Note: Descriptions are shown in the official language in which they were submitted.
CA 02442868 2003-10-02
Porous and non-porous matrices based on chitosan and
hydroxy carboxylic acids
Description
The invention relates to biocompatible matrices based
on chitosan and hydroxy carboxylic acids, to multilayer
systems comprising these matrices and to applications
of such matrices.
Considerable successes have been achieved in recent
years in the area of medical transplants. However,
problems arise through the small amounts of donor
organs available and through rejection reactions caused
by heterologous organs. A further problem is that
pathogens can also be transmitted with heterologous
donor organs. Attempts have therefore been made to
culture artificial organs from cell cultures on a
three-dimensional matrix which can be shaped according
to requirements, for example as an ear. This artificial
organ or body part can then be transplanted and, if
endogenous cells are used, no rejection reaction
occurs.
Chitosan has attracted increasing interest as a
promising matrix material. Chitosan is a partly
deacetylated chitin and is obtained from exoskeletons
of arthropods. It is an aminopolysaccharide (poly-I-4-
glucosamine) which is used for example in the medical
sector as suture material or for encapsulating drugs.
Its advantage is that it can be completely absorbed by
the body. Chitosan can be dissolved in water in the
slightly acid range (pH <6) through protonation of the
free amino groups. In the alkaline range (pH >7) it
precipitates again from the aqueous solution. Chitosan
can be purified and processed under mild conditions
through this pH-dependent mechanism.
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US 5,871,985 proposes a vehicle for transplantation
into a patient which consists of a matrix into which
cells have grown. This is done by firstly preparing a
solution of chitosan comprising living cells. This
solution is then enclosed in a semipermeable membrane
in order to form the carrier. The chitosan is
precipitated and forms an uncrosslinked matrix in which
the cells are dispersed.
Madihally et al. (Biomaterials 1999; 20(12), pages
1133-1142) describes a matrix for tissue generation.
Chitosan which is 85-90o deacetylated is for this
purpose dissolved in 0.2 M acetic acid to give
solutions having a chitosan content of from 1 to 3 o by
weight. The solution is frozen and the water and the
excess acetic acid are removed by lyophilization.
German patent application 199 48 120.2 discloses a
method for producing a biocompatible three-dimensional
matrix, where an aqueous solution of a chitosan and of
an acid, in particular a hydroxy carboxylic acid, which
is present in excess is frozen, and the water is
removed by sublimation under reduced pressure, with the
excess acid being removed, in particular neutralized,
before the freezing or after the removal of the water
by sublimation. In addition, a matrix which can be
obtained by the method and which can be used for
producing implants is disclosed.
Based on this knowledge, it was the object of the
present invention to provide novel matrix forms or/and
applications of a matrix based on chitosan and an acid,
in particular a hydroxy carboxylic acid.
A first aspect of the present invention therefore
relates to a biocompatible non-porous matrix based on
chitosan and an acid, in particular a hydroxy
carboxylic acid, which may be for example in the form
of a sheet or of a three-dimensional article, e.g. of a
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hollow article or of a roll. The non-porous matrix can
be obtained by:
- providing an aqueous solution of a chitosan and an
acid, in particular a hydroxy carboxylic acid,
which is present in excess,
- drying the solution without freezing and
- removing excess acids before or/and after drying,
preferably by neutralization.
The non-porous matrix can be used as carrier for a
porous three-dimensional matrix. It is thus possible to
provide biocompatible matrix systems which comprise at
least one biocompatible non-porous matrix as described
previously, and at least one biocompatible porous
matrix. The structure of the biocompatible porous
matrix is preferably based on chitosan and an acid, in
particular a hydroxy carboxylic acid. However, it is
also possible to use other porous biocompatible
matrices.
A biocompatible porous matrix as disclosed in German
application 199 48 120.2 is particularly preferred and
is obtainable by:
- providing an aqueous solution of a chitosan and of
an acid, in particular a hydroxy carboxylic acid,
which is present in excess,
- freezing and drying the solution, in particular by
sublimation under reduced pressure, and
- removing excess acid before or/and after the
freezing, in particular by neutralization with a
suitable base, e.g. NaOH.
