U.S. patent application number 16/108581 was filed with the patent office on 2018-12-13 for regenerative materials.
The applicant listed for this patent is LifeCell Corporation. Invention is credited to Melissa Richter Bowley, Goel Raghav, Wenquan Sun.
Application Number | 20180353644 16/108581 |
Document ID | / |
Family ID | 46001826 |
Filed Date | 2018-12-13 |
United States Patent
Application |
20180353644 |
Kind Code |
A1 |
Sun; Wenquan ; et
al. |
December 13, 2018 |
REGENERATIVE MATERIALS
Abstract
Methods of making tissue fillers are provided. In certain
embodiments, the tissue is flake-like and has regenerative
properties.
Inventors: |
Sun; Wenquan; (Warrington,
PA) ; Bowley; Melissa Richter; (Doylestown, PA)
; Raghav; Goel; (Plymouth, MN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LifeCell Corporation |
Madison |
NJ |
US |
|
|
Family ID: |
46001826 |
Appl. No.: |
16/108581 |
Filed: |
August 22, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15166848 |
May 27, 2016 |
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16108581 |
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13446422 |
Apr 13, 2012 |
9375513 |
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15166848 |
|
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61475378 |
Apr 14, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 2/087 20130101;
A61L 27/26 20130101; A61P 31/04 20180101; A61L 2/206 20130101; A61K
35/545 20130101; A61L 27/56 20130101; A61L 2/081 20130101; A61L
27/3691 20130101; A61L 27/362 20130101; A61L 27/3834 20130101; A61L
27/20 20130101; A61F 2/02 20130101; C08L 5/02 20130101; A61L 27/54
20130101; A61L 27/24 20130101; A61L 2430/34 20130101; A61L 27/20
20130101; A61K 38/18 20130101; A61K 35/35 20130101; A61L 27/3687
20130101; A61K 38/19 20130101 |
International
Class: |
A61L 27/24 20060101
A61L027/24; A61K 35/545 20060101 A61K035/545; A61K 38/19 20060101
A61K038/19; A61K 38/18 20060101 A61K038/18; A61K 35/35 20060101
A61K035/35; A61L 27/36 20060101 A61L027/36; A61L 27/54 20060101
A61L027/54; A61L 27/38 20060101 A61L027/38; A61L 27/26 20060101
A61L027/26; A61L 27/20 20060101 A61L027/20 |
Claims
1. A method of treatment, comprising: selecting an anatomical site;
and implanting a tissue filler into the anatomical site, the tissue
filler comprising a group of small, irregularly sized and shaped
tissue matrix flakes having a size distribution ranging from 0.2-5
mm in length, 0.2-3 mm in width, and 0.02-0.3 mm in thickness.
2. The method of claim 1, wherein the group of tissue matrix flakes
comprise fragments that have been cut from a collagen-containing
tissue matrix.
3. The method of claim 2, wherein the tissue matrix fragments
comprise acellular tissue matrix.
4. The method of claim 3, wherein the acellular tissue matrix
comprises an acellular dermal matrix.
5. The method of claim 3, wherein the acellular tissue matrix
comprises an acellular porcine dermal matrix.
6. The method of claim 1, wherein the size distribution of the
tissue matrix flakes prevents undesirable migration of the flakes
into areas surrounding the implantation site.
7. The method of claim 1, wherein the tissue matrix flakes are
created using a process comprising applying a cryoprotectant
solution prior to freezing.
8. The method of claim 7, wherein the cryoprotectant solution
comprises at least one of a maltodextrin, sucrose, polyethylene
glycol (PEG), or polyvinylpyrrolidone (PVP) solution, or a
combination thereof.
9. The method of claim 8, wherein the cryoprotectant solution
comprises a maltodextrin solution.
10. The method of claim 9, wherein the maltodextrin solution
comprises 5-50% (w/v) maltodextrin.
11. The method of claim 9, wherein the maltodextrin solution
comprises 15-25% (w/v) maltodextrin.
12. The method of claim 1, wherein the group of tissue matrix
flakes have been sterilized by application of at least one of
ethylene oxide, propylene oxide, gamma irradiation or e-beam
irradiation.
13. The method of claim 1, further comprising hydrating the tissue
matrix flakes.
14. The method of claim 13, further comprising hydrating the tissue
matrix flakes in a solution comprising a bioactive substance.
15. The method of claim 14, wherein the tissue matrix flakes are a
carrier for controlled delivery of the bioactive substance.
16. The method of claim 14, wherein the bioactive substance
comprises at least one of: an antimicrobial agent, a cytokine, a
growth factor, and adipose tissue.
17. The method of claim 1, wherein the anatomical site is at least
one of: a wound, a tissue void, a pressure ulcer, a diabetic foot
ulcer, a periosteal bone defect, or a wrinkle.
18. The method of claim 1, further comprising blending the tissue
matrix flakes to form a slurry prior to implanting the tissue
filler.
19. The method of claim 18, wherein the flakes in the slurry are
loosely intertwined and fibrillar.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 15/166,848, filed May 27, 2016, which is a continuation of U.S.
patent application Ser. No. 13/446,422, filed Apr. 13, 2012 (now
U.S. Pat. No. 9,375,513) which claims priority under 35 U.S.C.
.sctn. 119 to U.S. Provisional Application No. 61/475,378, which
was filed on Apr. 14, 2011.
[0002] The present disclosure relates to tissue fillers, and more
particularly, to methods of preparing tissue fillers having a
flake-like shape and tissue fillers prepared according to those
methods.
[0003] Various types of wound dressings and soft tissue fillers are
used to regenerate, repair, or otherwise treat diseased or damaged
tissues and organs. Such materials include hyaluronan-based gels,
solubilized and cross-linked collagen compositions, micronized
tissue matrices, and synthetic polymeric compositions in hydrogel
or other forms. Each of these materials has potential drawbacks if
used as bulk soft tissue fillers or deep wound dressings, including
limited suitability for deep wounds, inability to regenerate,
tendency to increase inflammatory response, or tendency to degrade
upon exposure to radiation.
[0004] According to certain embodiments, a method for preparing a
tissue matrix composition is provided. The method comprises
selecting a collagen-based tissue matrix, contacting the matrix
with a cryoprotectant solution, freezing the matrix, and cutting
the matrix, wherein the temperature of the tissue matrix ranges
from -10.degree. C. to -40.degree. C. for the cutting step.
[0005] In certain embodiments, a tissue matrix composition is
provided. The composition comprises a collagen-based tissue matrix,
wherein the matrix has been contacted with a cryoprotectant
solution and frozen thereafter, and wherein the matrix has been cut
at a temperature between -10.degree. C. and -40.degree. C. after
freezing.
[0006] In certain embodiments, a tissue matrix composition is
provided. The composition comprises a collagen-based tissue matrix,
wherein the tissue matrix comprises tissue particles having a size
distribution ranging from 0.2-5 mm in length, 0.203 mm in width,
and 0.02-0.3 mm in thickness.
