WO2015134936A1 - Amnion derived therapeutic compositions and methods of use - Google Patents

Amnion derived therapeutic compositions and methods of use Download PDF

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Publication number
WO2015134936A1
WO2015134936A1 PCT/US2015/019294 US2015019294W WO2015134936A1 WO 2015134936 A1 WO2015134936 A1 WO 2015134936A1 US 2015019294 W US2015019294 W US 2015019294W WO 2015134936 A1 WO2015134936 A1 WO 2015134936A1
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Prior art keywords
therapeutic composition
amniotic
fluid
therapeutic
amniotic membrane
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PCT/US2015/019294
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French (fr)
Inventor
Bruce WERBER
Terrell Suddarth
Christian Beaudry
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Amnio Technology Llc
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Priority to US201461949106P priority Critical
Priority to US201461949087P priority
Priority to US61/949,087 priority
Priority to US61/949,106 priority
Application filed by Amnio Technology Llc filed Critical Amnio Technology Llc
Publication of WO2015134936A1 publication Critical patent/WO2015134936A1/en
Priority claimed from EP15877280.6A external-priority patent/EP3242672A4/en
Priority claimed from US14/853,889 external-priority patent/US9814746B2/en
Priority claimed from US15/257,870 external-priority patent/US20160375064A1/en
Priority claimed from US15/381,044 external-priority patent/US10363278B2/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14Blood; Artificial blood
    • A61K35/16Blood plasma; Blood serum
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/44Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48Reproductive organs
    • A61K35/50Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/0019Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION, OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS, OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS, OR SURGICAL ARTICLES
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION, OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS, OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS, OR SURGICAL ARTICLES
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells

Abstract

Therapeutic composites are described for the treatment of joint, dermal, nerve and other orthopedic conditions. A therapeutic composite is a fluid that has micronized amniotic membrane particles and/or amniotic stem cells. A therapeutic composite may be a dispersion of micronized amniotic membrane combined with a fluid, such as plasma, saline, amniotic fluid, combinations thereof and the like. In another embodiment, the therapeutic composite is a mixture of micronized amniotic membrane particles combined with an amniotic rich stem cell fluid. An amniotic rich or concentrated stem cell fluid comprises at least 0.5 x 106 amniotic stem cells per milliliter of fluid or composition. A therapeutic composite may be used to treat any number of conditions through topical application, surgical introduction, and/or injection.

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. 81/949,106 to Amnio Technology LLC and U.S. provisional patent application no. 61/949,087 to Amnio Technology LLC, both filed on March 6, 2014 and entirety of both are incorporated herein by reference.

Field of the invention

[0002] The present invention relates to therapeutic compositions and method of use to treat orthopedic applications, dermal applications, including the treatment of wounds and scars.

Background

[0003] Amniotic membranes are being used in clinical trials to treat a wide range of conditions. Amniotic membranes are typically placed directly on a treatment location, such as a wound or scare, in many cases however, amniotic membranes Sack th proper architecture and cell viability to effectively provide the desired therapeutic responses, such as tissue regenerations, immunomodulation, anti-inflammatory and antifibroiic. 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 ceil viability. For example, many amniotic membranes are processed with a g!utaraidehyde which is known to significantly reduce celi viability. In many treatment applications, it is desirable to provide a high concentration and/or specific type or blend of stem cells, 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 fluid may comprises any number of celts, including stem cells, growth factors, proteins and the like. In one embodiment, a therapeutic fluid comprises an amniotic fluid that is aceSiu!ar. 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 fluid 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 fluid component.

[0005] In an exemplary embodiment, the therapeutic composition, as described herein, comprises a plurality of amniotic stem cells, and preferably at a high concentration, such as greater than 0.5 x 106 per milliliter of the therapeutic fluid component within the therapeutic composition. A therapeutic fluid component may also be ace!iu!ar, such as an aceliu!ar amniotic fiuid. An ace!luiar 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 simpl a fluid 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 fiuid component comprises a mixture of micronized amniotic membrane particles combined with an amniotic stem celt 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 cells in the therapeutic composite, as described herein, may b 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 cells such as plasma, Plasma Lyte-A, from Baxter Inc., saline and the like. The concentration of amniotic stems ceils in one milliliter of a fiuid component of an exemplar therapeutic composition, as described herein, may be about 0.5 x 10s or more, 1.0 x 10s or more, 5.0 x 106 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, mesenchymal stem cells, amniotic fluid stem cells, fibroblasts, proteins,

keritinocyfes, epithelial and/or epidermal cells, paratenacytes, keratinocytes, epithelial and/or epiderma! cells, paratenacytes, keratinocytes and growth factors, in some embodiments, protein markers for mesenchymal stem cells may be analyzed to quantify the various types of cells within the therapeutic composition. Flow cytometry may be used to identify proteins, CD44, CD105, GD73 and CD90. In one embodiment, a therapeutic composition comprises at least 30% of mesenchymal stem cells as identified by CD73. Mesenchymal stem ceils indicated by CD73 proteins may be more mobile and provide a more therapeutic effect that

mesenchymal stem cells identified by the other markers. A therapeutic fluid component, as described herein, may comprise anti-inflammatory nano-partides 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 multi otent progenitor cells and/or piuri potent progenitor cells. Progenitor cells may be derived from a patient to be treated, such as from a stromal vascular fraction. Vascular fraction cells and/or progenitor cells may be included with a therapeutic composite to further improve effectiveness. Progenitor cells may be autologous or allogeneic.

