U.S. patent application number 10/420561 was filed with the patent office on 2004-01-15 for engineered muscle.
Invention is credited to Bowlin, Gary L., Simpson, David G., Wnek, Gary.
Application Number | 20040009600 10/420561 |
Document ID | / |
Family ID | 23524908 |
Filed Date | 2004-01-15 |
United States Patent
Application |
20040009600 |
Kind Code |
A1 |
Bowlin, Gary L. ; et
al. |
January 15, 2004 |
Engineered muscle
Abstract
A muscle implant includes an extracellular matrix, tendon and
muscle cells. The extracellular matrix is made of a matrix of
electroaerosol polymer droplets. Cardiac and smooth muscles may be
formed by depositing an extracellular matrix onto a mandrel, the
extracellular matrix comprising a polymer helically wound around
the mandrel at predetermined pitches.
Inventors: |
Bowlin, Gary L.;
(Mechanicsville, VA) ; Wnek, Gary; (Midlothian,
VA) ; Simpson, David G.; (Mechanicsville,
VA) |
Correspondence
Address: |
JOHN S. PRATT, ESQ
KILPATRICK STOCKTON, LLP
1100 PEACHTREE STREET
SUITE 2800
ATLANTA
GA
30309
US
|
Family ID: |
23524908 |
Appl. No.: |
10/420561 |
Filed: |
April 22, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10420561 |
Apr 22, 2003 |
|
|
|
09386273 |
Aug 31, 1999 |
|
|
|
6592623 |
|
|
|
|
60121628 |
Feb 25, 1999 |
|
|
|
Current U.S.
Class: |
435/395 ;
424/484 |
Current CPC
Class: |
C08L 67/04 20130101;
C08L 89/06 20130101; A61L 27/3873 20130101; A61L 27/24 20130101;
A61L 27/34 20130101; A61L 2430/30 20130101; A61L 27/3817 20130101;
A61L 27/383 20130101; A61L 27/507 20130101; A61L 27/18 20130101;
A61L 27/18 20130101; A61L 27/34 20130101; A61F 2/08 20130101; A61L
27/3826 20130101 |
Class at
Publication: |
435/395 ;
424/484 |
International
Class: |
C12N 005/00 |
Claims
What is claimed is:
1. An extracellular matrix comprising a matrix of electroaerosol
droplets.
2. An extracellular matrix as described in claim 1, wherein the
droplets are discharged from one or more electrically charged
orifices onto a grounded substrate to form the matrix.
3. An extracellular matrix as described in claim 1, wherein the
matrix is treated with cross-linking agents whereby the droplets
are cross-linked.
4. An extracellular matrix as described in claim 1, wherein the
droplets are less than 10 .mu.m in diameter.
5. An extracellular matrix as described in claim 1, wherein the
droplets are comprised of collagen.
6. A method of manufacturing an extracellular matrix comprising:
streaming an electrically-charged polymer solution on to a grounded
target substrate under conditions effective to deposit polymer
droplets on said substrate to form an extracellular matrix.
7. A method of claim 6, wherein the polymer is extruded from a
capillary pipette.
8. A method of claim 6, wherein the polymer droplets form a
three-dimensional matrix.
9. A method of claim 6, wherein the polymer comprises collagen.
10. A method of claim 6, further comprising depositing a gel of
aligned collagen fibers on said extracellular matrix.
11. A method of forming a vascular prosthesis comprising: providing
a mandrel about which the prosthesis will be formed, forming an
extracellular matrix layer comprising winding a polymer fiber
around the mandrel in a helical manner at a predetermined pitch,
and depositing muscle cells onto the extracellular matrix.
12. The method described in claim 11, further comprising forming a
plurality of the extracellular matrix layers around the
mandrel.
13. The method described in claim 11, wherein the mandrel is
round.
14. The method described in claim 11, wherein the mandrel is in the
shape of a predetermined blood vessel.
15. The method described in claim 11, wherein the predetermined
pitch of the polymer fiber is different for each adjacent layer in
the prosthesis.
16. The method described in claim 11, wherein the extracellular
matrix comprises collagen gel.
17. The method described in claim 11, where in the extracellular
matrix comprises electrospun fibers.
18. The method described in claim 11, where in the extracellular
matrix comprises electroaerosol droplets.
Description
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 09/386,273, filed on Aug. 31, 1999. This
application also claims priority, in part, from U.S. Provisional
Application No. 60/121,628, filed on Feb. 25, 1999.
[0002] This invention relates to a muscle implant and an
extracellular matrix for the implant designed for transplantation.
The implant is both a functional and structural replacement for
dysfunctional muscle tissue.
BACKGROUND OF THE INVENTION
[0003] Muscle abnormalities are a fact of life whether they resuit
from a developmental anomaly or from a traumatic injury or for any
other reason. Structural defects to striated muscle tissue range
from relatively functionally benign to profoundly debilitating
disorders. In any circumstance, the condition can affect the
patient on a number of different levels. For example, structural
defects to the musculature of the face may have a minor impact on
the ability of a patient to survive. However, even minor cosmetic
defects of the muscle of the face can have substantial
psychological implications.
[0004] In addition to the striated muscle abnormalities noted
above, cardiovascular muscle is also subject to deterioration and
disease. Congenital malformations of the heart are also common.
Conventional surgical techniques are fundamentally unable to
adequately restore the subtle structural and functional
relationships that exist in a healthy heart. An intact heart has an
elaborate three-dimensional structure that insures the orderly
propagation of electrical signals and the coordinated contraction
of the ventricular wall. If the heart muscle is to be effectively
repaired, the three-dimensional organization must be addressed at
the cellular level.
[0005] Very few alternative technologies exist for the
reconstruction of dysfunctional skeletal muscle tissue. Attempts to
fabricate such tissue have been generally confined to experiments
in which skeletal muscle cells are trapped in a collagen gel. In
these experiments, the cells have been seeded onto the exterior of
a collagen gel or literally enveloped within the gel as it is
polymerized. Subsequently, the cells are allowed to differentiate
within the random, "three-dimensional" environment of the collagen
gel. The distribution of cells within these collagen gels
represents a limiting factor in these constructions. When muscle
cells are seeded onto the exterior of a collagen gel they typically
remain concentrated on the peripheral regions of the gel.
Experiments in which muscle cells are directly incorporated into a
collagen gel as it undergoes polymerization have yielded more
densely and uniformly populated cultures; however, these
constructions remain less dense than their in vivo counterparts.
More importantly, the implants produced in conventional tissue
culture are composed of muscle cells that lack a uniform alignment
or orientation. The random nature of the cells within these
sparsely populated implants limits the utility of that tissue and
its ability to function as an ordinary muscle.
[0006] Additional constraints that must be addressed in designing
an implant include the mechanical stability of the implant. The
implant must have enough structural integrity to withstand manual
manipulation, the surgical procedures and the mechanical
environment of the intact tissue. Intact skeletal muscle is
surrounded in vivo by multiple layers of a dense connective tissue
that compartmentalizes the muscle and reinforces the structure of
the tissue. Mimicking the specific structure of this arrangement in
vitro is difficult, because any dense, investing material will tend
to limit nutrient diffusion, oxygen transport and the removal of
metabolic waste products away from the cells. Components made from
artificial materials such as polyester mesh have been used with
some success to increase the strength of the cultures while
allowing them to retain a substantial portion of their elastic
properties. However, the incorporation of synthetic materials into
an implant can increase the likelihood that it will initiate an
inflammatory response in vivo.
[0007] Cardiac tissue lacks a dense connective tissue. However, the
muscle cells of the heart are organized into a complicated lattice.
The individual muscle cells of the heart have a rod-like cell
shape. Like skeletal muscle, they are oriented along a common axis
in a complex three-dimensional pattern. Each cell of the heart is
invested with a basement membrane and interconnected to its
neighbors by a complex matrix of collagen fibrils. The three
dimensional pattern of the cell layers within the heart is critical
for the orderly propagation of electrical signals and the
coordinate contraction of the ventricular wall.
[0008] Smooth muscle surrounds the supports of many of the hollow
organs. For example, in the gut it surrounds the stomach and
intestinal track. Contraction of this muscle mixes food and propels
it along the digestive track. In the cardiovascular system smooth
muscle cells surround the walls of the arteries and large veins and
functions to control the caliber of the vessels. Smooth muscle
lacks the nearly uniform cell shape and lattice like distribution
of skeletal and cardiac muscle cells. However, smooth muscle cells
do exhibit an elongated, bipolar cell shape. As a population they
are organized along a similar axis in a series of overlapping
cellular layers. This pattern of organization allows smooth muscle
to exert contractile forces in a complex pattern.
SUMMARY OF THE INVENTION
[0009] Accordingly, it is an object of the present invention to
overcome the foregoing drawbacks and to provide a muscle implant to
a host in need thereof. The implant can be used in a variety of
ways including to augment existing muscle, correct muscle
deficiencies or as a functional and structural replacement for
dysfunctional muscle tissue. Further, the invention includes a
method for manufacturing the muscle implant.
