`
`,
`
`PCP
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau .
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(51) International Patent Classification 5 :
`A61F 2/02
`(43) International Publication Date:
`10 June 1993 (10106.93)
`_.___.____.___.—_____—_—___—3
`
`
`(11) International Publication Number:
`
`W0 93/10722 ‘
`
`(21) International Application Number:
`
`PCT/US92/ 10165
`
`(22) International Filing Date:
`
`25 November 1992 (25.11.92)
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only) : SHENAQ, Saleh, M.
`[US/US]; 1911 North Boulevard, Houston, TX 77098,
`(US). GRAY, Kathy, Jo [US/US]; 4209 South Judson,
`Houston, TX 77098 (US).
`
`‘
`
`
`
`
`
`
`
` (54) Title: FETAL MEMBRANE TUBES FOR NERVE AND VESSEL GRAFTS
`
`(30) Priority data:
`07/799,517
`
`26 November 1991 (26.11.91) US
`
`(74) Agent: WEILER, James, F.; 1 Riverway, Suite 1560, Hous-
`ton, TX 77056 (US).
`
`(60) Parent Application or Grant
`(63) Related by Continuation
`US
`Filed on
`
`07/799,517 (CIP)
`26 November 1991 (26.11.91)
`
`(81) Designated States: AU, CA, Fl. JP, KR, NO, RU. US, Eu-
`ropean patent (AT, BE, CH, DE, DK, ES, FR, GB, GR,
`IE, IT, LU, MC, NL, PT, SE).
`
`(71) Applicant flor all designated States except US): RESEARCH
`DEVELOPMENT FOUNDATION [US/US];
`402
`North Division Street, Carson City, NV 89703 (US).
`
`Published
`Wit/10m international search report and to be republished
`upon receipl of that report.
`'
`
`l—______._.._.
`
`
`
`(57) Abstract
`
`Disclosed are cylinders having a wall formed of at least one layer of sterilized cross-linked Types 1, II, III collagen or com-
`binations thereof from placenta for nerve and blood vessel grafts, methods of manufacture and use. The nerve grafts promote ax-
`on regeneration therethrough. The nerve and blood vessel grafts are non-immunogenic, can be constructed into tubes of various
`lengths and diameters, are easily accessible and are patent or open in use.
`
`
`
`MTF Ex. 1009, pg. 1
`
`
`
`,
`
`’.
`
`Austria
`AT
`Australia
`AU
`Barbados
`BB
`Belgium
`BE
`Burkina Fasu
`BF
`Bulgaria
`BC
`Benin
`3.!
`' Brazil
`an
`Canada
`CA
`Central African Republic
`CF
`Congo
`CG
`Swiuerlaml
`CH
`[‘fite d'lvuirt:
`Cl‘
`(‘ameroun
`CM
`Czechoslovakia
`6
`(Ewell Republic
`CZ
`Germany
`DE
`Denmark
`DK
`Spain
`ES
`
`Ft
`Finland"
`
`-
`
`'
`
`FR
`CA
`68
`GN
`GR
`HU
`IE
`IT
`JP
`KP
`KR
`KZ
`Ll
`LK
`LU
`MC
`MG
`Ml.
`MN
`
`,
`
`France
`Gabon
`United Kingdom
`Guinea
`Greece
`Hungary
`Ireland
`Italy
`Japan
`Democratic People‘s Republic '
`of Korea
`7
`Republic of Korea
`Kamlzltstan
`Liechtenstein
`Sri lanka
`Luxembourg
`Monaco
`Madagascar
`Mali
`Mongolia
`
`
`
` FOR THE PURPOSES OF INFORMATION ONLY
`
`
`Codes used to identify States party to the PCI‘ on the front pages of pamphlets publishing international
`
`
`applications under the PCI‘.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`'
`
`MR
`
`Mauritania
`
`MW
`NL
`N0
`NZ
`PL
`PT
`R0
`RU
`SD
`SE
`SK
`, SN
`SU
`TD
`TC
`UA
`US
`VN
`
`Malawi
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Slovak Republic
`Senegal
`Soviet Union
`Chad
`'l‘ugo
`Ukraine
`United Statea of America
`Viet Nam
`
`.
`
`MTF Ex. 1009, pg. 2
`
`
`
`W0 93/ l 0722
`
`PCT/US92/101a65
`
`_l_
`
`FETAL MEMBRANE TUBES FOR
`
`NERVE AND VESSEL GRAFTS
`
`Field of the Invention
`
`The present invention is directed to fetal membrane
`
`tubes for nerve and vessel grafts.
