U.S. patent application number 14/255593 was filed with the patent office on 2014-10-09 for conjugated polymeric material and uses thereof.
This patent application is currently assigned to The Curators of the University of Missouri. The applicant listed for this patent is The Curators of the University of Missouri. Invention is credited to David GRANT, Sheila GRANT, Anthony HARRIS, Rebecca RONE, Jonathan THOMPSON.
Application Number | 20140302104 14/255593 |
Document ID | / |
Family ID | 44483792 |
Filed Date | 2014-10-09 |
United States Patent
Application |
20140302104 |
Kind Code |
A1 |
HARRIS; Anthony ; et
al. |
October 9, 2014 |
CONJUGATED POLYMERIC MATERIAL AND USES THEREOF
Abstract
Disclosed are compositions comprising collagen covalently bound
to particles, wherein covalent bonds are formed between reactive
groups of the collagen and reactive groups of the particles, and
wherein the particles have an average particle diameter ranging
from 20 to 1000 nanometers. Also disclosed are various methods that
utilize the compositions.
Inventors: |
HARRIS; Anthony; (Columbia,
MO) ; THOMPSON; Jonathan; (Cincinnati, OH) ;
RONE; Rebecca; (Columbia, MO) ; GRANT; Sheila;
(Columbia, MO) ; GRANT; David; (Columbia,
MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Curators of the University of Missouri |
Columbia |
MO |
US |
|
|
Assignee: |
The Curators of the University of
Missouri
Columbia
MO
|
Family ID: |
44483792 |
Appl. No.: |
14/255593 |
Filed: |
April 17, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13155111 |
Jun 7, 2011 |
|
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14255593 |
|
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|
|
61397100 |
Jun 7, 2010 |
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Current U.S.
Class: |
424/401 ;
424/423; 514/773; 530/356 |
Current CPC
Class: |
A61K 49/1866 20130101;
A61L 27/24 20130101; A61K 8/65 20130101; A61L 27/446 20130101; A61L
2430/34 20130101; A61Q 19/00 20130101; A61K 49/1833 20130101; A61L
27/047 20130101; A61K 2800/624 20130101; A61K 8/0241 20130101; A61K
8/19 20130101; A61Q 19/08 20130101; A61L 2400/16 20130101; A61L
27/446 20130101; C08L 89/06 20130101; A61K 8/02 20130101; A61L
27/60 20130101; A61K 47/42 20130101; A61K 2800/413 20130101; A61L
2400/12 20130101; A61P 19/00 20180101; A61P 17/00 20180101 |
Class at
Publication: |
424/401 ;
514/773; 424/423; 530/356 |
International
Class: |
A61K 47/42 20060101
A61K047/42; A61Q 19/08 20060101 A61Q019/08; A61K 8/65 20060101
A61K008/65 |
Claims
1-19. (canceled)
20. A method for bulking articular cartilage by increasing tissue
volume in a person, comprising administering to a person in need
thereof a composition comprising collagen covalently bound to
particles, wherein covalent amide bonds are formed between free
carboxylic acid groups of the collagen and amine reactive groups of
the particles, and wherein the particles have an average particle
diameter size ranging from 50 to 1000 nanometers, wherein the
composition is administered by injection into a joint capsule.
21. The method of claim 20, wherein the collagen is cross-linked
and porous and has an average pore size ranging from 500 nanometers
to 200 micrometers.
22. The method of claim 21, wherein the particles have an average
particle diameter between 50 and 150 nanometers.
23. The method of claim 20, wherein the reactive group is mercapto
ethyl amine or cystamine or both.
24. The method of claim 20, wherein the ratio of particles to
collagen is a range of 1.times.10.sup.9 particles per 1 mg of
collagen to 2.times.10.sup.10 particles per 1 mg of collagen.
25. The method of claim 20, wherein the composition is a gel,
solution, paste, or dehydrated rigid structure.
26. The method of claim 20, wherein 15 to 20% of the free
carboxylic acid groups of the collagen are covalently bound to the
particles through an amide bond.
27. The method of claim 20, wherein 2 to 4 mg of a carbodiimide
cross-linking agent per 30 mg of collagen is used to form the
covalent bonds.
28. The method of claim 20, wherein 0.5 to 0.2 mg of a carbodiimide
cross-linking agent per 1.times.10.sup.9-2.times.10.sup.10
particles is used to form the covalent bonds.
29. A method for filling voids, defects, or increasing tissue
volume in a person, comprising administering to a person in need
thereof a composition comprising collagen covalently bound to
particles, wherein covalent amide bonds are formed between free
carboxylic acid groups of the collagen and amine reactive groups of
the particles, and wherein the particles have an average particle
diameter size ranging from 50 to 1000 nanometers, wherein the
composition is administered by intradermal or subcutaneous
injection.
30. The method of claim 29, wherein the void is a facial fine line,
wrinkle, crease, pit, or nodule, and wherein the appearance of the
facial fine line, wrinkle, crease, pit, or nodule is reduced after
administration of the composition.
31. The method of claim 29, wherein the composition is administered
to a person's lip, and wherein the volume of the lip is increased
after administration of the composition.
32. A method of reducing collagen degradation in vitro or in vivo
by enzymatic breakdown comprising conjugating collagen with
particles having reactive amine groups and having an average
particle diameter ranging from 50-1000 nanometers, wherein covalent
amide bonds are formed between free carboxylic acid groups of the
collagen and the reactive amine groups on the particles, and
wherein degradation of the collagen by collagenase is thereby
reduced.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/397,100, filed Jun. 7, 2010. The contents of the
aforementioned application is incorporated by reference.
GRANT CLAUSE
[0002] None.
BACKGROUND OF THE INVENTION
[0003] A. Field of the Invention
[0004] The present invention relates to collagen covalently bound
to particles, which results in a material that is more resistant to
degradation such as collagenase degradation. This material can be
used in a wide range of applications.
[0005] B. Description of Related Art
[0006] The use of collagen in treating urinary incontinence, post
heart-attack congestive heart failure, joint fractures, and
congenital and age-related facial skin defects is limited by the
stability and integrity of the currently available collagen
materials. For example, collagen-based dermal fillers that are used
to treat facial ageing (e.g., improving facial contours,
ameliorating wrinkles, correction of scar depression, etc.) and to
augment lips are highly susceptible to breaking down over a period
of 12 months.
[0007] One proposed solution to the collagen breakdown issue is
crosslinking collagen by the formation of covalent bonds between
macromolecule collagen fibrils. However, the toxicity of the
chemicals utilized to crosslink collagen can be a concern. For
example, glutaraldehyde and hexamethylene diisocyanate become
incorporated within the collagen scaffold during crosslinking and
can release toxic residues into the body as the collagen is
degraded. Another problem is that too much cross-linking can create
a stiff and unusable collagen material.
