U.S. patent application number 12/850516 was filed with the patent office on 2011-10-20 for methods and compositions for treating intervertebral disc degeneration.
Invention is credited to Jeffrey William Moehlenbruck, John Paul Ranieri.
Application Number | 20110256106 12/850516 |
Document ID | / |
Family ID | 24176265 |
Filed Date | 2011-10-20 |
United States Patent
Application |
20110256106 |
Kind Code |
A1 |
Moehlenbruck; Jeffrey William ;
et al. |
October 20, 2011 |
Methods and Compositions for Treating Intervertebral Disc
Degeneration
Abstract
A fluid matrix comprising cross-linked remodelable collagen from
a donor vertebrate animal is useful for regenerating hydrodynamic
function in damaged intervertebral discs in vivo. The matrix may be
injectable and may comprise cells and a plurality of purified cell
growth factors. The matrix promotes cell growth and elaboration of
proteoglycans to facilitate regeneration of native tissues. The
collagen in the matrix may be cross-linked using photooxidative
catalysis and visible light, and purified cell growth factors are
preferably at least partly bone-derived.
Inventors: |
Moehlenbruck; Jeffrey William;
(Austin, TX) ; Ranieri; John Paul; (Austin,
TX) |
Family ID: |
24176265 |
Appl. No.: |
12/850516 |
Filed: |
August 4, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12476603 |
Jun 2, 2009 |
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12850516 |
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10812268 |
Mar 29, 2004 |
7556649 |
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12476603 |
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Current U.S.
Class: |
424/93.7 ;
204/157.68; 514/17.2; 514/8.8 |
Current CPC
Class: |
A61L 27/3683 20130101;
A61P 43/00 20180101; A61L 27/3817 20130101; A61L 27/50 20130101;
A61L 2400/06 20130101; A61L 27/3834 20130101; A61L 27/3658
20130101; A61L 2430/38 20130101; A61F 2002/444 20130101; A61P 19/00
20180101; A61L 27/3687 20130101; A61L 27/3895 20130101; A61P 19/08
20180101; A61L 27/3856 20130101; A61L 27/3612 20130101 |
Class at
Publication: |
424/93.7 ;
514/8.8; 514/17.2; 204/157.68 |
International
Class: |
A61K 35/12 20060101
A61K035/12; B01J 19/08 20060101 B01J019/08; A61P 19/00 20060101
A61P019/00; A61K 38/18 20060101 A61K038/18; A61K 38/39 20060101
A61K038/39 |
Claims
1. A matrix for treating a patient having degenerative disc
disease, the matrix comprising an injectable fluid comprising
digestion-resistant remodelable collagen, said collagen being
cross-linked through photooxidative catalysis and irradiation by
visible light; and a plurality of living cells dispersed within
said injectable fluid to form an injectable cell matrix for
treating degenerative disc disease, said cells having inherent
capability to elaborate proteoglycans in vivo.
2. The injectable matrix of claim 1, further comprising a plurality
of purified cell growth factors dispersed within said injectable
cell matrix to form an injectable disc regeneration fluid, said
living cells being responsive to said purified cell growth factors
by increased elaboration of proteoglycans in vivo.
3. The matrix of claim 1 wherein said cells are chondrocytes.
4. The matrix of claim 1 wherein said cells are mesenchymal stem
cells.
5. The matrix of claim 4 wherein said cells are human-derived.
6. The matrix of claim 1 wherein said collagen is cross-linked
using methylene blue as a photooxidative catalyst.
7. The matrix of claim 1 wherein said cells are cultured in vitro
to increase their response to said cell growth factors.
8. The injectable disc regeneration fluid of claim 2 wherein at
least two of said plurality of cell growth factors are
bone-derived.
9. An injectable disc regeneration fluid, comprising an injectable
cell matrix according to claim 7; and a plurality of cell growth
factors dispersed within said injectable cell matrix to form an
injectable disc regeneration fluid, said living cells being
responsive to said cell growth factors by increased elaboration of
proteoglycans in vivo.
10. A method of treating a patient presenting with degenerative
disc disease, the method comprising providing an injectable disc
regeneration fluid according to claim 2; and injecting said
injectable disc regeneration fluid into at least one of said
patient's intervertebral discs to treat degenerative disc disease
in said disc.
