The present studies show for the first time that demineralized bone re-calcifies rapidly when incubated at 37°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 seruminduced re-calcification of demineralized bone suggest that the serum calcification factor identified in these studies may participate in the normal calcification of bone.This study is a continuation of our investigations into the mechanisms that initiate the normal calcification of bone and the abnormal calcification of arteries and into the mechanisms that inhibit these calcifications. We recently proposed the hypothesis that artery calcification is linked to bone resorption (1, 2) to explain the association between increased bone resorption and increased artery calcification that has been seen in the vitamin D-treated rat (1), in the osteoprotegerin-deficient mouse (3), and in patients with postmenopausal osteoporosis (see Ref. 2 for references). One prediction of the hypothesis that artery calcification is linked to bone resorption is that inhibitors of bone resorption should inhibit artery calcification (2). In previous studies we tested this prediction using three different types of bone resorption inhibitors, each with an entirely different mode of action on the osteoclast, the amino bisphosphonates alendronate and ibandronate (2), the cytokine osteoprotegerin (4), and the V-H ϩ -ATPase inhibitor SB 242784 (5). Each bone resorption inhibitor potently inhibited artery calcification.Ibandronate, osteoprotegerin, and SB 242784 are each highly specific inhibitors of the osteoclast at the concentrations used in these studies and have no known actions on vascular cells. Their ability to potently inhibit vascular calcification is therefore hard to reconcile with the hypothesis that the process is initiated...
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 EDTAdecalcified 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 b-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.
Abstract. We investigated the evolutionary origin of a serum activity that induces calcification within a type I collagen matrix, an activity previously described in rat and bovine serum. Serum was obtained from vertebrates with calcified tissues (bony fish and shark), vertebrates without calcified tissues (lamprey and hagfish), and three invertebrates (marine worm, crab, and sea urchin). Serum from the bony fish and shark proved to contain a potent nucleator of collagen calcification; like the previously described calcifying activity in rat serum, the fish and shark activities are both able to recalcify a demineralized rat tibia when tested in DulbeccoÕs modified Eagle medium containing as little as 1.5% of the respective serum and have an apparent molecular weight of 50-150 kDa. No calcifying activity could be detected in any of several experimental tests of invertebrate or hagfish serum. Weak calcifying activity could be detected in lamprey serum, but calcification was restricted to the growth plate of the decalcified tibia, with no detectable calcification in the type I collagen of the midshaft. These studies reveal a correlation between the evolutionary timing of the appearance of calcified tissues in vertebrates and the appearance of the serum activity that initiates calcification within collagen and, therefore, support the hypothesis that this serum activity may play a role in normal calcification of bone. Key words: Calcification of bone -Serum nucleatorEvolutionary conservation -Bony fish -Cartilaginous fishWe recently showed that the type I collagen matrix of demineralized bone recalcifies when incubated in rat or bovine serum for 6 days at 37°C [1,2]. The incorporation of calcium and phosphate into demineralized rat tibias ceases after several weeks of incubation in successive volumes of serum, and the amount of calcium and phosphate introduced into the demineralized tibias at this point is comparable to that present in the same bone segment prior to demineralization [2]. These fully recalcified tibias are palpably hard, radiographs of the recalcified tibias are indistinguishable from tibias prior to demineralization, and the recalcified bone mineral is comparable to the original bone mineral in calcium and phosphate ratio and in Fourier transform infrared ratio (FTIR) and X-ray diffraction (XRD) spectra [2]. Early stages of bone recalcification in serum are characterized by the presence of numerous discrete foci of von Kossa staining, each 0.2-2 lm in diameter, which are present throughout the collagenous matrix of the demineralized bone. An identical pattern of calcification is seen when rat tail tendon is incubated for 6 days in serum [1,2], which shows that serum-induced calcification occurs in a type I collagen matrix that normally calcifies (demineralized bone) as well as in a type I collagen matrix that does not normally calcify in the rat (tendon).These studies further showed that the recalcification of demineralized bone during incubation in serum is attributable to the presence of a serum ca...
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