Calcification resistance, biostability, and low immunogenic potential of porcine heart valves modified by dye-mediated photooxidation

The calcification potential, biostability, and immunogenic response of materials intended for long-term in vivo use, such as in heart-valve bioprostheses, are essential components of device performance. Here we explore these properties in photooxidized porcine heart valves. To study immunological sensitization, we injected tissue extracts intradermally into guinea pigs. Test and control animals received a challenge patch of the appropriate extract and were scored for dermal reactions. Neither cottonseed oil nor sodium chloride extracts of photooxidized heart-valve tissues caused any dermal inflammatory response. After implantation in the rat subcutaneous model for 90 days, the calcium content of 48-h-treated photooxidized cusp tissue [0.04 +/- 0.00 mg/g wet weight (gww)] was comparable to that of unimplanted control tissues (usually <1 mg/gww) and much lower than that of glutaraldehyde-treated controls (71 +/- 15 mg/gww). The porcine aortic wall calcium content (49 +/- 31 mg/gww) was comparable to that of glutaraldehyde-treated controls (59 +/- 8 mg/gww). Histologically, a time-dependent decrease in inflammation and vascularization with increasing photooxidation time was noted in the rat model along with an increase in the stability and organization of collagen bundles. In summary, porcine valve tissues treated by dye-mediated photooxidation were resistant to calcification, were biostable, and demonstrated a low immunogenic response, indicating potential for use in heart-valve bioprostheses.     

Triglycidylamine crosslinking of porcine aortic valve cusps or bovine pericardium results in improved biocompatibility, biomechanics, and calcification resistance: chemical and biological mechanisms

We investigated a novel polyepoxide crosslinker that was hypothesized to confer both material stabilization and calcification resistance when used to prepare bioprosthetic heart valves. Triglycidylamine (TGA) was synthesized via reacting epichlorhydrin and NH(3). TGA was used to crosslink porcine aortic cusps, bovine pericardium, and type I collagen. Control materials were crosslinked with glutaraldehyde (Glut). TGA-pretreated materials had shrink temperatures comparable to Glut fixation. However, TGA crosslinking conferred significantly greater collagenase resistance than Glut pretreatment, and significantly improved biomechanical compliance. Sheep aortic valve interstitial cells grown on TGA-pretreated collagen did not calcify, whereas sheep aortic valve interstitial cells grown on control substrates calcified extensively. Rat subdermal implants (porcine aortic cusps/bovine pericardium) pretreated with TGA demonstrated significantly less calcification than Glut pretreated implants. Investigations of extracellular matrix proteins associated with calcification, matrix metalloproteinases (MMPs) 2 and 9, tenascin-C, and osteopontin, revealed that MMP-9 and tenascin-C demonstrated reduced expression both in vitro and in vivo with TGA crosslinking compared to controls, whereas osteopontin and MMP-2 expression were not affected. TGA pretreatment of heterograft biomaterials results in improved stability compared to Glut, confers biomechanical properties superior to Glut crosslinking, and demonstrates significant calcification resistance.     

Mechanisms of the in vivo inhibition of calcification of bioprosthetic porcine aortic valve cusps and aortic wall with triglycidylamine/mercapto bisphosphonate

Heart valve replacements fabricated from glutaraldehyde (Glut)-crosslinked heterograft materials, porcine aortic valves or bovine pericardium, have been widely used in cardiac surgery to treat heart valve disease. However, these bioprosthetic heart valves often fail in long-term clinical implants due to pathologic calcification of the bioprosthetic leaflets, and for stentless porcine aortic valve bioprostheses, bioprosthetic aortic wall calcification also typically occurs. Previous use of the epoxide-based crosslinker, triglycidyl amine (TGA), on cardiac bioprosthetic valve materials demonstrated superior biocompatibility, mechanics, and calcification resistance for porcine aortic valve cusps (but not porcine aortic wall) and bovine pericardium, vs. Glut-prepared controls. However, TGA preparation did not completely prevent long-term calcification of cusps or pericardium. Herein we report further mechanistic investigations of an added therapeutic component to this system, 2-mercaptoethylidene-1,1-bisphosphonic acid (MABP), a custom synthesized thiol bisphosphonate, which has previously been shown in a preliminary report to prevent bioprosthetic heterograft biomaterial calcification when used in combination with initial TGA crosslinking for 7 days. In the present studies, we have further investigated the effectiveness of MABP in experiments that examined: (1) The use of MABP after optimal TGA crosslinking, in order to avoid any competitive interference of MABP-reactions with TGA during crosslinking; (2) Furthermore, recognizing the importance of alkaline phosphatase (ALP) in the formation of dystrophic calcific nodules, we have investigated the hypothesis that the mechanism by which MABP primarily functions is through the reduction of ALP activity. Results from cell-free model systems, cell culture studies, and rat subcutaneous implants, show that materials functionalized with MABP after TGA crosslinking have reduced ALP activity, and in vivo have no significant calcification in long-term implant studies. It is concluded that bioprosthetic heart valves prepared in this fashion are compelling alternatives for Glut-prepared bioprostheses.     

