U.S. patent application number 13/476406 was filed with the patent office on 2012-09-13 for compositions and methods for soft tissue augmentation.
This patent application is currently assigned to Humacyte, Inc.. Invention is credited to Juliana Blum, Shannon Dahl, Geoffrey Erickson, Yuling Li, Laura Niklason, Frank Zeigler.
Application Number | 20120230950 13/476406 |
Document ID | / |
Family ID | 40295564 |
Filed Date | 2012-09-13 |
United States Patent
Application |
20120230950 |
Kind Code |
A1 |
Niklason; Laura ; et
al. |
September 13, 2012 |
Compositions and Methods for Soft Tissue Augmentation
Abstract
The present invention provides compositions comprising isolated
human collagen, isolated human elastin and a pharmaceutically
acceptable carrier wherein the human elastin is substantially
insoluble in water with a molecular weight greater than 100 kDa.
The present invention further provides methods and kits for soft
tissue augmentation.
Inventors: |
Niklason; Laura; (Greenwich,
CT) ; Li; Yuling; (Chapel Hill, NC) ; Blum;
Juliana; (Raleigh, NC) ; Dahl; Shannon;
(Durham, NC) ; Erickson; Geoffrey; (Westport,
CT) ; Zeigler; Frank; (Encinitas, CA) |
Assignee: |
Humacyte, Inc.
Morrisville
NC
|
Family ID: |
40295564 |
Appl. No.: |
13/476406 |
Filed: |
May 21, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12220420 |
Jul 24, 2008 |
8198245 |
|
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13476406 |
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60962289 |
Jul 27, 2007 |
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Current U.S.
Class: |
424/85.2 ;
206/571; 424/574; 424/85.1; 424/85.4; 424/93.7; 514/1.1; 514/13.5;
514/18.8; 514/21.92; 514/7.7; 514/8.2; 514/8.4; 514/8.5; 514/8.9;
514/9.1; 514/9.6 |
Current CPC
Class: |
A61K 38/39 20130101;
A61K 2800/91 20130101; A61K 8/64 20130101; A61K 8/65 20130101; A61K
8/735 20130101; A61P 17/10 20180101; A61P 27/00 20180101; A61P
17/00 20180101; A61P 43/00 20180101; A61P 17/02 20180101; A61K
2300/00 20130101; A61K 2800/86 20130101; A61K 8/00 20130101 |
Class at
Publication: |
424/85.2 ;
514/1.1; 424/574; 424/93.7; 514/9.6; 514/8.9; 514/8.2; 514/9.1;
514/13.5; 514/8.5; 424/85.1; 514/8.4; 424/85.4; 514/7.7; 514/21.92;
514/18.8; 206/571 |
International
Class: |
A61K 38/39 20060101
A61K038/39; A61K 35/36 20060101 A61K035/36; A61K 38/18 20060101
A61K038/18; A61K 38/19 20060101 A61K038/19; B65D 65/00 20060101
B65D065/00; A61K 38/21 20060101 A61K038/21; A61P 17/00 20060101
A61P017/00; A61P 27/00 20060101 A61P027/00; A61P 17/10 20060101
A61P017/10; A61P 17/02 20060101 A61P017/02; A61K 35/12 20060101
A61K035/12; A61K 38/20 20060101 A61K038/20 |
Claims
1. A composition comprising isolated human collagen, isolated human
elastin and a pharmaceutically acceptable carrier wherein the human
elastin is substantially insoluble in water with a molecular weight
greater than 100 kDa.
2. The composition of claim 1, wherein the isolated human collagen
is derived from engineered vascular tissue.
3. The composition of claim 1, wherein the isolated human collagen
is derived from micro-bead culture.
4. The composition of claim 1, wherein the isolated human elastin
is derived from engineered vascular tissue or native vascular
tissue.
5. The composition of claim 1, wherein the isolated human collagen
has a molecular weight of about 100 to about 500 kDa.
6. The composition of claim 1, wherein the isolated human elastin
is cross-linked.
7. The composition of claim 1, wherein the composition comprises
about 10-100 mg/ml of isolated human collagen.
8. The composition of claim 1, wherein the composition comprises
about 30 mg/ml of isolated human collagen.
9. The composition of claim 1, wherein the composition comprises
about 2 to about 60 mg/ml of isolated human elastin.
10. The composition of claim 1, wherein the composition comprises
about 3 to 30 mg/ml of isolated human elastin.
11. The composition of claim 1, wherein the composition further
comprises isolated human glycosaminoglycans.
12. The composition of claim 1, wherein the composition further
comprises adipose tissue.
13. The composition of claim 1 wherein the composition further
comprises dermal fibroblasts.
14. The composition of claim 1, wherein the composition further
comprises one or more active agents selected from the group
consisting of one or more anti-inflammatory agents, tissue
formation agents, adipose tissue formation agents, anesthetics,
antioxidants, heparin, epidermal growth factor, transforming growth
factor, transforming growth factor-.beta., platelet-derived growth
factor, fibroblast growth factor, connective tissue activating
peptides, .beta.-thromboglobulin, insulin-like growth factors,
tumor necrosis factors, interleukins, colony stimulating factors,
erythropoietin, nerve growth factors, interferons or combinations
thereof.
15. A dermal or subdermal filler comprising composition of claim
1.
16. The composition of claim 1, wherein said elastin is isolated
from human non-frozen vascular tissue and wherein the composition
does not induce calcification in vivo.
17. A method for soft tissue augmentation in a subject comprising,
administering a composition comprising isolated human collagen,
isolated human elastin and a pharmaceutically acceptable carrier
wherein the human elastin is substantially insoluble in water with
a molecular weight of above 100 kDa.
18. The method of claim 17, wherein the soft tissue augmentation
improves condition selected from the group consisting of lines,
folds, wrinkles, minor facial depressions, cleft lips, correction
of minor deformities due to aging or disease, deformities of the
vocal cords or glottis, deformities of the lip, crow's feet and the
orbital groove around the eye, breast deformities, chin
deformities, augmentation; cheek and/or nose deformities, acne,
surgical scars, scars due to radiation damage or trauma scars, and
rhytids.
19. The method of claim 17, wherein the soft tissue is located in
the pelvic floor, in the peri-urethral area, near the neck of the
urinary bladder, or at the junction of the urinary bladder and the
ureter.
20. The method of claim 17, wherein the soft tissue augmentation
increases tissue volume.
21. The method of claim 17, wherein the composition is injected
into the skin.
22. The method of claim 17, wherein the composition is injected
underneath the skin.
23. The method of claim 17, wherein the composition comprising
insoluble elastin derived from human vascular tissue does not
induce an inflammatory or immune response and does not induce
calcification.
24. A kit for augmentation of a soft tissue comprising composition
of claim 1, a syringe, a sterile wrapper surrounding said syringe
and providing a sterile environment for said syringe and any other
material/and or reagents necessary.
25. The kit of claim 24 wherein said reagents include agents
selected from the group consisting of heparin, epidermal growth
factor, transforming growth factor-alpha, transforming growth
factor-beta, platelet-derived growth factor, fibroblast growth
factor, connective tissue activating peptides,
.beta.-thromboglobulin, insulin-like growth factors, tumor necrosis
factors, interleukins, colony stimulating factors, erythropoietin,
nerve growth factors, interferons, osteogenic factors and bone
morphogenic proteins.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/220,420, filed on Jul. 24, 2008, which
claims the benefit of U.S. Provisional Application No. 60/962,289,
filed on Jul. 27, 2007, the contents of each of which are
incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates generally to compositions comprising
isolated human collagen and isolated human elastin, and generally
related to methods and kits for soft tissue augmentation using
these compositions.
BACKGROUND OF THE INVENTION
[0003] Natural skin is composed of many elements, including dermal
fibroblasts and keratinocytes, hair follicles, nerves and blood
vessels. Extracellular matrix components of skin, which are
responsible for the strength, elasticity and turgor of native,
healthy skin, include collagens, elastin and glycosaminoglycans.
Collagen molecules provide the bulk of the tensile properties of
all connective tissues in the human body, including skin. Elastin
is a very long-lived protein that nonetheless breaks down in the
skin of older individuals. Elastin breakdown contributes to skin
drooping and wrinkles. Hydration is retained in skin by the
presence of glycosaminoglycans, which act as "sponges" to retain
water and provide skin with its natural turgor. Without these
critical extracellular matrix components, skin becomes thin,
wrinkled, and weak.
[0004] Various forms of injectable products have been developed for
skin and other soft tissue augmentation. These products fall into
synthetic and "natural" categories, wherein natural materials are
derived from animal or human tissues. Synthetic materials that have
been used as tissue bulking agents include silicone, oils and
waxes, but these materials suffer from healing complications and
are very viscous and difficult to inject. Animal-derived materials
that have been described include bovine collagen in injectable
forms. However, bovine collagen induces occasional immune reactions
in recipients, due to the fact that bovine collagens are not
identical to human collagens and can serve as antigens for immune
reactivity. Other animal-derived extracellular matrix materials
include hyaluronic acid that is derived from rooster combs. This
material is quite viscous and also has the drawback of being of
non-human origin. Additionally, various preparations of elastin
currently in use have the drawback of inducing calcification upon
implantation.
[0005] The compositions and methods of the present invention
address these problems and fulfill a long felt need in the art.
SUMMARY OF THE INVENTION
[0006] The present invention provides compositions comprising
isolated human collagen, isolated human elastin and a
pharmaceutically acceptable carrier where the human elastin is
substantially insoluble in water with a molecular weight greater
than 100 kDa. The composition can comprise isolated human collagen
derived from engineered vascular tissue or derived from micro-bead
culture. The composition can comprise isolated human elastin
derived from engineered vascular tissue or native vascular tissue.
The isolated human elastin can be cross-linked.
[0007] The compositions can include about 10-100 mg/ml of isolated
human collagen, more preferably about 30 mg/ml of isolated human
collagen. The isolated human collagen can have a molecular weight
of about 100 to about 500 kDa. The compositions can include about 2
to about 60 mg/ml of isolated human elastin, preferably about 3 to
30 mg/ml of isolated human elastin.
