WO2007011644A2 - Compositions for regenerating defective or absent tissue - Google Patents

Abstract

The invention is to a composition for reconstruction, replacement or repair of a defect or damage in organ tissue comprising an emulsion, injectable solution, patch or other compositions or material forms comprising extracellular matrix. The organ can be any organ in the human body, for example, heart, brain, liver, kidney, pancreas or spleen. The ability to address organ damage or defects using extracellular matrix materials, including using minimally invasive surgery to contact the extracellular matrix with the damaged or defective organ tissue provide the opportunity to regenerate, replace or repair damaged or defective organ tissue that would otherwise remain defective or damaged or which would function suboptimally compared to healthy organ tissue.

Description

COMPOSITIONS FOR REGENERATING DEFECTIVE OR ABSENT TISSUE

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The present application claims priority from US application 11/182,551 filed July 15, 2005, and from US applications 11/334,319 and 11/334,631 both filed

January 18, 2006.

[0002] FIELD OF THE INVENTION

[0003] The invention relates to tissue engineering.

[0004] BACKGROUND OF THE INVENTION

[0005] Addressing defects or damage in organ tissue has presented a challenge to the medical community. Typically organ tissue can not regenerate so that defective or damaged organs can not return to full function, even after standard treatment which may include surgical repair. A notable example is brain tissue. Ischemic events in the brain can result in irreparable and permanent brain damage. Presently there is no treatment remedy to regenerate the ischemic tissue and so to restore the coordinate loss of function controlled by the region of the brain damaged during an ischemic attack. Tissue grafts from foreign hosts are rife with problems of viral and other host antigen contamination. Recipient immune rejection is also a significant problem following tissue grafting. Where the defect or damage to the organ is not capable of restoration, some patients can resort to transplantation of a donor organ in order to restore function to the body system. Organ transplantation has the primary drawback of the new foreign organ causing an immune response once it is placed in the recipient's body.

[0006] Congenital defects to kidney, liver, spleen and pancreas are presently treated with therapeutic drugs or surgery. Aging, trauma and chemical abuse can also be responsible for the need to medically address a failing or compromised organ. Trauma resulting in damage to an organ in the body is usually addressed with surgery to repair the physical damage. Diabetes remains a devasting condition, with tremendous cost in terms of human suffering and expense. A bioartificial pancreas has been created having encapsulated islet cells with a semipermeable membrane so that cells are protected from the host's immune system. Spinal cord injury is a medical event that changes the activity and health of the patient for life, as it is known that the spinal cord tissue does not regenerate.

[0007] Heart failure occurs in nearly 5 million people a year in the U.S. alone at a combined cost of about $40 billion annually for hospitalization and treatment of these patients. The results of all the effort and cost are disappointing with a 75% five year mortality rate for the heart failure victims. Treatments for chronic heart failure include medical management with pharmaceutical drugs, diet and exercise, transplantation for a few lucky recipients, and mechanical assist devices, which are costly and risk failure and infection. Thus the landscape for cardiac treatment is turning in recent years to transplantation of tissue or cells.

[0008] Medical researchers have transplanted human hematopoetic stem cells, mesenchymal stem cells, endothelial precursor cells, cardiac stem cells, and skeletal myoblasts or bone marrow cells to the myocardium, with however little or mixed success in satisfactory regeneration of the myocardium. Another protocol involved injecting transforming growth factor beta preprogrammed bone marrow stem cells to the myocardium, with greater success than transplantation of bone marrow stem cells alone, but without generation of contractile myocardium.

[0009] After myocardial infarction, injured cardiomyocytes are replaced by fibrotic tissue promoting the development of heart failure. On the basis that embryonic stem cells may be directed to differentiate into true cardiomyocytes, transplantation of embryonic stem cells to a site of myocardial infarction may yield success in myocardial tissue regeneration, though the experiments have not yet so proven. For a related challenge, to induce angiogenesis in ischemic myocardial tissue, transplanting endothelial progenitor cells, with or without angiogenic protein factors has been proposed to generate capillary blood vessels at the site of ischemia in the myocardium. As yet, the experiments to prove these theories have not worked sufficiently to be attempted in humans.

[00010] Meanwhile, typical structural abnormalities or damage to the heart that would lend itself to tissue regenerative therapies, were they available, include atrial septal defects, ventricular septal defects, right ventricular out flow stenosis, ventricular aneurysms, ventricular infarcts, ischemia in the myocardium, infarcted myocardium, conduction defects, conditions of aneurysmic myocardium, ruptured myocardium, and congenitally defective myocardium, and these defective conditions remain untreated in humans by any current tissue regenerative techniques.

[00011] Although tissue regeneration has been accomplished by transplantation in mammalian tissues such as the endocranium, the esophagus, blood vessels, lower urinary tract structures, and musculotendinous tissues, heart tissue regeneration by foreign tissue explant has remained a challenge. Recently, myocardium has been regenerated using xenogenic extracellular matrix patches in pigs and dogs, and the contractility achieved was at 90% of normal.

[00012] No experimentation has been conducted to date on regenerating mammalian myocardium using an emulsified or injectable extracellular matrix formulation. The only known experimental use of extracellular emulsions for tissue regeneration have been with gastroesophageal repair to prevent reflux and urinary bladder sphincter repair. Both of these experiments were conducted in non-human animals. Some veterinary use of extracellular matrix emulsions have been reported, but none of those uses were for the repair of myocardium. The disadvantage of using intact, non-emulsified extracellular matrix compositions such as patches or strips is that placement of the material requires open surgery, with its coordinate risk of infection, challenge of access to the site, and longer recovery for the patient postprocedure.

[00013] With regard to repair of intracardiac tissue, each year in the United

States alone over 600,000 open heart surgeries are conducted, and about 20% of these are on children and adults to address one or more defects in intracardiac tissue. Where a prosthetic device or material is used in these surgeries to correct the defect, many times the surgery will need to be repeated as the child grows, replacing the prosthetic item with a larger version to accommodate the growing organ in the growing child. Likewise, intracardiac defects in adults presently require open heart surgery, at a coordinate mortality risk due to the nature of the surgery alone. Repeat operations are common with heart valve replacements when the prosthetic valve needs to be replaced.

[00014] A function of the intracardiac tissue is electrical conduction of the heart. When the sino atrial or atrio ventricular nodes are not functioning properly, the electrical connections required for proper heart function are not being generated. The sino atrial node (often called the SA node or sinus node) serves as the natural pacemaker for the heart. Nestled in the upper area of the right atrium, it sends electrical impulse that triggers each heart beat. The impulse spreads through the atria, prompting the cardiac muscle tissue to contract in a coordinated wave-like manner. The impulse that originates from the sino atrial node strikes the atrio ventricular node (or AV node) which is situated in the lower portion of the right atrium. The atrio ventricular node in turn sends an impulse through the nerve network to the ventricles, initiated the same wave-like contraction of the ventricles. The electrical network serving the ventricles leaves the atrio ventricular node through the right and left bundle branches. These nerve fibers send impulses that cause the cardiac muscle tissue to contract. When the conduction pathway between the sino atrial and atrio ventricular nodes fails, or when either the sino atrial or atrio ventricular node repair is necessary, patients are implanted with pacemaker leads to re-establish the proper conduction impulses that cause the intracardiac muscle tissue to contract, and which may has been partially lost due to damage or defect to the intracardiac tissue in these regions. The damage can be due to aging. The patient with pace maker leads controlled by an external pacemaker is forever dependent on an external mechanical device (the pacemaker) to generate proper electrical conduction in the heart.

[00015] With regard to pericardial tissue repair, although the pericardium is usually described as a single sac, an examination of its structure shows that it consists essentially of two sacs intimately connected with one another, but totally different in structure. The outer sac, known as the fibrous pericardium, consists of fibrous tissue. The inner sac, or serous pericardium, is a delicate membrane which lies within the fibrous sac and lines its walls; it is composed of a single layer of flattened cells resting on loose connective tissue. The heart invaginates the wall of the serous sac from above and behind and practically obliterates its cavity, the space being merely a potential one.

[00016] Each year in the United States alone over 600,000 open heart surgeries are conducted, each involving opening the fibrous pericardial sac that surrounds the heart. Typically, the heart is accessed through the anterior portion of the pericardial sac. Current standard practice includes leaving the sac open after surgery. After coronary artery bypass or other open-heart procedures, the pericardium is usually not closed due to tension of the retracted edges and the compression the closure would cause on the underlying heart structures or bypass grafts. Adhesions form from the epicardium to the pericardium and from the heart to other structures such as the retro- sternum. These adhesions and the loss of the natural covering around the heart with scar formation can cause some loss of function and lead to increased mortality for future operations. Without the intact pericardium, opening the chest in a re-operation may likely cause damage to the heart or bypass grafts. It is estimated that some 10-

20% of all surgical procedures on the heart may require a second entry later, particularly in the case of operations on children having congenital heart defects where a prosthetic needs to be replaced with a larger version as the child grows. Many valve replacements also require second entries years later to replace the first valve.

[00017] A number of synthetic as well as animal based materials are currently being used as pericardial patches. These materials include expanded polytetrafluoroethylene (ePTFE), gluteraldehyde treated bovine pericardium, and polyglycolic acid (PGA). However, these materials have been associated with some tissue reaction and scar formation, limiting their application. Also Dacron or gortex patches have been used, with the disadvantage that they are foreign synthetic tissue that never assimilates into the live tissue that surround them. None of these materials work to achieve the goals of pericardial sac closure to the maximum benefit of the healing heart.

[00018] Many tissue types including myocardium, intracardiac tissue, the pericardial sac, and tissue of other organs such as kidney, pancreas, brain and spine would benefit from successful application of tissue regeneration to the site of damage or defect.

[00019] SUMMARY OF THE INVENTION

[00020] The invention is a composition for reconstruction, replacement or repair of a defect or damage in organ tissue, the composition comprising extracellular matrix.

[00021] The composition comprises a form selected from the group consisting of an emulsion, an injectable solution, a gel, a foam, a liquid, a paste, a powder, a spray, a vapor, a cream, a coating, a nanoparticle, a patch, a sheet, a laminate, a weave, a matrix, a fabric, a strand, a plurality of strands, a strip, a plurality of strips, a plug, a piece, and a plurality of pieces.

[00022] The extracellular matrix can comprise mammalian extracellular matrix.

The mammalian extracellular matrix can be selected from the group consisting of small intestine submucosa (SIS)₅ urinary bladder submucosa (UBS), urinary basement membrane (UBM), liver basement membrane (LBM), stomach submucosa (SS), collagen from animal sources, collagen from plant sources, synthesized extracellular matrix in cultures from cells (Matrigel™), dermal extracellular matrix, subcutaneous extracellular matrix, large intestine extracellular matrix, placental extracellular matrix, ornamentum extracellular matrix, heart extracellular matrix, and lung extracellular matrix.

[00023] The composition can further comprising a cell. The cell can be selected from the group consisting of a human embryonic stem cell, a mesenchymal stem cell, an autotransplanted expanded myocyte, an adipocyte, a totipotent cell, a pluripotent cell, a blood stem cell, a myoblast, an adult stem cell, a bone marrow cell, a mesenchymal cell, an embryonic stem cell, a parenchymal cell, an epithelial cell, an endothelial cell, a mesothelial cell, a fibroblast, a myofibroblast, an osteoblast, a chondrocyte, a cell derived from the organ being reconstructed, replaced or repaired, a precursor cell of an organ, an exogenous cell, an endogenous cell, a stem cell, a hematopoetic stem cell, a pluripotent stem cell, a bone marrow-derived progenitor cell, a progenitor cell, a myocardial cell, a skeletal cell, a fetal cell, an embryonic cell, an undifferentiated cell, a multi-potent progenitor cell, a unipotent progenitor cell, a monocyte, a skeletal myoblast, a macrophage, a capillary endothelial cell, a xenogenic cell, an allogenic cell, an adult stem cell, and a post-natal stem cell.

