U.S. patent application number 13/113599 was filed with the patent office on 2012-04-26 for compositions of adult disc stem cells and methods for the treatment of degenerative disc disease.
Invention is credited to Christopher Duntsch, Tatyana Ignatova, Valery KUKEKOV.
Application Number | 20120100607 13/113599 |
Document ID | / |
Family ID | 46673127 |
Filed Date | 2012-04-26 |
United States Patent
Application |
20120100607 |
Kind Code |
A1 |
Duntsch; Christopher ; et
al. |
April 26, 2012 |
COMPOSITIONS OF ADULT DISC STEM CELLS AND METHODS FOR THE TREATMENT
OF DEGENERATIVE DISC DISEASE
Abstract
This invention provides a tissue growth apparatus comprising one
or more discospheres and a method of producing nucleus pulposus
cells comprising the step of growing one or more discospheres on
the tissue growth apparatus. This invention also provides a
neo-engineered disc comprising nucleus pulposus cells, and related
methods of production and methods of use.
Inventors: |
Duntsch; Christopher;
(Memphis, TN) ; KUKEKOV; Valery; (Memphis, TN)
; Ignatova; Tatyana; (Memphis, TN) |
Family ID: |
46673127 |
Appl. No.: |
13/113599 |
Filed: |
May 23, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12216544 |
Jul 7, 2008 |
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13113599 |
|
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61442315 |
Feb 14, 2011 |
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60929792 |
Jul 12, 2007 |
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Current U.S.
Class: |
435/366 |
Current CPC
Class: |
C12N 2533/78 20130101;
C12N 2500/25 20130101; C12N 5/0662 20130101; C12N 2500/84 20130101;
C12N 5/066 20130101; A61K 35/32 20130101; C12N 2500/46 20130101;
C12N 2533/54 20130101; C12N 2501/11 20130101; C12N 2501/392
20130101; C12N 2500/90 20130101; C12N 5/0655 20130101; C12N
2501/115 20130101; A61K 35/12 20130101 |
Class at
Publication: |
435/366 |
International
Class: |
C12N 5/077 20100101
C12N005/077 |
Claims
1. A two dimensional tissue growth apparatus comprising one or more
discospheres.
2. The apparatus of claim 1, wherein said apparatus further
comprises media.
3. The apparatus of claim 1, wherein said apparatus comprises
gelatin.
4. The apparatus of claim 1, wherein said discospheres are seeded
at approximately 1 sphere/cm.sup.2.
5. (canceled)
6. The apparatus of claim 1, wherein said discospheres comprise
disc stem cells, disc progenitor cells, or a mixture thereof.
7. A three dimensional tissue growth apparatus comprising one or
more discospheres.
8. The apparatus of claim 7, wherein said apparatus further
comprises media.
9. The apparatus of claim 7, wherein said apparatus comprises
gelatin.
10. The apparatus of claim 7, wherein said discospheres are seeded
at approximately 1 sphere/cm.sup.2.
11. (canceled)
12. The apparatus of claim 7, wherein said discospheres comprise
disc stem cells, disc progenitor cells, or a mixture thereof.
13. A method of producing nucleus pulposus cells, comprising the
step of growing one or more discospheres on a two-dimensional
apparatus, thereby producing disc stem cells.
14. The method of claim 13, wherein said nucleus pulposus cells
express proteoglycans.
15. The method of claim 13, wherein said nucleus pulposus cells
express mucopolysaccharides.
16. The method of claim 13, wherein said nucleus pulposus cells are
motile.
17. The method of claim 13, wherein said nucleus pulposus cells are
human nucleus pulposus cells.
18. The method of claim 13, wherein said nucleus pulposus cells
comprise disc stem cells, disc progenitor cells, or a mixture
thereof.
19. The method of claim 13, wherein said apparatus further
comprises media.
20. The method of claim 13, wherein said apparatus comprises
gelatin.
21. The method of claim 13, wherein said discospheres are seeded at
approximately 1 sphere/cm.sup.2.
22.-40. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 61/442,315, filed Feb. 14, 2011, and is a
continuation-in-part of U.S. application Ser. No. 12/216,544 filed
Jul. 7, 2008, which claims the benefit of U.S. Provisional
Application Ser. No. 60/929,792, filed Jul. 12, 2007 which are
hereby incorporated by reference in their entirety.
FIELD OF INVENTION
[0002] This invention provides a tissue growth apparatus comprising
one or more discospheres and a method of producing nucleus pulposus
cells comprising the step of growing one or more discospheres on
the tissue growth apparatus. This invention also provides a
neo-engineered disc comprising nucleus pulposus cells, and related
methods of production and methods of use.
BACKGROUND OF THE INVENTION
[0003] Back pain resulting from degenerative disc disease is a
major cause of morbidity, disability, and lost productivity. Back
pain is the most frequent cause of activity limitation in people
under the age of 45, the second most frequent reason for physician
visits, the fifth-ranking reason for hospitalization, and the third
most common reason for surgical procedures. Additionally, chronic
back conditions that are both common and debilitating occur in 15
to 45 percent of people each year, and in 70 to 85 percent of
people at some time in their lives. The financial impact in terms
of health care dollars and lost work hours to society is between
$20 billion and $50 billion per year in the United States
alone.
[0004] Despite the continued improvements in non-operative and
operative treatment options for patients with lower back pain
secondary to degenerative disc disease, no treatment modalities
have served as the "magic bullet" to eliminate or consistently
improve this condition. Today, however, there are new and exciting
opportunities for the development of treatment modalities derived
from the merging of biomedical engineering and molecular science.
We are closer today then ever before to creating new treatment
modalities and devices for the treatment of degenerative disc
disease. Recent examples of advancements in bioscience and the
effect on clinical spine disease include the development of fusion
proteins, total disc arthroplasty and more recently nucleus
arthroplasty. Fusion proteins, such as recombinant human bone
morphogenetic protein-2 (rhBMP-2), are genetically produced
proteins that have the ability to stimulate new bone growth to
allow for a more reliable and rapid fusion of spinal vertebrae in
the context of surgical reconstruction.
[0005] The first total disc arthroplasty was performed by Fernstorm
in the late 1950's. Although initially there was a short period of
symptom relief, the prosthesis ultimately failed secondary to
subsidence of the implant within the spine vertebra. Although total
disc arthroplasty for the lumbar spine has been performed in Europe
since the late 1980's, its use in the United States did not begin
until March of 2000 with the introduction of the SB Charite III
(DePuy Spine, Raynham, Mass.). 10,11 Several other lumbar spine
prostheses have since been introduced, including the Maverick
(Medtronic Sofamor Danek, Memphis, Tenn.), the ProDisc-L (Spine
Solutions/Synthes, Paoli, Pa.), and FlexiCore (Stryker Spine,
Allendale, N.J.). Each of these prostheses differs in design with
respect to bearing surface, fixation to bone, number of
articulations, material, constraint, and mobility of the center of
rotation. In addition to the lumbar disc arthroplasty, as of last
year trials for cervical disc arthroplasty have begun in the United
States. Models of cervical disc arthroplasty include the Bryan
Cervical Disk (Medtronic Sofamor Danek), the Prestige ST (Medtronic
Sofamor Danek), the Porous Coated Motion artificial cervical disk
(Cervitech, Rockaway, N.J.), and the ProDisc-C (Spine
Solutions/Synthes).
[0006] Nucleus arthroplasty or nucleus replacement devices for
degenerative spine disease such as the PDN.RTM. Prosthetic Disc
Nucleus are similar in concept to TDA and have shown successful
results. The PDN.RTM. device consists of a hydrogel core center
encased in a polyethylene sleeve which shrinks and swells during
normal loading and unloading allowing for restoration of disc space
height and thus mimicking healthy human disc.
[0007] Although the total disc arthroplasty and nucleoplasty may
serve as an alternative to interbody spinal fusion, the procedure
is not without its complications. The most common complications
include adjacent level spinal disease, subsidence, and facet joint
arthrosis. Furthermore, recent studies from clinical trials have
demonstrated incidences of infection, vertebral body fracture,
implant malposition, subsidence, mechanical failure, and
paravertebral heterotopic ossification. More serious complications,
including anterior dislocation of the implant, have been reported.
Also, the issue of wear particles from the total disc arthroplasty
(TDA) and the potential effects on the spinal cord are still not
known. It is therefore evident that although the development of the
total disc arthroplasty is a step forward in the treatment of
degenerative disc disease, the ultimate goal should be the
development and replacement of a degenerative disc with a new
biologic disc which does not have the complications associated with
mechanical parts.
[0008] More than one million spine surgery procedures are performed
annually in United States. Furthermore, the lumbar fusion segment
of the spine surgery market is estimated at well over $1 billion in
annual revenue.
SUMMARY OF THE INVENTION
[0009] In one embodiment, the present invention provides a two
dimensional tissue growth apparatus comprising one or more
discospheres.
[0010] In another embodiment, the present invention provides a
three dimensional tissue growth apparatus comprising one or more
discospheres.
[0011] In another embodiment, the present invention provides a
method of producing nucleus pulposus cells, comprising the step of
growing one or more discospheres on a two-dimensional
apparatus.
[0012] In another embodiment, the present invention provides a
neo-engineered disc comprising nucleus pulposus cells.
[0013] In another embodiment, the present invention provides a
method of producing a neo-engineered disc, comprising the step of
growing one or more discospheres on a two-dimensional apparatus,
thereby producing a neo-engineered disc.
[0014] In another embodiment, the present invention provides a
method of treating a subject having a herniated disc, comprising
the step of administering to said subject a neo-engineered disc
comprising nucleus pulposus cells, thereby treating said subject
having a herniated disc.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The subject matter regarded as the invention is particularly
pointed out and distinctly claimed in the concluding portion of the
specification. The invention, however, both as to organization and
method of operation, together with objects, features, and
advantages thereof, may best be understood by reference to the
following detailed description when read with the accompanying
drawings in which:
[0016] FIG. 1 shows a microscopic histomorphological assessment
with various tissue stains of 3 month intervertebral disc cultures
after in vitro transplantation of human disc stem cells into
evacuated rabbit nucleus pulposus with bony end plates. Panel 1
shows photomicrographs of hematoxylin-eosin staining of rabbit disc
tissue in micrographs A, C, and E (control) and cultured
intervertebral disc in micrographs B, D, and F. Magnification:
1.25.times. (A and B), 10.times. (C and D), and 20.times. (E and
F). Photomicrographs C and D show the transition zone between the
inner nucleus pulposus and outer annulus. Photomicrograph E and F
show the inner zone of the nucleus pulposus and individual nucleus
pulposus cells. Panel 2 shows photomicrographs of safranin staining
of rabbit disc tissue in micrographs G, I, and K (control) and
cultured intervertebral disc in micrographs H, J, and L.
Magnification: 1.25.times. (G and H), 10.times. (I and J), and
20.times. (K and L). Photomicrograph I and J demonstrate the
transition zone between the inner nucleus pulposus and outer
annulus. Photomicrograph K and L demonstrate the inner zone of the
nucleus pulposus and individual nucleus pulposus cells. Panel 3
shows photomicrographs of Von Kossa staining of rabbit disc tissue
in micrographs M, O, and Q (control) and cultured intervertebral
disc in micrographs H, J, and L. Magnification: 1.25.times. (M and
N), 10.times. (O and P), and 20.times. (Q and R). Micrographs O and
P demonstrate the transition zone between the inner nucleus
pulposus and outer annulus. Micrographs Q and R demonstrate the
inner zone of the nucleus pulposus and individual nucleus pulposus
cells.
[0017] FIG. 2 shows a microscopic histomorphological assessment of
the expression of collagen type II in 3-month intervertebral disc
cultures after in vitro transplantation of human disc stem cells
into evacuated rabbit nucleus pulposus with bony end plates.
Photomicrographs of immunostaining for collagen type 2 of (A, C, E)
control (rabbit disc tissue) and (B, D, F) cultured intervertebral
disc. Magnification: 1.25.times. (A and B), 10.times. (C and D),
and 20.times. (E and F). Micrographs C and D demonstrate the
transition zone between the inner nucleus pulposus and outer
annulus. Micrographs E and F demonstrate the inner zone of the
nucleus pulposus and individual nucleus pulposus cells.
[0018] FIG. 3 shows a microscopic histomorphological assessment of
the expression of collagen type I in 3 month intervertebral disc
cultures after in vitro transplantation of human disc stem cells
into evacuated rabbit nucleus pulposus with bony end plates.
Photomicrographs of immunostaining for collagen type I of (A, C, E)
control (rabbit disc tissue) and (B, D, F) cultured intervertebral
disc (annulus matrix prepared in which the nucleus pulposus has
been chemically removed and human disc stem cell preparations have
been introduced). Magnification: 1.25.times. (A and B), 10.times.
(C and D), and 20.times. (E and F). Micrographs C and D demonstrate
the transition zone between the inner nucleus pulposus and outer
annulus. Micrographs E and F demonstrate the inner zone of the
nucleus pulposus and individual nucleus pulposus cells.
[0019] FIG. 4 shows a microscopic histomorphological assessment of
the expression of Ki-67 in 3 month intervertebral disc cultures
after in vitro transplantation of human disc stem cells into
evacuated rabbit nucleus pulposus with bony end plates.
Photomicrographs of immunostaining for Ki-67 of (A, C, E) control
(rabbit disc tissue) and (B, D, F) cultured intervertebral disc
(annulus matrix prepared in which the nucleus pulposus has been
chemically removed and human disc stem cell preparations have been
introduced). Magnification: 1.25.times. (A and B), 10.times. (C and
D), and 20.times. (E and F). Micrographs C and D demonstrate the
transition zone between the inner nucleus pulposus and outer
annulus. Micrographs E and F demonstrate the inner zone of the
nucleus pulposus and individual nucleus pulposus cells.
[0020] FIG. 5 depicts a schematic of the intervertebral disc
culture system with bony end plates. A--Single cell cultures are
prepared in media and conditions that promote growth of
discospheres (disc stem cell clusters). Discospheres are then
prepared and injected into the annulus of a healthy rabbit in which
all cells and nucleus pulposus tissue are removed from the disc.
B--Intervertebral disc annulus with bony end plates are then put
into a culture vessel with media and growth factors. At the end of
3 months, disc stem cells fill the previously empty annulus with a
disc like structure.
[0021] FIG. 6 describes the 2 D Tissue Engineering Process.
[0022] FIG. 7 shows 2D cell assay at 72 hours and cell
proliferation kinetics, including distance and velocity at 24-30
hours.
[0023] FIG. 8 shows central nucleus pulposus cells surrounded by
circular array after seeding spheres at 1.066 spheres/cm.sup.2 for
4 weeks.
[0024] FIG. 9 presents normal rabbit disc, enucleated rabbit disc,
and neoengineered disc tissue made without scaffolds using enriched
stem cells.
[0025] FIG. 10 shows the linear growth of discospheres over time
per passage.
[0026] FIG. 11 shows histology and proteoglycan assays of
discospheres plated on gelatin coated coverslips. Discosphere
remnants are surrounded by progeny that express low levels of
proteoglycans.
DETAILED DESCRIPTION OF THE INVENTION
[0027] In one embodiment, the present invention provides a two
dimensional tissue growth apparatus comprising one or more
discospheres.
[0028] In another embodiment, the present invention provides a
three dimensional tissue growth apparatus comprising one or more
discospheres.
[0029] In another embodiment, the present invention provides a
method of producing nucleus pulposus cells, comprising the step of
growing one or more discospheres on a two-dimensional
apparatus.
[0030] In another embodiment, the present invention provides a
method of producing nucleus pulposus cells, comprising the step of
growing one or more discospheres on a three-dimensional
apparatus.
