U.S. patent application number 15/708535 was filed with the patent office on 2018-01-04 for method of fabricating scaffold for tissue engineering.
The applicant listed for this patent is GC Corporation. Invention is credited to Yuuhiro SAKAI, Yusuke SHIGEMITSU, Katsuyuki YAMANAKA.
Application Number | 20180000988 15/708535 |
Document ID | / |
Family ID | 56321695 |
Filed Date | 2018-01-04 |
United States Patent
Application |
20180000988 |
Kind Code |
A1 |
YAMANAKA; Katsuyuki ; et
al. |
January 4, 2018 |
METHOD OF FABRICATING SCAFFOLD FOR TISSUE ENGINEERING
Abstract
A method of fabricating a scaffold for tissue engineering that
includes a frame structure including one of poly-D-lactic acid and
poly-L-lactic acid and a coating layer formed on a surface of the
frame structure and including a lactic acid-glycolic acid
copolymer. The method includes mixing a first granular porous
substance including one of poly-D-lactic acid and poly-L-lactic
acid with a second granular porous substance including the lactic
acid-glycolic acid copolymer to prepare a mixture, and pressurizing
and heating the mixture in a mold. In the heating, the mixture is
heated to a temperature greater than or equal to the melting point
of the lactic acid-glycolic acid copolymer and less than the
melting point of one of poly-D-lactic acid and poly-L-lactic
acid.
Inventors: |
YAMANAKA; Katsuyuki; (Tokyo,
JP) ; SAKAI; Yuuhiro; (Tokyo, JP) ;
SHIGEMITSU; Yusuke; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GC Corporation |
Tokyo |
|
JP |
|
|
Family ID: |
56321695 |
Appl. No.: |
15/708535 |
Filed: |
September 19, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
15168360 |
May 31, 2016 |
9795710 |
|
|
15708535 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 27/18 20130101;
A61L 2430/00 20130101; A61L 2/18 20130101; C08L 67/04 20130101;
A61L 27/54 20130101; C08L 67/04 20130101; C08L 67/04 20130101; A61L
27/18 20130101; C08L 67/04 20130101; A61L 27/56 20130101; A61L
27/34 20130101; A61L 27/34 20130101; A61L 2/18 20130101 |
International
Class: |
A61L 27/18 20060101
A61L027/18; A61L 27/34 20060101 A61L027/34; A61L 27/54 20060101
A61L027/54; A61L 27/56 20060101 A61L027/56 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 18, 2015 |
JP |
2015-123277 |
Claims
1. A method of fabricating a scaffold for tissue engineering that
includes a frame structure including one of poly-D-lactic acid and
poly-L-lactic acid, and a coating layer formed on a surface of the
frame structure and including a lactic acid-glycolic acid
copolymer, the method comprising: preparing the frame structure
including the one of poly-D-lactic acid and poly-L-lactic acid;
dispersing powder of the lactic acid-glycolic acid copolymer on the
surface of the frame structure; and heating the frame structure
after the dispersing, wherein in the heating, the frame structure
is heated to a temperature greater than or equal to a melting point
of the lactic acid-glycolic acid copolymer and less than a melting
point of the one of poly-D-lactic acid and poly-L-lactic acid.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a divisional application of U.S.
patent application Ser. No. 15/168,360 filed on May 31, 2016 and is
based upon and claims the benefit of priority of Japanese Patent
Application No. 2015-123277, filed on Jun. 18, 2015, the entire
contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0002] An aspect of this disclosure relates to a method of
fabricating a scaffold for tissue engineering.
2. Description of the Related Art
[0003] There exists a treatment where body tissue lost due to, for
example, surgery or an injury is regenerated using somatic cells or
stem cells, and the body tissue is transplanted into a patient to
recover the lost body tissue. To regenerate body tissue in this
treatment, a scaffold (matrix) is necessary to support inoculated
cells until the body tissue is regenerated by the cells.
[0004] For example, Japanese Laid-Open Patent Publication No.
2006-136673 discloses a block-shaped scaffold for tissue
engineering made of a bioabsorbable polymer. The disclosed
block-shaped scaffold for tissue engineering has a
three-dimensional porous open-pore structure with a pore diameter
of 5 to 50 .mu.m and irregular interconnected pores that occupy 20
to 80% of the cross-sectional area of the block-shaped scaffold for
tissue engineering. Also, the block-shaped scaffold for tissue
engineering has an elastic modulus of 0.1 to 2.5 MPa, and a modulus
of volume change of 95 to 105% when immersed in water for 24 hours.
Japanese Laid-Open Patent Publication No. 2006-136673 also
discloses polyglycolic acid and polylactic acid as examples of
bioabsorbable polymers.
[0005] Compared with a sponge-like scaffold for tissue engineering,
the disclosed block-shaped scaffold for tissue engineering has a
higher elastic modulus that provides excellent shape stability, and
does not greatly change in shape even when it absorbs water.