[00024] The composition can comprise a peptide, polypeptide or protein. The peptide, polypeptide or protein can be conjugated or cross-linked to the extracellular matrix.

[00025] The invention is also a composition for reconstruction, replacement or repair of a defect, or damage in organ tissue comprising extracellular matrix, wherein said composition comprises a form selected from the group consisting of an emulsion, an injectable solution, a gel, a foam, a liquid, a paste, a powder, a spray, a vapor, a cream, a coating, a nanoparticle, a patch, a sheet, a laminate, a weave, a matrix, a fabric, a strand, a plurality of strands, a strip, a plurality of strips, a plug, a piece, and a plurality of pieces, and further comprises an additional component selected from the group consisting of: a) a cell, b) a peptide, polypeptide, or protein, c) a vector having a DNA capable of targeted expression of a selected gene, and d) a nutrient, a sugar, a fat, a lipid, an amino acid, a nucleic acid, a ribo-nucleic acid, an organic molecule, an inorganic molecule, a small molecule, a drug, or a bioactive molecule, organ tissue is selected from the group consisting of myocardium, intracardiac tissue, pericardium, pancreas, kidney, liver, thyroid gland, adrenal gland, brain, spinal cord, ovary, prostate, testes, vocal cords, intestine, spleen, and stomach. [00026] The damaged or defective tissue can comprise intracardiac tissue having damage to or a defect in a conduction pathway in the intracardiac tissue.

[00027] The damaged or defective tissue can comprise intracardiac tissue having damage to or a defect in tissue of a sino atrial node, an atrio ventricular node, or a conduction pathway in between the two. [00028] The invention is also a patch for partial closure of an opening in a pericardial sac comprising mammalian extracellular matrix, the patch attachable to the opening at two or more points.

[00029] The invention is also a composition for regenerating defective or absent myocardium and restoring cardiac function comprising an emulsified or injectable extracellular matrix composition from a mammalian or synthetic source. [00030] The invention is also a composition for regenerating defective or absent myocardium and restoring cardiac function comprising an extracellular matrix derived from a mammalian or synthetic source, said composition further comprising an additional component selected from the group of: a) a cell, b) a peptide, polypeptide, or protein, c) a vector having a DNA capable of targeted expression of a selected gene, and d) a nutrient, a sugar, a fat, a lipid, an amino acid, a nucleic acid, a ribo-nucleic acid, an organic molecule, an inorganic molecule, a small molecule, a drug, or a bioactive molecule. [00031] The extracellular matrix can be formulated in a material form selected from the group consisting of a solid sheet, multilaminate sheets, a gel, an emulsion, an injectable solution, a fluid, a paste, a powder, a plug, a strand, a suture, a coil, a cylinder, a weave, a strip, a spray, a vapor, a patch, a sponge, a cream, a coating, a lyophilized material, and a vacuum-pressed material. [00032] The additional component can be formulated in a material form selected from the group consisting of a gel, an emulsion, an injectable solution, a fluid, a paste, a powder, a plug, a strand, a spray, a vapor, a cream, a coating, a lyophilized material and a vacuum-pressed material. [00033] The extracellular matrix can comprise an amount of the composition by weight selected from the group consisting of greater than 80%, greater than 82%, greater than 84%, greater than 86%, greater than 88%, greater than 90%, greater than 92%, greater than 94%, greater than 96%, greater than 98%, greater than 98.5%, greater than 99%, greater than 99.5%, and greater than 99.9% of the total composition by dry weight. [00034] The additional component comprises an amount of the composition by weight selected from the group consisting of greater than 0.1%, greater than 0.5%, greater than 1%, greater than 1.5%, greater than 2%, greater than 4%, greater than 5%, greater than 10%, greater than 12%, greater than 15%, and greater than 20%. [00035] Further embodiments of the invention are described herein.

[00036] BRIEF DESCRIPTION OF THE DRAWINGS

[00037] FIG.1 depicts cell-ECM interaction through the matrix proteoglycans, glycoaminoglycans and growth factors.

[00038] FIG.2 depicts cell-cell adhesions, and cell-matrix adhesions through specific structural and functional molecules of the ECM.

[00039] FIG.3 depicts a model of matrix scaffold structure including common collagen, proteoglycans, and glycoproteins.

[00040] FIG. 4 depicts a pericardial patch tacked in 4 places to cover a hole in the pericardial sac.

[00041] DETAILED DESCRIPTION OF THE INVENTION

[00042] The invention is a composition that regenerates defective or absent tissue arid restores the tissue function. For this purpose, an emulsified or injectable extracellular matrix composition can be derived from a mammalian or synthetic source. The composition can further include added cells or protein or both. An extracellular matrix composition of any formulation can include also an additional component such as: a) a cell, b) a peptide, polypeptide, or protein, or c) a vector expressing a DNA of a bioactive molecule, and d) other additives like nutrients or drug molecules. One additional component can be used in the composition or several. The composition is placed in contact with the defective or absent tissue, resulting in tissue regeneration and restoration of function to the tissue. The invention appreciates the importance of the presence of some amount and form of an extracellular matrix, or extracellular matrix-like scaffold, as a framework for the essential activities of cell-cell, matrix-cell, protein-cell, and protein-protein interactions that form the dynamic tissue regenerative process in vivo, potentially optimized by the presence of added cells, proteins, or other bioactive components. [00043] The invention provides compositions for addressing defects or damage in organ tissue of all types in the human body. Organs of the body can present with congenital defects. During the lifetime of a human, organs can receive damage requiring medical attention. Reconstruction, replacement or repair of these defects or damage to the organs can involve surgery, or administration of therapeutic drugs, or a combination of those two and also possibly including other alternate or combination therapies. The present invention provides compositions and methods for reconstruction, replacement or repair of a defect or damage in organ tissue. The composition comprises extracellular matrix. Placement of the extracellular matrix material at the site of defect or damage in the organ, preferably in contact with the defective or damaged organ tissue, provides an environment by which the organ can regenerate healthy organ tissue to effect a partial or full recovery from the defect or damage.

[00044] Organs for which tissue regeneration can be a significant benefit include any organ or tissue or tissue type in a human or mammalian body that it would be useful medically to ameliorate, correct, repair or heal the defect or damage in the organ. The term organ can be as minimally defined as a group of aggregate cells that perform a particular and specific function, such as islet cells in the pancreas. Organ can also mean an entire organ, perhaps including a combination of different tissues and tissue-types that make up the entire organ with a defined, or with many defined functions. Accordingly, the defect or damage can be located in a particular tissue within the organ, or several tissue-types within the organ. For example, the defect or damage can be in the cells or the outer surface of the organ, or can be within the organ in the cells of the inner regions of the organ. The defect or damage can also be in a part of the organ, for example, a lumen that runs through or connects with the organ and facilitates some organ function. Placement of a composition comprising extracellular matrix in contact with the region, site, tissue, tissue-type, cells or whole or part of the organ can facilitate a healing, reconstruction, replacement or repair of the region, site, tissue, tissue-type, cells or whole or part of the organ that is defective or damaged. [00045] Some organs that are commonly known are listed here as exemplary of the types of organs or tissue that can be treated with the compositions and methods of the invention. For example, heart (including myocardium, intracardiac tissue, and pericardial sac), pancreas, kidney, liver, thyroid gland, adrenal gland, brain, spinal cord, ovary, prostate, testes, vocal cords, intestine, spleen, and stomach organs can be treated with the compositions and methods of the invention. While there is no attempt to be exhaustive in a list of organs that can be reconstructed, replaced or repaired by the compositions and methods of the invention, some commonly known and studied organs can be listed here as exemplary of organs and tissue that can be treated by the invention. For example, regions, tissue, body parts or organs contemplated by the invention can include the pancreas, the thyroid, adrenals, pituitary, endocrine glands generally, liver, kidney, spinal cord, brain, other neurological tissues, reproductive organs including the prostate, ovaries, fallopian tubes, testes, digestive organs including intestines and stomach, and trachea, and other organs including lungs, heart (including the myocardium, the intracardiac tissue or the pericardial sac), sensory organs such as eyes and ears and tongue, membranes and linings such as mucous membranes and epithelial or endothelial linings of vessels, and vessels, connecting tubes, small subregions of organs, other glandular tissue such as lymph glands, connective tissues and covering or skin tissue such as dermis, epidermis, and parts or subparts of an organ such as a particular tissue within a specific organ.

[00046] Reconstruction, replacement or repair means reconstructing, replacing or repairing at least a part of the organ or at least a part of the tissue that is identified as defective or damaged in the organ. The terms reconstruction, replacement or repair can be used essentially synonymously here, indicating with the three different terms that the process by which healing occurs may be slightly different in different tissue, but that the broad result of partial or full healing or improvement in the quality of the tissue as a result of the composition or method occurs in the tissue regardless of which term is used. For example, reconstruction may be a more appropriate term to describe the process by which new tissue is rebuilt in the organ. Replacement may be more appropriate where it appears that new tissue replaces older damaged tissue.

Repair may be appropriate where as a result of the new tissue regenerated at a site, restored function results, as in a repair of the function of the organ. The use of the terms in various contexts is somewhat subjective, and providing multiple terms is meant to facilitate description as to the process of healing, without being limiting to exactly how that process occurs in the multitude of different contexts contemplated by the invention. In general, the terms reconstruction, replacement or repair are intended to encompass the process of generating new healthy functioning organ tissue at a site in the organ that was previously functioning suboptimally or not at all.

[00047] Likewise, the terms defective or damaged to describe the tissue or organ before treatment is an attempt to provide maximum flexibility and breadth to the nature of the compromised tissue being treated by the method, or contacted with the composition. For example, defect may be a birth defect, or an acquired defect through use. For example, damage may occur as with physical damage, for example, the patient experiences an ischemic attack such as a stroke or heart attack that damages the tissue. Damage can include any damage to the organs, for example, damage resulting from aging, trauma, or chemical abuse. There can be any number of debilitating biochemical or physiological conditions in the tissue or organ. Aging or drug abuse may cause damage in some tissues. Viral, bacterial or fungal infection are conditions from which treatment of the invention may be sought. Serosis of the liver may result from alcoholism, and may require liver repair. Diabetes may compromise kidney function, or by definition present with dysfunctional pancreas. Neurological conditions such as Parkinson's, Alzheimer's, or cerebral palsy are examples of conditions that leave organs or tissue compromised in function and quality. Accordingly, the terms defective or damaged are intended to provide description for the condition of the tissue or organs to be treated with the composition or methods of the invention with the broadest possible concept of a condition it would be a benefit to treat with the methods and compositions described here.

[00048] The composition can comprise mammalian extracellular matrix, or synthetic extracellular matrix, or a combination of both mammalian and synthetic extracellular matrix. The extracellular matrix composition can also further comprise an additional component. Additional components can include, e.g. cells, peptides, polypeptide, proteins, nucleic acids, or other additive moieties or molecules to enhance the healing function directed by placement of the extracellular matrix at the site of defect or damage. It is acknowledged that healing is a complicated biological process for which many biological actors may be needed or recruited in order for the healing process to be successful.

[00049] The additional components can be as few as a single molecule or a single cell with the entire extracellular matrix material, or may be many molecules or many cells. The molecule or molecules selected can be simply added to the extracellular matrix, or they can be more securely connected to the extracellular matrix by conjugation of the molecule to a molecule on the matrix, or by binding of a cell surface protein to a ligand that is part of the extracellular matrix. The extracellular matrix may be added to the site of damage first, and then a cell, or cells, or a protein or proteins can be added to that site, contacting the extracellular matrix in the process. Also, the cells or proteins can be added first at the site, followed by the extracellular matrix. Or, as just described the additional materials or components may be added to some form of the extracellular matrix (malleable or solid) first and then the entire composition placed at the site of damage or defect.

[00050] Specific applications of the invention include repair of myocardial tissue. After an ischemic attack or damage to the heart, an emulsion of extracellular matrix can be administered to the site. The extracellular matrix can be bare, or naked, without any additional components, or it may have additional components added into it within the emulsion. Administration can be subcutaneous (e.g. using a percutaneous delivery catheter) or as with other forms of the extracellular matrix the composition, open surgery may be required to deliver the extracellular matrix to the site.

