Detecting Multiple Damages in UHPFRC Beams through Modal Curvature Analysis.

Curvature-based damage detection has been previously applied to identify damage in concrete structures, but little attention has been given to the capacity of this method to identify distributed damage in multiple damage zones. This study aims to apply for the first time an enhanced existing method based on modal curvature analysis combined with wavelet transform curvature (WTC) to identify zones and highlight the damage zones of a beam made of ultra-high-performance fiber-reinforced concrete (UHPFRC), a construction material that is emerging worldwide for its outstanding performance and durability. First, three beams with a 2 m span of UHPFRC material were cast, and damaged zones were created by sawing. A reference beam without cracks was also cast. The free vibration responses were measured by 12 accelerometers and calculated by operational modal analysis. Moreover, for the sake of comparison, a finite element model (FEM) was also applied to two identical beams to generate numerical acceleration without noise. Second, the modal curvature was calculated for different modes for both experimental and FEM-simulated acceleration after applying cubic spline interpolation. Finally, two damage identification methods were considered: (i) the damage index (DI), based on averaging the quadratic difference of the local curvature with respect to the reference beam, and (ii) the WTC method, applied to the quadratic difference of the local curvature with respect the reference beam. The results indicate that the developed coupled modal curvature WTC method can better identify the damaged zones of UHPFRC beams.

Robust Synchronization of Ambient Vibration Time Histories Based on Phase Angle Compensations and Kernel Density Function.

The output-only modal analysis is ubiquitously used for structural health monitoring of civil engineering systems. The measurements for such applications require the use of multiple data acquisition systems (DAS) to avoid complicated meshes of cables in high-rise buildings, avoid traffic constriction on a bridge during measurements, or to avoid having limited channels in a single DAS. Nevertheless, such requirements introduce time synchronization problems which potentially lead to erroneous structural dynamic characterization and hence misleading or inconclusive structural health monitoring results. This research aims at proposing a system-identification-based time synchronization algorithm for output-only modal analysis using multiple DAS. A new procedure based on the compensation of the phase angle shifts is proposed to identify and address the time synchronization issue in ambient vibration data measured through multiple DAS. To increase the robustness of the proposed algorithm to the inherent inconsistencies in these datasets, the kernel density function is applied to rank multiple time-shift estimates that are sometimes detected by the algorithm when inaccuracies exist in the data arising from low signal-to-noise ratio and/or presence of colored noise in the ambient excitations. First, the synchronized ambient vibration dataset of a full-scale bridge is artificially de-synchronized and used to present a proof of concept for the proposed algorithm. Next, the algorithm is applied to ambient vibration data of a 30-story, reinforced concrete building, where the synchronization of the data could not be achieved using two DAS despite best efforts. The application of the proposed time synchronization algorithm is shown to both detect and correct the time synchronization discrepancies in the output-only modal analysis.

Carcinogenicity risk assessment of romosozumab: A review of scientific weight-of-evidence and findings in a rat lifetime pharmacology study.

Romosozumab is a humanized immunoglobulin G2 monoclonal antibody that binds and blocks the action of sclerostin, a protein secreted by the osteocyte and an extracellular inhibitor of canonical Wnt signaling. Blockade of sclerostin binding to low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6) allows Wnt ligands to activate canonical Wnt signaling in bone, increasing bone formation and decreasing bone resorption, making sclerostin an attractive target for osteoporosis therapy. Because romosozumab is a bone-forming agent and an activator of canonical Wnt signaling, questions have arisen regarding a potential carcinogenic risk. Weight-of-evidence factors used in the assessment of human carcinogenic risk of romosozumab included features of canonical Wnt signaling, expression pattern of sclerostin, phenotype of loss-of-function mutations in humans and mice, mode and mechanism of action of romosozumab, and findings from romosozumab chronic toxicity studies in rats and monkeys. Although the weight-of-evidence factors supported that romosozumab would pose a low carcinogenic risk to humans, the carcinogenic potential of romosozumab was assessed in a rat lifetime study. There were no romosozumab-related effects on tumor incidence in rats. The findings of the lifetime study and the weight-of-evidence factors collectively indicate that romosozumab administration would not pose a carcinogenic risk to humans.

The effects of bazedoxifene in the ovariectomized aged cynomolgus monkey.