[0006] Here, there is a demand for a scaffold for tissue
engineering that can maintain its structure even when used to
culture and differentiate cells over a long period of time, and has
an excellent cell differentiation potency.
SUMMARY OF THE INVENTION
[0007] In an aspect of this disclosure, there is provided a method
of fabricating a scaffold for tissue engineering that includes a
frame structure including one of poly-D-lactic acid and
poly-L-lactic acid and a coating layer formed on a surface of the
frame structure and including a lactic acid-glycolic acid
copolymer. The method includes mixing a first granular porous
substance including one of poly-D-lactic acid and poly-L-lactic
acid with a second granular porous substance including the lactic
acid-glycolic acid copolymer to prepare a mixture, and pressurizing
and heating the mixture in a mold. In the heating, the mixture is
heated to a temperature greater than or equal to the melting point
of the lactic acid-glycolic acid copolymer and less than the
melting point of one of poly-D-lactic acid and poly-L-lactic
acid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 is a schematic cross-sectional view of a scaffold for
tissue engineering according to an embodiment.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0009] According to research conducted by the inventors of the
present invention, a block-shaped scaffold for tissue engineering
fabricated according to Japanese Laid-Open Patent Publication No.
2006-136673 by using polylactic acid as a bioabsorbable polymer
material can maintain its structure even when used to culture cells
over a long time.
[0010] However, according to the research, a block-shaped scaffold
for tissue engineering fabricated by using polylactic acid as a
bioabsorbable polymer does not always have a sufficient cell
differentiation potency.
[0011] Embodiments of the present invention are described below.
However, the present invention is not limited to those embodiments,
and variations and modifications may be made without departing from
the scope of the present invention.
First Embodiment
[0012] An exemplary method of fabricating a scaffold for tissue
engineering according to a first embodiment is described below.
[0013] A scaffold for tissue engineering fabricated by the method
of the first embodiment may include a frame structure including
poly-D-lactic acid or poly-L-lactic acid, and a coating layer
formed on a surface of the frame structure and including lactic
acid-glycolic acid copolymer. The method may include the following
processes: [0014] A mixing process of mixing a first granular
porous substance including poly-D-lactic acid or poly-L-lactic acid
with a second granular porous substance including lactic
acid-glycolic acid copolymer to prepare a mixture. [0015] A
pressurizing-heating process of pressurizing and heating the
mixture placed in a mold.
[0016] In the pressurizing-heating process, the mixture may be
heated to a temperature that is greater than or equal to the
melting point of lactic acid-glycolic acid copolymer and less than
the melting point of poly-D-lactic acid or poly-L-lactic acid
included in the first granular porous substance.
[0017] First, a scaffold for tissue engineering that can be
fabricated by the method of the present embodiment is
described.
[0018] A scaffold for tissue engineering producible by the method
of the present embodiment may include a frame structure including
poly-D-lactic acid or poly-L-lactic acid, and a coating layer
formed on a surface of the frame structure and including lactic
acid-glycolic acid copolymer.
[0019] Thus, the scaffold for tissue engineering of the present
embodiment may include a frame structure including poly-D-lactic
acid or poly-L-lactic acid.
[0020] The frame structure has a three-dimensional porous open-pore
structure including irregular interconnected pores. According to
research conducted by the inventors of the present invention,
including poly-D-lactic acid or poly-L-lactic acid in a frame
structure makes it possible to improve the strength of the frame
structure and to achieve long-term structural stability of the
frame structure during cell culture.
[0021] As a type of polylactic acid, poly-DL-lactic acid, which
includes both D-lactic acid and L-lactic acid at the same time in
the molecular chain, is also known. However, compared with a frame
structure including poly-D-lactic acid or poly-L-lactic acid, a
frame structure including poly-DL-lactic acid does not have
sufficient long-term structural stability during cell culture. For
this reason, the frame structure of the present embodiment
preferably includes poly-D-lactic acid or poly-L-lactic acid.
[0022] More preferably, the frame structure of the present
embodiment is made of poly-D-lactic acid or poly-L-lactic acid.
However, the frame structure may include an inevitable component
that inevitably enters the frame structure during, for example, a
production process.
[0023] Next, the coating layer is described. The coating layer may
include lactic acid-glycolic acid copolymer. According to research
conducted by the inventors of the present invention, when used for
the frame structure, lactic acid-glycolic acid copolymer provides
less long-term structural stability during cell culture compared
with poly-D-lactic acid and poly-L-lactic acid. However, because
lactic acid-glycolic acid copolymer has excellent capability in
holding cells to be cultured, including lactic acid-glycolic acid
copolymer in the coating layer coating the surface of the frame
structure makes it possible to improve the differentiation potency
of the scaffold for tissue engineering.
