U.S. patent application number 10/081835 was filed with the patent office on 2002-10-24 for rapid preparation of stem cell matrices for use in tissue and organ treatment and repair.
Invention is credited to Capelli, Christopher, Chancellor, Michael B., Chung, Steve, Huard, Johnny, Sacks, Michael S..
Application Number | 20020155096 10/081835 |
Document ID | / |
Family ID | 23034861 |
Filed Date | 2002-10-24 |
United States Patent
Application |
20020155096 |
Kind Code |
A1 |
Chancellor, Michael B. ; et
al. |
October 24, 2002 |
Rapid preparation of stem cell matrices for use in tissue and organ
treatment and repair
Abstract
The present invention describes a rapid method for preparing
stem cell and physiologically acceptable matrix compositions for
use in tissue and organ repair. Compared with previous tissue
engineering materials, the stem cell-matrix compositions of the
present invention do not require long-term incubation or
cultivation in vitro prior to use in in vivo applications. The stem
cells can be from numerous sources and may be homogeneous,
heterogeneous, autologous, and/or allogeneic in the matrix
material. The stem cell-matrix compositions as described provide
point of service utility for the practitioner, wherein the stem
cells and matrix can be combined not long before use, thereby
alleviating costly and lengthy manufacturing procedures. In
addition, the stem cells offer unique structural properties to the
matrix composition which improves outcome and healing after use.
Use of stem cells obtained from muscle affords contractility to the
matrix composition.
Inventors: |
Chancellor, Michael B.;
(Pittsburgh, PA) ; Huard, Johnny; (Wexford,
PA) ; Capelli, Christopher; (Kenosha, WI) ;
Chung, Steve; (Pittsburgh, PA) ; Sacks, Michael
S.; (Pittsburgh, PA) |
Correspondence
Address: |
MORGAN & FINNEGAN, L.L.P.
345 Park Avenue
New York
NY
10154-0053
US
|
Family ID: |
23034861 |
Appl. No.: |
10/081835 |
Filed: |
February 22, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60271267 |
Feb 23, 2001 |
|
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|
Current U.S.
Class: |
424/93.7 ;
435/366 |
Current CPC
Class: |
A61L 27/3629 20130101;
A61K 35/12 20130101; C12N 2533/92 20130101; C12N 5/0658 20130101;
A61P 9/00 20180101; A61P 17/02 20180101; C12N 5/0068 20130101; C12N
2533/56 20130101; A61L 2430/22 20130101; A61L 27/3679 20130101;
C12N 2533/74 20130101; A61L 27/3834 20130101; A61L 27/225 20130101;
C12N 5/0668 20130101; A61P 21/00 20180101; C12N 2533/90 20130101;
C12N 5/0697 20130101; A61P 43/00 20180101 |
Class at
Publication: |
424/93.7 ;
435/366 |
International
Class: |
A61K 045/00; C12N
005/08 |
Goverment Interests
[0002] The U.S. Government has certain rights in the present
invention pursuant to Research Grant DK55387 from the National
Institutes of Health.
Claims
What is claimed is:
1. A rapid method of preparing a stem cell-biomatrix for use in
tissue and organ treatment or repair comprising: a) admixing a stem
cell preparation with a physiologically acceptable matrix material
to form a stem cell matrix; and b) incubating the stem-cell matrix
of step a in vitro for less than about 12 hours prior to use in
tissue or organ treatment or repair in a recipient.
2. The method according to claim 1, wherein the stem cells are
autologous to the recipient.
3. The method according to claim 1, wherein the stem cells are
allogeneic to the recipient.
4. The method according to claim 1, wherein the physiologically
acceptable matrix material is absorbable or non-absorbable.
5. The method according to claim 1 or claim 4, wherein the matrix
material is selected from the group consisting of small intestine
submucosa (SIS), crosslinked alginate, bioadhesives, hydrocolloid,
collagen gel, collagen sponge, polyglycolic acid (PGA) mesh,
polyglactin (PGL) mesh, fleeces and dead de-epidermized skin
equivalents in one or more layers.
6. The method according to claim 1, wherein homogeneous or
heterogeneous populations of stem cells are admixed with the matrix
material.
7. The method according to claim 1 or claim 6, wherein the stem
cells are obtained from tissues selected from the group consisting
of bone marrow, muscle, adipose, liver, heart, lung and nervous
system.
8. The method according to claim 7, wherein the tissues are
selected from the group consisting of adult, embryonic or fetal
tissues.
9. The method according to claim 1 or claim 6, wherein the stem
cells are obtained from muscle.
10. The method according to claim 9, wherein the stem cell matrix
is contractible.
11. The method according to claim 1, wherein the stem cells are
introduced into the physiologically acceptable matrix material so
as to deposit from about 2.5.times.103 to about 1.times.10.sup.6
stem cells in the matrix material.
12. The method according to claim 11, wherein the stem cells are
introduced into the physiologically acceptable matrix material so
as to deposit from about 5.times.10.sup.3 to about 1.times.10.sup.6
stem cells in the matrix material.
13. The method according to claim 1, wherein the stem cells are
introduced into the physiologically acceptable matrix material so
as to deposit about 1.times.10.sup.5 stem cells per 1 cm.sup.2 of
matrix.
14. The method according to claim 1, wherein the stem cells are
incubated with the matrix material in vitro for less than about 3
hours prior to being used for tissue or organ repair.
15. The method according to claim 1, wherein the stem cells are
incubated with the matrix material in vitro for less than about 1
hour prior to being used for tissue or organ repair.
16. The method according to claim 1, wherein the stem cells are
incubated with the matrix material in vitro for less than about 30
minutes prior to being used for tissue or organ repair.
17. The method according to claim 1, wherein the stem cells are
incubated with the matrix material in vitro from about 5 seconds to
about 30 minutes prior to being used for tissue or organ
repair.
18. The method according to claim 1, wherein the tissue or organ
repair are selected from the group consisting of wound healing,
surgical incision repair, tissue augmentation, organ augmentation,
smooth muscle repair, non-smooth muscle repair and blood vessel
repair.
19. The method according to claim 1, wherein the stem cells used
for tissue or organ repair are attached to the matrix material
using biological adhesives.
20. The method according to claim 1, wherein the stem cells alter
biomechanical properties of the matrix material.
21. A composition comprising stem cells and a physiologically
acceptable matrix material forming a stem cell matrix prepared
according to claim 1.
22. The composition according to claim 21, wherein the stem cell
matrix is contractible.
23. A rapid method of preparing a stem cell-biomatrix for use in
tissue and organ treatment or repair comprising: a) admixing a stem
cell preparation with a first physiologically acceptable matrix
material to form a first stem cell-matrix combination; b)
introducing the first stem cell-matrix combination of step a) onto
a second physiologically acceptable matrix material to form a
second stem cell-matrix material, wherein the first stem
cell-matrix combination and the second physiologically acceptable
matrix material are incubated in vitro for between about 5 seconds
and 1 hour; and c) applying the second stem cell-matrix material of
step b on or in a tissue or organ site in a recipient.
24. The method according to claim 23, wherein the stem cells are
autologous to the recipient.
25. The method according to claim 23, wherein the stem cells are
allogeneic to the recipient.
26. The method according to claim 23, wherein the first and second
physiologically acceptable matrix materials are absorbable or
non-absorbable.
27. The method according to claim 23, wherein the first
physiologically acceptable matrix material is selected from the
group consisting of crosslinked alginate, hydrocolloid, collagen
gel and bioadhesives; and wherein the second physiologically
acceptable matrix material is selected from the group consisting of
small intestine submucosa (SIS), collagen sponge, polyglycolic acid
(PGA) mesh, polyglactin (PGL) mesh, fleeces and dead de-epidermized
skin equivalents in one or more layers.
28. The method according to claim 23, wherein homogeneous or
heterogeneous populations of stem cells are admixed with the first
or second matrix material.
29. The method according to claim 23, wherein the stem cells are
obtained from tissues selected from the group consisting of bone
marrow, muscle, adipose, liver, heart, lung and nervous system.
30. The method according to claim 29, wherein the tissues are
selected from the group consisting of adult, embryonic and fetal
tissues.
