Porcine Bioprosthetic Heart Valves Research Articles

Biomimetic proteoglycans as a tool to engineer the structure and mechanics of porcine bioprosthetic heart valves.

The utility of bioprosthetic heart valves (BHVs) is limited to certain patient populations because of their poor durability compared to mechanical prosthetic valves. Histological analysis of failed porcine BHVs suggests that degeneration of the tissue extracellular matrix (ECM), specifically the loss of proteoglycans and their glycosaminoglycans (GAGs), may lead to impaired mechanical performance, resulting in nucleation and propagation of tears and ultimately failure of the prosthetic. Several strategies have been proposed to address this deterioration, including novel chemical fixatives to stabilize ECM constituents and incorporation of small molecule inhibitors of catabolic enzymes implicated in the degeneration of the BHV ECM. Here, biomimetic proteoglycans (BPGs) were introduced into porcine aortic valves ex vivo and were shown to distribute throughout the valve leaflets. Incorporation of BPGs into the heart valve leaflet increased tissue overall GAG content. The presence of BPGs also significantly increased the micromodulus of the spongiosa layer within the BHV without compromising the chemical fixation process used to sterilize and strengthen the tissue prior to implantation. These findings suggest that a targeted approach for molecularly engineering valve leaflet ECM through the use of BPGs may be a viable way to improve the mechanical behavior and potential durability of BHVs.

In Situ "Humanization" of Porcine Bioprostheses: Demonstration of Tendon Bioprostheses Conversion into Human ACL and Possible Implications for Heart Valve Bioprostheses.

This review describes the first studies on successful conversion of porcine soft-tissue bioprostheses into viable permanently functional tissue in humans. This process includes gradual degradation of the porcine tissue, with concomitant neo-vascularization and reconstruction of the implanted bioprosthesis with human cells and extracellular matrix. Such a reconstruction process is referred to in this review as “humanization”. Humanization was achieved with porcine bone-patellar-tendon-bone (BTB), replacing torn anterior-cruciate-ligament (ACL) in patients. In addition to its possible use in orthopedic surgery, it is suggested that this humanization method should be studied as a possible mechanism for converting implanted porcine bioprosthetic heart-valves (BHV) into viable tissue valves in young patients. Presently, these patients are only implanted with mechanical heart-valves, which require constant anticoagulation therapy. The processing of porcine bioprostheses, which enables humanization, includes elimination of α-gal epitopes and partial (incomplete) crosslinking with glutaraldehyde. Studies on implantation of porcine BTB bioprostheses indicated that enzymatic elimination of α-gal epitopes prevents subsequent accelerated destruction of implanted tissues by the natural anti-Gal antibody, whereas the partial crosslinking by glutaraldehyde molecules results in their function as “speed bumps” that slow the infiltration of macrophages. Anti-non gal antibodies produced against porcine antigens in implanted bioprostheses recruit macrophages, which infiltrate at a pace that enables slow degradation of the porcine tissue, neo-vascularization, and infiltration of fibroblasts. These fibroblasts align with the porcine collagen-fibers scaffold, secrete their collagen-fibers and other extracellular-matrix (ECM) components, and gradually replace porcine tissues degraded by macrophages with autologous functional viable tissue. Porcine BTB implanted in patients completes humanization into autologous ACL within ~2 years. The similarities in cells and ECM comprising heart-valves and tendons, raises the possibility that porcine BHV undergoing a similar processing, may also undergo humanization, resulting in formation of an autologous, viable, permanently functional, non-calcifying heart-valves.

