U.S. patent application number 17/316854 was filed with the patent office on 2022-03-31 for bone augmentation utilizing muscle-derived progenitor compositions in biocompatible matrix, and treatments thereof.
The applicant listed for this patent is University of Pittsburgh - Of the Commonwealth System of Higher Education. Invention is credited to Johnny Huard, Ronald Jankowski, Karin Payne, Thomas Payne, Arvydas Usas.
Application Number | 20220096714 17/316854 |
Document ID | / |
Family ID | 1000006016771 |
Filed Date | 2022-03-31 |
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United States Patent
Application |
20220096714 |
Kind Code |
A1 |
Usas; Arvydas ; et
al. |
March 31, 2022 |
BONE AUGMENTATION UTILIZING MUSCLE-DERIVED PROGENITOR COMPOSITIONS
IN BIOCOMPATIBLE MATRIX, AND TREATMENTS THEREOF
Abstract
The present invention provides muscle-derived progenitor cells
that show long-term survival following transplantation into body
tissues and which can augment non-soft tissue following
introduction (e.g. via injection, transplantation, or implantation)
into a site of non-soft tissue (e.g. bone) when combined with a
biocompatible matrix, preferably SIS. The invention further
provides methods of using compositions comprising muscle-derived
progenitor cells with a biocompatible matrix for the augmentation
and bulking of mammalian, including human, bone tissues in the
treatment of various functional conditions, including osteoporosis,
Paget's Disease, osteogenesis imperfecta, bone fracture,
osteomalacia, decrease in bone trabecular strength, decrease in
bone cortical strength and decrease in bone density with old
age.
Inventors: |
Usas; Arvydas; (Pittsburgh,
PA) ; Payne; Karin; (Pittsburgh, PA) ; Payne;
Thomas; (Pittsburgh, PA) ; Jankowski; Ronald;
(Pittsburgh, PA) ; Huard; Johnny; (Wexford,
PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Pittsburgh - Of the Commonwealth System of Higher
Education |
Pittsburgh |
PA |
US |
|
|
Family ID: |
1000006016771 |
Appl. No.: |
17/316854 |
Filed: |
May 11, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16293041 |
Mar 5, 2019 |
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17316854 |
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14499853 |
Sep 29, 2014 |
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16293041 |
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12543311 |
Aug 18, 2009 |
9199003 |
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14499853 |
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61166775 |
Apr 6, 2009 |
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61089798 |
Aug 18, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 27/3847 20130101;
A61L 27/3826 20130101; A61L 2430/40 20130101; A61K 35/38 20130101;
C12N 5/0659 20130101; A61L 27/365 20130101; A61L 2300/64 20130101;
A61L 27/3895 20130101; A61K 35/12 20130101; C12N 5/0658 20130101;
C12N 11/02 20130101; A61L 27/58 20130101; A61L 2430/02 20130101;
C12N 5/0652 20130101; A61L 27/3629 20130101; C12N 2533/92 20130101;
A61L 27/54 20130101 |
International
Class: |
A61L 27/38 20060101
A61L027/38; A61K 35/38 20060101 A61K035/38; C12N 5/077 20060101
C12N005/077; A61L 27/36 20060101 A61L027/36; A61L 27/54 20060101
A61L027/54; A61L 27/58 20060101 A61L027/58; C12N 11/02 20060101
C12N011/02 |
Goverment Interests
GOVERNMENT INTERESTS
[0002] This invention was made with Government support under Grant
No. R01-DE13420-01 awarded by the National Institutes of Health.
The Government has certain rights in this invention.
Claims
1.-34. (canceled)
35. A method of treating a bone disease, defect or pathology in a
human subject in need thereof, wherein the method comprises
administering to the human subject small intestine submucosa (SIS)
seeded with a cell population enriched for muscle derived
progenitor cells (MDCs), wherein the cell population enriched for
MDCs is isolated from skeletal muscle by a method comprising: a.
suspending skeletal muscle cells obtained from the human in a first
cell culture container for between 30 and 120 minutes to produce a
population of adherent cells and a population of non-adherent
cells; b. decanting media and the population of non-adherent cells
from the first cell culture container to a second cell culture
container; c. allowing the population of decanted, non-adherent
cells in the media to attach to the walls of the second cell
culture container; and d. isolating the population of cells from
the walls of the second cell culture container, wherein the
isolated population of cells is the cell population enriched for
MDCs.
36. The method of claim 35, wherein the isolation method further
comprises culturing the cell population enriched for MDCs to expand
their number before being used to seed the SIS.
37. The method of claim 36, wherein the expanded cell population
enriched for MDCs is frozen to a temperature below -30.degree. C.
after being cultured to expand their number and thawed prior to
being used to seed SIS.
38. The method of claim 35, wherein the isolation method further
comprises cooling the skeletal muscle cells obtained from the human
to a temperature below 10.degree. C. and storing for 1-7 days
before being suspended in a first cell culture container for
between 30 and 120 minutes.
39. The method of claim 35, wherein treating a bone disease, defect
or pathology comprises augmenting bone.
40. The method of claim 35, wherein treating a bone disease, defect
or pathology comprises increasing bone volume or bone density.
41. The method of claim 35, wherein treating a bone disease, defect
or pathology comprises treatment of osteoporosis, Paget's Disease,
osteogenesis imperfecta, bone fracture, osteomalacia, decrease in
bone trabecular strength, decrease in bone cortical strength, or
decrease in bone density with old age.
42. The method of claim 35, wherein the bone defect is a bone
fracture caused by trauma.
43. The method of claim 35, wherein the SIS seeded with a cell
population enriched for MDCs is administered by applying it to the
surface of the bone.
44. The method of claim 35, wherein the SIS seeded with a cell
population enriched for MDCs is positioned in the interior of the
bone.
Description
RELATED APPLICATIONS
[0001] This application claims priority to and is a continuation of
U.S. patent application Ser. No. 16/293,041, filed on Mar. 5, 2019,
which claims priority to and is a continuation of U.S. patent
application Ser. No. 14/499,853, filed on Sep. 29, 2014, which
claims priority to and is a continuation of U.S. patent application
Ser. No. 12/543,311, filed on Aug. 18, 2009 (now U.S. Pat. No.
9,199,003), which claims priority from U.S. Provisional Patent
Applications 61/089,798, filed on Aug. 18, 2008, and 61/166,775,
filed on Apr. 6, 2009, each of which is incorporated by reference,
herein, in their entireties for all purposes.
FIELD OF THE INVENTION
[0003] The present invention relates to muscle-derived progenitor
cells (MDCs) and compositions of MDCs with biologically compatible
matrix and their use with the augmentation of body tissues,
particularly bone. In particular, the present invention relates to
muscle-derived progenitor cells that show long-term survival
following introduction into bone used in combination with small
intestine sub-mucosa for the augmentation of human or animal bone.
The invention also relates to novel uses of muscle-derived
progenitor cells with biologically compatible matrix for the
treatment of cosmetic or functional conditions, such as
osteoporosis, Paget's Disease, osteogenesis imperfecta, bone
fracture, osteomalacia, decrease in bone trabecular strength,
decrease in bone cortical strength and decrease in bone density
with old age. The invention also relates to the novel use of MDCs
with biologically compatible matrix for the increase of bone mass
in athletes or other organisms in need of greater than average bone
mass.
BACKGROUND OF THE INVENTION
[0004] Myoblasts, the precursors of muscle fibers, are
mononucleated muscle cells that fuse to form post-mitotic
multinucleated myotubes, which can provide long-term expression and
delivery of bioactive proteins (T. A. Partridge and K. E. Davies,
1995, Brit. Med. Bulletin 51:123 137; J. Dhawan et al., 1992,
Science 254: 1509 12; A. D. Grinnell, 1994, Myology Ed 2, A. G.
