(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2004/0048796 A1
`(43) Pub. Date:
`Mar. 11, 2004
`Hariri et al.
`
`US 20040048796A1
`
`(54)
`
`(76)
`
`COLLAGEN BIOFABRIC AND METHODS OF
`PREPARATION AND USE THEREFOR
`
`Inventors: Robert J. Hariri, Florham Park, NJ
`(US); Aleksandr M. Kaplunovsky,
`RockaWay, NJ (US); Patricia A.
`Murphy, Hillsborough, NJ (US)
`
`Correspondence Address:
`JONES DAY
`222 EAST 41ST STREET
`NEW YORK, NY 10017 (US)
`
`(21)
`(22)
`
`Appl. No.:
`
`10/397,867
`
`Filed:
`
`Mar. 26, 2003
`
`Related US. Application Data
`
`(63)
`
`Continuation-in-part of application No. 10/106,653,
`?led on Mar. 26, 2002.
`
`Publication Classi?cation
`
`(51)
`
`Int. Cl.7 ........................... .. A61K 38/39; A61K 9/70
`
`(52) US. Cl. ........................... .. 514/12; 424/443; 442/123
`
`(57)
`
`ABSTRACT
`
`The present invention relates to collagenous membranes
`produced from amnion, herein referred to as a collagen
`biofabric. The collagen biofabric of the invention has the
`structural integrity of the native non-treated amniotic mem
`brane, i.e., the native tertiary and quaternary structure. The
`present invention provides a method for preparing a collagen
`biofabric from a placental membrane, preferably a human
`placental membrane having a chorionic and amniotic mem
`brane, by decellulariZing the amniotic membrane. In a
`preferred embodiment, the amniotic membrane is com
`pletely decellulariZed. The collagen biofabric of the inven
`tion has numerous utilities in the medical and surgical ?eld
`including for example, blood vessel repair, construction and
`replacement of a blood vessel, tendon and ligament replace
`ment, Wound-dressing, surgical grafts, ophthalmic uses,
`sutures, and others. The bene?ts of the biofabric are, in part,
`due to its physical properties such as biomechanical
`strength, ?exibility, suturability, and loW immunogenicity,
`particularly When derived from human placenta.
`
`

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`Patent Application Publication Mar. 11, 2004 Sheet 1 0f 5
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`US 2004/0048796 A1
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`FIG. 1
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`Patent Application Publication Mar. 11, 2004 Sheet 2 0f 5
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`US 2004/0048796 A1
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`FIG. 2A
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`

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`Patent Application Publication Mar. 11, 2004 Sheet 3 0f 5
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`US 2004/0048796 A1
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`

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`Patent Application Publication Mar. 11, 2004 Sheet 4 0f 5
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`US 2004/0048796 A1
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`FIG. 3
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`

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`Patent Application Publication Mar. 11, 2004 Sheet 5 0f 5
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`US 2004/0048796 A1
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`M esh
`Frame
`
`Tissue
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`Gauze
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`/
`
`FIG. 4
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`

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`US 2004/0048796 A1
`
`Mar. 11, 2004
`
`COLLAGEN BIOFABRIC AND METHODS OF
`PREPARATION AND USE THEREFOR
`
`[0001] This application is a continuation in part of US.
`application Ser. No. 10/106,653 ?led on Mar. 26, 2002,
`Which is incorporated herein by reference in its entirety.
`
`1. FIELD OF THE INVENTION
`
`[0002] The present invention relates to collagenous mem
`branes produced from amnion, herein referred to as a
`collagen biofabric. The collagen biofabric of the invention
`has the structural integrity of the native non-treated amniotic
`membrane, i.e., the native tertiary and quaternary structure.
