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Bioprosthetic heart valves: The need for a quantum leap
Abstract
More than 250,000 bioprosthetic heart valves are being implanted annually. Although the majority of recipients are elderly developed-world patients, the most urgent need for tissue valves is in younger patients, where rapid degeneration of contemporary prostheses remains a serious obstacle. After decades of empirical and mostly futile attempts to extend the longevity of tissue valve prostheses, new insights and solutions are on the horizon. Aetiologically, a shift of focus from mineralization to immune responses and inflammation emerges. On the development side, new engineering approaches to both selective extraction of tissue components and cross-links are increasingly defining the new direction. In order to dramatically improve the performance of bioprosthetic heart valves, these new developments need to lead to a broad consensus for a paradigm shift in a hitherto rather stagnant field of medical research.
... As insight into the correlation of crosslink density and mitigation of calcification grew, it became obvious that crosslink chemistry offered an additional lever in the quest to prevent calcific degeneration of bioprosthetic tissue. Increased crosslink density via increased concentrations of GA was previously confirmed by an increased modulus of the tissue [27], a decrease in free amines [7] and an increased resistance to protease digestion [7,27]. Augmenting GA crosslinks with diamines during conventional fixation reduced calcification in the particularly challenging aortic wall tissue by more than a third [9]. ...
... As insight into the correlation of crosslink density and mitigation of calcification grew, it became obvious that crosslink chemistry offered an additional lever in the quest to prevent calcific degeneration of bioprosthetic tissue. Increased crosslink density via increased concentrations of GA was previously confirmed by an increased modulus of the tissue [27], a decrease in free amines [7] and an increased resistance to protease digestion [7,27]. Augmenting GA crosslinks with diamines during conventional fixation reduced calcification in the particularly challenging aortic wall tissue by more than a third [9]. ...
Objectives:
While decellularization has previously significantly improved the durability of bioprosthetic tissue, remnant immunogenicity may yet necessitate masking through crosslinking. To alleviate the fears of reintroducing the risk of calcific degeneration, we investigated the application of rationally designed crosslinking chemistry, capable of abrogating mineralization in isolation, in decellularized tissue.
Methods:
Bovine and porcine pericardium were decellularized using the standard Triton X/sodium deoxycholate/DNAse/RNAse methodology and thereafter combined incrementally with components of a four-stage high-density dialdehyde-based fixation regimen. Mechanical properties prior to, and calcium levels following, subcutaneous implantation for 6 and 10 weeks in rats were assessed.
Results:
Enhanced four-stage crosslinking, independent of decellularization, or decellularization followed by any of the crosslinking regimens, achieved sustained, near-elimination of tissue calcification. Decellularization additionally resulted in significantly lower tissue stiffness and higher fatigue resistance in all groups compared to their non-decellularized counterparts.
Conclusions:
The dual approach of combining decellularization with enhanced crosslinking chemistry in xenogeneic pericardial tissue offers much promise in extending bioprosthetic heart valve longevity.
... 21,22 The biological solution to minimize stresses under these complex mechanical loading conditions is to use leaflets with three layers: the ventricularis, the spongiosa, and the fibrosa, as one moves from the ventricle to the aorta (Figure 1). 23 The fibrosa layer contains circumferentially aligned bundles of collagen fibers, whereas the ventricularis layer is formed by collagen and highly stretchable elastin. ...
... Replicating this elegant mechanism in bioinspired heart valves can potentially enhance their lifetime and biological function by tuning the stress distribution in the leaflets to minimize fatigue damage and provide the mechanical cues needed to keep cells alive and active in tissue-engineered constructs. 23,27 One such example in the literature is the ''BioTexValve,'' 28 which features a composite of 12 poly(L/DL)-lactide monofilaments manually laid and fixed in a planar frame before encapsulation using an electrospinning process. These composite sheets are placed in a 3D mold, and a cell seeded fibrin gel is introduced over the surface. ...
ABSTRACT
Synthetic implants made by traditional fabrication routes are not patient-specific and rarely match the performance of their biological counterparts. We present an additive manufacturing approach for the digital fabrication of tissue-like aortic heart valves featuring customizable geometry and leaflet architectures that resemble those of native valve tissue. Using biocompatible silicones with tunable mechanical properties, heart valves were fabricated by combining spray and extrusion-based Additive Manufacturing processes. Computer simulations showed that bioinspired leaflet architectures strongly affect the stress distribution throughout the valves, minimizing stresses on the leaflet during a cardiac cycle. Our computational analysis was complemented by in vitro experiments in a pulse duplicator to demonstrate the outstanding hemodynamic performance of the printed heart valves under physiological pressure cycles. The ability to fabricate synthetic implants with tailored designs at multiple length scales is a key contribution towards the digital fabrication of functional implants that perform on par with native body parts.
... Regenerative medicine aims to create living heart valves which would overcome the shortcomings of current alternatives. An ideal tissue engineered heart valve (TEHV) should encompass several characteristics: good hemodynamics, appropriate valve geometry, high durability, non-immunoge-nicity, non-inflammatory, non-thrombogenic and non-calcifying (1,3). Additionally, for young patients, it should have the ability to grow and adapt with the patient's somatic growth (2,(4)(5)(6). ...
... Once seeded within the scaffolds, these will offer the valve viability, growth potential and remodeling capacity. Cell-seeded structures can then be introduced in a bioreactor, which allows different modes of stimulation and mechanical preconditioning (8), or alternatively, can directly be implanted for in vivo conditioning and maturation (3). Evaluation of TEHV in vivo is typically performed in an animal model which resembles the physiologic and hemodynamic human characteristics. ...
: We hypothesized that an ideal heart valve replacement would be acellular valve root scaffolds seeded with autologous stem cells. To test this hypothesis, we prepared porcine acellular pulmonary valves, seeded them with autologous adipose derived stem cells (ADSCs) and implanted them in sheep and compared them to acellular valves.
: Fresh porcine pulmonary valve roots were decellularized with detergents and enzymes. ADSCs were isolated from subdermal fat and injected within the acellular cusps. Valves were then implanted in an extra-anatomic pulmonary position as RV to PA shunts: Group A (n=6) consisted of acellular valves and Group B (n=6) of autologous stem cell-seeded acellular xenografts. Sheep were followed up for 6 months by echocardiography and histologic analysis was performed on explanted valves.
: Early evolution was favorable for both groups. All Group A animals had physiologic growth without any signs of heart failure and leaflets were found with preserved structure and mobility, lacking signs of thrombi, inflammation or calcification. Group B sheep however expressed signs of right ventricle failure starting at one month, accompanied by progressive regurgitation and right ventricle dilatation, and the leaflets were found covered with host tissue. No cells were found in any Group A or B explants.
: Acellular stabilized xenogeneic pulmonary valves are reliable, stable, non-immunogenic, non-thrombogenic and non-calcifying scaffolds with excellent hemodynamics. Seeding these scaffolds with autologous ADSCs was not conducive to tissue regeneration. Studies aimed at understanding these novel observations and further harnessing the potential of stem cells are ongoing.
... Cow was the best source for the latter alternative, but it has become less popular after the bovine spongiform encephalopathy crisis, and most procedures have involved porcine-derived valves. These are treated in some way in order to remove infectivity and antigenicity (Zilla et al., 2004). For many years both these objectives were managed through the use of a glutaraldehyde fixation process. ...
... It has to be said that, in parallel with pharmaceutical approaches, medical device technologies (Zilla et al., 2004), have provided very powerful methods to alleviate the debilitating conditions associated with either the congenital malformation or deterioration of the heart and its constituent components. As good as they are, however, all have limitations in that they are not providing the optimal long-term solutions, for there is almost always a price to pay for the intervention. ...
The processes of tissue regeneration in adult humans are biologically feasible. The development of tissue engineering as a viable industry has to take into account the infrastructure surrounding the new concepts that are involved. There will be a number of critical pragmatic factors that control success. One of the most significant is the situation of unmet clinical need. It is highly unlikely that tissue engineering will succeed, at least initially, if it concentrates on the areas that are already well served with both clinical success and acceptable costs by alternative therapies. With the current models, it has been found that the development of tissue-engineering products requires investments that approach the astronomical amounts normally associated with the pharmaceutical industry but with financial returns more equivalent to those of the lower cost-based medical-device industry. This gap is unsustainable. The problem that this technology has faced is the separation of the tissue-engineering process into discrete phases.
... From this viewpoint it is an appropriate material to cover various medical devices, which are to be in contact with the internal environment of an organism [2]. In particular, there is a wellknown problem of calcinosis of bioprosthetic heart valves (BHV) prepared in a standard way [3]. According to the typical procedure, the pericardium xenotissue for BHV is pre-treated by glutaraldehyde in order to stabilize the material and to reduce the immune response on it [4]. ...
... The driving force of such complex behaviour is the increased density fluctuations of the solvent. When CO 2 is used in real applications as a SC solvent, typically solubility of polymer materials is rather poor in the proximity of the critical point (density of about 0.4 g/cm 3 ) and improves only when density is increased up to at least of about 1 g/cm 3 . In this region the dense SC CO 2 is still rather mobile and fluctuating fluid with peculiar properties. ...
Chitosan and its derivatives are promising materials for coating medical devices because this procedure improves their bio- and haemocompatibility. It is known that supercritical carbon dioxide attracts significant scientific interest in biomedical applications as well. Coatings deposited directly from solutions in supercritical carbon dioxide are expected to have particularly smooth and uniform morphology that should enhance their stability. We have tested the possibility of obtaining chitosan films using direct deposition from solutions in this fluid. In order to reveal benefits of this approach we modelled and studied the initial stage of formation of chitosan coatings with prototype system of depositing pioneer single polymer chains directly from such solutions on a model ultrasmooth mica substrate. We estimated achievable solubility of the chitosan materials in supercritical carbon dioxide and performed conformational analysis of the deposited chitosan chains on a substrate. AFM imaging directly demonstrated that the pioneer macromolecules adsorb as rather extended 2D coils from such solutions.
... It has been shown that these responses are controlled by antibodies generated against both galactose-α-1,3-galactose (gal) and non-gal antibodies 2 . Although fixed and unfixed xenogeneic biomaterials are commonly used in clinical practice, recipient graft-specific immune responses limit their long term durability and functional outcomes 3,4 . Clinical results achieved with such biomaterials are far from ideal, with both acute rejection (i.e., decellularized) 5 and chronic rejection (i.e., glutaraldehyde-fixation) responses being reported 6 . ...
Despite bovine pericardium (BP) being the primary biomaterial used in heart valve bioprostheses, recipient graft-specific immune responses remain a significant cause of graft failure. Consequently, tissue antigenicity remains the principal barrier for expanding use of such biomaterials in clinical practice. We hypothesize that our understanding of BP antigenicity can be improved by application of a combined affinity chromatography shotgun immunoproteomic approach to identify antigens that have previously been overlooked. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) analysis of affinity chromatography purified antigens resulted in identification of 133 antigens. Most importantly, antigens were identified from all subcellular locations, including 18 integral membrane protein antigens. Critically, isoforms of several protein families were found to be antigenic suggesting the possibility that shared epitope domains may exist. Furthermore, proteins associated with immune, coagulation, and inflammatory pathways were over-represented, suggesting that these biological processes play a key role in antigenicity. This study brings to light important determinants of antigenicity in a clinically relevant xenogeneic biomaterial (i.e. BP) and further validates a rapid, high-throughput method for immunoproteomic antigen identification.
... No data has been presented for such anticalcification treatments that further results in increased mechanical or biological durability. Marginal increases have been made to BHV implant life, if any, since the introduction of these technologies [9,27,48]. ...
Bioprosthetic heart valves (BHVs), derived from glutaraldehyde crosslinked (GLUT) porcine aortic valve leaflets or bovine pericardium (BP), are used to replace defective heart valves. However, valve failure can occur within 12–15 years due to calcification and/or progressive structural degeneration. We present a novel fabrication method that utilizes carbodiimide, neomycin trisulfate, and pentagalloyl glucose crosslinking chemistry (TRI) to better stabilize the extracellular matrix of BP. We demonstrate that TRI-treated BP is more compliant than GLUT-treated BP. GLUT-treated BP exhibited permanent geometric deformation and complete alteration of apparent mechanical properties when subjected to induced static strain. TRI BP, on the other hand, did not exhibit such permanent geometric deformations or significant alterations of apparent mechanical properties. TRI BP also exhibited better resistance to enzymatic degradation in vitro and calcification in vivo when implanted subcutaneously in juvenile rats for up to 30 days.
... 27 In contrast, manufactured collagen-based materials rely on cross-linking to increase their mechanical properties which significantly reduces the cellular degradation of the material. 60 The ability of cells to remodel their surrounding matrix is critical for regenerative success. Through the process of constructive remodeling, the degradable matrix template is gradually replaced by native tissue with the appropriate function, matrix organization, and cellular phenotype. ...
Tissue regeneration strategies have traditionally relied on designing biomaterials that closely mimic features of the native extracellular matrix (ECM) as a means to potentially promote site-specific cellular behaviors. However, inflammation, while a necessary component of wound healing, can alter processes associated with successful tissue regeneration following an initial injury. These processes can be further magnified by the implantation of a biomaterial within the wound site. In addition to designing biomaterials to satisfy biocompatibility concerns as well as to replicate elements of the composition, structure, and mechanics of native tissue, we propose that ECM analogs should also include features that modulate the inflammatory response. Indeed, strategies that enhance, reduce, or even change the temporal phenotype of inflammatory processes have unique potential as future pro-regenerative analogs. Here, we review derivatives of three natural materials with intrinsic anti-inflammatory properties and discuss their potential to address the challenges of inflammation in tissue engineering and chronic wounds.
