2. Brief Description of the Prior Art Significant advances have been made in the field of treatment of defective heart valves due to abnormalities during fetal development, or due to infectious or degenerative diseases. These surgical treatments most often require the use of biocompatible materials that can be either synthetic polymers or of biological origin, either from the patient (autologous), an individual of the same species (homologous), or different species (heterologous or xenograft). Defective heart valves are replaced with mechanical valves or tissue valves, such as cadaver or animal aortic valves (bioprosthesis). Because of their more-or-less predictable mechanical wear properties, the mechanical prostheses have been proven suitable for their intended purpose, primarily in younger patients. However, mechanical prostheses have their disadvantages because patients having them require long-term, constant and vigorous anti-coagulant therapy. In older patients however the bioprostheses have been favored mainly because they do not require anti-coagulation therapy, and in older patients they do not tend to undergo calcification as often as they tend to do in younger patients. Cryo- preserved homografts have been used widely in the western world during the recent years. However, these are hard and expensive to obtain, ship and store,

and their availability on a world-wide basis appears to be limited.

As is known, heart valve repair or replacement and many other implant operations require soft connective tissue which in preparation for implantation needs to be sized and cut into specific shapes. A substitute for such soft connective tissue of biological origin can be provided by flat sheets of certain synthetic materials. However, it is difficult to find synthetic materials which can match the compliance of the native tissue they are intended to replace, and which do not engender adverse reaction by the recipient of the implant.

Autologous tissues, such as pericardium, hold the promise for an ideal soft tissue replacement material in implants, and fresh autologous pericardium has been used in the prior art as a tissue source for repairing a variety of heart lesions, including heart valves. However, results with the use of such fresh untreated autologous tissue were less than satisfactory because tissue contraction distorted the repair, and in case of heart valves, tended to render the leaflets non-functional some time after operation. Generally speaking, the problem with fresh autologous soft connective tissue, such as pericardium, is that such tissues are often too soft and flexible to cut and otherwise handle especially during open heart surgery where an atmosphere of urgency prevails.

As an improvement Dr. Duran (one of the inventors of the present invention) developed a procedure in which the autologous tissue that has been freshly obtained from the patient operated on, is treated with 0.5% glutaraldehyde in a mold for 10 minutes. Thereafter, it is cut into the desired shape dictated by the mold and is placed in the patient as new replacement heart valve leaflets.

Although this procedure works reasonably well, the disadvantage of tissues treated by glutaraldehyde is that, similarly to xenografts, such tissues may well undergo calcification in long term implants.

The present invention provides an alternative to glutaraldehyde fixation of autologous tissues and yet eliminates the problems caused by contraction of fresh tissue and the difficulty of handling and manipulating soft tissue.

Because the invention avoids the above-noted problems by treating the autologous tissue with an aqueous solution of alcohols and other materials, prior art describing solutions and methods for treating biological tissues and specimens are thought to be of interest as background to the present invention.

Such prior art can be found in United States Patent Nos. 5,558,875; 5,296,514; 5,276,006; 4,323,358 and 4,329,492. Among the foregoing, the most recently issued United States Patent No. 5,558,875 describes a process of preparing a collagenous prosthesis by soaking tissue in an organic detergent for sufficient time to disrupt the cell membrane and to solubilize the cellular membrane proteins of the collagenous tissue and thereafter extracting and removing the cellular membrane proteins from the collagenous tissue by mechanical washing to obtain the prosthesis and thereafter preserving the prosthesis in alcohol. The process is said to preserve the elasticity of the prosthesis.

The following articles or scientific publications also provide background of interest to the present invention: Chachques et al., Ann. NY Acad Sci. 1988,529: 184; Love et al., J. Heart Valve Dis 1992: 1: 232-41; Chauvaud et al., J. Thorac Cardiovasc Surg. 1991,102: 171-8; Duran et al., J.

Thorac Cardiovasc Surg. 1995,11-511-6; Vyavahare et al., 4th Scientific Meeting International Association for Cardiac Biological Implants, Washington DC, May, 1997; Ritter et al., Plastic & Reconstructive Surgery.