[0007] In certain embodiments, a method for preparing a tissue
matrix composition is provided. The method comprises selecting a
collagen-based matrix, contacting the matrix with a cryoprotectant
solution, freezing the matrix, adjusting the temperature of the
tissue matrix and the cryoprotectant to between -10.degree. C. to
-40.degree. C., cutting the frozen tissue matrix, placing the cut
tissue matrix in a liquid to form a suspension, and freeze-drying
the suspension.
DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 is a diagram of a grating device for performing
methods of the present disclosure, according to certain
embodiments.
[0009] FIG. 2 is a diagram of a perfusion column for producing
tissue fillers of the present disclosure, according to certain
embodiments.
[0010] FIG. 3 is a flow chart summarizing the various steps that
may be used to produce tissue fillers, according to certain
embodiments of the disclosed method.
[0011] FIGS. 4A and 4B are photographs demonstrating the morphology
of various tissue pieces under light and SEM microscopy
respectively, according to certain embodiments, as described in
Example 1.
[0012] FIG. 5 is a chart showing the distribution of tissue flake
size according to certain embodiments, as described in Example
1.
[0013] FIG. 6 is a chart showing the mass fractions of ice and
amorphous tissue domain as a function of cryoprotectant
concentration, according to certain embodiments, as described in
Example 2.
[0014] FIG. 7 is a chart showing thermograms of tissue flakes
according to certain embodiments, as described in Example 4.
[0015] FIGS. 8A and 8B are charts showing the resistance of
processed tissue material, according to certain embodiments, to
collagenase and trypsin digestion, respectively, as described in
Example 4.
[0016] FIG. 9A is a photograph of rehydrated tissue flakes,
according to certain embodiments and FIG. 9B is a schematic of a
pressure testing pad, as described in Example 5.
[0017] FIGS. 10A and 10B are charts showing the width and length
size distribution, respectively, of processed tissue material,
according to certain embodiments, as described in Example 6.
[0018] FIGS. 11A through 11D are photographs of a soft tissue foam,
prepared according to certain embodiments, as described in Example
6.
[0019] FIGS. 12A through 12D show various histological sections of
various tissue matrices after implantation into an animal,
according to certain embodiments, as described in Example 6.
DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS
[0020] 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.
[0021] 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. It will be understood that
the benefits and advantages described above may relate to one
embodiment or may relate to several embodiments. Where appropriate,
aspects of any of the examples and embodiments described above may
be combined with aspects of any of the other examples described to
form further examples having comparable or different properties and
addressing the same or different problems.
[0022] 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.
[0023] As used herein, "tissue matrix" will refer to material
derived from animal tissue that includes a collagen-containing
matrix. Such tissue matrices can include intact tissues, tissues
that have been partially or completely decellularized, or synthetic
collagenous matrices (e.g., 3-D matrices formed from suspended or
otherwise processed tissues). As described further below, suitable
tissue matrices can be acellular. Any suitable tissue matrix can be
used, depending on the intended implantation site, so long as the
tissue is amenable for use with the methods described herein.
[0024] The cut tissue matrix, such as the flake-like tissue matrix
disclosed herein, possesses several properties that make it
suitable to use as a bulk soft tissue filler or deep wound
dressing. The flake-like tissue matrix is regenerative, permitting
host cell repopulation and revascularization. The large size of the
individual tissue flakes, relative to micronized tissue particles,
prevents the undesirable migration of the flakes into the areas
surrounding the original treatment site. The flake-like tissue
matrix disclosed herein has been found to be more stable and
resistant to enzymatic degradation when compared to micronized
tissue particles. The size and/or geometry of the tissue flakes
allows them to form a stable suspension without gelling or phase
separation, which makes the tissue fillers suitable for filling
large voids (tens to hundreds of milliliters). When used to form a
suspension that can be freeze-dried, the suspension of tissue
flakes can contain relatively large, inter-connected channels that
permit fluids to flow freely. When used as wound dressings and/or
with negative-pressure wound therapy systems, the channels can help
support host cell repopulation and revascularization. These
inter-connected channels can transduce pressure, making the
flake-like tissue matrix more amenable for the treatment of deep
wounds, while also being compatible with negative pressure
therapy.
[0025] According to certain embodiments, a method for preparing a
tissue matrix composition is provided. The method comprises
selecting a collagen-based tissue matrix, contacting the matrix
with a cryoprotectant solution, freezing the matrix, and cutting
the matrix, wherein the temperature of the tissue matrix ranges
from -10.degree. C. to -40.degree. C. for the cutting step. In
certain embodiments, an additional method for preparing a tissue
matrix composition is provided. The method comprises selecting a
collagen-based matrix, contacting the matrix with a cryoprotectant
solution, freezing the matrix, adjusting the temperature of the
tissue matrix and the cryoprotectant to between -10.degree. C. to
-40.degree. C., cutting the frozen tissue matrix, placing the cut
tissue matrix in a liquid to form a suspension, and freeze-drying
the suspension. In addition, in various embodiments, tissue
matrices produced according to the methods described herein are
provided. In certain embodiments, a tissue matrix composition is
provided. The composition comprises a collagen-based tissue matrix,
wherein the matrix has been contacted with a cryoprotectant
solution and frozen thereafter, and wherein the matrix has been cut
after freezing at a temperature between -10.degree. C. and
-40.degree. C. In certain embodiments, an additional tissue matrix
composition is provided. The composition comprises a collagen-based
tissue matrix, wherein the tissue matrix comprises particles having
a size distribution ranging from 0.2-5 mm in length, 0.2-3 mm in
width, and 0.02-0.3 mm in thickness.
[0026] After a tissue matrix has been selected, a cryoprotectant
solution is used to treat the tissue matrix prior to freezing. The
cryoprotectant solution prevent damage to the tissue from damage as
a result of freezing and/or thawing, reduce the amount of frozen
water in the tissue through osmotic dehydration, and can ensure the
formation of a high subzero temperature glassy matrix in the
tissue. The use of a cryoprotectant can also help maintain a
desired balance between ice content and non-frozen tissue after
freezing. A frozen tissue matrix containing too much ice can be
brittle and difficult to cut. Conversely, frozen tissue matrix with
insufficient ice is too soft and warms rapidly during cutting,
making it difficult to cut as well. Thus, the concentration of the
cryoprotectant in the cryoprotectant solution can be used to
control the ice content and hardness of the frozen matrix. In some
embodiments, the ice content of the frozen matrix (w/w) ranges from
40-60% (e.g., 40, 45, 50, 55, or 60%).
[0027] Any suitable cryoprotectant can be used in the
cryoprotectant solution. In certain embodiments, suitable
cryoprotectants can include maltodextrin, sucrose, polyethylene
glycol (PEG), and polyvinylpyrrolidone (PVP), or combinations
thereof. In some embodiments, the cryoprotectant comprises
maltodextrin. In some embodiments, the cryoprotectant solution
contains 5-50% (w/v) maltodextrin. In some embodiments, the
solution contains 15-25% (w/v) maltodextrin.
