An Improved Bioprosthetic Heart Valve are currently being used. These are Background ot~ the Invention mechanical and bioprosthetic heart valves. Mechanical heart valves are The human heart has four made of 100% synthetic materials.
chambers -two small upper chambers These valves are very durable, but (called atria) and t:wo large lower have several drawbacks. Mechanical chambers (called ventricles). Each valves are not blood compatible and ventricle has. a one-way inlet valve require the life - long use of and a one-way outlet valve. '.the ~ anticoagulants. A problem inherent tricuspid valve opens from the right with anticoagulation therapy is that atrium into t:he right ventricle, and women on anticoagulants are not the pulmonary valve opens from the able to undergo pregnancy and right ventricle into the pulmonary childbirth. Mechanical heart valves arteries. The :mitral valve opens from also have very poor hemodynamics the left atrium into the left ventricle, which can lead to other complications and the aortic valve opens from t:he (2). Bioprosthetic heart valves may, for left ventricle into the aorta (1). example, consist of porcine aortic valve cusps or bovine pericardium supported on All valves in a human heart are a rigid stent made of either metal or tricuspid - tihey have three cusps polymer covered by a Dacron shroud.
which are forced up and down to Their overall design is very similar to that of open and close the valve. natural heart valves so they have improved hemodynamics. Long term anticoagulant The heart valves c.an therapy is not required with bioprosthetic malfunction for a variety of reasons. heart valves(2).
Birth defects contribute a small number of valve failures. However, valves may ah>o fail either by leaking The main problem with (valve regurgit:ation) or by failing to bioprosthetic heart valves is that they open adequately (valve stenosis). fail within 10 to 15 years of Either problem can seriously interfere implantation (3). Consequently, even with the heart's ability to pump blood ~ though bioprosthetic heart valves and when serious enough, a provide patients with a better quality replacement heart valve may be of life, they are presently only used in needed older patients because of their limited life span.
Replacement Heart Valves The tyvo major causes of the Heart valve replacements are failure of bioprosthetic heart valves often used as a solution to heart valve are tearing (mechanical failure) and disease in both adults and children. calcification of the valve cusps (4).
~vo types of replacement heart valves Design In accordance with one Embodiment, the invention provides a material for use in a biopro:~thetic heart valve, the material comprising :heart tissue prepared by contacting the heart tissue for a suitable :period of time with dimethylsulphoxide (DMSO). The heart tissue may then be fixed with glutaraldehyde.
The heart tissue may be,, for example, aortic valve tissue such as porcine aortic valve cusp: or bovine pericardium. Porcine aortic valva cusps are preferred.
In accordance with a preferred embodiment, the invention provides a material for use in a bioprasthetic heart valve comprising :heart tissue prepared by contacting the heart tissue with DMSO at a concentration of about 4Oo, followed by fixing the heart tissue with glutaraldehyde at a concentration of about 0.5%.
In accordance with a further embodiment, the invention provides a method for preparing heart tissue for use in a bioprosthetic heart valve comprising contacting the heart tissue for a suitable period of time with DMSO. The method may further include the step of fixing the DMSO treated heart tissue with glutaraldehyde .
In accordance with a preferred embodiment, the invention provides a method for prepar~.ng heart tissue for use in a bioprosthetic heart valve connprising contacting the heart valve tissue with DMSO at a concentration of about 40o followed by fixing the heart valve tissue with c~lutaraldehyde at a concentration of about 0.50.
Heart tissue such as porcine aortic valve cusps or bovine pericardium treated by the method of the invention may then be used to fashion a bioprosthetic heart valve as previously described, for example, by support on a rigid stent of: metal or polymer covered by a Dacron shroud.
Effects of Glutaraldehyde -Causes of Calciffcation Treatment The causes of bioprosthetic To reduce the antigenicity of heart valve calcification are not porcine aortic valve cusps, they clearly known, but are believed are to be pretreated in glutaraldehyde beforerelated to lipids present in the tissue being incorporated into a (7). The heart valve cusp is made up bioprosthetic heart valve (5). of three layers: the . fibrosa, spongiosa, Glutaraldeh;yde is a five carbon and ventricularis (8). The fibrosa is on aliphatic molecule with an aldehydethe top surface of the cusp with the at each end of the chain. ?;;ach ventricularis on the bottom. In the aldehyde can react chemically fibrosa, large diameter collagen with - fibers , the amino groups on collagen to circumferentially oriented are form a network of cross-links. arranged in a corrugated manner Unfortunately, glutaraldeh.yde which allows the three cusps in a treatment lilberates within the valve to expand radially, typically valve to tissue, phospholipids, proteoglyc:ans50% strain, this expansion enables and cell debris which attract the three leaflets to come together' calcium ions (6). These calcium nucleationand seal off the aperture. The centers are believed to eventuallyventicularis provides the tensile lead recoil to tissue calcification. In addition,necessary to retain the folded shape glutaraldehy~de alters the mechanicalof the fibrosa. It consists of elastin properties of the tissue (4). fibers arranged radially, transverse Although to it slightly increases the tensilethe large collagen fibers. The strength of the tissue, its internalspongiosa has a gelatinous, watery shear properties dramatically consistency due to a loose decrease leading to shear stiffness.
