Note: Descriptions are shown in the official language in which they were submitted.
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SCAFFOLD FOR TISSUE ENGINEERING, ARTIFICIAL BLOOD VESSEL,
CUFF, AND BIOLOGICAL IMPLANT COVERING MEMBER
TECHNICAL FIELD
The present invention relates to a scaffold material
for tissue engineering, an artificial blood vessel, a
cuff and a biological implant covering member.
BACKGROUND ART
The present invention relates, in the first place,
to a porous scaffold for tissue engineering which allows
easy cell engraftment and cell culture and thus enables
stable organization, and to an artificial blood vessel
using this scaffold. The scaffold and the artificial
blood vessel of the present invention are effectively
used not only for basic studies on biotechnologies, but
also for biomedical materials used as artificial bone
structure substrates for substitute medicine by an
artificial internal organ or for regenerative medicine by
tissue engineering, and especially, for an artificial
blood vessel which can exhibit high patency rate even if
the inner diameter is small, less than 6 mm, as the
endothelial cells have a nature of being engrafted all
over the luminal surface.
Conventionally, as a scaffold material for tissue
engineering, substrates, such as polystyrene dish
(schale) or polyester mesh, coated by an extracellular
matrix such as collagen are employed commonly in
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monolayer culture. As another culture form other than
monolayer culture, there are spheroids by shake culture
or embedding culture using collagen gel. Especially the
embedding culture using collagen gel is advantageous
because it enables in vivo culture, that is, causes a
growth of cells in three dimensional structure, thus
enabling basic studies on cell function while it was
insufficient in monolayer culture.
In conventional artificial blood vessels, tubes
made of polyester resin mesh or PTFE resin mesh have been
in practical use from a long time ago, and works
challenging for smaller caliber or for better patency
rate has been proceeding. Primary techniques discussed
until today are segmented polyurethane tubes which have
been employed as antithrombotic material in practical use
and artificial blood vessel material having a surface to
which an antithrombotic material such as heparin is fixed
using graft chain and the like.
Collagen gels for embedding culture do not have a
porous structure such as three-dimensional network
structure, and there remains a problem that it is
impossible to obtain uniform cell engraftment on the
whole surface or impossible to adjust the distribution of
engraftment are not achieved. Although methods employing
salt or bubbles are known as method of preparing a porous
material having a three-dimensional network structure,
any has difficulty in strictly and discretionary
adjusting the pore diameter and pore density, so a
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scaffold comprising appropriate three-dimensional network
structure is still not fulfilled.
Cell engraftment structure achieved by collagen gel
embedding culture can not used for applications to be
subjected to mechanical load such as artificial blood
vessel while it is available for evaluating cell function
because collagen gel as the scaffold thereof does not
have physical strength.
Though artificial blood vessels as alternative
materials for autologous blood vessels are used in
clinical application broadly, smaller diameter artificial
blood vessels have poor patency rate. Therefore for the
current situation, autologous vein transplantations are
still employed for coronary bypass operations, peripheral
artery reconstorations requiring smaller diameter blood
vessels. For the present primary techniques discussing
smaller diameter pursuing only antithrombogenicity, only
a pannus is formed in these conventional artificial blood
vessel, but endodermis would not be formed. Accordingly,
artificial blood vessels having smaller diameter have low
patency rate. In addition, since wall does not have a
hole through which the cell would enter, even if the
pannus extends from the inosculated part, it would not be
bonded to the wall and would float and many cases of
resulting occlusion of blood vessels have been reported.
The invention, in the second place, relates to a
cuff which enables cell penetration from the native
tissue and enables a robust bonding to the native tissue,
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and especially relates to a cuff effective for blood
circulation method by ventricular assist device, which is
a treatment implanting a cannula or catheter
subcutaneously, peritoneal dialysis therapy, central
intravenous infusion nutrition, and for the implant part
of living skin for such as transcannula DDS,
transcatheter DDS, or the like.
The recently developed therapy such as ventricular
assist device or peritoneal dialysis employs cannula or
catheter which needs insertion under the skin and
placement within the living body unlike urethra catheters,
transgastrointestinal tract nutrition, and management of
airway. If the placement within the living body would be
of long period, for separation of living body from
outside of the body and preventing intrusion of germs
within the living body or evaporation of body fluid, a
cuff (also said as skin cuff) would be used to
artificially seal the insertion point. Conventionally, in
blood circulation method by ventricular assist device,
fabric velour typically made of polyester fiber would be
tied around the inserting cannula, and fixing by suturing
the fabric velour and subcutaneous tissue to place the
cannula. Also in peritoneal dialysis, fabric velour made
of polyester fiber or the like would be fixed as a cuff
at the location of insertion under the skin of catheter,
and subcutaneous tissue would be sutured as the cuff
being oppressed to place the catheter. There is fabric
velour impregnated with collagen and objected for robust
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bonding. In addition, there are methods fixing a cuff,
which is made of a biocompatible material, to
subcutaneous tissue of the insertion point.
However, in the blood circulation method using
ventricular assist device, since it is a therapy
assisting the blood circulation by a pulsating pump set
outside the body of the patient, vibrations corresponding
to 1.5Hz from the pulsating pump are transmitted to the
cannula. In other words, the insertion point of the
cannula always undergoes dynamic load by vibrations.
Moreover, stress occurs to denude the adhesive interface
between the subcutaneous tissue and the cuff by movement
of the cannula while the patient moves his or her body
position or the disinfection process to the insertion
point. Troubles that would be caused due to these
stresses causing a lowering of adhesion between the cuff
and the subcutaneous tissue include, as typical trouble,
infection trouble such as tunnel infection. In cases of
ventricular assist device therapy, such infection trouble
experiences are being very frequent. Under existing
conditions that there are a lot of cases that therapy has
to be aborted due to bacterial infection not due to
cardiac failure, it may be said that the therapy needs an
urgent task of developing a cuff capable of preventing
bacterial infection.
In peritoneal dialysis in which a catheter is
inserted under the skin and placed for a long period,
there remains a momentous problem on cuffs. That is, in
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this therapy, the catheter is placed within the abdominal
cavity in order to inject or discharge dialyzing fluid.
However, the living body recognizes the catheter as a
foreign substance and therefore acts to reject the
catheter so that the adhesion between the subcutaneous
tissue and the catheter would not be made, thus causing a
downgrowth phenomenon that the skin surface barges into
the abdominal cavity along the catheter. This pocket of
downgrowth makes reach of disinfectant difficult,
triggering inflammation of skin or tunnel infection and
finally resulting in induction of peritonitis.
Considering reports that patients experiencing frequent
peritonitis of Pseudomonas aeruginosa increased incidence
of SEP (sclerosing encapsulating peritonitis), the
improvement of cuff to prevent infection would be a
momentous object on the peritoneal dialysis therapy.
As described above, cuffs consisting primarily of
collagen have been developed. However, in the case of
this kind of cuff, the volume would decrease by absorbing
liquid such as normal saline solution, alcohol, Isodine,
blood and/or body fluid so that it is difficult to breed
the subcutaneous tissue on the location of the insertion
of the catheter. As a result, inhibiting effect of
downgrowth is not attained.
The invention, in the third place, relates to a
biological implant covering member, which covers the
surface of a biological implantation member such as
artificial heart valve, artificial heart valve ring,
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artificial blood vessel, artificial breast, artificial
bone, artificial joint and artificial heart or other
associated parts thereof, thereby reducing the foreign-
body reaction in the living body.
Conventionally, constituent materials for
biological implantation member such as artificial heart
valve, artificial heart ring, artificial blood vessel,
artificial breast, artificial bone, artificial joint and
artificial heart, and the like and other associated parts
thereof have been studied mainly with a focus on
materials that generate no or little eluate and is
chemically inactive causing no or little stimulation to
the surrounding tissue, and would be immunologically
neglected by the living body. Examples of those materials
include metal materials such as titanium, stainless steel
and platinum, ceramic materials such as hydroxyapatite
and polymeric materials such as polytetrafluoroethylene,
polyester and polypropylene, and are in practical use for
various applications. For instance, metallic materials
are used for intravascular stent, bone fixing bolt, and
artificial joint. Ceramic materials are used as, for
example, artificial joints and artificial bones for
filling or substituting deficient parts of joints and
bones. Polymeric materials have been put in practical use
as artificial blood vessel for retaining blood flow after
aneurysmectomy, suture thread for suturing a part which
needs incision once again for enabling suture removal,
artificial trachea, and artificial breast for prosthetic
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surgery of the lost breast caused by breast canser
incision or for breast enlargement in plastic surgery.
Metallic materials for biological implantation, for
example, a stent to be placed within the blood vessel
would consist primarily of good rust prevention stainless
steel. However, in case of long period placement within
the blood vessel, the stent is constantly exposed to
various electrolytes, protein, lipid containing blood so
that rust would form and possibly result in irritation of
the surrounding tissue.
Mainstream artificial breasts in practical use are
made of a silicone bag filled with normal saline solution
and the like. However, the loculated collagen tissue
would be thickened and contract on the surface after
subcutaneous implant, and in this case, there was a
problem that the silicone bag would deform within the
living body, compressing the surrounding tissue, evoking
inflammation reaction, or making breast cancer to recur.
