U3006152142A
`
`[19]
`United States Patent
`[11] Patent Number:
`6,152,142
`
`[45] Date of Patent:
`Tseng
`Nov. 28, 2000
`
`[54] GRAFTS MADE FROM AMNIOTIC
`MEMBRANE; METHODS OF SEPARATING,
`PRESERVING, AND USING SUCH GRAFTS
`IN SURGERIES
`
`[76]
`
`Inventor:
`
`Schefl'er C. G. Tseng, 10000 SW. 63rd
`P1., Pinecrest, Fla. 33156
`
`Sorsby, et a1., “Further Experience with Amniotic Mem-
`brane Grafts in Caustic Burns of the Eye," 31 Br J.
`Opthamology 409—18 (1947).
`Sorsby and Symons, “Amniotic Membrane Grafts in Caustic
`Burns of the Eye (Bums of the Second Degree),” 30 Br. J.
`Opt/tanzology 337—345 (1946).
`
`[21] Appl. No.: 09/027,109
`
`[22]
`
`Filed:
`
`Feb. 20, 1998
`
`[60]
`
`Related U.S. Application Data
`Provisional application No. 60/039,486, Feb. 28, 1997.
`
` [51] Int. Cl.7 ....................... A61B 19/00
`
`[52] U.S. Cl. ................... 128/898; 623/4
`[58] Field of Search .................................. 128/898; 623/4
`
`[56]
`
`References Cited
`PUBLICATIONS
`
`Badawy, et al. “Evaluation of Tissue Healing and Adhesion
`Formation After an Intraabdominal Amniotic Membrane
`Graft in the Rat,” 34 J. Reproductive Med. 198 (1989).
`Yokomori, et al., “Advantages and Pitfalls of Amnion Inver-
`sion Repair for the Treatment of Large Unruptured Ompha-
`locele: Results of 22 Cases," 23 Journal of Pediatric Sur-
`gery 882 (1992).
`
`Primary Examiner—V. Milliw
`Assistant Examiner—Kelly O’Hara
`Attorney, Agent, or Firm—Robert M. Schwartz; Gerald R.
`Hibnick
`
`[57]
`
`ABSTRACT
`
`Amethod for making, storing and using a surgical graft from
`human amniotic membrane;
`the resulting graft; and the
`storage solution. The amniotic membrane is obtained from
`human placenta, from which the chorion has been separated.
`Sheets of the amniotic membrane are cut
`to size and
`mounted on filter paper. The cells of the amniotic membrane
`are killed, preferably While being frozen and thawed in the
`storage solution. The storage solution comprises a culture
`medium and a hyperosmotic agent, wherein the hydration of
`the amniotic membrane is maintained. The membrane can be
`impregnated With therapeutic agents. prior to storage, for use
`in post surgical healing or other therapies.
`
`12 Claims, No Drawings
`
`MTF Ex. 1010. mm
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`

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`6,152,142
`
`1
`GRAFTS MADE FROM AMNIOTIC
`MEMBRAN ; METHODS OF SEPARATING,
`PRESERVING, AND USING SUCH GRAF’I‘S
`IN SURGERIES
`
`CROSS-REFERENCE TO RELATED
`APPI .ICAT‘IONS
`
`This application claims the benefit of U.S. Provisional
`Application No. 60/039,486, filed Feb. 28, 1997.
`BACKGROUND OF THE INVENTION
`
`1. Field of Invention
`
`This invention relates to amniotic membrane grafts espe-
`cially usable in the repair of injured eyes. This invention also
`encompasses: a method for separating and preserving amni-
`otic membrane for a graft; the graft that is created by such
`method; and methods of repairing eyes and other organs
`while using these grafts.
`2. The Prior Art
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`if there were failures of treatment
`period. Furthermore,
`during this time, they were not reported. This gap in research
`ended in 1972 with the research of Trelford and associates,
`cited above. Trelford, rising isolated amnion with an early
`form of preparation, showed that the orientation with stro-
`mal side down provided more consistent “take.” Robson and
`colleagues noted in 1972 that, when used in partial—thickness
`skin wounds, no “take” occurs, and the amnion peels olf. In
`1973 and later, Trelford and associates reported its use as a
`dressing on full-thickness skin wounds,
`to replace pelvic
`peritoneum, and to cover exposed deep surfaces in pedicle
`graft procedures, to treat non healing skin wounds in dia-
`betic patients, as a graft over the surgical defect of total
`glossectomy, as a biological dressing in omphalocele, and in
`the prevention of meningocerebral adhesions following head
`injury.
