Bending Assay Research Articles

Eucommia ulmoides extract regulates oxidative stress to maintain calcium homeostasis and improve diabetic osteoporosis.

Diabetic osteoporosis (DOP) is a secondary disease that severely affects the health and quality of life of patients with diabetes mellitus. This study aimed to explore the bone protective effect of aqueous extract of Eucommia ulmoides (EUL) in DOP mice. DOP mice were established using a high-sugar, high-fat diet and streptozotocin (STZ) (35 mg/kg for three consecutive days), and the EUL aqueous extract (2.5 g/kg/day) was orally administered for 6 weeks. The serum levels of oxidative stress-related factors, calcium, and phosphorus were assessed using biochemical assays. The osteoprotective effect of EUL was assessed using micro-computer tomography, three-point bending assay, histological analysis, and immunoblotting. Quantitative real-time polymerase chain reaction and western blotting were performed to detect the expression levels of calcium transport channel factors in the kidney and small intestine tissues. Furthermore, the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in the femur, kidney, and small intestine tissues were detected using western blotting and quantitative real-time polymerase chain reaction. EUL aqueous extract reduced blood glucose levels, increased body weight, and relieved symptoms in DOP mice (p < .05). It also increased bone mineral density, improved the bone microstructure, decreased the number of femoral osteoclasts, and increased the expression of femoral Runx2 and Bmp2 in DOP mice (p < .01). After 6 weeks of EUL aqueous extract administration, serum levels of SOD, CTA, calcium, and phosphorus were upregulated, whereas MDA levels were decreased (p < .01). The aqueous EUL extract also upregulated the expression of TRPV5, PMCA-1b, and CaBP-9 k in the kidney and small intestine of DOP mice (p < .01). Furthermore, the expression of Nrf2 and HO-1 in the kidney, small intestine, and femur tissues was increased (p < .01). EUL aqueous extract reduced blood glucose levels in DOP mice and regulated oxidative stress through the Nrf2/HO-1 pathway, thereby maintaining calcium homeostasis and ultimately improving bone quality. Our study suggested that EUL aqueous extract may be effective in the treatment of DOP.

OPN, BSP, and Bone Quality-Structural, Biochemical, and Biomechanical Assessment in OPN-/-, BSP-/-, and DKO Mice.

Osteopontin (OPN) and Bone Sialoprotein (BSP), abundantly expressed by osteoblasts and osteoclasts, appear to have important, partly overlapping functions in bone. In gene-knockout (KO, -/-) models of either protein and their double (D)KO in the same CD1/129sv genetic background, we analyzed the morphology, matrix characteristics, and biomechanical properties of femur bone in 2 and 4month old, male and female mice. OPN-/- mice display inconsistent, perhaps localized hypermineralization, while the BSP-/- are hypomineralized throughout ages and sexes, and the low mineralization of young DKO mice recovers with age. The higher contribution of primary bone remnants in OPN-/- shafts suggests a slow turnover, while their lower percentage in BSP-/- indicates rapid remodeling, despite FTIR-based evidence in this genotype of a high maturity of the mineralized matrix. In 3-point bending assays, OPN-/- bones consistently display higher Maximal Load, Work to Max. Load and in young mice Ultimate Stress, an intrinsic characteristic of the matrix. Young male and old female BSP-/- also display high Work to Max. Load along with low Ultimate Stress. Principal Component Analysis confirms the major role of morphological traits in mechanical competence, and evidences a grouping of the WT phenotype with the OPN-/- and of BSP-/- with DKO, driven by both structural and matrix parameters, suggesting that the presence or absence of BSP has the most profound effects on skeletal properties. Single or double gene KO of OPN and BSP thus have multiple distinct effects on skeletal phenotypes, confirming their importance in bone biology and their interplay in its regulation.

SiJunZi decoction ameliorates bone quality and redox homeostasis and regulates advanced glycation end products/receptor for advanced glycation end products and WNT/β-catenin signaling pathways in diabetic mice

