U.S. patent application number 14/057521 was filed with the patent office on 2014-04-24 for preservation of biomaterial properties and methods of storing.
This patent application is currently assigned to LIFELINE SCIENTIFIC, INC.. The applicant listed for this patent is LIFELINE SCIENTIFIC, INC.. Invention is credited to Kelvin G. M. BROCKBANK.
Application Number | 20140113273 14/057521 |
Document ID | / |
Family ID | 49551751 |
Filed Date | 2014-04-24 |
United States Patent
Application |
20140113273 |
Kind Code |
A1 |
BROCKBANK; Kelvin G. M. |
April 24, 2014 |
PRESERVATION OF BIOMATERIAL PROPERTIES AND METHODS OF STORING
Abstract
Described herein are enhanced compositions and methods for
storing biomaterials. In certain aspects, these biomaterials
include natural and engineered eukaryotic tissues. The methods
described herein include storing these biomaterials in such a
manner that reduces or prevents the loss of biomaterial properties
(e.g., extracellular matrix integrity, cell viability, or a
combination thereof) occurring either during storage or after
removal of the biomaterial from storage. In certain aspects, these
biomaterials will be stored in animal product-free solutions
containing an agent that prevents or reduces the loss of
extracellular matrix integrity.
Inventors: |
BROCKBANK; Kelvin G. M.;
(Charleston, SC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIFELINE SCIENTIFIC, INC. |
Itasca |
IL |
US |
|
|
Assignee: |
LIFELINE SCIENTIFIC, INC.
Itasca
IL
|
Family ID: |
49551751 |
Appl. No.: |
14/057521 |
Filed: |
October 18, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61715576 |
Oct 18, 2012 |
|
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Current U.S.
Class: |
435/1.1 |
Current CPC
Class: |
A01N 1/0226 20130101;
C12N 5/0655 20130101 |
Class at
Publication: |
435/1.1 |
International
Class: |
A01N 1/02 20060101
A01N001/02 |
Claims
1. A composition comprising a biomaterial placed in a solution that
includes at least one agent that reduces or prevents a loss of
biomaterial properties, wherein the solution is an animal
product-free solution, the biomaterial comprises chondrocytes in an
extracellular matrix or cartilage, and the at least one agent
comprises an enzyme inhibitor of a matrix metalloproteinase having
a concentration ranging from 1.0 nM to 1 mM.
2. The composition of claim 1, wherein the biomaterial properties
comprise extracellular matrix integrity, cell viability, or a
combination thereof.
3. The composition of claim 2, wherein extracellular matrix
integrity comprises extracellular matrix permeability,
extracellular matrix water content, extracellular matrix
glycosaminoglycan content, or any combination thereof.
4. The composition of claim 1, wherein the biomaterial comprises
cartilage.
5. The composition of claim 1, wherein the biomaterial comprises
chondrocytes in an extracellular matrix.
6. The composition of claim 1, wherein the solution does not
include fetal bovine serum.
7. The composition of claim 1, wherein the animal product-free
solution is an extracellular-type solution that is isotonic.
8. The composition of claim 1, wherein the animal product-free
solution is an intracellular-type solution that is isotonic.
9. The composition of claim 1, wherein the enzyme inhibitor
minimizes an enzymatic activity to reduce or prevent the loss of
biomaterial properties, wherein the biomaterial properties include
extracellular matrix integrity.
10. The composition of claim 1, wherein the matrix
metalloproteinase comprises one or more members selected from the
group consisting of MMP 1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP
11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP 19, MMP 20, MMP
21, MMP 23A, MMP23B, MMP24, MMP25, MMP26, MMP27, and MMP28.
11. The composition of claim 1, wherein the enzyme inhibitor is
selected from the group consisting of doxycycline, TIMPs, a
compound that up-regulates endogenous TIMPs, PCK3145, BB-2516, and
BB-94.
12. A method for storing a biomaterial comprising preparing the
composition of claim 1, wherein preparing the composition of claim
1 includes placing the biomaterial in a solution that includes at
least one agent that reduces or prevents a loss of biomaterial
properties.
13. The method of claim 12, further comprising storing the
biomaterial placed in the solution at a temperature ranging from
-25.degree. C. to +35.degree. C.
14. A composition comprising an animal product-free solution,
wherein the solution includes at least one matrix metalloproteinase
inhibitor at concentrations ranging from 1.0 nM to 1000 .mu.M, the
animal product-free solution is an intracellular-type solution that
does not include a cell culture media, and the matrix
metalloproteinase inhibitor reduces or inhibits enzymatic activity
of at least one of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10,
MMP11, MMP12, MMP13, MMP 14, MMP15, MMP16, MMP17, MMP19, MMP20,
MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28 or any
combination thereof.
15. The composition of claim 14, wherein the at least one matrix
metalloproteinase inhibitor reduces or inhibits enzymatic activity
of at least two of MMP1, MMP8, MMP9, and MMP13.
16. The composition of claim 14, wherein the at least one matrix
metalloproteinase inhibitor reduces or inhibits enzymatic activity
of at least three of MMP1, MMP8, MMP9, and MMP13.
17. The composition of claim 14, wherein the at least one matrix
metalloproteinase inhibitor is selected from the group consisting
of doxycycline, TIMPs, a compound that up-regulates endogenous
TIMPs, PCK3145, BB-2516, and BB-94.
18. The composition of claim 14, wherein doxycycline is the at
least one matrix metalloproteinase inhibitor and is present at a
concentration of from 1.0 nM to 1000 .mu.M.
19. The composition of claim 18, wherein the intracellular-type
solution further comprises a nutrient cocktail that includes at
least one of the following components: D-glucose, glycine,
L-arginine hydrochloride, L-cystine hydrochloride, L-glutamine,
L-histidine hydrochloride, L-isoleucine, L-leucine, L-lysine
hydrochloride, L-methionine, L-phenylalanine, L-serine,
L-threonine, L-tryptophan, L-tyrosine, L-valine, choline, D-calcium
pantothenate, folic acid, niacinamide, pyridoxine, riboflavin,
thiamine, inositol, any salt thereof, or any combination
thereof.
20. A method comprising storing a biomaterial at hypothermic
temperatures in the composition of claim 14, wherein the
composition includes at least one additive that promotes retention
of extracellular matrix integrity and cell viability, and the at
least one additive comprises an enzyme inhibitor, an amino acid, a
plurality of amino acids, a sugar, a plurality of sugars, a lipid,
a plurality of lipids, a vitamin, a plurality of vitamins, or any
combination thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This nonprovisional application claims the benefit of U.S.
Provisional Application No. 61/715,576 filed Oct. 18, 2012. The
disclosure of the prior application is hereby incorporated by
reference in its entirety.
TECHNICAL FIELD
[0002] This disclosure relates to methods for storing eukaryotic
biomaterials (e.g., cells in association with materials and
tissues) while reducing or preventing the loss of biomaterial
properties associated with storage. This disclosure further relates
to maintaining biomaterial (i) extracellular matrix integrity,
including extracellular matrix permeability, water content, and
glycosaminoglycan content, and (ii) cell viability during
storage.
BACKGROUND
[0003] Over the past few decades, storage methods and techniques
have been developed to preserve eukaryotic tissues and cells. These
storage methods and techniques are directed to storing various
eukaryotic cells in engineered extracellular matrices, engineered
tissues, and natural tissues for a period of time in a manner that
allows for the use of these stored tissues at a later date, such as
for implantation or transplantation into patients or for drug or
chemical screening bioassays.
[0004] Although these storage methods and techniques are widely
applicable both in basic research and translational research
settings, maintaining biomaterial properties (e.g., extracellular
matrix integrity and cell viability) during storage remains a
challenge. For example, significantly decreased extracellular
matrix permeability and tissue cell viability has been observed
using current techniques, and these decreases can lead to
inefficient biomaterial function after removal from storage.
[0005] In one example, chondrocytes and cartilage tissue are
preserved using various storage techniques, and are subsequently
removed from storage and used as osteochondral allografts. The
allografts can repair (1) trauma-induced cartilage defects and (2)
cartilage surfaces damaged by osteoarthritis. Use of chondrocytes
and cartilage as osteochondral allografts to treat osteoarthritis
is important because it is estimated that osteoarthritis currently
affects about 20 million people in the United States. Thus, as a
result, a large industry has grown to provide orthopedic implants
to treat people with defective joints, osteoporotic fractures, or
back problems resulting from a loss of endogenous cartilage or
resulting from the damage of endogenous cartilage.
