A.C. Vale

Rheumatoid Arthritis Bone Fragility Is Associated With Upregulation of IL17 and DKK1 Gene Expression

Our aim was to compare bone gene expression in rheumatoid arthritis (RA) and primary osteoporosis (OP) patients. Secondary aims were to determine the association of gene expression of the Wnt/β-catenin signaling pathway with inflammatory cytokines in the bone microenvironment and to assess the serum levels of Wnt/β-catenin proteins in both groups. RA patients referred for hip replacement surgery were recruited. Primary OP patients were used as controls. Gene expression of Wnt pathway mediators, matrix proteins, and pro-inflammatory cytokines were analyzed in bone samples. Bone turnover markers, inflammatory cytokines, and Wnt mediators were measured in serum. Twenty-two patients were included: 10 with RA and 12 with primary OP. The expressions of Wnt10b (p = 0.034), its co-receptor LRP6 (p = 0.041), and its negative regulator DKK1 (p = 0.008) were upregulated in RA bone. IL17 gene expression in bone was upregulated in RA patients (p = 0.031) and correlated positively with Wnt10b (r = 0.810, p = 0.015), DKK2 (r = 0.800, p = 0.010), and RANKL/OPG ratio (r = 0.762, p = 0.028). DKK2 (p = 0.04) was significantly decreased in RA serum compared with primary OP. In conclusion, bone fragility in RA patients is induced by an unbalanced bone microenvironment and is associated with a specific gene expression pattern, namely, the upregulation of IL17 and DKK1, suggesting that the modulation of these two pathways might prevent RA systemic bone loss.

Low osteocalcin/collagen type I bone gene expression ratio is associated with hip fragility fractures

INTRODUCTION Osteocalcin (OC) is the most abundant non-collagenous bone protein and is determinant for bone mineralization. We aimed to compare OC bone expression and serum factors related to its carboxylation in hip fragility fracture and osteoarthritis patients. We also aimed to identify which of these factors were associated with worse mechanical behavior and with the hip fracture event. METHODS In this case-control study, fragility fracture patients submitted to hip replacement surgery were evaluated and compared to a group of osteoarthritis patients submitted to the same procedure. Fasting blood samples were collected to assess apolipoproteinE (apoE) levels, total OC and undercarboxylated osteocalcin (ucOC), vitamin K, LDL cholesterol, triglycerides and bone turnover markers. The frequency of the apoε4 isoform was determined. Femoral epiphyses were collected and trabecular bone cylinders drilled in order to perform compression mechanical tests. Gene expression of bone matrix components was assessed by quantitative RT-PCR analysis. RESULTS 64 patients, 25 submitted to hip replacement surgery due to fragility fracture and 39 due to osteoarthritis, were evaluated. Bone OC/collagen expression (OC/COL1A1) ratio was significantly lower in hip fracture compared to osteoarthritis patients (p<0.017) adjusted for age, gender and body mass index. Moreover, OC/COL1A1 expression ratio was associated with the hip fracture event (OR ~0; p=0.003) independently of the group assigned, or the clinical characteristics. Apoε4 isoform was more frequent in the hip fracture group (p=0.029). ucOC levels were higher in the fracture group although not significantly (p=0.058). No differences were found regarding total OC (p=0.602), apoE (p=0.467) and Vitamin K (p=0.371). In hip fracture patients, multivariate analysis, adjusted for clinical characteristics, serum factors related to OC metabolism and gene expression of bone matrix proteins showed that low OC/COL1A1 expression ratio was significantly associated with worse trabecular strength (β=0.607; p=0.013) and stiffness (β=0.693; p=0.003). No association was found between ucOC and bone mechanics. Moreover, in osteoarthritis patients, the multivariate analysis revealed that serum total OC was negatively associated with strength (β=-0.411; p=0.030) and stiffness (β=-0.487; p=0.009). CONCLUSION We demonstrated that low bone OC/COL1A1 expression ratio was an independent predictor of worse trabecular mechanical behavior and of the hip fracture event. These findings suggest that in hip fracture patients the imbalance of bone OC/COL1A1 expression ratio reflects disturbances in osteoblast activity leading to bone fragility.

Antifungal activity of dental resins containing amphotericin B-conjugated nanoparticles.

