WO2015134951A1 - Amnion derived therapeutic compositions and methods of use - Google Patents

Abstract

A therapeutic composition may include a fluid component and/or a matrix component derived from amnion. A matrix component may be an amniotic membrane that is free of chorion. A fluid component may be a fluid having a concentration of stem cells, such as amniotic stem cells, and/or micronized amniotic membrane particles. A preferred fluid component includes micronized amniotic membrane particles dispersed in an amniotic stem cell concentrated fluid. An amniotic stem cell concentrated fluid has at least 0.1 x 10⁶ amniotic stem cells per milliliter of fluid or composition, A therapeutic composite may be used to treat any number of heart related conditions through topical application, injection or through introduction intravenously. A matrix component may be located on a treatment location and a fluid component may be subsequently introduced to the matrix component.

Description

AMNION DERIVED THERAPEUTIC COMPOSITIONS AND METHODS OF USE

BACKGROUND OF THE INVENTION

Cross Reference To Related Applications

[0001] This application claims the benefit of U.S. provisional patent application no. 61/949,066 to Amnio Cordis Technology LLC and U.S. provisional patent application no. 61/949,135 to Amnio Cordis Technology LLC, both filed on March 6, 2014; th entirety of both are incorporated herein by reference.

Field of the Invention

[0002] The present invention relates to therapeutic compositions derived from amnion materials and methods of use to treat the heart.

Background

[0003] Amniotic membranes are being used in clinical trials to treat a wide range of conditions. Amniotic membranes are typicaily placed directly on a treatment location, such as a wound or incision, in many cases however, amniotic membranes lack the proper architecture and ceil viability to effectively provide the desired therapeutic responses, such as tissue regenerations, immunomodulation, anti-inflammatory and antiftbrottc. Most amniotic membranes are dehydrated and cryogenicaily preserved. In other cases, the amniotic membranes are sterilized in a manner that damages the tissue and/or reduces cell viability. For example, many amniotic membranes are processed wit a gSutaraSdehyde which is known to significantly reduce ceil viability. In many treatment applications, it is desirable to provide a high concentration and/or specific type or biend of stem ceils. In addition, some therapeutic composites comprise components from two or more donors thereby limiting their use.

SUMMARY OF THE INVENTION

[0004] The invention is directed to therapeutic compositions that, in one embodiment, comprise a therapeutic fluid comprising amniotic fluid. An amniotic fiuid may comprise any number of cells, including stem ceils, growth factors, proteins

i and the like, in one embodiment, a therapeutic fluid comprises an amniotic fluid that is acellular. in another embodiment, a therapeutic composition comprises a matrix component, such as an amniotic membrane, in still another embodiment, a therapeutic composition comprises a matrix component and a fluid component, wherein a fiuid component may be imbibed into or coated onto one or more surfaces of the matrix component, in an exemplary embodiment, a therapeutic composition comprises an amniotic membrane in the matrix component and comprises amniotic fluid in the fiuid component.

[0005] in an exemplary embodiment, the therapeutic composition, as described herein, comprises a plurality of amniotic stem ceils, and preferably at a high concentration, such as greater than 0,1 x 10^δ per milliliter and more preferably above 0,6 x 106 per milliliter of the therapeutic fiuid component within the therapeutic composition, A therapeutic fluid component may aiso be aceiiuSar, such as an acellular amniotic fluid. An acellular amniotic fluid is described in U.S. application no 14/593,415 to Amnio Technology LLC; the entirety of which is incorporated by reference herein. A therapeutic fluid component may be referred to herein as simply a fiuid component for brevity. In another embodiment, a fluid component comprises amniotic membrane that has been micronized and dispersed in a fluid. In one embodiment, a fluid component is a dispersion of micronized amniotic membrane combined with a fluid, such as plasma, saline, amniotic fluid, combinations thereof and the like, In another embodiment, a fluid component comprises a mixture of micronized amniotic membrane particles combined with an amniotic stem cell concentrated fluid, in still another embodiment, a therapeutic fluid consists essentially of a concentrated amniotic fluid wherein the quantity of amniotic stem cells is increased. The amniotic stems ceils in the therapeutic composite, as described herein, may be derived from amniotic fluid and the stem ceils may be concentrated by a centrifuge process. Additional fluids and agents may be added to the amniotic stem celis such as plasma, Plasma Lyte-A, from Baxter Inc., saline and the like. The concentration of amniotic stems cells in one milliliter of a fluid component of an exemplary therapeutic composition, as described herein, may be about 0.5 X 10⁶ or more, 1 ,0 x 10⁶ or more, 5.0 x 10^s or more, 10 x 10⁶ or more and any range between and including the concentrations values provided, A high concentration of amniotic stems cells may greatly improve the effectiveness of the therapeutic composition for many applications. The therapeutic composition, as described herein, may comprise endothelial cells, mesenchyma! stem cells, amniotic fluid stem ceils, fibroblasts, proteins, keritinocytes, epithelial and/or epidermal ceils, paratenacytes, keratinocytes, epithelial and/or epidermal cells, paratenacytes, keratinocytes and growth factors, in some embodiments, protein markers for mesenchymal stem cells may be analyzed to quantify the various types of ceils within the therapeutic composition. Flow cytometry may be used to identify proteins, CD44, CD105, CD73 and GD9Q. In one embodiment, a therapeutic composition comprises at least 30% of mesenchymal stem cells as identified by CD73,

Mesenchymal stem cells indicated by CD73 proteins may be more mobile and provide a more therapeutic effect that mesenchyma! stem cells identified by the other markers. A therapeutic fluid component, as described herein, may comprise antiinflammatory nano-partic!es and/or statins, HMG-CoA reductase inhibitors to reduce inflation at a treatment location.

[0006] In some embodiments, a therapeutic composition is doped with progenitor cells and the progenitor cells may be multipotent progenitor cells and/or pluripotent progenitor cells. Progenitor cells may be derived from a patient to be treated, such as from a stromal vascular fraction. Vascular fraction ceiis and/or progenitor cells may be included with a therapeutic composite to further improve effectiveness. Progenitor cells may be autologous or allogeneic.

