Danielle Hirsch

Prevention of bioprosthetic heart valve calcification by ethanol preincubation: Efficacy and mechanisms

BACKGROUND Calcification of the cusps of bioprosthetic heart valves fabricated from either glutaraldehyde cross-linked porcine aortic valves or bovine pericardium frequently causes the clinical failure of these devices. Our investigations studied ethanol pretreatment of glutaraldehyde cross-linked porcine aortic valves as a new approach to prevent cuspal calcification. The hypothesis governing this approach holds that ethanol pretreatment inhibits calcification resulting from protein structural alterations and lipid extraction. METHODS AND RESULTS Results demonstrated complete inhibition of calcification of glutaraldehyde-pretreated porcine bioprosthetic aortic valve cusps by 80.0% ethanol in rat subdermal implants (60-day ethanol-pretreated calcium level, 1.87 +/- 0.29 micrograms/mg tissue compared with control calcium level, 236.00 +/- 6.10 micrograms/mg tissue) and in sheep mitral valve replacements (ethanol-pretreated calcium level, 5.22 +/- 2.94 micrograms/mg tissue; control calcium level, 32.50 +/- 11.50 micrograms/mg tissue). The mechanism of ethanol inhibition may be explained by several observations: ethanol pretreatment resulted in an irreversible alteration in the amide I band noted in the infrared spectra for both purified type I collagen and glutaraldehyde cross-linked porcine aortic leaflets. Ethanol pretreatment also resulted in nearly complete extraction of leaflet cholesterol and phospholipid. CONCLUSIONS Ethanol pretreatment of glutaraldehyde cross-linked porcine aortic valve bioprostheses represents a highly efficacious and mechanistically based approach and may prevent calcific bioprosthetic heart valve failure.

Onset and progression of calcification in porcine aortic bioprosthetic valves implanted as orthotopic mitral valve replacements in juvenile sheep

The purpose of this study was to characterize the onset and progression of mineralization in porcine bioprosthetic valves implanted in sheep and to test the hypothesis that such valves simulate calcification that is observed clinically and in other experimental models. Hancock I porcine aortic bioprosthetic valves (Medtronic Heart Valve Division, Irvine, Calif.) were implanted as orthotopic mitral valve replacements in juvenile sheep, retrieved after 1 to 124 days, and analyzed as follows: gross inspection, radiography, light, transmission, and surface scanning electron microscopy, and calcium analysis by absorption spectroscopy. Mineralization increased with increasing time after implantation in both valve cusps and adjacent aortic wall. Mean cuspal calcification was 80 micrograms/mg in valves removed after 3 to 4 months. Nevertheless, considerable variability among valves was apparent in the level of calcification noted at specific time intervals. Virtually all aspects of the morphologic characteristics were identical to those previously noted for clinical explants and experimental specimens, both subcutaneous and circulatory. In particular, ultrastructural examination revealed that the earliest calcific deposits were associated with devitalized cuspal connective tissue cells and their fragments. Collagen calcification was sparse. Both surface scanning and transmission electron microscopy indicated a lack of endothelial or blood-derived cells on the valves at all sampling times. We conclude that porcine bioprosthetic valves implanted as mitral valves in sheep provide a useful calcification model, simulating morphologic and pathobiologic events that occur clinically and in noncirculatory models. However, sufficient specimen replicates must be done to overcome variability in calcification among valves and sampling sites.

Colocalization of cholesterol and hydroxyapatite in human atherosclerotic lesions

SummaryCholesterol and calcium phosphate, the latter in the form of hydroxyapatite, accumulate in atherosclerotic lesions. In this report, we demonstrate that these organic and inorganic constitutents of lesions can accumulate together, closely associated in crystal agglomerates. Using the fluorescent cholesterol probe, filipin, we identified unesterified cholesterol that was associated with calcium granules in tissue sections of lesions. We also have shown that small crystallites of cholesterol can associate with preformed hydroxyapatite crystals in vitro. Scanning electron microscopy couple with energy-dispersive X-ray analysis demonstrated the physical association of many small crystallites of cholesterol with larger crystals of hydroxyapatite. These small crystallites of cholesterol associated with hydroxyapatite stained with filipin. This contrasted with the lack of filipin staining of unassociated larger cholesterol crystals or hydroxyapatite alone. How cholesterol and calcium come to be closely associated in crystal agglomerates within atherosclerotic lesions remains to be determined.

Prevention of calcification of glutaraldehyde-crosslinked porcine aortic cusps by ethanol preincubation: mechanistic studies of protein structure and water-biomaterial relationships.

