U.S. patent application number 14/600583 was filed with the patent office on 2015-06-04 for bioactive implant for myocardial regeneration and ventricular chamber restoration.
The applicant listed for this patent is Antonio BAYES GENIS, Juan Carlos CHACHQUES, Philippe JENNY, Manuel MONLEON PRADAS, Carlos Eduardo SEMINO, Nicole ZURNIEDEN. Invention is credited to Antonio BAYES GENIS, Juan Carlos CHACHQUES, Philippe JENNY, Manuel MONLEON PRADAS, Carlos Eduardo SEMINO, Nicole ZURNIEDEN.
Application Number | 20150150680 14/600583 |
Document ID | / |
Family ID | 43216442 |
Filed Date | 2015-06-04 |
United States Patent
Application |
20150150680 |
Kind Code |
A1 |
CHACHQUES; Juan Carlos ; et
al. |
June 4, 2015 |
BIOACTIVE IMPLANT FOR MYOCARDIAL REGENERATION AND VENTRICULAR
CHAMBER RESTORATION
Abstract
Bioactive implant for myocardial regeneration and ventricular
chamber support including an elastomeric microporous membrane. The
elastomeric microporous membrane being at least one non-degradable
polymer and at least one partially degradable polymer. The
non-degradable polymer is selected from polyethylacrylate and
polyethylacrylate copolymerized with a hydroxyethylacrylate
comonomer. The partially degradable polymer is selected from
caprolactone 2-(methacryloyloxy)ethyl ester and caprolactone
2-(methacryloyloxy)ethyl ester copolymerized with ethylacrylate.
The elastomeric microporous membrane further includes a nanofiber
hydrogel, and cells. The bioactive implant, having one or two
helical loops, contributes to the restauration of the heart conical
shape. Cardiac wrapping by ventricular support bioprostheses of the
present invention, having reinforcement bands spatially distributed
as helicoids, recovers the sequential contraction of the myocardium
resulting in the successive shortening and lengthening of the
ventricles, therefore improving the ejection (systolic function)
and suction of blood (diastolic function).
Inventors: |
CHACHQUES; Juan Carlos;
(Paris, FR) ; BAYES GENIS; Antonio; (Principal,
ES) ; MONLEON PRADAS; Manuel; (Valencia, ES) ;
SEMINO; Carlos Eduardo; (Barcelona, ES) ; ZURNIEDEN;
Nicole; (Riverside, CA) ; JENNY; Philippe;
(Pessac, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CHACHQUES; Juan Carlos
BAYES GENIS; Antonio
MONLEON PRADAS; Manuel
SEMINO; Carlos Eduardo
ZURNIEDEN; Nicole
JENNY; Philippe |
Paris
Principal
Valencia
Barcelona
Riverside
Pessac |
CA |
FR
ES
ES
ES
US
FR |
|
|
Family ID: |
43216442 |
Appl. No.: |
14/600583 |
Filed: |
January 20, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13660086 |
Oct 25, 2012 |
8968417 |
|
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14600583 |
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PCT/EP2011/056576 |
Apr 26, 2011 |
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13660086 |
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61327864 |
Apr 26, 2010 |
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Current U.S.
Class: |
623/3.16 ;
424/93.7; 435/173.1; 435/396 |
Current CPC
Class: |
A61L 27/52 20130101;
A61F 2210/0057 20130101; C12N 5/0657 20130101; A61L 27/16 20130101;
A61F 2220/0008 20130101; A61L 2300/252 20130101; A61L 2400/12
20130101; A61F 2240/001 20130101; A61F 2220/0016 20130101; A61L
27/3839 20130101; A61F 2002/0081 20130101; A61L 2300/602 20130101;
C08L 33/06 20130101; A61L 27/24 20130101; A61F 2/24 20130101; A61L
27/54 20130101; A61L 27/26 20130101; A61L 2300/44 20130101; A61F
2210/0004 20130101; C12N 2513/00 20130101; A61L 27/3834 20130101;
A61L 27/56 20130101; C12N 5/0663 20130101; A61L 27/58 20130101;
C12N 5/0667 20130101; A61L 2300/442 20130101; A61L 27/3873
20130101; A61L 2430/20 20130101; A61F 2/0077 20130101; A61L 27/26
20130101; C12N 2533/30 20130101; A61F 2/2481 20130101 |
International
Class: |
A61F 2/24 20060101
A61F002/24; C12N 5/0775 20060101 C12N005/0775; A61L 27/58 20060101
A61L027/58; A61L 27/26 20060101 A61L027/26; A61L 27/38 20060101
A61L027/38; A61F 2/00 20060101 A61F002/00; C12N 5/077 20060101
C12N005/077 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 30, 2010 |
EP |
10305851.7 |
Claims
1. A bioactive implant constituting a scaffold for myocardial
regeneration and ventricular chamber support, comprising: I. an
elastomeric microporous membrane comprising at least one
non-degradable polymer and at least one partially degradable
polymer, said membrane having a porosity comprised between 70% and
90%, the pores being interconnected and having diameters comprised
between 50 microns and 500 microns, wherein (a) Said non-degradable
polymer is selected from the group consisting of polyethylacrylate
and polyethylacrylate copolymerized with a 10% wt or a 20% wt
hydroxyethylacrylate comonomer; and (b) Said partially degradable
polymer is selected from the group consisting of caprolactone
2-(methacryloyloxy)ethyl ester and caprolactone
2-(methacryloyloxy)ethyl ester copolymerized with ethylacrylate in
weight proportions of this last comonomer comprised between 30% and
80%, wherein the percentage of non-degradable polymers versus
degradable polymers is comprised between 10% wt and 90% wt, II. a
nanofiber hydrogel and III cells.
2. (canceled)
3. (canceled)
4. (canceled)
5. (canceled)
6. (canceled)
7. (canceled)
8. (canceled)
9. (canceled)
10. (canceled)
11. (canceled)
12. (canceled)
13. (canceled)
14. (canceled)
15. (canceled)
16. (canceled)
17. (canceled)
18. (canceled)
19. (canceled)
20. A method for preparing the bioactive implant of claim 1,
comprising the steps of: filling an elastomeric microporous
membrane with a nanofiber hydrogel, so as to obtain a bioactive
construct; culturing said construct under biophysical, mechanical
conditions, and seeding or implanting cells onto or into said
bioactive construct.
21. The method of claim 20, wherein the seeding or the implantation
of the cells uses at least one of the following methods:
mechanical, chemical, and/or physical.
22. The method of claim 20, wherein the cells are myogenic,
cardiomyogenic, angiogenic or pluripotent stem cells.
23. A method for preparing the bioactive implant of claim 1,
comprising the steps of: (a) obtaining cells; (b) culturing said
cells in vitro; (c) mixing said cells with a nanofiber hydrogel;
and (d) filling an elastomeric microporous membrane with the cell
containing nanofiber hydrogel of step (c). Or (a) obtaining cells;
(b) culturing said cells in vitro; (c) filling an elastomeric
microporous membrane with the nanofiber hydrogel; (d) seeding or
implanting the said cells within the microporous membrane loaded
with the nanofiber hydrogel.
24. The method of claim 23 further comprising the step of (e)
culturing said bioactive construct under local in vitro
electrostimulation.
25. The method of claim 23, wherein lowered oxygen tension is used
during step (b).
26. The method of claim 23, wherein the bioactive implant is
manufactured so as to be adapted to left ventricular and/or right
ventricular support and regeneration, for partial or complete
ventricular wrappings.
27. An elastomeric microporous membrane for use in a bioactive
implant for myocardial regeneration and ventricular chamber
support, comprising at least one non-degradable polymer, at least
one partially degradable polymer, and at least one biomaterial of
nanoporous or nanoscale fiber dimensions, said membrane having a
porosity comprised between 70% and 90%, the pores being
interconnected and having diameters comprised between 50 microns
and 500 microns, wherein (a) said non-degradable polymer is
selected from the group consisting of polyethylacrylate and
polyethylacrylate copolymerized with a 10% wt or a 20% wt
hydroxyethylacrylate comonomer; and (b) said partially degradable
polymer is selected from the group consisting of caprolactone
2-(methacryloyloxy)ethyl ester andcaprolactone
2-(methacryloyloxy)ethyl ester copolymerized with ethylacrylate in
weight proportions of this last comonomer comprised between 30% and
80%, wherein the percentage of non-degradable polymers versus
degradable polymers is comprised between 10% wt and 90% wt.
28. The elastomeric microporous membrane of claim 27, being
surface-treated to graft adhesion molecules, said adhesion
molecules being selected from the group consisting of functional
peptides such as RGD peptides, functional sugars, lipids, and
proteins, wherein said proteins are laminin or laminin
fragments.
29. The elastomeric microporous membrane of claim 28 wherein said
functional peptides are RGD peptides.
30. A method for surgical myocardial repair, comprising: (a) mixing
cells with a nanofiber hydrogel, (b) positioning the elastomeric
microporous membrane of claim 27 at the intended location of the
body, and (c) injecting or spreading the mix obtained in step (a)
into or onto the positioned elastomeric microporous membrane.
31. A method for surgical myocardial repair, comprising: (a) mixing
cells with a nanofiber hydrogel, (b) injecting or spreading the mix
obtained in step (a) into or onto the elastomeric microporous
membrane of claim 27, so as to obtain a bioactive implant, and (c)
positioning the bioactive implant of step (b) at the intended
location of the body.
32. The method of claim 31 further comprising the step of injecting
cells through the epicardium.
33. The method of claim 31 wherein the injected cells are
autologous stem cells cultured in hypoxic conditions.
34. A method for treating a mammalian subject having injured
myocardial tissue comprising the steps of: implanting a bioactive
implant scaffold at a site of the injured myocardial tissue, said
scaffold including an elastomeric microporous membrane comprising
at least one non-degradable polymer and at least one partially
degradable polymer, a nanofiber hydrogel and cells, said membrane
having a porosity comprised between 70% and 90%, the pores being
interconnected and having diameters comprised between 50 microns
and 500 microns, said non-degradable polymer is selected from the
group consisting of polyethylacrylate and polyethylacrylate
copolymerized with 10% wt or 20% wt hydroxyethylacrylate comonomer;
said partially degradable polymer is selected from the group
consisting of caprolactone 2-(methacryloyloxy)ethyl ester and
caprolactone 2-(methacryloyloxy)ethyl ester copolymerized with
ethylacrylate in weight proportions of this last comonomer
comprised between 30% and 80%, the percentage of non-degradable
polymers versus degradable polymers is comprised between 10% wt and
90% wt.