Bazedoxifene (BZA) is a novel selective estrogen receptor modulator in clinical development for the prevention and treatment of postmenopausal osteoporosis. This preclinical study evaluated the efficacy and safety of BZA in preventing ovariectomy (OVX)-induced bone loss in aged cynomolgus monkeys. Animals (18 per group) underwent OVX and were administered BZA (0.2, 0.5, 1, 5, or 25 mg/kg/day) or vehicle, or were sham-operated and administered vehicle, by daily oral gavage for 18 months. Biochemical markers of bone turnover were assessed at 6, 12, and 18 months, along with bone densitometry using dual energy X-ray absorptiometry and peripheral quantitative computed tomography. Animals were killed after 18 months. Uterine and pituitary weights were determined, and histomorphometric and biomechanical measurements were performed. OVX vehicle controls showed increases in bone turnover associated with cancellous and cortical bone osteopenia (in vivo), and slight decreases (not statistically significant) in biomechanical strength parameters at the lumbar spine and femoral neck. BZA partially preserved cortical and cancellous bone mass by preventing the OVX-induced increases in bone turnover. Although the response was often similar among BZA-treated groups, the strongest efficacy was generally seen at 25 mg/kg/day. Treatment with BZA did not adversely affect measures of bone strength and was well tolerated; there was no evidence of uterotrophic activity, mammary tissue was unaffected, and there were no adverse effects on plasma lipids. Treatment of ovariectomized animals with BZA partially prevented changes in bone remodeling that correlated with increases in bone mineral density, while maintaining bone strength and a favorable safety profile.

Effects of pioglitazone and fenofibrate co-administration on bone biomechanics and histomorphometry in ovariectomized rats.

Pioglitazone, the peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonist is an effective therapy for type 2 diabetes, but has been associated with increased risk for bone fracture. Preclinical studies suggest that PPAR-α agonists (e.g., fenofibrate) increase bone mineral density/content, although clinical data on bone effects of fibrates are lacking. We investigated the effects of pioglitazone (10 mg/kg/day) and fenofibrate (25 mg/kg/day) on bone strength and bone histomorphometric parameters in osteopenic ovariectomized (OVX) rats. An additional group of rats received a combination of pioglitazone + fenofibrate to mimic the effects of a dual PPAR-α/γ agonist. The study consisted of a 13-week treatment phase followed by a 6-week treatment-free recovery period. Pioglitazone significantly reduced biomechanical strength at the lumbar spine and femoral neck compared with rats administered fenofibrate. Co-treatment with pioglitazone + fenofibrate had no significant effect on bone strength in comparison with OVX vehicle controls. Histomorphometric analysis of the proximal tibia revealed that pioglitazone suppressed bone formation and increased bone resorption at both cancellous and cortical bone sites relative to OVX vehicle controls. In contrast, fenofibrate did not affect bone resorption and only slightly suppressed bone formation. Discontinuation of pioglitazone treatment, both in the monotherapy and in the combination therapy arms, resulted in restoration of bone formation and resorption rates, demonstrating reversibility of effects. The above data support the concept that dual activation of PPAR-γ and PPAR-α attenuates the negative effects of PPAR-γ agonism on bone strength.

Inhibition of both sclerostin and DKK1 results in novel skull findings in the rat and non-human primate that is not observed with inhibition of sclerostin alone.

Sclerostin is an extracellular inhibitor of canonical Wnt signaling that inhibits bone formation and stimulates bone resorption. Anti-sclerostin antibodies (Scl-Ab) have been developed as bone-building agents. DKK1, another extracellular inhibitor of the pathway, is upregulated in osteocytes in response to sclerostin inhibition. To further enhance bone-forming effects, a bispecific antibody inhibiting both sclerostin and DKK1 was created (AMG 147). In nonclinical safety studies, AMG 147 resulted in novel skull findings. In the rat, there was increased thickness of skull bones of neural crest origin due to increased subperiosteal compact lamellar and intramembranous woven bone. Externally, subperiosteal fibroblastic/osteoblastic stromal cell proliferation with woven bone and hemorrhage was also observed. Scl-Ab alone resulted in increased skull thickness in the rat, like AMG 147, but without the stromal cell proliferation/woven bone formation. In contrast to embryonic flat bone development, intramembranous bone formed similar to plexiform bone. In the monkey, AMG 147 resulted in macroscopic skull thickening due to a diffuse increase in appositional lamellar bone and increased intramembranous bone on both periosteal surfaces of all skull bones. These data demonstrate that dual inhibition of sclerostin and DDK1 results in unique effects on the skull not observed with sclerostin inhibition alone.