`
`Background of the Invention
`have been
`Fetal membranes,
`amnion and Vchorion,
`selected as our starting material for several reasons.
`The
`
`fetal membranes preferably are human although animal membranes
`
`can be used.
`
`(1)
`
`In one aspect of
`
`the invention, we have
`
`demonstrated a low immunogenicity of the human amniotic and
`
`chorionic conduits in rats which was evidenced when different
`
`antigens in their membranes, mainly collagen types I, II, and
`
`III fibronectin, and laminin were tested by dot blot and ELISA
`
`techniques.
`
`(2) Human placentas are available in relatively
`
`unlimited quantities and at
`
`low cost.
`
`(3)
`
`For almost 90
`
`years amnion and chorion have been used for a wide variety of
`
`medical and surgical indications.
`to be capable of neovascularization.
`
`(4)
`
`They have been shown
`(5) They have also been
`
`placed in subcutaneous pockets in human without evidence of
`
`acute rejection for as long as 7 weeks.
`
`The fetal membranes
`
`are of a complex biochemical structure with unique physical
`
`characteristics.
`
`They can be modified physically by 'the
`
`processing technique later described herein into conduits
`
`which are semi-rigid,
`
`resilient,
`
`and of variable length,
`
`diameter and thickness.
`
`The biochemical components of the
`
`amnion and chorion membranes are mainly collagen types I, II,
`
`and
`
`III,
`
`laminin,
`
`fibronectin and other glycoproteins.
`
`Laminin has -been
`
`shown
`
`to promote
`
`axon
`
`extension by
`
`10
`
`15
`
`20
`
`25
`
`30
`
`MTF Ex. 1009, pg. 3
`
`
`
`WO 93/10722
`
`P(:T/1fl392/10165
`
`-2-
`
`ll-
`
`interacting with axonal glycoproteins that are members of the
`The immunocytochemical studies
`integrin family of receptors.
`cessing technique of the
`we conducted led us to modify our pro
`amnion and chorion membranes in order to preserve laminin as
`a significant component of these conduits, as later described
`herein.
`
`problem, and the
`
`are
`chorion membranes
`and
`amnion
`human
`Wh1le
`preferred, these membranes from other animals, such as bov1ne
`membranes, are another aspect of the present invention.
`Clinically, it is accepted that the use of a nerve
`autograft is the method employed to reconstruct a nerve gap.
`The disadvantages of nerve autografts are shown to be many,
`i. e. an additional surgical procedure is required, scarring
`with anesthesia or hyperesthesia at the donor site may be a
`re frequently are dimensional limitations of
`Although nerve allografts have overcome
`the donor grafts.
`rejection of
`the
`several disadvantages of the autografts,
`1 use
`graft remains a major problem and limits its clinica
`despite the use of immunosuppressive agents.
`The current interest and future directions in nerve
`research focus on the development of an ideal nerve conduit
`Several investigators have studied the use
`for clinical use.
`including silicone rubber,
`PTFE,
`,of different materials
`polyorthoester, polyglactin, mesothelial tubes, muscle basal
`lamina, and vein grafts as nerve conduits.
`Each of these
`n to
`materials mentioned has shortcomings and none have prove
`provide the ideal environment for the.regenerating nerve. An
`ideal nerve conduit should be readily available, of low cost,
`easily' manufactured, of different sizes, non-immunogenic,
`microporous, noncollapsible, biodegradable, and.of biochemical
`components
`that provide a
`favorable environment
`for
`the
`regenerating axons.
`As
`later described herein, we have
`nerve conduit using human amnidn membrane which
`developed a
`the characteristics of the proposed ideal
`
`possesses many of
`nerve'conduit.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`MTF EX. 1009, P9. 4
`
`
`
`W0 93/ 10722
`
`PCT/US92/10165
`
`-3-
`
`A number of investigators are currently exploring
`
`the use of collagen for nerve conduits.
`
`By necessity,
`
`this
`
`collagen is either allogenic or xenogeneic in origin and,
`
`thus, could stimulate an immune response that may inhibit
`
`nerve regeneration.
