Vitamin D hormone inhibits osteoclastogenesis in vivo by decreasing the pool of osteoclast precursors in bone marrow.

Previous observations that vitamin D hormone induces the expression of the receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL), thereby stimulating osteoclastogenesis in vitro, led to the widespread belief that 1alpha,25-dihydroxyvitamin D3 [1a,25(OH)2D3] is a bone-resorbing hormone. Here, we show that alfacalcidol, a prodrug metabolized to 1alpha,25(OH)2D3, suppresses bone resorption at pharmacologic doses that maintain normocalcemia in an ovariectomized (OVX) mouse model of osteoporosis. Treatment of OVX mice with pharmacologic doses of alfacalcidol does not increase RANKL expression, whereas toxic doses that cause hypercalcemia markedly reduce the expression of RANKL. When bone marrow (BM) cells from OVX mice were cultured with sufficient amounts of macrophage colony-stimulating factor (M-CSF) and RANKL, osteoclastogenic activity was higher than in sham mice. Marrow cultures from alfacalcidol- or estrogen-treated OVX mice showed significantly less osteoclastogenic potential compared with those from vehicle-treated OVX mice, suggesting that the pool of osteoclast progenitors in the marrow of vitamin D-treated mice as well as estrogen-treated mice was decreased. Frequency analysis showed that the number of osteoclast progenitors in bone marrow was increased by OVX and decreased by in vivo treatment with alfacalcidol or estrogen. We conclude that the pharmacologic action of active vitamin D in vivo is to decrease the pool of osteoclast progenitors in BM, thereby inhibiting bone resorption. Because of its unusual activity of maintaining bone formation while suppressing bone resorption, in contrast to estrogens that depress both processes, vitamin D hormone and its bone-selective analogs may be useful for the management of osteoporosis.     

Short-term effect of ovariectomy on osteoprogenitors in the healing rat mandibular incisor extraction socket

SUMMARY: OVX increased the percentage of AP-positive CFU-F in healing rat mandible. The increase of the number of osteoprogenitors was not significant in rat mandible-derived cultures but was in femur-derived ones. This suggests that the effect of OVX on osteoprogenitors is either smaller or develops later in mandible relative to femur. INTRODUCTION: Osteoprogenitors play an essential role in the regeneration process that leads to the successful integration of dental implants. However, it is unclear how systemic osteoporosis affects osteoprogenitors in oral bone. The present study was designed to determine the short-term effects of ovariectomy (OVX) on osteoprogenitors from the healing extraction socket in rat mandible. METHODS: Six-month-old rats were ovariectomized (n=8) and control rats were left intact (n=8). Two weeks post-OVX, the right mandibular incisor was extracted. Four weeks post-extraction, the basal mandibular bone between the 1st and 3rd molar in the healing extraction socket was used to determine the number of fibroblastic progenitors (CFU-F), alkaline phosphatase-positive fibroblastic progenitors (AP-positive CFU-F), Dex-dependent osteoprogenitors (CFU-O Dex) and Prog-dependent osteoprogenitors (CFU-O Prog) using colony assays (n=5). Osteocalcin mRNA expression was evaluated using in situ hybridization (n=3). Data were analyzed using two-way ANOVA or Student's t-test. RESULTS: OVX increased the percentage of AP-positive CFU-F in both mandible and femur. The number of CFU-O was increased only in femur. Osteocalcin mRNA expression in regenerating mandible was not statistically different between control and OVX animals. CONCLUSION: Our results suggest that the effect of OVX on osteoprogenitors is either smaller or develops later in mandible relative to femur.     

Age-related osteogenic potential of mesenchymal stromal stem cells from human vertebral bone marrow.

Mesenchymal stem cells (MSCs) residing in bone marrow (BM) are the progenitors for osteoblasts and for several other cell types. In humans, the age-related decrease in bone mass could reflect decreased osteoblasts secondary to an age-related loss of osteoprogenitors. To test this hypothesis, BM cells were isolated from vertebral bodies of thoracic and lumbar spine (T1-L5) from 41 donors (16 women and 25 men) of various ages (3-70 years old) after death from traumatic injury. Primary cultures were grown in alpha modified essential medium with fetal bovine serum for 13 days until adherent cells formed colonies (CFU-Fs). Colonies that stained positive for alkaline phosphatase activity (CFU-F/ALP+) were considered to have osteogenic potential. BM nucleated cells were plated (0.5, 1, 2.5, 5, or 10 x 106 cells/10-cm dish) and grown in dexamethasone (Dex), which promotes osteoblastic differentiation. The optimal plating efficiency using BM-derived cells from donors of various ages was 5 x 106 cells/10-cm dish. BM-derived cells were also grown in the absence of Dex at this plating density. At the optimal plating density, in the presence of Dex, the number of CFU-F/ALP+ present in the BM of the younger donors (3-36 years old) was 66.2 +/- 9.6 per 106 cells (mean +/- SEM), but only 14.7 +/- 2.6 per 106 cells in the older donors (41-70 years old). With longer-term culture (4-5 weeks) of these BM cells in medium containing 10 mM beta-glycerophosphate and 100 microg/ml ascorbic acid, the extracellular matrix mineralized, a result consistent with mature osteoblastic function. These results demonstrate that the number of MSCs with osteogenic potential (CFU-F/ALP+) decreases early during aging in humans and may be responsible for the age-related reduction in osteoblast number. Our results are particularly important in that the vertebrae are a site of high turnover osteoporosis and, possibly, the earliest site of bone loss in age-related osteoporosis.     

