Inhibited anabolic effect of insulin‐like growth factor‐I on stromal bone marrow cells in endothelial nitric oxide synthase‐knockout mice

Aims: Insulin‐like growth factor‐I (IGF‐I), parathyroid hormone (PTH) and PTH‐related protein (PTHrP) are hormones that have anabolic effects on bone formation. The aim of this study was to investigate whether production of nitric oxide (NO) is involved in the effect of IGF‐I and PTH/PTHrP on osteoblast‐like cells. Methods: Bone marrow stromal cells from adult endothelial nitric oxide synthase (eNOS)‐knockout (eNOSKO) mice and wild type (WT) counterparts were cultivated with osteogenic substances. The cells showed an osteoblastic phenotype measured as osteocalcin production and alkaline phosphatase activity. DNA synthesis was measured as [³H] thymidine incorporation in the bone marrow cells and in a human osteosarcoma cell‐line (SaOS‐2). Results: The stimulatory effect of IGF‐I on thymidine incorporation seen in WT animals was absent in eNOSKO mice. Addition of a NO donor to eNOSKO cells recovered the effect of IGF‐I on thymidine incorporation. PTH/PTHrP stimulated cell proliferation in both WT and eNOSKO mice. In SaOS‐2 cells, incubation with IGF‐I together with a NOS inhibitor resulted in an inhibition of the anabolic effect of IGF‐I on cell proliferation. Conclusions: The stimulatory effect of IGF‐I on WT cell proliferation was abolished in eNOSKO cells, recovered by an NO donor and inhibited in osteosarcoma cells by a NOS inhibitor. The results indicate that the effect of IGF‐I is dependent on NO production. The impaired IGF‐I response may contribute to the bone defect formation seen in eNOSKO animals.     

Inhibited anabolic effect of insulin-like growth factor-I on stromal bone marrow cells in endothelial nitric oxide synthase-knockout mice

AIMS: Insulin-like growth factor-I (IGF-I), parathyroid hormone (PTH) and PTH-related protein (PTHrP) are hormones that have anabolic effects on bone formation. The aim of this study was to investigate whether production of nitric oxide (NO) is involved in the effect of IGF-I and PTH/PTHrP on osteoblast-like cells. METHODS: Bone marrow stromal cells from adult endothelial nitric oxide synthase (eNOS)-knockout (eNOSKO) mice and wild type (WT) counterparts were cultivated with osteogenic substances. The cells showed an osteoblastic phenotype measured as osteocalcin production and alkaline phosphatase activity. DNA synthesis was measured as [3H] thymidine incorporation in the bone marrow cells and in a human osteosarcoma cell-line (SaOS-2). RESULTS: The stimulatory effect of IGF-I on thymidine incorporation seen in WT animals was absent in eNOSKO mice. Addition of a NO donor to eNOSKO cells recovered the effect of IGF-I on thymidine incorporation. PTH/PTHrP stimulated cell proliferation in both WT and eNOSKO mice. In SaOS-2 cells, incubation with IGF-I together with a NOS inhibitor resulted in an inhibition of the anabolic effect of IGF-I on cell proliferation. CONCLUSIONS: The stimulatory effect of IGF-I on WT cell proliferation was abolished in eNOSKO cells, recovered by an NO donor and inhibited in osteosarcoma cells by a NOS inhibitor. The results indicate that the effect of IGF-I is dependent on NO production. The impaired IGF-I response may contribute to the bone defect formation seen in eNOSKO animals.     

