中国组织工程研究

中国组织工程研究 ›› 2024, Vol. 28 ›› Issue (29): 4645-4651.doi: 10.12307/2024.588

• 材料生物相容性 material biocompatibility • 上一篇    下一篇

基于脱细胞方法制备3D植物肝组织工程支架及表征

胡婧婧1，2，何松霖1，张大旭1，2，赵  烁1，2，史潇楠1，2，李伟龙1，叶淑君1，王静怡1，郭全义1，2，阎  丽1   

  1. 1解放军总医院第二医学中心老年医学科，北京市  100853；2解放军总医院第一医学中心骨科研究所，骨科再生医学北京市重点实验室，全军骨科战创伤重点实验室，北京市  100853
  • 收稿日期:2023-11-17 接受日期:2023-12-23 出版日期:2024-10-18 发布日期:2024-03-22
  • 通讯作者: 阎丽，博士，主任医师，教授，解放军总医院第二医学中心老年医学科，北京市 100853 郭全义，博士，主任医师，教授，解放军总医院第二医学中心老年医学科，解放军总医院第一医学中心骨科研究所，骨科再生医学北京市重点实验室，全军骨科战创伤重点实验室，北京市 100853
  • 作者简介:胡婧婧，女，1996年生，汉族，解放军总医院在读硕士，主要从事肝脏再生智能化材料相关方向的研究。
  • 基金资助:
    国家重点研发计划课题(2019YFA0110600)，项目负责人：郭全义；国家自然科学基金项目(31971263)，项目负责人：阎丽

Preparation and characterization of 3D plant-based scaffold based on decellularization method in liver tissue engineering

Hu Jingjing1, 2, He Songlin1, Zhang Daxu1, 2, Zhao Shuo1, 2, Shi Xiaonan1, 2, Li Weilong1, Ye Shujun1, Wang Jingyi1, Guo Quanyi1, 2, Yan Li1   

  1. 1Second Medical Center and National Clinical Research Center of Geriatric Diseases, Chinese PLA General Hospital, Beijing 100853, China; 2Institute of Orthopedics, Beijing Key Lab of Regenerative Medicine in Orthopedics, Key Laboratory of Musculoskeletal Trauma & War Injuries PLA, Chinese PLA General Hospital, Beijing 100853, China
  • Received:2023-11-17 Accepted:2023-12-23 Online:2024-10-18 Published:2024-03-22
  • Contact: Yan Li, MD, Chief physician, Professor, Second Medical Center and National Clinical Research Center of Geriatric Diseases, Chinese PLA General Hospital, Beijing 100853, China Guo Quanyi, MD, Chief physician, Professor, Second Medical Center and National Clinical Research Center of Geriatric Diseases, Chinese PLA General Hospital, Beijing 100853, China; Institute of Orthopedics, Beijing Key Lab of Regenerative Medicine in Orthopedics, Key Laboratory of Musculoskeletal Trauma & War Injuries PLA, Chinese PLA General Hospital, Beijing 100853, China
  • About author:Hu Jingjing, Master candidate, Second Medical Center and National Clinical Research Center of Geriatric Diseases, Chinese PLA General Hospital, Beijing 100853, China; Institute of Orthopedics, Beijing Key Lab of Regenerative Medicine in Orthopedics, Key Laboratory of Musculoskeletal Trauma & War Injuries PLA, Chinese PLA General Hospital, Beijing 100853, China
  • Supported by:
    National Key Research and Development Plan Project, No. 2019YFA0110600 (to GQY); National Natural Science Foundation of China, No. 31971263 (to YL)

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摘要：


文题释义：

脱细胞方法：是一种通过物理、化学和生物酶法去除组织或器官中的细胞成分，以消除抗原性，同时保留器官的微观三维结构和含有生物活性的细胞外基质的技术。经过脱细胞处理获得的组织衍生脱细胞生物材料是组织工程应用中的理想选择。
植物脱细胞支架：经过脱细胞处理获得，保留了原有的三维结构和生物活性成分，能够很好地模拟体内微环境，具有良好的生物相容性。植物脱细胞支架来源丰富、制作简单，具有显著的经济发展及环境友好优势。


背景：组织工程为肝衰竭这一临床困境带来了新的希望，通过简单的制备方法制备植物脱细胞纤维支架对肝脏组织工程具有重要意义。

目的：采用新鲜苹果切片和十二烷基硫酸钠溶液制备苹果组织脱细胞支架材料，并评估其生物相容性。
方法：将新鲜苹果分别采用磷酸盐缓冲液和十二烷基硫酸钠溶液进行脱细胞处理，然后用磷酸盐缓冲液进行脱毒获得处理后的苹果组织和苹果脱细胞支架材料，然后在扫描电镜下观察苹果材料的脱细胞效果。实验从BALB/C小鼠腹股沟脂肪中提取脂肪干细胞，并且通过流式细胞术鉴定其干细胞相关标记物(CD45，CD34，CD73，CD90，CD105)的表达。然后分为无支架对照组和有支架组，在两组中接种等量脂肪干细胞，通过CCK-8、苏木精-伊红染色、鬼笔环肽骨架染色评价该脱细胞支架与脂肪干细胞的生物相容性，在光学显微镜、扫描电镜下观察细胞在支架上的黏附及生长情况。然后将支架分为非诱导组和成肝诱导组，将脂肪干细胞种植在脱细胞苹果支架上，分别在普通培养基和成肝诱导培养基中培养14 d后进行对比，通过肝细胞标记物(白蛋白、细胞角蛋白18、细胞色素P450家族1亚家族1)进行免疫荧光染色，通过酶联免疫吸附实验来检测白蛋白、甲胎蛋白的分泌情况，并通过扫描电镜观察支架上诱导培养的细胞形态，验证该脱细胞支架材料上肝细胞相关蛋白的表达。最后，实验通过将钴60照射消毒后的苹果脱细胞支架移植于小鼠肝脏表面，28 d后通过大体观及苏木精-伊红染色观察该支架的降解情况。

