Enzymatic treatment of tissues intended for implantation

Abstract

Methods of preparing a tissue for surgical use are described herein. The method may include providing a tissue and treating the tissue with a solution including at least one of serrapeptase or nattokinase. The use of the serrapeptase or nattokinase results in an improvement in the pliability and the porosity of the tissue and assists in the decellularization of the tissue.

Description

ENZYMATIC TREATMENT OF TISSUES INTENDED FOR IMPLANTATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of and priority to U.S. Provisional Patent Application No. 63 / 674,081, filed July 22, 2024, titled “ENZYMATIC TREATMENT OF TISSUES INTENDED FOR IMPLANTATION,” the disclosure of which is incorporated by reference herein in its entirety.TECHNICAL FIELD

[0002] The present disclosure relates generally to methods, systems, and apparatuses for the treatment of tissue matrices using enzymes. In particular, the present disclosure relates to methods of softening tissue for use in surgical applications.BACKGROUND

[0003] Various surgical grafts are prepared using human or animal tissue for use in surgical procedures. The surgical grafts often require decellularization to avoid invoking immune rejection or significant immune response in the surgical patient. Additionally, the mechanical properties of the surgical grafts, such as pliability and porosity, can make molding the grafts to the necessary shape and cellular growth within the patient difficult. To improve the pliability and porosity, enzymes have been used on the surgical grafts prior to implantation. Among the enzymes that have been explored for this purpose, bromelain, papain, ficin, and actinidin have shown particular promise. These enzymes, derived from various plant sources, have demonstrated the ability to break down specific protein structures within tissue matrices. This targeted enzymatic action allows for the modification of matrix properties, including porosity and pliability, of the grafts while maintaining desired mechanical properties such as the tensile, suture, or burst strength. However, the enzymes do not decellularize the tissue in the grafts or assist in improving fibrotic conditions. There exists a need for improved methods of preparing tissues for use as surgical grafts, including the use of other proteolytic enzymes that may be beneficial to a fibrotic environment.BRIEF DESCRIPTION OF THE DRAWINGS

[0004] FIG. 1 depicts a graphical representation of the measurement of the drapability of tissue sheets in accordance with an embodiment.

[0005] FIG. 2 and FIG. 3 depict images of draped treated and untreated tissues in accordance with several embodiments.

[0006] FIG. 4 and FIG. 5 depict images of tissues stained with hematoxylin and eosin (H&E) stain in accordance with several embodiments.

[0007] FIG. 6 depicts a graphical representation of the measurement of the DNA levels of tissue sheets in accordance with an embodiment.

[0008] FIG. 7 depicts a graphical representation of the measurement of the Galactose-a / / ?Aa-l ,3- galactose epitope levels of tissue sheets in accordance with an embodiment.SUMMARY

[0009] In some embodiments, a method for preparing a tissue for surgical use includes providing a tissue selected from the group consisting of dermis tissue, small intestinal submucosal (SIS) tissue, urinary bladder matrix (UBM) tissue, amnion tissue, peritoneum tissue, pericardium tissue, and combinations thereof; and treating the tissue with a first solution comprising serrapeptase, nattokinase, or a combination thereof.

[0010] In some embodiments, the first solution further comprises water, PBS, saline, Tris-HCl buffer, or a combination thereof.

[0011] In some embodiments, the first solution includes a pH of about 6 to about 9. In certain embodiments, the first solution includes a pH of about 7 to about 8.

[0012] In some embodiments, the method further includes pretreating the tissue to remove cellular material such as DNA, cellular membranes, and / or immunogenic components from the tissue.

[0013] In some embodiments, pretreating the tissue includes contacting the tissue with a decellularization solution selected from the group consisting of a detergent, DNase, a- galactosidase, and combinations thereof.

[0014] In some embodiments, the decellularization solution further comprises one or more of serrapeptase or nattokinase.

[0015] In some embodiments, the method further includes processing the tissue by a process selected from the group consisting of lyophilization, drying at room temperature, drying at a temperature greater than room temperature, freezing, mixing with a sterile saline solution, and combinations thereof.

[0016] In some embodiments, treating the tissue with the first solution comprises contacting the tissue with the first solution; incubating the tissue at a predetermined temperature; and washing the tissue with a deactivation solution configured to remove or inactivate enzymatic activity.

[0017] In some embodiments, the method further comprises mixing the tissue and the first solution during incubation.

[0018] In some embodiments, the deactivation solution includes one or more of triton wash or an inactivating agent.

[0019] In some embodiments, the method further comprises sterilizing the tissue.DETAILED DESCRIPTION

[0020] The invention, of which various embodiments and embodiments are described herein, is not limited strictly to the particular systems, devices, and methods described, as these may vary. The terminology used in the description is for the purpose of describing various embodiments and embodiments only and does not limit the scope of the invention, which is limited only by the appended claims.

[0021] As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Nothing in this disclosure is to be construed as an admission that the embodiments described in this disclosure are not entitled to antedate such disclosure by virtue of prior invention.

[0022] As used herein, the term “about” when immediately preceding a numerical value means a range of plus or minus 10% of that value and also includes exactly that value, e.g., “about 50” means 45 to 55, “about 25,000” means 22,500 to 27,500.

[0023] The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, non-recited elements or method steps. As used in this document, the term “comprising” means “including, but not limited to.” In contrast, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0024] As used herein, the term “surgical use” refers to use in medical or surgical applications, including implantation. In some instances, “surgical use” refers to implantation at or near a surgical site, or used during a surgical procedure. A surgical use may include any procedure or application performed by a surgeon or other medical professional.

[0025] The present disclosure relates to the field of medical technology, specifically to the modification of tissue matrices. This modification process involves the use of proteolytic enzymes, which are known for their ability to break down proteins into smaller peptides or amino acids. In the context of this disclosure, these enzymes are utilized to modify tissue matrices, potentially enhancing their suitability for various medical applications. Advantages of the present methods of modifying tissue matrices include improving pliability of the tissue matrix while maintaining relevant strength, increasing porosity of the tissue matrix to allow for better host cell infiltration, facilitating faster tissue integration with minimal chronic inflammation, allowingfaster rehydration time of the product for improved usability (if the product is in a dry / lyophilized form), and use of widely available enzymes with a track record of treating fibrosis.

[0026] The proteolytic enzymes may be applied to a wide range of tissue matrices, including but not limited to, mammalian tissues, plant tissues, and synthetic tissues. The modification process may result in tissue matrices that exhibit altered properties, such as improved biocompatibility, enhanced mechanical strength, or increased permeability. These modified tissue matrices may be used in a variety of medical applications, including tissue engineering, wound healing, and drug delivery.

