U.S. patent application number 15/347595 was filed with the patent office on 2017-03-23 for method for preparing porous scaffold for tissue engineering, cell culture and cell delivery.
The applicant listed for this patent is INSERM (Institut National de la Sante et de la Recherche Medicale), Universite Paris 7 - Denis Diderot. Invention is credited to Catherine Le Visage, Didier Letourneur.
Application Number | 20170080123 15/347595 |
Document ID | / |
Family ID | 38925478 |
Filed Date | 2017-03-23 |
United States Patent
Application |
20170080123 |
Kind Code |
A1 |
Le Visage; Catherine ; et
al. |
March 23, 2017 |
Method for Preparing Porous Scaffold for Tissue Engineering, Cell
Culture and Cell Delivery
Abstract
The present invention relates to a method for preparing a porous
scaffold for tissue engineering. It is another object of the
present invention to provide a porous scaffold obtainable by the
method as above described, and its use for tissue engineering, cell
culture and cell delivery. The method of the invention comprises
the steps consisting of: a) preparing an alkaline aqueous solution
comprising an amount of at least one polysaccharide, an amount of a
cross-linking agent and an amount of a porogen agent b)
transforming the solution into a hydrogel by placing said solution
at a temperature from about 4.degree. C. to about 80.degree. C. for
a sufficient time to allow the cross-linking of said amount of
polysaccharide and c) submerging said hydrogel into an aqueous
solution d) washing the porous scaffold obtained at step c).
Inventors: |
Le Visage; Catherine;
(Paris, FR) ; Letourneur; Didier; (Chatenay
Malabry, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSERM (Institut National de la Sante et de la Recherche
Medicale)
Universite Paris 7 - Denis Diderot |
Paris
Paris Cedex |
|
FR
FR |
|
|
Family ID: |
38925478 |
Appl. No.: |
15/347595 |
Filed: |
November 9, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12681682 |
Apr 5, 2010 |
9522218 |
|
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PCT/EP08/63671 |
Oct 10, 2008 |
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15347595 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 27/56 20130101;
C08J 2305/10 20130101; A61L 27/3804 20130101; C08B 37/003 20130101;
A61L 27/20 20130101; A61L 27/52 20130101; C08J 2305/12 20130101;
C08B 37/0018 20130101; C08J 9/28 20130101; A61L 27/507 20130101;
C08J 2201/0504 20130101; C08J 2305/04 20130101; C08B 37/0021
20130101; C08J 2305/00 20130101; A61L 27/54 20130101; C12N 2533/74
20130101; C08B 37/0072 20130101; C12N 2533/72 20130101; C08J
2201/026 20130101; A61L 27/3847 20130101; C08J 2207/10 20130101;
C08B 37/0075 20130101; C08B 37/0084 20130101; C08J 9/08 20130101;
C08J 9/283 20130101; C08J 2305/08 20130101; A61P 43/00 20180101;
C12N 2533/70 20130101; C12N 5/0068 20130101; A61L 27/3852 20130101;
A61L 27/3834 20130101; C08B 37/0063 20130101; C08J 2201/0484
20130101; C08J 2205/022 20130101; C08J 2305/02 20130101; C08B
37/0039 20130101; C08B 37/0054 20130101 |
International
Class: |
A61L 27/20 20060101
A61L027/20; A61L 27/38 20060101 A61L027/38; A61L 27/50 20060101
A61L027/50; A61L 27/52 20060101 A61L027/52; A61L 27/56 20060101
A61L027/56; A61L 27/54 20060101 A61L027/54 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 11, 2007 |
EP |
07301452.4 |
Claims
1.-19. (canceled)
20. A porous scaffold obtainable by a method comprising the
following steps: a) preparing an alkaline aqueous solution
comprising an amount of at least one polysaccharide selected from
the group consisting of dextran, agar, pullulan, inulin, heparin,
fucoidan, and mixtures thereof; an amount of a covalent
cross-linking agent selected from the group consisting of trisodium
trimetaphosphate (STMP), phosphorus oxychloride (POCl.sub.3),
epichlorohydrin, formaldehydes, and glutaraldehydes; and an amount
of a porogen agent selected from the group consisting of ammonium
carbonate, ammonium bicarbonate, calcium carbonate, sodium
carbonate, and sodium bicarbonate and mixtures thereof; b)
transforming the solution into a hydrogel by placing said solution
at a temperature from about 4.degree. C. to about 80.degree. C. for
a sufficient time to allow the cross-linking of said amount of
polysaccharide; c) submerging said hydrogel into an aqueous acidic
solution to form pores; and d) washing the porous scaffold obtained
at step c), wherein the porous scaffold is a hydrogel; the porogen
agent is dissolved in step a); the weight ratio of the
polysaccharide to the porogen agent is in the range from 6:1 to
1:1; and wherein the alkaline aqueous solution in a) does not
include a surfactant.
21. The porous scaffold according to claim 20 wherein the weight
ratio of the polysaccharide to the cross-linking agent is in the
range from 15:1 to 1:1.
22. The porous scaffold according to claim 20 wherein the solution
of step a) is poured in a mould before step b).
23. The porous scaffold according to claim 20 wherein said scaffold
is shaped.
24. The porous scaffold according to claim 20 wherein the porogen
agent used in step a) is selected from the group consisting of
ammonium carbonate, calcium carbonate, sodium carbonate, sodium
bicarbonate, and mixtures thereof.
25. The porous scaffold according to claim 20 wherein the size of
the pores is comprised between 1 .mu.m and 500 .mu.m.
26. The porous scaffold according to claim 20 wherein the porosity
is in the range from 4% to 50%.
27. The porous scaffold according to claim 20 loaded with an amount
of cells.
28. The porous scaffold according to claim 27 wherein the cells are
selected in the group consisting of yeast cells, mammalian cells,
insect cells, and plant cells.
29. The porous scaffold according to claim 28 wherein mammalian
cells are selected from the group consisting of chondrocytes;
fibrochondrocytes; osteocytes; osteoblasts; osteoclasts;
synoviocytes; bone marrow cells; epithelial cells, hepatocytes,
mesenchymal cells; stromal cells; muscle cells, stem cells;
embryonic stem cells; precursor cells derived from adipose tissue;
peripheral blood progenitor cells; stem cells isolated from adult
tissue; and genetically transformed cells.
