Conditional deletion of Bmpr1a in differentiated osteoclasts increases osteoblastic bone formation,increasing volume of remodeling bone in mice

Bone undergoes remodeling consisting of osteoclastic bone resorption followed by osteoblastic bone formation throughout life. Although the effects of bone morphogenetic protein (BMP) signals on osteoblasts have been studied extensively, the function of BMP signals in osteoclasts has not been fully elucidated. To delineate the function of BMP signals in osteoclasts during bone remodeling, we deleted BMP receptor type IA (Bmpr1a) in an osteoclast‐specific manner using a knock‐in Cre mouse line to the cathepsin K locus (Ctsk^Cre/+;Bmpr1a^flox/flox, designated as Bmpr1a^ΔOc/ΔOc). Cre was specifically expressed in multinucleated osteoclasts in vivo. Cre‐dependent deletion of the Bmpr1a gene occurred at 4 days after cultivation of bone marrow macrophages obtained from Bmpr1a^ΔOc/ΔOc with RANKL. These results suggested that Bmpr1a was deleted after formation of osteoclasts in Bmpr1a^ΔOc/ΔOc mice. Expression of bone‐resorption markers increased, thus suggesting that BMPRIA signaling negatively regulates osteoclast differentiation. Trabeculae in tibia and femurs were thickened in 3.5‐, 8‐, and 12‐week‐old Bmpr1a^ΔOc/ΔOc mice. Bone histomorphometry revealed increased bone volume associated with increased osteoblastic bone‐formation rates (BFR) in the remodeling bone of the secondary spongiosa in Bmpr1a^ΔOc/ΔOc tibias at 8 weeks of age. For comparison, we also induced an osteoblast‐specific deletion of Bmpr1a using Col1a1‐Cre. The resulting mice showed increased bone volume with marked decreases in BFR in tibias at 8 weeks of age. These results indicate that deletion of Bmpr1a in differentiated osteoclasts increases osteoblastic bone formation, thus suggesting that BMPR1A signaling in osteoclasts regulates coupling to osteoblasts by reducing bone‐formation activity during bone remodeling. © 2011 American Society for Bone and Mineral Research     

Effects of Vitamin K2 on Cortical and Cancellous Bone Mass, Cortical Osteocyte and Lacunar System, and Porosity in Sciatic Neurectomized Rats

The purpose of the present study was to examine the effects of vitamin K₂ on cortical and cancellous bone mass, cortical osteocyte and lacunar system, and porosity in sciatic neurectomized rats. Thirty-four female Sprague-Dawley retired breeder rats were randomized into three groups: age-matched control, sciatic neurectomy (NX), and NX + vitamin K₂ administration (menatetrenone, 30 mg/kg/day p.o., three times a week). At the end of the 8-week experiment, bone histomorphometric analysis was performed on cortical and cancellous bone of the tibial diaphysis and proximal metaphysis, respectively, and osteocyte lacunar system and porosity were evaluated on cortical bone of the tibial diaphysis. NX decreased cortical and cancellous bone mass compared with age-matched controls as a result of increased endocortical and trabecular bone erosion and decreased trabecular mineral apposition rate (MAR). Vitamin K₂ ameliorated the NX-induced increase in bone erosion, prevented the NX-induced decrease in MAR, and increased bone formation rate (BFR/bone surface) in cancellous bone, resulting in an attenuation of NX-induced cancellous bone loss. However, vitamin K₂ did not significantly influence cortical bone mass. NX also decreased osteocyte density and lacunar occupancy and increased porosity in cortical bone compared with age-matched controls. Vitamin K₂ ameliorated the NX-induced decrease in lacunar occupancy by viable osteocytes and the NX-induced increase in porosity. The present study showed the efficacy of vitamin K₂ for cancellous bone mass and cortical lacunar occupancy by viable osteocytes and porosity in sciatic NX rats.     