Reducing Immunogenicity of Porcine Bioprosthetic Heart Valves via GGTA1/β4GalNT2/CMAH Gene Targeting

Reducing Immunogenicity of Porcine Bioprosthetic Heart Valves via GGTA1/β4GalNT2/CMAH Gene Targeting

Reducing immunoreactivity of porcine bioprosthetic heart valves by genetically-deleting three major glycan antigens, GGTA1/β4GalNT2/CMAH

Bioprosthetic heart valves (BHVs) originating from pigs are extensively used for heart valve replacement in clinics. However, recipient immune responses associated with chronic calcification lead to structural valve deterioration (SVD) of BHVs. Two well-characterized epitopes on porcine BHVs have been implicated in SVD, including galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc) whose synthesis are catalyzed by α(1,3) galactosyltransferase (encoded by the GGTA1 gene) and CMP-Neu5Ac hydroxylase (encoded by the CMAH gene), respectively. It has been reported that BHV from αGal-knockout pigs are associated with a significantly reduced immune response by human serum. Moreover, valves from αGal/Neu5Gc-deficient pigs could further reduce human IgM/IgG binding when compared to BHV from αGal-knockout pigs. Recently, another swine xenoantigen, Sd(a), produced by β-1,4-N-acetyl-galactosaminyl transferase 2 (β4GalNT2), has been identified. To explore whether tissue from GGTA1, CMAH, and β4GalNT2 triple gene-knockout (TKO) pigs would further minimize human antibody binding to porcine pericardium, TKO pigs were successfully produced by CRISPR/Cas9 mediated gene targeting. Our results showed that the expression of αGal, Neu5G and Sd(a) on TKO pigs was negative, and that human IgG/IgM binding to pericardium was minimal. Moreover, the analysis of collagen composition and physical characteristics of porcine pericardium from the TKO pigs indicated that elimination of the three xenoantigens had no significant impact on the physical proprieties of porcine pericardium. Our results demonstrated that TKO pigs would be an ideal source of BHVs. Statement of significanceSurgical heart valve replacement is an established lifesaving treatment for diseased heart valve. Bioprosthetic heart valves (BHVs) made from glutaraldehyde-fixed porcine or bovine tissues are widely used in clinics but exhibit age-dependent structural valve degeneration (SVD) which is associated with the immune response against BHVs. Three major xenoantigens present on commercial BHVs, Galactosea α1,3 galactose (αGal), N-glycolylneuraminic acid (Neu5Gc) and glycan products of β-1,4-N-acetyl-galactosaminyl transferase 2 (β4GalNT2) are eliminated through CRISPR/Cas9 mediated gene targeting in the present study. The genetically modified porcine pericardium showed reduced immunogenicity but comparable collagen composition and physical characteristics of the pericardium from wild-type pigs. Our data suggested that BHVs from TKO pigs is a promising alternative for currently available BHVs from wild-type pigs.

Surgical Management of Ebstein’s Anomaly with Atrial Septal Defect in a Young Lady: A Case Report

Ebstein’s anomaly is a rare congenital heart defect accounting for <1% of all cases. It is commonly associated with other cardiac malformations particularly, 50% of the patients are associated with atrial septal defect. We report a 22-year-old lady diagnosed to have Ebstein’s anomaly with small atrial septal defect. She was surgically managed in Bangabandhu Sheikh Mujib Medical University by replacing the defective tricuspid valve withSt Jude Medical Epic porcine bio-prosthetic heart valve along with plication of atrialized portion of right ventricle and direct closure of the atrial septal defect.This resulted in excellent symptomatic improvement.University Heart Journal Vol. 12, No. 1, January 2016; 37-39

A Review on the Biomechanical Effects of Fatigue on the Porcine Bioprosthetic Heart Valve.

Characterization of the mechanisms of degeneration of porcine bioprosthetic heart valves (BHV) during long-term cyclic loading is required for predicting and ultimately preventing their failure. Isolation of purely mechanical effects from host biological ones is a necessary first step in understanding the fatigue process as a whole. Thus, in this review we focus on mechanical factors alone as a means of isolating their role in altering biomechanical properties and ultimately their contribution to the fatigue damage process. Mechanical evaluations included tension controlled biaxial, 3-point flexural, and uniaxial failure tests performed on cuspal tissue following 0, 50, 100, 200, and 300 × 106 in vitro accelerated test cycles. Overall, biaxial mechanical results indicate a decreasing radial extensibility that can be explained by stiffening of the effective collagen fiber network as well as a small decrease in the splay of the collagen fibers. Moreover, these results suggest that the loss in flexural rigidity with fatigue that we have previously measured (ASAIO 1999; 45:59-63) may not be because of loss of collagen stiffness alone, but also to fiber debonding and degradation of the amorphous extracellular matrix. We discuss the implications of these results that point toward the development of chemical-treatment methods that seek to maintain the integrity of the amorphous extracellular matrix to ultimately extend BHV long-term durability.

