The proliferation and differentiation of osteoblasts in co-culture with human umbilical vein endothelial cells: An improved analysis using fluorescence-activated cell sorting
Tóm tắt
The interaction of osteoblasts and endothelial cells plays a pivotal role in osteogenesis. This interaction has been extensively studied using their direct co-culture in vitro. However, co-culture experiments require clear discrimination between the two different cell types in the mixture, but this was rarely achieved. This study is the first to use fluorescence-activated cell sorting (FACS) for the separation and quantitative analysis of the proliferation and differentiation of MG-63 cells grown in direct co-culture with human umbilical vein endothelial cells (HUVECs). The cells of the MG-63 cell line have properties consistent with the characteristics of normal osteoblasts. We labeled HUVECs with fluorescent antibody against CD31 and used FACS to measure the proportions of each cell type and to separate them based on their different fluorescence intensities. The rate of proliferation of the MG-63 cells was estimated based on a count of the total viable cells and the proportion of MG-63 cells in the mixture. The mRNA expression levels of the osteoblast differentiation markers alkaline phosphatase (ALP), collagen type 1 (Coll-1) and osteocalcin (OC) in the MG-63 cells were measured via real-time PCR after the separation via FACS. We found that HUVECs stimulated the proliferation of the MG-63 cells after 72 h of co-culture, and inhibited it after 120 h of co-culture. The mRNA expression levels of ALP and Coll-1 significantly increased, whereas that of OC significantly decreased in MG-63 after co-culture with HUVECs. Using FACS for the quantitative analysis of the proliferation and differentiation of osteoblasts directly interacting with endothelial cells could have merit for further co-culture research.
Từ khóa
Tài liệu tham khảo
Sims, 2008, Bone remodeling : Multiple cellular interactions required for coupling of bone formation and resorption http dx org, Semin Cell Dev Biol, 2, 444, 10.1016/j.semcdb.2008.07.016
Unger, 2007, Tissue - like self - assembly in cocultures of endothelial cells and osteoblasts and the formation of microcapillary - like structures on three - dimensional porous biomaterials http dx org, Biomaterials, 26, 3965, 10.1016/j.biomaterials.2007.05.032
Guillotin, 2008, Interaction between human umbilical vein endothelial cells and human osteoprogenitors triggers pleiotropic effect that may support osteoblastic function http dx org, Bone, 14, 1080, 10.1016/j.bone.2008.01.025
Herzenberg, 1976, Fluorescence - activated cell sorting http dx org, Sci Am, 16, 234, 10.1038/scientificamerican0376-108
Lian, 1992, Concepts of osteoblast growth and differentiation : basis for modulation of bone cell development and tissue formation, Crit Rev Oral Biol Med, 33, 269, 10.1177/10454411920030030501
Bouletreau, 2002, Hypoxia and VEGF up - regulate BMP - mRNA and protein expression in microvascular endothelial cells : implications for fracture healing http dx org, Plast Reconstr Surg, 8, 2384, 10.1097/00006534-200206000-00033
Rausch, 2008, Differentiation and cytokine synthesis of human alveolar osteoblasts compared to osteoblast - like cells MG in response to titanium surfaces Dent http dx org, Mater, 18, 102, 10.1016/j.dental.2007.03.001
Jones, 2001, Sensitive determination of cell number using the CyQUANT cell proliferation assay http dx org, Immunol Methods, 28, 254, 10.1016/S0022-1759(01)00404-5
Villars, 2000, Effect of human endothelial cells on human bone marrow stromal cell phenotype : role of VEGF http dx org, Cell Biochem, 11, 672, 10.1002/1097-4644(20001215)79:4<672::AID-JCB150>3.0.CO;2-2
Boyan, 1998, Titanium surface roughness alters responsiveness of MG osteoblast - like cells to alpha OH Biomed http dx org, Mater Res, 21, 77
Herzenberg, 2002, The history and future of the fluorescence activated cell sorter and flow cytometry : a view from Stanford, Clin Chem, 15, 1819, 10.1093/clinchem/48.10.1819