Immunologic analyses of bovine bone treated with a novel tissue sterilization process

Abstract: Background: This study evaluates the efficacy of removal of xeno‐antigens from bovine bone using a patented BioCleanse^® process for decellularization of allograft tissues for clinical implantation. BioCleanse^® deploys a combination of chemicals and several high pressure rinses to achieve standardized sterility assurance levels. This method produces sterile grafts without reducing allograft bone biomechanical properties and effectively removes cells, lipids, and other sources of antigenic material from human allografts for clinical use. Methods: In this investigation, BioCleanse^® is evaluated for its potential in removing xenograft antigens from bovine bone grafts followed by immunologic evaluation in the subcutaneous pouch of immunocompetent rats. The alpha‐galactosyl (α‐gal) epitope with the structure Galα1‐3Galβ1‐4GlcNAc‐R constitutes a critical component of xenoantigens and its removal using BioCleanse^® from bovine bone was compared with tissue levels of unprocessed bone. The relative degree of antigen removal was also determined through measuring the pro‐inflammatory cytokine, tumor necrosis factor (TNF)‐α, and through the use of histologic grading of cellular infiltrates into bone. Results: Compact cortical bone inhibited immune cell migration but cancellous bone demonstrated cellular increase and bone resorption in the untreated control group. The α‐gal xenoantigen level was significantly lower in both cortical (P < 0.001) and cancellous bone (P < 0.001) compared with controls. TNF‐α levels were significantly (P < 0.001) reduced compared with untreated controls when human acute monocytic leukemia cells were exposed to cortical or cancellous bone. Conclusions: BioCleanse^® effectively removed xenoantigens and inflammatory markers justifying a follow up study in primates to determine these benefits in a model that is primed with preformed xeno‐antibodies responsible for hyperacute rejection in hard tissues.     

β‐Adrenergic Blockade and Leptin Replacement Effectively Mitigate Disuse Bone Loss

Our objective was to test effects of β‐adrenergic blockade on hindlimb unloading (HU)‐induced bone loss and serum leptin and to compare these responses with those observed with leptin replacement. Adult male rats were randomized into six groups (n = 10 each): HU rats treated with vehicle (VEHHU), leptin analog (LEPHU), or β‐blocker (BBHU) during a 28‐day HU and cage activity controls (CC) treated with the same three agents and pair‐fed to HU rats. On days 0 and 28, pQCT scans of proximal tibia and serum collections for leptin assays were performed, and histomorphometric measures of proximal tibia cancellous bone were assessed. The 20% decrease in cancellous vBMD observed in the VEHHU group was halved in BBHU rats and LEPHU rats. Bone formation rate (BFR) in BBHU rats, but not in LEPHU rats, was preserved. The 3‐fold increase in resorption surface with HU was abolished by BB and LEP treatments. The decrease in serum leptin after a 28‐day HU was attenuated in BBHU and LEPHU rats and was predictive of the decrease in BFR with HU. Blocking sympathetic adrenergic signaling by peripheral administration of a β‐blocker during HU mitigates disuse‐induced decreases in cancellous bone mass through stimulation of osteoblastic activity and suppression of osteoclastic activity. A direct effect of β‐adrenergic blockade on bone cells during HU may be enhanced by an indirect effect mitigating reductions in circulating leptin, possibly through disinhibition of leptin release from adipocytes.     

Blockade of receptor‐activated Gi signaling in osteoblasts in vivo leads to site‐specific increases in cortical and cancellous bone formation

Osteoblasts play a critical role in the maintenance of bone mass through bone formation and regulation of bone resorption. Targeted expression of a constitutively active engineered G_i‐coupled G protein–coupled receptor (GPCR) to osteoblasts in vivo leads to severe osteopenia. However, little is known about the role of endogenous receptor‐mediated G_i signaling in regulating osteoblast function. In this study, we investigated the skeletal effects of blocking G_i‐coupled signaling in osteoblasts in vivo. This was accomplished by transgenic expression of the catalytic subunit of pertussis toxin (PTX) under control of the collagen Iα 2.3‐kb promoter. These mice, designated Col1(2.3)⁺/PTX⁺, showed increased cortical thickness at the femoral midshaft at 12 weeks of age. This correlated with increased periosteal bone formation associated with expanded mineralizing surface observed in 8‐week‐old mice of both genders. The cancellous bone phenotype of the Col1(2.3)⁺/PTX⁺ mice was sexually dimorphic, with increases in fractional bone volume at the distal femur seen only in females. Similarly, while cancellous bone‐formation rates were unchanged in males, they could not be quantified for female Col1(2.3)⁺/PTX⁺ mice owing to the disorganized nature of the labeling pattern, which was consistent with rapid formation of woven bone. Alterations in osteoclast activity did not appear to participate in the phenotype. These data demonstrate that G_i‐coupled signaling by GPCRs endogenous to osteoblasts plays a complex role in the regulation of bone formation in a manner that is dependent on both gender and the anatomic site within bone. © 2011 American Society for Bone and Mineral Research.     

