Biological material pre-fixation treatment

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FIELD OF THE INVENTION

This invention relates to methods for rendering biological materials substantially acellular and for methods of treating biological materials to inhibit their mineralization after implantation into a human or animal. In one embodiment, this invention relates to a method of inhibiting post-implantation mineralization of a bioprosthetic heart valve.

BACKGROUND OF THE INVENTION

Disorders of the cardiac valves cause significant morbidity and mortality. These disorders affect persons of all ages and can result from congenital or degenerative conditions, as well as from the sequelae of infections. Stenosis and insufficiency of the aortic or mitral valves have a greater incidence than stenosis and insufficiency of the tricuspid and pulmonary valves.

Treatment of cardiac valvular disorders can require replacement of the defective valve with a prosthetic valve. There are two types of prosthetic heart valves. "Mechanical valves", the first type, are composed wholly of materials not derived from living organisms. Mechanical valves currently in use have either a ball-valve construction, a tilting disc construction or a hinged leaflet construction.

"Bioprosthetic valves", the second type of prosthetic heart valves, are composed in whole or in part of biological material. Bioprosthetic valves generally comprise a supporting stent and a plurality of leaflets. The leaflets generally comprise biological material, while the stent, if present, generally comprises non-biological material, at least in part. The biological material of the leaflets, can be of autologous tissues, such as pericardium, fascia lata or cardiac valves. Alternately, this material can be derived from homologous tissue, such as non-autologous human tissue for human implantation, or can be xenogeneic.

Each type of prosthetic heart valve has advantages and disadvantages. Mechanical heart valves are durable but they carry a significant risk of thrombus formation with secondary complications. Chronic anticoagulation therapy decreases the incidence of thrombotic related events to between 1% to 4% per patient year. (Criscitiello, M. and Levine, H.: Thromboembolism and Prosthetic Heart Valves. Hospital Practice. Dec. 15, 1992:69-96.) Chronic anticoagulation therapy, however, carries with it a risk of hemorrhage similar in incidence to that of the residual risk for thrombotic events. (Barnhart, G. et al.: Degeneration and Calcification of Bioprosthetic Cardiac Valves. American Journal of Pathology. 1982, 106/1:136-139.)

Bioprosthetic valves initially approximate the hemodynamic properties of the natural valve. They carry a smaller risk of complications secondary to thrombus formation than do mechanical valves. Thus, chronic anticoagulation therapy need not be instituted in most patients. Bioprosthetic valves, however, carry a significantly higher risk of calcification than mechanical valves.

Calcification of bioprosthetic valves develops more rapidly in children, which have an incidence of calcification of about 40% to 50% at 4 years, than it develops in adults, which have an incidence of calcification of between 5% to 20% at 10 years. (Carpentier, A. et al.: Techniques for Prevention of Calcification of Valvular Bioprostheses. Circulation 70 (suppl I). 1984, I-165 to I-168.) Calcification causes thickening, retraction and reduced mobility of the leaflets and can lead to stenosis, insufficiency or both. Hence, calcification is an important limitation on the useful life expectancy of the currently used bioprosthetic valves. Since treatment of a functionally compromised bioprosthetic heart valve frequently requires replacement with a new valve, limitations on the useful life expectancy of a bioprosthetic heart valve are both a serious medical problem for the patient and a financial drain on the medical system.

Several methods to decrease or prevent bioprosthetic heart valve mineralization have been described in several patents since the problem was identified. Generally, the methods involve treating the bioprosthetic valve with various substances prior to implantation. Among the substances reported to work are sulfated aliphatic alcohols, phosphate esters, amino diphosphonates, derivatives of carboxylic acid and various surfactants. Nevertheless, none of these methods have proven completely successful in solving the problem of post-implantation mineralization. Thus, there remains a need for the bioprosthetic heart valve resistant to post-implantation mineralization.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, there is provided a controlled autolysis method for making a biological material substantially acellular, wherein the biological material that is to be made substantially acellular has structural integrity and comprises cells and non-cellular structural components, and wherein the controlled autolysis method comprises the step of exposing the biological material, prior to any fixation thereof, to at least one buffered solution having a pH in the range from about 5.0 to 8.0 and a temperature in the range from about 12.degree. C. to 30.degree. C. for a sufficient period of time to render at least one region of the biological material substantially acellular while substantially preserving the structural integrity and non-cellular structural components of the biological material.

