U.S. patent application number 11/018343 was filed with the patent office on 2005-07-21 for tissue engineering scaffolds promoting matrix protein production.
This patent application is currently assigned to WILLIAM MARSH RICE UNIVERSITY. Invention is credited to Mann, Brenda K., West, Jennifer L..
Application Number | 20050158358 11/018343 |
Document ID | / |
Family ID | 22850332 |
Filed Date | 2005-07-21 |
United States Patent
Application |
20050158358 |
Kind Code |
A1 |
West, Jennifer L. ; et
al. |
July 21, 2005 |
Tissue engineering scaffolds promoting matrix protein
production
Abstract
The present invention provides tissue engineering scaffolds
capable of inducing extracellular matrix production by a cell
attached to the tissue engineering scaffolds, the tissue
engineering scaffolds comprising: a scaffold; a polymer tether
covalently coupled to the scaffold; and a TGF-.beta. molecule that
is covalently coupled to the polymer tether, wherein the TGF-.beta.
molecule is present at a concentration sufficient to elicit
production of extracellular matrix by the cell attached to the
tissue engineering scaffold without increasing cellular
proliferation of the attached cell.
Inventors: |
West, Jennifer L.;
(Pearland, TX) ; Mann, Brenda K.; (Upland,
CA) |
Correspondence
Address: |
Thomas M. Morrow
Baker Botts L.L.P.
910 Louisiana Street
Houston
TX
77002-4995
US
|
Assignee: |
WILLIAM MARSH RICE
UNIVERSITY
HOUSTON
TX
|
Family ID: |
22850332 |
Appl. No.: |
11/018343 |
Filed: |
December 21, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11018343 |
Dec 21, 2004 |
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09935168 |
Aug 21, 2001 |
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60226771 |
Aug 21, 2000 |
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Current U.S.
Class: |
424/423 ;
424/93.7 |
Current CPC
Class: |
A61P 19/00 20180101;
A61L 27/54 20130101; A61L 2300/414 20130101; A61P 17/02 20180101;
A61L 27/52 20130101; Y10S 530/816 20130101 |
Class at
Publication: |
424/423 ;
424/093.7 |
International
Class: |
A61K 045/00; A61K
009/70 |
Claims
We claim:
1. A tissue engineering scaffold capable of inducing extracellular
matrix production by a cell attached to the tissue engineering
scaffold without increasing cellular proliferation of the attached
cell, the tissue engineering scaffold comprising: a scaffold; a
polymer tether covalently coupled to the scaffold; and a TGF-.beta.
molecule that is covalently coupled to the polymer tether, wherein
the TGF-.beta. molecule is present at a concentration sufficient to
elicit production of extracellular matrix by the cell attached to
the tissue engineering scaffold without increasing cellular
proliferation of the attached cell.
2. The tissue engineering scaffold of claim 1 further comprising a
cell attached to the tissue engineering scaffold.
3. The tissue engineering scaffold of claim 2 wherein the cell is
attached to the tissue engineering scaffold by constraining the
cell within the scaffold.
4. The tissue engineering scaffold of claim 3 wherein the scaffold
is a hydrogel.
5. The tissue engineering scaffold of claim 2 wherein the cell is
selected from the group consisting of smooth muscle cells,
endothelial cells, fibroblasts, chondrocytes, and combinations
thereof.
6. The tissue engineering scaffold of claim 1 wherein the polymer
tether has a molecular weight of between about 200 and about
10,000.
7. The tissue engineering scaffold of claim 1 wherein the tether
has a molecular weight of between about 2,000 and about 6,000.
8. The tissue engineering scaffold of claim 1 wherein the scaffold
is formed from a biocompatible polymer selected from the group
consisting of a synthetic polymer, a natural polymer, an inorganic
material, and a combination thereof.
9. The tissue engineering scaffold of claim 1 wherein the scaffold
is formed from a biocompatible natural polymer, and wherein the
biocompatible natural polymer is selected from the group consisting
of a collagen, a hyaluronic acid, an albumin, and a combination
thereof.
10. The tissue engineering scaffold of claim 1 wherein the scaffold
is formed from a biocompatible inorganic material, and wherein the
biocompatible inorganic material is selected from the group
consisting of a hydroxyapatite, a silicone, and a combination
thereof.
11. The tissue engineering scaffold of claim 1 wherein the scaffold
is formed from a biocompatible synthetic polymer, and wherein the
biocompatible synthetic polymer is selected from the group
consisting of an ethylene vinyl acetate, a poly(meth)acrylate, and
a combination thereof.
12. The tissue engineering scaffold of claim 1 wherein the scaffold
is formed from a biocompatible biodegradable polymer.
13. The tissue engineering scaffold of claim 1 wherein the scaffold
is formed from a biocompatible biodegradable polymer, and wherein
the biocompatible biodegradable polymer is selected from the group
consisting of a polyhydroxyacid, a polylactic acid, a polyglycolic
acid, a polyanhydride, a polyorthoester, and a combination
thereof.
