Effect of bioactive glass particles on osteogenic differentiation of adipose-derived mesenchymal stem cells seeded on lactide and caprolactone based scaffolds.

Incorporation of bioactive glass (BG) particles to synthetic polymer scaffolds is a promising strategy to improve the bioactivity of bioinert materials and to stimulate specific cell responses. In this study, the influence of incorporating BG particles to lactide and caprolactone based porous scaffolds on osteogenic differentiation of adipose-derived stem cells (ASCs) was analyzed. Accordingly, ASCs were seeded on poly(L-lactide) (PLLA), poly(ε-caprolactone) (PCL), or poly(L-lactide-co-ε-caprolactone) (PLCL) scaffolds containing 15 vol % of BG particles in two culture conditions: standard versus osteogenic culture medium. In standard culture medium, incorporation of BG to a PLLA scaffold increased the ALP activity with respect to its unfilled counterpart (ca. 1.2- and a 1.6-fold increase over 7 and 14 days, respectively). Moreover, in all the studied polymers the incorporation of BG induced a slightly higher production of mineralized matrix by ASCs, but the differences observed were not statistically significant. In the osteogenic medium, the effect of BG was masked by the effect of osteogenic supplements in the long-term. However, in the short-term (day 7), BG particles induced an early ALP activity of predifferentiated osteoblasts on PLLA and PCL scaffolds and higher matrix mineralization on PCL scaffolds. In summary, the addition of BG particles to PLLA and PCL scaffolds sustains ASC osteogenic differentiation, facilitates mineralization and induces the formation of a hydroxyapatite layer on the surface of the polymer scaffolds.

Measurement of liver iron concentration by MRI is reproducible.

PURPOSE: The objectives were (i) construction of a phantom to reproduce the behavior of iron overload in the liver by MRI and (ii) assessment of the variability of a previously validated method to quantify liver iron concentration between different MRI devices using the phantom and patients. MATERIALS AND METHODS: A phantom reproducing the liver/muscle ratios of two patients with intermediate and high iron overload. Nine patients with different levels of iron overload were studied in 4 multivendor devices and 8 of them were studied twice in the machine where the model was developed. The phantom was analysed in the same equipment and 14 times in the reference machine. RESULTS: FeCl3 solutions containing 0.3, 0.5, 0.6, and 1.2 mg Fe/mL were chosen to generate the phantom. The average of the intramachine variability for patients was 10% and for the intermachines 8%. For the phantom the intramachine coefficient of variation was always below 0.1 and the average of intermachine variability was 10% for moderate and 5% for high iron overload. CONCLUSION: The phantom reproduces the behavior of patients with moderate or high iron overload. The proposed method of calculating liver iron concentration is reproducible in several different 1.5 T systems.

Pravastatin inhibits cell proliferation and increased MAT1A expression in hepatocarcinoma cells and in vivo models.

BACKGROUND: Statins may have therapeutic effects on hepatocarcinoma (HCC). This type of disorder is the most common malignant primary tumour in the liver. Our objective was to determine whether pravastatin had a therapeutic effect in vitro and in vivo models. METHOD: We design in vitro and in vivo model. In vitro we used PLC and determine cell proliferation. In vivo, we used and animal model to determined, PCNA and MAT1A expression and transaminases levels. RESULTS: We found that pravastatin decreases cell proliferation in vitro (cell proliferation in pravastatin group was 82%, in sorafenib group 51% and in combined group 40%) and in vivo (in pravastatin group 80%, in sorafenib group 76.4% and in combined group 72.72%). The MAT1A levels, was significantly higher in Pravastatin group (D 62%, P 94%, S 71%, P + S 91%). The transaminases levels, decreased significantly in Pravastatin group (GOT and GPT levels D 619.5 U/L; 271 U/L) (P 117.5 U/L; 43.5 U/L) (S 147 U/L; 59 U/L) (P + S 142 U/L; 59 U/L). CONCLUSION: The combination of pravastatin + sorafenib were more effective than Sorafenib alone.

Accurate fat fraction quantification by multiecho gradient-recalled-echo magnetic resonance at 1.5T in rats with nonalcoholic fatty liver disease.

