Functional response and predation rate of Cryptolaemus montrouzieri (Coleoptera: Coccinellidae) to Paracoccus marginatus (Hemiptera: Pseudococcidae) at different temperatures.

The ladybug, Cryptolaemus montrouzieri (Mulsant) (Coleoptera: Cocccinellidae)(Mulsant)(Coleoptera: Cocccinellidae), is a highly efficient predator in controlling mealybug populations and is considered an effective agent for controlling the papaya mealybugs (Paracoccus marginatus) (Williams & Granara de Willink) (Hemiptera: Pseudococcidae). Various criteria have been proposed for evaluating predator effectiveness, with the consumption rate of prey by individual predators, specifically the functional response, emerging as a common and crucial metric. This study evaluated the functional responses of third- and fourth-instar larvae, as well as male and female adults (<48 h old) of C. montrouzieri to adult females of P. marginatus at 3 different temperatures (22 °C, 28 °C, and 35 °C) with 70% ±â€…5% RH and a 12L:12D h photoperiod. Prey densities were 2, 4, 8, 16, 32, 45, or 60 papaya mealybugs per predator for all tests. The response to prey density by third- and fourth-instar larvae or both sexes of adult C. montrouzieri was a type II at all temperatures. The highest attack rate and lowest handling time were estimated at 28 °C in males and 35 °C in females, respectively. The highest daily prey consumption rate occurred at 35 °C in both the immature and adult stages of C. montrouzieri. These findings support the potential of C. montrouzieri in controlling the papaya mealybug, especially in tropical and subtropical regions, given its search efficiency at high temperatures tested in this study. However, additional field investigations are needed to ascertain the control efficacy of C. montrouzieri for this mealybug in biocontrol programs.

Effects of Elevated CO2 on the Fitness Parameters of Individually- and Group-Reared Agasicles hygrophila (Coleoptera: Chrysomelidae).

Agasicles hygrophila Selman and Vogt (Coleoptera: Chrysomelidae) is a natural enemy of Alternanthera philoxeroides (Mart.) Griseb (Amaranthaceae: Alternanthera), a worldwide invasive weed. Elevated atmospheric CO2 concentrations may have significant impacts plants, herbivorous insects, and natural enemies. To assess the concurrent effect of elevated CO2 on the development time, fecundity, and population parameters of A. hygrophila, the age-stage, two-sex life table was used to understand the fitness and population parameters of individually-reared and group-reared A. hygrophila under elevated CO2 concentration. In individually-reared population, the development time of preadults, adult pre-oviposition period, and total pre-oviposition period of A. hygrophila in the elevated CO2 (eCO2, 750 ppm) treatment were shorter than those in the ambient CO2 (aCO2, 420 ppm) treatment. In group-reared population, the developmental time of preadults, female adult longevity, female proportion, adult pre-oviposition period, and total pre-oviposition period of A. hygrophila in eCO2 were longer than those in aCO2. Additionally, in both individually-reared and group-reared population, fecundity and oviposition days of A. hygrophila in eCO2 were higher than those in aCO2, and a higher intrinsic rate of increase, finite rate of increase, and the net reproductive rate of A. hygrophila were observed at eCO2. Moreover, shorter preadult development time, adult pre-oviposition period, total pre-oviposition period, male adult longevity, and higher fecundity were found in group-reared cohort at both aCO2 and eCO2. The results indicates that elevated CO2 has effects on the growth and reproduction of A. hygrophila, and the population growth rate of group-reared was faster and produced more offspring.

Sublethal and Transgenerational Toxicities of Chlorfenapyr on Biological Traits and Enzyme Activities of Paracoccus marginatus (Hemiptera: Pseudococcidae).

Papaya mealybug, Paracoccus marginatus Williams and Granara de Willink (Hemiptera: Pseudococcidae), is an economically important, invasive insect that is now distributed worldwide. Chlorfenapyr has been demonstrated to have a significant control effect on P. marginatus. In order to evaluate the sublethal and transgenerational effects of chlorfenapyr on P. marginatus, the life table data of three consecutive generations were collected and analyzed by the age stage, two-sex life table method, and the enzyme activities were assayed using a spectrophotometer. The results showed that exposure to the insecticide had significant effects on the biological traits of subsequent generations of P. marginatus, and a higher intrinsic rate of increase (r), finite rate of increase (λ), net reproductive rate (R0), and a shorter mean generation time (T) were observed in the chlorfenapyr-treated F1 mealybugs. Enzyme activity assays showed that chlorfenapyr significantly inhibited the activities of catalase (CAT) and peroxidase (POD) while activating the activities of superoxide dismutase (SOD), which suggested that SOD, CAT, and POD may play an important role in the self-defense of P. marginatus against chlorfenapyr. These results conclusively demonstrated that exposure of P. marginatus to sublethal concentrations of chlorfenapyr induced hormetic effects on the F1 generation while having negative effects on the F0 and F3 generations.