31. The method according to claim 23, wherein the stem cells are
introduced into the physiologically acceptable matrix material so
as to deposit from about 2.5.times.10.sup.3 to about
1.times.10.sup.6 stem cells in the matrix material.
32. The method according to claim 31, wherein the stem cells are
introduced into the physiologically acceptable matrix material so
as to deposit from about 5.times.10.sup.3 to about 1.times.10.sup.6
stem cells in the matrix material.
33. The method according to claim 23, wherein the stem cells are
introduced into the physiologically acceptable matrix material so
as to deposit about 1.times.10.sup.5 stem cells per 1 cm.sup.2 of
matrix.
34. The method according to claim 23, wherein the second stem
cell-matrix material functions at the tissue or organ sites in
medical procedures selected from the group consisting of wound
healing, surgical incision repair, tissue augmentation, organ
augmentation, smooth muscle repair, non-smooth muscle repair and
blood vessel repair.
35. The method according to claim 23, wherein the stem cells alter
biomechanical properties of the second matrix material.
36. The method according to claim 23, wherein the stem cell matrix
is contractible.
37. The method according to claim 1 or claim 23, wherein the stem
cell-matrix is applied to a tissue or organ site by a mode selected
from the group consisting of spraying, painting, coating and
spreading.
38. A preparation of stem cells and a physiologically acceptable
substrate material forming an implantable and innervatable
three-dimensional scaffolding for tissue and organ repair.
39. The preparation according to claim 38, wherein the substrate
material is small intestine submucosa (SIS).
40. The preparation according to claim 38, wherein stem cells are
autologous to a recipient of the preparation.
41. The preparation according to claim 38, wherein the stem cells
are allogeneic to a recipient of the preparation.
42. The preparation according to claim 38, wherein the stem cells
are obtained from muscle.
43. The preparation according to claim 38, wherein the stem cells
are obtained from skeletal muscle.
44. The preparation according to claim 42 or claim 43, comprising a
three-dimensional muscle replacement having muscle
contractility.
45. The preparation according to claim 44, further wherein the
substrate material is small intestine submucosa (SIS).
Description
[0001] This application claims benefit of provisional application
U.S. Serial No. 60/271,267, filed Feb. 23, 2001, the contents of
which are hereby incorporated by reference its entirety.
FIELD OF THE INVENTION
[0003] The present invention generally relates to cellular-based
tissue engineering and methods of preparing cell and biologically
compatible matrix combinations. More specifically, the invention
relates to muscle-derived stem cell-based matrix compositions and
products and to a rapid method of producing and utilizing such
compositions and products at a tissue or organ site of need.
BACKGROUND OF THE INVENTION
[0004] Presently, all attempts at cellular-based tissue engineering
are being done at the manufacturing site where uniform and
continuous infiltration of cells into a matrix are prepared
together before shipping for implantation. The manufacturing of
cellular based medical devices is costly and time consuming. Part
of the lengthy time consumption results from the necessity of
incubating the cells in the matrix for long periods before in vivo
use. For example, in the overview of a company involved in the
preparation of cell scaffolds, it is stated that "after [the cells]
develop over a period of a few weeks, the cells on the scaffold are
kept under optimal conditions in bioreactors. The bioreactors are
challenging to build because they have to create perfect conditions
to grow the implants." (Advanced Tissue Sciences, Inc., La Jolla,
Calif.).
[0005] It is known that cellular tissue engineering requires
continuous biological devices that are pre-constructed prior to
implantation (L. Germain et al., 2000, Medical & Biological
Engineering & Computing, 38(2):232-40; W. W. Minuth et al.,
1998, Cell Tissue Res., 291; (1):1-11; G. K. Naughton et al., 1999,
Clin. in Plastic Surgery, 26(4):579-86, viii). Until the present
time using the invention described herein, most cell-matrices were
made using non-stem cells. However, these cells have proven to be
difficult to work with (F. Berthod and O. Damour, 1997, British
Journal of Dermatology, 136:809-816). In order to produce such
non-stem cell matrices, prolonged incubation times are needed after
the cells have been incorporated into the matrix. As a particular
example, it has been reported that at least 3 to 5 hours of
incubation time is needed for fibroblasts to attach to a scaffold
and another 2 to 3 weeks of incubation time is required to achieve
confluence (J. F. Hansbrough et al., 1991, Surgery,
111(4):438-446). Moreover, the use of collagen sponges requires
that the cells have to be incubated in the sponge for at least 24
hours, and another 8 to 10 days are needed for the cells to achieve
confluence (F. Berthod et al., 1993, Biomaterials,
14(10):749-754).
[0006] In addition, currently available cell-matrices require
careful monitoring. Cells on scaffolds are vigorously tested to
confirm and maintain the proper metabolic rate that will allow the
cells on scaffolds to grow and reproduce in a steady-state manner
resulting in optimal materials (Advanced Tissue Sciences, Inc. La
Jolla, Calif.).
[0007] For cell matrix products used in wound coverage, there has
been a perpetual belief that uniform layers of continuous cells are
needed for a functional product (F. A. Auger et al., 1988, Med.
Biol. Eng. Compu., 36: 801-812; S. T. Boyce, 1996, Tissue Eng., 2:
255-266; O. Damour, et al., 1997, "Cultured autologous epidermis
for massive burn wounds: 15 years of practice". Rouabhia, M. (Ed.):
Skin substitute production by tissue engineering: clinical and
fundamental applications. Landes, Austin, pp. 23-45). As an
example, the importance of continuous layers has been emphasized in
conjunction with the marketing of the product Apligraf.RTM.
(Organogenesis, Inc., Canton, Mass.), which is carefully cultured
on scaffold in a bilayer for over 20 days to produce a continuous
cell layer of dermis and epidermis.
[0008] Current methods of producing cell matrices for in vivo
tissue and organ repair are very costly and time consuming. Such
cell matrices are costly due to the specialized factories and/or
procedures needed to produce these products. Also, since
cell-matrix products involve living biological cells/tissue, a
tremendous loss of product occurs from shipping, the delays
associated therewith, and the like. Additionally, given the nature
of the products, obtaining regulatory approval for new products
that are based on living cells and a new matrix poses
difficulties.
[0009] Thus, there is a serious need for cell-matrix compositions
that are low in cost, that are versatile, and easily prepared
and/or manufactured. There is a further need for cell matrix
compositions that do not require extensive in vitro incubation or
cultivation periods after the cells have been incorporated into the
matrix. Those in the art have recognized that a major problem
remaining to be solved is the delay in producing the cell-matrix
product after initial preparation. Specifically, it has been stated
that there is a problem of a three week delay necessary to produce
a sufficient amount of autologous keratinocytes and fibroblasts for
the production of reconstructed skin. (F. Berthod and O. Damour,
1997, British Journal of Dermatology, 136: 809-816). The present
invention provides a solution for the above-mentioned problems and
delays currently extant in the art.
SUMMARY OF THE INVENTION
[0010] One aspect of the present invention provides methods of
preparing stem cell matrices, particularly biomatrices, for use in
tissue and organ repair. In accordance with the present invention,
stem cells are employed rather than other cell types (e.g.,
differentiated or non-stem cells). In addition, the stem cells and
the matrix material are admixed or combined shortly before or
immediately prior to use, thereby eliminating the need for
long-term incubations or culture of cells with matrix material.
Thus, the methods are rapid and allow the preparation of a stem
cell-based biomatrix material as needed.
[0011] In another aspect of this invention, the stem cell-biomatrix
material can be in the form of a sling, patch, wrap, such as are
employed in surgeries to correct, strengthen, or otherwise repair
tissues and organs in need of such treatment. As is appreciated by
the person skilled in the art, a sling can comprise a material
placed beneath deficient sphincter to provide support, e.g., a
pubvaginal sling to repair stress urinary incontinence. A patch can
comprise a material that is applied over a section of weak, thin,
or deficient organ or tissue that is either solid or hollow, e.g.,
a tissue engineering patch of heart or bladder. A wrap refers to a
circumferential patch, e.g., a material placed around a blood
vessel or gastroesophageal sphincter.
[0012] Another aspect of the present invention provides stem
cell-matrix compositions for in vivo tissue and/or organ repair, or
surgical or wound healing, that can be produced at the
"point-of-service", i.e., at the bedside or surgical suite just
before the medical procedure for tissue and/or organ treatment or
repair occurs. According to this invention, prolonged delays (as
well as increased costs) resulting from commercial preparation of a
cell matrix material are alleviated.
