U.S. patent application number 12/517891 was filed with the patent office on 2010-07-29 for three-dimensional porous hybrid scaffold and manufacture thereof.
This patent application is currently assigned to NANYANG TECHNOLOGICAL UNIVERSITY. Invention is credited to Kerm Sin Chian, Pamela E-Wei Gopal, Meng Fatt Leong, Buddy Dennis Ratner.
Application Number | 20100190254 12/517891 |
Document ID | / |
Family ID | 39492486 |
Filed Date | 2010-07-29 |
United States Patent
Application |
20100190254 |
Kind Code |
A1 |
Chian; Kerm Sin ; et
al. |
July 29, 2010 |
THREE-DIMENSIONAL POROUS HYBRID SCAFFOLD AND MANUFACTURE
THEREOF
Abstract
The present invention refers to a three-dimensional porous
hybrid scaffold for tissue engineering and methods of its
manufacture and use.
Inventors: |
Chian; Kerm Sin; (Singapore,
SG) ; Leong; Meng Fatt; (Singapore, SG) ;
Gopal; Pamela E-Wei; (Singapore, SG) ; Ratner; Buddy
Dennis; (Seattle, WA) |
Correspondence
Address: |
Pabst Patent Group LLP
1545 PEACHTREE STREET NE, SUITE 320
ATLANTA
GA
30309
US
|
Assignee: |
NANYANG TECHNOLOGICAL
UNIVERSITY
Singapore
SG
|
Family ID: |
39492486 |
Appl. No.: |
12/517891 |
Filed: |
December 5, 2007 |
PCT Filed: |
December 5, 2007 |
PCT NO: |
PCT/SG2007/000414 |
371 Date: |
November 16, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60872800 |
Dec 5, 2006 |
|
|
|
Current U.S.
Class: |
435/396 ;
156/325; 156/60 |
Current CPC
Class: |
A61L 27/58 20130101;
A61L 27/60 20130101; A61L 27/56 20130101; D01D 5/0076 20130101;
Y10T 156/10 20150115; A61L 27/3847 20130101; C08J 9/00
20130101 |
Class at
Publication: |
435/396 ;
156/325; 156/60 |
International
Class: |
C12N 5/00 20060101
C12N005/00; B32B 37/12 20060101 B32B037/12; B32B 37/16 20060101
B32B037/16 |
Claims
1. A three-dimensional hybrid scaffold for tissue engineering
comprising: a first layer made of a decellularized biological
material; a second porous layer connected to the surface of said
first layer, wherein said second layer is a porous bioadhesive; and
a third porous layer connected to the surface of said second layer
which is located opposite the surface to which said first layer is
connected, wherein said third layer is a three-dimensional porous
polymer scaffold.
2. The hybrid scaffold according to claim 1, wherein said third
layer is an electrospun three-dimensional porous scaffold.
3. The hybrid scaffold according to claim 2, wherein said
electrospun scaffold is obtained by providing an electrospinning
apparatus; forming crystals from a molecule or group of molecules,
which are in vapor phase comprised in the surrounding atmosphere,
at the surface of the collector of said electrospinning apparatus,
wherein the reaction chamber of said electrospinning apparatus has
a temperature which allows formation of crystals at said surface of
said collector; electrospinning a solution comprising at least one
polymer dissolved therein around the crystals; continuing the
formation of crystals and the electrospinning simultaneously; and
removing the crystals by sublimation.
4. The hybrid scaffold according to claim 3, wherein said collector
of said electrospinning apparatus is composed of said first and
second layer and thus said electrospun scaffold is electrospun
directly onto the surface of said second layer.
5. The hybrid scaffold according to claim 1, wherein said
biological material is selected from the group consisting of small
intestine, liver, pancreas, urinary bladder, stomach, bladder,
vascular system, bile duct, alimentary canal, respiratory tract,
kidney, spleen, heart, heart valve, bone, skin or fragments or
parts thereof.
6. The hybrid scaffold according to claim 1, wherein said
biological material is esophageal mucosa.
7. The hybrid scaffold according to claim 1, wherein said
biological material is derived from an individual which is selected
from the group consisting of mammal, reptiles and insects.
8. The hybrid scaffold according to claim 7, wherein said mammal is
selected from the group consisting of porcine, bovine, ovine,
rabbit, monkey and human.
9. The hybrid scaffold according to claim 1, wherein said
bioadhesive is selected from the group consisting of fibrin glue,
polyvinylpyrolidone, polyvinylpyrolidone/vinyl acetate copolymers,
polyethylene glycol, platelated gel, chitosan or
gelatin-resorcin-formaldehyde.
10. The hybrid scaffold according to claim 9, wherein said
bioadhesive is fibrin glue.
11. The hybrid scaffold according to claim 1, wherein the polymer
for said three-dimensional porous polymer scaffold is a
biodegradable and/or biocompatible polymer.
12. The hybrid scaffold according to claim 1, wherein said second
porous layer and said third layer have a pore size which is between
about 10 nm to about 500 .mu.m.
13. The hybrid scaffold according to claim 1, wherein the thickness
of said bioadhesive layer is about 10 to 1000 .mu.m before said
third layer is connected to said bioadhesive second layer.
14. A method of manufacturing a three-dimensional hybrid scaffold
according to claim 1, the method comprising: providing a first
layer made of a biological material which has been decellularized;
applying a second layer to the surface of said first layer, wherein
said second layer is a bioadhesive and wherein said second layer is
applied to said first layer at a temperature of the environment
which is at or below the freezing temperature of said bioadhesive;
and applying a third layer which is a three-dimensional porous
scaffold to a side of said second layer which is not the side
facing said first layer.
15. The method according to claim 14, wherein said third layer is
comprised of an electrospun scaffold.
16. The method according to claim 14, wherein said third layer is
comprised of an electrospun scaffold which is obtained by the
process referred to in claim 3.
17. The method according to claim 14, wherein said bioadhesive
layer is applied to said first layer using a method selected from
the group consisting of spraying, electrospraying, electrospinning,
spin coating, dip coating, casting and brushing.
18. The method according claim 14, wherein said third layer is
applied before the bioadhesive is fully set/crosslinked.
19. The method according to claim 14, further comprising
controlling the degradation time of said bioadhesive layer by
controlling the pore size of said bioadhesive layer which is
achieved by increasing or decreasing the temperature in the
environment when applying the bioadhesive layer, as long as said
temperature is still at or below the freezing point of said
bioadhesive.
20. The method according to claim 14, wherein said bioadhesive is
selected from the group consisting of fibrin glue,
polyvinylpyrolidone, chitosan and
gelatin-resorcin-formaldehyde.
21. The method according to claim 20, wherein said bioadhesive is
fibrin glue.
22. A medicament comprising a three-dimensional hybrid scaffold
according to claim 1 for autologous, allogenic, xenogenic
transplantation of tissue.
23. The medicament according to claim 22, wherein said tissue
bladder, vascular system, bile duct, alimentary canal, respiratory
tract, kidney, spleen, heart, heart valve, bone, skin or fragments
or parts thereof.
24. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority of U.S.
provisional application No. 60/872,800, filed Dec. 5, 2006, the
contents of it being hereby incorporated by reference in its
entirety for all purposes.
FIELD OF THE INVENTION
[0002] The present invention refers to a three-dimensional porous
hybrid scaffold for tissue engineering and methods of its
manufacture and use.
BACKGROUND OF THE INVENTION
[0003] Biological materials have been used in many tissue
engineering applications to control the function and structure of
engineered tissue by interacting with transplanted/host cells.
These materials include naturally derived materials (e.g. collagen
and alginate) and acellular tissue matrices (e.g. small intestinal
submucosa) among others. Biological materials have been proven to
support cell ingrowth and regeneration of damaged tissues with no
evidence of immunogenic rejection, and encourage the remodeling
process by stimulating cells to synthesize extracellular matrix
(ECM) proteins and secret ECM to aid in the healing process.
Extracellular matrix components preserved in these biological
materials are also able to influence the phenotypic differentiation
of cells through specific interactions with cell surface
markers.
[0004] However, since such isolated biological materials normally
show different mechanical properties upon re-implantation, their
use is limited since they do not resemble the same mechanical
properties as the original tissue they are derived from.
[0005] Thus, it is an object of the present invention to improve
the properties of biological materials for their use in tissue
engineering.
SUMMARY OF THE INVENTION
[0006] In a first aspect the present invention is directed to a
three-dimensional hybrid scaffold for tissue engineering
comprising: [0007] a first layer made of a decellularized
biological material; [0008] a second porous layer connected to the
surface of the first layer, wherein the second layer is a porous
bioadhesive; and [0009] a third porous layer connected to the
surface of the second layer which is located opposite the surface
to which the first layer is connected, wherein the third layer is a
three-dimensional porous polymer scaffold.
[0010] In another aspect, the present invention refers to a method
of manufacturing a three-dimensional hybrid scaffold according to
any of the preceding claims, the method comprising: [0011]
providing a first layer made of a biological material which has
been decellularized; [0012] applying a second layer to the surface
of the first layer, wherein the second layer is a bioadhesive and
wherein the second layer is applied to the first layer at a
temperature of the environment which is below the freezing
temperature of the bioadhesive; and [0013] applying a third layer
which is a three-dimensional porous scaffold to a side of the
second layer which is not the side facing the first layer.
[0014] In another aspect, the present invention is directed to the
use of a three-dimensional hybrid scaffold of the present invention
or a hybrid scaffold manufactured according to the method of the
present invention for autologous, allogenic, xenogenic
transplantation of tissue.
[0015] In another aspect, the present invention is directed to the
use of a three-dimensional hybrid scaffold of the present invention
or a hybrid scaffold manufactured according to the method of the
present invention for the manufacture of or use as a
medicament.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The invention will be better understood with reference to
the detailed description when considered in conjunction with the
non-limiting examples and the accompanying drawings, in which:
[0017] FIG. 1 shows SEM micrographs of the hybrid scaffold of the
present invention. FIG. 1a-b and d-e indicate different layers of a
hybrid scaffold. FIG. 1a shows a cross section of a hybrid scaffold
obtained by electrospinning onto fibrin coated porcine esophageal
tissue maintained at subzero temperatures. It consists of an open
cryogenic electrospun scaffold, a porous fibrin interface and a
decellularized porcine esophageal tissue, (b) the porous fibrin
interface of the hybrid scaffold, (c) top surface of the cryogenic
electrospun scaffold. FIG. 1(d) shows a cross section of a hybrid
scaffold obtained by electrospinning onto fibrin coated mucosa at
ambient temperature, (e) the non-porous fibrin interface
corresponding to (d), (f) top surface of the electrospun scaffold.
In FIG. 1 a hybrid scaffold of the present invention (FIG. 1 a-c)
is compared to a hybrid scaffold in which the bioadhesive layer was
applied to the first layer at ambient temperature (FIG. 1 d-e).
FIG. 1(b) shows the three different layers comprised in the hybrid
scaffold of the present invention. In this example, the layer on
the left side of the picture is a decelluarized porcine esophageal
tissue (ECM). It appears as a dense but porous collagenous layer
preserved in its native histo-architecture. The bonding interface
between the ECM and the bioadhesive is indicated with a
dotted-line. The bioadhesive interface shown in FIG. 1(b) is porous
with pores ranging between about 10 to 200 .mu.m. In addition, FIG.
1f shows an example of a third layer which was electrospun without
lowering the temperature of the surrounding atmosphere below the
freezing point of water vapour in the atmosphere. In contrast, a
hybrid scaffold in which the third layer was composed using
cryogenic electrospinning as described herein provides a much
larger pore size in the third layer (FIG. 1c).
[0018] FIG. 2a shows a schematic illustration of the basic setup
for conventional electrospinning. The insets show a drawing of the
electrified Taylor cone and a typical SEM image of a non-woven mat
deposited on a collector. FIG. 2b shows the setup for
electrospinning using a rotating mandrel as collector. The first
layer of the hybrid scaffold already connected to the second layer
can be connected to the surface of this collector so that the
second layer acts as the collector for electrospinning a scaffold
on the surface of the bioadhesive layer.
[0019] FIG. 3 shows a process flow of cryogenic electrospinning
process (CHIEF). FIG. 3 illustrates the principal of
electrospinning used in an example of the present invention for
applying the third layer at the surface of the second layer. In
FIG. 3 it is shown that the surface of the collector is chilled to
a temperature below the freezing temperature of the water comprised
in the surrounding atmosphere. In the method of the present
invention, the chilled mandrel is coated with the first and the
second layer of the hybrid scaffold. At those low temperatures ice
crystals form on the surface of the chilled mandrel. After the
first ice crystals have been formed electrospinning of the polymer
solution starts. A fibrous scaffold forms with ice crystals
embedded. After electrospinning is finished, a hybrid scaffold
connected to the mandrel is detached from the chilled mandrel and
freeze-dried to remove the ice crystals and to obtain the
three-dimensional porous hybrid scaffold.