`The present invention provides a method for preparing a
`collagen biofabric from a placental membrane, preferably a
`human placental membrane having a chorionic and amniotic
`membrane, by decellulariZing the amniotic membrane. In a
`preferred embodiment, the amniotic membrane is com
`pletely decellulariZed. The collagen biofabric of the inven
`tion has numerous utilities in the medical and surgical ?eld
`including for example, blood vessel repair, construction and
`replacement of a blood vessel, tendon and ligament replace
`ment, Wound-dressing, surgical grafts, ophthalmic uses,
`sutures, and others. The bene?ts of the biofabric are, in part,
`due to its physical properties such as biomechanical
`strength, ?exibility, suturability, and loW immunogenicity,
`particularly When derived from human placenta.
`
`2. BACKGROUND OF THE INVENTION
`
`[0003] 2.1 Amniotic Membrane: Anatomy and Histology
`[0004] The placental sac is composed of tWo layers inti
`mately connected by loose connective tissue. They are
`knoWn as the amniotic and chorionic layers (See FIG. 1).
`The amniotic layer is the most internal of the tWo layers and
`comes into contact With the amniotic ?uid that surrounds the
`fetus and together they form the amniotic sac. The amniotic
`layer is avascular and lined by simple columnar epithelium
`overlying a basal membrane and it measures 30-60 microns
`in thickness. The chorionic membrane is the outer layer of
`the sac and it is heavily cellulariZed. The vascular tree
`originates in the placenta and extends to the placental
`membranes through the chorionic layer. The chorionic layer
`is separated from the amniotic layer by loose connective
`tissue and combined, the tWo layers measure 120-180
`microns. The placental membranes have a collagen matrix
`that is heavily laden With mucopolysaccarides and they are
`believed to serve primarily as a protective sac for the
`developing fetus. The membranes also maintain a barrier for
`infectious and immnunologic agents present in the maternal
`circulation. Placental membranes have both active and pas
`sive transports. Most small molecules and proteins can
`travel freely through them but large proteins such as IgM
`cannot cross through the basal layer.
`[0005] Preservation of the placental membranes (Which
`are either from animal or human-sources) in either 95%
`ethyl alcohol or glycerol mixed in 50-50% proportions With
`tissue culture media has been utiliZed for preservation of the
`amniotic membrane prior to freeZing. These preservatives
`eliminate the vitality of the placental tissues making the
`nuclei pyknotic but the collagen matrix and basal mem
`branes are preserved. Interestingly, both forms of preserva
`tion also eliminate the antigenicity of the transplanted mem
`branes and also any potentially virulent agents. Preservation
`
`is usually accomplished after the amniotic membranes are
`carefully separated from the chorion. The side of the amni
`otic membrane With the basal lamina and epithelium is shiny
`and the opposite side facing the chorion is dull.
`
`[0006] 2.2 Previous Clinical Applications of Amniotic
`Membranes
`
`[0007] Possible potential problems With xenogenic tissues
`(tissues from other species) carrying Zoonotic diseases or
`causing cross-species rejection have made these tissues less
`desirable. Allogenic grafts, or grafts from different individu
`als of the same species, continue to be the preferred source
`for human graft materials. The scarcity of human donor
`tissues for grafting is a groWing problem that has stimulated
`the development of neW materials for tissue grafting. Most
`often these sources of biological raW material are scarce,
`dif?cult to obtain, and very costly. The collagen sheet from
`human amnion, hoWever, has desirable biomechanical char
`acteristics useful in tissue graft applications. Thus, amniotic
`membranes are a good source of allogenic graft material.
`
`[0008] The fetal membrane including the amniotic and
`chorionic membrane has been used in surgeries documented
`as early as 1910 and has been revieWed by Trelford and
`Trelford-Sauder in 1979 (See Trelford and Trelford-Sauder,
`1979, Am. J. Obstet. Gynecol. 833). In 1910 Davis Was the
`?rst to report the use of fetal membranes as a surgical
`material in skin transplantation for example on burned and
`ulcerated skins (Davis et al., 1910, Johns Hopkins Med. J.,'
`15: 307). These studies Were mainly in animals and human
`trials proved disappointing. Since then the use of amniotic
`membranes in surgery has been expanded (See, e.g., Stem et
`al., 1913, JAIVIA, 13: 973-4; Sabella et al., 1913 Med.