[00073 A fluid component, as described herein, may comprise particles and/or a concentration of amniotic stem cells. The partides 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 least 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 !yophilized have a great reduction in ceil 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 ma comprise stem cells and tissue that may substantially surround the stem cells. [0008] An amniotic membrane, or portion thereof, may be micronized while in a hyd rated state thereby improving the viability of ceils. The amniotic membrane particles may be derived from dehydrated and/or deoelluiarized amniotic tissue however. In addition, the amniotic membrane may be cryo-fractured, such as with a biunt 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 particle 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 Spm, no more than

Figure imgf000006_0001
no more than about 1pm, no more than about 0.5pm 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 (SE ). 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-miS!ing, 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 than about 10%, more than about 25%, more than about 50%, more than about 75%,o 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 mg/ml, more than about 5mg/ml, more than about lOmg/mS, more than about 50mg/ml, more than about lOOmg/ml, more than about 500mg/m!, and any range between and including the mass concentrations provided. The pariicies in the therapeutic composition may comprise collagen, growth factors, stem cells, amniotic stem cells, mesenchymal stem cells, progenitor celis, red blood ceils, white blood cells, proteins, fibroblasts, paratenacytes, keratinocytes and the like. [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 cell viability. Any suitable oxygen-carrier component or combination of components may be included into a therapeutic compositing including, but not iimited to, perfiuorocarbon such as perfluorotributyiamine (PFTBA), perfluorooctylbromide (PP08),

perfiuofodecy!bromide, perfiuoroperhydrophenanthrene and the tike. An oxygen- carrier may be bonded, such as cova!entiy 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

fiuofopoSymer, and an oxygen component is bonded thereto. Any 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 perfiuorocarbon 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 perfiuorocarbon phase.

100 i 1 j 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 be applied topically. The viscosity of the an Injectable fluid component may be no mor 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 50,000 mPa sec. In other embodiments, a fluid component may be provided for topical applications and the viscosity may 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.

[00123 In an exemplary embodiment, a therapeutic composition is a therapeutic composite and comprises any 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 material such as an amniotic membrane. In another embodiment, an amniotic membrane may be provided as a matrix component in a multilayered configuration or combined with any other suitable support layer for a desired application. For example, a therapeutic composite, as described herein, may comprise an amniotic membrane layer and a cover layer. A cover layer may be used to reduce 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 layer, 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 stit! another embodiment, a therapeutic composite comprises one or more layers of amniotic membrane that are tensilized, 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 ma 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 glutaraidehyde, radiation and the like. A therapeutic composite as described herein, may comprise anti -inflammatory nano-particles and/or statins, HMG-CoA reductase inhibitors to reduce inflation at a treatment location. An exemplary therapeutic composition, and in particular a fluid component, may comprise mannitol, saline, ringers lactate, vitamin 8 complex and the like,

[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 carrier fluid may be an amniotic stem cell concentrated fluid component. In an exemplary embodiment, the therapeutic 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 layer 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 ersabie a portion of the fluid component to be retained therein. Any suitable number and type of matrix o support layers may be configured in a therapeutic composite, as described herein, in one embodiment, a fiuid 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 vvi!i a!!ow 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), perfluoroaSkoxy (PFA) and the iike, or a metailic component, such as stainiess steel, titanium, gold and the like, A support layer may be porous and/or permeable. A support layer may be a membrane having a microstrttcture 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 o oieophobtc 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 pianar 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 deployable and removable balloon, !n 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 and/or second layer may comprise a fluid component and each layer may comprise a different composition of fluid component. A second matrix component layer may be substantially non-permeable to the fluid component thereby reducing wash-out o dilution of the fluid component from bodily fluid exposure.

10016] 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 fluid component as described herein. In an exemplary embodiment, stroma! vascular fraction is combined with micronized amniotic membrane and/or amniotic stem ceils to form a fluid component. In another embodiment, a stroma! vascular fraction is combined with a matrix component either before or after locating the matrix component over the treatment location. The stromal vascular fraction may contain any of the following: preadipocytes, mesenchymal stem cells (MSG), endothelial progenitor cells, T cells, 8 cells and mast cells as well as adipose tissue macrophages. In another embodiment, a therapeutic composition comprises bone marrow aspirate (BMA) 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, a exemplary fluid component comprising a concentrated amniotic stem ceil 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 ceil viability.

Table 1 : Donor Cell Vi ability Prior to Cell Viab Itty Post

Con trolled Rate Control! ed Rate

F ;reesing Free, dng

Sample 1 98.4% Sample 1 93.1%

1 Sample 2 98.5% Sample 2 9Q.Q

Sample 3 98.1% Sample 3 90.9%

Sample 1 97.3% Sample 1 93.1%

2 Sample 2 97.5% Sample 2 91.3%

Sample 3 94.7% Sample 3 92,5%

Sample 1 95.6% Sample 1 92,5%

3 Sample 2 95.1% Sample 2 95.5%

Sample 3 94,6% Sample 3 92.5%

[001 ] The viability of ceils was maintained after thawing a cryo-preserved concentrated amniotic fluid as reported in Table 1. A sma!! loss in viability was observed with a total viability after thawing a cryopreserved therapeutic composite of more than 90% in ail cases, A therapeutic composite, as described herein, may have a cell viability of about 70% or more, at least about 80% or more, about 00% or more and any range between and including the cell viability values provided,

[0020] An 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 regeneratson of new tissue. A fluid component of the therapeutic composition, as described herein, ma be injected directly into an affected area or introduced intravenously. It may be desirable to provide a fluid component comprising both amniotic stem cells 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 j A therapeutic composite, as described herein, may be used to treat any number of orthopedic conditions including, but not limited to, chondral defects, articular cartilage defects, arthritis, osteoarthritis, osteochondral defects or injuries, cartridge and tendon wear, tear and injury, tendinopathahies, i.e., tendonitis and tendinosis, and the like. For example, a therapeutic composite may be used to treat any Berndt & Hardy grading of osteochondritis dessicans, from undisplaced, partialiy detached, detached but not displace and detached and displaced or rotated. A therapeutic composite, as described herein, may be introduced to a joint, bone, tendon or cartilage to induce an immunomodulatory and/or a nti -inflammatory response. The therapeutic composite may be placed surgically or arthroscopicaliy into an affected area, such as a Joint, bursa, synovial membrane, synovium, intra articular, cartilage, tendon or between the tendon and partenon. In an exemplary embodiment, the therapeutic composite is introduced arthroscopicaSiy, or by small arthroiomy. The matrix component may be wrapped around a tendon with a defect, such as a tear, and the fluid component, as describe herein, may be injecting into or otherwise applied to the matrix component, prior to or after location of the matrix component on the tendon. A matrix component may be applied to a damaged or thinned area of cartilage or fractured, partialiy broken osteochondral injury, and subsequently the fluid component, as described herein may be applied to the matrix materiai. A matrix component may be imbibed with and or coated with a fluid component as described herein prior to positioning the therapeutic composite on a treatment location.

