Mineral Apposition Rate Research Articles

Mineral Apposition Rates in Coronal Dentine of Mandibular First Molars in Soay Sheep: Results of a Fluorochrome Labeling Study.

We studied the spatio-temporal variation of mineral apposition rate (MAR) in postnatally formed coronal dentine of mandibular first molars from Soay sheep repeatedly injected with different fluorochromes. MAR declined along the cuspal to cervical crown axis, and from early to late formed dentine, that is, from the dentine at the enamel-dentine-junction (EDJ) to the dentine adjacent to the dentine-pulp-interface (DPI). Highest mean MARs (about 21 µm/day) were recorded in cuspal dentine formed in the period of 28-42 days after birth. Lowest values (<2 µm/day) were recorded in late-formed (secondary) dentine close to the DPI. The high MARs recorded in the dentine of the cuspal crown portions enable the formation of a large tooth crown within a relatively short period of less than one year. The established MARs in the dentine of the different crown portions of sheep molars will allow a precise determination of the timing of stress events affecting dentine formation. They are also helpful for devising sampling protocols in studies of trace element or stable isotope distributions in sheep dentine aimed at assessing temporal variation of incorporation into forming dentine. Such data are useful in a variety of contexts, including, for example, the exposure to pollutants and the reconstruction of husbandry practices or feeding regimes. Anat Rec, 301:902-912, 2018. © 2017 Wiley Periodicals, Inc.

The protective effects of triptolide on age-related bone loss in old male rats

BackgroundPrevious studies have showed that triptolide have a critical role in inhibiting osteoclast formation, bone resorption and attenuating regional osteoporosis. However, the protective role of triptolide on age-related bone loss has not been investigated. In the study, we assessed the effect of triptolide supplementation on bone microstructure and bone remolding in old male rat lumbars. MethodsFifty-two 22-month-old male Sprague-Dawley rats were randomly assigned to either triptolide treatment group or control group. Triptolide (15 μg/kg/d) or normal saline was administered to the rats of assigned group for 8 weeks. Lumbar bone mineral density (BMD) and bone microstructure were analyzed by micro-CT. Fluorochrome labeling of the bones was performed to measure the mineral apposition rate (MAR) and bone formation rate (BFR). Osteoclast number was also measured by TRAP staining. Plasma level of osteocalcin and tartrate-resistant acid phosphatase 5b (Tracp 5b) was also analyzed. ResultsMicro-CT results revealed that triptolide-treated rats had significant higher BMD, bone volume over total volume (BV/TV), trabecular thickness (Tb.Th), bone trabecular number (Tb.N), and lower trabecular separation (Tb.Sp) compared to the control group. Although fluorochrome labeling result showed no significant difference in MAR and BFR between the groups, triptolide decreased osteoclast number in vivo. In addition, a significant higher level of plasma Tracp 5b was observed in the triptolide-treated rats. Furthermore, triptolide also reduced the expression of receptor for activation of NF-κB ligand (RANKL) and increased osteoprotegerin (OPG) expression in the lumbars. ConclusionThese results suggested that triptolide had a protective effect on age-related bone loss at least in part by reducing osteoclast number in elder rats. Therefore, triptolide might be a feasible therapeutic approach for senile osteoporosis.

Photoperiod-Induced Increases in Bone Mineral Apposition Rate in Siberian Hamsters and the Involvement of Seasonal Leptin Changes.

The adipokine leptin regulates energy balance, appetite, and reproductive maturation. Leptin also acts on bone growth and remodeling, but both osteogenic and anti-osteogenic effects have been reported depending on experimental conditions. Siberian hamsters (Phodopus sungorus) have natural variation in circulating leptin concentrations, where serum leptin is significantly decreased during the short day (SD)-induced winter state. In summer long day (LD) photoperiods, appetite and body adiposity increase with associated central leptin insensitivity. This natural change in leptin secretion was exploited to investigate leptin’s effect on bone growth. Hamsters were injected with calcium-chelating fluorescent dyes to measure bone mineral apposition rate (MAR). Measurements were initially obtained from 5-week and 6-month-old animals maintained in low leptin (SD) or high leptin (LD) states. A further study investigated effects of chronic administration of recombinant mouse leptin to hamsters housed in SD and LD conditions; growth plate thickness and bone density were also assessed. As expected, a reduction in body mass was seen in hamsters exposed to SD, confirming the phenotype change in all studies. Serum leptin concentrations were significantly reduced in SD animals in all studies. MAR was reproducibly and significantly increased in the femurs of SD animals in all studies. Vitamin D and growth plate thickness were significantly increased in SD animals at 6 months. No effect on bone density was observed in any study. Taken together these data suggest that bone growth is associated with the low leptin, winter, lean state. In leptin-treated animals, there was a significant interaction effect of leptin and photoperiod. In comparison to their vehicle counterparts, SD animals had decreased and LD animals had increased MAR, which was not apparent prior to leptin administration. In conclusion, increased MAR was associated with low serum leptin levels in early life and sustained over 6 months, implying that leptin has a negative effect on bone growth in this model. The unexpected finding that MAR increased after peripheral leptin administration in LD suggests that leptin exerts different effects on bone growth dependent on initial leptin status. This adds further weight to the hypothesis that leptin-treated LD animals display central leptin resistance.