[0031] In another embodiment, the present invention provides a
neo-engineered disc comprising nucleus pulposus cells. In another
embodiment, the present invention provides a neo-engineered disc
comprising disc stem cells.
[0032] In another embodiment, the present invention provides a
method of producing a neo-engineered disc, comprising the step of
growing one or more discospheres on a two-dimensional apparatus,
thereby producing a neo-engineered disc. In another embodiment, the
present invention provides a method of producing a neo-engineered
disc, comprising the step of growing one or more discospheres on a
three-dimensional apparatus, thereby producing a neo-engineered
disc.
[0033] In another embodiment, the present invention provides a
method of treating a subject having a herniated disc, comprising
the step of administering to said subject a neo-engineered disc
comprising nucleus pulposus cells, thereby treating said subject
having a herniated disc. In another embodiment, the present
invention provides a method of treating a subject having lower back
pain, comprising the step of administering to said subject a
neo-engineered disc comprising nucleus pulposus cells. In another
embodiment, the present invention provides a method of treating a
subject having a degenerative disc disease, comprising the step of
administering to said subject a neo-engineered disc comprising
nucleus pulposus cells.
[0034] In one embodiment, the present invention provides an
isolated disc stem cell population. In another embodiment, the
present invention provides a disc stem cells enriched population of
cells that can form discospheres. In another embodiment, the
present invention provides a disc stem cells enriched population of
cells that can give rise to disc progenitor cells. In another
embodiment, the isolated disc stem cell population of the present
invention comprises a human disc stem cell population. In another
embodiment, the isolated disc stem cell population of the present
invention comprises a non-human disc stem cell population. In
another embodiment, the isolated disc stem cell population of the
present invention comprises a mammal disc stem cell population. In
another embodiment, an isolated disc stem cell of the present
invention is derived from a nucleus pulposus of a subject. In
another embodiment, nucleus pulposus cells comprise disc stem
cells.
[0035] In another embodiment, the stem cells enriched cell
population of the present invention comprises a human disc stem
cell population. In another embodiment, the stem cells enriched
cell population of the present invention comprises a non-human disc
stem cell population. In another embodiment, the stem cells
enriched cell population of the present invention comprises a
mammal disc stem cell population. In another embodiment, the stem
cells enriched cell population of the present invention is derived
from a nucleus pulposus of a subject. In another embodiment,
nucleus pulposus cells comprise disc stem cells.
[0036] In another embodiment, a nucleus pulposus is a jelly-like
substance in the middle of the spinal disc. In another embodiment,
the nucleus pulposus comprises chondrocytes, collagen fibrils, and
proteoglycan aggrecans that have hyaluronic long chains which
attract water.
[0037] In another embodiment, nucleus pulposus cells of the present
invention comprise autograft nucleus pulposus cells. In another
embodiment, nucleus pulposus cells of the present invention
comprise allograft nucleus pulposus cells. In another embodiment,
nucleus pulposus cells of the present invention comprise xenograft
nucleus pulposus cells.
[0038] In another embodiment, nucleus pulposus cells of the present
invention comprise disc stem cells. In another embodiment, nucleus
pulposus cells of the present invention comprise disc progenitor
cells. In another embodiment, nucleus pulposus cells of the present
invention comprise mature disc cells. In another embodiment,
nucleus pulposus cells of the present invention comprise terminally
differentiated disc cells.
[0039] In another embodiment, the present invention provides a
method of isolating disc stem cells, comprising the step of
producing a discosphere culture. In another embodiment, the present
invention provides a method of isolating disc stem cells,
comprising the step of plating nucleus pulposus cells in a serum
free media. In another embodiment, the present invention provides a
method of producing a sphere comprising nucleus pulposus cells,
comprising the step of growing a culture of nucleus pulposus cells
in a serum free media, thereby producing a discosphere. In another
embodiment, the present invention provides that a discosphere
comprising nucleus pulposus cells is a free-floating structure
generated by nucleus pulposus stem cells in vitro. In another
embodiment, the present invention provides that a discosphere is a
free-floating structure generated by nucleus pulposus progenitor
cells in vitro. In another embodiment, the present invention
provides that a discosphere is a free-floating structure generated
by nucleus pulposus stem and progenitor cells in vitro.
[0040] In another embodiment, a disc stem cell of the present
invention is defined by its ability or capacity to form a
discosphere. In another embodiment, these disc stem cells when
grown in adherent culture have the capability to differentiate,
under appropriate differentiating conditions, to mature or fully
differentiate. In another embodiment, fully differentiated nucleus
pulposus cells secrete extra cellular matrix components. In another
embodiment, the terms "differentiate" or "differentiation" intended
to refer to the development of cells with specialized structure and
function from unspecialized or less specialized precursor cells,
and includes the development of cells that possess the structure
and function of nucleus pulposus cells from precursor cells. In
another embodiment, the terms "differentiate" or "differentiation"
intended to refer to the development of cells with specialized
structure and function from disc stem cells. In another embodiment,
the terms "differentiate" or "differentiation" intended to refer to
the development of cells with specialized structure and function
from disc progenitor cells. In another embodiment, appropriate
differentiating conditions comprise a media comprising serum.
[0041] In another embodiment, the methods of the present invention
provide that disc material is obtained from the nucleus pulposus of
a subject. In another embodiment, the methods of the present
invention provide that disc material is obtained surgically and
processed in the lab to create a single cell suspension of nucleus
pulposus cells (Example 1). In another embodiment, the methods of
the present invention provide that human disc material is obtained
surgically and processed in the lab to create a single cell
suspension of nucleus pulposus cells. In another embodiment, the
methods of the present invention provide that human nucleus
pulposus is obtained surgically and processed in the lab to create
a single cell suspension of nucleus pulposus cells.
[0042] In another embodiment, a heterogeneous population of nucleus
pulposus cells is obtained by scraping a nucleus pulposus of a
subject. In another embodiment, heterogeneous population of nucleus
pulposus cells comprises disc stem cells, disc progenitor cells,
and differentiated nucleus pulposus cells. In another embodiment, a
heterogeneous population of nucleus pulposus cells is scraped from
a nucleus pulposus of a human subject. In another embodiment, the
present invention provides that plating a heterogeneous population
of nucleus pulposus cells in a serum free media at low cell density
results in the survival of nucleus pulposus stem cells. In another
embodiment, the term survival of nucleus pulposus stem cells refers
to nucleus pulposus stem cells ability to maintain viability under
conditions which include a serum-free cell culture media. In
another embodiment, the present invention provides that the nucleus
pulposus cells (majority of the cells in the tissue) die away
because they cannot tolerate serum-free conditions, but the disc
stem cells (or nucleus pulposus stem cells, minority of the cells
in the tissue) grow into discospheres under theses conditions.
[0043] In another embodiment, the present invention provides that
plating a heterogeneous population of nucleus pulposus cells in a
serum free media at low cell density results in isolation of
nucleus pulposus stem cells. In another embodiment, the present
invention provides that plating a heterogeneous population of
nucleus pulposus cells in a serum free media at low cell density
results in enriching a nucleus pulposus cell population for disc
stem cells. In another embodiment, the present invention provides
that plating a heterogeneous population of nucleus pulposus cells
at low cell density in a serum free media, comprising a substance
the interferes with cell attachment results in the survival of
nucleus pulposus stem cells. In another embodiment, the present
invention provides that plating a heterogeneous population of
nucleus pulposus cells at low cell density in a serum free media,
comprising methylcellulose which interferes with cell attachment,
results in the survival of nucleus pulposus stem cells.
[0044] In another embodiment, a heterogeneous population of nucleus
pulposus cells is obtained from a biopsy specimen of nucleus
pulposus minced in pieces. In another embodiment, the pieces are
0.5-10 mm in size. In another embodiment, the pieces are 0.5-20 mm
in size. In another embodiment, the pieces are 0.5-3 mm in size. In
another embodiment, the pieces are 3-6 mm in size. In another
embodiment, the pieces are 6-12 mm in size. In another embodiment,
the pieces are 12-20 mm in size. In another embodiment, the pieces
are 1-6 mm in size. In another embodiment, the pieces are 3-5 mm in
size. In another embodiment, the pieces are 3-4 mm in size (Example
1).
[0045] In another embodiment, a heterogeneous population of nucleus
pulposus cells is obtained from a biopsy specimen of nucleus
pulposus by treating nucleus pulposus with a collagenase II
solution (Example 1). In another embodiment, a heterogeneous
population of nucleus pulposus cells is obtained from a biopsy
specimen of nucleus pulposus by treating nucleus pulposus with a
0.1%-1% clostridial collagenase (Worthington CLS II, 140 u/mg). In
another embodiment, a heterogeneous population of nucleus pulposus
cells is obtained from a biopsy specimen of nucleus pulposus by
treating nucleus pulposus with a collagenase II solution followed
by placing the specimen in a shaker thus obtaining a heterogeneous
population of nucleus pulposus cells.
[0046] In another embodiment, a heterogeneous population of nucleus
pulposus cells is obtained from a biopsy specimen of nucleus
pulposus by aspiration of a disc of a patient. In another
embodiment, a heterogeneous population of nucleus pulposus cells is
obtained from a biopsy specimen of nucleus pulposus by aspiration
of a disc of a donor animal. In another embodiment, a heterogeneous
population of nucleus pulposus cells is obtained from a biopsy
specimen of nucleus pulposus by aspiration of a nucleus pulposus of
a donor mammal. In another embodiment, a heterogeneous population
of nucleus pulposus cells is obtained from a biopsy specimen of
nucleus pulposus by aspiration of a healthy disc of a patient.
[0047] In another embodiment, the present invention provides a
method of producing a discosphere, comprising the step of growing a
culture of nucleus pulposus cells in a serum free media, thereby
producing a discosphere. In another embodiment, the present
invention provides that growing a primary culture of nucleus
pulposus cells in a serum free media results in selecting nucleus
pulposus stem cells. In another embodiment, the surviving isolated
culture of nucleus pulposus stem cells gives rise to discospheres
of the present invention. In another embodiment, the surviving disc
stem cells enriched culture of nucleus pulposus stem cells gives
rise to discospheres of the present invention.
[0048] In another embodiment, the supplemented serum free media of
the present invention enables only nucleus pulposus stem cells to
grow. In another embodiment, the methods of the present invention
provide that an enriched nucleus pulposus stem cell population is
produced when grown in a growth factor supplemented serum free
media of the present invention. In another embodiment, the methods
of the present invention provide that an enriched nucleus pulposus
stem cell population of the present invention comprises at least
60% nucleus pulposus stem cells. In another embodiment, the methods
of the present invention provide that an enriched nucleus pulposus
stem cell population of the present invention comprises at least
70% nucleus pulposus stem cells. In another embodiment, the methods
of the present invention provide that an enriched nucleus pulposus
stem cell population of the present invention comprises at least
80% nucleus pulposus stem cells. In another embodiment, the methods
of the present invention provide that an enriched nucleus pulposus
stem cell population of the present invention comprises at least
85% nucleus pulposus stem cells. In another embodiment, the methods
of the present invention provide that an enriched nucleus pulposus
stem cell population of the present invention comprises at least
90% nucleus pulposus stem cells. In another embodiment, the methods
of the present invention provide that an enriched nucleus pulposus
stem cell population of the present invention comprises at least
95% nucleus pulposus stem cells.
[0049] In another embodiment, a discosphere is derived from a
single nucleus pulposus stem cell. In another embodiment, only disc
stem cells grow when nucleus pulposus cells are plated in a serum
free media. In another embodiment, only disc stem cells grow when
nucleus pulposus cells are plated at low cell density. In another
embodiment, only disc stem cells grow when nucleus pulposus cells
are plated at low cell density in a serum free media. In another
embodiment, only nucleus pulposus stem cells can grow as free
floating solitary cells in the absence of serum.
[0050] In another embodiment, the present invention further
provides that disc stem cells are grown in a serum free media
comprising a compound which inhibits cell maturation. In another
embodiment, the present invention further provides that disc stem
cells are grown in a serum free media comprising FGF which inhibits
cell maturation. In another embodiment, the present invention
further provides that disc stem cells are grown in a serum free
media comprising a compound that maintains cell juvenility.
[0051] In another embodiment, the present invention further
provides that disc stem cells are grown in a media comprising a
TGF-.beta. superfamily member. In another embodiment, the present
invention further provides that disc stem cells are grown in a
media comprising a BMP. In another embodiment, the present
invention provides that a BMP of the invention inhibits
differentiation (Id) genes.
[0052] In another embodiment, the present invention further
provides that disc stem cells are grown in a media comprising an
IL6 cytokine family member. In another embodiment, the present
invention further provides that disc stem cells are grown in a
media comprising leukemia inhibitory factor (LIF).
[0053] In another embodiment, the present invention further
provides that disc stem cells are grown in a serum free media
comprising a compound which promotes cell proliferation. In another
embodiment, the present invention further provides that disc stem
cells are grown in a serum free media comprising EGF which promotes
cell proliferation. In another embodiment, the present invention
further provides that disc stem cells are grown in a serum free
media comprising interleukin-2 (IL-2). In another embodiment, the
present invention further provides that disc stem cells are grown
in a serum free media comprising interleukin-6 (IL-6). In another
embodiment, the present invention further provides that disc stem
cells are grown in a serum free media comprising a stem cell factor
(SCF). In another embodiment, the present invention further
provides that disc stem cells are grown in a serum free media
comprising leukemia inhibitory factor (LIF). In another embodiment,
the present invention further provides that disc stem cells are
grown in a serum free media comprising transforming growth
factor-.beta. (TGF-.beta.). In another embodiment, the present
invention further provides that disc stem cells are grown in a
serum free media comprising a compound that inhibits cell
differentiation (Example 1 and materials and methods).
[0054] In another embodiment, disc stem cells of the present
invention proliferate and give rise to additional stem cells. In
another embodiment, disc stem cells of the present invention
proliferate and give rise to disc progenitor cells. In another
embodiment, disc stem cells of the present invention proliferate
thus forming a discosphere. In another embodiment, a discosphere of
the present invention comprises nucleus pulposus stem cells and
nucleus pulposus progenitor cells arranged in a circular-spherical
structure. In another embodiment, a discosphere is a ball of cells
in which a single disc stem cell gives rise to clones of itself
(symmetric division) and to progenitor cells. In another
embodiment, a discosphere of the present invention comprises free
floating nucleus pulposus stem cells and nucleus pulposus
progenitor cells arranged in a circular-spherical structure. In
another embodiment, the nucleus pulposus cells comprising a
discosphere are attached to each other.
[0055] In another embodiment, the terms "nucleus pulposus stem
cells" and "disc stem cells" are used interchangeably. In another
embodiment, the terms "nucleus pulposus progenitor cells" and "disc
progenitor cells" are used interchangeably.
[0056] In another embodiment, the term "discosphere" comprises a
ball of cells in which a single disc stem cell gives rise to clones
of itself (symmetric division) and to progenitor cells. In another
embodiment, the term "progenitor cells" refer to immature stem-like
cells with plastic potential and high proliferation rates, which
can give rise to most if not all terminally differentiated tissue
cells, but is not by definition a disc stem cell.
[0057] In another embodiment, the methods of the present invention
provide that a single cell suspension is prepared for isolating a
disc stem cell by creating certain environmental conditions. In
another embodiment, the methods of the present invention provide
that a single cell suspension is prepared for producing a
discosphere by creating certain environmental conditions.