... kangaroo [1], equine pericardium [2] and porcine pericardium [3]) and fixation treatments [4], GA (glutaraldehyde)-fixed porcine aortic valves and bovine pericardium have remained the most commonly used materials in the fabrication of bioprosthetic heart valves over the past 35 years [5]. However, tissues treated with commercial concentrations of GA (0.2 or 0.625 %) remain prone to long-term failure due to calcification, inflammatory degradation, mechanical injury and pannus overgrowth [6]. ...
... We construct 3-dimensional model using the Pro/ENGNEER software. Firstly, we create a conic curved surface and a cylindrical curved surface, and then make them intersect [8]. Finally we get intersecting curves which are boundary curves of leaflets, as shown inFig. ...
In order to investigate the effect of the suture density on the mechanical properties of bioprosthetic heart valve, we establish the spherical leaflets models via computer aided design. Finite element analysis is used simulate the mechanical performance of bioprosthetic heart valve when leaflet is closed. The analysis results show that suture density has a significant effect to the stress state of valve, which may lead not only to different stress peak values, but also to different stress distributions. High or low density suture isn't appropriate for long-term durability of the bioprosthetic heart valve. Suture density tests are needed to perform on the valves to find satisfactory suture density for better mechanical properties of the bioprosthetic heart valve. (2012) Trans Tech Publications, Switzerland.
... kangaroo [1], equine pericardium [2] and porcine pericardium [3]) and fixation treatments [4], GA (glutaraldehyde)-fixed porcine aortic valves and bovine pericardium have remained the most commonly used materials in the fabrication of bioprosthetic heart valves over the past 35 years [5]. However, tissues treated with commercial concentrations of GA (0.2 or 0.625 %) remain prone to long-term failure due to calcification, inflammatory degradation, mechanical injury and pannus overgrowth [6]. ...
Despite indications that GA (glutaraldehyde)-crosslinked tissues remain prone to long-term degradation and calcification, it is still the reagent of choice in the fixation of bioprosthetic heart valves. We have shown previously that increased GA concentrations and diamine extension of cross-links with lysine incorporation lead to mitigated in vivo calcification, mainly of porcine aortic-wall tissue. The present study was performed to assess the correlation between the cross-link density of all three commonly used tissue types [PW (porcine aortic wall), PL (porcine aortic leaflet) and BP (bovine pericardium)] and tissue calcification in the subcutaneous rat model after GA treatment with or without lysine. The effect of lysine enhancement, and increased GA concentration in the presence of lysine, resulted in significant increases in tissue cross-linking in all three tissue types. Although increased GA concentration on its own resulted in decreased calcification without an increase in cross-link density, overall positive correlations were found between denaturation temperature and RPD (resistance towards protease degradation) [correlation coefficient (rho) values: rhoPW =0.922, rhoPL =0.783 and rhoBP =0.955], whereas negative correlations existed between RPD and calcification (rhoPW=-0.836, rhoPL=-0.929 and rhoBP=-0.579). The combination of lysine enhancement and an increase in GA concentration from 0.2 to 3% resulted in 79, 44 and 56% decreases in calcification in PW, PL and BP. In the case of BP, a decrease in calcification of 81% could be achieved merely by adding lysine extension to low-concentration (0.2 %) GA cross-linking. Thus it is concluded that the increase in cross-link density achieved by lysine incorporation, and by increased GA concentration in the presence of lysine, results in significant and marked decreases in calcification of all three types of tissues commonly used in bioprosthetic heart valves.
... 1,2 Most of the clinical biomaterials that are currently made of these scaffold-based materials (e.g., tissue-derived heart valves) are crosslinked to enhance their biomechanical performance, significantly reducing the materials' degradability, and thus removing the primary advantage of using these natural materials over synthetic nondegradable materials. 3 Decellularization is a processing method that is gaining interest in the clinical field of heart valve replacement, especially because it promises an approach by which natural biomaterials can have adequate mechanical properties, without exogenous chemical crosslinking, and thus remain degradable and capable of remodeling through cellular means. 4 With the increasing popularity of collagenous and decellularized scaffolds for tissue engineering, it becomes increasingly important to understand the in vivo cellular response to these innately degradable materials as a component of their biocompatibility. ...
Decellularized tissue-derived heart valves are an example of biomaterials derived from natural scaffolds. These types of implants are increasing in popularity although their in vivo performance is still only poorly understood and has, at times, been catastrophic. It is apparent that better understanding is required before these biomaterials can be used safely. In this study, the human monocyte-derived macrophage (MDM) response to decellularized bovine pericardium (DBP) was used as a model to predict the biological performance of these materials on implantation. Human monocytes differentiated on tissue culture polystyrene (TCPS) for 14 days were trypsinized and reseeded onto DBP, TCPS, and polydimethylsiloxane (PDMS) for 48 h. The MDMs on DBP contained less intracellular and extracellular esterase activity compared with MDMs on TCPS and PDMS, as well as less acid phosphatase activity than on TCPS. As well, morphologically, MDMs on DBP were less spread, less multinucleated and did not display many lamellipodia. Taken together, these data represent the first evidence of the MDM response to intact, native extracellular matrix, demonstrating that these cells reacted with an altered, possibly reduced foreign body response on this natural scaffold compared with the two control surfaces. This in vitro MDM cell model may provide a novel method for predicting and elucidating the biological performance of tissue-derived biomaterials, thereby directing a more rational design of biomaterials for tissue regeneration purposes.
... In the cuspal spongiosa layer, GAGs provide a " sliding gap " between the ventricularis and fibrosa. The hydrating nature of GAGs helps to absorb the shear and compressive stresses acting on the valve cusps [3,25,26]. Geometric and structural changes in the valves may affect the valves' biomechanical function when implanted [27]. GLUT, the commercially used fixative for BHVs, is an excellent fixative for the collagenous component of the valves, but it does not effectively stabilize GAGs and elastin, the other two major valve components. ...
Bioprosthetic heart valve (BHV) cusps have a complex architecture consisting of an anisotropic arrangement of collagen, glycosaminoglycans (GAGs) and elastin. Glutaraldehyde (GLUT) is used as a fixative for all clinical BHV implants; however, it only stabilizes the collagen component of the tissue, and other components such as GAGs and elastin are lost from the tissue during processing, storage or after implantation. We have shown previously that the effectiveness of the chemical crosslinking can be increased by incorporating neomycin trisulfate, a hyaluronidase inhibitor, to prevent the enzyme-mediated GAG degradation. In the present study, we optimized carbodiimide-based GAG-targeted chemistry to incorporate neomycin into BHV cusps prior to conventional GLUT crosslinking. This crosslinking leads to enhanced preservation of GAGs during in vitro cyclic fatigue and storage. The neomycin group showed greater GAG retention after both 10 and 50 million accelerated fatigue cycles and after 1 year of storage in GLUT solution. Thus, additional binding of neomycin to the cusps prior to standard GLUT crosslinking could enhance tissue stability and thus heart valve durability.
... shown that the current generations of decellularized biological scaffolds are subject to immunemediated rejection [1,[13][14][15][16][17]. The fundamental concept of decellularization has thus come under scrutiny with the focus shifting away from removal of visible cells and toward identification and removal of antigenic proteins [11,12,18]. ...
Bovine pericardium (BP) is an important biomaterial used in the production of glutaraldehyde-fixed heart valves and tissue-engineering applications. The ability to perform proteomic analysis on BP is useful for a range of studies, including investigation of immune rejection after implantation. However, proteomic analysis of fibrous tissues such as BP is challenging due to their relative low-cellularity and abundance of extracellular matrix. A variety of methods for tissue treatment, protein extraction, and fractionation were investigated with the aim of producing high-quality 2-DE gels for both water- and lipid-soluble BP proteins. Extraction of water-soluble proteins with 3-(benzyldimethylammonio)-propanesulfonate followed by n-dodecyl beta-D-maltoside extraction and ethanol precipitation for lipid-soluble proteins provided the best combination of yield, spot number, and resolution on 2-DE gels (Protocol E2). ESI-quadrupole/ion trap or MALDI-TOF/TOF MS protein identifications were performed to confirm bovine origin and appropriate subcellular prefractionation of resolved proteins. Twenty-five unique, predominantly cytoplasmic bovine proteins were identified from the water-soluble fraction. Thirty-two unique, predominantly membrane bovine proteins were identified from the lipid-soluble fraction. These results demonstrated that the final protocol produced high-quality proteomic data from this important tissue for both cytoplasmic and membrane proteins.
... Morphological and structural changes occur in BHVs, particularly in the extracellular matrix of valve leaflets, following implantation. Eventually, these modifications may lead to the demise of the implant via various mechanisms including: calcification, GAG loss, collagen bundle loosening, and degeneration [2,3,26,27]. The relationship between GAGs and calcification remains highly debated although previous work by our group as well as others have shown that the preservation of endogenous GAGs within valve leaflets may play a minor role in inhibiting calcification [4,28]. ...
Numerous crosslinking chemistries and methodologies have been investigated as alternative fixatives to glutaraldehyde (GLUT) for the stabilization of bioprosthetic heart valves (BHVs). Particular attention has been paid to valve leaflet collagen and elastin stability following fixation. However, the stability of glycosaminoglycans (GAGs), the primary component of the spongiosa layer of the BHV, has been largely overlooked despite recent evidence provided by our group illustrating their structural and functional importance. In the present study we investigate the ability of two different crosslinking chemistries: sodium metaperiodate (NaIO(4)) followed by GLUT (PG) and 1-Ethyl-3-(3 dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) followed by GLUT (ENG) to stabilize GAGs within BHV leaflets and compare resulting leaflet characteristics with that of GLUT-treated tissue. Incubation of fixed leaflets in GAG-degrading enzymes illustrated in vitro resistance of GAGs towards degradation in PG and ENG treated tissue while GLUT fixation alone was not effective in preventing GAG loss from BHV leaflets. Following subdermal implantation, significant amounts of GAGs were retained in leaflets in the ENG group in comparison to GLUT-treated tissue, although GAG loss was evident in all groups. Utilizing GAG-targeted fixation did not alter calcification potential of the leaflets while collagen stability was maintained at levels similar to that observed in conventional GLUT-treated tissue.
Over one million artificial heart valve transplantations are performed each year due to valvular stenosis or regurgitation. Among them, bioprosthetic heart valves (BHVs) are increasingly being used because of the absence of the need for lifelong anticoagulation. Almost all of the commercial BHVs are treated with Glutaraldehyde (GLUT). As GLUT-treated BHVs are prone to calcification and structural degradation, their durability is greatly reduced with a service life of only 12–15 years. The physiological structure and mechanical properties of the porcine aortic valve (PAV) are closer to that of a human heart valve, so in this study, PAV is used as the model to explore the comprehensive properties of the prepared BHVs by radical polymerization crosslinking method. We found that PAV treated by radical polymerization crosslinking method showed similar ECM stability and biaxial mechanical properties with GLUT-treated PAV. However, radical polymerization crosslinked PAV exhibited better cytocompatibility and endothelialization potential in vitro cell experiment as better anticalcification potential and reduced immune response than GLUT-treated PAV through subcutaneous animal experiments in rats. To conclude, a novel crosslinking method of non-glutaraldehyde fixation of xenogeneic tissues for the preparation of BHVs is expected.
Currently, the only treatment for moderate and severe aortic valve calcification disease is valve replacement. The clinical use of bioprosthetic heart valves (BHVs) is limited by device failure that is caused by structural degeneration, such as the calcification of BHV leaflets. In the present study, zwitterion (2-[methacryloyloxy]ethyl)dimethyl-(3-sulfopropyl) ammonium hydroxide (SBMA) was incorporated into poly(ethylene glycol) diacrylate (PEGDA) forming balanced charged networks to modify decellularized heart valves (DHV) and improve anti-calcification properties. The novel PEGDA-SBMA-DHV prostheses showed good hemodynamic properties (i.e., effective orifice area = 1.9 cm², regurgitant fraction = 1.5% for Ф25 mm surgical valve at heart stroke of 80 ml) complying with the requirements of ISO 5840-2 standards. Weight loss of the PEGDA-SBMA-DHV artificial heart valves was less than 5.5% after enzymatic hydrolysis for 12 hours. No obvious calcification was observed in the PEGDA-SBMA-DHV samples, whereas calcification was found in decellularized heart valves and the PEGDA-modified group by soaking in a calcification solution for 10 days. Furthermore, histological staining results indicated that the PEGDA-SBMA-DHV samples with balanced charged networks elicited negligible inflammation after subcutaneous implantation for one month. Thus, balanced charged networks of PEGDA-SBMA hydrogels with cationic and anionic groups can prevent the transportation of Ca²⁺ ions and enzyme molecules, conferring efficient anti-calcification properties to artificial heart valves.
For the past 50+ years, the heart valve replacement (HVR) industry has gone through multiple growth phases in terms of innovation and market growth, and is now a formidable submarket of the cardiovascular medical device space. HVRs started off as small, compact simplistic devices made from synthetic parts. Now, these archaic prototypes have evolved into a diverse product offering from a multitude of small to large healthcare firms that range from completely synthetic materials to crosslinked tissue-based HVRs to new research being performed to investigate engineered tissue valves. These innovative leaps in technology and growth in commerce would not have been possible without the collaboration of multidisciplinary investigators unified through their passion in pursuing one goal—a superior healthcare option that resulted in a better quality of life for individuals throughout the world needing a new heart valve. Everything from biomaterial design to micro/macro-biomechanics to build computational modeling to optimize valve design has been utilized to create the current product line and are currently still in use as our metrics and methods get more advanced to innovate the future of heart valves as well as cardiovascular device technology. The future of HVRs rests on academia and industry coming together to move technology forward to provide patients in dire need of a more durable HVR device. Therefore, the rest of the content covered in this book is a comprehensive review of current art and models in existence to design an effective HVR in the efforts of empowering individuals wishing to bring healthcare options to a patient segment in dire need of change. The following chapter will cover past and current approaches in designing and fabricating HVR materials, current performance of HVRs, material design considerations of next generation materials, and major research interests in the next generation of HVR materials. The rest of this publication will cover approaches in properly leveraging this base biomaterial in valve-specific design to innovate a more effective HVR.
