Care during freeze-drying of bovine pericardium tissue to be used as a biomaterial: A comparative study

Bovine pericardium (BP) tissue is widely used in the manufacture of bioprosthetics. The effects of freeze-drying on the BP tissue have been studied by some researchers in order to decrease their cytotoxicity due to preservation in formaldehyde solution, and to increase the lifetime of the product in storage. This study was undertaken in order to study the effect of freeze-drying in the structure of BP. To perform this study BP samples were freeze-dried in two different types of freeze-dryers available in our laboratory: a laboratory freeze-dryer, in which it was not possible to control parameters and a pilot freeze-dryer, wherein all parameters during freezing and drying were controlled. After freeze-drying processes, samples were analyzed by SEM, Raman spectroscopy, tensile strength, water uptake tests and TEM. In summary, it has been demonstrated that damages occur in collagen fibers by the loss of bulk water of collagen structure implicating in a drastic decreasing of BP mechanical properties due to its structural alterations. Moreover, it was proven that the collagen fibrils suffered breakage at some points, which can be attributed to the uncontrolled parameters during drying. (C) 2011 Elsevier Inc. All rights reserved.

A New Method to Prepare Porous Silk Fibroin Membranes Suitable for Tissue Scaffolding Applications

A new method to prepare porous silk fibroin (SF) membranes without dialysis proposed. Silk fibers were degummed to remove sericin and the resultant fibroin was dissolved in a CaCl(2)-CH(3)CH(2)OH-H(2)O ternary solvent. Rather than undergoing dialysis, a fibroin salty solution was diluted in water and then submitted to a mechanical agitation that led to a phase separation through foam formation on the solution surface. This foam was continually collected and then compacted between plates to remove the excess of water. The membranes presented large pores with diameters of greater than 100 pm (as shown by scanning electron microscopy - SEM), porosity of 68% and water content of 91% w/w. X-ray diffraction (XRD) and infrared spectroscopy (FTIR-ATR) indicated that the membranes present SF in a beta-sheet structure even before the ethanol treatment. A typical elastic deformation profile and degradation under temperature were observed using calorimetric analysis (DSC), thermal gravimetric analysis (TGA) and mechanical tests. As indicated by the in vitro cytotoxicity tests, these membranes present potential for use as scaffolds. (C) 2009 Wiley Periodicals, Inc. J Appl Polym Sci 114: 617-623, 2009

Ruthenium-nitrite complex as pro-drug releases NO in a tissue and enzyme-dependent way

Nitric oxide (NO) plays an important role in the control of the vascular tone and the most often employed NO donors have limitations due to their harmful side-effects. In this context, new NO donors have been prepared, in order to minimize such undesirable effects. cis-[Ru(bpy)(2)(py)NO(2)](PF(6)) (RuBPY) is a new nitrite complex synthesized in our laboratory that releases NO in the presence of the vascular tissue only. In this work the vasorelaxation induced by this NO donor has been studied and compared to that obtained with the well known NO donor SNP. The relaxation induced by RuBPY is concentration-dependent in denuded rat aortas pre-contracted with phenylephrine (EC(50)). This new compound induced relaxation with efficacy similar to that of SNP, although its potency is lower. The time elapsed until maximum relaxation is achieved (E(max) = 240 s) is similar to measured for SNP (210 s). Vascular reactivity experiments demonstrated that aortic relaxation by RuBPY is inhibited by the soluble guanylyl-cyclase inhibitor 1H-[1,2,4] oxadiozolo[4,3-a]quinoxaline-1-one (ODQ 1 mu M). In a similar way, 1 mu M ODQ also reduces NO release from the complex as measured with DAF-2 DA by confocal microscopy. These findings suggest that this new complex RuBPY that has nitrite in its structure releases NO inside the vascular smooth muscle cell. This ruthenium complex releases significant amounts of NO only in the presence of the aortic tissue. Reduction of nitrite to NO is most probably dependent on the soluble guanylyl-cyclase enzyme, since NO release is inhibited by ODQ. (C) 2011 Elsevier Inc. All rights reserved.

