Breast tumors induced by N‐methyl‐N‐nitrosourea are damaging to bone strength,structure, and mineralization in the absence of metastasis in rats

Current theory on the influence of breast cancer on bone describes metastasis of tumor cells to bone tissue, followed by induction of osteoclasts and bone degradation. Tumor influences on bone health in pre‐ or nonmetastatic models are unknown. Female rats (n = 48, 52 days old) were injected with N‐methyl‐N‐nitrosourea (MNU) to induce breast cancer. Animals were euthanized 10 weeks later, and tumors were weighed and classified histologically. Right femurs were extracted for testing of bone mineral density (BMD) by dual X‐ray absorptiometry (DXA), bone mechanical strength by three‐point bending and femoral neck bending tests, and structure by micro–computed tomography (µCT). Of 48 rats, 22 developed one or more tumors in response to MNU injection by 10 weeks. Presence of any tumor predicted significantly poorer bone health in 17 of 28 measures. In tumored versus nontumored animals, BMD was adversely affected by 3%, force at failure of the femoral midshaft by 4%, force at failure of the femoral neck by 12%, and various trabecular structural parameters by 6% to 27% (all p < .05). Similarly, greater tumor burden, represented by total tumor weight, adversely correlated with bone outcomes: r = ?0.51 for BMD, ?0.42 and ?0.35 for femur midshaft force and work at failure, and between 0.36 and 0.59 (absolute values) for trabecular architecture (all p < .05). Presence of MNU‐induced tumors and total tumor burden showed a negative association with bone health of the femur in rats in the absence of metastasis. Further study is required to elucidate mechanisms for this association. © 2011 American Society for Bone and Mineral Research.     

Inhibiting activin‐A signaling stimulates bone formation and prevents cancer‐induced bone destruction in vivo

Cancers that grow in bone, such as myeloma and breast cancer metastases, cause devastating osteolytic bone destruction. These cancers hijack bone remodeling by stimulating osteoclastic bone resorption and suppressing bone formation. Currently, treatment is targeted primarily at blocking bone resorption, but this approach has achieved only limited success. Stimulating osteoblastic bone formation to promote repair is a novel alternative approach. We show that a soluble activin receptor type IIA fusion protein (ActRIIA.muFc) stimulates osteoblastogenesis (p < .01), promotes bone formation (p < .01) and increases bone mass in vivo (p < .001). We show that the development of osteolytic bone lesions in mice bearing murine myeloma cells is caused by both increased resorption (p < .05) and suppression of bone formation (p < .01). ActRIIA.muFc treatment stimulates osteoblastogenesis (p < .01), prevents myeloma‐induced suppression of bone formation (p < .05), blocks the development of osteolytic bone lesions (p < .05), and increases survival (p < .05). We also show, in a murine model of breast cancer bone metastasis, that ActRIIA.muFc again prevents bone destruction (p < .001) and inhibits bone metastases (p < .05). These findings show that stimulating osteoblastic bone formation with ActRIIA.muFc blocks the formation of osteolytic bone lesions and bone metastases in models of myeloma and breast cancer and paves the way for new approaches to treating this debilitating aspect of cancer. © 2010 American Society for Bone and Mineral Research.     

Differential effects of biologic versus bisphosphonate inhibition of wear debris‐induced osteolysis assessed by longitudinal micro‐CT

Aseptic loosening of total joint replacements is caused by wear debris‐induced osteoclastic bone resorption, for which bisphosphonates (BPs) and RANK antagonists have been developed. Although BPs are effective in preventing metabolic bone loss, they are less effective for inflammatory bone loss. Because this difference has been attributed to the antiapoptotic inflammatory signals that protect osteoclasts from BP‐induced apoptosis, but not RANK antagonists, we tested the hypothesis that osteoprotegerin (OPG) is more effective in preventing wear debris‐induced osteolysis than zoledronic acid (ZA) or alendronate (Aln) in the murine calvaria model using in vivo micro‐CT and traditional histology. Although micro‐CT proved to be incompatible with titanium (Ti) particles, we were able to demonstrate a 3.2‐fold increase in osteolytic volume over 10 days induced by polyethylene (PE) particles versus sham controls (0.49 ± 0.23mm³ versus 0.15 ± 0.067mm³; p < 0.01). Although OPG and high‐dose ZA completely inhibited this PE‐induced osteolysis (p < 0.001), pharmacological doses of ZA and Aln were less effective but still reached statistical significance (p < 0.05). Traditional histomorphometry of the sagital suture area of calvaria from both Ti and PE‐treated mice confirmed the remarkable suppression of resorption by OPG (p < 0.001) versus the lack of effect by physiological BPs. The differences in drug effects on osteolysis were largely explained by the significant difference in osteoclast numbers observed between OPG versus BPs in both Ti‐ and PE‐treated calvaria; and linear regression analyses that demonstrated a highly significant correlation between osteolysis volume and sagittal suture area versus osteoclast numbers (p < 0.001). © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1340–1346, 2008     