[0028] After treatment with the cryoprotectant solution, the tissue
matrix is then frozen. In certain embodiments, the tissue matrix is
frozen at -80.degree. C. Freezing can be accomplished by using a
-80.degree. C. freezer. Before cutting, the temperature of the
tissue matrix can be adjusted if needed to a temperature ranging
from -10.degree. C. to -40.degree. C. for the cutting step. That
temperature range can help maintain the proper balance between ice
content and non-frozen tissue, which facilitates cutting.
[0029] After adjusting the temperature, the tissue can be cut. In
some embodiments, the tissue is cut into pieces of irregular shape
and size. In some embodiments, the irregular shape and size gives
the cut tissue a flake-like appearance. In other words, the pieces
of cut tissue encompass irregular shapes and sizes, and lack
consistency between pieces. In some embodiments, the cut tissue has
a selected size distribution. In certain embodiments, the cut
tissue has a size distribution ranging from 0.2-5 mm in length,
0.2-3 mm in width, and 0.2-0.3 mm in thickness. When hydrated, the
majority of cut tissue within this size distribution weighs between
0.5 and 2.0 mg. The irregular shape and size distribution of the
cut tissue facilitate the formation of large, interconnected
channels that permit body fluid to flow freely when the cut tissue
is placed in suspension, and thus aid host cell repopulation and
revascularization. In some uses, the large size of the cut tissue,
relative to smaller micronized tissue particles, prevents migration
of the cut tissues into surrounding anatomic sites when implanted
or placed in or on a wound. Cut tissue that is larger than the
disclosed size distribution may also be undesirable. If the cut
tissue is too large, the individual pieces may take up too much
space when in suspension and also impede formation of sufficiently
large channels.
[0030] Various methods can be used to cut the tissue and still be
in accordance with the disclosed method, provided the tissue is cut
into pieces of irregular shape and size. For example, the tissue
matrix can be cut manually, using scissors. In some embodiments,
cutting the tissue matrix comprises grating the tissue matrix. In
some embodiments, a grating device is used to perform the grating
step. Any grating device can be used in accordance with the
disclosed method, provided it cuts the tissue matrix into pieces of
irregular size and shape. In some embodiments, the grating device
is a grater, such as a MICROPLANE.RTM. grater. In other
embodiments, the grating device is a grating wheel.
[0031] In certain embodiments, the cutting step can be automated.
Automating reduces the amount of time necessary to prepare the cut
tissue and facilitates cutting larger amounts of tissue. In various
embodiments, one or more aspects of the cutting process can be
automated. For example, the grating device itself can be automated
so that manual effort is no longer required to cut the tissue
matrix. It is also possible to automate delivery of the tissue
matrix to the cutting device. Further, it is possible to automate
removal of tissue matrix once it has been cut. One example of an
automated cutting apparatus is illustrated in FIG. 1. As shown, the
apparatus comprises a grating wheel 101 that is fitted with blades
102 for cutting a matrix 107. Below the grating wheel is a
collection tray 103. The grating wheel 101 and collection tray are
enclosed in a housing unit 104, which protects the operator from
direct contact with the blades 102. The housing unit 104 is fitted
with a loading bay 106. In this example, the loading bay 106 is
inclined so that any samples loaded onto it will move toward the
grating wheel 101 by gravity. Tissue matrix 107 is placed onto the
loading bay 106 and moves toward the grating wheel 101. The blades
102 of the grating wheel 101 cut the tissue matrix 107 into flakes
108 which fall into the collection tray 103 below.
[0032] In various embodiments, the disclosed methods can further
comprise additional processing before or after cutting. For
example, in various embodiments, the disclosed methods comprise use
of a pre-processed acellular tissue matrix, which is described in
more detail below. In other embodiments, an intact, cellular tissue
may be used, which can be further processed to produce a suitable
acellular tissue matrix. The further processing may comprise
decellularization, DNA removal, and removal of .alpha.-gal epitopes
or other antigens. Decellularization, DNA removal, and .alpha.-gal
antigen removal are described in further detail below. Further
processing of the cut tissue may include disinfecting the tissue
matrix. In some embodiments, the flake-like tissue matrix is
disinfected with isopropyl alcohol (IPA) (e.g., at about 70%
IPA).
[0033] Processing of the tissue flakes is also amenable to
automation. Automation can include any process that does not
require the manual delivery and removal of the solutions that
facilitate processing. Automation of tissue processing reduces time
spent manually replacing processing solutions and minimizes
operator handling of the tissue flakes. In some embodiments,
automation of tissue processing comprises use of a closed,
low-pressure perfusion column. Decellularization, DNA removal,
.alpha.-gal antigen removal, and disinfection can all be performed
within the column, which allows for high-throughput processing of
the cut tissue. Cut tissue matrix is loaded into the column, and
air is purged from the column. Decellularization, DNA removal,
.alpha.-gal antigen removal, and disinfection are achieved by
stepwise perfusion of the appropriate solutions at or near
atmospheric pressure. Detergent, DNAse, and .alpha.-galactosidase
solutions are described in more detail below.
[0034] An example of an automated processing system is shown in
FIG. 2. The processing system comprises a solution pumping system
201 into which various processing solutions are placed. The pumping
system 201 sends an appropriate solution through tubing 202 into a
closed perfusion column 204, which has been fitted with a process
solution inlet 203 and outlet 205. The appropriate solution then
contacts the tissue flakes 108 inside the perfusion column 204 and
then leaves the column 204 through tubing 202 connected to the
process solution outlet 205 on one end and the solution pumping
system 201 on the other end.
[0035] The disclosed tissue fillers can be further treated to
produce substantially aseptic or sterile materials. Accordingly, in
various embodiments, the tissue fillers can be sterilized after
preparation. As used herein, a "sterilization process" can include
any process that reduces bioburden in a sample, but need not render
the sample absolutely sterile.
[0036] Certain exemplary processes include, but are not limited to,
a gamma irradiation process, an e-beam irradiation process,
ethylene oxide treatment, and propylene oxide treatment. Suitable
sterilization processes include, but are not limited to, those
described in, for example, U.S. Patent Publication No.
2006/0073592A1, to Sun et al.; U.S. Pat. No. 5,460,962 to Kemp;
U.S. Patent Publication No. 2008/0171092A1, to Cook et al. In some
embodiments, sterilization is performed in conjunction with
packaging of the flakes, while in other embodiments, sterilization
can occur after packaging.
[0037] After the tissue flakes are prepared by the disclosed
methods, they may be stored for some time before implantation in or
on a patient. In certain embodiments, the tissue filler may be
packaged in a Tyvek pouch for storage purposes. The tissue filler
may also be stored in different states. In some embodiments, the
flake-like tissue filler is freeze-dried after preparation. In
certain embodiments, freeze-drying of the flake-like tissue filler
is performed before or during packaging. In certain embodiments,
the tissue flakes are stored in a hydrated state. The tissue flakes
can be hydrated in various solutions, for example, an aqueous
preservation solution.