Reduction of parasitism tissue by treatment of mice chronically infected with Trypanosoma cruzi with lignano lactones

The reduction of parasitism tissue upon treatment with two lignano lactones, namely (-)- cubebin (CUB) and (-)-hinokinin (HNK), was evaluated in the chronic phase of Chagas` disease by quantifying the enzyme beta-galactosidase expressed by the CL B5 clone strain of Trypanosoma cruzi. Tissue karyometry was also performed. Treatment with the assessed lignans led to a larger reduction in parasitism tissue in all evaluated organs, compared with benznidazole (BZN). Oral treatment with CUB or HNK was more effective. Karyometry results demonstrated that the infected control animals had increased nuclear area compared with uninfected controls, indicating cellular hypertrophy. Results also revealed that use of CUB or HNK was able to significantly prevent this increase, and a slight decrease in the nuclear area was observed, compared with mice treated with BZN. Taken together, these data demonstrate that CUB and HNK could be considered as potential compounds for the development of new drugs for treatment of Chagas` disease.

Topical Delivery of Lycopene Using Microemulsions: Enhanced Skin Penetration and Tissue Antioxidant Activity

Topical delivery of lycopene is a convenient way to supplement cutaneous levels of antioxidants. In this study, lycopene was incorporated (0.05%, w/w) in two microemulsions containing BRIJ-propylene glycol (2:1, w/w, surfactant blend) but different oil phases: mono/diglycerides of capric and caprylic acids (MG) or triglycerides of the same fatty acids (TG). Microemulsions containing MG and TG were isotropic, fluid, and clear, with internal phase diameters of 27 and 52 nm, respectively. Both MG- or TG-containing microemulsions markedly increased lycopene penetration in the stratum corneum, (6- and 3.6-fold, respectively) and in viable layers of porcine ear skin 2 (from undetected to 172.6 +/- 41.1 and 103.1 +/- 7.2 ng/cm(2), respectively) compared to a control solution. To assure that lycopene delivered to the skin was active, the antioxidant activity of skin treated with MG-containing microemulsion was determined by CUPRAC assay, and found to be 10-fold higher than untreated skin. The cytotoxicity of MG-containing microemulsion in cultured fibroblasts was similar to propylene glycol (considered safe) and significantly less than of sodium lauryl sulfate (a moderate-to-severe irritant) at 1-50 mu g/mL. These results demonstrate that the MG-containing microemulsion is an efficient and safe system to increase lycopene delivery to the skin and the antioxidant activity in the tissue. (C) 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:1346-1357, 2010

HPLC-FLD methods to quantify chloroaluminum phthalocyanine in nanoparticles, plasma and tissue: application in pharmacokinetic and biodistribution studies

Analytical and bioanalytical methods of high-performance liquid chromatography with fluorescence detection (HPLC-FLD) were developed and validated for the determination of chloroaluminum phthalocyanine in different formulations of polymeric nanocapsules, plasma and livers of mice. Plasma and homogenized liver samples were extracted with ethyl acetate, and zinc phthalocyanine was used as internal standard. The results indicated that the methods were linear and selective for all matrices studied. Analysis of accuracy and precision showed adequate values, with variations lower than 10% in biological samples and lower than 2% in analytical samples. The recoveries were as high as 96% and 99% in the plasma and livers, respectively. The quantification limit of the analytical method was 1.12 ng/ml, and the limits of quantification of the bioanalytical method were 15 ng/ml and 75 ng/g for plasma and liver samples, respectively. The bioanalytical method developed was sensitive in the ranges of 15-100 ng/ml in plasma and 75-500 ng/g in liver samples and was applied to studies of biodistribution and pharmacokinetics of AlClPc. (C) 2011 Elsevier B.V. All rights reserved.

Preliminary study of human breast tissue using synchrotron radiation combining WAXS and SAXS techniques

Using synchrotron radiation, we combined simultaneously wide angle X-ray scattering (WAXS) and small angle X-ray scattering (SAXS) techniques to obtain the scattering profiles of normal and neoplastic breast tissu-es samples at the momentum transfer range 6.28 nm(-1) <= Q(=4 pi.sin(theta/2)lambda) <= 50.26 nm(-1) and 0.15 nm(-1) <= Q <= 1.90 nm(-1), respectively. The results obtained show considerable differences between the scattering profiles of these tissues. We verified that the combination of some parameters (ratio between glandular and adipose peak intensity and third-order axial peak intensity) extracted from scattering profiles can be used for identifying breast cancer. (c) 2009 Elsevier Ltd. All rights reserved.