As for an artificial trachea, products composing of
silicone tube have been put in practical use, however, it
has no affinity for living tracheas, and had a problem
that it would detach during long-term implant or cause
infection on the boundary face.
In the case of implantable artificial heart, for
example, the vibrational inertia of the driving motor
results in a problem of pocket infection, which is
occurred by the inflammation or infection on the native
tissue boundary surface.
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SUMMARY OF THE INVENTION
It is an object of the invention according to the
first aspect to provide a scaffold material for tissue
engineering which comprises a homogeneous porous body
having a three-dimensional network structure, allows
cells to be uniformly engrafted all over the inside of
the porous body thereof, is excellent in physical
strength, and is effectively used not only for basic
studies on biotechnologies, but also for an artificial
blood vessel which can exhibit high patency rate for a
long period of time even if the inner diameter is small,
less than 6 mm, and to provide an artificial blood. vessel
using this scaffold for tissue engineering.
A scaffold material for tissue engineering of the
present invention is a scaffold material for tissue
engineering made of thermoplastic resin forming a porous
three-dimensional network structure having communication
property, wherein the porous three-dimensional network
structure has an average pore diameter of from 100 to
650pm and an apparent density of from 0.01 to 0.5 g/cm3.
Since the scaffold for tissue engineering of the
present invention has the porous three-dimensional
network structure made of thermoplastic resin and having
the certain average pore diameter and the certain
apparent density mentioned above, cells and collagen
suspension are allowed to easily penetrate into pores of
the porous three-dimensional network structure. Therefore,
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cells can be seeded all over the porous three-dimensional
network structure. For example, an artificial peritoneal
composed of two layers of mesothelial cell and fibrocyte
can be obtained. It is expected that the scaffold is used
for analyzing the mechanism of glycosylation in
peritoneal dialysis and for basic study of the dialysis.
When the scaffold for tissue engineering is used as an
artificial blood vessel, vascular endothelial cell can be
present in the luminal surface of the artificial blood
vessel so that occlusion hardly occurs. As a result, it
is possible to achieve an artificial blood vessel of
small diameter.
The artificial blood vessel of the present
invention is composed of the scaffold of the present
invention, can exhibit high patency rate even if the
inner diameter is small, less than 6 mm, and is therefore
effectively applied to coronary bypass operations,
peripheral arterial reconstoration, and the like.
It is an object of the invention according to the
second aspect to provide a cuff which allows easy
infiltration of cells from living subcutaneous tissues,
easy engraftment of cells, and neovascularization of
capillary vessels so as to obtain robust bonding with
subcutaneous tissues, thereby inhibiting progression of
downgrowth, and therefore has none or little risk of
infection trouble such as tunnel infection.
A cuff of the present invention comprises a porous
three-dimensional network structure which is made of
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thermoplastic resin or thermosetting resin and has
communication property, wherein the porous three-
dimensional network structure has an average pore
diameter of from 100 to 10001lm and apparent density of
from 0.01 to 0.5g/cm3.
Since the cuff of the present invention has a
porous three-dimensional network structure which is made
of thermoplastic resin or thermosetting resin and has
communication property and which has the certain average
pore diameter and the certain apparent density mentioned
above, the cuff allows easy infiltration of cells into
pores of the porous three-dimensional structure and easy
engraftment of cells so as to obtain robust bonding with
living tissues.
It is an object of the invention according to the
third aspect to provide a biological implant covering
member which allows easy infiltration of cells from
living subcutaneous tissues, easy engraftment of cells,
and organization, thereby obtaining robust bonding with
native tissues and therefore protecting a living body
from adverse effect which may occur due to the insertion
of a biological implantation member into the living body.
A biological implant covering member of the present
invention comprises a porous three-dimensional network
structure which is made of thermoplastic resin or
thermosetting resin and has communication property,
wherein the porous three-dimensional network structure
has an average pore diameter of from 100 to 1000pm and
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apparent density of from 0.01 to 0.5g/cm3.
Since the biological implant covering member of the
present invention has a porous three-dimensional network
structure which is made of thermoplastic resin or
thermosetting resin and has communication property and
which has the certain average pore diameter and the
certain apparent density mentioned above, the biological
implant covering member allows easy infiltration of cells
into pores of the porous three-dimensional structure,
easy engraftment of cells, and neovascularization of
capillary vessels so as to obtain robust bonding with
native tissues.
The biological implant covering member of the
present invention has a porous three-dimensional network
structure which enables penetration and engraftment of
cells and neovascularization of capillary vessels.
Therefore, a biological implant covering member of
the present invention is used to cover the surface of a
biological implantation member such as artificial heart
valve, artificial heart valve ring, artificial blood
vessel, artificial breast, artificial bone, artificial
joint and artificial heart or other associated parts
thereof, thereby reducing the foreign-body reaction
against the biological implantation member by peripheral
tissues.
The biological implantation member means an object
to be implanted into a living body and includes a system
composed of various parts. Examples are, as for an
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artificial heart system, an actuator (energy converter)
as an in vivo driving unit,,left and right blood pumps as
pumps, an atrial cuff, an atrial connector, an artery
graft and an artery connector, an in vivo secondary coil
in a percutaneous energy transfer system, an in vivo unit
in a percutaneous information transfer system, an in vivo
battery in a buttery system, an in vivo control unit in a
control system, and a compliance chamber, a volume
displacement chamber, and a bent tube in a volume
displacement system. Beside these, there are examples a
device composed of a large number of parts such as in
vivo unit connecting cable and connector. In the present
invention, all of these are called as biological
implantation member.
The biological implant covering member may be used
for purposes other than clinical purposes and may be used
to cover the outer surface of a transmitter to be
implanted into an animal body for the purpose of
ecological survey, thereby reducing the foreign-body
reaction.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a SEM (scanning electron microscope)
picture (x20) showing the entire tubular structure of a
scaffold material made in Example 1;
Fig. 2 is a stereoscopic microscope picture (x100)
showing a fine structure inside the tubular structure of
the scaffold made in Example 1;
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Fig. 3 is a SEM picture (x20) showing a surface
layer of the inner wall of the tubular structure of the
scaffold made in Example 1;
Fig. 4 is a SEM picture (x20) showing a surface
layer of the outer periphery of the tubular structure of
the scaffold made in Example 1;
Fig. 5 is a SEM picture (x10) showing a porous
three-dimensional network structure containing cells made
in Example 2 after three days of incubation;
Fig. 6 is an optical microscope picture (x10)
showing that interior tissues are engrafted on entire
surface even after one week of additional incubation in
Example 2,
Fig. 7 is a picture showing a scene where
bloodstream is obtained by artificial blood vessels, thus
occurring heartbeat in Example 3;
Fig. 8 is a picture showing that no blood clot is
generated in the inside of the artificial blood vessels
after one week from implantation in Example 3;
Fig. 9 is a SEM picture (x50) showing a surface
layer of a tubular structure made in Comparative Example
1;
Fig. 10 is a SEM picture (x50) showing a fine
structure inside the tubular structure made iri
Comparative Example 1;
Fig. 11 is an optical microscope picture (xlO)
showing a tubular structural material containing cells
made in Comparative Example 2 after three days of
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incubation;
Fig. 12 is a SEM picture (x50) showing a surface of
a tissue contact side of a cuff made in Example 4;
Fig. 13 is a SEM picture (x50) showing an inner
section of the cuff made in Example 4;
Fig. 14 is a distribution chart obtained by
measuring distribution in pore diameter of the cuff made
in Example 4;
Fig. 15 is a picture just after an operation of
implanting a cuff made in Example 4 into an incised part
of chest of a goat and fixing the cuff by suturing
subcutaneous tissues; and
Fig. 16a is an enlarged picture showing tissues
surrounding the test piece after the cuff made in Example
4 was implanted into the incised part of chest of a goat
for two weeks and then removed and Fig. 16b is an
enlarged picture showing tissues surrounding the test
piece in case that the same test was conducted using a
fabric for comparison.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Hereinafter, embodiments of scaffolds for tissue
engineering and artificial blood vessels of the present
invention will be studied in detail.
The scaffold for tissue engineering of the present
invention is made of thermoplastic resin forming a three-
dimensional network structure. The three-dimensional
network structural layer is a porous three-dimensional
CA 02484012 2004-09-27
network structural layer which has an average pore
diameter of from 100 to 650pm and an apparent density of
from 0.01 to 0.5 g/cm3 and has communication property,
that is, has continuous pores. The three-dimensional
network structural layer may be formed to have allover
similar configuration from the inner wall to the outer
wall and may be formed such that the configuration at
portions near the inner wall is different from the
configuration at portions near the outer wall. In
addition, the average pore diameter and the apparent
density may vary partially. For example, the average pore
diameter may vary gradually from the inner wall to the
outer wall, that is, the three-dimensional network
structural layer may have anisotropy.
It should be noted that the "porous three-
dimensional network structural layer" is referred as --
porous three-dimensional network structure -- hereinafter.
As for the three-dimensional network structure
composed of the thermoplastic resin, the average pore
diameter is from 100 to 650pm and the apparent density is
from 0.01 to 0.5 g/cm3 as mentioned above. The average
pore diameter is preferably from 100 to 400pm, more
preferably from 100 to 300pm. The apparent density of
from 0.01 to 0.5 g/cm3 can provide well cell engraftment,
excellent physical strength, and elastic characteristics
similar to that of living body. The apparent density is
preferably from 0.01 to 0.2 g/cm3, more preferably from
0.01 to 0.1 g/cm3.