[0010] In one embodiment, a muscle implant includes an
extracellular matrix made of electrospun fibers and muscle cells
disposed on the matrix. In another embodiment, the muscle implant
comprises an extracellular matrix made of electrospun fibers for
supporting muscle, a tendon made of extruded fibers, and a muscle
cell layer that is disposed on the extracellular matrix. The muscle
cell layer can be multilayered. In other variations, the
electrospun fibers may be cross linked. Also, an oriented layer of
collagen can be deposited onto the extracellular matrix so that the
muscle cells are disposed onto the oriented layer of collagen.
[0011] In another embodiment, the invention includes an
extracellular matrix for supporting muscle comprising a matrix of
electrospun fibers. The fiber is discharged from an electrically
charged orifice onto a grounded substrate to form the matrix. The
matrix can also be treated with cross linking agents so that the
fibers are cross linked.
[0012] The invention also includes a method of manufacturing an
extracellular matrix comprising extruding electrically charged
polymer solution onto a grounded target substrate under conditions
effective to deposit polymer fibers on the substrate to form an
extracellular matrix. The extruded polymer may form a
three-dimensional matrix. The extracellular matrix may farther
include a gel of aligned collagen fibers deposited thereon.
[0013] In a further embodiment, the invention includes a method of
forming a muscle fascial sheath by providing an electrically
grounded substrate. There is further provided a reservoir of
collagen solution wherein the reservoir has an orifice that allows
the collagen solution to leave the reservoir. The collagen solution
is electrically charged and then streamed onto the substrate to
form a muscle fascial sheath.
[0014] In still a further embodiment, the invention includes a
method of layering muscle cells on an extracellular matrix. The
method includes providing an extracellular matrix and then placing
the extracellular matrix inside a rotating wall bioreactor. A
culture medium is loaded into the bioreactor wherein the medium
comprises muscle cells. The bioreactor is then run until muscle
cells attach to the extracellular matrix. Alternatively, the muscle
cells attached to the extracellular matrix form multiple
layers.
[0015] An additional embodiment of the invention includes an
extracellular matrix comprising a matrix of electroaerosol
droplets. The droplets are discharged from an electrically charged
orifice onto a grounded substrate to form the matrix. The matrix
can also be treated with cross linking agents so that the droplets
are cross linked. Additionally, the invention includes a method of
manufacturing an extracellular matrix comprising streaming an
electrically charged polymer solution onto a grounded target
substrate under conditions effective to deposit polymer droplets on
the substrate to form an extracellular matrix. The polymer droplets
may form a three dimensional matrix. The extracellular matrix may
further include a gel of aligned collagen fibers deposited
thereon.
[0016] Still further, the invention includes a method of forming a
vascular prosthesis comprising providing a mandrel about which the
prosthesis will be formed. An extracellular matrix is formed by
winding a polymer fiber around the mandrel in a helical manner at a
predetermined pitch. Muscle cells are then deposited onto the
extracellular matrix. This method may further comprise forming a
plurality of the extracellular matrix layers around the mandrel.
Further, the mandrel may be round or it may be in the shape of a
predetermined blood vessel.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is a scanning electron micrograph of an electrospun
matrix of fibers.
[0018] FIGS. 2A and 2B are schematic drawings of electrospinning
devices including the electrospinning equipment and a rotating wall
bioreactor.
[0019] FIG. 3 is a scanning electron micrograph of a thin gel of
aligned collagen.
[0020] FIG. 4A and 4B are scanning electron micrographs of muscle
cells deposited on aligned collagen gel and a random collagen gel
respectively.
[0021] FIGS. 5A and 5B are schematic representations of how a
muscle implant may be fabricated.
[0022] FIG. 6 is a schematic representation of one type of
hypertrophy mechanism.
[0023] FIG. 7 is a schematic representation of a form into which
the polymer stream may be directed.
[0024] FIG. 8 is a scanning electron micrograph of the external
surface of an electroaerosol PGA/PLA (50/50) scaffolding.
[0025] FIG. 9 is a scanning electron micrograph of a cross
sectional view of an electroaerosol PGA/PLA (50/50) scaffolding
produced on a 18 gauge needle.
[0026] FIG. 10 is a scanning electron micrograph cross sectional
view of the mid-wall of the electroaerosol PGA/PLA (50/50)
scaffolding produced on an 18 gauge needle. This figure illustrates
a high magnification of the same construct as shown in FIG. 9.
[0027] FIG. 11 is a scanning electron micrograph of the luminal
surface of an electroaerosol PGA/PLA (50/50) scaffolding produced
on an 18 gauge needle.
[0028] FIG. 12 is a schematic representation of five different
layers used to create one embodiment of a vascular prosthesis.
[0029] FIG. 13 illustrates a winding apparatus used to manufacture
a vascular prosthesis.
[0030] FIG. 14 is a schematic of a mandrel with a thin wall
tube.
[0031] FIG. 15 is a schematic of a mandrel holder and mandrel.
[0032] FIG. 16 is a schematic of a bioreactor composed of tubing
connectors, tubing and polycarbonate walls.
[0033] FIG. 17 is a scanning electron micrograph of a cross-section
of a biomimicking vascular prosthetic.
[0034] FIG. 18 is a scanning electron micrograph showing oriented
smooth muscle cells seeded on the external surface of a
biomimicking vascular prosthetic.
[0035] FIG. 19 is a scanning electron micrograph showing oriented
smooth muscle cells seeded on the external surface of a
biomimicking vascular prosthetic.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT
[0036] An engineered muscle implant can comprise one or more of the
following components. They are an engineered extracellular matrix,
an engineered tendon, and engineered muscle cells. Each component
will be discussed in detail separately and then in combination with
respect to their assembly.
[0037] A. Engineered Extracellular Matrix
[0038] In a normal human anatomy, muscles are bundles of oriented
muscle cells that are encased in an outer protective coating which
we refer to as an extracellular matrix. In skeletal muscle, this
outer coating is referred to as the fascial sheath. The fascial
sheath gives shape and support to the skeletal muscle. The fascial
sheath is the extracellular matrix or scaffolding that maintains
the integrity of the muscle. Smooth muscle and cardiac muscle are
also supported by an extracellular matrix. The cells of smooth and
cardiac tissue are both interconnected by a network of collagen
fibrils. Smooth muscle lacks a defined fascial sheath. The entire
surface of the heart is enclosed in a tough outer coating of
connective tissue composed of collagen called the pericardium.
[0039] Another type of extracellular matrix is a nerve guide. A
nerve guide (or nerve guidance channel) is a tube structure, small
bore, which is used to connect the distal and proximal stumps of
severed nerve segments to aid in the guidance of the regenerating
axons. A nerve guide allows for more direct rewiring of the axons,
a direct route for regeneration, and prevents the random axon
outgrowths from going off on a tangent and possibly not rewiring. A
nerve guide also allows for a controlled environment within the
lumen (neuroactive molecules released by the axon stumps) to
aid/promote axon regeneration at the same time preventing scar
tissue/surrounding tissue ingrowth. The scar tissue/tissue ingrowth
would obviously block/prevent axon regeneration.
[0040] The engineered extracellular matrix of the present invention
can be custom constructed to meet the requirements of skeletal,
smooth or cardiac muscles. In preferred embodiments, the
extracellular matrix is fabricated by electrospinning polymer
fibers or electroaerosoling polymer droplets (synthetic or natural)
to form a matrix directly onto a substrate; or to form a matrix
directed onto a substrate or form (mold), or other surface such as
the central cylinder of a RCCS Bioreactor (Synthecon).
[0041] There are a number of different kinds of bioreactors,
devices designed to provide a low-shear, high nutrient perfusion
environment, available on the market. Until recently, most of the
available bioreactors maintained cells in suspension and delivered
nutrients and oxygen by sparging, through the use of impellers, or
other means of stirring. The RCCS bioreactor is a rotating wall
bioreactor. It consists of a small inner cylinder, the substrate
for the electrospinning process, positioned inside a larger outer
cylinder. Although the electrospun or electroaerosol matrix can be
fabricated on the inner cylinder, other locations within the
bioreactor also may be used for placement of the matrix for
seeding. The gap between the inner and outer cylinders serves as
the culture vessel space for cells. Culture medium is oxygenated
via an external hydrophobic membrane. The low shear environment of
the Synthecon RCCS bioreactor promotes cell-cell and
cell-extracellular matrix (ECM) interactions without the damage or
"washing away" of nutrients that occurs with active stirring or
sparging. Typically, the RCCS device is operated at rotation rates
of 8 up to 60 RPM, as required to maintain cells in suspension, and
at less than 8 RM (preferably 2-3 RPM) for cultures immobilized
along the center shaft of the vessel. The Synthecon bioreactor can
be used in a standard tissue culture incubator.
[0042] 1. Electrospun Extracellular Matrix
[0043] The electrospinning process can be used to produce a dense,
mat-like matrix of oriented and/or unoriented polymer fibers (FIG.
