Note: Descriptions are shown in the official language in which they were submitted.
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TISSUE ENGINEERING DEVICES AND NIETHODS
FOR LUMINAL ORGANS
FIELD OF THE IlVVENTION
[0001] The present invention relates to methods and materials for tissue
reconstruction, repair, augmentation, and repla.cement. More specifically, the
present
invention provides for the treatmerxt of patients using an impIantable device
that is
oomprised of a biocompatible, biodegadable, synthetic or_natural polymeric
matrix
shaped to conform to at least a part of a luminal organ or tissue structure
and seeded with
minced tissue.
BACKGROUND
[0002] The human urinary bladder is a luminal organ constitutmg a
musculomembranous sac situated in the anterior portion of the pelvic cavity,
The bladder
serves as a reservoir for urine, which this organ receives through the ureters
and
discharges through the urethra. Iau huwoaaus,.th.e bladder is found inAfie
pelvis behind the
pelvic bone (pubis symphysis) and the urethra, which exits to the outside of
the body.
The bladder, ureters, and uxefta are all similarly constitated in that they
comprise
muscular structures lined with a riaezo,brane comprising urothelial cells
coated with mucus
that is impermeable to the normal soluble substances of the nrine. The trigone
of the
bladder (trigonum vesicae) is a smooth triangolar portion of the mucous
membrane at the
base of the bladder. 'Ihe bladder tissue is eiasti.c and compliant, i.e., the
bladder changes
shape and size aocordiug to the amount of urine it contains. A bladder
resembles a
deflated balloon wbten empty, but becomes somewhat pear-shaped and rises into
the
abdominal cavity whem the amount of urine increases.
[00031 The bladder wall has three main layers of tissues: the mucosa,
submuoosa, and detrusor. The mucosa, comprising urothelial cells, is the
innermost layer
and is composed of transitional cell epithelium. The submucosa lies
immediately beneath
the mucosa and its basement mentbrane. It is composed of blood vessels that
supply the
mucosa with nutrients and the lymph podes,
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which aid in the removal of waste products. The detrusor is a layer of smooth
muscle cells that expands to store urine and contracts to expel urine.
[0004] The bladder is subjected to numerous maladies and injuries
that cause deterioration in patients. For example, bladder deterioration may
result
from infectious diseases, neoplasms, and developmental abnormalities. Bladder
deterioration may also occur as a result of trauma from, for example, car
accidents
and sports injuries.
[0005] Although numerous biomaterials, including synthetic and
naturally derived polymers, have been employed for tissue reconstruction or
augmentation, no material has proven satisfactory for use in bladder
reconstruction. Attempts have usually failed due to mechanical, structural,
functional, or biocompatibility problems. Permanent synthetic materials have
been associated with mechanical failure and calculus formation.
[0006] Naturally derived materials such as lyophilized dura, de-
epithelialized bowel segments, and small intestinal submucosa have also been
proposed for bladder replacement. However, it has been reported that bladder
augmented with dura, peritoneum, and placenta and fascia contract over time.
De-
epithelialized bowel segments demonstrated an adequate urothelial covering for
use in bladder reconstruction, but difficulties remain with mucosal regrowth,
segment fibrosis, or both. It has been shown that de-epithelialization of the
intestinal segments may lead to mucosal regrowth, whereas removal of the
mucosa and submucosa may lead to retraction of the intestinal segment.
[0007] Other problems have been reported with the use of certain
gastrointestinal segments for bladder surgery, including stone formation,
increased mucus production, neoplasia, infection, metabolic disturbances, long-
term contracture, and resorption. These attempts have demonstrated that it is
not
easy to replace the permeability functions of the urothelium.
[0008] Due to the multiple complications associated with the use
of gastrointestinal segments for bladder reconstruction, alternate solutions
have
been sought. Recent surgical approaches have relied on native urological
tissue
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for reconstruction, including auto-augmentation and ureterocystoplasty.
However,
auto-augmentation has been associated with disappointing long-term results and
ureterocystoplasty is limited to cases in which a dilated ureter is already
present.