101 (1) : 142-6, Jan., 1998; and Vetter et al., J. Thorac Cardiovasc Surg, 35 (1) : 11-5, Feb. 1987.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a composition (solution) and method for treating autologous soft tissue so as to render it easier to handle and shape for implantation, while avoiding disadvantages caused by aldehyde treatment of such tissue.

It is another object of the present invention to provide a composition

(solution) and method that meets the foregoing objective and which treats the autologous soft tissue during a surgical procedure and while said procedure is in progress.

The foregoing and other objects and advantages are attained by exposing for approximately 2 to 8 minutes a fresh autologous tissue, such as pericardium, to an aqueous solution containing approximately 10 to 70 % by volume of a water-miscible non toxic polar solvent, such as ethyl alcohol, approximately 2 to 30 % by weight of polyethylene glycol of a molecular weight between approximately 6,000 to 15,000 D, and approximately 0.01 to 1.0 % by weight of heparin. The tissue preferably, and most frequently in accordance with the procedure is immersed in the above-described solution while placed in a suitable mold. In case of preparing the tissue for heart valve replacement the mold is configured to provide the appropriate shape and dimension for the replacement heart valve leaflets. The soft tissue implant treated in the foregoing manner temporarily becomes more rigid and easier to handle during surgical procedure than unprepared fresh tissue. However, within approximately the time taken to perform the surgical procedure of implantation the treated tissue regains its original physical properties, including its elasticity.

BRIEF DESCRIPTION OF THE DRAWING FIGURES Figure 1 is a schematic perspective view showing the general configuration of one cusp of a negative template of a mold used for shaping an aortic valve replacement from autologous tissue, utilizing the novel solution and method of the present invention.

Figure 2 is a schematic perspective view showing the general configuration of one cusp of a positive template of the mold used for shaping an aortic valve replacement from autologous tissue, utilizing the novel solution and method of the present invention.

Figure 3 is a top plan view of the negative cusp of Figure 1.

Figure 4 is an end plan view of the negative cusp of Figure 1.

Figure 5 is a side plan view of the negative cusp of Figure 1.

Figure 6 is a schematic top plan view showing the general configuration of three negative cusps of Figure 1 assembled to form a negative template used for shaping an aortic valve replacement from autologous tissue, utilizing the novel solution and method of the present invention.

Figure 7 is a front plan view of the negative template of Figure 6.

Figure 8 is a schematic perspective view of the negative template of Figure 6.

Figure 9 is a detailed top plan view of a first preferred embodiment of a negative template used for shaping a pericardial valve replacement from autologous tissue, utilizing the novel solution and method of the present invention.

Figure 10 is an end view of the first preferred embodiment of the negative template of Figure 9.

Figure 11 is a front plan view of the first preferred embodiment of the negative template of Figure 9, with part of the front material broken away.

Figure 12 is a detailed top plan view of a first preferred embodiment of a positive template used for shaping a pericardial valve replacement from autologous tissue, utilizing the novel solution and method of the present invention.

Figure 13 is an end view of the first preferred embodiment of the positive template of Figure 12.

Figure 14 is a front view of the first preferred embodiment of the positive template of Figure 12.

Figure 15 is a perspective view of the first preferred embodiment of the negative template of Figure 9.

Figure 16 is a perspective view of the first preferred embodiment of the

positive template of Figure 12.

Figure 17 is a top plan view showing the first preferred embodiment of the negative template of Figure 9 and the first preferred embodiment of the positive template of Figure 12 assembled to one another.

Figure 18 is a front plan view showing the first preferred embodiment of the positive template of Figure 9 and the first preferred embodiment of the negative template of Figure 12 assembled to one another.

Figure 19 is a partial cross-sectional view taken on lines 19,19 of Figure 18.

Figure 20 is a view showing the assembled mold of Figure 18 having autologous tissue and immersed in a solution in accordance with the present invention.

Figure 21 is a partial cross sectional view of the mold with autologous tissue, the cross-section being taken on lines 21,21 of Figure 20.