Basic fibroblast growth factor has rapid bone anabolic effects in ovariectomized rats

Basic fibroblast growth factor (bFGF) has a strong bone anabolic effect in intact and ovariectomized (OVX) rats treated for 7–14 days. Other growth factors such as IGF-I and TGF- have been implicated as potential mediators for this effect. The purpose of this study was to examine the early effects of bFGF therapy, in vivo, on bone formation and gene expression in OVX rats in order to determine whether upregulation of gene expression for IGF-I and/or TGF- precedes or coincides with the stimulatory effects of bFGF on bone formation. At 3 months of age, Sprague Dawley rats were OVX or sham-operated (SHAM), then maintained untreated for 3 months. One group of baseline OVX rats (BSL OVX) and BSL SHAM rats were then killed. Additional OVX groups were treated IV with bFGF at a daily dose of 200 g/kg and killed at 1–7 and 10 days. Another group of OVX rats was treated IV with vehicle daily for 10 days, then killed. Lumbar vertebrae were processed for cancellous bone histomorphometry or RNA isolation. Ovariectomy induced increased cancellous bone turnover and a significant decrease in vertebral bone mass. Treatment of OVX rats with bFGF resulted in a significant increase in bone formation. As early as 24 h after bFGF treatment of OVX rats, osteoblast surface, osteoid surface, and osteoid volume were more than double those in BSL OVX rats and continued to increase with time. These variables were also significantly higher in bFGF-treated OVX rats at 10 days compared with vehicle-treated OVX rats. Gene expression for IGF-I was not different between BSL OVX rats and bFGF-treated OVX rats at 1 day, but was significantly higher by approximately 50% in OVX rats treated with bFGF for 2 and 7 days, and was also significantly higher by nearly 75% in OVX rats treated for 10 days compared with OVX rats treated with vehicle. Gene expression for TGF-₁ was unchanged at early times and only significantly upregulated by a relatively modest 30% in OVX rats treated with bFGF for 10 days. The results indicate that the bone anabolic effects of bFGF in OVX rats begin as early as 24 h following the initial treatment, and increase with time. These early stages of the strong stimulatory effect of bFGF on bone formation were not associated with a large upregulation of gene expression for IGF-I and TGF-. The rapid increase in osteoblast surface in bFGF-treated OVX rats suggests that the growth factor induces conversion of bone lining cells to osteoblasts.     

Skeletal unloading induces resistance to insulin-like growth factor I on bone formation

Skeletal unloading results in an inhibition of bone formation associated with a decrease in osteoblast number, impaired mineralization of bone, and altered proliferation and differentiation of osteoprogenitor cells. Although such changes are likely to be mediated by multiple factors, resistance to the growth-promoting action of insulin-like growth factor I (IGF-I) has been hypothesized to play an important role. To determine whether skeletal unloading induces resistance to IGF-I on bone formation, we examined the response of unloaded (hindlimb elevation) and normally loaded tibia and femur to IGF-I administration. To eliminate the variable of endogenous growth hormone production and secretion during exogenous IGF-I administration, we used growth hormone-deficient dwarf rats (dw-4). The rats were given IGF-I (2.5 mg/kg/day) or vehicle during 7 and 14 days of unloading or normal loading. This significantly increased the serum level of IGF-I in both the normally loaded and unloaded rats. Unloading did not affect the serum level of IGF-I in the vehicle-treated rats. IGF-I markedly increased periosteal bone formation at the tibiofibular junction of normally loaded rats. Unloading decreased bone formation in the vehicle-treated rats, and blocked the ability of IGF-I to increase bone formation. On the other hand, IGF-I increased periosteal bone formation at the midpoint of the humerus (normally loaded in this model) in both hindlimb-elevated and normally loaded rats. IGF-I significantly increased osteogenic colony number, total ALP activity, and total mineralization in bone marrow osteoprogenitor (BMOp) cells of normally loaded rats. Unloading reduced these parameters in the vehicle-treated rats, and blocked the stimulation by IGF-I. Furthermore, IGF-I administration (10 ng/ml) in vitro significantly increased cell proliferation of the BMOp cells isolated from normally loaded bone, but not that of cells from unloaded bone. These results indicate that skeletal unloading induces resistance to IGF-I on bone formation.     