[0006] Although osteochondral allografts show promise for treating
cartilage-related medical conditions, chondrocyte viability and
extracellular matrix integrity of transplanted articular cartilage
largely determines the outcome (i.e., a successful surgical outcome
versus a failed surgical outcome, etc.) of osteochondral allograft
transplantation. Current preservation techniques do not acceptably
maintain extracellular matrix integrity of cartilage, and in
certain aspects, chondrocyte viability could be improved. For
example, conventional cryopreservation of chondrocytes and
cartilage includes freezing these cells and tissues in a solution
that includes dimethyl sulfoxide (DMSO), but these techniques
result in death of 80-100% of the chondrocytes in articular
cartilage plus extracellular matrix damage due to ice
formation.
[0007] The poor cryopreservation results discussed above ultimately
led to the practice of transplanting so-called "fresh" articular
segments (i.e., chondrocytes and/or cartilage allografts). For
example, donor-derived osteochondral tissue grafts are typically
harvested within 24 hours of donor death and banked at 4.degree. C.
for up to 42 days for repair of clinical cartilage defects. In
addition, commercially available fresh osteoarticular allografts
are stored for at least 17 days to allow serologic and
microbiologic testing prior to implantation to minimize potential
infection in the recipient.
SUMMARY
[0008] Although osteochondral allograft transplantation has been an
effective treatment for repairing (1) trauma-induced cartilage
defects and (2) cartilage surfaces damaged by osteoarthritis,
numerous challenges still exist for maintaining chondrocyte
viability and extracellular matrix integrity of cartilage during
storage. As demonstrated by recent research, it may be important to
maintain both chondrocyte viability and extracellular matrix
integrity to promote successful allograft transplantation. For
example, if either cell viability and/or matrix integrity decreases
during or after the removal from storage, the likelihood of a
successful transplantation may decrease. These challenges exist
with most eukaryotic cells in either engineered or natural tissues.
Thus, new eukaryotic tissue and cell preservation techniques would
be useful.
[0009] Described herein are compositions and methods for storing
biomaterials. In certain aspects, these biomaterials include
eukaryotic cells and eukaryotic tissues, such as chondrocytes and
cartilage. The methods described herein include storing these
biomaterials in a manner that reduces or prevents the loss of
biomaterial properties, such as extracellular matrix permeability
and chondrocyte viability, occurring either during storage or after
removal of the biomaterials from storage. In certain aspects, these
biomaterials are placed into a solution, which may include
animal-derived products, and are subsequently stored for later use.
In certain aspects, the solutions described herein contain an agent
that prevents or reduces the loss of biomaterial properties, and in
certain aspects, this agent can include an inhibitor of at least
one enzyme. For example, this agent can include a natural or
synthetic matrix metalloproteinase (MMP) inhibitor, which can
include but is not limited to endogenous tissue inhibitors of
metalloproteinase (TIMPs), compounds that regulate TIMP synthesis,
or doxycycline, respectively.
[0010] The advantages of this disclosure will be set forth in part
in the description that follows or may be learned by practice of
the aspects described below. The advantages described below will be
realized and attained by means of the elements and combinations
particularly pointed out in the appended claims. It is to be
understood that both the foregoing general description and the
following detailed description are exemplary and explanatory only
and are not restrictive.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a graph comparing chondrocyte viability and
proliferation after chondrocytes were stored for 28 days in four
different solutions. Viability and proliferation were quantified by
measuring relative fluorescence units (RFUs) of each sample.
[0012] FIG. 2 is a graph showing the correlation coefficient
(R.sup.2) between high cell viability and loss of cartilage matrix
permeability and conductivity occurring during cold storage in 4
different solutions. As shown in FIG. 2, the correlation
coefficient increased from 0.78 to 0.90 during 4 days of
post-storage recovery tissue culture.
[0013] FIG. 3 is a graph illustrating the impact of hypothermic
storage on cartilage permeability based on the electrical
conductivity of cartilage samples in hypotonic saline.
[0014] FIG. 4 is a schematic representation of a compression
chamber used to quantify mechanical properties (e.g., creep
compression) of cartilage.
[0015] FIG. 5 is a graph showing the impact of doxycycline
concentration on cartilage cell viability after various storage
intervals.
[0016] FIG. 6 is a graph showing the impact on porcine cartilage
plug electrical conductivity after one month of refrigerated
storage in various concentrations of doxycycline.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0017] The disclosed methods and compositions may be understood
more readily by reference to the following detailed description of
particular embodiments, the Examples included herein, and to the
Figures and their descriptions. The aspects described below are not
limited to specific compositions and/or methods as described which
may, of course, vary.
[0018] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties, as well as the publications included in the reference
list below, are hereby incorporated by reference into this
application to more fully describe the state of the art to which
this disclosure pertains. The references disclosed are also
individually and specifically incorporated by reference herein for
the specific portions that are referenced.
[0019] Concentrations, amounts, and other numerical data may be
expressed or presented herein in a range format. It is to be
understood that such a range format is used merely for convenience
and brevity and thus should be interpreted flexibly to include not
only the numerical values explicitly recited as the limits of the
range, but also to include all the individual numerical values or
sub-ranges encompassed within the ranges as if each numerical value
and sub-range is explicitly recited. As an illustration, a
numerical range of "about 1 to 5" should be interpreted to include
not only the explicitly recited values of about 1 to about 5, but
also include individual values such as 2, 3, and 4 and sub-ranges
such as from 1-3, from 2-4, and from 3-5, etc. as well as 1, 2, 3,
4, and 5, individually. The same principle applies to ranges
reciting only one numerical value as a minimum or maximum.
Furthermore, such an interpretation should apply regardless of the
breadth of the range or the characteristics being described.
[0020] In this specification and in the claims that follow,
reference will be made to a number of terms that shall be defined
to have the following meanings:
[0021] "Animal product-free" solution includes a solution that does
not include any animal product(s) or any products derived from
animals excluding the biomaterial described further below. "Animal
products" can include fetal bovine serum (FBS), which is an
animal-derived product that includes growth factors and is often
used in conventional cell culture. Thus, in one example, an "animal
product-free" solution can include a solution lacking FBS.
[0022] The term "biomaterial" includes non-plant, mammalian
eukaryotic cells and tissues.
[0023] Described herein are viable biomaterials and methods for
storing such biomaterials. In certain aspects, these biomaterials
include eukaryotic cells in both engineered and natural tissues,
and the methods described herein include storing these biomaterials
in such a manner that either reduces or prevents the loss of
biomaterial properties (e.g., reducing or preventing loss of
extracellular matrix integrity, tissue cell viability, or a
combination thereof) occurring either during storage or after
removal of the biomaterial from storage. In certain aspects, these
biomaterials are placed into a solution, which can include an
animal product-free solution, containing at least one agent that
reduces or prevents a loss of biomaterial properties. Subsequently,
the biomaterials placed into the solution containing at least one
agent are then stored at a particular temperature range until these
biomaterials are further needed. The concentration of the at least
one agent is optimized such that biomaterial properties (e.g.,
extracellular matrix integrity and cell viability) are
maximized.
[0024] In certain aspects, the biomaterials can include any
non-plant, mammalian eukaryotic cells and/or tissues including
primary cells (e.g., non-immortalized cells and/or tissues) and
immortalized cells. In certain aspects, the biomaterials can
include natural and engineered tissues and cells. Examples of
natural and engineered biomaterials can include, but are not
limited to, chondrocytes, cartilage, osteoblasts, osteoclasts,
bone, tissue plugs, allograft tissue plugs, cartilage tissue plugs,
a cornea, heart valves, blood vessels, a ureter, intestine, skin,
teeth, tumor biopsies, intervertebral discs or bodies, ligaments,
tendons, etc. In at least one aspect, the biomaterials include at
least chondrocytes, cartilage, or a combination thereof. In other
aspects, the biomaterials only include chondrocytes, cartilage, or
a combination thereof. In certain aspects, the biomaterials include
autograft tissues, allograft tissues, and xenograft tissues. For
example, with regard to suitable human graft tissues, the allograft
tissues and/or tissue plugs can be derived from a human donor.
Xenograft tissues can be derived from a porcine donor, a bovine
donor, an ovine donor, an equine donor, or any other species for
medical purposes. The tissues described herein may also be derived
from animal species for veterinary applications within the same
species; examples include dogs, cats, sheep, cows, and horses.
