U.S. patent application number 17/527498 was filed with the patent office on 2022-03-10 for methods and compositions to facilitate repair of avascular tissue.
The applicant listed for this patent is Regenerative Sciences, LLC. Invention is credited to Christopher J. Centeno.
Application Number | 20220072053 17/527498 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-10 |
United States Patent
Application |
20220072053 |
Kind Code |
A1 |
Centeno; Christopher J. |
March 10, 2022 |
METHODS AND COMPOSITIONS TO FACILITATE REPAIR OF AVASCULAR
TISSUE
Abstract
Compositions and methods are provided for repairing damaged
avascular zones, including intervertebral disc, in a patient in
need thereof.
Inventors: |
Centeno; Christopher J.;
(Broomfield, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Regenerative Sciences, LLC |
Broomfield |
CO |
US |
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|
Appl. No.: |
17/527498 |
Filed: |
November 16, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16441897 |
Jun 14, 2019 |
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17527498 |
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13132840 |
Jun 3, 2011 |
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PCT/US2009/066773 |
Dec 4, 2009 |
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16441897 |
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61120098 |
Dec 5, 2008 |
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61154874 |
Feb 24, 2009 |
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International
Class: |
A61K 35/28 20060101
A61K035/28; A61K 38/19 20060101 A61K038/19; A61K 35/19 20060101
A61K035/19; A61K 38/18 20060101 A61K038/18 |
Claims
1. A method for treating a degenerative intervertebral disc in a
patient in need thereof comprising: culturing harvested nucleated
cells in a culture medium under selective pressure of about 1% to
about 10% oxygen for 1-28 days; obtaining viable mesenchymal stem
cells capable of growth in the culture medium under selective
pressure of about 1% to about 10% oxygen; implanting the viable
mesenchymal stem cells in a posterior disc annulus of the
degenerative intervertebral disc; and implanting autologous
platelets or platelet lysate in the posterior disc annulus, either
separately or in combination with the viable mesenchymal stem
cells.
2. The method of claim 1 wherein the selective pressure includes
culturing the harvested nucleated cells in from about 3 to about 7%
oxygen.
3. The method of claim 1 wherein the selective pressure further
includes culturing the harvested nucleated cells in from about 2 to
about 10% carbon dioxide.
4. The method of claim 2 wherein the selective pressure further
includes culturing the harvested nucleated cells in from about 2 to
about 10% carbon dioxide.
5. The method of claim 1 further comprising harvesting autologous
platelets from the patient in need thereof.
6. The method of claim 1, wherein the autologous platelets are
implanted prior to, during or after implanting the viable
mesenchymal stem cells.
7. The method of claim 5 wherein the autologous platelets are
treated with thrombin and calcium chloride 1-7 days prior to
implanting in the posterior disc annulus.
8. The method of claim 7 wherein the amount of thrombin is 28.56
U/ml and the amount of calcium chloride is 2.86 mg/ml.
9. The method of claim 5 wherein the autologous platelets are
treated with thrombin, calcium chloride or its salts, thromboxane
A2, adenosine triphosphate and arachidonate.
10. The method of claim 1 further comprising administering one or
more compounds selected from the group consisting of growth
factors, cytokines, integrins, cadherins, molecules or drugs that
promote angiogenesis, molecules or drugs that promote
vasculogenesis, and molecules or drugs that promote
arteriogenesis.
11. The method of claim 10 wherein the compound is VEGF-A, PIGF,
VEGF-B, VEGF-C, VEGF-D, TGF-.beta., Ang-1, Ang-2, IGF, HGF, FGF,
Tie2, PDGF, CCL2, Alpha-V Beta-5, Alpha-5 Beta-1, VE-cadherin,
PECAM-1, plasminogen activator, or nitrogen oxide synthase.
12. The method of claim 1 further comprising administering one or
more growth factors before, during or after implanting the viable
mesenchymal stem cells in the posterior disc annulus.
13. The method of claim 12 wherein the one or more growth factor is
selected from the group consisting of: TGF-.beta., FGF, PDGF, and
IGF.
14. The method of claim 1 wherein the culturing of harvested
nucleated cells is performed in a basal culture media made from a
D-MEM base.
15. A method for treating a degenerative intervertebral disc in a
patient in need thereof comprising: culturing harvested nucleated
cells in a culture medium under selective pressure of about 3 to
about 10% oxygen for two to five passages; obtaining viable
mesenchymal stem cells capable of growth in the culture medium
under selective pressure of about 3% to about 10% oxygen; providing
the viable mesenchymal stem cells for implantation in a posterior
disc annulus of the degenerative intervertebral disc; and providing
autologous platelets or platelet lysate for implantation in the
posterior disc annulus.
16. The method of claim 15, wherein the autologous platelets or
platelet lysate is implanted either separately or in combination
with the viable mesenchymal stem cells.
17. The method of claim 15 wherein the selective pressure further
includes culturing the harvested nucleated cells in from about 2 to
about 10% carbon dioxide.
18. The method of claim 15 wherein the autologous platelets are
treated with thrombin and calcium chloride 1-7 days prior to
implanting in the posterior disc annulus.
19. The method of claim 15 wherein the autologous platelets are
treated with thrombin, calcium chloride or its salts, thromboxane
A2, adenosine triphosphate and arachidonate.
20. A method for treating an avascular zone in a patient in need
thereof comprising: culturing harvested nucleated cells in a
culture medium under selective pressure of about 3% to about 10%
oxygen for 1-28 days; obtaining viable mesenchymal stem cells
capable of growth in the culture medium under selective pressure of
about 3% to about 10% oxygen; implanting the viable mesenchymal
stem cells in the avascular zone; and implanting autologous
platelets or platelet lysate in the posterior disc annulus, either
separately or in combination with the viable mesenchymal stem
cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation application of
co-pending U.S. patent application Ser. No. 16/441,897, which is a
continuation application of U.S. patent application Ser. No.
13/132,840, which is a U.S. national stage application, filed under
35 U.S.C. .sctn.371, of International Application No.
PCT/US2009/066773, which was filed on Dec. 4, 2009, and which
claims priority to U.S. Provisional Application No. 61/120,098,
which was filed on Dec. 5, 2008 and U.S. Provisional Application
61/154,874, which was filed on Feb. 24, 2009. The contents of each
are incorporated by reference into this specification.
TECHNICAL FIELD
[0002] The present invention provides compositions and methods for
facilitating repair in a damaged avascular site, for example an
intervertebral disc; more particularly, the invention provides
applying environmentally conditioned autologous stem cells at
optimized locations within avascular sites or adjacent to avascular
sites in patients in need thereof.
BACKGROUND
[0003] Avascular transition zones and other hard to repair sites
are present in a number of key tissues of the body. These zones are
present where blood supply to the tissue, for example a disc, is
limited or lacking or where damage to the tissue has caused a harsh
environment that resists repair procedures. For example, when an
avascular tissue is damaged, the lack or limit of blood supply to
that tissue poses a significant hurdle to repair processes. This is
particularly true in the intervertebral disc, knee and hip, where
normal load issues make it difficult to facilitate repair and
healing.
[0004] One particularly important avascular transition zone in the
body is within the intervertebral disc, where there is no direct
blood supply. Nutrients to the disc typically arrive via small
capillary beds in the subchondral bone which diffuse throughout the
disc over the course of time. In addition, discs receive nutrients
via imbibitions, in other words by soaking up nutrients from
surrounding tissue during axial loading activities such as walking,
running and the like.
[0005] Intervertebral disc are shock absorbing pads that separate
any two vertebrae of the spine from one another. These discs
essentially provide three functions to a spine, first they act as
shock absorbers to carry axial load of the body while in an upright
position, second they act as a ligament to hold any two adjacent
vertebrae together, and third they act as pivot points for enhanced
bending and rotation of the spine.