[0007] A fluid component, as described herein, may comprise particles and/or a concentration of amniotic stem cells. The particles within the fluid component may comprise micronized amniotic membrane. The micronized amniotic membrane may comprise hydrated mammalian amniotic tissue having a percent hydration of at least about 25%, at feast about 50%, at least about 75% by weight or any range between the concentrations provided. Amniotic membrane maintained in a hydrated state may provide for more viable and regenerative properties. Amniotic membranes that are lyophilized have a great reduction in cell viability. The particles in the fluid component, as described herein, may consists essentially of amniotic membrane and be substantially free of chorion. The amnion layer may be removed from the chorion prior to processing. In one embodiment, the amniotic membrane particles consist essentially of epithelium wherein the concentration of the epithelium is about 70% or more, for example. The particles consisting essentially of epithelium may comprise stem ceiis and tissue that may substantially surround the stem cells. £0008] An amniotic membrane, or portion thereof, may be micronized while in a hydrated state thereby improving the viability of ceils. The amniotic membrane particles may be derived from dehydrated and/or decellularized amniotic tissue however. In addition, the amniotic membrane may be cryo-fractured, such as with a blunt object to minimize shear and damage to tissue, thereby improving therapeutic effectiveness. Particles of amniotic membrane may have any suitable particle size, average particle size and particl sized distribution. For example, the amniotic membrane derived particles, or micronized particles, may have a particle size, or an average particle size of no more than about 10pm, no more than about 5pm, no more than about 2pm, no more than about 1pm, no more than about O.Spm and any range between and including the average particle sizes provided. The particle size of the amniotic membrane particles can be determine through any suitable method, including image analysis, whereby a therapeutic composite is dried and imaged using a scanning electron micrograph (SEM).. The amniotic membrane derived particles may have an irregular shape and in some embodiments are planar having a first planar surface and a second planar surface. Cryo-fracturing of amniotic membrane with a blunt object provides particles with less shear and a more irregular shape than conventional cryo-miliing, thereby providing a higher surface area and more effective therapeutic effect.

[0009] The concentration of particles, such as micronized amniotic membrane, in the therapeutic composition and/or fluid component may be provided in any effective amount such as more than about 0,1%, more than about 0.5%., more than about 1%, more tha about 10%, more than about 25%, more than about 50%, more than about 75%,or more than about 90% by weight of therapeutic composition and any range between and Including the weight percentages listed. Likewise, the mass of particles, such as amniotic membrane particles, may be provided in a therapeutic fluid component of a therapeutic composition in any effective amount, such as more than about img/mi, more than about 5mg/ml, more than about 10mg/ml, more than about 50mg/mi, more than about lOOmg ml, more than about 500mg/mi, and any range between and including the mass concentrations provided. The particles in the therapeutic composition may comprise collagen, growth factors, stem cells, amniotic stem cells, mesenchymal stem cells, progenitor ceils, red blood ceils, white blood cells, proteins, fibroblasts, paratenacytes, keratinocytes and the tike. {0010] An exemplary therapeutic composition may comprise an oxygen-carrier component that may increase the effectiveness of the therapeutic composite by increasing oxygen availability and increase stem ceil viability. Any suitable oxygen- carrier component or combination of components may be included info a therapeutic compositing including, but not limited to, perfluorocarbon such as

perfSuorotributy!amine (PFTBA), perfluorooctyibromide (PFOS).

perf!uorodecyibromide, perfluoroperhydrophenanthrene and the like. An oxygen- carrier may be bonded, such as covalentiy bonded to a therapeutic composition, such as to a matrix component or to the micronized amniotic membrane, in one embodiment, a matrix component comprises a polymeric material, such a

fluoropoiymer, and an oxygen component is bonded thereto. An suitable means may be used to bond an oxygen component to a therapeutic composition component including, cross-linking agents, radiation, and the like. In still another embodiment, an oxygen-carrier component may form a emulsion, or micro-emulsion with another fluid component. A perfluorocarbon oxygen-carrier component is hydrophobic and when mixed with a fluid component that is hydrophilic or comprises water, an emulsion may be formed comprising an aqueous phase and a perfluorocarbon phase,

[001 1] Any of the fluid components described herein may be an injectable solution that will pass through a 20 gauge needle or a needle having a smaller diameter. In other embodiments, a fluid component Is provided in a thicker composition, such as a paste that may foe applied topically. The viscosity of the an injectable fluid component may be no more than about 1 mPa sec, no more than about 500 mPa sec, no more than about 1000 mPa sec, no more than 20.000 mPa sec, no more than 60,000 mPa sec. In other embodiments, a fluid component may be provided for topica! applications and the viscosity ma be more than about 20 Pa sec, more than about 50 Pa sec, more than about 100 Pa sec, more than about 250 Pa sec and any range between and including the viscosity values provided.

[0012] In an exemplary embodiment, a therapeutic composition is a

therapeutic composite and comprises an of the fluid components, as described herein, imbibed into or coated onto a matrix component, A matrix component is a sheet, block, tube or rod of material, for example, that may comprises porosity and pores for accepting a fluid component therein, A matrix component may be a biological materia! such as an amniotic membrane. In another embodiment, an amnioiic membrane may be provided as a matrix component in a muiti!ayered configuration or combined with any other suitable support iayer for a desired application. For example, a therapeutic composite, as described herein, may comprise an amniotic membrane iayer and a cover iayer. A cover iayer may be used to reduc the loss, wash-out, of a fluid component from the therapeutic composite. In another embodiment the therapeutic composite comprises an amniotic membrane and a support iayer, such as a polymer matrix materia! including, but not limited to, a bioresorbable or fluoropolymer membrane. A support layer may have a tensile break strength that is much greater, such as two times or more that of an amniotic membrane layer in a matrix component, in still another embodiment, a therapeutic composite comprises one or more layers of amniotic membrane that are tensiiized, whereby an amniotic membrane has been stretched in one or more directions to increase strength and/or area of the membrane. An amniotic membrane may be cross-linked, and a cross-linked amniotic membrane may be combined with a non- cross-linked amniotic membrane. Any suitable method, as known in the art of cross- linking an amniotic membrane may be used including chemical, such as treatment with g!utaraSdehyde, radiation and the like. A therapeutic composite as described herein, may comprise anti-inflammatory nano-particles and/or statins, H G-CoA reductase inhibitors to reduce inflation at a treatment location. An exemplary therapeutic composition, and in particular a fluid component, may comprise manriitol, saline, ringers lactate, vitamin B complex and the tike.

[0013] A therapeutic composite, as described herein, may be provided with the therapeutic fluid imbibed into, coated onto, or otherwise applied to a matrix component. For example, a therapeutic composite comprising an amniotic membrane may be provided with a therapeutic fluid component comprising micronized amniotic membrane particles dispersed in fluid component. This earner fluid may be an amniotic stem cell concentrated fluid component. In an exemplary embodiment, the therapeuiic fluid component and the amniotic membrane are from a single donor. In an exemplary embodiment, the amniotic membrane, the micronized amniotic membrane particles and the amniotic stem ceils in the fluid component are all from a single donor, in another exemplary embodiment,, a therapeutic composite comprises an amniotic membrane Iayer configured for direct application to a treatment location, a cover layer of a bioresorbable material and a therapeutic fluid component comprising a high concentration of amniotic stem cells. A portion of a bioresorbable material or other matrix layer of the therapeutic composite may be porous to enable a portion of the fluid component to be retained therein. Any suitable number and type of matrix or support layers may be configured in a therapeutic composite, as described herein. In one embodiment, a fluid component may be vacuum imbibed into a matrix component. Whereby a matrix component is submerged in a fluid component and vacuum is applied to remove substantially ail the air from the matrix component. This removal of air will allow the fluid component to more substantially fill the voids and porosity of the matrix component.

