Sweep imaging with Fourier transform as a tool with MRI for evaluating the effect of teriparatide on cortical bone formation in an ovariectomized rat model

Background Teriparatide (TPTD) is a drug for osteoporosis that promotes bone formation and improves bone quality. However, the effects of TPTD on cortical bone are not well understood. Sweep imaging with Fourier transform (SWIFT) has been reported as a useful tool for evaluating bound water of cortical bone, but it has yet to be used to investigate the effects of TPTD on cortical bone. This study aimed to evaluate the consequences of the effect of TPTD on cortical bone formation using SWIFT. Methods Twelve-week-old female Sprague-Dawley rats (n = 36) were reared after ovariectomy to create a postmenopausal osteoporosis model. They were divided into two groups: the TPTD and non-TPTD groups. Rats were euthanized at 4, 12, and 24 weeks after initiating TPTD treatment. Tibial bones were evaluated using magnetic resonance imaging (MRI) and bone histomorphometry. In MRI, proton density-weighted imaging (PDWI) and SWIFT imaging were performed. The signal-to-noise ratio (SNR) was calculated for each method. The same area evaluated by MRI was then used to calculate the bone formation rate by bone histomorphometry. Measurements were compared using the Mann-Whitney U-test, and a P-value of < 0.05 was considered significant. Results PDWI-SNR was not significantly different between the two groups at any time point (P = 0.589, 0.394, and 0.394 at 4, 12, and 24 weeks, respectively). Contrarily, SWIFT-SNR was significantly higher in the TPTD group than in the non-TPTD group at 4 weeks after initiating treatment, but it was not significantly different at 12 and 24 weeks (P = 0.009, 0.937, and 0.818 at 4, 12, and 24 weeks, respectively). The bone formation rate assessed by histomorphometry was significantly higher in the TPTD group than in the non-TPTD group at all timepoints (P < 0.05, all weeks). In particular, at 4 weeks, the bone formation rate was markedly higher in the TPTD group than in the non-TPTD group (P = 0.028, 1.98 ± 0.33 vs. 0.09 ± 0.05 μm3/μm2/day). Conclusions SWIFT could detect increased signals of bound water, reflecting the effect of TPTD on the cortical bone. The signal detected by SWIFT reflects a marked increase in the cortical bone formation rate.

Low bone turnover is associated with plain X-ray vascular calcification in predialysis patients

Background Vascular calcification (VC) is a common finding in chronic kidney disease (CKD) patients and predicts subsequent cardiovascular morbidity and mortality in this population. Vascular calcification is linked to disordered mineral metabolism and has been associated with bone histomorphometry changes in CKD. However, data on predialysis patients is scarce. Methods A cross-sectional study was conducted on a cohort of 56 CKD patients not yet on dialysis, who underwent a transiliac bone biopsy for histomorphometric evaluation after double tetracycline labeling. Patients had no previous exposure to calcium salts, vitamin D agents, steroids or bisphosphonates. Vascular calcification was assessed at the time of biopsy, using Kauppila (plain X-ray of the lateral lumbar spine) and Adragão (plain X-ray of the pelvis and hands) scores. Results Vascular calcification was seen in two-thirds of the cohort. Subjects with VC were more likely to be male and have diabetes, and had significantly higher sclerostin and osteoprotegerin circulating levels than those without VC. The histomorphometric analysis showed that bone formation rate was significantly lower in VC compared to non-VC patients. In the multivariable logistic regression analysis, bone formation rate was independently associated with the presence of VC. Conclusions Vascular calcification is highly prevalent in predialysis patients, especially in those with diabetes. The independent association between bone formation rate and VC provides evidence of an important interaction between bone and vessel in CKD. Our results suggest that low bone turnover is a non-traditional risk factor for cardiovascular disease in predialysis patients.

