TY - JOUR
T1 - Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures
T2 - An ultrastructural, compositional and comparative analysis with mouse bone
AU - Addison, W. N.
AU - Nelea, V.
AU - Chicatun, F.
AU - Chien, Y. C.
AU - Tran-Khanh, N.
AU - Buschmann, M. D.
AU - Nazhat, S. N.
AU - Kaartinen, M. T.
AU - Vali, H.
AU - Tecklenburg, M. M.
AU - Franceschi, R. T.
AU - McKee, M. D.
N1 - Funding Information:
We thank Lydia Malynowsky (McGill University, Montreal) for assistance with various microscopies, Line Mongeon and Dr. David Liu (Facility for Electron Microscopy Research, McGill University, Montreal) for assistance with electron microscopy. This work was primarily funded by the Canadian Institutes of Health Research (to MDM, and MDB and MTK; grants MOP-97858 , MOP-62801 and MOP-119403 ), and in part by the Natural Sciences and Engineering Research Council (to SNN; grant 341235 ). MMT acknowledges support in part from NIH grant R01 AR47969 . MDM, MTK, MDB and SNN are members of the McGill Centre for Bone and Periodontal Research, and the MDM and MTK are members of the FRQ-S Network for Oral and Bone Health Research.
Publisher Copyright:
© 2014 Elsevier Inc.
PY - 2015/2/1
Y1 - 2015/2/1
N2 - 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 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 (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, 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.
AB - 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 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 (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, 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.
KW - Biomineralization
KW - Cell culture
KW - Extracellular matrix
KW - Hydroxyapatite
KW - MC3T3-E1 cells
KW - Osteoblasts
UR - http://www.scopus.com/inward/record.url?scp=84912144863&partnerID=8YFLogxK
U2 - 10.1016/j.bone.2014.11.003
DO - 10.1016/j.bone.2014.11.003
M3 - Article
C2 - 25460184
AN - SCOPUS:84912144863
SN - 8756-3282
VL - 71
SP - 244
EP - 256
JO - Bone
JF - Bone
ER -