[00051] Specific applications of the invention also include repair of intracardiac tissue. Reconstruction, replacement or repair of damaged or defective intracardiac tissue typically requires open heart surgery, and where a prosthetic device or material is used, often (especially in the case of children and valve replacements for adults) second or subsequent open heart surgical procedures are necessary to continually revise upon and update the prosthetic device or material. The present invention provides compositions and methods that may provide the option for a single open heart surgical procedure without the need for second or subsequent procedures, or in some cases, depending on the composition used, may provide the opportunity to address the defect with a single minimally invasive surgical procedure alone. Intracardiac defects include valve defects, congential defects in the intracardium, and any other defect of the intracardium known in the art and identified as a defect in intracardial tissue and presently treated or treatable using prosthetic material or devices. Specific abnormalities of intracardiac tissue include, for example damage to the intracardiac tissue due to trauma or aging, and defects in the intracardiac tissue, for example congential defects, or developmental defects. Some specific abnormalities in intracardiac tissue include, but are not limited to, atrial septal defects, ventricular septal defects, right ventricular outflow, ventricular aneurism repair, patent foramen ovale, and other defects that occur either congentially, or with aging or damage to the tissue.

[00052] One such category of damage or defect to intracardiac tissue involves damage or defects of tissue in the conduction pathway of the heart. The conduction pathway runs from the sino atrial node to the atrio ventricular node. Damage or defect can occur at either node or in the pathway that runs between them. The result of damage or defect in the SA or AV nodes of the heart or in the conduction pathway of the heart is inadequate, inefficient, or insufficient electrical impulses. The compositions of the invention can be applied to (e.g. placed at or put in contact with or affixed to) either the SA or AV nodes or to tissue in the conduction pathway in order to restore the electrical conduction to the damaged or defective intracardiac tissue that is causing the failure of the electrical conduction of the heart.

[00053] The invention is a composition comprising extracellular matrix. The composition can be, for example, a patch, an emulsion, an injectable solution, a gel, a fluid, a paste, a powder, a strand, a strip, a spray, a vapor, a cream, or a coating. The composition can further comprise one or more additional components, including, for example, a cell, peptide, polypeptide, protein or other biological moieties. The additional moieties can be in a form such as, for example an emulsion, an injectable solution, a gel, a fluid, a paste, a powder, a strand, a strip, a spray, a vapor, a cream, or a coating. Where the composition is a patch, and the patch can be in a form selected from the group consisting of a sheet, a laminate, a weave, a polymer matrix, a plurality of strands, or one or more strips.

[00054] Another specific application of the invention is to repair of the pericardial sac. Partial closure of the pericardial sac falling short of complete closure can provide the optimal environment for the heart to heal after open heart surgery. Selection of the material to accomplish this goal is critical. The invention herein dictates use of extracellular matrix material in the form of a patch to provide a loose closure of the pericardial sac. The extracellular matrix yields itself to a healthy assimilation with the tissues that connect or surround it, and a patch of extracellular matrix tacked to the pericardial sac opening will model itself over time and upon recruitment of cells to the patch to form with the pericardial sac a loose closure around the heart. Without such a patch to partially close the pericardial sac, the fibrous tissue of the sac tends to retract and put pressure on the heart, which is particularly serious when pressure is placed on the grafts or other work that was the object of the surgery. The use of the patch is primarily in the context of cardiothoracic surgical procedures requiring reconstruction, replacement or repair of the pericardial sac after the procedure.

[00055] The invention is to a pericardial patch comprising extracellular matrix material, native or synthetic, or a combination of the two (e.g. a weave that integrates both native and synthetic strands). The patch can be made by standard techniques for extracellular matrix preparation, known in the art. The patch is tacked to the opening in the pericardial sac after manipulations on the heart have been completed. Tacking comprises generally at least 2 tacks, optimally 4 to 6 tacks and more or less if needed to provide a loose closure of the opening. Depending on the size of the opening, and the size of the patch, it is not unreasonable to expect up to 10, perhaps 12 tacks in some cases, or any number in between about 4 to 6 and up to about 10 or 12. See Fig. 4. The body is then closed, and the heart is allowed to heal within the sac. The healing of the sac with the extracellular matrix patch prevents or limits adhesions that can be formed between the heart tissue and neighboring tissue and bone. The patch, because it is made of extracellular matrix, a material naturally yielding to adaptation in the native tissue environment in which it is placed, assimilates into the pericardial tissue and prevents the pericardial sac from retracting. Attachments between the patch and the pericardial sac form tissue connections that secure the pericardial sac around the heart and protect it from contact with tissue with which it can adhere. Such a closure of the pericardial sac in a first open heart surgical operation, provides the opportunity for second and subsequent entries to the heart with greater safety and less scarring of the heart tissue. These advantages are particularly critical for children having congenital heart defects, patients having valve replacements, and in general any patient under 65 years of age who may be subject to second or subsequent open heart surgical procedure.

[00056] Extracellular matrix materials act as a natural scaffold for repairing soft tissues in the body. Animal studies have shown that the original extracellular matrix material remodels and is replaced by host tissue. Extracellular matrix (for example small intestinal submucosa or SIS) is a resorbable biomaterial which has been used successfully as a xenogenic tissue graft that induces constructive remodeling of a variety of animal tissues including blood vessels, urinary bladder, dura, abdominal wall, tendons and ligaments. The remodeling process includes rapid neovascularization and abundant accumulation of mesenchymal and epithelial cells that support extensive deposition of a new extracellular matrix. Two studies have demonstrated that the noncollagenous portion of the SIS extracellular matrix is composed of various glycoproteins, such as hyluronic acid, heparin, dermatan and chondroitin sulfate A, as well as FGF-2 and TGF-β growth factors.

[00057] After processing, the extracellular matrix retains many of the endogenous proteins which act as growth and differentiation factors. These factors stimulate the local environment to populate the extracellular matrix with cells that are then able to differentiate into the original tissue that the extracellular matrix is replacing. Research in rodents has shown that these materials attract pluripotential, marrow derived cells from the animal to regenerate and replace the tissue in a given location.

An intracardiac patch of extracellular matrix will act as a mechanical scaffold while the body recruits the necessary cells to remodel and repair the intracardiac tissue. [00058] Scaffold Requirements

[00059] A composition to accomplish regeneration of tissue needs to induce complex dynamic interactions and activities at the site of defect. The present invention provides a composition that creates an environment in vivo to allow these processes to occur. The processes needed to regenerate tissue include specific phenotypic changes in stem cells that are recruited to the defective site, establishment of cell-cell connections, establishment of vascular supply at the site, beginning of normal tissue specific metabolism, limiting new growth once new tissue is made, coupling electric conduction from new cells to existing cells and pathways, and establishment of cell-extracellular matrix connections by way of cell adhesions to the matrix proteins.

[00060] The expectations for the extracellular matrix scaffold are that it will organize the cells into tissues, both by recruiting endogenous cells and using cells that have been provided as additional components in the composition. The extracellular matrix scaffold then coordinates the function of the newly recruited or added cells, allowing also for cell migration within the matrix. The matrix allows and provides for normal metabolism to the cells once the vascular supply delivering nutrients to the cells is established. Additionally, signal transduction pathways for growth, differentiation, proliferation and gene expression are established.

[00061] The extracellular matrix of tissue is complex. There is a three- dimensional architecture established with proteoglycan molecules, with available cytokines in the microenvironment. Cell movement occurs using focal adhesions, and eventually permanent cell adhesions occur called hemidesmosomes. Environmental signals are transmitted, including specific cell signals from growth factors on cell surfaces and disposed within the matrix framework as well. The matrix itself has structural components and functional components and the line between the two sometimes blurs because some of the moieties of structural components signal and trigger protein activation, and activation of nearby cells. See FIG. 1 for an illustration of signaling, FIG. 2 for depiction of cell-cell, protein-cell, and matrix-cell interactions, and FIG. 3 for a diagrammatic view of three-dimensional ECM scaffold.

[00062] There has been much research recently to elucidate the properties and function of the extracellular matrix: its protein make-up, and its role in the body.

The extracellular matrix (ECM) is a scaffold matrix of polymerized "structural" proteins that fit into three groups: collagens, glycoproteins, and proteoglycans (which have glycosaminoglycan repeats throughout). These molecules actually polymerize to form the scaffold or matrix of proteins that exists in dynamic interaction with cells, and closely placed functional proteins (either on the cells, or bound to a structural protein). Thus the extracellular matrix also includes within its matrix scaffold "functional" proteins that interact with the structural proteins and with migrating or recruited cells, particularly stem cells in tissue regeneration. The matrix functional proteins also interact with protein expressing cells during the life and maintenance of the matrix scaffold itself as it rebuilds and maintains its components. Note that some proteins fall into both a structural protein classification and a functional protein classification, depending on the protein's configuration and placement in the whole matrix.

[00063] The extracellular matrix of myocardium, for example, is made up of collagen types I (which is predominant), III, IV, V, and VI, combined which are 92% of the dry weight of the matrix. Other tissue types have somewhat differing amounts of the collagen types, in a different tissue-defining balance. However, the myocardium is discussed here in an exemplary fashion. Glycosaminoglycans (GAGs) include chondroitin sulfate A and B, heparan, heparin, and hyaluronic acid.

Glycoproteins such as fibronectin and entactin, proteoglycans such as decorin and perlecan, and growth factors such as transforming growth factor beta (TGF-beta), fibroblast growth factor -2 (FGF-2) and vascular endothelial growth factor (VEGF)₅ are key players in the activity of a myocardium regenerating matrix. Furthermore, the precise chemical constitution of the matrix appears to play a role in its function, including for example what collagen type is prevalent in the matrix, the pore size established by the matrix scaffold, the forces transmitted to adhesion molecules and mechanoreceptors in the cell membranes of cells at the matrix, and the forces directed from the three-dimensional environment (for example the gene expression in the three-dimensional matrix scaffold environment is very different than in a monolayer environment). Thus, the outcome of any tissue regenerative processes is determined by the structural and functional components of the matrix scaffold that form the basis of the regenerative process.

[00064] More specifically, when in early regenerative processes, circulating cells or added cells are directed, initial temporary cell adhesion processes occur that result in embryogenesis of the cells, morphogenesis of the cells, regeneration of cell form, eventual maintenance of the cell, possible motility to another site, and organogenesis that further differentiates the cell. Facilitating these early cell adhesion functions are cell adhesion molecules (CAMs). The CAMs are available either endogenously, or added as an additional component of the composition. CAMs are glycoproteins lodged in the surface of the cell membrane or transmembrane connected to cytoskeletal components of the cell. Specific CAMs include cadherins that are calcium dependent, and more than 30 types are known. Also working as CAMs are integrins which are proteins that link the cytoskeleton of the cell in which they are lodged to the extracellular matrix or to other cells through alpha and beta transmembrane subunits on the integrin protein. See FIG. 2 for an illustration of these interactions. Cell migration, embryogenesis, hemostatis, and wound healing are so facilitated by the integrins in the matrix. Syndecans are proteoglycans that combine with ligands for initiating cell motility and differentiation. Immunoglobins provide any necessary immune and inflammatory responses. Selectins promote cell- cell interactions.

[00065] Specific requirements for the scaffold component of the invention, whether a native scaffold prepared for introduction into a mammal, or a synthetic scaffold formed by synthetic polymerizing molecules, or a combination of the two, are that the scaffold must be resorbable over time as the tissue regeneration ensues, and this resorbtion is at an appropriate degradation rate for optimal tissue regeneration and absence of scar tissue formation. The extracellular matrix scaffold must also be non-toxic, provide a three-dimensional construction at the site of defect in the tissue (once delivered to the site). The matrix scaffold is required to have a high surface area so that there is plenty of room for the biological activities required of the tissue regeneration process. The scaffold must be able to provide cellular signals such as those mentioned herein that facilitate tissue regeneration. Finally the scaffold needs to be non-immunogenic so that it is not rejected by the host, and it needs to be non-thrombogenic.

