U.S. patent application number 15/177708 was filed with the patent office on 2017-06-08 for methods of generating, repairing and/or maintaining connective tissue in vivo.
This patent application is currently assigned to Mesoblast, Inc.. The applicant listed for this patent is Peter Ghosh. Invention is credited to Peter Ghosh.
Application Number | 20170157181 15/177708 |
Document ID | / |
Family ID | 40340880 |
Filed Date | 2017-06-08 |
United States Patent
Application |
20170157181 |
Kind Code |
A1 |
Ghosh; Peter |
June 8, 2017 |
METHODS OF GENERATING, REPAIRING AND/OR MAINTAINING CONNECTIVE
TISSUE IN VIVO
Abstract
This invention relates to a method for generating, repairing
and/or maintaining connective tissue in a subject. In one
embodiment, the invention relates to a method for generating,
repairing and/or maintaining cartilage tissue in a subject. The
present invention also relates to a method of treating and/or
preventing a disease in a subject arising from degradation and
inflammation of connective tissue.
Inventors: |
Ghosh; Peter; (New South
Wales, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ghosh; Peter |
New South Wales |
|
AU |
|
|
Assignee: |
Mesoblast, Inc.
New York
NY
|
Family ID: |
40340880 |
Appl. No.: |
15/177708 |
Filed: |
June 9, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12452767 |
Apr 5, 2010 |
9381216 |
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PCT/AU2008/001137 |
Aug 6, 2008 |
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15177708 |
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61133111 |
Jun 25, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 17/06 20180101;
A61P 25/02 20180101; A61P 25/00 20180101; C12N 5/0663 20130101;
A61K 2300/00 20130101; A61K 31/726 20130101; A61K 31/737 20130101;
A61P 19/02 20180101; A61P 25/04 20180101; A61P 43/00 20180101; A61P
21/00 20180101; A61K 35/28 20130101; A61K 35/545 20130101; A61K
38/14 20130101; A61P 19/00 20180101; A61K 31/728 20130101; A61P
29/00 20180101; C12N 2501/905 20130101; A61K 2035/124 20130101;
A61P 19/04 20180101; A61K 9/4866 20130101; A61P 1/04 20180101; A61K
47/36 20130101; A61K 31/728 20130101; A61K 2300/00 20130101; A61K
35/28 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 35/545 20060101
A61K035/545; A61K 31/726 20060101 A61K031/726; A61K 35/28 20060101
A61K035/28; A61K 31/728 20060101 A61K031/728; A61K 47/36 20060101
A61K047/36; A61K 9/48 20060101 A61K009/48; A61K 31/737 20060101
A61K031/737; A61K 38/14 20060101 A61K038/14 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 6, 2007 |
AU |
2007904212 |
Claims
1-15. (canceled)
16. A therapeutic composition comprising a population of human
cells enriched for STRO-1.sup.bright mesenchymal precursor cells
(MPCs) or culture-expanded multipotent progeny thereof; and
chondroprotective agent.
17. The therapeutic composition of claim 16, wherein the
chondroprotective agent comprises an chondroprotective agent
selected from the group consisting of: pentosan polysulfate,
glycosaminoglycan polysufate ester, glyciamino-glycan-peptide
complex, and Hyaluronic Acid (HA).
18. The therapeutic composition of claim 17, wherein the
chondroprotective agent comprises HA.
19. The therapeutic composition of claim 16, further comprising a
cryoprotectant.
20. The therapeutic composition of claim 16, wherein the MPCs carry
at least one additional marker selected from the group of surface
markers consisting of THY-1, VCAM-1, STRO-2, TNAP, and CD146.
21. The therapeutic composition of claim 20, wherein the MPCs carry
the marker TNAP.
22. The therapeutic composition of claim 16, further comprising an
in situ-polymerizable gel.
23. The therapeutic composition of claim 22, wherein the in
situ-polymerizable gel comprises alginate.
24. The therapeutic composition of claim 16, wherein the population
is encapsulated.
25. The therapeutic composition of claim 16, wherein the population
is an allogeneic population.
26. The therapeutic composition of claim 16, wherein the population
is an autologous population.
27. The therapeutic composition of claim 16, wherein the population
is a genetically modified population.
28. The therapeutic composition of claim 27, wherein the cells in
the genetically modified population comprise an exogenous
polynucleotide that encodes a selective marker, a cytokine, or a
chemokine.
29. The therapeutic composition of claim 16, wherein the
STRO-1.sup.bright cells are STRO-1.sup.bright cells enriched from
bone marrow.
Description
FIELD OF THE INVENTION
[0001] This invention relates to a method for generating, repairing
and/or maintaining connective tissue in a subject. The present
invention also relates to a method of treating and/or preventing a
disease in a subject arising from degradation and inflammation of
connective tissue.
BACKGROUND OF THE INVENTION
[0002] Non-hematopoietic progenitor cells that reside in the body
and give rise to multipotential cells when isolated are referred to
as Mesenchymal Precursor Cells (MPCs). More specifically, purified
MPCs are capable of forming very large numbers of multipotential
cell colonies.
[0003] Simmons et al. (1994) describes enrichment of MPCs from
freshly harvested bone marrow cells by selecting for cells that
express the STRO-1 cell surface marker. As explained by the authors
at pages 272-273, it is known that bone marrow cells contain a
proportion of MPCs that are capable of giving rise to CFU-F. These
CFU-F in turn are capable of giving rise under appropriate
conditions to a broad spectrum of fully differentiated connective
tissue, including cartilage, bone, adipose tissue, fibrous tissue
and myelosupportive stroma.
[0004] MPCs and CFU-F are typically present at a very low incidence
in bone marrow cells (typically between 0.05%-0.001%) and this
rarity has been a major limitation to their study in the past. An
important finding discussed by Simmons et al. (1994) was the
identification that these MPCs could be enriched from freshly
isolated bone marrow cells to some extent by selecting for STRO-1
positive cells. In particular, the selection of STRO-1 positive
cells enabled isolation of MPCs (and resultant CFU-F) free of
contaminating hemopoietic progenitors.
[0005] WO 01/04268 provided a further important advance in the
enrichment of MPCs by identifying a subpopulation within this
fraction of STRO-1 positive cells that contains MPCs. In
particular, WO 01/04268 describes the sorting of the STRO-1
positive cell population into three subsets: STRO-1.sup.dull,
STRO-1.sup.intermediate and STRO-1.sup.bright. Clonogenic assays
for CFU-F in the different sorted subpopulations demonstrated that
the vast majority of the MPCs are contained within the
STRO-1.sup.bright fraction.
[0006] WO 2004/085630 discloses for the first time that MPCs are
present in perivascular tissue. One of the benefits of this finding
is that it greatly expands the range of source tissues from which
MPCs can be isolated or enriched and there is no longer an
effective restriction on the source of MPCs to bone marrow. The
tissues from which MPCs can be isolated according to the methods
described in WO 2004/085630 include human bone marrow, dental pulp,
adipose tissue, skin, spleen, pancreas, brain, kidney, liver and
heart. The MPCs isolated from perivascular tissue are positive for
the cell surface marker 3G5. They can therefore be isolated by
enriching for cells carrying the 3G5 marker, or by enriching for an
early developmental surface marker present on perivascular cells
such as CD146 (MUC18), VCAM-1, or by enriching for high level
expression of the cell surface marker STRO-1.
[0007] The avascular connective tissues are generally located at
anatomical sites within the musculoskeletal system that require
appreciable movement. These freely movable joints are responsible
for the majority of articulations in mammals. In synovial joints
the contact surfaces of two opposing bones are covered by hyaline
cartilages which glide effortlessly over each other because of the
presence of a low friction lubricant in synovial fluid produced by
the cells lining the joint capsule which overlays and connects the
long bones. In the spinal column articulation is achieved by
connection of the rigid vertebral bones by means of a flexible
fibrocartilagenous ring (the annulus fibrosus) that encapsulates a
hydrated gelatinous mass (the nucleus pulposus), populated by
chondrocyte like cells similar to those present in hyaline
cartilage. Irrespective of the type and location of these avascular
connective tissue they all contain cells which synthesise an
extracellular matrix which is rich in highly negatively charged
proteoglycans, which imbibe water molecules together with the
fibrous protein, type II collagen, which confers high tensile
strength.
[0008] Avascular connective tissues such as hyaline cartilage, the
inner two thirds of the meniscus and the intervertebral disc have
limited repair capabilities and when injured may respond by the
production of a functionally inferior fibrocartilagenous scar
tissue. Through a multitude of factors, dominated by aging,
genetics, hormonal status and physical injury these avascular
connectives often fail leading to the widespread clinical problems
of disc degeneration, back pain and osteoarthritis.
[0009] Current medical therapies normally used to treat the
symptoms arising from the failure of these connective tissues, for
the most part, do little to redress the underlying pathology
responsible for producing the symptoms and in many instances may
even exacerbate the problem by down regulating the capacity of the
resident cells to synthesis the structural components of the tissue
extracellular matrix. Ideally, therapeutic treatments should be at
least chondroprotective but even provide the conditions which
enhance matrix biosynthesis and effect repair and restoration of
the injured connective tissues.
SUMMARY OF THE INVENTION
[0010] The present inventors have now made the surprising finding
that intra-articular administration of MPCs provides a
chondroprotective effect in joints with pre-existing
osteoarthritis, and leads to generation and growth of cartilage
tissue in synovial joints and in the nucleus pulposus of the
intervertebral discs. This finding indicates that MPCs or their
progeny, or supernatant or soluble factors derived from these MPCs,
can be used to protect or repair damaged connective tissues as well
as generate new functional tissue at sites of degeneration or
injury.
[0011] Accordingly, the present invention provides a method of
treating and/or preventing a disease in a subject arising from
degradation and/or inflammation of connective tissue, the method
comprising administering to the subject MPCs and/or progeny cells
thereof and/or soluble factors derived therefrom.
[0012] In one embodiment of the invention, the connective tissue is
rich in proteoglycans. The connective tissue may be cartilage, for
example, hyaline cartilage. In another embodiment, the disease
results in a defect in the cartilage.
[0013] In another embodiment, the method comprises administering to
the subject MPCs and/or progeny cells thereof and/or soluble
factors derived therefrom, wherein the MPCs and/or progeny cells
and/or soluble factors are not directly administered into the
defect.
[0014] For example, administration may me made into a joint space
in order to treat or prevent defects in the cartilage on the
articular surfaces of bones that form that joint.
[0015] Similarly, administration may be made into an invertebral
disc space in order to treat or prevent defects in the surrounding
discs. In another example, administration is made intravenously at
a site near the cartilage defect.
[0016] The MPCs and/or progeny cells and/or soluble factors may be
administered by intra-articular injection. The intra-articular
injection may be made into any joint of the body which is near to a
site of a cartilage defect, or a potential cartilage defect. For
example, the intra-articular injection may be made into a knee
joint, hip joint, ankle joint, shoulder joint, elbow joint, wrist
joint, hand or finger joint or a joint of the foot, or an
invertebral disc joint.
[0017] In another embodiment of the invention, administration of
the MPCs and/or progeny cells and/or soluble factors results in
preservation or generation of cartilage that is rich in
proteoglygans and type II collagen. An example of a cartilage that
is rich in proteoglycans and type II collagen is hyaline cartilage.
Preferably the cartilage preserved or generated by the method of
the present invention is not fibrocartilage, which is rich in type
I collagen, very low in type II collagen and contains less
proteoglycan than hyaline cartilage.
[0018] Examples of diseases "arising from degradation and/or
inflammation of connective tissue" include, but are not limited to,
tendonitis, back pain, rotary cuff tendon degradation, Carpal
tunnel syndrome, DeQuervain's syndrome, degenerative cervical
and/or lumber discs, intersection syndrome, reflex sympathetic
dystrophy syndrome (RSDS), stenosing tenosynovitis, epicondylitis,
tenosynovitis, thoracic outlet syndrome, ulnar nerve entrapment,
radial tunnel syndrome, repetitive strain injury (RSI). Examples of
diseases that are associated with degradation and/or inflammation
of hyaline cartilage include, but are not limited to arthritis such
as osteoarthritis, rheumatoid arthritis, psoriatic arthritis, and
seronegative arthritis, arthritis associated with inflammatory
bowel disease or ankylosing spondylitis and degenerate invertebral
disc disorders.
[0019] In another preferred embodiment, the method further
comprises administering hyaluronic acid (HA). HA can be
administered in the same or different composition as the cells,
supernatant and/or factor(s).
[0020] The present invention also provides a composition comprising
MPCs and/or progeny cells thereof and hyaluronic acid.
[0021] The results presented herein indicate for the first time
that soluble factors released by the implanted cultured MPCs are
supportive of connective tissue protection, generation and
growth.
[0022] Accordingly, the present invention also provides a
composition comprising;
[0023] i) supernatant, or one or more soluble factors, derived from
mesenchymal precursor cells (MPCs) and/or progeny cells thereof,
and
[0024] ii) hyaluronic acid.
[0025] In a further aspect, the present invention provides for the
use of supernatant, or one or more soluble factors, derived from
mesenchymal precursor cells (MPCs) and/or progeny cells thereof for
treating and/or preventing a disease in a subject arising from
degradation and/or inflammation of connective tissue.
[0026] The present invention is applicable to a wide range of
animals. For example, the subject may be a mammal such as a human,
dog, cat, horse, cow, or sheep. In one embodiment the subject is a
human.
[0027] Throughout this specification the word "comprise", or
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated element, integer or step, or
group of elements, integers or steps, but not the exclusion of any
other element, integer or step, or group of elements, integers or
steps.
[0028] The invention is hereinafter described by way of the
following non-limiting Examples and with reference to the
accompanying figures.
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIG. 1. Means.+-.SD of femoral and tibial cartilage
morphology scores 12 weeks post-meniscectomy for joints injected
with Hyaluronan (HA) or HA plus different doses of Mesenchymal
Precursor Cells (MPC).
[0030] FIG. 2. Means.+-.SD of femoral and tibial osteophyte scores
12 weeks post-meniscectomy for joints injected with Hyaluronan (HA)
or HA plus different doses of Mesenchymal Precursor Cells
(MPC).
[0031] FIG. 3. Ratios [HA/(MPC+HA)] of cartilage morphology joint
scores for animals injected with different doses of Mesenchymal
Precursor Cells (MPC). When ratio=1 both treatments equally
effective. Ratios>1 indicate MPC+HA superior to HA.
