U.S. patent application number 10/722279 was filed with the patent office on 2004-06-03 for cardiac valve replacement.
Invention is credited to Hoffman-Kim, Diane, Hopkins, Richard A..
Application Number | 20040106991 10/722279 |
Document ID | / |
Family ID | 26891212 |
Filed Date | 2004-06-03 |
United States Patent
Application |
20040106991 |
Kind Code |
A1 |
Hopkins, Richard A. ; et
al. |
June 3, 2004 |
Cardiac valve replacement
Abstract
The invention provides a replacement heart valve which contains
an acellular matrix as a structural scaffold. The scaffold is
seeded with isolated myofibroblasts and/or endothelial cells prior
to implantation into a recipient mammal.
Inventors: |
Hopkins, Richard A.;
(Providence, RI) ; Hoffman-Kim, Diane;
(Providence, RI) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS,
GLOVSKY and POPEO, P.C.
One Financial Center
Boston
MA
02111
US
|
Family ID: |
26891212 |
Appl. No.: |
10/722279 |
Filed: |
November 24, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10722279 |
Nov 24, 2003 |
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09828768 |
Apr 9, 2001 |
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6652583 |
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60195673 |
Apr 7, 2000 |
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Current U.S.
Class: |
623/2.13 ;
435/325; 623/901 |
Current CPC
Class: |
A61L 27/3895 20130101;
A61F 2/2415 20130101; A61L 27/3804 20130101; A61L 27/3843 20130101;
A61L 27/507 20130101; A61L 2430/20 20130101 |
Class at
Publication: |
623/002.13 ;
623/901; 435/325 |
International
Class: |
A61F 002/24 |
Claims
What is claimed is:
1. A bioprosthetic heart valve comprising an acellular matrix and
isolated myofibroblasts wherein at least 60% of the total collagen
produced by said myofibroblasts is type I collagen.
2. The valve of claim 1, wherein said myofibroblasts produce at
least 2-fold greater type I collagen compared to type III
collagen.
3. The valve of claim 1, wherein said myofibroblasts produce one or
more extracellular matrix components selected from the group
consisting of fibronectin, elastin, and glycosaminoglycan.
4. The valve of claim 3, wherein said glycosaminoglycan is
chondroitin sulfate or hyaluronic acid.
5. A valve comprising an acellular matrix and an isolated
myofibroblast, wherein less than 25% of total collagen production
by said myofibroblast is type III collagen.
6. The valve of claim 5, wherein less than 20% of total collagen
production by said myofibroblast is type III collagen.
7. The valve of claim 5, wherein less than 15% of total collagen
production by said myofibroblast is type m collagen.
8. The valve of claim 5, wherein said myofibroblast is derived from
mammalian heart leaflet interstitial tissue.
9. The valve of claim 5, wherein said myofibroblast is derived from
a mammalian vascular or dermal tissue.
10. The valve of claim 5, wherein said myofibroblast is derived
from human heart leaflet interstitial tissue.
11. A method of enhancing production of type I collagen by an
isolated myofibroblast, comprising culturing said myofibroblast
under pulsatile flow conditions.
12. The method of claim 11, wherein said myofibroblast is cultured
in the presence of basic fibroblast growth factor.
13. The method of claim 11, wherein said myofibroblast is cultured
in endothelial cell-conditioned media.
14. The method of claim 11, wherein said myofibrobast is cultured
in the presence of an isolated endothelial cell.
15. A method of enhancing viability and contractile activity of
myofibroblasts in vitro comprising culturing said myofibroblast
under pulsatile flow conditions.
16. The method of claim 15, wherein said myofibroblast is cultured
in endothelial cell-conditioned media.
17. The method of claim 15, wherein said myofibrobast is cultured
in the presence of an isolated endothelial cell.
18. The method of claim 15, wherein said myofibroblast is cultured
in the presence of a purified endothelial cell-derived growth
factor, wherein said growth factor inhibits apoptosis of said
myofibroblast.
19. An isolated myofibroblast, wherein said myofibroblast is
genetically altered to increase type I collagen production relative
to type III collagen production.
20. A bioprosthetic heart valve comprising the myofibroblast of
claim 19.
21. A method of manufacturing an artificial heart valve, comprising
(a) providing an acellular matrix, (b) seeding said matrix with
isolated myofibroblasts; and (c) culturing said myofibroblasts
under pulsatile flow conditions.
22. The method of claim 21, wherein said myofibroblasts are derived
from an intended recipient from an intended recipient of said
centrifugal heart valve.
Description
[0001] This application claims priority to provisional patent
application U.S.S.N 60/195,673, the entire contents of which are
hereby incorporated by reference.
BACKGROUND
[0002] This invention relates to cardiac valve replacement.
[0003] Cardiac valve diseases are prevalent clinical problems,
usually requiring prosthetic replacement. Valves can become
diseased or damaged from a variety of causes. Congenital defects
may result in abnormally formed valves. Infections such as
rheumatic fever and bacterial endocarditis can lead to valve
damage.
[0004] The first prosthetic valvular device was implanted in 1952,
and a variety of mechanical, bioprosthetic, and homograft valves
are presently in use. Thromboembolic events and sudden structural
failure are problems associated with traditional mechanical valves.
Bioprosthetic xenograft replacement valves have been developed to
reduce the risk of such problems. Xenograft valves are typically
porcine or bovine. However, such valves are limited in their
durability, as calcification and fibrotic sheath formation often
lead to stenosis and regurgitation, with a 40% reoperation rate
8-10 years after implantation. Homograft valve transplants are
limited by immune and inflammatory recipient responses, limited
donor cell viability, and complex matrix issues resulting in
degradation of mechanical performance properties.
SUMMARY
[0005] The invention provides an improved replacement cardiac
valve. The bioprosthetic heart valve contains an acellular matrix
as a structural scaffold and isolated myofibroblasts. The acellular
matrix is preferably an acellular homograft, an acellular
xenograft, or a synthetic matrix. The matrix is contacted with
isolated myofibroblasts, which are allowed to cellularize the
matrix. The myofibroblasts are resistant to dedifferentiation
during culture prior to implantation and after implantation into a
recipient individual. At least 60% of the total collagen produced
by the myofibroblasts is type I collagen. Preferably, the
myofibroblasts produce at least 2-fold more type I collagen
compared to type III collagen. Reduced type III collagen production
is critical to minimizing scar tissue formation in the replacement
valve recipient. Accordingly, less than 25%, more preferably less
than 20%, and most preferably less than 15% of total collagen
production by valve myofibroblasts is type m collagen.
[0006] In addition to increased type I collagen production, the
myofibroblasts secrete extracellular matrix components, including
but not limited to, fibronectin, elastin, and glycosaminoglycans,
such as chondroitin sulfate or hyaluronic acid. The myofibroblast
cells are cultured in the presence of factors which inhibit
dedifferentiation. The cells are cultured in the presence or
absence of an acellular matrix or scaffold. For example, the cells
are maintained in an endothelial cell-conditioned media, or grown
in the presence of endothelial cells. The two cell types may be in
direct contact with one another, e.g., in a coculture, or separated
by a membrane which allows diffusion of soluble factors but
prevents cell-to-cell contact
[0007] The term "isolated" used in reference to a particular cell
type, e.g., a myofibroblast or endothelial cell, means that the
cell is substantially free of other cell types or compositions with
which it naturally occurs. For example, isolated myofibroblasts are
obtained from solid heart leaflet tissue but are separated from
other cell types which are present in heart leaflet interstitial
tissue. Cells are "isolated" when the particular cell type is at
least 60% of a cell population. Preferably, the cells represent at
least 75%, more preferably at least 90%, and most preferably at
least 99%, of the cell population. Purity is measured by any
appropriate standard method, for example, by fluorescence-activated
cell sorting (FACS) using cell type-specific markers described
herein. A population of cells used to cellularize an acellular
valve structure or synthetic structure may be a mixture of two or
more different cell types, each of which is isolated. For example,
valves are colonized with a mixture of isolated myofibroblasts and
isolated endothelial cells. An acellular or decellularized valve is
one which is synthetic (not derived from a living organism) or one
which has been treated to remove at least 85% of the cells with
which it is naturally associated. Preferably, 90%, 95%, 99% or 100%
of the cells with which the donor valve is associated in vivo are
removed.
