Mineral Fabrication and Golgi Apparatus Activity in the Mouse Calvarium


There is diverse opinion about the mechanism of bone mineralization with only intermittent reports of any direct organellar role played by the golgi apparatus (juxtanuclear body). Light and laser confocal microscopy was combined with electron microscopy and elemental EDX (energy dispersive X-ray microanalysis) in comparing “young” osteocytes in situ in fresh and “slam” frozen developing mouse calvarium, with similar cells (MC3T3-E1) maintained in vitro. The distribution of “nascent” electron dense mineral was examined histochemically (von Kossa, GBHA), including tetracycline (TC) staining as a fluorescent complex with bone salt, while golgi body activity was demonstrated by transfection with a specific green fluorescent construct (GFP/mannosidase II). In tissue culture golgi body activity and mineralization were both blocked by brefeldin A (an established golgi inhibitor) and restored by forskolin, enabling an association with mineral fabrication to be quantified as changing fluorescence intensity (AU) of GFP or TC markers. Results from osteocytes in situ supported previous descriptions of intracellular electron dense objects (microspheres and nanospheres) in a juxtanuclear pattern, containing Ca, P and transitory Si, in a spectrum recapitulated in the calcifying culture after 10 days, when GFP fluorophore surged from 71.7 ± 1.4SD to 133.7 ± 2.7SD AU by 14 days (p < 0.0001). At this stage TC fluorophore mean intensity was 23.8 ± 3.7SD AU (14 days) rising to 45.0 ± 5.1SD AU by 17 days, compared to its stationary 21.7 ± 3.6SD when treated 3 days previously with BFA golgi inhibitor (p < 0.0001), until forskolin reversal. It was concluded from the changing juxtanuclear morphology, Si mineralization mediation and the variably controlled activity versus stasis that the inorganic phase of bone is a complex golgi-directed fabrication with implications for bone matrix biology and evolution.

Share and Cite:

Fallon, V. , Carter, D. and Aaron, J. (2014) Mineral Fabrication and Golgi Apparatus Activity in the Mouse Calvarium. Journal of Biomedical Science and Engineering, 7, 769-779. doi: 10.4236/jbise.2014.79075.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Ross, G.D., Morrison, G.H., Sacher, R.F. and Staples, R.C. (1983) Freeze Substitution Sample Preparation for Ion Microscopy of Plant Tissue. Journal of Microscopy, 129, 221-228.
[2] Hunziker, E.B., Hermann, W., Schenk, R.K., Mueller, M. and Moor, H. (1984) Cartilage Ultrastructure after High Pressure Freezing, Freeze Substitution and Low Temperature Embedding. I. Chondrocyte Ultrastructure—Implications for the Theories of Mineralisation and Vascular Invasion. The Journal of Cell Biology, 98, 267-276.
[3] Carter, D.H., Hatton, P.V. and Aaron, J.E. (1997) The Ultrastructure of Slam-Frozen Bone Mineral. The Histochemical Journal, 29, 783-793.
[4] Kodama, H.-A., Amagai, Y., Sudo, H., Kasai, S. and Yamomoto, S. (1981) Establishment of a Clonal Osteogenic Cell Line from Newborn Mouse Calvaria. Japanese Journal of Oral Biology, 23, 899-901.
[5] Sudo, H., Kodama, H.A., Amagai, Y., Yamamoto, S. and Kasai, S. (1983) In Vitro Differentiation and Calcification in a New Clonal Osteogenic Cell Line Derived from Newborn Mouse Calvaria. The Journal of Cell Biology, 96, 191-198.
[6] Chang, Y.L., Stanford, C.M. and Keller, J.C. (2000) Calcium and Phosphate Supplementation Promotes Bone Cell Mineralization: Implications for Hydroxyapatite (HA)-Enhanced Bone Formation. Journal of Biomedical Materials Research, 52, 270-278.
[7] Aaron, J.E. and Pautard, F.G.E. (1973) A Cell Cycle in Bone Mineralization. In: Balls, M. and Billett, F.S., Eds., The Cell Cycle in Development and Differentiation, University Press, Cambridge, 325-330.
[8] Aaron, J.E. and Pautard, F.G.E. (1975) Tetracycline Staining of Bone in Normal and Pathological States. In: Kuhlencordt, F. and Kruse, H.P., Eds., Calcium Metabolism, Bone and Metabolic Bone Diseases, Springer-Verlag, Heidelberg, Berlin, New York, 211-217.
[9] Aaron, J.E., Makins, N.B. and Francis, R.M. (1984) Staining of the Calcification Front in Human Bone Using Contrasting Fluorochromes in Vitro. Journal of Histochemistry Cytochemistry, 32, 1251-1261.
[10] Perrin, D.D. (1965) Binding of Tetracyclines to Bone. Nature (London), 208, 787-789.
[11] Kaitila, I., Wartiovaara, J., Laitinen, O. and Saxon, L. (1970) The Inhibitory Effect of Tetracycline on Osteogenesis in Organ Culture. Journal of Embryology and Experimental Morphology, 23, 185-211.
[12] Moremen, K.W. and Touster, O. (1986) Topology of Mannosidase II in Rat Liver Golgi Membranes and Release of the Catalytic Domain by Selective Proteolysis. The Journal of Biological Chemistry, 261, 10945-10951.
[13] Kashiwa, H. (1970) Calcium Phosphate in Osteogenic Cells. Clinical Orthopaedics, 70, 200-211.
[14] Oda, K., Hirose, S., Takami, N., Misumi, Y., Takatsuki, A. and Lkehara, Y. (1987) Brefeldin A Arrests the Intracellular Transport of a Precursor of Complement C3 Before its Conversion Site in Rat Hepatocytes. Federation of European Biochemical Societies, 214, 135-138.
[15] Aaron, J.E. (1973) Osteocyte Types in the Developing Mouse Calvarium. Calcified Tissue Research, 12, 259-279.
[16] Aaron, J.E. (1974) The Development of the Bone Cell and its Role in Mineralization and Resorption. Ph.D. Dissertation, University of Leeds, Leeds.
[17] Fallon, V. (2006) The Fabrication of Mineral Particles by Bone Cells and Unicellular Organisms. Ph.D. Dissertation, University of Leeds, Leeds.
[18] Linton, K.M., Tapping, C.R., Adams, D.G., Carter, D.H., Shore, R.C. and Aaron, J.E. (2013) A Silicon Cell Cycle in a Bacterial Model of Calcium Phosphate Mineralogenesis. Micron, 44, 419-432.
[19] Davies, J.E., Chernecky, R., Lowenberg, B. and Shiga, A. (1991) Deposition and Resorption of Calcified Matrix in Vitro by Rat Marrow Cells. Cells Materials, 1, 3-15.
[20] Aaron, J.E. and Pautard, F.G.E. (1972) Ultrastructural Features of Phosphate in Developing Bone Cells. Israel Journal of Medical Sciences, 8, 625-629.
[21] Aaron, J.E., Oliver, B., Clarke, N. and Carter, D.H. (1999) Calcified Microspheres as Biological Entities and Their Isolation from Bone. The Histochemical Journal, 31, 445-470.
[22] Carter, D.H., Scully, A.J., Hatton, P.V., Davies, R.M. and Aaron, J.E. (2000) Cryopreservation and Image Enhancement of Juvenile and Adult Dentine Mineral. The Histochemical Journal, 32, 253-261.
[23] Wada, Y., Kataoka, H., Yokose, S., Ishizuya, T., Miyazono, K., Gao, Y.H., Shibasaki, Y. and Yamaguchi, A. (1998) Changes in Osteoblast Phenotype During Differentiation of Enzymatically Isolated Rat Calvaria Cells. Bone, 22, 479-485.
[24] Carter, D.H., Scully, A.J. and Aaron, J.E. (1998) Evidence for Phosphoprotein Microspheres in Bone. The Histochemical Journal, 30, 677-686.
[25] Shahtaheri, M.S. and Aaron, J.E. (2001) Immunolocalization of Osteocalcin in Calcified Microspheres in Adult Mouse Bone Using FITC-Labelling. Bone, 28, 108S.
[26] Carter, D.H., Scully, A.J., Heaton, D.A., Young, M.P.J. and Aaron, J.E. (2002) Effect of Deproteination on Mineral Morphology: Implications for Biomaterials and Aging. Bone, 31, 389-395.
[27] Gerdes, H. and Kaether, C. (1996) Green Fluorescent Protein: Applications in Cell Biology. Federation of European Biochemical Societies, 389, 44-47.
[28] Tamura, G., Ando, K., Suzuki, S., Takatsuki, A. and Arima, K. (1968) Antiviral Activity of Brefeldin A and Verrucarin A. The Journal of Antibiotics, 21, 160-161.
[29] Klausner, R.D., Donaldson, J.G. and Lippincott-Schhwartz, J. (1992) Brefeldin A: Insights into the Control of Membrane Traffic and Organelle Structure. The Journal of Cell Biology, 116, 1071-1080.
[30] Lu, Z., Joseph, D., Bugnard, E., Zaal, K.J.M. and Ralston, E. (2001) Golgi Complex Reorganization During Muscle Differentiation: Visualization in Living Cells and Mechanism. Molecular Biology of the Cell, 12, 795-808.
[31] Pautard, F.G.E. (1961) Calcium Phosphate and the Origin of Back-bones. New Scientist, 12, 364-366.
[32] Fallon, V., Garner, P.E. and Aaron J.E. (2014) Stress-Induced Golgi Activity and Mineralogenesis: Spirostomum ambiguum as a Protozoan Model for Bone? Submitted.

Copyright © 2023 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.