U.S. patent application number 14/552261 was filed with the patent office on 2015-03-19 for mesh enclosed tissue constructs.
The applicant listed for this patent is The Regents of the University of California. Invention is credited to Seyedhamed Alavi, Arash Kheradvar.
Application Number | 20150081012 14/552261 |
Document ID | / |
Family ID | 46877662 |
Filed Date | 2015-03-19 |
United States Patent
Application |
20150081012 |
Kind Code |
A1 |
Alavi; Seyedhamed ; et
al. |
March 19, 2015 |
MESH ENCLOSED TISSUE CONSTRUCTS
Abstract
A scaffold to form tissue membranes, comprising: at least one
layer of mesh having a first side and a second side, the layer of
mesh being either a woven wire metal mesh or a flat metal sheet
that is acid-etched such that the layer of mesh includes a network
of holes passing directly through the mesh from the first side to
the second side; and at least one layer of cells at each side of
the mesh enclosing the layer of mesh, such that the at least one
layer of cells on the first side interacts with the at least one
layer of cells on the second side through the network of holes to
provide for structure integration.
Inventors: |
Alavi; Seyedhamed; (Irvine,
CA) ; Kheradvar; Arash; (Irvine, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of California |
Oakland |
CA |
US |
|
|
Family ID: |
46877662 |
Appl. No.: |
14/552261 |
Filed: |
November 24, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13427843 |
Mar 22, 2012 |
8900862 |
|
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14552261 |
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61466882 |
Mar 23, 2011 |
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61496369 |
Jun 13, 2011 |
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61540330 |
Sep 28, 2011 |
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61559694 |
Jan 19, 2012 |
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Current U.S.
Class: |
623/2.14 ;
424/423; 424/93.7; 435/399 |
Current CPC
Class: |
A61L 27/54 20130101;
C12N 2533/10 20130101; A61L 27/3886 20130101; A61L 2400/18
20130101; A61L 27/34 20130101; A61L 27/56 20130101; C12N 5/0691
20130101; A61F 2/90 20130101; A61L 2430/20 20130101; C12N 5/0068
20130101; C12N 2501/15 20130101; A61L 27/446 20130101; A61K 35/12
20130101; A61L 27/34 20130101; A61L 27/3804 20130101; A61L 27/3808
20130101; A61L 27/3826 20130101; A61L 27/047 20130101; C08L 89/06
20130101; A61F 2/2418 20130101; A61F 2/07 20130101; C12N 2533/54
20130101; A61L 2300/414 20130101; A61F 2/2415 20130101; A61F
2240/001 20130101 |
Class at
Publication: |
623/2.14 ;
424/423; 424/93.7; 435/399 |
International
Class: |
A61F 2/24 20060101
A61F002/24; A61F 2/07 20060101 A61F002/07; A61F 2/90 20060101
A61F002/90; A61L 27/44 20060101 A61L027/44; A61K 35/12 20060101
A61K035/12 |
Claims
1. A scaffold to form tissue membranes, comprising: at least one
layer of mesh having a first side and a second side, the layer of
mesh being either a woven wire metal mesh or a flat metal sheet
that is acid-etched such that the layer of mesh includes a network
of holes passing directly through the mesh from the first side to
the second side; and at least one layer of cells at each side of
the mesh enclosing the layer of mesh, such that the at least one
layer of cells on the first side interacts with the at least one
layer of cells on the second side through the network of holes to
provide for structure integration.
2. The scaffold of claim 1, wherein the mesh becomes biologically
active when implanted in-vivo.
3. The scaffold of claim 1, further comprising a frame attached to
the layer of mesh with the at least one layer of cells at each side
of the mesh, wherein the frame is formed of a biocompatible metal
and is covered with a woven polyester cloth.
4. The scaffold of claim 1, wherein the mesh and at least one layer
of cells at each side of the mesh are formed as leaflets, such that
the scaffold is operable as a tissue heart valve.
5. The scaffold of claim 4, wherein the scaffold includes at least
two leaflets.
6. The scaffold of claim 5, further comprising a flexible frame
having a saddle-shaped base with at least two upstanding posts,
with the leaflets each having a peripheral free portion and a fixed
portion, such that the peripheral free portion extends between the
posts and the fixed portion is attached to the base.
7. The scaffold of claim 6, further comprising a frame having a
base with three upstanding posts, with the leaflets attached to the
frame and between the posts.
8. The scaffold of claim 4, further comprising a flexible frame
having a saddle-shaped base with at least two upstanding posts,
with the leaflets each having a peripheral free portion and a fixed
portion, such that the peripheral free portion extends between the
posts and the fixed portion is attached to the base.
9. The scaffold of claim 4, further comprising a frame having a
base with three upstanding posts, with the leaflets attached to the
frame and between the posts, and wherein the frame is formed of a
biocompatible metal and is covered with a woven polyester
cloth.
10. The scaffold of claim 1, wherein the layer of mesh is a tubular
wire mesh, wherein the at least one layer of cells at each side of
the mesh are formed around the mesh to completely or partially
conceal the mesh therein, whereby the scaffold is formed in the
shape of a vessel to operate as a vascular graft.