[0008] The myofibroblasts used to cellularize a valve matrix are
obtained from a variety of tissue sources, e.g., cardiac, vascular,
or dermal tissue. Preferably, the cells are derived from a human
donor. Preferably, the cells are derived from histocompatible
(e.g., autologous) mammalian heart leaflet interstitial tissue such
as human heart leaflet interstitial tissue. Alternatively, the
cells are derived from other tissue sources, e.g., dermal tissue,
and cultured under conditions which promote a myofibroblast-like
phenotype. The cells are syngeneic with respect to the intended
recipient of the replacement valve.
[0009] To inhibit dedifferentiation of myofibroblasts, the cells
are maintained in the presence of one or more cell signaling or
growth factors which favor the leaflet myofibroblast phenotype
(i.e., contractile and secretory function). The cells are
maintained in static culture conditions or subjected to pulsatile
flow culture conditions. Growth factors include basic fibroblast
growth factor (bFGF). As is discussed above, the cells are cultured
in endothelial cell-conditioned media or in physical contact with
endothelial cells. Myofibroblasts may be cultured in the presence
of a purified or recombinant growth factor. Preferably, the growth
factor is derived from an endothelial cell, e.g., purified from
endothelial cell conditioned media. The factor is purified using
methods known in the art such as standard chromatographic
techniques or recombinant cloning technology. A cell signaling
factor is distinguished from a growth factor in that a signaling
factor influences phenotype (e.g., secretory or contractile
activity) rather than growth rate.
[0010] Conditioned media is fractionated by size and charge. The
ability of each fraction to promote and maintain the myofibroblast
phenotype is assessed using methods known in the art, e.g.,
qualititative evaluation by immunocytochemistry and histology to
measure contractile and synthetic properties and quantitative
evaluation using assays for matrix components including collagen,
elastin and glycosaminoglycans. Proteins from the fraction(s) with
the highest activity are purified and sequences using known
methods. A secretory cell, e.g., one that has been genetically
modified to produce a signaling factor, a growth factor or matrix
component, is used in coculture with isolated myofibroblasts. For
example, the secretory cell is of non-endothelial and
non-myofibroblast origin.
[0011] Myofibroblast cells are cultured under pulsatile flow
conditions to enhance production of type I collagen and minimize
dedifferentiation. Cellularized valves cultured under such
conditions assume the functional anatomy of a native valve. For
example, the valve leaflets contain a monolayer of endothelial
cells on the external layer and myofibroblasts in the inner layers.
The leaflet interstitium contains a non-homogeneous matrix of one
or more layers with myofibroblasts present in all layers and with
collagen fibrils oriented in more than one direction. The cell
culture conditions inhibit apoptosis of a myofibroblast that has
been removed from a donor mammal, i.e., a harvested, cultured,
transformed or transplanted myofibroblast. The culture method
enhances viability and contractile activity of myofibroblasts in
vitro.
[0012] Also within the invention is a genetically-modified
myofibroblast. For example, the fibroblast is genetically modified
to confer a myofibroblast phenotype, e.g., matrix synthetic
capability, contractile capability The modified fibroblast produces
increased levels of collagen I (compared to a normal, untreated
fibroblast), fibronectin, or glycosaminoglycans. The cells may also
be modified to express recombinant actin and myosin or heparin.
Genetically-altered cells which have colonized a replacement heart
valve are useful as an in vivo recombinant protein delivery system
to deliver therapeutic polypeptides such as anticoagulant or
antithrombotic agents.
[0013] A method of manufacturing an artificial heart valve includes
the steps of (a) providing an acellular matrix, (b) seeding the
matrix with isolated myofibroblasts; and (c) culturing the
myofibroblasts under actual or biochemically simulated pulsatile
flow conditions. Optionally, the matrix is seeded with additional
cell types such as endothelial cells and/or secretory cells. The
tissue culture media includes growth and cell signaling factors,
e.g., those which are present in endothelial cell-conditioned
media. Alternatively, factors are isolated from conditioned media,
recombinant, or synthetic.
[0014] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described
herein. All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In the case of conflict, the present specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and are not intended to
be limiting.
[0015] Other embodiments and features of the invention will be
apparent from the following description thereof, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIGS. 1A-B are photomicrographs. FIG. 1A shows a native
leaflet, and FIG. 1B shows a decellularized leaflet.
[0017] FIG. 2 is a photomicrograph showing a decellularized
pulmonary valve 17 days after implantation showing the cusp region
of the valve. Loss of trilaminar histologic appearance is typical
of semilunar valves. Organizing thrombus is labeled on the outflow
surface of the cusp. Fibrin deposition was seen on the inflow
surface of the cusp as well as fibrin insudation into the
ventricuaris and spongiosa. There was no evidence of host cell
infiltration into the cusp. Movat pentachrome stain
(100.times.).
[0018] FIG. 3 is a photomicrograph of a decellularized pulmonary
valve homograft 17 days after implantation showing the basal region
of the valve. Residual cardiac myocytes have elicited an immune
response as demonstrated by the presence of plasma cells,
mononuclear cells, and macrophages at the site of the residual
cells. Movat pentachrome stain (100.times.).
[0019] FIGS. 4A-H are photomicrographs of cells derived from a
tricuspid valve biopsy. Figs. A-B show cells stained with an
antibody specific for alpha smooth muscle actin; FIGS. 4C-D show
cells stained with an antibody specific for vimentin; FIGS. 4E-F
show cells stained with an antibody specific for fibronectin; and
FIGS. 4G-H show cells stained with an antibody specific for
chondroitin sulfate. FIGS. 4A, C, E, and G are phase contrast
micrographs, and FIGS. 4B, D, F, and H are fluorescent
micrographs.
[0020] FIG. 5 is a line graph showing cell proliferation of cells
derived from a tricuspid valve biopsy.
[0021] FIGS. 6A-B are photomicrographs of a section of a Photofix
bovine pericardium seeded in vitro with myofibroblasts cultured
from the sheep tricuspid valve. FIG. 6A is a phase contrast
micrograph showing the results of staining with hematoxylin and
eosin (myofibroblasts are indicated with an arrow). FIG. 6B is a
fluorescent micrograph showing the results of labeling with an
anti-vimetin antibody.
DETAILED DESCRIPTION
[0022] The presence of viable functional myofibroblast cells in the
cardiac valve is essential for leaflet contractility, production of
extracellular matrix, and thus for maintenance of proper valve
function. Valves devoid of such cells lack the abilities to grow,
repair, and remodel. The replacement valve of the invention is a
"personal" valve, containing cells derived from histocompatible
tissue, such as from the valve recipient patient or neutered
non-antigenic cells. The cells dwell within a non-immunogenic
acellular matrix support from a donor. The donor valve scaffold is
derived from a syngeneic, allogeneic, or xenogeneic donor. The
methods are also applicable to matrices other than homografts.
