Heart Valve Decellularization Research Articles

A Systematic Review of Tissue Engineering of a Total Artificial Heart: Focus on Perfusion Decellularization

Introduction: A leading cause of death worldwide is heart disease, and in the United States alone, 6.2 million individuals live with heart failure. The recent ability to recreate the vascular network of cardiac Extracellular Matrix (ECM) suggests the feasibility of engineering whole heart constructs. Tissue engineering method such as perfusion decellularization allows for effective removal of nearly all cellular composition of a heart while maintaining the native mechanical integrity of the scaffold. This scaffold can be reseeded with stem cells in the hopes of generating a functional heart. The goal of this systematic review was to assess the various perfusion decellularization methods and its effects upon the biologic scaffold. Methodology: A comprehensive systematic literature review search was carried out through Pubmed, MEDLINE and Google Scholar using the search terms “whole heart decellularization”, “whole organ decellularization” and “perfusion decellularization”. The inclusion criteria for this research were as follows: scholarly or peer-reviewed studies, published within the last 20 years in the English language, and primary studies in which whole cardiac organ decellularization was performed. Chosen articles were those examining the various decellularization methods and its effects upon the biologic scaffold. Excluded were studies regarding heart valve decellularization, tissue decellularization, cardiac patch, tissue recellularization, articles in foreign languages, articles dated prior to 2001, literature reviews, systematic reviews, and duplicate articles. Results: Chemical agents such as acid and bases cause hydrolytic degradation of biomolecules which could reduce ECM strength and eliminate growth factors from the matrix. Compared to other detergents such as Triton X-100, sodium dodecyl sulfate yields a more complete removal of nuclear material. A combination of these various approaches has shown an increased efficacy of the decellularization process. The decellularization of a large solid organ such as the heart requires several sophisticated steps. Perfusion decellularization is achieved via anterograde or retrograde perfusion of the intrinsic vascular network of the heart by utilizing decellularizing agents (i.e., chemicals and/or enzymes). Hodgson et al., 2017 demonstrates a combined strategy in which the decellularization of porcine hearts is accomplished in 24 hours and results in 98% DNA removal with only 6 hours of detergent exposure. Conclusion: There have been significant advances to address the heart disease epidemic. A promising approach is perfusion decellularization which allows for complete DNA content removal while maintaining the ECM scaffold integrity. This scaffold is then re-cellularized with stem cells in the hopes of creating an artificial heart. There are several challenges that need to be surpassed before bio-artificial hearts can be used to replace in vivo function. Future research is directed towards optimizing types of cells and cell sources used to repopulate decellularized hearts, seeding strategies and bioreactor systems to provide in vitro conditions required for organ maturation.

Can Heart Valve Decellularization Be Standardized? A Review of the Parameters Used for the Quality Control of Decellularization Processes.

When a tissue or an organ is considered, the attention inevitably falls on the complex and delicate mechanisms regulating the correct interaction of billions of cells that populate it. However, the most critical component for the functionality of specific tissue or organ is not the cell, but the cell-secreted three-dimensional structure known as the extracellular matrix (ECM). Without the presence of an adequate ECM, there would be no optimal support and stimuli for the cellular component to replicate, communicate and interact properly, thus compromising cell dynamics and behaviour and contributing to the loss of tissue-specific cellular phenotype and functions. The limitations of the current bioprosthetic implantable medical devices have led researchers to explore tissue engineering constructs, predominantly using animal tissues as a potentially unlimited source of materials. The high homology of the protein sequences that compose the mammalian ECM, can be exploited to convert a soft animal tissue into a human autologous functional and long-lasting prosthesis ensuring the viability of the cells and maintaining the proper biomechanical function. Decellularization has been shown to be a highly promising technique to generate tissue-specific ECM-derived products for multiple applications, although it might comprise very complex processes that involve the simultaneous use of chemical, biochemical, physical and enzymatic protocols. Several different approaches have been reported in the literature for the treatment of bone, cartilage, adipose, dermal, neural and cardiovascular tissues, as well as skeletal muscle, tendons and gastrointestinal tract matrices. However, most of these reports refer to experimental data. This paper reviews the most common and latest decellularization approaches that have been adopted in cardiovascular tissue engineering. The efficacy of cells removal was specifically reviewed and discussed, together with the parameters that could be used as quality control markers for the evaluation of the effectiveness of decellularization and tissue biocompatibility. The purpose was to provide a panel of parameters that can be shared and taken into consideration by the scientific community to achieve more efficient, comparable, and reliable experimental research results and a faster technology transfer to the market.