[0024] The coating layer is preferably made of lactic acid-glycolic
acid copolymer. Even in this case, however, the coating layer may
include an inevitable component that inevitably enters the coating
layer during, for example, a production process.
[0025] Next, an exemplary structure of the scaffold for tissue
engineering of the present embodiment is described. FIG. 1 is a
schematic cross-sectional view of the scaffold for tissue
engineering of the present embodiment.
[0026] As described above, the scaffold for tissue engineering has
a three-dimensional porous open-pore structure including irregular
interconnected pores. In FIG. 1, for illustration purposes, the
size of the irregular interconnected pores is made larger than
their actual size with respect to the size of the cross section.
The actual size of the interconnected pores is very small.
[0027] As illustrated by FIG. 1, a scaffold for tissue engineering
10 of the present embodiment includes a frame structure 11 and a
coating layer 12 formed on a surface(s) of the frame structure 11.
The frame structure 11 includes irregular interconnected pores
13.
[0028] As illustrated by FIG. 1, the coating layer 12 may include
an outer coating layer 121 formed on an outer surface(s) of the
frame structure 11, as well as an inner coating layer 122 formed on
an inner surface(s) of the interconnected pores 13 in the frame
structure 11.
[0029] In the example of FIG. 1, the outer coating layer 121 is
formed on the entire outer surface of the frame structure 11, and
the inner coating layer 122 is formed on the entire inner surface
of the interconnected pores 13 in the frame structure 11. However,
the present invention is not limited to this example.
[0030] For example, the outer coating layer 121 and/or the inner
coating layer 122 may be formed such that a part of the surface of
the frame structure 11 is left uncovered or exposed.
[0031] As described above, including poly-D-lactic acid or
poly-L-lactic acid in a frame structure can improve the strength of
the frame structure and give long-term structural stability during
cell culture to the frame structure. However, the cell
differentiation potency of such a frame structure is still
insufficient. For this reason, in the present embodiment, the
coating layer 12 including lactic acid-glycolic acid copolymer is
provided on the frame structure 11 to improve the cell
differentiation potency. Even when the coating layer 12 does not
completely cover the surface of the frame structure 11, compared
with a configuration where a scaffold for tissue engineering is
composed only of a frame structure including poly-D-lactic acid or
poly-L-lactic acid, the configuration of the present embodiment
makes it possible to improve the cell differentiation potency of
the scaffold for tissue engineering 10 in addition to its long-term
structural stability during cell culture.
[0032] The proportion of the coating layer 12 in the scaffold for
tissue engineering 10 is not limited to any specific value, and can
be determined based on, for example, the type or purpose of cells
to be cultured. However, to improve the differentiation potency
during cell culture, the amount of the coating layer 12 in the
scaffold 10 is preferably greater than or equal to 1 mass %, and
more preferably greater than or equal to 3 mass %.
[0033] The maximum amount of the coating layer 12 in the scaffold
for tissue engineering 10 is not limited to any specific value.
However, the amount of the coating layer 12 in the scaffold for
tissue engineering 10 is preferably less than or equal to 50 mass
%, and more preferably less than or equal to 30 mass %.
[0034] Next, the method of fabricating the scaffold for tissue
engineering of the present embodiment is described in detail.
[0035] The method of fabricating the scaffold for tissue
engineering of the present embodiment may include the following
processes: [0036] A mixing process of mixing a first granular
porous substance including poly-D-lactic acid or poly-L-lactic acid
with a second granular porous substance including lactic
acid-glycolic acid copolymer to prepare a mixture. [0037] A
pressurizing-heating process of pressurizing and heating the
mixture placed in a mold.
[0038] Each of the processes is described below.
<Mixing Process>
[0039] In the mixing process, a first granular porous substance
including poly-D-lactic acid or poly-L-lactic acid is mixed with a
second granular porous substance including lactic acid-glycolic
acid copolymer to prepare a mixture.
[0040] The first granular porous substance may be any type of
granular substance that includes poly-D-lactic acid or
poly-L-lactic acid and has a porous structure. For example, the
first granular porous substance may be prepared as described
below.
[0041] First, poly-D-lactic acid or poly-L-lactic acid is dissolved
in an organic solvent (dissolution step). Any organic solvent that
can dissolve poly-D-lactic acid or poly-L-lactic acid may be used.
For example, the organic solvent preferably includes at least one
of chloroform, dichloromethane, carbon tetrachloride, acetone,
dioxane, and tetrahydrofuran.
[0042] When dissolving poly-D-lactic acid or poly-L-lactic acid in
the organic solvent, heat treatment or ultrasonic treatment may
also be performed. The concentration of poly-D-lactic acid or
poly-L-lactic acid in a solution obtained by dissolving
poly-D-lactic acid or poly-L-lactic acid is not limited to any
specific value, and may be determined so that poly-D-lactic acid or
poly-L-lactic acid can be uniformly dispersed in the organic
solvent. Preferably, the amount of poly-D-lactic acid or
poly-L-lactic acid in the organic solvent is greater than or equal
to 1 mass % and less than or equal to 20 mass %.