A system of progressive dilation for ureters and bladders has been proposed
though not yet attempted clinically. Sero-muscular grafts and de-
epithelialized
bowel segments, either alone or over a native urothelium, have also been
attempted. However, graft shrinkage and re-epithelialization of initially de-
epithelialized bowel segments have been recurring problems.
[0009] One significant limitation besetting bladder reconstruction
is directly related to the availability of donor tissue. The limited
availability of
bladder tissue prohibits the frequent routine reconstruction of bladder using
normal bladder tissue. The bladder tissue that is available and considered
usable
may itself include inherent imperfections and disease. For example, in a
patient
suffering from bladder cancer, the remaining bladder tissue may be
contaminated
with metastasis. The patient is thus predestined to less than perfect bladder
function.
[00010] Accordingly, a need exists in the art for improved methods
and materials for the reconstruction, repair, augmentation, and replacement of
luminal organs or tissue structures, such as the bladder. The deficiencies in
the
prior art are overcome by the present invention.
SUMMARY OF THE INVENTION
[00011] An embodiment of the present invention relates to an organ
reconstruction method comprising the steps of: providing a biodegradable
polymer matrix conforming to a portion of a laminarly arranged luminal organ;
obtaining autologous, allogeneic or xenogeneic tissue comprising multiple cell
populations; processing the tissue to obtain a minced tissue composition;
seeding
the matrix with the composition; and implanting into a patient the seeded
polymer
matrix.
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[00012] An embodiment of the present invention relates to an organ
reconstruction method comprising the steps of: providing a biodegradable
polymer matrix conforming to a portion of a laminarly arranged luminal organ;
obtaining autologous, allogeneic or xenogeneic tissue comprising multiple cell
populations; processing the tissue to obtain a first minced tissue composition
and
a second minced tissue composition; seeding a first area of the matrix with
the
first minced tissue composition, and seeding a second area of the matrix with
the
second minced tissue composition; and implanting into a patient the seeded
polymer matrix.
[00013] Yet another embodiment of the present invention relates to
an organ reconstruction device comprising an implantable, biodegradable
polymer
matrix conforming to a portion of a laminarly arranged luminal organ, wherein
said matrix is capable of being seeded with a processed tissue composition,
which
comprises minced autologous, allogeneic or xenogeneic tissue comprising
multiple cell populations.
BRIEF DESCRIPTION OF THE DRAWINGS
[00014] Some features and advantages of the invention are
described with reference to the drawings of certain preferred embodiments,
which
are intended to illustrate and not to limit the invention.
[00015] Figure 1 depicts the anatomy of a normal human bladder.
[00016] Figure 2 depicts the tissue layers of various cell types that
may be used in the minced tissue composition of the present invention.
[00017] Figure 3A, 3B, and 3C depicts the cell migration,
distribution and organization of urothelial and smooth muscle cells from
bladder
minced tissue into resorbable scaffolds. Arrows (1) point to urothelial cell
clusters
and layers; arrows (2) point to organization of smooth muscle like cells
around
the urothelial cells; and star denotes the cavity within the newly organized
urothelium and smooth muscle structures.
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DETAILED DESCRIPTION OF THE INVENTION
[00018] It should be understood that this invention is not limited to
the particular methodology, protocols, etc., described herein and, as such,
may
vary. The terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the present
invention,
which is defined solely by the claims.
[00019] As used herein and in the claims, the singular forms "a,"
"an," and "the" include the plural reference unless the context clearly
indicates
otherwise. Thus, for example, a reference to a cell may be a reference to one
or
more such cells, including equivalents thereof known to those skilled in the
art
unless the context of the reference clearly dictates otherwise. Unless defined
otherwise, all technical terms used herein have the same meaning as those
commonly understood to one of ordinary skill in the art to which this
invention
pertains. Other than in the operating examples, or where otherwise indicated,
all
numbers expressing quantities of ingredients or reaction conditions used
herein
should be understood as modified in all instances by the term "about." The
term
"about" when used in connection with percentages may mean 1%.
[00020] All patents and other publications identified are
incorporated herein by reference for the purpose of describing and disclosing,
for
example, the methodologies described in such publications that might be used
in
connection with the present invention. These publications are provided solely
for
their disclosure prior to the filing date of the present application. Nothing
in this
regard should be construed as an admission that the inventors are not entitled
to
antedate such disclosure by virtue of prior invention or for any other reason.
