U.S. patent application number 16/905185 was filed with the patent office on 2020-10-29 for processed adipose tissue.
The applicant listed for this patent is LifeCell Corporation. Invention is credited to Xianghong Liu, Wenquan Sun.
Application Number | 20200338234 16/905185 |
Document ID | / |
Family ID | 1000004945958 |
Filed Date | 2020-10-29 |
United States Patent
Application |
20200338234 |
Kind Code |
A1 |
Sun; Wenquan ; et
al. |
October 29, 2020 |
PROCESSED ADIPOSE TISSUE
Abstract
The present disclosure provides tissue products produced from
adipose-containing tissues, as well as methods for producing such
tissue products. The tissue products can comprise decellularized
and partially de-fatted tissues. In addition, the present
disclosure provides systems and methods for using such
products.
Inventors: |
Sun; Wenquan; (Warrington,
PA) ; Liu; Xianghong; (Hillsborough, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LifeCell Corporation |
Madison |
NJ |
US |
|
|
Family ID: |
1000004945958 |
Appl. No.: |
16/905185 |
Filed: |
June 18, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15159220 |
May 19, 2016 |
10709810 |
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16905185 |
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14036369 |
Sep 25, 2013 |
9370536 |
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15159220 |
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61705789 |
Sep 26, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61F 2/12 20130101; A61L
27/3604 20130101; A61L 2430/40 20130101; A61L 27/3695 20130101;
A61K 35/35 20130101 |
International
Class: |
A61L 27/36 20060101
A61L027/36; A61K 35/35 20060101 A61K035/35; A61F 2/12 20060101
A61F002/12 |
Claims
1. A method of producing a tissue product, comprising: providing a
sheet of tissue comprising adipose-containing transitional dermis;
treating the sheet of tissue to remove substantially all cellular
material from the tissue; and processing the sheet of tissue to
partially remove lipid components such that the tissue product
retains between about 30% and 50% lipid content as a percentage of
the overall tissue product by mass following processing.
2. The method of claim 1, wherein substantially all cellular
material is removed using one or more detergents.
3. The method of claim 2, wherein the one or more detergents
comprise at least a polyethylene glycol,
Tris[2-(dimethylamino)ethyl]amine, sodium dodecyl sulfate, sodium
deoxycholate, and polyoxyethylene (20) sorbitan mono-oleate.
4. The method of claim 1, wherein processing the sheet of tissue to
partially remove lipid components comprises exposing the sheet of
tissue to an elevated temperature of about 40-50.degree. C., to
ultrasonic energy, or a combination of the elevated temperature and
the ultrasonic energy.
5. The method of claim 4, wherein the adipose-containing tissue is
exposed to ultrasonic energy of about 20 to 2000 watts per square
meter at a frequency between about 20 to 400 kilohertz.
6. The method of claim 1, wherein the tissue product comprises at
least about 3% extracellular matrix components as a percentage of
the overall tissue product by mass following processing.
7. The method of claim 1, further comprising exposing the tissue
product to E-beam radiation.
8. The method of claim 1, further comprising storing the tissue
product in an aqueous solution or drying the tissue product for
storage.
9. The method of claim 1, wherein the drying includes
freeze-drying.
10. A method of treatment comprising: selecting a tissue site in a
patient; and implanting a tissue product comprising a sheet of
decellularized adipose-containing transitional dermal matrix,
wherein a portion of lipid components have been removed from the
sheet of decellularized adipose-containing transitional dermal
matrix such that the tissue product retains between about 30% and
50% lipid content as a percentage of the overall tissue product by
mass.
11. The method of claim 10, wherein the tissue product has
sufficient lipid content to provide a soft and malleable material
capable of molding or deforming to fill the tissue site while
having a reduced lipid content sufficient to avoid significant
inflammation following implantation.
12. The method of claim 10, wherein the tissue product comprises
sufficient extracellular matrix components to provide structural
support for the lipid components of the tissue product.
13. The method of claim 12, wherein the tissue product has at least
about 3% extracellular matrix components as a percentage of the
overall tissue product by mass.
14. The method of claim 10, wherein the implanted tissue product
promotes native adipose deposition, native cell migration, native
cell proliferation, or revascularization in or near the implanted
tissue product, while avoiding significant inflammation in or near
the implanted tissue product.
15. The method of claim 10, wherein the tissue product does not
harden significantly following implantation.
16. The method of claim 10, wherein the tissue site comprises
adipose tissue or soft tissue.
17. The method of claim 10, wherein the tissue product is implanted
following surgical removal of bulk tissue from a patient.
