免疫组织化学染色及蛋白质免疫印迹法检测显示DAT中保留了Ⅰ型和Ⅳ型胶原蛋白及层粘连蛋白。发现hAT-EV呈双层包膜球形，CD63、凋亡诱导因子6相互作用蛋白抗体、肿瘤易感基因101表达较高，97.9%的hAT-EV粒径范围为32.67 nm至220.20 nm，峰值为91.28 nm。共聚焦荧光显微镜及油红O染色显示hAT-EV被ADSCs吸收并诱导成脂分化。实验显示实验组新生成脂肪组织的湿重显著高于对照组（t = 2.278，P = 0.048）。HE染色显示实验组脂滴结构多于对照组，对照组胶原含量高于实验组。实验组新生成脂肪的比例显著高于对照组（t = 4.648，P = 0.017）。
携带hAT-EV的DAT可作为诱导脂肪组织再生的新方法，在软组织缺损修复中具有潜在的应用前景。
