U.S. patent application number 14/676289 was filed with the patent office on 2015-07-23 for method for processing tissues.
This patent application is currently assigned to LIFECELL CORPORATION. The applicant listed for this patent is Benjamin T. Kibalo. Invention is credited to Benjamin T. Kibalo.
Application Number | 20150202338 14/676289 |
Document ID | / |
Family ID | 43037218 |
Filed Date | 2015-07-23 |
United States Patent
Application |
20150202338 |
Kind Code |
A1 |
Kibalo; Benjamin T. |
July 23, 2015 |
Method for Processing Tissues
Abstract
Methods for processing tissue are provided. In some embodiments,
the methods comprise methods for decellularizing tissue samples by
applying high hydrostatic pressure to the tissues samples. In some
embodiments, the methods comprise methods for thawing tissue
samples and/or reducing the bioburden in a sample by applying high
hydrostatic pressure to the tissue samples.
Inventors: |
Kibalo; Benjamin T.;
(Columbia, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kibalo; Benjamin T. |
Columbia |
MD |
US |
|
|
Assignee: |
LIFECELL CORPORATION
Branchburg
NJ
|
Family ID: |
43037218 |
Appl. No.: |
14/676289 |
Filed: |
April 1, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12858065 |
Aug 17, 2010 |
9023273 |
|
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14676289 |
|
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61234681 |
Aug 18, 2009 |
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Current U.S.
Class: |
422/22 ; 422/1;
422/33 |
Current CPC
Class: |
A61L 2/0035 20130101;
A61K 35/36 20130101; A61L 2/0011 20130101; A61L 2/007 20130101;
A61L 2/0041 20130101; A61P 17/00 20180101; A61L 2/0088
20130101 |
International
Class: |
A61L 2/00 20060101
A61L002/00 |
Claims
1-58. (canceled)
59. A method for reducing the bioburden in a soft tissue sample,
said method comprising: providing a tissue comprising mammalian
skin; removing an epidermal layer from the skin; applying a
pressure to the tissue by placing the tissue in a liquid and
applying pressure to the liquid for a time sufficient to cause at
least a 5 log reduction in the bacterial concentration within the
tissue, wherein the pressure is applied at a rate to control the
temperature of the temperature of the tissue such that the
temperature of the tissue does not exceed 30.degree. C.
60. The method of claim 59, wherein the pressure applied to the
liquid is at least 500 MPa.
61. The method of claim 59, wherein the pressure applied to the
liquid is at least 300 MPa for at least 30 minutes.
62. The method of claim 59 wherein the pressure applied to the
liquid is at least 400 MPa for at least 10 minutes.
63. The method of claim 59, wherein the pressure applied to the
liquid is at least 400 MPa for at least 40 minutes.
64. The method of claim 59, wherein the liquid comprises an aqueous
salt solution.
65. The method of claim 64, wherein the liquid comprises phosphate
buffered saline.
66. The method of claim 59, further comprising performing at least
one additional sterilization process on the tissue.
67. The method of claim 66, wherein the sterilization process
comprises a gamma irradiation process.
68. The method of claim 66, wherein the sterilization process
comprises an e-beam irradiation process.
69. The method of claim 66, wherein the sterilization process
comprises a supercritical carbon dioxide sterilization process.
70. The method of claim 66, wherein the sterilization process
comprises a peracetic acid treatment process.
71. (canceled)
72. The method of claim 59, wherein the mammalian soft tissue
comprises porcine dermis.
73. (canceled)
74. The method of claim 59, wherein the temperature of the tissue
sample does not exceed 25.degree. C.
Description
[0001] This application claims priority under 35 U.S.C. .sctn.119
to U.S. Provisional Patent Application No. 61/234,681, which was
filed on Aug. 18, 2009.
[0002] Human and animal tissues can be used to produce a variety of
tissue products for patient use. The tissues are often processed to
remove certain cellular and/or non-cellular components and/or to
destroy pathogens present in the tissues. In addition, during
processing or storage, tissues may be frozen and thawed.
SUMMARY
[0003] According to certain embodiments, a method for
decellularizing a tissue sample is provided, which comprises
providing a tissue sample comprising a mammalian soft tissue in a
liquid; and applying a pressure to the liquid of at least 200 MPa
for a time sufficient to destroy substantially all of the native
tissue cells within the soft tissue, wherein destroying
substantially all of the cells includes disrupting the cell
membrane of the cells such that washing the tissue sample in a
saline solution allows removal of at least 95% of the native tissue
cells.