14. The tissue engineering scaffold of claim 1 wherein the scaffold
is formed from a biocompatible polymer that is not
biodegradable.
15. The tissue engineering scaffold of claim 1 wherein the scaffold
is formed from a biocompatible polymer that is a hydrogel.
16. The tissue engineering scaffold of claim 1 wherein the scaffold
is formed from a biocompatible polymer that is a polyethylene
glycol-diacrylate polymer hydrogel.
17. The tissue engineering scaffold of claim 1 wherein the scaffold
is formed from a biocompatible polymer that is an alginate
hydrogel.
18. The tissue engineering scaffold of claim 1 wherein the scaffold
is formed from a biocompatible polymer that is a malleable, ionic
hydrogel.
19. A tissue engineering scaffold capable of inducing extracellular
matrix production by a cell attached to the tissue engineering
scaffold without increasing cellular proliferation of the attached
cell, the tissue engineering scaffold comprising: a scaffold; a
polymer tether covalently coupled to the scaffold; and a TGF-.beta.
molecule that is covalently coupled to the polymer tether, wherein
the TGF-.beta. molecule is present at a concentration in the range
of from about 4.times.10.sup.-6 to about 4.times.10.sup.-3
nmol/mL.
20. The tissue engineering scaffold of claim 19, further comprising
a cell attached to the tissue engineering scaffold.
21. A tissue engineering scaffold capable of inducing extracellular
matrix production by a cell attached to the tissue engineering
scaffold without increasing cellular proliferation of the attached
cell, the tissue engineering scaffold comprising: a scaffold, at
least a portion of the scaffold comprising a hydrogel; a polymer
tether covalently coupled to the scaffold; and a TGF-.beta.
molecule that is covalently coupled to the polymer tether, wherein
the TGF-.beta. molecule is present at a concentration in the range
of from about 4.times.10.sup.-6 to about 4.times.10.sup.-3
nmol/mL.
22. The tissue engineering scaffold of claim 21, further comprising
a cell attached to the tissue engineering scaffold.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This Application is a divisional application of U.S. patent
application Ser. No. 09/935,168 entitled "Tissue Engineering
Scaffolds Promoting Matrix Protein Production," filed on Aug. 21,
2001, and claiming priority to U.S. application Ser. No.
60/226,771, filed Aug. 21, 2000.
SEQUENCE LISTING
[0002] The Sequence Listing in this application is identical to the
computer-readable copy of the Sequence Listing filed in U.S. patent
application Ser. No. 09/935,168 entitled "Tissue Engineering
Scaffolds Promoting Matrix Protein Production," filed on Aug. 21,
2001, and is incorporated herein by reference.
BACKGROUND
[0003] The present invention is generally in the field of improved
compositions for tissue engineering, specifically scaffolds
incorporating defined densities of matrix-enhancing molecules for
improving matrix protein production of cells, without inducing
excessive proliferation of the cells.
[0004] In fields where cell growth, maintenance, or production of
exogenous factors are important, such as in the field of tissue
engineering, cells are often grown on solid substrates or scaffolds
which provide a suitable substrate for cell adhesion and growth.
These scaffolds may be made of natural or synthetic materials.
[0005] Biomaterials developed for tissue engineering and
wound-healing applications need to support adequate cell adhesion
while being replaced by new tissue synthesized by those cells. In
order to maintain proper mechanical integrity of the tissue, the
cells must generate sufficient extracellular matrix (ECM).
Decreased ECM production by cells in tissue engineering scaffolds
may lead to reduced structural integrity of the developing
tissue.
[0006] In order to optimally promote adhesion to such materials,
researchers have investigated attachment of cell adhesion ligands,
such as the Arginine-Glycine-Aspartic acid (RGD) peptide, to
surfaces of biomaterials (Massia & Hubbell, Anal. Biochem.
187:292-301 (1990); Hern & Hubbell, J. Biomed. Mater. Res.
39:266-76 (1998); Dee, et al. J. Biomed. Mater. Res.
40:371-77(1998); Tong & Shoichet, J. Biomed. Mater. Res.
42:85-95 (1998); Zhang, et al., Biomaterials 20:1213-20 (1999)).
However, an increase in cell adhesion can adversely affect ECM
production (Mann, et al., Biomaterials 20:2281-86 (1999)). In
addition, there exists a substantial need to increase ECM
production, even in unmodified scaffolds, as the proteins in the
ECM largely determine the mechanical properties of the resultant
tissue and are often needed to replace the functions of a
biodegradable scaffold material. The mechanical properties of the
resultant tissue are particularly important in applications such as
tissue engineered vascular grafts and orthopedic tissue engineering
wherein failure can occur due to poor mechanical integrity.