[0025] When using the tissues and cells described herein with the
compositions and methods described herein, one objective is to
prevent loss of extracellular matrix integrity and/or reduce or
prevent the loss of cell viability. For example, extracellular
matrix integrity can be determined based on extracellular membrane
permeability, extracellular membrane water content, extracellular
membrane glycosaminoglycan content, or a combination thereof. In
certain aspects, one objective is to maintain at least one of
extracellular membrane permeability, extracellular membrane water
content, extracellular membrane glycosaminoglycan content, or any
combination thereof while storing the biomaterial to prevent or
reduce loss of extracellular matrix integrity. When determining
matrix integrity of the biomaterial, numerous techniques known in
the art can be used. These techniques include matrix electrical
conductivity assays that measure permeability, water content, and
glycosaminoglycan content, indentation tests, stress/strain tests,
elasticity, RAMAN spectroscopy, various microscopic methods (such
as laser scanning microscopy with second harmonic generation), etc.
As further stated above, another objective is to reduce or prevent
the loss of the biomaterial's cell viability. In certain aspects,
various types of cell death, including but not limited to, necrotic
cell death, apoptotic cell death, autophagic (Type II) cell death,
anoikis, and necroptosis can be reduced or prevented using the
compositions and methods described herein, and in certain aspects,
these types of cell death can be limited by the use of an agent as
described further below. In addition, metabolic activity assays
(e.g., a resazurin assay), various cellular staining techniques
(e.g., a Trypan Blue exclusion assay and live/dead stains),
immunohistochemistry, biochemistry and various gene expression
assays can be used.
[0026] In one aspect and when tissues containing a matrix are being
used as a biomaterial, preventing or reducing the loss of
extracellular matrix integrity and loss of cell viability is
important to maintain structural integrity and normal biological
function of the tissue. For example, cartilage contains
chondrocytes (i.e., cells) and an extracellular matrix, wherein the
extracellular matrix is primarily composed of collagen fibers,
proteoglycans, and elastin fibers. Both chondrocyte viability and
cartilage extracellular matrix integrity are important to maintain
normal, physiological biological function in in vivo, ex vivo, and
in vitro applications. For example, the extracellular matrix of
cartilage provides structural integrity and maintains a certain
level of rigidity in vivo, which functions in bone support, proper
joint mobility, etc. In certain aspects, the permeability of the
cartilage's extracellular matrix is of particular importance. For
example, cartilage permeability can be associated with and may play
an important role in maintaining the structural integrity of the
cartilage's extracellular matrix and aiding to maintain chondrocyte
viability as well. In certain aspects, decreased permeability of
the cartilage's extracellular matrix can be associated with
increased chondrocyte viability and decreased cartilage
extracellular matrix structural integrity. This increased viability
and decreased structural integrity due to production of cell
products, such as enzymes, can lead to a decreased likelihood of
successful transplantation when the stored cartilage is being
subsequently used for allograft transplantation. Thus, in certain
aspects, the methods and compositions described herein are used to
prevent or reduce the loss of cartilage extracellular matrix
integrity while reducing and/or preventing the loss of chondrocyte
viability, and in certain aspects, the methods and compositions
described herein are used to reduce and/or prevent the loss of
cartilage extracellular matrix integrity in an allograft while
optimizing chondrocyte viability.
[0027] The biomaterials described herein can be placed into a
solution that prevents or reduces the loss of biomaterial
properties (e.g., extracellular matrix integrity, cell viability,
or a combination thereof), and in certain aspects, this solution
can be either an animal product-free solution (e.g., excludes FBS)
or can contain animal products (e.g., includes FBS). It should be
noted that the below descriptions and embodiments also apply to
solutions containing animal products including the biomaterial. In
certain aspects, the biomaterial is at least partially submerged in
the solution, and in other aspects, the biomaterial is completely
submerged in the solution.
[0028] In one aspect, the solution can be an extracellular-type
solution including at least one agent that prevents or reduces the
loss of biomaterial properties (e.g., extracellular matrix
integrity, cell viability, or a combination thereof). For example,
extracellular-type solutions can include isotonic, plasma-like
solutions with ion complements that mimic the normal extracellular
environment of cells and tissues. These isotonic, plasma-like
solutions can include cell culture medium, which provide various
amino acids and metabolites to the biomaterial (e.g., cells and/or
tissues) for nutritional support. For example, cell culture medium
used for the extracellular-type solution can include, but are not
limited to, Dulbecco's Modified Eagle Medium (DMEM), .alpha.MEM,
Glasgow's MEM, Ham's F10, Ham's F-12, Leibovitz's L-15, Iscove's
Modified DMEM, DMEM/Ham's F-12, and derivatives thereof. The
extracellular-type solution can be animal product-free, such that,
before placing the biomaterial into the cell solution, the cell
solution contains no animal products. For example, when using cell
culture medium, the cell culture medium would not contain fetal
bovine serum (FBS) or any other product derived from an animal.
[0029] In certain aspects, the solution includes an
intracellular-type solution. The intracellular-type solution can
include, but is not limited to, an isotonic solution formulated to
restrict the passive exchange of water and ions between cells in
the biomaterial and intracellular-type solution during storage. For
example, an intracellular-type solution can include a
non-permeating anion such as lactobionate or gluconate to partially
replace chloride ions in the extracellular space, which provides
osmotic support to balance the intracellular oncotic pressure
generated by cytosolic macromolecules and their associated
counter-ions locked inside the cell. Intracellular-type solutions
can include, but are not limited to, VIASPAN.RTM. (i.e., Belzer's
Solution) and UNISOL.RTM. (e.g., SPS-1). Similar to the
extracellular-type solution described above, the intracellular-type
solution can be animal product-free.
[0030] Additional components can be added to the intracellular-type
solution to further supplement the intracellular-type solution and
to further promote biomaterial viability. For example, these
additional components provide additional nutritional support for
the biomaterial, which reduces or prevents the loss of viability of
the biomaterial. These additional components can include, but are
not limited to, a nutrient cocktail having non-animal derived
(i.e., synthetically derived) essential amino acids, synthetically
derived non-essential amino acids, synthetically derived vitamins,
synthetically derived lipids, synthetically derived carbohydrates,
or any combination thereof. Examples of the carbohydrates included
in the nutrient cocktail can further include monosaccharides (e.g.,
glucose, fructose, galactose), disaccharides (e.g., maltose,
lactose, etc.), or a combination thereof. Examples of amino acids
provided in the cocktail can include, but are not limited to, any
combination of glycine, L-arginine, L-cystine, L-glutamine,
L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,
L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine,
L-valine, or any salt thereof. Examples of vitamins provided in the
cocktail can include, but are not limited to, any combination of
choline, D-calcium, folic acid, niacinamide, pyridoxine,
riboflavin, thiamine, inositol, or any salt thereof.
