U.S. patent application number 12/589956 was filed with the patent office on 2010-05-13 for method and device for activating stem cells.
This patent application is currently assigned to Synthes USA, LLC. Invention is credited to Melissa Brown, Doug Buechter, Elliott Gruskin, Meredith Hans, Stephen Hornsby.
Application Number | 20100119492 12/589956 |
Document ID | / |
Family ID | 41426294 |
Filed Date | 2010-05-13 |
United States Patent
Application |
20100119492 |
Kind Code |
A1 |
Hans; Meredith ; et
al. |
May 13, 2010 |
Method and device for activating stem cells
Abstract
Invention embodiments described herein include methods and
devices for stimulating mesenchymal stem cells in a stem cell
source to differentiate into osteoblasts capable of forming bone.
Devices and methods described include exposing a stem cell source,
such as bone marrow aspirate, adipose tissue and/or purified
allogenic stem cells, to an active agent, in a manner effective to
form activated stem cells.
Inventors: |
Hans; Meredith; (Abington,
PA) ; Buechter; Doug; (Chester Springs, PA) ;
Gruskin; Elliott; (Malvern, PA) ; Hornsby;
Stephen; (Phoenixville, PA) ; Brown; Melissa;
(Reading, PA) |
Correspondence
Address: |
SCHWEGMAN, LUNDBERG & WOESSNER / SYNTHES
P.O. BOX 2938
MINNEAPOLIS
MN
55402
US
|
Assignee: |
Synthes USA, LLC
West Chester
PA
|
Family ID: |
41426294 |
Appl. No.: |
12/589956 |
Filed: |
October 30, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61152335 |
Feb 13, 2009 |
|
|
|
61110096 |
Oct 31, 2008 |
|
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Current U.S.
Class: |
424/93.7 ;
435/283.1; 435/286.1 |
Current CPC
Class: |
A61L 27/20 20130101;
C12N 2501/115 20130101; A61L 27/18 20130101; C12N 2501/135
20130101; A61L 27/3895 20130101; C12N 2506/1346 20130101; A61K
35/32 20130101; A61L 27/46 20130101; C12N 5/0654 20130101; C12N
2501/155 20130101; A61P 1/02 20180101; A61P 19/02 20180101; A61L
27/3847 20130101; C12N 5/0663 20130101; C12N 2501/15 20130101; A61L
27/3834 20130101; A61P 19/00 20180101; A61L 27/12 20130101; A61L
27/18 20130101; C08L 67/04 20130101; A61L 27/20 20130101; C08L 1/02
20130101 |
Class at
Publication: |
424/93.7 ;
435/283.1; 435/286.1 |
International
Class: |
A61K 35/12 20060101
A61K035/12; A61P 19/00 20060101 A61P019/00; C12M 1/00 20060101
C12M001/00; C12M 1/24 20060101 C12M001/24 |
Claims
1. A method for preparing a implant composition for promoting bone
growth in a mammal, comprising (a) contacting stem cells with one
or more active agents for 24 hours or less to prepare an activated
stem cells, (b) separating the active agents from the activated
stem cells to form an activated stem cell population that is
substantially free of active agents, and (c) mixing the activated
stem cell population that is substantially free of active agents
with a bone graft substitute to thereby prepare an implant
composition for promoting bone growth in a mammal, wherein at least
one active agent promotes differentiation of stem cells into
osteogenic cells or osteogenic precursor cells.
2. The method of claim 1, wherein the stem cells are contacted with
one or more active agents for 5 minutes to 1 hour.
3. The method of claim 1, wherein the stem cells are contacted with
one or more active agents for 5 minutes to 0.5 hours.
4. The method of claim 1, wherein the stem cells are from bone
marrow, adipose tissue, muscle tissue, umbilical cord blood,
embryonic yolk sac, placenta, umbilical cord, periosteum, fetal
skin, adolescent skin, or blood.
5. The method of claim 1, wherein the stem cells are autologous,
allogeneic or xenogeneic.
6. The method of claim 1, wherein the stem cells are embryonic,
post-natal or adult stem cells.
7. The method of claim 1, wherein the stem cells comprise
mesenchymal stem cells.
8. The method of claim 1, wherein the stem cells comprise
autologous bone marrow aspirate.
9. The method of claim 8, wherein the bone marrow aspirate is drawn
intraoperatively.
10. The method of claim 8, wherein cells in the bone marrow
aspirate are isolated or concentrated.
11. The method of claim 1, wherein the active agents can regulate
cellular growth and/or differentiation, and are selected from the
group consisting of small molecules, peptides, growth factors,
cytokines, ligands, hormones and combinations thereof.
12. The method of claim 1, wherein the active agents are selected
from the group consisting of transforming growth factor beta
(TGF-.beta.), fibroblast growth factor (FGF), platelet-derived
growth factor (PDGF), bone morphogenic protein (BMP), insulin
growth factor (IGF), interleukin-I (IL-I), interleukin-11 (IL-11),
simvastatsin, dexamethasone, oxysterol, sonic hedgehog, interferon,
tumor necrosis factor, nerve growth factor (NGF), fibronectin, RGD
peptide, integrin, epidermal growth factor (EGF), hepatocyte growth
factor (HGF), keratinocyte growth factor, osteogenic protein, and
combinations thereof.
13. The method of claim 1, wherein the active agents are selected
from the group consisting of BMP-2, TGF-beta3, PSGF-AB, PDGF-BB,
FGF-2, TGF-beta1, BMP-4, BMP-7, BMP-6, FGF-8, IL-11, simvastatsin,
dexamethasone, oxysterols, sonic hedgehog, and combinations
thereof.
14. The method of claim 1, wherein the active agents include
TGF-.beta. and FGFb.
15. The method of claim 14, wherein the active agents include
PDGF.
16. The method of claim 1, wherein the active agents are in
solution.
17. The method of claim 16, wherein the activated stem cells are
separated from the active agents by a procedure comprising
filtration, gel filtration, tangential flow filtration,
immunoprecipitation, immuno-absorption, affinity chromatography,
column chromatography or a combination thereof.
18. The method of claim 1, wherein the active agents are attached
to a solid support.
19. The method of claim 18, wherein at least some of the active
agents are attached to a solid support via a peptide, an antibody,
a chemical cross linker, or a combination thereof.
20. The method of claim 1, wherein the bone graft substitute
comprises a calcium salt.
21. The method of claim 20, wherein the calcium salt comprises
monocalcium phosphate monohydrate, .alpha.-tricalcium phosphate,
.beta.-tricalcium phosphate, calcium carbonate, or a combination
thereof.
22. The method of claim 20, wherein the bone graft substitute
further comprises demineralized bone, a sodium phosphate salt, a
polymer or a combination thereof.
23. The method of claim 22, wherein the polymer is collagen,
gelatin, hyaluronic acid, a hyaluronate salt,
hydroxypropylcellulose (HPC), carboxymethylcellulose (CMC),
hydroxypropyl methylcellulose (HPMC), hydroxyethylcellulose (HEC),
xantham gum, guar gum, alginate or a combination thereof.
24. The method of claim 1, wherein the method increases expression
of alkaline phosphatase and/or a bone morphogenetic protein (BMP)
receptor subunit in the stem cells.
25. The method of claims 1, further comprising implanting the
implant composition into a patient.
26. An implant composition prepared by the method of claim 1.
27. A method for treating a bone injury, disorder or condition in a
subject comprising administering the implant composition of claim
26 to a site of the bone injury, disorder or condition in the
subject.
28. The method of claim 27, wherein the bone injury, disorder or
condition comprises a broken bone, a bone defect, a bone
transplant, a bone graft, bone cancer, a joint replacement, a joint
repair, a bone fusion, a bone facet repair, bone degeneration, a
dental implants, a dental repair, arthritis, bone reconstruction,
or a combination thereof.
29. A device for activating stem cells comprising a solid support
and at least one active agent that promotes differentiation of stem
cells into osteogenic cells or osteogenic precursor cells, and the
device is adapted: (i) for incubating the stem cells with the at
least one active agent, and (ii) for separating the at least one
active agent from the stem cell after the incubating step (i).
30. The device of claim 29, wherein the at least one active agent
is selected from the group consisting of transforming growth factor
beta (TGF-.beta.), fibroblast growth factor (FGF), platelet-derived
growth factor (PDGF), bone morphogenic protein (BMP), insulin
growth factor (IGF), interleukin-I (IL-I), interleukin-11 (IL-11),
simvastatsin, dexamethasone, oxysterol, sonic hedgehog, interferon,
tumor necrosis factor, nerve growth factor (NGF), fibronectin, RGD
peptide, integrin, epidermal growth factor (EGF), hepatocyte growth
factor (HGF), keratinocyte growth factor, osteogenic protein, and
combinations thereof.
31. The device of claim 29, wherein the at least one active agent
is selected from the group consisting of BMP-2, TGF-beta3, PSGF-AB,
PDGF-BB, FGF-2, TGF-beta1, BMP-4, BMP-7, BMP-6, FGF-8, IL-11,
simvastatsin, dexamethasone, oxysterols, sonic hedgehog, and
combinations thereof.
32. The device of claim 29, wherein the at least one active agent
includes TGF-.beta. and FGFb.
33. The device of claim 32, wherein the at least one active agent
further includes PDGF.
34. The device of claim 29, wherein the at least one active agent
is attached to the solid support.
35. The device of claim 29, wherein the solid support comprises a
column matrix material, a filter, an culture plate, tube or dish, a
flask, a microtiter plate, a bead, a disk, or a combination
thereof.
36. The device of claim 29, wherein the solid support comprises
plastic, cellulose, cellulose derivatives, magnetic particles,
nitrocellulose, glass, fiberglass, latex, or a combination
thereof.
37. The device of claim 29, wherein the at least one active agent
is in solution.
38. The device of claim 29, wherein the solid support comprises a
container.
39. The device of claim 29, wherein the solid support comprises a
filter.
40. The device of claim 39, wherein the filter retains cells and
bone graft substitute materials but allows passage of the at least
one active agent.
41. The device of claim 39, wherein the filter retains the at least
one active agent but allows passage of the stem cells.
42. The device of claim 29, wherein the solid support comprises an
affinity matrix to remove the at least one active agent.
