Donor age and mechanosensitivity of human bone cells.

With increasing age the human skeleton decreases in density, thereby compromising its load-bearing capacity. Mechanical loading activates bone formation, but an age-dependent decrease in skeletal mechanoresponsiveness has been described in rats. In this paper we examine whether age-related bone loss is reflected by a decrease in the mechanosensitivity of isolated bone cells from human donors. Bone cell cultures were obtained from 39 donors (males and females) between 7 and 85 years of age. Cultures were challenged with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or mechanically stressed by treatment with pulsating fluid flow (PFF; 0.7 +/- 0.03 Pa at 5 Hz for 1 h). The growth capacity of the bone-derived cell population almost halved between 7 and 85 years of age. Basal alkaline phosphatase activity of the cells increased with donor age, while the response to 1,25(OH)2D3, measured as stimulated osteocalcin production, decreased with age. Together this suggests that the cell cultures from older donors represented a more mature, slower-growing cell population than the cultures from young donors. All cell cultures responded to mechanical stress with enhanced release of prostaglandin E2 (PGE2) and I2 (PGI2). The magnitude of the response was positively correlated with donor age, cell cultures from older donors showing a higher response than cultures from younger donors. There was also a positive correlation between time to reach confluency and mechanosensitivity, i.e., the PGE2 response to PFF treatment was higher in bone cell cultures with a slower growth rate. We conclude that bone cell cultures from older donors have a lower proliferative capacity and a higher degree of osteoblastic maturation than younger donors. The higher degree of osteoblastic maturation explains the higher response of the cultures to mechanical stress, in line with earlier studies on chicken bone cells. This study found no evidence for loss of mechanosensitivity with donor age. The reduced growth capacity might, however, be a factor in age-related bone loss.

Nitric oxide response to shear stress by human bone cell cultures is endothelial nitric oxide synthase dependent.

Bone cells, in particular osteocytes, are extremely sensitive to shear stress, a phenomenon that may be related to mechanical adaptation of bone. In this study we examined whether human primary bone cells produce NO in response to fluid shear stress and established by RT/PCR which NOS isoforms were expressed before and after application of shear stress. One hour pulsating fluid flow (PFF; 0.7 +/- 0.02 Pa, 5 Hz) caused a rapid (within 5 min) 2 to 4-fold increase in NO production. NO release was only transiently increased during the first 15 min of exposure to PFF, and remained at control levels during a 1-24 hr postincubation period. In both control and PFF-treated cells, mRNA was easily detected for ecNOS, but not nNOS, and only minimal amounts iNOS were found. mRNA levels for ecNOS increased 2-fold at 1 hr after 1 hr PFF treatment. These results suggest that the rapid production of NO by human bone cells in response to fluid flow results from activation of ecNOS. PFF also leads to an increase in ecNOS mRNA which is likely related to the shear stress responsive element in the promoter of ecNOS.

Response of normal and osteoporotic human bone cells to mechanical stress in vitro.

Bone adapts to mechanical stress, and bone cell cultures from animal origin have been shown to be highly sensitive to mechanical stress in vitro. In this study, we tested whether bone cell cultures from human bone biopsies respond to stress in a similar manner as animal bone cells and whether bone cells from osteoporotic patients respond similarly to nonosteoporotic donors. Bone cell cultures were obtained as outgrowth from collagenase-stripped trabecular bone fragments from 17 nonosteoporotic donors between 7 and 77 yr of age and from 6 osteoporotic donors between 42 and 72 yr of age. After passage, the cells were mechanically stressed by treatment with pulsating fluid flow (PFF; 0.7 +/- 0.03 Pa at 5 Hz for 1 h) to mimic the stress-driven flow of interstitial fluid through the bone canaliculi, which is likely the stimulus for mechanosensation in bone in vivo. Similar to earlier studies in rodent and chicken bone cells, the bone cells from nonosteoporotic donors responded to PFF with enhanced release of prostaglandin E2 (PGE2) and nitric oxide as well as a reduced release of transforming growth factor-beta (TGF-beta). The upregulation of PGE2 but not the other responses continued for 24 h after 1 h of PFF treatment. The bone cells from osteoporotic donors responded in a similar manner as the nonosteoporotic donors except for the long-term PGE2 release. The PFF-mediated upregulation of PGE2 release during 24 h of postincubation after 1 h of PFF was significantly reduced in osteoporotic patients compared with six age-matched controls as well as with the whole nonosteoporotic group. These results indicate that enhanced release of PGE2 and nitric oxide, as well as reduced release of TGF-beta, is a characteristic response of human bone cells to fluid shear stress, similar to animal bone cells. The results also suggest that bone cells from osteoporotic patients may be impaired in their long-term response to mechanical stress.

1,25-dihydroxyvitamin D3-mediated transforming growth factor-beta release is impaired in cultured osteoblasts from patients with multiple pituitary hormone deficiencies.

