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Mineralization of Decalcified Bone Occurs Under Cell Culture Conditions and Requires Bovine Serum But Not Cells

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Abstract

The purpose of this study was to develop an in vitro model system for bone matrix mineralization in the absence of cells. For this model, we utilized EDTA-decalcified new-born rat tibias with the cartilaginous ends intact, allowing us to visually determine the specificity of mineralization within the bone. Our results show that supplementation of DMEM culture medium with 10mM β-glycerophosphate and 15% fetal bovine serum (FBS) results in non-physiological mineral percipitation in the tibia because of the generation of supraphysiological (5mM) levels of inorganic phosphate in the medium. The same medium supplemented only with inorganic phosphate to a final concentration of 2mM failed to mineralize a decalcified tibia matrix. However, additional supplementation of this medium with as little as 5% FBS resulted in mineralization of those regions of the type I collagen where mineral was found prior to decalcification, with no evidence for mineralization in the cartilage at the bone ends or in the periosteum. Analysis of the mineral by Fourier-transform infrared spectroscopy and powder X-ray diffraction shows that tibias that have been decalcified and then remineralized contain an apatitic mineral that is strikingly similar to the mineral in normal bone. Tendon, a type I collagen matrix not normally mineralized in vivo , also mineralizes when incubated in DMEM containing 2mM Pi and as little as 1.5% FBS, but not when incubated in DMEM without serum. These data indicate that serum contains a nucleator of type I collagen matrix mineralization, and that mineralization of type I collagen under cell culture conditions requires serum but not living cells.

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Notes

  1. The failure of demineralized tibias to remineralize during incubation in serum-free DMEM clearly shows that serum-initiated remineralization is not due to the possible presence of calcium phosphate crystals that remained in the bone matrix after demineralization for 72 hours with 0.5 M EDTA. This conclusion is further supported by the observation that tibias that have been demineralized in 150 mM HCl for 24 hours also remineralize in DMEM, but only if serum is present (personal observations). The serum-free DMEM used in the present study did, however, sustain the growth of added hydroxyapatite crystals (data not shown).

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Acknowledgments

We are grateful to Dr. Murray Goodman (University of California, San Diego) for use of his FTIR spectrometer and to Matthew Williamson for critical review of the manuscript. N. Hamlin is partially supported by a GAANN (Graduate Assistance in Areas of National Need – Department of Education) fellowship. This work was supported in part by Grant AR25921 from The National Institute of Health

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  1. Division of Biological Sciences, University of California, La Jolla, San Diego, CA 92093-0368, USA

    N. J. Hamlin & P. A. Price

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  1. N. J. Hamlin
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  2. P. A. Price
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Correspondence to P. A. Price.

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Hamlin, N.J., Price, P.A. Mineralization of Decalcified Bone Occurs Under Cell Culture Conditions and Requires Bovine Serum But Not Cells. Calcif Tissue Int 75, 231–242 (2004). https://doi.org/10.1007/s00223-004-0190-1

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  • DOI: https://doi.org/10.1007/s00223-004-0190-1

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