[0038] The various steps described above can be combined, added,
deleted, or otherwise modified as necessary. For example, if the
starting material is a porcine hide, removing the epidermis and
subcutaneous fat may be necessary before cutting. However, removal
of those components is not required if the starting material is an
acellular tissue matrix. Further, if one is using porcine hide as
the starting material rather than an acellular tissue matrix,
additional processing may be necessary after cutting.
[0039] A sample protocol in accordance with the disclosed methods
is provided in FIG. 3, using porcine hides as the starting
material. Specific details regarding each step are provided
throughout the present disclosure. Fresh porcine hides are
collected with the hairs subsequently removed 301. The epidermis
and subcutaneous fat layers are then removed, leaving the dermal
tissue 302. The dermal tissue is then incubated in a cryoprotectant
solution prior to freezing to maintain the proper balance between
ice content and amorphous tissue during cutting 303. After
incubation in the cryoprotectant solution, the tissue is frozen,
brought to a temperature between -10.degree. C. and -40.degree. C.,
and cut to a desired size and shape. The flakes are then incubated
in an appropriate solution to decellularize the tissue 305. The
tissue is then treated with treated with enzymes to remove DNA and
.alpha.-gal epitopes 306. After enzyme treatment, the flakes are
disinfected with using an IPA solution 307 and washed in buffer
308.
[0040] After washing, the tissue flakes are packaged for storage.
Packaging can be performed in conjunction with freeze-drying 309 or
with sterilization 312. If the flakes are freeze-dried in
conjunction with packaging 309, they are then sterilized 310,
resulting in sterile, freeze-dried flakes 311. Alternatively, if
packaging and sterilization are performed together 312, the flakes
can be freeze-dried after 314, which also results in sterile,
freeze dried flakes 311. The flakes can also be hydrated after the
packaging and sterilization step 312, resulting in sterile,
hydrated tissue flakes 313.
[0041] The disclosed tissue matrix composition can be used in
various ways. In certain embodiments, the tissue matrix composition
can form a stable, non-gelling, low density suspension. In certain
embodiments, the disclosed tissue matrix composition can be used as
a bulk tissue filler for tissue regeneration and repair. Methods of
treatment using the composition include selecting an anatomical
site for treatment and implanting the tissue filler into the
treatment site. Examples include the direct application of the
flake-like tissue material to deep wounds and large soft tissue
voids that may occur during certain types of surgeries, such as
lumpectomies. The tissue filler may be also be used in the
treatment of pressure ulcers, diabetic foot ulcers, or periosteal
bone defects. The tissue filler can also be used for reconstructing
facial features as well as correcting facial defects, including
treatment of wrinkles, skin loss, or skin atrophy. In other
embodiments, the flake-like tissue material may be used as a
carrier for controlled delivery of other bioactive substances.
Examples of bioactive substances include, but are not limited to,
antimicrobial agents, cytokines, growth factors, and drugs.
Bioactive substances can also include non-collagenous tissue, such
as adipose tissue, or cells, including stem cells. In certain
embodiments, tissue flakes that have been subsequently freeze-dried
can be hydrated in solutions that contain bioactive substances, and
then applied to the sites as needed. In other embodiments, the
flake-like tissue filler can be applied as a slurry. If an aqueous
suspension of tissue flakes is blended briefly (30 to 300 seconds,
for example), it becomes a loosely intertwined fibrillar slurry
that is flowable for convenient application.
[0042] In certain instances, a tissue foam may be desired.
Depending on the surgical procedures and particular circumstances
of tissue repair, use of a tissue foam may be appropriate. Tissue
foams can be used to treat wounds or damaged tissues that are not
defined by voids with a particular boundary. For example, one could
use surgical adhesives or sutures to attach a tissue foam to
tissues or organs. In other cases, tissue flakes may be more
desirable when there is a need to fill voids of any shape, which
are defined by a particular boundary. For example, tissue flakes
are suitable when tumors are removed via laproscopic procedures or
cryosurgery, due to the small openings that result. Tissue foams
can also be made to have specific sizes and shapes, such as sheets,
spheres, and cubes, for certain well-defined surgeries. For
example, tissue foam sheets can be used to partially or completely
cover surgical implants to reduce the dramatic effect of initial
implant/body interactions and potentially slow capsule formation.
Further, tissue foams can be used as components of negative
pressure wound therapy systems, such as the VAC.RTM. system, which
is produced by Kinetic Concepts, Inc. Such systems can be used to
treat a variety of tissues sites and include, for example, a
negative pressure source such as a pump and one or more treatment
materials, which often include a porous foam or manifold. General
examples of such systems are described in U.S. Patent Publication
Number, 2010/0040687 A1, which was filed on Aug. 13, 2009.
[0043] The use of the disclosed tissue flakes facilitates the
preparation of tissue foam in several ways. The tissue flakes have
a small mass but large surface area, which facilitates further
processing, and the flakes are amenable to preparing a uniform
fiber suspension. In contrast to using ground or micronized tissue
particles or fibers, tissue flakes are unlikely to gel or cake
during decellularization. Finally, the process of preparing the
tissue flakes first adds an additional layer of size reduction,
which assists in the preparation of a uniform suspension.
[0044] Accordingly, the methods described herein can also be used
to prepare a regenerative foam using the cut tissue described above
as the starting material. As discussed above, a tissue matrix is
selected and a cryoprotectant is used to treat the tissue matrix
prior to freezing. The tissue matrix is then frozen and cut at a
temperature ranging from -10.degree. C. to -40.degree. C. after
freezing. As before, a pre-processed, acellular tissue matrix may
be used in conjunction with the disclosed method. In other
embodiments, the tissue matrix can be decellularized after cutting
as described above.
[0045] The cut tissue matrix is then placed in a liquid to form a
suspension. Any suitable liquid may be used, provided it does not
interfere with the regenerative properties of the tissue matrix. In
some embodiments, the cut tissue matrix is placed in an aqueous
solution. After being placed in solution, the tissue matrix can be
mixed within the solution. In various embodiments, the solution is
mixed until a stable tissue suspension is formed, and/or until the
tissue size distribution reaches a desired level. Mixing can be
accomplished by any suitable means that achieve these ends, such as
agitating, shaking, or vortexing the tissue matrix once it is in
solution. Blending can also be used to mix the tissue matrix in
solution. In certain embodiments, a blender is used to mix the
matrix. Mixing may also be achieved by using a pressure jet, an
ultrasound device, or a combination of the two. For example, after
decellularization, tissue flakes in solution can go through a
pressurized nozzle, where the tissue flakes break into fibers. One
can also place tissue flakes in solution into an ultrasonic field
to break the tissue flakes into a fiber suspension. A sonic nozzle
can also be used, which combines ultrasound and pressure to break
the tissue flakes into a fibrous suspension. The consistency of the
suspension can be controlled by how much cut tissue matrix is added
to the liquid. In some embodiments, the amount of cut tissue matrix
in the liquid ranges from 20-40% (w/v). In certain embodiments, the
amount of cut tissue matrix in the liquid is 25% (w/v).