Ethnopharmacological relevanceSiJunZi decoction (SJZD), one of the traditional Chinese medicine formulas, has been clinically and traditionally used to improve glucose and lipid metabolism and promote bone remodeling. Aim of the studyTo study the actions and mechanisms of SJZD on bone remodeling in a type 2 diabetes mouse model. Materials and methodsDiabetic mice generated with a high-fat diet (HFD) and streptozotocin (STZ) were subjected to SJZD treatment for 8 weeks. Blood glucose and lipid profile, redox status and bone metabolism were determined by ELISA or biochemical assays. Bone quality was evaluated by micro-CT, three-point bending assay and Fourier transform infrared spectrum (FTIR). Bone histomorphometry alterations were evaluated by Hematoxylin-Eosin (H&E), tartrate resistant acid phosphatase (TRAP) staining and Safranin O-fast green staining. The expressions of superoxide dismutase 1 (SOD1), advanced glycation end products (AGEs), receptor for advanced glycosylation end products (RAGE), phosphorylated nuclear factor kappa-B (p–NF–κB), NF-κB, cathepsin K, semaphorin 3A (Sema3A), insulin-like growth factor 1 (IGF1), p-GSK-3β, (p)-β-catenin, Runt-related transcription factor 2 (Runx2) and Cyclin D1 in the femurs and/or tibias were examined by Western blot or immunohistochemical staining. The main constituents in the SJZD aqueous extract were characterized by a HPLC/MS. ResultsSJZD intervention improved glucose and lipid metabolism and preserved bone quality in the diabetic mice, in particular glucose tolerance, lipid profile, bone microarchitecture, strength and material composition. SJZD administration to diabetic mice preserved redox homeostasis in serum and bone marrow, and prevented an increase in AGEs, RAGE, p–NF–κB/NF-κB, cathepsin K, p-GSK-3β, p-β-catenin expressions and a decrease in Sema3A, IGF1, β-catenin, Runx2 and Cyclin D1 expressions in tibias and/or femurs. Thirteen compounds were identified in SJZD aqueous extract, including astilbin, liquiritin apioside, ononin, ginsenoside Re, Rg1, Rb1, Rb2, Ro, Rb3, Rd, notoginsenoside R2, glycyrrhizic acid, and licoricesaponin B2. ConclusionsSJZD ameliorates bone quality in diabetic mice possibly via maintaining redox homeostasis. The mechanism governing these alterations are possibly related to effects on the AGEs/RAGE and Wnt/β-catenin signaling pathways. SJZD may offer a novel source of drug candidates for the prevention and treatment of type 2 diabetes and osteoporosis.

Zoledronic Acid Prevents Bone Resorption Caused by the Combination of Radium-223, Abiraterone Acetate, and Prednisone in an Intratibial Prostate Cancer Mouse Model.

An increased risk of non-pathological fractures in patients with prostate cancer and bone metastases has been associated with combination treatment with radium-223, abiraterone, and prednisone/prednisolone in the absence of bone-protecting agents. Here, we investigated possible mechanisms leading to this outcome using an intratibial LNCaP model mimicking prostate cancer bone metastases. Male NOD.scid mice were inoculated intratibially with LNCaP prostate cancer cells and treated with vehicle, radium-223, abiraterone, prednisone, zoledronic acid, or their combinations for 28 days. Serum TRACP 5b and PSA levels were measured. Bone structure, quality, and formation rate of non-tumor-bearing and tumor-bearing tibiae were analyzed by microCT, 3-point bending assay, and dynamic histomorphometry, respectively. Radium-223 incorporation into bone was also measured. Radium-223/abiraterone/prednisone combination treatment induced a transient increase in bone resorption indicated by elevated TRACP 5b levels, which was inhibited by concurrent treatment with zoledronic acid. Furthermore, radium-223/abiraterone/prednisone combination reduced periosteal and trabecular new bone formation and the number of osteoblasts, but bone structure or biomechanical quality were not affected. The abiraterone/prednisone treatment decreased radium-223 incorporation into tumor-bearing bone, possibly explaining the lack of additional antitumor efficacy. In conclusion, radium-223/abiraterone/prednisone combination increased bone resorption, which may have been one of the mechanisms leading to an increased fracture risk in patients with mCRPC.

A static magnetic field improves bone quality and balances the function of bone cells with regulation on iron metabolism and redox status in type 1 diabetes.

Osteoporosis is one of the chronic complications of type 1 diabetes with high risk of fracture. The prevention of diabetic osteoporosis is of particular importance. Static magnetic fields (SMFs) exhibit advantages on improvement of diabetic complications. The biological effects and mechanism of SMFs on bone health of type 1 diabetic mice and functions of bone cells under high glucose have not been clearly clarified. In animal experiment, six-week-old male C57BL/6J mice were induced to type 1 diabetes and exposed to SMF of 0.4-0.7 T for 4 h/day lasting for 6 weeks. Bone mass, biomechanical strength, microarchitecture and metabolism were determined by DXA, three-point bending assay, micro-CT, histochemical and biochemical methods. Exposure to SMF increased BMD and BMC of femur, improved biomechanical strength with higher ultimate stress, stiffness and elastic modulus, and ameliorated the impaired bone microarchitecture in type 1 diabetic mice by decreasing Tb.Pf, Ct.Po and increasing Ct.Th. SMF enhanced bone turnover by increasing the level of markers for bone formation (OCN and Collagen I) as well as bone resorption (CTSK and NFAT2). In cellular experiment, MC3T3-E1 cells or primary osteoblasts and RAW264.7 cells were cultured in 25 mM high glucose-stimulated diabetic marrow microenvironment under differentiation induction and exposed to SMF. SMF promoted osteogenesis with higher ALP level and mineralization deposition in osteoblasts, and it also enhanced osteoclastogenesis with higher TRAP activity and bone resorption in osteoclasts under high glucose condition. Further, SMF increased iron content with higher FTH1 expression and regulated the redox level through activating HO-1/Nrf2 in tibial tissues, and lowered hepatic iron accumulation by BMP6-mediated regulation of hepcidin and lipid peroxidation in mice with type 1 diabetes. Thus, SMF may act as a potential therapy for improving bone health in type 1 diabetes with regulation on iron homeostasis metabolism and redox status.