[0032] FIG. 4. Ratios [HA/(MPC+HA)] of osteophyte scores for
animals injected with different doses of MPC+HA relative to HA
alone. When ratio=1 both treatments equally effective. Ratios>1
indicate MPC+HA superior to HA.
[0033] FIG. 5. Means.+-.SE of histomorphometrically determined
regional thickness scores for cartilages of joints injected with
hyaluronan (HA) or 100 million MPC+HA twelve weeks post
meniscectomy. Combining all tibial cartilage regions HA+100 million
MPC >HA (p<0.05).
[0034] FIG. 6. Ratios [HA/(MPC+HA)] of mean.+-.SE total Mankin
Modified joint histopathology scores for animals injected with
different doses of Mesenchymal Precursor Cells (MPC). When ratio=1
both treatments equally effective. Ratios>1 indicate MPC+HA
superior to HA.
[0035] FIG. 7. Means.+-.SD of femoral and tibial cartilage
morphology scores for HA and HA+100 million MPC injected joints 12,
24 and 52 weeks post meniscectomy.
[0036] FIG. 8. Means.+-.SD of femoral and tibial osteophyte scores
for HA and HA+100 million MPC injected joints 12, 24 and 52 weeks
post meniscectomy.
[0037] FIG. 9. Ratios [HA/(MPC+HA)] of cartilage morphology joint
scores for animals injected with Mesenchymal Precursor Cells (MPC)
12, 24 and 52 weeks post meniscectomy. When ratio=1 both treatments
equally effective. Ratios>1 indicate MPC+HA superior to HA.
[0038] FIG. 10. Ratios [HA/(MPC+HA)] of osteophyte joint scores for
animals injected with Mesenchymal Precursor Cells (MPC) 12, 24 and
52 weeks post meniscectomy. When ratio=1 both treatments equally
effective. Ratios>1 indicate MPC+HA superior to HA.
[0039] FIG. 11. Ratios [HA/(MPC+HA)] of mean.+-.SE Modified Mankins
joint cartilage histopathology scores for animals injected with
Mesenchymal Precursor Cells (MPC) 12, 24 and 52 weeks post
meniscectomy. When ratios=1 both treatments equally effective.
Ratios>1 indicate MPC+HA superior to HA.
[0040] FIG. 12. Mean+/-SE of patella cartilage stiffness from
joints injected with hyaluronan (HA) or HA+different doses of
Mesenchymal Precursor Cells (MPC). *=p<0.05, **=p<0.01,
***=p<0.001, ****=p<0.0001.
[0041] FIG. 13. Mean.-+./-SE of patella cartilage stiffness from
joints injected with hyaluronan (HA) or 100 million Mesenchymal
Precursor Cells (MPC)+HA and sacrificed 12, 24 and 52 weeks post
meniscectomy. *=p<0.05, **=p<0.01, ***=p<0.001,
****=p<0.0001.
[0042] FIG. 14. Mean+/-SE of patella cartilage phase lag from
joints injected with hyaluronan (HA) or HA+different doses of
Mesenchymal Precursor Cells (MPC). *=p<0.05, **=p<0.01,
***=p<0.001, ****=p<0.0001.
[0043] FIG. 15. Mean+/-SE of patella cartilage phase lag from
joints injected with hyaluronan (HA) or HA +100 million Mesenchymal
Precursor Cells (MPC) and sacrificed 12, 24 and 52 weeks post
meniscectomy. *=p<0.05, **=p<0.01, ***=p<0.001,
****=p<0.0001.
[0044] FIG. 16. Comparison of joint cartilage morphology scores for
untreated castrated male sheep and ovariectomised ewes 12 weeks
post meniscectomy showing the significantly greater severity of OA
lesions in the female group.
[0045] FIG. 17. Comparison of joint osteophyte scores for untreated
castrated male sheep and ovariectomised ewes 12 weeks post
meniscectomy showing the significantly higher scores in the female
group.
[0046] FIG. 18. Mean.+-.SD of cartilage Modified Mankin
Histopathology scores 36 weeks post meniscectomy from joints of
ovariectomised ewes injected with Hyaluronan (HA) or HA+100 million
Mesenchymal Precursor Cells (MPC) 12 weeks post meniscectomy. P
values =HA versus MPC+HA. These results show that a single MPC
injection reduces abnormal histopathologic score of femoral hyaline
cartilage over 6 months to a greater extent than tibial
cartilage.
[0047] FIG. 19. Ratios (HA/HA+MPC) of cartilage Total Modified
Mankin Histopathology Scores for joints of ovariectomised ewes 36
weeks post meniscectomy administered intra-articular injections 12
weeks post meniscectomy. When ratio=1, MPC+HA equivalent to HA.
Ratio>1, shows MPC+HA more protective than HA alone. Data
=Means.+-.SEM. These results show that a single MPC injection
reduces abnormal histopathologic score of femoral hyaline cartilage
over 6 months to greater extent than tibial cartilage.
[0048] FIG. 20. Mean.+-.SD of femoral cartilage Modified Mankin
Histopathology scores 24 and 36 weeks post meniscectomy (MX) from
joints of ovariectomised ewes injected with Hyaluronan (HA) or 100
million Mesenchymal Precursor Cells (MPC) +HA 12 weeks post MX
compared with non-injected joints at 12 weeks post MX. P values are
for 12 wks NIL versus treatments. These results show that a single
MPC injection reduces abnormal histopathologic score over 6
months.
[0049] FIG. 21. Femoral cartilage histomorphometry data 36 weeks
post meniscectomy from joints of ovariectomised ewes injected with
Hyaluronan (HA) or 100 million Mesenchymal Precursor Cells (MPC)
+HA 12 weeks post meniscectomy. Data shown =Mean.+-.SEM. P
values=HA v MPC+HA. These results show that a single MPC injection
generates greater hyaline cartilage over 6 Months than hyaluronic
acid.
[0050] FIG. 22. Mean.+-.SEM histomorphometrically determined
femoral cartilage thickness of joints from untreated ewes
sacrificed 12 weeks post meniscectomy (Mx) or injected with
Hyaluronan (HA) or Mesenchymal Precursor Cells (MPC)+HA at 12 weeks
post Mx then sacrificed 12 or 24 weeks later. Data expressed as
Mean+SEM. P values relative to 12 week NIL treated. These results
show that a single MPC injection increases hyaline cartilage
thickness over 6 months.
[0051] FIG. 23. Histomorphometrically determined femoral cartilage
areas of joints from untreated ewes sacrificed 12 weeks post
meniscectomy (Mx) or injected with Hyaluronan (HA) or Mesenchymal
Precursor Cells (MPC)+HA at 12 weeks post Mx then sacrificed 12 or
24 weeks later. Data expressed as Mean.+-.SEM. P values relative to
12 week NIL treated. These results show that a single MPC injection
increases hyaline cartilage area over 6 months.
[0052] FIG. 24. Histomorphometrically determined Integrated
Grey-scale Density (IGD) as a measure of overall Proteoglycan (PG)
content of femoral cartilages from joints of untreated ewes
sacrificed 12 weeks post meniscectomy (Mx) or injected with
Hyaluronan (HA) or Mesenchymal Precursor Cells (MPC)+HA at 12 weeks
post Mx then sacrificed 12 or 24 weeks later. Data expressed as
Mean.+-.SEM. P values relative to 12 week NIL treated. These
results show that a single MPC injection generates significantly
more cartilage containing proteoglycan than hyaluronic acid
injection over 6 months.
[0053] FIG. 25. Schematic representation of the lumber spinal
levels treated with
[0054] Mesenchymal Precursor Cells (MPC) in all sheep Groups.
[0055] FIG. 26. Mean recovery in disc height three and six months
following injection of MPC and HA into the nuclei pulposi of
degenerate sheep discs.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
General Techniques and Selected Definitions
[0056] Unless specifically defined otherwise, all technical and
scientific terms used herein shall be taken to have the same
meaning as commonly understood by one of ordinary skill in the art
(e.g., in cell culture, stem cell biology, molecular genetics,
immunology, immunohistochemistry, protein chemistry, and
biochemistry).
[0057] Unless otherwise indicated, the recombinant protein, cell
culture, and immunological techniques utilized in the present
invention are standard procedures, well known to those skilled in
the art. Such techniques are described and explained throughout the
literature in sources such as, J. Perbal, A Practical Guide to
Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbour
Laboratory Press (1989), T. A. Brown (editor), Essential Molecular
Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991),
D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical
Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel
et al. (editors), Current Protocols in Molecular Biology, Greene
Pub. Associates and Wiley-Interscience (1988, including all updates
until present), Ed Harlow and David Lane (editors) Antibodies: A
Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.
E. Coligan et al. (editors) Current Protocols in Immunology, John
Wiley & Sons (including all updates until present).
[0058] As used herein, the terms "treating", "treat" or "treatment"
include administering a therapeutically effective amount of
supernatant, soluble factors and/or cells as defined herein
sufficient to reduce or eliminate at least one symptom of the
specified condition. As used herein, the terms "preventing",
"prevent" or "prevention" include administering a therapeutically
effective amount of supernatant, soluble factors and/or cells as
defined herein sufficient to stop or hinder the development of at
least one symptom of the specified condition.
[0059] As used herein, the term "derived from mesenchymal precursor
cells" refers to supernatant, and/or one or more soluble factors,
produced from the in vitro culturing of mesenchymal precursor cells
and/or progeny cells thereof.
[0060] As used herein, the term "supernatant" refers to the
non-cellular material produced following the in vitro culturing of
mesenchymal precursor cells, and/or progeny cells thereof, in a
suitable medium, preferably liquid medium. Typically, the
supernatant is produced by culturing the cells in the medium under
suitable conditions and time, followed by removing the cellular
material by a process such as centrifugation. The supernatant may
or may not have been subjected to further purification steps before
administration. In preferred embodiment, the supernatant comprises
less than 10.sup.5, more preferably less than 10.sup.4, more
preferably less than 10.sup.3 and even more preferably no live
cells.
[0061] As used herein, the term "one or more soluble" factors
refers to molecules, typically proteins, secreted by the MPCs,
and/or progeny cells thereof, during culture.
Mesenchymal Precursor Cells (MPCs) or Progeny Cells, and
Supernatant or one or more Soluble Factors Derived Therefrom
[0062] As used herein, "MPC" are non-hematopoietic STRO-1.sup.+
progenitor cells that are capable of forming large numbers of
multipotential cell colonies.
[0063] Mesenchymal precursor cells (MPCs) are cells found in bone
marrow, blood, dental pulp cells, adipose tissue, skin, spleen,
pancreas, brain, kidney, liver, heart, retina, brain, hair
follicles, intestine, lung, lymph node, thymus, bone, ligament,
tendon, skeletal muscle, dermis, and periosteum; and are capable of
differentiating into different germ lines such as mesoderm,
endoderm and ectoderm. Thus, MPCs are capable of differentiating
into a large number of cell types including, but not limited to,
adipose, osseous, cartilaginous, elastic, muscular, and fibrous
connective tissues. The specific lineage-commitment and
differentiation pathway which these cells enter depends upon
various influences from mechanical influences and/or endogenous
bioactive factors, such as growth factors, cytokines, and/or local
microenvironmental conditions established by host tissues.
Mesenchymal precursor cells thus non-hematopoietic progenitor cells
which divide to yield daughter cells that are either stem cells or
are precursor cells which in time will irreversibly differentiate
to yield a phenotypic cell.
[0064] In a preferred embodiment, the MPCs are enriched from a
sample obtained from a subject. The terms `enriched`, `enrichment`
or variations thereof are used herein to describe a population of
cells in which the proportion of one particular cell type or the
proportion of a number of particular cell types is increased when
compared with the untreated population.
[0065] In a preferred embodiment, the cells used in the present
invention are also TNAP.sup.+, VCAM-1.sup.+, THY-1.sup.+,
STRO-2.sup.+, CD45.sup.+, CD146.sup.+,3G5.sup.+or any combination
thereof. Preferably, the STRO-1.sup.+ cells are STRO-1.sup.bright.
Preferably, the STRO-1.sup.bright cells are additionally one or
more of VCAM-1.sup.+, THY-1.sup.+, STRO-2.sup.+ and/or
CD146.sup.+.
[0066] In one embodiment, the mesenchymal precursor cells are
perivascular mesenchymal precursor cells as defined in
WO2004/85630.
[0067] When we refer to a cell as being "positive" for a given
marker it may be either a low (lo or dim) or a high (bright, bri)
expresser of that marker depending on the degree to which the
marker is present on the cell surface, where the terms relate to
intensity of fluorescence or other colour used in the colour
sorting process of the cells. The distinction of lo (or dim or
dull) and bri will be understood in the context of the marker used
on a particular cell population being sorted. When we refer herein
to a cell as being "negative" for a given marker, it does not mean
that the marker is not expressed at all by that cell. It means that
the marker is expressed at a relatively very low level by that
cell, and that it generates a very low signal when detectably
labelled.
[0068] The term "bright", when used herein, refers to a marker on a
cell surface that generates a relatively high signal when
detectably labelled. Whilst not wishing to be limited by theory, it
is proposed that "bright" cells express more of the target marker
protein (for example the antigen recognised by STRO-1) than other
cells in the sample. For instance, STRO-1.sup.bri cells produce a
greater fluorescent signal, when labelled with a FITC-conjugated
STRO-1 antibody as determined by FACS analysis, than non-bright
cells (STRO-1.sup.dull/dim). Preferably, "bright" cells constitute
at least about 0.1% of the most brightly labelled bone marrow
mononuclear cells contained in the starting sample. In other
embodiments, "bright" cells constitute at least about 0.1%, at
least about 0.5%, at least about 1%, at least about 1.5%, or at
least about 2%, of the most brightly labelled bone marrow
mononuclear cells contained in the starting sample. In a preferred
embodiment, STRO-1.sup.bright cells have 2 log magnitude higher
expression of STRO-1 surface expression. This is calculated
relative to "background", namely cells that are STRO-1.sup.-. By
comparison, STRO-1.sup.dim and/or STRO-1.sup.intermediate cells
have less than 2 log magnitude higher expression of STRO-1 surface
expression, typically about 1 log or less than "background".