[0043] Next, particulates are added to and mixed with the solution
containing poly-D-lactic acid or poly-L-lactic acid (particulate
addition step). The particulates do not dissolve in the organic
solvent in the solution, but dissolve in a liquid that does not
dissolve poly-D-lactic acid or poly-L-lactic acid.
[0044] The particulates are preferably, but are not limited to, a
water-soluble organic/inorganic salt such as sodium chloride,
potassium chloride, calcium chloride, ammonium chloride, or
trisodium citrate.
[0045] The diameter of the particulates is preferably greater than
or equal to 100 .mu.m and less than or equal to 2000 .mu.m, and
more preferably greater than or equal to 200 .mu.m and less than or
equal to 1000 .mu.m.
[0046] The amount of added particulates with respect to the
solution containing poly-D-lactic acid or poly-L-lactic acid is not
limited to any specific value, and may be determined according to
the required density of a scaffold for tissue engineering to be
fabricated. The concentration of the particulates in the solution
containing poly-D-lactic acid or poly-L-lactic acid is preferably
greater than or equal to 1.0 g/cm.sup.3 and less than or equal to
1.5 g/cm.sup.3, and more preferably greater than or equal to 1.0
g/cm.sup.3 and less than or equal to 1.25 g/cm.sup.3.
[0047] With the concentration of the particulates set at a value
greater than or equal to 1.0 g/cm.sup.3, a poly-D-lactic
acid/poly-L-lactic acid structure including the particulates
becomes very hard, and this in turn makes it easier to grind the
poly-D-lactic acid/poly-L-lactic acid structure in a grinding step
described later. With the concentration of the particulates set at
a value less than or equal to 1.5 g/cm.sup.3, the proportion of
poly-D-lactic acid or poly-L-lactic acid in a poly-D-lactic
acid/poly-L-lactic acid structure including the particulates
obtained after a freeze-drying step (described later) becomes
sufficiently high, and this in turn makes it possible to increase
the yield.
[0048] For example, the particulates may be substantially uniformly
mixed with the solution containing poly-D-lactic acid or
poly-L-lactic acid by adding the particulates to the solution,
agitating the solution as necessary, and then pouring the solution
into a mold. Also, the particulates may be mixed with the solution
containing poly-D-lactic acid or poly-L-lactic acid by pouring the
solution into a mold containing the particulates. Further, the
particulates may be mixed with the solution containing
poly-D-lactic acid or poly-L-lactic acid by putting the
particulates into a mold containing the solution.
[0049] Next, the solution containing the particulates and
poly-D-lactic acid or poly-L-lactic acid is frozen and then dried
to remove the organic solvent (freeze-drying step).
[0050] As a result of the freeze-drying step, a porous
poly-D-lactic acid/poly-L-lactic acid structure including the
particulates is obtained. At this stage, the particulates are
included in the poly-D-lactic acid/poly-L-lactic acid structure in
the form of solid.
[0051] Next, the poly-D-lactic acid/poly-L-lactic acid structure
including the particulates is ground into granules (grinding step).
Because the particulates exist in the poly-D-lactic
acid/poly-L-lactic acid structure in the form of solid (particles),
the poly-D-lactic acid/poly-L-lactic acid structure becomes hard
and can be easily ground into granules with a desired diameter.
[0052] Next, from the granules obtained by grinding the
poly-D-lactic acid/poly-L-lactic acid structure including the
particulates, the particulates are removed by using a solution that
does not dissolve poly-D-lactic acid or poly-L-lactic acid
(particulate removing step).
[0053] The method of removing the particulates may vary depending
on the substance forming the particulates. However, when the
particulates are formed of a water-soluble organic/inorganic salt
such as sodium chloride, potassium chloride, calcium chloride,
ammonium chloride, or trisodium citrate, the particulates can be
dissolved and removed with water.
[0054] Through the above steps, a first granular porous substance
(granules) is prepared. The first granular porous substance
preferably includes poly-D-lactic acid or poly-L-lactic acid, and
more preferably made of poly-D-lactic acid or poly-L-lactic acid.
Still, however, the first granular porous substance may include an
inevitable component such as a residue of the particulates that
remains after the above steps.
[0055] After removing the particulates, the granules may be
screened (or sieved) to obtain the first granular porous substance
with a desired diameter.
[0056] The diameter of the first granular porous substance supplied
to a pressurizing-heating step described later is preferably
greater than or equal to 100 .mu.m and less than or equal to 3000
.mu.m.
