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Acellular scaffold production for liver transplant
Liver transplantation is the only potentially curative treatment for patients facing acute or end-stage hepatic disease. The surgical procedures include (i) orthotopic liver transplantation (OLT), in which the native liver is removed and replaced by the donor organ in the same anatomic position as the original liver, (ii) heterotopic auxiliary liver transplantation (HALT) which involves implanting the new liver graft in a non-anatomical location and (iii) auxiliary partial orthotopic liver transplantation (APOLT) where a partial liver graft is implanted in an orthotopic position after leaving behind a part of the native liver. However, transplantation is mainly limited by the supply of transplantable donor organs, which is far exceeded by the demand resulting in increasing transplantation waiting lists. In this context, tissue engineering offers hope to overcome this shortage. Producing an acellular hepatic extracellular matrix (ECM) that may act as an inductive template for recellularization is a first step in bioengineering a liver.
Objetivos - Metodologia - Resultados - Discussão dos Resultados/Objectives - Methodology - Results - Discussion of Results/Objetivos - Metodología - Resultados - Discusión de los resultados
Objective: The aim of this work was to optimize a surgical technique to remove livers from rat donors isolating the main vessels and to improve matrix stiffness to produce transplantable acellular liver scaffolds.
Methods: Donor animals received 100 µl of heparin (50 IU/ml) 15 min before surgery. Then under anesthesia (xylazine– 8mg/kg and ketamine-40 mg/kg) the animals were submitted to transverse abdominal incision and the portal vein (PV) was separated and cannulated. The infrahepatic vena cava (IVC) was dissected and a cuff Teflon tube was attached to the IVC and fixed with 6-0 silk ligation. The superior vena cava (SVC) was clamped. After removal, the donor liver was perfused via PV with 5ml of custodiol solution (organ storage solution) supplemented with 10 µl of heparin (50 IU/ml) and placed in a cold saline bath for 5 minutes. Then, the livers were transferred to be perfused through portal vein using an infusion pump at 3 ml/min with water for 1 hour followed by 1% Triton X-100 for 3 hours and SDS 1% for 24h. After total decellularization, livers were washed with distilled H2O for 7 days to remove residual SDS and then were preserved at 4 ºC for up to 1 day. To analyze the ECM integrity post decellularization protocol, DAPI, H&E, Sirius red, DNA quantification, electronic scanning microscopy and immunohistochemistry assays against collagen type I, III, IV, laminin and fibronectin, were performed. Toluidine blue was used to examine the vasculature integrity. To improve the decellularized ECM shape the matrix was perfused with 10 ml of rat blood diluted in 40 ml of custodiol solution for 1 hour and washed with 3 ml of phosphate saline solution (PBS) to remove excess of blood. Then, the matrix was analyzed by H&E staining.
Results: Toluidine blue showed that the vascular system was totally preserved. Macroscopy, microscopy and histological staining showed that the decellularization process preserves the structure and components of the ECM. After blood perfusion, the decellularized ECM shape resembled a native liver and H&E staining showed some cell retention on vessels of decellularized ECM.
Considerações Finais/Final considerations/Consideraciones finales
Conclusion: In the present study, we optimized a surgical technique isolating PV, IVC and SVC. Also, after blood perfusion, decellularized ECM shape resembled a native liver which may allow acellular rat liver scaffolds transplantable.
Sources of research support: CNPq, Capes, FAPERJ, Ministério da Saúde
Palavras-chave/Key words/Palabras clave
Key words: Bio-artificial liver, extracellular matrix, decellularization
MARLON LEMOS DIAS, Alexandre Cerqueira da Silva, Glaucia Julião, Cíntia Batista, Victoria Monteiro, Victor Secomandi, Lanuza Faccioli, Isalira Ramos, Michelle Christie, Regina Goldenberg