18. The method of claim 17, wherein the tissue site comprises a
breast.
19. The method of claim 10, wherein the tissue product is implanted
to augment a tissue site.
20. The method of claim 10, wherein the tissue site is a breast, a
face, a buttock, an abdomen, a hip, or a thigh.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/159,220, which was filed on May 19, 2016,
which is a continuation of U.S. patent application Ser. No.
14/036,369, which was filed on Sep. 25, 2013, now U.S. Pat. No.
9,370,536, which claims the benefit under 35 U.S.C. .sctn..sctn.
119 and 120 of U.S. Provisional Patent Application No. 61/705,789,
filed on Sep. 26, 2012, all of which are incorporated herein by
reference.
[0002] The present disclosure relates to tissue products, and more
particularly, to products containing extracellular tissue matrices
made from adipose tissue.
[0003] Various tissue-derived products are used to regenerate,
repair, or otherwise treat diseased or damaged tissues and organs.
Such products can include tissue grafts and/or processed tissues
(e.g., acellular tissue matrices from skin, intestine, or other
tissues, with or without cell seeding). Such products generally
have properties determined by the tissue source (i.e., the tissue
type and animal from which it originated) and the processing
parameters used to produce the tissue products. Since tissue
products are often used for surgical applications and/or as tissue
replacements or for augmentation, the products should support
tissue growth and regeneration and avoid excess inflammation, as
desired for the selected implantation site. The present disclosure
provides adipose tissue products that can provide for improved
tissue growth, revascularization, and regeneration in various
applications, while improving surgical handling and reducing
inflammation.
[0004] According to certain embodiments, methods for producing
tissue products are provided. The methods include selecting an
adipose-containing tissue; treating the tissue to remove
substantially all cellular material from the tissue, and further
processing the tissue to reduce the adipose content of the tissue.
In addition, tissue products made by the disclosed processes are
provided. The products can comprise a decellularized adipose
extracellular tissue matrix and a reduced lipid content. The tissue
product can be provided in a sheet format that is suitable for
surgical use and/or for further manipulation to prepare a desired
implant shape, or can be provided in any other desired shape.
[0005] Furthermore, methods of treatment are provided. The methods
can comprise placing an adipose tissue product into a surgical site
to replace, repair, regenerate, augment, and/or enhance a native
tissue. The tissue product can be formed into a predetermined
three-dimensional shape and implanted into the host tissue at the
desired location.
DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1 is a plot showing differential scanning calorimetry
data indicating the percentage of collagen denaturation in tissue
samples, prepared according to certain embodiments of the present
disclosure, after incubation at different temperatures. Tissue
samples were scanned from 2.degree. C. to 120.degree. C. at
4.degree. C./min.
[0007] FIG. 2 shows Hematoxylin and eosin (H&E) staining of
sections taken from adipose tissue products with different lipid
content (63%, 45% and 72%, from left to right) three months after
implantation in African green monkeys, according to certain
embodiments of the present disclosure. Sections were prepared using
tissues from the center of grafts, and the images are at 200.times.
magnification.
[0008] FIG. 3 shows systemic antibody (IgG) titer in African green
monkey serum over time following implantation of one of three
adipose tissue products with different lipid content (63%, 45% and
72% on a dry mass basis, respectively), according to certain
embodiments of the present disclosure.
DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS
[0009] 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.
[0010] 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.
[0011] 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.
[0012] Various human and animal tissues can be used to produce
products for treating patients. 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. Such products can include, for example,
acellular tissue matrices, tissue allografts or xenografts, and/or
reconstituted tissues (i.e., 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.
[0013] Although such materials are very useful for treating certain
types of conditions, materials having different biological and
mechanical properties may be desirable for certain applications.
For example, ALLODERM.RTM. and STRATTICE.TM. may not be ideal for
regeneration, repair, replacement, and/or augmentation of certain
soft tissues or adipose-containing tissues following the removal of
bulk tissue volume (e.g., a volume of at least about 1, 2, 5, 10,
20, 50, 100, 200, 1000 ml or more of tissue). Accordingly, the
present disclosure provides tissue products that can be placed into
a surgical site to replace, repair, regenerate, augment, and/or
enhance a native adipose-containing tissue or other soft tissue.
The present disclosure also provides methods for producing such
tissue products.