Fitness of the Papaya Mealybug, Paracoccus marginatus (Hemiptera: Pseudococcidae), after Transferring from Solanum tuberosum to Carica papaya, Ipomoea batatas, and Alternanthera philoxeroides.

The papaya mealybug, Paracoccus marginatus Williams and Granara de Willink (Hemiptera: Pseudococcidae), is a polyphagous invasive pest in China. The effect that the shifting of the host plant has on the fitness of a polyphagous pest is critical to its prevalence and potential pest control. In order to assess the fitness changes of P. marginatus after transferal from potato (Solanum tuberosum (Tubiflorae: Solanaceae)) to papaya (Carica papaya (Parietales: Caricacea)), sweet potato (Ipomoea batatas (Tubiflorae: Convolvulaceae)), and alligator weed (Alternanthera philoxeroides (Centrospermae: Amaranthaceae)), the life table data of three consecutive generations were collected and analyzed using the age-stage, two-sex life table method. The results showed that when P. marginatus was transferred from S. tuberosum to papaya, a higher intrinsic rate of increase (r) and finite rate of increase (λ) were observed. Paracoccus marginatus individuals transferred to I. batatas had the significantly lower population parameters than those on C. papaya; however, the fitness recovered for those on I. batatas after two generations. Paracoccus marginatus individuals were unable to complete development on A. philoxeroides. Our results conclusively demonstrate that P. marginatus individuals can readily adapt to C. papaya and I. batatas even after host plant shifting, and are capable of causing severe damage to these hosts.

Bioactive PLGA/tricalcium phosphate scaffolds incorporating phytomolecule icaritin developed for calvarial defect repair in rat model.

BACKGROUND/OBJECTIVES: For treatment of large bone defects challenging in orthopaedic clinics, bone graft substitutes are commonly used for the majority of surgeons. It would be proposed in the current study that our bioactive scaffolds could additionally serve as a local delivery system for therapeutic small molecule agents capable of providing support to enhance biological bone repair. METHODS: In this study, composite scaffolds made of poly (lactic-co-glycolic acid) (PLGA) and tricalcium phosphate (TCP) named by P/T was fabricated by a low-temperature rapid prototyping technique. For optimizing the scaffolds, the phytomolecule icaritin (ICT) was incorporated into P/T scaffolds called P/T/ICT. The osteogenic efficacies of the two groups of scaffolds were compared in a successfully established calvarial defect model in rats. Bone regeneration was evaluated by X-ray, micro-computerised tomography (micro-CT), and histology at weeks 4 and/or 8 post-implantation. In vitro induction of osteogenesis and osteoclastogenesis was established for identification of differentiation potentials evoked by icaritin in primary cultured precursor cells. RESULTS: The results of radiographies and decalcified histology demonstrated more area and volume fractions of newly formed bone within bone defect sites implanted with P/T/ICT scaffold than that with P/T scaffold. Undecalcified histological results presented more osteoid and mineralized bone tissues, and also more active bone remodeling in P/T/ICT group than that in P/T group. The results of histological staining in osteoclast-like cells and newly formed vessels indicated favorable biocompatibility, rapid bioresorption and more new vessel growth in P/T/ICT scaffolds in contrast to P/T scaffolds. Based on in vitro induction, the results presented that icaritin could significantly facilitate osteogenic differentiation, while suppressed adipogenic differentiation. Meanwhile, icaritin demonstrated remarkable inhibition of osteoclastogenic differentiation. CONCLUSION: The finding that P/T/ICT composite scaffold can enhance bone regeneration in calvarial bone defects through facilitating effective bone formation and restraining excessive bone resorption. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: The osteogenic bioactivity of icaritin facilitated PLGA/TCP/icartin composite scaffold to exert significant bone regeneration in calvarial defects in rat model. It might form an optimized foundation for potential clinical validation in bone defects application.