[0008] The compositions can further include isolated human
glycosaminoglycans. The compositions can further include one or
more active agents selected from the group consisting of one or
more anti-inflammatory agents, tissue formation agents, adipose
tissue formation agents, anesthetics, antioxidants, heparin,
epidermal growth factor, transforming growth factor, transforming
growth factor-.beta., platelet-derived growth factor, fibroblast
growth factor, connective tissue activating peptides,
.beta.-thromboglobulin, insulin-like growth factors, tumor necrosis
factors, interleukins, colony stimulating factors, erythropoietin,
nerve growth factors, interferons or combinations thereof. The
compositions can further comprise one or more cells or tissues,
preferably adipose tissue or dermal fibroblasts.
[0009] The present invention also provides dermal or subdermal
fillers including isolated human collagen, isolated human elastin
and a pharmaceutically acceptable carrier where the human elastin
is substantially insoluble in water with a molecular weight greater
than 100 kDa.
[0010] The compositions can further include elastin isolated from
human non-frozen vascular tissue which is substantially insoluble
in water. The compositions of the present invention do not induce
calcification in vivo.
[0011] The present invention also provides methods for soft tissue
augmentation in a subject comprising, administering a composition
comprising isolated human collagen, isolated human elastin and a
pharmaceutically acceptable carrier wherein the human elastin is
substantially insoluble in water with a molecular weight greater
than 100 kDa. The method of the soft tissue augmentation can
improve conditions including, but not limited to, lines, folds,
wrinkles, minor facial depressions, cleft lips, correction of minor
deformities due to aging or disease, deformities of the vocal cords
or glottis, deformities of the lip, crow's feet and the orbital
groove around the eye, breast deformities, chin deformities,
augmentation; cheek and/or nose deformities, acne, surgical scars,
scars due to radiation damage or trauma scars, and rhytids. The
soft tissue can be located in the pelvic floor, in the
peri-urethral area, near the neck of the urinary bladder, or at the
junction of the urinary bladder and the ureter. The method of soft
tissue augmentation can increase tissue volume. The compositions
may be injected into the skin or may be injected underneath the
skin. The compositions include insoluble elastin derived from human
vascular tissue that does not induce inflammatory or immune
response and does not induce calcification.
[0012] The present invention also include kits and methods of using
the kits for augmentation of a soft tissue. The present kits
include isolated human collagen, isolated human elastin and a
pharmaceutically acceptable carrier wherein the human elastin is
substantially insoluble in water with a molecular weight greater
than 100 kDa a syringe; a sterile wrapper surrounding said syringe
and providing a sterile environment for said syringe and any other
material/and or reagents necessary. The kits can also include
agents selected from the group consisting of heparin, epidermal
growth factor, transforming growth factor, transforming growth
factor-.beta., platelet-derived growth factor, fibroblast growth
factor, connective tissue activating peptides,
.beta.-thromboglobulin, insulin-like growth factors, tumor necrosis
factors, interleukins, colony stimulating factors, erythropoietin,
nerve growth factors, interferons, osteogenic factors and bone
morphogenic proteins.
[0013] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In the case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0014] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 illustrates the results of a polyacrylamide gel
showing the very high levels of collagen purity in this
preparation, as compared to the purified bovine collagen
control.
[0016] FIG. 2 illustrates the results of a polyacrylamide gel
showing immunoreactivity of isolated proteins with elastin
antibody, showing that elastin is isolated having molecular weights
in the range of approximately 100 kDa or greater.
[0017] FIG. 3 illustrates the low degree of calcification of
implanted elastin preparations in a juvenile rat model showing that
elastin isolated according to the present invention results in
calcification levels that are indistinguishable from vehicle
control.
[0018] FIG. 4 H&E and alizarin red staining of elastin compared
to commercial, purified bovine elastin, implanted into juvenile
rats showing that calcification of elastin that is isolated
according to the method of invention is negligible, while
calcification of bovine elastin is extensive. Panel A and Panel B
show H&E stain of explanted tissues 21 days after implantation
of human elastin that was isolated according to the present
invention ("Humacyte") (Panel A) and commercially obtained bovine
elastin ("Bovine") (Panel B). Panel C and Panel D show alizarin red
stain of explanted tissues 21 days after implantation of human
elastin that was isolated according to the present invention
("Humacyte") (Panel C) and commercially obtained bovine elastin
("Bovine") (Panel D).
[0019] FIG. 5 illustrate the results of H&E and alizarin red
staining of elastin compared to syngeneic rat aorta, implanted into
juvenile rats, showing that implanted elastin calcification
comparable to or less than that induced by syngeneic aorta. Panel A
and Panel B show H&E stain of explanted tissues 21 days after
implantation of human elastin that was isolated according to the
present invention ("Humacyte") (Panel A) and syngeneic rat aorta
containing elastin ("Rat") (Panel B). Panel C and Panel D show
alizarin red stain of explanted tissues 21 days after implantation
of human elastin that was isolated according to the present
invention ("Humacyte") (Panel C) and syngeneic rat aorta containing
elastin ("Rat") (Panel D).
[0020] FIG. 6 illustrates the results of H&E and alizarin red
staining of elastin compared to phosphate buffered saline carrier,
implanted into juvenile rats showing that implanted elastin
calcification is comparable to or less than that induced by saline
carrier. Panel A and Panel B show H&E stain of explanted
tissues 21 days after implantation of human elastin that was
isolated according to the present invention ("Humacyte") (Panel A)
and carrier ("PBS") (Panel B). Panel C and Panel D show alizarin
red stain of explanted tissues 21 days after implantation of human
elastin that was isolated according to the present invention
("Humacyte") (Panel C) and carrier ("PBS") (Panel D).
[0021] FIG. 7 illustrates the results of a polyacrylamide gel
stained with comassie blue for total protein showing that human
collagen isolated according to the present invention exhibit very
high purity.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The present invention provides compositions for the
augmentation of skin and other soft tissues. Preferably, the
compositions are formulated for injection. The compositions are
composed of extracellular matrix components that are derived from
vascular tissues, including, but not limited to, collagens, elastin
and glycosaminoglycans. The extracellular matrix elements are
combined in such a way as to improve their similarity to human skin
extracellular matrix components, and also to increase their
longevity in vivo and to minimize complications of administration.
Deriving extracellular matrix from vascular tissues produces a
"vascular-supporting" injectable formulation, which encourages host
blood vessels to infiltrate and support the injected product. Such
vascular-derived extracellular matrix compositions have the
advantage of incorporating more easily into the host, and of
stimulating the formation of nourishing blood vessels to the
treated skin or other soft tissue. The extracellular matrix
components are entirely of human origin, and may be derived from
engineered or from native tissues. Unlike other injectable
formulations for skin augmentation that contain only collagens or
only animal-derived hyaluronans, these formulations contain other
human extracellular matrix components that render them more similar
to native, healthy human skin.
Soft Tissue Augmentation
[0023] Augmentation of soft tissue, such as skin, can be an
important factor in recovering from injury or for cosmetic
purposes. For example, with normal aging, skin may become loose or
creases can form, such as nasal-labial folds. In the face, creases
or lines may adversely affect a person's self esteem or even a
career. Thus, there has been a need for compositions and methods
that can diminish the appearance of creases or lines.
[0024] Further, there are situations in which loss of tissue can
leave an indentation in the skin. For example surgical removal of a
dermal cyst, lipoatrophy or solid tumor can result in loss of
tissue volume. In other cases, injuries, such as gunshot wounds,
knife wounds, or other excavating injures may leave an indentation
in the skin. Regardless of the cause, it can be desirable to
provide a dermal filler that can increase the volume of tissue to
provide a smoother or more even appearance.
[0025] One example for needed support is dermal augmentation in the
face where dermal and subdermal volume is lost due to aging.
[0026] The term "soft tissue augmentation" includes, but is not
limited to, the following: dermal tissue augmentation; filling of
lines, folds, wrinkles, minor facial depressions, cleft lips and
the like, especially in the face and neck; correction of minor
deformities due to aging or disease, including in the hands and
feet, fingers and toes; augmentation of the vocal cords or glottis
to rehabilitate speech; hemostatic agent, dermal filling of sleep
lines and expression lines; replacement of dermal and subcutaneous
tissue lost due to aging; lip augmentation; filling of crow's feet
and the orbital groove around the eye; breast augmentation; chin
augmentation; augmentation of the cheek and/or nose; bulking agent
for periurethral support, filling of indentations in the soft
tissue, dermal or subcutaneous, due to, e.g., overzealous
liposuction or other trauma; filling of acne or traumatic scars and
rhytids; filling of nasolabial lines, nasoglabellar lines and
infraoral lines. Moreover, the present invention can be directed to
hard tissue augmentation. The term "hard tissue" includes but is
not limited to bone, cartilage and ligament.
[0027] The soft tissue can be located in the pelvic floor, in the
peri-urethral area, near the neck of the urinary bladder, or at the
junction of the urinary bladder and the ureter.
[0028] The term "augmentation" means the repair, decrease,
reduction or alleviation of at least one symptom or defect
attributed due to loss or absence of tissue, by providing,
supplying, augmenting, or replacing such tissue with the
compositions of the present invention. The compositions of the
present invention can also be used to prevent at least one symptom
or defect.
[0029] Dermal fillers are used to fill scars, depressions and
wrinkles. Dermal filler substances have various responses in the
dermis from phagocytosis to foreign body reactions depending on the
material (Lemperle et al., Aesthetic Plast. Surg. 27(5):354-366;
discussion 367 (2003)). One goal of dermal fillers is to
temporarily augment the dermis to correct the surface contour of
the skin without producing an unacceptable inflammatory reaction,
hypersensitivity reaction or foreign body reaction that causes
pain, redness or excessive scar formation for a period of time.
[0030] The ideal material for human skin augmentation would include
one or more of the critical extracellular matrix elements that
provide skin its mechanical properties. These elements include
collagen, elastin and glycosaminoglycans. In addition, to obviate
immune responses, these materials should optimally be of human
origin. Human materials will also induce less inflammatory reaction
than animal-derived materials, and hence will be likely to persist
longer after injection into the recipient, thereby extending and
improving the cosmetic effect of a formulation suitable for
injection.
[0031] Many types of dermal filling procedures can benefit from the
use of the compositions of the present invention. The uses of the
present invention are designed (but not limited) to be used to
provide increased volume of a tissue that, through disease, injury
or congenital property, is less than desired. Compositions can be
made to suit a particular purpose, and have desired retention times
and physical and/or chemical properties.