[0069] When used herein the term "TNAP" is intended to encompass
all isoforms of tissue non-specific alkaline phosphatase. For
example, the term encompasses the liver isoform (LAP), the bone
isoform (BAP) and the kidney isoform (KAP). In a preferred
embodiment, the TNAP is BAP. In a particularly preferred
embodiment, TNAP as used herein refers to a molecule which can bind
the STRO-3 antibody produced by the hybridoma cell line deposited
with ATCC on 19 Dec. 2005 under the provisions of the Budapest
Treaty under deposit accession number PTA-7282.
[0070] Furthermore, in a preferred embodiment, the MPCs are capable
of giving rise to clonogenic CFU-F.
[0071] It is preferred that a significant proportion of the
multipotential cells are capable of differentiation into at least
two different germ lines. Non-limiting examples of the lineages to
which the multipotential cells may be committed include bone
precursor cells; hepatocyte progenitors, which are multipotent for
bile duct epithelial cells and hepatocytes; neural restricted
cells, which can generate glial cell precursors that progress to
oligodendrocytes and astrocytes; neuronal precursors that progress
to neurons; precursors for cardiac muscle and cardiomyocytes,
glucose-responsive insulin secreting pancreatic beta cell lines.
Other lineages include, but are not limited to, odontoblasts,
dentin-producing cells and chondrocytes, and precursor cells of the
following: retinal pigment epithelial cells, fibroblasts, skin
cells such as keratinocytes, dendritic cells, hair follicle cells,
renal duct epithelial cells, smooth and skeletal muscle cells,
testicular progenitors, vascular endothelial cells, tendon,
ligament, cartilage, adipocyte, fibroblast, marrow stroma, cardiac
muscle, smooth muscle, skeletal muscle, pericyte, vascular,
epithelial, glial, neuronal, astrocyte and oligodendrocyte
cells.
[0072] In another embodiment, the MPCs are not capable of giving
rise, upon culturing, to hematopoietic cells.
[0073] The present invention also relates to use of supernatant or
soluble factors obtained derived from MPC and/or progeny cells
thereof (the latter also being referred to as expanded cells) which
are produced from in vitro culture. Expanded cells of the invention
may a have a wide variety of phenotypes depending on the culture
conditions (including the number and/or type of stimulatory factors
in the culture medium), the number of passages and the like. In
certain embodiments, the progeny cells are obtained after about 2,
about 3, about 4, about 5, about 6, about 7, about 8, about 9, or
about 10 passages from the parental population. However, the
progeny cells may be obtained after any number of passages from the
parental population.
[0074] The progeny cells may be obtained by culturing in any
suitable medium. The term "medium", as used in reference to a cell
culture, includes the components of the environment surrounding the
cells. Media may be solid, liquid, gaseous or a mixture of phases
and materials. Media include liquid growth media as well as liquid
media that do not sustain cell growth. Media also include
gelatinous media such as agar, agarose, gelatin and collagen
matrices. Exemplary gaseous media include the gaseous phase that
cells growing on a petri dish or other solid or semisolid support
are exposed to. The term "medium" also refers to material that is
intended for use in a cell culture, even if it has not yet been
contacted with cells. In other words, a nutrient rich liquid
prepared for bacterial culture is a medium. Similarly, a powder
mixture that when mixed with water or other liquid becomes suitable
for cell culture, may be termed a "powdered medium".
[0075] In an embodiment, progeny cells useful for the methods of
the invention are obtained by isolating TNAP+MPCs from bone marrow
using magnetic beads labelled with the STRO-3 antibody, and then
culture expanding the isolated cells (see Gronthos et al. (1995)
for an example of suitable culturing conditions). In one
embodiment, such expanded cells (progeny) (at least after 5
passages) can be TNAP-, CC9.sup.+, HLA class 1.sup.+, HLA class II,
CD14.sup.--, CD19.sup.--, CD3.sup.--, CD1 1a-c.sup.-, CD31.sup.-,
CD86.sup.-CD34.sup.-and/or CD80.sup.-. However, it is possible that
under different culturing conditions to those described herein that
the expression of different markers may vary. Also, whilst cells of
these phenotypes may predominate in the expended cell population it
does not mean that there is a minor proportion of the cells do not
have this phenotype(s) (for example, a small percentage of the
expanded cells may be CC9-). In one preferred embodiment, expanded
cells still have the capacity to differentiate into different cell
types.
[0076] In one embodiment, an expended cell population used to
obtain supernatant or soluble factors, or cells per se, comprises
cells wherein at least 25%, more preferably at least 50%, of the
cells are CC9+.
[0077] In another embodiment, an expended cell population used to
obtain supernatant or soluble factors, or cells per se, comprises
cells wherein at least 40%, more preferably at least 45%, of the
cells are STRO-1+.
[0078] In a further embodiment, the expanded cells may express
markers selected from the group consisting of LFA-3, THY-1, VCAM-1,
ICAM-1, PECAM-1, P-selectin, L-selectin, 3G5, CD49a/CD49b/CD29,
CD49c/CD29, CD49d/CD29, CD 90, CD29, CD18, CD61, integrin beta,
6-19, thrombomodulin, CD10, CD13, SCF, PDGF-R, EGF-R, IGF1-R,
NGF-R, FGF-R, Leptin-R, (STRO-2=Leptin-R), RANKL, STRO-
1.sup.bright and CD146 or any combination of these markers.
[0079] In one embodiment, the progeny cells are Multipotential
Expanded MPC Progeny (MEMPs) as defined in WO 2006/032092. Methods
for preparing enriched populations of MPC from which progeny may be
derived are described in WO 01/04268 and WO 2004/085630. In an in
vitro context MPCs will rarely be present as an absolutely pure
preparation and will generally be present with other cells that are
tissue specific committed cells (TSCCs). WO 01/04268 refers to
harvesting such cells from bone marrow at purity levels of about
0.1% to 90%. The population comprising MPC from which progeny are
derived may be directly harvested from a tissue source, or
alternatively it may be a population that has already been expanded
ex vivo.
[0080] For example, the progeny may be obtained from a harvested,
unexpanded, population of substantially purified MPC, comprising at
least about 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80 or 95% of
total cells of the population in which they are present. This level
may be achieved, for example, by selecting for cells that are
positive for at least one marker selected from the group consisting
of TNAP, STRO-1.sup.bright, 3G5.sup.+, VCAM-1, THY-1, CD146 and
STRO-2.
[0081] MEMPS can be distinguished from freshly harvested MPCs in
that they are positive for the marker STRO-1.sup.bri and negative
for the marker Alkaline phosphatase (ALP). In contrast, freshly
isolated MPCs are positive for both STRO-1.sup.bri and ALP. In a
preferred embodiment of the present invention, at least 15%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the administered cells
have the phenotype STRO-1.sup.bri, ALP-. In a further preferred
embodiment the MEMPS are positive for one or more of the markers
Ki67, CD44 and/or CD49c/CD29, VLA-3, .alpha.3.beta.1. In yet a
further preferred embodiment the MEMPs do not exhibit TERT activity
and/or are negative for the marker CD18.
[0082] The MPC starting population may be derived from any one or
more tissue types set out in WO 01/04268 or WO 2004/085630, namely
bone marrow, dental pulp cells, adipose tissue and skin, or perhaps
more broadly from adipose tissue, teeth, dental pulp, skin, liver,
kidney, heart, retina, brain, hair follicles, intestine, lung,
spleen, lymph node, thymus, pancreas, bone, ligament, bone marrow,
tendon and skeletal muscle.
[0083] It will be understood that in performing the present
invention, separation of cells carrying any given cell surface
marker can be effected by a number of different methods, however,
preferred methods rely upon binding a binding agent to the marker
concerned followed by a separation of those that exhibit binding,
being either high level binding, or low level binding or no
binding. The most convenient binding agents are antibodies or
antibody based molecules, preferably being monoclonal antibodies or
based on monoclonal antibodies because of the specificity of these
latter agents. Antibodies can be used for both steps, however other
agents might also be used, thus ligands for these markers may also
be employed to enrich for cells carrying them, or lacking them.
[0084] The antibodies or ligands may be attached to a solid support
to allow for a crude separation. The separation techniques
preferably maximise the retention of viability of the fraction to
be collected. Various techniques of different efficacy may be
employed to obtain relatively crude separations. The particular
technique employed will depend upon efficiency of separation,
associated cytotoxicity, ease and speed of performance, and
necessity for sophisticated equipment and/or technical skill.
Procedures for separation may include, but are not limited to,
magnetic separation, using antibody-coated magnetic beads, affinity
chromatography and "panning" with antibody attached to a solid
matrix. Techniques providing accurate separation include but are
not limited to FACS.
[0085] It is preferred that the method for isolating MPCs, for
example, comprises a first step being a solid phase sorting step
utilising for example MACS recognising high level expression of
STRO-1. A second sorting step can then follow, should that be
desired, to result in a higher level of precursor cell expression
as described in patent specification WO 01/14268. This second
sorting step might involve the use of two or more markers.
[0086] The method obtaining MPCs might also include the harvesting
of a source of the cells before the first enrichment step using
known techniques. Thus the tissue will be surgically removed. Cells
comprising the source tissue will then be separated into a so
called single cells suspension. This separation may be achieved by
physical and or enzymatic means.
[0087] Once a suitable MPC population has been obtained, it may be
cultured or expanded by any suitable means to obtain MEMPs.
[0088] In one embodiment, the cells are taken from the subject to
be treated, cultured in vitro using standard techniques and used to
obtain supernatant or soluble factors or expanded cells for
administration to the subject as an autologous or allogeneic
composition. In an alternative embodiment, cells of one or more of
the established human cell lines are used to obtain the supernatant
or soluble factors. In another useful embodiment of the invention,
cells of a non-human animal (or if the patient is not a human, from
another species) are used to obtain supernatant or soluble
factors.
[0089] The invention can be practised using cells from any
non-human animal species, including but not limited to non-human
primate cells, ungulate, canine, feline, lagomorph, rodent, avian,
and fish cells. Primate cells with which the invention may be
performed include but are not limited to cells of chimpanzees,
baboons, cynomolgus monkeys, and any other New or Old World
monkeys. Ungulate cells with which the invention may be performed
include but are not limited to cells of bovines, porcines, ovines,
caprines, equines, buffalo and bison. Rodent cells with which the
invention may be performed include but are not limited to mouse,
rat, guinea pig, hamster and gerbil cells. Examples of lagomorph
species with which the invention may be performed include
domesticated rabbits, jack rabbits, hares, cottontails, snowshoe
rabbits, and pikas. Chickens (Gallus gallus) are an example of an
avian species with which the invention may be performed.
[0090] Cells useful for the methods of the invention may be stored
before use, or before obtaining the supernatant or soluble factors.
Methods and protocols for preserving and storing of eukaryotic
cells, and in particular mammalian cells, are well known in the art
(cf., for example, Pollard, J. W. and Walker, J. M. (1997) Basic
Cell Culture Protocols, Second Edition, Humana Press, Totowa, N.J.;
Freshney, R. I. (2000) Culture of Animal Cells, Fourth Edition,
Wiley-Liss, Hoboken, N.J.). Any method maintaining the biological
activity of the isolated stem cells such as mesenchymal
stem/progenitor cells, or progeny thereof, may be utilized in
connection with the present invention. In one preferred embodiment,
the cells are maintained and stored by using cryo-preservation.
Administration and Compositions
Supernatant or Soluble Factors
[0091] The methods of the present invention may involve
administering MPC-derived supernatant or soluble factors,
topically, systematically, or locally such as within an implant or
device.
[0092] In one particular embodiment the invention involves
administering MPC-derived supernatant or soluble factors
systemically to the subject. For example, the supernatant or
soluble factors may be administered by subcutaneous or
intramuscular injection.
[0093] This embodiment of the invention may be useful for the
treatment of systemic degenerative diseases where generation or
repair of particular tissues is desirable. Examples of systemic
degenerative diseases that can be treated in this way include
osteoporosis or fractures, or degenerative diseases of
cartilage.
[0094] The MPC-derived supernatant or soluble factors may also be
used to treat patients requiring the repair or replacement of
cartilage tissue resulting from disease or trauma or failure of the
tissue to develop normally, or to provide a cosmetic function, such
as to augment facial or other features of the body. Treatment may
entail the use of the supernatant or soluble factors to produce new
cartilage tissue and/or maintain existing cartilage tissue. For
example, MPC-derived supernatant or soluble factors may be used to
treat a cartilage condition, for example, rheumatoid arthritis or
osteoarthritis or a traumatic or surgical injury to cartilage.
[0095] Suspensions comprising MPC-derived supernatant or soluble
factors may be prepared as appropriate oily suspensions for
injection. Suitable lipophilic solvents or vehicles include fatty
oils such as sesame oil; or synthetic fatty acid esters, such as
ethyl oleate or triglycerides; or liposomes. Suspensions to be used
for injection may also contain substances which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Optionally, the suspension may
also contain suitable stabilizers or agents which increase the
solubility of the compounds to allow for the preparation of highly
concentrated solutions.
[0096] Sterile injectable solutions can be prepared by
incorporating the supernatant or soluble factors in the required
amount in an appropriate solvent with one or a combination of
ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the supernatant or soluble factors into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying which yields a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof. In accordance with an
alternative aspect of the invention, the supernatant or soluble
factors may be formulated with one or more additional compounds
that enhance its solubility.
Cellular Compositions
[0097] In one embodiment, cellular compositions of the invention
are administered as undifferentiated cells, i.e., as cultured in
Growth Medium. Alternatively, the cellular compositions may be
administered following culturing.
[0098] The cellular compositions useful for the present invention
may be administered alone or as admixtures with other cells. Cells
that may be administered in conjunction with the compositions of
the present invention include, but are not limited to, other
multipotent or pluripotent cells or chondrocytes, chondroblasts,
osteocytes, osteoblasts, osteoclasts, bone lining cells, stem
cells, or bone marrow cells. The cells of different types may be
admixed with a composition of the invention immediately or shortly
prior to administration, or they may be co-cultured together for a
period of time prior to administration.
[0099] In some embodiments of the invention, it may not be
necessary or desirable to immunosuppress a patient prior to
initiation of therapy with cellular compositions. Accordingly,
transplantation with allogeneic, or even xenogeneic, MPCs or
progeny thereof may be tolerated in some instances.
[0100] However, in other instances it may be desirable or
appropriate to pharmacologically immunosuppress a patient prior to
initiating cell therapy. This may be accomplished through the use
of systemic or local immunosuppressive agents, or it may be
accomplished by delivering the cells in an encapsulated device. The
cells may be encapsulated in a capsule that is permeable to
nutrients and oxygen required by the cell and therapeutic factors
the cell is yet impermeable to immune humoral factors and cells.