SPATIO-TEMPORAL COMPLEXITY OF THE AORTIC SINUS VORTEX.

The aortic sinus vortex is a classical flow structure of significant importance to aortic valve dynamics and the initiation and progression of calific aortic valve disease. We characterize the spatio-temporal characteristics of aortic sinus voxtex dynamics in relation to the viscosity of blood analog solution as well as heart rate. High resolution time-resolved (2KHz) particle image velocimetry was conducted to capture 2D particle streak videos and 2D instantaneous velocity and streamlines along the sinus midplane using a physiological but rigid aorta model fitted with a porcine bioprosthetic heart valve. Blood analog fluids used include a water-glycerin mixture and saline to elucidate the sensitivity of vortex dynamics to viscosity. Experiments were conducted to record 10 heart beats for each combination of blood analog and heart rate condition. Results show that the topological characteristics of the velocity field vary in time-scales as revealed using time bin averaged vectors and corresponding instantaneous streamlines. There exist small time-scale vortices and a large time-scale main vortex. A key flow structure observed is the counter vortex at the upstream end of the sinus adjacent to the base (lower half) of the leaflet. The spatio-temporal complexity of vortex dynamics is shown to be profoundly influenced by strong leaflet flutter during systole with a peak frequency of 200Hz and peak amplitude of 4 mm observed in the saline case. While fluid viscosity influences the length and time-scales as well as the introduction of leaflet flutter, heart rate influences the formation of counter vortex at the upstream end of the sinus. Higher heart rates are shown to reduce the strength of the counter vortex that can greatly influence the directionality and strength of shear stresses along the base of the leaflet. This study demonstrates the impact of heart rate and blood analog viscosity on aortic sinus hemodynamics.

Heterotopic implantation of a porcine bioprosthetic heart valve in a dog with aortic valve endocarditis

A 5-year-old male German Shepherd Dog was evaluated because of a 5-month history of progressive lethargy, weight loss, and heart failure. On physical examination, bounding femoral pulses and systolic and diastolic murmurs were detected. Echocardiography revealed severe aortic valve insufficiency (AVI) and a large vegetative lesion on the aortic valve consistent with aortic valve endocarditis. The AVI velocity profile half-time was 130 milliseconds; the calculated peak systolic pressure gradient across the aortic valve was 64 mm Hg. Left ventricular diameter during diastole was 63.6 mm (predicted range, 40.2 to 42 mm) and during systole was 42.9 mm (predicted range, 25.4 to 27 mm). Systolic, diastolic, and mean arterial blood pressures were 120, 43, and 65 mm Hg, respectively. To palliate severe AVI, the descending aorta was occluded (duration, 16.75 minutes) and heterotopic implantation of a porcine bioprosthetic heart valve in that vessel was performed. After surgery, systolic, diastolic, and mean arterial blood pressures were 115, 30, and 61 mm Hg, respectively, in the forelimb and 110, 62, and 77 mm Hg, respectively, in the hind limb. Within 6 months, the AVI velocity profile half-time had increased to 210 milliseconds, indicating diminished severity of AVI. After 24 months, the dog was able to engage in vigorous exercise; no pulmonary edema had developed since surgery. Heterotopic bioprosthetic heart valve implantation into the descending aorta during brief aortic occlusion appears feasible in dogs and may provide substantial palliation for dogs with severe AVI.

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.