[0032] Exemplary uses of compositions of this invention can be
particularly desirable to fill facial tissue (e.g., nasolabial
folds), to increase the volume of the dermis in the lips, nose,
around the eyes, the ears and other readily visible tissue.
Additionally, the compositions can be desirably used to provide
bulk to increase the volume of skin secondary to excavating
injuries or surgeries. For example, the site around a dermal cyst
can be filled to decrease the appearance of a dimple at the site of
surgery.
[0033] As such, the present invention provides methods of skin
augmentation by administering the extracellular matrix compositions
of the invention to a subject in need thereof. Preferably, the
methods improve skin wrinkles and/or increase skin volume. The
subject or patient treated by the methods of the invention is a
mammal, more preferably a human. The following properties or
applications of these methods will essentially be described for
humans although they may also be applied to non-human mammals,
e.g., apes, monkeys, dogs, mice, etc. The invention therefore can
also be used in a veterinarian context.
Extracellular Matrix Protein Compositions
[0034] The present invention provides compositions comprising
isolated human collagen, isolated insoluble human elastin and a
pharmaceutically acceptable carrier. These compositions may include
additional proteins and active agents as described in further
detail herein.
[0035] The compositions of the present invention which combine
collagen with other elements of native skin, such as elastin, and
in some embodiments, glycosaminoglycans, provide superior tissue
augmentation, elasticity and turgor, as compared to compositions
comprising a single extracellular matrix component (e.g. collagen).
These compositions comprising isolated human collagen and elastin
provide increased persistence in vivo as compared to compositions
comprising collagen alone, due to the improved similarity of the
collagen/elastin matrix to natural human skin.
[0036] Further, as the extracellular matrix components are derived
from human vascular tissues that are subjected to decellularization
prior to isolation of extracellular matrix components, the
compositions provide longer persistence and retention in vivo (due
to less inflammatory breakdown), and will be less prone to
inflammation, calcification, and immune reaction, than components
derived from animal sources and isolated without a
decellularization step.
[0037] With respect to calcification, this complication is known to
exist for various purified forms of elastin, though the mechanism
that causes the calcification remains unclear (Lee, et al.,
American Journal of Pathology 2006; 168: 490-498; Daamenet al.,
Biomaterials 2005; 26: 81-92; Hollinger et al., Calcified Tissue
International 1988; 42: 231-236; Urry et al., Calcified Tissue
Research 1976; 21: 57-65). Competing hypotheses for elastin
calcification advanced by those skilled in the art include the
intrinsic nature of elastin pentapeptides to induce calcification,
the central role of metalloproteinases in inducing calcification
and the central role of microfibril impurities in elastin
calcification. However, the precise cause of elastin calcification
in vivo remains unknown.
Collagen
[0038] The compositions of the present invention include an
effective amount of isolated human collagen and a pharmaceutically
acceptable carrier. Preferably, the human collagen is derived from
engineered tissue in vitro and has a molecular weight of
approximately 100 kDa to approximately 500 kDa. Preferably, the
compositions of the present invention comprise about 10 mg/mL-100
mg/mL of isolated human collagen, preferably about 15 mg/ml-70
mg/ml of isolated human collagen, more preferably about 20 mg/ml-60
mg/ml of isolated human collagen and most preferably 30 mg/ml of
isolated human collagen.
[0039] To produce isolated human collagen, human vascular cells are
cultured in vitro so as to maximize their production of collagenous
matrix. This is accomplished by a combination of carefully selected
growth factors and culture medium components, combined with
physical stimuli of cells (such as stretching, shearing or
stirring) to increase collagen matrix synthesis (see, for example
U.S. Pat. No. 6,537,567). This cultured tissue is then subjected to
a decellularization process that removes cellular components and
leaves behind a mostly collagen-based extracellular matrix (see,
for example U.S. Pat. No. 6,962,814). The collagen in this matrix
can then isolated by one of several methods known in the art.
[0040] The collagen derived as described above has several
advantages over collagen derived from native tissues or using
previously-described methods to derive engineered collagen.
Engineered tissues are derived from cells that are banked and
highly screened for infectious agents, which makes this material
generally safer than materials derived from cadavers. Also, the
material is derived from vascular smooth muscle cells, resulting in
a "vascular-friendly" extracellular matrix material that supports
the formation of nourishing blood vessels. Further, this method for
collagen isolation incorporates a decellularization step, whereby
cellular components and proteins are actively removed from the
collagen matrix. This provides a highly pure collagen matrix
product (at least 70-80% purity as determined by any assay known in
the art, such as SDS PAGE analysis) and decreases the potential for
immune reaction to non-extracellular matrix components.
Elastin
[0041] The compositions of the present invention also include an
effective amount of isolated human elastin and a pharmaceutically
acceptable carrier. Preferably, the compositions of the present
invention comprise human elastin that is cross-linked and
insoluble. Further, it is preferable that the compositions of the
present invention comprise human elastin that has a molecular
weight of approximately 100 kDa, and more preferably greater then
100 kDa, as determined by any assay known in the art such as SDS
PAGE analysis. Moreover, the compositions of the present invention
comprise a particle size less than about 200 preferably less than
about 100 .mu.m, more preferably less than about 50 .mu.m. The
compositions comprise about 2-60 mg/ml of isolated human elastin,
preferably 3-30 mg/ml of isolated human elastin. The isolated
cross-linked elastin is substantially insoluble in water, wherein
the water-soluble elastin content is in the range of 0.1-10 wt %,
preferably in the range of 0.1-8 wt %, more preferably in range of
0.1-6 wt %, more preferably in the range of 0.1-4 wt %, more
preferably in the range of 0.1-2 wt % and most preferably in the
range of 0.1-1 wt %. Alternatively, the elastin is completely
insoluble in water. In some embodiments, it is preferable to have
elastin with amino acid length which permits the persistence of the
protein in vivo.
[0042] To produce isolated human elastin, human vascular cells are
cultured in vitro so as to maximize their production of
cross-linked elastin. The cross-linked elastin will be insoluble
and will permit the persistence of the protein in vivo. While there
are multiple reports of cells producing non-crosslinked
tropoelastin monomers in culture, it is known to be very difficult
to stimulate the formation of cross-linked elastin from human
vascular cells in vitro. However, the present invention provides
culture conditions whereby creation of insoluble elastin is
achieved, as documented by the presence of desmosine cross-links
that are specific to elastin. These tissues that contain elastin
may then be subjected to a decellularization process (as described
above for collagen), after which the elastin is collected from the
remaining matrix using any one of several standard techniques known
in the art.
[0043] The purity of elastin is typically assessed by the profile
of amino acids in the final product, and by the presence of
desmosine cross-links, which are specific for cross-linked and
insoluble elastin. The amino acid compositions of elastin from
various species have been reported (Starcher et al., Analytical
Biochemistry 1976; 74: 441-447). In particular, it is known that
alanine residue concentrations of greater than 200/1000 total
residues, and valine residues of greater than 70/1000 total
residues, are consistent with highly pure elastin (Daamen et al.,
Biomaterials 2001; 22: 1997-2005). However, many methods are
reported for the isolation of purified elastin, and no consensus
has been reached regarding the optimal method for elastin isolation
and implantation (Daamen, W. F., Hafmans, T., Veerkamp, J. H., van
Kuppevelt, T. H., "Isolation of intact elastin fibers devoid of
microfibrils", Tissue Engineering 2005; 11: 1168-1176).
[0044] The elastin derived as described above has several
advantages over previous reports of elastin isolation. The elastin
would be derived from human, and not animal, origin. Since the
cells used to produce the elastin are banked and derived from human
vascular tissue, this will result in lower immunogenicity in the
human recipients. The elastin thus generated is of a "vascular"
type, which should also promote the infiltration of blood vessels
into the treated area to support tissue reconstitution. The
decellularization process, as with the collagen production, removes
unwanted and potentially immunogenic cellular components from the
elastin matrix. This provides a highly pure elastin matrix product
(>70-80% purity and means that this elastin product has a lower
propensity for immune reaction, inflammation, and calcification,
which are known complications of implantation of xenogeneic
elastins. Preferably, the isolated human elastin of the present
invention comprises desmosine cross-links. More preferably, the
desmosine cross-links are present in insoluble elastin at a ratio
of above 10,000 picomoles per milligram of vascular tissue.
[0045] In addition to the methods described above, human elastin
may also be isolated from native human blood vessels by means that
ensure very high purity, and thus minimize chances for immune
reaction, inflammation, and calcification. Indeed, calcification is
one particular complication associated with elastin implantation,
and hence having species matching and highly pure formations, that
will minimize inflammatory response, are important to cosmetic
outcome and function.
[0046] An immune and inflammatory response can be measured by
various assays known in the art such as, but not limited to,
MHC-peptide tetramer, ELISPOT, intracellular cytokine assay. In
general, a 10-50% increase in T-lymphocytes over the base line
level (e.g., wild type normal state), preferably a 50% increase in
T-lymphocytes, more preferably a 40% increase in T-lymphocytes, and
most preferably a 30% increase in T-lymphocyte production indicates
a significant immune response.
[0047] Calcification levels can be measured by various assays known
in the art such as, but not limited to, atomic spectroscopy and
H&E and alizarin red staining. In general, 75-99% reduction in
calcification, preferably 80% reduction in calcification, more
preferably a 90% reduction in calcification, most preferably 95%
reduction in calcification, indicates significant reduction in
calcification with the compositions of the present invention as
compared to other control forms of elastin such as purified bovine
elastin (for example, from Elastin Products Co). That is, the
compositions of the present invention do not induce significant
calcification, e.g., calcification greater than 25%, preferably
calcification between 5-20%, more preferably between 10 and 15% as
compared to the vehicle control (or the wild type normal state in a
subject prior to administration). Alternatively, calcification
levels of elastin preparation are indistinguishable form vehicle
control.
[0048] In this embodiment, human blood vessels are treated with a
decellularization process that removes cellular components and
glycosaminoglycans. Preferably, the human blood vessels are
extracted from discarded human umbilical cords, but may be obtained
from other parts of the body, including the aorta or other major
arteries or veins.
[0049] The blood vessels thus treated consist primarily of collagen
and elastin. Collagen may be removed from such treated blood
vessels by any one of a number of methods known in the art,
including autoclave treatment, pepsin digestion, collagenase
digestion, high salt treatments, alkali treatments, etc. In this
way, collagen matrix is removed and elastin is retained.
