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Decellularized Cryopreserved Allografts as Off-the-Shelf Allogeneic Alternative for Heart Valve Replacement: In Vitro Assessment Before Clinical Translation

April 2017
Journal of Cardiovascular Translational Research 10(4)

April 2017
10(4)

DOI:10.1007/s12265-017-9738-0

Authors:

Laura Iop

University of Padua

Adolfo Paolin

Paola Aguiari

University of California, Los Angeles

Show all 6 authorsHide

Cryopreserved allogeneic conduits are the elective biocompatible choice among currently available substitutes for surgical replacement in end-stage valvulopathy. However, degeneration occurs in 15 years in adults or faster in children, due to recipient’s immunological reactions to donor’s antigens. Here, human aortic valves were decellularized by TRICOL, based on Triton X-100 and sodium cholate, and submitted to standard cryopreservation (TRICOL-human aortic valves (hAVs)). Tissue samples were analyzed to study the effects of the combined procedure on original valve architecture and donor’s cell removal. Residual amounts of nucleic acids, pathological microorganisms, and detergents were also investigated. TRICOL-hAVs proved to be efficaciously decellularized with removal of donor’s cell components and preservation of valve scaffolding. Trivial traces of detergents, no cytotoxicity, and abrogated bioburden were documented. TRICOL-hAVs may represent off-the-shelf alternatives for both aortic and pulmonary valve replacements in pediatric and grown-up with congenital heart disease patients.

Histological examination of native (a–d) and decellularized/cryopreserved (e–h) aortic wall after staining with H&E (a, b, e, f) and Masson trichrome (c, d, g, h). Note the maintenance of the trilaminated architecture, the typical distribution of ECM fibers, and the loss of nuclei after TRICOL decellularization/cryopreservation. Iintima, Mmedia, Aadventitia. Magnification bars: 200 μm (a, c, e, g) and 100 μm (b, d, f, h)

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Histological examination of native (a–d) and decellularized/cryopreserved (e–h) aortic valve cusp after staining with H&E (a, b, e, f) and Masson trichrome (c, d, g, h). Appropriate trilaminated architecture, typical distribution of ECM fibers, and loss of nuclei are appreciable after decellularization/cryopreservation. Vventricularis, Sspongiosa, Ffibrosa. Magnification bars: 200 μm (a, c, e, g) and 100 μm (b, d, f, h)

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Immunofluorescence on aortic wall pre-TRICOL/cryopreservation treatment. In wall section, nuclei were clearly visible with DAPI (blue; f). Collagen I (green; a) and elastin (green; c) fibers were well distributed with classic fibrous structure. Collagen IV (green; b) positivity showed the intact basal lamina. Leukocyte antigens (HLA class I and II) could be well identified respectively in d and e, expressed by endothelial and stromal cells. Iintima, Mmedia. Magnification bar: 250 μm

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Immunofluorescence on aortic valve cusp pre-TRICOL/cryopreservation treatment. Native cusps exhibited a dense nuclei distribution after DAPI staining (blue; f). Collagen I (green; a) and elastin (green; c) fibers were mainly distributed respectively in the fibrosa and the ventricularis. Collagen IV expression and distribution (green; b) revealed an intact basal lamina. Leukocyte antigens (HLA class I and II) could be well identified respectively in dand e, expressed by endothelial and stromal cells. Vventricularis, Sspongiosa, F fibrosa. Magnification bar: 250 μm

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Immunofluorescence on aortic wall post-TRICOL/cryopreservation treatment. General histoarchitecture of aortic wall was preserved after TRICOL decellularization and cryopreservation (a, c). Notably, the basal lamina elements were not removed from TRICOL treatment (b). Allogeneic cell components, as HLA I and II and nuclei, resulted to be absent from TRICOL-treated wall (d–f). Iintima, Mmedia. Magnification bar: 250 μm

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ORIGINAL ARTICLE

Decellularized Cryopreserved Allografts as Off-the-Shelf

Allogeneic Alternative for Heart Valve Replacement: In Vitro

Assessment Before Clinical Translation

Laura Iop

1,2

&Adolfo Paolin

&Paola Aguiari

1,2

&Diletta Trojan

&Elisa Cogliati

Gino Gerosa

1,2

Received: 22 September 2016 /Accepted: 2 February 2017 /Published online: 9 March 2017

