U.S. patent application number 16/799295 was filed with the patent office on 2020-08-06 for natural tissue scaffolds as tissue fillers.
The applicant listed for this patent is LifeCell Corporation. Invention is credited to Rick T. Owens, Wenquan Sun, Hua Wan, Hui Xu.
Application Number | 20200246508 16/799295 |
Document ID | / |
Family ID | 1000004722977 |
Filed Date | 2020-08-06 |
![](/patent/app/20200246508/US20200246508A1-20200806-D00001.png)
![](/patent/app/20200246508/US20200246508A1-20200806-D00002.png)
![](/patent/app/20200246508/US20200246508A1-20200806-D00003.png)
![](/patent/app/20200246508/US20200246508A1-20200806-D00004.png)
![](/patent/app/20200246508/US20200246508A1-20200806-D00005.png)
![](/patent/app/20200246508/US20200246508A1-20200806-D00006.png)
![](/patent/app/20200246508/US20200246508A1-20200806-D00007.png)
![](/patent/app/20200246508/US20200246508A1-20200806-D00008.png)
![](/patent/app/20200246508/US20200246508A1-20200806-D00009.png)
![](/patent/app/20200246508/US20200246508A1-20200806-D00010.png)
![](/patent/app/20200246508/US20200246508A1-20200806-D00011.png)
View All Diagrams
United States Patent
Application |
20200246508 |
Kind Code |
A1 |
Xu; Hui ; et al. |
August 6, 2020 |
NATURAL TISSUE SCAFFOLDS AS TISSUE FILLERS
Abstract
Tissue fillers derived from decellularized tissues are provided.
The tissue fillers can include acellular tissue matrices that have
reduced inflammatory responses when implanted in a body. Also
provided are methods of making and therapeutic uses for the tissue
fillers.
Inventors: |
Xu; Hui; (Plainsboro,
NJ) ; Sun; Wenquan; (Warrington, PA) ; Wan;
Hua; (Princeton, NJ) ; Owens; Rick T.;
(Stewartsville, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LifeCell Corporation |
Madison |
NJ |
US |
|
|
Family ID: |
1000004722977 |
Appl. No.: |
16/799295 |
Filed: |
February 24, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16123783 |
Sep 6, 2018 |
10610615 |
|
|
16799295 |
|
|
|
|
15344855 |
Nov 7, 2016 |
10092677 |
|
|
16123783 |
|
|
|
|
14741653 |
Jun 17, 2015 |
9504770 |
|
|
15344855 |
|
|
|
|
13560362 |
Jul 27, 2012 |
9089523 |
|
|
14741653 |
|
|
|
|
61512610 |
Jul 28, 2011 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 27/3633 20130101;
A61L 2430/00 20130101; A61L 27/362 20130101; A61L 27/505
20130101 |
International
Class: |
A61L 27/36 20060101
A61L027/36; A61L 27/50 20060101 A61L027/50 |
Claims
1. A tissue filler, comprising: an acellular tissue matrix having
the ability to maintain the same shape, size, and sponge property
after being implanted for one week as was observed prior to
application to a tissue site; and exogenous hyaluronic acid (HA) on
a surface of the acellular tissue matrix at a concentration that
reduces an inflammatory response or fibrosis when the tissue filler
is applied to the tissue site as compared to the same tissue filler
not comprising hyaluronic acid.
2. The tissue filler of claim 1, wherein a concentration of HA on
the acellular tissue matrix is between approximately 0.5 mg and
approximately 5.0 mg per gram of tissue filler.
3. The tissue filler of claim 1, further comprising at least one
growth factor.
4. The tissue filler of claim 3, wherein the at least one growth
factor is selected from at least one of FGF, VEGF, PDGF,
Angiopoitin-2, or Follistatin.
5. The tissue filler of claim 1, wherein the tissue filler has been
treated to reduce a bioburden.
6. The tissue filler of claim 1, further comprising an
antimicrobial agent.
7. The tissue filler of claim 6, wherein the antimicrobial agent
includes at least one of chlorhexidine (CHX) and silver.
8. The tissue filler of claim 7, wherein the CHX has a
concentration between approximately 0.1 mg and approximately 3.0 mg
per gram of tissue filler.
9. The tissue filler of claim 7, wherein the silver has a
concentration between approximately 0.1 mg and approximately 1.0 mg
per gram of tissue filler.
10. The tissue filler of claim 1, wherein the tissue filler is
capable of being compressed up to approximately 2/3 of its length
or width.
11. The tissue filler of claim 10, wherein the tissue filler is
capable of returning to its original dimensions after release of
compression.
12. The tissue filler of claim 1, wherein the acellular tissue
matrix has a stable three-dimensional structure.
13. The tissue filler of claim 1, wherein the acellular tissue
matrix comprises a dermal matrix.
14. The tissue filler of claim 13, wherein the acellular tissue
matrix is derived from skin.
15. The tissue filler of claim 13, wherein the acellular tissue
matrix is derived from dermis.
16. The tissue filler of claim 1, wherein the acellular tissue
matrix is derived from at least one of lung, liver, bladder,
muscle, and fat tissue.
17. The tissue filler of claim 1, wherein the acellular tissue
matrix is derived from porcine tissue.
18. The tissue filler of claim 1, wherein the acellular tissue
matrix comprises an adipose matrix.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/123,783, which was filed on Sep. 6, 2018,
which is a continuation of U.S. patent application Ser. No.
15/344,855, which was filed on Nov. 7, 2016, now U.S. Pat. No.
10,092,677, which is a continuation of U.S. patent application Ser.
No. 14/741,653, which was filed on Jun. 17, 2015, now U.S. Pat. No.
9,504,770, which is a continuation of U.S. patent application Ser.
No. 13/560,362, which was filed on Jul. 27, 2012, now U.S. Pat. No.
9,089,523, which claims priority to U.S. Provisional Application
No. 61/512,610, filed on Jul. 28, 2011, all of which are
incorporated herein by reference in their entirety.
[0002] The present disclosure relates generally to tissue fillers
and their use as implants and scaffolds for natural tissue regrowth
after removal of a portion of native tissue.
[0003] Currently, tissue fillers are often derived from temporary
hyaluronic acid or collagen-based materials. These materials lack
stability and biocompatibility, and may require complex harvesting
procedures. Their medical use is generally limited to temporarily
filling small sites of tissue removal. Thus, existing tissue
fillers are not suitable for long term removal of large volumes of
tissue, such as breast lumpectomies. In addition, existing tissue
fillers may not promote sufficient native tissue regrowth or limit
inflammation and the formation of scar tissue. Producing a tissue
filler having the texture and structural integrity of native tissue
that is also capable of promoting the regrowth of native tissue
while reducing inflammation and the formation of scar tissue would
therefore be desirable.
[0004] Accordingly, improved tissue fillers are provided herein. In
various embodiments, a tissue filler is provided, comprising an
acellular tissue matrix and at least one of exogenous hyaluronic
acid (HA) and exogenous decorin at a concentration sufficient to
reduce an inflammatory response or fibrosis, when the tissue filler
is implanted in a body. The acellular tissue matrix can be selected
from an acellular lung, liver, bladder, muscle, and fat matrix. In
further embodiments, the concentration of HA on the acellular
tissue matrix is between approximately 0.5 mg and approximately 5.0
mg per gram of tissue filler. In further embodiments, the
concentration of decorin on the acellular tissue matrix is between
approximately 0.3 mg and approximately 1.0 mg per gram of tissue
filler. In still further embodiments, the tissue filler elicits a
reduced inflammatory response, as compared to a tissue filler
lacking HA and/or decorin, when implanted in the body. In further
embodiments, the tissue filler reduces fibrosis and scar tissue
formation after removal of a native tissue, as compared to a tissue
filler lacking HA and/or decorin, when implanted in the body.
[0005] In various embodiments, the tissue filler further comprises
at least one growth factor. In further embodiments, the at least
one growth factor is FGF, VEGF, PDGF, angiopoitin-2, or
follistatin. In some embodiments, the tissue filler lacks
substantially all alpha-galactose moieties. In certain embodiments,
the tissue filler has been treated to reduce a bioburden. In
further embodiments, the tissue filler is sterile.
[0006] In some embodiments, the tissue filler further comprises an
antimicrobial agent. The antimicrobial agent can include at least
one of CHX and silver. The CHX can be at a concentration of between
approximately 0.1 mg and approximately 3.0 mg per gram of tissue
filler. The silver can be at a concentration of between
approximately 0.1 mg and approximately 1.0 mg per gram of tissue
filler.
