WO2011031827A2

WO2011031827A2 - Manufacture of extracellular matrix products using supercritical or near supercritical fluids

Info

Publication number: WO2011031827A2
Application number: PCT/US2010/048223
Authority: WO
Inventors: Jian-Lin Liu; Bruce J. Demars; Bhavin Shah
Original assignee: Cook Biotech Incorporated; Sabin Corporation
Priority date: 2009-09-09
Filing date: 2010-09-09
Publication date: 2011-03-17
Also published as: WO2011031827A3

Abstract

Described are methods for preparing extracellular matrix materials using treatment with a fluid under conditions at which the fluid is at least substantially supercritical. Preferred prepared extracellular matrix materials beneficially retain endogenous bioactive molecules and/or beneficial native mechanical properties even after treatment with the fluid. Also described are materials that can be prepared by such methods, and uses of such materials.

Description

MANUFACTURE OF EXTRACELLULAR MATRIX PRODUCTS USING SUPERCRITICAL OR NEAR SUPERCRITICAL FLUIDS

BACKGROUND Aspects of the present invention relate to products containing extracellular matrix materials obtained from animal sources, and to processes for their manufacture and use. In certain embodiments, the present invention relates to methods for processing animal-derived extracellular matrix materials using fluid at or near supercritical conditions and to products with unique mechanical, biochemical, and bioperformance properties that are obtainable by such methods.

A variety of extracellular matrix (ECM) materials have been proposed for use in medical grafting, cell culture, and other applications. For instance, medical grafts and cell culture materials containing submucosa derived from small intestine, stomach or urinary bladder tissues, have been proposed. See, e.g., U.S. Patent Nos. 4,902,508, 4,956, 178, 5,281 ,422, 5,554,389, 6,099,567 and 6,206,931 . In addition, Cook Biotech Incorporated, West Lafayette, Indiana, currently manufactures a variety of medical materials based upon small intestinal submucosa under the trademarks SURGISIS®, STRATASIS® and OASIS®.

Medical materials derived from liver basement membrane have also been proposed, for example in U.S. Patent No. 6,379,710. As well, ECM materials derived from amnion (see e.g. U.S. Patent Nos. 4,361 ,552 and 6,576,618) and from renal capsule membrane (see International PCT Patent Application No. WO 03/002165 published January 9, 2003) have been proposed for medical and/or cell culture applications. In this field, needs remain for improved or alternate materials that are useful for a wide variety of medical applications, and for methods for their manufacture. The present invention provides such materials, as well as methods of preparing and using the same.

SUMMARY

One feature of the present invention is the discovery that animal- derived extracellular matrix (ECM) materials can be processed with a supercritical fluid or near supercritical fluid to purify the materials while beneficially retaining both bioactivity and mechanical performance properties. Thus, in certain of its embodiments, the invention provides methods for making ECM products that include the steps of treating a collagenous ECM material retaining one or more endogenous bioactive components with an at least substantially supercritical fluid, and thereafter recovering the treated collagenous ECM material still retaining an amount of the one or more endogenous bioactive components. The one or more endogenous bioactive components can include at least one of an endogenous growth factor, an endogenous glycosaminoglycan, and an endogenous glycoprotein, and in preferred embodiments the treated collagenous ECM material includes a combination of all of these biomolecules. The treatment with the near supercritical fluid or supercritical fluid can be effective to remove undesired components of the ECM material, for example endogenous lipids. The ECM can be processed with one or more additional mediums, before and/or after treatment with the near/supercritical fluid, that enhance the biocompatibility of the finished ECM product. For example, the ECM can be treated with a decellularization medium separate from the near/supercritical fluid, preferably before treatment of the ECM with the near/supercritical fluid. The decellularization medium can be a peracetic acid medium in certain embodiments. A near/supercritical-fluid-treated ECM retaining the endogenous bioactive component(s) can be medically packaged and terminally sterilized to provide a medical product. Additional embodiments of the invention relate to other defined treatments of ECM materials with a near/supercritical fluid, and to ECM materials with enhanced properties and packaged end products that can, for example, be obtained by methods described herein.

Still further embodiments of the invention as well as features and advantages thereof will be apparent to those of ordinary skill in the art from the descriptions herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a digital image illustrating capillary development upon implantation with a collagenous extracellular matrix material (small intestine submucosa, SIS) that retains endogenous bioactive components.

FIG. 2 is a digital image illustrating capillary development upon implantation with a collagenous extracellular matrix material (SIS) that has been processed in such a fashion as to eliminate endogenous bioactive components.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of the invention, reference will now be made to certain embodiments thereof and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, and alterations and modifications in the specifically described embodiments, and further applications of the principles of the invention as illustrated herein are herein contemplated as would normally occur to one skilled in the art to which the invention relates.

As disclosed above, aspects of the present invention relate to the use of a supercritical or near supercritical fluid in the manufacture of useful extracellular matrix (ECM) materials. Additional embodiments of the invention relate to uniquely characterized ECM materials and their use in medical applications in humans or non-human animals. As terms or phrases are used herein in relation to these and other embodiments of the invention, the following definitions will apply unless context clearly indicates otherwise. "Supercritical", with reference to a fluid in general or to a specific compound, refers to such fluid or such compound at or above its critical temperature and at or above its critical pressure.