Optionally, the scaffold is modified to promote ingrowth and avoid
regurgitation. Alternatively, the scaffold is constructed in vitro.
Advantages of such a valve include elimination of need for immune
suppression when transplanting cells from a donor and elimination
of biocompatibility concerns which accompany the use of
biomaterials in tissue engineered valves.
[0023] For example, an adult or pediatric valve replacement is made
which approximates a normal, unstressed native valve, both in terms
of numbers and amounts as well as types of cells and matrix. The
valve is fully hemodynamically functional, without need for
anticoagulation or immunosuppression, with durability extending
potentially to the recipient's natural life-span.
[0024] The juvenile sheep chronic implant model of heart valve
transplantation is an art recognized model for human
transplantation. Ten cryopreserved sheep homograft valves and 5
cryopreserved human xenograft valves were transplanted into the
pulmonary position of sheep. All were evaluated via ECHO. The data
indicate that the homografts were predominantly acellular at 20
weeks. Cell culture of sheep leaflet interstitial cells are
cultured as described below and seeded onto valve scaffold. The
recellularized valves are evaluated using the sheep model.
[0025] Bioprosthetic Valve Components
[0026] A structure which acts as a scaffold is colonized by living
cells. The scaffold is obtained from a human homograft cardiac
valve (either pulmonary or aortic); a xenograft cardiac valve
(e.g., porcine or bovine). Alternatively, it is made from a
synthetic polymeric material, (e.g., polylactic/polyglycolic acid).
The structure is acellular and has the geometry of a native cardiac
valve. In the case of the homograft or xenograft valve, cells are
removed from the structure using methods known in the art, e.g., as
described in U.S. Pat. No. 5,843,182 or WO 96/03093. Acellular
human homograft cardiac valves that have been rendered acellular
are preferred because of their low antigenicity and similarity to
native human valve in geometry and molecular composition. Other
compositions such as plastic, metal, or cloth can be used as the
valve structure.
[0027] Mechanical Properties
[0028] The cardiac valve replacement approximates a native
mammalian heart valve with regard to hydrodynamics and durability.
The valve is strong, opens with a minimal transvalvular pressure
gradient, and exhibits minimal regurgitation upon closure. The
valve therefore produces a minimal transvalvular pressure gradient,
and minimal regurgitation, turbulence, shear stress, stagnation,
and resistance to flow. Values for each of the parameters listed
above are calculated using methods known in the art. Values for a
replacement valve are calculated relative to properties of a native
value. Using the properties of a native valve as a baseline, a
replacement valve is preferably characterized by numerical values
of each property in the range of 5-25% of the native valve
measurements (less than 5% being close to ideal). Instruments form
Dynatek Dalta (Galena, Mo.) are used to evaluate mechanical
properties of a valve.
[0029] Fatigue resistance is measured by applying cyclic loading to
the valve, and plotting stress versus logarithm of the number of
cycles to failure.
[0030] A pulse duplicator is used to duplicate physiological flow.
A pulsatile pressure gradient corresponding to flows of 2-7L/min.
at 70-160 cycles per minute is applied. Differential pressure
transducers, flowmeters, and laser Doppler anemometry instruments
are inserted into the path to allow measurement. Transvalvular
pressure drop, regurgitation, turbulence, stagnation, and high
shear in the flow path are evaluated.
[0031] An accelerated life-cycle tester is used to measure wear and
fatigue, to assess long term durability. At least 380 million
cycles are applied to approximate at least 10 years of life.
Physiologic pressure gradients are applied. Scanning electron
microscopy is used to evaluate wear depth.
[0032] Myofibroblasts and Other Cell Types for Cellularization of
Valve Structures
[0033] The biological valve is decellularized prior to culture with
isolated myofibroblasts. The decellularized valve contains other
extracellular matrix components such as collagen which confers upon
the valve general tensile strength, proteoglycans which absorb
stress, confer flexibility, regulate the extent of collagen fibrils
crosslinking, and elastin for leaflet coaptation or valve
closure.
[0034] Cells e.g., isolated myofibroblasts, are obtained from a
donor mammal. Preferably, the mammal is a human, and more
preferably, the tissue is obtained by biopsy from the individual to
be treated. For example, cells are surgically removed from heart
valve tissue, or elsewhere (e.g., artery, vein, dermis). The cells
are cultured ex vivo to expand the cells. Alternatively, cells are
obtained from human cadaver tissue, cultured to expand cell number,
and used to cellularize a valve scaffold.
[0035] The scaffold is contacted with cells (e.g., myofibroblasts
or myofibroblast-like cells, in the presence or absence of
endothelial cells). Replacement valves are colonized with cells in
a manner which resembles a naturally-occurring valve.
Naturally-occurring cardiac valve leaflets have three internal
layers--ventricularis, spongiosa, and fibrosa. Endothelial cells
are present in a single layer around the leaflet's blood-contacting
surface. Myofibroblasts are found throughout the 3 layers, with the
sparsest population in the fibrosa. Myofibroblasts are aligned with
the collagen fibrils in the matrix of the valve. The replacement
valve, recellularized as described herein, approximates a
naturally-occurring valve, as follows. The ventricularis contains
myofibroblasts, multidirectionally oriented collagen, and extensive
elastin, which is perpendicular to the free edge. The spongiosa
contains myofibroblasts, loosely arranged collagen, and
proteoglycans, including chondroitin sulfate and hyaluronic acid.
The fibrosa contains fewer myofibroblasts than the other layers, a
small number of elastic fibers, and dense collagen that is
circumferentially oriented, crimped when relaxed, and elongated
under pressure.
[0036] Cells incorporated within the valve matrix internal layers
are myofibroblasts, i.e., the cells have dual biological function:
matrix synthesis and contractility. Myofibroblast phenotype is
assessed by immunocytochemistry with the following antibodies:
monoclonal anti-alpha-smooth muscle actin, monoclonal
anti-vimentin, anti-desmin, monoclonal anti-light chain myosin,
monoclonal anti-alpha-tubulin, monoclonal anti-cellular
fibronectin, monoclonal anti-chondroitin sulfate (SIGMA, St. Louis,
Mo.), and monoclonal anti-prolyl-4-hydroxylase (Dako, Carpintera,
Calif.). The localization and the ability of the cells to
synthesize matrix components are assessed by standard histological
methods (e.g., Movat's pentachrome stanin), evaluation of mRNA for
collagen type I, collagen type III, and elastin, and by
incorporation of proline for collagen and sulfate or glucosamine
for proteoglycans.
[0037] Endothelial cell phenotype is assessed by
immunocytochemistry with antibodies to factor VIII (polyclonal
antibody, BioGenex, San Ramon, Calif.) and to CD31 (monoclonal
antibody, Dako, Carpintera, Calif.), and by incorporation of
DiI-labeled acetylated low density lipoprotein (Biomedical
Technologies, Stoughton, Mass.).
[0038] The acellular matrix is seeded prior to transplantation with
a secretory cell (alone, or in combination with other cell types
i.e. myofibroblasts, smooth muscle cells, endothelial cells). These
secretory cells (which may be modified genetically prior to
transplantation) function to attract autologous cells to migrate
into the matrix in vivo after transplantation. The cells secrete
signaling and growth factors (such as those derived from
endothelial cell-conditioned medium, as described) to attract and
maintain the differentiation of autologous cells such as the
recipient patient's myofibroblasts and endothelial cells.