30. The porous scaffold according to claim 20 for tissue
engineering, cell culture and cell delivery.
31. A vascular substitute made with a scaffold of claim 20.
32. Cartilage or bone implants made with a scaffold of claim
20.
33. A method for evaluating the toxicity and/or pharmacology of a
product comprising the use of a scaffold of claim 20.
34. A controlled release system of an active agent made with a
scaffold of claim 20.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method for preparing a
porous scaffold for tissue engineering. It is another object of the
present invention to provide a porous scaffold obtainable by the
method as above described, and its use for tissue engineering, cell
culture and cell delivery
BACKGROUND OF THE INVENTION
[0002] Tissue engineering is generally defined as the creation of
tissue or organ equivalents by seeding of cells onto or into a
scaffold suitable for implantation. The scaffolds must be
biocompatible and cells must be able to attach and proliferate on
the scaffolds in order for them to form tissue or organ
equivalents. These scaffolds may therefore be considered as
substrates for cell growth either in vitro or in vivo.
[0003] The attributes of an ideal biocompatible scaffold would
include the ability to support cell growth either in vitro or in
vivo, the ability to support the growth of a wide variety of cell
types or lineages, the ability to be endowed with varying degrees
of flexibility or rigidity required, the ability to have varying
degrees of biodegradability, the ability to be introduced into the
intended site in vivo without provoking secondary damage, and the
ability to serve as a vehicle or reservoir for delivery of drugs or
bioactive substances to the desired site of action.
[0004] A number of different scaffold materials have been utilized,
for guided tissue regeneration and/or as biocompatible surfaces.
Biodegradable polymeric materials are preferred in many cases since
the scaffold degrades over time and eventually the cell-scaffold
structure is replaced entirely by the cells. Among the many
candidates that may serve as useful scaffolds claimed to support
tissue growth or regeneration, are included gels, foams, sheets,
and numerous porous particulate structures of different forms and
shapes.
[0005] Among the manifold natural polymers which have been
disclosed to be useful for tissue engineering or culture, one can
enumerate various constituents of the extracellular matrix
including fibronectin, various types of collagen, and laminin, as
well as keratin, fibrin and fibrinogen, hyaluronic acid, heparin
sulfate, chondroitin sulfate and others.
[0006] Other common polymers that were used include
poly(lactide-co-glycolide) (PLG). PLG are hydrolytically degradable
polymers that are FDA approved for use in the body and mechanically
strong (Thomson R C, Yaszemski M J, Powers J M, Mikos A G.
Fabrication of biodegradable polymer scaffolds to engineer
trabecular bone. J Biomater Sci Polym Ed. 1995; 7(1):23-38; Wong W
H. Mooney D J. Synthesis and properties of biodegradable polymers
used as synthetic matrices for tissue engineering. In: Atala A,
Mooney D J, editors; Langer R, Vacanti J P, associate editors.
Synthetic biodegradable polymer scaffolds. Boston: Birkhauser:
1997. p. 51-82). However, they are hydrophobic and typically
processed under relatively severe conditions, which make factor
incorporation and entrapment of viable cells potentially a
challenge.
[0007] As an alternative, a variety of hydrogels, a class of highly
hydrated polymer materials (water content higher than 30% by
weight), have been used as scaffold materials. They are composed of
hydrophilic polymer chains, which are either synthetic or natural
in origin. The structural integrity of hydrogels depends on
cross-links formed between polymer chains via various chemical
bonds and physical interactions.
[0008] For example, document U.S. Pat. No. 6,586,246 B1 has
disclosed a method for preparing a porous hydrogel scaffold which
may be used as supports for tissue engineering or culture matrices.
The method of the document comprises the steps consisting of a)
dissolving a biodegradable synthetic polymer in an organic solvent
to prepare a polymeric solution of high viscosity b) adding a
porogen agent to this solution; c) casting the polymer into a mould
d) removing the organic solvent e) submerging the organic
solvent-free polymer/salt gel slurry in a hot aqueous solution or
acidic solution to cause the salt to effervesce at room temperature
to form the porous scaffold. However, this method of preparation of
a porous hydrogel involves the use of an organic solvent with a
synthetic polymer which renders the method according to this
invention weakly compatible with biological and therapeutic
purposes.
[0009] Therefore there is still an existing need in the art to
develop a method for preparing porous scaffold matrices that can be
used for biological and therapeutic purposes.
SUMMARY OF THE INVENTION
[0010] Therefore, it is an object of the present invention to
provide a method for preparing a porous scaffold which comprises
the steps consisting of: [0011] a) preparing an alkaline aqueous
solution comprising an amount of at least one polysaccharide, an
amount of a cross-linking agent and an amount of a porogen agent.
[0012] b) transforming the solution into a hydrogel by placing said
solution at a temperature from about 4.degree. C. to about
80.degree. C. for a sufficient time to allow the cross-linking of
said amount of polysaccharide and [0013] c) submerging said
hydrogel into an aqueous solution [0014] d) washing the porous
scaffold obtained at step c).
[0015] It is another object of the present invention to provide a
porous scaffold obtainable by the method as above described.
[0016] It is still further an object of the present invention to
provide the use of porous scaffold of the invention for tissue
engineering, cell culture and cell delivery.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0017] The term "polysaccharide", as used herein, refers to a
molecule comprising two or more monosaccharide units.
[0018] The term "alkaline solution", as used herein, denotes a
solution having a pH superior to 7.
[0019] The term "acidic solution", as used herein, denotes a
solution having a pH inferior to 7.
[0020] The term "aqueous solution", as used herein, refers to a
solution in which the solvent is water.
[0021] The term "cross-linking" refers to the linking of one
polymer chain to another one with covalent bonds.
[0022] The term "porogen agent" denotes any solid agent which has
the capability to form pores within a solid structure.
[0023] As used herein, a "scaffold" is defined as a semi-solid,
system comprising a three-dimensional network of one or more
species of polysaccharide chains. Depending on the properties of
the polysaccharide (or polysaccharides) used, as well as on the
nature and density of the network, such structures in equilibrium
can contain various amounts of water.
