Matthew F. Ogle

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.

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 μg/mg after 21 days versus control, unimplanted Ca 2+ = 0.16 ± 0.04 μg/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 μg/mg for glutaraldehyde crosslinked elastin versus Ca 2+ = 89.73 ± 9.84 μg/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 μg/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 β 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 .

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.

Calcification resistance with aluminum-ethanol treated porcine aortic valve bioprostheses in juvenile sheep

BACKGROUND Calcification of glutaraldehyde fixed bioprosthetic heart valve replacements frequently leads to the clinical failure of these devices. Previous research by our group has demonstrated that ethanol pretreatment prevents bioprosthetic cusp calcification, but not aortic wall calcification. We have also shown that aluminum chloride pretreatment prevents bioprosthetic aortic wall calcification. This study evaluated the combined use of aluminum and ethanol to prevent both bioprosthetic porcine aortic valve cusp and aortic wall calcification in rat subcutaneous implants, and the juvenile sheep mitral valve replacement model. METHODS Glutaraldehyde fixed cusps and aortic wall samples were pretreated sequentially first with aluminum chloride (AlCl3) followed by ethanol pretreatment. These samples were then implanted subdermally in rats with explants at 21 and 63 days. Stent mounted bioprostheses were prepared either sequentially as previously described or differentially with AlCl3 exposure restricted to the aortic wall followed by ethanol pretreatment. Mitral valve replacements were carried out in juvenile sheep with elective retrievals at 90 days. RESULTS Rat subdermal explants demonstrated that sequential exposure to AlCl3 and ethanol completely inhibited bioprosthetic cusp and aortic wall calcification compared with controls. However the sheep results were markedly different. The differential sheep explant group exhibited very low levels of cusp and wall calcium. The glutaraldehyde group exhibited little cusp calcification, but prominent aortic wall calcification. All sheep in the two groups previously described lived to term without evidence of valvular dysfunction. In contrast, animals in the sequential group exhibited increased levels of cusp calcification. None of the animals in this group survived to term. Pathologic analysis of the valves in the sequential group determined that valve failure was caused by calcification and stenosis of the aortic cusps. CONCLUSIONS The results clearly demonstrate that a combination of aluminum and ethanol reduced aortic wall calcification and prevented cuspal calcification. Furthermore, this study demonstrates that exclusion of aluminum from the cusp eliminated the cuspal calcification seen when aluminum and ethanol treatments were administered in a sequential manner.

High reactivity of alkyl sulfides towards epoxides under conditions of collagen fixation: a convenient approach to 2-amino-4-butyrolactones

Epoxy crosslinking agents have been investigated for use in the fabrication of bioprosthetic devices, such as heterograft heart valve prostheses. It has been generally assumed that epoxy crosslinking takes place via amino-epoxy reactions. The present study investigated the hypothesis that the reactions of methionine residues with epoxides also can occur in biomaterial crosslinking. A series of model reactions were studied in which a mono-epoxide was combined with individual alkyl sulfides. In the present studies epoxides rapidly alkylate aliphatic sulfides, including methionine derivatives, in buffered aqueous solutions at room temperature and pH close to neutral, forming sulfonium compounds, which are stable at pH 5-7 at temperatures up to 50 degrees C, except for cases in which methionine derivatives with non-protected carboxy groups are used. The rate of reaction remains practically unchanged within the range of pH from 5 to 12, whereas in strongly alkaline media the reverse reaction occurs. This discovery can provide a better understanding of processes occurring in the fixation of bioprosthetic tissues with polyepoxides. It can also develop into a site-specific method to label methionine residues in proteins. The carboxy group-containing sulfonium betaines derived from N-protected methionines undergo cyclization in unexpectedly mild conditions, which can be used as an efficient method for preparation of N-protected 2-amino-4-butyrolactones with sensitive protective groups.