中国组织工程研究

中国组织工程研究 ›› 2026, Vol. 30 ›› Issue (14): 3618-3625.doi: 10.12307/2026.048

• 复合支架材料 composite scaffold materials • 上一篇    下一篇

用于肝损伤修复的3D打印微结构丝素蛋白支架制备及表征

史潇楠1，2，吴  璇1，张大旭2，胡婧婧1，2，郑亚哲2，刘昱彤1，赵  烁1，2，李伟龙1，2，叶淑君1，王婧怡1，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:2025-03-10 接受日期:2025-04-19 出版日期:2026-05-18 发布日期:2025-09-11
  • 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
  • 作者简介:史潇楠，女，1998年生，山西省阳泉市人，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项目(2019YFA0110600)，项目参与人：阎丽；国家自然科学基金项目(31971263)，项目负责人：阎丽

Preparation and characterization of 3D printed microstructured silk fibroin scaffold for liver injury repair

Shi Xiaonan1, 2, Wu Xuan1, Zhang Daxu2, Hu Jingjing1, 2, Zheng Yazhe2, Liu Yutong1, Zhao Shuo1, 2, Li Weilong1, 2, Ye Shujun1, Wang Jingyi1, Yan Li1   

  1. 1Department of Geriatrics, Second Medical Center, Chinese PLA General Hospital, Beijing 100853, China; 2Institute of Orthopedics of First Medical Center, Beijing Key Lab of Regenerative Medicine in Orthopedics, Key Laboratory of Musculoskeletal Trauma & War Injuries PLA, Chinese PLA General Hospital, Beijing 100853, China
  • Received:2025-03-10 Accepted:2025-04-19 Online:2026-05-18 Published:2025-09-11
  • Contact: Yan Li, MD, Chief physician, Professor, Department of Geriatrics, Second Medical Center, Chinese PLA General Hospital, Beijing 100853, China
  • About author:Shi Xiaonan, Master candidate, Department of Geriatrics, Second Medical Center, Chinese PLA General Hospital, Beijing 100853, China; Institute of Orthopedics of First Medical Center, 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 YL); National Natural Science Foundation of China, No. 31971263 (to YL)

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摘要：

文题释义：
3D打印微结构丝素蛋白支架：通过物理及化学方法将丝素蛋白脱胶、接枝后，可以得到能够进行3D打印的生物墨水，采用数字光学处理3D打印机制备3D微结构丝素蛋白支架。
接枝：在脱胶后的丝素蛋白水溶液中加入甲基丙烯酸缩水甘油酯，丝素蛋白中的胺基可以与甲基丙烯酸缩水甘油酯中的活性环氧侧基偶联完成接枝反应，生成甲基丙烯酸丝素蛋白，形成生物墨水，用于3D打印。

背景：肝脏组织工程是治疗肝损伤的重要措施，前期研究采用脂肪干细胞联合植物纤维支架、静电纺丝丝素蛋白支架、脱细胞肝脏支架修复小鼠肝损伤取得了良好效果，但是支架材料存在机械性能差、微结构不规则、无法最大程度模拟肝脏微结构等问题。
目的：制备3D打印微结构丝素蛋白支架，评估该支架的生物相容性。
方法：①将蚕茧脱胶处理后得到的丝素纤维溶解于溴化锂溶液中，随后加入甲基丙烯酸缩水甘油酯进行接枝，将混合溶液透析、冻干后加入蒸馏水，配置3D打印生物墨水，加入光引发剂后进行3D打印，制备3D打印微结构丝素蛋白支架。检测3D打印支架的结构与压缩弹性模量。②将第3代小鼠脂肪干细胞分2组培养：对照组为平面培养，实验组为将细胞接种于3D打印微结构丝素蛋白支架中，CCK-8法检测支架的细胞毒性；活/死染色检测细胞增殖能力；扫描电镜观察细胞黏附情况；成肝诱导培养后，ELISA法检测诱导后细胞上清中白蛋白、甲胎蛋白分泌情况，RT-PCR检测诱导后细胞中白蛋白、细胞角蛋白18及细胞色素 P450 mRNA表达。③取9只BALB/c小鼠，暴露肝左叶后将3D打印微结构丝素蛋白支架缝合固定于肝脏表面，术后第2，7，28天观察支架降解情况与肝脏-支架接触部位组织形态。
结果与结论：①扫描电镜下见3D打印微结构丝素蛋白支架呈多孔状，孔隙结构规则、分布均匀，孔径为100 μm，支架的压缩弹性模量为11.96 kPa。②3D打印微结构丝素蛋白支架无细胞毒性，脂肪干细胞可以黏附在该支架上并进行增殖，在支架内均匀分布；成肝诱导培养后，与对照组相比，支架组细胞上清内白蛋白、甲胎蛋白分泌增加(P < 0.05)，白蛋白、细胞角蛋白18及细胞色素 P450 mRNA表达升高(P < 0.05)。③随着术后时间的延长，支架与肝脏表面逐渐贴合且支架逐渐降解；苏木精-伊红染色显示，术后第2天，支架与肝脏表面存在明显交界，交界处存在较多炎细胞浸润，细胞开始进入支架内部；术后第7天，支架与肝脏表面交界处开始融合，交界处炎细胞浸润减少，支架内形成较多空洞且有较多细胞；术后第28天，支架与肝脏表面无明显交界，交界处炎症反应基本消失，支架明显降解。④结果表明，3D打印微结构丝素蛋白支架具有良好的生物相容性，能促进脂肪干细胞的黏附、增殖与成肝细胞样细胞分化。