`Records NY, 83: 478-80; de Rotth et al., 1940 Arch. Opthal
`mol, 23: 522-5; Sorsby et al., 1946, Br J. Opthamlol. 30:
`337-45). It is noW utiliZed as a biological dressing for burned
`skin, skin Wounds, and chronic ulcers of the leg, as an
`adjunctive tissue in surgical reconstruction of arti?cial
`vagina, and for repairing omphaloceles (See e.g., Trelford
`and Trelford-Sauder, 1979, 134Am. J. Obstet. Gynecol. 833;
`Colocho et al., 1974 Arch. Surg. 109: 370-3; Faulk et al.
`1980, Lancet, 1156; Prasad et al., 1986, J. Trauma, 26:
`945-6; Subrahmanyan et al., 1995, J. Plastic Surg. 48:
`477-8; Gruss et al., 1978, J. Can. Med. Assoc. 118: 1237-46;
`Ward et al., 1984, Br J. Plastic Surg. 37: 191-3; Dhall, 1984,
`J. Obstet. Gynaecol. 91: 279-82). It has also been used to
`prevent tissue adhesion in surgical procedures of the abdo
`men, head and pelvis (Gharib et al., 1996, Pediatr Surg. Int.
`11: 96-9; Rennekampff et al., 1994, Invest. Surg. 7: 187-93).
`In the 1940s, several authors reported the bene?cial role of
`the amniotic membrane in treating a variety of ocular
`surface disorders (See e.g., de Rotth et al., 1940 Arch.
`Opthalmol, 23:522-5; Sorsby et al., 1946, Br J. Opthalmol.
`30:337-45; Lavery et al., 1946, Trans. Opthalmol. Soc. UK,
`66: 668).
`[0009] Numerous attempts in the ?eld to optimiZe the
`preparation and preservation of the amniotic membranes for
`use in transplantation have been previously described (see
`e.g., Dua et al., 1999, Br J. Opthalmol. 83: 748-52 (“Dua”)
`for a revieW). Various preparation of amniotic membranes
`have included preservation by saline and antibiotic mixtures,
`alcohol dehydration With or Without separation of the amni
`otic layer from the chorionic layer. HoWever, all of the
`methods described in Dua, and in the references described
`
`

`
`US 2004/0048796 A1
`
`Mar. 11, 2004
`
`above still carry shortcomings that need to be addressed by
`improvements in preparation and preservation of amniotic
`membranes.
`
`[0010] More recently, methods have been disclosed Which
`rely on freezing for preservation of the amniotic membrane
`for application in tissue graft surgical procedures to correct
`corneal epithelial defects. See e.g., US. Pat. Nos. 6,152,142
`and 6,326,019B1 (“Tseng”). Tseng discloses an amniotic
`membrane that is mounted on a substrate and preserved in a
`mixture of Dulbecco’s Modi?ed Eagle Medium and glycerol
`and frozen at —80° C. The process of freeZing the tissue at
`any time during its preparation makes the Tseng amniotic
`membrane brittle, and even more brittle after the steps of
`thaWing and activation. In addition, the thaWing and acti
`vation steps add time required for the handling of the
`amniotic membrane. Furthermore, because of the brittleness
`of the Tseng amniotic membrane caused by the freeZing step
`in the preservation and preparation process, a structural
`support or backing is required to ensure structural integrity
`of the Tseng amniotic membrane during storage. This pre
`sents the added dif?culty of separating the preserved amni
`otic membrane from the backing, Which, due to its brittle
`ness can be dif?cult to handle and separate intact. Separation
`of the amnion membrane from the backing thus increases the
`likelihood of rupture of the membrane, and increases the
`length of time required to activate the amniotic membrane to
`alloW for thorough impregnation of the activation solution
`into the froZen amniotic membrane prior to performing the
`surgical procedure, leading to increased preparation time in
`the surgical suite. Storage and shipping are also complicated
`by the requirement of —80° C. freeZing. Finally, the mem
`branes of Tseng are not generally decellulariZed; as a result,
`the amniotic membranes so prepared are typically opaque
`and do not have a uniform structural composition.