[0007] Researchers have also attached growth factors such as
transforming growth factor (TGF) to a tissue engineering matrix via
a polymeric tether such as a polyethylene glycol. See WO 96/27,657,
"Cell Growth Substrates with Tethered Cell Growth Effector
Molecules." There are a number of references that TGF-.beta. can be
bound to or dispersed within a synthetic or natural polymeric
carrier for controlled release of active growth factor. See, e.g.,
Schroeder-Tefft, et al., "Collagen and heparin matrices for growth
factor delivery," Journal of Controlled Release 49(2-3), 291-98
(1997); Nicoll, et al., "In vitro characterization of transforming
growth factor-beta-1-loaded composites of biodegradable polymer and
mesenchymal cells," Cells and Materials 5(3), 231-44 (1995). EP
00/42,8541, "Collagen Wound Healing Matrices and Process for their
Production" to Collagen Corporation; U.S. Pat. No. 6,013,853,
"Continuous release polymeric implant carrier" issued to
Athanasiou, et al. Additional references relate to the use of
TGF-.beta. in tissue engineering scaffolds to enhance cell or
tissue growth or proliferation, particularly of bone. See EP
0616814, "Ceramic and Polymer-Based Compositions for Controlled
Release of Biologically Active TGF-.beta. to Bone Tissue, and
Implants Using the Compositions," by Bristol-Myers Squibb
Company.
[0008] However, none of these disclosures disclose how one can
achieve enhanced production of extracellular matrix, while not
increasing cellular proliferation.
[0009] It is therefore an object of the present invention to
provide tissue engineering scaffolds which promote formation of
ECM, to enhance the formation of tissue with good mechanical
properties, on and within the tissue engineering scaffold, i.e.,
with little or no increase in cellular proliferation.
SUMMARY
[0010] It has been found that matrix-enhancing molecules, such as
TGF-.beta., can be conjugated to or immobilized on scaffolds to
increase ECM production by cells. The matrix-enhancing molecule is
conjugated to a polymer, such as polyethylene glycol (PEG)
monoacrylate, for attachment to a tissue engineering or cell growth
scaffold, useful in not only tissue engineering but also for tissue
regeneration and wound-healing applications. The matrix-enhancing
molecule retains activity after attachment to the scaffold and
causes cells growing in or on the scaffold to increase ECM
production, even when the scaffold additionally contains cell
adhesion ligands. This increase in ECM production is believed to be
due to an increase in gene expression, not cell proliferation. The
increased ECM produced by the cells aids in maintaining the
integrity of the scaffold, particularly when the scaffold is
degradable, either by hydrolysis or by enzymatic degradation.
[0011] The examples demonstrate that matrix production by vascular
smooth muscle cells (SMCs) grown in a polymer scaffold, which was
formed from PEG hydrogels containing covalently bound adhesive
ligands, was increased in the presence of 4.times.10.sup.-5 nmol
TGF-.beta./mL tethered to the scaffold even when no TGF-.beta. was
present. At the same time, cell proliferation was not increased,
which is advantageous since increased proliferation of SMCs could
lead to narrowing of a vessel lumen. Tethering TGF-.beta. to the
polymer scaffold resulted in a significant increase in
extracellular matrix production over the same amount of soluble
TGF-.beta. (see Example 1, FIG. 3). This is most likely due to
internalization of the soluble TGF-.beta. by the cells or diffusion
of the soluble TGF-.beta. from the hydrogels, making the TGF-.beta.
unavailable to the cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a graph of matrix production per cell by SMCs
growing on glass surfaces covalently coupled with RGDS (SEQ ID
NO:1), VAPG (SEQ ID NO:2), KQAGDV (SEQ ID NO:3), or RGES (SEQ ID
NO:4) with and without TGF-.beta. in the media. The "+" indicates
that TGF-.beta. was added to the media, while the "-" indicates
that TGF-.beta. was absent from the media.
[0013] FIG. 2 is a graph of matrix production per cell by SMCs
growing on RGDS (SEQ ID NO:1)-modified glass surfaces with no
TGF-.beta., soluble TGF-.beta. or acryloyl-PEG-TGF-.beta. at
4.times.10.sup.-5 nmol/mL in the hydrogel. The control is with no
TGF-.beta. in the hydrogel.
[0014] FIG. 3 is a graph of hydroxyproline production per ng of DNA
by SMCs growing in RGDS (SEQ ID NO:1)-containing hydrogels with no
TGF-.beta., soluble TGF-.beta., or acryloyl-PEG-TGF-.beta. at
4.times.10.sup.-5 nmol/mL in the hydrogel. The control is with no
TGF-.beta. in the hydrogel.
[0015] FIG. 4 is a graph of matrix production by SMCs growing on
RGDS (SEQ ID NO:1)-modified glass surfaces, as percent of control,
as a function of TGF-.beta. concentration (0, 1, and 5 ng/mL).
[0016] FIG. 5 is a graph of matrix production, as percent of
control, by auricular chondrocytes growing on tissue culture
polystyrene in the presence of varying amounts of TGF-.beta. (0,
0.1, 1, 5, 10, 25, and 100 ng/mL) in the media. The control is with
no TGF-.beta. in the media.
