U.S. patent application number 16/508783 was filed with the patent office on 2019-11-07 for electroporation of tissue products.
The applicant listed for this patent is LifeCell Corporation. Invention is credited to Martin J. Byrne, Gary Monteiro, Rick T. Owens.
Application Number | 20190336625 16/508783 |
Document ID | / |
Family ID | 50023878 |
Filed Date | 2019-11-07 |
United States Patent
Application |
20190336625 |
Kind Code |
A1 |
Monteiro; Gary ; et
al. |
November 7, 2019 |
ELECTROPORATION OF TISSUE PRODUCTS
Abstract
The present disclosure provides methods for reducing bioburden
on a tissue product, as well as the tissue products produced
according to the disclosed methods. In particular, the disclosure
relates to methods of electroporating tissue in the presence of one
or more bactericides in order to reduce bioburden. The methods
allow for reduced exposure to electrical energy and/or bactericide
while reducing bioburden.
Inventors: |
Monteiro; Gary; (Grosse
Pointe, MI) ; Byrne; Martin J.; (Hellertown, PA)
; Owens; Rick T.; (Stewartsville, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LifeCell Corporation |
Madison |
NJ |
US |
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|
Family ID: |
50023878 |
Appl. No.: |
16/508783 |
Filed: |
July 11, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15889473 |
Feb 6, 2018 |
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16508783 |
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14762235 |
Jul 21, 2015 |
9919066 |
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PCT/US14/10586 |
Jan 8, 2014 |
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15889473 |
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61755598 |
Jan 23, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 2/0011 20130101;
A01N 25/00 20130101; A01N 37/16 20130101; A61L 2/0088 20130101;
A61L 2/007 20130101 |
International
Class: |
A61L 2/00 20060101
A61L002/00; A01N 37/16 20060101 A01N037/16; A01N 25/00 20060101
A01N025/00 |
Claims
1-38. (canceled)
39. A method of reducing a bioburden in a tissue product,
comprising: providing a human or animal tissue; exposing the human
or animal tissue to one or more electrical pulses; and controlling
at least one of a pulse voltage, a pulse duration, or a number of
pulses, of the electrical pulses to regulate a pore size in the
human or animal tissue.
40. The method of claim 39, comprising controlling at least one of
the pulse voltage, the pulse duration, or the number of pulses, of
the electrical pulses to regulate an amount of time that pores in
the human or animal tissue remain open.
41. The method of claim 39, comprising contacting the human or
animal tissue with one or more bactericides.
42. The method of claim 39, comprising applying the one or more
electrical pulses for a long duration, at a high voltage or in
multiple pulses in rapid succession to create temporary or
permanent pores in lipid membranes of the human or animal
tissue.
43. The method of claim 41, wherein exposing the human or animal
tissue to the one or more electrical pulses drives the one or more
bactericides into the human or animal tissue.
44. The method of claim 41, wherein exposing the human or animal
tissue to the one or more electrical pulses evenly administers the
one or more bactericides across a thickness of the human or animal
tissue.
45. The method of claim 41, wherein the method produces a
substantial reduction in the bioburden on the human or animal
tissue.
46. The method of claim 45, wherein the bioburden on the human or
animal tissue is reduced by at least about 50%.
47. The method of claim 39, wherein the pulse duration, the pulse
voltage, or the number of pulses is controlled such that an
extracellular matrix of the tissue product is not damaged during
exposure to the one or more electrical pulses.
48. The method of claim 41, wherein a duration of exposure to and
concentration of the one or more bactericides are controlled such
that an extracellular matrix of the tissue product is not damaged
during exposure to the one or more bactericides.
49. The method of claim 39, wherein the pulse duration, the pulse
voltage, or the number of pulses is controlled such that a
temperature of the tissue product does not rise above about 42
degrees Celsius.
50. The method of claim 39, wherein the pulse duration, the pulse
voltage, or the number of pulses is controlled such that a pH of
the tissue product does not rise above about pH 8.
51. The method of claim 39, wherein the pulse voltage of the one or
more electrical pulses is about 1 volt (V) to about 10 kilovolts
(kV).
52. The method of claim 39, wherein the pulse voltage of the one or
more electrical pulses is about 40V or 80V.