[0013] Yet another aspect of the present invention provides stem
cell-biological, physiologically compatible adhesive (i.e.,
bioadhesive) and/or biological matrix (i.e., biomatrix)
combinations, or compositions thereof, for direct application to
the external surface of organs such as skeletal muscle and skin,
and smooth muscle, such as the diaphragm, bladder, intestine and
heart. In addition, these combinations, or compositions thereof,
can be applied to organs such as the liver, spleen, thymus, spinal
cord and bone. In accordance with the present invention, the stem
cells or progenitor cells can be autologous (obtained from the
recipient, including humans) or allogeneic (obtained from a host
source other than the recipient, including humans).
[0014] A further aspect of the present invention provides an
implantable, innervatable physiologically acceptable
three-dimensional scaffolding for tissue and organ repair
comprising a preparation of stem cells and a physiologically
acceptable biological substrate, preferably small intestine
submucosa (SIS). Preferably, the stem cells are obtained from
muscle. In accordance with the present invention, the stem
cell-biomatrix material, or stem cell-three-dimensional
scaffolding, as described herein, are capable of contractility,
particularly when the stem cells employed are obtained from
muscle.
[0015] Additional aspects, features and advantages afforded by the
present invention will be apparent from the detailed description
and exemplification hereinbelow.
DESCRIPTION OF THE FIGURES
[0016] FIG. 1 presents the results of experiments performed to test
areal strain of small intestine submucosa (SIS) containing muscle
stem cells (MDSC/SIS) compared with non-stem cell containing SIS
(control). (Example 8). The mean (.+-.S.D.) areal strain in the
control group (n=5) of non-incubated SIS was 20.80.+-.5.9. At 10
days of incubation, the mean (.+-.S.D.) areal strain in MDSC/SIS
(29.15.+-.5.4, n=5) was significantly higher (p=0.01) than that of
SIS alone (17.98.+-.n=6). Also at 20 days of incubation there were
significant differences between SIS alone and MDSC/SIS
preparations.
[0017] FIG. 2 presents results of experiments performed to test the
contractility of muscle stem cells incorporated into a SIS
scaffold. (Example 9). As observed in FIG. 2, none of the SIS
strips at any time point up to 8-weeks showed any contractile
activity. In the 8-week of MDSC/SIS preparations, spontaneous
contractile activity was observed cultures (8 of 8 specimens).
Also, in 8-week MDSC/SIS preparations, the frequency and amplitude
of spontaneous contractile activity were decreased by the addition
of 20 .mu.M succinylcholine.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The present invention involves methods of preparing stem
cell or progenitor cell matrices for use in tissue and organ
repair, comprising a medically- or physiologically-acceptable
matrix material and autologous and/or allogeneic stem cells,
preferably, muscle-derived stem cells. The invention involves
admixing, such as by inoculating or seeding the stem cells into the
medically- or physiologically-acceptable matrix material and using
the combined stem cell-matrix composition or product almost
immediately for in vivo tissue or organ treatment and repair.
[0019] According to this invention, the stem cell matrices are made
for in vivo use in tissue or organ treatment and repair without the
need for prolonged, prior in vitro incubation of the stem cell
matrices after the cells have been inoculated or introduced into a
given biomatrix. Unlike the present invention, previous cell
matrices made for in vivo tissue or organ repair have depended on a
prolonged incubation of the cells in the matrix prior to use. By
using stem cells in the stem cell matrix compositions of this
invention, rather than non-stem cells, or other types of cells, the
need for prolonged in vitro incubation is not required.
[0020] As a result of the present invention, methods of producing
stem cell-matrix compositions that can be used almost immediately
after preparation at the time of use are now possible. This is
important because stem cell-matrix compositions provided for in
vivo tissue and/or organ treatment or repair can be produced at the
"point-of-service", i.e., at the bedside or surgical suite just
before the medical procedure for tissue and/or organ treatment or
repair occurs. Prolonged delays (as well as increased costs)
resulting from sending cells to an outside laboratory for
incorporation and incubation into a matrix, and then waiting for
several weeks to receive the cell-matrix material for medical use
(e.g., for in vivo tissue or organ treatment or repair procedures)
are obviated and avoided in view of the present invention.
[0021] In accordance with the present invention, a short duration
mixture of stem cells on a scaffold with non-uniform or irregular
coverage of cells, on and/or within the scaffold, results in the
proliferation of stem cells in and on the matrix to result in
cellular differentiation, the release of factors by the stem cells,
and improved outcome. Although it was at first believed that
uniform and continuous layers of cells, as well as long periods of
careful incubation or culturing, were needed to produce cell-based
matrix compositions, it was surprisingly discovered that the use of
stem cells at the point of service resulted in the proliferation of
stem cells in the matrix that yielded smooth layers and improved
healing of the biological device. It was further discovered that
point of service application of stem cells with a biologic device,
e.g., a biological matrix, improved function. For example, the stem
cells were capable of releasing factors that allowed improved
therapy, treatment and overall function.
[0022] According to the present invention, the stem cells can be
mixed with the matrix material in vitro not long before application
to a tissue or organ site in vivo. Alternatively, the stem cells
can be mixed with, or inoculated onto, the matrix material just at
the time of use. In some cases, depending upon cell source, cell
concentration and matrix material, the admixing of stem cells and
matrix material, or the inoculation of stem cells onto matrix
material, needs no more time than the time that it takes to combine
the stem cells and the matrix at the point of use.
[0023] In accordance with the present invention, the in vitro
incubation of stem cells with matrix material is performed for from
about 5 seconds to less than about 12 hours, preferably for from
about 5 seconds to about 30 minutes. The in vitro incubation of
stem cells with matrix material according to this invention is
generally less than about 3 hours, preferably, less than about 1
hour, more preferably, less than about 30 minutes. Indeed, the
experiments described herein indicate that use of the stem cells
and matrix material combination can be used virtually immediately
after stem cells are mixed with or inoculated onto a biomatrix. No
long-term (e.g.,>about 12 hours, days, or weeks) of incubation
or culture time is necessary to achieve results using the
combination of stem cell-matrix material (i.e., stem cell-matrix
material composition) in a variety of applications. Such stem
cell-matrix combinations and compositions can be used in wound
healing; surgical procedures; the sealing of openings, fissures,
incisions, and the like; and the augmentation, filling, or
reconstitution of tissues and organs of the body, for example,
following surgery, or as the result of diseases, disorders,
conditions, accidents, or therapies.
[0024] The compositions of the invention can be used in treatments
for diseases such as impaired muscle contractility of the heart,
diaphragm, gastrointestinal tract, and genitourinary tract. Use of
the present invention is also made for a variety of treatments,
repair, augmentation, filling and healing of skin (dermis and
epidermis) and soft tissue, muscle, bone, ligaments, and the like,
so as to reduce scarring that results from conventional
techniques.
[0025] A variety of biological or synthetic solid matrix materials
(i.e., solid support matrices, biological adhesives or dressings,
and biological/medical scaffolds) are suitable for use in this
invention. The matrix material is preferably medically acceptable
for use in in vivo applications. Nonlimiting examples of such
medically acceptable and/or biologically or physiologically
acceptable or compatible materials include, but are not limited to,
solid matrix materials that are absorbable and/or non-absorbable,
such as small intestine submucosa (SIS), e.g., porcine-derived (and
other SIS sources); crosslinked or non-crosslinked alginate,
hydrocolloid, foams, collagen gel, collagen sponge, polyglycolic
acid (PGA) mesh, polyglactin (PGL) mesh, fleeces, foam dressing,
bioadhesives (e.g., fibrin glue and fibrin gel) and dead
de-epidermized skin equivalents in one or more layers. As an
exemplary bioadhesive, fibrin glue preparations have been described
in WO 93/05067 to Baxter International, Inc., WO 92/13495 to
Fibratek, Inc. WO 91/09641 to Cryolife, Inc., and U.S. Pat. Nos.
5,607,694 and 5,631,019 to G. Marx.