In matrix system of the invention it is possible for
non-porous matrices and porous matrices each to be
disposed alternately in layers. Examples of such
multilayer systems are depicted in Figure 1A, 1B and
1C. As an alternative, a non-porous matrix can also be
disposed between two porous matrices.
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The non-porous matrix of the invention or the matrix
system based thereon can be used for the in vitro
culturing of cells. In this case, the matrix system may
comprise additional factors for cell growth, e.g.
cytokines.
The matrix or the matrix system can be employed for
example for culturing cartilage tissue, for
reconstructing bone tissue, as filling material for
bioreactors for producing cells, proteins or viruses,
as microcarrier of filling material for bioreactors,
for generating capillaries and blood vessels, for
generating optionally multilayer skin systems, for
culturing blood stem cells, for regenerating nerve
tissues and for generating artificial organs.
A particularly preferred application of the multilayer
matrix system is the production of a base material for
generating a multilayer artificial skin system. In this
case, the matrix system may be colonized by
keratinocytes and, where appropriate, additionally by
fibroblasts. A further possibility is to generate a
vascularized skin system, in which case tubes are drawn
into the porous layers of the matrix system which,
after colonization with epithelial cells, contribute to
the vascularization of the artificial skin.
A further particularly preferred application of the
multilayer matrix system is the generation of an
artificial heart valve, in which case a non-porous
structure is incorporated between two porous
structures, to increase the mechanical stability, and
is then used for culturing muscle cells.
A further possibility is to employ the non-porous
matrix and the matrix system based thereon also as
implant without previous cell colonization, e.g. for
cartilage and bone defects, as substitute for
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microcapillaries or as surgical filling material, e.g.
for reconstructive surgery or cosmetic surgery.
A further aspect of the present invention relates to a
biocompatible matrix based on chitosan and an acid, in
particular a hydroxy carboxylic acid with anisotropic
structures, for example fibers or/and chambers in
parallel alignment. In this embodiment, the matrix is
preferably porous. The anisotropic matrix can be
obtained by:
- providing an aqueous solution of a chitosan and of
an acid, in particular a hydroxy carboxylic acid,
which is present in excess,
- anisotropic freezing and drying of the solution,
in particular by sublimation under reduced
pressure, and
- removing excess acid before or/and after freezing.
The anisotropic freezing preferably comprises a
freezing with use of structured cooling elements, e.g.
tubes in direct or indirect contact with the matrix
during the freezing process. The cooling elements may
be elongate in order to obtain for example fibers or
chambers in parallel alignment in the matrix. However,
it is also possible to use curved structures, e.g.
simulations of the organ to be shaped, as cooling
elements.
The anisotropic porous matrix can be employed in a
biocompatible matrix system together with another
matrix, for example with a biocompatible non-porous
matrix. The anisotropic matrix or the matrix system
based thereon can be employed for the in vitro
culturing of cells or as implant without previous cell
colonization in accordance with the aforementioned
applications.
Yet a further aspect of the invention is the use of a
biocompatible matrix based on chitosan and an acid, in
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particular a hydroxy carboxylic acid, as described in
DE 199 48 120.2, for culturing cartilage tissue, for
reconstructing bone tissue, as filling material for
bioreactors for producing cells, proteins or viruses,
as microcarrier of filling material for bioreactors,
for generating capillaries and blood vessels, for
generating optionally multilayer skin systems, for
culturing blood stem cells, for regenerating nerve
tissues, for generating artificial organs.
It has surprisingly been found that cells can be
cultured in a density of 106 or more cells per cm2 of
matrix. This cell density is an increase of more than
ten-fold compared with culturing in a culture dish.
The matrices of the invention based on chitosan and
acids are essentially produced by the method indicated
in German application 199 48 120.2 unless stated
otherwise. Preferably, first an aqueous solution of a
partially deacetylated chitosan and of an acid which is
present in excess is prepared. Excess means in this
connection that the pH of the aqueous solution is in
the acidic range, preferably below pH <_4. The free
amino groups of the chitosan are at least partially
protonated thereby, thus increasing the solubility in
water. The amount of acid is not critical. It needs
merely to be chosen so that the chitosan dissolves.