In recent years, the number of heart valve replacements has multiplied with valve diseases because of aging populations and the surge in rheumatic heart disease in young people. Among them, bioprosthetic heart valves (BHVs) have become increasingly popular. Transcatheter aortic valve implantation (TAVI) valve as an emerging BHV has been increasingly applied to patients. However, the current commercially used BHVs treated with glutaraldehyde (Glut) still face the problem of durability. BHVs derived from Glut-treated xenogenetic tissues would undergo structural degeneration and calcification sometimes even as short as less than 10 years. This issue has already become a big challenge considering more and more young patients at the age of 50–60 s are receiving the BHV replacement. In our study, an approach that is totally different from the previous techniques named by us as the radical polymerization-crosslinking (RPC) method was developed to improve extracellular matrix stability, prevent calcification, and reduce inflammatory response in BHVs. The porcine pericardium (PP) tissue was decellularized, functionalized with methacryloyl groups, and subsequently crosslinked by radical polymerization. We found that high-density RPC treatment remarkably improved the stability of collagen and elastin of PP, enhanced its endothelialization potential, and provided reliable biomechanical performance as compared to Glut treatment. The in vivo rat model also confirmed the increased componential stability and the reduced inflammatory response of RPC-treated PP. Moreover, the RPC-treated PP showed better in vivo anticalcification potential than Glut-treated PP.
Statement of significance
Bioprosthetic heart valves (BHVs) manufactured from glutaraldehyde (Glut)-treated xenogeneic tissues have been used to treat valve-related diseases for several decades. However, the durability of BHVs remains unresolved and becomes more pronounced particularly in younger patients. Although a number of new alternative methods for Glut crosslinking have been proposed, their overall performance is still far from ready to use in humans. In this study, radical polymerization was investigated for crosslinking the porcine pericardium (PP). This treatment was found to have advantages compared to Glut-treated PP in terms of stability, biocompatibility, and anticalcification potential with the hope of addressing the needs of more robust biomaterials for the fabrication of BHVs.
Current heart valve prostheses are associated with significant complications including aggressive immune response, limited valve life expectancy, and inability to grow in juvenile patients. Animal derived "tissue" valves undergo glutaraldehyde fixation to mask tissue antigenicity, however chronic immunological responses and associated calcification still commonly occur. A heart valve formed from an unfixed bovine pericardium (BP) extracellular matrix (ECM) scaffold, in which antigenic burden has been eliminated or significantly reduced, has potential to overcome deficiencies of current bio-prostheses. Decellularization and antigen removal methods frequently employ sequential solutions extrapolated from analytical chemistry approaches to promote solubility and removal of tissue components from resultant ECM scaffolds. However, the extent to which such pre-fractionation strategies may inhibit removal of antigenic tissue components has not been explored. We hypothesize that presence of magnesium in pre-fractionation steps, causes DNA precipitation and reduces removal of nuclear-associated antigenic proteins. Keeping all variables consistent bar the addition or absence of magnesium (2 mM magnesium chloride hexahydrate), residual BP ECM scaffold antigenicity and removed antigenicity were assessed, along with residual and removed DNA content, ECM morphology, scaffold composition and recellularization potential. Furthermore, we employed proteomic methods to determine the mechanism by which magnesium presence or absence affects scaffold residual antigenicity. This study demonstrates that absence of magnesium from antigen removal solutions enhances solubility and subsequent removal of antigenic nuclear-associated proteins from BP. We therefore conclude that the primary mechanism of action for magnesium removal during antigen removal processes is avoidance of DNA precipitation, facilitating solubilization and removal of nuclear-associated antigenic proteins. Future studies are necessary to further facilitate solubility and removal of nuclear-associated antigenic proteins from xenogeneic ECM scaffolds, in addition to an in vivo assessing of the material.
This paper constructs the bioprosthetic valve leaflets’ parametric model using computer aided design.A series of accurate parameters of the bioproshtetic heart valve, such as radius of the sutural ring, height of the supporting stent and inclination of the supporting stent are determined. Numerical simulation is used to determine the effect of different shape designs and suture density on the mechanical performance of the bioprosthetic valve leaflet. The dynamic behavior of the valve during diastolic phase is analyzed. The finite element analysis results show that the stress distribution of the ellipsoidal leaflet valve is good. The ellipsoidal leaflet valve has the following advantages over the cylindrical leaflet valve: lower peak von Mises-stress, smaller stress concentration area and relatively uniform stress distribution. The suture density also has a significant effect on the dynamic behavior of the valve as it can act to reduce the pressure and improve the stress distribution. It was found that the influence of suture density in the stress of the leaflet differs on the basis of different geometries considered in the model. The degree of influence of the suture density in the bioprosthetic heart valve may also be dependent on the geometries of the valves. This indicates the need to account for the attachment edge, when manufacturing such bioproshetic heart valves for long term durability. Further research is required to assess the effect of suture density on the bioprosthetic heart valve models.
This paper establishes parametric model of the biopresthetic heart valve via computer aided design. A series of accurate parameters of the biopresthetic heart valve, such as radius of the sutural ring, height of the supporting stent and inclination of the supporting stent are determined. The finite element analysis results show that the stress distribution of cylindrical and paraboloidal valve leaflets is quite different during the diastole period. The stress distribution of cylindrical and paraboloidal valve leaflets under the same load is analyzed as the leaflets are closed. Both the maximal primary stress of the two curved surface sorts appear on the bind area. Paraboloidal valve leaflets have the following advantages over cylindrical valves leaflets: the peak stress area of paraboloidal valve leaflets is comparatively far from seam position, the maximal primary stress of paraboloidal valve leaflets is lower than that of cylindrical valve leaflets. The unfolded algorithm on the paraboloidal valve leaflets is discussed in detail. The applied programs of the unfolded paraboloidal valve leaflets are designed based on finite element analysis. This work is very helpful to manufacture reasonable shaped valvular leaflets and to pro-lonq the lifetime of the bioprosthetic heart valve.
This paper analyzes the bioprosthetic heart valve's geometry design method and compares the stress distribution of the different shape leaflets based on the membrane theory and finite element analysis method in order to maximally reach the mechanics performance of human being's heart valve. According to both traditional design theories and modern design method, we choose the sphere and cylinder surfaces as valvular leaflets referenced frame and build the geometrical parametric model to analyze the stress distribution. After constructing parametric models of bioprosthetic heart valves via computer-aided design, a series of accurate dimension parameters are obtained to perform finite element analysis. The results indicate that force distribution of sphere valves leaflets is reasonable. The peak stress of sphere valve leaflets is comparatively far from seam position, and the maximal primary stress of sphere valve leaflets is less than that of cylindrical valves leaflets. It is also found that cylindrical valves leaflets have such obvious features as concentration of stress and non-uniform force distribution. Therefore, performance of sphere valves leaflets is superior to that of cylindrical valves leaflets. Meanwhile, it also indicates that the situation of stress distribution is not influenced obviously by the hyper-elasticity material of leaflets simplifying to imitative linear elastic material, but there is a certain tolerance. The present result provides a theoretical guide and useful information for the bioprosthetic heart valve designer.
Comparative study among aortic valves requires the use of an unbiased and relevant boundary condition. Pressure and flow boundary conditions used in literature are not sufficient for an unbiased analysis. We need a different boundary condition to analyze the valves in an unbiased, relevant environment. The proposed boundary condition is a combination of the pressure and flow boundary condition methods, which is chosen considering the demerits of the pressure and flow boundary conditions. In order to study the valve in its natural environment and to give a comparative analysis between different boundary conditions, a fluid-structure interaction analysis is made using the pressure and the proposed boundary conditions for a normal aortic valve. Commercial software LS-DYNA is used in all our analysis. The proposed boundary condition ensures a full opening of the valve with reduced valve regurgitation. It is found that for a very marginal raise in the ventricular pressure caused by pumping a fixed stroke volume, the cardiac output is considerably raised. The mechanics of the valve is similar between these two boundary conditions, however we observe that the importance of the root to raise the cardiac output may be overstated, considering the importance of the fully open nodule of arantius. Our proposed boundary condition delivers all the insights offered by the pressure and flow boundary conditions, along with providing an unbiased framework for the analysis of different valves and hence, more suitable for comparative analysis.
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In order to improve long-term durability of bioprosthetic heart valve, stress distribution of bioprosthetic heart valve leaflets with different shapes and suture density under the same load is analyzed and compared based on finite element method.The finite element analysis results are compared with each valve model, it shows that suture density has a significant effect on the dynamic behavior of the bioprosthetic heart valve, which may lead not only to different stress peak values, but also to different stress distributions and deformation. This work can be very helpful when manufacturing the bioprosthetic heart valve.
The paper constructs one type of bioprosthetic valve leaflets’ parametric model via computer aided design, a series of accurate parameters of the bioproshtetic heart valve, such as radius of the sutural ring, height of the supporting stent and inclination of the supporting stent, are determined. Numerical simulation is used to determine the effect of one shape design on the mechanical performance of the bioprosthetic valve leaflet. The stress with a shape under the same load is analysed and the thickness of the leaflets 0.5mm and 0.6mm is compared by us. We creat a ellipsoidal surface in accordance with geometrical features. The experimental results of the finite element analysis show that stress distribution of the same bioprosthetic heart valve leaflets with different thickness is different. The maximal primary stress with the thickness of 0.5mm is lower than the others. This work is very helpful to manufacture reasonable shaped valvular leaflets and to prolong the lifetime of the bioprosthetic heart valve.
The stress with different shapes under the same load is analysed and the thickness of the leaflets 0.2mm and 0.4mm is compared by us. We creat the spherical, paraboloidal and ellipsoidal curved surfaces in accordance with geometrical features. The experimental results of the finite element analysis show that stress distribution of the same bioprosthetic heart valve leaflets with different thickness is different. The maximal primary stress with the thickness of 0.4mm is lower than the others.With the same thickness,the stress of the ellipsoidal valve leaflets is lower. This work is very helpful to manufacture reasonable shaped valvular leaflets and to prolong the lifetime of the bioprosthetic heart valve.
This paper constructs the parametric model of the spherical heart valve via computer aided design, a series of accurate parameters of the bioprosthetic heart valve, such as the radius of the sutural ring, height of the supporting stent and inclination of the supporting stent are determined. The finite element method is used to determine the effect of the suture density on the dynamic properties of the bioprosthetic heart valve. The finite element analysis results show that the suture has a significant effect on the dynamic properties of the leaflets. The peak stress with different suture density is quite different and the stress distribution with higher suture density is more reasonable than that with lower suture density. In addition, the suture density has more effect at the top of the attachment edge than the other parts of the valve leaflets. This work is very helpful to manufacture the bioprosthetic heart valve with long term durability.
Current biological heart valves (BHVs) contain the major xenogeneic antigen Gal. Recipient anti-Gal antibody binding to such an implanted BHV may contribute to valve degeneration. The study aim was to compare, by implantation in non-human primates, the immune consequences of BHVs from Gal-positive wild-type (WT) pigs and those from alpha-galactosyltransferase knockout (GTKO) pigs.
Recipients were immunized prior to implant with keyhole limpet hemocyanin (KLH) conjugated to alphaGal to match the anti-Gal levels and isotypes found in humans. Stented glutaraldehyde-fixed BHVs from WT (n = 4) and GTKO (n = 3) pigs were commercially manufactured and implanted in the mitral position in non-human primates. Recipients were treated with enoxaparin (1 mg/kg b.i.d.) for five weeks which was tapered, and then discontinued. Serum antibody levels to Gal and KLH were measured using ELISA.
Overall anti-Gal and anti-KLH antibody levels were decreased in both WT and GTKO BHV recipients after implantation. Serum anti-Gal IgG levels in GTKO BHV recipients fell rapidly within one month, matching the loss of anti-KLH reactivity. There was no significant difference in retention of anti-KLH antibody between the groups. WT BHV recipients retained significantly elevated levels of anti-Gal IgG during the first year post implant. Area under the curve analysis showed that anti-Gal IgG was significantly increased in the WT BHV group compared to GTKO BHV recipients (p < 0.01).
Persistent and significantly (p < 0.01) elevated levels of anti-Gal IgG were observed in WT but not GTKO BHV non-human primate recipients, and indicated a continuing BHV-specific immune stimulation to the alphaGal antigen. These data support the hypothesis that the clinical use of Gal-positive xenogeneic bioprosthetic materials can induce an anti-Gal antibody response. Bioprosthetic devices prepared from GTKO pig tissue would eliminate immune stimulation to this major xenoreactive antigen, which may reduce the potential of immune-mediated injury and degeneration.
A modeling theory and technology of bioprosthetic heart valve is presented in this paper. Firstly, the geometric model of natural aortic valve is created according to its anatomy. Then, structure parameters described the geometric model are designed based on the shell theory. The equations are also presented to calculate the related parameters. In the end, a non-base artificial bioprosthetic aortic valve is designed and produced based on the theory and method proposed in this paper. Its perfect property has been confirmed by test results in vitro and clinical application in vivo.
In order to improve long-term durability of bioprosthetic heart valve, stress distribution of bioprosthetic heart valve leaflets with different shapes under the same load is analyzed and compared based on finite element method. Combining traditional design theories and modern design methods, we create the cylindrical, spherical, paraboloidal and ellipsoidal curved surfaces in accordance with the geometrical equations in the appropriate frame ordinal. Based on the stress analysis of two kinds of curved surfaces, we take turns to create relative inverse conic curved surfaces which satisfy the actual condition. Meanwhile, the space positions of boundary curves and important points are determined by the intersected curves and axis of revolution. Geometrical design and the finite element analysis could provide direct and useful information for the bioprosthetic heart valve designer. The experimental results of the finite element analysis reveal that stress distribution of different bioprosthetic heart valve leaflets is quite different in diastole time. Ellipsoidal valve leaflets have the following advantages over spherical, paraboloidal and cylindrical valves leaflets: one is that the peak stress area of ellipsoidal valve leaflets is comparatively far from seam position, the other is that the maximal primary stress of ellipsoidal valve leaflets is lower than that of spherical, paraboloidal and cylindrical valves leaflets. Therefore, mechanical properties of ellipsoidal valves leaflets are superior to those of spherical, paraboloidal and cylindrical valves leaflets.