Gender‐Specific Differences in the Skeletal Response to Continuous PTH in Mice Lacking the IGF1 Receptor in Mature Osteoblasts

The primary goal of this study was to determine whether the IGF1R in mature osteoblasts and osteocytes was required for the catabolic actions of continuous parathyroid hormone (cPTH). Igf1r was deleted from male and female FVN/B mice by breeding with mice expressing cre recombinase under control of the osteocalcin promoter (^0CNIgfr1^‐/‐). Littermates lacking the cre recombinase served as controls. PTH, 60 μg/kg/d, was administered continuously by Alzet minipumps for 4 weeks. Blood was obtained for indices of calcium metabolism. The femurs were examined by micro‐computed tomography for structure, immunohistochemistry for IGF1R expression, histomorphometry for bone formation rates (BFR), mRNA levels by qPCR, and bone marrow stromal cell cultures (BMSC) for alkaline phosphatase activity (ALP⁺), mineralization, and osteoblast‐induced osteoclastogenesis. Whereas cPTH led to a reduction in trabecular bone volume/tissue volume (BV/TV) and cortical thickness in the control females, no change was found in the control males. Although trabecular BV/TV and cortical thickness were reduced in the ^0CNIgfr1^‐/‐ mice of both sexes, no further reduction after cPTH was found in the females, unlike the reduction in males. BFR was stimulated by cPTH in the controls but blocked by Igf1r deletion in the females. The ^0CNIgfr1^‐/‐ male mice showed a partial response. ALP⁺ and mineralized colony formation were higher in BMSC from control males than from control females. These markers were increased by cPTH in both sexes, but BMSC from male ^0CNIgfr1^‐/‐ also were increased by cPTH, unlike those from female ^0CNIgfr1^‐/‐. cPTH stimulated receptor activator of NF‐κB ligand (RANKL) and decreased osteoprotegerin and alkaline phosphatase expression more in control female bone than in control male bone. Deletion of Igf1r blocked these effects of cPTH in the female but not in the male. However, PTH stimulation of osteoblast‐driven osteoclastogenesis was blocked by deleting Igfr1 in both sexes. We conclude that cPTH is catabolic in female but not male mice. Moreover, IGF1 signaling plays a greater role in the skeletal actions of cPTH in the female mouse than in the male mouse, which may underlie the sex differences in the response to cPTH. © 2015 American Society for Bone and Mineral Research.     

The Effects of Androgens on Murine Cortical Bone Do Not Require AR or ERα Signaling in Osteoblasts and Osteoclasts

In men, androgens are critical for the acquisition and maintenance of bone mass in both the cortical and cancellous bone compartment. Male mice with targeted deletion of the androgen receptor (AR) in mature osteoblasts or osteocytes have lower cancellous bone mass, but no cortical bone phenotype. We have investigated the possibility that the effects of androgens on the cortical compartment result from AR signaling in osteoprogenitors or cells of the osteoclast lineage; or via estrogen receptor alpha (ERα) signaling in either or both of these two cell types upon conversion of testosterone to estradiol. To this end, we generated mice with targeted deletion of an AR or an ERα allele in the mesenchymal (AR^f/y;Prx1‐Cre or ERα^f/f;Osx1‐Cre) or myeloid cell lineage (AR^f/y;LysM‐Cre or ERα^f/f;LysM‐Cre) and their descendants. Male AR^f/y;Prx1‐Cre mice exhibited decreased bone volume and trabecular number, and increased osteoclast number in the cancellous compartment. Moreover, they did not undergo the loss of cancellous bone volume and trabecular number caused by orchidectomy (ORX) in their littermate controls. In contrast, AR^f/y;LysM‐Cre, ERα^f/f;Osx1‐Cre, or ERα^f/f;LysM‐Cre mice had no cancellous bone phenotype at baseline and lost the same amount of cancellous bone as their controls following ORX. Most unexpectedly, adult males of all four models had no discernible cortical bone phenotype at baseline, and lost the same amount of cortical bone as their littermate controls after ORX. Recapitulation of the effects of ORX by AR deletion only in the AR^f/y;Prx1‐Cre mice indicates that the effects of androgens on cancellous bone result from AR signaling in osteoblasts—not on osteoclasts or via aromatization. The effects of androgens on cortical bone mass, on the other hand, do not require AR or ERα signaling in any cell type across the osteoblast or osteoclast differentiation lineage. Therefore, androgens must exert their effects indirectly by actions on some other cell type(s) or tissue(s). © 2015 American Society for Bone and Mineral Research.     