Mechanisms of bioprosthetic heart valve failure: fatigue causes collagen denaturation and glycosaminoglycan loss.

Bioprosthetic heart valve (BPHV) degeneration, characterized by extracellular matrix deterioration, remodeling, and calcification, is an important clinical problem accounting for thousands of surgeries annually. Here we report for the first time, in a series of in vitro accelerated fatigue studies (5-500 million cycles) with glutaraldehyde fixed porcine aortic valve bioprostheses, that the mechanical function of cardiac valve cusps caused progressive damage to the molecular structure of type I collagen as assessed by Fourier transform IR spectroscopy (FTIR). The cyclic fatigue caused a progressive loss of helicity of the bioprosthetic cuspal collagen, which was evident from FTIR spectral changes in the amide I carbonyl stretching region. Furthermore, cardiac valve fatigue in these studies also led to loss of glycosaminoglycans (GAGs) from the cuspal extracellular matrix. The GAG levels in glutaraldehyde crosslinked porcine aortic valve cusps were 65.2 +/- 8.66 microg uronic acid/10 mg of dry weight for control and 7.91 +/- 1.1 microg uronic acid/10 mg of dry weight for 10-300 million cycled cusps. Together, these molecular changes contribute to a significant gradual decrease in cuspal bending strength as documented in a biomechanical bending assay measuring three point deformation. We conclude that fatigue-induced damage to type I collagen and loss of GAGs are major contributing factors to material degeneration in bioprosthetic cardiac valve deterioration.     

染料介导光氧化固定牛带瓣颈静脉的生物特性评价

为了解染料介导光氧化固定牛颈静脉的生物稳定性,抗钙化性和免疫原性,新鲜的牛颈带瓣静脉经染料介导光氧化法固定,分别进行化学消化和胃蛋白酶消化,上清液进行梯度聚丙烯酰胺电泳来了解其生物稳定性;将氧化固定的材料埋植于SD大鼠皮下,3周后取出,测钙含量、进行钙染色以及组织学检查了解其抗钙化性和免疫原性;采用戊二醛固定以及新鲜的牛带瓣颈静脉作为对照。染料介导光氧化固定的牛带瓣颈静脉经化学和酶消化之后的上清液电泳之后出现极淡的蛋白条带,明显淡于新鲜的未处理的材料,戊二醛固定的材料几乎不出现蛋白条带。皮下埋植3周后,钙含量测定,染料介导光氧化组显著低于戊二醛组,统计学上差异有显著性意义;钙染色,戊二醛组可见大量的钙沉积,新鲜组也可见钙沉积,但比戊二醛组少,染料介导光氧化组只见少量钙沉积;组织学检查提示光氧化组细胞浸润明显少于其他两组。体外动物实验提示,染料介导光氧化固定的牛带瓣颈静脉具有生物稳定性,低的钙化性和低免疫原性。     

Periodate-mediated glycosaminoglycan stabilization in bioprosthetic heart valves

Bioprosthetic heart valves (BPHVs) derived from glutaraldehyde-crosslinked porcine aortic valves are frequently used in heart valve replacement surgeries. However, the majority of bioprostheses fail clinically because of calcification and degeneration. We have recently shown that glycosaminoglycan (GAG) loss may be in part responsible for degeneration of glutaraldehyde-crosslinked bioprostheses. In the present studies, we used a mild reaction of periodate-mediated crosslinking to stabilize glycosaminoglycans in the bioprosthetic tissue. We demonstrate the feasibility of periodate reaction by crosslinking major components of extracellular matrix of bioprosthetic heart valve tissue, namely type I collagen and hyaluronic acid (HA). Uronic acid assay of periodate-fixed HA-collagen matrices showed 48% of HA disaccharides were bound to collagen. Furthermore, we show that such reactions are also feasible to fix glycosaminoglycans present in the middle spongiosa layer of bioprosthetic heart valves. The periodate reactions were compatible with conventional glutaraldehyde crosslinking and showed adequate stabilization of extracellular matrix as demonstrated by thermal denaturation temperature and collagenase assays. Moreover, uronic acid assays of periodate-fixed BPHV cusps showed 36% reduction in the amount of unbound GAG disaccharides as compared with glutaraldehyde-crosslinked cusps. We also demonstrate that calcification of BPHV cusps was significantly reduced in the periodate-fixed group as compared with the glutaraldehyde-fixed group in 21-day rat subdermal calcification studies (periodate-fixed tissue Ca 72.01 +/- 5.97 microg/mg, glutaraldehyde-fixed tissue Ca 107.25 +/- 6.56 microg/mg). We conclude that periodate-mediated GAG fixation could reduce structural degeneration of BPHVs and may therefore increase the useful lifetime of these devices.     

