An Experimental Study On Treatment Of Liver Fibrosis In Situ/Splenic Pedicle By Vascular Engineering Liver Tissue In Rats

OBJECTIVE: To examine the effects of injection of hepatic constructs based onneonatal rat on the treatment of hepatic fibrosis，and to explore its mechanisms inrat model. To explore a new method of forming large-scale implanted engineeringliver tissue through injecting the liver tissue-engineering gel within splenicpedicle vascular bundles.METHODS:1.Cutted it into pieces,1d age newborn rat liver hepatocytes withmodified trypsin digestion method. Cultured routinely inverted microscopedynamic cell morphology was observed, Trypan Blue evaluation of cellactivity.2.Neonatal rat liver cells were separated and digested by conventionalmethod. Cells mixing with fibrin glue、chitosan gel、rat tail collagen、 hyaluronicacid scaffolds were seeded in culture plates respectively. The function of newbornrat liver cells was determined in vitro through regular light microscopeobservation、MTT determination、the supernatant albumin content、urea assay.3.The rat model of carbon tetrachloride (CCl4)-induced hepatic fibrosis was used toassess the effect of hepatic constructs on the treatment fibrosis. Hepatic fibrosiswas evaluated by survival、ultrasound、HE staining. The expression of TGF-βinliver tissue was examined by immunohistochemistry.4.2×10~7newborn rat liver cells were mixed with1ml fibrin glue to build engineered liver tissue gel. Afterthe spleen of SD rat was removed，the distal ligated, the proximal lapped，splenicpedicle vascular bundle was formed. The engineering liver tissue was injected intothe gel through the distal，forming an of≥1cm3volume of the spherical tissue;Liver engineering tissue was implanted under the thigh subcutaneous tissue as thecontrol group. After2weeks，compatibility evaluation was observed, and tissuesections were stained with HE.RESULTS:1. Neonatal rat hepatocytes isolated to approximately2×10~7/ratliver,99%of activity. The collected cell number and cell viability rate hasimproved significantly2. Liver cells showed three-dimensional formation in thefour groups. Neonatal rat liver cells showed different growth states in differentgels. In the fibrin glue and chitosan gel groups cell activity is good, value-addedcurve showing upward trend at early time. In the hyaluronic acid and rat tailcollagen groups cells grow slowly、reduced and dead gradually. Albumin and ureacontent also showed similar performance.3.Compared to controls，Histologicaland survival、ultrasound revealed that hepatic constructs arrested progression ofhepatic fibrosis. Furthermore, Multi-site injection of hepatic constructs reducedprotein expression of TGF-β.4. In the control group, the implants showed smallerresidual tissues(1.63±0.55mm diameter)with little blood vessels. In theexperiment group, the implants formed a larger tissue (7.33±2.4mm diameter)with a large quantity of blood vessel.CONCLUSION:1.The neonatal rat liver cells can obtain by cold modified trypsin digestion method. Cell numbers and activities were significantlyimproved.2. Of the injectable scaffolds, chitosan, and fibrin glue showed betterbiocompatibility to neonatal rat liver cells, and rat tail collagen and hyaluronicacid are poor. The former two scaffolds are suitable for engineering liver tissuebuilding.3.These results suggest that multi-site injection of hepatic constructsbased on neonatal rat liver cells ameliorates carbon tetrachloride-induced hepaticfibrosis in rats via inhibition of TGF-βexpression.4. Mixing with fibrin glue andnewborn rat liver cells in the spleen pedicle vascular bundles can form a largerengineering liver tissue having blood vessels.