Proliferative and non-proliferative lesions of the rat and mouse soft tissue, skeletal muscle and mesothelium.

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying lesions observed in the soft tissues including skeletal muscle as well as the mesothelium of rats and mice. The standardized nomenclature of lesions presented in this document is also available electronically on the Internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous developmental and aging lesions as well as those induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for lesions in soft tissues, skeletal muscle and mesothelium in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists. (DOI: 10.1293/tox.26.1S; J Toxicol Pathol 2013; 26: 1S-26S).

Proliferative and nonproliferative lesions of the rat and mouse mammary, Zymbal's, preputial, and clitoral glands.

The mammary gland of laboratory rodents is an important organ for the evaluation of effects of xenobiotics, especially those that perturb hormonal homeostasis or are potentially carcinogenic. Mammary gland cancer is a leading cause of human mortality and morbidity worldwide and is a subject of major research efforts utilizing rodent models. Zymbal's, preputial, and clitoral glands are standard tissues that are evaluated in animal models that enable human risk assessment of xenobiotics. A widely accepted and utilized international harmonization of nomenclature for mammary, Zymbal's, preputial, and clitoral gland lesions in laboratory animals will improve diagnostic alignment among regulatory and scientific research organizations and enrich international exchanges of information among toxicologists and pathologists.

Instilled or injected purified natural capsaicin has no adverse effects on rat hindlimb sensory-motor behavior or osteotomy repair.

BACKGROUND: A novel formulation of > or = 98% pure capsaicin (4975) is currently undergoing clinical investigation using novel routes of delivery to provide selective analgesia lasting weeks to months with a single dose. We conducted this study to assess the safety and effects of instilled and injected 4975 in rat models of wound healing osteotomy repair and sensory-motor nerve function. METHODS: Adult male and female Sprague-Dawley rats were used. To assess the effects of 4975 on nerve or muscle, 0.0083 or 0.025 mg 4975 or vehicle (25% polyethylene glycol-300) was applied to exposed sciatic nerve, or 0.1 mg 4975 or vehicle was injected into the surrounding muscle (Group 1). To assess the effect of 4975 on bone healing, an osteotomy was made in one femur and 0.5 mg of 4975 or vehicle was instilled into the site (Group 2). Behavioral testing was performed on both groups of rats and histological evaluation of the sciatic nerve, and surrounding soft tissue and bone was done at days 3, 14, and 28 after surgery. Femurs from osteotomy rats were assessed using peripheral quantitative computed tomography and biomechanical testing. Standard statistical tests were used to compare groups. RESULTS: Rats with direct application of 4975 to the sciatic nerve and surrounding muscle were no different from the controls in nociceptive sensory responses (F = 0.910, P = 0.454), grip strength (F = 0.550, P = 0.654), or histology of the muscle or sciatic nerve. In osteotomy rats, there were no statistical differences between 4975 and vehicle-treated rats for bone area (H = 2.858, P = 0.414), bone mineral content (F = 0.945, P = 0.425), or bone mineral density (F = 0.87, P = 0.462) and no difference in soft tissue healing. There were neither differences in bone stiffness (F = 1.369, P = 0.268) nor were there noticeable differences in the macro- or microscopic appearance of the right femur osteotomy healing site and surrounding soft tissues between the control group and the 4975-treated animals. CONCLUSION: A single, clinically relevant application of instilled or injected 4975 has no observable adverse effect on wound and bone healing after osteotomy or on the structural integrity of exposed muscle and nerve.

Neurotoxic effects of zoniporide: a selective inhibitor of the NA+/H+ exchanger isoform 1.