[0058] In another embodiment, the methods of the present invention
provide that a single cell suspension is incubated in a humidified
Incubator at 37.degree. C. In another embodiment, the methods of
the present invention provide that a single cell suspension is
incubated in a humidified Incubator at 35.degree. C. In another
embodiment, the methods of the present invention provide that a
single cell suspension is incubated in a humidified Incubator at
36.degree. C. In another embodiment, the methods of the present
invention provide that a single cell suspension is incubated in a
humidified Incubator at 38.degree. C. In another embodiment, the
methods of the present invention provide that a single cell
suspension is incubated in a humidified Incubator at 39.degree. C.
In another embodiment, the methods of the present invention provide
that a single cell suspension is incubated in a humidified
Incubator at 40.degree. C. In another embodiment, the methods of
the present invention provide that a single cell suspension is
incubated in a humidified Incubator at 41.degree. C. In another
embodiment, the methods of the present invention provide that a
single cell suspension is incubated in a humidified Incubator at
42.degree. C.
[0059] In another embodiment, the methods of the present invention
provide that a single cell suspension is incubated in an incubator
further maintaining 3-8% CO.sub.2. In another embodiment, the
methods of the present invention provide that a single cell
suspension is incubated in an incubator further maintaining 4%
CO.sub.2. In another embodiment, the methods of the present
invention provide that a single cell suspension is incubated in an
incubator further maintaining 5% CO.sub.2. In another embodiment,
the methods of the present invention provide that a single cell
suspension is incubated in an incubator further maintaining 6%
CO.sub.2.
[0060] In another embodiment, the methods of the present invention
provide that a single cell suspension is incubated in an incubator
further maintaining 60-100% humidity. In another embodiment, the
methods of the present invention provide that a single cell
suspension is incubated in an incubator further maintaining 70-100%
humidity. In another embodiment, the methods of the present
invention provide that a single cell suspension is incubated in an
incubator further maintaining 80-100% humidity. In another
embodiment, the methods of the present invention provide that a
single cell suspension is incubated in an incubator further
maintaining 90-100% humidity. In another embodiment, the methods of
the present invention provide that a single cell suspension is
incubated in an incubator further maintaining 95-100% humidity.
[0061] In another embodiment, the methods of the present invention
provide that a single cell suspension is plated at a final density
of less than 1.times.10.sup.6 cells/ml. In another embodiment, the
methods of the present invention provide that a single cell
suspension is plated at a final density of less than
5.times.10.sup.5 cells/ml. In another embodiment, the methods of
the present invention provide that a single cell suspension is
plated at a final density of less than 1.times.10.sup.5 cells/ml.
In another embodiment, the methods of the present invention provide
that a single cell suspension is plated at a final density of less
than 8.times.10.sup.4 cells/ml. In another embodiment, the methods
of the present invention provide that a single cell suspension is
plated at a final density of about 6.times.10.sup.4 cells/ml
(Example 1).
[0062] In another embodiment, the present invention provides a
composition comprising disc stem cells. In another embodiment, the
subject invention comprises a composition comprising a population
of nucleus pulposus cells enriched for nucleus pulposus stem cells.
In another embodiment, the composition further comprises an
appropriate environment, such as those described herein, wherein, a
disc stem cell can be induced to proliferate and generate disc stem
cells progeny. In another embodiment, the term environment in which
disc stem cells progeny are placed, refers to the combination of
external or extrinsic physical and/or chemical conditions that
affect and influence the growth and development of disc stem cells.
In another embodiment, the environment can be ex-vivo or in-vivo.
In another embodiment, a disc scaffold can serve as an in-vivo
environment that induces disc stem cells to generate progeny. In
another embodiment, the environment is ex-vivo and comprises disc
stem cells placed in cell culture medium in an incubator (Example
1).
[0063] In another embodiment, the present invention provides a
composition comprising disc stem cells and media. In another
embodiment, the media is a serum free media. In another embodiment,
the composition comprising disc stem cells further comprises
Epidermal Growth Factor (EGF) supplemented to the media. In another
embodiment, the composition comprising disc stem cells further
comprises 1-10 ng/ml EGF supplemented to the media. In another
embodiment, the composition comprising disc stem cells further
comprises 1-100 ng/ml EGF supplemented to the media. In another
embodiment, the composition comprising disc stem cells further
comprises 20-50 ng/ml EGF supplemented to the media. In another
embodiment, the composition comprising disc stem cells further
comprises 50-100 ng/ml EGF supplemented to the media. In another
embodiment, the composition comprising disc stem cells further
comprises 5-15 ng/ml EGF supplemented to the media. In another
embodiment, the composition comprising disc stem cells further
comprises 8-12 ng/ml EGF supplemented to the media.
[0064] In another embodiment, the composition comprising disc stem
cells further comprises FGF supplemented to the media. In another
embodiment, the composition comprising disc stem cells further
comprises Fibroblast Growth Factor 2 (FGF2) supplemented to the
media. In another embodiment, the composition comprising disc stem
cells further comprises 1-100 ng/ml FGF2 supplemented to the media.
In another embodiment, the composition comprising disc stem cells
further comprises 20-50 ng/ml FGF2 supplemented to the media. In
another embodiment, the composition comprising disc stem cells
further comprises 50-100 ng/ml FGF2 supplemented to the media. In
another embodiment, the composition comprising disc stem cells
further comprises 5-15 ng/ml FGF2 supplemented to the media. In
another embodiment, the composition comprising disc stem cells
further comprises 8-12 ng/ml FGF2 supplemented to the media.
[0065] In another embodiment, the composition comprising disc stem
cells further comprises insulin supplemented to the media. In
another embodiment, the composition comprising disc stem cells
further comprises 1-100 .mu.g/ml insulin supplemented to the media
(Example 1). In another embodiment, the composition comprising disc
stem cells further comprises 20-50 .mu.g/ml insulin supplemented to
the media. In another embodiment, the composition comprising disc
stem cells further comprises 50-100 .mu.g/ml insulin supplemented
to the media. In another embodiment, the composition comprising
disc stem cells further comprises 5-15 .mu.g/ml insulin
supplemented to the media. In another embodiment, the composition
comprising disc stem cells further comprises 8-12 .mu.g/ml insulin
supplemented to the media.
[0066] In another embodiment, the composition comprising disc stem
cells further comprises progesterone supplemented to the media
(Example 1). In another embodiment, the composition comprising disc
stem cells further comprises 1-200 ng/ml progesterone supplemented
to the media. In another embodiment, the composition comprising
disc stem cells further comprises 20-200 ng/ml progesterone
supplemented to the media. In another embodiment, the composition
comprising disc stem cells further comprises 50-150 ng/ml
progesterone supplemented to the media. In another embodiment, the
composition comprising disc stem cells further comprises 10-100
ng/ml progesterone supplemented to the media. In another
embodiment, the composition comprising disc stem cells further
comprises 20-80 ng/ml progesterone supplemented to the media. In
another embodiment, the composition comprising disc stem cells
further comprises 30-50 ng/ml progesterone supplemented to the
media.
[0067] In another embodiment, the composition comprising disc stem
cells further comprises putrescine supplemented to the media
(Example 1). In another embodiment, the composition comprising disc
stem cells further comprises 1-800 ng/ml putrescine supplemented to
the media. In another embodiment, the composition comprising disc
stem cells further comprises 1-100 ng/ml putrescine supplemented to
the media. In another embodiment, the composition comprising disc
stem cells further comprises 100-300 ng/ml putrescine supplemented
to the media. In another embodiment, the composition comprising
disc stem cells further comprises 300-500 ng/ml putrescine
supplemented to the media. In another embodiment, the composition
comprising disc stem cells further comprises 500-800 ng/ml
putrescine supplemented to the media. In another embodiment, the
composition comprising disc stem cells further comprises 150-250
ng/ml putrescine supplemented to the media. In another embodiment,
the composition comprising disc stem cells further comprises
180-220 ng/ml putrescine supplemented to the media.
[0068] In another embodiment, the composition comprising disc stem
cells further comprises transferrin supplemented to the media
(Example 1). In another embodiment, the composition comprising disc
stem cells further comprises 1-400 ng/ml transferrin supplemented
to the media. In another embodiment, the composition comprising
disc stem cells further comprises 1-100 ng/ml transferrin
supplemented to the media. In another embodiment, the composition
comprising disc stem cells further comprises 100-200 ng/ml
transferrin supplemented to the media. In another embodiment, the
composition comprising disc stem cells further comprises 200-400
ng/ml transferrin supplemented to the media. In another embodiment,
the composition comprising disc stem cells further comprises 20-150
ng/ml transferrin supplemented to the media. In another embodiment,
the composition comprising disc stem cells further comprises 80-200
ng/ml transferrin supplemented to the media. In another embodiment,
the composition comprising disc stem cells further comprises 80-120
ng/ml transferrin supplemented to the media.
[0069] In another embodiment, the composition comprising disc stem
cells further comprises sodium selenite supplemented to the media
(Example 1). In another embodiment, the composition comprising disc
stem cells further comprises 1-400 ng/ml sodium selenite
supplemented to the media. In another embodiment, the composition
comprising disc stem cells further comprises 1-100 ng/ml sodium
selenite supplemented to the media. In another embodiment, the
composition comprising disc stem cells further comprises 100-200
ng/ml sodium selenite supplemented to the media. In another
embodiment, the composition comprising disc stem cells further
comprises 200-400 ng/ml sodium selenite supplemented to the media.
In another embodiment, the composition comprising disc stem cells
further comprises 20-150 ng/ml sodium selenite supplemented to the
media. In another embodiment, the composition comprising disc stem
cells further comprises 40-180 ng/ml sodium selenite supplemented
to the media. In another embodiment, the composition comprising
disc stem cells further comprises 40-80 ng/ml sodium selenite
supplemented to the media.
[0070] In another embodiment, the composition comprising disc stem
cells further comprises methylcellulose supplemented to the media
(Example 1). In another embodiment, the composition comprising disc
stem cells further comprises 0.5-10% methylcellulose supplemented
to the media. In another embodiment, the composition comprising
disc stem cells further comprises 0.5-3% methylcellulose
supplemented to the media. In another embodiment, the composition
comprising disc stem cells further comprises 3-5% methylcellulose
supplemented to the media. In another embodiment, the composition
comprising disc stem cells further comprises 5-8% methylcellulose
supplemented to the media. In another embodiment, the composition
comprising disc stem cells further comprises 7-10% methylcellulose
supplemented to the media. In another embodiment, the composition
comprising disc stem cells further comprises 0.5-2.5%
methylcellulose supplemented to the media. In another embodiment,
the composition comprising disc stem cells further comprises 1-2.5%
methylcellulose supplemented to the media. In another embodiment,
the composition comprising disc stem cells further comprises
1.5-2.5% methylcellulose supplemented to the media.
[0071] In another embodiment, the composition comprising disc stem
cells further comprises an antibiotic supplemented to the media
(Example 1). In another embodiment, the antibiotic supplemented to
the media is penicillin-streptomycin. In another embodiment, the
composition comprising disc stem cells further comprises 1000-10000
U/ml penicillin-streptomycin supplemented to the media. In another
embodiment, the composition comprising disc stem cells further
comprises 1000-3000 U/ml penicillin-streptomycin supplemented to
the media. In another embodiment, the composition comprising disc
stem cells further comprises 3000-6000 U/ml penicillin-streptomycin
supplemented to the media. In another embodiment, the composition
comprising disc stem cells further comprises 6000-10000 U/ml
penicillin-streptomycin supplemented to the media. In another
embodiment, the composition comprising disc stem cells further
comprises 3000-8000 U/ml penicillin-streptomycin supplemented to
the media. In another embodiment, the composition comprising disc
stem cells further comprises 4000-6000 U/ml penicillin-streptomycin
supplemented to the media. In another embodiment, the composition
comprising disc stem cells further comprises 5000 U/ml
penicillin-streptomycin supplemented to the media.
[0072] In another embodiment, the composition comprising disc stem
cells further comprises KO serum replacer supplemented to the
media. In another embodiment, the composition comprising disc stem
cells further comprises 0.5-30% KO serum replacer supplemented to
the media. In another embodiment, the composition comprising disc
stem cells further comprises 5-30% knockout (KO) serum replacer
supplemented to the media. In another embodiment, the composition
comprising disc stem cells further comprises 3-5% KO serum replacer
supplemented to the media. In another embodiment, the composition
comprising disc stem cells further comprises 5-15% KO serum
replacer supplemented to the media. In another embodiment, the
composition comprising disc stem cells further comprises 15-30% KO
serum replacer supplemented to the media. In another embodiment,
the composition comprising disc stem cells further comprises 10-20%
KO serum replacer supplemented to the media. In another embodiment,
the composition comprising disc stem cells further comprises 15-25%
KO serum replacer supplemented to the media. In another embodiment,
the composition comprising disc stem cells further comprises 20% KO
serum replacer supplemented to the media.
[0073] In another embodiment, the composition comprising disc stem
cells further comprises non-essential Amino Acids supplemented to
the media. In another embodiment, the composition comprising disc
stem cells further comprises 0.1-10% non-essential Amino Acids
supplemented to the media. In another embodiment, the composition
comprising disc stem cells further comprises 0.1-1% non-essential
Amino Acids supplemented to the media. In another embodiment, the
composition comprising disc stem cells further comprises 1-5%
non-essential Amino Acids supplemented to the media. In another
embodiment, the composition comprising disc stem cells further
comprises 5-10% non-essential Amino Acids supplemented to the
media. In another embodiment, the composition comprising disc stem
cells further comprises 15-30% non-essential Amino Acids
supplemented to the media. In another embodiment, the composition
comprising disc stem cells further comprises 0.5-1% non-essential
Amino Acids supplemented to the media. In another embodiment, the
composition comprising disc stem cells further comprises 0.8-1.2%
non-essential Amino Acids supplemented to the media. In another
embodiment, the composition comprising disc stem cells further
comprises 1% non-essential Amino Acids supplemented to the
media.
[0074] In another embodiment, the composition comprising disc stem
cells further comprises L-glutamine supplemented to the media. In
another embodiment, the composition comprising disc stem cells
further comprises 0.1-10 mM L-glutamine supplemented to the media.
In another embodiment, the composition comprising disc stem cells
further comprises 0.1-5 mM L-glutamine supplemented to the media.
In another embodiment, the composition comprising disc stem cells
further comprises 5-10 mM L-glutamine supplemented to the media. In
another embodiment, the composition comprising disc stem cells
further comprises 5-8 mM L-glutamine supplemented to the media. In
another embodiment, the composition comprising disc stem cells
further comprises 0.5-2.5 mM L-glutamine supplemented to the media.
In another embodiment, the composition comprising disc stem cells
further comprises 1.5-3 mM L-glutamine supplemented to the media.
In another embodiment, the composition comprising disc stem cells
further comprises 0.5-1.5 mM L-glutamine supplemented to the media.
In another embodiment, the composition comprising disc stem cells
further comprises 0.8-1.2 mM L-glutamine supplemented to the
media.
[0075] In another embodiment, the composition comprising disc stem
cells further comprises b-mercaptoethanol supplemented to the
media. In another embodiment, the composition comprising disc stem
cells further comprises 0.01-1 mM b-mercaptoethanol supplemented to
the media. In another embodiment, the composition comprising disc
stem cells further comprises 0.01-0.5 mM b-mercaptoethanol
supplemented to the media. In another embodiment, the composition
comprising disc stem cells further comprises 0.5-1 mM
b-mercaptoethanol supplemented to the media. In another embodiment,
the composition comprising disc stem cells further comprises
0.5-0.8 mM b-mercaptoethanol supplemented to the media. In another
embodiment, the composition comprising disc stem cells further
comprises 00.5-0.25 mM b-mercaptoethanol supplemented to the media.