[0024] The term "cross-linking agent" includes any agent able to
introduce cross-link between the chains of the polysaccharides of
the invention.
[0025] "Biodegradable", as used herein, refers to materials that
degrade in vivo to non-toxic compounds, which can be excreted or
further metabolized.
Porous Scaffolds and Method for Preparing Thereof
[0026] A first object of the invention relates to a method for
preparing a porous scaffold which comprises the steps consisting
of: [0027] a) preparing an alkaline aqueous solution comprising an
amount of at least one polysaccharide, an amount of a covalent
cross-linking agent and an amount of a porogen agent [0028] b)
transforming the solution into a hydrogel by placing said solution
at a temperature from about 4.degree. C. to about 80.degree. C. for
a sufficient time to allow the cross-linking of said amount of
polysaccharide and [0029] c) submerging said hydrogel into an
aqueous solution [0030] d) washing the porous scaffold obtained at
step c).
[0031] In the present invention, any type of polysaccharide can be
used. Synthetic or natural polysaccharides may be alternatively
used for the purpose of the invention. For example, suitable
natural polysaccharides include, but are not limited to, dextran,
agar, alginic acid, hyaluronic acid, inulin, pullulan, heparin,
fucoidan, chitosan, scleroglucan, curdlan, starch, cellulose and
mixtures thereof. Monosaccharides that may be used to produce the
desired polysaccharide include but are not limited to ribose,
glucose, mannose, galactose, fructose, sorbose, sorbitol, mannitol,
iditol, dulcitol and mixtures thereof. Chemically modified
polysaccharides bearing for instance acidic groups (carboxylate,
sulphate, phosphate), amino groups (ethylene amine, diethylamine,
diethylaminoethylamine, propylamine), hydrophobic groups (alkyl,
benzyl) can be included. Many of these compounds are available
commercially from companies such as Sigma-Aldrich (St. Louis,
Mich., US).
[0032] The preferred weight-average molecular weight for the
polysaccharide is from about 10,000 Daltons to about 2,000,000
Daltons, more preferably from about 10,000 Daltons to about 500,000
Daltons, most preferably from about 10,000 Daltons to about 200,000
Daltons.
[0033] In one embodiment of the invention, the polysaccharide(s)
used to prepare the scaffold of the invention is a neutral
polysaccharide such as dextran, agar, pullulan, inulin,
scleroglucan, curdlan, starch, cellulose or a mixture thereof. In a
preferred embodiment, a mixture of pullulan and dextran is used to
prepare the scaffold of the invention. For example, said mixture
comprises 25% of dextran and 75% of pullulan.
[0034] In another embodiment of the invention, the
polysaccharide(s) used to prepare the scaffold of the invention is
a positively charged polysaccharide such as chitosan, DEAE-dextran
and mixtures thereof.
[0035] In another embodiment of the invention, the
polysaccharide(s) used to prepare the scaffold of the invention is
a negatively charged polysaccharide such as alginic acid,
hyaluronic acid, heparin, fucoidan and mixtures thereof.
[0036] In another embodiment of the invention, the
polysaccharide(s) used to prepare the scaffold of the invention is
a mixture of neutral and negatively charged polysaccharides,
wherein the negatively charged polysaccharides represents 1 to 20%,
preferably 5 to 10% of the mixture.
[0037] In a particular embodiment the covalent cross-linking agent
is selected from the group consisting of trisodium trimetaphosphate
(STMP), phosphorus oxychloride (POCl.sub.3), epichlorohydrin,
formaldehydes, hydrosoluble carbodiimides, glutaraldehydes or any
other compound that is suitable for crosslinking a polysaccharide.
In a preferred embodiment, the cross-linking agent is STMP. The
concentration of the covalent cross-linking agent in the aqueous
solution (w/v) is from about 1% to about 6%, more preferably from
about 2% to about 6%, most preferably from about 2% to about 3%. It
is preferred to use the cross-linking agent at such an amount that
the weight ratio of the polysaccharide to the cross-linking agent
is in the range from 20:1 to 1:1, preferably from 15:1 to 1:1 and
more preferably from 10:1 to 1:1.
[0038] Many of these compounds are available commercially from
companies such as Sigma-Aldrich (St. Louis, Mich., US).
[0039] The aqueous solution comprising the polysaccharide may
further comprise various additives depending on the intended
application. Preferably, the additive is compatible with the
polysaccharide and does not interfere with the effective
cross-linking of the polysaccharide(s). The amount of the additive
used depends on the particular application and may be readily
determined by one skilled in the art using routine
experimentation.
[0040] The aqueous solution comprising the polysaccharide may
optionally include at least one antimicrobial agent. Suitable
antimicrobial preservatives are well known in the art. Examples of
suitable antimicrobials include, but are not limited to, alkyl
parabens, such as methylparaben, ethylparaben, propylparaben, and
butylparaben; cresol; chlorocresol; hydroquinone; sodium benzoate;
potassium benzoate; triclosan and chlorhexidine. Other examples of
antibacterial agents and of anti-infectious agents that may be used
are, in a nonlimiting manner, rifampicin, minocycline,
chlorhexidine, silver ion agents and silver-based compositions.
[0041] The aqueous solution comprising the polysaccharide may also
optionally include at least one colorant to enhance the visibility
of the solution. Suitable colorants include dyes, pigments, and
natural coloring agents. Examples of suitable colorants include,
but are not limited to, alcian blue, fluorescein isothiocyanate
(FITC) and FITCdextran.
[0042] The aqueous solution comprising the polysaccharide may also
optionally include at least one surfactant. Surfactant, as used
herein, refers to a compound that lowers the surface tension of
water. The surfactant may be an ionic surfactant, such as sodium
lauryl sulfate, or a neutral surfactant, such as polyoxyethylene
ethers, polyoxyethylene esters, and polyoxyethylene sorbitan.