Acellular vascular tissues: natural biomaterials for tissue repair and tissue engineering

Various research groups around the world are actively investigating cardiovascular prostheses of biological origin. This review article discusses the need for such bioprosthetics and the potential role for natural tissues in cardiovascular applications such as cardiac valves and vascular grafts. Upon implantation, unmodified natural materials are subject to chemical and enzymatic degradation, seriously decreasing the life of the prosthesis. Therefore, methods such as glutaraldehyde and polyepoxide crosslinking treatments and dye-mediated photooxidation have been developed to stabilize the tissue while attempting to maintain its natural mechanical properties. Also, residual cellular components in a bioprosthetic material have been associated with undesired effects, such as calcification and immunological recognition, and thus have been the motivation for various decellularization processes. The effects of these stabilization and decellularization treatments on mechanical, biological and chemical properties of treated tissues have been investigated, specifically with regard to calcification, immunogenicity, and cytotoxicity concerns. Despite significant advances in the area of cardiovascular prostheses, there has yet to be developed a completely biocompatible, long-lasting implant. However, with the recent advent of tissue engineering, the possibility of applying selective cell seeding to naturally derived bioprosthetics moves us closer to a living tissue replacement.     

Onset and progression of experimental bioprosthetic heart valve calcification

Calcification, the major cause of bioprosthetic heart valve failures, is a serious clinical problem with uncertain pathogenesis. The objectives of the present study were to define the progressive chemical and morphologic sequence of mineralization in glutaraldehyde-treated porcine aortic valve cusps implanted subcutaneously in rats and to compare the pathology and pathophysiology of calcification in subcutaneous implants with that of orthotopic valve replacements in calves. Cusps were implanted subcutaneously in 3-week-old rats for 24 hours to 18 weeks. Cuspal calcium was 114 +/- 18 micrograms/mg of dry weight (mean +/- SEM) at day 21 and 218 +/- 6 at day 56 of implantation and unchanged thereafter. The earliest mineral deposits, noted at 48 hours, were associated with devitalized porcine connective tissue cells, but by 7 days, mineral deposits also involved collagen bundles. Scanning electron microscopy with energy-dispersive x-ray analysis demonstrated predominant accumulation in the spongiosa with a spongiosa to fibrosa energy-dispersive x-ray analysis count ratio of calcium of 15 at 21 days. In stent-mounted glutaraldehyde-preserved porcine valves implanted in five calves as mitral replacements for 69 to 142 days, cuspal calcium was 86 micrograms/mg (mean) (range 47 to 128). Calf implants also had cell oriented and collagen calcification predominating in the valvar spongiosa. In both rat subcutaneous and calf mitral valve models, early diffuse calcific microcrystals evolved into confluent nodules that disrupted tissue architecture. It is concluded that calcification of glutaraldehyde-preserved porcine aortic valves implanted subcutaneously in rats begins within 48 hours, earliest deposits are localized to residual porcine connective tissue cells, but latter deposits also involve collagen fibrils, mineralization is most prominent in the spongiosa, the pathology of calcification in rat subcutaneous implants and calf mitral replacements is comparable, suggesting a common pathophysiology, and calcific nodule formation most likely initiates clinical features.     