[0046] After formation of a suspension, the tissue suspension is
then freeze-dried. Freeze-drying can be performed under aseptic
conditions to prevent contamination of the tissue matrix. The
tissue suspension can also be aliquoted into an appropriate
container, so that upon freeze-drying, the tissue matrix will be
cast in the desired shape.
[0047] The process of mixing and freeze-drying results in a tissue
composition wherein small tissue filaments of various dimensions
are intertwined and interlocked with one another. In certain
embodiments, the process of mixing and freeze-drying the tissue
suspension results in a tissue foam. A foam can be produced, for
example, by blending an aqueous suspension freeze-dried tissue
flakes in a blender. After processing, the tissue composition
contains interconnected macropores due to the intertwined pieces of
small tissue, which permit the free flow of fluid and help support
cell repopulation and revascularization.
[0048] The tissue foam disclosed herein can be further processed as
needed. The tissue foam can be disinfected or sterilized as
described above. The freeze-dried material can also be further
treated to increase the strength of the tissue foam using heat and
vacuum conditions. Without being bound to theory, strengthening of
the tissue foam may occur through physical interlocking,
biochemical cross-linking, or a combination of the two. Physically,
final dehydration results in surface tension, which pulls tissue
fragments closer together and forms hydrogen bonds between the
hydroxyl groups of adjacent collagen fibers. Chemically, the
treatment results in amide formation between carboxyl and amino
groups, as well as esterification and glycation of collagen and
other extracellular matrix protein amino groups. In some
embodiments, the heat applied to the tissue matrix is limited to
avoid denaturation of the dry proteins contained in the tissue
matrix. Dry proteins in the tissue matrix typically denature
between 130.degree. C. and 170.degree. C. In one embodiment, the
strength of the tissue foam can be increased by treating the
freeze-dried material at a temperature above 30.degree. C. but
below the denaturation temperatures listed above under vacuum
conditions. In some embodiments, the strength of the tissue foam
can be increased by treating the freeze-dried material at
approximately 100.degree. C. under vacuum for a certain period of
time. In some embodiments, the dried material may be treated under
for vacuum for 24 hours. Other suitable periods of time can be
readily identified and tested by those skilled in the art.
[0049] After processing, the tissue foam may be stored for some
time before implantation in or on a patient. In certain
embodiments, the tissue foam may be packaged in a Tyvek pouch for
storage purposes. The tissue foam itself may also be stored in
different states. In some embodiments, the tissue foam is stored in
its already freeze-dried state. In other embodiments, the tissue
foam is stored in a hydrated state.
Tissue Matrices
[0050] As noted above, the methods described herein can be used to
produce flake-like tissue fillers using a variety of different
tissue types, so long as the tissue includes a collagen-containing
matrix amenable for use with the methods described above. Such
tissue matrices can include intact tissues, tissues that have been
partially or completely decellularized, or synthetic collagenous
matrices (e.g., 3-D matrices formed from suspended or otherwise
processed tissues).
[0051] The tissue matrix can be produced from a range of tissue
types. For example, the tissue matrix can be derived from fascia,
pericardial tissue, dura, umbilical tissue, placental tissue,
cardiac valve tissue, ligament tissue, tendon tissue, arterial
tissue, venous tissue, neural connective tissue, urinary bladder
tissue, ureter tissue, and intestinal tissue. In other embodiments,
the tissue matrix comprises a dermal tissue matrix. In certain
embodiments, the tissue matrix comprises porcine dermal matrix.
[0052] In certain embodiments, the tissues can include a mammalian
soft tissue. For example, in certain embodiments, the tissue can
include mammalian dermis. In certain embodiments, the dermis can be
separated from surrounding epidermis and/or other tissues, such as
subcutaneous fat. In certain embodiments, the tissue sample can
include small intestine submucosa. In certain embodiments, the
tissue samples can include human or non-human sources. Exemplary,
suitable non-human tissue sources include, but are not limited to,
pigs, sheep, goats, rabbits, monkeys, and/or other non-human
mammals.
[0053] The tissue matrices can be implanted at a variety of
different anatomic sites. For example, tissue matrices can be
implanted around breast implants; around or replacing vascular
structures; around or replacing luminal structures (e.g., ureters,
nerves, lymphatic tissues, gastrointestinal structures); on or
replacing heart valves, pericardium, or other cardiac structures;
in or on bony or cartilaginous materials (e.g., ears, noses,
articular surfaces, around dental structures, or along any short of
long bone); and/or surrounding, lining, supporting, or replacing
any body cavity (e.g., bladder, stomach).
[0054] Tissue matrices can be selected to provide a variety of
different biological and mechanical properties. For example, an
acellular tissue matrix can be selected to allow tissue ingrowth
and remodeling to assist in regeneration of tissue normally found
at the site where the matrix is implanted. For example, an
acellular tissue matrix, when implanted on or into fascia, may be
selected to allow regeneration of the fascia without excessive
fibrosis or scar formation. In certain embodiments, the tissue
matrix can be formed from ALLODERM.RTM. or STRATTICE.TM., which are
human and porcine acellular dermal matrices, respectively.
Alternatively, other suitable acellular tissue matrices can be
used, as described further below.
[0055] In some embodiments, the collagen-based material comprises
an acellular tissue matrix. In certain embodiments, these matrices
can be completely decellularized to yield acellular tissue matrices
to be used for patients. For example, various tissues, such as
skin, intestine, bone, cartilage, nerve tissue (e.g., nerve fibers
or dura), tendons, ligaments, or other tissues can be completely
decellularized to produce tissue matrices useful for patients.
Suitable processes for producing acellular tissue matrices are
described below.
[0056] In general, the steps involved in the production of an
acellular tissue matrix include harvesting the tissue from a donor
(e.g., a human cadaver or animal source) and cell removal under
conditions that preserve biological and structural function. In
certain embodiments, the process includes chemical treatment to
stabilize the tissue and avoid biochemical and structural
degradation together with or before cell removal. In various
embodiments, the stabilizing solution arrests and prevents osmotic,
hypoxic, autolytic, and proteolytic degradation, protects against
microbial contamination, and reduces mechanical damage that can
occur with tissues that contain, for example, smooth muscle
components (e.g., blood vessels). The stabilizing solution may
contain an appropriate buffer, one or more antioxidants, one or
more oncotic agents, one or more antibiotics, one or more protease
inhibitors, and/or one or more smooth muscle relaxants.
[0057] The tissue is then placed in a decellularization solution to
remove viable cells (e.g., epithelial cells, endothelial cells,
smooth muscle cells, and fibroblasts) from the structural matrix
without damaging the biological and structural integrity of the
collagen matrix. The decellularization solution may contain an
appropriate buffer, salt, an antibiotic, one or more detergents
(e.g., TRITON X100.TM., sodium deoxycholate, polyoxyethylene (20)
sorbitan mono-oleate), 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 1%
TRITON X-100.TM. in RPMI media with Gentamicin and 25 mM EDTA
(ethylenediaminetetraacetic acid). In some embodiments, the tissue
is incubated in the decellularization solution overnight at
37.degree. C. with gentle shaking at 90 rpm. For example, in some
embodiments, 2% sodium deoxycholate is added to the
decellularization solution.