"Near supercritical", with reference to a fluid in general or to a specific compound, refers to such fluid or such compound that is not supercritical but which is at about 95% or more of its critical temperature and at about 95% or more of its critical pressure.

"At least substantially supercritical", with reference to a fluid in general or to a specific compound, refers to such fluid or such compound which is near supercritical or supercritical. "Near/supercritical" is an abbreviation used at times herein referring to near supercritical or supercritical.

"Subcritical", with reference to a fluid in general or to a specific compound, refers to such fluid or such compound which is not supercritical.

"Decellularizing" refers to eliminating all or essentially all living endogenous cells from a tissue material, such as an ECM. "Decellularized", with reference to a tissue material such as an ECM, means free or essentially free from living endogenous cells.

"Acellular", with reference to a tissue material such as an ECM, means free or essentially free from any living cells.

"Substantially devoid of cells and cell components" means free or essentially free from cells (living or dead) and of cell membranes and other cell remnants. An ECM material substantially devoid of cells or cell components is intended to include the ECM material carrying cells or cell components at a level sufficiently low to be substantially nonimmunogenic when the material is implanted in a recipient, especially a recipient to which the cells or cell components are xenogenic or allogenic.

"Endogenous bioactive component", with reference to a tissue material such as an ECM, refers to a bioactive substance that occurs endogenously with the ECM. "Bioactive" in this context refers to the ability of a substance to induce, directly or indirectly, a cellular response such as a change in cell morphology, proliferation, growth, protein or gene expression. For purposes of the discussions herein, endogenous lipids are not considered to be endogenous bioactive components. Turning now to a discussion of ECM materials that can be used, suitable ECMs include collagenous ECM sheet materials comprising submucosa, renal capsule membrane, dermal collagen, dura mater, pericardium, fascia lata, serosa, peritoneum or basement membrane layers, including liver basement membrane. These and other similar animal-derived ECM tissue layers can be obtained and processed as described herein. Suitable submucosa materials include, for instance, intestinal submucosa, including small intestinal submucosa, stomach submucosa, urinary bladder submucosa, and uterine submucosa. Suitable animal sources for the ECM materials include either human donors (e.g. cadavers) or non-human animals such as ovine, bovine or porcine animals. Porcine ECM tissues are preferred. ECM tissues that exist as, and that as isolated provide, thin membranous connective tissue sheets, e.g. sheets having a thickness of less than about 1000 μιη, more preferably less than about 500 μιη, are preferred.

ECM materials as discussed above can be initially mechanically isolated containing endogenous bioactive components that can benefit the performance of the final ECM product. However, the bioactive nature of the ECM is relatively delicate and is subject to elimination by subsequent processing steps used to bring the ECM product to its final, biocompatible condition. Illustratively, FIGs. 1 and 2 provide a comparison of the performance of differentially processed ECM segments (both porcine SIS) upon implant. In particular, FIG. 1 provides a digital image showing capillary ingrowth into a bioactive SIS material (retaining a substantially preserved microarchitecture including endogenous FGF-2 and other growth factors, endogenous glycosaminoglycans and endogenous proteoglycans) 21 days after implant. The SIS material of FIG. 1 was prepared using relatively mild processing conditions that included mechanical separation of the SIS tissue (including the tunica submucosa, muscularis mucosa, and stratum compactum) from the remainder of the small intestine, disinfection by soaking in a 0.2% peracetic acid in 5% ethyl alcohol for about 2 hours, and sterilization with ethylene oxide. A segment of this SIS (prepared in a 4- layer construct by stacking and lyophilizing together the SIS layers) was used in a subcutaneous implant model in which 1 .5 cm-diameter discs were sandwiched between 0.22μιη filters and implanted subcutaneously in mice. The filters resist through-growth of tissue and thus new tissue development can be observed growing into the edges of the sandwiched SIS samples. At 21 days after implant, the mice were injected with fluorescent microbeads, which are then allowed to circulate. Images were then taken with confocal microscopy. As seen in FIG. 1 , capillary infiltration into the SIS was profuse and continued deep into the SIS layer. In a comparative test, 4-layer SIS samples prepared as described for FIG. 1 above and further chemically stripped (using strong sodium hydroxide) of essentially all components except collagen and a minor amount of elastin were subjected to similar subcutaneous implant testing and similarly imaged at 21 days. FIG. 2 shows the results. As is evident, capillary growth into the stripped SIS is highly stunted relative to that observed in the inductive angiogenic SIS. The new capillaries are seen to reverse their growth course back toward the disc edge where native tissues of the implanted animal and associated bioactive factors are readily available. FIGs. 1 and 2 thus demonstrate that mild treatments in the preparation of ECM products can result in beneficially bioactive angiogenic materials, whereas even relatively brief treatments with harsher extractants are shown to disrupt the enhanced bioactive angiogenic character of the ECM. In accordance with one aspect of the invention, it has been discovered that endogenously bioactive ECM starting materials can be treated with near/supercritical fluids, well-recognized aggressive penetrating media with high solvent power, and nonetheless beneficially retain endogenous bioactive components giving them a bioactive character. As set forth in greater detail in the illustrative Examples below, endogenously bioactive ECM materials were treated with supercritical carbon dioxide for two hours. The resultant treated ECM materials retained substantially the same levels of endogenous FGF-2 and sulfated glycosaminoglycans as the starting ECM materials, whereas the endogenous lipid content of the treated ECM materials was substantially reduced. Moreover, the supercritical fluid treatment of the ECM materials did not significantly alter their tensile strength as compared to non-treated controls. Near/supercritical fluid treatment of an endogenously bioactive ECM starting material can thus be used to process the material, for instance to reduce lipid content, while substantially retaining the endogenous bioactivity and mechanical properties of the ECM material such as high strength and/or excellent porosity.