[0022] A therapeutic composite, as described herein, may be used to treat any number of skin conditions and/or injuries including, but not limited to, keloid scaring, cuts, abrasions, infections, boils, surgical incisions and/or tumor removal, psoriasis, plastic surgery and the like. A therapeutic composite, as described herein, may be introduced into to treatment location to promote tissue regeneration and to reduce scaring. In one embodiment, a therapeutic composite is placed over an operative incision upon closure of the incision and may be place on the interior surface of the incision, and/or the exterior of the incision. Again, the inner wail of th incision may be treated with a therapeutic composite that incorporates both a matrix component and a fluid component, as described herein. The therapeutic composite may be antifibrotic and thereb reduce scarring.

[0023] In one embodiment, a therapeutic composition is used to treat keloids by application of a therapeutic fiuid to the keloid scar intra!esional!y and/or subcutaneous to the keloid scar. Application of the therapeutic fiuid ma be provided periodically to reduce the size and severity of the ke!oid scar through tissue regeneration and reformation. Any suitable dose of therapeutic fluid, as described herein may be used to treat a keloid scar.

[0024] A therapeuiic composition may be used to in breast reconstruction, or an piastic surgical repair. Strips of a therapeutic matrix component may be applied along exposed organs, nerves, and fascial planes. Typically, hydrostatic tension will hold the matrix component in place. However, in some anatomic locations, hydrostatic tension may not provide sufficient adhesion, A surgical glue or sutures may be used to retain the matrix component in position.

[0025] A therapeutic composition, as described herein, may be used to treat a nerve injury through injection of a therapeutic fluid in dose proximity to a damaged nerve or throug application of a matrix component to an exposed nerve, through surgery. A nerve may be exposed configured in a proper anatomic alignment and a matrix component may be configured around the damaged nerve to form a tunnel. Subsequently, a fluid component may be injection into the matrix component or into surrounding tissue. In one embodiment, a pudendal nerve may be treated with a therapeutic composition after prostrate surgery, for example.

[0026] A therapeutic composite, as described herein, may be used to treat digestive system conditions including application to reduce scarring of operative procedures including a stomach reduction or stomach by-pass surgery and to prevent intestinal strictures,

[0027] A therapeutic composite, as described herein, may be used to treat reduce scarring post piastic surgery, whereby the therapeutic composite is placed on the inside surface of the dermal tissue to reduce scarring. For example, when an incision is made in the abdomen, a therapeutic composite may be place on interior of the abdominal wall before closure of the incision,

[0028] A therapeutic composition as described herein may be used to treat burns, including second and third degree burns, A therapeutic composition, as described herein, may be applied over a burn location to reduce post burn scarring and to accelerate dermis and epidermal regeneration. A therapeutic fluid, or fluid component, as described herein, may be injected into and/or around a burn location. An exemplary fluid component for treatment of burns comprises amniotic fiuid and micronized amniotic membrane, such as 1 ml of fluid per about 8cm2 of micronlzed amniotic membrane. In an exemplary embodiment, injections of a fluid component are made around a bum location and a matrix component of amniotic membrane is placed over the burn. The amniotic membrane may comprise a therapeutic component, whereby the membrane is imbibed with or coated with a fluid component before application to the burn location. In an alternative embodiment, a fluid component may be sprayed onto the burn location with care being taken to prevent leaking or run-off of the fluid component from the burn location. A dressing may be positioned around the burn prior to spraying to catch any run-off. A fluid component may be applied to a burn, such as my injection or spraying, subsequent to the initial treatment. The severity of the burn or wound and the response of the patient to the treatment may dictated the time intervals} for additional application of fluid component.

[0029 J A venous stasis wound may be treated by the application of a matrix component comprising amniotic membrane and/or the application of a fluid component through injection, spraying and/or coating on the matrix component,

[0030] A therapeutic composite, as described herein, may be used to induce an immunomodulatory and/or anti-inflammatory response in an eye. For example, a therapeutic composite ma be used to reduce scaring and promote healing by application to the cornea. A fluid component may be applied periodically to a matrix component that is located on a portion of an eye. in addition, fluid component may be vacuum imbibed into a matrix component to produce a more transparent therapeutic composite. Vacuum imbibing of a fluid component info a matrix component may greatly Increase light transmission as many matrix components, including amniotic membranes are typically translucent,

[0031] A therapeutic composition, as described herein, may be used to treat muscle tears or muscle traumatic loss. An inflammatory signal may be created by needling techniques, use of infrared diode laser, radial pulse generator to the area, extra corporeal Shockwave i.e. dornier epos, epai device or surgical repair.

Therapeutic fluid may be administered by injection into the muscle treatment location. A second dose or treatments may be applied in approximately four to six weeks depending on patient response and recover.

[0032] A therapeutic composition, may be used to treat chronic tendiopathy, fascicpathy, and the like. Ultrasound imaging may be used to diagnosis the condition and to better understand the microvascular architecture of the tendon to be treated. An inflammatory signal may be created as described herein. Therapeutic fluid may be administered using ultrasound guidance around the paratenon of the injured tendon or fascia. Non-steroidal medications, steroid medications, and/or disease modifying medications may be discontinued, A second dose or treatments may be applied in approximately four to six weeks depending on patient response and recover,

[0033] A therapeutic composite, as described herein, may be used equine conditions including tendinitis, tendinosis, tendinopathy, osteoarthritis, iaminitis, A therapeutic composite may be used any of the ways described herein.

{ 0034] 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. An initial dose of fiuid component may be provided with a matrix component upon initial application of the matrix component to the treatment iocaiion. Subsequent doses of fluid component may be administered thereafter as required, and may be applied topically, such as in wound applications, or through injection, such as in deep applications, in one embodiment, a fluid component is injected into a specific treatment location through the use of a catheter, such as a steerable catheter and an injection implement configured on the introductory end of the catheter.