[0014] The tissue products of the present disclosure can include
adipose-containing tissues that have been processed to removal at
least some of the cellular components. In some cases, all (or
substantially all) cellular material is removed, while retaining
some or substantially all of the extracellular matrix components
(e.g., collagen, elastin, or other fibers, as well as
proteoglycans, polysaccharides and growth factors). In addition,
the tissue products can be further processed to remove some of the
extracellular and/or intracellular lipids. As described in further
detail below, the tissue product can be provided in sheet form or
any other desired three dimensional shapes. In addition, to allow
for treatment of a selected tissue site, the material can be
further processed (e.g., by E-beam or gamma irradiation) to reduce
bioburden on the tissue product.
[0015] As noted, the tissue products of the present disclosure are
derived from adipose-containing tissues. The adipose-containing
tissues can be from human or animal sources, and from any tissue
that contains adipose (e.g., a tissue containing a substantial
number of adipocytes, such as a tissue in which the lipid content
accounts for at least about 20% of the overall tissue mass). For
example, human adipose-containing 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). In addition, while the adipose-containing
tissue may be derived from one or more donor animals of the same
species as the intended recipient animal, this is not necessarily
the case. Thus, for example, the tissue product may be prepared
from an animal tissue and implanted in a human patient. Species
that can serve as donors and/or recipients of acellular tissue
include, without limitation, humans, 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.
[0016] If animal sources are used, the tissues may be further
treated (e.g., using enzymatic processes) to remove antigenic
components such as 1,3-alpha-galactose moieties, which are present
in, e.g., pigs, but not in humans or primates and may result in an
immune response following implantation. In addition, the
adipose-containing tissue can be obtained from animals that have
been genetically modified to remove antigenic moieties. See Xu,
Hui. et al., "A Porcine-Derived Acellular Dermal Scaffold that
Supports Soft Tissue Regeneration: Removal of Terminal
Galactose-.alpha.-(1,3)-Galactose and Retention of Matrix
Structure," Tissue Engineering, Vol. 15, 1-13 (2009). For further
descriptions of appropriate animals and methods of producing
transgenic animals for xenotransplantation, see U.S. patent
application Ser. No. 10/896,594 and U.S. Pat. No. 6,166,288, which
are hereby incorporated by reference in their entirety.
[0017] In certain embodiments, the adipose-containing tissue is
provided from transitional dermal tissue layers between the dermis
and the subcutaneous fat. In some embodiments, the
adipose-containing tissue comprises approximately 20-90% lipid
content by mass prior to the processing described below. In certain
embodiments, the adipose-containing tissue comprises 20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% lipid content by
mass prior to processing (or any percentage in between). In certain
embodiments, the adipose-containing tissue also comprises 1-10%
extracellular matrix (ECM) components (e.g., collagen, elastin, or
other fibers, as well as proteoglycans, polysaccharides and growth
factors) by mass prior to processing. In certain embodiments, the
adipose-containing tissue comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10% ECM components by mass prior to processing (or any percentage
in between).
[0018] In certain embodiments, the chosen adipose-containing tissue
is a dermal tissue (e.g., tissue from transitional tissue layers
between the dermis and subcutaneous fat) because these tissues
provide a sufficiently high ECM content as well as a high lipid
content suitable for further processing.
[0019] Once an adipose-containing tissue has been provided, the
tissue can be processed to form a tissue product. In various
embodiments, the processing includes partial or complete
decellularization and partial lipid removal (i.e., a partial
reduction in the lipid content). In some embodiments, both
processes are performed simultaneously. In other embodiments, the
adipose-containing tissue is first decellularized and then lipids
are partially removed, or vice versa.
[0020] In various embodiments, the adipose-containing tissue is
washed to remove any residual cryoprotectants, red blood cells,
and/or any other contaminants. Solutions used for washing can be
any physiologically-compatible solution. Examples of suitable wash
solutions include distilled water, phosphate buffered saline (PBS),
or any other biocompatible saline solution. In some embodiments,
the tissue is then decellularized by the addition of one or more
detergents to the wash solution in order to remove cells and
cellular material. 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.
[0021] In various embodiments, the general steps involved in the
production of an acellular or partially decellularized
adipose-containing tissue include providing adipose-containing
tissue from a donor (e.g., a human or animal source) and removing
cellular material under conditions that preserve some or all of the
biological and/or structural components of the extracellular
matrix.
[0022] In certain embodiments, the adipose-containing tissue can be
chemically treated to stabilize the tissue so as to avoid
biochemical and/or structural degradation before, during, or after
cell removal. In various embodiments, the stabilizing solution
arrests and prevents osmotic, hypoxic, autolytic, and/or
proteolytic degradation; protects against microbial contamination;
and/or reduces mechanical damage that may occur during
decellularization of the tissue. The stabilizing solution can
contain an appropriate buffer, one or more antioxidants, one or
more antibiotics, one or more protease inhibitors, and/or one or
more smooth muscle relaxants.