In another embodiment, the composition comprising disc stem cells
further comprises 0.15-0.3 mM b-mercaptoethanol supplemented to the
media. In another embodiment, the composition comprising disc stem
cells further comprises 0.05-0.15 mM b-mercaptoethanol supplemented
to the media. In another embodiment, the composition comprising
disc stem cells further comprises 0.08-0.12 mM b-mercaptoethanol
supplemented to the media.
[0076] In another embodiment, the composition comprising disc stem
cells further comprises a media comprising Dulbecco's Modified
Eagle's Medium (DMEM). In another embodiment, the composition
comprising a discosphere further comprises a media comprising
DMEM/F12. In another embodiment, the composition comprising disc
stem cells further comprises a media comprising Hamm's culture
media. In another embodiment, the composition comprising disc stem
cells further comprises a media comprising Hamm's/F12 culture
media. In another embodiment, the composition comprising disc stem
cells further comprises a media comprising ESGRO Complete.TM.
Accutase.TM.. In another embodiment, ESGRO Complete.TM.
Accutase.TM. is a cell detachment solution of proteolytic and
collagenolytic enzymes qualified for use for the detachment of stem
cells cultured in serum-free conditions with ESGRO Complete.TM.
Clonal Grade Medium. In another embodiment, 1.times. Accutase.TM.
enzymes in Dulbecco's PBS comprises 0.5 mM EDTA.4Na and 3 mg/L
Phenol. In another embodiment, the composition comprising disc stem
cells further comprises a media comprising HEScGRO hES cell medium
(Chemicon Temecula, Calif.).
[0077] In another embodiment, the composition comprising disc stem
cells is used for plating disc stem cells in ultra low attachment
plates. In another embodiment, the composition comprising disc stem
cells is used for plating disc stem cells in ultra low attachment
plates precoated with an anti-adhesive substance. In another
embodiment, the anti-adhesive substance is poly 2-hydroxyethyl
methacrylate.
[0078] In another embodiment, the present invention provides an
isolated discosphere. In another embodiment, a disc stem cell of
the present invention gives rise to an isolated discosphere. In
another embodiment, a discosphere of the present invention is the
result of stem cell proliferation which gives rise to additional
stem cells and progenitor cells. In another embodiment, a
discosphere is formed as a result of disc stem proliferation.
[0079] In another embodiment, an isolated discosphere of the
present invention comprises nucleus pulposus stem cells and nucleus
pulposus progenitor cells arranged in a circular-spherical
structure. In another embodiment, an isolated discosphere is a ball
of cells in which a single disc stem cell gives rise to clones of
itself (symmetric division) and to progenitor cells. In another
embodiment, a discosphere of the present invention is a free
floating conglomerate of nucleus pulposus stem cells and nucleus
pulposus progenitor cells arranged in a circular-spherical
structure. In another embodiment, a discosphere culture of the
present invention comprises solitary free floating
discospheres.
[0080] In another embodiment, the methods of the present invention
provide that isolating a discosphere of the present invention can
be readily preformed by one skilled in the art under a light
microscope.
[0081] In another embodiment, the methods of the present invention
provide that a single cell suspension is prepared for isolating a
disc stem cell by plating and incubating a disc stem cell in a
serum free media. In another embodiment, the methods of the present
invention provide that a single cell suspension is prepared for
producing a discosphere by plating and incubating a disc stem cell
in a serum free media.
[0082] In another embodiment, the present invention provides a
method of producing a discosphere, comprising the step of growing a
culture of nucleus pulposus cells in a serum free media, thereby
producing a discosphere. In another embodiment, the present
invention provides that growing a primary culture of nucleus
pulposus cells in a serum free media results in selecting nucleus
pulposus stem cells. In another embodiment, the remaining isolated
culture of nucleus pulposus stem cells gives rise to discospheres
of the present invention. In another embodiment, the remaining
enriched culture of nucleus pulposus stem cells gives rise to
discospheres of the present invention. In another embodiment,
discospheres according to the methods of the present invention grow
in the compositions of the present invention. In another
embodiment, discospheres according to the methods of the present
invention grow in the supplemented media of the present invention.
In another embodiment, discospheres according to the methods of the
present invention grow under conditions which do not permit
cell-substrate adhesion. In another embodiment, conditions which do
not permit cell-substrate adhesion comprise for example the
addition of about 0.2-2% methylcellulose to the cell culture media
of the present invention.
[0083] In another embodiment, the present invention provides a
composition comprising a discosphere. In another embodiment, a
composition of the present invention comprises a single
discosphere. In another embodiment, a composition of the present
invention comprises at least 1.times.10.sup.2 discospheres. In
another embodiment, a composition of the present invention
comprises at least 1.times.10.sup.3 discospheres. In another
embodiment, a composition of the present invention comprises at
least 1.times.10.sup.4 discospheres. In another embodiment, a
composition of the present invention comprises at least
1.times.10.sup.5 discospheres. In another embodiment, a composition
of the present invention comprises at least 1.times.10.sup.6
discospheres.
[0084] In another embodiment, the composition of the present
invention comprises a discosphere and media. In another embodiment,
the media is a serum free media. In another embodiment, the
composition comprising a discosphere further comprises EGF
supplemented to the media. In another embodiment, the composition
comprising a discosphere further comprises 1-10 ng/ml EGF
supplemented to the media. In another embodiment, the composition
comprising a discosphere further comprises 1-100 ng/ml EGF
supplemented to the media. In another embodiment, the composition
comprising a discosphere further comprises 20-50 ng/ml EGF
supplemented to the media. In another embodiment, the composition
comprising a discosphere further comprises 50-100 ng/ml EGF
supplemented to the media. In another embodiment, the composition
comprising a discosphere further comprises 5-15 ng/ml EGF
supplemented to the media. In another embodiment, the composition
comprising a discosphere further comprises 8-12 ng/ml EGF
supplemented to the media.
[0085] In another embodiment, the composition comprising a
discosphere further comprises FGF supplemented to the media. In
another embodiment, the composition comprising a discosphere
further comprises FGF2 supplemented to the media. In another
embodiment, the composition comprising a discosphere further
comprises 1-100 ng/ml FGF2 supplemented to the media. In another
embodiment, the composition comprising a discosphere further
comprises 20-50 ng/ml FGF2 supplemented to the media. In another
embodiment, the composition comprising a discosphere further
comprises 50-100 ng/ml FGF2 supplemented to the media. In another
embodiment, the composition comprising a discosphere further
comprises 5-15 ng/ml FGF2 supplemented to the media. In another
embodiment, the composition comprising a discosphere further
comprises 8-12 ng/ml FGF2 supplemented to the media.
[0086] In another embodiment, the composition comprising a
discosphere further comprises insulin supplemented to the media
(Example 2 and materials and methods). In another embodiment, the
composition comprising a discosphere further comprises 1-100
.mu.g/ml insulin supplemented to the media. In another embodiment,
the composition comprising a discosphere further comprises 20-50
.mu.g/ml insulin supplemented to the media. In another embodiment,
the composition comprising a discosphere further comprises 50-100
.mu.g/ml insulin supplemented to the media. In another embodiment,
the composition comprising a discosphere further comprises 5-15
.mu.g/ml insulin supplemented to the media. In another embodiment,
the composition comprising a discosphere further comprises 3-7
.mu.g/ml insulin supplemented to the media.
[0087] In another embodiment, the composition comprising a
discosphere further comprises progesterone supplemented to the
media. In another embodiment, the composition comprising a
discosphere further comprises 1-200 ng/ml progesterone supplemented
to the media. In another embodiment, the composition comprising a
discosphere further comprises 20-200 ng/ml progesterone
supplemented to the media. In another embodiment, the composition
comprising a discosphere further comprises 50-150 ng/ml
progesterone supplemented to the media. In another embodiment, the
composition comprising disc stem cells further comprises 10-100
ng/ml progesterone supplemented to the media. In another
embodiment, the composition comprising disc stem cells further
comprises 20-80 ng/ml progesterone supplemented to the media. In
another embodiment, the composition comprising disc stem cells
further comprises 15-25 ng/ml progesterone supplemented to the
media.
[0088] In another embodiment, the composition comprising a
discosphere further comprises putrescine supplemented to the media.
In another embodiment, the composition comprising a discosphere
further comprises 1-800 ng/ml putrescine supplemented to the media.
In another embodiment, the composition comprising a discosphere
further comprises 1-100 ng/ml putrescine supplemented to the media.
In another embodiment, the composition comprising a discosphere
further comprises 100-300 ng/ml putrescine supplemented to the
media. In another embodiment, the composition comprising a
discosphere further comprises 300-500 ng/ml putrescine supplemented
to the media. In another embodiment, the composition comprising a
discosphere further comprises 500-800 ng/ml putrescine supplemented
to the media. In another embodiment, the composition comprising a
discosphere further comprises 150-250 ng/ml putrescine supplemented
to the media. In another embodiment, the composition comprising a
discosphere further comprises 140-160 ng/ml putrescine supplemented
to the media.
[0089] In another embodiment, the composition comprising a
discosphere further comprises transferrin supplemented to the
media. In another embodiment, the composition comprising a
discosphere further comprises 1-400 ng/ml transferrin supplemented
to the media. In another embodiment, the composition comprising a
discosphere further comprises 1-100 ng/ml transferrin supplemented
to the media. In another embodiment, the composition comprising a
discosphere further comprises 100-200 ng/ml transferrin
supplemented to the media. In another embodiment, the composition
comprising a discosphere further comprises 200-400 ng/ml
transferrin supplemented to the media. In another embodiment, the
composition comprising a discosphere further comprises 20-150 ng/ml
transferrin supplemented to the media. In another embodiment, the
composition comprising a discosphere further comprises 80-200 ng/ml
transferrin supplemented to the media. In another embodiment, the
composition comprising a discosphere further comprises 30-70 ng/ml
transferrin supplemented to the media.
[0090] In another embodiment, the composition comprising a
discosphere further comprises sodium selenite supplemented to the
media. In another embodiment, the composition comprising a
discosphere further comprises 1-400 ng/ml sodium selenite
supplemented to the media. In another embodiment, the composition
comprising a discosphere further comprises 1-100 ng/ml sodium
selenite supplemented to the media. In another embodiment, the
composition comprising a discosphere further comprises 100-200
ng/ml sodium selenite supplemented to the media. In another
embodiment, the composition comprising a discosphere further
comprises 200-400 ng/ml sodium selenite supplemented to the media.
In another embodiment, the composition comprising a discosphere
further comprises 20-150 ng/ml sodium selenite supplemented to the
media. In another embodiment, the composition comprising a
discosphere further comprises 40-180 ng/ml sodium selenite
supplemented to the media. In another embodiment, the composition
comprising a discosphere further comprises 20-40 ng/ml sodium
selenite supplemented to the media.
[0091] In another embodiment, the composition comprising a
discosphere further comprises methylcellulose supplemented to the
media. In another embodiment, the composition comprising a
discosphere further comprises 0.5-10% methylcellulose supplemented
to the media. In another embodiment, the composition comprising a
discosphere further comprises 0.5-3% methylcellulose supplemented
to the media. In another embodiment, the composition comprising a
discosphere further comprises 3-5% methylcellulose supplemented to
the media. In another embodiment, the composition comprising a
discosphere further comprises 5-8% methylcellulose supplemented to
the media. In another embodiment, the composition comprising a
discosphere further comprises 7-10% methylcellulose supplemented to
the media. In another embodiment, the composition comprising a
discosphere further comprises 0.5-2.5% methylcellulose supplemented
to the media. In another embodiment, the composition comprising a
discosphere further comprises 1-2.5% methylcellulose supplemented
to the media. In another embodiment, the composition comprising a
discosphere further comprises 0.6-1% methylcellulose supplemented
to the media.
[0092] In another embodiment, the composition comprising a
discosphere further comprises an antibiotic supplemented to the
media. In another embodiment, the antibiotic supplemented to the
media is penicillin-streptomycin. In another embodiment, the
composition comprising a discosphere further comprises 1000-10000
U/ml penicillin-streptomycin supplemented to the media. In another
embodiment, the composition comprising a discosphere further
comprises 1000-3000 U/ml penicillin-streptomycin supplemented to
the media. In another embodiment, the composition comprising a
discosphere further comprises 3000-6000 U/ml
penicillin-streptomycin supplemented to the media. In another
embodiment, the composition comprising a discosphere further
comprises 6000-10000 U/ml penicillin-streptomycin supplemented to
the media. In another embodiment, the composition comprising a
discosphere further comprises 3000-8000 U/ml
penicillin-streptomycin supplemented to the media. In another
embodiment, the composition comprising a discosphere further
comprises 4000-6000 U/ml penicillin-streptomycin supplemented to
the media. In another embodiment, the composition comprising a
discosphere further comprises 5000 U/ml penicillin-streptomycin
supplemented to the media.
[0093] In another embodiment, the composition comprising a
discosphere further comprises KO serum replacer supplemented to the
media. In another embodiment, the composition comprising a
discosphere further comprises 0.5-30% KO serum replacer
supplemented to the media. In another embodiment, the composition
comprising a discosphere further comprises 5-30% KO serum replacer
supplemented to the media. In another embodiment, the composition
comprising a discosphere further comprises 3-5% KO serum replacer
supplemented to the media. In another embodiment, the composition
comprising a discosphere further comprises 5-15% KO serum replacer
supplemented to the media. In another embodiment, the composition
comprising a discosphere further comprises 15-30% KO serum replacer
supplemented to the media. In another embodiment, the composition
comprising a discosphere further comprises 10-20% KO serum replacer
supplemented to the media. In another embodiment, the composition
comprising a discosphere further comprises 15-25% KO serum replacer
supplemented to the media. In another embodiment, the composition
comprising a discosphere further comprises 20% KO serum replacer
supplemented to the media.
[0094] In another embodiment, the composition comprising a
discosphere further comprises non-essential Amino Acids
supplemented to the media. In another embodiment, the composition
comprising a discosphere further comprises 0.1-10% non-essential
Amino Acids supplemented to the media. In another embodiment, the
composition comprising a discosphere further comprises 0.1-1%
non-essential Amino Acids supplemented to the media. In another
embodiment, the composition comprising a discosphere further
comprises 1-5% non-essential Amino Acids supplemented to the media.
In another embodiment, the composition comprising a discosphere
further comprises 5-10% non-essential Amino Acids supplemented to
the media. In another embodiment, the composition comprising a
discosphere further comprises 15-30% non-essential Amino Acids
supplemented to the media. In another embodiment, the composition
comprising a discosphere further comprises 0.5-1% non-essential
Amino Acids supplemented to the media. In another embodiment, the
composition comprising a discosphere further comprises 0.8-1.2%
non-essential Amino Acids supplemented to the media. In another
embodiment, the composition comprising a discosphere further
comprises 1% non-essential Amino Acids supplemented to the
media.
[0095] In another embodiment, the composition comprising a
discosphere further comprises L-glutamine supplemented to the
media. In another embodiment, the composition comprising a
discosphere further comprises 0.1-10 mM L-glutamine supplemented to
the media. In another embodiment, the composition comprising a
discosphere further comprises 0.1-5 mM L-glutamine supplemented to
the media. In another embodiment, the composition comprising a
discosphere further comprises 5-10 mM L-glutamine supplemented to
the media. In another embodiment, the composition comprising a
discosphere further comprises 5-8 mM L-glutamine supplemented to
the media. In another embodiment, the composition comprising a
discosphere further comprises 0.5-2.5 mM L-glutamine supplemented
to the media. In another embodiment, the composition comprising a
discosphere further comprises 1.5-3 mM L-glutamine supplemented to
the media. In another embodiment, the composition comprising a
discosphere further comprises 0.5-1.5 mM L-glutamine supplemented
to the media. In another embodiment, the composition comprising a
discosphere further comprises 0.8-1.2 mM L-glutamine supplemented
to the media.