All
statements as to the date or representation as to the contents of these
documents is
based on the information available to the applicants and does not constitute
any
admission as to the correctness of the dates or contents of these documents.
[00021] The present invention provides for methods and materials
for the reconstruction, repair, augmentation, or replacement of shaped hollow
organs or tissue structures that exhibit a laminar segregation of different
cell types
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and that have a need to retain a general luminal shape. Luminal organs or
tissue
structures containing a smooth muscle cell layer to impart compliant or
contractible properties to the organ or structure are particularly well-suited
to the
methods and devices of the present invention.
[00022] One example of a luminal organ suitable for application of
the present invention is a bladder, which has an inner layer of a first cell
type that
comprises urothelial-tissue, a middle layer of submucosa, and an outer layer
of a
second cell type that comprises smooth muscle tissue. This organization is
also
present in other genitourinary organs and tissue structures such as the renal
pelvis
ureters and urethra. Laminarily organized organs or tissues refer to any organ
or
tissue made up of, or arranged in laminae, including ductal tissue. Other
suitable
laminarily organized luminal organs, tissue structure, or ductal tissues to
which
the present invention is directed include vas deferens, fallopian tubes,
lacrimal
ducts, trachea, stomach, intestines, vasculature, biliary duct, ductus
ejaclatoruis,
ductus epididymidis, ductus parotideus, ureters, urethras, and surgically
created
shunts.
[00023] The present invention may be suitable for the treatment of
such conditions as bladder extrophy, bladder volume insufficiency,
reconstruction
of bladder following partial or total cystectomy, repair of bladders damaged
by
trauma, and the like.
[00024] While reference is made herein to the reconstruction,
repair, augmentation, and replacement of the bladder, it will be understood
that
the methods and devices of the invention are useful for the reconstruction,
repair,
augmentation, and replacement of a variety of tissues and organs in a patient.
Thus, for example, organs or tissues such as bladder, ureter, urethra, renal
pelvis,
and the like, can be reconstructed, repaired, augmented, or replaced with
polymeric matrixes seeded with the appropriate minced tissue. The devices and
methods of the invention can be further applied to the reconstruction, repair,
augmentation, and replacement of vascular tissue (see, e.g., Zdrahala, R. J.,
J.
Biomater. Appl. 10(4): 309-29 (1996)), intestinal tissues, stomach (see, e.g.,
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Laurencin, C. T. et al., J. Biomed. Mater. Res. 30(2): 133-38 (1996)), and the
like.
The patient to be treated may be of any species of mammals, such as a dog,
cat,
pig, horse, cow, or human, in need of reconstruction, repair, augmentation, or
replacement of an organ or tissue structure.
[00025] The source of the minced tissue of the present invention
may be of the same or different tissue origin than that intended to be
reconstructed, repaired, augmented, and replaced. For example, the minced
tissue
may derive from urethral tissue to facilitate the reconstruction, repair,
augmentation, and replacement of bladder tissue. The morphologic similarity of
luminal organs, such as bladder and urethral tissue, for example, is known in
the
art, see Dass et al., 165 J. Urol. 1294-1299 (2001), and the use of bladder
tissue in
urethra reconstruction has been reported, A. Atala, 4 (Suppl. 6) Am. J. of
Transplantation 5873 (2004).
[00026] As stated earlier, one significant limitation besetting
bladder reconstruction is directly related to the availability of donor
tissue. The
limited availability of bladder tissue prohibits the frequent routine
reconstruction
of bladder using normal bladder tissue. The bladder tissue that is available
and
considered usable may itself include inherent imperfections and disease. For
example, in a patient suffering from bladder cancer, the remaining bladder
tissue
may be contaminated with metastasis. The patient is thus predestined to less
than
perfect bladder function.