[0023] In various embodiments, the adipose-containing tissue is
placed in a decellularization solution to remove viable and
non-viable cells (e.g., epithelial cells, endothelial cells, smooth
muscle cells, fibroblasts, etc.) and cellular components without
damaging the biological and/or structural integrity of the
extracellular matrix. For example, enzymes, detergents, and/or
other agents may be used in one or more steps to remove cellular
materials and/or other antigenic materials. The decellularization
solution may contain an appropriate buffer, salt, an antibiotic,
one or more detergents (e.g., TRITON X-100.TM.,
Tris[2-(dimethylamino)ethyl]amine, 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 about 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,
about 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 certain embodiments, the decellularization solution
comprises about 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 sodium deoxycholate and, optionally, about 1 mM, 2 mM,
3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13
mM, 14 mM, 15 mM, or 20 mM HEPES buffer
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) containing
about 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50
mM EDTA (or any concentrations in between). In some embodiments,
the tissue is incubated in the decellularization solution at about
20, 21, 22, 23, 24, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 degrees Celsius (or at
any temperature in between), and optionally, gentle shaking is
applied at about 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 about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 24, 36,
48, or 96 hours (or any time in between).
[0024] In various embodiments, the length of time of exposure to
the decellularization solution and/or the concentration of
detergent or other decellularizing agents can be adjusted in order
to partially or more fully decellularize the tissue. In some
embodiments, substantially all of the cellular material is removed
(e.g., at least about 80, 85, 90, 95, 98, 99, 99.5, or 99.9% of the
cellular material is removed). In certain embodiments, additional
detergents and combinations of detergents may be used to remove
cells from the adipose-containing tissue. For example, in certain
embodiments, a combination of sodium deoxycholate and TRITON
X-100.TM. are used. In various embodiments, the decellularization
process does not alter the structure and/or function of the
extracellular matrix in the adipose-containing tissue. For example,
the structure of the extracellular matrix in the decellularized
tissue can remain substantially unaltered when compared to
non-decellularized tissue. In some embodiments, further proteolytic
processing is employed to remove undesirable extracellular matrix
components. For example, alpha-galactosidase can be applied to
remove alpha-galactose moieties.
[0025] In certain embodiments, e.g., when xenogenic or allogenic
material is used, the decellularized tissue can optionally be
treated overnight at room temperature with a deoxyribonuclease
(DNase) solution. In some embodiments, the tissue sample is treated
with a DNase solution prepared in a DNase buffer (e.g., about 20 mM
HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 20 mM
CaCl.sub.2 and 20 mM MgCl.sub.2). Optionally, an antibiotic
solution (e.g., Gentamicin) may be added to the DNase solution. Any
suitable DNase buffer can be used, as long as the buffer provides
for suitable DNase activity.
[0026] In certain embodiments, after decellularization, viable
cells may optionally be seeded in the extracellular matrix of the
partially or completely decellularized adipose-containing tissue.
In some embodiments, viable cells may be added by standard in vitro
cell co-culturing techniques prior to transplantation, or by in
vivo repopulation following transplantation. In vivo repopulation
can be by the migration of native cells from surrounding tissue
into the ECM of a tissue product following implantation, or by
infusing or injecting viable cells obtained from the recipient or
from another donor into the tissue product in situ. Various cell
types can be used, including stem cells such as embryonic stem
cells and/or adult stem cells. Any other viable cells can also be
used. In some embodiments, the cells are mammalian cells. In
certain embodiments, the cells are histocompatible with the subject
in which they are implanted. Such cells can promote native cell
and/or tissue migration, proliferation, and/or revascularization.
In various embodiments, the cells can be directly applied to the
ECM of a tissue product just before or after implantation.
