Evidence for a serum factor that initiates the re-calcification of demineralized bone.

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.

SB 242784, a selective inhibitor of the osteoclastic V-H+ATPase, inhibits arterial calcification in the rat.

The present experiments were carried out to further test the hypothesis that arterial calcification is linked to bone resorption by determining whether the selective inhibition of bone resorption with SB 242784, a specific inhibitor of the osteoclastic V-H+-ATPase, will inhibit arterial calcification. Treatment for 96 hours with toxic doses of vitamin D caused widespread calcification in the aorta and in the femoral, mesenteric, hepatic, renal, and carotid arteries, and treatment with SB 242784 completely prevented the vitamin D-induced calcification of each of these arteries at a dose of 40 mg/kg per day and significantly reduced calcification at a dose of 10 mg/kg per day. Treatment with vitamin D also caused extensive calcification in the lungs, tracheal cartilage, and kidneys, and treatment with SB 242784 prevented or reduced calcification at each of these sites. Measurement of serum levels of cross-linked N-telopeptides, a specific measure of bone resorption activity, showed that treatment with vitamin D alone produced the expected 2.4-fold increase in bone resorption activity and that concurrent treatment with the 40-mg dose of SB 242784 reduced bone resorption activity to below control levels. With the inclusion of the present results, there are now three types of bone resorption inhibitors (each with an entirely different mode of action on the osteoclast) that share the ability to potently inhibit arterial calcification in the rat, the V-H+-ATPase inhibitor SB 242784, the cytokine osteoprotegerin, and the amino bisphosphonates alendronate and ibandronate.