Abstract
The present studies show for the first time that demineralized bone re-calcifies rapidly when incubated at 37 degrees C in rat serum: re-calcification can be demonstrated by Alizarin Red and von Kossa stains, by depletion of serum calcium, and by uptake of calcium and phosphate by bone matrix. Re-calcification is specific for the type I collagen matrix structures that were calcified in the original bone, with no evidence for calcification in periosteum or cartilage. Re-calcification ceases when the amount of calcium and phosphate introduced into the matrix is comparable to that present in the original bone prior to demineralization, and the re-calcified bone is palpably hard. Re-calcified bone mineral is comparable to the original bone mineral in calcium to phosphate ratio and in Fourier transform infrared and x-ray diffraction spectra. The serum activity responsible for re-calcification is sufficiently potent that the addition of only 1.5% serum to Dulbecco's modified Eagle's medium causes bone re-calcification. This putative serum calcification factor has an apparent molecular mass of 55-150 kDa and is inactivated by trypsin or chymotrypsin. The serum calcification factor must act on bone for 12 h before re-calcification can be detected by Alizarin Red or von Kossa staining and before the subsequent growth of calcification will occur in the absence of serum. The speed, matrix-type specificity, and extent of the serum-induced re-calcification of demineralized bone suggest that the serum calcification factor identified in these studies may participate in the normal calcification of bone.
Highlights
Serum contains a potent calcification factor that initiates the re-calcification of demineralized bone and provide evidence that this factor is a protein of 55–150 kDa molecular mass
The nature of the mineral phase introduced in the serum-initiated re-calcification of bone is comparable in XRD and FTIR spectra to that originally found in the bone prior to demineralization. These observations strongly suggest that serum-initiated re-calcification of demineralized bone could be identical to, or closely similar to, the process by which bone matrix is normally mineralized
Future studies will clearly be needed to identify the protein (or proteins) that are involved in serum-initiated bone re-calcification and to determine whether this protein (or proteins) participates in normal bone mineralization, ectopic calcifications, and/or other calcification processes
Summary
Materials—42-day-old, 22-day-old (weanling), and newborn albino rats (Sprague-Dawley derived) were purchased from Charles River Laboratories (Wilmington, MD). A single wet tibia or calvarium, or a rehydrated tendon (3 mg of dry weight) was added to 2 ml of rat serum, rat plasma, or physiological buffer and placed in the incubator for 6 days. The effect of adding different amounts of rat serum on the calcification of tibia and tendon was examined using 10 ml of DMEM in 100-mm culture dishes (Falcon 3803) in a humidified incubator at 37 °C and 5% CO2. Ten demineralized tibias were each separately incubated for 3 days at 37 °C in 2 ml of rat serum, washed twice for 15 min each with 20 ml of 25 mM HEPES buffer, pH 7.4, containing 1 mM calcium, 3 mM phosphate, 100 mM NaCl, 30 mM NaHCO3, and 150 g/ml fetuin (Sigma), and incubated in 20 ml of this buffer in a humidified incubator at 37 °C and 5% CO2. The resulting powders were first analyzed by XRD using a Scintag SDF 2000 x-ray diffractometer, and a portion of this powder was analyzed by FTIR
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