Engel and C. F. Armstrong, McGraw-Hill, Inc., 303 304; S. Jiao and
J. A. Wolff, 1992, Brain Research 575:143 7; H. Vandenburgh, 1996,
Human Gene Therapy 7:2195 2200).
[0005] Cultured myoblasts contain a subpopulation of cells that
show some of the self-renewal properties of stem cells (A. Baroffio
et al., 1996, Differentiation 60:47 57). Such cells fail to fuse to
form myotubes, and do not divide unless cultured separately (A.
Baroffio et al., supra). Studies of myoblast transplantation (see
below) have shown that the majority of transplanted cells quickly
die, while a minority survive and mediate new muscle formation (J.
R. Beuchamp et al., 1999, J. Cell Biol. 144:1113 1122). This
minority of cells shows distinctive behavior, including slow growth
in tissue culture and rapid growth following transplantation,
suggesting that these cells may represent myoblast stem cells (J.
R. Beuchamp et al., supra).
[0006] Myoblasts have been used as vehicles for gene therapy in the
treatment of various muscle- and non-muscle-related disorders. For
example, transplantation of genetically modified or unmodified
myoblasts has been used for the treatment of Duchenne muscular
dystrophy (E. Gussoni et al., 1992, Nature, 356:435 8; J. Huard et
al., 1992, Muscle & Nerve, 15:550 60; G. Karpati et al., 1993,
Ann. Neurol., 34:8 17; J. P. Tremblay et al., 1993, Cell
Transplantation, 2:99 112; P. A. Moisset et al., 1998, Biochem.
Biophys. Res. Commun. 247:94 9; P. A. Moisset et al., 1998, Gene
Ther. 5:1340 46). In addition, myoblasts have been genetically
engineered to produce proinsulin for the treatment of Type 1
diabetes (L. Gros et al., 1999, Hum. Gen. Ther. 10:1207 17); Factor
IX for the treatment of hemophilia B (M. Roman et al., 1992, Somat.
Cell. Mol. Genet. 18:247 58; S. N. Yao et al., 1994, Gen. Ther.
1:99 107; J. M. Wang et al., 1997, Blood 90:1075 82; G. Hortelano
et al., 1999, Hum. Gene Ther. 10:1281 8); adenosine deaminase for
the treatment of adenosine deaminase deficiency syndrome (C. M.
Lynch et al., 1992, Proc. Natl. Acad. Sci. USA, 89:1138 42);
erythropoietin for the treatment of chronic anemia (E. Regulier et
al., 1998, Gene Ther. 5:1014 22; B. Dalle et al., 1999, Gene Ther.
6:157 61), and human growth hormone for the treatment of growth
retardation (K. Anwer et al., 1998, Hum. Gen. Ther. 9:659 70).
[0007] Myoblasts have also been used to treat muscle tissue damage
or disease, as disclosed in U.S. Pat. No. 5,130,141 to Law et al.,
U.S. Pat. No. 5,538,722 to Blau et al., and application U.S. Ser.
No. 09/302,896 filed Apr. 30, 1999 by Chancellor et al. In
addition, myoblast transplantation has been employed for the repair
of myocardial dysfunction (C. E. Murry et al., 1996, J. Clin.
Invest. 98:2512 23; B. Z. Atkins et al., 1999, Ann. Thorac. Surg.
67:124 129; B. Z. Atkins et al., 1999, J. Heart Lung Transplant.
18:1173 80).
[0008] In spite of the above, in most cases, primary
myoblast-derived treatments have been associated with low survival
rates of the cells following transplantation due to migration
and/or phagocytosis. To circumvent this problem, U.S. Pat. No.
5,667,778 to Atala discloses the use of myoblasts suspended in a
liquid polymer, such as alginate. The polymer solution acts as a
matrix to prevent the myoblasts from migrating and/or undergoing
phagocytosis after injection. However, the polymer solution
presents the same problems as the biopolymers discussed above.
Furthermore, the Atala patent is limited to uses of myoblasts in
only muscle tissue, but no other tissue.
[0009] Thus, there is a need for other, different tissue
augmentation materials that are long-lasting, compatible with a
wide range of host tissues, and which cause minimal inflammation,
scarring, and/or stiffening of the tissues surrounding the implant
site. Accordingly, the muscle-derived progenitor cell
(MDC)-containing compositions of the present invention are provided
as improved and novel materials for augmenting bone. Further
provided are methods of producing muscle-derived progenitor cell
compositions that show long-term survival following
transplantation, and methods of utilizing MDCs and compositions
containing MDCs to treat various aesthetic and/or functional
defects, including, for example osteoporosis, Paget's Disease,
osteogenesis imperfecta, bone fracture, osteomalacia, decrease in
bone trabecular strength, decrease in bone cortical strength and
decrease in bone density with old age. Also provided are methods of
using MDCs and compositions containing MDCs for the increase of
bone mass in athletes or other organisms in need of greater than
average bone mass.
[0010] It is notable that prior attempts to use myoblasts for
non-muscle tissue augmentation were unsuccessful (U.S. Pat. No.
5,667,778 to Atala). Therefore, the findings disclosed herein are
unexpected, as they show that the muscle-derived progenitor cells
according to the present invention can be successfully transplanted
into non-muscle tissue, including bone tissue, and exhibit
long-term survival. As a result, MDCs and compositions comprising
MDCs can be used as a general augmentation material for bone
production. Moreover, since the muscle-derived progenitor cells and
compositions of the present invention can be derived from
autologous sources, they carry a reduced risk of immunological
complications in the host, including the reabsorption of
augmentation materials, and the inflammation and/or scarring of the
tissues surrounding the implant site.
[0011] Although mesenchymal stem cells can be found in various
connective tissues of the body including muscle, bone, cartilage,
etc. (H. E. Young et al., 1993, In vitro Cell Dev. Biol. 29A:723
736; H. E. Young, et al., 1995, Dev. Dynam. 202:137 144), the term
mesenchymal has been used historically to refer to a class of stem
cells purified from bone marrow, and not from muscle. Thus,
mesenchymal stem cells are distinguished from the muscle-derived
progenitor cells of the present invention. Moreover, mesenchymal
cells do not express the CD34 cell marker (M. F. Pittenger et al.,
1999, Science 284:143 147), which is expressed by the
muscle-derived progenitor cells described herein.
[0012] SIS is an acellular, naturally occurring collagenous
extracellular matrix material derived from the submucosa of porcine
small intestine, which contains bioactive molecules (TGF-.beta.,
bFGF) (Voytik-Harbin S, et al. J Cell Biochem, 1997). While SIS is
primarily used for the repair of soft tissues, its potential as a
bone graft material is still under debate. Only a few studies
reported that SIS had potential for bone regeneration (Suckow M, et
al. J Invest Surg, 1999, Voytik-Harbin S, et al. Trans First SIS
Symposium, 1996). Most recent report from Moore D, et al. J Biomed
Mater Res, 2004 suggests that SIS is not capable of inducing or
conducting new bone formation across a critical size segmental bone
defect.
[0013] Moreover, 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.
[0014] 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.
[0015] The description herein of disadvantages and problems
associated with known compositions, and methods is in no way
intended to limit the scope of the embodiments described in this
document to their exclusion. Indeed, certain embodiments may
include one or more known compositions, compounds, or methods
without suffering from the so-noted disadvantages or problems.