Preferably the encapsulant is hypoallergenic, is easily and stably
situated in a target tissue, and provides added protection to the
implanted structure. These and other means for reducing or
eliminating an immune response to the transplanted cells are known
in the art. As an alternative, the cells may be genetically
modified to reduce their immunogenicity.
General
[0101] A "therapeutically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired effect.
[0102] A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired prophylactic result, such as preventing or inhibiting
cell apoptosis or tissue damage.
[0103] The amount of supernatant or soluble factors, or MPCs or
progeny thereof to be administered may vary according to factors
such as the disease state, age, sex, and weight of the individual.
Dosage regimens may be adjusted to provide the optimum therapeutic
response. For example, a single bolus may be administered, several
divided doses may be administered over time or the dose may be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation. It may be advantageous to formulate
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. "Dosage unit form" as used
herein refers to physically discrete units suited as unitary
dosages for subjects to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier.
[0104] It will be appreciated that the supernatant or soluble
factors or MPCs or progeny thereof may be administered in the form
of a composition comprising a pharmaceutically acceptable carrier
or excipient.
[0105] As used herein "pharmaceutically acceptable carrier" or
"excipient" includes any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like that are physiologically
compatible. In one embodiment, the carrier is suitable for
parenteral administration. Alternatively, the carrier can be
suitable for intravenous, intraperitoneal, intramuscular,
sublingual or oral administration. Pharmaceutically acceptable
carriers include sterile aqueous solutions or dispersions and
sterile , powders for the extemporaneous preparation of sterile
injectable solutions or dispersion. The use of such media and
agents for pharmaceutically active substances is well known in the
art. Except insofar as any conventional media or agent is
incompatible with the active compound, use thereof in the
pharmaceutical compositions of the invention is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0106] Therapeutic compositions typically should be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure. The carrier can be a solvent
or dispersion medium containing, for example, water, ethanol,
polyol (for example, glycerol, propylene glycol, and liquid
polyethylene glycol, and the like), and suitable mixtures thereof.
The proper fluidity can be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of
surfactants. In many cases, it will be preferable to include
isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in the composition.
Prolonged absorption of the injectable compositions can be brought
about by including in the composition an agent which delays
absorption, for example, monostearate salts and gelatin. Moreover,
the stimulatory factor may be administered in a time release
formulation, for example in a composition which includes a slow
release polymer. The active compounds can be prepared with carriers
that will protect the compound against rapid release, such as a
controlled release formulation, including implants and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters,
polylactic acid and polylactic, polyglycolic copolymers (PLG). Many
methods for the preparation of such formulations are patented or
generally known to those skilled in the art.
[0107] The supernatant or soluble factors or cell compositions may
be administered in combination with an appropriate matrix, for
instance, to provide slow release of the soluble factors.
[0108] The choice of matrix material is based on biocompatibility,
biodegradability, mechanical properties, cosmetic appearance and
interface properties. Potential matrices for the compositions may
be biodegradable and chemically defined calcium sulfate, tricalcium
phosphate, hydroxyapatite, polylactic acid and polyanhydrides.
Other potential materials are biodegradable and biologically well
defined, such as bone or dermal collagen. Further matrices are
comprised of pure proteins or extracellular matrix components.
Other potential matrices are nonbiodegradable and chemically
defined, such as sintered hydroxyapatite, bioglass, aluminates, or
other ceramics. Matrices may be comprised of combinations of any of
the above mentioned types of material, such as polylactic acid and
hydroxyapatite or collagen and tricalcium phosphate. The
bioceramics may be altered in composition, such as in
calcium-aluminate-phosphate and processing to alter pore size,
particle size, particle shape, and biodegradability.
[0109] The MPC-derived supernatant or soluble factors, MPCs or
progeny thereof may be surgically implanted, injected, delivered
(e.g., by way of a catheter or syringe), or otherwise administered
directly or indirectly to the site in need of repair or
augmentation. Routes of administration of the MPC-derived
supernatant or soluble factors include intramuscular, ophthalmic,
parenteral (including intravenous), intraarterial, subcutaneous,
oral, and nasal administration. Particular routes of parenteral
administration include, but are not limited to, intramuscular,
subcutaneous, intraperitoneal, intracerebral, intraventricular,
intracerebroventricular, intrathecal, intracisternal, intraspinal
and/or peri-spinal routes of administration.
[0110] In some embodiments of the invention, the formulation
comprises an in situ polymerizable gel, as described, for example,
in US 2002/0022676; Anseth et al. (2002) and Wang et al.
(2003).
[0111] In some embodiments, the polymers are at least partially
soluble in aqueous solutions, such as water, buffered salt
solutions, or aqueous alcohol solutions, that have charged side
groups, or a monovalent ionic salt thereof. Examples of polymers
with acidic side groups that can be reacted with cations are
poly(phosphazenes), poly(acrylic acids), poly(methacrylic acids),
copolymers of acrylic acid and methacrylic acid, poly(vinyl
acetate), and sulfonated polymers, such as sulfonated polystyrene.
Copolymers having acidic side groups formed by reaction of acrylic
or methacrylic acid and vinyl ether monomers or polymers can also
be used. Examples of acidic groups are carboxylic acid groups,
sulfonic acid groups, halogenated (preferably fluorinated) alcohol
groups, phenolic OH groups, and acidic OH groups.
[0112] Examples of polymers with basic side groups that can be
reacted with anions are poly(vinyl amines), poly(vinyl pyridine),
poly(vinyl imidazole), and some imino substituted polyphosphazenes.
The ammonium or quaternary salt of the polymers can also be formed
from the backbone nitrogens or pendant imino groups. Examples of
basic side groups are amino and imino groups.
[0113] Alginate can be ionically cross-linked with divalent
cations, in water, at room temperature, to form a hydrogel matrix.
Due to these mild conditions, alginate has been the most commonly
used polymer for hybridoma cell encapsulation, as described, for
example, in U.S. Pat. No. 4,352,883. In the process described in
U.S. Pat. No. 4,352,883, an aqueous solution containing the
biological materials to be encapsulated is suspended in a solution
of a water soluble polymer, the suspension is formed into droplets
which are configured into discrete microcapsules by contact with
multivalent cations, then the surface of the microcapsules is
crosslinked with polyamino acids to form a semipermeable membrane
around the encapsulated materials.
[0114] Polyphosphazenes are polymers with backbones consisting of
nitrogen and phosphorous separated by alternating single and double
bonds. Each phosphorous atom is covalently bonded to two side
chains.
[0115] The polyphosphazenes suitable for cross-linking have a
majority of side chain groups which are acidic and capable of
forming salt bridges with di-or trivalent cations. Examples of
preferred acidic side groups are carboxylic acid groups and
sulfonic acid groups. Hydrolytically stable polyphosphazenes are
formed of monomers having carboxylic acid side groups that are
crosslinked by divalent or trivalent cations such as Ca.sup.2+ or
Al.sup.3+. Polymers can be synthesized that degrade by hydrolysis
by incorporating monomers having imidazole, amino acid ester, or
glycerol side groups.
[0116] For example, a polyanionic
poly[bis(carboxylatophenoxy)]phosphazene (PCPP) can be synthesized,
which is cross-linked with dissolved multivalent cations in aqueous
media at room temperature or below to form hydrogel matrices.
[0117] Biodegradable polyphosphazenes have at least two differing
types of side chains, acidic side groups capable of forming salt
bridges with multivalent cations, and side groups that hydrolyze
under in vivo conditions, e.g., imidazole groups, amino acid
esters, glycerol and glucosyl.
[0118] Hydrolysis of the side chain results in erosion of the
polymer. Examples of hydrolyzing side chains are unsubstituted and
substituted imidizoles and amino acid esters in which the group is
bonded to the phosphorous atom through an amino linkage
(polyphosphazene polymers in which both R groups are attached in
this manner are known as polyaminophosphazenes). For
polyimidazolephosphazenes, some of the "R" groups on the
polyphosphazene backbone are imidazole rings, attached to
phosphorous in the backbone through a ring nitrogen atom. Other "R"
groups can be organic residues that do not participate in
hydrolysis, such as methyl phenoxy groups or other groups shown in
the scientific paper of Allcock et al. (1977). Methods of synthesis
of the hydrogel materials, as well as methods for preparing such
hydrogels, are known in the art.
[0119] The MPC-derived supernatant or soluble factors, MPCs or
progeny thereof may be administered with other beneficial drugs or
biological molecules (growth factors, trophic factors). When
administered with other agents, they may be administered together
in a single pharmaceutical composition, or in separate
pharmaceutical compositions, simultaneously or sequentially with
the other agents (either before or after administration of the
other agents). Bioactive factors which may be co-administered
include anti-apoptotic agents (e.g., EPO, EPO mimetibody, TPO,
IGF-I and IGF-II, HGF, caspase inhibitors); anti-inflammatory
agents (e.g., p38 MAPK inhibitors, TGF-beta inhibitors, statins,
IL-6 and IL-1 inhibitors, PEMIROLAST, TRANILAST, REMICADE,
SIROLIMUS, and NSAIDs (non-steroidal anti-inflammatory drugs; e.g.,
TEPDXALIN, TOLMETIN, SUPROFEN); immunosupressive/immunomodulatory
agents (e.g., calcineurin inhibitors, such as cyclosporine,
tacrolimus; mTOR inhibitors (e.g., SIROLIMUS, EVEROLIMUS);
anti-proliferatives (e.g., azathioprine, mycophenolate mofetil);
corticosteroids (e.g., prednisolone, hydrocortisone); antibodies
such as monoclonal anti-IL-2Ralpha receptor antibodies (e.g.,
basiliximab, daclizumab), polyclonal anti-T-cell antibodies (e.g.,
anti-thymocyte globulin (ATG); anti-lymphocyte globulin (ALG);
monoclonal anti-T cell antibody OKT3)); anti-thrombogenic agents
(e.g., heparin, heparin derivatives, urokinase, PPack
(dextrophenylalanine proline arginine chloromethylketone),
antithrombin compounds, platelet receptor antagonists,
anti-thrombin antibodies, anti-platelet receptor antibodies,
aspirin, dipyridamole, protamine, hirudin, prostaglandin
inhibitors, and platelet inhibitors); and anti-oxidants (e.g.,
probucol, vitamin A, ascorbic acid, tocopherol, coenzyme Q-10,
glutathione, L-cysteine, N-acetylcysteine) as well as local
anesthetics. As another example, the MPC-derived supernatant or
soluble factors, MPCs or progeny thereof may be co-administered
with scar inhibitory factor as described in U.S. Pat. No.
5,827,735.
[0120] When treating and/or preventing a disease arising from
degradation and/or inflammation of connective tissue it is
preferred that the supernatant, soluble factors or cells are
administered with chondroprotective agents. Examples include, but
are not limited to, pentosan polysulfate (SP54 and Cartrophen),
glycosaminoglycan polysufate ester (Arteparon),
glyciamino-glycan-peptide complex (Rumalon) and hyaluronic acid
(Hyalgan). Further examples are described by Verbruggen (2005) and
Richette and Bardin (2004). In a preferred embodiment, the
chondroprotective agent is hyaluronic acid.
Fibrin Glue
[0121] Fibrin glues are a class of surgical sealants which have
been used in various clinical settings. As the skilled address
would be aware, numerous sealants are useful for the methods
defined herein. However, a preferred embodiment of the invention
relates to the use of fibrin glues.
[0122] When used herein the term "fibrin glue" refers to the
insoluble matrix formed by the cross-linking of fibrin polymers in
the presence of calcium ions. The fibrin glue may be formed from
fibrinogen, or a derivative or metabolite thereof, fibrin (soluble
monomers or polymers) and/or complexes thereof derived from
biological tissue or fluid which forms a fibrin matrix.
Alternatively, the fibrin glue may be formed from fibrinogen, or a
derivative or metabolite thereof, or fibrin, produced by
recombinant DNA technology.
[0123] The fibrin glue may also be formed by the interaction of
fibrinogen and a catalyst of fibrin glue formation (such as
thrombin and/or Factor XIII). As will be appreciated by those
skilled in the art, fibrinogen is proteolytically cleaved in the
presence of a catalyst (such as thrombin) and converted to a fibrin
monomer. The fibrin monomers may then form polymers which may
cross-link to form a fibrin glue matrix. The cross-linking of
fibrin polymers may be enhanced by the presence of a catalyst such
as Factor XIII. The catalyst of fibrin glue formation may be
derived from blood plasma, cryoprecipitate or other plasma
fractions containing fibrinogen or thrombin. Alternatively, the
catalyst may be produced by recombinant DNA technology.
[0124] The rate at which the clot forms is dependent upon the
concentration of thrombin mixed with fibrinogen. Being an enzyme
dependent reaction, the higher the temperature (up to 37.degree.
C.) the faster the clot formation rate. The tensile strength of the
clot is dependent upon the concentration of fibrinogen used.
[0125] Use of fibrin glue and methods for its preparation and use
are described by Hirsh et al. in U.S. Pat. No. 5,643,192. Hirsh
discloses the extraction of fibrinogen and thrombin components from
a single donor, and the combination of only these components for
use as a fibrin glue. Marx, U.S. Pat. No. 5,651,982, describes
another preparation and method of use for fibrin glue. Marx
provides a fibrin glue with liposomes for use as a topical sealant
in mammals. The preparation and use of a topical fibrinogen complex
(TFC) for wound healing is known in the field. International Patent
Publication No. WO96/17633, of The American Red Cross, discusses
TFC preparations containing fibrinogen, thrombin, and calcium
chloride.
[0126] Several publications describe the use of fibrin glue for the
delivery of therapeutic agents. For example, U.S. Pat. No.
4,983,393 discloses a composition for use as an intra-vaginal
insert comprising agarose, agar, saline solution
glycosaminoglycans, collagen, fibrin and an enzyme. Further, U.S.
Pat. No. 3,089,815 discloses an injectable pharmaceutical
preparation composed of fibrinogen and thrombin and U.S. Pat. No.
6,468,527 discloses a fibrin glue which facilitates the delivery of
various biological and non-biological agents to specific sites
within the body.