[0004] According to certain embodiments, a method for thawing a
tissue sample is provided, which comprises providing a tissue
sample comprising a mammalian tissue that is at least partially
frozen in a liquid; and applying a pressure to the liquid
sufficient to thaw the frozen tissue sample.
[0005] According to certain embodiments, a method for
decellularizing a tissue sample is provided, which comprises
providing a tissue sample comprising a mammalian tissue in a
container containing liquid; and applying a pressure to the liquid
for a time sufficient to destroy substantially all of the cells
within the soft tissue, wherein destroying substantially all of the
cells includes disrupting the cell membrane of the cells such that
washing the tissue sample in a saline solution allows removal of at
least 95% of the native tissue cells. and wherein the pressure is
applied at a rate such that the temperature of the tissue sample
does not exceed 30.degree. C.
[0006] According to certain embodiments, a method for reducing the
bioburden in a tissue sample is provided, which comprises providing
a tissue sample comprising a mammalian soft tissue in a container
containing liquid; and applying a pressure to the liquid for a time
sufficient to cause at least a 5 log reduction in the bacterial
concentration within the soft tissue, wherein during application of
the pressure, the temperature of the tissue sample does not exceed
30.degree. C.
DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 is a phase diagram for water.
[0008] FIG. 2A is bioburden test result data for whole porcine skin
samples, as described in Experiment 1.
[0009] FIG. 2B is bioburden test result data for porcine dermis, as
described in Experiment 1.
[0010] FIG. 3 is bioburden test result data for porcine dermis, as
described in Experiment 2.
[0011] FIG. 4 is the temperature vs. pressure profile curves for
the samples of Experiments 1 and 2.
DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS
[0012] Reference will now be made in detail to the 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.
[0013] 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.
[0014] As used herein, "high hydrostatic pressure" is understood to
refer to pressure applied to an object contained in a liquid, the
liquid being pressurized to exert force on the object. In certain
embodiments, high hydrostatic pressure can include pressures
applied to the liquid that are greater than 200 MPa.
[0015] As used herein, "bioburden" means the quantity of
microorganisms in a tissue sample, including, but not limited to,
bacteria, viruses, fungi, parasites, chlamydiae, rickettsiae,
mycoplasma, and protozoa.
[0016] As used herein, "tissue products" or "tissue-derived
products" means any product produced from a tissue that has been
altered in any way (e.g., but not limited to, by removing cells
from the tissue, removing certain chemicals from the tissue, or
sterilizing the tissue). As used herein, "tissue samples" include
both intact, unprocessed tissues and tissues that have been
processed to produce "tissue products" or "tissue-derived
products."
[0017] 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.
[0018] Various human and animal tissues can be used to produce
products for treating patients. For example, various tissue
products for regeneration, repair, augmentation, 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)
have been produced. Such products can include, for example, tissue
matrices and/or tissue-derived proteins or protein-containing
materials (e.g., glycosaminoglycans) that can be used alone or in
combination with other materials and/or chemicals.
[0019] In certain embodiments, these products can be completely or
partially decellularized to yield tissue matrices or extracellular
tissue materials to be used for patients. For example, various
tissues, such as skin, intestine, bone, cartilage, nerve tissue
(e.g., nerve fibers or dura), tendons, ligaments, or other tissues
can be completely or partially decellularized to produce tissue
products useful for patients. In some cases, these decellularized
products can be used without addition of exogenous cellular
materials (e.g., stem cells). In certain cases, these
decellularized products can be seeded with cells from autologous or
other sources to facilitate treatment.
[0020] Since tissue products are often implanted on or within a
patient's body, in certain embodiments, it is desirable to
sterilize such materials, or at least reduce the amount of bacteria
or other pathogens that might be in the products, to a level
acceptable for the selected use. In certain embodiments, various
tissues, tissue-derived products, and other implantable medical
devices are typically sterilized using processes such as
irradiation (e.g., gamma, E-beam or X-ray), treatment with
chemicals, or heat.