[0043] In a particular embodiment, the porogen agent may be an
agent that can be transformed into a gas in acidic conditions, with
pores being formed by the carbon dioxide molecules that leach out
from the polymer. Examples of such a porogen agent include but are
not limited to ammonium carbonate, ammonium bicarbonate, sodium
carbonate, and sodium bicarbonate, calcium carbonate and mixtures
thereof. It is preferred to use the porogen agent at such an amount
that the weight ratio of the polysaccharide to the porogen agent is
in the range from 6:1 to 1:1, preferably from 4:1 to 1:1, more
preferably to 2:1 to 1:1. Many of these compounds are available
commercially from companies such as Sigma-Aldrich (St. Louis,
Mich., US). In one embodiment, the ratio of the polysaccharide to
the porogen agent may be in the range from 6:1 to 0.5:1, preferably
from 4:1 to 0.5:1, more preferably to 2:1 to 0.5:1. In another
embodiment, while the polysaccharide is a positively charged
polysaccharide, the ratio of the polysaccharide to the porogen
agent may be in the range from 50:1 to 1:1, preferably from 20:1 to
1:1 and more preferably from 10:1 to 1:1.
[0044] In this particular embodiment, the aqueous solution of step
c) is an acidic solution. The acid may be selected from the group
consisting of citric acid, hydrochloric acid, acetic acid, formic
acid, tartaric acid, salicylic acid, benzoic acid, and glutamic
acid.
[0045] Alternatively, the porogen agent may be an inorganic salt
that can be dissolved once the cross-linked polysaccharide scaffold
is immersed in water. An example of such a porogen agent includes
saturated salt solution, which would be dissolved progressively. In
this particular embodiment, the aqueous solution of step c) is an
aqueous solution, preferably water, and more preferably distilled
water.
[0046] The concentration of the porogen agent affects the size of
the pores formed in the scaffolds, so that the pore size can be
under the control of the concentration of said porogen agent.
[0047] The average pore size of the scaffold is from about 1 .mu.m
to about 500 .mu.m, preferably from about 150 .mu.m to about 350
.mu.m, more preferably from about 175 .mu.m to about 300 .mu.m. The
density of the pores or porosity is from about 4% to about 75%,
preferably from about 4% to about 50%.
[0048] In another embodiment, the method of the invention may
comprise a further step consisting of freeze-drying the scaffold
obtained at step d). Freeze-drying may be performed with any
apparatus known in the art. There are essentially three categories
of freeze dryers: rotary evaporators, manifold freeze dryers, and
tray freeze dryers. Such apparatus are well known in the art and
are commercially available such as a freeze-dryer Lyovac (GT2,
STERIS Rotary vane pump, BOC EDWARDS). Basically, the vacuum of the
chamber is from 0.1 mBar to about 6.5 mBar. The freeze-drying is
performed for a sufficient time sufficient to remove at least 98.5%
of the water, preferably at least 99% of the water, more preferably
at least 99.5%.
[0049] In another embodiment, the method of the invention may
comprise a further step consisting of hydrating the scaffold as
prepared according to the invention.
[0050] Said hydration may be performed by submerging the scaffold
in an aqueous solution (e.g., de-ionized water, water filtered via
reverse osmosis, a saline solution, or an aqueous solution
containing a suitable active ingredient) for an amount of time
sufficient to produce a scaffold having the desired water content.
For example, when a scaffold comprising the maximum water content
is desired, the scaffold is submerged in the aqueous solution for
an amount of time sufficient to allow the scaffold to swell to its
maximum size or volume. Typically, the scaffold is submerged in the
aqueous solution for at least about 1 hour, preferably at least
about 2 hours, and more preferably about 4 hours to about 24 hours.
It is understood that the amount of time necessary to hydrate the
scaffold to the desired level will depend upon several factors,
such as the composition of the used polysaccharides, the size
(e.g., thickness) of the scaffold, and the temperature of the
aqueous solution, as well as other factors.
[0051] In a particular embodiment, the hydrated scaffold comprises
80% of water, preferably 90% of water, most preferably 95% of
water.
[0052] In another particular embodiment, the aqueous solution of
step a) may be poured in a mould before step b), so that the porous
scaffold obtained with the method of the invention can take a
desired form. Any geometrical moulds may be used according to the
invention. Different sizes may be also envisaged. For example,
typically, the aqueous solution may be poured in a tubular mould
with a central axis so that the porous scaffold may be tubular with
a desired external and internal diameter. The mould may be made of
any material, but preferred material includes non sticky surfaces
such as Teflon.
[0053] Alternatively, the scaffolds of the invention may be cut and
shaped to take a desired size and form.
[0054] The methods of the invention can further include the step of
sterilizing the scaffold using any suitable process. The scaffold
can be sterilized at any suitable point, but preferably is
sterilized before the scaffold is hydrated. A suitable irradiative
sterilization technique is for example an irradiation with Cesium
137, 35 Gray for 10 minutes. Suitable non-irradiative sterilization
techniques include, but are not limited to, UV-exposure, gas plasma
or ethylene oxide methods known in the art. For example, the
scaffold can be sterilized using a sterilisation system which is
available from Abtox, Inc of Mundelein, Ill. under the trade mark
PlazLyte, or in accordance with the gas plasma sterilization
processes disclosed in U.S. Pat. No. 5,413,760 and U.S. Pat. No.
5,603,895.
[0055] The scaffold produced by the methods of the invention can be
packaged in any suitable packaging material. Desirably, the
packaging material maintains the sterility of the scaffold until
the packaging material is breached.
[0056] In another embodiment, one or more biomolecules may be
incorporated in the porous scaffold. The biomolecules may comprise,
in other embodiments, drugs, hormones, antibiotics, antimicrobial
substances, dyes, radioactive substances, fluorescent substances,
anti-bacterial substances, chemicals or agents, including any
combinations thereof. The substances may be used to enhance
treatment effects, enhance visualization, indicate proper
orientation, resist infection, promote healing, increase softness
or any other desirable effect. In said embodiment, the scaffold of
the invention, comprising one or more biomolecules as described
here above, may be used as a controlled release system of an active
agent.