[00066] Particular study of the components of the native scaffolds facilitates design of compositions well-suited for regeneration of any tissue.

[00067] The Structural Proteins of the Extracellular Matrix Scaffold

[00068] Collagens, the most abundant components of ECM, are homo- or heterotrimeric molecules whose subunits, the alpha chains, are distinct gene products. To date 34 different alpha chains have been identified. The sequence of the alpha chains contains a variable number of classical GIy-X-Y repetitive motifs which form the collagenous domains and noncollagenous domains. The collagenous portions of 3 homologous or heterologous alpha chains are folded together into a helix with a coiled coil conformation that constitutes the basic structure motif of collagens.

[00069] Characteristically, collagens form highly organized polymers. Two main classes of molecules are formed by collagen polymers: the fibril-forming collagens (collagens type I₅ II, III, V₅ and XI) and the non-fϊbrillar collagens that are a more heterogeneous class. Fibril collagen molecules usually have a single collagenous domain repeated the entire length of the molecule, and non-fibrillar collagen molecules have a mixture of collagenous and noncollagenous domains. On this basis several more subgroups of the collagen family are identified: e.g. the basement membrane collagens (IV, VIII, and X). In addition, most all the different types of collagen have a specific distribution. For example, fibril forming collagens are expressed in the interstitial connective tissue. The most abundant component of basement membranes is collagen IV. The multiplexins, collagens XV and XVIII are also localized to the basement membranes. [00070] In the extracellular matrix of the heart, collagen types I and III predominate, together forming fibrils and providing most of the connective material for typing together myocytes and other structures in the myocardium, and thus these molecule types are involved in the transmission of developed mechanical force in the heart. Only collagen types I, II, III, V, and XI self assemble into fibrils, characterized by a triple helix in the collagen molecules. Some collagens form networks, as with the basement membrane, formed by collagen IV. Type III collagen dominates in the wall of blood vessels and hollow intestinal organs and copolymerizes with type I collagen. The extracellular matrix of other tissues vary from the myocardium, but function similarly with similar actors and similar results: a functional extracellular matrix component to the tissue.

[00071] Proteoglycans are grouped into several families, and all have a protein core rich in glycosoaminoglycans. They control proliferation, differentiation, and motility. The lecticans interact with hyaluronan and include aggrecan, versican, neurocan, and brevican. Versican stimulates proliferation of fibroblasts and chondrocytes through the presence in the molecule of EGF-like motifs. The second type of proteoglycans have a protein core with leucine-rich repeats, which form a horse shaped protein good for protein-protein interactions. Their glycosoaminoglycan side chains are mostly chondroitin/dermatan sulphate or keratin sulphate. Decorin, biglycan, fibromodulin, and keratocan are members of this family. Decorin is involved in modulation and differentiation of epithelial and endothelial cells. In addition, transforming growth factor beta (TGF beta) interacts with members of this family. There are part-time proteoglycans, comprising CD44 (a receptor for hyaluronic acid), macrophage colony stimulating factor, amyloid precursor protein and several collagens (IX, XII, XIV, and XVIII). The last family of proteoglycans is the heparan sulfate proteoglycans, some of which are located in the matrix, and some of which are on cell membranes. Perlecan and agrin are matrix heparan sulfate proteoglycans found in basement membranes. The syndecans and glypicans are membrane-associated heparan sulfate proteoglycans. Syndecans have a heparan sulfate extracellular moiety that binds with high affinity cytokines and growth factors, including fibroblast growth factor (FGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), heparin-binding epidermal growth factor (HB-EGF), and vascular endothelial growth factor (VEGF). The heparan sulfate proteoglycans have been implicated in modulation of cell migration, proliferation and differentiation in wound healing.

[00072] Glycoproteins are also structural proteins of ECM scaffold. The glycoprotein fibronectin (Fn) is a large dimer that attracts stem cells, fibroblasts and endothelial cells to a site of newly forming matrix. Tenascin is a glycoprotein that has Fn repeats and appears during early embryogenesis then is switched off in mature tissue. Tenascin reappears during wound healing. Other glycoprotein components of ECM include elastin that forms the elastic fibers and is a major structural component along with collagen; fibrillins which are a family of proteins consisting almost entirely of endothelial growth factor (EGF)-like domains. Small glycoproteins present in ECM include nidogen/entactin and fibulins I and II.

[00073] The glycoprotein laminin is a large protein with three distinct polypeptide chains. Together with type IV collagen, nidogen, and perlecan, laminin is one of the main components of the basement membrane. Laminin isoforms are synthesized by a wide variety of cells in a tissue-specific manner. Laminin I contains multiple binding sites to cellular proteins. Virtually all epithelial cells synthesize laminin, as do small, skeletal, and cardiac muscle, nerves, endothelial cells, bone marrow cells, and neuroretina. Laminins affect nearby cells, by promoting adhesion, cell migration, and cell differentiation. They exert their effects mostly through binding to integrins on cell surfaces. Laminins 5 and 10 occur predominantly in the vascular basement membrane and mediate adhesion of platelets, leukocytes, and endothelial cells.

[00074] The Functional Proteins of the Extracellular Matrix Scaffold

[00075] In addition to the structural matrix proteins just discussed, specific interactions between cells and the ECM are mediated by functional proteins of the ECM, including transmembrane molecules, mainly integrins, some members of the collagen family, some proteoglycans, glycosaminoglycan chains, and some cell- surface associated proteins. These interactions lead to direct or indirect control of cellular activities within the extracellular matrix scaffold such as adhesion, migration, differentiation, proliferation, and apoptosis.

[00076] Glycosaminoglycans (GAGs) are glycosylated post-translational molecules derived from proteoglycans. Well known GAGs include heparin, hyaluronic acid, heparan sulfate, and chondroitin sulfate A, B, and C. Heparin chains stimulate angiogenesis, and act as subunits in a proteoglycan to stimulate the angiogenic effects of fibroblast growth factor- 2 (FGF-2) (also known as basic FGF or bFGF). Chondroitin sulfate B (dermatan sulfate) interacts with TGF-beta to control matrix formation and remodeling. The proteoglycan form of chondroitin sulfate B regulates the structure of ECM by controlling collagen fibril size, orientation and deposition. Hyaluronic acid is associated with rapid wound healing and organized deposit of collagen molecules in the matrix. It is believed that hyaluronic acid binds TGF-betal to inhibit scar formation.

[00077] The ECM is also being remodeled constantly in the live animal. The proteins of the ECM are broken down by matrix metalloproteases, and new protein is made and deposited as replacement protein. Collagens are mostly synthesized by the cells comprising the ECM: fibroblasts, myofibroblasts, osteoblasts, and chondrocytes. Some collagens are also synthesized by adjacent parenchymal cells or also covering cells such as epithelial, endothelial, or mesothelial cells.

[00078] The extracellular part of integrins bind fibronectin, collagen and laminin, and act primarily as adhesion molecules. Integrin-ligand binding also triggers cascades of activity for cell survival, cell proliferation, cell motility, and gene transcription.

[00079] Tenascins include cytotactin (TN-C). Cell surface receptors for tenascins include integrins, cell adhesion molecules of the Ig superfamily, a transmembrane chrondroitin sulfate proteoglycan (phosphacan) and annexin II. TN-C also interacts with extracellular proteins such as fibronectin and the lecticans (the class of extracellular chondroitin sulphate proteoglycans including aggrecan, versican, and brevican). [00080] In addition to direct knowledge of protein cell interaction many of the proteins associated with the ECM can initiate binding to proteins that then activate to bind other proteins or cells, e.g. decorin binds Fn or thrombospondin and causes their cell adhesion promoting activity. Other proteoglycans control the hydration of the

ECM and the spacing between the collagen fibrils and network, which is believed to facilitate cell migration. Proteoglycans regulate cell function by controlling growth factor activity, e.g. decorin, biglycan, and fibromodulin bind to isoforms of transforming growth factor beta (TGF beta) and heparin sulfate proteoglycans bind and store fibroblast growth factor.

[00081] The matrix metalloproteases (MMPs) break down the collagen molecules in the ECM so that new collagen can be used to remodel and renew the ECM scaffold. It is also believed that the proteolytic activity of MMPs augment the bioavailability of growth factors sequestered within the ECM, and can activate latent secreted growth factors like TGF-beta and IGF from IGFBPs and cell surface growth factor precursors. MMPs can proteolytically cleave cell surface growth factors, cytokines, chemokine receptors and adhesion receptors, and thus participate in controlling responses to growth factors, cytokines, chemokines, as well as cell-cell and cell-ECM interactions.

[00082] Structural or functional matrix proteins that can comprise the compositions herein disclosed to facilitate tissue regeneration include, minimally, collagen I and III, elastin, laminin, CD44, hyaluronan, syndecan, bFGF, HGF, PDGF, VEGF, Fn, tenascin, heparin, heparan sulfate, chondroitin sulfate B, integrins, decorin, and TGF-beta.

[00083] Native Sources and Preparations

[00084] Native extracellular matrix scaffolds, and the proteins that form them, are found in their natural environment, the extracellular matrices of mammals. These materials are prepared for use in mammals in tissue grafts procedures. Small intestine submucosa (SIS) is described in USPN 5,275,826, urinary bladder submucosa (UBS) is described in USPN 5,554,389, stomach submucosa (SS) is described in USPN 6,099,567, and liver submucosa (LS) or liver basement membrane (LBM) is described in USPN 6,379,710, to name some of the extracellular matrix scaffolds presently available for explanting procedures. In addition, collagen from mammalian sources can be retrieved from matrix containing tissues and used to form a matrix composition. Extracellular matrices can be synthesized from cell cultures as in the product manufactured by Matrigel™. In addition, dermal extracellular matrix material, subcutaneous extracellular matrix material, large intestine extracellular matrix material, placental extracellular matrix material, omamentum extracellular matrix material, heart extracellular matrix material, and lung extracellular matrix material, may be used, derived and preserved similarly as described herein for the SIS, SS, LBM, and UBM materials. Other organ tissue sources of basement membrane for use in accordance with this invention include spleen, lymph nodes, salivary glands, prostate, pancreas and other secreting glands. In general, any tissue of a mammal that has an extracellular matrix can be used for developing an extracellular matrix component of the invention.

[00085] When using collagen-based synthetic ECMs, the collagenous matrix can be selected from a variety of commercially available collagen matrices or can be prepared from a wide variety of natural sources of collagen. Collagenous matrix for use in accordance with the present invention comprises highly conserved collagens, glycoproteins, proteoglycans, and glycosaminoglycans in their natural configuration and natural concentration. Collagens can be from animal sources, from plant sources, or from synthetic sources, all of which are available and standard in the art.

[00086] The proportion of scaffold material in the composition when native scaffold used will be large, as the natural balance of extracellular matrix proteins in the native scaffolds usually represents greater than 90% of the extracellular matrix material by dry weight. Accordingly, for a functional tissue regenerative product, the scaffold component of the composition by weight will be generally greater than 50% of the total dry weight of the composition. Most typically, the scaffold will comprise an amount of the composition by weight greater than 60%, greater than 70%, greater than 80%, greater than 82%, greater than 84%, greater than 86%, greater than 88%, greater than 90%, greater than 92%, greater than 94%, greater than 96%, and greater than 98% of the total composition.

[00087] Native extracellular matrices are prepared with care that their bioactivity for tissue regeneration is preserved to the greatest extent possible. Key functions that may need to be preserved include control or initiation of cell adhesion, cell migration, cell differentiation, cell proliferation, cell death (apoptosis), stimulation of angiogenesis, proteolytic activity, enzymatic activity, cell motility, protein and cell modulation, activation of transcriptional events, provision for translation events, inhibition of some bioactivities, for example inhibition of coagulation, stem cell attraction, and chemotaxis. Assays for determining these activities are standard in the art. For example, material analysis can be used to identify the molecules present in the material composition. Also, in vitro cell adhesion tests can be conducted to make sure that the fabric or composition is capable of cell adhesion.