[0020] FIG. 4 shows SEM micrographs of (a) conventionally
electrospun scaffold, (b) cryogenic electrospun scaffold (CES) and
(c) oblique view showing three-dimensional structure of a pore in a
cryogenic electrospun scaffold. Polymer scaffolds produced via
conventional electrospinning (FIG. 4a) are characterized by a
random polymer fiber mesh. However, the pore size between the
fibers in such a polymer fiber mesh is only between several
nanometer to a few micrometer. In contrast, the large pores
obtained using the cryogenic method for electrospinning described
herein can measure between about 50 to 500 .mu.m in size (FIG. 4b).
However, pores obtained with this method can exceed 500 .mu.m.
These pores are bounded by bundles of fibers that form a strut-like
support. As can be seen from a SEM picture taken at an oblique view
(FIG. 4c), the large pores have a three-dimensional spatial
structure and are interconnected via their thin fibrous walls,
which itself are porous like conventionally electrospun fibers.
[0021] FIG. 5 shows SEM micrographs of the cryogenic electrospun
scaffolds collected at different relative humidity of the chamber.
(a) 25%, (b) 40% and (c) 55%. It can be observed that as humidity
increases from 25% to 55%, the pores of the cryogenic electrospun
scaffolds become larger and more defined.
[0022] FIG. 6 shows the SEM micrographs of cryogenic electrospun
scaffolds collected at different temperatures to illustrate the
effect of the temperature on the pore size of an electrospun
scaffold. The two pictures in the top row show conventional
electrospun scaffolds which were electrospun at 23.degree. C. The
left picture in the top row shows the mandrel interface whereas the
right picture shows the air interface. For definition purposes it
should be mentioned that the "mandrel interface" is the side of the
scaffold facing the mandrel and the side of the scaffold facing the
air is called the "air interface". In the middle row of pictures,
the left picture shows the mandrel interface of a scaffold
electrospun at -15.degree. C. whereas the right picture shows the
air interface of this scaffold. In the bottom row of pictures, the
left picture shows the mandrel interface of a scaffold electrospun
at -30.degree. C. whereas the right picture shows the air interface
of this scaffold. It can be observed that a conventional dense
electrospun scaffold is obtained when the mandrel temperature is
kept at 23.degree. C. When the mandrel temperatures are -15.degree.
C. and -30.degree. C., large pore structures (>5 .mu.m) can be
observed on both the mandrel and air interfaces of the scaffold. As
illustrated in FIG. 3, ice crystals formed on the mandrel at
sub-zero temperatures are embedded within the electrospun mesh. The
subsequent removal of the ice crystals through freeze-drying forms
these pore structures within the electrospun mesh.
[0023] FIGS. 7 (a), (c) and (e) are SEM micrographs showing the
pore structures of cryogenic electrospun scaffold after 5, 10 and
15 minutes of spinning without any time interval in between
spinning respectively. FIGS. 7 (b), (d) and (f) are SEM micrographs
showing the pore structures of cryogenic electrospun scaffold after
5, 10 and 15 minutes of spinning with a time interval of 5 minutes
in between each cycle. From FIG. 7, both Samples A (FIGS. 7 (a),
(c) and (e)) & B (FIGS. 7 (b), (d) and (f)) are porous
throughout the thickness of the scaffold, as observed by the slow
building up over the 5, 10 and 15 minutes. However, pore sizes are
smaller in the early stage, becoming bigger as the spinning
proceeds. Sample B has pore structures that are larger in diameter
and shallower, as compared to Sample A.
[0024] FIG. 8 shows the results of in vivo experiments in which
decellularized porcine esophageal ECM, CES and conventional dense
electrospun scaffolds were implanted in rats. FIG. 8 shows the in
vivo cell infiltration and vascularization in scaffolds obtained
from those rats. FIG. 8 shows hematoxylin and eosin staining of
scaffolds implanted subcutaneously for 14 days. (a) Decellularized
porcine esophageal ECM at 100.times. magnification, (b)
Decellularized porcine esophageal ECM at 400.times. magnification,
(c) CES at 400.times. magnification and (d) Conventional dense
electrospun scaffold at 400.times. magnification. (S: Scaffold, B:
Capillaries). _ represents 200 .mu.m in (a) and 50 .mu.m in (b),
(c) and (d). The pictures lying next to each other in FIG. 8 are
identical but the left hand picture is a black & white print of
a color picture whereas the right hand picture is a grey scale
version of the color picture on the left hand side. Areas of cell
infiltration are circled with dotted lines.
DETAILED DESCRIPTION OF THE INVENTION
[0025] A method of manufacturing a three-dimensional hybrid
scaffold according to any of the preceding claims, the method
comprising: [0026] providing a first layer made of a biological
material which has been decellularized; [0027] applying a second
layer to the surface of the first layer, wherein the second layer
is a bioadhesive and wherein the second layer is applied to the
first layer at a temperature of the environment which is below the
freezing temperature of the bioadhesive; and [0028] applying a
third layer which is a three-dimensional porous scaffold to a side
of the second layer which is not the side facing the first
layer.
[0029] This hybrid scaffold has been shown to positively influence
cell-scaffold interaction such as cell attachment, migration,
proliferation and function. The hybrid scaffolds manufacture by
this method can be used to (1) tailor the mechanical properties of
the treated ECMs to comply with host tissues, (2) replace excised
tissue segments by providing a thick fibrous scaffold with large
pores for cell infiltration and vascularization and (3) organize
cells attachment and proliferation along aligned fibers.
[0030] Another important aspect of the third layer is that it can
be used to deliver drugs, growth factors and proteins. These
components can be applied together with the scaffold and the
scaffold can serve as carriers to these drugs or molecules. Some
possible applications are as follows. For example, the hybrid
scaffold can be used to make vascular grafts for replacement of
blocked coronary artery. This vascular graft consists of a
decellularized blood vessel (first layer), porous bioadhesive
(second layer) and electrospun fibers containing an anti-coagulant
drug (e.g. heparin) (scaffold forming third layer). The release of
the anti-coagulant reduces the thrombogenic reaction at the implant
site and this release can be controlled by the pore size of the
scaffold and the diameter of the fibers of the scaffold. The graft
can be used to replace diseased coronary artery with a
drug-releasing third layer on the adluminal side and the
decellularized vascular ECM on the abluminal side. Another possible
application is in promoting better wound healing. Transforming
growth factor beta-3 (TGF-.beta.3) has been shown to improve
scarring during wound healing (Ferguson, M. W. J., O'Kane, S.,
2004, vol. 359(1445), p. 839-850). TGF-.beta.3 can be incorporated
into the electrospun fibers in the scaffold forming the third
layer. Controlled release of TGF-.beta.3 during wound healing will
reduce scar formation when the hybrid scaffold is implanted. Hence,
the incorporation of a third layer is advantageous to the hybrid
design.
[0031] An additional advantage is that the bioadhesive layer of the
scaffold of the present invention is porous. Normally, bioadhesives
are applied to connect different components with each other. For
example, in US 2004/0005297 A1 fibrin glue has been used as
bioadhesive to seal different layers with each other. However, the
fibrin glue is applied in a way that seals and separates the
different layers from each other not allowing ingrowth of tissue in
this sealant layer. However, in the method and the hybrid scaffold
of the present invention the bioadhesive layer is porous which
allows cells to attach, migrate and proliferate through and over
this layer. Thus, the hybrid scaffold of the present invention
provides a continuous structure allowing development of new tissue
in every section as will be explained in more detail in the
following.
[0032] The term "three-dimensional porous scaffold" as used herein,
refers to an artificial structure capable of supporting a
three-dimensional tissue formation. Scaffolds are supposed to
resemble the connective tissue in an extracellular matrix. Thus,
scaffolds allow for cell attachment, migration and growing of cells
and synthesis of extracellular matrix components and biological
molecules specific to the tissue targeted for replacement. To
achieve those objects, a scaffold ideally provides a high porosity
and proper pore size, a high surface area, biodegradability, proper
degradation rate to match the rate of neotissue formation and it
should provide a sufficient mechanical integrity to maintain the
predesigned tissue structure. A scaffold should also not be toxic
to the cells (i.e. biocompatible) and should positively interact
with cells including enhanced cell adhesion, growth, migration, and
differentiated function (Ma, P. X., May 2004, Materials Today, p.
30-40).
[0033] "Biological material" composed of naturally occurring
extracellular matrix (ECM) has received significant attention for
their potential therapeutic application in tissue engineering. In
general, the biological material has been isolated from a part of
the body of an individual. The ECM is custom designed and
manufactured by the resident cells of each tissue and organ and is
in a state of dynamic equilibrium with its surrounding
microenvironment and thus the ideal source for the construction of
hybrid scaffolds. The structural and functional molecules of the
ECM provide the means by which adjacent cells communicate with each
other and with the external environment. The ECM is obviously
biocompatible since host cells produce their own matrix.
[0034] The ECM is obviously biocompatible since host cells produce
their own matrix. The ECM also provides a supportive medium or
conduit for blood vessels, nerves and lymphatics and for the
diffusion of nutrients from the blood to the surrounding cells. In
other words, the ECM possesses all of the characteristics of the
ideal tissue engineered scaffold or biomaterial. Various forms of
intact ECM have already been used as biological scaffolds to
promote the constructive remodeling of tissues and organs (Meyer,
T., Chodnewska, I., et al., 1998, Transplant Proc, vol. 30, p. 354
et seqq.; Robinson, K. A., Matheny, R. G., 2003, J Am Coll Cardiol,
vol. 41, no. 6, suppl. 2, p. 514 et seqq.; Schenke-Layland, K.,
Vasilevski, O., et al., 2003, J Struct Biol, vol. 143(3), p.
201-208; Sutherland, R. S., Baskin, L. S., et al., 1996, J Urol,
vol. 156, p. 571-577; Valentin, J. E., Badylak, J. S., et al.,
2006, J Bone Joint Surg Am, vol. 88(12), p. 2673-2686).
[0035] The constructive remodeling induced by ECM scaffold
materials and their widespread use across many clinical
applications are a consequence of their bio-inductive properties,
mechanical and material properties, the host tissue response to
naturally occurring ECM, and the degradation properties of the
material.
[0036] In general, a "biological material" can be from any tissue
of the body of an individual or different individuals. Related
cells joined together are collectively referred to as biological
tissue. The cells in a tissue are not identical, but they work
together to accomplish specific functions. A sample of tissue
removed for examination under a microscope (biopsy) contains many
types of cells. Connective tissue is the tough, often fibrous
tissue that binds the body's structures together and provides
support. It is present in almost every organ, forming a large part
of skin, tendons, and muscles. The characteristics of connective
tissue and the types of cells it contains vary, depending on where
it is found in the body.
[0037] The body's functions are conducted by organs. Each organ is
a recognizable structure--for example, the heart, lungs, liver,
eyes, and stomach--that performs specific functions. An organ is
made of several types of tissue and therefore several types of
cells. For example, the heart contains muscle tissue that contracts
to pump blood, fibrous tissue that makes up the heart valves, and
special cells that maintain the rate and rhythm of heartbeats. The
eye contains muscle cells that open and close the pupil, clear
cells that make up the lens and cornea, cells that produce the
fluid within the eye, cells that sense light etc. Even an organ as
apparently simple as the gallbladder contains different types of
cells, such as those that form a lining resistant to the irritative
effects of bile, muscle cells that contract to expel bile, and
cells that form the fibrous outer wall holding the sac
together.
[0038] The biological tissue referred to in the present invention
has been isolated from a part of the body of an individual and can
be from any tissue or organ or part of an organ of an individual.
It can for example be derived from the small intestine, liver,
pancreas, urinary bladder, stomach, bladder, vascular system, bile
duct, alimentary canal, respiratory tract, kidney, spleen, heart,
heart valve, bone, skin or fragments or parts thereof. In one
example, it is derived from the gastrointestinal tract, namely the
esophagus mucosa.
[0039] The term "hybrid scaffold" refers to a combination or
composition comprising a "biological material" and a "scaffold"
wherein these two components are connected to each other via a
bioadhesive layer.
[0040] The individual from which the biological tissue is derived
can be a mammal, reptile or insect. In general it can be taken from
any animal which tissue or organs are suitable to be implanted in a
host and which are not rejected by the immune system of the host or
show at least only little rejection which could be suppressed by
commercial drugs. In one example, the biological tissue is derived
from a mammal which can be of porcine, bovine or ovine origin or
can be a rabbit, monkey or human.
[0041] In one example, the biological material is decelluralized,
i.e. the material is completely acellular. Methods for
decelluralizing biological material are known in the art and are
described, for example in a review by Gilbert, T. W., Sellaro, T.