Bis‑enoxacin blocks alveolar bone resorption in rats with ovariectomy‑induced osteoporosis.

Postmenopausal osteoporosis is a common systemic skeletal disease that is associated with estrogen‑deficiency. Bone loss associated with bisphosphonates therapy can increase the risk of developing oral osteonecrosis. Recent studies have indicated that enoxacin may inhibit osteoclast formation and bone resorption via a different mechanism from that of bisphosphonates. Therefore, the authors hypothesized that the use of an enoxacin such as bis‑enoxacin (BE) in association with bisphosphonates may be effective in the treatment of postmenopausal osteoporosis‑associated alveolar bone resorption and reduce the risk of oral osteonecrosis by allowing the dose of bisphosphonates to be reduced. A total of 30 6‑month‑old female Sprague‑Dawley rats were randomly assigned to five groups: The Sham, Vehicle, zoledronic acid (ZOL), low concentrations of BE (BE‑L) and high concentrations of BE (BE‑H) groups. The results demonstrated that the ZOL, BE‑L and BE‑H groups had an increased bone volume/tissue volume, trabecular thickness, mineral apposition rate, mineralizing surface/bone surface and a decreased trabecular separation when compared with the Vehicle group. The microscopic evaluation of histological sections clearly supported the results of the micro‑computed tomography. The number of tartrate‑resistant acid phosphatase‑positive osteoclasts was markedly decreased in the ZOL, BE‑L and BE‑H groups, indicating that BE may inhibit osteoclast formation. The anti‑resorptive effect in the BE‑H group was close to or better than that exhibited by the ZOL group; however, this effect was poorer in the BE‑L group. In conclusion, BE has the potential to block alveolar bone resorption resulting from ovariectomy‑induced osteoporosis in rats in a dose‑dependent manner.

Beneficial effects of low-level laser irradiation on senile osteoporosis in rats.

To investigate the effect of low-level laser irradiation (LLLI) on bone mineral density (BMD), bone structures, bone biomechanical properties and bone metabolism in senile osteoporosis, and to explore a relatively more secure and effective way to prevent and treat osteoporosis. Sprague-Dawley (SD) male rats at different age stages (4 months old, 12 months old and 20 months old) were selected and randomly divided into six groups. The rats in the treatment group were treated with LLLI for 12 weeks, and then the microstructure of bones was analyzed by micro-computed tomography (micro-CT) scanning. The biomechanical indexes of the femur were detected by the three-point bending test. Levels of the blood calcium (Ca)2+, blood phosphorus (P)3+, urine Ca, urine P and urine creatinine (CREA) were detected using an automatic biochemical analyzer. The contents of serum osteocalcin (OCN) and bone alkaline phosphatase (BAP) were measured by enzyme-linked immunosorbent assay (ELISA). The bone formation rate (BFR) was analyzed by double fluorescent labeling with calcein and tetracycline. Hematoxylin and eosin (HE) staining and toluidine blue staining were used to analyze the number of bone marrow osteoblasts and adipocytes. Micro-CT results showed that compared with those in the young group, the bone mineral density (BMD) in the old group was significantly decreased, and the trabecular microstructure was seriously damaged. LLLI could significantly enhance the BMD and improve the damage to the trabecular microstructure; the three-point bending test revealed that LLLI could significantly improve the biomechanical properties and enhance the mechanical strength of the femur in the old group; the biochemical analysis showed that LLLI could significantly reduce Ca and P losses and elevate the levels of serum BAP and OCN; the bone histomorphology analysis results indicated that LLLI could increase BFR and mineral apposition rate (MAR), increase the number of osteoblasts and decrease the number of adipocytes in the bone marrow in the old group. LLLI can effectively improve osteoporosis, increase BMD, improve bone structure and improve bone biomechanical properties in old rats; at the same time, it increases the levels of serum BAP and OCN and the number of osteoblasts in the bone marrow, suggesting that the osteogenesis function of osteoblasts is enhanced.