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As for the concept of the average pore diameter,
the distribution of pore diameters is preferably
monodisperse and higher contribution ratio of pores of
150-300pm diameter (this pore size is important in
allowing cell infiltrate) is better. The contribution
ratio of pores of 150-300pm diameter is 10% or more,
preferably 20% or more, more preferably 30% or more,
particularly preferably 40% or more, especially
preferably 50% or more. Since such contribution ratio of
pores of 150-300pm diameter enables cells to easily
invade and allows the invaded cell to easily adhere and
grow, the three-dimensional network structure having such
contribution ratio is effective for application as a
scaffold material and an artificial blood vessel.
The contribution ratio of pores of 150-300}im
diameter in the average pore diameter of the porous
three-dimensional network structure denotes a ratio of
the number of pores of 150-300pm diameter relative to the
number of all pores in a measuring method of average pore
diameter in Example 1 described later.
By using this porous three-dimensional network
structure having the aforementioned average pore diameter,
apparent density, and pore diameter distribution, an
excellent scaffold can be obtained which allows
cell/collagen suspension culture solution to easily
penetrate into pores and allows easy adhesion and growth
of cells to porous layers. In case that the scaffold is
formed in a tubular shape, cells can be engrafted all
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over from the inner wall to the outer periphery, thereby
achieving an artificial blood vessel at a low risk of
occlusion and with high patency rate.
Examples of the thermoplastic resin composing the
scaffold for tissue engineering of the present invention
include polyurethane resin, polyamide resin, polylactide
resin, polyolefin resin, polyester resin, fluorocarbon
resin, acrylic resin, methacrylic resin, and derivatives
thereof. These may be used alone or in admixture of two
or more. Among these, polyurethane resin is preferable
and segmented polyurethane resin capable of providing an
artificial blood vessel which is excellent in
antithrombogenicity and physical property is especially
preferable.
The segmented polyurethane resin is prepared
synthetically from three components: a polyol, a
diisocyanate, and a chain elongation agent and thus has
elastomeric characteristics according to a so-called
block polymer structure having hard segments and soft
segments within molecule. Therefore, the scaffold and the
artificial blood vessel made using this segmented
polyurethane resin can be formed into a tubular structure
which exhibits an S-S curve (characteristics of high
compliance and low elasticity at low blood pressure range
and low compliance and high elasticity at high blood
pressure range) approximate to a living blood vessel in
elastic dynamics and is excellent in antithrombogenicity
and physical property.
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By using a thermoplastic resin having hydrolyzable
property or biodegradability, a resin substrate is
gradually dissolved and absorbed after implantation of an
artificial blood vessel into a living body and can be
finally removed from the living body with leaving
engrafted cells.
In the porous three-dimensional network structure
made of the thermoplastic resin, one or more selected
from a group composing of collagen Type I, collagen Type
II, collagen Type III, collagen Type IV, atelocollagen,
fibronectin, gelatin, hyaluronic acid, heparin, keratin
acid, chondroitin, chondroitin sulfate, condroitin
sulfate B, copolymer of hydroxyethyl methacrylate and
dimethylaminoethyl methacrylate, copolymer of
hydroxyethyl methacrylate and methacrylic acid, alginic
acid, polyacrylamide, polydimethylacrylamide, and
polyvinyl pyrrolidone may be held. Further in the porous
three-dimensional network structure, cytokines of one or
more kinds selected from a group composing of fibrocyte
growth factor, interleukin-1, tumor growth factor-R,
epidermal growth factor, and diploidic fibrocyte growth
factor may be held. Furthermore in the porous three-
dimensional network structure, cells of one or more kinds
selected from a group composing of embryo-stem cell,
vascular endothelial cell, mesodermal cell, smooth muscle
cell, peripheral vessel cell, and mesothelial cell may be
attached. The embryo-stem cell may be dividing cell.
The scaffold for tissue engineering of the present
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invention enables its skeleton made of the thermoplastic
resin constructing the porous three-dimensional network
structure to be provided with fine pores. These fine
pores make the skeleton to have complex irregular surface
not smooth surface. The irregular surface is effective in
holding collagen and cell growth factor, resulting in
increased cell engraftment. These fine pores are outside
the concept of calculating the average pore diameter of
the porous three-dimensional network structure as
employed in the present invention.
The configuration of the scaffold for tissue
engineering of the present invention is not particularly
limited. If taking a form of tubular structure, the
scaffold can be used as an artificial blood vessel.
In this case, the tubular structure is 0.3-15 mm in
inner diameter and 0.4-20 mm in outer diameter,
preferably 0.3-10 mm in inner diameter and 0.4-15 mm in
outer diameter, further preferably 0.3-6 mm in inner
diameter and 0.4-10 mm in outer diameter, particularly
preferably 0.3-2.5 mm in inner diameter and 0.4-10 mm in
outer diameter, especially preferably 0.3-1.5 mm in inner
diameter and 0.4-10 mm in outer diameter. Even in a case
of such small diameter artificial blood vessel, high
patency rate can be maintained.
The artificial blood vessel of the present
invention composed of the scaffold of the present
invention may be a tubular structure of which outside is
covered by another tubular structure. In case that the
CA 02484012 2004-09-27
impregnation density of collagen and the like into the
scaffold of the present invention is low and/or that the
thickness of the scaffold is small, a covering layer by
this tubular structure prevents leakage of blood for a
certain period after implantation and is absorbed in the
living body and is thus removed when there is no more
possibility of blood leakage after sufficient adhesion
and engraftment of cells. The tubular structure for
covering is not particularly limited and, for example,
may be a tube made of one or more selected from a group
composing of chitosan, polylactide resin, polyester resin,
polyamide resin, polyurethane resin, fibronectin, gelatin,
hyaluronic acid, keratin acid, chondroitin, chondroitin
sulfate, condroitin sulfate B, copolymer of hydroxyethyl
methacrylate and dimethylaminoethyl methacrylate,
copolymer of hydroxyethyl methacrylate and methacrylic
acid, alginic acid, polyacrylamide,
polydimethylacrylamide, and polyvinyl pyrrolidone, cross-
linked collagen, and fibroin. The thickness (difference
between the outer diameter and the inner diameter) of the
tubular structure for covering such as a chitosan tube is
preferably in the range of 5-500 pm.
Though the artificial blood vessel of the present
invention has novelty in that high patency can be
achieved so as to ensure stable blood flow even in a case
of a small-diameter vessel that has never been achieved
by any conventional technique, the artificial blood
vessel of the present invention can be adapted to a
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large-diameter vessel having an inner diameter of 6 mm or
more without any problems.
Hereinafter, an example of the method of producing
a porous three-dimensional network structure made of a
thermoplastic polyurethane resin for forming a scaffold
material or a tubular structure as an artificial blood
vessel of the present invention will be described.
However, the method of producing a porous three-
dimensional network structure made of a thermoplastic
polyurethane resin according to the present invention is
not limited to the following described method at all.
According to the following method, thermoplastic resin
substrates of three-dimensional network structure of
various configurations, such as a plane substrate,
required as the scaffold for tissue engineering can be
prepared.
To prepare a porous three-dimensional network
structure made of a thermoplastic polyurethane resin,
first a polymer dope is prepared by mixing a polyurethane
resin, a water-soluble polymer compound, as will be
described later, as a pore forming agent, and an organic
solvent as a good solvent for the polyurethane resin.
Specifically, after the polyurethane resin is mixed into
the organic solvent to have a homogeneous solution, a
water-soluble polymer compound is mixed and dissolved
into this homogeneous solution. Examples of the organic
solvent include N,N-dimethylformamide, N-methyl-2-
pyrrolidinone, and tetrahydrofuran. However, the organic
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solvent to be used is not limited thereto and may be any
organic solvent capable of solving the thermoplastic
polyurethane resin. In addition, the polyurethane resin
may be dissolved by heat with reduced amount of organic
solvent or without organic solvent and the pore forming
agent may be mixed to the dissolved polyurethane resin.
Examples of the water-soluble polymer compound as
pore forming agent include polyethylene glycol,
polypropylene glycol, polyvinyl alcohol, polyvinyl
pyrrolidone, alginic acid, carboxymethyl cellulose,
hydroxypropyl cellulose, methyl cellulose, and ethyl
cellulose. However, the water-soluble polymer compound to
be used is not limited thereto and may be any water-
soluble polymer compound capable of being homogeneously
dispersed with the thermoplastic resin to form a polymer
dope. In addition, depending on the kind of the
thermoplastic resin, the polymer compound is not limited
to water-soluble ones. For example, lipophilic compounds
such as phthalate ester and paraffin, and inorganic salts
such as lithium chloride and calcium carbonate may be
used. It is also available to use crystal-nucleation
agent for polymer so as to generate secondary particles
during coagulation, that is, to encourage skeletal
formation of porous body.
The polymer dope prepared from the thermoplastic
polyurethane resin, the organic solvent, and water-
soluble polymer compound is dipped in coagulation bath
containing a poor solvent of thermoplastic polyurethane
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resin so as to extract and remove organic solvent and
water-soluble polymer compound in the coagulation bath.