Effect of the immunosuppressants on hepatocyte proliferation and apoptosis in a young animal model of liver regeneration: An immunohistochemical study using tissue microarrays

Hepatocyte proliferation and apoptosis (programmed cell death) occur during the liver parenchyma regeneration and the liver size modeling is mainly controlled by hepatocyte apoptosis. The purpose of the present study was to verify the influence of immunosuppressant drugs on these phenomena by utilizing tissue microarray techniques. Thirty-six weaning rats (age 21-23 days, weight 30-50 g) were divided into six groups: control, sham, hepatectomy, hepatectomy plus solumedrol, hepatectomy plus CsA, and hepatectomy plus Tac. The animals were killed one day after hepatectomy, and the remnant livers were weighed and harvested for tissue microarray sections. Liver cell proliferation was evaluated by staining for PCNA and apoptosis was detected by the TUNEL method. It was verified that CsA promoted a decrease in the liver weight, Tac and CsA decreased the proliferation index of hepatocytes, and glucocorticoid had no significant effects. The apoptosis index was not altered by hepatectomy or immunosuppressants. Our data indicate that, in the growing rat, CsA and Tac have negative effects on hepatocyte proliferation and have no effect on the hepatocyte apoptosis.

Twelve-month Outcomes After Coronary Stenting With the Genous (TM) Bio-Engineered R Stent (TM) in Diabetic Patients from the e-HEALING Registry

Objectives: We compared 12-month outcomes, regarding ischemic events, repeat intervention, and ST, between diabetic and nondiabetic patients treated with the Genous (TM) EPC capturing R stent (TM) during routine nonurgent percutaneous coronary intervention (PCI) using data from the multicenter, prospective worldwide e-HEALING registry. Background: Diabetic patients have an increased risk for restenosis and stent thrombosis (ST). Methods: In the 4,996 patient e-HEALING registry, 273 were insulin requiring diabetics (IRD), 963 were non-IRD (NIRD), and 3,703 were nondiabetics. The 12-month primary outcome was target vessel failure (TVF), defined as target vessel-related cardiac death or myocardial infarction (MI) and target vessel revascularization. Secondary outcomes were the composite of cardiac death, MI or target lesion revascularization (TLR), and individual outcomes including ST. Cumulative event rates were estimated with the Kaplan-Meier method and compared with a log-rank test. Results: TVF rates were respectively 13.4% in IRD, 9.0% in NIRD, and 7.9% in nondiabetics (P < 0.01). This was mainly driven by a higher mortality hazard in IRD (P < 0.001) and NIRD (P = 0.07), compared with nondiabetics. TLR rates were comparable in NIRD and nondiabetics, but significantly higher in IRD (P = 0.04). No difference was observed in ST. Conclusion: The 1-year results of the Genous stent in a real-world population of diabetics show higher TVF rates in diabetics compared with nondiabetics, mainly driven by a higher mortality hazard. IRD is associated with a significant higher TLR hazard. Definite or probable ST in all diabetic patients was comparable with nondiabetics. (J Interven Cardiol 2011;24:285-294)

Clinical results after coronary stenting with the Genous (TM) Bio-engineered R stent (TM): 12-month outcomes of the e-HEALING (Healthy Endothelial Accelerated Lining Inhibits Neointimal Growth) worldwide registry

Aims: e-HEALING is a worldwide, internet-based registry designed to capture post marketing clinical data on the use of the Genous (TM) EPC capturing R stent (TM). Rapid restoration of a healthy endothelial layer after stent placement by capturing circulating endothelial progenitor cells may reduce both stent thrombosis (ST) and in-stent-restenosis. Methods and results: We planned a 5,000 patient registry with >= 1 lesion suitable for stenting. The 12-month primary outcome was target vessel failure (TVF), defined as target vessel-related cardiac death or myocardial infarction (MI) and target vessel revascularisation. Secondary outcomes were the composite of cardiac death, MI or target lesion revascularisation (TLR), and individual outcomes including ST. A total of 4,939 patients received >= 1 Genous stent between 2005 and 2007. Baseline characteristics showed a median age of 63 years, 79% males, 25% diabetics, and 37% with prior MI. A total of 49% of lesions treated were ACC/AHA type B2 or C; 1.1 stents per lesion were used. At 12 months, TVF occurred in 8.4% and the composite of cardiac death, MI or TLR in 7.9%. Twelve-month TLR and ST were 5.7% and 1.1%, respectively. Conclusions: Coronary stenting with the Genous results in good clinical outcomes, and low incidences of repeat revascularisation and ST.