43. The device of claim 29, wherein the solid comprises
streptavidin, biotin, a crosslinker, an antibody or peptide that
binds at least one active agent.
44. The device of claim 29, wherein the solid support material does
not bind or adversely interact with stem cells.
45. The device of claim 29, wherein the stem cells are from bone
marrow, adipose tissue, muscle tissue, umbilical cord blood,
embryonic yolk sac, placenta, umbilical cord, periosteum, fetal
skin, adolescent skin, or blood.
46. The device of claim 29, wherein the stem cells comprise
mesenchymal stem cells.
47. The device of claim 29, wherein the stem cells comprise
autologous bone marrow aspirate.
48. The device of claim 29, further comprising a timer for
controlling the time for incubating the stem cells with the at
least one active agent.
49. The device of claim 48, wherein the timer triggers separation
of the at least one active agent from the stem cell after the
incubating step (i).
50. The device of claim 48, wherein the timer controls the time for
incubating the stem cells with the at least one active agent to 24
hours or less.
51. The device of claim 48, wherein the timer controls the time for
incubating the stem cells with the at least one active agent to 5
minutes to 1 hour.
52. The device of claim 48, wherein the timer controls the time for
incubating the stem cells with the at least one active agent to 5
minutes to 0.5 hours.
Description
RELATED APPLICATIONS
[0001] This patent application claims the benefit of the filing
date of U.S. Provisional Patent Application Ser. No. 61/110,096,
filed Oct. 31, 2008, and entitled, "DEVICE FOR ACTIVATING BONE
MARROW ASPIRATE USING EX VIVO STIMULATION BY GROWTH FACTORS FOR
IMPROVED BONE FORMATION", and of U.S. Provisional Patent
Application Ser. No. 61/152,335, filed Feb. 13, 2009, and entitled,
"METHOD AND DEVICE FOR FORMING A BONE MARROW ASPIRATE PRODUCT", the
contents of which are incorporated herein by reference in their
entirety.
FIELD
[0002] Inventive subject matter described herein relates to devices
and methods for activating stem cells, including activating stem
cells in bone marrow aspirate using ex vivo stimulation. The
inventive subject matter also relates to implants containing such
activated stem cells.
BACKGROUND OF THE INVENTION
[0003] In order to provide for maximum bone formation, it is
desirable to transplant cells that already exhibit an osteoblastic
phenotype, because such cells likely to exhibit bone-forming
activity. However, in vitro differentiation of bone marrow stem
cells into osteoblasts involves culturing in osteogenic medium
(Jaiswal et al. 1997. J Cell Biochem 64: 295-312) and may lead to
decreased proliferation of such cells in vitro. Moreover, the use
of osteogenic medium involves addition of components to the cells
(e.g., growth factors) that can have unintended side effects if
those components are administered to a patient along with the
cells.
[0004] Hence, there exists a need in the art for a simple and
reliable method to produce osteoprogenitors, osteoblasts or
osteoblastic phenotypic cells in vitro from stem cells, for
example, human bone marrow stem cells, where the desirable cells
are readily separated from the factors used for generating the
osteoprogenitors, osteoblasts or osteoblastic phenotypic cells.
SUMMARY OF THE INVENTION
[0005] One aspect of the invention is a method for preparing a
implant composition for promoting bone growth in a mammal,
comprising (a) contacting stem cells with one or more active agents
for 24 hours or less to prepare an activated stem cells, (b)
separating the active agents from the activated stem cells to form
an activated stem cell population that is substantially free of
active agents, and (c) mixing the activated stem cell population
that is substantially free of active agents with a bone graft
substitute to thereby prepare an implant composition for promoting
bone growth in a mammal, wherein at least one active agent promotes
differentiation of stem cells into osteogenic cells or osteogenic
precursor cells. The osteogenic cells and/or osteogenic progenitor
cells can be osteoprogenitors, osteoblasts or osteoblastic
phenotypic cells. In some embodiments, the stem cells are contacted
with one or more active agents for 5 minutes to 1 hour. In other
embodiments, the stem cells are contacted with one or more active
agents for 5 minutes to 0.5 hours. The stem cells can, for example,
be obtained or isolated from bone marrow, adipose tissue, muscle
tissue, umbilical cord blood, embryonic yolk sac, placenta,
umbilical cord, periosteum, fetal skin, adolescent skin, or blood.
The stem cells can be embryonic, post-natal or adult stem cells. In
some embodiments, the stem cells are autologous, allogeneic or
xenogeneic. The stem cells can include mesenchymal stem cells. Such
mesenchymal stem cells can include autologous bone marrow aspirate.
The bone marrow aspirate can be drawn intraoperatively. After
obtaining the stem cells they can be concentrated so that
unnecessary liquid is removed. Alternatively, the stem cells can be
isolated from the tissue or liquid from which they were initially
obtained.
[0006] The active agents can, for example, regulate cellular growth
and/or differentiation, and may be selected from the group
consisting of small molecules, peptides, growth factors, cytokines,
ligands, hormones and combinations thereof. Examples of active
agents include active agents such as transforming growth factor
beta (TGF-.beta.), fibroblast growth factor (FGF), platelet-derived
growth factor (PDGF), bone morphogenic protein (BMP), insulin
growth factor (IGF), interleukin-I (IL-I), interleukin-11 (IL-11),
simvastatsin, dexamethasone, oxysterol, sonic hedgehog, interferon,
tumor necrosis factor, nerve growth factor (NGF), fibronectin, RGD
peptide, integrin, epidermal growth factor (EGF), hepatocyte growth
factor (HGF), keratinocyte growth factor, osteogenic protein, and
combinations thereof. In some embodiments, the active agents are
selected from the group consisting of BMP-2, TGF-beta3, PSGF-AB,
PDGF-BB, FGF-2, TGF-beta1, BMP-4, BMP-7, BMP-6, FGF-8, IL-11,
simvastatsin, dexamethasone, oxysterols, sonic hedgehog, and
combinations thereof. In other embodiments, the active agents
include TGF-.beta. and FGFb, and can further include PDGF. The
active agents can be from an autologous source. The active agents
can be used in the method while in solution. When the active agents
are used in the methods while in solution, the activated stem cells
are separated pursuant to step (b) from the active agents by a
procedure that includes filtration, gel filtration, tangential flow
filtration, immunoprecipitation, immuno-absorption, column
chromatography or a combination thereof. In other embodiments, the
active agents are attached to a solid support. For example, at
least some of the active agents can be directly or indirectly
attached to a solid support by covalent attachment, adsorption,
non-covalent interaction and/or combinations thereof. In some
embodiments, at least some of the active agents are attached to the
solid support via a peptide, an antibody, a chemical cross linker,
an alkylene chain or a combination thereof.
[0007] The bone graft substitute can include materials such as
calcium salts. Such calcium salts can, for example, include
monocalcium phosphate monohydrate, .alpha.-tricalcium phosphate,
.beta.-tricalcium phosphate, calcium carbonate, or a combination
thereof. The bone graft substitute can further include
demineralized bone, a sodium phosphate salt, a polymer or a
combination thereof. Such a polymer can be collagen, gelatin,
hyaluronic acid, a hyaluronate salt, hydroxypropylcellulose (HPC),
carboxymethylcellulose (CMC), hydroxypropyl methylcellulose (HPMC),
hydroxyethylcellulose (HEC), xantham gum, guar gum, alginate or a
combination thereof. In some embodiments, the methods increase
expression of alkaline phosphatase and/or a bone morphogenetic
protein (BMP) receptor subunit in the stem cells. Such methods can
further include implanting the implant composition into a
patient.
[0008] Another aspect of the invention is an implant composition
prepared by the methods described herein.
[0009] Another aspect of the invention is a method for treating a
bone injury, disorder or condition in a subject comprising
administering an implant composition described herein to a site of
the bone injury, disorder or condition in the subject. Such a bone
injury, disorder or condition can be a broken bone, a bone defect,
a bone transplant, a bone graft, bone cancer, a joint replacement,
a joint repair, a bone fusion, a bone facet repair, bone
degeneration, a dental implant, a dental repair, arthritis, bone
reconstruction, or a combination thereof.
[0010] Another aspect of the invention is a device for activating a
stem cell that includes a solid support and at least one active
agent that promotes differentiation of stem cells into osteogenic
cells or osteogenic precursor cells, wherein the device is adapted:
(i) for incubating the stem cell with the at least one active
agent, and (ii) for separating the at least one active agent from
the stem cell after the incubating step (i). For example, the at
least one active agent is selected from the group consisting of
transforming growth factor beta (TGF-.beta.), fibroblast growth
factor (FGF), platelet-derived growth factor (PDGF), bone
morphogenic protein (BMP), insulin growth factor (IGF),
interleukin-I (IL-I), interleukin-11 (IL-11), simvastatsin,
dexamethasone, oxysterol, sonic hedgehog, interferon, tumor
necrosis factor, nerve growth factor (NGF), fibronectin, RGD
peptide, integrin, epidermal growth factor (EGF), hepatocyte growth
factor (HGF), keratinocyte growth factor, osteogenic protein, and
combinations thereof.
[0011] In some embodiments, the at least one active agent is
selected from the group consisting of BMP-2, TGF-beta3, PSGF-AB,
PDGF-BB, FGF-2, TGF-beta1, BMP-4, BMP-7, BMP-6, FGF-8, IL-11,
simvastatsin, dexamethasone, oxysterols, sonic hedgehog, and
combinations thereof. In other embodiments, the at least one active
agent includes TGF-.beta. and FGFb, and can further include PDGF.
The at least one active agent can, for example, be from an
autologous source. The active agent(s) can be in solution within
the solid support or attached to the solid support. The active
agent can, for example, be attached to the solid support via an
antibody or peptide that binds at least one active agent.
[0012] The solid support can include a column matrix material, a
filter, an culture plate, tube or dish, a microtiter plate, a bead,
a disk, or a combination thereof. The solid support can be a
container. The solid support can include plastic, cellulose,
cellulose derivatives, magnetic particles, nitrocellulose, glass,
fiberglass, latex, or a combination thereof. The solid support can
also include an affinity matrix to remove the at least one active
agent. When a filter is present in the solid support, the filter
can retain cell and bone graft substitute materials but allow
passage of the at least one active agent. Alternatively, the filter
can retain the at least one active agent but allow passage of the
stem cells. In some embodiments, the solid support does not bind or
adversely interact with stem cells. In other embodiments, the solid
support can bind the stem cells without adversely interacting with
the stem cells.