[0027] In some embodiments, the proteolytic enzymes may be used in combination with other agents or treatments to further modify the tissue matrices. For example, the enzymes may be used in conjunction with cross-linking agents, sterilization processes, or other treatments known in the art. The combination of these treatments may result in tissue matrices with unique properties, potentially expanding their range of medical applications.

[0028] In some cases, the proteolytic enzymes may be used to selectively modify certain components of the tissue matrices, while leaving other components intact. This selective modification may allow for the creation of tissue matrices with specific properties tailored to particular medical applications. Overall, the use of proteolytic enzymes to modify tissue matrices may provide a versatile and effective approach to creating improved materials for a wide range of medical applications.

[0029] In some embodiments, the proteolytic enzymes utilized for tissue modification may include one or more of serrapeptase and nattokinase. Serrapeptase is an enzyme and metalloprotease that is found in Serratia marcescens and can be isolated from the intestine of the silkworm, Bombyx mori. Nattokinase is an enzyme that can be isolated from natto, made from soybeans fermented with bacterium such as Bacillus subtilis. Serrapeptase and nattokinase have previously been used in to treat inflammation, scar tissue, or fibrotic conditions such as fibrosis.

[0030] These enzymes may be used to treat human or animal tissue, potentially altering the mechanical properties of the tissue. For instance, the treatment with these enzymes may increase the pliability and porosity of the tissue after incubation. The tissue subjected to this enzyme treatment may originate from various sources, including but not limited to, porcine, bovine, or ovine sources. In some cases, the enzymes may be applied to specific types of tissue such as dermis tissue, but the application is not limited to this tissue type and may also include Small Intestine Submucosa (SIS), Urinary Bladder Matrix (UBM), Amnion, Peritoneum, and Pericardium.

[0031] In some cases, the tissue may be in its native form or may be decellularized prior to or following the enzyme treatment. Decellularization may involve the use of various chemicals or detergents known in the art. The enzymes may be used at various concentrations in different solutions or buffers, such as water, Phosphate-Buffered Saline (PBS), Hank’s Buffer solution, or Tris-HCl buffer.

[0032] In some embodiments, the enzymes may be used to alter the mechanical properties of the native tissue to affect handling properties of the tissue. For example, the enzyme treatment may improve the mechanical properties of the tissue such as softness, pliability, and drapability. While modifying the mechanical properties, the enzymes may still maintain the structural integrity of key extracellular matrix components, such as collagen proteins.

[0033] In some cases, the enzymes may also be a part of the decellularization protocol. One or more of the enzymes serrapeptase and nattokinase may be used to remove cellular material such as DNA and cellular membranes from human or animal tissue to create decellularized tissue matrices or scaffolds. This cellular removal process may remove immunogenic components of the native tissue.

[0034] In some embodiments, the enzymes may also remove other immunogenic components of the native tissue, such as Galactose-a / / ?Aa-l,3-galactose epitope (or “a-gal epitope”). Furthermore, the enzymes may improve tissue integration into the host by allowing for better and faster cellular infiltration with minimal chronic inflammation. The removal of both DNA and other potential immunogenic epitopes (such as a-gal epitope) is an advantage over other treatments which only be able to remove portions of immunogenic components.

[0035] In some embodiments, the concentration of the enzymes serrapeptase and nattokinase used for tissue treatment may vary. The concentration may be selected based on the desired effect on the tissue, the type of tissue being treated, or other factors. For instance, a higher concentration of enzymes may result in a greater increase in tissue pliability and porosity. However, the concentration should be selected such that the structural integrity of key extracellular matrix components, such as collagen proteins, is maintained.

[0036] In some cases, the enzymes may be used in different solutions or buffers for tissue treatment. These solutions or buffers may include, but are not limited to, water, Phosphate- Buffered Saline (PBS), Hank’s Buffer solution, or Tris-HCl buffer. The choice of solution or buffer may depend on the type of tissue being treated, the desired effect on the tissue, or other factors. For example, a buffer solution may be used to maintain a stable pH during the enzyme treatment, which may be beneficial for certain types of tissue or desired effects.

[0037] In some embodiments, the effect of the enzyme treatment on tissue pliability and porosity may depend on the concentration of the enzymes and the type of solution or buffer used. For instance, a higher concentration of enzymes in a buffer solution may result in a greater increase in tissue pliability and porosity compared to a lower concentration of enzymes in the same buffer solution. Similarly, the use of a particular solution or buffer may enhance the effect of the enzymes on tissue pliability and porosity compared to other solutions or buffers.

[0038] In some cases, the enzyme treatment may be performed at different temperatures or for different durations. The temperature and duration of the treatment may be selected based on the type of tissue being treated, the desired effect on the tissue, or other factors. For example, a longer duration of treatment at a higher temperature may result in a greater increase in tissue pliability and porosity compared to a shorter duration of treatment at a lower temperature. It is contemplated that the use of different concentrations of enzymes, different solutions or buffers, and different treatment conditions may allow for the customization of the enzyme treatment to achieve desired effects on tissue pliability and porosity.

[0039] In some embodiments, the tissue that may be treated with the enzymes serrapeptase and nattokinase can originate from various sources. These sources may include, but are not limited to, human and animal tissues. Animal tissues may be derived from a variety of species, such as porcine, bovine, or ovine sources. The choice of tissue source may depend on factors such as the intended application of the treated tissue, the availability of the tissue, or other considerations.

[0040] In some cases, the enzymes may be applied to specific types of tissue. For instance, the enzymes may be used to treat dermis tissue. However, the application of the enzymes is not limited to dermis tissue and may also include other tissue types. These other tissue types may include, but are not limited to, Small Intestine Submucosa (SIS), Urinary Bladder Matrix (UBM), Amnion, Peritoneum, and Pericardium. The choice of tissue type may depend on factors such as the desired properties of the treated tissue, the intended application of the treated tissue, or other considerations.

[0041] In some embodiments, the enzymes may be used to treat tissue in its native form. Alternatively, the enzymes may be used to treat tissue that has been decellularized. Decellularization may involve the use of various chemicals or detergents known in the art to remove cellular material from the tissue. The choice between native tissue and decellularized tissue may depend on factors such as the desired properties of the treated tissue, the intended application of the treated tissue, or other considerations.

[0042] In some embodiments, the enzymes serrapeptase and nattokinase may be used to alter the mechanical properties of the tissue, such as softness, pliability, and drapability. This alteration inmechanical properties may enhance the handling characteristics of the tissue, potentially making it more suitable for certain medical applications. For instance, a tissue that is more pliable and drapable may be easier to manipulate during surgical procedures, potentially improving the ease of use and effectiveness of the tissue in these applications.