11. The scaffold of claim 1, wherein the mesh has a shape of a
heart valve leaflet.
12. The scaffold of claim 11, wherein a plurality of leaflets are
attached together to form a heart valve shape.
13. The scaffold of claim 1, wherein the mesh is a Nitinol mesh
with a thickness between approximately 25 and 76 microns.
14. The scaffold of claim 1, wherein the at least one layer of
cells is selected from the group consisting of smooth muscle cells,
fibroblast/myofibroblast cells and vascular endothelial cells.
15. A method for forming a scaffold according to claim 1,
comprising: preparing a layer of mesh having a first side and a
second side, the layer of mesh being either a woven wire metal mesh
or a flat metal sheet that is acid-etched, such that the layer of
mesh comprises a network of holes passing directly through the mesh
from the first side to the second side, and growing a biological
matrix around the layer of mesh such that the biological matrix
comprises at least one layer of cells at each side of the mesh
enclosing the layer of mesh, such that the at least one layer of
cells on the first side interacts with the at least one layer of
cells on the second side through the network of holes to provide
for structure integration.
16. The method of claim 15, wherein the mesh is formed of stainless
steel wires, and wherein said preparing the layer of mesh further
comprises a preparation technique, or any combination thereof,
selected from the group consisting of: polishing the layer of mesh;
acid washing the layer of mesh; ultrasonic clean washing the layer
of mesh; and glow discharging the layer of mesh.
17. The method of claim 15, wherein said preparing the layer of
mesh comprises modifying the surface of the mesh by ion beam
surface modification to provide a smooth surface and to ensure
biocompatibility of the mesh and enhanced cell attachment to said
mesh.
18. The method of claim 15, wherein said growing a biological
matrix around the layer of mesh comprises modifying the surface of
the mesh with an additive to ensure tissue growth.
19. The method of claim 18, wherein the additive is collagen, which
applied to the layer of mesh to coat the layer of mesh to promote
development of an interconnected pore network.
20. The method of claim 15, wherein said growing a biological
matrix around the layer of mesh comprises seeding the at least one
layer of cells sequentially on each side of the layer of mesh.
21. The method of claim 20, wherein the sequential seeding of cell
layers further comprises adding cytokines, including TGF-.beta.1
with each sequentially seeded layer.
22. The method of claim 20, wherein, if more than one layer of
cells is seeded on each side of the mesh, a time interval of
approximately two weeks is provided between seeding of each of the
different layers of cells.
23. The method of claim 15, wherein the biological matrix is able
to further grow and develop when implanted in vivo.
24. The method of claim 15, wherein the biological matrix is grown
around the layer of mesh in vitro.
25. The method of claim 15, wherein the layer of mesh is a tubular
wire mesh, wherein the at least one layer of cells at each side of
the mesh are formed around the mesh to completely or partially
conceal the mesh therein, whereby the scaffold is formed in the
shape of a vessel to operate as a vascular graft.
26. The method of claim 15, comprising cutting the mesh to the
shape of a heart valve leaflet.
27. The method of claim 35, comprising attaching a plurality of
leaflets together to form a heart valve shape.
28. The method of claim 15, wherein the cells in the at least one
layer of cells at each side of the mesh are selected from the group
consisting of smooth muscle cells, fibroblast/myofibroblast cells,
and vascular endothelial cells.
Description
PRIORITY CLAIM
[0001] This application is continuation of U.S. application Ser.
No. 13/427,843, filed Mar. 22, 2014 which is a non-provisional
application of U.S. Provisional Application No. 61/466,882,
entitled "A SELF-REGENERATIVE HYBRID TISSUE STRUCTURE FOR 3D
FABRICATION OF HEART VALVES, BLOOD VESSELS AND OTHER CONSTRUCTS,"
filed on Mar. 23, 2011; and U.S. Provisional Application No.
61/496,369, entitled, "HYBRID TISSUE ENGINEERED HEART VALVE," filed
on Jun. 13, 2011; AND U.S. Provisional Application No. 61/540,330,
entitled, "Scaffold for Fabrication of Engineered Heart Valves and
Other Applications," filed on Sep. 28, 2011; and U.S. Provisional
Application No. 61/559,694, entitled, "METAL MESH SCAFFOLD FOR
TISSUE ENGINEERING OF MEMBRANES," filed on Jan. 19, 2012.
BACKGROUND OF THE INVENTION
[0002] (1) Technical Field
[0003] The invention pertains to methods for tissue engineering
and, more particularly, to the fabrication of a scaffold that is
Composed of multi-layered tissue enclosed on a metal mesh.