Magnesium and vitamin C supplementation attenuates steroid-associated osteonecrosis in a rat model.

Magnesium (Mg)-based biometal attracts clinical applications due to its biodegradability and beneficial biological effects on tissue regeneration, especially in orthopaedics, yet the underlying anabolic mechanisms in relevant clinical disorders are lacking. The present study investigated the effect of magnesium (Mg) and vitamin C (VC) supplementation for preventing steroid-associated osteonecrosis (SAON) in a rat experimental model. In SAON rats, 50 mg/kg Mg, or 100 mg/kg VC, or combination, or water control was orally supplemented daily for 2 or 6 weeks respectively. Osteonecrosis was evaluated by histology. Serum Mg, VC, and bone turnover markers were measured. Microfil-perfused samples prepared for angiography and trabecular architecture were evaluated by micro-CT. Primary bone marrow cells were isolated from each group to evaluate their potentials in osteoblastogenesis and osteoclastogenesis. The mechanisms were tested in vitro. Histological evaluation showed SAON lesions in steroid treated groups. Mg and VC supplementation synergistically reduced the apoptosis of osteocytes and osteoclast number, and increased osteoblast surface. VC supplementation significantly increased the bone formation marker PINP, and the combination significantly decreased the bone resorption marker CTX. TNFα expression and oxidative injury were decreased in bone marrow in Mg/VC/combination group. Mg significantly increased the blood perfusion in proximal tibia and decreased the leakage particles in distal tibia 2 weeks after SAON induction. VC significantly elevated the osteoblast differentiation potential of marrow cells and improved the trabecular architecture. The combination supplementation significantly inhibited osteoclast differentiation potential of marrow cells. In vitro study showed promoting osteoblast differentiation effect of VC, and anti-inflammation and promoting angiogenesis effect of Mg with underlying mechanisms. Mg and VC supplementation could synergistically alleviate SAON in rats, indicating great translational potentials of metallic minerals for preventing SAON.

Indirect Effect of Elevated CO2 on Population Parameters and Growth of Agasicles hygrophila (Coleoptera: Chrysomelidae), a Biocontrol Agent of Alligatorweed (Amaranthaceae).

Alligatorweed, Alternanthera philoxeroide (Mart.) Griseb. (Amaranthaceae) is an invasive weed in China that is often kept under control by the alligatorweed flea beetle, Agasicles hygrophila Selman and Vogt (Coleoptera: Chrysomelidae) introduced into China from Argentina in the 1980s. Elevated CO2 levels have been shown to have a direct effect on Ag. hygrophila. In order to fully evaluate the indirect effects of three different atmospheric concentrations of CO2 (420, 550, and 750 ppm) on the population parameters of Ag. hygrophila reared on Al. philoxeroides, we collected life table data for Ag. hygrophila using the age-stage, two-sex life table method. In general, there were no significant differences in the lengths of the preadult parameters among the three treatments. The adult duration and total longevity of males, however, did increase as CO2 increased in concentration. Although the adult preoviposition and total preoviposition periods decreased, the fecundity, oviposition days, eggs per oviposition day, net reproductive rate, intrinsic rate of increase, and finite rate of increase all increased significantly at the high CO2 concentration. Consequently, we determined that the Ag. hygrophila population size will potentially increase rapidly over a short period of time at elevated CO2 concentrations. Our results suggest that 550 and 750 ppm CO2 may also cause physiological changes in Al. philoxeroides that, in turn, provide enhanced nutrition for increasing reproduction in Ag. hygrophila by accelerating maturation of their reproductive system. These results indicate that the efficacy of Ag. hygrophila as a biological control agent against Al. philoxeroides will likely be increased at 550 and 750 ppm CO2.

Herbal Fufang Xian Ling Gu Bao prevents corticosteroid-induced osteonecrosis of the femoral head-A first multicentre, randomised, double-blind, placebo-controlled clinical trial.