The effect of glycosaminoglycan stabilization on tissue buckling in bioprosthetic heart valves

Bioprosthetic valves are used in thousands of heart valve replacement surgeries. Existing glutaraldehyde-crosslinked bioprosthetic valves fail due to either calcification or degeneration. Glutaraldehyde crosslinking does not stabilize valvular glycosaminoglycans (GAGs). GAGs, predominantly present in the medial spongiosa layer of native heart valve cusps, play an important role in regulating physico-mechanical behavior of the native cuspal tissue during dynamic motion. The primary objective of this study was to identify the role of cuspal GAGs in valve tissue buckling. Glutaraldehyde-crosslinked cusps showed extensive buckling compared to fresh, native cusps. Removal of GAGs by treatment with GAG-degrading enzymes led to a marked increase in buckling behavior in glutaraldehyde-crosslinked cusps. We demonstrate that the retention of valvular GAGs by carbodiimide crosslinking together with chemical attachment of neomycin trisulfate (a hyaluronidase inhibitor), prior to glutaraldehyde crosslinking, reduces the extent of buckling in bioprosthetic heart valves. Furthermore, following exposure to GAG-digestive enzymes, neomycin-trisulfate-bound cusps experienced no alterations in buckling behavior. Such moderate buckling patterns mimicked that of fresh, untreated cusps subjected to similar bending curvatures. Thus, GAG stabilization may subsequently improve the durability of these bioprostheses.     

Amyloid deposits in bioprosthetic cardiac valves after long-term implantation in man. A new localization of amyloidosis

Congo red staining with microscopic examination under polarized light was performed in 30 porcine bioprosthetic cardiac valves and one autologous fascia lata valve explanted from 31 patients in order to detect the presence of amyloid. Microdeposits of amyloid were present in the sewing ring of the fascia lata valve and in 10 porcine bioprostheses, and this finding was confirmed by transmission electron microscopy in 3 porcine bioprostheses. All amyloid-laden porcine valves had been implanted for at least 33 months before removal, and all except two showed dysfunction and/or severe degeneration of cuspal tissue. Statistical analyses failed to establish any correlation between the presence of amyloid and patient-related factors. In a majority of porcine bioprostheses amyloid was permanganate-sensitive and tryptophan-positive. The pathogenesis of this new form of heart valve amyloidosis might consist in penetration of human macrophages in deteriorated bioprosthetic cusps and their interaction with blood-borne amyloid precursors.     

Collagen fiber disruption occurs independent of calcification in clinically explanted bioprosthetic heart valves

The durability of bioprosthetic heart valves (BHV) is severely limited by tissue deterioration, manifested as calcification and mechanical damage to the extracellular matrix. Extensive research on mineralization mechanisms has led to prevention strategies, but little work has been done on understanding the mechanisms of noncalcific matrix damage. The present study tested the hypothesis that calcification-independent damage to the valvular structural matrix mediated by mechanical factors occurs in clinical implants and could contribute to porcine aortic BHV structural failure. We correlated quantitative assessment of collagen fiber orientation and structural integrity by small angle light scattering (SALS) with morphologic analysis in 14 porcine aortic valve bioprostheses removed from patients for structural deterioration following 5-20 years of function. Calcification of the explants varied from 0 (none) to 1+ (minimal) to 4+ (extensive), as assessed radiographically. SALS tests were performed over entire excised cusps using a 0.254-mm spaced grid, and the resultant structural information used to generate maps of the local collagen fiber damage that were compared with sites of calcific deposits. All 42 cusps showed clear evidence of substantial noncalcific structural damage. In 29 cusps that were calcified, structural damage was consistently spatially distinct from the calcification deposits, generally in a distribution similar to that noted in porcine BHV subjected to in vitro durability testing. Our results suggest a mechanism of noncalcific degradation dependent on cuspal mechanics that could contribute to porcine aortic BHV failure.     

Crosslinking of decellularized porcine heart valve matrix by procyanidins

Heart valve diseases have a significant high mortality, and the valve replacement using glutaraldehyde crosslinked porcine heart valves is one of the main curing techniques. But its application is limited due to poor durability, calcification of the valves and immunogenic reactions. The aim of this study was to evaluate the crosslinking effect of procyanidins on porcine heart valve matrix. After crosslinking of the decellularized porcine aortic heart valves by procyanidins, the tensile strength, the in vitro enzymatic degradation resistance, procyanidins release from the crosslinked materials and the cytotoxicity of procyanidins to heart valvular interstitial cells were examined. The results showed that the tensile strength of procyanidins crosslinked valve matrix was higher than that of glutaraldehyde crosslinked valve matrix. Valve matrix crosslinked by 10 mg/ml procyanidins could be stored in D-Hanks solution for at least 45 days without any decline in ultimate tensile strength and maintained the elasticity as the fresh valves. Furthermore, procyanidins was found to release when the crosslinked tissue stored in D-Hanks solution. The release rate was high during the first 4 days and then dramatically decreased thereafter. During releasing phase, the concentration of procyanidins was no toxicity to heart valve interstitial cells. In vitro enzymatic degradation revealed that crosslinked matrix could resist the enzymatic hydrolysis, and the resistant capacity was approximately the same as glutaraldehyde crosslinked valve matrix. This study shows that procyanidins can crosslink porcine heart valves effectively without toxicity. Our results suggested that this method might be a useful approach for preparation of bioprosthetic heart valve.     