SUMMARY OF THE INVENTION
[0008] The inventors have discovered a solution to the performance
issues limiting current collagen-based products. This solution
includes the use of particles having an average particle diameter
of 20 to 1000 nanometers that are capable of forming covalent bonds
with collagen. The conjugated material (e.g., compositions
comprising the material, conjugated collagen/particles, or
conjugated collagen fibril/particles) is more resistant to
degradation (e.g., by collagenase), biocompatible, and results in a
collagen matrix or scaffold that has an acceptable level of
porosity, thereby allowing for cellular in-growth. The cellular
in-growth is accelerated by the conjugated particles in the novel
material. In certain instances, the conjugated material also has
antimicrobial properties, which can be used to fight infection
after being administered to a patient.
[0009] In one instance, there is disclosed a material comprising
collagen covalently bound to particles, wherein the particles have
an average particle diameter ranging from 20 to 1000 nanometers. In
particular embodiments, the average particle diameter is between 50
to 1000 nanometers, although other diameter sizes and ranges are
contemplated as discussed below in this paragraph. The covalent
bond can be formed between reactive groups on the collagen (e.g.,
carboxylic acid and/or amine groups) and reactive groups on the
particles (e.g., amine-reactive groups, carboxylate-reactive
groups, thiol-reactive groups, and/or hydroxyl-reactive groups).
For example, and in one aspect, the covalent bonds can be formed
between free-carboxylic acid groups present on the collagen and
amine reactive groups on the particles, wherein amide bonds can be
formed between the carboxylic acid groups of the collagen and the
amine-reactive groups of the particles. As explained below,
particles can be functionalized to include reactive groups that are
capable of reacting with carboxylic acid or amine groups of the
collagen. In certain instances, the collagen can also be
cross-linked either by a cross linking agent such as a carbodiimide
(e.g., 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and/or by
the particles themselves (e.g., collagen can be cross-linked with
the particles, wherein at least one of the particles includes at
least two reactive groups, and wherein at least two covalent bonds
can be formed between, for example, carboxylic acid groups of the
collagen and the at least two reactive groups, wherein the two
reactive groups can be formed between, for example, amine groups.).
In certain aspects, the cross-linked collagen is porous and can
have an average pore size ranging from 500 nanometers to 200
micrometers (and any integer or range therein such as 550, 600,
650, 700, 750, 800, 850, 900, 1000, 1100, 1200, 1300, 1400, 1500,
1600, 1700, 1800, 1900 nanometers). In particular aspects, a pore
size range between 1 micrometer to 100 micrometers can be used (or
any integer or range therein such as 10, 20, 30, 40, 50, 60, 70,
80, or 90 micrometers). In certain instances, the particles have an
average particle diameter of 50, 60, 70, 80, 90, 100, 110, 120,
130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450,
500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000
nanometers. In particular embodiments, the average particle
diameter ranges from 60 to 900, 70 to 800, 80 to 700, 90 to 600,
100 to 500, 150 to 400, or 200 to 300 nanometers. In certain
aspects, the average particle diameter ranges from 50 to 150, 60 to
140, 70 to 130, 80 to 120, or 90 to 110 nanometers. The particles
can be made up of or comprise metallic material. The metallic
material can be gold, silver, platinum, titanium, nickel, or copper
or any combination thereof. In particular aspects, the metallic
material is gold or silver. The particles can also be made of or
comprise ceramic material or biodegradable material. In certain
embodiments, the ratio of particles to collagen can be a range of
1.times.10.sup.9 particles per mg of collagen to 2.times.10.sup.10
particles per mg of collagen, however broader ranges are
contemplated (e.g., 1.times.10.sup.4 to 1.times.10.sup.14 per mg of
collagen, and any range and integer therein). In some aspects, 2 to
4 mg of a carbodiimide cross-linking agent (e.g., EDC) per 30 mg of
collagen can be used to form the covalent bonds (in particular
aspects, the ratio can be 3.2 mg+/-0.8 mg of a carbodiimide
cross-linking agent such as EDC per 30 mg of collagen can be used).
Also, 0.5 to 0.2 mg of a carbodiimide cross-linking agent (e.g.,
EDC) per 1.times.10.sup.9-2.times.10.sup.10 particles can be used
to form the covalent bonds. In certain aspects, the material of the
present invention can further include cells that can be used to aid
in treatment options. Non-limiting examples of such cells include:
embryonic stem cells, adult stem cells, induced pluripotent stem
cells, and cells derived there from, cells of endodermal,
mesodermal or ectodermal orgin including but not limited to
epithelial cells, exocrine and endocrine cells, myoblasts,
fibroblasts, osteoblasts, chondroblasts, stromal cells,
hepatocytes, islet cells, neurobalsts keratinocytes, osteoclasts,
osteocytes, cardiac cells, chondrocytes, endothelial cells, and/or
muscle cells, and combinations thereof. The collagen that can be
used includes type I, II, III, IV or V collagen, or a combination
thereof. In particular embodiments, the material can be in a
gel-state, a solution, a paste, electrospun micron or nano
collagen, sheets of collagen, or a dehydrated rigid structure. The
material can be comprised in a syringe or in an injectable
solution. The material can be dermatologically acceptable
composition or a dermal or epidermal skin-equivalent. In certain
aspects, the amount of free carboxylic acid groups or free amine
groups that are present on the conjugated collagen/particle
material is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70,
80, or 90% less when compared with collagen that has not been
conjugated with a particle. Stated another way, at least 5, 10, 15,
20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90% or more of the free
carboxylic acid or free amine groups of the collagen are
conjugated.
[0010] Also contemplated is a collagen fibril covalently bound to
at least one particle, wherein the at least one particle has an
average particle diameter ranging from 20 to 1000 nanometers or 50
to 1000 nanometers. In particular aspects, the covalent bond can be
formed between a free carboxylic acid and/or amine group on the
collagen fibril and a reactive group present on the surface of the
particle. The reactive group can be, for example, an amine-reactive
group, a carboxylate-reactive group, a thiol-reactive group, and/or
a hydroxyl-reactive group. In one aspect, the covalent bond can be
formed between free-carboxylic acid groups present on the collagen
fibril and amine reactive groups on the particles, wherein amide
bonds can be formed between the carboxylic acid groups of the
collagen fibril and the amine-reactive groups of the particles.
[0011] In yet another embodiment, there is disclosed a method for
filling voids, defects, or increasing tissue volume in a mammal,
comprising administering to a patient or mammal in need thereof
(e.g., human, horse, cow, pig, dog, cat, rabbit, rat, mouse, etc.)
any one of the materials disclosed through this specification. The
materials, conjugated collagen/particles, or conjugated collagen
fibril/particles can be administered by intradermal or subcutaneous
injection. The void can be a fine line or wrinkle and the
appearance of the fine line or wrinkle can be reduced after
administration. The materials, conjugated collagen/particles, or
conjugated collagen fibril/particles can be administered to a lip
of the mammal, wherein the tissue volume of the lip is increased
after administration.
[0012] In a further embodiment, there is disclosed a method of
augmenting soft tissue or hard tissue in a mammal in need thereof
comprising administering or applying any one of the materials
disclosed through this specification to the soft or hard tissue.