Initially, it was believed that collagenous network and abundance the tissue stiffening was related of acid-mucopolysaccharides. It to the is collagen crysslinking but recent speculated that the spongiosa likely research sul;gests that dehydrationfaci I itates shearin g between the may also be an important factor. fibrosa and the ventricularis during Glutaraldehyde dehydrates v~~ve the strai htenin of the corru tissue as crosslinking occurs. ati Recen t g g g ons studies of the shear properties. (~). The largest calcific deposits of normal, hydrated, and dehydrated involve the spongiosa and sometimes valve tissue :have demonstrated extend into adjacent regions of that the maintaining hydration during fixationfjbrOSa and ventrlCUlarIS (9) .
can produce a more pliable tissue with a shear modulus more similar to fresh valve tissue. Hydration during fixation may produce a more flexible heart valve 'with better mechanical properties anal longer life span.
4 _ The present invention provides The present inventors have observed an improved bioprosthetic heart: the ability of the solvents ethanol and valve with a longer life span, DMSO to remove lipids from porcine better .
mechanical properties and greateraortic heart valve cusps, have studied resistance to calcification. A the mechanical properties of heart study looks at the effectiveness of valve cusps pretreated with different using . ethanol pretreatment in concentrations of ethanol or DMSO
removing lipids from the cusps by performing tensile and shear and examines the mechanical properties testing of the cusps, and have of the resulting tissue. It also looks at the effectiveness of dimethyl compared the mechan ical properties sulphoxide (DMS~O), a dipolar of heart valve cusps pretreated aprotic with solvent, in removing lipids in different concentrations of ethanol heart or valve tissue. DMSO was selected DMSO to fresh cusps and cusps fixed because it replaces hydrogen bonded with glutaraldehyde.
water in the tissue, and maintains tissue structural integrity and hydration during glutaraldehyde Materials aad Methods treatment. It is hypothesized that DMSO pretreatm~nt would produce A Use of Ethanol and DMSO to a lipid free, hydrated, heart valveDissoi~e irree Lipids tissue which is more pliable and has better mechaizical properties and betterdole hearts were obtained from freshly slaughtered pigs at the abbattoir.
calcification control than existingThey were d li d e bio vere rosthetic heart packed in ice within 24 hours l of Th p slaughter. The cusps were excised Va within 12 VeS.
e development of an improved hours, blotted dry and weighed.
They were bioprasthetic hf'.art Valve Wlth rinsed in Hanks solution. The a cusps were mounted on an optical light microscope longer hfe Spal7. wlll lmprOVe and a the ~~t~ of the location of lipids Cluahty of life and SurVlVa1 ratewas recorded.
of Th ree cusps were placed in each different cardiac patients. concentration of ethanol/DMSO
(20%) 40%, 80%, and 100%) for 24 hours and examined again for the location of lipids.
B Ethaaol and DMSO Pretreatment of Porcine Aortic Valve Cusps extension rates of 0.1) 0.2 and 1.0 mm/s in a cyclic manner(11).
Cusps were removed from pig hearts of freshly slaughtered pigs. They were blotted dry and Data AI181 S1S
rinsed in Hanka solution. Three cusps were placed in each different concentrations of ethanol/DMSO (20%) 40%, 80% and 1CI0%) Modules of Elasticity for 24 hours. The cusps were then blotted dry and transferred into a 0.5% Experimentally, for a given sample, a load glutaraldehyde/ Hank solution for 24 hours. versus extension data set was recorded.
The cusps were retrieved from the reaction Typical data is shown in Figures 1 and 2. To solution, blotted dry and stored in Hanks convert the data set into useful information solution in the refrigerator for subsequent on mechanical properties of the material, its tensile and shear testing. modules has to be determined.