11. A method of continuing treatment of a patient presenting with
degenerative disc disease, the method comprising treating the
patient according to the method of claim 10; and injecting a
plurality of cell growth factors into said at least one of said
patient's intervertebral discs after completion of the method of
claim 10 to continue treatment of a patient presenting with
degenerative disc disease.
12. A patient having a history of degenerative disc disease,
wherein the patient has been treated by the method of claim 11.
13. An intervertebral disc in vivo, said disc having been injected
with injectable disc regeneration fluid according to claim 2,
14. A method of treating a patient presenting with signs of
hydrodynamic intervertebral disc dysfunction, the method comprising
diagnosing hydrodynamic disc dysfunction in at least one
intervertebral disc of said patient; testing said at least one
intervertebral disc of said patient to establish cellular
proteoglycan production within said at least one disc; and
injecting a plurality of purified cell growth factors into said at
least one disc to treat a patient presenting with signs of
hydrodynamic intervertebral disc dysfunction.
15. The method of claim 14 wherein at least two of said plurality
of cell growth factors are bone-derived.
16. An injectable cell growth medium for intervertebral disc
regeneration, said medium comprising an injectable fluid comprising
digestion-resistant remodelable collagen, said collagen being
cross-linked through photooxidative catalysis and irradiation by
visible light; and a plurality of purified cell growth factors
dispersed within said fluid to form an injectable cell growth
medium.
17. The injectable cell growth, medium of claim 16 wherein at least
two of said plurality of cell growth factors are bone-derived.
18. An injectable disc regeneration fluid for intervertebral discs,
the material comprising injectable cell growth medium according to
claim 17; and cells responsive to said injectable cell growth
medium through proteoglycan elaboration in vivo.
19. The disc regeneration fluid of claim 18 wherein said cells are
chondrocytes.
20. The disc regeneration fluid of claim 18 wherein said cells are
mesenchymal stem cells.
21. The disc regeneration fluid of claim 20 wherein said cells are
human-derived.
22. The disc regeneration fluid of claim 20 wherein said cells are
cultured in vitro to increase their response to said cell growth
factors.
23. An injectable material for treating a patient for hydrodynamic
disc dysfunction, the material made by a process comprising
cross-linking collagen through photooxidative catalysis and
irradiation by visible light; purifying a plurality of bone-derived
cell growth factors; dispersing said purified bone-derived cell
growth factors within said cross-linked collagen; and dispersing
cells responsive to said purified plurality of bone-derived cell
growth factors within said cross-linked collagen to form an
injectable material for treating hydrodynamic disc dysfunction.
24. The injectable material of claim 23 wherein said cells are
chondrocytes.
25. The injectable material of claim 23 wherein said cells are
mesenchymal stem cells.
26. The injectable material of claim 24 wherein said cells are
human-derived.
27. The injectable material of claim 24 wherein said cells are
cultured in vitro to increase their response to said cell growth
factors.
28. A method of hydrating an intervertebral disc annulus fibrosus
in vivo, the method comprising testing said disc for cellular
proteoglycan production within said disc; and injecting cell growth
medium according to claim 16 into said disc to hydrate the annulus
fibrosus.
29. A method of reducing susceptibility to herniation of an
intervertebral disc in a patient having a history of intervertebral
disc herniation, the method comprising testing said disc for
cellular proteoglycan production within said disc; and injecting
cell growth medium according to claim 16 into said disc to reduce
susceptibility to herniation through hydration of the annulus
fibrosus.
30. A method of increasing the height of a patient presenting with
hydrodynamic disc dysfunction in at least one intervertebral disc,
the method comprising testing said at least one disc for cellular
proteoglycan production within said at least one disc; and
injecting cell growth medium according to claim 16 into said at
least one disc to increase the height of said patient by increasing
intervertebral spacing through increased proteoglycan production in
said at least one disc.
31. An injectable cell suspension for treating a patient having
degenerative disc disease, the suspension comprising an injectable
fluid comprising a plurality of purified cell growth factors; and a
plurality of living cells dispersed within said injectable fluid to
form an injectable cell suspension for treating degenerative disc
disease, said cells being responsive to said cell growth factors by
increased elaboration of proteoglycans.
