Note: Descriptions are shown in the official language in which they were submitted.
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CREATION OF THREE-DIMENSIONAL TISSUES
FIELD OF THE INVENTION
This invention relates to methods of tissue engineering, and
particularly to a method of creating a vascularized three-dimensional tissue
in
mammal.
BACKGROUND OF THE INVENTION
Despite advances in medical technology, tissue loss remains a serious
problem. Tissue damage can be caused by various reasons such as surgical
removal
of cancerous tissues and trauma. After the removal of the damaged tissue,
reconstruction is often desired. Another reason relates to organ
transplantation
where it is possible to replace a damaged organ with a healthy donor organ.
However, the organ transplantation approach has drawbacks. First, organs for
transplantation are scarce and one may have to wait for a long time before a
suitable
organ becomes available. Second, the immunological incompatibility between the
donor and donee tissues limits the ability of the transplanted organ to
survive and
function, and further aggravates the availability problem. Moreover, the
transplantation process itself is complex, invasive, and costly.
In contrast, a great number of cells can be obtained directly from the
patient. These autologous cells can further be propagated in cell culture
media.
Moreover, the use of autologous cells obviates the tissue incompatibility
problem.
Thus,viable three-dimensional tissues, generated from isolated cells, are
valuable for
use in repairing tissue damages.
Current approaches for generating three-dimensional tissues in vitro from
isolated
cells have not been very successful. Extensive research has been conducted to
develop techniques for transplanting cells directly into body tissues and
developing a
three-dimensional tissue from these cells in vivo. Cells seeded within three-
dimensional support constructs such as enclosed encapsulation membranes or
porous
matrices have been transplanted along with the constructs and rendered to
survive,
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grow, and function in vivo. (See, e.g., U.S. Patent Nos. 4,353,888; 4,487,758;
and
4,902,295.) However, there has been little success with respect to the
formation of
three-dimensional tissues, especially vascularized three-dimensional tissues,
from
transplanted cells.
Transplanted cells require nutrients, growth factors, hormones as well
as oxygen for survival. The transplanting techniques used heretofore generally
do
not provide an effective mechanism to support the transplanted cells. This is
especially problematic in the formation of vascularized tissues. These
techniques
are unable to establish adequate vascularized beds that provide necessary
nutrients to
the transplanted cells for the formation of three-dimensional tissues.
Furthermore, the approaches heretofore known generally require
invasive surgical procedures for delivering cells into body tissues. For
example,
U.S. Patent No. 5,71b,404 discloses a method for reconstruction or
augmentation of
breast tissue by implanting isolated cells with a polymeric matrix. The
polymeric
fibrous matrix used therein has a structure similar to the silicone implants
now used.
Complex and invasive surgery has to be performed to insert the matrix into
breast
tissue.
A major challenge in mammalian cell transplantation and developing
vascularized three-dimensional tissue is promoting long-term cell survival of
the
transplanted cells. The current approaches rely on transplanting cells on
surfaces
that have diffusional distances greater than 250 wm for nutrients which are
provided
by the surrounding host vasculature. Unfortunately, a necrotic core of tissue
develops within the construct because of an inadequate supply of nutrients and
gases. Therefore, there is need for an effective and substantially non-
invasive
method of creating vascularized three-dimensional tissue in vivo.
SUMMARY OF THE INVENTION
Another object of the present invention is to provide a method for
growing vascularized tissue in a human.
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Another object of the present invention is to provide a method to
deliver discrete tissue constructs containing cells into an area in vivo.
The discrete tissue construct acts as a functional unit, e.g., the growth
of the transplanted cells and the vascular ingrowth of the cells from the
surrounding
tissue forms small cellular "islands", interconnected with the surrounding
vasculature. As a result, a vascularized three-dimensional tissue is formed.
Thus, the invention is directed to methods for providing a
vascularized three-dimensional tissue in which transplanted cells are no
greater than
250 ~m away from a nutrient source, i.e., a developing vascular bed. A porous
construct containing cells for transplantation is delivered into an area of
interest in a
living subject where it is desired to build a vascularized three-dimensional
tissue.
The implanted porous construct supports the adhesion, growth and migration of
the
transplanted cells as well as the ingrowth of the tissue cells surrounding the
construct, thus forming a three dimensional discrete tissue. Preferably, the
delivery
of the porous construct is performed in a non invasive manner, for example, by
injection, or by delivering in a biocompatible pouch having an arteriole and
venule
connection from the outer pouch into the inner pouch. It is also preferred
that a
plurality of the porous constructs are delivered.
The porous construct is made from at least one biocompatible
material which supports cell adhesion, growth, and migration. Such
biocompatible
materials can be bioresorbable or non-resorbable. The cells to be transplanted
can be
seeded in the porous construct. The porous construct has a dimension in which
the
distance is about 50 ~m to about 500 ~.m from at least one point on the outer
surface
to the center of the construct, such that when cells are seeded within the
construct,
the innermost cell within the construct is no greater than about 250 ~m away
from
an outer surface of the construct. In an average tissue, the maximum distance
found
between a metabolically active cell and a capillary is approximately 250 ~,m.
This
distance has been established in vivo due to diffusional limitations of oxygen
and
nutrients from the capillary to the surface of the cell. The construct also
has
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interconnected pores having a pore size of from about 10 pm to about 300 ~,m.
The
porous construct may also contain a signal (e.g., a growth factor or an
extracellular
matrix protein) for modifying, preferably promoting cell adhesion, growth, or
migration.
In one embodiment of the invention, the porous construct containing
cells to be transplanted is delivered directly to the area of itnerest. In
another
embodiment of the invention, a vascular bed is formed in the area of interest
by
implanting, before the delivery of the porous construct, a second construct
which
promotes the formation of a vascularized bed in vivo. The porous construct
containing cells to be transplanted is then delivered onto the vascularized
bed for
formation of a three-dimensional tissue.