35. The method of claim 34 wherein the bioactive implant scaffold
further comprises a helical loop band including one or more loops,
said helical loop band being formed of a non-degradable or
semi-degradable material.
36. The method of claim 35 wherein said helical loop band is
integrated into the bioactive implant scaffold.
37. The method of claim 34 further comprising the step of: fixing a
helical loop band to said implanted bioactive implant, said helical
loop band including one or more loops, said helical loop band being
formed of a non-degradable or semi-degradable material.
38. The method of claim 34 wherein the bioactive implant scaffold
is a patch.
39. The method of claim 37 wherein the patch further includes a
helical loop band integrated into the patch.
40. The method of claim 34 wherein the scaffold is positioned at
the left and right ventricles, wherein the injured myocardial
tissue is in the right and left ventricles.
41. The method of claim 34 wherein the scaffold further comprises a
biodegradable portion, the biodegradable portion being formed of a
biodegradable or semi-degradable material.
42. The method of claim 41 wherein the biodegradable or
semi-degradable portion is positioned at the left ventricle,
wherein the injured myocardial tissue is the right ventricle.
43. The method of claim 41 wherein the biodegradable or
semi-degradable portion is positioned at the right ventricle,
wherein the injured myocardial tissue is the left ventricle.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of
PCT/EP2011/056576, filed 26 Apr. 2011, which claims the benefit of
U.S. Provisional Patent Application No. 61/327,864 filed 26 Apr.
2010 and EP 10305851.7 filed 30 Jul. 2010, the entirety of which
applications are hereby incorporated by reference into this
application.
[0002] The present invention generally relates to the field of
myocardial repair, more particularly to a method and to a bioactive
implant for repairing myocardium and support ventricular chamber
configuration and function.
BACKGROUND OF THE INVENTION
[0003] Heart failure (HF) is primarily a condition of the elderly,
and thus the widely recognized "aging of the population"
contributes to the increasing incidence of HF. The incidence of HF
approaches 10 per 1000 population after age 65 and approximately
80% of patients hospitalized with HF are more than 65 years
old.
[0004] Heart failure is a major and growing public health problem
in the developed countries. In the United States approximately 7
million patients have HF, and more than 550 000 patients are
diagnosed with HF for the first time each year. The disorder is the
primary reason for 12 to 15 million office visits and 6.5 million
hospital days each year.
[0005] Heart failure is the most common Medicare diagnosis-related
group (i.e., hospital discharge diagnosis), and more Medicare
dollars are spent for the diagnosis and treatment of HF than for
any other diagnosis.
[0006] In Europe, the epidemiology is not well known; it is
estimated that about 30 millions patients suffer from heart
failure.
[0007] Cell transplantation and tissue engineering to the diseased
heart are emerging as promising strategies to prevent or to treat
refractory heart failure that cannot successfully be treated by
conventional therapies. The advances in cellular biology, in
biological engineering and nanotechnologies give further advances
in this option. Implanting exogenous cells supported by scaffolds
in the myocardial scar tissue to replace the damaged or the
disabled cells is a safe and efficient therapeutic approach.
Stem Cell Niche and Cell Homing
[0008] After myocardial infarction, not only the changes affect the
contractile element of the myocardium (cardiomyocytes) but also the
extracellular matrix. The collagen type I percentage decreases from
80% to 40%, this collagen is responsible with the other elements of
the heart muscle of the normal ventricular geometry.
[0009] The efficiency of cell therapy to augment recovery after
myocardial ischemia depends on the sufficient recruitment of
applied cells to the target tissue. Homing to sites of active
neovascularization is a complex process depending on a timely and
spatially orchestrated interplay between chemokines (e.g. SDF-1),
chemokine receptors, intracellular signalling, adhesion molecules
(selectins and integrins) and proteases.
[0010] Until now, cell transplantation for cardiac support and
regeneration was limited by poor effects in ventricular function.
This can be due to the lack of gap junctions between the native
myocardium and the grafted cells. Also, cell transplantation seems
to be limited by the relocation of transplanted cells to remote
organs and noninfarcted myocardium and by the death of transplanted
cells. Most cell death occurs in the first few days
post-transplantation, likely from a combination of ischemia,
apoptosis and inflammation. Apoptosis can be induced by
anchorage-dependent cells detaching from the surrounding
extracellular matrix.
[0011] The cell niche, a specialized environment surrounding stem
cells, provides crucial support needed for cell maintenance.
Compromised niche function may lead to the selection of stem cells
that no longer depend on self-renewal factors produced by its
environment. Strategies for improving cell survival and
differentiation such as tissue engineering, has been developed.
Cardiac Tissue Engineering
[0012] Extra cellular matrix remodeling in heart failure (excessive
matrix degradation and myocardial fibrosis) contributes to Left
Ventricular (LV) dilatation and progressive cardiac dysfunction.
Myocardial tissue engineering should provide structural support to
the heart, specific scaffolds should help to normalize cardiac wall
stress in injured regions improving strain distribution.
Engineering materials requiring specific properties of stiffness
and resistance to deformation can be implanted or seeded into the
myocardial tissue. They are composed of natural or synthetic
structure capable of supporting 3D tissue formation. Survival and
engraftment of cells within the environment of the ischemic
myocardium represents a challenge for all types of cells,
regardless of their state of differentiation. Scaffolds
characteristics are critical to recapitulating the in vivo milieu
and allowing cells to influence their own microenvironments. Such
scaffolds serve at least one of the following purposes: allow cell
attachment and migration, deliver and retain cells and biochemical
factors, enable diffusion of vital cell nutrients and expressed
products, and exert certain mechanical and biological influences to
modify the behavior of the cell phase. In addition, development of
gap junctions within the new created tissue as well as with the
host myocardial tissue are of great functional interest.
Ventricular Chamber Restoration
[0013] Restoration of ventricular shape and geometry is a surgical
procedure designed to restore or remodel the left and/or right
ventricle to its normal, conical shape and size in patients with
akinetic segments of the heart, secondary to either post infarction
cardiomyopathy or dilated cardiomyopathy. The restoration procedure
can be performed during or after coronary artery bypass grafting
(CABG), mitral valve repair or replacement and other procedures
such as implantation of stem cells for myocardial regeneration.
Surgical ventricular restoration has been performed: 1) by partial
resection of the ventricular wall using cardiac arrest and
cardiopulmonary bypass (extracorporeal circulation), or 2) by
external ventricular remodelling, e.g. cardiac wrapping with
autologous tissues like the latissimus dorsi muscle flap.
Ventricular restoration procedure with bioactive implants avoids
cardiac arrest and extracorporeal circulation.
Ventricular Restraint Therapies
[0014] Heart failure patients develop oversized, dilated hearts due
to increased filling pressures. Over time the increased workload of
the heart can lead to a change called remodeling, which is the
enlargement and thinning of the ventricles. The failing cardiac
muscle need to be supported to decrease the ventricular wall
stress. Mesh wrap devices that are implanted around the heart have
been used. These devices are intended to prevent and reverse the
progression of heart failure by improving the heart's structure and
function, leading to improvements in the survival and quality of
patient's life. For example, implantable devices have been tested
for ventricular restraint therapy, like polyester netlike sack
designed for placement around the heart fabricated into a
multifilament mesh knit (CorCap device, Acorn). Also a nitinol mesh
for ventricular wrapping was investigated (HeartNet device,
Paracor). Permanent implantation experience of both devices showed
adverse effects like restriction in diastolic function and lack of
improvement of systolic function, without evidence of myocardial
healing. These results have limited its large clinical application,
including the "not to approve" U.S. Food and Drug Administration
(FDA) decision.
Translational Research
[0015] Experimental and clinical studies have been performed on
stem cell therapy and tissue engineered approaches for myocardial
support and regeneration. The results of these investigations tend
to demonstrate the interest of simultaneous intrainfarct stem cell
therapy with the fixation of cell-seeded matrices onto the
epicardium of infarcted ventricles.
[0016] Experimental studies suggest that simultaneous autologous
intramyocardial injection of stem cells and fixation of a
cell-seeded collagen matrix onto the epicardium is feasible.
However, the long-term efficacy of this approach is compromised by
the complete biodegradation of the grafted collagen matrix.
[0017] WO2006/036826 discloses a tissue-engineering scaffold
containing self-assembled-peptide hydrogels.
[0018] US2005/0095268 describes a cardiac wall tension relief with
cell loss management.
[0019] The article of Boublik et al. (Tissue engineering, 2005)
relates to the mechanical properties and remodelling of hybrid
cardiac constructs made from heart cells, fibrin, and
biodegradable, elastomeric knitted fabric.
[0020] In summary, the following problems are encountered in the
field of myocardial repair.
[0021] 1) It is difficult to repair a large myocardial scar.
[0022] 2) Cell bio-retention and engraftment within scar tissue is
too low.
[0023] 3) Mortality of implanted cells in ischemic myocardium is
high.
[0024] 4) Extracellular matrix remodeling in ischemic heart disease
(excessive matrix degradation and myocardial fibrosis) contributes
to LV dilatation and progressive cardiac dysfunction.
[0025] 5) The therapeutic limitation of heart dilatation and the
recovery of the native elliptical shape of ventricular chambers are
key prognostic factors for survival in HF patients.
[0026] 6) In cell transplantation, survival and engraftment within
the environment of the ischemic myocardium represents a challenge
for all types of cells, regardless of their state of
differentiation.
[0027] 7) Up to now, the optimal cell-matrix combination for robust
and sustained myocardial restoration has not been identified.
[0028] 8) The long-term efficacy of the approach-autologous
intramyocardial injection of stem cells and fixation of a
cell-seeded collagen matrix onto the epicardium--is compromised by
the complete biodegradation of the grafted collagen matrix.
[0029] 9) There are undesired effects of growth factor
administration.
[0030] 10) Tissue viability/evolution over time.
SUMMARY OF THE INVENTION
[0031] The present invention provides a bioactive implant for
repairing myocardium and support ventricular chamber configuration
and function, and a method for preparing such implant. The
bioactive implant is grafted onto and/or into the ventricular wall
for myocardial regeneration, for left or right ventricular support
and to restore the elliptical shape of ventricular chambers.
