Fibroblast growth factors (FGFs) appear to play an important role in human cranial osteogenesis. We therefore investigated the effects of recombinant human FGF-2 (rhFGF-2) on human calvaria (HC) osteoblastic cells. Immunocytochemical analysis showed that confluent HC cells express both FGF receptors -1 and -2. In short-term culture, rhFGF-2 (0.1-100 ng/ml, 2-5 days) increased HC cell growth and decreased alkaline phosphatase (ALP) activity and type I collagen (ColI) synthesis, as evaluated by P1CP levels. When HC cells were induced to differentiate in long-term culture in the presence of 50 g/ml ascorbic acid and 3 mM phosphate, HC cells initially proliferated, then ALP activity and ColI synthesis decreased and calcium content in the extracellular matrix increased. Continuous treatment with rhFGF-2 (50 ng/ml) for 1-28 days, or a transient rhFGF-2 treatment for 1-7 days, slightly increased DNA synthesis at 7 days, whereas a late treatment for 8 -28 days had no effect on cell growth. The continuous and transient treatments with rhFGF-2 decreased ALP activity, ColI synthesis, and matrix mineralization. This was associated with a transient fall in osteocalcin (OC) production at 7 days. In contrast, the late rhFGF-2 treatment for 8 -28 days only slightly inhibited ALP activity and increased matrix mineralization. In addition, both continuous and late treatments with rhFGF-2 increased OC production in more mature cells at 3-4 weeks of culture. We also found that the early and late treatments with rhFGF-2 had opposite effects on transforming growth factor ␤2 production in proliferating cells and more mature cells. The results show that rhFGF-2 slightly stimulates cell growth and reduces the expression of osteoblast markers in less mature cells, whereas it induces OC production and matrix mineralization in more mature cells, indicating that the effects of FGF-2 are differentiation stage specific and that FGF-2 may modulate HC osteogenesis by acting at distinct stages of cell maturation. (J Bone Miner Res 1998;13:645-654)

To investigate an eventual role of acidosis on hemodialysis osteodystrophy we prospectively studied 21 patients who were dialyzed with different amounts of bicarbonate in the dialysate for 18 months. According to the level of bone formation rate (BFR) on a prestudy bone biopsy, patients were split in two subgroups. Inside these two subgroups patients were randomly allocated to two therapeutics groups: 10 patients (group A) were dialyzed with the conventional amount of bicarbonate (33 +/- 2 mmol/liter) in the dialysate; the rest of the patients (group B, N = 11) had 7 to 15 mmol/liter sodium bicarbonate added to the dialysate to obtain 24 mEq predialysis bicarbonate plasma levels. An effective correction of acidosis was shown in group B by a higher predialysis plasma bicarbonate level (15.6 +/- 1 group A vs. 24.0 +/- 0.6 mEq/liter group B, P less than 0.005), which was reached three months after start of the study. Compared to the prestudy bone biopsy, osteoid and osteoblastic surfaces increased in group A but not in group B on the bone biopsies performed at the end of the study. Parathormone plasma level (iPTH), measured with an antiserum which cross reacts with the 44-68 region of PTH molecule, increased during the study in group A but not in group B. This finding suggested progression of secondary hyperparathyroidism (HPT) only in group A patients. Osteocalcin plasma values increased in both groups during the 18 months of the study. Consequently the two subgroups of patients formed on the basis of BFR level were evaluated separately.(ABSTRACT TRUNCATED AT 250 WORDS)

The systemic release of TNF-alpha and IL-6 during septic shock caused by generalized peritonitis was maximal on day 1 and decreased rapidly during the next days. No systemic release of IL-1 was observed. IL-6 serum concentrations remained higher in patients who subsequently died. Among the different features of peritonitis studied, only bacteremia influenced the systemic cytokine response (higher TNF-alpha).

Bone morphogenetic proteins (BMPs) are factors that promote osteoblastic cell differentiation and osteogenesis. It is unknown whether BMPs may act on human osteoblastic cells by increasing immature cell growth and/or differentiation. We investigated the short- and long-term effects of recombinant human (rh)BMP-2 on cell growth and osteoblast phenotype in a new model of human neonatal pre-osteoblastic calvaria cells (HNC). In short-term culture, rhBMP-2 (20-100 ng/ml) inhibited DNA synthesis and increased alkaline phosphatase (ALP) activity without affecting osteocalcin (OC) production. When cultured for 3 weeks in the presence of ascorbic acid and inorganic phosphate to induce cell differentiation, HNC cells initially proliferated, type 1 collagen mRNA and protein levels rose, and then decreased, whereas OC mRNA and protein levels, and calcium accumulation into the extracellular matrix increased at 2 to 3 weeks. A transient treatment with rhBMP-2 (50 ng/ml) for 1 to 7 days which affected immature HNC cells, decreased cell growth, increased ALP activity and mRNA, and induced cells to express ALP, osteopontin, and OC at 7 days, as shown by immunocytochemistry. At 2 to 3 weeks, matrix mineralization was markedly increased despite cessation of treatment, and although OC and Col 1 mRNA and protein levels were not changed. A continuous treatment with rhBMP-2 for 3 weeks which affected immature and mature cells reduced cell growth, increased ALP activity and mRNA at 1 week and increased OC mRNA and protein levels and calcium content in the matrix at 3 weeks, indicating complete osteoblast differentiation. These results indicate that the differentiating effects of BMP-2 on human neonatal calvaria are dependent on duration of exposure. Although long-term exposure led to complete differentiation of OC-synthesizing osteoblasts, the primary effect of rhBMP-2 was to promote osteoblast marker expression in immature cells, which was sufficient to induce optimal matrix mineralization independently of cell growth and type 1 collagen expression.

