U.S. patent application number 17/385862 was filed with the patent office on 2021-11-18 for scalable three-dimensional elastic construct manufacturing.
The applicant listed for this patent is ALLERGAN PHARMACEUTICALS INTERNATIONAL LIMITED. Invention is credited to Suzanne Marie MITHIEUX, Anthony Steven WEISS.
Application Number | 20210353825 17/385862 |
Document ID | / |
Family ID | 1000005738544 |
Filed Date | 2021-11-18 |
United States Patent
Application |
20210353825 |
Kind Code |
A1 |
WEISS; Anthony Steven ; et
al. |
November 18, 2021 |
SCALABLE THREE-DIMENSIONAL ELASTIC CONSTRUCT MANUFACTURING
Abstract
Tissue repair and restoration can be performed using an elastic
material formed from tropoelastin. The elastic material can be
formed by providing a solution of tropoelastin monomers, applying
the solution to a surface, and heating the solution on the surface
in absence of a cross-linking agent to enable the tropoelastin
monomers to bind to each other to form an elastic material that
does not dissociate into tropoelastin monomers when the elastic
material is contacted with an aqueous solution.
Inventors: |
WEISS; Anthony Steven;
(Sydney, NSW, AU) ; MITHIEUX; Suzanne Marie;
(Sydney, NSW, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALLERGAN PHARMACEUTICALS INTERNATIONAL LIMITED |
Dublin |
|
IE |
|
|
Family ID: |
1000005738544 |
Appl. No.: |
17/385862 |
Filed: |
July 26, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16937442 |
Jul 23, 2020 |
11077226 |
|
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17385862 |
|
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14650542 |
Jun 8, 2015 |
10842913 |
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PCT/AU2013/001435 |
Dec 10, 2013 |
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16937442 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C09D 189/00 20130101;
A61L 27/52 20130101; B05D 3/007 20130101; A61L 27/227 20130101;
C07K 14/78 20130101; A61K 38/00 20130101; A61L 27/22 20130101 |
International
Class: |
A61L 27/22 20060101
A61L027/22; C07K 14/78 20060101 C07K014/78; A61L 27/52 20060101
A61L027/52; B05D 3/00 20060101 B05D003/00; C09D 189/00 20060101
C09D189/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 10, 2012 |
AU |
2012905409 |
Claims
1. An elastic material comprising tropoelastin monomers, wherein
the tropoelastin monomers are not linked to one another by a
linker, and wherein the material does not dissociate into
tropoelastin monomers when the material is exposed to physiological
conditions.
2. The elastic material of claim 1, wherein the physiological
conditions comprise a pH between about 7.2 and about 7.5.
3. The elastic material of claim 1, wherein the physiological
conditions comprise a temperature of between about 36.degree. C.
and about 37.degree. C.
4. The elastic material of claim 1, wherein the physiological
conditions comprise a salt concentration of about 150 mM.
5. The elastic material of claim 1, wherein the physiological
conditions comprise a pH between about 7.2 and about 7.5, a
temperature of between about 36.degree. C. and about 37.degree. C.,
and a salt concentration of about 150 mM.
6. The elastic material of claim 1, wherein the material does not
dissociate into tropoelastin monomers when the material is exposed
to a pH of about 6.5 to about 8.0, a temperature of from about
30.degree. C. to about 45.degree. C., and a salt concentration of
about 75 mM to about 300 mM.
7. The elastic material of claim 1, wherein the elastic material is
biocompatible.
8. The elastic material of claim 1, wherein the tropoelastin
monomers comprise 2 or more individual tropoelastin isoforms.
9. An elastic material obtainable by a process including: applying
tropoelastin monomers to a surface; and heating the tropoelastin
monomers on the surface to a temperature to enable the monomers to
bind to each other to form a material that does not dissociate back
into monomers when the material is exposed to physiological
conditions, wherein the tropoelastin monomers are heated to a
temperature of from 60.degree. C. to 200.degree. C., thereby
forming the material.
10. The elastic material of claim 9, wherein the tropoelastin
monomers are heated to a temperature that is sufficient to enable
the tropoelastin monomers to bind to each other to form an elastic
material that does not dissociate into tropoelastin monomers when
the elastic material is exposed to physiological conditions.
11. The elastic material of claim 9, wherein the tropoelastin
monomers are heated to a temperature that is sufficient to enable
the tropoelastin monomers to bind to each other to form an elastic
material that does not dissociate into tropoelastin monomers when
the elastic material is contacted with an aqueous solution having a
pH of from about 6.5 to 8.0.
12. The elastic material of claim 9, wherein the tropoelastin
monomers are heated to a temperature that is sufficient to enable
the tropoelastin monomers to bind to each other to form an elastic
material that does not dissociate into tropoelastin monomers when
the elastic material is contacted with an aqueous solution having a
temperature of from about 30.degree. C. to about 45.degree. C.
13. The elastic material of claim 9, wherein the tropoelastin
monomers are heated to a temperature that is sufficient to enable
the tropoelastin monomers to bind to each other to form an elastic
material that does not dissociate into tropoelastin monomers when
the elastic material is contacted with an aqueous solution having a
salt concentration of about 75 mM to about 300 mM.
14. The elastic material of claim 9, wherein the surface is heated
for heating of the tropoelastin monomers.
15. The elastic material of claim 9, wherein the tropoelastin
monomers contain hydrophilic and hydrophobic domains of
tropoelastin.
16. The elastic material of claim 9, wherein the tropoelastin
monomers have a sequence that has at least 90% sequence identity
with an amino acid sequence of human tropoelastin across at least
50 consecutive amino acids.
17. The elastic material of claim 9, wherein the tropoelastin
monomers are recombinant tropoelastin monomers having an amino acid
sequence of a human tropoelastin isoform.
18. The elastic material of claim 9, wherein the tropoelastin
monomers are applied to the surface by spraying the tropoelastin
monomers onto the surface.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 16/937,442, filed on Jul. 23, 2020,
which is a continuation of U.S. patent application Ser. No.
14/650,542, filed on Jun. 8, 2015, now U.S. Pat. No. 10,842,913,
issued Nov. 24, 2020, which is a national stage entry of
International Application Serial No. PCT/AU2013/001435, filed Dec.
10, 2013, which claims the benefit of and priority to Australian
Patent Application No. 2012905409, filed Dec. 10, 2012, each of
which is incorporated herein by reference in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jun. 8, 2015, is named 122320-6036_Sequence_Listing_ST25.txt and
is 7097 bytes in size.
FIELD OF THE INVENTION
[0003] The invention relates to production of elastic materials
from tropoelastin, and especially to the formation of materials
into preferred three-dimensional shapes, and especially, although
not exclusively, to materials that can be used for tissue therapy
and repair.
BACKGROUND OF THE INVENTION
[0004] Reference to any prior art in the specification is not, and
should not be taken as, an acknowledgment or any form of suggestion
that this prior art forms part of the common general knowledge in
Australia or any other jurisdiction or that this prior art could
reasonably be expected to be ascertained, understood and regarded
as relevant by a person skilled in the art.
[0005] There is considerable, growing demand for three-dimensional
constructs that can be used for human tissue repair. Constructs
based on natural biomaterials (such as elastin) have emerged as
leading candidates for various tissue engineering applications due
to their remarkable properties including elasticity, self-assembly,
long-term stability, and biological activity.
[0006] Tropoelastin is the substrate material for formation of
elastin and elastic fibre. Elastin is formed from tropoelastin when
tropoelastin is cross-linked.
[0007] Tropoelastin is soluble in most aqueous solutions, and
indeed is soluble at physiological salt and pH. Tropoelastin can be
induced to precipitate from an aqueous solution by heating an
aqueous solution. The process is known as coacervation, in which
tropoelastin monomers associate with each other by contact of
hydrophobic regions of one tropoelastin monomer with the like
regions of another monomer. This association of monomers is
reversible, and the tropoelastin monomers in coacervated
tropoelastin may be dissociated, for example by pH, salt or
temperature modification, leading to dissolution of tropoelastin
monomers of the coacervate into the solution and disappearance of
the coacervate. What this means is that a coacervate of
tropoelastin is not sufficiently robust to form a preferred
three-dimensional elastic structure that is stable in physiological
conditions.
[0008] The cross-linking of tropoelastin monomers, whether in
coacervated form or otherwise, leads to a covalent bonding of
tropoelastin monomers that ostensibly represents an association of
tropoelastin monomers that cannot be dissociated by pH, salt or
temperature adjustment. Generally, cross-linked tropoelastin
monomers, as observed in elastin and elastic fibre, cannot be
dissociated from each other unless the monomers are hydrolysed, as
described in prior art processes for purification of elastin from
natural sources.
[0009] Given the generally irreversible association of tropoelastin
monomers observed in cross-linked tropoelastin as in elastin or
elastic fibre, cross-linking of tropoelastin has been proposed as a
solution to enable the formation of preferred three-dimensional
elastic structures. Examples of this technology are disclosed in
Miyamoto et al. (2009), WO 2008/033847 and WO 2009/099570 whereby
electrospun material is cross-linked into preferred stable
structures. Some of the cross-linking technologies require a
heating step whereby a tropoelastin-containing composition is
heated in the formation of a preferred elastic structure.
Generally, the heating step is required to evaporate solvent,
and/or to provide the required temperature condition for the
cross-linking reaction.
[0010] One problem with processes involving cross-linking is that
the cross-linking agents are not biocompatible in the context of
either tolerance of tissue to the chemistry of cross-linkers, nor
residual unreacted cross-linker, nor to elastic function of the
cross-linked material. Another problem is that it is difficult to
form a preferred structure from cross-linked material, because
after cross-linking, a material quickly solidifies into a type of
structure which cannot then be conformed to a preferred robust
shape. Therefore, there are limitations as to the extent to which
such processes can be used in forming structures by spraying
moulding technologies and the like.
[0011] Ultimately, what is required to form a stable preferred
three-dimensional structure from tropoelastin monomers is to link
the monomers with each other in such a way so as to prevent a
dissociation from one another that would result in loss of the
preferred structure or shape. Another approach to forming a stable
elastic three-dimensional construct is to use other molecules,
which ostensibly act as linkers for linking one tropoelastin
monomer with another. Examples include synthetic polymers generally
as discussed in WO 2009/099570. Another approach is to spray or
coat a water insoluble substrate with a solution of soluble elastic
monomers as in WO 2012/080706, WO 2011/127478 and WO 2007/029913.
In this latter approach, the insoluble substrate, such as a
nano-fibrous web, as in WO 2007/029913, or a tube, as in WO
2011/127478 ostensibly links the tropoelastin monomers to each
other so that they do not dissociate in aqueous conditions. The
problem with these approaches is that inevitably it is the
insoluble substrate that provides the preferred three-dimensional
shape, not the molecular components, which impacts on the overall
elastic profile of the structure and limits the ability to build
three-dimensional structures.
[0012] There is a need for new approaches to the formation of
elastic three-dimensional structures.
SUMMARY OF THE INVENTION
[0013] The invention seeks to address, or at least to provide an
improvement to, one or more of the above mentioned limitations,
needs or problems and in one embodiment provides a method for
forming an elastic material, including: [0014] providing a solution
of tropoelastin monomers; [0015] applying the solution to a
surface; [0016] heating the solution on the surface to a
temperature sufficient to enable the tropoelastin monomers to bind
to each other to form an elastic material that does not dissociate
into tropoelastin monomers when the material is contacted with an
aqueous solution, thereby forming the elastic material.
[0017] In another embodiment there is provided a method for forming
an elastic material including: [0018] providing a solution of
tropoelastin monomers; [0019] applying the solution to a surface;
[0020] heating the solution on the surface to a temperature within
a range defined by a minimum value and a maximum value;
[0021] wherein the minimum value is a temperature above which
tropoelastin monomers are bonded to each other to form a material
that does not dissociate in an aqueous solution; and
[0022] wherein the maximum value is a temperature above which a
non-elastic material is formed; [0023] thereby forming an elastic
material.
[0024] In another embodiment there is provided an elastic material
formed by a method described above.
[0025] In another embodiment there is provided a method for forming
an elastic hydrogel, including: [0026] forming an elastic material
according to a method described above; [0027] contacting the
elastic material with an aqueous solution.
[0028] In another embodiment there is provided an elastic hydrogel
formed by a method described above.
[0029] In another embodiment there is provided a construct, implant
or device including an elastic material or hydrogel described
above.
[0030] In other embodiments there are provided methods and uses of
the elastic material, hydrogel, device, implant or construct
described above for repairing and/or restoring biological tissue,
and for use of the elastic material in assay applications.
[0031] The present invention will now be more fully described with
reference to the accompanying examples and drawings. It should be
understood, however, that the description following is illustrative
only and should not be taken in any way as a restriction on the
generality of the invention described above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1. Heat-treated water-based tropoelastin solution. A.
After heating to 160.degree. C. B. After wetting in PBS.
[0033] FIG. 2. Heat-treated HFP-based tropoelastin solution. A.
After heating to 160.degree. C. B. After wetting in PBS.
[0034] FIG. 3. Heat-treated 70% ethanol-based tropoelastin
solution. A. Before heating to 160.degree. C. B. After heating to
160.degree. C. C. Side on view in PBS. D. After wetting in PBS.
[0035] FIG. 4. Heat-treated HFP-based tropoelastin solution used to
coat tubing. A. After heating to 160.degree. C. B. After wetting in
PBS.
[0036] FIG. 5. Scanning electron microscopy images of heat-treated
electrospun tropoelastin. A. After heating to 160.degree. C. B.
After wetting in PBS.
[0037] FIG. 6. Scanning electron microscopy image of fibroblasts
cultured on heat-treated electrospun tropoelastin.
[0038] FIG. 7. Images of VVG stained skin biopsies showing
persistence of heat-treated electrospun tropoelastin.
[0039] FIG. 8. Films made from a heat-treated water-based
tropoelastin solution A) after drying at 37.degree. C. for 16 h B)
after further heating to 160.degree. C. for 4 h.