#Springer Science+Business Media New York 2017

Abstract Cryopreserved allogeneic conduits are the elective bio-

compatible choice among currently available substitutes for surgi-

cal replacement in end-stage valvulopathy. However, degeneration

occurs in 15 years in adults or faster in children, due to recipient’s

immunological reactions to donor’s antigens. Here, human aortic

valves were decellularized by TRICOL, based on Triton X-100

and sodium cholate, and submitted to standard cryopreservation

(TRICOL-human aortic valves (hAVs)). Tissue samples were an-

alyzed to study the effects of the combined procedure on original

valve architecture and donor’s cell removal. Residual amounts of

nucleic acids, pathological microorganisms, and detergents were

also investigated. TRICOL-hAVs proved to be efficaciously

decellularized with removal of donor’s cell components and pres-

ervation of valve scaffolding. Trivial traces of detergents, no cyto-

toxicity, and abrogated bioburden were documented.

TRICOL-hAVs may represent off-the-shelf alternatives for both

aortic and pulmonary valve replacements in pediatric and

grown-up with congenital heart disease patients.

Keywords Heart valves .Decellularization .

Cryopreservation .Biocompatibility .Valv e r ep l ace men t .

Off-the-shelf solutions

Abbreviations

TRICOL Decellularization methodology (osmotic shock,

detergents (Triton X-100 and sodium cholate),

and aspecific nucleases)

hAV Human aortic valve

HLA Human leukocyte antigen

GUCH Grown-up with congenital heart disease

CVA Human cryopreserved valve allograft

SDS Sodium dodecyl sulfate

ECM Extracellular matrix

NHB Non-heart-beating

HPLC High-performance liquid chromatography

MS Mass spectrometry

Introduction

Congenital and acquired valvular diseases might require heart

valve replacement. The era of artificial heart valve substitution

started in 1952, when Dr. Charles Hufnagel implanted a caged

ball valve in the descending thoracic aorta of a patient with

aortic valve disease [1]. Despite remarkable progress has been

made over the last 60 years in the development of mechanical

valves, life-long anticoagulation is still required with statisti-

cally higher mortality and morbidity for treated patients [2].

Biological valve prostheses, from either human or animal tis-

sues, demonstrated more satisfactory hemocompatibility and

similar physiological performances when compared to native

valves. Xenogeneic (glutaraldehyde-treated) and allogeneic

valves are usually preferred for right ventricular outflow tract

reconstruction in children and adults, even if implantation in

pediatric population often results in faster graft degeneration

Laura Iop and Adolfo Paolin equally contributed to the study.

Associate Editor Adrian Chester oversaw the review of this article

Electronic supplementary material The online version of this article

(doi:10.1007/s12265-017-9738-0) contains supplementary material,

which is available to authorized users.

*Laura Iop

laura.iop@unipd.it

*Adolfo Paolin

apaolin@fbtv-treviso.org

Department of Cardiac, Thoracic and Vascular Sciences, University

of Padua, Via Giustiniani 2, 35128 Padua, Italy

Cardiovascular Regenerative Medicine Group, Venetian Institute of

Molecular Medicine, Via G. Orus 2, Padua 35129, Italy

Treviso Tissue Bank Foundation, Ca’Foncello Hospital, Piazzale

Ospedale, 31100 Treviso, Italy

J. of Cardiovasc. Trans. Res. (2017) 10:93–103

DOI 10.1007/s12265-017-9738-0

Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Development and In Vitro/In Vivo Comparative Characterization of Cryopreserved and Decellularized Tracheal Grafts

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Perspective Chapter: Role of Frozen Allografts in Aortic Valve Surgery

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Although, the mechanical and bioprosthetic valves, of good parameters, availability and easy of implantation, are universally applied as substitutes for failed aortic valve, the usefulness of aortic valve allografts (AVA); natural, viable, unstented human valves, is still considered. The essential technology for their preparation is cryopreservation, which allows for long-term storage. Hemodynamic functions of AVA are like of native valve, they do not produce hemolysis nor thromboembolism. Being markedly resistant for infection, AVA are recommended as the optimal grafts for severe endocarditis. Indeed, there exist some disadvantages, such as low availability, need for a specialized laboratories; implantation may be a challenge. Therefore, AVA are not recommended for routine use. Their important limitation is durability, affected with degenerative processes, characteristic of biological implants. Nevertheless, AVA presented satisfactory clinical results after 10, 20, and more years. This chapter have been discussed in detail the principal issues, connected with AVA, including preparation technologies, indications for use, surgical techniques, and first of all, clinical results.

