U.S. patent application number 16/068210 was filed with the patent office on 2019-09-26 for human placental tissue graft products, methods, and apparatuses.
The applicant listed for this patent is CryoLife, Inc.. Invention is credited to Steven GOLDSTEIN, Candace LAW, Adam MARTINEZ.
Application Number | 20190290802 16/068210 |
Document ID | / |
Family ID | 59274450 |
Filed Date | 2019-09-26 |
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United States Patent
Application |
20190290802 |
Kind Code |
A1 |
GOLDSTEIN; Steven ; et
al. |
September 26, 2019 |
HUMAN PLACENTAL TISSUE GRAFT PRODUCTS, METHODS, AND APPARATUSES
Abstract
Provided herein are tissue grafts, and in particular human
placenta-derived tissue grafts and methods and articles for the
manufacture and use thereof.
Inventors: |
GOLDSTEIN; Steven; (Atlanta,
GA) ; MARTINEZ; Adam; (Marietta, GA) ; LAW;
Candace; (Smyrna, GA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CryoLife, Inc. |
Kennesaw |
GA |
US |
|
|
Family ID: |
59274450 |
Appl. No.: |
16/068210 |
Filed: |
January 5, 2017 |
PCT Filed: |
January 5, 2017 |
PCT NO: |
PCT/US17/12384 |
371 Date: |
July 5, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62327857 |
Apr 26, 2016 |
|
|
|
62276655 |
Jan 8, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 27/54 20130101;
A61L 27/3604 20130101; A61L 27/60 20130101; F26B 5/04 20130101;
A61L 2300/236 20130101; A61L 2300/434 20130101; A61L 27/16
20130101; A61L 2430/40 20130101; A61L 2300/414 20130101; A61L 27/16
20130101; A61P 9/10 20180101; C08L 5/08 20130101; A61L 27/3695
20130101; A61L 27/24 20130101; C12M 21/08 20130101; A61P 17/02
20180101; A61L 2300/254 20130101 |
International
Class: |
A61L 27/36 20060101
A61L027/36; A61L 27/54 20060101 A61L027/54; C12M 3/00 20060101
C12M003/00; A61P 17/02 20060101 A61P017/02; A61P 9/10 20060101
A61P009/10; F26B 5/04 20060101 F26B005/04 |
Claims
1-15. (canceled)
16. A tissue graft comprising: a processed human fetal amniotic
membrane comprising an amnion layer and a chorion layer wherein
processing the human fetal amniotic membrane comprises: removing
maternal decidua cells from the membrane; compressing the membrane;
and dehydrating the membrane, wherein processing the membrane
provides a dense matrix having a compact structure and a reduced
amount of maternal decidua cells without a step of delaminating the
amnion layer from the chorion layer.
17-22. (canceled)
23. The tissue graft of claim 16, wherein the dense matrix allows
the releases bioactives in a controlled or extended manner.
24. (canceled)
25. The tissue graft of claim 16, wherein the dense matrix is
resistant to degradative enzymes.
26-31. (canceled)
32. The tissue graft of claim 16, wherein removing maternal decidua
cells is carried out by mechanical, chemical, osmotic, and/or
enzymatic treatment.
33-42. (canceled)
43. The tissue graft of claim 16, wherein processing the membrane
further comprises folding the membrane.
44-52. (canceled)
53. The tissue graft of claim 16, wherein processing the membrane
further comprises removing cells of a trophoblast layer from the
membrane.
54. The tissue graft of claim 53, wherein removing the cells of a
trophoblast layer is carried out by mechanical, chemical, osmotic,
or enzymatic treatment.
55-57. (canceled)
58. The tissue graft of claim 16, wherein processing the membrane
further comprises decontaminating the membrane to reduce its
bioburden.
59. (canceled)
60. The tissue graft of claim 58, wherein the membrane is
decontaminated with ethanol.
61-74. (canceled)
75. The tissue graft of claim 17, wherein the compressing the
membrane step and the dehydrating the membrane step produces
visibly distinguishable surfaces of the membrane.
76-77. (canceled)
78. The tissue graft of claim 16, wherein the membrane is
dehydrated to equal to or less than 10% by weight moisture
content.
79-85. (canceled)
86. The tissue graft of claim 16, wherein the process further
comprises terminally sterilizing the membrane.
87-97. (canceled)
98. The tissue graft of claim 16, wherein the membrane is secured
onto a backing.
99-104. (canceled)
105. The tissue graft of claim 98, wherein the backing comprises
multiple tabs configured to secure different sizes of the tissue
graft.
106-131. (canceled)
132. A method of treating a wound on a patient comprising: applying
to the wound, a tissue graft comprising a human fetal amniotic
membrane comprising an amnion layer and a chorion layer wherein the
two layers have not been delaminated, wherein the membrane is
prepared by a process comprising maintaining the membrane so that
the amnion and chorion layers are not delaminated; removing
maternal decidua cells from the membrane; and compressing and
dehydrating the membrane to result in a dense and compact
matrix.
133-135. (canceled)
136. An apparatus for compressing and dehydrating a tissue,
comprising: a chamber defining an opening at one end, the chamber
being in fluid connection with a vacuum source; a support platform
covering the opening of the chamber; and a sealing sheet positioned
adjacent the perforated support platform opposite the chamber, the
sealing sheet being configured to seal the chamber upon
depressurization of the chamber by the vacuum source, wherein the
apparatus is configured to receive the tissue between the support
platform and the sealing sheet, such that the tissue is dehydrated
upon depressurization of the chamber.
137. The apparatus of claim 136, wherein the support platform
comprises a perforated rigid support layer and a moisture, liquid
and vapor permeable material layer, the moisture, liquid and vapor
permeable material layer being positioned opposite the chamber.
138-140. (canceled)
141. The apparatus of claim 136, wherein the sealing sheet
comprises a gas impermeable, compliant or conformable polymer
sheet.
142. (canceled)
143. The apparatus of claim 136, further comprising an air source
in fluid connection with the chamber and configured to deliver air
to the sealed chamber, to augment the dehydration of the
tissue.
144-145. (canceled)
146. The apparatus of claim 136, wherein the chamber contains a
desiccant material.
147-148. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/276,655, filed Jan. 8, 2016, and U.S.
Provisional Application No. 62/327,857, filed Apr. 26, 2016, both
of which are hereby incorporated by reference in their
entireties.
BACKGROUND OF THE INVENTION
[0002] The present disclosure relates generally to tissue grafts,
and relates more particularly to human placenta-derived tissue
grafts and methods for the manufacture and use thereof.
[0003] Current advanced wound care technology includes
bioengineered skin substitutes that are effective as coverings for
healing chronic wounds, but are expensive to construct and may
require multiple applications of the constructs to the wound over
an extended healing period. Other advanced wound coverings include
human allograft based products, including cadaveric skin and fetal
membrane-derived tissue grafts. Commercial human fetal membrane
(also interchangeably referred to herein as "birth tissue," "human
placenta," "human placental," "human amniotic" or
"placenta-derived" tissue) allograft products include grafts of
processed placental tissue in single layer or laminate form. Such
processing includes drying, cryopreserving, and/or micronizing the
placental tissue. Such human allograft wound coverings result in a
shorter time to wound closure, more durably healed wounds, and a
higher wound healing percentage as compared to non-human tissue
wound care products. However, current processing of placental
tissue may diminish the retention of critical bioactives in the
tissue, such that the effectiveness of the placental tissue at
promoting cell in-migration, proliferation, and differentiation or
in limiting chronic inflammation is reduced. Additionally, such
human placental tissue can stimulate activity of allogeneic
lymphocytes, indicating presence of alloantigenicity, which may
result in inflammation and attenuated wound healing effect.
Moreover, current processing techniques may also contribute to
increased observed variability of physical and biochemical
properties among tissue grafts processed from different donors.
SUMMARY OF THE INVENTION
[0004] Described herein are methods of producing human placental
tissue grafts that improve retention of bioactives in the processed
tissue and reduce the antigenicity of the tissue, in addition to
human placental tissue grafts having improved healing and handling
properties.
[0005] In one aspect, described herein are tissue grafts
comprising: a processed human fetal amniotic membrane comprising an
amnion layer and a chorion layer wherein processing the human fetal
amniotic membrane comprises: removing maternal decidua cells and
cells of a trophoblast layer from the membrane; decontaminating the
membrane to reduce its bioburden; compressing the membrane; and
dehydrating the membrane, wherein processing the membrane provides
a dense matrix having a reduced amount of maternal decidua cells
without a step of delaminating the amnion layer from the chorion
layer. In some embodiments, removing the maternal decidua cells and
the cells of a trophoblast layer is carried out by mechanical,
chemical, osmotic, or enzymatic treatment. In some embodiments,
removing the cells of a trophoblast layer is carried out
concurrently to removal of the maternal decidua cells. In some
embodiments, the membrane is decontaminated with ethanol, peracetic
acid, one or more antibiotics, or combinations thereof. In some
embodiments, compressing the membrane comprises compacting the
membrane. In some embodiments, the compressing or compacting the
membrane step and the dehydrating the membrane step are carried out
simultaneously. In some embodiments, the compressing or compacting
step is subsequent to dehydrating. In some embodiments, the
compressing or compacting step is prior to dehydrating. In some
embodiments, the compressing or compacting the membrane step and
the dehydrating the membrane step produces visibly distinguishable
surfaces of the membrane. In some embodiments, the first surface of
the membrane is relatively shiny and the second surface of the
membrane is relatively matte. In some embodiments, the membrane is
dehydrated to equal to or less than 20% by weight moisture content.
In some embodiments, the membrane is dehydrated to a moisture
content amount that allows for at least six month stability at room
temperature. In some embodiments, the process further comprises
packaging the membrane under inert conditions. In some embodiments,
the process further comprises terminally sterilizing the membrane.
In some embodiments, the membrane is secured onto a backing.
[0006] In another aspect, described herein is a tissue graft
comprising a processed human fetal amniotic membrane comprising an
amnion layer and a chorion layer wherein processing the human fetal
amniotic membrane comprises: removing maternal decidua cells from
the membrane; compressing the membrane; and dehydrating the
membrane, wherein processing the membrane provides a dense matrix
having a reduced amount of maternal decidua cells without a step of
delaminating or separating the amnion layer from the chorion layer.
In some embodiments, processing the human fetal amniotic membrane
comprises removing the trophoblast cell layer. In some embodiments,
compressing the membrane comprises compacting the membrane.
[0007] In some embodiments, the membrane matrix is collagenous. In
some embodiments, the membrane matrix comprises bioactives. In some
embodiments, the membrane matrix comprises glycosaminoglycans. In
some embodiments, the membrane matrix comprises hyaluronic
acid.
[0008] In some embodiments, the membrane matrix is more resistant
to degradation at a wound or graft site as compared to an
uncompressed human amniotic membrane. In some embodiments, the
membrane matrix releases bioactives in a slow, controlled or
extended manner as compared to an uncompressed human amniotic
membrane. In some embodiments, the membrane matrix is resistant to
degradative enzymes as compared to an uncompressed human amniotic
membrane. In some embodiments, the release of bioactives is
extended over at least three days. In some embodiments, the release
of bioactives is extended over at least five days. In some
embodiments, the release of bioactives is extended over at least
seven days. In some embodiments, the release of bioactives is
extended over at least ten days. In some embodiments, the release
of bioactives is extended over at least fourteen days. In some
embodiments, the release of bioactives is extended over at least 30
days. In some embodiments, the membrane matrix acts as a barrier to
pathogens. In some embodiments, the membrane matrix allows for
slow, controlled, or extended release of active growth factors and
protease inhibitors at a wound or graft site.
[0009] In some embodiments, the membrane is not patterned or
labeled to enhance durability and handling.
[0010] In some embodiments, the membrane comprises active growth
factors, protease inhibitors or both. In some embodiments, the
membrane comprises cytokines, extracellular matrix proteins, or
combinations thereof. In some embodiments, the membrane comprises
basic fibroblast growth factor (bFGF), hepatocyte growth factor
(HGF), tissue inhibitor of metallopeptidase inhibitor-1 (TIMP-1),
or combinations thereof.
[0011] In some embodiments, removing maternal decidua cells is
carried out by mechanical, chemical, osmotic, and/or enzymatic
treatment. In some embodiments, the membrane is processed without
enzymatic treatment. In some embodiments, the maternal decidua
cells are treated with a hypotonic solution prior to removal. In
some embodiments, removing the maternal decidua cells reduces the
alloantigenicity of the membrane. In certain instances, wherein the
alloantigenicity is less than 1 peripheral blood mononuclear cell
(PBMC) Stimulation Index in a PBMC BrdU proliferation assay. In
some embodiments, the membrane is not antigenic.
[0012] In some embodiments, the process further comprises removing
cells from a trophoblast layer from the membrane. In certain
instances, removing the cells from trophoblast layer is carried out
by mechanical, chemical, osmotic, and/or enzymatic treatment. In
certain instances, processing comprises an enzymatic treatment. In
some embodiments, the enzyme is selected from the group consisting
of trypsin, thermolysin, collagenase, metalloproteinase, dispase,
hyaluronidase, papain, elastase, and pronase. In some embodiments,
the enzyme is collagenase. In some embodiments, the enzyme is
hyaluronidase. In some embodiments, the enzyme is elastase. In
certain instances, the membrane is processed without enzymatic
treatment. In certain instances, removing the cells from
trophoblast layer is carried out concurrently to removal of the
maternal decidua cells. In certain instances, the trophoblast layer
is treated with a hypotonic solution prior to its removal.
[0013] In some embodiments, removal of the maternal decidua cells
reduces the alloantigenicity of the membrane. In some embodiments,
the alloantigenicity is less than 1 peripheral blood mononuclear
cell (PBMC) Stimulation Index in a PBMC BrdU proliferation assay.
In some embodiments, the membrane is not antigenic.
[0014] In some embodiments, the process further comprises folding
the membrane. In some embodiments, the folded membrane has the
amnion layer on the outside of the folded membrane. In some
embodiments, the folded membrane has the chorion layer on the
outside of the folded membrane. In some embodiments, the membrane
is folded once. In some embodiments, the membrane is folded two to
seven times. In some embodiments, the membrane is folded along a
diagonal.
[0015] In some embodiments, the process further comprises stacking
at least two membranes. In some embodiments, stacking at least two
membranes results in a tissue graft having a chorion layer on the
outside. In some embodiments, stacking at least two membranes
results in a tissue graft having an amnion layer on the
outside.
[0016] In some embodiments, the process further comprises rolling
the membrane into a multilayered cylinder. In some embodiments, the
membrane is folded over or rolled along a diagonal.
[0017] In some embodiments, the process further comprises
decontaminating the membrane to reduce its bioburden. In certain
instances, the membrane is decontaminated with ethanol, peracetic
acid, one or more antibiotics, or combinations thereof. In certain
instances the membrane is decontaminated with ethanol. In certain
instances, the membrane is decontaminated with 60%-80% v/v ethanol.