https://orcid.org/0000-0001-7948-6119 (史潇楠) 
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: 3D打印, 支架, 组织工程, 丝素蛋白, 脂肪干细胞, 肝损伤, 肝细胞样细胞, 微结构

Abstract: BACKGROUND: Liver tissue engineering is an important strategy for treating liver injury. Previous studies have achieved good results in repairing mouse liver injury using adipose derived stem cells combined with plant fiber scaffolds, electrospun silk protein scaffolds, and decellularized liver scaffolds. However, the scaffold materials have poor mechanical properties, irregular microstructures, and cannot simulate the liver microstructure to the greatest extent.
OBJECTIVE: To fabricate microstructured silk fibroin scaffolds using 3D printing technology and evaluate their biocompatibility.
METHODS: (1) The silk fibroin fiber obtained after degummed silkworm cocoon was dissolved in lithium bromide solution, and then glycidyl methacrylate was added for grafting. The mixed solution was dialyzed, lyophilized, and added with distilled water to prepare 3D printing bio-ink. The microstructure and 
compressive elastic modulus of the scaffold were detected. (2) The third generation of mouse adipose derived stem cells were divided into two groups: the control group was cultured in flat plane, and the experimental group was seeded in 3D printed microstructured silk fibroin scaffold. The cytotoxicity of the scaffold was detected by CCK-8 assay. Cell proliferation was detected by live/dead staining. Cell adhesion was observed by scanning electron microscopy. After induction, the secretion of albumin and alpha-fetoprotein in the supernatant of the induced cells was detected by ELISA, and the expression of albumin, cytokeratin 18 and cytochrome P450 mRNA in the induced cells was detected by RT-PCR. (3) Nine BALB/c mice were selected, and the 3D printed microstructured silk fibroin scaffold was sutured and fixed to the surface of the liver after exposing the left lobe of the liver. The degradation of the scaffold and the tissue morphology of the liver-scaffold contact site were observed on days 2, 7, and 28 after surgery.
RESULTS AND CONCLUSION: (1) The 3D printed microstructured silk fibroin scaffold was porous under scanning electron microscopy, with regular and uniform pore structure, pore size of 100 μm, and the compressive elastic modulus of the scaffold was 11.96 kPa. (2) 3D printed microstructured silk fibroin scaffolds were non-cytotoxic, and adipose stem cells could adhere to the scaffolds and proliferate, and were evenly distributed in the scaffolds. After hepatogenic induction culture, compared with the control group, the secretion of albumin and alpha-fetoprotein in the scaffold group increased (P < 0.05), and the expression of albumin, cytokeratin 18, and cytochrome P450 mRNA increased (P < 0.05). (3) With the extension of postoperative time, the scaffold gradually adhered to the liver surface and the scaffold gradually degraded. Hematoxylin-eosin staining showed that on day 2 after surgery, there was a clear junction between the scaffold and the liver surface, and there were more inflammatory cell infiltrations at the junction, and cells began to enter the inside of the scaffold. On day 7 after surgery, the junction between the scaffold and the liver surface began to fuse, the inflammatory cell infiltration at the junction decreased, and more cavities and more cells were formed in the scaffold. On day 28 after surgery, there was no obvious junction between the scaffold and the liver surface, the inflammatory reaction at the junction basically disappeared, and the scaffold was significantly degraded. (4) The results showed that the 3D printed microstructured silk fibroin scaffold had good biocompatibility and could promote the adhesion, proliferation, and hepatocyte-like cell differentiation of adipose-derived stem cells.

Key words: 3D printing, scaffold, tissue engineering, silk fibroin, adipose derived stem cell, liver injury, hepatocyte-like cell, microstructure

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