Preventing extrinsic mechanisms of bioprosthetic degeneration using polyphenols.

OBJECTIVES: The purpose of this study was to evaluate the impact of a polyphenols-based treatment on the extrinsic mechanisms responsible for early bioprosthetic heart valve (BHV) degeneration. Structural degeneration can be driven by both extrinsic and intrinsic mechanisms. While intrinsic mechanisms have been associated with inherent biocompatibility characteristics of the BHV, the extrinsic ones have been reported to involve external causes, such as chemical, mechanical and hydrodynamic, responsible to facilitate graft damage. METHODS: The chemical interaction and the stability degree between polyphenols and pericardial tissue were carefully evaluated. The detoxification of glutaraldehyde in commercial BHVs models and the protective effect from in vivo calcification were taken into relevant consideration. Finally, the hydrodynamic and biomechanical features of the polyphenols-treated pericardial tissue were deeply investigated by pulse duplicator and stress-strain analysis. RESULTS: The study demonstrated the durability of the polyphenols-based treatment on pericardial tissue and the stability of the bound polyphenols. The treatment improves glutaraldehyde stabilization's current degree, demonstrating a surprising in vivo anti-calcific effect. It is able to make the pericardial tissue more pliable while maintaining the correct hydrodynamic characteristics. CONCLUSIONS: The polyphenols treatment has proved to be a promising approach capable of acting simultaneously on several factors related to the premature degeneration of cardiac valve substitutes by extrinsic mechanisms.

Bioengineered percutaneous heart valves for transcatheter aortic valve replacement: a comparative evaluation of decellularised bovine and porcine pericardia.

Glutaraldehyde-treated, surgical bioprosthetic heart valves undergo structural degeneration within 10-15 years of implantation. Analogous preliminary results were disclosed for percutaneous heart valves (PHVs) realized with similarly-treated tissues. To improve long-term performance, decellularised scaffolds can be proposed as alternative fabricating biomaterials. The aim of this study was to evaluate whether bovine and porcine decellularised pericardia could be utilised to manufacture bioengineered percutaneous heart valves (bioPHVs) with adequate hydrodynamic performance and leaflet resistance to crimping damage. BioPHVs were fabricated by mounting acellular pericardia onto commercial stents. Independently from the pericardial species used for valve fabrication, bioPHVs satisfied the minimum hydrodynamic performance criteria set by ISO 5840-3 standards and were able to withstand a large spectrum of cardiac output conditions, also during extreme backpressure, without severe regurgitation, especially in the case of the porcine group. No macroscopic or microscopic leaflet damage was detected following bioPHV crimping. Bovine and porcine decellularized pericardia are both suitable alternatives to glutaraldehyde-treated tissues. Between the two types of pericardial species tested, the porcine tissue scaffold might be preferable to fabricate advanced PHV replacements for long-term performance. CONDENSED ABSTRACT: Current percutaneous heart valve replacements are formulated with glutaraldehyde-treated animal tissues, prone to structural degeneration. In order to improve long-term performance, bovine and porcine decellularised pericardia were utilised to manufacture bioengineered replacements, which demonstrated adequate hydrodynamic behaviour and resistance to crimping without leaflet architectural alteration.

Vitrified Human Umbilical Arteries as Potential Grafts for Vascular Tissue Engineering.

BACKGROUND: The development of a biological based small diameter vascular graft (d < 6 mm), that can be properly stored over a long time period at - 196 °C, in order to directly be used to the patients, still remains a challenge. In this study the decellularized umbilical arteries (UAs) where vitrified, evaluated their composition and implanted to a porcine model, thus serving as vascular graft. METHODS: Human UAs were decellularized using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and sodium dodecyl sulfate (SDS) detergents. Then, vitrified with vitrification solution 55 (VS55) solution, remained for 6 months in liquid nitrogen and their extracellular matrix composition was compared to conventionally cryopreserved UAs. Additionally, total hydroxyproline, sulphated glycosaminoglycan and DNA content were quantified in all samples. Finally, the vitrified umbilical arteries implanted as common carotid artery interposition graft to a porcine animal model. RESULTS: Decellularized and vitrified UAs characterized by proper preservation of extracellular matrix proteins and tissue architecture, whereas conventionally cryopreserved samples exhibited a disorganized structure. Total hydroxyproline content was preserved, although sulphated glycosaminoglycan and DNA contents presented significantly alterations in all samples. Implanted UAs successfully recellularized and remodeled as indicated by the histological analysis. CONCLUSION: Decellularized and vitrified UAs retained their structure function properties and can be possible used as an alternative source for readily accessible small diameter vascular grafts.