Bone marrow ablation demonstrates that excess endogenous parathyroid hormone plays distinct roles in trabecular and cortical bone

Mice null for Cyp27b1, which encodes the 25-hydroxyvitamin D-1α-hydroxylase [1α(OH)ase(-/-) mice], lack 1,25-dihydroxyvitamin D [1,25(OH)(2)D] and have hypocalcemia and high parathyroid hormone (PTH) secretion. Intermittent, exogenous PTH is anabolic for bone. To determine the effect of the chronic excess endogenous PTH on osteogenesis and bone turnover, bone marrow ablations (BMX) were performed in tibiae and femurs of 6-week-old 1α(OH)ase(-/-) mice and in wild-type (WT) controls. Newly formed bone tissue was analyzed at 1, 2, and 3 weeks after BMX. BMX did not alter the higher levels of PTH in 1α(OH)ase(-/-) mice. In the marrow cavity, trabecular volume, osteoblast number, alkaline phosphatase-positive areas, type I collagen-positive areas, bone formation-related genes, and protein expression levels all increased significantly after BMX in 1α(OH)ase(-/-) mice, compared with WT. Osteoclast numbers and surface and ratio of RANKL/OPG-relative mRNA levels decreased significantly after BMX in 1α(OH)ase(-/-) mice, compared with WT. In the cortex, alkaline phosphatase-positive osteoblasts and osteoclast numbers increased significantly after BMX in 1α(OH)ase(-/-) mice, compared with WT. These results demonstrate that chronic excess endogenous PTH exerts an anabolic role in trabecular bone by stimulating osteogenic cells and reducing bone resorption, but plays a catabolic role in cortical bone by enhancing bone turnover with an increase in resorption.     

Modulation of ovariectomy-related bone loss by parathyroid hormone in rats

Studies were carried out to examine whether parathyroid hormone (PTH) will prevent the age-related bone loss that results from ovarian hormone deficiency and to explore the mechanism of its action. Ovariectomy caused a significant decrease in bone density in the distal metaphysis and mid-diaphysis, but not in the vertebra and proximal metaphysis. The decrease was prevented by PTH injection and in all the bones examined PTH administration increased bone density and bone calcium content above the levels in sham-operated controls. Similar findings were made in bone hydroxyproline levels. PTH treated ovariectomized animals had lower serum 25(OH)vitamin D and higher 1,25(OH)2 vitamin D levels than ovariectomized and sham operated animals that received solvent vehicle. Compared to the sham operated controls, ovariectomy caused a 4.5-fold increase in the number of tartrate resistant acid phosphatase (TRAP) positive multinuclear cells. This increase did not occur in PTH-treated animals. We conclude that PTH is effective in preventing ovarian hormone deficiency bone loss in rats. PTH may mediate this effect partly by stimulating osteoblastic bone formation and partly by increasing 1,25(OH)2 vitamin D-mediated calcium absorption. The data from TRAP positive multinuclear cells indicate that an etiologic component of ovarian hormone deficiency bone loss is the expansion of a pool of osteoclast progenitors and that the bone anabolic action of PTH involves, in part, a decrease in bone resorption as a result of the suppression of the proliferation of osteoclast progenitors.     

A potential therapeutic approach to overload-induced bone loss around implant: parathyroid hormone (PTH)

The clinical use of dental implants has a high success rate, but overload-induced bone loss around implant is not uncommon in patients with implant-supported denture especially those with long cantilever designs and greatly harmful to the long-term implant success. The mechanism underlying the bone loss is thought to be the imbalance of bone remodeling involving a detrimental positive feedback activated by overloading. While surgical regenerative treatments may be useful in promoting bone regeneration, extra suffering and risk of infection have to be fully recognized. To date, no optimal method is available to solve this problem. We hereby propose a novel therapy that may potentially improve this condition. Many studies have shown that parathyroid hormone (PTH), an anabolic agent targeting bone, is effective in reversing bone loss caused by osteoporosis with negative bone remodeling in clinical studies. Moreover, PTH has the potential to accelerate the bone healing in patients with fracture and fracture nonunion and improve osseointegration of implant inserted in pre-existed bone defect via its anabolic effect to increase bone formation in animal model studies. Specifically for alveolar bone, PTH is associated with effective bone regeneration in patients with severe periodontitis. What is more, PTH and mechanical loading has a synergistic effect on bone formation, which is in favor of bone healing under physiological loading. The mechanisms underlying its anabolic effect may involve increased osteoblasts activity, prolonged osteoblasts life-span and recruitment of new osteoblasts from marrow stromal cells. Furthermore, PTH could activate resting lining cells to initial de novo bone formation. Considering these actions of PTH, we hypothesize that PTH may be a potential treatment for overload-induced bone loss around implant.     