[0096] In another embodiment, the composition comprising a
discosphere further comprises b-mercaptoethanol supplemented to the
media. In another embodiment, the composition comprising a
discosphere further comprises 0.01-1 mM b-mercaptoethanol
supplemented to the media. In another embodiment, the composition
comprising a discosphere further comprises 0.01-0.5 mM
b-mercaptoethanol supplemented to the media. In another embodiment,
the composition comprising a discosphere further comprises 0.5-1 mM
b-mercaptoethanol supplemented to the media. In another embodiment,
the composition comprising a discosphere further comprises 0.5-0.8
mM b-mercaptoethanol supplemented to the media. In another
embodiment, the composition comprising a discosphere further
comprises 00.5-0.25 mM b-mercaptoethanol supplemented to the media.
In another embodiment, the composition comprising a discosphere
further comprises 0.15-0.3 mM b-mercaptoethanol supplemented to the
media. In another embodiment, the composition comprising a
discosphere further comprises 0.05-0.15 mM b-mercaptoethanol
supplemented to the media. In another embodiment, the composition
comprising a discosphere further comprises 0.08-0.12 mM
b-mercaptoethanol supplemented to the media.
[0097] In another embodiment, the composition comprising a
discosphere comprises a media comprising Dulbecco's Modified
Eagle's Medium (DMEM). In another embodiment, the composition
comprising a discosphere comprises a media further comprising
DMEM/F12. In another embodiment, the composition comprising a
discosphere comprises a media further comprising ESGRO Complete.TM.
Accutase.TM.. In another embodiment, ESGRO Complete.TM.
Accutase.TM. is a cell detachment solution of proteolytic and
collagenolytic enzymes qualified for use for the detachment of stem
cells cultured in serum-free conditions with ESGRO Complete.TM.
Clonal Grade Medium. In another embodiment, 1.times. Accutase.TM.
enzymes in Dulbecco's PBS comprises 0.5 mM EDTA.4Na and 3 mg/L
Phenol. In another embodiment, the composition comprising a
discosphere comprises a media comprising HEScGRO hES cell medium
(Chemicon Temecula, Calif.).
[0098] In another embodiment, the present invention provides that
discospheres obtained by the methods of the present invention are
further expanded. In another embodiment, the present invention
provides that discospheres are dissociated by incubation at
37.degree. C. in DMEM/F12 medium supplemented with collagenase. In
another embodiment, the present invention provides that the
dissociated cells are expanded by replating the same into
methylcellulose-based medium.
[0099] In another embodiment, the methods of the present invention
provide that the medium is supplemented with 8-20% fetal bovine
serum (FBS) when growing a disc tissue. In another embodiment, the
methods of the present invention provide that the medium is
supplemented with 8-20% fetal bovine serum (FBS) when growing a
disc tissue in a scaffold. In another embodiment, the medium
comprises 30-70% media derived from cultures of primary human
foreskin fibroblasts, or a combination thereof. In another
embodiment, the methods of the present invention provide that the
medium is free of serum when growing disc stem cells, disc
progenitor cells, or a combination thereof. In another embodiment,
the methods of the present invention provide that the medium is
free of a serum replacer when growing disc stem cells, disc
progenitor cells, or a combination thereof. In another embodiment,
the methods of the present invention provide that the medium is
free of FBS when growing disc stem cells. In another embodiment,
the methods of the present invention provide that the medium is
free of FBS when growing nucleus pulposus stem cells. In another
embodiment, the methods of the present invention provide that the
medium is free of FBS when growing progenitor cells. In another
embodiment, the methods of the present invention provide that the
medium is free of FBS when growing nucleus pulposus progenitor
cells.
[0100] In another embodiment, the methods of the present invention
provide that the medium is free of serum or a serum replacer when
isolating disc stem cells or disc progenitor cells. In another
embodiment, the methods of the present invention provide that the
medium is free of serum or a serum replacer when enriching a
heterogeneous population of cells for disc stem cells, disc
progenitor cells, or a combination thereof. In another embodiment,
the methods of the present invention provide that the medium is
free of serum or a serum replacer when growing discospheres
comprising disc stem cells, disc progenitor cells, or a combination
thereof. In another embodiment, the methods of the present
invention provide that the medium is free of serum or a serum
replacer when expanding a culture comprising disc stem cells, disc
progenitor cells, or a combination thereof. In another embodiment,
the methods of the present invention provide that the medium is
free of serum or a serum replacer when expanding a culture enriched
for disc stem cells, disc progenitor cells, or a combination
thereof.
[0101] In another embodiment, the present invention provides a disc
replacement device comprising nucleus pulposus cells. In another
embodiment, the present invention provides an artificial disc
comprising nucleus pulposus cells. In another embodiment, the disc
replacement device is an intervertebral disc replacement device. In
another embodiment, an intervertebral disc is located between the
concave articular surfaces of the adjacent vertebral body
endplates. In another embodiment, the disc replacement device of
the present invention permits movements such as flexion, extension,
lateral flexion, and rotation. In another embodiment, the disc
replacement device of the present invention is used to repair
and/or replace injured or damaged intervertebral discs. In another
embodiment, the disc replacement device of the present invention
provides a prosthetic disc that combines both stability to support
the high loads, of the patient's vertebrae and flexibility to
provide the patient with sufficient mobility and proper spinal
column load distribution.
[0102] In another embodiment, the disc replacement device comprises
a disc scaffold. In another embodiment, the scaffold comprises a
shape memory alloy. In another embodiment, a shape memory alloy may
be deformed during its martensitic phase, but will regain its
original shape when it is heated above a certain temperature, such
as an austenite phase temperature. In another embodiment, a shape
memory alloy of the present invention exhibits a superelastic
property, thereby able to absorb large deformations without
damaging its structure.
[0103] In another embodiment, the disc replacement device comprises
a rigid body that fits between the vertebrates with a protuberance
extending from a vertebral contacting surface and extends into the
vertebral body. In another embodiment, the disc replacement device
comprises a disc arthroplasty device for replacement of the spinal
disk. In another embodiment, the disc replacement device comprises
a ball-and-socket to enable rotation. In another embodiment, the
disc replacement device comprises an intermediate layer allowing
for movement between the upper joint piece and the lower joint
piece.
[0104] In another embodiment, the disc replacement device comprises
two endplates that are anchored to the top and bottom surfaces of
the spinal bones. In another embodiment, the disc replacement
device comprises two metal endplates that are anchored to the top
and bottom surfaces of the spinal bones. In another embodiment, the
metal is cobalt-chrome alloy. In another embodiment, the endplates
are coated with nucleus pulposus cell adhesion molecules. In
another embodiment, the endplates are coated with molecules
promoting nucleus pulposus cell growth.
[0105] In another embodiment, the disc replacement device comprises
ceramics. In another embodiment, the disc replacement device
comprises injectable fluids. In another embodiment, the disc
replacement device comprises hydrogels. In another embodiment, the
disc replacement device comprises a hydrogel core in a flexible,
inelastic, woven polyethylene jacket. In another embodiment, the
disc replacement device comprises a polyvinyl alcohol material. In
another embodiment, the disc replacement device comprises
inflatables. In another embodiment, the disc replacement device
comprises elastic coils. In another embodiment, the disc
replacement device comprises an elongated elastic memory-coiling
spiral. In another embodiment, the elongated elastic memory-coiling
spiral is made of polycarbonate urethane. In another embodiment,
the disc replacement device comprises a one-piece convex surfaced
ceramic or metal implant that anchors to the inferior vertebral
body as a hemiarthroplasty. In another embodiment, the disc
replacement device comprises a balloon-like implant made of
polyurethane. In another embodiment, the disc replacement device
comprises a protein hydrogel. In another embodiment, the disc
replacement device comprises a thermopolymer.
[0106] In another embodiment, the disc scaffold comprises an ECM
component. In another embodiment, the ECM component is a structural
protein. In another embodiment, the disc scaffold comprises
collagen. In another embodiment, the structural protein is elastin.
In some embodiments, the ECM component is a specialized protein. In
another embodiment, the specialized protein is fibrillin. In
another embodiment, the specialized protein is fibronectin. In
another embodiment, the specialized protein is laminin. In some
embodiments, the ECM component is a proteoglycan. In one
embodiment, proteoglycans are composed of a protein core to which
is attached long chains of repeating disaccharide units termed of
glycosaminoglycans (GAGs) forming extremely complex high molecular
weight component.
[0107] In another embodiment, collagen is collagen type I. In
another embodiment, collagen type I comprises [a1(I)]2[a(I)]
chains. In another embodiment, collagen type I is derived from
skin, tendon, or bone.
[0108] In another embodiment, collagen is collagen type II. In
another embodiment, collagen type II comprises [a1(II)]3 chains. In
another embodiment, collagen type II is derived from cartilage or
vitreous humor. In another embodiment, type II collagen fibrils are
cross-linked to proteoglycans in the matrix by type IX
collagen.
[0109] In another embodiment, collagen is collagen type III. In
another embodiment, collagen type III comprises [a1(III)]3 chains.
In another embodiment, collagen type III is derived from skin or
muscle, and is frequently found with type I.
[0110] In another embodiment, collagen is collagen type IV. In
another embodiment, collagen type IV comprises [a1(IV)2[a2(IV)]
chains. In another embodiment, collagen type IV is derived from
basal lamina.
[0111] In another embodiment, collagen is collagen type V. In
another embodiment, collagen type V comprises [a1(V)][a2(V)][a3(V)]
chains. In another embodiment, collagen type V is derived from an
interstitial tissue associated with type I collagen.
[0112] In another embodiment, collagen is collagen type VI. In
another embodiment, collagen type VI comprises
[a1(VI)][a2(VI)][a3(VI)] chains. In another embodiment, collagen
type VI is derived from an interstitial tissue associated with type
I collagen. In another embodiment, type VI collagen consists of
relatively short triple-helical regions about 60 nm long separated
by globular domains about 40 nm long. In some embodiments, fibrils
of pure type VI collagen form a structure similar to beads on a
string.
[0113] In one embodiment, collagen is collagen type VII. In one
embodiment, collagen type VII comprises [a1(VII)]3 chains. In
another embodiment, collagen type VII is derived from
epithelia.
[0114] In another embodiment, collagen is collagen type VIII. In
another embodiment, collagen type VIII comprises [a1(VIII)]3
chains. In another embodiment, collagen type VII is derived from
endothelial cells.
[0115] In another embodiment, collagen is collagen type IX. In
another embodiment, collagen type IX comprises
[a1(IX)][a2(IX)][a3(IX)] chains. In another embodiment, collagen
type IX is derived from cartilage associated with type II
collagen.
[0116] In another embodiment, collagen is collagen type X. In
another embodiment, collagen type X comprises [a1(X)]3 chains. In
another embodiment, collagen type X is derived from hypertrophic
and mineralizing cartilage.
[0117] In another embodiment, collagen is collagen type XI. In
another embodiment, collagen type XI comprises
[a1(XI)][a2(XI)][a3(XI)] chains. In another embodiment, collagen
type XI is derived from cartilage.
[0118] In another embodiment, collagen is collagen type XII. In
another embodiment, collagen type XII comprises a1(XII) chains. In
another embodiment, collagen type XII is derived from sites wherein
types I and III collagens are present.
[0119] In another embodiment, type I collagen molecules pack
together side-by-side, forming fibrils with a diameter of 50-200
nm. In some embodiments, fibrils, adjacent collagen molecules are
displaced from one another by 67 nm, about another -quarter of
their length. In some embodiments, collagens types I, II, III, and
V form rodlike triple helices to via side-by-side interactions.
[0120] In another embodiment, the collagen of the present invention
is derived from cows. In another embodiment, collagen of the
present invention is derived from patient's own fat or hyaluronic
acid.
[0121] In another embodiment, collagen is a collagen-like substance
which has been modified by dissolving collagen in water and
modifying the thusly dissolved collagen to render its surface
charge effectively more positive than prior to modification. In
another embodiment, this material is well known and is disclosed,
e.g., in U.S. Pat. No. 4,238,480. In another embodiment, modified
collagen is freeze-dried to form a solid mass of gelatin. In some
embodiments, the mass of gelatin may be formed in the shape of a
rod, strip, film or flake.
[0122] In another embodiment, other forms of collagen which are
suitable for use in the present invention include Semed F, a
collagen preparation manufactured in native fiber form without any
chemical or enzymatic modifications, and Semed S, a lyophilized
collagen powder extracted from fresh bovine hides. In another
embodiment, the Semed F material is a Type I collagen (greater than
95%), while the Semed S is a mixture of Type I and Type III
collagen macro-molecules in which the shape and dimension of
tropocollagen in its natural helical orientation is retained.
[0123] In another embodiment, the concentration of the collagen in
the liquid which is to be freeze-dried can range from 0.5-10% and
preferably 1-5%, with the lower concentrations forming less dense
or discontinuous solids. In another embodiment, at lower
concentrations of 0.5 to 1%, the Semed F forms a structure which
approximates dense cobwebs.
[0124] In another embodiment, native collagen film, wherein the
film strength is preserved and the triple-helix structure of the
collagen polymer is maintained intact, can also be used, either
alone or with a plasticizer incorporated therewith.
[0125] In another embodiment, gelatin or other water soluble forms
of collagen are utilized. In another embodiment, soluble forms of
collagen will readily polymerize at body temperatures to form a
stable subcutaneous gel. In another embodiment, when soluble forms
of collagen are implanted into the body, the polymerized material
will become rapidly populated by nucleus pulposus cells implanted
therein and host fibroblasts. In some embodiments, the material
becomes vascularized and can remain histologically stable. In
another embodiment, the material becomes vascularized and can
remain histologically stable for at least 4 months. In another
embodiment, the material becomes vascularized and can remain
histologically stable for at least 6 months. In another embodiment,
the material becomes vascularized and can remain histologically
stable for at least 8 months. In another embodiment, the material
becomes vascularized and can remain histologically stable for at
least 10 months. In another embodiment, the material becomes
vascularized and can remain histologically stable for at least 12
months. In another embodiment, the material becomes vascularized
and can remain histologically stable for at least 15 months. In
another embodiment, the material becomes vascularized and can
remain histologically stable for at least 18 months.
[0126] In another embodiment, the present invention provides
mixtures of the various types of collagen of the invention to
obtain the most desirable features of each grade.
[0127] In another embodiment, fibronectins are dimers of 2 similar
peptides. In another embodiment, each chain of a fibronectins is
60-70 nm long and 2-3 nm thick. In another embodiment, fibronectins
contain at least 6 tightly folded domains each with a high affinity
for a different substrate such as heparan sulfate, collagen
(separate domains for types I, II and III collagens), and fibrin
and cell-surface receptors.
[0128] In another embodiment, laminin molecule is a heterotrimer
assembled from .alpha., .beta., and .gamma.-chains. In some
embodiments, laminins form independent networks and are associated
with type IV collagen networks via entactin, and perlecan. In some
embodiments, laminins contribute to cell viability, attachment, and
differentiation, cell shape and movement, maintenance of tissue
phenotype, and promotion of tissue survival.
[0129] In another embodiment, proteoglycans comprise chondroitin
sulfate and dermatan sulfate chains. In another embodiment,
proteoglycans comprise heparin and heparan sulfate chains. In
another embodiment, proteoglycans comprise keratan sulfate chains.
In another embodiment, proteoglycans are aggrecans, the major
proteoglycan in cartilage. In another embodiment, proteoglycans are
versican, present in many adult tissues including blood vessels and
skin. In another embodiment, proteoglycans are small leucine rich
repeat proteoglycans (SLRPs). In another embodiment, SLRPs include
decorin, biglycan, fibromodulin, and lumican.