Highly porous FeNi 3D foams produced by one-step electrodeposition: Electrochemical stability and mechanical properties

Highly porous iron‑nickel foams, including the first-ever reported pure iron foam, were prepared by the dynamic hydrogen bubble template electrodeposition method and studied regarding their electrochemical stability and mechanical properties. Changes on the morphology, microstructure, and electrochemical resistance of the foams were investigated as a function of the Fe:Ni ratio by scanning electron microscopy (SEM), d.c. polarization and electrochemical impedance spectroscopy (EIS). Tensile and bending tests were carried out to evaluate the mechanical behavior of the prepared foams. Both crystalline structure and morphology are affected by the increase of Ni content, evolving from a highly porous foam with a body-centered cubic (bcc) structure to a denser material with open pores and face-centered cubic (fcc) structure. Polarization curves reveal an ennoblement of the corrosion potential as the nickel content increases. The impedance response revealed a dependence on the foam porosity. An increased number of pores was found more detrimental to the mechanical properties of the foams, as fractures seem to propagate, preferentially by cracks due to connecting pores. As result, foams with higher Ni contents displayed better mechanical performance for both tensile and bending assays. Results show that this innovative and powerfull technique enables to design iron foams with the adequate properties required for industrial applications. • Fe and FeNi nanofoams are prepared by Dynamic Hydrogen Bubble Template Electrodeposition. • Pure iron foam is obtained for the first time. • Foams morphology change for higher Ni contents. • Faradaic resistance depends on both %Fe and the pore availability. • Improved mechanical resistance is observed for the less porous foams.

Lycopene Improves Bone Quality and Regulates AGE/RAGE/NF-кB Signaling Pathway in High-Fat Diet-Induced Obese Mice.

Objective This study was aimed at examining the effects of lycopene on bone metabolism in high-fat diet (HFD)- induced obese mice and to identify the potential underlying mechanisms. Methods Mice were fed a HFD for 12 weeks and then continue with or without lycopene intervention (15 mg/kg) for additional 10 weeks. The effects of lycopene on blood glucose and lipid metabolism, as well as serum levels of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and malondialdehyde (MDA) were determined by biochemical assays. Bone histomorphological features and osteoclast activity were assessed by hematoxylin/eosin and tartrate-resistant acid phosphatase staining. Bone microstructure at the proximal tibial metaphysis and diaphysis was determined by microcomputed tomography. Tibial biomechanical strength and material profiles were measured by a three-point bending assay and Fourier transform infrared spectroscopy. Protein expressions involved in the AGE/RAGE/NF-кB signaling pathway were determined by western blot and/or immunohistochemical staining. Results Lycopene consumption reduced body weight gain and improved blood glucose and lipid metabolism in HFD-induced obese mice. In addition, lycopene treatment preserved bone biomechanical strength, material profiles, and microarchitecture in obese mice. Moreover, these alterations were associated with an increase in serum levels of T-AOC and SOD, and a decline in serum levels of MDA, as well as a reduction of AGEs, RAGE, cathepsin K, and p-NF-кBp65 and NF-кBp65 expressions in the femurs and tibias of obese mice. Conclusion Lycopene may improve bone quality through its antioxidant properties, which may be linked with the regulation of the AGE/RAGE/NF-кB signaling pathway in obese mice. These results suggest that lycopene consumption may be beneficial for the management of obesity-induced osteoporosis.

Examining the novel use of continuous compression implants in clavicle reconstruction: A biomechanical study

BackgroundCurrent implants for clavicle fractures are known to cause poor cosmesis and irritation, which may require implant removal. Low-profile shape-memory staples provide an attractive alternative, but their biomechanical utility in clavicle reconstruction is unknown. We hypothesized that shape-memory reconstructions would be more compliant compared to traditional constructs but would also outperform conventional plates during cyclic loading to failure. MethodsThis study was performed with 36 synthetic clavicles and 12 matched pairs of cadaveric specimens. The synthetic study tested four reconstructions: a single superiorly placed staple (n = 6), a single anteroinferiorly-placed staple (n = 6), a 3.5 mm reconstruction plate (n = 12), and two orthogonally placed staples (n = 12). The cadaveric study tested three constructs: reconstruction plate (n = 8), two orthogonal staples (n = 8), and a 2.7 mm reconstruction plate combined with a superior staple (n = 8). Non-destructive 4-point bending, compression, and torsion assays were performed prior to destructive cantilever bending and cyclic torsion tests. FindingsThe single staple and double staple groups demonstrated significantly decreased resistance to bending (p < 0.001) and torsion (p ≤ 0.027) when compared to reconstruction plate groups. The double staple group sustained significantly fewer cycles to failure than the reconstruction plate group in cyclic torsional tests (p = 0.012). The synthetic models produced higher stiffness and failure mechanisms that were completely different from cadaveric specimens. InterpretationShape memory alloy implants provided inadequate stiffness for clavicle fixation but may have utility in other orthopaedic applications when used as a supplementary compression device in conjunction with traditional plated constructs. Synthetic bones have limited capacity for modeling fragility fractures.