Cancellous bone formation response to simulated resistance training during disuse is blunted by concurrent alendronate treatment

The purpose of this study was to assess the effectiveness of simulated resistance training (SRT) exercise combined with alendronate (ALEN) in mitigating or preventing disuse‐associated losses in cancellous bone microarchitecture and formation. Sixty male Sprague‐Dawley rats (6 months old) were randomly assigned to either cage control (CC), hind limb unloading (HU), HU plus either ALEN (HU + ALEN), SRT (HU + SRT), or a combination of ALEN and SRT (HU + SRT/ALEN) for 28 days. HU + SRT and HU + SRT/ALEN rats were anesthetized and subjected to muscle contractions once every 3 days during HU (four sets of five repetitions, 1000 ms isometric + 1000 ms eccentric). Additionally, HU + ALEN and HU + SRT/ALEN rats received 10 µg/kg of body weight of ALEN three times per week. HU reduced cancellous bone‐formation rate (BFR) by 80%, with no effect of ALEN treatment (?85% versus CC). SRT during HU significantly increased cancellous BFR by 123% versus CC, whereas HU + SRT/ALEN inhibited the anabolic effect of SRT (?70% versus HU + SRT). SRT increased bone volume and trabecular thickness by 19% and 9%, respectively, compared with CC. Additionally, osteoid surface (OS/BS) was significantly greater in HU + SRT rats versus CC (+32%). Adding ALEN to SRT during HU reduced Oc.S/BS (?75%), Ob.S/BS (?72%), OS/BS (?61%), and serum TRACP5b (?36%) versus CC. SRT and ALEN each independently suppressed a nearly twofold increase in adipocyte number evidenced with HU and inhibited increases in osteocyte apoptosis. These results demonstrate the anabolic effect of a low volume of high‐intensity muscle contractions during disuse and suggest that both bone resorption and bone formation are suppressed when SRT is combined with bisphosphonate treatment. © 2011 American Society for Bone and Mineral Research     

High Bone Mass–Causing Mutant LRP5 Receptors Are Resistant to Endogenous Inhibitors In Vivo

Certain missense mutations affecting LRP5 cause high bone mass (HBM) in humans. Based on in vitro evidence, HBM LRP5 receptors are thought to exert their effects by providing resistance to binding/inhibition of secreted LRP5 inhibitors such as sclerostin (SOST) and Dickkopf homolog‐1 (DKK1). We previously reported the creation of two Lrp5 HBM knock‐in mouse models, in which the human p.A214V or p.G171V missense mutations were knocked into the endogenous Lrp5 locus. To determine whether HBM knock‐in mice are resistant to SOST‐ or DKK1‐induced osteopenia, we bred Lrp5 HBM mice with transgenic mice that overexpress human SOST in osteocytes (^8kbDmp1‐SOST) or mouse DKK1 in osteoblasts and osteocytes (^2.3kbCol1a1‐Dkk1). We observed that the ^8kbDmp1‐SOST transgene significantly lowered whole‐body bone mineral density (BMD), bone mineral content (BMC), femoral and vertebral trabecular bone volume fraction (BV/TV), and periosteal bone‐formation rate (BFR) in wild‐type mice but not in mice with Lrp5 p.G171V and p.A214V alleles. The ^2.3kbCol1a1‐Dkk1 transgene significantly lowered whole‐body BMD, BMC, and vertebral BV/TV in wild‐type mice and affected p.A214V mice more than p.G171V mice. These in vivo data support in vitro studies regarding the mechanism of HBM‐causing mutations, and imply that HBM LRP5 receptors differ in their relative sensitivity to inhibition by SOST and DKK1. © 2015 American Society for Bone and Mineral Research.     

Osteocyte control of osteoclastogenesis

Multiple lines of evidence support the idea that osteocytes act as mechanosensors in bone and that they control bone formation, in part, by expressing the Wnt antagonist sclerostin. However, the role of osteocytes in the control of bone resorption has been less clear. Recent studies have demonstrated that osteocytes are the major source of the cytokine RANKL involved in osteoclast formation in cancellous bone. The goal of this review is to discuss these and other studies that reveal mechanisms whereby osteocytes control osteoclast formation and thus bone resorption.This article is part of a Special Issue entitled "The Osteocyte".     