[0058] After the decellularization process, the tissue sample is
washed thoroughly with saline. In some exemplary embodiments, e.g.,
when xenogenic material is used, the decellularized tissue is then
treated overnight at room temperature with a deoxyribonuclease
(DNase) solution. In some embodiments, the tissue sample is treated
with a DNase solution prepared in DNase buffer (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 buffer can be used as long as the buffer provides suitable
DNase activity.
[0059] While an acellular tissue matrix may be made from the same
species as the acellular tissue matrix graft recipient, different
species can also serve as tissue sources. Thus, for example, an
acellular tissue matrix may be made from porcine tissue and
implanted in a human patient. Species that can serve as recipients
of acellular tissue matrix and donors of tissues or organs for the
production of the acellular tissue matrix include, without
limitation, mammals, such as humans, nonhuman primates (e.g.,
monkeys, baboons, or chimpanzees), pigs, cows, horses, goats,
sheep, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats,
or mice.
[0060] Elimination of the .alpha.-gal epitopes from the
collagen-containing material may diminish the immune response
against the collagen-containing material. The .alpha.-gal epitope
is expressed in non-primate mammals and in New World monkeys
(monkeys of South America) as well as on macromolecules such as
proteoglycans of the extracellular components. U. Galili et al., J.
Biol. Chem. 263:17755 (1988). This epitope is absent in Old World
primates (monkeys of Asia and Africa and apes) and humans, however.
Id. Anti-gal antibodies are produced in humans and primates as a
result of an immune response to .alpha.-gal epitope carbohydrate
structures on gastrointestinal bacteria. U. Galili et al., Infect.
Immun. 56:1730 (1988); R. M. Hamadeh et al., J. Clin. Invest.
89:1223 (1992).
[0061] Since non-primate mammals (e.g., pigs) produce .alpha.-gal
epitopes, xenotransplantation of collagen-containing material from
these mammals into primates often results in immunological
activation because of primate anti-Gal antibodies binding to these
epitopes on the collagen-containing material. 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).
Furthermore, xenotransplantation results in major activation of the
immune system to produce increased amounts of high affinity
anti-gal antibodies. Accordingly, in some embodiments, when animals
that produce .alpha.-gal epitopes are used as the tissue source,
the substantial elimination of .alpha.-gal epitopes from cells and
from extracellular components of the collagen-containing material,
and the prevention of re-expression of cellular .alpha.-gal
epitopes can diminish the immune response against the
collagen-containing material associated with anti-gal antibody
binding to .alpha.-gal epitopes.
[0062] To remove .alpha.-gal epitopes, after washing the tissue
thoroughly with saline to remove the DNase solution, the tissue
sample may be subjected to one or more enzymatic treatments to
remove certain immunogenic antigens, if present in the sample. In
some embodiments, the tissue sample may be treated with an
.alpha.-galactosidase enzyme to eliminate .alpha.-gal epitopes if
present in the tissue. In some embodiments, the tissue sample is
treated with .alpha.-galactosidase at a concentration of 300 U/L
prepared in 100 mM phosphate buffer at pH 6.0. In other
embodiments, the concentration of .alpha.-galactosidase is
increased to 400 U/L for adequate removal of the .alpha.-gal
epitopes from the harvested tissue. Any suitable enzyme
concentration and buffer can be used as long as sufficient removal
of antigens is achieved.
[0063] Alternatively, rather than treating the tissue with enzymes,
animals that have been genetically modified to lack one or more
antigenic epitopes may be selected as the tissue source. 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
co-pending U.S. application Ser. No. 10/896,594 and U.S. Pat. No.
6,166,288, the disclosures of which are incorporated herein by
reference in their entirety. In addition, certain exemplary methods
of processing tissues to produce acellular matrices with or without
reduced amounts of or lacking alpha-1,3-galactose moieties, are
described in 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), which is
incorporated by reference in its entirety.
[0064] After the acellular tissue matrix is formed,
histocompatible, viable cells may optionally be seeded in the
acellular tissue matrix to produce a graft that may be further
remodeled by the host. In some embodiments, histocompatible viable
cells may be added to the matrices 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 recipient's own cells migrating into the acellular tissue
matrix or by infusing or injecting cells obtained from the
recipient or histocompatible cells from another donor into the
acellular tissue matrix in situ. Various cell types can be used,
including embryonic stem cells, adult stem cells (e.g. mesenchymal
stem cells), and/or neuronal cells. In various embodiments, the
cells can be directly applied to the inner portion of the acellular
tissue matrix just before or after implantation. In certain
embodiments, the cells can be placed within the acellular tissue
matrix to be implanted, and cultured prior to implantation.
[0065] Although general process parameters for production of
acellular tissue matrices are described, a variety of
collagen-containing acellular materials are available, and the
methods of processing such materials to produce flake-like tissue
fillers may be used with any of those materials. For example, a
number of biological scaffold materials are described by Badylak et
al., and the methods of the present disclosure can be used to
produce flake-like tissue fillers using any of those materials, or
any other similar materials. Badylak et al., "Extracellular Matrix
as a Biological Scaffold Material: Structure and Function," Acta
Biomaterialia (2008), doi:10.1016/j.actbio.2008.09.013.
Example 1: Cryopreservation and Cryocutting of Split Porcine
Dermis
[0066] Fresh porcine hides were collected, and the hair was
removed. Dermis tissue was obtained from the hides by splitting off
the epidermis layer and subcutaneous fat (hypodermis) layer. The
porcine dermis was rinsed with Dulbecco's phosphate-buffered saline
(PBS), and incubated for 4 to 6 hours in a cryoprotectant solution
containing 50 mM sodium phosphate, 10 mM ethylenediamine
tetraacetic acid (EDTA) and 35% (w/v) maltodextrin (pH 7.0).
Cryoprotectant-treated dermis sheets were then frozen in a
-80.degree. C. freezer. Frozen dermis tissue was grated into tissue
flakes by using a medium-sized MICROPLANE.RTM. grater (grater
openings are approximately 2.2 to 3.2 mm wide and 0.2 mm
thick).
[0067] The tissue flakes were then washed for 10-15 minutes in PBS
to remove the cryoprotectant and then fixed in 2% glutaraldehyde
solution. The morphology of the tissue flakes was then observed
using light microscopy and scanning electron microscopy (SEM). For
examination under light microcopy, tissue samples were stained with
Sirius Red. For SEM examination, tissue samples were dehydrated
stepwise in ethanol solutions of increasing concentration (25%,
50%, 75%, 90%, 95%, 98% and 100%), dried with supercritical
CO.sub.2 in a critical point dryer, and sputter-coated with gold.
Tissue specimens were viewed under high vacuum and low electron
voltage (5 kV). As shown in FIGS. 4A and 4B, the morphology of the
cut pieces was observed to be flake-like under both types of
microscopy.