[0057] The scaffold for tissue engineering of the present
embodiment includes interconnected pores so that cells can be held
in the interconnected pores. This configuration makes it possible
to improve the efficiency of cell culture. According to research
conducted by the inventors of the present invention, a number of
interconnected pores sufficient to improve the efficiency of cell
culture can be formed in the scaffold for tissue engineering by
making the diameter of the first granular porous substance supplied
to the pressurizing-heating step greater than or equal to 100 .mu.m
and less than or equal to 3000 .mu.m.
[0058] The second granular porous substance may be any type of
granular substance that includes lactic acid-glycolic acid
copolymer and has a porous structure, and may be prepared by any
appropriate method. For example, the second granular porous
substance may be prepared by a method similar to the method of
preparing the first granular porous substance described above, by
dissolving lactic acid-glycolic acid copolymer instead of
poly-D-lactic acid or poly-L-lactic acid at the dissolution
step.
[0059] The second granular porous substance preferably includes
lactic acid-glycolic acid copolymer, and is more preferably made of
lactic acid-glycolic acid copolymer. Still, however, the second
granular porous substance may include an inevitable component such
as a residue of particulates that remains after the preparation
steps.
[0060] Then, a mixture is prepared by mixing the first granular
porous substance and the second granular porous substance prepared
as described above.
[0061] The proportions of the first granular porous substance and
the second granular porous substance in the mixture may be
determined at any appropriate values. For example, the proportion
of the second granular porous substance in the mixture is
preferably greater than or equal to 1 mass % and less than or equal
to 50 mass %, and more preferably greater than or equal to 3 mass %
and less than or equal to 30 mass %.
[0062] Setting the proportion of the second granular porous
substance at a value greater than or equal to 1 mass % makes it
possible to form a uniform coating layer and thereby makes it
possible to improve the cell differentiation potency. Also, setting
the proportion of the second granular porous substance at a value
less than or equal to 50 mass % makes it possible to improve the
strength and the long-term structural stability of the frame
structure.
[0063] The first granular porous substance and the second granular
porous substance may be mixed by any appropriate method for mixing
solids.
<Pressurizing-Heating Process>
[0064] Next, a pressurizing-heating process is described. In the
pressurizing-heating process, the mixture is placed in a mold, and
pressurized and heated.
[0065] Although the pressurization condition in the
pressurizing-heating process may vary depending on the shape and
size of a frame structure to be produced, the pressurization
condition is preferably greater than or equal to 500 g/cm.sup.2 and
less than or equal to 3000 g/cm.sup.2, and more preferably greater
than or equal to 1000 g/cm.sup.2 and less than or equal to 2000
g/cm.sup.2.
[0066] Setting the pressurization condition at a value greater than
or equal to 500 g/cm.sup.2 makes it possible to improve the shape
stability of the frame structure, and setting the pressurization
condition at a value less than or equal to 3000 g/cm.sup.2 makes it
possible to form a sufficient number of interconnected pores for
growing cells.
[0067] The heating temperature in the pressurizing-heating process
is preferably greater than or equal to the melting point of lactic
acid-glycolic acid copolymer and less than the melting point of
poly-D-lactic acid or poly-L-lactic acid included in the first
granular porous substance. More specifically, the heating
temperature is preferably greater than or equal to 40.degree. C.
and less than or equal to 105.degree. C., and more preferably
greater than or equal to 55.degree. C. and less than or equal to
95.degree. C.
[0068] By heating the mixture at a temperature greater than or
equal to the melting point of lactic acid-glycolic acid copolymer
and less than the melting point of poly-D-lactic acid or
poly-L-lactic acid included in the first granular porous substance,
it is possible to melt only lactic acid-glycolic acid copolymer in
the mixture while maintaining the frame structure. Consequently,
lactic acid-glycolic acid copolymer wets and spreads over the
surface of the frame structure and forms a coating layer. As a
result, a scaffold for tissue engineering, which includes the frame
structure including poly-D-lactic acid or poly-L-lactic acid and
the coating layer formed on the surface of the frame structure and
including lactic acid-glycolic acid copolymer, is produced.
[0069] As described above, the scaffold for tissue engineering
fabricated by the method of the present embodiment includes a frame
structure having long-term structural stability during cell
culture, and a coating layer that is formed on a surface of the
frame structure and can facilitate the differentiation of cells.
Thus, the method of the present embodiment can fabricate a scaffold
for tissue engineering that has both long-term structural stability
during cell culture and excellent cell differentiation potency.
Second Embodiment
[0070] An exemplary method of fabricating a scaffold for tissue
engineering according to a second embodiment is described
below.
[0071] A scaffold for tissue engineering fabricated by the method
of the second embodiment may include a frame structure including
poly-D-lactic acid or poly-L-lactic acid, and a coating layer
formed on a surface of the frame structure and including lactic
acid-glycolic acid copolymer. The method may include the following
processes: [0072] A frame structure preparation process of
preparing a frame structure including poly-D-lactic acid or
poly-L-lactic acid. [0073] A heating process of heating the frame
structure after dispersing powder of lactic acid-glycolic acid
copolymer on a surface of the frame structure.