In accordance with another aspect of the present invention, there is provided a bioprosthetic heart valve, comprising at least one leaflet that is adapted for reciprocal motion from an open position to a closed position upon blood flow through the valve, the at least one leaflet being formed, at least in part, of biological material that has been subjected to controlled autolysis, wherein the biological material that is subjected to controlled autolysis has structural integrity and comprises cells and non-cellular structural components.

In accordance with another aspect of the present invention, the bioprosthetic heart valve additionally comprises a generally tubular stent having an inflow end and an outflow end, wherein the at least one leaflet is positioned in relation to the stent such that the reciprocal motion of the at least one leaflet occurs as blood flows from the inflow end of the stent through the outflow end of the stent.

In accordance with another aspect of the present invention, there is provided a method of making a bioprosthetic heart valve, comprising the steps of (a) subjecting biological material to the controlled autolysis method of the present invention, wherein the biological material comprises a heart valve or a fragment of a heart valve, the heart valve or fragment of a heart valve comprising at least one leaflet, (b) fixing the biological material, and (c) fabricating the bioprosthetic heart valve from the biological material by the addition of non-biological material.

In accordance with another aspect of the present invention, there is provided a method of treating a mammal having a defective heart valve, comprising the steps of (a) providing a bioprosthetic heart valve according to claim 16, (b) removing the defective heart valve from the mammal, and (c) implanting the bioprosthetic heart valve in the mammal.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one figure executed in color. Copies of this patent with color figures will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

FIG. 1 is an outflow end elevational view of bioprosthetic heart valve.

FIG. 2 is an inflow end elevational view of bioprosthetic heart valve.

FIG. 3 is a photomicrograph of a cross-section of a porcine aortic valve leaflet before treatment at 20.times. (twenty times magnification).

FIG. 4 is a photomicrograph of a cross-section of a porcine aortic valve leaflet before treatment at 100.times. (one hundred times magnification).

FIG. 5 is a photomicrograph of a cross-section of a porcine aortic valve leaflet after treatment at 20.times. (twenty times magnification).

FIG. 6 is a photomicrograph of a cross-section of a porcine aortic valve leaflet after treatment at 40.times. (forty times magnification).

FIG. 7 is a photomicrograph of a cross-section of a porcine aortic valve leaflet 2 (two) weeks after implantation that had not been treated at 40.times. (forty times magnification).

FIG. 8 is a photomicrograph of a cross-section of a porcine aortic valve leaflet 20 (twenty) weeks after implantation that had not been treated at 40.times. (forty times magnification).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In one aspect, the present invention relates to an improved bioprosthetic heart valve having an improved resistance to post-implantation mineralization. In this aspect, the invention involves the use of specially treated biological material. The material is treated by exposing the biological material, prior to fixation, to at least one buffered solution having a preselected pH range and a preselected temperature range for a sufficient period of time to render at least one region of the biological material substantially acellular while substantially preserving the non-cellular structural components of the biological material. This treatment involves the process hereinafter referred to as "controlled autolysis" and described more fully below.

Selected Definitions:

As used herein, "biological material" comprises cells and non-cellular structural components derived from at least one of a living organism, a corpse, a carcass, an organ or part of an organ artificially maintained outside the living organism, a cell culture derived from a living organism, a corpse or a carcass, and a combination of the preceding. Biological material can be artificially manipulated prior to derivation from the above listed sources, such as by the introduction of special diets or drugs to the organism, or the introduction of gene sequences by an appropriate vector. Each biological material has a "structural integrity", as defined below.