Effects of fixation pressure on the biaxial mechanical behavior of porcine bioprosthetic heart valves with long-term cyclic loading

Zero transvalvular pressure fixation is thought to improve porcine bioprosthetic heart valve (BHV) durability by preserving the collagen fiber architecture of the native tissue, and thereby native mechanical properties. However, it is not known if the native mechanical properties are stable during long-term valve operation and thus provide additional durability. To address this question, we examined the biaxial mechanical properties of porcine BHV fixed at 0 and 4 mmHg transvalvular pressure following 0, 1×10 6, 50×10 6, and 200×10 6 in vitro accelerated test cycles. At 0 cycles, the extensibility and degree of axial cross-coupling of the zero-pressure-fixed cusps were higher than those of the low-pressure-fixed cusps. Furthermore, extensibility of the zero-pressure-fixed tissue decreased between 1×10 6 and 50×10 6 cycles, approaching that of the low-pressure-fixed tissue, whose extensibility was unchanged over 0–200×10 6 cycles. The decrease in extensibility of the zero-pressure-fixed tissue between 1×10 6 and 50×10 6 cycles may be attributable to the ability of its collagen fibers to undergo larger changes in orientation and crimp with cyclic loading. These observations suggest that the collagen fiber architecture of the 0-mmHg-fixed porcine BHV, although locked in place by chemical fixation, may not be maintained over a sufficient number of cycles to be clinically beneficial. This study further underscores that chemically treated collagen fibers can undergo conformational changes under long-term cyclic loading not associated with damage.

Aluminum Chloride Pretreatment of Elastin Inhibits Elastolysis by Matrix Metalloproteinases and Leads to Inhibition of Elastin-Oriented Calcification

Calcification of elastin occurs in many pathological cardiovascular diseases including atherosclerosis. We have previously shown that purified elastin when subdermally implanted in rats undergoes severe calcification and aluminum chloride (AlCl(3)) pretreatment of elastin inhibits calcification. In the present study we investigated whether matrix metalloproteinase (MMP) binding to elastin and elastin degradation is prevented by AlCl(3) pretreatment. Subdermal implantation of AlCl(3)-pretreated elastin showed significantly lower MMP-9 and MMP-2 activity surrounding the implant as compared to the control implants. AlCl(3) pretreatment also significantly inhibited elastin implant calcification at the seven-day implant period (AlCl(3)-pretreated 4.07 +/- 1.27, control 23.82 +/- 2.24 microg/mg; p<0.0001). Moreover, elastin gel zymography studies showed that gel pretreatment with AlCl(3) inhibited elastolysis by MMP-9. We also demonstrate significant suppression of MMP-2 activity in aortic wall segments of AlCl(3)-pretreated porcine bioprosthetic heart valve implants as compared to control valve implants in sheep mitral valve replacement studies. AlCl(3) pretreatment also significantly inhibited calcification of elastin in this model. Thus, we conclude that aluminum ion binding to elastin prevents MMP-mediated elastolysis and thus prevents elastin calcification.

Pulsatile flow studies of a porcine bioprosthetic aortic valve in vitro: PIV measurements and shear-induced blood damage

A two-dimensional particle image tracking velocimetry (PIV) system has been used to map the velocity vector fields and Reynolds stresses in the immediate downstream vicinity of a porcine bioprosthetic heart valve at the aortic root region in vitro under pulsatile flow conditions. Measurements were performed at five different time steps of the systolic phase of the cardiac cycle. The velocity vector fields and Reynolds stress mappings at different time steps allowed us to chart a time history of the stress levels experienced by fluid particles as they move across the aortic root. This Lagrangian description of the stresses experienced by individual blood cells enabled us to estimate the propensity of shear-induced damage to platelets and red blood cells. Coupled with flow visualization techniques, the hydrodynamic consequences of introducing a porcine bioprosthetic heart valve into the aortic root was examined. Although the PIV measurements may lack the accuracy of single point measuring systems, the overall view of the flow in the aortic root region compensates for the shortcoming.