The method of this invention can be used in the reconstruction of
many different tissues, such as breast tissue, pancreatic tissue, liver
tissue, neural
tissue, kidney tissue, muscle tissue and skin tissue. The present invention
provides a
minimally invasive procedure and effective method for development of three-
dimensional tissues.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other advantages and features of the invention, and
the manner in which the same are accomplished, will become more readily
apparent
upon consideration of the following detailed description of the invention
taken in
conjunction with the accompanying examples, which illustrate preferred and
exemplary embodiments.
Figure 1 A shows a trichrome stain of alg-RGD ( 1 X concentration of
RGD) beads implanted in the subcutaneous space of a rat and harvested after
one
week of implantation illustrating tissue ingrowth between the beads (40X
magnification);
Figure 1 B shows a formation of a vascular bed at the interface of alg-
RGD beads and tissue (200X magnification);
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Figure 2A shows tissue ingrowth between the beads (trichrome stain
of alg-RGD (100X concentration of RGD)) implanted in the subcutaneous space of
a
rat, harvested after two weeks of implantation (40X magnification);
Figure 2B shows the formation of a vascular bed at the interface of
the beads and the tissue (200X magnification);
Figure 3 shows Factor VIII localization in tissue that has grown
between alginate-RGD beads;
Figure 4 illustrates cross sections of collagen beads that were seeded
with smooth muscle cells;
Figure SA shows a trichrome stain of histological section (40X and
200X) of collagen beads harvested from a rat that had been implanted for 2
weeks;
Figure SB illustrates two Factor VIII stains from the same section as
Figure SA indicating that vascular endothelial cells are located throughout
the
collagen bead implant. (Top picture is 40X magnification and bottom picture is
200X magnification); and
Figure 6 illustrates the growth of aortic smooth muscle cells
implanted in collagen beads harvested after two weeks (400X magnification);
and
Figure 7 illustrates the cell growth of endothelial cells stained for
Factor VIII on a collagen bead harvested after two weeks.
Figure 8 is a bar graph comparing histological scoring of 2-week
subcutaneous implants of alginate rods or beads or collagen beads.
Figure 9 is a bar graph comparing histological scoring of alginate-
RGD bead subcutaneous implants at 2, 4 and 8 weeks from implant.
Figure 10 is a bar graph comparing area of alginate-RGD bead
implants at various time intervals.
Figure 11 is a bar graph comparing width of alginate-RGD bead
implant at different times.
Figure 12 is a bar graph comparing thickness of Alginate-RGD bead
implants at different times.
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Figure 13 is a bar graph comparing thickness of the capsule
surrounding alginate-RGD bead implants at different times.
DETAILED DESCRIPTION OF THE INVENTION
This invention provides a method for developing vascularized three-
dimensional tissue in a living subject such as a human. In one embodiment of
this
method, a porous construct seeded with cells for transplantation is created
and is
delivered into an area of interest in a living subject where it is desired to
build a
vascularized three-dimensional tissue. The implanted porous construct supports
the
adhesion, growth and migration of the transplanted cells as well as the
ingrowth of
the tissue cells surrounding the construct, thus forming a three-dimensional
discrete
tissue.
The porous construct of this invention can be in any three-
dimensional shape, so long as it has a dimension in which the distance between
an
outer surface of the construct and the center of the construct is from about
10 pm to
about S00 Vim, preferably from about 25 pm to about 400 pm, more preferably
from
about 50 pm to about 250 wm. For example, the porous construct can be in the
shape of spheres, particle beads, rods, triangles, threads, and cubes. In its
most
preferred form, the construct has a dimension such that the distance from each
and
every surface to the center of the construct has a length of about 250 pm. The
construct is configured so that the innermost cell situated in the center of
the
construct is no greater than 250 p.m from at least one surface of the
construct. In this
manner, it becomes more probable that, after the construct having cells
therein is
delivered to an area of interest in a living subject, all the cells within the
construct
are no greater than 250 ~,m from a nutrient source, e.g., a vascularized bed.
The distance into the construct at which cells are found is determined
primarily by the presence of nutrients and oxygen which diffuse into the
porous
construct to nourish the cells. The maximum diffusion distance will depend in
part
on cell type and metabolic state, as well as the porosity of the construct,
and may be
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up to about 400 p,m, or more typically 250-300 pm. For highly metabolic cells,
the
maximum diffusion distance may be about 150 pm. For particles with linear
dimensions larger than the maximum diffusion distance, cells will only
populate the
portion of the particle which may be reached by diffusion of oxygen and
nutrients.
Areas of the particle that are further from the surface than the maximum
diffusion
distance will be substantially free of cells. For particles with irregular
shapes, the
area populated by cells may include all portions of the particle which are
within the
maximum diffusion distance of at least one surface of the particle. This may
include
diffusion from the surface of macropores in macroporous particles.
The porous construct has an interconnected macroporous structure formed by
one or more biocompatible materials which constitutes the skeleton of the
structure.
There should be a plurality of macropores on the surfaces of the construct
connecting the macropores within the construct so that exchange of molecules
and
cell migration can occur between inside of the construct and the surrounding
environment. The diameter of the pores ranges from about 10 ~m to about 300
Vim,
preferably from about 25 ~,m to about 200 Vim, more preferably from about 50
~.m to
about 100 pm.