As used herein, the terms "bovine", "porcine", "ovine" "Macropodidae" and "non-human primate" refer respectively to at least one animal of or related to a cow or ox, pig or hog, sheep or goat, a kangaroo, and monkey or ape or gibbon or chimpanzee or lemur. The terms include, but are not limited to, an unborn animal, young animal, male animal, female animal and pregnant animal, whether occurring naturally, through selective breeding or artificial insemination, a carcass, a fragment thereof, and a combination of any of the preceding. As used herein, "human" includes an embryo, a fetus, an unborn individual, a corpse, a fragment or tissue of any of the foregoing and a combination thereof.

As used herein, a "buffered solution" refers to an aqueous solution having at least one substance which tends to preserve hydrogen ion concentration or pH.

As used herein, "cell" means the composite of the membrane structures enveloping protoplasm and distinguishing the enveloped protoplasm from the external environment, wherein the membranes are identifiable by visual inspection with the aid of light microscopy. The cell can be living or not living. "Cell" also includes remnants or ghosts of the composite of the membrane structures indicating the former presence of a living cell at or near the location of the remnant or ghost.

As used herein, "membrane structures" refer to lipid layers and lipid bilayers, with or without proteins, carbohydrates, glycoproteins, cholesterol or other substances incorporated into the layers, such as are found enveloping protoplasm from a living cell.

As used herein, "non-cellular structural components" comprises substances not enveloped by the composite of the membrane structures of a cell, even if derived from or secreted by cells. The substances include collagen, elastin, laminin, teninsin, actinin and proteoglycans.

As used herein, "fixation" or "fixing" refers to a process of treating biological material so as to preserve the material from natural decay, including decay by autolysis. Fixation includes methods such as exposing the biological material to glutaraldehyde or formaldehyde.

As used herein, "fragment" means any portion or amount less than the whole, including disjoined or non-contiguous portions.

As used herein, "heart valve" means at least one of the aortic valve, mitral valve, tricuspid valve and pulmonary valves in a human, an equivalent valve in non-human animals, with or without intimately related tissue, a fragment thereof and a combination thereof.

As used herein, "region", as applied to biological material, refers to the whole biological material or any fragment of the biological material having macroscopically identified boundaries. For example, a region of a harvested natural heart valve can be the whole valve, at least one leaflet, the stent, the adjunct myocardium and a combination thereof.

As used herein, "structural integrity" refers to the natural capacity of biological material to perform a physical, as opposed to chemical, function in the organism, such as a compression function, a valvular function or a support function.

As used herein, "substantially acellular" and "substantial acellularity" interchangeably mean having at least about 70% (seventy percent) fewer cells than the natural or living state of the biological material. Therefore, biological material that has been made substantially acellular according to the present invention, has had the absolute number of cells reduced by at least about 70% from the natural state.

For the purposes of determining substantial acellularity, only cells native to the biological material are counted. Blood borne cells including red cells and platelets, as well as cells from other organisms are not counted.

The number of cells present in biological material is determined by using visual inspection of the material at about 20.times. (twenty times) to 100.times. (one hundred times) magnification using light microscopy with or without stain, or an equivalent technique. Satisfactory stains include standard stains known to those with skill in the art, as is appropriate to the specific cell type of the biological material being examined.

Conditions for Controlled Autolysis.

Various aspects of the present invention utilize the process herein referred to as "controlled autolysis". "Controlled autolysis" means manipulating the physical conditions and storage solutions to which biological material is exposed to promote the breakdown of certain components of the biological material while substantially preserving other components due to properties of the autolytic enzymes found in the biological material. The treatment is performed prior to fixation.

Controlled autolysis can be used as a method of treating biological material that will be implanted into a human or animal to inhibit mineralization of regions of the biological mat