[0043] While the enzymes may alter the mechanical properties of the tissue, they may also maintain the structural integrity of key extracellular matrix components. These components, such as collagen proteins, are crucial for the structure and function of the tissue. By maintaining the integrity of these components, the enzymes may ensure that the tissue retains its essential characteristics, even as its mechanical properties are modified.

[0044] In some cases, the degree to which the enzymes alter the mechanical properties of the tissue may depend on various factors. These factors may include the concentration of the enzymes, the duration of the enzyme treatment, the type of solution or buffer used for the enzyme treatment, and the type of tissue being treated. By adjusting these factors, it may be possible to customize the enzyme treatment to achieve a desired balance between altered mechanical properties and maintained structural integrity.

[0045] In some embodiments, the enzymes may be used to treat a variety of tissue types. These tissue types may include, but are not limited to, dermis tissue, Small Intestine Submucosa (SIS), Urinary Bladder Matrix (UBM), Amnion, Peritoneum, and Pericardium. The effect of the enzymes on the mechanical properties and structural integrity of these tissue types may vary, potentially providing a range of modified tissues with different characteristics suitable for different medical applications.

[0046] In some embodiments, the enzymes serrapeptase and nattokinase may be utilized in decellularization protocols. These protocols may involve the use of these enzymes to remove cellular material from human or animal tissue, potentially creating decellularized tissue matrices or scaffolds. The cellular material that may be removed by these enzymes may include, but is not limited to, DNA and cellular membranes. The removal of this cellular material may result in the elimination of immunogenic components from the native tissue, potentially reducing the risk of immune response when the decellularized tissue is used in medical applications.

[0047] In some cases, the decellularization process may be performed using a variety of techniques known in the art. These techniques may involve the use of chemicals or detergents, in addition to the enzymes serrapeptase and nattokinase. The choice of decellularization technique may depend on factors such as the type of tissue being decellularized, the desired properties of the decellularized tissue, or other considerations.

[0048] In some embodiments, the decellularized tissue matrices or scaffolds created using the enzymes serrapeptase and nattokinase may exhibit altered properties compared to the native tissue. These altered properties may include increased porosity, which may enhance the ability of the decellularized tissue to support cell infiltration and tissue integration. This increased porosity may be beneficial for various medical applications, such as tissue engineering or wound healing.

[0049] In some cases, the decellularized tissue matrices or scaffolds may be used in their decellularized state for medical applications. Alternatively, the decellularized tissue may be further treated or modified, for instance, by seeding with cells, cross-linking, or sterilization, before being used in medical applications.

[0050] In some embodiments, the increased porosity of the tissue resulting from the enzyme treatment may also facilitate the addition of a drug layer to the tissue. This drug layer may provide a controlled release of therapeutic agents, potentially improving the efficacy and safety of the therapy. The addition of the drug layer may be performed while maintaining the desired mechanical and regenerative properties of the tissue, potentially expanding the range of medical applications for the treated tissue.

[0051] In some embodiments, the enzymes serrapeptase and nattokinase may be used to remove immunogenic components from the native tissue. These immunogenic components may include, but are not limited to, Galactose-a / / ?Aa-l,3-galactose epitope (or “a-gal epitope”). Galactose- alpha- 1,3 -galactose epitope is known to be immunogenic in certain individuals, and its removal from the tissue may reduce the risk of an immune response when the tissue is used in medical applications.

[0052] The removal of a-gal epitope and other immunogenic components may be achieved through the proteolytic activity of the enzymes serrapeptase and nattokinase. These enzymes may break down the immunogenic components into smaller peptides or amino acids, which may be more easily removed from the tissue. The removal of these components may be facilitated by the use of various solutions or buffers, such as water, Phosphate-Buffered Saline (PBS), Hank’s Buffer solution, or Tris-HCl buffer.

[0053] In some cases, the efficiency of the removal of immunogenic components may depend on various factors, including but not limited to the concentration of the enzymes, the duration of the enzyme treatment, the type of solution or buffer used for the enzyme treatment, and the type of tissue being treated. By adjusting these factors, it may be possible to increase the removal of immunogenic components from the tissue.

[0054] In some embodiments, the removal of immunogenic components from the tissue may enhance the biocompatibility of the tissue. This enhanced biocompatibility may make the tissuemore suitable for various medical applications, such as tissue engineering, wound healing, and drug delivery.

[0055] In some embodiments, the enzymes serrapeptase and nattokinase may contribute to improved tissue integration into the host. This improvement may be facilitated by the enzymes' ability to enhance cellular infiltration into the treated tissue. Cellular infiltration refers to the process by which cells migrate into the tissue matrix, a process that is crucial for tissue integration and healing. By enhancing this process, the enzymes may allow for better and faster tissue integration into the host.

[0056] In some cases, the enzymes may not only enhance cellular infiltration but also minimize chronic inflammation. Chronic inflammation is a prolonged and persistent inflammatory response that can lead to tissue damage and disease. By minimizing chronic inflammation, the enzymes may reduce the risk of adverse reactions and complications associated with tissue integration, potentially improving the safety and effectiveness of the treated tissue in medical applications.

[0057] In some embodiments, the degree to which the enzymes improve tissue integration and minimize chronic inflammation may depend on various factors. These factors may include the concentration of the enzymes, the duration of the enzyme treatment, the type of solution or buffer used for the enzyme treatment, and the type of tissue being treated. By adjusting these factors, it may be possible to enhance the effects of the enzymes on tissue integration and inflammation reduction.

[0058] In some cases, the enzymes may be used to treat a variety of tissue types for improved tissue integration into the host. These tissue types may include, but are not limited to, dermis tissue, Small Intestine Submucosa (SIS), Urinary Bladder Matrix (UBM), Amnion, Peritoneum, and Pericardium. The effect of the enzymes on tissue integration and inflammation may vary among these tissue types, potentially providing a range of treated tissues with different characteristics suitable for different medical applications.

[0059] In some embodiments, specific examples of enzyme concentrations, incubation times, and resulting tissue property changes may be provided. For instance, a concentration of serrapeptase and nattokinase ranging from about 0.1 to about 10 mg / mL, or any range or value contained within such a range, may be used. The enzymes may be incubated with the tissue for a duration ranging from about 1 to about 24 hours, or any range or value contained within such a range. The resulting tissue may exhibit increased pliability and porosity, potentially enhancing its suitability for various medical applications.

[0060] In some cases, the tissue may be incubated with the enzymes at a concentration of about 1 mg / mL for about 12 hours. This treatment may result in a significant increase in tissue pliabilityand porosity, potentially making the tissue more suitable for applications that require flexible and permeable materials.