C Tensile Testing Definition of Modules of Elasticity (E) Testing was can-ied out using E = FAA ( 1) a hydraulically powered MTS machine with a data oL/L
acquisition system and a special sample grip designed for thin strips of tissue. The sampleE - Modules of Elasticity was placed in a test tank filled with Hank solution(Young's Modules) and maintained at a temperature of F = force 37C) to mimic physiolol~ical temperature.A = area Strips of tissue 10 m:m x 5 mm were cut oL = change in length circumferentiall,y with a scalpelL = original length from the centre of each cusp. A custom built thickness gauge was used to measure the F/A = stress (o) thickness of each sample. Load-extension relationshipsoL/L = strain (E) were obtained at extension rates of 0.3, 3 and 30 mni/s to determine the stress-strainHence , relationships and elastic moduli E = stress = _o of the cuaps.
Strain E
D Shear Tenting Under tensile conditions:
The apparatus consisted of a high-preci::ionstress = tensile stress linear actuator ~uzd micro load E = tensile modules cell combined with a custom made tissue mount.
The linear actuator was a p:iezo electricallyWhen shear force is applied to driven motor the sample which could generate displacementsstress = shear stress with a precision of greater than 1 lum modules = shear modules (G) at speeda of between 0.1 mm/s and 1 mm/s over a travel of 2.5 em. Samples were prepared Modules is a measure of the strength by placing of a the cusps flat on. a rubber surfacematerial.
and using a stainless steel punch to obtain circular specimens of 6.a mm diameter. Testing under Tensile Conditions A thickness gauge was used to measure the thickness of the sample. Each sample was gluedIn order to calculate E) the data between must twa mounting blocks using absorbentbe converted from load vs. extension paper to stress soaked with cyanoacrylate adhesive.vs. strain. To convert load to '.Che stress, the load samples were mounted in such a at each interval must be divided way that the by the cross-deform ation w;is in a circumferentialsectional area, which can be calculated by direction. The tissue mount was multiplying the width and the then thickness of the attached to the shear testing sample. To convert extension to apparatus. Teats strain, the were performed at 37C in Hank change in extension at each interval solution. must be Load-extension tests were performeddivided by the original length, at which is measured at the beginning of the experunent. 'Ethanol concentrations of 40%, $0%
The data can then be re-plotted to give a ~d 100% and DMSO concentrations stress vs. stravz graph. Of 20%, 40%, 80% and 100°/a However) in order to calculate Young's removed lipids from CLISpS. HOWeVer, Modular, the :dope of the graph needs to be 20% ethanol was ineffective in determined. The raw data is fitted using the dissolving lipids from cusp samples.
empirical equation = Ae + B~ + c (2) Teasile Testiag = stress Stress-strain curves for the ethanol E = strain pretreated samples are shown in A, B) C, K = ad~lusdble parameters Figure 3 and compared to those for Once the data. is fitted with fresh and glutaraldehyde fixed this function) (fixed tissue) modulus can be calculated by taking;samples.
the derivative of the function:
da = A -~ KB~ (s) Cusps pretreated with DMSO are d E shown in Figure 4 and compared to those for fresh and gultaraldehyde Moduli were calculated at 0.1 fixed samples. It Can be seen :MPa) that which is the average blood pressure.40% DMSO had tenSlle properties This way) the strength of the cusps treated with different concentrations of ethanolVery Similar t0 fresh tlSSUe.
and DMSO
can be compared to fresh and faced tissue.
Shear Testiag Testiag wader Shear Coaditions Unlike tensile testing, the crossheadShear stress-strain curves for the m oves forwards, back to the originalethanol pretreated samples are ;spot) shown backwards and then back to the in Figure 5 and compared to those original spot. for The initial data recorded can fresh and gultaraldehyde fixed be graphed to give a load vs. extension graph in both the Samples. The Shear moduluS Of forward and the: reverse directions.
cusps pretreated with 20/a ethanol The shear curve is different fromiWere also Slmllar t0 those Of the fresh tensile curve and has four distincttissue.
sections :
rising positive, falling positive, rising negative and falling negative (Figure 2).
The data is converted from load vs. extensionshear StreSS-Strain CurVeS for to stress vs. the strain using eqadon (2). The modularDMSO pretreated Samples are Shown for .each pair of and a a~as calculated In Figure 6 and compared to those using the rising for positive section of the curve fresh alld gultaraldehyde fixed using equation samples. Cusps pretreated with 40/o DMSO had shear stress-strain curves Results that were similar in shape to fresh tissue.