[0004] (2) Description of Related Art
[0005] Engineering of the membrane-like tissue structures with
ability to remodel and regenerate is currently an unresolved
subject in the field of tissue engineering. Several attempts with
minimal success have been made to create functional viable membrane
tissues such as heart valve leaflet with the ability to grow,
repair, and remodel. Shinoka et al. fabricated single leaflet heart
valves by sequentially seeding ovine fibroblasts and endothelial
cells on a bioabsorbable polymer composed of a polyglactin woven
mesh surrounded by two nonwoven polyglycolic acid mesh sheets. (See
Shinoka, T., Breuer, C. K., Tanel, R. E., Zund, G., Miura, T., Ma,
P. X., Langer, R., Vacanti, J. P., and Mayer J. E. Tissue
engineering heart valves: Valve leafet replacement study in a lamb
model. Ann Thorac Surg, 60, 13, 1995). Hoerstrup et al. fabricated
a trileaflet heart valve using nonwoven polyglycolic acid mesh, a
bioabsorbable polymer, sequentially seeded with ovine
myofibroblasts and endothelial cells made using a pulse duplicator
in vitro system. (See Hoerstrup, S. P., Sodian, R., Daebritz, S.,
Wang, J., Bacha, E. A., Martin, D. P., Moran, A. M., Guleserian, K.
J., Sperling, J. S., Kaushal, S., Vacanti, J. P., Schoen, F. J.,
and Mayer, J. E. Jr. Functional living trileaflet heart valves
grown in vitro. Circulation, 102, 44, 2000). Sodian et al.
constructed trileaflet heart valve scaffolds fabricated from
seeding ovine arterial vascular cells on a polyhydroxyoctanoate
material. (See Sodian, R., Hoerstrup, S. P., Sperling, J. S.,
Daebritz, S., Martin, D. P., Moran, A. M., Kim, B. S., Schoen, F.
J., Vacanti, J. P., and Mayer, J. E. Jr. Early in vivo experience
with tissue-engineered trileaflet heart valves. Circulation, 102,
suppl III, 2000). Sutherland et al. created autologous semilunar
heart valves in vitro using mesenchymal stems cells and a
biodegradable scaffold made of polyglycolic acid and poly-L-lactic
acid. (See Sutherland, F. W., Perry, T. E., Yu, Y., Sherwood, M.
C., Rabkin, E., Masuda, Y., Garcia, A., McLellan, D. L., Engelmayr,
G. C., Sacks, M. S., Schoen, F. J., and Mayer J. E. Jr. From stem
cells to viable autologous semilunar heart valve. Circulation, 111,
2783, 2005). Drawbacks to the approaches described above include
structural vulnerability, short term functionality, and limited
mechanical properties of the membrane constructs.
[0006] Scaffolds are critical components of the engineered tissues
that allow them to be formed in vitro and remain secure in vivo
when implanted in a host. Several approaches have been taken to
develop scaffolds for tissue membranes. The most widely used method
involves biodegradable naturally-derived or synthetic polymers,
where the polymer eventually degrades by normal metabolic activity,
while the biological matrix is formed. To have viable tissue, the
rate of scaffold degradation should be proportional to the rate of
tissue formation to guarantee mechanical stability over time. The
poor control of enzymatic degradation and low mechanical
performance are two major limitations of naturally derived
polymers. In contrast, synthetic polymers can be prepared precisely
with respect to structure and function. However, most of them
produce toxic chemicals when they degrade in vivo, and due to lack
of receptor-binding ligands, they may not provide a good
environment for adhesion and proliferation of cells.
[0007] Another option for creating scaffolds is to use
decellularized xenogenic tissues, which has some advantages over
polymeric materials. Decellularized tissues provide a unique
scaffold, which is essentially composed of extracellular matrix
(ECM) proteins that serve as an intrinsic template for cells.
However, the process of decellularization cannot completely remove
the trace of cells and their debris. These remnants not only
increase the potential of an immunogenic reaction, but also result
in increased tissue susceptibility to calcification.
[0008] Another, albeit less developed, strategy involves creating a
scaffold with completely biological matrix components. This
approach has advantages over using polymeric materials or
decellularized xenogenic tissues. For example, large amounts can be
produced from xenogenic sources, which can readily accommodate
cellular ingrowth without cytotoxic degradation products. However,
this strategy is restricted due to mechanical fragility of the
scaffold and the low potentials for creating complex tissue
structures.
[0009] Thus, a continuing need exists for a tissue Construct that
is strong enough to resist forces that exist inside a body, while
possessing biocompatible surfaces.
SUMMARY OF THE INVENTION
[0010] The present invention is directed to a scaffold that is
strong enough to resist forces that exist inside a body, while
possessing biocompatible surfaces. The scaffold is formed of a
layer of mesh (e.g., Stainless Steel or Nitinol) that is tightly
enclosed by a multi-layer biological matrix. The biological matrix
can include any desired number of layers, such a first layer
(smooth muscle cells) formed directly on the metal mesh, a second
layer (fibroblast/myofibroblast cells) formed on the first layer,
and a third layer (endothelial cells) formed on the second
layer.
[0011] The scaffold can be formed to operate as a variety of
tissues, such as a heart valve or vascular graft. For example, the
mesh and corresponding biological matrix can be formed as leaflets,
such that the scaffold is operable as a tissue heart valve. In this
aspect, the scaffold includes a flexible frame having a
saddle-shaped base with at least two upstanding posts, with the
leaflets each having a peripheral free portion extending between
the posts and a fixed portion attached with the base.