Production of Genetically Modified Cells
[0127] In one embodiment, the cells used in the methods of the
invention, including for the production of supernatant or soluble
factors, are genetically modified. Preferably, the cells are
genetically modified to produce a heterologous protein. Typically,
the cells will be genetically modified such that the heterologous
protein is secreted from the cells. However, in an embodiment the
cells can be modified to express a functional non-protein encoding
polynucleotide such as dsRNA (typically for RNA silencing), an
antisense oligonucleotide or a catalytic nucleic acid (such as a
ribozyme or DNAzyme).
[0128] Genetically modified cells may be cultured in the presence
of at least one cytokine in an amount sufficient to support growth
of the modified cells. The genetically modified cells thus obtained
may be used immediately (e.g., in transplant), cultured and
expanded in vitro, or stored for later uses. The modified cells may
be stored by methods well known in the art, e.g., frozen in liquid
nitrogen.
[0129] Genetic modification as used herein encompasses any genetic
modification method which involves introduction of an exogenous or
foreign polynucleotide into a cell described herein or modification
of an endogenous gene within the cell. Genetic modification
includes but is not limited to transduction (viral mediated
transfer of host DNA from a host or donor to a recipient, either in
vitro or in vivo), transfection (transformation of cells with
isolated viral DNA genomes), liposome mediated transfer,
electroporation, calcium phosphate transfection or coprecipitation
and others. Methods of transduction include direct co-culture of
cells with producer cells (Bregni et al., 1992) or culturing with
viral supernatant alone with or without appropriate growth factors
and polycations.
[0130] An exogenous polynucleotide is preferably introduced to the
cell in a vector. The vector preferably includes the necessary
elements for the transcription and translation of the inserted
coding sequence. Methods used to construct such vectors are well
known in the art. For example, techniques for constructing suitable
expression vectors are described in detail in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,
N.Y. (3rd Ed., 2000); and Ausubel et al., Current Protocols in
Molecular Biology, John Wiley & Sons, Inc., New York
(1999).
[0131] Vectors may include, but are not limited to, viral vectors,
such as retroviruses, adenoviruses, adeno-associated viruses, and
herpes simplex viruses; cosmids; plasmid vectors; synthetic
vectors; and other recombination vehicles typically used in the
art. Vectors containing both a promoter and a cloning site into
which a polynucleotide can be operatively linked are well known in
the art. Such vectors are capable of transcribing RNA in vitro or
in vivo, and are commercially available from sources such as
Stratagene (La Jolla, Calif.) and Promega Biotech (Madison, Wis.).
Specific examples include, pSG, pSV2CAT, pXtl from Stratagene; and
pMSG, pSVL, pBPV and pSVK3 from Pharmacia.
[0132] Preferred vectors include retroviral vectors (see, Coffin et
al., "Retroviruses", Chapter 9 pp; 437-473, Cold Springs Harbor
Laboratory Press, 1997). Vectors useful in the invention can be
produced recombinantly by procedures well known in the art. For
example, WO94/29438, WO97/21824 and WO97/21825 describe the
construction of retroviral packaging plasmids and packing cell
lines. Exemplary vectors include the pCMV mammalian expression
vectors, such as pCMV6b and pCMV6c (Chiron Corp.), pSFFV-Neo, and
pBluescript-Sk+. Non-limiting examples of useful retroviral vectors
are those derived from murine, avian or primate retroviruses.
Common retroviral vectors include those based on the Moloney murine
leukemia virus (MoMLV-vector). Other MoMLV derived vectors include,
Lmily, LINGFER, MINGFR and MINT. Additional vectors include those
based on Gibbon ape leukemia virus (GALV) and Moloney murine
sarcoma virus (MOMSV) and spleen focus forming virus (SFFV).
Vectors derived from the murine stem cell virus (MESV) include
MESV-MiLy. Retroviral vectors also include vectors based on
lentiviruses, and non-limiting examples include vectors based on
human immunodeficiency virus (HIV-1 and HIV-2).
[0133] In producing retroviral vector constructs, the viral gag,
pol and env sequences can be removed from the virus, creating room
for insertion of foreign DNA sequences. Genes encoded by foreign
DNA are usually expressed under the control a strong viral promoter
in the long terminal repeat (LTR). Selection of appropriate control
regulatory sequences is dependent on the host cell used and
selection is within the skill of one in the art. Numerous promoters
are known in addition to the promoter of the LTR. Non-limiting
examples include the phage lambda PL promoter, the human
cytomegalovirus (CMV) immediate early promoter; the U3 region
promoter of the Moloney Murine Sarcoma Virus (MMSV), Rous Sacroma
Virus (RSV), or Spleen Focus Forming Virus (SFFV); Granzyme A
promoter; and the Granzyme B promoter. Additionally inducible or
multiple control elements may be used. The selection of a suitable
promoter will be apparent to those skilled in the art.
[0134] Such a construct can be packed into viral particles
efficiently if the gag, pol and env functions are provided in trans
by a packing cell line. Therefore, when the vector construct is
introduced into the packaging cell, the gag-pol and env proteins
produced by the cell, assemble with the vector RNA to produce
infectious virons that are secreted into the culture medium. The
virus thus produced can infect and integrate into the DNA of the
target cell, but does not produce infectious viral particles since
it is lacking essential packaging sequences. Most of the packing
cell lines currently in use have been transfected with separate
plasmids, each containing one of the necessary coding sequences, so
that multiple recombination events are necessary before a
replication competent virus can be produced. Alternatively the
packaging cell line harbours a provirus. The provirus has been
crippled so that although it may produce all the proteins required
to assemble infectious viruses, its own RNA cannot be packaged into
virus. RNA produced from the recombinant virus is packaged instead.
Therefore, the virus stock released from the packaging cells
contains only recombinant virus. Non-limiting examples of
retroviral packaging lines include PA12, PA317, PE501, PG13,
PSI.CRIP, RDI 14, GP7C-tTA-G10, ProPak-A (PPA-6), and PT67.
[0135] Other suitable vectors include adenoviral vectors (see, WO
95/27071) and adeno-associated viral vectors. These vectors are all
well known in the art, e.g., as described in Stem Cell Biology and
Gene Therapy, eds. Quesenberry et al., John Wiley & Sons, 1998;
and U.S. Pat. Nos. 5,693,531 and 5,691,176. The use of
adenovirus-derived vectors may be advantageous under certain
situation because they are not capable of infecting non-dividing
cells. Unlike retroviral DNA, the adenoviral DNA is not integrated
into the genome of the target cell. Further, the capacity to carry
foreign DNA is much larger in adenoviral vectors than retroviral
vectors. The adeno-associated viral vectors are another useful
delivery system. The DNA of this virus may be integrated into
non-dividing cells, and a number of polynucleotides have been
successful introduced into different cell types using
adeno-associated viral vectors.
[0136] In some embodiments, the construct or vector will include
two or more heterologous polynucleotide sequences. Preferably the
additional nucleic acid sequence is a polynucleotide which encodes
a selective marker, a structural gene, a therapeutic gene, or a
cytokine/chemokine gene.
[0137] A selective marker may be included in the construct or
vector for the purposes of monitoring successful genetic
modification and for selection of cells into which DNA has been
integrated. Non-limiting examples include drug resistance markers,
such as G148 or hygromycin. Additionally negative selection may be
used, for example wherein the marker is the HSV-tk gene. This gene
will make the cells sensitive to agents such as acyclovir and
gancyclovir. The NeoR (neomycin/G148 resistance) gene is commonly
used but any convenient marker gene may be used whose gene
sequences are not already present in the target cell can be used.
Further non-limiting examples include low-affinity Nerve Growth
Factor (NGFR), enhanced fluorescent green protein (EFGP),
dihydrofolate reductase gene (DHFR) the bacterial hisD gene, murine
CD24 (HSA), murine CD8a(lyt), bacterial genes which confer
resistance to puromycin or phleomycin, and .beta.-glactosidase.
[0138] The additional polynucleotide sequence(s) may be introduced
into the cell on the same vector or may be introduced into the host
cells on a second vector. In a preferred embodiment, a selective
marker will be included on the same vector as the
polynucleotide.
[0139] The present invention also encompasses genetically modifying
the promoter region of an endogenous gene such that expression of
the endogenous gene is up-regulated resulting in the increased
production of the encoded protein compared to a wild type cell.
EXAMPLES
Example 1
Expansion of Immunoselected MPCs and Collection of Supernatant
[0140] Bone marrow (BM) is harvested from sheep less than 2 years
old. Briefly, 40 ml of BM is aspirated from the anterior iliac
crest into lithium-heparin anticoagulant-containing tubes. BMMNC
are prepared by density gradient separation using Lymphoprep.TM.
(Nycomed Pharma, Oslo, Norway) as previously described (Zannettino
et al., 1998). Following centrifugation at 400.times.g for 30
minutes at 4.degree. C., the buffy layer is removed with a transfer
pipette and washed three times in "HHF", composed of Hank's
balanced salt solution (HBSS; Life Technologies, Gaithersburg,
Md.), containing 5% fetal calf serum (FCS, CSL Limited, Victoria,
Australia).
[0141] TNAP+ were subsequently isolated by magnetic activated cell
sorting as previously described (Gronthos et al., 2003; Gronthos et
al., 1995). Briefly, approximately 1-3.times.10.sup.8 BMMNC are
incubated in blocking buffer, consisting of 10% (v/v) normal rabbit
serum in HHF for 20 minutes on ice. The cells are incubated with
200 .mu.l of a 10 .mu.g/ml solution of STRO-3 mAb in blocking
buffer for 1 hour on ice. The cells are subsequently washed twice
in HHF by centrifugation at 400.times.g. A 1/50 dilution of goat
anti-mouse .gamma.biotin (Southern Biotechnology Associates,
Birmingham, UK) in HHF buffer is added and the cells incubated for
1 hour on ice. Cells are washed twice in MACS buffer (Ca.sup.2+-and
Mn.sup.2+-free PBS supplemented with 1% BSA, 5 mM EDTA and 0.01%
sodium azide) as above and resuspended in a final volume of 0.9 ml
MACS buffer.
[0142] One hundred .mu.l streptavidin microbeads (Miltenyi Biotec;
Bergisch Gladbach, Germany) are added to the cell suspension and
incubated on ice for 15 minutes. The cell suspension is washed
twice and resuspended in 0.5 ml of MACS buffer and subsequently
loaded onto a mini MACS column (MS Columns, Miltenyi Biotec), and
washed three times with 0.5 ml MACS buffer to retrieve the cells
which did not bind the STRO-3 mAb (deposited on 19 Dec. 2005 with
American Type Culture Collection (ATCC) under accession number
PTA-7282-see WO/2006/108229). After addition of a further 1 ml MACS
buffer, the column is removed from the magnet and the TNAP-positive
cells are isolated by positive pressure. An aliquot of cells from
each fraction can be stained with streptavidin-FITC and the purity
assessed by flow cytometry.
[0143] Primary cultures are established from the MACS isolated
TNAP+ cells by plating in .alpha.-MEM supplemented with 20% fetal
calf serum, 2mM L-glutamine and 100 .mu.m L-ascorbate-2-phosphate
as previously described (Gronthos et al., 1995).
[0144] Cells were cultured up to passage 5 at which point the
conditioned medium (supernatant) may be collected.
Example 2
Studies on the Dose Dependent Intra-Articular Effects of Allogeneic
Immunoselected Mesenchymal Precursors Cells (MPC) on Cartilage
Integrity in a Model of Early OA Induced by Bilateral Total Medial
Meniscectomy in Adult Castrated Male Sheep (Wethers).
[0145] The knee joint menisci, or semi-lunar cartilages, are
important weight bearing structures that also serve to improve
articular cartilage lubrication and provide lateral stabilization
during joint articulation. Surgical removal of a torn or degenerate
meniscus, i.e., meniscectomy, is a common orthopaedic procedure but
is known to be associated with an increased risk of osteoarthritis
(OA) in later years (Englund, 2004). Mechanical entrapment of the
joint synovium in the space previously occupied by the surgically
excised meniscus is known to lead to the partial regeneration of a
meniscus replica (Moon et al., 1984). However, the results of
experimental meniscectomy studies in dogs indicate that these
replacement structures consisted essentially of fibrous tissue with
far inferior biomechanical properties to the original menisci
(Ghosh et al., 1983). Furthermore, the extent of OA development in
the joints of these experimental animals 6 months post-meniscectomy
was relatively severe, confirming the limited functional protection
offered by the regrown structures on articular cartilage (Ghosh et
al. 1983a). Large and small animal models of OA have permitted
longitudinal evaluation of spatial and temporal changes in joint
tissues that occur during the development of this disease which is
difficult obtain using human patients (Smith and Ghosh, 2001). In
merino sheep, lateral or medial meniscectomy has been shown to
reliably reproduce biochemical, biomechanical and histopathological
alterations typical of OA (Smith and Ghosh, 2001). The ovine OA
model has also been extensively used to investigate the outcomes of
various modalities of post-operative treatments (Ghosh, 1991; Smith
and Ghosh, 2001) but to date has not been employed to evaluate
meniscal regrowth and the progression of OA and how these events
might be influenced by intra-articular mesenchymal precursor cell
(MPC) therapy.
[0146] Our previous studies had shown that Bilateral Total Medial
Meniscectomy (BTM) in merino sheep resulted in pathological changes
in articular cartilage (AC), subchondral bone and synovial tissues
that were progressive and simulated the development of early human
osteoarthritis (OA). We previously used this animal model to
evaluate potential disease-modifying OA drugs.
Methods
[0147] BTM was undertaken in 36 adult Merino wethers. Two weeks
post BTM, joints were randomly injected with either 2mL high MW
Hyaluronan (HA) or 2mL allogeneic Stro-3+ MPC suspended in 2mL HA.
Four doses of MPC were studied: Group A=10 million (mil) MPC [n=6];
Group B=25 mil MPC [n=6]; Group C=100 mil MPC [n=18] and Group
D=150 mil MPC [n=6]. Groups A, B and D were sacrificed 12 weeks
post-BTM while Group C were sacrificed 12 [n=6], 24 [n=6] and 52
[n=6] weeks post-BTM.
[0148] At necropsy, both medial compartments of BTM joints were
scored by 2 blinded observers for AC lesions and osteophytes (OP)
using a 0-4 scale. Synovial tissue and a 5 mm wide coronal
osteochondral slice were removed from the mid-line of the femur and
tibia and processed and scored for histopathological changes
(Little et al., 1997) and histomorphometric analyses (Cake et al.,
2003) using the methods cited.
