ABSTRACT
BACKGROUND@#Large-scale muscle tissue engineering remains a major challenge. An axial vascular pedicle and perfusion bioreactor are necessary for the development and maintenance of large-scale engineered muscle to ensure circulation within the construct. We aimed to develop a novel experimental model of a large-scale engineered muscle flap from an existing rat groin fat flap.@*METHODS@#A fat flap based on the superficial inferior epigastric vascular pedicle was excised from rats and placed into a perfusion bioreactor. The flaps were kept in the bioreactor for up to 7 weeks, and transdifferentiation of adipose to muscle tissue could have taken place. This system enabled myogenic-differentiation medium flow through the bioreactor at constant pH and oxygen concentration. Assessment of viability was performed by an immunofluorescence assay, histological staining, a calcein-based live/dead test, and through determination of RNA quantity and quality after 1, 3, 5, and 7 weeks.@*RESULTS@#Immunofluorescence staining showed that smooth muscle around vessels was still intact without signs of necrosis or atrophy. The visual assessment of viability by the calcein-based live/dead test revealed viability of the rat adipose tissue preserved in the bioreactor system with permanent perfusion. RNA samples from different experimental conditions were quantified by spectrophotometry, and intact bands of 18S and 28S rRNA were detected by gel electrophoresis, indicating that degradation of RNA was minimal.@*CONCLUSIONS@#Flow perfusion maintains the long-term viability of a rat groin engineered muscle flap in vitro, and a large-scale vascularized muscle could be engineered in a perfusion bioreactor.
Subject(s)
Animals , Male , Rats , Bioreactors , Groin , Perfusion , RNA , Rats, Inbred Lew , Surgical Flaps , Tissue EngineeringABSTRACT
<p><b>BACKGROUND</b>Large-scale muscle tissue engineering remains a major challenge. An axial vascular pedicle and perfusion bioreactor are necessary for the development and maintenance of large-scale engineered muscle to ensure circulation within the construct. We aimed to develop a novel experimental model of a large-scale engineered muscle flap from an existing rat groin fat flap.</p><p><b>METHODS</b>A fat flap based on the superficial inferior epigastric vascular pedicle was excised from rats and placed into a perfusion bioreactor. The flaps were kept in the bioreactor for up to 7 weeks, and transdifferentiation of adipose to muscle tissue could have taken place. This system enabled myogenic-differentiation medium flow through the bioreactor at constant pH and oxygen concentration. Assessment of viability was performed by an immunofluorescence assay, histological staining, a calcein-based live/dead test, and through determination of RNA quantity and quality after 1, 3, 5, and 7 weeks.</p><p><b>RESULTS</b>Immunofluorescence staining showed that smooth muscle around vessels was still intact without signs of necrosis or atrophy. The visual assessment of viability by the calcein-based live/dead test revealed viability of the rat adipose tissue preserved in the bioreactor system with permanent perfusion. RNA samples from different experimental conditions were quantified by spectrophotometry, and intact bands of 18S and 28S rRNA were detected by gel electrophoresis, indicating that degradation of RNA was minimal.</p><p><b>CONCLUSIONS</b>Flow perfusion maintains the long-term viability of a rat groin engineered muscle flap in vitro, and a large-scale vascularized muscle could be engineered in a perfusion bioreactor.</p>
ABSTRACT
The relationship between Kruppel-like factor 4 (KLF4) and the Notch pathway was determined to investigate the effect of KLF4 on the activation of hepatic stellate cells and underlying mechanisms. Fifty SPF BALB/c mice were randomly divided into two groups. A liver fibrosis model was established in 25 mice as the experimental group, and the remaining 25 mice served as controls. On the day 0, 7, 14, and 35, liver tissues were removed for immunofluorescent detection. The Notch pathway inhibitor DAPT was added to the primary original hepatic stellate cells, and KLF4 and Notch-associated factor expression was detected by qRT-PCR. Additionally, the hepatic stellate cell line LX-2 was used to establish control and experimental groups, and was cultured in vitro. LX-2 cells in the experimental groups were treated with DAPT and the Notch activator transforming growth factor-beta 1 separately, whereas those in the control group were given isotonic culture medium. After 48 h, KLF4 expression was examined by Western blotting. After transient transfection of LX-2 cells to increase KLF4, the expression of Notch factor was examined. Immunofluorescence analysis showed that, with the aggravation of liver fibrosis, the absorbance (A) values of KLF4 were decreased (day 0: 980.73±153.19; day 7: 1087.99±230.23; day 14: 390.95±93.56; day 35: 245.99±87.34). The expression of Notch pathway- related factors (Notch-1, Notch-2, and Jagged-1) in the hepatic stellate cell membrane was negatively correlated to KLF4 expression. With the increase of KLF4 expression, Notch-2 (0.73±0.13) and Jagged-1 (0.43±0.12) expression decreased, whereas Notch-1 level was not detectable. When the Notch pathway was inhibited, KLF4 levels generally increased (18.12±1.31). Our results indicate that KLF4 expression is negatively correlated to the Notch pathway in hepatic stellate cells, which may provide a reference for the treatment of hepatic fibrosis.
Subject(s)
Animals , Mice , Cell Line , Cells, Cultured , Hepatic Stellate Cells , Metabolism , Kruppel-Like Transcription Factors , Genetics , Metabolism , Liver Cirrhosis , Metabolism , Mice, Inbred BALB C , Receptors, Notch , Metabolism , Signal Transduction , Transforming Growth Factor beta1 , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To evaluate the value of cardiac troponin I (CTnI) measurement in predicting anthracycline-induced cardiotoxicity in patients with breast cancer.</p><p><b>METHODS</b>This study was conducted among 186 breast cancer patients receiving anthracycline-based chemotherapy. Serum cTnI concentrations before and after each cycle of the chemotherapy and the left ventricular ejection fraction (LVEF) before and at the 2nd, 4th and 6th months of the treatment were recorded. According to serum cTnI concentration, the patients were divided into CTnI+ group (with serum CTnI concentration of no less than 0.1 ng/ml, n=60) and CTnI- (<0.1 ng/ml) group (n=126).</p><p><b>RESULTS</b>No patients in this series experienced cardiac heart failure (CHF). The number of patients with a LVEF reduction by over 10% from the baseline was 16 (26.7%) in CTnI+ group, as compared to 7 (5.6%) in CTnI- group, showing a significant difference between the two groups (P<0.01).</p><p><b>CONCLUSION</b>CTnI can be a useful marker for early prediction of anthracycline-induced cardiotoxicity in patients with breast cancer.</p>