[0007] In various embodiments, the tissue filler is compressible.
In further embodiments, the tissue filler is capable of being
compressed up to approximately 2/3 of its length or width. In still
further embodiments, the tissue filler is capable of returning to
its original dimensions after release of compression.
[0008] In various embodiments, a method of treating a tissue after
removal of native tissue is provided, comprising implanting the
tissue filler described above into the tissue. In further
embodiments, the implanted tissue filler can swell to fill a region
of native tissue that has been removed. In still further
embodiments, the implanted tissue filler is selected to have the
same structural strength, texture and feel as the native tissue it
replaces. In even further embodiments, implanting the tissue filler
promotes the infiltration, migration, growth, and/or proliferation
of surrounding native tissue cells in the tissue filler, as well as
the revascularization of the tissue being treated.
[0009] In certain embodiments, the HA and/or decorin on the
implanted tissue filler elicits a reduced inflammatory response, as
compared to an implanted tissue filler lacking HA and/or decorin.
In further embodiments, the inflammatory response is reduced by at
least 10%. In still further embodiments, the HA and/or decorin on
the implanted tissue filler reduces scar tissue formation after
removal of a native tissue, as compared to an implanted tissue
filler lacking HA and/or decorin. In even further embodiments, the
decorin and/or HA remains on the tissue filler for the duration of
the implant.
[0010] In some embodiments, the method of treating a tissue further
comprising removing at least 20% by mass of a native tissue prior
to implanting a tissue filler. In certain embodiments, the tissue
being removed comprises a tumor. In some embodiments, the tissue
being removed comprises breast tissue.
[0011] In various embodiments, a method of preparing a tissue
filler is provided, comprising selecting a tissue, decellularizing
the tissue, and contacting the tissue with least one substance that
reduces inflammation and/or fibrosis when the tissue is implanted
in a body. The tissue can be selected from lung, liver, bladder,
muscle, and fat. In some embodiments, the at least one substance
that can reduce inflammation and/or fibrosis includes at least one
of hyaluronic acid (HA) and decorin. In some embodiments, when HA
is used, the method includes contacting the tissue filler with a
solution containing HA at a concentration of between approximately
1.0 mg/ml and approximately 10.0 mg/ml. In some embodiments, when
decorin is used, the method includes contacting the tissue filler
with a solution containing decorin at a concentration of between
approximately 0.1 mg/ml and approximately 3.0 mg/ml.
[0012] In various embodiments, the method of preparing a tissue
filler comprises contacting the tissue filler with at least one
detergent. The at least one detergent can include at least one of
sodium dodecyl sulfate, sodium deoxycholate, and Triton X-100. The
detergent concentration and detergent exposure time can be selected
to prevent removal of growth factors from the tissue filler. In
some embodiments, the method of preparing a tissue filler further
comprises removing alpha-galactose moieties from the tissue
filler.
[0013] In some embodiments, the method of preparing a tissue filler
comprises irradiating the tissue filler to reduce the bioburden of
the tissue filler. Irradiation can include exposing the tissue
filler to 15-17 kGy E-beam irradiation. In certain embodiments, the
method further comprises sterilizing the tissue filler. In some
embodiments, the method comprises contacting the tissue filler with
an antimicrobial agent. The antimicrobial agent can include at
least one of CHX and silver. In some embodiments, CHX is present at
a concentration of between approximately 0.1 mg and approximately
3.0 mg of CHX per gram of tissue filler. In other embodiments,
silver is present at a concentration of between approximately 0.1
mg and approximately 1.0 mg of silver per gram of tissue
filler.
[0014] In some embodiments, the method of preparing a tissue filler
comprises freeze-drying the tissue filler. In further embodiments,
the method comprises rehydrating the freeze-dried tissue filler
prior to implantation in a tissue.
[0015] In various embodiments, a tissue filler is provided,
prepared by the methods described above.
[0016] In various embodiments, a method of treatment is provided,
comprising removing a native tissue and implanting a tissue filler,
wherein the tissue filler comprises an acellular tissue matrix and
at least one of exogenous hyaluronic acid (HA) and exogenous
decorin at a concentration sufficient to reduce an inflammatory
response or fibrosis when the tissue filler is implanted in a body.
In some embodiments, the native tissue being removed comprises a
tumor. In certain embodiments, the native tissue is breast tissue.
In some embodiments, the tissue filler used in the method of
treatment is prepared according to any one of the methods described
above. In some embodiments, the tissue filler used in the method of
treatment comprises any one of the tissue fillers described
above.
[0017] In various embodiments, a method of enhancing a native
tissue is provided, comprising implanting a tissue filler into a
native tissue, wherein the tissue filler comprises an acellular
tissue matrix and at least one of exogenous hyaluronic acid (HA)
and exogenous decorin at a concentration sufficient to reduce an
inflammatory response or fibrosis when the tissue filler is
implanted in a body. In further embodiments, the native tissue is
breast tissue.
DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1A shows fresh porcine lung stained with hematoxylin
and eosin (H&E). FIG. 1B shows processed porcine lung stained
with H&E. FIG. 1C shows processed porcine lung stained with
Verhoeff's stain. FIG. 1D is a scanning electron micrograph of
processed porcine lung, as produced according to certain
embodiments.
[0019] FIG. 2A shows collagen type-I immunostaining of acellular
porcine lung. FIG. 2B shows collagen type-IV immunostaining of
acellular porcine lung. FIG. 2C shows fibronectin immunostaining of
acellular porcine lung, and FIG. 2D shows collagen type-III
immunostaining of acellular porcine lung.
[0020] FIG. 3A is an illustration of the compressibility of
acellular porcine lung. FIG. 3B is an illustration of the
elasticity of acellular porcine lung after the release of
compression.
[0021] FIG. 4A illustrates the shape of a porcine lung tissue
filler before freeze drying. FIG. 4B illustrates the shape of a
porcine lung tissue filler after freeze drying. FIG. 4C illustrates
the shape of a porcine lung tissue filler after rehydration.
[0022] FIG. 5 is a plot of the glycosaminoglycan (GAG)
concentration in extracellular matrices derived from porcine
vessel, dermal, liver, and lung tissue.
[0023] FIG. 6 shows thermogram plots for tissue fillers derived
from porcine lung and liver.
[0024] FIG. 7 is a plot showing the effect of collagenase digestion
on tissue fillers derived from porcine liver, lung, and dermal
tissue.
[0025] FIG. 8A shows alcian blue staining of tissue filler derived
from porcine lung. FIG. 8B shows alcian blue staining of tissue
filler derived from porcine lung that is coated in hyaluronic
acid.
[0026] FIG. 9 is a plot showing the concentration of HA in uncoated
porcine liver tissue filler (left), in porcine liver tissue filler
incubated with 5 mg/ml of hyaluronic acid sodium salt (right), and
in porcine liver tissue filler incubated with 5 mg/ml of hyaluronic
acid sodium salt and washed overnight (center).
[0027] FIG. 10A shows anti-human decorin staining of tissue filler
derived from porcine liver. FIG. 10B shows control serum staining
of tissue filler derived from porcine liver that is coated in human
decorin. FIG. 10C shows anti-human decorin staining of tissue
filler derived from porcine liver that is coated in human
decorin.
[0028] FIG. 11 is a plot showing the concentration of decorin in
uncoated porcine liver tissue filler (left) and in porcine liver
tissue filler incubated with 1 mg/ml of decorin and washed
overnight (right).
[0029] FIG. 12A is an H&E stain showing in vitro growth of rat
fibroblast in tissue fillers derived from porcine lung. FIG. 12B is
an H&E stain showing in vitro growth of rat fibroblast in
tissue fillers derived from porcine liver. FIG. 12C is an H&E
stain showing in vitro growth of rat stem cells in tissue fillers
derived from porcine lung. FIG. 12D is an H&E stain showing in
vitro growth of rat stem cells in tissue fillers derived from
porcine liver.
[0030] FIG. 13 illustrates inflammatory cytokine levels induced by
in vitro culturing of human blood mononuclear cells with tissue
fillers derived from porcine lung, liver and dermal tissue.
[0031] FIG. 14A illustrates the gross morphology of tissue fillers
derived from porcine lung two weeks after implantation in rat. FIG.