Glycosaminoglycans
[0050] The compositions of the present invention may also include
an effective amount of one or more isolated human
glycosaminoglycans and a pharmaceutically acceptable carrier. Human
glycosaminoglycans may be isolated from engineered tissues.
Engineered tissues, grown in serum-containing medium, produce an
extracellular matrix with a higher content of glycosaminoglycans
than corresponding native tissues. Thus, extracellular matrix
synthesized during culture contains high quantities of
glycosaminoglycans and is consequently more "watery" than native
tissues. Hence, engineered tissues are ideal for the production and
isolation of glycosaminoglycans, which bind water and confer tissue
turgor to connective tissues.
[0051] To produce isolated human glycosaminoglycans, human vascular
cells are cultured in medium containing high serum (i.e. >10% by
volume of serum), and after several weeks, tissues are removed from
culture and treated with hyaluronidase or other
glycosaminoglycan-cleaving enzymes. Supernatant from this digestion
is collected, containing high molecular weight glycosaminoglycans
that may be isolated using dialysis, centrifugation, or other
techniques known in the art. These glycosaminoglycans may then be
used to confer tissue turgor to a treated area.
[0052] In addition to the methods described above, human
glycosaminoglycans and human hyaluronic acid may be derived from
native vascular tissues. Native human blood vessels are treated
with a protease such as pepsin or collagenase, in order to break up
confining, fibrillar extracellular matrix. Preferably, the native
blood vessels are extracted from discarded human umbilical cords.
Such protease pre-treatment exposes glycosaminoglycans and
hyaluronans to aqueous solution and allows swelling.
Glycosaminoglycans and hyaluronans may then be collected from
vascular tissues by any of a variety of techniques known in the
art, including treatment with hyaluronidase, detergent treatment,
or treatment with other enzymes that cleave glycosaminoglycan
moieties.
[0053] The supernatant collected from this treatment can then be
purified for high molecular weight glycosaminoglycans by any of a
variety of methods, including dialysis, centrifugation, immune
isolation and precipitation, etc. Preferably, the
glycosaminoglycans have a MW greater than 100,000 kDa.
Additional Active Agents
[0054] The compositions of the present invention may also include
an effective amount of one or more active agents and a
pharmaceutically acceptable carrier. In some embodiments, it may be
useful to include one or more anti-inflammatory agents, tissue
formation agents, anesthetics, antioxidants and the like, or
combinations thereof.
[0055] Anti-inflammatory agents can include, but are not limited
to, naproxen, sulindac, tolmetin, ketorolac, celecoxib, ibuprofen,
diclofenac, acetylsalicylic acid, nabumetone, etodolac,
indomethacin, piroxicam, cox-2 inhibitors, ketoprofen, antiplatelet
medications, salsalate, valdecoxib, oxaprozin, diflunisal,
flurbiprofen, corticosteroids, MMP inhibitors and leukotriene
modifiers or combinations thereof.
[0056] Agents that increase formation of new tissues at the site of
application can include, but are not limited to, fibroblast growth
factor (FGF), transforming growth factor-beta (TGF-.beta.),
platelet-derived growth factor (PDGF) and/or fragments of
angiotensin II (A-II) or combinations thereof.
[0057] Anesthetics can include, but are not limited to, those used
in caudal, epidural, inhalation, injectable, retrobulbar, and
spinal applications, such as bupivacaine, lidocaine, benzocaine,
cetacaine, ropivacaine, and tetracaine, or combinations
thereof.
[0058] Antioxidants can include, but are not limited to, Vitamin C,
Vitamin A, Vitamin E, .beta.-carotene, superoxide dismutase,
catalase, selenoenzyme glutathione peroxidase,
ubiquinones/ubiquinols, thioredoxin reductase, propyl, octyl and
dodecyl esters of gallic acid, butylated hydroxyanisole (BHA),
butylated hydroxytoluene (BHT) and nordihydroguaiaretic acid or
combinations thereof.
[0059] Compositions used in the invention may additionally include
one or more biologically active agents to aid in the healing or
regrowth of natural tissue. For example, one may incorporate
factors such as heparin, connective tissue activating peptides,
.beta.-thromboglobulin, insulin-like growth factors, tumor necrosis
factors, interleukins, colony stimulating factors, erythropoietin,
nerve growth factors, interferons, osteogenic factors including
bone morphogenic proteins, and the like.
[0060] Any drug or other agent which is compatible with the
compositions and methods of manufacture may be used with the
present invention. Decisions to use such drug or agent are
typically made by the attending physician based on judgments about
the injury or defect being repaired.
Methods of Isolating and Purifying Extracellular Matrix
Proteins
[0061] There are numerous art recognized techniques that can be
used to extract extracellular matrix components from native and
engineered tissues. Specific enzymes that may be used to extract
collagen and elastin matrix components include, but are not limited
to, collagenase; pepsin; trypsin; elastase; matrix
metalloproteinases; dispase; serine proteases; other suitable
proteases; high concentrations of salts such as NaCl or other
salts; alkali treatment; acid treatment; Heat (for example,
autoclaving, boiling, or baking); detergents (for example, SDS or
CHAPS) and/or hypotonic treatment, (for example, water) or
combinations of these treatments.
[0062] There are numerous art recognized techniques that can be
used to isolate and purify the extracellular matrix components that
are extracted from the engineered or native vascular tissues. Such
methods may include, but are not limited to, centrifugation; salt
precipitation of proteins such as collagen; immunoprecipitation;
antibody-mediated binding to beads followed by cleavage to isolate
matrix component; isolation based upon
hydrophobicity/hydrophilicity (for example, extracting hydrophobic
elastin by adhesion to hydrophobic substrate such as polystyrene);
dialysis (to remove low molecular weight contaminants, enzymes,
salt, acid, for example); drying; altering pH of solution to induce
precipitation of extracellular components; inactivation of enzymes
that were used for isolation; and/or chromatographic methods (for
example, polyacrylamide gel electrophoresis or high performance
liquid chromatography that separate components based upon charge
and molecular weight); or combinations of these treatments.
[0063] There are numerous art recognized techniques that can be
used to decellularize engineered or native tissues prior to
extracellular matrix isolation, in order to increase the purity of
the extracted matrix, enhance its biocompatibility and persistence
in vivo, and to ease the isolation of selected matrix components.
In one example, aqueous hypotonic or low ionic strength solutions
facilitate cell lysis in engineered and native tissues through
osmotic effects. Such solutions may comprise deionized water or an
aqueous hypotonic buffer (e.g., at a pH of approximately 5.5 to 8,
preferably approximately 7 to 7.5). Decellularization may be
accomplished using a single decellularization solution, or the
construct may be incubated sequentially in two or more solutions.
Another approach involves immersing the construct in alternating
hypertonic and hypotonic solutions.
[0064] Preferred decellularization agents include, but are not
limited to, salts, detergent/emulsification agents and enzymes such
as proteases, and/or nucleases. Combinations of different classes
of detergents, e.g., a nonionic detergent such as Triton X-100
(tert-octylphenylpolyoxyethylene) and an ionic detergent such as
SDS (sodium dodecyl sulfate) may be employed. Preferably, one or
more decellularization solutions include Triton X-100, CHAPS
(3[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate), or
SDS in phosphate buffered saline (PBS). Other suitable detergents
include polyoxyethylene (20) sorbitan mono-oleate and
polyoxyethylene (80) sorbitan mono-oleate (Tween 20 and 80), sodium
deoxycholate, and octyl-glucoside. In certain preferred
embodiments, various additives such as metal ion chelators, e.g.,
EDTA (ethylenediaminetetraacetic acid) and/or protease inhibitors
are included in the decellularization solution. Suitable protease
inhibitors for use in decellularization solutions include, but are
not limited to, one or more of the following:
phenylmethylsulfonyl-fluoride (PMSF), aprotinin, leupeptin, and
N-ethylmaleimide (NEM).
[0065] Various enzymes that degrade cellular components may be
included in the decellularization solution. Such enzymes include
nucleases (e.g., DNAses such as DNAse I, RNAses such as RNAse A),
and phospholipases (e.g., phospholipase A or C). Certain proteases
such as dispase II, trypsin, and thermolysin may be of use in
decellularization. The decellularization solution preferably
includes a buffer. In general, a pH between about 5.5 and 8.0,
preferably between about 6.0 and 7.8, more preferably between about
7.0 and 7.5 is employed. Preferred buffers include organic buffers
such as Tris (hydroxymethyl) aminomethane (TRIS),
(N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid] (HEPES),
etc. Buffers including sodium phosphate, citrate, bicarbonate,
acetate, or glutamate may also be used.
[0066] Physical forces such as the formation of intracellular ice
may be employed as a primary means of accomplishing
decellularization or to augment the activity of decellularization
solutions. One such approach referred to as vapor phase freezing
involves placing the construct or tissue in an appropriate
solution, e.g., a standard cryopreservation solution such as
Dulbecco's Modified Eagle Medium (DMEM), 10% dimethylsulfoxide
(DMSO), 10% fetal bovine serum (FBS) and cooling at a slow rate,
e.g., 1-2.degree. C. Multiple freeze-thaw cycles may be employed.
Colloid-forming materials may be added to the solution to reduce
extracellular ice formation while allowing formation of
intracellular ice. Appropriate materials include
polyvinylpyrrolidone (10% w/v) and dialyzed hydroxyethyl starch
(10% w/v).
Pharmaceutical Compositions and Modes of Administration
[0067] The compounds of the present invention are administered to a
patient in the form of a pharmaceutical composition. A compound
that is administered in a pharmaceutical composition is mixed with
a pharmaceutically acceptable carrier or excipient such that a
therapeutically effective amount is present in the composition.
[0068] By "pharmaceutically acceptable" is meant a material that is
not biologically or otherwise undesirable, i.e., the material may
be incorporated into a pharmaceutical composition administered to a
patient without causing any undesirable biological effects or
interacting in a deleterious manner with any of the other
components of the composition in which it is contained. When the
term "pharmaceutically acceptable" is used to refer to a
pharmaceutical carrier or excipient, it is implied that the carrier
or excipient has met the required standards of toxicological and
manufacturing testing or that it is included on the Inactive
Ingredient Guide prepared by the U.S. Food and Drug administration.
