Osteopontin Concentrations Research Articles

Elevated osteopontin level of synovial fluid and articular cartilage is associated with disease severity in knee osteoarthritis patients

To investigate osteopontin (OPN) levels in both synovial fluid and articular cartilage of patients with primary knee osteoarthritis (OA) and their relationship with severity of the disease. Fifty patients aged 48-81 years with knee OA and 10 healthy controls were enrolled in this study. Anteroposterior knee radiographs or/and Mankin score were taken to determine the disease severity of the affected knee. The radiographic grading of OA in the knee was performed by using the Kellgren-Lawrence criteria. OPN levels in synovial fluid were measured using enzyme-linked immunosorbent assay. OPN levels in articular cartilage were assessed by immunohistochemical methods. Compared to healthy controls, OA patients had higher OPN concentration in synovial fluid (4519.60+/-1830.37, 95%CI 3999.42-5039.79 vs 1179.70+/-303.39, 95%CI 1035.53-1438.74 pg/ml, P<0.001)and articular cartilage(0.6+/-0.06, 95%CI 0.59-0.62 vs 0.43+/-0.07, 95%CI 0.38-0.48, P<0.01). In addition, synovial fluid OPN levels showed a positive correlation with articular cartilage OPN levels (r=0.411, 95%CI 0.150-0.619, P=0.003). Subsequent analysis showed that synovial fluid OPN levels significantly correlated with severity of disease (Spearman's rho=0.581, 95%CI 0.335-0.726, P<0.001). Furthermore, the articular cartilage levels of OPN also correlated with disease severity (Spearman's rho=0.675, 95%CI 0.500-0.808, P<0.001). OPN in synovial fluid and articular cartilage is associated with progressive joint damage and is likely to be a useful biomarker for determining disease severity and progression in knee OA.

Plasma Concentration of Osteopontin (OPN) in Children with Systemic Lupus Erythematosus: Relationship with Disease Activity

Background: Osteopontin (OPN) is an important bone matrix mediator found to have key roles in inflammation and immunity. OPN is a cytokine which can play a number of roles in promoting activation of T lymphocyte, regulating balance between T-helper 1 and T-helper 2, participating in cell-induced immunologic response and stimulating B lym- phocyte to express multi-clone antibodies. Overexpression of OPN has been associated with the development of the auto- immune/ lymphoproliferative syndrome. Objective: The aim of our present study was to analyze the possible correlation between the plasma concentration of OPN and disease activity in children with systemic lupus erythematosus (SLE). We also investigated the correlation between plasma IL-18 and OPN concentrations to further confirm the association of OPN with disease activity. Methods: We measured the plasma concentration of OPN, and the plasma proinflammatory IL-18 concentration in 40 SLE patients with or without renal disease (RSLE group and SLE group, respectively) and in 30 sex- and age-matched controls using enzyme immunoassay. Results: Plasma OPN concentrations were significantly higher in RSLE and SLE patients than in the controls (p = 0.000 and p = 0.002). Plasma OPN concentrations were significantly higher in RSLE patients than in the SLE patients (p = 0.000). Plasma OPN concentration correlated positively and significantly with SLE disease activity index in combined SLE patient groups (r = 0.34; p = 0.04). In RSLE patients, plasma OPN concentration showed a significant positive corre- lation with proinflammatory cytokine IL-18 concentration (r = 0.48; p = 0.004). Conclusion: The above results suggest that the production of OPN is associated with the inflammatory process and SLE development, and may serve as a potential disease marker of SLE.

A genetic polymorphism in transforming growth factor beta receptor‐2 is associated with serum osteopontin

Osteopontin (OPN) is a secreted glycoprotein demonstrated to play an important role in inflammation. Transforming growth factor beta and a related signalling pathway have been implicated in control of OPN secretion. We examined the relationship between transforming growth factor beta receptor-1 and -2 (TGFBR1 and 2) single nucleotide polymorphisms (SNP) and serum OPN in 296 men from the Health in Men Study. Serum concentrations of OPN and 58 SNPs for TGFBR1 and 2 were assessed. One SNP in TGFBR2 was associated with serum OPN (TGFBR2 g.20690C>T, SNP ID rs4522809, P = 0.0007) after adjusting for multiple testing. This study suggests that polymorphism in TGFBR2 are associated with altered secretion of OPN, supporting a role for transforming growth factor beta in OPN production.