Decellularized pig pulmonary heart valves-Depletion of nucleic acids measured by proviral PERV pol.

Decellularized human pulmonary heart valve (dhHV) scaffolds have been shown to be the gold standard especially for younger, adolescent patients. However, human heart valves are limited in availability. Xenogeneic decellularized pig heart valves (dpHV) may serve as alternative. The efficacy of DNA reduction processes upon decellularization of heart valves from German Landrace pigs was analyzed by measurements of remaining nucleic acids including proviral porcine endogenous retrovirus (PERV) sequences. Porcine pulmonary heart valves (pPHV) were decellularized by three different protocols and further treated with DNaseI or Benzonase, at varying incubation times. DNA isolated from valve associated muscle (m), valve cusp (c), and pulmonary artery (pa) was monitored by PCR and qRT-PCR using GAPDH and the PERV polymerase (pol) for read-out. Decellularization of pPHV led to a significant reduction of DNA (>99%) which could be further significantly increased for (m) and (pa) by nuclease treatment, reducing proviral PERV pol from approximately 5×107 to 5×103 copies/mg in nuclease treated tissues. Both nucleases demonstrated comparable activities. But DNaseI revealed to be less consistent for PERV, especially at muscular tissue. Noteworthy, remaining proviral sequences are still detectable by PCR; however, due to the absence of the cellular replication machinery the production of infectious particles is not expected. Decellularization and nuclease treatment of pPHV is an efficient procedure to reduce the DNA content including PERV, thus represents a valuable option to increase virus safety independently from the source animal background.

Heart valve tissue engineering: an overview of heart valve decellularization processes.

Despite recent advances in medicine and surgery, many people still suffer from cardiovascular diseases, which affect their life span and morbidity. Regenerative medicine and tissue engineering are novel approaches based on restoring or replacing injured tissues and organs with scaffolds, cells and growth factors. Scaffolds are acquired from two major sources, synthetic materials and naturally derived scaffolds. Biological scaffolds derived from native tissues and cell-derived matrix offer many advantages. They are more biocompatible with a higher affinity to cells, which facilitate tissue reconstruction. Interestingly, xenogeneic recipients generally tolerate their components. Therefore, heart valve tissue engineering is increasingly benefiting from naturally derived scaffolds. In this review, we investigated the different protocols and methods that have been used for heart valve decellularization.

Different approaches to heart valve decellularization: A comprehensive overview of the past 30years.

Xenogeneic decellularized heart valve scaffolds have the potential to overcome the limitations of existing bioprosthetic heart valves that have limited duration due to calcification and tissue degeneration phenomena. This article presents a review of 30years of decellularization approaches adopted in cardiovascular tissue engineering, with a focus on the use, either individually or in combination, of different detergents. The safety and efficacy of cell-removal procedures are specifically reported and discussed, as well as the structure and biomechanics of the treated extracellular matrix (ECM). Detergent residues within the ECM, production of hyaluronan fragments, safe removal of cellular debris, and the persistence of the alpha-Gal epitope after the decellularization treatments are of particular interest as parameters for the identification of the best tissue for the manufacture of bioprostheses. Special attention has also been given to key factors that should be considered in the manufacture of the next generation of xenogeneic bioprostheses, where tissues must retain the ability to be remodeled and to grow in weight along with body reshaping.

