Aluminum toxicity perturbs long bone calcification in the embryonic chick.

Long bone calcification in chick embryos acutely- or chronically-treated with aluminum (Al) citrate was investigated. Acutely treated embryos received 100 microl of 60 mM Al citrate, 60 mM sodium (Na) citrate, or 0.7% sodium chloride on day 8 of incubation. Chronically treated embryos received a daily 25 microl dose of the above solutions beginning on day 8. Following 2-8 days of additional incubation, blood was collected, embryos killed, hind limbs radiographed, and tibias collected. Radiography indicated that Al administration resulted in a persistent angulation in the mid-diaphysis of tibias and femurs and a transient mineralization defect during the 10- to 12-day period of incubation. Tibias from 10- to 12-day embryos which were administered Al contained significantly less (P < 0.005) bone calcium (Ca) compared with tibias from NaCl-treated embryos. By day 14 there were no significant differences among the Ca content of tibias from embryos acutely treated with Al citrate, Na citrate or NaCl. Similarly, the rate of (45)Ca uptake by tibias of embryos treated with Al was significantly lower on days 10 (acute) and 12 (chronic) with no significant differences in Ca uptake rate among the three treatment groups by day 16. In each treatment group bone alkaline phosphatase (ALPase) activity increased approximately tenfold between days 10 and 16. At all stages, bone ALPase activity was consistently higher and significantly different (chronic) compared with levels in NaCl-treated embryos. In contrast, Al had no significant effect on the rate of tibia collagen and noncollagenous protein synthesis or serum levels of procollagen carboxy-terminal propeptide (PICP), osteocalcin, and parathyroid hormone (PTH).

Matrix proteins associated with bone calcification are present in human vascular smooth muscle cells grown in vitro.

Atherosclerotic lesions are composed of cellular elements that have migrated from the vessel lumen and wall to form the cellular component of the developing plaque. The cellular elements are influenced by various growth-regulatory molecules, cytokines, chemoattractants, and vasoregulatory molecules that regulate the synthesis of the extracellular matrix composing the plaque. Because vascular smooth muscle cells (VSMC) constitute the major cellular elements of the atherosclerotic plaque and are thought to be responsible for the extracellular matrix that becomes calcified in mature plaques, immunostaining for collagenous and noncollagenous proteins typically associated with bone matrix was conducted on VSMC grown in vitro. VSMC obtained from human aorta were grown in chambers on glass slides and immunostained for procollagen type I, bone sialoprotein, osteonectin, osteocalcin, osteopontin, decorin, and biglycan. VSMC demonstrated an intense staining for procollagen type I, and a moderately intense staining for the noncollagenous proteins, bone sialoprotein and osteonectin, two proteins closely associated with bone mineralization. Minimal immunostaining was noted for osteocalcin, osteopontin, decorin, and biglycan. The presence in VSMC of collagenous and noncollagenous proteins associated with bone mineralization suggest that the smooth muscle cells in the developing atherosclerotic plaque play an important role in the deposition of the extracellular matrix involved in calcification of developing lesions.

Aluminum effects on blood chemistry and long bone development in the chick embryo.

Body growth, blood chemistry, and long bone development of 10- to 16-day chick embryos (Gallus gallus) treated with aluminum (Al) citrate, sodium (Na) citrate, or sodium chloride (NaCl) were investigated. Two administration protocols were used. Acutely-treated embryos received 6.0 mumol Al citrate or Na citrate on day 8 of incubation. Chronically-treated embryos received a daily dose of 1.5 mumol Al citrate or Na citrate beginning on day 8 of incubation. For both protocols, Al citrate and Na citrate had no significant influence on viability or body weight. Al citrate-treated embryos had: (a) significantly shorter mean tibia lengths by day 16 of incubation, (b) a consistently lower ratio of tibia length: body weight on all days investigated, and (c) a persistent mid-diaphyseal malformation (angulation) of the femur and tibia. Spatially correlated with the malformation was a calcification defect detected by alizarin red S staining of intact tibias and the accumulation of aluminum as demonstrated by acid solochrome azurine staining of histological sections. Aluminum was localized at the mineralization front of the osteogenic collar surrounding the cartilage core of the tibia. Aluminum citrate or Na citrate had no significant effect on serum total calcium, inorganic phosphorus, total alkaline phosphatase activity, or creatinine, except for a transitory hypercalcemia (day 10) and phosphatemia (days 10 and 12) in Al citrate-treated embryos. The concomitant localization of Al and the early calcification defect in the region of tibial malformation implicate aluminum in the pathogenesis of the skeletal abnormality.