[0074] In the heating process, the frame structure may be heated to
a temperature that is greater than or equal to the melting point of
lactic acid-glycolic acid copolymer and less than the melting point
of poly-D-lactic acid or poly-L-lactic acid included in the frame
structure.
[0075] Each of the processes is described below.
<Frame Structure Preparation Process>
[0076] First, the frame structure preparation process is described.
In the frame structure preparation process, a frame structure
including poly-D-lactic acid or poly-L-lactic acid is prepared.
[0077] The frame structure preferably has a three-dimensional
porous open-pore structure including irregular interconnected
pores.
[0078] For example, the frame structure may be prepared as
described below.
[0079] The frame structure may be prepared by placing porous
granules including poly-D-lactic acid or poly-L-lactic acid
(poly-D-lactic acid/poly-L-lactic acid granules) in a desired mold,
and pressurizing and heating the granules (pressurizing-heating
step).
[0080] The poly-D-lactic acid/poly-L-lactic acid granules may be
prepared by substantially the same method as the method of
preparing the first granular porous substance described in the
first embodiment, and therefore descriptions of the method are
omitted here.
[0081] Although the pressurization condition in the
pressurizing-heating step may vary depending on the shape and size
of a frame structure to be prepared, the pressurization condition
is preferably greater than or equal to 500 g/cm.sup.2 and less than
or equal to 3000 g/cm.sup.2, and more preferably greater than or
equal to 1000 g/cm.sup.2 and less than or equal to 2000
g/cm.sup.2.
[0082] Setting the pressurization condition at a value greater than
or equal to 500 g/cm.sup.2 makes it possible to improve the shape
stability of the frame structure, and setting the pressurization
condition at a value less than or equal to 3000 g/cm.sup.2 makes it
possible to form a sufficient number of interconnected pores for
growing cells.
[0083] The heating condition in this step may also vary depending
on the shape and size of the frame structure. When the
poly-D-lactic acid/poly-L-lactic acid granules are to be heated
under the above pressurization condition while maintaining their
volume, the heating condition is preferably greater than or equal
to 60.degree. C. and less than or equal to 200.degree. C.
[0084] Heating the poly-D-lactic acid/poly-L-lactic acid granules
at a temperature greater than or equal to 60.degree. C. makes it
possible to increase the bond between the poly-D-lactic
acid/poly-L-lactic acid granules, and thereby makes it possible to
improve the shape stability of the frame structure. Also, heating
the poly-D-lactic acid/poly-L-lactic acid granules at a temperature
less than or equal to 200.degree. C. makes it possible to prevent
denaturation of poly-D-lactic acid or poly-L-lactic acid.
<Heating Process>
[0085] Next, the heating process is described. In the heating
process, the frame structure is heated after dispersing powder of
lactic acid-glycolic acid copolymer on a surface of the frame
structure.
[0086] The particle diameter of powder of lactic acid-glycolic acid
copolymer dispersed on the surface of the frame structure is not
limited to any specific value. However, powder of lactic
acid-glycolic acid copolymer with a small particle diameter is
preferable because it can easily enter interconnected pores in the
frame structure and can form a uniform coating layer on the surface
of the frame structure.
[0087] For example, the average particle diameter of the powder of
lactic acid-glycolic acid copolymer is preferably greater than or
equal to 100 .mu.m and less than or equal to 710 .mu.m, and more
preferably greater than or equal to 150 .mu.m and less than or
equal to 500 .mu.m.
[0088] Here, the average particle diameter is represented by a
particle diameter at a cumulative volume of 50% in a particle size
distribution obtained by a laser diffraction and scattering
method.
[0089] The amount of the powder of lactic acid-glycolic acid
copolymer dispersed on the frame structure may be determined at any
appropriate value. For example, the proportion of the powder of
lactic acid-glycolic acid copolymer in the total mass of the frame
structure and the powder of lactic acid-glycolic acid copolymer is
preferably greater than or equal to 1 mass % and less than or equal
to 50 mass %, and more preferably greater than or equal to 3 mass %
and less than or equal to 30 mass %.
[0090] Setting the proportion of the powder of lactic acid-glycolic
acid copolymer at a value greater than or equal to 1 mass % makes
it possible to form a uniform coating layer and thereby makes it
possible to improve the cell differentiation potency. Also, setting
the proportion of the powder of lactic acid-glycolic acid copolymer
at a value less than or equal to 30 mass % makes it possible to
improve the strength and the long-term structural stability of the
frame structure.