In certain instances, the membrane is decontaminated with 60%, 61%,
62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, or 80% v/v ethanol. In certain instances,
the membrane is decontaminated with 60% v/v ethanol. In certain
instances, the membrane is decontaminated with 65% v/v ethanol. In
certain instances, the membrane is decontaminated with 70% v/v
ethanol. In certain instances, the membrane is decontaminated with
75% v/v ethanol. In certain instances, the membrane is
decontaminated with 80% v/v ethanol. In other instances, the
membrane is decontaminated with peracetic acid. In other instances,
the peracetic acid is at a concentration of about 0.01% to about 1%
v/v. In other instances, the peracetic acid is buffered. In other
instances, the buffer is at a pH of about 4.5 to about 7.5. In
other instances, the buffer is a phosphate, HEPES, MOPS, TES,
citrate, acetate, bicarbonate, PIPES, BES or Tris buffer. In other
instances, the membrane is decontaminated with one or more
antibiotics. In some instances, the bioburden is undetectable. In
some instances, the bioburden is zero. In some instances, the
bioburden is measured by an assay detecting microbial presence. In
some instances, the decontamination preserves the bioactives of the
membrane.
[0018] In some embodiments, the process is done under aseptic
conditions. In some embodiments, the process done under aseptic
conditions does not comprise a sterilization step.
[0019] In some embodiments, the compressing or compacting the
membrane step and the dehydrating the membrane step are carried out
simultaneously. In some embodiments, the compressing or compacting
step is subsequent to dehydrating. In some embodiments, the
compressing or compacting step is prior to dehydrating. In some
embodiments, the compressing or compacting the membrane step and
the dehydrating the membrane step produces visibly distinguishable
surfaces of the membrane. In some instances, the first surface of
the membrane is relatively shiny and the second surface of the
membrane is relatively matte. In some embodiments, the compressing
or compacting the membrane step and the dehydrating the membrane
step preserves the bioactives of the membrane.
[0020] In some embodiments, the membrane is dehydrated to equal to
or less than 20% by weight moisture content. In some embodiments,
the membrane is dehydrated to equal to or less than 10% by weight
moisture content. In some embodiments, the membrane is dehydrated
to equal to or less than 5% by weight moisture content. In some
embodiments, the membrane is dehydrated to a moisture content
amount that allows for at least six month stability at room
temperature. In some embodiments, the membrane is dehydrated to a
moisture content amount that allows for at least twelve month
stability at room temperature. In some embodiments, the membrane is
dehydrated to a moisture content amount that allows for at least 24
month stability at room temperature. In some embodiments, the
membrane is dehydrated to a moisture content amount that allows for
at least five year stability at room temperature.
[0021] In some embodiments, the process further comprises
terminally sterilizing the membrane. In some instances, the
sterilizing is by ionizing radiation, ethylene oxide, supercritical
carbon dioxide, peracetic acid, or combinations thereof.
[0022] In some embodiments, the membrane comprises at least 5,000
pg/mg of bFGF. In some embodiments, the membrane comprises at least
10,000 pg/mg of bFGF. In some embodiments, the membrane comprises
at least 100 pg/mg of HGF. In some embodiments, the membrane
comprises at least 300 pg/mg of HGF. In some embodiments, the
membrane comprises at least 5,000 pg/mg of TIMP-1. In some
embodiments, the membrane comprises at least 10,000 pg/mg of
TIMP-1. In some embodiments, the membrane comprises at least 7,000
ng/mg of HA. In some embodiments, the membrane comprises at least
15,000 ng/mg of HA. In some embodiments, the membrane comprises at
least 5,000 pg/mg of bFGF, at least 100 pg/mg of HGF, at least
5,000 pg/mg of TIMP-1, and at least 7,000 ng/mg of HA.
[0023] In some embodiments, the membrane is secured onto a backing.
In some instances, the membrane is adhered to a backing during the
dehydrating step. In some instances, the backing comprises a
material selected from high density polyethylene (HDPE), low
density polyethylene (LDPE), ethylene/vinyl alcohol copolymer
(EVOH), polypropylene (PP), polyethylene terephthalate (PET).
amorphous polyethylene terephthalate (APET), glycol modified
polyethylene terephthalate (PET-G), polyethylene naphthalate (PEN),
ethylene acrylic acid copolymer (EAA), and polyamide (PA),
polyvinyl chloride (PVC), polyvinylidene chloride (PVDC),
polychlorotrifluoroethylene (PCTFE), vinylidene chloride/methyl
acrylate copolymer, polyamide, polyester, polyurethane, silicone, a
metalized film, an oxide coated film, nitrocellulose, nylon, and
combinations thereof.
[0024] In some instances, the backing comprises at least one tab
configured to secure the tissue graft. In certain instances, the
tab is defined by a cut in the backing.
[0025] In some instances, the backing comprises at least two tabs
configured to secure the tissue graft. In some instances, the
backing comprises multiple tabs configured to secure different
sizes of the tissue graft.
[0026] In another aspect, also described herein is a tissue graft
comprising a processed human fetal amniotic membrane comprising an
amnion layer and a chorion layer wherein processing the human fetal
amniotic membrane comprises: removing maternal decidua cells from
the membrane; decontaminating the membrane with buffered peracetic
acid; compressing the membrane; and dehydrating the membrane,
wherein processing the membrane provides a dense matrix having a
reduced amount of maternal decidua cells without a step of
delaminating or separating the amnion layer and chorion layer.
[0027] In some embodiments, the peracetic acid is at a
concentration of about 0.01% to about 1% v/v. In some instances,
the peracetic acid is buffered with a phosphate, HEPES, MOPS, TES,
citrate, acetate, bicarbonate, PIPES, BES, or Tris buffer. In
certain instances, the peracetic acid is buffered at a range of
about 4.5 to about 7.5.
[0028] In another aspect, also described herein is a tissue graft
comprising: a processed human fetal amniotic membrane comprising an
amnion layer and a chorion layer wherein processing the human fetal
amniotic membrane comprises: removing maternal decidua cells from
the membrane; decontaminating the membrane with one or more
antibiotics; compressing the membrane; and dehydrating the
membrane, wherein processing the membrane provides a dense matrix
having a reduced amount of maternal decidua cells without a step of
delaminating the amnion layer and chorion layer.
[0029] In another aspect, also described herein is a tissue graft
comprising a processed human membrane wherein processing the human
membrane comprises: compressing the membrane; and dehydrating the
membrane, wherein processing the membrane provides a dense matrix.
In some embodiments, the human membrane is skin, dermis, small
intestine, small intestine submucosa, urinary bladder, pericardium,
peritoneum, placenta amnion, chorion, umbilical cord, or
fascia.
[0030] In another aspect, also described herein is a tissue graft
comprising a processed human membrane wherein processing the human
membrane comprises: decontaminating the membrane with buffered
peracetic acid; compressing the membrane; and dehydrating the
membrane, wherein processing the membrane provides a dense matrix.
In some embodiments, the human membrane is skin, dermis, small
intestine, small intestine submucosa, urinary bladder, pericardium,
peritoneum, placenta amnion, chorion, umbilical cord, or
fascia.
[0031] In another aspect, also described herein is a tissue graft
comprising a processed human tissue or organ wherein processing the
human tissue or organ comprises: decellularizing the tissue or
organ; and decontaminating the membrane with buffered peracetic
acid. In some embodiments, the tissue or organ is placenta, heart,
lung, kidney, liver, blood vessel, nerve, tendon, ligament,
skeletal muscle, smooth muscle, or bone.
[0032] In another aspect, also described herein is a tissue graft
product comprising: a processed human fetal amniotic membrane
comprising an amnion layer and a chorion layer, and a backing,
wherein processing the human fetal amniotic membrane comprises:
compressing or compacting the membrane, dehydrating the membrane;
and securing the membrane onto a backing, wherein processing the
membrane provides a dense matrix without a step of delaminating the
amnion layer from the chorion layer.
[0033] In another aspect, also described herein is a tissue graft
comprising a processed human fetal amniotic membrane comprising an
amnion layer and a chorion layer, and a backing wherein processing
the human fetal amniotic membrane comprises: decontaminating the
membrane with buffered peracetic acid; compressing or compacting
the membrane; dehydrating the membrane; and securing the membrane
onto a backing, wherein processing the membrane provides a dense
matrix without a step of delaminating or separating the amnion
layer from the chorion layer.
[0034] In another aspect, also described herein is a tissue graft
comprising a sterilized, decontaminated human fetal amniotic
membrane comprising an amnion layer and a chorion layer wherein the
two layers have not been delaminated, wherein the maternal decidua
cells and cells from the trophoblast layer are removed from the
membrane, and wherein the membrane is compressed and dehydrated
into a dense matrix.
[0035] In another aspect, also described herein is a tissue graft
comprising a sterilized, decontaminated human fetal amniotic
membrane and a backing, wherein the maternal decidua cells and
cells from the trophoblast layer are removed from the membrane, and
wherein the membrane is compressed and dehydrated into a dense
matrix.
[0036] In another aspect, also described herein is a tissue graft
comprising a sterilized, decontaminated human membrane and a
backing, and wherein the membrane is compressed and dehydrated into
a dense matrix.
[0037] In another aspect, also described herein is a tissue graft
comprising a sterilized, decontaminated human fetal amniotic
membrane comprising an amnion layer and a chorion layer wherein the
two layers have not been delaminated, wherein the maternal decidua
cells and cells from the trophoblast layer are removed from the
membrane, wherein the membrane is compressed and dehydrated into a
dense matrix, and wherein the membrane is folded. In some
embodiments, the folded membrane has amnion layer on the outside of
the folded membrane. In some embodiments, the folded membrane has
chorion layer on the outside of the folded membrane. In some
embodiments, the membrane is folded once. In some embodiments, the
membrane is folded two to seven times.
[0038] In another aspect, also described herein is a tissue graft
comprising a sterilized, decontaminated human fetal amniotic
membrane comprising an amnion layer and a chorion layer wherein the
two layers have not been delaminated, wherein the maternal decidua
cells and cells from the trophoblast layer are removed from the
membrane, wherein the membrane is compressed and dehydrated into a
dense matrix, and wherein the membrane is rolled into a multilayer
cylinder.
[0039] In another aspect, also described herein is a tissue graft
comprising a sterilized, decontaminated human fetal amniotic
membrane comprising an amnion layer and a chorion layer wherein the
two layers have not been delaminated, wherein the maternal decidua
cells and cells from the trophoblast layer are removed from the
membrane by enzymatic treatment; wherein the membrane is treated
with at least one enzyme; and wherein the membrane is compressed
and dehydrated into a dense matrix.
[0040] In another aspect, also described herein is a tissue graft
comprising a sterilized, decontaminated human fetal amniotic
membrane comprising an amnion layer and a chorion layer wherein the
two layers have not been delaminated, wherein the maternal decidua
cells and cells from the trophoblast layer are removed from the
membrane; wherein the membrane is compressed and dehydrated into a
dense matrix; and wherein the membrane releases bioactives in a
controlled or extended manner as compared to an uncompressed
membrane. In some embodiments, the release of bioactives is
extended over at least 3 to 14 days. In some embodiments, the
release of the bioactives is extended over at least 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, or 30 days.
[0041] In another aspect, also described herein is a tissue graft
comprising a sterilized, decontaminated human tissue and a backing,
wherein the backing comprises at least two tabs configured to
secure the tissue graft.
[0042] In another aspect, also described herein are methods of
preparing a tissue graft comprising: removing maternal decidua
cells and cells from the trophoblast layer from a human fetal
amniotic membrane; compressing the membrane; and dehydrating the
membrane; wherein the membrane results in a dense matrix and the
amnion and chorion layers are not delaminated or separated.
[0043] In another aspect, also described herein are methods
treating a wound on a patient comprising: applying to the wound, a
tissue graft comprising a human fetal amniotic membrane comprising
an amnion layer and a chorion layer wherein the two layers have not
been delaminated or separated, wherein the membrane is prepared by
a process comprising maintaining the membrane so that the amnion
and chorion layers are not delaminated; removing maternal decidua
cells from the membrane; and compressing and dehydrating the
membrane to result in a dense and compact matrix.
[0044] In another aspect, also described herein are methods
treating a wound on a patient comprising: applying to the wound, a
tissue graft comprising a sterile, decontaminated human fetal
amniotic membrane comprising an amnion layer and a chorion layer
wherein the two layers have not been delaminated from each other,
wherein the maternal decidua cells and cells from the trophoblast
layer are removed from the membrane, and wherein the membrane is
compressed and dehydrated into a dense matrix.
[0045] In another aspect, also described herein, are methods of
enhancing recovery following cardiac surgery on a patient
comprising: applying to a cardiac area, a tissue graft comprising a
human fetal amniotic membrane comprising an amnion layer and a
chorion layer wherein the two layers have not been delaminated,
wherein the membrane is prepared by a process comprising
maintaining the membrane so that the amnion and chorion layers are
not delaminated; removing maternal decidua cells from the membrane;
and compressing and dehydrating the membrane to result in a dense
and compact matrix.
[0046] In another aspect, also described herein, methods of
minimizing damage to the cardiovascular system caused by a
myocardial infarction in a patient comprising: applying to a
cardiac area, a tissue graft comprising a human fetal amniotic
membrane comprising an amnion layer and a chorion layer wherein the
two layers have not been delaminated, wherein the membrane is
prepared by a process comprising maintaining the membrane so that
the amnion and chorion layers are not delaminated; removing
maternal decidua cells from the membrane; and compressing and
dehydrating the membrane to result in a dense and compact
matrix.
[0047] In another aspect, also described herein are apparatuses for
compressing and dehydrating a tissue, comprising: a chamber
defining an opening at one end, the chamber being in fluid
connection with a vacuum source; a support platform covering the
opening of the chamber; and a sealing sheet positioned adjacent the
perforated support platform opposite the chamber, the sealing sheet
being configured to seal the chamber upon depressurization of the
chamber by the vacuum source, wherein the apparatus is configured
to receive the tissue between the support platform and the sealing
sheet, such that the tissue is dehydrated by depressurization of
the chamber.
[0048] In some embodiments, the support platform comprises a
perforated rigid support layer and a moisture, liquid, and vapor
permeable material layer, the moisture, liquid, and vapor permeable
material layer being positioned opposite the chamber. In some
instances, the perforated rigid support layer comprises a sheet
having a plurality of apertures therethrough. In some instances,
the perforated rigid support layer is stainless steel, titanium,
aluminum, or other suitable metal or a rigid plastic. In some
instances, the moisture, liquid, and vapor permeable material layer
comprises a porous polypropylene sheet with pores of from about 50
micron to about 200 micron.
[0049] In some embodiments, the sealing sheet comprises a gas
impermeable, compliant or conformable polymer sheet. In some
embodiments, the sealing sheet comprises silicone.
[0050] In some embodiments, the apparatuses further comprise an air
source in fluid connection with the chamber and configured to
deliver air to the sealed chamber, to augment the dehydration of
the tissue. In some instances, the air source is an inert gas. In
some instances, the air source is nitrogen, argon, helium, or
carbon dioxide.
[0051] In some embodiments, the chamber contains a desiccant
material. In some instances, the desiccant material is activated
alumina, aerogel, benzophenone, bentonite clay, calcium chloride,
calcium sulfate, cobalt(II) chloride, copper(II) sulfate, lithium
chloride, lithium bromide, magnesium sulfate, magnesium
perchlorate, potassium carbonate, potassium hydroxide, silica gel,
sodium, sodium chlorate, sodium chloride, sodium hydroxide, sodium
sulfate, sucrose, or combinations thereof.
[0052] In some embodiments, the apparatuses are configured without
a heating element.
INCORPORATION BY REFERENCE
[0053] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0054] Relevant features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0055] FIG. 1 depicts a cross section of a placental tissue with
its primary two layers of amnion and chorion tissue. (Adapted from
FIG. 2 of Uchide et al., "Possible Roles of Proinflammatory and
Chemoattractive Cytokines Produced by Human Fetal Membrane Cells in
the Pathology of Adverse Pregnancy Outcomes Associated with
Influenza Virus Infection," Mediators of Inflammation, Vol.