[0014] A support layer may comprise any suitable type of material including, but not limited to, a bioresorbable material, a non-bioresorbable polymer material, such a polyether ether keton (PEEK), or poiytetrafluoroethylene (PTFE), fluorinated ethylene propylene (FEP), perfiuoraalkoxy (PFA) and the like, or a metallic component, such as stainless steel, titanium, goid and the like. A support layer may be porous and/or permeable. A support layer may be a membrane having a

microstructure of pores, or a film, net, screen, woven and the like. A support layer may be substantially non-permeable to fluid and may be hydrophobic or oieophobic on at least one side. In an exemplary embodiment, a support layer is expanded PTFE, In an exemplary embodiment, a support layer is a sheet of material having a first substantially planar surface, a second substantially planar surface and a thickness.

[0015] A therapeutic composition may be introduced to a treatment location by direct topical application, such as by coating, applying, spraying, or placing over a treatment location and in some cases adhering a portion of the therapeutic composition with an adhesive, staples or sutures. In other embodiments, a therapeutic composition is delivered transcatheter and may be configured on any suitable implantable or delivery device, such as a stent or a depioyabSe and removable balloon. In some embodiments, the fluid component, as described herein, Is applied to the treatment location with both the matrix component and fluid component combined in a single step, whereby the fluid component is imbibed, coated or otherwise combined with the matrix component, in other embodiments, a matrix component is applied to a treatment location and a fluid component is subsequently added, such as by injection or topical application. For example, an amniotic membrane may be applied to a treatment location and a fluid component may subsequently be injected into the amniotic membrane and/or to the tissue under or around the location of the amniotic membrane, in still another embodiment, a first matrix component layer may be located on a treatment location and a second matrix component layer may be applied over the first matrix component layer. The first aod/or second layer may comprise a fluid component and each layer may comprise a different composition of fiuid component, A second matrix component layer may be substantially non-permeable to the fluid component thereby reducing wash-out or dilution of the fluid component from bodily fluid exposure,

|^'001 ] A therapeutic composition, as described herein, comprises other biological materials that are not amnion derived, in one embodiment, a therapeutic composition comprises a stromal vascular fraction (SVF) from a patient that is to be treated with the therapeutic composition. Stromal vascular fraction derived from adipose tissue of a patient, for example, may be combined with the matrix and/or fiuid component as described herein, in an exemplary embodiment, stromal vascular fraction is combined with micronized amniotic membrane and/or amniotic stem ceils to form a fluid component In another embodiment, a stromal vascular fraction is combined with a matrix component either before or after locating the matrix component over the treatment location. The stroma! vascular fraction may contain any of the following: preadipocytes, mesenchymal stem cells (MSG), endothelial progenitor cells, T cells, B ceils and mast cells as well as adipose tissue macrophages, in another embodiment, a therapeutic composition comprises bone marrow aspirate (B A) and/or platelet rich plasma (PRP).

[0017] The therapeutic composition, as described herein, may be

cryopreserved whereby the temperature of the therapeutic composite is lowered to a temperature of no more than -70°C, and preferable lower than about -80°C. The rate of cooling may be controlled to reduce damage and maintain viability of the cells upon thawing.

[0018] As shown in Table 1 below, an exemplary fluid component comprising a concentrated amniotic stem cell fluid and micronized amniotic membrane particles, as described in Example 1, retained a very high viability post controlled rate freezing. Maintaining the amniotic membrane in a hydrated state prior to cryo-fracturing and subsequent cryopreserving improves cell viability.

Table 1:

[001 ] The viability of ce!is was maintained after thawing a cryo-preserved concentrated amniotic fluid as reported in Table 1. A srnaSi loss in viability was observed with a totai viabiiiiy after thawing a cryopreserved therapeutic composite of more than 90% in ail cases. A therapeutic composite, as described herein, may have a ceil viability of about 70% or more, at least about 80% or more, about 90% or more and any range between and including the ceil viability values provided.

[0020] Any of the therapeutic compositions described herein may be used for a wide variety of treatment applications. A therapeutic compositions, as described herein, may be provided to any suitable treatment location of the body to induce an immunomodulatory and/or anti-inflammatory response. In another application, a therapeutic composition is introduced into a treatment location to reduce scaring and to promote healing, whereby the therapeutic composition aids in regeneration of new tissue. A fluid component of the therapeutic composition, as described herein, may be injected directly into an ffected area, introduced intravenously, through shunts, or ports. It may be desirable to provide a fluid component comprising both amniotic stem ceils and micronized amniotic membrane when tissue regeneration is desired. The micronized amniotic membrane particles may provide the architecture needed for more effective regeneration and tissue repair,

[0021 ] A therapeutic fluid, as described herein, may be provided intravenously to regulate systemic irnmun modulation post-surgery. An exemplary therapeutic fluid may comprise mannitol, saline, ringers lactate, vitamin B complex and the like. A therapeutic composition, as described herein, may be introduced into the heart, or the vascular system through transcatheter, or topical application, i some embodiment, a therapeutic composition, as described herein is configured onto a stent or is positioned by a balloon catheter.

[0022] A therapeutic composition may be used to treat an number of heart related conditions, including, but not limited to, restrictive and constrictive

cardiomyopathy, ischemic cardiomyopathy, Idiopathic cardiomyopathy, allograft vasculopathy, atherosclerosis, arrhythmia, post-operative atria! fibrillation, hypertension, pericarditis, indocardifis, myocarditis, acute myocardial infraction, carotid endarterectomy, chronic heart failure with scare, heart failure with Sow ejection fraction less than 35%, coronary artery disease with regional wail abnormalities, post-operative heart recovery and scaring and infective endocarditis. Post-operative atrial fibrillation may be treated with by placement of the therapeutic composition on the epicardium. In another embodiment, a therapeutic composition is used to treat a valve and/or leaflet defect. A therapeutic composition may be used to regenerate a portion of a valve or leaflet, for example. Treatment of these heart conditions may include Introduction of a therapeutic composition, as described herein, to a heart b topical application to a portion of a heart or epicardium for example, inter-artehaSly, intravenously, intra-arterially including intrarenal, intracoronary and intracarotid, and/or trans-bronchial!y. In one embodiment, a therapeutic composition comprising a matrix component is applied directly to the heart or vascular portion of the anatomy and attached by adhesive, sutures or staples, for example. In another embodiment, a therapeutic composition is wrapped around a portion of an artery or vein and may be adhered to itself to retain the therapeutic composition in place. For example, a therapeutic composition may be wrapped around the carotid artery in an endarterectomy procedure before or after suturing the carotid artery.

[0023] An exemplary method of treating a heart includes applying a therapeutic composition, as described herein after the concluding portion of a coronary bypass graft surgery (CABG) and after the grafts are completed, A therapeutic composition comprising an amniotic membrane matrix component, may be attached to the retracted pericardium or on the myocardium. A therapeutic matrix component may be attached to the heart with any suitable adhesive, sutured thereon, or held in place by hydrostatic tension. A matrix component attached to a portion of the heart may comprise a fluid component, containing, micro ized amnion membrane that inciudes a multitude of growth factors such as, insulin, or growth factor 1 , transforming growth factor b1 , cytokine proteins, collagen substrates, extracellular matrix proteins such as laminin, fibronecttn, annexin, vitronectin and the like. These components may produce a therapeutic effect on the heart, or the myocardium and reduce inflammation, prevent scarring and fibrosis. The effect of anti inflammation, and anti fibrosis, have been found to have a profound positive effect on the electrical activity function of the heart {myocardium}. An effective does of fluid component may be provided in one treatment or in several doses over a period of time. The specific treatment and dosing regime will depend on the type and severity of the condition to be treated,

0024 A study evaluating the therapeutic effectiveness of applying a therapeutic composition, as described herein is described in the American Journal of Medicine, Vol 128, No. 1 , "First in Man: Amniotic Patch Reduces Postoperative inflammation", by Dr. Zain Khalpey, et al; the entirety of which is incorporated by reference herein.