32. The injectable cell suspension of claim 31 wherein said cells
are chondrocytes.
33. The injectable cell suspension of claim 31 wherein said cells
are mesenchymal stem cells.
34. The injectable cell suspension of claim 33 wherein said cells
are human-derived.
35. The injectable cell suspension of claim 31 wherein said cells
are cultured in vitro to increase their response to said cell
growth factors.
36. The injectable cell suspension of claim 31 wherein at least two
of said plurality of cell growth factors are bone-derived.
37. A method of treating a patient presenting with degenerative
disc disease, the method comprising providing an injectable cell
growth medium according to claim 16; and injecting said injectable
cell growth medium into at least one of said patient's
intervertebral discs to treat degenerative disc disease in said
disc.
38. A method of treating a patient presenting with hydrodynamic
disc dysfunction, the method comprising providing an injectable
material according to claim 23; and injecting said injectable
material into at least one of said patient's intervertebral discs
to treat hydrodynamic disc dysfunction in said disc.
39. A method of treating a patient presenting with degenerative
disc disease, the method comprising providing an injectable cell
suspension according to claim 31; and injecting said injectable
cell suspension into at least one of said patient's intervertebral
discs to treat degenerative disc disease in said disc.
40. A method of cross-linking collagen to make digestion-resistant
remodelable cross-linked collagen, the method comprising providing
a hydrogel comprising collagen; containing said hydrogel within a
semipermeable membrane, said membrane being substantially
transparent to visible light and substantially permeable to at
least one photooxidative catalyst; transporting at least one
photooxidative catalyst through said semipermeable membrane and
into said hydrogel; and irradiating said hydrogel with visible
light to cross-link said collagen.
41. The method of claim 40 comprising an additional step between
said containing step and said transporting step, the additional
step being submerging said hydrogel-containing semipermeable
membrane in a high salt:high sucrose solution for about 24 to about
72 hours.
42. The method of claim 41 wherein said hydrogel comprises nucleus
pulposus tissue.
43. The method of claim 42 wherein said transporting step occurs at
a substantially constant hydrogel temperature of about 10.degree.
C.
44. Digestion-resistant remodelable cross-linked collagen made by
the method of claim 43.
45. The method of claim 41 wherein said hydrogel comprises
substantially Type II collagen.
46. The method of claim 43 wherein said semipermeable membrane
comprises dialysis tubing having a molecular weight cutoff of about
3500 Daltons.
47. The method of claim 43 wherein said transporting step and said
irradiating step are substantially simultaneous.
48. The method of claim 43 wherein said transporting step occurs
while said semipermeable membrane containing said hydrogel is
immersed in a solution comprising at least one photooxidative
catalyst.
49. The method of claim 43 comprising an additional step after said
irradiating step, the additional step being terminating said
transporting step and said irradiating step when said collagen
cross-linking is substantially complete.
50. The method of claim 49 comprising an additional step after said
terminating step, the additional step being extracting said
cross-linked collagen from said hydrogel.
51. The method of claim 43 wherein said at least one photooxidative
catalyst comprises methylene blue.
52. A fluid matrix for treating intervertebral disc disease in a
vertebrate, said fluid comprising nucleus pulposus tissue of a
donor vertebrate.
53. The fluid matrix of claim 52 wherein said nucleus pulposus
tissue is cross-linked.
54. The fluid matrix of claim 53 further comprising a growth
factor.
55. The fluid matrix of claim 54 further comprising a plurality of
living cells.
56. The fluid matrix of claim 55, wherein said plurality of living
cells comprise chondrocytes.
57. The fluid matrix of claim 55, wherein said plurality of living
cells comprise mesenchymal stem cells.
58. The fluid matrix of claim 55, wherein said plurality of living
cells are human-derived.
59. The fluid matrix of claim 52, wherein said nucleus pulposus
tissues are decellularized.
Description
FIELD OF THE INVENTION
[0001] This invention relates generally to methods and compositions
useful in treating intervertebral disc impairment in humans and
other mammals. More particularly, this invention concerns
compositions useful in restoring hydrodynamic function and
stimulating cell proliferation and extracellular matrix production
in intervertebral discs that have been compromised by injury,
degenerative disease, congenital abnormalities, and/or the aging
process.