结果与结论：①扫描电镜结果显示，脱细胞后的苹果支架材料具有约100 μm大小的孔隙结构，不存在残余细胞。②通过流式细胞学鉴定，提取培养的细胞为脂肪间充质干细胞。③CCK-8结果表明，所制备的苹果组织脱细胞支架材料无细胞毒性；苏木精-伊红染色和鬼笔环肽骨架染色观察到脂肪间充质干细胞能够在苹果组织脱细胞支架上黏附和聚集生长。④免疫荧光染色、酶联免疫吸附实验结果显示，培养在苹果组织脱细胞支架上的脂肪间充质干细胞高表达白蛋白、甲胎蛋白、细胞角蛋白18及细胞色素P450家族1亚家族1肝脏相关蛋白，说明被诱导分化为了具有肝细胞功能的肝细胞样细胞。⑤干预7 d时植入的苹果脱细胞支架与肝脏融合，部分支架已经降解，干预28 d时苹果脱细胞支架已完全降解，被新生组织替代。⑥结果表明，来源于苹果组织制备的脱细胞支架材料具有良好的生物相容性，可促进脂肪间充质干细胞的增殖、黏附及成肝细胞分化。 

https://orcid.org/0000-0001-8795-2466(胡婧婧)；https://orcid.org/0000-0001-8881-1921(阎丽)；https://orcid.org/0000-0001-7154-2227(郭全义)

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料；口腔生物材料；纳米材料；缓释材料；材料相容性；组织工程

关键词: 植物组织, 支架, 脱细胞, 肝衰竭, 脂肪间充质干细胞, 组织工程, 肝细胞样细胞

Abstract: BACKGROUND: Tissue engineering has brought new hope to the clinical challenge of liver failure, and the preparation of plant-derived decellularized fiber scaffolds holds significant importance in liver tissue engineering.
OBJECTIVE: To prepare apple tissue decellularized scaffold material by using fresh apple slices and a solution of sodium dodecyl sulfate, and assess its biocompatibility. 
METHODS: Fresh apples were subjected to decellularization using phosphate buffer saline and sodium dodecyl sulfate solution, separately. Afterwards, the decellularized apple tissues and apple decellularized scaffold materials were decontaminated with phosphate buffer saline. Subsequently, scanning electron microscopy was used to assess the effectiveness of decellularization of the apple materials. Adipose-derived mesenchymal stem cells were extracted from the inguinal fat BALB/C of mice, and their expression of stem cell-related markers (CD45, CD34, CD73, CD90, and CD105) was identified through flow cytometry. The cells were then divided into a scaffold-free control group and a scaffold group. Equal amounts of adipose-derived mesenchymal stem cells were seeded onto both groups. The biocompatibility of the decellularized scaffold with adipose-derived mesenchymal stem cells was evaluated using CCK-8 assay, hematoxylin-eosin staining, and phalloidine staining. Cell adhesion and growth on the scaffold were observed under light microscopy and scanning electron microscopy. Furthermore, the scaffold was subdivided into the non-induced group and the hepatogenic-induced group. Adipose-derived mesenchymal stem cells were cultured on the decellularized apple scaffold, and they were cultured for 14 days in regular culture medium or hepatogenic induction medium for comparison. Immunofluorescent staining using liver cell markers, including albumin, cytokeratin 18, and CYP1A1, was performed. Enzyme-linked immunosorbent assay was used to detect the secretion of alpha fetoprotein and albumin. Additionally, scanning electron microscopy was employed to observe the morphology of the induced cells on the scaffold, verifying the expression of liver cell-related genes on the decellularized scaffold material. Finally, the cobalt-60 irradiated and sterilized decellularized apple scaffolds were transplanted onto the surface of mouse liver and the degradation of the scaffold was observed by gross observation and hematoxylin-eosin staining after 28 days.
RESULTS AND CONCLUSION: (1) The scanning electron microscopy results revealed that the decellularized apple scaffold material retained a porous structure of approximately 100 μm in size, with no residual cells observed. (2) Through flow cytometry analysis, the cultured cells were identified as adipose-derived mesenchymal stem cells. (3) CCK-8 assay results demonstrated that the prepared decellularized apple tissue scaffold material exhibited no cytotoxicity. Hematoxylin-eosin staining and phalloidine staining showed that adipose-derived mesenchymal stem cells were capable of adhering and proliferating on the decellularized apple tissue scaffold. (4) The results obtained from immunofluorescence staining and enzyme-linked immunosorbent assay revealed that adipose-derived mesenchymal stem cells cultured on the decellularized apple scaffolds exhibited elevated expression of liver-specific proteins, including albumin, alpha-fetoprotein, cytokeratin 18, and CYP1A1. These results suggested that they were induced differentiation into hepatocyte-like cells possessing functional characteristics of liver cells. (5) The decellularized apple scaffold implanted at 7 days has integrated with the liver, with partial degradation of the scaffold observed. By 28 days, the decellularized apple scaffold has completely degraded and has been replaced by newly-formed tissue. (6) The results indicate that the decellularized scaffold material derived from apple tissue demonstrates favorable biocompatibility, promoting the proliferation, adhesion, and hepatic differentiation of adipose-derived mesenchymal stem cells.

Key words: plant tissue, scaffold, decellularization, liver failure, adipose-derived mesenchymal stem cell, tissue engineering, hepatocyte-like cell

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