`
`Immunological testing of the amniotic and
`
`chorionic nerve conduits of
`
`the present
`
`invention showed a
`
`minimal, nonsignificant immune response,
`
`thus, avoiding some
`
`of
`
`the
`
`inhibiting
`
`factors
`
`that
`
`could
`
`influence
`
`the
`
`regenerating nerve. Although amniotic and chorionic conduits
`
`showed signs of biodegradability and structural reorganization
`
`grossly and
`
`by
`
`electron microscopic
`
`examination,
`
`local
`
`inflammatory cells infiltration was slight, being limited to
`
`the area surrounding the conduit wall, and it did not produce
`
`changes similar to those of chronic nerve compression. This
`
`latter development is a major problem encountered with the use
`
`of silicone rubber conduits for nerve regeneration.
`
`Functional and morphological assessment of nerve
`
`'regeneration using amniotic and chorionic conduits proved its
`
`superiority to nerve autografts and silicone tubes.
`
`Also, there is a need for blood vessels which can be
`
`connected to their proximal and distal ends when blood vessels
`
`are severed such as when thrombosis occurs and blood vessel
`
`In bypass operations, veins stripping
`segments are removed.
`in the legs for these purposes can be avoided and similarly in
`
`subsequent bypass operations as the patient does not have
`
`adequate vein to take the place of the thrombosed arteries
`
`which are-
`
`removed.
`
`Also, vein graft wall collapse and
`
`obliteration of its lumen added to the donor’s site morbidity
`
`are significant problems which limit
`
`their use. Vascular
`
`conduits commonly used are vein grafts harvested from the
`
`patient’s legs or arms.
`
`The donor site morbidity and the
`
`unpredictable patency of vein grafts for coronary bypass and
`
`interposition bypass grafts are well
`peripheral~ vascular
`known.
`Several vascular conduits have been tried and in
`
`common use including preserved bovine vein grafts, dacron
`
`prosthetic grafts,
`
`teflon, and umbilical artery grafts.
`
`The
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`MTF Ex. 1009, pg. 5
`
`
`
`\N()93/10722
`
`P(3T/lfi592/10165
`
`-4-
`
`high i
`
`ncidence of the thrombosis and infection of these grafts
`To construct an ideal vascular graft, the
`is also a problem.
`graft
`should be non—thrombogenic,
`easily accessible,
`of
`extensible, and flexible.
`_variab1e.diameters and lengths,
`Amniotic and chorionic collagen tubes or conduits
`according to the present invention are non-immunogenic and can
`tubes of variable lengths and diameters
`be constructed into
`The amniotic and chorionic tubes of the
`readily and easily.
`invention are well suited for use as nerve grafts or
`present
`pass
`as bypass conduits for coronary bypass or vascular by
`surgery.
`
`rd side, which in the
`
`Summary of the Invention
`The present invention is directed to providing nerve
`and vessel tubes or conduits for grafting to bridge gaps in
`nerves and vessels. The physical.characteristics of the Types
`I,
`II,
`III collagen derived. from,
`the amnion and, chorion
`placentas in membrane form have been modified and the tubes
`constructed so that they are maintained patent and flexible so
`blood flows through their lumen and their walls can withstand
`and in which the fetal side or
`the interstitial pressure,
`shiny side of the membrane is the inwa
`case of nerve grafts promotes axon growth.
`to provide such a
`In one aspect of the invention,
`d chorion is obtained from fresh placentas,
`the amnion and chorion layers are separated
`cellular monolayer material
`membrane
`
`graft, amnion an
`
`preferably human,
`from the placenta and each other,
`overlying the basal lamina on the fetal side of the
`the
`is removed,
`such as by exposure to trypsin or pepsin,
`amnion and chorion is rinsed repeatedly with phosphate buffer
`solution or distilled water until clean, the amnion or chorion
`is then cross-linked either by exposure to gamma radiation or
`-linking such as with glutaraldehyde, which
`chemical cross
`provides- protection against. viral
`sterilizes the tissue,
`strengthens and permits remodeling the
`' disease transmission,
`material from sheet to conduit form.
`The amnion and chorion
`sheets are then wrapped in layers so that the fetal surface,
`
`10
`
`15
`
`20
`
`25
`
`3O
`
`35
`
`-
`
`ix
`
`MTF Ex. 1009, pg. 6
`
`
`
`W0 93/ 10722
`
`PCT/US92/10165
`
`-5-
`
`which is shiny,
`finished tube.