53. The method of claim 39, comprising administering the one or
more electrical pulses for the pulse duration of about 1
millisecond (ms) to about 1 second.
54. The method of claim 39, wherein the pulse duration of the one
or more electrical pulses is about 5 ms, about 10 ms, or about 15
ms.
55. The method of claim 39, wherein the number of pulses
administered is about 1 to 100,000 electrical pulses.
56. The method of claim 39, wherein the number of pulses
administered is about 1 to 500 electrical pulses.
57. The method of claim 39, comprising allowing the human or animal
tissue to cool and then exposing the human or animal tissue to one
or more additional electrical pulses.
58. The method of claim 57, comprising repeating the exposure to
one or more additional electrical pulses 2-5 times.
59. The method of claim 41, wherein the bactericide comprises one
or more microbial growth inhibitors, cytotoxic agents, oxidants,
and/or antibiotics.
60. The method of claim 41, wherein the bactericide comprises one
or more of a peroxide, oxidizer, antimicrobial metal, quaternary
ammonium compound, or charged bactericidal compound.
61. The method of claim 41, wherein the bactericide comprises one
or more of peracetic acid (PAA), ozone, hypochlorite, silver, zinc,
copper, benzalkonium chloride, cetylpyridinium chloride,
benzethonium chloride, cetyltrimethyl ammonium bromide, or
chitosan.
62. The method of claim 41, wherein the bactericide comprises
PAA.
63. The method of claim 62, wherein the PAA is at a concentration
of about 0.01%-2% weight/volume.
64. The method of claim 62, wherein the PAA is at a concentration
of about 0.2% weight/volume.
65. The method of claim 41, wherein the bactericide is applied to
the human or animal tissue at a concentration sufficient to
substantially reduce bioburden on the human or animal tissue.
66. The method of claim 39, comprising irradiating the human or
animal tissue using e-beam radiation.
67. The method of claim 39, further comprising decellularizing the
human or animal tissue.
68. The method of claim 67, wherein the human or animal tissue is
decellularized by contact with one or more detergents and by
exposure to the one or more electrical pulses.
Description
[0001] This application claims priority under 35 U.S.C. .sctn. 119
to U.S. Provisional Application No. 61/755,598, which was filed on
Jan. 23, 2013, and which is herein incorporated in its
entirety.
[0002] The present disclosure relates to methods for reducing
bioburden on tissue products, and more particularly, to methods of
reducing bioburden using electroporation, as well as tissue
products produced according to the disclosed methods.
[0003] Human and animal tissues can be used to produce a variety of
tissue products for patient use. When the tissues used in tissue
products are procured from tissue banks or animal sources, they may
contain undesirable levels of bacterial bioburden that must be
reduced using various procedures. One option for reducing bioburden
involves exposure to bactericides such as peracetic acid (PAA).
High concentration and long duration exposure to bactericide,
however, may be required to sufficiently reduce bioburden, which
could lead, in some instances, to undesirable consequences such as
damage to collagen networks or other tissue components. The
bactericides may also suffer from an inability to adequately
penetrate the bacterial cells or to reach bacteria growing
throughout the full thickness of the tissue, leading to potential
pockets of elevated bioburden in the final tissue product.
[0004] Accordingly, there is a need for improved methods of
sterilizing and/or reducing the bioburden on a tissue product.
Disclosed herein are methods comprising the use of electroporation
to reduce bioburden and to enhance the effectiveness of one or more
bactericides in reducing bioburden. In some instances,
electroporation allows for an effective method of reducing
bioburden and permits use of a lower concentration, duration,
and/or total volume of bactericide. Further, the use of
electroporation can allow for a more even administration of
bactericide across the full thickness of the tissue product.
Moreover, electroporation can also be used as part of a method of
decellularizing a tissue product while simultaneously reducing
bioburden.
[0005] In various embodiments, a method of reducing bioburden in a
tissue product is provided, comprising providing a human or animal
tissue, contacting the tissue with one or more bactericides, and
exposing the tissue to one or more electrical pulses. The method
can produce a substantial reduction in bioburden (e.g., a reduction
of at least about 50%). In some embodiments, the duration of the
electrical pulses, voltage of the electrical pulses, and number of
electrical pulses can be controlled such that the extracellular
matrix of the tissue product is not damaged during exposure to the
electrical pulses. In some embodiments the duration of exposure to
and concentration of the one or more bactericides are controlled
such that the extracellular matrix of the tissue product is not
damaged during exposure to the one or more bactericides. In certain
embodiments, the method further comprises allowing the tissue to
cool and then exposing the tissue to one or more additional
electrical pulses.