Excessive addition of acid is avoided as far as
possible because excess acid must be removed again, and
working up is impeded with large amounts of acid
thereby. Favorable amounts of acid result in a 0.05 to
1 N, preferably 0.1 to 0.5 N, in particular 0.1 to
0.3 N, solution. The amount of chitosan is preferably
chosen to result in a 0.01 to 0.5 M, preferably 0.1 to
0.3 M, solution. The structure of the matrix,
especially the pore size thereof, can be influenced via
concentration of the chitosan solution. It is possible
in this way to adjust the pore size of the matrix to
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the particular cell type of which the matrix is to be
colonized.
Because chitosan is produced from natural sources it
has no uniform molecular weight. The molecular weight
may be between 20 kDa to more than 1000 kDa depending
on the source and method of processing.
The chitosan for producing the three-dimensional matrix
is not subject to any restrictions in relation to its
molecular weight. The aqueous chitosan solution is
produced by using an acid which is an inorganic acid
or, preferably, an organic acid, particularly
preferably an alkyl or aryl hydroxy carboxylic acid.
Hydroxy carboxylic acids having 2 to 12 carbon atoms
are particularly suitable, it being possible for one or
more hydroxyl groups and one or more carboxyl groups to
be present in the molecule. Specific examples are
glycolic acid, lactic acid, malic acid, tartaric acid,
citric acid and mandelic acid. Lactic acid is
particularly preferred.
In producing a porous matrix, the solution of chitosan
and acid is initially at least partially neutralized by
adding base and then frozen or directly frozen without
previous neutralization. Neutralization before freezing
is preferred. The pH after the neutralization is
generally 5.0 to 7.5, preferably from 5.5 to 7.0 and in
particular from 6.0 to 7Ø
After the freezing, the water is removed by sublimation
under reduced pressure, for example in the pressure
range from 0.001 to 3 hPa.
To produce a non-porous matrix, the solution is not
subjected to freezing and sublimation, but is dried
without freezing at optionally elevated temperature
or/and reduced pressure, and is preferably neutralized
after drying. The resulting non-porous matrix has a
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high load-bearing capacity and extensibility in the
moist state.
The large number of amino and hydroxyl groups makes the
matrix modifiable as desired. In a preferred embodiment
of the three-dimensional matrix, ligands are covalently
or noncovalently bound to the chitosan matrix,
preferably to the free amino groups of chitosan.
Ligands which can be used are, for example, growth
promoters, proteins, hormones, heparin, heparan
sulfates, chondroit sulfates, dextran sulfates or a
mixture of these substances. The ligands preferably
serve to control and improve cell proliferation.
The ligands used in the matrix in a preferred
embodiment of the invention are nucleic acids, e.g. RNA
or DNA. The nucleic acids can be immobilized by
chemical coupling to the amino or/and hydroxyl groups
present in the chitosan. It is possible with a nucleic
acid-loaded matrix to achieve locally restricted
transient expression of heterologous genes in the body.
This is because when a matrix coupled in this way is
implanted in the body and colonized by endogenous cells
which dissolve the matrix, the cells also take up the
nucleic acids immobilized thereon and are able to
express the latter.
Cell growth on the matrix is further improved if the
matrix is cultured with autologous fibrin.
The three-dimensional matrix of the invention can be
used as solid phase in a culture reactor (Cell
Factory). The matrix shows a very high resistance in
the culture medium. It has also emerged that the matrix
promotes cell growth.
The matrix is further suitable for use as cell implant,
in particular for cartilage-forming cells. No
genetically modified cells must be used in this case.
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The cells are preferably taken from the patient by
biopsy and cultured on the cell matrix, and the cell
implant is then implanted into the patient. Transplant
rejection reactions are substantially precluded owing
to the colonization of the three-dimensional matrix
with endogenous stem cells (bone substitute) which,
stimulated by the respective growth factors of the
surrounding tissue, differentiate only at the site of
the transplant, or with cartilage cells for renewed
formation of hyaline cartilage.
The three-dimensional matrix can be colonized both by
human and by animal cells (for example from horse, dog
or shark). Shark cells are particularly suitable
because they induce negligible immunological response
in the recipient. Shark cells are already used as organ
replacement, e.g. for the lenses of eyes. Examples of
cells with which the matrices or matrix systems of the
invention can be colonized are chondrocytes,
osteocytes, keratinocytes, hepatocytes, bone marrow
stem cells or neuronal cells.