Lipid Solubility Discussion Lipid solubility results are shown in Table 1 Solvent concentrations tested . Lipid Solubility were 0I, 20I, 40/0, 80lo and 100/0.
Results in Table 1 show that DMSO~ Effect Of Pre-treatment On is a more effective solvent for dissolvingMechanical Properties Of Tissue lipids than fahanol. Since DMSO
is a dipolar aprotic solvent, it is Structurally, the heart valve expected cusp is to be more compatible with moleculesmade of three layers, the fibrosa, such as phospholipids and thereforespongiosa and ventricularis. The has better solubility as indicatedinternal structure of a fresh by cusp the present results. Hence for consists of materials making up better these control of c~~lcification, DMSO layers and is supported by a network would be the preferred pretreatment of hydrogen bonded water.
solvent.
Glutaraldehyde treatment crosslinks Tensile Aail Shear Testing the collagen in the cusps by removing the hydrogen bonded water. This The modulus of elasticity equationcauses the internal structure of the predicts that a plot of stress tissue to collapse and results versus in strain for a given material woulddrastically altered mechanical be linear with the slope of the curveproperties. Depending on the corresponding to the modulu;s concentration of ethanol used, of elasticity. As can be seen in ethanol pretreatment removes some Figures 3 and 4, the stress vs. strain curvesof the hydrogen bonded water from for all samples tested are not linear.the tissue. This causes a partial 'This implies that :E is not constant. collapse of the tissue structure., This is typical of animal tissue. As the similar to that of glutaraldehyde rnost important h values are those at fixation. Therefore, the mechanical average blood pressure ( 100-120 properties of tissue pre-treated with kpa), they are determined and ethanol are also very different from.
reported in Table 2. It can be fresh tissue. DMSO, being a dipolar seen , that tissue pretreated with 40% aprotic solvent, can substitute for the DMSO has the E value which is hydrogen bonded water in the tissue closest to that of fresh tissue. instead of removing it. It has been used for the cryopreservation of The shear stress-strain curves tissue. This indicates that DMSO
shown in Figures S and 6 exhibited pretreatment allows the tissue to nonlinear b~haviour similar to maintain its structural integrity the when ten site curves. Since the physiologicalb a i n g c r o s s 1 i n k a d w i t h shear stress is unknown, shear glutar~aldehyde. Therefore pretreating rnoduli (G) as a function of strain, are the tissue with a suitable calculated and compared in Table concentration of DMSO/water would 3.
The closest rnatch to fresh tissueresult in fixed tissue which has are the moduli of tissue pre-treated mechanical properties that are with more 40% DMSO and 20% ethanol. These similar to those of fresh tissue as moduli are also much closer to indicated by the present results.
that of fresh tissue than moduli of tissue treated by ghutaraldehyde alone.
Conclusions Furthermore, pretreatment with 40%
DMSO was effective in removing lipids 1. Lipid Solubility and reducing calcification.
DMSO at concentrations of 40/~ Therefore, heart valve tissue to 100% was effective in removing pretreated with 40% DMSO and fixed lipids and reducing calcification. in 0.5% glutaraldehyde would produce an improved bioprosthetic
2. Tensile: Testing heart valve that is more durable and has better mechanical properties and The stress-strain curve of for better calcification control than DMSO i s at 40% concentration showed good presently available commercially or in tensile properties very similar the research literature to frfsh tissue. This is a marked improvement over the tensile properties of glutaraldehydLe fixed tissue which.
is presently used for bioprosthetic heart valves. DMSO pretreatment of heart valve tissue at 40% DSMO concentration before fixation
3. Shear Testing in 0.5/ glutaraldehyde was effective in removing lipids and therefore reducing Cusps pretreated with 40% DMSO calcification and gave valves with better had shear stress-strain curves mechanical properties (shear and and tensile elastic moduli that were very properties) than valves pretreated similar with to fresh tissue. This is a significantethanol or any bioprosthetic heart valve improvement in the shear propertiespresently available commercially.
of valve cusps compared to those fixed in glutaraldehyde.