Time course of peri‐implant bone regeneration around loaded and unloaded implants in a rat model

The time‐course of cancellous bone regeneration surrounding mechanically loaded implants affects implant fixation, and is relevant to determining optimal rehabilitation protocols following orthopaedic surgeries. We investigated the influence of controlled mechanical loading of titanium‐coated polyether‐ether ketone (PEEK) implants on osseointegration using time‐lapsed, non‐invasive, in vivo micro‐computed tomography (micro‐CT) scans. Implants were inserted into proximal tibial metaphyses of both limbs of eight female Sprague–Dawley rats. External cyclic loading (60 or 100 μm displacement, 1 Hz, 60 s) was applied every other day for 14 days to one implant in each rat, while implants in contralateral limbs served as the unloaded controls. Hind limbs were imaged with high‐resolution micro‐CT (12.5 μm voxel size) at 2, 5, 9, and 12 days post‐surgery. Trabecular changes over time were detected by 3D image registration allowing for measurements of bone‐formation rate (BFR) and bone‐resorption rate (BRR). At day 9, mean %BV/TV for loaded and unloaded limbs were 35.5 ± 10.0% and 37.2 ± 10.0%, respectively, and demonstrated significant increases in bone volume compared to day 2. BRR increased significantly after day 9. No significant differences between bone volumes, BFR, and BRR were detected due to implant loading. Although not reaching significance (p = 0.16), an average 119% increase in pull‐out strength was measured in the loaded implants. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:997–1006, 2017.

     

Combination use of vitamin K<Subscript>2</Subscript> further increases bone volume and ameliorates extremely low turnover bone induced by bisphosphonate therapy in tail-suspension rats

 Bisphosphonate is a potent inhibitor of bone resorption, which results in the increase of bone volume. However, bisphosphonate treatment may lead to extremely low bone turnover and abnormal bone microstructure. In this study, we examined whether the combination of bisphosphonate with vitamin K₂ treatment may have beneficial effects on bone turnover and trabecular microstructure as well as on bone volume loss by using tail-suspension model rats. In these model rats, bone mineral density (BMD) decreased with histological evidence of enhanced bone resorption and suppressed bone formation. By bisphosphonate treatment, BMD was increased compared with that of tail-suspended rats. Osteoclast surface per bone surface (Oc.S/BS) and number of osteoclasts per bone perimeter (N.Oc/B.Pm) were reduced and mineral apposition rate (MAR) decreased, suggesting extreme suppression of bone turnover. However, trabecular structure examined by microfocus CT was apparently abnormal. By contrast, combination of bisphosphonate with vitamin K₂ leads to further increase of bone volume. MAR and BFR as well as Oc.S/BS and N.Oc/B.Pm were increased compared with those of the bisphosphonate-treated group. However, abnormal structure of trabeculae in secondary spongiosa was not completely ameliorated. These data suggested that concomitant use of vitamin K₂ with bisphosphonate excessively ameliorates too much suppression of bone turnover while more efficiently preventing bone volume loss. Received: January 30, 2002 / Accepted: November 6, 2002 RID="*" ID="*"  Present address: Department of Health Sciences, Oita University of Nursing and Health Sciences, Oita, Japan Acknowledgments. This work was supported in part by a Special Grant for Medical Research from Ministry of Post and Telecommunications, Japan (to M.F.), a grant in aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (#13671115 to M.F.), and by a grant from the Research Society for Metabolic Bone Disease (to M.F.). We are grateful to Miss Sachiko Suzuki for technical assistance. Offprint requests to: M. Fukagawa     

Early Changes in Bone Density,Microarchitecture, Bone Resorption,and Inflammation Predict the Clinical Outcome 12 Weeks After Conservatively Treated Distal Radius Fractures: An Exploratory Study