[0039] Tissue Culture Conditions
[0040] Myofibroblasts are harvested from histocompatible donor
tissue, e.g., valve leaflets or dermal tissue, and cultured
according to known methods, (e.g., Messier et al., 1994, J. Surg.
Res. 57:1-21) or by explant culture. For explant culture, leaflets
are scraped to remove endothelium and chopped into 1-3 mm3 pieces.
Pieces are plated in tissue culture flasks or dishes, and
myofibroblasts migrate out within 5-7 days. Fibroblasts from
vascular, dermal, or other tissue sources are cultured by the same
methods. Fibroblasts from these sources acquire the myofibroblast
phenotype with the use of dynamic tissue culture conditions and/or
cell signaling factors. Culture medium used to grow and maintain
myofibroblast cultures is M199, 5-15% fetal bovine serum,
penicillin-streptomycin.
[0041] Endothelial cells are cultured according to standard
protocols (e.g., Gimbrone, M. A., 1976, Culture of vascular
endothelium, Chapter 1 in Spaet, T. (ed.) Progress in hemostasis
and thrombosis. Vol. III. Grune and Stratton, Inc., pp. 1-28) from
femoral vein or artery, jugular vein or artery, or valve leaflet
biopsies. Cultures are generated by scraping endothelium,
collagenase treatment (0.1%), or explant cultures. Culture medium
is the same as for myofibroblast culture. Alternatively, cells are
cultured in serum-free medium with endothelial cell growth factor
(Gibco, Gaithersburg, Md.) added to promote proliferation. Collagen
or gelatin coating of tissue culture dishes or flasks is optionally
used to promote cell attachment.
[0042] Mixed cultures of myofibroblasts and endothelial cells are
cocultured with cell-cell contact. Cells for coculture are
generated by either collagenase treatment or explant culture of
unscraped leaflets. Cells are separated into isolated single-type
populations by flow cytometry using DiI-Ac-LDL to label endothelial
cells.
[0043] Cells are cultured in a mixed culture without cell-cell
contact, but with free diffusion of soluble factors. For example,
myofibroblasts and endothelial cells are separated by a cell
culture insert composed of a semipermeable membrane, i.e. cellulose
acetate. One cell type is grown on a tissue culture dish, while the
other grows on the cell culture insert. Alternatively, the two cell
types are grown on opposite sides of a semi-permeable membrane.
[0044] Conditioned medium from the co-cultures is collected and
separated into fractions by size and by charge using standard
protein and proteoglycan column isolation methods. Fractions are
analyzed for the ability to promote the myofibroblast phenotype,
and for the ability to promote recellularization of valve tissue.
Either purified myofibroblast-promoting factor or conditioned media
is used to promote growth and dedifferentiation of cells to be used
as the cellular component of replacement heart valves.
[0045] Cells are typically grown in culture prior to seeding for
1-4 weeks. The cells are maintained under standard static tissue
culture conditions, in a bioreactor (with or without rotation), or
in a pulsatile flow chamber. Cells cultured in a pulsatile flow
chamber are either myofibroblasts or mixed-type populations, e.g.,
a mixture of isolated myofibroblasts and isolated endothelial
cells.
[0046] Cells which have been genetically modified to produce
specific proteins (i.e. myofibroblast differentiation factors,
endothelial cell-myofibroblast cell signaling proteins,
extracellular matrix components) are cultured as described above.
Stem cells (whose phenotype is not permanently determined) are
cultured and driven toward a myofibroblast phenotype through
incubation with differentiation factors and/or pulsatile culture
conditions.
[0047] Endothelial cells are isolated by perfusion of vessels or
incubation of valve leaflets with collagenase. Culture medium is
changed 30 minutes after the initial culture to remove fibroblasts
and smooth muscle cells. Cells are cultured in endothelial basal
medium 131 with 10% fetal bovine serum and 2 ng/ml basic fibroblast
growth factor. Endothelial cells are identified by their uptake of
DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL).
[0048] Leaflet interstitial cells are the preferred source of
myofibroblasts. Leaflet tissue is dissected from the central third
of coronary cusps, beginning at the nodule of Arantius and ending
at a point 3-5 mm from the base. Excised tissue is incubated for 24
h in a 37 degrees C. humidified environment with 5% CO2, 95% air in
collagenase solution, then aspirated for thorough cell dispersion.
Cells are cultured in tissue culture flasks in M199 medium with 10%
FBS.
[0049] The ability of interstitial cells or isolated myofibroblast
cells to contract is assessed using known methods, e.g., Harris et
al., 1980, Science 208:177) Cells are cultured on a flexible
substrate composed of polydimethyl siloxane. The visible generation
of wrinkles on the surface of the rubber reveals cellular
contraction.
[0050] Methods for seeding the valves include diffusion of cells,
dynamic flow conditions, or direct injection. Cells cultured and
re-implanted are labeled with a fluorescent tracer prior to
implantation, to distinguish between cultured and native cells.
[0051] Genetic Modification of Cells for Matrix Seeding
[0052] DNA is incorporated into cells using standard recombinant
technology. Targeting cell specific vectors are preferred. For long
term stable transduction of recombinant DNA encoding polypeptides
to be expressed, retroviral vectors, e.g., murine leukemia viruses
such as Moloney murine leukemia virus, are used. A cell-specific
promoter such as a lysyl oxidase (LOX) promoter (Reynaud et al.,
1999, Cellular and Molecular Biol. 45:1237-1247) is used for
expression of recombinant proteins in myofibroblasts. Recombinant
polypeptides to be expressed in cells of the matrix include at
least part of the coding region of the following genes: human bFGF
(GENBANK Accession No. M27968 or J04513), human VEGF (GENBANK
Accession No. AF092127, AF092125, or M32977), human fibronectin
(GENBANK Accession No. M26179), human beta 1 integrin (GENBANK
Accession No. L24121 or U31518), human TGF-beta-1 (GENBANK
Accession No. J04431), human alpha 1 type I collagen (GENBANK
Accession No. U06669), human aortic-type smooth muscle alpha-actin
(GENBANK Accession No. M33216), or human myosin light chain 1
(GENBANK Accession No. M20642).
[0053] Signalling and Growth Factors
[0054] Soluble factors to be delivered to the matrix include
members of the families of transforming growth factors (e.g.,
TGF-beta), fibroblast growth factors (e.g., bFGF), and vascular
endothelial growth factors (e.g., VEGF). TGF-beta and bFGF are used
to promote myofibroblast growth (Khouw et al., 1999, Biomaterials
20:1815-1822), and VEGF is used to promote angiogenesis. Alpha
smooth muscle actin and myosin gene products are used to convert
fibroblasts into myofibroblasts. Extracellular matrix factors
(fibronectin, collagen, and integrins (a1b1 and a2b1 for collagen,
and a5b1 for fibronectin)) are used to promote myofibroblast
migration into the valve matrix. Ascorbic acid is used to regulate
collagen synthesis using known methods (e.g., Tajima et al., 1982,
Biochem. Biophys. Res. Commun. 106:632-7 and Grinnell et al., 1989,
Exp. Cell Res. 181:183-191)
[0055] Evaluation of Explanted Valves
[0056] The function and durability of replacement valves is
evaluated as follows. Once explanted, valves are placed either into
formalin or paraformaldehyde for evaluation at the histological,
cellular, and molecular levels, into glutaraldehyde for evaluation
by transmission electron microscopy, or into tissue culture medium
for specific cellular and molecular assays, tissue culture, or
mechanical evaluation.