Osteopontin is upregulated in malignant and inflammatory pleural effusions

Osteopontin (OPN) is an important mediator of inflammation and cancer progression. In the present study, we asked whether pleural fluid (PF) and serum OPN concentrations differed between patients with pleural effusions of different aetiologies, and whether assessment of OPN levels was useful for diagnostic purposes. One hundred and nine consecutive patients with pleural effusions of different aetiologies were recruited prospectively during daily clinics. OPN levels were measured by ELISA. PF OPN levels were 10-fold higher in exudates than in transudates and were significantly correlated with markers of pleural inflammation and vascular hyper-permeability, such as PF/serum LDH or protein ratios, PF protein and PF vascular endothelial growth factor levels. Patients with malignant pleural effusions had higher PF and lower serum OPN concentrations than those with benign disease. The diagnostic accuracies of PF and PF/serum OPN for malignancy were 71.5% (95% CI: 64-80) and 70.6% (95% CI: 62-80), respectively. OPN levels were elevated in exudative pleural effusions, as compared with the levels in blood or transudative pleural effusions. While PF and PF/serum OPN were higher in patients with malignancies, the diagnostic accuracy of the tests was not sufficient to permit routine use in clinical practice.

Role of osteopontin in induction of monocyte chemoattractant protein 1 and macrophage inflammatory protein 1β through the NF‐κB and MAPK pathways in rheumatoid arthritis

Osteopontin (OPN) is a proinflammatory protein with a critical role in leukocyte migration. Although OPN has been implicated in rheumatoid arthritis (RA), its underlying mechanism remains unknown. In this study, we investigated the role and molecular mechanism of OPN in the induction of 2 key chemokines, monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 1beta (MIP-1beta), in RA. Enzyme-linked immunosorbent assay and quantitative polymerase chain reaction were used to determine chemokine expression. Leukocyte migration in the presence of OPN was measured by chemotaxis assay. Signaling and molecular events were analyzed by immunoblotting and chromatin immunoprecipitation. The effect of OPN on inflammatory cell migration was mediated through its unique property of inducing the expression of MCP-1 and MIP-1beta in CD14+ monocytes. The concentration of OPN was significantly elevated in RA patients and appeared to correlate with the serum levels of inflammation markers and increased expression of MCP-1 or MIP-1beta in monocytes in RA patients. Endogenous production of OPN in RA synovial fluid was attributable to increased production of MCP-1 or MIP-1beta, and this effect could be blocked by an anti-OPN antibody. Furthermore, the structural motif responsible for this property resided within residues 50-83 of human OPN, sparing the known RGD or SVVYGLR sequences. It was evident that the effect of OPN on chemokine expression was mediated through both the NF-kappaB and MAPK pathways, involving the activation of IKKbeta, p38, and JNK. These results support a unique role of OPN in leukocyte migration, in the context of perpetuation of rheumatoid synovitis through the induction of MCP-1 and MIP-1beta.

Polymeric Osteopontin Employs Integrin α9β1 as a Receptor and Attracts Neutrophils by Presenting a de Novo Binding Site

Osteopontin (OPN) is a cytokine and ligand for multiple members of the integrin family. OPN undergoes the in vivo polymerization catalyzed by cross-linking enzyme transglutaminase 2, which consequently increases the bioactivity through enhanced interaction with integrins. The integrin alpha9beta1, highly expressed on neutrophils, binds to the sequence SVVYGLR only after intact OPN is cleaved by thrombin. The SVVYGLR sequence appears to be cryptic in intact OPN because alpha9beta1 does not recognize intact OPN. Because transglutaminase 2-catalyzed polymers change their physical and chemical properties, we hypothesized that the SVVYGLR site might also be exposed on polymeric OPN. As expected, alpha9beta1 turned into a receptor for polymeric OPN, a result obtained by cell adhesion and migration assays with alpha9-transfected cells and by detection of direct binding of recombinant soluble alpha9beta1 with colorimetry and surface plasmon resonance analysis. Because the N-terminal fragment of thrombin-cleaved OPN, a ligand for alpha9beta1, has been reported to attract neutrophils, we next examined migration of neutrophils to polymeric OPN using time-lapse microscopy. Polymeric OPN showed potent neutrophil chemotactic activity, which was clearly inhibited by anti-alpha9beta1 antibody. Unexpectedly, mutagenesis studies showed that alpha9beta1 bound to polymeric OPN independently of the SVVYGLR sequence, and further, SVVYGLR sequence of polymeric OPN was cryptic because SVVYGLR-specific antibody did not recognize polymeric OPN. These results demonstrate that polymerization of OPN generates a novel alpha9beta1-binding site and that the interaction of this site with the alpha9beta1 integrin is critical to the neutrophil chemotaxis induced by polymeric OPN.

