Homocysteine decreases chondrocyte-mediated matrix mineralization in differentiating chick limb-bud mesenchymal cell micro-mass cultures.

The differentiating chick limb-bud mesenchymal cell micro-mass culture system has been used as a model for monitoring the effects of matrix modification on cell-mediated calcification. In this study, we show that treating these micro-mass cultures with homocysteine (Hcys) impairs cartilage calcification. Cultures were treated from day 2 to day 7 with two nonphysiological concentrations of Hcys equivalent to 100x and 1000x avian serum levels (0.36 and 3.6 mmol/L), and from days 9-13 with one tenth the concentration. Mineralization assays were done at days 16, 19, and 21, and matrix and cell properties were examined between days 5 and 21. Mineral accretion, based on differential (45)Ca uptake (mineralizing minus control cultures), was significantly reduced in the high-Hcys-concentration group, and slightly reduced in the low-Hcys-concentration group. Electron microscopy at culture day 21 showed that the collagen matrix was less abundant and its banding pattern less obvious in the Hcys-treated groups than in the untreated cultures. Pyridinoline (Pyr) and deoxypyridinoline (d-Pyr) contents were not detectable in day 21 cultures with either 0.36 or 3.6 mmol/L homocysteine, whereas values in mineralizing and nonmineralizing controls ranged from 0.06 to 0.08 and 0.03 to 0.06 (moles/mole collagen) for Pyr and d-Pyr, respectively. Fourier transform infrared (FTIR) imaging also indicated a decreased content of pyridinoline cross-links. Hcys caused other matrix changes as well. Whereas at culture day 5 there was no significant difference in the number of chondrocyte nodules formed, by day 11 the proteoglycan content (measured by Alcian blue dye binding at 595 nm) was significantly reduced in both mineralizing and control cultures in the high- and low-Hcys groups. In contrast, there were no detectable differences in type X collagen and alkaline phosphatase staining in the mineralizing cultures with or without Hcys supplements. Because vital dye stains and electron microscopy studies indicated that cells in the control and experimental groups did not differ in terms of viability, the observed differences cannot be attributed to toxicity. Thus, Hcys treatment, which causes matrix disorganization, decreases the ability of the matrix to support mineralization.

Adenosine 5'-triphosphate promotes mineralization in differentiating chick limb-bud mesenchymal cell cultures.

When chick limb-bud mesenchymal cells are plated in micromass culture, they differentiate to form a mineralizable cartilage matrix. Previous studies have demonstrated that, when the total inorganic phosphate concentration of the medium is adjusted to 3-4 mM by adding inorganic phosphate to the basal medium, the mineralized matrix formed resembles that of chick calcified cartilage in ovo. When the high-energy phosphates adenosine 5'-triphosphate (ATP) or creatine phosphate are used as supplements in place of inorganic phosphate, the mineralized matrix as analyzed by electron microscopy and Fourier transform infrared microscopy is also similar to that in ovo. This is in marked contrast to the mineralized matrix formed in the presence of 2.5-5 mM beta-glycerophosphate, where mineral deposition is random and mineral crystal sizes in general are larger. This is also in contrast to the known ability of ATP to inhibit mineral deposition in solution in the absence of cells. In the differentiating mesenchymal cell culture system, ATP does not alter the rate of cell proliferation (DNA content), the rate of matrix synthesis (3H-leucine uptake), the mean crystallite length, or the rate of mineral deposition (45Ca uptake) when contrasted with cultures supplemented with inorganic phosphate. However, ATP does increase the mineral to matrix ratio, especially around the edge of the culture, where a type I collagen matrix is presented. It is suggested that ATP promotes mineral deposition by providing a high-energy phosphate source, which may be used to phosphorylate extracellular matrix proteins and to regulate calcium flux through cell membranes.

The pathophysiologic role of fat in dysbaric osteonecrosis.