In certain embodiments, ECM materials of and treated in accordance with the invention will include abundant collagen, most commonly being constituted at least about 80% by weight collagen on a dry weight basis. Such ECM materials can include collagen fibers that are non-randomly oriented, for instance occurring as generally uniaxial or multi-axial but regularly oriented fibers. When processed to retain endogenous bioactive components, the ECM material can retain these components interspersed as solids between, upon and/or within the collagen fibers. Particularly desirable ECM materials for use in the invention will include significant amounts of such interspersed, non-collagenous bioactive component solids that are readily ascertainable under light microscopic examination (with appropriate staining). Such non-collagenous solids can constitute a significant percentage of the dry weight of the ECM material in certain inventive embodiments, for example at least about 1 %, at least about 3%, and at least about 5% by weight in some embodiments of the invention. The above- described collagen fiber orientations and/or levels of non-collagenous bioactive component solids can be present both before and after near/supercritical fluid treatment as described herein, and in final products. The ECM material, both before and after the near/supercritical fluid treatment, may also exhibit the ability to induce angiogenesis in a host engrafted with the material. Angiogenesis is the process through which the body makes new blood vessels to generate increased blood supply to tissues. Thus, angiogenic materials, when contacted with host tissues, promote or encourage the formation of new blood vessels. Methods for measuring in vivo angiogenesis in response to biomaterial implantation have been developed. For example, one such method uses a subcutaneous implant model to determine the angiogenic character of a material. See, C. Heeschen et al., Nature Medicine 7 (2001 ), No. 7, 833-839. When combined with a fluorescence microangiography technique as discussed above, this model can provide both quantitative and qualitative measures of angiogenesis into biomaterials. C. Johnson et al., Circulation Research 94 (2004), No. 2, 262-268.

Additionally, the ECM material, both before and after the near/supercritical fluid treatment, can be fully bioremodelable. "Fully bioremodelable" as used herein with reference to an ECM material means that the ECM material is effective upon implant in an animal subject (including a human) to promote cellular invasion and ingrowth during a time in which the originally implanted ECM material is eliminated, such that the originally implanted ECM material is replaced by tissue of the host.

As noted above, the ECM material can retain any of a variety of growth factors or other beneficial bioactive components endogenous or native to the source tissue, both before and after processing with the near/supercritical fluid. For example, the ECM material can include one or more native growth factors such as basic fibroblast growth factor (FGF-2), transforming growth factor beta (TGF-beta), epidermal growth factor (EGF), connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF) and/or platelet derived growth factor (PDGF). As well, or alternatively, the ECM material can include other bioactive materials such as proteoglycans and/or sulfated glycosaminoglycans (sGAG), such as heparin sulfate, hyaluronic acid (HA), fibronectin and the like. In certain embodiments, the ECM material, both before and after the near/supercritical fluid treatment, will exhibit a component profile wherein the following non-collagen bioactive endogenous components are present in the stated amounts:

Component Preferred More Preferred

FGF-2 ≥ 2 ng/g ≥ 5 ng/g

HA ≥ 50 μg/g ≥ 100 μg/g sGAG: ≥ 1000 μg/g ≥ 2000 μg/g

Preferably also, after the near/supercritical fluid treatment, the ECM material will have an endogenous lipid content of less than about 4% by weight. A final, sterile ECM product ready for implantation, and optionally suitably packaged, can also include the endogenous FGF-2, HA, sGAG and/or endogenous lipids in the above-stated amounts.

Compounds with critical temperatures less than about 60⁵ C will be used with preference in generating the near/supercritical fluid used to treat the ECM materials. Compounds with critical temperatures in the range of about 0⁵ C to about 60⁵ C will typically be used, more preferably with critical temperatures in the range of about 0⁵ C to about 50⁵ C. The near/supercritical fluid treatment of the ECM material will desirably be conducted at a temperature at which no significant thermal damage occurs to the connective tissue (e.g. collagen) matrix of the ECM material, e.g. below the shrink temperature of a collagenous ECM material to be treated. For these purposes, the near/supercritical fluid can be maintained at a temperature less than about 60⁵ C during contact with the ECM material, typically in the range of about 0⁵ C to about 60⁵ C, and more preferably in the range of about 0⁵ C to about 50⁵ C. The pressure of the near/supercritical fluid during treatment of the ECM material will depend upon the temperature used and the specific compound(s) used to generate the fluid, but in typical embodiments will range from about 500 pounds per square inch (psi) to about 5000 psi. A variety of near/supercritical fluids can be used. Compounds which are gases under ambient conditions are useful for generating the near/supercritical fluids. These include, for instance, alkanes such as ethane or propane, alkenes such as ethylene, fluorocarbons such as chlorodifluoromethane or trifluoromethane, nitrous oxide, nitrogen, carbon dioxide, or mixtures of these compounds. Near/supercritical fluids that include near/supercritical carbon dioxide as the sole near/supercritical compound or in combination with one or more other near/supercritical compounds are preferred. In certain embodiments, the treatment medium consists or consists essentially of near/supercritical carbon dioxide.