`
`is directed toward the inner surface of the
`The number of wraps will depend upon the
`
`length and diameter of the tube. The tubes are then dried and
`
`placed in bottles which are sealed,
`
`labeled and, if desired,
`
`exposed to 2.5 M rads of gamma radiation to again sterilize
`
`and further cross-link the conduit collagen.
`
`If desired,
`
`the
`
`layers can be glued together by a suitable glue,
`
`such as a
`
`fibrin glue,
`
`to prevent delaminating, particularly in larger
`
`conduits, such as used for vascular grafts.
`
`The
`
`tubes or conduits
`
`can then be stored,
`
`for
`
`example at -20°C, until used.
`
`For nerve grafts, nerve promoting factors can be
`
`rused within the amnion and chorion tubes at
`
`the time of
`
`implantation,
`
`for
`
`example particles
`
`of
`
`basal
`
`lamina,
`
`fibronectin, collagen extract, nerve growth factors and other
`
`related growth factors.
`
`Accordingly,
`
`it
`
`is
`
`an object
`
`of
`
`the present
`
`invention to provide a tubular graft for joining proximal and
`
`distal stumps of a severed peripheral nerve or proximal and
`
`distalr ends
`
`of
`
`a vessel which
`
`avoids
`
`the
`
`foregoing
`
`disadvantages of
`mentioned above.
`
`the prior art
`
`and has
`
`the
`
`advantages
`
`It is a further object of the present invention to
`provide a graft for joining proximal and distal stumps of a
`
`severed peripheral nerve or proximal and distal ends of a
`
`vessel which comprises a cylindrical wall formed by layers of
`
`sheets of sterilized collagen, Types I, II, III,
`
`from human
`
`placenta from which its cellular material
`
`is substantially
`
`removed, which has its fetal side directed inwardly and its
`
`collagen cross—linked by
`
`irradiation or chemically,
`
`the
`
`cylinder having sufficient
`
`layers to maintain the cylinder
`
`patent or open, the cylinder having a length greater than the
`
`distance between the proximal and distal stumps or ends, and
`
`having a diameter at least equal to connecting tissues of the
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`distal stumps or ends.
`
`MTF Ex. 1009, pg. 7
`
`
`
`W0 93/10722
`
`PCT/US92/ 10165
`
`-5-
`
`invention is to
`A further object of the present
`provide such a graft in which the layers of amnion and/or
`chorion membranes are glued togethe-r effective to prevent
`delamination of the layers.
`the present
`It
`is still
`a further object of
`invention to provide such a chindrical graft in which its
`wall permits flow of interstitial fluid through it to provide
`and in the case of nerve
`early nourishment for the graft,
`grafts, to provide nourishment for growth of So
`the present
`It
`is a still further object of
`invention to provide such a cylindrical graft as a research
`model for inclusion of material for medication study of nerve
`
`hwann cells.
`
`regeneration in the field of neurology.
`It is a further object of the present invention to
`provide. a nerve graft
`comprised of
`an amniotic
`and/or
`chorionic tube containing related growth factors.
`It is a further object of the present invention to'
`provide a nerve graft.
`comprised of
`an amniotic and/or
`chorionic tube containing basal lamina particles.
`It is a further object of the present invention to
`provide such a graft which can be made in relatively long
`lengths, which will not kink, will retain its shape and in
`which the passageway remains patent or open.
`other and further objects, features and advantages
`'of the invention appear throughout.
`Description of Preferred Embodiments
`
`A. Amnion Harvesting and Preparation
`In this aspect of the invention, amnion is obtained
`from fresh human placentas.
`The placentas are from hospital
`labor and delivery within 24 hours of parturition. Placentas
`obtained only from mothers who have been screened for AIDS and
`hepatitis virus and who are not members of the high risk
`groups such as IV drug abusers are used. Care is taken to
`'avoid skin contact with blood and tissue and to minimize
`contamination of the work areas with these materials.
`
`H
`
`1"
`
`10
`
`15
`
`20
`
`25
`
`3O
`
`35
`
`MTF Ex. 1009, pg. 8
`
`
`
`W0 93/ 10722
`
`PCT/US92/ 10165
`
`-7-
`
`The amnion layer is separated from the placenta,
`
`such as by finger dissection.
`
`The largest possible pieces of
`
`amnion which are of uniform thickness are selected from all
`
`the amnion harvested.
`
`The selected pieces of membrane are
`
`thoroughly washed, preferably with phosphate buffered saline,
`
`or distilled water to remove all the blood and debris.