New Dual Monoclonal ELISA for Measuring Plasma Osteopontin as a Biomarker Associated with Survival in Prostate Cancer: Clinical Validation and Comparison of Multiple ELISAs

A previously developed monoclonal/polyclonal ELISA (Mono/Poly) to detect plasma concentrations of osteopontin (OPN) was shown to provide prognostic information in breast, prostate, and other cancers. Here we describe the clinical validation of a new dual monoclonal (Dual Mono) assay. We compared both assays with 4 assays that recognize defined regions of OPN protein (dual polyclonal systems 5-1, 4-1, 4-3 and polyclonal-monoclonal system 1-3). OPN sequences recognized by the monoclonal antibodies that make up the Dual Mono ELISA were identified by Pepscan CLIPS analysis. Using the 6 ELISAs, we measured OPN in plasma from 66 patients with castration-resistant prostate cancer, and we assessed the ability of each assay to predict patient survival. The assays varied in measured plasma OPN concentrations, with median values ranging from 112 to 1740 mug/L, and ability to predict patient survival. By Cox univariable regression of survival by tertiles of OPN, the Mono/Poly and Dual Mono ELISAs had the highest log-rank chi(2) values. After adjustment for risk factors independently associated with survival in our samples, OPN remained associated with survival only for the Mono/Poly and Dual Mono systems. OPN plasma values varied significantly depending on the assay used. Only the Mono/Poly and Dual Mono systems were independently associated with survival in a population of men with castration-resistant prostate cancer. The availability of a clinically validated, dual monoclonal-based ELISA will provide consistent reagents for studies of OPN plasma concentrations in cancer and other pathologies.

Characterization of zinc-releasing three-dimensional bioactive glass scaffolds and their effect on human adipose stem cell proliferation and osteogenic differentiation

While the addition of zinc ions to bioactive ceramics has been shown to enhance the proliferation and osteogenic differentiation of osteoblast-like cells, contradictory results have been found. Therefore, the effect of zinc-releasing ceramics on cell proliferation and differentiation into osteogenic lineages requires further clarification. The aim of this study was to evaluate the effects of zinc addition on the degradation profile of three-dimensional bioactive glass scaffold, and on the proliferation and osteogenesis of human adipose stem cells (hASCs) in these scaffolds. Bioactive glass scaffolds containing Na 2O, K 2O, MgO, CaO, B 2O 3, TiO 2, P 2O 5 and SiO 2 were prepared. The degradation was evaluated by weight loss measurement, scanning electron microscopy and elemental analysis. The degradation profile of bioactive glass was shown to slow down with the addition of zinc. Qualitative live/dead staining showed that zinc addition to bioactive glass inhibits cell spreading and proliferation of hASCs. However, zinc addition had no significant effect on DNA content, alkaline phosphatase activity and osteopontin concentration of hASCs when measured quantitatively. Our results suggest that the possible stimulatory effect of addition of zinc on hASC proliferation and osteogenesis was not detected because addition of zinc slowed down the degradation rate of the studied bioactive glass scaffolds.

Osteopontin and ‘Melanoma Inhibitory Activity’: Comparison of Two Serological Tumor Markers in Metastatic Uveal Melanoma Patients

Background: Evaluation of the protein osteopontin (OPN) as a potential new marker in comparison to melanoma inhibitory activity (MIA) for screening and detection of metastatic uveal melanoma. Methods: Plasma levels of 32 patients with uveal melanoma were analyzed for OPN and MIA by enzyme-linked immunosorbent assay (ELISA). Fourteen of these patients had clinically detectable liver metastases. Results: Median plasma concentration of OPN in patients with metastatic disease was 152.01 ng/ml compared to 47.39 ng/ml in patients without clinically detectable metastases (p < 0.001). The difference between the median MIA plasma levels in patients with (13.11 ng/ml) and patients without (5.64 ng/ml) metastatic disease was also statistically significant (p < 0.001). No correlation could be found between MIA or OPN levels and tumor height in patients without clinically detectable metastases. Conclusion: The proteins MIA and OPN seem to be promising tumor markers for the metastasis screening in patients with uveal melanoma.