L., et al. (2006, Biomaterials, vol. 27, p. 3675-3683), the content
of it is incorporated herein by reference in its entirety. Thus, in
one example of the method of the present invention, the method
further comprises decellularizing the biological material before
applying the bioadhesive second layer at the surface of the
biological material forming the first layer.
[0042] The goal of a decellularization protocol is to efficiently
remove all cellular and nuclear material while minimizing any
adverse effect on the composition, biological activity, and
mechanical integrity of the remaining ECM of the biological
material which forms the first layer in the hybrid scaffold of the
present invention.
[0043] Any processing step intended to remove cells will alter the
native three-dimensional architecture of the ECM. The most commonly
utilized methods for decellularization of tissues or parts of it
involve a combination of physical and chemical treatments. The
physical treatments can include agitation or sonication, mechanical
massage or pressure, or freezing and thawing. These methods disrupt
the cell membrane, release cell contents, and facilitate subsequent
rinsing and removal of the cell contents from the ECM. These
physical treatments are generally insufficient to achieve complete
decellularization and must be combined with a chemical treatment.
Enzymatic treatments, such as trypsin, and chemical treatment, such
as ionic solutions and detergents, disrupt cell membranes and the
bonds responsible for intercellular and extracellular
connections.
[0044] As previously mentioned, tissues are composed of both
cellular material and ECM arranged in variable degrees of
compactness depending on the source of the tissue. The ECM must be
adequately disrupt during the decellularization process to allow
for adequate exposure of all cells to the chaotropic agents and to
provide a path for cellular material to be removed from the tissue
leaving behind only the ECM, which is used as biological material
in the hybrid scaffold. The intent of most decellularization
processes is to minimize the disruption and thus retain native
mechanical properties and biological properties. The most robust
and effective decellularization protocols include a combination of
physical, chemical, and enzymatic approaches. A decellularization
protocol as described in the examples of the present application
generally begins with lysis of the cell membrane using physical
treatments or ionic solutions, followed by separation of cellular
components from the ECM using enzymatic treatments, solubilization
of cytoplasmic and nuclear cellular components using detergents,
and finally removal of cellular debris from the tissue. These steps
can be coupled with mechanical agitation to increase their
effectiveness. Following decellularization, all residual chemicals
should be removed to avoid an adverse host tissue response to the
chemical. The efficiency of decellularization and preservation of
the ECM can be assessed by several methods. The mechanisms of
physical, enzymatic, and chemical decellularization for a variety
of tissues are reviewed and displayed in Table 1 of the article of
Gilbert, T. W., Sellaro, T. L., et al. (2006, supra).
[0045] In an example of the present invention decellularization was
carried out using a multistep method. Firstly, the biological
material isolated from the body of an individual was rinsed in
saline solution to remove cellular debris. Secondly, the biological
material is left to immerse in deionized water at about 1 to 6
degrees Celsius for 30 min to 1 hour. Thirdly, a decellularizing
agent comprising hydrogen peroxide, glacial acetic acid and
deionized water in a ration of 20:40:40 vol. % is prepared. The
biological tissue was immersed in this solution at ambient
temperatures from 4.degree. C. to 40.degree. C. Afterwards, the
biological material is decellularized mechanically. Then the
biological material was neutralized. The next step is the treatment
with TritonX-100 solution in concentrations ranging from 0.05% to
5%. In another example, concentrations of 0.5% to 3% have been
used. The biological material was immersed in TritionX-100 for
about 6 to 48 hours. In one example, it has been treated for 12 to
24 hours. Gentle stirring helped to remove the remaining cells from
the biological material. Afterwards, the biological material is
rinsed to remove all traces of TritonX-100. A more detailed
description of an illustrative example can be found in the
experimental section of this application. This, method allows for
example also decellularization of whole organs, like kidney, liver,
esophagus etc.
[0046] Thus, the advantages of fabricating tissue engineering
scaffolds by decellularization of tissues are the preservation of
native topography of ECMs, inherent histo-architectures of
structural collagen and original configurations of functional
proteins. These are components necessary in promoting cell
attachment and proliferation. Acellular matrices are biodegradable
and they are generally resorbed during tissue remodeling processes.
In a particular case, it has been reported that the degraded
products of acellular matrices promoted migration of endothelial
cells, hence inducing angiogenesis within the matrices (Li, F., et
al., 2004, Endothelium, vol. 11, p. 199-206).
[0047] These decellularizing processes can weaken the mechanical
properties of the treated biological material. In addition, tissue
segments such as muscles, which are not intended for
decellularization, may be removed during the processes. These
tissues segments may need to be replaced with the acellular ECMs in
the reconstruction of the entire tissue. This is only one example
why the scaffold and the method of the present invention connect a
third layer via a second layer to the first layer, namely to
compensate the structural deficiencies of the first layer weakened,
for example, in a process of decellularization or upon removal of
the biological material from the body of the individual.
[0048] The second layer of the hybrid scaffold of the present
invention is made of a bioadhesive. In general, an adhesive is a
material capable of fastening two other materials together by means
of surface attachment. The words glue, mucilage, mastic, and cement
are synonymous with adhesive. In a generic sense, the word adhesive
implies any material capable of fastening by surface attachment,
and thus will include inorganic materials. In a practical sense,
however, adhesive implies the broad set of materials composed of
organic compounds, mainly polymeric, which can be used to fasten
two materials together. The materials being fastened together by
the adhesive are the adherents, and an adhesive joint or adhesive
bond is the resulting assembly. Adhesion is the physical attraction
of the surface of one material for the surface of another.
[0049] Examples of organic adhesives which can be used in the
method of the present invention for the manufacture of the scaffold
of the present invention are, for example, fibrin glue,
polyvinylpyrolidone, polyvinylpyrolidone/vinyl acetate copolymers,
cyanoacrylate gel, platelated gel, chitosan or
gelatin-resorcin-formaldehyde (GRFG). In one example, fibrin glue
is used to connect the first and third layer of the hybrid
scaffold.
[0050] For example, cyanoacrylate gel, fibrin glue and GRFG are
known as bioadhesives and are described, for example, in the
article of Albes, J. M., Krettek, C. et al. (1993, The annals of
Thoracic Surgery, vol. 56, p. 910-915).
[0051] Fibrin sealants or fibrin glues are a type of surgical
tissue adhesive derived from human and animal blood products
(Thorn, J. J., Sorensen, H., et al., 2004, Int. J. Oral Maxillofac.
Surg, vol. 33, p. 95-100). The ingredients in these sealants
interact during application to form a stable clot composed of a
blood protein called fibrin. Fibrin sealants are known since World
War II. All fibrin sealants in use as of 2003 have two major
ingredients, purified fibrinogen (a protein) and purified thrombin
(an enzyme) derived from human or bovine (cattle) blood. Many
sealants have two additional ingredients, human blood factor XIII
and a substance called aprotinin, which is derived from cows'
lungs. Factor XIII is a compound that strengthens blood clots by
forming cross-links between strands of fibrin. Aprotinin is a
protein that inhibits the enzymes that break down blood clots.
[0052] For preparation and application of fibrin sealants the
thrombin and fibrinogen are freeze-dried and packaged in vials that
must be warmed before use. The two ingredients are then dissolved
in separate amounts of water. Next, the thrombin and fibrinogen
solutions are loaded into a double-barreled syringe that allows
them to mix and combine as they are sprayed on the surface.
[0053] As the thrombin and fibrinogen solutions combine, a clot
develops in the same way that it would form during normal blood
clotting through a series of chemical reactions known as the
coagulation cascade. At the end of the cascade, the thrombin breaks
up the fibrinogen molecules into smaller segments of a second blood
protein called fibrin. The fibrin molecules arrange themselves into
strands that are then cross-linked by a blood factor known as
Factor XIII to form a lattice or net-like pattern that stabilizes
the clot. An example of a commercially obtainable fibrin sealant is
TISSEEL (Baxter International Inc.).
[0054] Recent developments of fibrin sealants include a delivery
system that forms a fibrin sealant from the individuals own blood
within a 30-minute cycle. The use of the individuals own blood
lowers the risk of allergic reactions to blood products derived
from animal or donated blood. Platelate gel is a similar autologous
glue where a platelet concentrate prepared from the individuals
plasma substituted the fibrinogen concentrate (Hood, A. G., Hill,
A. G., 1993, Proceedings of the American Academy of cardiovascular
perfusion, vol. 14, p. 126-129).
[0055] Another suitable group of adhesives is organic polymeric
compositions represented by the group of alkyd resins, polyvinyl
acetaldehydes, polyvinyl alcohols, polyvinyl acetates,
poly(ethylene oxide), polyacrylates, ketone resins,
polyvinylpyrolidone, polyvinylpyrolidone/vinyl acetate copolymer,
polyethylene glycols of 200 to 1000 molecular weight and
polyoxyethylene/polyoxopropylene block copolymers (Polyox),
silicone resins and silicone based pressure sensitive adhesives
such as those available from Dow Corning Company under the trade
designation BIO-PSA. The pressure sensitive adhesive (PSA) are well
known in the art and many are commercially available.
[0056] To achieve porosity of this bioadhesive second layer it is
important to apply the bioadhesive at a temperature which is at or
below the freezing temperature of the solvent of the bioadhesive.
If the bioadhesive layer is applied at ambient temperature, i.e. a
temperature above the freezing temperature, a dense layer is formed
which does not allow cell infiltration (see FIGS. 1 d and e). Such
a non-porous film also acts as a barrier to nutrient diffusion.
[0057] However, applying bioadhesives at a temperature at or below
the freezing temperature of the bioadhesive results in the
formation of crystals (from the solvent of the bioadhesive) in the
bioadhesive layer which form the porous system upon removal of the
crystals. Such crystals can be removed by sublimation which means
that the solid crystals pass directly into the gas phase without
becoming liquid beforehand. A method for achieving sublimation can
be freeze-drying. The pore size within the bioadhesive layer can be
in a range of about 1 nm to about 500 .mu.m. In another example,
the pore size is about 50 .mu.m to about 500 .mu.m or 100 .mu.m to
about 300 .mu.m. Which range will be chosen depends also on the
desired tissue which is supposed to be replaced. Thus, further
ranges which can be used will be mentioned further below. The pore
size of the second and third porous layer can be the same or
different.
[0058] FIGS. 1 a and b depict hybrid scaffolds in which the
bioadhesive second layer has been formed at or below the freezing
temperature of the bioadhesive. As can be seen, the bioadhesive
layer forms a porous network with pores having a size of about 10
to 200 .mu.m. This is a positive step towards attaining a wholly
porous hybrid scaffold that would be advantageous in nutrient
diffusion and cell infiltration in in vitro and in vivo systems.
Another advantage of the porous interface is that it will promote
cell infiltration and vascularization and thus facilitates
remodeling of the scaffold construct in vivo. The bioadhesive layer
can be applied using any method which allows freezing the
bioadhesive layer upon application. Examples of such methods
include for example spraying, electrospraying, electrospinning,
spin coating, dip coating, casting and brushing.
[0059] Electrospraying is a method in which a liquid (in this case,
a solution of bioadhesive) is atomized into a mist of droplets by
electrical forces and is described, for example, in the article of
Huang, J., Jayasinghe, S. N., et al. (2004, Journal of Materials
Sciences, vol. 39(3), p. 1029-1032). Dip coating is a method in
which the substrate (e.g. ECM) can be dipped into a solution of
bioadhesive in a process similar to that described, for example, in
the article of Liu, Z. F., Jin, Z. G., et al. (2006, Journal of
Sol-Gel Science and Technology, vol. 40(1), p. 25-30). In dip
coating, the thickness of the bioadhesive coating depends on the
viscosity of the solution, the speed of substrate withdrawal from
the solution and the time which the substrate is immersed in the
solution, all of which are known in the prior art. Spin coating is
a method in which a dilute solution of the bioadhesive is applied
on the ECM and the ECM is spun at high speeds (e.g. 3000 to 5000
rpm) to obtain a thin coat of bioadhesive on the surface of the
ECM. The process is described, for example, in the article of
Dupont-Gillain, C. C. and Rouxhet, P. G. (2001, Nano Letters, vol.
1(5), p. 245-251). Casting is a method in which the bioadhesive is
spread on the substrate (e.g. ECM) to obtain a uniform layer of
bioadhesive on the ECM in a process similar to that described, for
example, in the article of Smith, S., Stolle, D. (2000, vol. 40(8),
p. 1870-1877).
[0060] In general, the third layer needs to be applied before the
bioadhesive is fully set/crosslinked in order to ensure a strong
connection between the different layers of the hybrid scaffold. The
surface of the scaffold of the third layer facing the bioadhesive
layer should be wetted with the bioadhesive in order to form
sufficient bonding with the setting and freezing bioadhesive. A
person skilled in the art can easily determine the point of time
before the bioadhesive is fully set/crosslinked because the
bioadhesives turn from a liquid phase into a gelling liquid and
eventually to a solid film when cured. The third layer should be
applied during the initial or gelling phase of the bioadhesive
layer.