1). "Electrospinning" means a process in which fibers are formed
from a solution or melt by streaming an electrically charged
polymer solution or melt through an orifice. Electrospinning has
been used in the textile industry to produce ultra thin layers of
fiber fabrics (continuous multi filaments) and dense mats of
material. The polymer fibers formed by this technique are in the
40-500 nanometer diameter range. The mechanical properties(i.e.,
strength), porosity, and weight of the fabrics produced by
electrospinning can be controlled by regulating the processing
conditions, the materials used in the fabrication process and the
thickness of the deposited material. Gibson, P. W., et al.,
Electrospun Fiber Mats: Transport Properties, 1998 AIchE J.; Deshi,
J., et al., Electrospinning Process and Applications of Electrospun
Fibers, 1996 J. Electrostatics 35:151.
[0044] An extracellular matrix of electrospun fibers in accordance
with the present invention can be produced analogously. While any
polymer can be used, it is preferable to electrospin natural
polymer fibers such as collagen fibers. Various effective
conditions can be used to electrospin a collagen matrix. While the
following is a description of a preferred method, other protocols
can be followed to achieve the same result. Referring to FIGS. 2A
and 2B, in electrospinning collagen fibers, micropipettes 10 are
filled with a solution of collagen and suspended above a grounded
target 11, for instance, a metal ground screen placed inside the
central cylinder of the RCCS bioreactor. A fine wire 12 is placed
in the solution to charge the collagen solution in each pipette tip
13 to a high voltage. At a specific voltage determined for each
solution and apparatus arrangement, the collagen solution suspended
in the pipette tip is directed towards the grounded target. This
stream 14 of collagen forms a continuous filament that, upon
reaching the grounded target, collects and dries to form a
three-dimensional, ultra thin, interconnected matrix of collagen
(fabric). Minimal electrical current is involved in this process,
and, therefore, the streaming process does not denature the
collagen, because there is no expected temperature increase in the
collagen solution during the procedure.
[0045] 2. Electroaerosol Extracellular Matrix
[0046] Like the electrospinning process, an electroaerosoling
process can be used to produce a dense, matte-like matrix of
polymer droplets. "Electroaerosoling" means a process in which
droplets are formed from a solution or melt by streaming an
electrically charged polymer solution or melt through an orifice.
The electroaerosoling process is a modification of the
electrospinning process in that the electroaerosol process utilizes
a lower concentration of the polymer during the procedure. Instead
of producing a polymeric splay (fibers) at the charge tip of the
splay nozzle, small droplets are formed. These droplets then travel
from the charged tip to the grounded substrate to form a sponge
like matrix composed of fused polymeric droplets--all on the order
of less than 10 microns in diameter. FIGS. 8 to 11 are scanning
electron micrographs of an electroaerosol matrix of 50:50 PGA/PLA
extracellular matrix.
[0047] As with the electrospinning process described earlier, the
electroaerosol process can be carried out using various effective
conditions. The same apparatus that is used in the electrospinning
process, for instance as shown in FIGS. 2A and 2B, is utilized in
the electroaerosol process. The differences from electrospinning
include the concentration of the polymer placed in solution in the
micropipette reservoir and/or the voltage used to create the stream
of droplets. Obviously, those of skill in the art recognize that
changes in the concentration of polymer solution would require
modification of the specific voltage to obtain the formation and
streaming of droplets from the tip of a pipette.
[0048] 3. Electrospin/Electroaerosol Process Variations
[0049] Various polymers can be used alone, or in combination, to
produce the electrospun and/or electroaerosol matrices. In
preferred embodiments, collagen is used to form the extracellular
matrix. Any suitable collagen can be used, including, types I
through XIV. In preferred embodiments, types I and III are used.
Collagens are available from commercial sources, or they can be
prepared according to methods known in the art.
[0050] A variety of material can be supplemented into the
electrospinning or electroaerosoling solution. DNA coding for
desired products (vectors) can be mixed into the polymeric solution
for incorporation into the tissue-engineered scaffold. Upon
consumption/reorganization of the scaffolding by the seeded cells,
they may incorporate the vector (i.e. genetic engineering) into
their DNA and produce a desired affect. The DNA can be in any form
which is effective to enhance its uptake into cells. For example,
it can be naked (e.g., U.S. Pat. Nos. 5,580,859; 5,910,488) or
complexed or encapsulated (e.g., U.S. Pat. Nos. 5,908,777;
5,787,567). Similar to adding DNA, it may be possible to
incorporate growth factors or other chemotaxins such as angiogenic
factors into the electrospun or electroaerosol matrix to aid in
tissue regeneration.
[0051] The electrospinning or electroaerosoling process can be
manipulated to meet the specific requirements for any given
application. The micropipettes can be mounted on a frame that moves
in the x, y and z planes with respect to the grounded substrate.
The micropipettes may be mounted all around a grounded substrate
for instance a tubular mandrel. In this way, the collagen or other
polymer streamed from the micropipette can be specifically aimed or
patterned. Although the micropipettes can be moved about manually,
preferably, the frame onto which the micropipettes are mounted is
controlled by a microprocessor and a motor that allows the pattern
of streaming collagen to be predetermined by a person making a
specific matrix. For instance, collagen fibers or droplets can be
oriented in a specific direction, they can be layered, or they can
be programmed to be completely random and unoriented.
[0052] In the electrospinning process, the polymer stream can
branch out to form fibrils of the polymer. The degree of branching
can be varied by many factors including, but not limited to,
voltage, ground geometry, distance from micropipette tip to the
substrate, diameter of micropipette tip, polymer concentration,
etc. These variables are well-known to those of skill in the art of
electrospinning microfiber textile fabrics.
[0053] The geometry of the grounded target can be modified to
produce a desired matrix. In a preferred embodiment, a rotating
wall bioreactor is used. The grounded target is a cylinder that
fits inside the inner cylinder in the electrospinning or
electroaerosoling process. By varying the ground geometry, for
instance having a planar or linear or multiple points ground, the
direction of the streaming collagen can be varied and customized to
a particular application. For instance, a grounded target
comprising a series of parallel lines can be used to orient
electrospun collagen in a specific direction. The grounded target
may be a cylindrical mandrel whereby a tubular matrix is formed.
Most preferably, the ground is a variable surface that can be
controlled by a microprocessor that dictates a specific ground
geometry that is programmed into it Alternatively, for instance,
the ground may be mounted on a frame that moves in the x, y, and z
planes with respect to a stationary micropipette tip streaming
collagen. The grounded target 11 in FIG. 2B is shown as being able
to oscillate along its longitudinal axis.
[0054] The substrate onto which the collagen is streamed can be the
grounded target itself or it can be placed between the micropipette
tip and the grounded target. The substrate can be specifically
shaped, for instance in the shape of a heart or a part thereof or a
vascular graft, to substitute or replace a specifically shaped
muscle.
[0055] Through modification of a substrate shape and by programming
the specific orientation and density of the electrospun or
electroaerosol polymer, a very complex muscle pattern can be
replicated to enable a specific muscle form for a specific
application to be created. Alternatively, for example, collagen may
be streamed into a preselected form. The thickness and attributes
of the matrix may be preselected to form cartilage, dentin packing,
or other similar prosthesis. A schematic example of this type of
application is shown in FIG. 7.
[0056] Other variations on electrospinning and electroaerosoling
include:
[0057] 1. Using different solutions (e.g.,. collagen I and III) to
produce two or more different fibers or droplets simultaneously
(matrix fiber or droplet array). In this case, the single component
solutions can be maintained in separate reservoirs.
[0058] 2. Using mixed solutions (e.g., collagen I and III) in the
same reservoir(s) to produce fibers or droplets composed of
multiple polymers (fiber composition "blends"). Nonbiological but
biologically compatible material can be mixed with a biological
molecule such as collagen, e.g., PVA, PLA, PGA, PEO, etc.
[0059] 3. Utilizing multiple potentials applied for the different
solutions or even the same solutions.
[0060] 4. Having two or more different geometric grounded targets
(i.e. small and large mesh screens).
[0061] All these variations can be done separately or in
combination with each other to produce a wide variety of
electrospun and electroaerosol extracellular matrices.
[0062] Additionally, a matrix can be formed that includes both
electrospun and electroaerosol polymers. In other words, a
combination of fibers and droplets may be beneficial for some
applications envisioned by a particular structure to be mimicked.
This combination of fibers and droplets can be obtained by using
the same micropipette and solution in varying the electrical
charge; varying the distance from the grounded substrate; varying
the polymer concentration in the reservoir; using multiple
micropipettes--some from streaming fibers and others for streaming
droplets; or any other variations to the method that could be
envisioned by those of skill in this art. The fibers and droplets
could be layered or mixed together in same layers. The possible
combinations of the electrospinning and electroaerosoling processes
are virtually unlimited.