[0035] 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] 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

[0037] Figure 1 shows a cross-sectional diagram of amniotic membrane surrounding a fetus in utero. [0038] Figure 18 shows a cross-section diagram of the layers of the amnion and chorion.

[0039] Figure 2A show a transmission electron micrograph (TEM) of the epithelium layer of the amniotic membrane having a singie layer of amniotic stem cells. The TEM is at 2500 X magnification.

[0040] Figure 2B show a TE : of the epithelium iayer of the amniotic membrane having a single layer of amniotic stem ceils. The TEM is at 8200 X magnification.

[0041 ] Figure 3A is a scanning electron micrograph (S£M) of an amniotic membrane having amniotic stem cells.

[0042] Figure 3B is a SE of cryo-fractured amniotic membrane particles.

[0043] Figure 4 is a scanning electron micrograph (SEM) of an amniotic membrane having pores between the amniotic membrane tissue.

[0044] Figure 5A is a representation of an exemplary tensilized amniotic membrane.

[0045] Figure 58 is a representation of two exemplary tenssiized amniotic membranes being layered together.

[0046] Figure 8 shows a diagram of an exemplary method to apply a therapeutic composition, as described herein.

[0047] Figure 7 shows a diagram of a process to produce a fluid component comprising micronized amniotic membrane particles.

[0048] Figure 8 shows a diagram of a process to produce a fluid component comprising a concentrated stem ceil fluid.

[0049] Figure 9 shows a cross-sectional representation of an exemplary amniotic membrane configured over a treatment location.

[0050] Figure 10 shows a cross-sectional representation of an exemplary therapeutic composition comprising an amniotic membrane and fluid component configured over a treatment location.

[0051 ] Figure 11 shows a cross-sectional representation of an exemplary therapeutic composite configured over a treatment location wherein the therapeutic composition comprises an amniotic membrane matrix component imbibed with a fluid component and a cover layer configured there over.

[0052] 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 layer of amniotic membrane! a second matrix layer of a fluid component reservoir, and a third matrix layer that is a cover layer,

[0053] Figure 13 shows a cross-sectional representation of an exemplary therapeutic composite configured over a treatment location wherein the therapeutic composite comprises a first matrix layer of amniotic membrane imbibed with fluid component and a second matrix layer that is a support layer comprising

bioresorbable material.

[0054] Figure 14 shows a cross-sectional representation of an exemplary iherapeutic composite configured over a treatment location wherein the therapeutic composite comprises a first matrix layer of amniotic membrane imbibed with fluid component, a second matrix layer that is a support layer and a third matrix layer that comprises amniotic membrane,

[0055] Figure 15 shows a knee joint having an exemplary therapeutic composite configured therein and a syringe injecting fluid component into the matrix component.

[0056] Figure 16 shows an exemplary therapeutic composite configured around a constrictabie body part, and a fluid component being injected therein.

[0057] Figure 17 shows an exemplary fluid component being drawn from an enclosure by a syringe,

[0058] Figure 18 shows flow cytometry analysis data for amniotic fluid as received and amniotic stem cell concentrated fluid.

[0059] Figure 19A shows a picture of a wound on a diabetic person's foot prior to treatment.

[0060] Figure 19B shows a picture of the wound shown in FIG. 19A after 57 days of treatment with an exemplary therapeutic composite as described herein.

[00611 Figure 20A shows an x-ray of an osteochondral defect in an ankle, prior to treatment.

[0062] Figure 208 shows an x-ray of an osteochondral defect in an ankle, prior to treatment.

[0063] Figure 21 shows a cross-sectional view of an eye.

[0064 ] Figures 22A-22C show black and white photographs of a wound on a heel and the progression of healing with application of a therapeutic composition, as described herein. [0065] Figure 23A shows a biack and white photograph of a patient with late- stage fibrosis and Figure 23B shows the therapeutic effect of application of a therapeutic composition to the scar.

[0066] Figures 24A and 24B show X-rays of a patient with arthroscopy, chondroplasty and Figures 24C and 24D shows X-rays of said patient six months post operation in which a therapeutic composition was applied,

[00673 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 details 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.

[00683 s L,se herein, the terms "comprises," "comprising," "includes," "including," "has," "having" or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of elements is not necessariiy 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 genera! sense of the scope of the invention. This description should be read to Inciude one or at least one and the singular also includes the p!ura! unless it is obvious that it is meant otherwise.

[0069] 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.

[0070] As shown if FIG. 1 A the amniotic membrane surround a fetus in utero. As shown in FIG. 18, 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.

£0071 3 As shown in FIGS, 2A and 2B, the epithelium layer of the amniotic membrane 20 has a single iayer of amniotic stem cells 46. The tissue around the amniotic stem ceils may protect and enhance the viability of these stem cells when the epithelium is cryo-fractured to produce particles for the therapeutic composition.

[0072] As shown in FIG. 3A, an amniotic membrane 20 comprises a plurality of amniotic stem cells 46.

[00733 As shown in FIG, 3B, 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 2pm. There are no particles shown that are larger than 2 m and substantially all of the particles are less than 1 pm in size. The SEM shows that the micronszed amniotic membrane particles are irregularly shaped. As shown, some of the particles have a planar surface,

[0074 j 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 tensiSize the tissue. A fertilized amniotic membrane may have a higher matrix tensile strength than an original un-tensilized amniotic membrane. In addition, a plurality of layers of amniotic membrane may be utilized to build strength in one or more directions.

[0075] As shown in FIG. 5A, an amniotic membrane 20 has been stretched in one direction to form an elongated and mor aligned amniotic tissue orientation. As shown in FIG. 5A, 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 tensiiizing. The tensilized 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 iensiiized amniotic membrane may be stretched over a long 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, 8 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 hydrated and or submerged in amniotic fluid or a p!asticizing fluid. An amniotic membrane may be cross-Ssnked after being stretched. The load applied to tensliize an amniotic membrane may be a portion: of the maximum tensile load required to fracture the amniotic membrane at a rate of i Omm/second for a 2.54cm by 15.2cm sample having a 5cm gap. For example, a tensilizing ioad applied may be no more than about 80%, no more than about 6G%, no more than about 50%, no more than about 25% of the maximum tensile load.