[0012] In another aspect, the scaffold is formed as a vascular
graft. In this aspect, the layer of mesh is a tubular wire mesh,
with the biological matrix formed around the mesh to completely
conceal the mesh therein.
[0013] As can be appreciated by one skilled in the art, the present
invention is also directed to the method of forming the scaffold
described herein. The method includes a plurality of acts, such as
preparing a layer of mesh and growing a biological matrix around
the layer of mesh such that the biological matrix tightly encloses
the layer of mesh.
[0014] In another aspect, the act of preparing the layer of mesh
further comprises a preparation technique, or any combination
thereof, selected from a group consisting of polishing the layer of
mesh; acid washing the layer of mesh; ultrasonic clean washing the
layer of mesh; and glow discharging the layer of mesh.
[0015] Additionally, the act of preparing the layer of mesh further
comprises an act of ion beam surface modification to provide a
smooth surface and ensure the biocompatibility and enhanced cell
attachment.
[0016] In yet another aspect, growing a biological matrix around
the layer of mesh further comprises an act of providing collagen as
an additive to coat the layer of mesh to ensure development of an
interconnected pore network.
[0017] In another aspect, wherein growing a biological matrix
around the layer of mesh further comprises an act of sequentially
seeding three different types of cells on the layer of mesh. In
sequentially seeding three different types of cells on the layer of
mesh, the three different types of cells are smooth muscle cells,
fibroblast/myofibroblast cells, and endothelial cells. Further,
protein, including TGF-.beta.1, can be added to the collagen in
each layer. Thus, as described above, the present invention is
directed to a scaffold and various methods for forming such a
scaffold.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The objects, features and advantages of the present
invention will be apparent from the following detailed descriptions
of the preferred aspect of the invention in conjunction with
reference to the following drawings where:
[0019] FIG. 1A shows a representation of a scaffold of one aspect
of the present invention;
[0020] FIG. 1B is a diagram showing the three layers of cells of a
scaffold that mimic heart valve tissue structure of one aspect of
the present invention;
[0021] FIG. 2 is a schematic showing the steps in the
three-dimensional (3D) cell culture method to develop a tissue;
[0022] FIG. 3A is an image of a stainless steel mesh with a surface
area of about 1 cm.sup.2;
[0023] FIG. 3B is a view of the engineered tissue after three
months of cell culture;
[0024] FIG. 4A is a scanning electron micrograph of the first layer
on the mesh showing that smooth muscle cells are attached over the
mesh;
[0025] FIG. 4B is a expanded view of FIG. 4A;
[0026] FIG. 5A is a scanning electron microscropy image taken after
culturing the second layer of cells containing fibroblasts and
myofibroblasts;
[0027] FIG. 5B shows the formation of extracellular matrix and a
layer of cells formed on the metal mesh, the black arrow indicates
a single fibroblast cell;
[0028] FIG. 6A shows a top view of cell culture without addition of
TGF-.beta.;
[0029] FIG. 6B shows a top view of cell culture without addition of
TGF-.beta.;
[0030] FIG. 6C shows the top view of the cell culture with
TGF-.beta. added to the cell culture;
[0031] FIG. 6D shows the top view of the cell culture with
TGF-.beta. added to the cell culture;
[0032] FIG. 7A is a scanning electron microscopy, image that show
layers of tissue tightly enclosing the stainless steel mesh;
[0033] FIG. 7B is a scanning electron microscopy image that show
three layers of tissue tightly enclosing the stainless steel
mesh;
[0034] FIG. 7C is a scanning electron microscopy image that show
three layers of tissue tightly enclosing the stainless steel
mesh;
[0035] FIG. 7D is a scanning electron microscopy image that show
three layers of tissue tightly enclosing the stainless steel
mesh;
[0036] FIG. 8A is an illustration depicting a size comparison of a
one-centimeter by one-centimeter Nitinol mesh in relation to a
United States Penny;
[0037] FIG. 8B shows the engineered tissue on Nitinol mesh after
the months of cell culture;
[0038] FIG. 9A is an illustration of a heart valve depicting the
Nitinol mesh scaffolding;
[0039] FIG. 9B is an illustration of a heart valve with heart
leaflets that are made of tissue described in this application;
[0040] FIG. 9C is an illustration of a heart valve with heart
leaflets that are made of tissue described in this application;
[0041] FIG. 9D is an illustration depicting schematic parts of a
tri-leaflet scaffold that can be used as a heart valve;
[0042] FIG. 9E is an illustration that includes various view-point
illustrations of the heart valve;
[0043] FIG. 9F is an image of the tri-leaflet scaffold that is
depicted in FIGS. 9A and 9D;
[0044] FIG. 10A is a schematic representation of a blood vessel;
and
[0045] FIG. 10B is a schematic representation of a blood vessel
formed from the tissue described in this application.