`
`Then
`
`the membranes are further washed until
`
`they are white and
`
`transparent.
`
`The cellular monolayer overlying the basal lamina on
`
`the fetal side of the membrane is removed, such as by exposure
`
`to trypsin. Membranes are immersed for two hours at
`
`room
`
`temperature in 1:1 solution of distilled water and trypsin.
`The trypsin used preferably is from the procine pancreas at a
`
`25%-concentration without calcium or magnesium.
`
`Following
`
`treatment with trypsin,
`
`the amnion is rinsed repeatedly,
`
`preferably with phosphate buffered saline, or with distilled
`
`water until clean, white membranes with no trace of pink
`
`trypsin are obtained.
`
`'
`
`Rinsed amnion sheets are bottled in distilled water
`
`and exposed to 500,000 rads of gamma radiation.
`
`Irradiating
`
`the amnion cross-links the collagen, sterilizes the tissue,
`
`provides animal protection against viral disease transmission
`
`and subsequent
`
`remodeling of
`
`the material
`
`from sheet
`
`to
`
`conduit or tubular form.
`
`The bottles are then stored in a
`
`freezer at -80°C.
`
`If desired, the amnion sheets may be cross-
`
`linked chemically such as with glutaraldehyde.
`
`B.
`
`Conduit Manufacturing
`
`Amnion is removed from frozen storage and then
`
`thawed for 3
`
`to 4 hours at
`
`room temperature.
`
`The amnion
`
`sheets are examined under the operating microscope for defects
`
`and determination of the fetal or shiny side of the membrane.
`
`By using a rolling machine, sheets of the amnion wide enough
`
`for
`
`a desired conduit
`
`length and diameter are carefully
`
`collected.
`
`The amnion sheets are oriented so that the fetal
`
`surface, which is shiny, would be directed towards the inner
`
`surface of the finished tube.
`
`10
`
`15
`
`2O
`
`25
`
`3O
`
`35
`
`MTF Ex. 1009, pg. 9
`
`
`
`
`
`VV()93/10722
`
`PCIF/lfl392/10165
`
`—8—
`
`minutes.
`
`The tubes are then
`
`Preferably, wrapping of the amnion sheets in layers
`is effected by using a highly polished stainless steel stent
`of the appropriate diameter, although the amnion sheets can be
`wrapped in any desired manner.
`The number of wraps or layers of the amnion sheets'
`necessary to maintain the. amnion. cylinder’s shape,
`avoid
`kinking and to keep it patent or open depend upon the length
`and diameter of the tube or cylinder.
`For example, conduits
`of 1.6-1.8 inches in diameter require approximately 10 wraps
`of amnion and 15 wraps are satisfactory for a diameter of 2.5.
`The conduits can be of any desired length.
`The tubes which
`stents are then dried in 40-60°C oven for about 30
`are on the
`Drying allows the tubes to be removed from the
`If desired, to prevent delamination, a suitable
`stents easily.
`n.be used to glue the
`adhesive or glue, such as fibrin glue, ca
`layers or wraps of amnion sheets together.
`and exposed to
`placed in bottles which are sealed,
`labeled,
`2,000,000 rads of gamma radiation to again sterilize and
`further cross-link the conduits collagen. After that,
`the
`conduits are stored in -20°C until used.
`Example 1
`In this example, the basement membrane integrity and
`lamina content of
`human
`amnion before,
`and after,
`the
`construction
`of
`the
`conduits
`were
`evaluated'
`by
`immunocytochemical methods.
`Human
`amniotic
`conduits
`according to the invention have low immunogenicity which was
`evidenced when different antigens in the amnion membrane,
`mainly collagen type 1, fibronectin, and laminin were tested
`by dot blot and ELISA techniques. For almost 90 years amnion
`has been used for a wide variety of medical and surgical
`indications
`and
`has
`been
`shown '_to
`be
`capable
`of
`neOvascularization.
`It has also been placed in subcutaneous
`pockets in human without evidence of acute rejection for as
`long as 7 weeks.
`,
`The fetal membranes are ofra complex biochemical
`structure with unique physical characteristics.
`They are
`
`4,,
`
`U-
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`MTF Ex. 1009. pg. 10
`
`
`
`WO 93/10722
`
`PCT/US92/10165
`
`-9-
`
`modified physically in the present
`
`invention by the process
`
`technique described herein into conduits which are semi-rigid,
`
`'resilient,
`
`and of variable length, diameter and thickness.