[0061] In some embodiments, different tissue types may respond differently to the enzyme treatment. For example, dermis tissue may exhibit a greater increase in pliability and porosity when treated with a higher concentration of enzymes for a longer duration. On the other hand, Small Intestine Submucosa (SIS) may exhibit a similar increase in pliability and porosity when treated with a lower concentration of enzymes for a shorter duration.

[0062] In some cases, the enzyme treatment may be adjusted for each tissue type. For instance, Urinary Bladder Matrix (UBM) may be treated with a concentration of about 2 mg / mL of enzymes for about 6 hours to achieve a desired increase in pliability and porosity. Similarly, Amnion may be treated with a concentration of about 0.5 mg / mL of enzymes for about 3 hours to achieve a similar increase in pliability and porosity. The preceding values are non-limiting examples and other concentrations and times may be used.

[0063] In some embodiments, the enzyme treatment may be adjusted based on the desired properties of the treated tissue. For example, if a greater increase in pliability is desired, a higher concentration of enzymes may be used. If a greater increase in porosity is desired, a longer incubation time may be used.

[0064] In some embodiments, methods for assessing tissue properties before and after enzyme treatment may be used. These methods may include mechanical testing and cellular content analysis. Mechanical testing may be used to evaluate changes in the mechanical properties of the tissue, such as softness, pliability, and drapability. This testing may involve the use of various techniques known in the art, such as tensile testing, compression testing, or shear testing. The choice of mechanical testing technique may depend on the type of tissue being tested, the desired information about the tissue, or other factors.

[0065] In some cases, cellular content analysis may be used to evaluate changes in the cellular content of the tissue. This analysis may involve the use of various techniques known in the art, such as histological staining, immunohistochemistry, or DNA quantification. The choice of cellular content analysis technique may depend on the type of tissue being analyzed, the desired information about the tissue, or other factors.

[0066] In some embodiments, the results of the mechanical testing and cellular content analysis may be used to assess the effect of the enzyme treatment on the tissue. For instance, an increase in tissue pliability and porosity as determined by mechanical testing may indicate a successful enzyme treatment. Similarly, a decrease in cellular content as determined by cellular content analysis may indicate a successful decellularization process.

[0067] In some cases, the results of the mechanical testing and cellular content analysis may be used to optimize the enzyme treatment. For instance, if the desired increase in tissue pliability and porosity is not achieved, the concentration of the enzymes or the duration of the enzyme treatment may be adjusted. Similarly, if the desired decrease in cellular content is not achieved, the decellularization process may be modified.

[0068] In some embodiments, the mechanical testing and cellular content analysis may be performed at various stages of the tissue modification process. For example, these tests may be performed before the enzyme treatment to establish baseline properties of the tissue, after the enzyme treatment to assess the effect of the treatment, and after any subsequent treatments to assess the overall effect of the modification process.

[0069] In some embodiments, the enzymes serrapeptase and nattokinase may be used in combination with other decellularization methods. These methods may include, but are not limited to, the use of chemicals or detergents known in the art for decellularization. The enzymes and these other decellularization methods may be used in a sequential or simultaneous manner. For instance, the tissue may be treated with the enzymes first, followed by treatment with chemicals or detergents. Alternatively, the tissue may be treated with the enzymes and the chemicals or detergents at the same time.

[0070] In some cases, the order of the enzyme treatment and the other decellularization methods may be selected based on the type of tissue being treated, the desired properties of the treated tissue, or other factors. For example, for certain types of tissue, it may be beneficial to perform the enzyme treatment first to increase the tissue's pliability and porosity, followed by the use of chemicals or detergents to remove cellular material. In other cases, it may be beneficial to perform the enzyme treatment and the use of chemicals or detergents simultaneously to achieve a more efficient decellularization process.

[0071] In some embodiments, the combination of the enzyme treatment with other decellularization methods may result in tissue matrices with unique properties. These properties may include increased pliability and porosity, reduced immunogenicity, and enhanced suitability for various medical applications. The specific properties achieved may depend on the type of tissue being treated, the specific enzymes and other decellularization methods used, and the order and conditions of their application.

[0072] In some embodiments, the enzyme-treated tissues may find potential applications in various medical and surgical procedures. For instance, the increased pliability and porosity of the tissue, resulting from the enzyme treatment, may enhance its suitability for use in surgical procedures such as grafting, suturing, or implantation. The treated tissue may be easier tomanipulate and conform to the surgical site, potentially improving the ease of use and effectiveness of the tissue in these procedures.

[0073] In some cases, the enzyme-treated tissues may be used in tissue engineering applications. The increased porosity of the tissue may enhance cell infiltration and tissue integration, potentially making the tissue more suitable as a scaffold for tissue regeneration. The treated tissue may support the growth and differentiation of various cell types, potentially facilitating the regeneration of damaged or diseased tissues.

[0074] In some embodiments, the enzyme-treated tissues may be used in wound healing applications. The increased pliability and porosity of the tissue may enhance its ability to conform to the wound site and support the infiltration of cells necessary for wound healing. The treated tissue may provide a protective barrier over the wound, while also promoting the healing process.

[0075] In some cases, the enzyme-treated tissues may be used in drug delivery applications. The increased porosity of the tissue may enhance its ability to load and release therapeutic agents, potentially making the tissue more suitable as a drug delivery system. The treated tissue may provide a controlled release of therapeutic agents, potentially improving the efficacy and safety of the therapy.

[0076] In some embodiments, the enzyme-treated tissues may be used in other medical applications, such as in the creation of medical devices or prosthetics. The increased pliability and porosity of the tissue may enhance its suitability for use in these applications, potentially improving the performance and biocompatibility of the devices or prosthetics.

[0077] In some embodiments, the enzyme-treated tissues may be stored and handled in such a way to maintain their modified properties. For instance, the tissues may be stored at a specific temperature or in a specific solution or buffer to preserve their increased pliability and porosity. The choice of storage conditions may depend on factors such as the type of tissue, the specific modifications made to the tissue, or other considerations.

[0078] In some cases, the enzyme-treated tissues may be stored in a refrigerated environment. This cold storage may help to slow down any enzymatic reactions or other processes that could potentially alter the properties of the tissue. The specific temperature of the refrigerated environment may be selected based on the type of tissue, the specific modifications made to the tissue, or other factors.

[0079] In some embodiments, the enzyme-treated tissues may be stored in a solution or buffer. This solution or buffer may help to maintain the hydration of the tissue, potentially preserving its increased pliability and porosity. The choice of solution or buffer may depend on the type of tissue,the specific modifications made to the tissue, or other factors. For example, the tissues may be stored in Phosphate-Buffered Saline (PBS), Hank’s Buffer solution, or Tris-HCl buffer.