[0031] As indicated above, an agent that prevents or reduces the
loss of biomaterial properties (e.g., extracellular matrix
integrity, cell viability, or a combination thereof) can be
included in the solution. In certain aspects, the agent can prevent
or reduce the loss of extracellular matrix integrity. For example,
agents that prevent or reduce the loss of extracellular matrix
integrity can include small organic compounds, inorganic compounds,
biological molecules (e.g., proteins, polypeptides, peptides,
nucleic acids, nucleic acid aptamers, peptide aptamers), or any
combination thereof that inhibits or reduces the loss of
extracellular matrix integrity in the solution when, for example,
compared to a control. In certain aspects, the agent can reduce the
loss of the biomaterial's properties (e.g., extracellular matrix
integrity) by, for example, 5% or more, 10% or more, 20% or more,
30% or more, 40% or more, 50% or more, 60% or more, 70% or more,
80% or more, 90% or more, or 99% or more when compared to, for
example, a control. Stated another way, the agent can substantially
or completely inhibit the loss of a biomaterial's properties by,
for example, at least 80%, at least 85%, at least 90%, at least
95%, at least 99%, or 100% when compared to, for example, a
control. In certain aspects, the solution includes an agent at
concentrations ranging from 1 pM to 1000 .mu.M, 1 pM to 500 .mu.M,
1 pM to 30 .mu.M, 1 pM to 1000 nM, 1 pM to 500 nM, 1 pM to 250 nM,
100 pM to 750 .mu.M, 100 pM to 500 .mu.M, 100 pM to 20 .mu.M, 100
pM to 1000 nM, 1 pM to 750 nM, 1 pM to 500 nM, 1 pM to 250 nM, 1 pM
to 1 nM, 500 pM to 500 .mu.M, 500 pM to 250 .mu.M, 500 pM to 100
.mu.M, 500 pM to 10 .mu.M, 500 pM to 1000 nM, 500 pM, to 750 nM,
500 pM to 500 nM, 500 pM to 250 nM, 500 pM to 100 nM, 500 pM to 1
nM, 1 nM to 1000 .mu.M, 1 nM to 750 .mu.M 1 nM to 500 .mu.M, 1 nM
to 250 .mu.M, 1 nM to 100 .mu.M, 1 pM to 1 .mu.M, 100 nM to 1000
.mu.M, 100 nM to 750 .mu.M, 100 nM to 500 .mu.M, 100 nM to 250
.mu.M, 100 nM to 100 .mu.M, 100 pM to 1 .mu.M, 250 nM to 1000
.mu.M, 250 nM to 750 .mu.M, 250 nM to 500 .mu.M, 250 nM to 250
.mu.M, 250 nM to 100 .mu.M, 250 nM to 1 .mu.M, 500 nM to 1000
.mu.M, 500 nM to 750 .mu.M, 500 nM to 500 .mu.M, 500 nM to 250
.mu.M, 100 nM to 100 .mu.M, 500 nM to 1 .mu.M, 750 nM to 1000
.mu.M, 750 nM to 750 .mu.M, 750 nM to 500 .mu.M, 750 nM to 250
.mu.M, 750 nM to 100 .mu.M, 750 nM to 1 .mu.M, 0.5 .mu.M to 1000
.mu.M, from 10 .mu.M to 950 .mu.M, from 20 .mu.M to 900 .mu.M, from
30 .mu.M to 850 .mu.M, from 40 .mu.M, to 800 .mu.M, from 50 .mu.M
to 750 .mu.M, from 60 .mu.M to 700 .mu.M, from 70 .mu.M to 650
.mu.M, from 80 .mu.M to 600 .mu.M, from 90 .mu.M to 550 .mu.M, from
100 .mu.M to 500 .mu.M, from 110 .mu.M to 450 .mu.M, from 120
.mu.M, to 400 .mu.M, from 130 .mu.M to 350 .mu.M, from 140 .mu.M to
300 .mu.M, from 150 .mu.M to 250 .mu.M, from 160 .mu.M to 200
.mu.M, from 0.5 .mu.M to 100 .mu.pM, from 1 .mu.M to 90 .mu.M, from
5 .mu.M to 90 .mu.M, from 10 .mu.M to 85 .mu.M, from 10 .mu.M to 75
.mu.M, from 20 .mu.M to 85 .mu.M, from 20 .mu.M to 65 .mu.M, from
30 .mu.M to 70 .mu.M, from 30 to 50 .mu.M, from 40 .mu.M to 80
.mu.M, or from 40 .mu.M to 50 .mu.M, wherein any concentration
occurring within the above ranges can also serve as an endpoint for
a range.
[0032] In one aspect, it is believed that the agent inhibits or
reduces the activity of an enzyme that affects the biomaterial's
properties (e.g., extracellular matrix integrity). Thus, the agent
can act as an enzyme inhibitor of a specific enzyme associated with
promoting damage of the biomaterial (e.g., extracellular matrix
damage). In this aspect, the enzyme inhibitor can be used in the
ranges described above to inhibit or reduce the activity of an
enzyme and to increase retention of biomaterial properties (e.g.,
retention of extracellular matrix integrity). In one aspect, this
enzyme inhibitor can specifically include but is not limited, to a
matrix metalloproteinase (MMP) inhibitor.
[0033] For example, in certain aspects MMPs can adversely affect
biomaterial properties (e.g., extracellular matrix integrity) via
enzymatic degradation of at least a portion of the biomaterial and
potentially lead to inefficient biomaterial function after storage.
Thus, in certain aspects, it is desired to reduce or inhibit MMP
enzyme activity by using an MMP inhibitor. For example, the MMP
inhibitor can inhibit or reduce the enzymatic activity of MMP1,
MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14,
MMP15, MMP16, MMP17, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24,
MMP25, MMP26, MMP27, MMP28 or any combination thereof. In certain
aspects, the MMP inhibitor reduces or inhibits the enzymatic
activity of at least one of MMP1, MMP8, MMP9, MMP13, or any
combination thereof. In certain aspects, the MMP inhibitor reduces
or inhibits the enzymatic activity of at least two of MMP1, MMP 8,
MMP9, MMP13, or any combination thereof. In certain aspects, the
MMP inhibitor reduces or inhibits the enzymatic activity of at
least three of MMP1, MMP 8, MMP9, MMP13, or any combination
thereof. Furthermore, the MMP inhibitor can include but is not
limited to natural or synthetic matrix metalloproteinase (MMP)
inhibitors. Synthetic MMP inhibitors generally contain a chelating
group that tightly binds the catalytic zinc atom at an MMP's active
site. Common chelating groups include hydroxamates, carboxylates,
thiols, and phosphinyls. In certain aspects, hydroxymates are
particularly potent inhibitors of MMPs due to their bidentate
chelation of zinc atoms. Zinc chelators can include
diethyldithiocarbamate (DEDTC) and calcium
ethylenediaminetetraacetic acid (EDTA). In certain aspects, the
inhibitors described herein can include, but are not limited to,
doxycycline, PCK3145 (a synthetic peptide corresponding to amino
acids 31-45 of prostate secretory protein 94), BB-2516
(Marimastat), BB-94(i.e., batimastat, which is
(2R,3S)-N.sup.4-Hydroxy-N1-[(1S)-2-(methylamino)-2-oxo-1-(phenylmethyl)et-
hyl]-2-(2-methylpropyl)-3-[(2-thienylthio)methyl]butanediamide),
compounds that regulate endogenous tissue inhibitors of
metalloproteinase (TIMPs) (e.g., compounds that regulate TIMP
synthesis), or any combination thereof. For example, genipin, a
natural compound, has been shown to upregulate the expression of
TIMP-1. Without wishing to be bound by theory, genipin induced
upregulation of TIMP-1 reduces or inhibits MMP-2 activity, and in
certain aspects, genipin can be used to inhibit or reduce MMP
enzyme activity in the methods and compositions described herein.
Furthermore, transforming growth factor-.beta. (TGF-.beta.)
signaling has been shown to play a pivotal role in extracellular
matrix deposition by stimulating collagen production and other
extracellular matrix proteins and by inhibiting matrix degradation
by up-regulation of the TIMP-1 gene. Therefore, compounds that
regulate TGF-.beta. signaling and ultimately regulate expression
TIMP expression (e.g., TIMP-1 expression) and MMP inhibition may be
used as an inhibitor with the methods described herein.
[0034] In certain aspects, the biomaterial is placed into a
solution that includes at least one agent that reduces or prevents
a loss of extracellular matrix integrity of the biomaterial and at
least one or more additional agents that promote retention of cell
viability.
[0035] After placing the biomaterial into any of the solutions
described above, the biomaterial can then be stored. For example,
after placing the biomaterial into the solution including at least
one agent, this mixture can be stored at various temperatures to
further promote preservation of the biomaterial's extracellular
matrix and to further prevent or reduce a loss of viability of the
biomaterial. For example, these temperatures can include, but are
not limited to, hypothermic temperatures and normothermic
temperatures. When storing the biomaterial in hypothermic
temperatures, it is preferred to reduce or prevent ice nucleation.
In certain aspects, hypothermic temperatures can include
temperatures ranging -25.degree. C. from to +35.degree. C., ranging
from -15.degree. C. from to +30.degree. C., ranging from -5.degree.
C. to +25.degree. C., ranging from -5.degree. C. to +20.degree. C.,
ranging from -5.degree. C. to +15.degree. C., ranging from
-5.degree. C. to +10.degree. C., ranging from -5.degree. C. to
+5.degree. C., ranging from 0.degree. C. to +10.degree. C., ranging
from 0.degree. C. to +9.degree. C., ranging from 0.degree. C. to
+8.degree. C., ranging from 0.degree. C. to +7.degree. C., ranging
from 0.degree. C. to +6.degree. C., ranging from 0.degree. C. to
+5.degree. C., ranging from 0.degree. C. to +5.degree. C., ranging
from 0.degree. C. to +4.degree. C., ranging from 0.degree. C. to
+3.degree. C., ranging from 0.degree. C. to +2.degree. C., ranging
from +1.degree. C. to +8.degree. C., ranging from +1.degree. C. to
+6.degree. C., ranging from +1.degree. C. to +4.degree. C., ranging
from +1.degree. C. to +3.degree. C., ranging from +2.degree. C. to
+9.degree. C., ranging from +2.degree. C. to +6.degree. C., ranging
from +2.degree. C. to +4.degree. C., ranging from +3.degree. C. to
+8.degree. C., ranging from +3.degree. C. to +6.degree. C., ranging
from +3.degree. C. to +5.degree. C., ranging from +4.degree. C. to
+8.degree. C., ranging from +4.degree. C. to +6.degree. C., ranging
from +5.degree. C. to +9.degree. C., ranging from +5.degree. C. to
+7.degree. C., ranging from +6.degree. C. to +10.degree. C.,
ranging from +6.degree. C. to +8.degree. C., ranging from
+7.degree. C. to +9.degree. C., and ranging from +8.degree. C. to
+10.degree. C. In certain aspects and depending on the biomaterial,
hypothermic temperatures may be preferred. For example, if
chondrocytes and/or cartilage are the biomaterial, the chondrocytes
and/or cartilage can be preserved using hypothermic temperatures
described above. For example, if chondrocytes and/or cartilage are
the biomaterial, hypothermic temperatures ranging preferably from
-25.degree. C. to +35.degree. C., ranging more preferably from
-5.degree. C. from to +25.degree. C., and most preferably 0.degree.