[0040] FIG. 9. Micropatterned films made from a heat-treated
water-based tropoelastin solution. Groove patterns are 500 nm deep
and 3.5 .mu.m wide.
[0041] FIG. 10. Calculation of moduli at 0-105% and 105-19%
extension.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0042] Reference will now be made in detail to certain embodiments
of the invention. While the invention will be described in
conjunction with the embodiments, it will be understood that the
intention is not to limit the invention to those embodiments. On
the contrary, the invention is intended to cover all alternatives,
modifications, and equivalents, which may be included within the
scope of the present invention as defined by the claims.
[0043] A person skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practice of the present invention. The present
invention is in no way limited to the methods and materials
described.
[0044] It will be understood that the invention disclosed and
defined in this specification extends to all alternative
combinations of two or more of the individual features mentioned or
evident from the text or drawings. All of these different
combinations constitute various alternative aspects of the
invention.
[0045] All of the patents and publications referred to herein are
incorporated by reference in their entirety.
[0046] The present work demonstrates, for the first time, that a
biocompatible material having desirable properties such as strength
and elasticity can be synthesized by a simple process through
heating tropoelastin. Accordingly, the present invention provides a
reliable, scalable, inexpensive path to manufacturing biocompatible
three-dimensional elastic materials. The invention is amenable to
high throughput production and uses protein to produce a versatile
range of biomaterials (such as sheets, tubes and fibres) that are
useful in therapeutic and in vitro assay applications. The
materials produced by the process of the present invention possess
the properties of elasticity and strength that are the hallmarks of
native elastin but are devoid of chemical contaminants and toxic
by-products that are commonly found in, or associated with,
constructs formed from the use of cross-linking agents.
[0047] The advantageous properties of the materials of the present
invention are discussed throughout the present specification, and
in particular, are exhibited in the Examples, which show that the
materials of the present invention can be made in a simple manner
by heating of a solution of tropoelastin, and that the materials
formed possess the required properties of biocompatibility,
strength, resilience, cell binding and extracellular matrix
interactions that enable them to be used in tissue engineering
applications, as well as in the construction of in vitro
assays.
[0048] As mentioned above, scaffolds based on biomaterials have
been used for tissue engineering applications because of their
biocompatibility and mechanical properties. However, prior art
methods of synthesising three-dimensional biomaterial-based
constructs are inefficient, slow and restricted, and the
alternative, traditional tissue engineering technologies
overwhelmingly require the use of slow and expensive methods (which
can take weeks to produce a construct), are generally
diffusion-constrained to several hundred microns thickness, and are
regularly burdened by toxicity component or by-product issues that
demand regulatory compliance.
[0049] Thus, in one embodiment there is provided a method for
forming an elastic material, including: [0050] providing an aqueous
solution of tropoelastin monomers; [0051] applying the solution to
a surface; [0052] heating the solution on the surface to a
temperature sufficient to enable the tropoelastin monomers to bind
to each other to form an elastic material that does not dissociate
into tropoelastin monomers when the material is contacted with an
aqueous solution, thereby forming the elastic material.
[0053] An important finding of the invention is that the heating
step enables the association of the tropoelastin monomers with such
affinity that the monomers do not substantially dissociate when the
aggregate is contacted with an aqueous solution. The heating forms
an elastic aggregate, mass or material that is different to a
coacervate to the extent that it does not dissociate into
individual monomers in physiological conditions, and different to
elastic fibre or other material in the sense that it does not
require cross-linking of monomers with toxic cross-linking agents
such as glutaraldehyde, or use of a solvent having a basic pH, to
maintain itself in a solid phase, or to retain the permanency of
the shape in which it is formed. The advantage is that the process
more or less permanently assembles biocompatible monomers into a
permanent structure or shape that can be used in tissue
applications without toxicity concerns.
[0054] Typically the solution is heated to a temperature that is
sufficient to enable the tropoelastin monomers to bind to each
other to form an elastic material that does not dissociate into
tropoelastin monomers when the material is exposed to physiological
conditions, especially human physiological conditions. In
particular, the aggregate does not dissociate under physiological
conditions of temperature and pH. Advantageously, the aggregate
does not dissociate under the following conditions of: [0055]
temperature (from about 30 to about 45.degree. C.); [0056] salt
(concentration of about 75 mM to about 300 mM); [0057] pH (of about
6.5 to about 8.0).
[0058] Therefore, this material is suitable for use not only under
physiological conditions, but also in other applications, such as
in vitro assays, where it may be exposed to other, more demanding
conditions. Notably, this material is achieved without having to
perform any cross-linking of the tropoelastin monomers and without
using a scaffold to bind assemblies of the peptide thereto.
[0059] The material formed by the process of the present invention
has a number of advantages. Firstly, the tropoelastin monomers
remain bound with each other so that the three-dimensional shape of
the elastic material formed from the aggregate is retained in an
aqueous environment. Secondly, the properties of the starting
material that make tropoelastin so useful in tissue engineering
applications (for example, elasticity, strength, resilience, and
biocompatibility) are retained in the end-product. Thirdly, while
the aggregate may take up water in an aqueous solution to form a
hydrogel, in doing so the aggregate does not dissociate, thereby
maintaining the three-dimensional structure of the elastic
material.
[0060] In one embodiment the solution is heated to a temperature
that is sufficient to enable the tropoelastin monomers to bind to
each other to form an elastic material that does not dissociate
into tropoelastin monomers when the material is contacted with an
aqueous solution having a pH of from about 6.5 to about 8.0. As
used herein a wording defining the limits of a range of length such
as, for example, "from 1 to 5" means any integer from 1 to 5, i. e.
1, 2, 3, 4 and 5. In other words, any range defined by two integers
explicitly mentioned is meant to include and disclose any integer
defining said limits and any integer included in said range. For
example, the solution may have a pH of about 6.6, about 6.7, about
6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about
7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or
about 8.0.
[0061] In one embodiment the solution is heated to a temperature
that is sufficient to enable the tropoelastin monomers to bind to
each other to form an elastic material that does not dissociate
into tropoelastin monomers when the material is contacted with an
aqueous solution having a temperature of from about 30 to about
45.degree. C. For example, the temperature may be about 31.degree.
C., about 32.degree. C., about 33.degree. C., about 34.degree. C.,
about 35.degree. C., about 36.degree. C., about 37.degree. C.,
about 38.degree. C., about 39.degree. C., about 40.degree. C.,
about 41.degree. C., about 42.degree. C., about 43.degree. C.,
about 44.degree. C., or about 45.degree. C.
[0062] In one embodiment the solution is heated to a temperature
that is sufficient to enable the tropoelastin monomers to bind to
each other to form an elastic material that does not dissociate
into tropoelastin monomers when the material is contacted with an
aqueous solution having a salt concentration of about 75 mM to
about 300 mM. For example, the salt concentration may be about 75
mM, about 80 mM, about 85 mM, about 90 mM, about 95 Mm, about 100
mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about
150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM,
about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240
mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about
290 mM, or about 300 mM.
[0063] In one embodiment, the solution is heated to a temperature
that is sufficient to enable the tropoelastin monomers to bind to
each other to form an elastic material that does not dissociate
into tropoelastin monomers when the material is exposed to
physiological conditions (e.g. pH of between about 7.2 and about
7.5, a temperature of between about 36 and about 37.degree. C., and
a salt concentration of about 150 m M).
[0064] In accordance with the process, the solution is heated to
form the elastic material of the present invention. As discussed
above, the purpose of the heating step is to form a material
containing associated tropoelastin monomers, more specifically, to
enable the tropoelastin monomers to bind to each other to form an
elastic material that does not then dissociate into tropoelastin
monomers when the elastic material is contacted with aqueous
solution. The heating step is to be carried out at a temperature
sufficient to enable the tropoelastin monomers in the concentrate
to bind to each other to form an aggregate or material including
tropoelastin monomers. Typically, heating will be carried out at a
temperature of about 100.degree. C. or greater, for example from
100.degree. C. to 160.degree. C. For example, the temperature of
the heating step may be 110.degree. C. or greater, 120.degree. C.
or greater, 130.degree. C. or greater, 140.degree. C. or greater,
150.degree. C. or greater, 160.degree. C. or greater, 170.degree.
C. or greater, or 180.degree. C. or greater. Preferably, the
temperature is between about 120.degree. C. and about 180.degree.
C., between about 130.degree. C. and about 170.degree. C., or
between about 140.degree. C. and about 160.degree. C. Most
preferably, the temperature is about 160.degree. C.
[0065] In another embodiment there is provided a method for forming
an elastic material including: [0066] providing a solution of
tropoelastin monomers; [0067] applying the solution to a surface;
[0068] heating the solution on the surface to a temperature within
a range defined by a minimum value and a maximum value;
[0069] wherein the minimum value is a temperature above which
tropoelastin monomers are bonded to each other to form a material
that does not dissociate in an aqueous solution; and wherein the
maximum value is a temperature above which a non-elastic material
is formed.
[0070] According to the embodiment, below the minimum value, the
elastic material of the invention is not formed. That is to say
that what is formed is dissociable in aqueous solution,
particularly in physiological conditions. Therefore, below the
minimum value, something more resembling a coacervate may be
formed. Below the maximum value, the material retains the elastic
properties discussed herein. Above the maximum value, the material
may lose properties of elasticity.
[0071] Suitable lengths of time over which the heating of the
solution should be carried out include about 10 minutes or more,
about 20 minutes or more, about 30 minutes or more, about 40
minutes or more, about 50 minutes or more, about 1 hour or more,
about 2 hours or more, about 3 hours or more, about 4 hours or
more, or about 5 hours or more. However, a person skilled in the
art will understand that the temperature to which the solution
should be heated as well as the time over which the solution should
be heated will vary depending on various factors, such as: [0072]
the type of heating method employed (for example, dry heating,
flash heating, etc.); [0073] the concentration of the tropoelastin
monomers in the solution; [0074] the volume of solution; [0075] the
composition of the tropoelastin monomers; [0076] the degree of
association desired in the aggregate or elastic material; [0077]
the relative humidity during heating.
[0078] In certain embodiments, heating from 8 to 16 hours may be
used to provide a substance which is more crystalline and yet
retains elastic properties.
[0079] Generally, the humidity during heating may be from about 20
to about 80%, preferably about 35, 45, 55, 65 or 75% relative
humidity.
[0080] As described herein, the heating step may result in the
formation of an elastic material that develops a colour change.
Thus, in certain embodiments, one may test for the formation of an
elastic material, or to check for completion of the heating step,
by determining whether the material has developed a colour change.
A colour change is generally a change from the normal translucent
appearance of elastin to a colour which may be yellow or brownish.
It is not necessary that the whole of the material develops a
colour change. Generally, the colour change may be reduced in the
elastic material by hydration.
[0081] A person skilled in the art will also be aware that, by
utilising different heating methods, aggregates with different
internal structures can be obtained. For example, flash-heating
would involve subjecting the concentrate to an intense source of
heat for only a very limited amount of time. Accordingly, heating
will occur for a sufficient amount of time to associate the
monomers to form the aggregate, but will be too fast for all
solvent trapped in the aggregate to evaporate from the aggregate,
thereby forming an aggregate having a vacuole-type structure.
Further, the aggregate could be heated again such that the trapped
solvent evaporates, thereby forcing the vacuoles to expand, and
resulting in the formation of a porous aggregate. A person skilled
in the art will also be aware that solvent may not be present
internally in the aggregate, but may be present on the external
surface of the aggregate (in addition to or as an alternative to
the solvent present inside the aggregate).
[0082] One particularly important advantage of the process is that
in certain embodiments the material formed by the process may be
ostensibly gas impermeable, resulting from a close alignment of
protein molecules, which is retained by the heating step. This
enables the material to be blown into a particular shape, much the
same as occurs in glass blowing techniques.
[0083] Regardless of which heating method is used, a person skilled
in the art will understand that the aggregate formed by heating of
the solution may therefore include water, to varying degrees. For
example, the aggregate may include a significant amount of water
(for example, more than about 60% w/w water), making it essentially
a hydrogel. Alternatively, water may be present in the aggregate in
an amount of only about 10% w/w. Because water content influences
elasticity, the elasticity of the aggregate (and therefore the
material) can be varied by varying the water content of the
aggregate, which, in turn, can be varied by changing various
factors such as the amount of water present in the concentrate
prior to heating, as well as the heating time, method and
temperature.
[0084] In one embodiment, the elastic material has a solvent
content of from greater than about 0 to about 50% (w/w) of the
material at the completion of the heating step. For example, the
elastic material may have a solvent content of from about 0.5%
(w/w), about 1% (w/w), about 2% (w/w), about 3% (w/w), about 4%
(w/w), about 5% (w/w) about 10% (w/w), about 15% (w/w), about 20%
(w/w), about 25% (w/w), about 30% (w/w), about 35% (w/w), about 40%
(w/w), about 45% (w/w), or about 50% (w/w). In one embodiment, the
solvent is water.
[0085] The solution may be heated by directly heating the solution,
or by heating the surface onto which the solution is placed. In the
latter embodiment, the surface may be heated before the solution is
applied to it, or it may be at room temperature at the time of
application of the solution and then heated to the relevant
temperature. Therefore, in one embodiment, the surface is heated
for heating of the solution.
[0086] As also discussed above, a major advantage of the process of
the present invention is that biocompatible materials can be formed
because the process does not require the use of agents such as
cross-linkers to effect the polymer formation. Accordingly, in one
embodiment, the process of the present invention excludes the use
of cross-linking agents.
[0087] In another embodiment, the process may exclude the use of
salts, or other coacervation agents, to assist in the formation of
tropoelastin-based polymers.
[0088] In another embodiment, the process may exclude the use of
pH-modifying agents that effect irreversible aggregation of the
tropoelastin monomers. In particular, in one embodiment the heating
step is carried out at a pH that is not an alkaline pH, for
example, the pH may be generally less than 8.5 or 8.0.