14B illustrates the gross morphology of tissue fillers derived from
porcine liver two weeks after implantation in rat. FIG. 14C
illustrates the gross morphology of tissue fillers derived from
porcine liver and coated in CHX, two weeks after implantation in
rat. FIG. 14D illustrates the gross morphology of tissue fillers
derived from porcine liver and coated in hyaluronic acid, two weeks
after implantation in rat. FIG. 14E illustrates the gross
morphology of tissue fillers derived from porcine liver and coated
in decorin, two weeks after implantation in rat.
[0032] FIG. 15A shows H&E staining of tissue fillers derived
from porcine liver. FIG. 15B shows H&E staining of tissue
fillers derived from porcine liver coated in chlorhexidine (CHX).
FIG. 15C shows H&E staining of tissue fillers derived from
porcine liver coated in hyaluronic acid. FIG. 15D shows H&E
staining of tissue fillers derived from porcine liver coated in
decorin.
[0033] FIG. 16A shows H&E staining of tissue fillers derived
from porcine liver. FIG. 16B shows H&E staining of tissue
fillers derived from porcine liver coated in chlorhexidine (CHX).
FIG. 16C shows H&E staining of tissue fillers derived from
porcine liver coated in hyaluronic acid. FIG. 16D shows H&E
staining of tissue fillers derived from porcine liver coated in
decorin.
[0034] FIG. 17A shows H&E staining of tissue fillers derived
from porcine liver coated in HA. FIG. 17B shows H&E staining of
tissue fillers derived from porcine liver coated in decorin.
[0035] FIG. 18A shows immunostaining of fibroblast cells in a
porcine liver tissue filler two weeks after implantation in rat.
FIG. 18B shows anti-vWF immunostaining of neo-vessel formation in a
porcine liver tissue filler two weeks after implantation in rat.
FIG. 18C shows anti-SMC-.alpha.-actin immunostaining of
myofibroblast cells in a porcine liver tissue filler two weeks
after implantation in rat.
[0036] FIG. 19A shows anti-vimentin immunostaining of fibroblast
cells in a porcine liver tissue filler two weeks after implantation
in rat. FIG. 19B shows anti-vimentin immunostaining of fibroblast
cells in a porcine liver tissue filler coated in hyaluronic acid,
two weeks after implantation in rat. FIG. 19C shows anti-vimentin
immunostaining of fibroblast cells in a porcine liver tissue filler
coated in decorin, two weeks after implantation in rat.
[0037] FIG. 20A shows anti-human decorin immunostaining of tissue
fillers derived from porcine liver two weeks after implantation in
rat. FIG. 20B shows control serum staining of tissue fillers
derived from porcine liver coated in human decorin, two weeks after
implantation in rat. FIG. 20C shows anti-human decorin
immunostaining of a tissue filler derived from porcine liver and
coated in human decorin, two weeks after implantation in rat.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0038] 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.
[0039] In this application, the use of the singular includes the
plural unless specifically stated otherwise. Also 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," are not limiting.
Any range described herein will be understood to include the
endpoints and all values between the endpoints.
[0040] 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. To the
extent publications and patents or patent applications incorporated
by reference contradict the invention contained in the
specification, the specification will supersede any contradictory
material.
[0041] In various embodiments, tissue fillers are provided. The
tissue filler can comprise an acellular tissue matrix and at least
one substance that can reduce inflammation and/or fibrosis when the
filler is implanted in the body. The substance can be hyaluronic
acid (HA) and/or decorin, at a concentration sufficient to reduce
inflammation or fibrosis after implantation in the body.
[0042] The acellular tissue matrix in a tissue filler can be
derived from various organ sources. Organs having a compact three
dimensional shape are preferable, such as lung, liver, bladder,
muscle, or fat, as they provide a spongy acellular matrix after
decellularization. This spongy tissue filler can be molded and used
as an implant to fill the void left by the removal of native
tissue. In certain embodiments, the acellular tissue matrix is an
acellular lung or liver tissue matrix. In some embodiments, the
acellular tissue matrix is an acellular porcine lung or liver
tissue matrix.
[0043] In various embodiments, the tissue fillers are useful as
implants following removal of native tissue from a recipient organ
or tissue, or as implants for cosmetic enhancement purposes. Tissue
fillers derived from organs such as lung, liver, bladder, muscle,
or fat provide the texture and structural strength of native
tissue, while also providing a biological scaffold in which native
cells and vasculature can migrate and proliferate. Furthermore,
adding at least one substance such as HA and/or decorin to the
acellular tissue can help reduce undesirable inflammation and/or
fibrosis following implantation of a tissue filler into a recipient
organ or tissue.
[0044] In certain embodiments, tissue fillers can be produced by
decellularizing an organ tissue and coating the tissue in a
solution containing an anti-inflammatory and/or an anti-fibrotic
substance such as HA and/or decorin. In other embodiments, the
presently described tissue fillers can be further processed into
desired shapes and stored either fresh or freeze-dried prior to
implantation in a recipient organ. The tissue fillers can be
produced as aseptic or sterile materials. In some embodiments, the
tissue filler is in strips, balls, or molded into other shapes that
provide the desired size, shape, or structural features necessary
for a given tissue filler.
[0045] As noted, the tissue fillers can comprise acellular tissue
matrices, providing natural tissue scaffolds on which native tissue
can grow and regenerate. As used herein, "native" cells or tissue
means the cells or tissue present in the recipient organ or tissue
prior to implantation of a tissue filler. Tissue fillers derived
from organs having a compact three dimensional shape, such as lung,
liver, bladder, muscle, or fat, are preferable as they provide a
spongy acellular tissue matrix after decellularization. These
spongy acellular matrices can be molded to fill the void left by
removal of a native tissue while providing the texture and
durability of native tissue. Furthermore, the acellular tissue
matrix provides a natural tissue scaffold for native cell growth.
The scaffold may consist of collagen, elastin, or other fibers, as
well as proteoglycans, polysaccharides and growth factors. Tissue
fillers may retain all components of the extracellular matrix, or
various undesirable components may be removed by enzymatic or
genetic means prior to implantation. The exact structural
components of the extracellular matrix will depend on the tissue
selected and the processes used to prepare the tissue scaffold. The
natural tissue scaffold in a tissue filler provides a structural
network of fibers, proteoglycans, polysaccharides, and growth
factors on which native tissue and vasculature can migrate, grow,
and proliferate.
[0046] Tissue fillers can contain acellular tissue matrices derived
from various tissues and animal sources. In certain embodiments,
the acellular tissue is taken from human cadaver, cow, horse, or
pig. In some embodiments, tissue fillers contain acellular tissue
matrices derived from lung, liver, bladder, muscle, or fat tissue
in order to approximate the soft and spongy property of a native
soft tissue. In some embodiments, the tissue is acellular porcine
liver or lung tissue.
[0047] In various embodiments, tissue fillers comprise at least one
substance that can reduce inflammation and/or fibrosis after
implantation into the body (i.e., into a recipient tissue or
organ), as compared to tissue matrices lacking such substances. In
certain embodiments, the substance is exogenous HA and/or exogenous
decorin. Decorin is a proteoglycan commonly found in connective
tissue and implicated in fibrilogenesis. HA is a glycosaminoglycan
commonly found in epithelial tissue. In further embodiments, the
hyaluronic acid is at a concentration of, e.g., 0.5, 1.0, 1.5, 2.0,
2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mg of hyaluronic acid per gram of
tissue filler (or any value in between). In other embodiments, the
decorin is at a concentration of, e.g., 0.3, 0.35, 0.4, 0.45, 0.5,
0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1.0 mg of
decorin per gram of tissue filler (or any value in between). In
some embodiments, tissue fillers containing decorin and/or HA can
reduce inflammation and/or fibrosis after implantation in a
recipient tissue, as compared to an implanted tissue filler lacking
HA and/or decorin.
[0048] In some embodiments, the tissue fillers contain natural
tissue scaffolds, and these can be used to replace the tissue
scaffold lost after removal of native tissue. Any natural tissue
scaffold can be used that approximates the consistency, texture, or
structural integrity of the native tissue that it is replacing. The
texture and structural properties of a given tissue filler will
depend on the tissue source selected, as well as on the method
chosen to process the harvested tissue. In various embodiments,
lung, liver, bladder, muscle, or fat tissue is used to provide
effective natural tissue scaffolds because they provide the
consistency, biocompatibility, and structural integrity of native
tissue. In some embodiments, lung or liver tissue is used.
[0049] In various embodiments, the tissue fillers are compressible.