[0013] The device can further include a timer for controlling the
time for incubating the stem cells with the at least one active
agent. For example, the timer can trigger separation of the at
least one active agent from the stem cell after the incubating step
(i). In some embodiments, the device with the timer controls the
time for incubating the stem cells with the at least one active
agent to 24 hours or less. In other embodiments, the device with
the timer controls the time for incubating the stem cells with the
at least one active agent to 5 minutes to 1 hour.
[0014] Another aspect of the invention is a device for bone
formation comprising a first component for handling a stem cell
source; and a second component for exposing the stem cell source to
an active agent in a manner effective to stimulate mesenchymal stem
cells in the stem cell source to differentiate into osteoblasts,
wherein the osteoblasts can be incorporated into an implant
composition useful for repair and/or generation of bone. The stem
cell source can be bone marrow aspirate, including autologous bone
marrow aspirate, adipose tissue and/or purified allogenic stem
cells.
[0015] The active agent can include, but is not limited to BMP-2,
TGF-beta3, PSGF-AB, PDGF-BB, FGF-2, TGF-beta1, BMP-4, BMP-7, BMP-6,
FGF-8, IL-11, simvastatsin, dexamethasone, oxysterols and/or sonic
hedgehog. The active agent may be directly attached to a solid
support of the device or tethered to the solid support, for
example, through a linker. In one embodiment, in the tether is
selected from an alkylene chain, a peptide, an antibody, a chemical
cross linker, and combinations thereof.
[0016] In one embodiment, the active agent is in solution, and the
second component can further comprise a filter component or an
affinity matrix (e.g., with a peptide or antibody) for removing the
active agent from the mesenchymal stem cells.
[0017] One embodiment includes a device for activating bone marrow
aspirate. The device comprises a component for mixing a bone graft
substitute and a bone marrow aspirate drawn from a patient to form
a mixture. The device also includes a component for transiently
exposing the mixture to a fixed or tethered active agent in a
manner effective for triggering mesenchymal stem cells to enhance
an osteoblast phenotype.
[0018] Another embodiment includes a device for activating bone
marrow aspirate which includes a component for handling bone marrow
aspirate drawn from a patient. The device also includes a component
for exposing the bone marrow aspirate to an active agent in a
manner effective for triggering mesenchymal stem cells to enhance
an osteoblast phenotype.
[0019] Another device for activating bone marrow aspirate is
described, including a component for mixing a bone graft substitute
and a bone marrow aspirate drawn from a patient to form a mixture.
The device further includes a component for exposing the mixture to
a fixed or tethered active agent in a manner effective for
triggering mesenchymal stem cells to enhance an osteoblast
phenotype. Following exposure, BMA is separated from tethered or
fixed active agent and can be mixed with a bone graft
substitute.
[0020] In one embodiment, a method comprising exposing a stem cell
source (e.g., bone marrow aspirate, including autologous bone
marrow aspirate) to an active agent, wherein mesenchymal stem cells
in the stem cell source are stimulated to differentiate into
osteoblasts. In one embodiment, the bone marrow aspirate is drawn
intraoperatively and/or used in a form as drawn from the patient.
Alternatively, the bone marrow aspirate can further be concentrated
after it is drawn from the patient. The method can also include
mixing stimulated or activated stem cells (e.g., mesenchymal stem
cells) with a synthetic bone graft substitute to form a mixture,
wherein the exposing comprises transiently exposing the mixture to
a fixed or tethered active agent in a manner effective for
triggering mesenchymal stem cells to enhance an osteoblast
phenotype.
[0021] In one embodiment, the active agent is bonded to a substrate
to form a bonded substrate. In this embodiment, the stem cell
source is incubated with the bonded active agent for a period of
time, such as between 5 minutes and 24 hours, or between 5 minutes
and 1 hour, or between 15 minutes and 1 hour. In one embodiment,
the incubating causes upregulation in a bone morphogenetic protein
(BMP) receptor subunit in the bone.
[0022] In one embodiment, the exposing of a stem cell source to an
active agent occurs in a stem cell solution, and the method can
further comprise removing the active agent from the stem cell
solution, such as with formation of an affinity matrix and/or
filtration.
[0023] In another embodiment, a method is provided for forming
progenitor cells capable of stimulating bone formation. Method
operations include mixing bone marrow aspirate (BMA) with an active
agent, and using the active agent to trigger mesenchymal stem cells
(MSCs) to enhance an osteoblast phenotype. Such a method can
trigger mesenchymal stem cells to enhance or develop an osteoblast
phenotype. The method can also include mixing a bone graft
substitute and a bone marrow aspirate drawn from a patient to form
a mixture; and transiently exposing the mixture to a fixed or
tethered active agent in a manner effective for triggering
mesenchymal stem cells to enhance or develop the osteoblast
phenotype.
[0024] In some embodiments, the method further includes implanting
stimulated mesynchemal stem cells in a patient, which may include
implanting the active agent together with the stem cell source.
DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 graphically illustrates the levels of alkaline
phosphatase expressed by primary human MSC's in embodiments of the
present invention. The cells were cultured in differentiation media
following a 1 hour treatment with fibroblast growth factor
(hereinafter "FGF-2") (100 ng/ml) or TGF beta3 (250 ng/ml). The
levels of alkaline phosphatase expressed by the cells were then
measured using the assay described in Example 1.
[0026] FIG. 2 graphically illustrates relative mRNA copy number for
three types of bone morphogenetic protein (hereinafter "BMP")
receptor units, namely BMPR-1A ("1A"), BMPR-1B ("1B") and BMPR-II
("II") in primary human mesenchymal cells (hereinafter "MSC's")
after the cells were exposed to active agents for 24 hours. The
active agents used were platelet-derived growth factor (hereinafter
"PDGF BB"), fibroblast growth factor (hereinafter "FGF b"), and
transforming growth factor (hereinafter "TGF beta3"), at
concentrations of 1 ng/ml, 10 ng/ml and 100 ng/ml, with exposure
for 24 hours. Cells were harvested and subjected to a quantitative
polymerase chain reaction (hereinafter "PCR"), for all three BMP
receptor units, namely -1A, -1B and II as described in Example
1.
[0027] FIG. 3 graphically illustrates relative mRNA copy number of
three BMP receptor units (namely -1A, -1B and II) after primary
MSCs were exposed to PDGF BB, FGF b and TGF beta3 for one (1) hour
using concentrations of 1 ng/ml, 10 ng/ml and 100 ng/ml. Cells were
harvested and subjected to a quantitative PCR for all three BMP
receptor units (namely -1A, -1B and II) as described in Example
1.
DETAILED DESCRIPTION
[0028] In the current state of the art, a bone marrow aspirate
(hereinafter "BMA") is drawn from a patient intraoperatively, mixed
with a bone graft substitute and re-implanted in the surgical site
to promote bone healing. However, while bone marrow aspirate may
contain some connective tissue progenitor cells that may produce
osteoblasts, there is a wide patient to patient variation in the
number and type of osteogenic cells that can actually promote bone
formation.
[0029] Described herein are methods, devices and implants that
improve the performance of stem cells (e.g., BMA), either by
increasing in cell number or by an increasing in the osteogenic
potential of the progenitor cells in the BMA or stem cell mixture.
Embodiments described herein include methods and devices to
briefly, intraoperatively expose stem cells ex vivo, to one or more
exogenous active agents, to thereby generate progenitor cells that
are capable of stimulating bone growth, where the active agent(s)
are then removed from the stem cells (i.e., progenitor cells
activated by the active agents). The active agent(s) can be, for
example, a small molecule, a peptide, a growth factor, cytokine,
ligand or other factor. The active agent(s) can be captured from an
autologous source, be obtained from a commercial source or be
manufactured (e.g., by recombinant procedures).
[0030] In some embodiments, the active agents are capable of
increasing the expression of bone morphogenetic protein,
hereinafter BMP, receptor subunit(s) in the progenitor cells of the
stem cell mixture. This treatment by the active agents helps, for
example, to potentiate the ability of the progenitor cells to
respond to endogenous BMPs at the site of bone injury or bone
surgery. Such treatment with active agents can also drive the stem
cells to differentiate down an osteoblast pathway. While stem cells
have been activated by treatment with cytokines for extended
periods of time (e.g., several days), as described herein, exposure
of stem cells to active agents for such extended periods of time is
not necessary: stem cells can be activated and exhibit osteogenic
potential after exposure to active agents for only about 15 minutes
to about 3 hours.
[0031] Moreover, some studies indicate that the existence of an
accessory cell population that might be necessary for the outgrowth
of bone precursor cells in vitro. Thus, highly purified bone
precursor cells may fail to multiply, even in the presence of a
cocktail of osteogenic cytokines. Thus, extended culturing of stem
cells may not be advantageous. The methods and devices described
herein do not involve extended periods of culturing cells and are
faster and thereby avoid the potential for contamination and the
expenses associated with extended cell culture.
[0032] As described herein potentiation, activation and/or
differentiation of stem cells to have osteogenic potential can be
accomplished by tethering the active agent(s) onto or within a
device so that cells contained within stem cell mixture can be
activated, and easily separated from the active agent(s), for
example, by removing the cells from the device. Separating the
active agents from the cells before implantation of the cells
reduces the potential for unintended side effects from the active
agents themselves (e.g., stimulating undesired responses or
inducing harmful immune responses).
Stem Cell Sources
[0033] The methods, devices and implants described herein can
employ stem cells from any convenient source. However, stem cells
that have osteogenic potential or that can be treated (e.g.,
differentiated) to generate cells with osteogenic potential are
preferred.
[0034] Sources of stem cells that can be used in the methods,
devices and implants described herein include bone marrow, adipose
tissue, muscle tissue, ex vivo cultured autologous mesenchymal stem
cells, allogeneic off-the-shelf mesenchymal stem cells, umbilical
cord blood, embryonic yolk sac, placenta, umbilical cord,
periosteum, fetal and adolescent skin, and blood. In some
embodiments, the stem cells are mesenchymal stem cells or a mixture
of cells that include mesenchymal stem cells (e.g., bone marrow
aspirate). The stem cells can be autologous, allogeneic or from
xenogeneic sources. The stem cells can be embryonic or from
post-natal or adult sources.