[0027] In various embodiments, the adipose-containing tissue in a
tissue product can be processed to partially remove lipid
components. For example, the adipose-containing tissue can be
partially de-fatted by exposing the tissue to an elevated
temperature, to ultrasonic energy, or to a combination of the two
in order to melt or otherwise remove a desired percentage of
lipids. For example, the tissue can be exposed to temperatures of
about 40-50.degree. C. (e.g., about 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, or 50.degree. C.) for up to about 24 hours (e.g., about 1,
2, 3, 4, 5, 10, 15, 20, or 24 hours, or any time period in between)
in order to remove a desired percentage or type of fat, especially
unsaturated fat species. In some embodiments, the temperature used
or the length of exposure can be increased in order to increase the
amount of lipids removed, or can be decreased in order to reduce
the amount of lipids removed. The adipose-containing tissue can
also be exposed to ultrasonic energy in order to remove lipids. For
example, the tissue can be exposed to ultrasonic energy of about 20
to 2000 watts per square meter (e.g., about 20, 40, 60, 80, 100,
200, 500, 1000, or 2000 watts per square meter, or any value in
between). The ultrasonic energy can be at a frequency of about 20
to 400 kilohertz (e.g., about 20, 40, 60, 80, 100, 150, 200, 250,
300, 350, or 400 kHz), and the exposure duration can be about 30
seconds to 8 hours (e.g., 30 seconds, 45 seconds, or 1, 5, 10, 30,
or 60 minutes, or 1, 2, 3, 4, 5, 6, 7, or 8 hours, or any time
period in between). The tissue can be exposed to ultrasonic energy
alone or in combination with high temperatures, in order to remove
a desired percentage of lipids. In some embodiments, the energy
level used or the length of exposure can be increased in order to
increase the amount of lipids removed, or can be decreased in order
to reduce the amount of lipid removed. In some embodiments, a
combination of high temperature and ultrasonic energy can be used.
In certain embodiments, one or more detergents, such as sodium
dodecyl sulfate or Tris[2-(dimethylamino)ethyl]amine, can be used
in combination with high temperature and/or ultrasonic energy to
assist in lipid removal.
[0028] In various embodiments, the decellularization and lipid
removal processes can occur simultaneously. Alternatively,
decellularization can be carried out first or lipid removal can be
done first.
[0029] In some embodiments, after decellularization and partial
lipid removal, the adipose-containing tissue is washed thoroughly.
Any physiologically compatible solutions can be used for washing.
Examples of suitable wash solutions include distilled water,
phosphate buffered saline (PBS), or any other biocompatible saline
solution. In some embodiments, the wash solution can contain a
disinfectant. In certain embodiments, the disinfectant is peracetic
acid (PAA), for example, at a concentration of about 0.05, 0.1,
0.15, 0.2, 0.25, 0.3, 0.4, or 0.5% (or any percentage in
between).
[0030] In some embodiments, following the decellularization and
partial lipid removal processes, the tissue product can contain
about 20-70% lipid content (as a percentage of the overall tissue
product by mass), and preferably contains about 30-50% lipid
content by mass. In some embodiments, the tissue product contains
about 20, 30, 40, 50, 60, or 70% lipid content by mass following
processing (or any percentage in between).
[0031] In various embodiments, the tissue product is processed to
remove sufficient lipids such that the product can avoid
significant inflammatory and/or immunologic responses following
implantation (e.g., by removing lipids such that the tissue product
comprises less than about 60% lipid content). Significant
inflammation encompasses any inflammation that would hinder the
long-term ability of the implant to promote native cell
repopulation and host tissue repair, regeneration, treatment, or
healing. Inflammation can be evaluated, for example, by measuring
the level of one or more inflammatory marker in a sample taken from
a patient (e.g., the level of one or more inflammatory cells,
cytokines, immunoglobulins, or other inflammatory molecules in a
blood or tissue sample) and comparing that level to one or more
reference levels.
[0032] In certain embodiments, the tissue product retains
sufficient lipid content such that the product can provide a soft
and malleable material suitable for filling the potentially
irregular shape of an implant site (e.g., by retaining at least
about 20% lipid content in the tissue product).
[0033] In some embodiments, following decellularization and partial
lipid removal, the tissue product contains an increased amount of
ECM as a percentage of the overall tissue product by mass. In
certain embodiments the tissue product contains about 3-20% ECM by
mass. In some embodiments, the tissue product contains about 3, 4,
5, 6, 7, 8, 9, 10, 15, or 20% ECM by mass (or any percentage in
between). In certain embodiments, the tissue product comprises
sufficient ECM following decellularization and partial lipid
removal such that the ECM can provide structural support and
integrity for the lipid components of the tissue product (e.g.,
sufficient structural support such that the tissue product
comprises a solid material rather than a shapeless and greasy mass
of adipose). For example, the ECM can provide structural support
such that the tissue product can be provided in sheets, thereby
allowing for improved surgical handling and manipulation before
and/or during implantation. In some embodiments, the ECM in an
adipose tissue product also provides a scaffold into which native
cells and vasculature can migrate and proliferate from tissue
surrounding an implant after surgical implantation into a host.