[0057] The scaffold produced by the methods of the invention is
free from growth factors and other growth stimulants. In one
embodiment, the biomolecule may comprise chemotactic agents,
antibiotics, steroidal or non-steroidal analgesics,
antiinflammatories, immunosuppressants, anti-cancer drugs, various
proteins (e.g., short chain peptides, bone morphogenic proteins,
glycoprotein and lipoprotein); cell attachment mediators;
biologically active ligands; integrin binding sequence; ligands;
various growth and/or differentiation agents (e.g., epidermal
growth factor, IGF-I, IGF-II, TGF-[beta], growth and
differentiation factors, stromal derived factor SDF-1; vascular
endothelial growth factors, fibroblast growth factors, platelet
derived growth factors, insulin derived growth factor and
transforming growth factors, parathyroid hormone, parathyroid
hormone related peptide, bFGF; TGF[beta] superfamily factors;
BMP-2; BMP-4; BMP-6; BMP-12; sonic hedgehog; GDF5; GDF6; GDF8;
PDGF); small molecules that affect the upregulation of specific
growth factors; tenascin-C; hyaluronic acid; chondroitin sulfate;
fibronectin; decorin; thromboelastin; thrombin-derived peptides;
heparin-binding domains; heparin; heparan sulfate; DNA fragments,
DNA plasmids, Si-RNA, transfection agents or any combination
thereof.
[0058] In one embodiment growth factors include heparin binding
growth factor (HBGF), transforming growth factor alpha or beta
(TGF.beta.), alpha fibroblastic growth factor (FGF), epidermal
growth factor (TGF), vascular endothelium growth factor (VEGF), and
SDF-1, some of which are also angiogenic factors. In another
embodiment factors include hormones such as insulin, glucagon, and
estrogen. In some embodiments it may be desirable to incorporate
factors such as nerve growth factor (NGF) or muscle morphogenic
factor (MMF). In one embodiment, TNF alpha/beta, or Matrix
metalloproteinases (MMPs) are incorporated.
[0059] Additionally, scaffolds of the invention may optionally
include anti-inflammatory agents, such as indomethacin, salicylic
acid acetate, ibuprofen, sulindac, piroxicam, and naproxen;
thrombogenic agents, such as thrombin, fibrinogen, homocysteine,
and estramustine; and radio-opaque compounds, such as barium
sulfate, gold particles and iron oxide nanoparticles (USPIOs).
[0060] Additionally, scaffolds of the invention may optionally
comprise anti-thrombotic agents such as antivitamin K or aspirin,
antiplatelet agents such as aspirin, thienopyridine, dipyridamole
or clopidogrel (that selectively and irreversibly inhibits
adenosine diphosphate (ADP)-induced platelet aggregation) or
anticoagulant agent such as heparin or fucoidan. The combination of
heparin (anticoagulant) and tirofiban (antiplatelet agent) has been
shown to be effective in reducing both thrombus and thromboemboli
and may be incorporated. Genistein, a potential isoflavone which
possesses dose-dependent antiplatelet and antiproliferative
properties and inhibits collagen-induced platelet aggregation
responsible for primary thrombosis, may also be incorporated.
Methods for Using the Scaffolds of the Invention
[0061] Scaffolds of the invention are especially suited for tissue
engineering, repair or regeneration. A difference in porosity may
facilitate migration of different cell types to the appropriate
regions of the scaffold. In another embodiment, a difference in
porosity may facilitate development of appropriate cell-to-cell
connections among the cell types comprising the scaffold, required
for appropriate structuring of the
developing/repairing/regenerating tissue. For example, cell
processes extension may be accommodated more appropriately via the
varied porosity of the scaffolding material. Therefore, the
scaffold may comprise cells of any tissue.
[0062] In particular embodiment, the cells are seeded on said
scaffold. In another embodiment, the scaffolds of the invention are
submerged in a culture solution comprising the desired cells for an
amount of time sufficient to enable penetration of the cells
throughout the scaffold.
[0063] In another embodiment, scaffold of the invention is capable
of supporting the viability and the growth of seeded cells in
culture over long periods of time without inducing
differentiation.
[0064] In another embodiment, scaffold of the invention provides an
environment for unstimulated cell growth (without activation by
growth stimulants)
[0065] In another embodiment, scaffold of the invention can be used
to study physiological and pathological processes such as tissue
growth, bone remodeling, wound healing, tumorigenesis (including
migration and invasion), differentiation and angiogenesis. Scaffold
allows the creation of defined and controlled environments where
specific processes can be modulated and studied in a controlled
manner free of endogenous factors.
[0066] In particular, scaffold of the invention can be used for 3D
culture for diagnostic or toxicological dosages. In this
embodiment, the scaffold of the invention would allow evaluation of
the toxicity of a product directly on cells present in a 3D
environment. In said embodiment, the scaffold of the invention is
used for cultivating cells useful for the evaluation of the
toxicity and/or pharmacology of a product, such as hepatocytes,
embryonic stem cells, epithelial cells, keratinocytes, or induced
pluripotent stem cells (iPS cells).
[0067] In another embodiment, scaffold of the invention is capable
of supporting growth and differentiation of cell types in vitro and
in vivo.
[0068] In another embodiment, the cells are stem or progenitor
cells. In another embodiment the cells may include but are not
limited to chondrocytes; fibrochondrocytes; osteocytes;
osteoblasts; osteoclasts; synoviocytes; bone marrow cells;
mesenchymal cells; epithelial cells, hepatocytes, muscle cells;
stromal cells; stem cells; embryonic stem cells; precursor cells
derived from adipose tissue; peripheral blood progenitor cells;
stem cells isolated from adult tissue; induced pluripotent stem
cells (iPS cells); genetically transformed cells; a combination of
chondrocytes and other cells; a combination of osteocytes and other
cells; a combination of synoviocytes and other cells; a combination
of bone marrow cells and other cells; a combination of mesenchymal
cells and other cells; a combination of stromal cells and other
cells; a combination of stem cells and other cells; a combination
of embryonic stem cells and other cells; a combination of
progenitor cells isolated from adult tissue and other cells; a
combination of peripheral blood progenitor cells and other cells; a
combination of stem cells isolated from adult tissue and other
cells; and a combination of genetically transformed cells and other
cells.