Adaptation of diaphyseal structure to aging and decreased mechanical loading in the adult rat: a densitometric and histomorphometric study

Nine-month-old female rats were subjected to right hindlimb immobilization or served as controls for 0, 2, 10, 18, and 26 weeks. They were double-labeled with bone markers prior to sacrifice. Experimental unloading was produced by immobilizing the right limb against the abdomen with an elastic bandage. Single-photon absorptiometry was performed on the intact femurs; static and dynamic histomorphometry were performed on 20-micron thick toluidine blue-stained, undecalcified cross sections of the tibial shafts. Changes in the continuously immobilized tibiae were compared to those in both tibiae of age-matched controls. Unloading shut off nearly all periosteal bone formation and accelerates bone marrow expansion over that which occurs in age-related controls. The effect of unloading appeared to be mediated by recruiting fewer osteoblasts which showed inhibited activity. Furthermore, unloading increased endocortical percentage eroded surface. These histological changes lowered cortical bone mass by inhibiting diaphyseal cross sectional expansion and enlarging the bone marrow cavity. The results support Frost's suggestion that decrease mechanical usage depresses bone modeling-dependent bone gain by decreasing activation of modeling in the formation mode. It also stimulates bone remodeling-dependent bone loss by increasing activation of remodeling in the resorption mode.     

Inhibitory effect of CGRP on osteoclast formation by mouse bone marrow cells treated with isoproterenol

The present study was designed to elucidate the mode of action of isoproterenol (Isp; adrenergic beta-agonist) and to characterize the effect of the calcitonin gene-related peptide (CGRP; sensory neuropeptide) on osteoclast formation induced by Isp in a mouse bone marrow culture system. Treatment of mouse bone marrow cells with Isp generated tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNCs) capable of excavating resorptive pits on dentine slices, and caused an increase in receptor activator of NF-kappaB ligand (RANKL) and a decrease in osteoprotegerin (OPG) production by the marrow cells. The osteoclast formation was significantly inhibited by OPG, suggesting the involvement of the RANKL-RANK system. CGRP inhibited the osteoclast formation caused by Isp or soluble RANKL (s-RANKL) but had no influence on RANKL or OPG production by the bone marrow cells treated with Isp, suggesting that CGRP inhibited the osteoclast formation by interfering with the action of RANKL produced by the Isp-treated bone marrow cells without affecting RANKL or OPG production. This in vitro data suggest the physiological interaction of sympathetic and sensory nerves in osteoclastogenesis in vivo.     

Various evaluation techniques of newly formed bone in porous hydroxyapatite loaded with human bone marrow cells implanted in an extra-osseous site

The purpose of this work was to develop qualitative methods for in situ analysis of bone formation in an osteoconductive hydroxyapatite matrix (ENDOBON), loaded with human bone marrow cells (HBMSC) implanted subcutaneously in athymic mice. Samples were taken before implantation (T0), 1, 2, 4 and 6 weeks after implantation. Bone-biomaterial interaction were investigated on undecalcified sections by histological, cytochemical, immunological and molecular biology methodologies. Histological observations were performed in order to observe inflammatory cells, vessels, newly formed bone, woven and lamellar bone. Enzymohistochemistry was carried out to detect positive tartrate resistant acid phosphatase activity (TRAP+). Immunohistochemistry using antibodies against type I collagen and osteocalcin permitted us to characterize the content of the matrix elaborated within the implant. Moreover, in situ hybridization was carried out to discriminate, the implanted human cells from the murine cells, and to evaluate the function of these human cells in osteogenesis. Results demonstrated an early formation of lamellar bone only in the pores of the studied HAP loaded with HBMSC. This bone contained a matrix showing positive reaction for type I collagen and osteocalcin. In situ hybridization identified some of these cells as human cells. At 6 weeks, examination of histological results showed persistance of lamellar bone in the implants. We only found TRAP+ activity in the materials loaded with human bone marrow cells. Molecular hybridization no longer revealed positive cells for the human DNA probe. All these results indicate that the various evaluation techniques performed on undecalcified sections, permit us to evaluate the response of human bone marrow cells in HAP implanted into mice.     