In particular, macroporous constructs have pores of sufficient dimension to
permit the passage of cells by migration through the pores. For macroporous
constructs, vascular ingrowth proceeds into the large diameter pores, as well
as the
spaces between discrete constructs. Thus, the vascular bed formed by the
ingrowth
can provide nutrients and oxygen to cells within particles having overall
diameters
much larger than the 250~,m suggested by the diffusion in microporous
spherical
particles. The preferred constructs of the present invention are microscopic
particulates with macroporous structure. A plurality of these particles forms
a mass
with a abundance of channels both within and between particles, through which
nutrient-containing medium (plasma, lymph, cell culture medium, etc.) may
reach
cells in the particles and through which vascular ingrowth may occur. The
overall
dimensions of these preferred particulates are typically on the order of 1
millimeter,
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although particle sizes greater or smaller than this are also contemplated for
the
discrete, macroporous constructs of this invention.
By "biocompatible" is intended that the material used for making the
porcus construct does not substantially adversely affect any desired
characteristics of
the cells to be seeded within the construct and of the cells and tissues in
the area of a
living subject where the construct is to be delivered. It is also intended
that the
material used does not cause any substantially medically undesirable effect in
any
other area of the living subject. The materials are chosen such that the
porous
construct supports the adhesion, growth, or migration of cells. Thus, the
biocompatible material used must allow cell adhesion, growth, and migration.
Preferably, the biocompatible material should also be mechanically strong
enough to
support cells and to substantially maintain a dimension. Generally, the
methods for
testing a material's biocompatibility and mechanical strength is well known in
the
art. The biocompatible material may be synthetic or natural.
The biocompatible material may be a material that is non-resorbable
such that it will remain once delivered inside the body of a living subject.
More
preferably the biocompatible material is bioresorbable and will be gradually
degraded after the porous construct is delivered to an area of interest inside
the body
of a living subject. Such degradation should be in a slow and gradual fashion
and
preferably should take place after sufficient cells have grown within the
construct to
form an organized three-dimensional tissue. In this manner, the degradation of
the
bioresorbable material does not interfere with the formation of a three-
dimensional
tissue. Preferably, the bioresorbable material degrades within about 1 to 3
months
after transplantation.
Examples of suitable bioresorbable materials include but are not
limited to polyesters, polyethylene glycols, and hydrogels. Examples of
suitable
non-resorbable materials include but not limited to polytetrafluoroethylenes
(Teflon), nylon, polycarbonates, and polyethylenes. Suitable examples also
include
but not limited to polysaccharides such as dextran, dextrin, starch,
cellulose, agarose,
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carrageenan, alginate (a carboxylated seaweed polysaccharide), and the like;
synthetic polymers such as polyvinyl alcohols, polyacrylamides, polyamides,
polyacrylates, polyesters, polymethacrylates, polyurethanes, polyphosphazene,
copolymers of lactic acid and glycolic acid, copolymers of lysine and lactic
acid,
S copolymers of lysine RGD and lactic acid, and the like. Proteins such as
collagens,
copolymers of collagen and chondroitin sulfate (a proteoglycan component), and
the
like can also be used.
The construct may also contain a signal for modifying cell adhesion,
growth, or migration, preferably stimulating or promoting the adhesion,
growth, or
migration of the desired cells, and/or inhibiting the adhesion, growth, or
migration of
the undesired cells. The signals may be selected from growth factors,
hormones,
extracellular matrix proteins and other cellular adhesion peptides identified
in the
extracellular matrix protein. Growth factors include for example, epithelial
growth
factor (EGF}, acidic or basic fibroblast growth factor (FGF), vascular
endothelial
growth factor {VEGF), hepatocyte growth factor (HGF), heparin binding growth
factor (HGBF), transforming growth factor {TGF), nerve growth factor (NGF),
muscle morphogenic factor (MMP), and platelet derived growth factor. Examples
of
extracellular matrix proteins include fibronectin, collagens, laminins, and
vitronectins; and the tri-peptide RGD {arginine-glycine-aspartate) that is
found in
many of the extracellular matrix proteins. A signal can also be included to
induce
the ingrowth of the desired cells, e.g., smooth muscle cells and epithelial
cells,
surrounding the construct in the area within the body of a living subject.
Preferably,
compounds which inhibit undesired cells, such as cancerous cells and
inflammatory
cells, can also be included. The signals can be covalently linked to a
biocompatible
material in the porous construct or associated with the porous construct by
affmity.or
linked to a material that can be covalently linked to or associated by
affinity with a
biocompatible material in the construct. See, e.g., U.S. Pat. Nos. 4,517,686;
4,792,525; and S, 330,911, incorporated herein by reference.
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The method of providing the porous constructs has been successfully
applied to the development of many different three-dimensional tissues in
mammals.
The selection of the cells contained in the porous construct for delivery into
an area
of interest in the body of a living subject depends on the location and the
type of
5 three-dimensional tissue desired. For example, if a three-dimensional
pancreatic
tissue is desired, the porous construct may contain pancreatic cells. In
another
example, if the development of constructs to replace absent breast tissue is
wanted,
cells such as vascular endothelial cells and smooth muscle cells can be used.
Other
cells, such as mesenchymal cells which include fibroblasts, chondrocytes, and
10 adipocytes can also be used alone or in conjunction with endothelial or
smooth
muscle cells. Other cell types that may be used in this invention include
hepotocytes, renal tubular cells, Sertoli cells, thyroid cells, islet cells,
skeletal muscle
cells, neural cells, cardiac muscle cells, osteocytes, stem cells, and the
like.
Various techniques for isolating cells from suitable sources are
generally known in the art. The cells used in this invention are preferably
autologous, i.e., obtained from the living subject itself. The cells can also
be
allogeneic, i.e., obtained from a subject of same species as the subject of
interest or
xenogeneic, i.e., from a subject of different species. In addition, the cells
can also be
treated in vivo or in vitro, and before or after being incorporated into the
porous
construct. For example, the cells may be cultured to expand in number or
modified
to change one or more characteristics. For example, it is generally known in
the art
to genetically modify cells in vitro by introducing a desired gene.or by
replacing an
undesired gene with a desired one, thus improving the characteristics of the
cells.