[0006] Humans have 23 discs in their spine, i.e., 6 in the cervical
region, 12 in the thoracic region, and 5 in the lumbar region. Each
disc is composed of a nucleus pulposus, annulus fibrosus and
vertebral end-plates. The nucleus pulposus is water-rich and
gelatinous and comprises the center region of a disc. The annulus
fibrosus is fibrous in nature, being made of collagen and includes
little water (as compared to the nucleus pulposus) and surrounds
the nucleus pulposus. A series of lamellae are arranged in the
annulus fibrosus in order to contain the pressurized nucleus
pulposus. In addition, vertebral end-plates act to attach each disc
to adjacent vertebral bodies.
[0007] As discussed above, tissue repair and regeneration have
proven difficult in damaged disc due to harsh environmental aspects
of the disc (avascular, high pressure, adverse pH, etc) and the
difficult mechanical requirements placed on a disc during repair
(stress and strain associated with bipedal movement). Conventional
repair methodologies that utilize stem cell technology have focused
on direct implantation of stem cells (typically obtained from
pre-existing non-autologous cell lines) into the nucleus pulposus,
typically in the presence of a carrier material. While these
methodologies have provided some hopeful results in animal models,
these repairs have yet to be demonstrated in humans with
degenerative disc disease (DDD) or other like conditions. The
promising results in these animal models are likely due to the
acute nature of the disc degeneration model used in animal
research. For example, the discs in these animal models are newly
degenerated with a better blood supply than a long standing
degenerative disc in a human. In addition, the animals used in
these models are generally quadrapedal versus humans who are
bipedal and as such load discs differently. Finally, the animals
used in these DDD models tend to be young and healthy, equivalent
in age to patients who are much younger than the cohort commonly
seen clinically with DDD. In general therefore, conventional
methodologies are based on repair and regeneration in damaged disc
environments potentially very different from those found in human
patients in need of stem cell therapy.
[0008] Issues relevant to tissue repair and regeneration in the
disc are also prevalent in the hip, knee, and shoulder (including
rotator cuff). In each of these tissues, harsh environmental
aspects are often established upon injury or aging, where avascular
transition zones and difficult mechanical requirements combine to
establish scenarios of low stem cell repair success.
[0009] The present invention is directed toward overcoming one or
more of the problems discussed above.
SUMMARY OF THE EMBODIMENTS
[0010] The present invention provides compositions and methods for
use in repair of damaged tissue having one or more poor nutritional
zones or hostile environments, i.e., avascular zones. Poor
nutritional zones or hostile environments are typically located
where the tissue has limited or lacks vascular blood supply, i.e.,
termed avascular transition zones or avascular zones herein. Zones
or environments in this light include: intervertebral disc, hip
(labrum), shoulder (including rotator cuff) and other like sites.
Embodiments herein include procurement of stem cells, for example
mesenchymal stem cells, from a patient in need of repair in an
avascular zone. Procured cells are then conditioned, in vitro, in
an environment that allows for optimization of cells capacity to be
used in repair of the patient's avascular zone. When a sufficient
number of optimized, conditioned cells are present, cells are
placed in target sites of the avascular zone in need of repair. In
some embodiments, platelets or platelet lysate (typically
autologous) and/or supplement treatments are placed in combination
with the conditioned cells to enhance blood flow/nutrient flow to
the site. Timing of the platelet and/or supplement treatment is
typically just prior, during or just after placement of conditioned
cells, although other timing regiments are contemplated. Procedures
can be repeated to ensure repair of site, including repeat of only
conditioned cell placement or platelet/platelet lysate/supplement
treatment(s).
[0011] In one embodiment compositions and methods are provided for
use in repair of damaged intervertebral discs in a patient in need
thereof. In one aspect, methods are provided for procurement and
culturing of stem cells under conditions which optimize the cells
capacity to be used in repair and/or regeneration of a damaged
disc. In another aspect, methods are provided for placement of
these conditioned stem cells in targeted sites of the damaged disc
to optimize growth and regeneration of the damaged disc tissue. In
yet another aspect, methods are provided for placement of the
conditioned stem cells in targeted sites within the patient in
combination with treatment to the patient using epidural supplement
treatment to enhance blood flow to and through the damaged disc.
Additionally, the present invention provides improved compositions
for stem cell culture dedicated toward selection of cells capable
of repair and/or regeneration of a damaged disc. Individually,
and/or in combination, the methods and compositions of the present
invention provide surprising and unexpected advancements in the
field of disc repair and regeneration (as compared to other
conventional technologies).
[0012] In another embodiment, stem cells (mesenchymal stem cells,
for example) are harvested from a patient in need of disc repair
and cultured under conditions based on selecting and expanding
cells able to withstand a poor nutritional environment, an
otherwise hostile environment (for example one where pH is not in
the ranges normally consistent with promoting healthy cell growth),
a hypoxic environment, and/or an environment exhibiting elevated
carbon dioxide levels within a damaged disc. These conditioned
cells are then implanted in the fibrous posterior disc annulus (as
compared to conventional methodologies which typically call for
implantation in the nucleus pulposus). In some cases the patient is
then treated with epidural supplements (growth factors, cytokines,
integrins, cadherins, etc.) to facilitate blood flow to the
posterior disc annulus. Each aspect of the embodiment increases and
selects for stem cells capable of viability and expansion in the
damaged disc environment as well as facilitates the disc
environment to provide enhanced nutrition and oxygen to the
implanted cells. In addition, autologous platelet or platelet
lysate compositions can be administered separately or in
combination with the selected stem cells to facilitate stem cell
viability and expansion within the damaged disc. Individually or in
combination the approaches herein enhance the repair process and
results of autologous stem cell based disc repair.
[0013] In some instances, harvested stem cells from the patient in
need of disc repair are cultured in vitro under 1-10% oxygen and
more typically under 3 to 7% oxygen for a period of from 1 to 28
days. This can represent approximately 1/3 to all of the time the
cells are cultured prior to implantation within the patient.
Surviving/viable cells, i.e., cells capable of growth under hypoxic
conditions, are selected for viability and expanded under these
hypoxic conditions to procure enough cells for implantation into
the damaged disc. Selected cells have enhanced capability to
survive, expand and ultimately repair within the oxygen deficient
environment of a damaged disc.
[0014] In other instances, harvested stem cells from the patient in
need of disc repair are cultured in vitro under elevated carbon
dioxide and more typically under 2 to 10% carbon dioxide for a
period of from 1 to 28 days. This can represent approximately 1/3
to all of the time the cells are cultured prior to implantation
within the patient. Surviving/viable cells, i.e., cells capable of
growth under elevated carbon dioxide conditions, were selected for
viability and expanded under these conditions to procure enough
cells for implantation into the damaged disc. Selected cells have
enhanced capability to survive, expand and repair within the higher
carbon dioxide conditions of the environment of a damaged disc.
This same approach can be used to select for stem cells used to
repair/regenerate damaged (through injury or aging) hip and/or
shoulder avascular sites.
[0015] In other instances, harvested stem cells from the patient in
need of disc repair are cultured in vitro under both hypoxic and
elevated carbon dioxide conditions. In vitro culture conditions can
be maintained for a period of up to 1/3 to all of the total culture
time of the cells. Selected cells have enhanced capability to
survive, expand and repair within the lower oxygen and higher
carbon dioxide conditions typically found in damaged intervertebral
disc. This same approach can be used to select for stem cells used
to repair/regenerate damaged (through injury or aging) hip and/or
shoulder avascular sites.
[0016] In still other instances, harvested stem cells from the
patient in need of disc repair are cultured under nutrient poor
conditions to select for viability and are expanded under these
poor nutrient environments. Culture conditions include use of a
basal cell culture media prepared from Dulbecco's Modified
Essential Medium (DMEM) (or other like basal media) supplemented
with sugars, amino acids, lipids, minerals, proteins, or other
substances intended to facilitate stem cell growth. Growth media
may or may not contain serum such as fetal calf serum, human whole
serum, platelet rich plasma, platelet lysate, etc . . . However,
these preparations are specifically designed to mimic the local
environment of a human degenerated disc such as hypoxia, altered
pH, or certain limited nutrient availability. This same approach
can be used to select for stem cells used to repair/regenerate
damaged (through injury or aging) hip (for example in labrum)
and/or shoulder avascular sites.
