WO2018167536A1

WO2018167536A1 - Implantable material and method for preserving

Info

Publication number: WO2018167536A1
Application number: PCT/IB2017/051504
Authority: WO
Inventors: Marina Strasly
Original assignee: Sorin Group Italia S.R.L.
Priority date: 2017-03-15
Filing date: 2017-03-15
Publication date: 2018-09-20

Abstract

An implantable material includes decellularized, sterilized mammalian tissue having an extracellular matrix, wherein a plurality of interstitial spaces of the extracellular matrix include a solution of one or more polyols other than glycerol and, optionally, glycerol.

Description

IMPLANTABLE MATERIAL AND METHOD FOR PRESERVING

TECHNICAL FIELD

[0001] The present invention relates to implantable materials. More specifically, the invention relates to decellularized implantable materials or tissue engineered implantable materials and methods for producing and preserving such implantable materials.

BACKGROUND

[0002] Biological prostheses are medical devices that use mammalian donor tissues. Examples of suitable mammalian tissues include, for example, bovine, porcine, ovine, equine, and human tissues. Depending on the various medical uses of a biological prosthesis, the donor tissue may include, for example, cardiac valves, pericardium, tendons, ligaments, dura mater, skin, and veins.

[0003] Currently, tissues used in biological prostheses, especially cardiac valves, undergo a series of chemical treatments in order to give biological and mechanical stability to the tissue itself and to sterilize the final device that incorporates the tissue. These chemical treatments typically involve aldehydes able to crosslink the tissue to alter its mechanical properties, reduce in vivo enzymatic digestion and immune response after implant and, in combination with other chemicals, to sterilize the device. Aldehyde treatments result in a stable device that is resistant to remodeling or degradation by the patient's body after implant. This stability in general is seen as an advantage, but for certain advanced applications it represents a drawback. For example, a biological heart valve implanted within a child able to be remodeled and grow with the child may reduce the need to replace the heart valve as the child grows. Recent technical development in tissue engineering and in native tissue decellularization are devoted to producing new tissues that are not fixed so that the recipient organism can remodel donor tissue in some way, by repopulating it with the recipient's own cells.

[0004] Despite the significant improvements provided with these innovative techniques, a number of critical issues remain. For example, in the case of decellularized tissue, before use in a biological prosthesis, the donor tissues are subjected to numerous washing steps and mechanical action to remove donor cells. However, even after such decellularization processes, some cells or cellular residues may remain trapped within the tissue. After a biological prosthesis incorporating the tissue is implanted in the patient, any remaining cells may cause an immune response that can lead to the degradation or destruction of the prosthesis. Other relevant issues to be taken into account are how to insure sterility and how to store the bioprosthesis. Thus, it is important to address these issues, but without the use of aldehydes-based sterilization processes that would destroy their potential to remodel in vivo.

SUMMARY

[0005] Example 1 is an implantable material including decellularized sterilized mammalian tissue having an extracellular matrix, wherein a plurality of interstitial spaces of the extracellular matrix include a solution of one or more polyols other than glycerol and, optionally, glycerol.

[0006] In Example 2, the implantable material of Example 1 , wherein the glycerol is present in an amount of about 1 vol. % to about 50 vol. % of the mixture and the one or more polyols other than glycerol are present in an amount of about 50 vol. % to about 99 vol. % of the mixture.

[0007] In Example 3, the implantable material of either of Examples 1 or 2, wherein the solution in the plurality of interstitial spaces of the extracellular matrix consists essentially of the one or more polyols other than glycerol and glycerol.

[0008] In Example 4, the implantable material of any of Examples 1 -3, wherein the one or more polyols other than glycerol includes at least one of 1 ,2 propanediol, 1 ,2 octanediol, 1 ,4 butanediol, fructose, xylitol, mannitol, sorbitol, lactulose, lactic acid, maltose, glucose, galactose, erythritol, lactobionic acid or its salts, and hyaluronic acid.

