A New Decellularization Protocol of Porcine Aortic Valves Using Tergitol to Characterize the Scaffold with the Biocompatibility Profile Using Human Bone Marrow Mesenchymal Stem Cells

Abstract

The most common aortic valve diseases in adults are stenosis due to calcification and regurgitation. In pediatric patients, aortic pathologies are less common. When a native valve is surgically replaced by a prosthetic one, it is necessary to consider that the latter has a limited durability. In particular, current bioprosthetic valves have to be replaced after approximately 10 years; mechanical prostheses are more durable but require the administration of permanent anticoagulant therapy. With regard to pediatric patients, both mechanical and biological prosthetic valves have to be replaced due to their inability to follow patients’ growth. An alternative surgical substitute can be represented by the acellular porcine aortic valve that exhibits less immunogenic risk and a longer lifespan. In the present study, an efficient protocol for the removal of cells by using detergents, enzyme inhibitors, and hyper- and hypotonic shocks is reported. A new detergent (Tergitol) was applied to replace TX-100 with the aim to reduce toxicity and maximize ECM preservation. The structural integrity and efficient removal of cells and nuclear components were assessed by means of histology, immunofluorescence, and protein quantification; biomechanical properties were also checked by tensile tests. After decellularization, the acellular scaffold was sterilized with a standard protocol and repopulated with bone marrow mesenchymal stem cells to analyze its biocompatibility profile.

Tergitol Based Decellularization Protocol Improves the Prerequisites for Pulmonary Xenografts: Characterization and Biocompatibility Assessment

“…Primarily, the thickness of each tissue sample was measured using Mitutoyo digital Caliber model ID-C112XB (Aurora, IL, USA), and later, the test for the uniaxial tensile property was performed with a customized apparatus (IRS, Padova, Italy) aided by a LabVIEW software (National Instruments, Austin, TX, USA). Tests were carried out as previously described [ 26 , 27 ]. Briefly, the samples were preloaded to 0.1 N at room temperature, then stretched until ruptured at the rate of 0.2 mm/s.…”

“…In order to better characterize the non-linear stress-strain curves obtained from each sample, two tensile moduli, E1 and E2, were calculated as the slope of the linear portion of the curve between 0–10% (elastin region) and 60–100% deformation (collagen region), respectively [ 26 , 28 ]. A t -test (GraphPad Prism Software, San Diego, CA, USA) was applied to calculate the statistical analysis.…”

“…The specific aim of the present study is to assess the preservation of the ECM scaffold of the decellularized porcine pulmonary valve by using a new eco-friendly detergent, i.e., Tergitol. Our group has reported Tergitol application for the decellularization of the aortic valve [ 26 ].…”

A New Detergent for the Effective Decellularization of Bovine and Porcine Pericardia

“…Each pericardium was isolated as described by Aguiari et al [ 37 , 38 ] and then decellularized following the TRICOL method published by Spina et al [ 39 ], but using Tergitol instead of Triton X-100 with the same concentration (1–0.1% v / v ) [ 40 , 41 ]. Tergitol was proposed by the supplier (Sigma Aldrich, Saint Louis, MO, USA) as an alternative detergent: its effectiveness was assessed by treating some biological samples (e.g., cardiovascular tissues).…”

Small intestinal submucosa-derived extracellular matrix as a heterotopic scaffold for cardiovascular applications

“…Triton-X 100 has been removed from the market since January 2021 for its endocrine-disrupting properties (ECHA, REACH), and a new protocol has been developed by our group, which is based on a new detergent called Tergitol 15-S 9. This is a non-ionic, linear-chain surfactant belonging to the series of ethoxylated secondary alcohols, which has received promising preliminary results in the decellularization process ( Faggioli et al, 2022 ; Todesco et al, 2022 ; Casarin et al, 2022 ). Unlike Triton X-100, Tergitol 15-S 9 is easily biodegradable, does not induce cytotoxicity, and, due to its chemical structure, has a much faster micelle formation mechanism, resulting in better wettability.…”