In order to improve long-term durability of bioprosthetic heart valve, stress distribution of bioprosthetic heart valve leaflets with different shapes under the same load are analysed and empared based on finite element method. After constructing parametric models of bioprosthetic heart valves via computer aided design, a series of accurate dimension parameters are obtained. The experimental result of the finite element analysis reveal that stress distribution of different bioprosthetic heart valve leaflets is quite different in diastole time. Spherical valve leaflets have the following advantages over paraboloidal and cylindrical valves leaflets: one is that the peak stress area of spherical valve leaflets is comparatively far from seam position, the other is that the maximal primary stress of spherical valve leaflets is lower than that of paraboloidal and cylindrica valves leaflets. Therefore, mechanical properties of spherical valves leaflets are superior to those of paraboloid and cylindrical valves leaflets.
An approach is offered here to assist the pathologist with the actual dissection of the operated heart at autopsy. The challenge
to the pathologist is to define any underlying cardiac pathology, recognize changes in morphology brought about by prior surgical
intervention, and correlate the gross cardiac findings with the hemodynamic status of the deceased prior to death. Even without
detailed clinical records, one can still perform the dissection in a consistent and careful manner that allows an accurate
assessment of the heart regardless of postoperative adhesions and complex surgical reconstructions. Developing the plane of
dissection between the heart and adherent parietal pericardium should be done with a scalpel, using a methodical technique.
As one examines the external surface of the heart, one may observe clues that the surgeon had used cardiopulmonary bypass
to rest the heart during surgery and that specific surgical procedures had been performed. By removal of the coronary arteries
with any bypass grafts off the heart and subsequent careful examination of the chambers and great vessels of the heart, the
pathologist can then begin to understand the “real story.”
The finite element software is often applied to stress analysis, which is also crucial to the design of anti-fatigue and anti-calcification of artificial heart valve. The material properties and the boundary conditions of bioprosthetic valve leaflets are defined and geometrical models are established. The linear material and the non-linear material of leaflets with different shapes are analyzed. After valve leaflets with the finite element models have been loaded, the finite element analysis results can be got .It is showed that the maximal primary stress of the valve leaflets with elliptic sphere is lower than that of the other valve leaflets and the stress distribution of the valve leaflet with elliptic sphere is comparatively reasonable. The work is helpful to make optimization design for the bioprosthetic heart valve and prolongs the lifetime of the synthetic heart valve.
This paper establishes parametric model of the bioprosthetic heart valve via computer aided design. Dynamic finite element analysis is used along with a geometrically accurate model of the bioprosthetic heart valve. The stress distribution of cylindrical and paraboloidal valve leaflets under the same load is analyzed as the leaflets are closed. The finite element analysis results show that the stress distribution of cylindrical and paraboloidal valve leaflets is quite different during the diastole peroid. The stress concentration of two kind valves occurs not only at the free margin but also at the belly. Mechanical properties of paraboloidal valve leaflets are superior to those of cylindrical valves leaflets. Paraboloidal valve leaflets have the following advantages over cylindrical valves leaflets: lower peak von-Mises stress, smaller stress concentration area and range of high-stress region, relatively uniform stress distribution. The complex stress distribution can be effectively analyzed using a sophisticated dynamic finite element model. This work is very helpful to manufacture valvular leaflets with reasonable shapes and to prolong the lifetime of the bioprosthetic heart valve. 2010 IEEE.
This paper constructs three types of bioprosthetic valve leaflets' parametric model via computer aided design, a series of accurate parameters of the bioproshtetic heart valve, such as radius of the sutural ring, height of the supporting stent and inclination of the supporting stent, are determined. Numerical simulation is used to determine the effect of different shape designs on the mechanical performance of the bioprosthetic valve leaflet. The dynamic behavior of the valve during diastolic phase is analyzed. The finite element analysis results show the stress distribution of the ellipsoidal and spherical valve leaflets are comparatively reasonable. The ellipsoidal and spherical valve leaflets have the following advantages over the cylindrical leaflet valve, lower peak von-Mises stress, smaller stress concentration area, and relatively uniform stress distribution. The ellipsoidal and spherical valve leaflets may contribute to the long term durability of the valve. This work is very helpful to manufacture valvular leaflets with reasonable shapes and to prolong the lifetime of the bioprosthetic heart valve. (2011) Trans Tech Publications.
This paper constructs the bioprosthetic valve leaflets' parametric model via computer aided design, a series of accurate parameters of the bioproshtetic heart valve, such as such as radius of the sutural ring, height of the supporting stent and inclination of the supporting stent, are determined. Numerical simulation is used to determine the effect of different shape designs and attachment edge fixed ways on the mechanical performance of the bioprosthetic valve leaflet. The dynamic behavior of the valve during diastolic phase is analyzed. The finite element analysis results show that the stress distribution of the ellipsoidal leaflet valve is comparatively reasonable. The ellipsoidal leaflet valve has the following advantages over the cylindrical leaflet valve, lower peak von Mises-stress, smaller stress concentration area, and relatively uniform stress distribution. The different ways of the attachment edge fixed also have a significant effect on the dynamic behavior of the valve. Attachment edge with some degrees of rotation when seamed to the stent can act to reduce the pressure and make the stress distribution reasonable. Reasonable attachment edge fixation may contribute to the long term durability of the valve. This indicates the need to account for the attachment edge seamed way, when manufacturing the bioproshetic heart valve with long term durability. This work is very helpful to manufacture valvular leaflets with reasonable shapes and to prolong the lifetime of the bioprosthetic heart valve. (2012) Trans Tech Publications, Switzerland.
This paper constructs four types of bioprosthetic heart valve's parametric model via computer aided design, a series of accurate parameters of the bioprosthetic heart valve, such as radius of the sutural ring, height of the supporting stent and inclination of the supporting stent are determined. The finite element method is used to analyze the mechanical properties of the bioprosthetic heart valve in which geometric non-linearity and material non-linearity are all taken into account. The finite element analysis results show that the shape of the bioprosthetic has a significant effect on the mechanical performance of the valve. The stress distribution of ellipsoidal valve leaflets is comparatively reasonable. It has lower peak von-Mises, smaller stress concentration area than the other three types of valve leaflets. This work is very helpful to manufacture valvular leaflets with reasonable shapes and to prolong the lifetime of the bioprosthetic heart valve. (2012) Trans Tech Publications, Switzerland.
Decellularized tissues have been successfully used in a variety of tissue engineering/regenerative medicine applications, and more recently decellularized organs have been prepared utilized in the first stages of organ engineering. The protocols used to decellularize simple tissues versus intact organs differ greatly. Herein, the most commonly used decellularization methods for both surgical mesh materials and whole organs are described, with consideration given to how these different processes affect the extracellular matrix and the host response to that scaffold. J. Cell. Biochem. (c) 2012 Wiley Periodicals, Inc.
Glutaraldehyde crosslinked bioprosthetic heart valves (BHVs) have two modalities of failure: degeneration (cuspal tear due to matrix failure) and calcification. They can occur independently as well as one can lead to the other causing co-existence. Calcific failure has been extensively studied before and several anti-calcification treatments have been developed; however, little research is directed to understand mechanisms of valvular degeneration. One of the shortcomings of glutaraldehyde fixation is its inability to stabilize all extracellular matrix components in the tissue. Previous studies from our lab have demonstrated that neomycin could be used as a fixative to stabilize glycosaminoglycans (GAGs) present in the valve to improve matrix properties. But neomycin fixation did not prevent cuspal calcification. In the present study, we wanted to enhance the anti-calcification potential of neomycin fixed valves by pre-treating with ethanol or removing the free aldehydes by sodium borohydride treatment. Ethanol treatment has been previously used and found to have excellent anti-calcification properties for valve cusps. Results demonstrated in this study suggest that neomycin followed by ethanol treatment effectively preserves GAGs both in vitro as well as in vivo after subdermal implantation in rats. In vivo calcification was inhibited in neomycin fixed cusps pretreated with ethanol compared to glutaraldehyde (GLUT) control. Sodium borohydride treatment by itself did not inhibit calcification nor stabilized GAGs against enzymatic degradation. Neomycin fixation followed by ethanol treatment of BHVs could prevent both modalities of failure, thereby increasing the effective durability and lifetime of these bioprostheses several fold.
Valvular heart disease is an important cause of morbidity and mortality, and currently available substitutes for failing hearts have serious limitations. A new promising alternative that may overcome these shortcomings is provided by the relatively new field of tissue engineering (TE). TE techniques involve the growth of autologous cells on a 3D matrix that can be a biodegradable polymer scaffold, or an acellular tissue matrix. These approaches provide the potential to create living matrix valve structures with an ability to grow, repair and remodel within the recipient. This article provides an appraisal of artificial heart valves and an overview of developments in TE that includes the current limitations and challenges for creating a fully functional valve. Biomaterials and stem cell technologies are now providing the potential for new avenues of research and if combined with advances in the rapid prototyping of biomaterials, the engineering of personalized, fully functional, and autologous tissue valve replacements, may become a clinical alternative.
This work was concerned with the numerical simulation of the behaviour of aortic valves whose material can be modelled as non-linear elastic anisotropic. Linear elastic models for the valve leaflets with parameters used in previous studies were compared with hyperelastic models, incorporating leaflet anisotropy with pronounced stiffness in the circumferential direction through a transverse isotropic model. The parameters for the hyperelastic models were obtained from fits to results of orthogonal uniaxial tensile tests on porcine aortic valve leaflets. The computational results indicated the significant impact of transverse isotropy and hyperelastic effects on leaflet mechanics; in particular, increased coaptation with peak values of stress and strain in the elastic limit. The alignment of maximum principal stresses in all models follows approximately the coarse collagen fibre distribution found in aortic valve leaflets. The non-linear elastic leaflets also demonstrated more evenly distributed stress and strain which appears relevant to long-term scaffold stability and mechanotransduction.
Investigation of the properties of Ca²⁺-ATPase of sarcoplasmic reticulum cross-linked at the active site with glutaraldehyde showed that ATP binding affinity and rate of ATP-dependent phosphorylation and Ca²⁺ occlusion were decreased 2-3 orders of magnitude compared with the native enzyme. Cross-linkage had little effect on or marginally increased the rate of acetyl phosphate- and p-nitrophenyl phosphate-supported Ca²⁺ occlusion. Ca²⁺ binding or Ca²⁺-induced changes in tryptophan fluorescence were unaffected. High levels of phosphoenzyme (up to 4 nmol/mg of protein) were obtained, with 2 mol of Ca²⁺ occluded/mol of E-P. Dephosphorylation and deocclusion occurred together at a slow rate (k = 0.01 s⁻¹) and were stimulated in a monophasic manner up to 20-fold by ADP. Cross-linking inhibited E2-P formation from Pi in 30% (v/v) dimethyl sulfoxide by more than 95%. Induction of turnover of the native ATPase, under conditions designed to yield high steady state levels of E1 ∼ P(2Ca), results in a 3-4-fold increase in reactivity of active site residues to glutaraldehyde. The results show that cross-linkage sterically impairs nucleotide binding, changing ATP and ADP into relatively poor substrates, slowing nucleotide-dependent phosphoryl transfer and Ca²⁺ occlusion and deocclusion. The forward reaction with smaller substrates is unaffected. Another major effect of the cross-link is to inhibit E2-P formation, causing accumulation of E1 ∼ P(2Ca) during enzyme turnover and preventing phosphorylation by Pi in the reverse direction. We suggest that occlusion and deocclusion of cations at the transport site of the native enzyme are linked to a two-step cleft closure movement at the active site and that the crosslink stabilizes occluded forms of the pump because it blocks part of this tertiary structural change. The latter could normally be propagated through linking helices to the distal side of the pump to destabilize the cations and open the transport sites to the lumen.