[0032] The scaffolds are created by the combination of a membrane
which is a mix of biodegradable materials (biological or synthetic)
with non-biodegradable (biostable) synthetic materials, with
hydrogel and cells. During the procedure, cells, e.g. stem cells
mixed with hydrogel, are seeded into/onto the membrane, i.e. a
template form, and immediately or secondarily grafted onto diseased
myocardial tissue.
[0033] The method of the invention comprises the steps of creating
a scaffold combining biodegradable with non-biodegradable
materials, obtaining autologous cells or cells from a donor,
implanting the cells into the matrix and grafting the composite
cellular scaffolds onto the heart.
[0034] The implant and method of the present invention aims to
improve ventricular function, to limit chronic dilation of
ventricular chambers and to restore the native elliptical shape of
the heart as a new modality in the treatment of heart failure.
[0035] The advantages of the objects of the present invention are
the following:
[0036] a) Stem cell transplantation induces myocardial angiogenic
and/or myogenesis improving myocardial viability and reducing scar
fibrosis.
[0037] b) Matrix scaffolds grafting improves stem cell niche and
cell homing, consequently increasing the thickness of the infarct
scar with viable tissues. This composite material helps to
normalize cardiac wall stress in injured regions. In addition, new
vessels formation from the epicardium and from the surrounding well
irrigated myocardium contribute to the reduction of the fibrosis
and size of infarction scars, inducing the regeneration of
contracting cells and extracellular collagen matrix.
[0038] c) Synthetic cardiac support material onto the heart brings
long-term beneficial impact on ventricular chamber size and shape
reducing tension and promoting limitation of adverse remodelling.
In addition, this material helps to normalize cardiac wall stress
in injured regions improving strain distribution, avoiding scar
dyskinesia and the risk of formation of ventricular aneurysms,
ventricular wall rupture and mitral valve insufficiency.
[0039] d) Adapted ventricular wrapping. The bioactive implants of
the present invention are designed for left ventricular and/or
right ventricular support and regeneration, including different
sizes for partial or complete ventricular wrappings. The implant
characteristics (mechanical, physical, chemical, biological) are
adapted for the left or the right ventricle geometry, physiology
and pathology.
[0040] e) Maintenance and survival of the implanted cells in situ.
Preparation and maintenance of the cellular population of the
bioactive implants is obtained by cardiac cell therapy before,
during or after grafting "Bioactive Implants" onto the heart. Cell
transplantation is performed using either catheter-based approaches
via the endocardium (endoventricular), via an intravascular
procedure (through coronary arteries or veins) or injecting the
cells through the epicardium during cardio-thoracic surgery,
thoracoscopy or computer-robotic assisted procedures. Additionally,
lowered oxygen tension (e.g., 5% to 15%) is used during cell growth
as a preconditioning procedure to improve cell survival following
patch implantation in ischemic myocardium.
[0041] f) Prevention of LV dilatation and progressive cardiac
dysfunction. According to an aspect of the invention, the entire
organ is contained with the elastomeric membrane of the bioactive
implant to prevent heart dilatation. Thus, with decreased
ventricular wall tension the complementary treatment of grafting
biological tissue (e.g. peptides and stem cells) can successfully
achieve myocardial regeneration. Additionally, pacing electrodes
can be used in the method of the invention and also incorporated
into the Bioactive Implants and the native myocardium for
synchronous electrostimulation of the implanted tissue and other
electrophysiological treatments (defibrillation, resynchronization,
etc).
[0042] g) Regenerative treatment in association with implants in
view of survival and engraftment. Cell-based myocardial
regenerative treatments can be associated, i.e. intramyocardial and
intrainfarct stem cell transplantation, with the implantation of
bioactive implants onto the heart. Associated method for seeding or
implanting stem cells into or onto the Bioactive Implants using the
following methods: mechanical (shaking, centrifugation), chemical
(electrophoresis), physical (electroporation), etc. Seeded or
implanted cells that can be angiogenic, cardio-myogenic or
pluripotents. Additionally, Bioactive Implants can be labelled with
products (dies, microspheres, radioisotopes, iron-particles, etc)
for evaluation of biodegradation, integration, proliferation,
differentiation and function, using radiologic,
ultrasound-echocardiographic, radioisotopic, metabolic (PET), RMI,
CT Scan and bio-luminescence-fluorescence methods (etc.).
[0043] h) Adjusted composition of the bioactive implants. The
composition of the bioactive implants has a percentage of
non-biodegradable (synthetic) versus biodegradable (biological or
synthetic) components, which ranges from 10% to 90%.
[0044] i) To obviate the undesired systemic effect of growth
factors, the synthetic material is designed to locally release
angiogenic factors such as VEGF, HBEGF, bFGF. Additionally,
according to an embodiment of the present invention, Bioactive
Implants are endowed with a system for the controlled release of
angiogenic and antiapoptotic factors.
[0045] j) Assessment of the tissue growth and viability. Sensing
Electrodes are incorporated in the bioactive implants and connected
to a bioelectrical impedance measuring device. The goal of this
implantable monitor is to assess by telemetry the evolution of
engineered tissue in cardiac regeneration and to detect early
pulmonary oedema in heart failure patients.
[0046] The invention will be more fully described by reference to
the following drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] FIG. 1A is a schematic diagram of a bioprosthesis for
biventricular heart failure patients.
[0048] FIG. 1B is a schematic diagram of a bioprosthesis for right
ventricular heart failure patients.
[0049] FIG. 1C is a schematic diagram of a bioprosthesis for left
ventricular heart failure patients.
[0050] FIGS. 2A and 2B are schematic diagrams of a single helical
loop used to reinforce the bioprosthesis.
[0051] FIG. 3 is a schematic diagram a double loop used to
reinforce the bioprosthesis.
[0052] FIGS. 4A and 4B are schematic diagrams of a bioactive
patch.
[0053] FIG. 5 of the bioprosthesis fixed with epicardial
interrupted sutures.
DETAILED DESCRIPTION OF THE INVENTION
[0054] While the making and using of various embodiments of the
present invention are discussed in detail below, it should be
appreciated that the present invention provides many applicable
inventive concepts that can be embodied in a wide variety of
specific contexts. The specific embodiments discussed herein are
merely illustrative of specific ways to make and use the invention
and do not delimit the scope of the invention.
[0055] To facilitate the understanding of this invention, a number
of terms are defined below. Terms defined herein have meanings as
commonly understood by a person of ordinary skill in the areas
relevant to the present invention. Terms such as "a", "an" and
"the" are not intended to refer to only a singular entity, but
include the general class of which a specific example may be used
for illustration. The terminology herein is used to describe
specific embodiments of the invention, but their usage does not
delimit the invention, except as outlined in the claims.
[0056] The present invention provides a bioactive implant for
myocardial regeneration and cardiac support.
[0057] The bioactive implant of the invention constitutes a
scaffold.
[0058] As used herein, a scaffold is a material acting as a
template for cells to grow and produce new tissue. The scaffold of
the invention can be provided in a desired size and shape dependent
on the contemplated use. Indeed each ventricle has different wall
thickness, wall tension and chamber pressure. To reply to the
variety of size and shape, the implant of the invention can notably
be provided in the form of a cone or in the form of a square
depending on the intended and location use. It can be of a size of
from 1 cm.sup.2 to 20 cm.sup.2. As the bioactive construct of the
invention is implanted in the body of a mammal, for example a
human, the material of the membrane is chosen to be physiologically
inert to avoid rejection or other negative inflammatory
response.
[0059] According to the present invention, the bioactive implant
comprises
[0060] I. an elastomeric microporous membrane (patch) comprising at
least: [0061] a. one non-degradable synthetic polymer and [0062] b.
one partially degradable synthetic polymer
[0063] II. a peptide nanofiber hydrogel and
[0064] III. cells.
[0065] As used herein, a membrane is a material having one of its
three dimensions (its thickness) much smaller than its other two
dimensions (its length and width), these latter being comparable in
magnitude. The term patch may be used equivalently instead of the
term membrane. An elastomer is a crosslinked macromolecular
material which in working conditions is above its glass transition
temperature and thus is able to rapidly recover its original
unstressed dimensions after cessation of mechanical loading not
exceeding a critical value. An elastomeric membrane is a membrane
made out of an elastomer. A microporous elastomeric membrane is an
elastomeric membrane in which the elastomer configures a system of
interconnected void spaces, the pores, throughout the bulk of the
membrane, the pores having linear dimensions in the range of
several tens to a few hundreds of micrometers. The pores are
intended to host cells and the extracellular matrix produced by
them. The pores of the microporous elastomeric membrane may also be
filled with the peptide nanofiber hydrogel. Alternatively or
additionally, the peptide nanofiber hydrogel may be placed on top
of the elastomeric membrane. The microporous membrane of the
invention configures a three-dimensional environment able to convey
mechanical stimuli to the cells and to facilitate three-dimensional
cell-to-cell interactions.
[0066] According to the present invention, the porosity of the
membrane is comprised between 70% and 90%, said pores being
interconnected and having preferred diameters comprised between 50
microns and 500 microns, for example between 80 microns and 150
microns.
[0067] As used herein, a polymer is a macromolecule consisting in
the repeat of a few different units (if one, a homopolymer; if more
than one, a copolymer). A synthetic polymer is a polymer not
present naturally in biological system. A non-degradable polymer is
a polymer which remains chemically unaltered in vivo. The term
biostable and the term non-degradable may be used interchangeably.
A degradable polymer is a polymer which in vivo undergoes
depolymerization (scission) reactions whose products can be toxic
or non-toxic, metabolized or non-metabolized by the tissues or
organs of the host. The term biodegradable, the term degradable and
the term bioresorbable may be used interchangeably. The term
biodegradable refers to material that degrade or break down, with
time, upon interaction with a physiological environment into
components that can be metabolised or excreted by the body. A
partially degradable polymer is a polymer composed of at least one
non-degradable polymer and at least one degradable polymer.
[0068] More specifically, the non-degradable or biostable synthetic
polymer a. is selected from the group consisting of
polyethylacrylate or polyethylacrylate copolymerized with a 10% wt
or a 20% wt hydroxyethylacrylate co-monomer. Scaffolds made of this
polymer are advantageously produced by the template leaching
method, using as template an arrangement of sintered spheres and/or
fiber fabrics. The partially degradable synthetic polymer b. is
selected from the group consisting of caprolactone
2-(methacryloyloxy)ethyl ester or caprolactone
2-(methacryloyloxy)ethyl ester copolymerized with ethylacrylate in
weight proportions of this last co-monomer comprised between 30%
and 80%. Using caprolacton as a component of the partially
degradable polymer is particularly advantageous. Indeed since
caprolacton is produced by chemical synthesis, it does not produce
antigenic reaction, by comparison with collagen.