去势大鼠5种骨转换生化指标的动态变化

目的： 对去势大鼠骨质疏松动物模型形成过程中5种骨转换指标的变化及其相关性进行研究。方法: 将3月龄SD雌性大鼠分为3组：切除卵巢组(OVX)，假手术组(sham)和对照组(control)，术前和术后分别于1、1.5、2、2.5、3和4月检测血清骨钙素(OC)、碱性磷酸酶(ALP)、 骨碱性磷酸酶(BALP)、抗酒石酸酸性磷酸酶(TRAP)和羟脯氨酸(HYP)水平，并作大鼠胫骨病理切片检查。结果: OVX组血清OC、ALP、BALP、TRAP和HYP水平均明显高于sham组，其变化顺序依次为：TRAP/HYP→OC→ALP/BALP；5种指标之间呈显著正相关；术后3月OVX组大鼠胫骨小梁结构有病理改变。结论: 去势大鼠属于高转换型骨质疏松；在模型形成过程中，骨吸收指标的变化早于骨形成指标的改变；骨转换指标是反映绝经后早期骨量丢失的灵敏指标。     

The role of BMP-6, IL-6, and BMP-4 in mesenchymal stem cell-dependent bone development: effects on osteoblastic differentiation induced by parathyroid hormone and vitamin D(3)

Human bone marrow-derived mesenchymal stem cells (MSCs) represent an ideal source for cell therapy for inherited and degenerative diseases, bone and cartilage repair, and as target for gene therapy. The role of the combination of human parathyroid hormone (PTH) and vitamin D(3) in bone formation and mineralization has been established in several osteoblast cell culture studies. The aim of the present study was to evaluate the role of this hormonal combination alone and in the presence of bone morphogenetic protein-4 (BMP-4) or-6 (BMP-6) in inducing osteogenic differentiation of human MSC. Human MSC derived from adult normal bone marrow that are positive for CD29, CD44, CD105, and CD166 and negative for CD14, CD34, and CD45, were treated with the PTH and 1,25-dihydroxyvitamin D(3) in the presence and absence of recombinant human BMP-4 or BMP6. PTH and vitamin D(3) induced high levels of expression of two key markers of bone formation: osteocalcin and alkaline phosphatase by MSCs. BMP-6 but not BMP-4 increased osteocalcin expression induced by PTH and vitamin D(3). Both BMPs enhanced calcium formation in MSC cultures and this response was potentiated by PTH and vitamin D(3). The present results revealed a novel potent effect of PTH and vitamin D(3) plus BMPs in inducing bone development by human MSCs. These results may facilitate therapeutic utility of MSCs for bone disease and help clarify mechanisms involved in stem cell-mediated bone development.     

Osteosclerotic prostate cancer metastasis to murine bone are enhanced with increased bone formation