`Previous Methods of Preparation and Preservation:
`All of the above-mentioned applications appeared to have
`used live tissues or tissue removed and preserved “lively” in
`a special solution before use. For example, de Rotth put the
`fetal membrane, amnion and chorion together without
`separation, in “tepid Locke solution”, and one to fifteen
`hours after cesarean section the tissue was implanted to
`patients. Reports which appeared after 1980 refer to live
`amniotic membranes having been used (See 34 J. REPROV
`DUCTIVE MED. 198 (1989) and 27 J. PEDIATRIC SURGERY 882
`(1992)). For “amnioplastin”, Chao and associates isolated
`the amnion, placed it in 70% alcohol, and then dried it in an
`oven prior to use. Robson and associates rinsed the mem-
`brane in a 0.025% NaOH solution and showed that
`it
`remained sterile up to six weeks when stored in saline
`containing penicillin at 4° C. Trelford and associates found
`that amniotic membranes stored at 4° C. in 0.5N saline to
`which polymyxin, ampicillin, gentamicin, and amphotericin
`B was added were sterile at the end of four hours and
`remained so for at least 48 hours.
`
`SUMMARY OF THE INVENTION
`
`Human amniotic membrane, obtained and preserved in a
`new way is made into a graft which is effective in: promot-
`ing healing of persistent corneal epithelial defects with
`ulceration; reduction of inflamation, angiogenesis and sear-
`ring; restoration of the epithelal phenotype; numerous fur-
`her uses in ocular surface reconstruction; and as a substrate
`alternative to conjunctival autograft during the “bare sclera”
`removal of pterygia.
`In addition, when combined with
`imbal allografts, amniotic membrane transplantation is use—
`ul for ocular surface reconstruction in patients with
`advanced ocular cicatricial pemphigoid, Stevens-Johnson
`syndrome, chemical and thermal burns, aniridia, atopic
`(cratitis and idiopathic limb al stem cell deficiency. After the
`alacenta is obtained and cleaned, the amnion is separated
`mm the chorion by blunt dissection, flattened onto filter
`Japer with the epithelium surface facing away from the
`3aper, and cut into small sheets. These sheets are stored in
`a media composed, for example, in Dulbecco’s Modified
`Eagle Medium and glycerol at the ratio of 1:1 (V/V), and
`rozen at —80° C. until just prior to use as a graft. When
`hawed to room temperature the day of use, the cells of the
`graft membrane have been killed, probably by ice crystals
`mm the surrounding storage medium. The side of the
`membrane adherent to the filter paper is opposed to the
`surgical site.
`
`
`
`OBJECTS OF TIIE INVENTION
`
`It is an object of this invention to prepare grafts made
`from amniotic membrane.
`
`MTF Ex.1010,P9.2
`
`Terminology:
`An amniotic membrane has two major components: the
`basement membrane and stroma. The side of the amniotic
`membrane dominated by the basement membrane is referred
`to as the “basement membrane side”. The side of the
`amniotic membrane dominated by the stroma is referred to
`as the “stroma side”. An autograft is a tissue transplant from
`the same recipient. When used in subcutaneous tunnels,
`autografts of the amnion become a permanent structure. In
`contrast, allografts are replaced by hyaline—hke material. An
`allograft is a tissue transplant to a recipient from a donor of
`another individual of the same species.
`Previous Clinical Applications
`The fetal membrane including amnion (amniotic
`membrane) and chorion has been used in surgeries docu-
`mented as early as 1910 and has been reviewed by Trelford
`and 'l'relford-Sauder in 1979. See 'I'relford and 'l'relford-
`Sauder, The Amnion in Surgery, Past and Present, 134 AM
`J. OBS'I'H‘I'. GYNH(I()I.833 (1979). In the beginning, the fetal
`membrane was used by Davis in 1910 on burned and
`ulcerated skins with additional coverage of warm paraffin
`and dressing. In 1940, De Rotth used fetal membrane for
`ophthalmic reconstruction of symblepharon, and noted a
`success in one out of six cases. See De Rotth, Plastic Repair
`of Conjunctival Defects with Fetal Membranes, 23
`ARCHIVES OF OPTHAMOLOGY 522 (1940). In 1952, Douglas
`thought chorion might be more useful for skin use. Massee
`and colleagues in 1962 used the fetal membrane in dogs to
`treat pelvic basins after total exenteration; however,
`the
`human trials proved disappointing.