Bone adaptation to mechanical loading in a mouse model of reduced peripheral sensory nerve function

Underlying mechanisms contributing to the imbalance in bone turnover during osteoporosis remain only partially explained. Reduced sensory nerve function may contribute to this imbalance, as sensory neuropeptides affect the activity of osteoblasts and osteoclasts in vivo, especially during bone adaptation. In this study, we investigated bone adaptation in mice following two weeks of tibial compression (peak magnitude 3 N or 7 N). To induce decreased sensory nerve function, mice were treated with capsaicin as neonates. We hypothesized that decreased sensory nerve function would diminish the adaptation of bone to mechanical loading, assessed with μCT and dynamic histomorphometry. We found that tibial compression induced significant changes in cortical microarchitecture that depended on compression magnitude and location along the length of the tibia; in contrast, there was no effect of loading on trabecular bone of the tibial metaphysis. Tibial compression significantly increased periosteal, and decreased endosteal, bone formation. Contrary to our initial hypothesis, capsaicin-treated mice generally displayed a similar, if not larger, adaptive response to mechanical loading, including greater increases in bone mineral content and mineral apposition rate. To integrate mechanical loading of bone with sensory nerve activation, we examined whether concentration of the neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP) in bone were affected following 1 or 5 days of 5 N tibial compression or hindlimb unloading. We found that 1 day of tibial compression significantly increased CGRP concentrations in bone, and hindlimb unloading also exhibited a trend toward increased CGRP in bone. These results may suggest a role of sensory nerves in the bone adaptation response to the mechanical environment, though this remains unclear.

Effect of Deconditioning on Cortical and Cancellous Bone Growth in the Exercise Trained Young Rats

Exercise enhances bone growth and increases peak bone mass. The aim of this study was to determine whether or not 4 weeks of deconditioning after 8 weeks of exercise in growing rats would result in a decrease in bone gain or reverse the benefits of exercise. Fifty 4-week-old female Sprague-Dawley rats were randomized by a stratified weight method into 5 groups with 10 rats in each group: 8 weeks exercise (8EX), 8 weeks sedentary control (8S), 12 weeks exercise (12EX), 8 weeks exercise followed by 4 weeks sedentary (8EX4S), and 12 weeks sedentary control (12S). The exercise consisted of running on a treadmill with a 5 degrees slope at 24 m/minute for 1 h/day and 5 days/week. After each period of exercise, cancellous and cortical bone histomorphometry were performed on double fluorescent labeled 5-microm-thick sections of the proximal tibia and 40-microm-thick sections of the tibial shaft, respectively. Eight and 12 weeks of exercise resulted in a significant increase in the body weight and gastrocnemius muscle weight by two-way analysis of variance (ANOVA). The femoral wet weight (mg; mean +/- SD; 8EX, 781 +/- 45.1 vs. 8S, 713 +/- 40.5; p < 0.05; 12EX, 892 +/- 41.6 vs. 12S, 807 +/- 19.8; p < 0.05) was significantly higher in the exercise group than that in the respective control groups. The femoral wet weight and bone volume (BV) of the 8EX4S group (818 +/- 46.2 mg and 531 +/- 31.2 microl, respectively) were significantly lower than those of the 12EX group (p < 0.05) and did not differ significantly from those of the 12S groups. The cancellous BV was significantly higher in the 8EX and 12EX groups than that in the respective sedentary groups (p < 0.05). The cortical bone area of the tibial shaft was also significantly higher in the 12EX than that in the 12S group (p < 0.05). The increase in the cancellous BV or cortical bone area was caused by an increase in the mineral apposition rate (MAR), without a significant effect in the labeled perimeter. The bone formation rate (BFR; microm3/microm2 per day) in the cancellous bone (12EX, 27.9 +/- 7.74 vs. 12S, 15.4 +/- 4.56; p < 0.05) or periosteal surface (12EX, 127.6 +/- 27.7 vs. 12S, 79.5 +/- 18.6; p < 0.05) was significantly higher in the exercised groups than that in the respective control group (p < 0.05). Again, deconditioning resulted in a decrease in the cancellous BFR, BV, periosteal BFR, and cortical bone area to levels not significantly different from the 12S group. In conclusion, our findings showed that exercised growing rats, when deconditioned, lost the benefits gained through exercise and their bone parameters were reduced to levels not different from the sedentary control. Thus, continued exercise is required to maintain high bone mass.