[0061] For example, the components of the bioadhesive fibrin which
are fibrinogen and thrombin are reconstituted separately into
solutions according to procedures described in the instruction for
use, for example, of the Tisseel Kit (Baxter International Inc.).
Both of these solutions should be clear or slightly opalescent. On
mixing the two solutions, the mixture turns cloudy and curing
begins. After change of the phase, from solution form to a gelling
liquid the scaffold should be applied. When electrospinning is used
for the third layer (see further below) the fibers can be deposited
during the solution and gelling liquid phase, when bonding between
the bioadhesive and fibers may occur. The curing time of the
bioadhesive can be shortened by either increasing the thrombin
concentration, decreasing the aprotinin concentration or increasing
the temperature of the mixture, all of which are known in the prior
art.
[0062] In another aspect, the method of the present invention
further comprises controlling the degradation time of the
bioadhesive layer by controlling the pore size of the bioadhesive
layer which is achieved by increasing or decreasing the temperature
in the environment when applying the bioadhesive layer, as long as
the temperature is still at or below the freezing point of the
bioadhesive. Lower temperatures result in faster rate of crystal
formation and smaller solid crystals. That means that at a lower
pore size within the bioadhesive layer, the degradation of the
bioadhesive takes longer because the enzymes and cells growing in
the area surrounding the scaffold, which is seeded with cells and
possibly already implanted into the body of an individual, take
longer to degrade the bioadhesive.
[0063] Controlling the degradation time of the bioadhesive can be
an advantage, when one wished to synchronize the degradation rate
of the bioadhesive layer to the rate of remodeling by the
infiltrating host cells. If the bioadhesive degrades too fast, the
first layer might delaminate from the third layer in the host;
conversely, if the bioadhesive or the entire hybrid scaffold
degrades too slowly, remodeling takes place with scarring around
and within the construct. The hybrid scaffold might not be
integrated with the host for its intended application due to the
formation of excessive collagenous scar.
[0064] In one example, the pore size of a bioadhesive can be
changed by varying the water content in the bioadhesive solution.
For example for fibrin glue, the pore size can be influenced by
changing the concentration of fibrinogen and thrombin in the
aqueous medium, thus changing the amount of ice within the fibrin
layer. The more water in the bioadhesive solution the larger the
ice crystals grow.
[0065] Another factor influencing the degradation time of the
bioadhesive layer is the thickness of the bioadhesive second layer.
The thicker the layer the longer the degradation of the whole layer
takes.
[0066] In one example, the thickness of the bioadhesive second
layer can be about 10 to 1000 .mu.m or 20 to 600 .mu.m before the
third layer is applied onto the second layer. In another example
the thickness is about 20 to 300 .mu.m. However, bioadhesive layers
having a thickness that exceeds 1000 .mu.m, namely 2, 3 or even
more millimeter up to 1 cm would also be possible.
[0067] The third layer is a porous three-dimensional scaffold.
Scaffolds are widely known in the area of tissue engineering and
every scaffold which can be connected to the first layer via the
bioadhesive layer can be used in for the hybrid scaffold and in the
method of the present invention. The scaffolds used in the present
invention comprise a reticulated structure of interconnected pores.
The third layer can be for example a scaffold which is obtained via
electrospinning, i.e. an electrospun scaffold.
[0068] Depending on the use of the claimed method, scaffold
material can be biodegradable. To use biodegradable material is
especially advantageous, e.g., for tissue engineering wherein the
scaffolds containing the cells are used to repair defect sites in
living tissue, e.g. bone. A high variety of scaffolds can be used
dependent on the application. Scaffolds comprise or are can be made
from agarose, polycaprolactone (Endres, M. et al., Tissue
Engineering, 2003, Vol. 9, No. 4, P. 689-702), niobium coated
carbon, chitosan, hydroxyapatite-tricalcium phosphate (Harris, C.
T. and Cooper, L. F., Comparison of matrices for hMSC delivery,
2004, P. 747-755), collagen, hyaluronic acid, calcium phosphate,
starch, hydroxyapatite, fibrin, alginate, poly-glycolic acid,
carbon nano fibres, polytetrafluoroethylene, polylactic acid
(Moran, J. et al., Tissue Engineering, 2003, Vol. 9, No. 1, P.
63-70) and mixtures thereof, for example. Foam scaffold as those
described in U.S. Pat. No. 6,231,879 which are based on
thermoplastic elastomers such as polyamide, polyester, polyethylene
polyvinylidene fluoride, polyethyurethane or silicone can also be
used in the present invention.
[0069] The scaffold can have a regular or an irregular (outer)
shape. If the scaffolds are, e.g., used in tissue engineering the
shape of the scaffold will fit the shape of the defect side in
which the scaffold will be implanted or at least will fill up the
part not already covered by the biological material of the hybrid
scaffold. A scaffold with a regular shape can be rectangular, a
square, or of polyhedric or spherical shape. Scaffold of a
rectangular shape usually have a length in their largest dimension
of about 1 mm to about 5 cm or even 20 cm.
[0070] The shape of the scaffold forming the third layer depends in
particular on the shape and form of the biological material forming
the first layer. The third layer advantageously rounds out the
missing characteristics of the first layer. For example, the
scaffold of the third layer provides the mechanical stability
within a defect side which the biological material of the first
layer cannot provide any more. The scaffolds made of the materials
listed above can be manufactured in any specific size and form
depending of the application.
[0071] Even though any commercially available scaffold can be used
as third layer, it is also possible to create the scaffold directly
on the surface of the bioadhesive second layer which is already
attached to the first layer. Thus, in one aspect of the present
invention, the scaffold is created by direct electrospinning on the
surface of the bioadhesive second layer.
[0072] Unlike other methods for generating nanostructures, the
formation of a thin fiber for a scaffold via electrospinning is
based on the uniaxial stretching (or elongation) of a viscoelastic
jet derived from a polymer solution or melt. This technique is
similar to the commercial processes for drawing microscale fibers
except for the use of electrostatic repulsions between surface
charges (rather than a mechanical or shear force) to continuously
reduce the diameter of a viscoelastic jet or a glassy filament.
Compared with mechanical drawing, electrostatic spinning is better
suited for generating fibers with much thinner diameters, since the
elongation can be accomplished via a contactless scheme through the
application of an external electric field. Like mechanical drawing,
electrospinning is also a continuous process and therefore should
work well for high-volume production (Li, D. & Xia, Y. N.,
2004, supra).
[0073] In electrospinning, a solid fiber is generated as the
electrified jet (composed of a highly viscous polymer solution, see
further below) is continuously stretched due to the electrostatic
repulsions between the surface charges and the evaporation of
solvent. As the fiber travels toward the surface of the collector,
evaporation of the solvent in which the polymer is dissolved occurs
and the fiber is typically dry when arriving at the surface of the
collector (see FIGS. 2 a and b).
[0074] Therefore, the terms "electrospinning" or "electrospun" as
used herein refer to any method where materials are streamed,
sprayed, sputtered or otherwise transported in the presence of an
electric field. The electrospun solution comprising at least one
polymer can be deposited form the direction of a charged container
towards a grounded collector, or from a grounded container in the
direction of a charged collector.
[0075] Thus, the third layer can be made of a scaffold which is
directly electrospun on the bioadhesive second layer. However, it
should be noted that also the second layer can be applied on the
first layer using electrospinning. Thus, the following arguments
regarding the options of modifying electrospun scaffolds can also
be transferred to the electrospinning of the bioadhesive second
layer.
[0076] However, polymer scaffolds produced via conventional
electrospinning, which can certainly be used herein as third layer,
are often characterized by a random polymer fiber mesh as
illustrated by the SEM picture of such a mesh in FIG. 2a and FIG.
4a. However, the pore size between the fibers in such a polymer
fiber mesh is only between several nanometer to a few
micrometer.
[0077] But some applications require a bigger pore size in order to
enable cells seeded in the hybrid scaffold to grow and develop the
tissue they are supposed to resemble. For example, Klawitter et al.
reported (1976, J Biomed Mater Res, vol. 10(2), p. 311-323) that
for adequate bone regeneration to occur in a scaffold, scaffold
pore size needs to be at least 100 micrometer. It is generally
known in the art that optimal bone regeneration occurs for pore
sizes between 300 to 600 micrometer. For other applications the
following pore sizes have been reported to be optimal for the
specific applications: about 5 .mu.m for neovascularization,
between about 5 to about 15 .mu.m for fibroblast ingrowth, about 20
.mu.m for hepatocyte ingrowth, between about 20 to 125 .mu.m for
skin regeneration, between about 70 to 120 .mu.m for chondrocyte
ingrowth, between about 40 to 150 .mu.m for fibroblast binding,
between about 45 to 150 .mu.m for liver tissue regeneration,
between about 60 to 150 .mu.m for vascular smooth muscle cell
binding, between about 100 to 300 .mu.m for bladder smooth muscle
cell adhesion and ingrowth, between about 100 to 400 .mu.m for bone
regeneration and between about 200 to 350 .mu.m for
osteoconduction.
[0078] Thus, the pore size of the two layers which are not natural,
namely the bioadhesive second layer and the third layer made of an
artificial three-dimensional scaffold should match or come close to
the pore size required for replacing a certain type of tissue or
whole organ.
[0079] For example, the dense ECM (first layer) can be used as a
barrier for cells (e.g. epithelial cells, endothelial cells) that
need a basal membrane for attachment, proliferation and
differentiation. The open porous structure of the porous
electrospun (third layer) can be used to promote cell infiltration
and vascularization and is important in reconstruction of thick
tissues (e.g. muscle). Some examples of cells or combination of
cells that can be grown on the hybrid for tissue replacement are
described in the following. Esophagus--Epithelial cells on
decellularized esophageal ECM (first layer) and smooth muscle cells
in porous electrospun scaffold (third layer). Blood
vessels--Endothelial cells on decellularized blood vessel (first
layer) and smooth muscle cells in porous electrospun scaffold
(third layer). Skin--Keratinocytes on decellularized dermis and
dermal fibroblasts in porous electrospun scaffold (third layer).
Bladder--Urothelial cells on decellularized bladder (first layer)
and smooth muscle cells in porous electrospun scaffold (third
layer).
[0080] Obtaining pore sizes as described in paragraph 68 using
electrospinning is possible due to the following method. In this
method a three-dimensional scaffold for tissue engineering is
manufactured using an apparatus for electrospinning comprising a
high-voltage power supply; at least one spinneret connected to at
least one container comprising a solution with at least one polymer
dissolved therein; and a collector which can already be connected
to the first and second layer of the hybrid scaffold which are
already connected to each other. The method comprises: [0081]
forming crystals from a molecule or group of molecules in vapor
phase comprised in the surrounding atmosphere at the surface of the
second layer, wherein the atmosphere in the reaction chamber of the
electrospinning apparatus comprising the collector or the first and
second layer already connected to each other has a temperature
which allows formation of crystals at the surface of the second
layer; [0082] electrospinning the solution comprising at least one
polymer dissolved therein around the crystals; [0083] continuing
the formation of crystals and the electrospinning simultaneously;
and [0084] removing the crystals by sublimation.
[0085] In case the reaction chamber already contains the first and
second layer which are already connected to each other, the
bioadhesive second layer will form the collector for
electrospinning.
[0086] It should be noted that this specific technique of obtaining
an electrospun scaffold, which can be used as third layer, can also
be carried out separately to obtain the cryogenic scaffold. After
obtaining such a cryogenic scaffold, the scaffold can be connected
to the surface of said second layer.
[0087] Formation of solid crystals in the above method can be
achieved by either cooling the collector of the electrospinning
apparatus itself to create a temperature gradient above the first
and second layer connected to the collector or by cooling the
atmosphere in the reaction chamber of the electrospinning apparatus
to an extent that solid crystals are formed at the surface of the
second layer.
[0088] This method of electrospinning provides for the design of
electrospun scaffolds at the surface of the bioadhesive second
layer having different distributions of pore sizes to cater for
different requirements in basal membrane formation (nano-scale),
tissue remodeling and regeneration (nano to micro scale),
vascularization (micro scale) and cell in-growth (micro-scale).
[0089] When referring to "pores" in connection with the pores of
the electrospun scaffold, it is not referred to the pores which
might be formed in the fibers which are spun but the size of the
three-dimensional pores formed by the fibers as can bee seen, for
example, in FIGS. 1a, 3, 4b, 4c, 5a, 5b and 5c. A conventional
electrospun scaffold as shown, for example, in FIGS. 1f and 4a
shows a highly porous network of non-woven submicron fibers in a
planar orientation. The pores are bounded by individual fibers, and
measure only between several nanometer to a few micrometer. In
contrast, the large pores obtained using the above method for
electrospinning of a scaffold on the second layer of the hybrid
scaffold measure in this illustrative example between about 50 to
500 .mu.m in size (see FIG. 4b). However, it should be noted that
such pores can exceed 500 .mu.m. These pores are bounded by bundles
of fibers that form a strut-like support. As can be seen from a SEM
picture taken at an oblique view (FIG. 4c), the large pores have a
three-dimensional spatial structure and are interconnected via
their thin fibrous walls, which itself are porous like
conventionally electrospun fibers.