`The isolated amnion alone was first used by Brindeau in
`1935 and Burger in 1937 as a graft in forming artificial
`vaginas. Between 1941 and 1948, Kubanyi used “live”
`amnion in patients with burns, traumatic skin wounds, and .
`enterocutaneous llstula secondary to surgery for lysis of
`adhesions. The isolated amnion, with preservation in a
`technique termed “amnioplastin”, was first reported by Chao
`and associates in 1940. Chao used amnioplastin for con—
`tinual dural repair, peripheral nerve injuries, conjunctiva]
`graft and flexor and tendon repair. In the Russian literature,
`this technique was also used for fresh trauma by Pikin in
`1942.
`Although all reports were enthusiastic, mention of
`“amnioplastin" disappeared from the literature with no real
`explanation. No critical reports regarding isolated, non-
`liVing amnion with preservation were found for a thirty-year
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`6,152,142
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`3
`is another object of this invention to prepare grafts made
`I
`from amniotic membrane that can be stored for long periods
`of time.
`I
`is another object to this invention to prepare grafts that
`have been treated so that the grafts contain agents that can
`be delivered to the recipient when attached to the recipient.
`I
`is another object of this invention to provide an
`improved substrate alternative to conjunctival autograft dur-
`ing the “bare sclera” removal of pterygia.
`I
`is another object of the invention to provide an
`improved substrate alternative to conjunctival flaps to pro—
`mote healing of corneal epithelial defects with ulceration.
`I
`is another object of this invention to provide an
`improved method for conjunctival surface reconstruction for
`symbelpharon lysis.
`I
`is another object of this invention to provide an
`improved method for surgical removal of tumors, lesions, or
`scar tissue from the conjunctiva] or corneal surface.
`I
`is another object of this invention to reduce the corneal
`haze induced by excimer laser photerefractive/therapeutic
`keratectomy.
`I
`is another object of this invention to promote successful
`glaucoma surgeries by correcting bleb leakage.
`I
`is another object of this invention to prevent recurrence
`of band keratopathy.
`Other objects of the invention and advantages over the
`prior art, as well as differences from the prior art, will
`become fully appreciated from the following discussion of:
`Embryogcncsis and Histology; Components; Applications in
`Basic Research; and the Description of the Preferred
`Embodiments, along with description of numerous proposed
`uses of the resulting, improved graft.
`Embryogenesis and Histology
`Early in the process of blastocyst implantation, a space
`develops between the embryonic mass and adjacent tropho-
`blasts. The amniotic epithelium is derived from fetal ecto—
`derm (the embryonic disc). In addition to the epithelial cells,
`a layer of fibroblast-like cells might also be derived from the
`embryo, but this is not certain. The important “missing”
`elements of human amnion are smooth muscle cells, nerves,
`lymphatics, and, most important, blood vessels. The human
`amnion likely develops about the 7th and 8th day of devel—
`opment of the normal blastocyst. Amnion fuses with chorion
`during the 10th and 12th weeks of pregnancy, when the
`amniotic cavity expands. The amnion remains avascular till
`term. This is the reason why separation between amnion and
`chorion is possible via the interface, i.e., the intermediate
`zone. The normal amnion is 0.2 to 0.5 mm in thickness.
`Histologically, the amnion is comprised of five layers.
`The inner surface consists of a simple cuboidal epithelium,
`which lies on the basement membrane. The avascular stro-
`mal contains fetal mesenchyme and includes the compact
`layer, fibroblastic layer and spongy layer. Four distinct
`anatomic portions of the amnion exist. First, the reflected
`amnion is that portion that is contiguous with the chorion .
`laeve. Second, the placenta amnion overlies the fetal surface
`of the placenta, which is directly contiguous with the adven-
`titial surface of the chorionic vessels. Third, the amnion also
`covers the umbilical cord, that is contiguous with Wharton
`jelly. Wharton Jelly is the extracellular matrix through which
`the umbilical vessels traverse. Fourth,
`in diamnioin-
`dichorionic twin pregnancy, the amnion are “fused”, which
`in the former is contiguous with fused chorion laeve and in
`the la ter amnion is fused with amnion.
