Osteonectin inhibiting de novo formation of apatite in the presence of collagen.

The effect of bone matrix protein of osteonectin on de novo formation of apatite was studied in a wide range of calcium phosphate solutions in the presence of collagen. In every solution, from which amorphous calcium phosphate, octacalcium phosphate, or apatite precipitated as a possible initial phase, osteonectin at concentrations less than 1 microM retarded the precipitation, subsequent transformation to apatite, and ripening crystal growth of apatite. Collagen present as either reconstituted or denatured form had no effect on the osteonectin-associated reactions as well as osteonectin-free reactions, and no structural correlation was observed between collagen fibrils and any of the calcium phosphates that appeared in our system. Direct measurement of free calcium levels in the solutions suggested that the reduction in calcium activity due to complexing with osteonectin hardly explained the inhibitory activity of osteonectin in retarding the formation of apatite. Instead, our transmission electron microscopic (TEM) observation strongly suggested that the primary mechanism for osteonectin to inhibit the formation of apatite is to block growth sites of calcium phosphates nucleated. The apatite thus formed in the presence of osteonectin showed less resolved X-ray diffraction patterns, partly because of smaller crystallites as suggested by TEM.

Ion-translocating properties of calcifiable proteolipids.

De novo formation of calcium hydroxyapatite in biological systems occurs on membrane surfaces through specific interactions of Ca, Pi, phospholipids, calcifiable proteolipids, and ion flux to and from the nucleating site. This paper reports an in vitro model demonstrating an ion transport function for calcifiable proteolipid. Bacterionema matruchotii proteolipid was incubated with a radiolabeled H+-channel inhibitor, 14C-dicyclohexyl-carbodiimide, and binding characterized by displacement studies with DCCD or ethyldimethylaminopropylcarbodiimide. A carboxyl binding site was suggested by displacement of DCCD by the nucleophile, glycine ethyl ester. The displacement studies indicated that proteolipid bound DCCD via carboxyl group interaction in a hydrophobic region of the protein. SDS-polyacrylamide gel electrophoresis showed that all label was associated with a single band of 8500 Mr. No non-specific binding of 14C-DCCD to phospholipids occurred, since all bound label was associated with protein following Sephadex LH-20 chromatography of crude proteolipid. Phospholipid liposomes were prepared containing bacteriorhodopsin and proteolipid or proteolipid-14C-DCCD, via cholate dialysis. Transmembrane pH changes established by the bacteriorhodopsin H+ pump were measured in the presence and absence of added proteolipid. Proteolipid had an effect similar to those of uncouplers such as tetraphenylboron. Both the rate and extent of proton translocation increased following addition of proteolipid to BR-liposomes. 14C-DCCD abolished the proteolipid-augmented ion transport. When tetraphenylboron was used to abolish the transmembrane electrical potential, calcifiable proteolipid did not augment proton transport.(ABSTRACT TRUNCATED AT 250 WORDS)

Mineralization in in vitro cultures of rabbit marrow stromal cells.

Localized regions of mineralization were found in confluent cultures of rabbit marrow fibroblastic cells. The mineralized tissue developed within clusters of giant fat cells in the spaces between the cells. Investigations with light and electron microscopy demonstrated that in these sites there was some differentiation of the fibroblastic cells in an osteogenic direction, shown by changes to more polygonal shapes, and the synthesis of well-banded collagen similar to that found in bone tissue. Differentiation may be due, in part, to increased cell density in a confined space. Growth of the mineralized tissue was observed in the living cultures with a fluorescence microscope. Electron probe microanalysis confirmed that the mineral formed was hydroxyapatite. Initiating sites of mineralization included membranous vesicular bodies, lipid, and products of cellular degeneration. Once initiated, mineralization appeared to spread rapidly into adjacent collagenous and other structures, suggesting the appearance of a mixture of skeletal-type and dystrophic mineralization.

Proteolipid-lipid relationships in normal and vitamin D-deficient chick cartilage.

Previous studies have shown that in vitro calcification of chick epiphyseal cartilage matrix vesicles is proteolipid-dependent. The purpose of this research is to examine the role of proteolipid in cartilage calcification in vivo by comparing the proteolipid concentration of normal and vitamin D-deficient chick epiphyseal cartilage, the relationship of proteolipid to other tissue lipids, and its ability to support in vitro apatite formation. Proteolipid was isolated from the upper growth centers (reserve cell zone, upper proliferative zone) and lower growth centers (lower proliferative, hypertrophic, and calcified cartilage zones) of long-bone epiphyses from 3-week-old normal and rachitic male white leghorn chicks by Sephadex LH-20 chromatography of the crude phospholipid component of the total lipid extract. In both normal and rachitic tissue the proteolipid/dry weight and proteolipid/total lipid ratios were greater in the lower growth center than in the upper zones. No statistically significant change in the proteolipid/total lipid ratio in rachitic tissues relative to comparable cell zones in normal cartilages was observed. However, there was an increase in the nonproteolipid phospholipid content of rachitic tissues, altering the relative proteolipid/phospholipid composition. Whereas proteolipids from normal tissue supported in vitro calcification, proteolipids from rachitic tissues did not, indicating a direct effect of vitamin D on proteolipid structure. These data support the hypothesis that failure of rachitic cartilage to calcify in vivo may be due in part to alterations in phospholipid and proteolipid metabolism.

The synthesis and characterization of N-sulfo-2-amino tricarballylate: an analog of phosphocitrate and inhibitor of calcification.

The synthesis of N-sulfo-2-amino tricarballylate, a sulfamate analog of phosphocitrate, is described using pyridine-sulfur trioxide to sulfonate 2-amino tricarballylate. The product was purified using selective precipitation and chromatographic techniques and characterized by a variety of physical and chemical means. In particular, its ability to inhibit hydroxyapatite formation and calcium oxalate crystallization was assessed by comparison with known inhibitors including phosphocitrate and pyrophosphate. On the basis of these results, this new compound may ultimately have a role in preventing abnormal biological calcification.

Calcific diseases. A concept.

New observations portray calcification processes as similar whether occurring normally or pathologically. Most forms of calcification are initiated by membranous organelles, ie, extracellular, calcifying "matrix vesicles" or intracellular mitochondria. Matrix vesicles promote calcification through calcium-binding phospholipids and phosphatase activity. Mitochondria use a forceful, inwardly directed Ca and phosphate transport mechanism. After mineral initiation, the proliferation of mineral crystals is dependent on regulatory factors, such as extracellular Ca2+ and PO4(3-) and other mineral inhibitors and promoters. Calcific diseases are defined as those in which (1) Ca uptake is early, (2) calcification is importantly related to dysfunction, and (3) the control of calcification may lead to decreased morbidity or enhanced diagnostic capability. Calcific diseases include such well-known entities as crystal deposition arthritis, atherosclerosis, calcific valvular sclerosis, tumor calcification, dental plaque, and dysfunctional calcification occurring in implanted cardiovascular devices.