SUMMARY OF THE INVENTION
[0016] It is an object of the present invention to provide uses for
MDCs and compositions comprising MDCs with biologically compatible
matrix for the augmentation of non-muscle tissue, including bone,
without the need for polymer carriers or special culture media for
transplantation. Such uses include the administration of MDC
compositions with biologically compatible matrix by introduction
into bone, for example by direct injection into or on the surface
of the tissue, wherein the tissue as been previously administered a
biologically compatible matrix. Preferably, this matrix is small
intestine submucosa (SIS).
[0017] It is yet another object of the present invention to provide
uses for MDCs for augmenting bone, following injury, wounding,
surgeries, traumas, non-traumas, or other procedures that result in
fissures, openings, depressions, wounds, and the like.
[0018] The invention provides the use of SIS seeded with MDCs for
treating a bone disease, defect or pathology or improving at least
one symptom associated with a bone disease, defect or pathology in
a mammalian subject in need thereof wherein the MDCs are isolated
from skeletal muscle, wherein the MDCs express desmin and wherein
the MDCs are able to form bone tissue. In one embodiment, the MDC
seeded SIS is administered by applying it to the surface of the
bone. In another embodiment, the MDC seeded SIS is positioned in
the interior of the bone. In another embodiment, the mammal is a
human. In some embodiments, the symptom is selected from the group
consisting of decreased bone density and decreased bone mass.
[0019] In another specific embodiment, the MDCs are cultured to
expand their number before being used to seed the SIS. Preferably,
the MDCs are frozen to a temperature below -30.degree. C. after
being cultured to expand their number and thawed prior to being
used to seed SIS.
[0020] In another embodiment, the skeletal muscle cells are
isolated from the human subject before the bone disease, defect or
pathology begins in the human subject. Preferably, when the bone
defect, disease or pathology is a bone defect the bone defect is a
bone fracture caused by trauma.
[0021] In other preferred embodiments, the MDCs are isolated by a
method comprising: isolating skeletal muscle cells from a mammal,
suspending the mammalian skeletal muscle cells in a first cell
culture container for between 30 and 120 minutes; decanting the
media from the first cell culture container to a second cell
culture container; allowing the remaining cells in the media to
attach to the walls of the second cell culture container; isolating
the cells from the walls of the second cell culture container,
wherein the isolated cells are MDCs; providing small intestine
submucosa (SIS); seeding the SIS with MDCs; and administering the
MDC seeded SIS to a bone suffering from the bone defect, disease or
pathology of the mammalian subject.
[0022] Preferably, the mammalian skeletal muscle cells are cooled
to a temperature below 10.degree. C. and stored for 1-7 days after
being isolated and before being suspended in a first cell culture
container between 30 and 120 minutes
[0023] In other preferred embodiments, the MDCs are isolated by a
method comprising: plating a suspension of skeletal muscle cells
from mammalian skeletal muscle tissue in a first container to which
fibroblast cells of the skeletal muscle cell suspension adhere,
re-plating non-adherent cells from step (a) in a second container,
wherein the step of re-plating is after 15-20% of cells have
adhered to the first container; repeating step (b) at least once;
and isolating the non-adherent cells wherein the isolated cells are
MDCs; providing small intestine submucosa (SIS); seeding the SIS
with MDCs; and administering the MDC seeded SIS to a bone suffering
from the bone defect, disease or pathology of the mammalian
subject.
[0024] Additional objects and advantages afforded by the present
invention will be apparent from the detailed description and
exemplification hereinbelow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The appended drawings of the figures are presented to
further describe the invention and to assist in its understanding
through clarification of its various aspects.
[0026] FIG. 1 shows 3D reconstruction of untreated (A,D),
SIS-treated (B,E) and SIS-hMDC-treated (C,F) calvarial defects at 4
weeks (A,B,C) and 10 weeks (D,E,F) after surgery.
[0027] FIG. 2 is a bar graph showing new bone formation in
calvarial defects at 4 and 10 weeks.
[0028] FIG. 3 is a bar graph showing a bone bridging score for mice
treated with SIS with and without MDCs at 4 and 10 weeks.
[0029] FIG. 4A is a bar graph showing the volume of bone matrix
formation on SIS with and without hMDCs and with osteogenic or
proliferation medium (OSM and PM, respectively) at 7, 10, 14, 21
and 28 days.
[0030] FIG. 4B is a bar graph showing the density of bone matrix
formation on SIS with and without hMDCs and with OSM or PM at 7,
10, 14, 21 and 28 days.
[0031] FIG. 5 is a 3D reconstruction of the SIS with and without
hMDCs and with OSM or PM at 28 days.
[0032] FIG. 6A is a bar graph showing the volume of bone matrix
formation on cell pellets at 7, 10, 14, 21 and 28 days.
[0033] FIG. 6B is a bar graph showing the density of bone matrix
formation on cell pellets at 7, 10, 14, 21 and 28 days.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The invention provides methods of treating bone disorders
including incontinence osteoporosis, Paget's Disease, osteogenesis
imperfecta, bone fracture, osteomalacia, decrease in bone
trabecular strength, decrease in bone cortical strength and
decrease in bone density with old age. The isolation of human
muscle-derived cells (MDCs) from adult tissue are capable of
achieving increased bone density and bone volume within human
subjects administered these cells in combination with a
biologically compatible matrix.
Muscle-Derived Cells and Compositions
[0035] The present invention provides MDCs comprised of early
progenitor cells (also termed muscle-derived progenitor cells or
muscle-derived stem cells herein) that show long-term survival
rates following transplantation into body tissues, preferably bone.
To obtain the MDCs of this invention, a muscle explant, preferably
skeletal muscle, is obtained from an animal donor, preferably from
a mammal, including humans. This explant serves as a structural and
functional syncytium including "rests" of muscle precursor cells
(T. A. Partridge et al., 1978, Nature 73:306 8; B. H. Lipton et
al., 1979, Science 205:12924).
[0036] Cells isolated from primary muscle tissue contain mixture of
fibroblasts, myoblasts, adipocytes, hematopoietic, and
muscle-derived progenitor cells. The progenitor cells of a
muscle-derived population can be enriched using differential
adherence characteristics of primary muscle cells on collagen
coated tissue flasks, such as described in U.S. Pat. No. 6,866,842
of Chancellor et al. Cells that are slow to adhere tend to be
morphologically round, express high levels of desmin, and have the
ability to fuse and differentiate into multinucleated myotubes U.S.
Pat. No. 6,866,842 of Chancellor et al.). A subpopulation of these
cells was shown to respond to recombinant human bone morphogenic
protein 2 (rhBMP-2) in vitro by expressing increased levels of
alkaline phosphatase, parathyroid hormone dependent 3',5'-cAMP, and
osteogenic lineage and myogenic lineages (U.S. Pat. No. 6,866,842
of Chancellor et al.; T. Katagiri et al., 1994, J. Cell Biol.,
127:1755 1766).
[0037] In one embodiment of the invention, a preplating procedure
may be used to differentiate rapidly adhering cells from slowly
adhering cells (MDCs). In accordance with the present invention,
populations of rapidly adhering MDC (PP1-4) and slowly adhering,
round MDC (PP6) were isolated and enriched from skeletal muscle
explants and tested for the expression of various markers using
immunohistochemistry to determine the presence of pluripotent cells
among the slowly adhering cells (Example 1; patent application U.S.
Ser. No. 09/302,896 of Chancellor et al.). As shown in Table 2,
Example 3 herein, the PP6 cells expressed myogenic markers,
including desmin, MyoD, and Myogenin. The PP6 cells also expressed
c-met and MNF, two genes which are expressed at an early stage of
myogenesis (J. B. Miller et al., 1999, Curr. Top. Dev. Biol. 43:191
219; see Table 3). The PP6 showed a lower percentage of cells
expressing M-cadherin, a satellite cell-specific marker (A.
