The in vitro behaviour of canine osteoblasts derived from different bone types.

BACKGROUND: Our understanding of the biology of osteoblasts is important as they underpin bone remodelling, fracture healing and processes such as osseointegration. Osteoblasts isolated from human humeral samples display distinctive biological activity in vitro, which relates to the samples' bone types (subchondral (S), trabecular (T), cortical (C)). Our aim was to isolate primary osteoblast cultures from different bone types from the proximal femur of a clinical population of dogs presented for total hip replacement and compare the behaviour of the osteoblasts derived from different bone types, to identify a preferred bone type for isolation. RESULTS: No differences were found for osteoblast doubling time (median for S = 2.9, T = 3.1 and C = 2.71 days, respectively; p = 0.33), final cell number (median for S = 54,849, T = 49,733, C = 61,390 cells/cm2; p = 0.34) or basal tissue non-specific alkaline phosphatase (TNAP) activity (median for S = 0.02, T = 0.02, C = 0.03 U/min/mg protein; p = 0.81) between bone types after 6 days of culture in basal media. There were no differences in mineralizing TNAP activity (S = 0.02, T = 0.02, C = 0.03 U/min/mg protein, p = 0.84) or in mineralized area (S = 0.05, T = 0.04, C = 0.04%, p = 0.92) among cells from different bone types. CONCLUSIONS: There is no significant difference in mean doubling time, basal or mineralizing TNAP activity or mineralized area in osteoblasts derived from subchondral, cortical, or trabecular bone types from the canine femoral head. However, there appears to be a high level of inter-animal variability in the studied parameters, which was independent of age, body mass, and sex. Trabecular isolate osteoblasts have the least variation of the bone types studied, and therefore should be considered a preferred source for primary osteoblast cultures. The work here provides baselines for canine osteoblast function, which has utility for future comparative studies.

Isolation and Generation of Osteoblasts.

This chapter describes the isolation, culture, and staining of osteoblasts. The key advantages of this assay are that it allows direct measurement of bone matrix deposition and mineralization, as well as yielding good quantities of osteoblasts at defined stages of differentiation for molecular and histological analysis. An additional focus of this chapter will be the culture of osteoblasts from less conventional animal species.

Corrigendum: Ankylosing Spondylitis Patients Have Impaired Osteoclast Gene Expression in Circulating Osteoclast Precursors.

[This corrects the article on p. 5 in vol. 4, PMID: 28191455.].

Effect of Tumor Necrosis Factor Inhibitor Therapy on Osteoclasts Precursors in Rheumatoid Arthritis.

Objective. Tumor necrosis factor (TNF) increases circulating osteoclast (OC) precursors numbers by promoting their proliferation and differentiation. The aim of this study was to assess the effect of TNF inhibitors (TNFi) on the differentiation and activity of OC in rheumatoid arthritis (RA) patients. Methods. Seventeen RA patients treated with TNFi were analyzed at baseline and after a minimum follow-up period of 6 months. Blood samples were collected to assess receptor activator of nuclear factor kappa-B ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers, in vitro OC differentiation assays, and qRT-PCR for OC specific genes was performed. Results. After TNFi therapy, patients had reduced RANKL surface expression in B-lymphocytes and the frequency of circulating classical CD14brightCD16- monocytes was decreased. Serum levels of sRANKL, sRANKL/OPG ratio, and CTX-I were reduced in RA patients after TNFi treatment. Moreover, after exposure to TNFi, osteoclast differentiation and activity were decreased, as well as the expression of TRAF6 and cathepsin K. Conclusion. We propose that TNFi arrests bone loss and erosion, through two pathways: direct reduction of osteoclast precursor numbers and inhibition of intracellular signaling pathways acting through TRAF6.

Ankylosing Spondylitis Patients Have Impaired Osteoclast Gene Expression in Circulating Osteoclast Precursors.

INTRODUCTION: Ankylosing spondylitis (AS) is typically characterized by focal bone overgrowth and also by systemic bone loss. We hypothesize that the increased osteoproliferation found in AS might be partially due to reduced ability of osteoclast precursors (OCPs) to differentiate into osteoclasts (OCs). Therefore, our aim was to characterize bone remodeling and pro-osteoclastogenesis inflammatory environment, monocytes' phenotype, and in vitro osteoclast differentiation in AS patients. METHODS: Patients with active AS without any ongoing therapy and age- and gender-matched healthy donors were recruited. Receptor activator of nuclear factor-κß (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations were assessed. Quantification of serum levels of bone turnover markers and cytokines, in vitro OC differentiation assay and quantitative reverse transcription real-time PCR for OC-specific genes were performed. RESULTS: Pro- and anti-inflammatory cytokine serum levels were higher in AS patients than in controls. RANKL neutrophil expression was higher in AS patients when compared to healthy donors, but CD51/CD61 expression was lower in the classical monocyte subpopulation. Concerning osteoclastogenesis, we found no differences in the in vitro osteoclast differentiating potential of these cells when compared to healthy donors. However, we observed low expression of CSF1R, RANK, and NFATc1 in AS OCPs. CONCLUSION: Despite the high levels of pro-inflammatory cytokines present in AS patients, no differences in the number of OC or resorbed area were found between AS patients and healthy donors. Moreover, we observed that OCPs have low OC-specific gene expression. These findings support our hypothesis of an impaired response of OCPs to pro-osteoclastogenic stimuli in vivo in AS patients.