[0069] Also the degradation of a part of the implant reduces a
possible risk of chronic restriction of the diastolic function.
Indeed, all prosthetic materials implanted around the heart usually
produce chronic inflammatory reaction resulting in fibrosis,
responsible for the restriction of the diastolic function. The
amount of fibrosis is related with the characteristics and the
amount of the implanted material. Using a bioactive implant
according to the present invention, that is to say an implant
containing a biodegradable portion, allows a decrease of the
inflammatory reaction with time and thus reduces the possible risk
of a restriction of the diastolic function.
[0070] Also, it is to be noted that the scaffold membrane of the
invention is made of a combination of degradable polymers and
partially non-degradable polymers.
[0071] The bioactive implant provides a suitable environment for
cell homing, growth and differenciation (myocardial repair), as
well as mechanical support to the heart. The combination of
degradable polymers and partially non-degradable polymers is
advantageous because cells implanted in niches will organize,
connect and contract more easily with time if they are surrounded
by material that degrade with time and if not directly surrounded
by a synthetic prosthetic material. However some definitive
prosthetic fibres are necessary to avoid progressive heart
dilatation. The combination of both polymers, degradable and
non-degradable ones, allows a good cells implantation and to keep
the scaffold structure.
[0072] This polymer is advantageously produced by the template
leaching method, using as template an arrangement of sintered
spheres and/or fiber fabrics. The percentage of non-degradable
polymers versus degradable polymers is comprised between 10 and 90%
wt; it is preferably comprised between 10 and 48% wt. The membrane
of the present invention is thus a combination of a degradable
component and a non-degradable component. Although the membrane
comprises at least one polymer that is partially biodegradable, the
implant made from such membrane (and thus from such polymer) must
maintain the structural integrity for a time required for the
intended use.
[0073] The membrane of the invention may additionally comprise
biomaterials of nanoporous or nanoscale fiber dimensions, for
example a coating of hyaluronic acid, independently from the
composition and presence of the hydrogel.
[0074] As used herein, a hydrogel is a macromolecular material, by
which either physical or chemical crosslink interactions produce a
macromolecule base component, able to retain large amounts of water
molecules. A nanofiber hydrogel is a hydrogel made of nanoporous or
nanoscale fibers that percolates above a defined concentration to
form a network.
[0075] According to an aspect of the invention, the nanofiber
hydrogel can be degradable, biologically or chemically, or
non-degradable. In certain embodiments, the hydrogel includes
natural molecules such as protein, peptide, oligosaccharide,
polysaccharide, or proteoglycan derived matrices such as collagens,
fibrins, alginates, chitosans, hyaluronic acid, and/or any
synthetic molecule that will develop into a nanofiber network with
gel/hydrogel properties, such a peptide nanofiber hydrogel
scaffold; a class of self-complementary amphiphilic peptides that
self-assemble into nanofibers illustrates such peptides. The
following peptide AcN-RADARADARADARADA-COONH.sub.2 commercially
available by the name of PURAMATRIX is an example of this peptide
class.
[0076] In an aspect of the invention, the nanofiber hydrogel
comprises at least one self-assembling peptide (SAPs).
[0077] A self-assembling peptide is a peptide with
self-complementary properties able to undergo spontaneously a phase
transition from a disorder sol state to a more ordered state, where
the final ordered state consists of a crystal-like structures or a
collapsed amorphous material. The transition is triggered by
environmental parameters such as a pH or pK threshold, temperature,
etc. A self-assembling peptide gel is the spontaneous assembly of
self-complementary peptides developing into ordered chain or
domains with elongated shapes and dimensions in the range of a few
to tens of nanometers, and are thus referred to as nanofibers. An
example of SAP is RAD16-I. Other examples are: RAD16-II
(AcN-RARADADARARADADA-COONH.sub.2) and KLD12
(AcN-KLDLKLDLKLDL-COONH.sub.2.
[0078] In a preferred embodiment, the nanoporous or nanoscale fiber
hydrogel either completely fills the pores of the elastomer
membrane or partially fills the pores by forming a layer coating to
the inner surfaces of the membrane's pores.
[0079] In a specific aspect of the invention, the membrane is
surface-treated to graft adhesion molecules such as functional
peptides like as RGD peptides (Arg-Gly-Asp), functional sugars or
lipids, and proteins such as laminin or laminin fragments.
[0080] The bioactive implant of the invention is designed to
feature mechanical properties to be elastic enough to match the
myocardium contraction-distraction activity to allow deep
structural and functional bio integration.
[0081] The implant of the invention has an elastomeric membrane
which contains the entire organ to prevent heart dilatation (i.e. a
decreased ventricular wall tension).
[0082] The bioactive implant, in a particular aspect of the
invention, additionally comprises a system for the controlled
release or absorbance of active molecules such as any organic
molecule, such as small molecule, peptide, lipid, sugar, protein,
proteoglycan, with angiogenic, antiangiogenic, pro-regenerative,
anti-regenerative, apoptotic, necrotic, antiapoptotic and
antinecrotic activity, such as VEGF, IL-6, IL-10, IGF-1, FGF-2,
HBEGF, bFGF and chitosan.
[0083] Chitosan, a natural polymer of glucosamine and N-acetyl
glucodamine, is widely used in the pharmaceutical and tissue
engineering fields due to its biocompatibility, biodegradability,
and antimicrobial properties. Addition of chitosan improves the
physical properties of bioactive implants, and enhances their
ability to support endothelial cells and angiogenesis for use in
cardiovascular tissue engineering applications.
[0084] The release or absorbance system may consist in:
[0085] (a) the molecule encapsulated in degradable microparticles
made of such polymers as chitosan, hyaluronic acid, complexes of
these last two polymers, or a degradable polyester, such as
polyglycolic acid, or polylactic acid, or polycaprolactone; the
said microparticles embedded in the gel filling or coating the
membrane's pores;
[0086] (b) the molecule included in the gel filling or coating the
membrane's pores associated non-specifically or specifically to the
structure of the gel filling material;
[0087] (c) the molecule covalently or non-covalently bonded to the
self-assembling peptide filling or coating the membrane's
pores.
[0088] (d) the molecule with absorbance capacity to eliminate any
organic molecule with antiangiogenic, anti-regenerative, apoptotic
or necrotic activity.
[0089] The bioactive implant of the invention, in another aspect,
additionally comprises cytokines and angiogenic antiapoptotic
peptides.
[0090] It has binding capacity of components secreted by the
necrotic tissue. Accordingly, it has the ability to modulate and
neutralize the effect of components such as Midkine (MDK), a
negative regulator of angiogenesis.
[0091] The implant of the invention can be functionalized with
biological active motifs (peptides and glycopeptides) to promote
cellular responses, in particular, myocardial instruction to
maintain phenotype, allow cell-cell contact and establishment of
gap-junctions.
[0092] The bioactive implant of the invention can be designed for
elliptical or conical heart shape restoration, in which both
ventricles are completely wrapped by the device. The structure of
this device consists in special reinforcements at the level of the
anatomical bands, forming two helical loops of "non-degradable
polymers" for conical shape restoration. The geometric disease in
ischemic dilated cardiomyopathy is the spherical chamber, which is
different than the elliptical or conical normal heart shape. The
use of two helical reinforcement bands allows a three-dimensional
recovery of the original ventricular elliptical shape.
[0093] The bioactive implant of the present invention comprises
cells.
[0094] The cells can be myogenic or cardiomyogenic cells. According
to this embodiment, the cells are selected from the group
consisting of skeletal myoblasts, smooth muscle cells, fetal and
neonatal cardiomyocytes, adult ventricular cardiomyocytes,
cardiospheres and epicardial progenitors.
[0095] The cells can alternatively be angiogenic cells, such as
bone marrow and peripheral blood mononuclear fraction, bone marrow
and peripheral blood endothelial progenitors, endothelial cells,
mesothelial cells from omentum, adipocyte derived stem cells, stem
cells from adipose epicardial tissue and multipotent menstrual
blood stromal cells.
[0096] The cells can also be pluripotent stem cells. In this
embodiment, the cells are selected from the group consisting of
embryonic cells, animal embryonic cells, adult stem cells, fetal
stem cells, neonatal stem cells, non-human stem cells, umbilical
cord cells, induced pluripotent stem cells (iPSCs), bone-marrow
mesenchymal stem cells (MSCs), adult testis pluripotent stem cells
and human amniotic fluid stem cells (hAFSCs). In this regard the
bioactive implant is designed to host educated or trained cells
expected to grow, multiply, differentiate and organize into a
nanoscale fiber within the microporous structure of the patch and
to connect with the native myocardium. The combination of the
element of the bioactive implant along with the cells is such that
it presents a decreased ventricular wall tension and can
successfully achieve myocardial regeneration.
[0097] According to an aspect of the invention, a mixture of cells
is used as component of the bioactive implant. These cells can for
example be selected among the following: myogenic cells,
cardiomyogenic cells, angiogenic cells and pluripotent stem cells,
with the above-given definitions.
[0098] Suitable sources of cells for bioactive implant seeding and
intrainfarct injection will depend on the types of diseases to be
treated. For recent myocardial infarction, angiogenic cells that
reduce myocardial necrosis and augments vascular blood flow will be
desirable. For chronic heart failure, cells that replace or promote
myogenesis, reverse apoptotic mechanisms and reactivate dormant
cell processes will be useful. For chronic ischemic cardiomyopathy,
both angiogenic and cardiomyogenic cells will be associated.
[0099] According to the present invention, it is possible to embed
cells in a 3-dimensional structure replicating the extracellular
matrix, which is crucial for full tissue restoration and prevention
of ventricular remodeling. The clinical translation of cell therapy
requires avoidance of potentially harmful drugs and cytokines, and
rapid off-the-shelf availability of cells. The combination of
pre-differentiated cells within a functionalized scaffold, locally
releasing molecules tailored to promote in situ completion of
differentiation and improve homing, survival, and functions,
circumvents the potential undesired systemic effects of growth
factor administration and improve tissue restoration.
