Pressurized Perfusion System for Obtaining Completely Acellular Pulmonary Valve Scaffolds for Tissue Engineering

Abstract

Introduction. Xenogeneic tissues decellularization represents the obtaining process of extracellular matrix derived scaffolds. Most antigens being cell based, non-immunogenicity is obtained by cells removal. Scaffolds are temporary structures with biologic and mechanical role. Scaffolds, stem cells and bioreactors represent premise of regenerative medicine, aiming towards the ideal valvular substitute. In previous studies, we decellularized pulmonary valves root by immersion histology revealing cellular residue, requiring a more efficient approach. We hypothesized that immersion is insufficient and thus a pressure gradient was added. Material and Method. This is part of a grant approved by the UMFTS. Eleven porcine pulmonary valves were included in the study: n=6 underwent immersion decellularization and n=5 were cyclically perfused with a 20-25mmHg pressure gradient during a 10-day protocol. The acellular valves obtained underwent a quality control using DAPI (4′,6-diamidino-2-phenylindol) nuclear staining, histological Haematoxylin-Eosin, DNA extraction and quantification, harvested from different structural levels: arterial wall, sinus, cusp. Results. Histological assessments highlighted integrity of extracellular matrix in both groups and overall cells absence at the different levels of valvular structures analyzed. Immersion decellularized valves exhibited DAPI positive structures identified as potential residual nucleic material. Comparatively, the perfusion decellularized valves, lacked in those structures, result confirmed by DNA extraction and quantitation procedure. Conclusions. Perfusion decellularization represents a feasible approach to obtain acellular cardiac valvular scaffolds derived from the extracellular matrix, being superior to immersion decellularization method. Their nonimmunogenic potential is underlined by total absence of nuclei. The process is fast, allowing production of an abundant number of valvular biomaterials in a short time.

Preclinical Testing of Living Tissue-Engineered Heart Valves for Pediatric Patients, Challenges and Opportunities

“…In this project, we proposed a translational scenario (Figure 1) based on our previous experience with acellular valve scaffolds, which performed very well hemodynamically in the right ventricular outflow tract (RVOT) in sheep (22,23). The decellularization protocol proved to be effective in removing all nucleic materials while preserving the integrity and architecture of the ECM, as reported earlier in pilot studies (15). Acellular valve scaffolds have several advantages: they preserve the natural 3D architecture, have excellent hemodynamics and biomechanics, are highly biocompatible, are not antigenic, FIGURE 3 | Differentiation of adipose-derived stem cells.…”

“…The decellularization system was perfused by a peristaltic pump with a 30-s off/3-min on cycle over 12 days under a pressure gradient of 4 mmHg (between the intraluminal and the external environment), the last step ending in sterile conditions. Studies performed previously pointed to improved cell removal from the valve roots when using perfusion methods in comparison to only immersion of the heart valves (14,15). The decellularization procedure started with 24 h of 0.02% NaN 3 in H 2 O (Sigma-Aldrich Chemistry, St. Louis, MO, USA) followed by 1 h perfusion of 0.05M NaOH (Lach-Ner, Neratovice, Czech Republic) and rising with H 2 O.…”

“…Three batches of freshly prepared decellularization solution containing 0.05% sodium dodecyl sulfate (Sigma-Aldrich Chemistry, St. Louis, MO, USA), 0.5% TRITON X-100 (PanReac AppliChem, Castellar del Vallès, Spain), 0.5% sodium deoxycholate (Sigma-Aldrich Chemistry, St. Louis, MO, USA), 0.2% ethylenediaminetetraacetic acid (PanReac AppliChem, Spain) in 10 mM hydroxymethyl aminomethane TRIS (Sigma-Aldrich Chemistry, St. Louis, MO, USA) were circulated for 48 h each, followed by enzymatic treatments with 720 mU/ml deoxyribonuclease (PanReac AppliChem, Spain) and 720 mU/ml ribonuclease (PanReac AppliChem, Spain) for 48 h, at 37 • C, applied twice. In the last day of the protocol, valves were treated with 70% ethanol and then 2 h with 0.2% peracetic acid for sterilization (Merck KgaA, Darmstadt, Germany) and storage in sterile phosphate-buffered saline (PBS) at 4 • C. Further details can be found in our previous publication (14,15).…”

“…Histology with 4 ′ ,6-diamidino-2fenilindol (DAPI) nuclear staining on cryosections (Thermo Fisher, Waltham, MA, USA) and hematoxylin/eosin assessed the extracellular matrix architecture and integrity and the absence of cells nuclei. DNA was also extracted from n = 6 tissue samples from each decellularization batch, quantified by NanoDrop spectrophotometry and analyzed by EthBr agarose gel electrophoresis, as described before (15). All valves underwent sterility testing as follows: decellularized valve fragments were collected in aseptic conditions, immersed in approximately 3 ml sterile saline, in sterile 15-ml tubes, and transported to the Microbiology Department of UMFST for further processing.…”

Challenges in Perioperative Animal Care for Orthotopic Implantation of Tissue-Engineered Pulmonary Valves in the Ovine Model

“…The valves were subjected to a perfusion decellularization protocol described in our previous work on porcine pulmonary valves. Randomly chosen valves were used for a microbiological sterility assay and the efficiency of the decellularization process was assessed by DAPI staining and DNA extraction [6].…”