[0069] In another embodiment, any of these cells for use in the
scaffolds and methods of the invention, may be genetically
engineered to express a desired molecule, such as for example green
fluorescent protein (GFP), reporter gene (luciferase, phosphatise
alkaline), heparin binding growth factor (HBGF), transforming
growth factor alpha or beta (TGF.beta.), alpha fibroblastic growth
factor (FGF), epidermal growth factor (TGF), vascular endothelium
growth factor (VEGF) and SDF-1, some of which are also angiogenic
factors. In another embodiment expressed factors include hormones
such as insulin, glucagon, and estrogen. In another embodiment
factors such as nerve growth factor (NGF) or muscle morphogenic
factor (MMF), or in another embodiment, TNF alpha/beta are
expressed.
[0070] In a particular embodiment, scaffolds of the invention are
suitable to prepare vascular substitutes to replace compromised
arteries as described for example, in Chaouat et al. (Chaouat M, Le
Visage C, Autissier A, Chaubet F, Letourneur D. The evaluation of a
small-diameter polysaccharide-based arterial graft in rats.
Biomaterials. 2006 Nov.; 27(32):5546-53. Epub 2006 Jul. 20.). Such
substitutes may be prepared according to the methods of the
invention by using a mould as above described. Such substitutes may
then comprise a population of cells to reconstruct in vitro or in
vivo a vessel. In another embodiment the cells may include but are
not limited to Mesenchymal Stem Cells (MSC), Endothelial Progenitor
cells (EPCs), endothelial cells, fibroblastic cells and smooth
muscle cells.
[0071] In another particular embodiment, scaffolds of the invention
are suitable to prepare cartilage or bone implants. In such a way,
the scaffolds of the invention may be loaded with chondrocytes,
osteocytes; osteoblasts; osteoclasts; vascular cells or mixtures
thereof, and may be cultured in presence of differentiating
agents.
[0072] The site of implantation is dependent on the
diseased/injured tissue that requires treatment. For example, to
treat structural defects in articular cartilage, meniscus, and
bone, the cell-seeded composite scaffold will be placed at the
defect site to promote repair of the damaged tissue.
[0073] In case of central nervous system (CNS) injuries, the
composite scaffold can be seeded with a combination of adult
neuronal stem cells, embryonic stem cells, glial cells and Sertoli
cells. In the preferred embodiment, the composite scaffold can be
seeded with Sertoli cells derived from transformed cell lines,
xenogeneic or allogeneic sources in combination with neuronal stem
cells. The Sertoli cells can be cultured with the composite
scaffold for a period before addition of stem cells and subsequent
implantation at the site of injury. This approach can circumvent
one of the major hurdles of cell therapy for CNS applications,
namely the survival of the stem cells following transplantation. A
composite scaffold that entraps a large number of Sertoli cells can
provide an environment that is more amenable for the survival of
stem cells.
[0074] Accordingly, the porous polysaccharide scaffold, which is
prepared according to the present invention, can be effectively
used as a raw material for fabricating artificial tissues or organs
such as artificial blood vessels, artificial bladder, artificial
esophagus, artificial nerves, artificial hearts, prostatic heart
valves, artificial skins, orthopedic implants, artificial muscles,
artificial ligaments, artificial respiratory organs, etc. Further,
the porous polysaccharide scaffold of the present invention can be
prepared in the form of a hybrid tissue by blending or
incorporating on or into other types of biomaterials and with
functional cells derived from tissues or organs. It may have
various biomedical applications, for example, to maintain cell
functions, tissue regeneration, etc.
[0075] Alternatively scaffolds of the invention may be used for
cell delivery. Actually, scaffolds of the invention may be used as
a raw material for preparing cell delivery systems that can be
administered to a subject for therapeutic or diagnostic purposes.
In a particular embodiment, scaffolds of the invention may be used
to prepare a patch, a biofilm or a dressing that can be loaded with
cells. For example, scaffolds of the invention may used to prepare
a dressing that can be applied on the skin, for reconstructing or
healing the skin. Alternatively, said dressing may used to be
applied on the heart of a subject for treating ischemia (myocardial
infarction). In those embodiments, the cells that are entrapped in
the scaffold can thus migrate into the targeted tissue or
organ.
[0076] In another embodiment, scaffolds of the invention may be
used for culturing cells. Cells may then be stimulated to undergo
growth of differentiation or other physiological processes by the
addition of appropriate growth factors. Culture medium containing
one or more cytokines, growth factors, hormones or a combination
thereof, may be used for maintaining cells in an undifferentiated
state, or for differentiating cells into a particular pathway.
[0077] More particularly, the scaffold of the invention may be used
for producing molecules of interest. Actually, scaffolds of the
invention may be used to provide a biological environment for the
anchorage of cells in a bioreactor, so that the cells can produced
the desired molecules. The scaffolds of the invention provide
mechanical and biochemical protection of the cultured cells.
[0078] The scaffolds may thus serve as a cell reservoir for
producing desired molecules such as proteins, organic molecules,
and nucleotides. For example, proteins of interest include but are
not limited to growth factors, hormones, signal molecules,
inhibitors of cell growth, and antibodies. Scaffolds of the
invention are particularly interesting for producing monoclonal
antibodies. Scaffolds of the invention may be also suitable to
produce organic molecules such as flavours, therapeutic molecules .
. . .
[0079] In this purpose, the scaffolds of the invention may be
loaded with any type of cells, including prokaryotic and eukaryotic
cells. For examples, scaffolds of the invention may be load with
bacteria, yeast cells, mammalian cells, insect cells, plant cells,
etc. Specific examples include E. coli, Kluyveromyces or
Saccharomyces yeasts, mammalian cell lines (e.g., Vero cells, CHO
cells, 3T3 cells, COS cells, etc.) as well as primary or
established mammalian cell cultures (e.g., produced from
lymphoblasts, fibroblasts, embryonic cells, epithelial cells,
nervous cells, adipocytes, etc.). More particularly, the invention
contemplates the use of established cell lines such as hybridomas.
Alternatively, the cells may be genetically engineered to express a
desired molecule as described above.
[0080] The scaffold of the invention may be loaded with cells,
cultured for a certain period of time then the cells can be
retrieved/extracted/separated from the scaffold for further use,
such as therapeutic or diagnostic applications or cell analysis.
