Pluripotent mesenchymal stem cells in bone marrow differentiate to osteoblast progenitor cells. When the bone marrow cells are cultured in vitro, they form colony-forming units-fibroblastic (CFU-Fs) with exhibiting osteoblastic features such as expression of alkaline phosphatase (ALP) and formation of calcified nodules ex vivo. This article describes the effect of growth, maturation, and aging of the skeleton on human CFU-Fs harvested from human iliac bone marrow. Human bone marrow cells were harvested from the ilia of 49 women, and were cultured ex vivo for examination. The 49 subjects ranged in age from 4 to 88 years and were without metabolic bone disease. These aspirated bone marrow cells from human ilium exhibited osteoblastic phenotype such as alkaline phosphatase (ALP) activity, expression of osteocalcin (OSC) and parathyroid hormone-receptor (PTH-R) mRNA, and the formation of calcified nodules in vitro. The number of ALP-positive CFU-Fs and the ALP activity were quantified. The highest levels of ALP-positive CFU-Fs were observed in the young group, particularly in those under 10 years of age. The levels of ALP-positive CFU-Fs declined sharply after 10 years of age; those above 20 years of age exhibited a lower number of ALP-positive CFU-Fs, with a gradual decline with increasing age. These results indicate that change in the number of ALP-positive CFU-Fs may be associated with skeletal growth and maturation. The results also show that osteoblastic features such as ALP activity and capability of formation of calcification nodules were maintained even in the older subjects. These findings suggest that decreased activity of bone formation in the aged subjects could be, in part, caused by the decreased number of osteoprogenitor cells differentiating into osteoblasts because the number of ALP-positive CFU-Fs was one of the indices exhibiting bone-forming activity in the human marrow stromal cells.

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Recently, an imaging technique using microcomputed tomography (micro-CT) has emerged as a method for nondestructively assessing the microarchitecture of unprocessed surgical bone biopsy specimens. Using micro-CT, two-dimensional (2D) axial images were obtained from undecalcified transiliac bone biopsies which were taken from 15 patients with various metabolic bone diseases. Total area, bone area, and bone perimeter were determined, from which the bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) were calculated semiautomatically and instantaneously. To evaluate the validity of this technique as a useful tool, the results were compared with those obtained from conventional histomorphometry. There were significant correlations between the two techniques for all parameters, with correlation coefficients ranging from 0.759 (Tb.N, P < 0.005) to 0.949 (BV/TV, P < 0.0001). Different resolutions seem to lead to major differences in perimeter values measured by the two methods. These factors may explain why the correlation coefficients of Tb.N and Tb.Th estimated from the perimeter and area is lower than that of BV/TV. Our results show that the micro-CT based on 2D images is a useful tool for imaging and nondestructively quantifying the microarchitecture of trabecular bone in unprocessed surgical bone specimens.

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Changes of bone formation markers lagged behind those of histomorphometric parameters in cancellous bone because changes of cortical bone were observed later and were incomplete compared with those of cancellous bone.

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Transforming growth factor-beta1 (TGF-beta1) has opposite effects on osteoblastic cells in vitro, namely an inhibitory or stimulatory effect on cell differentiation. Because these effects are dependent on TGF-beta1 concentration or culture condition, we investigated whether the in vivo effects of TGF-beta1 on bone formation in infant rat calvaria were affected by the dose or the injection site. Human platelet-derived TGF-beta1 was injected subcutaneously onto the periosteal surface of parietal bone of 4-week-old rats at doses of 5 or 20ng/100microl per animal for 14 days, and the local effect on bone formation was examined by bone histomorphometry. TGF-beta1 treatment for 7 days decreased the mineral apposition rate, bone formation rate, and elongated mineralization lag time at the injection site. This change became more prominent when treatment continued for 14 days. These changes were restricted to the TGF-beta1-exposed area. Multiple subcutaneous injections of a relatively high dose (200ng/100microl per animal) of TGF-beta1 induced woven bone formation, in addition to marked inhibition of bone formation rate and prolongation of mineralization lag time. On the other hand, direct exposure of TGF-beta1 in the subperiosteal layer induced woven bone with periosteal cell proliferation even at a single injection of a low dose (5 or 50ng/25 microl) of TGF-beta1. In conclusion, the in vivo effects of TGF-beta1 on bone formation varied depending on its concentration and injection site. Also, subcutaneous injection of relatively low doses of TGF-beta1 inhibited local lamellar bone formation.