2012.)
[0056] FIG. 2 (left) depicts an exemplary histological cross
section of human fetal tissue membrane after post-shipping and
post-dissection from the placenta and prior to removal maternal
decidua cells and cells in the fetal trophoblast layer. FIG. 2
(right) depicts an exemplary histological cross section of human
fetal tissue membrane after post-cell removal of maternal decidua
cells and cells in the fetal trophoblast layer.
[0057] FIG. 3 depicts an exemplary histological cross section of
human fetal tissue membrane prior to dehydrating and compressing
(left) and post dehydrating and compressing (right).
[0058] FIG. 4 shows a histological cross section of an exemplary
human fetal tissue membrane graft (panels B and D) and a
comparative commercial fetal tissue graft (panels A and C, adapted
from Koob et al., J Biomed Mater Res B Appl. Biomater, 2014, 102(6)
1353-62) stained with hematoxylin and eosin (panels A and B) and
Alcian blue (panels C and D).
[0059] FIG. 5 depicts an exemplary vacuum assisted drying
apparatus: 1 Vacuum assisted drying apparatus, 2 handles for
transporting apparatus, 3 ribbed supports, 4 lower ledge for
holding support platform, 5 upper ledge for sealing, 6 vacuum
outflow port.
[0060] FIG. 6 depicts an exemplary perforated rigid support
layer.
[0061] FIG. 7 depicts an exemplary moisture, liquid and vapor
permeable material layer.
[0062] FIG. 8 depicts an exemplary sealing sheet.
[0063] FIG. 9 depicts another view of an exemplary drying and
compressing apparatus.
[0064] FIG. 10 depicts an exemplary flexible backing with multiple
tabs cut into the flexible backing material to secure and hold
various sizes of a tissue graft.
[0065] FIG. 11 depicts an assay of bioactive concentrations (bFGF,
HGF, and TIMP-1) of an exemplary tissue graft as compared to a
commercially available product (Koob et al., J Biomed Mater Res B
Appl. Biomater, 2014, 102(6) 1353-62).
[0066] FIG. 12 depicts the percent reduction in wound area versus
time with an exemplary tissue graft or an absorbent dressing
control material in a diabetic animal wound healing model.
[0067] FIG. 13 depicts the percent reduction in wound perimeter
versus time with an exemplary tissue graft or an absorbent dressing
control material in a diabetic animal wound healing model.
[0068] FIG. 14 depicts suture retention strength versus various
dosages of ionizing radiation or control (zero kGy).
[0069] FIG. 15 depicts suture retention strength of an exemplary
tissue graft that is pulled in either in a x-direction or a
y-direction.
[0070] FIG. 16 shows tensile stress in non-irradiated versus
irradiated exemplary tissue grafts.
[0071] FIG. 17 shows tensile strain in non-irradiated versus
irradiated exemplary tissue grafts.
[0072] FIG. 18 shows tensile stress in an exemplary tissue graft
that is pulled in either in a x-direction or a y-direction.
[0073] FIG. 19 shows tensile strain in an exemplary tissue graft
that is pulled in either in a x-direction or a y-direction.
[0074] FIG. 20 is a photograph an exemplary tissue graft.
[0075] FIG. 21 shows an exemplary vacuum assisted drying apparatus:
1 Vacuum assisted drying apparatus, 2 handles for transporting
apparatus, 3 ribbed supports, 4 lower ledge for holding support
platform, 5 upper ledge for sealing, 6 vacuum outflow port, 7 air
inflow port.
[0076] FIG. 22 shows the amount of hyaluronic acid released into
extraction medium following sequential extractions, rocking at
4.degree. C. and 37.degree. C. for 48 hours each.
DETAILED DESCRIPTION OF THE INVENTION
[0077] The present disclosure relates to human placenta-derived
tissue grafts that display high bioactives content and reduced or
no alloantigenicity and display improved healing properties. As
used herein, the term "bioactives" includes, but is not limited to,
cell binding motifs, extracellular matrix (ECM) components,
collagen, elastin, hyaluronic acid, laminin, vimentin, fibronectin,
growth factors, glycosaminoglycans (GAGs), proteoglycans,
proteases, collagenases, gelatinases, protease inhibitors,
cytokines, matricryptins, matrikines, ground substance components,
and the like. The placenta-derived wound coverings described
herein, in some embodiments, also display improved handling and
performance properties, such as compactness, flexibility, suture
retention, and resistance to enzymatic attack, slower or extended
release of bioactives, and longer residence time at a wound or
other implantation sites.
[0078] While the present disclosure refers to the use of these
tissue graft products as "wound coverings" or "tissue grafts," it
should be understood that the use and applications of such grafts
are not limited to the treatment of wounds. Rather, these tissue
graft products are contemplated for use in a variety of
applications and treatments, including but not limited to use as
anti-adhesion barriers, or for use in a variety of surgical
procedures from closures to suspensory slings as well as to reduce
atrial fibrillation post cardiac surgery or reduces pathogenic
cardiac remodeling and scarring after myocardial infarction, as is
discussed in greater detail herein. It is also contemplated that
the tissue grafts products are used in scaffolding for tissue and
organ regeneration, as an enhanced environment for stem cell
attachment and function, and the like.
[0079] Generally, as shown in FIG. 1 (adapted from FIG. 2 of Uchide
et al., "Possible Roles of Proinflammatory and Chemoattractive
Cytokines Produced by Human Fetal Membrane Cells in the Pathology
of Adverse Pregnancy Outcomes Associated with Influenza Virus
Infection," Mediators of Inflammation, Vol. 2012), placental tissue
has two primary layers of tissue: amnion, chorion as well as a
layer of maternal decidua. The amnion is the innermost layer of the
placenta (i.e., the layer that faces the fetus), and consists of
epithelial cells, a basement membrane composed of thin reticular
fibers, a thick compact layer, and a fibroblast layer. The chorion
is the maternal side of the placenta and consists of a reticular
layer/basement membrane composed of a layer of dense connective
tissue, and a trophoblast layer. The trophoblast layer of the
chorion is in intimate contact with the maternal decidua of the
uterine wall. An intermediate spongy layer connects the amnion and
chorion layers.
[0080] It is contemplated that fetal membrane tissue grafts
generally provide shorter time to wound closure and higher wound
healing percentage by redirecting chronic, non-healing wounds into
a healing pattern by promoting cell in-migration, proliferation,
and differentiation, along with anti-inflammatory action and
antimicrobial action. However, conventional placental tissue
grafts, in some embodiments, display variable, decreased, or
impeded retention of the bioactives thought to be critical or
important to such healing, due to aggressive, harmful, and/or
disruptive processing techniques. Therefore, placental processing
that improves retention of such bioactives, including growth
factors and other factors important to the promotion of wound
healing, would provide improved wound healing properties of these
products. The present disclosure therefore relates to processes and
apparatuses for preparing tissue grafts while maintaining such
critical bioactives, as well as the tissue grafts prepared
therefrom and the use of such products in would healing and other
treatments.
[0081] Additionally, many conventional placental processing
techniques retain the maternal cells derived from the endometrium
along with the fetal membrane as well as maternal cells within as
the trophoblast layer of the chorion, all of which can produce an
immune response in the recipient, stimulating activity of
allogeneic lymphocytes, indicating presence of alloantigenicity,
which in some cases results in a pro-inflammatory effect or graft
rejection. As such, the present disclosure relates to processes and
apparatuses for preparing tissue grafts in which at least a portion
of the maternal decidua and trophoblast cells are removed, as well
as the tissue grafts prepared therefrom and the use of such
products in wound healing and other treatments.
[0082] Furthermore, due to harsh processing techniques and
retention of the maternal cells, conventional placental tissue
grafts typically are non-uniform in appearance and structure and
brittle, or inflexible, in handling, making it difficult to
effectively align and apply the product to a deep and/or uneven
wound surface.
[0083] In certain embodiments, the tissue grafts of the present
disclosure are room temperature stable (i.e., the products need not
be cryopreserved, frozen, or refrigerated for stable transport
and/or storage).
[0084] Tissue grafts and methods and apparatuses for preparing and
using the same are described in greater detail below. Such tissue
grafts in some embodiments display one or more superior
handling/performance properties as compared to traditional
placental tissue grafts, such as compactness, flexibility, suture
retention, enhanced stability, extended shelf life, high bioactives
content, reduced alloantigenicity, resistance to enzymatic attack,
slower or extended release of bioactives as compared to
commercially available tissue grafts, longer residence time at a
wound or other implantation sites, and/or improved healing. As will
be discussed, such superior properties in some embodiments are
related to or achieved by one or a combination of the following
processing features of the disclosed tissue grafts: maintenance of
the native bilayer architecture of the amnion and chorion
throughout processing and in the resulting tissue graft, removal of
maternal exogenous cells from the tissue, decontamination while
limiting or avoiding the leaching of bioactives, application of
pressure to the tissue during drying, dehydration of the tissue,
processing or packaging the tissue such that the faces of the
tissue graft are visibly distinguishable from one another, and
terminal sterilization of the tissue graft.
Tissue Grafts & Methods for Preparing Placental Tissue
Grafts
Tissue Procurement & Transport
[0085] Placental tissue is obtained from donated placenta, and in
some cases, is harvested after Caesarean section or vaginal birth.
In certain embodiments, the placental tissue is obtained from a
donated placenta harvested from a birth at or after 36 weeks of
gestation, such as prior to 39 weeks of gestation. In other
embodiments, placental tissue from a premature birth, i.e., prior
to 36 weeks of gestation, is used; however, the amounts of
bioactives and enzymes in such tissue, in some cases, differ from
tissue from a placenta at or after 36 weeks of gestation. In
certain embodiments, donated placentas are screened to exclude
those from certain donors such as those with gestational
diabetes.
[0086] In certain embodiments, the donated placenta is harvested,
chilled, and rinsed, then placed in a container for shipping to the
tissue processing facility. In some embodiments, the harvested,
donated placenta is chilled and then rinsed. In some embodiments,
the harvested, donated placenta is rinsed and then chilled. In some
embodiments, the harvested, donated placenta is chilled for
transport and rinsed at the tissue processing facility. In some
embodiments, the membrane is left attached to the placenta during
rinsing and shipping. In another embodiment, the membrane is
dissected away from the placenta at the acquisition site, and then
processed as described below with reference to processing the
placenta. Removal of the membrane at the placenta acquisition site
advantageously decreases the amount of material to be shipped,
providing easier control of the material. However, if the membrane
is shipped without the placenta, it should be placed on a backing
or other structure to stabilize the tissue in its native structure
and prevent shear of the membrane layers or their
configuration.
Tissue Processing--Removal of Maternal Cell Matter
[0087] Placenta or placental membrane is provided to a processing
facility, e.g., following procurement and shipping. In embodiments
in which the placenta with membrane attached is provided, the
membrane is removed from the placenta by gross dissection. In
certain embodiments, the adherent area of amnion and chorion layers
that is dissected from the placenta is from about 50 cm.sup.2 to
about 500 cm.sup.2, or larger, depending on the size of the
placenta. In some embodiments, the adherent area of amnion and
chorion layers that is dissected from the placenta is from about
400 cm.sup.2 to about 800 cm.sup.2. In some embodiments, the
adherent area of amnion and chorion layers that is dissected from
the placenta is about 600+/-200 cm.sup.2. In certain embodiments,
the adherent area of amnion and chorion layers that is dissected
from the placenta is from about 50 cm.sup.2, about 100 cm.sup.2,
about 150 cm.sup.2, about 200 cm.sup.2, about 250 cm.sup.2, about
300 cm.sup.2, about 350 cm.sup.2, about 400 cm.sup.2, about 450
cm.sup.2 about 500 cm.sup.2, about 550 cm.sup.2, about 600
cm.sup.2, about 650 cm.sup.2, about 700 cm.sup.2, about 750
cm.sup.2, or about 800 cm.sup.2.
[0088] Thus, the human fetal (i.e., placental) tissue membrane at
this stage (i.e., post-shipping and post-dissection from the
placenta, but pre-processing) comprises the amnion, chorion, and
maternal decidua/fetal trophoblast layers in the native, or
natural, architecture, as shown in the pre-cell removal (left-hand
side) histology of FIG. 2.
[0089] In certain embodiments, the human fetal support tissue
membrane is then contacted with a hypotonic solution to osmotically
swell the cell matter at the maternal side of the membrane. For
example, the hypotonic solution, in some embodiments, is sterile
water, a suitable saline solution, such as a hypotonic saline
solution, or other biocompatible hypotonic solutions having an
osmotic concentration of from about 0 to about 50 mOsm/L. It was
discovered that if the tissue is exposed to hypertonic or isotonic
solutions, as in some conventional placental tissue processing, the
removal is more difficult and the chorion is more likely to be torn
resulting in loss of bilayer tissue. The present methods, which
instead involve contacting the placental tissue with a hypotonic
solution, advantageously avoid the issues of separation and tearing
of the amnion and chorion associated with the use of
hypertonic/isotonic solutions when removing certain cells.
[0090] After the tissue membrane is contacted with the hypotonic
solution to swell the cell matter on the maternal side of the
membrane, the swollen decidua and trophoblast cell matter, in some
embodiments, is then removed from the chorion connective, support
tissue layer of the membrane to produce an isolated human fetal
support tissue membrane which comprises amnion and the chorion
tissue layers, in which amnion and chorion layers are in an
original, undisrupted connective architecture, as shown in the
post-cell removal (right-hand side) histology of FIG. 2.
[0091] For example, in some cases, removing the swollen decidua and
trophoblast cell matter is performed by mechanical blunt removal or
scraping, such as with a scalpel or other suitable instrument,
and/or by manually peeling the swollen layers from the chorion. In
certain embodiments, the step of removing the swollen trophoblast
and decidua cell matter from the chorion results in removing
substantially all of the trophoblast and decidua cell matter from
the human fetal support tissue membrane. Thus, the resulting
modified membrane, in some embodiments, includes the complete
amnion layer, the reticular layer/basement membrane of the chorion
layer, with the immunogenic maternal origin decidua and
trophoblasts, which are fetal cells that, in some cases, also
contain maternal cells (e.g., macrophages), substantially removed
from the stromal collagen layer of the chorion. In certain
embodiments, the step of removing substantially all of the
trophoblast and decidua cell matter results in a non-antigenic
human fetal support tissue membrane. Advantageously, the connective
architecture of the amnion and chorion is maintained during this
process, which is believed to provide beneficial mechanical and
performance properties of the resulting products, as is discussed
in more detail herein.