[0017] Other selection conditions can be used to expand harvested
stem cells including: pH, use of spent media, co-culture with
nucleus pulposus cells, where the target site is in a damaged disc
(thereby providing the environmental factors present from being in
proximity to the ultimate target site for delivery of the cultured
cells), and the like. Note also, in some instances two or more of
the selection conditions described herein can be used to identify
and expand stem cells for use in avascular site repair. So for
instance, poor nutritional media and hypoxic conditions can be used
to select for the stem cells used in a first patient, while pH and
carbon dioxide conditions may be used for selection in a second
patient. This may be based on actual measurements of the local
micro environment in any given patient.
[0018] In still further instances, platelets, from the same patient
having the damaged site, are procured (harvested) and treated with
thrombin and calcium chloride (CaCl.sub.2). Treated platelets are
combined with cultured and selected stem cells for implantation
into the damaged site. Note that treatment of the platelets can
extend from one to seven days and more particularly from 5 to 7
days prior to implantation into damaged site. Additionally,
platelets can be implanted just prior to, during or after
implantation of the selected stem cells. Such preconditioned
platelets are capable of releasing targeted growth factors into the
avascular zone environment useful in facilitating stem cell
survival and expansion within the damaged site. Alternatively, or
in combination, growth factors, cytokines, integrins, etc can be
directly administered with the selected stem cells into the damaged
site. In one embodiment, these growth factors are administered
around the exterior of, for example, a damaged disc such as placed
into the epidural space.
[0019] These and various other features and advantages of the
invention will be apparent from a reading of the following detailed
description and a review of the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is an axial lumbar view with superimposed path of
needle placement of stem cells into the vascular and transitional
vascular zones of the posterior disc annulus using embodiments
described herein.
[0021] FIG. 2 shows MSCs grown in monolayer culture according to a
technique provided herein.
[0022] FIG. 3 is an exemplary fluoroscopy image of the injection
location of MSCs and platelet derived VEGF supernatant into the
posterior disc annulus of the L5-S1 disc (subject ML). Note the
concentration of contrast in the posterior disc annulus (contrast
flow enhanced with blue).
[0023] FIG. 4 demonstrates exemplary epidural flow attained with
the platelet derived VEGF supernatant injections that were
performed after the stem cell transplant.
[0024] FIG. 5 provides ML Short Tau Inversion Recovery (STIR) image
taken less than 1 month prior to procedure. This sagittal slice is
chosen as it represents the maximum extent of the contained L5-S1
disc extrusion. ET=6, TR=4816.7, TE=48.1 with an imaging time of
day of 1:01 p.m. This image demonstrates a 0.7 cm disc extrusion at
L.sub.5-S1. L5-S1 disc height measured at central disc is 0.5 cm
with L4-L5 measuring at 0.7 cm.
[0025] FIG. 6 provides ML 1 month post procedure matching sagittal
slice using the same STIR parameters. ET=6, TR=4816.7, TE=48.1.
Imaging time of day was 11:01 a.m. This image demonstrates a 0.3 cm
disc extrusion at L5-S1. Note disc heights 0.5 cm at L5-S1 and 0.7
cm at L4-L5.
[0026] FIG. 7 provides ML 5 month post procedure matching sagittal
slice using the same STIR parameters. ET=6, TR=4816.7, TE=48.3.
Imaging time of day was 10:23 a.m. This image demonstrates a 0.3 cm
disc extrusion at L5-S1. Note disc heights 0.5 cm at L5-S1 and 0.7
cm at L4-L5.
[0027] FIG. 8 provides MJM pre-procedure sagittal slice through the
maximum extent of the contained L4-L5 disc extrusion. Image was
taken at 12:15 pm. ET=6, TR=4816.7, TE=48.1. The L4-L5 disc
extrusion is measured at 6 mm. Disc heights measured at the
mid-portion of the disc on this slice were: L4-L5=8 mm, L5-S1=7 mm,
S1-S2=5 mm.
[0028] FIG. 9 provides MJM 2 months post-procedure matching
sagittal STIR slice with same imaging parameters. ET=6, TR=4816.7,
TE=48.1. Image was taken at 12:35 pm. The L4-L5 disc extrusion is
measured at 3 mm. Disc heights measured the same as pre-procedure:
L4-L5=8 mm, L5-S1=7 mm, S1-S2=5 mm.
[0029] FIG. 10 provides MJM 4.5 months post-procedure. This is
matching sagittal STIR slice with same imaging parameters. ET=6,
TR=4833.3, TE=48.2. Image was taken at 12:27 pm. The L4-L5 disc
extrusion is measured at 3 mm. Disc heights measured the same as
pre-procedure: L4-L5=8 mm, L5-S1=7 mm, S1-S2=5 mm.
[0030] FIG. 11 provides HO pre-procedure sagittal STIR slice
through the maximum extent of the L5-S1 disc protrusion. ET=6,
TR=4816.7, TE=48.3. Image time of day was 11:26 a.m. The L5-S1 disc
protrusion is measured at 9 mm. Disc heights measure: L4-L5=6 mm,
L5-S1=8 mm.
[0031] FIG. 12 provides HO 6 week post procedure sagittal matching
STIR slice. Imaging parameters kept constant at ET=6, TR=4816.7,
TE=48.3. Image time of day was 11:25 a.m. The L5-S1 disc protrusion
is measured at 8 mm. Disc heights measure: L4-L5=6 mm, L5-S1=8
mm.
[0032] FIG. 13 provides HO 3.5 month post procedure matching
sagittal STIR slice. Imaging parameters kept constant at ET=6,
TR=4816.7, TE=48.3. Image time of day was 1:10 p.m. The L5-S1 disc
protrusion is measured at 9 mm. Disc heights measure: L4-L5=6 mm,
L5-S1=8 mm.
[0033] FIG. 14 shows contrast flow in HO (enhanced in blue) was
more typical of a nucleogram than the intended target which was the
posterior disc annulus.
DETAILED DESCRIPTION
[0034] The present invention provides compositions and methods for
use in repair of damaged tissue having one or more poor nutritional
zones or hostile environments, i.e., avascular zones. Poor
nutritional zones or hostile environments are typically located
where the tissue has limited or lacks vascular blood supply, i.e.,
termed avascular transition zones herein. Zones or environments in
this light include: intervertebral disc, hip, shoulder (including
rotator cuff) and other like sites. Embodiments herein include
procurement of stem cells, for example mesenchymal stem cells, from
a patient in need of repair in an avascular zone. Procured cells
are then conditioned, in vitro, in an environment that allows for
optimization of cells capacity to be used in repair of the
patient's avascular zone. When a sufficient number of optimized,
conditioned cells are present, cells are placed in target sites of
the avascular zone in need of repair. In some embodiments,
supplement treatments are placed in combination with the
conditioned cells to enhance blood flow/nutrient flow to the site.
Timing of the supplement treatment is typically just prior, during
or just after placement of conditioned cells, although other timing
regiments are contemplated. Procedures can be repeated to ensure
repair of site, including repeat of only conditioned cell placement
or supplement treatment.
[0035] Avascular zone conditions for stem cell selection herein
generally include: 1-10% oxygen, 2-10% carbon dioxide, altered pH,
altered nutrition, and combinations of the like. Selected cells can
be placed into repair sites in combination with supplements, e.g.,
growth factors, cytokines, integrins, cadherins, and the like,
and/or with treated autologous platelets.
Definitions
[0036] The following definitions are provided to facilitate
understanding of certain terms used frequently herein and are not
meant to limit the scope of the present disclosure.
[0037] "Stem cell(s)" as used herein refers to cells possessing the
properties of self-renewal and potency. With regard to mesenchymal
stem cells, these cells are multipotent and have the capability to
differentiate into osteoblasts, chondrocytes, myocytes, adipocytes,
and other like cells.
[0038] Disc Bulge as used herein refers to a protrusion of the
nucleus pulposis into the annulus fibrosis of the disc.