Moderate-intensity 4 mT static magnetic fields prevent bone architectural deterioration and strength reduction by stimulating bone formation in streptozotocin-treated diabetic rats

Type 1 diabetes mellitus (T1DM) has been associated with deterioration of bone microarchitecture and strength, resulting in increased fracture risk. Substantial studies have revealed the capacity of moderate-intensity static magnetic fields (SMF) on promoting osteoblastogenesis in vitro and stimulating bone growth and bone regeneration in vivo, whereas it is unknown whether SMF can resist T1DM-associated osteopenia/osteoporosis. We herein investigated the potential effects of whole-body SMF exposure with 4mT on bone loss in streptozotocin-induced T1DM rats. We found that SMF exposure for 16weeks inhibited architectural deterioration of trabecular bone and cortical bone and mechanical strength reduction in T1DM rats, as evidenced by the MicroCT and 3-point bending findings. Our serum biochemical, bone histomorphometric and PCR results revealed that SMF induced higher serum osteocalcin, mineral apposition rate and osteoblast number of trabecular bone, and higher skeletal osteocalcin, BMP2 and Runx2 gene expression in T1DM rats, whereas SMF showed no significant alteration in serum CTX, skeletal osteoclast number, or osteoclastogenesis-related RANKL-RANK signaling gene expression. Together, our findings suggest that moderate SMF prevented bone architectural deterioration and strength reduction by inhibiting the reduction of bone formation in T1DM rats, and indicate that SMF might become a promising biophysical countermeasure for T1DM-related osteopenia/osteoporosis.

High dose teriparatide (rPTH1-34) therapy increases callus volume and enhances radiographic healing at 8-weeks in a massive canine femoral allograft model.

Small animal studies have demonstrated significant high-dose recombinant parathyroid hormone1-34 (rPTH1-34) effects on intercalary allograft healing. Towards a human adjuvant therapy to decrease non-unions, we evaluated rPTH1-34 safety and efficacy in a clinically relevant canine femoral allograft model. Adult female mongrel hounds (n = 20) received a 5cm mid-diaphyseal osteotomy reconstructed with a plated allograft, and were randomized to: 1) Placebo (n = 5; daily saline), 2) Continuous rPTH1-34 (n = 7; 5 μg/kg/day s.c. from day 1–55 post-op), or 3) Delayed rPTH1-34 (n = 8; 5 μg/kg/day s.c. from day 14–28 post-op). Safety was assessed by physical behavior and blood calcium monitoring. Cone beam CT (CB-CT) was performed on days 14, 28 and 56 post-op to assess 2D cortical healing, 3D bone volume, and Union Ratio. Biomechanical testing and dynamic histomorphometry were also performed. The high drug dose was poorly tolerated, as most dogs receiving rPTH1-34 had to be given intravenous saline, and one dog died from hypercalcemia. Continuous rPTH1-34 significantly increased 2D healing and callus volumes at 4-weeks versus Placebo, and sustained the significant increase in cortical union at 8-week (p<0.05). These rPTH1-34 effects were confirmed by histomorphometry, revealing significant increases in mineral apposition rates (MAR) on host bone and graft-host junctions (p<0.05). Delayed rPTH1-34 significantly increased callus volume and MAR at 8 weeks (p<0.05). Although no biomechanical differences were observed, as expected for early healing, the results demonstrated that 2D RUST scoring significantly correlated with torsional biomechanics (p<0.01). In conclusion, 8-weeks of intermittent high-dose rPTH1-34 treatment significantly increases callus formation and accelerates bony union of intercalary massive allografts in a clinically relevant canine model, but with serious side-effects from hypercalcemia.

Low-1 level mechanical vibration improves bone microstructure, tissue mechanical properties and porous titanium implant osseointegration by promoting anabolic response in type 1 diabetic rabbits

Type 1 diabetes mellitus (T1DM) is associated with reduced bone mass, increased fracture risk, and impaired bone defect regeneration potential. These skeletal complications are becoming important clinical challenges due to the rapidly increasing T1DM population, which necessitates developing effective treatment for T1DM-associated osteopenia/osteoporosis and bone trauma. This study aims to investigate the effects of whole-body vibration (WBV), an easy and non-invasive biophysical method, on bone microstructure, tissue-level mechanical properties and porous titanium (pTi) osseointegration in alloxan-diabetic rabbits. Six non-diabetic and twelve alloxan-treated diabetic rabbits were equally assigned to the Control, DM, and DM with WBV stimulation (WBV) groups. A cylindrical drill-hole defect was established on the left femoral lateral condyle of all rabbits and filled with a novel non-toxic Ti2448 pTi. Rabbits in the WBV group were exposed to 1h/day WBV (0.3g, 30Hz) for 8weeks. After sacrifice, the left femoral condyles were harvested for histological, histomorphometric and nanoindentation analyses. The femoral sample with 2-cm height above the defect was used for qRT-PCR analysis. The right distal femora were scanned with μCT. We found that all alloxan-treated rabbits exhibited hyperglycemia throughout the experimental period. WBV inhibited the deterioration of cancellous and cortical bone architecture and tissue-level mechanical properties via μCT, histological and nanoindentation examinations. T1DM-induced reduction of bone formation was inhibited by WBV, as evidenced by elevated serum OCN and increased mineral apposition rate (MAR), whereas no alteration was observed in bone resorption marker TRACP5b. WBV also stimulated more adequate ingrowths of mineralized bone tissue into pTi pore spaces, and improved peri-implant bone tissue-level mechanical properties and MAR in T1DM bone defects. WBV mitigated the reductions in femoral BMP2, OCN, Wnt3a, Lrp6, and β-catenin and inhibited Sost mRNA expression but did not alter RANKL or RANK gene expression in T1DM rabbits. Our findings demonstrated that WBV improved bone architecture, tissue-level mechanical properties, and pTi osseointegration by promoting canonical Wnt signaling-mediated skeletal anabolic response. This study not only advances our understanding of T1DM skeletal sensitivity in response to external mechanical cues but also offers new treatment alternatives for T1DM-associated osteopenia/osteoporosis and osseous defects in an economic and highly efficient manner.