ORIGINAL ARTICLE: Endometrial Osteopontin mRNA Expression and Plasma Osteopontin Levels are Increased in Patients with Endometriosis

Problem The aim of this study was to evaluate osteopontin (OPN) mRNA expression in eutopic endometrium and plasma OPN levels in patients with endometriosis.Method of study A total of 79 patients with histologically confirmed endometriosis and 43 patients without endometriosis participated in this study. OPN mRNA expression in endometrial tissues was measured by real‐time quantitative polymerase chain reaction (PCR) and plasma concentrations of OPN were quantified using a specific commercial sandwich enzyme‐linked immunosorbent assays (ELISA).Results Osteopontin mRNA expression in endometrial tissue was significantly higher in women with endometriosis than in controls (P = 0.010). The mean plasma levels of OPN (mean ± S.E.M.) in patients with endometriosis and controls were 407.31 ± 37.80 ng/mL and 165.84 ± 19.29 ng/mL, respectively (P &lt; 0.001). Receiver operating characteristic (ROC) analysis for plasma OPN revealed an area under the curve (AUC) of 0.894, with a sensitivity of 93.0%, specificity of 72.4%, positive likelihood ratio of 3.37, and negative likelihood ratio of 0.1 using a cut‐off value of 167.68 ng/mL.Conclusion Osteopontin may be involved in the pathogenesis of endometriosis and plasma OPN may be a useful non‐invasive marker for the diagnosis of endometriosis.

Correlation of plasma and synovial fluid osteopontin with disease severity in knee osteoarthritis

Objectives The purposes of this study were to examine osteopontin levels in both plasma and synovial fluid of patients with primary knee osteoarthritis (OA) and to investigate their relationship with severity of the disease. Design and methods Thirty-two patients aged 53–83 years with knee OA and 15 healthy controls were enrolled in this study. Anteroposterior knee radiographs were taken to determine the disease severity of the affected knee. The radiographic grading of OA in the knee was performed by using the Kellgren–Lawrence criteria. Osteopontin levels in the plasma and synovial fluid were measured using enzyme-linked immunosorbent assay. Results The mean plasma osteopontin concentration of the knee OA patients was significantly higher compared with that of healthy controls (168.8 ± 15.6 vs 67.2 ± 7.7 ng/mL, P < 0.0001). Osteopontin levels in synovial fluid were significantly higher with respect to paired plasma samples (272.1 ± 15.0 vs 168.8 ± 15.6 ng/mL, P < 0.001). In addition, plasma osteopontin levels showed a positive correlation with synovial fluid osteopontin levels ( r = 0.373, P = 0.035). Subsequent analysis showed that plasma osteopontin levels significantly correlated with severity of disease ( r = 0.592, P < 0.001). Furthermore, the synovial fluid levels of osteopontin also correlated with disease severity ( r = 0.451, P = 0.01). Conclusion The data suggest that osteopontin in plasma and synovial fluid is related to progressive joint damage in knee OA. Osteopontin may serve as a biochemical marker for determining disease severity and could be predictive of prognosis with respect to the progression of knee OA.

Osteopontin Genetic Polymorphism and Serum Levels in Patients with Systemic Lupus Erythematosus

Objective: A recent study suggested that a single nucleotide polymorphism (SNP) at position nt 9250 (C to T) in exon 7 of the osteopontin (OPN) gene is strongly associated with the susceptibility to systemic lupus erythematosus (SLE). This study examined the possible association between a single nucleotide polymorphism (SNP) at position nt 9250 (C to T) and SLE and measured the serum levels of OPN in Korean patients with SLE. Methods: A total of 39 patients with SLE and 104 healthy controls were enrolled in this study. SNP located at position 9250 in the OPN gene were genotyped using the restriction fragment length polymorphism (RFLP). The serum levels of OPN in 39 patients with SLE and 20 healthy controls were determined by enzyme-linked immunosorbent assay. Results: The allele frequencies of C and T at this position in patients with SLE were 34.6 and 65.4, whereas those in the controls were 20.7 and 79.3 (p＜0.05). The serum levels of OPN in 39 patients with SLE were significantly higher than that in 20 healthy controls (49.13±26.71 versus 28.49±18.39 ng/ml, p＜0.05). The increase in OPN concentration was associated with the SLE disease activity index (SLEDAI) score in all SLE patients (r=0.337, p＜0.05). Conclusion: The allele frequencies of Eta-1/osteopontin were significantly associated with SLE. Moreover, the increased serum level of OPN is associated with the SLE disease activity.