[0025] Stents are known to be configured for almost every portion of the vascular system and are becoming smaller and more versatile in their applications. Cardiac stents, thoracic aortic stents, abdominal aortic stents, coronary stents, endoprosthesis and any other stent for the vascula system may be configured with a therapeutic composition, as described herein. A matrix component may be configured around an exterior portion of the stent to provide better adhesion to the treatment location,

[0026] In one embodiment, a fluid component is injected into a specific treatment location through the use of a catheier, such as a steerable catheter and an injection implement configured on the introductory end of the catheter. For example, a catheter having an injection implement may be introduced into the femora! artery, inserted to position the injection implement in proximity to the heart, whereby a dose of therapeutic composite is administered info the tissue of the heart.

[002?] The summary of the invention is provided as a general introduction to some of the embodiments of the invention, and is not intended to be limiting.

Additional example embodiments including variations and alternative configurations of the invention are provided herein, BRl EF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention, and together with the description serve to explain the principles of the invention,

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

[ 0029] Figure 1 A shows a cross-sectional diagram of amniotic membrane surrounding a fetus in utero.

[0030] Figure 1 B shows a cross-section diagram of the layers of the amnion and chorion.

[0031 ] Figure 2A show a transmission electron micrograph (TEM) of the epithelium layer of the amniotic membrane having a single layer of amniotic stem cells. The TEM is at 25D0 X magnification.

[0032] Figure 2B show a TEM of the epithelium layer of the amniotic membrane having a single layer of amniotic stem cells. The TE is at 8200 X magnification.

[0033] Figure 3A is a scanning electron micrograph (SEM) of an amniotic membrane having amniotic stem cells.

[0034] Figure 3B is a SEM of cryo-fraeiured amniotic membrane particles.

[0035] Figure 4 is a scanning electron micrograph (SEM) of an amniotic membrane having pores between the amniotic membrane tissue.

[0036] Figure 5A is a representation of an exemplary tenssiized amniotic membrane.

[0037] Figure SB is a representation of two exemplary tensi!ized amniotic membranes being layered together.

[0038] Figure 6 shows a diagram of an exemplary method to apply a therapeutic composition, as described herein.

[0039] Figure 7 shows a diagram of a process to produce a fluid component comprising micronized amniotic membrane particles.

[0040] Figure 8 shows a diagram of a process to produce a fluid component comprising a concentrated stem cell fluid. {0041 Figure 9 shows a cross-sectional representation of an exemplary amniotic membrane configured over a treatment Iocation.

[0042] Figure 10 shows a cross-sectional representation of an exemplary therapeutic composition comprising an amniotic membrane and fluid component configured over a treatment iocation,

[0043 J Figure 11 shows a cross-sectional representation of an exemplary therapeutic composite configured over a treatment iocation wherein the therapeutic composition comprises an amniotic membrane matrix component imbibed with a fluid component and a cover Iayer configured there over.

[0044] Figure 12 shows a cross-sectional representation of an exemplary therapeutic composite configured over a treatment location wherein the therapeutic composite comprises a first matrix iayer of amniotic membrane, a second matrix layer of a fluid component reservoir, and a third matrix Iayer that is a cover iayer.

[0045] Figure 13 shows a cross-sectional representation of an exemplary therapeutic composite configured over a treatment iocation wherein the therapeutic composite comprises a first matrix iayer of amniotic membrane imbibed with fluid component and a second matrix Iayer that is a support layer comprising

bioresorbable material.

[0046] Figure 14 shows a cross-sectional representation of an exemplary therapeutic composite configured over a treatment iocation wherein the therapeutic composite comprises a first matrix layer of amniotic membrane imbibed with fluid component, a second matrix Iayer that is a support Iayer and a third matrix layer that comprises amniotic membrane.

[004?] Figure 15 shows an exemplary matri component of a therapeutic composite configured around an artery and a fluid component being injected therein.

[0048] Figure 16 shows a diagram of the anatomy and various organs within the body.

[0049] Figure 17 shows a diagram of the circulatory system.

[0050] Figure 18 shows an exemplary fluid component being drawn from an enclosure by a syringe.

[0051] Figure 19 shows an exemplary catheter inserted through the femoral artery with the proximal end located at the heart.

[0052] Figure 20 shows flow cytometry analysis data for amniotic fluid as received and amniotic stem ceil concentrated fluid. {0053] Figure 21 shows a cross-sectional view of a heart.

[0054] Figure 22 shows a cross-sectional view of a heart with an exemplary therapeutic composite placed on the epicardium.

[0055] Corresponding reference characters indicate corresponding parts throughout the several views of the figures. The figures represent an illustration of some of the embodiments of the present invention and are not to be construed as limiting the scope of the invention in any manner. Further, the figures are not necessariiy to scale, some features may be exaggerated to show details of particular components. Therefore, specific structural and functional detaiis disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ the present invention,

[0056] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having" or any other variation thereof, are intended to cover a non-exclusiv Inclusion. For example, a process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Also, use of "a" or "an" are employed to describe elements and components described herein. This is done merely for convenience and to give a general sense of the scope of the invention. This description should be read to include one or at least one and the singular also includes the piurai unless it is obvious that it is meant otherwise,

[0057] Certain exemplary embodiments of the present invention are described herein and illustrated in the accompanying figures. The embodiments described are only for purposes of illustrating the present invention and should not be interpreted as limiting the scope of the invention. Other embodiments of the invention, and certain modifications, combinations and improvements of the described

embodiments, will occur to those skilled in the art and all such alternate

embodiments, combinations, modifications, improvements are within the scope of the present invention.

[0058] As shown if FIG. 1 A the amniotic membrane surround a fetus in utero.

As shown in FIG. 1 B, the amniotic membrane comprises an amnion portion and a chorion portion. As described herein, the amnion portion may be separated from the chorion, in an exemplary embodiment, the epithelium, or inner most layer of the amniotic membrane, is removed and used to produce particles for the therapeutic composite, as described herein. The particles may consists essentially of the epithelium, consists essentially of the epithelium and base membrane, consist essentially of the epithelium, base membrane and compact layer, or consist essentially of epithelium, base membrane, compact layer, and fibroblast layer,

[0059] As shown in FIGs, 2A and 2B, the epithelium layer of the amniotic membrane 20 has a single layer of amniotic stem cells 46. The tissue around the amniotic stem cells may protect and enhance the viability of these stem cells when the epithelium is cryo-fractured to produce particles for the therapeutic composition,

[0060] As shown in FIG. 3A, an amniotic membrane 20 comprises a plurality of amniotic stem cells 46.