Spontaneous development of osteoblastic lesions of prostate cancer (PCa) in mice is modeled by orthotopic (intraprostatic) deposition of neoplastic cells followed by an extremely long latency associated with low incidence of spontaneous bone metastasis. Intracardial injection results in overt bone metastases only with osteoclastic PCa cells (i.e., PC-3). Herein, we report that androgen independent osteoblastic PCa cells readily colonize bone when in a high remodeling state. SCID/Beige mice were subjected to periods of intermittent human parathyroid hormone 1–34 (hPTH) exposure, followed by an intracardiac infusion of osteoblastic C4-2 PCa cells. At the time of PCa infusion, analysis of bone turnover markers from mice treated with hPTH revealed significant increases in osteocalcin (55.06 ± 7.5 vs. 74.01 ± 18.5 ng/ml) and TRAcP-5b (3.3 ± 0.6 vs. 4.81 ± 0.8 U/l), but no change in type I collagen C-terminal teleopeptide levels relative to control mice. Analysis of femoral cancellous bone architecture revealed significant increases in bone mineral density, trabecular thickness (0.056 ± 0.002 vs. 0.062 ± 0.001 mm) and porosity, but significant decreases in connectivity density and trabecular number in hPTH treated mice relative to controls. By 8 weeks post-infusion, 70% of mice pre-treated with hPTH demonstrated detectable serum prostate specific antigen (PSAs) ranging between 2 and 18.8 ng/ml. Immuno-histochemical labeling of femurs for PSA and pan-Cytokeratin revealed the presence of significant tumor cell nests in marrow and trabecular spaces. These results suggest that: (1) local bone physiology is an important factor for developing osteoblastic/sclerotic PCa bone metastases in murine hosts; (2) the establishment of osteosclerotic PCa bone metastases in mice is enhanced by alterations that drive bone formation.     

Effect of beclomethasone dipropionate nasal aerosol on serum markers of bone metabolism in children with seasonal allergic rhinitis

Thirty-nine children with grass pollen hay fever were randomly treated with nasal inhaled beclomethasone dipropionate (BDP) 200 or 400 microg/day or sodium cromoglycate (SCG) 30 mg/day for 2 months during the pollen season. Serum osteocalcin (OC), parathyroid hormone (PTH), total alkaline phosphatase (AP), bone alkaline phosphatase (BAP) and type I collagen telopeptide (ICTP) were measured immediately before, 1 and 2 months after treatment and 1 week after stopping the therapy. No significant changes in OC, PTH, AP, BAP and ICTP serum level occurred within each group. Minor and probably clinically insignificant between group differences were occasionally found. Our study shows that BDP nasal spray has no significant effect on common markers of bone metabolism.     

Co-secretion of parathyroid hormone and parathyroid-hormone-related protein via a regulated pathway in human parathyroid adenoma cells.

Parathyroid-hormone-related protein (PTHrP) is widely expressed not only in malignant tumors but also in both epithelial and nonepithelial cells of normal tissues. Secreted PTHrP is suspected to act as a paracrine or autocrine regulator. However, little is known about its secretory pathway. To cast light on this question, we studied the intracytoplasmic distribution of parathyroid hormone (PTH) and PTHrP immunohistochemically and immunoelectron microscopically in 10 surgically resected parathyroid adenomas. Double immunostaining was performed using anti-PTH antibody and a newly established anti-PTHrP antibody to reveal the relationship between their two distributions. Additional examination by cell immunoblot assay was performed to determine whether both PTH and PTHrP are secreted simultaneously. Both PTH and PTHrP were actually secreted from individual parathyroid cells simultaneously on cell immunoblot assay. Immunohistochemically, there were two different types of adenoma cells, i.e., one positive only for PTH and the other positive for both PTH and PTHrP. PTH was distributed linearly or fine granularly along the cytoplasmic membrane, whereas PTHrP was distributed diffusely or coarse granularly in the cytoplasm. The intracytoplasmic distributions of PTH and PTHrP often overlapped. Immunoelectron microscopical examination demonstrated that PTHrP co-localized with PTH in the same secretory granules. The results clearly demonstrated that PTHrP can be co-secreted with PTH via a regulated pathway using secretory granules.     