[0034] After decellularization and partial lipid removal, a 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 host tissue. For
example, a spherical or cylindrical shape can be provided where the
tissue product will be implanted following removal of a similarly
shaped volume of native tissue.
[0035] In some embodiments, the adipose tissue product can be
treated to reduce bioburden (i.e., to reduce the number of
microorganisms growing on the tissue). In some embodiments, the
treated tissue product lacks substantially all bioburden (i.e., the
tissue product is aseptic or sterile). Suitable bioburden reduction
methods are known to one of skill in the art, and may include
exposing the tissue product to radiation. Irradiation may reduce or
substantially eliminate 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 order to reduce or substantially
eliminate bioburden. 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.
[0036] In certain embodiments, one or more additional agents can be
added to the adipose tissue product. In some embodiments, the
additional agent can comprise an anti-inflammatory agent, an
analgesic, or any other desired therapeutic agent. In certain
embodiments, the additional agent can comprise at least one added
growth or signaling factor (e.g., a cell growth factor, an
angiogenic factor, a differentiation factor, a cytokine, a hormone,
and/or a chemokine). In some embodiments, these additional agents
can promote native tissue migration, proliferation, and/or
vascularization within the ECM of a tissue product following
implantation. In some embodiments, the growth or signaling factor
is encoded by a nucleic acid sequence contained within an
expression vector. Preferably, the expression vector is in one or
more of the viable cells that can be added, optionally, to the
tissue product. As used herein, the term "expression vector" refers
to any nucleic acid construct that is capable of being taken up by
a cell, contains a nucleic acid sequence encoding a desired
protein, and contains the other necessary nucleic acid sequences
(e.g. promoters, enhancers, termination codon, etc.) to ensure at
least minimal expression of the desired protein by the cell.
[0037] In various embodiments, the tissue products described above
have the ability to support the migration and proliferation of
native cells into the extracellular matrix in the tissue product
following implantation, as well as the ability to promote the
regeneration, revascularization, repair, and/or treatment of native
tissue when implanted in or on a patient. In addition, the tissue
products have the ability to act as a carrier for and support the
growth of cells, including stems cell, such as adipose-derived stem
cells. Accordingly, in certain embodiments, the processes discussed
above should not alter the extracellular matrix proteins of the
adipose-containing tissue (e.g., by damaging protein structures
and/or removing important glycosaminoglycans and/or growth
factors). In some embodiments, the products will have normal
collagen banding as evidenced by transmission electron
microscopy.
[0038] In some embodiments, an adipose tissue product can be stored
in a suitable aqueous solution or can be freeze-dried for long-term
storage. The specific freeze drying protocol can vary depending on
the solvent used, sample size, and/or to optimize processing time.
One suitable freeze-drying process can include freezing the tissue
product to -35.degree. C. over a 45 minute period; holding the
samples at -35.degree. C. for 90 minutes to insure complete
freezing; applying a vacuum; raising the temperature to -10.degree.
C. and holding for 24 hours; raising the temperature to 0.degree.
C. and holding for 24 hours; and raising the temperature to
20.degree. C. and holding for 12 hours. The freeze-dried samples
can then be removed from the freeze-dryer and packaged in foil
pouches under nitrogen.
Use of Tissue Products
[0039] The adipose tissue products described herein can be used to
treat a variety of different anatomic sites. For example, the
tissue products can be implanted to fill a void space in a native
tissue (e.g., following injury or surgical removal of a bulk volume
of native tissue, such as after surgical removal of a tumor).
Similarly, the tissue products can be used as implants or in
conjunction with polymeric implants for use in cosmetic procedures
to augment or enhance a native tissue. For example, the tissue
products of the present disclosure are produced from
adipose-containing tissues. Accordingly, it is believed that the
adipose tissue products will provide superior regenerative
capabilities when implanted in certain tissue sites, as compared to
materials produced from other tissue types. For example, the
retained lipid components in the partially de-fatted tissue
products are believed to promote the deposition and storage of
lipids within and around the implanted product, while the ECM
components of the implanted product provide a scaffold for the
migration and proliferation of native cells within the implant,
thereby allowing for the regeneration of more natural looking
and/or feeling tissue around the implant site. The implanted tissue
products can also promote revascularization. Accordingly, in
certain embodiments, the tissue products disclosed herein can be
implanted in tissue sites in a host human or other animal that
predominantly or significantly comprise adipose tissue, and the
implanted products can promote the repair, regeneration, treatment,
augmentation, and/or enhancement of the host tissue.