[00088] The matrices are generally decellularized in order to render them non- immunogehic. A critical aspect of the decellularization process is that the process be completed with some of the key protein function retained, either by replacement of proteins incidentally extracted with the cells, or by adding exogenous cells to the matrix composition after cell extraction, which cells produce or carry proteins needed for the function of tissue regeneration in vivo.

[00089] Exemplary to the invention, myocardial tissue has been regenerated in vivo in non-humans using native xenogenic extracellular matrix scaffolds in the form of intact patches derived and prepared from mammals, so it can be presumed that at least some of the components required for myocardial tissue regeneration are to be found in these xenogenic patch matrices. Prudent practice may dictate that the cell extract from the patches be tested for its protein make-up, so that if necessary proteins are removed they can be place back into the matrix composition, perhaps using exogenous proteins at approximately the same amount as those detected in the extraction solution. Replacing lost essential proteins may also be necessary with emulsions or injectable solutions of extracellular matrix, particularly those emulsified from mammalian sources. Another option would be that the proteins extracted during the cell extraction process can simply be added back after the cell extraction is complete, thus preserving the desired bioactivity in the material.

[00090] The bioactivity of extracellular matrix material can be mimicked in tissue regeneration experiments with combinations of native and synthetic extracellular matrices explanted together, also optionally with additional components such as proteins or cells, in order to provide an optimal myocardial tissue regenerative composition and environment in vivo. What works as the best composition for myocardial tissue regeneration in patients, particularly humans can be tested first in other mammals by standard explanting procedures to determine whether tissue regeneration is accomplished and optimized by a particular composition. See Badylak et al, The Heart Surgery Forum, Extracellular Matrix for Myocardial Repair 6(2) E20-E26 (2003). Other organs can be treated similarly.

[00091] When adding proteins to the extracellular matrix composition, be it an emulsified composition, or another formulation of matrix, the proteins may be simply added with the composition, or each protein may be covalently linked to a molecule in the matrix. Standard protein-molecule linking procedures may be used to accomplish the covalent attachment.

[00092] For decellularization when starting with a whole organ, whole organ perfusion process can be used. The organ is perfused with a decellularization agent, for example 0.1% peractic acid rendering the organ acellular. The organ can then be cut into portions and stored (e.g. in aqueous environment, liguid nitrogen, cold, freeze-dried, or vacuum-pressed) for later use. Any appropriate decellularizing agent may be used in whole organ perfusion process.

[00093] With regard to submucosal tissue, extractions may be carried out a near neutral pH (in a range from about pH 5.5 to about pH 7.5) in order to preserve the presence of growth factor in the matrices. Alternatively, acidic conditions (i.e. less than 5.5 pH) can be used to preserved the presence of glycosaminoglycan components, at a temperature in a range between 0 and 50 degrees centrigrade. In order to regulate the acidic or basic environment for these aqueous extractions, a buffer and chaotropic agent (generally at a concentration from about 2M to about 8M) are selected, such as urea (at a concentration from about 2M to 4M), guanidine (at a concentration from about 2M to about 6M₅ most typically about 4M), sodium chloride, magnesium chloride, and non-ionic or ionic surfactants. Urea at 2M in pH 7.4 provides extraction of basis FGF and the glycoprotein fibronectin. Using 4M guanidine with pH 7.4 buffer yields a fraction having transforming growth factor beta. (TGF-beta). Accordingly, it may behoove a practitioner to decellularize one portion of a matrix, and extract desired proteins to add back in from other different portions.

[00094] Because of the collagenous structure of basement membrane and the desire to minimize degradation of the membrane structure during cell dissociation, collagen specific enzyme activity should be minimized in the enzyme solutions used in the cell-dissociation step. For example, liver tissue is typically also treated with a calcium chelating agent or chaotropic agent such as a mild detergent such as Triton

100. The cell dissociation step can also be conducted using a calcium chelating agent or chaotropic agent in the absence of an enzymatic treatment of the tissue. The cell- dissociation step can be carried out by suspending liver tissue slices in an agitated solution containing about 0.05 to about 2%, more typically about 0.1 to about 1% by weight protease, optionally containing a chaotropic agent or a calcium chelating agent in an amount effective to optimize release and separation of cells from the basement membrane without substantial degradation of the membrane matrix.

[00095] After contacting the liver tissue with the cell-dissociation solution for a time sufficient to release all cells from the matrix, the resulting liver basement membrane is rinsed one or more times with saline and optionally stored in a frozen hydrated state or a partially dehydrated state until used as described below. The cell- dissociation step may require several treatments with the cell-dissociation solution to release substantially all cells from the basement membrane. The liver tissue can be treated with a protease solution to remove the component cells, and the resulting extracellular matrix material is further treated to remove or inhibit any residual enzyme activity. For example, the resulting basement membrane can be heated or treated with one or more protease inhibitors. [00096] Basement membrane or other native ECM scaffolds may be sterilized using conventional sterilization techniques including tanning with glutaraldehyde, formaldehyde tanning at acidic pH, ethylene oxide treatment, propylene oxide treatment, gas plasma sterilization, gamma radiation, and peracetic acid sterilization.

A sterilization technique which does not significantly weaken the mechanical strength and biotropic properties of the material is preferably used. For instance, it is believed that strong gamma radiation may cause loss of strength in the graft material. Preferred sterilization techniques include exposing the graft to peracetic acid, low dose gamma irradiation and gas plasma sterilization; peracetic acid sterilization being the most preferred method.

[00097] Synthetics

[00098] Synthetic extracellular matrices can be formed using synthetic molecules that polymerize much like native collagen and which form a scaffold environment that mimics the native environment of mammalian extracellular matrix scaffolds. According, such materials as polyethylene terephthalate fiber (Dacron), polytetrafluoroethylene (PTFE), glutaraldehyde-cross linked pericardium, polylactate (PLA), polyglycol (PGA), hyaluronic acid, polyethylene glycol (PEG), polyethelene, nitinol, and collagen from non-animal sources (such as plants or synthetic collagens), can be used as components of a synthetic extracellular matrix scaffold. The synthetic materials listed are standard in the art, and forming hydrogels and matrix-like materials with them is also standard. Their effectiveness can be tested in vivo as sited earlier, by testing in mammals, along with components that typically constitute native

ECMs, particularly the growth factors and cells responsive to them.

[00099] The ECM-like materials are described generally in the review article "From CeIl-ECM Interactions to Tissue Engineering" Rosso et al, Journal of Cellular Physiology 199: 174- 180 (2004). In addition, some ECM-like materials are listed here. Particularly useful biodegradable and/or bioabsorbable polymers include polylactides, poly-glycolides, polycarprolactone, polydioxane and their random and block copolymers. Examples of specific polymers include poly D,L-lactide, polylactide-co-glycolide (85:15) and polylactide-co-glycolide (75:25). Preferably, the biodegradable and/or bioabsorbable polymers used in the fibrous matrix of the present invention will have a molecular weight in the range of about 1,000 to about 8,000,000 g/mole, more preferably about 4,000 to about 250,000 g/mole. The biodegradable and/or bioabsorbable fiberizable material is preferably a biodegradable and bioabsorbable polymer. Examples of suitable polymers can be found in Bezwada, Rao S. et al. (1997) Poly(p-Dioxanone) and its copolymers, in Handbook of Biodegradable Polymers, A. J. Domb, J. Kost and D. M. Wiseman, editors, Hardwood Academic Publishers, The Netherlands, pp. 29-61. The biodegradable and/or bioabsorbable polymer can contain a monomer selected from the group consisting of a glycolide, lactide, dioxanone, caprolactone, trimethylene carbonate, ethylene glycol and lysine. The material can be a random copolymer, block copolymer or blend of monomers, homopolymers, copolymers, and/or heteropolymers that contain these monomers. The biodegradable and/or bioabsorbable polymers can contain bioabsorbable and biodegradable linear aliphatic polyesters such as polyglycolide (PGA) and its random copolymer poly(glycolide-co- lactide- ) (PGA-co-PLA). The FDA has approved these polymers for use in surgical applications, including medical sutures. An advantage of these synthetic absorbable materials is their degradability by simple hydrolysis of the ester backbone in aqueous environments, such as body fluids. The degradation products are ultimately metabolized to carbon dioxide and water or can be excreted via the kidney. These polymers are very different from cellulose based materials, which cannot be absorbed by the body.

[000100] Other examples of suitable biocompatible polymers are polyhydroxyalkyl methacrylates including ethylmethacrylate, and hydrogels such as polyvinylpyrrolidone, polyacrylamides, etc. Other suitable bioabsorbable materials are biopolymers which include collagen, gelatin, alginic acid, chitin, chitosan, fibrin, hyaluronic acid, dextran, poly amino acids, polylysine and copolymers of these materials. Any glycosaminoglycan (GAG) type polymer can be used. GAGs can include, e.g., heparin, chondroitin sulfate A or B, and hyaluronic acid, or their synthetic analogues. Any combination, copolymer, polymer or blend thereof of the above examples is contemplated for use according to the present invention. Such bioabsorbable materials may be prepared by known methods.

[000101] Nucleic acids from any source can be used as a polymeric biomaterial. Sources include naturally occurring nucleic acids as well as synthesized nucleic acids.

Nucleic acids suitable for use in the present invention include naturally occurring forms of nucleic acids, such as DNA (including the A, B and Z structures), RNA (including mRNA, tRNA, and rRNA together or separated) and cDNA, as well as any synthetic or artificial forms of polynucleotides. The nucleic acids used in the present invention may be modified in a variety of ways, including by cross linking, intra- chain modifications such as methylation and capping, and by copolymerization. Additionally, other beneficial molecules may be attached to the nucleic acid chains. The nucleic acids may have naturally occurring sequences or artificial sequences. The sequence of the nucleic acid may be irrelevant for many aspects of the present invention. However, special sequences may be used to prevent any significant effects due to the information coding properties of nucleic acids, to elicit particular cellular responses or to govern the physical structure of the molecule. Nucleic acids may be used in a variety of crystalline structures both in finished biomaterials and during their production processes. Nucleic acid crystalline structure may be influenced by salts used with the nucleic acid. For example, Na₅ K, Bi and Ca salts of DNA all have different precipitation rates and different crystalline structures. Additionally, pH influences crystalline structure of nucleic acids.

[000102] The physical properties of the nucleic acids may also be influenced by the presence of other physical characteristics. For instance, inclusion of hairpin loops may result in more elastic biomaterials or may provide specific cleavage sites. The nucleic acid polymers and copolymers produced may be used for a variety of tissue engineering applications including to increase tissue tensile strength, improve wound healing, speed up wound healing, as templates for tissue formation, to guide tissue formation, to stimulate nerve growth, to improve vascularization in tissues, as a biodegradable adhesive, as device or implant coating, or to improve the function of a tissue or body part. The polymers may also more specifically be used as sutures, scaffolds and wound dressings. The type of nucleic acid polymer or copolymer used may affect the resulting chemical and physical structure of the polymeric biomaterial.

[000103] Emulsions and Iniectables

[000104] The extracellular matrix can be emulsified for administration to the defective or absent myocardium. The matrix may also be otherwise liquefied or made into an injectable solution, such as an emulsion, or a liquid, or injectable gel, or semi-gel, other injectable formulation that can be administered with a percutaneous catheter, or other device capable of delivering an injectable formulation.

[000105] An emulsion of mammalian or synthetic extracellular matrix material can be accomplished as is standard for tissue or polymer emulsification in general. Generally, the emulsion will be maintained in an emulsified state by control of some component of the composition, for example the pH. Upon delivery of the emulsion the pH is altered to allow the molecules of the matrix to polymerize into a three- dimensional scaffold.

[000106] An emulsified extracellular matrix material comprising also cells can have the cultured cells simply added into the matrix emulsion, or the cells may be co- cultured with the matrix for a time before administration to the patient. Standard procedures for culturing or co-culturing cells can be used.