[0100] The cells seeded into matrix scaffold and supported by a
synthetic ventricular support device treated with adhesion
molecules ameliorate functional recovery of infarcted hearts and
improve long-term evolution by providing myocardial regeneration
and gentle support.
[0101] The cell-matrix combination associated with a ventricular
constraint non-absorbable material such as, mesh cardiac wrap,
positioned over the diseased myocardium improves ventricular
function and reduces adverse chamber remodelling.
[0102] The present invention combines a regenerative biological
approach with a prosthetic cardiac support device. Stem cells
associated with a tissue engineered matrix scaffold and combined
with a mesh cardiac wrap should reduce post-ischemic fibrosis and
assist the recovery of myocardial viability and compliance. This
procedure can be proposed for the treatment of ischemic heart
disease, associating a regenerative biological approach with a
prosthetic support device.
[0103] The present invention constitutes a unique platform for
engineering highly efficient contractile tissues and enhancing cell
therapy.
[0104] For the present invention, the cells can be obtained from
any suitable source. They can be purchased or they can be isolated
from a suitable source by methods well known to those skilled in
the art. They may be cultured according to methods known to those
skilled in the art. For example, the cells can be added to culture
medium which may additionally comprise growth factors, serum,
antibiotics or any of a variety of cell culture components known to
those skilled in the art.
[0105] Ischemic cardiomyopathy induces geometric alteration of the
ventricular cavity, which changes from an elliptical to a spherical
shape. The geometric disease in ischemic dilated cardiomyopathy is
the spherical chamber, which is different than the elliptical or
conical normal heart shape. The sphericity index quantifies this
geometric form alteration by comparing the transverse ventricular
(short) and long axis; an ellipse has a 0.5 ratio (the length is
twice the width) and a sphere is 1.0 due to similar transverse and
longitudinal dimensions.
[0106] According to one embodiment of the invention, the structure
of the bioactive implant of the present invention consists in
special reinforcements at the level of the anatomical bands,
forming two helical loops of non-degradable biostable synthetic
polymers. This embodiment of the present invention is intended to
cover both ventricles for ischemic dilated cardiomyopathy. Helical
ventricular artificial bands for LV elliptical shape restoration
using bioactive implants is useful in the context of the
biosurgical strategies indicated to manage patients with advanced
myocardial diseases. An important advantage of this technique is
the fact that the surgery is performed without the risk of opening
the ventricular chambers, i.e. without extracorporeal
circulation.
[0107] In another aspect, the present invention deals with a method
for preparing the bioactive implant of the invention, comprising
the steps of filling an elastomeric microporous membrane with a
nanofiber hydrogel, so as to obtain a bioactive construct. In this
respect, the filling is for example made by placing a syringe into
the membrane, said syringe being filled with the gel and gently
evacuating the air in the pores; according to another step, said
construct is cultured under biophysical, mechanical conditions
(i.e. compression and elongation); then a step of seeding or
implanting cells onto or into said bioactive construct using the
following methods: mechanical (shaking, centrifugation), chemical
(electrophoresis), physical (electroporation). The cells that can
be used are myogenic, cardiomyogenic, angiogenic or pluripotent
stem cells.
[0108] Another method for preparing the bioactive implant of the
invention comprises the steps of obtaining cells, for example,
myogenic, cardiomyogenic, angiogenic or pluripotent stem; culturing
said cells in vitro; mixing said cells with a nanofiber hydrogel;
and filling an elastomeric microporous membrane with said
cell-containing nanofiber hydrogel, so as to obtain a bioactive
construct. The method of the invention may also comprise the step
of culture of said bioactive construct under local in vitro
electrostimulation.
[0109] In a specific aspect of the method, the cells are cultured
under lowered oxygen tension.
[0110] In a still specific aspect of said method, the bioactive
implant is cultured so as to be adapted to left ventricular and/or
right ventricular support and regeneration, for partial or complete
ventricular wrappings.
[0111] The present invention also deals with a method for repairing
the myocardium of an individual comprising the steps of preparing a
bioactive implant according to the invention and implanting the
bioactive implant into and/or onto the myocardium.
[0112] In a more specific aspect, the method for repairing the
myocardium comprises an additional step consisting in injecting
cells through the epicardium during cardio-thoracic surgery,
thoracoscopy or computer-robotic assisted procedures. The injected
cells can be autologous stem cells cultured in hypoxic
conditions.
[0113] The present invention also deals with an elastomeric
microporous membrane, comprising at least one non-degradable
polymer, at least one partially degradable polymer, and at least
one biomaterial of nanoporous or nanoscale fiber dimensions, said
membrane having a porosity comprised between 70% and 90%, the pores
being interconnected and having diameters comprised between 50
microns and 500 microns, for example between 80 microns and 300
microns, preferably between 80 microns and 150 microns, wherein
[0114] a. the non-degradable polymer is selected from the group
consisting of poly(ethylenglycol diacrylate), polyethylacrylate and
polyethylacrylate copolymerized with a 10% wt or a 20% wt
hydroxyethylacrylate comonomer; and
[0115] b. the partially degradable polymer is selected from the
group consisting of polycaprolactone, caprolactone
2-(methacryloyloxy)ethyl ester and caprolactone
2-(methacryloyloxy)ethyl ester copolymerized with ethylacrylate in
weight proportions of this last comonomer comprised between 30% and
80%,
[0116] wherein the percentage of non-degradable polymers versus
degradable polymers is comprised between 10% wt and 90% wt.
[0117] This membrane may be used in a bioactive implant for
myocardial regeneration and ventricular chamber support.
[0118] In a more specific aspect, the elastomeric microporous
membrane can be surface-treated to graft adhesion molecules. The
adhesion molecules being selected from the group consisting of
functional peptides such as RGD peptides, functional sugars or
lipids, and proteins such as laminin or laminin fragments.
[0119] The present invention also deals with a method for surgical
myocardial repair, comprising the steps of:
[0120] a) mixing cells with a nanofiber hydrogel,
[0121] b) positioning the elastomeric microporous membrane of the
invention at the intended location of the body, and
[0122] c) injecting or spreading the mix obtained in step a) into
or onto the positioned elastomeric microporous membrane.
[0123] The present invention also concerns a method for surgical
myocardial repair, comprising the steps of:
[0124] a) mixing cells with a nanofiber hydrogel,
[0125] b) injecting or spreading the mix obtained in step a) into
or onto the elastomeric microporous membrane of the invention, so
as to obtain a bioactive implant, and
[0126] c) positioning the bioactive implant of step b) at the
intended location of the body.
[0127] FIG. 1A-1C illustrate adapted ventricular wrapping. FIGS.
1A-1C show devices for complete ventricular wrapping. FIG. 1A is
used for biventricular heart failure patients. Bioprosthesis 10 is
made of the same material for both right ventricle 12 and left
ventricles 14. Bioprosthesis 10 can be formed of the elastomeric
microporous membrane. FIG. 1B is used for right ventricular failure
patients. Bioprosthesis 20 is made of high rate [60 to 80%] of
biodegradable material in left heart side 14 and the elastomeric
microporous membrane on the right heart side 12. FIG. 1C is used
for left ventricular failure patients. Bioprosthesis 30 is made of
high rate of biodegradable material in right heart side 12 and the
elastomeric microporous membrane on the left heart side 14.
[0128] Bioprosthesis 10, 20, 30 can be reinforced by helical loops
made of non-degradable or semidegradable polymers. These materials
can be the following: polyethylacrylate (PEA) copolymerized with
hydroxyethylacrylate comonomer, semi degradable
methacrylate-endcapped caprolactone (CLMA), polyethylene
terephthalate (PET), polypropylene, polydioxanone, polyglecaprone,
e-caprolactone, poly-L-lactide polymers, poly salicylic acid
polymer, poly desaminotyrosyl-trypsine ethyl ester polymer,
polycarbonate urethane, polyurethanes, poly(glycerol sebacate)
(PGS), elastin, silk.
[0129] Loops are made of a band of 30 mm to 40 mm width with 1 mm
to 2 mm uniform thickness, or made with thickness progressively
increased from 1 mm to 3 mm.
[0130] Helical loops follow the anatomical heart configuration,
where muscular ventricular bands begin at the insertion of the
pulmonary artery in the right ventricle and ending at the aortic
valve annulus (LV). The role of myocardial band is to limit
ventricular dilatation, preserving elliptical shape, and contribute
to systolic contraction and diastolic filling (suction
mechanism).
[0131] In one approach, a helical loop can be integrated into the
ventricular bioprosthesis structure during manufacturing.
[0132] In another approach, a helical loop is a complement of a
bioactive patch fixed onto a myocardial pathologic zone, during
surgery or thorascocopic approach.
[0133] In another approach, a helical loop is fixed around the
heart as a complement of the ventricular support bioprosthesis,
implanted to cover the ventricles during surgery or thorascocopic
approach.
[0134] In another approach, a helical loop can be used as a single
therapeutic procedure.
[0135] For moderate heart dilatation, single apical loop 50 is used
to wrap ventricles, starting at the level of the left atrial
appendage 52 and ending at the aortic root 53, as shown in FIG. 2.
(See FIG. 2B showing the position of the left atrial appendage
52).
[0136] For severe heart dilatation, double basal and apical
ventricular helical loops 60a, 60b are used, starting at the level
of pulmonary artery root 54 and ending at the level of the aortic
root 53, as shown in FIG. 3. Fixation of the loop/band onto the
heart, onto the bioactive patch and onto the ventricular support
bioprosthesis can be made by surgical sutures and/or surgical clips
and/or glue of biological or synthetic origin.
[0137] The method for implantation of small bioactive patches or
large cardiac support bioprostheses can be performed during
cardio-thoracic surgery, thoracoscopy or computer-robotic assisted
procedures. After gaining access, the pericardium is open to expose
the heart.
[0138] In a bioactive patch implantation procedure, scaffold 70 is
positioned onto the pathologic myocardial lesion, for example
covering the infarct and peri-infarct zones. It is fixed to the
epicardium by single interrupted sutures 72 (4-0 or 5-0) and
covered by the pericardium, as shown in FIGS. 4A and 4B.