Influence of short-term aluminum exposure on demineralized bone matrix induced bone formation.

The effects of aluminum exposure on bone formation employing the demineralized bone matrix (DBM) induced bone development model were studied using 4-week-old Sprague-Dawley rats injected with a saline (control) or an aluminum chloride (experimental) solution. After 2 weeks of aluminum treatment, 20-mg portions of rat DBM were implanted subcutaneously on each side in the thoracic region of the control and experimental rats. Animals were killed 7, 12, or 21 days after implantation of the DBM and the developing plaques removed. No morphological, histochemical, or biochemical differences were apparent between plaques from day 7 control and experimental rats. Plaques from day 12 control and experimental rats exhibited cartilage formation and alkaline phosphatase activity localized in osteochondrogenic cells, chondrocytes, osteoblasts, and extracellular matrix. Unlike the plaques from control rats that contained many osteoblastic mineralizing fronts, the plaques from the 12-day experimental group had a preponderance of cartilaginous tissue, no evidence of mineralization, increased levels of alkaline phosphatase activity, and a reduced calcium content. Plaques developing for 21 days in control animals demonstrated extensive new bone formation and bone marrow development, while those in the experimental rats demonstrated unmineralized osteoid-like matrix with poorly developed bone marrow. Alkaline phosphatase activity of the plaques continued to remain high on day 21 for the control and experimental groups. Calcium levels were significantly reduced in the experimental group. These biochemical changes correlated with histochemical reductions in bone calcification.(ABSTRACT TRUNCATED AT 250 WORDS)

Radioautographic localization of 3H-fucose incorporation into bone cells in cultured calvaria.

Radioautographic procedures were used to examine the incorporation of 3H-fucose into macromolecular material in bone organ cultures. Radioautography demonstrated that silver grains were first apparent in the paranuclear area of osteoclasts, and continued to increase in number over osteoclasts throughout the culture period. Silver grains associated with osteoclasts were later found on the side adjacent to bone and the adjacent bone surface. Osteoblasts, pre-osteoblasts and fibroblasts of the fibrous periosteum incorporated some 3H-fucose as evidenced by labelling in the later time periods. At later incubation periods, labelled material was found in the underlying bone below osteoblasts, demonstrating the active elaboration of bone matrix glycoproteins. Osteocytes were labelled only after the longest period of incubation.

Differentiation of mononuclear cells into multinucleated osteoclast-like cells.

Experimental evidence accumulated in recent years suggests that the osteoclast is derived from the fusion of mononuclear precursors which are of hematopoietic origin. Mononuclear cells were isolated from the spleen and bone marrow of young rats in order to examine osteoclast formation. The isolated cells were placed in diffusion chambers containing devitalized bone fragments freed of soft tissue, and the chambers sealed and placed in the peritoneal cavity of host rats. The host animals were killed after 4 days, and the bone removed from the chambers for examination. Light-microscopic examination demonstrated two types of cells adjacent to the bone surface, one a flattened and elongated mononuclear cell, and the other a larger and frequently multinucleated cell which had the morphological appearance of an osteoclast. Scanning electron microscopy demonstrated numerous flattened and elongated cells adjacent to the bone surface, as well as a second cell type which had dorsal membrane specializations and numerous lateral microprojections attaching to the bone surface. The second cell type was thought to correspond to the osteoclast-like cells seen with light microscopy. The observations suggest that osteoclast-like cells differentiate from mononuclear precursor cells of hematopoietic tissue.

Inhibition of bone resorption and increased incorporation of 3H-glucosamine into hyaluronate in bone organ cultures treated with dibutyryl cyclic AMP and colchicine.