[0068] To determine the size (mass) distribution of the tissue
flakes, glutaraldehyde-fixed tissue pieces were washed in distilled
water to remove glutaraldehyde residue. The wet weight of greater
than 200 tissue pieces was determined after blot-drying surface
water with surgical gauze. FIG. 5 shows the mass-based size
distribution of the tissue flakes. More than 70% of the tissue
fragments weighed between 0.5 and 2.0 mg, with an average weight of
1.3 mg. Few tissue pieces weighed over 4 mg. The tissue fragment
size was in the expected range after using a medium microplane (2
to 3 mm wide and 0.2 mm thick, nominal tissue volume of 0.8 to 1.8
mm.sup.3).
Example 2: Control of Ice Content in Frozen Porcine Dermis
[0069] The proper ratio between ice and amorphous phase frozen
dermis tissue facilitates cutting dermis sheets into a flake-like
tissue matrix. Frozen dermis sheets that were not treated in
cryoprotectant solution contained too much ice, were too brittle,
and were difficult to cut. On the other hand, frozen dermis sheets
with low ice content were soft and warmed rapidly during cutting,
also making them difficult to cut. The ice content and the hardness
of the frozen dermis sheets were controlled by pretreating the
dermis sheets in cryoprotectant solutions that reduced ice
formation while facilitating formation of an amorphous tissue
matrix with a high subzero glass transition temperature.
[0070] Split porcine dermis was prepared as described in Example 1.
Maltodextrin solutions containing 0% to 35% (w/v) maltodextrin were
made with phosphate buffer solution (50 mM sodium phosphate, 10 mM
EDTA, pH 7.0). Samples of split porcine dermis were incubated in
the cryoprotectant solutions overnight. After incubation, water and
cyroprotectant content in dermis tissue samples were determined.
Ice contents of frozen tissue samples were determined by
differential scanning calorimetry according to their ice melting
enthalpies. FIG. 6 shows the mass fractions of ice and amorphous
tissue domain in the frozen tissue pretreated in different
cryoprotectant solutions. In the absence of maltodextrin (PBS
buffer alone), porcine dermis had 75% (w/v) water and 25% dry mass.
Upon freezing, .about.93% of dermis water crystallized as ice,
resulting in a mass fraction of 70% ice and a mass fraction of 30%
freeze-concentrated amorphous (non-frozen) domain. The
corresponding volume fractions were 77% and 23% for ice and
non-frozen domain, respectively. With increasing maltodextrin
concentrations, the ice fraction of incubated porcine dermis tissue
decreased and the fraction of the non-frozen domain increased. For
porcine dermis incubated in 35% maltodextrin solution, the mass
fractions of ice and non-frozen domain were reduced to 34% and
increased to 66%, respectively (corresponding volume fractions of
42% and 58%, respectively). Reduced ice formation was due to both
osmotic tissue dehydration and maltodextrin penetration into the
extracellular matrix of the dermis tissue. As a result of
maltodextrin penetration into the dermis extracellular matrix, the
glass transition temperature of the frozen dermis amorphous domain
increased to -17.degree. C. Porcine tissue treated with 15-35%
maltodextrin solutions could be easily grated into tissue
flakes.
Example 3: Processing of Dermal Tissue Flakes
[0071] Tissue flakes prepared as described in Example 1 were
further processed to remove cellular components and
.alpha.-galactosyl epitopes. The process included the following
steps: (i) rinsing off the cryoprotectants, (ii) decellularization,
(iii) enzyme treatment, (iv) disinfection, (v) freeze drying, (vi)
sterilization, and (vii) secondary packaging.
[0072] (i) Removal of cryoprotectants. Tissue flakes were placed
into 225 mL conical centrifuge bottles (.about.30 g/bottle). The
material was washed with 150 mL of sterile PBS for about 10
minutes. The tissue suspension was centrifuged at 500 rpm for 3
minutes, and the supernatant was discarded. The tissue pellet was
re-suspended with 150 mL sterile distilled water, and centrifuged
again at 500 rpm for 3 minutes to collect the tissue flakes.
[0073] (ii) Decellularization. Tissue material was decellularized
at ambient temperature with agitation in 150 ml of 2% (w/v) sodium
deoxycholate dissolved in 10 mM HEPES buffer with 10 mM EDTA (pH
7.8). Decellularization solution was changed after 1 hour through
centrifugation at 500 rpm for 3 minutes. After fresh solution was
added, the tissue material was allowed to incubate for another 4
hours. Decellularization solution was drained after another round
of centrifugation.
[0074] (iii) Enzyme treatment. Decellularized tissue flakes were
washed twice for 30 minutes at a time with 10 mM HEPES buffer
containing 5 mM EDTA (pH 7.3). Enzyme treatment was carried out for
4 hours in 150 mL of HEPES buffer with 2 mM MgCl.sub.2, 2 mM
CaCl.sub.2, 1 mg/L dornase alfa, and 1 mg/L .alpha.-galactosidase.
Enzyme solution was drained after centrifugation at 500 rpm for 3
minutes.
[0075] (iv) Disinfection. Enzyme-treated tissue flakes were
re-suspended and washed twice with 10 mM HEPES buffer with 5 mM
EDTA. The first wash was for 60 minutes and the second wash was
conducted overnight. After centrifugation, the tissue material was
rinsed with 100 mL sterile distilled water for 30 minutes. For some
bottles, 100 mL isopropyl alcohol (IPA) solution (70% w/v) was
added to the tissue suspension. After about 10 minutes, the
suspension was centrifuged at 500 rpm for 3 minutes. Old IPA
solution was drained, and fresh 70% IPA solution was added to
re-suspend the material. The material was treated with IPA for at
least 4 to 6 hours before being packaged in Tyvek bags for freeze
drying. For other bottles, tissue flakes were directly packaged in
Tyvek bags for freeze-drying without IPA disinfection.
[0076] (v) Freeze drying. Processed tissue material was
freeze-dried. Freeze-drying consisted of 3 stages: (a) cooling the
material from room temperature to -35.degree. C. at -1.degree.
C./minute, then holding at -35.degree. C. for 10 minutes; (b)
ramping up to -10.degree. C. at -1.degree. C./minute under 40 mT,
then holding for 16 hours; and (c) ramping up to 20.degree. C. at
-1.degree. C./minute under 20 mT and then holding for 8 hours.
[0077] (vi) Sterilization. Freeze-dried tissue samples that were
not disinfected with IPA were sterilized with ethylene oxide (EO).
EO sterilization included (a) conditioning at 52.degree. C. to
63.degree. C. and 55 to 75% Relative Humidity for 30 to 45 minutes,
(b) EO exposure with a gas concentration of 600.+-.50 mg/L for 4
hours, and (c) aeration at 38 to 54.degree. C. for at least 12
hours.
[0078] (vii) Secondary packaging. IPA-treated samples were packaged
immediately into foil-to-foil bags after freeze-drying. EO treated
samples were packaged in foil-to-foil bags following EO
treatment.
[0079] The processed tissue flakes were acellular. Both IPA-treated
and EO-treated materials tested to be sterile.
