Effect of basic fibroblast growth factor on the growth and differentiation of adult stromal bone marrow cells: enhanced development of mineralized bone-like tissue in culture.

Rat stromal bone marrow cells (SBMC) were shown to produce mineralized bone-like tissue in culture in the presence of dexamethasone, ascorbic acid, and beta-glycerophosphate. The addition of 3 ng/ml of basic fibroblast growth factor (bFGF) resulted in a significant increase in formation of mineralized tissue. The present study was aimed at assessing the effect of bFGF on the proliferation and differentiation of SBMC and on the sequential development of mineralized bone-like tissue in culture. Transmission electron microscopy of bFGF-treated cultures demonstrated the development of a multilayered structure resembling mineralized bone tissue consisting of cell layers embedded within a heavy extracellular matrix. The matrix was rich in bundles of collagen fibers associated with extensive mineral deposits consisting of hydroxyapatite as determined by infrared spectrophotometry. The addition of 3 ng/ml of bFGF resulted in significant enhancement of [3H]thymidine and [3H]proline incorporation and protein accumulation by 12-, 2.5-, and 2.5-fold, respectively. bFGF treatment increased cAMP responsiveness, alkaline phosphatase activity, osteocalcin level, 45Ca2+ deposition, and mineralized-like tissue formation and induced the earlier expression of these markers in the treated culture. A biphasic sequence of events was observed during the development of mineralized bone-like tissue in bFGF-treated and control cultures. The first phase is characterized by cell proliferation and matrix accumulation and is reflected by a progressive increase in [3H]thymidine and [3H]proline incorporation until day 11. The second phase, which follows, is characterized by a sharp decline in cell proliferation and matrix accumulation and a concomitant expression of osteoblast differentiation as reflected by the progressive increase in alkaline phosphatase activity, mineral deposition, and osteocalcin expression. Treatment of cultures with bFGF accentuated this biphasic sequence of events. These results indicate that bFGF has the capacity to stimulate both the growth and the biochemical functions of SBMC obtained from a young adult animal.

Effect of short-term hyperthyroxinemia on vitamin D metabolism in congenital hypothyroidism.

The circulating concentrations of vitamin D metabolites were measured in nine children (four to ten years of age) with congenital hypothyroidism on L-thyroxine therapy, before and after a short term increase (33%) in dosage. The concentrations of 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D were not altered, but the concentration of 1,25 dihydroxyvitamin D was significantly higher in the serum of the children after three weeks of hyperthyroxinemia. This was associated with an increase in urinary calcium excretion. The increases in serum concentration of 1,25 dihydroxyvitamin D cannot be explained by differences in serum levels of calcium, phosphorus or parathyroid hormone. These findings differ from data obtained in adults.

Blood levels of vitamin D metabolites in gonarthrosis.

The blood levels of the active metabolites of vitamin D3 25-hydroxycholecalciferol [25(OH)D3]. 1,25-dihydroxycholecalciferol [1,25(OH)2D3], and 24,25-dihydroxycholecalciferol [24,25(OH)2D3] were determined in 27 patients suffering from arthrosis of the knee, including 4 patients with non-insulin-dependent diabetes mellitus. The blood level of 24,25-dihydroxycholecalciferol was found to be significantly lower in patients with gonarthrosis than in patients with coxarthrosis. With the exclusion of the diabetic patients, the mean value for this metabolite was lower than in the coxarthrosis group, but the difference was not significant statistically.

Blood levels of active metabolites of vitamin D3 in fracture repair in humans. A preliminary report.

Blood levels of the active metabolites of vitamin D3, 25-hydroxycholecalciferol [25(OH)D3], 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25(OH)2D3] were determined in seven patients. Two subjects suffered from delayed union of tibial fractures; one showed a delayed union after a proximal tibial osteotomy; one patient suffered from bilateral femoral neck fractures, of which one failed to unite and the other united late; two patients had multiple fractures that united normally; and one patient exhibited staged bilateral femoral neck fractures whose occurrence was separated by a short interval and which united without undue delay. The blood levels of 25(OH)D3 were within the normal range. A relative decrease in 24,25(OH)2D3 values was noted in all patients, whereas in three subjects the decrease was absolute, to non-detectable levels. A decrease in 1,25(OH)2D3 levels was noted in only two patients. We postulate that these changes reflect the consumption of these metabolites during healing at the fracture site.

Basic fibroblast growth factor enhances the capacity of bone marrow cells to form bone-like nodules in vitro.