[0130] In another embodiment, the extracellular matrix components
are morselized. In another embodiment, morselization of the
extracellular matrix proteins increases the surface area for
nucleus pulposus cells attachment. In another embodiment,
morselization of the extracellular matrix proteins increases the
surface area for discospheres attachment. In another embodiment,
morselization of the extracellular matrix proteins increases the
surface area for disc stem cells attachment. In another embodiment,
morselization of the extracellular matrix proteins increases the
surface area for disc progenitor cells attachment. In another
embodiment, morselization of the extracellular matrix proteins
increases the surface area thus aiding diffusion of nutrients and
waste products to the implant and from the implant. In another
embodiment, morselization of the extracellular matrix proteins
allows the introduction of nucleus pulposus cells into the disc
scaffold through a needle or a small cannula. In another
embodiment, morselization of the extracellular matrix proteins
allows the introduction of discospheres into the disc scaffold
through a needle or a small cannula. In another embodiment, small
holes could be drilled into the disc scaffold for cell
attachment.
[0131] In another embodiment, the present invention provides that
the disc scaffold is obtained from an animal or human. In another
embodiment, the present invention provides that the disc scaffold
is an intervertebral disc with the vertebral endplates left intact.
In another embodiment, the present invention provides that the disc
scaffold is a rabbit intervertebral disc with the vertebral
endplates left intact (Example 3). In another embodiment, the
present invention provides that the disc scaffold is a dog
intervertebral disc with the vertebral endplates left intact. In
another embodiment, the present invention provides that the disc
scaffold is a horse intervertebral disc with the vertebral
endplates left intact. In another embodiment, the present invention
provides that the disc scaffold is a monkey intervertebral disc
with the vertebral endplates left intact. In another embodiment,
the present invention provides that the disc scaffold is a pig
intervertebral disc with the vertebral endplates left intact. In
another embodiment, the present invention provides that the disc
scaffold is a cow intervertebral disc with the vertebral endplates
left intact. In another embodiment, the present invention provides
that the disc scaffold comprising collagen further comprises
additional material such as ceramics or metals.
[0132] In another embodiment, the present invention provides that
the disc replacement device comprises nucleus pulposus cells. In
another embodiment, the present invention provides that the disc
replacement device comprises human nucleus pulposus cells. In
another embodiment, the present invention provides that the disc
replacement device comprises nucleus pulposus stem cells. In
another embodiment, the present invention provides that the disc
replacement device comprises nucleus pulposus progenitor cells. In
another embodiment, the present invention provides that the disc
replacement device comprises discospheres of the present
invention.
[0133] In another embodiment, the disc replacement device further
comprises media. In another embodiment, the media comprises cell
culture media of the present invention (Example 3).
[0134] In another embodiment, the present invention provides a
method of producing an artificial disc, comprising the step of
growing discospheres in a disc scaffold. In another embodiment, the
present invention provides a method of producing an intervertebral
disc replacement device, comprising the step of growing
discospheres in a disc scaffold. In another embodiment,
discospheres are administered onto a disc scaffold. In another
embodiment, discospheres are administered into a layer comprising
collagen in the disc scaffold. In another embodiment, discospheres
are administered onto a layer comprising collagen in the disc
scaffold. In another embodiment, discospheres are injected into a
disc scaffold (Example 4). In another embodiment, discospheres are
injected onto a disc scaffold. In another embodiment, discospheres
are injected into a layer comprising collagen in the disc scaffold.
In another embodiment, discospheres are injected onto a layer
comprising collagen in the disc scaffold. In another embodiment,
the discospheres of the present invention are applied or injected
into or onto the disc scaffold together with a composition of the
present invention. In another embodiment, the discospheres of the
present invention are applied or injected into or onto the disc
scaffold together with a DMEM/F12 medium supplemented with 10%
FCS.
[0135] In another embodiment, the present invention provides a
method of producing an intervertebral disc replacement device,
comprising the step of growing nucleus pulposus cells in a disc
scaffold. In another embodiment, the present invention provides a
method of producing a spinal disc tissue, comprising the step of
growing discospheres in a disc scaffold, thereby producing a spinal
disc tissue. In another embodiment, a spinal disc tissue of the
present invention comprises a disc scaffold of the present
invention. In another embodiment, a spinal disc tissue of the
present invention comprises nucleus pulposus cells of the present
invention. In another embodiment, a spinal disc tissue of the
present invention comprises a disc scaffold of the present
invention. In another embodiment, a spinal disc tissue of the
present invention comprises nucleus pulposus cells of the present
invention grown on a disc scaffold of the present invention. In
another embodiment, a spinal disc tissue of the present invention
comprises a disc scaffold of the present invention. In another
embodiment, a spinal disc tissue of the present invention comprises
matured nucleus pulposus cells derived from discospheres of the
present invention attached to a disc scaffold of the present
invention. In another embodiment, a spinal disc tissue of the
present invention comprises matured nucleus pulposus cells derived
from disc stem cells of the present invention attached to a disc
scaffold of the present invention. In another embodiment, a spinal
disc tissue of the present invention comprises fibroblasts and
matured nucleus pulposus cells derived from discospheres of the
present invention attached to a disc scaffold of the present
invention.
[0136] In another embodiment, the present invention provides a
method of producing an intervertebral disc replacement device,
comprising coating the disc scaffold of the present invention with
nucleus pulposus cells growth factors. In another embodiment, the
present invention provides a method of producing an intervertebral
disc replacement device, comprising coating the disc scaffold of
the present invention with nucleus pulposus cells adhesion factors.
In another embodiment, the present invention provides a method of
producing an intervertebral disc replacement device, comprising
coating the disc scaffold of the present invention with nucleus
pulposus cells differentiation factors. In another embodiment, the
present invention provides a method of producing an intervertebral
disc replacement device, comprising placing the disc scaffold of
the present invention in a media comprising nucleus pulposus cells
growth factors, adhesion factors, and differentiation factors. In
another embodiment, the present invention provides a method of
producing an intervertebral disc replacement device, comprising
placing the disc scaffold of the present invention in a cell
culture media comprising nucleus pulposus cells growth factors,
adhesion factors, and differentiation factors. In another
embodiment, the present invention provides a method of producing an
intervertebral disc replacement device, comprising placing the disc
scaffold of the present invention in a media comprising DMEM/F12
medium. In another embodiment, the present invention provides a
method of producing an intervertebral disc replacement device,
comprising placing the disc scaffold of the present invention in a
media comprising DMEM/F12 medium and 10% fetal calf serum (FCS)
(Example 3).
[0137] In another embodiment, the present invention provides a
method of producing an intervertebral disc replacement device,
comprising incubating the disc scaffold of the present invention in
a media of the invention at 35-42.degree. C. In another embodiment,
the present invention provides a method of producing an
intervertebral disc replacement device, comprising incubating the
disc scaffold of the present invention in a media of the invention
at 36-38.degree. C. In another embodiment, the present invention
provides a method of producing an intervertebral disc replacement
device, comprising incubating the disc scaffold of the present
invention in a media of the invention at 37.degree. C.
[0138] In another embodiment, the present invention provides a
method of producing an intervertebral disc replacement device,
comprising incubating the disc scaffold of the present invention in
a media of the invention while maintaining 4-10% CO.sub.2. In
another embodiment, the present invention provides a method of
producing an intervertebral disc replacement device, comprising
incubating the disc scaffold of the present invention in a media of
the invention while maintaining 4-8% CO.sub.2. In another
embodiment, the present invention provides a method of producing an
intervertebral disc replacement device, comprising incubating the
disc scaffold of the present invention in a media of the invention
while maintaining 5% CO.sub.2.
[0139] In another embodiment, the present invention provides a
method of producing an intervertebral disc replacement device,
comprising incubating the disc scaffold of the present invention in
a media of the invention for 2-12 hours in an incubator. In another
embodiment, the present invention provides a method of producing an
intervertebral disc replacement device, comprising incubating the
disc scaffold of the present invention in a media of the invention
for 3-10 hours in an incubator. In another embodiment, the present
invention provides a method of producing an intervertebral disc
replacement device, comprising incubating the disc scaffold of the
present invention in a media of the invention for 6-10 hours in an
incubator. In another embodiment, the present invention provides a
method of producing an intervertebral disc replacement device,
comprising incubating the disc scaffold of the present invention in
a media of the invention for 8 hours in an incubator.
[0140] In another embodiment, the present invention provides that
incubating the disc scaffold of the present invention in a media of
the invention in an incubator comprises a step for preparing the
disc scaffold before nucleus pulposus cells are applied into or
onto the disc scaffold. In another embodiment, the present
invention provides that incubating the disc scaffold of the present
invention in a media of the invention in an incubator enable
nucleus pulposus cells of the invention to adhere, grow and
differentiate on the disc scaffold. In another embodiment, the
present invention provides that incubating the disc scaffold of the
present invention in a media of the invention in an incubator
enable discospheres of the invention to adhere, grow and
differentiate on the disc scaffold. In another embodiment, the
present invention provides that incubating the disc progenitor
cells of the present invention in a media of the invention in an
incubator enable nucleus pulposus cells of the invention to adhere,
grow and differentiate on the disc scaffold. In another embodiment,
the present invention provides that incubating the disc scaffold of
the present invention in a media of the invention in an incubator
enable disc stem cells of the invention to adhere, grow and
differentiate on the disc scaffold. In another embodiment, the
present invention provides that incubating the disc scaffold of the
present invention in a media of the invention in an incubator
enable nucleus pulposus cells, disc stem cells, disc progenitor
cells, discospheres, or any combination thereof to adhere, grow and
differentiate on the disc scaffold.
[0141] In another embodiment, the present invention provides that
autograft nucleus pulposus cells are harvested, cultured, and
injected to the center of a disc scaffold (Example 4). In another
embodiment, the present invention provides that alloograft nucleus
pulposus cells are harvested, cultured, and injected to the center
of a disc scaffold. In another embodiment, the present invention
provides that xenograft nucleus pulposus cells are harvested,
cultured, and injected to the center of a disc scaffold.
[0142] In another embodiment, the present invention provides that
nucleus pulposus stem cells, nucleus pulposus progenitor cells,
discospheres, or a combination thereof are implanted into the disc
scaffold to form a living nucleus pulposus. In another embodiment,
nucleus pulposus stem cells, nucleus pulposus progenitor cells,
discospheres, or a combination thereof obtained from a cell culture
are implanted into the disc scaffold to form a living nucleus
pulposus.
[0143] In another embodiment, disc stem cells are administered onto
a disc scaffold. In another embodiment, disc stem cells are
administered into a layer comprising collagen in the disc scaffold.
In another embodiment, disc stem cells are administered onto a
layer comprising collagen in the disc scaffold. In another
embodiment, disc stem cells are injected into a disc scaffold. In
another embodiment, disc stem cells are injected onto a disc
scaffold. In another embodiment, disc stem cells are injected into
a layer comprising collagen in the disc scaffold. In another
embodiment, disc stem cells are injected onto a layer comprising
collagen in the disc scaffold. In another embodiment, the disc stem
cells of the present invention are applied or injected into or onto
the disc scaffold together with a composition of the present
invention. In another embodiment, the disc stem cells of the
present invention are applied or injected into or onto the disc
scaffold together with a DMEM/F12 medium with 10% FCS.
[0144] In another embodiment, the present invention provides a
method of producing an intervertebral disc replacement device,
comprising the step of growing nucleus pulposus primary cells in a
disc scaffold. In another embodiment, disc primary cells are
administered onto a disc scaffold. In another embodiment, disc
primary cells are administered into a layer comprising collagen in
the disc scaffold. In another embodiment, disc primary cells are
administered onto a layer comprising collagen in the disc scaffold.
In another embodiment, disc primary cells are injected into a disc
scaffold. In another embodiment, disc primary cells are injected
onto a disc scaffold. In another embodiment, disc primary cells are
injected into a layer comprising collagen in the disc scaffold. In
another embodiment, disc primary cells are injected onto a layer
comprising collagen in the disc scaffold. In another embodiment,
the disc primary cells of the present invention are applied or
injected into or onto the disc scaffold together with a composition
of the present invention. In another embodiment, the disc primary
cells of the present invention are applied or injected into or onto
the disc scaffold together with a DMEM/F12 medium with 10% FCS.
[0145] In another embodiment, the present invention provides a
method of producing an intervertebral disc replacement device,
comprising the step of collecting the discospheres, disc stem
cells, disc progenitor cells, or a mixture thereof from cell
culture media of the present invention by methods known to a person
with skill in the art and placing the cells in DMEM/F12. In another
embodiment, the present invention provides that discospheres, disc
stem cells, disc progenitor cells, or a mixture thereof are first
washed free of cell-substrate adhesion inhibitory factor. In
another embodiment, the present invention provides that
discospheres, disc stem cells, disc progenitor cells, or a mixture
thereof are first washed free of methylcellulose.
[0146] In another embodiment, the present invention provides that
washed discospheres, disc stem cells, disc progenitor cells, or a
mixture thereof substantially free of cell-substrate adhesion
inhibitory factors are placed in a cell culture media. In another
embodiment, the present invention provides that washed
discospheres, disc stem cells, disc progenitor cells, or a mixture
thereof substantially free of cell-substrate adhesion inhibitory
factors are placed in DMEM. In another embodiment, the present
invention provides that washed discospheres, disc stem cells, disc
progenitor cells, or a mixture thereof substantially free of
cell-substrate adhesion inhibitory factors are placed in DMEM/F12.
In another embodiment, the present invention provides that washed
discospheres, disc stem cells, disc progenitor cells, or a mixture
thereof substantially free of cell-substrate adhesion inhibitory
factors are placed in DMEM/F12 comprising serum.
[0147] In another embodiment, discospheres, disc stem cells, disc
progenitor cells, or a mixture thereof are introduced to the disc
scaffold in a cell culture media of the invention. In another
embodiment, discospheres, disc stem cells, disc progenitor cells,
or a mixture thereof are introduced to the disc scaffold in a cell
culture media comprising recombinant generated morphogenetic
proteins. In another embodiment, discospheres, disc stem cells,
disc progenitor cells, or a mixture thereof are introduced to the
disc scaffold in a cell culture media comprising PDGF. In another
embodiment, discospheres, disc stem cells, disc progenitor cells,
or a mixture thereof are introduced to the disc scaffold in a cell
culture media comprising TGF-.beta.. In another embodiment,
discospheres, disc stem cells, disc progenitor cells, or a mixture
thereof are introduced to the disc scaffold in a cell culture media
comprising EGF/TGF-.alpha.. In another embodiment, discospheres,
disc stem cells, disc progenitor cells, or a mixture thereof are
introduced to the disc scaffold in a cell culture media comprising
IGF-I. In another embodiment, discospheres, disc stem cells, disc
progenitor cells, or a mixture thereof are introduced to the disc
scaffold in a cell culture media comprising .beta.FGF. In another
embodiment, discospheres, disc stem cells, disc progenitor cells,
or a mixture thereof are introduced to the disc scaffold in a cell
culture media comprising hydrogels. In another embodiment,
discospheres, disc stem cells, disc progenitor cells, or a mixture
thereof are introduced to the disc scaffold in a cell culture media
comprising absorbable or non-resorbable synthetic or natural
polymers such as but not limited to collagen, fibrin, polyglycolic
acid, polylactic acid, or polytetrafluoroethylene. In another
embodiment, discospheres, disc stem cells, disc progenitor cells,
or a mixture thereof are introduced to the disc scaffold in a cell
culture media comprising antibiotics. In another embodiment,
discospheres, disc stem cells, disc progenitor cells, or a mixture
thereof are introduced to the disc scaffold in a cell culture media
comprising anti-inflammatory medication. In another embodiment,
discospheres, disc stem cells, disc progenitor cells, or a mixture
thereof are introduced to the disc scaffold in a cell culture media
comprising immunosuppressive medications.