[0026] In an embodiment of the present invention, the stem
cell-biomatrix material can be in the form of a sling, patch, wrap,
such as are employed in surgeries to correct, strengthen, or
otherwise repair tissues and organs in need of such treatment. As
is appreciated by the person skilled in the art, a sling can
comprise a material placed beneath deficient sphincter to provide
support, e.g., a pubvaginal sling to repair stress urinary
incontinence. A patch can comprise a material that is applied over
a section of weak, thin, or deficient organ or tissue that is
either solid or hollow, e.g., a tissue engineering patch of heart
or bladder. A wrap refers to a circumferential patch, e.g., a
material placed around a blood vessel or gastroesophageal
sphincter.
[0027] The stem cells or progenitor cells can be autologous
(obtained from the recipient, including humans) or allogeneic
(obtained from a donor source other than the recipient, including
humans). For allogeneic stem cell or progenitor cell sources, the
closest possible immunological match between donor and recipient is
desired. If an autologous source is not available or warranted,
donor and recipient Class I and Class II histocompatibility
antigens can be analyzed to determine the closest match available.
This minimizes or eliminates immune rejection and reduces the need
for immunosuppressive or immunomodulatory therapy. If required,
immunosuppressive or immunomodulatory therapy can be started
before, during, and/or after the matrix is applied or introduced
into a patient. For example, cyclosporin A, or other
immunosuppressive drugs, can be administered to the recipient.
Immunological tolerance may also be induced prior to
transplantation by alternative methods known in the art (D. J. Watt
et al., 1984, Clin. Exp. Immunol. 55:419; D. Faustman et al., 1991,
Science 252:1701).
[0028] According to the present invention, stem cells or progenitor
cells are prepared, isolated or obtained from a variety of sources.
For example, the stem cells or progenitor cells may be from bone
marrow or from muscle (e.g., skeletal muscle). Preferred are
muscle-derived stem cells (MDSC), also called muscle stem cells
(MSC) herein, which have been isolated as described in WO 99/56785
(University of Pittsburgh) and in U.S. Serial Nos. 09/302,896,
filed Apr. 30, 1999, and 09/549,937, filed Apr. 14, 2000, to M.
Chancellor et al., the contents of which are hereby incorporated by
reference herein in their entireties.
[0029] The stem cells utilized in the stem cell matrix compositions
can be combinations of different types of stem cells, e.g.,
heterogeneous populations of stem cells obtained from autologous or
allogeneic donor sources, or they can be homogeneous stem cell
populations (from autologous or allogeneic sources). Combinations
of stem cells of different origins (e.g., a combination of bone
marrow and muscle stem cells) are also envisioned. The stem cells
can be obtained from animal tissues, including human tissue, such
as muscle, adipose, liver, heart, lung and the nervous system, as
non-limiting examples. In addition, the tissues may be adult,
fetal, or embryonic tissues.
[0030] The stem cell-bioadhesive or biomatrix combinations, or
compositions thereof, can be directly applied to the external
surface of organs such as skin, skeletal muscle and smooth muscle,
e.g., the diaphragm, bladder, intestine and heart. In addition,
these combinations, or compositions thereof, can be applied to
organs such as the liver, spleen, thymus, spinal cord and bone.
[0031] In addition, the stem cells can be genetically modified to
contain an expression vector, e.g., plasmid or viral, containing
one or more heterologous genes which are expressed and whose
expression products are produced at the site at which the stem
cell-matrix is applied or introduced in vivo. Accordingly, the
cells may be genetically engineered to contain one or more nucleic
acid sequence(s) encoding one or more active biomolecules, and to
express these biomolecules, including proteins, polypeptides,
peptides, hormones, metabolites, drugs, enzymes, and the like. The
stem cell-matrix composition or product can serve as a long-term
local delivery system for a variety of treatments, for example, for
the treatment of various diseases and pathologies, such as cancer,
tissue regeneration and reconstitution, and to deliver a gene
product, such as a therapeutic agent, e.g., hormone or factor, to a
tissue or organ site.
[0032] The stem cells may be genetically engineered by a variety of
molecular techniques and methods known to those having skill in the
art, for example, transfection, infection, transduction, or direct
DNA injection. Transduction as used herein commonly refers to cells
that have been genetically engineered to contain a foreign or
heterologous gene via the introduction of a viral or non-viral
vector into the cells. Viral vectors are preferred. Transfection
more commonly refers to cells that have been genetically engineered
to contain a foreign gene harbored in a plasmid, or non-viral
vector. The stem cells can be transfected or transduced by
different vectors and thus can serve as gene delivery vehicles to
allow the gene products to be expressed and produced at the tissue
or organ site.
[0033] Although viral vectors are preferred, those having skill in
the art will appreciate that the genetic engineering of cells to
contain nucleic acid sequences encoding desired proteins or
polypeptides, cytokines, and the like, may be carried out by
methods known in the art, for example, as described in U.S. Pat.
No. 5,538,722, including fusion, transfection, lipofection mediated
by precipitation with DEAE-Dextran (Gopal, 1985) or calcium
phosphate (Graham and Van Der Eb, 1973, Virology, 52:456-467; Chen
and Okayama, 1987, Mol. Cell. Biol. 7:2745-2752; Rippe et al.,
1990, Mol. Cell. Biol., 10:689-695); gene bombardment using high
velocity microprojectiles (Yang et al., 1990, Proc. Natl. Acad.
Sci. USA, 87:9568-9572); microinjection (Harland and Weintraub,
1985, J. Cell Biol., 101:1094-1099); electroporation (Tur-Kaspa et
al., 1986, Mol. Cell. Biol., 6:716-718; Potter et al., 1984, Proc.
Natl. Acad. Sci. USA, 81:7161-7165); DNA (vector)-loaded liposomes
(Fraley et al., 1979, Proc. Natl. Acad. Sci. USA, 76:3348-3352);
lipofectamine-DNA complexes; cell sonication (Fechheimer et al.,
1987, Proc. Natl. Acad. Sci. USA, 84:8463-8467); receptor-mediated
transfection (Wu and Wu, 1987, J. Biol. Chem., 262:4429-4432; Wu
and Wu, 1988, Biochemistry, 27:887-892); and the like.
[0034] Preferred viral vectors are typically derived from
non-cytopathic eukaryotic viruses in which non-essential genes have
been replaced with the nucleic acid sequence(s) of interest.
Non-cytopathic viruses include retroviruses, which replicate by
reverse transcription of genomic viral RNA into DNA with subsequent
proviral integration into host cellular DNA. Retroviruses have been
approved for human gene therapy trials. In general, the
retroviruses are replication-deficient, i.e., capable of directing
synthesis of the desired proteins, but incapable of manufacturing
an infectious particle. Retroviruses from which the retroviral
plasmid vectors may be derived include, but are not limited to,
Moloney murine leukemia virus, spleen necrosis virus, retroviruses
such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis
virus, gibbon ape leukemia virus, human immunodeficiency virus,
adenovirus, myeloproliferative sarcoma virus, and mammary tumor
virus. In general, the retroviruses used to create a viral vector
are preferably debilitated or mutated in some respect to prevent
disease transmission.
[0035] Standard protocols for producing replication-deficient
retroviruses, including the steps of 1) incorporating exogenous
genetic material into a plasmid, 2) transfecting a packaging cell
line with plasmid, production of recombinant retroviruses by the
packaging cell line, 3) collecting viral particles from tissue
culture media, and 4) infecting the target cells with viral
particles, are provided in M. Kriegler, 1990, "Gene Transfer and
Expression, A Laboratory Manual," W. H. Freeman Co., N.Y.; and E.
J. Murry, Ed., 1991, "Methods in Molecular Biology," vol. 7, Humana
Press, Inc., Clifton, N.J.
[0036] Expression vectors containing all the necessary elements for
expression are commercially available and known to those skilled in
the art. See, e.g., Sambrook et al., 1989, Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.; F. M. Ausubel et al. (eds), 1995,
Current Protocols in Molecular Biology, John Wiley & Sons,
Inc., New York, N.Y.; D. N. Glover (ed), 1985, DNA Cloning: A
Practical Approach, Volumes I and II; M. L. Gait (ed), 1984,
Oligonucleotide Synthesis; Hames and Higgins (eds), 1985, Nucleic
Acid Hybridization; Hames and Higgins (eds), 1984, Transcription
and Translation; R. I. Freshney (ed), 1986, Animal Cell Culture;
Immobilized Cells and Enzymes, 1986, (IRL Press); Perbal, 1984, A
Practical Guide to Molecular Cloning; The Series, Methods in
Enzymology, Academic Press, Inc.; J. H. Miller and M. P. Calos
(eds), 1987, Gene Transfer Vectors for Mammalian Cells, Cold Spring
Harbor Laboratory; Wu and Grossman (eds), Methods in Enzymology,
Vol. 154; Wu (ed), Methods in Enzymology, Vol. 155.