BACKGROUND/OBJECTIVE: This is a multicentre, randomised, double-blind, placebo-controlled clinical trial to investigate the safety and efficacy of Chinese herbal Fufang Xian Ling Gu Bao (XLGB) with antiadipogenic compounds for the prevention of corticosteroid (CS)-induced osteonecrosis of femoral head (ONFH). METHODS: Patients of both genders, aged between 18 and 65 years, with diseases such as systemic lupus erythematosus, nephrosis, dermatosis and rheumatoid arthritis indicated for CS treatment and who did not show magnetic resonance imaging of ONFH at baseline were recruited into the study and then randomised into either XLGB group (n = 129) with daily oral administration of XLGB or placebo group (n = 146). RESULTS: Magnetic resonance imaging revealed a total of 30 ONFH cases at 6 months after CS treatment, with 6.98% (9 of 129 cases) and 14.4% (21 of 146 cases) in the XLGB group and placebo group, respectively, (p < 0.05), i.e., a 2-fold significantly less ONFH identified in the XLGB treatment group. Blood tests suggested that XLGB significantly inhibited the elevation of activated protein C resistance induced by CS treatment. CONCLUSION: This is the first multicentre clinical study to demonstrate that the antiadipogenic compounds-rich herbal Fufang (formula) XLGB is effective in preventing CS-associated ONFH in patients with immune-inflammatory diseases under CS treatment. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: The translation potential of this clinical trial is that the initially officially approved clinical indication for XLGB for treatment of osteoporosis has been now also proven to be effective for a new clinical application.

Steroid-associated osteonecrosis animal model in rats.

OBJECTIVE: Established preclinical disease models are essential for not only studying aetiology and/or pathophysiology of the relevant diseases but more importantly also for testing prevention and/or treatment concept(s). The present study proposed and established a detailed induction and assessment protocol for a unique and cost-effective preclinical steroid-associated osteonecrosis (SAON) in rats with pulsed injections of lipopolysaccharide (LPS) and methylprednisolone (MPS). METHODS: Sixteen 24-week-old male Sprague-Dawley rats were used to induce SAON by one intravenous injection of LPS (0.2 mg/kg) and three intraperitoneal injections of MPS (100 mg/kg) with a time interval of 24 hour, and then, MPS (40 mg/kg) was intraperitoneally injected three times a week from week 2 until sacrifice. Additional 12 rats were used as normal controls. Two and six weeks after induction, animals were scanned by metabolic dual energy X-ray absorptiometry for evaluation of tissue composition; serum was collected for bone turnover markers, Microfil perfusion was performed for angiography, the liver was collected for histopathology and bilateral femora and bilateral tibiae were collected for histological examination. RESULTS: Three rats died after LPS injection, i.e., with 15.8% (3/19) mortality. Histological evaluation showed 100% incidence of SAON at week 2. Dual energy X-ray absorptiometry showed significantly higher fat percent and lower lean mass in SAON group at week 6. Micro-computed tomography (Micro-CT) showed significant bone degradation at proximal tibia 6 weeks after SAON induction. Angiography illustrated significantly less blood vessels in the proximal tibia and significantly more leakage particles in the distal tibia 2 weeks after SAON induction. Serum amino-terminal propeptide of type I collagen and osteocalcin were significantly lower at both 2 and 6 weeks after SAON induction, and serum carboxy-terminal telopeptide was significantly lower at 6 weeks after SAON induction. Histomorphometry revealed significantly lower osteoblast surface and higher marrow fat fraction and oedema area in SAON group. Hepatic oedema appeared 2 weeks after SAON induction, and lipid accumulation appeared in the liver of SAON rats 6 weeks after SAON induction. CONCLUSION: The present study successfully induced SAON in rats with pulsed injection of LPS and MPS, which was well simulating the clinical feature and pathology. Apart from available large animal models, such as bipedal emus or quadrupedal rabbits, our current SAON small model in rats could be a cost-effective preclinical experimental model to study body metabolism, molecular mechanism of SAON and potential drugs developed for prevention or treatment of SAON. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: The present study successfully induced SAON in a small animal model in rats with pulsed injection of LPS and MPS. The evaluation protocols with typical histopathologic ON features and advanced evaluation approaches to identify the metabolic disorders of SAON could be used in future rat SAON studies. The SAON rat model is a suitable and cost-effective animal model to study molecular mechanism of SAON and potential drugs developed for prevention and treatment of SAON.

Role of mesenchymal stem cells in osteoarthritis treatment.