Irintchev et al., 1994, Development Dynamics 199:326 337), but a
higher percentage of cells expressing Bcl-2, a marker limited to
cells in the early stages of myogenesis (J. A. Dominov et al.,
1998, J. Cell Biol. 142:537 544). The PP6 cells also expressed
CD34, a marker identified with human hematopoietic progenitor
cells, as well as stromal cell precursors in bone marrow (R. G.
Andrews et al., 1986, Blood 67:842 845; C. I. Civin et al., 1984,
J. Immunol. 133:157 165; L. Fina et al, 1990, Blood 75:2417 2426;
P. J. Simmons et al., 1991, Blood 78:2848 2853; see Table 3). The
PP6 cells also expressed Flk-1, a mouse homologue of human KDR gene
which was recently identified as a marker of hematopoietic cells
with stem cell-like characteristics (B. L. Ziegler et al., 1999,
Science 285:1553 1558; see Table 3). Similarly, the PP6 cells
expressed Sca-1, a marker present in hematopoietic cells with stem
cell-like characteristics (M. van de Rijn et al., 1989, Proc. Natl.
Acad. Sci. USA 86:4634 8; M. Osawa et al., 1996, J. Immunol.
156:3207 14; see Table 3). However, the PP6 cells did not express
the CD45 or c-Kit hematopoietic stem cell markers (reviewed in L K.
Ashman, 1999, Int. J. Biochem. Cell. Biol. 31:1037 51; G. A.
Koretzky, 1993, FASEB J. 7:420 426; see Table 3).
[0038] In one embodiment of the present invention is the PP6
population of muscle-derived progenitor cells having the
characteristics described herein. These muscle-derived progenitor
cells express the desmin, CD34, and Bcl-2 cell markers. In
accordance with the present invention, the PP6 cells are isolated
by the techniques described herein (Example 1) to obtain a
population of muscle-derived progenitor cells that have long-term
survivability following transplantation. The PP6 muscle-derived
progenitor cell population comprises a significant percentage of
cells that express progenitor cell markers such as desmin, CD34,
and Bcl-2. In addition, PP6 cells express the Flk-1 and Sca-1 cell
markers, but do not express the CD45 or c-Kit markers. Preferably,
greater than 95% of the PP6 cells express the desmin, Sca-1, and
Flk-1 markers, but do not express the CD45 or c-Kit markers. It is
preferred that the PP6 cells are utilized within about 1 day or
about 24 hours after the last plating.
[0039] In a preferred embodiment, the rapidly adhering cells and
slowly adhering cells (MDCs) are separated from each other using a
single plating technique. One such technique is described in
Example 2. First, cells are provided from a skeletal muscle biopsy.
The biopsy need only contain about 100 mg of cells. Biopsies
ranging in size from about 50 mg to about 500 mg are used according
to both the pre-plating and single plating methods of the
invention. Further biopsies of 50, 100, 110, 120, 130, 140, 150,
200, 250, 300, 400 and 500 mg are used according to both the
pre-plating and single plating methods of the invention.
[0040] In a preferred embodiment of the invention, the tissue from
the biopsy is then stored for 1 to 7 days. This storage is at a
temperature from about room temperature to about 4.degree. C. This
waiting period causes the biopsied skeletal muscle tissue to
undergo stress. While this stress is not necessary for the
isolation of MDCs using this single plate technique, it seems that
using the wait period results in a greater yield of MDCs.
[0041] According to preferred embodiments, tissue from the biopsies
is minced and centrifuged. The pellet is resuspended and digested
using a digestion enzyme. Enzymes that may be used include
collagenase, dispase or combinations of these enzymes. After
digestion, the enzyme is washed off of the cells. The cells are
transferred to a flask in culture media for the isolation of the
rapidly adhering cells. Many culture media may be used.
Particularly preferred culture media include those that are
designed for culture of endothelial cells including Cambrex
Endothelial Growth Medium. This medium may be supplemented with
other components including fetal bovine serum, IGF-1, bFGF, VEGF,
EGF, hydrocortisone, heparin, and/or ascorbic acid. Other media
that may be used in the single plating technique include InCell
M310F medium. This medium may be supplemented as described above,
or used unsupplemented.
[0042] The step for isolation of the rapidly adhering cells may
require culture in flask for a period of time from about 30 to
about 120 minutes. The rapidly adhering cells adhere to the flask
in 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 minutes. After they
adhere, the slowly adhering cells are separated from the rapidly
adhering cells from removing the culture media from the flask to
which the rapidly adhering cells are attached to.
[0043] The culture medium removed from this flask is then
transferred to a second flask. The cells may be centrifuged and
resuspended in culture medium before being transferred to the
second flask. The cells are cultured in this second flask for
between 1 and 3 days. Preferably, the cells are cultured for two
days. During this period of time, the slowly adhering cells (MDCs)
adhere to the flask. After the MDCs have adhered, the culture media
is removed and new culture media is added so that the MDCs can be
expanded in number. The MDCs may be expanded in number by culturing
them for from about 10 to about 20 days. The MDCs may be expanded
in number by culturing them for 10, 11, 12, 13, 14, 15, 16, 17, 18,
19 or 20 days. Preferably, the MDCs are subject to expansion
culture for 17 days.
[0044] As an alternative to the pre-plating and single plating
methods, the MDCs of the present invention can be isolated by
fluorescence-activated cell sorting (FACS) analysis using labeled
antibodies against one or more of the cell surface markers
expressed by the MDCs (C. Webster et al., 1988, Exp. Cell. Res.
174:252 65; J. R. Blanton et al., 1999, Muscle Nerve 22:43 50). For
example, FACS analysis can be performed using labeled antibodies
that specifically bind to CD34, Flk-1, Sca-1, and/or the other
cell-surface markers described herein to select a population of
PP6-like cells that exhibit long-term survivability when introduced
into the host tissue. Also encompassed by the present invention is
the use of one or more fluorescence-detection labels, for example,
fluorescein or rhodamine, for antibody detection of different cell
marker proteins.
[0045] Using any of the MDCs isolation methods described above,
MDCs that are to be transported, or are not going to be used for a
period of time may be preserved using methods known in the art.
More specifically, the isolated MDCs may be frozen to a temperature
ranging from about -25 to about -90.degree. C. Preferably, the MDCs
are frozen at about -80.degree. C., on dry ice for delayed use or
transport. The freezing may be done with any cryopreservation
medium known in the art.
Muscle-Derived Cell-Based Treatments
[0046] In one embodiment of the present invention, the MDCs are
isolated from a skeletal muscle source and introduced or
transplanted into a muscle or non-muscle soft tissue site of
interest, or into bone structures. Advantageously, the MDCs of the
present invention are isolated and enriched to contain a large
number of progenitor cells showing long-term survival following
transplantation. In addition, the muscle-derived progenitor cells
of this invention express a number of characteristic cell markers,
such desmin, CD34, and Bcl-2. Furthermore, the muscle-derived
progenitor cells of this invention express the Sca-1, and Flk-1
cell markers, but do not express the CD45 or c-Kit cell markers
(see Example 1).
[0047] MDCs and compositions comprising MDCs of the present
invention can be used to repair, treat, or ameliorate various
aesthetic or functional conditions (e.g. defects) through the
augmentation of bone. In particular, such compositions can be used
for the treatment of bone disorders. Multiple and successive
administrations of MDC are also embraced by the present
invention.