Figure 22 is a partial schematic view, schematically showing the trimming of excess autologous tissue to form a replacement heart valve, in accordance with the present invention.

Figure 23 is a cross-sectional view taken on lines 23,23 of Figure 22.

Figure 24 is a partial cross sectional view of the trimmed autologous tissue.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is practiced in conjunction with a surgical procedure wherein a heart valve, aortic, pulmonary, tricuspid or mitral, or other biological membrane is replaced or repaired. Because its most frequent use is in conjunction with replacement of defective membranes in the heart, the present invention is described here primarily as it pertains to replacement of defective heart valves. In accordance with the present invention the operating surgeon excises a membrane-like fresh autologous tissue from the patient and by application of the solution of the present invention changes the

physical properties of the fresh autologous tissue to be better suited for trimming, handling, and manipulation during implantation into the patient.

Membrane-like tissues which are suitable to be handled and implanted in accordance with the present invention include the peritoneum, pericardium, gut, dermis pleura and tendon. For open heart surgery and replacement of defective heart valves the use of the patient's pericardium is preferred, and therefore the invention is described herein primarily in connection with the use of pericardium as the membrane-like autologous tissue.

In accordance with the invention, the freshly obtained pericardium (or other membrane-like autologous tissue) is treated with an aqueous solution containing approximately 10 to 70 % by volume of a water-miscible non-toxic organic solvent, approximately 2 to 30 % by weight of polyethylene glycol of a molecular weight between approximately 6,000 to 15,000 D, and approximately 0.01 to 1.0 % by weight of heparin, the rest of the solution being water. Examples of suitable water-miscible organic solvents or liquids are lower alkyl, especially C, to C3 alcohols, such as methanol, ethanol and iso-propanol, and acetone, acetonitrile and methyl ethyl ketone. A more preferred range of the components in the solution in accordance with the present invention is approximately 15 to 60 % by volume of the water- miscible organic liquid, 2 to 10 % by weight of polyethylene glycol, and 0.1 to 0.7 % by weight of heparin.

Preferably the organic solvent is ethyl alcohol, and in the presently most preferred embodiment of the solution there is approximately 50 % by volume ethanol, approximately 5 % by weight of polyethylene glycol having a molecular weight of approximately 8,000 D, and approximately 0.5 % by weight of heparin. The biological membrane is thoroughly exposed to the solution for sufficient time to provide the desired results of rendering the membrane more rigid and therefore easier to trim, suture and otherwise handle. However, usually a time limit is set to this exposure by the fact that

the process occurs while the patient is undergoing surgery, usually open heart surgery. It was found in practice that approximately 2 to 8 minutes of exposure of the biological membrane to the solution is sufficient.

Nevertheless, under circumstances where the surgical procedure per se does not represent a time-limiting factor, the biological membrane can be kept in the solution for indefinite length of time provided the solution is kept under sterile condition. Treatment by this solution kills the living cells in the membrane although treatment with the solution containing organic solvent at the lower end of the above-described range may only kill cells on the surface of the membrane and merely retards the biological response of cells in the interior. Nevertheless, unlike treatment with glutaraldehyde, treatment with the solution of the present invention does not result in any cross-linking of the membrane materials. The biological membrane or tissue becomes more rigid or stiff during exposure to the solution partly because of the hypertonic, dehydrating nature of the solution.

Hardening or stiffening of the membranes is temporary, however, because after sufficient rinsing with saline or upon equilibration with isotonic biological fluids, such as blood, the biological membranes regain virtually completely their original physical properties, and as a result are well suited for their intended function as replacement of natural membranes, primarily as heart valves.