205 EFFECT OF OSTEOPONTIN ON CLEAVAGE AND BLASTOCYST RATES IN BUFFALO SPECIES (BUBALUS BUBALIS)

It was previously reported that osteopontin (OPN), an acidic single-chain phosphorylated glycoprotein found in the oviductal fluid in cattle (Gabler C et al. 2003 Reproduction 126, 721–729), is able to facilitate fertilization in this species (Gasparrini B et al. 2008 Reprod. Fertil. Dev. 20(Suppl. I), 180 abst). The present study aimed to investigate whether the addition of OPN to the fertilization medium would affect both cleavage and postfertilization embryo development in the buffalo. To assess the influence of OPN on cleavage and blastocyst rates, in vitro-matured oocytes were fertilized in modified Tyrode’s albumin lactate pyruvate medium (Lu KH et al. 1987 Vet. Rec. 121, 259–260) supplemented with penicillamine, hypotaurine, and heparin, in the presence of 0.0 (n = 258), 0.1 (n = 263), 1 (n = 261), and 10 μg mL–1 (n = 264) of OPN. In vitro fertilization was carried out with frozen–thawed spermatozoa from a bull already tested for IVF. After 20 to 22 h of co-incubation at 38.5°C and 5% CO2 in air, putative zygotes were gently pipetted to remove cumulus cells, washed, and transferred, 10 per droplet, into 20 μL of SOF medium including essential and nonessential amino acids and BSA (Tervit HR et al. 1972 J. Reprod. Fertil. 30(3), 493–497), in a controlled gas atmosphere consisting of 5% CO2, 7% O2, and 88% N2, in humidified air, at 38.5°C. The culture medium was changed on Day 5 (Day 0 = day of insemination), when cleavage rate was assessed and embryos were moved into fresh medium for an additional 2 days. On Day 7, development rates into blastocysts of superior quality were recorded. Differences in the percentages of both cleavage and blastocyst rates among groups were analyzed by chi-square test. Significantly higher cleavage rates (59.3, 70.3, 71.6, and 42.4%, respectively, in the control group and in the groups with 0.1, 1, and 10 μg mL–1 of OPN; P &lt; 0.01) were observed in the groups with 0.1 and 1 μg mL–1 of OPN compared with the other groups. Likewise, higher blastocyst rate percentages (17.4, 27.4, 29.9, and 9.5%, respectively, in the control group and in the groups with 0.1, 1, and 10 μg mL–1 of OPN; P &lt; 0.01) were observed in the groups with 0.1 and 1 μg mL–1 of OPN compared with the other groups. In conclusion, these results showed that addition of low concentrations of OPN in the fertilization medium improved both cleavage and postfertilization embryo development in the buffalo, whereas the higher concentration resulted in impaired late-stage embryo development.

Simple Obesity Is Associated with Reduced Breast Arterial Calcification and Increased Plasma Osteopontin Level

We undertook this study to evaluate whether simple obesity is associated with breast arterial calcifications. Mammograms and patient records of 1309 women who underwent screening or diagnostic mammography were reviewed retrospectively. Patients were divided into two groups according to body mass index. Mammograms were evaluated for the presence of arterial calcification and results were coded. Plasma osteopontin levels were measured. The prevalence of breast arterial calcification of patients classified with simple obesity was lower than the non-obese group. Obese group exhibited significantly increased circulating osteopontin concentrations as compared with non-obese group. Plasma osteopontin level is an independent protective factor for the presence of breast arterial calcification. Simple obesity is associated with reduced breast arterial calcification. Our study indicates the use of breast artery calcification as an indicator of cardiovascular disease or arterial status should be reconsidered.