[0057] Histology
[0058] Valves are sectioned with a cryostat, a microtome, or an
ultramicrotome, depending on the evaluation procedure. Histological
stains include hematoxylin and eosin, Miller's elastin stain,
Movat's pentachrome stain, and von Kossa stain. Valves are examined
for the presence of an intact endothelium and a stroma containing
matrix proteins and cells. The presence of elastin, collagen,
fibronectin, and glycosaminoglycans are assessed.
[0059] Transmission Electron Microscopy (TEM)
[0060] Cultured cells and valve sections are examined by TEM for
general morphology, cell and matrix types, and cell viability.
Cells are examined for the presence of cellular organelles
appropriate for contractile and synthetic cell types (i.e.
cytoskeletal filaments, endoplasmic reticulum), as well as
intercellular communicative junctions.
[0061] Mechanical Properties
[0062] To monitor calcification, calcium content of explanted
valves is assessed via atomic absorption spectroscopy. Evaluation
of valve mechanical properties, (e.g., tests for strength,
flexibility, low-strain-rate tensile fracture, high-strain-rate
extensibility, stress-relaxation, and forced vibration) are carried
out using known methods.
[0063] Cell and Matrix Properties
[0064] Endothelial cell function is assessed by immunocytochemistry
for von Willebrand factor, and CD31, as well as by uptake of
ac-LDL. Markers of contractile properties include smooth muscle
actin and myosin light chain by immunohistochemistry, as well as
staining with Texas red-phalloidin, which selectively labels
F-actin and has the advantages of stoichiometric binding and
negligible non-specific staining. Cytoskeletal markers include
vimentin, desmin, and tubulin, by immunohistochemistry. The ability
of myofibroblast cells to synthesize fibronectin and chondroitin
sulfate proteoglycans and to modify collagen types I and III is
determined by immunohistochemistry and in situ hybridization.
[0065] The presence of fibronectin, collagen types I and I, and
chondroitin sulfate proteoglycans (core proteins as well as
glycosaminoglycan chains) is assessed by immunohistochemistry and
Western blotting. Collagen levels are evaluated by hydroxyproline
assays known in the art, as well as with the Sircol dye assay
(Accurate Scientific, Westbury, N.J.).
[0066] Seeding of Cells Into Replacement Valve Structure
[0067] For seeding purposes, harvested primary cells are cultured
and used within culture passages 1-5 to preserve phenotype.
Myofibroblasts are seeded to populate a valve structure at cell
numbers of 10,000-150,000 per ml. The matrix or valve structure is
optionally treated prior to cell seeding to promote cell
attachment, and during the seeding process to promote migration
into internal layers, proliferation and maintenance of valve cell
phenotype. Coating compositions include cell signaling factors,
growth factors, and extracellular matrix components that were
removed from a donor valve tissue during decellularization. Such
matrix components are typically not present in the case of a
synthetic polymer valve matrix, or may need to be augmented to
facilitate seeding. For example, the structure is coated with basic
fibroblast growth factor, platelet derived growth factor,
endothelial cell growth factor, fibronectin, integrins, collagen
type I, chondroitin sulfate, hyaluronic acid, and heparan sulfate.
Factors also include cell signaling and differentiation factors
isolated from cocultures of myofibroblasts and endothelial cells,
cultured under pulsatile flow conditions.
[0068] The valve scaffold matrix is seeded with myofibroblasts
first, followed by endothelial cells. Alternatively, the matrix is
seeded with myofibroblasts, then incubated with factors to attract
endothelial cells in vivo, or the matrix is seeded with a mixed
population of myofibroblasts and endothelial cells. Alternatively,
the matrix is modified first mechanically and/or biochemically
(e.g., unique packaging and attractant vehicles are used to retain
signaling factors in contact with cells during cell growth,
migration, and differentiation). For example, the matrix is first
incubated with signaling or growth factors prior to the addition of
cells such as myofibroblasts and endothelial cells. Alternatively,
signaling proteins produced in response to energy dissipation,
which regulate increased production of alpha-smooth muscle actin
and related contractile compounds, are introduced directly (e.g.,
by contacting the matrix with a factor) or via a secretory cell (by
seeding the matrix with secretory cells expressing the factor) to
induce and/or maintain a myofibroblast phenotype.
[0069] Once cells have attached (3-24 hours), the recellularized
valve matrix is incubated under pulsatile flow conditions designed
to duplicate the cyclic opening and closing under pressure of a
native valve. Typical flow values approximate a cardiac output of
2-7.5 liters/min, with a frequency of 60-120 cycles/min and
resistances configured to duplicate back pressures of up to 120 mm
Hg for aortic valve according to standard methods. For example,
isolated myofibroblasts (in the presence or absence of endothelials
cells) are cultured with a valve structure under normal blood flow
conditions. Frequency is 70 bpm with a diastolic pressure of 70 mg
Hg and a flow rate of 5 L/min.
[0070] Pulsatile flow conditions promote and/or maintain a
myofibroblast phenotype. As is discussed above, myofibroblasts are
distinguished phenotypically by their content of alpha-smooth
muscle actin. Pulsatile flow culture conditions also promote
elevated synthesis of Type I collagen by myofibroblasts. Pulsatile
flow culture conditions for fibroblasts and myofibroblasts are
known in the art, e.g., U.S. Pat. No. 5,899,937.
[0071] Methods of Enhancing Type I Collagen Production
[0072] Myofibroblasts and/or myofibroblast/endothelial mixtures are
cultured as described above to increase type I collagen production
relative to type III collagen production. The amount of collagen in
the valve leaflet is assessed by using known methods, e.g., the
4-hydroxy-proline assay, and also by the Biocolor Sircol dye assay
(Biocolor; Accurate Scientific, Westbury, N.J.). The proportions of
collagens type I and III in the valve leaflet is an important
measure of the health of the tissue. Collagen III is present in
scar tissue or healing tissue, and amounts exceeding 15-20% are not
appropriate for a functional valve. Types of collagen are evaluated
by interrupted gel electrophoresis and by transmission electron
microscopy (TEM).
[0073] Interrupted gel electrophoresis resolves type I, III, and V
collagen, the main subtypes found in leaflets. After radiolabeling
with .sup.3H-proline, the tissue is electrophoresed on nonreducing
5% SDS polyacrylamide gel in the presence of 0.05M urea until the
dye front has migrated approximately 1/3 of the total run distance.
Then 20 microliters of b-mercaptoethanol is added to each well.
Since type III collagen is disulfide bonded, its migration is
retarded relative to the a chains of type I and V collagen until
the reducing agent is added. Therefore, the a1(III) chain can be
resolved from the a1 (1) chain. The a1(V) and a2(V) chains migrate
between the a1(I) and thea1(III) chains. The gel is soaked in 10
volumes of sodium salicylate (pH 6.0) for 30 min to enhance 3H
emission intensity and exposed to Kodak SB X-ray film for
fluorography. The ratio of type III to type I collagen will be
determined by scanning densitometry. Type I and type III collagen
are distinguished as follows: collagen I fibrils are 50-100 nm in
diameter, and collagen III fibrils are 25-40 nm (analyzed by TEM).