Fluorescence‐guided surgery of human prostate cancer experimental bone metastasis in nude mice using anti‐CEA DyLight 650 for tumor illumination

The present report demonstrates efficacy of fluorescence‐guided surgery (FGS) to resect and prevent recurrence of experimental skeletal metastasis in a nude‐mouse model of human prostate cancer. Green fluorescent protein (GFP)‐expressing PC‐3 human prostate cancer cells were injected into the intramedullary cavity of the tibia in 25 nude mice. One week after implantation, monoclonal antibodies, specific for carcinoembryonic antigen (CEA), labeled with DyLight 650, were injected into the tail vein of 13 mice. Thirteen mice underwent FGS and 12 mice underwent bright‐light surgery (BLS). Weekly GFP fluorescence imaging of the mice was performed to observe tumor recurrence. The extent of residual tumor after BLS was 13‐fold greater than after FGS (p < 0.001). Time‐course imaging visualized rapid growth of the residual tumor in the BLS group, whereas the FGS group showed only slight tumor growth and significantly improved disease‐free survival of the treated mice. Our study demonstrated that FGS significantly reduced residual tumor as well as the recurrence of experimental prostate‐cancer bone metastasis. The present results suggest that FGS will be effective for resection of skeletal metastases in selected patients with prostate cancer. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:559–565, 2016.     

TNF is required for the induction but not the maintenance of compression‐induced BME signals in murine tail vertebrae: Limitations of anti‐TNF therapy for degenerative disc disease

While bone marrow edema (BME) is diagnostic of spondyloarthropathy, its nature remains poorly understood. In contrast, BME in ankylosing spondylitis is caused by tumor necrosis factor (TNF)‐induced vascular and cellular changes. To investigate the relationship between chronic compression and TNF signaling in compression‐induced BME we utilized a tail vertebrae compression model with WT, TNF‐Tg, and TNFR1&2?/? mice to evaluate: (i) healing following release of chronic compression, (ii) induction of BME in the absence of TNFR, and (iii) efficacy of anti‐TNF therapy. Compression‐induced normalized marrow contrast enhancement (NMCE) in WT was significantly decreased threefold (p < 0.01) within 2 weeks of release, while the NMCE values in TNF‐Tg vertebrae remained elevated, but had a significant decrease (p < 0.05) by 6 weeks after the release of compression. TNFR1&2?/? mice were resistant to compression‐induced BME. Anti‐TNF therapy did not affect NMCE versus placebo. Histological examination revealed that NMCE values significantly correlated with marrow vascularity and cellularity (p < 0.05), which account for 76% of the variability of NMCE. Collectively, these data demonstrate a critical role for TNF in the induction of chronic compression‐induced BME, but not in its maintenance. Amelioration of BME is achieved through biomechanical stability, but is not affected by anti‐TNF therapy. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1367–1374, 2011     

Effects of repetitive loading on the growth‐induced changes in bone mass and cortical bone geometry: A 12‐month study in pre/peri‐ and postmenarcheal tennis players

Pre‐ and early puberty may be the most opportune time to strengthen the female skeleton, but there are few longitudinal data to support this claim. Competitive female premenarcheal (pre/peri, n = 13) and postmenarcheal (post, n = 32) tennis players aged 10 to 17 years were followed over 12 months. The osteogenic response to loading was studied by comparing the playing and nonplaying humeri for dual‐energy X‐ray absorptiometry (DXA) bone mineral content (BMC) and magnetic resonance imaging (MRI) total bone area (ToA), medullary area (MedA), cortical area (CoA), and muscle area (MCSA) at the humerus. Over 12 months, growth‐induced gains (nonplaying arm) in BMC, ToA, and CoA were greater in pre/peri (10% to 19%, p < .001) than in post (3% to 5%, p < .05 to .001) players. At baseline, BMC, ToA, CoA, and MCSA were 8% to 18% greater in the playing versus nonplaying arms in pre/peri and post players (all p < .001); MedA was smaller in the playing versus nonplaying arms in post only players (p < .05). When comparing the annual gains in the playing arm relative to changes in the nonplaying arm, the increases in ToA and CoA were greater in pre/peri than post players (all p < .05). The smaller the side‐to‐side differences in BMC and CoA at baseline, the larger the exercise benefits at 12 months (r = ?0.39 to ?0.48, p < .01). The exercise‐induced change in MCSA was predictive of the exercise benefits in BMC in pre/peri players only (p < .05). In conclusion, both pre/peri‐ and postmenarcheal tennis players showed significant exercise‐induced skeletal benefits within a year, with greater benefits in cortical bone geometry in pre/perimenarcheal girls. © 2011 American Society for Bone and Mineral Research.     