For example, the tissue fillers may be compressed up to
approximately 2/3 of their initial length or width. In still
further embodiments, the tissue filler is capable of returning to
its original dimensions after the release of compression. In even
further embodiments, a tissue filler derived from decellularized
porcine lung is capable of being compressed up to approximately 2/3
of its length or width and then returning to substantially the same
original dimensions after the release of compression (see FIG. 3).
In various embodiments, lung or liver tissue is used as a tissue
filler because, after decellularization and subsequent processing
(as described below), the filler exhibits substantial ability to
stretch or compress.
[0050] Tissue fillers derived from lung and liver contain abundant
glycosaminoglycans when compared to other porcine tissues. In
addition, in certain embodiments, tissue fillers derived from lung
and liver tissue retain the major growth factors present in
unprocessed lung or liver tissue. For example, lung and liver
tissue fillers can retain FGF, VEGF, PDGF, angiopoitin-2 and/or
follistatin (among other growth factors). In certain embodiments,
10, 20, 30, 40, 50, or 60 ng of FGF are present per gram of dried
tissue filler (or any value in between). In other embodiments, 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 ng of VEGF are present per gram of
dried tissue filler (or any value in between). In some embodiments,
0.5, 1.0, 1.5, or 2.0 ng of PDGF are present per gram of dried
tissue filler (or any value in between). In some embodiments, 0.05,
0.1, or 0.2 ng of angiopoitin-2 are present per gram of dried
tissue filler (or any value in between) where the tissue filler is
made from processed lung tissue. In some embodiments, 0.5, 1.0, or
1.5 ng of follistatin are present per gram of dried tissue filler
(or any value in between).
[0051] In certain embodiments, the tissue fillers lack certain
antigens. For example, certain animal tissues contain
alpha-galactose (.alpha.-gal) epitopes that are known to elicit
reactions in humans. Therefore, tissue fillers produced from animal
tissues can be produced or processed to lack certain antigens, such
as .alpha.-gal. In some embodiments, tissue fillers lack
substantially all .alpha.-gal moieties. Elimination of the
.alpha.-gal epitopes from the natural tissue scaffold may diminish
the immune response against the tissue filler, as the .alpha.-gal
epitope is absent in humans. U. Galili et al., J. Biol. Chem. 263:
17755 (1988). Since non-primate mammals (e.g., pigs) produce
.alpha.-gal epitopes, xenotransplantation of tissue filler material
from these mammals into primates may result in rejection because of
primate anti-Gal binding to the .alpha.-gal epitopes on the tissue
filler material. The binding results in the destruction of the
tissue filler material by complement fixation and by
antibody-dependent cell cytotoxicity. U. Galili et al., Immunology
Today 14: 480 (1993); M. Sandrin et al., Proc. Natl. Acad. Sci. USA
90: 11391 (1993); H. Good et al., Transplant. Proc. 24: 559 (1992);
B. H. Collins et al., J. Immunol. 154: 5500 (1995).
[0052] As described in detail below, in various embodiments, the
tissues fillers can be processed to remove antigens such as
.alpha.-gal, e.g., by enzymatic treatment. Alternatively, the
tissue fillers can be produced from animals that have been
genetically modified to lack those epitopes.
[0053] In various embodiments, tissue fillers have reduced
bioburden (i.e., a reduced number of microorganisms growing on the
tissue filler). In some embodiments, tissue fillers lack
substantially all bioburden (i.e., the tissue fillers are aseptic
or sterile). In certain embodiments, the tissue fillers further
comprise an antimicrobial agent to eliminate microbial growth
and/or prevent microbial growth when implanted. The antimicrobial
agent can include, for example, chlorhexidine (CH) or silver. In
certain embodiments, the concentration of CHX or silver is adjusted
to remove substantially all bioburden and/or to prevent microbial
growth. Effective concentrations of CHX capable of substantially
reducing bioburden on tissue fillers may include 0.1 mg, 0.5 mg,
0.7 mg, 0.9 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, or 3.0 mg per gram
of tissue filler (or any value in between). Effective
concentrations of silver capable of substantially reducing
bioburden on tissue fillers may include 0.1 mg, 0.2 mg, 0.3 mg, 0.4
mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, or 1.0 mg per gram of
tissue filler (or any value in between). As used herein,
"substantially all bioburden" means tissue fillers in which the
concentration of microorganisms growing on the filler is less than
1%, 0.1%, 0.01%, 0.001%, or 0.0001% of that growing on untreated
fillers.
[0054] The tissue fillers, as described above, can be used after
surgical removal of native tissue and/or to augment existing native
tissues. In certain embodiments, the method of use comprises
removing a native tissue and implanting a tissue filler. As used
herein, the "native tissue" being removed can be a portion,
fragment, or entirety of a tissue found in a body. The tissue
filler can comprise an acellular tissue matrix and at least one
substance capable of reducing inflammation and/or fibrosis. In
certain embodiments, the substance is exogenous HA and/or exogenous
decorin at a concentration sufficient to reduce an inflammatory
response when the tissue filler is implanted in the body. In
various embodiments, the tissue filler used after removal of a
native tissue comprises the tissue filler described above, or is
prepared as described below. In some embodiments, the tissue being
removed comprises a tumor. In certain embodiments, the tissue is
breast tissue.
[0055] In various embodiments, the tissue fillers described above
are used after removal of a native soft tissue because they have a
sponge-like consistency and can swell to fill the region of tissue
that has been removed. In addition, the tissue fillers can retain
the structural strength, texture, and/or feel of native soft
tissue. Thus, the tissue fillers can preserve the shape of the
excised natural tissue. For instance, tissue fillers derived from
lung or liver can be compressed by up to approximately 2/3 of their
length or width before returning to their original shape after
release of the compressing force, thereby simulating the texture
and elasticity of the native soft tissue that has been removed.
Furthermore, in certain embodiments, tissue fillers containing
anti-inflammatory and/or anti-fibrotic substances can reduce
inflammation and/or scar tissue formation following implantation
into a recipient organ. In various embodiments, the
anti-inflammatory and/or anti-fibrotic substances are HA and/or
decorin.
[0056] In certain embodiments, tissue fillers produced from lung,
liver, bladder, muscle, or fat tissue are implanted after removal
of native tissue. In some embodiments, these tissue fillers are
rich in elastin and collagen, as well as glycosaminoglycans and
growth factors. The tissue fillers thus provide natural tissue
scaffolds that approximate the structure, texture, and cellular
growth conditions of the tissue that has been removed. In further
embodiments, porcine lung or liver tissue is used because its
extracellular matrix has a well-organized sponge structure that
approximates the texture of a native soft tissue.
[0057] In various embodiments, tissue fillers produced from lung,
liver, bladder, muscle, or fat tissue do not elicit a significant
inflammatory response when implanted in a tissue, as compared to
implants derived from other tissues or from non-biologic sources.
For example, tissue fillers produced from lung or liver tissue do
not induce significant increases in cytokine secretion after
implantation when compared tissue fillers produced from other
tissue sources, such as dermis. Suitable cytokines to measure in
evaluating the inflammatory response include IL-1, IL-6, IL-8, or
IL-10. Similarly, any other indicator of the inflammatory response
known to one of skill can be used to measure the inflammatory
response. The inflammatory response can be assayed by various
techniques, including in vitro incubation of tissue fillers with
mononuclear blood cells or in vivo implantation of tissue fillers
into a host tissue. Either method may involve cytokine immune
staining or direct cytokine quantification. Other suitable
techniques for assaying the inflammatory response are known in the
art and may be used.
[0058] To further reduce inflammation after implantation into a
recipient tissue, tissue fillers can be coated with or otherwise
contain substances that reduce inflammation. As used herein, a
"coated" tissue filler is one that has been contacted with an
anti-inflammatory reagent or a solution containing the
anti-inflammatory reagent on one or more surfaces of the tissue
filler or within the tissue filler. For example, tissue fillers can
be coated in decorin and/or hyaluronic acid (HA). In some
embodiments, tissue fillers containing decorin and/or HA reduce
inflammation after implantation in a tissue, as compared to an
implanted tissue filler lacking HA and/or decorin. In further
embodiments, coating a tissue filler in decorin and/or HA reduces
inflammatory T cell, B cell, and/or macrophage infiltration into
the tissue by 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% (or any
value in between) after implantation into a recipient organ, as
compared to infiltration after an uncoated tissue filler is
implanted. In further embodiments, the decorin and/or HA coating
remains on the tissue filler for the duration of the implant. In
still further embodiments, the coating of decorin and/or HA remains
on the tissue filler 5 days, 10 days, 15 days, 20 days, or 25 days,
1 month, or 2 months (or any value in between) after implantation
in a tissue.