[0009] In Example 5, the implantable material of any of Examples 1 -4, wherein the one or more polyols other than glycerol are selected from the group consisting of 1 ,2 propanediol, 1 ,4 butanediol, lactobionic acid and its salts, and combinations thereof.

[0010] In Example 6, the implantable material of any of Examples 1 -5, wherein the one or more polyols other than glycerol consists essentially 1 ,2 propanediol. [0011] In Example 7, the implantable material of any of Examples 1 -6, wherein the implantable material is substantially free of water.

[0012] In Example 8, the implantable material of any of Examples 1 -6, wherein the implantable material is non-fixed.

[0013] In Example 9, the implantable material of any of Examples 1 -8, wherein the implantable material forms a portion of an engineered heart valve.

[0014] Example 10 is method for making an implantable material. The method includes decellularizing mammalian tissue, treating the decellularized mammalian tissue with a solution; drying the treated tissue, and sterilizing the dried tissue. The treatment solution includes one or more polyols other than glycerol, glycerol and, optionally, water.

[0015] In Example 1 1 , the method of Example 10, wherein the solution includes the glycerol at a concentration from about 15 vol. % to about 35 vol. %, the one or more polyols other than glycerol at a concentration of about 65 vol. % to about 85 vol. % of the solution, and the balance water.

[0016] In Example 12, the method of either of Examples 10 or 1 1 , wherein the solution consists essentially of the one or more polyols other than glycerol, glycerol, and, optionally, water.

[0017] In Example 13, the method of any of Examples 10-12, wherein sterilizing includes at least one of exposing the dried tissue to ethylene oxide gas, exposing the dried tissue to ionizing radiation, and exposing the dried tissue to ultraviolet light.

[0018] In Example 14, the method of any of Examples 10-13, wherein drying the treated tissue includes drying until substantially all water is removed from the treated tissue.

[0019] In Example 15, the method of any of Examples 10-14, wherein treating the tissue includes immersing the tissue in the solution.

[0020] In Example 16, the method of Example 15, wherein the tissue is immersed in the solution for a time ranging from 2 to 100 hours and at a temperature ranging from 5°C to 35°C.

[0021] In Example 17, the method of either of Examples 15 or 16, wherein the tissue is immersed in the solution more than once. [0022] In Example 18, the method of Examples 10-14, wherein treating the tissue includes one of spraying the solution on the tissue and spreading the solution on the tissue.

[0023] In Example 19, the method of any of Examples 10-18, wherein the one or more polyols other than glycerol includes at least one of 1 ,2 propanediol, 1 ,2 octanediol, 1 ,4 butanediol, fructose, xylitol, mannitol, sorbitol, lactulose, lactic acid, maltose, glucose, galactose, erythritol, lactobionic acid or its salts, and hyaluronic acid.

[0024] In Example 20, the method of any of Examples 10-19, wherein the one or more polyols other than glycerol are selected from the group consisting of 1 ,2 propanediol, 1 ,4 butanediol, lactobionic acid, and combinations thereof.

[0025] In Example 21 , the method of any of Examples 10-20, wherein solution includes the glycerol at a concentration from about 15 vol. % to about 35 vol. %, the one or more polyols other than glycerol at a concentration of about 65 vol. % to about 85 vol. % of the solution, and the balance ethanol.

[0026] In Example 22, the method of any of Examples 10-21 , wherein the tissue is not treated with a fixing agent.

[0027] Example 23 is a preserved tissue engineered heart valve including decellularized, non-fixed, sterilized mammalian tissue having an extracellular matrix, wherein a plurality of interstitial spaces of the extracellular matrix includes a solution consisting essentially of one or more polyols other than glycerol and glycerol, wherein the glycerol is no more than 50 vol. % of the solution.