[0076] As shown in FIG, 56, 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 aiiemative embodiment, a plurality of layers of iensiiized 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 tensiiized amniotic membrane.

[0077] Figure 6 shows a diagram of an exemplary method to apply a therapeuiic 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.

[0078] As shown in FSG. 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 biunt 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 cell fiuid and amniotic stem cells, in another embodiment, the micronized particles are dispersed in a concentrated amniotic stem cell fiuid. [0079] 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 exempSary embodiment, as described in FIG. 5, the amniotic fluid is cenfrifuged to remove debris and excess liquid and concentrate the amniotic stem cells in the therapeutic composition.

[00803 As shown in FIG. 9, an exemplary therapeutic composition 11 is a therapeutic composite 10. The therapeutic composite 10 comprises an amniotic membrane 20, as a matrix component ^configured over a treatment location 18. The matrix components 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.

[00813 As shown in FIG.10, an exemplary therapeutic composite 10 comprises an amniotic membrane 20 and a fluid component 1 imbibed therein, configured over a treatment location 18. The fluid component 14 comprises mlcronized amniotic membrane particles 40 and amniotic fluid 43. However, any suitable fluid carrier may be used to disperse the micronized amniotic membrane particles and or amniotic stem cells 48.

[0082 j As shown in FIG. 11 , an exemplary therapeutic composite 10 is configured over a treatment location 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 iayer is configured over said first matrix Iayer and comprises an overhang portion 38 that extends outside of the first matrix iayer. The second matrix layer is attached to the tissue 19 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 location, whereby a outer area of the matrix component can be affixed to tissue. A cover layer may fully cover a first or 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 poSytetraf!uoroethylene membrane may have a mean flow pore size as measure by a Coulter Porometer (PM! Industries}, of less than 50um, less than 40um, less than 10um, less than 1um and any range between and including the pore sizes provided. In one embodiment, fhe pores are sized to a!Sow fluid to flow but retain cells, such as stem cells within the matrix component.

[0083] 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 matrix 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 porosit containing a fluid component 14', as described herein. As shown, a first fluid component 14 is configured within the first matrix layer 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 cells may be drawn from the reservoir iayer as they are needed.

[00843 As shown in FIG, 13, a therapeutic composite 10 is configured over a treatment location 18 wherein the matrix component 12 comprises a first matrix layer 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 thai 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 be hydrophobic to reduce fluid ingress into the therapeutic composite. Bodily fluid ingress into a therapeutic composite may dilute a fluid component comprises therein.

[0085] 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 layer 30 of amniotic membrane 20 imbibed with fluid component 14, a second matrix layer 32 that is a support iayer 22 and a third matrix Iayer 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.

[0086] As shown in FIG. 15, a knee joint has an exemplary therapeutic composition 11 configured therein comprising a matrix component and a fluid component 14, therein forming a therapeutic composite 10, A syringe 80 is injecting a fluid component 14 into the matrix component 12. As described herein, a fluid component may be applied into or around a matrix component during or after an initial therapeutic procedure to position a matrix component on a treatment location. The fiuid component 14 shown in FIG, 15 comprises mscronized amniotic membrane particles 40, and amniotic stem cells 46 dispersed in a fiuid 42. A syringe may be used to inject a fluid component periodically after an initial procedure,

[0087] As shown in FIG, 16, an exemplary therapeutic composite 10 is configured around a constrictabie body part 80, such as a nerve or tendon, for example, and a fiuid component 14 is being injected therein. This type of procedure may reduce and/or eliminate strictures. 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 portion of the urinar or digestive system. In another embodiment a matrix component sheet is applied externally over a treatment location in a patient's dermal tissue.

[0088] Figure 17 shows an exemplary fluid component 14 being drawn from an enclosure 70 by a syringe SO. 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 iarger than a 20 gauge needle.

[0089] Figure 19 shows flow cytometry analysis data for amniotic fluid as received and amniotic stem ceil concentrated fluid as described herein. Flow cytometry was performed on four different liquid samples from different donors. The analyses shows that the expression level of mesenchymal stem cell surface antigens is consistent between donors with CD44 being positive and CD73 being strongly positive while CD90 and CD105 are low positive. The level of expression is maintained between the processed samples concentrated sample 1 and

concentrated sample 2 and unprocessed samples {Fresh Amniotic Fluid 1&2), suggesting no eel! loss during the manufacturing process and preservation of potency. What is also interesting is that CD73 is expressed the most, it has been reported that mesenchyma! stem cell migration ss controlled by CD73 and therefore if is speculated that a high level of CD73 expression promotes cell migration and the ability of the cells to home to tissue sites of repair or to participate in healing responses.

{ 0090] As shown in FIG. 19A a wound on a diabetic person's foot prior has a length of approximately 11mm and width of approximately 7mm. As shown in FIG. 19B, the wound has healed considerably after 57 days of treatment with an exemplary therapeutic composite as described herein. A therapeutic composite of amniotic membrane was placed over the wound along and a fluid component comprising micronlzed amniotic membrane and a concentrated amniotic stem ceil fiuid was applied topically. The fluid component described was applied topicaily over a 57 day period, over which the wound healed as shown in FIG. 198

[0091] As shown in FIG. 20A a patient has an osteochondral defect in an ankle, with some bone degradation. A therapeutic composite was applied over the defect and a fluid component was then applied to the treatment site.

[0092] Figure 2GB shows the improvement in the defect including tissue regeneration and reduction of the defect area and volume.

[0093] Figure 21 shows a cross-sectional diagram of an eye and some of the treatment locations for a therapeutic composite, as described herein. For example, a therapeutic composite 10, as described herein, may be applied topically over the cornea and a fluid component may be added periodically to promote healing and reduce scaring.