[0021] For use in various medical or surgical applications, tissues
or tissue products should possess desired biologic properties,
depending on the intended use. For example, tissue products used
for tissue regeneration should generally be capable of supporting
or inducing cellular ingrowth and/or regeneration. However, certain
tissue processing techniques can damage some tissues and/or remove
portions of the tissue that may be desirable for certain biologic
functions. For example, in certain embodiments, tissue
decellularization processes can include the use of various enzymes,
detergents, and/or chemicals that may damage or remove various cell
signal molecules or extracellular matrix proteins desired for
regeneration or growth of certain tissues. In addition, in certain
embodiments, sterilization techniques, such as gamma irradiation,
can alter tissue products by causing breakdown and/or chemical
alteration of such products.
[0022] The present disclosure provides methods of processing tissue
samples that maintain certain desired biologic properties of tissue
products produced using such methods. In some embodiments, the
methods comprise a method for decellularizing a tissue sample. In
certain embodiments, the methods comprise a method for thawing a
tissue sample. In some embodiments, the methods comprise a method
for reducing the bioburden of a tissue sample.
[0023] In some embodiments, the methods for processing tissue
samples can include application of a high hydrostatic pressure to a
tissue. In certain embodiments, high hydrostatic pressure can be
applied to a tissue sample by placing a tissue sample in a liquid
or providing a tissue in a liquid. In certain embodiments, pressure
can be applied to the liquid, thereby controlling the pressure
applied to the tissue sample. In various embodiments, the pressure
applied to the tissue sample, the time that the pressure is
applied, and/or the rate of pressure increase and/or decrease can
be controlled to decellularize the tissue sample, reduce the
bioburden in the tissue sample, and/or thaw the tissue sample.
[0024] In various embodiments, the pressure applied to the tissue
sample can be selected based on a variety of factors. In some
embodiments, the pressure is selected based on the type of tissue
sample to be processed. In some embodiments, the pressure is
selected to allow decellularization of the tissue sample while
maintaining certain desired biologic properties of the tissue
sample. In some embodiments, the pressure is selected to allow the
tissue to be thawed without raising the tissue sample above a
selected temperature and/or to allow thawing within a selected
time.
[0025] In various embodiments, the methods of the present
disclosure can be used to process a variety of different tissue
sample types. Exemplary mammalian tissues samples include, but are
not limited to, bone, skin, intestine, urinary bladder, tendon,
ligament, muscle, fascia, neurologic tissue, liver, heart, lung,
kidney, cartilage, and/or other mammalian tissue. In certain
embodiments, the tissue sample can include a mammalian soft tissue
sample. For example, in certain embodiments, the tissue sample can
include mammalian dermis. In certain embodiments, the dermis can be
separated from surrounding epidermis and/or other tissues, such as
subcutaneous fat. In certain embodiments, the tissue sample can
include small intestine submucosa. In certain embodiments, the
tissue samples can include human or non-human sources. Exemplary,
suitable non-human tissue sources include, but are not limited to,
pigs, sheep, goats, rabbits, monkeys, and/or other non-human
mammals.
[0026] Various types of high hydrostatic pressure application
systems can be used to process tissue samples according to certain
embodiments. In certain embodiments, a high hydrostatic pressure
application system will include a rigid vessel or container formed
of steel or other hard material. In certain embodiments, a tissue
sample to be treated is placed in the vessel along with a fluid
(e.g., water). In certain embodiments, the tissue sample may be
packaged in a flexible container that also contains fluid, and the
flexible package may be placed in a vessel containing fluid. In
certain embodiments, after the vessel is loaded with the tissue
sample and fluid, pressure is applied to the fluid in the vessel.
In certain embodiments, the pressure can be applied in a number of
ways. For example, in certain embodiments, the pressure can be
applied using a pneumatic piston to compress the fluid in the
vessel, or a pump can force additional fluid into the vessel until
the pressure in the vessel reaches a desired level.
[0027] In certain embodiments, a tissue sample is packaged in a
flexible container containing a liquid, and pressure is applied to
the container. In some embodiments, a tissue sample is placed in a
rigid pressurization container containing a liquid and pressure is
applied to the liquid in the rigid container. In some embodiments,
the tissue sample is packaged in a flexible container containing a
liquid, and the flexible container is placed in a rigid
pressurization container containing a liquid, and pressure is
applied to the liquid in the rigid container. In various
embodiments, the pressure can be applied to the fluid by
compressing the fluid using, for example, a piston, or by pumping
addition fluid into a container with a fixed volume. In certain
embodiments, when the tissue sample is packaged in a flexible
container, generally, the flexible container is sealed so that only
the fluid inside the flexible container contacts the tissue
sample.