海藻酸钙/枸杞多糖凝胶微球对小鼠骨质疏松的作用研究

目的研究海藻酸钙(ALG-Ca)/枸杞多糖(LBPs)凝胶微球对小鼠骨质疏松的作用。方法采用静电液滴法制备ALG-Ca凝胶微球、ALG-Ca/LBPs凝胶微球,用扫描电镜观察微球结构。建立去卵巢小鼠骨质疏松模型,随机分为假手术组、模型组、单纯ALG-Ca微球组、单纯LBPs组、ALG-Ca/LBPs凝胶微球组,每组8只,每日灌胃1次,干预时间为12周,对各组小鼠股骨行HE染色观察骨组织形态变化,Micro-CT检测并经三维重建获得骨组织微观结构,对小鼠血清骨钙素(BGP)、血清碱性磷酸酶(BALP)、1型胶原羧基末端肽(CTX-1)进行检测。结果扫描电镜观察示凝胶微球呈均匀一致的微球形结构;OVX+LBPs、OVX+ALG-Ca/LBPs组的BGP、BALP、CTX-1均低于OVX+PBS组(P<0.05);OVX+LBPs、OVX+ALG-Ca/LBPs组骨体积分数(BV/TV)、骨小梁数目(Tb.N)及骨小梁厚度(Tb.Th)均高于OVX+PBS组(P<0.05),骨小梁间隙(Tb.Sp)小于OVX+PBS组(P<0.05),以OVX+ALG-Ca/LBPs组为最佳(P<0.01)。结论ALG-Ca凝胶微球是LBPs治疗骨质疏松的良好药物缓释载体;LBPs为天然植物成分,通过LBPs/ALG-Ca凝胶微球可以改善骨质疏松小鼠的骨代谢及骨微结构。     

Role of IGF-I signaling in regulating osteoclastogenesis.

We showed that IGF-I deficiency impaired osteoclastogenesis directly and/or indirectly by altering the interaction between stromal/osteoblastic cells and osteoclast precursors, reducing RANKL and M-CSF production. These changes lead to impaired bone resorption, resulting in high BV/TV in IGF-I null mice. INTRODUCTION: Although IGF-I has been clearly identified as an important growth factor in regulating osteoblast function, information regarding its role in osteoclastogenesis is limited. Our study was designed to analyze the role of IGF-I in modulating osteoclastogenesis using IGF-I knockout mice (IGF-I(-/-)). MATERIALS AND METHODS: Trabecular bone volume (BV/TV), osteoclast number, and morphology of IGF-I(-/-) or wildtype mice (IGF-I(+/+)) were evaluated in vivo by histological analysis. Osteoclast precursors from these mice were cultured in the presence of RANKL and macrophage-colony stimulating factor (M-CSF) or co-cultured with stromal/osteoblastic cells from either genotype. Osteoclast formation was assessed by measuring the number of multinucleated TRACP+ cells and pit formation. The mRNA levels of osteoclast regulation markers were determined by quantitative RT-PCR. RESULTS: In vivo, IGF-I(-/-) mice have higher BV/TV and fewer (76% of IGF-I(+/+)) and smaller osteoclasts with fewer nuclei. In vitro, in the presence of RANKL and M-CSF, osteoclast number (55% of IGF-I(+/+)) and resorptive area (30% of IGF-I(+/+)) in osteoclast precursor cultures from IGF-I(-/-) mice were significantly fewer and smaller than that from the IGF-I(+/+) mice. IGF-I (10 ng/ml) increased the size, number (2.6-fold), and function (resorptive area, 2.7-fold) of osteoclasts in cultures from IGF-I(+/+) mice, with weaker stimulation in cultures from IGF-I(-/-) mice. In co-cultures of IGF-I(-/-) osteoblasts with IGF-I(+/+) osteoclast precursors, or IGF-I(+/+) osteoblasts with IGF-I(-/-) osteoclast precursors, the number of osteoclasts formed was only 11% and 48%, respectively, of that from co-cultures of IGF-I(+/+) osteoblasts and IGF-I(+/+) osteoclast precursors. In the long bones from IGF-I(-/-) mice, mRNA levels of RANKL, RANK, M-CSF, and c-fms were 55%, 33%, 60%, and 35% of that from IGF-I(+/+) mice, respectively. CONCLUSIONS: Our results indicate that IGF-I regulates osteoclastogenesis by promoting their differentiation. IGF-I is required for maintaining the normal interaction between the osteoblast and osteoclast to support osteoclastogenesis through its regulation of RANKL and RANK expression.     

Climbing exercise enhances osteoblast differentiation and inhibits adipogenic differentiation with high expression of PTH/PTHrP receptor in bone marrow cells

We developed previously a mouse voluntary climbing exercise model as a physiological mechanical loading model and reported that climbing exercise increased bone formation, but its effect on adipogenesis is unknown. We assessed the effects of loading and PTH/PTHrP receptor (PTHR1) on bone marrow adipocyte differentiation in relation with osteoblast differentiation. 8-week-old C57BL/6J male mice were divided into ground control (GC) and climbing exercise (EX) group. Mice were housed in 100-cm towers and climbed up toward a bottle placed at the top of the cage to drink water. The values of bone volume and osteoblast number were significantly higher while those of marrow adipocyte volume and number were significantly lower in the 28dayEX group than 28dayGC group. The mRNA expression levels of adipocyte differentiation genes CCAAT/enhancer-binding proteins (C/EBP) beta and delta were lower in 4dayEX mice, while the adipocyte specific genes fatty acid binding protein (aP2) and phosphoenolpyruvate carboxykinase (PEPCK) expressions were lower in 7dayEX mice. In primary bone marrow cell cultures, the number of alkaline phosphatase-positive colony forming units-fibroblastic (ALP+ CFU-f) and Oil-red-O-positive cells were both increased in the 4dayEX group. Climbing exercise transiently increases both osteogenic and adipogenic potential in bone marrow stromal cells, and inhibits terminal adipocyte differentiation and promotes osteoblast differentiation. Immunoreactivity for the PTHR1 was intense on osteoblastic cell lineage in the endosteal tibial metaphysis. PTHR1 mRNA expression was increased in 4dayEX mice and PTHR1-positive cells were increased after 7 days in the experimental group. Ex vivo addition of PTHR1 antibody decreased and that of PTHrP(1-34) increased the number of ALP+ CFU-f in bone marrow cell cultures obtained at 4 days after the exercise, while the addition of PTHR1 antibody increased and PTHrP(1-34) decreased the number of Oil-red-O-positive cells. Our results indicate that climbing exercise enhanced osteoblast differentiation and inhibited terminal differentiation of adipocyte progenitors with high expression of PTHR1 in bone marrow cells.     