[0089] As discussed above, the materials of the present invention
are formed by heating a solution of tropoelastin on a surface (such
as, for example, in a shaped mold). Without wishing to be bound by
any theory or mode of action, the present inventors believe that in
a concentrated solution of tropoelastin, the tropoelastin monomers
are closely packed. This close-packing facilitates bonding between
the monomers upon heating of the solution, thereby producing an
elastic material that does not dissociate into separate
tropoelastin monomers when put in an aqueous environment.
[0090] The solution forming the solvent of the tropoelastin
monomers may be an aqueous solution or a non-aqueous solution.
[0091] As used herein, the term "aqueous solution" refers to a
water-containing solution. An aqueous solution may include other
components, such as buffers, and pharmaceutically acceptable
excipients, and may also include other organic, water-miscible
solvents, such as methanol, ethanol and hexafluoropropanol, and
combinations thereof. Where the aqueous solution includes other
solvents, a person skilled in the art will understand that water
will be the major solvent component and the other solvent(s) will
make up the minor portion of the solvent component. The use of an
aqueous solution is particularly advantageous because it means that
the tropoelastin concentrate, and therefore the material, is formed
from a composition that does not contain any components that are
non-biocompatible or toxic, or that may degrade in the body to form
toxic or undesirable by-products. Accordingly, preferably, the
aqueous solution does not contain any components (solvents,
buffers, etc.) that are toxic or non-biocompatible and/or that form
toxic or non-biocompatible species when the material is in use (for
example, in the body or in an assay).
[0092] As used herein, the term "non-aqueous solution" refers to a
solution that either does not contain water, or that contains water
as a minor solvent component. Examples of non-aqueous solvents
include HFP, for example, as exemplified in the examples here. One
advantage of using a non-aqueous solvent to form a non-aqueous
solution is that generally the solvent may have a lower boiling
point than water. This would enable the solvent to be evaporated as
required during the process without the substantial addition of
heat.
[0093] In one embodiment, the solution is formed by a process
including the steps of: [0094] providing a solution of tropoelastin
monomers; [0095] increasing the concentration of tropoelastin
monomers in the solution.
[0096] The end product may be referred to as a "concentrate". The
concentrate may be obtained by any method known to be suitable to a
person skilled in the art. It is believed that in the concentrate,
tropoelastin monomers are brought into close contact with each
other, such that, upon heating, the monomers will form an aggregate
or mass, which can conform to the shape of the surface on which it
is formed, and which does not dissociate or experience significant
disruption of the linkages formed during heating when the aggregate
is placed in an aqueous environment.
[0097] The concentrate may be obtained by evaporating the solvent
from the solution by, for example, heating the solution, or blowing
air or nitrogen over the solution. Thus, in one embodiment, the
concentration of tropoelastin monomers is increased by evaporating
solvent from the solution.
[0098] The solvent may be evaporated from the solution when the
solution is applied to the surface. The solvent may be evaporated
enabling concentration of the tropoelastin monomers as the solution
is heated on the surface to a temperature enabling the tropoelastin
monomers to bind to each other to form an elastic material that
does not dissociate into tropoelastin monomers when the material is
contacted with an aqueous solution.
[0099] In one embodiment, the concentration of tropoelastin
monomers is increased by separating tropoelastin monomers from
solvent. The tropoelastin monomers may be separated from the
solvent by electrospinning of the tropoelastin monomers.
"Electrospinning" is a process in which fibers are formed from a
solution or melt by streaming an electrically charged solution or
melt through a hole across a potential gradient. In one embodiment,
the solution has a concentration of tropoelastin monomers from
about 1% to about 40% (w/v) at the time that the solution is
applied to the surface. For example, the concentration of
tropoelastin monomers in the solution may be about 2%, about 3%,
about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about
10%, about 12%, about 14%, about 16%, about 18%, about 20%, about
22%, about 24%, about 26%, about 28%, about 30%, about 32%, about
34%, about 36%, about 38%, or about 40% (w/v).
[0100] "Electrospun material" is any molecule or substance that
forms a structure or group of structures (such as fibers, webs, or
droplets), as a result of the electrospinning process. Generally,
this material may be natural, synthetic, or a combination of these
but in the present invention it is preferred that tropoelastin is
used.
[0101] The matrix material may be deposited on the textile template
using electrospinning. This platform technology is widely used in
tissue engineering to fabricate scaffolds composed of nano- and
micro-fibrous architecture (Li et al. (2006) and Li et al.
(2005)).
[0102] The process of electrospinning involves placing a polymer or
monomer-containing fluid (for example, a polymer or monomer
solution, polymer or monomer suspension, or polymer or monomer
melt) in a reservoir equipped with a small orifice, such as a
needle or pipette tip, and a metering pump. One electrode of a high
voltage source is placed in electrical contact with the fluid or
orifice, while the other electrode is placed in electrical contact
with a target (typically a collector screen or rotating mandrel).
During electrospinning, the fluid is charged by the application of
high voltage to the solution or orifice (for example, about 3 to
about 15 kV) and then forced through the small orifice by the
metering pump, providing a steady flow. While the fluid at the
orifice normally would have a hemispherical shape due to surface
tension, the application of the high voltage causes the otherwise
hemispherically shaped fluid at the orifice to elongate to form a
conical shape known as a Taylor cone. With sufficiently high
voltage applied to the fluid and/or orifice, the repulsive
electrostatic force of the charged fluid overcomes the surface
tension and a charged jet of fluid is ejected from the tip of the
Taylor cone and accelerated towards the target, which typically is
biased between -2 to -10 kV. A focusing ring with an applied bias
(for example, 1 to 10 kV) may be optionally used to direct the
trajectory of the charged jet of fluid. As the charged jet of fluid
travels towards the biased target, it undergoes a complicated
whipping and bending motion. If the fluid is a monomer or polymer
solution or suspension, the solvent typically evaporates during
mid-flight, leaving behind a polymer or monomer fiber on the biased
target. If the fluid is a polymer or monomer melt, the molten
monomer/polymer cools and solidifies in mid-flight and is collected
as a monomer/polymer fiber on the biased target. As the
polymer/monomer fibers accumulate on the biased target, a porous
mesh is formed on the biased target.
[0103] The properties of the electrospun matrix may be tailored by
varying the electrospinning conditions. For example, when the
template is relatively close to the orifice, the resulting
electrospun mesh tends to contain unevenly thick fibers, such that
some areas of the fiber have a "bead-like" appearance. However, as
the template is moved further away from the orifice, the fibers of
the mesh tend to be more uniform in thickness. Moreover, the
template may be moved relative to the orifice. In certain
embodiments, the template is moved back and forth in a regular and
periodic fashion, such that fibers of the mesh are substantially
parallel to each other. When this is the case, the resulting mesh
may have a higher resistance to strain in the direction parallel to
the fibers, compared to the direction perpendicular to the fibers.
In other embodiments, the biased target is moved relative to the
orifice in a two- or three-dimensional pattern to create a mesh
comprising one or more patterned layers with similar or different
strand orientation, thickness, etc. In other embodiments, the
template is moved randomly relative to the orifice, so that the
resistance to strain in the plane of the mesh is isotropic. The
properties of the electrospun matrix may also be varied by changing
the magnitude of the voltages applied to the electrospinning
system. In a non-limiting example, the electrospinning apparatus
includes an orifice biased to 20 kV. In another non-limiting
example, the electrospinning apparatus includes a template biased
to -7 kV. In yet another non-limiting example, the electrospinning
apparatus includes a focusing ring biased to 3 kV.
[0104] In one embodiment, the concentration of tropoelastin in the
solution is between about 10 and about 350 mg/mL at the time that
the solution is applied to the surface. For example, the
concentration of tropoelastin is about 15 mg/mL, about 20 mg/mL,
about 25 mg/mL, about 30 mg/mL, about 35 mg/mL, 40 mg/mL, about 45
mg/mL, about 50 mg/mL, about 55 mg/mL, about 60 mg/mL, about 65
mg/mL, about 70 mg/mL, about 75 mg/mL, about 80 mg/mL, about 85
mg/mL, about 90 mg/mL, about 95 mg/mL, about 100 mg/mL, about 110
mg/mL, about 120 mg/mL, about 130 mg/mL, about 140 mg/mL, about 150
mg/mL, about 160 mg/mL, about 170 mg/mL, about 180 mg/mL, about 190
mg/mL, about 200 mg/mL, about 210 mg/mL, about 220 mg/mL, about 230
mg/mL, about 240 mg/mL, about 250 mg/mL, about 260 mg/mL, about 270
mg/mL, about 280 mg/mL, about 290 mg/mL, about 300 mg/mL, about 310
mg/mL, about 320 mg/mL, or about 340 mg/m L.
[0105] In one embodiment, the concentration of tropoelastin in the
solution may depend on the type of solvent and the temperature of
the solvent at which the tropoelastin is added to it.
[0106] The solution or concentrate of tropoelastin that is applied
to the surface may have a range of viscosities. It may include
precipitated non-cross linked tropoelastin, such as a
coacervate.
[0107] In a certain embodiment, the solution applied to the surface
may also include coacervated tropoelastin monomers.
[0108] In one embodiment there is provided a method for forming an
elastic material, including: [0109] providing a solution of
tropoelastin monomers; [0110] increasing the concentration of
tropoelastin monomers in the solution to form a concentrate of
tropoelastin; [0111] applying the concentrate to a surface; [0112]
heating the concentrate on the surface to a temperature sufficient
to enable tropoelastin in the concentrate to bind to each other to
form an elastic material that does not dissociate into tropoelastin
monomers when the material is contacted with an aqueous solution,
thereby forming the elastic material. In one embodiment, the
concentrate is heated to a temperature within a range defined by a
minimum value and a maximum value;
[0113] wherein the minimum value is a temperature above which
tropoelastin monomers are bonded to each other to form a material
that does not dissociate in an aqueous solution; and
[0114] wherein the maximum value is a temperature above which a
non-elastic material is formed;
thereby forming an elastic material. The method may involve the
step of heating the concentrate on the surface to enable a water
loss of from about 1 to 20% water (w/w), preferably about 15% water
(w/w) of the concentrate.
[0115] Tropoelastin is a monomeric protein encoded by the elastin
(ELN) genomic sequence (or gene). Tropoelastin monomers are
approximately 60-70 kDa in size. There are about 36 small domains
in tropoelastin and each weighs about 2 kDa. Within the exons,
there are alternating hydrophobic domains rich in non-polar amino
acids such as glycine, valine, proline, isoleucine and leucine
(which domains often occur in repeats of three to six peptides such
as GVGVP, GGVP and GVGVAP), and hydrophilic domains rich in lysine
and alanine. The hydrophilic domains often consist of stretches of
lysine separated by two or three alanine residues such as
AAAKAAKAA. Additionally, tropoelastin ends with a hydrophilic
carboxy-terminal sequence containing its only two cysteine
residues. Tropoelastin does not undergo cleavage during assembly
and forming the microfibril is achieved by a self-association
process termed coacervation.
[0116] Tropoelastin aggregates at physiological temperature due to
interactions between hydrophobic domains. This process is
reversible and thermodynamically controlled. The coacervate is
stabilized by cross-linking via lysyl oxidase. The coacervate then
becomes insoluble and the process is irreversible. It then
condenses to form a cross-linked structure of two residues or four
residues in either desmosine or isodesmosine.
[0117] In certain embodiments the tropoelastin monomer that is used
in the present invention includes both hydrophilic and hydrophobic
domains. Hydrophilic domains contribute to elastic function (by,
for example, binding to water). They also contribute to a wider
variety of biological functions including binding to cells and to
the extra-cellular matrix. The hydrophobic domains are believed to
be important for providing the elasticity that is a feature of the
materials of the present invention.
[0118] Some examples of amino acid sequences that may be present in
a tropoelastin monomer are as follows:
TABLE-US-00001 GGVPGAIPGGVPGGVFYP GVGLPGVYP GVPLGYP PYTTGKLPYGYGP
GGVAGAAGKAGYP TYGVGAGGFP KPLKP ADAAAAYKAAKA GAGVKPGKV GAGVKPGKV
TGAGVKPKA QIKAPKL VAPGVG VPGVG AAAAAAAKAAAK AAAAAAAAAAKAAKYGAAAGLV
EAAAKAAAKAAKYGAR EAQAAAAAKAAKYGVGT AAAAAKAAAKAAQFGLV
GGVAAAAKSAAKVAAKAQLRAAAGLGAGI GALAAAKAAKYGAAV AAAAAAAKAAAKAA
AAAAKAAKYGAA CLGKACGRKRK.
[0119] The tropoelastin for use in the present invention may, in
certain embodiments, include or consist of, any one of the above
described sequences. In one embodiment the tropoelastin for use in
the present invention includes or consists of a sequence shown
below:
TABLE-US-00002 VXPGVG
[0120] where X is any amino acid residue or no residue
TABLE-US-00003 ZXPGZG
[0121] wherein Z is an aliphatic residue
TABLE-US-00004 VXP(I/L/V)V(I/L/V)
[0122] wherein (I/LN) is isoleucine, leucine or valine.
[0123] In one embodiment, the tropoelastin monomers contain
hydrophilic and hydrophobic domains of tropoelastin.
[0124] Other suitable tropoelastin sequences are known in the art
and include CAA33627 (Homo sapiens), P15502 (Homo sapiens),
AAA42271 (Rattus norvegicus), AAA42272 (Rattus norvegicus),
AAA42268 (Rattus norvegicus), AAA42269 (Rattus norvegicus),
AAA80155 (Mus musculus), AAA49082 (Gallus gallus), P04985 (Bos
taurus), ABF82224 (Danio rerio), ABF82222 (Xenopus tropicalis) and
P11547 (Ovis aries). In a preferred embodiment, the tropoelastin
monomers for use in the present invention are derived from human
tropoelastin. In one embodiment, they have the sequence
corresponding to amino acid residues 27-724 of GenBank entry
AAC98394. As stated herein, the present invention also includes
variants, for example species variants or polymorphic variants, of
tropoelastin.