Small airway remodeling in acute respiratory distress syndrome: a study in autopsy lung tissue

Introduction: Airway dysfunction in patients with the Acute Respiratory Distress Syndrome (ARDS) is evidenced by expiratory flow limitation and dynamic hyperinflation. These functional alterations have been attributed to closure/obstruction of small airways. Airway morphological changes have been reported in experimental models of acute lung injury, characterized by epithelial necrosis and denudation in distal airways. To date, however, no study has focused on the morphological airway changes in lungs from human subjects with ARDS. The aim of this study is to evaluate structural and inflammatory changes in distal airways in ARDS patients. Methods: We retrospectively studied autopsy lung tissue from subjects who died with ARDS and from control subjects who died of non pulmonary causes. Using image analysis, we quantified the extension of epithelial changes (normal, abnormal and denudated epithelium expressed as percentages of the total epithelium length), bronchiolar inflammation, airway wall thickness, and extracellular matrix (ECM) protein content in distal airways. The Student`s t test or the Mann-Whitney test was used to compare data between the ARDS and control groups. Bonferroni adjustments were used for multiple tests. The association between morphological and clinical data was analyzed by Pearson rank test. Results: Thirty-one ARDS patients (A: PaO(2)/FiO(2) <= 200, 45 +/- 14 years, 16 males) and 11 controls (C:52 +/- 16 years, 7 males) were included in the study. ARDS airways showed a shorter extension of normal epithelium (A:32.9 +/- 27.2%, C:76.7 +/- 32.7%, P < 0.001), a larger extension of epithelium denudation (A:52.6 +/- 35.2%, C:21.8 +/- 32.1%, P < 0.01), increased airway inflammation (A:1(3), C:0(1), P = 0.03), higher airway wall thickness (A:138.7 +/- 54.3 mu m, C:86.4 +/- 33.3 mu m, P < 0.01), and higher airway content of collagen I, fibronectin, versican and matrix metalloproteinase-9 (MMP-9) compared to controls (P = 0.03). The extension of normal epithelium showed a positive correlation with PaO(2)/FiO(2) (r(2) = 0.34; P = 0.02) and a negative correlation with plateau pressure (r(2) = 0.27; P = 0.04). The extension of denuded epithelium showed a negative correlation with PaO(2)/FiO(2) (r(2) = 0.27; P = 0.04). Conclusions: Structural changes in small airways of patients with ARDS were characterized by epithelial denudation, inflammation and airway wall thickening with ECM remodeling. These changes are likely to contribute to functional airway changes in patients with ARDS.

Severe and refractory myositis in mixed connective tissue disease: a description of a rare case

Mixed connective tissue disease (MCTD) is a rare disease that includes clinical and laboratorial manifestations of systemic lupus erythematosus, scleroderma and polymyositis that is associated with high titers of anti-U1RNP antibodies. In general, muscle involvement is subclinical, usually appearing as an increase in muscle enzyme levels that tends to be a characteristic of the initial phases of the disease. Severe clinical muscle weakness is not observed in this disease. The objective of this study is to report a rare case of a patient who presented a severe onset of myositis characterized by dysphagia, an increase in myopathy and a weakening of the cervical musculature. While there was no response to the administration of an initial dose of corticosteroids, improvement was observed after increasing the dose of corticosteroids, in addition to the initiation of pulse therapy with methylprednisolone accompanied by methotrexate treatment. The authors emphasize that there is only one previously reported case regarding a child with MCTD and severe clinical myopathy on electromyography and muscle biopsy, and they report in this article one adult female patient who presented severe myositis and was refractive to corticotherapy. Lupus (2010) 19, 1659-1661.