Sequential treatment with basic fibroblast growth factor and parathyroid hormone restores lost cancellous bone mass and strength in the proximal tibia of aged ovariectomized rats.

This study was designed to determine whether sequential treatment with basic fibroblast growth factor (bFGF) and parathyroid hormone (PTH) can restore lost cancellous bone mass and strength at a severely osteopenic skeletal site in aged ovariectomized (OVX) rats. Female Sprague-Dawley rats were subjected to sham surgery or ovariectomy at 3 months of age and maintained untreated for the first year after surgery. At 15 months of age, groups of baseline control and OVX rats were killed and catheters were inserted in the jugular veins of all remaining rats. Two groups of OVX rats were injected intravenously (iv) daily with bFGF for 14 days at a dose of 200 microg/kg body weight. At the end of bFGF treatment, one group was killed whereas the other group was subjected to 8 weeks of treatment with synthetic human PTH 1-34 [hPTH(1-34)] consisting of subcutaneous (sc) injections 5 days/week at a dose of 80 microg/kg. Another group of OVX rats was treated iv with vehicle for 2 weeks followed by treatment with PTH alone for 8 weeks. Other groups of sham-operated control rats and OVX rats were treated iv and sc with vehicle alone. The right proximal tibia from each rat was processed undecalcified for quantitative bone histomorphometry and the left proximal tibia was subjected to biomechanical testing. Baseline and vehicle-treated OVX rats were severely osteopenic because their tibial cancellous bone volumes were less than 5% compared with mean values of 20.3% and 15.0% in baseline and vehicle-treated control rats, respectively. Treatment of OVX rats for 2 weeks with bFGF alone did not significantly increase tibial cancellous bone volume but induced marked increases in osteoid volume, osteoblast surface, and osteoid surface. Sequential treatment of aged OVX rats with bFGF and PTH increased tibial cancellous bone volume (15.1%) and load to failure to at least the level of vehicle-treated control rats. Tibial cancellous bone volume (10.8%) and load to failure also were significantly increased by treatment with PTH alone, and these variables were not significantly different from those of OVX rats treated with bFGF + PTH. However, tibial ash density was significantly greater in OVX rats treated sequentially with bFGF and PTH compared with OVX rats treated with PTH alone. Our findings suggest that sequential treatment with bFGF and PTH may be useful for restoration of lost cancellous bone in the severely osteopenic, estrogen-deplete skeleton, but it cannot be concluded with certainty that this sequential treatment has a greater bone restorative effect than treatment with PTH alone.     

Ovariectomized murine model of postmenopausal calcium malabsorption.

Two experiments were carried out to examine whether the ovariectomized mouse is a potential in vivo model of intestinal calcium malabsorption as occurs in postmenopausal osteoporosis. In the first experiment, we compared the effects of ovariectomy and 17 beta-estradiol (E2) therapy on calcium absorption in C3H/HeJ (C3H) and C57BL/6J (C57BL) mice, which have high and low peak bone mass, respectively. For each strain of mice, three groups were studied: sham operated, ovariectomized, (OVX), and OVX + E2 (60 micrograms/kg of body weight [bw]/day). Therapy was continued for 35 days and calcium absorption measured. In the C3H mice, ovariectomy caused an increase in fecal calcium (17.6%), and a decrease in the amount (12.8%) and percentage (12.5%) of calcium absorbed. The decrease was prevented by E2 therapy, but the differences in the calcium absorption parameters among the groups were not statistically significant. In contrast, in the C57BL mice, ovariectomy caused a marked increase in fecal calcium (84.5%, p < 0.01), and a marked decrease in the amount (55%, p < 0.01) and percentage (34.8%, p < 0.001) of calcium absorbed, and the decrease in the percentage of calcium absorbed was partially prevented by E2 therapy (p < 0.05). In experiment 2, a dose-response study of the effects of E2 therapy on calcium absorption in OVX C57BL mice was carried out. Five groups of mice were studied: Group 1, sham operated; Group 2, OVX; Group 3, OVX + 60 micrograms of E2/kg of bw/day; Group 4, OVX + 120 micrograms of E2/kg of bw/day; Group 5, OVX + 240 micrograms of E2/kg of bw/day. Therapy was continued for 28 days and calcium absorption measured. Ovariectomy caused a marked increase in fecal calcium (50%, p < 0.0001) and urinary calcium (31%, p < 0.0001) and a marked decrease in calcium absorption (44.9%, p < 0.0001), and these changes were prevented by E2 therapy. The highest level of calcium absorption (109%, p < 0.0001, vs. OVX) was observed in the 120 micrograms of E2 group. Ovariectomy and E2 slightly increased plasma 1,25-dihydroxyvitamin D (1,25(OH)2D) levels but there was no significant correlation between 1,25(OH)2D levels and calcium absorption. The findings in the C57BL mice suggest that estrogen is a physiological regulator of calcium absorption in this strain of mice. Furthermore, these findings share many characteristics with intestinal calcium malabsorption and its reversal by estrogen therapy in hypoestrogenic women. We propose that the OVX C57BL mice warrants further characterization as a potential animal model for investigating issues related to calcium malabsorption in postmenopausal women.     