[000107] In addition, where proteins such as growth factors, or any other protein, including protein forms such as peptides or polypeptides, or protein fragments, are added into the extracellular matrix, the protein molecules may be added into the matrix composition, or the protein molecules may be covalently linked to a molecule in the matrix. The covalent linking of protein to matrix molecules can be accomplished by standard covalent protein linking procedures known in the art. The protein may be covalently linked to one or more matrix molecules. The covalent linking may result in an integration of the protein molecules in the matrix scaffold formation once the emulsion converts from the emulsified form to the scaffold form of the extracellular matrix. [000108] Additional Components: Cells

[000109] Unlike skeletal myocytes, cardiomyocytes withdraw from cell cycle shortly after birth, and adult mammalian cardiomyocytes lack the potential to proliferate. Therefore, in order to regenerate myocardium, the right cells may have to be added to the composition, or the site, or the right molecules to attract the right cells will have to be added to the composition or the site. Transplantation cell sources for the myocardium include allogenic, xenogenic, or autogenic sources. Accordingly, human embryonic stem cells, neonatal cardiomyocytes, myofibroblasts, mesenchymal cells, autotransplanted expanded cardiomyocytes, and adipocytes can be used as additive components to accompany the scaffold.

[000110] Embryonic stem cells begin as totipotent cells, differentiate to pluripotent cells, and then further specialization. They are cultured ex vivo and in the culture dish environment differentiate either directly to heart muscle cells, or to bone marrow cells that can become heart muscle cells. The cultured cells are then transplanted into the mammal, either with the composition or in contact with the scaffold and other components.

[000111] Myoblasts are another type of cell that lend themselves to transplantation into myocardium, however, they do not always develop into cardiomyocytes in vivo. Adult stem cells are yet another species of cell that work in the context of tissue regeneration. Adult stem cells are thought to work by generating other stem cells (for example those appropriate to myocardium) in a new site, or they differentiate directly to a cardiomyocyte in vivo. They may also differentiate into other lineages after introduction to organs, such as the heart. The adult mammal provides sources for adult stem cells in circulating endothelial precursor cells, bone marrow-derived cells, adipose tissue, or cells from a specific organ. It is known that mononuclear cells isolated from bone marrow aspirate differentiate into endothelial cells in vitro and are detected in newly formed blood vessels after intramuscular injection. Thus, use of cells from bone marrow aspirate may yield endothelial cells in vivo as a component of the composition. [000112] Yet another viable option for cells to use in the invention are the mesenchymal stem cells administered with activating cytokines. Subpopulations of mesenchymal cells have been shown to differentiate toward myogenic cell lines when exposed to cytokines in vitro.

[000113] Once a type of cell is chosen, the number of cells needed is determined. Their function and anticipated change upon implantation, as well as their viability during the process of transplantation need to be considered to determine the number of cells to transplant. Also the mode of transplantation is to be considered: several modes including intracoronary, retrograde venous, transvascular injection, direct placement at the site, thoracoscopic injection and intravenous injection can be used to put the cells at the site or to incorporate them with the composition either before delivery or after delivery to the defective myocardium. In all cases, the mode of delivery and whether the cells are first mixed with the other components of the composition is a decision made based on what will provide the best chance for viability of the cells, and the best opportunity for their continued development into cells that can function in the scaffold in vivo in order to signal and promote tissue regeneration.

[000114] The following list includes some of the cells that may be used as additional cellular components of the composition of the invention: a human embryonic stem cell, a fetal cardiomyocyte, a myofibroblast, a mesenchymal stem cell, an autotransplanted expanded cardiomyocyte, an adipocyte, a totipotent cell, a pluripotent cell, a blood stem cell, a myoblast, an adult stem cell, a bone marrow cell, a mesenchymal cell, an embryonic stem cell, a parenchymal cell, an epithelial cell, an endothelial cell, a mesothelial cell, a fibroblast, a myofibroblast, an osteoblast, a chondrocyte, an exogenous cell, an endogenous cell, a stem cell, a hematopoetic stem cell, a pluripotent stem cell, a bone marrow-derived progenitor cell, a progenitor cell, a myocardial cell, a skeletal cell, a fetal cell, an embryonic cell, an undifferentiated cell, a multi-potent progenitor cell, a unipotent progenitor cell, a monocyte, a cardiomyocyte, a cardiac myoblast, a skeletal myoblast, a macrophage, a capillary endothelial cell, a xenogenic cell, an allogenic cell, an adult stem cell, and a postnatal stem cell.

[000115] In particular, human embryonic stem cells, fetal cardiomyoctes, mesenchymal stem cells, adipocytes, bone marrow progenitor cells, embryonic stem cells, adult stem cells, or post-natal stem cells together with growth factors or alone with matrix scaffold optimize myocardium regeneration in vivo.

[000116] Cells can be seeded directly onto matrix scaffold sheets under conditions conducive to eukaryotic cell proliferation. The highly porous nature of extracellular matrices in particular will allow diffusion of cell nutrients throughout the membrane matrix. Thus, cells can be cultured on or within the matrix scaffold itself. With the emulsified extracellular matrix compositions, or with some of the other formulations, the cells can be co-cultured with the extracellular matrix material before administration of the complete composition to the patient.

[000117] Additional Components: Peptides. Polypeptides, or Proteins

[000118] In addition to a native ECM scaffold, or a synthetic scaffold, or a mixture of the two, peptides, polypeptides or proteins can be added. Such components include extracellular structural and functional proteins in admixture so as to mimic either heart ECM, or other native ECMs that are capable of regenerating at least some reasonable percentage of the defective myocardium, for example at least 30%, preferably more than 50%. Effective regeneration of the myocardium relies on the extracellular matrix scaffold by its structure and components. Mimicking the native explant material as closely as possible thus optimizes the opportunity for regeneration using a composition comprising some native ECM, albeit treated, but also with additional components.

[000119] The peptides, polypeptides or proteins that can be added to the scaffold are: a collagen, a proteoglycan, a glycosaminoglycan (GAG) chain, a glycoprotein, a growth factor, a cytokine, a cell-surface associated protein, a cell adhesion molecule (CAM), an angiogenic growth factor, an endothelial ligand, a matrikine, a matrix metalloprotease, a cadherin, an immunoglobin, a fibril collagen, a non-fibrillar collagen, a basement membrane collagen, a multiplexin, a small-leucine rich proteoglycan, decorin, biglycan, a fibromodulin, keratocan, lumican, epiphycan, a heparan sulfate proteoglycan, perlecan, agrin, testican, syndecan, glypican, serglycin, selectin, a lectican, aggrecan, versican, nuerocan, brevican, cytoplasmic domain-44

(CD-44), macrophage stimulating factor, amyloid precursor protein, heparin, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate A, heparan sulfate, hyaluronic acid, fibronectin (Fn), tenascin, elastin, fibrillin, laminin, nidogen/entactin, fibulin I, fibulin II, integrin, a transmembrane molecule, platelet derived growth factor (PDGF)₅ epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), transforming growth factor beta (TGF-beta), fibroblast growth factor-2 (FGF-2) (also called basic fibroblast growth factor (bFGF)), thrombospondin, osteopontin, angiotensin converting enzyme (ACE), and vascular epithelial growth factor (VEGF).

[000120] Typically, the additional peptide, polypeptide, or protein component will comprise an amount of the composition by weight selected from the group consisting of greater than 0.1%, greater than 0.5%, greater than 1%, greater than 1.5%, greater than 2%, greater than 4%, greater than 5%, greater than 10%, greater than 12%, greater than 15%, and greater than 20%.

[000121] Whether a particular protein component or combination of components is effective for tissue regeneration can be tested by contacting the composition with defective myocardium in a test animal, for example a dog, pig, or sheep, or other common test mammal. Tissue regeneration and function are both indicia to measure the success of the composition and procedure, by procedures standard in the art. In addition, a small sampling of the regenerated tissue can be made to determine that new extracellular matrix and new tissue has been made. As to what balance between structural extracellular matrix proteins and functional ones to use in a given composition, nature provides direction. Most ECMs are predominantly made up of structural proteins by dry weight. Thus only a small portion of functional proteins by weight are needed for effective tissue regeneration. [000122] Peptides, polypeptides or proteins for the composition may be formulated as is standard in the art for the particular class of protein, and that formulation may be added to the extracellular matrix material (of whatever formulation) for delivery into the patient.

[000123] Alternatively, the protein molecules may be covalently linked to an appropriate matrix molecule of any of the matrix formulations. Covalent linking of the protein molecules to molecules of the matrix may be accomplished by standard covalent linking methods known in the art.

[000124] Additional Components: Vector Expressing DNA, Nutrients, Drug Molecules

[000125] Some of the proteins required for the composition can be genetically synthesized in vivo with DNA and vector constituents. Thus a vector having a DNA capable of targeted expression of a selected gene can contribute a bioactive peptide, polypeptide, or protein to the composition. Standard in vivo vector gene expression can be employed.

[000126] In addition, other additives such as a nutrient, a sugar, a fat, a lipid, an amino acid, a nucleic acid, a ribo-nucleic acid, may provide support to the regenerative process in vivo in the composition. Finally, also a drug, such as a heart regenerating or angiogenesis promoting drug may be also added to the composition, in such a form as, for example, an organic molecule, an inorganic molecule, a small molecule, a drug, or any other drug-like bioactive molecule.

[000127] Formulations

[000128] A formulation of extracellular matrix material can be an emulsified or injectable material derived from mammalian or synthetic sources. The extracellular matrix material can be emulsified or made into an injectable formulation by standard procedures in the art, and maintained as an emulsion or injectable until delivered to the patient. Once delivered to the patient, an environment is established (by some change such as a change in pH) so that the extracellular matrix molecules (be they mammalian or synthetic) polymerize to form a matrix scaffold.

[000129] Depending on the nature of the scaffold selected, and depending on which additional components are used, the scaffold component and the additional component can be formulated together in the same way, or in different ways that are however but delivery-compatible with each other for delivery purposes. Options for formulation of the scaffold include a solid sheet, multilaminate sheets, a gel, an emulsion, an injectable solution, a fluid, a paste, a powder, a plug, a strand, a suture, a coil, a cylinder, a weave, a strip, a spray, a vapor, a patch, a sponge, a cream, a coating, a lyophilized material, or a vacuum pressed material, all of which are standard in the art.

[000130] Formulation of the additional components, when they are not scaffold-like is generally accomplished using some form of an injectable, semi-gel, or emulsified material, although powdered forms may also then be combined with a hydration- promoting solution at delivery. Thus, formulations for the additional components will generally comprise formulations of the nature of a gel, an emulsion, an injectable solution, a fluid, a paste, a spray, a vapor, a cream, and a coating. Dried materials that are hydrated either at delivery or just before delivery are powders, such as lyophilized materials.

[000131] Cells can be added in from a culture, or can be co-cultured with the matrix component of the composition. Proteins can be added into the composition, or covalently linked to matrix molecules. DNA can be added in with their vectors for expressing proteins in vivo. Other additives can be combined with the matrix component as is practical for the delivery of the composition (for example, as an injectable or a composition administered with a percutaneous catheter) and as is practical for maintaining bioactivity of the molecules or components in vivo.

[000132] Fluidized forms of native extracellular matrices are described, e.g. in USPN 5,275,826. The conminuted fluidized tissue can be solubilized by enzymatic digestion including the use of proteases, such as trypsin or pepsin, or other appropriate enzymes such as a collagenase or a glycosaminoglycanase, or the use of a mixture of enzymes, for a period of time sufficient to solubilize said tissue and form a substantially homogeneous solution.

[000133] The present invention also contemplates the use of powder forms of extracellular matrix scaffolds. In one embodiment a powder form is prepared by pulverizing basement membrane submucosa tissue under liquid nitrogen to produce particles ranging in size from 0.1 to 1 mm.sup.2. The particulate composition is then lyophilized overnight and sterilized to form a solid substantially anhydrous particulate composite. Alternatively, a powder form of basement membrane can be formed from fluidized basement membranes by drying the suspensions or solutions of comminuted basement membrane. The dehydrated forms have been rehydrated and used as cell culture substrates without any apparent loss of their ability to support cell growth.