The role of basic fibroblast growth factor (bFGF) in the proliferation and differentiation of rat bone marrow cells in culture was studied. bFGF stimulated [3H]thymidine incorporation into these cells by 4-fold at a concentration of 0.3 ng/ml and half-maximal effect was observed at a concentration of 15 pg/ml. In addition to its mitogenic effect, bFGF stimulated alkaline phosphatase activity by 3.6-fold. Continuous treatment with bFGF (for 21 days) resulted in a 6.3-fold increase in the culture dish surface area covered by bone-like mineralized tissue. Maximal bone-like tissue formation was observed in the presence of 3 ng/ml bFGF with half-maximal effect at a concentration of 0.3 ng/ml. These results indicate the possible role of bFGF in the proliferation of osteogenic rat bone marrow cells and their differentiation into cells of osteoblast-like phenotype.

Active metabolites of vitamin D in patients with coxarthrosis and with aseptic loosening of total hip endoprostheses.

The blood levels of the active metabolites of vitamin D--25-hydroxycholecalciferol (25(OH)D), 1,25 dihydroxycholecalciferol (1,25(OH)2D), and 24,25 dihydroxycholecalciferol (24,25(OH)2D)--were determined in 15 patients suffering from arthrosis of the hip and in 13 patients with aseptic loosening of total hip endoprostheses. Normal values were found in all but one patient with aseptic loosening, in whom 24,25(OH)2D was not detectable. The difference between the two groups of patients was not statistically significant.

Effect of cholecalciferol derivatives on the mechanical properties of chick bones.

Chicks were depleted of vitamin D, divided into groups, and treated daily with (a) cholecalciferol, (b) 1 alpha-hydroxycholecalciferol [1 alpha (OH)-D3], (c) 24R, 25-dihydroxycholecalciferol [24R,25-(OH)2D3], or (d) 1 alpha (OH)D3 and 24R,25(OH)2D3. Two additional groups of chicks were studied, one that was continuously depleted of vitamin D, and another that was continuously supplemented with the vitamin, since day 1. After killing, the tibiae were removed and tested for their mechanical properties. Bending load was applied to the midshaft, and the intrinsic properties of this site, its quantity and geometry were analyzed. From a mechanical point of view, the weakest bones found were of birds depleted of vitamin D, whereas the strongest were of those treated with 1 alpha (OH)D3. Only the bones of the 24R,25(OH)2D3-treated or the 1 alpha (OH)D3 and 24R,25(OH)2D3-treated groups of birds showed mechanical properties comparable to those obtained with vitamin D-replete chicks.

The effect of toxic doses of 1,25-dihydroxycholecalciferol on dental tissues in the rat.

Vitamin D-depleted rats 4-weeks old were divided into three groups and given daily for 5 weeks cholecalciferol (0.25 microgram) or 1,25(OH)2D3 (0.075 microgram). The third group received no treatment with vitamin D sterols. A fourth control group was fed a diet containing vitamin D. The animals were killed after 5 weeks, plasma was prepared for calcium analysis, and incisors and molars were taken for histology. Growth was monitored throughout. Plasma calcium, body weight and the physical condition of the 1,25(OH)2D3-treated animals indicated that they were toxemic. The pulp-dentine complex of their incisors showed premature aging of fibroblasts and odontoblasts, disturbances in the dentinal matrix and osteodentine formation. That of molars was not affected. There was hypercementosis and bone-like tissue formation in the periodontal-ligament which in the incisors was considerably enlarged; some molars were ankylosed. The pulp-dentine complex of the incisors and molars of the rats in the remaining three groups appeared normal except for zones of hypomineralization in incisors of the third group. The supporting tissues of the teeth of the rats in the other three groups were within normal limits. Thus toxic doses of 1,25(OH)2D3 affected the dental tissues of both developing and mature teeth.

1,25-dihydroxyvitamin D3 and the regulation of macrophage function.

Vitamin D3 deficient (D-) mice show a depressed inflammatory response and both inflammatory peritoneal macrophages and bone marrow polymorphonuclear leukocytes of D- mice exhibit a decreased spontaneous migration under agarose. The impaired phagocytic response of peritoneal macrophages from D- mice can be corrected by incubation with 1,25-dihydroxyvitamin D3 and is not affected by interaction with other vitamin D3 metabolites. Transfer of mice from the D- to the D+ state results in correction of both the inflammatory and the phagocytic response. Intactness of phagocyte function is thus directly dependent on vitamin D3 metabolism.

Absorption and excretion of cholecalciferol and of 25-hydroxycholecalciferol and metabolites in birds.

The absorption and excretion in vivo of cholecalciferol or 25-hydroxycholecalciferol (25-HCC) were determined in chicks (Gallus domesticus) and turkeys (Meleagris gallopavo). The overall net cholecalciferol or 25-HCC absorption in chicks and cholecalciferol in turkey poults was 66.5 +/- 3.3, 74.9 +/- 3.7 and 83.6 +/- 7.1% of the intake, respectively. The absorption of cholecalciferol or 25-HCC in chicks and turkeys occurred at the upper part of the intestine. 25-HCC, esters and non-polar metabolites of cholecalciferol or 25-HCC, and their polar metabolites, were secreted in the duodenum of chicks and turkeys but were partially reabsorbed at the upper part of the jejunum.