Mice lacking the integrin beta5 subunit have accelerated osteoclast maturation and increased activity in the estrogen-deficient state.

Integrin alphavbeta5 is expressed on osteoclast precursors and is capable of recognizing the same amino acid motif as alphavbeta3. Three-month-old beta5(-/-) female OVX mice had increased osteoclastogenesis ex vivo, and microCT assessment of trabecular bone volume was 53% lower than WT-OVX animals. These preliminary data suggest alphavbeta5 integrin's presence on osteoclast precursors may inhibit of osteoclast formation. INTRODUCTION: Osteoclasts are unique resorptive skeletal cells, capable of degrading bone on contact to the juxtaposed matrix. Integrin alphavbeta5 is expressed on osteoclast precursors, structurally similar to alphavbeta3, and capable of recognizing the same amino acid motif. Given the structural relationship and reciprocal regulation of alphavbeta3 and alphavbeta5, the purpose of this study was to evaluate how alphavbeta5 might contribute to osteoclast maturation and activity. MATERIALS AND METHODS: Three-month-old wildtype (WT) and beta5(-/-) female mice had ovariectomy (OVX) or sham operations. The osteoclastogenic capacity of marrow-derived precursors, the kinetic, the circulating, and structural parameters of bone remodeling, was determined after 6 weeks of paired feeding. RESULTS AND CONCLUSIONS: OVX increased osteoclastogenesis ex vivo and in vivo. Osteoclast formation and prolonged pre-osteoclast survival were substantially enhanced in cultures containing beta5(-/-) cells whether obtained from sham-operated or OVX mice. Expression of cathepsin K, beta3 integrin subunit, and calcitonin receptor were accelerated in cultured beta5(-/-)osteoclasts. beta5(-/-) osteoclasts from OVX animals showed a 3-fold enhancement of net resorptive activity, with quantitative muCT showing trabecular bone volume loss after OVX 53% greater in beta5(-/-) OVX compared with similarly treated WT OVX mice (p < 0.05). alpha5beta3 seems to be an inhibitor of osteoclast formation, in contrast to alphavbeta3. In addition, loss of alphavbeta5 seems to accelerate osteoclast formation in the OVX model. Further examination of alphavbeta5 signaling pathways may enhance our understanding of the activation of bone resorption.     

Number and proliferative capacity of osteogenic stem cells are maintained during aging and in patients with osteoporosis.

Decreased bone formation is an important pathophysiological mechanism responsible for bone loss associated with aging and osteoporosis. Osteoblasts (OBs), originate from mesenchymal stem cells (MSCs) that are present in the bone marrow and form colonies (termed colony-forming units-fibroblastic [CFU-Fs]) when cultured in vitro. To examine the effect of aging and osteoporosis on the MSC population, we quantified the number of MSCs and their proliferative capacity in vitro. Fifty-one individuals were studied: 38 normal volunteers (23 young individuals [age, 22-44 years] and 15 old individuals [age, 66-74 years]) and 13 patients with osteoporosis (age, 58-83 years). Bone marrow was aspirated from iliac crest; mononuclear cells were enriched in MSCs by magnetic activated cell sorting (MACS) using STRO-1 antibody. Total CFU-F number, size distribution, cell density per CFU-F, number of alkaline phosphatase positive (ALP+) CFU-Fs, and the total ALP+ cells were determined. In addition, matrix mineralization as estimated by alizarin red S (AR-S) staining was quantified. No significant difference in colony-forming efficiency between young individuals (mean +/- SEM; 87 +/- 12 CFU-Fs/culture), old individuals (99 +/- 19 CFU-Fs/culture), and patients with osteoporosis (129 +/- 13 CFU-Fs/culture; p = 0.20) was found. Average CFU-F size and cell density per colony were similar in the three groups. Neither the percentage of ALP+ CFU-Fs (66 +/- 6%, 65 +/- 7%, and 72 +/- 4% for young individuals, old individuals, and patients with osteoporosis, respectively) nor the percentage of ALP+ cells per culture (34 +/- 5%, 40 +/- 6%, and 41 +/- 4%) differed between groups. Finally, mineralized matrix formation was similar in young individuals, old individuals, and patients with osteoporosis. Our study shows that the number and proliferative capacity of osteoprogenitor cells are maintained during aging and in patients with osteoporosis and that other mechanisms must be responsible for the defective osteoblast (OB) functions observed in these conditions.     