OBJECTIVES To evaluate the antifungal activity, biocompatibility and mechanical properties of dental resins containing silica nanoparticles functionalized with amphotericin B (SNP-DexOxAmB) against five species of Candida. METHODS Dental resin composites (Spectrum, Dentsply DeTrey, GmbH, Germany) having 2% (w/w) of SNP-DexOxAmB (SNPs of 5 and 80nm, denoted as SNP5 and SNP80) were aged for 10, 20 and 30 days at 37°C, in phosphate buffer saline buffer pH 7.4 (PBS). At different time, the antifungal activity was evaluated by a direct contact assay against 1×10(4)cells of Candida. The biocompatibility of the resins was tested against human fibroblasts, endothelial cells and red blood cells. RESULTS Dental resins containing SNP5-DexOxAmB have high (1×10(4)cells killed in 5h by ∼70mg of dental resin composite containing 2% (w/w) of SNP-DexOxAmB) and durable (for at least 1 month) antifungal activity against five strains of Candida. The incorporation of the nanoparticles (NPs) had no significant change in the mechanical properties of the resin, specifically the flexural strength and modulus. Our results further show that the antifungal activity is mainly mediated by direct contact and not by leaching of NPs from the resin. Resins incorporating SNP5-DexOxAmB have longer-term antifungal activity than SNP80-DexOxAmB. The antimicrobial activity of resins with SNP5-DexOxAmB persists after 4 cycles of re-use and it is superior to the activity obtained for dental resins containing silver NPs. In addition, dental resins incorporating SNP5-DexOxAmB are non-cytotoxic against human skin fibroblasts and human umbilical vein endothelial cells, and non-hemolytic against human red blood cells. SIGNIFICANCE The incorporation of SNP5-DexOxAmB in dental resins resulted in a non-cytotoxic composite with high and durable antifungal activity.

Arthritis Induces Early Bone High Turnover, Structural Degradation and Mechanical Weakness

Background We have previously found in the chronic SKG mouse model of arthritis that long standing (5 and 8 months) inflammation directly leads to high collagen bone turnover, disorganization of the collagen network, disturbed bone microstructure and degradation of bone biomechanical properties. The main goal of the present work was to study the effects of the first days of the inflammatory process on the microarchitecture and mechanical properties of bone. Methods Twenty eight Wistar adjuvant-induced arthritis (AIA) rats were monitored during 22 days after disease induction for the inflammatory score, ankle perimeter and body weight. Healthy non-arthritic rats were used as controls for compar-ison. After 22 days of disease progression rats were sacrificed and bone samples were collected for histomorphometrical, energy dispersive X-ray spectroscopical analysis and 3-point bending. Blood samples were also collected for bone turnover markers. Results AIA rats had an increased bone turnover (as inferred from increased P1NP and CTX1, p = 0.0010 and p = 0.0002, respectively) and this was paralleled by a decreased mineral content (calcium p = 0.0046 and phos-phorus p = 0.0046). Histomorphometry showed a lower trabecular thickness (p = 0.0002) and bone volume (p = 0.0003) and higher trabecular sepa-ration (p = 0.0009) in the arthritic group as compared with controls. In addition, bone mechanical tests showed evidence of fragility as depicted by diminished values of yield stress and ultimate fracture point (p = 0.0061 and p = 0.0279, re-spectively) in the arthritic group. Conclusions We have shown in an AIA rat model that arthritis induc-es early bone high turnover, structural degradation, mineral loss and mechanical weak-ness.

Effect of the Strain Rate on the Twisting of Trabecular Bone from Women with Hip Fracture

As one of the major functions of bone is to provide structural support for the musculoskeletal system, it is important to evaluate its mechanical strength. Bones may be subjected to multiaxial stresses due to bone pathologies, accidental loads which may lead to hip, wrist fracture, or to a prosthetic joint replacement. Twist loading may lead to fractures, especially involving long bones from lower limbs. The aim of this work was to study the effect of the strain rate on the shear properties of trabecular bone samples from women with hip fracture (from 65 to 100 years). Cylindrical samples were core drilled from human femoral heads along the primary trabecular direction. The cylinders ends were polished and embedded in blocks of polymeric material which fit the grips of the testing device. Deformation rates of 0.005, 0.01, 0.015, and 0.05 s⁻¹ were applied. Twisting tests were conducted with or without an applied axial load of 500 N. From the torque-angular displacement curves, the shear stress-strain curves were obtained. The maximum shear strength and the shear modulus (i.e. the slope of the linear region) were determined. A large scatter of the results of the shear strength and the shear modulus was found, which is probably related to the heterogeneity of nonhealthy human bone samples. There is no significant effect of the strain rate on the maximum shear stress and the shear modulus, either in tests undertaken with or without the application of an axial load. The effect of strain rate on nonhealthy bone trabecular twisting properties did not follow the trend observed on the effect of strain rate in healthy bone, where an increase is detected.

Correction: Arthritis Induces Early Bone High Turnover, Structural Degradation and Mechanical Weakness.