[0139] In ventricular support bioprothesis implantation procedure,
for a device choice, the size of the heart is measured by a
circumferential tape. Adequate size of bioprothesis 10, 20, 30 is
chosen and placed around right ventricle 12 and left ventricle 14,
as shown in FIG. 5. Bioprosthesis is placed around the ventricles
by sliding it gently into position (arrow), from the apex of the
heart to the atrio-ventricular groove Bioprothesis 10, 20, 30 is
fixed with epicardial interrupted sutures 82 (4-0) to heart 80 at
the level of the A-V groove, starting at the most posterior
location. For example, fixation sutures are placed every 2 cm to 3
cm.
[0140] The sequential contraction of the ventricular myocardium
results in the successive shortening and lengthening of the
ventricles. These movements may determine the ejection and suction
of blood, respectively. The shape and duration of ventricular
filling/emptying mechanism can be compared to a stroke action
induced by a piston water pump. Surgical interventions for heart
failure like reduction ventriculectomy have not proven surgically
efficacious. Removal of apical or basal ventricular segments and
the muscle bands seems to interfere with the natural sequence of
myocardial contraction and diastolic filling. Cardiac wrapping by
ventricular support bioprostheses of the present invention, having
bands spatially distributed as helicoids, is an advantageous
physiological therapeutic method.
EXAMPLES
Example 1
Biological Evaluation of Elastomeric Scaffold Membranes
Quantification of Cell Proliferation.
[0141] MTT Assay
[0142] The MTT system is a simple, accurate, reproducible means of
measuring the activity of living cells via mitochondrial
dehydrogenase activity. The key component is
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide or
MTT. Solutions of MTT solubilised in tissue culture media or
balanced salt solutions, without phenol red, are yellowish in
color. Mitochondrial dehydrogenases of viable cells cleave the
tetrazolium ring, yielding purple MTT formazan crystals which are
insoluble in aqueous solutions. The crystals can be dissolved in
acidified isopropanol. The resulting purple solution is
spectrophotometrically measured. An increase in cell number results
in an increase in the amount of MTT formazan formed and an increase
in absorbance.
[0143] Material and Method
[0144] Pieces of elastomeric microporous membranes of polyethyl
acrylate (PEA100 in what follows) and a copolymer of ethyl acrylate
and hydroxyethyl acrylate with a 90:10 mass ratio of both monomers
(hereafter PEA90) were employed. The membranes had been cut into
pieces of dimensions 25 mm.times.25 mm, with an approximate
thickness of 1.0 mm (PEA100 and PEA90A) and of 0.7 mm (PEA90B). The
porosity of the membrane was 70%. The pores of the membranes
consisted in layers of orthogonal families of parallel cylindrical
pores, with pore diameter of 150 microns and pore separation of 300
microns. They were generated by letting the precursors of the
polymers react inside a mould with a template of layers of porogen
orthogonal fibers, and dissolving the template afterwards to give
place to said pores.
[0145] Scaffolds Conditioning Protocol
[0146] Due both to their hydrophobic nature and to their
microporous structure, the elastomeric membrane needs to be
pre-hydrated before cell seeding. The conditioning procedure
consists in a 24 h immersion in a PBS solution. Vacuum might be
necessary to improve fluid penetration into the pores, putting the
sample in a tube sealed hermetically with a cap pierced by a
syringe's needle, and performing vacuum with the syringe. The
pre-hydrated sample is then immersed in the culture medium. If the
pH changes, the medium is renewed until the reference pH value
remains stable.
[0147] Cell Seeding
[0148] Bone marrow mesenchymal stem cells (BMC) were isolated under
sterile conditions from femur and tibia bones of Wistar rats. After
2 weeks of in vitro cultures in DMEM complete medium with L
glutamine, sodium pyruvate and 15% Fetal Bovine Serum (1.sup.st
passage), 10,000 cells diluted in 0.5 ml medium were seeded into
PEA90 and PEA100 scaffolds and into 3D Collagen type I matrix (n=5
for each sample). After careful cell seeding using a micropipette,
elastomeric scaffolds and collagen matrix were maintained 20
minutes without motion, to start cell adhesion. At the following
step, and in order to promote a regular distribution of BMC into
the matrix pores, Petri dishes containing the elastomeric and
collagen scaffolds/matrices were shaken continuously for 10 minutes
at 80 g using an Orbital Shaker (Stuart Scientific, Stone,
Staffordshire, UK). Afterwards cell seeded scaffolds were incubated
one hour at 37.degree. C. Finally DMEM complete medium was added to
the Petri dish and the cell seeded scaffolds/matrix were cultured
during 3 weeks at 37.degree. C., 5% CO.sub.2.
[0149] Quantification of Cell Propagation
[0150] Cultures were removed from the incubator into a laminar flow
hood. The supernatant was removed and then the scaffolds were
washed with PBS two times. The scaffolds were transferred into new
tubes (15 ml Falcon). Aseptically the MTT solution was added in an
amount equal to 10% of the culture volume (1800 microliter phenol
red free medium+180 microliter MTT) and cultures were incubated for
3 hours at 37.degree. C. in a 5% CO2 humidified atmosphere. Two ml
of solubilisation solution or solvent were added and then vortexed
for 5 min. This provoked the release from the scaffold of MTT which
was actively reduced by viable cells acquiring a yellow colouring.
Each sample was centrifuged at 15,000 g for 5 min and the
supernatant was read at 570 nm using a multiwell
spectrophotometer.
[0151] Results
[0152] Spectrophotometer assessments showed optical density (OD)
values of 0.13+/-0.02 for collagen matrix; 0.22+/-0.04 for PEA90A
scaffolds; 0.11+/-0.03 for PEA90B scaffolds; and 0.34+/-0.05 for
PEA100 scaffolds.
[0153] These results showed that cell proliferation was well
developed in the elastomeric scaffolds, presenting a better
proliferation than the 3D collagen scaffolds. Until now collagen
scaffolds have been used in experimental and clinical myocardial
tissue engineering as a gold standard.
Example 2
Electrophysiological Evaluation of the Elastomeric Scaffold
Membranes
[0154] Measurements of Electrical Impedance
[0155] Electrical Conduction
[0156] Myocardial electrical impedance has shown to be an effective
indicator of myocardial tissue characteristics and electrode tissue
interface. Significant modifications have been demonstrated during
tissue ischemia.
[0157] Electrical resistivity (also known as specific electrical
resistance or volume resistivity) is a measure of how strongly a
material opposes the flow of electric current. A low resistivity
indicates a material that readily allows the movement of electrical
charge. The SI unit of electrical resistivity is the ohm [2].
[0158] Material and Methods
[0159] Pieces of microporous membranes of polyethyl acrylate
(PEA100 in what follows) and a copolymer of ethyl acrylate and
hydroxyethyl acrylate with a 90:10 mass ratio of both monomers
(hereafter PEA90) were employed. The membranes had been cut into
pieces of dimensions 25 mm.times.25 mm, with an approximate
thickness of 1.0 mm (PEA100 and PEA90A) and of 0.7 mm (PEA90B). The
pores of the membranes consisted in layers of orthogonal families
of parallel cylindrical pores, with pore diameter of 150 microns
and pore separation of 300 microns. They were generated by letting
the precursors of the polymers react inside a template of the
porous structure, and dissolving the template afterwards.
[0160] Scaffolds Pre-Hydration
[0161] Elastomeric scaffolds need 2 days of pre-hydration as
follows: 24 Hs immersion in a PBS solution and 24 Hs immersion in
culture medium. Vacuum could be necessary to improve tissue
hydration, putting the sample in tube with cap and performing
vacuum with a syringe. Once pH change is observed, the samples
should be overnight in fresh culture medium.
[0162] Electrophysiological Studies
[0163] Two electrodes having curved needles for easy insertion were
sutured into the opposites borders of the elastomeric scaffolds and
of 3D collagen type I matrix (n=5 for each sample). These
electrodes were conceived to be implanted for temporary
postoperative cardiac pacing in heart surgery. Scaffolds and
implanted electrodes were immersed in Petri dishes containing DMEM
cell culture medium. After 30 minutes electrophysiological studies
were performed connecting the electrodes to a Pacing System
Analyzer Model 5311 (Medtronic Inc.). Bipolar charge balanced
electrostimulation was delivered using the following parameters:
pulse amplitude 1 Volt, pulse width 0.5 ms, frequency of
stimulation 70 pulses per minute (ppm). Electrostimulation was
delivered just for testing. Afterwards electrical impedance within
the scaffolds was assessed.
[0164] Results
[0165] Electrical measurements were performed in each preparation
group, i.e. cell medium alone, collagen matrix, PEA90A scaffold,
PEA90B scaffolds, PEA100 scaffolds. Each group consisted of 5
samples.
[0166] Impedance measurements showed the following values: cell
culture medium 292+/-25 ohms, collagen matrix 230+/-21 ohms; PEA90A
scaffolds 321+/-34 Ohms; PEA90B scaffolds 345+/-33 ohms; PEA100
scaffolds 340+/-29 ohms.
TABLE-US-00001 Cell Collagen PEA90A PEA90B PEA100 medium matrix
scaffold scaffold scaffold Impedance [.OMEGA.] 292 230 321 345 340
Current [mA] 3.42 4.35 3.11 2.90 2.94
[0167] Pacing pulse; 1.0 V, 0.5 ms
[0168] These results showed that all the evaluated materials
present electrical conduction properties, i.e. resistance, similar
to those encountered with cardiac tissues, thus these scaffolds
have the potential to be used for myocardial substitution.
Example 3
[0169] The failing cardiac muscle needs to be chronically supported
to decrease ventricular wall stress and also to be regenerated to
improve ventricular function. This Example demonstrates that the
association of stem cells with a collagen matrix and a polyester
mesh for cardiac wrap provides better results than the implantation
of polyester mesh alone.
[0170] To illustrate this embodiment, fifteen sheep underwent 1
hour of surgical myocardial ischemia followed by reperfusion. Three
groups were created: Group 1: coronary occlusion without treatment
(control group). Group 2: LV constraint using a polyester mesh for
cardiac wrap. Group 3: the ischemic area was treated associating
stem cells, a collagen matrix and a polyester mesh. Autologous
adipose tissue derived stem cells (ASC) cultured in hypoxic
conditions (5%) were labelled with BrdU and injected into the
infarct area and into a collagen matrix. At 3 months animals were
evaluated with echocardiography and histopathological studies.
[0171] Biopsy Extraction
[0172] In 15 female Rambouillet sheep weighing 32 to 37 kg (mean
35.+-.2.2 Kg), subcutaneous fat tissue was removed for stem cell
isolation and expansion.