Development and characterisation of a full-thickness acellular porcine bladder matrix for tissue engineering.

The aim of this study was to produce a natural, acellular matrix from porcine bladder tissue for use as a scaffold in developing a tissue-engineered bladder replacement. Full-thickness, intact porcine bladders were decellularised by distention and immersion in hypotonic buffer containing 0.1% (w/v) SDS and nuclease enzymes. Histological analysis of the resultant matrices showed they were completely acellular; that the major structural proteins had been retained and that there were some residual poorly soluble intracellular proteins. The amount of DNA per mg dry weight of fresh porcine bladder was 2.8 (+/-0.1) microg/mg compared to 0.1 (+/-0.1) microg/mg in decellularised bladder and biochemical analysis showed proportional differences in the hydroxyproline and glycosaminoglycan content of the tissue before and after decellularisation. Uniaxial tensile testing indicated that decellularisation did not significantly compromise the ultimate tensile strength of the tissue. There was, however, an increase in the collagen and elastin phase slopes indicating decreased extensibility. Cytotoxicity assays using porcine smooth muscle cell cultures excluded the presence of soluble toxins in the biomaterial. In summary, a full-thickness natural acellular matrix retaining the major structural components and strength of the urinary bladder has been successfully developed. The matrix is biocompatible with bladder-derived cells and has potential for use in urological surgery and tissue-engineering applications.

Review: tissue engineering of the urinary bladder: considering structure-function relationships and the role of mechanotransduction.

A variety of conditions encountered in urology result in bladder dysfunction and the need for bioengineered tissue substitutes. Traditionally, a number of synthetic materials and natural matrices have been used in experimental and clinical settings. However, the production of functional bladder tissue replacements remains elusive. The urinary bladder sustains considerable structural deformation during its normal function and represents an ideal model tissue in which to study the effects of biomechanical simulation on tissue morphogenesis, differentiation, and function. However, the actual role of mechanical forces within the bladder has received little attention. A strategy in which in vitro-generated tissue constructs are conditioned by exposure to the same mechanical forces as they would encounter in vivo could potentially be used both in the development of functional tissue replacements and to further study the role of biomechanical signalling. The purpose of this review is to examine the role and structure-function relationship of the urinary bladder and, through consultation of the literature available on mechanotransduction and tissue engineering of alternative tissues, to determine the factors that need to be considered when biomechanically engineering a functional bladder.

Development and characterization of an acellular human pericardial matrix for tissue engineering.

This study aimed to produce an acellular human tissue scaffold with a view to recellularization with autologous cells to produce a tissue-engineered pericardium that can be used as a patch for cardiovascular repair. Human pericardia from cadaveric donors were treated sequentially with hypotonic buffer, SDS in hypotonic buffer, and a nuclease solution. Histological analysis of decellularized matrices showed that the human pericardial tissue retained its histioarchitecture and major structural proteins. There were no whole cells or cell fragments. There were no significant differences in the hydroxyproline (normal and denatured collagen) and glycosaminoglycan content of the tissue before and after decellularization (p > 0.05). There were no significant changes in the ultimate tensile strength after decellularization (p > 0.05). However, there was an increased extensibility when the tissue strips were cut parallel to the visualized collagen bundles (p = 0.005). No indication of contact or extract cytotoxicity was found when using human dermal fibroblasts and A549 cells. In summary, successful decellularization of the human pericardium was achieved producing a biocompatible matrix that retained the major structural components and strength of the native tissue.

Biocompatibility and recellularization potential of an acellular porcine heart valve matrix.