Dysbaric osteonecrosis (DON) can occur in humans and sheep after a single hyperbaric air exposure with inadequate decompression. The authors hypothesize that DON does not result from primary embolic or compressive effects of nitrogen bubbles on the osseous vasculature, but by secondary injury to the marrow adipose tissue by rapidly expanding nitrogen gas that triggers local, and possibly systemic, intravascular coagulation. A 28-year-old scallop diver remained at a depth of 92 feet in sea water for 4.5 hours on surface-supplied compressed air. Decompression sickness occurred after a no-stop ascent to the surface, and he died 70 minutes later. Autopsy showed multiple gas bubbles, not only within the great vessels, but in the fatty marrow of his femoral and humeral heads. Lipid and platelet aggregates were found on the surface of marrow bubbles. Fibrin-platelet thrombi were detected within dilated venous sinusoids adjacent to bubbles, and in veins, capillaries, and arterioles. Since pulmonary, renal, and intraosseous (subchondral) fat embolism and fibrin thromboses were observed, it is suggested that injured marrow adipocytes can release liquid fat, thromboplastin, and other vasoactive substances, which conceivably can also play a systemic procoagulant role in triggering disseminated intravascular coagulation and additional DON.

FT-IR microscopic mappings of early mineralization in chick limb bud mesenchymal cell cultures.

Chick limb bud mesenchymal cells differentiate into chondrocytes and form a cartilaginous matrix in culture. In this study, the mineral formed in different areas within cultures supplemented with 4 mM inorganic phosphate, or 2.5, 5.0, and 10 mM beta-glycerophosphate (beta GP), was characterized by Fourier-transform infrared (FT-IR) microscopy. The relative mineral-to-matrix ratios, and distribution of crystal sizes at specific locations throughout the matrix were measured from day 14 to day 30. The only mineral phase detected was a poorly crystalline apatite. Cultures receiving 4 mM inorganic phosphate had smaller crystals which were less randomly distributed around the cartilage nodules than those in the beta GP-treated cultures. beta GP-induced mineral consisted of larger, more perfect apatite crystals. In cultures receiving 5 or 10 mM beta GP, the relative mineral-to-matrix ratios (calculated from the integrated intensities of the phosphate and amide I bands, respectively) were higher than in the cultures with 4 mM inorganic phosphate or in the in vivo calcified chick cartilage.

Requirement of vitamin C for cartilage calcification in a differentiating chick limb-bud mesenchymal cell culture.

Mesenchymal cells isolated from stage 21-24 chick limb-buds plated in a micro-mass culture differentiate to form chondrocytes and synthesize a calcifiable matrix. In the presence of inorganic phosphate (4 mM), hydroxyapatite mineral deposits around cartilage nodules. Ascorbic acid is, in general, an essential co-factor for extracellular matrix synthesis in culture, since it is required for collagen synthesis. In this study we demonstrate that in the absence of ascorbic acid supplementation in the mesenchymal cell cultures, mineral deposition (indicated by X-ray diffraction, measurement of Ca:hydroxyproline ratio, and 45Ca uptake) does not occur. Concentrations of 10-50 micrograms/ml ascorbate were compared to find the "optimal" concentration for cell mediated mineralization; 25 micrograms/ml was selected as optimal based on matrix appearance at the EM level and the rate of 45Ca uptake. High concentrations of ascorbic acid (greater than 75 micrograms/ml), while increasing the amount of hydroxyproline in the matrix synthesized, caused some cell death and hence less cell-mediated mineralization. This study demonstrates both the need for viable cells and a proper matrix for in vitro cell-mediated mineralization, and shows that varying the concentration of L-ascorbate (vitamin C) in the medium can have a marked effect on mineralization in vitro.

Concentration-dependent effects of dentin phosphophoryn in the regulation of in vitro hydroxyapatite formation and growth.

The effect of dentin phosphophoryn on hydroxyapatite formation and growth was studied in an in vitro gelatin gel diffusion system. Phosphophoryn, in low concentrations (0.010-1 microgram/ml) promoted de novo hydroxyapatite formation; at a higher concentration (100 micrograms/ml) in the same system, the dentin matrix protein inhibited hydroxyapatite growth. Similar inhibition of hydroxyapatite growth was seen in solution. The intact phosphophoryn was not essential for either inhibition of seeded growth or promotion of mineralization, since the formic acid degraded protein was comparably effective. Transmission electron microscopy of the precipitates formed at 7 days showed no significant differences in crystallite size distribution in the presence and absence of phosphophoryn. However there was a dose-dependent decrease in the number of mineral clusters formed in the presence of increasing amounts of phosphophoryn, suggesting inhibition of secondary nucleation. These data provide support for the postulated 'multifunctional' role of the dentin phosphoprotein in the mineralization process.