Summary Humanitarian Considerations At present the two main causes of ~ Proved bioprosthetic heart valve bioprosthetic heart valve failure are has important humanitarian values.
tearing (mechanical failure) arid It would provide patients with a better calcification. quality of life free from the need for life long anticoagulant therapy. With The results o:f the present project a sufficiently durable bioprosthetic demonstrated that pretreating heart heart valve these benefits could be valve tissue wiah 40% DMSO befora extended to younger patients and fixation with glutaraldehyde produced flow women with heart valve a heart valve tissue that has much replacements to forgo anticoagulant better mechanical properties (tensile ~erapy and bear children. An and shear) than the glutaraldehyd.e improved bioprosthetic heart valve fixed tissue that is presently being would also reduce the need for used for bioprosthetic heart valves, repeated heart surgery and improve the survival rate of cardiac patients.Calcification of Bovine Pericardium Used in Cardiac:
Ecoaomic Considerations V a 1 v a B i o p r o s t h a s a s Implications for the Aside from the important Mechanisms of Bioprosthetic humanitarian values of an improvedTissue Mineralization. Am. J
bioprosthetic heart valve there Path. 1986;123:134-145 are also economic values. There its 7. forge-Herrero E, Fernandez a market for an improved bioprostheticP, Gutierrez M, Castillo-heart valve. :Each year there Olivares JL: Study of the are over 300,000 heart valve replacements Calcification of Bovine worldwide at. an estimated cost Pericardium: Analysis of the of $2 billion annually. As the size implication of Lipids and of the aging population increases there Proteoglycans. Biomaterials.
would be an increased demand for 1991;12:683-689 replacement heart valves in the 8. Dunmore-Buyze J, Boughner near future. D, Macris N, Vesely I: A
Comparison of Macroscopic References lipid Content within Porcine Pulmonary and aortic Valves. J
1. Merck Maaual of Medical Thorac Card. Surg.
Infora~atioa - Home Edition. 1995;110:1756-61 Chapter 19 (found on the 9. Levy RJ, Sehoea FJ, Golomb internet) G: Bioprosthetic Heart Valve 2. Lefrak, Edward A, Starr A: Calcification. CRC Critical Cardiac Valve Prostheses. Reviews in Biocompatibility Appleton - Century - Crofts. 1VY.1986;2:147-187 1979. 10. Vyavahare N, Hirsch D, 3. Gabba~,~ S, Kadam P, Factor Lerner E, Baskia JZ:
S:
Do Heart Valve Bioprostheaes Prevention of Bioprosthetic Degenerate for Metabolic or Heart Valve Calcification by Mechanical Reasons. J Thorac Ethanol Preincubation.
Card Surg. 1988;95:208-15 Circulation. 1997;95:479-488
4. Talma:n E, Boughner D: 11. Talmaa E, Boughaer D: A
Glutaraldehyde Alters the Versatile Apparatus for Shear Internal Shear Properties of Testing of Soft Tissues. JP
orcine Aortic Heart V~~lve Robarts Research Institute.
Tissue. Ann Thorac Surg. 1994 1995;60: S369-73
5. Jayakrishnaa A, Jameela SR:
Glutar~sldehyde as a fixative in Biopro;stheses and Drug DeluveryMatrices(review) .
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6. Schoea FJ, Tsao JW, Leroy RJ:
Table 1 Lipid Solub' 'ty in Different Concentrations of Ethanol and DMSO
Solvent Concentration Lipids Present After 24hrs?
Yes No DMSO 20% _ 40% '/
80%
100%
Ethanol 20% 1/
40% f/
80%
100%
Hank's Control ~/
Table 2 Elastic Moduli of Cusps Pretreated with Different Concentrations of Solvents Determined At Average Blood Pressure X0.11 Mpaj DM SO Ethanol ~'LSC~d~'~i~ 20% ,40% 80% 100% 20% 40% 80% 1.00%
_ Tts~ ""~c~~5~~.
Vie.
Elastic Modulus ~g 1 ~ 67 4.67 6.1~ 6.61 6.14 4.71 3.02 2.68 4.24 M a Table 3 Shear Moduli of Cusps Pretreated with Different Concentrations of Solvents Fr~s~:. ~=~xei~.DMSO Ethanol G k: G 1 a a Strain G (~- Ea ( 20% ~~~'jc>80% 100% ~93~~~ 40% 80% 100%
a - o~) 0.1. - 14.~ 4.0 1g 4.1 16.8 1.~ 11.1 0-22 1.2 ! 7.$ 6.6 2~ 4.9 16.9 1.~ 15.0 _0. - 23=2. 10.7 ~: 6 6.1 18.2 2.2 21.4 ~~
0.4 201 ~ 17.4 ~~- 6.2 * 3~0 0_. ~ 28.3 8r3 8.1 * ~..~.
~~
O. EE. ~. ~' * l 2.9 10. * 7~ A
0. i' 8~3 '~ * 203 15.4 * I ( .5 o.~ 1a.9 ~ * ~ * * ~~
T curves ctict not reach strain