人原代成骨细胞和骨细胞胶原凝胶共培养模型。

In Vitro Co-culture Model of Primary Human Osteoblasts and Osteocytes in Collagen Gels.

机构信息

Centre for Translational Bone, Joint and Soft Tissue Research, Medical Faculty and University Hospital, Technische Universität, 01307 Dresden, Germany.

出版信息

Int J Mol Sci. 2019 Apr 23;20(8):1998. doi: 10.3390/ijms20081998.

DOI:10.3390/ijms20081998

PMID:31018582

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6514924/

Abstract

BACKGROUND

Osteocytes are the key regulator cells in bone tissue, affecting activity of both osteoblasts and osteoclasts. Current in vitro studies on osteocyte-osteoblast interaction are invariably performed with rodent cells, mostly murine cell lines, which diminishes the clinical relevance of the data.

OBJECTIVE

The objective of the present study was to establish an in vitro co-culture system of osteoblasts and osteocytes, which is based solely on human primary cells.

METHODS

Three different approaches for the generation of human primary osteocytes were compared: direct isolation of osteocytes from bone tissue by multistep digestion, long-time differentiation of human pre-osteoblasts embedded in collagen gels, and short time differentiation of mature human osteoblasts in collagen gels. Co-cultivation of mature osteoblasts with osteocytes, derived from the three different approaches was performed in a transwell system, with osteocytes, embedded in collagen gels at the apical side and osteoblasts on the basal side of a porous membrane, which allowed the separate gene expression analysis for osteocytes and osteoblasts. Fluorescence microscopic imaging and gene expression analysis were performed separately for osteocytes and osteoblasts.

RESULTS

All examined approaches provided cells with typical osteocytic morphology, which expressed osteocyte markers E11, osteocalcin, phosphate regulating endopeptidase homolog, X-linked (PHEX), matrix extracellular phosphoglycoprotein (MEPE), sclerostin, and receptor activator of NF-κB Ligand (RANKL). Expression of osteocyte markers was not significantly changed in the presence of osteoblasts. In contrast, osteocalcin gene expression of osteoblasts was significantly upregulated in all examined co-cultures with differentiated osteocytes. Alkaline phosphatase (ALPL), bone sialoprotein II (BSPII), and RANKL expression of osteoblasts was not significantly changed in the co-culture.

CONCLUSION

Interaction of osteoblasts and osteocytes can be monitored in an in vitro model, comprising solely primary human cells.

摘要

背景

成骨细胞是骨组织中的关键调节细胞，影响成骨细胞和破骨细胞的活性。目前关于成骨细胞-成骨细胞相互作用的体外研究均使用啮齿动物细胞进行，主要是鼠系细胞系，这降低了数据的临床相关性。

目的

本研究旨在建立一种仅基于人原代细胞的成骨细胞和骨细胞体外共培养系统。

方法

比较了三种生成人原代骨细胞的方法：通过多步消化直接从骨组织中分离骨细胞、将嵌入胶原凝胶中的人前成骨细胞进行长期分化，以及在胶原凝胶中对成熟的人成骨细胞进行短期分化。在 Transwell 系统中将成熟的成骨细胞与三种不同方法获得的骨细胞进行共培养，将嵌入胶原凝胶中的骨细胞置于顶层，成骨细胞置于多孔膜的底层，这样可以分别对骨细胞和成骨细胞进行单独的基因表达分析。分别对骨细胞和成骨细胞进行荧光显微镜成像和基因表达分析。

结果

所有检查的方法都提供了具有典型骨细胞形态的细胞，这些细胞表达骨细胞标志物 E11、骨钙素、磷酸调节内肽酶同源物 X 连锁（PHEX）、基质外磷酸糖蛋白（MEPE）、骨硬化蛋白和核因子κB 受体激活剂配体（RANKL）。在存在成骨细胞的情况下，骨细胞标志物的表达没有明显变化。相比之下，在所有检查的与分化骨细胞的共培养中，成骨细胞的骨钙素基因表达均显著上调。成骨细胞碱性磷酸酶（ALPL）、骨涎蛋白 II（BSPII）和 RANKL 的表达在共培养中没有明显变化。