Vascularization of poly(hydroxybutyrate-co-hydroxyoctanoate) osteochondral scaffold in vitro

doi:10.3969/j.issn.2095-4344.2014.08.007

Abstract

Abstract:

BACKGROUND: The poly(hydroxybutyrate-co-hydroxyoctanoate) osteochondral scaffold which has been constructed in previous experiments has good biocompatibility and biodegradability and generates non-toxic degradation products.
OBJECTIVE: To observe the vascularization of rabbit renal microvascular endothelial cells co-cultured with poly(hydroxybutyrate-co-hydroxyoctanoate) osteochondral scaffold.
METHODS: The poly(hydroxybutyrate-co-hydroxyoctanoate) osteochondral scaffold having a three-layer structure (layer of bone/bone and cartilage interface layer/layer of cartilage) was prepared by solvent casting/particle leaching method. The renal microvascular endothelial cells at passage 3 were seeded onto the scaffold of bone layer. The proliferation of the renal microvascular endothelial cells growing on the scaffolds was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method, the growth of cells in the scaffold was observed by hematoxylin-eosin staining under electron microscope after 10 days.
RESULTS AND CONCLUSION: The integrated osteochondral scaffold had a clear appearance of three-layer structure, which had closed connections between the three layers. Porous bone layer was visible as well as uniform and interlinked pores, and the porosity was 78%. The renal microvascular endothelial cells seeded onto the scaffold proliferated well and presented a three-dimensional growth after 10 days of co-culture, but there were no cells on the interface layer. Cells which adhered and grew between the pores of the bone layer were observed through hematoxylin-eosin staining. Cells showed a luminal-like structure growing on the scaffold with the porous structure, but they did not grow into the interface layer of bone and cartilage.

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

全文链接：

Key words: biocompatible materials, neovascularization, physiologic, 3-hydroxybutyric acid, tissue engineering

CLC Number:

R318

Xiao Wei, Ren Wei, Zhang Yong-hong, Zhao Liang-qi. Vascularization of poly(hydroxybutyrate-co-hydroxyoctanoate) osteochondral scaffold in vitro[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(8): 1185-1190.

Figures/Tables 1

2.1 PHBHOx一体化骨软骨支架外形、超微结构与孔隙率检测 PHBHOx一体化骨软骨支架外观具备明显的3层结构，各层之间连接紧密，骨层疏松多孔，骨软骨界面层致密，软骨层相对致密，同时 PHBHOx一体化骨软骨支架具有较强的可塑性，可利用不同形状的模具以及不同大小的致孔剂制备出不同外形和不同孔径的支架(图1A)。扫描电镜可见PHBHOx一体化骨软骨支架各层支架孔隙均匀且相通，孔壁也有较多的微孔，各层之间通过不同孔径的孔隙紧密连接，其中骨层支架由于添加羟基磷灰石，孔隙表面粗糙，孔径150-450 µm，软骨层支架孔径150-250 µm，骨软骨中间界面层支架孔径≤60 μm(图1B)。经测定，PHBHOx一体化骨软骨支架骨层孔隙率为87%，骨软骨界面层孔隙率为68%，软骨层孔隙率为79%，一体化支架孔隙率为78%。 2.2 兔肾微血管内皮细胞的观察及鉴定细胞接种48 h，大部分肾微血管内皮细胞已贴壁，呈短胖梭形，7-12 d细胞增殖较多，镶嵌排列，互不重叠，发生接触抑制呈典型的铺路鹅卵石形(图2A)。第Ⅷ因子相关抗原测定，肾微血管内皮细胞的胞浆和核膜周围染成棕褐色(图2B)，未加一抗的阴性对照之肾微血管内皮细胞胞浆和核膜无染色，仅细胞核呈淡蓝色(图2C)。 2.3 肾微血管内皮细胞在一体化支架上的黏附和增殖肾微血管内皮细胞与支架复合培养10 d后，扫描电镜可见细胞在骨层和骨层与中间界面层连接处立体生长，细胞在骨层支架的孔壁及孔底黏附生长良好，并连接成片，同时在骨软骨界面层内未发现细胞长入(图3A，B)。肾微血管内皮细胞在骨层支架上生长增殖活跃，复合培养2，4，6，8，10 d，MTT值分别为0.444，0.461，0.555，0.727及0.812，表明细胞在支架上分裂增殖良好(图4)。 2.4 细胞支架复合物苏木精-伊红染色结果 10 d后的细胞支架复合物切片苏木精-伊红染色可见肾微血管内皮细胞被染成红色，未被染色的支架材料呈乳白色，细胞黏附生长于骨层支架孔隙间，依附支架的多孔结构生长形成连接并形成管腔样结构(图3C)。"

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