Fracture healing is an active process with early changes in bone and inflammation. We performed an exploratory study evaluating the association between early changes in densitometric, structural, biomechanical, and biochemical bone parameters during the first weeks of fracture healing and wrist‐specific pain and disability at 12 weeks in postmenopausal women with a conservatively treated distal radius fracture. Eighteen patients (aged 64 ± 8 years) were evaluated at 1 to 2 and 3 to 4 weeks postfracture, using high‐resolution peripheral quantitative computed tomography (HR‐pQCT), micro‐finite element analysis, serum procollagen type‐I N‐terminal propeptide (P1NP), carboxy‐terminal telopeptide of type I collagen (ICTP), and high‐sensitive C‐reactive protein (hsCRP). After 12 weeks, patients rated their pain and disability using Patient Rated Wrist Evaluation (PRWE) questionnaire. Additionally, Quick Disability of the Arm Shoulder and Hand (QuickDASH) questionnaire and active wrist range of motion was evaluated. Linear regression models were used to study the relationship between changes in bone parameters and in hsCRP from visit 1 to 2 and PRWE score after 12 weeks. A lower PRWE outcome, indicating better outcome, was significantly related to an early increase in trabecular bone mineral density (BMD) (β ?0.96 [95% CI ?1.75 to ?0.16], R² = 0.37), in torsional stiffness (?0.14 [?0.28 to ?0.004], R² = 0.31), and to an early decrease in trabecular separation (209 [15 to 402], R² = 0.33) and in ICTP (12.1 [0.0 to 24.1], R² = 0.34). Similar results were found for QuickDASH. Higher total dorsal and palmar flexion range of motion was significantly related to early increase in hsCRP (9.62 [3.90 to 15.34], R² = 0.52). This exploratory study indicates that the assessment of early changes in trabecular BMD, trabecular separation, calculated torsional stiffness, bone resorption marker ICTP, and hsCRP after a distal radius fracture provides valuable information regarding the 12‐week clinical outcome in terms of pain, disability, and range of motion and validates its use in studies on the process of early fracture healing. © 2014 American Society for Bone and Mineral Research.     

Relationships between osteocyte density and bone formation rate in human cancellous bone

Iliac cancellous osteocyte density decreases with age in deep bone but not in superficial bone, most likely because of remodeling. It has been suggested that osteocytes can inhibit bone remodeling. Accordingly, we examined the relationship between osteocyte density and bone formation rate in 92 healthy women. In superficial bone (<25 μm from the surface), we found a weak but significant (p < 0.03) inverse correlation between BFR/BS and Ot. N/B.Ar that was unaffected by menopause and independent of age. A weaker positive relationship with empty lacunar density improved significance. The data appear to suggest a negative feedback loop, but osteocytes explain only 10% of the variance in BFR/BS, and 97% of the variance in osteocyte density is explained by total lacunar density. This measure of initial osteocyte density during bone formation has a high coefficient of variation (20%) indicating large individual differences. We conclude that: (1) our data support the proposal that osteocytes can inhibit bone remodeling; (2) osteocyte density in superficial bone depends mainly on initial osteocyte density during bone formation and is maintained but not regulated by bone remodeling; and (3) the inverse relationship between BFR/BS and osteocyte density may reflect the homeostatic need to maintain calcium exchangeability in the lining cell–osteocyte syncytium.     

Exogenous prostacyclin,but not prostaglandin E2, produces similar responses in both G6PD activity and RNA production as mechanical loading,and increases IGF-II release,in adult cancellous bone in culture

Summary Cyclic mechanical loadingin vivo that leads to new bone formation is also associated in osteocytes and surface bone cells with almost immediate increases in G6PD activity, and later increases in RNA production. Both these early, loading-related, responses can be reproduced in organ culture of adult cancellous bone, and both are abolished by the presence of indomethacin in the culture medium at the time of loading. The implication that prostaglandins (PGs) are involved in the control of loading-related osteogenesis is supported by increases in prostacyclin (PGIZ) and PGE₂ release from cores of cancellous bone during loading. In the experiments reported here, PGE₂ and PGI₂ were added exogenously (10^–6 M) to perfusable cores of adult canine cancellous bone to determine whether they would simulate the loading-related responses in G6PD activity and RNA synthesis. PGE₂ increased GOD activity in surface cells and osteocytes within 8 minutes but had no effect on [³H]-uridine incorporation at 6 hours. PGI₂ stimulated both G6PD activity and [³H]-uridine incorporation equally in osteocytes and surface cells. Neither PG produced any significant change in medium concentrations of IGF-I, and PGE₂ had no effect on IGF-II. In contrast, PGI₂ elevated the medium concentration of IGF-II threefold. IGF-I and IGF-II were localized immunocytochemically to osteocytes and surface cells in both treated and untreated cores. Prostacyclin, but not PGE₂, appears to imitate the early loading-related increases in G6PD activity and RNA synthesis in bone cellsin situ. Prostacyclin, but not PGE₂, also stimulates the early release of IGF-II.     