[0125] The tropoelastin monomers for use in the present invention
may be obtained from recombinant sources. They can also be
extracted from natural sources or synthesised (by, for example,
solid-phase synthesis techniques). Tropoelastin monomers are also
commercially available.
[0126] There are a number of isoforms of tropoelastin and therefore
the exact number of amino acids that make up the tropoelastin
polypeptide will vary. The term "polypeptide" or "polypeptide
chain" refers to a polymer of amino acids, usually linked together
by amide bonds. A functionally active polymer of amino acids is
generally referred to as a "protein". The present invention also
includes variants of tropoelastin, for example species variants or
polymorphic variants. The present invention is intended to cover
all functionally active variants of tropoelastin that exhibit the
same activity (i.e. biocompatibility and elasticity). This also
includes apo- and holo-forms of tropoelastin, post-translationally
modified forms, as well as glycosylated or de-glycosylated
derivatives. Such functionally active fragments and variants
include, for example, those having conservative amino acid
substitutions.
[0127] In one embodiment, the monomers are recombinant tropoelastin
monomers having the sequence of a human tropoelastin isoform.
[0128] The term "functionally active" in relation to a fragment or
variant of tropoelastin means the fragment or variant (such as an
analogue, derivative or mutant) that is capable of forming an
elastic material, as discussed further below. Such variants include
naturally occurring variants and non-naturally occurring variants.
Additions, deletions, substitutions and derivatizations of one or
more of the amino acids are contemplated so long as the
modifications do not result in loss of functional activity of the
fragment or variant. A functionally active fragment can be easily
determined by shortening the amino acid sequence, for example using
an exopeptidase, or by synthesizing amino acid sequences of shorter
length, and then testing for elastic material formation ability
such as by the methods illustrated in the examples below.
[0129] Where non-natural variations occur, the fragment may be
called a peptidomimetic, which are also within the scope of the
invention. For example, synthetic amino acids and their analogues
may be substituted for one or more of the native amino acids
providing construct-forming activity as described further
below.
[0130] A "peptidomimetic" is a synthetic chemical compound that has
substantially the same structure and/or functional characteristics
of a tropoelastin for use in the present invention. A
peptidomimetic generally contains at least one residue that is not
naturally synthesised. Non-natural components of peptidomimetic
compounds may be according to one or more of: a) residue linkage
groups other than the natural amide bond ("peptide bond") linkages;
b) non-natural residues in place of naturally occurring amino acid
residues; or c) residues which induce secondary structural mimicry,
i.e., to induce or stabilize a secondary structure, for example, a
beta turn, gamma turn, polyproline turn, beta sheet, alpha helix
conformation, and the like.
[0131] Peptidomimetics can be synthesized using a variety of
procedures and methodologies described in the scientific and patent
literatures (for example, Gilman et al., al-Obeidi et al. (1998),
Hruby et al. (1997) and Ostergaard & Holm (1997)).
[0132] Preferably, the functionally active fragment is about 100
amino acids in length. Generally, the shortest fragment for use in
the present invention will be about 10 amino acids in length.
Therefore, the fragment may be between about 10 and about 100 amino
acids in length. Shorter fragments are advantageous where, for
example, the fragments are sought to be made by synthetic
techniques because the preparation of long fragments by, for
example, solid-phase synthesis, can be difficult to achieve.
Fragments are generally synthesised in vitro where very pure
products are desired to be obtained. The advantage of longer
fragments is that the hydrophobic/hydrophilic nature of the
fragment can be more easily fine-tuned, as can its elastic
properties. Preferably, the functionally active fragment or variant
has at least approximately 60% identity to a peptide such as
described above, more preferably at least approximately 65%, 66%,
67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,
80%, 81%, 82%, 83%, 84% or 85% identity, even more preferably 90%
identity, even more preferably at least approximately 95%, 96%,
97%, 98%, 99% or 100% identity. The functionally active fragment or
variant may correspond to, or have identity with, a contiguous
sequence of amino acids from the tropoelastin, however it is also
contemplated that a functionally active fragment corresponds to, or
has identity with, sequences of amino acids that are clustered
spatially in the three-dimensional structure of the
tropoelastin.
[0133] Such functionally active fragments and variants include, for
example, those having conservative amino acid substitutions. Those
skilled in the art can determine appropriate parameters for
measuring alignment, including any algorithms (non-limiting
examples described below) needed to achieve maximal alignment over
the full-length of the sequences being compared. When amino acid
sequences are aligned, the percent amino acid sequence identity of
a given amino acid sequence A to, with, or against a given amino
acid sequence B (which can alternatively be phrased as a given
amino acid sequence A that has or includes a certain percent amino
acid sequence identity to, with, or against a given amino acid
sequence B) can be calculated as: percent amino acid sequence
identity=(X/Y).times.100, where X is the number of amino acid
residues scored as identical matches by the sequence alignment
program's or algorithm's alignment of A and B, and Y is the total
number of amino acid residues in B. If the length of amino acid
sequence A is not equal to the length of amino acid sequence B, the
percent amino acid sequence identity of A to B will not equal the
percent amino acid sequence identity of B to A.
[0134] In calculating percent identity, exact matches are counted.
The determination of percent identity between two sequences can be
accomplished using a mathematical algorithm. A non-limiting example
of a mathematical algorithm utilized for the comparison of two
sequences is the algorithm of Karlin and Altschul (1990), modified
as in Karlin and Altschul (1993). Such an algorithm is incorporated
into the BLASTN and BLASTX programs of Altschul et al. (1990). To
obtain gapped alignments for comparison purposes, Gapped BLAST (in
BLAST 2.0) can be utilized as described in Altschul et al. (1997).
Alternatively, PSI-Blast can be used to perform an iterated search
that detects distant relationships between molecules. See Altschul
et al. (1997) supra. In one preferred embodiment, utilizing BLAST,
Gapped BLAST, and PSI-Blast programs, the default parameters of the
respective programs (for example, BLASTX and BLASTN) are used.
Alignment may also be performed manually by inspection. Another
non-limiting example of a mathematical algorithm utilized for the
comparison of sequences is the ClustalW algorithm (Higgins et al.
(1994)). ClustalW compares sequences and aligns the entirety of the
amino acid or DNA sequence, and thus can provide data about the
sequence conservation of the entire amino acid sequence. The
ClustalW algorithm is used in several commercially available
DNA/amino acid analysis software packages, such as the ALIGNX
module of the Vector NTI Program Suite (Invitrogen Corporation,
Carlsbad, Calif.). After alignment of amino acid sequences with
ClustalW, the percent amino acid identity can be assessed. A
non-limiting example of a software program useful for analysis of
ClustalW alignments is GENEDOC.TM. or JalView
(http://www.jalview.org/). GENEDOC.TM. allows assessment of amino
acid (or DNA) similarity and identity between multiple proteins.
Another non-limiting example of a mathematical algorithm utilized
for the comparison of sequences is the algorithm of Myers and
Miller (CABIOS 1988; 4: 11-17). Such an algorithm is incorporated
into the ALIGN program (version 2.0), which is part of the GCG
Wisconsin Genetics Software Package, Version 10 (available from
Accelrys, Inc., 9685 Scranton Rd., San Diego, Calif., USA). In one
preferred embodiment, utilizing the ALIGN program for comparing
amino acid sequences, a PAM 120 weight residue table, a gap length
penalty of 12, and a gap penalty of 4 is used when assessing
percentage identity.
[0135] The term "conservative amino acid substitutions" refers to
the substitution of an amino acid by another one of the same class,
the classes being as follows:
[0136] Non-polar: Ala, Val, Leu, Ile, Pro, Met, Phe, Trp
[0137] Uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln
[0138] Acidic: Asp, Glu
[0139] Basic: Lys, Arg, His.
[0140] Other conservative amino acid substitutions may also be made
as follows:
[0141] Aromatic: Phe, Tyr, His
[0142] Proton Donor: Asn, Gln, Lys, Arg, His, Trp
[0143] Proton Acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gln.
[0144] In one embodiment, the monomers have a sequence that has at
least 90% sequence identity with the amino acid sequence of human
tropoelastin across at least 50 consecutive amino acids.
[0145] In one embodiment, the monomers have a sequence that has at
least 80% sequence identity with the sequence of human tropoelastin
across a consecutive amino acid sequence consisting of VPGVG.
[0146] One type of tropoelastin monomer may be used in the present
invention, or combinations of different tropoelastin monomers may
be used. For example, the combination of tropoelastin monomers can
include 1, 2, 3, 4, 5, 6, 7, 9, 10, or more, different types of
tropoelastin monomers. In another embodiment, at least 2, at least
3, at least 4, at least 5, at least 6, at least 7, at least 8, at
least 9, at least 10 or more, different tropoelastin monomers can
be used. In another embodiment, 1 or more, 2 or more, 3 or more, 4
or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or
10 or more different types of tropoelastin monomers can be
used.
[0147] In addition, in other embodiments, the tropoelastin monomers
are any number or combination of human and/or non-human (e.g.
primate, bovine, equine, sheep, goat, pig, dog, cat, or rodent)
tropoelastin monomers.
[0148] Further, it will be appreciated that varying the ratio
and/or identity of each of the tropoelastin monomers present in a
combination can generate tropoelastin-based hydrogels with desired
elasticity, tensile strength, and shapeability, and that the
strength, elasticity, cross-linking potential and other physical
and biochemical behavior of tropoelastin polymers can therefore be
varied, and possibly controlled, by incorporating various
polymorphic forms of tropoelastin into polymeric scaffolds.
[0149] In addition, the ratio and/or identity of each of the
tropoelastin monomers present in a combination can be varied so as
to match the tropoelastin monomers present in the tissue being
repaired, replaced, or regenerated.
[0150] In one embodiment, the solution is applied to the surface by
spraying the solution onto the surface.
[0151] The term "surface," as used herein, refers to any object or
device that can be used to make a tropoelastin-based polymer
construct of complementary shape. For example, the surface may be a
flat surface, such that the aggregate forms as a flat film thereon,
or may be a mold. Molds are generally understood to be objects or
devices that include a hollowed-out portion. This portion can be
filled with the solution of tropoelastin monomers, such that when
the concentrate is heated, it hardens or sets inside the mold,
adopting its shape. The mold may be of any shape desired by a
person skilled in the art. For example, the mold may be shaped such
that the construct formed therefrom is in the shape of a particular
biological tissue to be repaired and/or replaced (for example,
cartilage, vascular tissue or bone) or may include a pattern (of
channels, grooves, and the like, as discussed further below) that
can be used for assay applications. Accordingly, in one embodiment,
the surface is provided in the form of a die, mold or cast enabling
the elastic material formed by the process to be shaped into a
predefined shape.
[0152] In one embodiment, an elastic material may form a "surface"
to which the solution of tropoelastin monomers may be applied
according to the above described process. For example, a first
application may result in the formation of an elastic material on a
non-proteinaceous surface. A second application may be made to that
elastic material formed on the non-proteinaceous surface, resulting
in the formation of an elastic material on the elastic material
derived from the first application. The process can be repeated
multiple times, enabling building of structures, for example by
drop-wise application of solutions of tropoelastin monomers.
[0153] The present invention also relates to an elastic material
formed by a process including: [0154] providing a solution of
tropoelastin monomers; [0155] applying the solution to a surface;
and [0156] heating the solution on the surface to a temperature
sufficient to enable the tropoelastin monomers in the solution to
bind to each other to form an aggregate of tropoelastin
monomers.
[0157] The present invention also relates to a construct including
an elastic material formed by heat-assisted association of
tropoelastin monomers.
[0158] An elastic material may be a three-dimensional polymeric
structure that can be used to repair, augment or replace (at least
a portion of) a natural tissue of a subject (for example, for
veterinary or medical (human) applications). In addition, the
elastic material may be incorporated into, or form a part of, a
three-dimensional construct. For example, the aggregate may be
incorporated, as a layer, into a construct that is used for
cartilage repair, or may be incorporated into a stent.
[0159] It will be understood by a person skilled in the art that
the degree of contact between the tropoelastin monomers in the
solution before the heating step can also affect the material's
properties. For example, the more concentrated the solution is (in
terms of the quantity of tropoelastin present in the concentrate)
prior to heating, the more tropoelastin monomers will interact to
form the aggregate, and the less elastic the resulting material
will be. Therefore, in certain embodiments, the concentration of
tropoelastin monomer in the solution may be directly correlated to
the polymerisation degree. Other factors may also contribute to the
properties of the material and these include, for example, the type
of tropoelastin monomer used, the temperature at which the heating
step is carried out, and the time over which the heating is carried
out, as discussed above.
[0160] As mentioned above, the materials described herein may be
porous, i.e., the materials may have porosity i.e. a fractional
volume of the material may be composed of open space, for example,
pores or other openings. Therefore, porosity measures void spaces
in a material and is a fraction of volume of voids over the total
volume, as a percentage between 0 and 100% (or between 0 and 1)
(see, for example, Coulson et al. (1978)). Determination of matrix
porosity is well known to a person skilled in the art, for example,
using standardized techniques, such as mercury porosimetry and gas
adsorption (such as nitrogen adsorption). Generally, porosity of
the material can range from 0.5 to 0.99, from about 0.75 to about
0.99, or from about 0.8 to about 0.95. Preferably, porosity of the
material is at least 0.75, more preferably at least 0.8, and most
preferably at least 0.9.