C. to +10.degree. C. In certain aspects, the biomaterial can be
stored for hours, days, months or years. For example, it may be
preferable to store chondrocytes and/or cartilage (e.g., cartilage
tissue plugs) from a few hours up to three months, from a few hours
up to two months, from a few hours up to one month, etc.
[0036] In certain aspects, the animal product-free solution of the
stored biomaterial can be replaced at various desired time
intervals. For example, the animal product-free solution can be
replaced twice weekly, one a week, every two weeks, once a month,
once every two month, etc. throughout the duration of biomaterial
storage and until the stored biomaterial is removed from storage
for further use.
[0037] When using the methods and compositions described above, in
certain aspects, the biomaterial includes chondrocytes and/or
cartilage. One objective of this disclosure includes reducing or
preventing the loss of extracellular matrix material properties and
optimizing retention of cell viability of the biomaterial during
storage for later use. In this aspect, the chondrocytes and/or
cartilage are placed into a solution that includes at least one
agent that at least reduces or prevents a loss of extracellular
matrix integrity. The chondrocytes and/or cartilage can be placed
into the extracellular-type solution, wherein the
extracellular-type solution includes a MMP inhibitor at a
concentration as described above, and this mixture can be
subsequently stored at a hypothermic temperature ranging from
-25.degree. C. to +30.degree. C. for a period of time. In another
aspect, the chondrocytes and/or cartilage can be placed into the
intracellular-type solution, wherein the intracellular-type
solution includes a MMP inhibitor at a concentration as described
above, and in certain aspects, this intracellular-type solution
optionally further includes the nutrient cocktail described above
to promote retention of cell viability. The chondrocytes and/or
cartilage placed into the intracellular-type solution are
subsequently stored at a hypothermic temperature ranging from
-25.degree. C. to +25.degree. C. for a period of time.
[0038] In one aspect, it is desirable to determine which solution
best reduces or prevents a loss of biomaterial properties (e.g.,
extracellular matrix integrity including extracellular matrix
permeability, water content, cell viability, etc.) during storage.
While further determining the methods and compositions that best
reduce or prevent a loss of biomaterial properties identical
biomaterials can be placed into two different solutions as
described above (i.e., the intracellular-type and the
extracellular-type). The two solutions will both contain an agent
that reduces or prevents the loss of extracellular matrix
properties of the biomaterial, and the intracellular-type solution
can optionally contain a nutrient cocktail. After placing the
identical biomaterials into the two different solutions, the
biomaterials in the two different solutions will be stored in a
similar manner (i.e., at the same temperature, for the same
duration of time, etc.). After a period of time, the identical
biomaterials that were placed in two different solutions can be
removed from storage and biomaterial properties will be tested
(e.g., cell viability, extracellular matrix permeability, etc.) and
compared to determine which methods and compositions best reduce or
prevent the loss of viability of the biomaterial. In certain
aspects, these methods and techniques will be applied to
chondrocytes and/or cartilage to further determine which solution
best reduces or prevents a loss of biomaterial integrity during
storage.
[0039] In some embodiments, the present disclosure relates to a
composition comprising a biomaterial placed in a solution that
includes at least one agent that reduces or prevents a loss of
biomaterial properties. The biomaterial properties may comprise
extracellular matrix integrity, cell viability, or a combination
thereof. The extracellular matrix integrity may include, for
example, extracellular matrix permeability, extracellular matrix
water content, extracellular matrix glycosaminoglycan content, or
any combination thereof. The biomaterial may include an eukaryotic
tissue. In some embodiments, the biomaterial may comprise
cartilage. In some embodiments, the biomaterial comprises
chondrocytes in an extracellular matrix. In some embodiments, the
biomaterial comprises an allograft material having viable cells. In
some embodiments, the biomaterial may comprise an allograft
material having viable cells and an extracellular matrix, and
wherein the agent reduces or prevents the loss of extracellular
matrix integrity, cell viability, or a combination thereof. In such
embodiments, the allograft material may be cartilage. In
embodiments, the solution may be an animal-product free solution,
such as a solution that does not include fetal bovine serum. In
some embodiments, the solution may be an extracellular-type
solution, such as an isotonic extracellular-type isotonic. In some
embodiments, the solution may be an intracellular-type solution,
such as an isotonic intracellular-type solution.
[0040] In some embodiments, the at least one agent that reduces or
prevents a loss of biomaterial properties is present in the
solution at a concentration ranging from 100 pM to 1 mM. In some
embodiments, the at least one agent is an enzyme inhibitor, such as
an enzyme inhibitor that minimizes an enzymatic activity to reduce
or prevent the loss of biomaterial properties, wherein the
biomaterial properties include extracellular matrix integrity. For
example, the enzyme inhibitor may inhibit at least one matrix
metalloproteinase, such as one or more of MMP 1, MMP2, MMP3, MMP7,
MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17,
MMP 19, MMP 20, MMP 21, MMP 23A, MMP23B, MMP24, MMP25, MMP26,
MMP27, MMP28 or any combination thereof. In some embodiments, the
enzyme inhibitor may be present at a concentration in the solution
ranging from 1.0 nM to 1000 .mu.M. In some embodiments, the enzyme
inhibitor may be selected from the group consisting of doxycycline,
TIMPs, a compound that up-regulates endogenous TIMPs, PCK3145,
BB-2516, and BB-94.
[0041] In some embodiments, the present disclosure relates to a
composition comprising a biomaterial placed in a solution that
includes at least one agent that reduces or prevents a loss of
biomaterial properties, where the solution is an animal
product-free solution that comprises an extracellular-type solution
that is isotonic, wherein the biomaterial comprises chondrocytes in
an extracellular matrix or cartilage, and wherein the at least one
agent comprises an enzyme inhibitor of a matrix metalloproteinase
having a concentration ranging from 1.0 nM to 1 mM. In some
embodiments, the present disclosure relates to a composition
comprising a biomaterial placed in a solution that includes at
least one agent that reduces or prevents a loss of biomaterial
properties, where the solution is an animal product-free solution
that comprises an intracellular-type solution that is isotonic,
wherein the biomaterial comprises chondrocytes in an extracellular
matrix or cartilage, and wherein the at least one agent comprises
an enzyme inhibitor of a matrix metalloproteinase having a
concentration ranging from 1.0 nM to 1 mM.
[0042] In some embodiments, the present disclosure relates to a
method for storing a biomaterial comprising placing the biomaterial
in a solution that includes at least one agent that reduces or
prevents a loss of biomaterial properties. In some embodiments, the
biomaterial may comprise a natural or engineered eukaryotic tissue.
In some embodiments, the biomaterial may comprise cartilage. In
some embodiments, the biomaterial may comprise chondrocytes in an
extracellular matrix. In some embodiments, the biomaterial may
comprise an allograft material having viable cells. In some
embodiments, the biomaterial may comprise an allograft material
having viable cells and an intact extracellular matrix. In some
embodiments, the allograft material may be cartilage. In some
embodiments, the solution may be an animal product-free solution.
In some embodiments, the solution may be an extracellular-type
solution, such as an extracellular-type solution that is isotonic.
In some embodiments, the solution may be an intracellular-type
solution, such as an intracellular-type solution that is isotonic.