Clinical usage of bioprosthetic heart valves (BPHVs) fabricated from glutaraldehyde-pretreated porcine aortic valves is restricted due to calcification-related failure. We previously reported a highly efficacious ethanol pretreatment of BPHVs for the prevention of cuspal calcification. The aim of the present study is to extend our understanding of the material changes brought about by ethanol and the relationship of these material effects to the ethanol pretreatment anticalcification mechanism. Glutaraldehyde-crosslinked porcine aortic valve cusps (control and ethanol-pretreated) were studied for the effects of ethanol on tissue water content and for spin-lattice relaxation times (T1) using solid state proton NMR. Cusp samples were studied for protein conformational changes due to ethanol by ATR-FTIR spectroscopy. The changes in cuspal tissue-cholesterol (in vitro) interactions also were studied. Cusp material stability was assessed in terms of residual glutaraldehyde content and collagenase degradation. Water content of the cusp samples was decreased significantly due to ethanol pretreatment. The cuspal collagen conformational changes (per infrared spectroscopy) brought about by ethanol pretreatment were persistent even after rat subdermal implantation of cusp samples for 7 days. In vitro cholesterol uptake by cusps was greatly reduced as a result of ethanol pretreatment. Ethanol pretreatment of cusps also resulted in increased resistance to collagenase digestion. Cuspal glutaraldehyde content was not changed by ethanol pretreatment. We conclude that ethanol pretreatment of bioprosthetic heart valve cusps causes multi-component effects on the tissue/material and macromolecular characteristics, which partly may explain the ethanol-pretreatment anticalcification mechanism.

Current Progress in Anticalcif ication for Bioprosthetic and Polymeric Heart Valves

The use of bioprosthetic valves fabricated from fixed heterograft tissue (porcine aortic valves or bovine pericardium) in heart valve replacement surgery is limited because of calcification-related failures. The mechanism of calcification of bioprosthetic valves is quite complex and has a variety of determinants, including host factors, tissue fixation conditions, and mechanical effects. Currently, there is no effective therapy to prevent calcification in clinical settings. This article reviews a variety of anticalcification strategies that are under investigation either in advanced animal models or in clinical trials. Bisphosphonates, such as ethan hydroxybisphosphonate (EHBP), inhibit calcium phosphate crystal formation. However, because of their systemic toxicity, they are used as either tissue treatments or polymeric site-specific delivery systems. Detergent treatment, such as sodium dodecyl sulfate (SDS), extracts almost all phospholipids from bioprosthetic heart valve cuspal tissue. Procedures, such as amino oleic acid pretreatment, inhibit calcium uptake. Polyurethane trileaflet valves, investigated as alternatives to bioprosthetic or mechanical valve prostheses, undergo intrinsic and thrombus-related calcification and degradation. Calcification- and thrombus-resistant polyurethanes synthesized in our laboratory by covalent linking of EHBP or heparin (either in bulk or on surface) by unique polyepoxidation chemistry are attractive candidates for further research. Tissue-engineered heart valves may have an important place in the future.

Effects of metallic ions and diphosphonates on inhibition of pericardial bioprosthetic tissue calcification and associated alkaline phosphatase activity

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.

Synergistic inhibition of the calcification of glutaraldehyde pretreated bovine pericardium in a rat subdermal model by FeCl3 and ethanehydroxydiphosphonate: pre-incubation and polymeric controlled release studies

Calcification is a frequent cause of the clinical failure of bioprosthetic heart valves fabricated from glutaraldehyde-pretreated porcine aortic valves or glutaraldehyde-pretreated bovine pericardium (GPBP). We investigated the hypothesis that ferric chloride (FeCl3) and sodium-ethanehydroxydiphosphonate (EHDP) may act synergistically to prevent bioprosthetic tissue calcification. Pre-incubations and controlled release systems were studied individually. FeCl3-EHDP polymeric controlled release matrices were formulated using silicone rubber and evaluated for in vitro release kinetics at pH 7.4 and 37 degrees C. The effects of Fe-EHDP synergism on GPBP calcification were investigated with 21 d subdermal implants in 3 wk-old male rats. Results demonstrated that levels of Fe3+ and EHDP uptake, measured in GPBP tissues pre-incubated first in an FeCl3 solution (10(-5) M) followed by an EHDP solution (0.1 M), were higher than in the reverse order of incubation. In the first series of rat implants, GPBP was pre-incubated in either FeCl3 or Na2EHDP solutions, or sequential pre-incubations of first FeCl3 and then Na2EHDP solutions, or the reverse. The inhibition of calcification was greatest when FeCl3 (first pre-incubation, 10(-5) M) was combined with Na2EHDP (second pre-incubation, 0.1 M) (1.78 +/- 0.2 micrograms of Ca2+/mg of dried tissue) compared with the other pre-incubation groups: EHDP (first pre-incubation) combined with FeCl3 (second pre-incubation) (21.7 +/- 6.4), FeCl3 solution alone at 10(-5) M (27.9 +/- 10.7), Na2EHDP solution alone at 0.1 M (52.3 +/- 11.9) and the control group (72.3 +/- 10.2). In a second series of implants, GPBP specimens were co-implanted with individual controlled release systems containing one of the following formulations (weight percentage in silicone rubber): 1% FeCl3, 20% CaEHDP, 20% protamine sulphate, 1% FeCl3-20% CaEHDP, and 1% FeCl3-20% protamine sulphate. The 1% FeCl3-20% CaEHDP silicone-rubber matrices were the most effective for inhibiting GPBP mineralization (13.7 +/- 3.0 micrograms Ca2+/mg of dried tissue) compared with non-drug silicone co-implant controls (74.7 +/- 5.58 micrograms Ca2+/mg of dried tissue) and other polymeric treatment groups (32.3 +/- 2.3-80.0 +/- 19.7). No adverse effects on bone or overall growth of any treatment protocols were noted. Thus, combinations of FeCl3 and EHDP, using either pre-incubations or polymeric controlled release, were synergistic for inhibiting GPBP calcification.