[0090] As can be seen from FIG. 3, this method of electrospinning
involves in this example a reaction chamber cooled to subzero
temperatures. In one example, a hollow rotating mandrel (see FIG.
2b) is used as collector. The first layer is directly connected to
the collector and the bioadhesive second layer is applied on the
first layer and cooled to provide a porous structure. In one
example, the temperature used for freezing the bioadhesive second
layer is the same temperature which is used for the cryogenic
electrospinning as long as the temperature is equal or below the
freezing temperature of the bioadhesive as well as the solvent in
the polymer solution used for electrospinning. Subsequently, solid
crystals made, for example, of water (H.sub.2O) from the
surrounding atmosphere of the collector form on the surface of the
second layer and serve as a negative template around which
electrospun fibers are deposited. After the scaffold reaches the
desired form, the scaffold can be subsequently freeze-dryed to
remove ice crystals, leaving behind a cryogenic electrospun
scaffold (CES) with large pores (see FIGS. 4b and 4c). Further
details for this illustrative example are given in the experimental
section of this application.
[0091] Thus, this cryogenic electrospinning technique enables the
fabrication of an electrospun scaffold with large pores, while
retaining the nanofibrous structure that mimics the physical
environment of the targeted tissue or organ to be replaced and
which is required for cell growth, vascular ingrowth and tissue
development.
[0092] The crystals which form at the surface of the second layer
can be made of any molecule or group of molecules in vapor phase
which is/are comprised in the atmosphere surrounding the collector
and which is/are deposited at the surface of the second layer in
form of solid crystals when the temperature at the surface of the
second layer is lowered to or below the freezing point of the
molecule or group of molecules which is/are comprised in the
atmosphere surrounding the second layer. Since the question of
forming crystals depends also on the pressure in the .surrounding
atmosphere, the pressure can be increased or decreased to support
formation of crystals even at higher temperatures. Which pressure
and temperature is most suitable to ease the freezing step of a
certain group of molecules can be easily determined by a person
skilled in the art when looking at the phase diagram of the
molecule which shall form crystals at the surface of the
collector.
[0093] In one example, the crystals are ice crystals formed from
water (H.sub.2O) comprised in the surrounding atmosphere. After the
scaffold reaches its final dimensions the crystals are removed by
freeze-drying. Instead of water D.sub.2O can also be used. In
another example, the crystals are formed from CO.sub.2 comprised in
the surrounding atmosphere. Other than the water ice crystals which
are removed by freeze-drying, the CO.sub.2 crystals are removed by
sublimation. The sublimation temperature of carbon dioxide
(CO.sub.2) is -78.5.degree. C. at atmospheric pressure. If the
electrospinning environment is filled with vapor CO.sub.2,
deposition of solid CO.sub.2 crystals on the surface of the
collector can be achieved if the collector is maintained at
temperatures below -78.5.degree. C. After formation of the
electrospun scaffold the atmosphere surrounding the crystals are
removed from the hybrid scaffold through sublimation of CO.sub.2
into vapor phase at room temperature.
[0094] The shape and thus the size of crystals depends also on the
temperature at the surface of the second layer or the surrounding
of the layer. For example, water has a freezing point of 0.degree.
C. at atmospheric pressure. Just below freezing, at temperatures
near T=-2.degree. C., the growth of ice crystals is plate-like,
with thick plates at lower supersaturations, thinner plates at
intermediate supersaturations, and plate-like dendritic structures
at high supersaturations. For temperatures near T=5.degree. C., the
growth is columnar, with stout columns at the lower
supersaturations, more slender, often hollow columns at
intermediate supersaturations, and clusters of thin, needle-like
crystals at higher supersaturations. Colder still, near
T=-15.degree. C., the growth again becomes plate-like, and again
one sees increasing structure with increasing supersaturation.
Finally, at the lowest temperatures the growth becomes a mixture of
thick plates at low supersaturations and columns at higher
supersaturations. Growth of heavy water (D.sub.2O) crystals from
the vapour phase produces similar morphologies as a function of
temperature, except shifted by approximately four degrees, in
keeping with the isotopic shift in the freezing point between
D.sub.2O and H.sub.2O (Libbrecht, K. G., 2005, Reports on Progress
in Physics, vol. 68, p. 855-895). This principle regarding the
shape and the size of crystals applies also to the structure of the
porous network of the bioadhesive second layer.
[0095] Thus, this method also comprises increasing or decreasing
the temperature of the second layer or the surrounding atmosphere
of the layer as long as the temperature allows freezing of the
molecules or group of molecules from the surrounding atmosphere at
the surface of the second layer.
[0096] In one example, crystals can be formed by depositing water
at the surface of the second layer at a temperature of the second
layer of about -15.degree. C. or -30.degree. C. When the molecule
to be deposited at the surface of the second layer is water then
the temperature should be below 0.degree. C. at atmospheric
pressure or between about 0.degree. C. to about -100.degree. C. In
another example, the temperature is between about 0.degree. C. or
-1.degree. C. to about -30.degree. C.
[0097] It is also important to note that the crystal growth in this
method can be enhanced when one let flow air over a growing surface
of crystals, a phenomenon called the ventilation effect.
[0098] From the previous comments it also becomes obvious that not
only the temperature can influence the crystal formation and thus
the size and structure of the pores formed in the scaffold but also
the saturation of the atmosphere with the elements or group of
elements which are to be deposited on the surface of the
collector.
[0099] Thus, the method also comprises increasing or decreasing the
saturation of the atmosphere with the molecules or group of
molecules which are to be deposited on the surface of the
collector.
[0100] As demonstrated in the examples (see also FIGS. 5a to 5c),
when increasing the saturation of the atmosphere surrounding the
collector with water (i.e. humidity), the size of the
three-dimensional pores is larger and more defined.
[0101] The "atmosphere" surrounding the collector can be varied to
contain, for example, the specific molecules or group of molecules
which are to be deposited on the surface of the second layer or to
vary the saturation of the atmosphere with the specific molecules
or group of molecules to be deposited at the surface of the second
layer. In one example, normal air forms the atmosphere surrounding
the collector. In another example, mentioned herein, nitrogen
(N.sub.2) is added to the atmosphere to control the water content
in the air. A pure CO.sub.2 atmosphere would for example also be
possible.
[0102] Thus, the method of electrospinning can also comprise
increasing or decreasing the flow rate of the solution comprising
the at least one polymer dissolved therein. The flow rate of the
solution or in other words the solution feeding rate can be changed
by increasing or decreasing the pressure in the at least one
container comprising the at least one polymer dissolved therein. In
general, a higher feeding rate for the solution leads to the
formation of thicker fibers.
[0103] In general, the diameter of fibers affects the surface area
of the fibers, which in turn affects the rate of degradation of the
scaffold material. The mechanical properties of the electrospun
mesh can also be affected by the diameter of the fibers. Varying
solution flow rate is also in changing the pore size and porosity
of the eventual scaffold. To elaborate, if one increases solution
flow rate while keeping all other parameters constant, there is
more throughput of polymer through the spinneret which will occupy
more space with respect to the deposited crystals. In this way, the
pore size and the porosity of the scaffold will decrease with
increasing flow rate of the solution.
[0104] In still another aspect the method can further comprise
varying the time interval between electrospinning and the formation
of crystals. It can be easily imagined that the rates of crystal
formation and fiber deposition are two competing factors that can
have an effect on the pore structure of the cryogenic electrospun
scaffolds. As more time passes between different electrospinning
steps as larger the crystals can grow on the collector which means
that the resulting pores of the electrospun scaffold are growing
larger. To demonstrate the effect of the relative rates of crystal
formation and fiber deposition on the pore structure of the
cryogenic electrospun scaffold, the time interval between fiber
deposition has been varied in an example. A greater time interval
between spinning steps allows ice crystals to grow in size, hence
resulting in larger pores in the cryogenic electrospun scaffold
formed on the second layer of the hybrid scaffold.
[0105] Thus, by varying the time intervals between different
spinning steps, an electrospun scaffold with different layers
having different pore sizes formed by the fibers can be
manufactured.
[0106] The method for electrospinning can also comprise varying at
least one of the parameters selected from the group consisting of
needle size and design, voltage and the concentration of the at
least one polymer in the solution.
[0107] Changing the voltage applied during electrospinning can
influence the diameter of the fibers produced. As described by
Megelski S. et al. (2002, Macromolecules, vol. 35, no. 22, p.
8456-8466) and Pham, Q. P. et al. (2006, Tissue Engineering, vol.
12, no. 5, p. 1197-1211) the diameter of a fiber can be decreased
with increasing the spinning voltage, whereas decreasing the
spinning voltage increases the diameter of a fiber. At low voltage
of field strengths, a drop of the solution comprising the at least
one polymer dissolved therein is typically suspended at the tip of
the spinneret, i.e. needle tip. A jet will originate from the
Taylor cone ("Taylor cone" refers to the droplet produced at the
tip of the needle (see FIG. 2a) when an electric field is applied.
G. I. Taylor showed 1969 that this droplet is a cone-shaped and the
jets are ejected from the vertices of the cone) producing spinning
(assuming that the force of the electric field is sufficient to
overcome the surface tension of the solution). Using laser
diffraction, it has also been shown that increased voltages can
produce jets with larger diameters and ultimately lead to the
formation of several jets (Demir, M. M., Yilgor, I. et al., 2002,
Polymer, vol. 43, p. 3303 et seqq.)
[0108] In this method of electrospinning, the voltage applied can
be in a range of 0 kV to 50 kV or 7 kV up to 35 kV. In one example,
a voltage between 10 and 35 kV has been applied. The voltage across
the electrodes is usually varied together with other parameters of
the electrospinning process. For example, when the distance between
the spinneret and second layer is increased, the voltage has to be
increased to sustain the Taylor cone at the spinneret.
[0109] Several designs and configurations of needle tips have been
investigated for the electrospinning process (Pham, Q. P., Sharma,
U. et al., 2006, supra). For example, a coaxial, two-capillary
spinneret was designed. Using polymer feeds consisting of two
immiscible liquids, it was possible to produce hollow nanofibers.
With this two-capillary spinneret it was also possible to prepare
blends of polymers. The use of multiple tips has also been
investigated as a way to increase the throughput and production
rate of electrospinning of poly(ethylene oxide) (PEO). Multiple
needle tips have also been used to prepare blends of polyvinyl
alcohol) (PVA) and cellulose acetate. Using four tips and varying
the number containing PVA and cellulose acetate allowed for fibers
with various weight ratios of PVA and cellulose acetate to be
produced. Using two tips and a collector that could move
transversely, mixes of PEO and polyurethane fibers have been spun.
The transverse motion of the collector allowed for more uniform
distribution of each polymer.
[0110] The concentration and thus the viscosity of the solution
comprising the at least one polymer has also been examined.
Megelski S. et al. (2002, supra) reported that the fiber diameter
increases with increasing solution concentration. Both
concentration and viscosity of the polymer solution are parameters
that can be changed to provide for a steady Taylor cone and
consequently, a stable electrospinning process. Changing the
concentration of the solution can also affect the morphology of the
electrospun fibers. In general, increasing the solution
concentration while keeping all other parameters for
electrospinning constant result in a slower flow rate of the
solution.
[0111] The method for electrospinning is not limited to a specific
kind of polymer for electrospinning. Every known polymer which is
suitable for electrospinning or can be made suitable for
electrospinning can be used in this method for electrospinning. A
list of electrospun polymers which is suitable is listed for
example in Table I of the article of Subbiah, T. and Bhat, G. S. et
al. (2005, Journal of Applied Polymer Science, vol. 96, p.
557-569).
[0112] Due to the use of the scaffolds in cell biology and tissue
engineering, the polymers used for electrospinning can also be
biocompatible and/or biodegradable. "Biodegradable" refers to
material that can be absorbed or degraded in a patient's body.
"Biocompatible" refers to materials that do not have toxic or
injurious effects on biological functions.
[0113] A large number of suitable biocompatible polymers is known
and can be selected from the group consisting of poly(urethanes),
poly(siloxanes), poly(silicones), poly(ethylene), poly(vinyl
pyrrolidone), poly(2-hydroxy ethyl methacrylate), poly(N-vinyl
pyrrolidone), poly(methyl methacrylate), poly(vinyl alcohol) (PVA),
poly(acrylic acid), poly(vinyl acetate), polyacrylamide,
poly(ethylene-co-vinyl acetate), poly(ethylene glycol),
poly(methacrylic acid), polylactic acid (PLA), polyglycolic acids
(PGA), poly(lactide-co-glycolides) (PLGA), nylons, polyamides,
polyanhydrides, poly(ethylene-co-vinyl alcohol) (EVOH),
polycaprolactone, poly(vinyl acetate), polyvinylhydroxide,
poly(ethylene oxide) (PEO) and polyorthoesters. It is also possible
to use blends of different polymers listed above. In one example
PLA is used which has been dissolved in
1,1,1,3,3,3-hexafluoroisopropanol (HFIP).