[0063] The stability, rigidity, and other attributes of the
electrospun or electroaerosol matrix can be regulated by the degree
to which it is chemically modified. The electrospun or
electroaerosol matrix may be used in its unmodified state, or it
may be modified in accordance with the requirements of a specific
application. Modifications to the matrix can be made during the
electrospinning or electroaerosoling process or after it is
deposited. Cross-linking agents such as carbodiimide EDC
(1-ethyl-3(3 dimethyl aminopropyl)), carbodiimide hydrochloride,
NHS (n-hydroxysuccinimide), or UV light can be-used, e.g., to
stabilize the fascial sheath against proteolytic attack, and/or to
increase the stability of collagen gels. See, e.g., Van Wachem, et
al., 1996 Myoblast seeding in a collagen matrix evaluated in vitro,
J. Biomedical Materials Res. 30:353-60.
[0064] 4. Biomimicking a Vascular Prosthesis
[0065] One type of engineered extracellular matrix and/or
engineered muscle (media component) that can be fabricated is a
vascular prosthesis. A successful prosthesis may be accomplished
best if the prosthesis biomimics the artery or other vessel or
branch that is to be replaced. One of the keys to creating this
prosthesis is the orientation and layering of the extracellular
matrix that will be the scaffolding for the prosthesis.
[0066] The vascular prosthesis includes winding thin collagen
fibers (<150 .mu.m) in a collagen gel and/or in a matrix of
electrospun or electroaerosol polymer(s). Although any polymer may
be used, natural polymers are preferable. In a specific example
using electrospun PLA fibers, a matrix of the electrospun fibers
are formed around a thin mandrel. The collagen fibers are then
wound in a helical manner around (embedded in) the matrix of
electrospun fibers. The composite of collagen fiber and electrospun
matrix creates a residual stress environment similar to that found
in native arteries. Multiple layers of the wound fibers and
electrospun matrices can be assembled with each layer of the
multi-layer prosthesis oriented at a specific pitch to mimic the
specific vasculature being replaced. The specific application will
also determine the number and thickness of the layers. In one
embodiment addressed to building a small diameter artery, each
layer is cylindrical with each possessing a collagen fibril
bundle/smooth muscle cell structure wound (spiral arrangement) at
varying pitches (FIG. 12 displays a 5 layer prostheses). All
historical attempts at developing a vascular construct from
collagen and cellular components have been unsuccessful for two
reasons: 1) the collagen was added in the form of a gel containing
smooth muscle cells and allowed to form around a solid mandrel (no
specific orientation) and 2) the procedures were maintained at
static conditions.
[0067] The polymer (preferably collagen and/or other natural
polymers) that makes up each layer can be oriented using the
electrospinning and/or electroaerosoling techniques described
herein. Alternatively, as described in Example 4, the layers may be
formed on a mandrel by mechanical winding of fibers about the
mandrel at specified pitches. Still further alternatively, a
combination of mechanical winding and
electrospinning/electroaerosoling may be used to assemble the
extracellular matrix.
[0068] It has been discovered that the mechanical winding of
polymer fibers in the prosthesis has an unexpected effect on muscle
cells subsequently seeded on the extracellular matrix. The residual
stress in the wound polymer (collagen) creates an energy in the
prosthesis that promotes growth of the muscle cells in the
direction of the spiral wound polymer. This residual stress also
mimics the actual stress found in natural arteries. FIGS. 17-19
display a matrix and wound fiber composite as well as the
orientation of muscle cells seeded onto the prosthesis.
[0069] Also, the specific embodiment described in Example 4 is
directed to a simple, tubular vascular graft. More complicated
shapes including tapered and/or branched vessels may also be
constructed. All that is necessary is a different-shaped mandrel to
wind the large fibers around or to orient the
electrospun/electroaerosol polymer around.
[0070] B. Engineered Tendon.
[0071] The engineered tendon, or the connective tissue struts that
anchor the engineered muscle to bone, can be assembled from
extruded collagen fibers or other suitable materials. Collagen
fibers are preferred, because collagen is less likely to be
rejected by a recipient's immune system. These fibers function in
combination with the extracelluar matrix to stabilize the overall
structural integrity of the muscle implants. Collagen fibers for
the fabrication of the engineered tendon can be extruded after
known methods. Kato, Y. P. and Silver, F. H., Formation of
Continuous Collagen Fibers: Evaluation of Biocompatibility and
Mechanical Properties, 1990 Biomaterials 11:169-75; Kato, Y. P., et
al., Mechanical Properties of Collagen Fibers: A Comparison of
Reconstituted Rat Tendon Fibers, 1989 Biomaterials 10:38-42; and
U.S. Pat. Nos. 5,378,469 and 5,256,418 to Kemp, et al..
[0072] A preferred collagen extrusion apparatus comprises a syringe
pump, microbore tubing, a dehydration trough, recirculation pump,
rinsing trough, drying chamber, heating air dryer, and a collagen
fiber winder. The syringe is filled with degassed collagen and
mounted onto a syringe pump. The collagen solution is then extruded
from the syringe, through the microbore tubing, and into a
dehydration bath (Polyethylene glycol in PBS). The formed collagen
fiber is subsequently guided through a rinsing bath (phosphate
buffered saline, PBS) and attached to a winding system within a
dryer. Once the initial fiber has been formed and attached to the
winding element, the process becomes automated and continuous. At
an extrusion rate of approximately 8 cm/minute, the extrusion
apparatus can produce fiber 1-10 meters in length and 50-250 .mu.m
in diameter. After production, the fiber diameter can be verified
through scanning electron and light microscopic evaluation. Varying
the reaction conditions controls the diameter of the collagen fiber
that is polymerized. The physical properties of the engineered
collagen fiber can be further modified and controlled by regulating
the composition of the extrusion material. The elastic properties
of the engineered tendon can be modulated by incorporated elastin,
fibrin or man made material into the collagen solution as it is
extruded. Prior to use in the engineered implant the collagen
fibers are sterilized by peracetic acid sterilization.
[0073] C. Engineered Muscle Cells
[0074] Any type of muscle cells can be used in the present
invention, including cell culture strains, transformed cells,
primary muscle cells, embryonic muscle cells, neonatal muscle
cells, embryonic stem cells, etc. Preferred cells are stem cells or
muscle cells (or muscle precursor cells) which are obtained from a
host into which the muscle will be transplanted. Barrofio, A., et
al., Identification of Self-Renewing Myoblasts in the Progeny of
Single Human Muscle Satellite Cells, 1996 Differentiation 66:47-57;
Blau, H. M. and Webster, C., Isolation and Characterization of
Human Muscle Cells, 1981 Proc. Natl. Acad. Sci 78:5623-27. The term
"primary myocytes" means muscle cells which are obtained directly
from a host animal muscle which retain the ability to differentiate
and which have been passed a minimum number of times in culture.
Such cells generally are not transformed.
[0075] The cell type to be used in the implant depends upon use and
the site of implantation that is to be reconstructed repaired or
otherwise augmented by the engineered muscle. A variety of cell
types can be used and include but are not limited to; embryonic
stem cells, bone marrow stem cells, satellite muscle cells from the
striated muscle beds, cardiac muscle cells, smooth muscle cells,
muscle cell lines, transformed cell lines and genetically
engineered cell lines. Cells isolated from fetal, neonatal and
adult tissue may be used. Fetal cardiac myocytes can be used in the
construction of the cardiac prosthesis. Cells of the C2C12 muscle
cell line can be used for the fabrication of skeletal muscle
implants. In the long run a stem cell population (adult or
embryonic) will be the ideal cell source for the fabrication of the
muscle. Stem cells are attractive for this use because they can be
engineered to become nearly any type of cell (e.g., smooth muscle,
cardiac muscle, skeletal muscle, cartilage, bone, etc). They can
but do not have to come from the patient to be treated with the
muscle implant, because even if they come from some other source
they will not invoke an immune response.
[0076] For skeletal, muscle, satellite muscle cells are derived
from a suitable, and unobtrusive, donor site on the subject who is
to receive the muscle implant. Muscle biopsies are isolated, and
the connective tissue removed by dissection. Various protocols can
be utilized to isolate satellite or other cell types from the
muscle for engineering the implant. For instance, a protocol can be
as follows: Isolated muscle tissue is minced and dissociated into a
single cell suspension, by trypsin-EDTA digestion (or other
suitable enzymes) under constant stirring. At the conclusion of the
enzymatic dissociation procedure, the digestion media is quenched
with the addition of 10% serum. Cell suspensions are washed by
centrifugation, and a sample is stored in liquid nitrogen for
future use. The balance of the isolated cells are enriched in
satellite cells by flow cytometery or through the use of
differential adhesion or through immunoseparation methods. Flow
cytometery, can be used to separate cells by size, by cell cycle
stage, or, if labeled, by cell surface markers. In differential
adhesion, cells are placed into a culture vessel for a short
incubation period to allow contaminating fibroblasts to seed out of
the solution, thus enriching the remaining cell population in
satellite cells. A longer interval of differential adhesion can be
used to seed out satellite cells so they can be purified away from
contaminating debris and muscle cells. Enriched cell fractions that
contain myogenic satellite cells can be stored at -70.degree. C. in
liquid nitrogen. Regardless of the technique used to isolate and
purify satellite cells the samples are thawed and plated into
culture vessels and assayed for myogenic potential. Cell lots that
are competent to undergo differentiation are used in the
fabrication of the muscle implant. After partial purification,
clones are assayed for myogenic potential, e.g., by plating cells
onto collagen--coated dishes and observing whether adherent cells
display the characteristics of muscle cells.