The effect of elastin damage on the mechanics of the aortic valve

Porcine bioprosthetic heart valves degenerate and fail mechanically through a mechanism that is currently not well understood. It has been suggested that damage to the elastin component of prosthetic valve cusps could be responsible for changes in the mechanical function of the valve that would predispose it to increased damage and ultimate failure. To determine whether damage to elastin can produce the structural and mechanical changes that could initiate the process of bioprosthetic valve degeneration, we developed an elastase treatment protocol that fragments elastin and negates its mechanical contribution to the valve tissue. Valve cusps were mechanically tested before and after digestion to measure the mechanical changes resulting from elastin damage. Elastin damage produced a decrease in radial and circumferential extensibility (from 43 to 18% strain radially and 12 to 7% strain circumferentially), with a slight increase in stiffness (1.3–2.6 kN/m for radial and 10.6–11.9 kN/m for circumferential directions). Digestions with trypsin, which does not cleave elastin, confirmed that the changes in mechanics of the circumferential samples were likely due to the nonspecific removal of proteoglycans by elastase, while the changes in the radial samples were indeed due to elastin damage. Removing the mechanical contribution of elastin alters the mechanical behavior of the aortic valve cusp, primarily in the radial direction. This finding implies that damage to elastin will distend the cusps, reduce their extensibility, and increase their stiffness. Damage to elastin may therefore contribute to the degeneration and failure of prosthetic valves.

The Biomechanical Effects of Fatigue on the Porcine Bioprosthetic Heart Valve

Characterization of the mechanisms of degeneration of porcine bioprosthetic heart valves (BHV) during long-term cyclic loading is required for predicting and ultimately preventing their failure. Isolation of purely mechanical effects from host biological ones is a necessary first step in understanding the fatigue process as a whole. Thus, in this review we focus on mechanical factors alone as a means of isolating their role in altering biomechanical properties and ultimately their contribution to the fatigue damage process. Mechanical evaluations included tension controlled biaxial, 3-point flexural, and uniaxial failure tests performed on cuspal tissue following 0, 50, 100, 200, and 300 x 10(6) in vitro accelerated test cycles. Overall, biaxial mechanical results indicate a decreasing radial extensibility that can be explained by stiffening of the effective collagen fiber network as well as a small decrease in the splay of the collagen fibers. Moreover, these results suggest that the loss in flexural rigidity with fatigue that we have previously measured (ASAIO 1999; 45:59-63) may not be because of loss of collagen stiffness alone, but also to fiber debonding and degradation of the amorphous extracellular matrix. We discuss the implications of these results that point toward the development of chemical-treatment methods that seek to maintain the integrity of the amorphous extracellular matrix to ultimately extend BHV long-term durability.

Development of bioprosthetic heart valve calcification in vitro and in animal models: morphology and composition

While calcification of bioprosthetic valves presents a clinical problem, the mechanism of formation of calcific deposits in the leaflets remains unclear. A new method for in vitro calcification is employed, in parallel with an in vivo (subcutaneous animal) model. The nature of crystal phases grown by both methods on porcine bioprosthetic heart valves was investigated. Light microscopy, scanning electron microscopy (SEM), X-ray energy dispersive spectroscopy (SEM-EDS) and stoichiometric chemical analysis were used as tools for the qualitative and quantitative assessment of the calcification process. Photomicrographs from leaflets calcified early in vitro (24–48 h) and in vivo (5–10 days) confirmed that calcification initiated in the central region of the tissue, mainly in fibrosa, and extended with time (after 28 and 56 days) in vivo towards the outer surface of the tissue. SEM micrographs from tissue sections of early in vivo and in vitro calcified leaflets showed coexistence of different sizes (1–30 μm) of crystal phases in contact with tissue fibers. EDS analysis confirmed that the deposits were calcium phosphate salts. Chemical analysis of samples calcified in vivo for longer time periods, showed that the content of the salts was mainly calcium and phosphate with a Ca/P molar ratio 1.78 and 1.94 after 28 and 56 days, respectively. These results suggested that the calcification process is followed by a sequential hydrolytic transformation of CaHPO 4·2H 2O (DCPD) to Ca 4H(PO 4) 3·2.5H 2O (OCP) and to Ca 5(PO 4) 3OH (HAP). Calcium phosphate crystal phases grown in vitro under controlled conditions and at constant supersaturation provide a successful simulation of the calcification process in the animal model and may be applied in screening new biomaterials with respect to their potential for calcification.