Increased Tartrate-Resistant Acid Phosphatase Expression in Osteoblasts and Osteocytes in Experimental Osteoporosis in Rats

Tartrate-resistant acid phosphatase (TRAP) is known as an osteoclast marker, but osteoblasts and osteocytes in the vicinity of bone remodeling sites also express TRAP. Cell culture studies suggest that osteoblasts endocytose osteoclastic TRAP for inactivation. To evaluate whether changes in osteoclast activity could alter TRAP expression in osteoblasts and/or osteocytes in vivo, we studied the ovariectomized and vitamin D-deficient rat (Ovx-D) and rats healing from rickets. Bone sections were analyzed for TRAP gene expression by in situ hybridization, TRAP protein by immunogold labeling, and TRAP enzyme activity using the fluorescent substrate ELF97. Osteoblasts and osteocytes close to intracortical remodeling sites and bone surfaces demonstrated TRAP, most prominently in cancellous bone and osteocytes. Intracellular TRAP was located to electron-dense vesicles with similar morphology in both cell types. Ovx-D increased osteoclast activity (p < 0.001) and ELF97⁺ osteocytes (p < 0.05) in cancellous bone, but no corresponding increase was observed in the osteocyte lacunar area. The level of TRAP⁺ vesicles in cortical osteoblasts (p < 0.01) in Ovx-D rats was also increased. Enhanced osteoclast activity was noted in healing rickets after 72 h (p < 0.05), but no differences in TRAP expression were detected in osteoblasts or osteocytes. Thus, increased osteoclast activity does not affect TRAP expression in osteoblasts and osteocytes, favoring the notion that increased TRAP in these cells is rather due to increased synthesis. Although the role of TRAP in osteoblasts and osteocytes remains elusive, we speculate that the function is related to the capability of the enzyme to regulate the phosphorylation of proteins known to be expressed by these cells.     

Changes in cortical width with bone turnover in the three different endosteal envelopes of the ilium in postmenopausal osteoporosis.

Histological indicators of bone turnover were compared in the three endosteal envelopes (cancellous, endocortical, and intracortical) of iliac bone specimens obtained from 82 osteoporotic women, to assess the correlation between bone turnover and bone volume in different remodeling sites. Although there was a significant but weak correlation between the mineral apposition rate (MAR), a histological indicator of bone formation at the basic multicellular unit (BMU) level, and the three endosteal envelopes, the bone formation rate corrected for bone surface (BFR/BS) and mineralizing surface (MS/BS), indicators of the rate of bone formation reflecting activation frequency, in the cancellous and endocortical envelopes was more closely related to the rate in the intracortical envelope. The endocortical BFR/BS and MS/BS were higher than the rate in the cancellous envelope (1.6-2.1 times and 2.0-2.4 times, respectively), indicating a higher turnover rate in the endocortical envelope. According to stepwise regression analysis of the significant determinants contributing to bone mass, several histological determinants relating to bone turnover were identified: (1) trabecular thickness (Tb.Th) was a positive determinant, whereas age and cancellous bone volume referent BFR (BFR/BV) were negatively correlated determinants of the cancellous bone volume (BV/TV) (R2 = 0.50, p < 0.001); and (2) the endocortical wall thickness (W.Th) of the given side and the cortical width (Ct.Wi) of the opposite side were positive determinants, whereas the cancellous osteoid surface (OS/BS), cancellous MAR, and endocortical eroded surface (ES/BS) of the given side were the negatively correlated determinants of the Ct.Wi of the thicker cortex (R2 = 0.62, p < 0.001). In the thinner cortex, the endocortical W.Th of the given side and Ct.Wi of the opposite side were only used as the positive determinants of the Ct.Wi of the given side (R2 = 0.55, p < 0.001). In addition: (3) a significant but weak correlation was found using the intracortical BFR/BV as a positively correlated determinant of the cortical porosity (Ct.Po) in the thicker cortex (R2 = 0.17, p < 0.01). Although these histological determinants do not fully explain the mechanisms of bone loss, an increased rate of bone turnover contributes to bone loss not only in the cancellous and intracortical envelopes, but also in the endocortical envelope, indicating increased endocortical bone resorption in osteoporosis.     