[0149] Intact patellae from all joints were subjected to
topographical biomechanical indentation studies to deterine the
stiffness and phase lag of the articular cartilage (Appleyard et
al., 2003).
[0150] Statistical analysis for treatment effect was undertaken
using Kruskal-Wallis nonparametric analysis and for specific
between group comparisons using Mann Whitney U nonparametric
analysis with p<0.05 considered significant.
[0151] Statistical analysis for comparison between group means for
MPC+HA injected and HA injected joints of each group was undertaken
using the equal variance two tailed Student's T-Test with p<0.05
considered significant.
[0152] Statistical analysis for comparison between patella
cartilages from MPC+HA injected (Treated) and HA injected joints of
each group was undertaken using an independent T-Test with
p<0.05 considered significant.
Results
[0153] Gross morphological scores 12 wks post BTM showed a
dose-dependent effect of MPC on AC integrity and OP formation; 100
mil MPC emerging as the most effective chondroprotective dose
relative to HA alone (FIGS. 1 and 2). Total AC score ratios
(HA+MPC)/(HA) showed 100>150>25=10 while OP ratios were
100=25>10>150 mil MPC (FIGS. 3 and 4). Statistically
significant (SS) lower score were observed for total femoral &
tibial AC (p=0.02) while p=0.052 was observed for Group C MPC
femoral cartilages compared to HA alone (FIG. 1).
[0154] Histomorphometric analysis of Group C MPC+HA tibial plateau
revealed that AC was thicker than the corresponding HA-AC in the
middle (p=0.057) and outer regions (p=0.028); all regions (p=0.01)
(FIG. 5). Mean modified Mankin scores for AC sections from Group C
MPC+HA joints were less than corresponding HA sections but were not
SS. In addition, when the ratios of the total Mankin scores for the
HA injected and contralateral HA+MPC injected joints from each
group were calculated and plotted it was clearly evident that the
100 million dose of MPC was the most efficacious (FIG. 6).
[0155] The question of the sustainability of the 100 million MPC
dose in preserving joint cartilage integrity was addressed by
studying the morphological, histological and biomechanical
properties of the tissues 22 and 50 weeks post injection ie, 24 and
52 weeks post meniscectomy. As is evident from FIGS. 7 and 8 the
difference between the mean values for morphological scores for HA
and HA+100 million MPC diminish over this time, although there is
some evidence of a therapeutic effect at 24 weeks. This view is
supported by the HA/MPC+HA data which indicated a stronger effect
of the cells in suppressing osteophyte scores for up to 52 weeks
(FIGS. 10 and 11). On the other hand, similar plots for the Mankin
histopathology scores showed that by 52 weeks the protective
effects of the MPC was lost (FIG. 11).
[0156] Biomechanical indentation studies on the patella cartilages
from joints of all the animal groups were generally consistent with
the morphological and histological data. However, the stiffness of
the cartilage is influenced by the thickness of the cartilage which
in the early phases of injury may be hypertrophic but normalize
with time. This situation may be occurring in the present model
since the patella cartilage stiffness determined for the 25 and 100
million MPC groups were significantly less than the 10 and 150
million MPC groups which, from other studies exhibited the most
damage tissues (FIGS. 12 and 13). This interpretation was supported
by the phase lag data which was significantly lower for the
patellae from the 100 million MPC group both relative to the
corresponding HA injected joints and the 150 million MPC dose (FIG.
14). Moreover, the mean phase lag values observed at 12 weeks were
found to significantly increase at 24 and 52 weeks post
meniscectomy confirming the loss of a useful therapeutic effect of
the injected MPC beyond 6-12 months (FIG. 15). Phase lag reflects
the molecular assembly of the the cartilage extracellular matrix
and the lower the angle (Phase) the greater the elasticity and thus
ability to recover from deformation (Cake et al., 2005).
[0157] The chondroprotective effects observed for the 100 mil MPC
injected joints diminished with time; the positive effects noted at
12 and 24 weeks BTM being lost by 52 weeks.
[0158] There was no evidence of synovial histopathology modulation.
Clinical and gross organ pathology conducted on these animals has
not shown any evidence of systemic adverse effect of MPC.
Conclusions
[0159] This is the first report, as far as we are aware, of a
beneficial therapeutic effect of allogeneic MPC on cartilage
integrity in a model of early OA. MPCs are known to release growth
factors and cytokines and also suppress the production of TNF-alpha
by other cells, while up-regulating anti-inflammatory cytokines
(eg. IL-4, IL-10). These paracrine activities of MPC could
stimulate chondrocyte biosynthesis of new matrix but also attenuate
local production and activity of catabolic mediators. The finding
in this study that 100 million MPC were chondroprotective was
consistent with such a mechanism of action. The data generated in
these sheep studies indicate that the duration of the
chondroprotective effect mediated by a single intra-articular
injection of 100 million MPC is between 6-12 months post treatment
suggesting that multiple injections may be required for the long
term management of the OA patient.
[0160] While intra-articular injections of HA are widely used for
the treatment of knee osteoarthritis there is limited evidence that
this therapy is chondroprotective (Ghosh et al., 2002). However,
intra-articular HA therapy is reported to provide symptomatic
relief in OA which is of slow onset, but more sustained than with
intra-articular corticosteroids (Bellamy et al., 2006).
Example 3
Relative Therapeutic Effects of Intra-Articular Injection of
Hyaluronan (HA) or 100 Million Mesenchymal Precursor Cells (MPC)
+HA on Cartilage Integrity in a Model of Severe Osteoarthritis
Induced by Bilateral Total Medial Meniscectomy in Stifle Joints of
Ovariectomized Ewes.
[0161] The knee joint meniscus performs an important role in
protecting articular cartilage (AC) against damage during normal
joint articulation (Arnoczky et al., 1988). Total or partial
excision of the meniscus in humans following its injury generally
results in premature degeneration of AC and progression to
osteoarthritis (OA) (Jorgensen et al., 1987; Roos et al., 1998 and
McNicholas et al., 2000). Experimental studies have shown that
unilateral or bilateral total meniscetomy in sheep also leads to
premature breakdown of AC and the early onset of OA (Ghosh et al,
1990; Appleyard et al., 1999 and Ghosh et al., 1993c).
[0162] Since the failure of AC in meniscectomised joints is a
consequence of the imposition of high focal and shearing stress on
cartilage, bilateral meniscectomy was found to induce a more rapid
progression of cartilage degeneration than unilateral meniscectomy
where supportive pain-free weight bearing can be accommodated by
use of the contralateral non-operated hind limb (Ghosh et al.,
1993a and 1993b; Appleyard et al., 2003; Little et al., 1997 and
Oakley et al., 2004). Furthermore, ovariectomised ewes subjected to
bilateral meniscectomy have also been shown to undergo a more
progressive OA than adult castrated males (wethers), largely due to
the depletion from their circulation of the cartilage protective
hormone, oestrogen (Parker et al., 2003). For these reasons
ovariectomised and bilaterally meniscectomised ewes are favoured as
a large animal model of OA to study the disease modifying
activities of anti-OA agents (Ghosh et al., 1993; Smith et al.,
1997; Burkhardt et al., 2001; Hwa et al., 2001 and
[0163] Cake et al., 2000). The ovariectomised/bilaterally
meniscectomised sheep model of OA was therefore selected for the
present investigation--the purpose of which was to evaluate the
effects of intra-articularly (IA) administered allogeneic
Mesenchymal Precursor Cells (MPC) on induction of growth or
regeneration of proteoglycan-rich cartilage and on
chondroprotection relative to a currently used anti-OA therapy, IA
Hyaluronan (HA).
Methods
[0164] Bilateral total medial meniscectomy (BTM) was undertaken in
18 adult Merino ewes that had undergone ovariectomy 3 months
previously using a published method (Cake et al., 1004). The
surgical procedure and post-operative regimen used for BTM was
identical to that described for the castrated male sheep BTM
studies described in Example 2.
[0165] Twelve weeks post BTM, 6 ewes were sacrificed while the
stifle (knee) joints of the remaining 12 meniscectomised ewes were
randomly injected with either 2mL high MW HA or 100 million MPC
suspended in 2mL Profreeze.RTM. plus 2mL HA. This dose of MPC+HA
was shown in Example 2 to afford the most beneficial
chondroprotective effects in the BTM male sheep model. The
meniscectomised and injected ewes were divided into two groups of 6
that were sacrificed 24 and 36 weeks post-BTM, i.e. 12 and 24 weeks
post HA or MPC+HA intra-articular injection. To determine the
effects of gender on the response of AC joint destabilization 6
untreated castrated male sheep were also subjected to BTM and
sacrificed 12 weeks post-meniscectomy.
[0166] At necropsy, joints were opened, menisci removed and the
medial femoral and tibial plateux photographed. The recorded images
were scored by 2 blinded observers for gross morphological changes
to cartilage using a 0-4 scale. Synovium from the suprapatellar
fold and a 5mm wide coronal osteochondral slice were removed from
the mid-line of the femur and tibia of each joint and processed for
preparation of histological sections. Cartilage histopathology was
assessed by two blinded observers using a modified Mankin Scoring
system as described previously (Little et al., 1997). Synovial
histopathology was scored using the criteria recently described by
Cake et al., 2008.
[0167] Histological serial sections from the same cartilage blocks
as used for Mankin Scoring were also utilized for histomorphometric
analysis as described previously (Caket et al., 2000; Cake et al.,
2004). This technique employs computer-aided image analysis
(ImagePro Plus v.3.0, Media Cybernetics) to generate quantitative
data on the dimensions and an index of the proteoglycan content of
Toluidine blue stained AC. In brief, images of the stained sections
were acquired via a Microtek Slidescanner 35t plus (Microtek Model
No. PTS-1950) at a resolution of 1300 dpi and then analysed using
Image J.RTM. software (http://rsb.info.nih.gov/ij/) on a personal
computer. The digital images of the femoral and tibial sections
were subdivided into inner, middle and outer regions, each region
representing approximately one third of the total area of the
cartilage sections. Spatial calibration of the system was achieved
by scanning a 10.times.10 mm high precision reticule. This scale
was then used to quantify the length (mm) and area (mm.sup.2) in of
each region of the imported images. The average thickness of the
sections was determined by dividing the area by the length. The
optical density (OD) of the TB stained cartilage sections was
obtained as the mean grey value (MGV) (sum grey values/number of
pixels) and was taken as an index of proteoglycan (PG) content. The
integrated grey-scale density (IGD) was calculated as MGV x
regional area of section. Although the grey scale system used was
not independently calibrated against TB stained sections of known
PG content, all histological sections were cut on the same
microtome, were the same thickness and were processed as a group
using the same staining protocol. Differences in cartilage staining
are therefore relative rather than absolute. Intact patellae from
all joints were removed within 1 hour of sacrifice and immediately
frozen and stored prior to topographical biomechanical indentation
studies to determine the stiffness and phase lag of the articular
cartilage (Appleyard et al., 2003).
[0168] Statistical analysis to identify differences in treatments
outcomes (HA versus HA+MPC) or treatments versus untreated 12 week
post BTM controls, as assessed by the morphological and
histological scoring systems, was undertaken using Kruskal-Wallis
nonparametric analysis and for differences between group
comparisons using Maim Whitney U nonparametric analysis with
p<0.05 considered significant.
[0169] Data generated by the histomorphometric analysis of
digitised histological sections were evaluated using the equal
variance Two Tailed Student's T-Test with p<0.05 considered to
be significant. Statistical analysis of patella cartilages
biomechanical parameters with respect to different treatments and
between time post-BTM was calculated using an independent T-Test
with p<0.05 considered significant.
Results
[0170] The gross morphological assessment of cartilage erosions and
osteophyte formation in joints from the untreated ewes 12 weeks
post-BTM confirmed that this model of OA represented a more
aggressive and severe form of the disease compared with
meniscectomised castrated males subjected to the same surgical
procedure. For this untreated female control group the mean
cartilage morphological score for the femur was 87% and for the
tibia 75% of the maximum scores used to assess this parameter. The
gross morphology scores obtained for the joints derived from the
untreated 12 week post meniscectomised castrated males subjected to
the same surgical procedure was significantly less than for the
ovariectomised ewes (FIGS. 16 and 17) a finding which was
consistent with previous observations using bilateral lateral
meniscectomy (Parker et al., 2003; Cake et al., 2004).
[0171] While both treatments resulted in lower mean femoral
morphological cartilage scores at 24 and 36 weeks than the baseline
untreated 12 week post-meniscectomised ewes (data not shown), no
significant differences were detected between the MPC and HA
treated joints. We interpret this to mean that in this model of
severe OA, the severity of the gross morphologic lesions (erosion
and osteophyte scores) make these parameters too insensitive to
detect therapeutic differences.
[0172] Modified Mankin histopathology scores for cartilages from
the untreated 12 weeks post-BTM group were found to be consistent
with the extent of cartilage damage as assessed morphologically
(FIGS. 16 and 17). In contrast to morphologic parameters, at 36
weeks the total mean modified Mankin score for the femoral
cartilages in the ovariectomised ewes who received MPC+HA was lower
than the corresponding score for the joints that received HA alone
and showed a significantly lower cell number (p=0.01) and a trend
(p=0.06) for stronger inter-territorial Toluidine Blue (IT TB)
staining for proteoglycans than HA alone (FIG. 18). These effects
were less pronounced for the tibial cartilages (FIG. 18).
[0173] The lower Modified Mankin histopathology cartilage score
observed for the MPC+HA injections at 36 weeks post meniscectomy
relative to the HA injected joints was highlighted when the ratio
of the mean total Modified Mankin scores for the two
intra-articular treatments were determined (FIG. 19). As each ratio
was obtained from the two treated joints of the same animal a
ratio=1 would indicate that both treatments were equally effective.
However, for the ratios>1 the MPC+HA treatment can be said to be
more beneficial. As is evident from FIG. 19 the mean of the ratios
obtained for the femoral cartilages were significantly higher
(1.71) than unity at 36 weeks post-BTM while the tibial cartilage
ratios (1.12) for the two treatments were only slightly in favor of
the MPC+HA injected group (FIG. 19).