[0173] Autologous cells were used throughout in order to avoid any
problem of histocompatibility. Adipose tissue biopsies were
obtained by subcutaneous fat tissue removal (40-60 grams) from the
right thoracic wall and stored in phosphate buffered saline (PBS)
at room temperature until processing.
[0174] Isolation and Hypoxic Culture of Adipose Derived Stem Cells
(ASC)
[0175] The tissue samples were finely minced and digested by
incubation in a 0.14 Wiinsch units/mL Liberase Blendzyme 2 (Roche
Applied Science, Hvidovre, Denmark) solution at 37.degree. C. for
two hours. The digests were centrifuged at 400 g for 10 min and the
top fluid and fat layers were discarded. Contaminating erythrocytes
were lyzed by resuspension of the pellet in sterile milli-Q water
for 20 seconds, after which the salt concentration was adjusted
through addition of 10.times.PBS. The cells were filtered through a
100 .mu.m cell strainer, centrifuged at 400 g for 10 min, and
resuspended in 25 mL growth medium, consisting of minimum essential
medium alpha (A-MEM) (GIBCO/Invitrogen) supplemented with 10% fetal
bovine serum (FBS), and penicillin (10 U/ml), streptomycin (10
mg/ml), gentamicin (10 mg/ml) (all from GIBCO/Invitrogen). The
cells were seeded in a T75 flask and transferred to a CO.sub.2
incubator overnight, after which non-adherent cells were removed.
The flasks were then transferred to a hypoxic workbench/incubator
(Xvivo; Biospherix, Lacona, N.Y.), allowing for uninterrupted cell
culture and passaging in a controlled atmosphere of 5% O.sub.2 and
5% CO.sub.2 balanced with nitrogen. During expansion of the cells,
the media was changed twice a week. When cells were 90% confluent,
the cells were detached from the culture flasks using 0.125%
trypsin/0.01% EDTA and transferred to new flasks.
[0176] Labeling with Bromodeoxyuridine
[0177] For each sample, the cells were expanded until eight T175
culture flasks were 75% confluent, then the cells were labeled with
bromodeoxyuridine (BrdU). Briefly, cells were incubated with growth
media containing 10 micrograms BrdU (Sigma) for 48 hours, and then
the cells were washed several times with PBS and frozen in aliquots
of approximately 10.times.10.sup.6 cells.
[0178] Experimental Myocardial Injury
[0179] After preoperative medication and induction of anaesthesia
(same protocol as fat tissue biopsies) animals were intubated and
mechanically ventilated with an Aestiva/5 system (Datex-Ohmeda,
Helsinki, Finland). The electrocardiogram was monitored during
operation. A central venous line was placed through the external
jugular vein for administration of fluid and drug infusions. Left
thoracotomy was performed at the level of the 5th intercostal
space, and the heart was exposed. To reduce the risk of ventricular
fibrillation, a continuous IV perfusion (2 mg/kg per hour) of
Xylocaine 1% (Lidocaine, AstraZeneca) was performed during the
entire surgical procedure. In all animals a LV myocardial ischemia
was surgically created by transitory ligation (60 minutes) of the
main diagonal branch of the left coronary artery, followed by
reperfusion. A 4-0 non-absorbable Prolene suture was passed
underneath the coronary artery branch, the flow was interrupted
using a Teflon pledget compressed by a polyurethane occluder. This
occluder was released after 60 minutes, thus the myocardial
ischemic territory was reperfused. Significant EKG changes,
including widening of the QRS complex and elevation of the ST
segment, and colour and kinetics changes of the area at risk were
considered indicative of coronary occlusion.
[0180] Treatment Groups
[0181] Animals were randomized in 3 groups:
[0182] Group 1 (n=5): myocardial ischemia without treatment
(control group).
[0183] Group 2 (n=5): post-ischemia implantation of a mesh
ventricular wrap device.
[0184] Group 3 (n=5): post-ischemia intrainfarct injection of stem
cells, implantation of a collagen matrix as interface and
implantation of the mesh ventricular wrap device.
[0185] Cell Injection and Collagen Matrix Implantation
[0186] In Group 3 animals, at 1 hour of infarction cell were
injected into the infarct zone by using a 27-gauge needle.
Injections consisted of 99+/-12 million cells, 50% (2 mL) injected
into the infarction and 50% (5 mL) seeded into a 3D collagen type I
matrix.
[0187] For myocardial treatment, six injection needle points were
used in each animal, bulging over the myocardial infarction area
was confirmed in every case after injection. Criteria to guide the
epicardial injections were the ventricular surface discoloration
and hypokinesia.
[0188] Collagen Matrix Preparation
[0189] Collagen matrix was prepared from a commercially available
CE Mark collagen kit (Pangen 2, Urgo Laboratory, Chenove, France).
This 3D biodegradable matrix (size: 5.times.7.times.0.6 cm) was
manufactured using a lyophilised, non denatured, native type I
collagen. The matrix pores measured 50 to 100 .mu.m. In the
operating room and under high sterility conditions, matrix was
placed into a Petri dish; afterward, the cell suspension (50.+-.6
million cells diluted in 5 mL medium) was seeded onto the matrix.
To promote a regular distribution of ASC into the matrix pores,
Petri dishes containing the collagen matrix was shaken continuously
for 10 minutes at 160 g using an Orbital Shaker (Stuart Scientific,
Stone, Staffordshire, UK).
[0190] Mesh Cardiac Wrap
[0191] To avoid hemodynamic instability and arrhythmias during
implantation, we start to fix the mesh cardiac wrap (CorCap
polyester device) before creation of myocardial ischemia. The
CorCap model Gen2 CSD Size B (Acorn Cardiovascular Inc, St Paul,
Minn., USA) was chosen in all cases, then was opened
longitudinally, slid behind the ventricles and fixed with 2 lateral
epicardial sutures (Prolene 4-0). Afterwards the ischemia was
created followed by reperfusion. One hour later the cells were
injected, the collagen matrix implanted and the anterior part of
the CorCap was closed using a continuous suture (Prolene 2-0). The
fixation of the device was completed by multiple single sutures
over the atrio-ventricular anterior groove.
[0192] Results
[0193] No mortality was observed. The hypoxic treatment for cell
cultures demonstrated a quite dramatic improvement of proliferation
rate: under hypoxia cells grown faster. Echocardiography showed a
limitation of LVED (Left Ventricular End-diastolic Dimension)
volume in both treated groups (polyester mesh alone 35.6.+-.5 mL
and with cell therapy 32.6.+-.4 mL) vs. control (65.+-.6.3 mL,
p<0.01 for both comparisons). EF (Ejection Fraction) was
significantly greater in the hearts treated with the polyester
mesh+cells/collagen (55.8.+-.3.8%) compared with those receiving
polyester wrapping-only (44.1.+-.2.3%) (p=0.04) or without
treatment (34.8.+-.3.6%) (p=0.01). Doppler-derived mitral valve
deceleration time (DT) improved from 140.+-.6.3 ms to 195.+-.9.5 ms
(p=0.03) in the cell-collagen CorCap group but not in the other
groups. Histology showed in the cell treated group multifocal
ischemic areas much less prominent than in other groups, with
focuses of angiogenesis and viable grafted cells. Minimal fibrosis
interface between the polyester mesh and the epicardium was
observed in Group 3, probably due to the interposition of the
cell-seeded collagen.
[0194] Comments
[0195] In an ischemic model, stem cells associated with a collagen
matrix and a polyester mesh for cardiac wrapping improves EF and
diastolic function, reducing adverse remodelling and fibrosis. This
procedure associating a regenerative biological approach with a
prosthetic support device seems to be appropriate for the treatment
of advanced ischemic heart failure.
Example 4
[0196] This clinical Example demonstrates that a cell-seeded
collagen matrix associated with intra-infarct cell therapy provides
better results than stem cell alone.
[0197] Matrix Preparation
[0198] A 3D biodegradable matrix (size: 5.times.7.times.0.7 cm)
manufactured using lyophilized bovine type I collagen was prepared.
The matrix pores measured 50 to 100 .mu.m. In the operating room
and under high sterility conditions, matrix was placed into a Petri
dish; afterward, the cell suspension (250.+-.28 million cells
diluted in 10 ml medium) was seeded onto and into the matrix. To
promote a regular distribution of cells into the matrix, Petri
dishes containing the matrix were shaken continuously for 10
minutes at 160 g using an Orbital Shaker (Stuart Scientific, Stone,
Staffordshire, UK).
[0199] Surgical Procedure
[0200] In 10 patients (mean age 52.6 y), after sternotomy, a single
OP-CABG (off pump-coronary artery bypass graft) was performed using
the left internal mammary artery (LIMA). At the end of surgery,
250.+-.28 million cells diluted in 5 ml medium were injected in the
infarcted area and borderzone, using a 25 G.times.40 mm retrobulbar
ophthalmic needle. Then the cell seeded matrix was placed covering
the infarcted area and fixed to the epicardium with 6 PDS sutures
(6-0).
[0201] In another group of 10 patients (mean age 56.8 y), a single
OP-CABG was performed. Stem cells were injected into the infarction
scar but no seeded matrix was used in this group.
[0202] Results
[0203] There was no mortality and any related adverse events
(follow-up 10.+-.3.5 months). NYHA FC improved in both groups from
2.3.+-.0.5 to 1.3.+-.0.5 (matrix, p=0.0002) vs 2.4.+-.0.5 to
1.5.+-.0.5 (no matrix, p=0.001). LV end-diastolic volume evolved
from 142.4.+-.24.5 to 112.9.+-.27.3 mL (matrix, p=0.02) vs
138.9.+-.36.1 to 148.7.+-.41 mL (no matrix, p=0.57), LV filling
deceleration time improved significantly in the matrix group from
162.+-.7 ms to 198.+-.9 ms (p=0.01) vs no-matrix group (from
159.+-.5 ms to 167.+-.8 ms, p=0.07). Scar area thickness progress
from 6.+-.1.4 to 9.+-..+-.1.1 mm (matrix, p=0.005) vs 5.+-.1.5 to
6.+-.0.8 mm (no matrix, p=0.09). EF improved in both groups, from
25.3.+-.7.3 to 32.+-.5.4% (matrix, p=0.03) versus 27.2.+-.6.9 to
34.6.+-.7.3% (no matrix, p=0.031).