Pharmacologically Inactive Bisphosphonates as an Alternative Strategy for Targeting Osteoclasts: In Vivo Assessment of 5‐Fluorodeoxyuridine‐Alendronate in a Preclinical Model of Breast Cancer Bone Metastases

Bisphosphonates have effects that are antiresorptive, antitumor, and antiapoptotic to osteoblasts and osteocytes, but an effective means of eliciting these multiple activities in the treatment of bone metastases has not been identified. Antimetabolite‐bisphosphonate conjugates have potential for improved performance as a class of bone‐specific antineoplastic drugs. The primary objective of the study was to determine whether an antimetabolite‐bisphosphonate conjugate will preserve bone formation concomitant with antiresorptive and antitumor activity. 5‐FdU‐ale, a highly stable conjugate between the antimetabolite 5‐fluoro‐2'‐deoxyuridine and the bisphosphonate alendronate, was tested for its therapeutic efficacy in a mouse model of MDA‐MB231 breast cancer bone metastases. In vitro testing revealed osteoclasts to be highly sensitive to 5‐FdU‐ale. In contrast, osteoblasts had significantly reduced sensitivity. Tumor cells were resistant in vitro but in vivo tumor burden was nevertheless significantly reduced compared with untreated mice. Sensitivity to 5‐FdU‐ale was not mediated through inhibition of farnesyl diphosphate synthase activity, but cell cycle arrest was observed. Although serum tartrate‐resistant acid phosphatase (TRAP) levels were greatly reduced by both drugs, there was no significant decrease in the serum bone formation marker osteocalcin with 5‐FdU‐ale treatment. In contrast, there was more than a fivefold decrease in serum osteocalcin levels with alendronate treatment (p < 0.001). This finding is supported by time‐lapse micro–computed tomography analyses, which revealed bone formation volume to be on average 1.6‐fold higher with 5‐FdU‐ale treatment compared with alendronate (p < 0.001). We conclude that 5‐FdU‐ale, which is a poor prenylation inhibitor but maintains potent antiresorptive activity, does not reduce bone formation and has cytostatic antitumor efficacy. These results document that conjugation of an antimetabolite with bisphosphonates offers flexibility in creating potent bone‐targeting drugs with cytostatic, bone protection properties that show limited nephrotoxicity. This unique class of drugs may offer distinct advantages in the setting of targeted adjuvant therapy and chemoprevention of bone diseases. © 2016 American Society for Bone and Mineral Research.     

Levels of serotonin,sclerostin, bone turnover markers as well as bone density and microarchitecture in patients with high‐bone‐mass phenotype due to a mutation in Lrp5

Patients with an activation mutation of the Lrp5 gene exhibit high bone mass (HBM). Limited information is available regarding compartment‐specific changes in bone. The relationship between the phenotype and serum serotonin is not well documented. To evaluate bone, serotonin, and bone turnover markers (BTM) in Lrp5‐HBM patients, we studied 19 Lrp5‐HBM patients (T253I) and 19 age‐ and sex‐matched controls. DXA and HR‐pQCT were used to assess BMD and bone structure. Serum serotonin, sclerostin, dickkopf‐related protein 1 (DKK1), and BTM were evaluated. Z‐scores for the forearm, total hip, lumbar spine, forearm, and whole body were significantly increased (mean ± SD) between 4.94 ± 1.45 and 7.52 ± 1.99 in cases versus ?0.19 ± 1.19 to 0.58 ± 0.84 in controls. Tibial and radial cortical areas, thicknesses, and BMD were significantly higher in cases. In cases, BMD at the lumbar spine and forearm and cortical thickness were positively associated and trabecular area negatively associated with age (r = 0.49, 0.57, 0.74, and ?0.61, respectively, p < .05). Serotonin was lowest in cases (69.5 [29.9–110.4] ng/mL versus 119.4 [62.3–231.0] ng/mL, p < .001) and inversely associated with tibial cortical density (r = ?0.49, p < .05) and directly with osteocalcin (OC), bone‐specific alkaline phosphatase (B‐ALP), and procollagen type 1 amino‐terminal propeptide (PINP) (r = 0.52–0.65, p < .05) in controls only. OC and S‐CTX were lower and sclerostin higher in cases, whereas B‐ALP, PINP, tartrate‐resistant acid phosphatase (TRAP), and dickkopf‐related protein 1 (DKK1) were similar in cases and controls. In conclusion, increased bone mass in Lrp5‐HBM patients seems to be caused primarily by changes in trabecular and cortical bone mass and structure. The phenotype appeared to progress with age, but BTM did not suggest increased bone formation. © 2011 American Society for Bone and Mineral Research     