[000134] Delivery Modes

[000135] The mode used for delivery of the compositions of the invention to the defective myocardium may be critical in establishing tissue regeneration in vivo. Standard delivery to myocardial sites can be used for injectable, fluidized, emulsified, gelled, or otherwise semi-fluid materials, such as direct injecting (e.g. with a needle and syringe), or injecting with a percutaneous catheter. For materials that have been rendered wholly or partially vaporized, force-driven delivery of the material can be used, for example, CO₂ powering emission of fine emulsion, micronizing an injectable solution, ink jet delivery, spray with a conventional atomizer or spray unit, or other type of vaporized delivery. Some of these vaporized formulations can be delivered using a percutaneous catheter adapted for delivery of a vaporized formulation.

[000136] For materials that are essentially solid, such as some of the native or synthetic scaffolds, physically depositing the material will be the most prudent mode of delivery. For example a patch, sponge, strip, weave, or other geometrically defined material form should be placed at the site of deposit either during surgery, or with a percutaneous minimally invasive catheter capable of depositing all or portions of solid material at the site. Preferred modes of delivery will be minimally invasive delivery procedures, which reduce the risk of infection and provide an easier recovery for the patient.

[000137] Where the scaffold component is in a different material form than the additional components, care must be taken to orchestrate an effective delivery of both components to the site. For example, where the scaffold is a solid sheet, and cells have been cultured and proteins hydrolyzed, the cells and proteins may be added to the scaffold prior to delivery and the composition is then delivered in surgery.

Alternatively, also in surgery, the solid sheet of scaffold may be delivered and the emulsified agents deposited on the sheet before closure. Where both the scaffold component and the additional components can be emulsified, with complete retention of functionality, the composition can be delivered together by direct injection or percutaneous catheter delivery.

[000138] In all cases, before a mode is used to treat a patient, the feasibility and effectiveness of any one delivery mode or combination of modes can be tested in a test mammal prior to actual use in humans.

[000139] Methods of Use

[000140] A site of defective myocardium is identified and the appropriate composition of a scaffold component and additional components is made and formulated. The formulated composition is delivered by an appropriate means to the site of defect. The site and mammal are observed and tested for regeneration of the defective myocardium to determine that an effective amount of the composition has been delivered, particularly to observe new tissue growth, and also to determine that the new tissue has the contractility necessary for it to function usefully as myocardium. Tissue growth and contractility can be tested and observed by standard means, for example as described in Badylak et al, The Heart Surgery Forum, Extracellular Matrix for Myocardial Repair 6(2) E20-E26 (2003). [000141] Goals for contractility in the defective myocardium include observed and measured contractility in an amount measured against contractility of a normal heart selected from the group consisting of greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, and greater than 95% of normal myocardial contractility in vivo.

[000142] The method step of contacting the defective myocardium or site of absent myocardium with a composition of the invention can be accomplished by means discussed in the delivery section, including, delivering the composition by injecting, suturing, stapling, injecting with a percutaneous catheter, CO₂ powering emission of fine emulsion, micronizing an injectable solution, inkjet delivery, physically depositing a sponge, physically depositing a patch, physical depositing a strip, or physically depositing a formed scaffold of any shape.

[000143] A complementary method of use of the compositions of the invention include a method of inducing angiogenesis in myocardium at a site of ischemia by similarly contacting said ischemic myocardium with a composition of the invention in an amount effective to induce angiogenesis in the myocardium at the site of ischemia. Effectiveness can be measured by measuring vascularization at the site, using standard biomedical procedures for such analysis.

[000144] Example

[000145] EXAMPLE l

[000146] A set of strips intended for placement in damaged kidney tissue is made of extracellular matrix scaffold derived from porcine small intestinal submucosa (SIS). SIS was developed from a select layer of tissue that is recovered from porcine small intestine. During processing, the inner and outer muscle layers of the material are removed, leaving an intact submucosa with a portion of the tunica propria layer attached to the outer surface. Following processing, the remaining acellular extracellular material is cut to specific shapes and sizes, lyophilized, and terminally sterilized using ethylene oxide gas. The kidney strips are supplied in four-ply sheets of various dimensions, which can be cut to size as the physician deems necessary for the procedure. The strips are provided to the customer in the lyophilized, sterile state. The strips product is sterilized by ethylene oxide (EtO). The strips can be packaged in a sterile, double, tyvek pouch and then placed inside a paperboard box for shipment to the customer.

[000147] EXAMPLE 2

[000148] A plug for liver tissue is tested for viral inactivation. Viral inactivation studies are performed to assess the safety and effectiveness of the device. Viral Inactivation Testing is performed in accordance with the Good Laboratory Practices regulations, 21 CFR Section 58, to validate the inactivation of viral contamination during disinfection processing of the SIS material comprising the plug. The methods used are based on the European Committee for Standardization, prEN 12442-3: 1996,

Animal tissues and their derivatives utilized in the manufacture of medical devices - Part 3: Validation of the elimination and/or inactivation of viruses and other transmissible agents. Results will demonstrate that the disinfection process reduces viral load to a SAL of at least 10^"6. Inactivation of Spiked Parvovirus and Reovirus During Reduced-Scale Processing/Disinfection of Porcine Small Intestine Sheets;

Inactivation of Spike Murine Leukemia Retrovirus and Porcine Pseudorabies (Herpes) Virus During Reduced-Scale Processing/Disinfection of Porcine Small Intestine Sheets; Probe Burst Strength Test of Four-layer, Lyophilized, SIS; Suture Retention Strength of Multilayer (4) Lyophilized SIS; Suture Retention Strength of Multilayer (4) Lyophilized SIS; Tensile Strength and Thickness of 4-layer, Freeze- dried, High Strength, SIS Sheet.

[000149] The plug is EtO sterilized to a sterility assurance level of 10^"6. EtO sterilization is considered a traditional method of sterilization for medical devices. The liver plug comprising lyophilized material can be labeled shelf-life of 18 -22 months.

[000150] EXAMPLE 3 [000151] A child presents with a damage to brain tissue due to ischemic events occurring at birth. A composition comprising an emulsion of native extracellular matrix is prepared and a percutanous catheter is loaded with the emulsion. A minimally invasive procedure is conducted to deliver the emulsion to the site of brain defect in the child, guided by a visualization technic such as radiography following a radio-opaque portion of the catheter to determine that the catheter is positioned to deliver the emulsion to the proper site in the brain. The emulsion is released at the site, and the catheter withdrawn. The catheter entry site is closed and the patient monitored for response to the procedure and return of brain function upon regeneration of the ischemic brain tissue.

[000152] EXAMPLE 4

[000153] A intracardiac patch is made of extracellular matrix scaffold derived from porcine small intestinal submucosa (SIS). SIS was developed from a select layer of tissue that is recovered from porcine small intestine. During processing, the inner and outer muscle layers of the material are removed, leaving an intact submucosa with a portion of the tunica propria layer attached to the outer surface. Following processing, the remaining acellular extracellular material is cut to specific shapes and sizes, lyophilized, and terminally sterilized using ethylene oxide gas. The intracardiac patch is supplied in four-ply sheets of various dimensions, which can be cut to size as the physician deems necessary for the procedure. The patch is provided to the customer in the lyophilized, sterile state. The patch product is sterilized by ethylene oxide (EtO). The patch can be packaged in a sterile, double, tyvek pouch and then placed inside a paperboard box for shipment to the customer.

[000154] EXAMPLE 5

[000155] The intracardiac patch is tested for viral inactivation. Viral inactivation studies are performed to assess the safety and effectiveness of the device. Viral Inactivation Testing is performed in accordance with the Good Laboratory Practices regulations, 21 CFR Section 58, to validate the inactivation of viral contamination during disinfection processing of the SIS material comprising the percardial patch. The methods used are based on the European Committee for Standardization, prEN12442-3: 1996, Animal tissues and their derivatives utilized in the manufacture of medical devices - Part 3: Validation of the elimination and/or inactivation of viruses and other transmissible agents. Results will demonstrate that the disinfection process reduces viral load to a SAL of at least 10^"6. Inactivation of Spiked Parvovirus and Reovirus During Reduced-Scale Processing/Disinfection of Porcine Small Intestine Sheets; Inactivation of Spike Murine Leukemia Retrovirus and Porcine Pseudorabies (Herpes) Virus During Reduced-Scale Processing/Disinfection of Porcine Small Intestine Sheets; Probe Burst Strength Test of Four-layer, Lyophilized,

SIS; Suture Retention Strength of Multilayer (4) Lyophilized SIS; Suture Retention Strength of Multilayer (4) Lyophilized SIS; Tensile Strength and Thickness of 4- layer, Freeze-dried, High Strength, SIS Sheet.

[000156] The patch is EtO sterilized to a sterility assurance level of 10^"6. EtO sterilization is considered a traditional method of sterilization for medical devices. The intracardial patch comprising lyophilized sheets can be labeled shelf-life of 18 - 22 months.

[000157] EXAMPLE 6

[000158] A child is presented with a congenital defect in intracardiac tissue. A composition comprising an emulsion of native extracellular matrix is prepared and a percutanous catheter is loaded with the emulsion. A minimally invasive procedure is conducted to deliver the emulsion to the site of defect in the child, guided by a visualization technic such as radiography following a radio-opaque portion of the catheter to determine that the catheter is positioned to deliver the emulsion to the proper site in the intracardiac tissue. The emulsion is released at the site, and the catheter withdrawn. The catheter entry site is closed and the patient monitored for response to the procedure.

[000159] EXAMPLE 7 [000160] A pericardial patch was made of extracellular matrix scaffold derived from porcine small intestinal submucosa (SIS). SIS was developed from a select layer of tissue that is recovered from porcine small intestine. During processing, the inner and outer muscle layers of the material were removed, leaving an intact submucosa with a portion of the tunica propria layer attached to the outer surface.

Following processing, the remaining acellular ECM material was cut to specific shapes and sizes, lyophilized, and terminally sterilized using ethylene oxide gas. The pericardial patch was supplied in four-ply sheets of various dimensions, which can be cut to size as the physician deems necessary for the procedure. The pericardial patch was provided to the customer in the lyophilized, sterile state. The available sizes include the following in 4-ρly thickness: 1. 7 x 20 ; 2. 7 x 10; 3. 5 x 10; 4. 5 x 7

[000161] The patch product was sterilized by ethylene oxide (EtO). The patch can be packaged in a sterile, double, tyvek pouch and is then placed inside a paperboard box for shipment to the customer.

[000162] EXAMPLE 8

[000163] The pericardial patch was tested for viral inactivation. Viral inactivation studies were performed to assess the safety and effectiveness of the device. Viral

Inactivation Testing was performed in accordance with the Good Laboratory Practices regulations, 21 CFR Section 58, to validate the inactivation of viral contamination during disinfection processing of the SIS material comprising the percardial patch. The methods used were based on the European Committee for Standardization, prEN12442-3: 1996, Animal tissues and their derivatives utilized in the manufacture of medical devices - Part 3: Validation of the elimination and/or inactivation of viruses and other transmissible agents. Results demonstrate that the disinfection process reduces viral load to a SAL of at least 10^"6. Inactivation of Spiked Parvovirus and Reovirus During Reduced-Scale Processing/Disinfection of Porcine Small Intestine Sheets; Inactivation of Spike Murine Leukemia Retrovirus and Porcine Pseudorabies (Herpes) Virus During Reduced-Scale Processing/Disinfection of Porcine Small Intestine Sheets; Probe Burst Strength Test of Four-layer, Lyophilized, SIS; Suture Retention Strength of Multilayer (4) Lyophilized SIS; Suture Retention Strength of Multilayer (4) Lyophilized SIS; Tensile Strength and Thickness of 4-layer, Freeze-dried, High Strength, SIS Sheet. [000164] The results were as follows: [000165] Burst Strength [N] 126.6 ± 30.2 [000166] Suture Retention Strength [gξ]

[000167] Longitudinal 774.9 ± 196.3

[000168] Transverse 1000.1 ± 203.8

[000169] The pericardial patch is EtO sterilized to a sterility assurance level of 10^"6. EtO sterilization is considered a traditional method of sterilization for medical devices. The pericardial patch comprising lyophilized sheets has a labeled shelf-life of 18 -22 months.