We previously have been able to show that fixation at increasing concentrations of glutaraldehyde (GA) leads to mitigated rather than facilitated tissue calcification. The purpose of the present study was to introduce additional crosslinks and provide evidence that crosslink density may be an underlying inhibitory principle. Entire aortic roots were chosen to verify the concept on the challenging aortic wall tissue. Porcine aortic roots were crosslinked with 0.2% GA, 3%GA, and 3% GA containing an interim step that introduced diamine bridges. Crosslink efficiency was determined on the basis of shrinkage temperature (SrT°), resistance to protease digestion (RPD), residual amine analysis (RA), and tensile modulus (E10). Calcium levels, calcification patterns, and inflammation were assessed after 6 and 24 weeks of implantation in a sheep circulatory model. Crosslink efficiency in aortic wall tissue was moderately affected by increasing the fixative concentration from 0.2% GA to 3% GA (SrT° from 85.7° ± 0.3° to 87.5° ± 0.3°C, p < 0.002; RPD from 24.2 ± 1.2 to 29.1 ± 0.7%, p < 0.003; RA from 0.069 ± 0.004 to 0.058 ± 0.003 μmol/mg, p < 0.03, and E10 from 1.9 ± 0.11 to 2.94 ± 0.34 MPa, p < 0.01), but it was distinctly enhanced when diamine bridges were introduced (SrT° from 87.5° ± 0.3° to 93.4° ± 0.3°C, p << 0.0001; RPD from 29.1 ± 0.7 to 68.4 ± 1.8%, p << 0.0001; and E10 from 2.94 ± 0.34 to 6.80 ± 0.61 MPa, p < 0.0003). Aortic wall calcification was reduced significantly by increasing the GA concentration from 0.2 to 3% [37.8%, p = 0.076 (6 weeks) and 34.0%, p = 0.008 (24 weeks)] and further reduced by the introduction of additional diamine [84.0%, p = 0.006 (6 weeks) and 29.8%, p = 0.037 (24 weeks)]. The combined effect of increased GA concentration plus an interim diamine step on aortic wall tissue resulted in a 90% and 53.7% reduction of calcification after 6 weeks and 24 weeks, respectively. The correlation coefficients between calcification and SrT°, RDP, and E10 was −0.9767, −0.9460, and −0.9740, respectively (6 weeks). The inflammatory host reaction regularly found in 0.2% fixed tissue was practically abolished through the introduction of diamine bridges. Our study demonstrated a distinct correlation between the mitigation of aortic wall calcification and three parameters used to assess crosslink density. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res 56: 56–64, 2001
Intramolecular crosslinking of the active site of the sarcoplasmic reticulum Ca(2+)-ATPase with glutaraldehyde results in substantial inhibition of ATPase activity and stabilization of the ADP-sensitive E1 approximately P(2Ca) intermediate (E, enzyme) with occluded Ca2+ [Ross, D. C., Davidson, G. A. & McIntosh, D. B. (1991) J. Biol. Chem. 266, 4613-4621]. We show here, using conditions of low passive vesicle permeability and absence of ADP, that Ca2+ "deoccludes" more rapidly than it leaks out of the vesicle lumen. Deocclusion is paralleled by dephosphorylation. Therefore, turnover of crosslinked E1 approximately P(Ca) (approximately 5 nmol/min per mg of protein at 25 degrees C) involves Ca2+ release to the vesicle exterior and concomitant phosphoenzyme hydrolysis. Ca2+ release to the lumen, the normal pathway, is apparently blocked completely. In the presence of ADP, Ca2+ is also released to the vesicle exterior, and this release is coupled to the synthesis of ATP. The results suggest that a tertiary structural change at the active site follows phosphorylation and is an absolute requirement for Ca2+ release from the native enzyme to the vesicle lumen.
The human tissue nonspecific alkaline phosphatase (TNAP) is found in liver, kidney, and bone. Mutations in the TNAP gene can lead to Hypophosphatasia, a rare inborn disease that is characterized by defective bone mineralization. TNAP is 74% homologous to human placental alkaline phosphatase (PLAP) whose crystal structure has been recently determined at atomic resolution (Le Du, M. H., Stigbrand, T., Taussig, M. J., Ménez, A., and Stura, E. A. (2001) J. Biol. Chem, 276, 9158-9165). The degree of homology allowed us to build a reliable TNAP model to investigate the relationship between mutations associated with hypophosphatasia and their probable consequences on the activity or the structure of the enzyme. The mutations are clustered within five crucial regions, namely the active site and its vicinity, the active site valley, the homodimer interface, the crown domain, and the metal-binding site. The crown domain and the metal-binding domain are mammalian-specific and were observed for the first time in the PLAP structure. The crown domain contains a collagen binding loop. A synchrotron radiation x-ray fluorescence study confirms that the metal in the metal-binding site is a calcium ion. Several severe mutations in TNAP occur around this calcium site, suggesting that calcium may be of critical importance for the TNAP function. The presence of this extra metal-binding site gives new insights on the controversial role observed for calcium.
Glutaraldehyde-tanned bovine pericardium was tested for its ability to induce immunologic responses in vivo. Sections of glutaraldehyde-tanned bovine pericardium were implanted between the abdominal muscles of rats and guinea pigs. Control animals received Dacron implants. Lymphocytes and sera from animals were isolated at 2 and 4 weeks after implantation (four animals per group per time). Tritiated thymidine incorporation and an enzyme-linked immunosorbent assay were used to measure T- and B-lymphocyte responses to glutaraldehyde-tanned bovine pericardium antigens. At the same time points, implants and surrounding tissue from all animals were processed for histologic data. Results show that T-lymphocytes from animals with glutaraldehyde-tanned bovine pericardium implants responded significantly (p < 0.001) to glutaraldehyde-tanned bovine pericardium antigens in vitro but not to Dacron. In contrast, lymphocytes from animals with Dacron implants failed to respond to glutaraldehyde-tanned bovine pericardium or Dacron preparations. Results of enzyme-linked immunosorbent assay show that animals with glutaraldehyde-tanned bovine pericardium implants produced antibody directed against glutaraldehyde-tanned bovine pericardium antigens. Histologic study revealed a dense mononuclear and multinuclear giant cell infiltrate at the interface between glutaraldehyde-tanned bovine pericardium and surrounding host tissues, with focal degradation of implant collagen. Dacron elicited a nonspecific lymphocytic and foreign body-type reaction. These results indicate that glutaraldehyde-tanned bovine pericardium can induce immunologic responses in vivo consistent with a host-versus-graft reaction.
Blood flow in arteries is dominated by unsteady flow phenomena. The
cardiovascular system is an internal flow loop with multiple branches in
which a complex liquid circulates. A nondimensional frequency parameter,
the Womersley number, governs the relationship between the unsteady and
viscous forces. Normal arterial flow is laminar with secondary flows
generated at curves and branches. The arteries are living organs that
can adapt to and change with the varying hemodynamic conditions. In
certain circumstances, unusual hemodynamic conditions create an abnormal
biological response. Velocity profile skewing can create pockets in
which the direction of the wall shear stress oscillates. Atherosclerotic
disease tends to be localized in these sites and results in a narrowing
of the artery lumena stenosis. The stenosis can cause turbulence and
reduce flow by means of viscous head losses and flow choking. Very high
shear stresses near the throat of the stenosis can activate platelets
and thereby induce thrombosis, which can totally block blood flow to the
heart or brain. Detection and quantification of stenosis serve as the
basis for surgical intervention. In the future, the study of arterial
blood flow will lead to the prediction of individual hemodynamic flows
in any patient, the development of diagnostic tools to quantify disease,
and the design of devices that mimic or alter blood flow. This field is
rich with challenging problems in fluid mechanics involving
three-dimensional, pulsatile flows at the edge of turbulence.
Collagens of most connective tissues are subject to continuous remodelling and turnover, a phenomenon which occurs under both
physiological and pathological conditions. Degradation of these proteins involves participation of a variety of proteolytic
enzymes including members of the following proteinase classes: matrix metalloproteinases (e.g. collagenase, gelatinase and
stromelysin), cysteine proteinases (e.g. cathepsin B and L) and serine proteinases (e.g. plasmin and plasminogen activator).
Convincing evidence is available indicating a pivotal role for matrix metalloproteinases, in particular collagenase, in the
degradation of collagen under conditions of rapid remodelling, e.g. inflammation and involution of the uterus. Under steady
state conditions, such as during turnover of soft connective tissues, involvement of collagenase has yet to be demonstrated.
Under these circumstances collagen degradation is likely to take place particularly within the lysosomal apparatus after phagocytosis
of the fibrils. We propose that this process involves the following steps: (i) recognition of the fibril by membranebound
receptors (integrins?), (ii) segregation of the fibril, (iii) partial digestion of the fibril and/or its surrounding noncollagenous
proteins by matrix metalloproteinases (possibly gelatinase), and finally (iv) lysosomal digestion by cysteine proteinases,
such as cathepsin B and/or L. Modulation of this pathway is carried out under the influence of growth factors and cytokines,
including transforming growth factor β and interleukin 1α.
Background:
Glutaraldehyde has been said to be responsible in part for the calcification of glutaraldehyde-treated tissues after implantation in animals or humans. We investigated whether the origin of the tissue, autologous or heterologous, could have a more prominent role in the process of calcification.
Methods:
Three-month-old sheep received sheep pericardial samples (n = 133) and human pericardial samples (n = 123) implanted subcutaneously. Samples were treated with 0.6% glutaraldehyde for 5, 10, or 20 minutes or 7 days and then rinsed thoroughly before implantation. Samples were then retrieved after 3 months. Calcium content was assessed by spectrophometry.
Results:
The results show a low calcium content in the autologous group (mean 1.14+/-2.07) and a high calcium content in the heterologous group (mean 38.97+/-26). These results were the same regardless of the duration of the treatment.
Conclusions:
Glutaraldehyde treatment (0.6%) does not play a significant role in the calcification of glutaraldehyde-treated tissue regardless of the origin, autologous or heterologous, of the tissue. Glutaraldehyde-treated autologous tissues are associated with an incidence of calcification lower than heterologous tissues.
Morphologic changes in six glutaraldehyde-fixed porcine heterografts recovered from five patients were studied with light and scanning electron microscopy. Light microscopy showed minimal changes in the valves recovered after 2 months of implantation. Fibrin deposits, red blood cell trapping, disruption of the valve matrix and interstitial edema were noted in heterografts 2 months and 2 years after implantation. In valves examined with scanning electron microscopy, 6 hours after implantation the surface changes varied from normal to patchy disruptions. Two months after inplantation, subendothelial fibers were conspicuous and the geometry of endothelial cells was changed. In valves examined after 6 months in place, many nuclei were burst and fibrin strands were seen over the surfaces. Bacterial infection was observed in two cases. The endothelial cells in the valve implanted for 2 years were completely obscured and some areas were densely covered with fibrillar material where blood cells were trapped in the mesh.
Scanning and transmission electron microscopic studies were made of (1) 12 glutaraldehyde-treated porcine valvular heterografts that had been implanted in patients for 2 days to 76 months; (2) 3 unimplanted commercially processed porcine aortic valves; and (3) 1 unprocessed porcine aortic valve. Comparison of unprocessed porcine valves and unimplanted commercially processed valves showed loss of endothelium and acid mucopolysaccharides during preimplantation processing. Short-term (less than 2 months) changes after implantation consisted of insudation of plasma proteins, penetration of erythrocytes into surface crevices, formation of a thin surface layer of fibrin, and deposition of macrophages, giant cells and a few platelets. Longer-term (more than 2 months) changes were proportional to the time interval after implantation and consisted of progressive disruption of collagen, erosion of the valve surfaces, formation of aggregates of platelets and accumulation of lipid. The surfaces of the leaflets did not become covered with endothelium or with a fibrous sheath. Calcific deposits were found in one valve and bacterial organisms in another. Thus, progressive breakdown of collagen appears to be a critical factor in determining the long-term durability of glutaraldehyde-treated porcine valvular heterografts.
The mechanism of opening of the aortic valve was investigated in dogs by attaching radiopaque markers to the commissures and the leaflets. Analysis of abnormal cardiac cycles demonstrated that, when the ventricular pressure first equalled the aortic pressure, the intercomissural distances increased 9 percent, and the valve opened with a stellate orifice without forward flow and without a rise in aortic pressure. Further opening of the aortic valve was dependent on forward flow over a narrow range. A new mechanism of aortic valve opening is proposed. This mechanism results in minimal flexion stresses on the leaflets and is important for the longevity of the normal aortic valve. It can occur only if the leaflets arise from an expansile aortic root.
Degenerative alterations of two different glutaraldehyde (GA)-fixed bioprosthetic heart valve materials were investigated in subcutaneous rat implants: Bovine pericardium, prepared according to clinically used bioprosthetic heart valve material (BHV) was compared to alternatively preserved pericardium (APHV), which was fixed in GA and treated with L-glutamic acid. Following 63 days of subcutaneous implantation, calcification of APHV implants was significantly lower as compared to BHV implants (13 +/- 6 versus 158 +/- 18 micrograms Ca/mg dry weight tissue; p less than 0.05). In BHV implants ultrastructural investigations showed nucleation of plate-shaped hydroxyapatite crystals at the surface of collagen fibrils and in remnants of connective tissue cells; no signs of calcification could be detected in APHV implants. The time-course of the inflammatory reaction was determined by quantification of immunohistochemical stained mononuclear host-cells invading the implants. In both preparation groups inflammatory reaction reached maximum 42 days after implantation. However, infiltration rate of inflammatory cells was markedly decreased in APHVs as compared to BHVs (p less than 0.05).
A marked release of glutaraldehyde from commercially available pericardial bioprosthetic heart valve (BHV) material in washing solutions was found by high performance liquid chromatography (up to 1.8 ppm of glutaraldehyde per gram of dry tissue). In vitro endothelial cell proliferation rate was impaired dose-dependently in the presence of increasing glutaraldehyde concentrations of the cultivation medium (r = 0.9; p less than 0.05). Cultivation of endothelial cells was impossible on the surface of commercially available BHV material, but successful and uninhibited when toxic glutaraldehyde ligands of the BHV material were antagonized by treatment with L-glutamic acid.
The elastic response of aortic valve cusps is a summation of its fibrous components. To investigate the micromechanical function of valve leaflet constituents, we separated the fibrosa and the ventricularis from fresh and glutaraldehyde-fixed leaflets and tested them individually. The ventricularis was stiffer circumferentially than radially (7.41 kPa vs 3.68 kPa, p less than 0.00001) and was more extensible radially (62.7% vs 21.8% strain to high modulus phase, p less than 0.00001). The fibrosa was also stiffer circumferentially than radially (13.02 kPa vs 4.65 kPa, p less than 0.0008), but had uniform extensibility. Glutaraldehyde fixation did not affect the circumferential elastic modulus of the fibrosa, but reduced its radial modulus from 4.65 kPa to 2.32 kPa (p less than 0.0078). The elastic modulus of the ventricularis remained unchanged. Fixation also reduced the extensibility of the ventricularis circumferentially (from 21.8% to 15.2% strain, p less than 0.018), but not radially, and increased the radial extensibility of the fibrosa from 27.7% to 46.1% (p less than 0.0048). These data show that while the ventricularis contains a large amount of elastin, the amount of radially oriented collagen is similar to that of the fibrosa. The fibrosa, by itself, has the same extensibility in both directions (about 23% strain), but can extend much more radially when connected to the rest of the leaflet because it is attached to the ventricularis in a highly folded configuration. The two layers therefore complement each other during aortic valve function, and become detrimentally altered by fixation in glutaraldehyde.