As the most common form of joint disorder, osteoarthritis (OA) imposes a tremendous burden on health care systems worldwide. Without effective cure, OA represents a unique opportunity for innovation in therapeutic development. In contrast to traditional treatments based on drugs, proteins, or antibodies, stem cells are poised to revolutionize medicine as they possess the capacity to replace and repair tissues and organs such as osteoarthritic joints. Among different types of stem cells, mesenchymal stem cells (MSCs) are of mesoderm origin and have been shown to generate cells for tissues of the mesoderm lineage, thus, raising the hope for them being used to treat diseases such as OA. However, given their ability to differentiate into other cell types, MSCs have also been tested in treating a myriad of conditions from diabetes to Parkinson's disease, apparently of the ectoderm and endoderm lineages. There are ongoing debates whether MSCs can differentiate into lineages outside of the mesoderm and consequently their effectiveness in treating conditions from the ectoderm and endoderm lineages. In this review, we discuss the developmental origin of MSCs, their differentiation potential and immunomodulatory effects, as well as their applications in treating OA. We suggest further investigations into new therapies or combination therapies that may provide more effective treatment for bone and joint diseases. Furthermore, cell-based therapy and its associated safety and effectiveness should be carefully evaluated before clinical translation. This review provides updated information on recent approval of clinical trials and related applications of MSCs, and discusses additional efforts on cell-based therapy for treating OA and other joint and bone diseases.

Validity of leptin receptor-deficiency (db/db) type 2 diabetes mellitus mice as a model of secondary osteoporosis.

This study aimed to evaluate the validation of the leptin receptor-deficient mice model for secondary osteoporosis associated with type 2 diabetes mellitus (T2DM) at bone micro-architectural level. Thirty three 36-week old male mice were divided into four groups: normal control (db/m) (n = 7), leptin receptor-deficient T2DM (db/db) (n = 8), human C-reactive protein (CRP) transgenic normal control (crp/db/m) (n = 7), and human CRP transgenic T2DM (crp/db/db) (n = 11). Lumber vertebrae (L5) and bilateral lower limbs were scanned by micro-CT to analyze trabecular and cortical bone quality. Right femora were used for three-point bending to analyze the mechanical properties. Trabecular bone quality at L5 was better in db/db or crp/db/db group in terms of bone mineral density (BMD), bone volume fraction, connectivity density, trabecular number and separation (all p < 0.05). However the indices measured at proximal tibia showed comparable trabecular BMD and microarchitecture among the four groups. Femur length in crp/db/db group was significantly shorter than db/m group (p < 0.05) and cortices were thinner in db/db and crp/db/db groups (p > 0.05). Maximum loading and energy yield in mechanical test were similar among groups while the elastic modulus in db/db and crp/db/db significantly lower than db/m. The leptin-receptor mice is not a proper model for secondary osteoporosis associated with T2DM.

Demography and Population Projection of Flea Beetle, Agasicles hygrophila (Coleoptera: Chrysomelidae), Fed on Alligator Weed Under Elevated CO 2.

The flea beetle, Agasicles hygrophila Selman and Vogt, was introduced into China in 1987. For a more comprehensive understanding of the effect of elevated CO 2 concentration on the population dynamics, we collected the life table data of the flea beetle, A. hygrophila , at two different CO 2 concentration conditions, i.e., ambient (420 µl/liter) and elevated (750 µl/liter). The raw data were analyzed using the age-stage, two-sex life table theory. At 750 µl/liter CO 2 , shorter developmental durations of the egg, first instar, and pupa were observed, while the duration of the third instar and the total developmental duration of the larva were prolonged. The generation length of A. hygrophila was significantly shortened at the higher concentration. It was observed that the intrinsic rate of increase ( r ), finite rate (λ), and net reproduction rate ( R0 ) were higher and the mean generation time ( T ) was shorter at 750 µl/liter compared with that at 420 µl/liter. The bootstrap techniques were adopted to estimate the variances and standard errors of the developmental time, longevity, fecundity, and the population parameters. The bootstrap technique generated a normal distribution that was consistent with the central limit theorem and critical for following statistical analysis and comparison. Population projections based on age-stage, two-sex life tables could reveal the stage structure of A. hygrophila population and the leaf consumption capacity. Data collected in this study can potentially be used to evaluate the efficacy of A. hygrophila as a biological control agent of the alligator weed.

Src siRNA prevents corticosteroid-associated osteoporosis in a rabbit model.

