Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures: An ultrastructural, compositional and comparative analysis with mouse bone

被引：87

作者：

Addison, W. N. ^{[1
]}

Nelea, V. ^{[1
]}

Chicatun, F. ^{[2
]}

Chien, Y-C ^{[1
]}

Tran-Khanh, N. ^{[3
]}

Buschmann, M. D. ^{[3
]}

Nazhat, S. N. ^{[2
]}

Kaartinen, M. T. ^{[1
]}

Vali, H. ^{[4
]}

Tecklenburg, M. M. ^{[5
]}

Franceschi, R. T. ^{[6
]}

McKee, M. D. ^{[1
,4
]}

机构：

[1] McGill Univ, Fac Dent, Montreal, PQ H3A 0C7, Canada

[2] McGill Univ, Dept Min & Mat, Montreal, PQ H3A 0C7, Canada

[3] Ecole Polytech, Dept Chem Engn, Montreal, PQ H3C 3A7, Canada

[4] McGill Univ, Dept Anat & Cell Biol, Montreal, PQ H3A 0C7, Canada

[5] Cent Michigan Univ, Dept Chem, Mt Pleasant, MI 48859 USA

[6] Univ Michigan, Sch Dent, Dept Periodont & Oral Med, Ann Arbor, MI 48109 USA

来源：

BONE | 2015年 / 71卷

基金：

加拿大健康研究院; 加拿大自然科学与工程研究理事会;

关键词：

Cell culture; Biomineralization; Hydroxyapatite; Osteoblasts; MC3T3-E1; cells; Extracellular matrix; TRANSFORM INFRARED-SPECTROSCOPY; CALCIUM-PHOSPHATE CRYSTALS; IN-VITRO; CELL-CULTURE; CHEMICAL CHARACTERISTICS; COLLAGEN-SYNTHESIS; OSTEOPONTIN; CARBONATE; DIFFERENTIATION; CALCIFICATION;

D O I：

10.1016/j.bone.2014.11.003

中图分类号：

R5 [内科学];

学科分类号：

100201 [内科学];

摘要：

Bone cell culture systems are essential tools for the study of the molecular mechanisms regulating extracellular matrix mineralization. MC3T3-E1 osteoblast cell cultures are the most commonly used in vitro model of bone matrix mineralization. Despite the widespread use of this cell line to study biomineralization, there is as yet no systematic characterization of the mineral phase produced in these cultures. Here we provide a comprehensive, multi-technique biophysical characterization of this cell culture mineral and extracellular matrix, and compare it to mouse bone and synthetic apatite mineral standards, to determine the suitability of MOT3-E1 cultures for biomineralization studies. Elemental compositional analysis by energy-dispersive X-ray spectroscopy (EDS) showed calcium and phosphorus, and trace amounts of sodium and magnesium, in both biological samples. X-ray diffraction (XRD) on resin-embedded intact cultures demonstrated that similar to 1-month-old mouse bone, apatite crystals grew with preferential orientations along the (100), (101) and (111) mineral planes indicative of guided biogenic growth as opposed to dystrophic calcification. XRD of crystals isolated from the cultures revealed that the mineral phase was poorly crystalline hydroxyapatite with 10 to 20 nm-sized nanocrystallites. Consistent with the XRD observations, electron diffraction patterns indicated that culture mineral had low crystallinity typical of biological apatites. Fourier-transform infrared spectroscopy (FUR) confirmed apatitic carbonate and phosphate within the biological samples. With all techniques utilized, cell culture mineral and mouse bone mineral were remarkably similar. Scanning (SEM) and transmission (TEM) electron microscopy showed that the cultures had a dense fibrillar collagen matrix with small, 100 nm-sized, collagen fibril-associated mineralization foci which coalesced to form larger mineral aggregates, and where mineralized sites showed the accumulation of the mineral-binding protein osteopontin. Light microscopy, confocal microscopy and three-dimensional reconstructions showed that some cells had dendritic processes and became embedded within the mineral in an osteocyte-like manner. In conclusion, we have documented characteristics of the mineral and matrix phases of MC3T3-E1 osteoblast cultures, and have determined that the structural and compositional properties of the mineral are highly similar to that of mouse bone. (C) 2014 Elsevier Inc. All rights reserved.

引用

页码：244 / 256

页数：13

共 43 条

[1]

Pyrophosphate inhibits mineralization of osteoblast cultures by binding to mineral, up-regulating osteopontin, and inhibiting alkaline phosphatase activity [J].