[0006] In various embodiments, the bactericide comprises one or
more microbial growth inhibitors, cytotoxic agents, oxidants,
and/or antibiotics. In some embodiments, the bactericide comprises
one or more of a peroxide, oxidizer, antimicrobial metal,
quaternary ammonium compound, or charged bactericidal compound. In
some embodiments, the bactericide comprises one or more of
peracetic acid (PAA), ozone, hypochlorite, silver, zinc, copper,
benzalkonium chloride, cetylpyridinium chloride, benzethonium
chloride, cetyltrimethyl ammonium bromide, or chitosan. For
example, the bactericide can comprise PAA, e.g., at a concentration
of about 0.01%-2% weight/volume.
[0007] In various embodiments, the bactericide is applied to the
tissue at a concentration sufficient to substantially reduce
bioburden on the tissue. In some embodiments, the method of
reducing bioburden further comprises irradiating the tissue using
e-beam radiation. In some embodiments, the method further comprises
decellularizing the tissue, for example by contacting the tissue
with one or more detergents and/or by exposure to one or more
electrical pulses.
[0008] In various embodiments, a tissue product is provided,
comprising a human or animal tissue that has been contacted with
one or more bactericides and exposed to one or more electrical
pulses. In some embodiments, the bioburden on the tissue has been
reduced by at least about 50%. In some embodiments, the tissue
product comprises an intact extracellular matrix. In some
embodiments, the tissue product has been irradiated using e-beam
radiation. In some embodiments, the tissue product comprises
decellularized tissue.
DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1A-C show hematoxylin and eosin (H&E) staining of 1
cm by 1 cm samples of porcine dermis after exposure for 15
milliseconds to one electrical pulse of 0V (FIG. 1A), 40V (FIG.
1B), or 80V (FIG. 10), according to certain embodiments of the
present disclosure.
DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS
[0010] Reference will now be made in detail to certain exemplary
embodiments according to the present disclosure, certain examples
of which are illustrated in the accompanying drawings. Wherever
possible, the same reference numbers will be used throughout the
drawings to refer to the same or like parts.
[0011] In this application, the use of the singular includes the
plural unless specifically stated otherwise. In this application,
the use of "or" means "and/or" unless stated otherwise.
Furthermore, the use of the term "including", as well as other
forms, such as "includes" and "included," is not limiting. Any
range described herein will be understood to include the endpoints
and all values between the endpoints.
[0012] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents, or portions of documents, cited in
this application, including but not limited to patents, patent
applications, articles, books, and treatises, are hereby expressly
incorporated by reference in their entirety for any purpose.
[0013] Various human and animal tissues can be used to produce
tissue products. For example, various tissue products have been
produced for regeneration, repair, reinforcement, and/or treatment
of human tissues that have been damaged or lost due to various
diseases and/or structural damage (e.g., from trauma, surgery,
atrophy, and/or long-term wear and degeneration). Likewise, such
products have been used to augment or enhance various tissues.
These products can include, for example, acellular tissue matrices,
tissue allografts or xenografts, and/or reconstituted tissues
(e.g., at least partially decellularized tissues that have been
seeded with cells to produce viable materials). For example,
ALLODERM.RTM. and STRATTICE.TM. (LifeCell Corp., Branchburg, N.J.)
are two dermal acellular tissue matrices made from human and
porcine dermis, respectively. In preparing these tissue products,
there can be a need to reduce bioburden.
[0014] Disclosed herein are methods for reducing bioburden in a
tissue product using electroporation, as well as the tissue
products produced according to the disclosed methods. The method
can involve providing a tissue from an animal (including a human
tissue). The tissue can be processed to prepare a desired tissue
product (e.g., by manually cutting, shaping, or molding the tissue
product to a desired shape, by decellularizing the tissue, and/or
by any other desired processing procedure). The tissue is treated
to reduce bioburden before, at the same time, or after the
processing step(s).