[0035] Bone marrow aspirate is one source of stem cells useful in
the methods, devices and implants described herein. While such bone
marrow aspirate can be autologous, allogeneic or from xenogeneic
sources, in some embodiments the bone marrow aspirate is
autologous.
[0036] Bone marrow aspirate contains a complex mixture of
hematopoietic stem cells, red and white blood cells and their
precursors, mesenchymal stem and progenitor cells, stromal cells
and their precursors, and a group of cells including fibroblasts,
reticulocytes, adipocytes, and endothelial cells which form a
connective tissue network called "stroma." Cells from the stroma
morphologically regulate the differentiation of hematopoietic cells
through direct interaction via cell surface proteins and the
secretion of growth factors and are involved in the foundation and
support of the bone structure. Studies indicate that bone marrow
contains "pre-stromal" cells which have the capacity to
differentiate into cartilage, bone, and other connective tissue
cells. Beresford "Osteogenic Stem Cells and the Stromal System of
Bone and Marrow", Clin. Orthop., 240:270, 1989. Recent evidence
indicates that these cells, called pluripotent stromal stem cells
or mesenchymal stem cells, have the ability to generate into
several different types of cell lines (i.e., osteocytes,
chondrocytes, adipocytes, etc.) upon activation. However,
mesenchymal stem cells are often present in bone marrow aspirates
in very minute amounts with a wide variety of other cells (i.e.,
erythrocytes, platelets, neutrophils, lymphocytes, monocytes,
eosinophils, basophils, adipocytes, etc.). In addition, their
ability to differentiate into an assortment of connective tissues
depends not only on the presence of bioactive factors in the
aspirate, which can vary, but is also, to some extent, dependent
upon the age of the donor. The methods and devices described herein
address these problems by improving the numbers of cells in the
stem cell sample and the potential for the stem cells to
differentiate into osteoblasts.
[0037] In some embodiments, the stem cells include mesenchymal stem
cells. Mesenchymal stem cells can be identified by procedures
available to those of skill in the art. For example, mesenchymal
stem cells can be identified via colony forming unit assays (CFU-f)
or via flow cytometry using markers that are typically expressed by
mesenchymal stem cells. Mesenchymal stem cells generally express
such markers as CD271.sup.+, CD105.sup.+, CD73.sup.+, but exhibit a
CD34.sup.- and CD45.sup.- phenotype.
[0038] When bone marrow cells are employed, these cells may be
obtained from iliac crest, femora, tibiae, spine, rib or other
medullary spaces. In some embodiments, the stem cells are from an
autologous fluid (e.g., bone marrow aspirate). Bone marrow aspirate
is a good source of mesenchymal stem cells.
[0039] The stem cells can, in some embodiments, be subjected to a
separation process such as centrifugation, size filtration,
immunomagetic selection, etc., in order to either screen out
"irrelevant" cells, and improve the efficiency of the activation
step, or to preselect for mesenchymal stem cells to facititate bone
formation in implant materials. While it may not be necessary to
separate the cell types and/or purify the mesenchymal stem cells,
it some embodiments it may be desirable.
[0040] When separation of cell types is desired, a biological
sample, for example, comprising bone marrow can be centrifuged to
separate the components of the sample into various fractions based
on density, including a fraction rich in connective tissue growth
promoting components such as mesenchymal stem cells. The fraction
rich in connective tissue growth promoting components can then be
isolated. In addition, the biological sample that is centrifuged
can be free from cell culture medium materials. In some
embodiments, the biological sample that is centrifuged can consist
essentially of tissue material (e.g. bone marrow material
optionally in combination with blood or other tissue material) from
a patient into which the resulting isolated and activated stem
cells will later implanted.
Active Agents
[0041] Active agents are used in the methods and devices described
herein to promote the formation and/or differentiation of stem
cells into osteogenic cells. Such active agents can be, for
example, small molecules, peptides, growth factors, cytokines,
ligands, hormones, and other molecules that regulate growth and
differentiation. The active agent(s) can be captured from an
autologous source, be obtained from a commercial source, or can be
manufactured (e.g., by recombinant procedures).
[0042] Examples of active agents that can be employed include TGF,
FGF, PDGF, BMP, IGF, interleukins, IL-I, IL-11, TGF, NGF, EGF, HGF,
simvastatsin, dexamethasone, oxysterols, sonic hedgehog,
interferon, fibronectin, "RGD" or integrin peptides and/or protein,
keratinocyte growth factor, osteogenic proteins, MSX1, NFKB1,
RUNX2, SMAD1, SMAD2, SMAD3, SMAD4, SOX9, TNF, TWIST1, VDR., AHSG,
AMBN, AMELY, BGLAP, ENAM, MINPP1, STATH, TUFT1, BMP1, COL11A1,
SOX9, ALPL, AMBN, AMELY, BGLAP, CALCR, CDH11, DMP1, DSPP, ENAM,
MINPP1, PHEX, RUNX2, STATH, TFIP11, TUFT1, BGLAP, BMP3, BMP5, BMP6,
COL10A1, COL12A1, COL1A1, COL1A2, COL2A1, COMP, FGFR1, GDF10, IGF1,
IGF2, MSX1, ANXA5, CALCR, CDH11, COMP, DMP1, EGF, MMP2, MMP8,
COL10A1, COL14A1, COL15A1, COL3A1, COL4A3, COL5A1, EGFR, FGF1,
FGF3, IGF1R, TGFB2, VEGFA, VEGFB, COL4A3, CSF3, FLT1, IGF1, IGF1,
IGF2, PDGFA, SMAD3, TGFB1, TGFB2, TGFB3, TGFBR2, VEGFA, VEGFB,
BMP1, CSF2, CSF3, FGFR1, FGFR2, FLT1, GDF10, IGF1, IGF1R, IGF2,
PDGFA, TGFB1, TGFB2, TGFB3, TGFBR1, TGFBR2, VEGFA, VEGFB, AHSG,
SERPINH1, CTSK, MMP10, MMP9, PHEX, AMBN, AMELY, ENAM, STATH, TUFT1,
BGN, COMP, DSPP, GDF10, CDH11, ICAM1, ITGB1, VCAM1, ITGA1, ITGA2,
ITGA3, ITGAM, ITGB1, CD36, COMP, SCARB1, AMH, GDF2 (BMP9), GDF3
(Vgr-2), GDF5 (CDMP-1), GDF6, GDF7, IGFBP3, IL6, INHA (inhibin a),
INHBA (inhibin BA), LEFTY1, LTBP1, LTBP2, LTBP4, NODAL, ACVR1
(ALK2), ACVR2A, ACVRL1 (ALK1), AMHR2, BMPR1A (ALK3), BMPR1B (ALK6),
BMPR2, ITGB5 (integrin B5), ITGB7 (integrin B7), LTBP1, NR0B1,
STAT1, TGFB1I1, TGFBR1, (ALK5) TGFBR2, TGFBR3, TGFBRAP1, CDC25A,
CDKN1A (p21WAF1/p21CIP1), CDKN2B (p15LNK2B), FOS, GSC (goosecoid),
IGFBP3, ITGB5 (integrin B5), ITGB7 (integrin B7), JUN, JUNB, MYC,
SERPINE 1 (PAI-1), TGFB1I1, TSC22D1 (TGFB1I4), TGIF1, DLX2, ID1,
ID2, JUNB, SOX4, STAT1, BAMBI, BMPER, CDKN2B (p15LNK2B), CER1
(cerberus), CHRD (chordin), CST3, ENG (Evi-1), EVI1, FKBP1B, HIPK2,
NBL1 (DAN), NOG, PLAU (uPA), RUNX1 (AML1), SMURF1 and other
molecules that regulate growth and differentiation, as well as
combinations of these factors.
[0043] In some embodiments, the active agents include transforming
growth factor beta (TGF-.beta.), fibroblast growth factor (FGF,
including acid or basic fibroblast growth factor (FBFa or FBFb),
and/or fibroblast growth factor-8 (FGF-8)), platelet-derived growth
factor (PDGF), bone morphogenic protein (BMP) family (such as
BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and/or BMP-7), members of
the insulin growth factor (IGF) family (e.g., insulin like growth
factor-I and/or II), interleukin-I (IL-I), IL-11, simvastatsin,
dexamethasone, oxysterols, sonic hedgehog, interferon, tumor
necrosis factor, nerve growth factor (NGF), fibronectin, "RGD" or
integrin sequences, epidermal growth factor (EGF), hepatocyte
growth factor (HGF), keratinocyte growth factor, osteogenic
proteins (Ops; such as OP-1, OP-2, OP-3), and other molecules that
regulate growth and differentiation, as well as combinations of
these factors.
[0044] Peptide active agents can also be employed that elicit the
same activiation response as the full protein. For example, an
amino acid fragment of a protein or a peptide with a similar action
to the TGF-.beta., FGFb and/or PDGF can be used. Peptide active
agents from any of the active agents described herein or known to
have utility for activating stem cells can be employed. In other
embodiments, a small molecule activator can be employed to activate
the stem cells.
[0045] In many embodiments, members of TGF-.beta. family are
included as active agents in the methods and devices described
herein. The TGF-.beta. family encompasses a group of structurally
related proteins, which affect a wide range of differentiation
processes during embryonic development. Inclusion in the TGF.beta.
family is based on primary amino acid sequence homologies including
conservation of seven cysteine residues. The family includes, for
example, Mullerian inhibiting substance (MIS), which is required
for normal male sex development (Behringer, et al., Nature,
345:167, 1990), Drosophila decapentaplegic (DPP) gene product,
which is required for dorsal-ventral axis formation and
morphogenesis of the imaginal disks (Padgett, et al., Nature,
325:81-84, 1987), the Xenopus Vg-1 gene product, which localizes to
the vegetal pole of eggs (Weeks, et al., Cell, 51:861-867, 1987),
the activins (Mason, et al., Biochem, Biophys. Res. Commun.,
135:957-964, 1986), which can induce the formation of mesoderm and
anterior structures in Xenopus embryos (Thomsen, et al., Cell,
63:485, 1990), and the bone morphogenetic proteins (BMP's, such as
BMP-2 to BMP-15) which can induce de novo cartilage and bone
formation (Sampath, et al., J. Biol. Chem., 265:13198, 1990). The
TGF-.beta. gene products can influence a variety of differentiation
processes, including adipogenesis, myogenesis, chondrogenesis,
hematopoiesis, and epithelial cell differentiation (for a review,
see Massague, Cell 49:437, 1987), which is incorporated herein by
reference in its entirety.