[0077] Candidate clones from the primary cell isolate are grown
under sparse culture condition (i.e. low cell density) in an
appropriate media, e.g., containing 10-15% serum, to accumulate an
adequate number of cells from which the implant can be fashioned.
Once a sufficient number of cells have been obtained (dependent
upon the size of the implant to be fabricated), they are prepared
for insertion into the bioreactor for the assembly of the
prosthetic muscle. Muscle cell differentiation in the bioreactor
can be induced by replacing the high serum content media (10-15%
serum) with low serum media.
[0078] Cells utilized in the fabricated muscle are readily amenable
to genetic manipulation. For example, genes encoding angiogentic
factors, growth factors or structural proteins can be incorporated
into the isolated cells. This can be accomplished before, during or
after the fabrication of the muscle construct. Useful genes
include, e.g., VEGF, FGF, and related genes. For introducing genes
into cells, any effective method can be used, including viral
vectors, such as adenovirus, also DNA, plasmids, etc. can be
used.
[0079] D. Assembly of an Engineered Skeletal Muscle Implant
[0080] The muscle implant can comprise three distinct components,
the extracellular matrix, the engineered tendon and the population
of muscle cells. As detailed earlier, an extracellular matrix
composed of a matrix of collagen fibers or other biologically
compatible material is prepared on the outer surface of the inner
cylinder of an RCCS bioreactor (or suitable substitute). The
structural properties of this mat of fibers are regulated by the
diameter of fibers produced, the relative concentration of
materials used in the reaction (e.g. concentration of type I to
type III collagen, or other incorporated materials), and other
reaction conditions.
[0081] In one preferred embodiment, a thin gel matrix of collagen
or other suitable matrix material can be applied over the surface
of the extracellular matrix to enhance muscle cell adhesion,
differentiation, and/or alignment. The gel matrix can be applied in
any suitable manner including electrospinning, spraying, dipping,
spreading, dropping, etc. Simpson, et al., Modulation of Cardiac
Phenotype in vitro by the Composition and Organization of the
Extracellular matrix, 1994 J. Cell Physiol. 161:89-105. In a
preferred embodiment, the collagen fibers in the thin gel are
aligned along a common axis. For example, the aligned matrix can be
produced by dipping the central cylinder core of the RCCS
bioreactor, with its electrospun coating of collagen, end-on into a
ice cold neutral stock solution-of collagen (1 mg/ml) (Type I or
type III or a mixture thereof). After a very brief interval (1-3
secs), the cylinder is removed from the solution and the excess
collagen is allowed to drain by gravity off of the distal end of
the cylinder. The orientation of the cylinder is maintained
constant throughout this process, i.e. perpendicular to the
collagen solution in which it was dipped. This allows the excess
collagen to drain off the long axis of the cylinder. The cylinder
is then placed into an incubator, e.g., set for 37.degree. C., to
allow the collagen to polymerize, e.g., sixty minutes or more.
After polymerization is complete, the aligned collagen fibers are
allowed to dry down on to the underlying facial sheath. These
procedures result in a thin layer of aligned collagen fibrils
arrayed along the axis the cylinder was drained. See FIG. 3. Other
methods for aligning the collagen may be employed, for instance,
using the described electrospinning system or using a centrifuge
after dipping the core in the collagen solution. Regardless of how
the collagen is aligned, at the conclusion of this step, the
central RCCS cylinder has a mat-like coating of electrospun
collagen fibers (the extracellular matrix) covered or coated with a
thin layer of aligned collagen.
[0082] If desired, the extracellular matrix can incorporate other
materials as well, such as polyester mesh and other synthetic
materials.
[0083] Also, as discussed earlier, the need for the thin gel of
collagen fibers may be obviated if the electrospun matrix is
sufficiently oriented during the electrospinning process. In other
words, the additional thin gel layer of oriented collagen is only
necessary if the extracellular matrix (fascial sheath in the
example of skeletal muscle) of collagen or other polymer is
unoriented.
[0084] Large diameter, extruded collagen fibers (engineered
tendons) are then applied over the aligned collagen gel. The
mechanical properties of the implant are controlled in this step at
two separate sites. First, by the thickness of the individual
extruded fibers and the number of these filaments added to the
implant. Second, by the orientation of these fibers with respect to
the long axis of prosthesis. The implant can be made more or less
stiff by applying these fibers in an undulating pattern. The large
fibers can also be attached to the matrix by only overlapping the
matrix at the distal ends, i.e., not necessarily running the entire
length of the engineered muscle. Regardless of the orientation
used, the ends of extruded fibers are allowed to project from the
distal ends of the implant. At the conclusion of this step, the
large diameter collagen fibers are allowed to dry down onto the
fibers of the aligned collagen gel. Alternative fabrication
processes can be used to further customize the mechanical
properties of the implant. For example, large diameter collagen
fibers may be laid down first followed by collagen fibers deposited
by electrospinning, followed by another layer of large diameter
collagen fibers, the aligned collagen gel and the satellite cells.
Other permutations on this assembly process are also possible.
[0085] A tendon can also be created in situ by combining tendon
fibroblasts with the synthetic muscle bed. For example, tendon
fibroblasts may also be harvested from a recipient's own tendon.
These cells are placed on the end of the muscle bed synthesized as
described herein. The tendon is allowed to grow with the muscle bed
in the bioreactor. The tendon fibroblasts are encouraged to grow in
an oriented fashion by use of the aligned substrate herein or by
other orientation methods. If this method is chosen, the extruded
collagen tendons described herein become unnecessary, although a
combination of extruded and cultured tendons may be desired for
certain applications.
[0086] In the final step of the fabrication process, the inner
cylinder with its engineered fascial sheath and overlaying layers
of aligned collagen and large diameter collagen fibers is loaded
into a RCCS bioreactor. Muscle cells, such as satellite myoblasts
isolated from the subject or compatible donor, are loaded into the
chamber and allowed to interact with the collagen-based substrate.
The RCCS bioreactor is preferably used in this step because it
provides high nutrient profusion in a very low shear environment.
However, other culture vessels can be used. Under these conditions,
it is possible to assemble a muscle cell culture comprising
multiple layers (8-12 layers in 48 hours) of aligned cells. In the
assembly of the muscle implant, cells are gradually depleted from
suspension culture and plated onto the collagen matrix, either
directly on the electrospun matrix or on the collagen gel coating,
to form the three dimensional arrangement of the engineered tissue.
Additional satellite cells are added as need to the bioreactor to
assemble additional cell layers. Once the desired mass of cells has
been plated onto the fascial sheath, they are allowed to
differentiate into myotubes, e.g., by transfer to a serum media
Also, there are artificial oxygen carriers that can be used in
vitro to increase oxygen delivery to tissues or cells in culture.
They would be mixed into the reactor with the satellite cells as
the muscle is fabricated. They basically function like red blood
cells in the sense these carriers increase the oxygen content of
the culture media.
[0087] Any suitable culture media can be used to grow the cells,
including medias comprising serum and other undefined constituents,
defined medias, or combinations thereof, RPMI, etc.
[0088] With the completion of the differentiation process, the
skeletal muscle implant is ready for transplantation into the site
of reconstruction in the subject. The implant is removed from the
central cylinder of the bioreactor. The size and thickness of the
implant is controlled at this stage of the process at two different
levels--first, by the number of cell layers assembled onto the
central cylinder and, second, by the size of the sheet of tissue
used to construct the muscle implant. This latter procedure
involves trimming (for instance, into a rectangular sheet--FIG.
5A), stacking, and rolling (FIG. 5B) the engineered muscle into the
desired configuration. Alternatively, the engineered muscle can be
directly implanted or layered for the reconstruction of, for
instance, facial muscles as a flat sheet. If the engineered muscle
is to be used to reconstruct a muscle bed of the axial skeleton, it
may be attached to the implantation site through the large diameter
collagen fibers 20 that protrude from the ends of engineered muscle
21, through the distal ends of the fascial sheath itself or through
a combination of these methods.
[0089] If the tissue is to be used to reconstruct a congenital
heart defect or repair an otherwise dysfunctional region of
myocardium or reconstruct a muscle of facial expression it can be
sutured or affixed in place with fibrin glue. By modifying the
assembly process, implants for the reconstruction of cardiac muscle
or smooth muscle can be assembled. In general, the major
modification may be in the relative pattern of the engineered
extracellular matrix and connective struts or tendons described in
this application. Cardiac tissue and smooth muscle lack tendons.
