Vascularization is fundamental for bone formation and bone tissue homeostasis. However, in human subjects, a direct molecular relationship has not been identified between angiogenesis and agents that promote bone disease or factors related to age. Osteopenia is a condition in which bone mineral density is lower than normal, and it represents a sign of normal aging. Here we tested whether the type H vessel, which was recently identified as strongly positive for CD31 and Endomucin (CD31hiEmcnhi) in mice, is an important indicator of aging and osteopenia in human subjects. We found that age-dependent losses of type H vessels in human bone sections conform to the observations in aged mice. The abundance of human type H vessels and osteoprogenitors may be relevant to changes in the skeletal microarchitecture and advanced osteopenia. Furthermore, ovariectomized mice, a widely used model for postmenopausal osteoporosis, exhibited significantly reduced type H vessels accompanied by reduced osteoprogenitors, which is consistent with impaired bone microarchitecture and osteoporosis, suggesting that this feature is an indicator of bone mass independent of aging. More importantly, administration of desferrioxamine led to significantly increased bone mass via enhanced angiogenesis and increased type H vessels in ovariectomized mice. Altogether, these data represent a novel finding that type H vessels are regulated in aged and osteopenia subjects. The abundance of human type H vessels is an early marker of bone loss and represents a potential target for improving bone quality via the induction of type H vessels.

Iron overload has recently been connected with bone mineral density in osteoporosis. However, to date, the effect of iron overload on osteoblasts remains poorly understood. The purpose of this study is to examine osteoblast biological activity under iron overload. The osteoblast cells (hFOB1.19) were cultured in a medium supplemented with different concentrations (50, 100, and 200 μM) of ferric ammonium citrate as a donor of ferric ion. Intracellular iron was measured with a confocal laser scanning microscope. Reactive oxygen species (ROS) were detected by 2,7-dichlorofluorescin diacetate fluorophotometry. Osteoblast biological activities were evaluated by measuring the activity of alkaline phosphatase (ALP) and mineralization function. Results indicated that iron overload could consequently increase intracellular iron concentration and intracellular ROS levels in a concentration-dependent manner. Additionally, ALP activity was suppressed, and a decline in the number of mineralized nodules was observed in in vitro cultured osteoblast cells. According to these results, it seems that iron overload probably inhibits osteoblast function through higher oxidative stress following increased intracellular iron concentrations.

Bone metabolism has a close relationship with iron homeostasis. To examine the effects of iron excess and iron deficiency on the biological activities of osteoblast in vitro, human osteoblast cells (hFOB1.19) were incubated in a medium supplemented with 0-200 μmol/L ferric ammonium citrate and 0-20 μmol/L deferoxamine. The intracellular iron was measured by a confocal laser scanning microscope. Proliferation of osteoblasts was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptotic cells were detected using annexin intervention V/PI staining with a flow cytometry. Alkaline phosphatase (ALP) activity was measured using an ALP assay kit. The number of calcified nodules and mineral area was evaluated by von Kossa staining assay. The expressions of type I collagen and osteocalcin of cultured osteoblasts were detected by reverse transcriptase polymerase chain reaction and Western blot. Intracellular reactive oxygen species (ROS) was measured using the oxidation-sensitive dye 2,7-dichlorofluorescin diacetate by flow cytometry. The results indicated that excessive iron inhibited osteoblast activity in a concentration-dependent manner. Low iron concentrations, in contrast, produced a biphasic manner on osteoblasts: mild low iron promoted osteoblast activity, but serious low iron inhibited osteoblast activity. Osteogenesis was optimal in certain iron concentrations. The mechanism underlying biological activity invoked by excessive iron may be attributed to increased intracellular ROS levels.

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117 postmenopausal women were divided into Normal, Bone loss (BL), and Osteoporosis group. Compared with Normal group (120.96 ± 43.18 μg/L), the serum ferritin (Fer) in BL (223.37 ± 130.27 μg/L) and Osteoporosis group (307.50 ± 161.48 μg/L) was significantly increased (p < 0.05). Fer level was negatively correlated with BMD (p < 0.01). TRACP levels in Osteoporosis group (4.37 ± 1.69 U/L) were significantly higher than Normal group (4.10 ± 1.60 U/L, p < 0.05). ALP levels in Osteoporosis group (112.06 ± 62.05 U/L) were significantly upregulated compared with Normal group (80.22 ± 14.94 U/L, p < 0.05). β-CTX and PINP were the degradation products of type I collagen. β-CTX levels in Osteoporosis group (667.90 ± 316.55 ng/L) were significantly increased compared with Normal group (406.06 ± 112.12 ng/L, p < 0.05). PINP levels in Osteoporosis group (78.03 ± 37.31 μg/L) were significantly higher than Normal group (37.60 ± 13.17 μg/L, p < 0.01). More importantly, there was a positive correlation between serum Fer and PINP (p < 0.01). Serum Fer showed a positive correlation of serum β-CTX (p < 0.01). The overloaded iron improved the degradation of type I collagen.