[0161] The porous materials can have any pore size. As used herein,
the term "pore size" refers to a diameter or an effective diameter
of the cross-sections of the pores. The term "pore size" can also
refer to an average diameter or an average effective diameter of
the cross-sections of the pores, based on the measurements of a
plurality of pores. The effective diameter of a cross-section that
is not circular equals the diameter of a circular cross-section
that has the same cross-sectional area as that of the non-circular
cross-section. The pores can be filled with a fluid such as water
or air. In some embodiments, the pores of the material can have a
pore size distribution ranging from about 50 nm to about 1000
.mu.m, from about 250 nm to about 500 .mu.m, from about 500 nm to
about 250 .mu.m, from about 1 .mu.m to about 200 .mu.m, from about
10 .mu.m to about 150 .mu.m, from about 15 .mu.m to about 125
.mu.m, from about 20 .mu.m to about 100 .mu.m, or from about 40
.mu.m to about 65 .mu.m. In some embodiments, the material can have
a pore size of about 12 .mu.m, about 25 .mu.m, about 45 .mu.m,
about 50 .mu.m, or about 65 .mu.m. In some embodiments, the
material can have a pore size of 11.7.+-.3.3 .mu.m, 23.4.+-.5.8
.mu.m, or 51.+-.9 .mu.m.
[0162] It will be understood by a person skilled in the art that
pores can exhibit a distribution of sizes around the indicated
"size." Unless otherwise stated, the term "size" as used herein
refers to the mode of a size distribution of pores, i.e., the value
that occurs most frequently in the size distribution.
[0163] The pores can be substantially round cross-section or
opening. What is meant by "substantially round" is that the ratio
of the lengths of the longest to the shortest perpendicular axes of
the pore cross-section is less than or equal to about 1.5.
Substantially round does not require a line of symmetry. In some
embodiments, the ratio of lengths between the longest and shortest
axes of the pore cross-section is less than or equal to about 1.5,
less than or equal to about 1.45, less than or equal to about 1.4,
less than or equal to about 1.35, less than or equal to about 1.30,
less than or equal to about 1.25, less than or equal to about 1.2,
less than or equal to about 1.15 less than or equal to about
1.1.
[0164] Advantageously, the materials of the present invention are
elastic. An "elastic" material is one that returns to a particular
shape or conformation after a force (such as compression or
extension) that has been applied to it has been withdrawn. It is
also referred to as resiliently compressible and extendible,
mechanically durable, or pliable material of relatively low
hysteresis. This material may be referred to as stretchable,
tensile, resilient or capable of recoil. For example, the material
can have an extensibility of from about 20 to about 400%.
[0165] In some embodiments, the material can have an elastic
modulus in the range about 1 kPa to about 10.sup.3 kPa. As used
herein, the term "elastic modulus" refers to an object's or
substance's tendency to be deformed elastically (i.e.,
non-permanently) when a force is applied to it. Generally, the
elastic modulus of an object is defined as the slope of its
stress-strain curve in the elastic deformation region. Specifying
how stress and strain are to be measured, including directions,
allows for many types of elastic moduli to be defined. Young's
modulus (E) describes tensile elasticity, or the tendency of an
object to deform along an axis when opposing forces are applied
along that axis; it is defined as the ratio of tensile stress to
tensile strain. It is often referred to simply as the elastic
modulus. It will also be appreciated that the elastic material
formed by the process responds elastically to compression. In some
embodiments, the material can have an elastic modulus in the range
from about 1 kPa to about 1000 kPa. In some embodiments, the
material can have an elastic modulus of about 10 kPa, about 100
kPa, or about 200 kPa.
[0166] A higher Young's modulus for a given material according to
the invention can be achieved by any one of the following: [0167]
heating for a longer period of time, for example 8 to 16 hours
[0168] addition of silk prior to solubilisation of tropoelastin and
heating [0169] addition of linkers
[0170] These adjustments result in a material having a Young's
Modulus of up to 10 mega pascal.
[0171] The material of the present invention may be added to water
to form a hydrogel. Accordingly, the present invention relates to a
hydrogel including an elastic material, wherein the elastic
material is formed by a process including: [0172] providing a
solution of tropoelastin monomers; [0173] applying the solution to
a surface; and [0174] heating the solution on the surface to a
temperature sufficient to enable the tropoelastin monomers in the
concentrate to bind to each other to form an aggregate of
tropoelastin monomers, thereby forming the elastic material.
[0175] A hydrogel is generally understood as a network of polymer
chains (that are hydrophilic) in which water is the dispersion
medium. Hydrogels are highly absorbent--they can contain over 99.9%
water--and possess a degree of flexibility very similar to natural
tissue, due to their significant water content.
[0176] Accordingly, a hydrogel including an elastic material or
aggregate of the present invention will typically contain a
substantial amount of water. However, the amount of water into
which the aggregate is added or immersed to form a hydrogel will
depend on factors such as the degree of elasticity desired in the
hydrogel. That is, the amount of water added to the aggregate may
be an amount that is only just sufficient to impart elasticity.
Alternatively, a significant amount of water may be added to make
the resultant hydrogel highly elastic. A person skilled in the art
will understand that the amount of water used will also depend on
the elasticity of the aggregate itself (i.e. if the aggregate is
already quite elastic, a smaller amount of water will need to be
added than if the aggregate is less elastic).
[0177] A person skilled in the art will understand that the
discussions herein relating to additional components (e.g. cells,
pharmaceutically active ingredients, and the like) of the materials
of the present invention, as well as forms of the materials of the
present invention (e.g. as tissue engineering constructs and
assays) also apply to the constructs and hydrogels comprising the
elastic material of the present invention.
[0178] Materials described herein can be used for tissue
engineering applications. In some embodiments, tissue engineering
aims to replace, repair and/or regenerate tissue and/or organ
function or to create artificial tissues and organs for
transplantation. In general, scaffolds used in tissue engineering
mimic the natural extracellular matrix (ECM) and provide support
for cell adhesion, migration, and proliferation. Ideally, they
allow for differentiated function, new tissue generation, and its
three-dimensional organization. Desired characteristics of
scaffolds include physical parameters such as mechanical strength
and degradability, while biological properties include
biocompatibility and the ability to provide a biologically relevant
microenvironment. Biodegradable materials are advantageous in some
applications (such as tissue regeneration) because after tissue is
grown, the resulting structures are made entirely or almost
entirely from biological components.
[0179] In some embodiments, the materials described herein can be
used for many tissue-engineering applications, including growth
and/or replacement of vascular tissues, cardiac tissues, bladder,
skin, lung, ligament, tendon, endocrine glands, liver, renal
tissue, lymph nodes, pancreas, bone, cartilage, and other tissues.
In some embodiments, the materials can be used to deliver signals
to cells, act as support structures for cell growth and function,
and provide space filling.
[0180] Exemplary desired shapes of the elastic material, include,
but are not limited to sheets, tubes, and any other
three-dimensional shape. Elastic materials formed in the shape of a
sheet can be used in the preparation of implants, constructs and
grafts to provide reparative, replacement, and/or regenerative
therapy for dermal tissues, membranes for tooth root coverage
procedures, membranous tissues, and the like. Elastic materials
formed in the shape of a tube can be used in the preparation of
implants, constructs and grafts to provide reparative, replacement,
and/or regenerative therapy for arteries, veins, ureters, urethras,
nerves, long bones, and the like. Elastic materials formed in the
shape of any other three-dimensional object can be used in the
preparation of implants, constructs and grafts to provide
reparative, replacement, and/or regenerative therapy for organ
transplants, bone remodelling or mending, dental implants, or for
muscle, tendon, ligament, and cartilage grafts.
[0181] In one embodiment, the elastic material may be formed in a
shape enabling its use as a pre-cast patch, which may then be
sutured or otherwise adhered onto a surface. Examples include a
cardiac patch, a dermal patch, or a patch suitable for the
cornea.
[0182] A biocompatible elastic material formed, cast or molded in
the shape of a sheet can be a flat sheet, or a sheet having
curvatures to closely match the contours of the injured, damaged,
or diseased tissue or organ being repaired, replaced, or
regenerated. The sheets may be of any geometrical shape, including
but not limited to squares, rectangles, trapezoids, triangles,
circles, ellipses, and the like.
[0183] Exemplary areas of the sheets include areas of about 1
mm.sup.2 to about 1 m.sup.2, about 1 mm.sup.2 to about 50 cm.sup.2,
about 1 mm.sup.2 to about 25 cm.sup.2, about 1 mm.sup.2 to about 10
cm.sup.2, about 1 mm.sup.2 to about 1 cm.sup.2, about 1 cm.sup.2 to
about 1 m.sup.2, about 1 cm.sup.2 1 cm.sup.2 to about 500 cm.sup.2,
1 cm.sup.2 to about 250 cm.sup.2, 1 cm.sup.2 to about 200 cm.sup.2,
1 cm.sup.2 to about 150 cm.sup.2, to about 100 cm.sup.2, about 1
cm.sup.2 to about 50 cm.sup.2, about 1 cm.sup.2 to about 25
cm.sup.2, about 1 cm.sup.2 to about 10 cm.sup.2, about 1 cm.sup.2
to about 5 cm.sup.2, about 1 cm.sup.2 to about 2.5 cm.sup.2, about
10 mm.sup.2 to about 10 cm.sup.2, about 0.1 cm.sup.2 to about 10
cm.sup.2, about 0.1 cm.sup.2 to about 1 cm.sup.2, or any
intervening range thereof. For example, the range of areas of 1
cm.sup.2 to 100 cm.sup.2 of an exemplary sheet includes about areas
of about 1 cm.sup.2, about 5 cm.sup.2, about 10 cm.sup.2, about 20
cm.sup.2, about 30 cm.sup.2, about 40 cm.sup.2, about 50 cm.sup.2,
about 60 cm.sup.2, about 70 cm.sup.2, about 80 cm.sup.2, about 90
cm.sup.2, and about 100 cm.sup.2.
[0184] Exemplary degrees of thickness of an elastic material
formed, cast or molded in the shape of a sheet, include a range of
about 0.1 mm to about 10 mm, about 0.25 mm to about 7.5 mm, about
0.5 mm to about 5 mm, about 0.75 mm to about 2.5 mm, about 1 mm to
about 2 mm or any intervening range thereof.
[0185] In another embodiment, the thickness can be about 0.1 mm,
about 0.25 mm, about 0.5 mm, about 0.75 mm, about 1 mm, about 2 mm,
about 3 mm, about 4 mm, about 5 mm, about 7.5 mm, or about 10 mm or
more.
[0186] An elastic material formed, cast or molded in the shape of a
tube can have any desired length, diameter, and thickness such that
the size of the scaffold is suitable to repair, replace, and/or
regenerate an injured, damaged, or diseased tissue or organ.
Exemplary lengths of the tube include about 0.5 cm, about 1 cm,
about 2.5 cm, about 5 cm, about 10 cm, about 25 cm, about 50 cm,
about 100 cm, about 150 cm, about 200 cm, about 250 cm, about 300
cm, about 350 cm, about 400 cm, about 450 cm, about 500 cm, or
longer. Exemplary diameters of the tube include about 0 mm (e.g., a
solid fiber), 0.5 mm, about 1 mm, about 1.5 mm, about 2 mm, about
2.5 mm, about 3 mm, about 3.5 mm, about 4 mm, about 4.5 mm, about 5
mm, about 5.5 mm, about 6 mm, about 6.5 mm, about 7 mm, about 7.5
mm, about 8 mm, about 8.5 mm, about 9 mm, about 9.5 mm, about 10
mm, about 11 mm, about 12 mm or more mm in diameter. In a preferred
embodiment, a tube of the invention has about 1 mm to about 10 mm
diameter.
[0187] An elastic material formed, cast or molded in the shape of
other three-dimensional objects can have any desired volume
and/shape such that the size of the scaffold is suitable to repair,
replace, and/or regenerate an injured, damaged, or diseased tissue
or organ.
[0188] Exemplary volumes of the three-dimensional shape scaffolds
of about 100 mm.sup.3 to about 5 m.sup.3, about 100 mm.sup.3 to
about 1000 cm.sup.3, about 1 cm.sup.3 to about 1000 cm.sup.3, about
1 cm.sup.3 to about 100 cm.sup.3, about 1 cm.sup.3 to about 10
cm.sup.3, about 10 cm.sup.3 to about 1000 m.sup.3, about 10
cm.sup.3 to about 100 cm.sup.3, about 500 cm.sup.3 to about 1000
cm.sup.3, about 100 mm.sup.3 to about 5 cm.sup.3, about 100
mm.sup.3 to about 2.5 cm.sup.3, about 1 cm.sup.3 to about 5
cm.sup.3, about 1 cm.sup.3 to about 2.5 cm.sup.3, about 750
cm.sup.3 to about 1250 cm.sup.3, about 850 cm.sup.3 to about 1150
cm.sup.3, about 950 cm.sup.3 to about 1050 cm.sup.3, about 900
cm.sup.3 to about 1000 cm.sup.3, or any intervening range thereof.
For example, the range of volumes of 1 cm.sup.33 to 10 cm.sup.3 of
an exemplary three-dimensional shape includes about volumes of
about 1 cm.sup.3, about 2 cm.sup.3, about 3 cm.sup.3, about 4
cm.sup.3, about 5 cm.sup.3, about 6 cm.sup.3, about 7 cm.sup.3,
about 8 cm.sup.3, about 9 cm.sup.3, and about 10 cm.sup.3. In one
embodiment, the scaffold may have a volume of from about 1 to about
100 microlitres.
[0189] In some embodiments, the elastic material is in the form of
a film. The thickness of the film can range from nanometers to
millimeters. For example, film thickness can range from about 1 nm
to about 1000 mm. In some embodiments, the film thickness can be
from about 1 nm to 1000 nm, from about 1 .mu.m about 1000 .mu.m,
from about 1 mm to about 1000 mm. In some embodiments, the film
thickness can be from about 500 nm to about 750 .mu.m, from about
750 nm to about 500 .mu.m, from about 1000 nm to about 250 .mu.m,
from about 10 .mu.m to about 100 .mu.m, from about 25 .mu.m to
about 75 .mu.m. In some embodiments, film thickness ranges from
about 10 nm to about 1 mm. In some embodiments, the film thickness
can be about 50 .mu.m.