`
`[0011] More recently, Yui et al., U.S. Pat. Nos. 5,618,312,
`(the “’312 Patent”) described the preparation of collagen
`sheets from stratum compactum of tissue membrane.
`Although the material described is primarily collagen, it is
`Weak enough due to its processing to require a separate step
`of crosslinking in order to render it strong enough for
`medical use, e.g., suturable. One method for cross-linking
`described in the ’312 Patent employs high heat treatment,
`preferably at 100° to 110° C., Which is believed to adversely
`affect the native conformation of collagen. Yui et al., US.
`Pat. No. 5,876,451 describe a similar collagen material
`derived from placenta. This material, hoWever, is treated
`during preparation With proteases as part of a decellulariZa
`tion step, Which likely results in destruction and/or disrup
`tion of native conformation of the components of the matrix;
`thus, the resulting collagen matrix does not maintain its
`native conformation.
`
`[0012] There thus remains a need in the art for an
`improved amniotic membrane for use in medical, diagnos
`tics, and cosmetic applications, Which has improved struc
`tural characteristics. The present invention provides such a
`biofabric, comprising collagen that, unlike prior collagen
`based biofabrics, retains the native collagen quaternary
`structure. As a result, the biofabric of the present invention
`is easily prepared, easily used, and is strong enough for
`medical and surgical purposes, and provides a superior
`substrate for Wound healing.
`
`3. SUMMARY OF THE INVENTION
`
`[0013] The present invention relates, in part, to the dis
`covery by the inventors of a novel method of preparation of
`a collagen biofabric from placenta, preferably a human
`placenta, that results in a novel collagen biofabric With
`improved physical and biophysical properties. Preferably
`the method of preparation involves minimal manipulation of
`the amniotic membrane. The collagen biofabric of the inven
`tion unlike those described in the prior art, due to the Way
`by Which it is processed, has an intact native tertiary and
`quaternary structure The present invention also provides a
`placental-derived amniotic membrane or biofabric having
`superior characteristics of increased tensile strength, sutur
`ability, and reduced immunogenicity resulting in reduced
`host-graft rejection. The present invention also provides a
`placental-derived amniotic membrane or biofabric that can
`be stored as dehydrated sheets Without freeZing or cryo
`preservation. Preferably, the placental-derived amniotic
`membrane is derived from a human placenta for use in
`human patients. HoWever, the same methods can be
`employed using placentas from various animal species for
`veterinary use in animal patients.
`
`[0014] The present invention provides a method of pre
`paring a collagen biofabric comprising providing a placenta,
`preferably a human placenta, separating the amnionic and
`chorionic layers from each other, and decellulariZing the
`amniotic membrane While preserving the architecture of the
`underlying extracellular matrix. The method further entails
`Washing and drying the decellulariZed membrane. This
`method yields a dehydrated, decellulariZed biofabric that
`can remain stable under sterile storage conditions at room
`temperature and that is subsequently rehydrated and grafted
`to or implanted into a subject.
`
`[0015] In a speci?c embodiment, the placenta is from a
`human female Who has undergone a cesarean section deliv
`ery or natural delivery. In preferred embodiments, common
`seriological and bacteriological assays knoWn to one skilled
`in the art are used to determine if the placenta is from a
`donor that is free of a communicable disease. In a speci?c
`embodiment, the placenta has been tested to be free of at
`least one communicable disease. In another embodiment, the
`source of the placenta is knoWn including medical history,
`blood type, immunologic data, and genotype characteristics
`of the donor. Although, the placenta can be from any
`mammal, preferably the donor is a human female. In some
`embodiments, the placenta is exsanguinated using common
`methods knoWn to one skilled in the art prior to separating
`the amniotic membrane from the chorionic membrane.
