WO1999058166A1

WO1999058166A1 - Improved bioprosthetic heart valve

Info

Publication number: WO1999058166A1
Application number: PCT/CA1999/000420
Authority: WO
Inventors: Wan Kei Wan; Derek R. Boughner
Original assignee: The University Of Western Ontario
Priority date: 1998-05-08
Filing date: 1999-05-10
Publication date: 1999-11-18
Also published as: CA2237229A1

Abstract

Animal heart tissue with improved mechanical properties for use in a bioprosthetic heart valve is prepared by pre-treatment with dimethyl sulphoxide (DMSO) prior to glutaraldehyde fixation.

Description

IMPROVED BIOPROSTHETIC HEART VALVE

Field of the Invention

This invention relates to bioprosthetic valves and, more particularly, to novel methods and materials for the preparation of bioprosthetic valves.

Background of the Invention

The human heart has four chambers, two small upper chambers or atria and two large lower chambers or ventricles. Each ventricle has a one-way inlet valve and a one-way outlet valve. The tricuspid valve opens from the right atrium into the right ventricle, and the pulmonary valve opens from the right ventricle into the pulmonary arteries. The mitral valve opens from the left atrium into the left ventricle, and the aortic valve opens from the left ventricle into the aorta. All valves in the human heart are tricuspid; the three cusps are forced up and down to open and close the valve.

The heart valves can malfunction for a variety of reasons. Birth defects contribute a small number of valve failures. Valves may also fail either by leaking (valve regurgitation) or by failing to open adequately (valve stenosis). Either problem can interfere with the heart's ability to pump blood and in serious cases, a replacement heart valve may be needed.

Artificial heart valves are used as replacements for diseased heart valves in both adults and children. Two types of replacement heart valves are currently being used. The first type, mechanical valves, are constructed of synthetic rigid materials such as plastic or metal. Their use is associated with thrombogenesis, requiring valve recipients to be on long term anticoagulation.

The second type, tissue valves or bioprosthetic valves, consist of valve leaflets of preserved animal tissue either mounted on an artificial support or stent or in the case of stentless bioprosthetic valves, attached to and supported by the aortic root of the animal.

While bioprosthetic heart valves offer the advantage of not requiring long term anticoagulant therapy, their durability is limited to about 10-15 years. The limitations in the long term performance of bioprosthetic heart valves are largely due to mechanical failure, due to tearing of the valve tissue and deteriorated performance due to calcification of the valve cusps. To reduce the antigenicity of animal tissue, it is pretreated in glutaraldehyde before being incorporated into a bioprosthetic heart valve (Jayakrishnan et al., (1996), Biomaterials, v. 17, pp. 471 -484). Glutaraldehyde reacts chemically with the amino groups on collagen to form a network of crosslinks. Glutaraldehyde treatment, however, liberates within the valve tissue phospholipids, proteoglycans and cell debris which attract calcium ions (Schoen et al., (1986), Am. J. Path., v. 123, pp. 134-135). These calcium nucleation centers are believed to eventually lead to tissue calcification. In addition, glutaraldehyde alters the mechanical properties of the tissue. Although glutaraldehyde treatment slightly increases the tensile strength of the tissue, its internal shear properties dramatically decrease, leading to shear stiffness. There remains a need for improved bioprosthetic valves with greater durability, obviating the need for long term anticoagulant therapy while avoiding the need for repeat heart surgery.

Summary of the Invention

The invention provides a method for preparing animal heart tissue suitable for use in bioprosthetic heart valves and with improved mechanical properties, by pre-treating the tissue with dimethyl sulphoxide (DMSO) before fixation of the tissue with glutaraldehyde. Heart tissue such as porcine aortic valve cusps or bovine percardium 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 (Bartek et al., (1974), Thorax, v. 29, p. 51 ). In accordance with one embodiment, the invention provides a method for preparing an animal heart tissue for use in a bioprosthetic heart valve by 3

treatment of the tissue with glutaraldehyde, characterised in that the animal heart tissue is contacted for a suitable period of time with dimethyl sulphoxide (DMSO) before treatment with glutaraldehyde.

In accordance with a further embodiment, the invention provides animal heart tissue for use in a bioprosthetic heart valve prepared by a method comprising:

(a) contacting the tissue for a suitable period of time with DMSO; and

(b) contacting the tissue with glutaraldehyde.