[0190] In some embodiments, the elastic material is a foam. Foams
can be made from methods known in the art, including, for example,
freeze-drying and gas foaming in which water is the solvent or
nitrogen or other gas is the blowing agent, respectively.
[0191] In some embodiments, the materials can be used to construct
complex delivery devices capable of precisely defined release
profiles. This could be achieved through combining drugs or drug
delivery devices (i.e. nanoparticles or microparticles) with the
materials described herein and using these to construct more
complex drug delivery systems. To give but one example, the
materials described herein can additionally include a therapeutic
agent to be delivered (for example a small molecule, nucleic acid,
protein, lipid and/or carbohydrate drug). Such materials can be
useful for delivering a drug to a site that has been targeted for
tissue regeneration. For example, a material comprising
osteoinductive cells, which is administered to a subject for
purposes of regenerating new bone, can additionally include one or
more bone morphogenetic proteins (BMPs) which, upon their release,
can help further stimulate the growth of new bone.
[0192] The elastic material described herein can be combined with
another material, for example a biomaterial, to form a composite
material. The term "biomaterial" as used herein refers in general
to biocompatible naturally occurring materials. Exemplary
biomaterials include, but are not limited to, biopolymers, sponges,
silk, decellularized tissues, and gelatin. The term "biopolymer" as
used herein refers to either a naturally occurring polymer, or a
synthetic polymer that is compatible with a biological system or
that mimics naturally occurring polymers. Exemplary biopolymers
include, but are not limited to, oligosaccharides, polysaccharides
such as glycosaminoglycans, peptides, proteins, oligonucleotides,
nucleic acids, polyketides, peptoids, hydrogels, poly(glycols) such
as poly(ethylene glycol), collagen, silk, and polylactates.
[0193] In one embodiment the elastic material may be combined with
a salt, or with polyvinyl pyrolidone.
[0194] The elastic material of the present invention may also
include other components such as pharmaceutically acceptable
excipients and biologically active agents (for example drugs,
vitamins and minerals), to assist in repair and/or re-generation of
the target tissue, and/or to provide a method of achieving targeted
delivery of biologically active compounds. Such components may be
added to the tropoelastin solution prior to heating (so that they
are incorporated into the elastic material as it forms) or they may
be placed into the elastic material after it has formed. In
addition, the components may be present in the aqueous solution
used to form a hydrogel from the elastic material. A person skilled
in the art will understand that where the components to be added
are not stable under the conditions required to form the elastic
material, the components should be added after the elastic material
has already been formed.
[0195] Any biologically active agent known to a person skilled in
the art to be of benefit in the diagnosis, treatment or prevention
of a disease is contemplated as a therapeutic agent in the context
of the present invention. Therapeutic agents include hormones,
growth factors, enzymes, DNA, plasmid DNA, RNA, siRNA, viruses,
proteins, lipids, pro-inflammatory molecules, antibodies,
antibiotics, anti-inflammatory agents, anti-sense nucleotides and
transforming nucleic acids or combinations thereof. Any of the
therapeutic agents can be combined to the extent such combination
is biologically compatible.
[0196] Suitable growth factors and cytokines include, but are not
limited, to stem cell factor (SCF), granulocyte-colony stimulating
factor (G-CSF), granulocyte-macrophage stimulating factor (GM-CSF),
stromal cell-derived factor-1, steel factor, VEGF, TGF.beta.,
platelet derived growth factor (PDGF), angiopoeitins (Ang),
epidermal growth factor (EGF), bFGF, HNF, NGF, bone morphogenic
protein (BMP), fibroblast growth factor (FGF), hepatocye growth
factor, insulin-like growth factor (IGF-1), interleukin (IL)-3,
IL-1.alpha., IL-1.beta., IL-6, IL-7, IL-8, IL-11, and IL-13,
colony-stimulating factors, thrombopoietin, erythropoietin,
fit3-ligand, and tumor necrosis factor .alpha. (TNF.alpha.). Other
examples are described in Dijke et al. (1989); Mulder et al.
(1998); Ziegler et al. (1997). Suitable hormones include, but are
not limited to, antimullerian hormone (or mullerian inhibiting
factor or hormone), adiponectin, adrenocorticotropic hormone (or
corticotropin), angiotensinogen and angiotensin, antidiuretic
hormone (or vasopressin, arginine vasopressin), atrial-natriuretic
peptide (or atriopeptin), calcitonin, cholecystokinin,
corticotropin-releasing hormone, erythropoietin,
follicle-stimulating hormone, gastrin, ghrelin, glucagon,
gonadotropin-releasing hormone, growth hormone-releasing hormone,
human chorionic gonadotropin, human placental lactogen, growth
hormone, insulin-like growth factor 1, insulin-like growth factor
(or somatomedin), leptin, luteinizing hormone, melanocyte
stimulating hormone MSH, orexin, oxytocin, parathyroid hormone,
prolactin, relaxin, secretin, somatostatin, thrombopoietin,
thyroid-stimulating hormone (or thyrotropin), and
thyrotropin-releasing hormone.
[0197] Exemplary pharmaceutically active compounds (for example,
therapeutic agents) include, but are not limited to, those found in
Harrison et al., Physicians Desk Reference, Pharmacological Basis
of Therapeutics (1990), United States Pharmacopeia, current edition
of Goodman and Oilman's The Pharmacological Basis of Therapeutics;
and current edition of The Merck Index.
[0198] In another embodiment, the elastic material (or hydrogel
formed therefrom) includes a population of multipotent or
pluripotent stem cells (discussed further below), and hormones,
growth factors, cytokines, morphogens (e.g., retinoic acid etc.),
extracellular matrix materials (e.g., fibronectin, laminin,
collagen, etc.) or other materials (e.g., DNA, viruses, other cell
types, etc.) that facilitate the differentiation of the cell
population along a particular developmental pathway once the
elastic material or hydrogel has been implanted in the patient.
Alternatively, or in addition, the cells may be differentiated in
vitro during cell culturing with the elastic material or
hydrogel.
[0199] The bioactive agent can be covalently linked to the elastic
material through a linker. The linker can be a cleavable linker or
non-cleavable linker, depending on the application. As used herein,
a "cleavable linker" refers to linkers that are capable of cleavage
under various conditions. Conditions suitable for cleavage can
include, but are not limited to, pH, UV irradiation, enzymatic
activity, temperature, hydrolysis, elimination and substitution
reactions, redox reactions, and thermodynamic properties of the
linkage. In many cases, the intended nature of the conjugation or
coupling interaction, or the desired biological effect, will
determine the choice of linker group.
[0200] Pharmaceutically acceptable excipients include any and all
solvents, dispersion media, diluents, or other liquid vehicles,
dispersion or suspension aids, surface active agents, isotonic
agents, thickening or emulsifying agents, preservatives, solid
binders, lubricants and the like, as suited to the particular
dosage form desired. Gennaro (2006) discloses various excipients
used in formulating pharmaceutical compositions and known
techniques for the preparation thereof. Except insofar as any
conventional excipient is incompatible with a substance or its
derivatives, such as by producing any undesirable biological effect
or otherwise interacting in a deleterious manner with any other
component(s) of the hydrogel, its use is contemplated to be within
the scope of this invention.
[0201] Pharmaceutically acceptable excipients used in the
manufacture of pharmaceutical compositions include, but are not
limited to, inert diluents, dispersing and/or granulating agents,
surface active agents and/or emulsifiers, disintegrating agents,
binding agents, preservatives, buffering agents, lubricating
agents, and/or oils. Such excipients may optionally be included in
the tropoelastin-containing solutions. Excipients such as colouring
agents, coating agents, sweetening, flavouring, and perfuming
agents can be present in the solution, according to the judgment of
the formulator. General considerations in the formulation and/or
manufacture of pharmaceutical agents may be found, for example, in
Gennaro (2006).
[0202] The amount of tropoelastin and biologically active agent
present in the material will necessarily depend upon the particular
drug and the condition to be treated. A person skilled in the art
will be aware of appropriate agents and amounts to use to treat the
condition.
[0203] A therapeutically effective amount of a material of the
present invention may be delivered to a patient and/or organism
prior to, simultaneously with, and/or after diagnosis with a
disease, disorder, and/or condition. In some embodiments, a
therapeutically effective amount of a material of the present
invention is delivered to a patient and/or organism prior to,
simultaneously with, and/or after onset of symptoms of a disease,
disorder, and/or condition.
[0204] The term "therapeutically effective amount," as used herein,
refers to an amount of the material of the present invention that
is sufficient to treat, alleviate, ameliorate, relieve, delay onset
of, inhibit progression of, reduce severity of, and/or reduce
incidence of one or more symptoms or features of the disease,
disorder, and/or condition.
[0205] As mentioned above, materials of the present invention can
be used for tissue engineering applications. In some embodiments,
tissue engineering aims to replace, repair and/or regenerate tissue
and/or organ function or to create artificial tissues and organs
for transplantation. In general, scaffolds used in tissue
engineering (for example hydrogel scaffolds) mimic the natural ECM
and provide support for cell adhesion, migration, and
proliferation. Ideally, they allow for differentiated function, new
tissue generation, and its three-dimensional organization. Desired
characteristics of elastic scaffolds include physical parameters
such as mechanical strength and degradability, while biological
properties include biocompatibility and the ability to provide a
biologically relevant microenvironment. Biodegradable materials are
advantageous because after tissue is grown, the resulting
structures are made entirely or almost entirely from biological
components.
[0206] In some embodiments, materials to be utilized for drug
delivery can be altered in ways that result in enhanced residence
times, sustained drug delivery and/or targeted drug delivery. The
material properties, such as permeability (for example,
sustained-release applications), enviro-responsive nature (for
example, pulsatile-release applications), surface functionality
(for example, PEG coatings for stealth release), biodegradability
(for example, bioresorbable applications), and surface
biorecognition sites (for example, targeted release and bioadhesion
applications), can be altered and/or optimized for controlled
drug-delivery applications. For example, by controlling
tropoelastin chain length, tropoelastin composition and/or
tropoelastin concentration, it is possible to control the density
of the material. Control over the density provides, among other
things, control over sustained-release properties of the resulting
material.
[0207] In some embodiments, enzymes can be encapsulated within the
materials to create drug delivery systems that are responsive to
biological analytes.
[0208] The elastic materials described herein can additionally
include one or more additives. Additives can be resolving
(biodegradable) polymers, mannitol, starch sugar, inosite,
sorbitol, glucose, lactose, saccharose, sodium chloride, calcium
chloride, amino acids, magnesium chloride, citric acid, acetic
acid, hydroxyl-butanedioic acid, phosphoric acid, glucuronic acid,
gluconic acid, poly-sorbitol, sodium acetate, sodium citrate,
sodium phosphate, zinc stearate, aluminium stearate, magnesium
stearate, sodium carbonate, sodium bicarbonate, sodium hydroxide,
polyvinylpyrolidone, polyethylene glycols, carboxymethyl
celluloses, methyl celluloses, starch, micro-particles,
nano-particles, aprotinin, Factor XIII, or their mixtures. Without
wishing to be bound by a theory, one or more additives in the
material can alter (for example reduce or increase) the rate of
degradation of the material.
[0209] In some embodiments, the materials described herein can be
utilized for in vitro tissue culture applications. In certain
embodiments, the materials described can be utilized to develop
assays that are useful for drug discovery and biological studies
(for example, assemble arrays of well-defined materials for
high-throughput drug screening). For example, the presence of
feeder cells (for example, endothelial cells or fibroblasts) in the
presence of functional cells (for example, hepatocytes) can be used
to increase the maintenance of the functional cell type. Thus, it
is possible to generate three-dimensional structures that mimic the
native structure of functional organs that can be subsequently used
for drug discovery and/or diagnostics assays.
[0210] A person skilled in the art will understand that where the
cells to be incorporated into the elastic material are not stable
under the conditions required to form the elastic material, the
cells should be added after the elastic material has already been
formed. For example, the cells may be present in the aqueous
solution used to form a hydrogel from the elastic material.
[0211] In some embodiments, the materials described herein can be
utilized for toxicity assays that can test the toxicity of a test
substance (for example, utilizing materials in which liver cells
have been encapsulated).
[0212] In some embodiments, the materials described herein can be
used to make and coat various structures, such as microfluidic
channels. In this approach, the walls of the microchannels can be
made from construct assemblies instead of from more commonly used
materials such as polystyrene, glass and PDMS. Microfluidic
channels made from construct assemblies could be useful for many
purposes, for example, in applications where it is desirable for
the walls of the microfluidic channel to attract and bind
cells.
[0213] In some embodiments, the materials described herein can be
used for diagnostic applications. To give but one example,
cell-laden materials can be used for generating tissue-like
materials and/or material assemblies that can be used in assays
which test for the presence of one or more particular microbes. For
example, if a microbe (for example, bacteria, viruses, fungi, etc.)
were known to specifically bind to a particular tissue, then
tissue-like materials could be fabricated that would test for the
presence of the microbe in the sample.
[0214] The materials described herein may be patterned (e.g. a
micropatterned elastic material). Micropatterned materials can be
prepared using, for example, a method including contacting a
tropoelastin solution with a surface of a mold, the mold including,
on at least one surface thereof, a three-dimensional negative
configuration of a predetermined micropattern to be disposed on and
integral with at least one surface of the elastic material, and
heating the solution while in contact with the micropatterned
surface of the mold, thereby providing a micropatterned elastic
material. Elastic materials prepared this way include a
predetermined and designed micropattern on at least one surface of
the material, which pattern is effective to facilitate cell
alignment, tissue repair, growth or regeneration, or is effective
to provide delivery of a protein or a therapeutic agent. The
micropattern geometry can be controlled using the molds of the
appropriate pattern or size. Further, the micropattern can be
characterized for surface morphology by known techniques, such as
field emission scanning electron and atomic force microscopy.