[0048] For MDC-based treatments, a skeletal muscle explant is
preferably obtained from an autologous or heterologous human or
animal source. An autologous animal or human source is more
preferred. MDC compositions are then prepared and isolated as
described herein. To introduce or transplant the MDCs and/or
compositions comprising the MDCs according to the present invention
into a human or animal recipient, a suspension of mononucleated
muscle cells is prepared. Such suspensions contain concentrations
of the muscle-derived progenitor cells of the invention in a
physiologically-acceptable carrier, excipient, or diluent. For
example, suspensions of MDC for administering to a subject can
comprise 10.sup.8 to 10.sup.9 cells/ml in a sterile solution of
complete medium modified to contain the subject's serum, as an
alternative to fetal bovine serum. Alternatively, MDC suspensions
can be in serum-free, sterile solutions, such as cryopreservation
solutions (Celox Laboratories, St. Paul, Minn.). The MDC
suspensions can then be introduced e.g., via injection, into one or
more sites of the donor tissue.
[0049] In certain embodiments, the described cells are administered
as a pharmaceutically or physiologically acceptable preparation or
composition containing a physiologically acceptable carrier,
excipient, or diluent, and administered to the tissues of the
recipient organism of interest, including humans and non-human
animals. In other embodiments, the MDC-containing composition are
prepared by resuspending the cells in a suitable liquid or solution
such as sterile physiological saline or other physiologically
acceptable injectable aqueous liquids. The amounts of the
components to be used in such compositions can be routinely
determined by those having skill in the art.
[0050] According to the invention, the MDCs or compositions thereof
can be administered by placement of the MDC suspensions onto a
biocompatible matrix, e.g., small intestine submucosa (SIS). In
some embodiments, the MDCs are inserted into the biocompatible
matrix and then the MDC-containing matrix into or onto the site of
interest. Alternatively, the MDCs can be administered by parenteral
routes of injection, including subcutaneous, intravenous,
intramuscular, and intrasternal to the desired tissue that has
already been administered the biocompatible material.
[0051] To optimize transplant success, the closest possible
immunological match between donor and recipient is desired. If an
autologous source is not available, 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 transplant procedure. For example, cyclosporin A or other
immunosuppressive drugs can be administered to the transplant
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).
[0052] Consistent with the present invention, the MDCs can be
administered to body tissues, including bone in the presence of a
biocompatible matrix. The number of cells in an MDC suspension and
the mode of administration may vary depending on the site and
condition being treated. From about 1.0.times.10.sup.5 to about
1.times.10.sup.8 MDCs may be administered according to the
invention. As a non-limiting example, in accordance with the
present invention, about 0.5-3.0.times.10.sup.6 MDCs. Preferably
2.0.times.10.sup.6 MDCs are administered in combination with a
biocompatible matrix.
[0053] For bone augmentation or treatment of bone disorders, the
MDCs are prepared as described above and are administered, e.g. in
combination with a biocompatible matrix at the site of treatment or
via injection, onto, into or around bone tissue pretreated with the
biocompatible matrix to provide additional bone density and/or
volume. As is appreciated by the skilled practitioner, the number
of MDC introduced is modulated to provide varying amounts of bone
density and/or bone volume, as needed or required. In certain
embodiments, about 1.0-3.0.times.10.sup.6 MDCs are injected for the
augmentation of bone in combination with a biocompatible matrix.
Thus, the present invention also embraces the use of MDC of the
invention in treating bone disorders or enhancing bone density
and/or bone volume. Bone disorders include osteoporosis, Paget's
Disease, osteogenesis imperfecta, bone fracture, osteomalacia,
decrease in bone trabecular strength, decrease in bone cortical
strength and decrease in bone density with old age. The invention
also relates to the novel use of MDCs for the increase of bone mass
in athletes or other organisms in need of greater than average bone
mass.
Biocompatible Matrices
[0054] According to some embodiments of the present invention, MDCs
are mixed with the biocompatible matrix material in vitro not long
before application to a tissue or organ site in vivo.
Alternatively, MDCs can be mixed with, or inoculated onto, the
biocompatible matrix material just at the time of use. In some
cases, depending upon cell source, cell concentration and matrix
material, the admixing of MDCs and biocompatible matrix material,
or the inoculation of stem cells onto matrix material, needs no
more time than the time that it takes to combine the MDCs and the
biocompatible matrix at the point of use.
[0055] In accordance with the present invention, the in vitro
incubation of MDCs with biocompatible 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 MDCs 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. In some
embodiments of the invention, long-term (e.g., >about 12 hours,
days, or weeks) of incubation or culture time is necessary to
achieve results using the combination of MDC-biocompatible matrix
material.
[0056] The compositions of the invention can be used in treatments
for bone disorders include osteoporosis, Paget's Disease,
osteogenesis imperfecta, bone fracture, osteomalacia, decrease in
bone trabecular strength, decrease in bone cortical strength and
decrease in bone density with old age.
[0057] 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 as the
biocompatible matrix of the invention. The biocompatible 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. Preferably, the biocompatible matrix material
is SIS.
[0058] In an embodiment of the present invention, the biocompatible
matrix material can be in the form of a sling, patch, wrap, such as
are employed in surgeries to correct, strengthen, or otherwise
repair tissues in need of such treatment.
[0059] In another embodiment, the biocompatible matrix, either
combined with MDCs or alone, can be applied through a minimally
invasive fiberoptic scope (e.g., laparoscope) to bone. In another
embodiment, the biocompatible matrix, either combined with MDCs or
alone, is 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.
Genetically Engineered Muscle-Derived Cells
[0060] In another aspect of the present invention, the MDCs of this
invention may be genetically engineered to contain a 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. Such
MDCs may be histocompatible (autologous) or nonhistocompatible
(allogeneic) to the recipient, including humans. These cells can
serve as long-term local delivery systems for a variety of
treatments, for example, for the treatment of bone diseases and
pathologies, including, but not limited to osteoporosis, Paget's
Disease, osteogenesis imperfecta, bone fracture, osteomalacia,
decrease in bone trabecular strength, decrease in bone cortical
strength and decrease in bone density with old age.
[0061] Preferred in the present invention are autologous
muscle-derived progenitor cells, which will not be recognized as
foreign to the recipient. In this regard, the MDC used for
cell-mediated gene transfer or delivery will desirably be matched
vis-a-vis the major histocompatibility locus (MHC or HLA in
humans). Such MHC or HLA matched cells may be autologous.
Alternatively, the cells may be from a person having the same or a
similar MHC or HLA antigen profile. The patient may also be
tolerized to the allogeneic MHC antigens. The present invention
also encompasses the use of cells lacking MHC Class I and/or II
antigens, such as described in U.S. Pat. No. 5,538,722,
incorporated herein by reference.
[0062] The MDCs may be genetically engineered by a variety of
molecular techniques and methods known to those having skill in the
art, for example, transfection, infection, or transduction.
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.
Transfection more commonly refers to cells that have been
genetically engineered to contain a foreign gene harbored in a
plasmid, or non-viral vector. MDCs can be transfected or transduced
by different vectors and thus can serve as gene delivery vehicles
to transfer the expressed products into muscle.
[0063] 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 the use of liposomes, electroporation, precipitation with
DEAE-Dextran or calcium phosphate, particle bombardment
(biolistics) with nucleic acid-coated particles (e.g., gold
particles), microinjection, and the like.
[0064] Vectors for introducing heterologous (i.e., foreign) nucleic
acid (DNA or RNA) into muscle cells for the expression of bioactive
products are well known in the art. Such vectors possess a promoter
sequence, preferably, a promoter that is cell-specific and placed
upstream of the sequence to be expressed. The vectors may also
contain, optionally, one or more expressible marker genes for
expression as an indication of successful transfection and
expression of the nucleic acid sequences contained in the
vector.