[0174] Next we examined the effect of the treatments on Mankin
scores over time. Significant differences in effects on femoral
cartilage over time were found between the MPC+HA and the HA alone
treatment arms at 24 and 36 weeks post meniscectomy, i.e. 12 and 24
weeks post-injection (FIG. 20). In the group receiving MPC+HA, mean
scores at 24 and 36 weeks were progressively lower than at the 12
week baseline. This was due to reduced scores and improvement in
cell cloning (P=0.01) at 24 weeks, and in cell numbers (P=0.04) and
inter-territorial Toluidine Blue staining for proteoglycans (PGs)
(P=0.04) at 36 weeks relative to the 12 week untreated group (FIG.
20). No such improvements were seen in the HA alone group. No
significant differences were observed between the synovial
pathology scores for any of the groups or intra-articular
treatments.
[0175] The analysis of cartilage thickness, area and intensity of
TB staining as an index of PG content for the 3 regions (inner,
middle and outer) of the femoral condyles from the injected joints
at 36 week post-BTM using histomorphometric methods of analysis are
shown in FIG. 21. By 36 weeks post-BTM significant differences
between treatments groups were evident. Femoral cartilages from the
MPC+HA injected joints were significantly thicker (FIG. 21A) and
occupied a significantly larger area (FIG. 21B) than the
corresponding cartilages of HA injected joints. The larger volume
of the femoral cartilages from the MPC+HA injected joints was
accompanied by a higher content of proteoglycans as determined from
the integrated grey-scale density of the TB stained sections (FIG.
21C).
[0176] Again comparing these parameters in a time-based analysis,
significant differences in effects on femoral cartilage over time
were found between the MPC+HA and the HA alone treatment arms at 24
and 36 weeks post meniscectomy, i.e. 12 and 24 weeks
post-injection. Using the same histomorphometric methodology, we
were able to demonstrate that the MPC+HA injection administered 12
weeks post meniscectomy resulted in progressively greater
proteoglycan-rich femoral cartilage growth or regeneration 12 and
24 weeks later (i.e. at 24 and 36 weeks post-BTM) than HA alone
(FIGS. 22 to 24). Thus, femoral cartilages at 24 and 36 weeks
post-BTM from joints of meniscectomised ewes injected with MPC+HA
at 12 weeks were significantly thicker (FIG. 22) and generally had
larger areas (FIG. 23) than the baseline values from untreated
joints at 12 weeks post-meniscectomy. The corresponding regions
scanned from sections of femoral cartilage derived from HA injected
joints failed to demonstrate statistically significant changes
relative to the 12 week untreated controls (FIGS. 22 & 23). The
integrated grey-scale density as a measure of PG content of
sections of femoral cartilages was significantly higher for both HA
and MPC+HA injected joints relative to the same cartilage regions
of joints from the untreated 12 week post-BTM group but the
magnitude of the MPC+HA induced change was significantly greater
than HA alone (FIG. 24). Whereas the MPC+HA group developed almost
60% greater proteoglycan-rich femoral tissue at 36 weeks compared
with baseline (P<0.001), and this rate of cartilage growth had
not reached a plateau phase, the HA only group had reached a
plateau phase and developed only about 30% greater tissue. This
indicated that treatment with MPC+HA stimulated significantly
greater increase in proteoglycan-rich cartilage over the 24 week
period of follow-up (i.e. growth and/or regeneration of cartilage)
relative to both baseline and to any temporal effects of HA
treatment alone.
[0177] The results of the indentation studies on the patella
cartilages from the injected joints failed to demonstrate any
difference in the biomechanical properties of the cartilages for
the two treatments but changes were identified with respect to time
elapsed post meniscectomy and the untreated 12 week post-BTM group.
The stiffness of the patella cartilages from the MPC+HA at 24 weeks
post-meniscectomy was significantly higher than at 12 weeks
(P=0.05) and 36 weeks (P<0.01) (data not shown). However, both
treatments produced thicker patella cartilage 36 weeks compared to
24 weeks (P=0.001) that was also lower than the non-treated 12 week
control (P=0.01). Patella cartilage phase-lag for both treatment
groups at 24 and 36 weeks were higher than the untreated 12 week
controls (P=0.001) (data not shown).
Discussion
[0178] The present studies have shown that bilateral medial
meniscectomy in ovariectomised ewes induced pathological changes in
joint articular cartilage after 3 months that were consistent with
progressive and severe OA. Thus the gross morphology scores for the
femoral and tibial cartilages were 87-70% of the maximum score.
Interestingly, castrated males subjected to the same surgical
procedure and sacrificed at the same time (12 weeks) showed less
severe cartilage lesions than the observed for the ovariectomised
females. The extent of cartilage pathology was also reflected in
the high aggregate Modified Mankin histopathology scores observed
for this group that were consistent with the assignment of early OA
(Little et al., 1997). Although previous studies had identified a
strong association of OA in postmenopausal females, which was
explained by the depletion of estrogen from the circulation (Roos
et al., 2001; Pelletier et al., 2007 and Nevitt et al., 1996) and
was supported by studies in ovariectomised ewes (Parker et al.,
2003 and Cake et al., 2004), other more recent studies suggests
that the adipose derived hormone, Leptin, may play a more
significant role in mediating cartilage breakdown and OA (Dumond et
al, 2003 and Teichtahl et al., 2005). The 3 months post BTM period
was therefore taken as the starting point for the evaluation of the
relative effects of intra-articular injections of HA or MPC+HA on
the rate of progression of cartilage pathology 12 and 24 weeks
following the administration of these agents.
[0179] The results of this study indicated that a single
intra-articular injection of 100 million MPC dispersed in 2mL HA
and 2 mL Profreeze.RTM. (a commercial cryoprotectant) into joints
with established, severe OA can, over an intervening period of 24
weeks, slow the progression of joint pathology and enhance growth
and/or regeneration of proteoglycan-rich cartilage to a greater
extent than a single injection of 2 mL HA. Surprisingly, the
growth/regenerative and chondroprotective effects mediated by the
MPC were observed to be more significant 24 weeks after
administration than after 12 weeks in the majority of parameters
examined, indicating progressive effects which had not yet reached
a plateau phase. The reasons for this finding are presently unclear
however, it is possible that the growth factors such as members of
the TGF-beta superfamily, eg BMPs, released by the MPC (Ahrens et
al., 1993; Aggarwal et al., 2005) were supportive of the anabolic
(compensatory) phase of cartilage to the altered mechanical
stresses imposed across the joint by medial meniscectomy. This view
was supported by the histomorphometric data that demonstrated the
presence of higher volumes and more intense staining for
proteoglycans in the MPC injected groups than at the commencement
of treatment at 12 weeks post-BTM. These matrix changes are
consistent with increase chondrocyte biosynthesis. Significantly,
the magnitude of the anabolic parameters was generally found to be
greater in the cartilages of animals who received the MPC+HA rather
than HA alone. The ability of MPC to preserve and even enhance this
cartilage response to mechanical overload contrasts with the known
inhibitory effects on chondrocyte metabolism mediated by many
traditional treatments of OA, including many of the steroidal and
non-steroidal anti-inflammatory drugs (NSAIDs) (McKenzie et al.,
1976; Ghosh, 1988; Brandt, 1993 and 1993a; Huskisson et al.,
1995).
[0180] Multiple intra-articular HA injections have been used as a
therapy for the management of knee OA for more than 30 years.
Although the consensus is that this form of treatment does provide
symptomatic relief clinically, a recent review and a meta-analysis
of published HA clinical trials have questioned the validity of
this conclusion on the basis of the stronger placebo effects
associated with intra-articular injections, difficulty of blinding
investigators and publication biases (Brandt et al., 2000; Lo et
al., 2003). Whether intra-articular HA exhibits any
chondroprotective or cartilage regenerative activity is also
controversial. However, extensive animal investigations have shown
that HA does exhibit analgesic, anti-inflammatory and disease
modifying effects in rabbit and ovine models of OA induced by
uni-lateral and bilateral meniscectomy as well as anterior cruciate
ligament transection in dogs. A discussion of these data together
with preclinical and laboratory based clinical studies with HAs of
different molecular weight has been reviewed (Ghosh et al.,
2002).
[0181] In the present study only a single intra-articular injection
of HA, either alone or in combination with MPC, was evaluated. On
the basis of our own data we conclude that the long-lasting growth
and regenerative, as well as chondroprotective, effects afforded by
the MPC+HA combination was mediated by the MPC. In this regard it
is important to note that the design of this study allowed each
animal to act as its own control since one joint received HA while
the contra-lateral joint received the same quantity of HA plus the
MPC in the cryoprotectant, Profreeze.RTM.. Since both knee joints
were surgically de-stabilised in the present study and were
injected at the same time we are confident that the magnitude and
nature of the weight-bearing mechanical stresses acting on the
articular cartilages was the same on both joints.
[0182] From the present studies we conclude that a single
intra-articular administration of MPC+HA into ovine joints with
pre-existing, severe OA results in growth or regeneration of
proteoglycan-rich cartilage as manifest by increased cartilage
extracellular matrix 24 weeks post treatment relative to baseline
pre-treatment and to HA injected controls.
Example 4
Ovine Disc Re-Generation Studies Using Immunoselected MPC
Methods
[0183] Thirty-six age-matched, Merino wethers (approximately 18 to
24 months old) were used for this study. In all 36 sheep three
adjacent lumbar discs (L3-L4, L4-L5, L5-L6) were injected with 1.0
IU chondroitinase ABC (Seikagaku Corporation, Japan) in
approximately 0.1 ml sterile normal saline to breakdown and remove
the PGs of the NP. The remaining lumbar discs (L1-L2 and L2-L3)
were not injected with chondroitinase ABC and served as controls.
Fifteen weeks (.+-.3 weeks) following administration of
chondroitinase ABC, injections MPCs (0.5.times.10.sup.6 cells) in
ProFreeze.TM. Freezing Medium (NAO) or ProFreeze.TM. NAO alone
(Lonza Walkersville Ltd.) mixed with an equal volume of hyaluronic
acid (Euflexxa.RTM.,
[0184] (Ferring Pharmaceuticals) were administered directly into
the chondroitinase ABC treated nuclei pulposi of the intervertebral
discs identified schematically in FIG. 25. The respective
experimental groups were sacrificed 3 and 6 months later as
summarized in Table 1.
TABLE-US-00001 TABLE 1 Study Design Summary 15 .+-. 3 weeks
Baseline Group No. Disc before Baseline Day 0 Sacrifice Analysis at
Sacrifice 1 n = 6 L1-L2 No injection No injection 3 mths
Compositional/Histology L2-L3 No injection No injection 3 mths
Compositional/Histology L3-L4 Chondroitinase MPCs 0.5 .times.
10.sup.6 3 mths Compositional/Histology L4-L5 Chondroitinase No
injection 3 mths Compositional/Histology L5-L6 Chondroitinase HA
and NAO 3 mths Compositional/Histology 2 n = 6 L1-L2 No injection
No injection 6 mths Compositional/Histology L2-L3 No injection No
injection 6 mths Compositional/Histology L3-L4 Chondroitinase MPCs
0.5 .times. 10.sup.6 6 mths Compositional/Histology L4-L5
Chondroitinase No injection 6 mths Compositional/Histology L5-L6
Chondroitinase HA and NAO 6 mths Compositional/Histology
[0185] Animals had lateral plain radiographs taken of the lumbar
spine under induction anaesthesia at the following time points: Day
0 (Injection of chondroitinase ABC (Seikagaku Corporation, Japan),
Day of Test Article administration (15.+-.3 weeks following
induction of lumbar disc degeneration) and 3 months and 6 months
following implantation of the Test Article. Evaluation of the
radiographs was undertaken using an index of intervertebral height
(DHI) calculated by averaging the measurements from the anterior,
middle and posterior parts of the IVD and dividing it by the
average of the adjacent intervertebral body heights as described by
(Masuda et al., 2004).
[0186] The MRIs were taken of the lumbar spine under induction
anaesthesia at the following time points: Day Zero (injection of
chondroitase ABC [Seikagu Corp Japan]), Day of test article
administration (15+3 weeks following induction of lumbar disc
degeneration), 3 months and 6 months following implantation of test
article. Disc were graded from the MRI scans using the Pfirrmann
Classification System (Pfirrmann et al., 2001).
[0187] Spinal motion segments that were designated for
histochemical and biochemical analysis were isolated by cutting
through the cranial and caudal vertebral bodies close to the
cartilaginous endplates using a bone saw. These spinal sections
were fixed en bloc in Histochoice.RTM. for 56 h and decalcified in
several changes of 10% formic acid in 5% Neutral Buffered Formalin
for 2 weeks with constant agitation until complete decalcification
was confirmed using a Faxitron HP43855A X-ray cabinet (Hewlett
Packard, McMinnville, USA).
[0188] Multiple sagittal slices of the decalcified specimens,
approximately 5 mm thick, were dehydrated through graded ethanol
solutions by standard histological methods and embedded in paraffin
wax. Paraffin sections 4 .mu.m thick were mounted on Superfrost
Plus glass microscope slides (Menzel-Glaser), dried at 85.degree.
C. for 30 min then at 55.degree. C. overnight. The sections were
then deparaffinised in xylene (4 changes.times.2 min) and
rehydrated through graded ethanol washes (100-70% v/v) to tap
water. One section from all blocks prepared from the sagittal
slices was stained with haematoxylin and eosin. The coded section
was examined by an independent histopathologist who compared the
histological characteristics of those levels that were subjected to
enzyme injection only with those that were enzyme-injected and
subsequently received MPCs. A four-point semi-quantitative grading
system was used to assess the microscopic features of the entire
disc as shown in Table 2. Additional tinctorial stains including
Alcian Blue (for general glycosaminoglycan species) and Safranin O
(specific for chondroitin sulphate species) were also prepared to
demonstrate the extent of disc matrix synthesis.
[0189] The immunohistochemistry procedures were also performed
using a Sequenza cassette and disposable Coverplate immunostaining
system as described previously (Melrose et al., 2003; Melrose et
al., 2002; Melrose et al., 2000; Melrose et al., 2002a; Melrose et
al., 1998; Panjabi et al., 1985; Race et al., 2000; Smit, 2002).