[000170] An emulsion of urinary bladder submucosa (UBS) is prepared using standard emulsifying techniques. The emulsion is free of endogenous cells. This preparation is maintained as an emulsion by controlling the pH during storage of the emulsion before it is admininstered to the patient. In a minimally invasive procedure, a percutaneous catheter device is loaded with sufficient quantity of the emulsified UBS to address a defect in a human heart, the defect having been identified previously by imaging. The catheter is directed to the site of the myocardium in need of tissue regeneration using sonographic or radiographic imaging. Upon contact with the site, the emulsion is released and the catheter is withdrawn. The tissue regeneration process is monitored by sonography for several weeks or months post- delivery of the emulsion.

[000171] EXAMPLE lO

[000172] An emulsion of decellularized immunogenic liver basement membrane (LBM) is prepared using standard known techniques. While maintaining the emulsion state of the LBM, adult stem cells are co-cultured with the emulsion using standard stem cell culturing techniques. When the cells are ready, the entire composition is loaded into a catheter for percutaneous delivery to a human patient in need of tissue regeneration at a site of defective or absent myocardium. The emulsion with the co-cultured cells is delivered to the patient: a percutaneous catheter is loaded with the emulsion and directed to the site of the myocardium in need of tissue regeneration using sonographic or radiographic imaging. Upon contact with the site, the emulsion is released and the catheter is withdrawn. The tissue regeneration process is monitored by sonography for several weeks or months post- delivery of the emulsion.

[000173] EXAMPLE I l

[000174] An injectable emulsion of decellularized immunogenic stomach submucosa (SS) is prepared using standard known techniques. An aliquot of glycoaminoglycan (GAG) protein is covalently linked to some of the molecules of the matrix emulsion using standard covalent linking procedures for proteins. While maintaining the emulsive state of the SS, bone marrow progenitor cells are co- cultured with the emulsion using standard progenitor cell culturing techniques. An aliquot of transforming growth factor protein is added to the co-culturing composition before delivery to the human in need of tissue regeneration. The emulsion complete with cells and proteins is loaded into a percutaneous catheter which is directed to the site of the myocardium in need of tissue regeneration using sonographic or radiographic imaging. Upon contact with the site, the emulsion is released and the catheter is withdrawn. The tissue regeneration process is monitored by sonography for several weeks or months post-delivery of the emulsion.

[000175] All references cited are incorporated in their entirety. Although the foregoing invention has been described in detail for purposes of clarity of understanding, it will be obvious that certain modifications may be practiced within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A composition for reconstruction, replacement or repair of a defect or damage in organ tissue, the composition comprising extracellular matrix.

2. The composition of claim 1, wherein the composition comprises a form selected from the group consisting of an emulsion, an injectable solution, a gel, a foam, a liquid, a paste, a powder, a spray, a vapor, a cream, a coating, a nanoparticle, a patch, a sheet, a laminate, a weave, a matrix, a fabric, a strand, a plurality of strands, a strip, a plurality of strips, a plug, a piece, and a plurality of pieces.

3. The composition of claim 2, wherein in the extracellular matrix comprises mammalian extracellular matrix.

4. The composition of claim 3, wherein the mammalian extracellular matrix is selected from the group consisting of small intestine submucosa (SIS), urinary bladder submucosa (UBS), urinary basement membrane (UBM), liver basement membrane (LBM), stomach submucosa (SS), collagen from animal sources, collagen from plant sources, synthesized extracellular matrix in cultures from cells (Matrigel™), dermal extracellular matrix, subcutaneous extracellular matrix, large intestine extracellular matrix, placental extracellular matrix, ornamentum extracellular matrix, heart extracellular matrix, and lung extracellular matrix.

5. The composition of claim 1, further comprising a cell.

6. The composition of claim 1, wherein the cell is a stem cell.

7. The composition of claim 5, wherein the cell is selected from the group consisting of a human embryonic stem cell, a mesenchymal stem cell, an autotransplanted expanded myocyte, an adipocyte, a totipotent cell, a pluripotent cell, a blood stem cell, a myoblast, an adult stem cell, a bone marrow cell, a mesenchymal cell, an embryonic stem cell, a parenchymal cell, an epithelial cell, an endothelial cell, a mesothelial cell, a fibroblast, a myofibroblast, an osteoblast, a chondrocyte, a cell derived from the organ being reconstructed, replaced or repaired, a precursor cell of an organ, an exogenous cell, an endogenous cell, a stem cell, a hematopoetic stem cell, a pluripotent stem cell, a bone marrow-derived progenitor cell, a progenitor cell, a myocardial cell, a skeletal cell, a fetal cell, an embryonic cell, an undifferentiated cell, a multi-potent progenitor cell, a unipotent progenitor cell, a monocyte, a skeletal myoblast, a macrophage, a capillary endothelial cell, a xenogenic cell, an allogenic cell, an adult stem cell, and a post-natal stem cell.

8. The composition of claim 1, further comprising a peptide, polypeptide or protein.

9. The composition of claim 8, wherein the peptide, polypeptide or protein is conjugated or cross-linked to the extracellular matrix.

10. The composition of claim 9, wherein the peptide, polypeptide or protein is a growth factor.

11. The composition of claim 8, wherein the peptide, polypeptide or protein is selected from the group consisting of a collagen, a proteoglycan, a glycosaminoglycan (GAG) chain, a glycoprotein, a growth factor, a cytokine, a cell-surface associated protein, a cell adhesion molecule (CAM), an angiogenic growth factor, an endothelial ligand, a matrikine, a matrix metalloprotease, a cadherin, an immunoglobin, a fibril collagen, a non-fibrillar collagen, a basement membrane collagen, a multiplexin, a small-leucine rich proteoglycan, decorin, biglycan, a fibromodulin, keratocan, lumican, epiphycan, a heparan sulfate proteoglycan, perlecan, agrin, testican, syndecan, glypican, serglycin, selectin, a lectican, aggrecan, versican, nuerocan, brevican, cytoplasmic domain-44 (CD-44), macrophage stimulating factor, amyloid precursor protein, heparin, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate A, heparan sulfate, hyaluronic acid, fibronectin (Fn), tenascin, elastin, fibrillin, laminin, nidogen/entactin, fibulin I, fibulin II, integrin, a transmembrane molecule, platelet derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), transforming growth factor beta (TGF-beta), fibroblast growth factor-2 (FGF-2) (also called basic fibroblast growth factor (bFGF)), thrombospondin, osteopontin, angiotensin converting enzyme (ACE), and vascular epithelial growth factor (VEGF).

12. A composition for reconstruction, replacement or repair of a defect, or damage in organ tissue comprising extracellular matrix, wherein said composition comprises a form selected from the group consisting of an emulsion, an injectable solution, a gel, a foam, a liquid, a paste, a powder, a spray, a vapor, a cream, a coating, a nanoparticle, a patch, a sheet, a laminate, a weave, a matrix, a fabric, a strand, a plurality of strands, a strip, a plurality of strips, a plug, a piece, and a plurality of pieces, and further comprises an additional component selected from the group consisting of: a) a cell, b) a peptide, polypeptide, or protein, c) a vector having a DNA capable of targeted expression of a selected gene, and d) a nutrient, a sugar, a fat, a lipid, an amino acid, a nucleic acid, a ribonucleic acid, an organic molecule, an inorganic molecule, a small molecule, a drug, or a bioactive molecule.

13. The composition of claim 12, wherein the extracellular matrix comprises mammalian extracellular matrix.

14. The composition of claim 13, wherein the mammalian extracellular matrix is selected from the group consisting of small intestine submucosa (SIS), urinary bladder submucosa (UBS), urinary basement membrane (UBM), liver basement membrane (LBM), stomach submucosa (SS), collagen from animal sources, collagen from plant sources, synthesized extracellular matrix in cultures from cells (Matrigel™), dermal extracellular matrix, subcutaneous extracellular matrix, large intestine extracellular matrix, placental extracellular matrix, ornamentum extracellular matrix, heart extracellular matrix, and lung extracellular matrix.

15. The method of claim 1 , wherein the organ tissue is selected from the group consisting of myocardium, intracardiac tissue, pericardium, pancreas, kidney, liver, thyroid gland, adrenal gland, brain, spinal cord, ovary, prostate, testes, vocal cords, intestine, spleen, and stomach.

16. The composition of claim I , wnerein tne damaged or defective tissue comprises intracardiac tissue having damage to or a defect in a conduction pathway in the intracardiac tissue.

17. The composition of claim 1, wherein the damaged or defective tissue comprises intracardiac tissue having damage to or a defect in tissue of a sino atrial node, an atrio ventricular node, or a conduction pathway in between the two.

18. A patch for partial closure of an opening in a pericardial sac comprising mammalian extracellular matrix, the patch attachable to the opening at two or more points.

19. The patch of claim 18, wherein the mammalian extracellular matrix comprises one selected from the group consisting of small intestinal submucosa (SIS), urinary bladder submucosa (UBS), small-intestine submucosa (SS), liver submucosa (LS), liver basement submucosa (LBM)₅ dermis, facia, pericardium, and other collagen scaffolds from mammalian sources.

20. A composition for regenerating defective or absent myocardium and restoring cardiac function comprising an emulsified or injectable extracellular matrix composition from a mammalian or synthetic source.

21. The composition of claim 20, wherein the source of extracellular matrix is mammalian and the mammalian source comprises small intestine submucosa (SIS), urinary bladder submucosa (UBS), urinary basement membrane (UBM), liver basement membrane (LBM), stomach submucosa (SS), collagen from animal sources, collagen from plant sources, synthesized extracellular matrix in cultures from cells (Matrigel™), dermal extracellular matrix material, subcutaneous extracellular matrix material, large intestine extracellular matrix material, placental extracellular matrix material, ornamentum extracellular matrix material, heart extracellular matrix material, and lung extracellular matrix material.

22. A composition for regenerating defective or absent myocardium and restoring cardiac function comprising an extracellular matrix derived from a mammalian or synthetic source, said composition further comprising an additional component selected from the group of: a) a cell, b) a peptide, polypeptide, or protein, c) a vector having a DNA capable of targeted expression of a selected gene, and d) a nutrient, a sugar, a fat, a lipid, an amino acid, a nucleic acid, a ribonucleic acid, an organic molecule, an inorganic molecule, a small molecule, a drug, or a bioactive molecule.

23. The composition of claim 22, wherein said extracellular matrix is formulated in a material form selected from the group consisting of a solid sheet, multilaminate sheets, a gel, an emulsion, an injectable solution, a fluid, a paste, a powder, a plug, a strand, a suture, a coil, a cylinder, a weave, a strip, a spray, a vapor, a patch, a sponge, a cream, a coating, a lyophilized material, and a vacuum-pressed material.

24. The composition of claim 22, wherein said additional component is formulated in a material form selected from the group consisting of a gel, an emulsion, an injectable solution, a fluid, a paste, a powder, a plug, a strand, a spray, a vapor, a cream, a coating, a lyophilized material and a vacuum-pressed material.

25. The composition of claim 22, wherein the extracellular matrix comprises an amount of the composition by weight selected from the group consisting of greater than 80%, greater than 82%, greater than 84%, greater than 86%, greater than 88%, greater than 90%, greater than 92%, greater than 94%, greater than 96%, greater than 98%, greater than 98.5%, greater than 99%, greater than 99.5%, and greater than 99.9% of the total composition by dry weight.

26. The composition of claim 22, wherein the additional component comprises an amount of the composition by weight selected from the group consisting of greater than 0.1%, greater than 0.5%, greater than 1%, greater than 1.5%, greater than 2%, greater than 4%, greater than 5%, greater than 10%, greater than 12%, greater than 15%, and greater than 20%.