[0046] The proteins of the TGF-.beta. family are initially
synthesized as a large precursor protein, which subsequently
undergoes proteolytic cleavage at a cluster of basic residues
approximately 110-140 amino acids from the C-terminus. The
C-terminal regions of the proteins are all structurally related and
the different family members can be classified into distinct
subgroups based on the extent of their homology. Although the
homologies within particular subgroups range from 70% to 90% amino
acid sequence identity, the homologies between subgroups are
significantly lower, generally ranging from only 20% to 50%. In
each case, the active species appears to be a disulfide-linked
dimer of C-terminal fragments. For most of the family members that
have been studied, the homodimeric species has been found to be
biologically active, but for other family members, like the
inhibins (Ung, et al., Nature, 321:779, 1986) and the TGF-.beta.'s
(Cheifetz, et al., Cell, 48:409, 1987), heterodimers have also been
detected, and these appear to have different biological properties
than the respective homodimers.
[0047] Members of the superfamily of TGF-.beta. genes include
TGF-.beta.3, TGF-.beta.2, TGF-.beta.4 (chicken), TGF-.beta.1,
TGF-.beta.5 (Xenopus), BMP-2, BMP-4, Drosophila DPP, BMP-5, BMP-6,
Vgr1, OP-1/BMP-7, Drosophila 60A, GDF-1, Xenopus Vgf, BMP-3,
Inhibin-.beta.A, Inhibin-.beta.B, Inhibin-.alpha., and MIS. These
genes are discussed in Massague, Ann. Rev. Biochem. 67:753-791,
1998, which is incorporated herein by reference in its
entirety.
[0048] In some embodiments, the member of the family of TGF-.beta.
employed in the devices and methods described herein is
TGF-.beta.3.
[0049] Fibroblast Growth Factors and their Receptors Fibroblast
growth factors (FGFS) comprise a family of evolutionarily conserved
polypeptides involved in a variety of biological processes
including morphogenesis, angiogenesis, and tissue remodeling as
well as in the pathogenesis of numerous diseases (reviewed in
Ornitz, Bioessays 22: 108, 2000, specifically incorporated by
reference herein in its entirety). The various members of this
family stimulate the proliferation of a wide spectrum of cells,
ranging from mesenchymal to epithelial and neuroectodermal origin
in vitro and in vivo. FGFs are expressed in a strict temporal and
spatial pattern during development and have important roles in
patterning and limb formation (Ornitz, Bioessays 22:108, 2000).
[0050] All members of the FGF family share a homology core domain
of about 120 amino acids, where 28 amino acid residues are highly
conserved and six are identical. Structural studies on several FGFs
identified 12 antiparallel .beta. strands each one adjacent to
.beta.-loops comprising the core region, conserved throughout the
family. The core domain comprises the primary FGFR and heparin
binding sites. Receptor binding regions are distinct from heparin
binding regions (reviewed in Ornitz and Itoh, Gen. Biol. 2, 3005.1,
2001).
[0051] In some embodiments, the member of the family of FGF
employed in the devices and methods described herein is FGF-2.
[0052] Platelet-derived growth factor (PDGF) from human platelets
contains two polypeptide sequences--the PDGF-B and PDGF-A
polypeptides (Antoniades, H. N. and Hunkapiller, M., Science
220:963-965, 1983). PDGF-B is encoded by a gene localized on
chromosome 7 (Betsholtz, C. et al., Nature 320:695-699), and PDGF-A
is encoded by the sis oncogene (Doolittle, R. et al., Science
221:275-277, 1983) localized on chromosome 22 (Dalla-Favera, R.,
Science 218:686-688, 1982). The sis gene encodes the transforming
protein of the Simian Sarcoma Virus (SSV), which is closely related
to PDGF-2 polypeptide. The human cellular c-sis also encodes the
PDGF-A chain (Rao, C. D. et al., Proc. Natl. Acad. Sci. USA
83:2392-2396, 1986). Because the two polypeptide chains of PDGF are
coded by two different genes localized in separate chromosomes,
human PDGF may consist of a disulfide-linked heterodimer of PDGF-B
and PDGF-A, or a mixture of the two homodimers (PDGF-BB homodimer
and PDGF-AA homodimer), or a mixture of the heterodimer and the two
homodimers.
[0053] PDGF may be obtained commercially, or from human tissues or
cells, e.g., platelets, by solid phase peptide synthesis, or by
recombinant DNA technology. Mammalian cells in culture infected
with the Simian Sarcoma Virus, which contains the gene encoding the
PDGF-A chain, can synthesize the PDGF-A polypeptide and to process
it into a disulfide-linked homodimer (Robbins et al., Nature
305:605-608, 1983). In addition, the PDGF-A homodimer reacts with
antisera raised against human PDGF and the functional properties of
the secreted PDGF-A homodimer are similar to those of
platelet-derived PDGF. The recombinant PDGF-B homodimer can be
obtained by the introduction of cDNA clones of c-sis/PDGF-B gene
into mouse cells using an expression vector. A c-sis/PDGF-B clone
used for such expression has been obtained from normal human
cultured endothelial cells (Collins, T., et al., Nature
216:748-750, 1985).
[0054] While many active agents have utility for activating
osteogenic stem cells, data generated by the inventors indicates
that in some embodiments TGF-.beta.3, FGF-2 and various forms of
PDGF are useful. In other embodiments, the devices and methods for
activating stem cells include at least TGF-.beta.3 and FGF-2 as
active agents.
[0055] In some embodiments, the active agents are capable of
increasing the expression of bone morphogenetic protein receptor
subunit(s) in the progenitor cells of the stem cell mixture.
Treatment with such active agents helps, for example, to potentiate
the ability of the progenitor cells to respond to endogenous bone
morphogenetic proteins at a site of bone injury or bone surgery.
Such treatment with active agents can also drive the stem cells to
differentiate down an osteoblast pathway.
[0056] Bone morphogenic proteins (BMPs) not only induce bone and
cartilage formation but are multifunctional cytokines having a wide
range of effects on numerous cell types (Hogan et al., Genes Dev.
10:1580-1594 (1996); Reddi et al., Cytokine Growth Factor Rev.
8:11-20 (1997)). BMPs are members of the TGF.beta. superfamily.
There are approximately 15-20 BMPs genes in man, three BMP
receptors, and a number of BMP associated proteins that function as
BMP antagonists (Yamashita et al. Bone 19:569-574 (1996)). BMP
functions through the Smad signal transduction pathway via three
BMP receptors, BMPR-IA, -IB, and II. When a BMP dimer binds the
type II receptor it complexes and phosphorylates the type I
receptor which activates the Smad pathway.
[0057] Exposure of primary osteoblasts to exogenous growth factors
can modulate the expression of these receptors. Singhatanadgit et.
al. (J. Cell Physiol. 209(3): 912-22 (2006)) tested TGF-beta1,
FGF-2, PDGF-AB, and BMP-2 treatment of primary osteoblasts, and
some effects of these growth factors onintracellular receptors to
the cell surface. Yeh et. al. (J. Cell. Physiol. 190(3): 322-31
(2002); J. Cell Physiol. 191(3): 298-309 (2002)) observed a
differential regulation pattern of receptor subunit mRNA in fetal
rat calvarial cells after exposure to OP-1. Xu et. al. (Growth
Factors 24(4): 268-78 (2006)) tested the effects of TGFbeta3 on
BMPR-IB. While the factors involved in the signaling pathway of
BMPs bound to their respective receptors are generally understood,
the regulation and expression patterns of their receptor subunits
has not been fully elucidated. Moreover, researchers have not
appreciated that treatment of stem cells (e.g., bone marrow) with
growth factors for short periods of time, followed by removal of
the growth factors, can stimulate the formation of osteogenic cells
and/or osteogenic precursors.
Activating Stem Cells
[0058] As illustrated herein, stem cells can be osteogenically
activated by transient exposure to active agents. Such activated
stem cells differentiate into osteoprogenitors, osteoblasts and/or
osteoblastic phenotypic cells. Moreover, removal of the active
agents from the activated stem cells yields a mixture of cells that
does not include growth factors and cytokines that may have
unintended side effects when transplanted into a subject. Hence,
there is no need for several days of stem cell culture in a
cocktail of biologically active molecules, which can result in
ongoing pain and immobility for a patient waiting for treatment,
additional surgery for insertion of an implant after initial repair
of a bone injury, contamination of the cultured cells, growth of
undesirable cell types in the stem cell population or bone
aspirate, as well as the additional time and expense of maintaining
the culture and caring for the injured patient.
[0059] Instead, stem cells can be activated for implantation and
stimulation of bone growth by incubation with the active agent(s)
for short time periods. Thus, for example, when a patient is
admitted for treatment of a bone injury or condition, autologous or
allogenic stem cells (e.g., bone marrow aspirate) can be activated
while the patient is undergoing surgery and the activated stem
cells can immediately be implanted (along with a bone graft
substitute, if desired).
[0060] The phrase "activating stem cells" as used herein means that
the stem cells are induced to differentiate into osteogenic
precursor cells, capable of proliferating and subsequently
differentiating into bone-forming cells. Such bone-forming cells
include osteoblasts and osteoblastic progenitors. Bone-forming
cells can be recognized by their expression of osteospecific
markers such as alkaline phosphatase, osteocalcin, osteopontin and
BMP receptors,
[0061] As indicated herein, activation of stem cells can be for
just a short period of time, for example, time periods ranging from
5 minutes to 24 hours. Other optimal time frames for exposing stem
cells to active agent range from 10 minutes to 2 hours, or 15
minutes to 1 hour. In some embodiment, the stem cells are contacted
with one or more active agents for 5 minutes to 1 hour, or the stem
cells are contacted with one or more active agents for 5 minutes to
0.5 hours. As illustrated herein, exposure of bone marrow aspirate
to active agents for just one hour leads to upregulation in the
expression of alkaline phosphatase and BMP receptor subunit(s).
