Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures: An ultrastructural,compositional and comparative analysis with mouse bone |
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| Affiliation: | 1. Faculty of Dentistry, McGill University, Montreal, Quebec, Canada;2. Department of Mining and Materials, McGill University, Montreal, Quebec, Canada;3. Department of Chemical Engineering, École Polytechnique, Montreal, Quebec, Canada;4. Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada;5. Department of Chemistry, Central Michigan University, Mount Pleasant, MI, USA;6. Department of Periodontics and Oral Medicine, School of Dentistry, University of Michigan, Ann Arbor, MI, USA;1. Marmara University, Department of Pediatric Endocrinology, Istanbul, Turkey;2. Division of Experimental Paediatric Endocrinology and Diabetes, University of Luebeck, Germany;3. Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, USA;1. University of British Columbia, Vancouver, Canada;2. CaMos National Coordinating Centre, McGill University, Montreal, Canada;3. McGill University, Montreal, Canada;4. Memorial University, St John''s, Canada;5. Dalhousie University, Halifax, Canada;6. McMaster University, Hamilton, Canada;7. Queen''s University, Kingston, Canada;8. University of Toronto, Toronto, Canada;9. Laval University, Quebec City, Canada;10. University of Manitoba, Winnipeg, Canada;1. Hokkaido University, Department of Orthopedic Surgery, School of Medicine, Sapporo, Japan;2. Hokkaido University, Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Sapporo, Japan;3. Hokkaido University, Department of Biochemistry, School of Medicine, Sapporo, Japan;4. Institute of Biological Chemistry, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei 11529, Taiwan;1. Department of Orthopaedics Surgery, Wuhan General Hospital of Guangzhou Command, Wuhan 430000, PR China;2. Department of Orthopaedics Surgery, Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China;1. Skeletal Biology Laboratory, School of Biological and Population Health Sciences, College of Public Health and Human Sciences, Oregon State University, Corvallis, OR 97331, USA;2. Division of Neuroscience, Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR 97006, USA;3. Division of Biostatistics, Office of Surveillance and Biometrics, Center for Devices and Radiological Health, Food and Drug Administration, Silver Spring, MD 20993, USA;4. Center for Healthy Aging Research, Oregon State University, Corvallis, OR 97331, USA |
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| 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 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. |
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