[0039] Disc Hernation as used herein refers to an extrusion of the
nucleus pulposis beyond the confines of the annulus fibrosis.
[0040] Contained disc herniation as used herein refers to an
extrusion of the nucleus pulposis beyond the confines of the
annulus fibrosis and still confined by the posterior longitudinal
ligament.
[0041] "Patient" as used herein refers to a mammal, and more
typically a human, having one or more damaged or aged avascular
sites, for example damaged intervertebral discs. With regard to a
damaged disc, damage may include herniated disc, bulging disc,
fractured disc, disc protrusion, disc extrusion, disc sequestration
and other like disc ailments.
[0042] "Platelet and platelet lysate" are used interchangeably
herein and include the combination of natural growth factors
contained in platelets that have been released through lysing of
the platelets. This can be accomplished through chemical means
(i.e. CaCl.sub.2), osmotic means (use of distilled H.sub.2O), or
through freezing/thawing procedures. Platelet lysates of the
invention can also be derived from whole blood and can be prepared
as described in U.S. Pat. No. 5,198,357, which is incorporated by
reference herein.
[0043] "Repair" or "regeneration" are used interchangeably and
refer to partial or complete replacement of a damaged area within a
target avascular zone or a zone adjacent to the avascular zone. For
example, repair of an intervertebral disc includes partial or
complete repair or replacement of the tissue within the disc.
Repair or regeneration can also refer to repair or regeneration of
an avascular zone in a normally aging patient, i.e., repair damage
to a zone induced by age.
[0044] "Environment" as used herein refers to the entire set of
conditions that effect or influence cells in vitro or in vivo.
[0045] "Hypoxia or hypoxic" as used herein refers to an in vitro or
in vivo condition having 10% or less oxygen in the environment.
[0046] "Supplement" or supplement treatments include growth
factors, cytokines, integrins, including: VEGF-A, PIGF, VEGF-B,
VEGF-C, VEGF-D, FGF, Ang1, Ang2, MCP-1 endoglin, TGF-.beta., CCL2,
VE-cadherin, etc.
[0047] Embodiments in accordance with the present invention include
methods and compositions useful in repair and regeneration of
damaged avascular zones, e.g., intervertebral discs. Embodiments
herein are predicated on the unexpected finding that harvest and
expansion of autologous stem cells under poor nutritional and
hypoxic conditions provide more capable cells for repair of an
avascular zone. Further, implantation of conditioned cells with
autologous platelets, as well as facilitating blood flow to the
damaged site yield dramatically enhanced repair. Implantation can
also include one or more supplement treatment(s) (with or without
platelets). Finally, embodiments herein include administering these
conditioned cells and ancillary materials at optimized sites within
the damaged site to further facilitate repair and/or
regeneration.
Stem Cells
[0048] Mesenchymal stem cells (MSCs) hold great promise as
therapeutic agents in regenerative medicine. Alhadlaq, A. and J. J.
Mao, Mesenchymal stem cells: isolation and therapeutics. Stem Cells
Dev, 2004. 13(4): p. 436-48. Barry, F. P., Mesenchymal stem cell
therapy in joint disease. Novartis Found Symp, 2003. 249: p. 86-96;
discussion 96-102, 170-4, 239-41. Bruder, S. P., D. J. Fink, and A.
I. Caplan, Mesenchymal stem cells in bone development, bone repair,
and skeletal regeneration therapy. J Cell Biochem, 1994. 56(3): p.
283-94. Cha, J. and V. Falanga, Stem cells in cutaneous wound
healing. Clin Dermatol, 2007. 25(1): p. 73-8. Gangji, V., M.
Toungouz, and J.P. Hauzeur, Stem cell therapy for osteonecrosis of
the femoral head. Expert Opin Biol Ther, 2005. 5(4): p. 437-42.
These adult stem cells can be easily isolated from many sources in
the body. Alhadlaq, A. and J.J. Mao, Mesenchymal stem cells:
isolation and therapeutics. Stem Cells Dev, 2004. 13(4): p. 436-48.
In addition, they have demonstrated in numerous animal studies, the
ability to differentiate into muscle, bone, cartilage, nerves,
tendon, and various internal organs cells. Lumbar disc degeneration
and pathology are major causes of significant disability and
medical expense. Dagenais, S., J. Caro, and S. Haldeman, A
systematic review of low back pain cost of illness studies in the
United States and internationally. Spine J, 2008. 8(1): p. 8-20.
Surgical treatments such as discectomy, fusion, and disc
replacement have been utilized in clinical practice, with strong
potential for significant morbidity. de Kleuver, M., F. C. Oner,
and W. C. Jacobs, Total disc replacement for chronic low back pain:
background and a systematic review of the literature. Eur Spine J,
2003. 12(2): p. 108-16. Gotfryd, A. and 0. Avanzi, A systematic
review of randomized clinical trials using posterior discectomy to
treat lumbar disc herniations. Int Orthop, 2008. Katonis, P., et
al., Postoperative infections of the thoracic and lumbar spine: a
review of 18 cases. Clin Orthop Relat Res, 2007. 454: p. 114-9. As
a result, the ability to repair the Intervertebral disc (IVD)
rather than surgical alteration or removal is an attractive
treatment option. Saki and others have shown that MSC's are capable
of lumbar disc repair in animal studies using a puncture model of
simulated disc degeneration. Sakai, D., et al., Regenerative
effects of transplanting mesenchymal stem cells embedded in
atelocollagen to the degenerated intervertebral disc. Biomaterials,
2006. 27(3): p. 335-345. Sakai, D., et al., Differentiation of
mesenchymal stem cells transplanted to a rabbit degenerative disc
model: potential and limitations for stem cell therapy in disc
regeneration. Spine, 2005. 30(21): p. 2379-87. Sakai, D., et al.,
Transplantation of mesenchymal stem cells embedded in Atelocollagen
gel to the intervertebral disc: a potential therapeutic model for
disc degeneration. Biomaterials, 2003. 24(20): p. 3531-41. The
inventors recognized that there are many physiologic differences
between animal and human IVD models. These include different forces
created with quadrupeds in ovine, porcine and murine animals,
versus bipedal mechanics in humans. In addition, the popular
puncture model of degenerative disc disease (DDD), used by many
animal researchers creates the scientific equivalent of an acutely
injured disc. Human DDD is often present for decades prior to the
patient seeking medical or surgical treatment. Embodiments herein
(see Examples) show that MSC's percutaneously deployed into a
posterior disc annulus of human subjects with VEGF enriched,
platelet derived supernatant provides significant disc repair. The
decision to place cells into the posterior disc annulus was based,
partly, on the higher vascularization of this area versus the well
defined avascular, low density nutrient environment within the
nucleus pulposus.
[0049] Embodiments of the invention include harvest of stem cells
from the patient in need of avascular site repair. Stem cells in
accordance with the invention are as described above in the
definitions section. In some embodiments the stem cells are
mesenchymal stem cells, i.e., multipotent cells capable of
differentiating into, among other cell types, osteoblasts,
chondrocytes, myocytes, adipocytes and pancreatic islet cells. Note
that for purposes of the invention many embodiments are described
in relation to mesenchymal stem cells, although other stem cell
types can also be used and are within the scope of the present
invention.
[0050] Stem cell harvest in accordance with aspects of the present
invention include those described in U.S. patent application Ser.
No. PCT/us08/68202 which are incorporated by reference in there
entirety. Additionally, methods and compositions as described in
U.S. Pat. Nos. 5,486,359, 6,387,367 and 5,197,985 are incorporated
by reference herein in their entirety.
[0051] In more detail, mesenchymal stem cells are multipotent stem
cells located in the bone marrow, peripheral blood, adipose tissue
and other like sources. MSCs have the capacity to differentiate
into a number of cell types, including osteoblasts, chondrocytes,
myocytes, adipocytes, and beta-pancreatic islet cells.