"Pharmacologically active" (or simply "active") as in a
"pharmacologically active" derivative or analog, refers to a
derivative or analog having the same type of pharmacological
activity as the parent compound and approximately equivalent in
degree.
[0069] The terms "effective amount" or "therapeutically effective
amount" refers to an amount of the compound that is nontoxic and
necessary to achieve a desired endpoint or therapeutic effect
(e.g., act as a dermal or subdermal filler).
[0070] A variety of preparations can be used to formulate the
compositions or active agents of the present invention to render
the most appropriate pharmaceutical compositions. Techniques for
formulation and administration may be found in "Remington: The
Science and Practice of Pharmacy, Twentieth Edition," Lippincott
Williams & Wilkins, Philadelphia, Pa. For human administration,
preparations should meet sterility, pyrogenicity, general safety
and purity standards as required by the FDA. Administration of the
pharmaceutical composition can be, performed in a variety of ways,
as described herein.
[0071] The active agent may be administered, if desired, in the
form of a salt, ester, amide, prodrug, derivative, or the like,
provided the salt, ester, amide, prodrug or derivative is suitable
pharmacologically. Salts, esters, amides, prodrugs and other
derivatives of the active agents may be prepared using standard
procedures known to those skilled in the art of synthetic organic
chemistry and described, for example, by J. March, Advanced Organic
Chemistry: Reactions, Mechanisms and Structure, 4th Ed. (New York:
Wiley-Interscience, 1992).
[0072] The amount of active agent (e.g., collagen, elastin, etc.)
administered will depend on a number of factors and will vary from
subject to subject and depend on the particular drug administered,
the particular disorder or condition being treated, the severity of
the symptoms, the subject's age, weight and general condition, and
the judgment of the prescribing physician. The minimum amount of
drug is determined by the requirement that sufficient quantities of
drug must be present in a device or composition to maintain the
desired rate of release over the given period of application. The
maximum amount for safety purposes is determined by the requirement
that the quantity of drug present cannot exceed a rate of release
that reaches toxic levels. Generally, the maximum concentration is
determined by the amount of agent that can be received in the
carrier without producing adverse histological effects such as
irritation, an unacceptably high initial pulse of agent into the
body, or adverse effects on the characteristics of the delivery
device such as the loss of tackiness, viscosity, or deterioration
of other properties.
[0073] The term "dosage form" denotes any form of a pharmaceutical
composition that contains an amount of active agent sufficient to
achieve a therapeutic effect with a single administration. When the
formulation is an injection, the dosage form is usually one such
injection. The frequency of administration that will provide the
most effective results in an efficient manner without overdosing
will vary with the characteristics of the particular active agent,
including both its pharmacological characteristics and its physical
characteristics.
[0074] The compositions of the present invention can also be
formulated for controlled release or sustained release. The term
"controlled release" refers to a drug-containing formulation or
fraction thereof in which release of the drug is not immediate,
i.e., with a "controlled release" formulation, administration does
not result in immediate release of the drug into an absorption
pool. The term is used interchangeably with "nonimmediate release"
as defined in Remington: The Science and Practice of Pharmacy,
Nineteenth Ed. (Easton, Pa.: Mack Publishing Company, 1995). In
general, the term "controlled release" as used herein includes
sustained release and delayed release formulations.
[0075] The term "sustained release" (synonymous with "extended
release") is used in its conventional sense to refer to a drug
formulation that provides for gradual release of a drug over an
extended period of time, and that preferably, although not
necessarily, results in substantially constant blood levels of a
drug over an extended time period.
[0076] The present formulations may also include conventional
additives such as opacifiers, colorants, gelling agents, thickening
agents, stabilizers, surfactants, and the like. Other agents may
also be added, such as antimicrobial agents, to prevent spoilage
upon storage, i.e., to inhibit growth of microbes such as yeasts
and molds. Suitable antimicrobial agents are typically selected
from the group consisting of the methyl and propyl esters of
p-hydroxybenzoic acid (i.e., methyl and propyl paraben), sodium
benzoate, sorbic acid, imidurea, and combinations thereof.
[0077] Administration of a compound of the invention may be carried
out using any appropriate mode of administration. Thus,
administration can be, for example, oral, parenteral, topical,
transdermal, transmucosal (including rectal and vaginal),
sublingual, by inhalation, or via an implanted reservoir in a
dosage form.
[0078] Depending on the intended mode of administration, the
pharmaceutical formulation may be a solid, semi-solid or liquid,
such as, for example, a tablet, a capsule, a caplet, a liquid, a
suspension, an emulsion, a suppository, granules, pellets, beads, a
powder, or the like, preferably in unit dosage form suitable for
single administration of a precise dosage. Suitable pharmaceutical
compositions and dosage forms may be prepared using conventional
methods known to those in the field of pharmaceutical formulation
and described in the pertinent texts and literature, e.g., in
Remington: The Science and Practice of Pharmacy (Easton, Pa.: Mack
Publishing Co., 1995).
[0079] The dose regimen will depend on a number of factors that may
readily be determined, such as severity of the condition and
responsiveness of the condition to be treated, but will normally be
one or more doses per day, with a course of treatment lasting from
several days to several months, or until a cure is effected or a
diminution of disease state is achieved. One of ordinary skill may
readily determine optimum dosages, dosing methodologies, and
repetition rates. In general, it is contemplated that the
formulation will be applied one to four times daily. With a skin
patch, the device is generally maintained in place on the body
surface throughout a drug delivery period, typically in the range
of 8 to 72 hours, and replaced as necessary.
[0080] Preferably, the pharmaceutical compositions of the present
invention can be administered parenterally to a subject/patient in
need of such treatment. The term "parenteral" as used herein is
intended to include subcutaneous (dermal or subdermal),
intravenous, and intramuscular injection or implantation (e.g.,
subcutaneously or intramuscularly or by intramuscular
injection).
[0081] Preparations according to this invention for parenteral
administration include sterile aqueous and nonaqueous solutions,
suspensions, and emulsions. Injectable aqueous solutions contain
the active agent in water-soluble form. Examples of nonaqueous
solvents or vehicles include fatty oils, such as olive oil and corn
oil, synthetic fatty acid esters, such as ethyl oleate or
triglycerides, low molecular weight alcohols such as propylene
glycol, synthetic hydrophilic polymers such as polyethylene glycol,
liposomes, and the like. Parenteral formulations may also contain
adjuvants such as solubilizers, preservatives, wetting agents,
emulsifiers, dispersants, and stabilizers, and aqueous suspensions
may contain substances that increase the viscosity of the
suspension, such as sodium carboxymethyl cellulose, sorbitol, and
dextran. Injectable formulations are rendered sterile by
incorporation of a sterilizing agent, filtration through a
bacteria-retaining filter, irradiation, or heat. They can also be
manufactured using a sterile injectable medium. The active agent
may also be in dried, e.g., lyophilized, form that may be
rehydrated with a suitable vehicle immediately prior to
administration via injection.
[0082] The quantity of active ingredient and volume of composition
to be administered depends on the host animal to be treated.
Precise amounts of active compound required for administration
depend on the judgment of the practitioner and are peculiar to each
individual.
[0083] A minimal volume of a composition required to disperse the
active compounds is typically utilized. Suitable regimes for
administration are also variable, but would be typified by
initially administering the compound and monitoring the results and
then giving further controlled doses at further intervals.
[0084] A carrier for parenteral administration can be a solvent or
dispersion medium containing, for example, water, ethanol, polyol
(for example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), suitable mixtures thereof, and vegetable
oils. The proper fluidity can be maintained, for example, by the
use of a coating, such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. The prevention of the action of microorganisms can be
brought about by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal,
and the like. In many cases, it will be preferable to include
isotonic agents, for example, sugars or sodium chloride. Prolonged
absorption of the injectable compositions can be brought about by
the use in the compositions of agents delaying absorption, for
example, aluminum monostearate and gelatin. It is also advantageous
to include one or more cells or tissues which may supplement the
use of the composition of the present invention. For example, it is
preferred to include adipose tissue or cells, dermal fibroblasts or
combination of thereof.
[0085] Suitable preservatives for use in solution include
benzalkonium chloride, benzethonium chloride, chlorobutanol,
thimerosal and the like. Suitable buffers include boric acid,
sodium and potassium bicarbonate, sodium and potassium borates,
sodium and potassium carbonate, sodium acetate, sodium biphosphate
and the like, in amounts sufficient to maintain the pH at between
about pH 6 and pH 8, and preferably, between about pH 7 and pH 7.5.
Suitable tonicity agents are dextran 40, dextran 70, dextrose,
glycerin, potassium chloride, propylene glycol, sodium chloride,
and the like, such that the sodium chloride equivalent of the
ophthalmic solution is in the range 0.9 plus or minus 0.2%.
Suitable antioxidants and stabilizers include sodium bisulfate,
sodium metabisulfite, sodium thiosulfite, thiourea and the like.
Suitable wetting and clarifying agents include polysorbate 80,
polysorbate 20, poloxamer 282 and tyloxapol. Suitable
viscosity-increasing agents include dextran 40, dextran 70,
gelatin, glycerin, hydroxyethylcellulose,
hydroxmethylpropylcellulose, lanolin, methylcellulose, petrolatum,
polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone,
carboxymethylcellulose and the like.
[0086] The compositions of the invention can be formulated for
parenteral administration by dissolving, suspending or emulsifying
in an aqueous or nonaqueous solvent. Vegetable (e.g., sesame oil,
peanut oil) or similar oils, synthetic aliphatic acid glycerides,
esters of higher aliphatic acids and propylene glycol are examples
of nonaqueous solvents. Aqueous solutions such as Hank's solution,
Ringer's solution or physiological saline buffer can also be used.
In all cases the form must be sterile and must be fluid to the
extent that easy syringability exists. It must be stable under the
conditions of manufacture and storage and must be preserved against
the contaminating action of microorganisms, such as bacteria and
fungi.
[0087] Solutions of active compounds as free base or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0088] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0089] The preparation of more, or highly, concentrated solutions
for subcutaneous or intramuscular injection is also contemplated.
In this regard, the use of DMSO as solvent is preferred as this
will result in extremely rapid penetration, delivering high
concentrations of the active compound(s) or agent(s) to a small
area.