[0080] Methods

[0081] The present disclosure provides methods for preparing a tissue for surgical use. In some embodiments, the method may include providing a tissue and treating the tissue with a first solution to achieve an improved level of pliability or porosity. In some embodiments, the first solution includes one or more of serrapeptase or nattokinase. The serrapeptase or nattokinase allow for the softening of the tissue by increasing the pliability, drapability, and the porosity of the tissue. This enables the tissue to be better molded into surgical sites and around implants and devices. The increased porosity additionally allows for a faster rehydration of the tissue in case of lyophilization as compared to tissues with lower porosity. The serrapeptase or nattokinase also assist in the decellularization of the tissue including the removal of DNA, cellular membranes and other immunogenic components of the tissue, such Galactose-a / / ?Aa-l,3-galactose epitope (or “a- gal epitope”). The serrapeptase or nattokinase improve the mechanical properties of the tissue while maintaining the tensile, suture, or burst strength of the tissue as well as the structural integrity of key extracellular matrix components, such as collagen proteins.

[0082] A method for preparing a tissue for surgical use includes providing a tissue. The tissue may include any tissue effective for use as a surgical graft. In some embodiments, the tissue includes one of dermis tissue, small intestinal submucosal (SIS) tissue, urinary bladder matrix (UBM) tissue, amnion tissue, peritoneum tissue, or pericardium tissue. In some embodiments, the tissue includes one of human, porcine, bovine, or ovine tissue. In some embodiments, providing the tissue includes cutting the tissue to a size and shape effective for use as a surgical graft. In some embodiments, the tissue is cut to a size and shape effective to fit into a surgical site. In some embodiments, is cut to a size and shape effective to fit around one of an implant or an implantable device. In some embodiments, the provided tissue is decellularized. In some embodiments, the provided tissue is in a native form and is not decellularized.

[0083] The method may further include treating the tissue with a first solution including an enzyme effective for increasing the pliability and porosity of the tissue. In some embodiments, the first solution includes one or more of serrapeptase or nattokinase. The first solution may further include one or more of water, phosphate buffer solution (PBS), saline, Hank’s Buffer solution, or Tris-HCl buffer. The first solution may have any pH effective for increasing the pliability and porosity of the tissue. In some embodiments, the first solution has a pH of about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9,about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, or any value or range of values between any two of these values. In some embodiments, the first solution has a pH of about 6.0 to about 9.0 or about 7.0 to about 8.0.

[0084] In some embodiments, treating the tissue with the first solutions may include contacting the tissue with the first solution. In some embodiments, treating the tissue with the first solutions may include submerging the tissue in the first solution. In some embodiments, treating the tissue with the first solution may include incubating the tissue at a predetermined temperature. The predetermined temperature may be any temperature effective for enabling the first solution to increase to pliability and porosity of the tissue. In some embodiments, the predetermined temperature is about -10 °C, about -9 °C, about -8 °C, about -7 °C, about -6 °C, about -5 °C, about -4 °C, about -3 °C, about -2 °C, about -1 °C, about 0 °C, about 1 °C, about 2 °C, about 3 °C, about 4 °C, about 5 °C, about 6 °C, about 7 °C, about 8 °C, about 9 °C, about 10 °C, about 11 °C, about 12 °C, about 13 °C, about 14 °C, about 15 °C, about 16 °C, about 17 °C, about 18 °C, about 29 °C, about 30 °C, or any value or range of values between any two of these values.

[0085] The tissue may be incubated for any amount of time effective for increasing the porosity and pliability of the tissue. In some embodiments, the tissue is incubated until the tissue reaches a predetermined porosity, pliability, or decellularization amount. In some embodiments, the predetermined porosity includes an average pore size of about 500 nm, about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, about 800 nm, about 900 nm, about 950 nm, about 1,000 nm, about 1,050 nm, about 1,100 nm, about 1,150 nm, about 1,200 nm, about 1,250 nm, about 1,300 nm, about 1,350 nm, about 1,400 nm, about 1,450 nm, about 1,500 nm, about 1,550 nm, about 1,600 nm, about 1,650 nm, about 1,700 nm, about 1,750 nm, about 1,800 nm, about 1,850 nm, about 1,900 nm, about 1,950 nm, about 2,000 nm, or any value or range of values between any two of these values. In some embodiments, the predetermined porosity includes a tissue porosity of about 5 %, about 10 %, about 15 %, about 20 %, about 25 %, about 30 %, about 35 %, about 40 %, about 45 %, about 50 %, about 55 %, about 60 %, about 65 %, about 70 %, about 75 %, about 80 %, about 85 %, about 90 %, about 95 %, about 98 %, about 99 %, or any value or range of values between any two of these values.

[0086] In some embodiments, the predetermined decellularization amount is measured by the amount of removal of cellular material such as DNA, cellular membranes, and immunogenic components from the tissue. In some embodiment, the predetermined decellularization amount is about 90%, about 95%, about 98%, about 99%, 99.9%, or any value or range of values between any two of these values. In some embodiment, the predetermined decellularization amount includes removal of substantially all of the cellular material such as DNA, cellular membranes,and immunogenic components. In some embodiments, the immunogenic components include a- gal epitope.

[0087] In some embodiments, treating the tissue may further include washing the tissue with a deactivation solution. The deactivation solution may include any solution known to one of skill in the art effective to remove or inactivate the enzymatic activity. In some embodiments, the deactivation solution includes one or more of triton wash or an inactivating agent. In some embodiments, treating the tissue may further include mixing the tissue and the first solution during incubation. In some embodiments the mixing is performed by one or more of a shaker, a rocker, or a stir plate.

[0088] In some embodiments, the method may further include pretreating the tissue to remove cellular material such as DNA, cellular membranes, and immunogenic components from the tissue. In some embodiments, pretreating the tissue includes contacting the tissue with a decellularization solution including a decellularization agent. In some embodiments, the decellularization agent includes one or more of a detergent, DNase, deoxycholate, or a- galactosidase. In some embodiments, the decellularization agent include one or more of serrapeptase or nattokinase. In some embodiments, the tissue is pretreated to a predetermined decellularization amount measured by the amount of removal of cellular material such as DNA, cellular membranes, and immunogenic components from the tissue. In some embodiment, the predetermined decellularization amount is about 90%, about 95%, about 98%, about 99%, 99.9%, or any value or range of values between any two of these values. In some embodiment, the predetermined decellularization amount is substantially all of the cellular material such as DNA, cellular membranes, and immunogenic components.