The matrices or matrix systems as described previously
can be employed in the human medical and veterinary
sectors. Further areas of application are the use as
disposable article as in vitro test system for
investigating active pharmaceutical ingredients. For
this purpose, for example, blood stem cells or
hepatocytes can be cultured on the matrix. This system
can be used to investigate the activity of test
substances from a chemical or/and biological substance
library, where appropriate in a high-throughput method.
The matrix and the matrix system are sterilized before
use in the cell culture, in order to guarantee freedom
from germs. The sterilization can take place by thermal
treatment, e.g. by autoclaving, steam treatment etc.
or/and by irradiation, e.g. gamma-ray treatment. The
sterilization is preferably carried out in a
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physiologically tolerated buffered solution, e.g. in
PBS, in order to ensure thorough wetting of the matrix
with liquid and the absence of larger air inclusions.
When the cells are cultured, the matrix is degraded
within a period of about 5-8 weeks. The degradation
time can be adjusted via the degree of the
deacetylation of the chitosan and the concentration of
the material.
The invention is further to be explained by the
following examples.
Example 1: Production of a non-porous sheet
A mixture of chitosan and lactic acid is prepared by
the method described in Example 3 of DE 199 48 120.2.
The solution is poured into a Petri dish and dried at
50°C and, after a glass-clear film has resulted,
neutralized to a pH of 7 with 1 M sodium hydroxide
solution. The resulting sheet has a high load-bearing
capacity and extensibility in the moist state.
Example 2: Growth of Hep-G2 cells in the matrix
Two defined initial amounts, 1 x 105 and 1 x 106, of
Hep-G2 hepatocytes were injected into a piece, 1.5 cm2
in size, of porous matrix (produced as in Example 3 of
DE 199 48 120 . 2 ) , and cell growth was observed at four
points in time for a maximum of 33 days. A continuous
cell growth was observable in this case.
The maximum cell count per matrix after 33 days was
1.6 x 10' cells (Figure 2). This means the cell count
was able to increase further by one power of ten on the
small basic area of 1.5 cm2. The cell density of a
confluent, conventional culture dish with a basic area
of 80 cm2 is stated by the Deutsche Sammlung von
Mikroorganismen and Zellkulturen (DSMZ) to be 2.5-3.0 x
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10' Hep-G2. This amount is, when apportioned to the
basic area of the matrix, about 25 times less than the
cell count determinable in the matrix after 33 days.
Example 3: Effect of the matrix on cell proliferation
The intention of this experiment was to show whether
substances present in the matrix have an unfavorable
influence on cell growth. It was intended in this case
to assess not the growth of the cells on the matrix,
but only the influence of potential soluble substances
possibly released into the medium. For this purpose, a
piece, 1.5 cmz in size, of a matrix (produced as in
Example 3 of DE 199 48 120.2) was preincubated in 3 ml
of cell culture medium at 37°C and 5% COZ for 6 days.
The medium was then analyzed with control media, which
had likewise been preincubated, in a standard
proliferation assay (XTT). In this assay, a tetrazolium
salt is converted by metabolically active cells into a
colored formazan salt which can subsequently be
detected by photometry. No influence on cell growth was
observable in this case. Hep-G2 was used as cell line,
and 5o DMSO was added to the medium as positive
control. The assay was repeated three times and gave
the same result in all three cases.
Example 4: Growth of other cell lines in the matrix
and cell morphology
Besides Hep-G2, two other cell lines were seeded on the
matrix in order to observe whether they grow in the
matrix. Both Hela and the CHO-K1 cell line is able to
grow in the matrix.
An altered morphology compared with cells growing in
normal culture dishes is observable with all three cell
lines. The cells are distinctly rounded and also grow
in the third dimension and thus show more resemblance
to cells in natural three-dimensional tissues. As
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example, Figure 3 shows two pictures of the hepatocyte
line Hep-G2 with Figure 3A showing the cells after
culturing from a cell culture dish and Figure 3B
showing the cells after culturing in a matrix.