Effect of Tumor Necrosis Factor Inhibitor Therapy on Osteoclasts Precursors in Ankylosing Spondylitis.

INTRODUCTION: Ankylosing Spondylitis (AS) is characterized by excessive local bone formation and concomitant systemic bone loss. Tumor necrosis factor (TNF) plays a central role in the inflammation of axial skeleton and enthesis of AS patients. Despite reduction of inflammation and systemic bone loss, AS patients treated with TNF inhibitors (TNFi) have ongoing local bone formation. The aim of this study was to assess the effect of TNFi in the differentiation and activity of osteoclasts (OC) in AS patients. METHODS: 13 AS patients treated with TNFi were analyzed at baseline and after a minimum follow-up period of 6 months. 25 healthy donors were recruited as controls. Blood samples were collected to assess receptor activator of nuclear factor kappa-B ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers and cytokines, in vitro OC differentiation assay and qRT-PCR for OC specific genes were performed. RESULTS: RANKL+ circulating lymphocytes (B and T cells) and IL-17A, IL-23 and TGF-ß levels were decreased after TNFi treatment. We found no differences in the frequency of the different monocyte subpopulations, however, we found decreased expression of CCR2 and increased expression of CD62L after TNFi treatment. OC number was reduced in patients at baseline when compared to controls. OC specific gene expression was reduced in circulating OC precursors after TNFi treatment. However, when cultured in OC differentiating conditions, OC precursors from AS TNFi-treated patients showed increased activity as compared to baseline. CONCLUSION: In AS patients, TNFi treatment reduces systemic pro osteoclastogenic stimuli. However, OC precursors from AS patients exposed to TNFi therapy have increased in vitro activity in response to osteoclastogenic stimuli.

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/ß-catenin signaling pathway with inflammatory cytokines in the bone microenvironment and to assess the serum levels of Wnt/ß-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 (ß=0.607; p=0.013) and stiffness (ß=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 (ß=-0.411; p=0.030) and stiffness (ß=-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.

TNF promoter -308 G>A and LTA 252 A>G polymorphisms in Portuguese patients with systemic lupus erythematosus.

The polymorphism of the tumor necrosis factor (TNF) promoter gene at position -308 and that of the lymphotoxin alpha (LTA) gene at position 252 have been implicated as genetic risk factors for systemic lupus erythematosus (SLE) in some populations. In a nested case-control study, we investigated the possible association of these polymorphisms with susceptibility to SLE and with phenotypic disease features in Portuguese Caucasian patients. TNF-308 G>A and LTA 252 A>G polymorphisms were determined by restriction fragment length polymorphism analysis in a cohort of 115 SLE patients and 152 unrelated healthy controls, and the magnitude of the association between genotypes and SLE diagnosis was calculated. For SLE patients, we also tested the association between disease characteristics and genotypes. No significant differences in genotype or allele frequencies could be identified between SLE cases and controls. Lupus nephritis (OR = 2.84; 95%CI 1.14-7.03, P = 0.02) and the presence of anti-Sm antibodies (OR = 3.11; 95%CI 1.08-8.94; P = 0.03) were significantly more prevalent among lupus patients possessing the TNF-308 A allele. The occurrence of nephritis was also higher in LTA 252 G allele carriers (OR = 2.90; 95%CI 1.12-7.54; P = 0.02). Our results do not support a major role of either the TNF-308 G>A or the LTA 252 A>G polymorphisms as genetic risk factors for SLE. Nevertheless, these polymorphisms appear to associate with the risk of renal lupus and distinct immunological features.

Lymphotoxin-α 252 A>G polymorphism: a link between disease susceptibility and dyslipidemia in rheumatoid arthritis?