Treatment With a Soluble Bone Morphogenetic Protein Type 1A Receptor (BMPR1A) Fusion Protein Increases Bone Mass and Bone Formation in Mice Subjected to Hindlimb Unloading.

ABSTRACTPrevious work has shown that the soluble murine BMPR1A–fusion protein (mBMPR1A‐mFc) binds to BMP2 and BMP4 with high affinity, preventing downstream signaling. Further, treatment of intact and ovariectomized mice with mBMPR1A‐mFc leads to increased bone mass, and improved bone microarchitecture and strength, via increased bone formation and reduced resorption. In this study, we tested the effects of mBMPR1A‐mFc on disuse‐induced bone loss caused by 21 days of hindlimb unloading (HLU) via tail suspension versus cage controls (CONs). Adult female C57BL/6J mice (12 weeks old) were assigned to one of four groups (n = 10 each): CON‐VEH; CON‐mBMPR1A‐mFc; HLU‐VEH; and HLU‐mBMPR1A‐mFc. Mice were injected subcutaneously with VEH or mBMPR1A‐mFc (4.5 mg/kg, 2×/week). Leg BMD declined in the HLU‐VEH group (–5.3% ± 1.3%), whereas it was unchanged in HLU‐mBMPR1A‐mFc (–0.3% ± 0.9%, p < 0.05 versus HLU‐VEH). Leg BMD increased significantly more in CON‐mBMPR1A‐mFc than CON‐VEH (10.2% ± 0.6% versus 4.4% ± 0.8%). In the femur, trabecular, and cortical bone microarchitecture was worse in the HLU‐VEH compared to CON‐VEH mice, whereas mBMPR1A‐mFc treatment for 3 weeks led to greater Tb.BV/TV, Tb.Th, and midshaft Ct.Th in both the HLU and CON groups compared to comparable VEH‐treated counterparts (p < 0.05). HLU‐mBMPR1A‐mFc mice also had 21% greater failure load (p < 0.05) compared to their VEH‐treated counterparts. Dynamic histomorphometry indicated that treatment with mBMPR1A‐mFc led to significantly greater mineralizing surface and mineral apposition rate, resulting in a 3.5‐fold and fivefold higher bone formation rate in the mBMPR1A‐mFc‐treated CON and HLU animals versus VEH groups, respectively. mBMPR1A‐mFc‐treated mice had a similar osteoblast surface but significantly lower osteoclast surface than VEH‐treated animals in both the CON and HLU groups. Altogether, these findings suggest that treatment with the soluble BMPR1A fusion protein may be useful for maintenance of skeletal integrity in the setting of disuse‐induced bone loss. © 2017 The Authors JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Effects of Nano-Hydroxyapatite/Polyetheretherketone-Coated, Sandblasted, Large-Grit, and Acid-Etched Implants on Inflammatory Cytokines and Osseointegration in a Peri-Implantitis Model in Beagle Dogs.