Interstitial fluid pressure as an alternate regulator of angiogenesis independent of hypoxia driven HIF‐1α in solid tumors

We previously showed that interstitial fluid pressure (IFP) may be an alternate regulator of angiogenesis in solid tumors. Given the accepted link between hypoxia‐induced factor and angiogenesis this study investigated the effect of IFP on hypoxia‐inducible factor (HIF‐1α) and vascular endothelial growth factor (VEGF) in human osteosarcoma xenografts in SCID mice and in different hypoxic environments. Tumors were grown either at heterotopic (flank) or orthotopic (medullary canal of the proximal tibia) sites in the host animal. Microfluidic probes determined pH, O₂‐saturation, IFP, and peripheral blood flow perfusion continuously. We assessed tumor growth in the orthotopic site (n = 15) by softex radiographs weekly, 3D microCT, histological evaluation, and for molecular responses. An increased cytoplasmic immunohistostaining of cells for HIF‐1α (p = 0.03) and VEGF‐A (p = 0.004) on the outer periphery was noted compared to the tumor center, with VEGFR2 uniformly stained throughout. This paralleled a raised state of interstitial hypertension (p = 0.007) in the tumor center relative to the peripheral surface but was inconsistent with a state of hypoxia (p = 0.03) in the tumor center. In vitro culture of human osteosarcoma cell lines (HOS, U2OS) and a human osteoblast control at 0‐ and 20‐mmHg of hydrostatic pressure revealed suppression of HIF‐1α (p = 0.02) and VEGF‐A (p = 0.02) gene expression when IFP was raised, while the effect on VEGFR1 was equivocal. This study proposes an alternative regulatory angiogenic pathway via the influence of IFP on cancer cell function. The identification of a mechanistic cellular link to the physical parameter becomes an important tool to evaluate cancer cell growth within solid tumors. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:2038–2045, 2012     

Intraarticular injection autologous platelet‐rich plasma and bone marrow concentrate in a goat osteoarthritis model

To evaluate the effects of intraarticular injections of autologous platelet‐rich plasma (PRP) or bone marrow concentrate (BMC) on osteoarthritis (OA), 24 adult goats were equally divided into control (Ctrl), saline (NS), PRP, and BMC groups, and OA was induced by surgery in NS, PRP, and BMC groups. Autologous PRP and BMC were obtained from whole blood and bone marrow aspirates, respectively. The data revealed, platelets were increased in BMC by 1.8‐fold, monocytes by 5.6‐fold, TGF‐β1 by 7.7‐fold, and IGF‐1 by 3.6‐fold (p < 0.05), and platelets were increased in PRP by 2.9‐fold, and TGF‐β1 by 3.3‐fold (p < 0.05). From the sixth week post‐operation, saline, PRP, and BMC were administered by intraarticular injection once every 4 weeks, three consecutive times. After the animals were sacrificed, inflammatory cytokines in the synovial fluid was measured, and bone and cartilage degeneration progression was observed by macroscopy, histology, and immunohistochemistry. Compared with the NS group, the level of inflammatory cytokines was reduced in the PRP and BMC groups (p < 0.05). Histologically, delayed cartilage degeneration and higher levels of extracellular matrix (ECM) were observed in both PRP and BMC treated groups (p < 0.05). Furthermore, the BMC group showed greater cartilage protection and less ECM loss than the PRP group (p < 0.05). In summary, this study showed that intraarticular injection of autologous PRP and BMC has therapeutic efficacy in a goat osteoarthritis model, with the greater benefit in terms of cartilage protection being observed in the BMC‐treated group than PRP. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2140–2146, 2018.

     

Application of High‐Resolution Skeletal Imaging to Measurements of Volumetric BMD and Skeletal Microarchitecture in Chinese‐American and White Women: Explanation of a Paradox