Summary of the Drawings

Certain embodiments of the invention are described, reference being made to the accompanying drawings, wherein:

Figure 1 shows a graph of tensile load (Y axis) against extension (X axis) for porcine heart valve cusps pre-treated with 20% ethanol. Figure 2 shows a graph of shear load (Y axis) against extension (X axis) for porcine heart valve cusps pre-treated with 20% ethanol.

Figure 3 shows a graph of stress (Y axis) against strain (X axis) for porcine heart valve cusps pretreated with the indicated concentrations of ethanol. Figure 4 shows a graph of stress (Y axis) against strain (X axis) for porcine heart valve cusps pretreated with the indicated concentrations of DMSO.

Figure 5 shows a graph of shear stress (Y axis) against shear strain (X axis) for porcine heart valve cusps pretreated with the indicated concentrations of ethanol.

Figure 6 shows a graph of shear stress (Y axis) against shear strain (X axis) for porcine heart valve cusps pretreated with the indicated concentrations of DMSO.

Detailed Description of the Invention

Animal tissue used for bioprosthetic heart valves includes porcine aortic 4

valve cusps and bovine pericardium which is fashioned into valve leaflets; these porcine valve cusps or leaflets fashioned from bovine pericardium have to be mounted on a support or stent to form a bioprosthetic heart valve. Also used is the complete porcine aortic root bearing the porcine aortic valve cusps, which may be employed as a stentless bioprosthetic valve.

In all of these cases, the animal tissue is treated with glutaraldehyde, which fixes the tissue and reduces its antigenicity in humans, before it is incorporated into a bioprosthetic valve.

Suitable glutaraldehyde fixation conditions are well known in the art and have been described, for example, in Jayakrishnan et al., (supra) and Chan and Gallucci, Ed., (1982) "Cardiac Prostheses", Yorke Medical Books, N.Y.

The mechanical properties of the fresh heart tissue are altered by the glutaraldehyde treatment; for example, stiffening of the tissue occurs. It is desirable to maintain the mechanical properties of the tissue as close as possible to those of the fresh tissue, for optimum functioning of the bioprosthetic valve.

The present inventors have found that pre-treatment of animal heart tissue with DMSO, prior to glutaraldehyde fixation, provides better maintenance of the mechanical properties of fresh tissue than previously described methods for preparation of animal heart tissue for use in bioprosthetic heart valves.

Treatment with DMSO may be carried out at a concentration in the range of about 20% to about 80%. A concentration of 30% to 50% is preferred and a DMSO concentration of about 40% is especially preferred.

Treatment of the heart tissue with DMSO is carried out at a temperature in the range 10°C to 30°C for about 30 minutes to about 50 hours. Treatment at ambient temperature (about 20°C) for about 24 hours is preferred.

Following DMSO treatment, the tissue is treated with glutaraldehyde as previously described (Jayakrishnan et al., and Chan (supra)). A glutaraldehyde concentration in the range of 0.2% to 1 % is frequently employed. A concentration of about 0.5% is preferred.

The DMSO- and glutaraldehyde-treated tissue is used for fashioning bioprosthetic heart valves as previously described for glutaraldehyde treated tissue.

The heart valve cusp is made up of three layers: the fibrosa, spongiosa, and ventricularis. The fibrosa is on the top surface of the cusp with the ventricularis on the bottom. In the fibrosa, large diameter collagen fibers, circumferentially oriented, are arranged in a corrugated manner, which allows the three cusps in a valve to expand radially, typically to 50% strain, this expansion enabling the three leaflets to come together and seal off the aperture. The ventricularis provides the tensile recoil necessary to retain the folded shape of the fibrosa. It consists of elastin fibers arranged radially, transverse to the large collagen fibers. The spongiosa has a gelatinous, watery consistency due to a loose collagenous network and an abundance of acid- mucopolysaccharides. It is speculated that the spongiosa facilitates shearing between the fibrosa and the ventricularis during the straightening of the corrugations. The largest calcific deposits involve the spongiosa and sometimes extend into adjacent regions of the fibrosa and ventricularis. The causes of bioprosthetic heart valve calcification are not clearly known, but are believed to be related to lipids present in the tissue.