[0037] Illustrative examples of vehicles or vector constructs for
transfection or infection of the stem cells of the present
invention include replication-defective viral vectors, DNA virus or
RNA virus (retrovirus) vectors, such as adenovirus, herpes simplex
virus and adeno-associated viral vectors. Preferred are adenovirus
vectors.
[0038] Such vectors will include one or more promoters for
expressing the bioactive molecule. Suitable promoters which may be
employed include, but are not limited to, adenoviral promoters,
such as the adenoviral major late promoter; or heterologous
promoters, such as the cytomegalovirus (CMV) promoter; the
respiratory syncytial virus (RSV) promoter; inducible promoters,
such as the MMT promoter, the metallothionein promoter; heat shock
promoters; the albumin promoter; the ApoAl promoter; human globin
promoters; viral thymidine kinase promoters, such as the Herpes
Simplex thymidine kinase promoter; retroviral LTRs (including the
modified retroviral LTRs herein above described); the .beta.-actin
promoter; and human growth hormone promoters. The promoter also may
be the native promoter that controls the nucleic acid sequence
encoding the polypeptide.
[0039] The vectors are typically substantially free of any
prokaryotic DNA and may comprise a number of different functional
nucleic acid sequences. Examples of such functional sequences
include nucleic acid, e.g., DNA or RNA, sequences comprising
transcriptional and translational initiation and termination
regulatory sequences, including promoters (e.g., strong promoters,
inducible promoters, and the like) and enhancers which are active
in esophagus or small intestine cells. Also included as part of the
functional sequences is an open reading frame (nucleic acid
sequence) encoding a protein, polypeptide, or peptide of interest.
Flanking sequences may also be included for site-directed
integration. In some situations, the 5'-flanking sequence will
allow for homologous recombination, thus changing the nature of the
transcriptional initiation region, so as to provide for inducible
or noninducible transcription to increase or decrease the level of
transcription, as an example.
[0040] In general, the nucleic acid sequence desired to be
expressed by the stem cell in the biological matrix is that of a
structural gene, or a functional fragment, segment or portion of
the gene, which is heterologous to the cell serving as delivery
vehicle and which encodes a desired protein or polypeptide product.
The encoded and expressed product may be intracellular, i.e.,
retained in the cytoplasm, nucleus, or an organelle of a cell, or
may be secreted by the cell. For secretion, the natural signal
sequence present in the structural gene may be retained, or a
signal sequence that is not naturally present in the structural
gene may be used. When the polypeptide or peptide is a fragment of
a protein that is larger, a signal sequence may be provided so
that, upon secretion and processing at the processing site, the
desired protein will have the natural sequence. Examples of genes
of interest for use in accordance with the present invention
include genes encoding cell growth factors, suppressor molecules,
cell differentiation factors, cell signaling factors and programmed
cell death factors.
[0041] Preferably, a marker is present for the selection of cells
containing the vector construct. The marker may be an inducible or
non-inducible gene and will generally allow for positive selection
under induction, or without induction, respectively. Examples of
commonly used marker genes include neomycin, dihydrofolate
reductase, glutamine synthetase, and the like. The vector employed
also generally includes an origin of replication and other genes
that are necessary for replication in the host cells, as routinely
employed by those having skill in the art. As an example, the
replication system comprising the origin of replication and any
proteins associated with replication encoded by a particular virus
may be included as part of the construct.
[0042] The replication system is preferably selected so that the
gene(s) encode products that are necessary for replication, but do
not ultimately transform the stem cells. Such replication systems
are represented by replication-defective adenovirus constructed as
described, for example, by G. Acsadi et al., 1994, Hum. Mol. Genet.
3:579-584, and by Epstein-Barr virus. Examples of replication
defective vectors, particularly, retroviral vectors that are
replication defective, are described by Price et al., 1987, Proc.
Natl. Acad. Sci. USA, 84:156; and Sanes et al., 1986, EMBO J.,
5:3133.
[0043] It will be understood that the final gene construct may
contain one or more genes of interest, for example, a gene encoding
a bioactive metabolic molecule or a gene encoding a suppressor
molecule, such as p53. In addition, cDNA, synthetically produced
DNA or chromosomal DNA may be employed utilizing methods and
protocols known and practiced by those having skill in the art. A
vector may be transduced into the cells through any means known in
the art. Non-limiting methods include electroporation, liposomes,
and calcium phosphate precipitation. In one alternative, the
retroviral or plasmid vector can be encapsulated into a liposome,
or coupled to a lipid, and then introduced into a cell. The cells
are preferably engineered to contain a plasmid or viral vector in
an ex vivo approach.
[0044] The stem cells can be admixed with, or introduced into, the
biologically compatible matrix by a number of methods known to
those having skill in the art. For example, inoculation can be used
if the matrix comprises a solid or semi-solid material that does
not readily mix with cells in suspension. As another example, a
suspension of the stem cells can mixed with a suitable biological
or synthetic adhesive (bioadhesive) matrix and the combination can
be spread, sprayed, painted, or otherwise applied onto a tissue or
organ site where the stem cell matrix forms, e.g., by gelling or
solidifying in situ. Accordingly, the stem cell-biomatrix
combination may be applied by spreading, painting or coating using
a spreading or coating means, such as a small brush, spatula,
knife, or other device suitable for medically coating a surface,
such as a tissue or organ surface. In addition, compressed air may
be used to spray or foam a stem cell-biomatrix mixture or
suspension onto a wound or biological surface.
[0045] In a further aspect of the invention, the stem cells are
attached to, introduced into, or applied to a biomatrix using
another type of biomatrix material, for example, a biological
adhesive (bioadhesive), such as, but not limited to, fibrin glues
or gels. Some bioadhesives and fibrin glues can be photoactivated,
or activated by temperature or calcium and are also suitable for
use. Further, from about 2.5.times.10.sup.3 to about
1.times.10.sup.6, preferably about 5.times.10.sup.3 to about
1.times.10.sup.6 stem cells can be used for admixing with,
inoculating, seeding, or otherwise introducing onto or into, the
medically or physiologically acceptable matrix material.
Preferably, about 1.times.10.sup.5 stem cells are deposited per 1
cm.sup.2 of matrix material.
[0046] In an embodiment of the present invention, a unique
processing of stem cells with a biological adhesive and unique
application for stem cell tissue engineering are provided. A
significant limitation of present tissue engineering approaches,
especially heart and blood vessel tissue engineering, is the need
for either systemic vascular injection of cells or direct needle
inoculation of specific sites in the heart. Each of the
aforementioned procedures has significant disadvantages. According
to the present invention, a method is provided which allows the
attachment of stem cells to the external surface of the target
organ, as exemplified and described in the examples herein. Thus,
the invention overcomes the limitations that currently exist for
heart and blood vessel tissue engineering procedures.
[0047] In another embodiment, the stem cell and biologic matrix
composition can be applied through a minimally invasive fiberoptic
scope (e.g., laparoscope) to multiple sites including, but not
limited to, bone, cartilage, ligaments. spinal cord, brain, heart,
lung, kidney, digestive and genitourinary organs. For stem cell
tissue engineering, muscle stem cells (MSC) and bioadhesive, or
biomatrix material, are applied, for example, through a laparoscope
to spray or coat kidney or ureter surgical anastomoses to enhance
healing and prevent stricture. As another example, a combination of
MSC and bioadhesive, or biomatrix material, are applied via
orthopedic endoscopy to coat the outside of damaged or weakened
bone or disc to promote and/or improve healing and strength, and/or
to prevent degeneration. Also encompassed in this embodiment are
specifically engineered stem cells which can deliver the expression
products of genes encoding bone factors or growth factors at the
site during the treatment, repair, or healing process.