The soft tissue can be cardiac muscle, smooth muscle, skeletal
muscle, menisci tissue, cartilage, tendons, ligaments, fascia,
skin, blood vessels, fibrous tissue, or extracellular matrix. For
instance, the materials can be used to support myocardial muscle to
a patient that is susceptible or that already has had a heart
attack. With respect to hard tissue, non-limiting examples include
bones or teeth. The materials can be used to treat bone fractures
or can be used to enhance boney in-growth by applying the materials
to bone fractures or to bones where an increase in boney in-growth
is desired.
[0013] In a particular embedment there is disclosed a method for
bulking articular cartilage by increasing tissue volume in a
person, comprising administering to a person in need thereof any
one of the materials or compositions described throughout the
specification into a joint capsule.
[0014] In one aspect, there is disclosed a method of reducing
collagen degradation in vitro or in vivo by enzymatic breakdown
comprising conjugating collagen with particles having an average
particle diameter ranging from 20 to 1000 nanometers or 50 to 1000
nanometers, wherein covalent bonds are formed between the collagen
and particles, and wherein degradation of the collagen by
collagenase is thereby reduced when compared with collagen that is
not conjugated with particles. The conjugation can be through a
covalent bond between free carboxylic acid groups or free amine
groups of collagen and reactive groups present on the surface of
the particles. The reactive groups can be, for example,
amine-reactive groups, carboxylate-reactive groups, thiol-reactive
groups, and/or hydroxyl-reactive groups. In one aspect, the
covalent bond can be formed between free-carboxylic acid groups
present on the collagen and amine reactive groups on the particles,
wherein amide bonds can be formed between the carboxylic acid
groups of the collagen and the amine-reactive groups of the
particles. The method can further include administering the
conjugated collagen to a mammal (e.g., intradermal or subcutaneous
injection or topical application).
[0015] In yet another embodiment, there is disclosed a method of
increasing cell attachment in vitro or in vivo comprising
conjugating collagen with particles having an average particle
diameter ranging from 20 to 1000 nanometers or 50 to 1000
nanometers, wherein covalent bonds are formed between the collagen
and particles, and wherein the surface area to volume ratio of the
nanoparticles attract cell re-population and collagen synthesis.
The conjugation can be through a covalent bond between free
carboxylic acid groups or free amine groups of collagen and
reactive groups present on the surface of the particles. The
reactive groups can be, for example, amine-reactive groups,
carboxylate-reactive groups, thiol-reactive groups, and/or
hydroxyl-reactive groups. In one aspect, the covalent bond can be
formed between free-carboxylic acid groups present on the collagen
and amine reactive groups on the particles, wherein amide bonds can
be formed between the carboxylic acid groups of the collagen and
the amine-reactive groups of the particles. The method can further
include administering the conjugated collagen to a mammal (e.g.,
intradermal or subcutaneous injection or topical application).
[0016] In still a further embodiment, there is disclosed a method
for generating tissue comprising seeding any one of the materials
disclosed through this specification with embryonic stem cells,
adult stem cells, induced pluripotent stem cells, and cells derived
there from or seeding with cells of endodermal, mesodermal or
ectodermal orgin including but not limited to epithelial cells,
exocrine and endocrine cells, myoblasts, fibroblasts, osteoblasts,
chondroblasts, stromal cells, hepatocytes, islet cells, neurobalsts
keratinocytes, osteoclasts, osteocytes, cardiac cells,
chondrocytes, endothelial cells, and/or muscle cells. The method
can further include administering the conjugated collagen to a
mammal.
[0017] The materials disclosed throughout the specification can
also be used to treat urinary diseases (e.g., urinary incontinence)
by administering to a mammal in need thereof said materials,
conjugated collagen/particles, or conjugated collagen
fibril/particles. By way of example, the materials can be formed
into a pelvic sling or can be used with an existing pelvic sling.
Alternatively, the materials can be in an injectable form and can
be used as a bulking agent to reduce or prevent urinary
incontinence by injecting said material into the mammal.
[0018] Also disclosed is a method for clotting blood comprising
administering to a mammal in need thereof the materials disclosed
throughout the specification to a site where blood clotting is
desired (e.g. internal or external wounds). Non-limiting examples
of external wounds include bed sores, cuts, scrapes, incisions,
open wounds, loss of limbs etc.
[0019] In one particular embodiment, there is disclosed a method
for treating osteoarthritis comprising administering to a mammal in
need thereof any one of the materials disclosed throughout the
specification. For instance, the materials can be administered to a
joint capsule or cartilage as a bulking agent to promote re-growth
and decrease pain.
[0020] In still another particular embodiment, there is disclosed a
method for enhancing nerve growth comprising administering to a
mammal in need thereof any one of the materials disclosed
throughout the specification. For instance, the materials can be
administered to nerves as conduits for nerve growth or
re-growth.
[0021] Also disclosed is a method for making collagen/particle
conjugated material of the present invention. Such a process
includes 1) functionalizing the preselected particles and 2)
crosslinking the functionalized particles with the soluble collagen
fibers in the presence bioconjugate reagent. The process can
further include an incubation period for polymerization following
the crosslinking step. In one aspect, the process includes: (1)
obtaining functionalized particles (e.g. metal particles such as
gold functionalized with cysteamine); (2) add functionalized
particles to a solution comprising EDC and NHS and optionally
buffer; and (3) add collagen to the solution with mixing. In
certain embodiments, the ratio of particles to collagen can be a
range of 1.times.10.sup.9 particles per mg of collagen to
2.times.10.sup.10 particles per mg of collagen, however broader
ranges are contemplated (e.g., 1.times.10.sup.4 to
1.times.10.sup.14 per mg of collagen, and any range and integer
therein). Also, 2 to 4 mg of a carbodiimide cross-linking agent
(e.g., EDC) per 30 mg of collagen can be used to form the covalent
bonds (in particular aspects, the ratio can be 3.2 mg+/-0.8 mg of a
carbodiimide cross-linking agent such as EDC per 30 mg of collagen
can be used), and/or 0.5 to 0.2 mg of a carbodiimide cross-linking
agent (e.g., EDC) per 1.times.10.sup.9-2.times.10.sup.10 particles
can be used to form the covalent bonds.
[0022] In another embodiment, there is disclosed a method for
increasing cellularity, promoting an influx of cells, promoting
cell adhesion, or promoting cell migration into a collagen implant
or a collagen-based bulking agent, comprising using anyone of the
materials or compositions disclosed throughout this specification
to make a collagen implant or the collagen-based bulking agent.
Also disclosed is a method for increasing cellularity, an influx of
cells, promoting cell adhesion, or promoting cell migration into a
collagen implant or a collagen-based bulking agent, comprising
covalently binding collagen to particles to form the collagen
implant or the collagen-based bulking agent, wherein covalent amide
bonds are formed between free carboxylic acid groups of the
collagen and amine reactive groups of the particles, and wherein
the particles have an average particle diameter size ranging from
50 to 1000 nanometers. Such methods can further include
administering the collagen implant or collagen-based bulking agent
to a person in need thereof.