[00027] As a result, others have tried a cell culturing approach
(Atala et al.) where the smooth muscle cells and the urothelium cells are
isolated
from a biopsy, cultured separately in vitro, and then added onto a bladder
substrate. However, this process is long and time consuming where a patient
has
to wait for at least eight weeks before the next implantation of a tissue
engineered
scaffold. Other tissues have also been evaluated as a source of cells for
bladder
augmentation for buccal tissue, for example. See El-Sherbiny et al.,
"Treatment of
Urethral Defects: Skin, Buccal or Bladder Mucosa, Tube or Patch? An
Experimental Study in Dogs," 167 J. Urol. 2225-2228 (2002).
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[00028] The methods of the present invention provide a
biocompatible synthetic or natural polymeric matrix that is shaped to conform
to
its use as a part or all of the bladder structure to be repaired,
reconstructed,
augmented or replaced. A biocompatible material is any substance not having
toxic or injurious effects on biological function. As used herein the term
"synthetic polymer" refers to polymers that are not found in nature, even if
the
polymers are made from naturally occurring biomaterials. The term "natural
polymer" refers to polymers that are naturally occurring. The shaped,
synthetic or
natural polymeric matrix is preferably porous to allow for cell deposition and
migration both on and in the pores of the matrix. It can be made from various
scaffolding materials such as lyophilized foams, nonwoven scaffolds, or melt-
blown scaffolds.
[00029] Lyophilization, or freeze-drying, removes a solvent from a
polymer-solvent solution through sublimation, leaving behind a porous solid.
More specifically, the process separates a solvent from a frozen solution
through a
solid to gas phase transition. This transition, called sublimation, removes
the
solvent without it ever entering a liquid state. The final construct is a
porous solid
structure made out of the remaining solute often described as a foam.
[00030] Liquid solution comprising any natural or synthetic
biocompatible, biodegradable polymer, or any blend of such polymers, dissolved
in a solvent that can be removed through sublimation, is poured into an open-
ended, hinged mold and mechanically rotated during freezing. In the first
step, the
mold is hinged shut and partially filled with solution. During filling, some
of the
mold's volume remains empty. After lyophilization, the volume of solution
poured into the mold will make up the scaffold volume whereas the empty
volume will make up the hollow void. After filling, the mold may be rotated in
a
number of ways. When the mold is held vertically and spun quickly, a
centrifugal
force acts on the liquid solution, pushing it away from the mold's center and
up
upon its sides. The spinning mold may then be cooled slowly or flash frozen by
submersion in liquid nitrogen. The mold may also be held horizontally and
rotated
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slowly whereby gravity allows the polymer to settle upon one side of the mold.
Assuming that the temperature of the mold is lower than the temperature of the
ambient air, a layer of frozen liquid will gradually build up on the mold's
interior,
resulting in an internal frozen skin. Both methods will produce a frozen
construct
that has a shape and texture consistent with the mold's internal geometry.
Once
fully frozen, the construct is placed in a vacuum for sublimation.
[000311 A variety of absorbable polymers can be used to make
foams. Examples of suitable biocompatible, bioabsorbable polymers that could
be
used include polymers selected from the group consisting of aliphatic
polyesters,
poly(amino acids), copoly(ether-esters), polyalkylene oxalates, polyamides,
poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamindoesters,
polyoxaesters containing amine groups, poly(anhydrides), polyphosphzenes,
biomolecules (i.e., biopolymers such as collagen, elastin, bioabsorbable
starches,
etc.), and blends thereof.
[00032] Suitable solvents include but are not limited to solvents
selected from a group consisting of formic acid, ethyl formate, acetic acid,
hexafluoroisopropanol (HFIP), cyclic ethers (i.e., THF, DMF, and PDO),
acetone,
acetates of C2 to C5 alcohol (such as ethyl acetate and t-butylacetate), glyme
(i.e.,
monoglyme, ethyl glyme, diglyme, ethyl diglyme, triglyme, butyl diglyme, and
tetraglyme), methylethyl ketone, dipropyleneglycol methyl ether, lactones
(such
as y-valerolactone, b-valerolactone, (3-butyrolactone, y-butyrolactone), 1,4-
dioxane, 1,3-dioxolane, 1,3-dioxolane-2-one (ethylene carbonate),
dimethylcarbonate, benzene, toulene, benzyl alcohol, p-xylene, naphthalene,
tetrahydrofuran, N-methyl pyrrolidone, dimethylformamide, chloroform, 1,2-
duchloromethane, morpholine, dimethylsulfoxide, hexafluoroacetone
sesquihydrate (HFAS), anisole and mixtures thereof. A homogenous solution of
the polymer in the solvent is prepared using standard techniques.