However, the use of large diameter collagen fiber may still be
desirable to lend mechanical strength to the implant. In the case
of cardiac implants, the large fibers may be used as a delivery
system to assemble or to manipulate the implants. The key common
feature to assembly of these implants is the ability to fabricate a
multi-layer implant composed of cells in an in vivo pattern of
organization.
[0090] Vascularization of the implanted muscle tissue will occur in
situ several days after surgery It can be stimulated further, as
mentioned above, by angiogenetic and growth-promoting factors,
either administered as peptides or as gene therapy. Another
alternative for supplying engineered tissue with a vascular supply
is to temporarily transplant the muscle into the omentum. The
omentum has an extensive and rich vascular supply that could be
tapped and used like a living incubator for the support of
engineered tissue. The engineered tissue would be removed from a
bioreactor and wrapped in the omentum and would be supported by the
diffusion of nutrients and oxygen from the surrounding tissue.
Alternatively or in addition to this approach, engineered tissue
could be connected directly to the endogenous vascular supply of
the omentum. A blood vessel might be partially perforated or cut or
left simply dissected free of the omentum. The engineered tissue
could then be wrapped around the vessel. The engineered tissue
would be supported by nutrients leaking from the perforated vessel
or by the simple diffusion of nutrients if the vessel was left
intact. Regardless of strategy, the engineered tissue would be
surrounded by the omentum and its rich vascular supply. It is also
possible to engineer muscle with an endogenous vascular system.
This vascular system might be composed of artificial vessels or
blood vessels excised from a donor site on the transplant
recipient. The engineered tissue would then be assembled around the
vessel. By enveloping such a vessel with the muscle during or after
assembly of the engineered muscle, the engineered tissue would have
a vessel that could be attached to the vascular system of the
recipient. In this example, a vessel in the omentum would be cut.
The vessel of the engineered muscle would be inserted and connected
to the two free ends of the omental vessel. Blood would pass from
the omental vessel into the vascular system of the muscle, then
pass through the muscle and drain back into the omentum vessel. By
wrapping the tissue in the omentum and connecting it to a omental
blood vessel, the engineered tissue would be supported by the
diffusion of nutrients from the omentum and the vessel incorporated
into the muscle during its fabrication. After a suitable period of
time the muscle would be removed from the omental "incubator" and
placed in the correct site in the recipient. By using this type of
strategy the engineered muscle could be supported in a nutrient
rich environment during the first several days following its
removal from the bioreactor. The environment of the omentum also
has the capacity to promote the formation of new blood vessels in
implanted tissue. This omental incubator strategy could also be
combined with the other angiogenic strategies discussed
earlier.
[0091] The engineered muscle described above is advantageous in
several respects. First, the connective backbone support of the
implant comprises natural materials. This material has low
antigenic potential and its structural properties can be regulated
at many different sites, including, but not limited to; the
relative concentration of different collagen isoforms used to
produce the sheath, the thickness of the fibers used and, the
degree of chemical cross-linking present in the matrix. Next, the
implant uses large diameter collagen fibers to further modify the
structural properties of the implant and provide a means to anchor
the engineered muscle to the site of transplantation. These fibers
are very similar to the fibers used to manufacture catgut for
surgical sutures (>250 .mu.m), however, the extrusion process
allows for better control of fiber diameter and the fabrication of
fibers that are much smaller in diameter than conventional catgut
(50 to 200 .mu.m depending upon reaction condition). Preliminary
studies from other laboratories indicate the efficacy of using
extruded collagen fibers in the production of tendons in the rat
and in the formation of woven sheets for the repairs of
experimental abdominal wounds in the rat. The implantation of large
diameter, extruded collagen fibers did not induce inflammation
beyond background levels in these experiments.
[0092] The stem cells or muscle cells used to construct the implant
can be isolated from the subject, or other compatible donor, that
requires muscle reconstruction. This has the obvious advantage of
using cells that will not induce an immune response, because they
originated with the subject (autologous tissue) requiring the
reconstruction. Relatively small muscle biopsies can be used to
obtain a sufficient number of cells to construct the implant. This
minimizes functional deficits and damage to endogenous muscle
tissues that serves as the donor site for satellite muscle
cells.
[0093] Another factor unique to this muscle prosthesis is the shape
of the individual muscle cells and their three-dimensional
arrangement. The cultures are composed of linear, three-dimensional
arrays of muscle cells distributed along a common axis in an in
vivo-like pattern of organization. This makes it possible to
install an implant that can produce contractile force along a
defined direction, allowing for the structural and functional
repair of a dysfunctional muscle bed. This feature is especially
critical in cardiac muscle where it is essential to restore both
the mechanical and electrical properties of the damaged muscle.
Cultures prepared on, or, in a random gel of collagen lack this
uniform alignment. Compare FIG. 4A (aligned) with FIG. 4B (random).
Random cultures are unsuitable for the use in reconstruction
because they lack the clearly defined orientation that is
characteristic of intact skeletal muscle and the individual
cellular layers of the heart. The highly polarized nature of intact
muscle allows it to effectively and efficiently apply mechanical
force along a defined axis during contraction. This property also
facilitates the conduction of electrical impulses through the
tissue.
[0094] Strategies must be implemented to promote the formation of
vascular elements within the implant. Several options are
available. First, the implants can be seeded with angioblasts
and/or endothelial cells to accelerate the formation of vascular
elements once the engineered tissue is placed in situ. Second,
angiogenic peptides can be introduced into the engineered tissue
via an osmotic pump. The use of an osmotic pump makes it possible
to deliver active peptides or, as noted, angiogenic peptides or
growth factors directly to the site of interest in a biologically
efficient and cost-effective manner. Experimentation in the
vascular bed of ischemic skeletal muscles has demonstrated the
efficacy of this approach (Hopkins et al., Controlled delivery of
vascular endothelial growth factor promotes neovascularization and
maintains the limb function in a rabbit model of ishemia, 1997 J.
of Vascular Surgery 27:886-95). VEGF delivered to ischemic hind
limbs of rabbits accelerated capillary bed growth, increased
vascular branching and improved muscular performance with respect
to ischemic controls. Upon initial implantation, an early phase of
muscle degeneration of intact muscle implants (Faulkner et al.,
Revascularization of skeletal muscle transplanted into the hamster
cheek pouch: Interavital and light microscopy, 1983 Microvasular
Res. 26:49-64) suggests that it may be desirable to implant
engineered muscle tissue at a time just prior to muscle
differentiation. An alternative approach is to "seed" fully
differentiated muscle constructs with additional satellite cells
and/or endothelial cells and or angioblasts shortly before they are
implanted in situ. Also, use of the omentum "incubator" was
discussed earlier.
[0095] In vivo deneravation of skeletal muscle promotes the
evolution of atrophy in the effected tissue. To a great extent,
this response appears to develop because deneravation reduces the
amount of resting tension observed in the affected muscle (Thomsen
and Luco 1944; Gutman et al., 1971). In vitro, the effects of
denervation may be substantially overcome by applying tension to
the denervated muscle. Cardiac muscle is also very sensitive to its
surrounding mechanical environment. Muscle mass may be initially
retained in an engineered prosthesis by placing it under tension at
the time it is implanted. Different methods of promoting
hypertrophy or stretching of the implant are available. A
mechanical stretcher can be used in the bioreactor by attachment of
the "tendons" on either end of the implant to ring clamps to
tighten or loosen the implant. FIG. 6 illustrates how the fibers
will be held in place (at desired orientations longitudinally) on
the inner cylinder surface of the bioreactor by two end supports
30. FIGS. 6 also illustrates the use of motor driven screw 31
drives to be added to the bioreactor to allow mechanical stretching
(well defined percentage of stretch) for preconditioning particular
tissue (muscle, blood vessels, and intestines) during the initial
cell seeding/development stage. The stretching makes bigger,
thicker and stronger cells/tissue that are less likely to tear
after implantation. The stretching can also be used to further
align the muscle cells. Electrical pacing or pharmacological
stimulation can also be used. Electrical pacing, in particular, is
very effective and easy to control.
[0096] The second level of control that is imparted by the central
nervous system on skeletal muscle is more fundamental. Neural
inputs directly control the action of the tissue. In order to
achieve a fully functional muscle prosthesis it is necessary to
bring it under the control of the central nervous system.
Preferably, the engineered implant can be transplanted into a
muscle bed adjacent to the area of interest and allowed to adapt to
the in vivo environment. After a period of adaptation the
autologous implant would be mobilized, perhaps with a portion of
the motor units arising from the original transplant site, and
repositioned within the site requiring reconstruction. It may also
be possible to induce the ingrowth of motor neurons through the use
of growth peptides delivered by osmotic pumps, or other means, to
the implant tissue. Cardiac tissue mass is not subject to much
regulation by the central nervous system. However, it is sensitive
to changes in mechanical activity. By pre-stressing by stretching,
electrical stimulation or using pharmacological agents to promote
cardiac muscle hypertrophy an implant of cardiac muscle during or
following the fabrication process, it can be better prepared for
the region of the in vivo environment.