The inventors have found that DMSO is a more effective solvent for dissolving lipids than ethanol, as seen from the results in Table 1. Since DMSO is a dipolar aprotic solvent, it is expected to be more compatible with molecules such as phospholipids and would be the preferred pretreatment solvent for prevention of calcification.

Tensile and Shear Testing

The modulus of elasticity equation predicts that a plot of stress versus strain for a given material would be linear with the slope of the curve corresponding to the modulus of elasticity. As can be seen in Figures 3 and 4, the stress vs. strain curves for all samples tested are not constant. This is typical of animal tissue. The most important E values (Young's Modulus) are those at average blood pressure (100-120 kpa), as seen in Table 2. It can be 6

seen that at all concentrations of DMSO tested, the E value was closer to that of fresh tissue than for conventionally fixed tissue. Tissue pretreated with 40% DMSO had the E value closest to that of fresh tissue.

The shear stress-strain curves shown in Figures 5 and 6 exhibit nonlinear behavior similar to the tensile curves. Since the physiological shear stress is unknown, shear moduli (G) as a function of strain was calculated and compared in Table 3. DMSO concentrations from 20% to 80% gave moduli closer to fresh tissue than that of conventionally fixed tissue. The closest match to fresh tissue was the moduli of tissue pre-treated with 40% DMSO. This is much closer to the moduli of fresh tissue than moduli of tissue treated by glutaraldehyde alone.

Effect of Pre-treatment on Mechanical Properties of Tissue

The internal structure of a fresh cusp is supported by a network of hydrogen-bonded water. Glutaraldehyde treatment crosslinks the collagen in the cusps by removing the hydrogen-bonded water. This causes the internal structure of the tissue to collapse and results in drastically altered mechanical properties. Depending on the concentration of ethanol used, ethanol pretreatment removes some of the hydrogen-bonded water from the tissue. This causes a partial collapse of the tissue structure, similar to that of glutaraldehyde fixation. Therefore, the mechanical properties of tissue pre-treated with ethanol are different from fresh tissue. DMSO, being a dipolar, aprotic solvent, may substitute for the hydrogen-bonded water in the tissue instead of removing it, thus allowing the tissue to maintain its structural integrity when being crosslinked with glutaraldehyde. Pretreating the tissue with a suitable concentration of DMSO results in fixed tissue which has mechanical properties that are more similar to those of fresh tissue.

DMSO at concentrations of 40% to 100% was effective in removing lipids, believed to be the source of calcium nucleation centres, leading to calcification. This DMSO treatment therefore will protect the tissue from calcification, thus prolonging the life of the bioprosthetic valve. The stress-strain curve of cusps treated with DMSO before glutaraldehyde fixation showed tensile properties very similar to fresh tissue. This is a marked improvement over the tensile properties of the glutaraldehyde- fixed tissue which is presently used for bioprosthetic heart valves. Cusps pretreated with DMSO also had shear stress-strain curves and elastic moduli that were very similar to fresh tissue. This is a significant improvement over the shear properties of the glutaraldehyde-fixed tissue presently used in bioprosthetic heart valves.

Animal heart tissue treated by the method of the invention therefore offers improved mechanical properties and improved durability of bioprosthetic heart valves.

Examples

The examples are described for the purposes of illustration and are not intended to limit the scope of the invention.

Example 1

Whole hearts were obtained from freshly slaughtered pigs at the abbattoir. They were delivered packed in ice within 24 hours of slaughter. The cusps were excised within 12 hours, blotted dry and weighed. Cusps were then rinsed in Hanks solution and mounted on an optical microscope and a sketch of the location of lipids was recorded.

Three cusps were placed in each of a series of concentrations of ethanol (20%, 40%, 80% and 100%) or of DMSO (20%, 40%, 80% and 100%) for 24 hours and then were re-examined microscopically for the presence of lipids. The results are shown in Table 1. Ethanol concentrations of 40%, 80% and 100% and DMSO concentrations of 20%, 40%, 80% and 100% removed lipids from cusps. However, 20% ethanol was ineffective in dissolving lipids from cusps. 8

Example 2

Cusps were removed from hearts of freshly slaughtered pigs, blotted dry and rinsed in Hanks solution. Three cusps were placed in each of a series of concentrations of ethanol (20%, 40%, 80% and 100%) or of DMSO (20%, 40%, 80% and 100%o) for 24 hours. The cusps were then blotted dry and transferred into a 0.5% glutaraldehyde/Hanks solution for 24 hours. The cusps were then blotted dry and stored in Hanks solution in the refrigerator for subsequent tensile and shear testing.