[0090] The present invention also provides kits for performing soft
tissue augmentation. Such kits can be prepared from readily
available materials and reagents and can come in a variety of
embodiments. For example, such kits can comprise, in an amount
sufficient for at least one treatment, any one or more of the
following materials: human elastin and collagen isolated by methods
of the present invention, sterilized buffers (e.g., phosphate
buffered salt) or water, other reagents necessary or helpful to
perform the method, and instructions. Typically, instructions
include a tangible expression describing reagent concentration or
at least one method parameter, such as the amount of reagent to be
used, maintenance time periods for reagents, and the like, to allow
the user to carry out the methods described above. In a preferred
embodiment of the invention, a kit comprises a means for delivery.
Such means can include, by way of illustration and not limitation,
a small syringe (22 to 27-gauge), a large syringe (13 to 19-gauge)
and equipment used in endoscopic or percutaneous discectomy
procedures. The reagents can be provided in solution, as
suspensions, or as a substantially dry powder, e.g., in lyophilized
form, either independently or in a mixture of components to improve
ease of use. Where a degradable reagent is provided, conditions are
chosen so as to stabilize the reagent, e.g., storage at lower
temperature, addition of stabilizing agents (e.g., glycerol or a
reducing agent). Unstable reagents can be provided together with or
separately from the more stable components of the kit.
[0091] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.
[0092] The present invention is further illustrated by the
following examples that should not be construed as limiting in any
way.
EXAMPLES
Example 1
Isolation of Collagen from Engineered Vascular Tissue
[0093] Vascular tissues are engineered from human vascular smooth
muscle cells according to methods as previously described (Niklason
et al., Science 284(5413):489-93, 1999). Briefly, vascular smooth
muscle cells from screened and banked human vascular cell sources
are seeded onto a tubular synthetic fibrous scaffolding comprising
polyglycolic acid fibers. The tubular seeded scaffold is threaded
over distensible silicone tubing within a sterile bioreactor. The
bioreactor is filled with culture medium that supports the
synthesis of collagen by vascular cells. Specifically, this medium
comprises Dulbecco's Modified Eagles Medium (DMEM) supplemented
with 20% fetal bovine serum or other serum, ascorbic acid (50
mg/L), growth factors such as platelet derived growth factor (10
ng/mL), basic fibroblast growth factor (10 ng/mL), epidermal growth
factor (3 ng/mL), proline 50 mg/L, glycine 50 mg/L, alanine 20
mg/L, copper sulfate 3 ng/mL Other medium components that support
growth of cells and/or extracellular matrix production may also be
included in the culture medium. Cyclic pulsatile radial strain may
be administered to the tubular constructs over the silicone tubing
by pumping fluid through the tubing, with distensions of 1-5% being
most preferable. Alternatively, cyclic strain may be omitted during
culture, in order to simplify the culture system. Culture is
maintained for 2-10 weeks, during which time collagenous matrix is
synthesized by the vascular smooth muscle cells.
[0094] At the conclusion of culture, the engineered vascular tissue
is decellularized using techniques similar to those reported in the
art (Dahl, Cell Transplant 12(6):659-66, 2003). Specifically,
detergent-based decellularization can incorporate two different
treatment solutions. Solution 1 includes 8 mM CHAPS, 1.0 M NaCl,
and 25 mM EDTA in PBS. Engineered vascular tissues are exposed to
this solution for one hour. Following rinses in PBS, engineered
vascular tissues are then exposed to Solution 2 for one hour.
Solution 2 includes 1.8 mM sodium dodecyl sulfate, 1.0 M NaCl, and
25 mM EDTA in PBS. Engineered vessels are then rinsed in PBS and
are rendered acellular by this process. All treatments are
performed at room temperature or at about 37.degree. C.
[0095] The following steps are used to isolate and purify the
collagen from the decellularized, engineered human vascular
tissues:
[0096] 1. Begin with a celluar engineered material. Cut, slice,
blend, chop into small pieces.
[0097] 2. Digest tissue material in pepsin (0.5 to 2.0 mg/ml pepsin
concentration dissolved in a low pH solution) at 4-20.degree. C.
Agitation during digest will aid the process.
[0098] 3. Once digestion has completed, centrifuge briefly to
remove any undigested material.
[0099] 4. Remove supernatant and raise the pH of the solution to
about pH 8.5 by slowly adding NaOH to inactivate pepsin.
[0100] 5. Using HCl, bring the pH of the solution back to about pH
3.5.
[0101] 6. Clarify collagen solution using diatomaceous earth.
[0102] 7. Precipitate clarified collagen by adding NaCl to the
solution. Precipitate at 4.degree. C. for >24 hrs.
[0103] 8. Collect precipitated collagen by chilled centrifugation
at high speeds for .about.30 minutes.
[0104] 9. Aspirate supernatant carefully and resuspend precipitated
collagens in ice-cold HCl. Allow for collagen molecules to
completely solubilized.
[0105] 10. Dialyze solution to further purify collagen.
[0106] 11. Concentrate collagen to desired level.
[0107] 12. Store this purified collagen in solution.
[0108] 13. Add sterile-filtered sodium diphosphate solution to
concentrated, purified, collagen, until final concentration of
about 20-50 mM and about pH 7.4 is reached. Incubate at
22-37.degree. C. for >24 hours.
[0109] 14. An opaque white fibrous precipitate will form,
containing large macromolecular collagen fibrils.
[0110] 15. Centrifuge to obtain the resulting high concentration of
fibrillar collagen for injection, discarding supernatant.
[0111] Using these steps, collagen is extracted from engineered,
decellularized tissues. Extracted collagen is then run on a
polyacrylamide gel to assess preservation of chain morphology and
purity. FIG. 1 shows the very high levels of collagen purity in
this preparation, as compared to the purified bovine collagen
control. Hence, the methods in this example produce highly pure,
collagen from engineered vascular tissues.
Example 2
Isolation of Elastin from Engineered Vascular Tissue
[0112] Synthesis of cross-linked, insoluble elastin in cultured
cells is typically quite difficult. While many reports exist of
synthesis of non-crosslinked tropoelastin monomers, creation of
documented, cross-linked elastin is extremely rare. Most reports of
production of insoluble elastin utilize cells that have been
genetically engineered, for example to express high levels of
tropolelastin protein, or to express variants of versican that
stimulate elastin deposition. Alternatively, rodent cells derived
from neonatal animals have been reported to synthesize elastin.
However, non-human elastin is associated with risks of immune
rejection if injected into a human recipient. Further, utilizing
genetically modified cells to generate human elastin carries
intrinsic risks of passing on transgene material to any eventual
recipient of the extracellular matrix material.
[0113] Hence, it is advantageous to devise methods to generate
crosslinked, insoluble elastin from cultured human cells, without
the use of genetic engineering. In particular, the use of human
vascular cells that are banked and have been screened for
infectious agents also helps to reduce any infectious risk of
resultant elastin that is produced.
[0114] The methods of the present invention culture human vascular
tissues to produce measurable amounts of insoluble, crosslinked
elastin, as indicated by desmosine anlaysis. Such tissues may then
be decellularized and the elastin within these tissues extracted
and purified. In one example, human vascular smooth muscle cells
are seeded onto polyglycolic acid scaffolds in bioreactors,
analogously to the procedure described in Example 1. Cyclic strain
may be applied via the luminal silicone tubing, or may be omitted,
in order to simplify the culture conditions. Total culture time may
vary from 2-10 weeks. In this example, total culture time is 3
weeks. Culture medium that is designed to stimulate elastin
synthesis for the first two weeks of culture contains the following
components:
[0115] 1. 400 mL DMEM-low glucose
[0116] 2. 100 mL Human Serum
[0117] 3. 5 mL (50,000 U) of Penicillin G
[0118] 4. 2.5 mg Insulin,
[0119] 5. 0.5 .mu.g CuSO.sub.4
[0120] 6. 5 ml aliquot of Glycine/Alanine/Valine/Proline (30 mg/18
mg/17.5 mg/11.5 mg)
[0121] 7. Dexamethasone 10.sup.-8-10.sup.-10 M
[0122] 8. 2.5 .mu.g TGF-beta
[0123] In order to further stimulate elastin synthesis by cultured
human vascular smooth muscle cells within the engineered tissue,
the following medium is used during the final week of culture:
[0124] 1. 475 mL DMEM-low glucose
[0125] 2. 25 mL Human Serum
[0126] 3. 5 mL (50,000 U) of Penicillin G
[0127] 4. 2.5 mg Insulin, (5 .mu.g/ml)
[0128] 5. 1.5 .mu.g CuSO.sub.4
[0129] 6. 5 ml aliquot of Glycine/Alanine/Valine/Proline (30 mg/18
mg/17.5 mg/11.5 mg)
[0130] 7. Dexamethasone 10.sup.-8-10.sup.-10 M
[0131] 8. 2.5 .mu.g TGF-beta (5 ng/mL)
[0132] Using these culture conditions, engineered vascular tissues
containing elastin, cells and other matrix components are
generated. At the conclusion of culture, engineered vascular
tissues are subjected to the decellularization process as described
in Example 1. Subsequent to this decellularization process, the
intact and decellularized tissues are subjected to analysis for
desmosine, which is a covalent cross-link component in mature
elastin. Presence of desmosine indicates the production of mature
elastin by engineered vascular tissues. Table 1 contains desmosine
results for a variety of engineered vascular tissues, both intact
and decellularized.
TABLE-US-00001 TABLE 1 pmol Samples pmol Desmosine/mg Protein
Desmosine/mg Tissue Fresh-1 32 25 Fresh-2 29 27 Fresh-3 37 20
Fresh-4 49 35 Fresh-5 57 26 Fresh-6 67 29 Fresh-7 41 12
Decellutarized-1 53 29 Decellutarized-2 31 34 Decellutarized-3 39
19 Decellutarized-4 55 46 Decellutarized-5 37 25 Decellutarized-6
28 35 Decellutarized-7 64 51
[0133] Table 1 shows that desmosine elastin cross-links are present
in intact engineered tissues, and are retained after the
decellularization process. Hence, it is feasible to engineer
cross-linked elastin that is stable following removal of cellular
constituents. Isolation of elastin from engineered and
decellularized vascular tissues may then be accomplished by any one
of several methods known in the art, including, but not limited to,
autoclaving, alkali or acid treatment, pepsin or collagenase
digestion, or combinations of these treatments.