[0148] In another embodiment, the present invention provides that
the collagen fibers of the annulus fibrosis are arranged in 5-50
layers or lamella. In another embodiment, the present invention
provides that the collagen fibers of the annulus fibrosis are
arranged in 10-40 layers or lamella. In another embodiment, the
present invention provides that the collagen fibers of the annulus
fibrosis are arranged in 20-30 layers or lamella.
[0149] In another embodiment, the present invention provides that
the fibers of the lamella alternate direction between layers. In
another embodiment, the present invention provides that a blunt
tipped needle or cannula could be forced through the annulus. In
another embodiment, the present invention provides that upon
withdraw of the needle, after injecting the transplanted nucleus
pulposus cells or discospheres, the separated fibers of the lamella
would return to their normal position, sealing the annulus. In
another embodiment, the present invention provides that the needle
would be inserted into the anterior or lateral portion of the disc
scaffold. In another embodiment, the present invention provides
that those skilled in the art will realize that the needle could be
directed into the lateral portion of the disc percutaneously with
fluoroscopic guidance and into the anterior portion of the disc
laparoscopically.
[0150] In another embodiment, the present invention provides that
the recipient of the nucleus pulposus cells of the present
invention is the donor. In another embodiment, the present
invention provides that the recipient of the nucleus pulposus cells
of the present invention may function at least in part as a donor.
In another embodiment, the present invention provides that the
donor of nucleus pulposus cells of the present invention is a
single donor. In another embodiment, the present invention provides
that multiple donors provide nucleus pulposus cells of the present
invention to a single recipient. In another embodiment, the present
invention provides that multiple donors provide nucleus pulposus
cells of the present invention to multiple recipients. In another
embodiment, the present invention provides that fetal sources are
used. In another embodiment, the present invention provides that
the donor or donors of the nucleus pulposus cells of the present
invention is or are preferably having a familial relationship to
the recipient in order to minimize or avoid immunosuppression. In
another embodiment, the present invention provides that the donor
or donors of the nucleus pulposus cells of the present invention is
or are preferably having a familial relationship to the recipient
in order to minimize or avoid the need for immunosuppressive
substances. In another embodiment, the present invention provides
guidelines for tissue procurement including surgical techniques of
removal, number of hours between death of the donor and tissue
procurement, and testing of the donor for infectious disease, are
well known to one of skill in the art.
[0151] In another embodiment, the present invention provides that
nucleus pulposus cells injected into or onto the disc scaffold
deposit extracellular matrix components. In another embodiment, the
present invention provides that discospheres injected into or onto
the disc scaffold deposit extracellular matrix components of the
disc. In another embodiment, the present invention provides that
these extracellular matrix components shape the discs' subsequent
physiological functions. In another embodiment, the present
invention provides that these extracellular matrix components shape
the discs' subsequent biomechanical functions. In another
embodiment, the present invention provides that by the 2.sup.nd
week of incubation, the disc tissue demonstrates resistance to
pressure force. In another embodiment, the present invention
provides that by the 3.sup.rd week of incubation, the disc tissue
demonstrates resistance to pressure force. In another embodiment,
the present invention provides that resistance to pressure force
indicates that the disc is matured. In another embodiment, the
present invention provides that resistance to pressure force
indicates that the disc acquired tensile properties. In another
embodiment, the present invention provides that by the 8.sup.th
week, the disc tissue demonstrates maximal thickness and resistance
to compressive forces. In another embodiment, the present invention
provides that by the 9.sup.th week, the disc tissue demonstrates
maximal thickness and resistance to compressive forces. In another
embodiment, the present invention provides that by the 10.sup.th
week, the disc tissue demonstrates maximal thickness and resistance
to compressive forces.
[0152] In another embodiment, the present invention provides a
method for total disc replacement. In another embodiment, the
present invention provides a method for partial disc replacement.
In another embodiment, the method for partial disc replacement
comprises replacement of the nucleus pulposus.
[0153] In another embodiment, the present invention provides that
the ruptured disc is removed in a minimally invasive manner through
a 10-25 mm paraspinal incision. In another embodiment, the present
invention provides that the ruptured disc is removed in a minimally
invasive manner through a 10-20 mm paraspinal incision. In another
embodiment, the present invention provides that the ruptured disc
is removed in a minimally invasive manner through a 15-18 mm
paraspinal incision. In another embodiment, the present invention
provides that the ruptured disc is removed in a minimally invasive
manner through a 16-20 mm paraspinal incision.
[0154] In another embodiment, the present invention provides that
pre-prepared scaffold is inserted into the disc space. In another
embodiment, the present invention provides that pre-prepared
scaffold comprising collagen is inserted into the disc space. In
another embodiment, the present invention provides that
pre-prepared scaffold is inserted into the disc space and expanded
to fill the space.
[0155] In another embodiment the recipient receives the disc
replacement device of the present invention. In another embodiment
the recipient receives nucleus pulposus cells of the present
invention. In another embodiment the recipient receives local
anesthesia. In another embodiment the recipient receives general
anesthesia. In another embodiment the precise anesthesia protocol
will be determined by one of skill in the art.
[0156] In another embodiment a damaged disc is removed from the
recipient by methods known to one of skill in the art. In another
embodiment, the disc replacement device of the present invention
replaces the damaged disc. In another embodiment, a pre-treated
disc scaffold of the present invention replaces the damaged disc.
In another embodiment, a pre-treated disc scaffold of the present
invention comprising collagen replaces the damaged disc. In another
embodiment, a pre-treated disc scaffold of the present invention
comprising various collagens of the invention replaces the damaged
disc. In another embodiment, a pre-treated disc scaffold of the
present invention comprising various ECM components replaces the
damaged disc.
[0157] In another embodiment, nucleus pulposus cells are
administered to a disc scaffold of the present invention after the
disc scaffold is surgically placed in the recipient. In another
embodiment, the term "nucleus pulposus cells" comprise disc stem
cells, disc progenitor cells, discospheres, or a combination
thereof. In another embodiment, nucleus pulposus cells are
administered via a blunt tipped needle. In another embodiment,
nucleus pulposus cells are administered via a cannula. In another
embodiment, nucleus pulposus cells are forced through the annulus.
In another embodiment, nucleus pulposus cells are administered via
a needle inserted into the anterior or lateral portion of the disc.
In another embodiment, one skilled in the art will realize the
needle could be directed into the lateral portion of the disc
percutaneously with fluoroscopic guidance and into the anterior
portion of the disc laparoscopic ally.
[0158] In another embodiment, nucleus pulposus cells of the present
invention are added to the patient's nucleus pulposus. In another
embodiment, the patient's disc is removed with standard techniques.
In another embodiment, the patient's disc nucleus could be removed
with standard enzymatic techniques. In another embodiment, the
patient's disc nucleus could be removed with chymopapain. In
another embodiment, the patient's disc nucleus could be removed
with the aid of a laser. In another embodiment, the patient's disc
nucleus could be removed with the aid of a suction device. In
another embodiment, the patient's disc nucleus could be removed
with the aid of a shaver. In another embodiment, the patient's disc
nucleus could be removed with the aid of a any other useful
surgical instrument. In another embodiment, if the nucleus is
removed the hole in the annulus must be small and closed at the end
of the procedure.
[0159] In another embodiment, additional therapeutic substances are
added to the transplanted nucleus. In another embodiment,
additional therapeutic substances are added to the transplanted
disc scaffold. In another embodiment, additional therapeutic
substances are added to the transplanted disc replacement device of
the present invention.
[0160] In another embodiment, additional resorbable culture medium
is added to the transplanted nucleus. In another embodiment,
additional tissue growth or factors are added to the transplanted
nucleus. In another embodiment, additional tissue differentiation
factors are added to the transplanted nucleus. In another
embodiment, additional recombinant generated morphogenetic proteins
are added to the transplanted nucleus. In another embodiment,
additional PDGF is added to the transplanted nucleus. In another
embodiment, additional TGF-.beta. is added to the transplanted
nucleus. In another embodiment, additional EGF/TGF-.alpha. are
added to the transplanted nucleus. In another embodiment,
additional IGF-I is added to the transplanted nucleus. In another
embodiment, additional FGF is added to the transplanted nucleus. In
another embodiment, additional hydrogels are added to the
transplanted nucleus. In another embodiment, additional
non-resorbable synthetic or natural polymers are added to the
transplanted nucleus. In another embodiment, additional collagen is
added to the transplanted nucleus. In another embodiment,
additional fibrin is added to the transplanted nucleus. In another
embodiment, additional polyglycolic acid is added to the
transplanted nucleus. In another embodiment, additional
polytetrafluoroethylene is added to the transplanted nucleus. In
another embodiment, additional antibiotics are added to the
transplanted nucleus. In another embodiment, additional
anti-inflammatory medications are added to the transplanted
nucleus. In another embodiment, additional immunosuppressive
medications are added to the transplanted nucleus.
[0161] In another embodiment, additional resorbable culture medium
is added to the transplanted disc scaffold. In another embodiment,
additional tissue growth or factors are added to the transplanted
disc scaffold. In another embodiment, additional tissue
differentiation factors are added to the transplanted disc
scaffold. In another embodiment, additional recombinant generated
morphogenetic proteins are added to the transplanted disc scaffold.
In another embodiment, additional PDGF is added to the transplanted
disc scaffold. In another embodiment, additional TGF-.beta. is
added to the transplanted disc scaffold. In another embodiment,
additional EGF/TGF-.alpha. are added to the transplanted disc
scaffold. In another embodiment, additional IGF-I is added to the
transplanted disc scaffold. In another embodiment, additional FGF
is added to the transplanted disc scaffold. In another embodiment,
additional hydrogels are added to the transplanted disc scaffold.
In another embodiment, additional non-resorbable synthetic or
natural polymers are added to the transplanted disc scaffold. In
another embodiment, additional collagen is added to the
transplanted disc scaffold. In another embodiment, additional
fibrin is added to the transplanted disc scaffold. In another
embodiment, additional polyglycolic acid is added to the
transplanted disc scaffold. In another embodiment, additional
polytetrafluoroethylene is added to the transplanted disc scaffold.
In another embodiment, additional antibiotics are added to the
transplanted disc scaffold. In another embodiment, additional
anti-inflammatory medications are added to the transplanted disc
scaffold. In another embodiment, additional immunosuppressive
medications are added to the transplanted disc scaffold.
[0162] In another embodiment, additional resorbable culture medium
is added to the transplanted disc replacement device. In another
embodiment, additional tissue growth or factors are added to the
transplanted disc replacement device. In another embodiment,
additional tissue differentiation factors are added to the
transplanted disc replacement device. In another embodiment,
additional recombinant generated morphogenetic proteins are added
to the transplanted disc replacement device. In another embodiment,
additional PDGF is added to the transplanted disc replacement
device. In another embodiment, additional TGF-.beta. is added to
the transplanted disc replacement device. In another embodiment,
additional EGF/TGF-.alpha. are added to the transplanted disc
replacement device. In another embodiment, additional IGF-I is
added to the transplanted disc replacement device. In another
embodiment, additional FGF is added to the transplanted disc
replacement device. In another embodiment, additional hydrogels are
added to the transplanted disc replacement device. In another
embodiment, additional non-resorbable synthetic or natural polymers
are added to the transplanted disc replacement device. In another
embodiment, additional collagen is added to the transplanted disc
replacement device. In another embodiment, additional fibrin is
added to the transplanted disc replacement device. In another
embodiment, additional polyglycolic acid is added to the
transplanted disc replacement device. In another embodiment,
additional polytetrafluoroethylene is added to the transplanted
disc replacement device. In another embodiment, additional
antibiotics are added to the transplanted disc replacement device.
In another embodiment, additional anti-inflammatory medications are
added to the transplanted disc replacement device. In another
embodiment, additional immunosuppressive medications are added to
the transplanted disc replacement device.
[0163] In another embodiment, additional resorbable culture medium
is added to the transplanted discospheres, disc stem cells, disc
progenitor cells, or a mixture thereof of the present invention. In
another embodiment, additional tissue growth or factors are added
to the transplanted discospheres, disc stem cells, disc progenitor
cells, or a mixture thereof of the present invention. In another
embodiment, additional tissue differentiation factors are added to
the transplanted discospheres, disc stem cells, disc progenitor
cells, or a mixture thereof of the present invention. In another
embodiment, additional recombinant generated morphogenetic proteins
are added to the transplanted discospheres, disc stem cells, disc
progenitor cells, or a mixture thereof of the present invention. In
another embodiment, additional PDGF is added to the transplanted
discospheres, disc stem cells, disc progenitor cells, or a mixture
thereof of the present invention. In another embodiment, additional
TGF-.beta. is added to the transplanted discospheres, disc stem
cells, disc progenitor cells, or a mixture thereof of the present
invention. In another embodiment, additional EGF/TGF-.alpha. are
added to the transplanted discospheres, disc stem cells, disc
progenitor cells, or a mixture thereof of the present invention. In
another embodiment, additional IGF-I is added to the transplanted
discospheres, disc stem cells, disc progenitor cells, or a mixture
thereof of the present invention. In another embodiment, additional
FGF is added to the transplanted discospheres, disc stem cells,
disc progenitor cells, or a mixture thereof of the present
invention. In another embodiment, additional hydrogels are added to
the transplanted discospheres, disc stem cells, disc progenitor
cells, or a mixture thereof of the present invention. In another
embodiment, additional non-resorbable synthetic or natural polymers
are added to the transplanted discospheres, disc stem cells, disc
progenitor cells, or a mixture thereof of the present invention. In
another embodiment, additional collagen is added to the
transplanted discospheres, disc stem cells, disc progenitor cells,
or a mixture thereof of the present invention. In another
embodiment, additional fibrin is added to the transplanted
discospheres, disc stem cells, disc progenitor cells, or a mixture
thereof of the present invention. In another embodiment, additional
polyglycolic acid is added to the transplanted discospheres, disc
stem cells, disc progenitor cells, or a mixture thereof of the
present invention. In another embodiment, additional
polytetrafluoroethylene is added to the transplanted discospheres,
disc stem cells, disc progenitor cells, or a mixture thereof of the
present invention. In another embodiment, additional antibiotics
are added to the transplanted discospheres, disc stem cells, disc
progenitor cells, or a mixture thereof of the present invention. In
another embodiment, additional anti-inflammatory medications are
added to the transplanted discospheres, disc stem cells, disc
progenitor cells, or a mixture thereof of the present invention. In
another embodiment, additional immunosuppressive medications are
added to the transplanted discospheres, disc stem cells, disc
progenitor cells, or a mixture thereof of the present
invention.
[0164] In another embodiment, a matrix formulated disc stem cell
preparation loaded with key nutrients is injected into the disc
space and will grow into a disc tissue structure over time
restoring the damaged disc (Example 4). In another embodiment, a
matrix formulated disc stem cell preparation further comprises
resorbable culture medium. In another embodiment, a matrix
formulated disc stem cell preparation further comprises tissue
growth or factors. In another embodiment, a matrix formulated disc
stem cell preparation further comprises tissue differentiation
factors. In another embodiment, a matrix formulated disc stem cell
preparation further comprises recombinant generated morphogenetic
proteins. In another embodiment, a matrix formulated disc stem cell
preparation further comprises PDGF. In another embodiment, a matrix
formulated disc stem cell preparation further comprises TGF-.beta..