[0017] FIG. 6 is a graph of cell number, as percent of control, by
auricular chondrocytes growing on tissue culture polystyrene in the
presence of varying amounts of TGF-.beta. (0, 0.1, 1, 5, 10, 25,
and 100 ng/mL) in the media. The control is with no TGF-.beta. in
the media.
[0018] FIG. 7 is a graph of matrix production, as percent of
control, by auricular chondrocytes and aortic smooth muscle cells
growing on tissue culture polystyrene in the presence of 0 and 50
.mu.g/mL ascorbic acid in the media. The control is with no
ascorbic acid in the media.
[0019] FIG. 8 is a graph of cell number, as percent of control, by
auricular chondrocytes and aortic smooth muscle cells growing on
tissue culture polystyrene in the presence of 0 and 50 .mu.g/mL
ascorbic acid in the media. The control is with no ascorbic acid in
the media.
DESCRIPTION
[0020] Tissue engineering is performed using a scaffold material
that allows for attachment of cells. The scaffold material contains
a matrix-enhancing molecule. As described herein, the
matrix-enhancing molecule should promote the production of
extracellular matrix proteins, but should not promote cell
proliferation.
[0021] Scaffold Materials
[0022] In the preferred embodiment, the scaffold is formed of
synthetic or natural polymers, although other materials such as
hydroxyapatite, silicone, and other inorganic materials can be
used. The scaffold may be biodegradable or non-biodegradable.
[0023] There are a number of biocompatible polymers, both
degradable and non-degradable. Representative synthetic
non-biodegradable polymers include ethylene vinyl acetate and
poly(meth)acrylate. Representative biodegradable polymers include
polyhydroxyacids such as polylactic acid and polyglycolic acid,
polyanhydrides, polyorthoesters, and copolymers thereof. Natural
polymers include collagen, hyaluronic acid, and albumin.
[0024] A preferred material is a hydrogel. A particularly preferred
hydrogel-forming material is a polyethylene glycol-diacrylate
polymer, which is photopolymerized. Other hydrogel materials
include calcium alginate and certain other polymers that can form
ionic hydrogels that are malleable and can be used to encapsulate
cells.
[0025] Formation of Scaffolds
[0026] Scaffolds can be formed in situ or in vitro. In a preferred
embodiment for formation of joint linings, the scaffold material is
sprayed in a dilute solution onto the joint, then polymerized, so
that the polymer forms a hydrogel coating bonded onto the surface
of the joint tissues. Cells can be dispersed within the polymer, or
seeded onto the polymeric matrix. Scaffolds may also be formed of
fibers of polymer, woven or non-woven into meshes that can be used
to support cell attachment and growth. These scaffolds can be
formed by casting, weaving, salt leaching, spinning, or molding. In
still another embodiment, scaffolds can be formed using molds
formed by micromachining and photolithographic techniques, where
the cells can be seeded into the scaffold while in the molds or
after removal of the scaffold. In a preferred embodiment, a liquid
cell-polymer solution is placed in a mold and photopolymerized,
converting the liquid to a hydrogel with the cells seeded within
the hydrogel.
[0027] The scaffolds can be seeded at the time of or before
implantation at a site where tissue is desired. Meshes should
preferably be sufficiently open to allow free diffusion of
nutrients and gases throughout the scaffold.
[0028] Matrix-Enhancing Molecules
[0029] Matrix-enhancing molecules which promote increased
production of ECM can be attached to the scaffold material to
induce production of matrix proteins, such as glycoproteins,
elastin, and collagen, without substantially increasing cell
proliferation. These matrix-enhancing molecules include TGF-.beta.,
angiotensin II, insulin-like growth factors, and ascorbic acid.
[0030] TGF-.beta. is known to increase production of extracellular
matrix proteins by vascular SMCs growing in culture (Amento, et
al., Arterioscler. Thromb. 11:1223-30 (1991); Lawrence, et al., J.
Biol. Chem. 269:9603-09 (1994); Plenz, et al., Atherosclerosis
144:25-32 (1999)). TGF-.beta., through production by SMCs naturally
during vessel injury or by gene transfer, can also increase ECM
production by SMCs in vivo (Majesky, et al., J. Clin. Invest.
88:904-10 (1991); Nabel, et al., Proc. Natl. Acad. Sci. USA
90:10759-63 (1993)). Cultured fibroblasts have also been shown to
increase collagen synthesis (Clark, et al., J. Cell Sci.
108:1251-61 (1995); Eickelberg, et al., Am. J. Physiol. 276:L814-24
(1999)) and proteoglycan synthesis (Heimer, et al., J. Mol. Cell
Cardiol. 27:2191-98 (1995)) in the presence of TGF-.beta.. Further,
topical delivery of TGF-.beta. (Puolakkainen, et al., J. Surg. Res.