[0028] While multiple embodiments are disclosed, still other embodiments of the present invention will become apparent to those skilled in the art from the following detailed description, which shows and describes illustrative embodiments of the invention. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 is a photograph of a non-fixed decellularized tissue sample treated with pure glycerol. [0030] FIG. 2 is a photograph of a non-fixed decellularized tissue sample treated with pure 1 ,2 propanediol, according to embodiments.

[0031] FIG. 3 is a photograph of a non-fixed decellularized tissue sample treated with a solution of 1 ,2 propanediol and glycerol, according to embodiments.

[0032] FIG. 4 is a photograph of a stented valve made of non-fixed decellularized pericardium and treated with a solution of 1 ,2 propanediol and glycerol, according to embodiments.

[0033] FIG. 5 is a photograph of a fixed tissue sample treated with pure glycerol.

[0034] FIG. 6 is a photograph of a fixed tissue sample treated with pure 1 ,2 propanediol, according to embodiments.

[0035] FIG. 7 is a photograph of a fixed tissue sample treated with a solution of 1 ,2 propanediol and glycerol, according to embodiments.

[0036] FIG. 8 is a photograph of a fixed tissue sample treated with a solution of 1 ,4 butanediol and glycerol, according to embodiments.

[0037] FIGS. 9A and 9B are photographs showing opposite sides of a heart valve made of fixed tissue treated with pure glycerol.

[0038] FIGS. 10A and 10B are photographs showing opposite sides of a heart valve made of fixed tissue treated with pure 1 ,2 propanediol, according to embodiments.

[0039] FIGS. 1 1A and 1 1 B are photographs showing opposite sides of a heart valve made of fixed tissue treated with a solution of 1 ,2 propanediol and glycerol, according to embodiments.

[0040] While the invention is amenable to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and are described in detail below. The intention, however, is not to limit the invention to the particular embodiments described. On the contrary, the invention is intended to cover all modifications, equivalents, and alternatives falling within the scope of the invention as defined by the appended claims.

DETAILED DESCRIPTION

[0041] Many processes and chemicals employed to treat tissue for a bioprosthesis result in crosslinking of the extracellular collagen matrix within the tissue. Crosslinking or "fixing" of the extracellular matrix improves the strength and biological durability of the tissue. Aldehyde based processes are a typical example of this approach. Aldehyde based processes are also used to provide chemical sterilization to the bioprosthesis. Applied to a decellularized tissue or to a material obtained via tissue engineering, these crosslinking/sterilization methods reduce the ability of the tissue to remodel. In some applications, crosslinking or "fixing" of the extracellular matrix improves the strength and biological durability of the tissue, but the use of the crosslinking agent usually destroys the potential for the patient's body to favorably remodel the tissue.

[0042] However, in some applications, such as tissue engineering, it is desirable for the implanted biological prosthesis to be remodeled by the host's body. Remodeling refers to the action by the host body to replace some or all of the extracellular matrix of the implanted tissue, with new tissue produced by host cells of the patient's body repopulating the implanted tissue. In tissue engineering, the extracellular matrix acts as a scaffold to guide the growth of the new tissue. For example, a tissue engineered heart valve implanted within a child may be remodeled and grow with the child, reducing the need to replace the heart valve as the child grows. Fixed (i.e., crosslinked) tissue cannot be remodeled by the body because of the structural fixation and/or cytotoxicity imparted by crosslinking the collagen of the extracellular matrix of the donor tissue.