[0015] Treating a tissue to reduce bioburden can comprise
administering one or more electrical pulses to the tissue. In some
embodiments, the exposure to electrical pulses alone is sufficient
to reduce, and/or substantially reduce, bioburden. Electroporation
can be administered to a tissue that has been contacted with one or
more chemical or biological bactericide agents. For example, a
tissue product can be immersed in a solution containing PAA, and
then one or more electrical pulses can be administered to the
tissue product. In some embodiments, pulses of long duration, high
voltage, and/or multiple pulses in rapid succession can create
temporary or permanent pores in the lipid membranes of bacterial
cell walls, resulting in cell death directly or through the
subsequent penetrance of one or more bactericides into the
microbial cell. A minimum voltage can be required to establish
pores in bacterial cell walls. In addition, the pulse voltage,
pulse duration, and/or number of pulses administered can regulate
the size of pores and the amount of time that pores in bacterial
cell walls remain open, with higher voltage, duration, and/or pulse
number resulting in larger and more long-lasting pores.
[0016] In some embodiments, the use of electroporation allows for
an efficient reduction in bioburden while using a lower
concentration, volume, and/or exposure time of the one or more
bactericide. For instance, the use of electrical pulses can destroy
microbes on the tissue surface by degrading the microbial cell
membranes. Likewise, the electrical pulses can open pores in the
bacterial cell membranes through which one or more bactericide
(e.g., PAA) can enter and destroy the microbes, allowing for a
reduction in bioburden while using a reduced concentration of
bactericide and/or shorter duration exposure to bactericide. In
addition, electroporation may drive bactericide into the tissue,
allowing for a reduction in bioburden more consistently along the
full thickness of the tissue.
[0017] In some embodiments, the electroporation methods described
above can be used in conjunction with a decellularization
procedure. For instance, a tissue can be decellularized by exposure
to one or more detergents and by exposure to electrical pulses,
either simultaneously or sequentially. Both the electrical pulses
and the detergent can be used to destroy and remove cellular
material from the tissue, while at the same time the exposure to
electrical pulses and bactericide can also reduce bioburden in the
tissue undergoing processing.
[0018] In various embodiments, electroporation can include the
administration of one or more high voltage pulses. The high voltage
pulses can have a duration of about 1 millisecond (ms) to 1 second.
For example, pulses of about 1-50 ms can be used (e.g., about 1, 2,
3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 ms, or any time
period in between). In certain embodiments, pulses of about 5 ms,
10 ms, or 15 ms in duration are used. In some embodiments, longer
pulses are used in order to increase the percentage reduction in
bioburden. The one or more pulses can be delivered at a voltage of
about 1V-10 kV. For example, pulses of about 10V-100V can be used
(e.g., about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 V, or any
voltage in between). In certain embodiments, pulses of about 40V or
80V are used. In some embodiments, higher voltage pulses are used
to increase the percentage reduction in bioburden. Both direct
current and alternating current can be used with the
electroporation methods disclosed herein. In some embodiments where
alternating current pulses are administered, frequencies of about
1-10 KHz can be used. In some embodiments, about 1-100,000 pulses
are delivered. For example, about 1-500 pulses can be delivered
(e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 100, 200,
300, 400, or 500 pulses, or any value in between). In some
embodiments, a higher number of pulses are used in order to
increase the percentage reduction in bioburden.
[0019] In various embodiments, the upper limit on the voltage,
duration, and/or number of electrical pulses applied to a tissue
will depend on the sensitivity of the chosen tissue to the electric
field (e.g., the amount of electrical energy that the tissue can
absorb without damage). In various embodiments, the lower limit on
the voltage, duration, and/or number of pulses is based upon the
sensitivity of the targeted bacterial organism to the electric
field (e.g., the amount of energy required to kill or otherwise
inactivate the targeted bacteria). In some embodiments, the pulse
duration, pulse voltage, and/or number of pulses are controlled to
avoid damage to the collagen and other extracellular networks in
the tissue, to prevent an excessive elevation in temperature or pH,
and/or to prevent any other damage to the tissue. In some
embodiments, an excessive elevation in temperature is an increase
above about 42 degrees Celsius (e.g., above about 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, or 50 degrees Celsius). In some
embodiments, an excessive increase in pH is an increase above about
7.5 (e.g., above about 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0). In
certain embodiments, the voltage, duration, and/or number of pulses
required to reduce bioburden will depend on the thickness of the
tissue, with thicker and/or larger tissues requiring more joules of
energy in order to reduce bioburden by a desired percentage.