In another embodiment, a matrix formulated disc stem cell
preparation further comprises EGF/TGF-.alpha.. In another
embodiment, a matrix formulated disc stem cell preparation further
comprises IGF-I. In another embodiment, a matrix formulated disc
stem cell preparation further comprises FGF. In another embodiment,
a matrix formulated disc stem cell preparation further comprises
hydrogels. In another embodiment, a matrix formulated disc stem
cell preparation further comprises non-resorbable synthetic or
natural polymers. In another embodiment, a matrix formulated disc
stem cell preparation further comprises collagen. In another
embodiment, a matrix formulated disc stem cell preparation further
comprises fibrin. In another embodiment, a matrix formulated disc
stem cell preparation further comprises polyglycolic acid. In
another embodiment, a matrix formulated disc stem cell preparation
further comprises polytetrafluoroethylene. In another embodiment, a
matrix formulated disc stem cell preparation further comprises
anti-inflammatory medications. In another embodiment, a matrix
formulated disc stem cell preparation further comprises
antibiotics. In another embodiment, a matrix formulated disc stem
cell preparation further comprises immunosuppressive
medications.
[0165] In another embodiment, the present invention provides a
method of treating a subject having a herniated disc, comprising
the step of administering to a subject an artificial disc
comprising nucleus pulposus cells. In another embodiment, the
subject is a human subject. In another embodiment, the subject is a
farm animal. In another embodiment, the subject is a pet
animal.
[0166] In another embodiment, the present invention provides that
administering to a subject an artificial disc comprises
transplanting to a subject an artificial disc. In another
embodiment, the present invention provides that the replacement
device comprises processed biological tissues from a single donor.
In another embodiment, the present invention provides that the
replacement device comprises processed biological tissues from a
single donor which is the patient in need of an artificial disc. In
another embodiment, the present invention provides that the
replacement device comprises processed biological tissues in
combination with men made materials. In another embodiment, the
present invention provides that the replacement device comprises
processed biological tissues in combination with plastic based
materials. In another embodiment, the present invention provides
that the replacement device comprises processed biological tissues
in combination with ceramics. In another embodiment, the present
invention provides that the replacement device comprises processed
biological tissues in combination with metals.
[0167] In another embodiment, the present invention provides a
method of treating a subject having a herniated disc. In another
embodiment, the present invention provides a method of treating a
subject having a degenerative disc disease (DDD). In another
embodiment, the present invention provides a method of treating a
subject having a DDD at one level in the lumbar spine (from L3-S1).
In another embodiment, the present invention provides a method of
treating a subject having no more than Grade 1 spondylolisthesis.
In another embodiment, the present invention provides a method of
treating a subject having more than Grade 1 spondylolisthesis. In
another embodiment, the present invention provides a method of
treating a subject having no more than Grade 1 spondylolisthesis
that have had no relief from pain after at least six months of
non-surgical treatment.
[0168] In another embodiment, the present invention provides that
administering to a subject an artificial disc restores disc height.
In another embodiment, the present invention provides that
administering to a subject an artificial disc may reduce pain. In
another embodiment, the present invention provides that
administering to a subject an artificial disc restores movement at
the level where it is implanted. In another embodiment, the present
invention provides posterolateral annulotomy after discectomy.
EXPERIMENTAL DETAILS SECTION
Materials and Methods
[0169] Methylcellulose-Based Medium for Expanding Disc
Stem/Progenitor Cells into Discospheres Comprising.
[0170] The methylcellulose-based (medium for expanding discospheres
comprising disc stem/progenitor cells contained a base DMEM/F12
medium supplemented with 2% Methylcellulose, 10 .mu.g/ml insulin,
40 nM progesterone, 200 .mu.M putrescine, 100 .mu.g/ml transferrin,
60 nM sodium selenite, 10 ng/ml recombinant FGF2, and 10 ng/ml
recombinant EGF.
[0171] Methylcellulose-Based Medium for Expanding Discospheres
Comprising Disc Stem/Progenitor Cells
[0172] The Methylcellulose-based medium for expanding discospheres
comprising disc stem/progenitor cells contained a base DMEM/F12
medium supplemented with 0.8% Methylcellulose, 5 .mu.g/ml insulin,
20 nM progesterone, 100 .mu.M putrescine, 50 .mu.g/ml transferrin,
and 30 nM sodium selenite. 10 ng/ml FGF2 and 10 ng/ml EGFb were
added every 3.sup.rd day.
[0173] Histochemistry
[0174] Hematoxilin-Eosin Staining
[0175] Hematoxilin-Eosin staining on disc biopsies obtained from
the discs produced by the procedures disclosed in Example 3 were
preformed as follows: Formalin fixed paraffin embedded tissue
sections (5 .mu.m) were sequentially deparaffinized and rehydrated.
Then slides were stained with Harris' haematoxylin for 10 minutes,
washed and blue in running tap water for 1 minute, differentiated
in acid alcohol (1% hydrochloric acid in 70% alcohol) for 10
seconds, washed and blue in running tap water for 5 minutes,
stained with eosin for 4 minutes, and finally washed in tap water,
dehydrated through graded alcohol and cleared in xylene.
[0176] von Kossa Staining
[0177] von Kossa Staining on disc biopsies obtained from the discs
produced by the procedures disclosed in Example 3 were preformed as
follows: Formalin fixed paraffin embedded tissue sections (5 .mu.m)
were sequentially deparaffinized and rehydrated. Sections were
incubated with 1% silver nitrate solution in a clear glass coplin
jar placed under ultraviolet light for 20 minutes. Then sections
were rinsed in several changes of distilled water followed by the
removal of un-reacted silver with 5% sodium thiosulfate for 5
minutes. Then sections were rinsed in several changes of distilled
water and counterstained with nuclear fast red for 5 minutes.
Finally, sections were rinsed in several changes of distilled
water, dehydrated through graded alcohol and cleared in xylene.
[0178] Immunohistochemical Identification of Collagen Type I,
Collagen Type II, or Ki67 in Tissue
[0179] Immunohistochemical staining for collagen type I, collagen
type II, or Ki67 on disc biopsies from Example 3 were preformed as
follows: Formalin fixed paraffin embedded tissue sections (5 .mu.m)
were sequentially deparaffinized, rehydrated, and blocked for
endogenous peroxidase activity following a 95.degree. C. degree, 25
minutes antigen retrieval in Trilogy unmasking solution (Cell
Marque, Hot Springs Ark.). Slides were biotin blocked, serum
blocked and immunostained using a goat ABC Elite Kit (Vector Labs,
Burlingame, Calif.) Antibodies to collagen type I (cat. #: 63170,
MP Biomedicals, Solon, Ohio), collagen type II (cat. #: MAB 1330,
Chemicon, Billerica, Mass.), or Ki67 (cat. #: MAB4062, Chemicon,
Billerica, Mass.) were applied at 1:100 dilution for one hour at
room temperature. Positive staining was detected with DAB
(3,3'-Diaminobenzidene) Immuno-reactivity was visualized with a
Bio-Rad confocal microscope and images collected on a computer for
later analysis.
[0180] Safranin O Staining for Cartilage
[0181] This method was used for the detection of cartilage on
formalin-fixed, paraffin-embedded tissue sections. The cartilage
was stained orange to red, and the nuclei will were stained black.
The background was stained green. Weigert's Iron Hematoxylin
Solution was prepared from two stock solutions. Stock Solution A: 1
g Hematoxylin, 100 ml 95% alcohol. Stock Solution B: 4 ml 29%
Ferric chloride in water, 95 ml distilled water, 1 ml Hydrochloric
acid. Equal parts of stock solution were mixed resulting in
Weigert's Iron Hematoxylin Solution.
[0182] 0.1% Safranin O Solution was prepared by mixing 0.1 g
Safranin O, C.I. 50240 and 100 ml distilled water. Then slides were
deparaffinized and hydrated to distilled water followed by staining
the slides with Weigert's iron hematoxylin working solution for 10
minutes. Followed by washing the slides in running tap water for 10
minutes and staining with fast green (FCF) solution for 5 minutes,
rinsing quickly with 1% acetic acid solution for 10 seconds, and
staining in 0.1% safranin O solution for 5 minutes. Slides were
then dehydrated and cleared with 95% ethyl alcohol, absolute ethyl
alcohol, and xylene, using 2 changes each, 2 minutes each. Finally
slides were mounted using resinous medium.
Example 1
A Method of Growing Discospheres
[0183] A biopsy specimen of human nucleus pulposus was minced into
pieces approximately 2-3 millimeters in size and transferred to a
50 ml falcon tube containing 30 ml of Phosphate buffered saline
(PBS) supplemented with standard antibiotics and antimycotics
(standard penicillin/streptomycin solution (GIBCO BRL) in
concentration 1:100).
[0184] PBS was aspirated and 30 ml of Dulbecco's Modified Eagle
Media with F12 (DMEM/F12) medium containing 300 U/ml of Collagenase
II solution was added to the 50 ml tube.
[0185] The tube was placed in a horizontal position in a shaker
incubator at 37.degree. C. at 100 RPM for 2-3 hours until fragments
were completely dissociated.
[0186] The cell suspension was filtered through a nylon mesh into a
50 ml falcon tube and triturated with a fire-polished pasteur
pipette to form a single-cell suspension. A cell count was
performed at this point to determine the cell concentration.
[0187] The cell suspension was then centrifuged at room temperature
for 4 minutes (min.) at 400 g, followed by the removal of the
supernatant by aspiration.
[0188] Cells were resuspended in DMEM/F12 medium supplemented with
insulin (10 ug/ml), progesterone (40 nM), putrescine (200 uM),
transferrin (100 ug/ml), sodium selenite (60 nM) to a final density
of 120,000 cells/ml.
[0189] A volume of a 2% solution of methylcellulose in DMEM/F12
medium equal to final volume obtained previously was added to the
cell suspension and mixed by vortexing.
[0190] Growth factors EGF and FGF2 were added to final
concentration 10 ng/ml and mixed again.
[0191] Finally, the cell/media suspension was added to 6-well
plates at approximately 2 ml/well comprising about 120,000 cells
per well, and incubated at 37.degree. C. in 5% CO2. Each well was
pre-coated with an anti-adhesive substance (e.g. poly
2-hydroxyethyl methacrylate (#P-3932 Sigma) anti-adhesive coating)
according to manufacturer's recommendations.
[0192] Growth factors were added every 3.sup.rd day.
[0193] After approximately 2 weeks, discospheres had formed in the
culture.
Example 2
A Method of Expanding Discospheres Cell Culture
[0194] Discospheres obtained by the method disclosed in Example 1
were dissociated by incubation at 37.degree. C. in DMEM/F12 medium
supplemented by collagenase II (300 U/ml).
[0195] Dissociated cells were expanded in 6-well plates according
by passaging the cells using the same plating and culture
techniques as described in Example 1.
Example 3
A Method of Obtaining a Spinal Disc Collagen Scaffold (Annulus)
[0196] A postmortem (rabbit cadaver) intervertebral disc was
removed by dissection with the vertebral endplates left intact. The
intervertebral disc sample was soaked in 4 M guanidine thyocyonate
for 24 hours at room temperature to remove intradisc biomaterial.
After 24 hours, the intradisc biomaterial was liquidfied.
[0197] The liquid was aspirated, and the remaining disc scaffold
was washed 3 times with room temperature PBS.
[0198] At this stage the disc scaffold can be stored in PBS at
4.degree. C. up to one year.
Example 4
A Method of Obtaining an Artificial Disc
[0199] The disc scaffold obtained according to the method disclosed
in Example 3 was placed in tissue culture vessel and washed 3 times
with DMEM/F12 medium with 10% FCS and incubated at 37.degree. C. in
5% CO2 for 8 hours.
[0200] Discospheres were pooled from culture and collected in
DMEM/F12. Then discospheres were washed free of methylcellulose
with DMEM/F12, and suspended in 200 .mu.l DMEM/F12 medium.
[0201] The suspended discosphere were injected into the center of
the scaffold incubated at 37.degree. C. in 5% CO.sub.2 for 8
hours.
[0202] The disc tissue culture vessel was then filled with DMEM/F12
medium and incubated at 37.degree. C. in 5% CO2.
[0203] The media was changed every 3.sup.rd day.
[0204] Results
[0205] Nucleus pulposus cells were harvested from a donor patient
and prepared as a single cell suspension as described in Example 1.
After approximately 2 weeks, discospheres were collected and
prepared for injection into the pre-processed rabbit annulus
fibrosis. This disc scaffold containing the disc stem cell
preparation was then placed in a tissue culture vessel for 3
months. The media was changed every third day. Each day, a downward
pressure was applied to each disc tissue to induce biomechanical
regulated differentiation programs.
[0206] Biomechanical Properties
[0207] Disc cells laid down extracellular matrix components of the
disc, which in turn, shaped the discs' subsequent physiological and
biomechanical functions. By the 3.sup.rd week, the disc tissue
began to demonstrate resistance to pressure force, indicating its
maturation and acquisition of tensile properties. By the 10th week,
the disc tissue demonstrated maximal thickness and resistance to
compressive forces.
[0208] Comparative Histology
[0209] After 3 months of culture, the disc tissues were removed
from culture and sectioned with a cryostat. Basic histological
analyses were completed using selected tissue stains and
immunohistochemistry.
[0210] As shown in FIG. 1 (Panel 1), Hematoxilin-Eosin staining
revealed that the gross structure and cellular morphology of the
human disc tissue grown from disc stem cells was comparative to
that derived from healthy rabbit disc tissue. Additionally,
safranin staining (FIG. 1, Panel 2) demonstrated that a rich
cartilage matrix of sulfated proteoglycans was secreted into the
extracellular matrix by the disc stem cells and was comparable to
healthy rabbit disc tissue at the time of analysis. Von Kossa
staining (FIG. 1, Panel 3) demonstrated the absence of any
osteogenic differentiation of in vitro disc stem cells in this
culture system. Finally, immunohistochemical staining with collagen
type II (FIG. 2) and type 1 (FIG. 3) demonstrated high and low
expression respectively indicating maturation of the disc tissue
and again was found to be comparable to healthy controls.
[0211] Demonstration of Lack of Proliferation in the Tissue
[0212] As a further indicator that the disc tissue was mature and
thus did not contain any immature and/or proliferating cells, Ki67
(marker of proliferation) immunostaining was performed on the
tissues. As shown in FIG. 4, no proliferating cells were noted in
the control tissues or the disc tissue grown from human disc stem
cells.
Example 5
Two-Dimensional Tissue Engineering
[0213] Discospheres were seeded at 1.0666 spheres/cm.sup.2 onto
gelatin-coated coverslips. Discospheres went through attachment and
differentiation (FIG. 6). Cells proliferated, had changes in
morphology, and demonstrated motility (FIG. 7), traveling an
average distance of 525 microns at a mean velocity of 7.3
microns/hour. Seeding with engineered spheres organized into a
particular structure composed of central nucleus pulposus cells
surround by circular arrays, which was not observed after random
cell seeding (FIG. 8).
Example 6
Open Two-Dimensional/Three-Dimensional Tissue Engineering
[0214] Neo-engineered disc tissue was made using enriched stem
cells and without scaffolds. After 12 weeks, tissue with the
neo-engineered disc was similar to normal rabbit disc (FIG. 9).
Example 7
[0215] Using discospheres to grow disc stem cells, disc stem cells
demonstrated linear growth, with fractional losses at each passage
(FIG. 10). This method is therefore a good source of stem cells for
additional studies and therapy. Discospheres plated on gelatin
coated coverslips were exposed to chrondrogenic conditions with
serum and attached to gelatin coated surfaces. The discosphere were
cultures for 7 days and stained with H&E, toluidine blue, and
alician blue (FIG. 11). Disc stem cells generate progency that
produce proteoglycans and mucopolysaccharides. Interestingly,
extracellular matrix production occurred primarily in matured disc
remnants (FIG. 11).
* * * * *