By eliminating a part or all of organic solvent and
water-soluble polymer compound, a porous three-
dimensional network structural material of polyurethane
resin is obtained. Examples of the poor solvent used
herein include water, lower alcohol, and low carbon
number ketones. The coagulated polyurethane resin is
finally washed with water or the like to remove remaining
organic solvent and pore forming agent.
Hereinafter, examples and comparative examples will
be described, but the present invention is not limited by
the following examples at all without departing from the
scope of the invention.
f Example 1]
A thermoplastic polyurethane resin (MIRACTRAN
E98OPNAT available from Nippon Miractran Co., Ltd.) was
dissolved into N-methyl-2-pyrrolidinone (reagent for
peptide synthesis, NMP available from Kanto Kagaku) by
using a dissolver (about 2000 rpm) at room temperature to
obtain 5.0% solution (weight/weight). 1.0kg of this NMP
solution was measured and entered into a planetary mixer
(PLM-2 type, capacity 2.0 liters, available from Inoue
Mfg., Inc.) and was mixed with methylcellulose (reagent,
25cp grade, available from Kanto Kagaku) of an amount
corresponding to the amount of polyurethane resin at a
temperature of 40 C for 20 minutes. With the agitation
being continued, the defoaming was conducted by reducing
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CA 02484012 2004-09-27
the pressure to 20 mmHg (2.7kPa) for 10 minutes, thereby
obtaining polymer dope.
A tube forming jig was prepared which comprised
cylindrical paper tube of 3.5 mmO in inner diameter, 4.6
mmO in outer diameter, and 60 mm in length made of a
chemical experimental paper filter (Qualitative filter
paper No. 2, available from Toyo Roshi Kaisha, Ltd.), a
mandrel of 1.2 mmO in diameter made of SUS440, and a
cylindrical airtight stopper made of biomedical
polypropylene resin capable of fixing the mandrel at the
center of the paper tube. The polymer dope was injected
into the tube forming jig by using a needle of 23 gauges.
After that, the tube forming jig was tightly stopped and
then entered into methanol under refluxing condition. The
refluxing was continued for 72 hours to extract and
remove NMP solution from inside through the surface of
the paper tube, whereby the polyurethane resin was
coagulated. During this, the methanol was replaced with
new one as needed with keeping the refluxing condition.
After 72 hours, the tube forming jig was moved to a
methanol bath at room temperature from the methanol under
the refluxing condition without being dried. The content
was extracted from the tube forming jig within the bath
and was washed in purified water of the Japanese
Pharmacopoeia for 72 hours to extract and remove
methylcellulose, methanol, and remaining NMP. The water
for washing was replaced with new one as needed. The
washed content was depressurized (20 mmHg (2.7kPa)) at
CA 02484012 2011-01-06
room temperature for 24 hours and dried, thereby
obtaining a tubular scaffold of porous three-dimensional
network structure which can be used as an artificial
blood vessel.
Figs. 1 through 4 are pictures of this scaffold
taken by a scanning electron microscope (SEM, JMS-5800LV,
available from JEOL Ltd.) or by a stereoscopic microscope
(VH-6300 available from Keyence Corporation). Apparent
from Figs. 1 through 4, the substrate of the obtained
scaffold is a porous three-dimensional network structure
of about 200pm in pore diameter, 1.2 mmO in inner
diameter, and 3.2 mm O in outer diameter in which the
inside of the structure (Fig. 2), the surface layer of
the inner wall (Fig. 3), and the surface layer of the
outer periphery (Fig.4) are substantially the same and is
an entirely homogeneous porous body.
For the obtained scaffold, the average pore
diameter and the apparent density were measured according
to-the following methods. In the measurements of the
average pore diameter and the apparent density, specimens
were cut by using a.twin bladed razor (HighStainless
available from FEATHER Safety Razor Co., Ltd) at room
temperature.
[Measurement of average pore diameter]
By using a picture of a plane (cutting surface) of
specimen, cut by the twin bladed razor, taken by a
stereoscopic microscope (VH-6300 available from Keyence
Corporation), image processing was conducted to take
* Trade-mark
26
CA 02484012 2011-01-06
respective pores on the same plane as figures surrounded
by skeleton of three-dimensional network structure (using
LUXEX AP available from NIRECO Corporation as an image
processing unit and LE N50 available from SONY
Corporation as a CCD camera for taking images) and the
areas of the respective figures were measured. The areas
were converted to areas of real circles. The diameters of
the corresponding circles were obtained as the pore
diameters. Measurement was conducted only for through
pores on the same plane in disregard for micropores bored
in the porous skeleton, with the result that the average
pore diameter was obtained as 169 55 pm. The contribution
ratio of pores of 150-3001im diameter in the pore
distribution was obtained as 71.2% so that it was
recognized that the specimen was a porous body mainly
having pores effective in cell adhesion.
(Measurement of apparent density)
The scaffold was cut into a specimen of about 10 mm
in length by the twin bladed razor. The volume of the
specimen was obtained from dimensions measured by a
projector (V-12, Nikon). As a result that the weight was
divided by the volume, the apparent density was obtained
as 0.077 0.002gfcm3.
The three-dimensional network structure as a
characteristic of the present invention is a structure
which is excellent in pore-to-pore communication. The
water permeability as indicator of this communication
property was evaluated as follows.
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27
CA 02484012 2004-09-27
[Evaluation of water permeability]
First, a specimen of 10 mm in length was prepared
by cutting the material as mentioned above. While the end
of one side of the specimen was tightly stopped, a needle
of 0.3 mmO in inner diameter, 1.2 mmO in outer diameter,
40 mm in length was inserted into the specimen at the
other side in such a manner as to obtain 0.50 mm in
length as the effective permeability area of a tubular
body of the specimen. A silicone tube of 50 mm in length
and 5 mm4 in diameter and a gator of 20 mmO and 90 mm in
length filled with 25g of water were connected to the
needle so as to measure the permeability of distilled
water at a temperature of 25 C. The water permeation rate
was 13.47 0.33g/60 sec. and 24.64 0.35g/120 sec. Since
the water permeation rate with no specimen, i.e. in the
unloaded state, was 13.70/60 sec. and 24.87/120 sec., it
was recognized that the scaffold was a three-dimensional
network structure having excellent water permeability
with high communication property.
[Example 2]
DMEM (culture component) solution (containing FCS
(cow embryo blood serum) 10%) of smooth muscle cells from
cow' s blood vessel (cell density: 6x106 cells/mL) and
collagen type I solution (0.3% acid solution available
from Koken Co., Ltd.) are mixed in equivalent quantities
while being cooled on ice, thereby preparing suspension
solution of smooth muscle cells (cell density: 3x106
cells/mL).
28
CA 02484012 2004-09-27
The scaffold of tubular porous three-dimensional
network structure (inner diameter: 1.2 mm 0, outer
diameter: 3.2 mmO, length: 2 cm) prepared in Example 1
was clamped at its one end and the suspension solution of
smooth muscle cells (1 mL) was injected at the other end
into the scaffold until leaking out through a side wall
of the tubular structure. All of the injection operation
was conducted on ice. By repeating the injection
operation several times, the collagen solution containing
smooth muscle cells well penetrated all over the tubular
structure including the inside thereof. After that, the
clamping was cancelled, a mandrel of 1.2 mmO made of
SUS440 was inserted into the tubular body of the scaffold
at the center thereof, and incubation was conducted in an
incubator at a temperature of 37 C, thereby obtaining a
porous three-dimensional network structure containing
cells.
The porous three-dimensional network structure
containing cell obtained as mentioned above underwent
three days of incubation. Fig. 5 is a picture showing
sectional tissue of the porous three-dimensional network
structure observed by an optical microscope after three
days of incubation. From Fig. 5, it is found that the
cells are distributed all over the inside of the obtained
structure. It was observed that tissues inside the
structure containing cells engrafted without necrosing
even after one week of additional incubation (Fig. 6).
[Example 3]
29
CA 02484012 2004-09-27
The scaffold of tubular porous three-dimensional
network structure (inner diameter: 1.2 mm 0, outer
diameter: 3.2 mmO, length: 2 cm) prepared in Example 1
was clamped at its one end and the collagen type I
solution (0.15 wt. %) was injected at the other end into
the scaffold until the collagen solution penetrated all
over the scaffold including the inside thereof. After
that, the clamping was cancelled, a mandrel of 1.2 mm O
made of SUS440 was inserted into the tubular body of the
scaffold at the center thereof, and the tubular structure
of the scaffold was held inside an incubator at a
temperature of 37 C to make the collagen solution to gel,
thereby obtaining a tubular body of which network
structure was filled with collagen gel.
A piece of about 3cm was exfoliated from aorta
abdominalis of a rat and was clamped at its both ends to
block the blood stream. After that, a middle portion of
the aorta was cut. The tubular body was inserted between
the cut ends of the aorta and the ends of the tubular
body are connected to the corresponding cut ends. As the
blood stream was reactivated after canceling the clamping
of both ends, beat starts. Therefore, the tubular body
functioned as an artificial blood vessel (Fig. 7). The
artificial blood vessel was removed after one week. As
the lumen surface of the tubular tissue body was observed,
blood clot was not attached nor formed on the lumen
surface so that the lumen surface was really smooth (Fig.