[0114] In matrices composed of electrospun elastin (for
elasticity), electrospun collagen (to promote cell infiltration and
lend mechanical integrity), and other components, such as PLGA,
PEO, PVA, or other blends, the relative ratio of the different
components in the matrix can be tailored to specific applications
(e.g. more elastin, less collagen depending on the tissue to be
engineered).
[0115] Representative materials for forming the biodegradable
material include natural or synthetic polymers, such as, collagen,
poly(alpha esters) such as poly(lactate acid), poly(glycolic acid),
polyorthoesters, polyanhydrides and their copolymers, which degrade
by hydrolysis at a controlled rate and are reabsorbed. These
materials provide the maximum control of degradability,
manageability, size and configuration.
[0116] Other biodegradable materials can be selected from the group
consisting of cellulose ether, cellulose, cellulose ester,
chitosan, gelatin, fluorinated polyethylene, poly-4-methylpentene,
polyacrylonitrile, polyamide, polyamideimide, polyacrylate,
polybenzoxazole, polycarbonate, polycyanoarylether, polyester,
polyestercarbonate, polyether, polyetheretherketone,
polyetherimide, polyetherketone, polyethersulfone, polyethylene,
polyfluoroolefin, polyimide, polyolefin, polyoxadiazole,
polyphenylene oxide, polyphenylene sulfide, polypropylene,
polystyrene, polysulfide, polysulfone, polytetrafluoroethylene,
polythioether, polytriazole, polyurethane, polyvinyl,
polyvinylidene fluoride, regenerated cellulose, silicone,
urea-formaldehyde, or copolymers or physical blends of these
materials. The material of the electrospun scaffold forming the
third layer as well as the first and second layer of the hybrid
scaffold may be impregnated with suitable antimicrobial agents and
may be colored by a color additive to improve visibility and to aid
in surgical procedures.
[0117] In one example, the polymer used for the polymer solution
for electrospinning can be polylactides (PLA), polyglycolides
(PGA), polycaprolactones (PCL), copolymers of PLA-PGA, PGA-PCL,
PLA-PCL or a terpolymer of PLA-PGA-PCL. Another group of polymers
that can be used in the method of the present invention are
polymers having amino acids (e.g. lysine or RGD sequence) or
peptides (polypeptides or polypeptides co-polymers) added to the
polymer backbone or grafted onto the surface of the polymers to
promote cell material interactions. Such materials are described
for example in the article of Deng, X. M., Liu., et al. (2002,
European Polymer Journal, vol. 38(7), p. 1435-1441). One example of
such a polymer would be poly(DL-lactic acid)-co-poly(ethylene
glycol)-co-poly(L-lysine)copolymer.
[0118] As limited by their molecular weights and/or solubilities,
some functional polymers are not suitable for use with
electrospinning. One strategy for solving this problem is to blend
them with polymers that are well-suited for electrospinning. Based
on this approach, Kaplan and co workers (2002, Biomacromolecules,
vol. 3, p. 1233 et seqq.) have successfully fabricated
protein-carrying fibers by adding the proteins to the solution of a
conventional polymer. Fibers consisting of blends between
polyaniline (or polythiophene) with conventional organic polymers
have also been investigated. Blending was found to be fruitful in
improving some properties or applications associated with fibers.
For instance, it has been demonstrated that the physical and
biological properties (e.g., biodegradation rate and
hydrophilicity) of PLA fibers could be finely tuned by simply
controlling the compositions of polymer blend solutions used for
electrospinning (Kim, K. Yu, M. et al., 2003, Biomaterials, vol.
24, p. 4977 et seqq.)
[0119] The use of biocompatible and/or biodegradable polymers will
depend on given applications and specifications required. A more
detailed discussion of such polymers and types of polymers can also
be found in Brannon-Peppas, Lisa, "Polymers in Controlled Drug
Delivery," Medical Plastics and Biomaterials, November 1997, which
is incorporated herein by reference.
[0120] Sometimes it is necessary to dissolve a polymer before it
can be used for electrospinning. Therefore, the at least one
polymer used in the method of electrospinning can be dissolved in
an aqueous solvent or an organic solvent. Exemplary solvents which
are known in the prior art can be selected from the group
consisting of acetone, N,N-dimethylformamide (DMF),
water/chloroform, water, methylethylketone, silk aqueous solution,
acetic acid, formic acid, ethanol, diethylformamide,
hexa-fluoro-2-propanol, methylene chloride together with dimethyl
formamide, dimethyl formamide:toluene (1:9), water/ethanol or NaCl,
hydrochloric acid, camphorsulfonic acid, dichloromethane mixed with
trifluoroacetic acid, chloroform, dimethylacetamide, dimethyl
formamide:tetrahydrofuran (1:1), dichloromethane, tetrahydrofuran
(THF), N,N-dimethyl acetamide (DMAc), 1,1,1,3,3,3-hexa
fluoro-2-propanol (HFIP), HFIP mixed with DMF, isopropyl alcohol
(IPA), sulphuric acid, hexafluoro isopropanol, and mixtures
thereof.
[0121] Those solvents evaporate during electrospinning. In one
example HFIP is used to dissolve PLA. In Table 1 of a review
article of Huang, Z.-M., Kotaki, M. and Ramakrishna, S. (2003,
Composites Science and Technology, vol. 63, p. 2223-2253) at page
2226-2230, a list of polymers together with a possible solvent is
given. Another example is the list referred to in the article of
Subbiah, T., et al. (2005, Table I, supra). These articles and in
particular the content of Table I is incorporated by reference into
the present application. It should be noted that these lists
illustrate only exemplary combinations of polymers and solvents and
that a person skilled in the art would know how to create further
or different combinations than the one mentioned in these
articles.
[0122] Organic solvents could be, for example, acetone,
N,N-dimethylformamide (DMF), diethylformamide, chloroform,
methylethylketone, acetic acid, formic acid, ethanol,
1,1,1,3,3,3-hexa fluoro-2-propanol (HFIP), tetrafluoroethanol,
dichlormethan (DCM), tetrahydrofuran (THF), trifluoroacetic acid
(TFA), camphorsulfonic acid, dimethylacetamide, isopropyl alcohol
(IPA) and mixtures thereof. Examples of mixtures would be DCM with
DMF, DMF:Toluene (1:9), ethanol/NaCl, DCM mixed with TFA, DMF:THF
(1:1) and HFIP mixed with DMF.
[0123] Inorganic solvents could be, for example, water,
hydrochloric acid, sulphuric acid and mixtures thereof. Examples of
mixtures of inorganic solvents would be water/NaCl and
water/chloroform.
[0124] For some applications it might be of some advantage not to
use solvents. In these cases polymer melts can be used
alternatively. Thus, it is also possible to use a polymer melt for
electrospinning. The use of polymer melts is not limited to a
specific polymer melt. For example, polymers, such as polyethylene
(PE), polypropylene (PP), nylon 12 together with PA-12,
polyethylene terephthalate (PET), polyethylene naphthalate (PEN),
polyethylene terephthalate together with polyethylene naphthalate,
and polycaprolactone together with poly(ethylene
oxide-block-.epsilon.-caprolactone) (PEO-b-PCL), or
poly(D,L-lactide) (PLA), poly(glycolide) (PGA),
poly(lactides-co-glycolides) (PLGA) and further blends or mixtures
thereof can be used in melted forms.
[0125] Polymer melts are known to a person skilled in the art and
are described, for example, in the article of Huang, Z.-M., Kotaki,
M. and Ramakrishna, S. (2003, supra), Table II and Dalton, P. D.,
Klinkhammer, K. et al. (2006, Biomacromolecules, vol. 7, no. 3, p.
686-690).
[0126] The processing temperature for polyethylene (PE) is about
200 to 220.degree. C., about 200 to 240.degree. C. for
polypropylene (PP), about 220.degree. C. for nylon 12 together with
PA-12, about 270.degree. C. for polyethylene terephthalate (PET),
about 290.degree. C. for polyethylene naphthalate (PEN), about
290.degree. C. for polyethylene terephthalate together with
polyethylene naphthalate (75/25 or 25/75 (wt. %)), and about
85.degree. C. for polycaprolactone together with poly(ethylene
oxide-block-.epsilon.-caprolactone) (PEO-b-PCL) (20:80). The
melting temperature of PLA melt is about 200.degree. C. For
example, PLA is commonly used in biomedical applications and is
described for example by Zhou, H. J., Green, T. B., et al. (2006,
Polymers, vol. 47(21), p. 7497-7505).
[0127] It is further possible to enhance attachment of cells to the
biocompatible or biodegradable substrate of the different layers of
the hybrid scaffold by coating the matrix with compounds such as
basement membrane components, agar, agarose, gelatin, gum arabic,
collagens, fibronectin, laminin, glycosaminoglycans, mixtures
thereof, and other materials having properties similar to
biological matrix molecules known to those skilled in the art of
cell culture. Mechanical and biochemical parameters ensure the
matrix (three-dimensional structure of the scaffold or biological
material) provide adequate support for the cells with subsequent
growth and proliferation. Factors, including nutrients, growth
factors, inducers of differentiation or dedifferentiation, products
of secretion, immunomodulators, inhibitors of inflammation,
regression factors, biologically active compounds which enhance or
allow vascular ingrowth or ingrowth of the lymphatic network or
nerve fibers, and drugs, can be incorporated into the matrix of the
scaffold or provided in conjunction with the matrix. Similarly,
polymers containing peptides such as the attachment peptide RGD
(Arg-Gly-Asp) can be synthesized for use in forming matrices.
[0128] In general it is also possible to add a variety of
functional components directly to the solution for electrospinning
to obtain fibers with a diversified range of compositions and
well-defined functionalities. In particular components which can
support the ingrowth of cells into the scaffold can be
incorporated. Those components can be applied either by mixing them
directly with the solution or by applying the additional components
simultaneously or subsequently through another container onto the
scaffold. Those substances can also be applied to the first and
second layer of the hybrid scaffold of the present invention.
[0129] For example, biological molecules such as fibronectin and
laminin can be incorporated to promote cellular activities such as
attachment and migration. The final freeze-drying step serves to
lyophilize the molecules, which might help to preserve the proteins
in the porous mesh. In order to integrate cells into porous mesh of
the hybrid scaffold, the freeze drying step is preferably excluded.
This means that the process has to use other vapor molecules as the
templating crystals. For example, a technique that uses CO.sub.2
could be used as the crystals are removed by sublimation at room
temperature without freeze drying. In this way, the viability of
the cells can be easily maintained.
[0130] The sublimation step is carried out to remove the solid
crystals formed and which are responsible for forming the pores
within the second and third layer of the hybrid scaffold.
Sublimation can be carried out by freeze-drying (also known as
lyophilization). Freeze drying works by freezing the material and
then reducing the surrounding pressure and adding enough heat to
allow the frozen compound in the material to sublime directly from
the solid phase to gas.
[0131] Different kinds of freeze dryers can be used, such as rotary
evaporators, manifold freeze dryers, or tray freeze dryers.
[0132] Optionally it is possible to include a further step of
drying after the sublimation step to remove residual solvent which
still remained in the scaffold.
[0133] FIG. 2a shows a schematic illustration of the basic setup of
an apparatus for electrospinning. FIG. 2a shows the four major
components: a high-voltage power supply, a spinneret (i.e. a
metallic needle), and a collector (a grounded conductor). Direct
current (DC) power supplies are usually for electrospinning
although the use of alternating current (AC) potentials is also
feasible.
[0134] The spinneret is connected to a syringe in which the polymer
solution (or melt is hosted). It is possible to use more than one
spinneret, for example, 1, 2, 3 or 4 spinnerets, which are
connected to a syringe or container comprising the polymer solution
or melt. More than one spinneret is of specific advantageous if
several kinds of polymers are ought to be applied together and
those polymers are not dissolvable in the same solvent.
[0135] With the use of a syringe pump, the solution can be fed
through the spinneret at a constant and controllable rate. When a
high voltage (usually in the range of 1 to 35 kV) is applied, the
pendent drop of polymer solution at the nozzle of the spinneret
will become highly electrified and the induced charges are evenly
distributed over the surface. As a result, the drop will experience
two major types of electrostatic forces: the electrostatic
repulsion between the surfaces charges; and the Coulomb force
exerted by the external electric field. Under the action of these
electrostatic interactions, the liquid drop will be distorted into
a conical object commonly known as the Taylor cone. Once the
strength of the electric field has surpassed a threshold value, the
electrostatic forces can overcome the surface tension of the
polymer solution and thus force the ejection of a liquid jet from
the nozzle. This electrified jet then undergoes a stretching and
whipping process, leading to the formation of a long and thin
thread. As the liquid jet is continuously elongated and the solvent
is evaporated, its diameter can be greatly reduced from hundreds of
micrometers to as small as tens of nonometers. Attracted by the
grounded collector placed under the spinneret, the charged fiber is
deposited on the collector.