58:321-29 (1995)) and delivery to TGF-.beta. through a collagen
scaffold (Pandit, et al., J. Invest. Surg. 12:89-100 (1999)) have
been shown to enhance wound healing.
[0031] All of the above-referenced studies have examined effects in
the presence of soluble TGF-.beta.. As demonstrated by the
following examples, it has now been shown that tethered TGF-.beta.
can also be used to induce formation of ECM by cells, including
cells such as smooth muscle cells and chondrocytes.
[0032] Tethers
[0033] For the matrix-enhancing molecules to induce formation of
ECM, it is necessary for the molecule to be tethered to the
scaffold by a tether. These tethers have a molecular weight of
preferably between about 200 and 10,000, most preferably between
about 2,000 and 6,000. The tether is preferably a linear polymer,
such as polyethylene glycol. The matrix-enhancing molecule may be
coupled to the tether or, for that matter, to the scaffold
material, by any method known to those of skill in the art,
preferably covalently coupled using a reagent such as
N-hydroxysuccinimide, carbodiimide, diisocyanate,
carbonyldiimidazole, or tosyl chloride.
[0034] Density of Matrix-Enhancing Materials
[0035] The density of the matrix-enhancing materials is important
in eliciting ECM production with little or no cellular
proliferation. The amount of ECM production that is most desirable
is that which results in formation of tissue with good mechanical
properties, on and within the tissue engineering scaffold. The
optimal density will depend on the type of cells to be attached to
the scaffold. In the case of TGF-.beta., optimal concentrations to
induce ECM production is in the range of between one and five ng
TGF-.beta./mL for aortic smooth muscle cells and between 5 and 100
ng TGF-.beta./mL for auricular chondrocytes, which is equivalent to
between 4.times.10.sup.-6 and 4.times.10.sup.-3 nmol/mL.
[0036] Source of Cells
[0037] Cells can be obtained directly from a donor, from a culture
of cells from a donor, or from established cell culture lines. In
the preferred embodiment, cells of the same species and, preferably
having the same or a similar immunological profile, are obtained by
biopsy, either from the patient or a close relative, which can then
be grown in culture using standard conditions. If cells that are
likely to elicit an immune reaction are used, such as human muscle
cells from an immunologically distinct individual, then the
recipient can be immunosuppressed as needed, for example, using a
schedule of steroids and other immunosuppressant drugs, such as
cyclosporine.
[0038] In the preferred embodiments, cells are obtained directly
from a donor, washed and implanted directly in combination with the
polymeric material. The cells are cultured using techniques known
to those skilled in the art of tissue culture. Cells obtained by
biopsy are harvested and cultured, passaging as necessary to remove
contaminating cells.
[0039] Preferred cells for formation of vascular tissue include
smooth muscle cells, endothelial cells, and fibroblasts. Preferred
cells for formation of connective tissue include chondrocytes,
fibroblasts, and other types of cells that differentiate into bone
or cartilage.
[0040] Methods of Using the Scaffolds
[0041] The scaffolds are used to produce new tissue, such as
vascular tissue, cartilage, tendons, and ligaments. The scaffold is
typically seeded with the cells; the cells are cultured; and then
the scaffold implanted. Alternatively, as noted above, the scaffold
is sprayed into or onto a site such as a joint lining, and seeded
with cells, and then the site is closed surgically. Liquid
polymer-cell suspensions can also be injected into a site, such as
within a joint, where the material may be polymerized.
[0042] Applications include the repair and/or replacement of organs
or tissues, such as blood vessels, cartilage, joint linings,
tendons, or ligaments, or the creation of tissue for use as
"bulking agents," which are typically used to block openings or
lumens, or to shift adjacent tissue, as in treatment of reflux.
[0043] The present invention will be further understood by
reference to the following non-limiting examples.
EXAMPLES
Example 1
Comparison of Effect of Soluble and Bound TGF-.beta. in Combination
with PEG-Diacrylate Hydrogel
[0044] It was determined whether TGF-.beta. can counteract the
decrease in ECM synthesis caused by immobilized cell adhesion
ligands. SMCs were grown on both peptide-modified glass substrates
and in hydrogels containing tethered cell adhesion ligands.
Further, TGF-.beta. was covalently tethered to a polymer scaffold
and shown that it retains its ability to increase ECM
production.
Materials & Methods
[0045] Cell Maintenance
[0046] Chemicals were obtained from Sigma Chemical Co. (St. Louis,
Mo.) unless otherwise stated. SMCs from the thoracic aorta of
Wistar-Kyoto rats were isolated and characterized as previously
described by Scott-Burden, et al., Hypertension 13:295-05 (1989).
Human aortic smooth muscle cells (HASMCs) were obtained from
Clonetics (San Diego, Calif.). Both SMCs and HASMCs were maintained
on Minimal Essential Medium Eagle supplemented with 10% fetal
bovine serum (FBS; Bio Whittaker, Walkersville, Md.), 2 mM
L-glutamine, 500 units penicillin and 100 mg/L streptomycin (MEM).