The ECM material can be treated with a pure near/supercritical fluid as a single near/supercritical substance or a mixture of near/supercritical substances, or with a near/supercritical fluid mixed with another component to facilitate the treatment, such as: a diluent; an entrainer; a surfactant; a detergent; an alcohol such as ethanol or n-propanol; a ketone such as acetone; an ester; dimethylsulfoxide (DMSO); dimethylformamide (DMF); a hydrocarbon; a disinfectant, for instance an oxidizing agent disinfectant such as hydrogen peroxide or an organic peroxy compound, more preferably a peracid such as peracetic acid, perpropioic acid, or perbenzoic acid; a chelating agent such as ethylene diamine tetraacetic acid (EDTA); an acidic aqueous medium (e.g. with a pH of 2 to 6) or a basic aqueous medium (e.g. with a pH of above 7 to about 9); or a mixture including two, three or more of these components, potentially along with others. In certain embodiments, the near/supercritical fluid treatment can be conducted to remove nucleic acid components, in which case the near/supercritical fluid can be mixed with a nucleic acid solubilizing medium such as a basic aqueous medium having a pH of above 7 to about 9, more preferably of about 8 to 8.5, desirably also including a biocompatible buffer such as tris(hydroxymethyl)aminomethane (TRIS) and a chelating agent such as (EDTA). In certain embodiments, the near/supercritical fluid treatment can be conducted so as to sterilize the ECM material, an in such cases the presence of a disinfectant as noted above can assist in achieving sterility. Also, an ECM material can be sequentially contacted with different near/supercritical fluid mediums designed to enhance removal of different undesired components of the ECM material. For example, the ECM material can be contacted with a first near/supercritical fluid medium to remove lipids, a second near/supercritical fluid to remove nucleic acids (e.g. with the near/supercritical fluid mixed with a nucleic acid solubilizing medium as identified above), and with a third near/supercritical fluid to sterilize the ECM material (e.g. with the near/supercritical fluid mixed with a disinfecting agent as identified above). Such sequential treatments with near/supercritical fluid mediums of differing compositions and function can be conducted sequentially in batch mode, or can be conducted in a continuous flow system in which a near/supercritical fluid is continuously passed through a chamber containing the ECM material and the composition of the near/supercritical fluid is changed over time by introducing the differing additives at different times in advance of the chamber. These and other variations in near/supercritical fluid treatments of ECM materials will be apparent to those of ordinary skill in the art from the descriptions herein.

In addition to or as an alternative to removal or inactivation of undesired materials in the ECM material, such as lipids and/or endotoxins, the near/supercritical fluid can include substances to be added to the ECM material. Such substances to be added can be soluble in, or dispersed or suspended in, the near/supercritical fluid. Upon contact of the near/supercritical fluid with the ECM material, the additive substances are incorporated in the ECM material. Upon separation of the near/supercritical fluid from the ECM material, amounts of the additive substances remain incorporated in the ECM material. In this regard, a variety of additive substances can be used, alone or in combination.

Illustrative additive substances include, for example, physiologically or pharmacologically active substances that act locally or systemically in a subject in which the ECM material is implanted. These substances may include antiviricides, antimicrobials and/or antibiotics such as erythromycin, bacitracin, neomycin, penicillin, polymycin B, tetracyclines, biomycin, Chloromycetin, and streptomycins, cefazolin, ampicillin, azactam, tobramycin, clindamycin, gentamycin, rifampin, minocycline, silver colloids or salts, etc.; biocidal/biostatic sugars such as dextran, glucose, etc.; amino acids; peptides; vitamins; inorganic elements; co-factors for protein synthesis; hormones; enzymes such as alkaline phosphatase, collagenase, peptidases, oxidases, etc.; angiogenic agents and polymeric carriers containing such agents; antigenic agents; cytoskeletal agents; natural extracts; DNA delivered by plasmid, viral vectors or other means; bioadhesives; bone morphogenic proteins (BMPs); osteoinductive factor (IFO); fibronectin (FN); endothelial cell growth factor (ECGF); vascular endothelial growth factor (VEGF); cementum attachment extracts (CAE); ketanserin; human growth hormone (HGH); animal growth hormones; epidermal growth factor (EGF); interleukins, e.g., interleukin-1 (IL-1 ), interleukin-2 (IL-2); human alpha thrombin; transforming growth factor (e.g. TGF-beta); insulin-like growth factors (IGF-1 , IGF-2); platelet derived growth factors (PDGF); fibroblast growth factors (FGF, BFGF, etc.); periodontal ligament chemotactic factor (PDLGF); growth and differentiation factors (GDF); hedgehog family of proteins; protein receptor molecules; small peptides derived from growth factors above; bone promoters; cytokines; somatotropin; bone digesters; antitumor agents; cellular attractants and attachment agents; immunosuppressants; permeation enhancers, e.g., fatty acid esters such as laureate, myristate and stearate monoesters of polyethylene glycol, enamine derivatives, alpha-keto aldehydes, etc.; nucleic acids; or mixtures including two, three or more of these additives. Given the teachings herein and using routine experimentation, it will be within the purview of those of ordinary skill in the art to incorporate effective amounts of the additive(s) into the ECM material using near/supercritical fluid processing. In certain embodiments, a drug, growth factor, or other additive substance to be incorporated into the ECM material can be water-insoluble. "Water-insoluble" as used herein refers to substances that are either totally insoluble in water or are so poorly soluble in water that preparing an aqueous solution of the substance is practically impossible. In certain embodiments, the water-insoluble substance will have that have a solubility in water at 25 of less than 0.5% by weight. The water-insoluble additive can nonetheless be solvated in the near/supercritical fluid used to treat the ECM material. In preferred embodiments, the near/supercritical fluid is or comprises near/supercritical carbon dioxide, and the additive substance is soluble therein. By efficiently incorporating water-insoluble substances in the ECM material while retaining the desirable bioactive components and character of the ECM material as discussed herein, the near/supercritical fluid treatment increases the range of modified, bioactive ECM materials available for medical and other applications, while providing the potential for otherwise retaining native bioactivity and mechanical properties of the ECM materials.