EXAMPLE 1
[0097] (Electrospinning an Extracellular Matrix)
[0098] An extracellular matrix was made of
poly-lactic/poly-glycolyic acid (PLA/PGA; 50/50-RESOMER.RTM. RG
503, Boehringer Ingelheim, Germany) and poly(ethylene-co-vinyl)
acetate (Aldrich Chemical Company, Inc., Milwaukee, Wis.) polymers.
The concentration of the two polymers dissolved in dichloromethane
(Sigma-Aldrich, St. Louis, Mo.) were 0.19 g/ml RESOMER.RTM. RG 503
and 0.077 g/ml poly(ethylene-co-vinyl) acetate. The electrospinning
set-up consists of a glass pipet (overall length approximately 21
cm with a tapered tip with an opening estimated at 0.3 mm, no exact
measurement obtained, 0.32 mm diameter silver-coated copper wire,
20.times.20 mesh 316 stainless steel screen, two large clamp
holders (polymeric coated), base support, and a Spellman CZE1000R
power supply (0-30,000 volts, Spellman High Voltage Electronic
Corp., Hauppauge, N.Y.). The physical set-up had the top clamp
holder containing the glass pipet at approximately 12 inches from
the base with the pipet tip pointing (pipet at approximately at 45
angle to base) toward the base. The wire was then placed in the top
of the glass pipet and inserted until reaching the pipet tip where
it remained during the procedure. The second clamp holder was
placed at approximately 6 inches above the base for holding the
screen (grounded target) approximately perpendicular to the axis of
the glass pipet. The distance between the pipet tip and the
grounded screen was approximately 10 cm. The positive lead from the
high voltage power supply was attached to the wire hanging out the
top end of the glass pipet while the negative lead (ground) was
attached directly to the stainless steel screen. The glass pipet
was then filled with the appropriate solution and the power supply
turn on and adjusted until electrospinning was initiated (i.e.
fibers shooting from the tip of the glass pipet). This stream
(splay) of solution begins as a monofilament which between the
pipet tip and the grounded target is converted to multifilaments
(electric field driven phenomena). This allows for the production
of a "web-like" structure to accumulate at the target site. Upon
reaching the grounded target, the multifilaments collect and dry to
form the 3-D interconnected polymeric matrix (fabric). The
apparatus described is conceptually the same as the set-up
illustrated in FIGS. 2A and 2B. All described studies and solutions
are at room temperature. The fibers produced by these preliminary
studies ranged from 1-100 microns in diameter with both polymeric
solutions evaluated. The thickness of the matrices produced was not
measured. Although, the thickness of the matrix that can be
produced is dependent on the amount of polymer solution (spinning
time) utilized and allowed to accumulate in a particular region.
Thus, allowing the ability to produce a matrix with varying
thickness across the sample. A scanning electron micrograph of the
fiber forming the matrix is shown in FIG. 1.
EXAMPLE 2
[0099] (Fabrication of a Three Dimensional Segment of Skeletal
Muscle)
[0100] An aligned collagen gel was prepared after the methods of
Simpson et al., 1994 (Journal of Cell Physiology) on a silastic
membrane (Speciality Manufacturing). Silastic membranes were
sterilized in an autoclave and exposed to 2 minutes of electrical
discharge to make the rubber more hydrophilic. Aligned collagen was
then applied over the surface of the treated silastic rubber. In
brief, 500 ul of 0.2 N HEPES was mixed with 500 ul of 10.times. MEM
in a 50 ml centrifuge tube and placed on ice. Under sterile
conditions 3.5 mls of Type I collagen (3 mg/ml in 0.012 HCL,
Collagen Corporation) was layered over the top of the
HEPES/10.times. MEM solution, mixed by inversion and diluted to a
final volume of 10 mls with ice cold Phosphate Buffered Saline. A
sterile and treated silastic membrane (70 mm.times.30 mm) was
placed in a 100 mm culture dish. One milliliter of ice cold
collagen solution (final concentration 1.05 mg collagen/ml
solution) was applied to the one end of the rectangular piece of
silastic membrane. The collagen was pulled in a single continuous
stroke across the long axis of the silastic membrane with a sterile
cell scraper. The dish containing the silastic membrane was then
tipped and the collagen was allowed to drain across the membrane
along the axis that it was applied. The dish was covered and placed
into a 37 degree Celsius incubator for 1 hour to allow the collagen
to undergo polymerization. These procedures resulted in a thin
layer of aligned collagen fibrils on the silastic membrane. The
membranes were then allowed to dry in a moist atmosphere for 12-24
hours. This allows the collagen to partially dry down without
pooling the collagen and disturbing the aligned collagen fibrils.
The silastic membranes were then allowed to completely dry for an
additional 30-60 minutes under a sterile laminar flow hood.
Complete drying of the collagen anchors the fibrils to the rubber
for further manipulation. Silastic membranes were used in these
experiments solely to provide a support surface that could be
easily manipulated for the fabrication of the engineered
muscle.
[0101] A segment of silastic membrane (22 mm.times.22 mm)
containing uniformly arrayed collagen fibrils was cut and
transferred to a sterile 35 mm culture dish. Cells of the mouse
c2c12 skeletal muscle cell line were placed onto the silastic
membranes and cultured for 3-5 days in DMEM-F12 (50:50 DMEM:F12
mix, supplemented with 10% Horse serum, 5% FBS plus penstrep and
gentimysin) and allowed to form a confluent culture of uniformly
arrayed cells. These cultures served as a template layer for the
further assembly of the cultures. A thin layer of silicon grease
was placed on the underside of a piece of silastic rubber
containing a culture of aligned c2c12 cells. The silicon grease
serves as a non-toxic adhesive to anchor the culture to different
locations within the culture chamber of a RCCS Bioreactor
(Synthecon, Inc). In preliminary experiments several different
locations in the bioreactor culture vessel were used in attempts to
fabricate a three dimensional array of aligned muscle.
[0102] An aligned culture of c2c12 cells was placed on the back
wall of the rotating bioreactor culture vessel, cell side up and
held in place by a thin layer of silicon grease. The vessel was
closed and then filled with culture media (50:50; DMEM:F12 mix,
supplemented with 10% Horse serum, 5% FBS plus penstrep and
gentimysin). The syringe ports were opened and 1 million c2c12
cells were added as a single cells suspension to the bioreactor
vessel. The device was mounted on the control axle that controls
the rotational rate of the vessel. The entire device was then
placed into a C02 incubator (37.degree. C.) and set to rotate at 3
revolutions per minute. At 24 hour intervals an additional 1
million c2c12 cells were added to the reactor chamber.
[0103] By setting the rate of rotation at a slow rate the cells
added in suspension were gradually seeded out onto the template
culture. After 48 hours of mixing in the vessel, the template
cultures were isolated and prepared for electron microscopic
examination. The cultures prepared in the RCCS reactor were
composed of multiple layers of c2c12 cells arrayed along a common
axis. Several different locations and conditions within the
bioreactor were assayed for the ability to fabricate the
multi-layered cultures. In this experimental run, it was found that
cells arrayed on the back wall of the reactor in an orientation
that was perpendicular to the direction of rotation was most
effective. Other sites within the vessel also promoted the assembly
of multilayered cultures, including the outer wall, central core
and outer cylindrical wall. Aligned cultures that were oriented
with the direction flow were also capable of promoting multilayer
assembly, although not as effectively as the cultures oriented
perpendicular to the direction of rotation.
[0104] In other experiments, an aligned collagen gel was prepared
on a silastic membrane as described and placed directly into the
RCCS bioreactor chamber (i.e. the experiments were designed to
determine if the aligned collagen fibrils could promote
multilayered assembly without first growing a confluent template
layer of cells). Templates consisting of aligned collagen alone
were about as equally effective as the templates containing
confluent cell layers at promoting the assembly of the multilayered
cultures. Again, these collagen templates were placed onto the
bioreactor in different locations. They behaved identically as the
confluent cell layers, i.e. collagen coated membranes of the back
wall oriented perpendicular to the axis of rotation were most
effective at promoting multilayer assembly.
[0105] A scanning electron micrograph of the muscle cells deposited
on the aligned collagen is shown in FIG. 4A.