Artificial neural networks in computer-assisted classification of heart sounds in patients with porcine bioprosthetic valves

The paper describes the design, training and testing of a three-layer feedforward back-propagation neural network for the classification of bioprosthetic valve closure sounds. Forty-seven patients with a porcine bioprosthetic valve inserted in the aortic position were involved in the study. Twenty-four of them had a normal bioprosthetic valve, and the other 23 had a degenerated valve. Five features extracted from the Fourier spectra and 12 linear predictive coding (LPC) coefficients of the sounds were used separately as the input of two neural-network classifiers. The performance of the classifiers was tested using the leave-one-out method. Results show that correct classifications were 85 per cent using the spectral features, and 89 per cent using the LPC coefficients. The study confirms the potential of artificial networks for the classification of bioprosthetic valve closure sounds. Clinical use of this method, however, still requires further investigation.     

Controlled release of diphosphonates from synthetic polymers to inhibit calcification

Calcification is the most frequent cause of the failure of bioprosthetic heart valves fabricated from glutaraldehyde pretreated porcine aortic valves or bovine pericardium. Formulation and evaluation of controlled-release drug delivery system to inhibit bioprosthetic heart valve calcification is reviewed.     

New concepts in the design and use of biological prosthetic valves

The natural aortic valve is a structure that has thus far eluded all attempts at duplication with synthetic materials. Real success in the replacement of the aortic valve has come about primarily through the use of biological devices, such as the porcine aortic valve xenograft. In the future, bioprostheses based more closely on the natural aortic valve may ultimately succeed where synthetic approaches have failed. Some recent advances in the design and development of bioprosthetic heart valves, such as the absence of a stent and the better preservation of the valve's natural biomechanical properties, show considerable promise in improving the long term durability of these devices. With a greater understanding of the structure/function relationship of the aortic valve at the micromechanical level, the future of bioprostheses may be even more biologically oriented than it is today.     

Glutaraldehyde-fixed kangaroo aortic wall tissue: histology,crosslink stability and calcification potential

Stentless aortic heart valve substitutes, manufactured from biological tissues, are fixed with glutaraldehyde to cross-link collagen, reduce antigenicity, and sterilize the tissue. Despite improved cross linking, reduced antigenicity, and various anticalcification measures, the aortic wall tissue present in these prostheses tends to calcify. The aim of this study was to assess the morphology, collagen cross-link stability, and calcification potential of glutaraldehyde-preserved kangaroo aortic wall tissue as opposed to porcine aortic wall tissue. Porcine and kangaroo aortic wall tissues were fixed in 0.625% buffered glutaraldehyde. Histology and cross-link stability were examined. Calcification potential was determined in the subcutaneous rat model. Kangaroo aortic wall tissue was significantly (p < 0.01) less calcified than porcine aortic wall tissue (26.67 +/- 6.53 versus 41.959 +/- 2.75 microg/mg tissue) at 8 weeks. In conclusion, the histological differences between kangaroo and porcine aortic wall tissue correlate well with the reduced calcification potential of kangaroo aortic wall tissue. The reduced calcification potential could result in improved long-term durability of stentless kangaroo heart valves as bioprostheses.     

改造天然生物组织为血管支架材料的预处理方法

目前 ,临床对血管替代物的需求量越来越大 ,传统的来源已不能满足需要。组织工程化血管的出现使得这一问题有望得到解决。构建组织工程化血管就必然涉及血管支架的预制。生物支架材料是血管支架中的一大类 ,它在细胞黏附及促细胞生长等方面优于人工合成的支架材料。由于生物性材料自活体取出后即开始降解 ,同时不同材料间也存在着种群差异 ,不宜保存和直接应用 ,故需采用一些预处理方法来解决这些问题 ,预处理目的就是在移植生物性材料前 ,降低其抗原性、提高其抗酶降解能力 ,并较长时间地保持其良好的力学性能和组织结构。这些处理方法包括运用戊二醛、多聚环氧化合物、碳化二亚胺、京尼平及原花色素等化学试剂进行交联的化学方法和应用光氧化等进行交联的物理方法。本文详细地叙述了各种预处理方法的机理及相应各种材料处理前后免疫原性、生物稳定性、力学性能、细胞毒性、抗钙化能力等特性的变化 ,并对各种方法的优缺点做一简要的评述。总之 ,生物性材料预处理的发展趋势是继续深入研究和开发细胞毒性小的天然交联剂 ,完善并拓宽光氧化交联的应用     

Inflammation and infection in nine surgically explanted Medtronic Freestyle stentless aortic valves.