[0028] A variety of liquids can be used to contact the liquid
during application of high hydrostatic pressure. For example,
various aqueous solutions can be used. In certain embodiments, the
liquid can include an aqueous salt. In certain embodiments, the
liquid can include a saline solution, such as a phosphate buffered
saline.
[0029] In some embodiments, the tissue sample can be processed to
destroy some or substantially all of the native tissue cells of the
tissue sample. In certain embodiments, determination that
destruction of the native tissue cells has been accomplished can be
performed by washing a tissue sample that has been treated with
high hydrostatic pressure with a liquid that does not damage the
cells significantly (e.g., PBS) and analyzing the samples to
determine how much, if any, of the native tissue cells remain. For
example, in some embodiments, after high hydrostatic pressure
treatment to destroy cells, simple washing with a saline solution
can remove cell remnants, and the washed sample can be evaluated to
determine if the cells have been removed, thereby indicating
destruction of the cells. Certain suitable methods for evaluating
the samples to determine if cells have been destroyed and removed
are well known, and include for example, but are not limited to,
light microscopy of frozen or fixed tissue cells. In certain
embodiments, the presence of cells or cell remnants can be
evaluated using reagents that indicate that DNA is present, for
example, PICOGREEN.RTM. DNA quantification kits can be used.
[0030] As used herein, destruction of substantially all of the
cells will be understood to mean that at least 95% to 100%,
including the endpoints and all percentages between those end
points, of the native tissue cells of a tissue sample that has been
treated with high hydrostatic pressure and washed in a saline
solution are not present when evaluated using conventional
histology (e.g., light microscopy).
[0031] In some embodiments, tissue samples may be treated with high
hydrostatic pressure to remove some or substantially all of the
cells in the tissue sample, and the tissue sample may be treated
further with other processes to remove remaining cells. For
example, as noted above, in various embodiments, various enzymes,
detergents, and/or other chemicals are used to remove cells from
tissues, but such treatments may alter tissue extracellular
matrices. Therefore, to reduce the amount of treatment with
enzymes, detergents, and/or other chemicals, tissue samples may
first be treated with a high hydrostatic pressure treatment,
thereby removing some or substantially all of the cells, and the
tissue sample may be treated further with at least one additional
decellularization process to remove additional cells, if any are
present in the tissue sample. Suitable reagents and methods for
performing decellularization include, but are not limited to, those
described in, for example, U.S. Pat. No. 5,336,616, to Livesey et
al.
[0032] In some embodiments, the tissue sample may be processed to
produce an acellular tissue matrix. In some embodiments, the
acellular tissue matrix can include an extracellular matrix. For
example, in various embodiments, the tissue matrix can include a
collagen matrix derived from a variety of different mammalian soft
tissues. In certain embodiments, the tissue matrix can include one
or more additional extracellular matrix proteins and/or molecules,
including, but not limited to, various GAGs, cell-signaling
molecules, or other chemicals desired for effecting various
biologic functions, such as cell binding, adhesion, growth,
differentiation, and/or remodelling.
[0033] In some embodiments, a method for decellularizing a tissue
sample comprises providing a tissue sample comprising a mammalian
soft tissue in a liquid and applying a pressure to the liquid for a
time sufficient to destroy substantially all of the native tissue
cells within the soft tissue. In some embodiments, the pressure is
applied at a minimum pressure to destroy substantially all of the
native tissue cells within the soft tissue. In various embodiments,
the pressure is at least 200 MPa, at least 300 MPa, at least 400
MPa, or at least 500 MPa. In various embodiments the pressure is
between 300 MPa and 500 MPa. In various embodiments, the pressure
is applied for a time sufficient to destroy substantially all of
the native tissue cells within the soft tissue. In various
embodiments, the pressure is applied for at least 30 minutes to at
least 60 minutes. In certain embodiments, the pressure applied to
the liquid is at least 400 MPa for at least 10 minutes. In certain
embodiments, the pressure applied to the liquid is at least 400 MPa
for at least 30 minutes. In certain embodiments, the pressure
applied to the liquid is at least 500 MPa for at least 30 minutes.