[0052] Source MSCs of the invention are typically harvested from
the iliac crest of the patient in need (or other source such as the
IVD, periosteum, synovial fluid, or the vertebral body or pedicle)
of the restorative/replacement therapy (or a suitable donor), such
patient is referred to herein as a "patient in need or patient in
need thereof" (note that other sources, such as adipose tissue,
synovial tissue, and connective tissue have recently been
identified and are also considered as MSC sources within the scope
of the present invention). In one embodiment, approximately 10-100
cc of bone marrow is harvested and "isolated" using methods
described in U.S. patent application Ser. No. 60/761,441 to Centeno
or through adherence to plastic, as described in U.S. Pat. No.
5,486,359 to Caplan et al. Each of these references is incorporated
herein in their entirety for all purposes.
[0053] As described in more detail below embodiments of the present
invention may also require some level or amounts of platelets. As
such, this invention incorporates changes to standard marrow draw
procedures to allow appropriate nucleated cell number yield to use
platelets or platelet lysate techniques. In addition, these
platelets can be obtained from whole blood. Since the vast majority
of the published research is again performed in healthy humans or
animals, the application of this technique to humans with various
disease states has never been tested. Note that, the use of an
altered technique drawing three small 2-3 cc marrow aliquots on
each side (total of 6 aliquots), produced the required nucleated
cell yield which was successfully expanded in 20% platelet
lysate.
[0054] Platelets and platelet lysate for use herein is prepared
from the bone marrow harvest using the method of Doucet (Doucet,
Ernou et al., 2005 J. Cell Physiol 205(2): 288-36), which is
incorporated by reference herein in its entirety. Typical lysates
include from about tens of millions to 100's of billions platelets.
As shown by Martineau et al., Biomaterials, 2004 25(18) p4489-503
(incorporated herein by reference in its entirety), platelet
lysates inherently include the growth factors required to
facilitate consistent MSC growth. In typical embodiments the
platelet lysate and MSC are autologous and are in amounts useful
for effective and consistent expansion of the MSCs (described more
fully below). In particular, it should be noted that while the
levels of growth factors such as TGF-beta are much lower in
platelet lysate than those commonly used to expand MSC's, it is
believed that there are significant synergistic effects when all of
the low level growth factors contained in platelet lysate are used
together.
Stem Cell Selection (Selective Pressure)
[0055] Harvested stem cells are cultured to select for stem cells
(typically mesenchymal stem cells) and ultimately for stem cells
that are viable and expand under environmental conditions similar
to those conditions found in a site in need of repair, for example
a disc in need of repair. Selective pressure as it relates to
intervertebral disc repair is discussed in greater detail below,
but similar conditions are present and ascertainable for conditions
required for hip, shoulder and the like.
[0056] As discussed in Urban et al., Nutrition of the
intervertebral Disc. Spine, 2004. 29 (23): p2700-9, (incorporated
herein by reference in its entirety), intervertebral disc that have
suffered injury and are degenerative provide a poor nutritional as
well as oxygen environment. This environment is distinct from the
environment of a healthy intervertebral disc. In fact, studies
performed to determine viability of transplanted mesenchymal stem
cells in injured disc show poor cell viability results, with few
cells capable of expanding to provide the necessary numbers of
cells needed for enhanced disc repair (Wuertz et al., Behavior of
mesenchymal stem cells in the chemical microenvironment of the
intervertebral disc. Spine, 2008. 33(17): p 1843-9, incorporated
herein by reference in its entirety).
[0057] In more detail, harvested stem cells from a patient in need
of disc repair or restoration are placed under culture conditions.
In one embodiment, the culture medium is a basal cell culture
medium prepared from DMEM or other like media. Culture medium can
be supplemented with sugars, amino acids, lipids, minerals,
proteins, or other like substances intended to facilitate stem cell
expansion.
[0058] In addition, embodiments herein can include culturing
harvested and expanding mesenchymal stem cells under various
atmospheric conditions that simulate a damaged disc's environment.
In one embodiment, harvested stem cells are cultured in vitro under
1 -15% oxygen. In some cases the harvested stem cells are cultured
under 3 to 10% oxygen and in other cases the harvested stem cells
are cultured under 3 to 7% oxygen. These lower oxygen conditions
replicate the hypoxic conditions present in typical damaged disc
environments.
[0059] Hypoxic conditions can be present for part or all of the
stem cell expansion period but is typically present for at least
1/3 of the time that cells are cultured in vitro. Selection occurs
as cells are cultured, with viable cells that are able to survive
and ultimately expand having an advantage when implanted into a
disc having a hypoxic environment.
[0060] In other embodiments, harvested stem cells are cultured in
vitro under elevated carbon dioxide conditions, typically from
2-10% carbon dioxide. Harvested cells can additionally be cultured
in a combined elevated carbon dioxide and hypoxic environment,
where conditions include from 2-10% carbon dioxide and from 3-10%
oxygen. As above, selection occurs as cells are cultured, with
viable cells that are able to survive and ultimately expand having
an advantage when implanted into a disc having an elevated carbon
dioxide environment or an elevated carbon dioxide environment
combined with a hypoxic environment.
[0061] In other embodiments, harvested stem cells are cultured and
expanded in combination with harvested and cultured nucleas
pulposis cells (NP cells) or annulus fibrosis cells (AF cells). The
nucleas pulposis (NP) cells for co-culture with stem cells can be
harvested from the patient in need of disc repair via a needle
aspirate or other like technique. These NP cells or AF cells can be
either autologous or non-autologous. In typical embodiments,
approximately 10.sup.3 to 10.sup.9 NP or AF cells are co-cultured
with the harvested stem cells and are allowed to provide an
environment useful for selection of stem cells that respond to NP
cell and/or AF cell released factors and waste products. Co-culture
conditions can include poor nutritional environment, hypoxia,
elevated carbon dioxide and other disclosed embodiments described
herein. In some embodiments, the NP cells are cultured in a
separate in vitro flask (or other like container) from the stem
cells. The spent media from the NP cell culture can then be
combined with media conditions above during stem cell culture or
can be used exclusively to expand and select for stem cells able to
maintain viability and ultimately expand under such conditions.
[0062] In other embodiments, harvested stem cells are cultured and
expanded under modified pH conditions similar to those found in a
damaged intervertebral disc. For example, in vitro culture media
(as described herein) can be modified to have a pH of from 6.6-7.0,
and more typically from 6.7 to 6.9. Modified pH can be combined
with any of the culture conditions discussed herein to facilitate
selection of stem cells for use in disc repair and
regeneration.
[0063] In other embodiments, harvested stem cells are cultured and
expanded under modified osmolarity conditions similar to those
found in a damaged intervertebral disc. For example, in vitro
culture media (as described herein) can be modified to have an
osmolarity of from 350-600 mOsm, and more typically from 450 to 500
MOsm. Modified osmolarity can be combined with any of the culture
conditions discussed herein to facilitate selection of stem cells
for use in disc repair and regeneration.
[0064] In other embodiments, viability and expansion of stem cells
under one or more selection conditions can be modified by inclusion
of one or more growth factors. In these cases, cells under
selection are cultured in the presence of TGF-beta FGF, PDGF, IGF
and/or HIF-1 alpha, including mixtures thereof and other like
factors.
[0065] Note that for each of the above stem cell culture based
embodiments, the condition(s) can be gradually incorporated into
cells standard culture environment. For example, harvested stem
cells may be initially cultured under 10% oxygen for one or two
passages, then moved to 9% oxygen conditions for one or two
passages, and cultured under decreasing levels of oxygen until a
target hypoxic condition is obtained. Under this procedure, cells
are gradually allowed to adapt to an environment present in a
damaged disc.
[0066] The following treatment procedure is described in relation
to treatment of a damaged disc, although treatment of other
avascular zones are envisioned to be within the scope of the
present invention.
Treatment of Damaged Intervertebral Disc
[0067] Stem cells having been selected for by at least one of the
above discussed damaged disc modifiers are allowed to expand until
a sufficient number of cells are present for implantation into a
patient's damaged disc. In typical embodiments, from about 10.sup.5
to10.sup.9 selected mesenchymal stem cells are required for
implantation into the damaged disc.