In certain embodiments, in addition to the retention of native bioactive components from the source tissue for the ECM material, non-native bioactive components such as those synthetically produced by recombinant technology or other methods, may be incorporated into the ECM material using near/supercritical fluid processing. In some forms, the non-native bioactive components may be naturally-derived or recombinantly produced proteins that correspond to those natively occurring in the ECM tissue, but potentially of a different species (e.g. human proteins applied to collagenous ECMs from other animals, such as pigs). The non-native bioactive components may also be drug substances such as any of those pharmacologically active substances disclosed hereinabove.

When adding a substance to the treated ECM, a substance that is solvated in the near/supercritical fluid can be precipitated from the fluid so as to be entrained or incorporated into the ECM material. This can be accomplished by adjusting the conditions of the near/supercritical fluid to reduce its solvating power for the additive substance(s). Such reduction can be achieved by reducing the pressure and/or temperature of the fluid, for instance by adjusting pressure and/or temperature to convert a supercritical fluid to a non-supercritical fluid (e.g. liquid), and/or by adding other liquids or fluids to the treatment medium to decrease its overall solvating power for the additive substance(s) in use. These and other variations will be apparent to those skilled in the art from the descriptions herein.

In other embodiments in which additive substance(s) are being added to the ECM material, the solvating power of the near/supercritical fluid for the substance(s) can remain unchanged, and the fluid can simply be removed, such as by evaporation, to leave amounts of the additives (e.g. as a precipitate, gel or liquid) incorporated in the ECM material.

As to modes of incorporation of the additive substance in the ECM tissue material, the additive can for example be associated with the material through specific or non-specific interactions, or covalent or noncovalent interactions. Examples of specific interactions include those between a ligand and a receptor, an epitope and an antibody, etc. Examples of nonspecific interactions include hydrophobic interactions, electrostatic interactions, magnetic interactions, dipole interactions, van der Waals interactions, hydrogen bonding, etc. In certain embodiments, the additive is attached to the ECM material using a linker so that the additive is free to associate with its receptor or site of action in vivo. In other embodiments the additive is either covalently or non-covalently attached to the tissue. In certain embodiments, the additive may be attached to a chemical compound such as a peptide that is recognized by the tissue. In another embodiment, the additive is attached to an antibody, or fragment thereof, that recognizes an epitope found within the tissue. An additive may be provided within the tissue in a sustained release format. For example, the additive may be encapsulated or otherwise incorporated within particles such as biodegradable nanospheres, microspheres, or the like.

In addition to treatment by contact with the near/supercritical fluid, the ECM material can be processed in a number of additional ways before and/or after the near/supercritical fluid treatment. Illustratively, the ECM material can be treated with a detergent medium, DNA-solubilizing, lipid- solubilizing and/or disinfecting agent prior to or after treatment with the near/supercritical fluid.

In certain embodiments, the ECM material will be treated with a detergent solution, such as an ionic or nonionic detergent solution, and preferably a mild detergent solution. A low concentration of detergent enables retention of a substantial level of desired native components of the ECM material, such as those as noted above. In certain modes of operation, the ECM material will be treated with an aqueous solution of sodium dodecyl sulfate (SDS) or another ionic or nonionic detergent at a detergent concentration of about 0.05% to about 1 %, more preferably about 0.05% to about 0.3%. This treatment can be for a period of time effective to disrupt cell and nuclear membranes in the ECM material and to reduce the immunoglobulin (e.g. IgA) content of the ECM material, typically in the range of about 0.1 hour to about 1 0 hours, more typically in the range of about 0.5 hours to about 2 hours. Processing the isolated ECM material in this manner preferably disrupts cell and nuclear membranes and results in a material with a substantially reduced its IgA content, thus reducing the immunogenicity of the material. For example, a final ECM material product of the invention can have a native IgA content of no greater than about 20 ug/g. In preferred embodiments, a final ECM material of the invention can have a native IgA content of no greater than 15 μ9/9, no greater than 10 μ9Λ3, or even no greater than 5 μg/g. In certain embodiments, the processed ECM material includes essentially no native IgA. By "essentially no IgA" is meant that the isolated ECM material includes IgA below detectable levels. Means for detecting IgA are well known in the art and include, for example, enzyme- linked immunosorbent assay (ELISA). It will be understood in this regard that ECM materials obtained from different sources may have differing immunoglobulins that predominate in the tissue. It is expected that the processing techniques disclosed herein will be effective to reduce the content of ECM materials in other immunoglobulins, including those that predominate in the source tissue. Accordingly, other aspects of the invention relate to final ECM materials that have substantially reduced levels (e.g. less than about 20 μg/g) of (i) the predominant immunoglobulin in the source tissue, or (ii) the total immunoglobulin content (the sum of all immunoglobulins in the tissue).