ABSTRACT

We studied the biochemical effects of calcium supplementation during a 2-mo course in postmenopausal women (x +/- SD: 64 +/- 5 y of age and 14.5 +/- 6.7 y since menopause). The effects on calcium homeostasis and bone remodeling were assessed after 1 and 2 mo of daily administration of either calcium carbonate (1200 mg elemental Ca/d, n = 60) or a placebo (n = 56). The daily dietary calcium intake assessed before the beginning of calcium supplementation was 786 mg/d. We found a significant inverse relation between baseline intact parathyroid hormone (iPTH) and dietary calcium intake before supplementation (r = -0.48, P = 0.0002). A significant increase in urinary excretion of pyridinoline was observed when the dietary calcium intake was lower than the median value. Calcium supplementation resulted in a significant increase in 24-h urinary calcium (39%, P < 0.02) and a significant reduction of bone alkaline phosphatase at 2 mo and of all bone-resorption markers (hydroxyproline, pyridinoline, and deoxypyridinoline) at I and 2 mo without significant changes in 44-68 PTH fragments or iPTH concentrations. When the dietary calcium intake was low (mean +/- SD: 576 +/- 142 mg/d), calcium supplementation was responsible for a greater increase in urinary calcium excretion and a greater decrease in markers of bone turnover. The greatest variations were observed for deoxypyridinoline at 1 and 2 mo (-18.5%, P < 0.05) and for pyridinoline at 1 mo (-16.3%, P < 0.01). Two months of calcium supplementation in postmenopausal women was efficient in reducing markers of bone turnover, with a greater effect in women with a low dietary calcium intake.

Increased bone resorption is a mechanism contributing to bone loss in the postmenopausal period. Cytokines are involved in osteoclastic differentiation and, therefore, may play a role in the regulation of bone resorption. Several previous works showed the implication of interleukin-1 (IL-1), IL-6, and tumor necrosis factor-alpha (TNF alpha) in the modulation of bone remodeling. This study determines the concomitant production of the three cytokines and tests the bone-resorbing activity of peripheral monocyte supernatants. Four groups of women were studied: premenopausal women (n = 13; mean age, 47 +/- 0.9 yr), untreated postmenopausal women (n = 21; mean age, 52 +/- 0.6 yr), postmenopausal women treated with estrogens (n = 14; mean age, 54.2 +/- 1.1 yr), or postmenopausal women treated with ethanehydroxydiphosphonate (n = 12; mean age, 53.2 +/- 2 yr). Assignment to clinical groups was verified by plasma FSH and estradiol determinations. Lumbar spine bone mineral density was significantly higher in the premenopausal women group than in the three postmenopausal groups. Peripheral blood monocytes were cultured for 48 h with 20% autologous plasma, and after stimulation with lipopolysaccharides. IL-1, IL-6, and TNF alpha levels were measured by RIA in the monocyte surpernatants. The three cytokines were highly correlated to each other, IL-1 with IL-6 (r = 0.76; P < 0.001), IL-1 with TNF alpha (r = 0.89; P < 0.001), and IL-6 with TNF alpha (r = 0.89; P < 0.001). The mean levels of the three cytokines could not be compared because of the variations in the values. However, a trend toward lower levels in the three cytokines was noted in estrogen-treated women compared to the untreated postmenopausals. The bone-resorbing activity of monocyte supernatants, assessed by fetal long bone-resorbing assay, increased in untreated postmenopausal compared to that in premenopausal women (1.22 +/- 0.08 vs. 0.87 +/- 0.11; P < 0.05). In estrogen-treated patients, this activity decreased to premenopausal levels (0.89 +/- 0.04 vs. 0.87 +/- 0.11; P = NS). The resorbing activity was correlated to IL-1 (r = 0.28; P = 0.03), IL-6 (r = 0.52; P < 0.01), and TNF alpha (r = 0.48; P < 0.01). The addition of cytokine inhibitors and IL-1 receptor antagonist and TNF alpha antibodies to the supernatant bone culture medium induced a significant decrease in the calcium release. Those data show the involvement of several cytokines in the bone resorption process after estrogen deficiency.

To determine whether abnormal bone cell recruitment or differentiation may be involved in the development of aplastic bone lesion in renal osteodystrophy we have compared histomorphometric parameters of bone formation and in vitro behavior of osteoblastic cells isolated from the trabecular bone surfaces in 37 dialysis patients with osteitis fibrosa, normal bone formation rate, or aplastic bone lesion. The bone cell responses to human PTH-(1-34) (20 nmol/L), as evaluated by intracellular cAMP production, and to 1,25-dihydroxyvitamin D (10 nmol/L), as assessed by osteocalcin synthesis, were not different from normal in patients with low, normal, or high bone formation rates. Osteoblastic cells isolated from patients with a high bone formation rate and markedly elevated serum iPTH and osteocalcin values had a higher than normal DNA replication in primary culture. The peak of [3H]thymidine incorporation, the maximal DNA synthesis, and the area under the growth curve were 4.4- to 6.3-fold increased in osteitis fibrosa compared to those in normal bone cells obtained from age-matched individuals. By contrast, [3H]thymidine incorporation in bone cells from aplastic patients was about 25% of normal and only 5% of the value in osteitis fibrosa. The decreased DNA replication of cultured bone cells in aplastic patients was unrelated to trabecular bone aluminum staining, but was associated with low serum immunoreactive PTH values compared to those in other groups of patients. These results show that high bone formation in uremic osteoitis fibrosa is associated with higher than normal [3H]thymidine incorporation in bone cells in vitro, whereas low bone formation in aplastic patients results from lower than normal DNA replication and suggest that the defective osteoblastic recruitment in aplastic patients may be related to factors other than aluminum, including inappropriate PTH secretion.