[0048] Rapid methods of preparing a stem cell matrix for use in
tissue and organ treatment and repair are embodied by the present
invention. In one aspect, a method of the present invention
involves admixing a stem cell preparation with a physiologically
acceptable matrix material to form a stem cell matrix and
incubating the stem-cell matrix in vitro for about 5 seconds to
less than about 12 hours prior to use in the tissues or organs of a
recipient. In this method, any suitable physiologically acceptable
matrix material can be used, e.g., alginate, fibrin glue, fibrin
gel, small intestine submucosa (SIS).
[0049] In another aspect, a method of the present invention
involves admixing a stem cell preparation with a first
physiologically acceptable matrix material to form a first stem
cell-matrix combination; introducing the first stem cell-matrix
combination onto a second physiologically acceptable matrix
material to form a second stem cell-matrix material, wherein the
first stem cell-matrix combination and the second physiologically
acceptable matrix material are incubated in vitro for between about
5 seconds to less than 12 hours; and applying the second stem
cell-matrix material on or in a tissue or organ site in a
recipient. In this method, the first medically acceptable matrix
material is preferably a bioadhesive such as fibrin glue or gel.
The stem cell-fibrin glue biomatrix is then applied to a second
medically acceptable biomatrix material, e.g., SIS and the like.
(Example 3). A preparation or suspension of stem cells can be
simultaneously applied with the bioadhesive material to the second
medically acceptable biomatrix material, such as via a syringe, and
then spread, or allowed to coat this material, which is then used
to treat a tissue or organ site. Alternatively, the stem cells and
the bioadhesive material can be mixed together and then the stem
cell-bioadhesive mixture can be applied to the second matrix
material.
[0050] The present invention also provides methods which are not
likely to be hampered by the usual regulatory barriers. There is a
need for a cost effective combination therapy comprising medical
devices and cellular therapy. Keeping the medical devices and stem
cells separate until combined at the point of service (e.g.,
bedside or site of use) provides the solution to the need. A
significant advantage of the present invention is moving biological
stem cell tissue engineering out of the site of commercial
manufacture, e.g., the factory, to the point of service, where the
stem cell-matrix product is prepared and used "on location".
[0051] The stem cell-biological matrix product according to the
present invention is rapidly prepared just before, or at the time
of, use. There is no requirement for the cells and the matrix
material to be incubated or cultured for long durations, e.g., days
or weeks, prior to application or introduction at the tissue or
organ site by the practitioner. In addition, the stem cell-matrix
material, after combination, allows new structures to form, which
was unexpected. The stem cells in the biomatrix combination can
change the biomechanical properties of the biological scaffold and
create new 2-dimensional and 3-dimensional tissue, muscle and organ
structures. For example, muscle stem cells were shown to seed
porcine small intestine submucosa (SIS) at the point of service so
as to improve the SIS tissue properties (Example 2), for vascular
intervention. The stem cells seeding the biological or synthetic
matrix afford advantages to the use of the present invention for
numerous surgical and treatment procedures.
[0052] In accordance with the present invention, muscle stem cells
can be added onto a scaffold, which has been demonstrated to behave
in a manner similar to that of muscle, providing innervation with
neuromuscular receptors. (Examples 8-10). Thus, the present
invention allows 3-dimensional muscle repair to a variety of
tissues and organs, e.g., sphincters, including the urethra,
gastroesophageal sphincter, anal sphincter, as well as wrap and
patch including the bladder, intestine and stomach, blood vessels
and heart, diaphragm, tendons, muscle and ligaments, thereby
extending the advantages of the invention to include not only the
tissue engineering of an MSC/SIS patch, for tissue repair, for
example, but also the engineering of 3-dimensional repair
scaffolding utilizing muscle stem cells
EXAMPLES
[0053] The examples described below are provided to illustrate the
present invention and are not included for the purpose of limiting
the invention.
Example 1
Muscle Stem Cell/Alginate Dressing Composition
[0054] Muscle stem cells (MSC) were harvested from rat hindleg
muscle using the pre-plate technique (see WO 99/56785). After
obtaining a late plate (i.e., post PP5, preferably, PP6) cell
population, 100,000 cells, transduced with a retrovirus vector
containing LacZ, were then suspended in 200 microliters of Hank's
Buffered Salt Solution (HBSS; Gibco BRL, Grand Island, N.Y.) for
use. A 1 cm.sup.2 piece of Alginate (Johnson & Johnson Medical,
Arlington, Tex.) was cut, dipped into the MSC suspension to prepare
the Alginate+MSC composition and immediately placed on a 1 cm.sup.2
full-thickness wound defect on the upper dorsum of rat. After 1
week, the completely healed wound was harvested and stained. The
results demonstrated that the Alginate+MSC healed with better
cosmesis than did Alginate alone. After a week's time, the wound
was completely covered with normal skin. Upon histological
examination, the Alginate+MSC, showed better wound closure with
areas of fibrosis within the deeper wound. In addition, at the
dermal and epidermal layers, there appeared to be "new" dermal and
epidermal cell formation. Such a finding indicates that the MSC
actually differentiate and create epidermal and dermal cells in an
organized fashion when used to repair a wound according to this
invention.
Example 2
Muscle Stem Cell/SIS Composition
[0055] Single layer SIS (Cook Biologic, Inc., Indianapolis, Ind.)
was initially incubated in Hank's Buffered Salt Solution for one
hour at 37.degree. C. 100,000 late preplate (e.g., PP6), (See, WO
99/56785; and U.S. Serial Nos. 09/302,896, filed Apr. 30, 1999, and
09/549,937, filed Apr. 14, 2000, to M. Chancellor et al.) rat MSC
cells (transduced with a retroviral vector containing Lac Z) were
placed onto 1 cm diameter circular SIS to form an MSC-SIS matrix
composition. The MSC-SIS matrix was placed into a 24-well culture
plate. Different preparations of SIS and MSC were then incubated
for 3 days, 1 week, and 2 weeks, respectively, to assess cell
viability at 37.degree. C., with daily media changes with
Dulbecco's Modified Eagle Media (DMEM, Gibco BRL, Grand Island,
N.Y.) supplemented to contain 10% horse serum and 10% fetal bovine
serum. At appropriate time intervals, the SIS+MSC matrix
combination was harvested and sectioned for staining. At all time
intervals, cell viability was evident. The MSC continued to
proliferate and form myotubes (evident with Myosin Heavy Chain
Staining) at all time intervals. These experiments confirmed that
MSC grow on SIS and SIS is not toxic to these cells. Late preplate
cells grew in an organized confluent manner.
Example 3
Muscle Stem Cell/Fibrin Glue Composition
[0056] Rat MSC from a late preplate (PP6) were obtained using the
preplate technique (see, WO 99/56785; and U.S. Serial Nos.
09/302,896, filed Apr. 30, 1999, and 09/549,937, filed Apr. 14,
2000, to M. Chancellor et al.). Fibrin glue was obtained from
Baxter Healthcare Corporation (Glendale, Calif.). Fibrin glue is an
FDA approved sealant that is composed of human thrombin, calcium
chloride, bovine fibrinolysis inhibitor solution, and human sealer
protein concentrate. The elements are combined prior to use and
injected using a needle syringe. Using 100,000 cells and 0.5 cc of
fibrin glue simultaneously, the stem cells and fibrin glue were
inoculated onto SIS without using barrier inserts. One day later,
the system was harvested and sectioned. After immediate placement
of fibrin glue and MSC simultaneously on the SIS, not only did the
MSC attach to SIS more quickly, but cell viability was maintained.
The fibrin glue did not affect the viability of SIS. This
experiment showed the feasibility of using fibrin glue and stem
cells to allow for more immediate attachments to matrices and
scaffolds.
Example 4
Muscle Stem Cell/Artery Composition
[0057] Rat MSC were obtained using the pre-plate technique (see, WO
99/56785; and U.S. Serial Nos. 09/302,896, filed Apr. 30, 1999, and
09/549,937, filed Apr. 14, 2000, to M. Chancellor et al.) Single
layered SIS was seeded with MSC. After a one day incubation in
vitro, the MSC/SIS matrix composition was folded into vessel-like
lumen. Cell viability was intact after prolonged incubation time
(i.e., two weeks) with seeding throughout the vessel lumen.