Example 3
Isolation of Elastin from Native Human Umbilical Vessels
[0134] Elastin is isolated from native human blood vessels, such as
those isolated from discarded umbilical cords. Vessels are
decellularized and then elastin is isolated from the resultant
extracellular matrix. The solutions for decellularization process
are those described in Example 1. Steps used in the
decellularization and elastin isolation process are as follows:
[0135] 1. Thaw frozen umbilical artery or vein overnight at
4.degree. C.
[0136] 2. Record vessel length and weight.
[0137] 3. Decellularize vessel in Solution 1 for 12 hr.
[0138] 4. Rinse 2 times with phosphate buffered saline, 5 min
each.
[0139] 5. Decellularize in Solution 2 for 12 hr.
[0140] 6. Rinse 3 times with phosphate buffered saline, 5 min
each.
[0141] Digest decellularized umbilical artery or vein with pepsin
or collagenase at 37.degree. C. or room temperature for 1-5 hr with
gentle shaking. This removes non-elastin extracellular matrix
components.
[0142] 7. Alternatively, decellularized vessels may be autoclaved
for 3-4 cycles at 121.degree. C. for 30 min or 115.degree. C. for
20 min, to remove non-elastin extracellular matrix components.
[0143] 8. Rinse digested tissue or autoclaved tissue with distilled
water.
[0144] 9. Snap freeze tissue in dry ice.
[0145] 10. Lyophilize tissue overnight.
[0146] 11. Subject resultant elastin to amino acid analysis to
evaluate purity.
[0147] 12. Further digest the purified elastin with pepsin to
produce injectable particle size.
[0148] 13. Alternatively, mechanically break down elastin using
mortar/pestle or a mill or homogenizer, to produce particles of a
size appropriate for injectable products, preferably less than
about 200 .mu.m, more preferably less than about 100 .mu.m, most
preferably less than about 50 .mu.m.
[0149] Human umbilical vessels are treated according to the steps
in this example, and are analyzed for amino acid content to
determine elastin purity (step 12 above). Table 2 indicates the
exact preparation steps from the above list that are used for each
sample.
TABLE-US-00002 TABLE 2 Samples 1 2 3 4 5 6 7 8 NaCI extraction y y
Autoclave 115 C. 115 C. 115 C. 115 C. 121 C. 121 Delipid Y y y y y
y decell y y y Pepsin 37 C. RT
[0150] Table 3 shows results of the amino acid analysis of the
resultant purified elastins.
TABLE-US-00003 TABLE 3 Values are Residues/1000 Ex- Sample # pect-
1 2 3 4 5 6 7 8 ed *CVS 16 0 24 2 23 3 0 0 3 asx 93 73 92 88 96 38
94 79 2 thr 38 23 45 42 43 21 38 28 14 ser 31 24 34 31 34 13 37 30
9 glx 111 98 120 116 125 51 100 88 3 pro 87 104 76 84 68 113 94 144
129 gly 202 352 145 157 134 286 273 326 312 ala 96 120 100 107 99
197 97 110 239 val 73 37 76 81 78 123 55 41 137 *met 6 0 5 14 0 0 0
0 0 ile 44 24 48 53 50 31 52 28 24 leu 76 40 85 84 88 66 69 45 65
*tyr 0 0 3 2 0 0 0 0 23 phe 28 15 33 34 35 26 29 20 24 his 1 0 8 6
23 0 0 0 0 lys 50 36 60 57 59 25 33 29 9 arg 47 54 46 43 44 8 30 32
9
[0151] In Table 3, the "Expected" column indicates the number of
amino acid residues per 1000 amino acid residues that would be
expected in the case of completely pure human elastin. It is clear
from Table 3 that Sample 6 contains the most highly pure isolated
elastin. This sample was prepared using decellularization and
autoclave extraction (see Table 2). Hence, in contrast to
previously reported techniques that claim to isolate pure elastin
from other types of tissues, it had been found that human vascular
tissues require an additional decellularization step in order to
isolate elastin of sufficient purity. This finding is in contrast
to multiple reports in the literature that claim that autoclave
treatment alone, when applied to other tissues such as bovine
ligamentum nuchae, produces a highly pure elastin preparation (Lee
et al., American Journal of Pathology 2006; 168: 490-498). From
Table 3, it is clear that standard methods such as autoclaving,
without an additional decellularization step, produce highly impure
elastin products when applied to native human umbilical cord
vascular material.
Example 4
Isolation of Elastin from Native Human Aorta
[0152] Elastin is also purified from human aorta. The process
involves a salt-based decellularization step, followed by boiling
in 0.1 N NaOH and then extraction in hydrophobic solvents. Elastin
isolation according to this example has unexpected properties when
implanted in vivo, as shown in Example 5 (below). Steps for
purifying elastin from aorta according to the present invention are
as follows:
[0153] Obtain wet weight of aorta. Aorta is preferably fresh or
non-frozen.
[0154] 1. Shred aorta using a blender or some other device in
distilled water.
[0155] 2. Extract shredded tissues at 1-hour intervals in 0.9% NaCl
solution at 4.degree. C. with shaking.
[0156] 3. Repeat NaCl extraction until protein assay shows no
soluble protein extraction.
[0157] 4. Suspend samples in boiling 0.1 N NaOH solution, boil for
40-45 minutes.
[0158] 5. Discard NaOH solution, then rinse with distilled
water.
[0159] 6. Extract elastin 3 times, 30 minutes each, with 100%
ethanol at room temperature.
[0160] 7. Extract elastin in 50% ethanol/50% diethyl ether for 1
hour at room temperature.
[0161] 8. Extract elastin in 100% diethyl ether for 1 hour at room
temperature.
[0162] 9. Decant ether, dry overnight. Obtain final weight.
[0163] 10. Grind or pulverize to create injectable and insoluble
elastin particles.
[0164] The amino acid analysis is performed, along with the RIA
analysis for desmosine cross-links, of elastin that is purified
from human aorta using the above method. For these experiments, a
total of 6 different aortas are treated using this protocol, and
amino acid analysis is performed on 4 of the 6 samples. Desmosine
quantification is performed on all samples as summarized in Table
4.
TABLE-US-00004 TABLE 4 AA analysis and Desmosine for elastin from
human aorta: (per 1.000 total residues) Amino Acid Sample 54 Sample
55 Sample 56 Sample 57 Expected *cys 0 0 3 asx 7 7 5 6 2 thr 9 8 5
5 14 ser 6 6 3 3 9 glx 21 21 20 19 3 pro 116 116 111 111 129 gly
332 331 353 353 312 ala 263 263 261 259 239 val 114 116 127 127 137
*met 0 0 0 0 0 ile 23 25 21 21 24 leu 60 61 55 58 65 *tyr 13 14 5 3
23 phe 24 25 19 21 24 his 0 0 0 0 0 lys 6 7 11 9 9 arg 5 4 5 5 9
Des 12332 13291 19793 11904 (pM/mg) *Cys, Tyr and Met are partially
destroyed during acid hydrolysis Desmosine units are in (pico
Moles/mg Protein)
[0165] As shown in Table 4, the values of alanine are well above
200 residues per 1,000 total residues, and values of valine are
well above 70 residues per 1,000 residues. These are consistent
with high purity elastin protein. In addition, values of Desmosine
cross-links are very high, and are comparable to or higher than
those reported for elastin preparations from a variety of species
(see Table 5):
TABLE-US-00005 TABLE 5 Desmosine from aortas of different species
(pM/mg protein) picomole/mg protein Cow 12573 Pig 13934 Monkey
10948 Rat 6266 Dog 12942
[0166] An alternative method in accordance with the present
invention is to isolate elastin from human aorta using a pepsin
digestion. FIG. 2 shows an immunoblot of proteins isolated from
human aorta using pepsin digestion, and reacted with anti-elastin
antibody. For digestion times ranging from 2-5 hours, extensive
protein is liberated that reacts with elastin antibody, having
molecular weights (MW) in the approximate range of 100-500 kDa
(i.e., greater than 100 kDa).
Example 5
Implantation of Purified Elastin In Vivo
[0167] One drawback of other elastin preparations is a tendency to
calcify in vivo. Implantation into juvenile (i.e. 21 day-old) rats
is an extremely sensitive assay for calcification. In order to
determine the propensity of human elastin that is isolated
according to the present invention to calcify, the elastin was
implanted subdermally into rats. The human elastin was isolated
from aorta according to the present invention using salt-based
decellularization followed by NaOH extraction as described in
Example 4. Calcification was compared to that induced by purified
bovine elastin (purchased commercially), syngeneic rat aorta
(containing rat elastin), and injection of phosphate buffered
saline control. Implants remained in situ for 21 days and then were
explanted. Calcification was assessed histologically, using the
alizarin red stain, which produces a reddish-brown color in
calcified tissues. In addition, calcium accumulation at implant
sites was determined quantitatively by atomic absorption
spectroscopy.
[0168] FIG. 3 shows the atomic absorption spectroscopy of calcium
content of explanted tissues from juvenile rats that contained
various elastin implants or control implants. Error bars are
standard deviation of the mean. Various samples were analyzed:
juvenile rat subcutaneous tissue, negative control (SubCut Tissue);
phosphate buffered saline carrier (PBS); syngeneic rat aorta,
containing syngeneic rat elastin (Rat Elastin); human elastin
isolated according to the present invention, from fresh aorta
(E56); human elastin isolated according to the present invention,
from fresh aorta, sterilized by gamma radiation (E56 gamma); human
elastin isolated according to the present invention, from frozen
aorta (E59); human elastin isolated according to the present
invention, from frozen aorta, sterilized by gamma radiation (E59
gamma); purified bovine elastin obtained from Elastin Products Co.
(B-elastin); purified bovine elastin from Elastin Products Co,
sterilized by gamma radiation (B-elastin gamma); injectable form of
bovine elastin obtained from Elastin Products Co. (B-elastin
Injection); injectable form of bovine elastin obtained from Elasin
Products Co, sterilized by gamma radiation (Injection B-elastin
Gamma); and phosphate buffered saline carrier (Injection PBS).