DETAILED DESCRIPTION OF THE INVENTION
[0046] Reference will now be made in detail to embodiments of the
present invention, examples of which are illustrated in the
accompanying drawings. While the invention will be described in
conjunction with these embodiments, it will be understood that they
are not intended to limit the invention to these embodiments. On
the contrary, the invention is intended to cover alternatives,
modifications, and equivalents, which may be included with the
spirit and scope of the invention as defined by the appended
claims. Furthermore, in the following detailed description of
embodiments of the present invention, numerous specific details are
set forth in order to provide thorough understanding of the present
invention. However, it will be recognized by one of ordinary skill
in the art that the present invention may be practiced without
these specific details. In other instances, well-known methods,
procedures, and components have not been described in detail as not
to unnecessarily obscure aspects of the embodiments of the present
invention.
[0047] As noted above and as shown in FIG. 1A, the present
invention is directed to a scaffold 100 that is composed of
multi-layered tissue enclosed on a metal mesh. This is further
illustrated in FIG. 1B, which illustrates that the scaffold 100 is
made of an extra layer of metal mesh 102 enclosed by a biological
matrix, such as layers (e.g., three layers) of cells (e.g.,
different cell types). It should be understood that while the
present invention is described as scaffold 100 that includes three
layers of different cell types, it is not intended to be limited
thereto as the scaffold 100 can be formed with a single layer, or
any suitable number of layers, and, further, with a single or
different cell types. Additionally, while the mesh 102 is described
as being covered with biological materials or a biological matrix,
the invention is not limited thereto as the mesh 102 can also be
enclosed by synthetic materials that are known to one skilled in
the art (such as polymers, etc.) As a non-limiting example, the
synthetic material can be molded onto the mesh.
[0048] However, desirably, the three layers of biological materials
include a first layer 104 of smooth muscle cells. The second layer
106 may be composed of fibroblast and myofibroblast cells and the
third layer 108 (which can is the outer layer) may comprise of
endothelial cells. These three layers wrap around the metal mesh
102 in three-dimensions so that each layer fully envelopes the
metal mesh 102. This approach is intended to retain all the
advantages of using biological scaffolds while developing a strong
extracellular matrix (ECM) backbone composed of the mesh 102 that
can withstand various types of loads after implantation inside the
body. Additionally, such a mesh pattern ensures structure
integration of the formed tissue and allows cells and ECM
components on both sides of the mesh 102 to interact with each
other. The formed tissue is intended to be biomechanically
resilient against the physiological stresses inside the body. In
one aspect, the scaffold 100 is a living tissue, able to
continually remodel and mature in vitro and in vivo. For example,
the scaffold 100 has living tissue (as described below) that can
continue to grow and mature, with the mesh 102 becoming
biologically active when implanted in-vivo.
[0049] In one aspect, the three layers of cells of the scaffold 100
may mimic the heart valve structure. These three layers mimic
ventricularis, spongiosa and fibrosa layers of a heart valve
leaflet. This type of scaffold can be used in any membrane tissue
fabrication, such as heart valve leaflets, vascular grafts,
etc.
[0050] While the present invention is directed to a unique hybrid
scaffold 100 as shown in FIGS. 1A and 1B, the present invention
also includes the method of making the novel scaffold (made of an
extra layer of metal mesh enclosed by three layers of different
cell types). For example, FIG. 2 shows a schematic diagram of a
method for producing the multilayered tissue. Through the
three-dimensional cell culture technique detailed in this
application, all layers of the cells were seeded on
rectangular-shaped Stainless Steel meshes to produce ECM or
connective tissue.
[0051] The method of making the multilayered tissue is as follows.
The first step in creating the scaffold is preparation of the metal
mesh scaffold. The metal mesh is any suitable material that can
operate as scaffolding for a tissue. As a non-limiting example, the
metal mesh may be a flat mesh of T316 Stainless Steel woven from
0.0037'' round wires, targeting at 80 end per inch (EPI).times.80
pick per inch (PPI) that possesses an opening size of 0.0088''. A
non-limiting example of such a mesh is that sold by TWP, Inc.,
located at 2831 Tenth Street, Berkeley, Calif. 94710 USA. The metal
mesh was heated at 520.degree. C. for 5 min, followed by water
quenching. The oxidized film was removed at multiple stages; by
polishing the surface, using hydrochloric acid wash, ultrasonic
cleaning wash in ethanol for 15 min and glow discharging for 40
seconds. Finally, the mesh was cut into pieces with area of one
square centimeter to be used for cell culture.
[0052] After the metal was cleaned and cut into pieces, an ion beam
surface modification method was used to get a smooth surface and
ensure the biocompatibility and enhanced cell attachment for the
Stainless Steel meshes. The meshes were mechanically polished with
wetted metallographic polishing high-grade Silicon Carbide (SiC)
papers. Afterward, the meshes were acid-washed, degreased in an
ultrasonic vibrobath, and rinsed with distilled water. Prior to
cell culture, the samples were irradiated by He.sup.+ ion beam at
energy of 150 keV with fluences of 1.times.10.sup.14
ions/cm.sup.2.