In an established steroid-associated osteonecrosis (SAON) rabbit model we found recently that blockage Src by siRNA could improve reconstructive repair of osteonecrosis via enhancing osteogenesis and inhibiting bone resorption. The current study investigated if blocking Src was able to prevent steroid-associated osteoporosis (SAOP) in the same SAON animal model. Rabbits were treated with pulsed lipopolysaccharide (LPS) and corticosteroid methylprednisolone (MPS). At 2, 4, and 6weeks after induction, Src siRNA, control siRNA and saline were intramedullary injected into proximal femur, respectively. Two fluorescent dyes xylenol orange and calcein green were injected before sacrificing the animals for in vivo labeling of the newly formed bone. At week 6 after induction, proximal femora of rabbits were dissected for micro-CT and histological analysis. Results showed significant bone loss in the metaphysis of femoral head in the control rabbits after SAON induction. Src siRNA treatment was able to prevent steroid-associate bone loss in trabecular bone and increase cortical bone thickness at femoral neck. Histomorphometry showed that Src siRNA increased the osteoblastic bone formation and decreased the eroded bone surfaces suggesting decreased osteoclastic bone resorption. This was the first study to report bone loss after SAON induction in rabbit model that could be prevented by knocking down Src by siRNA.

Comparative study of two types of herbal capsules with different Epimedium species for the prevention of ovariectomised-induced osteoporosis in rats.

BACKGROUND/OBJECTIVE: Epimedii Folium is the most important osteogenic herb formulated for the traditional Chinese Medicine Xian Ling Gu Bao (XLGB) capsule. The present study compared XLGB capsules containing two different Epimedium species, i.e., either Epimedium pubescens (XEP) or Epimedium koreanum (XEK), with the focus being on the chemical constituents and antiosteoporotic efficacy. METHODS: Ultra performance liquid chromatography was used to demonstrate the different chemical constituents. Biomechanical tests, histological, and cytological evaluation were performed to characterise and compare the bone mineral density, bone strength, microstructure of bone tissue, and biological activity between XEP and XEK using an established ovariectomised (OVX) rat model. RESULTS: Six flavonoids with different contents between XEK and XEP were identified. As compared with the OVX group, significantly higher bone mineral density, elastic-modulus, and compressive strength were found in both the XEK group and XEP group (p < 0.05 for all, n = 8). Histomorphometric data presented significantly higher osteoblast surface ratio and osteoid area accompanied by significantly lower values of erosion surface and adiopocytes area in two treatment groups (p < 0.05, n = 6). XLGB Fufang with either XEK or XEP all showed significant preventive effects in OVX-induced osteoporosis and deterioration of bone mechanical properties. CONCLUSION: The significance of the current preclinical experimental study was that these two Epimedium species used for formulating XLGB capsules were equally effective for the prevention of oestrogen-depletion induced osteoporosis.

Sclerostin Antibody Treatment Increases Bone Formation, Bone Mass, and Bone Strength of Intact Bones in Adult Male Rats.

We investigated the systemic effect of sclerostin monoclonal antibody (Scl-Ab) treatment on intact non-operated bones in an open osteotomy male Sprague Dawley (SD) rat model. Six-month-old male SD rats were subjected to transverse osteotomy at the right femur mid-shaft. Rats were injected subcutaneously with vehicle or Scl-Ab (25 mg/kg, 2 times per week) treatment for 9 weeks. Compared with vehicle control, Scl-Ab treatment significantly improved trabecular and cortical bone mass and microarchitecture at L5 vertebrae and left femora by micro-CT at week 6 and 9. Mechanical testing showed that Scl-Ab treatment resulted in significantly higher stiffness, energy to failure and ultimate load at the femora at week 9. Mineral apposition rate, mineralizing surface and bone formation rate on the trabecular bone in the distal femora was significantly increased in Scl-Ab group at week 6 and 9. The administered Scl-Ab was localized in the osteocytes and beta-catenin was strongly expressed in osteoblasts. Scl-Ab treatment significantly increased serum P1NP level and there was no between-group difference in serum level of CTX-1. In conclusion, Scl-Ab treatment could induce rapid and sustained increase in bone formation, bone mass and bone strength in non-operated bones. Sclerostin inhibition might be advantageous to prevent secondary fracture(s).

Blockage of Src by Specific siRNA as a Novel Therapeutic Strategy to Prevent Destructive Repair in Steroid-Associated Osteonecrosis in Rabbits.

