Toshinori Ishizuya

Parathyroid hormone exerts disparate effects on osteoblast differentiation depending on exposure time in rat osteoblastic cells.

It has been reported that PTH exerts bone-forming effects in vivo when administered intermittently. In the present study, the anabolic effects of PTH(1-34) on osteoblast differentiation were examined in vitro. Osteoblastic cells isolated from newborn rat calvaria were cyclically treated with PTH(1-34) for the first few hours of each 48-h incubation cycle. When osteoblastic cells were intermittently exposed to PTH only for the first hour of each 48-h incubation cycle and cultured for the remainder of the cycle without the hormone, osteoblast differentiation was inhibited by suppressing alkaline phosphatase activity, bone nodule formation, and mRNA expression of alkaline phosphatase, osteocalcin, and PTH/PTHrP receptor. Experiments using inhibitors and stimulators of cAMP/protein kinase A (PKA) and Ca2+/PKC demonstrated that cAMP/PKA was the major signal transduction system in the inhibitory action of PTH. In contrast, the intermittent exposure to PTH for the first 6 h of each 48-h cycle stimulated osteoblast differentiation. Both cAMP/ PKA and Ca2+/PKC systems appeared to be involved cooperatively in this anabolic effect. Continuous exposure to PTH during the 48-h incubation cycle strongly inhibited osteoblast differentiation. Although both cAMP/PKA and Ca2+/PKC were involved in the effect of continuous exposure to PTH, they appeared to act independently. A neutralizing antibody against IGF-I blocked the stimulatory effect on alkaline phosphatase activity and the expression of osteocalcin mRNA induced by the 6-h intermittent exposure. The inhibitory effect induced by the 1-h intermittent exposure was not affected by anti-IGF-I antibody. These results suggest that PTH has diverse effects on osteoblast differentiation depending on the exposure time in vitro mediated through different signal transduction systems. These in vitro findings explain at least in part the in vivo action of PTH that varies with the mode of administration.

Changes in Osteoblast Phenotype During Differentiation of Enzymatically Isolated Rat Calvaria Cells

Osteoblasts enzymatically isolated from newborn rat calvariae show various phenotypes including formation of mineralized bone nodules in culture. We investigated the temporal changes in osteoblast phenotype in these cells up to day 20 in culture. These cells formed unmineralized nodules by day 5. Mineralization was observed at the center of nodules by day 10, and nodules became larger on day 15. The nodules were surrounded by numerous alkaline phosphatase (ALP)-positive cells. ALP activity gradually increased by day 20. Parathyroid hormone (PTH) responsiveness increased with time in culture. Osteoblasts produced no osteocalcin by day 10, but its synthesis was detected from day 15. These cells expressed substantial levels of ALP and PTH/PTHrP receptor mRNAs as early as day 5 in culture, but very weak expression of osteocalcin mRNA on day 5. The levels of expression of these transcripts increased with time in culture. In situ hybridization demonstrated that PTH/PTHrP receptor and osteocalcin mRNAs were strongly expressed in nodules, but the former appeared much earlier than the latter. BMP-2 and BMP-4 mRNAs also appeared in the cells forming nodules. Immunohistochemical analysis demonstrated that cells expressing either BMP-2/4 or their receptors (BMPR-IA, BMPR-IB, and BMPR-II) preferentially appeared in nodules. These observations suggested that BMPs play an important role in the formation of mineralized bone nodules in an autocrine and/or paracrine fashion in these cells. The present study confirmed that osteoblasts enzymatically isolated from newborn rat calvariae are a useful tool for studying the differentiation process of osteoblasts.

Administration frequency as well as dosage of PTH are associated with development of cortical porosity in ovariectomized rats

To investigate whether the administration frequency of parathyroid hormone (PTH) is associated with the development of cortical porosity, this study established 15 dosage regimens of teriparatide [human PTH(1–34), TPTD] with four distinct concentrations and four distinct administration frequencies of TPTD to 16-week-old ovariectomized rats. Our analyses demonstrated that the bone mineral density, mechanical properties, and bone turnover were associated with the total amount of TPTD administered. Our observations further revealed that the cortical porosity was markedly developed as a result of an increased administration frequency with a lower concentration of total TPTD administration in our setting, although the highest concentration also induced cortical porosity. Deconvolution fluorescence tiling imaging on calcein-labeled undecalcified bone sections also demonstrated the development of cortical porosity to be closely associated with the bone site where periosteal bone formation took place. This site-specific cortical porosity involved intracortical bone resorption and an increased number and proximity of osteocytic lacunae, occasionally causing fused lacunae. Taken together, these findings suggested the involvement of local distinctions in the rate of bone growth that may be related to the site-specific mechanical properties in the development of cortical porosity induced by frequent and/or high doses of TPTD.