[0020] In some embodiments, multiple rounds of electroporation are
delivered to the tissue to reduce bioburden. In some embodiments,
the tissue is allowed to cool to room temperature between rounds of
electroporation.
[0021] In various embodiments, the electroporation methods
disclosed herein comprise applying one or more bactericides (e.g.,
1, 2, 3, 4, 5, or more) to a tissue and administering one or more
high voltage pulses to the tissue. The bactericide can comprise any
chemical or biological agent suitable for reducing bioburden
(including microbial growth inhibitors, cytotoxic agents, oxidants,
and/or antibiotics). In some embodiments, the bactericide is
peracetic acid (PAA). In some embodiments the bactericide is any
chemical or biological agent that functions through bacterial
membrane perturbation or acts intracellularly on a microbe.
Examples of suitable bactericides include peroxides (e.g., PAA),
oxidizers (e.g., ozone, hypochlorite), antimicrobial metals (e.g.,
silver, zinc, copper), quaternary ammonium compounds (e.g.,
benzalkonium chloride, cetylpyridinium chloride, benzethonium
chloride, cetyltrimethyl ammonium bromide), and/or other charged
bactericidal compounds such as chitosan that can be driven by
electroporation through the pores formed in bacterial cell
membranes.
[0022] In some embodiments, the bactericide is applied to the
tissue at a concentration sufficient to reduce bioburden, and/or to
substantially reduce bioburden. In some embodiments, PAA is applied
to the tissue at a concentration sufficient to reduce bioburden,
and/or to substantially reduce bioburden. In certain embodiments,
PAA is applied to the tissue at a concentration of about 0.01%-2%
weight/volume to reduce bioburden on the tissue (e.g., about 0.01,
0.02, 0.03, 0.04, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45,
0.5, 0.55, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0%, or any percentage
in between). In certain embodiments, PAA is applied to the tissue
at a concentration of about 0.2% (w/v).
[0023] In various embodiments, any device known in the art for the
delivery of electrical energy can be used to electroporate a
tissue. For example, conductive electroporation plates can be
placed in parallel on either side of a tissue and one or more
electrical pulses can be passed between the plates. In some
embodiments, the electroporation plates comprise a conductive metal
and are positioned in parallel around a tissue, with each plate
about 1 mm from the edge of the tissue. In certain embodiments, the
electroporation plates comprise a conductive metal having
dimensions of about 1 cm.times.2 cm and each plate is positioned
about 1 mm from the edge of a 1 mm thick sample of tissue.
[0024] In various embodiments, the administration of electrical
pulses in combination with one or more bactericide can allow for a
method of reducing bioburden while also reducing the concentration
of bactericide and the voltage/duration of electricity. For
instance, a high concentration or long duration exposure to PAA or
other bactericides can be associated with undesirable damage to the
collagen networks in a tissue product. Likewise, exposure to high
voltage or prolonged exposure to an electrical field can damage a
tissue product. In certain embodiments, these negative effects on
the quality of a tissue can be avoided by using a bactericide in
combination with electroporation. Accordingly, in some embodiments,
electroporation can be used in combination with a bactericide to
enable a reduction in bioburden while using a lower concentration
or shorter duration exposure to bactericide. Likewise, in certain
embodiments, electroporation can be used in combination with a
bactericide to enable a reduction in bioburden while using shorter
duration or lower energy pulses, or fewer total pulses. For
example, a substantial reduction in bioburden can be achieved by
contacting a tissue with at least about 0.1% PAA and then
administering one or more electrical pulses of at least about 40V
and at least about 5 ms in duration.
[0025] In various embodiments, the electroporation methods
discussed above can be used to reduce bioburden on a tissue (i.e.,
to reduce the number of microorganisms growing on the tissue). In
some embodiments, the methods substantially reduce bioburden. As
used herein, a tissue that has "substantially reduced bioburden"
means a tissue on which the concentration of growing microorganisms
is less than 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 1%, 0.1%,
0.01%, 0.001%, or 0.0001% of that growing on untreated tissue.