[0068] Cultured cells are washed using PBS or other like buffer to
obtain a cell population that does not include materials not
intended for implantation into the patient's body, i.e., media
constituents, waste products, etc. Washed stem cells can include NP
cells, although it is contemplated that where stem cells are
co-cultured with NP cells, that the NP cell population can be
removed via cell sorting techniques or affinity chromatography.
Stem cells are now ready for implantation into the patient in need
thereof.
[0069] In one embodiment, the washed stem cells are implanted
directly into the posterior annulus of the damaged disc. This is an
unexpected location for implantation of stem cells, as conventional
methodologies show implantation of cells into the nucleas pulposis.
Cells are implanted into the posterior annulus of the damaged disc
via known techniques in the art, including via percutaneous x-ray
guided or surgical IVD access. One or more iteration of cell
implantation can be used in repair procedure for a damaged disc,
although, a period of 14 to 180 days is typical between
treatments.
[0070] In another embodiment, autologous platelets from the patient
in need of therapy are pretreated with thrombin and CaCl.sub.2 for
approximately one to seven days. This treatment preconditions these
platelets to preferentially express vascular endothelial growth
factor (VEGF). In some embodiments, the harvested platelets are
pretreated with approximately 28.56 U/ml thrombin and approximately
2.86 mg/ml CaCl.sub.2. In additional embodiments, autologous
platelets from the patient in need thereof are pretreated with a
combination of thrombin, calcium or its' salts, thromboxane A2,
adenosine triphosphate, and arichidonate. As above, pretreatment
can be from one to seven days prior to implantation into the
patient in need thereof. The preconditioned autologous platelets
are then implanted before, during or after implantation of the
previously discussed selected conditioned stem cells of the
invention. Platelets are generally implanted in the same location
as the implanted stem cells.
[0071] In another embodiment, harvested and selected stem cells of
the invention are implanted with one or more supplement or
supplement treatments, including growth factors, cytokines,
integrins, cadherins, or molecules or drugs known to promote
angiogenesis, vasculogenesis or aerteriogenesis, including: VEGF-A,
PIGF, VEGF-B, VEGF-C, VEGF-D, TGF-.beta., Ang-1, Ang-2, IGF, HGF,
FGF, Tie2, PDGF, CCL2, Alpha-V Beta-5, Alpha-5 Beta-1, VE-cadherin,
PECAM-1, plasminogen activator and nitrogen oxide synthase. In
alternative embodiments, stem cells for use in implantation in
patients in need thereof are co-implanted with a combination of
growth factors including TGF-.beta., FGF, PDGF, IGF or other like
growth factors intended to promote stem cell and/or mesenchymal
stem cell stemness or proliferation. In some embodiments autologous
platelets or platelet lysates can be implanted in combination with
the before mentioned supplements.
[0072] FIG. 1 illustrates the utility of one embodiment of the
invention showing an axial lumbar view with a superimposed needle
placement. Conditioned stem cells using embodiments described
herein were placed in the vascular and transition vascular zones of
the posterior disc annulus.
EXAMPLES
Example 1
Percutaneously Implanted Autologous Mesenchymal Stem Cells
Methods:
Subjects:
[0073] Three patient subjects were selected based on willingness to
participate in an IRB (Spinal Injury Foundation, Westminster,
Colo.) approved MSC implantation protocol. Each subject signed an
IRB approved consent form. Subjects were selected based on the
following inclusion/exclusion criterion: [0074] Inclusion Criteria:
[0075] 1. 18-65 years of age [0076] 2. Failure of conservative
management [0077] 3. Lumbar degenerative disc disease with a disc
protrusion or contained disc extrusion (subligamentous) [0078] 4.
Selective nerve root blocks that confirmed the disc
protrusion/nerve to be treated as the pain generator (>75%
relief of major pain complaint) or discography which confirmed the
disc as a P2 pain generator [0079] 5. At least 75% of normal disc
height with or without dehydration on T2 weighted MRI images [0080]
6. Unwillingness to pursue surgical options [0081] Exclusion
criteria: [0082] 1. Active inflammatory or connective tissue
disease (i.e. lupus, fibromyalgia, RA) [0083] 2. Active
non-corrected endocrine disorder potentially associated with
symptoms (i.e. hypothyroidism, diabetes) [0084] 3. Active
neurologic disorder potentially associated with symptoms (i.e.
peripheral neuropathy, multiple sclerosis) [0085] 4. Severe cardiac
disease [0086] 5. Pulmonary disease requiring medication usage
[0087] 6. A history of dyspnea or other reactions to transfusion of
autologous blood products
Pre-Procedure Data Collection:
[0088] 1. CBC and blood chemistries were obtained within 3 months
of the MSC implantation to rule out unknown medical conditions.
[0089] 2. Pre-procedure MRI
[0090] 3. Pre-procedure outcomes measures
Isolation and Expansion of Mesenchymal Stem Cells (MSCs):
[0091] For one week prior to the marrow harvest procedure the
patient was restricted from taking corticosteroids or NSAIDs.
Coincident with the marrow harvest procedure, heparinized (Abraxis
Pharmaceuticals) IV venous blood was drawn to be used for platelet
lysate (PL). Platelet lysate was prepared via centrifugation at 200
g to separate platelet rich plasma (PRP) from red blood cells
(RBCs). PRP volume was aliquoted and stored at -20.degree. C. to
produce PL. Platelet lysate was supplemented in cell culture media
at 10-20%.
[0092] A platelet derived VEGF rich supernatant was also prepared
based on the method described by Martineau. (Martineau, I., E.
Lacoste, and G. Gagnon, Effects of calcium and thrombin on growth
factor release from platelet concentrates: kinetics and regulation
of endothelial cell proliferation. Biomaterials, 2004. 25(18): p.
4489-502). Using the same PRP isolation steps as above, the PRP was
drawn off and an aliquot was activated with 28.56 U/mL of human
thrombin (Johnson and Johnson) and 2.85mg/mL of Calcium Chloride
(CaCl.sub.2, American Regent) for 6 days at 37.degree. C. and 5%
CO.sub.2. Activated PRP samples were centrifuged at 3,000 rpm for 6
minutes, supernatant was draw off and stored at -80.degree. C. This
was later used as both injectate to be mixed with culture expanded
MSC's as well as for supplement injections to be delivered via
epidural injection.
[0093] Coincident with the whole IV blood draw, the patient was
then placed prone on an operating room (OR) table and the area to
be harvested was anesthetized with 1% Lidocaine, and a sterile
disposable trocar was used to draw 10 cc of marrow blood from the
right PSIS area and 10 cc from the left PSIS area, in heparinized
syringes. If the patient reported pain during the marrow draw that
was not easily controlled by local anesthetics, a caudal epidural
with anesthetic only was added.
[0094] Whole marrow was centrifuged at 200g for 4-6 minutes to
separate the nucleated cells from the RBCs. The nucleated cells
were removed and placed in a separate tube. Samples were
centrifuged at 1000 g for 6 minutes to pellet. The nucleated cells
were washed once in phosphate buffered saline (PBS, GibCo),
counted, and then re-suspended in Dulbecco's modified eagle medium
(DMEM, GibCo) with 10-20% PL, 5 ug/mL doxycyline (Bedford Labs),
and 2 IU/mL heparin (Abraxis Pharmaceuticals). Nucleated cells were
seeded at 1.times.10.sup.6 cells/cm.sup.2 in a tissue culture
flask. Cultures were incubated at 37.degree. C./5% CO.sub.2/5%
O.sub.2 in a humidified environment. The culture medium was changed
after 48-72 hours, removing the majority of the non-adherent cell
population. The MSC colonies developed in 6-12 days and then were
harvested with animal origin-free trypsin like enzyme (TrypLE
Select, GibCo) such that only the colony-forming MSCs detached. To
expand the MSCs, they were plated at a density between 6-12,000
cells/cm.sup.2 in alpha-modified eagle medium (AMEM, GibCo) with
10-20% PL, 5 ug/mL doxycyline, and 2IU/mL of heparin, and grown to
near confluence at 37.degree. C./5% CO.sub.2/5% O.sub.2. Primary
cells derived from the bone marrow were designated as passage 0 and
each subsequent subculture of MSCs was considered one further
passage. See FIG. 2 for an example of the MSC morphology grown with
this monolayer cell culture technique. After MSC's had been sub
cultured to the 2nd-5th passage, they were harvested, washed, and
suspended in the activated PRP for injection.
