整合素介导的人成骨细胞对多孔无定形磷酸钙/聚（L-乳酸）复合材料的粘附增强。

Enhanced integrin-mediated human osteoblastic adhesion to porous amorphous calcium phosphate/poly (L-lactic acid) composite.

作者信息

Huang Xin, Qi Yiying, Li Weixu, Shi Zhongli, Weng Wenjian, Chen Kui, He Rongxin

机构信息

Department of Orthopaedic Surgery, Second Affiliated Hospital, Medical College of Zhejiang University, Hangzhou, Zhejiang 310009, China.

Department of Materials Science and Engineering, Zhejiang University, Hangzhou, Zhejiang 310027, China.

出版信息

Chin Med J (Engl). 2014;127(19):3443-8.

PMID:25269911

Abstract

BACKGROUND

The initial osteoblastic adhesion to materials characterizes the first phase of cell-material interactions and influences all the events leading to the formation of new bone. In a previous work, we developed a novel amorphous calcium phosphate (ACP)/poly(L-lactic acid) (PLLA) material that demonstrated morphologic variations in its microstructure. The aim of this study was to investigate the initial interaction between this material and osteoblastic cells. Cellular attachment and the corresponding signal transduction pathways were investigated.

METHODS

A porous ACP/PLLA composite and PLLA scaffold (as a control) were incubated in fetal bovine serum (FBS) containing phosphate-buffered saline (PBS), and the protein adsorption was determined. Osteoblastic MG63 cells were seeded on the materials and cultured for 1, 4, 8, or 24 hours. Cell attachment was evaluated using the MTS method. Cell morphology was examined using scanning electron microscopy (SEM). The expression levels of the genes encoding integrin subunits α1, α5, αv, β1, focal adhesion kinase (FAK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined using real-time reverse transcription polymerase chain reaction (RT-PCR).

RESULTS

The ACP/PLLA material significantly increased the protein adsorption by 6.4-fold at 1 hour and 2.4-fold at 24 hours, compared with the pure PLLA scaffold. The attachment of osteoblastic cells to the ACP/PLLA was significantly higher than that on the PLLA scaffold. The SEM observation revealed a polygonal spread shape of cells on the ACP/ PLLA, with the filopodia adhered to the scaffold surface. In contrast, the cells on the PLLA scaffold exhibited a spherical or polygonal morphology. Additionally, real-time RT-PCR showed that the genes encoding the integrin subunits α1, αv, β1, and FAK were expressed at higher levels on the ACP/PLLA composite.

CONCLUSIONS

The ACP/PLLA composite promoted protein adsorption and osteoblastic adhesion. The enhanced cell adhesion may be mediated by the binding of integrin subunits α1, αv, and β1, and subsequently may be regulated through the FAK signal transduction pathways.

摘要

背景

成骨细胞对材料的初始黏附是细胞与材料相互作用的第一阶段，影响着所有导致新骨形成的事件。在之前的一项工作中，我们开发了一种新型的无定形磷酸钙（ACP）/聚（L-乳酸）（PLLA）材料，其微观结构呈现出形态变化。本研究的目的是探究这种材料与成骨细胞之间的初始相互作用。研究了细胞黏附及相应的信号转导途径。

方法

将多孔ACP/PLLA复合材料和PLLA支架（作为对照）置于含有磷酸盐缓冲盐水（PBS）的胎牛血清（FBS）中孵育，测定蛋白质吸附情况。将成骨MG63细胞接种于材料上，培养1、4、8或24小时。使用MTS法评估细胞黏附情况。用扫描电子显微镜（SEM）检查细胞形态。使用实时逆转录聚合酶链反应（RT-PCR）测定编码整合素亚基α1、α5、αv、β1、黏着斑激酶（FAK）和甘油醛-3-磷酸脱氢酶（GAPDH）的基因表达水平。

结果

与纯PLLA支架相比，ACP/PLLA材料在1小时时蛋白质吸附显著增加6.4倍，在24小时时增加2.4倍。成骨细胞在ACP/PLLA上的黏附明显高于在PLLA支架上的黏附。SEM观察显示，ACP/PLLA上的细胞呈多边形铺展形态，丝状伪足黏附于支架表面。相比之下，PLLA支架上的细胞呈现球形或多边形形态。此外，实时RT-PCR显示，编码整合素亚基α1、αv、β1和FAK的基因在ACP/PLLA复合材料上表达水平更高。