[0092] In other embodiments, the decidua and trophoblast cell
matter is removed from the membrane by enzymatic degradation using
a suitable enzyme. For example, in some embodiments, proteases are
utilized to facilitate cellular dissociation either singly or in
mixtures. Suitable enzymes include, but are not limited to, trypsin
(a serine protease used for tissue dissociation), thermolysin,
collagenase, and other metalloproteinases (which release cells by
degradation of the structural protein collagen in the extracellular
matrix), dispase (a neutral protease which cleaves amino terminal
bonds of non-polar amino acids and digests fibronectin an
extracellular matrix cell binding protein), hyaluronidase (degrades
hyaluronic acid, a component of the extracellular matrix often
found associated with collagen), papain (a cysteine protease that
can be used to degrade extracellular matrix), elastase (a serine
proteinase used to degrade the extracellular matrix of tissues with
significant elastin content), and pronase (a mixture of proteolytic
enzymes from Streptomyces griesus having varied specificities). In
some embodiments, the decidua and trophoblast cell matter is
removed from the membrane by a collagenase. In some embodiments,
the trophoblast and decidua cell matter is removed from the
membrane by a hyaluronidase. In some embodiments, the decidua and
trophoblast cell matter is removed from the membrane by an
elastase. It was observed in some embodiments that enzymatic
degradation resulted in high levels of bioactives as compared to
other types of removal of trophoblast and decidua cell matter from
the human fetal amniotic membrane. FIG. 22 shows the amount of
hyaluronic acid contained within the human fetal amniotic membrane
before and after enzymatic treatment.
[0093] In other embodiments, the trophoblast and decidua cell
matter is removed through use of divalent metal cation chelators
that facilitate the removal of cells from the membrane. Suitable
divalent metal cation chelators include, but are not limited to,
ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic
acid (EGTA), and citrate buffers.
[0094] In other embodiments, the trophoblast and decidua cell
matter is removed through use of a detergent. Exemplary suitable
detergents include, but are not limited to, non-ionic detergents
such as Triton X-100.TM., sodium dodecyl sulfate (SDS), and
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
(CHAPS).
[0095] In some embodiments, combinations of these cell removal
techniques are used. For example, in some embodiments, the decidua
and trophoblast cell matter are removed by a hypotonic sterile
water rinse and EDTA solution (e.g., approximately 10 mM), to
separate the cell layer from the stromal collagen layer.
[0096] The present processing advantageously maintains the amnion
and chorion layers of the fetal membrane in their natural,
connective architecture, as shown in the post-cell removal
(right-hand side) histology of FIG. 2, while also providing for
removal of the decidua and trophoblast layers from the chorion.
That is, unlike other placental membrane processing methods, the
present process does not involve separation of the amnion from the
chorion at any time, and thereby does not result in loss of the
endogenous matrix components and possible damage to the membrane
that occurs when the amnion and chorion are separated, which is
believed to provide beneficial mechanical and performance
properties of the resulting membranes. Accordingly, no reassembly
or lamination of the amnion and chorion layers is required during
any stage of processing. Thus, in embodiments, the present graft is
not a structure formed by lamination. Rather, the natural anatomic
bilayer relationship of the amnion and chorion layers is maintained
from procurement of the tissue through application of the resulting
tissue graft at a patient site.
[0097] Additionally, in conventional processes in which the amnion
and chorion are separated, the intermediate spongy layer swells and
beneficial glycosaminoglycans, as well as other biological
components present in the spongy layer, are lost. Moreover, as will
be discussed in more detail below, the layers of reassembled
membranes (i.e., amnion and chorion that are laminated together
after separation), in some embodiments, do not compact as tightly
as the intact (non-separated fetal membrane) membranes.
[0098] Again, removal of the decidua is desirable because the
decidua cells are present in their own matrix that is highly
vascularized, making it susceptible to severe blood staining, the
thickness of the decidua is highly variable and non-uniform (i.e.,
heterogeneous) in thickness, and local architecture across the
membrane and between donors, and the decidua contains maternal
cells of many types including inflammatory cells (leukocytes) and
other cells (e.g., stromal fibroblasts and stem cells) as well as
other cells that, in some cases, cause immunogenicity. Removal of
the trophoblast layer is desirable because while this layer is of
fetal origin, it also contains maternal inflammatory or immunogenic
cells. Thus, tissue grafts prepared by the processes described
herein have a greater uniformity of thickness (i.e., more
homogeneous) and have no patches of discoloration or staining,
resulting in homogenous appearance and handling. Additionally,
maternal cells can present a potential for an antigenic response in
the graft recipient. Maternal macrophages are often found within
the decidua cell layer, further exacerbating the potential
antigenic response of the decidua layer. In conventional processes,
this issue is usually addressed by chemical treatment of the
chorion layer or by complete removal of the chorion layer, or the
issue is overlooked and the antigenic maternal cells are retained
in the membrane. However, removal of the chorion to limit implant
antigenicity results in the removal of important amounts of
bioactive molecules from the tissue as well as leading to a thinner
graft with less mechanical strength and increased potential for
degradation. Without the chorion, bioactives can also elute or
leach out of the amnion more rapidly.
[0099] In some embodiments, the epithelial cells of the amnion are
removed, such as by enzymatic means as discussed above in reference
to removal of the decidua. In other embodiments, the amnion is
decellularized (i.e., the epithelial cells are removed) by any
suitable chemical or physical means known in the art. In other
embodiments, the complete amnion layer is maintained with the
epithelium intact (i.e., the amnion is not decellularized).
Tissue Processing--Aseptic Conditions
[0100] In certain embodiments, the human fetal support tissue
membrane is obtained and processed under aseptic conditions,
thereby not requiring a sterilization step. Aseptic conditions
comprising aseptic technique is known by those of skill in the art
and includes use of filtered airflow, such as a surgical suite or a
laminar air flow cabinet; use of sterile tools, such as sterilized
forceps, sterilized scalpels, sterilized scissors, sterilized
pipettes, sterilized gloves; and use of sterile solutions and
buffers that have been sterile filtered and/or autoclaved; amongst
other methods known by one of skill in the art. Sterilizing
techniques, include but are not limited to autoclaving, UV
radiation exposure, gamma radiation exposure, rinsing in 70%
alcohol, ethylene oxide, 10% bleach, and/or iodine solutions.
Exemplary aseptic conditions for processing of a human fetal
support tissue membrane comprises obtaining the human fetal support
tissue in a sterile hospital environment, using sterile surgical
tools, wearing sterile gloves, a face mask, hair cover, shoe
covers, and sterile lab coat, and placing the human fetal support
tissue membrane into a sterile container, such as a sterilized
pouch, transporting the human fetal support tissue membrane to a
facility with a laminar air flow cabinet that has been disinfected
with 70% ethanol and/or UV radiation, and conducting the remaining
processing steps in the decontaminated laminar air flow cabinet or
other suitable decontaminated environment.
Tissue Processing--Decontamination
[0101] In certain embodiments, the human fetal support tissue
membrane is decontaminated by contacting the membrane with a
suitable disinfectant. In certain embodiments, the isolated human
fetal support tissue membrane which comprises amnion and the
chorion connective, supportive tissue layer, which amnion and
chorion are in an original, undisrupted connective architecture, is
contacted with a disinfecting concentration of a suitable
disinfectant. The disinfectant is used, in some embodiments, to
decontaminate the tissue such that the bioburden is reduced to
undetectable levels, i.e., below the threshold of detection by an
assay detecting microbial presence, while also reducing damage or
loss of endogenous matrix components and bioactive factors. In some
embodiments, the bioburden is reduced to zero.
[0102] In certain embodiments, the disinfectant is ethanol or
peracetic acid. For example, in some embodiments, the ethanol is an
aqueous solution of about 50 to about 100 percent ethanol, of about
65 to about 75 percent ethanol, or of about 70 percent ethanol (all
by volume). The ethanol is selected to achieve particular tissue
membrane properties, such as reduced water content, a less adherent
chorionic side compared to aqueous solutions (e.g., adherence
between chorion and plastic backing, such as LDPE), favorable
handling properties (e.g., stiffness, robustness), and an
unmeasurable bioburden allowing for a sterilization requirement of
substantially lower dosage of ionizing radiation. For example, it
was found that the use of ethanol at concentrations greater than
75%, by volume, results in a brittle tissue membrane product.
Specifically, it was found that after drying the tissue in the
drying process described below, tissue treated with ethanol from
75% to 100%, by volume, was stiff and brittle, as compared to
tissue treated with lower concentrations of ethanol.
[0103] In some embodiments, the peracetic acid is buffered.
Suitable buffering agents include but are limited to phosphate,
HEPES, MOPS, TES, citrate, acetate, bicarbonate, PIPES, BES, or
Tris buffers. In some embodiments, the buffered peracetic acid
contains ethanol. In other embodiments, the buffered peracetic acid
does not contain ethanol. In some embodiments, the buffered
peracetic acid has a pH of from about 4.5 to about 7.5. In one
embodiment, the buffered peracetic acid is an aqueous solution of
at least 30 percent peracetic acid, by volume. It was discovered
that buffering the peracetic acid to a neutral pH of 4.5 to 7.5
enables a higher retention of bioactive molecules (i.e., HGF, KGF,
and TIMP-1) compared to non-buffered peracetic acid while still
decontaminating the tissue.
[0104] In certain embodiments, decontaminating the tissue membrane
involves contacting the membrane with disinfectant for a period of
at least about 15 minutes. In certain embodiments, decontaminating
the tissue membrane involves contacting the membrane with
disinfectant for a period of about 15 minutes. In certain
embodiments, the tissue is contacted with disinfectant more than
once, e.g., for two periods of 15 minutes. For example, in some
embodiments, it is beneficial to contact the tissue with
disinfectant more than once because the tissue contains residual
moisture from the prior processing steps.
[0105] In certain embodiments, other disinfectants, such as
suitable antibiotics or surfactants are used. However, care should
be taken to avoid delamination of the tissue layer, loss of
bioactives, that can occur with both high processing temperatures
(e.g., 37.degree. C.) and long treatment time (e.g., 3 hours or
more). Delamination as used herein refers to separation of the
amnion tissue layer from the chorion tissue layer in the fetal
membrane. Delamination does not refer to removal of certain cells
in either the amnion tissue layer or the chorion tissue layer.
Additionally, conventional surfactants, in some cases, undesirably
lead to a reduction in bioactives or an alteration of the tissue
layer structure, and/or they leave an unwanted residue on the
tissue. Thus, it is possible that the ethanol and peracetic acid
treatments described above could provide certain benefits over
other such disinfecting processes. For example, decontamination
using these ethanol and/or peracetic acid treatments minimizes or
limits or prevent leaching of bioactives during the decontamination
step. That is, preservation of bioactives present in the original
amnion and chorion layers is achieved by decontaminating the tissue
membrane using these ethanol and/or peracetic acid treatments.
[0106] The described decontamination step advantageously reduces
the bioburden at or near the start of tissue processing to limit
tissue damage during subsequent processing steps, while maximizing
retention of endogenous bioactive constituents. Additionally, these
steps achieve a reproducible low or undetectable bioburden level to
allow reduction of terminal sterilization levels (i.e., in some
cases, a less severe sterilization process is needed to effect the
desired tissue membrane sterility). With proper handling or aseptic
conditions, sterilization may not be necessary. In certain
embodiments, processing of the membrane is conducted under aseptic
conditions and no final decontamination steps are used.
Tissue Processing--Folding, Rolling, or Stacking
[0107] In certain embodiments, the human fetal support tissue
membrane is folded, rolled, or stacked to create a tissue graft
that is uniform on each side. This feature has the advantage that a
user, such as a physician or a patient, does not need to rely upon
a marking on the human fetal support tissue membrane in order to
place it correctly to administer treatment. An additional advantage
is that the human fetal support tissue membrane does not need to be
marked or labeled during processing. In some embodiments, the human
fetal support tissue membrane that is folded, rolled, or stacked
has a higher density of therapeutic factors than a single layer
human fetal support tissue membrane. In some embodiments, the human
fetal support tissue membrane that is folded, rolled, or stacked
has an amnion layer on each side. In some embodiments, the human
fetal support tissue membrane that is folded, rolled, or stacked
has a chorion layer on each side. In some embodiments, the human
fetal support tissue membrane is folded. In some embodiments, the
human fetal support tissue membrane is folded once. In some
embodiments, the human fetal support tissue membrane is folded
twice, three times, four times, five times, six times, or seven
times. In some embodiments, the human fetal support tissue membrane
is folded into a square. In some embodiments, the human fetal
support tissue membrane is folded into a rectangle. In some
embodiments, the human fetal support tissue membrane is folded into
a triangle. In some embodiments, the human fetal support tissue
membrane is folded into a shape corresponding to the area or tissue
to be treated. In some embodiments, the human fetal support tissue
membrane is rolled. In some embodiments, the human fetal support
tissue membrane is folded or rolled along a diagonal of the human
fetal support tissue membrane. In some embodiments, the human fetal
support tissue membrane comprises at least two human fetal support
tissue membranes stacked on top of each other such that the amnion
layers face toward the center and the chorion layers face towards
the outside of the tissue membrane. In some embodiments, the human
fetal support tissue membrane comprises at least two human fetal
support tissue membranes stacked on top of each other such that the
chorion layers face toward the center and the amnion layers face
towards the outside of the tissue membrane. In some embodiments, at
least three, four, five, six, seven, eight, nine, ten, eleven,
twelve, or more human fetal support tissue membranes are stacked on
top of each other with an amnion layer or a chorion layer facing
toward the outside of the stacked human fetal support tissue
membrane.
Tissue Processing--Dehydrating
[0108] In certain embodiments, the isolated human fetal support
tissue membrane is dehydrated to a moisture content (used
interchangeably with "water content" herein) of less than 20
percent by weight. As used herein, the term "dehydrated" refers to
the tissue having decreased amount of water, water mixtures with
miscible organic solvents, and/or non-aqueous solvents. For
example, in some cases, the tissue membrane is dehydrated to a
water content of less than 20 percent by weight, of less than 10
percent by weight, or less than 5 percent by weight. Particular
apparatuses and methods of dehydrating tissue are described in
further detail below.
[0109] Dehydration (also referred to interchangeably as "drying"
herein) of the tissue membrane, in some embodiments, advantageously
extends the shelf life of the resulting tissue membrane product,
enhances the adhesion between the membrane layers, and enhances the
mechanical properties and handling properties of the tissue. As
shown in the histology of FIG. 3, after drying (shown in right-hand
side), the isolated tissue membrane is significantly more compact
and dense than before drying (shown in left-hand side). As can be
seen in FIG. 3, the hydrated (i.e., pre-drying) membrane has a
visible anatomical distinction between amnion and chorion layers,
while the dehydrated (i.e., post-drying) membrane has very little
visible anatomical distinction between the layers (i.e., it is
histologically difficult to differentiate the layers using
conventional techniques).
[0110] It has been found that decreasing the moisture content of
the tissue membrane to less than 20 percent by weight is important
when balancing the biological activity of the endogenous bioactives
with the desire for long-term storage at room temperature
conditions. While it is traditionally believed that sterilization
of a product of decreased moisture content lowers the recoverable
content of bioactives, it was surprisingly found that decreasing
the moisture content of the tissue membrane in fact resulted in
more favorable stability of bioactives over time, as is discussed
further in relation to sterilization of the tissue membrane.
[0111] In some embodiments, the dehydrating step involves
dehydrating the isolated human fetal support tissue membrane in a
desiccator chamber. In some embodiments, as discussed in greater
detail below, the dehydrating step involves positioning the
isolated human fetal support tissue membrane between a support
platform and a sealing sheet, the support platform covering an
opening of a chamber, and depressurizing the chamber, such that the
sealing sheet seals the chamber, to dehydrate the isolated human
fetal support tissue membrane.
[0112] In certain embodiments, the drying step involves embossing
or otherwise imparting a visibly distinguishing surface
characteristic to at least one surface of the tissue membrane, to
facilitate determination of the amnion surface versus chorion
surface, for example by the physician handling the tissue membrane
product in the process of applying it to a tissue site of a
patient.