BackgroundThis study explored the effects of nano-hydroxyapatite/polyetheretherketone (n-HA/PEEK)-coated sandblasted, large-grit, and acid-etched (SLA) implants on inflammatory cytokines and osseointegration in peri-implantitis model beagle dogs.Material/MethodsPeri-implantitis models were established. Eight beagle dogs were randomly and evenly assigned into SLA tied, SLA + n-HA/PEEK tied, SLA untied, or SLA + n-HA/PEEK untied groups. A special periodontal probe was used to detect the plaque index (PLI), probing depth (PD), and modified Sulcus Bleeding Index (mSBI). Gingival crevicular fluid was collected and an ELISA kit was utilized to detect IL-1, IL-6, and IL-17 levels. The colony-forming units were counted and the maximum shear strength of implants was tested using the axial pullout test. HE staining was used to detect the inflammation of peri-implant bone tissues. Osseointegration was observed through toluidine blue staining. Bone-to-implant contact (BIC) was obtained through histological observation and the mineral apposition rate (MAR) was calculated after immune fluorescent double staining.ResultsThe SLA tied group demonstrated higher levels of PLI, PD, mSBI, IL-1, IL-6, and IL-17 and a higher degree of inflammation than the SLA + n-HA/PEEK tied group. The tied groups also displayed similar results over the untied groups at the same time point. The maximum shear strength, BIC, and MAR in the SLA tied group were significantly lower than in the SLA + n-HA/PEEK tied group.ConclusionsOur findings demonstrate that SLA + n-HA/PEEK implants can promote osseointegration and relieve the inflammation response of peri-implantitis in beagle dogs.

Clcn7F318L/+ as a new mouse model of Albers-Schönberg disease

Dominant negative mutations in CLCN7, which encodes a homodimeric chloride channel needed for matrix acidification by osteoclasts, cause Albers-Schönberg disease (also known as autosomal dominant osteopetrosis type 2). More than 25 different CLCN7 mutations have been identified in patients affected with Albers-Schönberg disease, but only one mutation (Clcn7G213R) has been introduced in mice to create an animal model of this disease. Here we describe a mouse with a different osteopetrosis-causing mutation (Clcn7F318L). Compared to Clcn7+/+ mice, 12-week-old Clcn7F318L/+ mice have significantly increased trabecular bone volume, consistent with Clcn7F318L acting as a dominant negative mutation. Clcn7F318L/F318L and Clcn7F318L/G213R mice die by 1month of age and resemble Clcn7 knockout mice, which indicate that p.F318L mutant protein is non-functional and p.F318L and p.G213R mutant proteins do not complement one another. Since it has been reported that treatment with interferon gamma (IFN-G) improves bone properties in Clcn7G213R/+ mice, we treated Clcn7F318L/+ mice with IFN-G and observed a decrease in osteoclast number and mineral apposition rate, but no overall improvement in bone properties. Our results suggest that the benefits of IFN-G therapy in patients with Albers-Schönberg disease may be mutation-specific.

RETRACTED ARTICLE: Tridax procumbens flavonoids: a prospective bioactive compound increased osteoblast differentiation and trabecular bone formation

BackgroundThe Tridax procumbens extracts (TPE) are known for their ethno-medicinal properties to increase osteogenic functioning in mesenchymal stem cells. Recently, we found that the T. procumbens flavonoids (TPF) significantly suppressed the RANKL-induced osteoclasts differentiation and bone resorption. The TPF also promoted osteoblasts differentiation and bone formation demonstrated by increasing bone formation markers in cultured mouse primary osteoblasts. However, the effects of the TPF on in vivo bone formation remain unclear. In this study, we investigated the effects of the TPF on in vivo bone formation, injected the TPF (20 mg/kg) twice a day in the low calcium diet mice and killed them after 21 day. Radiographic and histomorphometric analyses were performed on the dissected bones to determine the anabolic effects of the TPF.ResultsBone mineral density and bone mineral content of the TPF-treated mice were significantly increased compared to the control mice. Bone formation-related indices like osteoblast number, osteoblast surface, bone volume, mineralizing surface, mineral apposition rate and bone formation rate were significantly increased in the TPF-treated mice compared to the control mice.ConclusionOur findings point towards the stimulation of bone formation by TPF, suggested that the TPF could be a potential natural anabolic agent to treat patients with bone loss-associated diseases such as osteoporosis.

Effects of low-intensity pulsed electromagnetic fields on bone microarchitecture, mechanical strength and bone turnover in type 2 diabetic db/db mice

Type 2 diabetic patients have impaired bone quality, leading to increased fracture risk. Substantial evidence demonstrates that pulsed electromagnetic fields (PEMF) could resist osteopenia/osteoporosis induced by estrogen deficiency and disuse. However, the effects of PEMF on osteopenia/osteoporosis associated with diabetes, especially for more prevalent type 2 diabetes, remain poorly understood. We herein investigated the skeletal effects and mechanisms of PEMF (15 Hz, 20 Gs) on leptin receptor-deficient db/db mice with typical type 2 diabetic symptoms. Our µCT results showed that 12-week PEMF exposure significantly improved both cancellous and cortical bone microarchitecture in db/db mice. Three-point bending and biomechanical indentation testing demonstrated that PEMF improved whole-bone structural properties and tissue-level material properties in db/db mice. PEMF significantly promoted bone formation in db/db mice evidenced by increased serum osteocalcin and bone mineral apposition rate, whereas PEMF exerted no observable alteration in bone resorption. Real-time PCR showed that PEMF upregulated tibial gene expression of osteoblastogenesis-related of canonical Wnt/β-catenin signaling but not osteoclastogenesis-related RANKL-RANK signaling in db/db mice. Our findings demonstrate that PEMF improved bone quantity and quality with obvious anabolic activities in db/db mice, and imply that PEMF might become a clinically applicable treatment modality for improving bone quality in type 2 diabetic patients.