Effect of ovariectomy on dexamethasone- and progesterone-dependent osteoprogenitors in vertebral and femoral rat bone cell populations

We have found previously that the skeleton of adult female rats contains dexamethasone (Dex)- and progesterone (Prog)-dependent osteoprogenitors, and that estrogen treatment in vitro upregulates proliferation and differentiation of the Prog-dependent but not of the Dex-dependent osteoprogenitors (Bone 1997;20:17-25). The purpose of the present study was to determine whether ovariectomy (OVX) would have different effects on these two classes of osteoprogenitors. Six-month-old Sprague-Dawley rats underwent OVX and the lumbar vertebrae and proximal femurs were collected 1.5, 3, and 6 months after OVX. Cells were obtained from outgrowths of explant cultures and grown in alpha-MEM with 10% FBS, 50 microg/ml ascorbic acid, and 5 mM beta-glycerophosphate. Osteoprogenitors were identified by their ability to generate a colony of osteoblastic cells forming bone (bone nodule). We also evaluated the number of colony-forming units-fibroblast (CFU-F) and of alkaline phosphatase (AP)-positive CFU-F. In cell populations obtained from vertebrae of rats ovariectomized for 1.5, 3, and 6 months and their corresponding control rats, both Dex (1-100 nM) and Prog (1-10 microM) dose-dependently stimulated nodule formation. Both Dex- and Prog-induced nodule formation were higher in cell populations from control rats than in those from ovariectomized rats (P < 0.001). Numbers of CFU-F and AP-positive CFU-F were also higher in cell populations from control rats compared with those from ovariectomized rats. Estrogen (10 nM) enhanced Prog-dependent bone nodule formation but decreased Dex-dependent bone nodule formation in populations from both control and ovariectomized rats. In femoral populations, the responses to Dex (10 nM), Prog (3 microM), and estrogen (10 nM) were similar to those of the vertebral populations in both control and ovariectomized rats. Our results demonstrate that ovariectomy in rats results in a dramatic decrease in the number of both Dex- and Prog-dependent osteoprogenitors in cell populations from vertebrae and proximal femurs. In addition, we confirmed our previous observation that estrogen upregulated proliferation and differentiation of Prog-dependent progenitors, but found here that estrogen clearly downregulated proliferation and differentiation of the Dex-dependent progenitors.     

miR-187-5p 对骨髓间充质干细胞成骨分化能力的调控作用

目的利用小鼠骨髓间充质干细胞(BMSCs)的成骨分化模型,探讨miR-187-5p在成骨分化中的表达趋势及调控作用。方法通过切除雌性小鼠双侧卵巢构建小鼠骨质疏松模型;应用qRT-PCR技术检测组织和细胞中miR-187-5p的表达;应用基因转染技术观察过表达或敲减miR-187-5p对BMSCs向成骨分化的影响;应用茜素红和碱性磷酸酶染色检测BMSCs中矿化结节的数量和矿化区域的染色面积。结果 qRT-PCR结果显示miR-187-5p在骨质疏松模型小鼠的骨组织及BMSCs中均表达下降。过表达miR-187-5p可提高ALP、Collagen-1、Runx2、BMP4、OCN和OPN等成骨分化相关基因mRNA的表达,促进BMSCs向成骨分化;而敲减miR-187-5p降低ALP等成骨分化相关基因mRNA的表达,抑制BMSCs向成骨分化。在体实验同样证实,过表达miR-187-5p可以显著促进骨质疏松小鼠BMSCs向成骨分化,改善小鼠的骨质疏松表型。结论过表达miR-187-5p促进BMSCs向成骨分化,敲减miR-187-5p抑制BMSCs向成骨分化。     

17 beta estradiol stimulation of endosteal bone formation in the ovariectomized mouse: an animal model for the evaluation of bone-targeted estrogens.

To evaluate the potential of an animal model as a means to identify bone-targeted estrogens, we have studied the response of the skeleton of ovariectomized mice to prolonged estrogen treatment. Seventy female Swiss-Webster mice were randomly divided into ten groups, with nine groups undergoing bilateral ovariectomy and one group a sham procedure. Mice were injected subcutaneously once per week for nine weeks with one of the following doses of estrogen (17 beta-E2) in oil vehicle: 19.3, 38.5, 75, 150, 300, 500, 1000, or 3000 micrograms. One group of ovariectomized (OVX) mice and the sham operated animals received vehicle injections only. At the end of the nine-week experimental protocol, there were no significant differences in body weights among any treatment groups. However, when compared to control values, spleen weights in vehicle-treated OVX mice and in mice treated with 1000 micrograms or 3000 micrograms of 17 beta-E2 were significantly elevated (p less than .01). Liver weights in the OVX mice treated with 1000 or 3000 micrograms 17 beta-E2 were also increased significantly (p less than .05). Comparisons of uterine weights and cortical bone areas were strongly correlated with 17 beta-E2 dose (r2 = .86 and .94, respectively), with maximal increases observed at estradiol doses in excess of 500 micrograms per week. Furthermore, based on bone histomorphometry of in vivo fluorochrome labels, increases in cortical bone area could be attributed to accelerated rates of endosteal mineral apposition and bone formation. These results indicate that the comparison of the response of endosteal bone and uterine tissue in the OVX mice to chronic estrogen treatment offers the potential to identify estrogen and/or estrogen-like compounds with bone-specific activity.     