[0091] After the powder of lactic acid-glycolic acid copolymer is
dispersed on the surface of the frame structure, the frame
structure is heated. The heating temperature in the heating process
is preferably greater than or equal to the melting point of lactic
acid-glycolic acid copolymer and less than the melting point of
poly-D-lactic acid or poly-L-lactic acid included in the frame
structure. More specifically, the heating temperature is preferably
greater than or equal to 40.degree. C. and less than or equal to
105.degree. C., and more preferably greater than or equal to
55.degree. C. and less than or equal to 95.degree. C.
[0092] By heating the frame structure at a temperature greater than
or equal to the melting point of lactic acid-glycolic acid
copolymer and less than the melting point of poly-D-lactic acid or
poly-L-lactic acid included in the frame structure, it is possible
to melt only lactic acid-glycolic acid copolymer dispersed on the
surface of the frame structure while maintaining the frame
structure itself. Consequently, lactic acid-glycolic acid copolymer
wets and spreads over the surface of the frame structure and forms
a coating layer. As a result, a scaffold for tissue engineering,
which includes the frame structure including poly-D-lactic acid or
poly-L-lactic acid and the coating layer formed on the surface of
the frame structure and including lactic acid-glycolic acid
copolymer, is produced.
[0093] The scaffold for tissue engineering fabricated by the method
of the second embodiment has substantially the same configuration
as that of the scaffold for tissue engineering fabricated by the
method of the first embodiment. Accordingly, the scaffold for
tissue engineering fabricated by the method of the second
embodiment also includes a frame structure having long-term
structural stability during cell culture, and a coating layer that
is formed on a surface of the frame structure and can facilitate
the differentiation of cells. Thus, the method of the second
embodiment can also fabricate a scaffold for tissue engineering
that has both long-term structural stability during cell culture
and excellent cell differentiation potency.
EXAMPLES
[0094] Examples below are provided to facilitate understanding of
the present invention. However, the present invention is not
limited to those examples.
Example 1
[0095] A scaffold for tissue engineering was fabricated as
described below.
(Mixing Process)
[0096] A first granular porous substance made of poly-L-lactic acid
was prepared through the steps described below.
[0097] First, poly-L-lactic acid (about 250,000 weight-average
molecular weight) was added to 1,4-dioxane such that the
concentration of poly-L-lactic acid would become 6 mass %, and the
resulting mixture was agitated with an agitator to dissolve
poly-L-lactic acid and obtain a 1,4-dioxane solution containing
poly-L-lactic acid (dissolution step).
[0098] Next, trisodium citrate powder (with a particle diameter
between 200 .mu.m and 500 .mu.m) was substantially uniformly mixed
with the 1,4-dioxane solution containing poly-L-lactic acid such
that the concentration of trisodium citrate powder would become
about 1.02 g/cm.sup.3 (particulate addition step), and the
resulting solution was poured into a mold.
[0099] The solution was frozen at -30.degree. C. using a freezer
(SANYO Electric Co., Ltd., product name: MDF-0281AT). Then, the
frozen solution was dried for 48 hours under reduced pressure using
a vacuum dryer (Yamato Scientific Co., Ltd., product name: DP43) to
remove 1,4-dioxane and obtain a polymer material of poly-L-lactic
acid substantially uniformly containing trisodium citrate powder
(freeze-drying step).
[0100] The polymer material was cut into pieces, and the pieces
were ground for 20 minutes using a planetary pot mill (grinding
step).
[0101] The ground polymer material was put into a flask, distilled
water was added to the flask, and the resulting mixture was
agitated to remove trisodium citrate powder (particulate removing
step). After removing trisodium citrate powder, the ground polymer
material was placed in a petri dish and was dried for 48 hours
using a vacuum drier. Then, the ground and dried polymer material
was screened to obtain a first granular porous substance (granules)
made of poly-L-lactic acid and having a particle diameter between
700 .mu.m and 1400 .mu.m and an average pore diameter of about 5
.mu.m.
[0102] A second granular porous substance was prepared by a method
similar to the above-described method of preparing the first
granular porous substance except that lactic acid-glycolic acid
copolymer was added to 1,4-dioxane at a concentration of 6 mass %
in the dissolution step.
[0103] A mixture was prepared by mixing the first granular porous
substance and the second granular porous substance such that the
proportion of the second granular porous substance in the mixture
would become 10 mass %.
(Pressurizing-Heating Process)
[0104] The prepared mixture was placed in a glass container with an
inside diameter of 9 mm and a height of 10 mm up to a height of
about 7 mm. Then, the mixture was heated at 50.degree. C. for 20
minutes, while maintaining the volume of the mixture being
pressurized at 1500 g/cm.sup.2 by a titanium rod having a diameter
of 9 mm. As a result, a columnar scaffold for tissue engineering
with a diameter of 9 mm and a height of about 7 mm was
obtained.
Example 2
[0105] In Example 2, a scaffold for tissue engineering was
fabricated and evaluated in substantially the same manner as in
Example 1 except that a first granular porous substance made of
poly-D-lactic acid was used.