AIM: To assess the diagnostic accuracy of a new reconstruction technique for gradient-recalled-echo magnetic resonance (MR) sequences that provides a full decomposition of the water and fat content inside a voxel for nonalcoholic fatty liver disease (NAFLD) in rats. MATERIAL AND METHODS: Rats were randomized into two groups. A control group (n = 10) was given free access to regular dry rat chow for 4 weeks. The steatosis (n = 40) group was given free access to feed and water 4 days per week, and fasted for the remaining 3 days for 4 weeks. All rats were killed at 4 weeks and assessed for fatty infiltration and biochemical method. RESULTS: The average fat content using the gold standard method was 2.65 g (2.20-3.05) of fat/100g liver for the control group and 4.14 g (1.95-8.60) of fat/100g of liver for the overfed group (p<0.05). The average fat-fraction obtained from the MR was 0.016 (0.01-0.02) for the control group and 0.057 (0.00-0.18) for the overfed group. The Pearson correlation coefficient between the samples was r(2) = 0.87. CONCLUSION: Multi-echo MR is a good technique to quantify liver fat in rats.

Escherichia coli translocation in experimental short bowel syndrome: probiotic supplementation and detection by polymerase chain reaction.

BACKGROUND: After massive bowel resection, bacterial overgrowth is frequent and favors the occurrence of Gram-negative intestinal bacterial translocation (BT). Probiotics have been recommended in several diseases and may also have beneficial effects on BT. Conversely, polymerase chain reaction (PCR) technique has shown better sensitivity than conventional methods in bacterial detection and has not been investigated in experimental models of short bowel syndrome and BT. OBJECTIVE: To test the hypothesis that Bifidobacterium lactis (BL) administration decreases Escherichia coli bacterial translocation (ECBT) in experimental short bowel syndrome and to confirm the better sensitivity of PCR technique to detect ECBT. METHODS: Forty-eight adult Wistar rats, orally fed with standard rat chow and tap water ad libitum, were maintained in individual metabolic cages for 10 days and divided into three groups: Control group (n = 15): non-manipulated animals. RES group (n = 15): 80% gut resection. 1 ml of sterile water was administered daily after orogastric intubation. RES-PRO group (n = 18): same resection as RES group and daily administration of 7.8 × 10(9) BL (CFU). At the end of the study, portal blood, peripheral blood and mesenteric lymph node (MLN) samples were recovered and cultured. Also, genomic DNA from E. coli was detected by PCR technique. RESULTS: In conventional culture there was no ECBT in control animals whereas 73% of RES and 33% of RES-PRO animals showed it. PCR detected ECBT in 47, 87 and 33%, respectively, showing higher sensitivity.

Biochemical determination of lipid content in hepatic steatosis by the Soxtec method.

AIM: To establish a quantitative method to measure the amount of lipids. METHODS: The livers of 53 male Wistar rats (225 g) with different degrees of hepatic steatosis were studied. This model of hepatic steatosis was based on a high carbohydrate, fat-free modified diet. Biopsies were classified into four grades depending on fat accumulation, using the Kleiner and Brunt classification. Total fat was studied by the Soxtec method (Soxtec 2050 Auto Fat Extraction System), and agreement between both assays was assessed by calculating the kappa coefficient. RESULTS: According to the histological classification, 38% of rats presented grade 0, 21% grade 1, 22% grade 2 and 20% grade 3. The amount of fat per 100 g tissue was 2.60 +/- 0.64 g for grade 0, 3.87 +/- 1.59 g for grade 1, 5.82 +/- 1.37 g for grade 2 and 8.68 +/- 2.30 g for grade 3. Statistically significant differences were found between the mean values for each of the histological grades (P < 0.05). The correlation for the quantification of fat in the liver between both assays was moderate (kappa = 0.60). CONCLUSION: The biochemical quantification of fat in liver tissue by the Soxtec method was correlated with the histological classification, although the agreement between the two tests was only moderate.

Phospholipase Cbeta4 isozyme is expressed in human, rat, and murine heart left ventricles and in HL-1 cardiomyocytes.