Detergent Decellularization of Heart Valves for Tissue Engineering: Toxicological Effects of Residual Detergents on Human Endothelial Cells

Detergents are powerful agents for tissue decellularization. Despite this, the high toxicity of detergent residua can be a major limitation. This study evaluated the efficacy of detergent removal from decellularized pulmonary valves (PVs) and the consequences of repopulation with human endothelial cells (HECs). Porcine PVs were treated with 1% sodium deoxycholate (SDC), group A; 1% sodium dodecyl sulfate (SDS), group B; and a mixture of 0.5% SDC/0.5% SDS, group C (n = 5 each). After each of 10 succeeding wash cycles (WCs), samples of the washing solution (WS) were analyzed by solid phase extraction and high performance liquid chromatography for the presence of detergents. Metabolic activity of HEC was also assessed in the WS samples (cytotoxicity and MTS assays). Decellularized and washed PVs were reseeded with HEC. Histological analysis demonstrated efficient tissue decellularization in all groups. Detergents' concentration in all WSs decreased exponentially and was below 50 mg/L after 6, 8, and 4 WCs in groups A, B, and C, respectively. This concentration resulted in no significant toxic influence on cell cultures, and scaffolds could be efficiently reseeded with HEC. In conclusion, intensive washing of detergent decellularized valvular scaffolds lowers the residual contamination below a hazardous threshold and allows their successful repopulation with HEC for tissue engineering purposes.

Impact of heart valve decellularization on 3-D ultrastructure, immunogenicity and thrombogenicity

Decellularized xenogeneic tissue represents an interesting material for heart valve tissue engineering. The prospect objective is removal of all viable cells while preserving extracellular matrix (ECM) integrity. The major concerns of all decellularization protocols remain ECM disruption, immunogenicity and thrombogenicity. Accordingly the aim of this study was visualization of ultrastructural ECM disruption and human immune response and thrombogenicity using different decellularization protocols of porcine heart valves. Porcine pulmonary leaflets were decellularized with four different protocols: sodium deoxycholate, sodium dedecylsulfate, trypsin/EDTA, and trypsin–detergent–nuclease. Then the tissues were processed for histology and two-photon laser scanning microscopy (LSM). For thrombogenicity and immunogenicity testing tissues were incubated with human blood. The histological examination revealed no remaining cells and no significant differences in the ECM histoarchitecture in any group. LSM detected significant ECM alterations in all groups except sodium deoxycholate group with an almost completely preserved ECM. There was no increased immunogenicity between fresh and decellularized tissue. Compared to GA-fixed tissue however significantly increased immune responses and thrombogenicity was observed in all protocols. From our experiment, sodium deoxycholate enables cell removal with almost complete preservation of ECM structures. And all of these four decellularization protocols affected human immunological response and increased thrombogenicity.

Mid-Term Clinical Results Using a Tissue-Engineered Pulmonary Valve to Reconstruct the Right Ventricular Outflow Tract During the Ross Procedure

The Ross procedure is mainly limited by the durability of the valve prostheses used to reconstruct the right ventricular outflow tract. This study was performed to collect prospective safety and effectiveness data of the Ross procedure using a tissue-engineered heart valve to reconstruct the right ventricular outflow tract. Between May 2000 and February 2003, 23 patients received tissue-engineered heart valves. Two to four weeks before the Ross operation, a piece of forearm or saphenous vein was harvested to isolate, characterize, and expand endothelial cells. A pulmonary allograft (n = 11) or xenograft (n = 12) was decellularized, coated with fibronectin, and seeded with autologous vascular endothelial cells, using a specially developed bioreactor. Follow-up was performed by clinical evaluation, transthoracic echocardiography, magnetic resonance imaging, and multislice computed tomography. The patient mean age was 44.0 +/- 13.7 years. Cell seeding density was 1.1 x 10(5) +/- 0.5 x 10(5) cells/cm2, with a viability of 90.2% +/- 8.9%. All patients survived the operation. One patient died during follow-up, and 1 patient required reoperation. All surviving patients are currently in New York Heart Association functional class I. Transthoracic echocardiographic evaluation of the tissue-engineered heart valve showed a mean flow velocity of 0.9 +/- 0.4 m/s at 5 years. Multislice computed tomography showed no calcification up to 5 years postoperatively. Tissue-engineered heart valves showed excellent hemodynamic performance during mid-term follow-up. Decellularization of heart valves and seeding with autologous vascular endothelial cells may prevent tissue degeneration and improve valve durability.

Detergent decellularization of heart valves for tissue engineering: Toxic effects of residual detergents on human endothelial cells

Introduction: In tissue engineering detergents were used for cardiac valve decellularization. Detergent remnants, however, are toxic and inhibit a successful cell reseeding. Here we evaluated the efficacy of detergent elimination and susceptibility of treated matrices to recellularization with human endothelial cells (HEC).