[0026] The electroporation methods discussed above can, optionally,
be used in combination with one or more additional methods for
reducing bioburden, such as exposure to radiation ("irradiation").
Irradiation can be used to further reduce bioburden. In some
embodiments, an absorbed dose of about 14-18 kGy of e-beam
radiation or 25-30 kGy of gamma irradiation is delivered. In
various embodiments, a tissue product is exposed to between about 5
Gy and 50 kGy of radiation (e.g., about 5, 10, 15, 20, 25, 30, 35,
40, 45, or 50 kGy, or any value in between). Suitable forms of
radiation can include gamma radiation, E-beam radiation, and X-ray
radiation. In some embodiments, E-beam irradiation is used. Other
irradiation methods are described in U.S. Application 2010/0272782,
the disclosure of which is hereby incorporated by reference in its
entirety. Irradiation can be applied before, simultaneously, or
after electroporation.
[0027] Various tissues can be used with the methods disclosed
herein. For example, human tissue can be obtained from one or more
cadavers, e.g., from dermal or subdermal sources. Suitable human
tissue can also be obtained from live donors (e.g., with an
autologous tissue). Other species that can serve as donors of
acellular tissue include, without limitation, nonhuman primates
(e.g., monkeys, baboons, or chimpanzees), pigs, cows, horses,
goats, sheep, dogs, cats, rabbits, guinea pigs, gerbils, hamsters,
rats, or mice. In some embodiments, tissue from more than one donor
animal can be used. Tissue from animals genetically modified to
lack one or more antigens, such as the immunogenic antigen alpha
galactose, can also be used.
[0028] In various embodiments, the tissue can come from one or more
of fascia, pericardial tissue, dura, umbilical cord tissue,
placental tissue, cardiac valve tissue, ligament tissue, tendon
tissue, arterial tissue, venous tissue, neural connective tissue,
urinary bladder tissue, ureter tissue, skin, dermal tissue, muscle
tissue, heart tissue, lung tissue, liver tissue, or intestinal
tissue. In some embodiments, the tissue is dermis. In certain
embodiments, the tissue is human or porcine dermis. In certain
embodiments, the tissue is ALLODERM.RTM. or STRATTICE.TM. (LifeCell
Corp., Branchburg, N.J.).
[0029] The electroporation methods described above can be used in
conjunction with additional tissue processing procedures, such as a
decellularization procedure. In some embodiments, the
electroporation procedure is conducted before the decellularization
procedure, while in other embodiments the electroporation procedure
is conducted after the decellularization. In some embodiments,
electroporation procedures are conducted both before and after
decellularization. In some embodiments, the two procedures are
conducted simultaneously. In some embodiments, the electroporation
procedure can disintegrate the cellular material from a tissue
product, and can therefore be used as part of a decellularization
procedure, as well as a bioburden reduction procedure.
[0030] In various embodiments, the decellularization procedure that
is used in combination with an electroporation procedure comprises
placing the tissue in a decellularization solution to remove viable
cells (e.g., epithelial cells, endothelial cells, smooth muscle
cells, and fibroblasts, etc.) from the extracellular matrix in the
tissue without damaging the biological and/or structural integrity
of the extracellular matrix. The decellularization solution may
contain an appropriate buffer, salt, an antibiotic, one or more
detergents (e.g., TRITON X-100.TM., sodium dodecyl sulfate, sodium
deoxycholate, polyoxyethylene (20) sorbitan mono-oleate, etc.), one
or more agents to prevent cross-linking, one or more protease
inhibitors, and/or one or more enzymes. In some embodiments, the
decellularization solution comprises 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,
1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, or 5.0% (or any
percentage in between) of TRITON X-100.TM. and, optionally, 10 mM,
15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM EDTA
(ethylenediaminetetraacetic acid) (or any concentration in
between). In some embodiments, the tissue is incubated in the
decellularization solution at 25, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, or 42.degree. C. (or any temperature in between),
and optionally with gentle shaking at 10, 20, 30, 40, 50, 60, 70,
80, 90, 100, 110, 120, 130, 140, or 150 rpm (or any rpm in
between). The incubation can be for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 15, 20, 24, 36, or 48 hours (or any time in between). The
length of time or concentration of detergent can be adjusted in
order to partially or more fully decellularize the tissue.
