title: Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures: an ultrastructural, compositional and comparative analysis with mouse bone.
abstract: 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 MC3T3-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 20nm-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 (FTIR) 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, 100nm-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.
authors.affiliation: Faculty of Dentistry, McGill University, Montreal, Quebec, Canada.
authors.forename: W N
authors.initials: WN
authors.lastname: Addison
authors.affiliation: Faculty of Dentistry, McGill University, Montreal, Quebec, Canada.
authors.forename: V
authors.initials: V
authors.lastname: Nelea
authors.affiliation: Department of Mining and Materials, McGill University, Montreal, Quebec, Canada.
authors.forename: F
authors.initials: F
authors.lastname: Chicatun
authors.affiliation: Faculty of Dentistry, McGill University, Montreal, Quebec, Canada.
authors.forename: Y-C
authors.initials: YC
authors.lastname: Chien
authors.affiliation: Department of Chemical Engineering, École Polytechnique, Montreal, Quebec, Canada.
authors.forename: N
authors.initials: N
authors.lastname: Tran-Khanh
authors.affiliation: Department of Chemical Engineering, École Polytechnique, Montreal, Quebec, Canada.
authors.forename: M D
authors.initials: MD
authors.lastname: Buschmann
authors.affiliation: Department of Mining and Materials, McGill University, Montreal, Quebec, Canada.
authors.forename: S N
authors.initials: SN
authors.lastname: Nazhat
authors.affiliation: Faculty of Dentistry, McGill University, Montreal, Quebec, Canada.
authors.forename: M T
authors.initials: MT
authors.lastname: Kaartinen
authors.affiliation: Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada.
authors.forename: H
authors.initials: H
authors.lastname: Vali
authors.affiliation: Department of Chemistry, Central Michigan University, Mount Pleasant, MI, USA.
authors.forename: M M
authors.initials: MM
authors.lastname: Tecklenburg
authors.affiliation: Department of Periodontics and Oral Medicine, School of Dentistry, University of Michigan, Ann Arbor, MI, USA.
authors.forename: R T
authors.initials: RT
authors.lastname: Franceschi
authors.affiliation: Faculty of Dentistry, McGill University, Montreal, Quebec, Canada; Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada. Electronic address: marc.mckee@mcgill.ca.
authors.forename: M D
authors.initials: MD
authors.lastname: McKee
mesh_terms: D000818:Animals; D001842:Bone and Bones / Q000502:physiology* / Q000648:ultrastructure*; D002113:Calcification, Physiologic; D002478:Cells, Cultured; D005109:Extracellular Matrix / Q000378:metabolism*; D051379:Mice; D008903:Minerals / Q000378:metabolism; D010006:Osteoblasts / Q000502:physiology* / Q000648:ultrastructure*; D013052:Spectrometry, X-Ray Emission; D017550:Spectroscopy, Fourier Transform Infrared; D014732:Vibration; D014961:X-Ray Diffraction
chemical_list: D008903:Minerals
keywords: Biomineralization; Cell culture; Extracellular matrix; Hydroxyapatite; MC3T3-E1 cells; Osteoblasts