Zoniporide, an inhibitor of the Na+-H+ exchanger-1, was administered by continuous intravenous infusion to rats and dogs for up to 1 month. In 1-month studies, histological and functional changes were observed in select portions of the peripheral nervous system; however, these findings were not detected in 2-week studies at similar or higher doses. In the 1-month rat study, there was dose-dependent, minimal, focal, or multifocal nerve fiber (axonal) degeneration in the spinal cord and/or sciatic nerve. In a follow-up rat study, findings included slowing of caudal nerve conduction velocity and axonal degeneration in the spinal cord (dorsal funiculus), dorsal roots, dorsal root ganglia (DRG), radial, sciatic, and tibial nerves. In the 1-month dog study, there was impairment of the patellar reflex and associated postural reaction changes, minimal to marked proximal nerve fiber degeneration in the DRG, and minimal nerve fiber degeneration in the dorsal roots and funiculi of the spinal cord. Minimal nerve fiber degeneration of equivocal significance was noted in various peripheral nerves. Taken together, these findings were consistent with a specific effect on peripheral sensory nerve fibers. These studies demonstrated that zoniporide produces clinical, electrophysiologic, and microscopic evidence of peripheral sensory axonopathy and establishes the importance of careful preclinical evaluation of neurological function.

Studies on articular and general toxicity of orally administered ozenoxacin in juvenile rats and dogs.

AIM: Ozenoxacin is a nonfluorinated quinolone antibacterial approved for topical treatment of impetigo. Because quinolones have known chondrotoxic effects in juvenile animals, the potential toxicity of ozenoxacin was assessed in preclinical studies. MATERIALS & METHODS: Ozenoxacin or ofloxacin (300 mg/kg/day for 5 days, for each compound) was orally administered to juvenile rats, and oral ozenoxacin (10-100 mg/kg/day for 14 days) was administered to juvenile dogs. RESULTS: In juvenile rats, ozenoxacin showed no chondrotoxicity, whereas ofloxacin produced typical quinolone-induced lesions in articular cartilage in three of ten rats. Oral ozenoxacin administration to juvenile dogs showed no chondrotoxicity or toxicologically relevant findings in selected target organs. CONCLUSION: Ozenoxacin was generally well-tolerated in juvenile rats and dogs, with no evidence of quinolone-induced arthropathy.

One Year of Abaloparatide, a Selective Activator of the PTH1 Receptor, Increased Bone Formation and Bone Mass in Osteopenic Ovariectomized Rats Without Increasing Bone Resorption.

Abaloparatide is a novel 34-amino acid peptide selected to be a potent and selective activator of the parathyroid hormone receptor (PTH1R) signaling pathway with 41% homology to PTH(1-34) and 76% homology to PTHrP(1-34). A 12-month treatment study was conducted in osteopenic ovariectomized (OVX) rats to characterize the mechanisms by which abaloparatide increases bone mass. Sprague-Dawley (SD) rats were subjected to OVX or sham surgery at age 6 months and left untreated for 3 months to allow OVX-induced bone loss. Ten OVX rats were euthanized after this bone depletion period, and the remaining OVX rats received daily subcutaneous injections of vehicle (n = 18) or abaloparatide at 1, 5, or 25 µg/kg/d (n = 18/dose level) for 12 months. Sham controls (n = 18) received vehicle daily. Bone densitometry and biochemical markers of bone formation and resorption were assessed longitudinally, and L3 vertebra and tibia were collected at necropsy for histomorphometry. Abaloparatide increased biochemical bone formation markers without increasing bone resorption markers or causing hypercalcemia. Abaloparatide increased histomorphometric indices of bone formation on trabecular, endocortical, and periosteal surfaces without increasing osteoclasts or eroded surfaces. Abaloparatide induced substantial increases in trabecular bone volume and density and improvements in trabecular microarchitecture. Abaloparatide stimulated periosteal expansion and endocortical bone apposition at the tibial diaphysis, leading to marked increases in cortical bone volume and density. Whole-body bone mineral density (BMD) remained stable in OVX-Vehicle controls while increasing 25% after 12 months of abaloparatide (25 µg/kg). Histomorphometry and biomarker data suggest that gains in cortical and trabecular bone mass were attributable to selective anabolic effects of abaloparatide, without evidence for stimulated bone resorption. © 2016 American Society for Bone and Mineral Research.

One year of abaloparatide, a selective peptide activator of the PTH1 receptor, increased bone mass and strength in ovariectomized rats.