In various embodiments, the methods of decellularization are
performed without causing excessive heating of the tissue sample,
as described below.
[0034] In various embodiments, the methods of the present
disclosure allow application of high hydrostatic pressure to a
tissue sample without causing significant heating of the tissue
sample. In various embodiments, heating of certain tissue samples
may damage various tissue extracellular matrix proteins, thereby
diminishing desired biologic functions of the tissue samples when
used for tissue repair, replacement, or regeneration. Therefore,
certain embodiments herein can allow tissue decellularization,
tissue thawing, and/or reduction in tissue bioburden without
heating the tissue samples to a temperature or for a time that may
damage tissue extracellular matrix proteins. In certain
embodiments, high hydrostactic pressure is applied at a rate and to
a maximum pressure such that the tissue sample does not reach a
temperature greater than 30.degree. C. In certain embodiments, the
temperature does not exceed 25.degree. C.
[0035] In certain embodiments, application of pressure in a
hydrostatic pressure vessel causes adiabatic compression of the
materials within the vessel (i.e., the liquid), which causes the
temperature of the compressed materials to increase. However,
certain pressurization vessels allow some heat transfer through the
walls of the vessel, and therefore, such systems are not truly
adiabatic. Therefore, in various embodiments, the amount of
pressure increase is related to the rate of compression (i.e.,
pressure increase) and heat transfer to or from the vessel walls.
In addition, in various embodiments, phase changes of the water
within the vessel can also affect the temperature within the
vessel. Therefore, in some embodiments, the temperature of the
sample being treated with high hydrostatic pressure can be
controlled by controlling the rate of pressure increase in the
treatment vessel.
[0036] In certain embodiments, the tissue sample, liquid contained
in a pressurization vessel, and/or pressurization equipment can be
cooled before and/or during application of high hydrostatic
pressure. In some embodiments, ice may be placed in the liquid
contained in the pressurization vessel, and/or the walls of the
pressurization vessel can be cooled.
[0037] In various embodiments, to prevent tissue damage, breakdown,
and/or microbial growth, it is often desirable to freeze tissue
samples during processing, transport, and/or storage. In various
embodiments, during subsequent processing or use, the tissue sample
is thawed. But, in certain instances, thawing by heating the tissue
sample can damage tissue extracellular matrix components and/or
promote microbial growth. Further, in certain embodiments, thawing
tissue samples under relatively cool conditions (e.g. under
refrigeration or just above the freezing point of water in the
sample) can be time consuming, especially for larger tissue
samples.
[0038] In certain embodiments, a method for thawing a tissue sample
comprises providing a tissue sample comprising a mammalian tissue
that is at least partially frozen in a liquid and applying a
pressure to the liquid sufficient to thaw the frozen tissue sample.
In some embodiments, thawing occurs within a limited time and/or
with only limited elevation of the tissue sample temperature.
[0039] FIG. 1 provides a phase diagram for solid and liquid phases
of water. As shown, the melting point of various ice phases
decreases at higher pressures. Therefore, in certain embodiments,
application of elevated hydrostatic pressures to samples containing
ice can cause conversion of the solid state water to liquid without
significant heating of the tissue sample.
[0040] In various embodiments, a tissue sample can contain water
that is partially or entirely solid state (i.e., ice). In various
embodiments, thawing the tissue sample comprises causing a portion
or substantially all of the solid-state water in the sample to be
converted to liquid. In some embodiments, thawing the frozen tissue
sample comprises causing greater than 50% of the solid state water
in the tissue sample to undergo a phase transformation to a liquid
state. In some embodiments, thawing the frozen tissue comprises
causing substantially all of the solid state water in the tissue
sample to undergo a phase transformation to a liquid state. In
various embodiments, between 50% to 100% of the ice in the sample
undergoes a phase transformation to a liquid state.