`
`[0016] The decellulariZation of the amniotic membrane in
`accordance With the methods of the invention comprises
`removing all visible cellular material and cellular debris
`from the amniotic membrane, e.g., from the maternal side of
`the amniotic membrane and the fetal side of the amniotic
`membrane. The decellulariZation of the amniotic membrane
`should not result in disruption of the structural integrity of
`the amniotic membrane or alter the biochemical composi
`tion. Accordingly, decellulariZing the amniotic membrane in
`accordance With the methods of the invention does not
`constitute freeZing at any point in preparation of the amni
`otic membrane or contacting the membrane With a protease.
`Preferably, the amniotic membrane is decelluariZed using a
`Weak detergent containing solution, e.g., a solution com
`
`

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`US 2004/0048796 A1
`
`Mar. 11, 2004
`
`prising 0.01-1.0% deoXycholic acid sodium salt monohy
`drate, a nonionic detergents, Triton X-100, an anionic deter
`gent, or sodium dodecyl sulfate or a combination thereof.
`[0017] Once the amniotic membrane is decellulariZed in
`accordance With the methods of the invention, the mem
`brane may be Washed and dried, preferably With loW heat
`under vacuum.
`[0018] In some embodiments, the invention provides a
`collagen biofabric comprising a dehydrated, decellulariZed
`and substrate-free amniotic membrane, so that the mem
`brane has native tertiary and quaternary structure. In other
`embodiments, the invention provides a collagen biofabric
`comprising a decellulariZed substrate-free amniotic mem
`brane, comprising of collagen, elastin and ?bronectin. In yet
`other embodiments, the invention provides a collagen bio
`fabric comprising a dehydrated, decellulariZed, uniform,
`translucent, and substrate-free amniotic membrane, With the
`provision that the amniotic membrane has never been con
`tacted With a protease.
`[0019] In some embodiments, the biofabric further com
`prises one or more biomolecules, e.g., therapeutic agents,
`including but not limited to, antibiotics, hormones, groWth
`factors, anti-tumor agents, anti-fungal agents, anti-viral
`agents, pain medications, anti-histamines, anti-in?ammatory
`agents, anti-infectives, Wound healing agents, Wound seal
`ants, cellular attractants and scaffolding reagents, and the
`like. In a speci?c eXample, the collagen biofabric may be
`impregnated With one or more groWth factors, for eXample,
`?broblast groWth factor, epithelial groWth factor, etc. The
`biofabric may also be impregnated With one or more small
`molecules, including but not limited to small organic mol
`ecules such as speci?c inhibitors of particular biochemical
`processes e.g., membrane receptor inhibitors, kinase inhibi
`tors, groWth inhibitors, anti-cancer drugs, antibiotics, etc. In
`some embodiments, the collagen biofabric is impregnated
`With a biomolecule, during production or during preparation
`for surgery depending on its intended use.
`[0020] In some embodiments, the invention encompasses
`a laminate comprising at least tWo layers of the biofabric of
`the invention, and methods of preparing same. In other
`embodiments, the invention encompasses shaping the lami
`nates into complex three dimensional scaffolds depending
`on the intended use, including but not limited to sheets,
`?bers, spheres, tubes.
`[0021] In one embodiment, the invention encompasses a
`method of preparing an amniotic membrane laminate com
`prising: providing a placenta comprising an amniotic mem
`brane and a chorionic membrane; separating the amniotic
`membrane from the chorionic membrane; and decellulariZ
`ing the amniotic membrane. In another embodiment, the
`method further comprises Washing the decellulariZed amni
`otic membrane at least once; layering at least tWo of the
`decellulariZed amniotic membranes in contact With each
`other so that an amniotic membrane laminate is formed; and
`drying the decellulariZed amniotic membrane laminate.
`Alternatively, in another embodiment, the method for pre
`paring an amniotic membrane laminate comprises, drying at
`least tWo amniotic membranes prepared in accordance With
`the methods of the invention, and layering the at least tWo
`amniotic membranes in contact With each other so that an
`amniotic membrane laminate is formed.