Asian women have lower rates of hip and forearm fractures despite lower areal BMD (aBMD) by DXA compared with white women and other racial groups. We hypothesized that the lower fracture rates may be explained by more favorable measurements of volumetric BMD (vBMD) and microarchitectural properties, despite lower areal BMD. To address this hypothesis, we used high‐resolution pQCT (HRpQCT), a new method that can provide this information noninvasively. We studied 63 premenopausal Chinese‐American (n = 31) and white (n = 32) women with DXA and HRpQCT. aBMD by DXA did not differ between groups for the lumbar spine (1.017 ± 0.108 versus 1.028 ± 0.152 g/cm²; p = 0.7), total hip (0.910 ± 0.093 versus 0.932 ± 0.134 g/cm²; p = 0.5), femoral neck (0.788 ± 0.083 versus 0.809 ± 0.129 g/cm²; p = 0.4), or one‐third radius (0.691 ± 0.052 versus 0.708 ± 0.047 g/cm²; p = 0.2). HRpQCT at the radius indicated greater trabecular (168 ± 41 versus 137 ± 33 mg HA/cm³; p = <0.01) and cortical (963 ± 46 versus 915 ± 42 mg HA/cm³; p < 0.0001) density; trabecular bone to tissue volume (0.140 ± 0.034 versus 0.114 ± 0.028; p = <0.01); trabecular (0.075 ± 0.013 versus 0.062 ± 0.009 mm; p < 0.0001) and cortical thickness (0.98 ± 0.16 versus 0.80 ± 0.14 mm; p < 0.0001); and lower total bone area (197 ± 34 versus 232 ± 33 mm²; p = <0.001) in the Chinese versus white women and no difference in trabecular number, spacing, or inhomogeneity before adjustment for covariates. Similar results were observed at the weight‐bearing tibia. At the radius, adjustment for covariates did not change the direction or significance of differences except for bone, which became similar between the groups. However, at the tibia, adjustment for covariates attenuated differences in cortical BMD and bone area and accentuated differences in trabecular microarchitecture such that Chinese women additionally had higher trabecular number and lower trabecular spacing, as well as inhomogeneity after adjustment. Using the high‐resolution technology, the results provide a mechanistic explanation for why Chinese women have fewer hip and forearm fractures than white women.     

Effect of Two‐Year Caloric Restriction on Bone Metabolism and Bone Mineral Density in Non‐Obese Younger Adults: A Randomized Clinical Trial

Although caloric restriction (CR) could delay biologic aging in humans, it is unclear if this would occur at the cost of significant bone loss. We evaluated the effect of prolonged CR on bone metabolism and bone mineral density (BMD) in healthy younger adults. Two‐hundred eighteen non‐obese (body mass index [BMI] 25.1 ± 1.7 kg/m²), younger (age 37.9 ± 7.2 years) adults were randomly assigned to 25% CR (CR group, n = 143) or ad libitum (AL group, n = 75) for 2 years. Main outcomes were BMD and markers of bone turnover. Other outcomes included body composition, bone‐active hormones, nutrient intake, and physical activity. Body weight (–7.5 ± 0.4 versus 0.1 ± 0.5 kg), fat mass (–5.3 ± 0.3 versus 0.4 ± 0.4 kg), and fat‐free mass (–2.2 ± 0.2 versus –0.2 ± 0.2 kg) decreased in the CR group compared with AL (all between group p < 0.001). Compared with AL, the CR group had greater changes in BMD at 24 months: lumbar spine (–0.013 ± 0.003 versus 0.007 ± 0.004 g/cm²; p < 0.001), total hip (–0.017 ± 0.002 versus 0.001 ± 0.003 g/cm²; p < 0.001), and femoral neck (–0.015 ± 0.003 versus –0.005 ± 0.004 g/cm²; p = 0.03). Changes in bone markers were greater at 12 months for C‐telopeptide (0.098 ± 0.012 versus 0.025 ± 0.015 μg/L; p < 0.001), tartrate‐resistant acid phosphatase (0.4 ± 0.1 versus 0.2 ± 0.1 U/L; p = 0.004), and bone‐specific alkaline phosphatase (BSAP) (–1.4 ± 0.4 versus –0.3 ± 0.5 U/L; p = 0.047) but not procollagen type 1 N‐propeptide; at 24 months, only BSAP differed between groups (–1.5 ± 0.4 versus 0.9 ± 0.6 U/L; p = 0.001). The CR group had larger increases in 25‐hydroxyvitamin D, cortisol, and adiponectin and decreases in leptin and insulin compared with AL. However, parathyroid hormone and IGF‐1 levels did not differ between groups. The CR group also had lower levels of physical activity. Multiple regression analyses revealed that body composition, hormones, nutrients, and physical activity changes explained ～31% of the variance in BMD and bone marker changes in the CR group. Therefore, bone loss at clinically important sites of osteoporotic fractures represents a potential limitation of prolonged CR for extending life span. Further long‐term studies are needed to determine if CR‐induced bone loss in healthy adults contributes to fracture risk and if bone loss can be prevented with exercise. © 2015 American Society for Bone and Mineral Research.     