`
`
`
`Components:
`
`
`
`
`Di erent tissues of the body have di erent components of
`collagen and glycosaminoglycans (hereinafter “GAGs”).
`
`
`
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`The amniotic stroma has been shown to contains collagens
`I and III and fibronectin, and has a perilaminal distribution
`of collagen types V and VII. The basal lamina of the amnion
`contains a network of type IV collagen fibrils, laminin, and
`1eparin sulfate proteoglycan. 'l‘he collagenous fibers of the
`amnion are closely distributed with no ground substances,
`orming a thick collagenous layer. In human and monkey
`alacenta, hyaluronic acid (hereinafter “HA”) is thought to be
`he only GAGs found, but this finding is not shown in the
`amnion yet. This compositional feature is in part responsible
`‘or the tensile strength noted during pregnancy.
`The basement membrane side of the amniotic membrane
`can be used to support epithelial growth to maintain epithe-
`ial polarity. When growing human fibroblasts or A431
`epidermal carcinoma cells, the stromal side of the amniotic
`membrane is found to be comparable to isolated collagen,
`3ut greater than a plain plastic surface in culturing cell
`growth. When sandwiched in the Boyden chamber,
`this
`membrane can be used for studies of polymorphous nuclear
`cells emigration (hereinafter “PMN emigration”) in the
`rocess of leukodiapedisis, vascular endothelial invasion,
`and tumor cell metastasis, through the basement membrane.
`Although the amnion is not
`innervated, avian amnion
`express at
`least eleven different
`types of receptors for
`neurotransmitters, including: acetylcholine, norepinephrine,
`histamine, 5-hydroxytryptamine, VIP, urotensin II,
`neurotensin, and somatostatin—28.
`Applications in Basic Research:
`Taking advantage of its basement membrane content,
`human amnion can be used as a substrate to culture periph—
`eral and central nervous system neurons and to promote
`
`axonal regeneration when implanted in the central nervous
`
`
`
`system. This e ect appears not dependent on the live cells
`and is mediated by the amniotic matrix, which promotes the
`host regenerative power, and can be further enhanced when
`added with nerve growth factor (hereinafter “NGF”) in
`regeneration of a severed peripheral nerve.
`DESCRIPTION OF THE PREFERRED
`EMBODIMENTS
`
`Method for Selection, Preparation and Preservation of the
`Graft:
`
`To prepare and preserve grafts from human amniotic
`membrane, the following method should be used.
`First, the placenta is taken as soon as possible after the
`delivery. Preferably, the placenta is taken immediately fol-
`lowing the elective cesarean section (C/S) delivery of a
`normal healthy baby.
`To avoid any potential blood—transmittable diseases, the
`pregnant female is prescreened for HIV-1, HIV-2, HTLV-l,
`hepatitis B and C viruses and syphilis, using conventional
`serological tests. Only those placentas of which the maternal
`bloods reveal negative serological results are used for this
`method to produce the amniotic graft.
`Human placenta that meet the above selection criteria is
`transferred to the laboratory in a sterile plastic bag stored in
`an ice bucket. The following procedures are performed
`under sterile conditions, as routinely used for tissue cultures.
`1. Under a lamellar—flow hood, the placenta is rinsed several
`times with balanced salt saline to remove excessive blood
`clots. Balanced saline solution is available, for example
`under the trademark BSS® from Alcon Inc., 6201 South
`Freeway, Fort W'orth, Tex, 76101. The rinse also should
`contain antibiotics to aid in the cleaning and preserving.
`An example of an effective antibiotic formulation con-
`tains 50 rig/ml penicillin, 50 ,ug’ml streptomycin, 100
`
`MTF Ex. 1010. pg. 3
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`

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`6,152,142
`
`5
`In
`rig/m1 neomycin, and 2.5 jig/ml amphotericin B.
`addition, the membrane can be treated so that it contains
`other substances that would be transferred to the recipient
`once the graft is placed. Examples of substance which can
`be impregnated into the graft membrane are: therapeutics,
`hormones, polypeptides; to aid healing of the surgical area
`or other therapies.
`2. With two sets of forceps, the placenta’s amniotic mem-
`brane is separated easily from the remaining chorion by
`blunt dissections, while immersed in the above
`antibiotics-containing balanced saline solution.