[0136] The orifice of the metallic needle (spinneret) can be of
different size depending on the final diameter of the fiber which
is desired to deposit on the bioadhesive second layer. In general,
the size of the needle used for electrospinning depends on the
concentration and viscosity of the solution. A person skilled in
the art is able to select the correct needle size to obtain the
optimal conditions for electrospinning. In one example, the needle
sizes range from 21 to 26 gauges. In another example a needle size
of 26 G is used.
[0137] This needle(s) can be connected to one or more container
depending on how many polymer streams are supposed to be fed
through one needle. Preferably, each needle is connected to one
container comprising a polymer solution.
[0138] Fibers have also been collected using a rotating cylindrical
drum rather than a stationary target (see FIG. 2b). The first and
second layer of the hybrid scaffold are fixed on this drum. Thus,
the collector used in the apparatus of the present invention can
also be rotatable around at least one axis. In another variation, a
thin, steel pin was used as a counter electrode and was placed
behind a rotating, non-conductive cylindrical collector. The
rotating drum can also be combined with the multiple field
method.
[0139] For the method of the present invention it can be important
that the hybrid scaffold provides a certain shape which resembles
the shape of the defect side in which the hybrid scaffold is
supposed to be implanted after manufacturing. For example, in case
of bone or skin reconstruction it might be necessary to reconstruct
a certain specific part of the tissue with specific measurements.
When reconstructing specific tissues, such as whole organs or blood
vessels, a specific structure of the scaffold will be necessary to
mimic the original extracellular membrane (ECM) of the tissue like
in the biological material as good as possible.
[0140] In another aspect, the present invention is directed to a
three-dimensional hybrid scaffold for tissue engineering obtained
by the method of the present invention. A hybrid scaffold of the
present invention comprises: [0141] a first layer made of a
biological material which has been decellularized; [0142] a second
porous layer connected to the surface of the first layer, wherein
the second layer is a porous bioadhesive; and [0143] a third porous
layer connected to the surface of the second layer which is located
opposite the surface to which the first layer is connected, wherein
the third porous layer is a scaffold.
[0144] In one aspect, the scaffold of the third layer is an
electrospun three-dimensional porous scaffold. In one example this
electrospun scaffold has been manufactured and applied/connected to
the surface of the bioadhesive layer at a temperature which is at
or below the freezing temperature of the solvent in which the
polymer has been dissolved.
[0145] In a further aspect, the present invention refers to the use
of a three-dimensional hybrid scaffold of the present invention
which has been manufactured according to the method of the present
invention for autologous, allogenic, xenogenic transplantation of
tissue.
[0146] In a further aspect, the hybrid scaffold is used for the
transplantation of small intestine, liver, pancreas, urinary
bladder, stomach, bladder, vascular system, bile duct, alimentary
canal, respiratory tract, kidney, spleen, heart, heart valve, bone,
skin or fragments or parts thereof. In one example, the hybrid
scaffold is used for transplantation of esophageal mucosa.
[0147] In still another aspect, the present invention is directed
to the use of a three-dimensional hybrid scaffold of the present
invention which has been manufactured according to the method of
the present invention for the manufacture of or use as a
medicament. The use as a medicament includes replacing damaged part
of a tissue or organ. The hybrid scaffold is supposed to accelerate
the healing process by providing a porous ECM like structure
allowing ingrowth of newly formed tissue and vascularization.
[0148] By "consisting of" is meant including, and limited to,
whatever follows the phrase "consisting of". Thus, the phrase
"consisting of" indicates that the listed elements are required or
mandatory, and that no other elements may be present.
[0149] By "comprising" it is meant including, but not limited to,
whatever follows the word "comprising". Thus, use of the term
"comprising" indicates that the listed elements are required or
mandatory, but that other elements are optional and may or may not
be present.
[0150] The inventions illustratively described herein may suitably
be practiced in the absence of any element or elements, limitation
or limitations, not specifically disclosed herein. Thus, for
example, the terms "comprising", "including", "containing", etc.
shall be read expansively and without limitation. Additionally, the
terms and expressions employed herein have been used as terms of
description and not of limitation, and there is no intention in the
use of such terms and expressions of excluding any equivalents of
the features shown and described or portions thereof, but it is
recognized that various modifications are possible within the scope
of the invention claimed. Thus, it should be understood that
although the present invention has been specifically disclosed by
preferred embodiments and optional features, modification and
variation of the inventions embodied therein herein disclosed may
be resorted to by those skilled in the art, and that such
modifications and variations are considered to be within the scope
of this invention.
[0151] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
invention. This includes the generic description of the invention
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein.
[0152] Other embodiments are within the following claims and
non-limiting examples. In addition, where features or aspects of
the invention are described in terms of Markush groups, those
skilled in the art will recognize that the invention is also
thereby described in terms of any individual member or subgroup of
members of the Markush group.
EXAMPLES
[0153] In the following section the general setup of the examples
for the manufacture of a hybrid scaffold of the present invention
is described in more detail before referring to some specific
examples carried out.
[0154] The purpose of producing a hybrid scaffold is to combine the
advantages of both biocompatible polymers with that of acellular
matrices for tissue engineering application.
[0155] FIGS. 2a and b show a schematic diagram of the
electrospinning process. In this process, long and fine threads are
drawn from droplets of polymer by the application of a high voltage
electric field. The resulting nano-fibers are deposited onto a
rotating mandrel (collector) to form a tubular interconnected
porous network. The collector can be in any geometrical shapes
other than the cylindrical mandrel used in this particular
embodiment. Many different materials have been electrospun alone or
in combinations using different solvents. Some examples are given
in the following reviews (Huang, Z.-M., Kotaki, M. and Ramakrishna,
S., 2003, supra; Li, D., Xia, Y. N., 2004, Advanced Materials, vol.
16, p. 1151-1170). In all examples, the polymer used is
poly(D,L-lactide) (PLA) dissolved in
1,1,1,3,3,3-hexafluoroisopropanol (HFIP) in various
concentrations.
[0156] For the purposes of this disclosure, the biological material
used is a freeze-dried decellularized porcine esophageal mucosa
(hereinafter known as decellularized mucosa). While references are
made to porcine esophageal mucosa, it is understood that other
naturally occurring matrices (e.g. stomach, bladder, bile duct,
alimentary, respiratory), from other sources (e.g. bovine, ovine,
human) are within the scope of this disclosure. The decellularized
mucosa is prepared by mechanically separating the mucosal and
submucosal layers from the remaining tissue (consisting of
muscularis externa and serosa/adventitia).
[0157] The mucosa is treated with dispase solution to detach the
epithelium from the basal membrane.
[0158] This is followed by treatment with Triton-X, peracetic acid
solution and mechanical abrasion to remove cellular components from
the tissue, rendering it acellular, while attempting to preserve
the important structural and functional proteins. The resulting
acellular ECM is then subjected to freeze drying process to remove
the liquid within the hydrated scaffold via sublimation.
Alternatively, the liquid may be removed by subjecting the sample
to critical point drying (CPD).
[0159] Biodegradable and biocompatible adhesives, such as fibrin
glue as the bonding interface between the acellular ECM forming the
first layer and, for example, an electrospun scaffold as the third
layer. More importantly, the the bioadhesive is made porous by
applying it to the biological material which is maintained below
subzero temperatures such that ice crystals formed within the
bioadhesive. The hybrid scaffold is subsequently formed by
electrospinning biocompatible polymer directly onto the biological
material coated with the the bioadhesive. This is followed by
lyophilization to remove the ice crystals, forming a wholly porous
hybrid scaffold.
[0160] Bioadhesives can be applied to either side (adluminal and
abluminal) of the decellularized mucosa by casting. Other methods
such as spraying, electrospraying, electrospinning, spin coating,
dip coating and brushing can be used. After the application of the
bioadhesive on the decellularized mucosa, electrospinning of the
fibers begins before the bioadhesive is fully set/crosslinked. This
is to ensure that the depositing electrospun fibers are wetted with
the bioadhesive in order to form sufficient bonding with the
setting bioadhesive.
[0161] In one example, the fibrin coated decellularized mucosa is
attached to a chilled mandrel. Ice crystals formed on the fibrin
serve as a negative template around which electrospun fibers are
deposited. In addition, the water component within the fibrin
freezes to form ice within the crosslinking fibrin. The hybrid
scaffold is subsequently freeze-dried to remove ice crystals,
leaving behind a construct consisting of cryogenic electrospun
scaffold (CES) with large pores and a porous fibrin interface. This
wholly porous construct can be used to support cell infiltration
and vascularization within the scaffold. This is useful in
replacing thick segments of the tissues that have been removed
during the decellularization processes. In comparison, the
non-porous fibrin film acts as a barrier to nutrient diffusion and
cell infiltration. Another advantage of the porous interface is
that the degradation products of fibrin are angiogenic and the
porous fibrin layer will facilitate vascular ingrowth in vivo. By
promoting cell infiltration and vascularization, the porous fibrin
layer also facilitates remodeling of the construct in vivo. The
infiltrated fibroblasts can deposit collagen and start the
remodeling process while the porous fibrin layer degrades. In
comparison, the non-porous- fibrin interface degrades without
collagen deposited from the host cells within the interface, which
might result in delamination of the hybrid scaffold upon complete
degradation of the fibrin. Lastly, the degradation of fibrin can be
controlled by controlling the pore size of the interface. The pore
size can be varied by changing the concentration of fibrinogen and
thrombin in the aqueous medium, thus changing the amount of ice
within the fibrin layer.
[0162] Thus, a hybrid scaffold of the present invention adds more
versatility to native acellular matrices. It can be used to (1)
tailor the mechanical properties of the treated ECMs to comply with
host tissues, (2) replaced excised tissue segments by providing a
thick fibrous hybrid scaffold with large pores for cell
infiltration and vascularization and (3) organize cells attachment
and proliferation along aligned fibers.
[0163] Hybrid Scaffold Consisting of Cryogenic Electrospun Scaffold
and Decellularized Mucosa with Porous Fibrin Layer
[0164] Decellularization of Esophageal Mucosa
[0165] A sample of length 5 cm is harvested from the mid-section of
freshly harvested porcine esophagus. It is rinsed in saline
solution to remove cellular debris for 5 minutes at room
temperature, and the sample slit opened longitudinally. The mucosal
and submucosal layers are then mechanically separated from the
remaining tissue (consisting of muscularis externa and serosa), and
left to immerse in deionized water at 4 degrees Celsius for 30
minutes.
[0166] The decellularization reagent is prepared using a
formulation of 31% hydrogen peroxide, glacial acetic acid and
deionized water in the ratio of 20% volume:40% volume:40% volume.
The typical concentrations of the reagent utilized in this
particular invention ranged from 1%-20%, with 1%-10% preferred. 30
ml of the reagent is then poured into a 100 mm glass Petri dish.
Individual samples of the mucosal and submucosal layers are then
immersed in the reagent at ambient temperature from 4 to 40.degree.
C., and both sides delaminated using mechanical tools including,
but not limited to, gauze, brush, and the edge of microscopic
slides.
[0167] Mechanical delamination cycles ranged from about 50 to about
300 cycles. 150 to about 300 cycles are also suitable. After
mechanical delamination in the reagent, the samples are then
neutralized to physiological pH with the use of sterile phosphate
buffered saline (PBS) by placing them on the rotor shaker for 15
minutes at ambient temperature. Typically, it took 5 to 6 changes
of sterile PBS before the samples are neutralized at physiological
pH. The sample is then immersed in a Triton X-100 solution, typical
concentrations ranging from about 0.05% to 5% or about 0.5% to 3%.
Time immersion of the sample in Triton X-100 ranged from 6 hours to
48 hours or from 12 to 24 hours. Mechanical agitation was provided
in the form of gentle stirring with methods including magnetic
stirring, but not limited to mechanical agitation such as rotor
shaking, paddle stirrer and rotation shaking. After the treatment
with Triton X-100, samples are rinsed of residual detergent by
mechanical stirring by the above means, with rotor shaking
preferred. Typically, it takes about 6 changes of sterile PBS to
remove all traces of Triton X-100 from the samples. The sterile
acellular matrices are then stored in sterile PBS supplemented with
antibiotics (penicillin/streptomycin) at 4.degree. C. till further
use.