8) .
CA 02484012 2004-09-27
[Comparative Example 1]
thermoplastic polyurethane resin (MIRACTRAN
E980PNAT available from Nippon Miractran Co., Ltd.) was
heated at a temperature of 60 C to lyse into
tetrahydrofuran (THF available from Wako Pure Chemical
Industries, Ltd.), thereby obtaining 5.0% solution
(weight/weight) thereof. 12g of NaCl particles (having
particle diameters ranging from 100pm to 200pm which were
selected by filtering procedure) were dispersed into 16mL
of the THE solution, thus preparing suspension. A mandrel
of 1.2 mmO in diameter made of SUS440 was immersed in
the suspension and was dried, whereby the periphery of
the mandrel was coated with tubular coating of
polyurethane containing NaCl particles. After the mandrel
with coating was sufficiently dried, the mandrel was
washed enough with ion-exchange water to remove NaCl
contained in the tubular coating. The washed mandrel was
depressurized (20 mmHg (2.7kPa)) at room temperature for
24 hours and dried, thereby obtaining a porous tubular
body of 1.2 mm4 in inner diameter and 3.2 mm4 in outer
diameter.
The average pore diameter and the apparent density
of this porous tubular body were measured in the same
manner as Example 1. While the average pore diameter was
121 65 pm, the contribution ratio of pores of 150-300pm
diameter was 31.8%. The apparent density was
0.086 0.004g/cm3.
As a result of appearance observation by the SEM,
31
CA 02484012 2004-09-27
while Example 1 had a three-dimensional structure in
which the outer layer and the inside are the same, this
comparative example had a structure in which the outer
layer and the inside are quite different from each other
because closely-spaced layers were generated in the outer
layer (Fig. 9) and spherical pores were gathered in the
inside structure so that at contact portions between
adjacent pores, pore walls were provided with penetrated
holes, that is, this structure was not a three-
dimensional network structure (Fig. 10).
The water permeation rate was also measured in the
same manner as Example 1, with the result of
11.22 0.46g/60 sec. and 20.08 0.96g/120 sec. These values
were lower than those of Example 1. It can be concluded
that this is because the communication property between
pores in the outer layer is low and the closely-spaced
layer in the outer layer affects.
[Comparative Example 2]
Suspension solution of smooth muscle cells (cell
density: 3x106 cells/mL) prepared in the same manner as
Example 2 was injected into the porous tubular body
(inner diameter: 1.2 mmO, outer diameter: 3.2 mm O,
length: 2 cm) prepared in Comparative Example 1 in the
same manner as Example 2. After that, incubation was
conducted, thereby obtaining a tubular structural
material containing cells.
The tubular structural material containing cells
obtained as mentioned above underwent three days of
32
CA 02484012 2004-09-27
incubation. Fig. 11 is a picture showing sectional tissue
of the tubular structural material containing cells
observed by an optical microscope after three days of
incubation. From Fig. 11, it is found that little cells
exist inside the obtained structure and cells exist only
on the luminal surface.
As described in the above, the present invention
can provide a scaffold material for tissue engineering
which comprises a homogeneous porous body having a three-
dimensional network structure, allows cells to be
uniformly engrafted all over the inside of the porous
body thereof, is excellent in physical strength, and is
effectively used not only for basic studies on
biotechnologies, but also for an artificial blood vessel
which can exhibit high patency rate for a long period of
time even if the inner diameter is small, less than 6 mm,
and the present invention can provide an artificial blood
vessel using this scaffold for tissue engineering.
Hereinafter, preferred embodiments of the cuff of
the present invention will be described in detail.
The cuff of the present invention is composed of a
three-dimensional network structure having well
communication property made of thermoplastic resin or
thermosetting resin. The three-dimensional network
structure is a porous three-dimensional network structure
having an average pore diameter from 100 to 1000pm and
apparent density from 0.01 to 0.5g/cm3. In the cutting
surfaces in the depth direction, the surfaces may be
33
CA 02484012 2004-09-27
entirely similar or one side surface is different from
the other side surface. The average pore diameter and/or
the apparent density may partially vary. For example, the
average pore diameter may vary gradually from the one
side surface to the other side surface, that is, the
three-dimensional network structure may have anisotropy.
The three-dimensional network structure may be provided,
in the contact surface with native tissues, with pores
having a large pore diameter which is extremely larger
than the average pore diameter. It is preferable that
these pores are pores having a pore diameter in the range
of 500-2000pm. These pores existing near the outer layer
on the side of native tissues facilitate extracellular
matrix such as collagen to homogeneously penetrate deep
parts and effectively act on infiltration of cells from
tissues and neovascularization of capillary vessels. It
should be noted that such large diameter pores are
outside the concept of calculating the average pore
diameter of the porous three-dimensional network
structure as employed in the present invention.
As for the porous three-dimensional network
structure, the average pore diameter is from 100 to
1000pm and the apparent density is from 0.01 to 0.5 g/cm3.
The average pore diameter is preferably from 200 to 600pm,
more preferably from 200 to 500pm. The apparent density
in the range of from 0.01 to 0.5 g/cm3 can provide well
cell engraftment, excellent physical strength, and
elastic characteristics similar to subcutaneous tissues
34
CA 02484012 2004-09-27
when cells infiltrate, are sufficiently grown and tightly
interconnected. The apparent density is preferably from
0.05 to 0.3 g/cm3, more preferably from 0.05 to 0.2 g/cm3.
As for the distribution of pore diameter with the
same average pore diameter, higher contribution ratio of
pores of 150-400 m diameter that is important in allowing
cell infiltrate is better. The contribution ratio of
pores of 150-400 m diameter is 10% or more, preferably
20% or more, more preferably 30% or more, particularly
preferably 40% or more, especially preferably 50% or more.
Such contribution ratio is preferable because it enables
cells to easily invade and allows the invaded cell to
easily adhere and grow.
The contribution ratio of pores of 150-400 m
diameter in the average pore diameter of the porous
three-dimensional network structure denotes a ratio of
the number of pores of 150-400 m diameter relative to the
number of all pores in a measuring method of average pore
diameter in Example 4 described later.
The porous three-dimensional network structure
having the average pore diameter, the apparent density,
and the distribution of pore diameters as mentioned above
allows easy infiltration of cells into pores and allows
easy adhesion and growth of cells onto the porous three-
dimensional network structure, thereby constructing
capillary vessels. Therefore, with this porous three-
dimensional network structure, an excellent cuff which
can provide a robust bonding between subcutaneous tissue
CA 02484012 2004-09-27
and a catheter or cannula at a portion of the cuff
insertion can be obtained.
The porous three-dimensional structure may have a
thickness ranging from 0.2 mm to 500 mm. The thickness is
preferably from 0.2 to 100 mm, more preferably from 0.2
to 50 mm, particularly preferably from 0.2 to 10 mm,
especially preferably from 0.2 to 5 mm. Such a thickness
as mentioned provides a high level of satisfaction in
physical strength required as a cuff, infiltrate of cells,
organization, bonding with subcutaneous tissue, and
antibacterial property.
The thermoplastic resin or thermosetting resin
composing the porous three-dimensional network structure
may be one or more of polyurethane resin, polyamide resin,
polylactide resin, polyolefin resin, polyester resin,
fluorocarbon resin, urea resin, phenol resin, epoxy resin,
polyamide resin, acrylic resin, methacrylic resin, and
derivatives thereof. The preferable one is polyurethane
resin, especially segmented polyurethane resin.
The segmented polyurethane resin is prepared
synthetically from three components: a polyol, a
diisocyanate, and a chain elongation agent and thus has
elastomeric characteristics according to a block polymer
structure having hard segments and soft segments within
molecular. Therefore, the elasticity achieved by using
this segmented polyurethane resin was expected to exhibit
an effect of attenuating the stress generated at
interface between subcutaneous tissue and the cuff when
36
CA 02484012 2004-09-27
the patient, the catheter, or the cannula moves or when
skin around the portion of the cuff insertion is moved
during the disinfection process.
The cuff of the present invention may comprise a
layer having the said specific porous three-dimensional
network structure as a first layer and a second layer,
laminated on the first layer, having a structure
different from that of the first layer. The second layer
may be a fiber aggregation, a flexible film, or a porous
three-dimensional network structure of which the average
pore diameter and the apparent density are different from
those of the porous three-dimensional network structure
of the first layer.
The fiber aggregation may be, for example, unwoven
fabric or woven fabric, of which thickness is from 0.1 to
100 mm, preferably from 0.1 to 50 mm, more preferably
from 0.1 to 10 mm, particularly preferably from 0.1 to 5
mm. The thickness in this range is preferable because
well flexibility is maintained when laminated on the
porous three-dimensional network structure and robust
bonding with subcutaneous tissue is obtained.
Porosity of the unwoven fabric or woven fabric is
preferably in the range of from 100 to 5000 cc/cm2/min in
view of flexibility, connecting strength with
subcutaneous tissue, and the like. It should be noted
that "porosity" used here is a value measured according
to JIS L 1004 and is sometimes called as air permeability
or ventilation volume.