Phospholipase C-beta (PLCbeta) isozymes (PLCbeta(1) and PLCbeta(3)) have been extensively characterized in cardiac tissue, but no data are available for the PLCbeta(4) isozyme. In this study, PLCbeta((1-4)) isozymes mRNA relative expression was studied by real-time PCR (RT-PCR) in human, rat, and murine left ventricle and the presence of PLCbeta(4) isozyme at the protein level was confirmed by Western blotting in all species studied. Confocal microscopy experiments carried out in HL-1 cardiomyocytes revealed a sarcoplasmic subcellular distribution of PLCbeta(4). Although there were unexpected significant interspecies differences in the PLCbeta((1-4)) mRNA expression, PLCbeta(4) mRNA was the main transcript expressed in all left ventricles studied. Thus, whereas in human and rat left ventricles PLCbeta(4) > PLCbeta(3) > PLCbeta(2) > PLCbeta(1) mRNA pattern of expression was found, in murine left ventricle the pattern of expression was different, i.e., PLCbeta(4) > PLCbeta(1) > PLCbeta(3) > PLCbeta(2). However, results obtained in mouse HL-1 cardiomyocytes showed PLCbeta(3) approximately PLCbeta(4) > PLCbeta(1) > PLCbeta(2) pattern of mRNA expression indicating a probable cell type specific expression of the different PLCbeta isozymes in cardiomyocytes. Finally, RT-PCR experiments showed a trend, even though not significant (P = 0.067), to increase PLCbeta(4) mRNA levels in HL-1 cardiomyocytes after angiotensin II treatment. These results demonstrate the presence of PLCbeta(4) in the heart and in HL-1 cardiomyocytes showing a different species-dependent pattern of expression of the PLCbeta((1-4)) transcripts. We discuss the relevance of these findings in relation to the development of cardiac hypertrophy.

Resveratrol inhibits nonalcoholic fatty liver disease in rats.

BACKGROUND: The prevalence of nonalcoholic fatty liver disease (NAFLD) is high. NAFLD is linked to obesity, diabetes mellitus, and hypertriglyceridemia. Approximately 20% of patients with NAFLD will eventually develop cirrhosis. Our purpose was to investigate whether resveratrol decreased hepatic steatosis in an animal model of steatosis, and whether this therapeutic approach resulted in a decrease in tumor necrosis factor alpha (TNF-alpha) production, lipid peroxidation and oxidative stress. METHODS: Male Wistar CRL: Wi (Han) (225 g) rats were randomized into three groups. A control group (n = 12) was given free access to regular dry rat chow for 4 weeks. The steatosis (n = 12) and resveratrol (n = 12) groups were given free access to feed (a high carbohydrate-fat free modified diet) and water 4 days per week, and fasted for the remaining 3 days for 4 weeks. Rats in the resveratrol group were given resveratrol 10 mg daily by the oral route. All rats were killed at 4 weeks and assessed for fatty infiltration and bacterial translocation. Levels of TNF-alpha in serum, hepatic malondialdehyde (MDA), oxidative stress (superoxide dismutase, glutathione peroxidase, catalase and nitric oxide synthase) and biochemical parameters were measured. RESULTS: Fat deposition was decreased in the resveratrol group as compared to the steatosis group (Grade 1 vs Grade 3, P < 0.05). TNF-alpha and MDA levels were significantly increased in the steatosis group (TNF-alpha; 33.4 +/- 5.2 vs 26.24 +/- 3.47 pg/ml and MDA; 9.08 +/- 0.8 vs 3.17 +/- 1.45 muM respectively, P < 0.05). This was accompanied by increased superoxide dismutase, glutathione peroxidase and catalase and decreased nitric oxide synthase in the liver of resveratrol group significantly (P < 0.05 vs steatosis group). Bacterial translocation was not found in any of the groups. Glucose levels were decreased in the group of rats given resveratrol (P < 0.05). CONCLUSION: Resveratrol decreased NAFLD severity in rats. This effect was mediated, at least in part, by TNF-alpha inhibition and antioxidant activities.