[0061] As shown in FIG. 38, particles of cryo-fractured amniotic membrane particles 40 are on the order of 0.2 to 0.5pm in size. The average particle size shown is less than 2p.m. There are no particles shown that are larger than 2pm and substantially all of the particles are less than 1 pm in size. The SE shows that the micronized amniotic membrane particles are irregularly shaped. As shown, some of the particles have a planar surface.

[0062] As shown in FIG. 4 an amniotic membrane 20 comprises pores 29 between the amniotic membrane tissue. This porosity may be imbibed with a fluid component. In addition, an amniotic membrane may be stretched in one or more direction to tensilize the tissue. A tensiiized amniotic membrane may have a higher matrix tensile strength than an original un-tensilized amniotic membrane. In addition, a pSurality of layers of amniotic membrane may be utilized to build strength in one or more directions.

[0063] As shown in FIG. 5A, an amniotic membrane 20 has been stretched in one direction to form an elongated and more aligned amniotic tissue orientation. As shown in FIG. 5A_t oriented tissue 23 is aligned horizontally and connecting tissue interconnects the oriented tissue. A tensiiized amniotic membrane 21 may be stronger by unit weight in the oriented direction and may have a much higher elongation to break in the cross-oriented direction than a precursor amniotic membrane, before tenstiizing. The tensiiized amniotic membrane 21 may be stretched as much as 120%, 150%, 175%, 200% of the original membrane length.

The amniotic membrane may neck or narrow in the opposing direction of stretch. A stretched or tensiiized amniotic membrane may be stretched over a Song period of time to minimize tissue fracture. For example, an amniotic membrane may have a low load applied and may be stretched over a period of 10 minutes or more, 30 minutes or more, 1 hour or more, 6 hours or more, 1 day or more, 2 days more and any range between and including the durations provided, in addition, an amniotic membrane may be stretched while being hyd rated and or submerged in amniotic fluid or a pfasticizing fluid. An amniotic membrane may be cross-linked after being stretched. The load applied to tensiiize an amniotic membrane may be a portion of the maximum tensile load required to fracture the amniotic membrane at a rate of 10mm/second for a 2.54cm by 15.2cm sample having a 5cm gap. For example, a fertilizing load applied may be no more fhan about 80%, no more than about 60%, no more than about 50%, no more than about 25% of the maximum tensile load.

[0064] As shown in FIG. 58, a first tensiiized amniotic membrane 20 is configured at a 90 degree offset from a second amniotic membrane 20'. This orientation of layering may provide for a much stronger therapeutic composite, in an alternative embodiment a plurality of layers of tensi!ized amniotic membrane may be aligned with the oriented tissue of a first layer being aligned with the oriented tissue of a second layer. A matrix component or a therapeutic composite, as described herein, may consist essentially of tensilized amniotic membrane.

[0065] Figure 8 shows a diagram of an exemplary method to apply a therapeutic composite as described herein. As described herein, a fluid component may be configured with a matrix component or may be applied after application of the matrix component to a treatment location.

[0066] As shown in FIG. 7, a process to produce a therapeutic composition, as described herein, comprises the steps of cryo-fracturing amniotic membrane fragments. As described, the amniotic membrane fragments may be cryo-fractured with a blunt object, such as a bar, that reduces shear and damage to the particles, in a preferred embodiment, the fragments are cryo-fractured with an object having substantially no sharp edges. The micronized particles are combined with any suitable carrier fluid to produce a therapeutic composite. In an exemplary embodiment, the micronized particles are dispersed in a fluid comprising stem ceil fluid and amniotic stem cells, in another embodiment, the micronized particles are dispersed in a concentrated amniotic stem cell fluid.

[0067] As shown in FIG. 8, a process to produce a therapeutic composition, as described herein, comprises the steps concentrating amniotic stem cells in an amniotic fluid. An amniotic fluid may be processed in any suitable way to concentrate the amniotic stem ceils in the fluid. In an exemplary embodiment, as described in F!G. 5, the amniotic fluid is centrifuged to remove debris and excess liquid and concentrate the amniotic stem ceils in the therapeutic composition.

[0068] As shown in FIG. 9, an exemplary therapeutic composition 1 1 is a therapeutic composite 10, The therapeutic composite 10 comprises an amniotic membrane 20, as a matrix component 2,configured over a treatment location 18. The matrix component"! 2 in this embodiment consists essentially of amniotic membrane 20 and a fluid component 14 is coated onto the treatment surface 50 of the therapeutic composite. The fluid component 14 is not present on the outer surface 52 of the therapeutic composite 10.

[0069] As shown in FIG.10, an exemplary therapeutic composite 10 comprises an amniotic membrane 20 and a fluid component 14 imbibed therein, configured over a treatment location 18. The fluid component 14 comprises micronized amniotic membrane particles 40 and amniotic fluid 43. However, any suitable fluid carrier may be used to disperse the micronized amniotic membran particles and or amniotic stem cells 46.

[0070] As shown in FIG. 11 , an exemplary therapeutic composite 10 is configured over a treatment Iocation 18 wherein the therapeutic composite comprises an amniotic membrane 20 imbibed with a fluid component 14 and a cover layer 24 is configured there over. The matrix component 12 comprises a first matrix layer 30 and a second matrix layer 32, The second matrix layer is configured over said first matrix layer and comprises an overhang portion 36 that extends outside of the first matrix layer. The second matrix layer is attached to the tissue 9 by a attachment component 38, such as a staple, glue and/or sutures, for example. A matrix component or a layer of a matrix component may be configured to extend beyond a treatment iocation, whereby an outer area of the matrix component can be affixed to tissue. A cover layer may fully cover a first o under layer of matrix component or may only cover a portion of an layer thereunder. A cover layer may be a net or mesh or strands that extend across and over an under-layer, for example. An exemplary cover layer comprises pores or apertures 28 that allow fluid transfer to and from the treatment location. Apertures may be small slits, holes, in an otherwise solid and Impermeable matrix component or layer, or they may be pores in porous matrix component or layer. For example, an expanded po!ytetrafluoroethylene membrane may have a mean flow pore size as measure by a Coulter Porometer {PMI Industries), of less than 50um, less than 40um, less than 10urn, less than 1um and any range between and including the pore si2es provided, !n one embodiment, the pores are sized to allow fluid to flow but retain ceils, such as stem cells within the matrix component.

[0071] As shown in FIG. 12, an exemplary therapeutic composite 10 is configured over a treatment location 18 wherein the therapeutic composite comprises a matrix component 12 comprising a first matri layer 12 of amniotic membrane 20, a second matrix layer 32 of a fluid reservoir layer 25, and a third matrix layer 34 that is a cover layer 24, The fluid reservoir layer comprises a matrix having porosity containing a fluid component 14', as described herein. As shown, a first fluid component 14 is configured within the first matrix iayer 30. It is be noted that different compositions of a first and second fluid component may be configured in a matrix component 12. A first fluid component may comprise an amniotic stem ceil concentrated fluid and a second fluid component may comprise micronized amniotic membrane dispersed in a fluid, for example. A reservoir layer may comprise a fluid component having stem cells, and these stem celis may be drawn from the reservoir iayer as they are needed.