[0059] A major challenge after removal of native tissue is the
formation of undesirable scar tissue. After excision of a large
volume of tissue, dense fibrosis will form due to the cross-linking
of collagen during wound healing. Preventing or reducing the amount
of scar tissue that forms after bulk tissue removal is therefore
desirable in order to preserve the appearance and texture of the
tissue. Thus, in various embodiments, tissue fillers are first
coated in anti-fibrosis reagents before being implanted in a
tissue, thereby reducing the amount of scar tissue formed after
implantation. As used herein, a "coated" tissue filler is one that
has been contacted with an anti-fibrosis reagent or a solution
containing the anti-fibrosis reagent on one or more surfaces of the
tissue filler or within the tissue filler.
[0060] In certain embodiments, the anti-fibrosis reagent is HA or
decorin or a combination of the two. For example, decorin can
stabilize the structure of native cell colonies growing in the
tissue filler while preventing the infiltration of excessive
fibrous connective tissue. HA and/or decorin can also reduce the
inflammation caused by implantation of a tissue filler, thereby
further reducing the amount of scar tissue formation. In further
embodiments, coating a tissue filler in decorin and/or HA reduces
fibrosis by 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% (or any value
in between) after implantation into a recipient organ, as compared
to fibrosis after an uncoated tissue filler is implanted. In
further embodiments, the decorin and/or HA coating remains on the
tissue filler for the duration of the implant. In still further
embodiments, the coating of decorin and/or HA remains on the tissue
filler 5 days, 10 days, 15 days, 20 days, or 25 days, 1 month, or 2
months (or any value in between) after implantation in a
tissue.
[0061] In certain embodiments, tissue fillers produced from lung or
liver tissue are more resistant to collagenase digestion after
implantation in a tissue when compared to tissue fillers produced
from dermal tissue. Collagenases are enzymes which break the
peptide bonds in collagen and can therefore degrade the
extracellular matrix structures of implanted tissue fillers. Thus
in some embodiments, tissue fillers produced from lung or liver
tissue provide natural tissue scaffolds that retain their
structural integrity and ability to promote native cell
repopulation for longer periods of time after implantation when
compared to tissue fillers derived from other tissue sources, such
as dermal tissue.
[0062] In certain embodiments, tissue fillers retain their shape
and structural integrity after implantation for one week, two
weeks, three weeks, 1 month or 2 months (or any time period in
between). In other embodiments, the tissue fillers retain their
structural integrity for the duration of therapeutic use.
[0063] The ability of tissue fillers derived from tissue sources
such as lung, liver, bladder, muscle, or fat to hold and retain
desired shapes (due to their spongy extracellular matrix structure)
allows for the use of tissue fillers after removal of large tissue
defects (e.g., large tumor removals). For example, the removal of
more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (or any
percentage in-between) of a tissue mass creates a large void that
requires the use of a filler to replace the lost tissue and to
provide structural integrity for the remaining tissue or organ.
Existing filler substances are generally used only to fill in small
areas (e.g., as cosmetic fillers to remove skin wrinkles) and also
lack the durability and biocompatibility needed after removal of a
large volume of native tissue. In certain embodiments, a tissue
filler as described above is implanted after removal of more than
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (or any percentage
in-between) of native tissue. The spongy extracellular matrix of
the tissue fillers allows them to fill the void left by the removed
tissue, providing a biocompatible and durable scaffold that
persists for a sufficient length of time to enable the migration
and proliferation of native tissue and vasculature into the
scaffold. In some embodiments, the tissue being removed and
replaced with tissue filler is breast tissue. In other embodiments,
the tissue being removed is muscle. In still further embodiments,
the tissue being removed is liver tissue.
[0064] Tissue fillers that can provide structural support are
useful to prevent visible detection of a tissue removal, i.e. for
aesthetic purposes. Similarly, a tissue filler having a consistency
similar to that of a removed soft tissue allows the tissue filler
to provide the texture and feel the tissue had before surgical
removal. For example, bulk tissue removal in breast cancer results
in a disfigured breast. Implanting a tissue filler having the
structural strength and elasticity of natural breast tissue can
assist in breast reconstruction after surgery. Thus, in some
embodiments, tissue fillers are used as implants after removal of a
native soft tissue in order to retain the appearance and feel the
tissue had prior to soft tissue removal. In further embodiments,
the tissue removed is a tumor. In further embodiments, the tumor is
a breast tumor.
[0065] In other embodiments, tissue fillers are implanted after
loss or removal of large amounts of muscle tissue, for example due
to muscle wasting or tumor growth. In other embodiments, tissue
fillers are used as natural tissue scaffolds for liver repair or
regeneration after liver resection.
[0066] In various embodiments, implanting a tissue filler can
promote the repair or regeneration of a tissue after bulk tissue
removal. Tissue repair or regeneration can include, for example,
the infiltration of native cells from the surrounding tissue into
the natural tissue scaffold of the tissue filler. Other examples of
tissue repair and regeneration include the growth and proliferation
of native cells in the natural tissue scaffold. In some
embodiments, the tissue filler is able to support at least 50%,
60%, 70%, 80%, 90%, 95% or 99% of the cell proliferation supported
by the native tissue. Still further examples of tissue repair and
regeneration include revascularization of the tissue in the region
where native tissue has been removed. In further embodiments, the
preservation of growth factors, including FGF, VEGF, PDGF,
angiopoitin-2 or follistatin (among others), in tissue fillers can
enhance angiogenesis or revascularization at the implant site. In
certain embodiments, implanting a tissue filler derived from lung,
liver, bladder, muscle, or fat leads to fibroblast cell
infiltration, growth, and/or proliferation. In further embodiments,
implanting a tissue filler derived from lung, liver, bladder,
muscle, or fat leads to neo-vessel formation.
[0067] In certain embodiments, tissue fillers as disclosed herein
can also be used outside the reconstruction context. For example,
tissue fillers can be used as implants for aesthetic enhancement
purposes. Tissue fillers can be implanted into a native tissue to
enhance the shape, look, or feel of a native tissue. The aesthetic
tissue targets can include breast, lip, cheek, and buttocks
implants, among others. In certain embodiments, implanting tissue
fillers for aesthetic purposes can also lead to native tissue cell
infiltration, growth, proliferation, or vascularization of the
implanted tissue filler.
Production of Tissue fillers
[0068] Tissue fillers can be produced by processing tissue from
various animal sources. In certain embodiments, the tissue is taken
from human cadaver, cow, horse, or pig. In some embodiments, the
tissue is lung, liver, bladder, muscle, or fat tissue. In further
embodiments, the tissue is porcine lung or liver tissue. In some
embodiments, an entire organ is used to prepare a tissue filler. In
other embodiments, portions of the organ are processed into tissue
fillers. In further embodiments, the organ portions may include
strips, balls, or other tissue fragments that provide the desired
size, shape, or structural features necessary for a given tissue
filler.
[0069] Tissue can be subjected to multiple rounds of freeze/thaw to
disrupt the tissue and improve the decellularization process. In
addition, bronchi or large blood vessels can be removed from tissue
by manual dissection, and the tissue can be washed to remove blood
cells. Any suitable washing solution can be used, including
distilled water, HEPES buffer, or phosphate buffered saline, among
others.
[0070] Next, the tissue is decellularized in order to remove cells
from the remaining natural tissue scaffold. Various detergents can
be used to decellularize, including sodium dodecyl sulfate, sodium
deoxycholate, and Triton X-100. Other examples of suitable
decellularization detergents and decellularization procedures are
described in Cortiella et al, Tissue Engineering 16: 2565-2580
(2010), hereby incorporated in its entirety.
[0071] The concentration of detergent used to decellularize can be
adjusted in order to preserve desirable matrix proteins and prevent
protein damage during the decellularization process. The detergent
may be used, for example, at a concentration of approximately 0.1%,
0.2%, 0.5%, 1%, 2%, 3%, 4%, or 5% and tissue can be incubated with
detergent for 2 hours, 3 hours, 5 hours, 10 hours, 12 hours, 24
hours, 2 days, 3 days, 5 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5
weeks, or 6 weeks (or any concentration or time period in between).