Fructus Ligustri Lucidi aqueous extract promotes calcium balance and short-chain fatty acids production in ovariectomized rats

Ethnopharmacological relevanceFructus Ligustri Lucidi (FLL) is an edible herb with anti-osteoporotic activity, yet whether and how the aqueous extract of this herb affect calcium metabolism in preservation of bone quality remain unclear. Aim of the studyTo investigate the effects of FLL aqueous extract on calcium balance and short-chain fatty acids (SCFAs) production in ovariectomized (OVX) rats. Materials and methodsOVX rats were daily and orally administrated with FLL aqueous extract (3.5 g/kg) for 14 weeks. The levels of N-terminal propeptide of type I collagen (PINP) and C-terminal telopeptide of type I collagen (CTx-I) in rat serum were evaluated by ELISA assays. The concentration of calcium in serum, urine, and feces were determined by biochemical assays. Bone quality was determined by Micro-CT, a three-point bending assay, and Fourier Transform Infrared (FTIR) Spectrometry. The expressions of Calbindin D28K and Calcium-sensing receptor (CaSR) in kidney as well as the Vitamin D receptor (VDR), the transient receptor potential vanilloid receptor 6 (TRPV6), Calbindin D9k in the duodenum were measured by immunohistochemistry, western blotting, or real-time PCR. The short-chain fatty acids (SCFAs) levels in the feces of the cecum were tested by gas chromatograghy. ResultsThe administration of FLL to OVX rats resulted in a significant improvement in bone mineral density and biomechanical strength as well as in maintaining bone microstructures and material quality. Meanwhile, the decreased levels of PINP and increased levels of CTx-I in OVX rats were restored by FLL treatment. Additionally, FLL treatment increased calcium absorption, upregulated VDR, TRPV6, Calbindin D9k expressions in the duodenum, Calbindin D28K in kidney, and down-regulated CaSR expression in the kidney, as well as enhanced SCFAs levels in the feces of OVX rats. ConclusionsFLL aqueous extract may preserve bone quality through regulation of the calcium balance and intestinal SCFAs production in OVX rats. This offers translational value of FLL into osteoporosis clinical trial.

The Effects of 8-Week Resistance and Endurance Trainings on Bone Strength Compared to Irisin Injection Protocol in Mice.

Background:Osteoporosis is a prevalent elderly complication that is characterized by decreased bone mineral density and increased fracture risk because of dysregulation in bone mineralization and resorption. Physical activity can enhance bone strength by exerting mechanical forces and myokines. Irisin is a myokine that is increased following physical exercise and can affect bones. In this study, 8 weeks of resistance and endurance exercises are applied in mice compared to irisin injection to assess the contribution of the protocols and this myokine to bone strength.Materials and Methods:Thirty-five male NMRI mice were separated into five groups; control, placebo, irisin injection, resistance exercise, and endurance exercise. 8-week of exercise protocols and irisin injection protocol (100 μg/kg/week) was applied. Plasma irisin concentration and bone strength were measured using enzyme-linked immunoassay and 3-point bending assay, respectively. Statistical analyses were done through one-way ANOVA and Tukey test, and P < 0.05 was considered the significant difference.Results:Serum irisin concentration and bone strength in resistance exercise and irisin-injected groups were significantly higher than control and placebo groups (P < 0.0001). Serum irisin concentration, but not bone strength, of the endurance exercise group was also significantly higher than control and placebo groups (P < 0.0001) but lower than resistance and irisin-injected groups.Conclusion:Resistance exercise and irisin injection, but not endurance exercise, are likely to be effective in increasing bone strength. There may be a threshold for plasma irisin level to affect bones which the applied protocols of irisin injection and resistance exercise but not endurance exercise can reach.

Pulsed Electromagnetic Fields Ameliorate Skeletal Deterioration in Bone Mass, Microarchitecture, and Strength by Enhancing Canonical Wnt Signaling-Mediated Bone Formation in Rats with Spinal Cord Injury.

Spinal cord injury (SCI) leads to extensive bone loss and high incidence of low-energy fractures. Pulsed electromagnetic fields (PEMF) treatment, as a non-invasive biophysical technique, has proven to be efficient in promoting osteogenesis. The potential osteoprotective effect and mechanism of PEMF on SCI-related bone deterioration, however, remain unknown. The spinal cord of rats was transected at vertebral level T12 to induce SCI. Thirty rats were assigned to the control, SCI, and SCI+PEMF groups (n = 10). One week after surgery, the SCI+PEMF rats were subjected to PEMF (2.0 mT, 15 Hz, 2 h/day) for eight weeks. Micro-computed tomography results showed that PEMF significantly ameliorated trabecular and cortical bone microarchitecture deterioration induced by SCI. Three-point bending and nanoindentation assays revealed that PEMF significantly improved bone mechanical properties in SCI rats. Serum biomarker and bone histomorphometric analyses demonstrated that PEMF enhanced bone formation, as evidenced by significant increase in serum osteocalcin and P1NP, mineral apposition rate, and osteoblast number on bone surface. The PEMF had no impact, however, on serum bone-resorbing cytokines (TRACP 5b and CTX-1) or osteoclast number on bone surface. The PEMF also attenuated SCI-induced negative changes in osteocyte morphology and osteocyte survival. Moreover, PEMF significantly increased skeletal expression of canonical Wnt ligands (Wnt1 and Wnt10b) and stimulated their downstream p-GSK3β and β-catenin expression in SCI rats. This study demonstrates that PEMF can mitigate the detrimental consequence of SCI on bone quantity/quality, which might be associated with canonical Wnt signaling-mediated bone formation, and reveals that PEMF may be a promising biophysical approach for resisting osteopenia/osteoporosis after SCI in clinics.