In addition or as an alternative to treating an ECM material with a detergent medium, the ECM material can be contacted with other agents that participate in achieving a desired ECM component profile, either before or after the near/supercritical fluid processing. For example, the ECM material can be treated with an aqueous medium, preferably basic, in which DNA is soluble. Such a medium can in certain forms have a pH in the range of above 7 to about 9, with pH's in the range of about 8 to about 8.5 proving particularly beneficial in some embodiments. The basic aqueous medium can include a buffer, desirably a biocompatible buffer such as tris(hydroxymethyl)aminomethane (TRIS), and/or a chelating agent such as ethylene diamine tetraacetic acid (EDTA). In one preferred form, the nucleic acid solubilizing medium is a TRIS-borate-EDTA (TBE) buffer solution. In another preferred form, the nucleic acid solubilizing medium is a solution of ammonium hydroxide. This treatment with a DNA solubilizing medium can be for a period of time effective to reduce the DNA content of the ECM material, typically in the range of about 0.1 hour to about 10 hours, more typically in the range of about 0.5 hours to about 2 hours.

In addition or as an alternative to treatment with detergent and/or DNA-solubilization media, methods of preparing ECM materials can involve treatment with a liquid medium that results in a substantial reduction of the level of lipid components of the ECM material, either before or after the near/supercritical fluid processing. For example, the native lipid content of a final ECM material product can be reduced to no greater than about 4% in certain embodiments. This can be accomplished, for example, by a preparative process that involves a step of treating the ECM material with a liquid organic solvent in which the lipids are soluble. Suitable such organic solvents include for example water-miscible solvents, including polar organic solvents. These include low molecular weight (e.g. C1 to C4) alcohols, e.g. methanol, ethanol, isopropanol, and butanols, acetone, chloroform, and others. Additional organic solvents include nonpolar solvents such as hexane, benzene, toluene and the like. In more preferred embodiments, the processed ECM material will be processed to have a native lipid content no greater than about 3%, or no greater than about 2.5%. This treatment with a lipid-removing medium can be for a period of time effective to reduce the lipid content of the ECM material, typically in the range of about 0.1 hour to about 1 0 hours, more typically in the range of about 0.1 hours to about 1 hours. In certain embodiments, multiple (two or more) such treatments will be conducted. Additionally, treatment with the lipid-reducing medium as discussed above can be carried out before or after treatment with a detergent medium and/or aqueous (preferably basic) DNA-reducing medium as discussed above. In certain preferred embodiments, treatment with the lipid-reducing medium will occur before treatment with the detergent medium and/or the aqueous (preferably basic) medium. In regard to lipid reduction, it will be understood that the near/supercritical fluid processing can also serve to reduce lipid content in the ECM material, alone or combined with a separate, non-near/supercritical fluid treatment regimen. To this end, lipid- reducing organic solvents such as those mentioned above, e.g. methanol, ethanol, isopropanol, butanol, acetone, chloroform, and the like, can optionally be used as co-solvents in a near/supercritical fluid to aid in lipid extraction and/or for other purposes.

The ECM material can also optionally be treated with a disinfecting solution, before or after the near/supercritical fluid processing. Preferred disinfecting agents are oxidizing agents such as peroxy compounds, preferably organic peroxy compounds, and more preferably peracids. As to peracid compounds that can be used, these include peracetic acid, perpropioic acid, or perbenzoic acid. Peracetic acid is the most preferred disinfecting agent for purposes of the present invention. Such disinfecting agents are desirably used in a liquid medium, preferably a solution, having a pH of about 1 .5 to about 10, more preferably a pH of about 2 to about 6, and most preferably a pH of about 2 to about 4. In methods of the present invention, the disinfecting agent will generally be used under conditions and for a period of time which provide the recovery of purified ECM materials, preferably exhibiting a bioburden of essentially zero and/or essential freedom from pyrogens. Certain desirable disinfecting treatments involve immersing the ECM material (e.g. by submersing or showering) in a liquid medium containing the disinfecting agent for a period of at least about 5 minutes, typically in the range of about 5 minutes to about 40 hours, and more typically in the range of about 0.5 hours to about 5 hours. When used as a disinfecting agent, peracetic acid is desirably diluted into about a 2% to about 50% by volume of alcohol solution, preferably ethanol. The concentration of the peracetic acid may range, for instance, from about 0.05% by volume to about 1 .0% by volume. Most preferably, the concentration of the peracetic acid is from about 0.1 % to about 0.3% by volume. When hydrogen peroxide is used, the concentration can range from about 0.05% to about 30% by volume. More desirably the hydrogen peroxide concentration is from about 1 % to about 10% by volume, and most preferably from about 2% to about 5% by volume. The solution may or may not be buffered to a pH from about 5 to about 9, with more preferred pH's being from about 6 to about 7.5. These concentrations of hydrogen peroxide can be diluted in water or in an aqueous solution of about 2% to about 50% by volume of alcohol, most preferably ethanol.

