Perfusion Decellularization of Extrahepatic Bile Duct Allows Tissue-Engineered Scaffold Generation by Preserving Matrix Architecture and Cytocompatibility

Yolik Ramírez-Marín,Francisco Drusso Martínez-García,David M Giraldo-Gomez,Dulce Maria Puente-Guzmán,Jorge Luna Del Villar-Velasco,M Cristina Piña-Barba,Miguel Ángel Mercado,Norma S Pérez-Gallardo,Leonardo Alejandro Rodríguez-López,Gonzalo Torres-Villalobos,Martin Conrad Harmsen,David Eduardo Abad-Contreras,Lorena Villafuerte-García,Jesús Tapia-Jurado,Martha Ustarroz-Cano

doi:10.3390/ma14113099

Abstract

Reconstruction of bile ducts damaged remains a vexing medical problem. Surgeons have few options when it comes to a long segment reconstruction of the bile duct. Biological scaffolds of decellularized biliary origin may offer an approach to support the replace of bile ducts. Our objective was to obtain an extracellular matrix scaffold derived from porcine extrahepatic bile ducts (dECM-BD) and to analyze its biological and biochemical properties. The efficiency of the tailored perfusion decellularization process was assessed through histology stainings. Results from 4’-6-diamidino-2-phenylindole (DAPI), Hematoxylin and Eosin (H&E) stainings, and deoxyribonucleic acid (DNA) quantification showed proper extracellular matrix (ECM) decellularization with an effectiveness of 98%. Immunohistochemistry results indicate an effective decrease in immunogenic marker as human leukocyte antigens (HLA-A) and Cytokeratin 7 (CK7) proteins. The ECM of the bile duct was preserved according to Masson and Herovici stainings. Data derived from scanning electron microscopy (SEM) and thermogravimetric analysis (TGA) showed the preservation of the dECM-BD hierarchical structures. Cytotoxicity of dECM-BD was null, with cells able to infiltrate the scaffold. In this work, we standardized a decellularization method that allows one to obtain a natural bile duct scaffold with hierarchical ultrastructure preservation and adequate cytocompatibility.

Highlights

Cholecystectomy or gall bladder removal is an intraabdominal surgical procedure with 750,000 patients treated annually, solely in the United States
In this work, we describe a method for porcine bile duct decellularization, going from tissue harvesting to evaluation of the microarchitecture, and demonstrating the biological properties of decellularized extracellular matrix from pig bile ducts (dECM-BD) scaffolds aimed at tissue engineering
The selection of decellularization reagents was based on modifying preexistent protocols presented by Giraldo et al and Cheng et al [9,13], briefly described as follows: All solutions were perfused via the bile duct lumen connecting each bile duct to a caterer number 14 attached by a 2-0 silk suture

Summary

Introduction

Cholecystectomy or gall bladder removal is an intraabdominal surgical procedure with 750,000 patients treated annually, solely in the United States These patients generally develop life-threatening iatrogenic-induced bile duct injuries [1,2,3], which require surgical reconstruction procedures like Roux-en-Y hepaticojejunostomy [4,5]. This is a major surgical procedure that frequently causes complications such as stenosis, recurrent cholangitis, and secondary biliary cirrhosis [6]. Decellularization is a process that generates scaffolds of natural origin, with the aim to replace or repair diseased organs or tissues. Acellular scaffolds derived from healthy bile ducts of animal sources (e.g., porcine origin) may provide a low-cost and widely accessible off-the-shelf replacements [9]. Using porcine decellularized extrahepatic bile duct scaffolds may tackle the disadvantages before-mentioned, due to the anatomical similarities between species

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