Tensile Testing

Testing was carried out using a hydraulically powered MTS machine with a data acquisition system and a special sample grip design for thin strips of tissue. A cusp sample was placed in a test tank filled with Hanks solution and maintained at a temperature of 37°C, to mimic physiological temperature. 10 mm x 5 mm strips of tissue were cut circumferential ly with a scalpel from the center of each cusp. A custom-built thickness gauge was used to measure the thickness of each sample. Load-extension relationships were obtained at extension rates of 0.3, 3 and 30mm/s to determine the stress-strain relationships and elastic moduli of the cusps, as described by Leeson-Dietrich et al., (1995), J. Heart Valve Disease, v. 4, p. 88.

Shear Testing

The apparatus consisted of a high-precision linear actuator and micro load cell combined with a custom made tissue mount. The linear actuator was a piezo electrically driven motor which could generate displacements with a precision of greater than 1 μm at speeds of between 0.1 mm/s and 1 mm/s over a travel of 2.5 cm. Samples were prepared by placing the cusps flat on a rubber surface and using a stainless steel punch to obtain circular specimens of 6.3 mm diameter. A thickness gauge was used to measure the thickness of the sample. Each sample was glued between two mounting blocks using absorbent paper soaked with cyanoacrylate adhesive. The samples were mounted in such a way that the deformation was in a circumferential direction. The tissue mount was then attached to the shear testing apparatus. Tests were performed at 37°C in Hank solution. Load-extension tests were performed at extension rates of 0.1 , 0.2 and 1.0 mm/s in a cyclic manner, as described by Talman et al., (1995), Ann. Thoracic Surgery, v. 60, p. S369 and Talman et al., (1996), J. Heart Valve Disease, v. 5, p. 152.

Modulus of Elasticity

Experimentally, for a given sample, a load versus extension data set was recorded. Typical data are shown in Figures 1 and 2. To covert the data set into useful information on mechanical properties of the material, its modulus was determined.

Definition of Modulus of Elasticity (E) E E = = = FF//AA (1 ) ΔL/L

E = = Modulus of Elasticity (Young's Modulus) F = = force

A = = area ΔL = change in length

L : = original length F/A = stress (_σ)

ΔL/L = strain (_e) Hence,

E = stress = strain _<≡

Under tensile conditions: stress = tensile stress E = tensile modulus When shear force is applied to the sample stress = shear stress modulus = shear modulus (G) 10

Testing under Tensile Conditions

In order to calculate E, the data must be converted from load vs. extension to stress vs. strain. To convert load to stress, the load at each interval must be divided by the cross-sectional area, which can be calculated by multiplying the width and thickness of the sample. To convert extension to strain, the change in extension at each interval must be divided by the original length, which is measured at the beginning of the experiment. The data can be re-plotted to give a stress vs. strain graph. However, in order to calculate Young's Modulus, the slope of the graph needs to be determined. The raw data is fitted using the empirical equation σ = A_e + B^K€ + C (2) σ = stress e = strain

A, B, C, K = adjustable parameters

Once the data is fitted with this function, modulus can be calculated by taking the derivative of the function:

dri = A + KB^h (3)

Moduli were calculated at 0.11 Mpa, the average blood pressure, so that the strength of the cusps treated with different concentrations of ethanol and DMSO can be compared to fresh tissue.

Testing under Shear Conditions

Unlike tensile testing, the crosshead moves forwards, back to the original spot, backwards and then back to the original spot. The initial data recorded can be graphed to give a load vs. extension graph in both the forward and the reverse directions.

The shear curve is different from the tensile curve and has four distinct sections: rising positive, falling positive, rising negative and falling negative 11

(Figure 2). The data is converted from load vs. extension to stress vs. strain using equation (2). The modulus for each pair of G and e was calculated using the rising positive section of the curve using equation (3).