[0053] In one aspect, the growth of the tissue may be aided by the
addition of growth factors and materials. For example, a mixture
containing bovine and rat tail collagen may be used to coat the
mesh to ensure development of an interconnected pore network, which
is essential for cell growth, nutrient supply, and removal of
metabolic waste products. In addition, the culture media may be
supplemented with additives, including, but not limited to,
ascorbic acid to promote matrix production. Moreover, proteins
(cytokines), including TGF-.beta.1, may be added to the collagen
gels in each layer to increase the rate of extracellular matrix
production. For the biological part of the scaffold any collagen
type by itself or in mixture as well as the other biological
scaffold such as fibrin or even synthetic scaffolds can be used.
Growth factors depending on the target tissue and the cells that
have been used can be different, such as vascular endothelial
growth factor (VEGF) if endothelial progenitor cells are used
instead of endothelial cells.
[0054] After the mesh has been prepared, the three-dimensional
tissue scaffold was constructed by sequential seeding of three
different types of cells on the metal mesh. As a non-limiting
example, three different cell types were isolated and used for
preliminary assay, as follows: smooth muscle cells and fibroblast
and myofibroblast cells to fulfill the role of valvular
interstitial cells (VICs) and endothelial cells to act as the
valvular endothelial cells. The basal media for culturing cells
contained DMEM (e.g., Dulbecco's Modified Eagle Medium, Gibco,
produced by Invitrogen Corporation, located at 1600 Faraday Ave.,
Carlsbad, Calif. 92006, USA), 10% fetal bovine serum (HyClone,
Rockford, Ill.), 1% penicillin/streptomycin (Gibco, Carlsbad,
Calif.) and 1% L-glutamine (Gibco, Carlsbad, Calif.), with
appropriate growth factors added to it for enhancement of growth
and proliferation. Cultured cells were fed every two to three days,
and split 1 to 3 at confluence. Cells were used on the passages 3
to 5 for the experiment.
[0055] Each mesh was coated with a mixture of bovine and rat tail
collagen (Gibco, Carlsbad, Calif.) in a tissue culture hood with an
aligned appearance. The liquid collagen mixture was neutralized
using NaOH. Cell-seeded collagen constructs were prepared by first
casting an acellular collagen solution and then adding a total of
3.times.10.sup.6 cells for each cell type to it, before the
collagen had set. After placing the Stainless Steel meshes among
the solutions, the constructs were incubated at 37.degree. C. in a
5% CO.sub.2 humidified incubator for polymerization. This method
ensures that collagen constructs have uniform cell density
(3.times.10.sup.6 cells/cm.sup.2) after gel formation. The tissue
constructs were cultured at 37.degree. C. with replacement of
culture media every two days. To achieve a phenotype similar to the
natural valve leaflets in-vivo, the cells in the next layers were
plated over the constructs at time intervals of two weeks and the
next layer was constructed around the deeper layer in a similar
method that has been described in the beginning of this paragraph.
The media was also supplemented with ascorbic acid (e.g., produced
by Sigma-Aldrich Inc., located at 3050 Spruce Street, St. Louis,
Mo. 63103, USA) as an additive to promote matrix production. To
increase the rate of extracellular matrix production, 10 ng/ml of
TGF-.beta.1 (e.g., produced by R&D Systems Inc., located at 614
McKinley Place Northeast, Minneapolis, Minn. 55413, USA) was added
to the collagen gels in each layer. These cultures were later on
compared to the control group with no TGF-.beta.
supplementation.
[0056] In one aspect, the tissue may be suitable for applications
in which strong composition of the membrane is essential, including
but not limited to, heart valves and vascular grafts. For further
understanding, FIGS. 3A and 3B provide images that depict the scale
and size of the mesh and corresponding tissue. For example, FIG. 3A
is an image of a stainless steel mesh 102 with a surface area of
about one square centimeter Additionally, FIG. 3B is a macroscopic
view of the engineered tissue 100 after three months of cell
culture. The outer surface shown in FIG. 3B is the endothelial
layer or the third layer. Seeding the third layer completely
concealed the mesh 102 and formed a smooth, confluent surface
around the construct. Although the third layer concealed the mesh
102, the metallic mesh 102 can still be seen inside the tissue.
[0057] FIG. 4A and FIG. 4B are scanning electron micrographs (SEM)
images of the first layer of cells. FIG. 4A shows the smooth muscle
cells 400 as being attached over the mesh 102. FIG. 4B shows the
first layer of tissue (i.e., the smooth muscle cells 400) compacted
during the culture period, which confirmed the expression of
alpha-SMA, as its expression.
[0058] FIG. 5A is a top-view of the SEM image taken after culturing
the second layer of cells containing fibroblasts/ myofibroblasts.
Formation of ECM and a confluent layer around the construct are
visible. Alternatively, FIG. 5B shows a side-view of the SEM image.
The arrow in FIG. 5B indicates a single fibroblast cell 500. Both
FIG. 5A and FIG. 5B show fibroblast cells 500 in the second layer.
Addition of TGF-.beta. increased the number of cells with either
fibroblasts or myofibroblasts in the second layer.