[0002] Compositions of the invention may be injectable, and may
include growth factors, bioactive agents, and living cells. The
compositions are useful for restoring, improving, or augmenting
hydrodynamic function of the intervertebral disc, increasing
intervertebral disc height, and stimulating cell proliferation
and/or extracellular matrix production in intervertebral discs.
BACKGROUND
[0003] The human vertebral column (spine) comprises a plurality of
articulating bony elements (vertebrae) separated by soft tissue
intervertebral discs. The intervertebral discs are flexible joints
which provide for flexion, extension, and rotation of the vertebrae
relative to one another, thus contributing to the stability and
mobility of the spine within the axial skeleton.
[0004] The intervertebral disc is comprised of a central, inner
portion of soft, amorphous mucoid material, the nucleus pulposus,
which is peripherally surrounded by an annular ring of layers of
tough, fibrous material known as the annulus fibrosus. The nucleus
pulposus and the annulus fibrosus together are bounded on their
upper and lower ends (i.e., cranially and caudally) by vertebral
end plates located at the lower and upper ends of adjacent
vertebrae. These end plates, which are composed of a thin layer of
hyaline cartilage, are directly connected at their peripheries to
the lamellae of the inner portions of the annulus fibrosus. The
lamellae of the outer portions of the annulus fibrosus connect
directly to the bone at the outer edges of the adjacent
vertebrae.
[0005] The soft, mucoid nucleus pulposus contains chondrocytes,
which produce fibrils of collagen (primarily Type II collagen, but
also Types IX, XI, and others) and large molecules of negatively
charged, sulfated proteoglycans, as depicted in FIG. 1. The term
matrix as used herein refers to a composition which provides
structural support for, and which facilitates respiration and
movement of nutrients and water to and from, an intervertebral
disc. The collagenous components of the nucleus pulposus
extracellular matrix comprise a scaffold that provides for normal
cell (i.e., chondrocyte) attachment and cell proliferation. The
negatively charged proteoglycan component of the nucleus pulposus
extracellular matrix attracts water to form a hydrated gel, which
envelops the collagen fibrils and chondrocyte cells. In the normal
healthy nucleus pulposus, water comprises between 80-90% of the
total weight
[0006] The nucleus pulposus thus plays a central role in
maintaining normal disc hydrodynamic function. The large molecular
weight proteoglycans are contained within the nucleus pulposus by
the annulus fibrosus and by the, vertebral end plates, and they
attract water into the nucleus through sieve-like pores in the end
plates. The resulting osmotic pressure within each disc tends to
expand it axially (i.e., vertically), driving the adjacent
vertebrae further apart. On the other hand, mechanical movements
resulting in axial compression, flexion, and rotation of the
vertebrae exert forces on the intervertebral discs, which tends to
drive water out of the nucleus pulposus. Water movements into and
out of an intervertebral disc under the combined influence of
osmotic gradients and mechanical forces constitute hydrodynamic
functions important for maintaining disc health.
[0007] Movement of solutes in the water passing between discs and
vertebrae during normal hydrodynamic function facilitates
chondrocyte respiration and nutrition within the discs. This
function is critical to chondrocyte survival since nucleus pulposus
tissues of intervertebral discs are avascular (the largest such
avascular structures in the human body). Maintaining sufficient
water content in the nucleus pulposus is also important for
absorbing high mechanical (shock) loads, for resisting herniation
of nucleus pulposus matter under such loads, and for hydrating the
annulus fibrosus to maintain the flexibility and strength needed
for spine stability.
[0008] Normal hydrodynamic functions are compromised in
degenerative disc disease (DDD). DDD involves deterioration in the
structure and function of one or more intervertebral discs and is
commonly associated with aging and spinal trauma. Although the
etiology of DDD is not well understood, one consistent alteration
seen in degenerative discs is an overall decrease in proteoglycan
content within the nucleus pulposus and the annulus fibrosus. The
loss in proteoglycan content results in a concomitant loss of disc
water content. Reduced hydration of disc structures may weaken the
annulus fibrosus, predisposing the disc to herniation. Herniation
frequently results in extruded nucleus pulposus material impinging
on the spinal cord or nerves, causing pain, weakness, and in some
cases permanent disability.