[0040] In some embodiments, the tissue sites for implantation of an
adipose tissue product can include a breast (e.g., for
augmentation, enhancement, replacement of resected tissue, or
placement around an implant). In addition, a site in any other
adipose or soft tissue can be selected. For example, the tissue
products may be used for reconstructive or cosmetic purposes in the
face, neck, buttocks, abdomen, hips, thighs, and/or any other site
comprising adipose or soft tissue where reconstruction or
augmentation is desired using a tissue product having a structure
and/or feel that approximates that of native adipose. In any of
those sites, the tissue may be used to reduce or eliminate
wrinkles, sagging, or undesired shapes.
[0041] When used as implants to repair, regenerate, treat, augment,
and/or enhance adipose or other soft tissues, the tissue products
disclosed herein can provide advantages over other implanted
natural and synthetic products. For example, although some tissue
implants allow for native cell ingrowth and tissue formation (e.g.,
implants from non-adipose tissue sources that comprise an
extracellular matrix), those implants may induce the formation of
fibrotic tissue that does not mimic normal texture and/or feel of
adipose or other soft tissues, and may appear abnormal on
radiologic imaging. Since the tissue products of the present
disclosure are formed from adipose-containing tissues, they may
avoid or reduce the extent of fibrotic tissue formation.
Furthermore, since the tissue products retain some lipid components
following partial lipid removal, it is believed that the implanted
products promote the deposition of native adipose and are less
likely to harden over time, thereby retaining the look and/or feel
of native adipose tissue. In contrast, adipose tissue implants that
lack substantially all lipid components (e.g., less than 20% of the
adipose present prior to processing) may result in stiff implant
materials that lack sufficient malleability for use as soft tissue
fillers, and which may also harden further over time.
In addition, as discussed above, the tissue products disclosed
herein can be provided in sheets or other desired three-dimensional
shapes that retain structural integrity and provide for ease of
surgical manipulation. The tissue products disclosed herein do not,
in certain embodiments, require micronization, homogenization, or
further processing (e.g., freeze drying and/or cross-linking) in
order to provide for malleable yet structurally stable tissue
implants that do not induce significant immune and/or inflammatory
responses, in contrast to certain full-fat implants. Such full-fat
implants may have a viscous consistency and cannot retain a desired
shape, and may also have an increased possibility of inducing an
immune and/or inflammatory response. In contrast, the partially
de-fatted tissue products disclosed herein promote native lipid
deposition while avoiding the inflammatory and immunologic
responses that may be associated with implanted adipose tissues
that have not been de-fatted.
EXAMPLES
[0042] The following examples serve to illustrate, and in no way
limit, the present disclosure.
Example 1: Determining Lipid Content
[0043] To determine lipid content, tissue samples were washed with
0.9% NaCl, and then with mini-Q water. Washed tissue was
freeze-dried. Lipid from the freeze-dried samples was extracted
with chloroform. Extracted tissue samples were vacuum-dried. The
loss of sample mass due to extraction was used to determine lipid
content. The lipid content was calculated using the formula:
Lipid Content (%)=(Initial Dry Weight-Extracted Dry
Weight)/(Initial Dry Weight).times.100
Example 2: Decellularization and Partial Lipid Removal of Porcine
Dermis
[0044] Layers of porcine adipose tissue at a depth of between 2.75
mm and 4.20 mm was obtained for processing. The lipid content of
the adipose tissue samples was determined to be 85.9.+-.6.8%
(mean.+-.SD, N=8) on a dry mass basis. The loose fat on the tissue
surface was scraped manually, and the tissue was pre-incubated for
22 hours with gentle agitation in 35% maltodextrin solution
containing 0.24 g/L cefoxitin, 0.12 g/L licomycin, 0.03 g/L
vancomycin and 0.1 g/L polymyxin B sulfate. The scrapping and
incubation reduced the tissue lipid content to 74.8.+-.12.6%
(mean.+-.SD, N=7) on a dry mass basis. The tissue was stored at
-80.degree. C. until used.