[00033] As will be appreciated by those skilled in the art, the
applicable polymer concentration or amount of solvent which may be utilized
will
vary with each system. Suitable phase diagram curves for several systems have
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already been developed. However, if an appropriate curve is not available,
this
can be readily developed by known techniques. The amount of polymer will
depend to a large extent on the solubility of the polymer in a given solvent
and the
final properties of the foam desired.
[00034] A parameter that may be used to control foam structure is
the rate of freezing of the polymer-solvent solution. The type of pore
morphology
that gets locked in during the freezing step is a function of the solution
thermodynamics, freezing rate, temperature to which it is cooled,
concentration of
the solution, homogeneous or heterogeneous nucleation, etc. Detailed
description
of such phase separation phenomenon can be found in the references provided
herein. See A. T. Young, "Microcellular foams via phase separation," J. Vac.
Sci.
Technol. A 4(3), May/June 1986; S. Matsuda, "Thermodynamics of Formation of
Porous Polymeric Membrane from Solutions," Polymer J. Vol. 23, No. 5, pp 435-
444, 1991).
[00035] A foam scaffold may also be constructed by a two-step
mold where one part of the mold consists of a hollow section and another part
consists of a core. This design is similar to that used in a typical injection
molding
process. The solution can be filled via the space between the cavity and the
core.
The space can be determined by the thickness of the final construct. Once the
filling is complete, the solution can be frozen by the steps above.
[00036] Another embodiment of the present invention may include
nonwoven scaffolds. Preferred nonwoven materials include flexible, porous
structures produced by interlocking layers or networks of fibers, filaments,
or
film-like filamentary structures. Such nonwoven materials can be formed from
webs of previously prepared/formed fibers, filaments, or films processed into
arranged networks of a desired structure.
[00037] Generally, nonwoven materials are formed by depositing
the constituent components (usually fibers) on a forming or conveying surface.
These constituents may be in a dry wet, quenched, or molten state. Thus, the
nonwoven can be in the form of a dry laid, wet laid, or extrusion-based
material,
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or hybrids of these types of nonwovens can be formed. The fibers or other
materials from which the nonwovens can be made are typically polymers, either
synthetic or naturally occurring.
[00038] Dry laid scaffolds may include those nonwovens formed by
gameting, carding, and/or aerodynamically manipulating dry fibers in the dry
state. In addition, wet laid nonwovens may be formed from a fiber-containing
slurry that is deposited on a surface, such as moving conveyor. The nonwoven
web can be formed after removing the aqueous component and drying the fibers.
Extrusion-based nonwovens may include those formed from spun bond fibers,
melt blown fibers, and porous film systems. Hybrids of these nonwovens can be
formed by combining one or more layers of different types of nonwovens by a
variety of lamination techniques. The nonwoven may also be reinforced with a
woven, knit or mesh fabric.
[00039] The nonwovens of the present invention preferably have a
density designed to obtain mechanical characteristics ideal for augmenting
bladder repair. The density may be measured by determining the felt dimensions
(length and width), for example, obtaining two measurements in each direction
to
calculate the average length and width for each nonwoven felt. The trimmed
felt
may be weighed, and the weight recorded. The average thickness of each
nonwoven felt may be obtained using a Shirley gauge. The density may be
calculated by the following formula:
[00040] Density = (weight of felt (W) (grams))/(lengthXwidth
(cm2)) = ((WX 1000 (mg/cm2))/((thickness (mm))/10 (mm/cm))
[00041] Additionally, scaffolds may be manufactured by use of
melt-blowing technology whereby fibrous webs from molten polymer resin are
extruded from spinnarettes onto a rotating collapsible object in the presence
of a
porogen. The collapsible object can be made to rotate or otherwise move
therefore
allowing a coating of extruded polymer to layer itself substantially evenly on
the
collapsible object. Continuous rotation of the surface will produce an
increasingly
thick or dense layer due to more polymer being deposited. The use of a
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collapsible object creates seamless, three-dimensional shapes of polymer web.