Abstract 6286: The effects of prednisone, abiraterone acetate and radium-223 dichloride on bone in the intratibial LNCaP prostate cancer model

Abstract Radium-223 dichloride (Ra-223) is a targeted alpha therapy that binds to newly formed bone matrix in bone metastases and induces double-stranded DNA breaks in cancer cells, osteoblasts, and osteoclasts. Ra-223 showed survival benefit in patients with bone-metastatic castration-resistant prostate cancer in a pivotal phase 3 trial. Recently, in a phase 3 trial of Ra-223 in combination with abiraterone acetate (AA) and prednisone/prednisolone (P), an increased risk of fractures was observed, which could be largely diminished by concurrent administration of bone resorption-inhibiting bisphosphonates or denosumab. The aim of this study was to investigate the effects of vehicle, Ra-223, AA, P, AA/P, and Ra-223/AA/P on bone using a mouse model mimicking osteoblastic prostate cancer bone metastases. Male NOD.scid mice were injected intratibially with LNCaP cells. Following stratification based on serum PSA and total bone volume of the tumor-bearing tibia, mice were treated for 28 days. Serum TRACP 5b and PSA levels were measured at multiple time points. At sacrifice, hind limbs were collected to analyze healthy and tumor-bearing bone structure, quality and formation rate by microCT, 3-point bending assay and dynamic histomorphometry, respectively. Also, Ra-223 incorporation was measured. Treatment with AA/P reduced Ra-223 incorporation to bone. PSA levels were similarly reduced at sacrifice upon Ra-223, AA, AA/P, and Ra-223/AA/P treatment indicating the absence of triple combination-specific additive anti-tumor effects. Bone resorption marker TRACP 5b levels increased transiently during the first two weeks of Ra-223/AA/P treatment. Neither bone structure or bone biomechanical quality in healthy or tumor-bearing tibia were affected by Ra-223/AA/P. Regarding dynamic bone formation, trabecular mineralizing surface (MS) was reduced in all groups containing Ra-223, while periosteal MS was reduced in all groups receiving P. However, only Ra-223/AA/P reduced both trabecular and periosteal MS. In line with the inhibition of dynamic bone formation, Ra-223/AA/P treatment decreased the number of osteoblasts. The AA/P treatment-induced reduction of Ra-223 incorporation may mediate the lack of additive anti-tumor effects in the Ra-223/AA/P group. Regarding bone health, Ra-223/AA/P specifically induced a transient increase in bone resorption and inhibited both periosteal and trabecular new bone formation but did not lead to structural or biomechanical bone changes. This preclinical mechanism may nevertheless contribute to the increased fragility observed in the clinics and may explain the beneficial clinical effects of bone resorption-inhibiting bisphosphonates and denosumab. However, several species- and model-related factors may limit the translational relevance of these findings. Citation Format: Mari I. Suominen, Matias Knuuttila, Birgitta Sjöholm, Esa Alhoniemi, Dominik Mumberg, Jussi M. Halleen, Sanna-Maria Käkönen, Arne Scholz. The effects of prednisone, abiraterone acetate and radium-223 dichloride on bone in the intratibial LNCaP prostate cancer model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6286.

Making Paving Stones from Copper Mine Tailings as Aggregates.

Copper mining, the central axis of Chile’s economic development, produces a large number of tailings, which become a potential environmental risk. This study aims to evaluate the mechanical properties resulting from the making of Portland cement mixtures with tailings as aggregates so that they can be eventually used in paving stones for building inactive tailings dams. Tailings coming from two dams at a concentration plant located in Taltal (Chile) were used. Currently, Dam 1 is inactive, while Dam 2 is active. The tailings samples obtained from both dams were granulometrically characterized by sieving. In addition, pH, humidity, Eh, and mineralogical assays (sulfides, oxides, sulfates, carbonates, phosphates, and silicates) were measured. The fines content of the tailings from Dams 1 and 2 with a sieve size of N°200 ASTM were 76.2% and 29.6%, respectively. Therefore, owing to their high percentage of fines, they cannot be as used as concrete aggregates. Aggregates must contain a maximum percentage of fines so that mortars and concrete can meet Chilean standards. In this paper, to comply with a 7% and 15% fines content lower than 0.075 mm, tailings materials were mixed with conventional aggregates containing very little fines. In addition, a reference mixture was made with only tailings aggregates with and without a superplasticizer additive. To measure the mixtures of cement, aggregates, and tailings, bending and compression strength assays were made of the specimens after a 28-day curing, according to the Chilean standard. The results of the study show that the addition of only part of the tailings to the mixture increases bending strength by 26% and compression strength by 180% compared with the reference mortar, with a fines content lower than 0.075 mm in the 7% mixture, thus allowing paving stone manufacture with tailings materials. In addition, it was possible to increase the workability of the reference mixture by using superplasticizers as additives.