EXAMPLE 3
[0106] (Electroaerosol Production of Extracellular Matrix)
[0107] An extracellular matrix in the form of a tube was made. Like
the electrospinning described in Example 1, the electroaerosol
process includes a polymer reservoir, spray nozzle and grounded
mandrel. (See FIGS. 2A and 2B). In this experiment, the polymer
reservoir and spray nozzle was a 1.0 ml syringe (minus plunger) and
a simple plastic pipette tip (Gel Loading Tip, Fisher Scientific),
respectively. The grounded mandrel was composed of stainless stell
needle (18 gauge, length -8 cm). Note: Prior to aerosol/matrix
production, the mandrel was treated with a hexane solution
saturated with Vaseline to allow easy removal of the formed
construct from the mandrel. The polymeric solution used was
polylactic/polyglycolyic acid (PLA/PGA; 50/50) at a concentration
of 0.189 g/ml in methylene chloride. A fine wire was placed into
the pipette tip as far as it would go. With this tip, the wire
could not pass all the way through, thus approximately a quarter
inch of the tip was cut-off at the point where the wire had passed
through and became lodged. The wire in this experiment was charged
to 12,000 volts (Spellman High Voltage Power Supply). Upon applying
the electrical potential, the polymer aerosol began at the pipette
tip and was directed towards the grounded mandrel. The aerosol was
then collected around the mandrel. A total of 4 ml of the polymeric
solution was used to create an extracellular matrix that could be
used to construct, for instance, a nerve guide. Step one was to
fill the reservoir/syringe with 1 ml of polymeric solution, charge
the solution and allowing aerosol production. Upon emptying the
reservoir, the mandrel was rotated 90 degrees (step 2) and step one
was repeated. These steps were then repeated 4 time for the
complete construct/nerve guide. FIGS. 8 to 11 are scanning electron
micrographs of the extracellular matrix produced by this
procedure.
[0108] The polymers evaluated--polylactic/polyglycolyic acids
(PLA/PGA; 50/50), give a soft, sponge yet fairly rigid construct
with completed dried. As noted, the mechanical properties will
depend on the polymer used, aerosol particle diameter, and overall
mesh (matrix packing) structure.
[0109] Upon working with different material and mandrels, it should
be noted that this technique could be able to form seamless
constructs with bifurcations or other complex geometries (i.e.
heart valves, vascular grafts, intestine, and cartilage (knee,
nose, ear).
EXAMPLE 4
[0110] (Construction of a Vascular Prosthesis)
[0111] This example focuses on the development of a small diameter
vascular prosthesis from collagen threads (Plain gut
suture--collagen), arterial cellular components (smooth muscle
cells (SMC), fibroblast cells (FB), and endothelial cells (EC)),
collagen (Type I, III & IV), and fibronectin (All major
components of arteries). Small diameter arteries such as the one
described herein are composed of cylindrical layers (3-6 layers) of
smooth muscle cells which are arranged in spirals of varying
pitches. The arrangement of one example of a vascular prosthesis
has the layers presented in FIG. 12.
[0112] Winding Apparatus
[0113] The winding apparatus (FIG. 13) will be composed of two end
supports 50, thin walled tubing 51, glass rod 52, bathing chamber
53, suture movement system 54, and a mandrel rotation system 55.
The two end supports 50 will be bored centrally to match the
external diameter of the thin walled tubing 51. The glass rod 52
will be placed through the thin walled tubing 51 during the
collagen thread winding to maintain the graft cylindrical shape and
be removed once in a bioreactor. The mandrel rotational system 55
will allow rotation of the graft mandrel at a specific speed. The
suture movement and guidance system 54 will be, mounted above the
mandrel and will be composed of a rail and motor drive system to
move the thread at a specific speed across the mandrel. The
combination of the thread guidance speed and mandrel rotation speed
will allow the control of the pitch of the collagen threads in
layers 2-4 of the media. The pitch of layers 1 and 5 are high and
will be prefabricated by weaving the suture between the mandrel end
supports which will be designed to produce the desired pitch. The
bathing chamber will allow the mandrel to be maintained in a saline
or collagen solution during the winding procedure. The entire
apparatus will be built from aluminum, 304 stainless steel (304SS),
glass, and polycarbonate to allow sterilization by steam or
ethylene oxide.
[0114] Prosthetic Mandrel, Mandrel Holder, and Bioreactor
[0115] The mandrel 60 (FIG. 14) will be composed of a glass rod 52,
thin walled tubing 51, and two end supports 50. The end supports 50
will be composed of 304 SS. The exact composition (or whether it
needs to be used at all) of the thin walled tubing 51 is not known
at this time. The mandrel holder 65 (FIG. 15) will maintain the
vascular construct when placed within the bioreactor. It is
primarily an end support system which doubles as a bathing chamber.
The mandrel holder 65 will be filled with either saline or
Medium-199. The mandrel holder 65 will be composed of polycarbonate
which can be sterilized by either steam or ethylene oxide. The
bioreactor (FIG. 16) will be composed of tubing connectors 70,
tubing 71, polycarbonate walls 72, and a pulsatile pump (not
shown).
[0116] Methodology
[0117] The internal cylinder (lumen region) of the media will be
developed by stringing (weaving) plain gut suture (0.23 mm
diameter, L=6 m; H. Hess & Co.) between the end supports at a
high pitch (75.degree., almost parallel to the long axis of the
vessel) with a 0.2-0.5 mm space between each suture. After weaving
the plain gut suture, the mandrel will be placed into the
bioreactor containing a suspension of human umbilical artery SMCs
(UASMC). The suspension will be composed of 2.times.10.sup.6
UASMC/ml, collagen solution (46% type I & 54% type III;
composition of normal vascular tissue; Imedex), 4.times. PBS, and
distilled water to make a collagen concentration of 3 mg/ml. After
5 minutes at 37.degree. C., the gelification around the mandrel
will have occurred and the mandrel transferred to its holder within
the bioreactor. The well of the mandrel holder will contain a
Medium-199 (M-199) (10% FBS) solution containing 2.times.10.sup.6
UASMC/ml. The collagen mixture will be allowed to form the initial
cylindrical layer for 24 hours. During the 24 hour period in the
bioreactor, the graft will have a pulsatile flow (flow rate: 90
ml/min; frequency 60 Hz; amplitude <5% of graft outer diarneter)
through the thin walled tubing of the mandrel for the first media
layer. During subsequent media layers, the thin walled tubing will
be removed to allow perfusion (nutrient delivery) to the media
layers as well as direct mechanical forces. The advantage of this
pulsatile flow during the media and adventitia development is that
the cellular components will be exposed to circumferential
(stretch-relaxation) forces. This is hoped to contribute to
significant mechanical structural properties when compared to the
vascular prostheses developed on mandrels under static conditions.
After 24 hours, the second cylindrical layer will begin by winding
the plain gut suture at a low pitch (15.degree.; essentially
perpendicular to the long axis of the vessel) with a spacing of
approximately 0.2-0.5 mm. After the suture winding, the protocol is
exactly as described for the initial layer. Layers 3-5 also follow
the described protocol with suture pitches of -15.degree.,
15.degree., and -75.degree., respectively (FIG. 12). All procedures
will be performed under a laminar flow sterile workstation. The
bioreactor apparatus (pulsatile flow system) will be housed in an
incubator at 37.degree. C., 5% CO.sub.2 and constant humidity. No
complications are expected due to the use of a bioabsorbable suture
as the scaffolding. It has been shown that bioabsorbable suture
(compared to non-absorbable) exhibits little to no anastomotic
thrombus or hyperplasia along with less inflammation and scar
tissue. It has been demonstrated that in vascular anastomoses,
complete hydrolytic decomposition (@ 7 mo.) of the absorbable
suture was followed by almost complete tissue regeneration of the
vessel including the connective tissue of the media.
[0118] The adventitia equivalent structure will be developed by
following the media development with fibroblast cells
(FB)(5.times.10.sup.5FB/ml) and collagen type I and III (3 mg/ml).
The fibroblasts to be used will be human dermal fibroblasts (HDF).
Three layers of the FB and collagen will be added to the media
construct to build the adventitial layer. In small diameter
arteries the adventitia can occupy up to half the thickness of the
vascular wall. Thus, the necessity for multiple layers of the
adventitial components.
[0119] The intima will be developed by endothelial cell (EC)
seeding of the adventitia-media construct Prior to EC seeding, the
adventitia-media construct will be removed from the mandrel end
supports and cannulated. The first step in EC seeding, neointimal
development, is to coat the lumen surface with human fibronectin
and collagen type IV. The EC seeding will be accomplished by
injecting ECs (5.times.10.sup.6 EC/ml) suspended in M-199 (20% FBS)
into the construct's lumen. The cannulas will then be sealed and
the construct placed in a tube containing M-199 and 20% FBS
overnight to allow adherence of the ECs to the construct. The tube
and construct will be rotated at 1/8 r.p.m. during this time to
allow an even distribution of the ECs on the luminal surface. After
the EC adhesion period, the complete vascular prosthesis will be
placed back in the bioreactor and exposed directly to physiologic
pulsatile flow for 10 days (flow rate: 90 ml/min; frequency 60 Hz;
amplitude <5% of graft outer diameter). The vascular construct
will be bathed externally in M-199 and 10% FBS while the
circulating media will be M-199 and 20% FBS. The circulating and
bathing media will be replenished every two days.
[0120] Without further elaboration, it is believed that one skilled
in the art can, using the proceeding description, utilize the
present invention to its fullest extent. The preceding preferred
specific embodiments are, therefore, to be construed as merely
illustrative, and not limiting the remainder of the disclosure in
any way whatsoever. The entire disclosure of all applications,
patents, and publications, cited above and in the figures are
hereby incorporated by reference in their entirety.
* * * * *