NMUR1 in the NMU-Mediated Regulation of Bone Remodeling

Neuromedin-U (NMU) is an evolutionarily conserved peptide that regulates varying physiologic effects including blood pressure, stress and allergic responses, metabolic and feeding behavior, pain perception, and neuroendocrine functions. Recently, several lines of investigation implicate NMU in regulating bone remodeling. For instance, global loss of NMU expression in male and female mice leads to high bone mass due to elevated bone formation rate with no alteration in bone resorption rate or observable defect in skeletal patterning. Additionally, NMU treatment regulates the activity of osteoblasts in vitro. The downstream pathway utilized by NMU to carry out these effects is unknown as NMU signals via two G-protein-coupled receptors (GPCRs), NMU receptor 1 (NMUR1), and NMU receptor 2 (NMUR2), and both are expressed in the postnatal skeleton. Here, we sought to address this open question and build a better understanding of the downstream pathway utilized by NMU. Our approach involved the knockdown of Nmur1 in MC3T3-E1 cells in vitro and a global knockout of Nmur1 in vivo. We detail specific cell signaling events (e.g., mTOR phosphorylation) that are deficient in the absence of NMUR1 expression yet trabecular bone volume in femora and tibiae of 12-week-old male Nmur1 knockout mice are unchanged, compared to controls. These results suggest that NMUR1 is required for NMU-dependent signaling in MC3T3-E1 cells, but it is not required for the NMU-mediated effects on bone remodeling in vivo. Future studies examining the role of NMUR2 are required to determine the downstream pathway utilized by NMU to regulate bone remodeling in vivo.

Epo/EpoR signaling in osteoprogenitor cells is essential for bone homeostasis and Epo-induced bone loss

High erythropoietin (Epo) levels are detrimental to bone health in adult organisms. Adult mice receiving high doses of Epo lose bone mass due to suppressed bone formation and increased bone resorption. In humans, high serum Epo levels are linked to fractures in elderly men. Our earlier studies indicated that Epo modulates osteoblast activity; however, direct evidence that Epo acts via its receptor (EpoR) on osteoblasts in vivo is still missing. Here, we created mice lacking EpoR in osteoprogenitor cells to specifically address this gap. Deletion of EpoR in osteoprogenitors (EpoR:Osx-cre, cKO) starting at 5 weeks of age did not alter red blood cell parameters but increased vertebral bone volume by 25% in 12-week-old female mice. This was associated with low bone turnover. Histological (osteoblast number, bone formation rate) and serum (P1NP, osteocalcin) bone formation parameters were all reduced, as were the number of osteoclasts and TRAP serum level. Differentiation of osteoblast precursors isolated from cKO versus control mice resulted in lower expression of osteoblast marker genes including Runx2, Alp, and Col1a1 on day 21, whereas the mineralization capacity was similar. Moreover, the RANKL/OPG ratio, which determines the osteoclast-supporting potential of osteoblasts, was substantially decreased by 50%. Similarly, coculturing cKO osteoblasts with control or cKO osteoclast precursors produced significantly fewer osteoclasts than coculture with control osteoblasts. Finally, exposing female mice to Epo pumps (10 U·d−1) for 4 weeks resulted in trabecular bone loss (−25%) and increased osteoclast numbers (1.7-fold) in control mice only, not in cKO mice. Our data show that EpoR in osteoprogenitors is essential in regulating osteoblast function and osteoblast-mediated osteoclastogenesis via the RANKL/OPG axis. Thus, osteogenic Epo/EpoR signaling controls bone mass maintenance and contributes to Epo-induced bone loss.