[0168] Manufacture of Hybrid Scaffold
[0169] The PLA solution is placed in a 30 ml syringe fitted with a
26 gauge metal needle, which is in contact with the earthed plate
(needle sizes can vary from 18G to 28G). The environmental
conditions are controlled with an ambient temperature between
20-28.degree. C. and a relative humidity between 25-80%. The
mandrel is chilled to subzero temperatures. The decellularized
mucosa is attached to the mandrel with the abluminal surface facing
up. The fibrinogen and thrombin solutions are constituted, mixed
thoroughly and applied onto the top surface. Constant pneumatic
pressure is applied to the syringe to sustain a droplet at the
needle tip. A voltage between 10 to 35 kV is applied to the mandrel
until a stable Taylor cone formed and a constant polymer jet is
ejected. Electrospun fibers are deposited while ice crystals
simultaneously formed on the chilled fibrin bioadhesive. The
aqueous solvent in the fibrin layer froze, forming ice crystals
within the bioadhesive. When electrospinning is completed, the
hybrid scaffold is freeze-dried overnight to remove the embedded
ice crystals.
[0170] FIGS. 1a and b depict the cross section of the hybrid
scaffold obtained by electrospinning onto a fibrin coated mucosa
maintained at subzero temperatures. As a comparison, FIGS. 1d and e
show the cross section of the hybrid scaffold by electrospinning
onto a fibrin coated mucosa at ambient temperature. As a result of
the subzero temperatures in which cryogenic electrospinning is
carried out, the formation of a porous fibrin interface is observed
(FIG. 1b). This is a positive step towards attaining a wholly
porous hybrid scaffold that would be advantageous in nutrient
diffusion and cell infiltration in in vitro and in vivo systems. In
comparison, the non-porous fibrin film shown in FIG. 1e acts as a
barrier to nutrient diffusion and cell infiltration. Another
advantage of the porous interface is that the degradation products
of fibrin are angiogenic and the porous fibrin layer will
facilitate vascular ingrowth in vivo. By promoting cell
infiltration and vascularization, the porous fibrin layer also
facilitates remodeling of the construct in vivo. The infiltrated
fibroblasts can deposit collagen and start the remodeling process
while the porous fibrin layer degrades. In comparison, the
non-porous fibrin interface degrades without collagen deposited
from the host cells within the interface, which might result in
delamination of the hybrid scaffold upon complete degradation of
the fibrin. Lastly, the degradation of fibrin can be controlled by
controlling the pore size of the interface. The pore size can be
varied by changing the concentration of fibrinogen and thrombin in
the aqueous medium, thus changing the amount of ice within the
fibrin layer.
[0171] FIG. 1c shows the top surface of the cryogenic electrospun
scaffold which corresponds to FIGS. 1a and b. The large pores of
these scaffolds will be useful in inducing cell ingrowth (e.g.
smooth muscle cells) and vascularization when reconstructing the
muscularis externa. In comparison, the dense scaffold shown in FIG.
1f acts as a barrier to cell infiltration and may not be useful in
constructing thick tissues.
[0172] Method for Cryogenic Electrospinning
[0173] The polymer used in this example is poly(D,L-lactide) (PLA,
LASIA H100J, T.sub.g 58.degree. C., T.sub.m 165.degree. C.), and
the solvent is 1,1,1,3,3,3-hexafluoroisopropanol (HFIP, analytical
grade, Merck Singapore).
[0174] Cryogenic electrospun scaffolds (CES) can be fabricated as
follows. PLA is dissolved in HFIP at a concentration of 15 wt./vol.
%. The PLA solution is then placed in a 30 ml syringe fitted with a
26 gauge metal needle, which is in contact with the earthed plate.
The environmental conditions are controlled with an ambient
temperature between 20.degree. C.-28.degree. C. and a relative
humidity between 25%-80%. The mandrel is chilled to subzero
temperatures. Constant pneumatic pressure is applied to the syringe
to sustain a droplet at the needle tip. A voltage between 10 to 35
kV is applied to the mandrel until a stable Taylor cone is formed
and a constant polymer jet is ejected towards the collector.
Electrospun fibers are deposited while ice crystals simultaneously
form on the chilled rotating mandrel. When electrospinning is
completed, the fibrous mesh is freeze-dried (Freeze-dryer, Alpha
1-2, Germany) overnight to remove the embedded ice crystals. The
scaffold is then oven-dried (Thermoline VORD-460-D, Australia) to
remove residual solvent. A schematic presentation of this method is
illustrated in FIG. 3.
[0175] Effect of Relative Humidity of the Environment on Pore
Structure of Cryogenic Electrospun Scaffold
[0176] The effect of the relative humidity of the environment on
the pore structure of cryogenic electrospun scaffold used in the
hybrid scaffold of the present invention as third layer is
investigated. The relative humidity of the electrospinning chamber
is decreased by introducing dry gaseous nitrogen (N.sub.2) into the
chamber or is increased by introducing water vapour with a
humidifier. Three data points, 25% RH, 40% RH and 55% RH
(RH=relative humidity) are collected. The mandrel temperature is
maintained at -30.degree. C. and all other parameters are kept
constant.
[0177] FIG. 5 shows the SEM micrographs for the three data points
(FIG. 5: 25% RH, FIG. 5b: 40% RH and FIG. 5c: 55%). It can be
observed that as humidity increases from 25% to 55%, the pores of
the cryogenic electrospun scaffolds become larger and more defined.
This accord with the observation that simple plate-like structures
are formed at low saturations, while higher saturations produce
dendritic ice crystals (Libbrecht, K. G., 2005, supra). These
complex structures aggregate more readily and occupy more space,
resulting in the larger pore size of the cryogenic electrospun
scaffold at higher humidities. Moreover, the rate of ice crystal
formation increases with humidity; hence the proportion of ice
crystals to fiber is increased resulting in a more open
structure.
[0178] Based on these findings, it can be postulated that
parameters that governs ice crystal formation on the surface of the
second layer, such as the temperature of the second layer,
temperature and relative humidity of the environment, affects the
pore structures of the cryogenic electrospun scaffold. Conditions
that favor aggregation and formation of ice crystals result in
higher volumes of ice crystals between bundles of electrospun
fibers; hence resulting in larger pores of the cryogenic
electrospun scaffold.
[0179] Effect of Time Interval Between Fiber Deposition on Pore
Structure of Cryogenic Electrospun Scaffold
[0180] Ice crystal formation and fiber deposition are two events
that happen simultaneously. It follows that the rates of ice
crystal formation and fiber deposition are two competing factors
that can affect the pore structure of the cryogenic electrospun
scaffolds. To demonstrate the effect of the relative rates of ice
crystal formation and fiber deposition on the pore structure of the
cryogenic electrospun scaffold, the time interval (X) between fiber
deposition is varied. The longer the time interval, the slower the
rate of fiber deposition.
[0181] The environmental relative humidity (RH) is maintained at
55% RH, and the mandrel temperature is maintained at -30.degree. C.
All other parameters are kept constant.
[0182] For this study, two waiting periods were used as follows:
[0183] (i) Sample A--X=0 minute [0184] (ii) Sample B--X=5
minutes
[0185] Samples were labeled as follows: [0186] (i) Sample A: A-5
(spin for 5 mins, X=0), A-10 (spin for 10 mins, X=0), A-15 (spin
for 15 mins, X=0) [0187] (ii) Sample B: B-5 (spin for 5 mins, X=5
min), B-10 (spin for 10 mins, X=5 mins), B-15 (spin for 15 mins,
X=5 mins)
[0188] FIG. 7 illustrates the result of this experiment. FIGS. 7
(a), (c) and (e) are SEM micrographs showing the pore structures of
cryogenic electrospun scaffold after 5, 10 and 15 minutes of
spinning without any time interval in between spinning
respectively. FIGS. 7 (b), (d) and (f) are SEM micrographs showing
the pore structures of cryogenic electrospun scaffold after 5, 10
and 15 minutes of spinning with a time interval of 5 minutes in
between each cycle. From FIG. 6, both Samples A (FIGS. 7 (a), (c)
and (e)) & B (FIGS. 7 (b), (d) and (f)) are porous throughout
the thickness of the scaffold, as observed by the slow building up
over the 5, 10 and 15 minutes. However, pore sizes are smaller in
the early stage, becoming bigger as the spinning proceeds. Sample B
has pore structures that are larger in diameter and shallower, as
compared to Sample A. Both of these observations can be attributed
to the time interval X. The waiting period between spinning allows
ice crystals to grow in size, hence resulting in larger pores in
the cryogenic electrospun scaffold.
[0189] Effect of Mandrel Temperature on the Pore Structure of
Cryogenic Electrospun Scaffold
[0190] The effect of the mandrel temperature on the pore structure
of the cryogenic electrospun scaffold is investigated. The
temperature of the mandrel which is used in this experiment can be
varied by, but not limited to, packing different mass of dry ice
inside the hollow mandrel. Three data points (23.degree. C.,
-15.degree. C., -30.degree. C.) are collected. All other parameters
are kept constant.
[0191] FIG. 6 shows the SEM micrographs of the cryogenic
electrospun scaffolds collected at different mandrel temperatures.
For definition purposes it should be mentioned that the "mandrel
interface" is the side of the scaffold facing the mandrel and the
side of the scaffold facing the air is called the "air interface".
It can be observed that a conventional dense electrospun scaffold
is obtained when the mandrel temperature is kept at 23.degree. C.
When the mandrel temperatures are -15.degree. C. and -30.degree.
C., large pore structures (>5 .mu.m) can be observed on both the
mandrel and air interfaces of the scaffold. As illustrated in FIG.
3, ice crystals formed on the mandrel at sub-zero temperatures are
embedded within the electrospun mesh. The subsequent removal of the
ice crystals through freeze-drying forms these pore structures
within the electrospun mesh.
[0192] In addition, for scaffolds formed at sub-zero temperatures,
it can be observed that the mandrel interface of the scaffold have
less defined and smaller pore structures as compared to the air
interface but still much larger pores than scaffolds obtained using
classical electrospinning at room temperature.
[0193] In vivo Cell Infiltration for Decellularized Porcine
Esophageal ECM and Cryogenic Electrospun Scaffolds (CES) Implanted
Subcutaneously
[0194] The decellularized sterile porcine esophageal ECM and the
CES scaffold are prepared according to the procedures described
above. Dense electrospun scaffold prepared by conventional
electrospinning technique is used as a comparison for the CES.
Prior to use, both ECM and all electrospun scaffolds are rinsed 5
times for 15 minutes with 40 ml of phosphate buffered saline (PBS)
per rinse.
[0195] Wistar rats weighing 300 to 350 g are used for the
subcutaneous implantation study. Implantation is performed in an
aseptic manner under a laminar hood. The rats are anaesthetized
with inhalational isoflurane and oxygen, administered via a
facemask. A patch of skin on the dorsum is shaved and cleansed with
chlorhexidine and iodine. A single 3 cm dorsal midline incision is
made. Subcutaneous pockets are created by blunt dissection. The
decellularized ECM and electrospun scaffolds are implanted in
separate rats. The decellularized ECM is inserted into the pocket,
ensuring that placement is flat. A second pocket is left empty as a
negative control for normal healing response. For the electrospun
scaffolds, three subcutaneous pockets are created by blunt
dissection. The cryogenic electrospun scaffolds (CES) and dense
conventionally electrospun scaffolds are inserted into two of the
pockets, ensuring that placement is flat and that the scaffolds
remained separate from each other. The last pocket is left empty as
a negative control for normal healing response. The incisions are
closed with interrupted 3/0 polypropylene sutures. Postoperatively,
an injection of tolfedine 0.1 ml is administered intramuscularly in
the thigh for pain relief. Sutures are removed on the tenth
post-operative day.
[0196] At 14 days post implantation, the rats are euthanized by
carbon dioxide inhalation. The dorsum is shaved and the previous
incision reopened and extended to visualize all the pockets. Each
of the scaffold and the empty pocket are retrieved with the
surrounding tissue. The samples are fixed overnight in 10% buffered
formalin, embedded in paraffin and 7 .mu.m sections obtained using
a microtome (Leica RM2125RT, Germany). These are then stained with
hematoxylin and eosin to assess cellular infiltration.
[0197] The entire decellularized ECM is infiltrated with host cells
by Day 14 (FIGS. 8 (a) and (b)). A second observation is the
presence of capillaries containing intraluminal red blood cells
within the ECM. Both these phenomena are important as they show
that the ECM (first layer of invention) supports in vivo cell
infiltration and vascularization such that remodeling of the
scaffold can take place. FIGS. 8(c) and (d) show the difference
between the CES and conventional dense electrospun scaffold in
promoting cell infiltration. There is markedly better cell
infiltration in the CES with macrophages and collagen-producing
fibroblasts penetrating deep into the scaffold at Day 14 (FIG.
8(c)). In contrast, cells are limited to the periphery of the
conventional dense electrospun scaffold (FIG. 8(d)). This shows
that the CES, with its open porous structure, promote better cell
infiltration as a third layer of the invention.
* * * * *