The following information is missing from the Funding section: This study was supported by an unrestricted grant from UCB Pharma (www.ucb.com). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors have also provided an updated Competing Interests statement: This study was supported by an unrestricted grant from UCB Pharma. Joao Eurico Cabral da Fonseca received this grant as the head of Rheumatology Research Unit at Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

A4.5 Arthritis induces early bone high turnover, structural degradation and mechanical weakness

Background and objectives We have previously found in the chronic SKG mouse model of arthritis that long standing (5 and 8 months) inflammation directly leads to high collagen bone turnover, disorganisation of the collagen network, disturbed bone microstructure and degradation of bone biomechanical properties. The main goal of the present work was to study the effects of the first days of the inflammatory process on the microarchitecture and mechanical properties of bone. Methods Twenty eight Wistar adjuvant-induced arthritis (AIA) rats were monitored during 22 days after disease induction for the inflammatory score, ankle perimeter and body weight. Healthy non-arthritic rats were used as controls for comparison. After 22 days of disease progression rats were sacrificed and bone samples were collected for histomorphometrical, energy dispersive X-ray spectroscopical analysis and 3-point bending. Blood samples were also collected for bone turnover markers. Results AIA rats had an increased bone turnover (as inferred from increased P1NP and CTX1, p = 0.0010 and p = 0.0002, respectively) and this was paralleled by a decreased mineral content (calcium p = 0.0046 and phosphorus p = 0.0046). Histomorphometry showed a lower trabecular thickness (p = 0.0002) and bone volume (p = 0.0003) and higher trabecular separation (p = 0.0009) in the arthritic group as compared with controls. In addition, bone mechanical tests showed evidence of fragility as depicted by diminished values of yield stress and ultimate fracture point (p = 0.0061 and p = 0.0279, respectively) in the arthritic group. Conclusions We have shown in an AIA rat model that arthritis induces early bone high turnover, structural degradation, mineral loss and mechanical weakness.

THU0034 Rheumatoid arthritis bone fragility is associated with increased DKK1 expression and disturbances in the bone turnover regulating genes

Background Rheumatoid arthritis (RA) and primary osteoporosis (OP) induce bone fragility. In this study we aimed at identifying differences in the mechanisms involved in bone fragility by comparing gene expression between RA and OP bone samples with similar fracture risk factors. Methods Patients with RA submitted to hip replacement surgery were recruited. Trabecular bone microarchitecture was assessed by micro-computed tomography and bone mechanical behavior by compression tests. Bone cell activity was analyzed by studying gene expression. RA patients compared with OP patients were matched for bone mineral density (BMD) and major clinical fracture risk factors (age, gender, BMI, FRAX). Results Seventeen patients were included, ten with RA and seven with primary established OP. Bone microarchitecture did not differ between the groups, but mechanical bone properties were similarly decreased in RA and primary OP patients. RA bone microenvironment, compared to primary OP, had a gene expression profile characterized by upregulated pro-osteoclastogenic cytokines and DKK1, increased RANKL/OPG ratio, paralleled by raised expression of factors that promote osteoblastic activity, but with low COL1A1 expression. Conclusions Bone fragility in RA patients is induced by an unbalanced bone turnover that is qualitatively different from the pathobiologic phenomena that occur in primary OP. The type of bone gene disturbances is suggestive of a pivotal role for DKK1 in this process, suggesting that it could be used as a therapeutic target to prevent RA bone damage. Disclosure of Interest None Declared

Rheumatoid arthritis is associated with increased DKK1 expression and disturbances in the bone turnover regulating genes

Materials and methods RA patients submitted to hip replacement surgery were recruited. They were matched to a group of primary OP patients for bone mineral density and major clinical fracture risk factors (age, gender, BMI). Trabecular bone microarchitecture was assessed by micro-computed tomography and bone mechanical behavior by compression tests. Bone cell activity was analyzed by studying gene expression.

Apolipoprotein E and undercaboxylated osteocalcin are associated with bone fragility but not with bone mineral density in osteoarthritis patients

Apolipoprotein E and undercaboxylated osteocalcin are associated with bone fragility but not with bone mineral density in osteoarthritis patients Ana Rodrigues, Joana Caetano-Lopes, Ana Lopes, Ana C Vale, Ines Aleixo, Ines P Perpetuo, Ana S Pena, Alexandra Faustino, Alexandre Sepriano, Joaquim Polido-Pereira, Elsa Vieira-Sousa, Bruno Vidal, Jose C Romeu, Pedro M Amaral, Luis G Rosa, Jose A Pereira da Silva, Jacinto Monteiro, Maria F Vaz, Joao E Fonseca, Helena Canhao