[0094] As shown in FIG 22A, a patient had a deep cut in their heel. A therapeutic composition comprising a fluid component comprising amniotic fluid and micronized amniotic membrane particles, as generally described in Example 1 , was injected around the cut. The wound was fully evaluated to insure no osteomyelitis. The wound was cleaned to remove any debris and then covered with a dressing. The therapeutic composition was injected in approximately equal amounts around the periphery of the cut. Approximately 1m! of therapeutic composition having approximately 8sq cm of micronized membrane was mixed with an equal volume of saiine. Four injections were made, about equally spaced, around the perimeter of the wound. The injections were made approximately 6 to 8mm from the edge of the wounds and in a direction toward the wound. About 0.5ml of the therapeutic composition and saiine mixture was dispensed in each injection location. The wound was then covered with a non-stick dressing,

[0095] After approximately 30 hours, the dressing was removed from the wound and the wound was washed and covered with a new dressing.

[0096] The wound shown in FIG, 22B was substantially dosed after only 13 days from the injection. However, in the event that the wound in not closed another injection may be administered as need, such as after 5 days, or after 10 days o after 20 days.

[0097] In an alternative embodiment, an amniotic membrane may be applied over a cut or wound and a fluid component, such as that described in Example 1 , may be injected around the wound as described herein.

[0098] Figure 23A shows a black and white photograp of a patient with late- stage fibrosis and Figure 23B shows the therapeutic effect of application of a therapeutic composition to the scar. FIG 23A, shows an image of a person's chest having an involuted scar at the Iocation of a previous tube insertion. The patient had a iot of pain from this scar and internal adhesions, A therapeutic fluid was injected into the scar area and the scar adhesions were released, the tissue was remodeled, and pain was reduced. Figure 238 shows the dramatic improvement of the scar.

[0099] Figures 24A and 24B show X-rays of a patient with stage 4

osteoarthritis. Figures 24C and 240 shows X-rays of said patient 6 months post anthroscopy chondropiasty procedure in which a therapeutic composition was introduced to the joint. Figure 24A shows pre-operative image of the tibia, talus and the calcaneus bone o bone affected area. After application of a therapeutic composition, as described in detail in Example 2, the joint between was widened and restored as shown in FIG. 24D and 24D.

[00100] 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

Example 1 00101 } Three fiuid components were made and ce!! viability was measured as reported in Table 1. Three amniotic membrane samples, obtained from three separate donors, were cryo-fractured and dispersed in fluid io create a fluid components, as described herein.

f 00.1021 A fluid component of the therapeutic composite was prepared by concentrations amniotic stem cells in a ceil suspension solution. A 1mi sample of an unprocessed amniotic fluid was used to measure initial ceil count and viability. The amniotic fiuid was then separated into 50ml steril centrifuge tubes and centnfuged two times at 400xg for 10 minutes at ambient temperature. Cell pellet from each tube was washed with 20ml of Plasma Lyte-A, from Baxter inc., between

centrifugation. Supernatant was removed and cells were re-suspended in a predetermined volume of cell suspension solution, Plasma Lyte-A, to obtain a final product ceil concentration of 1 x 106 cell/ml.

[00103] Cryo-fractured particles of amniotic membrane were prepared for dispersion in the fiuid component. Three amniotic membranes were obtained and rinsed using Plasma Lyte-A and transferred to a cutting board. Using blunt 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 steef ruier. The amount of amniotic membrane needed to obtain a concentration of 1cm2/mi of therapeutic solution was retained and placed on a sterile drying rack and allowed to dry for one hour. Th amniotic membrane was then cut into small pieces, less than a 1cmk' and placed Inside a milling chamber containing a blunt impactor. The cryo-miii used was from SPEX Sample Prep Inc., 6970EF Enclosed Freezer/Mill Mode! 6970D.

[00104] The milling chambers were placed inside the cryomill and the amniotic membrane was micronized. The frequency of the impactor was S cycles per second, the precooling time was five minutes, the grinding time was three minutes and the intermediate cooling time was two minutes. After the micronlzation of the amniotic membrane was complete, the chambers were removed from th cryomill 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 final therapeutic composite was prepared by combining 100ml or the fluid component and micronized amniotic membrane with equal volume (100m!)t of cryprotectant solution, CryoStor 10, available from Sigma-Aldrich. Using a repealer pipet, cryoviais were then filled at the desired volume. The therapeutic solution was maintained at 4°C during the vial filling process to preserve ce!i viability.

OOtOS j 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°C/min until chamber reached - G°C, warm at a rate of 10.0°C/min until chamber reached -12°C, cool at rate of 1.0°C/rnm until chamber reached -40°C, and coo! 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,

{00106] Thawing of the cryoviais was performed and ceil viability was again measured. Ceil viability pre and post cryopreservation Is reported in Table 1. The cryoviais 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 1 ml sample. An equal amount of cold Plasma 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. Cell count and viability was assessed using Trypan Blue

Example 2: Prophetic example

{00107] Osteoarthritis is treated with a therapeutic composition, as described herein. Arthroscopic synovectomy was performed on the joint to remove debris, excess synovium and repair ligamentous instability. Arthroscopic chondrop!asty was performed around to remove any free floating or unstable osteo-cartilage fragments. The subchondral bone was perforated with a drill or kirschner wire {.035inched or less) multiple times around and in the area of cartilage injury, allowing bone bleeding. Small perforations in the subchondral plate was necessary as larger disruptions will cause irregular Joint cartilage to develop. The arthroscope was removed, and the portal to the joint was enlarged, converting to a small arthrotomy. An amniotic membrane was wrapped around an insert instrument, and after the insert instrument was inserted into the joint, the amniotic membrane was unfurled into the joint. The amniotic membrane was positioned over the injured and perforated cartilage. The insert instrument was removed and the portal to the joint was closed with traditional surgical techniques. After the portal was closed, a fluid component, comprising 1 m! of micronized amniotic membrane In concentrated amniotic fluid having viable amniotic stem cells and 1m! of saline, was injected into t e operaied joini using a 18-22 gauge needle on a syringe. X-rays of the affected joint before this procedure are shown in FIGS. 24A and 24B. X-rays were taken of six months after the operation and the joint was widened and the tissue was remode!ed.