Vascular hyperpermeability and highly upregulated bone resorption in the destructive repair progress of steroid-associated osteonecrosis (SAON) are associated with a high expression of VEGF and high Src activity (Src is encoded by the cellular sarcoma [c-src] gene). This study was designed to prove our hypothesis that blocking the VEGF-Src signaling pathway by specific Src siRNA is able to prevent destructive repair in a SAON rabbit model. Destructive repair in SAON was induced in rabbits. At 2, 4, and 6 weeks after SAON induction, VEGF, anti-VEGF, Src siRNA, Src siRNA+VEGF, control siRNA, and saline were introduced via intramedullary injection into proximal femora for each group, respectively. Vascularization and permeability were quantified by dynamic contrast-enhanced (DCE) MRI. At week 6 after SAON induction, proximal femurs were dissected for micro-computed tomography (µCT)-based trabecular architecture with finite element analysis (FEA), µCT-based angiography, and histological analysis. Histological evaluation revealed that VEGF enhanced destructive repair, whereas anti-VEGF prevented destructive repair and Src siRNA and Src siRNA+VEGF prevented destructive repair and enhanced reparative osteogenesis. Findings of angiography and histomorphometry were consistent with those determined by DCE MRI. Src siRNA inhibited VEGF-mediated vascular hyperpermeability but preserved VEGF-induced neovascularization. Bone resorption was enhanced in the VEGF group and inhibited in the anti-VEGF, Src siRNA, Src siRNA+VEGF groups as determined by both 3D µCT and 2D histomorphometry. FEA showed higher estimated failure load in the Src siRNA and Src siRNA+VEGF groups when compared to the vehicle control group. Blockage of VEGF-Src signaling pathway by specific Src siRNA was able to prevent steroid-associated destructive repair while improving reconstructive repair in SAON, which might become a novel therapeutic strategy.

Src blockage by siRNA inhibits VEGF-induced vascular hyperpemeability and osteoclast activity - an in vitro mechanism study for preventing destructive repair of osteonecrosis.

INTRODUCTION: Destructive repair is the pathological feature of ONFH characterized with the elevated vascular permeability and persistent bone resorption, which is associated with higher VEGF expression, activated c-Src, and vascular leakage. Activated c-Src also participates in mediating endothelial permeability and osteoclasts activity. However, the molecular mechanism of the VEGF and c-Src contributing to the destructive repair process remains unknown. The purpose of this study is to delineate the role of VEGF and c-Src in triggering destructive repair of osteonecrosis in vitro, as well as to elucidate if VEGF mediating vascular permeability and osteoclastic bone resorption are Src dependent. METHODS: We employed pharmacological VEGF to induce higher endothelial permeability and osteoclasts activity for simulating related pathological features of destructive repair in vitro. Src specific pp60(c-src)siRNA was used for determining the contribution of VEGF and Src to destructive repair. The primary endothelial cells and osteoclasts were treated with 50ng/ml VEGF and/or transfected with the pp60(c-src)siRNA, while equivalent PBS and non-targeting siRNA were treated in the control groups. RESULTS: VEGF enhanced Src bioactivity through promoting dephosphorylation of Src at Y527 and phosphorylation of Src at Y416. Meanwhile, Src specific pp60(c-src)siRNA significantly reduced Src expression in both cells. VEGF destroyed the junctional integrity of endothelial cells resulting in higher endothelial permeability. However, Src blockade significantly relieved VEGF induced actin stress and inhibited caveolae and VVOs formation, meanwhile further stabilized the complex ß-catenin/VE-cadherin/Flk-1 through decreasing phosphorylation of VE-cadherin, ultimately decreasing VEGF-mediating higher vascular permeability. In addition, VEGF promoted osteoclasts formation and function without affecting the adhesion activity and cytoskeleton. We further found that Src blockade significantly impaired cytoskeleton resulting in a lower adhesion activity through down-regulation of phosphorylation of Src, Pyk2 and Cbl, and ultimately inhibited osteoclasts formation and function. CONCLUSIONS: These findings provide a new insight into VEGF and c-Src mode of reaction in triggering destructive repair of osteonecrosis and further indicate that VEGF mediating vascular permeability and osteoclasts activity are Src-dependent. Blockade of Src may have great potential as an effective therapy targeting destructive repair in osteonecrosis.

Steroid-associated hip joint collapse in bipedal emus.