[0041] In various embodiments, the amount of solid state ice in the
sample before and after application of a high hydrostatic pressure
treatment can be determined in several ways. For example, in
various embodiments, the presence of ice in a sample can be
determined using small samples for differential scanning
calorimetry (DSC). For larger samples, in various embodiments, ice
can be identified by placing a sample in a thermally insulated
liquid at a known temperature and supplying heat to the system. In
certain embodiments, samples can be compressed (pressurized) in an
adiabatic system, and the sample temperature or the temperature of
a fluid media surrounding the sample can be measured during
compression. Samples that have no ice will be expected to increase
their temperature in an adiabatic system at a constant rate related
to the pressurization rate. In certain embodiments, for samples
containing ice, the temperature of the samples will plateau at a
temperature near the melting point of ice. In some embodiments, if
the temperature of the fluid surrounding the sample is measured,
the fluid temperature will increase more slowly for samples
containing ice than for samples that do not contain ice. The
plateau in sample temperature and/or decrease in the rate of
temperature rise will be dependent on the amount of ice
present.
[0042] In some embodiments, the temperature of a tissue sample
undergoing high hydrostatic pressure treatment is maintained below
an upper limit. In some embodiments, the upper limit is based on
the initial temperature of the sample. For example, in some
embodiments, the thawing of the tissue is performed without
increasing the temperature of the tissue sample more than
10.degree. C. In some embodiments, the thawing is performed without
increasing the temperature above 30.degree. C. In some embodiments,
the thawing is performed without increasing the temperature above
25.degree. C. In certain embodiments, the thawing is performed
without increasing the temperature of the tissue sample above
between about 25.degree. C. and 30.degree. C.
[0043] In some embodiments, the thawing is performed without
increasing the temperature above an upper limit, and within a
certain time. For example, in some embodiments, thawing occurs
within 30 minutes. In certain embodiments, thawing occurs within 60
minutes. In various embodiments, thawing occurs in between about 30
minutes and about 60 minutes.
[0044] In various embodiments, the high-hydrostatic pressure
treatment is performed to obtain a certain level of reduction in
sample bioburden. For example, in various embodiments, high
hydrostatic pressure may be applied at a pressure and time
sufficient to cause a pressure and time sufficient to cause at
least a 5 log reduction, a 6 log reduction, a 7 log reduction, or
an 8 log reduction in the bacterial load of a sample. In some
embodiments, high hydrostatic pressure may be applied at a pressure
and time sufficient to reduce the bioburden to a particular
level.
[0045] In various embodiments, the bioburden of a tissue sample can
be measured by extracting microbes from a tissue sample and
culturing or quantifying a particular type of organism. A suitable
method for extracting microbes from a sample includes washing a
sample with a sterile liquid and culturing a portion or all of the
liquid used to wash the sample in order to quantify the amount of
any particular microbe or microbes in a sample. In various
embodiment, the washing fluid can be selected based on the type of
microbe to be quantified and/or to prevent damage to the tissue. In
some embodiments, the bioburden reduction is performed without
increasing the temperature above 30.degree. C. In some embodiments,
the bioburden reduction is performed without increasing the
temperature above 25.degree. C. In certain embodiments, the
bioburden reduction is performed without increasing the temperature
of the tissue sample above between about 25.degree. C. and
30.degree. C.
[0046] In some embodiments, a sterilization process can be
performed before or after applying high hydrostatic pressure to a
tissue sample. For example, in some embodiments, application of
high hydrostatic pressure will at least partially reduce the
bioburden of a tissue sample, and a tissue sterilization process
can be performed to further reduce the bioburden in the sample. In
some embodiments, the sterilization process can be a terminal
sterilization process that is performed just before or after
packaging a tissue sample. As used herein, a "sterilization
process" can include any process that reduces the bioburden in a
sample, but need not render the sample absolutely sterile.
[0047] Certain exemplary processes include, but are not limited to,
a gamma irradiation process, an e-beam irradiation process, a
supercritical carbon dioxide sterilization process, and a peracetic
acid treatment process. In various embodiments, such processes may
damage some tissue components, and, therefore, to produce tissues
having desired biologic properties, it may be desirable to limit
the time or intensity (e.g., radiation dose or pH) of the
sterilization process. In certain embodiments, application of high
hydrostatic pressure to a sample to partially reduce the bioburden
can therefore reduce the dose of subsequent sterilization processes
used to achieve a desired level of sterility. Suitable
sterilization processes include, but are not limited to, those
described in, for example, U.S. Patent Publication No.
2006/0073592A1, to Sun et al.; U.S. Pat. No. 5,460,962, to Kemp;
U.S. Patent Publication No. 2008/0171092A1, to Cook et al.