`3. The separated amniotic membrane, as a sheet, then is
`mounted/apposed onto a substrate, for example, a sterile
`nitrocellulose filter, so that the epithelial surface is kept
`facing up when flattened. Thus, the stromal/fibroblistic
`layer lies on the filter. The basement membrane lies above
`the stromal layer, and the epithelial lies above the base-
`ment membrane. The filter should be inert with respect to
`the amniotic membrane and the next discussed storage
`medium. The filter should not contain bleach or chlorine
`and should be stable when in the storage medium, espe—
`cially when subject to freezing and thawing therein. One
`source of such a filter is Millipore, Inc. and is sold as
`product number 162—0180. Other useful substrates are
`nitro cellulose membranes 162—0115, supported nitro cel—
`lulose membrane 162-0090 and blot absorbent filter paper
`162-0118, all from Biorad.
`Sheets of amniotic membranes that have been adhered/
`mounted onto the nitrocellulose filter are cut to different
`sizes, for example, 2.5—3.0 cm.><2.0—2.5 cm. and 1.2—1.5
`cm.><1.75—2.0 cm. and are stored in a culture medium at
`temperatures below freezing. It is important to appreciate
`that the freezing of the grafts (the cut up and filter mounted
`sheets of amniotic membrane) and their subsequent thawing,
`soon prior to the use, results in the killing of the cells of the
`membrane, probably by ice crystals formed in the membrane
`from the liquid in the culture medium. By killing the cells,
`the resulting graft
`thereby is not rejected after surgery.
`However,
`the integrity of the extracellular matrix is not
`altered despite the freezing and thawing. Although the cells
`could be killed by other, conventional, means, the use of
`freezing-thawing does not introduce agents or conditions
`which might adversely affect the stored membrane or the
`completed graft,
`to cause it not to “take” or be rejected,
`impair the healing of the eye, etc. As previously noted, the
`prior art used media to keep the cells vital (alive) and did not
`recognize the great advantage of killing the cells. While any
`temperature below freezing should work, a temperature of
`
`
`—80° C. has been used typically to store the grafts.
`
`
`Furthermore, this method of preservation is e ‘ective with
`respect to sterility and efficacy for long-term storage beyond
`one year, even when the thus stored and frozen cut sheets of
`membrane are shipped long distances in dry ice.
`An example of an effective medium is comprised by 50%
`Dubecco Modified Eagle’s Medium (hereinafter “DMEM
`medium”) (from GIBCO) and 50% glycerol (V/V). A range .
`of 30% to 50% glycerol
`is usable. The function of the
`glycerol is to maintain the hydration state of the amniotic
`membrane; too little or too much hydration is detrimental,
`60% to 90% hydration is effective for the intended purpose.
`The glycerol acts as a high oneotic or hyperosmotic agent;
`another term for which is a plasma expander. Examples of
`other usable hyperosmotic agents are: dextran, albumin, and
`mannitol. The general purpose of the storage medium, such
`as DMEM, is to provide nutrients to and maintain electrolyte
`balance for the amniotic membrane. Other examples of a
`suitable storage medium are: Liebowitz’s medium, MEM,
`and NCTC, all manufactured by Life Technologies.
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`If the sheets of amniotic membrane, on the filter paper, are
`to be used “near term”, within twenty-four hours of
`harvesting, then the use of a storage medium is not essential;
`however, the cells do have to be killed, as previously stated.
`Graft prepared according to the previously-stated method:
`A graft comprising amnion, from human placenta, which
`has had the chorion removed, that is prepared and preserved
`according to the previously-stated method. This graft has
`been rinsed with balanced salt solution and antibiotics. The
`graft can contain therapeutic substances that have been
`absorbed into it. The graft
`is mounted onto filter paper,
`having suitable characteristics, as previously stated. The
`graft is stored in a composition of culture medium and a high
`oncotie agent at freezing temperatures. Prior to use, the graft
`is thawed.
`Method of using prepared amniotic membrane as a sur—
`gical graft:
`In eyes with persistent epithelial defect and ulceration,
`after retrobulbar anesthetic injection, the base of the ulcer is
`debrided with surgical sponges (an example of a suitable
`surgical sponge is sold under the trademark MICRO-
`SPONGE® from Alcon Surgical, Inc., 6201 South Freeway,
`Fort Worth, Tex. 76134—2099) and 0.12 forceps, and the
`poorly adherent epithelium adjacent to the edge of the ulcer
`also is removed to the area where the epithelium becomes
`quite adherent. The amniotic membrane, which recently was
`
`
`removed from the storage medium and thawed to room
`
`
`temperature, is peeled o
`from the nitrocellulose filter paper,
`
`
`transferred to the recipient eye, with the stromal surface
`
`
`facing the eye and fitted to cover the defect by trimming o
`the excess edges of the membrane/graft sheet. In other
`instances, the opposite side can be used. This fashioned
`membrane then is secured to the edge of the defect by
`interrupted 10-0 nylon sutures, and in some cases by a
`running 10—0 nylon suture. After the knots are buried, the
`corneal surface becomes smoothed as a result of the well—
`ap aroximated amniotic membrane filling in the ulcer bed.
