Tatsuhiko Tanizawa

Number of osteoprogenitor cells in human bone marrow markedly decreases after skeletal maturation.

Abstract: 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.

Increased bone formation by intermittent parathyroid hormone administration is due to the stimulation of proliferation and differentiation of osteoprogenitor cells in bone marrow

In order to examine the mechanism of the anabolic effect of parathyroid hormone (PTH) on bone formation, human PTH(1-34) [hPTH(1-34)] (30 micrograms/kg) was injected subcutaneously to 9-week-old rats 5 times a week for 1 or 3 weeks. Trabecular bone volume (BV/TV) in the tibial metaphysis was not significantly different between the PTH- and vehicle-treated groups, but the parameters related to bone formation, including osteoid surface (OS/BS), mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS), were significantly increased as early as 1 week after PTH treatment. And the parameters related to bone resorption including eroded surface (ES/BS) and osteoclast number (N.Oc/BS) were also significantly increased as early as 1 week after PTH treatment. Treatment with PTH for 1 week induced no significant increase in bone mineral density at the femoral metaphysis, whereas the same treatment for 3 weeks induced a significant increase. When bone marrow cells isolated from femora and tibiae of either PTH- or vehicle-treated rats were cultured at a high density (2 x 10(7) cells/one well of 24-multiwell plate), cellular alkaline phosphatase (ALP) activity was significantly increased in the cells isolated from PTH-treated rats compared with vehicle-treated rats. When bone marrow cells were cultured at a low density (4 x 10(6) cells/a one well of 6-multiwell plate) to generate colonies (colony forming unit-fibroblastic, CFU-F), PTH induced apparent increases in both the total number of CFU-F and the number of ALP-positive CFU-F.(ABSTRACT TRUNCATED AT 250 WORDS)

A Morphometric Comparison of Trabecular Structure of Human Ilium Between Microcomputed Tomography and Conventional Histomorphometry

Abstract. 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.

Three-dimensional microstructural analysis of human trabecular bone in relation to its mechanical properties

The purpose of this preliminary study is to explore the relationship between elastic modulus, bone mineral density (BMD), and trabecular microstructure in three dimensions. Twenty cubes of trabecular bone were processed from two lumbar vertebrae obtained from one individual. The BMD of each cube was measured by dual-energy X-ray absorptiometry and peripheral quantitative computed tomography. Each cube was serially scanned by microcomputed tomography to produce three-dimensional data sets. By analyzing these data sets, three-dimensional trabecular microstructural indices of connectivity density and fractal dimension were calculated as well as histomorphometric parameters. The cubes were tested mechanically in a nondestructive manner for measurement of their elastic modulus. This preliminary study showed that: (1) bone mass index is correlated with mechanical properties, with coefficients of correlation ranging from 0.552 to 0.601; and (2) when controlling for BMD, no association could be detected between measures of structural complexity (connectivity density and fractal dimension) and elastic modulus in the craniocaudal direction of human vertebral bodies.

Local effects of transforming growth factor-β1 on rat calvaria: Changes depending on the dose and the injection site

Abstract: Transforming growth factor-β1 (TGF-β1) has opposite effects on osteoblastic cells in vitro, namely an inhibitory or stimulatory effect on cell differentiation. Because these effects are dependent on TGF-β1 concentration or culture condition, we investigated whether the in vivo effects of TGF-β1 on bone formation in infant rat calvaria were affected by the dose or the injection site. Human platelet-derived TGF-β1 was injected subcutaneously onto the periosteal surface of parietal bone of 4-week-old rats at doses of 5 or 20 ng/100 μl per animal for 14 days, and the local effect on bone formation was examined by bone histomorphometry. TGF-β1 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-β1-exposed area. Multiple subcutaneous injections of a relatively high dose (200 ng/100 μl per animal) of TGF-β1 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-β1 in the subperiosteal layer induced woven bone with periosteal cell proliferation even at a single injection of a low dose (5 or 50 ng/25 μl) of TGF-β1. In conclusion, the in vivo effects of TGF-β1 on bone formation varied depending on its concentration and injection site. Also, subcutaneous injection of relatively low doses of TGF-β1 inhibited local lamellar bone formation.

Bone mineral density and bone histomorphometric assessments of postpregnancy osteoporosis: A report of five patients

Reports of five young women who developed vertebral fractures associated with pregnancy and lactation are presented (Fig. 1). Ages ranged from 24 to 37 (mean 30) years. All five patients have osteoporosis with two to nine vertebral fractures at presentation postpartum. Bone mineral density (BMD) was measured by single-photon absorptiometry, quantitative computer tomography, and dual-energy X-ray absorptiometry. BMD of the trabecular bone was less than normal values and it remained apparently low even several years after pregnancy. Histological findings of bone biopsy identified the bone loss with increasing bone resorption. Our present findings suggest that postpregnancy osteoporosis affects mainly the trabecular bone site, and the patients might have low peak bone mass and poor reversibility probably due to a low rate of remodeling.