Endogenous peroxidase activity was initially blocked by incubating
the tissue sections with 3% H.sub.2O.sub.2. They were then
pre-digested with combinations of chondroitinase ABC (0.25 U/ml) in
20 mM Tris-acetate buffer pH 8.0 for 1 h at 37.degree. C., bovine
testicular hyaluronidase 1000 U/ml for 1 h at 37.degree. C. in
phosphate buffer pH 5.0, followed by three washes in 20 mM Tris-HCl
pH 7.2 0.5M NaCl (TBS) or proteinase-K (DAKO S3020) for 6 min at
room temperature to expose antigenic epitopes. The tissues were
then blocked for 1 h in 20% normal swine serum and probed with a
number of primary antibodies to large and small proteoglycans and
collagens (Table 3). Negative control sections were also processed
either omitting primary antibody or substituting an irrelevant
isotype matched primary antibody for the authentic primary antibody
of interest. Commercial (DAKO) isotype matched mouse IgG (DAKO Code
X931) or IgM (DAKO Code X942) control antibodies (as appropriate)
were used for this step. The DAKO products X931 and X942 are mouse
monoclonal IgG.sub.1 (clone DAK-GO1) and monoclonal IgM (clone
DAK-G08) antibodies directed against Aspergillus niger glucose
oxidase, an enzyme that is neither present nor inducible in
mammalian tissues. Horseradish peroxidase or alkaline phosphatase
conjugated secondary antibodies were used for the detection using
0.05% 3, 3'-diaminobenzidene dihydrochloride and 0.03%
H.sub.2O.sub.2 in TBS, Nova RED, nitroblue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate/iodo
nitrotetrazolium violet (NBT/BCIP/INT) or New Fuchsin as
substrates. The stained slides were examined by bright field
microscopy and photographed using a Leica MPS 60 photomicroscope
digital camera system.
TABLE-US-00002 TABLE 2 Grading system of histologic changes in
lower lumbar discs (BEP bony end-plate, CEP cartilaginous
end-plate) Grade Annulus fibrosis Nucleus pulposus Cartilage
end-plate Margins/subchondral bone 1 Intact lamellae Homogeneity
Uniform thickness Even thickness of BEP Narrow inter-lamellar
matrix Absence of clefting Intact attachment to bone Lamellar bone
only Intact annulus attachment Uniform calcification <1/5 of
depth Distinct junction with CEP Vessels only in outer 1/3 Uniform
cell distribution Few vascular intrusions into CEP 2 Minor lamellar
splitting and Minor clefting Minor cartilage-thinning Slightly
uneven BEP disorganisation. Minor widening of Minor cell necrosis
Small transverse fissures Schmorl's nodes matrix Minor
disorganisation of Minor posterior displacement of Irregular
thickening of calcified Minimal remodelling of BEP attachment Rim
lesion without annulus zone Small marginal osteophytes reparative
reaction Minor chondrone formation Few invading vascular channels
Small chondrones 3 Moderate widening of matrix Moderate clefting
Marked cartilage thinning Moderately uneven BEP moderate fissuring
of attachment Moderate cell necrosis Marked thickening of calcified
Vascularised Schmorl's nodes Radiating tears not involving outer
Cystic degeneration zone Moderate trabecular thickening 1/3 minimal
chondroid metaplasia Posterior displacement within Many transverse
fissures Defect in bone lamellae Cystic degeneration Vessels in
annulus Many vascular channels Minimal fibrosis tissue outewr and
middle 1/3 rim lesion Centripetal extension of collagen Many
chondrones in marrow spaces with minor reparative reaction Moderate
chondrone formation Medium-size osteophytes 4 Extensive lamellar
disorganisation Complete loss of nucleus Total loss of cartilage
Marked uneven BEP Radiating tears extending into outer Loose body
formation Calcification of residual cartilage Ossified Schmorl's
nodes 1/3 Marked chondrone formation Widespread fissuring Large
osteophytes Extensive chondroid metaplasia Marked trabecular
thickening Vessels in all zones Marked fibrosis of Rim lesion with
marked reparative marrow spaces reaction Cartilage formation
TABLE-US-00003 TABLE 3 Primary antibodies to proteoglycan and
collagen core protein epitopes Primary antibody epitope Clone
(isotype) References Large PGs Aggrecan AD 11-2A9 (IgG) 26, 30
Versican 12C5 (IgG) 26, 28 Collagen Type I I8H5 (IgG.sub.1) 23, 28
Type II II-4CII (IgG.sub.1) 28 Type IV CIV-22 (IgG.sub.1) 28 Type
VI Rabbit polyclonal 28 Type IX Mouse monoclonals D1-9 (IgG.sub.1),
35 B3-1 (IgG.sub.2b)
[0190] Samples of annulus fibrosus and nucleus pulposus were
dissected from the processed blocks finely diced and representative
portions of the tissue zone of known wet weight were freeze dried
to constant weight. Triplicate portions (1-2 mg) of the dried
tissues were hydrolysed in 6M HCl at 110.degree. C. for 16 h and
aliquots of the neutralised digests assayed for hydroxyproline as a
measure of the tissue collagen content (Sakai et al., 2005).
Triplicate portions of dried tissues (.about.2 mg) will also be
digested with papain and aliquots of the solubilised tissue assayed
for sulphated glycosaminoglycan using the metachromatic dye 1,
9-dimethylmethylene blue as a measure of PGs (Sakai et al.,
2005).
[0191] The motion segments were wrapped in saline-soaked gauze,
sealed in double thickness polythene bags and frozen at -30.degree.
C. until biomechanical testing. This treatment has been shown not
to alter the biomechanical characteristics of the tissue (Panjabi
et al.,1985). Biomechanical testing was undertaken to measure the
stiffness of each disc in axial compression, flexion, extension,
lateral bending and axial torsion under defined computer-controlled
conditions approximating physiological loading (Panjabi et
al.,1985; Race et al., 2000; Smit, 2002; Wilke et al., 1999). Full
details of the testing protocol are documented elsewhere (Panjabi
et al.,1985; Race et al., 2000; Smit, 2002; Wilke et al., 1999).
The specimens for testing (functional spinal units, FSUs) comprised
two adjacent vertebrae, the intervening disc and associated
ligaments. Three FSUs per spine were tested: a level that was only
degraded with C-ABC only, one in which the disc was degraded with
C-ABC and which was subsequently treated with hyaluronic acid only
and the central level that was degraded with C-ABC and which was
subsequently treated with hyaluronic acid and with MPCs. Each FSU
was mounted in two aluminium alloy cups and secured with three
bolts and cold cure polymethyl methacrylate dental cement (Vertex
SC Self Curing, Dentimex BV, Zeist, Holland). Care was taken to
ensure that the midline of the intervertebral disc is positioned
horizontally. The motion segments will be centred in the cups by
placing a dowel through the vertebral canal into a hole in one of
the cups. All tests were conducted in a saline water bath
maintained at 37.degree. C. Prior to the commencement of testing
each FSU will be preloaded to a stress of 0.5 MPa until a
reproducible state of hydration is achieved. This was used as the
baseline prior to each test. The preload stress of 0.5 MPa
simulates relaxed standing and was based on in vivo measurement of
intradiscal pressure.
[0192] Mechanical tests were performed using a Model 8511 Dynamic
Servohydraulic Materials Testing Machine (INSTRON Pty Ltd, High
Wycombe, UK) equipped with a `six degrees of freedom` load cell to
allow the simultaneous monitoring and control of forces in all
three planes. The machine was controlled by a personal computer and
custom-designed software that also records and analyses the data.
Test data was acquired in stable hysteresis from the final of five
sinusoidal 0.1 Hz loading cycles in either axial load or torsion
control. The tests were performed are pure axial compression, left
and right lateral bending, combined flexion/extension and pure
axial torsion.
[0193] Pure axial compression to 200N was produced in the FSU with
little or no bending or flexion accompanying the load. All
compressive tests were performed using point contact on the cranial
cup surface. The neutral axis of bending (NAB) is determined by
applying a cyclic load to the joint through a point on the
aluminium alloy cup holding the specimen to achieve negligible
bending. This trial and error process enables as close to pure
axial compression as possible using a rigid point load contact.
Despite slight variability between specimens this point is found on
the sagittal plane approximately 10 mm anterior to the spinal canal
but slightly posterior to the disc centroid. Marks were placed 10
mm anterior and posterior and to the left and right of the NAB to
position the offset loads for the bending tests. A maximum
compressive load of 200 N was applied at each point to produce 2 Nm
of bending and 200 N of axial compression.
[0194] Conservative bending and compressive loads were chosen to
ensure that the disc, posterior elements, endplates and other
ligamentous structures were not e damaged. Pure bending was not
produced using this loading method. Instead a combination of
bending and axial compression was present for the combined
flexion/extension and lateral bending tests. We believe this was
justified given that in vivo loading would seldom produce pure
bending but rather a combination of compression and bending. In
either load case, all loads were applied consistently to each
specimen allowing direct comparisons of the mechanical
response.
[0195] For the torsion tests 5 Nm of pure axial torsion will be
applied. This was within the physiological range of torques
estimated from, and applied in, other studies. A novel custom
designed torsion testing system will be used to apply pure torsion
to each FSU. This system uses a ballscrew/thrust plate mechanism to
convert the axial displacement of the Instron actuator into pure
rotation. An X-Y bearing table ensures that the FSU does not have a
fixed centre of rotation imposed on it during testing. This is
important, as the centre of rotation is not constant during axial
rotation. The inferior cup was fixed to a torque transducer with
the superior cup fixed to the X-Y bearing table and
ballscrew/thrust plate mechanism.
[0196] All tests were conducted on the intact FSU initially. Once
completed the disc were isolated by cutting through the posterior
elements using a small hacksaw blade passed through the neural
foramen and cutting posteriorly. This cut through the zygapophysial
joints and the interspinous and supraspinous ligaments, leaving the
intervertebral disc, the posterior and the anterior longitudinal
ligaments intact. The cut was made in a wedge fashion increasing
posteriorly to ensure no contact between the zygapophyseal joints.
All tests were then repeated on the isolated disc.
[0197] Data analysis included parameters such as stiffness in the
linear region during the fifth loading cycle, hysteresis and strain
energy and the extent of the neutral zone. Data from the control
levels was compared with the degenerated/MPC-injected levels and
repeated measures analysis of variance was conducted on each of the
biomechanical parameters.
Results
[0198] All animals in the MPC injected groups maintained normal
body weights and showed no evidence of adverse side effects over
the duration of the experiment.
[0199] In the Chondroitinase-ABC injected discs the depletion of
PGs by this enzyme resulted in a 38% decrease in disc height index
(DHI) in all injected discs after 3 months. This loss of disc
height confirmed the degenerate status of the nucleus pulposus
prior to treatments and hitherto is referred to as the pre-MPC DHI.
Three months post HA or MPC+HA injection into the degenerate discs
failed to produce any significant increase in DHI relative to the
pre-MPC DHI (FIG. 26). However, by 6 months post treatment, discs
injected with MPC+HA showed a mean increase of 52% in DHI relative
to the corresponding 3 month scores (Group 1) (FIG. 26 and Table
4). In contrast, discs injected with HA alone only showed a 23.1%
mean improvement in the DHI scores over the same period (FIGS. 26
and Table 4). Significantly, the mean DHI of the low MPC+HA
injected discs were comparable 6 months post treatment to the DHA
scores for the non-chondroitinase ABC injected (ie, non-degenerate)
control discs (FIG. 26).
[0200] A statistical analysis for the DHI for 6 versus 3 months
post HA or MPC+HA injection is shown in Table 4.
[0201] Administration of ovine MPC together with a suitable
carrier, such as high molecular weight hyaluronic acid (HA), into
the nucleus pulposus of experimentally created degenerate IVDs has
been shown in the present experiments to accelerate the
regeneration of the disc extracellular matrix as assessed
radiographically by the recovery of disc height. This
interpretation is based on the assumption that in the loaded spinal
column the disc height is maintained by the presence within the NP
and inner-annulus of high concentrations of matrix proteoglycans
that together with their bound water molecules confer a high
swelling pressure to this structure. Indeed, the use of
chondroitinase-ABC to induce disc degeneration at the commencement
of these experiments relied on the ability of this enzyme to
degrade and remove the majority of the proteoglycans from the NP
extracellular matrix.
[0202] The data obtained to date suggests that the therapeutic
effect mediated by the MPC is a relatively slow process. In the
present study, the dose of 0.5.times.10.sup.6 MPC was particularly
effective.
[0203] Although the present experiments were terminated 6 months
after the MPC were injected into the disc, the level of disc height
recovery obtained for the low dose MPC injections was found to be
close to the values observed for the non-chondroitinase ABC
injected internal controls, suggesting that the maximum extent of
NP reconstitution was achieved over this period.
[0204] It will be appreciated by persons skilled in the art that
numerous variations and/or modifications may be made to the
invention as shown in the specific embodiments without departing
from the spirit or scope of the invention as broadly described. The
present embodiments are, therefore, to be considered in all
respects as illustrative and not restrictive.
[0205] All publications discussed and/or referenced herein are
incorporated herein in their entirety.
[0206] Any discussion of documents, acts, materials, devices,
articles or the like which has been included in the present
specification is solely for the purpose of providing a context for
the present invention. It is not to be taken as an admission that
any or all of these matters form part of the prior art base or were
common general knowledge in the field relevant to the present
invention as it existed before the priority date of each claim of
this application.
TABLE-US-00004 TABLE 4 Extent of disc height restoration 3 and 6
months post intra-discal injection of Mesenchymal Precursor Cells
(MPC) + Hyaluronan (HA) or HA alone into degenerate sheep lumber
discs PRE-MPC INJECTION 3 MONTHS POST MPC DHI INJECTION DHI
Non-injected cABC only cABC + HA cABC + HA + MPC Non-injected cABC
only cABC + HA MEAN 0.054 0.04 0.04 0.04 0.055 0.0416667 0.0433333
Std Deviation 0.015 0.01 0.01 0.01 0.01 0.014 0.008 % Change from 3
months to 6 months Statistical Significance (P values) P < 0.05
= significant DHI = Disc Height cABC = Chondroitinase ABC Index 3
MONTHS POST MPC INJECTION DHI 6 MONTHS POST MPC INJECTION DHI cABC
+ HA + MPC Non-injected cABC only cABC + HA cABC + HA + MPC MEAN
0.0383333 0.0566667 0.0516667 0.0533333 0.05833333 Std Deviation
0.014 0.015 0.017 0.008 0.007 % Change from 0.2 23.9 23.1 52.174 3
months to 6 months Statistical 0.83 0.31 0.059 0.0142 Significance
(P values) P < 0.05 = significant cABC = Chondroitinase ABC
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