Addition of dibutyryl adenosine 3':5'-monophosphate (DBCAMP), colchicine or dbcAMP/colchicine to bone organ cultures markedly inhibited bone resorption. Calvaria of newborn mice labeled with 45Ca 3 days before sacrifice were removed and cultured in medium containing 3H-glucosamine. Both agents alone and in combination inhibited 45Ca release. DEAE-cellulose chromatography of the papain-digested medium and bone resolved the 3H-glucosamine-labeled macromolecular material into four peaks. A marked increase in radioactivity was observed in the hyaluronate fraction (peak III) of the chromatographed bone samples. The results indicate that dbcAMP does not mimic the effect of parathyroid hormone on bone resorption, but that it is similar to colchicine in both the inhibition of bone resorption and the increased incorporation of 3H-glucosamine into hyaluronate. Although the role of hyaluronate, if any, in this experimental system remain elusive, the increased radioactivity associated with the hyaluronate fraction may be due to decreased degradation resulting from decreased hyaluronidase synthesis.

3H-fucose incorporation into bone following exposure to parathyroid hormone, dibutyryl cyclic-AMP and colchicine.

Radioautographic and scintillation counting procedures were used to examine the effect of parathyroid hormone (PTH), dibutyryl cyclic-AMP (DB-cAMP), and colchicine on the incorporation of 3H-fucose into macromolecular material in organ cultures of bone. Radioautography demonstrated 3H-fucose incorporation into bone cells, with the heaviest uptake occurring in osteoclasts. A minimal incorporation occurred in pre-osteoblasts and osteoblasts of the osteogenic periosteum, and in fibroblasts of the fibrous periosteum. PTH appeared to produce a heavier label in association with osteoclasts while decreasing the limited labeling associated with cells of the osteogenic and fibrous periosteum. DB-cAMP and colchicine both markedly reduced the labeling associated with osteoclasts, while the minimal labeling of other bone cells remained. By contrast, scintillation counting results indicated that PTH had little or no effect on 3H-fucose incorporation, while DB-cAMP and colchicine considerably reduced the amount of labeled macromolecular material. The incorporation of 3H-fucose into glycoproteins and the role of glycoproteins are discussed.

The effect of cyclic nucleotides on the incorporation of 3H-glucosamine into hyaluronate in bone organ culture.

Addition of dibutyryl cyclic AMP or parathyroid hormone to bone organ cultures markedly increased the incorporation of 3H-glucosamine into non-dialyzable macromolecules. Other cyclic nucleotides or their dibutyryl derivatives did not stimulate glucosamine incorporation. DEAE-cellulose chromatography of the papain-digested calvaria and culture medium resolved the labeled material into four peaks. A four-fold increase in radioactivity was observed in peak III. Previous studies of peak III have identified the labeled material as hyaluronic acid. The results suggest that the parathyroid hormone stimulated increase in glucosamine incorporation is mediated via the adenylate cyclase-cyclic AMP system, and that the increased amount of radioactivity is due to an increased amount of hyaluronic acid. Turnover studied of the labeled material suggest that the release of proteoglycans into the culture medium is not inhibited in the cultures treated with dibutyryl cyclic AMP. The role of hyaluronate in this experimental system remains to be elucidated.

Demonstration of adenosine triphosphate hydrolysis in the enamel organ of the mouse incisor.

The hydrolysis of adenosine triphosphate in the mandibular enamel organ demonstrated that the Mg++-activated ATPase was destroyed by pre-treatment with either heat or alcohol, substrate specific for ATP, stimulated by the addition of glutathione or dinitrophenol, and inhibited by oligomycin. The distribution of reaction product was the same with Mg++, Mn++ or Zn++ as the activating cation. Omission of Mg++ from the incubation medium, or replacement with Ca++ or Sr++ resulted in marked hydrolysis of ATP in the cells associated with enamel matrix formation, with loss of enzyme activity in the cells of the zone of enamel matrix maturation. Hydrolysis of ATP by the cells of the stratum intermedium, stellate reticulum and papillary layer was dependent upon Mg++, Mn++, or Zn++.