Effects of Time on Ultrastructural Integrity of Parathyroid Tissue Before Cryopreservation

Cryopreservation of parathyroid tissue is used in the surgical treatment of secondary hyperparathyroidism. After surgical resection, the tissue is temporarily maintained in a cell culture solution until it arrives at the specialized laboratory where the cryopreservation process will take place. The present study evaluates the time that the human hyperplastic parathyroid gland tissue can wait before cryopreservation, based on parathyroid cell ultrastructural integrity. This prospective study included 11 patients who underwent total parathyroidectomy with heterotopic autotransplantation and cryopreservation of parathyroid tissue fragments. Part of the tissue was kept in cell culture solution at 4A degrees C. Five time periods between 2 and 24 h were defined, and parathyroid fragments were kept in the solution for that length of time. At the end of each period, the fragments were removed from the transport solution, fixed, and prepared for ultrathin sections. Of the 11 cases studied, 10 showed ultrastructural findings consistent with cellular viability in tissue fragments that remained in the transport solution up to 12 h. Electron microscopy revealed that cell adhesion and the integrity of plasma membranes, nuclei, and mitochondria were preserved in one case for up to 24 h. Changes in mitochondrial structure represented the most constant ultrastructural damage seen in the cases studied, in addition to the presence of edema and cell vacuoles. Analysis of the ultrastructure of hyperplastic parathyroid gland tissue showed that ultrastructural integrity was in most cases properly maintained in fragments stored up to 12 h in a cell culture solution at 4A degrees C.

cDNA microarray analysis of differentially expressed genes in penile tissue after treatment with tadalafil

To evaluate differential gene expression in penile tissue after treatment with the phosphodiesterase 5 (PDE5) inhibitor tadalafil, as of the three clinically available PDE5 inhibitors (sildenafil, tadalafil, and vardenafil) used for the treatment of erectile dysfunction (ED), tadalafil has a long half-life and low incidence of side-effects. In all, 32 adult rats were divided into two groups. The control group received 0.5 mL of drinking water alone, while the tadalafil group was treated with tadalafil at a dose of 0.27 mg/kg. At 4 h after treatment with water or tadalafil the rats were killed and the penile tissue was removed. The total RNA was isolated from the penile tissue from both groups and differentially expressed genes were identified by cDNA microarray analysis. To validate the expression data from the microarray analysis, quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry were used. In all, 153 genes were differentially expressed between the control group and the tadalafil group. We validated the microarray results by quantitative PCR for the insulin-like growth factor binding protein 6 (IGFBP-6) gene and the neuronal calcium sensor 1 (NCS-1) gene, both of which were up-regulated in the tadalafil group, and for the natriuretic peptide receptor 1 (NPR-1) gene that was down-regulated in this group. Immunohistochemistry showed localization of the NCS-1 protein in sinusoid trabeculae of the corpus cavernosum in control and tadalafil-treated rats. There was differential expression in 153 genes after tadalafil treatment. Some of these genes such as IGFBP-6, NPR-1 and NCS-1, might result in new targets in the treatment of ED.

Potassium in Hemorrhagic Shock: A Potential Marker of Tissue Hypoxia

Background: This study was designed to evaluate serum potassium level variation in a porcine model of hemorrhagic shock ( HS). Methods: Eight pigs were studied in a controlled hemorrhage model of HS. Blood withdrawal began at a 50 mL/min to 70 mL/min rate, adjusted to reach a mean arterial pressure ( MAP) level of 60 mm Hg in 10 minutes. When MAP reached 60 mm Hg, the blood withdrawal rate was adjusted to maintain a MAP decrease rate of 10 mm Hg every 2 minutes to 4 minutes. Arterial and mixed venous blood samples were collected at MAP levels of 60 mm Hg, 50 mm Hg, 40 mm Hg, 30 mm Hg, 20 mm Hg, and 10 mm Hg and analyzed for oxygen saturation, PO(2), PCO(2), potassium, lactate, bicarbonate, hemoglobin, pH, and standard base excess. Results: Significant increase in serum potassium occurred early in all animals. The rate of rise in serum potassium and its levels accompanied the hemodynamic deterioration. Hyperkalemia ( K >5 mmol/L) incidence was 12.5% at 60 mm Hg and 50 mm Hg, 62.5% at 40 mm Hg, 87.5% at 30 mm Hg, and 100% at 20 mm Hg. Strong correlations were found between potassium levels and lactate ( R = 0.82), SvO(2) ( R = 0.87), Delta pH ( R = 0.83), and Delta PCO(2) ( R = 0.82). Conclusions: Serum potassium increase accompanies the onset of HS. The rise in serum potassium was directly related to the hemodynamic deterioration of HS and strongly correlated with markers of tissue hypoxia.