The sequential effects of a multifactorial detergent based decellularization process on bovine pericardium

Abstract

Decellularization is a promising method for obtaining extracellular matrix scaffolds (ECM) to be used as replacement material in reconstructive procedures. The effectiveness of decellularization and the alterations to the ECM vary, depending on several factors, including the tissue source, composition and density. With an optimized decellularization process, decellularized scaffolds can preserve the spatial and temporal ECM microenvironment, which play an integral role in modulating cell migration, proliferation and differentiation. The exploration of a variety of decellularization protocols has led to mixed outcomes and comparisons between decellularization protocols could not attribute these differences to any single step in a multiple-step process. This study aimed to characterize the effects of each step of a multifactorial decellularization method on the scaffold structure and mechanical integrity of bovine pericardium. Each step of the decellularization process and the effect on the tissue was assessed using hematoxylin and eosin staining, electron microscopy, total protein, ECM protein and triglyceride quantification. The biomechanical properties were assessed using uniaxial tensile strength testing. Cell lysis occurred mainly during the detergent and alcohol steps. Collagen structural damage occurred during the detergent and alcohol steps, with no significant decreased in collagen concentration. No significant damage to elastin could be shown throughout the process, however glycosaminoglycans were significantly removed by detergent treatment. Triglycerides were removed mostly by the alcohol treatment. The strength of the pericardium decreased somewhat after each step of the protocol. It is important to characterize each decellularization protocol with regards to the decellularization efficiency and the effect on the ECM proteins structure and function to accurately evaluate in vivo outcomes.

Monomeric glutaraldehyde fixation and amino acid detoxification of decellularized bovine pericardium for production of biocompatible tissue with tissue-guided regenerative potential

“…ECM proteins are conserved among different species and can serve as a scaffold promoting host tissue regeneration through cell attachment, migration, and proliferation [ 43 ]. In a subsequent study, the decellularization technique used in this study was also shown to remove lipids [ 30 ], which can further contribute to decreasing the calcification potential of the tissue [ 44 ]. In another study by our group, the decellularized bovine pericardium, processed using this proprietary method, was implanted into the main pulmonary artery and the descending aorta of juvenile sheep.…”

“…The SDS and Triton-X100 group and SDS, TritonX-100 and ammonium group had better cytobiocompatibility, with the porosity structure maintained in all groups [ 47 ]. The addition of ammonium might have the added benefit of retaining the glycosaminoglycan content (GAGs) [ 48 ], which was found to significantly decreased in bovine pericardium processed using our proprietarary multi-detergent based decellularization process [ 44 ].…”

Development and testing of a transcatheter heart valve with reduced calcification potential

“…Decellularization can be obtained through various methods (physical, enzymatic, or chemical) but needs to be tailored for each tissue type depending on cellularity, density, lipid content, and thickness ( 75 ). Each step of the process has different effects on the outcome ( 36 ), and related techniques may yield different results depending on small variations in the technique. Our decellularization methodology has been developed over various iterations, and the results reported here are from tissues with encouraging performance in the subcutaneous rat model and aortic and pulmonary artery ovine implants ( 38 ).…”

“…These processes can be broadly divided into decellularization, fixation, and detoxification. After evaluating the contributions of various decellularization steps and combinations, we have developed a unique method to decellularize bovine pericardium (BP) without significantly altering its strength and collagen structure ( 36 , 37 ). This decellularized (DE) tissue performed well in 6-month implants in ovine aortas and pulmonary arteries, with less pannus formation, limited calcification, and adequate strength ( 38 ).…”

Impact of Three Different Processing Techniques on the Strength and Structure of Juvenile Ovine Pulmonary Homografts

“…However, the decellularization protocol they used reduced the GAG content, with resultant increased extensibility and changes in the relaxation behavior of the pulmonary valve leaflets [ 44 ]. The method currently used led to a reduction of the GAG content when used for the decellularization of bovine pericardium [ 48 ]. The GAG content was not evaluated in the current study, and the effect of this decellularization method on the GAG content of the pulmonary homografts should be investigated.…”