[0215] In some embodiments, the micropattern is in the forms of
grooves or channels. The groove size (width) can range from about
500 nm to about 500 .mu.m. In some embodiments, the groove size can
range from about 1 .mu.m to about 250 .mu.m, from about 10 .mu.m to
about 100 .mu.m, or from about 20 .mu.m to about 75 .mu.m. In some
embodiments, the groove size is about 50 .mu.m or about 20
.mu.m.
[0216] The spacing between the grooves can also be optimized for
desired use. For example, spacing between the grooves can range
from about 500 nm to about 500 .mu.m. In some embodiments, the
distance between the grooves can range from about 1 .mu.m to about
250 .mu.m, from about 10 .mu.m to about 100 .mu.m, or from about 20
.mu.m to about 75 .mu.m. In some embodiments, the distance between
the grooves is about 50 .mu.m or about 20 .mu.m.
[0217] The groove thickness depth can range from about 250 nm to
about 500 .mu.m. In some embodiments, groove thickness can range
from about 500 nm to about 250 .mu.m, or from about 750 nm to about
1000 nm.
[0218] As mentioned above, the elastic materials described herein
can be used in tissue engineering and repair. As used herein, the
term "repair" refers to any correction, reinforcement,
reconditioning, remedy, making up for, making sound, renewal,
mending, patching, or the like that restores function. Accordingly,
the term "repair" can also mean to correct, to reinforce, to
recondition, to remedy, to make up for, to make sound, to renew, to
mend, to patch or to otherwise restore function.
[0219] It will be understood by a person skilled in the art that
hydrogels formed from the elastic materials of the present
invention can be used in tissue engineering and repair as well.
Therefore, where the elastic material of the present invention is
mentioned in these contexts, it will be understood that, where
appropriate, hydrogels formed from the materials can be utilised in
addition to, or as an alternative to, the elastic materials
themselves. It will also be understood by a person skilled in the
art that hydrogels can form from the elastic materials simply by
contact of the elastic materials with physiological conditions by
virtue of the elastic material absorbing water from the surrounding
environment.
[0220] By "treatment," "prevention," or "amelioration" is meant
delaying or preventing the onset, reversing, alleviating,
ameliorating, inhibiting, slowing down or stopping the progression,
aggravation, deterioration, or the progression of severity of a
condition associated with a disease or disorder.
[0221] The elastic materials of the present invention may be
administered using any amount and any route of administration
effective for treatment. The exact amount required will vary from
subject to subject, depending on the species, age, and general
condition of the subject, the severity of the infection, the
particular hydrogel, its mode of administration, its mode of
activity, and the like.
[0222] In another embodiment, the elastic materials described
herein are used in regenerative medicine for osteopathic
applications, including, but not limited to craniofacial, odontic,
and periodontic applications. In one embodiment, a construct or
device including an elastic material (or hydrogel formed from an
elastic material), is provided for use in reconstruction and
regeneration of oral and craniofacial tissues.
[0223] In particular embodiments, the elastic material (or hydrogel
formed from the elastic material) includes one or more tropoelastin
monomers, and human collagen. The resulting materials and hydrogels
are engineered for the desired surface topography, porosity,
strength and elasticity. In some embodiments, the elastic material
or hydrogel does not contain proteins or polypeptides other than
tropoelastin.
[0224] In one embodiment, the elastic material is cast in the form
of a sheet and can be used as a regenerative membrane in various
clinical applications, e.g., guided tissue regeneration (GTR) or
root coverage procedures. In one embodiment, the elastic material
is cast as a sheet and seeded with periodontal ligament cells (PDL)
forming an implant or graft that is suitable for use in a root
coverage procedure. Once the implant has formed, a surgeon engrafts
the implant in a root coverage procedure using methods known to a
person skilled in the art.
[0225] In another embodiment, the elastic material is cast in a
three-dimensional shape for use as a bone filling material.
Virtually any shape can be achieved because the pre-heated solution
is in a shapeable form. Once placed into a mold or into the desired
area, the solution can be "hardened" by heating. In addition, the
material can support unique clinical applications in periodontal
medicine for guided bone regeneration (GBR) procedures and
eliminate the need for a bone filler and a membrane to contain the
bone graft.
[0226] In a particular embodiment, the elastic material (or
hydrogel formed from the material), or an implant comprising the
elastic material or hydrogel formed from the material, is molded
into a desired shape, and includes one or more populations of
cells.
[0227] In general, cells to be used in accordance with the present
invention are any types of cells. The cells should be viable when
incorporated in the elastic materials of the present invention (or
hydrogels formed from the elastic materials). In some embodiments,
suitable cells include, but are not limited to, mammalian cells
(for example human cells, primate cells, mammalian cells, rodent
cells, etc.), avian cells, fish cells, insect cells, plant cells,
fungal cells, bacterial cells, and hybrid cells. In some
embodiments, exemplary cells include stem cells, totipotent cells,
pluripotent cells, and/or embryonic stem cells. In some
embodiments, exemplary cells include, but are not limited to,
primary cells and/or cell lines from any tissue. For example,
cardiomyocytes, myocytes, hepatocytes, keratinocytes, melanocytes,
neurons, astrocytes, embryonic stem cells, adult stem cells,
hematopoietic stem cells, hematopoietic cells (for example
monocytes, neutrophils, macrophages, etc.), ameloblasts,
fibroblasts, chondrocytes, osteoblasts, osteoclasts, neurons, sperm
cells, egg cells, liver cells, epithelial cells from lung,
epithelial cells from gut, epithelial cells from intestine, liver,
epithelial cells from skin, etc., and/or hybrids thereof, may be
used in accordance with the present invention.
[0228] Exemplary mammalian cells include, but are not limited to,
human umbilical vein endothelial cells (HUVEC), Chinese hamster
ovary (CHO) cells, HeLa cells, Madin-Darby canine kidney (MDCK)
cells, baby hamster kidney (BHK cells), NSO cells, MCF-7 cells,
MDA-MB-438 cells, U87 cells, A172 cells, HL60 cells, A549 cells,
SP10 cells, DOX cells, DG44 cells, HEK 293 cells, SHSY5Y, Jurkat
cells, BCP-1 cells, COS cells, Vero cells, GH3 cells, 9L cells, 3T3
cells, MC3T3 cells, C3H-10T1/2 cells, NIH-3T3 cells, and C6/36
cells.
[0229] In a certain embodiment, the one or more cell populations
include bone marrow stem cells, mesenchymal stem cells, or
pre-osteoblast cells to facilitate tissue or bone regeneration.
Additionally, the osteogenic potential of the
material/hydrogel/implant can be used as a sole therapy or in
combination with currently available commercial bone filler
products or primary autologous bone harvests. A person skilled in
the art will recognize that any type of bones can be repaired,
replace, or regenerated using the foregoing techniques.
[0230] In some embodiments, the conditions under which cells are
included in the elastic materials (or hydrogels formed therefrom)
are altered in order to maximize cell viability. In some
embodiments, conditions (for example pH, ionic strength, nutrient
availability, temperature, oxygen availability, osmolarity, etc.)
of the surrounding environment may need to be regulated and/or
altered to maximize cell viability.
[0231] Cell viability can be measured by monitoring one of many
indicators of cell viability. In some embodiments, indicators of
cell viability include, but are not limited to, intracellular
esterase activity, plasma membrane integrity, metabolic activity,
gene expression, and protein expression. To give but one example,
when cells are exposed to a fluorogenic esterase substrate (for
example, calcein AM), live cells fluoresce green as a result of
intracellular esterase activity that hydrolyzes the esterase
substrate to a green fluorescent product. To give another example,
when cells are exposed to a fluorescent nucleic acid stain (for
example ethidium homodimer-1), dead cells fluoresce red because
their plasma membranes are compromised and, therefore, permeable to
the high-affinity nucleic acid stain.
[0232] In general, the percent of cells in the material (or the
hydrogel formed therefrom) is a percent that allows for the
formation of elastic materials and/or hydrogels in accordance with
the present invention. In some embodiments, the percent of cells
that is suitable ranges between about 0.1% w/w and about 80% w/w,
between about 1.0% w/w and about 50% w/w, between about 1.0% w/w
and about 40% w/w, between about 1.0% w/w and about 30% w/w,
between about 1.0% w/w and about 20% w/w, between about 1.0% w/w
and about 10% w/w, between about 5.0% w/w and about 20% w/w, or
between about 5.0% w/w and about 10% w/w. In some embodiments, the
percent of cells in a solution that is suitable for forming elastic
materials in accordance with the present invention is approximately
5% w/w. In some embodiments, the concentration of cells in an
aqueous solution that is suitable for forming hydrogels in
accordance with the invention ranges between about 1.times.10.sup.5
cells/mL and 1.times.10.sup.8 cells/mL or between about
1.times.10.sup.6 cells/mL and 1.times.10.sup.7 cells/mL. In some
embodiments, a single elastic material r hydrogel formed therefrom
includes a population of identical cells and/or cell types. In some
embodiments, a single elastic material or hydrogel formed therefrom
includes a population of cells and/or cell types that are not
identical. In some embodiments, a single elastic material or
hydrogel formed therefrom may include at least two different types
of cells. In some embodiments, a single elastic material or
hydrogel formed therefrom may include 3, 4, 5, 10, or more types of
cells.
[0233] Any of a variety of cell culture media, including complex
media and/or serum-free culture media, that are capable of
supporting growth of the one or more cell types or cell lines may
be used to grow and/or maintain cells. Typically, a cell culture
medium contains a buffer, salts, energy source, amino acids (for
example, natural amino acids, non-natural amino acids, etc.),
vitamins, and/or trace elements. Cell culture media may optionally
contain a variety of other ingredients, including but not limited
to, carbon sources (for example, natural sugars, non-natural
sugars, etc.), cofactors, lipids, sugars, nucleosides,
animal-derived components, hydrolysates, hormones, growth factors,
surfactants, indicators, minerals, activators of specific enzymes,
activators inhibitors of specific enzymes, enzymes, organics,
and/or small molecule metabolites. Cell culture media suitable for
use in accordance with the present invention are commercially
available from a variety of sources, for example, ATCC (Manassas,
Va.). In certain embodiments, one or more of the following media
are used to grow cells: RPMI-1640 Medium, Dulbecco's Modified
Eagle's Medium, Minimum Essential Medium Eagle, F-12K Medium,
Iscove's Modified Dulbecco's Medium.
[0234] As discussed above, one significant advantage of the
invention is the development of materials (and corresponding
hydrogels) with unique properties, e.g., tensile strength,
elasticity, and flexibility/stiffness, generated by combining 2, 3,
4, 5, 6, 7, 8, 9, 10 or more individual tropoelastin isoforms,
themselves having unique properties. Such unique materials (and the
corresponding hydrogels) can be tailored for use at locations in
the body where their unique properties are the most advantageous.
For example, the strongest fibers can be used to repair muscles,
the most elastic fibers can be used to construct bladders and other
flexible organs (e.g. blood vessels and cardiac tissues), and the
stiffest fibers can be used in cartilage repair.
[0235] The present invention also relates to a method of repairing
and/or restoring biological tissue, the method comprising
administration of a therapeutically effective amount of an elastic
material of the present invention to a subject in need thereof.
[0236] The present invention also relates to the use of a
therapeutically effective amount of an elastic material of the
present invention, for repairing and/or restoring biological
tissue.
[0237] In one embodiment, the invention provides an elastic
material of the present invention, when used in a method of
repairing and/or restoring biological tissue.
[0238] The present invention also relates to the use of a
therapeutically effective amount of an elastic material of the
present invention, for the repair and/or restoration of biological
tissue. The invention also includes use of this material for the
manufacture of a medicament for the repair and/or restoration of
biological tissue.
[0239] As mentioned above, it will be appreciated that, in these
embodiments, a hydrogel formed from the elastic material of the
present invention can be used as an alternative to the elastic
material, provided that it is then treated appropriately (by, for
example, exposure to water) to form a hydrogel.
[0240] The present invention also relates to a method of repairing
and/or restoring biological tissue comprising the steps of: [0241]
identifying a subject having tissue injury; and [0242]
administering to the subject a therapeutically effective amount of
the elastic material of the present invention, [0243] administering
to the subject a therapeutically effective amount of a hydrogel
formed from the elastic material of the present invention, or
[0244] administering to the subject an amount of the elastic
material of the present invention to form a therapeutically
effective amount of the hydrogel, followed by treating the elastic
material of the present invention to form the hydrogel.
[0245] The present invention also relates to a method of
accelerating repair and/or restoration of biological tissue
comprising administering to a subject in need thereof: [0246] a
therapeutically effective amount of the elastic material of the
present invention, [0247] a therapeutically effective amount of a
hydrogel formed from the elastic material of the present invention,
or [0248] an amount of the elastic material of the present
invention to form a therapeutically effective amount of the
hydrogel, followed by treating the elastic material to form the
hydrogel.
[0249] Elastic materials of the present invention, and hydrogels
formed therefrom, are typically formulated in dosage unit form for
ease of administration and uniformity of dosage. It will be
understood, however, that the total daily usage of the materials
and/or hydrogels of the present invention will be decided by the
attending physician within the scope of sound medical judgment.
[0250] The specific therapeutically effective dose level for any
particular subject or organism will depend upon a variety of
factors including the disorder being treated and the severity of
the disorder; the activity of the specific active ingredient
employed; the specific polymer and/or cells employed; the age, body
weight, general health, sex and diet of the subject; the time of
administration, route of administration, and rate of excretion of
the specific active ingredient employed; the duration of the
treatment; drugs used in combination or coincidental with the
specific active ingredient employed; and like factors well known in
the medical arts.
[0251] The materials of the present invention (and hydrogels formed
therefrom) may be administered by any route. In some embodiments,
the materials of the present invention are administered by a
variety of routes, including direct administration to an affected
site. For example, materials (and/or hydrogels formed therefrom)
may be administered locally near a site which is in need of tissue
regeneration.