Increased cortical porosity is associated with daily, not weekly, administration of equivalent doses of teriparatide

INTRODUCTION The pharmacokinetic profile of parathyroid hormone (PTH) determines its effects on bone resorption and formation. When administered intermittently, anabolic effects are favored in comparison with the continuous treatment. Among the intermittent treatment regimens, lower frequency of administration may have a lower effect on bone remodeling. We therefore hypothesized that weekly administration of teriparatide will produce less increase in intracortical remodeling and porosity than reported using daily treatment. METHODS We treated 17 female New Zealand white rabbits aged 6months for 1month with teriparatide [human PTH(1-34)] as follows. (i) Vehicle-treated Control (n=4); (ii) 20μg/kg daily (n=3); (iii) 40μg/kg daily (n=3); (iv) 140μg/kg weekly (n=3); (v) 280μg/kg weekly (n=4). Proximal femurs were imaged ex vivo using micro-CT (Scanco Viva CT-40) at 15μmvoxel size. Areas, pore size, and porosity were analyzed on the total, compact cortex (CC), and transitional zones in a 10mm length region of interest (ROI) starting at the midshaft using StrAx1.0. RESULTS Compared to controls, the 20μg/kg daily was associated with 3.0% higher porosity in the transitional zone (p=0.09) while the 40μg/kg daily was associated with a higher porosity in the cortex (8.7%; p=0.04) and in the transitional zone (5.7%; p=0.007). The daily regimens were also associated with a greater proportion of porosity due to pores >15μm2; particularly in the transitional zone where 20 and 40μg/kg daily increased porosity 2 fold (p=0.06) and 5 fold (p=0.04) relative controls respectively. The 140 and 280μg/kg weekly were not associated with an increase in porosity. There was no difference in total, compact or transitional zone cross sectional areas between the groups. CONCLUSION Effects of intermittent teriparatide depend on the dose and frequency of administration. Daily dosing, particularly the higher dose, but not weekly dosing, increased cortical porosity. Work is needed to investigate the effects of the regimens on bone formation.

Acute development of cortical porosity and endosteal naïve bone formation from the daily but not weekly short-term administration of PTH in rabbit

Teriparatide [human parathyroid hormone (1–34)], which exerts an anabolic effect on bone, is used for the treatment of osteoporosis in patients who are at a high risk for fracture. That the once-daily administration of teriparatide causes an increase in cortical porosity in animal models and clinical studies has been a matter of concern. However, it is not well documented that the frequency of administration and/or the total dose of teriparatide affect the cortical porosity. The present study developed 4 teriparatide regimens [20 μg/kg/day (D20), 40 μg/kg/day (D40), 140 μg/kg/week (W140) and 280 μg/kg/week (W280)] in the rabbit as a model animal with a well-developed Haversian system and osteons. The total weekly doses were equivalent in the low-dose groups (D20 and W140) and in the high-dose groups (D40 and W280). After the short-term (1 month) administration of TPDT, micro-CT, histomorphometry and three-dimensional second harmonic generation (3D-SHG) imaging to visualize the bone collagen demonstrated that daily regimens but not weekly regimens were associated with the significant development of cortical porosity and endosteal naïve bone formation by marrow fibrosis. We concomitantly monitored the pharmacokinetics of the plasma teriparatide levels as well as the temporal changes in markers of bone formation and resorption. The analyses in the present study suggested that the daily repeated administration of teriparatide causes more deleterious changes in the cortical microarchitecture than the less frequent administration of higher doses. The findings of the present study may have some implications for use of teriparatide in clinical treatment.

Short-term intermittent administration of parathyroid hormone facilitates osteogenesis by different mechanisms in cancellous and cortical bone

Intermittent administration of human parathyroid hormone (1–34)[hPTH(1–34)] induces anabolic action on the bones. To understand the mechanism underlying the early phase of hPTH(1–34)-induced anabolic action, we investigated the expression profiles of osterix and sclerostin after short-term intermittent administration of hPTH(1–34) using immunohistochemistry in adult rats. In the cancellous bone, hPTH(1–34) administration greatly increased the number of osterix-positive cells in the bone marrow on day 1, but the cells gradually decreased on days 3 and 5. Injections of hPTH(1–34) induced no significant changes in the number of sclerostin-positive osteocytes in the cancellous bone. In the cortical bone, intermittent administration of hPTH(1–34) significantly reduced the number of sclerostin-positive osteocytes. The serum sclerostin level was downregulated and the osteocalcin level was upregulated on day 5 after intermittent administration of hPTH(1–34). Intermittent hPTH(1–34) injections increased osteoblast surface, osteoid thickness, and osteoid surface in cancellous bone, but not in cortical bone. This study suggested that the increase in osterix-positive osteoprogenitors in cancellous bone and the decrease in sclerostin-positive osteocytes in cortical bone play important roles in anabolic action on osteogenesis induced by short-term administration of hPTH(1–34).