Specifically, the final product may be a hollow shape with a single outlet
from
which the collapsed shape has been removed. More complex geometries may be
achieved by using suitably shaped tooling such as a mold or mandrel to guide
the
formation of the melt-blown filaments into a specific shape. This method is
described in detail by Keeley et al. in US Patent Application serial number
11/856,743.
[00042] Melt-blown technology is able to incorporate synthetic
biopolymers, such as PGA, PLA or their respective copolymers, and natural
polymers. A scaffold constructed of either material is both biocompatible and
resorbable but may not be sufficiently porous to facilitate optimal
proliferation of
cells or advanced tissue ingrowth. To overcome this obstacle, a porogen may be
added during the fabrication of the non-woven web. Porogens such as salt or
glucose spheres can be dusted or blown onto the molten fibers during their
extrusion. Gelatin microspheres can also be used. The resulting scaffold's
porosity can be controlled by the amount of porogen added, while the pore size
is
dependent on the size of the spheres. As these particles enter the turbulent
air,
they are randomly incorporated into the web. Because the filaments in the melt-
blown structure will typically shrink due to crystallization as they age, the
porous
structure may undergo an annealing process with the porogen material in place.
Once the porogen-fiber composite is annealed, the entire construct may then be
submerged in water so that the porogens dissolve or leach out of the web. The
resulting matrix contains polymer fibers but with increased distance between
them
to effect porosities. In one embodiment, the matrix has more porogen and
hence,
more porosity, the porosity in excess of 90%.
[00043] The polymers or polymer blends that are used to form the
biocompatible, biodegradable scaffold may also contain pharmaceutical
compositions. The previously described polymer may be mixed with one or more
pharmaceutical prior to forming the scaffold. Alternatively, such
pharmaceutical
compositions may coat the scaffold after it is formed. The variety of
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pharmaceuticals that can be used in conjunction with the scaffolds of the
present
invention includes any known in the art. In general, pharmaceuticals and/or
biologics that may be administered via the compositions of the invention
include,
without limitation: anti-infectives such as antibiotics and antiviral agents;
chemotherapeutic agents; anti-rejection agents; analgesics and analgesic
combinations; anti-inflammatory agents; hormones such as steroids; growth
factors; and other naturally derived or genetically engineered (recombinant)
proteins, polysaccharides, glycoproteins, or lipoproteins.
[00044] Scaffolds containing these materials may be formulated by
mixing one or more agents with the polymer used to make the scaffold or with
the
solvent or with the polymer-solvent mixture. Alternatively, an agent could be
coated onto the scaffold, preferably with a pharmaceutically acceptable
carrier.
Any pharmaceutical carrier may be used that does not substantially degrade the
scaffold. The pharmaceutical agents may be present as a liquid, a finely
divided
solid, or any other appropriate physical form. Typically, but optionally, they
will
include one or more additives, such as diluents, carriers, excipients,
stabilizers or
the like. In addition, various biologic compounds such as antibodies, cellular
adhesion factors, growth factors, and the like, may be used to contact and/or
bind
delivery agents of choice (e.g., pharmaceuticals or other biological factors)
to the
scaffold of the present invention.
[00045] Synthetic polymers can also be modified in vitro before
use, and can carry growth factors and other physiologic agents such as peptide
and steroid hormones, which promote proliferation and differentiation. The
polyglycolic acid polymer undergoes biodegradation over a four month period;
therefore as a cell delivery vehicle it permits the gross form of the tissue
structure
to be reconstituted in vitro before implantation with subsequent replacement
of
the polymer by an expanding population of engrafted cells.