Wang, 1997, Anabolic effects of dihydroxyvitamin D on osteoblasts are enhanced by vascular endothelial growth factor produced by osteoblasts and by growth factors produced by endothelial cells http dx org, Endocrinology, 5, 2953, 10.1210/en.138.7.2953
Stein, 1993, Molecular mechanisms mediating proliferation / differentiation interrelationships during progressive development of the osteoblast phenotype, Endocr Rev, 31, 424, 10.1210/edrv-14-4-424
Finkenzeller, 2006, Gene expression profiling reveals platelet - derived growth factor receptor alpha as a target of cell contact - dependent gene regulation in an endothelial cell - osteoblast co - culture model http dx org, Tissue Eng, 9, 2889, 10.1089/ten.2006.12.2889
Collin, 1994, Role of vascular endothelial cells in bone biology http dx org, Cell Biochem, 4, 304, 10.1002/jcb.240550306
Billiard, 2003, Transcriptional profiling of human osteoblast differentiation http dx org, Cell Biochem, 30, 389, 10.1002/jcb.10514
Ng, 2005, The challenge to measure cell proliferation in two and three dimensions http dx org, Tissue Eng, 27, 182, 10.1089/ten.2005.11.182
Jonsson, 1999, Three isolation techniques for primary culture of human osteoblast - like cells : a comparison http dx org, Acta Orthop Scand, 23, 365, 10.3109/17453679908997826
Martinez, 1999, del Influence of skeletal site of origin and donor age on osteoblastic cell growth and differentiation http dx org, Calcif Tissue Int, 25, 280, 10.1007/s002239900619
Olsen, 2000, Bone http dx org, development Annu Rev Cell Dev Biol, 1, 191, 10.1146/annurev.cellbio.16.1.191
Birch, 1999, Differential regulation of syndecan expression by osteosarcoma cell lines in response to cytokines but not osteotropic hormones http dx org, Bone, 22, 571, 10.1016/S8756-3282(99)00088-5
Aubin, 2001, Regulation of osteoblast formation and function http dx org, Rev Endocr Metab Disord, 32, 81, 10.1023/A:1010011209064
Ducy, 1997, a transcriptional activator of osteoblast differentiation http dx org, Cell, 34, 747, 10.1016/S0092-8674(00)80257-3
Streeten, 1990, Biology of bone endothelial cells http dx org, Bone Miner, 6, 85, 10.1016/0169-6009(90)90084-S
Sun, 2007, In vitro and in vivo effects of rat kidney vascular endothelial cells on osteogenesis of rat bone marrow mesenchymal stem cells growing on polylactide - glycoli acid scaffolds http dx org, Biomed Eng Online, 35, 41, 10.1186/1475-925X-6-41
Grellier, 2009, Role of vascular endothelial growth factor in the communication between human osteoprogenitors and endothelial cells http dx org, Cell Biochem, 12, 390, 10.1002/jcb.22018
Kaigler, 2005, Endothelial cell modulation of bone marrow stromal cell osteogenic potential, FASEB J, 13, 665, 10.1096/fj.04-2529fje
Meury, 2006, Human endothelial cells inhibit BMSC differentiation into mature osteoblasts in vitro by interfering with osterix expression http dx org, Cell Biochem, 36, 992, 10.1002/jcb.20818
Fuchs, 2007, and Microvessel - like structures from outgrowth endothelial cells from human peripheral blood in - dimensional and - dimensional co - cultures with osteoblastic lineage cells http dx org, Tissue Eng, 17, 2577
Declercq, 2004, der De De Isolation , proliferation and differentiation of osteoblastic cells to study cell / biomaterial interactions : comparison of different isolation techniques and source http dx org, Biomaterials, 24, 757, 10.1016/S0142-9612(03)00580-5
Villars, 2002, Effect of HUVEC on human osteoprogenitor cell differentiation needs heterotypic gap junction communication, Am J Physiol Cell Physiol, 10, 775, 10.1152/ajpcell.00310.2001
Moreau, 1997, Pharmacological and biochemical evidence for the regulation of osteocalcin secretion by potassium channels in human osteoblast - like MG - cells http dx org, Bone Miner Res, 20, 1984, 10.1359/jbmr.1997.12.12.1984
Finkenzeller, 2010, Post - transcriptional regulation of osteoblastic platelet - derived growth factor receptor - alpha expression by co - cultured primary endothelial cells, Cells Tissues Organs, 29, 10.1159/000276590
Clarkin, 2008, Evaluation of VEGF - mediated signaling in primary human cells reveals a paracrine action for VEGF in osteoblast - mediated crosstalk to endothelial cells http dx org, Cell Physiol, 7, 537, 10.1002/jcp.21234