[0062] Thus, one aspect of the invention is a method of making an
implant for promoting bone growth in a mammal. The method involves
exposing stem cells to one or more active agents for 24 hours or
less (e.g., about 5 minutes to 24 hours, or about 10 minutes to 2
hours, or about 15 minutes to 1 hour) to form activated stem cells,
separating the activated stem cells from the one or more active
agents to form an activated stem cell population that is
substantially free of active agents, and mixing the activated stem
cell population that is substantially free of active agents with a
bone graft substitute to thereby make an implant for promoting bone
growth in a mammal.
[0063] The stem cells are exposed to concentrations of one or more
active agents that are sufficient to activate the stem cells to an
osteogenic or osteogenic precursor phenotype. One of skill in the
art can readily determine what such concentrations are, for
example, by observed what concentrations give rise to increases or
upregulation in the expression of alkaline phosphatase and BMP
receptor subunit(s). Example, of appropriate concentrations of
active agents include use of the active agent(s) at concentration
of about 0.01 ng/ml to about 1 .mu.g/ml. In some embodiments, the
active agents are used in concentrations of about 0.1 ng/ml to
about 500 ng/ml, or about 1 ng/ml to about 100 ng/ml.
[0064] As described herein, the stem cells can be from any
convenient source. However, stem cells that have osteogenic
potential or that can be treated (e.g., differentiated) to generate
cells with osteogenic potential are preferred. Sources of stem
cells that can be used in the methods, devices and implants
described herein include bone marrow, adipose tissue, umbilical
cord blood, embryonic yolk sac, placenta, umbilical cord,
periosteum, fetal and adolescent skin, and blood. In some
embodiments, the stem cells are mesenchymal stem cells or a mixture
of cells that include mesenchymal stem cells. The stem cells can be
autologous, allogeneic or from xenogeneic sources. The stem cells
can be embryonic or from post-natal or adult sources. In some
embodiments, the stem cells are an autologous or allogenic bone
marrow aspirate.
[0065] In general, it is not necessary to separate the stem cells
from non-stem cells, or to purify the activated (osteogenic) stem
cells from other cell types. However, if one of skill in the art
wishes to purify stem cells from non-stem cells, or the activated,
osteogenic stem cells from non-activated stem cells and other cell
types, the person of skill in the art can do so by any convenient
procedure. For example, bone marrow can be centrifuged to separate
the components of an aspirate into various fractions based on
density, and a fraction rich in mesenchymal stem cells can be
obtained. The cells can also be subjected to immunopurification
using antibodies that recognize and bind to factors expressed on
the cell surface of activated osteogenic stem cells (e.g., BMP
receptors).
[0066] As indicated herein, the active agents used in the methods
and devices described herein for activating the stem cells can be
any active agent that can activate the osteogenic potential of stem
cells. Examples are recited and illustrated herein.
[0067] When the stem cells are activated, they begin to express
factors characteristic of osteogenic progenitor cells. For example,
as illustrated herein, the levels of alkaline phosphatase, an early
marker of osteoblast differentiation, expressed by primary human
mesenchymal stem cells, are increased. See, for example, FIG. 1, in
which increased alkaline phosphatase expression was observed over
time in mesenchymal stem cells treated with TGF.beta.3 (triangles)
or FGF-2 (squares) for just 1 hour compared to untreated control
cells, indicating that the treated cells exhibited a more potent
differentiation response.
[0068] In some embodiments, stem cell activation using the methods
and devices described herein can also increase the levels of the
BMP receptor subunits. As illustrated herein, the mRNA copy number
of three BMP receptor units (namely BMPR-1A, BMPR-1B and BMPR-II)
in primary mesenchymal stem cells was increased after exposure to
active agents (PDGF BB, FGF b and TGF beta3 at 1 ng/ml, 10 ng/ml
and 100 ng/ml) for just one (1) hour (FIG. 3) or for 24 hours (FIG.
2). Thus, treatment of stem cells (e.g., mesenchymal stem cells)
with active agents for just a short period of time prior to
implantation in a surgical site may potentiate the cells for a more
robust response after implantation to endogenous BMP.
[0069] The activated stem cells are separated from the one or more
active agents to which they have been exposed by any convenient
method to thereby form an activated stem cell population that is
substantially free of active agents. The stem cell population is
substantially free of active agents when a composition containing
the stem cells (e.g., an implant composition) does not exhibit side
effects from the active agent(s) that would preclude administration
of the stem cell composition (or implant composition). Thus, small
amounts of active agents may remain in the activated stem cell
population (or implant composition) so long as the amounts of
active agents are, for example, less than 10 ng/ml, or less than 1
ng/ml, or less than 0.1 ng/ml or less than 0.01 ng/ml.
[0070] Procedures for separating cells from small and large
molecules are available to one of skill in the art. For example,
the stem cells can be washed by suspending the cells in media or
saline and collecting the cells by centrifugation. Several such
washes yield an activated stem cell population that is
substantially free of active agents. In another example, the cells
can be separated from active agents by passing the cells through a
column that retains the active agents but allows the cells to pass
through. Such a column can have a matrix that binds or retains the
active agent(s); for example, the column can be a gel filtration
column, an affinity purification column, or an ion-exchange
column.
[0071] Thus, the active agent(s) can be introduced to the stem cell
source (i.e., bone marrow aspirate) in solution. After a given
incubation time, the activated stem cells are separated from the
active agents by a procedure that involves filtration, gel
filtration, immunoprecipitation, immune-absorption, column
chromatography or a combination thereof. For example, the active
agents can be removed using an antibody or binding protein or
peptide to immobilize the active agents and allow removal or
separation of the active agent from the stem cells. In some
embodiments, active agents in solution may be removed from the
cells via an intraoperative filtration process, such as tangential
flow filtration, with the appropriate molecular weight cutoff so as
to allow for the intraoperative removal of the active agent from
solution.
[0072] In another embodiment, the isolated stem cells are
incorporated into a bone graft substitute and then exposed to the
active agent(s) as described herein. The active agents can be
removed from the complex of the stemcells/bone graft substitute by
any convenient procedure, for example, by several rounds of
sedimentation of the complex of the stemcells/bone graft substitute
with removal of a liquid supernatant wash that contains the active
agent(s).
[0073] In some embodiments, the activated stem cells are separated
from the one or more active agents to which they have been exposed
by using the device described herein.
Devices for Activating Stem Cells
[0074] Another aspect of the invention is a device that activates
stem cells, for example, to differentiate into cells that can
stimulate bone growth (e.g., osteogenic progenitor cells,
osteoblasts and the like). Use of the device in the methods
described herein permits incubation of stem cells, stem cell
mixtures and stem cell compositions (e.g., implant compositions)
with at least one active agent for a time sufficient to activate
the stem cells, and then allows separation of the activated stem
cells from the at least one active agent, to yield activated stem
cells and/or activated stem compositions that are substantially
free of active agents.
[0075] Thus, for example, one aspect of the invention is a device
that includes a solid support and one or more active agents
attached to the solid support. The active agent(s) can be directly
attached to the solid support (e.g., by adsorption or via a
covalent bond) or the active agent(s) can be indirectly attached to
the solid support (e.g., via a linker, antibody, peptide, aptamer,
alkylene chain, biotin-streptavidin, etc.).
[0076] The solid support can be any material to which an active
agent can be directly or indirectly attached where the material
does not bind or adversely interact with stem cells. Thus, the
solid support can be a column matrix material, a filter, an culture
plate, tube or dish, a microtiter plate (or the wells of the
microtiter plate), a bead (e.g., magnetic beads), a disk, and other
materials compatible with stem cells. The solid support can be made
from a variety of materials such as plastic, cellulose, cellulose
derivatives, magnetic particles, nitrocellulose, glass, fiberglass,
latex, and other substrate materials. If desired the solid support
can be coated with a substance that inhibits binding of the stem
cells or that reduces the reactivity of the materials in the solid
support.
[0077] The active agent(s) may be attached to the solid support
using a variety of techniques known to those of skill in the art,
which are amply described in the patent and scientific literature.
In the context of the present invention, the term "attached" or
"attachment" refers to both noncovalent association, such as
adsorption, covalent attachment (which may be via direct linkage
between the active agent and functional groups on the support or
may be via indirect linkage).
[0078] Adsorption onto some solid support materials (e.g., plastic)
may be achieved by contacting the active agent, in a suitable
buffer, with the solid support for a suitable amount of time. The
contact time varies with temperature, but is typically between
about 1 hour and about 1 day. In general, for example, contacting a
well of a plastic microtiter plate (such as polystyrene or
polyvinylchloride) with an amount of active agent ranging from
about 10 ng to about 10 .mu.g, and preferably about 100 ng to about
1 .mu.g, is sufficient to immobilize an adequate amount of the
active agent.
[0079] Covalent attachment of active agent to a solid support may
generally be achieved by first reacting the support with a
bifunctional reagent that will react with both the support and a
functional group, such as a hydroxyl or amino group, on the active
agent. For example, the active agent may be covalently attached to
supports having an appropriate polymer coating using benzoquinone
or by condensation of an aldehyde group on the support with an
amine and an active hydrogen on the binding partner (see, e.g.,
Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).
The bifunctional reagent can be a cross linking agent, a linker
with two functional groups, a peptide, an alkylene chain, an
aptamer, or other bifunctional molecules.
[0080] The active agent may be non-covalently attached to the solid
support via a binding agent such as an antibody where the antibody
is attached or adsorbed to the solid support and binds the active
agent non-covalently. Alternatively, the active agent may be
non-covalently attached to the solid support via
biotin-streptavidin, where either the biotin or the streptavidin is
attached to the solid support. When the streptavidin is attached to
the solid support, biotin is attached to the active agent. The
biotin linked to the active agent will bind to the streptavidin,
thereby immobilizing the active agent onto the solid support.
[0081] In one example, a column matrix or filter material is
included, to which the active agent is covalently bound. The stem
cells (e.g., bone marrow aspirate) can then be passed through, or
incubated in, this substrate for some period of time, for example
time frames ranging from 5 minutes to 24 hours with optimal time
frames ranging from 15 minutes to 1 hour.