[0045] For further processing, frozen tissue material was thawed at
4.degree. C. over a period of 65 hours. After washing twice with 20
mM HEPES buffer (pH 8.0) to remove the maltodextrin solution, the
tissue was decellularized for .about.20 hours in 1% (w/v) sodium
deoxycholate dissolved in 10 mM HEPES buffer (pH 8.1) containing
0.3% (w/v) Triton X100, with agitation. Decellularized tissue was
rinsed with 10 mM HEPES buffer (pH 7.2) containing 10 mM MgCl.sub.2
and 10 mM CaCl.sub.2. DNAse and alpha galactosidase were then added
at 4 mg/L and 2 mg/L, respectively for treatment for 20 hours. The
resultant tissue matrix was washed three times in HEPES buffer (pH
7.2) over 8 hours to remove residual enzymes. The processing steps
resulted in a further reduction of lipid content. Processed tissue
was stored in 4 mM citrate-phosphate buffer (pH 6.5) containing 12%
(w/v) glycerol, and terminally sterilized by 26 kGy gamma
irradiation.
[0046] The average thickness of sterilized adipose tissue matrix
sheets was 1.0.+-.0.2 mm, which is thinner than the starting
material due to partial lipid removal during processing. The soft
adipose matrix had a moderate tensile strength of 2.7.+-.1.6 MPa
(mean.+-.SD, N=48). Residual DNA content was 0.073.+-.0.041 .mu.g/g
(mean.+-.SD, N=10) on a dry mass basis, indicating the removal of
greater than 99.5% of the DNA in the tissue. Immunostaining with
lectin was negative for the presence of alpha-gal antigen. Lipid
content, non-fat tissue matrix density, and water content of the
sterilized adipose tissue matrices were measured to be
37.0.+-.6.2%, 11.8.+-.2.5%, and 51.3.+-.3.8% (mean.+-.SD, n=5),
respectively.
Example 3: Ultrasound-Facilitated Decellularization
[0047] Ultrasound was used to aid in the process of
decellularization and partial lipid removal. The first method
involved treatment of tissue before decellularization in sodium
deoxycholate solution. The tissue was exposed to high ultrasonic
energy for 30 seconds (.about.95 Watts per square inch).
Ultrasound-treated tissue was then decellularized in 1% (w/v)
sodium deoxycholate solution.
[0048] The second method involved decellularization of tissue
material in a lower energy ultrasonic water bath (Bransonic
ultrasonic cleaner, 44 kilohertz, .about.1.0 Watt per square inch)
for up to 8 hours. Porcine dermal tissue was decellularized in two
different solutions: (a) 1% sodium deoxycholate +0.5% Triton X-100
in 10 mM HEPES buffer (pH 8.0) and (b) 1% sodium dodecyl sulfate 10
mM HEPES buffer (pH 8.0).
Example 4: Control of Temperature During Ultrasonic
Decellularization
[0049] Ultrasonic treatment generates heat and could lead to an
increase in temperature of decellularization solution. To avoid
denaturation of adipose tissue material, the solution temperature
was controlled to keep it below a threshold above which collagen
denaturation may occur. To determine the appropriate temperature,
tissue samples were incubated for 60 minutes at different
temperatures between 44.degree. C. and 60.degree. C. After
incubation, the extent of collagen denaturation was measured with
differential scanning calorimeter. During the calorimetric test,
tissue samples were scanned from 2.degree. C. to 120.degree. C. at
4.degree. C/min. FIG. 1. No denaturation was observed for tissues
incubated at temperatures below 50.degree. C. Thus, the tissue
processing temperature can be raised above 40.degree. C. to
accelerate the decellularization process.
Example 5: In Vivo Performance
[0050] The in vivo performance of processed adipose tissue grafts
with high lipid content of between about 45% and 75% on a dry mass
basis was evaluated using a primate functional abdominal wall
repair model (African green monkey). Three such grafts with
different lipid content (45%, 63%, and 72%) were implanted for
three months, and blood samples were taken at 0, 1, 2, 4, 6, 8, and
12 weeks after implantation. There was no herniation in any of the
animals, and all three grafts integrated well with surrounding
animal tissues. Histological analysis of explanted materials
demonstrated host cell repopulation and re-vascularization. FIG. 2
shows the H&E stained sections of the three grafts after
explantation at 3 months. No inflammation was observed in the graft
with 45% lipid content (on a dry weight basis). Significant
inflammation was observed only in the graft with about 70% lipid
content (on a dry weight basis). The tests of blood samples taken
after implantation showed that the quantity of IgG antibodies was
low with a transient increase (<128 folds) following surgery,
indicating that grafts induced insignificant immunological
reactions (FIG. 3).
[0051] The in vivo primate evaluation demonstrated that tissue
grafts with high lipid content were able to integrate into host
tissues and to support cell repopulation and re-vascularization. As
lipid content increased (>about 70%), however, inflammation
became more severe. The primates did not have significant foreign
body reactions to the grafts.
[0052] 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.
* * * * *