[0204] Comments
[0205] This clinical study showed that cell transplantation
associated with a collagen cell-seeded matrix increased the
thickness of the infarct scar with viable tissues and help to
normalize cardiac wall stress in injured regions (scaffold effect),
thus limiting ventricular remodelling and improving diastolic
function. Patients treated without the cell-seeded collagen matrix
didn't show limitation of post ischemic remodelling and
improvements in diastolic function.
Example 5
Preparation of an Hybrid Material for Three-Dimensional Culture
with Improved Mechanical Properties Filling Elastomeric Membranes
with Self-Assembling Synthetic Peptides Resuspended in Water
[0206] Mechanical Properties of Three Dimensional Scaffolds
[0207] During the last decades cellular cardiomyoplasty has become
a state of art for cardiac affects. It consists in introducing
myocardial or stem cells (with and without three-dimensional
matrices) in the infarcted ventricles trying to recover the lost
function. The drawback is that it was proved that there were a low
number of cells capable of surviving in these conditions; partly
because they cannot stand the mechanical forces of the receptor
tissue.
[0208] Three-dimensional scaffolds as RAD16-I (self-assembling
peptides resuspended in water) allow the cells to form a functional
network in the .beta.-sheet scaffold formed, but additionally it is
indispensable that the scaffold could stand the beat of the heart.
Elastomeric membranes can offer these mechanical properties.
[0209] Congo Red Staining
[0210] Congo red staining is a simple method to appreciate the
formation of typical RAD16-I self-assembly .beta.-sheet. The
reactive is as sodium salt of
benzidinediazo-bis-1-naphthylamine-4-sulfonic acid (formula:
C.sub.32H.sub.22N.sub.6Na.sub.2O.sub.6S.sub.2) and its
configuration permits hydrogen bonding of the azo and amine groups
of the dye to similarly spaced hydroxyl radicals giving a red
coloration.
[0211] Material and Methods
[0212] Pieces of microporous membranes of polyethyl acrylate
(PEA100 in what follows) and a copolymer of ethyl acrylate and
hydroxyethyl acrylate with a 90:10 mass ratio of both monomers
(hereafter PEA90) were employed as elastomeric membranes. The
membranes had been cut into pieces of dimensions 0.5 cm.times.0.5
cm, with an approximate thickness of 1.0 mm (PEA100 and PEA90). The
pores of the membranes consisted in layers of orthogonal families
of parallel cylindrical pores, with pore diameter of 150 microns
and pore separation of 300 microns. They were generated by letting
the precursors of the polymers react inside a template of the
porous structure, and dissolving the template afterwards.
[0213] The self assembling peptide RAD16-I was used as
three-dimensional scaffold. The stock was prepared in 1% solution
of sucrose 10% avoiding the self-assembling produced by the
increase of the ionic strength. The stock solution is diluted to
the desired concentration in sucrose 10% for each experiment.
[0214] Scaffolds Pre-Hydration
[0215] The elastomeric scaffolds needed to be pre-conditioned
before the peptide addition. Initially the membranes were
sterilized using three washes with EtOH 70% and letting them dry in
air during 10 min. After this pre-treatment the scaffolds were
hydrated as follows: 30 min of immersion in a PBS solution with
vacuum and three washes with sucrose 10%. The vacuum was necessary
to ensure that all the pores were filled with the aqueous solution,
and the isotonic solution was necessary to avoid the self-assembly
during the first contact between the membranes and the peptide.
After this treatment, the membranes were dried to moist since the
complete drying would return the membranes to their initial
hydrophobicity.
[0216] Filling of the Membranes with RADI6-I Peptide
[0217] The pre-treated membranes were introduced inside a
9-mm-diameter cell culture insert (PICM01250, Millipore, Billerica,
Mass.). Then RAD16-I peptide 0.15% in sucrose 10% was loaded,
carefully, on the top of the membrane using a micropipette. After
the loading of the peptide, 500 .mu.L of DMEM complete medium with
L-glutamine, sodium pyruvate and 15% Fetal Bovine Serum was placed
out of the insert. The peptide was let to self-assemble in the flow
cabinet during 20 min. At this point the medium penetrates in the
insert from the bottom membrane inducing a bottom-to-top
self-assembly of RAD16-I inside the membrane. To wash out the
remaining sucrose, medium was added in sequential steps on the top
of the ensemble and allowed to infiltrate. Finally 500 L were
loaded inside the insert and 2.5 mL in the well outside the
insert.
[0218] Results and Comments
[0219] Both, PEA100 and PEA90 membranes are filled with RAD16-I
peptide. Each group consisted on 2 samples. It is therefore
considered a composite material: elastomeric
membranes+self-assembling peptides.
[0220] The results showed that PEA100 membrane allows RAD16-I to
fill the porous easily than PEA90 membrane. Thus, it seems that the
most hydrophobic PEA100 polymer is in principle preferable in order
to obtain the combined system with improved mechanical properties
compared with those of the peptide gel, that would permit to hold
the heartbeat.
Example 6
[0221] The goal of this example is to show the viscoelasticity
evaluation of elastomer matrix scaffolds for ventricular support
and myocardial regeneration.
[0222] Viscoelastic properties of myocardial tissue has been
recently identified as a major determinant of contraction and
relaxation coupling. The goal of our approach is to develop tissue
engineered implants for ventricular support and myocardial
regeneration, using nanobiomaterials associated with stem cell
grafting. In the present study viscoelastic properties of several
nanobiopolymers developed were assessed by applying a constant
stress. Their stress-strain responses as well as their temporal
dependencies mimicked the behavior of the classical Kelvin Standard
Linear Solid Model which combines a Voigt system (hookean spring E2
in parallel with a viscous dashpot n2), and a hookean spring (E1)
in series of the Voigt system. Thus, under a constant stress, the
materials instantaneously deform to some strain, which is the
elastic part of the strain, and after that it will continue to
deform and asymptotically approach a steady-state strain. This last
part is the viscous component of the strain.
[0223] Methods
[0224] We evaluated viscous and elastic properties of 3 types of
porous membranes:
[0225] A) a non-degradable copolymer of ethyl acrylate and
hydroxyethyl acrylate with a 90:10 mass ratio of monomers (PEA
90),
[0226] B) partially degradable polymer: methacrylate-endcapped
caprolactone (CLMA) membranes, and
[0227] C) native collagen matrices of bovine origin (control
group).
[0228] The elastic modulus E1 and E2 (in mN/mm) and the viscosity
coefficient (n2 in mN/mms) were calculated using the load clamp
technique. Pieces of 12 mm.times.1 mm, thickness 1 mm of PEA 90
matrices were studied in water (8 days) (1), in water (1 hour) (2)
or only in air (3); same protocol for CLMA matrices: (4), (5), (6);
and same protocol for collagen matrices: (7), (8), (9).
[0229] Results
[0230] All samples showed linear stress-strain relationship,
simplifying the evaluation of viscoelasticity. In all groups, E1
ranged from 20 to 40 mN/mm, except in (6) where E1 was about 180
mN/mm. In all groups, E2 ranged from 10 to 100 mN/mm, except in (6)
where E1 was about 400 mN/mm. In all groups, n2 ranged from 1 to 5
mN/mms, except in (6) where n2 was about 18 mN/mms. All studied
matrices exhibited a viscoelastic behavior similar to the Kelvin
Standard Linear Solid Model. However, all viscoelastic coefficients
E1, E2 and n2 were higher in CLMA in air than in all other
groups.
[0231] Conclusions
[0232] Viscous and elastic properties of "bioactive implants" of
the present invention match the characteristics of myocardial
contraction and relaxation activity at both structural and
functional bio integrative levels. Bioactive implants specially
conditioned for the recovery of left and/or right ventricular
myocardium may reduce adverse chamber remodelling and fibrosis.
Example 7
Tensile Properties of Partially Degradable Polymer Matrices
Employed for the Fabrication of the Membranes
[0233] This example aims to give representative information about
the swelling capacity and the tensile mechanical properties of
three possible polymer compositions employed for the elastomer
membrane fabrication, as a function of their weight fraction of
degradable and non-degradable parts.
[0234] Polyethylacrylate (PEA) was chosen as the non-degradable
polymer, and poly(caprolactonemethacryloyloxyethyl ester) (PCLMA)
as the partially degradable polymer. Three systems containing both
polymers were prepared by radical polymerization of the monomers in
mass ratios of 15:85, 50:50 and 85:15 (see table). Sheets of the
three bulk polymers of 0.3 mm thickness were obtained in this way,
and samples of dimensions 0.3 mm.times.30 mm.times.6 mm were cut
from those sheets in order to perform mechanical tests. The table
gives, for each system prepared, the weight fraction of
non-degradable part, of partially degradable component, and of
degradable part.
[0235] Stress-strain measurements were made in tensile mode in a
Microtest SCM3000 95 apparatus by stretching the specimen at a
constant strain rate of 0.01/min and simultaneously measuring the
force applied to the specimen. The tests were continued until the
samples broke. The tests were carried out on samples equilibrated
in phosphate buffer saline (PBS). At least 3 replicates of each
sample were tested. The weight increase after 48 h immersion in PBS
was referred to the dry weight of the sample to define the
"swelling mass increase".
TABLE-US-00002 mass ratio mass ratio mass ratio of of non- of
partially swelling System degradable degradable degradable mass
breaking strain Young PEA:PCLMA part part polymer increase strength
at break modulus mass ratio (%) (%) (PCLMA) (%) (%) (KPa) (%) (Pa)
15:85 40.1 59.9 85 6.83 .+-. 0.15 1982 .+-. 117 19.8 .+-. 1.8 11961
.+-. 1295 50:50 23.6 76.4 50 5.32 .+-. 0.13 1904 .+-. 402 23.7 .+-.
5.0 10214 .+-. 1368 85:15 7.1 92.9 15 1.92 .+-. 0.40 3445 .+-. 299
55.8 .+-. 0.8 9816 .+-. 472
[0236] Table. Tensile and Swelling Properties of the Three Polymer
Matrices.
[0237] It is to be understood that the above-described embodiments
are illustrative of only a few of the many possible specific
embodiments, which can represent applications of the principles of
the invention. Numerous and varied other arrangements can be
readily devised in accordance with these principles by those
skilled in the art without departing from the spirit and scope of
the invention.
* * * * *