[0089] In some embodiments, the method may further include processing the tissue by one or more of lyophilization, drying at room temperature, drying at a temperature greater than room temperature, freezing, or mixing with a sterile saline solution. In some embodiments, the tissue may be stored at -20° C to 10° C after processing. In some embodiments, the method further includes sterilizing the tissue. The tissue may be sterilized by any method known to one of skill in the art. In some embodiments, the tissue is sterilized by irradiation.

[0090] Tissue products

[0091] Enzyme-treated tissues having improved properties as compared to untreated tissues may be assembled using the above-described methods.

[0092] In some embodiments, an enzyme-treated tissue may have improved properties as compared to untreated tissues. The tissue may include any tissue effective for use as a surgical graft. In some embodiments, the tissue includes one of dermis tissue, small intestinal submucosal(SIS) tissue, urinary bladder matrix (UBM) tissue, amnion tissue, peritoneum tissue, or pericardium tissue. In some embodiments, the tissue includes one of human, porcine, bovine, or ovine tissue.

[0093] In some embodiments, the enzyme treatment may involve exposing the tissue to a first solution comprising serrapeptase, nattokinase, or a combination thereof. In some cases, the enzyme solution may include added chemicals such as magnesium sulfate to provide metal ions, which may enhance enzymatic activity.

[0094] Enzyme-treated tissues may have an increased pliability as compared to untreated tissues. In some embodiments, the enzyme -treated tissue may have a drapability coefficient of about 0.75, about 0.70, about 0.65, about 0.60, about 0.55, about 0.50, about 0.45, about 0.40, about 0.35, about 0.30, about 0.25, about 0.20, about 0.15, about 0.10, about 0.05, or any value or range of values between any two of these values. In some embodiments, the enzyme-treated tissue may have a reduced drapability coefficient as compared to an untreated tissue by about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or any value or range of values between any two of these values. This increased pliability may allow the tissue to better conform to surgical sites or around implants and devices.

[0095] The porosity of the enzyme-treated tissues may be increased as compared to untreated tissues. Increased porosity may facilitate faster rehydration of the tissue, particularly in cases where the tissue has been lyophilized. In some embodiments, the enzyme-treated tissue may have an average pore size of about 500 nm, about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, about 800 nm, about 900 nm, about 950 nm, about 1,000 nm, about 1,050 nm, about 1,100 nm, about 1,150 nm, about 1,200 nm, about 1,250 nm, about 1,300 nm, about 1,350 nm, about 1,400 nm, about 1,450 nm, about 1,500 nm, about 1,550 nm, about 1,600 nm, about 1,650 nm, about 1,700 nm, about 1,750 nm, about 1,800 nm, about 1,850 nm, about 1,900 nm, about 1,950 nm, about 2,000 nm, or any value or range of values between any two of these values. In some embodiments, the enzyme-treated tissue may have a tissue porosity of about 5 %, about 10 %, about 15 %, about 20 %, about 25 %, about 30 %, about 35 %, about 40 %, about 45 %, about 50 %, about 55 %, about 60 %, about 65 %, about 70 %, about 75 %, about 80 %, about 85 %, about 90 %, about 95 %, about 98 %, about 99 %, or any value or range of values between any two of these values.

[0096] Enzyme treatment may result in significant decellularization of the tissue. In some embodiments, the enzyme-treated tissue may have a DNA level of less than about 50 ng / mg of dry tissue weight, less than about 45 ng / mg of dry tissue weight, less than about 40 ng / mg of drytissue weight, less than about 35 ng / mg of dry tissue weight, less than about 30 ng / mg of dry tissue weight, less than about 25 ng / mg of dry tissue weight, less than about 20 ng / mg of dry tissue weight, less than about 15 ng / mg of dry tissue weight, less than about 10 ng / mg of dry tissue weight, less than about 5 ng / mg of dry tissue weight, or any value or range of values between any two of these values. In some embodiments, the enzyme-treated tissue may have a reduced DNA level as compared to an untreated tissue of about 80 %, about 85 %, about 90 %, about 95 %, about 98 %, about 99 %, about 99.9 %, or any value or range of values between any two of these values. This reduction in cellular content may help minimize the risk of immune rejection when the tissue is used in medical procedures.

[0097] Enzyme treatment may result in a decrease in the amount of immunogenic factors, such as Galactose-a / / ?Aa-l,3-galactose (or “a-gal epitope”), in the tissue. In some embodiments, the enzyme-treated tissue may have a reduction in a-gal as compared to an untreated tissue of about 50 %, about 55 %, about 60 %, about 65 %, about 70 %, about 75 %, about 80 %, about 85 %, about 90 %, about 95 %, about 98 %, about 99 %, or any value or range of values between any two of these values.

[0098] Mechanical testing may be performed to assess the properties of enzyme-treated tissues. This testing may include measurements of tensile strength, burst strength, and suture retention strength. In some embodiments, the enzyme treatment may improve certain mechanical properties, such as the pliability and porosity of the tissue, while maintaining the structural integrity of key extracellular matrix components, such as collagen proteins. In some embodiments, the enzyme treated tissue may have a tensile strength within about 10 %, about 9 %, about 8 %, about 7 %, about 6 %, about 5 %, about 4 %, about 3 %, about 2 %, about 1 %, or the same as compared to an untreated tissue. In some embodiments, the enzyme treated tissue may have a ball burst strength within about 10 %, about 9 %, about 8 %, about 7 %, about 6 %, about 5 %, about 4 %, about 3 %, about 2 %, about 1 %, or the same as compared to an untreated tissue. In some embodiments, the enzyme treated tissue may have a suture retention strength within about 10 %, about 9 %, about 8 %, about 7 %, about 6 %, about 5 %, about 4 %, about 3 %, about 2 %, about 1 %, or the same as compared to an untreated tissue.

[0099] In some embodiments, the enzyme-treated tissue may be sterilized to ensure suitability for medical use. The enzyme -treated tissue may be sterilized by any method known to one of ordinary skill in the art. In some embodiments, the tissue may be sterilized by irradiation. The sterilization process may be selected to effectively eliminate potential pathogens while preserving the beneficial properties of the enzyme-treated tissue.

[0100] The following numbered embodiments may be combined to form new embodiments and may be used with any of the embodiments disclosed herein.

[0101] Embodiment 1 : A method for preparing a tissue for surgical use, the method comprising: providing a tissue selected from the group consisting of dermis tissue, small intestinal submucosal (SIS) tissue, urinary bladder matrix (UBM) tissue, amnion tissue, peritoneum tissue, pericardium tissue, and combinations thereof; and treating the tissue with a first solution comprising serrapeptase, nattokinase, or a combination thereof.

[0102] Embodiment 2: The method of embodiment 1 , wherein the tissue comprises human tissue, porcine tissue, bovine tissue, ovine tissue, or a combination thereof.