Bone Structural, Biomechanical and Histomorphometric Characteristics of the Postcranial Skeleton of the Marsh Rice Rat (Oryzomys palustris)

INTRODUCTIONThe rice rat (Oryzomys palustris) is a non-conventional laboratory rodent species used to model some human bone disorders. However, no studies have been conducted to characterize the postcranial skeleton. Therefore, we aimed to investigate age- and gender-related features of the appendicular skeleton of this species.METHODSWe used femurs and tibiae from 94 rats of both genders aged 4-28 wks. Bone mineral content (BMC), bone mineral density (BMD), and biomechanical properties were determined in femurs. In addition, bone histomorphometry of tibiae was conducted to assess bone cells activities and bone turnover over time.RESULTSBodyweight, bone length, total metaphysis BMC/BMD, cortical BMC/BMD, cortical thickness, and cortical area progressively augmented with age. Whereas the increase in these parameters plateaued at age 16-22 wks in female rats, they continued to rise to age 28 wks in male rats. Furthermore, bone strength parameters increased with age, with few differences between genders. We also observed a rapid decrease in longitudinal growth between ages 4-16 wks. Whereas young rats had a greater bone formation rate and bone turnover, older rice rats had greater bone volume and trabecular thickness, with no differences between genders.CONCLUSIONS1) Sexual dimorphism in the rice rat becomes grossly evident at age 16 wks; 2) the age-related increases in bone mass, structural cortical parameters, and in some biomechanical property parameters plateau at an older age in male than in female rats; and 3) bone growth and remodeling significantly decreased with age indistinctive of the gender.

Sweep Imaging with Fourier Transform as a Tool for Evaluating the Effect of Teriparatide on Cortical Bone Turnover in an Ovariectomized Rat Model

Background: Teriparatide (TPTD) is a drug for osteoporosis that promotes bone formation and improves bone turnover. However, the specific effects of TPTD on cortical bone are not well understood. Sweep imaging with Fourier transform (SWIFT) has been reported as a useful tool for evaluating cortical bone, but it has yet to be used to investigate the effects of TPTD on cortical bone. This study aimed to evaluate the effects of TPTD on cortical bone turnover using SWIFT in rats. Methods: Twelve-week-old female Sprague-Dawley rats (n=36) were reared for 12 weeks after ovariectomy to create a postmenopausal osteoporosis model. They were divided into two groups: the TPTD and non-TPTD groups. Rats were euthanized at 4, 12, and 24 weeks after initiating TPTD treatment. Tibial bones were extracted and evaluated using magnetic resonance imaging (MRI) and bone histomorphometry. In MRI, proton density-weighted imaging (PDWI) and SWIFT imaging were performed. The signal-to-noise ratio (SNR) was calculated for each method. The same area evaluated by MRI was then used to calculate for the bone formation rate by bone histomorphometry . Measurements were compared using the Mann-Whitney U-test, and a P-value of <0.05 was considered significant. Results: PDWI-SNR was not significantly different between the two groups at any time point (P = 0.589, 0.394, and 0.394 at 4, 12, and 24 weeks, respectively). Contrarily, SWIFT-SNR was significantly higher in the TPTD group than in the non-TPTD group at 4 weeks after initiating treatment, but it was not significantly different at 12 and 24 weeks (P = 0.009, 0.937, and 0.818 at 4, 12, and 24 weeks, respectively). The bone formation rate was significantly higher in the TPTD group than in the non-TPTD group at all timepoints (P < 0.05, all weeks). In particular, at 4 weeks, the bone formation rate was markedly higher in the TPTD group than in the non-TPTD group (1.98±0.33 vs. 0.09±0.05 μm3/μm2/day).Conclusions: The marked increase of the bone formation rate in the cortical bone by TPTD could be measured using SWIFT. SWIFT could be an effective tool for evaluating the effects of TPTD on cortical bone turnover as images.