[0009] Because adequate disc hydration is important for stability
and normal mobility of the spine, effective treatment of DDD would
ideally restore the disc's natural self-sustaining hydrodynamic
function. Such disc regeneration therapy may require substantial
restoration of cellular proteoglycan synthesis within the disc to
maintain the hydrated extracellular matrix in the nucleus pulposus.
Improved hydrodynamic function in such a regenerated disc may
result in restoration and reestablishment of intervertebral disc
height. It may also provide for improved hydration of the annulus
fibrosus, making subsequent herniation less likely.
[0010] Prior art approaches to intervertebral disc problems fail to
restore normal self-sustaining hydrodynamic function, and thus may
not restore normal spinal stability and/or mobility under high
loads. One approach to reforming intervertebral discs using a
combination of intervertebral disc cells and a bioactive,
biodegradable substrate is described in U.S. Pat. No. 5,964,807 to
Gan et al., incorporated herein by reference. The biodegradable
substrate disclosed in Gan et al., including bioactive glass,
polymer foam, and polymer foam coated with sol gel bioactive
material, is intended to enhance cell function, cell growth and
cell differentiation. The bioactive glass contains oxides of
silicon, sodium, calcium and phosphorus. The polymer foam is
described as biocompatible and includes polyglycolide (PGA), poly
(D,L-lactide) (D,L-PLA), poly(L-lactide) (L-PLA),
poly(D,L-lactide-co-glycolide) (D,L-PLGA),
poly(L-lactide-co-glycolide) (L-PLGA), polycaprolactone (PCL),
polydioxanone, polyesteramides, copolyoxalates, and polycarbonates.
Gan et al. describes application of this approach to intervertebral
disc reformation in mature New Zealand rabbits, concluding with
ingrowth of cells and concurrent degradation of implanted material
with little or no inflammation. However, degradation of portions of
the implanted material, such as acidic breakdown of PLAs, PGAs and
PLGAs, may adversely affect cell growth, cell function and/or cell
differentiation.
[0011] A somewhat analogous disclosure relating to tissues for
grafting describes matrix particulates comprising growth factors
that may be seeded with cells; see U.S. Pat. No. 5,800,537 to Bell,
incorporated herein by reference. The matrix and cells are applied
to scaffolds, which include biodegradable polymers,
microparticulates, and collagen which has been cross-linked by
exposure to ultraviolet radiation and formed to produce solids of
foam, thread, fabric or film. The matrix particulates are derived
from tissue from which cells and cell remnants have been removed
without removing factors necessary for cell growth, morphogenesis
and differentiation. Bell specifically avoids the use of reagents
like high salt, or deliysidation reagents such as butanol/ether or
detergents. Such reagents are unfavorably characterized as being
responsible for removing from the source tissue factors essential
for stimulating repair and remodeling processes. Alternative
approaches, in which such factors are obtained from other sources
rather than being retained in the tissue, are not addressed.
[0012] Still another disclosure related to regeneration of
cartilage is found in U.S. Pat. No. 5,837,235 to Mueller et al.,
incorporated herein by reference. Mueller et al. describes
comminuting small particles of autologous omentum or other fatty
tissue for use as a carrier, and adding to the carrier growth
factors such as Transforming Growth Factor Beta and Bone
Morphogenic Protein. Mueller et al. does not teach cross-linking
tissues to create a cross-linked matrix.
[0013] The Gan et al. patent above is representative of past
attempts to restore or regenerate substantially natural
hydrodynamic disc function to intervertebral discs, but such
techniques have not been proven in clinical trials. Similarly, the
approaches of Bell and Mueller et al. have not been widely adapted
for disc regeneration, and better approaches are still needed
because low back pain sufficient to prevent the patient from
working is said to affect 60% to 85% of all people at some time in
their life. In the absence of safer and more efficacious treatment,
an estimated 700,000 discectomies and 550,000 spinal fusions are
performed worldwide each year to treat these conditions. Several
prosthetic devices and compositions employing synthetic components
have also been proposed for replacement of degenerated discs or
portions thereof. See, for example, U.S. Pat. Nos. 4,772,287,
4,904,260, 5,047,055, 5,171,280, 5,171,281, 5,192,326, 5,458,643,
5,514,180, 5,534,028, 5,645,597, 5,674,295, 5,800,549, 5,824,093,
5,922,028, 5,976,186, and 6,022,376.