`[0022] In some embodiments, the amniotic membrane
`layers produced in accordance With the methods of the
`
`invention may be placed in contact With each other in the
`presence of an adhesive to form an amniotic membrane
`laminate. The adhesive used in accordance With the methods
`and compositions of the invention may be any biological
`glue knoWn to one skilled in the art, preferably a biocom
`patible glue, including but not limited to, natural glue, e.g.,
`?bronectin, ?brin, synthetic glue. In other embodiments, the
`amniotic membrane layers prepared in accordance to the
`methods of the invention are cross-linked to each other to
`form an amniotic membrane laminate. Any cross-linking
`reagent and method knoWn to one skilled in the art is Within
`the scope of the present invention, including but not limited
`to, chemical cross-linking, peptide cross-linking, UV cross
`linking, radiation cross-linking, ?bronectin cross-linking,
`?brinogen cross-linking, hydrogel cross-linking. In other
`embodiments, the amniotic membrane laminates produced
`in accordance With the methods of the invention do not
`comprise an adhesive.
`[0023] In some embodiments, the invention encompasses
`using the collagen biofabric as a surgical graft. In a speci?c
`embodiment, the invention encompasses use of the surgical
`graft in a surgical procedure in a subject, preferably a
`human, comprising placing the graft directly on the surgical
`site.
`[0024] The invention provides a collagen biofabric, an
`amniotic membrane laminate, or a three-dimensional scaf
`fold further comprising one or more hydrogel compositions,
`and methods of preparing same. The hydrogel composition
`may comprise a polymer inlcuding but not limited to poly
`vinyl alcohol, polyethylene glycol, hyaluronic acid, and
`derivative and analogs thereof.
`[0025] In some embodiments, the collagen biofabric of the
`invention, aminates, three-dimensional scaffolds, or hydro
`gel compositions thereof may be further populated With
`cells, such as stem cells, differentiated adult cells, progenitor
`cells, and the like, preferably human so that the cells are
`uniform and con?uent.
`[0026] The invention provides methods of high level pro
`duction of the collagen biofabric, laminates thereof, three
`dimensional scaffolds thereof, and hydrogel compositions
`thereof, particularly but not limited to, commercial scale
`production. The invention solves dif?culties in producing
`large-scale quantities of amniotic membranes for use in
`clinical trials and commercial sales.
`[0027] The invention encompasses compositions compris
`ing a collagen biofabric of the invention suitable for drug
`delivery; tissue engineering; urological related uses, e.g.,
`correction of urinary incontinence; ocular uses, e.g., for the
`treatment of an ocular surface disorder, and as an ophthalmic
`surgical graft; vascular uses, e.g., blood vessel repair, con
`struction and replacement of a blood vessel; cardiological
`uses, e.g., as a prosthetic device in constructing diseased
`valves; neuronal-related uses, e.g., repair of injured nerves,
`especially severed peripheral nerves, as a dural substitute,
`and as a prostheses around nerve anastosmosis; bone related
`uses, e.g., for the treatment of orthopedic defects, as a bone
`replacement; dermatological uses, e.g., for the treatment of
`Wounds (external and internal), acute and chronic Wounds,
`congenital Wounds, and burns; for the treatment of skin
`conditions, e.g., skin lesions, aged skin, Wrinkles, ?ne lines,
`thinning, reduced skin elasticity, rough skin, and sun dam
`aged skin; as a Wound dressing; and for the treatment of
`Wound infections.
`
`

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`US 2004/0048796 A1
`
`Mar. 11, 2004
`
`[0028] In a speci?c embodiment, the invention encom
`passes a method for treating and/or preventing an eye related
`disease or disorder, e.g., ocular surface disease, in a subject,
`comprising using the collagen biofabric of the invention, for
`eXample, by placing the biofabric as a surgical graft on the
`diseased corneal surface of the subject. In another embodi
`ment, the invention encompasses a method of treating and/or
`preventing a skin condition in a subject, comprising using a
`biofabric of the invention for eXample, by contacting the
`skin With the biofabric. In yet another embodiment, the
`invention encompasses treating a Wound and/or burn in a
`subject comprising contacting the Wound and/or burn With a
`biofabric of the invention. In another speci?c embodiment,
`the invention encompasses a method of correcting urinary
`incontinence in a subject, preferably, a human, comprising
`using a collagen biofabric of the invention as an implant.