[0043] Embodiments of this disclosure include an implantable material including decellularized, non-fixed mammalian tissue that is sterilized and preserved sterile and dry without impairing the ability of the extracellular matrix to be remodeled. Other embodiments include an implantable material including decellularized mammalian tissue that has been fixed. In either case, the decellularized tissue may be made by removing the cells from mammalian tissue, such as a porcine heart valve, leaving behind the extracellular matrix. Other mammalian tissues may include bovine pericardium, equine pericardium or other porcine tissues such as the intestinal mucosa. Alternatively, the tissue may be made by seeding a polymeric mold with mammalian cells which grow along the polymeric mold to produce the extracellular matrix of collagen or of other structural proteins. Once completed, the cells are removed, leaving behind the extracellular matrix. [0044] After decellularization, the decellularized tissue (or polymer with attached extracellular matrix, in alternative embodiments) is treated with a solution in order to reduce the retained water in the tissue. In some embodiments, the treatment solution includes one or more polyols, other than glycerol. As shown below, embodiments treated with solutions described herein are less swollen and rigid than those treated with glycerol alone. In some embodiments, the one or more polyols can include at least one of 1 ,2 propanediol, 1 ,2 octanediol, 1 ,4 butanediol, fructose, xylitol, mannitol, sorbitol, lactulose, lactic acid, maltose, glucose, galactose, erythritol, lactobionic acid or its salts, and hyaluronic acid of various molecular weights and crosslinked to various degrees or not crosslinked. In some embodiments, the one or more other polyols are selected from the group consisting of 1 ,2 propanediol, 1 ,4 butanediol, and lactobionic acid, or combinations thereof. For example, the treatment solution may consist of about 50 volume percent (vol. %) 1 ,2 propanediol and about 50 vol. % 1 ,4 butanediol; or the treatment solution may consist of 1 ,2 propanediol and lactobionate at saturation in the solution. In some embodiments, the one or more polyols consists essentially of 1 ,2 propanediol.

[0045] In some embodiments, the treatment solution may further include glycerol. In some embodiments, glycerol is present in the treatment solution at a concentration ranging from about 1 volume percent (vol. %) to about 50 vol. %, and the one or more other polyols are present at a concentration of about 50 vol. % to about 99 vol. %. For example, in one embodiment, the treatment solution consists of 90 vol. % 1 ,2 propanediol and about 10 vol. % glycerol. In another embodiment, the treatment solution consists of 85 vol. % 1 ,4 butanediol and about 15 vol. % glycerol. In other embodiments, the glycerol is present in the solution at a concentration from about 20 vol. % to about 50 vol. %, and the one or more polyols are present at a concentration from about 50 vol. % to about 80 vol. %. For example, in one embodiment, the treatment solution consists of about 50 vol. % 1 ,2 propanediol and about 50 vol. % glycerol.

[0046] In some embodiments, the treatment solution may further include water. In some embodiments, water is present in the treatment solution at concentration of about 1 vol. % to about 20 vol. %. In some embodiments, the treatment solution can include glycerol ranging from about 15 vol. % to about 35 vol. %, one or more other polyols ranging from about 65 vol. % to about 85 vol. %, and the balance water. For example, in one embodiment, the treatment solution includes about 10 vol. % water, about 30 vol. % glycerol, about 60 vol. % 1 ,2 propanediol, and lactobionate at saturation in the solution.

[0047] In some embodiments, the treatment solution may include ethanol. In some embodiments, ethanol is present in the treatment solution at concentration of about 1 vol. % to about 20 vol. %. In some embodiments, the treatment solution can include glycerol ranging from about 15 vol. % to about 35 vol. %, one or more other polyols ranging from about 65 vol. % to about 85 vol. %, and the balance ethanol. For example, in one embodiment, the treatment solution includes about 10 vol. % ethanol, about 30 vol. % glycerol, about 60 vol. % 1 ,2 propanediol, and lactobionate at saturation in the solution.

[0048] In some embodiments, the decellularized tissue (or polymer with attached extracellular matrix, in alternative embodiments) is soaked in the treatment solution as described below. Excess treatment solution can be mechanically removed from the tissue, which helps remove the major part of the retained water. In some embodiments, the tissue is stored dry and used for fabrication or manufacturing of a bioprosthesis, for example, a heart valve. The foregoing process can be applied, for example, to decellularized pericardium of a mammalian donor for use in heart valve manufacturing. The valve may then be sterilized using ethylene oxide gas, ionizing radiation, and/or ultraviolet (UV) light.