`
`The biochemical components of the amnion membrane are mainly
`
`5
`
`collagen type I and type III,
`
`laminin, fibronectin and other
`
`glycoproteins.
`
`Laminin has been shown
`
`to promote axon
`
`' extension by interacting with axonal glycoproteins that are
`
`members of the integrin family of receptors.
`
`The methods of
`
`processing the amnion membrane of
`preserved laminin
`as
`a
`significant
`
`the present
`component
`
`invention
`of
`these
`
`10 _
`
`components. The present method of using gamma irradiation has
`
`several advantages.
`
`It has been shown that all bacteria,
`
`fungi, and viruses (including AIDS) are destroyed at
`
`.20 M
`
`rads. Preferably, a dose of 2.5 M rads is used in order to
`
`15
`
`ensure complete sterility of the final material.
`
`This dose
`
`alsor
`
`increases
`
`amniotic
`
`collagen
`
`cross-linking which
`
`strengthens and thus enables the manufacture of the amniotic
`
`conduits in a semi-rigid form while retaining some resiliency
`
`to maintain patency after
`
`implantation.
`
`As
`
`indicated
`
`20
`
`previously, however, chemical cross—linking by known methods,
`
`such
`
`as
`
`exposure
`
`to glutaraldehyde,
`
`is
`
`effective
`
`and
`
`satisfactory.
`
`By immunological testing, the amniotic nerve conduit
`
`W
`
`showed
`
`a minimal, nonsignificant
`
`immune
`
`response,
`
`thus,
`
`25
`
`avoiding some of the inhibitory factors that could influence
`
`the regenerating nerve. Although amniotic conduits showed
`
`signs
`
`of, biodegradability and
`
`structural
`
`reorganization
`
`grossly and
`
`by electron microscopic
`
`examination,
`
`local
`
`inflammatory cells infiltration was slight, being limited to
`
`30
`
`the area surrounding the conduit wall, and it did not produce
`
`changes of chronic nerve compression. This latter development
`
`is a major problem encountered with the use of silicone rubber
`
`conduits
`
`for nerve
`
`regeneration recently presented for
`
`clinical use.
`
`35
`
`Long-term studies of the amniotic conduits showed
`
`major structural changes as evidenced by Schwann-like cells
`
`MTF Ex. 1009. pg. 11
`
`
`
`W0 93/10722
`
`PCT/U592/10165
`
`-10-
`
`'fé‘
`
`and collagen
`conduit.
`infiltration through the layers of
`indicating a phenomenon of biodegradability
`istic unique to the amniotic
`
`reorganization,
`and reorganization, a character
`
`conduit.
`
`the amniotic tube of the
`As previously mentioned,
`present invention can be used as a carrier for many materials
`that promote nerve regeneration, such as lamina, fibronectin,
`Collagen extract, nerve growth factors,
`and other related
`Collagen extract is collagen extracted from human
`factors.
`described in U.S. Patent No.
`
`such as
`fetal membranes
`5,002,071.
`For example, basal lamina can be used as freeze
`dried ground, polarized, or preserved in any preservative
`solution within the amnion tube at the time of implantation.
`Example 2
`
`nerve regeneration through a
`In this example,
`conduit. according to they present
`invention. was
`compared
`morphologically and functionally with autographs and other
`.types of nerve tubes in an experimental animal model.
`Eleven cats Were divided into five groups to assess
`nerve regeneration through a 4 cm gap of the tibial nerve.
`Group 1
`(3 animals) amnion tube.
`Group 2
`(3 animals) amnion tube and basal lamina as
`a neurotrophic factor extracted form muscle.
`Group 3
`(3 animals) nerve autograft.
`Group 4
`(1 animal)
`sham operation as a control.
`Group 5 (1 animal) no repair.
`The animals were followed for six months when they
`were harvested,
`and
`the
`nerve
`segments were
`studied
`
`morphologically and histologically.
`The cats were
`anesthetized using Demerol
`and
`phenobarbital intramuscular and a mixture of Halothane and No2
`by inhalation.
`The tibial nerve was exposed,
`and a 4 cm
`segment was excised and repaired as following:
`In group 1 (amnion) an amnion tube was sutured to
`the distal and proximal stumps ensuring a gap of 4 cm.