Thyroid-stimulating hormone restores bone volume, microarchitecture, and strength in aged ovariectomized rats.

We show the systemic administration of low levels of TSH increases bone volume and improves bone microarchitecture and strength in aged OVX rats. TSH's actions are mediated by its inhibitory effects on RANKL-induced osteoclast formation and bone resorption coupled with stimulatory effects on osteoblast differentiation and bone formation, suggesting TSH directly affects bone remodeling in vivo. INTRODUCTION: Thyroid-stimulating hormone (TSH) receptor haploinsufficient mice with normal circulating thyroid hormone levels have reduced bone mass, suggesting that TSH directly affects bone remodeling. We examined whether systemic TSH administration restored bone volume in aged ovariectomized (OVX) rats and influenced osteoclast formation and osteoblast differentiation in vitro. MATERIALS AND METHODS: Sprague-Dawley rats were OVX at 6 months, and TSH therapy was started immediately after surgery (prevention mode; n = 80) or 7 mo later (restoration mode; n = 152). Hind limbs and lumbar spine BMD was measured at 2- or 4-wk intervals in vivo and ex vivo on termination at 8-16 wk. Long bones were subjected to microCT, histomorphometric, and biomechanical analyses. The direct effect of TSH was examined in osteoclast and osteoblast progenitor cultures and established rat osteosarcoma-derived osteoblastic cells. Data were analyzed by ANOVA Dunnett test. RESULTS: In the prevention mode, low doses (0.1 and 0.3 microg) of native rat TSH prevented the progressive bone loss, and importantly, did not increase serum triiodothyroxine (T3) and thyroxine (T4) levels in aged OVX rats. In restoration mode, animals receiving 0.1 and 0.3 microg TSH had increased BMD (10-11%), trabecular bone volume (100-130%), trabecular number (25-40%), trabecular thickness (45-60%), cortical thickness (5-16%), mineral apposition and bone formation rate (200-300%), and enhanced mechanical strength of the femur (51-60%) compared with control OVX rats. In vitro studies suggest that TSH's action is mediated by its inhibitory effects on RANKL-induced osteoclast formation, as shown in hematopoietic stem cells cultivated from TSH-treated OVX rats. TSH also stimulates osteoblast differentiation, as shown by effects on alkaline phosphatase activity, osteocalcin expression, and mineralization rate. CONCLUSIONS: These results show for the first time that systemically administered TSH prevents bone loss and restores bone mass in aged OVX rats through both antiresorptive and anabolic effects on bone remodeling.     

Teriparatide [PTH(1-34)] strengthens the proximal femur of ovariectomized nonhuman primates despite increasing porosity.

OVX monkeys treated for 18 months with 1 or 5 microg/kg/d teriparatide [PTH (1-34)] had significantly stronger proximal femora relative to ovariectomized controls. Teriparatide enhancement of cortical area, cortical width, and trabecular bone volume seemed to more than compensate for the dose-dependent increase in cortical porosity. Beneficial effects of teriparatide treatment on the proximal femur persisted beyond the treatment period and may extend to the marrow. INTRODUCTION: We conducted a detailed quantitative analysis of the effects of teriparatide on the proximal femur of ovariectomized monkeys. Teriparatide increased bone mass, enhanced structural architecture, and strengthened the hip, despite increasing cortical porosity. MATERIALS AND METHODS: Monkeys were treated with vehicle (sham or OVX controls), 1 microg/kg/day teriparatide [parathyroid hormone (1-34); PTH1], or 5 microg/kg/day teriparatide (PTH5) for 18 months or for 12 months followed by 6 months of treatment withdrawal (PTH1W and PTH5W, respectively). Excised proximal femora were analyzed by microCT, conventional histomorphometry, and biomechanics.RESULTS AND CONCLUSIONS: The femoral neck showed significant reduction in trabecular bone volume (BV/TV) for OVX compared with sham, whereas PTH1 BV/TV was restored to sham levels and PTH5 BV/TV was greater than sham and OVX. The withdrawal groups had BV/TVs intermediate between sham and OVX. PTH1 had trabecular number (Tb.N) greater than OVX, and PTH5 Tb.N was greater than sham and OVX. The withdrawal groups had Tb.Ns intermediate between sham and OVX. No differences between groups were observed for trabecular orientation or trabecular thickness. Teriparatide dose-dependently increased bone formation rate and activation frequency in the femoral neck. Cellular composition analyses suggested a tendency of ovariectomy to increase adiposity of marrow by 100%, whereas PTH tended to reduce adipocyte number and increase osteoblast number compared with OVX. Analyses of the cortex showed dose-dependent elevation of cortical porosity, which was consistent with enhanced bone turnover with treatment. Cortical porosity was reduced after withdrawal of teriparatide, because PTH1W cortical porosity was lower than OVX, whereas PTH5W cortical porosity was intermediate between sham and OVX. Increased cortical porosity did not weaken the proximal femora. Biomechanics showed that ovariectomy weakened proximal femora compared with sham, but PTH1, PTH5, and PTH1W were stronger than OVX and not different from sham. PTH5W strength was intermediate between sham and OVX. Therefore, teriparatide had beneficial effects on the proximal femur, despite increasing cortical porosity. Cortical porosity did not adversely affect the mechanical integrity of the proximal femora, because enhanced cortical area and trabecular bone volume more than compensated for the porosity. Much of the beneficial effects of teriparatide were retained after 6 months withdrawal from treatment. PTH effects on the femoral neck were not limited to bone but may include inhibition of OVX-stimulated adiposity of the marrow.     