[0065] Illustrative examples of vehicles or vector constructs for
transfection or infection of the muscle-derived 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. Adeno-associated
virus vectors are single stranded and allow the efficient delivery
of multiple copies of nucleic acid to the cell's nucleus. Preferred
are adenovirus vectors. The vectors will normally be substantially
free of any prokaryotic DNA and may comprise a number of different
functional nucleic acid sequences. Examples of such functional
sequences include polynucleotide, 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 muscle cells.
[0066] Also included as part of the functional sequences is an open
reading frame (polynucleotide sequence) encoding a protein of
interest; flanking sequences may also be included for site-directed
integration. In some situations, the 5'-flanking sequence will
allow 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.
[0067] In general, the nucleic acid sequence desired to be
expressed by the muscle-derived progenitor cell is that of a
structural gene, or a functional fragment, segment or portion of
the gene, that is heterologous to the muscle-derived progenitor
cell and encodes a desired protein or polypeptide product, for
example. 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, cell differentiation
factors, cell signaling factors and programmed cell death factors.
Specific examples include, but are not limited to, genes encoding
BMP-2 (rhBMP-2), IL-1Ra, Factor IX, and connexin 43.
[0068] As mentioned above, a marker may be present for 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.
[0069] The vector employed will generally also include 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. The
replication system must be selected so that the genes encoding
products necessary for replication do not ultimately transform the
muscle-derived 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 BAG, described by Price et al., 1987, Proc. Natl. Acad. Sci.
USA, 84:156; and Sanes et al., 1986, EMBO J., 5:3133. 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. 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.
[0070] If desired, infectious replication-defective viral vectors
may be used to genetically engineer the cells prior to in vivo
injection of the cells. In this regard, the vectors may be
introduced into retroviral producer cells for amphotrophic
packaging. The natural expansion of muscle-derived progenitor cells
into adjacent regions obviates a large number of injections into or
at the site(s) of interest.
[0071] In another aspect, the present invention provides ex vivo
gene delivery to cells and tissues of a recipient mammalian host,
including humans, through the use of MDC, e.g., early progenitor
muscle cells, that have been virally transduced using an adenoviral
vector engineered to contain a heterologous gene encoding a desired
gene product. Such an ex vivo approach provides the advantage of
efficient viral gene transfer, which is superior to direct gene
transfer approaches. The ex vivo procedure involves the use of the
muscle-derived progenitor cells from isolated cells of muscle
tissue. The muscle biopsy that will serve as the source of
muscle-derived progenitor cells can be obtained from an injury site
or from another area that may be more easily obtainable from the
clinical surgeon.
[0072] It will be appreciated that in accordance with the present
invention, clonal isolates can be derived from the population of
muscle-derived progenitor cells (i.e., PP6 cells or "slowly
adhering" cells using the single plate procedure) using various
procedures known in the art, for example, limiting dilution plating
in tissue culture medium. Clonal isolates comprise genetically
identical cells that originate from a single, solitary cell. In
addition, clonal isolates can be derived using FACS analysis as
described above, followed by limiting dilution to achieve a single
cell per well to establish a clonally isolated cell line. An
example of a clonal isolate derived from the PP6 cell population is
mc13, which is described in Example 1. Preferably, MDC clonal
isolates are utilized in the present methods, as well as for
genetic engineering for the expression of one or more bioactive
molecules, or in gene replacement therapies.
[0073] The MDCs are first infected with engineered viral vectors
containing at least one heterologous gene encoding a desired gene
product, suspended in a physiologically acceptable carrier or
excipient, such as saline or phosphate buffered saline, and then
administered to an appropriate site in the host. Consistent with
the present invention, the MDCs can be administered to body
tissues, including bone, as described above. The desired gene
product is expressed by the injected cells, which thus introduce
the gene product into the host. The introduced and expressed gene
products can thereby be utilized to treat, repair, or ameliorate
the injury, dysfunction, or disease, due to their being expressed
over long time periods by the MDCs of the invention, having
long-term survival in the host.
[0074] In animal model studies of myoblast-mediated gene therapy,
implantation of 10.sup.6 myoblasts per 100 mg muscle was required
for partial correction of muscle enzyme defects (see, J. E. Morgan
et al., 1988, J. Neural. Sci. 86:137; T. A. Partridge et al., 1989,
Nature 337:176). Extrapolating from this data, approximately
10.sup.12 MDCs suspended in a physiologically compatible medium can
be implanted into muscle tissue for gene therapy for a 70 kg human.
This number of MDC of the invention can be produced from a single
100 mg skeletal muscle biopsy from a human source (see below). For
the treatment of a specific injury site, an injection of
genetically engineered MDC into a given tissue or site of injury
comprises a therapeutically effective amount of cells in solution
or suspension, preferably, about 10.sup.5 to 10.sup.6 cells per
cm.sup.3 of tissue to be treated, in a physiologically acceptable
medium.
EXAMPLES
Example 1. MDC Enrichment, Isolation and Analysis According to the
Pre-Plating Method
[0075] MDCs were prepared as described (U.S. Pat. No. 6,866,842 of
Chancellor et al.). Muscle explants were obtained from the hind
limbs of a number of sources, namely from 3-week-old mdx
(dystrophic) mice (C57BL/10ScSn mdx/mdx, Jackson Laboratories), 4-6
week-old normal female SD (Sprague Dawley) rats, or SCID (severe
combined immunodeficiency) mice. The muscle tissue from each of the
animal sources was dissected to remove any bones and minced into a
slurry. The slurry was then digested by 1 hour serial incubations
with 0.2% type XI collagenase, dispase (grade II, 240 unit), and
0.1% trypsin at 37.degree. C. The resulting cell suspension was
passed through 18, 20, and 22 gauge needles and centrifuged at 3000
rpm for 5 minutes. Subsequently, cells were suspended in growth
medium (DMEM supplemented with 10% fetal bovine serum, 10% horse
serum, 0.5% chick embryo extract, and 2% penicillin/streptomycin).
Cells were then preplated in collagen-coated flasks (U.S. Pat. No.
6,866,842 of Chancellor et al.). After approximately 1 hour, the
supernatant was removed from the flask and re-plated into a fresh
collagen-coated flask. The cells which adhered rapidly within this
1 hour incubation were mostly fibroblasts (Z. Qu et al., supra;
U.S. Pat. No. 6,866,842 of Chancellor et al.). The supernatant was
removed and re-plated after 30-40% of the cells had adhered to each
flask. After approximately 5-6 serial platings, the culture was
enriched with small, round cells, designated as PP6 cells, which
were isolated from the starting cell population and used in further
studies. The adherent cells isolated in the early platings were
pooled together and designated as PP1-4 cells.
[0076] The mdx PP1-4, mdx PP6, normal PP6, and fibroblast cell
populations were examined by immunohistochemical analysis for the
expression of cell markers. The results of this analysis are shown
in Table 1.
TABLE-US-00001 TABLE 1 Cell markers expressed in PP1-4 and PP6 cell
populations. mdx PP1-4 mdx PP6 nor PP6 cells cells cells
fibroblasts desmin +/- + + - CD34 - + + - Bcl-2 (-) + + - Flk-1 na
+ + - Sca-1 na + + - M-cadherin -/+ -/+ -/+ - MyoD -/+ +/- +/- -
myogenin -/+ +/- +/- -
[0077] Mdx PP1-4, mdx PP6, normal PP6, and fibroblast cells were
derived by preplating technique and examined by immunohistochemical
analysis. "-" indicates less than 2% of the cells showed
expression; "(-)"; "-/+" indicates 5-50% of the cells showed
expression; "+/-" indicates .about.40-80% of the cells showed
expression; "+" indicates that >95% of the cells showed
expression; "nor" indicates normal cells; "na" indicates that the
immunohistochemical data is not available.