For example, the ratio of collagen is I:III:V=85:15:5. Orientation
of collagen fibrils varies, e.g., crimped vs. elongated, depending
on layer and pressure conditions. Collagen crosslinking is
evaluated for extent of crosslinking and type of crosslinking
(reducible vs. non-reducible, typical of load-bearing tissues).
[0074] Quantification of Collagen
[0075] Tissue is homogenized in the presence of protease
inhibitors. Quantity of collagen is measured using a standard
4-hydroxyproline assay. Alternatively, collagen content is
quantified using the Sircol dye binding assay (Biocolor, Accurate
Scientific, Westbury, N.J.) Th Sircol dye contains Sirius Red, an
anionic dye with sulphonic acid side chain groups. These groups
react with side chain groups of the basic amino acids present in
collagen. Binding is highly specific because, under the assay
conditions, elongated dye molecules become aligned in parallel with
long, rigid, helical structure of collagen.
[0076] RNA Analysis of Collagen
[0077] Collagen content is measured by detecting collagen gene
transcripts or the gene product itself.
[0078] A cDNA probe for collagen type I is subcloned into a
transcribable vector, e.g., pGEM7Z (insert: Hf677, site I: EcoR1;
site II: EcorR1). Competent E. coli are transformed and replicated
in culture to produce additional plasmid and inserts.
[0079] The probe is labeled with a detectable marker using standard
methods. For example, the probe is labeled with digoxigenin-II-UTP,
and the labeled double stranded DNAs are generated with using a
random priming reaction. In vitro transcription of DNA is used to
synthesize labeled RNA probes, both antisense and sense (via SP6
and T7 RNA polymerase promoters in the pGEM7Z plasmid).
[0080] For Northern analyis of collagen transcripts, total RNA is
isolated from native cardiac valves and from cultured cells using
methods known in the art (e.g., by a SDS lysis/acid phenol
technique. RNA is separated by electrophoresis on a 1%
agarose-formaldehyde gel, blotted onto a charged nylon membrane,
and hybridized with the collagen I cDNA probe described above.
After high stringency washes, the blots are reacted with an
anti-digoxigenin antibody, alkaline phosphatase, and BCIP/NBT.
Blots are analyzed by scanning densitometry.
[0081] In situ hybridization is also used to measure collagen
content. Cells to be analyzed (e.g., cultured cells,
genetically-modified cells, or cryosections of valve tissue) are
plated or mounted onto slides under RNAse-free conditions. The
cells or tissue sections are tixed with 4% paraformaldehyde.
Prehybridization is carried out at 37 degrees in a solution
containing formamide, Ficoll, polyvinylpyrrolidone, bovine serum
albumin, EDTA, salmon sperm DNA, yeast tRNA, and
beta-mercaptoethanol) to block nonspecific binding. Both cDNA
probes and RNA probes are used for hybridization to optimize MRNA
detection. The slides are washed under high stringency wash
conditions, and the transcripts detected as described above for
Northern blot analysis.
[0082] Quantification of Calcium
[0083] Atomic absorption spectroscopy is used for elemental
determination of calcium.
[0084] Juvenile Sheep Model of for Human Aortic Valve
Replacement.
[0085] Domestic sheep (Ovis aries): Rambouillet, Dorset, Hampshire,
Suffolk Breed mix (30, 6 per valve type) either male or female (20
to 40 weeks of age with body weight 40-50 kg) are commercially
available. Prior to implantation, the animals are certified to be
free from disease.
[0086] The sheep is given Amikacin (10 mg/kg IM) and amoxicillin
(5-10 mg/kg IM) and fasted from its daily standardized diet from
the evening prior to surgery. On the morning of surgery, the animal
is weighed, surgical sites are sheared, and the animal is
anesthetized with using standard methods.
[0087] The sheep is secured to the operating table in the left side
up lateral position. Total volume is maintained at 10 ml/kg body
weight of 99% oxygen with a 50-100 m. compensation for dead space
at a rate of 12-14 cycles per minute (ABG's checked at 15
minutes-30 minutes). Left thoracotomy is performed and the chest
entered through the fifth intercostal space. A bypass Heparin
bonded shunt is inserted from the right atrium to the distal
pulmonary artery with a roller pump head in the circuit. The
pulmonary artery is mobilized and a vascular clamp is applied just
proximal to the bifurcation and below the level of the insertion of
the inflow shunt tubing from the roller pump. The native pulmonary
valve is excised with the right ventricle being kept empty as a
consequence of the right heart bypass circuit as described above.
The tissue engineered valve or control is sutured as an
interposition graft. The proximal and distal end-to-end anastomoses
is accomplished with running 4-0 Prolene suture. The bypass shunt
is occluded and the vascular clamps removed. The homograft or
unstented bioprosthesis is oriented with the base closest to the
heart to ensure antegrade flow through the graft.
[0088] Fresh valves are harvested from one sheep and implanted into
another sheep. Valves to be frozen are harvested from an abbatoir
as a byproduct of meat packing. They are treated with antibiotics,
antifingal agents and cryopreserved. No disease transmission has
ever been documented following such treatment. Decellularized
valves is carried out using methods known in the art.
[0089] Native pulmonary valves are explanted, placed in sterile
tissue culture media, and transported in a sealed, autoclaved
container to the site of the surgery.
[0090] Following implantation and prior to closing, the thoracic
cavity is ravaged with warm saline. The fluid from the thoracic
cavity is evacuated. The pericardium is closed with Vicryl sutures.
The ribs are approximated with Vicryl sutures. The muscle and
fascia layers of the chest are approximated with Vicryl sutures
(running). The air and blood are evacuated from the thoracic cavity
via chest tube. The skin layer of the chest is closed with 2-0
Vicryl subcuticular suture. When the animal is able to breath
spontaneously, mechanical ventilation is discontinued and
supplemental oxygen substituted.
[0091] The animal is placed in the position of sternal recumbency
in intensive care. To permit the expulsion of ruminal gas, this
position is maintained until the animal regains consciousness. The
chest tube is aspirated regularly following the operation and is
removed 6 hours after surgery. The animal is awakened with the
assistance of a sling and then walked into a pen. Animals receive
weekly trans-thoracic ECHO evaluations. Trans-thoracic Doppler
echocardiography will be performed weekly to assess for stenosis or
regurgitation. Euthanasia is by overdose of pentobarbitol (360
mg/kg).
EXAMPLE 1
Tricuspid Valve as a Source of Cells for Tissue-Engineered Cardiac
Valve Replacement
[0092] Cardiac valve replacements are designed to be
non-obstructive when open, competent when closed, non-thrombogenic,
non-immunogenic, free from calcification, durable, flexible, and
strong. Pevious cardiac valve replacements, which are devoid of
cells, lack the ability to grow, repair, and remodel as a native
valve. Long term growth, repair, and remodeling functions are
especially crucial for pediatric patients. The tissue-engineered
valve replacements described herein are capable of such functions.
The replacements are living valves, containing cells derived from
the patient, dwelling within a non-immunogenic acellular matrix
support from a donor. Such valves eliminate the need for immune
suppression. In addition, the donor valve provides an inherently
biocompatible, heterogeneous matrix for optimal support of the
patient's own cells.
[0093] To design the tissue engineered valve replacement, donor
cells are removed from a cadaveric donor homograft valve.
Decellularization successfully removes greater than 90% leaflet
cells from valve leaflets (FIGS. 1A-B). The decellularized
homograft valve is repopulated using cells from the patient. An
optimal source of autologous cells for repopulation is the
tricuspid valve.