Example 1
Reduction in Tissue Bioburden
[0048] Porcine skin was used. The tissue was provided either as
whole skin with hair intact or as dermal layers that were isolated
from the epidermis and subdermal fat layers. The dermal layer was
isolated by cutting the subdermal fat and a thin layer (1-2 mm) of
the lower dermis from the dermis, and by cutting a thin layer
(0.25-1 mm) of the epidermis and upper dermis from the dermis. Hair
was mechanically removed before isolating the dermis. Both sample
types were previously frozen, and to increase the bioburden levels
in isolated dermis tissue, all of the tissue was stored together
(whole skin and isolated dermis) for several days after thawing
under refrigerated conditions. Each piece was individually packaged
using a DENI.TM. Magic Vac food saver device. Each piece was sealed
within three vacuum sealed pouches to prevent exposure of porcine
tissue to the pressurization vessel. The samples were packaged with
a minimum amount of fluid in the sealed pouch such that the package
closely conformed to the sample.
[0049] A 13 liter pressurization system made by ElmHurst Research,
Inc (Albany, N.Y.) was used for the experiments. The system had a
fixed volume and applied pressure by pumping fluid into the vessel.
The temperature was measured using a thermocouple that protruded
from the cap of the vessel into the pressurization chamber to
measure bulk fluid pressure.
[0050] Small pieces of both whole skin and isolated dermis were
tested first (runs 1 and 2), and large non-dehaired pieces were
exposed to the same conditions (runs 3 and 4). Table 1 summarizes
these conditions. The pressure vessel had no temperature control
system, so the maximum temperature was dependant mainly on the
initial temperature and maximum pressure. The rate of pressure
increase was at a single speed of 350 PSI/sec. After runs 1 and 2,
the small pieces of tissue that were exposed to high hydrostatic
pressure were examined by eye and touch for obvious signs of
degradation. No obvious signs of degradation were seen, so the
large pieces of tissue were processed in runs 3 and run 4.
TABLE-US-00001 TABLE 1 Run Conditions Pressure Time Start Temp Max
Temp Run # (PSI) (min) (C.) (C.) 1 and 3 60,000 5 27.0 and 25.3
38.7 and 38.8 2 and 4 75,000 10 26.8 and 25.7 39.9 and 40.5
[0051] After exposure, the tissue samples were stored in
refrigerated conditions (1-10.degree. C.) for less than 1 week.
Samples of the whole skin and isolated dermis and were submitted
for bioburden testing. The samples were agitated in a PBS solution
to extract the bacteria from the samples, and the PBS solution was
plated on an agar plate and incubated. Bacterial colonies were
counted. Control samples of untreated tissue (both isolated dermis
and whole skin) were also subjected to the same bioburden testing
before exposure to high hydrostatic pressure.
[0052] After refrigeration, some remaining isolated dermal tissue
samples were also subjected to DSC. DSC was performed using 12-23
mg of sample on a TA Differential Scanning calorimeter (TA
Instruments, New Castle, Del.).
[0053] Bioburden test results are shown in FIGS. 2A and 2B. The
data is shown as colony forming units (CFU) per tissue sample, in
LOG.sub.10 scale. The results show at least a 1 to 3 LOG.sub.10
reduction for non-dehaired tissue and a 4 to 5 LOG.sub.10 reduction
for isolated dermal tissue. The results show that higher pressure
and longer pressure hold times reduced the overall bioburden more
than a lower pressures and shorter times.
[0054] There was a reduction in bacterial deactivation with whole
skin tissue, compared to isolated dermal tissue at each pressure
tested (60 kPSI and 75 kPSI). Therefore, for dermal grafts, cutting
the tissue sample to remove non-dermal components before high
hydrostatic pressure treatment can provide improved reduction in
bioburden.
[0055] In this work, the inactivation data showed excellent results
for the short periods tested. However, the DSC properties for
processed samples indicated some thermal tissue damage. For
example, samples subjected to either 65 kPSI for 5 minutes or 70
kPSI for 10 minutes had thermal onset values on DSC that indicated
a high level of denatured collagen. Therefore, experiments were
performed to assess the effect of high hydrostatic pressure for
decellularization, thawing, or bioburden reduction when the
processing temperature was controlled.