Percutaneous Implantation Procedure
[0095] Subjects were positioned prone on an x-ray table and prepped
using betadine swab with sterile gloves and drapes. An AP
fluoroscopy view (Siemens Iso-C) was obtained with an ipsilateral
oblique orientation. The superior endplate of the targeted level to
be injected was visualized "on end" by adjusting cephalic-caudal
tilt. Using sterile technique, a 22 gauge 7 inch quinke needle was
guided under bi-planar fluoroscopy to the superior articular
process of the lumbar facet joint of the level to be treated and
advanced past the facet into the posterior disc. Once the disc
access was obtained, under lateral view, the needle was positioned
to maximize contrast flow (Omnipaque 300 mg/ml-NDC 0407-1413-51
diluted 50% with PBS) into the posterior annulus as close to the
anatomic position of the disc protrusion as possible, using
pre-treatment MM imaging for approximation (see FIG. 3). Culture
expanded autologous MSC's in platelet derived VEGF supernatant were
then injected and the needle was extracted.
[0096] Following MSC implantation, at week 1 and week 2 the subject
returned for additional transformainal epidurals performed with
VEGF derived supernatant at the target levels. Epidural procedures
were performed with the same preparation. Epidural access was
obtained using a 25 gauge 3.5 inch quince needle which was
manipulated under biplanar fluoroscopy and directed toward the
subpedicular recess of the target level being injected. Once good
epidural dye flow extending upwards and under the pedicle was
visualized, the VEGF derived supernatant plus 4% lidocaine was
injected and the needle was extracted (see FIG. 4). Post-op
treatment protocol consisted of lumbar traction in physical therapy
or at home at 3 times a week for four weeks.
Imaging and Patient Follow-up:
[0097] A GE 3.0 Tesla Excite HD was used to image the lumbar spine.
Imaging sequences included a sagittal Short Tau Inversion Recovery
(STIR), Fast Gradient Recall Echo (FGRE) sagittal, and a Gradient
Recall Echo Fast Spin (GRE-FS). Images were viewed and measured in
E-Film Workstation Version 1.5.3 (Merge Healthcare). Sagittal short
tau inversion recovery (STIR) and gradient recall echo (GRE) fast
spin images with matching TR/TE and matching imaging planes were
used. This was performed to reduce the likelihood of interpretation
error of serial images in the same patient. To reduce diurnal
effects, the imaging center was instructed to perform the serial
films as close to the same time of day as possible.
[0098] Follow-up questionnaires were initiated via phone and
obtained from the patient concerning function and symptoms.
Modified VAS scores were obtained with regard to low back pain and
a "Functional Rating Index" (FM) was also obtained. This
questionnaire focuses on patient function. (Feise, R. J. and J.
Michael Menke, Functional rating index: a new valid and reliable
instrument to measure the magnitude of clinical change in spinal
conditions. Spine, 2001. 26(1): p. 78-86; discussion 87). Office
visits were also used for post-op examinations. These were
initiated at the following intervals: [0099] 1. At 6 weeks post
procedure. [0100] 2. At 12 weeks post procedure
Results:
[0101] Medical histories and outcomes of the three subjects
enrolled are described below:
Patient 1:
[0102] HO was a 19 year old white female, college athlete with a 7
year history of low back pain thought to be secondary to lifting
trauma and her chosen sport. She had undergone extensive
conservative care for four years prior to presentation, including
evaluation and treatment with an interventional pain management
physician The lumbar facets were ruled out as pain generators with
negative response to intra-articular injections., Mill showed an
L5-S1 disc protrusion abutting both 51 nerve roots, worse on the
right side. Discography revealed a symptomatic posterior annular
tear with concordant reproduction of pain at the L5-S1 disc.
Pre-procedure the subject described constant pain of variable
severity, with numbness and tingling in the S1 distribution of the
right foot with any physical activity, resulting in significant
functional limitations.
[0103] The subject reported up to 75% improvement through 12 weeks.
At 6 months post procedures, the subject showed little change in
her reported modified VAS pain scores and Functional Rating Index.
There was a corresponding lack of change seen in the post-treatment
Mill imaging results. A 1 mm reduction in maximum protrusion size
was seen at 6 weeks, but this returned to pre-procedure size at 3.5
months. All disc height measurements remained the same across all
imaging sessions and the maximum time of day interval for all
images taken was 1 hour and 45 minutes. Results for the HO are
shown in FIGS. 11, 12 and 13.
Patient 2:
[0104] MJM was a 35 year old white male with a 15 year history of
low back pain prior to presentation. He reported failed
conservative care and increasing frequency of severe pain episodes
due to activity. MM revealed a lumbarized S1-S2 segment, a
broad-based disc bulge with moderate facet encroachment and
moderate foraminal narrowing at L5-S1. Additionally, there was a
right greater than left posterior contained protrusion at L4-L5.
The physical examination revealed no active radiculopathy, but the
patient reported intermittent radicular symptoms associated with
exacerbations. Surgery was recommended after failure on
conservative management, but was declined. The L4-L5 disc was the
focus of this treatment with the dehydrated L5-S1 disc left as a
control.
[0105] Six months post procedure, the patient improvement of
modified VAS from 3 to a 0 with a drop in frequency of pain by more
than 80% (see Table 1). The FM score for this patient increased by
more than 60% and he reported overall symptom improvement at 50%.
L4-L5 disc bulge sagittal STIR image measured 6 mm at its maximum
extent in the pre-op MR images (see FIG. 8). In both the 2 month
and 4.5 month follow-up films (matching image sequence and
slice-see FIGS. 9 and 10) the L4-L5 bulge was found to be reduced
to 3 mm with no change in disc heights measured at any of the L4-S2
discs. The time of day when the images were acquired varied by no
more than 20 minutes.
Patient 3:
[0106] ML was a 24 year old white female with a traumatic low back
injury associated with a military training exercise. At the time of
presentation she had been symptomatic for three years. Her
complaints consisted of electric shooting pain down one and
sometimes both legs, low back pain with prolonged sitting or
standing, and pain with bending or stooping. She had failed
conservative management consisting of physical therapy. The
pre-procedure MM demonstrated decreased disc height but somewhat
preserved T2 signal in the L5-S1 disc (see FIG. 5). This disc also
had an extrusion of 0.7 cm that was contained by the posterior
longitudinal ligament. The posterior annulus was disrupted and
there was a high intensity zone from the nucleus pulposis to the
posterior longitudinal ligament. The L4-L5 disc had less T2 signal
and was dehydrated, but had preservation of disc height. There was
a protrusion at this level measuring 0.4 cm with a high intensity
zone seen in the inferomedial portion of the disc. Based on the
history, examination and MM findings, intermittent traversing nerve
root irritation at L5 and S1 roots was suspected at both L4-L5 and
L5-S1 disc levels. Discogram was performed showing low pressure
concordant pain (P2) at both L4-L5 and L5-S1, both with posterior
annular tears. The decision was made to treat only the L5-S1 disc
with the MSC injection, leaving L4-L5 to serve as an untreated
control.
[0107] At 6 months post procedure (see Table 1) ML reported
improvement of approximately 40% in low back modified VAS (1-10
scale) with a decrease of greater than 60% in frequency of pain.
Function improved as measured by FRI by more than 70%. She self
reported symptom improvement of 60%. Her post-procedure lumbar MRI
imaging demonstrated that the size of the L5-S1 disc protrusion
decreased from 0.7 cm pre-procedure to 0.3 cm at 1 month post
procedure and 0.4 cm at 5 months post procedure (see FIGS. 6 and
7). Additionally, the size of the HIZ area in the posterior disc
annulus decreased in both follow-up films. The time of image
acquisition differed by a no more than 1.5 hours.
