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Ultrastructural and Mineral Phase Characterization of the Bone-Like Matrix Assembled in F-OST Osteoblast Cultures

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Abstract

Cell cultures are often used to study bone mineralization; however, not all systems achieve a bone-like matrix formation. In this study, the mineralized matrix assembled in F-OST osteoblast cultures was analyzed, with the aim of establishing a novel model for bone mineralization. The ultrastructure of the cultures was investigated using scanning electron microscopy, atomic force microscopy, and transmission electron microscopy (TEM). The mineral phase was characterized using conventional and high-resolution TEM, energy-dispersive X-ray spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and solid-state 31P and 1H nuclear magnetic resonance. F-OST osteoblast cultures presented a clear nodular mineralization pattern. The chief features of the mineralizing nodules were globular accretions ranging from about 100 nm to 1.5 μm in diameter, loaded with needle-shaped crystallites. Accretions seemed to bud from the cell membrane, increase in size, and coalesce into larger ones. Arrays of loosely packed, randomly oriented collagen fibrils were seen along with the accretions. Mineralized fibrils were often observed, sometimes in close association with accretions. The mineral phase was characterized as a poorly crystalline hydroxyapatite. The Ca/P atomic ratio was 1.49 ± 0.06. The presence of OH was evident. The lattice parameters were a = 9.435 Å and c = 6.860 Å. The average crystallite size was 20 nm long and 10 nm wide. Carbonate substitutions were seen in phosphate and OH sites. Water was also found within the apatitic core. In conclusion, F-OST osteoblast cultures produce a bone-like matrix and may provide a good model for bone mineralization studies.

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Acknowledgments

The authors gratefully acknowledge COPPE/UFRJ for the SEM facilities and technical support, LNLS for the HRTEM facilities, M. M. Medeiros (ICB/UFRJ) for her support in electron microscopic analysis, F. P. Almeida (IMPPG/UFRJ) for his assistance in SEM analyses, J. Gomes Filho (CBPF) for his contribution in AFM analysis, V. C. A. Moraes and V. A. Ferraz (CBPF) for their support in the XRD analyses, and C. L. R. Fragoso for his assistance in the FTIR analyses. This study was supported by CNPq, CAPES, FAPERJ, and FINEP Brazilian agencies.

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Authors and Affiliations

  1. Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, 21941-902, Brazil

    W. Querido, L. G. Abraçado, A. L. Rossi, R. Borojevic & M. Farina

  2. Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, 21941-902, Brazil

    W. Querido

  3. Divisão de Metrologia de Materiais, Instituto Nacional de Metrologia, Normalização e Qualidade Industrial, Duque de Caxias, RJ, 25250-020, Brazil

    A. P. C. Campos

  4. Grupo de Biomateriais: Preparação, Caracterização, Modelagem Teórica e Aplicações Biomédicas, Centro Brasileiro de Pesquisas Físicas, Rio de Janeiro, RJ, 22290-180, Brazil

    A. M. Rossi

  5. Laboratório Multiusuário de RMN de Sólidos Professora Adelina Costa Neto, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, 21941-909, Brazil

    R. A. S. San Gil

  6. Centro de Terapia Celular e Bioengenharia Ortopédica, Instituto Nacional de Traumatologia e Ortopedia, Rio de Janeiro, RJ, 20230-024, Brazil

    A. Balduino

  7. Centro de Ciências da Saúde, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, bloco F, sala F2-027, Cidade Universitária, Rio de Janeiro, RJ, 21941-902, Brazil

    M. Farina

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  1. W. Querido
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  8. A. Balduino
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Correspondence to M. Farina.

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Querido, W., Abraçado, L.G., Rossi, A.L. et al. Ultrastructural and Mineral Phase Characterization of the Bone-Like Matrix Assembled in F-OST Osteoblast Cultures. Calcif Tissue Int 89, 358–371 (2011). https://doi.org/10.1007/s00223-011-9526-9

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