[0072] As shown in FIG. 13, a therapeutic composite 10 is configured over a treatment location 18 wherein the matrix component 2 comprises a first matrix iayer 30 of amniotic membrane 20 imbibed with fluid component 14 and a second matrix layer 32 that is a support iayer 22 comprising bioresorbable material 26, The support layer may be substantially impermeable to the fluid component configured in the first matrix component that is proximate a treatment location, in addition, an outer surface 52 of a matrix component 12, or the surface facing away a treatment location, may foe hydrophobic to reduce fluid ingress into the therapeutic composite, Bodii fluid ingress into a therapeutic composite may dilute a fluid component comprises therein.

[0073] As shown in FIG. 14, an exemplary therapeutic composite 10 is configured over a treatment location 18 wherein the matrix component 12 comprises a first matrix Iayer 30 of amniotic membrane 20 imbibed with fluid component 14, a second matrix Iayer 32 that is a support layer 22 and a third matrix layer 34 that comprises amniotic membrane 20. A support layer is configured between amniotic membranes in this embodiment. As described herein, a matrix component may be provided with multiple layers attached and ready for orientation on a treatment location, or a plurality of matrix components may be applied, one after another, during the treatment procedure.

[0074] Any number of combinations of matrix components layers have been envisioned and are within the scope of the present invention. In addition, any number of different fluid components may be incorporated into a therapeutic composite as described herein.

[0075] As shown in FIG, 15, an exemplary therapeutic composite 10 is configured around an artery 64 and a fluid component 14 is being injected therein. This type of procedure may reduce and/or eliminate aneurisms. A matrix component may be a sheet of material having a substantially planar top and bottom surface and substantially uniform thickness therebetween, A sheet of matrix composite may be supple and may be configured around a cylindrical treatment location, such as a artery or vein. In another embodiment, a matrix component sheet is applied externally over a treatment location, such as to the eplcardium.

[0076] Figure 16 shows a diagram of the anatomy and various organs withi the body that may be treated with a therapeutic composite as described herein. A therapeutic composite, as described herein, may be introduced into any anatomy shown in FIG. 18 by open surgery, topical application, or transcatheter. A deliver vehicle such as a stent or balloon may be used with a therapeutic composite, as described herein. For example, a therapeutic composite may be introduced into any portion of the urinary or digestive system, including the bladder, ureter, urethra, small intestine, large intestine, stomach, esophagus, mouth, tongue, colon, rectum, and the like,

[0077] Figure 17 shows a diagram of the circulatory system where a therapeutic composite ma be introduced into the body through transcatheter.

[0078] Figure 18 shows an exemplary fluid component 14 being drawn from an enclosure 70 by a syringe 80. The fluid component comprises micronized

particles 40 of amniotic membrane 20 and stromal vascular fraction 48 in a concentrated amniotic stem cell fluid 44. The needle may be any suitable size, however in a preferred embodiment the needle is no larger than a 20 gauge needle.

[0079] As shown in FIG. 19, a catheter is inserted into the femora! artery and the proximal end of the catheter is located at the heart. A therapeutic composite may be introduced through a catheter to a treatment location within the body. A catheter may be configured with an injection impiement at the proximal end to enable the therapeutic composite to be injected into tissue, such as heart tissue,

[0080] Figure 20 shows flow cytometry anaiysis data for amniotic fluid as received and amniotic stem ceil concentrated fluid as described herein. Flow cytometry was performed on four different Siquid samples from different donors. The analyses shows thai the expression level of mesenchymal stem celi surface antigens is consistent between donors with CD44 being positive and CD73 being strongly positive while CD90 and CD 05 are low positive. The levei of expression is maintained between the processed samples concentrated sample 1 and

concentrated sample 2 and unprocessed samples (Fresh Amniotic Fluid 1 &2)_r suggesting no celi ioss during the manufacturing process and preservation of potency. What is aiso interesting is that CD73 is expressed the most. It has been reported that mesenchymal stem ceil migration is controlled by CD73 and therefore it is speculated that a high levei of CD73 expression promotes cell migration and the ability of the ceils to home to tissue sites of repair or to participate in heaiing responses.

[0081] Figure 21 and 22 show cross-sections of a heart and some of the treatment locations for a therapeutic composite, as described herein. For example, therapeutic composite 10 may be piaced on the epicardium, as shown in FiG, 22, myocardium, and/or the endocardium to treat arrhythmia or post-operative atrial fibrillation, for example. The therapeutic composite may have a fluid component, or a fluid component may be applied subsequent to placement of the matrix component on the heart.

EXAMPLE 1

[0082] Three fluid components were made and ceil viability was measured as reported in Table 1. Three amniotic membrane sampies. obtained from three separate donors, were cryo-fractured and dispersed in fluid to create a fluid components, as described herein.

[0083] A fluid component of the therapeutic composite was prepared by concentrations amniotic stem ceils in a cell suspension solution. A 1 mi sample of an unprocessed amniotic fluid was used to measure initial celi count and viability. The amniotic fluid was then separated into 50m! sterile centrifuge tubes and centrifuged two times at 400xg for 10 minutes at ambient temperature. Ceil peiiet from each tube was washed with 20m! of Plasma Lyte-A, from Baxter inc., between centrif ligation. Supernatant was removed and ceils were re-suspended in a predetermined volume of ceil suspension solution, Piasma Lyte-A, to obtain a final product cell concentration of 1 x 10⁶ cell/m!.

[0084] Cryo-fractured particles of amniotic mernbrane were prepared for dispersion in the fluid component. Three amniotic membranes were obtained and rinsed using Plasma Lyte-A and transferred to a cutting board. Using b!unt

dissection, chorion was removed from the amniotic membrane and any remaining debris/blood was removed using sterile laps. The amniotic membrane dimensions were measured using a sterile stainless steei ruier. The amount of amniotic membrane needed to obtain a concentration of 1cm²/ml of therapeutic soiution was retained and placed on a sterile drying rack and allowed to dry for one hour. The amniotic membrane was then cut into smaiS pieces, less than a 1cm² and placed inside a miiiing chamber containing a blunt smpactor. The cryo-mill used was from SPEX Sample Prep Inc., 6970EF Enclosed Freezer/Mill Mode! 6970D.

[0085] The milling chambers were placed inside the cryomi!l and the amniotic membrane was micronized. The frequency of the impactor was 8 cycies per second, the preceding time was five minutes, the grinding time was three minutes and the intermediate cooling time was two minutes. After the micronization of the amniotic membrane was compiete, the chambers were removed from the cryomiii and allowed to warm at room temperature for one hour. The cryo-fractured amniotic membrane was then dispersed in 100ml of fluid component prepared as described In this example. The finai therapeutic composite was prepared by combining 100ml or the fluid component and micronized amniotic membrane wit equal volume (100ml), of cryprotectant soiution, CryoStor 10, available from Sigma-Aldrich. Using a repeater pipe!, cryoviais were then filled at the desired volume. The therapeutic soiution was maintained at 4°C during the via! fiiiing process to preserve ceii viabiiity.