Skeletal Effects of Bone-Targeted TGFbeta Inhibition in a Mouse Model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease that is frequently associated with secondary osteoporosis. Previous studies have shown that TGFbeta inactivating antibody improves the muscle phenotype in mdx mice, a model of DMD. In the present study, we assessed the skeletal effects of treatment with a bone-targeted TGFbeta antibody (PCT-011) in mdx mice. Micro-computed tomography showed that 8 weeks of intraperitoneal administration of PCT-011 (10 mg per kg body mass, 3 times per week) was associated with more than twofold higher trabecular bone volume at the distal femur, which was explained by a higher trabecular number. At the femoral midshaft, PCT-011 exposure increased cortical thickness but did not significantly affect the results of three-point bending tests. Histomorphometric analyses of the lumbar vertebra 4 showed that PCT-011 treatment led to a lower bone formation rate. In conclusion, treatment with the TGFbeta antibody PCT-011 had a positive effect on bone development in mdx mice. Inhibiting TGFbeta activity thus appears to be a promising approach to treat bone fragility in the context of DMD.

Effects of Autologous Bone Marrow Mesenchymal Stem Cells and Platelet-Rich Plasma on Bone Regeneration and Osseointegration of A Hydroxyapatite-Coated Titanium Implant

Bone regeneration remains one of the major clinical needs in orthopedics, and advanced and alternative strategies involving bone substitutes, cells, and growth factors (GFs) are mandatory. The purpose of this study was to evaluate whether the association of autologous bone marrow mesenchymal stem cells (BMSC), isolated by ‘one-step surgical procedure’, and activated platelet rich plasma (PRP) improves osseointegration and bone formation of a hydroxyapatite-coated titanium (Ti-HA) implant, already in clinical use, in a rabbit cancellous defect. The GFs present in plasma, in inactivated and activated PRP were also tested. At 2 weeks, histology and histomorphometry highlighted increased bone-to-implant contact (BIC) in Ti-HA combined with BMSC and PRP in comparison to Ti-HA alone and Ti-HA + PRP. The combined effect of BMSC and PRP peaked at 4 weeks where the BIC value was higher than all other treatments. At both experimental times, newly formed bone (Trabecular Bone Volume, BV/TV) in all tested treatments showed increased values in comparison to Ti-HA alone. At 4 weeks Ti-HA + PRP + BMSC showed the highest BV/TV and the highest osteoblasts number; additionally, a higher osteoid surface and bone formation rate were found in Ti-HA + BMSC + PRP than in all other treatments. Finally, the analyses of GFs revealed higher values in the activated PRP in comparison to plasma and to non-activated PRP. The study suggests that the combination of autologous activated PRP, as a carrier for BMSCs, is a promising regenerative strategy for bone formation, osseointegration, and mineralization of bone implants.

Short-term glucocorticoid excess blunts abaloparatide-induced increase in femoral bone mass and strength in mice

Glucocorticoids (GCs), such as prednisolone, are widely used to treat inflammatory diseases. Continuously long-term or high dose treatment with GCs is one of the most common causes of secondary osteoporosis and is associated with sarcopenia and increased risk of debilitating osteoporotic fragility fractures. Abaloparatide (ABL) is a potent parathyroid hormone-related peptide analog, which can increase bone mineral density (aBMD), improve trabecular microarchitecture, and increase bone strength. The present study aimed to investigate whether GC excess blunts the osteoanabolic effect of ABL. Sixty 12–13-week-old female RjOrl:SWISS mice were allocated to the following groups: Baseline, Control, ABL, GC, and GC + ABL. ABL was administered as subcutaneous injections (100 μg/kg), while GC was delivered by subcutaneous implantation of a 60-days slow-release prednisolone-pellet (10 mg). The study lasted four weeks. GC induced a substantial reduction in muscle mass, trabecular mineral apposition rate (MAR) and bone formation rate (BFR/BS), and endocortical MAR compared with Control, but did not alter the trabecular microarchitecture or bone strength. In mice not receiving GC, ABL increased aBMD, bone mineral content (BMC), cortical and trabecular microarchitecture, mineralizing surface (MS/BS), MAR, BFR/BS, and bone strength compared with Control. However, when administered concomitantly with GC, the osteoanabolic effect of ABL on BMC, cortical morphology, and cortical bone strength was blunted. In conclusion, at cortical bone sites, the osteoanabolic effect of ABL is generally blunted by short-term GC excess.