[0169] The results of atomic absorption spectroscopy show that
calcium levels in explants having elastin that is isolated from
non-frozen human aorta according to the present invention are not
different from calcium levels in tissues that are injected with PBS
carrier. However, the results show that when elastin was isolated
according to the present invention from frozen aorta, tissue
calcification was significantly increased. Overall, there does not
appear to be any impact of sterilization by gamma irradiation on
the degree of tissue calcification for any of the forms of elastin
that is tested (see FIG. 3 for the atomic absorption spectroscopy
results and Table 6 for summary of the corresponding quantitative
values of calcium in tissue explants)
[0170] FIG. 4 shows the staining of explanted tissue specimens from
juvenile rats implanted with elastin that was isolated according to
the present invention, and with bovine elastin. A,B: Hematoxylin
& eosin (H&E) stain of explanted tissues 21 days after
implantation of human elastin that was isolated according to the
present invention ("Humacyte") and commercially obtained bovine
elastin ("Bovine"). C,D: Alizarin red stain of explanted tissues 21
days after implantation of human elastin that was isolated
according to the present invention ("Humacyte") and commercially
obtained bovine elastin ("Bovine"). Arrows in panel D indicate
areas of visible calcification.
[0171] FIG. 5 shows the staining of explanted tissue specimens from
juvenile rats implanted with elastin that was isolated according to
the present invention, and with syngeneic rat elastin from rat
aorta. A,B: Hematoxylin & eosin (H&E) stain of explanted
tissues 21 days after implantation of human elastin that was
isolated according to the present invention ("Humacyte") and
syngeneic rat aorta containing elastin ("Rat"). C,D: Alizarin red
stain of explanted tissues 21 days after implantation of human
elastin that was isolated according to the present invention
("Humacyte") and syngeneic rat aorta containing elastin ("Rat").
Arrows in panel D indicate areas of likely calcification.
[0172] FIG. 6 shows the staining of explanted tissue specimens from
juvenile rats implanted with elastin that was isolated according to
the present invention, and with phosphate buffered saline carrier.
A,B: Hematoxylin & eosin (H&E) stain of explanted tissues
21 days after implantation of human elastin that was isolated
according to the present invention ("Humacyte") and carrier
("PBS"). C,D: Alizarin red stain of explanted tissues 21 days after
implantation of human elastin that was isolated according to the
present invention ("Humacyte") and carrier ("PBS"). Arrows in panel
D indicate areas of possible calcification.
[0173] The H&E staining together with an alizarin red staining
of explanted tissue from juvenile rats for calcification in FIGS.
4-6 confirms the results from atomic absorption spectroscopy in
FIG. 3 and Table 6 regarding the degree of tissue calcification,
and shows that human elastin that is isolated according to the
present invention from non-frozen tissue does not induce
calcification in vivo, using an extremely sensitive implantation
model system.
TABLE-US-00006 TABLE 6 Calcium levels in explanted samples: Calcium
from Explant Tissue Group Elastin source Average St Dev N Rat
SubCut Tissue N/A 0.25 0.160421877 4 PBS carrier N/A 0.49
0.383992568 6 Rat elastin Fresh aorta 2.80 3.808000837 2
E56-Humacyte Fresh human aorta 0.47 0.195592829 3 E-56 Humacyte
Fresh human aorta 0.44 0.081904605 3 gamma E59-Humacyte Frozen
human aorta 23.73 6.610771334 3 (frozen) E59-Humacyte Frozen human
aorta 46.36 17.10482952 3 (frozen) gamma Bovine elastin Elastin
Products Co. 22.26 4.662817486 6 Bovine elastin, Elastin Products
Co. 28.97 11.63379649 6 gamma Bovine injectable Elastin Products
Co. 4.76 5.485544335 4 Bovine injectable, Elastin Products Co. 9.95
9.181395142 6 gamma PBS carrier N/A 0.46 0.172634466 5
Example 6
Isolation of Human Collagen from SMCs on Micro-Carrier Beads
[0174] Human vascular smooth muscle cells can also be cultured on
micro-carrier beads in a suspension culture as described herein.
During the period of culturing, the smooth muscle cells replicate
on the surface of the beads, and deposit collagenous extracellular
matrix. The collagenous matrix is then harvested and purified
according to the present invention. Specific steps in this process
are as follows:
[0175] 1. Culture human smooth muscle cells in a standard culture
flask under conditions suitable for growth of the cells, in
complete culture medium containing at least 10% serum.
[0176] 2. Sterilize spinner flask by autoclaving.
[0177] 3. Weigh out 2.0 g Cytodex-1 micro-carrier beads and mix
with 500 mL of PBS, then sterilize by autoclaving.
[0178] 4. Pipet 10 mL of micro-carrier bead slurry into spinner
flask reactor.
[0179] 5. Trypsinize vascular smooth muscle cells and seed onto
beads in spinner flask a total of 5 million cells in 25 mL of cell
growth medium.
[0180] 6. Culture cells on beads in the presence of DMEM medium
containing at least 10% serum, ascorbic acid (50 mg/L), growth
factors such as platelet derived growth factor (10 ng/mL), basic
fibroblast growth factor (10 ng/mL), epidermal growth factor (3
ng/mL), proline 50 mg/L, glycine 50 mg/L, alanine 20 mg/L, copper
sulfate 3 ng/mL
[0181] 7. Spin the flask at low speed, preferably not more than 10
revolutions per minute, during culture.
[0182] 8. Supplement vitamin C twice per week, and replace cultures
with fresh medium once per week.
[0183] 9. After 4-12 weeks of culture of smooth muscle cells on
beads, decant off culture medium supernatant and retain beads
contains cells and collagenous matrix.
[0184] 10. Digest bead and tissue material in pepsin (0.5 to 2.0
mg/ml pepsin concentration dissolved in a low pH solution) at
4-20.degree. C. Agitation during digest will aid the process.
[0185] 11. Once digestion has completed, centrifuge briefly to
remove any undigested material by filtration, to remove
micro-carrier beads.
[0186] 12. Remove supernatant and raise the pH of the solution to
about pH 8.5 by slowly adding NaOH to inactivate pepsin.
[0187] 13. Using HCl, bring the pH of the solution back to about pH
3.5.
[0188] 14. Clarify collagen solution using diatomaceous earth.
[0189] 15. Precipitate clarified collagen by adding NaCl to the
solution. Precipitate at 4.degree. C. for >24 hrs.
[0190] 16. Collect precipitated collagen by chilled centrifugation
at high speeds for .about.30 minutes.
[0191] 17. Aspirate supernatant carefully and re-suspend
precipitated collagens in ice-cold HCl. Allow for collagen
molecules to completely solubilized.
[0192] 18. Dialyze solution to further purify collagen.
[0193] 19. Concentrate collagen to desired level.
[0194] 20. Store this purified collagen in solution.
[0195] 21. Add sterile-filtered sodium diphosphate solution to
concentrated, purified, collagen, until final concentration of
about 20-50 mM and about pH 7.4 is reached. Incubate at
22-37.degree. C. for >24 hours.
[0196] The purity of the resultant solubilized product produced as
described herein was compared to other purified human collagens
from commercial resources by polyacrylamide gel electrophoresis as
shown in FIG. 7. Lane 1 shows 20 micrograms of PureCol Human
Collagen, Inamed Biomaterials. Lane 2 shows 10 micrograms of
collagen derived from human dermal fibroblasts. Lane 3 shows 10
micrograms of human collagen derived from vascular smooth muscle
cells and purified as described in the instant example. The gel was
stained with coomassie blue for protein detection. Typical collagen
bands alpha, beta and gamma were present in all samples. The
results in FIG. 7, Lane 3, demonstrate the high purity of collagen
produced by the instant methods when compared with other samples of
highly purified human collagen.
Example 7
Formulation of Injectable Collagen Material
[0197] Collagen can be formulated into an injectable product that
can be delivered to patients. Soluble collagen is collected from
engineered vascular tissue using the procedure contained in Example
1. Precipitated collagen is centrifuged and the supernatant removed
by aspiration. The precipitated collagen is then re-suspended in a
physiological saline buffer solution that is pharmaceutically
acceptable (such as 0.9% sodium chloride solution) that can contain
0.3% lidocaine. The mixture is agitated to ensure uniform mixing,
and pH adjusted to approximately 7.0. The volume of re-suspension
solution is titrated such that the final concentration of collagen
is approximately 30 mg/mL. The collagen solution is then dispensed
into sterile syringes and packaged in sterile fashion, for clinical
applications.
Example 8
Formulation of Injectable Collagen and Elastin Composite
Material
[0198] Collagen and elastin can be combined into a composite
product that is injectable. Soluble collagen is collected from
engineered vascular tissue using the procedure contained in Example
1. Precipitated collagen is centrifuged and the supernatant removed
by aspiration. The precipitated collagen is then re-suspended in a
physiological saline buffer solution that is pharmaceutically
acceptable (such as 0.9% sodium chloride solution) that can contain
0.3% lidocaine. The mixture is agitated to ensure uniform mixing,
and pH adjusted to approximately 7.0. The volume of re-suspension
solution is titrated such that the final concentration of collagen
is approximately 20 mg/mL.
[0199] To generate the collagen-elastin composite injectable
formulation, elastin is isolated from non-frozen aortic tissue
according to Example 4. Insoluble elastin isolated after ether
extraction is pulverized (with or without a prior freezing step to
aid in particle formation) and then is sieved under sterile
conditions to select particles that are less than 50 microns. Dry
particles are then admixed and suspended within collagen-containing
solution, in order to create the collagen-elastin composite
solution. The collagen-elastin composite is then dispensed into
sterile syringes and packaged in sterile fashion, for clinical
applications.
[0200] Another means by which the elastin may be rendered suitable
for injection is by digestion in pepsin or some other protease with
elastase activity. Digestion of purified elastin with pepsin at
room temperature or at 37.degree. C. for between 1-5 hours
generates elastin fragments of molecular weight greater than
100,000. Elastin fragments are then purified from residual pepsin
utilizing size-exclusion dialysis membranes, and then concentrated
to a final concentration of approximately 10 mg/mL or greater in a
physiologically acceptable carrier such as 0.9% saline. Suspended
elastin fragments are then combined with precipitated collagen or
collagen-containing solution to produce a final product with a
concentration of collagen 20 mg/mL, and a concentration of elastin
10 mg/mL, in saline with 0.3% lidocaine. The collagen-elastin
composite is then dispensed into sterile syringes and packaged in
sterile fashion, for clinical applications.
* * * * *