Elastin Calcification and its Prevention with Aluminum Chloride Pretreatment

Elastin, an abundant structural protein present in the arterial wall, is prone to calcification in a number of disease processes including porcine bioprosthetic heart valve calcification and atherosclerosis. The mechanisms of elastin calcification are not completely elucidated. In the present work, we demonstrated calcification of purified elastin in rat subdermal implants (Ca(2+) = 89.73 +/- 9.84 microgram/mg after 21 days versus control, unimplanted Ca(2+) = 0.16 +/- 0.04 microgram/mg). X-ray diffraction analysis along with resolution enhanced FTIR spectroscopy demonstrated the mineral phase to be a poorly crystalline hydroxyapatite. We investigated the time course of calcification, the effect of glutaraldehyde crosslinking on calcification, and mechanisms of inhibition of elastin calcification by pretreatment with aluminum chloride (AlCl(3)). Glutaraldehyde pretreatment did not affect calcification (Ca(2+) = 89.06 +/- 17.93 microgram/mg for glutaraldehyde crosslinked elastin versus Ca(2+) = 89.73 +/- 9.84 microgram/mg for uncrosslinked elastin). This may be explained by radioactive ((3)H) glutaraldehyde studies showing very low reactivity between glutaraldehyde and elastin. Our results further demonstrated that AlCl(3) pretreatment of elastin led to complete inhibition of elastin calcification using 21-day rat subdermal implants, irrespective of glutaraldehyde crosslinking (Ca(2+) = 0.73-2.15 microgram/mg for AlCl(3) pretreated elastin versus 89.73 +/- 9.84 for untreated elastin). The AlCl(3) pretreatment caused irreversible binding of aluminum ions to elastin, as assessed by atomic emission spectroscopy. Moreover, aluminum ion binding altered the spatial configuration of elastin as shown by circular dichroism (CD), Fourier transform infrared (FTIR), and (13)C nuclear magnetic resonance (NMR) spectroscopy studies, suggesting a net structural change including a reduction in the extent of beta sheet structures and an increase in coil-turn conformations. Thus, it is concluded that purified elastin calcifies in rat subdermal implants, and that the AlCl(3)-pretreated elastin completely resists calcification due to irreversible aluminum ion binding and subsequent structural alterations caused by AlCl(3).

Effects of mechanical fatigue on the bending properties of the porcine bioprosthetic heart valve.

The mechanisms underlying the failure of porcine bioprosthetic aortic heart valves are not well understood. One possible explanation is that delaminations of the layered leaflet structure occur through flexion, leading to calcification and further delaminations, and finally resulting in valve failure. We investigated the changes in flexural rigidity of the belly of aortic valve cusps subjected to accelerated durability testing. We used three-point bending wherein a load was applied to the center of each specimen by a thin stainless steel bar calibrated to a known load-displacement relationship. Ten circumferential and 15 radial specimens from valves fatigued to 0, 50, 100, and 200 million cycles were flexed both with and against the curvature of the cusp. Linear beam theory was applied as a means to compare the relative bending stiffness between groups. Although specimens aligned to the circumferential direction were stiffer when bent against the cuspal curvature, the radial oriented specimens exhibited no bending directional dependence. Both the radial and circumferential specimens experienced a significant decrease in the bending stiffness with an increased number of accelerated test cycles. Overall, our results suggest that it is the fibrosa that experiences the greatest loss of stiffness with mechanically induced fatigue damage.