Appropriate concentrations and duration of detergent incubation can
be adjusted depending on the detergents used and the desired
harshness of decellularization.
[0072] In certain embodiments, the concentration of detergents
and/or detergent incubation times are reduced in order to retain a
higher level of growth factors in the extracellular matrix after
decellularization. For example, a less harsh decellularization
process may lead to the retention of increased levels of FGF, VEGF,
PDGF, angiopoitin-2, and follistatin. In another example, the
concentration of detergents and/or incubation times are increased
in order to more completely remove cells from the extracellular
matrix. Further, decellularization can be performed so as to remove
substantially all viable cells from the extracellular matrix. As
used herein, "substantially all viable cells" means tissue fillers
in which the concentration of viable cells is less than 1%, 0.1%,
0.01%, 0.001%, or 0.0001% of the cells found in the tissue or organ
from which the tissue filler is made.
[0073] In certain embodiments, the major growth factors present in
unprocessed tissue are preserved by the decellularization process.
For example, FGF, VEGF, PDGF, angiopoitin-2 and/or follistatin can
be preserved in the decellularized tissue. In some embodiments, 10,
20, 30, 40, 50, or 60 ng of FGF are present after decellularization
per gram of dried tissue filler (or any value in between). In some
embodiments, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0
ng of VEGF are present after decellularization per gram of dried
tissue filler (or any value in between). In some embodiments, 0.5,
1.0, 1.5, or 2.0 ng of PDGF are present after decellularization per
gram of dried tissue filler (or any value in between). In some
embodiments, 0.05, 0.1, or 0.2 ng of angiopoitin-2 are present
after decellularization per gram of dried tissue filler (or any
value in between) when the tissue filler is made from processed
lung tissue. In some embodiments, 0.5, 1.0, or 1.5 ng of
follistatin are present after decellularization per gram of dried
tissue filler (or any value in between).
[0074] In various embodiments, the decellularization process does
not alter the extracellular matrix of the harvested tissue. For
example, the extracellular matrix in decellularized tissue can
remain substantially unaltered when compared to non-decellularized
tissue. The extracellular matrix can consist of collagen, elastin,
fibronectin, and proteoglycans, among other extracellular proteins.
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, as described below.
[0075] In some embodiments, after decellularization, tissue is
treated with DNase to remove cellular DNA. In further embodiments,
approximately 10, 20, 30, 40, or 50 units/ml of DNase are used (or
any value in between). In certain embodiments, RNase is added to
the DNase solution. In further embodiments, approximately 10, 20,
30, 40, or 50 units/ml of RNase are used (or any value in between).
In some embodiments, at least one antibiotic is added to the DNase
and/or RNase solution that is applied to decellularized tissue.
Appropriate antibiotics may include, for example, gentamicin,
streptomycin, penicillin, and amphotericin. In further embodiments,
the antibiotic is added at a concentration of approximately 20, 30,
40, 50, 60, 70, 80, 90, or 100 .mu.g/ml (or any value in between).
In various embodiments, treatment of decellularized tissue with
DNase, RNase and/or antibiotics can be for 1 hour, 2 hours, 5
hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, or 5 days (or
any time period in between). Appropriate duration of application
and effective concentrations will depend on the type of tissue and
on the DNase, RNase, and/or antibiotics selected to process the
tissue.
[0076] In certain embodiments, the tissue filler is coated in at
least one substance that can reduce inflammation and/or fibrosis
after implantation into a recipient tissue. As used herein,
"coating" a tissue filler in at least one substance means
contacting one or more surfaces of the tissue filler, or an
internal portion of the tissue filler, with the at least one
substance, or a solution containing the substance. In further
embodiments, the at least one substance is HA and/or decorin. For
example, tissue fillers can be incubated in a solution containing
approximately 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml,
7 mg/ml, 8 mg/ml, 9 mg/ml, or 10 mg/ml of hyaluronic acid (or any
value in between). Incubation can be for, e.g., 1, 2, 3, 4, 5, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 36, or
48 hours (or any time period in between.)
[0077] After incubation, hyaluronic acid-coated tissue fillers are
washed for, e.g., 1, 6, 12, 15, 20, 24, 36, or 48 hours (or any
time period in between). After incubation and washing, tissue
fillers are coated in hyaluronic acid at a concentration of, e.g.,
0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mg of
hyaluronic acid per gram of tissue filler (or any value in
between). In another example, tissue fillers can be incubated in a
solution containing approximately 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml,
0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml,
or 1.0 mg/ml, 1.5 mg/ml, 2.0 mg/ml, 2.5 mg/ml, or 3.0 mg/ml of
decorin (or any value in between). Incubation can be for, e.g., 1,
2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 30, 36, or 48 hours (or any time period in between). After
incubation, decorin-coated tissue fillers are washed for, e.g., 1,
6, 12, 15, 20, 24, 36, or 48 hours (or any time period in between).
After incubation and washing, tissue fillers are coated in decorin
at a concentration of, e.g., 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, or
0.6 mg of decorin per gram of tissue filler (or any value in
between.)
[0078] In further embodiments, tissue fillers are treated with
alpha-galactosidase to remove alpha-galactose (.alpha.-gal)
moieties. In some embodiments, to enzymatically remove .alpha.-gal
epitopes, after washing the tissue thoroughly with saline, the
tissue may be subjected to one or more enzymatic treatments to
remove .alpha.-gal antigens, if present in the sample. In some
embodiments, the tissue may be treated with an
.alpha.-galactosidase enzyme to eliminate .alpha.-gal epitopes. In
further embodiments, the tissue is treated with
.alpha.-galactosidase at a concentration of 0.2 U/ml prepared in
100 mM phosphate buffered saline at pH 6.0. In other embodiments,
the concentration of .alpha.-galactosidase is reduced to 0.1 U/ml
or increased to 0.3 or 0.4 U/ml (or any value in between.) In other
embodiments, any suitable enzyme concentration and buffer can be
used as long as sufficient antigen removal is achieved. In
addition, certain exemplary methods of processing tissues to reduce
or remove alpha-1,3-galactose moieties are described in Xu et al.,
Tissue Engineering, Vol. 15, 1-13 (2009), which is incorporated by
reference in its entirety.
[0079] Alternatively, in certain embodiments animals that have been
genetically modified to lack one or more antigenic epitopes may be
selected as the tissue source for a tissue filler. For example,
animals (e.g., pigs) that have been genetically engineered to lack
the terminal .alpha.-galactose moiety can be selected as the tissue
source. For descriptions of appropriate animals, see U.S.
application Ser. No. 10/896,594 and U.S. Pat. No. 6,166,288, which
are incorporated herein by reference in their entirety.
[0080] In various embodiments, tissue fillers are processed to
reduce bioburden (i.e., to reduce the number of microorganisms
growing on the tissue filler). In some embodiments, tissue fillers
are processed to remove substantially all bioburden (i.e., to
sterilize the tissue filler). Suitable bioburden reduction methods
are known to one of skill in the art, and may include irradiation.
Irradiation may reduce or substantially eliminate bioburden. In
further embodiments, 15-17 kGy E-beam radiation is used. In other
embodiments, tissue fillers are coated in chlorhexidine (CHX) or
silver to reduce or prevent bioburden. As used herein, "coating" a
tissue filler in CHX or silver means contacting one or more
surfaces of the tissue filler, or an internal portion of the
filler, with CHX or silver or a solution containing CHX or silver.
In certain embodiments, the concentration of CHX or silver is
adjusted to remove substantially all bioburden. As used herein,
"substantially all bioburden" means tissue fillers in which the
concentration of microorganisms growing on the filler is less than
1%, 0.1%, 0.01%, 0.001%, or 0.0001% of that growing on untreated
fillers. Effective concentrations of CHX may include 0.1 mg, 0.5
mg, 0.7 mg, 0.9 mg, 1 mg, 1.5 mg, 2.0 mg, 2.5 mg, or 3.0 mg per
gram of tissue filler (or any value in between). In some
embodiments, the tissue filler may be coated with a solution
containing silver at any concentration between 10 .mu.g/ml to 500
.mu.g/ml of solution. Effective concentrations of silver may
include 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8
mg, 0.9 mg, or 1.0 mg per gram of tissue filler (or any value in
between).