Tissue Processing--Compressing
[0113] In certain embodiments, the isolated human fetal support
tissue membrane is compressed to substantially collapse the tissue
membrane and reduce its void spaces, i.e., open volume. The
isolated human fetal support tissue membrane is, in certain
embodiments, compressed to have an open volume of less than 15
percent, of less than 10 percent, or less than 5 percent. In
certain embodiments, the compression comprises compaction. It is
contemplated that the compact, collapsed nature of the tissue
membrane advantageously provides a resistance to enzymatic
breakdown and release of bioactives from the membrane, resulting in
longer graft residence time in a wound and less product required to
heal.
[0114] In certain embodiments, the compressed and dehydrated
isolated human fetal support tissue membrane is a compact or dense
matrix. As used herein, the terms "compact" and "dense" are used
interchangeably and refer to the compressed and dehydrated isolated
human fetal support tissue membrane being substantially collapsed
and substantially free of voids. In certain embodiments, the tissue
membrane is substantially nonporous. As used herein, the term
"nonporous" refers to the compressed and dehydrated isolated human
fetal support tissue membrane having substantially no visible open
volume or space, as determined via histological analysis.
[0115] In some embodiments, the compression is from positive
pressure exerted onto the tissue membrane. In other embodiments,
the compression is from negative pressure, e.g., from a vacuum
assisted apparatus as described below.
Tissue Processing--Sizing and Packaging
[0116] In certain embodiments, the dehydrated and compressed tissue
membrane is cut to a desired size and then packaged. For example,
in some embodiments, the tissue membrane is cut to size using a
scalpel, scissors, a die (e.g., a pneumatic die), punch, or other
suitable cutting tool before packaging the tissue membrane in a
suitable packaging. In some cases, the tissue membrane is packaged
in an air- and moisture-impermeable package. In some cases, the
tissue membrane is packaged in an air- and moisture-permeable
package.
[0117] Because accurate sizing the hydrated membrane is difficult
and, in some cases, results in pieces that are not uniform, the
present method advantageously sizes the dehydrated membrane. In
particular, manual cutting or cutting using a press, in some cases,
causes an unequal strain on the amnion and chorion layers,
resulting in a lengthening of the amnion layer at the cut edges, if
the membrane is cut while hydrated. Cutting the membrane after
dried therefore allows for sizing the membrane to reproducible
dimensions (e.g., 2 cm squares, 6 cm squares).
[0118] In certain embodiments, the tissue membrane is packaged in a
pouch, such as a pouch having one side that is flashspun
high-density polyethylene (HDPE, e.g., Tvyek.RTM., manufactured by
DuPont) and one side that is low-density polyethylene (LDPE). In
some embodiments, this pouch is placed within a moisture resistant
outer pouch. In some embodiments, this pouch is placed within a
moisture permeable outer pouch.
[0119] As will be discussed in greater detail below, the tissue
membrane, in some embodiments, is placed onto a backing material
either during the drying process or after the drying process. For
example, the backing material, in some embodiments, is a nylon
mesh, flashspun high-density polyethylene (HDPE, e.g., Tvyek,
manufactured by DuPont), or low-density polyethylene (LDPE)
material.
[0120] In other embodiments, the dehydrated tissue membrane is
micronized or otherwise further processed or packaged for uses
other than as a planar tissue graft.
[0121] In preparation of the tissue membranes described herein, the
packaging of the tissue membrane is, in some embodiments, carried
out in an environment containing an inert gas, i.e., under inert
packaging conditions, to lower the moisture and oxygen
concentration in the packaging. Under inert packaging conditions
also include the use of flushing or blanketing a packaging
container (e.g., pouch) with an inert gas. The use of an inert gas
(e.g., nitrogen, argon, CO.sub.2, helium, xenon, neon and the like)
limits exposure of the tissue membrane to moisture and oxygen and
possible degradation. Moreover, packaging the dehydrated tissue
membrane under an inert atmosphere, such that the moisture content
is maintained at a low level, in some cases, advantageously allows
for terminal sterilization without detectable reduction of
bioactives content, such as growth factors.
Tissue Processing--Terminal Sterilization
[0122] After packaging, in some embodiments, the tissue membrane is
sterilized to minimize the risk of infecting the recipient and to
reduce the burden of aseptic tissue processing. In certain
embodiments, the tissue membrane is sterilized with radiation in an
amount of from about 15 kGy to about 35 kGy, such as 25 kGy. In
some embodiments, the tissue membrane is sterilized with radiation
in an amount from about 22.5 to about 31.5 kGy. For example, in
some embodiments, the radiation is e-beam or gamma radiation.
[0123] It was surprisingly found that sterilization with e-beam
radiation in the range of about 15 kGy to about 35 kGy yielded
sterile tissue with no quantifiable change in immunoassayable
growth factors (i.e., the tissue membrane retains a majority of
endogenous bioactives from the amnion and chorion after
processing). It is contemplated that the low moisture content
(i.e., dehydration level or extent of dehydration) of the packaged
tissue membrane that is subjected to irradiation minimizes the
amount of oxygen present in the packaged tissue membrane that, in
some cases, damages bioactives during irradiation. Thus, no
decrease in the amount of certain bioactives after to irradiation
sterilization was observed, despite the conventional understanding
that bioactives decrease during irradiation.
[0124] In certain embodiments, the sterilization is performed at a
controlled temperature.
[0125] In other embodiments, other suitable sterilization methods
are used, such as treating the membrane with supercritical
CO.sub.2, peracetic acid, or ethylene oxide.
[0126] The membrane resulting from the processes described herein,
in some embodiments, constitutes a dry, easy to store, and ready to
use sheet. To this end, minimal processing methods (e.g., no
separation of the amnion and chorion layers of the membrane)
coupled with benign processing parameters are employed to maximize
the concentration of endogenous bioactives in the implantable
product. The method also includes the non-chemical or biochemical
removal of maternal cells from the fetal membrane; these cells can
produce an immune response in the recipient which is not desirable
in a wound covering product. The resulting intact bilayer of amnion
and chorion does not stimulate proliferation or other activity of
allogeneic lymphocytes indicating lack of alloantigenicity. That
is, the resulting intact bilayer of amnion and chorion displays no
detectable antigenicity.
[0127] In certain embodiments, the sterilized tissue can be stored
at room temperature (e.g., 25.+-.10.degree. C.) for up to six
months. In certain embodiments, the sterilized tissue can be stored
at room temperature for up to twelve months. In certain
embodiments, the sterilized tissue can be stored at room
temperature for up to 24 months. In certain embodiments, the
sterilized tissue can be stored at room temperature for up to 5
years.
[0128] In certain embodiments, the aseptically processed, dry
tissue, has not been sterilized and can be stored at room
temperature (e.g., 25.+-.10.degree. C.) for up to six months. In
certain embodiments, the aseptically processed, dry tissue can be
stored at room temperature for up to twelve months. In certain
embodiments, the aseptic, dry tissue can be stored at room
temperature for up to 24 months. In certain embodiments, the
aseptically processed, dry tissue can be stored at room temperature
for up to 5 years.
Tissue Grafts & Packaged Tissue Grafts
[0129] Tissue grafts produced by the methods described herein are
also provided. These tissue grafts, in some embodiments, display
beneficial mechanical, handing, and performance properties,
including flexibility/non-brittleness, compact structure, and
maintained bioactives/growth factors. In certain embodiments, a
tissue graft includes a dehydrated isolated human fetal support
tissue membrane which contains amnion tissue and an associated
chorion connective, supportive tissue layer, which amnion and
chorion are in an original, native bilayer architecture, wherein
the human fetal support tissue membrane is substantially free of
trophoblast and decidua cell matter. In some embodiments, a tissue
graft includes a dehydrated isolated human fetal support tissue
membrane which contains amnion tissue and an associated chorion
connective, supportive tissue layer in their anatomic bilayer
configuration, wherein the human fetal support tissue membrane is
substantially free of maternal cells and is in a dense, compact
collagenous and glycosaminoglycan matrix form.
[0130] In some embodiments, the amnion may maintain the epithelium.
In some embodiments, the amnion is de-epithelialized.
Advantageously, the tissue grafts retain the spongy layer
connecting the amnion and chorion, which is believed to contribute
to beneficial characteristics of the grafts, such as flexibility
and the ability to achieve a compact structure during drying and
compressing steps as well as retaining significant amounts of
glycosaminoglycans such as hyaluronic acid.
[0131] Additionally, the present methods provide a solution to the
problem of competing processing goals to achieve retention of
growth factors and a tissue that can be stored at room temperature.
Specifically, the present methods are designed to exploit the
intrinsic value of the fetal membrane and to maximize retention of
bioactives in the processed tissue and to reduce the antigenicity
of that tissue, while preserving the connective structure of the
native membrane (i.e., without laminating previously separated
amnion and chorion layers together).
[0132] In certain embodiments, the dehydrated isolated human fetal
tissue membrane retains effective levels of bioactives from the
amnion, chorion, and spongy layer between the amnion and chorion.
That is, the levels of bioactives in the tissue membrane product
are not be statistically different from that of the natural tissue
from which the product is derived. In certain embodiments, the
dehydrated isolated human fetal support tissue membrane retains a
majority of endogenous bioactives from the amnion and chorion. In
some embodiments, the dehydrated isolated human fetal support
tissue membrane comprises basic fibroblast growth factor (bFGF) in
an amount of at least 5,000 pg/mg, or in an amount of at least
10,000 pg/mg. In some embodiments, the dehydrated isolated human
fetal support tissue membrane comprises hepatocyte growth factor
(HGF) in an amount of at least 100 pg/mg or in an amount of at
least 450 pg/mg. In some embodiments, the dehydrated isolated human
fetal support tissue membrane comprises TIMP metallopeptidase
inhibitor-1 (TIMP-1) in an amount of at least 7,000 pg/mg, or in an
amount of at least 10,000 pg/mg. In some embodiments, the
dehydrated isolated human fetal support tissue membrane comprises
hyaluronic acid (HA) in an amount of at least 5,000 ng/mg, or in an
amount of at least 10,000 ng/mg. It is believed that this HA
content is associated with the maintenance of the spongy
intermediate layer between the amnion and chorion, and provides
flexibility in the resulting product. Low HA content in
conventionally processed tissue grafts is believed to result in
brittleness.
[0133] In certain embodiments, the tissue graft is configured for
use as a wound covering or implant, which, in use, is not
antigenic. In certain embodiments, the tissue graft is configured
for use as a wound covering and is stable at room temperature. In
certain embodiments the tissue graft is configured for use as a
wound covering or implant, which, in use, is contemplated to
display a resistance to biodegradation at an implant site due to
namely, in part, to its dense, compact structure and high content
of protease inhibitors such as TIMP-1. In certain embodiments, the
tissue graft is configured for use as a wound covering or implant,
which, in use, acts a barrier to certain molecules, such as
proteases and degradative enzymes. In certain embodiments, the
tissue graft is configured to have a sufficiently small pore size
(e.g., 0.2 .mu.m) to act as a barrier to infectious agents, such as
bacteria and fungi. In certain embodiments, the tissue graft is
configured for use as a wound covering or implant, which, in use,
permits release of low molecular weight growth factors into
surrounding tissue. In certain embodiments, the tissue graft is
configured for use as a wound covering or implant, which, in use,
acts as a barrier to fluid flux, limiting dehydration of
surrounding tissues (e.g., limiting wound dehydration). In certain
embodiments, the tissue graft is configured for use as a wound
covering or implant, which, in use, provides a release rate of
bioactives including but not limited to growth factors (e.g., bFGF)
that is slower than a release rate of bioactives from a non-compact
tissue graft. In certain embodiments, the tissue graft is
configured for use as a wound covering or implant, which, in use,
provides increased hydrophobicity of the dense, compact structure
of the tissue membrane, by limiting the hydration state of the
tissue matrix and thereby increasing the effective concentration of
bioactives and protease regulatory factors within the matrix.
[0134] In certain embodiments, the dehydrated isolated human fetal
support tissue membrane has a water content of less than 20 percent
by weight, such as a water content of less than 10 percent by
weight, or a water content of less than 5 percent by weight.
[0135] In certain embodiments, a first face of the tissue membrane
is shiny and an opposed second face of the membrane is relatively
matte, such that the first and second faces of the tissue membrane
are visibly distinguishable from one another. Such visibly
distinguishable membrane faces, in some cases, facilitate
determination of the chorion versus amnion faces of the tissue
graft, to assist in proper orientation and placement of the tissue
graft onto a patient in use. The shiny and relatively matte faces,
in some cases, are established during the drying process, as is
discussed in more detail below.
[0136] In certain embodiments, the tissue graft is room temperature
stable, and is not cryopreserved or lyophilized. Thus, the tissue
graft, in some embodiments, does not require special storage
conditions other than room temperature conditions. The tissue
grafts described herein therefore do not require thawing or rinsing
prior to application to the wound environment, but also provide
improved membrane handling properties. For example, the tissue
graft is alternatively pre-wet before application to the wound or
it is applied in the dry state then moistened.
[0137] In certain embodiments, the tissue graft is provided in the
form of a flat sheet. For example, providing the tissue graft in a
flat or planar form, in some cases, provides for improved Van der
Waals adhesion of the membrane to a backing material. In other
embodiments, the tissue graft is provided in the form of another
suitable graft shape, including but not limited to a tube, a rod, a
fragment, or a wedge. In other embodiments, the tissue graft is
combined with other materials, such as amniotic fluid, cells
including stem cells, pharmaceuticals, and the like.
Apparatuses and Methods of Dehydrating and Compressing Tissue
[0138] Apparatuses and methods of dehydrating and compressing
tissue, such as fetal support tissue, are also provided herein.
While the apparatuses and methods are generally described with
reference to drying isolated fetal support tissue, it should be
understood that the apparatuses and methods are used to dry a
variety of tissues, including whole or partial placenta,
pericardium, fascia, and peritoneum, among others.
[0139] In certain embodiments, the apparatuses and methods are used
to dehydrate the tissue to a water content of less than about 20
percent by weight, such as less than about 10 percent by weight, to
achieve the desired water content for storage, stabilization, or
other purposes. In some embodiments, the apparatuses and methods
are used to dehydrate the tissue to a water content of less than 5
percent by weight. These apparatuses and methods, in some cases,
are further be utilized to achieve the desired dense, compact, or
collapsed tissue structure. These apparatuses and methods also are
used to achieve consistent planarity that is free of wrinkles to
improve surgical handling and use.
[0140] In certain embodiments, a method of preparing a compact
tissue membrane includes simultaneously compressing and dehydrating
the tissue membrane to form a dehydrated, compact tissue membrane.
For example, in some cases, compressing the tissue membrane
involves positioning the tissue membrane on a support platform and
pulling a vacuum across the platform and/or applying a load to a
surface of the tissue membrane, such as the surface opposite any
support platform.
[0141] In certain embodiments, as shown in FIGS. 5-9 and FIG. 21,
an apparatus 1 for dehydrating a tissue, includes a chamber
defining an opening at one end, the chamber being in fluid
connection with a vacuum source 6, a support platform comprising
FIG. 6 and FIG. 7 covering the opening of the chamber, and a
sealing sheet FIG. 8 positioned adjacent the support platform FIG.
6 and FIG. 7 opposite the chamber, the sealing sheet FIG. 8 being
configured to seal the chamber upon depressurization of the chamber
by the vacuum source 6. The apparatus is configured to receive the
tissue between the support platform FIG. 6 and FIG. 7 and the
sealing sheet FIG. 8, such that the tissue is dehydrated upon
depressurization of the chamber. FIG. 21 further comprises an air
inflow port 7.