Combination therapy in the Col1a2G610C mouse model of Osteogenesis Imperfecta reveals an additive effect of enhancing LRP5 signaling and inhibiting TGFβ signaling on trabecular bone but not on cortical bone.

Enhancing LRP5 signaling and inhibiting TGFβ signaling have each been reported to increase bone mass and improve bone strength in wild-type mice. Monotherapy targeting LRP5 signaling, or TGFβ signaling, also improved bone properties in mouse models of Osteogenesis Imperfecta (OI). We investigated whether additive or synergistic increases in bone properties would be attained if enhanced LRP5 signaling was combined with TGFβ inhibition. We crossed an Lrp5 high bone mass (HBM) allele (Lrp5A214V) into the Col1a2G610C/+ mouse model of OI. At 6-weeks-of-age we began treating mice with an antibody that inhibits TGFβ1, β2, and β3 (mAb 1D11), or with an isotype-matched control antibody (mAb 13C4). At 12-weeks-old, we observed that combining enhanced LRP5 signaling with inhibited TGFβ signaling produced an additive effect on femoral and vertebral trabecular bone volumes, but not on cortical bone volumes. Although enhanced LRP5 signaling increased femur strength in a 3-point bending assay in Col1a2G610C/+ mice, femur strength did not improve further with TGFβ inhibition. Neither enhanced LRP5 signaling nor TGFβ inhibition, alone or in combination, improved femur 3-point-bending post-yield displacement in Col1a2G610C/+ mice. These pre-clinical studies indicate combination therapies that target LRP5 and TGFβ signaling should increase trabecular bone mass in patients with OI more than targeting either signaling pathway alone. Whether additive increases in trabecular bone mass will occur in, and clinically benefit, patients with OI needs to be determined.

Mechanism of maslinic acid of reducing the inflammatory reaction and oxidative stress in acute liver injury in mice

Objective To study the protective effect and mechanism of maslinic acid (MA) on acute liver injury (ALI) in rats. Methods Fifty BALB/c mice were randomly divided into control group, model group, and low (12.5 mg/kg), medium (25.0 mg/kg) and high doses (50.0 mg/kg) of MA, with 10 rats in each group. The control group was intraperitoneally injected with normal saline. The other groups were intraperitoneally injected with lipopolysaccharide (LPS) (50 mg/kg) and D-Gal N (500 mg/kg) to prepare mouse ALI model. The MA groups were administered with 12.5, 25.0, 50.0 mg/kg MA 1 h before model establishment, respectively. All the mice were sacrificed 6 h after model establishment, and serum and liver tissues were collected. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured. Hematoxylin-eosin staining was used to observe the pathological changes of liver tissue. Thiobarbituric acid method was used to determine malondialdehyde (MDA). H2O2 reaction product colorimetric was used to determine the content of myeloperoxidase (MPO). The tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in serum and liver tissue was detected by enzyme-linked immunosor- bent assay, respectively. Western Blot was conducted to detect the expression of nuclear factor E2 related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and the activation of nuclear factor-kappa B (NF-κB) pathway. Results Compared with the model group, the liver histopathology in the low, medium and high doses MA groups was significantly improved. The serum ALT and AST levels were decreased, and the differences were statistically significant (all P<0.05). The contents of MDA and MPO in liver tissues were decreased, and the differences were statistically significant (all P<0.05). The protein contents of Nrf2 and HO-1 were increased, the differences were statistically significant (all P<0.05). The NF-κB pathway was inhibited, and the differences were statistically significant (all P<0.05). The levels of TNF-α and IL-6 in serum and liver tissues were decreased, and the differences were statistically significant (all P<0.05). Conclusions MA has a protective effect on LPS/D-Gal N-induced ALI, and its mechanism is related to inhibition of NF-κB pathway and activation of Nrf2/HO-1 pathway. Key words: Maslinic acid; Acute liver injury; Lipopolysaccharide; D-galactosamine; Nuclear factor erythroid 2-related factor 2

Aqueous Extract of Mori Folium Exerts Bone Protective Effect Through Regulation of Calcium and Redox Homeostasis via PTH/VDR/CaBP and AGEs/RAGE/Nox4/NF-κB Signaling in Diabetic Rats.