[0086] The cryoviais were then cryopreserved using a controlled rate freezer. The controlled freezing protocol: cool at a rate of 1 .0°C/min until chamber reached ~ 4°C, cool at rate of 25.0^oC/min until chamber reached -40°C, warm at a rate of

10.0°C/min until chamber reached -12°C, cooi at rate of 1 .0°C/min until chamber reached ~40°C, and cool at rate of 10.0°C/min until chamber reached -90°C.

Cryoviais were then placed into cryo-boxes and transferred to a ~80.0°C freezer

[00871 Thawing of the cryoviais was performed and ceil viabiiity was again measured. Cell viability pre and post cryopreservation is reported in Table 1 . The cryoviaSs were removed from the -80.0°C freezer and allowed to thaw at room temperature until the fluid components in the vial had a slushy consistency, or approximately three minutes for a 1mS sample. An equal amount of cold Piasma Lyte-A was added to the sample for a 1 ;2 dilution. Samples were mixed and a small aliquot was used to perform ceil count and viability enumeration, Celi count and viability was assessed using Trypan Blue.

EXAMPLE 2

iOOSSj A prepared therapeutic composition, as described herein, comprising an amniotic membrane matrix component and a fluid component comprising amniotic fluid and micronized amniotic membrane was used to treat a heart after a coronary bypass graft surgery (CABG), During the concluding portion of a CABG and after the grafts were completed, a prepared amniotic membrane about 4cm x 8cm in dimension, was placed onto the myocardium and held in place by hydrostatic tension. The fluid component contained a multitude of therapeutic components including growth factors including insulin, growth factor 1 , transforming growth factor b1, cytokine proteins, collagen substrates, extracellular matrix proteins such as iaminin, fibronectin annexin, vitronectin, and the like. These therapeutic components reduced inflammation, scarring and fibrosis of the myocardium. This reduced inflammation and fibrosis acted to ensure improved electrical activity of the heart (myocardium). This effect was documented on postoperative MR! that demonstrated the decreased fluid buildup, elimination of atrial fibrillation. The reduction of inflammation allowed normal electrical nerve impulses to be transmitted, and long term MRI imaging at eight weeks revealed minimal scarring around the surgical site, DEFINITIONS

[0089] Micronized particles, such as micronized amniotic membrane particles, as used herein, means that the particles have an average particle size of less than 1 μηι, and in some cases have an average particle size of less than G.5pm. Particle size may be measured by analysis of scanning electron micrograph.

[0090] It will be apparent to those skilled in the art that various modifications, combinations and variations can be made in the present invention without departing from the spirit or scope of the invention. Specific embodiments, features and elements described herein may be modified, and/or combined in any suitable manner. Thus, it is intended that the present invention cover the modifications, combinations and variations of this invention provided they come within the scope of the appended claims and their equivalents.

Claims

What is claimed is;

1. A therapeutic composition comprising a fluid component comprising amniotic fluid.

2. The therapeutic composition of claims 1 , wherein the fluid component

comprises a concentration of amniotic stem cells of at least 0.1 x 10⁶ per milliliter of said fluid component.

3- The therapeutic composition of claims 1 , wherein the fluid component

comprises a concentration of amniotic stem ceils of at least 0.5 x 10⁶ per milliliter of said fluid component,

4. The therapeutic composition of claim 1 , wherein the fluid component

comprises a concentration of amniotic stern cells of at least 1 x 10⁶ per milliliter of said fluid component.

5. The therapeutic composition of claim 1 , wherein the fluid component

comprises a concentration of amniotic stem ceils of at least 5 x 10^s per milliliter of said fluid component,

6. The therapeutic composition of claim 1 , wherein the fluid component

comprises a concentration of amniotic stem cells of at least 10 x 10^fi per milliliter of said fluid component

7. The therapeutic composition of claims , wherein the fluid component

comprises an acelluSar fluid component

8. The therapeutic composition of claims 1 , wherein the fluid component

comprises an ace!lu!ar amniotic fluid,

9. The therapeutic composiiion of claims 1 , wherein the fluid component consists of an aceiiuSar amniotic fluid.

10. The therapeutic composition of claims 1 , wherein the fluid component has a viscosity of no more than about 50 Pa sec.

11.The therapeutic composition of claims 1 , wherein said fluid component is a paste having a viscosity of more than about 50 Pa sec.

12, The therapeutic composition of claims 1 , wherein the fluid component

comprises a plurality of protein markers including CD44, CD105, CD73, and CD90 proteins.

13, The therapeutic composition of claims 1 , wherein the fluid component

comprises: a. growth factors; and

b. proteins.

, The therapeutic composition of claims 1 to 13, further comprising micronized amniotic membrane particles,

, he therapeutic composition of claims 14, wherein the particles consist essentially of micronized amniotic membrane particles.

, The therapeutic composition of claim 14. wherein the micronized amniotic membrane particles consist essentially of amnion and are essentially free of chorion,

, The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles consist essentially of epithelium .

, The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles consist essentially of epithelium and amnion basement membrane.

, The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles consist essentially of epithelium, amnion basement membrane and compact layer.

, The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles consist essentially of epithelium, amnion basement membrane, compact layer and fibroblast layer.

, The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles are hydrated and have a percent hydration of a least 20 percent.

, The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles are dece!Sularized particles.

, The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles have an average particle size of no more than about 250pm.

, The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles have an average particle size of no more than about 150pm.

, The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles are irregularly shaped. 28, The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles are planar in shape, having a first planar surface and a second planar surface,

27. The therapeutic composition of claim 14, having a concentration of micronized amniotic membrane particles of at least about 1.Omg/mi of therapeutic composition,

28. The therapeutic composiiion of claim 14, having a concentration of micronized amniotic membrane particles of at least about 5.Qmg/mi of therapeutic composition.

29. The therapeutic composition of claim 14, having a concentration of micronized amniotic membrane particles of at least about IG.Qmg/ml of therapeutic composition,

30. The therapeutic composition of claim 14. wherein the micronized amniotic membrane particles comprise:

a. collagen; and

b. growth factors.

31. A therapeutic composition of ciaims 1 to 30 comprising;

a. a matrix component comprising amniotic membrane;

32. The therapeutic composition of claim 31 , wherein the amniotic membrane consists essentially of amnion and is essentiaiiy free of chorion.

33. The therapeutic composition of ciaim 31 , wherein the amniotic membrane consists essentiaiiy of epithelium,

34. The therapeutic composition of claim 31 , wherein the amniotic membrane consists essentiaiiy of epithelium and amnion basement membrane.

35. The therapeutic composition of claim 31 , wherein the amniotic membrane consists essentiaiiy of epithelium, amnion basement membrane and compact iayer,

36. The therapeutic composition of ciaim 31 , wherein the amniotic membrane consists essentiaiiy of epithelium, amnion basement membrane, compact Iayer and fibroblast layer.