[0049] Sterilization with ethylene oxide gas, ionizing radiation, or UV light does not impair remodeling because neither method induces tissue fixation. Also, these sterilization methods are more robust and less complex from an industrial perspective than traditional aldehyde based sterilization processes, that involve set up and maintenance of controlled environments, with critical operating procedures.

[0050] Alternatively, the treatment solution can be applied to a bioprosthesis, for example a heart valve made of fresh decellularized tissue or of tissue produced via tissue engineering. In some embodiments, the valve is soaked in the treatment solution one or more times for several hours, as described below. When removed from the treatment solution, a small amount of the treatment solution remaining in the valve is easily removed. Some polyols, such as 1 ,2 propanediol and 1 ,4 butanediol, are less viscous than glycerol, which helps in removing them from the valve. The valve may be sterilized using ethylene oxide, ionizing radiation, and/or UV light and then stored dry until use.

[0051] In some embodiments, the treatment with the solution includes immersing the tissue in the treatment solution at a temperature ranging from about 5°C to about 35°C for a time ranging from about 2 hours to about 100 hours. In other embodiments, treatment with the solution includes repeated immersions two to three times, each immersion time ranging from about 1 hour to about 20 hours. In other embodiments, the treatment with the solution may include spreading the treatment solution on the tissue or spraying it on the tissue.

[0052] In some embodiments, during the treatment with the solution, the tissue and the treatment solution can be held under pressure greater than about 100 kPa to enhance movement of the treatment solution into the tissue. In some embodiments, the pressure under which the tissue and the treatment solution are held during the treatment can range from about 200 kPa to about 10,000 kPa. In some embodiments, the pressure under which the tissue and the treatment solution are held during the treatment can range from about 300 kPa to about 1 ,000 kPa.

[0053] After treatment with the treatment solution, the excess treatment solution can be removed mechanically from the treated tissue by, for example, contact with an absorbent fabric. In embodiments in which the treatment solution includes water, this results in reduced water content in the treated tissue. The treatment solution may remain, for example, within interstitial spaces of the extracellular matrix. The treated tissue may be completely dried by exposing it to air for a time ranging from about 2 hours to about 96 hours at a temperature ranging from about 20°C to about 45°C. In some embodiments, the treated tissue of the implantable material may be dried to remove substantially all of the water, so that the implantable material is substantially free of water. The treatment solution of one or more polyols and, optionally, glycerol, remains within the interstitial spaces of the extracellular matrix, and preserves the integrity and flexibility of the implantable material without the need of storing it in a water based storage solution.

[0054] After drying, the dried tissue can be sterilized. In some embodiments, sterilizing the dried tissue may include exposing the dried tissue to ethylene oxide gas, avoiding temperatures above 50°C in order not to damage the tissue itself. In some embodiments, sterilizing the dried tissue may include exposing the dried tissue to ionizing radiation, such as gamma radiation. In some embodiments, sterilizing the dried tissue may include exposing the dried tissue to UV light. In some embodiments, the dried tissue may be sterilized by exposure to any combination of ethylene oxide gas, ionizing radiation, and UV light. As noted above, none of these sterilization methods induces tissue fixation, so remodeling is not impaired.

[0055] Once sterilized, the decellularized or tissue engineered, tissue is preserved in a stable, pliable state and may be stored for an extended period of time. The methods described herein provide an alternative to aldehydes for sterilization and storage of fixed tissue, and increase the shelf life of non-fixed tissue. Non-fixed tissue not treated according to the methods provided herein must be used within a few days or weeks from production or it quickly degenerates. Advantageously, tissue/devices of the present disclosure can be sterilized in its final packaging and then stored at room temperature. By allowing non-chemical sterilization (e.g., ethylene oxide gas, ionizing radiation, and/or UV light), methods of the present disclosure provide tissue and/or bioprostheses that may be stored for long time periods in a simplified way. The preserved implantable material may be made ready for implanting by immersing the implantable material in a physiologic saline solution. Alternatively, the preserved implantable material may be implanted as is and the host's blood and body fluids relied upon to rehydrate the material.

