[1]丁晓明,张凯三,刘婷婷,等.丝素蛋白-羟基磷灰石三维大孔支架复合脂肪干细胞体外构建组织工程骨的可行性研究[J].中医正骨,2021,33(12):1-6.
　DING Xiaoming,ZHANG Kaisan,LIU Tingting,et al.A feasibility study of three-dimensional silk fibroin-hydroxyapatite macroporous scaffold-adipose-derived stem cells complex in constructing tissue-engineered bone in vitro[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2021,33(12):1-6.
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丝素蛋白-羟基磷灰石三维大孔支架复合脂肪干细胞体外构建组织工程骨的可行性研究()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第33卷
期数:
2021年12期
页码:
1-6
栏目:
基础研究
出版日期:
2021-12-20

文章信息/Info

Title:
A feasibility study of three-dimensional silk fibroin-hydroxyapatite macroporous scaffold-adipose-derived stem cells complex in constructing tissue-engineered bone in vitro
作者:
丁晓明1; 张凯三1; 刘婷婷2; 朱明1; 王祥杰1; 潘月兴1; 刘玉田1; 郭振光1; 徐开民1; 杨彬1
(1.日照市中医医院,山东 日照 276800; 2.日照市人民医院,山东 日照 276827)
Author(s):
DING Xiaoming1; ZHANG Kaisan1; LIU Tingting2; ZHU Ming1; WANG Xiangjie1; PAN Yuexing1; LIU Yutian1; GUO Zhenguang1; XU Kaimin1; YANG Bin1
1.Rizhao Hospital of TCM, Rizhao 276800,Shandong,China2.People’s Hospital of Rizhao,Rizhao 276827,Shandong,China
关键词:
组织工程;  丝心蛋白;  羟基磷灰石类;  脂肪干细胞;  骨诱导
Keywords:
tissue engineering;  fibroins;  hydroxyapatites;  adipose derived stem cell;  osteoinduction
摘要:
目的:探讨丝素蛋白(silk fibroin,SF)-羟基磷灰石(hydroxyapatite,HA)三维大孔支架复合脂肪干细胞(adipose-derived stem cells,ADSCs)体外构建组织工程骨的可行性。方法:以SF和HA为原料,采用石蜡微球沥滤技术制备三维大孔支架,以micro-CT扫描重建观察其结构,并测定支架的弹性模量。将兔ADSCs接种到支架上,给予成骨诱导液培养,分别取样本进行扫描电子显微镜观察、HE染色、细胞增殖情况测定(CCK8)、Von Kossa染色、Ⅰ型胶原免疫组化染色及Ⅰ型胶原含量测定(Elisa),评估SF-HA三维大孔支架的生物相容性和ADSCs在支架上的成骨分化情况。结果:①SF-HA三维大孔支架性能评估结果。支架内部可见均匀排列的大孔结构,连通性好。支架孔径(365.30±27.10)μm,孔隙率(85.30±1.80)%,弹性模量(54.93±5.44)kPa。②SF-HA三维大孔支架生物相容性评估结果。扫描电子显微镜图像显示细胞能够很好地在支架孔壁上黏附和伸展,细胞通过孔隙之间的连通结构长入孔隙内或在孔隙边缘呈拉网样覆盖,细胞周围可见基质分泌。SF-HA三维大孔支架ADSCs复合体HE染色结果显示,细胞核深染、胞浆红染,均匀黏附在支架孔隙内壁上,随成骨诱导培养时间延长,细胞和基质明显增多。CCK8检测结果显示,培养21 d时SF-HA三维大孔支架上的细胞数量较培养7 d时增加(1.14±0.08,1.98±0.11,t=13.810,P=0.000)。③ADSCs在SF-HA三维大孔支架上的成骨分化情况评估结果。Von Kossa染色和Ⅰ型胶原免疫组化染色可观察到钙沉积和Ⅰ型胶原积累,而且随时间延长不断增多。成骨诱导培养1 d、7 d、21 d时,SF-HA三维大孔支架ADSCs复合体上Ⅰ型胶原含量比较,差异有统计学意义(0.90±0.24,1.26±0.27,3.89±0.82,F=49.780,P=0.000); 成骨诱导培养21 d时的Ⅰ型胶原含量高于成骨诱导培养1 d、7 d时(P=0.000; P=0.000); 成骨诱导培养1 d、7 d时的Ⅰ型胶原含量比较,差异无统计学意义(P=0.059)。结论:SF-HA三维大孔支架复合成骨诱导培养的ADSCs具有在体外构建组织工程骨的可能。
Abstract:
Objective:To explore the feasibility of constructing tissue-engineered bone in vitro with the three-dimensional(3D)silk fibroin(SF)-hydroxyapatite(HA)macroporous scaffold-adipose-derived stem cells(ADSCs)complex.Methods:With SF and HA as raw materials,a 3D macroporous scaffold was prepared using the paraffin microsphere-leaching method,and its structure was reconstructed by micro-CT scanning,followed by the determination of its modulus of elasticity.The rabbit ADSCs were incubated onto this scaffold and cultured with the osteogenic induction fluid.The samples were then observed under the scanning electron microscope(SEM),followed by HE staining,cell proliferation assay(CCK8),Von Kossa staining as well as immunohistochemical staining of type I collagen for its content determination(Elisa)for assessing the biocompatibility of 3D SF-HA composite macroporous scaffold and the osteogenic differentiation of ADSCs on the scaffold.Results:①The performance evaluation results of 3D SF-HA macroporous scaffold.The homogeneously arranged macroporous structure was present inside the scaffold with good connectivity.The aperture,porosity and modulus of elasticity of the scaffold were 365.30±27.10 μm,85.30±1.80% and 54.93±5.44 kPa respectively.②Biocompatibility evaluation results of 3D SF-HA macroporous scaffold.As demonstrated by the SEM images,the cells displayed good adhesion and extension on the pore wall of the scaffold,and grew into the pores through the interconnected structure between the pores or covered the edges of the pores like a stretched mesh,with matrix secretion seen around the cells.The HE staining results of the 3D SF-HA macroporous scaffold-ADSCs complex showed that the deeply-stained cell nuclei and red-stained cytoplasm were uniformly adhered to the pore wall of the scaffold,and the number of cells and matrix increased significantly with the prolongation of osteogenic culture time.The CCK8 assay proved that the number of cells on 3D SF-HA macroporous scaffold increased on day 21 compared with that on day 7(1.14±0.08 vs 1.98±0.11,t=13.810,P=0.000).③The osteogenic differentiation evaluation results of ADSCs on 3D SF-HA macroporous scaffold.The calcium deposition and type I collagen accumulation were observed after Von Kossa staining and immunohistochemical staining of type I collagen,which were further enhanced over time.The differences in content of type I collagen in 3D SF-HA macroporous scaffold-ADSCs complex were statistically significant on day 1,7 and 21 osteogenic induction(0.90±0.24,1.26±0.27,3.89±0.82,F=49.780,P=0.000).The content of type I collagen was higher on day 21 compared with that on day 1 and 7(P=0.000; P=0.000),and there was no statistical difference between day 1 and day 7(P=0.059).Conclusion:It is possible to construct tissue-engineered bone in vitro with 3D SF-HA macroporous scaffold-osteogenic-induced ADSCs complex.

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备注/Memo

备注/Memo:
基金项目:山东省医药卫生科技发展计划项目(2016WS0330)
通讯作者:杨彬 E-mail:rzgkyangbin@163.com
更新日期/Last Update: 1900-01-01