[0023] It is contemplated that the materials disclosed throughout
the specification can be comprised within a dermatologically
acceptable vehicle, a pharmaceutically-acceptable vehicle, or
pharmacologically acceptable vehicle. Such vehicles are ones that
do not produce prohibitive toxicity, incompatibility, instability,
allergic response, and/or the like, when administered to a mammal
such as a human. Further, such compositions can be in powdered
form, dehydrated, electrospun, liquid form, gel-form, a semi-solid,
or solid. In this regard, compositions of the present invention can
have a viscosity range between 10 up to 100,000,000 cps, as
measured on a Brookfield Viscometer using a TC spindle at 2.5 rpm
at 25.degree. C. In particular aspects, a range of 150,000 to
250,000 can be used.
[0024] Routes of administering the materials and compositions of
the present invention can vary with the location and nature of the
condition to be treated. By way of example, topical application,
intradermal, parenteral, intramuscular, subcutaneous, percutaneous,
intratracheal, intraperitoneal, direct injection (e.g., an
injectable solution), and surgical (e.g., through incision and
placing in target area).
[0025] It is contemplated that any embodiment discussed in this
specification can be implemented with respect to any method or
composition of the invention, and vice versa. Furthermore,
compositions of the invention can be used to achieve methods of the
invention.
[0026] "Injectable collagen" includes collagen pastes, gels,
solutions, or suspensions, homogeneous or heterogeneous, which are
contained in syringes, tubes or other containers equipped with
appropriate plungers or systems, designed to extrude the collagen
through a needle or a nozzle. Injectable collagen is designed for
injection, surgical application through a trocar, or direct
application on a wound surface.
[0027] "Mammals" includes humans, horse, cow, pig, dog, cat,
rabbit, rat, mouse, etc.
[0028] "Keratinous tissue" includes keratin-containing layers
disposed as the outermost protective covering of mammals and
includes, but is not limited to, skin, hair and nails.
[0029] "Topical application" means to apply or spread a composition
onto the surface of keratinous tissue. "Topical skin composition"
includes compositions suitable for topical application on
keratinous tissue. Such compositions are typically
dermatologically-acceptable in that they do not have undue
toxicity, incompatibility, instability, and the like, when applied
to skin. Topical skin care compositions of the present invention
can have a selected viscosity to avoid significant dripping or
pooling after application to skin.
[0030] The term "about" or "approximately" are defined as being
close to as understood by one of ordinary skill in the art, and in
one non-limiting embodiment the terms are defined to be within 10%,
preferably within 5%, more preferably within 1%, and most
preferably within 0.5%.
[0031] The terms "inhibiting" or "reducing" or any variation of
these terms, when used in the claims and/or the specification
includes any measurable decrease or complete inhibition to achieve
a desired result.
[0032] The term "effective," as that term is used in the
specification and/or claims, means adequate to accomplish a
desired, expected, or intended result.
[0033] The use of the word "a" or "an" when used in conjunction
with the term "comprising" in the claims and/or the specification
may mean "one," but it is also consistent with the meaning of "one
or more," "at least one," and "one or more than one."
[0034] The words "comprising" (and any form of comprising, such as
"comprise" and "comprises"), "having" (and any form of having, such
as "have" and "has"), "including" (and any form of including, such
as "includes" and "include") or "containing" (and any form of
containing, such as "contains" and "contain") are inclusive or
open-ended and do not exclude additional, unrecited elements or
method steps.
[0035] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the examples, while indicating specific embodiments
of the invention, are given by way of illustration only.
Additionally, it is contemplated that changes and modifications
within the spirit and scope of the invention will become apparent
to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented below.
[0037] FIG. 1. A schematic diagram for covalently conjugating
collagen (designated "1") with a gold particle (designated "2") via
the formation of an amide bond between a free carboxylic acid group
of the collagen and a reactive amine group of the functionalized
particle with mercaptoethylamine (MEA).
1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)
and N-hydroxysulfosuccinimide (Sulfo-NHS) are used to facilitate
conjugation.
[0038] FIG. 2. UV spectrum illustrating gold particles
functionalized with .beta.-mercaptoethylamine (MEA).
[0039] FIG. 3. SEM of a gold particle conjugated gel scaffold.
[0040] FIG. 4. EDS image demonstrating that crosslinked particles
are gold particles within the scaffold structure.
[0041] FIG. 5. Bar diagram illustrating the improved resistance to
collagenase degradation of collagen conjugated with gold
particles.
[0042] FIG. 6. Bar diagram illustrating cell viability in the
presence of collagen conjugated with gold particles.
[0043] FIG. 7. Fourier Transform Infrared Spectroscopy of collagen
with and without gold particles illustrating an 18% decrease in
free carboxylic acid groups in conjugated collagen/gold particle
material when compared with collagen alone.
[0044] FIG. 8. DNA concentrations per scaffold group and treatment
over time. Day 7 is left bar, and Day 14 is right bar for each
group, respectively.
[0045] FIG. 9. Glycosaminoglycans (GAG) concentrations per scaffold
group and treatment over time. Day 7 is left bar, and Day 14 is
right bar for each group, respectively.
[0046] FIG. 10. Live/Dead staining of all groups at Day 7
demonstrating high viability and cellularization.
[0047] FIG. 11. Cellularization of the groups at two time points
demonstrating surface proliferation at Day 7 with more elaborate
interior penetration at Day 14. Cellularization of the AuNP
associated channels and cavities was noted.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0048] The inventors have discovered that by covalently binding
particles to collagen at free carboxylic acid groups of collagen or
collagen fibrils, degradation of collagen can be reduced. This
results in a collagen-based material that is more stable when
administered to a mammal to treat or prevent a particular disease
or skin condition. Further, by using particles having an average
particle diameter of 20 to 1000 nanometers, 50 to 1000 nanometers,
or even 50 to 150 nanometers, the resulting collagen/particle
material creates an environment which promotes cellular growth and
infiltration (e.g., cells that are either present within the
patient or cells that are incorporated into the material are
attracted to the particles, which allows for a more sustained and
vibrant growth of the cells than was to be expected when compared
with collagen that does not include such particles) while
exhibiting reduced toxicity when compared with particles that are
less than 20 nanometers or less than 50 nanometers. That is to say,
the inventors have discovered an effective way to stabilize
collagen by reducing collagen degradation while also promoting
cellular growth without the risk of toxic side effects that are
currently seen in existing collagen-based materials.
[0049] Without wishing to be bound by theory, it is believed that
covalent bonds formed on the free carboxylic sites of collagen
hindered and/or block some of the collagenase binding sites, while
the particle size provides a sufficient surface area and surface
energy which allows for cellular adherence, increased cellularity,
and protein adsorption, thereby promoting cellular proliferation
and growth. Additionally, metallic particles may provide
anti-oxidative effects which reduces reactive oxygen species and
other free radicals that can damage cells, and metallic particles
may provide anti-microbial effects. Further, the particle size is
sufficient to reduce toxicity in the surrounding environment by
preventing or reducing cellular uptake of the particles.
[0050] These and other aspects of the present invention are
described in further non-limiting detail below.