Separation of the cells from the scaffold may involve the use of
enzymes that could degrade the scaffold, such as pullulanase and/or
the use of enzymes that could detach the cells such as collagenase,
elastase, trypsin or cell-detaching solutions such as EDTA.
[0081] The invention will further be illustrated in view of the
following figures and examples.
FIGURES
[0082] FIG. 1: A porous scaffold obtained as in Example 1 (Scale: 6
mm)
[0083] FIG. 2: A porous scaffold obtained as in Example 1: scanning
Electron Microscopy analysis of the scaffold (right image, scale:
200 microns).
[0084] FIG. 3: Formazan absorbance (570 nm) at day 1 as a function
of the initial number of cells seeded on porous scaffolds.
EXAMPLES
Example 1
Polysaccharides-Based Scaffolds Preparation
[0085] Polysaccharide-based scaffolds were prepared using a mixture
of pullulan/dextran 75:25 (pullulan, MW 200,000, Hayashibara Inc.,
Okayama, Japan; dextran MW 500,000, Pharmacia). A polysaccharide
solution was prepared by dissolving 9 gr of pullulan and 3 gr of
dextran into 40 mL of distilled water. Sodium carbonate (8 g) was
then added to the polysaccharide solution and stirring was
maintained until a homogeneous mixture was obtained. Chemical
cross-linking of polysaccharide was carried out using the
cross-linking agent trisodium trimetaphosphate STMP (Sigma, St
Louis) under alkaline condition. Briefly, one milliliter of 10M
sodium hydroxide was added to 10 g of the polysaccharide solution,
followed by the addition of one milliliter of water containing 300
mg of STMP. The mixture was then poured into petri dishes
(Nunclon.RTM., #150288) and incubated at 50.degree. C. for 15 min.
Resulting hydrogels were immediately immersed into a large beaker
containing a 20% acetic acid solution, for at least 30 minutes.
Resulting scaffolds were washed extensively with phosphate buffer
saline pH 7.4 then with distilled water for at least 2 days. After
a freeze-drying step, porous scaffolds were stored at room
temperature until use. Scanning Electron Microscopy analysis
confirmed the porosity of the scaffolds (FIGS. 1 and 2).
Example 2
Types of Polysaccharides
[0086] Porous scaffolds were prepared as described in example 1,
using different types and ratios of polysaccharides, while keeping
the total amount of polysaccharide at a constant value.
Polysaccharides were either pullulan, dextran 500, fucoidan LMW
(Low Molecular Weight) and fucoidan HMW (High Molecular
Weight).
TABLE-US-00001 Dextran Fucoidan Fucoidan Pullulan 500 LMW HMW
Solubilization Viscosity 100% +++ +++ 100% +/- + 50% 50% ++ ++ 75%
25% ++ ++ 75% 25% +/- +++ 75% 25% + +
[0087] Solubilization (+++ indicates a complete solubilization of
the polysaccharides) and viscosity of the resulting polysaccharide
solution (+++ indicates a very high viscosity of the solution) were
visually assessed. In all cases, porous scaffolds were obtained at
the end of the protocol.
Example 3
Porogen Amount
[0088] Porous scaffolds were prepared as described in example 1,
while varying the amount of the porogen agent. Briefly, 2, 4 or 8
gr of sodium carbonate were added to the pullulan/dextran
solution.
TABLE-US-00002 Porogen agent Solubilization Viscosity Porosity 2 g
++ ++ + 4 g ++ ++ ++ 8 g ++ ++ ++
[0089] Solubilization (++ indicates a complete solubilization of
the polysaccharides), viscosity of the resulting polysaccharide
solution (+++ indicates that a very high viscosity of the solution)
and porosity were visually assessed. For scaffolds prepared with
the lowest amount of porogen (2 g), the effervescence process was
moderate, as compared to the effervescence obtained with 4 g and 8
g of porogen agent. In all cases, porous scaffolds were obtained at
the end of the protocol.
Example 4
Cross-Linker Concentration
[0090] Porous scaffolds were prepared as described in example 1,
while varying the amount of the cross-linking agent from 200 mg to
500 mg.
TABLE-US-00003 Cross-linking agent Solubilization Viscosity
Porosity 200 mg ++ ++ ++ 300 mg ++ ++ ++ 400 mg ++ +++ ++ 500 mg ++
+++ +
[0091] Solubilization (+++ indicates a complete solubilization of
the polysaccharides), viscosity of the resulting polysaccharide
solution (+++ indicates that a very high viscosity of the solution)
and porosity were visually assessed. In all cases, porous scaffolds
were obtained at the end of the protocol.
Example 5
Cell Loading into the Porous Scaffolds
[0092] Human bone marrow Mesenchymal Stem Cells (hMSC) were
cultured on scaffolds prepared as in Example 1. A circular punch
was used to cut 6 mm diameter and 1 mm thickness round-shaped
porous scaffolds. Culture medium consisted of low glucose DMEM
(Gibco, Life Technology, New York) with 10% fetal bovine serum and
1% penicillin/streptomycin (Sigma). After cell trypsinization,
rehydration of the dried scaffold was performed with 20 .mu.L of
cell suspension (10.sup.6 cells/scaffold). Samples were then
maintained in 1 mL of culture medium for up to 1 week. Non-seeded
porous scaffolds incubated in culture medium were used as
controls.
[0093] A metabolic assay (MTT,
3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide,
Sigma) was performed to assess the cell viability. Briefly, a 5
mg/mL stock solution of MU (Sigma) was mixed 1:10 with DMEM.
Scaffolds were incubated for 3 h at 37C with 1 mL of the reagent
solution. After washing the scaffolds with PBS, the formazan
crystals were solubilized in 0.3 mL of Isopropranol/HCl 0.04M.
Absorbance was recorded at 590 nm with a microplate reader
(Multiskan, Thermo Electron Corporation, Waltham, Mass.).
Absorbance at day 1 was directly proportional to the initial number
of cells seeded in the scaffolds (FIG. 3).
[0094] Similar experiments were successfully carried out with other
cell types such as primary vascular smooth muscle cells and
endothelial cells from animal and human origin.