37
CA 02484012 2004-09-27
The fiber aggregation may be made of synthetic
resin composing of one or more selected from a group
composing of polyurethane resin, polyamide resin,
polylactide resin, polyolefin resin, polyester resin,
fluorocarbon resin, acrylic resin, methacrylic resin, and
derivatives thereof. The fiber aggregation may also be
made of naturally-occurring fibers composing of one or
more selected from a group composing of fibroin, chitin,
chitosan, and cellulose, and derivatives thereof. In
addition, mixture of synthetic fibers and naturally-
occurring fibers may also be used.
The flexible film may be a thermoplastic resin film,
especially a film made of one or more selected from a
group composing of polyurethane resin, polyamide resin,
polylactide resin, polyolefin resin, polyester resin,
fluorocarbon resin, urea resin, phenol resin, epoxy resin,
polyimide resin, acrylic resin, methacrylic resin, and
derivatives thereof. The flexible film is preferably a
film made of one or more selected from a group composing
of polyester resin, fluorocarbon resin, polyurethane
resin, acrylic resin, vinyl chloride, fluorocarbon resin,
and silicone resin.
Thickness of the flexible film ranging from 0.1 to
500 mm makes a cuff which is advantageous in view of
flexibility and physical strength. The thickness of the
flexible film is preferably from 0.1 to 100 mm, more
preferably from 0.1 mm to 50 mm, furthermore preferably
from 0.1 mm to 10 mm.
38
CA 02484012 2004-09-27
The flexible film may be not only a solid film but
also a porous film or a foamed film. By laminating a
solid flexible film, a cuff which has excellent
antibacterial property and is therefore advantageous in
transmission maintenance is obtained.
When a porous three-dimensional network structure
of which the average pore diameter and the apparent
density are different from that of the porous three-
dimensional network structure of the first layer is used
as the second layer, the second layer may be a porous
three-dimensional network structure having an average
pore diameter of from 0.1 to 200 pm and an apparent
density of from 0.01 to 1.0 g/cm3. The thickness of the
porous three-dimensional network structure of the second
layer preferably ranges from 0.2 mm to 20 mm.
As for the method of laminating the second layer
onto the porous three-dimensional network structure, when
the second layer is a fiber aggregation, a flexible film,
or a porous three-dimensional network structure of which
the average pore diameter and/or the apparent density are
different from those of the porous three-dimensional
network structure of the first layer, a bonding method
using adhesives, particularly, a method of inserting a
hot-melt unwoven fabric between the first layer and the
second layer and pressing them under heating condition
may be employed. The hot-melt unwoven fabric may be a
polyamide type hot-melt adhesive sheet such as PA1001
available from Nitto Boseki Co., Ltd. or the like.
39
CA 02484012 2004-09-27
Alternatives are a method of bonding by melting an outer
layer of a contact surface with a solvent, a method of
bonding by melting an outer layer with heating, and a
method using ultrasonic sound or high frequency wave.
Further, during the preparation of the first layer, the
fiber aggregation or the flexible film may be laminated
on the polymer dope. In this manner, the second layer can
be laminated and formed in a continuous fashion.
The second layer may be formed of two or more of
the fiber aggregation, the flexible film, and the porous
three-dimensional network structure. The cuff may be a
three layer structure in which another porous three-
dimensional network structure same as the first layer may
also be laminated via the second layer.
In the porous three-dimensional network structure
of the cuff of the present invention, one or more
selected from a group composing of collagen Type I,
collagen Type II, collagen Type III, collagen Type IV,
atelocollagen, fibronectin, gelatin, hyaluronic acid,
heparin, keratin acid, chondroitin, chondroitin sulfate,
condroitin sulfate B, elastin, heparan sulfate, laminin,
thrombospondin, hydronectin, osteonectin, entactin,
copolymer of hydroxyethyl methacrylate and
dimethylaminoethyl methacrylate, copolymer of
hydroxyethyl methacrylate and methacrylic acid, alginic
acid, polyacrylamide, polydimethylacrylamide, and
polyvinyl pyrrolidone may be held. Further in the porous
three-dimensional network structure, one or more selected
CA 02484012 2004-09-27
from a group composing of platelet-derived growth factor,
epidermal growth factor, transforming growth factor-a,
insulin-like growth factor, insulin-like growth factor
binding proteins, hepatocyte growth factor, vascular
endothelial proliferation growth factor, angiopoietin,
nerve growth factor, brain-derived neurotrophic factor,
ciliary neurotrophic factor, transforming growth factor-13,
latent form transforming growth factor-13, activin, bone
plasma proteins, fibrocyte growth factor, tumor growth
factor-R, diploid fibrocyte growth factor, heparin-
binding epidermal growth factor-like growth factor,
schwannoma-derived growth factor, anfillegrin,
betacellulin, epi.llegrin, lymphotoxin, erythropoietin,
tumor necrosis factor-a, interleukin-1(3, interleukin-6,
interleukin-8, interleukin-17, interferon, antivirotic,
antimicrobial agent, and antibacterial agent may be held.
Furthermore in the porous three-dimensional network
structure, cells of one or more kinds selected from a
group composing of embryo-stem cell (which may be
dividing cell), vascular endothelial cell, mesodermal
cell, smooth muscle cell, peripheral vessel cell,. and
mesothelial cell may be attached.
The cuff of the present invention enables its
skeleton made of the thermoplastic resin or the
thermosetting resin constructing the porous three-
dimensional network structure to be provided with fine
pores. These fine pores make the skeleton to have complex
irregular surface not smooth surface. The irregular
41
CA 02484012 2004-09-27
surface is effective in holding collagen and cell growth
factor, resulting in increased cell engraftment. These
fine pores are outside the concept of calculating the
average pore diameter of the porous three-dimensional
network structure as employed in the present invention.
Hereinafter, an example of method for preparing the
porous three-dimensional network structure made of
thermoplastic polyurethane resin constructing the cuff of
the present invention will be described, but the
preparing method of the cuff of the present invention is
not limited to the following method at all.
To prepare a porous three-dimensional network
structure made of a thermoplastic polyurethane resin,
first a polymer dope is prepared by mixing a polyurethane
resin, a water-soluble polymer compound, as will be
described later, as a pore forming agent, and an organic
solvent as a good solvent for the polyurethane resin.
Specifically, after the polyurethane resin is mixed into
the organic solvent to have a homogeneous solution, a
water-soluble polymer compound is mixed and dissolved
into this homogeneous solution. Examples of the organic
solvent include N,N-dimethylformamide, N-methyl-2-
pyrrolidinone, and tetrahydrofuran. However, the organic
solvent to be used is not limited thereto and may be any
organic solvent capable of solving the thermoplastic
polyurethane resin. In addition, the polyurethane resin
may be dissolved by heat with reduced amount of organic
solvent or without organic solvent and the pore forming
42
CA 02484012 2004-09-27
agent may be mixed to the dissolved polyurethane resin.
Examples of the water-soluble polymer compound as
pore forming agent include polyethylene glycol,
polypropylene glycol, polyvinyl alcohol, polyvinyl
pyrrolidone, alginic acid, carboxymethyl cellulose,
hydroxypropyl cellulose, methyl cellulose, and ethyl
cellulose. However, the water-soluble polymer compound to
be used is not limited thereto and may be any water-
soluble polymer compound capable of being homogeneously
dispersed with the thermoplastic resin to form a polymer
dope. In addition, depending on the kind of the
thermoplastic resin, the polymer compound is not limited
to water-soluble ones. For example, lipophilic compounds
such as phthalate ester and paraffin, and inorganic salts
such as lithium chloride and calcium carbonate may be
used. It is also available to use crystal-nucleation
agent for polymer so as to generate secondary particles
during coagulation, that is, to encourage skeletal
formation of porous body.
The polymer dope prepared from the thermoplastic
polyurethane resin, the organic solvent, and water-
soluble polymer compound is dipped in coagulation bath
containing a poor solvent of thermoplastic polyurethane
resin so as to extract and remove organic solvent and
water-soluble polymer compound in the coagulation bath.
By removing a part or all of organic solvent and water-
soluble polymer compound, a porous three-dimensional
structural material of polyurethane resin is obtained.
43
CA 02484012 2004-09-27
Examples of the poor solvent used herein include water,
lower alcohol, and low carbon number ketones. The
coagulated polyurethane resin is finally washed with
water or the like to remove remaining organic solvent and
pore forming agent.
Hereinafter, a preferred embodiment of the
biological implant covering member of the present
invention will be described.
The biological implant covering member of the
present invention is composed of a three-dimensional
network structure having well communication property made
of thermoplastic resin or thermosetting resin. The three-
dimensional network structure is a porous three-
dimensional network structure having an average pore
diameter of from 100 to 1000pm and apparent density of
from 0.01 to 0.5g/cm3. In the cutting surfaces in the
depth direction, the surfaces may be entirely similar or
one side surface is different from the other side surface.
The average pore diameter and/or the apparent density may
partially vary. For example, the average pore diameter
may vary gradually from the one side surface to the other
side surface, that is, the three-dimensional network
structure may have anisotropy. The three-dimensional
network structure may be provided, in the contact surface
with native tissues, with pores having a large pore
diameter which is extremely larger than the average pore
diameter. It is preferable that these pores are pores
having a pore diameter in the range of from 500 to 2000pm.