Abaloparatide is a novel 34 amino acid peptide selected to be a potent and selective activator of the parathyroid hormone receptor 1 (PTHR1) signaling pathway. The effects of 12months of abaloparatide treatment on bone mass, bone strength and bone quality was assessed in osteopenic ovariectomized (OVX) rats. SD rats were subjected to OVX or sham surgery at 6months of age and left untreated for 3months to allow OVX-induced bone loss. Eighteen OVX rats were sacrificed after this bone depletion period, and the remaining OVX rats received daily s.c. injections of vehicle (n=18) or abaloparatide at 1, 5 or 25µg/kg/d (n=18/dose level) for 12months. Sham controls (n=18) received vehicle daily. Bone changes were assessed by DXA and pQCT after 0, 3, 6 or 12months of treatment, and destructive biomechanical testing was conducted at month 12 to assess bone strength and bone quality. Abaloparatide dose-dependently increased bone mass at the lumbar spine and at the proximal and diaphyseal regions of the tibia and femur. pQCT revealed that increased cortical bone volume at the tibia was a result of periosteal expansion and endocortical bone apposition. Abaloparatide dose-dependently increased structural strength of L4-L5 vertebral bodies, the femur diaphysis, and the femur neck. Increments in peak load for lumbar spine and the femur diaphysis of abaloparatide-treated rats persisted even after adjusting for treatment-related increments in BMC, and estimated material properties were maintained or increased at the femur diaphysis with abaloparatide. The abaloparatide groups also exhibited significant and positive correlations between bone mass and bone strength at these sites. These data indicate that gains in cortical and trabecular bone mass with abaloparatide are accompanied by and correlated with improvements in bone strength, resulting in maintenance or improvement in bone quality. Thus, this study demonstrated that long-term daily administration of abaloparatide to osteopenic OVX rats led to dose-dependent improvements in bone mass, geometry and strength.

Prevention and treatment of experimental osteomyelitis in dogs with ciprofloxacin-loaded crosslinked high amylose starch implants.

Crosslinked high amylose starch (CLHAS) matrix was used as a biodegradable drug delivery implant for the prevention and treatment of osteomyelitis. Thirty-two dogs underwent the femoral insertion of a screw inoculated with Staphylococcus aureus and were then randomly assigned to four groups: (A) prevention with ciprofloxacin-CLHAS implants, (B) surgical debridement (positive control), (C) surgical debridement and oral ciprofloxacin treatment and (D) surgical debridement and treatment with ciprofloxacin-CLHAS implants. At week 4 the osteomyelitis was confirmed, the infected site debrided and respective treatments initiated for groups B, C and D. Radiographs, macroscopic evaluations, bacterial cultures and histopathological examinations were used to evaluate the femora at week 10. Femora from preventive group A were almost normal. Dogs of both ciprofloxacin treatment groups C and D showed better bone healing, less periosteal reaction and less screw mobility than dogs from group B. Eradication of infection was observed at proximal/distal sites in B: 25%/12%, C: 37%/62% and D: 62%/75%. Both ciprofloxacin treated groups improved radiographically from week 4 to week 10. Periosteal and marrow neutrophilic and lymphoplasmocytic infiltrations were less severe in groups C and D versus group B. These data suggest that biodegradable ciprofloxacin-CLHAS implants are a safe and efficient modality for the prevention and treatment of osteomyelitis.

Infant cynomolgus monkeys exposed to denosumab in utero exhibit an osteoclast-poor osteopetrotic-like skeletal phenotype at birth and in the early postnatal period.