Methods are also known in the art to modify the immunological character of
allogeneic or xenogeneic cells so that the cells are not substantially
rejected by the
host tissue when they are delivered to the area of interest. Immunologically
inert
cells, such as stem cells and embryonic cells, are preferably used to avoid
immunological incompatibility.
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The cells to be incorporated in the porous construct of the invention
can be homogeneous cells from one source, or from different sources. Thus,
heterogous cells are used, the porous construct may have two or more
biocompatible
materials and two or more signals discussed above, each supporting a different
type
of cell in the construct. The number of cells to be incorporated into the
porous
construct can range from 10 cells/cmz to 1x108 cellslcm2.
The porous construct can be made in many methods known in the art
for making a porous scaffold supporting cell growth, with certain
modifications of
these methods to obtain the distinct features of the porous construct of the
present
invention as specified above. Such modifications will be apparent to an
ordinarily
skilled person in the art apprised of the present disclosure.
The macroporous lattice structure of the porous construct is formed
during the process of polymerization of one or more polymers using
conventional
methods for making macroporous structures. Alternatively, the macropores in
the
construct are formed by dissolution of a polymer or removal of one material
after
polymerization. In another method, the construct is formed from a porous
matrix of
polymers in which the pores are formed by dissolution of one of the polymers.
In
that method, two polymerization precursors, a matrix polymer precursor (e.g.,
collagen, fibrin, etc.) and a reversible gel polymer precursor (e.g.,
alginate, gums,
agarose, etc.) are polymerized in an aqueous solution to form a shape-
retaining solid
matrix comprising viable cells, matrix polymer and reversible gel polymer. The
reversible gel polymer is then dissolved and removed to form an insoluble,
porous
matrix containing viable cells. This methodology is used to construct small
discrete
tissue constructs as described herein with the requisite size constraints and
the
appropriate macroporous materials. An alternative method is described in U.S.
provisional application entitled: "Methods of Preparing Porous Hydrogel
Materials
and Products of the Methods", filed on even date herewith under Attorney
Docket
No. UMICH 8VI, and incorporated herein by reference.
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The porous construct is made by any known technique of producing
small beads or other discrete structures. Suitable techniques include but are
not
limited to pressure or air shear spraying, extrusion, emulsification, and
droplet
formation techniques such as electrostatic droplet formation, droplet
formation by
gravity, droplet formation by centrifugal forces, droplet formation using
Raleigh
liquid jet instability techniques, and droplet formation using inertial
forces. For
example, suitable porous constructs are prepared from collagen materials by
solidifying a collagen solution or dispersion into dry beads using any known
drying
techniques including but not limited to spray drying and freeze drying. Also,
beads
having larger sizes are reduced to small sizes by, for example, grinding.
For example, for the development of autologous breast tissue, smooth
muscle cells can be seeded and grown on porous polyglycolide beads or other
porous
biodegradable or naturally occurring materials (such as porous type I collagen
beads). Once the smooth muscle cells reach a specified density, a coating of a
hydrogel, for example an alginate RGD containing vascular endothelial cells is
polymerized on the beads. The alginate with endothelial cells encompasses the
beads, and intercalates within the beads. This provides a close contact
between the
endothelial cells and the smooth muscle cells causing the necessary cell
communication to occur. The smooth muscle beads provide the physical structure
necessary to create this tissue.
The porous construct should be sterilized before use. Examples of
such techniques include but are not limited to UV irradiation, gamma
irradiation, e-
beam sterilization and sterilization using chemicals such as ethylene oxide.
The
sterilization method used must not substantially adversely affect the
structure of the
porous construct and its ability to support the formation of a vascularized
three-
dimensional tissue.
It is also preferred to coat the porous construct with materials that
promote cell adhesion and attachment to the structure of the porous construct.
Examples of such materials include but not limited to fibronectin,
vitronectin,
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collagens, polylysine, laminins, polypeptides derived from these extra-
cellular
matrices, and other cell adhesion molecules. Such coating can be done at any
time,
for example, coating on polymer precursors, coating of a prepared construct
before
seeding cells in vitro, or after seeding in vitro but before delivering the
construct into
the body of a living subject of interest. An example of coating is to use
porous
beads such as porous polyglycolide beads and porous collagen beads, prepared
as
described above and coated with a hydrogel, for example, an alginate RGD, by
polymerizing the hydrogel onto the beads. The hydrogel not only encompasses
the
beads, but also intercalates within the beads.
The cells to be transplanted with the porous construct can be
incorporated therein while the porous construct is formed, for example, during
the
process of polymerization. Methods of incorporating cells during the formation
of a
porous structure are well known in the art. Alternatively, the cells can be
seeded
into a pre-formed porous construct. Since the pores of the porous construct
are fairly
large as described above, cell seeding can be easily done, for example, by
immersing
the porous construct for a period of time in a cell culture medium having
cells to be
transplanted floating therein. The cell density in the medium and the period
of time
can be easily controlled to allow a desired number of cells to attach within
the
porous construct.
Optionally, the cells in the porous construct are allowed to grow in
vitro for a period of time before they are transplanted along with the porous
construct into the body of a subject of interest. Methods for doing so will be
apparent to those skilled in the art. For example, a porous construct with
cells
attached therein can be conveniently immersed in a suitable culture medium. To
promote the growth and differentiation of the cells, suitable signal molecules
which
modify cell adhesion, growth, and migration, such as growth factors and
extracellular matrix proteins, are added to the culture medium.