Serum Levels of a Cathepsin‐K Generated Periostin Fragment Predict Incident Low‐Trauma Fractures in Postmenopausal Women Independently of BMD and FRAX

Periostin is a matricellular protein involved in bone formation and bone matrix organization, but it is also produced by other tissues. Its circulating levels have been weakly associated with bone microstructure and prevalent fractures, possibly because periostin measured by the current commercial assays does not specifically reflect bone metabolism. In this context, we developed a new ELISA for a periostin fragment resulting from cathepsin K digestion (K‐Postn). We hypothesized that circulating K‐Postn levels could be associated with bone fragility. A total of 695 women (age 65.0 ± 1.5 years), enrolled in the Geneva Retirees Cohort (GERICO), were prospectively evaluated over 4.7 ± 1.9 years for the occurrence of low‐trauma fractures. At baseline, we measured serum periostin, K‐Postn, and bone turnover markers (BTMs), distal radius and tibia microstructure by HR‐pQCT, hip and lumbar spine aBMD by DXA, and estimated fracture probability using the Fracture Risk Assessment Tool (FRAX). Sixty‐six women sustained a low‐trauma clinical fracture during the follow‐up. Total periostin was not associated with fractures (HR [95% CI] per SD: 1.19 [0.89 to 1.59], p = 0.24). In contrast, K‐Postn was significantly higher in the fracture versus nonfracture group (57.5 ± 36.6 ng/mL versus 42.5 ± 23.4 ng/mL, p < 0.001) and associated with fracture risk (HR [95%CI] per SD: 2.14 [1.54 to 2.97], p < 0.001). After adjustment for aBMD, FRAX, bone microstructure, or BTMs, K‐Postn remained significantly associated with fracture risk. The performance of the fracture prediction models was improved by adding K‐Postn to aBMD or FRAX (Harrell C index for fracture: 0.70 for aBMD + K‐Post versus 0.58 for aBMD alone, p = 0.001; 0.73 for FRAX + K‐Postn versus 0.65 for FRAX alone, p = 0.005). Circulating K‐Postn predicts incident fractures independently of BMD, BTMs, and FRAX in postmenopausal women. Hence measurement of a periostin fragment resulting from in vivo cathepsin K digestion may help to identify subjects at high risk of fracture. © 2017 American Society for Bone and Mineral Research     

Patients with high‐bone‐mass phenotype owing to Lrp5‐T253I mutation have low plasma levels of serotonin

The Lrp5 gene is a major determinant of bone mass accrual. It has been demonstrated recently to achieve this function by hampering the synthesis of gut‐derived serotonin, which is a powerful inhibitor of bone formation. In this study we analyzed plasma serotonin levels in patients with a high‐bone‐mass (HBM) phenotype owing to gain‐of‐function mutation of Lrp5 (T253I). A total of 9 HBM patients were compared with 18 sex‐ and age‐matched controls. In HBM patients, the serotonin concentrations in platelet‐poor plasma were significantly lower than in the controls (mean ± SEM: 2.16 ± 0.28 ng/mL versus 3.51 ± 0.49 ng/mL, respectively, p < .05). Our data support the hypothesis that circulating serotonin levels mediate the increased bone mass resulting from gain‐of‐function mutations in Lrp5 in humans. © 2010 American Society for Bone and Mineral Research.     

Low‐magnitude high‐frequency mechanical signals accelerate and augment endochondral bone repair: Preliminary evidence of efficacy

Fracture healing can be enhanced by load bearing, but the specific components of the mechanical environment which can augment or accelerate the process remain unknown. The ability of low‐magnitude, high‐frequency mechanical signals, anabolic in bone tissue, are evaluated here for their ability to influence fracture healing. The potential for short duration (17 min), extremely low‐magnitude (25 µm), high‐frequency (30 Hz) interfragmentary displacements to enhance fracture healing was evaluated in a mid‐diaphyseal, 3‐mm osteotomy of the sheep tibia. In a pilot study of proof of concept and clinical relevance, healing in osteotomies stabilized with rigid external fixation (Control: n = 4), were compared to the healing status of osteotomies with the same stiffness of fixation, but supplemented with daily mechanical loading (Experimental: n = 4). These 25‐µm displacements, induced by a ferroactive shape‐memory alloy (“smart” material) incorporated into the body of the external fixator, were less than 1% of the 3‐mm fracture gap, and less than 6% of the 0.45‐mm displacement measured at the site during ambulation (p < 0.001). At 10‐weeks post‐op, the callus in the Experimental group was 3.6‐fold stiffer (p < 0.03), 2.5‐fold stronger (p = 0.05), and 29% larger (p < 0.01) than Controls. Bone mineral content was 52% greater in the Experimental group (p < 0.02), with a 2.6‐fold increase in bone mineral content (BMC) in the region of the periosteum (p < 0.001). These data reinforce the critical role of mechanical factors in the enhancement of fracture healing, and emphasize that the signals need not be large to be influential and potentially clinically advantageous to the restoration of function. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 922–930, 2009     

Prospective study of combined treatment with interferon‐alpha and active vitamin D3 for Japanese patients with metastatic renal cell carcinoma