Bone turnover is altered in transgenic rats overexpressing the P2Y2 purinergic receptor.

It is now widely recognized that purinergic signaling plays an important role in the regulation of bone remodeling. One receptor subtype, which has been suggested to be involved in this regulation, is the P2Y2 receptor (P2Y2R). In the present study, we investigated the effect of P2Y2R overexpression on bone status and bone cell function using a transgenic rat. Three-month-old female transgenic Sprague Dawley rats overexpressing P2Y2R (P2Y2R-Tg) showed higher bone strength of the femoral neck. Histomorphometry showed increase in resorptive surfaces and reduction in mineralizing surfaces. Both mineral apposition rate and thickness of the endocortical osteoid layer were higher in the P2Y2R-Tg rats. μCT analysis showed reduced trabecular thickness and structural model index in P2Y2R-Tg rats. Femoral length was increased in the P2Y2R-Tg rats compared to Wt rats. In vitro, there was an increased formation of osteoclasts, but no change in total resorption in cultures from P2Y2R-Tg rats. The formation of mineralized nodules was significantly reduced in the osteoblastic cultures from P2Y2R-Tg rats. In conclusion, our study suggests that P2Y2R is involved in regulation of bone turnover, due to the effects on both osteoblasts and osteoclasts and that these effects might be relevant in the regulation of bone growth.

Inhibition of FGFR Signaling Partially Rescues Hypophosphatemic Rickets in HMWFGF2 Tg Male Mice.

Transgenic mice harboring high molecular weight fibroblast growth factor (FGF)2 isoforms (HMWTg) in osteoblast lineage cells phenocopy human X-linked hypophosphatemic rickets (XLH) and Hyp murine model of XLH demonstrating increased FGF23/FGF receptor signaling and hypophosphatemic rickets. Because HMWFGF2 was upregulated in bones of Hyp mice and abnormal FGF receptor (FGFR) signaling is important in XLH, HMWTg mice were used to examine the effect of the FGFR inhibitor NVP-BGJ398, now in clinical trials for cancer therapy, on hypophosphatemic rickets. Short-term treatment with NVP-BGJ398 rescued abnormal FGFR signaling and hypophosphatemia in HMWTg. Long-term treatment with NVP-BGJ398 normalized tail, tibia, and femur length. Four weeks NVP-BGJ398 treatment significantly increased total body bone mineral density (BMD) and bone mineral content (BMC) in HMWTg mice; however, at 8 weeks, total body BMD and BMC was indistinguishable among groups. Micro-computed tomography revealed decreased vertebral bone volume, trabecular number, and increased trabecular spacing, whereas femur trabecular tissue density was increased; however, NVP-BGJ398 rescued defective cortical bone mineralization, increased thickness, reduced porosity, and increased endosteal perimeter and cortical tissue density in HMWTg. NVP-BGJ398 improved femur cancellous bone, cortical bone structure, growth plate, and double labeling in cortical bone and also increased femur trabeculae double labeled surface, mineral apposition rate, bone formation rate, and osteoclast number and surface in HMWTg. The decreased NPT2a protein that is important for renal phosphate excretion was rescued by NVP-BGJ398 treatment. We conclude that NVP-BGJ398 partially rescued hypophosphatemic rickets in HMWTg. However, long-term treatment with NVP-BGJ398 further increased serum FGF23 that could exacerbate the mineralization defect.

Stanozolol-soaked grafts enhance new bone formation in rat calvarial critical-size defects