Purpose: The present study is aimed to explore whether the aqueous extract of Mori Folium (MF) exhibits bone protective effect by regulating calcium and redox homeostasis in diabetic rats, and to identify the signaling pathways involved in this process.Methods: Diabetic rats were established using high-sugar and high-fat diet and streptozotocin (STZ) (30 mg/kg for 3 consecutive days). The serum levels of osteocalcin (OC), insulin-like growth factor-1 (IGF-1), tartrate-resistant acid phosphatase (TRAP), phosphorus (P), calcium (Ca), 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], parathormone (PTH), advanced glycation end products (AGEs), superoxide dismutase (SOD), and malondialdehyde (MDA), total antioxidant capacity (TAC), 8-hydroxy-2′-deoxyguanosine (8-OH-dG), and interleukin 6 (IL-6) were determined by ELISA or biochemical assays. Histopathological alterations in the femurs were evaluated by the stainings of hematoxylin-eosin (H&E) and alizarin red S. In addition, femoral strength was detected by a three-point bending assay, bone microstructure was detected with micro-computer tomography. Bone material properties were examined by Fourier-transform infrared spectroscopy. Furthermore, the expressions of IGF-1, runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), receptor activator of nuclear factor kappa-B ligand (RANKL), cathepsin K, AGEs, receptor of advanced glycation end products (RAGE), NADPH oxidase 4 (Nox4), and nuclear factor kappa-B (NF-κB) in the femurs and tibias, and the alterations in the levels of calcium-binding protein-28k (CaBP-28k), transient receptor potential V6 (TRPV6), and vitamin D receptor (VDR) in the kidneys and duodenums were determined by western blot and immunohistochemical analysis.Results: Treatment of diabetic rats with MF aqueous extract induces an increase in the levels of OC and IGF-1 as well as a decrease in TRAP level in serum. MF treatment also upregulates the expression of OPG, downregulates the expressions of AGEs, RAGE, Nox4, NF-κB, and RANKL, which leads to improve bone microstructure and strength exhibited by an increase in cortical area ratio, cortical thickness, and trabecular area ratio as well as ultimate load, elastic modulus, and bending stress in the femurs and tibias of diabetic rats. In addition, MF aqueous extract preserves bone material properties by decreasing the ratio of fatty acid/collagen and increasing the ratio of mineral/matrix in the femurs of diabetic rats. Moreover, MF treatment increases the levels of P, Ca, and 1,25(OH)2D3, and decreases the level of PTH in the serum, as well as upregulates the expressions of TRPV6 and VDR in the duodenums and CaBP-28k in the kidneys of diabetic rats. Additionally, MF has ability of rebuilding redox homeostasis and eliminating inflammatory stress by increasing the levels of SOD and TAC as well as decreasing the levels of IL-6, AGEs, MDA, and 8-OH-dG.Conclusions: MF treatment may improve bone quality through maintenance of calcium homeostasis via regulating the PTH/VDR/CaBP signaling, and elimination of oxidative stress via regulating the AGEs/RAGE/Nox4/NF-κB signaling. These results may suggest the potential of MF in preventing the development of diabetic osteoporosis.

Rehmanniae Radix Preparata suppresses bone loss and increases bone strength through interfering with canonical Wnt/β-catenin signaling pathway in OVX rats.

Determine the anti-osteoporotic effect of RRP in ovariectomized (OVX) rats and identify the signaling pathway involved in this process. OVX rats were treated with RRP aqueous extract for 14weeks. The serum levels of tartrate-resistant acid phosphatase (TRAP), receptor activator of nuclear factor kappa-Β ligand(RANKL), alkaline phosphatase (ALP), and osteoprotegerin (OPG) were determined by ELISA. Bone histopathological alterations were evaluated by H&E, Alizarin red S, and Safranin O staining. Bone mineral density (BMD) and bone microstructure in rat femurs and lumbar bones were determined by dual-energy X-ray absorptiometry and micro-computed tomography. Femoral bone strength was detected by a three-point bending assay. The expression of Phospho-glycogen synthase kinase 3 beta (p-GSK-3β), GSK-3β, Dickkopf-related protein 1 (DKK1), cathepsin K, OPG, RANKL, IGF-1, Runx2, β-catenin, and p-β-catenin was determined by western blot and/or immunohistochemical staining. Treatment of OVX rats with RRP aqueous extract rebuilt bone homeostasis demonstrated by increasing the levels of OPG as well as decreasing the levels of TRAP, RANKL, and ALP in serum. Furthermore, RRP treatment preserved BMD and mechanical strength by increasing cortical bone thickness and epiphyseal thickness as well as improving trabecular distribution in the femurs of OVX rats. In addition, RRP downregulated the expression of DKK1, sclerostin, RANKL, cathepsin K, and the ratio of p-β-catenin to β-catenin, along with upregulating the expression of IGF-1, β-catenin, and Runx2 and the ratio of p-GSK-3β to GSK-3β in the tibias and femurs of OVX rats. Echinacoside, jionoside A1/A2, acetoside, isoacetoside, jionoside B1, and jionoside B2 were identified in the RRP aqueous extract. RRP attenuates bone loss and improves bone quality in OVX rats partly through its regulation of the canonical Wnt/β-catenin signaling pathway, suggesting that RRP has the potential to provide a new source of anti-osteoporotic drugs.

Beta Palmitate Improves Bone Length and Quality during Catch-Up Growth in Young Rats.