BACKGROUND AND AIM OF THE STUDY: Tissue-engineered heart valves have the potential to overcome the limitations of present heart valve replacements. The study aim was to investigate the biocompatibility and recellularization potential of an acellular porcine valve matrix. METHODS: Acellular porcine valve matrix contact and extract cytotoxicity was tested against porcine fibroblasts and smooth muscle cells (SMC). Porcine cells were incubated with decellularized aortic valve leaflets and aortic wall, and then assessed for changes in morphology and contact inhibition of growth. Soluble tissue extracts were prepared from decellularized leaflets and aortic wall, and assessed for their effect on the viability of cultured porcine cells. Acellular leaflets were seeded with either fibroblasts or SMC at 1 x 10(3) to 1 x 10(6) cells/cm2 for 24 h, or 5 x 10(4) cells/cm2 for 1-4 weeks. Cell attachment onto, and migration into, the acellular matrix was assessed by scanning electron microscopy and histology. RESULTS: No contact inhibition of growth, or changes in fibroblast or SMC morphology, were observed following contact with the acellular valve matrix. No soluble extract cytotoxicity was found. Intermediate cell-seeding densities (2.5 x 10(4) to 7.5 x 10(4) cells/cm2) of both cell types produced confluent cell attachment; at the lowest concentration (1 x 10(3) cells/cm2) cell attachment was sparse, and at the highest (1 x 10(6) cells/cm2) it was multilayered. The SMC migrated throughout the leaflet matrix over four weeks, but there was no fibroblast migration into the matrix. CONCLUSION: The absence of contact and extract cytotoxicity indicated that the acellular valve matrix was biocompatible in vitro. The failure of porcine fibroblasts to grow on, or infiltrate into, the matrix suggested that the SMC may be the preferred cell type for future leaflet recellularization studies in the development of a tissue-engineered heart valve replacement.

Tissue engineering of cardiac valve prostheses I: development and histological characterization of an acellular porcine scaffold.

BACKGROUND AND AIMS OF THE STUDY: Several deficiencies in current heart valve prostheses make them problematic for use in younger patients. Tissue valve substitutes are non-viable with a life expectancy of only 10-15 years, while mechanical valves require long-term anti-coagulation therapy. A solution to these problems would be to develop a tissue-engineered heart valve containing autologous cells, enabling the valve to maintain its biochemical and mechanical properties, yet grow with the patient. The study aim was to optimize a protocol to produce a porcine acellular matrix scaffold for use in developing a tissue-engineered heart valve. METHODS: Fresh porcine aortic valve leaflets were treated with Triton X-100, sodium dodecyl sulfate (SDS), sodium deoxycholate, MEGA 10, TnBP, CHAPS, and Tween 20, over a range of concentrations, in the presence of protease inhibitors for up to 72 h. Histological analysis was used to detect the major structural proteins of the heart valve, collagen I, elastin and glycosaminoglycans. RESULTS: After 72 h, most protocols resulted in the retention of large numbers of whole cells and cell fragments. Only SDS (0.03-1%) or sodium deoxycholate (0.5-2%) resulted in total decellularization at 24 h. Histological analysis of acellular matrices showed that the major structural proteins had been retained and appeared to be intact. CONCLUSION: Protocols utilizing SDS or sodium deoxycholate were successful for leaflet decellularization, and histological analysis showed that the major structural components of the valve matrix had been maintained. These methods are being developed further with a view to reseeding with autologous cells to produce tissue-engineered solutions for clinical implantation.

Tissue engineering of cardiac valve prostheses II: biomechanical characterization of decellularized porcine aortic heart valves.

BACKGROUND AND AIMS OF THE STUDY: For both young patients with congenital heart disease and young, growing adults there is a need for replacement heart valves that will develop with the patient. Tissue-engineered heart valves coupled with in-vitro recellularization have this potential. One approach is to use acellular tissue matrices, but the decellularization treatment must not affect the biomechanical integrity of the valvular matrix. This study investigated the effect of 0.03% (w/v) and 0.1% (w/v) sodium dodecyl sulfate (SDS) on the mechanical integrity of porcine aortic valve leaflets. METHODS: Left coronary porcine leaflets were treated with SDS (0.03% or 0.1%, w/v) in hypotonic or isotonic buffer and buffer alone. SDS in hypotonic buffer produced accellularity. Circumferential and radial specimens of treated leaflets were subjected to uniaxial tensile testing, and the effect of the buffer on leaflet morphology was assessed. Whole porcine aortic roots were also treated with 0.1% (w/v) SDS and subjected to function testing. RESULTS: SDS treatment significantly increased extensibility of the leaflet specimens, which was greater in the circumferential than radial direction. This was seen as a significantly decreased slope of both the elastic and collagen phases of the stress-strain behavior. The ultimate tensile strength and transition stress were not affected significantly; nor was there any significant difference between hypotonic buffer and hypotonic buffer + SDS treatments. Study of the leaflet morphology suggested that the increased extensibility was due to shrinkage as well as to increased hydration of the treated leaflets caused by the hypotonic buffer. CONCLUSION: SDS treatment produced a more extensible tissue with equal strength compared with the fresh aortic valve. Functionality experiments with SDS-treated whole aortic roots showed complete valve leaflet competence under physiological pressures (120 mmHg) as well as physiological leaflet kinematics.