EXAMPLES

[0056] The present invention is more particularly described in the following examples that are intended as illustrations only, since numerous modifications and variations within the scope of the present invention will be apparent to those of skill in the art. Unless otherwise noted, all parts, percentages, and ratios reported in the following examples are on a weight bases, and all reagents used in the examples were obtained, or are available, from the chemical suppliers, or may be synthesized by conventional techniques.

Example 1

[0057] In one example, freshly harvested bovine pericardium tissue was decellularized by immersion in a solution of 0.5% deoxycholate and 0.5% sodium dodecyl sulfate for 24 hours at 22°C with continuous shaking. Then the tissue was washed with phosphate buffered saline solution (PBS) for 24 hours (10 solution changes) at 22°C with continuous shaking (ref.: Cebotari, S. et al., Detergent Decellularization of Heart Valves for Tissue Engineering: Toxicological Effects of Residual Detergents on Human Endothelial Cells, Artificial Organs, 34: 206-210 (2010)). Other decellularization methods or tissue engineering techniques can be used.

[0058] Samples of the decellularized tissue, a tissue sample 10, a tissue sample 20, and a tissue sample 30, were submitted to different treatments A, B, and C, respectively. In treatment A, the tissue sample 10 was immersed in pure glycerol for 16 hours at room temperature. In treatment B, the tissue sample 20 was immersed in pure 1 ,2 propanediol for 16 hours at room temperature. In treatment C, the tissue sample 30 was immersed in a solution of about 50 vol. % 1 ,2 propanediol and about 50 vol. % glycerol for 16 hours at room temperature.

[0059] At the end of the immersion time, each of the three tissue samples 10, 20, and 30 were blotted on adsorbent fabric and put as a sandwich between two sheets of adsorbent polymeric fabric and gently squeezed under a press to mechanically remove excess liquid. Then the three tissue samples were dried at room temperature for 24 hours, sealed in Tyvek® pouches, and sterilized by exposure to ethylene oxide gas.

[0060] The three tissue samples after sterilization are shown in FIGS. 1 -3 for treatments A, B, and C, respectively. The tissue sample 10 that received treatment A was observed to be more swollen and rigid with respect to the other two samples, but was still pliable. The tissue sample 20 that received treatment B and the tissue sample 30 that received treatment C were perfectly pliable, soft and elastic. All the three tissue samples 10, 20, and 30 preserved their initial dimension and were soft enough to sew into a heart valve bioprosthesis. When put in physiologic saline solution they were observed to immediately recover their initial appearance.

Example 2

[0061] In another example, decellularized bovine pericardium tissue was obtained as described above in Example 1 . This tissue was then used to prepare a stented valve 40.

[0062] The valve 40 was then immersed in a solution of about 70 vol. % 1 ,2 propanediol and about 30 vol. % glycerol for 16 hours at room temperature. During immersion the valve leaflets were kept in the correct shape using soft polymer clips.

[0063] At the end of the immersion the excess of polyols mixture was removed by a smooth spatula and gently blotted with polymeric fabric to mechanically remove excess liquid. The valve 40 was left to dry in an oven at about 35°C for 3 days and then was sterilized by exposure to ethylene oxide gas.

[0064] The valve 40 after sterilization is shown in FIG. 4. After sterilization, the valve 40 was observed to be perfectly pliable and in a good shape.

Example 3

[0065] In another example, patches of freshly harvested bovine pericardium tissue was washed several times in saline solution and selected to obtain suitable tissue for sewing tissue heart valves. The patches were then submitted to an initial treatment with a glutaraldehyde solution at low concentration 0.1 - 0.3% w/v for few hours at room temperature to fix the tissue.