TABLE-US-00001 TABLE 1 Pre-procedure and 6 month post procedure
reported outcomes for low back modified VAS and frequency.
Frequency of 1.0 equals constant pain. Functional Rating Index
measurements as well as self report of percentage change in
condition. VAS-pre VAS-post VAS-pre AVG VAS-post AVG Self-Reported
Subject AVG AVG frequency frequency FRI-pre FRI-post Outcome ML 4 2
1.00 0.38 44 13 60% improvement MJM 3 1 1.00 0.13 23 9 50%
improvement HO 7 4 1.0 1.0 27 25 No lasting improvement
TABLE-US-00002 TABLE 2 Total MSC yields and time in culture.
Subject Final MSC Yield in Millions Days in Culture ML 14.3 18 MJM
33.0 17 HO 28.5 17
Discussion:
[0108] Two of the three treated subjects demonstrated a decrease in
the size of the treated disc protrusion and reported sustained
subjective and functional improvement. A single subject
demonstrated a temporary decrease in symptoms and a small transient
change in the size of the disc protrusion, followed by regression
to pretreatment baseline measures. Of interest, placing cells
preferentially into the posterior disc annulus of this patient was
technically difficult, with sub-optimal flow of contrast into the
posterior annulus. The inventors question possible correlation
between this suboptimal placement and the subject's lack of
sustained response. Koga et al. has demonstrated that MSC's
injected nonspecifically into the intra-articular space failed to
repair cartilage lesions, but those applied directly on the defect,
allowing attachment at the target tissue were capable of repair.
Note that HO's contrast flow and subsequent MSC flow (see FIG. 14)
was primarily into the nucleus pulposus. Our own unpublished
clinical experience in placing mesenchymal stem cells directly into
the nucleus pulposus of other patients failed to initiate any
observable MRI changes. These observations would also support the
concept that location of stem cell adherence may be critical to the
success of treatment. In particular, the posterior disc annulus
maintains some vascular perfusion, while the avascular nucleus
pulposus has a well documented suboptimal nutritional environment,
which may result in a lack of sustainability of implanted MSC's.
Martin, M. D., C. M. Boxell, and D. G. Malone, Pathophysiology of
lumbar disc degeneration: a review of the literature. Neurosurg
Focus, 2002. 13(2): p. E1. Also of note, HO had the least prolific
stem cell yield, producing significantly fewer cells over a longer
culture period than the other subjects. (see Table 2).
[0109] Since MSC's have a fibroblastic morphology in monolayer
culture (see FIG. 2) the observed results could have been due to
fibroblastic differentiation of the MSC's placed preferentially
into the posterior disc annulus. Awad, H. A., et al., In vitro
characterization of mesenchymal stem cell-seeded collagen scaffolds
for tendon repair: effects of initial seeding density on
contraction kinetics. J Biomed Mater Res, 2000. 51(2): p. 233-40.
Delorme, B. and P. Charbord, Culture and characterization of human
bone marrow. Mesenchymal stem cells. Methods Mol Med, 2007. 140: p.
67-81. Xiang, Y., et al., Ex vivo expansion and pluripotential
differentiation of cryopreserved human bone marrow mesenchymal stem
cells. J Zhejiang Univ Sci B, 2007. 8(2): p. 136-46.
[0110] Alternatively, the other variable which may have contributed
to a therapeutic effect was the follow up platelet supernatant
epidural injections. Martineau et al. has shown that a platelet
supernatant prepared using specific calcium and thrombin
preconditioning, produces a maximal burst of VEGF degranulation
from platelets (as well as a host of other growth factors).
Martineau, I., E. Lacoste, and G. Gagnon, Effects of calcium and
thrombin on growth factor release from platelet concentrates:
kinetics and regulation of endothelial cell proliferation.
Biomaterials, 2004. 25(18): p. 4489-502. VEGF is known to cause
angiogenesis and the human degenerative intervertebral disc (IVD)
is known to suffer from poor vascular perfusion. Maroudas, A., et
al., Factors involved in the nutrition of the human lumbar
intervertebral disc: cellularity and diffusion of glucose in vitro.
J Anat, 1975. 120(Pt 1): p. 113-30. Wallace, A.L., et al., Humoral
regulation of blood flow in the vertebral endplate. Spine, 1994.
19(12): p. 1324-8. Pandya, N. M., N. S. Dhalla, and D. D. Santani,
Angiogenesis--a new target for future therapy. Vascul Pharmacol,
2006. 44(5): p. 265-74.
[0111] An alternative explanation for the decrease in IVD
protrusion in these patients may have been attributed to diurnal
effects. Park et al. demonstrated changes in the size of disc
bulges in 8 asymptomatic volunteers at L4-L5 when MRIs were
performed in the morning and evening. Park, C. O., Diurnal
variation in lumbar MRI. Correlation between signal intensity, disc
height, and disc bulge. Yonsei Med J, 1997. 38(1): p. 8-18. While
this effect could have occurred in these subjects, two of the
patients had untreated control discs that did not change in size.
In addition, the single imaging center responsible for the MRI
studies was instructed to perform follow-up scans as close as
possible to the same time of day as the initial scans. As a result,
the maximum time of day variance between images for any patient was
less than 2 hours. Finally, treated and control disc heights were
measured and no significant changes were noted for all subjects. If
diurnal changes had been present, one would expect significant
variation in disc height due to the effects of imbibition. An
alternative explanation for reduction in disc protrusion size could
be a healing effect induced directly by needle trauma. Korecki et
al. investigated this effect in an in-vitro animal model and found
the opposite to be true, needle puncture likely caused
biomechanical injury to the disc and a reduction in its overall
function. Korecki, C. L., J. J. Costi, and J. C. Iatridis, Needle
puncture injury affects intervertebral disc mechanics and biology
in an organ culture model. Spine, 2008. 33(3): p. 235-41.
Additionally, it should be noted that the standard model for
inducing DDD in animals is via puncture injury. Niinimaki, J., et
al., Quantitative magnetic resonance imaging of experimentally
injured porcine intervertebral disc. Acta Radiol, 2007. 48(6): p.
643-9.
Conclusions:
[0112] This Example demonstrates that percutaneously implanted
autologous MSC's with platelet supernatant epidural supplementation
is capable of reducing the size of contained lumbar disc
protrusion. Although it is unclear why one of the patients did not
respond to the therapy, it is reasonable to hypothesize that lack
of response was attributable to comparatively low MSC yield, as
well as the sub-optimal placement of the MSCs.
[0113] The data in this Example shows the utility of one embodiment
of the invention where procedures described herein provide a
surprising improvement in repair over other procedures described in
the art. Data with regard to other avascular repair sites, e.g.,
shoulder, hip, etc, is expected to show similar levels of
improvement.
[0114] Various embodiments of the disclosure could also include
permutations of the various elements recited in the claims as if
each dependent claim was multiple dependent claim incorporating the
limitations of each of the preceding dependent claims as well as
the independent claims. Such permutations are expressly within the
scope of this disclosure.
[0115] While the invention has been particularly shown and
described with reference to a number of embodiments, it would be
understood by those skilled in the art that changes in the form and
details may be made to the various embodiments disclosed herein
without departing from the spirit and scope of the invention and
that the various embodiments disclosed herein are not intended to
act as limitations on the scope of the claims.
[0116] The description of the present invention has been presented
for purposes of illustration and description, but is not intended
to be exhaustive or limiting of the invention to the form
disclosed. The scope of the present invention is limited only by
the scope of the following claims. Many modifications and
variations will be apparent to those of ordinary skill in the art.
The embodiment described and shown in the figures was chosen and
described in order to best explain the principles of the invention,
the practical application, and to enable others of ordinary skill
in the art to understand the invention for various embodiments with
various modifications as are suited to the particular use
contemplated.
[0117] This specification contains numerous citations to patent,
patent application, and publications. Each is hereby incorporated
by reference for all purposes.
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