Reduction in bone formation and elevated bone resorption in ovariectomized rats with special reference to acute inflammation

Changes in bone modeling and remodeling in the tibia of growing rats within 30 days of ovariectomy (ovx) were evaluated by histomorphometric, mechanical; and biochemical means. Three days after ovx, suppressed bone formation was seen. This was shown by reduced osteoid volume, osteoblast surface, and bone formation rate in the secondary spongiosa, and a reduced longitudinal growth rate in the growth plate. In addition, the alkaline phosphatase and tartrate-resistant acid phosphatase activity in bone marrow supernatants was suppressed in conjunction with elevated serum sialic acid levels, indicating inflammation. Although estrogen deprivation itself may provoke the inflammatory process, the serum sialic acid level in the ovx group returned to the baseline level within 5 days after surgery, while that of estradiol in the ovx group remained consistently lower. This suggests that surgical stress, not estrogen deprivation, is the primary cause of the inflammatory response shortly after ovx. A significant difference (p < 0.01) between the ovx and sham rats was seen in the osteoclast surface, which peaked on day 7 in the ovx rats. On day 14 postovariectomy, the bone formation rate peaked and remained constant until day 30. In the ovx rats, there was a sustained reduction in the serum albumin level until day 30. Estrogen deprivation may be the primary cause of these changes, because both surgical ovx and medical oophorectomy with gonadotropin-releasing hormone agonist (G(nRHa) reduce the serum albumin level. In numerous studies dealing with changes after ovx in rats, we have observed: 1) a transient reduction in bone formation in relation to inflammatory changes evoked by ovx surgery, and 2) a sustained reduction in the serum albumin level for at least 30 days after ovx that is possibly due to estrogen deprivation.

Effects of intermittent administration of low dose human PTH(1–34) on cancellous and cortical bone of lumbar vertebral bodies in adult beagles

This study assessed the effect of low dose human parathyroid hormone [hPTH(1-34)] administration on cancellous and cortical bone of lumbar vertebrae in intact male beagles. 16 19-20-month-old beagle dogs were randomized into four groups: in group 1, the vehicle control group, saline was injected daily; in group 2, the sequential group, 0.375 microg/kg of PTH was injected daily for 4 weeks, then off 8 weeks, and this sequence was once repeated for another 4 and 8 weeks; in group 3, the same dose of PTH was injected once per week for 24 weeks; and, in group 4, PTH was injected three times per week for 24 weeks. Histomorphometric assessment on cancelllous and cortical bone (both ventral and dorsal shell) and two-dimensional node-strut analysis were done on the fifth lumbar vertebral bodies after calcein double bone labeling. In intact adult beagles, on the group treated with 0.375 microg/kg per day three times per week (group 4): (1) had a higher mean value in cancellous bone formation parameters [osteoid surface (+74%), osteoid volume (twofold), mineral apposition rate (+21%), and bone formation rate (twofold)]; (2) exhibited no effect on cortical thickness and porosity in both the ventral and dorsal shell; and (3) showed a lower mean value of node to termini (0.11 +/- 0.02 vs. 0.22 +/- 0.09) and a higher mean value of cortex to node (0.18 +/- 0.06 vs. 0.08 +/- 0.02), but not in trabeculae to trabeculae node, than age-related controls. In conclusion, we found that a low dose of PTH administration: (1) stimulated cancellous bone formation; (2) improved connectivity of trabeculae joined to the cortex; (3) did not decrease cortical thickness; and (4) did not increase cortical porosity in both ventral and dorsal cortexal shell of the lumbar vertebrae during this dosage and period in intact male beagles.

Peripheral Quantitative Computed Tomography of the Femoral Neck in 60 Japanese Women

Abstract Peripheral quantitative computed tomography (pQCT) is able to evaluate trabecular and cortical bone separately, and to determine geometric properties from cross-sectional images for noninvasive assessments of mechanical strength. In order to assess the diagnostic value of pQCT of the femoral neck, 60 healthy women were examined with a new pQCT machine, XCT-3000 (Norland-Stratec, Germany), which is suitable for direct measurement of the hip. The region of interest chosen was the center of the femoral neck. pQCT of the distal radius and dual energy X-ray absorptiometry (DXA) of the lumbar spine and femoral neck were also performed.The study demonstrated that total bone mineral density (BMD) (femoral MD) and trabecular BMD (femoral-TBD) decreased with advancing age. Percent cortical area showed a small but significant decrease with advancing age and % trabecular area increased slightly. Both the endosteal perimeter and the periosteal perimeter were relatively constant with aging. Bone strength index (BSI) and stress-strain index (SSI), which reflect the mechanical strength of bone, declined with advancing age, especially after menopause. Femoral TBD correlated strongly with femoral neck BMD by DXA and L2-L4 BMD by DXA but femoral-CBD did not correlate with femoral neck BMD by DXA. Volumetric BMD of the femoral neck and distal radius were closely correlated. It is concluded that (1) cortical thinning occurs with aging by endocortical resorption and loss of femoral-TBD; (2) loss of femoral-CBD occurred at a slower rate than radial CBD, perhaps due to the weight-bearing effect; (3) biomechanical parameters such as the BSI and SSI may reflect increasing fragility of the femoral neck in pre- and postmenopausal women; (4) pQCT of the femoral neck had diagnostic value at least equivalent to that of DXA or pQCT of the distal radius.

Maintenance of bone mass by physical exercise after discontinuation of intermittent hPTH(1–34) administration

Human PTH(1-34) has been recognized for its marked anabolic effect on bone, but that effect has been reported to be lost after cessation of PTH treatment. The objective of this study was to determine the fate of hPTH-stimulated bone and whether this anabolic effect of PTH could be maintained by daily exercise. Eleven-week-old Sprague-Dawley rats were ovariectomized (OVX) and human PTH(1-34) (30 micrograms/kg) was injected subcutaneously three times per week for 12 weeks beginning one week after surgery. After the cessation of PTH treatment, treadmill exercises were performed for 8 weeks (15.7 m/min, 1 h/day, 5 days/week). The results of histomorphometric assessment in the proximal tibial metaphysis demonstrated that hPTH treatment partially prevented OVX-induced cancellous bone loss. Eight weeks following the cessation of PTH treatment, PTH-stimulated bone mass went back to the OVX control level. Daily exercise did maintain PTH-stimulated bone mass; however, this exercise did not increase the bone mass in PTH-untreated OVX rats.