RANKL is a key regulator of bone resorption and osteoclastogenesis. Denosumab is a fully human IgG2 monoclonal antibody that inhibits bone resorption by binding and inhibiting the activity of RANKL. To determine the effects of denosumab on pre- and postnatal skeletal growth and development, subcutaneous injections of 0 (control) or 50 mg/kg/month denosumab were given to pregnant cynomolgus monkeys from approximately gestation day (GD) 20 until parturition (up to 6 doses). For up to 6 months postpartum (birth day [BD] 180/181), evaluation of the infants included skeletal radiographs, bone biomarkers, and oral examinations for assessment of tooth eruption. Infant bones were collected at necropsy for densitometry, biomechanical testing, and histopathologic evaluation from control and denosumab-exposed infants on BD1 (or within 2 weeks of birth) and BD181, and from infants that died or were euthanized moribund from BD5 to BD69. In all denosumab-exposed infants, biomarkers of bone resorption and formation were markedly decreased at BD1 and BD14 and slightly greater at BD91 vs. control, then similar to control values by BD181. Spontaneous long bone fractures were detected clinically or radiographically in 4 denosumab-exposed infants at BD28 and BD60, with evidence of radiographic healing at ≥BD60. In BD1 infants exposed to denosumab in utero, radiographic evaluations of the skeleton revealed decreased long bone length; a generalized increased radio-opacity of the axial and appendicular skeleton and bones at the base of the skull with decreased or absent marrow cavities, widened growth plates, flared/club-shaped metaphysis, altered jaw/skull shape, and reduced jaw length; and delayed development of secondary ossification centers. Densitometric evaluations in these infants demonstrated a marked increase in bone mineral density at trabecular sites, but cortical bone mineral density was decreased. Histologically, long bone cortices were attenuated and there was an absence of osteoclasts. Bones with active endochondral ossification consisted largely of a dense network of retained primary spongiosa with reduced marrow space consistent with an osteopetrotic phenotype. A minimal increase in growth plate thickness largely due to the expansion of the hypertrophic zone was present. Retained woven bone was observed in bones formed by intramembranous ossification, consistent with absence of bone remodeling. These changes in bone tissue composition and geometry were reflected in reduced biomechanical strength and material properties of bones from denosumab-exposed infants. Material property changes were characterized by increased tissue brittleness reflected in reductions in calculated material toughness at the femur diaphysis and lack of correlation between energy and bone mass at the vertebra; these changes were likely the basis for the increased skeletal fragility (fractures). Although tooth eruption was not impaired in denosumab-exposed infants, the reduced growth and increased bone density of the mandible resulted in dental abnormalities consisting of tooth malalignment and dental dysplasia. Radiographic changes at BD1 persisted at BD28, with evidence of resumption of bone resorption and remodeling observed in most infants at BD60 and/or BD90. In 2 infants euthanized on BD60 and BD69, there was histologic and radiographic evidence of subphyseal/metaphyseal bone resorption accompanied by multiple foci of ossification in growth plates that were markedly increased in thickness. In infants necropsied at BD181, where systemic exposure to denosumab had been below limits of quantitation for approximately 3months, there was largely full recovery from all bone-related changes observed earlier postpartum, including tissue brittleness. Persistent changes included dental dysplasia, decreased bone length, reduced cortical thickness, and decreased peak load and ultimate strength at the femur diaphysis. In conclusion, the skeletal and secondary dental effects observed in infant monkeys exposed in utero to denosumab are consistent with the anticipated pharmacological activity of denosumab as a monoclonal antibody against RANKL and inhibitor of osteoclastogenesis. The resulting inhibition of resorption impaired both bone modeling and remodeling during skeletal development and growth. The skeletal phenotype of these infant monkeys resembles human infants with osteoclast-poor osteopetrosis due to inactivating mutations of RANK or RANKL.

Eldecalcitol, a vitamin D analog, reduces bone turnover and increases trabecular and cortical bone mass, density, and strength in ovariectomized cynomolgus monkeys.

Vitamin D insufficiency is common in elderly people worldwide, and intake of supplementary calcium and vitamin D is recommended to those with a high risk of fracture. Several clinical studies and meta-analyses have shown that calcium and vitamin D supplementation reduces osteoporotic fractures, and a strong correlation exists between vitamin D status and fracture risk. Vitamin D supplementations improve calcium balance in the body; however, it remains unclear whether vitamin D directly affects bone metabolism. Recently, eldecalcitol (ELD), an active form of vitamin D analog, has been approved for the treatment of osteoporosis in Japan. A 3-year clinical trial showed ELD treatment increased lumbar spine bone mineral density (BMD) and reduced fracture risk in patients with osteoporosis. To evaluate the mechanism of ELD action in bone remodeling, ovariectomized cynomolgus monkeys were treated with 0.1 or 0.3µg/day of ELD for 6months. This treatment increased lumbar BMD by 4.4% and 10.2%, respectively, and suppressed ovariectomy-induced increases in bone turnover markers compared to OVX-vehicle control. Histomorphometric analysis of bone revealed that both bone formation parameters and bone resorption parameters in the trabecular bone of the lumbar vertebrae were suppressed by ELD treatment. ELD treatment also improved biomechanical properties of the lumbar vertebrae and the femoral neck in the ovariectomized cynomolgus monkeys. These results indicate that, in a bone-remodeling animal model, ELD increases BMD and improves bone biomechanical properties by normalizing bone turnover. Therefore, ELD has a direct and potentially beneficial effect on bone metabolism.