[0066] The tissue patches, a tissue patch 50, at tissue patch 60, a tissue patch 70, and a tissue patch 80, were submitted to different treatments A, B, C, and D, respectively. In treatment A, the tissue patch 50 was immersed in pure glycerol for 16 hours at room temperature. In treatment B, the tissue patch 60 was immersed in pure 1 ,2 propanediol for 16 hours at room temperature. In treatment C, the tissue patch 70 was immersed in a solution of about 50 vol. % 1 ,2 propanediol and about 50 vol. % glycerol for 16 hours at room temperature. In treatment D, the tissue patch 80 was immersed in a solution of about 85 vol. % 1 ,4 butanediol and about 15 vol. % glycerol for 16 hours at room temperature.

[0067] At the end of the immersion time, the four tissue patches 50, 60, 70 and 80 were blotted on adsorbent fabric and put as a sandwich between two sheets of adsorbent polymeric fabric and gently squeezed under a press to mechanically remove excess liquid. Then the three tissue patches were dried at room temperature for 24 hours, sealed in Tyvek® pouches, and sterilized by exposure to ethylene oxide gas.

[0068] The four tissue patches 50, 60, 70, and 80 after sterilization are shown in FIGS. 5-8 for treatments A, B, C, and D, respectively. The tissue patches 50, 60, 70, and 80 were all observed to be soft and pliable.

Example 4

[0069] In another example, patches of freshly harvested bovine pericardium tissue was washed several times in saline solution and selected to obtain suitable tissue for sewing tissue heart valves. The patches were then submitted to an initial treatment with a glutaraldehyde solution at low concentration 0.1 - 0.3% w/v for few hours at room temperature to fix the tissue. These patches were then used for heart valve sewing.

[0070] Three different heart valves, a valve 90, a valve 100, and a valve 1 10, made from the patches were submitted to different treatments A, B, and C, respectively. In treatment A, the valve 90 was immersed in pure glycerol for 16 hours at room temperature. In treatment B, the valve 100 was immersed in pure 1 ,2 propanediol for 16 hours at room temperature. In treatment C, the valve 1 10 was immersed in a solution of about 50 vol. % 1 ,2 propanediol and about 50 vol. % glycerol for 16 hours at room temperature. During immersion the valve leaflets were kept in the correct shape using soft polymer clips.

[0071] At the end of the immersion the excess of polyols mixture was removed from each of the valves 90, 100, and 1 10 by a smooth spatula and gently blotted with polymeric fabric to mechanically remove excess liquid. The valves were left to dry in an oven at about 35°C for 3 days and then were sterilized by exposure to ethylene oxide gas. [0072] The heart valves 90, 100, and 1 10 after sterilization are shown in FIGS. 9A-1 1 B. FIGS. 9A and 9B are photographs showing opposite sides of the valve 90 that received Treatment A. FIGS. 10A and 10B are photographs showing opposite sides of the valve 100 that received Treatment B. FIGS. 1 1A and 1 1 B are photographs showing opposite sides of the valve 1 10 that received Treatment C. After sterilization, the valves 90, 100, and 1 10 were observed to be perfectly pliable and in a good shape.

[0073] Various modifications and additions can be made to the exemplary embodiments discussed without departing from the scope of the present invention. For example, while the embodiments described above refer to particular features, the scope of this invention also includes embodiments having different combinations of features and embodiments that do not include all of the described features. Accordingly, the scope of the present invention is intended to embrace all such alternatives, modifications, and variations as falling within the scope of the claims, together with all equivalents thereof.

Claims

CLAIMS I claim:

1. An implantable material comprising: decellularized sterilized mammalian tissue having an extracellular matrix,

wherein a plurality of interstitial spaces of the extracellular matrix include a solution of one or more polyols other than glycerol and, optionally, glycerol.