Preliminary In Vitro Assessment of Decellularized Porcine Descending Aorta for Clinical Purposes

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Conduit substitutes are increasingly in demand for cardiovascular and urological applications. In cases of bladder cancer, radical cystectomy is the preferred technique: after removing the bladder, a urinary diversion has to be created using autologous bowel, but several complications are associated with intestinal resection. Thus, alternative urinary substitutes are required to avoid autologous intestinal use, preventing complications and facilitating surgical procedures. In the present paper, we are proposing the exploitation of the decellularized porcine descending aorta as a novel and original conduit substitute. After being decellularized with the use of two alternative detergents (Tergitol and Ecosurf) and sterilized, the porcine descending aorta has been investigated to assess its permeability to detergents through methylene blue dye penetration analysis and to study its composition and structure by means of histomorphometric analyses, including DNA quantification, histology, two-photon microscopy, and hydroxyproline quantification. Biomechanical tests and cytocompatibility assays with human mesenchymal stem cells have been also performed. The results obtained demonstrated that the decellularized porcine descending aorta preserves its major features to be further evaluated as a candidate material for urological applications, even though further studies have to be carried out to demonstrate its suitability for the specific application, by performing in vivo tests in the animal model.

Biomaterials for Regenerative Medicine in Italy: Brief State of the Art of the Principal Research Centers

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Porcine Small Intestinal Submucosa (SIS) as a Suitable Scaffold for the Creation of a Tissue-Engineered Urinary Conduit: Decellularization, Biomechanical and Biocompatibility Characterization Using New Approaches

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Reconstruction of the human nipple–areolar complex: a tissue engineering approach

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Pericardial patches are currently used as reconstructive material in cardiac surgery for surgical treatment of cardiac septal defects. Autologous pericardial patches, either treated with glutaraldehyde or not, can be used as an alternative to synthetic materials or xenograft in congenital septal defects repair. The availability of an allogenic decellularized pericardium could reduce complication during and after surgery and could be a valid alternative. Decellularization of allogenic tissues aims at reducing the immunogenic reaction that might trigger inflammation and tissue calcification over time. The ideal graft for congenital heart disease repair should be biocompatible, mechanically resistant, non-immunogenic, and should have the ability to growth with the patients. The aim of the present study is the evaluation of the efficacy of a new decellularization protocol of homologous pericardium, even after cryopreservation. The technique has proven to be suitable as a tissue bank procedure and highly successful in the removal of cells and nucleic acids content, but also in the preservation of collagen and biomechanical properties of the human pericardium.

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Circulatory Mitochondrial DNA Is a Pro-Inflammatory Agent in Maintenance Hemodialysis Patients

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Decellularized Allogeneic Heart Valves Demonstrate Self-Regeneration Potential after a Long-Term Preclinical Evaluation

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Jun 2014
PLOS ONE

Tissue-engineered heart valves are proposed as novel viable replacements granting longer durability and growth potential. However, they require extensive in vitro cell-conditioning in bioreactor before implantation. Here, the propensity of non-preconditioned decellularized heart valves to spontaneous in body self-regeneration was investigated in a large animal model. Decellularized porcine aortic valves were evaluated for right ventricular outflow tract (RVOT) reconstruction in Vietnamese Pigs (n = 11) with 6 (n = 5) and 15 (n = 6) follow-up months. Repositioned native valves (n = 2 for each time) were considered as control. Tissue and cell components from explanted valves were investigated by histology, immunohistochemistry, electron microscopy, and gene expression. Most substitutes constantly demonstrated in vivo adequate hemodynamic performances and ex vivo progressive repopulation during the 15 implantation months without signs of calcifications, fibrosis and/or thrombosis, as revealed by histological, immunohistochemical, ultrastructural, metabolic and transcriptomic profiles. Colonizing cells displayed native-like phenotypes and actively synthesized novel extracellular matrix elements, as collagen and elastin fibers. New mature blood vessels, i.e. capillaries and vasa vasorum, were identified in repopulated valves especially in the medial and adventitial tunicae of regenerated arterial walls. Such findings correlated to the up-regulated vascular gene transcription. Neoinnervation hallmarks were appreciated at histological and ultrastructural levels. Macrophage populations with reparative M2 phenotype were highly represented in repopulated valves. Indeed, no aspects of adverse/immune reaction were revealed in immunohistochemical and transcriptomic patterns. Among differentiated elements, several cells were identified expressing typical stem cell markers of embryonic, hematopoietic, neural and mesenchymal lineages in significantly higher number and specific topographic distribution in respect to control valves. Following the longest follow-up ever realized in preclinical models, non-preconditioned decellularized allogeneic valves offer suitable microenvironment for in vivo cell homing and tissue remodeling. Manufactured with simple, timesaving and cost-effective procedures, these promising valve replacements hold promise to become an effective alternative, especially for pediatric patients.