[0031] Exemplary methods for decellularizing tissue are disclosed
in U.S. Pat. No. 6,933,326 and U.S. Patent Application
2010/0272782, which are hereby incorporated by reference in their
entirety.
[0032] After electroporation and optional tissue processing, the
resulting tissue product can be further processed to provide a
desired three dimensional shape (e.g., a sheet of tissue product).
In some embodiments, a tissue product can be further processed to
provide an anatomical shape useful for implanting into a targeted
host tissue. For example, a spherical or cylindrical shape can be
provided when the tissue product will be implanted following
removal of a similarly shaped volume of native tissue.
EXAMPLES
[0033] The following examples serve to illustrate, and in no way
limit, the present disclosure.
Example 1: Effect of Pulse Voltage on Electroporation
[0034] To determine the impact of pulse voltage on bacteria present
on tissue samples, 1 cm by 1 cm pieces of porcine dermis (1 mm
thickness) were electroporated in 2 ml of 0.2% peracetic acid (PAA)
at three different pulse voltages: 0V, 40V and 80V. The 0V samples
were used as a control condition. Electroporation plates (1 cm by 2
cm conducting metal plates) were set 2 mm apart and a 1 mm thick
piece of tissue was placed between them. Electrical pulses were
delivered for 10 ms. All conditions were tested in triplicates.
[0035] Following electroporation each piece of tissue was removed
and rinsed in a PAA neutralizing wash for 5 minutes and then placed
in 10 ml of phosphate buffered saline (PBS). The purpose of the
neutralizing wash was to prevent further bacterial kill from
residual PAA following electroporation that could confound the
results.
[0036] Stomaching was used to extract the bacteria from each piece
of tissue and bacteria counts were performed following incubation
of culture plates for 2 days at 37.degree. C. Stomaching is a
mechanical extraction method used to remove microorganisms from
samples of foods, fabrics, swabs, or other soft materials such as
human or animal tissue. The sample and diluents (water/PBS or any
other buffer that does not impact microorganism viability) are
placed in a sterile bag which is vigorously agitated on its outer
surfaces by paddles inside a stomaching machine. The resulting
compression and shearing forces elute deep-seated bacteria. Once
the agitation process is completed, samples of the eluent are taken
for microbial enumeration and subsequent identification.
[0037] The tissue samples exposed to 40V and 80V pulse voltages had
0 bacteria. The bacterial colonies from the 0V condition were too
numerous to count. Porcine dermis was also stained using
hematoxylin and eosin (H&E). See FIG. 1. Histological
examination of the stained samples demonstrated potential changes
in the collagen structure following exposure to 80 volts, whereas
there was no detectable change after exposure to 40 volts.
Example 2: Effect of Pulse Length on Electroporation
[0038] To determine the effect of pulse length on bioburden, 1 cm
by 1 cm pieces of porcine dermis (1 mm thickness) were
electroporated in 2 ml of 0.2% PAA at one of four pulse lengths: 0
ms, 5 ms, 10 ms, and 15 ms. 0 ms pulses were used as a control
condition. All conditions were tested in triplicates.
[0039] Electroporation plates (1 cm by 2 cm conducting metal
plates) were set 2 mm apart and a 1 mm thick piece of tissue was
placed between them. Pulse voltage was set at 80V. Following
electroporation each piece of tissue was removed and rinsed in a
PAA neutralizing wash for 5 minutes and then placed in 10 ml of
PBS.
[0040] Stomaching was used to extract the bacteria from each piece
of tissue and bacteria counts were performed following incubation
of culture plates for 2 days at 37.degree. C. As shown in Table 1
below, increasing the pulse length resulted in a decrease of
detectable bioburden, with reductions in observable bioburden
ranging between 2 and 4 log. Minimal bacterial colony forming units
(CFU) were detected on tissue samples following 15 ms pulse.
TABLE-US-00001 TABLE 1 Pulse Length Viable Cell Count (ms) # of
Pulses (CFU) 15 1 <10 10 1 140 5 1 300 1 100 430 0 -- 26000
[0041] The preceding examples are intended to illustrate and in no
way limit the present disclosure. Other embodiments of the
disclosed devices and methods will be apparent to those skilled in
the art from consideration of the specification and practice of the
devices and methods disclosed herein.
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