Rat-specific decreases in platelet count caused by a humanized monoclonal antibody against sclerostin.

LY2541546 is a humanized monoclonal antibody (IgG(4)) that has been optimized for neutralizing activity against sclerostin. In 5-week and 6-month nonclinical safety studies in rats, LY2541546 caused dose-dependent reversible decreases in platelet counts accompanied by accelerated platelet production, increased megakaryocytes, and altered megakaryocyte morphology. These treatment-related effects resulted in altered primary hemostasis as manifested by prolonged bleeding after phlebotomy or incidental toenail break. In some cases, the defects in hemostasis were sufficient to result in death of the affected rats. There was no evidence in rats of general bone marrow suppression or processes (e.g., disseminated intravascular coagulopathy) that may result in thrombocytopenia. Cynomolgus monkeys given LY2541546 for 5 weeks or 9 months had no changes in platelet count or megakaryocytes. In vitro cross-reactivity studies in rats, cynomolgus monkeys, and humans revealed LY2541546-bound rat but not cynomolgus monkey or human platelets and megakaryocytes. These data taken together demonstrated that the platelet and megakaryocyte effects in rats had a species-specific pathogenesis which likely involved LY2541546 binding of a rat-specific antigen on the surface of platelets and megakaryocytes resulting in the increased clearance of platelets and megakaryocyte hyperplasia. The species-specific nature of these reversible toxicological findings combined with the ease of clinical monitoring using standard hematology enabled the safe initiation of clinical studies in human volunteers.

Skeletal effects of bazedoxifene paired with conjugated estrogens in ovariectomized rats.

A novel approach to menopausal therapy is the tissue selective estrogen complex (TSEC) that partners bazedoxifene (BZA) with conjugated estrogens (CE). We examined the effects of daily treatment with BZA 0.3mg/kg, CE 2.5mg/kg, or combined BZA/CE (BZA 0.1, 0.3, or 1.0mg/kg with CE 2.5mg/kg) over 12months on bone mass, bone architecture and strength, and biochemical markers of bone turnover in ovariectomized (OVX) female Sprague-Dawley rats vs OVX control rats. Total cholesterol and uterine weights were also evaluated. All BZA/CE dose combinations prevented ovariectomy-induced increases in bone turnover and significantly increased bone mineral density (BMD) at the lumbar spine, proximal femur, and tibia compared with OVX controls. All BZA/CE doses evaluated also prevented many of the ovariectomy-induced changes of the static and dynamic parameters of the cortical compartment of the tibia and the cancellous compartment of the L1 and L2 vertebrae. All BZA/CE doses increased biomechanical strength at the lumbar spine (L4) compared with OVX animals. The co-administration of BZA 0.3 and 1.0mg/kg/day with CE 2.5mg/kg/day showed a dose-dependent reduction in uterine wet weight compared with administration of CE alone. All BZA/CE doses significantly lowered total cholesterol levels compared with OVX controls. In conclusion, 12months of treatment with BZA/CE in OVX rats effectively maintained BMD, bone microstructure, and bone quality; and the pairing of BZA with CE prevented CE-induced uterine stimulation.

Potential of skin fibroblasts for application to anterior cruciate ligament tissue engineering.

Fibroblasts isolated from skin and from anterior cruciate ligament (ACL) secrete type I and type III collagens in vivo and in vitro. However, it is much easier and practical to obtain a small skin biopsy than an ACL sample to isolate fibroblasts for tissue engineering applications. Various tissue engineering strategies have been proposed for torn ACL replacement. We report here the results of the implantation of bioengineered ACLs (bACLs), reconstructed in vitro using a type I collagen scaffold, anchored with two porous bone plugs to allow bone-ligament-bone surgical engraftment. The bACLs were seeded with autologous living dermal fibroblasts, and grafted for 6 months in goat knee joints. Histological and ultrastructural observations ex vivo demonstrated a highly organized ligamentous structure, rich in type I collagen fibers and cells. Grafts' vascularization and innervation were observed in all bACLs that were entirely reconstructed in vitro. Organized Sharpey's fibers and fibrocartilage, including chondrocytes, were present at the osseous insertion sites of the grafts. They showed remodeling and matrix synthesis postimplantation. Our tissue engineering approach may eventually provide a new solution to replace torn ACL in humans.