Cells were incubated at 37.degree. C. in a 5% CO.sub.2
environment.
[0047] Surface Modification
[0048] Peptides used in this study were RGDS (SEQ ID NO:1), VAPG
(SEQ ID NO:2), and KQAGDV (SEQ ID NO:3) (Research Genetics,
Huntsville, Ala.). RGES (SEQ ID NO:4) was used as a non-adhesive
control peptide. The peptides were acetylated and coupled to
aminophase glass slides as previously described by Mann, et al.,
Biomaterials 20:2281-86 (1999). Briefly, aminophase slides were
prepared by incubating glass slides with
3-aminopropyltriethoxysilane in dry acetone at 37.degree. C.
overnight. Acetylated peptides were then coupled to the slides
using 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDAC)
chemistry. Slides were sterilized under UV light overnight prior to
use.
[0049] Preparation of Acryloyl-PEG-TGF-.beta.
[0050] TGF-.beta. was conjugated to polyethylene glycol (PEG) by
reacting TGF-.beta. with acryloyl-PEG-N-hydroxysuccinimide
(acryloyl-PEG-NHS, 3,400 Da; Shearwater Polymers, Huntsville, Ala.)
in 50 mM TRIS buffer (pH 8.5) for 2 hours. The mixture was then
lyophilized and stored frozen. Gel permeation chromatography
equipped with UV/Vis (260 nm) and evaporative light scattering
detectors was used to analyze the resulting acryloyl-PEG-TGF-.beta.
and PEG standards (Polymer Laboratories, Amherst, Mass.).
[0051] Evaluation of Matrix Protein Production on Surfaces
[0052] Matrix protein production was evaluated as previously
described by Mann (1999). Suspensions of SMCs were prepared in MEM
supplemented with 5 .mu.g/mL ascorbic acid at a concentration of
40,000 cells/mL. For samples receiving TGF-.beta., 0.04 pmol/L (1
ng/mL) unmodified TGF-.beta. or 0.04 pmol/mL
acryloyl-PEG-TGF-.beta. was added to the media. Cell suspensions to
be used for measurement of ECM protein production were also
supplemented with 1 .mu.Ci/mL .sup.3H-glycine (40 Ci/mmol). The
glass slides were attached to FlexiPerm strips in QuadriPerm Cell
Culture Vessels (Heraeus, Osterode am Harz, Germany) to create
eight wells (1.11.times.0.79.times.0.79 cm) on each slide. Four of
the wells on each slide were utilized to measure ECM production,
while the remaining four wells were utilized for cell number
determination and were cultured in the absence of .sup.3H-glycine.
Cell number was determined after 2 days of culture by preparing
single cell suspensions using trypsin and counting cells using a
Coulter counter (Multisizer #0646, Coulter Electronics, Hialeah,
Fla.).
[0053] To evaluate synthesis of ECM proteins, the cell growth media
was supplemented with .sup.3H-glycine, as described above. Two days
following .sup.3H-glycine addition, cells were removed
non-enzymatically with 25 mM ammonium hydroxide, then rinsed with
70% ethanol and dried. This process leaves intact the ECM
elaborated by cells during culture (Jones, et al., Proc. Natl.
Acad. Sci. USA 76:353-57 (1979)). Sequential enzyme digestion was
used as previously described by Mann et al. (1999) to determine the
gross composition of the ECM proteins. Briefly, the ECM was
digested first with trypsin, followed by elastase, and then
collagenase. After the final enzymatic digestion, any material
remaining on the substrate was dissolved by incubation with 1 N
NaOH. Aliquots were taken from each step of the digest for
scintillation counting.
[0054] Preparation of Acryloyl-PEG-RGDS (SEQ ID NO: 1)
[0055] RGDS (SEQ ID NO:1) was conjugated to acryloyl-PEG-NHS in the
same manner as TGF-.beta..
[0056] Preparation of PEG-Diacrylate
[0057] PEG-diacrylate was prepared by combining 0.1 mmol/mL dry PEG
(6,000 Da; Fluka, Milwaukee, Wis.), 0.4 mmol/mL acryloyl chloride,
and 0.2 mmol/mL triethylamine in anhydrous dichloromethane and
stirring under argon overnight. The resulting PEG-diacrylate was
then precipitated with ether, filtered, and dried in a vacuum
oven.
[0058] Preparation of Hydrogels
[0059] Hydrogels were prepared by combining 0.4 g/mL
PEG-diacrylate, 1.4 .mu.mol/mL acryloyl-PEG-RGDS (SEQ ID NO:1), and
0.3 mmol/mL triethanolamine in 10 mM HEPES-buffered saline (pH 7.4,
HBS). This aqueous polymer solution was sterilized by filtration
(0.8 .mu.m prefilter and 0.2 .mu.m filter) and added to an equal
volume of a suspension of SMCs at 2.times.10.sup.6 cells/mL, such
that the resulting polymer-cell solution contained 1.times.10.sup.6
cells/mL. For hydrogels containing TGF-.beta., 0.04 pmol/mL (1
ng/mL) unmodified TGF-.beta. or 0.04 pmol/mL
acryloyl-PEG-TGF-.beta. was added to the polymer-cell solution.