Effects of accelerated testing on porcine bioprosthetic heart valve fiber architecture

We undertook the following study to quantitatively assess the changes in porcine bioprosthetic heart valve (PBHV) fiber architecture to increasing levels of fatigue damage using an in vitro accelerated test model. PBHVs were subjected to 0–500 million test cycles at 16 Hz, and small-angle light scattering (SALS) was used to quantify the gross fiber structure of the cusps. The degree of gross fiber alignment remained essentially constant from 0 to 500 million cycles over the entire cusp. Increasing fiber orientation randomness, indicative of local damage, was observed only in the vicinity of the nodulus of Arantii after 50 million cycles. The SALS data from the damaged regions suggested shearing between fiber layers, which may be part of the failure process and accelerates valve failure. Histological analysis revealed a relatively intact gross fiber structure with the collagen fiber crimp remaining, although delamination and de-registration of the crimp was also observed. Accelerated tested PBHVs also demonstrated a pronounced ‘sagging’, which began at the earliest cycle number tested (1.4 million cycles) and whose rate decreased logarithmically with cycle number. Results of this study suggest that PBHV cusps can alter their shape without any visually apparent material yielding or fiber failure under continual cyclic loading. Further, while most of the 4 mmHg pressure fixed PBHV’s gross fiber architecture remains unchanged after 500 million cycles of accelerated testing, localized accumulated fiber damage can occur on a sub-visual structural level as early as 50 million cycles.

Calcification Potential of Small Intestinal Submucosa in a Rat Subcutaneous Model

Glutaraldehyde treatment of collagen biomaterials promotes calcification, poor host–tissue incorporation, and ultimately mechanical failure of bioprotheses. Porcine small-intestinal submucosa (SIS) is a biomaterial which has been investigated for several applications including arterial and venous grafts and repair of tendon, ligament, body wall, and urinary bladder defects. The calcification potential of peracetic acid (PAA)-sterilized SIS was studied. Four test samples, (1) native (cleaned, untreated) SIS, (2) SIS sterilized with 0.1% PAA, (3) SIS treated with 0.25% glutaraldehyde for 20 min, and (4) commercially available glutaraldehyde-preserved porcine bioprosthetic heart valve cusp segments (GPV), were each implanted subcutaneously in each of 24 weanling rats. Six rats were euthanatized at 1, 2, 4, and 8 weeks. Evaluation of calcium concentration by atomic absorption spectroscopy and extent of mineralization and fibrosis by light microscopy were performed. Atomic absorption revealed no calcification in native or peracetic acid-treated SIS at any time point compared with preimplant calcium concentration. Statistically significant (P< 0.0001) calcification occurred in glutaraldehyde-treated materials (SIS and GPV) at each evaluation as compared to native and peracetic acid-treated samples. Histopathology indicated native and peracetic acid-treated SIS showed no implant mineralization (P< 0.0001) and little peri-implant fibrosis (P< 0.0001). Results suggested that native and peracetic acid-treated SIS have a low calcification potential and further study of this biomaterial is warranted.

Differential calcification of cusps and aortic wall of failed stented porcine bioprosthetic valves

In this study, we examined separately calcification of cusps (C) and associated aortic wall (AW) of 38 (13 aortic and 25 mitral) porcine bioprosthetic heart valves explanted from 37 patients (ages 25-80 years, mean 59) for structural dysfunction, following 54-210 months (mean 125 months aortic, 119 months mitral). Valves were sectioned into C and corresponding AW components; calcification was assessed by atomic absorption spectroscopy for calcium and histologic examination. Overall, AW calcification was half that of C (33.3 ± 5.4 vs. 65.9 ± 6.3 μg/mg, mean ± standard error of the mean respectively, p = 0.002). Correlation of calcification in individual C/AW pairs was weak (r2 = 0.34). Calcification in C was nodular, largely in the valve fibrosa, but AW calcification predominated in the cells between elastic lamellae; large nodules were sparse. We conclude that since AW calcification in these failed porcine valves was neither prominent nor clinically significant, this process should rarely if ever be a limiting factor in the function of stented porcine valves, and that development of anticalcification therapies directed toward the AW of stented valves should be of low priority. However, in stent-free valves, the AW is not covered by prosthetic material, and the level of calcification could be greater and more likely to cause clinical problems through stiffening, embolism, and/or protrusion into the lumen of calcific masses. © 1997 John Wiley & Sons, Inc.