[0142] For example, the opening of the chamber has an area that is
at least the size of a tissue to be dehydrated (e.g., a 2 cm.sup.2,
a 6 cm.sup.2, 10 cm.sup.2, 20 cm.sup.2, 30 cm.sup.2, 40 cm.sup.2,
50 cm.sup.2, 60 cm.sup.2, 70 cm.sup.2, 80 cm.sup.2, 90 cm.sup.2,
100 cm.sup.2, 150 cm.sup.2, 200 cm.sup.2, 250 cm.sup.2, 300
cm.sup.2, 350 cm.sup.2, 400 cm.sup.2, 450 cm.sup.2, 600 cm.sup.2,
650 cm.sup.2, 700 cm.sup.2, 750 cm.sup.2, 800 cm.sup.2, 850
cm.sup.2, 900 cm.sup.2, 950 cm.sup.2, or 1000 cm.sup.2). In some
embodiments, the opening of the chamber has an area the size of an
array of at least two tissue grafts to be dehydrated positioned in
a side by side arrangement. For example, the chamber opening, in
some embodiments, has an area of from about 2 cm.sup.2 to about 1
m.sup.2. For example, the chamber, in some embodiments, has a
height from a base of the chamber to the opening of from about 2 mm
to about 5 cm. The vacuum source 6, in some embodiments, is in
fluid connection with this chamber such that the volume of the
chamber is depressurized during dehydration. That is, the vacuum
source is in fluid connection with the open space that extends
across the chamber opening. For example, in some cases, the vacuum
source is connected to the chamber via a vacuum port provided in a
sidewall of the chamber.
[0143] In some embodiments, the support platform includes a
perforated rigid support layer FIG. 6 and a moisture, liquid and
vapor permeable material layer FIG. 7. The moisture, liquid and
vapor permeable material layer FIG. 7 is positioned between the
perforated rigid support layer FIG. 6 and the tissue to be
dehydrated, and it functions to keep the tissue from being pulled
into the perforations of the perforated rigid support layer FIG. 6
while also facilitating passage of moisture from the tissue through
the perforations and into the chamber. In a preferred embodiment,
the moisture, liquid and vapor permeable material layer FIG. 7
prevents the perforations of the perforated rigid support layer
FIG. 6 from imparting their shapes to the surface of the dehydrated
tissue, and instead keeps the surface of the dehydrated tissue
relatively planar.
[0144] In other embodiments, the support platform may be a single
structure that provides both the rigid structural support and
suitable permeability, to accomplish the dehydration. In various
embodiments, the single structure may be formed of a rigid woven or
non-woven material, or a porous rigid sheet, which may be formed
for example of a sintered metal, ceramic, polymer, or a composite
thereof.
[0145] For example, the moisture, liquid, and vapor permeable
material layer FIG. 7 may be any suitable material that is
moisture, liquid, and vapor permeable, to allow moisture from the
hydrated tissue to travel through the moisture, liquid, and vapor
permeable material layer FIG. 7 upon depressurization of the
chamber by the vacuum. In one embodiment, the moisture, liquid, and
vapor permeable material layer is a porous polypropylene sheet with
pores of from about 50 micron to about 200 micron. For example, the
moisture, liquid, and vapor permeable material layer may be 0.125''
thick polypropylene with 125-195 micron pores.
[0146] For example, the perforated rigid support layer FIG. 6 may
be any suitable rigid support layer that allows for the passage of
moisture and provides adequate structural support for the moisture,
liquid, and vapor permeable material layer FIG. 7, tissue to be
dried and sealing sheet FIG. 8, upon depressurization. In some
embodiments, the perforated rigid support layer FIG. 6 is a
stainless steel sheet having a plurality of apertures therethrough
(e.g., 0.06'' thick stainless steel with 0.25'' diameter holes). In
certain embodiments, the moisture, liquid, and vapor permeable
material layer FIG. 7 is provided between the tissue and the
perforated rigid support layer FIG. 6, to prevent embossing of the
apertures of the rigid support layer FIG. 6 into the tissue.
[0147] In some embodiments, the sealing sheet FIG. 8 is a gas
impermeable, compliant or conformable polymer sheet, such as a
silicone sheet (e.g., 0.0625'' thick, 12''.times.12'' FDA-compliant
or conformable silicone rubber) or an LDPE or polyurethane sheet.
For example, upon depressurization of the chamber, the sealing
sheet may create a sealed chamber. In other embodiments, the
sealing sheet may have some degree of gas permeability, to provide
limited air inflow.
[0148] In some embodiments, the apparatus optionally includes an
air source 7 in fluid connection with the chamber and configured to
deliver air to the sealed chamber, to further dehydrate the tissue
by driving an exchange of moisture off the tissue. For example, the
air may be a dry or pure inert gas, such as nitrogen, argon, or
helium.
[0149] In some embodiments, the apparatus fully assembled as in
FIG. 9 includes a perforated steel support, a porous support
substrate and a silicone sealing sheet, with the tissue between
Tyvek, polyethylene, and/or Poly layers.
[0150] In some embodiments, the chamber of the apparatus contains a
desiccant material, such as silica gel (e.g., .about.2-5 mm
diameter granules), to act as a moisture sink for moisture from the
tissue.
[0151] While any suitable apparatus design providing suitable
pressure on the tissue and depressurization to achieve dehydration
of the tissue may be used, a particular embodiment is illustrated
in FIGS. 5-9, and further includes handles 2 for transporting the
apparatus, ribbed supports 3 and lower ledge 4 for holding the
support platform FIG. 6 and FIG. 7, and the sealing sheet FIG. 8
off the chamber, and upper ledge 5 for vacuum sealing down the
sealing sheet.
[0152] In certain embodiments, a method of dehydrating a tissue
membrane includes positioning the tissue membrane between a support
platform FIG. 6 and FIG. 7, and a sealing sheet FIG. 8, the support
platform covering an opening of a chamber, and depressurizing the
chamber, such that the sealing sheet FIG. 8 seals the chamber, to
dehydrate the tissue membrane. In some embodiments, the tissue
membrane is an isolated human fetal support tissue membrane that
has been decontaminated. For example, the tissue membrane may be
dehydrated to a moisture content of less than 20 percent by
weight.
[0153] In some embodiments, prior to positioning the tissue
membrane between the support platform and the sealing sheet, but
after decontamination, the tissue membrane is placed within a
flexible pouch. In one embodiment, the flexible pouch has at least
one side that is a nonwoven, flashspun, or other porous material
(e.g., Tyvek.RTM.), such that the nonwoven, flashspun, or other
porous material may be positioned in contact with the support
platform FIG. 6 and FIG. 7, to provide for the flow of moisture
from the tissue through the support platform FIG. 6 and FIG. 7.
That is, the nonwoven, flashspun, or other porous material side of
the pouch faces the support platform. In some embodiments, the
tissue membrane is an isolated human fetal support tissue membrane
and is positioned in the pouch with the amnion side of the tissue
membrane facing the nonwoven, flashspun, or other porous material
side of the pouch. In some embodiments, the tissue membrane is an
isolated human fetal support tissue membrane and is positioned in
the pouch with the chorion side of the tissue membrane facing the
nonwoven, flashspun, or other porous material side of the pouch. In
such embodiments, the pouch may be used to temporarily transport
the tissue to and from the drying apparatus and to impart "shiny"
and "dull" "matte" or "relatively matte" surface finishes to the
tissue membrane, to allow for visual distinguishing of the two
sides. In particular, the shiny surface may be imparted by the LDPE
pouch side and the "relatively matte" surface may be imparted by
the porous pouch side.
[0154] In certain embodiments, a second side of the pouch is formed
of a moisture impermeable material, such as LDPE. In such
embodiments, wherein the tissue membrane is an isolated human fetal
support tissue membrane, the membrane is positioned in the pouch
with the chorion side facing the LDPE. In some embodiments, the
membrane is positioned in the pouch with the amnion side facing the
LDPE.
[0155] In certain embodiments, after the tissue membrane is placed
in the pouch, the excess edges of the pouch are cut away, so that
the maximum number of pouched membranes may be placed between the
sealing sheet and support platform to be dried simultaneously.
[0156] In certain embodiments, depressurizing the chamber involves
operating a vacuum pump to draw at least about 10 inHg from the
chamber, such as about 25 inHg from the chamber. In certain
embodiments, the drying method optionally includes delivering air
to the sealed chamber to further dehydrate the tissue membrane. For
example, the air may be an inert gas, such as nitrogen, argon, or
helium. In one embodiment, inert gas is delivered to the chamber at
a rate of from about 20 mL/min to about 4 L/min, such as at a rate
of about 300 mL/min. In certain embodiments, depressurizing the
chamber involves operating a vacuum pump, and delivering air to the
sealed chamber occurs after operation of the vacuum pump, such that
the depressurizing and air delivery does not occur simultaneously.
In other embodiments, depressurizing and air delivery occur
simultaneously.
[0157] In certain embodiments, the tissue membrane is maintained at
the depressurized chamber (i.e., between the sealing sheet and the
support platform) for a period of from about 16 to about 24 hours.
After this drying step, the tissue has a compact, collapsed
structure, and a moisture content of as low as 5 percent or less,
by weight; however, the tissue may increase in moisture content
after removal from the drying apparatus, if not maintained in a dry
(or low humidity) room (or other vessel or location) e.g., in a
dry, inert gas atmosphere.
[0158] In certain embodiments, after removal from the drying
apparatus, the tissue may be dissected, or cut, to size. Cutting
the tissue to size is discussed in more detail above. In certain
embodiments, the tissue is cut to size using a scalpel and a
stencil. In other embodiments, the tissue is cut to size using a
pneumatic press and a die. During the cutting process, if not
conducted in a dry room or inert atmosphere, the tissue may
increase to a water content of up to about 15 percent, by weight.
Thus, in certain embodiments, a second drying step may be
required.
[0159] In some embodiments, the tissue membrane is removed from the
sealed chamber and then, possibly after an intermediate cutting
step, the tissue membrane is positioned in a desiccator chamber
with a desiccant material and the desiccator chamber is
depressurized. For example, depressurizing the desiccator chamber
may involve operating a vacuum pump to draw at least about 10 inHg
from the desiccator chamber, or operating a vacuum pump to draw
about 25 inHg from the desiccator chamber. In some embodiments, the
desiccant material is silica gel (e.g., up to about 100 g of silica
gel). In some embodiments, the tissue membrane is maintained in the
depressurized desiccator chamber for a period of at least 24 hours.
In some embodiments, the tissue membrane is maintained in the
depressurized desiccator chamber for a period of 12, 24, 36, 48,
60, 72, or more hours. In some embodiments, the tissue membrane is
maintained in the depressurized desiccator chamber for a period of
at least 24 hours.
[0160] In certain embodiments, the tissue membrane is releasably
secured onto a backing material before or after some level of
dehydration. For example, the tissue membrane may be releasably
bonded onto the backing material during a drying process.
[0161] In certain embodiments, the tissue membrane is releasably
secured on a flexible backing material comprising at least one tab
defined by the flexible backing material, wherein the membrane is
releasably secured on the flexible backing material via the at
least one tab. The tissue membrane may be secured to the backing
material between the first and second drying steps, i.e., after
drying in the apparatus to form the compact structure and prior to
secondary drying in the desiccator chamber. For example, the tissue
membrane may be secured to the backing material after sizing. After
being secured on the backing material, the backing material with
secured tissue membrane may be placed in an unsealed pouch prior to
secondary drying.
[0162] The secondary drying step, if needed, may bring the moisture
content of the tissue to about 5 percent moisture, by weight. It is
contemplated that a moisture content of 3 percent or less may
result in brittleness of the dried tissue. Thus, a moisture content
of up to 8 percent, or more, may be desired based on the necessary
performance characteristics and shelf life.
[0163] In certain embodiments, either or both drying steps are
performed at an elevated temperature.
[0164] The above described drying apparatus and methods
advantageously utilize vacuum pressure on the tissue and optional
air flow below the tissue within a completely closed environment to
produce a product that is compact, dry, and wrinkle free. It is
believed that drying while imparting a force on one side of the
tissue structure while also applying a vacuum at the opposed side
of the tissue structure yields the collapsed tissue structure that
has reduced porosity. Furthermore, the vacuum applies pressure and
changes the histologic appearance of the tissue, such that no
discernible layers (i.e., amnion/chorion) are apparent in the dried
product. That is, the resulting membrane structure, while including
the amnion and chorion in their native connective structure, is
monolithic in appearance. Thus, compared to conventional drying
methods in convection oven-like systems in which no force is
applied to a surface of the tissue, the present methods result in a
much more compact structure (see FIG. 4, comparative cross-section
of tissue membranes dried in presently described apparatus (panels
B and D) and commercial tissue graft (panels A and C)).
[0165] As discussed above, it is hypothesized that this structure
reduces the rate of proteolysis and retards the elution of
bioactives. Specifically, it is believed that the denser tissue
structure allows extended delivery (i.e., diffusion based) of
bioactives and extended life of the ECM in the wound bed because
the compact structure is more resilient to enzymatic protease
infiltration into the membrane system and the resulting degradation
of matrix and bioactive molecules.
[0166] Additionally, the result of drying the tissue in between a
nonporous, ultra-smooth material and a nonwoven yet porous material
is a final product that is shiny on one side, relatively matte on
the other, and completely planar. Moreover, the final water content
achieved using sequential vacuum and desiccation steps yields a
lower water content than is achieved from evaporation alone (i.e.,
consistently less than 10%). Thus, the tissue grafts dried using
these apparatuses and methods display better handling properties
due to their compact structure, as compared to laminated products
and products dried through other methods.
Tissue Backing Material
[0167] In certain embodiments, the tissue material is secured onto
a backing material for packaging the tissue and providing improved
handling and maintenance of the planar tissue structure.
Additionally, the backing may further help to identify the
orientation of the tissue membrane, by providing a consistent
orientation for secured tissue membranes (e.g., amnion faces out,
away from backing) in addition to the optional shiny/relatively
matte surface feature, to assist physicians in application of the
membrane to a patient site.
[0168] In certain embodiments, as shown in FIG. 10, a flexible
backing material includes at least one tab defined by the flexible
backing material (i.e., the tab is formed by a cut in the flexible
backing material). In certain embodiments, the flexible backing
material is a suitable medical grade flexible polymer material,
such as high density polyethylene (HDPE), low density polyethlyene
(LDPE), ethylene/vinyl alcohol copolymer (EVOH), polypropylene
(PP), polyethylene terephthalate (PET). amorphous polyethylene
terephthalate (APET), glycol modified polyethylene terephthalate
(PET-G), polyethylene naphthalate (PEN), ethylene acrylic acid
copolymer (EAA), polyamide (PA), polyvinyl chloride (PVC),
polyvinylidene chloride (PVDC), polychlorotrifluoroethylene
(PCTFE), vinylidene chloride/methyl acrylate copolymer, polyamide,
polyester, polyurethane, silicone, a metalized film, an oxide
coated film, nitrocellulose, and combinations thereof. In certain
embodiments, the flexible backing material includes two opposed
tabs configured to secure opposed ends of the tissue membrane on
the backing material. For example, the tissue membrane may be
rectangular in shape and a pair of opposed tabs may be provided in
the backing material to secure opposed ends of the membrane to the
backing.