OBJECTIVE: Rheumatoid arthritis (RA) is associated with higher levels of inflammatory mediators and with a more atherogenic lipid profile. Dyslipidemia can be present years before arthritis develops. Lymphotoxin-α (LTA) is a cytokine that mediates proinflammatory responses while also participating in lipid homeostasis, and its transcriptional activity is in part genetically determined. We examined the role of the single-nucleotide polymorphism at position 252 of the LTA gene in the genetic background of RA and dyslipidemia. METHODS: The association between the LTA 252 A>G polymorphism and disease status was examined in a nested case-control study of 388 patients with RA and 269 unrelated healthy controls, all white. Demographics and disease features were assessed, fasting lipids measured, and the use of lipid-lowering agents evaluated. RESULTS: The LTA 252 A allele was more frequent in cases compared to controls (70.5% and 64.3%, respectively; p = 0.018, OR 1.325, 95% CI 1.049-1.675), as well as the A/A genotype (50.8% vs 43.5%; p = 0.025). The A/A genotype was independently associated with dyslipidemia in patients, but not in controls. Patients with RA who had the LTA 252 G/G genotype were younger at disease onset and had higher C-reactive protein (CRP) levels. CONCLUSION: We found the LTA 252 A allele to be associated with an increased risk for developing RA in whites. The LTA 252 A/A genotype translates to a phenotype more prone to dyslipidemia, and the G/G genotype to a phenotype with earlier onset of disease and higher levels of CRP, when RA does occur. These observations highlight a possible common genetic predisposition to RA and dyslipidemia.

Upregulation of inflammatory genes and downregulation of sclerostin gene expression are key elements in the early phase of fragility fracture healing.

BACKGROUND: Fracture healing is orchestrated by a specific set of events that culminates in the repair of bone and reachievement of its biomechanical properties. The aim of our work was to study the sequence of gene expression events involved in inflammation and bone remodeling occurring in the early phases of callus formation in osteoporotic patients. METHODOLOGY/PRINCIPAL FINDINGS: Fifty-six patients submitted to hip replacement surgery after a low-energy hip fracture were enrolled in this study. The patients were grouped according to the time interval between fracture and surgery: bone collected within 3 days after fracture (n = 13); between the 4(th) and 7(th) day (n = 33); and after one week from the fracture (n = 10). Inflammation- and bone metabolism-related genes were assessed at the fracture site. The expression of pro-inflammatory cytokines was increased in the first days after fracture. The genes responsible for bone formation and resorption were upregulated one week after fracture. The increase in RANKL expression occurred just before that, between the 4(th)-7(th) days after fracture. Sclerostin expression diminished during the first days after fracture. CONCLUSIONS: The expression of inflammation-related genes, especially IL-6, is highest at the very first days after fracture but from day 4 onwards there is a shift towards bone remodeling genes, suggesting that the inflammatory phase triggers bone healing. We propose that an initial inflammatory stimulus and a decrease in sclerostin-related effects are the key components in fracture healing. In osteoporotic patients, cellular machinery seems to adequately react to the inflammatory stimulus, therefore local promotion of these events might constitute a promising medical intervention to accelerate fracture healing.

Chronic arthritis leads to disturbances in the bone collagen network.

INTRODUCTION: In this study we used a mice model of chronic arthritis to evaluate if bone fragility induced by chronic inflammation is associated with an imbalance in bone turnover and also a disorganization of the bone type I collagen network. METHODS: Serum, vertebrae and femur bones were collected from eight-month-old polyarthritis SKG mice and controls. Strength of the femoral bones was evaluated using three-point bending tests and density was assessed with a pycnometer. Bone turnover markers carboxy-terminal collagen cross-linking telopeptides (CTX-I) and amino-terminal propeptide of type I procollagen (PINP) were measured in serum. The organization and density of bone collagen were analyzed in vertebrae using second-harmonic generation (SHG) imaging with a two-photon microscope and trabecular bone microstructure was assessed by scanning electron microscope (SEM). RESULTS: Femoral bones of SKG mice revealed increased fragility expressed by deterioration of mechanical properties, namely altered stiffness (P = 0.007) and reduced strength (P = 0.006), when compared to controls. Accordingly, inter-trabecular distance and trabecular thickness as observed by SEM were reduced in SKG mice. PINP was significantly higher in arthritic mice (9.18 +/- 3.21 ng/ml) when compared to controls (1.71 +/- 0.53 ng/ml, P < 0.001). Bone resorption marker CTX-I was 9.67 +/- 3.18 ng/ml in arthritic SKG mice compared to 6.23 +/- 4.11 ng/ml in controls (P = 0.176). The forward-to-backward signal ratio measured by SHG was higher in SKG animals, reflecting disorganized matrix and loose collagen structure, compared to controls. CONCLUSIONS: We have shown for the first time that chronic arthritis by itself impairs bone matrix architecture, probably due to disturbed bone remodeling and increased collagen turnover. This effect might predispose patients to bone fragility fractures.