2. The implantable material of claim 1 , wherein the glycerol is present in an amount of about 1 vol. % to about 50 vol. % of the mixture and the one or more polyols other than glycerol are present in an amount of about 50 vol. % to about 99 vol. % of the mixture.

3. The implantable material of claim 2, wherein the solution in the plurality of interstitial spaces of the extracellular matrix consists essentially of the one or more polyols other than glycerol and glycerol.

4. The implantable material of claim 1 , wherein the one or more polyols other than glycerol includes at least one of 1 ,2 propanediol, 1 ,2 octanediol, 1 ,4 butanediol, fructose, xylitol, mannitol, sorbitol, lactulose, lactic acid, maltose, glucose, galactose, erythritol, lactobionic acid or its salts, and hyaluronic acid.

5. The implantable material of claim 1 , wherein the one or more polyols other than glycerol are selected from the group consisting of 1 ,2 propanediol, 1 ,4 butanediol, lactobionic acid and its salts, and combinations thereof.

6. The implantable material of claim 1 , wherein the one or more polyols other than glycerol consists essentially 1 ,2 propanediol.

7. The implantable material of claim 1 , wherein the implantable material is substantially free of water.

8. The implantable material of claim 1 , wherein the implantable material is non- fixed.

9. The implantable material of claim 1 , wherein the implantable material forms a portion of an engineered heart valve.

10. A method for making an implantable material, the method comprising:

decellularizing mammalian tissue;

treating the decellularized mammalian tissue with a solution including one or more polyols other than glycerol, glycerol and, optionally, water;

drying the treated tissue; and

sterilizing the dried tissue.

1 1 . The method of claim 10, wherein the solution includes the glycerol at a concentration from about 15 vol. % to about 35 vol. %, the one or more polyols other than glycerol at a concentration of about 65 vol. % to about 85 vol. % of the solution, and the balance water.

12. The method of claim 10, wherein the solution consists essentially of the one or more polyols other than glycerol, glycerol, and, optionally, water.

13. The method of claim 10, wherein sterilizing includes at least one of: exposing the dried tissue to ethylene oxide gas; exposing the dried tissue to ionizing radiation; and exposing the dried tissue to ultraviolet radiation.

14. The method of claim 10, wherein drying the treated tissue includes drying until substantially all water is removed from the treated tissue.

15. The method of claim 10, wherein treating the tissue includes immersing the tissue in the solution.

16. The method of claim 15, wherein the tissue is immersed in the solution for a time ranging from 2 to 100 hours and at a temperature ranging from 5°C to 35°C.

17. The method of claim 15, wherein the tissue is immersed in the solution more than once.

18. The method of claim 10, wherein treating the tissue includes one of spraying the solution on the tissue and spreading the solution on the tissue.

19. The method of claim 10, wherein the one or more polyols other than glycerol includes at least one of 1 ,2 propanediol, 1 ,2 octanediol, 1 ,4 butanediol, fructose, xylitol, mannitol, sorbitol, lactulose, lactic acid, maltose, glucose, galactose, erythritol, lactobionic acid or its salts, and hyaluronic acid.

20. The method of claim 10, wherein the one or more polyols other than glycerol are selected from the group consisting of 1 ,2 propanediol, 1 ,4 butanediol, lactobionic acid, and combinations thereof.

21 . The method of claim 10, wherein solution includes the glycerol at a concentration from about 15 vol. % to about 35 vol. %, the one or more polyols other than glycerol at a concentration of about 65 vol. % to about 85 vol. % of the solution, and the balance ethanol.

22. The method of claim 10, wherein the tissue is not treated with a fixing agent.

23. A preserved tissue engineered heart valve comprising: decellularized, non-fixed, sterilized mammalian tissue having an extracellular matrix, wherein a plurality of interstitial spaces of the extracellular matrix includes a solution consisting essentially of one or more polyols other than glycerol and glycerol, wherein the glycerol is no more than 50 vol. % of the solution.