[0094] Using the juvenile sheep models, a technique that reliably
and safely biopsies the tricuspid valve was developed. The same
techniques is used to obtain patient cells to be used for
repopulation of a decellularized donor valve. Biopsies of the
tricuspid valve were cultured and evaluated as sources of cells for
valve repopulation. The tricuspid valve was accessible for biopsy
under ECHO guidance, with excellent animal survival. Post-operative
ECHO revealed no detectable effect on leaflet function. Enzymatic
digestion of biopsied tissue gave rise to endothelial cells
identified by uptake of DiI-acetylated low density lipoprotein, and
myofibroblasts identified by immunoreactivity for smooth muscle
actin, chondroitin sulfate, vimentin, and fibronectin. These cells
were expanded in culture and banked for future use. The data
indicate that tricuspid leaflet cells provide an effective source
of autologous cells for recellularization of a decellularized
homograft cardiac valve and production of a tissue engineered valve
replacement.
EXAMPLE 2
Cell Harvest and Repopulation
[0095] A decellularized matrix was obtained by removing donor cells
from a homograft valve. The matrix was repopulated with cells from
the intended replacement valve recipient. Since the repopulating
cells are autologous, the valve resists immunogenic destruction.
The anatomy and physiology are essentially identical to the native
valve. There is little or no need for anticoagulation.
[0096] The valve replacement requires a reliable source of cardiac
valve leaflet cells. Interstitial cells are harvested from the
recipient yielding an adequate supply of cells for repopulation of
the graft. Interstitial cells are safely obtained from the
recipient's tricuspid valve. The valve is accessible and contains
the cells of interest. Expansion of the harvested cells in culture
is possible and provides an ideal source of leaflet interstitial
cells.
[0097] Surgical methods were carried out using standard protocols.
Ten sheep were studied: nine female sheep and one male. The ages of
the animals varied from 20 to 60 weeks and their weights varied
from 30 to 60 kilograms. At the time of surgery, each animal was
sedated, intubated and anesthetized. The animal was placed into the
left side down position and the right neck was shaved.
Trans-thoracic ECHO was performed to document normal anatomy and
physiology. The animal was then transferred to the operating suite
where appropriate monitors, including pulse oximetry and
electrocardiography (EKG), were placed. A standard Betadine prep
was performed and the animal was sterilely draped.
[0098] A small incision was made in the right neck overlying the
carotid sheath. The tissues were divided until the carotid artery
and internal jugular (IJ) vein were identified. The IJ was
encircled with a vessel loop both proximally and distally. A small
venotomy was performed and a 0.8 cm gastrointestinal biopsy forceps
was inserted into the vein. The forceps was advanced until
resistance was felt. At this point, ECHO was used to identify the
tricuspid valve. In some cases, the forceps was visualized as it
was advanced, but in most cases it was not. The biopsy was taken by
advancing the forceps with closed jaws just past the point of
resistance. This was considered to be the passage through the
tricuspid valve. The jaws were opened and closed and the forceps
were pulled back. In the cases where ECHO was available, tethering
of the tricuspid valve was seen as the forceps were pulled back. As
the instrument was brought back up through the venotomy, hemostasis
was maintained. Three to six biopsies were taken from each animal.
The pieces were grossly evaluated, held in sterile media and placed
into cell culture. Once adequate specimens were obtained, the
venotomy was repaired with a continuous 4-0 Prolene and the
incision was closed. Anesthesia was discontinued and the animal was
allowed to recover. At the time of explantation, an ECHO was
performed. The animal was then euthanized with an overdose of
pentabarbitol. The heart was harvested and examined.
[0099] Cell culture was carried out using standard methods. Each
biopsy piece was incubated for two hours at 37.degree. C. in 1.6%
collagenase solution. Digested tissue was pelleted, resuspended and
cultured in a single well of a 24-well tissue culture flask. The
media was changed every three to five days. When approximately 85%
confluence was achieved, the cells were subcultured by incubation
with 0.25% trypsin-EDTA at 37.degree. C. for 10-15 minutes. For
immunocytochemistry, cells were fixed in 4% paraformaldehyde for 20
minutes, rinsed in 0.1M phosphate buffered saline (PBS), and
incubated with primary antibody overnight at 4C. Primary antibodies
were diluted 1:100 for alpha smooth muscle actin and vimentin,
1:400 for chondroitin sulfate, in 0.2% Triton X-100, 5% normal goat
serum in PBS. Cells were rinsed with 0.2% Triton X-100 and
incubated with rhodamine or fluorescein-conjugated goat anti-mouse
antibody diluted 1:200 in PBS.
[0100] The animals were sacrificed at several different time
points. One animal expired during the immediate post-operative
period. After four biopsies were performed, the anesthesia was
turned off and the animal was allowed to wake up. There was a delay
in extubation secondary to poor respiratory effort, although oxygen
saturation remained good at over 90% and EKG tracings were normal.
The animal was arousable, but had a decreased level of
consciousness and labored breathing. Three hours after extubation
the animal expired. Upon necropsy, it was noted that the cordae
tendineae had ruptured. The posterior leaflet was destroyed and the
valve was obviously incompetent.
[0101] Two animals were sacrificed acutely to demonstrate that the
tricuspid valve was indeed the cardiac tissue that was being
biopsied. During these two procedures, the biopsy forceps was
advanced until resistance was met. At this position it was sutured
in place and left in situ. The animal was euthanized and a right
thoracotomy was performed. Necropsy was performed with the biopsy
forceps in place. In both cases, biopsy of the tricuspid valve was
demonstrated. The posterior leaflet was noted to be between the
jaws of the forceps. There was no evidence of deviation of the
instrument into the atrial appendage. The other five animals were
survived for either 4 weeks (n=3), 5 weeks (n=1) or 15 weeks (n=1).
Prior to sacrifice each animal underwent ECHO. All animals
demonstrated normal anatomy and function of the tricuspid valve.
There was no evidence of regurgitation through this valve and the
valvular apparatus was intact. After the animal was euthanized, the
hearts were explanted and examined. All five of these animals
demonstrated normal anatomy. There was no discontinuity of the
tricuspid valve leaflets. No thickening or scarring was noted. The
cordae tendinae were normal and there was no evidence to suggest
that the endocardium had been disrupted or biopsied.
[0102] Collagenase digestion of the biopsy pieces yielded cells
that were expanded through at least 5 passages. Growth curves
demonstrated varied growth rates, dependent upon biopsy size and
plating density (FIG. 5). Cells exhibited a typical myofibroblast
morphology and stained positive for alpha smooth muscle actin,
fibronectin, vimentin, and chondroitin sulfate (FIGS. 4A-H).
[0103] These data indicate successful harvesting of leaflet
interstitial cells (e.g., myofibroblasts) from the prospective
recipient's tricuspid valve. The procedure is simple, safe and
reliable. Under controlled circumstances it is done with sedation
and local anesthetic. With the aid of ECHO, the procedure was
visualized so that the tricuspid valve leaflet can be biopsied
under guidance. Visualization makes the procedure safer by
eliminating the complication of biopsying the cordae tendinae and
disrupting the valvular apparatus. Up to 15 weeks from the time of
biopsy, there were no changes in leaflet integrity and subsequent
cardiac function.
[0104] The myofibroblast cells that are grown in culture exhibit a
normal growth pattern. A single biopsy specimen of approximately
0.8 cm can be expanded to on the order of 75 million cells after
two months in culture. Immunohistochemistry with markers for
contractile and synthetic properties demonstrates the presence of
myofibroblasts through at least ten passages.