A preferred manner of practicing the present invention, together with molds that are used for shaping pericardium or other biological membranes to provide aortic and pericardial heart valve replacements are illustrated in the drawing figures. Referring now back to the Brief Description of the Drawing Figures, Figures 1 through 8 schematically illustrate the basic geometry of a mold comprising a negative 30 and a positive 32 template for forming an aortic valve replacement in accordance with the present invention. Figures 1 through 5 schematically illustrate the basic geometry of the individual negative

34 and positive 36 cusps of the templates 30 and 32 that together form the mold. The templates 30 and 32 are made from thin plastic material, and are configured and dimensioned to provide the aortic heart valve replacement for the individual patient who is being operated on. As it will be readily understood by those skilled in the art of cardiology and related cardiac surgery, primarily echocardiograms of the patient provide the information as to what size heart valve replacement is needed. Edges of the templates 30 and 32 are beveled or rounded in order to facilitate trimming of excess tissue with a surgical knife.

In accordance with one manner of practicing the invention, the pericardium (or other suitable biological membrane) is placed into the mold between the negative and postive templates and treated for approximately 5 minutes with the solution of the invention by immersion in the solution.

Thereafter it is very quickly (for less than 5 seconds) rinsed with saline solution containing approximately 250 unit per ml heparin. This step of treating the tissue with heparin solution for a very brief period of time is not necessary for the successful practice of the invention and is therefore optional.

In any event, after removal from the mold and having been trated with the solution of the invention the tissue is more rigid than the native untreated pericardium, and is easier to handle. Excess tissue is then removed by trimming with a surgical knife, the tissue in the shape of heart valve leaflets is removed from the mold, and is thereafter surgically implanted. The increased rigidity or stiffness of these replacement leaflets renders the implantation procedure easier to handle. After suturing is completed, the tissue is irrigated with saline solution, whereupon it regains its original physical properties. As noted above, the biological membrane treated in accordance with the present invention regains its original physical properties upon adequate rinsing with saline, or achieving equilibrium with isotonic aqueous fluid, such as blood.

Figures 9 through 24 illustrate in more detail an actual mold comprising a negative template 38 and a positive template 40 adapted for shaping a flat biological membrane, such as pericardium, to form replacement leaflets for a pericardial valve. The two templates 38 and 40 of this mold are made of thin plastic material having beveled edges, and the templates are dimensioned to provide replacement leaflets of appropriate size for the patient who is undergoing the open heart surgery. For use in conjunction with the present invention, the negative 38 and the positive template 40 both are provided with a plurality of apertures 42. When this mold is used in the practice of the present invention, the autologous biological membrane, preferably pericardium excised from the patient who is undergoing open heart surgery, is placed between the templates, 38 and 40, that is into the mold.

Accordingly, the pericardium, shown in Figures 21 through 24 as 44 is sandwiched between the two templates 38 and 40. The two templates of the mold are held together with suitable plastic clips 46, shown in Figure 20.

The mold including the pericardium 44 is then immersed for approximately 5 minutes in the solution 48 of the invention, as is schematically shown in Figure 20. During this time the solution 48 percolates through the apertures 42 into the pericardium 44 and renders the pericardium 44 more rigid than in its natural native state. After removal from the solution, the tissue 44 is trimmed with a surgical instrument along the beveled edges of the mold. The surgical instrument is schematically shown in Figure 22 and bears the reference numeral 50. The resulting replacement heart leaflets (not shown) are then removed from the mold, and may be quickly (less than 5 seconds) rinsed with saline solution containing approximately 250 unit per ml heparin. This optional quick rinsing does not yet decrease the rigidity of the tissue which was the result of treatment with the solution. Surgical implantation of the replacement leaflets is greatly facilitated by its increased rigidity or stiffness.

After implantation, the replacement leaflets are irrigated with saline and regain

their original physical properties. A substantial advantage of the heart valve replacements obtained in accordance with the present invention is that, unlike replacement valves made of autologous tissues in the prior art, the replacement valves of the invention do not contract or shrink after implantation. Generally speaking, implants of autologous tissues which have been treated in accordance with the present invention are highly resistant or immune to thickening, contraction or fibrin deposition after the implants are placed into the bloodstream of the host. The above described process of exposing the biological membrane to the solution of the invention while the membrane is held in an appropriately configured and dimensioned mold is the presently preferred mode of practicing the invention.

Specific Examples Fresh autologous tissues were dissected from the sheep and placed on molds which were used as templates for cutting the tissues into a shape which appeared as three half-moons joined together at the base of the half-moon.