Example 6
Confocal Analysis of Cell Behavior within the Porous Scaffolds
[0095] Fluorescent scaffolds were prepared as in example 1, by
adding a small amount (5 mg) of FITC-dextran to the polysaccharide
solution. Fluorescent scaffolds were seeded as in Example 5, with
hMSC labeled with a fluorescent marker (PKH26, SIGMA P9691)
according to the manufacturer's instructions). Confocal imaging
confirmed the porous structure of the scaffold.
Example 7
Cell Viability by Live and Dead Assay
[0096] Confocal imaging was used to assess the cell viability with
a live/dead assay (Calbiochem, San Diego, Calif.), based on the use
of two fluorescent probes that measure the cell membrane
permeability: a cell-permeable green fluorescent dye to stain live
cells (calcein AM) and a cell nonpermeable red fluorescent dye
(propidium iodide) to stain dead cells. At day 7, most of the cells
were live cells, with only few dead cells found within the
scaffolds.
Example 8
Influence of the Porogen Agent on Scaffold Porosity
[0097] Porous scaffolds were prepared as described in example 1,
while varying the amount and the nature of the porogen agent. For
confocal analysis of fluorescent porous scaffolds, 5 mg of
FITC-dextran were added to the polysaccharide solution. Optical
sections were acquired using a Zeiss LSM 510 confocal microscope
(Carl Zeiss, Oberkochen, Germany), equipped with a 10.times.
Plan-NeoFluar objective lens (numerical aperture of 0.3) (Carl
Zeiss). FITC-dextran was excited at 488 nm with an argon laser and
its fluorescent emission was selected by a 505-530 nm bandpass
filter. Pore size was assessed with ImageJ.RTM. software. Void
volume was calculated with a statistics/volume measurement module
from Amira.RTM. software and results are expressed as a percentage
of the scaffold volume.
TABLE-US-00004 Mean Void diameter volume Polysaccharides Porogen
agent (.mu.m) (%) Pullulan (9g) + Sodium Carbonate 195 37% dextran
500 (3g) (8 g) Pullulan (9g) + Sodium Carbonate 207 71% dextran 500
(3g) (8 g) + Sodium Chloride (2 g) Pullulan (9g) + Sodium Carbonate
272 59% dextran 500 (3g) (8 g) + Sodium Chloride (8 g)
Example 9
Positively Charged Polysaccharide
[0098] Positively charged porous scaffolds were prepared using
DEAE-Dextran as the only polysaccharide. Briefly, DEAE-dextran
solution was prepared by dissolving 1 g of DEAE-dextran (Fluka
reference #30461) into 1.5 mL of distilled water. Sodium carbonate
(100 mg) was then added to the polysaccharide solution and stirring
was maintained until a homogeneous mixture was obtained. Chemical
cross-linking of polysaccharide was carried out using the
cross-linking agent trisodium trimetaphosphate STMP (Sigma, St
Louis) under alkaline condition. Briefly, 150 .mu.L of 10M sodium
hydroxide was added to the polysaccharide solution, followed by the
addition of 150 .mu.L of water containing 45 mg of STMP. The
mixture was then poured into petri dishes (Nunclon.RTM., #150288)
and incubated at 50.degree. C. for 15 min. Resulting hydrogels were
immediately immersed into a large beaker containing a 20% acetic
acid solution, for at least 30 minutes. Resulting scaffolds were
washed extensively with phosphate buffer saline pH 7.4 then with
distilled water for at least 2 days. After a freeze-drying step,
porous scaffolds were obtained and stored at room temperature until
use.
Example 10
Negatively Charged Polysaccharide
[0099] Negatively charged porous scaffolds were prepared by adding
fucoidan (Sigma reference #F5631) to a pullulan/dextran mixture.
Briefly, a polysaccharide solution was prepared by dissolving 9 g
of pullulan and 3 g of dextran into 40 mL of distilled water, then
adding 1.2 g of fucoidan into the polysaccharide solution. Sodium
carbonate (8 g) was then added to the polysaccharide solution and
the cross-linking process was carried out as described in Example 1
to obtain a 3D scaffold that contains a negatively charged
polysaccharide.
Example 11
Differentiation of Human Mesenchymal Stem Cells into
Chondrocyte-Like Cells in 3D Scaffolds
[0100] Human bone marrow Mesenchymal Stem Cells (hMSC) were
cultured on scaffolds prepared as in Example 1 in serum-free
chondrogenic medium. Chondrogenic medium consisted of DMEM
supplemented with 10 ng/ml TGF-.beta.3 (Oncogene, Cambridge,
Mass.), 100 nM dexamethasone (Sigma, St Louis, Mo.), 170 .mu.M
ascorbic acid 2-phosphate (Sigma, St Louis, Mo.) and 5 mL of
ITS-plus (Collaborative Biomedical Products, Bedford, Mass.). After
3 weeks of culture, seeded scaffolds were fixed in formaldehyde 10%
then cryosectioned. Frozen sections were stained with either 0.05%
(w/v) toluidine blue or with 0.1% safranin O solution. A strong
positive staining for extracellular matrix synthesis was observed,
indicating MSC differentiation into cartilage cells.
Example 12
3D Culture of Hepatocytes
[0101] HepG2 cells, human hepatocellular carcinoma cells, were
cultured in low glucose DMEM (Gibco, Life Technology, New York,
USA) with 10% fetal bovine serum and 1% penicillin/streptomycin
(Sigma) on scaffolds prepared as in Example 1. A circular punch was
used to cut 6 mm diameter and 1 mm thickness round-shaped porous
scaffolds.
[0102] After cell trypsinization, rehydration of the dried scaffold
was performed with 20 .mu.L of cell suspension (85,000
cells/scaffold). Samples were then maintained in 1 mL of culture
medium for up to 1 week. Non-seeded porous scaffolds incubated in
culture medium were used as controls. Hepatocyte spheroids
formation was observed after 4 days of culture. Cell viability in
spheroids was assayed using Calcein AM (Calbiochem, San Diego
Calif., USA) which is a polyanionic dye hydrolyzed by live cells
thus producing an intense uniform green fluorescence (wavelength
485-535 nm), according to the manufacturer's instructions. The
seeded scaffolds contained living hepatocytes suitable for
pharmaco-toxicological assays.
* * * * *