Then, 40 .mu.l of 2,2-dimethyl-2-phenyl-acetophenone in
N-vinylpyrrolidone (600 mg/mL) was added, and 0.25 mL of the
solution was placed in a disk-shaped mold (20 mm diameter, 2 mm
thickness). This liquid polymer-cell solution was then exposed to
UV light (365 nm, 10 mW/cm.sup.2) for 20 seconds to convert the
liquid polymer-cell solution to a hydrogel with homogeneously
seeded cells. Hydrogels were incubated in MEM containing 10% FBS
for 7 days at 37.degree. C. with 5% CO.sub.2. Media was changed
every 3 days.
[0060] DNA and Hydroxyproline Determination in Hydrogels
[0061] After 7 days of culture, hydrogels were removed from the
culture media, weighed, and digested with 1 mL 0.1 N NaOH overnight
at 37.degree. C. Digested hydrogels were then neutralized with 1 mL
0.1 N HCl. DNA content of the digested, neutralized hydrogels was
determined using a fluorescent DNA binding dye, Hoechst 33258
(Molecular Probes, Eugene, Oreg.). Fluorescence of the samples was
determined using a fluorometer (VersaFluor, Bio-Rad Laboratories,
Hercules, Calif.) with excitation filter at 360 nm and emission
filter at 460 nm, and compared to fluorescence of calf thymus DNA
standards.
[0062] Hydroxyproline concentration was determined by oxidation
with chloramine T (ICN Biomedicals, Aurora, Ohio) and development
with p-dimethylaminobenzaldehyde (ICN Biomedicals) (Woessner, et
al., Arch. Biochem. Biophys. 93:440-47 (1961)). Hydroxyproline is a
marker for collagen production, and thus was used as an indication
of matrix synthesis.
[0063] Mechanical Testing of Hydrogels
[0064] Hydrogels were prepared as described above, except that 2 mL
of the liquid polymer-cell solution was placed in a 3 mm thick
rectangular mold (42 mm.times.14 mm). For this experiment, HASMCs
were used at a final cell density of 3.5.times.10.sup.5 cells/mL.
Hydrogels contained either no TGF-.beta. or 0.04 pmol/mL
acryloyl-PEG-TGF-.beta..
[0065] Following photopolymerization, the hydrogels were placed in
QuadriPerm Cell Culture Vessels with 10 mL media containing 10% FBS
and incubated at 37.degree. C. with 5% CO.sub.2. Media was changed
every 3 days. After 7 days of culture, the hydrogels were cut into
3 sections (14 mm.times.14 mm), and mechanical testing was
performed using a Vitrodyne V-1000 Universal Tester (Chatillon,
Greensboro, N.C.) at a strain rate of 100 .mu.m/s using a 150 g
loading cell.
[0066] Statistical Analysis
[0067] Data sets were compared using two-tailed, unpaired t-tests.
P-Values less than 0.05 were considered to be significant.
Results
[0068] The goal of the current study was to determine whether
TGF-.beta. can enhance the rate of ECM synthesis by cells grown on
or in biomaterials, particularly materials that have been modified
with cell adhesion ligands. FIG. 1 shows the matrix protein
production on a per cell basis for cells grown with either no
TGF-.beta. or 0.04 pmol/mL (1 ng/mL) soluble TGF-.beta. in the
media. With no TGF-.beta. in the media, more matrix was produced by
cells growing on the non-adhesive control, RGES (SEQ ID NO:4), than
on the adhesive peptides. When TGF-.beta. was added to the media,
matrix production increased on the adhesive surfaces over that
produced when no TGF-.beta. was added (224% increase on RGDS (SEQ
ID NO:1) surfaces, 20% increase on VAPG (SEQ ID NO:2) surfaces,
104% increase on KQAGDV (SEQ ID NO:3) surfaces). Matrix production
on the RGDS (SEQ ID NO:1) and KQAGDV (SEQ ID NO:3) modified
surfaces in the presence of TGF-.beta. increased over that seen
with the non-adhesive control.
[0069] Cells seeded onto RGDS (SEQ ID NO:1)-modified glass surfaces
were also grown in the presence of 0.04 pmol/mL
acryloyl-PEG-TGF-.beta. to determine if TGF-.beta. could be
covalently bound to a polymer (covalently attached to a soluble
polymer chain but not tethered to a three-dimensional structure)
and retain its ability to increase ECM production. FIG. 2 shows the
matrix production by cells grown with no TGF-.beta., soluble
TGF-.beta., or acryloyl-PEG-TGF-.beta. in the media. SMCs produced
greater amounts of matrix in the presence of either soluble or
polymer-conjugated TGF-.beta. over that produced in the absence of
TGF-.beta.. However, less matrix was produced when
polymer-conjugated TGF-.beta. was used than when unmodified
TGF-.beta. was used.