This particular shape was designed for the purpose of aortic or pulmonary heart valve cusp extension operation. Pericardial tissues cut according to this design can be directly implanted into the heart of the individual patient where the tissue is derived from. The tissues in the mold were immersed into a solution containing 50 % by volume of alcohol, 5 % by weight of polyethylene glycol (MW=8,000) and 0.5 % by weight of heparin for five minutes at room temperature. The tissues in the mold were removed from the solution and after the removal of excess liquid outside the tissues, the tissues were rinsed in saline containing 250 unit/ml of heparin for less than 5 seconds.

Tissues treated with the solution mentioned above appeared to be slightly translucent and natural in color. Unlike the fresh untreated tissues, the treated tissues were stiffer so that it was relatively easy to lift the tissues without the tissues folding onto themselves. The treated tissues were easily spread on a flat or curved surface with different markings for cutting the

tissues. Since the cut tissues maintained their shape, they were easily implanted as replacement leaflets of heart valves. Yet, once the suturing of the tissues was completed and a small amount of saline was irrigated onto the implanted tissues, the tissues became soft quickly. Within the time required to close the open heart, the physical properties of the tissues became indistinguishable from the fresh untreated tissues. Therefore the resulting implants function perfectly as repaired heart valves.

Human fibroblasts and umbilical cord vein endothelial cells were cultured on the treated tissues after there were rinsed in saline containing 250 unit/ml of heparin to study their biocompatibility. Round discs of the tissues were cut to fit the bottom of the wells of a 24 well culture plate. TygonR flexible rings were placed on top of the tissues to ensure a good seal at the edge of the tissues. Cells were seeded on the tissues in normal culture media for one week. At the end of the incubation period, tissues were recovered and processed for histology. Both human umbilical cord vein endothelial cells and human skin fibroblasts attached and proliferated on the treated tissues as evidence that after rinsing in saline the treated tissue is not cytotoxic and biocompatible for host cells to attach and proliferate. The attachment and proliferation of endothelial cells and other connective tissue cells on cardiac implants is potentially important for the long term survival of the implant.

Integrity of the collagen fibers in the treated tissues was examined by melting temperature measurements. Tissues were heated in phosphate buffered saline from 37° C until they shrunk. The shrinkage temperature of the treated tissues after they were rinsed in saline was 64i1 °C which is identical to untreated fresh tissue indicating that the collagen fibers remained intact throughout the treatment and saline-rinse process.

Efficacy of the treated tissues as useful cardiovascular implants was tested by implanting the treated autologous tissues in the descending aorta of sheep in different configurations. A piece of pericardium was dissected and

divided into three pieces with different shapes, namely a trapezoid, a strip made into a conduit and a square. These pieces of tissues were implanted serially in the descending aorta of sheep. The trapezoid shaped tissue was implanted upstream as a patch on the aortic wall, next to it down-stream the short conduit was implanted and further down-stream a square shaped tissue was placed as a semi-free flap across the lumen inside the aorta with two edges of the square attached to opposite side of the inner wall of the aorta. When fresh autologous tissues were implanted under the same condition, the semi- free flap in the aorta lumen became fibrotic and contracted within 30 days.

However the patch and the conduit upstream, that was implanted in accordance with the present invention did not show the same reaction. There was also evidence of thrombus formation and fibrin deposition on the surfaces of the fresh implants. When the treated implants (not rinsed in saline) were implanted in sheep in the same manner all implants remained intact after 30 days without any evidence of fibrotic reaction and tissue contraction.

Thrombus and fibrin deposition were minimal or absent on these implants.

In still other further examples fresh bovine pericardial tissues were treated using the solution of the invention. The treated tissues were cut and trimmed to sizes and shapes suitable for valve repairs. The treated and trimmed tissues were sutured in the aortic roots of isolated human and porcine hearts. The whole hearts were than mounted on a pulse duplicator to examine the competency of the repair valve. The treated tissues were very flexible and the reconstructed valves functioned normally as competent aortic valves.