[0169] In certain embodiments, a single backing contains multiple
tabs for securing tissue membranes of various sizes thereon. As
shown in FIG. 10, an exemplary single backing contains six pairs of
opposed tabs, such that a tissue membrane may be secured by the
pair of tabs having a distance therebetween that is slightly longer
than the distance between the ends of the membrane to be secured.
It is contemplated herein that a single backing contains,
additional or fewer pairs of opposed tabs, for example one, two,
three, four, five, six, seven, eight, nine, ten, or more pairs of
opposed tabs, such that a tissue membrane may be secured. In other
embodiments, a single backing contains one pair of tabs for
securing a membrane having a particular size thereon.
[0170] In certain embodiments, the tabs are defined by a cut in the
flexible backing material, such as a cut having filleted ends,
which may prevent damage to the tissue membrane. In some
embodiments, the tabs are integral with the backing material. In
other embodiments, the tabs are a separate material from the
backing material.
[0171] In certain embodiments, an isolated human fetal support
tissue membrane having a water content of less than 20 percent by
weight is mounted on a flexible backing material having at least
one tab defined by the flexible backing material, wherein the
membrane is releasably secured on the flexible backing material via
the at least one tab. Thus, the tissue membrane may be mounted on
the backing after a primary drying step (e.g., after drying in the
apparatus discussed above). In some embodiments, the isolated human
fetal support tissue membrane is releasably secured to the flexible
backing material such that the chorion connective, supportive
tissue layer contacts the flexible backing material and the amnion
tissue is opposite the backing material. After the membrane is
mounted on the backing material, the backing with secured membrane
may be positioned within a pouch prior to terminal packaging or a
second drying step.
[0172] It was determined that securing a dried membrane to the
backing with tabs prevented the membrane from becoming adhered to
the backing, as in conventional placental tissue grafts having a
backing, which makes removal of the membrane from the backing
easier. The flexible nature of the backing also allows for a
physician to control the placement of the tissue membrane at a
patient site and to facilitate separation of the tissue membrane
from the backing, while avoiding contact with the tissue membrane
itself. In some embodiments, the backing is larger than the tissue
membrane, such that a gripping area is provided around the
periphery of the tissue membrane, so that a user can avoid contact
with the tissue membrane itself.
[0173] In certain embodiments, a packaged tissue graft includes a
flexible backing material having at least one tab defined by the
flexible backing material and a tissue membrane as described herein
releasably secured on the flexible backing material via the at
least one tab. In one embodiment, the flexible backing material
extends at least 1/8 inch (3.175 mm) past the perimeter of the
tissue membrane. In one embodiment, the chorion connective,
supportive tissue layer contacts the flexible backing material,
such that the amnion tissue is opposite the backing material.
[0174] The foregoing packaging systems (flexible backing and pouch)
for a tissue membrane can be used with tissue grafts other than
those derived from placental tissues. For example, it is envisioned
that that packaging systems can be used with tissue grafts derived
from fascial, pericardial, or peritoneal tissues or other similar
natural sheet or sheets derived from organs such as heart, lung,
stomach, intestine, bladder, skin and the like from human or other
mammalian donors.
Applications and Methods of Use of Placental Tissue grafts
[0175] The tissue grafts described herein may be used in a variety
of medical applications, for example in wound covering and healing,
as an anti-adhesion barrier, in regenerative medicine, and in
post-operative atrial fibrillation, among others. Advantageously,
the tissue grafts may be effective to redirect chronic, non-healing
wounds into a healing pattern by promoting cell in-migration,
proliferation, and differentiation, along with an anti-inflammatory
action.
[0176] In particular, the tissue grafts may retain beneficial
bioactives present in the placental tissue, including growth
factors and cytokines that promote fibroblast proliferation and
angiogenesis (bFGF) and epithelialization (KGF, HGF),
anti-fibrotic/anti-inflammatory factors that inhibit expression of
pro-inflammatory cytokines (IL-1.alpha., IL-2, IL-8, INF-.gamma.,
etc.) and/or protect from oxidative stress, and antimicrobial
peptides, and may be effective to inhibit allo-reactive immune
cells such as macrophages and proliferating T-cells. The tissue
grafts may also include the ECM matrix, which beneficially provides
a sacrificial matrix for endogenous wound bed proteases, contains
hyaluronic acid, which suppresses TGF-.beta. signaling reducing
proliferation of fibroblasts and limits the migration of
inflammatory cells, and provides cell binding motifs that enable
interstitial cell migration, adhesion, differentiation, de novo
matrix synthesis, and proliferation.
[0177] In some embodiments, tissue grafts herein release bioactives
for an extended period of time compared to commonly available or
commercially available tissue grafts. For example, tissue grafts
herein, in some embodiments, release bioactives for at least three
days. In some embodiments, tissue grafts herein release bioactives
for at least five days. In some embodiments, tissue grafts herein
release bioactives for at least seven days. In some embodiments,
tissue grafts herein release bioactives for at least 14 days, 21
days, 28 days, 30 days, or longer.
[0178] As a wound covering or for wound healing, the tissue grafts
described herein may be used to treat chronic wounds, such as
diabetic, venous, and arterial wounds, as well as acute non-healing
wounds, such as surgical and injury-related wounds, and pressure
ulcers. For example, the tissue grafts described herein may be used
in the treatment of a diabetic foot ulcer or a leg ulcer. For
example, the tissue graft described herein may be used in the
treatment of foot and lower limb ulcers resulting from diabetes and
peripheral vascular diseases. In certain embodiments, the tissue
graft is applied to a wound area for a period of at least 7 days.
In certain embodiments, the tissue graft is applied to a wound area
for a period of at least 14 days. In certain embodiments, the
tissue graft is applied to a wound area for a period of at least 30
days. In certain embodiments, the tissue graft is applied to a
wound area for a period of one, two, three, four, five, six, seven,
eight, nine, ten, eleven, or twelve weeks. In some instances, the
tissue graft is applied as singular application. In other
instances, multiple tissue grafts are applied periodically to a
wound area, e.g., an application of two, three, four, or more
tissue grafts over a wound, where each application is every 7, 10,
14, 30 days.
[0179] As an anti-adhesion barrier, the tissue grafts described
herein may be used in cardiac, thoracic, abdominal, foot, and ankle
surgery. In some embodiments, the grafts are used in cardiac
surgery. In some embodiments, the grafts are used in thoracic
surgery. In some embodiments, the grafts are used in abdominal
surgery. In some embodiments, the grafts are used in foot surgery.
In some embodiments, the grafts are used in ankle surgery. It is
contemplated that the tissue grafts described herein are used to
fashion tubes for construction of vascular conduits or to repair
heart valve leaflets.
[0180] In certain embodiments, the tissue grafts described herein
may be used in the treatment of cardiac tissue, including the
valves and arteries.
[0181] In regenerative medicine, the tissue grafts described herein
may be used as a scaffold for tissue reconstruction and
regeneration including tendon and other connective tissue repair
and reconstruction, as a delivery vehicle for cells and bioactive
factors. In certain embodiments, the tissue grafts described herein
may be used in the treatment of an injured tendon.
[0182] In post-operative atrial fibrillation, the tissue grafts
described herein are used in the prevention of atrial fibrillation
following open heart surgery (e.g., valve repair, valve
replacement, atrial septal defect, or ventricular septal defect,
etc.), coronary artery bypass grafting, and transmyocardial laser
revascularization, among others. In particular, these tissue grafts
are believed to provide an anti-inflammatory action at the
application site, along with pericardial closure to protect the
surface of the heart. In myocardial infarction, or other cardiac
disease, the tissue grafts described herein are used to prevent or
reduce size and/or severity of scarring occurring due to a
myocardial infarction or other cardiac disease.
[0183] In cardiac surgery, the tissue grafts described herein are
used in enhancing recovery following cardiac surgery (e.g., valve
repair, valve replacement, atrial septal defect, or ventricular
septal defect, coronary artery bypass grafting, and transmyocardial
laser revascularization, among others) by placing the tissue grafts
described herein into the cardiac area. "Cardiac area" as used
herein, refers to the region around the heart including but not
limited to the heart, the pericardial cavity, the pericardium, and
the coronary arteries and veins. In particular, these tissue grafts
are believed to provide an anti-inflammatory action at the
application site. In myocardial infarction, or other cardiac
disease, the tissue grafts described herein are used to limit
adverse cardiac remodeling (e.g., scarring occurring due to a
myocardial infarction or other cardiac disease). In some
embodiments, these tissue grafts are also used to assist in
pericardial closure to protect the surface of the heart.
[0184] In myocardial infarction, the tissue grafts herein in some
embodiments are used to minimize damage to the cardiovascular
system caused by a myocardial infarction. In particular, these
grafts can be placed in individuals experiencing an acute
myocardial infarction and undergoing a coronary artery bypass
procedure. Tissue grafts described herein limit the size and/or
severity of scarring following a myocardial infarction.
[0185] Advantageously, the tissue grafts described herein may have
one or more improved handling or performance characteristics, such
as flexibility, non-brittleness, and/or structural integrity, that
make the tissue graft particularly suitable for certain uses or
applications. For example, a highly flexible and non-brittle tissue
graft may be desired for tendon wrapping or deep wound healing,
because of the wound morphology or anatomic features of such
treatment sites. Additionally, improved handling and structural
integrity may provide medical practitioners with improved control
over placement of the tissue graft at a patient wound or other
treatment site. Additionally, it is contemplated that the increased
mechanical performance of these tissue grafts (i.e. suture
retention strength) facilitates the anchoring of the tissue graft
into deep wound beds and allow for a suture stitch on the proximal
and distal ends of a graft wrap.
EXAMPLES
Example 1--Tissue Graft Preparation
[0186] Placental tissue grafts were prepared in accordance with the
present disclosure, by obtaining a human fetal support tissue
membrane obtained from donated placenta, contacting the donated
human fetal support tissue membrane with a hypotonic solution to
osmotically swell cell matter at a maternal side of the human fetal
support tissue membrane, and removing swollen trophoblast and
decidua cell matter from a chorion connective, supportive tissue
layer of the human fetal support tissue membrane to produce an
isolated human fetal support tissue membrane which comprises amnion
and the chorion connective, supportive tissue layer, which amnion
and chorion are in an original, undisrupted connective
architecture. The isolated human fetal support tissue membrane was
decontaminated with ethanol, dried in the apparatus disclosed
herein, and terminally sterilized with radiation. The resulting
tissue membrane was analyzed as described below, to determine
various properties of the membrane. The tests conducted to measure
certain characteristics of the tissue grafts are described in more
detail below.
Example 2--Analysis of Membrane Layer Structure and Compactness
[0187] As discussed above, histological analysis, as shown in FIGS.
2 and 3, was performed to confirm that the maternal cell matter was
removed during processing and to analyze the effect of the drying
process on the tissue membrane. From the histological analysis, it
was determined that the hypotonic removal of cell material was
effective at removing the maternal decidua and trophoblast layers,
while maintaining the amnion and chorion in the native, or natural,
connective architecture. Additionally, the histological analysis
showed that the drying apparatus and method disclosed herein was
effective to produce a compact membrane having very little
anatomical distinction between the amnion and chorion layers (i.e.,
it was histologically difficult to differentiate the layers).
[0188] Data on the porosity void fraction, of the compact tissue
was collected using an image analysis test method, in which it was
determined that the sample tissue membranes from 5 donors display
an overall compactness or closed spaces of 97.9.+-.1.5%. In brief,
samples were processed using standard histological processing
methods. Samples were then sectioned (5 .mu.m thick) and mounted on
slides. The samples were then stained using hematoxylin and eosin
(H&E) and cover slipped (see FIG. 4, panel B). The stained
slides were imaged using a microscope with a 20.times. objective.
The images were then imported into image analysis software (ImageJ)
and a macro was run to look at portions of the tissue that did not
stain. These areas are considered pores within the tissue and
calculated as a percent open area based on pixels.
[0189] FIG. 4, panel B, shows one of the H&E stained tissue
graft samples used to determine the open volume of the processed
tissue graft, while FIG. 4, panel A, shows a commercially available
laminated amnion-chorion (i.e., separated layers reassembled)
tissue graft that has also been H&E stained. (FIG. 4, panel A,
taken from Koob, Thomas J., et al. "Properties of dehydrated human
amnion/chorion composite grafts: implications for wound repair and
soft tissue regeneration." J. Biomedical Materials Research Part B:
Applied Biomaterials 102.6 (2014): 1353-62.) From this image, the
commercially available laminate product was determined to have a
compactness of 82.9 percent. Thus, the tissue grafts made by the
process disclosed herein displays greater compactness, which is
hypothesized to reduce the rate of proteolysis and retard the
elution of bioactives. Specifically, it is believed that the denser
(i.e., less porous) tissue structure allows extended delivery
(i.e., diffusion based) of bioactives and extended life of the ECM
in the wound bed because the compact structure is more resistant to
enzymatic protease infiltration into the membrane system and the
resulting degradation.
[0190] It is contemplated that the maintenance of the connective
structure of the amnion and chorion and/or vacuum drying on one
side of the tissue to which a force is being applied, each may
provide these improved structural properties of the tissue grafts,
as opposed to those commercially available tissue grafts in which
the amnion and chorion are separated and which are believed to be
dried using a convection oven-type apparatus.
Example 3--Retention of Bioactives
[0191] First, an enzyme-linked immunosorbent assay (ELISA)
assessment of growth factors/cytokines, protease inhibitors, and
glycosaminoglycans was performed on the implant-ready tissue
grafts. In brief, tissue grafts were extracted in either
physiological relevant solutions or detergents. The samples were
then diluted to ensure the endpoints would be within the standard
curve for each respective assay. The results are shown below in
Table 1, which gives the minimal amount of selected moieties
isolated from any given donor as well as the average and standard
deviations of multiple donors.
TABLE-US-00001 TABLE 1 6 donors 20 Sections Avg SD Min bFGF pg/mg
13641.42 6985.92 6235.31 HGF pg/mg 337.63 216.01 67.06 TIMP-1 pg/mg
13351.64 5789.32 4842.19 HA ng/mg 16844.93 8112.73 5665.08
[0192] Comparative data on a commercially available laminated
amnion-chorion (i.e., layers separated then reassembled) tissue
graft was obtained from publicly available articles and is given in
Table 2 below. (Data from Koob, Thomas J., et al. "Biological
properties of dehydrated human amnion/chorion composite graft:
implications for chronic wound healing." International Wound
Journal 10.5 (2013): 493-500; and Koob, Thomas J., et al.
"Angiogenic properties of dehydrated human amnion/chorion
allografts: therapeutic potential for soft tissue repair and
regeneration." Vasc Cell 6.10 (2014).)
TABLE-US-00002 TABLE 2 Koob - Angiogenic . . . (2014) Koob -
Biological . . . (2013) Avg SD Donor # Avg SD Donor # bFGF (pg/mg
dry tissue) 0.717 0.225 8 1649.4 925.4 56 HGF (pg/mg dry tissue)
245.418 103.302 8 N/A N/A N/A TIMP-1 (pg/mg dry tissue) N/A N/A N/A
6356.8 3410.1 55
[0193] While no published data on the HA content of these
comparative laminated tissue grafts was found, qualitative images
based on Alcian blue staining of histological sections was found,
and is shown in FIG. 4, panel C. (Image from Koob, Thomas J., et
al. "Properties of dehydrated human amnion/chorion composite
grafts: implications for wound repair and soft tissue
regeneration." J. Biomedical Materials Research Part B: Applied
Biomaterials 102.6 (2014): 1353-62.) A comparative image of a
tissue graft sample prepared using the methods disclosed herein and
stained with Alcian blue is shown at FIG. 4, panel D. In FIG. 4,
panel D, it is noted that the exemplary tissue graft contains a
significant amount of HA as depicted from the bright staining. In
contrast, the comparative laminated tissue graft has much less HA
content as depicted from the light stain from FIG. 4, panel C.