37. The therapeutic composiiion of claim 31 , wherein the amniotic membrane Is hydrated and has a percent hydration of at least 20 percent. 38, The therapeutic composition of claim 31, wherein the amniotic membrane are is a deceiluiarized amniotic membrane.

39, The therapeutic composition of claim 31 , wherein the amniotic membrane is tensilized in one direction to produce oriented amniotic tissue,

40, The therapeutic composition of claim 31 _» wherein the amniotic membrane is tensilized bi-axialiy.

41.The therapeutic composition of claim 31 to 40, comprising a first layer of amniotic membrane and a second matrix component layer.

42. The therapeutic composition of claim 41 , wherein the second matrix

component layer is configured for placement over said first layer of amniotic membrane,

43. The therapeutic composition of claim 41 , wherein the second matrix

component layer is an amniotic membrane.

44. The therapeutic composition of claim 41 , wherein the second matrix

component layer is a bioresorbable materia!.

45. The therapeutic composition of claim 41 , wherein the second matrix

component layer is a non-bioresorbable polymer material.

46. The therapeutic composition of claim 41 , wherein the second matrix

component layer is substantially non-permeable to a fluid component retained in the first layer of amniotic membrane.

47. The therapeutic composition of claim 41 , wherein the second matrix

component layer is a fluid reservoir layer, whereby said second matrix component layer has porosity that is at least 50% filled with a fluid

component.

48. The therapeutic composition of claim 41 , wherein the second matrix

component layer is a support layer, having a tensile break strength of at least two times that of a first layer of amniotic membrane tensile break strength.

49. The therapeutic composition of claim 41 , wherein the first layer of amniotic membrane is a tensilized amniotic membrane and a second matrix

component layer a tensiiiized amniotic membrane.

50. The therapeutic composition of claims 1 to 49, further comprising a plurality of progenitor cells.

51.The therapeutic composition of claims 1 to 49, further comprising a plurality of vascular fraction cells.

52, The therapeutic composition of claims 1 to 49, further comprising a plurality of progenitor ceils derived from vascular fraction eel is.

53, The therapeutic composition of claims 1 to 49, further comprising

preadipocytes, mesenchymal stem ceils and endothelial progenitor cells from a stromal vascuiar fraction.

54, The therapeutic composition of claims 1 to 49, further comprising

mesenchymal stem cells,

55, The therapeutic composition of claims 1 to 49, further comprising bone

marrow aspirate,

56, The therapeutic composition of claims 1 to 49, further comprising platelet rich plasma.

57, The therapeutic composition of claim claims 1 to 49, further comprising an oxygen-carrier component.

58, The therapeutic composition of claim 57, wherein the oxygen-carrier

component comprises a perfluorocarbon.

59, The therapeutic composition of claim 57, wherein the oxygen-carrier

component comprises a perfluoroperhydrophenanthrene.

60, The therapeutic composition of claim 57, wherein the oxygen-carrier

component comprises is bonded to a matrix component,

61 , The therapeutic composition of claims 57, wherein the oxygen-carrier is a perfluorocarbon and wherein the fluid component is an emulsion,

62, The therapeutic composition of claim 1 to 81 _« wherein the therapeutic

composition is a thawed therapeutic composition from a cryopreserved state,

83. A method of treating post-operative atria! fibrillation by introducing a

therapeutic composition as described in any of claims 1 to 62 to the heart.

64, A method of inducing immunomodulatory response to a heart by introducing a therapeutic composition as described in any of claims 1 to 62 to said heart.

65. A method of inducing an anti-inflammatory response of a heat by introducing a therapeutic composite as described in any of claims 1 to 62 to said heart. 68, A method of inducing a repair of a scare on a heart by introducing a therapeutic composition as described in any of claims 1 to 82 to said heart.

87. A method of treating pulmonary hypertension by introducing a therapeutic composition as described in any of claims 1 to 62 to an affected heart.

88, A method of treating ischemic cardiomyopathy by introducing a therapeutic composition as described in any of claims 1 to 62 to an affected heart.

69, A method of treating idiopathic cardiomyopathy by introducing a therapeutic composition as described in any of claims 1 to 62 to an affected heart.

70, A method of treating dilated cardiomyopathy by introducing a therapeutic composition as described in any of claims 1 to 62 to an affected heart.

71.A method of treating restrictive cardiomyopaihy by introducing a therapeutic composition as described in any of claims 1 to 62 to an affected heart,

72. A method of treating constriciive cardiomyopathy by introducing a therapeutic composition as described in any of claims 1 to 62 to an affected heart.

73. A method of treating allograft vascuiopathy by introducing a therapeutic

composition as described in any of claims 1 to 62 to an affected heart.

74. A method of treating arrhythmia by introducing a therapeutic composition as described in any of claims 1 to 62 to an affected heart.

75. A method of treating pericarditis by introducing a therapeutic composition as described in any of claims 1 to 62 to an affected heart.

76. A method of treating endocarditis by introducing a therapeutic composition as described in any of claims 1 to 62 to an affected heart,

77. A method of treating myocarditis by introducing a therapeutic composition as described in any of claims 1 to 62 to an affected heart.

78. A method of treating acute myocardial infraction by introducing a therapeutic composition as described in any of ciaims 1 to 62 to an affected heart.

79. A method of treating chronic heart failure with scare by introducing a

iherapeutic composition as described in any of claims 1 to 62 to an affected heart.

80. A method of treating heart failure with low ejection fraction less than 35% by introducing a therapeutic composition as described in any of claims 1 to 62 to an affected heart.

81. A method of ireaimg coronary artery disease with regional wail abnormaiities b introducing a therapeutic composition as described in any of claims 1 to 82 to an affected heart.

82. A method of treating coronary artery infective endocarditis by introducing a therapeutic composition as described in any of claims 1 to 62 to an affected heart.

83. A method of treating an endarterectomy by applying a therapeutic

composition as described in any of claims 1 to 62 over a cut in the carotid artery,

84. A method of claims 73 to 83, wherein the therapeutic composition is

introduced through an inter-arterial artery,

85. A method of claims 73 to 83, wherein the therapeutic composition is

introduced through an intra-arteriaS artery.

86. A method of claims 73 to 83, wherein the therapeutic composition is

introduced through an intramyocardia!,

87. A method of claims 73 to 83, wherein the therapeutic composition is

introduced onto an epicedium.

88. A method of claims 73 to 83, wherein the therapeutic composition is

introduced through an intrarenal.

89. A method of claims 73 to 83, wherein the therapeutic composition is

introduced through an intracoronary.

90. A method of claims 73 to 83, wherein the therapeutic composition is

introduced through an intracarotid.

91. A method of claims 73 to 83, wherein the therapeutic composition is

introduced intravenously,

92. A method of claims 73 to 83, wherein the therapeutic composition is

introduced transbronchiai!y,

93. A method of claims 73 to 83, wherein the therapeutic composition is

introduced by a topical application,

94. A method of claims 73 to 83, wherein the therapeutic composition is

introduced by topical application and subsequent injection of a fluid component.