Palmitic acid (PA) is the most abundant saturated fatty acid in human milk, where it is heavily concentrated in the sn-2-position (termed beta palmitate, BPA) and as such is conserved in all women, regardless of their diet or ethnicity, indicating its physiological and metabolic importance. We hypothesized that BPA improves the efficiency of nutrition-induced catch up growth as compared to sn-1,3 PA, which is present in vegetable oil. Pre-pubertal male rats were subjected to a 17 days food restriction followed by re-feeding for nine days with 1,3 PA or BPA-containing diets. We measured bone length, epiphyseal growth plate height (EGP, histology), bone quality (micro-CT and 3-point bending assay), and gene expression (Affymetrix). The BPA-containing diet improved most growth parameters: humeri length and EGP height were greater in the BPA-fed animals. Further analysis of the EGP revealed that the hypertrophic zone was significantly higher in the BPA group. In addition, Affymetrix analysis revealed that the diet affected the expression of several genes in the liver and EGP. Despite the very subtle difference between the diets and the short re-feeding period, we found a small but significant improvement in most growth parameters in the BPA-fed rats. This pre-clinical study may have important implications, especially for children with growth disorders and children with special nutritional needs.

Bactericidal dental nanocomposites containing 1,2,3-triazolium-functionalized POSS additive prepared through thiol-ene click polymerization

ObjectiveDeterioration of mechanical strength for the dental composite containing ionic bactericidal compounds restricts the widespread utilization of this class of useful materials. This problem is originated from the reduction of the intermolecular interaction of polymeric network due to plasticization effect of absorbed water molecules penetrated between the chain segments. The main goal of this study is the synthesis of the highly efficient bactericidal additive with low hydrophilicity and consequently the least adverse effect on the final mechanical strength of the dental composite. MethodsThe bactericidal 1, 2, 3-triazolium functional groups were chemically anchored on the surface of hydrophobic POSS nanoparticles (Triazolium-POSS) and incorporated into a dental restorative system composed of a ternary thiol-allyl ether-methacrylate resin and glass fillers. A similar system was also prepared, in which the POSS additive was replaced with quaternized dimethyl aminoethyl methacrylate monomer (DMAEMA-BC). The chemical structure of POSS derivatives was evaluated by 1HNMR and FTIR spectra. The water uptake of dental composites was evaluated at days 1 and 14 after immersion into water. The bactericidal activity of composite specimens against Streptococcus mutans (ATCC 35668) was determined based on ASTM E 2180 – 07. The flexural properties of samples were investigated through three-point bending assay and the shrinkage-strain of photo-cured resins was measured using the bonded-disk technique. The degree of conversion (DC %) of methacrylate functions was followed by FTIR spectroscopy. MTT assay was performed to investigate the cytocompatibility of samples. ResultsRegardless of the partial increase in water uptake for Triazolium-POSS-containing sample, this parameter was much favor than the composite made from DMAEMA-BC. Therefore, the lower decline in flexural properties was recorded under the wet condition for the former system. Incorporation of Triazolium-POSS had no significant effect on shrinkage strain and cytocompatibility of composite specimen, meanwhile, a higher degree of conversion of methacrylate functional groups was recorded. The Triazolium-POSS-containing nano composite showed significantly higher bactericidal activity against Streptococcus mutans than another studied model system. SignificanceThe new derivative of bactericidal POSS nanoparticles decorated with 1, 2, 3-Triazolium moieties is a highly efficient bactericidal compound. If Triazolium-POSS is incorporated into a proper dental resin formulation, it can provide a strong bactericidal activity for dental materials; in the meantime, it leads to minimum deterioration of their mechanical strength due to its low water uptake.

Differential effects of IGF-1 deficiency during the life span on structural and biomechanical properties in the tibia of aged mice.

Advanced aging is associated with the loss of structural and biomechanical properties in bones, which increases the risk for bone fracture. Aging is also associated with reductions in circulating levels of the anabolic signaling hormone, insulin-like growth factor (IGF)-1. While the role of IGF-1 in bone development has been well characterized, the impact of the age-related loss of IGF-1 on bone aging remains controversial. Here, we describe the effects of reducing IGF-1 at multiple time points in the mouse life span--early in postnatal development, early adulthood, or late adulthood on tibia bone aging in both male and female igf (f/f) mice. Bone structure was analyzed at 27 months of age using microCT. We find that age-related reductions in cortical bone fraction, cortical thickness, and tissue mineral density were more pronounced when IGF-1 was reduced early in life and not in late adulthood. Three-point bone bending assays revealed that IGF-1 deficiency early in life resulted in reduced maximum force, maximum bending moment, and bone stiffness in aged males and females. The effects of IGF-1 on bone aging are microenvironment specific, as early-life loss of IGF-1 resulted in decreased cortical bone structure and strength along the diaphysis while significantly increasing trabecular bone fraction and trabecular number at the proximal metaphysis. The increases in trabecular bone were limited to males, as early-life loss of IGF-1 did not alter bone fraction or number in females. Together, our data suggest that the age-related loss of IGF-1 influences tibia bone aging in a sex-specific, microenvironment-specific, and time-dependent manner.