[0070] SMCs were then homogeneously seeded into polyethylene glycol
(PEG) hydrogels containing covalently tethered RGDS (SEQ ID NO:1).
The hydrogels contained either no TGF-.beta., unmodified (soluble)
TGF-.beta., or PEG-tethered TGF-.beta.. In these photopolymerized
hydrogels, the tethered peptides of TGF-.beta. are covalently bound
to the hydrogel structure via a highly flexible PEG chain. This
gives the tethered moieties conformational freedom to interact with
their receptors while causing them to be retained in the hydrogel
material. After 7 days of culture, the hydrogels were digested and
assayed for DNA and hydroxyproline. Since hydroxyproline is a
marker for collagen, it is an indication of how much extracellular
matrix has been produced.
[0071] The results for cells grown in the presence of 0.04 pmol/mL
of TGF-.beta. are presented in FIG. 3. More hydroxyproline, and
thus more collagen, was produced by SMCs grown in the presence of
either soluble or tethered TGF-.beta. than when no TGF.beta. was
present. Additionally, significantly more hydroxyproline was
produced when TGF-.beta. was tethered onto the hydrogels than when
soluble TGF-.beta. was used.
[0072] Additionally, the mechanical properties of hydrogels made
with and without TGF-.beta. were examined. Young's modulus, a
measure of the stiffness of the scaffold, was significantly higher
when TGF-.beta. was tethered to the scaffolds than when no
TGF-.beta. was used (66.6.+-.3.7 kPa with tethered TGF-.beta.
versus 58.5.+-.1.8 kPa with no TGF-.beta., p=0.03).
Example 2
Dose Response of Aortic Smooth Muscle Cells to TGF-.beta.
[0073] Aortic smooth muscle cells were grown on aminophase glass
that had 0.5 nmol/cm.sup.2 RGDS (SEQ ID NO:1) covalently coupled to
the glass. TGF-.beta. was added to the media at 0, 1, or 5 ng/mL
(0, 4.times.10.sup.-5, 2.times.10.sup.-4 nmol/mL). ECM protein
production by the cells over a 2-day time period was determined by
examining the amount of .sup.3H-glycine incorporated into the ECM
elaborated by the cells.
[0074] As seen in FIG. 4, ECM protein production per cell (% of
control) was increased when TGF-.beta. was added to the media at
both 1 and 5 ng/mL. Further, cell numbers did not increase over the
2 days, despite changes in matrix production per cell, indicating
that the presence of TGF-.beta. did not increase proliferation of
the SMCs, as seen in the hydrogels.
Example 3
Dose Response of Auricular Chondrocytes to TGF-.beta.
[0075] Auricular chondrocytes were grown on tissue culture
polystyrene with varying amounts of TGF-.beta. added to the media:
0, 1, 5, 10, 25, or 100 ng/mL. ECM protein production by the cells
over a 2-day time period was determined by examining the amount of
.sup.3H-glycine incorporated into the ECM elaborated by the
cells.
[0076] As seen in FIG. 5, ECM protein production per cell was
increased when TGF-.beta. was added to the media at concentrations
above 1 ng/mL, with an optimal concentration of 25 ng/mL
(1.times.10.sup.-3 nmol/mL). Further, cell numbers did not increase
over the 2 days (see FIG. 6), despite changes in matrix production
per cell, indicating that the presence of TGF-.beta. did not
increase proliferation of the chondrocytes.
Example 4
Increase in Matrix Production in the Presence of Ascorbic Acid
[0077] Aortic smooth muscle cells and auricular chondrocytes were
grown on tissue culture polystyrene with and without 50 .mu.g/mL
ascorbic acid added to the media. ECM protein production by the
cells over a 2-day time period was determined by examining the
amount of .sup.3H-glycine incorporated into the ECM elaborated by
the cells.
[0078] As seen in FIG. 7, ECM protein production per cell was
increased in the presence of ascorbic acid for both SMCs (light
gray) and chondrocytes (dark gray). Further, cell numbers did not
increase over the 2 days (FIG. 8), despite changes in matrix
production per cell, indicating that the presence of ascorbic acid
did not increase proliferation of the smooth muscle cells (light
gray) or the chondrocytes (dark gray).
[0079] Modifications and variations of these scaffolds and method
for preparation and use thereof will be obvious to those skilled in
the art and are intended to be encompassed by the following claims.
Sequence CWU 1
1
4 1 4 PRT artificial sequence cell adhesion ligand 1 Arg Gly Asp
Ser 1 2 4 PRT artificial sequence cell adhesion ligand 2 Val Ala
Pro Gly 1 3 6 PRT artificial sequence cell adhesion ligand 3 Lys
Gln Ala Gly Asp Val 1 5 4 4 PRT artificial sequence cell adhesion
ligand 4 Arg Gly Glu Ser 1
* * * * *