[0252] In certain embodiments, the elastic materials of the present
invention (and/or hydrogels formed therefrom) may be administered
such that included cells and/or therapeutic agents to be delivered
are released at concentrations ranging from about 0.001 mg/kg to
about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from
about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30
mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1
mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg,
of subject body weight per day, one or more times a day, to obtain
the desired therapeutic effect. The desired dosage may be
delivered, for example, three times a day, two times a day, once a
day, every other day, every third day, every week, every two weeks,
every three weeks, or every four weeks. In certain embodiments, the
desired dosage may be delivered using multiple administrations (for
example, two, three, four, five, six, seven, eight, nine, ten,
eleven, twelve, thirteen, fourteen, or more administrations).
[0253] In some embodiments, the present invention encompasses
"therapeutic cocktails" comprising the elastic materials of the
present invention (and/or hydrogels formed therefrom). In some
embodiments, the materials include a single cell type and,
optionally, a therapeutic agent. In some embodiments, materials
include multiple different cell types and, optionally, a
therapeutic agent.
[0254] It will be appreciated that cell-laden elastic materials in
accordance with the present invention (and hydrogels formed
therefrom) can be employed in combination therapies. The particular
combination of therapies (therapeutics or procedures) to employ in
a combination regimen will take into account compatibility of the
desired therapeutics and/or procedures and the desired therapeutic
effect to be achieved. It will be appreciated that the therapies
employed may achieve a desired effect for the same purpose (for
example, a hydrogel comprising a certain cell type to be used to
promote tissue growth may be administered concurrently with another
therapeutic agent used to stimulate growth of the same tissue), or
they may achieve different effects (for example, control of any
adverse effects, such as inflammation, infection, etc.).
[0255] The invention provides a variety of kits comprising one or
more of the materials of the present invention. For example, the
invention provides a kit comprising an elastic material and
instructions for use. A kit may include multiple different elastic
materials. A kit may optionally include tropoelastin monomers, a
concentrated solution of tropoelastin monomers, associated
tropoelastin monomers, biologically active compounds, and the like.
A kit may include any of a number of additional components or
reagents in any combination. All of the various combinations are
not set forth explicitly, but each combination is included in the
scope of the invention. A few exemplary kits that are provided in
accordance with the present invention are described in the
following paragraphs.
[0256] According to certain embodiments of the invention, a kit may
include, for example, (i) a solution of tropoelastin monomers; (ii)
a mold; and (iii) instructions for heating and forming an elastic
material from the solution.
[0257] A kit may also include, for example, (i) concentrate of
tropoelastin monomers; (ii) a mold; and (iii) instructions for
forming an elastic material from the concentrate.
[0258] Kits may further include other materials desirable from a
commercial and user standpoint, including other buffers, diluents,
filters, needles, and syringes.
[0259] Kits typically include instructions for use of the materials
of the present invention. Instructions may, for example, include
protocols and/or describe conditions for production of elastic
materials, administration of the materials to a subject in need
thereof, production of material assemblies, etc. Kits will
generally include one or more vessels or containers so that some or
all of the individual components and reagents may be separately
housed. Kits may also include a means for enclosing individual
containers in relatively close confinement for commercial sale, for
example, a plastic box, in which instructions, packaging materials
such as styrofoam, etc., may be enclosed.
[0260] The kit or "article of manufacture" may include a container
and a label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, blister
packs, etc. The containers may be formed from a variety of
materials such as glass or plastic. The label or package insert
indicates that the construct or composition is used for treating
the condition of choice. In one embodiment, the label or package
insert includes instructions for use and indicates that the
therapeutic composition can be used to repair or regenerate
tissue.
EXAMPLES
Example 1--Use of Water as Solvent for Tropoelastin
[0261] 100 mg tropoelastin dissolved in 333 .mu.l water at
4.degree. C. Used a 1 ml 31 gauge syringe to place a drop of
tropoelastin solution onto a glass slide. Placed at 160.degree. C.
for 1 minute. Added a further drop of tropoelastin; left for 1 min
before adding a further drop. Repeated approximately 10 times. Left
at 160.degree. C. for 4 h. Material turned glassy and darkish brown
(A). Placed in PBS--slowly wetted, did not dissolve and became
quite elastic (B).
Example 2--Use of HFP as Solvent for Tropoelastin
[0262] 100 mg tropoelastin dissolved in 500 .mu.L
1,1,1,3,3,3-hexafluoro-2-propanol (HFP) overnight at room temp.
Used a 1 mL 31 gauge syringe to place drops of tropoelastin
solution onto a glass slide sitting on top of a heating block set
at 70.degree. C. Placed at 160.degree. C. for 4 h. Material
appeared to bubble in oven and turned glassy and brown (A). Placed
in PBS--slowly wetted, became soft and elastic, appeared to have
gas bubbles caught within the material (B).
Example 3--Use of 70% EtOH as Solvent for Tropoelastin
[0263] Dissolved 100 mg tropoelastin in 650 .mu.l 70% EtOH (154
mg/mL). Used a 1 ml 31 gauge syringe to place drops of tropoelastin
solution onto a glass slide sitting on top of a heating block set
at 85.degree. C. Could build up 3D structure of drop upon drop by
waiting .about.1 min between each drop. Placed at 160.degree. C.
oven for 4 h. Material appeared to bubble in oven and turned glassy
and darkish brown.
Example 4--Coating an Inanimate Object
[0264] 20% w/v tropoelastin in HFP used to coat piece of Tygon
tubing by repeated dipping into the solution. Coated tube placed at
160.degree. C. for 4 h. Tropoelastin solution became hard and glass
like and could not be removed from tubing. Wet with PBS. Material
became soft and elastic, it could be peeled off the tubing and did
not dissolve.
Example 5--Electrospinning
[0265] Electrospun 20% (w/v) tropoelastin in HFP, 1 mL/h, .about.17
cm from syringe tip to collector, 20 kV(+)/grounded, 0.1 ml
solution, collector-aligned wires 2 cm apart 4 cm long. Placed at
160.degree. C. for 24 h. Wet with PBS did not dissolve; went
gel-like, maintained shape. Checked by SEM.
Example 6--Dermal Human Fibroblast Growth In Vitro on Heat-Treated
Electrospun Tropoelastin
[0266] 20% (w/v) tropoelastin in HFP was electrospun as described
above.
[0267] Human neonatal dermal fibroblasts (NHF8909; 5.times.10.sup.5
cells/well) were seeded onto heat-treated electrospun aligned
fibers that were anchored to plastic coverslips within 6 well
plates. Following 48 h culture in DMEM+10% FBS+Pen/Strep at
37.degree. C. in 5% CO.sub.2 the samples were prepared for SEM
analysis. Samples were fixed with 2% glutaraldehyde in 0.1 M sodium
cacodylate/0.1 M sucrose, post-fixed with 1% osmium, dehydrated in
increasing concentrations of ethanol mounted and gold coated.
Heat-treated electrospun tropoelastin supported cell attachment,
spreading and proliferation.
Example 7--Subcutaneous Implantation of Heat-Treated Electrospun
Tropoelastin in Mice
[0268] Non-aligned electrospun tropoelastin constructs were
prepared using 20% tropoelastin in HFP. Samples were spun at 20 kV
onto a round collector (non-aligned) at a distance of 17 cm, 1
mL/hr rate. 0.2 ml solution was used per construct. Placed at
160.degree. C. for 22 h.
[0269] Each mouse was implanted with one heat-treated non-aligned
electrospun tropoelastin construct and one Integra control. Two
mice for each time point at 1 week, 3 weeks and 6 weeks.
Subcutaneous implantation was performed with two 10 mm incisions
which were made on the back of each mouse and dissected to create
subcutaneous pouches. Electrospun scaffolds or Integra scaffold
(Integra LifeSciences Corporation) without an outer silicone layer
were inserted into each pouch. The wounds were then closed with 6-0
silk sutures and covered using IV3000 wound dressings (Smith &
Nephew) for 5 days. Carprofen (5 mg/kg) was given at the time of
anesthesia and then on the following day post surgery for
analgesia. After surgery, each mouse was caged individually for the
first two days and then two mice per cage thereafter with free
access to water and food. Skin biopsies were collected for
histological analysis at 1, 3 and 6 weeks post-implantation.
Explanted scaffolds and surrounding skin were stained with
Verhoeff-Van Gieson (VVG), demonstrating the elastic nature of the
implant.
[0270] Heat-treated electrospun tropoelastin persisted in mice for
a minimum of 6 weeks post implantation.
Example 8--Heat-Treated Water-Based Tropoelastin Films
[0271] 100 mg tropoelastin dissolved in 1 ml water at 4.degree. C.
Solution pipetted into wells of an 8-well glass chamber slide.
Solution was concentrated and dried by placing at 37.degree. C. for
16 h. Samples were further heated to 160.degree. C. for 4 h. After
heating at 37.degree. C. the scaffolds are translucent and light
brown. Following 160.degree. C. heating the samples are still
translucent but darker in colour.
Example 9--Micropatterned Heat-Treated Water-Based Tropoelastin
Films
[0272] 70 mg tropoelastin dissolved in 1 ml water at 4.degree. C.
Solution pipetted onto a PDMS (polydimethylsiloxane) mould
containing 3.5 .mu.m wide, and 500 nm, deep ridges. Solution was
concentrated and dried by placing at 37.degree. C. for 16 h.
Samples were further heated to 160.degree. C. for 4 h. Images were
obtained using a light microscope with 20.times. and 40.times.
objectives.
Sequence CWU 1
1
3115PRTArtificial SequencePeptide sequence 1Gly Val Gly Val Pro1
524PRTArtificial SequencePeptide sequence 2Gly Gly Val
Pro136PRTArtificial SequencePeptide sequence 3Gly Val Gly Val Ala
Pro1 549PRTArtificial SequencePeptide sequence 4Ala Ala Ala Lys Ala
Ala Lys Ala Ala1 5518PRTArtificial SequencePeptide sequence 5Gly
Gly Val Pro Gly Ala Ile Pro Gly Gly Val Pro Gly Gly Val Phe1 5 10
15Tyr Pro69PRTArtificial SequencePeptide sequence 6Gly Val Gly Leu
Pro Gly Val Tyr Pro1 577PRTArtificial SequencePeptide sequence 7Gly
Val Pro Leu Gly Tyr Pro1 5813PRTArtificial SequencePeptide sequence
8Pro Tyr Thr Thr Gly Lys Leu Pro Tyr Gly Tyr Gly Pro1 5
10913PRTArtificial SequencePeptide sequence 9Gly Gly Val Ala Gly
Ala Ala Gly Lys Ala Gly Tyr Pro1 5 101010PRTArtificial
SequencePeptide sequence 10Thr Tyr Gly Val Gly Ala Gly Gly Phe Pro1
5 10115PRTArtificial SequencePeptide sequence 11Lys Pro Leu Lys
Pro1 51212PRTArtificial SequencePeptide sequence 12Ala Asp Ala Ala
Ala Ala Tyr Lys Ala Ala Lys Ala1 5 10139PRTArtificial
SequencePeptide sequence 13Gly Ala Gly Val Lys Pro Gly Lys Val1
5149PRTArtificial SequencePeptide sequence 14Gly Ala Gly Val Lys
Pro Gly Lys Val1 5159PRTArtificial SequencePeptide sequence 15Thr
Gly Ala Gly Val Lys Pro Lys Ala1 5167PRTArtificial SequencePeptide
sequence 16Gln Ile Lys Ala Pro Lys Leu1 5176PRTArtificial
SequencePeptide sequence 17Val Ala Pro Gly Val Gly1
5185PRTArtificial SequencePeptide sequence 18Val Pro Gly Val Gly1
51912PRTArtificial SequencePeptide sequence 19Ala Ala Ala Ala Ala
Ala Ala Lys Ala Ala Ala Lys1 5 102022PRTArtificial SequencePeptide
sequence 20Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Lys Ala Ala Lys
Tyr Gly1 5 10 15Ala Ala Ala Gly Leu Val 202116PRTArtificial
SequencePeptide sequence 21Glu Ala Ala Ala Lys Ala Ala Ala Lys Ala
Ala Lys Tyr Gly Ala Arg1 5 10 152217PRTArtificial SequencePeptide
sequence 22Glu Ala Gln Ala Ala Ala Ala Ala Lys Ala Ala Lys Tyr Gly
Val Gly1 5 10 15Thr2317PRTArtificial SequencePeptide sequence 23Ala
Ala Ala Ala Ala Lys Ala Ala Ala Lys Ala Ala Gln Phe Gly Leu1 5 10
15Val2429PRTArtificial SequencePeptide sequence 24Gly Gly Val Ala
Ala Ala Ala Lys Ser Ala Ala Lys Val Ala Ala Lys1 5 10 15Ala Gln Leu
Arg Ala Ala Ala Gly Leu Gly Ala Gly Ile 20 252515PRTArtificial
SequencePeptide sequence 25Gly Ala Leu Ala Ala Ala Lys Ala Ala Lys
Tyr Gly Ala Ala Val1 5 10 152614PRTArtificial SequencePeptide
sequence 26Ala Ala Ala Ala Ala Ala Ala Lys Ala Ala Ala Lys Ala Ala1
5 102712PRTArtificial SequencePeptide sequence 27Ala Ala Ala Ala
Lys Ala Ala Lys Tyr Gly Ala Ala1 5 102811PRTArtificial
SequencePeptide sequence 28Cys Leu Gly Lys Ala Cys Gly Arg Lys Arg
Lys1 5 10296PRTArtificial SequencePeptide
sequencemisc_feature(2)..(2)Xaa can be any naturally occurring
amino acid 29Val Xaa Pro Gly Val Gly1 5306PRTArtificial
SequencePeptide sequencemisc_feature(2)..(2)Xaa can be any
naturally occurring amino acid 30Glx Xaa Pro Gly Glx Gly1
53110PRTArtificial SequencePeptide sequencemisc_feature(2)..(2)Xaa
can be any naturally occurring amino acid 31Val Xaa Pro Ile Leu Val
Val Ile Leu Val1 5 10
* * * * *
References