[0078] It is noted that both mdx and normal mice showed identical
distribution of all the cell markers tested in this assay. Thus,
the presence of the mdx mutation does not affect the cell marker
expression of the isolated PP6 muscle-cell derived population.
[0079] MDCs were grown in proliferation medium containing DMEM
(Dulbecco's Modified Eagle Medium) with 10% FBS (fetal bovine
serum), 10% HS (horse serum), 0.5% chick embryo extract, and 1%
penicillin/streptomycin, or fusion medium containing DMEM
supplemented with 2% fetal bovine serum and 1% antibiotic solution.
All media supplies were purchased through Gibco Laboratories (Grand
Island, N.Y.).
Example 2. MDC Enrichment, Isolation and Analysis According to the
Single Plate Method
[0080] Populations of rapidly- and slowly-adhering MDCs were
isolated from skeletal muscle of a mammalian subject. The subject
may be a human, rat, dog or other mammal. Biopsy size ranged from
42 to 247 mg.
[0081] Skeletal muscle biopsy tissue is immediately placed in cold
hypothermic medium (HYPOTHERMOSOL.RTM. (BioLife) supplemented with
gentamicin sulfate (100 ng/ml, Roche)) and stored at 4.degree. C.
After 3 to 7 days, biopsy tissue is removed from storage and
production is initiated. Any connective or non-muscle tissue is
dissected from the biopsy sample. The remaining muscle tissue that
is used for isolation is weighed. The tissue is minced in Hank's
Balanced Salt Solution (HBSS), transferred to a conical tube, and
centrifuged (2,500.times.g, 5 minutes). The pellet is then
resuspended in a Digestion Enzyme solution (Liberase Blendzyme 4
(0.4-1.0 U/mL, Roche)). 2 mL of Digestion Enzyme solution is used
per 100 mg of biopsy tissue and is incubated for 30 minutes at
37.degree. C. on a rotating plate. The sample is then centrifuged
(2,500.times.g, 5 minutes). The pellet is resuspended in culture
medium and passed through a 70 .mu.m cell strainer. The culture
media used for the procedures described in this Example was Cambrex
Endothelial Growth Medium EGM-2 basal medium supplemented with the
following components: i. 10% (v/v) fetal bovine serum, and ii.
Cambrex EGM-2 SingleQuot Kit, which contains: Insulin Growth
Factor-1 (IGF-1), Basic Fibroblast Growth Factor (bFGF), Vascular
Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF),
Hydrocortisone, Heparin, and Ascorbic Acid. The filtered cell
solution is then transferred to a T25 culture flask and incubated
for 30-120 minutes at 37.degree. C. in 5% CO.sub.2. Cells that
attach to this flask are the "rapidly-adhering cells".
[0082] After incubation, the cell culture supernatant is removed
from the T25 flask and placed into a 15 mL conical tube. The T25
culture flask is rinsed with 2 mL of warmed culture medium and
transferred to the aforementioned 15 mL conical tube. The 15 mL
conical tube is centrifuged (2,500.times.g, 5 minutes). The pellet
is resuspended in culture medium and transferred to a new T25
culture flask. The flask is incubated for .about.2 days at
37.degree. C. in 5% CO2 (cells that attach to this flask are the
"slowly-adhering cells"). After incubation, the cell culture
supernatant is aspirated and new culture medium is added to the
flask. The flask is then returned to the incubator for expansion.
Standard culture passaging is carried out from here on to maintain
the cell confluency in the culture flask at less than 50%.
Trypsin-EDTA (0.25%, Invitrogen) is used to detach the adherent
cells from the flask during passage. Typical expansion of the
"slowly-adhering cells" takes an average of 17 days (starting from
the day production is initiated) to achieve an average total viable
cell number of 37 million cells.
[0083] Once the desired cell number is achieved, the cells are
harvested from the flask using Trypsin-EDTA and centrifuged
(2,500.times.g, 5 minutes). The pellet is resuspended in BSS-P
solution (HBSS supplemented with human serum albumin (2% v/v, Sera
Care Life)) and counted. The cell solution is then centrifuged
again (2,500.times.g, 5 minutes), resuspended with Cryopreservation
Medium (CryoStor (Biolife) supplemented with human serum albumin
(2% v/v, Sera Care Life Sciences)) to the desired cell
concentration, and packaged in the appropriate vial for cryogenic
storage. The cryovial is placed into a freezing container and
placed in the -80.degree. C. freezer. Cells are administered by
thawing the frozen cell suspension at room temperature with an
equal volume of physiologic saline and injected directly (without
additional manipulation). The lineage characterization of the
slowly adhering cell populations shows: Myogenic (87.4% CD56+,
89.2% desmin+), Endothelial (0.0% CD31+), Hematopoietic (0.3%
CD45+), and Fibroblast (6.8% CD90+/CD56-).
[0084] Following disassociation of the skeletal muscle biopsy
tissue, two fractions of cells were collected based on their rapid
or slow adhesion to the culture flasks. The cells were then
expanded in culture with growth medium and then frozen in
cryopreservation medium (3.times.10.sup.5 cells in 15 .mu.l) in a
1.5 ml eppendorf tube. For the control group, 15 .mu.l of
cryopreservation medium alone was placed into the tube. These tubes
were stored at -80.degree. C. until injection. Immediately prior to
injection, a tube was removed from storage, thawed at room
temperature, and resuspended with 15 .mu.l of 0.9% sodium chloride
solution.
[0085] Cell count and viability was measured using a Guava flow
cytometer and Viacount assay kit (Guava). CD56 was measured by flow
cytometry (Guava) using PE-conjugated anti-CD56 antibody (1:50, BD
Pharmingen) and PE-conjugated isotype control monoclonal antibody
(1:50, BD Pharmingen). Desmin was measured by flow cytometry
(Guava) on paraformaldehyde-fixed cells (BD Pharmingen) using a
monoclonal desmin antibody (1:100, Dako) and an isotype control
monoclonal antibody (1:200, BD Pharmingen). Fluorescent labeling
was performed using a Cy3-conjugated anti-mouse IgG antibody
(1:250, Sigma). In between steps, the cells were washed with
permeabilization buffer (BD Pharmingen). For creatine kinase (CK)
assay, 1.times.10.sup.5 cells were plated per well into a 12 well
plate in differentiation-inducing medium. Four to 6 days later, the
cells were harvested by trypsinization and centrifuged into a
pellet. The cell lysis supernatant was assayed for CK activity
using the CK Liqui-UV kit (Stanbio).
Example 3. Small Intestine Submucosa Alleviates the Repair of a
Critical Size Calvarial Defect in Mice
[0086] The purpose of this study was to investigate the bone
regenerative potential of single-layer SIS scaffold transplanted
into critical size calvarial defect in mice. We also preconditioned
SIS grafts by seeding them with human muscle-derived cells (hMDCs),
prepared as detailed in Example 2, above, in order to test
osteogenic potential of this construct in response to natural
fracture environment.