In this study we established a bipedal animal model of steroid-associated hip joint collapse in emus for testing potential treatment protocols to be developed for prevention of steroid-associated joint collapse in preclinical settings. Five adult male emus were treated with a steroid-associated osteonecrosis (SAON) induction protocol using combination of pulsed lipopolysaccharide (LPS) and methylprednisolone (MPS). Additional three emus were used as normal control. Post-induction, emu gait was observed, magnetic resonance imaging (MRI) was performed, and blood was collected for routine examination, including testing blood coagulation and lipid metabolism. Emus were sacrificed at week 24 post-induction, bilateral femora were collected for micro-computed tomography (micro-CT) and histological analysis. Asymmetric limping gait and abnormal MRI signals were found in steroid-treated emus. SAON was found in all emus with a joint collapse incidence of 70%. The percentage of neutrophils (Neut %) and parameters on lipid metabolism significantly increased after induction. Micro-CT revealed structure deterioration of subchondral trabecular bone. Histomorphometry showed larger fat cell fraction and size, thinning of subchondral plate and cartilage layer, smaller osteoblast perimeter percentage and less blood vessels distributed at collapsed region in SAON group as compared with the normal controls. Scanning electron microscope (SEM) showed poor mineral matrix and more osteo-lacunae outline in the collapsed region in SAON group. The combination of pulsed LPS and MPS developed in the current study was safe and effective to induce SAON and deterioration of subchondral bone in bipedal emus with subsequent femoral head collapse, a typical clinical feature observed in patients under pulsed steroid treatment. In conclusion, bipedal emus could be used as an effective preclinical experimental model to evaluate potential treatment protocols to be developed for prevention of ON-induced hip joint collapse in patients.

Phytoestrogenic molecule desmethylicaritin suppressed adipogenesis via Wnt/ß-catenin signaling pathway.

Epimedium flavonoids inhibit extravascular lipid deposition during prevention of steroid-associated osteonecrosis. Desmethylicaritin is a bioactive metabolite of Epimedium flavonoids in serum. As it is well known that estrogen inhibits aidpogenesis, so we hypothesized that desmethylicaritin as a phytoestrogen might have the potential to inhibit lipid deposition. This study was designed to investigate the effect of desmethylicaritin on adipogenesis and its underlying mechanism in vitro. Adipogenesis was assessed by Oil Red O staining in 3T3-L1 preadipocytes. Bromodeoxyuridine was used to test the clonal expansion. Further, the mRNA level and protein expression of adipgenic and related factors were detected by qRT-PCR and western blot, respectively. The nuclear location of ß-catenin was identified using immunofluoresence assay. Our results showed that desmethylicaritin suppressed the adipogenesis in 3T3-L1 cells in a dose-dependent manner. In addition, desmethylicaritin inhibited clonal expansion during adipogenesis. Desmethylicaritin did not affect CCAAT/enhancer binding protein δ and ß mRNA expression, but decreased the mRNA expression of CCAAT/enhancer binding protein α, peroxisome proliferator-activated receptor γ, adipocyte lipid-binding protein and lipoprotein lipase. Desmethylicaritin up-regulated the mRNA expression of Wnt10b that was however down-regulated after adipogenic induction. Desmethylicaritin increased the protein expression of ß-catenin both in the cytoplasm and nuclei and immunofluorescence results confirmed that desmethylicaritin increased nuclear translocation of ß-catenin. Above findings implied that desmethylicaritin was able to inhibit adipogenesis and Wnt/ß-catenin signaling pathway was regulated by desmethylicaritin in the process of suppression of adipogenesis. Above findings supported desmethylicaritin as a novel phytochemical agent for potential prevention of disorders involving lipid metabolism.

Triterpenoid saponins from Dipsacus asper and their activities in vitro.

Two new triterpenoid saponins 1 and 2, along with six known saponins 3-8, were isolated from the roots of Dipsacus asper. The structures of new compounds were established as 3-O-ß-d-glucopyranosyl-(1 â†’ 4)-[α-l-rhamnopyranosyl-(1 â†’ 3)]-ß-d-glucopyranosyl-(1 â†’ 3)-α-l-rhamnopyranosyl-(1 â†’ 2)-α-l-arabinopyranosyl-hederagenin-28-O-ß-d-glucopyranoside (dipsacus saponin J, 1) and 3-O-α-l-arabinopyranosyl-hederagenin-28-O-ß-d-glucopyranosyl-(1 â†’ 6)-ß-d-glucopyranosyl-(1 â†’ 6)-ß-d-glucopyranoside (dipsacus saponin K, 2). The structures were determined by extensive analysis of their spectroscopic data. Compounds 6 and 7 could significantly stimulate UMR106 cell proliferation and increase alkaline phosphatase activities in UMR106 cell at the concentration of 4 µM.