Tensile Testing

Stress-strain curves for the ethanol-pretreated samples are shown in Figure 3 and compared to those for fresh and glutaraldehyde-fixed samples.

Stress-strain curves for cusps pretreated with DMSO are shown in Figure 4 and compared to those for fresh and glutaraldehyde-fixed samples. It can be seen that 40% DMSO-treated tissue had tensile properties very similar to those of fresh tissue.

Shear Testing

Shear stress-strain curves for the ethanol-pretreated samples are shown in Figure 5 and compared to those for fresh and glutaraldehyde fixed samples. The shear modulus of cusps pretreated with 20% ethanol was similar to those of fresh tissue.

Shear stress-strain curves for the DMSO pretreated samples are shown in Figure 6 and compared to those for fresh and glutaraldehyde fixed samples. Cusps pretreated with 40% DMSO gave shear stress-strain curves that were similar to those obtained with fresh tissue.

Table 1 00

Os

Lipid Solubility in Different Concentrations of Ethanol and DMSO

Solvent Concentration Lipids Present After 24 hrs?

Yes No

0) DMSO 20% y c

00 40% y 0)

H 80% y

H

C 100% H Ethanol 20% y

40% y x m m 80% y H 100% y 3 c Hank's Control y

r m κ>

O

H

O

>

≥ o e

- m- oo

Table 2

Elastic Moduli of Cusps Pretreated with Different Concentrations of Solvents Determined At Average Blood Pressure (0.11 Mpa)

DMSO Ethanol

Fixed Fresh 20% 40% 80% 100% 20% 40% 80% 100% Tissue Tissue

Elastic 3.81 5.67 4.67 6.10 6.61 6.14 4.71 3.02 2.68 4.24 H

U)

Modulus

(Mpa)

n H O

> o o *-» β

00 Os

Table 3 Shear Moduli of Cusps Pretreated with Different Concentrations of Solvents

Fresh Fixed DMSO G (kpa) Ethanol G (kpa)

Strain G G 20% 40% 80% 100% 20% 40% 80% 100%

(kpa) (kpa)

0.1 - 14.9 4.0 1.8 4.1 16.8 .6 11.1 * *

0.2 1.2 17.8 6.6 2.5 4.9 16.9 1.9 15.0 * *

0.3 - 23.2 10.7 3.6 6.1 18.2 2.2 21.4 * *

0.4 2.1 * 17.4 5.4 6.2 * 3.0 * * *

0.5 - * 28.3 8.3 8.1 * 4.4 * * *

0.6 3.9 * * 12.9 10.9 * 7.0 * * *

0.7 8.3 * * 20.3 15.4 * 11.5 * * *

0.8 10.9 * * * * *

9.3 * * *

* curves did not reach strain

"a o

H O > o o t *--> o

Claims

15We claim:

1. A method for preparing an animal heart tissue for use in a bioprosthetic heart valve by treatment of the tissue with glutaraldehyde, characterised in that the animal heart tissue is contacted for a suitable period of time with dimethyl sulphoxide (DMSO) before treatment with glutaraldehyde.

2. The method of claim 1 wherein the animal heart tissue is selected from the group consisting of porcine aortic valve tissue, porcine aortic root and bovine pericardium.

3. The method of claim 1 wherein the animal heart tissue is porcine aortic valve cusps.

4. The method of any of claims 1 to 3 wherein the animal heart tissue is contacted with DMSO at a concentration in the range of about 20% to about 80%.

5. The method of any of claims 1 to 4 wherein the animal heart tissue is contacted with DMSO at a concentration of about 40%.

6. The method of any of claims 1 to 5 wherein the animal heart tissue is contacted with DMSO for about 24 hours.

7. The method of any of claims 1 to 6 wherein the animal heart tissue is contacted with glutaraldehyde at a concentration of about 0.5% after being contacted with DMSO.

8. Animal heart tissue for use in a bioprosthetic heart valve prepared by a method comprising:

(a) contacting the tissue for a suitable period of time with DMSO; and 16

(b) contacting the tissue with glutaraldehyde.

9. The tissue of claim 9 wherein the tissue is selected from the group consisting of porcine aortic valve tissue, porcine aortic root and bovine pericardium.

10. The tissue of claim 9 wherein the tissue is porcine aortic valve cusps.