44
CA 02484012 2004-09-27
These pores existing near the outer layer on the side of
native tissues facilitate extracellular matrix such as
collagen to homogeneously penetrate deep parts and
effectively act on infiltration of cells from tissues and
neovascularization of capillary vessels. It should be
noted that such large diameter pores are outside the
concept of calculating the average pore diameter of the
porous three-dimensional network structure as employed in
the present invention.
As for the porous three-dimensional network
structure, the average pore diameter is from 100 to
1000pm and the apparent density is from 0.01 to 0.5 g/cm3.
The average pore diameter is preferably from 200 to 600pm,
more preferably from 200 to 500pm. The apparent density
in the range of from 0.01 to 0.5 g/cm3 can provide well
cell engraftment, excellent physical strength, and
elastic characteristics similar to subcutaneous tissues
when cells infiltrate, are sufficiently grown and tightly
interconnected. The apparent density is preferably from
0.05 to 0.3 g/cm3, more preferably from 0.05 to 0.2 g/cm3.
As for the distribution of pore diameter with the
same average pore diameter, higher contribution ratio of
pores of 150-400pm diameter that is important in allowing
cell infiltrate is better. The contribution ratio of
pores of 150-400pm diameter is 10% or more, preferably
20% or more, more preferably 30% or more, particularly
preferably 40% or more, especially preferably 50% or more.
Such contribution ratio is preferable because it enables
CA 02484012 2004-09-27
cells to easily invade and allows the invaded cell to
easily adhere and grow.
The contribution ratio of pores of 150-400pm
diameter in the average pore diameter of the porous
three-dimensional network structure denotes a ratio of
the number of pores of 150-400pm diameter relative to the
number of all pores in a measuring method of average pore
diameter in Example 4 described later.
The porous three-dimensional network structure
having the average pore diameter, the apparent density,
and the distribution of pore diameters as mentioned above
allows easy infiltration of cells into pores and allows
easy adhesion and growth of cells onto the porous three-
dimensional network structure, thereby constructing
capillary vessels. Therefore, with this porous three-
dimensional network structure, an excellent biological
implant covering member which can provide a robust
bonding to subcutaneous tissue at a portion where it is
inserted can be obtained.
The porous three-dimensional structure may have a
thickness ranging from 0.5 mm to 500 mm. The thickness is
preferably from 0.5 to 100 mm, more preferably from 0.5
to 50 mm, particularly preferably from 0.5 to 10 mm,
especially preferably from 0.5 to 5 mm. Such a thickness
as mentioned provides a high level of satisfaction in
physical strength required as a biological implant
covering member, infiltrate of cells, organization, and
bonding with subcutaneous tissue.
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CA 02484012 2004-09-27
The thermoplastic resin or thermosetting resin
composing the porous three-dimensional network structure
may be one or more of polyurethane resin, polyamide resin,
polylactide resin, polymalate resin, polyglycolate resin,
polyolefin resin, polyester resin, fluorocarbon resin,
urea resin, phenol resin, epoxy resin, polyimide resin,
acrylic resin, methacrylic resin, and derivatives thereof.
The preferable one is polyurethane resin, especially
segmented polyurethane resin.
The segmented polyurethane resin is prepared
synthetically from three components: a polyol, a
diisocyanate, and a chain elongation agent and thus has
elastomeric characteristics according to a so-called
block polymer structure having hard segments and soft
segments within molecular. Therefore, the elasticity
achieved by using this segmented polyurethane resin was
expected to exhibit an effect of attenuating the stress
generated at interface between subcutaneous tissue and
the biological implantation member.
The biological implant covering member of the
present invention may comprise a layer having the said
specific porous three-dimensional network structure as a
first layer and a second layer, laminated on the first
layer, having a structure different from that of the
first layer. The second layer may be a porous three-
dimensional network structure of which the average pore
diameter and the apparent density are different from
those of the porous three-dimensional network structure
47
CA 02484012 2004-09-27
as the first layer.
In the porous three-dimensional network structure
of the biological implant covering member, one or more
selected from a group composing of collagen Type I,
collagen Type II, collagen Type III, collagen Type IV,
atelocollagen, fibronectin, gelatin, hyaluronic acid,
heparin, keratin acid, chondroitin, chondroitin sulfate,
condroitin sulfate B, elastin, heparan sulfate, laminin,
thrombospondin, hydronectin, osteonectin, entactin,
copolymer of hydroxyethyl methacrylate and
dimethylaminoethyl methacrylate, copolymer of
hydroxyethyl methacrylate and methacrylic acid, alginic
acid, polyacrylamide, polydimethylacrylamide, and
polyvinyl pyrrolidone may be held. Further in the porous
three-dimensional network structure, one or more selected
from a group composing of platelet-derived growth factor,
epidermal growth factor, transforming growth factor-a,
insulin-like growth factor, insulin-like growth factor
binding proteins, hepatocyte growth factor, vascular
endothelial proliferation growth factor, angiopoietin,
nerve growth factor, brain-derived neurotrophic factor,
ciliary neurotrophic factor, transforming growth factor-R,
latent form transforming growth factor-1i, activin, bone
plasma proteins, fibrocyte growth factor, tumor growth
factor-R, diploid fibrocyte growth factor, heparin-
binding epidermal growth factor-like growth factor,
schwannoma-derived growth factor, anfillegrin,
betacellulin, epillegrin, lymphotoxin, erythropoietin,
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CA 02484012 2004-09-27
tumor necrosis factor-a, interleukin-113, interleukin-6,
interleukin-8, interleukin-17, interferon, antivirotic,
antimicrobial agent, and antibacterial agent may be held.
Furthermore in the porous three-dimensional network
structure, cells of one or more kind selected from a
group composing of embryo-stem cell (which may be
dividing cells), vascular endothelial cell, mesodermal
cell, smooth muscle cell, peripheral vessel cell, and
mesothelial cell may be attached.
The biological implant covering member of the
present invention enables its skeleton made of the
thermoplastic resin or the thermosetting resin
constructing the porous three-dimensional network
structure to be provided with fine pores. These fine
pores make the skeleton to have complex irregular surface
not smooth surface. The irregular surface is effective in
holding collagen and cell growth factor, resulting in
increased cell engraftment. These fine pores are outside
the concept of calculating the average pore diameter of
the porous three-dimensional network structure as
employed in the present invention.
Hereinafter, an example of method for preparing the
porous three-dimensional network structure made of
thermoplastic polyurethane resin constructing the
biological implant covering member of the present
invention will be described, but the preparing method of
the biological implant covering member of the present
invention is not limited to the following method at all.
49
CA 02484012 2004-09-27
To prepare a porous three-dimensional network
structure made of a thermoplastic polyurethane resin,
first a polymer dope is prepared by mixing a polyurethane
resin, a water-soluble polymer compound, as will be
described later, as a pore forming agent, and an organic
solvent as a good solvent for the polyurethane resin.
Specifically, after the polyurethane resin is mixed into
the organic solvent to have a homogeneous solution, a
water-soluble polymer compound is mixed and dissolved
into this homogeneous solution. Examples of the organic
solvent include N,N-dimethylformamide, N-methyl-2-
pyrrolidinone, and tetrahydrofuran. However, the organic
solvent to be used is not limited thereto and may be any
organic solvent capable of solving the thermoplastic
polyurethane resin. In addition, the polyurethane resin
may be dissolved by heat with reduced amount of organic
solvent or without organic solvent and the pore forming
agent may be mixed to the dissolved polyurethane resin.
Examples of the water-soluble polymer compound as
pore forming agent include polyethylene glycol,
polypropylene glycol, polyvinyl alcohol, polyvinyl
pyrrolidone, alginic acid, carboxymethyl cellulose,
hydroxypropyl cellulose, methyl cellulose, and ethyl
cellulose. However, the water-soluble polymer compound to
be used is not limited thereto and may be any water-
soluble polymer compound capable of being homogeneously
dispersed with the thermoplastic resin to form a polymer
dope. In addition, depending on the kind of the
CA 02484012 2004-09-27
thermoplastic resin, the polymer compound is not limited
to water-soluble ones. For example, lipophilic compounds
such as phthalate ester and paraffin, and inorganic salts
such as lithium chloride and calcium carbonate may be
used. It is also available to use crystal-nucleation
agent for polymer so as to generate secondary particles
during coagulation, that is, to encourage skeletal
formation of porous body.
The polymer dope prepared from the thermoplastic
polyurethane resin, the organic solvent, and water-
soluble polymer compound is dipped in coagulation bath
containing a poor solvent of thermoplastic polyurethane
resin so as to extract and remove organic solvent and
water-soluble polymer compound in the coagulation bath.
By eliminating a part or all of organic solvent and
water-soluble polymer compound, a porous three-
dimensional structural material of polyurethane resin is
obtained. Examples of the poor solvent used herein
include water, lower alcohol, and low carbon number
ketones. The coagulated polyurethane resin is finally
washed with water or the like to remove remaining organic
solvent and pore forming agent.
As described above, the biological implant covering
member of the present invention allows easy infiltration
of cells from native tissues, easy engraftment of cells,
and organization, thereby obtaining robust bonding with
native tissues and therefore protecting a living body
from adverse effect which may occur due to the insertion
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of a biological implantation member into the living body.