[0194] The tissue grafts made by the presently disclosed methods
were also tested using the protocols outlined in Koob, Thomas J.,
et al. "Properties of dehydrated human amnion/chorion composite
grafts: implications for wound repair and soft tissue
regeneration." J. Biomedical Materials Research Part B: Applied
Biomaterials 102.6 (2014): 1353-62, with the comparative bioactives
content data being given in FIG. 11 for three moieties.
[0195] Overall, from the images of the stained tissue samples and
comparative bioactives data collected, it is clear that the tissue
grafts made from the methods described herein maintain a
significantly greater content of bioactives as compared to
commercially available laminated amnion-chorion (i.e., layers
separated and then reassembled) tissue graft. Without intending to
be bound by a particular theory, it is believed that the benign
processing conditions and/or maintenance of the connective
structure of the amnion and chorion as taught herein, provides the
improved maintenance of desirable bioactives, which it is believed
will result in more effective healing and promotion of cell
in-migration, proliferation, and differentiation along with an
anti-inflammatory action.
[0196] Additionally, the distribution and presence of growth
factors and structural proteins were also assessed via
immunohistochemistry (IHC) in further studies. In these studies,
control and processed tissue was formalin fixed and 5 .mu.m
sections were then placed on slides using standard histologic
means. Using standard IHC techniques the amount and location of the
biological motifs were assessed.
[0197] The protein components of this tissue were also assessed
using gel electrophoresis. The intensity and the molecular weight
(MW) of the dye stained proteins were analyzed. This technique
showed the no major alterations on the composition of extracts
derived from control (unprocessed) tissues and processed
tissues.
[0198] Finally, tissue grafts and extracts of tissue grafts
containing the material's endogenous bioactives were used as the
media for cell culture studies to determine their effect on the
viability and proliferative response of multiple types of human
derived cell lines.
Example 4--Alloantigenicity Detection
[0199] A mixed lymphocyte reaction (MLR) was used to explore the
allogenicity of the tissue grafts disclosed herein. In brief, fully
processed membranes were cultured with allogeneic peripheral blood
mononuclear cells (PBMCs) from three different donors in a MLR and
the proliferation of lymphocytes was assessed by BrdU. Table 3
lists the Stimulation Index of tissue graft samples with PBMCs from
the three donors. Table 4 shows the Stimulation Index of mixes of
PBMCs.
TABLE-US-00003 TABLE 3 Tissue graft Donor 1 Donor 2 Donor 3 T1-A
0.03 0.04 0.03 T1-B 0.06 0.18 0.25 T2-A 0.24 0.32 0.40 T2-B 0.05
0.26 0.89 T3-A 0.14 0.16 0.00 T3-B 0.00 0.14 0.50
TABLE-US-00004 TABLE 4 PBMC mix SI Donor 1 .times. Donor 2 11.2
Donor 1 .times. Donor 3 6.5 Donor 2 .times. Donor 3 8.5
[0200] The results demonstrated that the processed membranes did
not generate an increase in lymphocyte number.
[0201] Additionally, feasibility studies have demonstrated that the
tissue graft does not stimulate allogeneic PBMCs and that it does
enhance the rate of closure of dermal wounds in a diabetic mouse
model.
Example 5--Bioburden Analysis
[0202] The bioburden of processed tissue grafts after ethanol
decontamination and drying was assessed via an external laboratory.
Test articles 1-10 were 14 mm disc membranes and test articles
12-21 were 5 mm.times.6 mm rectangular membranes. The bioburden
test results confirmed minimal bioburden present on the processed
membranes, as shown in Table 5 below.
TABLE-US-00005 TABLE 5 Results Unit # Test Article Aerobic
Anaerobic Fungal 1 #1A <4 <4 <4 2 #2A <4 <4 <4 3
#3A <4 <4 <4 4 #4A <4 <4 <4 5 #5A <4 <4
<4 6 #7A <4 <4 <4 7 #9A <4 <4 <4 8 #10A <3
<4 <3 9 #11A <4 <4 <4 10 #13A <4 <4 <4
Averages <3.9 <3.9 <4.0 Recovery Efficiency UTD 12 #1B
<4 <4 <4 13 #2B <4 <4 <4 14 #3B <4 <4 <4
15 #4B <4 <4 <4 16 #5B <4 <4 <4 17 #7B <4
<4 <4 18 #9B <4 <4 <4 19 #10B <4 <4 <4 20
#11B <4 <4 <4 21 #13B <4 <4 <4 Averages <3.9
<4.0 <4.0 Recovery Efficiency UTD <=No Organisms Detected
Note: The results are reported as colony forming units per test
article
Example 6--Water Content Analysis
[0203] Water content of the prepared tissue grafts was determined
by gravimetric analysis. In brief, the sample was weighed, heated
to a temperature above the boiling point of water, and weighed
again. As discussed above, it was determined that the drying
apparatus disclosed herein was capable of consistent dehydration of
tissue samples to 5 percent water content, or less. The two-step
drying process disclosed above achieved a desired dehydration level
of 10 percent water content, or less, depending on properties
desired. The first drying step generally dries the tissue graft to
a water content of between 15 and 10 percent to enable cutting and
dicing of the tissue. The secondary drying step is then used to dry
the tissue after it is exposed to the room and to further dry the
tissue to enhance stability for room temperature storage.
Example 7--Stability Studies
[0204] The stability of the tissue grafts prepared by the disclosed
methods was determined by analysis of structural proteins with
differential scanning calorimetry, glycosaminoglycans GAGs with
electrophoresis, and cell proliferation assays.
Example 8--Use of Buffered Peracetic Acid as a Decontaminant
[0205] It was discovered that buffering peracetic acid to a neutral
pH of 4.5 to 7.5 enables a higher retention of bioactive molecules
(i.e., HGF, KGF, and TIMP-1) compared to non-buffered peracetic
acid. Specifically, using commercially available peracetic acid
concentration test strips (i.e., test strips that determined the
potency level of peracetic acid in a solution), greater stability
of peracetic acid was observed during dilution of 40% peracetic
acid in acetic acid into a buffered solution with certain
parameters (i.e., molarity of the buffer, initial pH). These
specific bioactives (HGF, KGF, and TIMP-1) may advantageously
characterize a tissue with different indications than the one
resulting from treatment with ethanol.
Example 9--Suture Retention, Membrane Tensile Strength and
Bending
[0206] First, a suture loop (4-0 polypropylene monofilament) was
placed through the end of a tissue sample (2 mm from short edge of
10 mm.times.20 mm tissue coupon), looped over a fixture attached to
a 10 N load cell, and pulled under uniaxial tension until failure
occurred while monitoring the applied load at a fixed elongation
rate of 50 mm/min. The samples came from three donors each with
samples irradiated at 0 (Control), 22.5, and 30 kGy, were each
tested in triplicate. The results are given in FIG. 14 and indicate
that suture retention strength declines from Control values
following irradiation at either 22.5 kGy or 30 kGy (p-value=0.001
by ANOVA). However, these differences may be due to the
directionality of testing as some samples appear to rip straight
out of the tissue and some pull and create a horizontal tear
first.
[0207] Second, a suture loop (4-0 polypropylene monofilament) was
placed through the end of a tissue sample (2 mm from short edge of
10 mm.times.20 mm tissue) and pulled under uniaxial tension until
failure occurred while monitoring the applied load at a fixed
elongation rate of 50 mm/min. The samples came from one donor,
non-irradiated, tested eight times, with four replicates cut in one
direction and four cut in the perpendicular direction. The results
are given at FIG. 15, and indicate that suture retention strength
is significantly different within the same section of tissue
depending on the pulling direction (p-value=0.025 by ANOVA). Two of
four samples pulled in the x-direction ripped straight out of the
tissue while the other two and all four samples pulled in the
y-direction created a horizontal tear first.
[0208] Third, a 5 mm.times.20 mm strip of sheet tissue was pulled
under uniaxial tension until failure occurred while monitoring the
applied load with a 10 N load cell at a fixed elongation rate of 50
mm/min. The samples were from two donors each with samples
irradiated at 0 (control), 22.5 (only one donor), and 30 kGy,
tested in triplicate. The results are shown in FIGS. 16 and 17 and
indicate that there is no significant difference between irradiated
and non-irradiated tissue with regard to ultimate tensile stress
and strain at break (p-value=0.231 and 0.058 respectively by
ANOVA).
[0209] Fourth, a 3 mm wide dog bone shaped piece of sheet tissue
was pulled under uniaxial tension until failure occurred while
monitoring the applied load at a fixed elongation rate of 50
mm/min. The samples were from one donor, non-irradiated, tested six
times, three cut in one direction and three cut in the
perpendicular direction. The results are shown in FIGS. 18 and 19,
and indicate that ultimate tensile stress and strain at break show
a difference between samples tested in one direction compared to
the perpendicular direction. The tensile stress is not
significantly different while the strain values are statistically
different (p-value=0.068 and 0.049 respectively, by ANOVA).
[0210] Fifth, a strip of dried sheet tissue at least 10 mm wide was
manually rolled around a fixed mandrel of 2 mm diameter until the
ends were parallel and the tissue was examined for cracking,
breakage, or tearing. The samples were from three donors with
samples tested three times each. A photograph of a sample during
testing is shown at FIG. 20. No samples failed the radius of
curvature test on a 2 mm diameter mandrel. Furthermore, most tissue
samples dried in accordance with the disclosed embodiments were
able to be folded completely and pressed to crease and remain
unbroken. Once rehydration is initiated, any possibility of
cracking and breakage was eliminated.
[0211] In summary, human placenta-derived tissue grafts have been
developed that display high bioactives content and reduced
alloantigenicity compared to known placental tissue grafts, and
therefore may display improved healing properties. The
placenta-derived products may display improved handling and
performance properties, such as compactness, flexibility, suture
retention, resistance to enzymatic breakdown, slow release of
bioactives, and longer residence time at a wound or implantation
site.
[0212] Generally, it is believed that because the native
configuration of the membrane layers has never been violated (i.e.,
the amnion and chorion are not separated and/or treated in a
separate manner), improved retention of bioactive elements present
within and between these layers is achieved. To minimize both the
potential inflammatory response in the recipient and the transfer
of elements in the maternal blood, the decidua layer (cells of
maternal origin) is removed along with a substantial portion of the
trophoblast cell population which can contain maternal macrophages.
That is, the goal of the processing is to maintain the native
composition with three exceptions, the decidua layer, trophoblast
cell population and the bulk of the residual blood in the membrane.
Benign processing conditions that have a neutral pH and do not
include of aggressive components, such as certain detergents, are
believed to maintain bioactives in levels not seen with presently
commercially available placental tissue grafts.
[0213] To lower the bioburden of the tissue, the tissue is
decontaminated, such as with an ethanolic solution. To enable
planar dried products with reproducible cross-sectional
architecture and minimal open space, the tissue is placed in
between a porous membrane and a nonporous material and dried under
vacuum. To facilitate stable storage, the membrane is desiccated to
a final moisture content lower than 10 percent, as determined by
loss on drying testing. The membrane is set onto a backing material
with tabs that will enable stability during shipment and storage
and to provide an easy means of delivery to the wound site. The
membrane is terminally sterilized by ionizing radiation while in a
dry state and at a minimal dosage commensurate with retention of
activity of the contained bioactives and low bioburden of the input
tissue following decontamination. Thus, tissue grafts prepared by
most or all of these steps display improved properties over known
placental tissue grafts.
Example 10--Preparation of Folded Membrane Tissue Grafts
[0214] Placental tissue grafts are prepared in accordance with the
present disclosure, by obtaining a human fetal support tissue
membrane obtained from donated placenta, contacting the donated
human fetal support tissue membrane with a hypotonic solution to
osmotically swell cell matter at a maternal side of the human fetal
support tissue membrane, and removing swollen trophoblast and
decidua cell matter from a chorion connective, supportive tissue
layer of the human fetal support tissue membrane to produce an
isolated human fetal support tissue membrane which comprises amnion
and the chorion connective, supportive tissue layer, which amnion
and chorion are in an original, undisrupted connective
architecture. The isolated human fetal support tissue membrane is
decontaminated with ethanol. The decontaminated isolated human
fetal support tissue membrane is then folded so that the amnion
layer is on the outside of the folded membrane and the chorion
layer is in the center. The folded human fetal support tissue
membrane is then dried in the apparatus disclosed herein, and
terminally sterilized with radiation. The resulting tissue membrane
is analyzed as described below, to determine various properties of
the membrane. The tests conducted to measure certain
characteristics of the tissue grafts are described in more detail
below.
Example 11--Treatment of Wounds in a Diabetic Animal Wound Healing
Model
[0215] Full-thickness acute excisional wounds were created on the
dorsal flank of db/db (BKS.Cg-m Dock7m+/+Leprdb/J mice) mice (in
vivo model of type II diabetes impaired wound healing). Placental
tissue grafts prepared in accordance with the present disclosure
were implanted in the wound bed of the test animals (CryoLife). The
implants were covered with a non-adherent dressing (Tegaderm
Contact, 3M) and Bioclusive Film Dressing (Systegenix), the later
to limit wound closure due to skin contraction. Control wounds
(Standard of Care) received only Tegaderm and Bioclusive dressings.
Only one application of the placental tissue graft was used. Wound
closure (expressed as percent reduction in wound area) and length
of wound margins (wound perimeter) were monitored at the indicated
times following wounding by digital measurements (Image J, NIH) of
digital images obtained after removal of the Tegaderm and
Bioclusive dressings to visualize the wounds.
Example 12--Treatment of Myocardial Infarction in a Rat Model
[0216] Rats were acutely implanted with a placental tissue graft
prepared in accordance with the present disclosure after inducing a
myocardial infarction by permanent coronary artery ligation in the
rats. Six weeks after the induced myocardial infarction, the rats
were sacrificed and the heart and the chest cavity were examined.
Rats treated with the placental tissue graft as described herein
had reduced myocardial fibrosis, likely due to reduced cell death,
and no evidence of epicardial adhesions to the chest wall. Rats
treated with a control graft experienced cardiac adhesions with the
chest wall.
Example 13--Treatment of Myocardial Infarction
[0217] Patients who have experienced myocardial infarction are
treated with placental tissue grafts prepared in accordance with
the present disclosure. Placental tissue grafts are placed on the
affected cardiac tissue. Patients treated with the placental grafts
described herein experience reduced incidence of post-operative
atrial fibrillation compared to patients not treated with the
graft.
Example 14--Treatment of Post-Operative Atrial Fibrillation
[0218] Patients experiencing open heart surgery are treated with
placenta tissue grafts prepared in accordance with the present
disclosure. Placental tissue grafts are placed on the affected
cardiac tissue. Patients treated with the placental grafts as
described herein experience reduced incidence of post-operative
atrial fibrillation compared to patients not treated with the
graft.
[0219] The examples and embodiments described herein are for
illustrative purposes only and various modifications or changes
suggested to persons skilled in the art are to be included within
the spirit and purview of this application and scope of the
appended claims.
* * * * *