A. Collagen
[0051] Collagen is a type of protein found in mammals that connects
and supports bodily tissues, such as skin, bone, tendons, muscles,
and cartilage. It also provides support for internal organs and is
present in teeth. There are more than 25 types of collagens that
naturally occur in the body, all of which can be used in the
context of the present invention. The more prevalent collagens
include Types I (found in skin, tendon, vascular, ligature, organs,
bone), II (found in cartilage), III (found in reticular fibers), IV
(forms bases of cell basement membrane) and V (found in cell
surfaces, hair, and placenta). Some of the more prevalent
structural features of collagen include an abundance of glycine,
proline, hydroxyproline, free carboxylic acid groups, and free
amine groups See Collagen Structure and Mechanics (2008).
[0052] With respect to skin, collagen provides the skin with
strength, flexibility, and resilience. It also provides a framework
for the growth of cells and blood vessels in skin. Collagen
degradation (e.g., in aged skin, diseased, damaged skin such as
scars, sun damage, acne, etc.) leads to the presence of fine lines,
wrinkles, pits, nodules, creases, and the like in skin. One way to
reduce the appearance of these skin defects is to inject collagen
into skin, which results in filling-in the skin defects, hence a
"dermal filler." Collagen also has several medical uses ranging
from increasing joint mobility, treating burns and other open skin
wounds, treating osteogenesis imperfect (i.e., brittle bone
disease), and other medical uses disclosed and claimed throughout
this specification.
[0053] Collagen that can be used in the context of the present
invention can be extracted from a wide range of sources (e.g.,
porcine, bovine, human, fish, rat tail, etc.). Non-limiting
collagen materials that can be used include recombinant human
collagen, tissue engineered human-based collagen, porcine collagen,
human placental collagen, bovine collagen, autologous collagen,
collagen fibers, and human tissue collagen matrix. Further collagen
and collagen-based products that can also be used are commercially
available, non-limiting example of which are listed in
International Cosmetic Ingredient Dictionary and Handbook,
12.sup.th edition, volume 1, page 656 (2008), which is incorporated
by reference. Additional non-limiting examples of commercially
available collagen products that can be used in the context of the
present invention include Cosmoderm.RTM. 1 and 2, CosmoPlast.RTM.,
Zyderm.RTM., and Zyplast.RTM., all of which are manufactured by
Inamed Corp., Santa Barbara Calif. Evolence.RTM.. In particular
embodiments, porcine collagen is used.
B. Particles and Covalent Bond Formation with Collagen
[0054] As explained above, particles having an average particle
diameter size of 20 to 1000 nanometers, 50 to 1000 nanometers, or
50 to 150 nanometers can be used in the context of the present
invention. The average particle diameter size can be determined by
Dynamic Light Scattering (DLS). DLS is a technique that provides
the size distribution profile of particles in suspension. The
average particle size can be determined from the size distribution
profile (Thomas (1987)) In addition, there are several resources
available by which one can purchase or obtain particles having a
particular diameter size (e.g., PELCO.RTM. NanoXact & BioPure
Gold and Silver Colloids from Ted Pella, Inc. (Redding, Calif.);
Accurate Spherical Gold Nanoparticles, Gold Nanorodz, Microgold,
Gold Nanobeads, Gold Nanowires, Platinum, Palladium, and
Trimetallic Nanoparticles from NanoPartz, Inc. (Loveland, Colo.);
and Gold Nanoparticles, Silver Nanoparticles, Platinum
Nanoparticles, Palladium Nanoparticles, and Green Nanoparticles
from Nanoparticle Biochem Inc, (Columbia, Mo.)).
[0055] The particles that can be used can include or be made up of
either metallic material, ceramic material, and/or biodegradable
material or a combination thereof. With respect to metallic
particles, non-limiting examples include gold, silver, platinum,
titanium, nickel, and/or copper. In particular instances, the
material used for the particles (e.g., gold or silver) can have
antimicrobial properties, which can be useful to reduce the
likelihood of infection. Further, such particulate material can
function as an electron acceptor and can therefore reduce
free-radical damage caused by reactive oxygen species ("ROS").
[0056] The particles that are used in the context of the present
invention can include reactive groups, non-limiting examples of
which include amine-reactive groups, carboxylate-reactive groups,
thiol-reactive groups, carboxylic acid reactive groups, or
hydroxyl-reactive groups, or any combination thereof. Such
functionalized particles are commercially available and can be made
by a person having ordinary skill in the art. Further, the use of
cross-linking agents can be used to promote formation of covalent
bonds between collagen and the particles and can also be used to
promote cross linking between the collagen itself (e.g.,
cross-linking of the collagen can occur via the particles when the
particles have at least two functional groups present where one of
the functional groups forms a covalent bond with collagen and the
other function group forms a second covalent bond with collagen or
in instances with the cross-linking agent forms covalent bonds
between the collagen itself). A non-limiting process is provided
below.
[0057] In particular embodiments, the particles include amine
reactive groups that are capable of forming an amide bond with free
carboxylic acid groups present in the collagen. By way of example,
FIG. 1 describes such an embodiment. In particular, FIG. 1
illustrates that the carboxylic acid functional group on collagen
fiber 1, is first activated by EDC, then though nucleophilic
addition to generate amide bond between collagen fiber and the
metallic nanomaterial 2. EDC forms an active ester functional group
with carboxylate groups on the collagen fibrils; but hydrolysis
occurs rapidly and thus EDC is typically coupled with sulfo-NHS to
form a sulfo-NHS ester intermediate. The ester intermediates then
react with amine groups on the metallic nanoparticles. The
EDC-sulfo-NHS facilitates an amide bond between the collagen and
MEA attached to the particle with release of an isourea by-product.
NHS is commonly added to the EDC to enhance stability and
binding.
[0058] The toxicity of the chemicals utilized to promote formation
of covalent bonds between collagen and the particles and to
crosslink collagen should be considered. Glutaraldehyde,
hexa-methylene diisocyanate, and EDC are all commonly used
crosslinkers, but only the carbodiimide is non-toxic and does not
become incorporated within the collagen scaffold during
crosslinking (see Shanmugam (2006), Lee (2001), Rault (1996),
Grtzer (2001), Chan (2005), Billiar (2001), Pieper (1999),
Haidekker (2006)). Conversely, glutaraldehyde and hexamethylene
diisocyanate do become incorporated within the scaffold and may
release toxic residues into the body as the scaffold is degraded.
Additionally, excessive crosslinking may drastically change the
microstructure and render the scaffold so resistant to degradation
that it becomes encapsulated by a fibrous layer and is never
replaced by healthy tissue.
C. Process for Making Conjugates
[0059] The following procedure is a non-limiting way to make the
conjugated materials of the present invention: [0060] (1) Obtain
non-polymerized collagen: [0061] a. Mix 30 mg of lyophilized
collagen with 1 mL of acetic acid (10 mM). [0062] b. Dissolve for 3
hours at room temperature by turning the vial slowly. [0063] (2)
Prepare concentrated functionalized Nanomaterials: [0064] a. Spin
1.344 mL of 100 nm gold nano-particles suspension (AuNP
concentration 5.6.times.10.sup.9 particles/mL for 5 min at 7,000
rpm. [0065] b. Remove 1.144 mL of water leaving 0.2 mL of AuNP in
water suspension. [0066] c. Add 9.1 uL of 0.12M cysteamine
(=beta-mercaptoethylamin; MEA) to 0.2 mL of AuNP suspension. [0067]
d. Mix to yield functionalized nanomaterials by pipette, turning
over 3 times, or by vortex for 5 seconds in room temperature.