Hereinafter, the cuff and the biological implant
covering member composing the surface thereof will be
described in detail with reference to the following
examples, but the present invention is not limited by the
following examples at all without departing from the
scope of the invention.
[Example 4]
A thermoplastic polyurethane resin (MIRACTRAN
E980PNAT available from Nippon Miractran Co., Ltd.) was
dissolved into N-methyl-2-pyrrolidinone (reagent for
peptide synthesis, NMP available from Kanto Kagaku) by
using a dissolver (about 2000 rpm) at room temperature to
obtain 7.5% solution (weight/weight). 1.0kg of this NMP
solution was measured and entered into a planetary mixer
(PLM-2 type, capacity 2.0 liters, available from Inoue
Mfg., Inc.) and was mixed with methylcellulose (reagent,
50cp grade, available from Kanto Kagaku) of an amount
corresponding to half the amount of polyurethane resin at
a temperature of 40 C for 20 minutes. With the agitation
being continued, the defoaming was conducted by reducing
the pressure to 20 mmHg (2.7kPa) for 10 minutes, thereby
obtaining polymer dope.
Two Teflon plates of 3 mm in thickness and of 150
mm x 150 mm were prepared and each inner section of 140
mm x 140 mm was punched in each plate, thereby forming
two square frames. The two square frames were superposed
and a chemical experimental paper filter (Qualitative
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CA 02484012 2004-09-27
filter paper No. 1, available from Toyo Roshi Kaisha,
Ltd.) was inserted and fixed therebetween so as to form a
Teflon frame unit. The said polymer dope was filled into
the frame unit and wiped by glass bar in order to drain
excess dope off by using a glass bar. After that, a
chemical experimental paper filter (Qualitative filter
paper No. 1, available from Toyo Roshi Kaisha, Ltd.) was
put on as a cover sheet and fixed to the frame unit to
hold the filled polymer dope. The frame unit was entered
into methanol under refluxing condition. The refluxing
was continued for 72 hours to extract and remove NMP
solution through the chemical experimental paper filters
on both sides of the frame unit, whereby the polyurethane
resin was coagulated. During this, the methanol was
replaced with new one as needed with keeping the
refluxing condition.
After 72 hours, the solidificated polyurethane
resin was removed from the Teflon frame unit and was
washed in purified water of the Japanese Pharmacopoeia
for 72 hours to extract and remove methylcellulose,
methanol, and remaining NMP. The water for washing was
replaced with new one as needed. The washed content was
depressurized.(20 mmHg) at room temperature for 24 hours
and dried, thereby obtaining a porous three-dimensional
network structural material made of thermoplastic
polyurethane resin. The porous three-dimensional network
structural material was a biological implant covering
member of the present invention.
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Fabric velour made of polyester of 140 mm x 140 mm
(Bobeiky Double Velour Fabric, having a porosity of 3800
cc/cm2/min and a thickness of 1.5 mm, available from Bird
Company) was impregnated with tetrahydrofuran (reagent of
superfine quality available from Kanto Kagaku) and was
wrung by two rollers to have impregnated amount of
0.104 0.002g/cm2. The said porous three-dimensional
network structural material (the biological implant
covering member) was superposed onto the fabric velour
impregnated with tetrahydrofuran and was pressed by a
load of 1.0 kg/cm2, thereby obtaining a cuff of the
present invention.
Fig. 12 and Fig. 13 are pictures of the biological
implant covering member on the surface of the cuff taken
by a scanning electron microscope (SM200 available from
TOPCON Corporation). From these pictures, it is found
that the biological implant covering member on the
surface of the obtained cuff is a porous three-
dimensional network structure of 350pm in pore diameter.
As for the porous three-dimensional network
structure portion (i.e. the biological implant covering
member) of 2.3 mm in thickness of the obtained cuff, the
average pore diameter and the apparent density were
measured in the following methods. The results are shown
in Table 1. In the measurements of the average pore
diameter and the apparent density, specimens were cut by
using a twin bladed razor (HighStainless available
FEATHER Safety Razor Co., Ltd) at room temperature.
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[Measurement of average pore diameter]
By using a picture of a plane (cutting surface) of
specimen, cut by the twin bladed razor, taken by an
electron microscope (SM200 available from TOPCON
Corporation), image processing was conducted to take
respective pores on the same plane as figures surrounded
by skeleton of three-dimensional network structure (using
LUXEX AP available from NIRECO Corporation as an image
processing unit and LE N50 available from SONY
Corporation as a CCD camera for taking images) and the
areas of the respective figures were measured. The areas
were converted to areas of real circles. The diameters of
the corresponding circles were obtained as the pore
diameters. Measurement was conducted only for through
pores on the same plane in disregard for micropores bored
in the porous skeleton. At the same time, the
distribution of pore diameters regarding all measured
pores was measured and shown in Fig. 14. The contribution
ratio of pores of 150-400pm diameter was obtained from
the measurement result of pore diameter distribution.
[Measurement of apparent density]
The three-dimensional network structure prepared in
Example 4 before lamination of a second layer was cut
into a cubic specimen of about 10 mmx 10 mmx 3 mm by the
twin bladed razor. The volume of the specimen was
obtained from dimensions measured by a projector (V-12,
Nikon). The apparent density was obtained by dividing the
weight by the volume.
CA 02484012 2004-09-27
=
Table 1
Contribution Apparent Thickness
Average pore ratio of 150-400 density (g/cm3) (mm)
diameter (gm) gm pores (%)
Porous three-
dimensional network 329 160 62.2 0.117 0.008 2.3
structure as first
layer
It is apparent from Table 1 that the porous three-
dimensional network structure as the first layer is a
porous three-dimensional network structure mainly having
pores effective in cell adhesion.
[Example 5]
An adult goat (female, weight 54 kg) was prepared
as an analyte and a portion of shaved skin from left
thoracic part to abdominal part was used as a test
substance. During operation, the analyte was rapidly
inserted with an endotracheal tube in a left supine
position of in an ordinal technical manner and was
maintained under general anesthesia by isoflurane. The
surface of a portion including the thoracic part and
abdominal part was sterilized with Isodine. After that,
the surface was incised 20 mm and a half of the specimen
of the cuff prepared in Example 4 was implanted and fixed
by suturing subcutaneous tissue (Fig. 15). The cuff was
cut into a specimen of 10 mm x 10 mm and was subjected to
ethylene oxide gas sterilization. After the operation,
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the test substance was sterilized with acid water or
Isodine twice a day. The analyte drunk water freely and
was supplied with a suitable amount (about 1 kg) of
haycubes as fodder five times a day. After two weeks from
the operation, the specimens previously implanted and
peripheral tissues were removed from the analyte under
general anesthesia. The specimen and the peripheral
tissue were engrafted tightly so that exfoliation
therebetween was difficult, and there were no evidences
of infection, inflammation, and the like in peripheries.
Fig. 16a is a picture of the surface of the cuff
(i.e. the biological implant covering member) showing an
engrafted portion enlarged by a loupe. A poorly-
demarcated milk-white layer, indicated by an arrow in Fig.
16a, extended to the inside of the cuff and the inside of
the cuff was filled with transparent tissues. From this,
it was recognized that granulation tissues were
infiltrated.
Fig. 16b is a picture enlarged by a loupe in case
that the same test was conducted using a woven fabric
(fabric velour made of polyester (Bobeiky Double Velour
Fabric available from Bird Company) used in Example 4)
alone. A milk-white layer was infiltrated along the
surface of the fabric only in the depth direction not
near the outer surface, that is, the downgrowth
phenomenon was confirmed.
Unlike this, in case of the cuff of the present
invention, it was found that the milk-white layer
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continuously extended to near the outer skin so that the
downgrowth phenomenon was inhibited.
After the tests, the extracted specimens were fixed
promptly by 10% neutral buffered formalin and HE stained
samples were prepared in ordinary method. The samples
were observed by an optical microscope. As a result, it
is recognized that granulation tissues mainly comprising
extracellular matrix such as fibrocyte, macrophage, and
collagen fibril extending from the surrounding tissues
were infiltrated and vascularization was observed.
It was recognized from the samples obtained by the
same procedure after four weeks that many granulation
tissues extended and further grown bonding tissues were
formed on the embedded specimens. That is, it was
observed that the organization further advanced.
As described above, the cuff of the present
invention enables further organization by the
infiltration of living cells into the porous three-
dimensional network structure and ensures separation of a
wounded portion from the outside, thereby protecting
against exacerbation factors such as bacterial infection
on healing.
As described above in detail, the cuff of the
present invention allows easy infiltration of cells from
living subcutaneous tissues, easy engraftment of cells,
and neovascularization of capillary vessels so as to
obtain robust bonding with subcutaneous tissues. As a
result, separation of a wounded portion from the outside
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is ensured, thereby blocking exacerbation factors such as
bacterial infection on healing and inhibiting progression
of downgrowth. That is, the invention provides a cuff
with none or little infection trouble such as tunnel
infection.
The cuff of the present invention as mentioned
above can be suitably used for blood circulation method
by ventricular assist device, which is a treatment
implanting a cannula or catheter subcutaneously,
peritoneal dialysis therapy, central venous nutrition
method, and for the implant part of living skin for such
as transcannula DDS, transcatheter DDS, or the like.
59