Adipose Tissue Lymphocytes and Macrophages in Obesity and Insulin Resistance

Obesity, and more specifically accumulation of adipose tissue in the visceral and subcutaneous abdominal locations, is a major risk factor for the development of cardiovascular pathologies including hypertension and atherosclerosis, as well as metabolic disorders such as type 2 diabetes. During recent years, “metaflammation” or metabolically-triggered inflammation1 has emerged as a key process involved in the clustering of those conditions. Although several metabolically active organs such as the liver, muscle, and, recently, the intestine2 certainly play major roles, the white adipose tissue appears as a central and primary player as both a source and site of inflammation. Accumulation of adipose tissue macrophages (ATMs) has been well-described in obese conditions in mice and humans.3–5 Moreover, the ATM proinflammatory phenotype has been linked to the development of insulin resistance in mice,4 although the exact nature of the proinflammatory myeloid cells, ie, macrophages or dentritic cells, remains to be determined.6 Nevertheless, the causal link between inflammation and insulin resistance was further strengthened by the specific knock-out of the inflammation coordinator IkappaB kinase beta of myeloid cells, which gave protection against insulin resistance.7 The study of Kintscher and al in this issue8 extends those original observations to cells of adaptative immunity. The authors suggest that the accumulation of T-lymphocytes, assessed mainly through gene expression analyses and immunohistochemistry, occurs in the perigonadal adipose tissue of mice on …

Elevated Osteopontin Levels in Patients with Peripheral Arterial Disease

This study was carried out to compare concentrations of osteopontin (OPN) and osteoprotegerin (OPG) in peripheral arterial disease (PAD). The study population consisted of 200 consecutive subjects in whom both OPN/OPG and ankle-brachial index were measured. It was found that OPN levels, but not OPG levels, were significantly more increased in patients with PAD than those without PAD. Serum OPN levels were significantly lower in subjects with angiotensin converting enzyme inhibitors or angiotensin II receptor blockers than those without these agents. In this study, it has been demonstrated for the first time that serum OPN levels are related to PAD. Inhibition of renin- angiotensin system could decrease OPN levels and prevent the progression of PAD.

Osteopontin and basic fibroblast growth factor are associated with prognosis in advanced non-small cell lung cancer (JMTO LC 0004)

19025 Background: A prospective randomized phase III trial for advanced non-small cell lung cancer (NSCLC) compared paclitaxel (P) plus carboplatin (C) vs. vinorelbine (V), gemcitabine (G) followed by docetaxel (D) in the Japan-Multinational Trial Organization (JMTO LC0003) (Kawahara ASCO 06). This trial was conducted jointly with SWOG 0003 using a common arm of PC. Pharmacogenomic studies of samples from both trials demonstrated significant associations between genotype and clinical outcome (Gandara ASCO 07). Previous analysis of SWOG 0003 samples showed that elevated osteopontin (OPN) plasma levels were highly prognostic (Mack ASCO 05). Our correlative study (JMTO LC 0004) of JMTO LC 0003 was originally designed to assess β-tubulin mutations, which was determined to be insignificant. We elected to study and validate OPN as a prognostic marker, using stock samples under additional approval of JMTO IRB. Method: A total of 20 ml of blood was collected prior to treatment from patients enrolled in JMTO LC0003. Serum concentrations of OPN and basic fibroblast growth factor (bFGF) were measured by ELISA. The relationships between OPN, bFGF levels and patients characteristics were examined using χ2 test, including treatment arm, age, smoking status, histology, clinical stage, performance status, weight loss and LDH. Effect of OPN and bFGF levels on progression-free survival (PFS) and overall survival (OS) were examined by multivariate analysis adjusted by treatment arm, smoking status, clinical stage, performance status, weight loss and LDH. Results: Seventy-one samples were obtained, including 32 specimens from the PC arm and 39 from the VGD arm. There were no significant relationships between either OPN or bFGF levels with patient characteristics. However, smokers tended to have higher OPN levels (p=0.083). In analysis of clinical outcome, low OPN levels correlated with better PFS (p<0.001), and high bFGF correlated with better OS (p=0.003). Conclusion: Consistent with findings from SWOG 0003, low OPN serum levels were significantly associated with a favorable prognosis in the JMTO LC 0004. Additionally, high bFGF levels were associated with improved survival. No significant financial relationships to disclose.