Example 4: Stability of Processed Tissue Flakes
[0080] Thermal stability of processed tissue flakes was tested
using differential scanning calorimetry (DSC). Both IPA-treated and
EO-treated materials were rehydrated in PBS saline (pH 7.5).
Samples were scanned at a heating rate of 3.degree. C./min from 2
to 125.degree. C. (DSC Q200, TA Instruments). Both IPA-treated and
EO-treated tissue had a small low temperature peak (.about.30 to
32.degree. C.). As shown in FIG. 7, the onset temperature of the
major collagen denaturation peaks was 53.3.+-.0.2.degree. C. and
58.0.+-.0.2.degree. C. (N=4) for EO-treated tissue flakes and
IPA-treated tissue flakes, respectively. The onset temperature of
IPA-treated tissue flakes was similar to the fresh dermis samples.
The data indicated that EO sterilization destabilizes tissue matrix
compared to IPA treatment.
[0081] The susceptibility of processed, freeze-dried tissue flakes
to enzyme degradation was tested with collagenase and trypsin
assays. FIGS. 8A and 8B show the resistance of processed tissue
material to collagenase and trypsin digestion, respectively.
IPA-disinfected tissue flakes resisted collagenase and trypsin
digestion fairly well. EO-sterilized material had increased
susceptibility to proteolysis compared to the IPA-disinfected
material.
Example 5: Suitability of Tissue Flakes to Permit Fluid Flow and
Pressure Transduction
[0082] Upon rehydration, tissue flakes form a stable suspension
without gelling or phase separation, allowing the tissue flakes to
be used for filling large voids or defects (tens to hundreds of
milliliters). The suspension of tissue flakes contains large,
inter-connected channels that permit fluid to flow freely, and thus
aid cell repopulation and revascularization when the flakes are
used as tissue fillers. The ability of the tissue flake suspension
to permit the flow of fluids and the transduction of pressure
differentials was investigated. Rehydrated tissue flakes were
placed onto Organza mesh and spread out over a 3''.times.3''
pressure distribution pad with 36 sensor ports, as shown in FIGS.
9A and 9B. A piece of GranuFoam.TM. (3''.times.3'', and 1.5''
thick) was placed on the top of the tissue flake material and a
6''.times.6'' V.A.C..RTM. was attached to the dressing assembly
along with a T.R.A.C..RTM. pad. The assembly was connected to the
V.A.C..RTM. ATS therapy unit to produce a continuous pressure at
125 mm Hg. After 5 minutes of equilibrium, the negative pressure
detected at the 36 ports was noted. Thereafter, 4 of the ports were
disconnected from the pressure sensors and connected to a reservoir
of dyed 0.9% saline solution for infusion at a rate of 500 mL per
day via a peristaltic pump. The pressure at the 32 remaining ports
was then monitored over time. The average pressure detected was
approximately 110-112 mm Hg. The negative pressure was 111.4.+-.0.9
(mean.+-.SD), 110.1.+-.1.4, and 110.6.+-.1.3 mm Hg after 1, 2, and
3 hours respectively. The consistency indicated that pressure was
distributed evenly across the entire dressing assembly, and the
inter-connected channels were able to transduce pressure
differentials well.
Example 6: Reconstructive Tissue Foam Made From Flake-Like Tissue
Material
[0083] An acellular tissue matrix derived from porcine dermis was
frozen at -80.degree. C. and used to make tissue flakes aseptically
with a medium size MICROPLANE.RTM. cheese grater. Approximately 50
g of tissue sample was suspended in 200 mL sterile water and then
mixed using a RETSCH.RTM. blender at 4000 rpm in one minute
intervals for a total of 5 cycles. As shown in FIGS. 10A and 10 ft
blending reduced the size of the tissue flake material. Blending
the tissue flakes also resulted in a stable and consistent tissue
suspension. The tissue suspension was distributed in 80 cm.sup.2
plastic petri dishes at 25 mL suspension per dish and freeze-dried
aseptically. The freeze-drying process included the controlled
cooling of the tissue suspension from room temperature to
-30.degree. C. within 60 minutes and drying at a chamber pressure
of 100 mT and a shelf temperature of 20.degree. C. for 24
hours.
[0084] The process of blending and freeze-drying resulted in small
tissue pieces of various dimensions intertwined and interlocked
with one another, forming a soft tissue foam with interconnected
macropores and preserved extracellular tissue matrix structures, as
shown in FIGS. 11A through 11D. FIG. 11A presents a gross
appearance of the tissue foam. FIG. 11B shows the interconnected
macropores of the soft tissue foam under SEM microscopy. FIG. 11C
is a histogram of the extracellular matrix structure of the foam
using a hematoxylin and eosin stain. FIG. 11D is an enlarged view
of a section found within FIG. 11C. As shown in FIGS. 11C and 11D,
the tissue matrix of the foam has a fibrous, filamentous nature
after blending and freeze-drying. The aseptically freeze-dried foam
had a dry tissue mass of 9.9.+-.0.3% (w/v, N=5).
[0085] Some of the freeze-dried tissue material was further treated
at -100.degree. C. under vacuum for 24 hours to increase the
strength of the tissue foam. calorimetric measurement detected an
onset denaturation temperature of 62.2.+-.0.1.degree. C. and a
denaturation enthalpy of 60.5 J/g tissue mass, indicating no tissue
collagen denaturation.
[0086] In a separate experiment using the freeze-dried material,
the in vivo response of reconstructive tissue foam made from
flake-like tissue material was investigated using an athymic rat
model (Rattus norvegicus, nude rat). Tissue specimens (10
mm.times.10 mm, and -3 mm thick) were prepared from the tissue foam
and rehydrated in 0.9% saline solution. After rehydration, the
tissue specimens were then implanted subcutaneously in athymic
rats. For each rat, four separate incisions were made on the right
and left side of the back through the skin and parallel to the
lumbar region of the vertebral column. Pockets were formed by blunt
dissection in the subcutaneous tissue in which the tissue material
was introduced. Two specimens were implanted on the right side of
the vertebral column and two specimens were implanted on the right,
with the skin closed thereafter. Animals were euthanized either
four or eight weeks after implantation. Implanted specimens with
attached adjacent soft tissue were excised and the excised tissue
samples were fixated in 10% formalin and processed for histological
evaluation using hematoxylin and eosin stains. Histological slides
were assessed microscopically for evidence of host cell
repopulation and re-vascularization. As shown in FIG. 12,
significant cell repopulation and revascularization were observed
in 4-week implants (FIGS. 12A and 12B), while implanted tissue
foams were fully repopulated in 8-weeks (FIGS. 12C and D).
Inflammation was observed to be mild in 4-week explants, and
subdued in 8-week explants.
[0087] Other embodiments of the invention will be apparent to those
skilled in the art from consideration of the specification and
practice of the invention disclosed herein. It is intended that the
specification and examples be considered as exemplary only, with a
true scope and spirit of the invention being indicated by the
following claims.
* * * * *