[0082] This exposure to the active agent(s) has been shown to
increase BMP receptor subunit and alkaline phosphatase expression.
For example, FIGS. 2 and 3 illustrate that three active agents
(PDGF BB, FGF b, and TGF-.beta.3) upregulate the BMPR-IB subunit,
and at specific concentrations, the BMPR-IA and BMPR-II subunits
are increased over background. Exposure to such active agents
serves to activate or trigger the mesenchymal stem cells to enhance
the osteoblast phenotype.
[0083] The active agent can therefore be FGF b, TGF-.beta.3 and/or
PDGF BB. In other embodiments, the active agents on the solid
support can include BMP polypeptides. Other active agents can be
attached onto the solid support as well, for example, any of the
active agents listed herein. The active agent can be from an
autologous source, and allogenic or may be manufactured (e.g. via
recombinant technology).
[0084] Several or many active agents can be attached onto the same
solid support. For example, it is generally accepted that BMPs are
more powerful at heterodimers (i.e. BMP-2/BMP-7 combination) than
in the homodimeric formulation in which they are currently
commercially available. Consequently, for the device and method
embodiments described herein the active agents can be attached to a
solid support and subsequently separated from the BMA prior to
reimplantation. Thus, there is added flexibility as to the
possibility of using multiple active agents that can be employed
for the current device and in the methods described herein.
[0085] In some embodiments, the device is adapted to expose an
implant composition to one or more active agents for a selected
time (e.g., 24 hours or less, and/or other times described herein)
in order to activate stem cells in the implant composition. Such
devices are adapted to allow incubation of the implant composition
with at least one of the active agents described herein, and then
to allow separation of the active agent(s) from the implant
composition. Thus, the implant material (e.g., bone graft
substitute) and the stem cells within the implant composition can
be retained in the device while the active agents are removed. Use
of the device yields an implant composition that is substantially
free of active agents.
[0086] The devise can therefore include a means for separating the
stem cells and/or bone graft substitute from the active agent(s).
For example, the devise can include a filter that excludes larger
materials such as the stem cells and/or the bone graft substitute,
but that permits the active agent(s) in solution to pass through
the filter material, thereby separating the stem cells and/or bone
graft substitute from the active agents. After incubation of the
implant composition with the active agent(s), the incubation
chamber holding implant composition and the active agent(s) can be
drained and rinsed with an appropriate medium (e.g., a buffer,
saline, buffered saline, culture medium). Thus, the device can
yield a stem cell composition (e.g., an implant composition) that
contains activated stem cells and that is substantially free of
active agent(s).
[0087] The devices described herein can further include a timer for
controlling the time for incubating the stem cells with the at
least one active agent. For example, the timer can trigger
separation of the at least one active agent from the stem cell
after the incubating step (i). Thus, for example, the timer can
initiate drainage or removal of a solution containing at least one
active agent. In addition, or alternatively, the timer can initiate
addition of a solution to wash the stem cells and/or bone graft
substitute materials. In some embodiments, the device with the
timer controls the time for incubating the stem cells with the at
least one active agent to 24 hours or less. In other embodiments,
the device with the timer controls the time for incubating the stem
cells with the at least one active agent to 5 minutes to 1
hour.
Implants
[0088] In one embodiment, activated stem cells (e.g., bone marrow
aspirate) prepared as described herein are further combined with
synthetic bone graft substitutes, such as beta tricalcium phosphate
(beta-TCP) to form an implant composition. In one example, a
composite of activated stem cells (e.g., activated bone marrow
aspirate) and synthetic bone graft substitute is used in place of a
tissue graft. Stem cells can be combined with the synthetic bone
graft substitute either before or after the stem cells are
activated by exposure to the active agent(s). However, prior to
implantation, the stem cells are exposed or contacted with active
agent(s) so that the stem cells in the implant composition are
activated stem cells.
[0089] The bone graft substitute can be a solid material which,
when placed in, or in juxtaposition to, living bone under suitable
conditions, serves as a scaffold for the formation of new bone by
bone-forming activated stem cells. Examples of bone graft
substitutes that can be employed are described in U.S. Pat. Nos.
5,383,931; 6,461,632; 7,044,972; 7,494,950; and US application
publication number 20060008504, which are each specifically
incorporated by reference herein in their entireties.
[0090] The bone graft substitute can include a calcium
salt-containing component. For example, the bone graft substitute
can include monocalcium phosphate monohydrate, .alpha.-tricalcium
phosphate, calcium carbonate, demineralized bone, sodium phosphate
salt and, optionally, a polymer. The polymer can be a resorbable
polymer. In some embodiments, the polymer includes homopolymer or
copolymer fibers having a fiber length of not more than about 15
mm, an aspect ratio from about 50:1 to about 1000:1, or both (and
optionally also include continuous reinforcing fibers).
[0091] The polymer can be collagen, gelatin, hyaluronic acid, a
hyaluronate salt, hydroxypropylcellulose (HPC),
carboxymethylcellulose (CMC), hydroxypropyl methylcellulose (HPMC),
hydroxyethylcellulose (HEC), xantham gum, guar gum, and/or
alginate.
[0092] Examples of bone graft substitutes that can be employed
include, without limitation, beta-TCP (e.g., chronOS made by
Synthes), collagen, bioglass (e.g., 45S5 BioGlass), BioOss (calcium
phosphate-based bone graft substitute), Pepgen P-15 (synthetic P-15
peptide bound to a natural form of hydroxylapatite) and AlloGraft
(demineralized bone matrix, allograft-based bone graft
substitute).
[0093] In general, the activated stem cells are incubated or mixed
with the bone graft substitute to form an implant composition. In
some embodiments, the implant composition is a putty; in other
embodiments the implant composition is sufficiently fluid to flow
through a syringe needle. For example, the implant composition can
have a ratio of liquid components to solid components from about
0.3 to about 0.0 or about 0.41 to about 0.55.
[0094] Another embodiment includes use of an active agent in
combination with a stem cell source (i.e. BMA), ex vivo, for a
short period of time (i.e., 5 min to 60 min) in order to stimulate
the stem cells down an osteoblastic pathway. After the ex vivo
incubation, the combination of stem cells and active agent are
implanted together.
Methods of Treatment
[0095] The implant compositions containing the activated stem cells
and the bone graft substitute are useful for repairing and treating
bone injuries, disorders and conditions. Such bone injuries,
disorders or conditions are characterized by bone loss (osteopenia
or osteolysis) or by bone damage or injury. Such bone injuries,
disorders and conditions include but are not limited to broken
bones, bone defects, bone transplant, bone grafts, bone cancer,
joint replacements, joint repair, fusion, facet repair, bone
degeneration, dental implants and repair, bone defects resulting
from disease (e.g., arthritis), bone defects resulting from
reconstructive surgeries, and other conditions associated with bone
and boney tissue. Examples of bone defects include but are not
limited to a gap, deformation or a non-union fracture in a bone.
Examples of bone degeneration include but are not limited to
osteopenia or osteoporosis. In one embodiment, the bone defect is
due to dwarfism. The compositions are also useful for joint
replacement or repair wherein the joint is vertebral, knee, hip,
tarsal, phalangeal, elbow, ankle, sacroiliac or other
articulating/non-articulating joint.
[0096] The implant composition can be administered by pressing or
incorporating the composition into a bone site, or by injection of
the implant composition. When administered by injection, the
syringe may have a needle with a gauge of from about 12 to about 18
where the maximum injection pressure employed is not more than
about 40 pounds, In one embodiment, the composition also includes
continuous reinforcing fibers.
[0097] The following nonlimiting Examples further illustrate
certain aspects of the invention.
Example 1
Materials and Methods
[0098] The following materials and methods were used to develop
certain aspects of the invention.
Differentiation of Cells
[0099] Human mesenchymal stem cells (Lonza, Walkersville, Md.) at
passage 3 were seeded in basal medium (Stem Cell Technologies,
Vancouver, Canada) at a density of 6.times.10.sup.4 cells/35 mm
well and incubated for 2 days at 37.degree. C. Cells were rinsed
with PBS, activated with 100 ng/ml of growth factor (FGF-2 or
TGF.beta.3, R&D Systems, Minneapolis, Minn.) in fresh basal
medium for 1 hour, rinsed with PBS and then incubated in either
fresh basal medium or osteogenic differentiation medium (Stem Cell
Technologies, Vancouver, Canada) for 7-14 days at 37.degree. C.
Real time PCR
[0100] Total RNA was prepared from cells treated with various
active agents using RNeasy Plus Mini Kit and QIA shredder Mini Spin
columns (Qiagen). Total RNA was also prepared from untreated cells
as a control. cDNA was generated using random hexamer and Oligo dT
following the TaqMan Reverse Transcription Kit (Applied
Biosystems). Primer and probe sets for real-time were as
follows:
TABLE-US-00001 huBMPR1A_2 fwd (SEQ ID NO: 1):
5'-TAACCAGTATTTGCAACCCAC ACT-3'. huBMPR1A_2 rev (SEQ ID NO: 2):
5'-GAGCAAAACCAGCCATCGAA-3'. huBMPR1A_2 Probe (SEQ ID NO: 3): 5'-CCC
CCT GTT GTC ATA GGT CCG TTT TTT GAT-3' (FAM/TAMRA). huBMPR1B_1 fwd
(SEQ ID NO: 4): 5'-CCA AAG GTC TTG CGT TGT AAA TG-3'. huBMPR1B_1
rev (SEQ ID NO: 5): 5'-CAT CGT GAA ACAATA TCC GTC TGT-3'.
huBMPR1B_1 Probe (SEQ ID NO: 6): 5'-CCA CCA TTG TCC AGA AGA CTC AGT
CAA CAA-3' (FAM/TAMRA) huBMPR2_2 fwd (SEQ ID NO: 7): 5'-TGC CCT GGC
TAC CAT GGA-3' huBMPR2_2 rev (SEQ ID NO: 8): 5'-CGC ACA TAG
CCGTTCTTGATT-3' huBMPR2_2 Probe (SEQ ID NO: 9): 5'-TCA GCA CTG CGG
CTG CTT CGC-3' (FAM/TAMRA)
Samples were run on an Applied Biosystems 7500 Fast Real-Time PCR
System as a multiplex reactions with beta 2 microglobulin
endogenous controls (VIC/TAMRA) (Applied Biosystems).