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Once delivered to an area inside the body of a subject of interest, the
porous construct also allows the cells in the area surrounding the construct
to
migrate into the construct, promoting the formation of a three-dimensional
tissue.
In accordance with the present invention, at least one porous
construct having cells therein is delivered into an area inside the body of a
living
subject where a three-dimensional tissue is desired to be built. A plurality
of porous
constructs from 0.1 ml of volume to S00 ml of volume may be placed in the
body.
Each such discrete construct will develop and become interconnected with the
surrounding vasculature and with other developed constructs, mimicking the
functional units in natural tissue, thus facilitating the development of a
three-
dimensional tissue.
The delivery of the porous constructs is preferably performed in a
substantially non-invasive manner, avoiding any complex surgical procedures.
For
example, the constructs could be fashioned to be easily injected into the
tissue of
interest. More than one injection may be desired. Alternatively, these
constructs are
delivered in a biocompatible pouch that has an arteriole and venule connection
from
the outer pouch into the inner pouch.
In one particular embodiment, a second construct is also used which
may or may not contain cells. Once delivered into an area in the body of a
living
subject of interest, this second construct is capable of stimulating the
formation of a
vascularized bed surrounding it. Once the vascularized bed is formed, the
porous
construct as described above having therein cells to be transplanted is
delivered onto
or near the vascularized bed. The pre-developed vascularized bed can better
supply
the necessary nutrients required by the transplanted cells.
Preferably, a porous construct as described herein is used as the
second construct for pre-formation of a vascularized bed. However, so long as
it can
induce the formation of a vascularized bed, the second construct can be made
from
any biocompatible material and in virtually any shape or dimension, and is not
limited by the features of the porous construct. Preferably, small spheres of
about 5
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wm are used to help create the vascularized tissue. In addition, the second
construct
need not be macroporous. Preferably, the second construct contains signals
which
stimulate the formation of a vascularized bed. An example of such signal is
the tri-
peptide arginine-glycine-aspartate (RGD). This tri-peptide can be optimally
linked
to the second construct.
An example of such second construct is solid sodium alginate-RGD
macro-beads of about 1 to 2 mm in diameter. These beads were shown to be
capable
of inducing the development of a vascularized bed inside the body of a living
subject
in a relatively short period. The second construct may be delivered by the
same
10 methods used for the delivery of the porous construct as described above.
The present invention can be used in many different applications in
mammals, particularly in human. Many types of vascularized tissues can be
developed using the method of this invention, including but not limited to
three-dimensional breast tissue, pancreatic tissue, liver tissue, kidney
tissue, muscle
1 S tissue, skin tissue, etc.
This method can be used in reconstructive therapy, reconstructing a
body part that is injured or from which undesired tissue has been removed. In
particular, the method of this invention may be used for reconstruction or
repair of
soft tissue defects. Typically, a plurality of discrete, macroporous
constructs seeded
with cells selected from chondrocytes, fibroblasts, endothelial cells, smooth
muscle
cells, etc., will be implanted to replace missing soft tissue, forming a
tissue mass to
fill a depression or produce a protrusion, or to otherwise provide a bulky
mass of
tissue where it is needed. For example, using the method of this invention, a
soft
vascularized breast tissue can be built in the area where cancerous breast
tissue has
been removed, thereby reconstructing the breast. To do so, porous constructs
containing both normal vascular endothelial cells and smooth muscle cells
isolated
from the patient can be created and delivered to the pocket of the expanded
breast
tissue in accordance with the present invention.
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Further, the method of the present invention can also be used to treat
certain diseases in mammals.
For example, to treat insulin-deficient diabetes, three-dimensional
pancreatic islets which release a desirable level of insulin can be
transferred into
relatively vascular sites such as the subcutaneous space and in combination
with the
creation of a three dimensional vascular bed using these discrete tissue
constructs.
In summary, it has been provided an effective and non-invasive
method of creating a vascularized three-dimensional tissue in an area of
interest in a
mammal such as humans. A plurality of macroporous constructs are constructed
from biocompatible materials and contain cells to be transplanted for the
development of the three-dimensional tissue. The porous constructs are
delivered
into the desired area in a substantially non-invasive manner. The dimension of
porous constructs and the size of their macropores are made such that the
transplanted cells are no greater than 250 ~,m apart from a nutrient source in
the
body. As a result, adequate support is provided for the adhesion, growth and
migration of the transplanted cells as well as the ingrowth of the tissue
cells
surrounding the construct, thus forming a three-dimensional discrete tissue.
The present invention is further illustrated by the following non-
limiting examples. This invention may, however, be embodied in many different
forms and should not be construed as limited to the embodiments set forth
herein;
rather, this embodiment is provided so that this disclosure will be thorough
and
complete and will fully convey the scope of the invention to those skilled in
the art.
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EXAMPLE 1 Formation of A Vascularized Bed Induced by Sodium
Alginate RGD Macro beads.
Sodium alginate covalently coupled with arginine, glycine and
aspartic acid tri-peptide (RGD) (alg-RGD) was mixed in calcium-free, magnesium
free phosphate buffered saline (Gibco/BRL) at a concentration of 2 gm/100 mL
(2%). After thorough mixing the alg-RGD was filtered sterilized using a 0.45
~,m
filter (Nalgene unit). The alg-RGD solution was then aseptically dropped into
a
sterile filtered solution of 1.5% calcium chloride solution (1.5 ~.m of
calcium
chloride mixed with 100 ~m of Milipore reverse osmosis water) using a syringe
pump set at a flow rate of approximately 50 mL/hr. Beads of approximately 1 to
2
mm in diameter were formed during this process. A subcutaneous pocket was then
created on the back of a Fisher Rat (female) by using a purse string suturing
technique (a SO mL conical tube cap was used to mark the 39 mm circumference
of
the suturing plane, a 4.0 prolene suture was used to follow the outline of the
circle,
and the skin in the center of the circle was picked up using forceps), and one
mL of a
bead slurry was transplanted into this subcutaneous space.
