PCL/β-TCP Composite Scaffolds Exhibit Positive Osteogenic Differentiation with Mechanical Stimulation

We investigated the use of Polycaprolactone (PCL)/β-tricalcium phosphate (β-TCP) composites with applied mechanical stimulation as scaffold for bone tissue engineering. PCL-based three-dimensional (3D) structures were fabricated in a solvent-free process using a 3D-printing technique. The mass fraction of β-TCP was varied in the range 0–30%, and the structure and compressive modulus of the specimens was characterized. The shape and interconnectivity of the pores was found to be satisfactory, and the compressive modulus of the specimens was comparable with that of human trabecular bone. Human mesenchymal stem cells were seeded on the composites, and various biological evaluations were performed over 9 days. With a mass fraction of β-TCP of 30%, differentiation began earlier; however, the cell proliferation rate was lower. Through the use of mechanical stimulation, however, the proliferation rate recovered, and was comparable with that of the other groups. This stimulation effect was also observed in ECM generation and other biological assays. With mechanical stimulation, expression of osteogenic markers was lower on samples with a β-TCP content of 10 wt% than without β-TCP; however, with mechanical stimulation, the sample with a β-TCP content of 30 wt% exhibited significantly greater expression of those markers than the other samples. We found that mechanical stimulation and the addition of β-TCP interacted closely, and that a mass fraction of β-TCP of 30% was particularly useful as a bone tissue scaffold when accompanied by mechanical stimulation.

Relationship between Tetrahydrobiopterin and Portal Hypertension in Patients with Chronic Liver Disease

Tetrahydrobiopterin (BH4) is an essential cofactor in NO synthesis by endothelial nitric oxide synthase (eNOS) enzymes. It has been previously suggested that reduced intrahepatic BH4 results in a decrease in intrahepatic NO and contributes to increased hepatic vascular resistance and portal pressure in animal models of cirrhosis. The main aim of the present study was to evaluate the relationship between BH4 and portal hypertension (PHT). One hundred ninety-three consecutive patients with chronic liver disease were included in the study. Liver biopsy, measurement of BH4 and hepatic venous pressure gradient (HVPG) were performed. Hepatic fibrosis was classified using the Laennec fibrosis scoring system. BH4 levels were determined in homogenized liver tissues of patients using a high performance liquid chromatography (HPLC) system. Statistical analysis was performed to evaluate the relationship between BH4 and HVPG, grade of hepatic fibrosis, clinical stage of cirrhosis, Child-Pugh class. A positive relationship between HVPG and hepatic fibrosis grade, clinical stage of cirrhosis and Child-Pugh class was observed. However, the BH4 level showed no significant correlation with HVPG or clinical features of cirrhosis. BH4 concentration in liver tissue has little relation to the severity of portal hypertension in patients with chronic liver disease.

Detection of Mitochondrial ATP-Sensitive Potassium Channels in Rat Cardiomyocytes

Mitochondrial ATP-sensitive potassium (mitoKATP) channels play a role in early and late ischemic preconditioning. Nevertheless, the subunit composition of mitoKATP channels remains unclear. In this study, we investigated the subunit composition of mitoKATP channels in mitochondria isolated from rat cardiac myocytes. Mitochondria were visualized using the red fluorescence probe, Mitrotracker Red, while mitoKATP channels were visualized using the green fluorescence probe, glibenclamide-BODIPY. The immunofluorescence confocal microscopy revealed the presence of Kir6.1, Kir6.2 and SUR2 present in the cardiac mitochondria. Western blot analysis was carried to further investigate the nature of mitoKATP channels. For SUR proteins, a 140-kDa immunoreactive band that corresponded to SUR2, but no SUR1 was detected. For Kir6.2, three bands (~4, ~6, and ~0 kDa) were detected, and a specific ~6-kDa immunoreactive band corresponding to Kir6.1 was also observed. These observations suggest that the subunits of mitoKATP channels in rat myocytes include Kir6.1, Kir6.2, and a SUR2-related sulfonylurea-binding protein.

Identification of a Marker Protein for Cardiac Ischemia and Reperfusion Injury by Two-Dimensional Gel Electrophoresis and Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

The purpose of the present study was to evaluate the expression of cardiac marker protein in rabbit cardiac tissue that was exposed to ischemic preconditioning (IPC), or ischemiareperfusion injury (IR) using two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). We compared 2DE gels of control (uninjured) cardiac tissue with those of IPC and IR cardiac tissue. Expression of one protein was detected in IR heart tissue, however the protein was not detected in the samples of control and IPC tissue. To further characterize the detected protein molecule, the protein in the 2D gel was isolated and subjected to trypsin digestion, followed by MALDI-MS. The protein was identified as myoglobin, which was confirmed also by Western blot analysis. These results are consistent with previous studies of cardiac markers in ischemic hearts, indicating myoglobin as a suitable marker of myocardial injury. In addition, the present use of multiple techniques indicates that proteomic analysis is an appropriate means to identify cardiac markers in studies of IPC and IR.