Mapping quantitative trait loci that influence blood levels of alkaline phosphatase in MRL/MpJ and SJL/J mice

To examine the hypothesis that serum alkaline phosphatase (ALP) levels have a heritable component, we analyzed blood from two inbred strains of mice, MRL/MpJ and SJL, which exhibit 90% difference in total serum ALP activity (268+/-26 vs. 140+/-15 U/l, respectively, P<0.001). A genome-wide scan was carried out using 137 polymorphic markers in 518 F2 female mice. Serum ALP activity in the F2 progeny showed a normal distribution with an estimated heritability of 56%. Genome-wide scan for cosegregation of genetic marker data with serum ALP activity revealed three major quantitative trait loci (QTL), one each on chromosomes 2 (LOD score 3.8), chromosome 6 (LOD score 12.0), and chromosome 14 (LOD score 3.7). In addition, there was one suggestive QTL on chromosome 2 (LOD score of 3.3). In aggregate, these QTLs explain 22.5% of variance in serum ALP between these two strains. Serum ALP showed a moderate but significant correlation with body weight adjusted total body bone mineral density (r=0.12, P=0.0108) and periosteal circumference at midshaft tibia (r=0.15, P=0.0006) in F2 mice. The chromosome 6 locus harboring the major serum ALP QTL also contains a major BMD and bone size QTL, identified earlier, between these two strains of mice; in addition, this QTL is also close to the locus that regulates IGF-I levels (LOD score 8-9) in C3HB6 F2 mice. These common QTLs indicate that the observed difference in ALP and BMD or bone size may be regulated by same loci (or genes). Accordingly, the osteoblast cells isolated from femur and tibia of MRL mice showed a significantly higher number of ALP +ve cells/colony and two- to threefold higher ALP activity (P<0.001) as compared to the cells isolated from SJL mice, thus suggesting that differences in serum ALP between MRL and SJL reflect difference in ALP expression from osteoblasts from these strains of mice. These data suggest that serum ALP levels are genetically determined and correlate with cellular mechanisms that differentiate BMD accrual in these two strains of mice. The findings that ALP and BMD traits share the same loci on chromosome 6 suggest a role for genetic determinants of bone formation in overall BMD accretion.     

Effect of simulated weightlessness on osteoprogenitor cell number and proliferation in young and adult rats

Experiments with rats flown in space or hind limb unloaded (HU) indicate that bone loss in both conditions is associated with a decrease in bone volume and osteoblast surface in cancellous and cortical bone. We hypothesize that the decrease in osteoblastic bone formation and osteoblast surface is related to a decrease in the number of osteoprogenitors and/or decreased proliferation of their progeny. We tested this hypothesis by evaluating the effect of 14 days of HU on the number of osteoprogenitors (osteoblast colony forming units; CFU-O), fibroblastic colony forming units (CFU-F), and alkaline phosphatase-positive CFU (CFU-AP) in cell populations derived from the proximal femur (unloaded) and the proximal humerus (normally loaded) in 6-week-old and 6-month-old rats. To confirm the effect of unloading on bone volume and structure, static histomorphometric parameters were measured in the proximal tibial metaphysis. Effects of HU on proliferation of osteoprogenitors were evaluated by measuring the size of CFU-O. HU did not affect the total number of progenitors (CFU-F) in young or adult rats in any of the cell populations. In femoral populations of young rats, HU decreased CFU-O by 71.0% and mean colony size was reduced by 20%. HU decreased CFU-AP by 31.3%. As expected, no changes in CFU-O or CFU-AP were seen in cell populations from the humerus. In femoral cell populations of adult rats, HU decreased CFU-O and CFU-AP by 16.6% and 36.6%, respectively. Again, no effects were seen in cell populations from the humerus. In 6-week-old rats, there was a greater decrease in bone volume, osteoblast number, and osteoblast surface in the proximal tibial metaphysis than that observed in adult rats. Both trabecular thickness and trabecular number were decreased in young rats but remained unaffected in adults. Neither osteoclast number nor surface was affected by unloading. Our results show that the HU-induced decrease in the number of osteoprogenitors observed in vitro parallels the effects of HU on bone volume and osteoblast number in young and old rats in vivo, suggesting that the two may be interdependent. HU also reduced CFU-O colony size in femoral populations indicating a diminished proliferative capacity of osteoblastic colonies.