Maintenance of cancellous bone in ovariectomized, human parathyroid hormone [hPTH(1-84)]-treated rats by estrogen, risedronate, or reduced hPTH

This study compares effects of maintenance doses of human parathyroid hormone [hPTH(1-84)], 17beta-estradiol (E2), and risedronate on distal femur bone mineral density and proximal tibia cancellous bone histomorphometry in ovariectomized (ovx), osteopenic rats previously administered a higher dose of hPTH. Nine groups (n = 8) of 3.5-month-old ovx or intact Sprague-Dawley rats were left untreated for 11 weeks to allow for the development of cancellous osteopenia in the ovx groups. Next, the ovx rats received subcutaneous injections of hPTH (75 microg/kg per day, three times per week) or vehicle for 12 weeks. Treatments were then changed to E2 (10 microg/kg per day, two times per week), risedronate (Ris; 3 microg/kg per day, three times per week), low-dose hPTH(1-84) (LowPTH; 25 microg/kg per day, three times per week), or vehicle, and administered for 36 weeks. The intact control group remained untreated for the duration of study. Femora and tibiae were collected at weeks -11 (baseline); 0 (ovx effect); 12 (hPTH effect), and 24, 36, and 48 (maintenance effects). Endpoints evaluated included distal femur bone mineral density (BMD) and proximal tibia cancellous bone volume (BV/TV), osteoclast surface (Oc.S), mineralizing surface (MS), mineral apposition rate (MAR), and bone formation rate (BFR). Ovariectomy had a negative effect on distal femur BMD and proximal tibia BV/TV. Treatment of ovx rats with hPTH for 12 weeks resulted in higher BMD in comparison to intact controls, and higher cancellous BV/TV in comparison to ovx controls. Discontinuation of hPTH resulted in loss of gained BMD within 24 weeks and loss of gained BV/TV within 12 weeks. Treatment of ovx rats with hPTH for 12 weeks followed by E2 treatment left BMD and BV/TV similar to vehicle-treated ovx rats by week 48 (36 weeks after commencement of the E2 maintenance treatment). Maintenance treatment with risedronate resulted in BMD and BV/TV similar to that of intact controls. Maintenance treatment with low-dose hPTH resulted in greater BMD and similar BV/TV in comparison to intact controls. MS and BFR were highest after low-dose hPTH administration. MS and BFR were lowest after E2 or risedronate, whereas Oc.S was lowest after risedronate administration. Thus, in osteopenic rats, the increment in distal femur BMD and proximal tibia BV/TV gained by 12 weeks of hPTH treatment was lost within 24 and 12 weeks of treatment termination, respectively. Low-dose hPTH maintained BMD and BV/TV after hPTH treatment by stimulating bone formation, whereas risedronate maintained BMD and BV/TV by reducing bone resorption. E2 in a maintenance dose failed to maintain BMD and BV/TV after withdrawal of hPTH treatment.     

Ibuprofen inhibits localized bone resorption in the middle ear

Summary Localized osteoclastic bone resorption plays a significant role in the pathogenesis of several diseases of the middle ear as well as orthodontic tooth movement and long bone remodeling. The mechanisms of control of localized bone loss and systemic bone resorption may be different but both may be mediated by a final common pathway which includes prostaglandins. Prostaglandins seem to have a predominantly stimulatory effect on bone resorption, although the exact mechanism is poorly understood. Ibuprofen, a nonsteroidal antiinflammatory drug, is known to inhibit the synthesis of prostaglandins. It is likely that ibuprofen, through its inhibition of prostaglandin synthesis, would decrease the localized osteoclastic bone resorption in a previously described animal model system. Mongolian gerbils were divided into three groups: low dose ibuprofen (10 mg/kg per day), high dose ibuprofen (30 mg/kg per day), and a control group. Following surgical implantation of catheters to the right bullae of each gerbil, pressure was applied for 8 days, stimulating osteoclastic bone resorption. After killing the animals and histomorphometric analysis of the bullae from each, comparisons were made between each group using osteoclast surface (percentage of bone area covered by osteoclasts), osteoclast number (number of osteoclasts/mm bone length), and osteoclast profile area (in μm²). Significantly lower osteoclast surface (Oc. S/BS) was found in pressurized bullae from both treatment groups when compared with pressurized bullae from controls (P<0.05) and significantly lower osteoclast number (N.Oc/T.L) in pressurized bullae from both treatment groups when compared with pressurized bullae from controls (P<0.05). These differences were found to be dose-dependent. No significant differences in individual osteoclast profile area were found in either treatment group when compared with controls.