[0068] (3) Prepare 10.times. phosphate buffer saline (PBS) solution
[0069] (4) Dissolve 0.0032 g of EDC
(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and
0.00424 g sulfo-NHS (N-hydroxysuccinimide) in 0.2 mL of
10.times.PBS buffer. [0070] (5) Add all 0.209 ml, of functionalized
nanomaterials to 0.2 mL of EDC and NHS in 10.times.PBS buffer if
rat collagen is used. If human collagen is used then the same
buffer system can be used or a different one can be used (e.g.,
sodium phosphate dibasic buffer system. [0071] (6) Add 0.045 mL of
1M NaOH to nanomaterials in PBS buffer. [0072] (7) Mix 0.454 mL of
functionalized nanomaterials EDC, NHS in 10.times.PBS buffer with
NaOH to 1 mL of collagen solution at 30 g/L. [0073] (8) Pipette up
and down 5-10 times to ensure mixture. [0074] (9) Place in
incubator at 37.degree. C. for 90 minutes to polymerize. [0075]
(10) Remove the newly formed scaffold from the incubator and
condition to be injected out of 30 Ga needle or prepare scaffold in
other forms.
[0076] As noted above, this process is a non-limiting example of
one way to make a particle/collagen conjugate within the context of
the present invention. Modifications and variations are
contemplated and can be made to prepare a desired end-product for a
particular treatment option.
D. Compositions of the Present Invention
[0077] As noted above, the conjugated materials of the present
invention (e.g., conjugated collagen/particles, or conjugated
collagen fibril/particles) can be included in compositions such as
injectable compositions, topical compositions, implantable
compositions, and can take a variety of forms (e.g., liquid,
powdered, dehydrated, semi-solid, gel, solid, rigid, etc.). The
compositions can also include additional ingredients such as
cosmetic ingredients (both active and non-active) and
pharmaceutical ingredients (both active and non-active) depending
on the nature of the route of administration and/or the particular
disease to be treated.
[0078] The CTFA International Cosmetic Ingredient Dictionary and
Handbook (2008), 12.sup.th Edition, describes a wide variety of
non-limiting cosmetic ingredients that can be used in the context
of the present invention. Examples of these ingredient, which can
be useful for topical products include adsorbents, emulsifiers,
stabilizers, lubricants, solvents, moisturizers (including, e.g.,
emollients, humectants, film formers, occlusive agents, and agents
that affect the natural moisturization mechanisms of the skin),
water-repellants, vitamins (e.g., A, B, C, D, E, and K), botanical
extracts, anti-microbial agents, antioxidants (e.g., BHT and
tocopherol), chelating agents (e.g., disodium EDTA and tetrasodium
EDTA), and preservatives.
[0079] Non-limiting examples of pharmaceutical ingredients that can
also be used include analgesics, anesthetics, anti-inflammatory
agents including non-steroidal anti-inflammatory drugs,
antibiotics, antifungals, antivirals, antimicrobials, anti-cancer
actives, antipsoriatic agents, antiseborrheic agents, biologically
active proteins and peptides, burn treatment agents, cauterizing
agents, skin protectant/barrier agents, steroids including hormones
and corticosteroids, wound treatment agents, wound healing agents,
etc.
E. Kits
[0080] Kits are also contemplated as being used in certain aspects
of the present invention. For instance, a material or composition
of the present invention can be included in a kit. A kit can
include a container. Containers can include a bottle, a metal tube,
a laminate tube, a plastic tube, a syringe, a dispenser, a
pressurized container, a barrier container, a package, a
compartment, or other types of containers such as injection or
blow-molded plastic containers into which the materials or
compositions are retained. A kit can also include instructions for
using the kit and/or compositions. Instructions can include an
explanation of how to apply, use, and maintain the
compositions.
EXAMPLES
[0081] The following examples are included to demonstrate certain
non-limiting aspects of the invention. It should be appreciated by
those of skill in the art that the techniques disclosed in the
examples which follow represent techniques discovered by the
inventor to function well in the practice of the invention.
However, those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Functionalized AuNP
[0082] Gold nanoparticles (AuNP) having an average particle
diameter size of 20 nanometers were functionalized with 15 uM of
2-mercaptoethylamine (MEA). FT-IR spectrometry confirms the
presence of the functionalize groups on the AuNP. Additionally, the
optimal concentration of MEA is determined through the use of
UV-Vis spectroscopy before and after the addition of an electrolyte
(10% NaCl). The optimal concentration was defined as the
concentration of MEA that stabilized the AuNPs, preventing
aggregation and maintaining dispersion even after the addition of
10% NaCl. As shown in FIG. 2, the UV/Vis spectrum undergoes a shift
in absorbance peak when functionalized with MEA. The functionalized
nanomaterials are then mixed with 2 mM EDC
(1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide) and 5 mM sulfo-NHS
(sulfo-N-Hydroxysuccinimide) in order to facilitate covalent
binding to the carboxyl groups on the collagen fibrils.
Example 2
AuNP-Collagen Conjugated Material
[0083] To form AuNP-collagen gel scaffolds, 2.5 mL rat tail
collagen (concentration of 9 mg/ml) was added to a mixture of 0.5
mL 10.times.PBS, 0.057 mL 1M NaOH, 4.0 mg EDC, 5.3 mg sulfo-NHS and
0.5 mL of functionalized AuNP solution (9.408.times.10 9
particles). Next, the matrix was placed in an incubator at
37.degree. C. for 90 minutes for polymerization and crosslinking.
The ratio between the number of nanoparticles and collagen solution
is 3.8.times.10 9 AuNP per 9 mg rat tail collagen.
[0084] FIG. 3, which is an SEM of an exemplary collagen gel
scaffold with 20 nm AuNP attached through a cysteine EDC/NHS
crosslinker. SEM characterizes the distribution and density of the
gold nanoparticles in the collagen gel scaffolds. As shown in FIG.
3 displaying an SEM of the AuNP-collagen material at 100.times.,
AuNPs are present throughout the scaffold, which indicates that the
AuNPs are binding to the collagen fibrils. The gels undergo
extensive washing which removes any unbound AuNPs from the collagen
scaffold (Haidekker (2006)).
[0085] While the SEM micrograph shown in FIG. 3 confirms the
attachment of the nanoparticles, FIG. 4 confirms that the attached
particles are gold particles. FIG. 4 is an EDS (Energy Dispersive
Spectroscopy) image of the gold nanoparticles covalently
immobilized to the collagen scaffold.