`
`[0029] In other embodiments, the invention encompasses
`a method of delivering a therapeutic agent to a subject
`comprising contacting the subject With a collagen biofabric
`of the invention. The invention further encompasses meth
`ods of delivering cells to a subject comprising populating a
`collagen biofabric or laminate of the invention With living
`cells for eXample in tissue engineering.
`[0030] The invention provides a surgical graft comprising
`the biofabric of the invention for use in a surgical procedure.
`The surgical graft may be applied to an internal or external
`site of the subject, preferably a human.
`
`4. BRIEF DESCRIPTION OF THE FIGURES
`
`[0031] FIG. 1 The chorion and amniotic membrane of a
`human placenta.
`[0032] FIG. 2 PHOTOMICROGRAPH OF THE COL
`LAGEN BIOFABRIC
`
`[0033] A. BEFORE PROCESSING
`
`[0034] B. AFTER PROCESSING
`
`[0035] FIG. 3 THE COLLAGEN BIOFABRIC. The col
`lagen biofabric of the invention is exempli?ed having a
`uniform translucent surface With an embossed pattern.
`
`[0036] FIG. 4 MESH FRAME AND THE BIOFABRIC
`BEING DRIED THEREIN
`
`5. DETAILED DESCRIPTION OF THE
`INVENTION
`
`[0037] The present invention provides a collageneous
`membrane or biofabric derived from the placenta of a
`mammal, preferably of a human. The collagen biofabric is
`prepared so as to retain the native collagen conformation,
`i.e., the native tertiary and quaternary conformation, in the
`?nal product. In addition to the collagen biofabric, the
`present invention also provides methods of making the
`collagen biofabric, and of using the biofabric in a medical
`setting.
`[0038] The present invention provides a collagen biofabric
`comprising a dehydrated, decellulariZed and substrate-free
`amniotic membrane so that the amniotic membrane has a
`native tertiary and quaternary structure. In some embodi
`ments the invention provides a decellulariZed and substrate
`free collagen biofabric comprising of collagen, elastin, and
`?bronectin.
`
`[0039] In some embodiments, the invention provides an
`amniotic membrane laminate comprising a collagen biofab
`ric of the invention. The amniotic membrane laminate
`prepared in accordance With the methods of the invention
`comprises at least tWo layers of the collagen biofabric that
`are placed in contact With each other to form the amniotic
`membrane laminate. In other embodiments, the invention
`provides a three dimensional scaffold, such as a tube,
`comprising a collagen biofabric of the invention. In yet other
`embodiments, the invention provides a collagen biofabric of
`the invention, a laminate thereof, or a three dimensional
`scaffold thereof further comprising a hydrogel composition.
`[0040] The invention thus provides various forms and
`con?gurations of the collagen biofabric including but not
`limited to laminates, three-dimensional scaffolds, and
`hydrogel compositions. The invention provides any medical
`useful form of the compositions of the invention. Regardless
`of the particular form or con?guration, the compositions of
`the invention may further comprise one or more biomol
`ecules, preferably a therapeutic agent. The compositions of
`the invention comprising a biomolecule have numerous
`utility in the medical ?eld as described in detail herein. In
`some embodiments, the invention encompasses populating
`the compositions of the invention With living cells so that the
`cells are uniform and con?uent. The compositions of the
`invention populated With cells have numerous utility in the
`medical and dental ?eld for eXample for tissue engineering
`purposes.