To evaluate the osteoblastic function in patients with multiple pituitary hormone deficiencies (M-PHD) and with isolated growth hormone deficiency (I-GHD), bone cells were cultured and the effects of 10(-8) M 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) on parameters of cell proliferation, osteoblastic differentiation, and local paracrine regulation were measured. Three days of 1,25(OH)2D3 treatment increased alkaline phosphatase activity and osteocalcin release but inhibited [3H]thymidine incorporation in all cell cultures from patients as well as from controls. In addition, 1,25(OH)2D3 increased the release of both total and active transforming growth factor-beta (TGF-beta) in bone cells from controls by, respectively, 4.9- and 3.2-fold and in bone cells from I-GHD by 5.1- and 1.5-fold, respectively. However, in bone cells from M-PHD, the stimulation of total TGF-beta release was significantly lower (1.3-fold) than in control and I-GHD cells, and active TGF-beta release was not stimulated at all. One year of supplementation with human growth hormone did not improve this deficient TGF-beta release in bone cells from M-PHD. We conclude that cultured bone cells from I-GHD and M-PHD show a normal response to 1,25(OH)2D3 regarding cell proliferation and osteoblastic differentiation, which implicates a normal 1,25(OH)2D3-receptor function. In cells from controls and I-GHD, 1,25(OH)2D3 enhanced both total and active TGF-beta release. However, bone cells from M-PHD showed a deficient TGF-beta response to 1,25(OH)2D3. These results suggest that the regulation of TGF-beta production is a major paracrine factor involved in hypopituitarism.

Mechanical loading stimulates the release of transforming growth factor-beta activity by cultured mouse calvariae and periosteal cells.

We have shown earlier that mechanical stimulation by intermittent hydrostatic compression (IHC) inhibits bone resorption and stimulates bone formation in cultured fetal mouse calvariae (Klein-Nulend et al., 1986, Arthritis Rheum., 29: 1002-1009). The production of soluble bone factors by such calvariae is also modified (Klein-Nulend et al., 1993, Cell Tissue Res., 271:513-517). Transforming growth factor-beta (TGF-beta) is an important local regulator of bone metabolism and is produced by osteoblasts. In this study, the release of TGF-beta activity as a result of mechanical stress was examined in organ cultures of neonatal mouse calvariae, in primary cultures of calvariae-derived osteoprogenitor (OPR) cells, and in more differentiated osteoblastic (OB) cells. Whole calvariae and calvariae-derived cells were cultured in the presence or absence of IHC for 1-7 days and medium concentrations of active as well as total TGF-beta were measured using a bioassay. IHC (maximum 13 kPa, maximal pressure rate 32.5 kPa/sec) was generated by intermittently (0.3 Hz) compressing the gas phase above the cultures. We found that mechanical loading by IHC stimulated the release of TGF-beta activity from cultured calvariae by twofold after 1 day. IHC also stimulated the release of TGF-beta activity from calvariae-derived cells after 1 and 3 days. The absolute amounts of TGF-beta activity released were lower in OPR cells than in OB cells, but the stimulatory effect of IHC was greater in OPR cells. Total TGF-beta (active and bound) released into the medium was not affected by IHC. IHC did not change the dry weight of the organ cultures, nor the DNA or protein content of the cell cultures. These data show that mechanical perturbation of bone cells, particularly OPR cells, enhances the activation of released TGF-beta. We conclude that modulation of TGF-beta metabolism may be part of the response of bone tissue to mechanical stress.

Inhibitory effect of high protein intake on nephrocalcinogenesis in female rats.

Increased intakes of protein have been shown to reduce kidney calcification (nephrocalcinosis) in female rats. Two questions were addressed in the present study. First, can protein-induced inhibition of nephrocalcinosis be demonstrated when the diets used are balanced for calcium, magnesium and phosphorus in the added protein? Second, can the protein effect be explained by the frequently observed magnesiuria after giving high-protein diets? Nephrocalcinosis was induced in female rats by giving purified diets containing 151 g casein/kg and either an increased concentration of P (6 v. 2 g/kg) or a decreased concentration of Mg (0.1 v. 0.4 g/kg). To these diets 151 g ovalbumin/kg was added at the expense of glucose, and the diets were balanced for Ca, Mg and P in ovalbumin. The diets were given for 29 d. In rats fed on the diet containing 151 g protein/kg, an increased intake of P or a decreased intake of Mg caused nephrocalcinosis as measured chemically by analysis of kidney Ca as well as histologically by scoring kidney sections stained according to Von Kossa's method. The addition of ovalbumin to the diet prevented the induction of nephrocalcinosis. High P intake and low Mg intake with the low-protein diets induced enhanced loss of albumin in urine, suggesting that nephrocalcinosis caused kidney damage. Increased protein intake with a non-calcinogenic diet also caused increased albumin excretion in urine. Irrespective of the composition of the background diet, increased protein intake caused increased urinary excretion of Mg. When all dietary groups were considered, differences in nephrocalcinosis and urinary Mg output were not proportionally related.