Figure lA shows the histological results of a trichrome stain after one
week of implantation. Figure 1 B shows the formation of a vascular bed at the
interface of the beads and the tissue that forms. The results indicate that a
vasculature can develop in a relatively short time period and may be induced
by the
shape of the materials. However, this material was not designed to be
macroporous
and did not provide a means for cellular in growth into the tissue construct.
When
larger solid implants were used in a similar animal model, a capsule was
formed
around the entire perimeter of the implant without the formation of a vascular
bed
that would support cellular survival.
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EXAMPLE 2 Formation of A Vascularized Bed Induced by Sodium
Alginate-RGD Macro-beads.
Sodium alginate covalently coupled with arginine, glycine and
aspartic acid tripeptide (RGD)(alg-RGD) (100X more concentrated than Example
1)
was mixed in 0.8 g NaCI, 0.2g Na-hexametaphosphate in 100 mL of reverse
osmosis
water at a concentration of 1.2 gm/100 mL (1.2%). After thorough mixing, the
alg-RGD was filter sterilized using a 0.22 ~m filter. The alg-RGD solution was
then
aseptically dropped into a sterile-filtered solution of 1.5% calcium chloride
solution
(1.5 gm of calcium chloride mixed with 100 Ml of reverse osmosis water) using
a
syringe pump set at a flow rate of approximately 50 mL/hour. Beads of
approximately 1 to 2 mm in diameter were formed during this process. A
subcutaneous pocket was then created on the back of a Fisher Rat (female) by
using
a purse string suturing technique (a 50 mL conical tube cap was used to mark
the 39
mm circumference of the suturing plane, a 4.0 prolene suture was used to
follow the
outline of the circle, and the skin in the center of the circle was picked up
using
forceps), and one mL of a bead slurry was transplanted into this subcutanecus
space.
Figures 2A and 2B show the histological results after two weeks of
implantation.
Figure 3 is histological section stained for Factor VIII which is a protein
that is
located on vascular endothelial cells. The material was implanted in a purse
string
subcutaneous space and harvested after two weeks. The dark open circles in the
tissue have been positively identified as vascular endothelial cells.
EXAMPLE 3 Creation of A Vascular Three-dimensional Tissue with a Porous
Construct Made from Collar eon.
The previous examples indicated that the transplantation of bead
structures of an appropriate material would provide the impetus for vascular
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19
ingrowth into an avascu1ar, subcutaneous pocket. However, these materials were
not
porous and the size of the beads were approximately 1 to 2 mm in diameter.
To determine if a macroporous bead would enhance vascular
ingrowth into these structures, type I collagen (bovine collagen) microspheres
purchased from Cellex, Inc., Minneapolis, MN was transplanted. The beads,
ranging
in size from 400 to 700 pm in diameter and having a nominal pore size of SO to
100
pm in size, were designed to entrap and grow mammalian cells in vitro for the
production of biologics. A subcutaneous pocket was created on the back of a
Fisher
Rat (female) by using a purse string suturing technique (a 50 mL conical
tubecap
was used to mark the 39 mm circumference of the suturing plane, a 4.0 prolene
suture was used to fallow the outline of the circle, and the skin in the
center of the
circle was picked up using forceps), and one mL of a bead slurry was
transplanted
into this subcutaneous space. Figure 4 is a hematoxylin and eosin section of
the
collagen beads that were seeded with rat aortic smooth muscle cells. Note the
highly
porous structure of the beads (dark bands in the figure) and how the cells are
growing within the beads after a six day period (lighter stained area).
Figures SA
and SB show the histological results after two weeks of implantation. Figure
SA is a
histological section of collagen stain of the collagen beads implanted in a
purse
string subcutaneous space after two weeks implantation. Figure SB is
histological
section of the collagen beads staining for Factor VIII which is an antigen
expressed
on the surface of endothelial cells. This is a marker for vascularization.
Note the
staining that has occurred within the collagen bead.
In a related experiment aortic smooth muscle cells were implanted in
collagen beads. The histological results after two weeks of implantation are
indicated by the dark stain of expressed oc-actin. In Figure 7 there is shown
the
growth endothelial cells stained for Factor VIII on a collagen bead. The brown
staining shows that cells are in the interstices of the bead, i.e., the cells
migrated
from the host.
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EXAMPLE 4 Histolo~ical Observation of Two-week Subcutaneous Implants.
Many tissues and organs are organized as discrete tissue segments.
These segments are generally arranged as islands of parenchyma) or stromal
cells
that are surrounded by an intricate capillary bed. The size of these discrete
tissue
constructs is dictated by the metabolic need of the cells and diffusional
distances for
nutrients, gasses, and waste products to and from the cells.
The development of a three-dimensional vascular bed is imperative
for the successful implantation of large tissue structures that have been
created ex
vivo. In order to develop a soft tissue that will replace the large mass of
tissue that is
10 removed from a patient following a lumpectomy, a strategy has been
developed for
the creation of a vascular bed to support cellular survival.
Alginate is a natural hydrogel that has previously been used for cell
encapsulation and wound dressings. However, this material does not support
cellular adhesion, as is required of any tissue engineering scaffold
materials. Hence,
15 a process was developed to couple the cellular adhesion tripeptide RGD to
the
alginate. The next task was to process this material into constructs of
varying
morphologies and therefore diffusional constraints.