[0105] Biopsy of the tricuspid valve is an effective method for
obtaining recipient cardiac valve leaflet cells. The biopsy of the
leaflet itself does not compromise function and animal survival is
good. Interstitial cells can be harvested and expanded in culture.
The technique described herein is safe and reliable and is an ideal
source for harvesting leaflet cells that will later be used to
populate a heart valve scaffold.
EXAMPLE 3
In vitro seeding of Photofix Bovine Pericardium
[0106] Sheets of Photofix bovine pericardium (Sulzer Carbomedics)
were seeded in vitro with myofibroblasts cultured from the sheep
tricuspid valve. Cells from passages 7-9 were placed onto the
surface of the Photofix material (approx. 7 mm.times.5 mm), which
was cultured on a Transwell cell culture insert. The seeded
Photofix pieces were cultured for 2-6 weeks in vitro in M199 media
with 10% fetal bovine serum at 37.degree. C. in a humidified tissue
culture incubator with 5% CO.sub.2.
[0107] Hematoxylin and eosin staining revealed the presence of up
to 5 layers of cells on top of the seeded Photofix material, with
cellular infiltration of the middle layers of tissue in some cases.
Cells were found to be immunoreactive for alpha smooth muscle actin
and vimentin (FIGS. 6A-B).
EXAMPLE 4
Evaluation of Valve Conduits: A Comparison of Cryopreserved
Sheep
[0108] Pulmonary Valve Allografts and Human Cryopreserved Aortic
Valve Xenografts An animal model was used to evaluate the
suitability of implanting a cryopreserved allograft or xenograft
interposed in the pulmonary artery. The art-recognized model
described above was used for the evaluation of biological valves
and emerging tissue engineered valve designs. To assess the
suitability of this model the following items were considered: 1)
the ability to assess the surgical handling characteristics of an
investigational valve conduit; 2) the ability to evaluate
hemodynamic performance using ECHO; 3) postoperative survival for
20 weeks without somatic growth compromising valve conduit
performance; 4) no anticoagulation or antiplatelet medications
required, and 5) the ability to assess valve conduit related
pathology. The model was evaluated using sheep cryopreserved
pulmonary valve allografts and human cryopreserved aortic valve
xenografts.
[0109] The cryopreservation, thawing and washing techniques were
identical to those utilized clinically. Under general anesthesia,
15 sheep, Ovis Aries, 20-40 weeks, 40-50 kg, underwent a left
thoracotomy. Cardiopulmonary bypass was initiated, native leaflets
were removed and the cryopreserved pulmonary valve allograft (n=10)
or the human cryopreserved aortic valve xenograft (n=5) was
interposed in the pulmonary artery. Transesophageal ECHO studies
were conducted on a weekly basis throughout the study. After 20
weeks the animals were sacrificed, the valve grafts were explanted
and histologic studies were conducted.
[0110] All 15 animals survived for the duration of the study. All
animals gained weight during the course of the study. There were no
anatomic constraints or surgical handling characteristics that
confounded the implantation of these grafts. After 20 weeks of
implantation the following hemodynamic findings were observed: 1)
mean transvalvular gradients for the allografts were 1.36 mm Hg at
2 weeks and 1.56 mm Hg at 20 weeks 2) mean transvalvular gradients
for the xenografts were 1.38 mm Hg at 2 weeks and 1.89 mm Hg at 20
weeks 3) regurgitation was not present in either graft type.
Mononuclear cells, histologically resembling histiocytes, were
present only within the xenograft cuspal tissue. Histologic studies
of both types of valves demonstrated: the loss of cuspal cells and
trilaminar architecture, calcification limited to the arterial
wall, fibrous sheath formation with endothelialization (primarily
on the inflow aspect of the cusps), no endothelial cells present on
the cuspal surfaces, and the absence of valve-related thrombosis,
cuspal perforations or tears.
[0111] These data demonstrate that the interposition of a
cryopreserved allograft or xenograft in the pulmonary artery
position is feasible in sheep and demonstrates excellent animal
survival. The findings also indicate that it is possible to assess
surgical handling characteristics of biological valves, long-term
hemodynamic performance and valve-related pathology in this animal
model. Twenty weeks of implantation, histological evaluation
indicated that cryopreserved grafts remain largely acellular with
calcium deposition noted to be limited to the graft wall.
EXAMPLE 5
Assessment of a Decellularized Sheep Pulmonary Valve Homogaft In
Vivo
[0112] A pulmonary valve homograft was harvested from a juvenile
sheep, stored in Lactated Ringers' solution, and decellularized as
described above. The decellularized homograft was stored in a
humidified tissue culture incubator with 5% CO.sub.2 for 47 days
prior to implantation. Standard tissue culture media, e.g., M199
media, was changed every 1-2 days for the first 15 days, then once
per week for the next 21 days. The homograft was then stored in
Lactated Ringers solution, with weekly changes until implantation.
The homograft was implanted into the pulmonary position using
standard surgical procedures.
[0113] ECHO at 7 and 17 days following implantation showed normal
hemodynamic function. The homograft was explanted at 17 days
following implantation and fixed in Macdowell Trump fixative.
Histologic evaluation was carried out using known methods.
[0114] With respect to leaflets, trilaminar structure was reduced
or gone. FIG. 2 shows the trilaminar histologic appearance of a
decellularized pulmonary valve homograft, and FIG. 3 shows the
appearance of the basal region of a decellularized valve 17 days
after transplantation. The matrix was acellular but collagen crimp
was preserved. Outflow surface of the leaflet had adherent thrombus
formation. Inflow surface of the leaflets had eosinophilic fibrin
infiltration. The opened spaces that had been seen in in vitro
decellularized valve tissue were not apparent in the explanted
valve, suggesting compression of these spaces and/or occupation of
portions of the spaces by the fibrin.
[0115] The pulmonary wall was examined and found to be acellular.
There was fibrous connective tissue outside and a capsule or bursa
formation. Myofibroblasts were present as detected by various
myofibroblast markers (FIGS. 4A-H). There was no giant cell
formation or evidence of macrophage inflammatory response.
[0116] Basal region of the cusp was also evaluated. In the region
of the cusp, there were a few donor cardiomyocytes retained. In
contrast to the more mechanical response to the pulmonary artery
wall, these residual donor cells invoked a positive cellular
inflammatory response. These data suggests that retained cellular
material invokes an immune response.
[0117] These data indicate that decellularization obviates the
immune response. The process of decellularization described herein
is reliable and effective in removing cells and cellular debris
from donor valves. Some of the mechanical changes seen in vitro in
the unloaded valve leaflet were reversed by exposure to loading
conditions in vivo. The fibrin infiltration from the ventricularis
side suggests that pore size is being maintained and that there are
pathways for infiltration of viable myofibroblasts in vivo.
Presence of pores and pathways for infiltration allow effective in
vitro recellularization of valves.
[0118] While the collagen was not viewed as foreign protein, it did
engender a sterile capsule or bursa type of response. Despite the
significant changes in the fibrous connective tissue, collagen
crimp was preserved. The fibrin infiltration suggested that there
are pathways for inserting cells. The decellularization process
does not obliterate a positive inflammatory response to any
residual cellular debris. The thrombus formation in the outflow
surface suggested that the collagen is viewed as inert, but
nevertheless thrombogenic, suggesting that anticoagulation should
be maintained until recellularization has been completed and
stabilized.
[0119] Other embodiments are within the following claims.
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