`Two
`stitches were placed 180° apart on each side and went through
`
`10
`
`15
`
`20
`
`25
`
`3O
`
`35
`
`.MWW
`
`MTF EX. 1009. P9. 12
`
`
`
`W0 93/ 10722
`
`PCP/US92/10165
`
`_11_
`
`the Whole thickness of the tube containing only the epineurium
`
`of the nerve stump.
`
`(In group 2
`
`(amnion tube - basal
`
`lamina)
`
`- same as
`
`above, a 4 cm segment of the nerve was excised, and the gap
`
`was bridged by an amniotic tube filled with basal lamina.
`
`In group 3
`
`(nerve autograft)
`
`a 4
`
`cm segment was
`
`excised,
`
`then reattached using epineural technique.
`
`In group 4
`left undisturbed.
`
`(sham)
`
`the tibial nerve was explored and
`
`In group 5 (no repair) a 4 cm segment of the tibial
`
`nerve was excised, and no repair was ensured by suturing the
`
`nerve stumps to the underlying muscle.
`
`The animals were followed for six months, and no
`
`functional improvement was noticed in any of the experimental
`
`groups excluding the sham.
`At
`six months
`
`the
`
`cats were
`
`sacrificed and
`
`morphological and histological studies were performed.
`
`The
`
`results recommend amnion basal
`
`lamina as a strong candidate
`
`for nerve regeneration and show that the addition of muscle
`
`basal
`
`lamina to the amniotic collagen tube enhances nerve
`
`regeneration.
`
`In axonal diameter histograms
`
`the amnion basal
`
`lamina group showed a distribution comparable to the sham with
`
`even larger axons and a considerable percentage of the axons
`
`(9%) fell in the range of from 1.5 to 2.25 microns.
`
`In the
`
`case of nerve autograft, the histogram was comparable with the
`
`sham but with more percentage of axons
`
`(37%) falling in the
`
`range from 0.5 to 0.75 microns.
`
`In the amnion tube group the
`
`axonal diameters ranged between 0.1 to 1 micron with most of
`
`the axons (58%) falling in the range 0.25 to 0.5 microns. The
`
`axonal diameter histograms showed that basal lamina helped in
`
`rendering larger axons and provided to be the closest to the
`normal.
`
`The conclusions from this example are that human
`
`amniotic conduits are strong substitutes for nerve grafting
`
`and muscle basal
`
`lamina proved to be a good neurotrophic
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`MTF Ex. 1009. pg. 13
`
`
`
`W0 93/10722
`
`PCT/US92/10165
`
`-12.—
`
`«A
`
`a)
`
`autografts.
`for nerve
`
`Amnion
`material when added to the amniotic collagen tube.
`to nerve
`conduits filled with. basal
`lamina are. superior
`Human amniotic collagen tubes are good conduits
`used to house
`any
`
`regeneration and
`
`can be
`
`5
`
`neurotrophic material.
`
`Example 3
`
`In this example, human chorion was substituted for
`human amnion.
`The chorion membrane was separated from the
`amnion membrane and was then harvested and treated the same as
`in paragraph A, and conduits formed the same as in paragraph
`B has properties similar to those set forth in Examples 1 and
`2, and is satisfactory for both nerve growth and vascular
`tubes. The step of using trypsin or pepsin may be omitted if
`the chorion is separated from the amnion.
`
`7
`'Example 4
`-
`Intthis example, collagen Types I, II, and II, and
`thereof derived
`from bovine
`amniotic
`and 7
`combinations
`chorionic membranes were substituted for human membranes and
`'prepared.and formed into conduits or tubes as described above
`and provide similar and satisfactory results for both nerve
`growth and vascular tubes as set forth above for the human
`membranes.
`
`The methods of the invention comprise joining the
`proximal and distal stumps or ends of a severed nerve or
`proximal and distal ends of a vessel with a tube comprised of
`at least one layer of sterilized cross-linked.membrane free of
`cellular or epithelial material comprising Types I, II, III
`collagen or mixtures thereof,
`laminin, fibronectin and other
`glycoproteins, having its fetal side directed inwardly.
`The
`membrane is sterilized and cross—linked by irradiation or
`chemically and has sufficient layers to maintain them patent
`in place.
