[Decellularization technology application in whole liver reconstruct biological scaffold].

OBJECTIVE: To explore an innovative method for preparation of a whole-liver reconstruct scaffold with intact three-dimensional geometry, vasculature and bile duct by decellularization technology. METHODS: The portal vein was annulated and perfused sequentially with 1%Triton X-100 and 1%SDS for about 4 h, and then was perfused with phosphate buffered saline to dilute SDS residue. The retained structure was evaluated by histological analyses, including macroscopic, Hematoxylin-Eosin staining, Masson's trichrome staining, orcein staining and SEM. The liquid polymer preparation 8% - 10%, which was made of chlorinated poly vinyl chloride (CPVC as solute), acetone (as solvent) and pigment, was injected into portal vein and bile duct to demonstrate the integrity of the portal vein and bile duct. The scaffold was cut into slices with the thickness of about 50 microm and cocultured with C3A cell line. RESULTS: Macroscopic examination showed that the decellularized liver was transparent and intrahepatic Glisson's system could be observed. H&E staining of slices of decellularized liver demonstrated no intact cells or nuclei existed. Masson trichrome staining revealed collagen retained. Orcein staining showed that there were elastic fibers. SEM showed the network of ECM was intact. C3A-to-scaffold co-culture revealed the scaffold of good biocompatibility. CONCLUSION: Perfusion with detergents through portal vein for liver decellularization was an efficient method to obtain a completely whole-liver scaffold which can be used for hepatic organ reconstruction.

Quantification of liver fibrosis via second harmonic imaging of the Glisson's capsule from liver surface.

Liver surface is covered by a collagenous layer called the Glisson's capsule. The structure of the Glisson's capsule is barely seen in the biopsy samples for histology assessment, thus the changes of the collagen network from the Glisson's capsule during the liver disease progression are not well studied. In this report, we investigated whether non-linear optical imaging of the Glisson's capsule at liver surface would yield sufficient information to allow quantitative staging of liver fibrosis. In contrast to conventional tissue sections whereby tissues are cut perpendicular to the liver surface and interior information from the liver biopsy samples were used, we have established a capsule index based on significant parameters extracted from the second harmonic generation (SHG) microscopy images of capsule collagen from anterior surface of rat livers. Thioacetamide (TAA) induced liver fibrosis animal models was used in this study. The capsule index is capable of differentiating different fibrosis stages, with area under receiver operating characteristics curve (AUC) up to 0.91, making it possible to quantitatively stage liver fibrosis via liver surface imaging potentially with endomicroscopy.

DNAzyme-based probe for circulating microRNA detection in peripheral blood.

BACKGROUND: The recent discovery of microRNAs (miRNAs) and their extracellular presence suggest a potential role of these regulatory molecules in defining the metastatic potential of cancer cells and mediating the cancer-host communication. This study aims to improve the sensitivity of miRNA detection via DNAzyme-based method and enhance the selectivity by using the DNAzyme-based probe to reduce nonspecific amplification. METHODS: The miRNA probes were chemically synthesized with a phosphate at the 5' end and purified by polyacrylamide gel electrophoresis. Exosomal RNA from peripheral blood was isolated. Carboxylated magnetic microsphere beads (MBs) were functionalized with streptavidin (SA) according to a previously reported method with some modification. T capture probe-coated SA-MBs (DNA-MBs) were also prepared. The fluorescent spectra were measured using a spectrofluorophotometer. RESULTS: We designed an incomplete DNAzyme probe with two stems and one bubble structure as a recognition element for the specific detection of miRNA with high sensitivity. The background effects were decreased with increase of the added of DNA-MBs and capturing times. Therefore, 20 minutes was selected as the optimal concentration in the current study. The fluorescence intensity increases as the hybridization time changed and reached a constant level at 40 minutes, and 1 µM is the optimum signal probe concentration for self-assembled DNA concatemers formation. In the presence of miRNA, the fluorescence of the solution increased with increasing miRNA concentration. There is no obvious fluorescence in the presence of 10 mM of other nontarget DNA. CONCLUSION: A simple, rapid method with high performance has been constructed based on identified circulating miRNA signatures using miRNA-induced DNAzyme. This assay is simple, inexpensive, and sensitive, enabling quantitative detection of as low as 10 fM miRNA.

In vivo label-free quantification of liver microcirculation using dual-modality microscopy.

Microcirculation lesion is a common symptom of chronic liver diseases in the form of vasculature deformation and circulation alteration. In acute to chronic liver diseases such as biliary atresia, microcirculation lesion can have an early onset. Detection of microcirculation lesion is meaningful for studying the progression of liver disease. We have combined wide-field fluorescence microscopy and a laser speckle contrast technique to characterize hepatic microcirculation in vivo without labeling in a bile-duct ligation rat fibrosis model of biliary atresia. Through quantitative image analysis of four microcirculation parameters, we observed significant microcirculation lesion in the early to middle stages of fibrosis. This bimodal imaging method is useful to assess hepatic microcirculation lesion for the study of liver diseases.

Hepatic stellate cell-targeted delivery of hepatocyte growth factor transgene via bile duct infusion enhances its expression at fibrotic foci to regress dimethylnitrosamine-induced liver fibrosis.

