A macromolecular inhibitor of in vitro calcification of tendon matrix

Abstract

Bovine and human tendon tissue do not induce calcification in vitro. However, extraction of those tissues with 3% Na2HPO4 converts them to calcifiable matrices. The supernatant fraction derived from the extraction contains a nondialyzable, perchloric acid soluble component that inhibits calcification of the extracted matrix. This inhibitory substance is characterized by a molecular weight in the range of 85,000-100,000. Exposure to pronase or hyaluronidase did not alter the inhibitory potency but did render the inhibitor dialyzable. Commercial sources of hyaluronic acid, chondrotitin-6-sulfate, chrondroitin-4-sulfate, dermatan sulfate, heparin and lysozyme did not inhibit calcification of the extracted matrix. Phosvitin, a phosphoglycoprotein is a potent inhibitor. Although phosvitin and the tendon extract also inhibit calcification of previously calcified matrix, they have no detectable effect on the rate of decalcification. We conclude that the mechanism of inhibition is characterized by a degree of specificity and that phosvitin and a macromolecular component of tendon tissue blocks conversion of an intermediate matrix-bound CaP complex to crystalline apatite. It seems reasonable that the tendon inhibitor could function in situ and possibly in vivo to control calcification of tendon tissue.

Calcification inhibitors in human ligamentum flavum

“…As Quittner and Wadkins pointed out (24), this change in the ligamentum flavum can be explained by the following two hypotheses: (a) phosphate specifically promotes dissociation from the tissue of substances that are calcification-inhibitory in situ, and (b) phosphate alone, or in combination with endogenous Ca2+, binds at appropriate sites of the matrix and thereby provides centers for nucleation and growth of calcium phosphate crystals. Although no evidence has been presented to indicate that the extract inhibits through the binding to the nucleation site of the matrix, the results of the present study suggest that the extract does have an inhibitory activity for matrix calcification in vitro.…”

“…It has been reported that the calcification inhibitor in bovine Achilles tendon has a peptidoglycan structure with a molecular weight of 85,000-100,000 (24). In the present study, although identification and measurement of the molecular weight was not possible because of the limited number of specimens, it was presumed to be greater than 8,000 from the exclusion limit of the cellulose dialysis tubing used, and the molecular weight of the protein obtained in the present method probably was smaller than that of protein extracted by use of protease inhibitors.…”

“…The radioactivity was then measured with a liquid scintillation counter (LS 3801; Beckman, Fullerton, CA, U.S.A.) to determine the uptake of calcium by the matrix. With the use of extracted matrices, changes in radioactivity in the reaction system were measured in the presence of 16 mg of the ligamentum flavum extract (the weight of tendon matrix extract used by Quittner and Wadkins [24]), 1 mM NaF (32), and 250 pgiml of phosvitin (24). The concentration of inorganic phosphate in the solution was measured by a pmethylaminophenol reduction method (6).…”

“…Two thousand micrograms of streptomycin and 2,000 units of penicillin were added to all incubations. At 2,4,6,8,12,18,24,36, and 48 h of incubation under stirring, a 1 ml aliquot of the soluble phase was obtained after centrifugation at 3,000 G for 5 min. The radioactivity was then measured with a liquid scintillation counter (LS 3801; Beckman, Fullerton, CA, U.S.A.) to determine the uptake of calcium by the matrix.…”

Calcific Tendonitis : A Model

“…The interesting observation that normal tendon matrix does not mineralize in vitro [17] implies that changes in the structure or composition of tendon extracellular matrix are necessary for the development of hydroxyapatite deposits. Aging, diabetes, and injury likely alter tendon extracellular matrix so that calcification of matrix vesicles might occur.…”