Type I bovine collagen or sodium alginate was implanted into the
subcutaneous spaces of Lewis rats. Type I bovine collagen was acquired as
porous
20 spheres (0.4 to 0.7 mm in diameter), whereas sodium alginate coupled with
RGD
tripeptide was fashioned either into rods, solid beads ( 1.0 to 3.0 mm in
diameter), or
porous beads (2.7 to 3.2 mm in diameter). The volume of each implanted
material
was one mL. The implants were harvested after two weeks implantation and
characterized as to capsule formation, vascular ingrowth, tissue ingrowth, and
inflammatory response. Table I outlines the experiment.
The Institutional Animal Care and Use Committee (IACUC)
approved all animal procedures. A subcutaneous pocket was created on the back
of
a female Lewis rat using either a flank incision for the rods or a purse
string suturing
technique for the beads. The implants were harvested after 2 weeks and
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21
Mstologically processed. Briefly, the animals were perfused with either Bouins
fixative or Z-fix formalin to preserve the tissue biomaterial interface. After
at least
24 hours in a formalin fixative, the tissue samples were carefully cut in half
at the
mid-line to preserve the tissue biomaterial interface. These small tissue
blocks were
then processed using a paraffin embedding method. Four micron sections were
cut,
and the tissues were stained with either hematoxylin and eosin, Masson's
trichrome
for the detection of collagen bundles, or stained with an antibody against Von
willebrand's factor VIII (Dako, Cupertino, CA) for the detection of
endothelial cells.
Three to six animals were evaluated for each implanted material type.
At least two independent readers histogically scored the samples using 0 as a
minimal and 4 as a maximal response. The histology samples were evaluated for
vascular ingrowth, capsule formation, tissue ingrowth, and inflammation. The
mean
and standard deviations were calculated for all implanted materials, and an
ANOVA
analysis using Fisher's criteria was calculated using StatView 4.5 (Aacus,
Berkeley,
CA)
Table 1
Material Dimensions Number Number of
Implanted Recovered Implants
(2 weeks)
Alginate-RGD Implant volume = 6 3
1 mL
Solid Rod Diameter = 1 cm
and
length = 1 cm
Alginate-RGD Implant volume = 6 6
1 mL
Solid Bead Diameter = 1.5 -
3.0 mm I
Alginate-RGD Implant volume = 6 6
mL
Porous Bead Diameter = 2.5 -
3.0 mm I
Cellex Type I Implant volume = 5 5**
1 mL
Bovine Collagen Diameter = 400 -
700
Porous Beads mm
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Table 1: Experimental layout for biomaterials implanted into a Lewis rat
model.
The number of samples analyzed for the implanted alginate rods was reduced due
to
the difficulty in preserving the tissue material interface during histological
processing.
*3 implants were lost during histological fixation due to the lack of tissue
ingrowth
into the material.
**One animal died post-transplant.
15 Solid Rods and Beads:
Alginate rods were formed by injecting a 1.2% alg-RGD solution into
Spectra/Por dispodialyzers. These dialyzer tubes were Sml volume and l Omm
width, with a molecular weight cut-off of 300,000 and were pre-filled with
sterile
water. The water in the dispodialyzer case was poured out and replaced with
sterile
1.5% CaCl2 and then 3.3 mls of alg-RGD (in PBS) was placed inside the dialysis
tube which was embedded in the case. The alginate-RGD was allowed to gel for
one
hour, and then the CaCl2 was replaced with DMEM. At the time of surgery, the
dispodialyzer tube membrane was cut away with small scissors, and the cylinder
of
alginate was placed into a petri dish. A ruler was placed under the dish, and
the
alginate was cut into 1-cm rods with a sterile scalpel. The diameter was also
1 cm,
making the implant approximately 1 ml, similar to the volume of the bead
implants.
For rod placement, an incision was made on the flank of the rat, blunt
dissection
used to form a pocket, the rod was placed inside the pocket, and the incision
was
stapled.
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For solid beads, sodium alginate (Pronova, Lot #411-256-O5,
city/state) was covalently coupled with arginine, glycine, and aspartic acid
tri-
peptide (RGD). The alginate-RGD (alg-RGD) was mixed in calcium-free,
magnesium-free phosphate buffered saline (GibcoBRL) at a concentration of 2
gm/100 mL (2%). After thorough mixing, the alg-RGD was filtered using a 0.45
mm filter (Nalgene). The alg-RGD solution was then aseptically dropped into a
sterile-filtered solution of 1.5% calcium chloride solution (Sigma Chemicals,
St.
Louis, MO) using a 60 mL syringe (Becton Dickinson) and a 30 gauge needle. A
Harvard syringe pump (Harvard Industries) was used with a flow rate of 50
mL/hr.
Beads of approximately 1 to 3 mm in diameter were formed in this manner.
A subcutaneous pocket was created on the back of a female Lewis rat
using a purse string suturing technique for the beads. The purse string was
formed
by using a 50-mL conical tube cap to mark a 39-mm circumference of the
suturing
plane, and then a 4.0 prolene suture was used to follow the outline of the
circle. A
1.5 cm incision was made slightly off center in order to bluntly dissect the
subcutaneous space before implanting a 1-mL bead slurry (either alginate or
collagen beads), the incision was closed with Vicryl suture (Ethicon, Inc.,
Cincinnati, Ohio), and the prolene suture was tightened and tied to form a one
mL
subcutaneous pocket. All materials were pretreated overnight in a DMEM media
containing (2%) fetal bovine serum.