Liver fibrosis generates fibrotic foci with abundant activated hepatic stellate cells and excessive collagen deposition juxtaposed with healthy regions. Targeted delivery of antifibrotic therapeutics to hepatic stellate cells (HSCs) might improve treatment outcomes and reduce adverse effects on healthy tissue. We delivered the hepatocyte growth factor (HGF) gene specifically to activated hepatic stellate cells in fibrotic liver using vitamin A-coupled liposomes by retrograde intrabiliary infusion to bypass capillarized hepatic sinusoids. The antifibrotic effects of DsRed2-HGF vector encapsulated within vitamin A-coupled liposomes were validated by decreases in fibrotic markers in vitro. Fibrotic cultures transfected with the targeted transgene showed a significant decrease in fibrotic markers such as transforming growth factor-ß1. In rats, dimethylnitrosamine-induced liver fibrosis is manifested by an increase in collagen deposition and severe defenestration of sinusoidal endothelial cells. The HSC-targeted transgene, administered via retrograde intrabiliary infusion in fibrotic rats, successfully reduced liver fibrosis markers alpha-smooth muscle actin and collagen, accompanied by an increase in the expression of DsRed2-HGF near the fibrotic foci. Thus, targeted delivery of HGF gene to hepatic stellate cells increased the transgene expression at the fibrotic foci and strongly enhanced its antifibrotic effects.

Hepatocyte function within a stacked double sandwich culture plate cylindrical bioreactor for bioartificial liver system.

Bioartificial liver (BAL) system is promising as an alternative treatment for liver failure. We have developed a bioreactor with stacked sandwich culture plates for the application of BAL. This bioreactor design addresses some of the persistent problems in flat-bed bioreactors through increasing cell packing capacity, eliminating dead flow, regulating shear stress, and facilitating the scalability of the bioreactor unit. The bioreactor contained a stack of twelve double-sandwich-culture plates, allowing 100 million hepatocytes to be housed in a single cylindrical bioreactor unit (7 cm of height and 5.5 cm of inner diameter). The serial flow perfusion through the bioreactor increased cell-fluid contact area for effective mass exchange. With the optimal perfusion flow rate, shear stress was minimized to achieve high and uniform cell viabilities across different plates in the bioreactor. Our results demonstrated that hepatocytes cultured in the bioreactor could re-establish cell polarity and maintain liver-specific functions (e.g. albumin and urea synthesis, phase I&II metabolism functions) for seven days. The single bioreactor unit can be readily scaled up to house adequate number of functional hepatocytes for BAL development.

[Artificial and bioartificial liver support systems for acute and acute-on-chronic liver failure: a meta-analysis].

OBJECTIVE: To evaluate the effect of artificial and bioartificial liver support systems for management of acute and acute-on-chronic liver failure. METHODS: Articles documenting randomized clinical trials concerning any liver support systems vs standard conservative therapy, published between January, 1970 and June, 2008, were retrieved by database searching. Of the 1134 articles retrieved, 12 randomized trials involving 479 patients were included. The data were extracted and the trial quality was assessed by 2 independent reviewers. The primary outcome measure was all-cause mortality, and the results were combined on the risk ratio (RR) scale. RESULTS: Of the 12 trials included, 10 assessed artificial liver support systems for acute or acute-on-chronic liver failure, and 2 assessed bioartificial systems for acute liver failure. Overall, the liver support systems had moderate effect on mortality compared with standard conservative therapy (RR=0.80; 95% CI 0.664-0.969, P=0.022). Meta-regression indicated that the effect of the support systems depended on the type of liver failure (P=0.00). In stratified meta-analyses, the support systems appeared to reduce the mortality by 43% in acute-on-chronic liver failure (RR=0.57; 95% CI 0.39-0.84, P=0.004), but not in acute liver failure (RR=0.899; 95% CI 0.72-1.12, P=0.361). CONCLUSION: Artificial liver support systems reduce the mortality of acute-on-chronic liver failure as compared with standard conservative therapy, but have no significant effect on the mortality of acute liver failure. Bioartificial liver support systems lower the mortality rates in both acute and acute-on-chronic liver failure, and should be the future focus of development.

[Preparation of whole-kidney acellular matrix in rats by perfusion].

OBJECTIVE: To prepare rat whole-kidney acellular matrix (ACM) scaffolds using fluid perfusion method. METHODS: The kidneys with ureters and renal vessels were harvested from 12-week-old Wistar rats. Intravenous catheters were inserted through the renal arteries to establish channels for whole-kidney retrograde perfusion successively with heparinized PBS, 1% SDS, deionized water, 1% TritonX-100 and antibiotic-containing PBS under a pressure of 100 cmH2O. After decellularization, the scaffolds were observed under microscope with HE staining, scanning electron microscope, and fluorescence microscope with DAPI fluorescence staining. RESULTS: No cell residue was found in the scaffolds under microscope. Scanning electron microscope identified reticular structures consisting of basilar membrane and collagen without normal cellular structures in the scaffolds, and no strong fluorescence due to the binding of DAPI to the cell nuclei was observed under fluorescence microscope. CONCLUSION: Fluid perfusion is simple and reliable to prepare rat whole-kidney acellular matrix, which may serve as an ideal cell-free scaffold.