Alkaline Phosphatase Assay
[0101] Human mesenchymal stem cells (Lonza, Walkersville, Md.) at
passage 3 were seeded in basal medium (Stem Cell Technologies,
Vancouver, Canada) at a density of 6.times.10.sup.4 cells/35 mm
well and incubated for 2 days at 37.degree. C. Cells were rinsed
with PBS, activated with 100 ng/ml of growth factor (FGF-2 or
TGF.beta.3, R&D Systems, Minneapolis, Minn.) in fresh basal
medium for 1 hour, rinsed with PBS then incubated in either fresh
basal medium or osteogenic differentiation medium (Stem Cell
Technologies, Vancouver, Canada) for 7-14 days at 37.degree. C. At
the time of assay, cells were rinsed twice with PBS, harvested in
100 .mu.l/35 mm well lysis buffer and frozen/thawed twice in liquid
nitrogen. Alkaline phosphatase activity was determined by
incubation of 20 .mu.l lysate with 20 .mu.l 1 mg/ml p-nitrophenyl
phosphate for 3 minutes and measurement of the resulting
luminescence at 405 nm. Alkaline phosphatase activity was
normalized according to cell content, as determined by CyQuant
(Invitrogen, Carlsbad, Calif.) DNA quantification.
Immobilization
[0102] For initial studies, active agents (e.g. growth factors)
were immobilized onto microtiter wells as follows.
[0103] Biotinylated anti-growth factor antibody (500 ng) (R&D
Systems, Minneapolis, Minn.) was incubated in 200 .mu.l PBS/well of
a streptavidin-coated 96-well plate for 30 minutes, rinsed 3 times
with PBS and then bound to increasing quantities of growth factor
in 200 .mu.l PBS for 30 minutes. After 3 rinses with PBS, the
presence of the growth factor was detected by incubation with 1
.mu.g of a second, unlabeled anti-growth factor antibody in 200
.mu.l PBS with 0.1% BSA for 30 minutes. The wells were washed three
times with PBS, incubated with 1 .mu.g of horseradish
peroxidase-conjugated secondary antibody in 200 .mu.l PBS with 0.1%
BSA for 30 minutes, and rinsed again with PBS three times. The
immobilized antibody-growth factor-secondary antibody was then
incubated with 200 .mu.l enhanced chemiluminescent substrate for 1
minute. Quantification of the amount of immobilized horseradish
peroxidase-conjugated secondary antibody was performed by
measurement of chemiluminescent emission at visible
wavelengths.
Activity Assays
[0104] To determine the bioactivity of tethered active agents prior
to the stem cell activation step, the following assays were formed.
For studies involving FGF-2, Swiss Albino 3T3 cells (ATCC,
Manassas, Va.) were seeded in 10% serum basal medium at a density
of 4.times.10.sup.5/35 mm well, incubated for 1 day at 37.degree.
C., rinsed 3 times with PBS and then synchronized in 0.5% serum
basal medium for 1 day at 37.degree. C. FGF2 (R&D Systems,
Minneapolis, Minn.) and a 50 fold excess of biotinylated anti-FGF2
antibody (R&D Systems, Minneapolis, Minn.) were complexed in
0.5% serum basal medium for 15 minutes. The synchronized cells were
rinsed 3 times with PBS then treated with the FGF2/biotinylated
anti-FGF2 antibody complex for 30 minutes at 37.degree. C. At the
endpoint of the assay, cells were rinsed 3 times with PBS,
harvested in SDS sample buffer and heated for 10 minutes at
90.degree. C. The resulting lysates were fractionated by SDS-PAGE,
transferred to a PVDF membrane and sequentially probed with a
1:10000 dilution of anti-phospho-ERK (Cell Signaling Technology,
Beverly, Mass.) antibody, followed by a 1:10000 dilution of a
horseradish peroxidase-conjugated secondary antibody (Santa Cruz
Biotechnology, Santa Cruz, Calif.). Horseradish peroxidase activity
was determined by exposure to enhanced chemiluminescent substrate
for 1 minute, visualized by CCD camera and densitometrically
quantified using ImageJ analysis program.
[0105] For the TGF.beta.3 activity assay, Mv1Lu mink lung cells
(ATCC, Manassas, Va.) were seeded in basal medium at a density of
4.times.10.sup.3 cells/well of a 96-well plate and incubated for 1
day at 37.degree. C. TGF.beta.3 (R&D Systems, Minneapolis,
Minn.) and a 50 fold excess of biotinylated anti-TGF.beta.3
antibody (R&D Systems, Minneapolis, Minn.) were complexed in
basal medium for 15 minutes. After 3 rinses with PBS, cells were
treated with the TGF.beta.3/biotinylated anti-TGF.beta.3 antibody
complex for 3 days at 37.degree. C. At the time of assay,
TGF.beta.3 treatment medium was supplemented with CellTiter-Glo ATP
detection reagent (Promega, Madison, Wis.), incubated 5 minutes and
cell number was quantified by measurement of chemiluminescent
emission at visible wavelengths.
Example 2
Results
[0106] Primary human mesenchymal stem cells are capable of
differentiating down an osteoblastic lineage. This is demonstrated
in vitro by culturing cells in an osteogenic cocktail containing,
but not limited to, dexamethasone, ascorbic acid and
.beta.-glycerophosphate. Under these culture conditions cells show
an upregulation of osteoblast differentiation markers, the most
common of which is the early marker alkaline phosphatase.
[0107] FIG. 1 shows a slight upregulation in alkaline phosphatase
activity in the untreated mesenchymal stem cells (circles) from
days 10 to 12, as expected. However, when the mesenchymal stem
cells were pretreated with TGF.beta.3 (triangles) or FGF-2
(squares) for 1 hour, followed by removal of the agents and culture
in differentiation media, the level of alkaline phosphatase
activity increased significantly 2-3 fold.
[0108] These data indicate that just a 1 hour treatment of
mesenchymal stem cells with either TGF.beta.3 or FGF-2 on day 0 can
impact osteoblast differentiation such that the markers of
osteoblast formation are upregulated 10+ days post treatment.
[0109] Mesenchymal stem cells respond to bone morphogenetic
proteins both in vitro and in vivo to induce an osteoblast
phenotype. BMPs act via a receptor complex made up of three
subunits, BMPR-IA, BMPR-IB, and BMPR-II. When MSCs were treated
with selected active agents (PDGF-BB, TGFbeta3, and FGF-2) for 24
hours, the relative copy number of the BMPR-IB gene increased
significantly over untreated cells. However, FIG. 3 shows that a
similar response was observed after just 1 hour of treatment.
[0110] These data indicate that an intraoperative time frame of
just 1 hour is sufficient to treat MSCs with an active agent such
that the level of receptors for the BMP ligand can be increased
over untreated cells. The presence of increased levels of BMP
receptors on mesenchymal stem cells potentiates these cells for a
BMP-2 osteogenic response in vivo and results in more robust bone
healing.
[0111] All patents and publications referenced or mentioned herein
are indicative of the levels of skill of those skilled in the art
to which the invention pertains, and each such referenced patent or
publication is hereby specifically incorporated by reference to the
same extent as if it had been incorporated by reference in its
entirety individually or set forth herein in its entirety.
Applicants reserve the right to physically incorporate into this
specification any and all materials and information from any such
cited patents or publications.
[0112] The specific methods and compositions described herein are
representative of preferred embodiments and are exemplary and not
intended as limitations on the scope of the invention. Other
objects, aspects, and embodiments will occur to those skilled in
the art upon consideration of this specification, and are
encompassed within the spirit of the invention as defined by the
scope of the claims. It will be readily apparent to one skilled in
the art that varying substitutions and modifications may be made to
the invention disclosed herein without departing from the scope and
spirit of the invention. The invention illustratively described
herein suitably may be practiced in the absence of any element or
elements, or limitation or limitations, which is not specifically
disclosed herein as essential. The methods and processes
illustratively described herein suitably may be practiced in
differing orders of steps, and that they are not necessarily
restricted to the orders of steps indicated herein or in the
claims. As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural reference unless the
context clearly dictates otherwise. Thus, for example, a reference
to "an antibody" includes a plurality (for example, a solution of
antibodies or a series of antibody preparations) of such
antibodies, and so forth. Under no circumstances may the patent be
interpreted to be limited to the specific examples or embodiments
or methods specifically disclosed herein. Under no circumstances
may the patent be interpreted to be limited by any statement made
by any Examiner or any other official or employee of the Patent and
Trademark Office unless such statement is specifically and without
qualification or reservation expressly adopted in a responsive
writing by Applicants.
[0113] The terms and expressions that have been employed are used
as terms of description and not of limitation, and there is no
intent in the use of such terms and expressions to exclude any
equivalent of the features shown and described or portions thereof,
but it is recognized that various modifications are possible within
the scope of the invention as claimed. Thus, it will be understood
that although the present invention has been specifically disclosed
by preferred embodiments and optional features, modification and
variation of the concepts herein disclosed may be resorted to by
those skilled in the art, and that such modifications and
variations are considered to be within the scope of this invention
as defined by the appended claims and statements of the
invention.
[0114] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
invention. This includes the generic description of the invention
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein.
[0115] Other embodiments are within the following claims. In
addition, where features or aspects of the invention are described
in terms of Markush groups, those skilled in the art will recognize
that the invention is also thereby described in terms of any
individual member or subgroup of members of the Markush group.
Sequence CWU 1
1
9124DNAHomo sapiens 1taaccagtat ttgcaaccca cact 24220DNAHomo
sapiens 2gagcaaaacc agccatcgaa 20330DNAHomo sapiens 3ccccctgttg
tcataggtcc gttttttgat 30423DNAHomo sapiens 4ccaaaggtct tgcgttgtaa
atg 23524DNAHomo sapiens 5catcgtgaaa caatatccgt ctgt 24630DNAHomo
sapiens 6ccaccattgt ccagaagact cagtcaacaa 30718DNAHomo sapiens
7tgccctggct accatgga 18821DNAHomo sapiens 8cgcacatagc cgttcttgat t
21921DNAHomo sapiens 9tcagcactgc ggctgcttcg c 21
* * * * *