Example 5
Muscle Stem Cell/Alginate Dressing with Varying Soaking Times
[0058] Experiments were performed to assess the effects of soaking
MSC in pieces of alginate (1-2 cm.sup.2) for 1 minute, 5 minutes, 1
hour, 6 hours, or 12 hours prior to application and use at a tissue
or organ site. It was determined that the longer the MSC were
soaked in the alginate, the weaker the alginate became, and caused
the alginate to dissolve. Weakening of the alginate began as soon
as it became hydrated and eventually showed evidence of dissolution
within 6 hours of soaking. The results of these studies indicated
that immediate or brief soaking with MSC at the point of service
was all that was necessary for successful physiological
results.
[0059] The experiments described in this Example showed that the
use of a stem cell concentration of from about 10,000 to 500,000
cells per cm.sup.2 of alginate matrix material was sufficient for
achieving physiological results. Using the stem cell-biomatrix
technique, a large variability in cell concentration and an uneven
distribution of cells throughout the matrix material still allowed
good physiological results and improvement.
Example 6
Muscle Cells Compared with Fibroblasts Soaked in Alginate
[0060] Experiments were conducted to compare the effectiveness of
muscle stem cells (MSC) combined with alginate with the
effectiveness of fibroblasts combined with alginate in wound
healing. A full thickness 1 cm.sup.2 wound was made at the dorsum
aspect of the upper torso of an anaesthetized rat. All of the
experiments were performed by immediately soaking alginate with
either the muscle stem cells or fibroblasts. 100,000 late-plate MSC
cells (i.e., PP6, see, WO 99/56785; and U.S. Serial Nos.
09/302,896, filed Apr. 30, 1999, and 09/549,937, filed Apr. 14,
2000, to M. Chancellor et al.) and 100,000 early-plate (i.e., PP1)
cells were used with two 1 cm.sup.2 pieces of alginate. After two
full thickness wounds were created, two, 1 cm.sup.2 alginate pieces
were soaked with the PP1 cells or the PP6 MSC cells for 5 seconds.
This system was then directly placed onto the wound defects and
sutured in. The results of these experiments demonstrated that the
MSC-alginate combination healed the wound with better cosmoses. In
contrast, the fibroblast-alginate composition did not change the
healing appearance, but it did heal more quickly than control
(alginate alone). Thus, according to the invention, prolonged
incubations of cells and the alginate matrix material were not
required to effect improved healing of wounds.
Example 7
In Vitro Muscle Stem Cell/SIS Composition
[0061] Experiments were performed to assess the adhesion and
persistence of late preplate MSC (e.g., PP6, see, WO 99/56785; and
U.S. Serial Nos. 09/302,896, filed Apr. 30, 1999, and 09/549,937,
filed Apr. 14, 2000, to M. Chancellor et al.) to an SIS biological
scaffold (both 1 and 4 collagen layered SIS). The results
demonstrated superior adhesion of the late preplate MSC (i.e., PP6)
compared with early preplate cells (i.e., PP1-4).
[0062] Using stem cell concentrations of 100,000 per cm.sup.2, cell
viability was verified after 3 days, 1 week, and 2 weeks for the
early and late plate cells. The early plate cells+SIS matrix did
not form myotubes well because of the many fibroblasts in the cell
population. This created less confluency of the cells on the SIS.
The late-plate stem cells (MSC)+SIS proliferated well homogeneously
and formed myotubes on the SIS. Cell growth, differentiation into
myotubes, and confluency were the desired results achieved by the
late plate MSC cells compared with the early plate cells.
Increasing the cell count to 1,000,000 / cm.sup.2 for the early
plate cells did not change the results on SIS; however, this
increase in cell number for the late-plate MSC cells allowed for
the growth of the MSC on SIS in multiple layers.
Example 8
Muscle Stem Cells Alter and Improve SIS Biomechanical
Properties
[0063] The experiments carried out in this example demonstrated
that muscle stem cells (MSC), isolated as described in the
aforementioned examples, remodeled SIS and changed the
biomechanical properties compared with native SIS. Biomechanical
properties of the SIS (and MSC+SIS matrix) were assessed using
biaxial testing. For biaxial testing, calculated stresses are
applied on all four sides of the SIS using four fine hooks
connected to two pulley systems on each side, using procedures
known to those having skill in the pertinent art. The stresses
applied cause straining to occur within the SIS sample. The data
are analyzed and biomechanical properties of the material, such as
elasticity, creep, and compliance, can be easily obtained. Upon
biaxial testing, native SIS (i.e., SIS with no muscle stem cells)
showed decreased horizontal/cross sectional elasticity versus the
longitudinal direction. By contrast, a composition containing a
combination of MSC and SIS increased the elasticity of the SIS in
the horizontal/cross section direction so that it nearly equaled
that in the longitudinal direction. The studies were carried out
using SIS+MSC cultured for 10 and 20 days in in vitro culture
dishes. Thus, these results demonstrate evidence of active
remodeling of SIS by MSC.
[0064] More particularly, thirty SIS.TM. sheets (soft-tissue
freeze-dried graft, Cook Biotech Incorporated, West Lafayette,
Ind.) were prepared for this experiment. The SIS material was kept
sealed until testing. Prior to testing, the sheets were cut into
the appropriate size to fit into specifically designed sterilized
cell culture wells. MSC (1.times.10.sup.5-6) were placed onto the
SIS and were fed with culture medium (SMEM). The culture medium was
refreshed every 24 hours. The cell culture inserts were incubated
at 37.degree. C. for 10 or 20 days. After this time, the specimens
were cut into 25 mm square patches and placed into the biaxial
mechanical testing setup. (see, e.g., K. L. Billiar and M. S.
Sacks, 2000, "Biaxial mechanical properties of the natural and
glutaraidehyde treated aortic valve cusp--Part I: Experimental
results", J. Biomech. Eng., 122(1):23 and M. S. Sacks, 1999, "A
method for planar biaxial mechanical testing that includes in-plane
shear", J. Biomech. Eng., 121(5):551-555).
[0065] For biaxial mechanical testing, each side of the test
specimen was attached to the motor carriages of the biaxial
mechanical testing device with sutures looped around two small
pulleys on each side of a horizontal common axle, which was
connected, in turn, to a vertical pivoting rod, thus allowing
near-frictionless rotation in three dimensions. A surgical staple
was connected to both ends of each suture line for attachment to
the specimen, resulting in a total of four staples per specimen
side. Each pulley ensured that the force on each line end was
equal; the pivoting rod ensured that the forces were the same on
each pair. Small floats were attached to each staple to make the
mounted sample neutrally buoyant. Load was monitored in two
orthogonal axes by two load cells and the in-plane strain was
determined by calculating the centroids of four black markers
affixed to the surface of the specimen. The Green's strain (E)
along each test axis was calculated from stretch ratio (.lambda.)
using the following equation:
E=(.lambda..sup.2-1).div.2
[0066] Tissue deformations were measured by monitoring the
real-time movements of four graphite markers forming a 5-mm square
region using real-time video marker tracking. From the marker
displacements the 2D in-plane Green's finite strain tensor was
computed, where EL and ET denote the Green's strain along the
longitudinal and transverse directions, respectively. Both the load
and deformation in both axes were continuously recorded at 12-15 Hz
during testing. All preparations were tested in Hank's buffered
saline solution (HBSS) at room temperature. Each test involved 10
contiguous cycles with a period of 20-30 seconds, with a total of
seven runs. Testing began with equi-biaxial preconditioning to the
maximum stress level. Thereafter, five consecutive tests were
conducted, and the axial ratios of the stresses were maintained at
values of 0.5:1, 0.75:1, 1:1, 1:0.75, and 1:0.5. These ratios were
chosen to determine the mechanical behavior over a wide range of
stress states. A final equi-biaxial test was conducted to confirm
that the mechanical behavior was not changed during the tests.
Total testing time was approximately 60 minutes for each
specimen.
[0067] The positions of the optical markers at three different
stages were recorded during mechanical testing. The first
measurement was obtained in the unloaded conditions, with the
specimen free-floating in the bath. The second set of marker
positions was taken after the sample had attached to the device and
a 0.5 gm load was applied to both axes. The preconditioned
reference state produced the most stable stress-strain response,
and was considered as the most physiological-like state. Thus the
marker positions recorded after the first preconditioning run were
used for all subsequent strain computations.