[00046] The polymeric matrix may be shaped into any number of
desirable configurations to satisfy any number of overall systems, geometries,
or
space restrictions. For example, in the use of the polymeric matrix for
bladder
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reconstruction, the matrix may be shaped to conform to the dimensions and
shapes of the whole or a part of a bladder. Furthermore, the polymeric matrix
may
be shaped in different sizes and shapes to conform to the bladders of
differently
sized patients. Optionally, the polymeric matrix should be shaped such that
after
its biodegradation, the resulting reconstructed bladder may be collapsible
when
empty in a fashion similar to a natural bladder. The polymeric matrix may also
be
shaped in other fashions to accommodate the special needs of the patient. For
example, a previously injured or disabled patient may have a different
abdominal
cavity and may require a bladder reconstructed to adapt to fit it.
Furthermore, the
portion of a laminarly arranged luminal organ to which the polymeric matrix
can
be conformed may be relatively minor. For example, 70% to 80%, or more, of the
luminal organ could be replaced using the methods and materials of the present
invention.
[00047] Recent publications have discussed seeding a supporting
matrix with cells for purposes of tissue regeneration in such organs as the
bladder.
A. Atala, in "Tissue Engineering for Bladder Substitution," World J. Urol. 18:
364-70, 365 (2000), refers to techniques all involving the use of "cells that
are
dissociated and expanded in vitro, reattached to a matrix, and implanted."
Specifically, the article describes a "system ... which does not use any
enzymes
or serum and has a large expansion potential." J. Yoo et. al., in "Bladder
Augmentation Using Allogeneic Bladder Submucosa Seeded with Cells,"
Urology 51:221-225 (1998), used urothelial and smooth muscle cells that were
harvested and expanded from dog to seed allogeneic bladder submucosa. U.S.
Patent No. 6,576,019 discloses methods and devices involving "cell
populations"
that have been isolated and cultured in vitro to increase the number of cells
available for seeding. These approaches are not based on directly seeding a
polymeric matrix with use minced tissue that has not been cultured in vitro.
Patent
No. EP1410811 discusses the use of minced tissue to seed a biocompatible
scaffold for purposes of repairing and or regenerating diseased or damaged
tissue.
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Nowhere in the patent, however, is the invention applied to the regeneration
of
full organs.
[00048] The polymeric matrix of the present invention includes a
biocompatible scaffold having at least a portion in contact with a minced
tissue
suspension. The minced tissue suspension can be disposed on the outer surface
of
the scaffold, on an inner region of the scaffold, and any combination thereof,
or
alternatively, the entire scaffold can be in contact with the minced tissue
suspension.
[00049] The tissue can be obtained using any of a variety of
conventional techniques, such as for example, by biopsy or surgical removal.
Preferably, the tissue sample is obtained under aseptic conditions. Once a
sample
of living tissue has been obtained, the sample can then be processed under
sterile
conditions to create a suspension having at least one minced, or finely
divided,
tissue particle. The particle size and shape of each tissue fragment can vary,
for
example, the tissue size can be in the range of about 0.1 and 3 mm3, in the
range
of about 0.5 and 1 mm3, in the range of about 1 to 2 mm3, or in the range of
about
2 to 3 mm3, but preferably the tissue particle is less than 1 mm3. The shape
of the
tissue fragments can include slivers, strips, flakes or cubes as examples.
Some
methods include mechanical fragmentation or optical/laser dissections.
[00050] The tissue samples used in the present invention are
obtained from a donor (autogeneic, allogeneic, or xenogeneic) using
appropriate
harvesting tools. The tissue samples can be finely minced and divided into
small
particles either as the tissue is collected, or alternatively, after it is
harvested and
collected outside the body. Mincing the tissue can be accomplished by a
variety of
methods. In one embodiment, the mincing is accomplished with two sterile
scalpels using a parallel direction, and in another embodiment, the tissue can
be
minced by a processing tool that automatically divides the tissue into
particles of
a desired size. In one embodiment, the minced tissue can be separated from the
physiological fluid and concentrated using any of a variety of methods known
to
those having ordinary skill in the art, such as for example, sieving,
sedimenting or
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centrifuging. In embodiments where the minced tissue is filtered and
concentrated, the suspension of minced tissue preferably retains a small
quantity
of fluid in the suspension to prevent the tissue from drying out. In another
embodiment, the suspension of minced tissue is not concentrated, and the
minced
tissue can be directly delivered to the site of tissue repair via a high
concentration
tissue suspension or other carrier such as for example, a hydrogel, fibrin
glue, or
collagen. In this embodiment, the minced tissue suspension can be covered by
any
of the biocompatible scaffolds described above to retain the tissue fragments
in
place.