Example 3
Degradation Assay
[0086] As noted above, the inventors believe that by blocking a
portion of the carboxylic acid binding sites on collagen fibrils
with particles, a decrease in collagenase activity and resulting
decrease in degradation rates of collagen would occur. This has
been confirmed experimentally (see data in FIG. 5). As illustrated
in FIG. 5, the effects of the nanomaterials on collagen degradation
at different concentrations are examined and compared with the
samples without nanomaterials. The diameter size of the gold
nanoparticles (AuNP) was constant for each sample at 100 nm and the
process by which the conjugated material was made is that described
in Section C ("Process for Making Conjugates") of the Description
of Illustrative Embodiments of this specification, which is
incorporated into this example by reference. The concentration
varied between samples (1.times., 2.times., 4.times.).
Concentration of the zero length crosslinker 1-ethyl-3-(3-dimethyl
aminopropyl) carbodiimide (EDC) also varied between samples. The
control sample contained rat tail collagen crosslinked by ECD
(1.times.) without nanoparticles. A collagenase assay was performed
to test the biological stability of the crosslinked samples. The
biological stability through degradation of the samples was
measured by the amount of hydroxyproline release. The percent of
the degraded matrix is reported relative to the control (EDC
1.times. no nanoparticles) in FIG. 5. The error bars represent
standard deviation calculated from eight samples. Simply doubling
the concentration of the zero length crosslinker ECD significantly
decreased the degradation of the scaffold by 30% (p<0.001).
Adding gold nanoparticles to the matrix also had a significant
effect of decreasing the degradation of the matrix (p<0.001). A
1.times. concentration of 100 nm gold nanoparticles reduced
degradation of 50% while a 2.times. concentration was significantly
reduced to as little as at 7% degradation (p<0.01). There are no
significant differences between AuNP (2.times.) and AuNP (4.times.)
as well as AuNP (1.times.) with an increase of EDC to 2.times.
concentration. The results indicate that addition of 100 nm gold
nanoparticles aids in the proteolytic resistance of the collagen
and increase the biological stability of the matrix. The results
also indicate that the attachment of the nanomaterials to the
collagenase binding sites along the collagen fibrils decreases the
degradation rate of the scaffold. The results further indicate that
a range of sized and shaped nanomaterials, such as nanorods with
diameters between about 20 to about 1000 nm can be utilized.
[0087] Moreover, through functionalizing the nanomaterials with
amine groups (MEA), the number of bonds formed between the
nanomaterial and collagen may be maximized. Additionally, each
nanoparticle may provide multiple (more than two) sites of
attachment, while most crosslinking agents typically provide a
two-point link between collagen fibrils. This approach may enable
fabrication of specific pre-determined collagen matrix pore sizes
optimal for tissue ingrowth and native collagen deposition. Since
gold nanomaterials act as free radical scavengers, the scaffold
will also contribute to antioxidant effects while also provide
antimicrobial effects.
[0088] Other proteins may be conjugated to the nanomaterials to
facilitate specific interactions once inserted into the body. For
example, fibrin may be added to the nanomaterial with MEA to assist
in clotting of blood during wound healing.
Example 4
Cell Viability Assay
[0089] FIG. 6 provides data showing the effect of the gold
nanoparticles used on cell viability via an WST-1 viability assay.
Particularly, collagen scaffolds with gold nanoparticles as
prepared in Example 3 in concentrations of 1.times., 2.times.,
4.times., and 8.times. were incubated with cells for 3 days. The
viability of the cells was determined by conversion of WST-1 to an
absorbance value recorded with UV-Vis. The results shown in FIG. 3
indicate viability of the control is not significantly higher than
cell viability in the presence of nanoparticles. Therefore,
nanomaterials have a very low cytotoxicity. With a greater
absorbance reading from the higher concentration of gold
nanoparticles, it is suggested that there was a larger turnover of
cells in the presence of gold nanoparticles to convert more WST-1
or the number of binding sites at 2.times. concentration of AuNP is
saturated leaving gold nanoparticles in the media and interfering
with the UV-Vis absorbance values.
Example 5
Carboxylic Acid Binding Analysis
[0090] The collagen scaffolds as prepared in Example 3 was analyzed
to determine the amount of free carboxylic acid groups remaining on
the scaffolds. In particular, Fourier Transform Infrared
Spectroscopy was used on the scaffold with and without the gold
particles. This technique is used to indicate a reduction in peak
at the carboxylic acid sites showing the binding of the gold to the
COOH on the collagen. As illustrated in FIG. 7, a decrease in peak
at 1125-920 nm area was observed, which is indicative of a
reduction in the C--OH bond (free COOH groups). The area under the
curve went from 0.3680 to 0.3, which is an 18% decrease in free
carboxylic acid groups on the collagen/particle conjugate
scaffold.
Example 6
In Vitro Assessment of Cellularity Cellular Retention, and
Extracellular Matrix Production
[0091] This example provides data showing the effects of conjugated
collagen/particle material on the cellularity, cellular viability,
extracellular matrix production, and cellular distribution when
compared to untreated controls.
Materials and Methods
[0092] Scaffold Group Assignments:
[0093] Five different combinations of gold nanoparticales and
collagen gels were evaluated. The groups were numbered 1 through 5
and are outlined in Table 1. A total of 10 constructs were seeded
with dermal fibroblasts and incubated for 7 and 14 days. A total of
50 samples were analyzed.
TABLE-US-00001 TABLE 1 Group Group 1: Group 2: Group 3: Group 4:
Group 5: Control Collagen + Collagen + Collagen + Collagen +
Collagen + EDC + 2x EDC + 1x EDC + 4x 1/2 EDC + 2x EDC AuNP AuNP
AuNP AuNP Day 7 n = 5 n = 5 n = 5 n = 5 n = 5 Day 14 n = 5 n = 5 n
= 5 n = 5 n = 5
[0094] Fibroblast Harvest and Culture:
[0095] Skin dermis was harvested from dogs humanely euthanized by
an overdose of barbiturate for reasons unrelated to this study.
Tissue was placed in Dulbecco's Modified Eagle's Media with 10%
fetal bovine serum, 0.008% Hepe's buffer, 0.008% non-essential
amino acids, 0.002% Penicillin 100 IU/mL streptomyic 100 ug/mL,
amphoterocin B 25 ug/mL, 0.002% L-ascorbate, 0.01% L-glutamine
(DMEM+FBS) for transport. The dermal tissue was sectioned into 2
mm.times.2 mm pieces using a #10 scalpel blade under sterile
technique. The tissue fragments were combined with sterile Type IA
clostridial collagenase solution (Sigma, USA), at a concentration
of 7.5 mg/mL of RPMI 1640 solution. The mixture was agitated in an
incubator at 37.degree. C., 5% CO2, 95% humidity for 6 hours. The
digested solution was centrifuged at 1000 RPM for 10 minutes. The
supernatant was decanted and the cellular pellet re-suspended in 5
mL of DMEM+FBS. The flasks were incubated at 37.degree. C., 5% CO2,
95% humidity with sterile medium change performed every 3 days.
Fibroblasts were monitored for growth using an inverted microscope
until observance of 95% cellular confluence per tissue culture
flask. Cells were transferred to 75 mL tissue culture flasks
through subculturing until the 3rd passage is achieved and then
frozen for future use. Cells were subsequently thawed, released
from monolayer and put into solution prior to use.