[0081] In various embodiments, processed tissue fillers are cut or
molded into desired shapes. Shapes may be selected to conform to
the contours of the tissue into which the filler will be implanted,
or to conform to the contours of the void left by removal of native
tissue. For example, tissue fillers may be cut into strips or
molded into balls. Optimal shapes will be familiar to one of skill
in the art and will depend on the specific tissue into which a
filler is being implanted and on the size and/or shape of the bulk
tissue that has been removed. For example, the desired shape of the
tissue filler may depend on the shape and size of a tumor that has
been removed from a patient.
[0082] In various embodiments, tissue fillers are cryopreserved for
storage by freeze-drying. In one example, the tissue filler is
placed in a cryopreservation solution that includes an organic
solvent or water, to protect against damage during freeze drying.
In further embodiments, following incubation in the
cryopreservation solution, the tissue filler is placed in a sterile
vessel that is permeable to water vapor but impermeable to
bacteria. The vessel is cooled to a low temperature at a specified
rate that is compatible with the specific cryoprotectant
formulation to minimize the freezing damage. The tissue filler is
then dried at a low temperature under vacuum conditions. At the
completion of the drying, the vacuum of the freeze drying apparatus
is reversed with a dry inert gas such as nitrogen, helium or argon.
The tissue filler is then sealed in an impervious container and
stored until use. While the example above describes one method for
cryopreservation, one of skill in the art will recognize that other
such methods are well known in the art and may be used to
cryopreserve and store tissue fillers.
[0083] In some embodiments, a tissue filler that has been freeze
dried is rehydrated prior to implantation in a tissue. In further
embodiments, the rehydrated tissue filler retains the spongy
character and also substantially the same stretchability and
compressibility as found in tissue filler that has not been
freeze-dried. In certain embodiments, rehydrated tissue filler
derived from decellularized porcine lung retains substantially the
same shape and spongy character as found in tissue filler that has
not been freeze-dried (see FIG. 4).
[0084] The following examples serve to illustrate, and in no way
limit, the present disclosure.
EXAMPLES
Example 1
Decellularization of Porcine Lung and Liver
[0085] To obtain lung or liver scaffolds, tissue was harvested from
3-6 month-old pigs in the slaughterhouse and shipped immediately to
the laboratory for processing. Porcine tissue was freeze-thawed
twice at -80.degree. C. and then washed with distilled water for 2
days. The bronchi or large blood vessels were removed by manual
dissection. Porcine lung and liver tissues were decellularized at
room temperature (22 to 25.degree. C.) for 5 days in a 10 mM HEPES
buffer solution (pH 8.0) containing 2% sodium deoxycholate, 0.1%
(w/v) Triton x-100 and 10 mM EDTA. Bottles were gently agitated on
a shaker. Decellularized organ matrices were washed with 0.9%
saline to remove detergent until foam was no longer observed in
solution. Tissues were then treated at room temperature (22 to
25.degree. C.) for 24 hours in a second HEPES buffer solution (10
mM, pH 7.4) containing 30 units/ml DNase, 50 .mu.g/ml gentamicin,
20 mM calcium chloride and 20 mM magnesium chloride. The DNase
solution was discarded, and tissue was washed three times with 0.9%
saline (30 min per wash). Decellularized tissues were further
treated in phosphate-buffered saline (pH 6.5) containing 0.2
unit/ml .alpha.-galactosidase and 50 mM EDTA. Tissues were
sterilized with 0.1% PAA and 15-17 kGy E-beam. Tissues were stored
in hydrated form or freeze-dried.
Example 2
Evaluation of Acellular Porcine Organ Matrices
[0086] To confirm the removal of cellular components,
decellularized tissues were digested with proteinase K and DNA
content was measured by Quant-iT PicoGreen dsDNA Kit (Molecular
Probes, Inc.), following the manufacturer's instructions.
Decellularized tissues were also processed for histology (H&E,
Verhoeff's, Alcian blue) and evaluated using a scanning electron
micrograph (SEM). Histological evaluation (H&E stain)
demonstrated that the decellularization process completely
decellularized porcine organs while preserving a well-organized
collagen network. FIGS. 1A & 1B show that the well-organized
collagen network in porcine lung was preserved by the
decellularization process. FIG. 1C shows that processed porcine
lung contains abundant elastin (Verhoeff's staining) and FIG. 1D
shows the well-organized sponge ultrastructure of the
decellularized lung (SEM).
[0087] To further characterize the organ matrices, extracellular
matrix molecules (ECM) were measured using anti-collagen type I,
Ill, IV, fibronectin and laminin antibodies. FIGS. 2A, 2B & 2C
show that decellularized porcine lung tissue contains some type I
collagen and abundant type IV collagen and fibronectin. FIG. 2D
shows that type III collagen, as well as laminin (data not shown),
is not a major component of the acellular lung matrix. Evaluation
of fresh porcine lung tissue with the above antibodies demonstrated
similar staining patterns, suggesting that the tissue processing
method preserved the structure of native lung tissue matrices.
Example 3
Bioburden Quantification
TABLE-US-00001 [0088] TABLE 1 Samples CFU (Processed) CFU
(Sterilized) Porcine Liver 2.85 .times. 10.sup.4 0 Porcine Lung
>10.sup.6 0
[0089] To ensure matrix sterility, the bioburden of tissue matrices
was measured before and after a sterilization step. Table 1 shows
the Bioburden quantification of acellular porcine liver and lung
matrices before and after sterilization. The bioburden was
extracted from tissue matrices in 50 ml saline solution followed by
stomacher 400 circulator at 150 rpm for 2 minutes and collection on
a 0.45 micron filter. The filter was placed on solid growth media
and incubated at 37.degree. C. for 3 days to allow for microbial
colony formation. The microbial colonies were then counted to
quantify the microorganisms in terms of colony forming units (CFU).
Table 1 shows that no bioburden was detected after
sterilization.
[0090] Acellular lung and liver matrices were also coated with the
antimicrobial reagent Chlorhexidine (CHX) or silver. CHX is an
antimicrobial used in many consumer products, such as mouthwash and
contact lens solutions. The concentration of CHX coated on the
acellular lung and liver matrices was determined by high
performance liquid chromatography (HPLC) to be 1.0 mg per gram of
tissue. The concentration of silver coated on the acellular
matrices was determined by ICP to be 0.22 mg per gram of tissue.
Tissue matrices coated with CHX were measured for bioburden. Table
2 shows that the CHX coating efficiently reduced bioburden.
TABLE-US-00002 TABLE 2 Samples CFU (Processed) CFU (Sterilized)
Porcine Liver 2.85 .times. 10.sup.4 0 Porcine Lung >10.sup.6
0
Example 4
Mechanical Property of Porcine Lung and Liver
[0091] The softness of acellular porcine lung or liver tissues was
measured using a durometer (Table 3). A durometer measures the
indentation resistance of elastomeric or soft materials based on
the depth of penetration of a conical indenter. Hardness values
range from 0 to 100. A lower number indicates a softer material,
whereas a higher number indicates that the material is harder. The
data from the durometer shows that porcine lung and liver have
similar softness to human breast tissue.
TABLE-US-00003 TABLE 3 Porcine Porcine Porcine Porcine Human breast
muscle liver lung dermis (neck) tissue 29.4 .+-. 4.9 2.33 .+-. 0.92
5.63 .+-. 0.91 40.0 .+-. 8.6 5* (N = 4 ) (n = 7) (n = 7) (N = 24)
*Reference
Example 5
Tissue Integrity Testing
[0092] To measure the retention of matrix integrity in processed
lung and liver tissue, glycosaminoglycan concentrations were
analyzed by a Sulfated Glycosaminoglycan Assay (Biocolor Ltd.)
following the manufacturer's instructions (FIG. 5). Porcine
acellular liver and lung matrices contain abundant GAG when
compared to porcine acellular dermal tissue (PADM).
[0093] The effect of tissue processing on collagen stability was
measured by modulated temperature differential scanning calorimetry
(TA Instruments, New Castle, DE), as previously described. Gouk et
al., J. Biomed. Mat. Res. Part B: Appl. Biomat. 84B: 205-217
(2007). The onset temperature (T.sub.m) and enthalpy (.DELTA.H) of
collagen denaturation were determined by analysis of thermograms
using Universal Analysis 2000 software (version 4.0) with dry
tissue samples (FIG. 6). Thermograms of both porcine lung and liver
acellular tissue matrices had unfolding onset temperatures for
collagen molecules around 59-60.degree. C., and peak unfolding
temperature around 62-64.degree. C. The data demonstrate that the
integrity of native lung and liver matrices is preserved after
processing.