`
`[0041] The invention also relates to methods for preparing
`a collagen biofabric, a laminate thereof, a three-dimensional
`scaffold thereof, or a hydrogel composition. In a speci?c
`embodiment, the invention provides a method of preparing
`a collagen biofabric from a placenta having an amniotic
`membrane and a chorionic membrane comprising: separat
`ing the amniotic membrane from the chorionic membrane;
`and decellulariZing the amniotic membrane so that the
`amniotic membrane is not contacted With an enZyme, e.g., a
`protease. In other embodiments, the method further entails
`Washing and drying the decellulariZed amniotic membrane.
`In another speci?c embodiment, the invention provides a
`method of preparing an amniotic membrane laminate from
`a placenta having an amniotic membrane and a chorionic
`membrane comprising: separating the amniotic membrane
`from the chorionic membrane; decellulariZing the amniotic
`membrane; and layering at least tWo of the decellulariZed
`amniotic membranes in contact With each other so that an
`amniotic membrane laminate is formed. In a speci?c
`embodiment, the. decellulariZed amniotic membrane is dried
`prior to layer. In another speci?c embodiment, the decellu
`lariZed amniotic membrane is dried after is layered so that at
`least tWo of the decellulariZed amniotic membranes are in
`contact With each other.
`
`[0042] The invention provides a method of using the
`collagen biofabric of the invention, laminates thereof, three
`dimensional scaffolds thereof, or hyrdogel compositions
`thereof in a medical, dental and surgical setting. In fact, it is
`eXpected that the compositions of the invention have an
`enhanced therapeutic and clinical utility relative to the other
`biomaterials knoWn in the art. In some embodiments, the
`invention provides a method of treating and/or preventing an
`eye related disease or disorder in a subject using a compo
`sition of the invention. In a speci?c embodiment, the inven
`tion provides a method of treating and/or preventing an eye
`
`

`
`US 2004/0048796 A1
`
`Mar. 11, 2004
`
`related disease or disorder in a subject comprising placing
`the biofabric on a diseased eye surface of the subject.
`
`[0043] In other embodiments, the invention provides a
`method of treating and/or preventing a skin condition in a
`subject, preferably a human, using a composition of the
`invention. In a speci?c embodiment, the method comprises
`contacting the skin of the subject With the composition. In
`another speci?c embodiment, the composition is placed
`directly on the surface of the skin of the subject, Which is the
`site of the skin condition.
`
`[0044] The invention also provides a method for treating
`a Wound or a burn in a subject, preferably a human,
`comprising contacting the composition at the site of the
`Wound or burn.
`
`[0045] In other embodiments, the invention provides using
`a composition of the invention (e.g., a collagen biofabric, an
`amniotic membrane laminate) in a surgical procedure, such
`as ophthalmic surgery; cardiovascular surgery; periodontal
`surgery; neurological surgery, dental surgery, and orthopedic
`surgery.
`
`[0046] The invention provides a method for using a com
`position of the invention for delivering a biomolecule,
`preferably a therapeutic agent to a subject, preferably a
`human. The invention also encompasses a method of deliv
`ering cells using a composition of the invention to a subject,
`preferably a human, Wherein the composition has been
`further populated With cells.
`
`[0047] 5.1 Collagen Biofabric
`[0048] The invention provides a collagenous amniotic
`membrane, herein referred to as a collagen biofabric. The
`collagen biofabric of the invention maintains the structural
`integrity of the native, non-treated amniotic membrane, i.e.,
`retains the tertiary and quaternary structure of the structural
`proteins in its compositions such as collagen, elastin, and
`possibly ?bronectin. Thus, the collagen biofabric of the
`invention is composed of the same structural proteins as the
`native or non-treated amniotic membranes. Prior art meth
`ods of producing amniotic membranes require the use of
`proteases or high heat treatment, as a result these membranes
`do not maintain the tertiary and quaternary structure of the
`structural proteins in their composition.
`
`[0049] In a speci?c embodiment, the present invention
`provides a dehydrated, decellulariZed, and substrate-free
`(i.e., no ?lter backing) amniotic membrane such that the
`amniotic membrane has