Definitions;

[00108] icronized particles, such as micronized amniotic membrane particles, as used herein, means that the particles have an average particle size of iess than 1pm, and in some cases have an average particle size of iess than Q,5pm. Particle size may be measured by analysis of scanning electron micrographs

[00 S 09] it wi!i 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 thai 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 i , 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 cells of at least 0,5 x 10b per milliliter of said fluid component.
4. The therapeutic composition of claim 1 , wherein the fluid component
comprises a concentration of amniotic stem 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 cells of at least 5 10δ 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 10s per milliliter of said fluid component.
7. The therapeutic composition of claims 1 , wherein the fluid component
comprises an aceiiular fluid component.
8. The therapeutic composition of claims 1 , wherein the fluid component
comprises an aceiiular amniotic fluid.
9. The therapeutic composition of claims 1 , wherein the fluid component consists of an aceiiular amniotic fluid.
10. The therapeutic composition of claims 1 , wheresn the fluid component has a viscosity of no more than about 50 Pa sec.
11.The therapeutic composition of daims 1 , wherein said fluid component is a paste having a viscosity of more than about 50 Pa sec.
12. The therapeutic composition of claims 11 wherein the fluid component
comprises a plurality of protein markers including CD44, CD1G5, CD73, and CD30 proteins.
13. The therapeutic composition of claims 1 , wherein the fiuid component
comprises: a, growth factors; and
b, proteins.
14. The therapeutic composition of claims 1 to 13, further comprising micronized amniotic membrane particles.
5. he therapeutic composition of claims 14, wherein the particles consist essentially of micronized amniotic membrane particles.
16. The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles consist essentially of amnion and are essentially free of chorion.
17. The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles consist essentially of epithelium.
18. The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles consist essentially of epithelium and amnion basement membrane.
19. The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles consist essential!y of epithelium, amnion basement membrane and compact layer.
20. The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles consist essentially of epithelium , amnion basement membrane, compact layer and fibroblast layer.
21. The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles are hyd rated and have a percent hydration of a least 20 percent.
22. The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles are decelluSan'2ed particles.
23. The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles have an average particle size of no more than about 250pm.
24. The therapeuiic composition of claim 14, wherein the micronized amniotic membrane particles have an average particle size of no more than about 150pm,
25. The therapeutic composition of claim 14, wherein the micronized amniotic membrane particles are irregularly shaped.
26. The therapeutic composiiion 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/mS of therapeutic composition.
28. The therapeutic composition of ciaim 14, having a concentration of micronized amniotic membrane particles of at least about 5.0mg/mi of therapeutic composition.
29. The therapeutic composition of claim 14, having a concentration of micronized amniotic membrane particles of at least about IQ.Omg/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 claims 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 essentially free of chorion.
33. The therapeutic composition of claim 31 , wherein the amniotic membrane consists essentially of epithelium.
34. The therapeutic composition of claim 31, wherein the amniotic membrane consists essentially of epithelium and amnion basement membrane.
35. The therapeutic composition of claim 31, wherein the amniotic membrane consists essentially of epithelium, amnion basement membrane and compact layer.
36. The therapeutic composition of claim 31 , wherein the amniotic membrane consists essentially of epithelium, amnion basement membrane, compact layer and fibroblast layer.
37. The therapeutic composition of claim 31„ wherein the amniotic membrane is hydrated and has a percent hydration of at feast 20 percent.
38. The therapeutic composiiion 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 s wherein the amniotic membrane is tensilized bi~axtaiiy.
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 iayer is configured for placement over said first iayer of amniotic membrane.
43. The therapeutic composition of claim 41, wherein the second matrix
component layer is a amniotic membrane.
44. The therapeutic composition of claim 41 , wherein the second matrix
component layer is a bioresorbable materiai.
45. The therapeutic composiiion of claim 41 , wherein the second matrix
component Iayer is a non-bioresorbabie polymer material,
46. The therapeutic composition of claim 41, wherein the second matrix
component iayer is substantially ποπ-permeab!e to a fiuid component retained in the first Iayer of amniotic membrane.
47. The therapeutic composition of claim 41 , wherein the second matrix
component layer is a fiuid reservoir layer, whereby said second matrix component Iayer has porosity that is at least 50% filled with a fluid
component.
48. The therapeutic composition of claim 41, wherein the second matrix
component iayer is a support layer, having a tensi!e break strength of at ieast two times that of a first iayer of amniotic membrane tensiie break strength,
49. The therapeutic composition of claim 41 , wherein the first iayer of amniotic membrane is a tensiiized amniotic membrane and a second matrix
component Iayer a tensiiized amniotic membrane.
50. The therapeutic composition of claims 1 to 49, further comprising a plurality of progenitor ceils. 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 cells derived from vascular fraction cells,
53. The therapeutic composition of claims 1 to 49, further comprising
preadipocytes, mesenchymal stern cells and endothelial progenitor cells from a stromal vascular 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 i to 49, further comprising an oxygen-carrier component.
58. The therapeutic composition of claim 57, wherein the oxygen-carrier
component comprises a perf!uorocarbon,
59. The therapeutic composition of claim 57. wherein the oxygen-carrier
component comprises a perfiuoroperhydrophenanthrene.
80. 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 perfiuorocarbon and wherein the fluid component is an emulsion.
02. The therapeutic composition of claim 1 to 61 , wherein the therapeutic
composition is a thawed therapeutic composition from a cryopreserved state.
83. A process of making a therapeutic composition comprising the steps of:
a. providing an amniotic membrane from a donor;
b. cutting a portion of said amniotic membrane into membrane fragments and retaining an amniotic membrane matrix component portion;