In some embodiments, the solution does not include fetal bovine
serum. In some embodiments, the at least one agent may be present
in the solution at a concentration ranging from 1.0 nM to 1 mM. In
some embodiments, the at least one agent may be an enzyme
inhibitor, such as an enzyme inhibitor minimizes an enzymatic
activity to reduce or prevent loss of biomaterial properties,
wherein the biomaterial properties include extracellular matrix
integrity. In some embodiments, the enzyme inhibitor may inhibit at
least one matrix metalloproteinase, such as at least one matrix
metalloproteinase selected from the group consisting of MMP1, MMP2,
MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15,
MMP16, MMP17, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25,
MMP26, MMP27, MMP28 and any combination thereof. In some
embodiments, the enzyme inhibitor may be selected from the group
consisting of doxycycline, TIMPs, a compound that up-regulates
endogenous TIMPs, PCK3145, BB-2516, and BB-94. In some embodiments,
the method may further comprise storing the biomaterial placed in
the solution at a temperature ranging from -25.degree. C. to
+35.degree. C. In some embodiments, the solution is an animal
product-free solution that comprises an extracellular-type
solution, wherein the extracellular-type solution is isotonic,
wherein the biomaterial is chondrocytes in an extracellular matrix
or cartilage, and wherein the at least one agent comprises an
enzyme inhibitor of a matrix metalloproteinase having a
concentration ranging from 1.0 nM to 1 mM. In some embodiments, the
solution is an animal product-free solution that comprises an
intracellular-type solution, wherein the intracellular-type
solution is isotonic, wherein the biomaterial is chondrocytes in an
extracellular matrix or cartilage, and wherein the at least one
agent comprises an enzyme inhibitor of a matrix metalloproteinase
having a concentration ranging from 1.0 nM to 1 mM.
[0043] In some embodiments, the present disclosure relates to a
composition comprising an animal product-free solution, wherein the
solution includes at least one matrix metalloproteinase inhibitor.
In some embodiments, the animal product-free solution includes a
cell culture media. In some embodiments, the animal product-free
solution is an intracellular-type solution that does not include a
cell culture media. In some embodiments, the matrix
metalloproteinase inhibitor reduces or inhibits enzymatic activity
of at least one of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10,
MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20,
MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28 or any
combination thereof. In some embodiments, the at least one matrix
metalloproteinase inhibitor reduces or inhibits enzymatic activity
of at least one of MMP1, MMP8, MMP9, MMP13, or any combination
thereof. In some embodiments, the at least one matrix
metalloproteinase inhibitor reduces or inhibits enzymatic activity
of at least two of MMP1, MMP8, MMP9, MMP13, or any combination
thereof. In some embodiments, the at least one matrix
metalloproteinase inhibitor reduces or inhibits enzymatic activity
of at least three of MMP1, MMP8, MMP9, MMP13, or any combination
thereof. In some embodiments, the at least one matrix
metalloproteinase inhibitor is present in the animal-product free
solution at concentrations ranging from 1.0 nM to 1000 .mu.M. In
some embodiments, the at least one matrix metalloproteinase
inhibitor is present in the animal-product free solution at
concentrations ranging from 100 nM to 100 .mu.M. In some
embodiments, the at least one matrix metalloproteinase inhibitor is
present in the animal-product free solution at concentrations
ranging from 1 pM to 30 .mu.M. In some embodiments, the at least
one matrix metalloproteinase inhibitor is present in the
animal-product free solution at concentrations ranging from 100 pM
to 20 .mu.M. In some embodiments, the at least one matrix
metalloproteinase inhibitor is present in the animal-product free
solution at concentrations ranging from 500 pM to 10 .mu.M. In some
embodiments, the at least one matrix metalloproteinase inhibitor is
present in the animal-product free solution at concentrations
ranging from 1 .mu.M to 5 .mu.M. In some embodiments, the at least
one matrix metalloproteinase inhibitor is selected from the group
consisting of doxycycline, TIMPs, a compound that up-regulates
endogenous TIMPs, PCK3145, BB-2516, and BB-94. In some embodiments,
the at least one matrix metalloproteinase inhibitor is selected
from the group consisting of doxycycline, TIMPs, a compound that
up-regulates endogenous TIMPs, PCK3145, BB-2516, and BB-94. In some
embodiments, the one matrix metalloproteinase inhibitor is
doxycycline ranging from 1.0 nM to 1000 .mu.M. In some embodiments,
the intracellular-type solution further comprises a nutrient
cocktail that includes at least one of the following components:
D-glucose, glycine, L-arginine hydrochloride, L-cystine
hydrochloride, L-glutamine, L-histidine hydrochloride,
L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine,
L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine,
L-valine, choline, D-calcium pantothenate, folic acid, niacinamide,
pyridoxine, riboflavin, thiamine, inositol, any salt thereof, or
any combination thereof. In some embodiments, the
intracellular-type solution further comprises a nutrient cocktail
that includes at least one of the following components: D-glucose,
glycine, L-arginine hydrochloride, L-cystine hydrochloride,
L-glutamine, L-histidine hydrochloride, L-isoleucine, L-leucine,
L-lysine hydrochloride, L-methionine, L-phenylalanine, L-serine,
L-threonine, L-tryptophan, L-tyrosine, L-valine, choline, D-calcium
pantothenate, folic acid, niacinamide, pyridoxine, riboflavin,
thiamine, inositol, any salt thereof, or any combination
thereof.
[0044] In some embodiments, the present disclosure relates to a
composition comprising a biomaterial in a solution that promotes
retention of extracellular matrix integrity and cell viability,
wherein the solution includes an enzyme inhibitor. In some
embodiments, the present disclosure relates to a method comprising
storing a biomaterial at hypothermic temperatures in an
intracellular-type solution with at least one additive that
promotes retention of extracellular matrix integrity and cell
viability. In some embodiments, the at least one additive comprises
an enzyme inhibitor, an amino acid, a plurality of amino acids, a
sugar, a plurality of sugars, a lipid, a plurality of lipids, a
vitamin, a plurality of vitamins, or any combination thereof.
[0045] The foregoing is further illustrated by reference to the
following examples, which are presented for purposes of
illustration and are not intended to limit the scope of the present
disclosure.
EXAMPLES
[0046] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compositions, and methods described and
claimed herein are made and evaluated, and are intended to be
purely exemplary and are not intended to limit the scope of what
the inventors regard as their invention. Efforts have been made to
ensure accuracy with respect to numbers (e.g., amounts,
temperature, etc.) but normal errors and deviations should be
accounted for. Unless indicated otherwise, parts are parts by
weight, temperature is in .degree. C. or is at ambient temperature,
and pressure is at or near atmospheric. There are numerous
variations and combinations of reaction conditions, e.g., component
concentrations, desired solvents, solvent mixtures, temperatures,
pressures and other reaction ranges and conditions that can be used
to optimize the product purity and yield obtained from the
described process.
[0047] Preliminary Research
[0048] Samples were hypothermically stored in various solutions
(e.g., DMEM, SPS, PBS, and UHK) for 28 days. These samples were
subsequently removed from storage and cartilage chondrocyte
viability and permeability (i.e., the cartilage's extracellular
matrix integrity) were evaluated. As shown in FIG. 1, chondrocyte
viability and proliferation were evaluated in samples stored for 28
days in four different solutions. Cells stored in DMEM demonstrated
considerably higher chondrocyte viability than the samples stored
in SPS, PBS, and UHK as shown by assessing viability with the
resazurin reduction metabolic assay. Specifically, the data of FIG.
1 is expressed as the mean RFU/6 mm plug.+-.1 se and * indicates
significant differences at p<0.05. Statistically significant
differences in cell viability were observed between cells stored in
DMEM and the other solutions starting at day 2. DMEM achieved
control levels after 4 days in culture. Untreated control values
are shown as the mean (dashed line).+-.1 se (hatched) at the top of
the figure. The correlation coefficient (R.sup.2) between these
results and loss of cartilage matrix permeability (shown in FIG. 3)
increased over 4 days in post-storage recovery tissue culture (FIG.
1). These data demonstrated that complex extracellular-type culture
media (e.g., DMEM) are best for maintaining chondrocyte functions
(FIG. 1), which correlates with cell survival (i.e., cell
viability). Furthermore, storage of these samples for approximately
one month in both intracellular-type solutions (i.e., SPS and UHK)
resulted in less metabolic during post-recovery proliferation under
physiologic tissue culture conditions (FIG. 1). Similarly, storage
of these samples in phosphate buffered saline (PBS), an
extracellular formulation without nutrients, also demonstrated less
proliferation during post-recovery tissue culture. These
observations suggest that nutrients are responsible for the
significantly better performance (i.e., chondrocyte viability) of
cartilage plugs stored in DMEM (FIG. 1).