`Except for deep ulcers, one layer of membrane generally is
`su icient. But it is also feasible to use two or more layers.
`A bandage contact lens is applied together with a topical
`ophthalmic antibiotic ointment comprising neomycin, poly-
`myxin b sulfate and dexamethasone. An example of such a
`sui able ophthalmic antibiotic ointment is sold under the
`trademark MAXITROL® from Alcon Laboratories, Inc.,
`6201 South Freeway, Fort Worth, Tex. 76134.
`The inventions herein are more effective than that of De
`
`
`
`Ro th in conjunctiva] surface reconstruction for symble—
`pharon lysis.
`In addition,
`the invention can be used to
`reconstruct the conjunctival surface which is damaged dur-
`ing surgical removal of tumor, lesion, or scar tissue. Impres-
`sion cytology can prove that the reconstructed conjunctiva
`regain normal epithelial phenotype with goblet cells of
`which the number is greater than the normal control. Goblet
`cells secret mucin and are shaped like a goblet. Amembrane
`produced through the invented method, when used as a
`patch, can reduce corneal haze, a form of scarring, induced
`by excimer laser photo refractive keratectomy and therapeu-
`tic keratectomy, a procedure presently used in patients to
`correct myopia and astigmatism, and to remove the diseased
`part of corneas, respectively. Also this method and product
`can be used by itself or in conjunction with stem cell
`transplantate to reconstruct surfaces damaged by various
`causes leading to nimbal stem cell deficiency.
`This amniotic graft can be used for promoting successful
`glaucoma surgeries by correcting bleb leakage. The surgical
`use of amniotic grafts made according to the inventions
`herein can prevent recurrence of band keratopathy, prevent
`
`
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`MTF Ex.1010,P9.4
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`adhesion during muscle surgeries, and help orbit reconstruc—
`tion in oculoplastic surgeries. Band keratopathy is the depo-
`sition of calcium on the corneal surface.
`In addition to the mentioned ophthalmic uses, it is envi-
`sioned that the amniotic membrane of this invention also can
`be used: as a graft or dressing to cover burned or surgical
`skin wounds;
`to prevent adhesion in all intra peritoneal
`surgeries or other reconstruction on the serosal surfaces
`covering the abdomen, chest cavity and pericardium;
`to reconstruct all mocosal surfaces lining the oral and
`nasal cavities, respiratory tracts, gastrointestinal tracts, and
`urogenital tracts; as a substrate to support dural repair in
`brain surgeries; as a substrate to promote nerve regeneration
`in the central and peripheral nervous systems; and to recon—
`struct soft tissues to prevent adhesion in joint or tendon
`repairs.
`It is believed that surgeons, scientists and researchers will
`benefit from the information provided in the following
`papers:
`1. Kim J C, Tseng S C G. Transplantation of preserved
`human amniotic membrane for surface reconstruction in
`severely damaged rabbit corneas. Cornea. 1995;14:473—84.
`2. Tsubota K, Satake Y, Ohyama M, et al. Surgical
`reconstruction of the ocular surface in advanced ocular
`cicatricial pemphigoid and Stevens—Johnson syndrome. Am
`J Ophthalmol. 1996;122:38—52.
`3. Lee S. Tseng S C G. Amniotic membrane transplanta-
`tion for persistent epithelial defects with ulceration. Amn J
`Ophthalmol. 1997;123:303—312.
`4. Prabhasawat P, Barton K, Burkett G, Tseng S C G.
`Comparison of conjunctival autografts, amniotic membrane
`grafts and primary closure for pterygium excision. Ophthal-
`mology. 1997;104:974—985.
`5. Tseng S C G, Prabhasawat F, Lee S. Amniotic mem—
`brane transplantation for conjunctival surface reconstruc-
`tion. Am J Ophthalmol. 1997; December
`issue,
`124:765—774.