Co-crystallization of cholesterol and calcium phosphate as related to atherosclerosis

Abstract Calcification of atherosclerotic plaques occurs very frequently and aggravates the disease. In biological systems, epitaxial relationships between crystal structures may be important in nucleating the deposit of a solid phase. The biologically preferred calcium phosphate species, apatite, and cholesterol crystal have structurally compatible crystallographic faces which allow epitaxial growth of one crystal upon another. The present study describes a new approach to explore, in vitro, the crystallization processes of calcium phosphate (CaP) with cholesterol (CS) and cholestanol (CN) which are related to atherosclerosis. Aqueous solutions containing calcium and phosphate ions or CaP crystals as hydroxyapatite were added into saturated ethanolic solutions of CS or CS and 10% CN. After precipitation, crystals were collected and analyzed by nuclear magnetic resonance (NMR), infra-red (IR), X-ray, scanning electron microscope (SEM-LINK), differential scanning calorimeter (DSC) and atomic absorption. The principal result is the well-formed crystals precipitation when an aqueous solution and CaP seed crystals were added to saturated solutions of CS and 10% CN. Cholesterol-cholestanol dihydrate (CC2W) crystals precipitated in the presence of CaP seeds were compared to the CC2W crystals obtained without the mineral compound. The results of this comparison indicate a special link between crystals of CaP and CC2W, and support the epitaxial relationship between the two kinds of crystals. The potential of CC2W crystals to be precipitated by CaP seed crystals prove likewise the possible significant role of the cholestanol metabolite in the process of cholesterol crystallization and calcification in the arteries.

An extension of the concept of epitaxial growth

Abstract The experimental conditions to maintain metastability in a solution of calcium oxalate during 15 min were defined. Seed crystals of uric acid and sodium urate were added and the decrease of calcium ion concentration was measured by a calcium selective electrode. Sodium urate crystals caused a decrease of about 14% while uric acid seeds were inactive. The growth of calcium oxalate on sodium urate surface was confirmed by X-ray emission analysis. When glumatic acid was added at 4–5 ppm concentration to the metastable solution containing uric acid seeds, a ca. 28% calcium concentration decrease was observed. The precipitation of calcium oxalate on uric acid was confirmed by X-ray emission analysis. The results show that a structural fit at an atomic level between two solids, which is a necessary condition for epitaxis, is not sufficient for epitaxial growth to occur. An additional factor, namely attraction at an ionic or molecular level, is also needed. It may be furnished by a mediating agent compatible with both solids.

Liposome interactions with hydroxyapatite crystals: A possible mechanism in the calcification of atherosclerotic plaques

SummarySome stages in the calcification of atherosclerotic plaques may involve associations between lipids and hydroxyapatite (HA) by surface interactions. Liposomes, artifical membranous lipid vesicles, have been used in this study as model structures for biological calcification processes. Liposome (containing cholesterol and phosphatidylcholine in most cases) suspensions were prepared by sonication, after which HA seed crystals were added to the suspensions and stirred at 37°C. Aliquots of the liposome suspensions were analyzed for particle size distribution and by transmission electron microscopy and electron diffraction. The results showed that HA induced aggregation of liposomes and modifications of the microscopic shapes of the liposomes in the aggregates. These data can be explained by the electron diffraction pattern where superimposition of liposome reflection and crystal reflection exists and may suggest organic-inorganic interaction. The potential of HA crystals to induce formation of liposome aggregates may be seen as a step in atherosclerotic plaques calcification.