[0059] FIG. 6A through FIG. 6D show confocal microscopy images of
the cell culture at the end of the eighth week, with and without
addition of TGF-.beta.. FIG. 6A shows the control group from a
top-view, without TGF-.beta. added. FIG. 6B shows the control group
from a side-view without TGF-.beta. added. Alternatively, FIG. 6C
is a top-view image of the cell culture with TGF-.beta. added to
the cell culture. FIG. 6D is a side-view image, showing the cell
culture with TGF-.beta. added to the cell culture. As shown between
FIGS. 6A through 6D, greater extracellular matrix deposition is
observed when TGF-.beta. is added, in comparison to control groups.
DAPI (i.e., 4',6-Diamidino-2-Phenylindole, Dihydrochloride)
staining of nuclei in the construct shows that the number of cells
at the surface of the mesh increased progressively in TGF-.beta.
groups, and the groups treated with TGF-.beta. eventually formed a
thicker tissue around the mesh.
[0060] FIGS. 7A through 7D show SEM images taken after eight weeks,
depicting the three layers of tissue tightly enclosing the
stainless steel mesh. FIG. 7A shows the endothelial surface layer,
the smooth structures 108, covering the construct in a confluent
manner. FIG. 7B shows that after eight weeks, the tissue shows
three different cell layers in sequence, 108 is the surface
endothelial layer, 106 is the middle fibroblast and myofibroblast
layer, and 104 is the base layer of smooth muscle cells. FIG. 7C
and FIG. 7D show that the mesh 102 is tightly integrated with the
tissue membrane, with FIG. 7C further illustrating that the cells
104 are penetrating through the mesh 102 opening holes. It can be
observed that adding the second and the third layers improves
production of the ECM (mainly collagen and glycosaminoglycans) that
covers the mesh, forming a confluent smooth surface with
endothelial cell lining in both experimental groups.
[0061] As noted above, the metal mesh is any suitable material that
can operate as scaffolding for a tissue. Further, the mesh can be
in any form, non-limiting examples of which include being braided
or flat (e.g., the mesh is fabricated as sheet of punched wire mesh
or with a woven pattern). In another aspect, a Nitinol metal mesh
scaffold may be used instead of stainless steel metal mesh for the
scaffold. For scale comparison, FIG. 8A shows multiple sheets of
one centimeter by one centimer Nitinol mesh 800 in relation to a
United States one cent coin 802. In production of the tissue, the
Nitinol metal mesh 800 is etched with acid in the same process used
for the Stainless Steel metal mesh. In this non-limiting example,
the mesh 800 is made of a superelastic Nitinol sheet with the
thickness of 76 microns etched as a network of holes with 240
microns diameter and the central distance of 320 microns. For the
heart valve leaflet application, a sheet that is 25 microns thick
is used, which provides the desired elastic recoil of the leaflets.
In this aspect, the mesh 800 is cut to the shape of a heart valve
leaflet. The Nitinol mesh is seeded with cells in the same manner
as the described for the Stainless Steel mesh. An example of the
resulting scaffold 100 that is grown for 3 months is shown in FIG.
8B.
[0062] As noted above, the scaffold of the present invention can be
incorporated into any suitable tissue based item, a non-limiting
example of which includes a vascular graft. As another non-limiting
example and as shown in FIGS. 9A through 9C, the scaffold may be
incorporated into a tissue heart valve that mimicks the natural
heart valve. The tissue heart valve comprises a flexible frame
having a saddle-shaped base 901 and at least two upstanding posts
902 (or three as depicted), which divide the base into at least two
portions (or three as depicted), together with tissue leaflets 903
formed from the tissue described in this application. The posts 902
can be formed at opposite ends of a diameter of an undistorted base
or, as depicted three (or more) posts 902 are placed at regular
intervals around the base.
[0063] The tissue leaflets 903 each having a periphery consisting
of a free portion 906 extending between the tips of posts 902 and a
fixed portion secured, sealed or sutured to corresponding sides of
the posts 902 and the adjacent portion of the base 901. The
leaflets 903 are made of a mesh material, such as but not limited
to superelastic Nitinol mesh (or Stainless Steel or any other
suitable mesh material). The superelastic mesh acts as a structure
that defines the shape of the leaflets 903 and can be a structure,
such as but not limited to a mesh with arranged or unarranged
holes. The mesh can be fabricated, such as but not limited to a
sheet of punched wire mesh or with a woven pattern.
[0064] To use the heart valve shown in FIGS. 9A through 9C, the
saddle-shaped base 901 is attached to the circumference of the
auriculoventricular orifice, preferably through an intermediate
suture ring 904, whereby the base can deform from a substantially
circular shape to the shape of the orifice simultaneously, as is
the case with the natural heart valve. In a valve replacement, the
posts 902 may be disposed at regular intervals round the
undistorted base, or at other intervals as needed, for example, by
the anatomical requirements of coronary ostia in aortic valve
replacement.