Histologic analyses of tissue sections containing the solid alginate-
RGD beads and rods were compared after 2 weeks of implantation. A fibrous
capsule had formed around the rods and the smaller bead implants with no
discernible tissue ingrowth into the material. This lack of tissue ingrowth
into the
alginate rods made it difficult to maintain the tissue-biomaterial interface
during
histological processing. There was a minimal presence of giant and
polymorphonuclear cells (PMNs) in both of these implant sites. At high
magnification, the capsule that formed around the alginate-RGD beads could be
seen. The capsule appeared to be highly vascularized. An immunohistochemical
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24
section identified the presence of Factor VIII throughout the capsule region.
(This
marker is used to identify endothelial cells.)
The ability to form a highly vascularized capsule around the beads
and rods is an important factor for the delivery of nutrients. However, the
size of
these constructs (greater than 1.5 mm) is an order of magnitude greater than
the
diffusional distance of nutrients and oxygen (0.15 mm).; therefore, cells
delivered in
these materials would not receive an adequate nutrient supply.
Porous Collagen Beads:
A porous bovine Type I collagen bead was chosen to determine if a
three-dimensional vascular bed could be established with the requisite
dimensions to
support transplanted cells. Type I collagen beads were purchased from Cellex
(Minnesota). The beads were glutaraldehyde fixed, with an average diameter of
0.4
- 0.7 mm and pore size ranging from 0.05 to 1 mm. The beads were highly
porous,
ranging in diameter from 0.4 to 0.7 mm, with a pore size range of 0.05 to 0.1
mm,
and they promoted cellular attachment. Type I collagen beads were implanted on
the back of female Lewis rats using the purse string implant technique
described
above. Histological examination of a section of the implanted Type I porous
collagen beads showed that, unlike the alginate beads, there was a cellular
ingrowth
within the bead structure. However, there was also an increased presence of
giant
cells and PMNs throughout the tissue construct. Upon staining for Factor VIII
within these transplanted areas, it was observed that vascular endothelial
cells are
located throughout the implanted area.
Macroporous Alginate-RGD Beads
Mimicking this microarchitecture, a macroporous alginate-RGD bead
was developed with a diameter range between 2.7 and 3.2 mm, and an
undetermined
pore size range. Porous alginate-RGD beads were made using 3% alginate-RGD
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mixed with a 2 M sodium bicarbonate and a 1.5% BSA solution ( 1:1:1 ). After
approximately 15 minutes of stirring on a stir plate at medium rotation, the
alginate-
RGD became foamy and thick. This foamy material was located into a 10 mL
syringe and dripped into a calcium chloride solution (0.5 M calcium chloride
5 solution mixed in glacial acetic acid at 9:1 volume ratio) at a rate of
approximately 1
bead per second. The beads were rinsed in sterile double distilled water, and
the
beads were placed in 700 ml of 0.33 M calcium chloride solution. This bead
mixture was then placed on a Buchi (Buchi, Switzerland) rotavapor, and vacuum
was applied until the beads sank to the bottom of the vessel. The beads were
10 sterilized using gamma irradiation. The porous alginate-RGD beads were
implanted
on the back of female Lewis rats using the purse string implant technique
described
above. Histological cross-section of a porous alginate-RGD bead after 2 weeks
implantation showed that a vascular bed is present throughout the bead
implant.
15 Summary:
Histological sections of the implanted alginate-RGD and Type I
collagen beads were graded according to capsule formation, presence of
vasculature
within the implant site, tissue ingrowth, and the inflammatory response, and
the
results are presented in bar graph format in Figure 8. A scale of 0 to 4 was
assigned
20 to each of these observations, with a 0 being a minimal and a 4 being a
maximal
quantity. As indicated in Figure 8, both implanted materials provided a
mechanism
for the development of a vascular bed. However, the solid alginate beads did
not
permit tissue ingrowth into the material, whereas the Type I collagen beads
and the
porous alginate-RGD beads supported tissue ingrowth throughout the implant
site.
25 The alginate-RGD beads had a minimal presence of giant cells and PMNs,
while the
Type I collagen beads showed a moderate to severe inflammatory reaction. The
inflammatory response to the Type I collagen beads may have been due to the
glutaraldehyde fixation method used to covalently modify the material.
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The animal model employed in this experiment provided the ability
to test whether a vascular bed could be established in a large defect. On
average, the
distance from the host vascular bed to the center of the bead implant was 2.0
mm
post-histological processing. The ability to create a three-dimensional
vascular bed
with this thickness will allow for soft tissue reconstruction. It is also
feasible
through multiple step implants of a similar thickness to serially increase
tissue
volumes. This approach could be used to develop the larger tissue masses
needed
for breast reconstruction following a lumpectomy.
EXAMPLE 5 Histolo~ical Observation of Subcutaneous Implants at 2. 4,
and 8 Weeks.
Macroporous alginate-RGD beads were prepared and implanted as
described in Example 4, and histological observations were taken as described
at 2,
4, and 8 weeks after the implants. The results are shown in Figures 9-13. The
porous alginate-RGD beads allowed tissued ingrowth with the presence of a
vascular
bed throughout the implanted material. Figure 13 shows that the maximum
thickness of the surrounding capsule (corresponding to scar tissue) occurs at
two
weeks (on the side of the implant facing muscle) or four weeks (on the side of
the
implant facing skin) and decreases by eight weeks.
Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of understanding,
it will
be obvious that certain changes and modifications may be practiced within the
scope
of the appended claims.
In the specification, there has been set forth a preferred embodiment
of the invention and, although specific terms are employed, the terms are used
in a
generic and descriptive sense only and not for purpose of limitation, the
scope of the
invention being set forth in the following claims.
All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the art to which
this
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27
invention pertains. All publications and patent applications are herein
incorporated
by reference to the same extent as if each individual publication or patent
application
was specifically and individually indicated to be incorporated by reference.
Likewise, the parent application of this application is incorporated herein by
reference.