[0106] The first granular porous substance made of poly-D-lactic
acid was prepared by a method similar to the method of preparing
the first granular porous substance made of poly-L-lactic acid in
Example 1 except that in the dissolution step, poly-D-lactic acid
(about 250,000 weight-average molecular weight) was added to
1,4-dioxane such that the concentration of poly-D-lactic acid would
become 6 mass % and the resulting mixture was agitated with an
agitator.
Example 3
[0107] A scaffold for tissue engineering was fabricated as
described below.
(Frame Structure Preparation Process)
[0108] A frame structure preparation process was performed as
described below to prepare a frame structure including
poly-L-lactic acid.
[0109] First, poly-L-lactic acid (about 250,000 weight-average
molecular weight) was added to 1,4-dioxane such that the
concentration of poly-L-lactic acid would become 6 mass %, and the
resulting mixture was agitated with an agitator to dissolve
poly-L-lactic acid and obtain a 1,4-dioxane solution containing
poly-L-lactic acid (dissolution step).
[0110] Next, trisodium citrate powder (with a particle diameter
between 200 .mu.m and 500 .mu.m) was substantially uniformly mixed
with the 1,4-dioxane solution containing poly-L-lactic acid such
that the concentration of trisodium citrate powder would become
about 1.02 g/cm.sup.3 (particulate addition step), and the
resulting solution was poured into a mold.
[0111] The solution was frozen at -30.degree. C. using a freezer
(SANYO Electric Co., Ltd., product name: MDF-0281AT). Then, the
frozen solution was dried for 48 hours under reduced pressure using
a vacuum dryer (Yamato Scientific Co., Ltd., product name: DP43) to
remove 1,4-dioxane and obtain a polymer material of poly-L-lactic
acid substantially uniformly containing trisodium citrate powder
(freeze-drying step).
[0112] The polymer material was cut into pieces, and the pieces
were ground for 20 minutes using a planetary pot mill (grinding
step).
[0113] The ground polymer material was put into a flask, distilled
water was added to the flask, and the resulting mixture was
agitated to remove trisodium citrate powder (particulate removing
step). After removing trisodium citrate powder, the ground polymer
material was placed in a petri dish and was dried for 48 hours
using a vacuum drier. Then, the ground and dried polymer material
was screened to obtain porous poly-L-lactic acid granules with a
diameter between 700 .mu.m and 1400 .mu.m and an average pore
diameter of about 5 .mu.m.
[0114] The obtained porous poly-L-lactic acid granules were placed
in a glass container with an inside diameter of 9 mm and a height
of 10 mm up to a height of about 7 mm. The porous poly-L-lactic
acid granules were heated at 180.degree. C. for 30 minutes, while
maintaining the volume of the porous poly-L-lactic acid granules
being pressurized at 1500 g/cm.sup.2 by a glass rod having a
diameter of 9 mm (pressurizing-heating step), to obtain a columnar
frame structure with a diameter of 9 mm and a height of about 4
mm.
[0115] The obtained frame structure was observed with an electron
microscope, and it was confirmed that the frame structure had a
three-dimensional porous open-pore structure having pores in
partitioning walls and also including irregular interconnected
pores.
(Heating Process)
[0116] Next, the obtained frame structure was placed in a heating
furnace for heat processing, and powder of lactic acid-glycolic
acid copolymer with an average particle diameter of 200 .mu.m was
dispersed on the surface of the frame structure. The powder of
lactic acid-glycolic acid copolymer was dispersed such that the
proportion of the powder of lactic acid-glycolic acid copolymer in
the total mass of the frame structure and the powder of lactic
acid-glycolic acid copolymer would become 20 mass %.
[0117] Then, the frame structure with the powder of lactic
acid-glycolic acid copolymer was heated at 95.degree. C. for 20
minutes to obtain a scaffold for tissue engineering.
Example 4
[0118] In Example 4, a scaffold for tissue engineering was
fabricated and evaluated in substantially the same manner as in
Example 3 except that a frame structure including poly-D-lactic
acid was used.
[0119] The frame structure including poly-D-lactic acid was
prepared in substantially the same manner as in Example 3 except
that in the dissolution step of the frame structure preparation
process, poly-D-lactic acid (about 250,000 weight-average molecular
weight) was added to 1,4-dioxane such that the concentration of
poly-D-lactic acid would become 6 mass % and the resulting mixture
was agitated with an agitator.
[0120] An aspect of this disclosure makes it possible to provide a
method of fabricating a scaffold for tissue engineering that has
both long-term structural stability during cell culture and
excellent cell differentiation potency.
[0121] Methods of fabricating a scaffold for tissue engineering
according to embodiments are described above. However, the present
invention is not limited to the specifically disclosed embodiments,
and variations and modifications may be made without departing from
the scope of the present invention.
* * * * *