Increase of Osteopontin Plasma Concentrations After Bariatric Surgery Independent from Inflammation and Insulin Resistance

Osteopontin (OPN) is a multifunctional matrix glycoprotein associated with bone metabolism and has been linked to chronic inflammation, insulin resistance, and atherosclerosis. Diet-induced weight loss decreases elevated OPN concentrations in obese patients. The aim of the current study was to investigate the role of OPN after bariatric surgery, where not only improvements of chronic inflammation, insulin resistance and comorbidities, but also malabsorption and altered bone metabolism have been reported. OPN plasma concentrations were determined in 31 morbidly obese patients (5 men, 26 women, BMI 46.2+/-7.1 kg/m2, age 41+/-11 years; mean+/-SD) before and 18 months after bariatric surgery, together with parameters of bone metabolism and inflammation. OPN concentrations increased by +20.3+/-26.6 ng/ml (mean+/-SD, p<0.01), concomitant to a weight loss of -38+/-22 kg, and a decrease in BMI by -13.1+/-7.7 kg/m2 (both p<0.01). HOMA-index improved from 5.2+/-3.4 to 1.5+/-1.0 (p<0.01). Calcium concentrations slightly decreased, and phosphate increased (-0.06+/-0.13 mmol/l and +0.08+/-0.16 mmol/l, respectively; both p<0.05), while 25-OH-Vitamin D3 remained unchanged and PTH tended to increase (+5.1+/-14.0 pg/ml, p=0.054). Monocyte chemoattractant protein 1 and interleukin 18 were significantly decreased and associated with HOMA both before and after bariatric surgery. DeltaOPN was correlated with DeltaPTH, but not with other parameters. OPN plasma concentrations increased concomitant to weight loss after bariatric surgery, which was independent from an improvement of insulin sensitivity and a decrease of inflammatory markers. Further studies are needed to differentiate whether these changes in bone metabolism after bariatric surgery are secondary to calcium deficiency or an adaptation to weight loss.

High calcium concentration and calcium oxalate crystals cause significant inaccuracies in the measurement of urinary osteopontin by enzyme linked immunosorbent assay

Strong evidence that osteopontin (OPN) is a determinant of urolithiasis has prompted studies comparing the protein's urinary excretion in healthy subjects and stone formers. However, reported mean urinary values have varied widely, from <1 microg/mL to more than 20 times that value. Since OPN binds to CaOx crystals, the presence of crystals in urine may cause underestimation of its urinary levels. Using a commercial ELISA, we measured urinary OPN levels in the presence of endogenous or exogenous CaOx monohydrate (COM) and dihydrate (COD) crystals. OPN concentrations decreased in the presence of endogenous and exogenous CaOx crystals, but never below 2 microg/mL. Increasing the urinary calcium concentration decreased detectable OPN levels, possibly as a result of changes in the three-dimensional conformation of the protein. Because calcium concentration and the formation of CaOx crystals cannot be controlled in urine, the use of urinary OPN levels as a biomarker for any human pathology must be seriously questioned, but particularly for the investigation of stone formers in whom hypercalciuria and crystalluria are more common than in healthy subjects.

Resorbable polymeric scaffolds for bone tissue engineering: The influence of their microstructure on the growth of human osteoblast‐like MG 63 cells

Degradable three-dimensional porous scaffolds applicable as cell carriers for bone tissue engineering were developed by an innovative solvent casting/particulate leaching technique from poly(L-lactide-co-glycolide) (PLG). Three types of PLG scaffolds were prepared, and these had the same high porosity (83%) but increasing diameter of the pores (180-200 microm, 250-320 microm, and 400-600 microm) and increasing pore interconnectivity. The colonization of the scaffolds with human osteoblast-like MG 63 cells was then studied in vitro in a conventional static cell culture system. The number of cells growing on the scaffolds on days 1 and 7 after seeding was highest in the material with the largest pore diameter, but on day 15, the differences among the scaffolds disappeared. Confocal microscopy revealed that on day 1 after seeding, the cells penetrated to a depth of 490 +/- 100 microm, 720 +/- 170 microm, and 720 +/- 120 microm into the scaffolds of small, medium, and large pore size, respectively. Incorporation of bromodeoxyuridine into newly synthesized DNA and the concentration of vinculin, beta-actin, osteopontin, and osteocalcin in cells on the scaffolds of all pore sizes were similar to the values obtained on standard tissue culture polystyrene, which indicated good biocompatibility of the scaffolds. These results suggest that all scaffolds could serve as good carriers for bone cells, although the quickest colonization with cells was found in the scaffolds with the largest pore diameter from 400 to 600 microm.