It will be understood that the ECM material also can be rinsed at various stages throughout its preparation (e.g., with tap water, high purity water or buffer) so as to remove introduced chemical residues that remain in or on the material.

Treatments such as those described herein and/or other chemical and/or mechanical treatments, will decellularize the ECM tissue, desirably resulting in a processed ECM tissue material that is decellularized, and more preferably that is substantially devoid of cells and cell components. Such a processed ECM material can be used in an acellular form, or can be subsequently used to culture cells for studies conducted entirely in vitro, or be cultured with or otherwise seeded with cells, including for example stem cells, prior to implantation in a patient.

Individual ECM sheet layers isolated and processed to biocompatibility as described herein can optionally be used to form multilaminate constructs, e.g. to prepare an overall mult-layer device having increased thickness and strength. Multi-laminate constructs having from 1 to about 8 layers of the isolated ECM material will be typical. In such constructs, the layers can be fused or bonded to one another using any suitable technique, including for example dehydrating the layers in contact with one another (e.g. by lyophilization or vacuum pressing) and/or with the use of chemical crosslinking agents to aid in bonding the layers to one another through covalent linkages. Such constructs can be prepared before after near/supercritical fluid processing as described herein. ECM materials of the invention can be used in a wide variety of medical indications, including for example in wound care, soft tissue support (e.g. hernia repair), soft tissue defect, and bone repair applications. In these or other applications, the ECM material can be used as a solid device (e.g. a sheet-form device or plug) or as a fluidized material such as a suspension of ECM material particles and/or a gel formed from a digest of the ECM material.

The final product ECM material of the invention can be packaged or otherwise stored in a dehydrated or hydrated state. Dehydration of the ECM material can be achieved by any means known in the art. In certain embodiments, dehydration is accomplished by either lyophilization or vacuum pressing, although other techniques, for example air drying, can also be used. In certain embodiments, the near/supercritical fluid processing will result in a dried product which can then be packaged in such state. When stored in a dry state, it will often be desirable to rehydrate the processed ECM material prior to use. In this regard, any suitable wetting medium can be used to rehydrate the medical material, including as examples water or buffered saline solutions.

In order to promote a further understanding of certain aspects of the invention, the following specific Example is provided. It will be understood that this Example is illustrative, and not limiting, of the invention. EXAMPLE

Experimental Methods

SIS was prepared from a segment of whole small intestine from a mature adult hog. The intestine was rinsed with water, and then treated in a 0.2 percent by volume peracetic acid in a 5 percent by volume aqueous ethanol solution (pH approximately 2.6) for a period of two hours with agitation. The high-strength ECM sheet including the submucosa layer (SIS) was then delaminated from the remainder of the intestinal tissue in a disinfected casing machine. The SIS was then rinsed with sterile water. The wet SIS was arranged into 4-SIS-layer stacks, all air bubbles smoothed from the stacks, and lyophilized. The lyophilization of the stacked SIS resulted in 4-ply lyophilized laminate sheets.

Six 4-ply SIS sheets were initially provided from the above SIS lot, sized 7 cm X 20 cm. Each 4-ply sheet was cut in half to provide two 7 cm x 10 cm sheets and labeled as Test samples or sheet matched Controls. Pretreatment weights for the Test samples were recorded after stabilization for greater than 18 hours in a desiccator. The Test samples were exposed, with agitation, to supercritical C0₂ at 4000 psi and 40⁵ C for 2 hours followed by gradual dissipation of pressure and then stabilization of the samples for greater than 18 hours in a desiccator. Final weight of the Test samples was recorded and samples were then subjected to mechanical and biochemical component testing.

For mechanical testing, five dog-bone shaped samples with a test area width of 5 mm were cut in the transverse direction (to the longitudinal axis of the intestine from which the sheets were obtained) from C0₂ treated Test sheets and Control sheets. Samples were rehydrated for at least 3 minutes prior to testing to failure on an Instron® tensile strength tester.

For biochemical component testing, samples of Test and Control sheet were taken and assessed for the presence of FGF-2, sGAG, IgA and lipid. For content values, weights were recorded and all calculations were based on content divided by the initial weight of the sample.

Results

The supercritical C0₂ treatment of the SIS Test samples significantly reduced the weight of the samples, reflecting an effective extraction of material from the samples. The average weight loss for the six Test samples assessed was 10.3%, with a standard deviation of 2.7%.

The supercritical C0₂ treatment did not significantly alter the tensile strength of the SIS Test samples as compared to the Controls. The ultimate tensile force at failure for the Test samples was 12.8 ± 2.5 Newtons, whereas that for the Control samples was 12.3 ± 2.3 Newtons.