c. cryo-fracturing said membrane fragments to produce a plurality of particles consisting essentially of mieronized hydrated amniotic membrane; d. dispersing said particles with a fluid to produce a fiuid component; e. combining said fluid component with said amniotic membrane matrix component portion to produce a therapeutic composite.
84, The process of ciaim 83, wherein the step of cryo-fracturing said membrane fragments comprises the steps of:
a. reducing the temperature of the membrane fragments to no more than -50°C
65. The process of ciaim 63, wherein the step of cryo-fracturing said membrane fragments comprises the steps of:
a. contacting said membrane fragments with a blunt fracturing implement
66, The process of claim 63, further comprising the step of washing said amniotic membrane with a PH neutral saline solution.
67, The process of claim 62, wherein the fiuid comprises an amniotic fluid
comprising amniotic stem cells, whereby said amniotic fluid is derived from the donor.
68. The process of claim 63, further comprising the step of concentrating the amniotic stem ceils in the amniotic fluid to a concentration of at least 0,5 x 10& milliliters.
89. The process of claim 63, further comprising the step of cr opreserving said therapeutic composite,
70. The process of ciaim 63, further comprising the step of thawing the
cryopreserved therapeutic composite to produce a thawed therapeutic composite whereby said thawed therapeutic composite comprises at !east 0.5 x 106 viable amniotic stem cells per ml of fluid.
71. The process of claim 83 to 70 further comprising the step of adding a stromal vascular fraction.
72. A process of making a therapeutic composition comprising the steps of:
a. providing an amniotic membrane from a donor;
b. providing a amniotic fiuid comprising amniotic stem cells, whereby said amniotic fluid is from said same donor; c, concentrating said amniotic stem cells to produce an amniotic stem cell concentrated fluid having a concentration of said amniotic stem ceils of at least 0.5 x 106 per ml of said fluid, to produce a fluid component; d. combining said fluid component with said amniotic membrane to
produce a therapeutic composite.
73. The process of ciaim 72, wherein the fluid component comprises a
concentration of amniotic stem cells of at least 1 x 106 per milliliter of said fluid component.
74. he process of claim 72, wherein the fluid component comprises a
concentration of amniotic stem cells of at least 5 x 10s per milliter of said fluid component.
75. The process of ciaim 72. wherein the fluid component comprises a
concentration of amniotic stem cells of at least 10 x 106 per milliliter of said fluid component.
76. The process 0f ciaim 72, further comprising the steps of vacuum imbibing the fluid component into the matrix component.
77. The process of ciaim 72, further comprising the step of washing said amniotic membrane with a PH neutral saline solution.
78. The process of claim 72, wherein the step of concentrating the amniotic fluid comprises the step of eentrifuging said amnioiic fluid.
79. The process of ciaim 72, further comprising the step of cryopreserving said therapeutic composite.
80. The process of ciaim 79, further comprising the step of thawing the
cryopreserved therapeutic composite to produce a thawed therapeutic composite that is a fluid, whereby said thawed therapeutic composite comprises at least 1 x to6 viable amniotic stem ceils per ml of said thawed therapeutic composite,
81. The process of ciaim 72 to 80 further comprising the step of adding a stromai vascular fraction.
82. A method of treating chondral defects by introducing any suitable therapeutic composition as described in any of claims 1 to 82 to a joint.
83. A method of treating arthritis by introducing any suitable therapeutic composition as described in any of claims 1 to 62 to a joint,
84. A method of treating osetoarthritis by introducing any suitable therapeutic composition as described in any of claims 1 to 62 to a joint.
85. A method of treating articular cartilage defects by introducing any suitable therapeutic composition as described in any of claims 1 to 82 to a portion of said cartilage,
88. A method of treating tendon defects by introducing any suitable therapeutic composition as described in any of claims 1 to 62 to a portion of said tendon.
87. The method of ciaims 88 wherein the therapeutic composite is configured around a tendon.
88. A method of claims 82 to 87, wherein the therapeutic composite is introduced arthroscopically.
89. A method of claims 82 to 87, wherein a matrix component is introduced to a treatment location and a fluid component is subsequently introduced into said matrix component.
90. A method of claims 82 to 87, further comprising the step of stimulating the treatment location.
91.The method of claim 90 wherein the step of stimulation is selected from the group consisting of; laser, needling, extra corporeal Shockwave therapy, radial pulse therapy, and activ rehabilitation .
92. A method of treating equine tendinitis by introducing any suitable therapeutic composition as described in any of claims 1 to 62 to an affected tendon.
93. A method of treating equine tendinopathy by introducing any suitable
therapeutic composition as described in any of claims 1 to 62 to an affected tendon.
94. A method of treating equine osteoarthritis by introducing any suitable
therapeutic composition as described in any of claims 1 to 62 to an affected Joint
95. A method of treating Saminitis b introducing any suitable therapeutic
composition as described in an of claims 1 to 62 to an affected hoof.
96. A method of treating a damaged nerve by introducing any suitable therapeutic composition, as described in any of ciaims 1 to 82 to a damaged nerve.
97. The method of claim 96 wherein the therapeutic composition comprises a matrix component that is wrapped around the damaged nerve,
98. The method of ciaim 96, wherein the nerve is a pudendal nerve,
99. A method of treating an ocuiar condition by introducing any suitable
therapeutic composition as described in any of ciaims 1 to 62 to an eye.
100. A method of inducing immunomodulation to an eye by introducing any suitable therapeutic composition as described in any of claims 1 to 82 to a portion of an eye.
101. A method of inducing an anti-inflammatory response of a portion of an eye by Introducing any suitable therapeutic composition as described in any of claims 1 to 62 to a portion of an eye.
102. A method of claims 99 to 101, wherein the therapeutic composite Is introduced topically to an eye.
103. A method of claims 99 to 101, wherein the therapeutic composite is introduced into an intraocular.
104. A method of ciaims 99 to 01 , wherein the therapeutic composite is introduced by a topical application.
105. A method of claims 99 to 101, wherein the therapeutic composite is introduced by topical application and subsequent injection of a fluid
component.
PCT/US2015/019294 2014-03-06 2015-03-06 Amnion derived therapeutic compositions and methods of use WO2015134936A1 (en)

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PCT/US2015/050046 WO2016111726A1 (en) 2014-06-15 2015-09-14 Amnion derived therapeutic composition and process of making same
US15/257,870 US20160375064A1 (en) 2014-06-15 2016-09-06 Amnion derived therapeutic compositions and methods of use
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