`(J. Prabhasawat P, Tseng S C G. Impression cytology study
`of epithelial phenotype of ocular surface reconstructed by
`preserved human amniotic membrane. Arch Ophthalmol.
`1997; November issue, 115:1360—1367.
`7. Barton K, Budenz D L, Khaw P T, Tseng S C G.
`Amniotic membrane transplantation in glaucoma surgery.
`Invest Ophthalmol Vis Sci 1997; 38:8473.
`8. Wang M, Gray T, Prabhasawat P, Ma X, Ding F-y,
`Hernandez E, Sanabria O, Culbertson W', Hanna K, Forster
`R K, Tseng S C G. Corneal haze is reduced by amniotic
`membrane matrix in excimer laser photoablation in rabbits.
`Invest Ophthalmol Vis Sci 1997; 3815405.
`9. Tseng S C G, Prabhasawat P, Barton K, Gray T B,
`Meller D. Amniotic membrane transplantation with or With-
`out limbal transplantation for corneal surface reconstruction
`in patients with limbal stem cell deficiency. Arch
`Ophthalmol, in press 1998 (April).
`While there has been described preferred embodiments of
`this invention’s methods and products, and there has been >
`mentioned modifications thereto; other changes, variations
`and modifications can be made within the scope of the
`appended claims, without departing from the spirit and
`scope of this invention.
`What I claim is:
`1. Amethod for obtaining human amniotic membrane and
`preparing it to be used as a surgical graft, comprising the
`steps of:
`obtaining human placenta and cleaning it;
`separating said placenta’s amniotic membrane from its
`chorion, said amniotic membrane having cells and an
`extracellular matrix;
`
`50
`
`60
`
`65
`
`
`
`8
`mounting said amniotic membrane onto a substrate, with
`the epithelial surface of said membrane facing away
`from said substrate; and
`killing the cells of said membrane prior to using said
`membrane as a surgical graft and maintaining the
`integrity of said extracellular matrix.
`2. A method according to claim 1, in which;
`sai
`step of killing said cells is accomplished by freezing
`said amniotic membrane in liquid; and
`thawing said membrane prior to using it as a giaft.
`3. A method according to claim 2, in which;
`saic freezing step is accomplished after said mounting
`S 6P;
`and said thawing step is just prior to use of said membrane
`as a graft.
`4. A method according to claim 2, in which;
`sai
`liquid is a culture medium,
`5. A method according to claim 4, in which;
`saic liquid is a hyperosmotic reagent.
`6. A method according to claim 2, in which;
`saic liquid is DMEM medium and glycerol.
`7. A method according to claim 1, in which;
`saic substrate is a sterile nitrocellulose filter further
`including the steps of;
`separating said amniotic membrane from said filter; and
`securing said membrane to a surgical site as a graft, with
`the stromal layer of said membrane facing said site.
`8. A method according to claim 7, in which;
`said surgical site is a human eye.
`9. A method according to claim 8, and employing said
`graft with associated medical/surgical steps for any one of:
`an autograft during the bare sclera removal of pterygium;
`promoting healing of corneal epithelial defects with ulcer—
`ation;
`conjunctive surfacere construction for symbelpharom
`lysis;
`reducing scaring;
`maintaining a normal epithelial phenotype;
`removing tumors, lesions, scar tissue from conjunctiva] or
`corneal surfaces;
`reducing corneal haze induced by excimer lasers; promot—
`ing correct bleb leakage;
`preventing recurrence of band keratopathy; and
`helping orbit reconstruction in oculoplastic surgeries;
`reconstructing corneal surface damaged by Iibal stem cell
`deficiency as a result of various causes.
`10. A method according to claim 1, and employing said
`amniotic membrane graft with associated medical/surgical
`steps for any one of:
`a dressing over skin wounds;
`preventing adhesion in surgeries;
`reconstructing mocosal surfaces;
`supporting, as a substrate, dural repair;
`promoting nerve repair as a substrate;
`reconstructing soft tissues; and reducing scaring.
`11. A method according to claim 1, and providing said
`amniotic membrane with at least one of a therapeutic, a
`hormone, a polypeptide, for becoming part of said graft.
`12. A method according to claim 1, in which;
`said obtaining is from a human female during a cesarean
`section delivery.
`
`MTF Ex. 1010. pg. 5
`
`