[0087] Materials and Methods
[0088] In this study a total of 24 SCID mice were used. All animal
experiments were approved by institutional ARCC. Surgical procedure
was performed under general anesthesia. Critical size calvarial
bone defect was created using a 5-mm-diameter trephine burr. Human
muscle-derived cells (hMDCs) isolated from a 35 year old male
patient were provided. Animals were divided into 3 groups according
to the treatment they received. A control group consisted of
untreated mice with a calvarial defect void of cells or SIS. The
second group consisted of mice receiving 5.times.5 mm single layer
of SIS sheet (Cook Biotech, Inc) without cells that was placed on
top of the defect. The third group consisted of mice receiving
5.times.5 mm single layer SIS sheet that was seeded with
2.times.10.sup.6 human muscle-derived cells hMDCs twelve hours
before transplantation. Microcomputed tomography (vivaCT40, Scanco)
of the calvaria was performed on the following day after the
surgery for each animal. Four animals in each group were sacrificed
at 4 and 10 weeks and harvested calvaria were evaluated by microCT
for a new bone formation. Specimens were fixed in 10% neutral
buffered formalin and preserved for later histological
analysis.
[0089] Results
[0090] 3D reconstruction of the untreated calvaria did not revealed
any substantial bone formation within the defects at 4 and 10 weeks
(FIGS. 1A and 1D). Bone regeneration was seen only along the rim of
the defect which remained entirely open and did not contain any
islands of new bone. At 4 weeks the calvarial defects that were
treated with SIS sheet without cells contained very small or
undetectable bone formation mostly along the edge of the defect
(FIG. 1B). At the same time defects treated with SIS sheet seeded
with hMDCs contained obvious islands of newly formed bone (FIG.
1C). At 10 weeks we detected large islands of new bone in both SIS,
and SIS-hMDC-treated calvarial defects (FIGS. 1E and 1F).
Quantification of new bone within volume of interest (VOI) using
Scanco imaging software revealed difference between
control-untreated and SIS-treated defects at 4 and 10 weeks (FIG.
2). At 4 weeks the new bone volume was 0.01.+-.0.005 mm.sup.3 in
the control group, 0.16.+-.0.15 mm.sup.3 in the SIS-treated group,
and 0.4.+-.0.27 mm.sup.3 in the SIS-hMDC-treated group. At 10 weeks
the new bone volume increased up to 0.02.+-.0.02 mm.sup.3 in the
control group, 1.11.+-.0.73 mm.sup.3 in the SIS-treated group, and
1.38.+-.1.02 mm.sup.3 in the SIS-hMDC-treated group. The SIS-hMDC
treatment group had significantly more bone at 4 and 10 weeks
compared to the empty (untreated) group. Also, there was
significant increase in bone volume in the SIS-treated group at 10
weeks compared to the 4 week time point. FIG. 3 contains important
information and supports our previous results showing significant
difference in bony bridging score between the SIS-hMDC-treated
group and the empty group at 4 and 10 weeks. (Patel et al. Bone,
43:931-940 (2008), provides methods for determining a bony bridging
score and is incorporated herein by reference in its entirety). The
data suggests that the combination of MDCs with SIS administered to
subjects leads to faster healing of bone.
[0091] Discussion
[0092] This study demonstrated that SIS grafts function as a
regenerative matrix scaffold, guiding the attachment of host cells
and supporting formation of new bone. Enhanced bone formation was
observed in SIS-treated calvarial defects in mice, while control
untreated defects showed only minimal calcification Bone formation
in SIS-treated calvarias was already visible after 4 weeks and
gradually increased over 10 week period. Addition of human
muscle-derived cells to the SIS grafts apparently enhanced
calvarial defect healing.
Example 4. hMDCs Seeded on SIS Undergo Osteogenesis In Vitro
Methods
[0093] 2.times.10.sup.6 human muscle-derived cells were seeded on
pre-cut 6 mm diameter 4 layer SIS disks and incubated for 28 days
in either proliferation medium (n=3) containing phenol red-free
Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen) supplemented
with 110 mg/L sodium pyruvate (Sigma-Aldrich), 584 mg/L
L-Glutamine, 10% fetal bovine serum (FBS), 10% horse serum (HS), 1%
penicillin/streptomycin (all from Invitrogen), and 0.5% chick
embryo extract (Accurate Chemical Co.), or osteogenic medium (n=6)
containing phenol-red free DMEM, 10% FBS, 1%
Penicillin/streptomycin, 10-7 M dexamethasone, 5.times.10.sup.-5 M
ascorbic-acid-2-phosphate, 10.sup.-2 M .beta.-glycerophosphate]. at
36.degree. C. in the presence of 5% CO.sub.2 with medium change
every 2-3 days. SIS scaffolds without cells were used for the
control and cultivated similarly in either osteogenic (n=6) or
proliferation (n=4) medium. The same human cells were used to make
four cell pellets (250,000 cells/pellet) that were incubated for 28
days in osteogenic medium. All scaffolds and cell pellets undergo
micro-CT scanning at 7, 10, 14, 21, and 28 days and were evaluated
for mineralized matrix volume and density.
[0094] Results:
[0095] 3D reconstruction by micro-CT revealed presence of
mineralization as early as 7 days in human cell-populated SIS
scaffolds cultured in osteogenic medium. The mineralized matrix
volume in this group progressively increased from 0.112.+-.0.09
mm.sup.3, as observed at 7 days, to 4.673.+-.0.72 mm.sup.3, as
detected at 28 days (FIG. 4A). No matrix mineralization during the
entire culture period was detected in SIS scaffolds containing
human cells that were placed in proliferation medium. Empty SIS
scaffolds containing no cells also exhibited mineral deposition at
21 days (0.162.+-.0.19 mm.sup.3) and 28 days (1.329.+-.0.8
mm.sup.3) when cultured in osteogenic medium, but did not conduce
to mineralization when cultivated in proliferation medium. The
mineralized matrix density in SIS scaffolds with human cells
cultivated in osteogenic medium was 222.31.+-.35.7 mm HA/ccm at 7
days, and slightly decreased to 200.05.+-.25.4 mm HA/ccm at 28 days
(FIG. 4B). Density of empty SIS scaffolds cultured in OSM was
157.09.+-.7.2 mm HA/ccm at 21 day and 170.05.+-.20.12 mm HA/ccm at
28 days (FIG. 4B). FIG. 5 shows 3D micro-CT reconstruction of SIS
and SIS-hMDC scaffolds (5 samples in each group) cultured in
osteogenic medium on day 28. It demonstrates that hMDC-seeded SIS
scaffolds have more intense mineralization than SIS scaffolds
without cells suggesting that hMDCs accelerated the formation of
mineralized matrix on SIS sheets.
[0096] Micro-CT scanning of human cell pellets cultured in
osteogenic medium revealed matrix mineralization to a lesser
extent. The initial matrix volume detected at 7 days
(0.221.+-.0.004 mm.sup.3) was merely increased at 28 days
(0.31.+-.0.06 mm.sup.3) (FIG. 6A). However, mineralized matrix
density in pellet cultures increased noticeably. It was
252.2.+-.9.96 mg HA/ccm at 7 days, 445.34.+-.22.55 mg HA/ccm at 14
days, and 609.01.+-.42.82 mm HA/ccm at 28 days (FIG. 6B).
[0097] We do not wish to be limited by theory, however, the effect
of increased volume produced when using SIS and increased density
when using pellets could be caused by the difference of the cells
being spread out on SIS as opposed to compacted into a tight
pellet. The cells on SIS are spread out and are simply creating
bone over the entire SIS area. Thus, the effect seen is increased
bone volume during the period of evaluation. Whereas, the cells in
pellet are compacted into a small area and therefore are just
increasing in density over the period of evaluation. Arguably,
there is really no room for much volume increase since they are
already in a pellet.
* * * * *