Investigation of the properties of Ca2+-ATPase of sarcoplasmic reticulum cross-linked at the active site with glutaraldehyde showed that ATP binding affinity and rate of ATP-dependent phosphorylation and Ca2+ occlusion were decreased 2-3 orders of magnitude compared with the native enzyme. Cross-linkage had little effect on or marginally increased the rate of acetyl phosphate- and p-nitrophenyl phosphate-supported Ca2+ occlusion. Ca2+ binding or Ca2+-induced changes in tryptophan fluorescence were unaffected. High levels of phosphoenzyme (up to 4 nmol/mg of protein) were obtained, with 2 mol of Ca2+ occluded/ mol of E-P. Dephosphorylation and deocclusion occurred together at a slow rate (k = 0.01 s-1) and were stimulated in a monophasic manner up to 20-fold by ADP. Cross-linking inhibited E2-P formation from Pi in 30% (v/v) dimethyl sulfoxide by more than 95%. Induction of turnover of the native ATPase, under conditions designed to yield high steady state levels of E1 ∼ P(2Ca), results in a 3-4-fold increase in reactivity of active site residues to glutaraldehyde. The results show that cross-linkage sterically impairs nucleotide binding, changing ATP and ADP into relatively poor substrates, slowing nucleotide-dependent phosphoryl transfer and Ca2+ occlusion and deocclusion. The forward reaction with smaller substrates is unaffected. Another major effect of the cross-link is to inhibit E2-P formation, causing accumulation of E1 ∼ P(2Ca) during enzyme turnover and preventing phosphorylation by Pi in the reverse direction. We suggest that occlusion and deocclusion of cations at the transport site of the native enzyme are linked to a two-step cleft closure movement at the active site and that the cross-link stabilizes occluded forms of the pump because it blocks part of this tertiary structural change. The latter could normally be propagated through linking helices to the distal side of the pump to destabilize the cations and open the transport sites to the lumen.
The principal cause of the clinical failure of bioprosthetic heart valves fabricated from glutaraldehyde-pretreated porcine aortic valves is calcification. Other prostheses composed of tissue-derived and polymeric biomaterials also are complicated by deposition of mineral. In this study the authors focused on the relationship of AP to early events in calcification, and the inhibition of both calcification and AP activity by FeCl3 and AlCl3 preincubations. The results of these studies are as follows: (a) Extractable AP activity is present in fresh but not glutaraldehyde-pretreated bovine pericardial tissue. (b) Extrinsic AP is rapidly adsorbed following implantation, with peak activity at 72 h (424 ± 67.2 nm pnpp/mg protein/min enzyme activity [units]), but markedly lesser amounts at 21 days (96.8 ± 3.9 units). (c) Simultaneously to the AP activity maximum, bulk calcification is initiated, with GPBP calcium levels rising from 1.2 ± 0.1.
Following explanation from the mitral position because of primary tissue failure, 30 human antibiotic sterilised stent mounted aortic valve allografts and 28 glutaraldehyde treated porcine xenografts were examined for evidence of tissue detachment from the stents. These grafts had been in situ for 34 to 166 months. Graft detachment had occurred from one or two stent posts with displacement of the commissures and central valvular incompetence in 12 (67%) of 18 allografts supported on rigid stainless steel stents, in six (50%) of 12 allografts mounted on flexible acetal copolymer stents, but in only one (4%) of 28 xenografts mounted on polypropylene stents. In regions of detachment the aortic remnant of the graft was infiltrated by components of blood and phagocytic cells which had removed not only fibrin but also graft tissue. Detachment did not occur from stent posts where the graft margin had become coated by collagenous host tissue. This intimal fibrous sheath appeared not only to strengthen the attachment of the graft but also to limit the entry of fibrin and phagocytes into the graft tissue.
The microstructure of 33 spontaneously degenerated porcine bioprosthetic valves was assessed by scanning electron microscopy in order to gather insight regarding the degenerative process. Twenty-four mitral and 9 aortic valves were removed from 32 patients. The duration of insertion was 7.7 +/- 2.4 years (mean +/- SD). All valves showed denudation of endothelial cells and exposure of the subendothelial fibrous tissue. Fibroblastlike cells were occasionally seen. Platelet deposits were observed on 22 of 33 valves (67%). Leukocytes were observed on the surface of 27 of 33 valves (82%). Mononuclear leukocytes were the most common category of cells (66%). Crystalline material was present on the surface of some leukocytes, suggesting that they may serve as a nidus for calcification. Transmission electron microscopy showed leukocytes in the process of phagocytizing collagen fibers. Macrophages, by exerting their scavenger function seem to contribute to destruction of the collagen framework of the valves. Whether the observed lymphocytes and plasma cells reflect an immunological involvement is unclear.
Glutaraldehyde crosslinking of native or reconstituted collagen fibrils and tissues rich in collagen significantly reduces biodegradation. Other aldehydes are less efficient than glutaraldehyde in generating chemically, biologically, and thermally stable crosslinks. Tissues crosslinked with glutaraldehyde retain many of the viscoelastic properties of the native collagen fibrillar network which render them suitable for bioprostheses. Implants of collagenous materials crosslinked with glutaraldehyde are subject long-term to calcification, biodegradation, and low-grade immune reactions. We have attempted to overcome these problems by enhancing crosslinking through bridging of activated carboxyl groups with diamines and using glutaraldehyde to crosslink the epsilon-NH2 groups in collagen and the unreacted amines introduced by aliphatic diamines. This crosslinking reduces tissue degradation and nearly eliminates humoral antibody induction. Covalent binding of diphosphonates, specifically 3-amino-1-hydroxypropane-1, 1-diphosphonic acid (3-APD), and chondroitin sulfate to collagen or to the crosslink-enhanced collagen network reduces its potential for calcification. Platelet aggregation is also reduced by glutaraldehyde crosslinking and nearly eliminated by the covalent binding of chondroitin sulfate to collagen. The cytotoxicity of residual glutaraldehyde--leaching through the interstices of the collagen fibrils or the tissue matrix--and of reactive aldehydes associated with the bound polymeric glutaraldehyde can be minimized by neutralization and thorough rinsing after crosslinking and storage in a nontoxic bacteriostatic solution.
A noncalcifying drug, aminodiphosphonate, was immobilized on pericardial collagen to inhibit calcification. The parameters of ADP binding were optimized by using labeled tracer. The effects of the detergents, Triton X-100 and sodium dodecyl sulfate, pH, and temperature on ADP binding to collagen in fresh pericardium, and stabilization of ADP-collagen bond by borohydride (BH) reduction, were optimized. These studies indicate that pretreatment of pericardium with SDS and TX-100 increases ADP binding three- to fivefold. Three-step cross-linking via Schiff-base reactions between collagen, ADP, and cross-linking via Schiff-base reactions between collage, ADP, and glutaraldehyde gives a high value of 30 to 35 molecules of ADP per collagen in pericardium. Twenty-five millimeter tissue valves were made with detergent (1% SDS, 1% TX-100) treated ADP-bound pericardium and implanted in the mitral annuli in calves, which were killed at 60 days post implantation. Regional calcium deposition was measured with atomic absorption spectrometry. The calcium level (micrograms/mg of tissue) in components of control and four processed valves were determined. Regional platelet deposition on zones of leaflets and components of tissue valves were quantified with 111In-labeled autologous platelets. The calcium levels in thrombus and flexion zones of treated valves are lower than that in the control valve. SEM studies of leaflet surfaces at 60 days indicate that these treatment processes reduce calcification and promote spontaneous cell coverage on leaflets around the smooth surface of the outflow tract.
With 111In-labeled platelets and 125I-labeled bovine fibrinogen, regional mapping of platelet and fibrinogen deposition on leaflets and sewing rings was obtained. Ten Holstein calves received 25-mm mitral valves (ISLM) and were killed 1, 14, and 30 days after implantation. Twenty-four hours before the calves were killed, 350 to 450 microCi of 111In-labeled platelets and 200 to 250 microCi of 125I-labeled bovine fibrinogen were administered intravenously. The components of the tissue valves, i.e., three leaflets and sewing rings, were separated. Each leaflet was cut into four sections: free edge, central zone, flexion zone, and attachment zone. From the radioactivity in blood, leaflet zones, sewing rings, area of leaflet zones, platelet count, and fibrinogen level in blood, the mean regional density of adherent platelets, fibrinogen-fibrin, and fibrinogen/platelet were calculated. The density of platelets and fibrinogen deposited on the components of the valves decreases with time postimplantation. The number of fibrinogen molecules per platelet is fivefold to 20-fold higher than that of the receptor concentration on platelets on leaflet zones, suggesting the heterogeneity of fibrinogen-fibrin in thrombus and components of the valve.
From August 1977 to June 1981, 221 patients received a Hancock porcine valve and 133 an Ionescu-Shiley bovine (I-S) pericardial valve as aortic valve substitutes. No special selection or randomization was used and no patient with either of these types of valves was excluded. Preoperative data show no differences between the groups influencing the appearance of primary tissue failure. Hospital survivors were followed until June 1984 and those with an uneventful history at least 36 months. Patients who died late postoperatively or who underwent reoperation for causes other than primary dysfunction were considered at risk until death or reoperation. Primary tissue failure occurred in 8 patients in the I-S group from 36 to 70 months postoperatively and in 6 patients of the Hancock group from 24 to 83 months. Linearized rates of primary failure were 0.61 valves per 100 patient-years for the Hancock and 1.70 valves per 100 patient-years for the I-S group. Mean age of patients with failing valves was 38 years (range 25 to 55) for Hancock valves and 39 years (range 15 to 62) for I-S valves. Actuarial analysis shows a lower rate of primary dysfunction in the Hancock group since the fourth year, which is statistically significant in the sixth and seventh years (96.5 +/- 1.5% vs 79.6 +/- 7.6% in the sixth year and 93.1 +/- 3.6 vs 79.6 +/- 7.6% in the seventh year). Microscopically, calcium and collagen degeneration were consistently associated and present on failing bioprostheses.(ABSTRACT TRUNCATED AT 250 WORDS)
The details of heart valve prosthesis-associated problems are not widely known. This study investigated the etiologies of the failures of 91 valves, 33 mechanical prostheses and 58 bioprostheses, obtained at reoperation (83) or autopsy (eight) at the Brigham and Women's Hospital during the 42-month period from mid- 1980 through 1983, one to 264 months (mean, 72 months) after valve replacement. Analysis was by gross, histologic, radiographic, and microbiologic examination, as well as review of clinical records. Overall causes of failure included paravalvular leak (15 per cent), thrombosis (7 per cent), tissue overgrowth (8 per cent), degeneration or mechanical failure (43 per cent), and endocarditis (19 per cent). Endocarditis and paravalvular leak were equally frequent with mechanical prostheses and bioprostheses. In addition, thrombosis (18 per cent), tissue overgrowth (21 per cent), and structural failure (12 per cent) were all important failure modes for mechanical prostheses. Sterile degeneration was the overwhelming cause of failure for bioprostheses, accounting for the failure of 35 of 58 (60 per cent) of those recovered. Sterile degeneration took several forms: calcification, with or without cuspal tears (27 cases, 47 per cent of bioprostheses; mean, 77 months, range, 44 to 108 months) and cuspal defects without calcification (eight cases, 14 per cent; mean, 59 months, range, eight to 122 months). In general, calcification increased with time after implantation, but the propensity for the mineralization of bioprostheses varied widely among patients. Four torn valves that had been in place for more than six years had radiographically undetectable calcific deposits. The results of this study indicate that paravalvular leak and endocarditis are frequent causes of failure for all valve types. No clear failure mode predominates with mechanical valve prostheses, although some designs have specific inherent limitations. In contrast, degeneration, especially that related to mineralization, is the most important cause of the late failure of contemporary bioprostheses.
The mechanism of calcification of glutaraldehyde-preserved porcine aortic valves (GPV) is incompletely understood, but pretreatment of the valve with the protein crosslinking reagent glutaraldehyde appears to be essential. Experimental bioprosthetic heart valve calcification has been studied with subcutaneous implantations of the cusps in young rats. Calcified subcutaneous retrievals are virtually identical to clinically recovered bioprostheses both pathologically and biochemically. Since collagen calcification is an important feature of bioprosthesis mineralization, the study reported here was undertaken to study the fate of rat subcutaneous implants of purified collagen, both untreated and pretreated with either glutaraldehyde or formaldehyde.
This investigation used light microscopy and transmission and scanning electron microscopy to study native human and bovine parietal pericardium, glutaraldehyde-fixed bovine pericardial patch-grafts, and bovine pericardial cusps of unimplanted Incor or Puig - Zerbini heart valves. The primary objective was to obtain a data base for the future evaluation of postimplantation structural alteration in this valve and in other cardiac valvular bioprostheses constructed of pericardium. The mesothelial cell layer in normal pericardium was best preserved in the bovine tissue. In both bovine patch-grafts and Puig - Zerbini valvular cusps, the serosal surface usually was completely devoid of mesothelial cells and revealed an underlying, finely fibrillar basal lamina. The fibrosa varied in thickness and organization, both within and between the two species, but similar nervous, vascular, and connective tissue components were observed in each. The epipericardial surface was smoother, had fewer elastic fibers, and possessed more surface cells in the human tissue than in the bovine tissue. No evidence of significant tissue degeneration or remodeling was noted in either the stored patch-grafts or heart valves when compared with control bovine and human pericardial tissue layers.