[0049] Interestingly, cartilage stored in DMEM demonstrated the
highest cell viability (i.e., RFU Viability values) but the lowest
electrical conductivity (mS/cm) after 4 days of post-storage
recovery. Although this result indicated that DMEM promoted the
highest cell viability, this result also indicated that the
greatest loss of cartilage matrix permeability occurred while
cartilage was stored in DMEM. This observation led to the
hypothesis that retention of cell viability resulted in release of
cell-derived materials that impacted extracellular matrix
permeability. Specifically, these studies demonstrated a strong
correlation (R.sup.2=0.90) between retention of cell viability and
loss of cartilage matrix permeability (FIG. 2). FIG. 2 specifically
shows the correlation coefficient (R.sup.2) between high cell
viability and loss of cartilage matrix permeability and
conductivity, due to cold storage in 4 different solutions
increased from 0.78 to 0.90 during 4 days of post-storage recovery
tissue culture.
[0050] Based on the data of FIGS. 1 and 2, cartilage permeability
was further evaluated in samples stored in the different solutions.
The samples stored in DMEM for 28 days (i.e., DMEM 28) exhibited
significantly lower conductivity than samples stored in the other
solutions (i.e., PBS, UHK, and SPS). FIG. 3 demonstrates the impact
of hypothermic storage of cartilage permeability assessed by
measuring electrical conductivity in hypotonic saline. The data is
expressed as the mean.+-.1 se and * indicates significant
differences at p<0.05 between a DMEM control at day one compared
with storage groups after 28 days, n=5 samples per porcine donor.
The day 28 DMEM group was significantly less in four independent
experiments. Thus, these data further demonstrate the correlation
between high cell viability and low permeability when cartilage was
stored in DMEM.
EXAMPLES
Example 1
[0051] Assessing Impact of Matrix Metalloproteinase (MMP)
Inhibition on Cartilage Properties During Storage in
Extracellular-type Solution.
[0052] Animal product-free culture medium are formulated with
varying concentrations of an MMP inhibitor (e.g., Doxycycline).
Biomaterial properties including chondrocyte viability, cartilage
chemistry, permeability and other biomaterial properties are
compared over hypothermic storage periods of at least one month.
Biomaterial testing is performed using established methods [Yao,
2002; Gu, 2004; Brockbank, 2011 (see reference list below)].
[0053] Doxycycline is used clinically for the treatment of
periodontal disease and is the only MMP inhibitor widely available
for clinical use. Doxycycline has been shown to have beneficial in
vivo effects on cartilage such as reducing MMP 8, 9, and 13
activity in animal models and humans Furthermore, in vitro studies
suggest that Doxycycline may inhibit MMP synthesis as well as MMP
activity.
[0054] Additional MMP inhibitors including, for example, TIMPs,
PCK3145, a synthetic peptide corresponding to amino acids 31-45 of
prostate secretory protein 94, and Marimastat (BB-2516) may also be
effective. Both PCK3145 and Marimastat have been well tolerated in
early Phase clinical studies. It is also likely that solution
exchange at weekly intervals is not needed, however the ratio of
cartilage mass to solution volume may need to be explored. High
Doxycycline concentrations may be needed for long-term storage
without solution exchange.
[0055] Experimental Design:
[0056] Porcine cartilage plugs are obtained, and these plugs are
stored at 4.degree. C. (hypothermic conditions) in animal
product-free DMEM supplemented with 0 to 300 .mu.M Doxycycline. In
certain samples, media is changed weekly, as in prior studies
(FIGS. 1-3), and for other samples, media is not changed during
storage. These two sample sets (i.e., (1) media changed weekly and
(2) no media change) are compared. In certain aspects, it is
desirable to minimize the need for handling of allografts once they
are placed in storage.
[0057] Methods:
[0058] Pig knees are procured post-mortem from adult domestic
Yorkshire cross-farm pigs (25 Kg). After procuring the knees, the
knees are placed in zip lock bags with iodine solution and
transported on ice to the lab for aseptic dissection. Femoral head
cartilage disc-shaped plugs are prepared using sterile punches.
Groups of 5 plugs are placed in storage solution in sterile
containers with and without weekly media exchange for 1-2
months.
[0059] Metabolic Activity:
[0060] A rezasurin reduction assay is used to evaluate the
metabolic activity of control and treated cartilage plugs [O'Brien,
2000; Brockbank, 2011]. Tissue plugs (n=5/experiment/donor) are
incubated in 2 ml of DMEM+10% FBS culture medium for one hour to
equilibrate followed by the addition of 20% resazurin reduction
assay solution under standard cell culture conditions for 3 hours.
The resazurin reduction assay reagent is a fluorometric indicator
based on detection of metabolic activity. The amount of
fluorescence is measured in duplicates by the multimode microplate
reader at an excitation wavelength of 544 nm and an emission
wavelength of 590 nm. This evaluation is performed daily for
several days to allow characterization of re-warmed cells in
tissues (FIG. 2). Resazurin is not cytotoxic at the concentration
employed, so the same tissue samples can be tested on multiple
occasions. Results shortly after rewarming (day 0) demonstrate cell
viability, after 1-2 days a decrease indicates cell death due to
apoptosis, and increases measure cell proliferation. Tissue plugs
are then dried to obtain the dry weight. For each experimental
group and untreated controls, cell metabolic activity are expressed
as relative fluorescence units (RFU) per mg of dry weight or per
tissue plug.
[0061] Other Viability Assessment Methods:
[0062] The metabolic assay described above is the primary viability
assessment method; however, additional viability assessment assays
may be performed. For example, cell viability can be further
determined by fluorescent live/dead staining of cells. The cells
can also be assessed after release from tissue plugs by enzyme
digestion and assessed using the membrane integrity-based Trypan
Blue exclusion assay [Brockbank, 2011]. Cells may also be cultured
in DMEM for at least one week to verify that the cells,
chondrocytes, are actually able to adhere and proliferate in vitro.
Cell counts and digital image analysis may be performed on the
cultures.
[0063] Water, proteoglycan, and collagen contents: After material
property measurements, samples may be lyophilized to determine
water (porosity), proteoglycan (S-GAG), and collagen
(hydroxyproline) contents. Samples are analyzed for porosity based
on Archimedes' principle [Gu, 2004], the S-GAG using a method
described by Farndale (1982), and for hydroxyproline content using
the method of Bergman and Loxley (1970).
[0064] Electrical Conductivity:
[0065] Tissue conductivity is measured at zero fluid flow condition
using a standard apparatus [Gu 2002a; 2002b] which consists of
current and voltage electrodes placed around a Plexiglas chamber
containing each specimen. Employing a combination of a 4-wire
method and a Keithley Source Meter, the resistance (R) across the
specimen is measured at a very low current density of 0.015
mA/cm.sup.2. A current sensing micrometer is used to measure
specimen dimensions, the corresponding electrical conductivity is
generated using the following equation:
.chi.=h/(RA) (1)
[0066] where A is the cross sectional area, and h is the thickness
of the tissue specimen. Electrical conductivity measurements are
performed in either isotonic or hypotonic phosphate buffered saline
(PBS, pH 7.4) at room temperature (22.degree. C.).
[0067] Solute Diffusivity:
[0068] Under a zero fluid flow condition, the electrical
conductivity (.chi.) of a tissue in NaCl solution is related to
Na.sup.+ and Cl.sup.- diffusivities (D.sup..alpha., .alpha.=+, -)
by Maroudas (1968):
.chi.=F.sub.c.sup.2.phi..sup.w(c.sup.+D.sup.++c.sup.-D.sup.-)/RT,
(2)
[0069] where F.sub.c is the Faraday constant, .phi..sup.w is the
volume faction of water (porosity), c.sup.+ is the cation
(Na.sup.+) concentration, and c.sup.- is the anion (Cl.sup.-)
concentration, R is the gas constant, T is the temperature. The
c.sup.+ and c.sup.- can be calculated using Donnan equation
[Maroudas, 1975]:
c.sup.+=(c.sup.F+ {square root over
((c.sup.F).sup.2+4c*.sup.2)})/2, c.sup.-=(-c.sup.F {square root
over ((c.sup.F).sup.2+4c*.sup.2)})/2 (3)
[0070] Here c.sup.F is the tissue fixed charge density (FCD) and c*
is the NaCl concentration of the bathing solution. The tissue FCD
is determined from the measured proteoglycan content. Using the
data of the electrical conductivity, FCD, porosity, and the
concentration of the bathing solution, the ion diffusivities can be
calculated from equation 3. This method can be used for studying
porcine and bovine cartilage tissues [Gu, 2004; Jackson, 2006].