[0103] Embodiment 3: The method of embodiment 1 or embodiment 2, wherein the first solution further comprises water, PBS, saline, or Tris-HCl buffer, or a combination thereof.

[0104] Embodiment 4: The method of any of embodiments 1 to 3, wherein the first solution has a pH of about 6 to about 9.

[0105] Embodiment 5: The method of any of embodiments 1 to 3, wherein the first solution has a pH of about 7 to about 8.

[0106] Embodiment 6: The method of any of embodiments 1 to 5, further comprising pretreating the tissue to remove cellular material from the tissue.

[0107] Embodiment 7: The method of embodiment 6, wherein the cellular material comprises DNA, cellular membranes, immunogenic components, or combinations thereof.

[0108] Embodiment 8: The method of embodiment 6 or embodiment 7, wherein pretreating the tissue comprises contacting the tissue with a decellularization solution selected from the group consisting of a detergent, DNase, a-galactosidase, and combinations thereof.

[0109] Embodiment 9: The method of embodiment 8, wherein the decellularization solution further comprises serrapeptase, nattokinase, or a combination thereof.

[0110] Embodiment 10: The method of any of embodiments 1 to 9, further comprising processing the tissue by a process selected from the group consisting of lyophilization, drying at room temperature, drying at a temperature greater than room temperature, freezing, mixing with a sterile saline solution, and combinations thereof.

[0111] Embodiment 11: The method of any of embodiments 1 to 10, wherein treating the tissue with the first solution comprises: contacting the tissue with the first solution; incubating the tissue at a predetermined temperature; and washing the tissue with a deactivation solution configured to remove or inactivate enzymatic activity.

[0112] Embodiment 12: The method of any of embodiments 1 to 11, further comprising mixing the tissue and the first solution during incubation.

[0113] Embodiment 13: The method of any of embodiments 1 to 12, wherein the deactivation solution comprises one or more of triton wash and an inactivating agent.

[0114] Embodiment 14: The method of any of embodiments 1 to 13, further comprising sterilizing the tissue.

[0115] Embodiment 15: The method of any of embodiments 1 to 14, wherein the method results in an increase in porosity of the tissue.

[0116] Embodiment 16: The method of any of embodiments 1 to 15, wherein the method results in an increase in pliability of the tissue.

[0117] Embodiment 17: The method of any of embodiments 1 to 16, wherein the method results in a decellularization amount of at least about 90%.

[0118] Embodiment 18: A tissue prepared by the method of any of embodiments 1 to 17.

[0119] Examples illustrating particular implementations of the various embodiments described herein are discussed below.EXAMPLES

[0120] Example 1 : Preparation of enzymes

[0121] Serrapeptase was added as a powder to water or a buffer solution such as PBS, Hank’s buffer solution, or Tris-HCl with added chemicals such as magnesium sulfate to provide metal ions. The solution was mixed until fully dissolved. The amount of the enzyme powder added was sufficient to fully dissolve in the solution. The amount of enzyme powder added depends on the specific activity of the isolated powder and the type of tissue to be treated. The preferable starting pH range of the solution is pH 7-8.

[0122] Nattokinase was added as a powder to water or a buffer solution such as PBS, Hank’s buffer solution, or Tris-HCl with added chemicals such as magnesium sulfate to provide metal ions. The solution was mixed until fully dissolved. The amount of the enzyme powder added was sufficient to fully dissolve in the solution. The amount of enzyme powder added depends on the specific activity of the isolated powder and the type of tissue to be treated. The preferable starting pH range of the solution is pH 7-8.

[0123] Alternatively, the two enzyme powders may be mixed together or combined with chemicals or solutions involved in decellularization such as detergents, DNase, or a-galactosidase.

[0124] Example 2 : Enzymatic treatment of tissue

[0125] Enzymatic solutions such as those described in Example 1 were incubated with porcine dermis tissue samples that were cut to size and placed in a sealable container. The enzyme solutions were added to the container in a volume sufficient to fully submerge the tissue. Thecontainer was sealed and maintained at the proper temperature (e.g., in either an incubator or refrigerated unit). Optionally, the tissue may be also placed on a shaker or rocker to mix the container during treatment. When the tissue reached the desired physical state and / or level of decellularization, it was removed from the enzyme solution and washed with appropriate solutions (e.g., triton wash and / or other inactivating reagent) to remove and / or inactivate the enzymatic activity.

[0126] After the tissue was treated with the enzyme solution and other solutions to support decellularization and DNA removal, the resulting decellularized dermal tissue was lyophilized into sheets with a size of up to about 18 cm x about 20 cm, having a thickness of about 0.9 to about 1.1 mm. Optionally, the decellularized dermal tissue may also be further processed for end use by methods including drying at room temperature or elevated temperatures, freezing, and / or mixing with sterile saline solution.

[0127] Example 3: Mechanical testing of treated tissue

[0128] Mechanical testing was performed to determine the pliability and drapability of tissues treated with enzymes compared to native tissue. Enzymatically treated porcine dermal tissue was prepared using the method described in Example 2 and by using the enzyme serrapeptase. Samples were prepared from the enzymatically treated tissue as well as native untreated porcine dermal tissue.

[0129] The drapability of each tissue was measured using the Cusick Drape Test (recognized by International Standard ISO 9073-9). The tissue was cut into a circle of appropriate size and draped over a support. Using a light source, parabolic mirror, and a specialized apparatus, a shadow of the draping tissue was captured and quantified. As a comparator, the untreated tissue (which is stiff and non-drapable) was cut into the same circle size of the test tissue and quantified. The resulting drapability coefficient is the ratio of the area of the shadow of the treated tissue to the control comparator tissue.

[0130] The results of this testing clearly show that the enzymatically treated tissue had improved drapability compared to the native tissue, with drapability coefficients of about 0.4 and about 1.0, respectively. Results of the drapability coefficient testing are provided in FIG. 1, with images of the draped treated tissue and native tissue provided in FIG. 2 and FIG. 3, respectively.

[0131] Example 4: Tissue porosity and DNA levels

[0132] Samples of the treated and untreated tissue were tested for the porosity and DNA levels. Samples of the treated tissue were prepared as described in Example 3. The treated and untreated samples were stained using hematoxylin and eosin (H&E) stain used to detect DNA in the tissue.Images were taken of both the treated and untreated samples to determine the porosity of the samples. The images of the untreated and treated samples are provided in FIG. 4 and FIG. 5, respectively. The porosity of the tissue post-enzyme treatment was greater than the porosity of the native tissue. This porosity allows for faster rehydration time and increased pliability allowing for improved ease of use. Furthermore, the collagen fibers of the enzyme treated tissue remained intact, indicating preservation of collagen morphology.