Gross anatomic, histologic, and transmission and scanning electron microscopic observations were made of 29 bioprosthetic valves that had been implanted in patients for up to 115 months. On the basis of these morphologic data, no significant evidence of tissue rejection was seen. However, the durability of these valve bioprostheses is still questionable. Our observation primarily emphasize three factors: (1) disruption of the endothelial cell barrier and the lack of significant host endothelialization even 115 months after transplantation; (2) increased permeability that eased diffusion of circulating host plasma proteins into valve tissue, and increased activity of infiltration processes, eg. calcification and lipid accumulation; and (3) biodegradation of the collagen framework. Each of these factors may contribute further to valve dysfunction. Development of an intimal fibrous sheath seems to occur in porcine bioprostheses that have been implanted for the longest periods of time, but the rate of host tissue ingrowth varies.
Gross, histologic and ultrastructural studies were made of 14 porcine valve bioprostheses that were found to contain calcific deposits at the time of removal either at reoperation (13 patients) or at necropsy (1 patlent). Eleven bioprostheses had been in the mitral position, 1 fn the aortic, 1 in the tricuspid and 1 in a pulmonary condult. The ages of the patients at the time of implantation ranged from 2.5 to 65 years (average 32), and the bioprostheses had been in place from 3 to 94 months (average 39). Analysis of these 14 bioprostheses and review of reports concerning 37 other calcified porcine bioprostheses suggest the following conclusions: (1) Calcific deposits occur commonly in bioprostheses implanted in patients of all ages, but are more likely to become severe and clinically significant in children and in young adults than in older patients. (2) Calcific deposits can lead to prosthetic valve stenosis, because they can limit the mobility of the cusps; however, they can also be associated with prosthetic valve regurgitation. (3) Metabolic disorders that could contribute to bioprosthetic calcification are not identifiable in most patients having calcified prosthetic tissue valves. (4) The two main sites of deposition of calcium phosphate in porcine valve bioprostheses are the connective tissue in the cusps, particularly in the spongiosa, and small thrombi on the surfaces. (5) Calcification of connective tissue first involves the collagen fibrils; calcification of thrombi involves the mitochondria in platelets and leukocytes trapped in the mesh of fibrin. From these two sites, calcific deposits can grow and spread into other areas of the cusps.
To obtain a basis for the evaluation of postimplantation changes in bioprostheses made of parietal pericardium, we conducted comparative histologic, scanning and transmission electron microscopic studies of the structure of (1) normal bovine parietal pericardium, (2) glutaraldehyde-treated pericardial patches to be used for repair of cardiac defects, and (3) pericardial tissue cusps of unimplanted Ionescu-Shiley valves. Bovine parietal pericardium has three layers: (1) the serosa, or mesothelial cell layer; (2) the fibrosa, formed by diversely oriented, wavy bundles of collagen and by elastic fibers, and (3) the epipericardial connective tissue layer, which is partly continuous with the pericardiosternal ligaments. Pericardial patches and pericardial bioprosthetic cusps differ from normal pericardium by being denuded of mesothelium but they have normal degrees of waviness in their collagen. In Ionescu-Shiley valves, the inflow and outflow surfaces of each cusp correspond to the epipericardial and serosal surfaces of parietal pericardium, respectively. The inflow surfaces have a coarse texture, characterized by large bundles of collagen, and the outflow surfaces have numerous grooves, 10 to 30 mu in width and 20 mu in depth, which probably result from pressure exerted on the cuspidal surfaces by cotton material either during manufacturing or packing of the valves. Comparisons of the structure of bioprosthetic pericardial cusps and porcine aortic valve cusps show that the latter have reduced degrees of collagen waviness and a different layered structure: A layer similar to the spongiosa of aortic valve cusps is not present in pericardium. The functional implications of these observations are discussed in detail.
The aortic valve cusp is a three-layered structure, composed of differing amounts of collagen, elastin, and glycosaminoglycans. Little quantitative information is presently available on the amount, location, orientation, and overall structure of these constituents, particularly of elastin. We developed a technique to isolate aortic valve elastin in a morphologically intact state. Whole leaflets were digested in 0.1 N sodium hydroxide solution at a temperature of 75 degrees C. Both scanning electron microscopy and computerized three-dimensional reconstructions of serial sections showed a well-defined honeycomb or spongelike structure, suggesting that elastin forms a matrix that surrounds and links the collagen fiber bundles. This relationship between collagen and elastin is further supported by the naturally wavy configuration of the valve cusps, permitting elongations of 40%, even though collagen fibrils typically strain to 1% to 2% before fracture. Elastin likely acts to return collagen fibers back to their undeformed state, maintaining rest geometry.
The use of biologic heart valve prostheses is decreasing because of the high incidence of failure of these bioprostheses resulting from tissue degeneration or tearing. Immunologic reactions might play a decisive role in this process. The present experimental and clinical studies were conducted to investigate the relevance of immunologic reactions to the tissue failure of glutaraldehydetanned bovine pericardial and porcine valves. Specimens of the two different types of valve material were implanted in the abdominal muscles of rats. Enzyme-linked immunosorbent assays and tritiated thymidine incorporation tests were performed to detect specific antibodies and activated T cells. All specimens were studied histologically. Identical enzyme-linked immunosorbent assays and tritiated thymidine incorporation tests were performed in 29 patients with bioimplants and in 48 controls. Twenty explanted bioprostheses were investigated using histologic and immune histologic methods. The results of the enzyme-linked immunosorbent assays and lymphocyte proliferation tests showed that glutaraldehyde-tanned bovine pericardial valves can provoke cellular and humoral immunologic reactions in rats and human beings. In explanted bovine valves, macrophages were found invading and degrading implant collagen, starting from surface lesions. The combination of the formation of mechanical lesions, the development of cellular infiltrates, and collagen disruption strongly indicates that initial surface lesions initiate the immunologic reactions in bovine pericardial valves as the result of the exposure of incompletely tanned collagen. These immune responses might accelerate tissue degeneration. Porcine valves do not provoke immunologic reactions.
The sarcoplasmic reticulum Ca(2+)-ATPase loses hydrolytic activity and the ability to be phosphorylated by Pi following incubation with EDC [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide]. 4 nmol of tempamine per mg SR protein can be coupled to either a glu or an asp side chain through the EDC reaction. Mg2+ protects against loss of activity and tempamine labeling with a mid-point of about 3 mM in the absence of Ca2+. This is similar to the Kd for a Mg2+ that serves as a cofactor in enzyme phosphorylation. The Mg2+ protection constant is lowered by an order of magnitude when Ca2+ is bound to the transport sites. It is suggested that control of the Mg2+ binding site affinity may be part of the mechanism of enzyme activation by Ca2+.
Chordal preservation during mitral valve replacement is thought to greatly preserve left ventricular function. Recently a stentless mitral valve (Biocor) became available for clinical use. It is a porcine mitral valve with the entire chordal apparatus, the sewing ring being reinforced by a bovine pericardial ring and the chordae being sutured together onto two pericardial patches. During a six months period, seven patients undergoing mitral valve replacement for mitral insufficiency (four cases), mitral stenosis (two cases) or mixed lesions (one case) received a stentless mitral valve. Their mean age was 66.3 +/- 4.8. The valve was implanted by suturing the pericardial patches onto the papillary muscles and the sewing ring onto the mitral annulus. Echocardiography control immediately after surgery showed good valve function. Three valves failed after a period of seven days, seven and 12 months respectively. The valve lesions were similar and consistent with an excessive tension on the chordae and on the valvular tissue. There were tears along the posterior annulus and at the level of the commissures, often accompanied by chordal rupture. Histology and scanning electron microscopy showed good integration of the prosthesis with the patients own tissues. There were areas of overgrowing tissue without endothelial cells especially in the area of papillary muscle -pericardial patch interaction. The remaining four patients continue to do well after a mean follow up of two years. The apparent excessive tension on the valve tissues leading to the premature failure of the prosthesis could be due to the variability in the distance between mitral annulus and papillary muscles.(ABSTRACT TRUNCATED AT 250 WORDS)
Calcification is a frequent cause of the clinical failures of glutaraldehyde-pretreated bioprosthetic heart valves (BPHV) fabricated from glutaraldehyde-cross-linked porcine aortic valves. 2-Amino oleic acid (AOA) has been shown in previous in vivo studies to be a promising anticalcification agent. Our objective was to investigate the mechanism of calcification inhibition mediated by AOA pretreatment of porcine aortic valve bioprostheses.
BPHV tissues were treated with an AOA solution for 72 hours before experimentation. The diffusion of AOA across both cusp and aortic wall was evaluated. The lag time for AOA to diffuse across the aortic wall was prolonged compared with that of the cusp. An extraction study was performed to determine the stability of AOA binding; the results indicated that the binding was relatively stable regardless of solvent extraction conditions. The interaction between ionic calcium and AOA on treated tissue also was investigated by evaluating the patterns of calcium diffusion across both treated and untreated tissues. The results showed that AOA significantly reduced the diffusion of calcium. AOA inhibition of aortic valve calcification (calcium level, 5.5 +/- 3.0 mg/g of tissue compared with control; calcium level, 91.2 +/- 19.5 mg/g of tissue) but not aortic wall (calcium level, 158.7 +/- 10.3 mg/g of tissue compared with control; calcium level, 157.5 +/- 7.9 mg/g of tissue) was demonstrated on representative specimens from valves implanted in left ventricular apicoaortic shunts explanted after 150 days.
AOA covalently binds to glutaraldehyde-pretreated bioprosthetic heart valve tissue, presumably as the result of an aldehyde-amino reaction. Covalently bound AOA diminishes Ca2+ diffusion compared with non-AOA-pretreated bioprosthetic tissues. This may explain in part the anticalcification mechanism of AOA. Furthermore, AOA inhibits calcification of porcine BPHV cusps in the circulation.
Studies utilizing a quantitative assay, radioimmunoassay, and a biological activity assay, the murine thymocyte proliferation assay, to analyze IL-1 beta cytokine production by monocytes/macrophages on biomedical polymers have been carried out. Results indicate that the quantitative analysis of IL-1 beta on biomedical polymers and protein-adsorbed biomedical polymers is not indicative of and does not correlate with the results of the biological activity assay. IL-1 beta secreted from human monocytes/macrophages on Biomer, polydimethylsiloxane (PDMS), Dacron, polyethylene, expanded polytetrafluoroethylene, and control polystyrene with and without the preadsorption of physiological concentrations of human IgG, fibrinogen, and/or fibronectin was assayed. Quantitative levels of IL-1 beta suggest a greater functional response than that observed in the biological thymocyte proliferation assay when the polymers were studied directly or preadsorbed with IgG. On the other hand, preadsorption with fibrinogen or fibronectin resulted in high functional activity for IL-1 beta with low quantitative levels of IL-1 beta. The lack of correlation between the functional and biological activity assays suggests the presence of other cytokines or antagonists which modulate the biological activity of IL-1 beta.
This study focused on the association of extrinsic alkaline phosphatase (AP) activity with both early and advanced calcification of glutaraldehyde-pretreated bovine pericardial bioprosthetic (GPBP) tissue, and the inhibition of both calcification and AP activity by pre-incubation in diphosphonates (sodium-ethanehydroxydiphosphonate [NaEHDP], aminopropanehydroxydiphosphonate [APD]) and metallic salts (FeCl3, Ga(NO3)3, AICI3). GPBP specimens were implanted subcutaneously in 3 wk old male rats after pre-incubation. Following explantation of the tissue at 72 h and 21 d, calcification was assessed morphologically by light microscopy and chemically by atomic adsorption spectroscopy for calcium content and by molybdate complexation for phosphorus. AP activity was characterized by enzymatic hydrolysis of paranitrophenyl phosphate and by histochemical studies. In both control and pretreated groups, AP levels were greater in 72 h explants than 21 d retrievals, which demonstrated extensive calcification in control explants. All pre-incubations that resulted in inhibition of calcification after 21 d, except for APD, were associated with 72 h AP content which was lower than control specimens. The typical time of initiation of calcification was 72 h, as determined by previous studies with this model system. Covalently bound APD inhibited calcification. Increased AP activity in the APD group may be due to the toxicity of this agent with resultant acute inflammation, or other incompletely understood effects of diphosphonates on calcification and AP. Furthermore, EHDP and Ga3+ incubations were also associated with decreased GPBP AP at 72 h compared to control, but were not effective for inhibiting calcification after 21 d. We concluded that inhibition of peak GPBP AP activity is not necessarily associated with the prevention of GPBP mineralization.
Due to its superb crosslinking activity, glutaraldehyde (GA) is still the most widely used fixative for bioprosthetic heart valves. At the same time, however, GA is also believed to be partly responsible for tissue calcification and the lack of surface re-endothelialization, both of which may contribute to valve degeneration. Although excess GA has previously been extracted from thin leaflet tissue, this treatment proved insufficient for the detoxification of thick aortic wall tissue of stentless valves or root prostheses.
In order to establish a detoxification procedure which thoroughly extracts biologically active GA from aortic wall tissue, we used a highly sensitive bioassay where endothelial cells were seeded onto glutaraldehyde-fixed aortic wall discs following various detoxification procedures. Absolute cell numbers and morphologic shape were correlated with shrinkage temperature and shrinkage extent of the tissue to determine the potential of the treatments to reverse crosslinks. To optimize treatment conditions, pH (3.2 versus 4.5), temperature (22 degrees C versus 37 degrees C) and incubation time (48 h versus one week) were varied. In order to identify an optimal detoxification agent, 12 different amino-reagents from four chemical groups were compared: low pKa aromatic amines, amino acids, low pKa N-heterocyclic compounds and amino sugars.