BACKGROUND: The Medtronic Freestyle valve is fixed in glutaraldehyde at zero pressure on the cusps and treated with alpha-amino oleic acid. This valve reportedly has excellent clinical and hemodynamic results, but little has been reported about its long-term pathology. METHODS AND RESULTS: Nine Freestyle valves explanted between 2003 and 2005 were reviewed to assess the reasons for bioprosthesis failure (six implanted at our institution). All valves were examined in detail, using histochemistry and immunohistochemistry to identify the cellular response. One Freestyle valve, explanted for mitral valve endocarditis on the fifth postoperative day, was excluded from analysis. Average implant duration was 52.8+/-35.5 months. Four valves were explanted for infective endocarditis, three for aortic insufficiency, two for aortic stenosis with cusp calcification seen in five valves, pannus and thrombus in all valves and a chronic inflammatory reaction involving the xenograft arterial wall seen in eight of nine valves. This was associated with significant damage to the porcine aortic wall in seven cases, and cusp myocardial shelf damage in six cases. CONCLUSIONS: In this series of valves, we found (1) infective endocarditis; (2) pannus, thrombus, and calcification; and (3) unusual and significant inflammatory reaction and aortic tissue damage, which could by itself lead to aortic incompetence.     

In vivo behavior of epoxy-crosslinked porcine heart valve cusps and walls

Calcification limits the long-term durability of xenograft glutaraldehyde-crosslinked heart valves. In this study, epoxy-crosslinked porcine aortic valve tissue was evaluated after subcutaneous implantation in weanling rats. Non-crosslinked valves and valves crosslinked with glutaraldehyde or carbodiimide functioned as control. Epoxy-crosslinked valves had somewhat lower shrinkage temperatures than the crosslinked controls, and within the series also some macroscopic and microscopic differences were obvious. After 8 weeks implantation, cusps from non-crosslinked valves were not retrieved. The matching walls were more degraded than the epoxy- and control-crosslinked walls. This was observed from the higher cellular ingrowth with fibroblasts, macrophages, and giant cells. Furthermore, non-crosslinked walls showed highest numbers of lymphocytes, which were most obvious in the capsules. Epoxy- and control-crosslinked cusps and walls induced lower reactions. Calcification, measured by von Kossa-staining and by Ca-analysis, was always observed. Crosslinked cusps calcified more than walls. Of all wall samples, the non-crosslinked walls showed the highest calcification. It is concluded that epoxy-crosslinked valve tissue induced a foreign body and calcification reaction similar to the two crosslinked controls. Therefore, epoxy-crosslinking does not represent a solution for the calcification problem of heart valve bioprostheses.     

Stability and function of glycosaminoglycans in porcine bioprosthetic heart valves

Glycosaminoglycans (GAGs) are important structural and functional components in native aortic heart valves and in glutaraldehyde (Glut)-fixed bioprosthetic heart valves (BHVs). However, very little is known about the fate of GAGs within the extracellular matrix of BHVs and their contribution to BHV longevity. BHVs used in heart valve replacement surgery have limited durability due to mechanical failure and pathologic calcification. In the present study we bring evidence for the dramatic loss of GAGs from within the BHV cusp structure during storage in saline and both short- and long-term Glut fixation. In order to gain insight into role of GAGs, we compared properties of fresh and Glut-fixed porcine heart valve cusps before and after complete GAG removal. GAG removal resulted in significant morphological and functional tissue alterations, including decreases in cuspal thickness, reduction of water content and diminution of rehydration capacity. By virtue of this diminished hydration, loss of GAGs also greatly increased the "with-curvature" flexural rigidity of cuspal tissue. However, removal of GAGs did not alter calcification potential of BHV cups when implanted in the rat subdermal model. Controlling the extent of pre-implantation GAG degradation in BHVs and development of improved GAG crosslinking techniques are expected to improve the mechanical durability of future cardiovascular bioprostheses.