[0133] DNA levels of the treated and untreated tissue were measured using a PicoGreen dsDNA assay. DNA from solubilized porcine dermis was isolated and purified using the PureLink Genomic DNA Kit (Invitrogen Corp., Carlsbad, CA). Tissue was solubilized in a Proteinase K solution and then bound to the membrane of a spin column, washed, and then eluted into a set volume of buffer. DNA amounts were quantified using a set of standards and the Quant-iT PicoGreen reagent kit (Invitrogen Corp., Carlsbad, CA) that could detect lower than 1 ng / mL of DNA in the sample solution.

[0134] The results of the testing showed a significant decrease in the DNA levels of the treated samples as compared to the untreated samples. The treated samples had a measured DNA level of about 20 ng DNA per mg of dry tissue weight, while the untreated samples had a measured DNA level of about 180 ng DNA per mg of dry tissue weight. Results of the DNA measurements are provided in FIG. 6.

[0135] The Galactose-a / / ?Aa- 1,3 -galactose (“a-gal epitope”) level of each prepared sample was tested as well. The a-gal epitope levels were measured by western blot analysis. 50 pg of isolated protein solution from tissue samples or controls were run on an SDS page gel and then transferred to a nitrocellulose membrane. The transfer was confirmed with Ponceau S staining (Thermo Fisher, Waltham, MA). The membrane was blocked with 5% BSA in TBS with 0.1% Tween-20 for 1 hour at room temperature and then incubated overnight at 4°C with a biotinylated primary anti-alpha-gal monoclonal IgM antibody (Clone M86, Enzo Life Sciences, Germany) diluted 1:50 in the blocking buffer. After washing, the membrane was incubated with a fluorescent-conjugated anti-Mouse secondary antibody diluted to 1:5000 for 1 hour at room temperature. After another round of washes, a-Gal bands were detected and quantified using a SpectraMax iD5e Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA).

[0136] The results of the a-gal epitope level measurements showed a significant decrease in the treated samples as compared to the untreated samples. The treated samples had an a-gal epitope level of about 0.1, while the untreated samples had an a-gal epitope level of about 1.25. Results of the a-gal epitope level measurements are provided in FIG. 7. This removal of DNA and a-gal epitope provides a reduced risk of immune response when the decellularized tissue is used inmedical applications.

[0137] While various illustrative embodiments incorporating the principles of the present teachings have been disclosed, the present teachings are not limited to the disclosed embodiments. Instead, this application is intended to cover any variations, uses, or adaptations of the present teachings and use its general principles. Further, this application is intended to cover such departures from the present disclosure that are within known or customary practice in the art to which these teachings pertain.

[0138] In the above detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the present disclosure are not meant to be limiting. Other embodiments may be used, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that various features of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

[0139] The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as illustrations of various features. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds, compositions, or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0140] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity.

[0141] It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (for example, the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” et cetera). While various compositions, methods, and devices are described in terms of “comprising” various components or steps (interpreted as meaning “including, but not limited to”), the compositions, methods, anddevices can also “consist essentially of’ or “consist of’ the various components and steps, and such terminology should be interpreted as defining essentially closed-member groups.

[0142] In addition, even if a specific number is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (for example, the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, et cetera” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (for example, “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, et cetera). In instances where a convention analogous to “at least one of A, B, or C, et cetera” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (for example, “a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, et cetera). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, sample embodiments, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0143] In addition, where features of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0144] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, et cetera. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, et cetera. As will also be understood by one skilled in the art all language such as “up to,” “at least,” and the like include the number recited and refer to ranges that can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 components refer to groups having 1,2, or 3 components. Similarly, a group having 1-5 components refer to groups having 1, 2, 3, 4, or 5 components, and so forth.

[0145] Various of the above-disclosed and other features and functions, or alternatives thereof, may be combined into many other different systems or applications. Various presently unforeseen or unanticipated alternatives, modifications, variations, or improvements therein may be subsequently made by those skilled in the art, each of which is also intended to be encompassed by the disclosed embodiments.

Claims

CLAIMSWhat is claimed is:

1. A method for preparing a tissue for surgical use, the method comprising: providing a tissue selected from the group consisting of dermis tissue, small intestinal submucosal (SIS) tissue, urinary bladder matrix (UBM) tissue, amnion tissue, peritoneum tissue, pericardium tissue, and combinations thereof; and treating the tissue with a first solution comprising serrapeptase, nattokinase, or a combination thereof.

2. The method of claim 1, wherein the tissue comprises human tissue, porcine tissue, bovine tissue, ovine tissue, or a combination thereof.

3. The method of claim 1 or claim 2, wherein the first solution further comprises water, PBS, saline, or Tris-HCl buffer, or a combination thereof.

4. The method of any of claims 1 to 3, wherein the first solution has a pH of about 6 to about 9.

5. The method of any of claims 1 to 3, wherein the first solution has a pH of about 7 to about 8.

6. The method of any of claims 1 to 5, further comprising pretreating the tissue to remove cellular material from the tissue.

7. The method of claim 6, wherein the cellular material comprises DNA, cellular membranes, immunogenic components, or combinations thereof.

8. The method of claim 6 or claim 7, wherein pretreating the tissue comprises contacting the tissue with a decellularization solution selected from the group consisting of a detergent, DNase, a-galactosidase, and combinations thereof.

9. The method of claim 8, wherein the decellularization solution further comprises serrapeptase, nattokinase, or a combination thereof.

10. The method of any of claims 1 to 9, further comprising processing the tissue by a process selected from the group consisting of lyophilization, drying at room temperature, drying at a temperature greater than room temperature, freezing, mixing with a sterile saline solution, and combinations thereof.

11. The method of any of claims 1 to 10, wherein treating the tissue with the first solution comprises: contacting the tissue with the first solution; incubating the tissue at a predetermined temperature; and washing the tissue with a deactivation solution configured to remove or inactivate enzymatic activity.

12. The method of any of claims 1 to 11, further comprising mixing the tissue and the first solution during incubation.

13. The method of any of claims 1 to 12, wherein the deactivation solution comprises one or more of triton wash and an inactivating agent.

14. The method of any of claims 1 to 13, further comprising sterilizing the tissue.

15. The method of any of claims 1 to 14, wherein the method results in an increase in porosity of the tissue.

16. The method of any of claims 1 to 15, wherein the method results in an increase in pliability of the tissue.

17. The method of any of claims 1 to 16, wherein the method results in a decellularization amount of at least about 90%.

18. A tissue prepared by the method of any of claims 1 to 17.

Images