绝经后妇女骨质疏松患者血清IGF-1、IGFBP-3与骨密度及骨代谢指标的关系

目的： 探讨胰岛素样生长因子-1（IGF-1）和胰岛素样生长因子结合蛋白-3（IGFBP-3）与绝经后妇女骨密度及骨代谢指标之间的关系。方法： 通过检测90例绝经后妇女骨质疏松患者及70例绝经后骨量正常的健康对照组血清IGF-1、IGFBP-3、骨钙素（BGP）、I型胶原异构C端肽（β-CTX）、雌激素（E₂）、降钙素（CT）、甲状旁腺激素（PTH）、钙（Ca）、磷（P）等指标，然后同用双能X线骨密度仪检测的两组研究对象的腰椎（L2-L4）侧位、左股骨颈骨密度进行比较。结果： 绝经后骨质疏松组妇女腰椎、股骨颈骨密度显著低于对照组（均P<0.01）；血清IGF-1、IGFBP-3、E₂、CT、BGP水平均低于对照组（均P<0.01）；血清β-CTX、PTH均高于对照组（均P<0.01），血清Ca、P两组之间无差异（均P>0.05）。骨质疏松组和对照组腰椎侧位、左股骨颈BMD均与IGF-1、IGFBP-3、E₂、BGP、CT水平呈正相关，与β-CTX、PTH水平呈负相关，而与血钙、血磷无明显关系。结论： IGF-1、IGFBP-3、E₂、BGP、CT、β-CTX、PTH血清水平与腰椎、左股骨质具有明显的相关性，通过检测上述指标可考虑作为筛查绝经后妇女是否容易患有骨质疏松症的一项有价值的生化参考指标。     

Seasonal variation of bone turnover markers in top-level female skiers

Different levels of weight-bearing activities imply different levels of anabolic effects on skeletal tissue and this can be assessed by measuring biochemical markers reflecting bone metabolism. With this study we wanted to determine how the serum levels of bone turnover markers change during different phases of annual training in elite female skiers. Fourteen top-level Caucasian athletes, from the Italian Women’s Alpine Ski Team (slalom and giant slalom), were tested at the end of the relative rest period (T1), the pre-competitive season (T2) and the competitive season (T3). Serum levels of bone-specific alkaline phosphatase (BAP) and tartrate-resistant acid phosphatase (TRAP5b) activities and of osteocalcin (OC), and crosslaps (the carboxyterminal crosslinked telopeptide of type I collagen—β-CTx), were assayed together with the determination of 25(OH)D levels. The formation markers, BAP and OC and the resorption marker TRAP5b significantly increased from T2 to T3, while crosslaps showed no significant changes. The peculiar trends of bone formation markers correlated one to each other at T2 versus T3, and this was probably linked to the highly demanding period of competitions when, in athletes performing weight-bearing exercise, bone is more stimulated by mechanical forces. 25(OH)D levels, instead, changed from T1 to T2 and from T1 to T3 and its trend do not show any correlation with that of bone markers. In conclusion, we found that both the bone formation markers and TRAP5b, marker of resorption, are significantly increased from the pre-competitive season to the competitive season.     

Ursolic acid derivative ameliorates streptozotocin-induced diabestic bone deleterious effects in mice

Objective: This study was performed to investigate bone deteriorations of diabetic mice in response to the treatment of ursolic acid derivative (UAD). Methods: The biomarkers in serum and urine were measured, tibias were taken for the measurement on gene and protein expression and histomorphology analysis, and femurs were taken for the measurement on bone Ca and three-dimensional architecture of trabecular bone. Results: UAD showed a greater increase in bone Ca, BMD and significantly increased FGF-23 and OCN, reduced PTH and CTX in diabetic mice. UAD reversed STZ-induced trabecular deleterious effects and stimulated bone remodeling. The treatment of STZ group with UAD significantly elevated the ratio of OPG/RANKL. Moreover, insulin and IGF-1 showed a negative correlation with both FBG and Hb1Ac in STZ group. We attributed down-regulating the level of Hb1Ac in diabetic mice to that ursolic acid derivative could primely control blood sugar levels. After analyzing of two adipocyte markers, PPARγ and aP2, increased expression in the tibias of diabetic mice, and UAD could improve STZ-induced adipocyte dysfunction. Conclusions: These results demonstrated that UAD could ameliorate STZ-induced bone deterioration through improving adipocyte dysfunction and enhancing new bone formation and inhibiting absorptive function of osteoclast in the bone of diabetic mice.