Example 2
Control of Process Temperature
[0056] Porcine skin tissue was obtained and dermal tissue was
isolated as described above in Example 1. The tissue was stored at
-80.degree. C. prior to use. The samples were then thawed in a
convective incubator held at 7.degree. C. for up to 36 hours. All
of the samples were cut into approximately 7 cm.times.7 cm square
pieces. Packages were made using the DENI.TM. Magic Vac packaging
to closely fit the dimensions of the tissue.
[0057] Tissue samples were placed individually within pre-made
packages. PBS was then added to almost fill the package (at least
50 mL on average). The PBS was degassed prior to placement in the
packages using a vacuum pull down with agitation for several hours
prior to use. Degassing was considered complete when air bubbles
were no longer forming around the stir-bar. As much air as possible
was removed from the package by squeezing the package, and the open
end of the package was sealed with a heat sealer.
[0058] Ice was used to cool the high hydrostatic pressure vessel.
Approximately 50 lbs of ice was required for a total of three runs.
The ice was added to the vessel prior to pressurization, both above
and below the tissue. The same pressurization system described in
Example 1 was used for the experiments described in Example 2.
[0059] Table 2 summarizes the conditions for each run of Example 2.
Ice was not used equally because there was a concern that there
would be vessel seal leakage at lower temperatures. More ice was
used as confidence in the vessel's integrity at low temperature
increased. Therefore, the starting temperature of each run was
lower as because more ice used.
TABLE-US-00002 TABLE 2 Run Conditions for Experiment 2 Pressure
Time Start Temp Max Temp Run # (PSI) (min) (C.) (C.) 1 50,000 10
11.1 23.9 2 50,000 30 8.6 21.3 3 75,000 10 6.7 25.3
[0060] After high hydrostatic pressure application the tissue was
stored under refrigerated conditions (1-10.degree. C.) for no more
than 24 hours. To control for effects of the PBS solution on tissue
during the time between packaging, treatment, and bioburden
testing, untreated control samples were held in PBS and were tested
with the treated samples. The samples were cut after high
hydrostatic pressure exposure under sterile conditions, which may
have affected bioburden results. Bacterial contamination was
assessed as in Experiment 1.
[0061] Bioburden testing was performed as described in Example 1.
The bioburden test results are shown in FIG. 3. Only isolated
dermal tissue was tested in this study, and the y-axis represents
CFUs on a logarithmic scale. The reduction in bioburden improved
with longer hold times and higher pressures. For example, Runs 1
and 3 were both performed with 10 minute hold times, but Run 3,
which was performed at higher pressure, resulted in increased
bioburden reduction. In addition, Runs 1 and 2 were both performed
at 50 kPSI, but Run 2, which was performed for a longer time,
resulted in increased bioburden reduction compared to Run 1.
[0062] FIG. 4 is a graphical record of the pressure versus
temperature during the three runs in this experiment and the four
runs of Example 1. In the current experiment, the sample
temperatures did not exceed approximately 25.degree. C. In Example
1, the sample temperature exceeded 35.degree. C., and even
40.degree. C. for the higher pressures. The flat region at the top
of each curve is a cooling plateau likely due to heat transfer to
or from the fluid to the vessel walls during the high pressure hold
step. The steel walls of the high pressure vessel started at
ambient, and the mass of the steel walls of the vessel provided a
massive heat sink or source, depending on the thermal gradient.
Example 2 has these cooling plateaus as well, but they are less
pronounced.
[0063] DSC testing was also performed on each sample using a TA
Differential Scanning calorimeter. The DSC results are shown in
Table 3. A control, untreated dermal sample is also shown. In
contrast to Example 1, the samples in Example 2 did not show low
thermal onset values indicative of collagen denaturation. Rather,
the thermal onset values for Runs 1 to 3 of Example 2, were
approximately 59-60.degree. C., which is similar to the control
sample. Therefore, application of high hydrostatic pressure while
controlling the sample temperature was effective at reducing the
sample bioburden without causing significant collagen
denaturation.
TABLE-US-00003 TABLE 4 DSC Results for Experiment 2 Sample Onset
Denaturation Temperature/Enthalphy Control 60.47/59.17 1
59.84/52.32 2 59.92/59.83 3 60.05/44.43
* * * * *