In the biochemical component testing, neither the FGF-2 content nor the sGAG content of the SIS Test samples was reduced by the supercritical C0₂ treatment. The FGF-2 content for the Control samples was 1 1 1 ,000 ± 1 1 ,000 picogram/gram and for the treated Test samples was 123,000 ± 14,000 picogram/gram. The sGAG content for the Control samples was 3.6 ± 0.72 μg/g and for the treated Test samples was 4.1 1 ± 0.08 μ9/9. The IgA content of the SIS Test samples was not reduced by the treatment. The IgA content for the Control samples was 21 .6 ± 3.0 ng/mg and for the treated Test samples was 34.4 ± 5.5 ng/mg. The slight increase in the observed FGF-2, sGAG and IgA levels for the treated Test samples is likely due at least in part to the decrease in overall weight of the samples caused by the treatment. Cell nucleus remnants, as assessed using fluorescent staining and microscopy, were not significantly altered in the treated Test samples versus Controls.

The lipid content of the treated Test samples was significantly reduced as compared to the Controls. The lipid level for the Control samples was 9.3 ± 1 .9% by weight and for the treated Test samples was 1 .6 ± 0.4% by weight.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non- claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein. Of course, variations of those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. In addition, all publications cited herein are indicative of the abilities of those of ordinary skill in the art and are hereby incorporated by reference in their entirety as if individually incorporated by reference and fully set forth.

Claims

1 . A method for preparing a purified, bioactive extracellular matrix material, the method comprising:

(a) obtaining an extracellular matrix starting material from a mammalian tissue source, the extracellular matrix starting material including (i) endogenous cells from the mammalian tissue source; (ii) endogenous bioactive components from the mammalian tissue source including one or more of an endogenous glycosaminoglycan, an endogenous glycoprotein, and an endogenous growth factor; and (iii) endogenous lipids from the mammalian tissue source;

(b) treating the extracellular matrix starting material with a decellularizing agent so as to remove the endogenous cells and form a decellularized extracellular matrix material retaining at least some of the endogenous bioactive components;

(c) contacting the decellularized extracellular matrix material with an least substantially supercritical fluid under conditions effective to produce a purified extracellular matrix material having a reduced endogenous lipid content while retaining at least some of the endogenous bioactive components in the extracellular matrix material;

(d) evaporating the fluid to recover the purified extracellular matrix material retaining at least some of the endogenous bioactive components; and

(e) after the evaporating, sterilizing the purified extracellular matrix material retaining at least some of the endogenous bioactive components.

2. The method of claim 1 , wherein said contacting step comprises contacting the decellularized extracellular matrix material with at least substantially supercritical carbon dioxide.

3. The method of claim 1 , also comprising agitating said fluid during said contacting step.

4. The method of any of claims 1 -3, wherein the extracellular matrix starting material, the decellularized extracellular matrix material, and purified extracellular matrix material all comprise submucosal tissue.

5. The method of claim 4, wherein the submucosal tissue comprises small intestinal submucosal tissue.

6. The method of any of claims 1 -5, wherein the purified extracellular matrix material retains at least some of the endogenous glycosaminoglycan, endogenous glycoprotein, and endogenous growth factor.

7. The method of any of claims 1 -6, wherein said sterilizing comprises treating the purified extracellular matrix material with ethylene oxide.

8. A method for preparing a modified, bioactive extracellular matrix material, the method comprising:

(a) providing a decellularized extracellular matrix material including endogenous bioactive components from a mammalian tissue source for the material, the bioactive components including one or more of an endogenous glycosaminoglycan, an endogenous glycoprotein, and an endogenous growth factor;

(b) contacting the decellularized extracellular matrix material with an at least substantially supercritical fluid containing an additive under conditions effective to incorporate the additive into the extracellular matrix material while retaining at least some of the endogenous bioactive components in the extracellular matrix material;

(c) evaporating the fluid to recover the extracellular matrix material retaining at least some of the endogenous bioactive components and an amount of the additive; and

(d) after the evaporating, sterilizing the extracellular matrix material retaining at least some of the endogenous bioactive components and an amount of the additive.

9. The method of claim 8, wherein said contacting step comprises contacting the decellularized extracellular matrix material with at least substantially supercritical carbon dioxide.

10. The method of claim 8 or 9, wherein the additive is a pharmacologically active agent.

1 1 . The method of any of claims 8-10, wherein the additive is a water- insoluble substance.

12. The method of any of claims 8-1 1 , wherein the decellularized extracellular matrix material comprises submucosal tissue.

13. The method of claim 12, wherein the submucosal tissue comprises small intestinal submucosal tissue.

14. The method of any of claims 8-13, wherein the extracellular matrix material is a membranous sheet having a thickness of less than 1000 μιη.

15. The method of any of claims 8-14, wherein after said sterilizing, the extracellular matrix material retains at least some of the endogenous glycosaminoglycan, endogenous glycoprotein, and endogenous growth factor.

16. A material obtained or obtainable by a process according to any of claims 1 -15.

17. A method for treating a patient, comprising grafting the patient with a material according to claim 16.

18. A method of claim 17, wherein the material is in sheet form.

19. A method of claim 1 7 or 18, wherein the material is effective to induce angiogenesis in the patient.

20. A method of claim 19, wherein the material comprises endogenous fibroblast growth factor-2.