Metabolism of cholecalciferol in land snails.

1. Radioactively labelled cholecalciferol was injected into the land snails Levantina hiersolyma and Theba pisana. Three metabolites (C, D and E), more polar than cholecalciferol, were found. 2. Metabolite C was found to be identical with 25-hydroxycholecalciferol. On injection of 25-hydroxy[26,27-3H]cholecalciferol, metabolite E was predominantly formed. Metabolite D was predominantly formed from cholecalciferol. Metabolites D and E differ from any known cholecalciferol metabolites. 3. The intestine was found to be the tissue capable of carrying out the transformation of 25-hydroxycholecalciferol into metabolite E. 4. 25-Hydroxycholecalciferol and metabolite E were localized in the digestive gland of the snail, the tissue responsible for the absorption of Ca2+ and its storage. Metabolite D was not localized in any specific tissue.

Synthesis of 1 alpha-hydroxy[7-3H]cholecalciferol and its metabolism in the chick.

1. 1 alpha-Hydroxy[7-3H]cholecalciferol (specific radioactivity of 2-Ci/mmol) was synthesized, and its metabolism in chicks studied. 2. 1 alpha-Hydroxy[7-3H]cholecalciferol was metabolized very rapidly in the chick to 1 alpha,25-dihydroxy[7-3H]cholecalciferol and to a metabolite less polar than 1 alpha-hydroxycholecalciferol. Intestine exhibited highest accumulation of 1 alpha-25-dihydroxy[7-3H]cholecalciferol, and liver exhibited highest accumulation of the non-polar metabolite. 3. Tissue uptake of 1 alpha-hydroxy[7-3H]cholecalciferol and its metabolites in chicks that were dosed continuously for 16 days with 1 alpha-hydroxy[7-3H]cholecalciferol did not exceed by very much that observed in tissues obtained from chicks that were dosed with a single injection of 1 alpha-hydroxy[7-3H]cholecalciferol 24 h before killing, except for liver and kidney. 4. Lowest accumulation of metabolites was noted in muscle and bone, and for the latter, highest uptake of 1 alpha,25-dihydroxy[7-3H]cholecalciferol was noted in the epiphysial periosteum and the metaphysis. 5. Formation of 1 alpha,24,25-trihydroxy[7-3H]cholecalciferol was not observed in the chicks that were dosed continuously with 1 alpha-hydroxy[7-3H]cholecalciferol, despite the fact that plasma calcium and phosphorus were normal and despite the presence of renal 24-hydroxylase activity. 6. The vitamin D status of the chicks did not appear to affect the metabolic profile of the administered 1 alpha-hydroxy[7-3H]cholecalciferol.

The parotid gland: a new target organ for vitamin D action.

Radioactively labelled cholecalciferol was administered continuously to rats which were fed a vitamin D-deficient diet. It has been possible to show that all the metabolites of the cholecalciferol which normally occur in known target tissues of vitamin D are present in the parotid gland, and the pattern resembled that obtained for the kidney, a known target tissue for vitamin D action. The accumulation of cholecalciferol metabolites in the parotid gland was shown to be functional, as a calcium-binding protein was found to be present in the gland, possessing similar properties to the renal vitamin D-dependent calcium-binding protein.

Vitamin D metabolism and expression in rats fed on low-calcium and low-phosphorus diets.

1. Cholecalciferol, radioactively labelled with both (14)C and (3)H, was administered weekly for 7 weeks to rats that had been depleted of vitamin D for 4 weeks before repletion with the radioactive vitamin. This permitted measurement of the steady-state effect on vitamin D metabolism of low-calcium and low-phosphorus regimens, as compared with a normal mineral intake. These dietary manoeuvres were carried out during the last 3 weeks of repletion. Cholecalciferol, 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol were determined in plasma, intestine, kidney and bone. Ca(2+)-binding-protein content was measured in intestine and kidneys of comparable animals. 2. In rats on the low-calcium diets, 1,25-dihydroxycholecalciferol concentration was elevated in plasma, bone, kidney and intestine, and intestinal Ca(2+)-binding protein was increased to over twice the concentration found in the control animals. 3. The low-phosphorus regimens led to a decrease in plasma phosphate and 1,25-dihydroxycholecalciferol in all tissues studied, for the latter to the point where it was undetectable in plasma and bone. Intestinal and renal concentrations of Ca(2+)-binding protein were unchanged in the low-phosphate-intake group and decreased in the very-low-phosphate-intake group. 4. It is concluded that in the rat, unlike in the chick, hypophosphataemia is not associated with a stimulation of the production of 1,25-dihydroxycholecalciferol or its expression in the synthesis of Ca(2+)-binding protein. Therefore the plasma phosphate concentration does not appear to be directly involved in the regulation of the functional metabolism of vitamin D.