Amino-reagent treatment required warm temperature (37 degrees C), prolonged reaction time (one week) and a pH of 4.5 to achieve long-term cell growth on glutaraldehyde-fixed aortic wall. All 12 amino-reagents were able to detoxify aortic tissue satisfactorily; and all mildly reversed crosslinks, although there were differences between candidates. When summarized data were ranked correlating cell growth and quality with shrinkage temperature and shrinkage extent, seven reagents had a rank sum above the overall mean value, and five below with statistically significant differences between candidates. The additional stabilization of the detoxification reaction through borohydride-reduction had no further effect on tissue biocompatibility and crosslinks.
Efficient detoxification of thick aortic wall tissue is possible if a one-week incubation in an acetic acid buffer-based amino-reagent is carried out at 37 degrees C.
This study was performed in order to: (i) determine whether a similar reduction of tissue calcification as seen after prolonged storage can be achieved through higher concentrations of glutaraldehyde (GA); and (ii) verify that well-preserved tissue integrity can suppress calcification.
Before fixation in 0.2% GA (PBS, 4 degrees C, seven days) porcine aortas were kept on ice for 48 h. Alternatively, tissue was immediately fixed at the abattoir in 0.2%, 1.0% or 3% glutaraldehyde (PBS, 4 degrees C, seven days). A second group of immediately fixed tissue (0.2%, 1.0%, 3.0% GA) (PBS, 4 degrees C, two days) had an interim step of L-lysine treatment (0.1M, 37 degrees C, acetic acid buffer, two days) in order to enhance cross-linking followed by warm-temperature fixation (PBS, 37 degrees C, five days). Two animal models were compared: subcutaneous implantation in rats (12 weeks) and vascular implantation in non-human primates, Chacma baboons (six weeks).
In both animal models the highest level of calcification was found in the group with delayed fixation in 0.2% GA. In the rat model there was an inverse correlation between tissue calcification and the GA concentration used, with 3% GA-fixed tissue showing the lowest level of tissue calcium. Overall, increasing GA concentration had a significant benefit on calcification (p < 0.0001; two-factor analysis of variance). Enhancement of cross-linking with L-lysine further abrogated tissue calcium levels at all GA concentrations (p < 0.0001; two- factor analysis of variance). Although the short-term baboon model showed lower tissue calcium levels, the trend seen in the rat model was confirmed.
Our results demonstrate the detrimental effect of delayed fixation and further suggest that, against previous beliefs, fixation at higher glutaraldehyde concentrations reduces the calcification tendency of cross-linked aortic tissue.
Poor ultrastructural tissue preservation of bioprosthetic heart valves is associated with a higher propensity for calcification. In spite of this realization, commercial valve fixation remains suboptimal.
In an attempt to maintain tissue integrity through improved cross-linking procedures, transmission electron microscopy and a 21-point damage score were applied to assess the ultrastructural preservation of aortic wall tissue-the main component of contemporary aortic valve bioprostheses. An ideal glutaraldehyde (GA) concentration was assessed by immediate tissue fixation at 4 degrees C comparing 0.2%, 0.5%, 0.65%, 1.0%, 2.0%, 3.0% and 4.0% GA in phosphate-buffered saline (PBS). Subsequently, an optimal concentration of 3.0% GA was used to determine the effect of fixation temperature (4 degrees, 22 degrees and 37 degrees C). Finally, the superior glutaraldehyde concentration (3.0%) and cross-linking temperature (4 degrees C) were used to assess tolerance towards delayed fixation.
When different GA concentrations were used almost identical damage scores of 6.3 and 5.8 were found for 0.2% and 0.65% fixation. The first significant improvement was found at a concentration of 1.0% (score 3.3; p < 0.01) followed by a further improvement at 3.0% (score 2.6; p = 0.05). The optimal fixation temperature was 4 degrees C (3.7) with the worst results obtained at room temperature (score 9.2; p < 0.03). When fixation was delayed, the most significant damage occurred during the initial 30 min after slaughter (from 2.3 to 7.4; p < 0.02) followed by another significant deterioration between 4 and 16 h (from 5.6 to 9.7; p < 0.02).
In summary, the prerequisites for an ideal ultrastructural preservation of bioprosthetic aortic wall tissue are immediate fixation (within 30 min), high GA concentrations (> 1.0%) and cold-temperature fixation (4 degrees C).
The authors recommend changes to the paradigm employed in the current ISO Heart Valve Standard (ISO 5840) so that future patients who receive a heart valve prosthesis are assured of a device that will function with minimal complications for at least 25 years. Based on valve failures of the past decade, it is clear that current standards are inadequate because present-day Standards and Regulatory Agencies operate in a manner which inhibits innovation and creativity. Thus, engineers and scientists in this field react to problems, rather than proact. As we approach the new millennium, the authors consider it time to rethink the ground rules.
Enhanced fixation of bioprosthetic tissue by both increased concentrations of glutaraldehyde (GA) and the introduction of additional cross-links with L-lysine significantly reduces calcification. We have previously reported that prolonged exposure to high-volume amino-compounds under warm, acidic conditions leads to thorough, non-rebounding GA detoxification. The aim of the present study was to prove that removal of excess GA can amplify the benefits of enhanced GA cross-linking with regard to bioprosthetic tissue calcification.
Porcine ascending aortas and leaflet tissue, and bovine pericardium were immediately fixed using three GA concentrations (0.2%, 1.0%, 3.0% (v/v)) for seven days at 4 degrees C. Samples were allocated to nine groups. Groups I to III received no further treatment (one at each GA concentration); groups IV to IX underwent an additional L-lysine interim step (48 h/37 degrees C/0.1 M) two days before completion of standard seven-day GA fixation; and groups VII to IX were additionally treated with a GA extraction process using high-volume urazole solution (acetic acid buffer, pH 4.5, 37 degrees C, one week) followed by NaBH4 reduction (2 days, 37 degrees C). Samples were implanted subcutaneously in rats (six per group) for six weeks. Tissue calcium was measured by atomic absorption spectrophotometry and examined histologically after von Kossa staining.
Calcification was reduced in all three tissue types by enhanced cross-linking and by extraction of excess GA. Increasing the GA concentration from 0.2% to 3.0% led to a reduction in calcification of 11.5% (p = 0.074; Student's t-test) in leaflets; 63.6% (p <0.0001) in pericardium; and 17.5% (p = 0.034) in aortic wall tissue. The introduction of additional cross-links with L-lysine resulted in a significant reduction of calcium in all tissues (maximally 42.5%, p = 0.0003 in leaflets; 79.3%, p = 0.005 in pericardium; and 49.6%, p <0.0001 in aortic wall; Student's t-test). Optimal reduction in calcification could be achieved with the combined effect of 3.0% GA fixation, L-lysine enhancement and urazole detoxification. When compared with 0.2% GA-fixed tissue, calcification could be reduced by 99.1% in leaflets, 95.9% in pericardium, and 90.8% in aortic wall tissue (p <0.0001 for all tissue types; Student's t-test).
Removal of excess GA from fixed bioprosthetic tissue was capable of markedly improving the anti-calcific effect of enhanced GA cross-linking.
Fixation at high glutaraldehyde (GA) concentrations mitigated bioprosthetic calcification in the rat model. The present study intended to verify this observation in the circulatory sheep model.
Porcine aortic roots were either fixed in 0.2%, 1.0%, or 3.0% GA. Eight roots per group were implanted in the distal aortic arch of sheep. After six weeks and six months calcification and inflammation were quantitatively and qualitatively assessed.
By increasing the GA concentration from 0.2% to 3.0%, aortic wall calcification could be reduced by 38% after 6 weeks and 34% after 6 months of implantation (p < 0.01). Mineralization coincided with the presence of elastin although calcium was predominantly found in cell nuclei and membranes. Leaflet calcification was absent in all groups after 6 weeks but in a few leaflets presented as heterogeneous, nodular spongiosa deposits after 6 months. Overall, differences between 0.2%-, 1.0%-, and 3.0%-fixed tissue were quantitative but not qualitative regarding distribution patterns. There was no significant difference in inflammatory host reaction between all groups.
We have shown in the circulatory sheep model that the anticalcific effect of better cross-linking seems to outweigh the intrinsic pro-calcific effect of GA accumulation in bioprosthetic aortic wall tissue.
The mild inflammatory response against stented bioprosthetic heart valves in the sheep model is often opposed by a more distinct response in failing human implants. With the emergence of stentless root prostheses with their significantly larger proportion of tissue interacting with the immune system of the host, a more relevant animal model than the sheep may be needed.
Valved, porcine aortic roots of 5 cm length were fixed in 0.2% glutaraldehyde and implanted in the upper descending aorta of Merino sheep (n = 5; 43+/-3 kg) and Chacma baboons (n = 5; 17+/-3 kg). After 6 weeks of tissue calcification, pannus outgrowth and inflammation were assessed by atomic absorption spectrophotometry, histologic damage scoring (0 to 3), image analysis, and transmission electron microscopy.
The main difference between the two animal models was in aortic wall calcification (64.8+/-39.8 microg/mg in the sheep model versus 4.1+/-5.9 microg/mg in the primate model; p > 0.005). In both models, leaflet calcification was negligible (2.6+/-2.4 microg/mg in the sheep versus 2.5+/-1.9 microg/mg in the primate), and the overall extent of inflammation was comparable (1.2+/-0.8 versus 0.98+/-0.7; p = 0.18 in the sheep and the primate, respectively). Qualitatively, the sheep demonstrated a macrophage-dominated reaction whereas the inflammatory demarcation often resembled a granulocyte-dominated xenograft response in the primate. Pannus outgrowth was comparable in length (8.4+/-2.3 mm versus 9.1+/-4.3 mm proximally and 7.1+/-3.4 mm versus 7.4+/-5.1 mm distally, in the sheep and baboon, respectively; p > 0.05).
Our results confirm the sheep as a significantly stronger calcification model for stentless aortic heart valves than the primate. Remaining antigenicity of porcine tissue as a result of incomplete cross-linking, however, elicits a distinctly stronger xenograft-type reaction in the primate model.
The role of an immune response in the failure of bioprosthetic heart valves is poorly understood and disregarded by many. To elucidate the nature of the immune response to glutaraldehyde-treated tissue and the possible role of graft-specific antibody in graft mineralization, we performed immune-calcification studies in the rabbit and correlated those results with the analysis of specific antibodies.
Aortic wall buttons (6 mm) were punched from porcine aortic wall tissue fixed with 0.2% glutaraldehyde and detoxified with urazole and then subsequently perforated under sterile conditions. The perforated buttons were then incubated with either immune serum prepared by immunization of New Zealand White rabbits (n = 5) with Freund's incomplete adjuvant emulsions of tissue homogenates of similarly treated aortic wall tissue, or incubated with the corresponding control preimmune sera obtained before immunization of the same animals. The tissue was then implanted subdermally on the back of unrelated New Zealand White rabbits (n = 8) for a period of 3 weeks. After the buttons were explanted, tissue calcium levels were determined by atomic absorption spectroscopy.
Tissue calcium was increased in all five immune serum-treated replicates (range, 61.8% to 431.2%; mean, 225.9%+/-73.2%) when compared with control samples treated with preimmune sera. Overall, the mean calcium level was significantly increased (p < 0.0001) when tissue was treated with immune sera (66.0+/-10.0 microg/mg versus 22.6+/-4.8 microg/mg in control tissue). Graft specificity of immune sera was confirmed by Western blot analysis.
These results strongly suggest a role of circulating graft-specific antibody in the disease of bioprosthetic graft calcification.
It has been established previously that immediate fixation and increased glutaraldehyde (GA) concentrations are required to prevent severe autolytic tissue damage during bioprosthetic aortic root production. The study aim was to verify that structure-preserving fixation also reduces aortic wall calcification.
Porcine aortic roots were fixed either instantly or after being kept on ice for 48 h (phosphate-buffered saline, PBS). Two concentrations of GA (0.2% and 3.0%) were chosen (4 degrees C, seven days, PBS). Discs of aortic wall tissue (1.2 cm diameter) were implanted subcutaneously in rats for 60 days (n = 10 per group), while aortic roots were implanted in the distal aortic arch of sheep for six weeks (n = 3 per group) and six months (n = 4 per group). Calcification was assessed by atomic absorption spectrophotometry and light microscopy. Fixation-related tissue damage was determined by transmission electron microscopy, and correlated with calcification.
No significant difference in calcification was found between immediate and delayed fixation if tissue was fixed with 0.2% GA. In the 3.0% GA group, both animal models showed a significantly lower level of calcification if tissue was immediately fixed. In the subcutaneous rat model, immediate fixation reduced calcification by 26% (p <0.0001). In the circulatory sheep model immediate fixation did not affect calcification in the short-term six-week implants, but markedly lowered it by 37% (p = 0.035) after six months. Ultrastructurally, there was a significant correlation between membrane damage, vacuolization and vesicle shedding on the one hand, and calcification on the other.
Coincidental fixation-related ultrastructural damage and increased calcification was demonstrated in bioprosthetic aortic wall tissue.
In an attempt to avoid the destructive process of bioprosthetic heart-valve calcification associated with the use of glutaraldehyde, valves are today prepared using low concentrations of the crosslinking reagent. In this review, we summarize our findings and those of others that confirm that the immunogenicity of such tissue is not sufficiently masked and that a defined humoral response is indeed mounted against a repertoire of antigens unrelated to those associated with vascularized and non-cross-linked xenograft organs. We demonstrate the need for increased cross-linking of tissue to satisfactorily mitigate that response; furthermore, we examine the impact of increased cross-link density on the macrophage as antigen presenting cell with respect to its involvement in both tissue erosion and pannus overgrowth. Finally we present evidence for a role of circulating antibodies in bioprosthesis calcification.
The glutaraldehyde fixation technique for bioprosthetic tissue valves has been used for about 30 years. It is more or less generally recognized, however, that a solution to the ongoing problems of calcification and immunomodulated inflammation can only be achieved with the development of alternative fixation techniques that differ from fixation with glutaraldehyde. This review reports on the chemistry behind newly developed tissue-fixation processes and the preliminary results achieved with these.