[0068] The areal strain was used to assess the compliance of the
specimen. Areal strain, which is a measure of tissue compliance
under biaxial loading, represents the change in tissue area when
the tissue is equally loaded along two directions. Areal strain was
measured using the following formula:
Areal Strain=(U.sub.1.times.U.sub.2.times.1).times.100
[0069] (where, U.sub.1: stretch in X.sub.1 axis; and U.sub.2:
stretch in X.sub.2 axis)
[0070] The mean (.+-.S.D.) areal strain in the control group (n=5)
of non-incubated SIS was 20.80.+-.5.9 (see FIG. 1). At 10 days of
incubation, the mean (.+-.S.D.) areal strain in MSC/SIS
(29.15.+-.5.4, n=5) was significantly higher (p=0.01) than that of
SIS alone (17.98.+-.n=6) (see FIG. 1). Also at 20 days of
incubation there were significant differences between SIS alone and
the MSC/SIS preparations. These results demonstrate that the
MSC/SIS preparations have better compliance properties at 10 and 20
days than SIS alone.
[0071] Additionally, when the stretch changes in fiber and
non-fiber direction were compared, the increase in stretch change
in MDSC/SIS was more prominent in the non-fiber direction than
fiber direction axis of the specimens. Thus the biaxial testing
results exhibited the classic biological non-linear stress-strain
response. The specimens were in a state of nearly pure biaxial
strain with neglible shear.
Example 9
Contractility of Muscle Stem Cells (MSC) Incorporated into a SIS
Scaffold
[0072] Twenty five preparations of MDSC/SIS and twenty five control
preparations of SIS only were incubated at 37.degree. C. for 1, 4,
or 8 weeks. The specimens were then mounted in a thermally
controlled bath of 5 ml at 37.degree. C. in Krebs solution (mmol/L:
NaCl, 113; KCl, 4.7; CaCl.sub.2, 1.25; MgSO.sub.4, 1.2;
NaHCO.sub.3, 25; KH.sub.2PO.sub.4, 1.2 and glucose, 11.5) with a
mixture of 95% O.sub.2 and 5% CO.sub.2. The frequency and amplitude
of isometric contractions were measured with strain gauge
transducers coupled with a TBM4 strain gauge amplifier and recorded
on a computer using a data acquisition program (Windaq, DATAQ
Instruments Inc. Akron, Ohio). After 60 minutes of equilibration,
electrical field stimuli (10 Hz, 150 volt, 0.5 ms, 60 sec) were
applied in the bath. Pharmacological evaluations were performed
with succinylcholine (S-Chol), (4, 10 and 20 .mu.M); carbachol (10
and 20 .mu.M); KCl, (7%, 1M); Ca.sup.++-free Krebs solution with
EGTA (200 .mu.M); or distilled water, were each added to the bath
sequentially at 30 minute intervals. The frequency, amplitude, and
pattern of contraction of the specimens were compared among the
different groups.
[0073] All MDSC/SIS specimens were curved in shape, thus suggesting
tonic contractile activity. In contrast, the specimens having SIS
alone did not have a curved shape (FIG. 2). All of the SIS only
control groups of the 1 (n=11), 4 (n=6), and 8-week (n=8) specimens
did not show any activity in the bath. In the 4-week (5 of 6
specimens) and 8-week cultures (8 of 8 specimens) of MDSC/SIS
preparations, spontaneous contractile activity was observed.
However, in the 1-week MDSC/SIS cultures no spontaneous
contractility was observed (0 of 11 specimens).
[0074] In the 8-week MDSC/SIS preparations, the frequency and
amplitude of spontaneous contractile activity (SCA) were decreased
in all (8 of 8) specimens by S-Chol (10 .mu.M) and were blocked in
5 of 8 specimens after administration of S-Chol (20 .mu.M). In the
4-week MDSC/SIS preparations, the frequency and amplitude of SCA
were decreased in 2 of 5 specimens by S-Chol (10 .mu.M) and were
decreased in 5 of 5 specimens and blocked in 2 of 5 specimens by
S-Chol (20 .mu.M). The suppressant effects observed were reversible
after washout of the drug. S-Chol (4 .mu.M) did not alter the
frequency, amplitude, or pattern of spontaneous activity of any of
the preparations.
[0075] In all of the MDSC/SIS specimens, spontaneous contractile
activity was blocked by both Ca.sup.++-free Krebs solution with
EGTA (200 .mu.M) and distilled water. However, electrical field
stimulation, carbachol, and KCI did not alter the frequency,
amplitude, and pattern of the spontaneous contractile activity in
MDSC/SIS specimens. Accordingly, the 8-week results suggest that
the MDSC/SIS scaffold is able to generate contractile activity
which is Ca++ -dependent and modulated by nicotinic receptors.
(FIG. 2).
Example 10
Effectiveness of MSC/SIS Suburethral Sling Placement in a Rat
[0076] To evaluate the effectiveness of the MDSC/SIS suburethral
sling placement in a female rat, the bilateral proximal sciatic
nerve transection (PSNT) model of stress urinary incontinence (SUI)
was used. Both bilateral pudendal nerve transection, or pudendal
nerve crush, result in alterations in behavioral voiding patterns
and significant histological changes of urethral striated muscle
atrophy. In initial studies for SIS sling placement, more proximal
nerve transection at the level of the sciatic nerve (before the
branching of the pudendal nerves) was used, combined with the
vertical tilt table and intravesical pressure clamp method of leak
point pressure (LPP) testing.
[0077] Five halothane anesthetized rats underwent bilateral sciatic
nerve transection and periurethral sling placement using a SIS/MDSC
patch. The sling comprised an SIS strip with dimensions of 14 mm in
length and 3 mm in width. This sling was placed posterior to the
urethra via a transabdominal approach, and was sutured bilaterally
into the pubic bone using 4.0 prolene, 6 mm lateral to the pubic
symphysis. An intraurethral catheter (PE20) was inserted prior to
tying the sling sutures in place to ensure that the sling would not
produce obstruction. Care was taken not alter the natural
bladder/urethral angle. Sham rats (n=2) underwent bilateral
proximal sciatic nerve transection and identical periurethral
dissection without SIS/MDSC sling placement. The rats were
subsequently monitored for signs of urinary retention and given
antibiotic therapy once every 3 days.
[0078] Two weeks following surgery, the rats underwent leak point
pressure (LPP) testing using the vertical tilt/intravesical
pressure clamp method. (see, O. Matthew et al., 2000, "Creation of
a new stess incontinence model in the rat by using vertical tilt
table and surgical or pharmacological manipulation of internal and
external sphincter activity with measurement of leak point pressure
(LPP)", 2000 Annual Meeting of the AUA, J. Urol., 163:76). Control
rats (n=5) also underwent the same LPP testing, but without prior
treatment. It was determined that LPPs in PSNT rats with slings
were not significantly different from those of control rats
(51.23.+-.1.60 versus 46.11.+-.1.83, respectively). Importantly, no
PSNT-sling rat demonstrated signs of urinary retention.
(28.6.+-.0.8 cm H.sub.2O for 1 week LPP in denervated rats). Thus,
these findings demonstrated that the SIS/MDSC sling restored LPP in
the PSNT rat model of SUI to nearly that of LPP in normal control
rats. MDSC/SIS according to the present invention restores muscular
function and innervation to the damaged sphincter. It is therefore
an added advantage of this invention not only to prepare a scaffold
with MDC, but also to achieve a type of 3-dimensional muscle
replacement for in vivo use in tissue and organ repair using the
MDSC/SIS that behaves as a muscle, including innervation and
neuromuscular receptors.
[0079] All patent applications, published applications, patents,
texts, and literature references cited in this specification are
hereby incorporated herein by reference in their entirety to more
fully describe the state of the art to which the present invention
pertains.
[0080] As various changes can be made in the above methods and
compositions without departing from the scope and spirit of the
invention as described, it is intended that all subject matter
contained in the above description, shown in the accompanying
drawings, or defined in the appended claims be interpreted as
illustrative, and not in a limiting sense.
* * * * *