`The tube has a length at
`least equal to the
`distance between the proximal and distal stumps or ends, and
`has a diameter at least equal and preferably slightly larger
`than connective tissue of the proximal and distal stumps or
`For nerve grafts the tube can contain nerve growth
`
`ends.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`MTF EX. 1009. P9. 14
`
`
`
`W0 93/ 10722
`
`PCT/ US92/ 1 01 65
`
`_13_
`
`factors, such as basal lamina, fibronectin or collagen extract
`
`or combinations of them, as previously mentioned. The tube is
`
`sutured or otherwise secured to the connective tissue adjacent
`
`the nerve stumps or vessel ends.
`
`The grafts are storable for extended periods of time
`
`providing a ready available source of graft material.
`
`The
`
`grafts can be formed of any required diameter and length, and
`
`the layers of
`
`the graft can be glued together to prevent
`
`delamination of the layers in use.
`
`Accordingly, grafts for bridging a gap between
`
`proximal and distal ends of a severed nerve or of a vessel
`
`comprising a cylinder having a wall
`
`formed of at
`
`least one
`
`layer of sterilized cross-linked membrane derived from Types
`
`I, II, III collagen or combinations thereof from placenta are
`
`suitable and satisfactory.
`
`Accordingly,
`
`the present
`
`invention is well suited
`
`and adapted to attain the objects
`
`and ends
`
`and has
`
`the
`
`features and advantages mentioned as well as others inherent
`
`therein.
`
`While presently preferred embodiments
`
`of
`
`the
`
`invention have been given for the purposes of disclosures,
`
`changes and modifications can be made within the spirit of the
`
`invention as defined by the scope of the appended claims.
`
`What is claimed is:
`
`MTF Ex. 1009. pg. 15
`
`
`
`W0 93/10722
`
`PCI'/USQZ/10165
`
`-14-
`
`Claims
`
`w
`
`A graft for bridging a gap between proximal and
`1.
`distal ends of a severed nerve and proximal and.dista1 ends of ,
`a vessel comprising,
`,
`a cylinder having a length at least equal to
`the distance between the proximal and distal ends of
`the severed nerve or vessel and a diameter at least
`equal
`to the diameters of the proximal and the
`distal ends of the Severed nerve or vessel,
`said cylinder having a wall formed of at least
`one layer of sterilized cross-linked membrane,
`said membrane comprising Collagen selected from the
`group consisting of Type I, Type II, Type III, and
`mixtures thereof,
`7
`said collagen being derived from the group
`consisting of amnion of a placenta, chorion of a
`
`placenta, and combinations thereof.
`
`2.
`
`The graft of Claim 1 wherein,
`said wall comprising at least twa layers of
`said membrane in sheet form glued together effective
`to prevent delamination of the layers in use.
`
`3.,
`
`7
`The graft of Claim 1 where,
`the graft permits flow of interstitial fluid
`
`through its cylindrical wall.
`
`4. The'graft of Claims 1, 2 or 3 where,
`the graft
`is a nerve graft and contains at
`least one nerve growth factor.
`
`5.
`
`The graft of Claims 1, 2 or 3 where,7
`the graft is a nerve graft and contains basal
`lamina,
`fibronectin, amniotic collagen extract or
`combinations thereof.
`
`MTF EX. 1009. P9. 16
`
`10
`
`15
`
`20
`
`25
`
`30
`
`
`
`W0 93/ 10722
`
`PCT/ US92/ 10165
`
`-15-
`
`6.
`
`A method for restoring nerve function of
`
`a
`
`severed move comprising,
`
`joining the proximal and distal ends of
`
`the
`
`severed nerve with the graft of Claims 1,
`
`2 or 3.
`
`5
`
`7.
`
`A method for restoring nerve function of a
`
`severed nerve comprising,
`
`joining the proximal and distal ends of
`
`the
`
`severed nerve with the graft of Claims 1,
`
`2 or 3,
`
`the nerve graft containing at least one nerve
`
`10
`
`growth factor.
`
`8.
`
`A method for restoring nerve function of a
`
`severed nerve comprising,
`
`joining the proximal and distal ends of
`
`the
`
`severed nerve with the graft of Claims 1,
`
`2 or 3
`
`15
`
`containing basal lamina.
`
`9. A method of replacing a blood vessel comprising,
`
`joining the proximal and distal ends of
`
`the
`
`blood vessel with the graft of Claims 1,
`
`2 or 3.
`
`20
`
`10.
`
`The graft of Claims 1, 2, 3, 4, or 5 where,
`the amnion or chorion membrane is from a human
`
`rplacenta.
`
`MTF Ex. 1009. pg. 17
`
`
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