[0096] Scaffold Seeding:
[0097] Collagen gels 250 .mu.l in approximate volume were fashioned
from each of group and treatments. Ten (n=10) constructs of each
group were placed in individual wells of a tissue culture plate in
PBS for 24 hours, placed inside sterile incubators at 37.degree.
C., 5% CO2, 95% humidity as a pre-soaking conditioning. Previous
microbial culture and sensitivity examinations confirmed no growth
after 3 days of culturing of the constructs for a period of 3 days.
After pre-soaking, media was removed from each well and replaced
with the fibroblast cell solution at a concentration of
1.times.10.sup.6 cells/ml. Constructs were cultured statically with
the cell solution for 24 hours, at which time the cell solution was
replaced with DMEM+FBS culture media for the duration of the
study.
[0098] Construct Harvest and Assessment:
[0099] Five (n=5) constructs were harvested from each group at days
7 and 14. Cross sections were taken from each construct for
cellular viability and distribution assessment. Cell viability was
determined with the use of ethidium homodimer-1 (4 uL/ml PBS) and
Calcein AM (acetoxymethylester) (0.4 ul/ml PBS) fluorescent stains
(LIVE/DEAD Viability/cytotoxicity Kit, Molecular Probes Co.) and
the use of ultraviolet microscopy. One millimeter sections were
made and incubated with the staining agents for 20 minutes at room
temperature, placed on a glass microscope slide, moistened with
several drops of PBS, and stained using the fluorescent double
labeling technique. The sections were examined under 10.times.
magnification. Images of each section were digitally captured by an
Olympus DP-70 (Olympus, Melville, N.Y.) digital camera and saved as
Tiff files. The remainder of each construct was lyophilized and a
dry weight obtained and then mixed with 1 ml Papin Solution.
Portions of each digest were used to determine GAG content by the
dimethylmethylene blue assays, and collagen content by determining
hydroxyproline concentrations. The remaining solution was incubated
at 60.degree. C. in a water bath for 4 hours. The Quant-iT
PicoGreen.TM. double stranded DNA quantification assay (Invitrogen)
was used to determine the cellularity of the remaining scaffold.
Double stranded DNA extracted from bovine thymus was mixed with TE
buffer (Invitrogen) to create standard DNA concentrations of 1,000,
100, 10 and 1 ng/ml. The standards and 100 ul of each papain
digested sample (used in the above GAG and hydroxyproline assays)
were added to a 96 well plate. 100 uL of 2 ug/ml of Pico Green
reagent was added to each well and the plate incubated for 5
minutes. Sample fluorescence was read at 485 nm excitation/528 nm
emission by the Syngergy HT-KC-4 spectrophotometric plate reader
(BioTec, Winooski Vt.). Absorbances were converted to ng/1
concentrations and total double stranded DNA yield expressed in ng
using FT4 software (BioTec, Winooski Vt.).
[0100] Each data set was examined and outliers were determined by
those values that were more or less than 2 standard deviations
outside of the remaining data set, and those values discarded.
Differences within and between groups were analyzed statistically
with a one-way ANOVA test with difference between individual groups
determined by various post-hoc all-pairwise examinations with
statistical significance set at p<0.05.
Results
[0101] Ds DNA Assessment as a Measure of Cellularity:
[0102] As illustrated in FIG. 8, Day 7: Group 1 possessed
significantly higher amounts of DNA than groups 2,3 and 5. No other
significant differences were detected. Day 14: Group 2 possessed
significantly higher amounts of DNA than group 5. No other
significant differences were detected. Groups 1 showed a
significant decline in DNA content over time, whereas Groups 2 and
3 showed an increase in DNA between the two time points. No other
significant differences were detected.
[0103] Glycosaminoglycans (GAG) Assessment:
[0104] As illustrated in FIG. 9, Day 7: Group 1 possessed
significantly higher amounts of GAG than group 5. No other
significant differences were detected. Day 14: Group 2 possessed
significantly higher amounts of GAG than groups 1 and 5. No other
significant differences were detected. Group 1 showed a significant
decline in GAG content over time, whereas Groups 2 and 3 and 5
showed an increase in GAG between the two time points. No other
significant differences were detected.
[0105] Cellular Viability/Integration Assessment:
[0106] Cellular viability was subjectively >95% in all groups at
all time points (FIG. 11). Marked cell rafting was evident in all
groups making specific viability quantification impossible via
computer image analysis due to the overwhelming confluence of
viable cellularity. No group demonstrated what would be interpreted
as indications of cellular death. Each group demonstrated cellular
adherence, retention and proliferation (FIG. 12). The day 7 groups
demonstrated more evidence of cellular surface proliferation in
large rafts, whereas by day 14, deeper penetration into the
interior of the gel constructs was witnessed in each group.
Subjectively, no difference could be detected in the degree or
extent of cellular penetration between groups. In those sections
where the AuNP-associated cavities in the gel were witnessed,
cellular proliferation was noted to be abundant along the channels
(see Day 7, Groups 2 and 5).
CONCLUSIONS
[0107] These data in Example 6 suggest that although initial
cellularization of collagen gels appeared to be most optimal in the
non-treated gels, longer term analysis revealed that, in general,
the AuNP treated groups appeared to either retain cells or foster
their proliferation better than non-treated gel constructs. It
should be noted that these observations are largely based on trends
only as at Day 14, the only statistical difference regarding DNA
content among groups was Group 2 possessing more cellularity that
Group 5. The difference in treatment between these two groups was
doubling the EDC concentration in Group 5 which may impart a
deleterious effect on cellular retention or proliferation. But
examining the two time points within each treatment, Group 1 is the
only group which demonstrated a significant decline in cellularity
over time, whereas Groups 2 and 3 showed increases. Although
cellular mitogenesis or proliferation was not specifically examined
here, this increase in cellularity in those groups was likely a
result (in part) of increasing cellularity as no additional cells
were added at any time point. All groups demonstrated the ability
to retain cells and foster their integration into the interior of
the gel constructs over time without evidence of detectable cell
death. Based on the paired dsDNA/cell viability data, it appears as
though cells were less successfully retained (but did not
necessarily undergo increasing amounts of cell death) in Group 1
between days 7 and 14, thus implying that the treated groups also
favored cellular retention better, especially in Groups 2 and 3
(2.times. and 1.times. AuNP concentrations). This increase in
cellularity was likely responsible for a corresponding large
increase in GAG production in Group 2 at Day 14. With respect to
the examination of hydroxyproline as a determinant of collagen
production, the activity of Groups 1,2 and 3 was very similar at
both time points. Interestingly, Group 4 (4.times. AuNP)
demonstrated lower levels of HP concentrations, especially at Day
14. Group 5 showed an initial spike in HP content which declined
significantly over time.
[0108] All of the materials, compositions, or methods disclosed and
claimed in this specification can be made and executed without
undue experimentation in light of the present disclosure. While the
materials, compositions, or methods of this invention have been
described in terms of particular embodiments, it will be apparent
to those of skill in the art that variations may be applied to the
materials, compositions, or methods without departing from the
concept, spirit and scope of the invention
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