利用鸡胚模型半体内评价多孔磷酸钙骨修复材料血管化的研究

doi:10.15541/jim20160535

无机材料学报 ›› 2017, Vol. 32 ›› Issue (6): 649-654.DOI: 10.15541/jim20160535 CSTR: 32189.14.10.15541/jim20160535

利用鸡胚模型半体内评价多孔磷酸钙骨修复材料血管化的研究

肖雯¹, 刘玉梅¹, 任凯歌², 匙峰¹, 李焰², 智伟¹, 翁杰¹, 屈树新¹

(1. 西南交通大学材料先进技术教育部重点实验室, 材料科学与工程学院, 成都610031; 2. 成都军区总医院口腔科, 成都610083)

收稿日期:2016-09-22 修回日期:2016-11-02 出版日期:2017-06-20 网络出版日期:2017-05-27
作者简介:肖雯(1993–), 女, 硕士研究生. E-mail: 18728428596@163.com
基金资助:
国家重点基础研究发展计划(973)项目(2012CB933602);国家自然科学基金(51372210);四川省应用基础(重点)项目(2016JY0011);教育部博士点基金(20130184110023);中央高校基本科研业务费专项基金(2682016YXZT11);四川省高校科研创新团队建设计划项目(14TD0050)

Evaluation of Vascularization of Porous Calcium Phosphate by Chick Chorioallantoic Membrane Model ex vivo

XIAO Wen¹, LIU Yu-Mei¹, REN Kai-Ge², SHI Feng¹, LI Yan², ZHI Wei¹, WENG Jie¹, QU Shu-Xin¹

(1. Key Lab of Advanced Technologies of Materials, Ministry of Education, School of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China; 2. Department of Stomatology, Chengdu Army General Hospital, Chengdu 610083, China)

Received:2016-09-22 Revised:2016-11-02 Published:2017-06-20 Online:2017-05-27
About author:XIAO Wen. E-mail: 18728428596@163.com
Supported by:
National Basic Research Program of China (973 Program, 2012CB933602);National Natural Science Foundation of China (51372210);Basic Research Foundation Key Project of Sichuan Province (2016JY0011);Research Fund for the Doctoral Program of Higher Education of China (20130184110023);Fundamental Research Funds for the Central Universities (2682016YXZT11);The Construction Program for Innovative Research Team of University in Sichuan Province (14TD0050)

摘要/Abstract

摘要：

利用鸡胚绒毛尿囊膜(CAM)中丰富的血管建立半体内模型, 研究多孔磷酸钙骨修复材料的血管化。将多孔羟基磷灰石陶瓷(HA)和具有部分孔隙结构的磷酸钙骨水泥(CPCs)分别植入CAM中共培养, 利用体式显微镜和扫描电子显微镜(SEM)观察CPCs表面血管的生长, 并利用Image-Pro Plus和Nano Measurer定量血管密度、直径和数量; 对CAM中培养的HA采用不脱钙组织学处理和观察。体式显微镜观察及定量结果显示多孔结构对血管生长影响显著, 随着培养时间延长, 多孔部分血管密度和数量均有明显增加, 而无孔部分血管生长缓慢。CPCs表面血管以出芽方式生长, 形成血管网络, 直径一般小于50 μm。SEM观察显示血管紧贴CPCs表面生长。苏木精-伊红(H&E)染色显示血管通过孔隙结构长入HA内部。实验结果说明三维孔隙结构利于血管的生长和长入, 此实验可简便、高效、低成本地研究多孔磷酸钙骨修复材料的血管化, 为快速评价生物材料血管化寻找一种新途径。

关键词: 鸡胚绒毛尿囊膜, 多孔磷酸钙骨修复材料, 血管化, 半体内

Abstract:

In this study, ex vivo chick chorioallantoic membrane (CAM) model with abundant blood vessels was developed to investigate the vascularization of porous calcium phosphate. Porous hydroxyapatite ceramic (HA) and calcium phosphate cement with segmental porous structure (CPCs) were implanted into CAM, respectively. Stereomicroscope and scanning electron microscope (SEM) were employed to observe the angiogenesis on the surface of CPCs. The vascular density, diameters and numbers were quantified by Image-Pro Plus and Nano Measurer. Furthermore, undecalcified histological staining was conducted to observe the inner angiogenesis of porous HA. The vascular density and number in the porous part of CPCs both increased significantly with the increase of culture time, while the angiogenesis was tardily in the part of CPCs without pores. Most of the blood vessels less than 50 μm on the porous part of CPCs grew in budding manner to form vascular network. SEM images showed that blood vessels grew tightly on the surface of CPCs. Histological staining demonstrated that some blood vessels grew into the inside of porous HA. These results suggest that three-dimensional porous structure may promote the angiogenesis of porous calcium phosphate and the present CAM model may provide a convenient, efficient and with low-cost approach to evaluate vascularization of porous biomaterials.

Key words: chick chorioallantoic membrane, porous calcium phosphate materials, vascularization, ex vivo

中图分类号:

TB321

肖雯, 刘玉梅, 任凯歌, 匙峰, 李焰, 智伟, 翁杰, 屈树新. 利用鸡胚模型半体内评价多孔磷酸钙骨修复材料血管化的研究[J]. 无机材料学报, 2017, 32(6): 649-654.

XIAO Wen, LIU Yu-Mei, REN Kai-Ge, SHI Feng, LI Yan, ZHI Wei, WENG Jie, QU Shu-Xin. Evaluation of Vascularization of Porous Calcium Phosphate by Chick Chorioallantoic Membrane Model ex vivo[J]. Journal of Inorganic Materials, 2017, 32(6): 649-654.

图/表 6

图1 预培养8 d的CAM血管网络(a), 以及植入CPCs (b)和HA (c)后的CAM

Fig. 1 Vascular network of CAM after pre-incubated for 8 d (a), CAM after implanted CPCs (b) and HA (c)

图2 CPCs植入CAM模型中培养2 d(a)、6 d(b)和8 d(c)后其表面的血管生长, 以及培养8 d后放大CPCs表面不同区域(1 (d)、2 (e)和3 (f))的血管生长的体式显微镜照片

Fig. 2 Angiogensis on the surface of CPCs after implanted in CAM model for 2 d (a), 6 d (b) and 8 d (c). Angiogensis of different parts (1 (d), 2 (e) and 3 (f)) on the surface of CPCs after implanted for 8 d

图3 培养2、6和8 d后CPCs表面血管总密度(a), 以及CPCs表面多孔和无孔部分血管密度所占百分比(b)

Fig. 3 Total density of blood vessels on CPCs after implanted in CAM model for 2, 6 and 8 d (a). The percentage of vascular densities of CPCs, with pores and without pores (b)**p<0.01, n=3

图4 培养2、6和8 d后CPCs表面血管的直径分布(a), CPCs表面不同直径血管(Φ<50 μm, 50 μm<Φ<100 μm和Φ>100 μm)总数量(b), 以及CPCs表面多孔和无孔部分中不同直径血管的数量所占百分比(c)

Fig. 4 The distribution of vascular diameters (a), numbers of different vascular diameters (Φ<50 μm, 50 μm<Φ<100 μm and Φ>100 μm) (b) and the percentage of different diameters in different parts (with pores and without pores) (c) on the surface of CPCs after implanted for 2, 6 and 8 d*p<0.05, **p<0.01, n=3

图5 植入CAM 8 d后CPCs表面的SEM照片

Fig. 5 SEM images of the surface on CPCs after implanted in CAM model for 8 d

图6 多孔羟基磷灰石陶瓷植入CAM 4 d后不同部位的H&E染色

Fig. 6 H&E staining images of porous hydroxyapatite ceramic after implanted for 4 d in CAM modelBlack arrow: red blood cell grew into the inner pore of HA, white arrow: blood vessel grew into the inner pore of HA

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利用鸡胚模型半体内评价多孔磷酸钙骨修复材料血管化的研究

Evaluation of Vascularization of Porous Calcium Phosphate by Chick Chorioallantoic Membrane Model ex vivo

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