Objectives: To assess the safety and efficacy of combined therapy with interferon‐alpha (INF‐α) and active vitamin D₃ for metastatic renal cell carcinoma (RCC). Methods: Sixteen patients with metastatic RCC were enrolled in this prospective study. All received oral alfacalcidol (1 µg once daily) and INF‐α (Sumiferon; 3 million units, three times a week). The primary endpoint was the response rate (defined as complete + partial remission). Secondary endpoints were cancer‐specific survival and toxicity. The median follow‐up period was 17 months (range: 5–49 months). Results: The median age of the patients was 68 years (range: 41–73 years). The sites of metastases were: lung in 13 patients, bone in one, lung and bone in one, and lung, bone, and lymph nodes in one. Four patients (25%) had a partial response (PR), 10 patients (62.5%) showed no change (NC), and two patients (12.5%) had progressive disease (PD). The median cancer‐specific survival time was 45 months. One patient had to discontinue vitamin D₃ because of hypercalcemia. Kaplan‐Meier survival analysis revealed that metastasis at the time of initial diagnosis and older than average age were significant predictors of poor survival (P < 0.05). Conclusions: Combined treatment with INF‐α and active vitamin D₃ has shown to be safe and effective for metastatic RCC patients.     

Bisphosphonate Induces Osteonecrosis of the Jaw in Diabetic Mice via NLRP3/Caspase‐1‐Dependent IL‐1β Mechanism

Diabetes mellitus is an established risk factor associated with bisphosphonate‐related osteonecrosis of the jaw (BRONJ). Sustained activation of Nod‐like receptor (NLR) family, pyrin domain‐containing protein 3 (NLRP3) inflammasome contributes to the persistent inflammation and impaired cutaneous wound healing in diabetic mice and human. We have recently demonstrated a compelling linkage between M1 macrophages and BRONJ conditions in both murine and human diseases. The aim of this study was to determine whether NLRP3 inflammasome activation is involved in BRONJ development in diabetic mice. We showed an increased incidence of delayed oral wound healing and bone necrosis of extraction sockets in db/db mice compared with those in nondiabetic db/+ controls, which correlated with an elevated expression of NLRP3, caspase‐1, and IL‐1β in macrophages residing at local wounds. Constitutively, bone marrow‐derived macrophages from db/db mice (db/db BMDMs) secrete a relatively higher level of IL‐1β than those from db/+ mice (db/+ BMDMs). Upon stimulation by NLRP3 activators, the secretion of IL‐1β by db/db BMDMs was 1.77‐fold higher than that by db/+ BMDMs (p < 0.001). Systemic treatment of mice with zoledronate (Zol), a nitrogen‐containing bisphosphonate, resulted in a 1.86‐ and 1.63‐fold increase in NLRP3/caspase‐1‐dependent IL‐1β secretion by db/+ and db/db BMDMs, respectively, compared with BMDMs derived from nontreated mice (p < 0.001). Importantly, systemic administration of pharmacological inhibitors of NLRP3 activation improved oral wound healing and suppressed BRONJ formation in db/db mice. Mechanistically, we showed that supplementation with intermediate metabolites of the mevalonate pathway, inhibitors of caspase‐1 and NLRP3 activation, an antagonist for P2X₇R, or a scavenger of reactive oxygen species (ROS), robustly abolished Zol‐enhanced IL‐1β release from macrophages in response to NLRP3 activation (p < 0.001). Our findings suggest that diabetes‐associated chronic inflammatory response may have contributed to impaired socket wound healing and rendered oral wound susceptible to the development of BRONJ via NLRP3 activation in macrophages. © 2015 American Society for Bone and Mineral Research.     

Osteocyte Apoptosis Caused by Hindlimb Unloading is Required to Trigger Osteocyte RANKL Production and Subsequent Resorption of Cortical and Trabecular Bone in Mice Femurs

Osteocyte apoptosis is essential to activate bone remodeling in response to fatigue microdamage and estrogen withdrawal, such that apoptosis inhibition in vivo prevents the onset of osteoclastic resorption. Osteocyte apoptosis has also been spatially linked to bone resorption owing to disuse, but whether apoptosis plays a similar controlling role is unclear. We, therefore, 1) evaluated the spatial and temporal effects of disuse from hindlimb unloading (HLU) on osteocyte apoptosis, receptor activator of NF‐κB ligand (RANKL) expression, bone resorption, and loss in mouse femora, and 2) tested whether osteocyte apoptosis was required to activate osteoclastic activity in cortical and trabecular bone by treating animals subjected to HLU with the pan‐caspase apoptosis inhibitor, QVD (quinolyl‐valyl‐O‐methylaspartyl‐[‐2,6‐difluorophenoxy]‐methylketone). Immunohistochemistry was used to identify apoptotic and RANKL‐producing osteocytes in femoral diaphysis and distal trabecular bone, and µCT was used to determine the extent of trabecular bone loss owing to HLU. In both cortical and trabecular bone, 5 days of HLU increased osteocyte apoptosis significantly (3‐ and 4‐fold, respectively, p < 0.05 versus Ctrl). At day 14, the apoptotic osteocyte number in femoral cortices declined to near control levels but remained elevated in trabeculae (3‐fold versus Ctrl, p < 0.05). The number of osteocytes producing RANKL in both bone compartments was also significantly increased at day 5 of HLU (>1.5‐fold versus Ctrl, p < 0.05) and further increased by day 14. Increases in osteocyte apoptosis and RANKL production preceded increases in bone resorption at both endocortical and trabecular surfaces. QVD completely inhibited not only the HLU‐triggered increases in osteocyte apoptosis but also RANKL production and activation of bone resorption at both sites. Finally, µCT studies revealed that apoptosis inhibition completely prevented the trabecular bone loss caused by HLU. Together these data indicate that osteocyte apoptosis plays a central and controlling role in triggering osteocyte RANKL production and the activation of new resorption leading to bone loss in disuse. © 2016 American Society for Bone and Mineral Research.     