Androgen hormones play a significant role in regulating bone morphogenesis and in maintaining bone homeostasis throughout life. This study aimed to investigate the local effects of the non-aromatizable androgen stanozolol (ST) on bone regeneration in rats. Bilateral critical-size defects were created in the parietal bone of 26 male Wistar rats: the defect on one side was filled with a deproteinized bovine bone scaffold (DBB) soaked in ST solution (test) and the contralateral with DBB alone (control). Samples were collected at one month and three months. Histomorphometry revealed a significantly higher new bone formation (NB) (24.41% ± 4.14% versus 15.01% ± 2.43%, p < 0.05) and mineral apposition rate (MAR) (9.20 μm/day ± 0.37 versus 6.50 μm/day ± 1.09, p < 0.05) in the test versus control group at one month. Accordingly, real time-polymerase chain reaction revealed a consistently higher Runx2 expression in test samples (fold change test/control: 4.50 ± 1.17, p ≤ 0.05). No morphometrical differences between groups were detected at three months (p > 0.05). However, test samples were characterized by an increase in blood capillary density from one month (11.43 n mm−2 ± 2.01) to three months (28.26 n mm−2 ± 5.62), providing evidence of a vital remodeling tissue. Control samples presented a decrease of anti-Osterix (SP7)/anti-osteocalcin (BGLAP) (3.9 n mm−2 ± 0.32 versus 1.01 n mm−2 ± 0.20) and alkaline phosphatase (ALP) (12.14 n mm−2 ± 6.29 versus 6.29 n mm−2 ± 2.73) immunohistochemical-positive elements, which was suggestive of a stabilized healing phase. Based on these observations, local ST administration boosted bone regeneration in rat calvarial critical-size defects at one month. This study showed the potential of local steroid delivery in bone regeneration.

Reduced bone loss in a murine model of postmenopausal osteoporosis lacking complement component 3.

The growing field of osteoimmunology seeks to unravel the complex interdependence of the skeletal and immune systems. Notably, we and others have demonstrated that complement signaling influences the differentiation of osteoblasts and osteoclasts, the two primary cell types responsible for maintaining bone homeostasis. However, the net effect of complement on bone homeostasis in vivo was unknown. Our published in vitro mechanistic work led us to hypothesize that absence of complement component 3 (C3), a central protein in the complement activation cascade, protects against bone loss in the ovariectomy-based model of postmenopausal osteoporosis. Indeed, we report here that, when compared to their C57BL/6J (WT) counterparts, ovariectomized C3 deficient mice experienced reduced bone loss at multiple sites and increased stiffness at the femoral neck, the latter potentially improving mechanical function. WT and B6;129S4-C3tm1Crr /J (C3-/- ) mice were either ovariectomized or sham-operated at 6 weeks of age and euthanized at 12 weeks. MicroCT on harvested bones revealed that the trabecular bone volume fraction in the metaphyses of both the proximal tibiae and distal femora of ovariectomized C3-/- mice is significantly greater than that of their WT counterparts. Lumbar vertebrae showed significantly greater osteoid content and mineral apposition rates. Mechanical testing demonstrated significantly greater stiffness in the femoral necks of ovariectomized C3-/- mice. These results demonstrate that C3 deficiency reduces bone loss at ovariectomy and may improve mechanical properties. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:118-128, 2018.

Sustained Notch2 signaling in osteoblasts, but not in osteoclasts, is linked to osteopenia in a mouse model of Hajdu-Cheney syndrome

Individuals with Hajdu-Cheney syndrome (HCS) present with osteoporosis, and HCS is associated with NOTCH2 mutations causing deletions of the proline-, glutamic acid-, serine-, and threonine-rich (PEST) domain that are predicted to enhance NOTCH2 stability and cause gain-of-function. Previously, we demonstrated that mice harboring Notch2 mutations analogous to those in HCS (Notch2HCS) are severely osteopenic because of enhanced bone resorption. We attributed this phenotype to osteoclastic sensitization to the receptor activator of nuclear factor-κB ligand and increased osteoblastic tumor necrosis factor superfamily member 11 (Tnfsf11) expression. Here, to determine the individual contributions of osteoclasts and osteoblasts to HCS osteopenia, we created a conditional-by-inversion (Notch2COIN ) model in which Cre recombination generates a Notch2ΔPEST allele expressing a Notch2 mutant lacking the PEST domain. Germ line Notch2COIN inversion phenocopied the Notch2HCS mutant, validating the model. To activate Notch2 in osteoclasts or osteoblasts, Notch2COIN mice were bred with mice expressing Cre from the Lyz2 or the BGLAP promoter, respectively. These crosses created experimental mice harboring a Notch2ΔPEST allele in Cre-expressing cells and control littermates expressing a wild-type Notch2 transcript. Notch2COIN inversion in Lyz2-expressing cells had no skeletal consequences and did not affect the capacity of bone marrow macrophages to form osteoclasts in vitro In contrast, Notch2COIN inversion in osteoblasts led to generalized osteopenia associated with enhanced bone resorption in the cancellous bone compartment and with suppressed endocortical mineral apposition rate. Accordingly, Notch2 activation in osteoblast-enriched cultures from Notch2COIN mice induced Tnfsf11 expression. In conclusion, introduction of the HCS mutation in osteoblasts, but not in osteoclasts, causes osteopenia.