[0065] The flexible frame (i.e., saddle-shaped base 901 and at
least two upstanding posts 902) is formed of any suitably flexible
yet durable material. As a non-limiting example, the flexible frame
is desirably formed of Elgiloy covered with a woven polyester cloth
912 (such as but not limited to Dacron cloth, or any other suitable
covering material), with the differential flexibility afforded by
differing thicknesses of the frame material to either side of the
posts and/or differing thicknesses of Eligiloy at each portion of
the posts. It is designed to be compliant at the orifice and
commissures to reduce the closing loading shocks at the commissure
tips and free margin of the leaflets. The suture ring 904 can
contain inserts of silicone rubber and non-woven polyester. At
least two contrasting marking sutures 905 are located on the suture
ring 904. The marking sutures 905 are intended to aid in the proper
orientation for implanting the prosthesis. The posts 902 desirably
merge at each side into the respective arcuate portions of the
saddle-shaped base 901, with the merging preferably being by way of
a continuous curve from the rounded tip of one post 902 to the
rounded tip of the other post 902.
[0066] For example in a tri-leaflet valve, the shape of each
leaflet 903 preferably corresponds to a portion of a surface of a
cone, which portion is defined by the intersections on the conical
surface of three flat planes with sixty degree angles together. The
three flat panes having peripheries on the conical surface
corresponding in length respectively to the circumference of the
saddle-shaped base and the distance between the tips of the posts
of the frame. A forth intersection is included on the conical
surface of a curved plane that is concave towards the apex of the
cone and intersects the three mentioned flat planes at opposite
sides of the cone. The spacing of the flat planes and the curvature
of the curved plane are such that the development of the curved
plane on the conical surface matches in length and curvature a
continuously blending of the curve of one arcuate portion of the
saddle-shaped base and the adjacent sides of the posts, so that no
moulding or stress-fixing of the leaflet material is required.
[0067] For further understanding of the scaffold nature of the
heart valve, FIG. 9A depicts the heart valve with the mesh (such as
Nitinol mesh 800) that is the underlying base structure of the
leaflets 903. Specifically, FIG. 9A illustrates the heart valve and
its scaffold without the biological matrix. FIG. 9A includes an
enlarged view 910 of the mesh 800 to illustrate a non-limiting
example of a mesh pattern and the holes therethrough. Further, as
shown in FIG. 9B, the three layers are grown on top of the Nitinol
mesh 800. Specifically, shown is the first layer 104 of smooth
muscle cells, the second layer 106 of fibroblast and myofibroblast
cells and the third layer 108 of endothelial cells. Finally, FIG.
9C illustrates a resulting heart valve, where the outer layer of
each leaflet 903 is the third layer 108 (or endothelial cells).
[0068] For further understanding of a suitable base structure, FIG.
9D illustrates components of the heart valve as depicted in FIG.
9A. Shown in FIG. 9D is the flexible frame that includes the
saddle-shaped base 901 and at least two upstanding posts 902. The
suture ring 904 is also depicted in FIG. 9D, along with the suture
material 914. Further, the leaflets 903 are shown, including an
enlarged view 910 of the mesh to illustrate an example of the mesh
pattern.
[0069] As shown, the leaflets 903 can be attached together to form
a dimensionally stable and consistent coapting leaflet subassembly
916 when subjected to physiological pressures. Then each of the
leaflets 903 of the subassembly 916 is aligned with and
individually sewn to the frame (i.e., the saddle-shaped base 901
and posts 902), typically from one commissure tip (i.e., post 902),
uniformly around the leaflet 903 cusp perimeter, to the tip of an
adjacent commissure tip (post 902). The frame (base 901 and 902) is
usually covered with cloth but can alternatively be covered with
biologic tissue. The sewed sutures 914 act like similarly aligned
staple, all of which equally take toe loading force acting along
the entire cusp of each of the pre-aligned leaflets 903. The
resulting structural assembly (i.e., the heart valve 918 depicted
at the top of FIG. 9D and also shown in FIG. 9A) thereby formed
reduces stress and potential fatigue at the leaflet suture
interface by distributing stress evenly over the entire leaflet
cusp from commissure to commissure. Thus, unlike some bioprosthetic
valves wherein leaflets are attached individually and the
peripheral stitching of the cusps terminates before the tips of the
commissures, producing a potential stress point, the produced valve
assembly has uniform stitching from commissure tip to commissure
tip and consistently aligned coapting leaflet mating edges. This is
further illustrated in FIG. 9E, which provides various view-point
illustrations of the tri-leaflet heart valve to clearly illustrate
the shape of the valve assembly (i.e., tri-leaflet heart valve) and
its leaflet mating edges. Finally and for further illustration,
FIG. 9F provides an illustration of the tri-leaflet scaffold that
is depicted in FIGS. 9A and 9D.
[0070] FIG. 10A and FIG. 10B provide yet another example of a
tissue based item that can be adapted or formed to incorporate the
scaffold. For example, FIG. 10A is a schematic representation of a
blood vessel, depicting the various components of an actual blood
vessel. Alternatively, FIG. 10B illustrates the scaffold formed as
a blood vessel. As shown, the scaffold in this example includes the
base Nitinol mesh 800 that is provided in a tubular wire mesh form
to mimic the shape of a blood vessel. The corresponding tissue is
grown around the Nitinol mesh 800. Thus, as can be appreciated, the
present invention enables for the generation of a variety of
scaffolds that are strong enough to resist forces that exist inside
a body, while possessing biocompatible surfaces.
* * * * *