Antioxidant impregnated ultra‐high molecular weight polyethylene wear debris particles display increased bone remodeling and a superior osteogenic:osteolytic profile vs. conventional UHMWPE particles in a murine calvaria model

Periprosthetic osteolysis remains a major limitation of long‐term successful total hip replacements with ultra‐high molecular weight polyethylene (UHMWPE) bearings. As intra and extracellular reactive oxygen species are know to contribute to wear debris‐induced osteoclastic bone resorption and decreased osteoblastic bone formation, antioxidant doped UHMWPE has emerged as an approach to reduce the osteolytic potential of wear debris and maintain coupled bone remodeling. To test this hypothesis in vivo, we evaluated the effects of crosslinked UHMWPE wear debris particles (AltrX^?), versus similar wear particles made from COVERNOX^? containing UHMWPE (AOX^?), in an established murine calvaria model. Eight‐week‐old female C57B/6 mice (n = 10/Group) received a pre‐op micro‐CT scan prior to surgical implantation of the UHMWPE particles (2mg), or surgery without particles (sham). Dynamic labeling was performed by intraperitoneal injection of calcein on day 7 and alizarin on day 9, and the calvaria were harvested for micro‐CT and histology on day 10. Surprisingly, we found that AOX particles induced significantly more bone resorption (1.72‐fold) and osteoclast numbers (1.99‐fold) vs. AltrX (p < 0.001). However, AOX also significantly induced 1.64‐fold more new bone formation vs. AltrX (p < 0.01). Moreover, while the osteolytic:osteogenic ratio of both particles was very close to 1.0, which is indicative of coupled remodeling, AOX was more osteogenic (Slope = 1.13 ± 0.10 vs. 0.97 ± 0.10). Histomorphometry of the metabolically labeled undecalcified calvaria revealed a consistent trend of greater MAR in AOX vs. AltrX. Collectively, these results demonstrate that anti‐oxidant impregnated UHMWPE particles have decreased osteolytic potential due to their increased osteogenic properties that support coupled bone remodeling. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:845–851, 2016.

     

Comparison of accuracy of 14‐, 18‐ and 20‐G needles in ex‐vivo renal mass biopsy: a prospective,blinded study

Study Type – Diagnostic (exploratory cohort)
Level of Evidence 2b

OBJECTIVE

To prospectively determine the accuracy of 14‐, 18‐ and 20‐G core needle biopsies to render the appropriate histological diagnosis of solid, enhancing renal masses, using a controlled, ex‐vivo biopsy technique.

PATIENTS AND METHODS

From March 2007 to September 2007, 31 patients undergoing partial or radical nephrectomy were randomly selected for biopsy. After extirpative surgery, three ex‐vivo biopsies were taken from each lesion with 14‐, 18‐ and 20‐G biopsy needles. One experienced genitourinary pathologist, unaware of patient identifiers and final pathology results, determined the biopsy histology and tumour grade, based on standard haematoxylin and eosin (H&E) techniques and immunohistochemistry.

RESULTS

The final pathological evaluation classified 21 masses (68%) as clear cell renal cell carcinoma (RCC), three (10%) as papillary RCC, three (10%) as chromophobe RCC, three (10%) as oncocytoma and one (3%) as a benign lymphoid infiltrate. The biopsy histology correlated with the final pathology in 29/31 cases (94%) with the 14‐G, 30/31 cases (97%) with the 18‐G and 25/31 cases (81%) with the 20‐G needles. In two cases chromophobe RCC was misdiagnosed with oncocytoma, and vice versa.

CONCLUSION

In this study a minimum of an 18‐G biopsy needle was the most accurate in determining the histological diagnosis. Clear cell and papillary RCCs were accurately diagnosed on biopsy using an 18‐G, whereas oncocytoma and chromophobe RCC were difficult to differentiate using standard H&E techniques and immunohistochemistry.