[0071] Compressive Aggregate Modulus and Hydraulic
Permeability:
[0072] The mechanical properties of the cartilage samples are
determined using confined compression creep test. The test is
applied in the load bearing axial direction on a Dynamic Mechanical
Analyzer (Q800, TA Instruments, New Castle, Del.). The specimen is
allowed to equilibrate in PBS at its initial height measured under
a minute compressive tare load in a confined chamber (FIG. 4).
After equilibrium, the swelling stress is recorded at the initial
height and the specimen is subjected to a constant compressive
stress for three hours. Creep data is curve-fitted to the biphasic
theory to obtain the aggregate modulus H.sub.A and hydraulic
permeability [Yao, 2002].
Experimental Data
[0073] In a cold storage experiment using the methods described
immediately above in Example 1, 45 pieces of pig cartilage plugs
were harvested from one pig. 20 pieces of cartilage plugs were
included in the viability test (FIG. 5). In this viability test, 4
plugs were used per Doxycycline concentration. Another 25 plugs
were used for mechanical test (FIG. 6).
[0074] For the data shown in FIG. 5, each plug diameter was 6 mm.
An injectable form of Doxycycline (DOXY 100.TM.) was obtained from
APP Pharmaceuticals, LLC, (Schaumberg, Ill.) with a molecular
weight of 1,025.89 daltons. The storage solutions contained DMEM,
1.44 mg/ml ascorbic acid, manitol 0.9 mg/ml, and different
concentrations of doxycycline (0 uM, 10 uM, 30 uM, 100 uM, 300 uM).
The storage temperature was 4.degree. C. As shown in FIG. 5, the
viability tests were tracked from week 0 to week 4. As shown in
FIG. 6, the mechanical test was only performed at week 4.
[0075] Viability Assessment: Chondrocyte metabolic activity was
assessed using the resazurin reduction method. The resazurin
reduction assay, commonly known as the alamarBlue assay,
incorporates a water soluble fluorometric viability
oxidation-reduction (REDOX) indicator which detects metabolic
activity by both fluorescing and changing color in response to
chemical reduction of the growth medium. Metabolically active cells
reduce resazurin to fluorescing resorufin. Fresh control and
hypothermically stored tissue samples were placed in 37.degree. C.
culture conditions for 1 hour to permit adjustment to tissue
culture conditions in DMEM plus 10% FBS. The tissues were then
incubated for three hours with resazurin working solution, after
which aliquots of medium were placed in microtiter plate wells and
read on a microtiter plate spectrofluorometer at a wavelength of
590 nm The data is expressed as the mean.+-.1 se relative
fluorescent units.
[0076] Biomaterial Testing: Cartilage plugs were also evaluated for
permeability by measuring their electrical conductivity to
determine if cartilage matrix characteristics were being altered
during storage. Specimens were prepared by cutting a 5 mm
cylindrical plug using a corneal trephine from the stored 6 mm
diameter cartilage discs. The samples were tested after 0 and 1
month of storage, the cartilage surfaces were trimmed manually
using a sharp blade. Then conductivity was tested in hypotonic
saline. The height of each specimen was measured with an electrical
current sensing micrometer. All electrical conductivity
measurements were performed in hypotonic saline at room temperature
(22.degree. C.). Electrical conductivity is a material property of
biological tissues. Its value is related to the diffusivity of
small ions inside the tissue, which depend on tissue composition
and structure.
[0077] Statistical methods: One-way ANOVA (p<0.05 being
considered significant) was conducted to determine differences in
mean values of cell fluorescence units and electrical
conductivity.
[0078] The viability was impacted by the presence of doxycycline in
a dose dependent manner. As shown in FIG. 5, the zero group (i.e.,
the group having no doxycycline added) was significantly higher
than all treatment groups at all time points (p<0.05). As
further shown in FIG. 5, the 10 .mu.M group was significantly
higher than all other groups from week 2-4 (FIG. 5; p<0.05).
This data is expressed as the mean.+-.1 standard error of the mean,
n=4 using one way analysis of variance (ANOVA).
[0079] As shown in FIG. 6, the electrical conductivity was lower in
the 0 .mu.M group at one month and all doxycycline groups (i.e., 10
.mu.M, 30 .mu.M, 100 .mu.M, and 300 .mu.M) were similar to the time
zero group (FIG. 6; p<0.05). This data is expressed as the
mean.+-.1 standard error of the mean, n=5 using one way analysis of
variance (ANOVA).
[0080] The results of this experiment with doxycycline demonstrate
that inhibition of MMPs promote retention of electrical
conductivity and permeability, in the presence of viable cells. For
example, this is demonstrated by the 10 .mu.M group viability (FIG.
5) and conductivity results (FIG. 6).
Example 2
[0081] Determining Impact of Key Culture Medium Components on
Cartilage Stored in an Intracellular-type Hypothermic Solution.
[0082] Step I: Animal product-free hypothermic storage solution is
formulated with and without the primary nutrients in Dulbecco's
Modified Eagle Medium (DMEM). Step II: Various concentrations of
Doxycycline are added to the new intracellular-type storage
formulation of Example 2 to minimize MMP activity.
[0083] Experimental Design:
[0084] Step I: A nutrient cocktail based upon the DMEM formulation
is added to Belzer's solution, the lead clinical organ preservation
formulation marketed as SPS-1 (Organ Recovery Systems, Itasca,
Ill.). The nutrient cocktail consists of D-glucose and amino acids
(glycine, L-arginine hydrochloride, L-cystine 2HCl, L-glutamine,
L-histidine hydrochloride-H20, L-isoleucine, L-leucine, L-lysine
hydrochloride, L-methionine, L-phenylalanine, L-serine,
L-threonine, L-tryptophan, L-tyrosine disodium salt dehydrate and
L-valine) at concentrations used for DMEM (Mediatech, Manassas,
Va., Cat #10-014-CM). The modified formulation is compared with the
original formulation. Step II: 0-300 .mu.M Doxycycline is added to
the modified Belzer's solution. Cell viability, biomaterial
properties, and cartilage biochemistry is performed on cartilage
plugs over a period of at least one month of hypothermic storage.
The best Doxycycline dose is selected for comparison with the
optimized extracellular solution from Example 1. Solution change
schedule is also assessed as described in Example 1.
[0085] Belzer's solution (SPS-1) and Doxycycline are selected
because they are FDA cleared products. Step I: The higher viability
values obtained employing DMEM in the preliminary data (FIG. 2) is
most likely due to nutritional components supporting the low level
of metabolism (<10%) anticipated at 4.degree. C. during
hypothermic storage. Step II: This step may be useful because
increased MMP synthesis can occur when chondrocyte viability is
improved by nutrient supplementation in Step I.
Example 3
[0086] Comparing of Extracellular-type and Intracellular-type
Solutions.
[0087] Experimental Design:
[0088] Cartilage plug properties are assessed after storage in the
extracellular-type and intracellular-type preservation formulations
described in Example 1 and Example 2, Plugs are evaluated over a
period of 2 months (e.g., time 0 and after 1, 4 and 8 weeks of
storage) and gene expression is assessed in addition to the assays
used in the earlier examples. Two samples are assessed at each time
point for each group and the experiment is repeated four times (7
groups.times.2 replicates.times.4 experiments=56 samples).
[0089] The purpose of this example is to compare the
extracellular-type solution of Example 1 with the
intracellular-type solution of Example 2. In addition to the cell
viability and ECM assays described above, analysis of gene
expression is conducted to ensure that the chondrocytes are
expressing appropriate pro-cartilage genes relative to chondrocytes
in fresh untreated cartilage.
[0090] Methods:
[0091] Gene Expression: Samples are snap frozen in liquid nitrogen
and stored at -80.degree. C. Total cellular RNA is isolated (RNeasy
Kit, Qiagen, Calif.), reverse-transcribed into cDNA (Omniscript RT
kit, Qiagen, Calif.), evaluated for quality and changes in gene
expression due to storage is quantified using real time PCR.
Retention of phenotype (and/or loss of phenotype) is assessed by
evaluating expression of Sox9, aggrecan, collagen type II (versus
dedifferentiation marker collagen type I), cartilage oligomeric
matrix, ECM resorption marker (MMP-9) plus protein and hypertrophic
marker genes (collagen type 10 and alkaline phosphatase).
[0092] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed invention belongs.
[0093] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
claims.
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