International Heart Valve Bank Survey: A Review of Processing Practices and Activity Outcomes

Article

Full-text available

Jan 2013

A survey of 24 international heart valve banks was conducted to acquire information on heart valve processing techniques used and outcomes achieved. The objective was to provide an overview of heart valve banking activities for tissue bankers, tissue banking associations, and regulatory bodies worldwide. Despite similarities found for basic manufacturing processes, distinct differences in procedural details were also identified. The similarities included (1) use of sterile culture media for procedures, (2) antibiotic decontamination, (3) use of dimethyl sulfoxide (DMSO) as a cryoprotectant, (4) controlled rate freezing for cryopreservation, and (5) storage at ultralow temperatures of below -135°C. Differences in procedures included (1) type of sterile media used, (2) antibiotics combination, (3) temperature and duration used for bioburden reduction, (4) concentration of DMSO used for cryopreservation, and (5) storage duration for released allografts. For most banks, the primary reasons why allografts failed to meet release criteria were positive microbiological culture and abnormal morphology. On average, 85% of allografts meeting release criteria were implanted, with valve size and type being the main reasons why released allografts were not used clinically. The wide variation in percentage of allografts meeting release requirements, despite undergoing validated manufacturing procedures, justifies the need for regular review of important outcomes as cited in this paper, in order to encourage comparison and improvements in the HVBs' processes.

The Mechanism of Interaction of Sodium Dodecyl Sulfate with Elastic Fibers

Article

Jun 1995

Sodium dodecyl sulfate (SDS), an anionic hydrophobic ligand, is known to alter the mechanical properties of elastic fibers. In order to analyze the mechanism of the alteration, two forms of fibrous elastins, “solid” and “powder” elastins, which consisted of fascicular elastic fibers and single or oligomeric elastic fibers, respectively, were prepared from bovine aorta, and the interactions of SDS with these elastins in the presence and absence of 0.15 M NaCl were studied. The solid elastin was able to retain 1.2- to 1.4-fold larger amounts of SDS than the powder elastin under both conditions, and both elastins retained 1.2-fold or larger amounts of SDS in the presence of NaCl than in its absence. Whereas both elastins released the retained SDS gradually on repeated washing with an SDS-free buffer, the release rates from the solid elastin, especially the rate in the presence of NaCl, were much smaller than those from the powder elastin, and the solid elastin retained approximately 40% of the bound SDS under conditions where the powder elastin lost most of its SDS. The SDS-binding capacities of both elastins were significantly lower than those of soluble x-elastin and serum albumin, which bound SDS homogeneously on the polypeptide chains. When the washed SDS-bound solid elastin was incubated with methylene blue and examined under a microscope, most of the methylene blue-SDS complex was located at the interfiber spaces of the elastic fibers. These results suggest that SDS alters the mechanical properties of elastic fibers by binding to the interfiber spaces and surfaces of the fibers rather than by binding to the internal polypeptide chains.

Late Durability of Decellularized Allografts for Aortic Valve Replacement: a Word of Caution

Article

Mar 2016

Sodium Deoxycholate Hydrogels: Effects of Modifications on Gelation, Drug Release, and Nanotemplating

Article

Jun 2015
J PHYS CHEM B

In the present study, sodium deoxycholate (NaDC) was used to produce gelation of tris(hydroxymethyl)amino-methane (TRIS) solutions above, below, and near the pKa of NaDC, respectively, which yielded a neutral gelator, a charged gelator, and a mixture of each. Impacts of ionic interactions on gel formation were studied in detail and showed that pH can be used to modify many hydrogel properties including sol-gel temperature, crystallinity, and mechanical strength. Several formulations yielded a unique rheological finding of two stable regions of elastic modulus. Release of a small molecule has been investigated under different hydrogel conditions and at variable shear rate, suggesting utility as a drug delivery vehicle. It was also observed that pH modification of the hydrogels affected nanoparticle formation. Nanoparticles derived from a Group of Uniform Materials Based on Organic Salts (nanoGUMBOS), specifically cyanine based NIR dyes, were templated within the hydrogel network for potential applications in tissue imaging. These nanoGUMBOS were found to be size-tunable, although material dependent. Further understanding of NaDC/TRIS gelation has broadened the tunability and multidimensional applications of these tailored hydrogel systems.

Decellularized Cryopreserved Allografts as Off-the-Shelf Allogeneic Alternative for Heart Valve Replacement: In Vitro Assessment Before Clinical Translation

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