[0094] The susceptibility of collagen in lung and liver matrices to
enzyme digestion was evaluated in vitro by type I collagenase
(Sigma-Aldrich, St. Louis, Mo.) and proteinase K (Fisher
Scientific, Fair Lawn, N.J.) following the protocol described
previously (Gouk, 2007). Briefly, samples were incubated with
collagenase for varying lengths of time at 37.degree. C. The
digested tissues were then washed and freeze-dried. The resistance
to enzyme digestion was calculated as a percentage of the dry
weight of tissue remaining at various time points (FIG. 7). FIG. 7
suggests that liver and lung acellular matrices are more resistant
to collagenase digestion when compared to porcine dermal
tissue.
Example 6
Anti-Fibrosis Coatings on Acellular Lung and Liver Matrices
[0095] To reduce the possibility of fibrosis tissue formation,
acellular tissue matrices were coated with anti-fibrosis reagents,
such as hyaluronan or recombinant human decorin. For hyaluronan
coating, tissue matrices were incubated with 5 mg/ml of hyaluronic
acid (HA) sodium salt (Fluka 53747) at room temperature for 16
hours. Binding of HA to tissue matrices was confirmed by Alcian
blue staining (FIG. 8). FIG. 8 shows that lung matrices were
successfully coated with HA. The concentration of HA coated on the
tissue matrices was determined by a DMMB colorimetric assay.
Briefly, the HA coated tissue matrices were digested with
collagenase and the supernatants were quantified to determine the
GAG concentration using dimethylmethylene blue (DMMB) staining.
FIG. 9 shows that HA was effectively coated onto the tissues and
persisted after washing overnight with 3 changes.
[0096] For decorin coating, tissue matrices were incubated with 1
mg/ml of recombinant human decorin (DCN) at room temperature for 16
hours and then washed overnight to remove unbound decorin. Binding
of human decorin to the tissue matrices was confirmed by an
anti-human decorin antibody that does not react with porcine
decorin (FIG. 10). The decorin concentration coated on the tissue
was determined with a RayBio Human Decorin ELISA kit (Ray Biotech
Inc). FIG. 11 shows that human decorin was effectively coated onto
liver matrices and persisted after washing overnight with 3
changes.
Example 7
In Vitro Cell Growth and Inflammation
[0097] Both rat fibroblast cells (ATCC, CRL-1213) and human
fibroblast cells (ATCC, CRL-2522) were cultured in minimal
essential medium (MEM) supplemented with 10% fetal bovine serum
(ATCC, MD). Human monocytes were cultured in macrophage-serum-free
medium (SFM) (Gibco, Calif.). Rat bone marrow or adipose
mesenchymal stem cells (MSC) were cultured in MSC expansion medium
(Millipore, Mass.). Acellular lung and liver tissues were washed in
saline (supplemented with 50 .mu.g/ml gentamicin) for 12 hours with
shaking at room temperature (6 changes) and then placed at the
bottoms of the wells in 24-well plates (0.5.times.0.5 cm of tissue
per well). One milliliter of cells (5-10.times.10.sup.4cells/ml)
was applied to the tissues. Co-cultures of tissues with cells were
then fixed and stained for H&E at 1, 2 and 3 weeks of culture.
FIG. 12 shows that porcine lung and liver matrices support growth,
migration and proliferation of rat MSC and fibroblast cells.
[0098] Fresh human mononuclear cells were isolated from human donor
blood by Ficoll separation. One million cells in 1 ml
macrophage-SFM (Gibco, Caif.) were applied to acellular tissue
matrices (0.5.times.0.5 cm) placed at the bottoms of the wells in
24-well plates. The culture supernatant was collected 7 days post
co-culture and centrifuged. The supernatant was analyzed for
cytokines (IL-1, -2, -4, -6, -8, -10, IFN-g and TNF-a) with an
Elisa kit for human cytokines. FIG. 13 shows that porcine liver and
lung matrices did not induce significant inflammatory cytokines
when the matrices were cultured with human blood mononuclear
cells.
Example 8
Growth Factors in Processed Lung or Liver Tissue Matrices
[0099] Growth factors retained in processed lung and liver tissue
matrices were determined using a Bio-Plex Pro Assay (BioRad).
Briefly, processed tissue was washed with saline and then
freeze-dried and cryo-milled. Cryo-milled tissue (100 mg) was
extracted in 1 ml tissue extraction reagent I (invitrogen) at
4.degree. C. overnight. The supernatant was used for Bio-Plex Pro
Assays (Human Angiogenesis Panel). The data shows that the
decellularizing and processing of lung and liver tissues preserve
the key growth factors (FGF, VEGF, PDGF) found in porcine organ
matrices (table 4).
TABLE-US-00004 TABLE 4 ng/g dried tissue FGF VEGF PDGF
Angiopoitin-2 Follistatin Processed 18.43 2.04 0.72 undetectable
0.72 Liver Processed 42.7 8.44 1.89 0.11 0.58 Lung
Example 9
In Vivo Testing
[0100] To assess the biological response to processed tissue
matrices in vivo, processed porcine lung and liver tissue matrices
(1.0.times.1.0.times.0.5 cm) were subcutaneously implanted in rats
for 14 days. Processed acellular matrices were compared to matrices
coated with an anti-microbial agent, HA, or human decorin. The
explants were grossly evaluated for their shape, hardness, and size
(Table 5 & FIG. 14). After two weeks, implanted porcine lung
matrices had the same shape, size and sponge property as observed
prior to implantation. CHX-coated liver tissue matrices were firm
and seemed encapsulated, while HA or decorin-coated liver tissue
matrices were soft with less surrounding connective tissues.
TABLE-US-00005 TABLE 5 Gross observation of the explants Explants
Shape change Hardness Size Liver matrix No Slightly firm 1 .times.
1 .times. 0.6 cm Liver-CHX No Firm 1.5 .times. 1 .times. 0.8 cm
Liver-HA No Soft 1 .times. 1 .times. 0.8 cm Liver-Decorin No Soft 1
.times. 1 .times. 0.8 cm
[0101] The processed porcine lung and liver tissue matrix explants
were also evaluated histologically for cell infiltration,
inflammation and encapsulation. Histology demonstrated cellular
infiltration in all grafts. Matrices coated with CHX appeared to be
encapsulated and showed less fibroblast cell repopulation (FIG.
15). Matrices coated with HA and decorin had minimal inflammation
(FIG. 16). This data suggests that coating matrices in HA and human
decorin reduces inflammation and encapsulation. Histology also
demonstrated cellular repopulation and revascularization in the
implanted matrices. Human decorin-coated grafts appeared to have
fewer fibroblast cells than did the uncoated or HA-coated grafts,
indicating regulatory effects of decorin on fibroblast
proliferation (FIG. 17).
[0102] To assess the host biological response to the implants, two
week explants of acellular liver matrix grafts were immunostained
with anti-vimentin to detect fibroblasts, anti-vWF to detect
neo-vessel formation, and anti-alpha-smooth muscle cell actin to
detect myofibroblast cells (myofibroblast cells have been reported
to be involve in fibrosis formation). Fibroblast cell repopulation
was confirmed by immunostaining and neo-vessel formation was shown
by vWF staining (FIGS. 18A & B). SMC-.alpha.-Actin staining did
not suggest induction of myofibroblast cells (FIG. 18C). The grafts
coated with human decorin appeared to have fewer fibroblast cells
when compared to non-coated or HA-coated grafts (FIG. 19).
[0103] To further characterize the inflammatory cells in the liver
and lung grafts, T cells, B cells and Macrophages at the graft site
were stained using antibodies. The level of inflammation was scored
as follows: 0=none, 1=mild, 2 moderate, 3=significant. The data in
Table 6 indicate that the porcine acellular lung and liver matrices
do not induce significant inflammation in a rat subcutaneous
implantation model.
TABLE-US-00006 TABLE 6 T cells B cells Macrophages Lung 1 1 1
Lung-HA 0.5 0.5 0.5 Lung-decorin 0.5 0.5 0.5 Liver 1 1 1 Liver-HA
0.5 0.5 0.5 Liver-decorin 0.5 0.5 0.5
[0104] To assess if coated decorin remained on acellular matrices
over time, acellular liver matrices coated with decorin were
stained with anti-human decorin antibody two weeks after rat
subcutaneous implantation (FIG. 20). The results suggest that
coated human decorin was present on the implant 2-weeks
post-implantation. 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.
* * * * *