[00051] The minced tissue can then be distributed onto a scaffold
using a cell spreader or other tools known in the art. The minced tissue can
be
dispersed onto a scaffold in one of several ways. In one example, a biopsy of
tissue sample comprising of full thickness of the bladder can be obtained.
Tissue
can be minced as a whole and distributed on the scaffold. In a second example,
a
partial thickness biopsy of tissue sample can be obtained and minced as a
whole
and distributed on the scaffold. The difference in these two methods is the
proportion of the urothelial cells to other cells, for example, smooth muscle
cells.
A third example includes separating the urothelial layer and seromuscular
layer
and subsequently mincing the layers separately before distributing each onto
to
surfaces of the scaffold. In a fourth example, the urothelial minced tissue
can be
distributed on a scaffold seeded with isolated smooth muscle cells. In a fifth
example, the minced smooth muscle tissue can be combined with a scaffold
seeded with isolated urothelial cells. In a sixth example, the urothelial and
or
smooth muscle minced tissue can be combined with stem cells seeded on the
scaffold.
[00052] The minced tissue has at least one viable cell that can
migrate from the tissue fragment onto the scaffold. The tissue contains an
effective amount of cells that can migrate from the tissue fragment and begin
populating the scaffold. In one embodiment, the minced tissue particles can be
formed as a suspension in which the tissue particles are associated with a
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physiological buffering solution. Suitable physiological buffering solutions
include, but are not limited to, saline, phosphate buffer solution, Hank's
balanced
salts, Tris buffered saline, Hepes buffered saline and combinations thereof.
In
addition the tissue can be minced in any standard cell culture medium known to
those having ordinary skill in the art, either in the presence or absence of
serum.
Prior to depositing the suspension of minced tissue on the scaffold or at the
site of
tissue/organ injury, the minced tissue suspension can be filtered and
concentrated,
such that only a small quantity of physiological buffering solution remains in
the
suspension.
[00053] The minced tissue fragments may be contacted with a
matrix-digesting enzyme to facilitate cell migration out of the extracellular
matrix
and into the scaffold material. Suitable matrix-digesting enzymes that can be
used
in the present invention include, but are not limited to, collagenase,
chondroitinase, trypsin, elastase, hyaluronidase, peptidase, thermolysin, and
protease.
EXAMPLE
[00054] Example 1: Healthy intact bladder tissue was be obtained
from a porcine source. The bladder tissue was dissected open, and
intravesicular
fluid within the bladder was aspirated out. The bladder tissue was then rinsed
three times with phosphate buffered saline (PBS), and partial thickness
biopsies
were obtained from the bladder consisting of the urothelium layer, submucosa
and
a portion of the smooth muscle layer. The biopsied tissue was minced to a fine
paste. This tissue paste was then distributed evenly on a 5mm punch of
bioresorbable scaffold such that the minced tissue paste completely covered
the
scaffold. The scaffold loaded with minced tissue was implanted subcutaneously
into severe combined immunodeficiency (SCID) mice for 4 weeks. Hematoxylin
and Eosin (H/E) stained histological sections were analyzed for cell
migration,
distribution and organization within and around the scaffolds, and for the
nature
of matrix formed. Figure 3 shows the extent of cell migration into the polymer
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scaffolds from the minced bladder tissue fragments. Clusters of urothelial
cells are
observed surrounded by smooth muscle cells. The size of the organized clusters
range from small ones with central urothelial clusters (Fig. 3A), to larger
ones
with a central cavity (Fig. 3B). As these clusters grew they also began to
coalesce
to form a larger structure (Fig. 3C) with well organized urothelial cell
layers
surrounded by smooth muscle like cell layer with a central cavity. These
structures resemble the organization seen in typical normal bladder. These
figures
demonstrate that the cells are able to migrate from the minced tissue into the
scaffolds and are able to segregate and reorganize themselves into bladder
like
structures.
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