[0014] A portion of the disc prostheses referenced above comprise
hydrogels which are intended to facilitate hydrodynamic function
similar in some respects to that of healthy natural discs. See, for
example, U.S. Pat. No. 6,022,376 (Assell et al.). These prosthetic
hydrogels, however, are not renewed through cellular activity
within the discs. Thus, any improvement in disc hydrodynamic
function would not be self-sustaining and would decline over time
with degradation of the prosthetic hydrogel. Healthy intervertebral
discs, in contrast, retain their ability to hydrodynamically
cushion axial compressive forces in the spine over extended periods
because living cells within the discs renew the natural hydrogel
(i.e., extracellular matrix) component.
[0015] Restoration of a clinically useful degree of normal
hydrodynamic function in degenerated intervertebral discs is an
object of the present invention, and the methods and compositions
described herein have been shown to induce and/or enhance such
regeneration.
SUMMARY OF THE INVENTION
[0016] The present invention comprises methods and compositions for
intervertebral disc regeneration. In preferred embodiments, the
compositions comprise a three-dimensional fluid matrix of
digestion-resistant, cross-linked nucleus pulposus tissue from a
donor vertebrate. The donor may be the patient or another animal of
the same or different species. Cross-linking of donor nucleus
pulposus tissue for the present invention is preferably achieved
through use of one or more photooxidative catalysts which
selectively absorb visible light. See U.S. Pat. Nos. 5,147,514,
5,332,475, 5,817,153, and 5,854,397, all incorporated herein by
reference. Other cross-linking approaches may be used without
departing from the scope of the invention, however.
[0017] Prior to cross-linking the tissues, chondrocytes of the
donor vertebrate are preferably destroyed, fragmented, and/or
removed (i.e., decellularized). A preferred decellularization
approach involves soaking the tissue in a solution having high
concentrations of salt (preferably NaCl) and sugar (preferably
sucrose). Such high-salt, high-sugar solutions are referred to as
HSHS solutions. Other decellularization approaches may be used,
however. After the tissues are decellularized and cross-linked, the
resulting fluid matrix may be lyophilized for sterilization and
storage, and then rehydrated prior to use. FIG. 2 illustrates a
process for producing a preferred embodiment of the fluid matrix of
the present invention.
[0018] The fluid matrix of the present invention is biocompatible,
substantially non-immunogenic, and resistant to degradation in
vivo. As such, it is capable of providing important internal
structural support for an intervertebral disc undergoing
regeneration during a period of accelerated proteoglycan synthesis.
The cross-linked matrix may be delivered to the intervertebral disc
space by injection through a syringe (as depicted in FIG. 2), via a
catheter, or other methods known in the art.
[0019] The three-dimensional fluid matrix of the present invention
may be used alone or in combination with growth factors and/or
living cells to facilitate regeneration of the structures of a
degenerated disc. In patients having sufficient viable endogenous
disc cells (chondrocytes) and cell growth factors, the
three-dimensional cross-linked matrix alone may substantially
contribute to the regeneration of hydrodynamic function in an
intervertebral disc in vivo by providing improved mechanical
stability of the disc and a more favorable environment for cellular
growth and/or metabolism. Conversely, in another embodiment of the
invention, a combination of the three-dimensional matrix and one or
more purified, preferably bone-derived, cell growth factors may
also be used to treat DDD in discs containing viable chondrocytes
in a depleted proteoglycan hydrogel matrix. In this case, the
cross-linked collagen provides an expanded remodelable
three-dimensional matrix for the existing (native) chondrocytes
within a disc, while the cell growth factors induce accelerated
proteoglycan production to restore the hydrogel matrix of the
patient. The combination of the three-dimensional matrix and one or
more purified cell growth factors is referred to as a cell growth
medium. The present invention may also comprise an injectable cell
growth medium. Individual purified cell growth factors may be
obtained by recombinant techniques known to those skilled in the
art, but a preferred plurality of bone-derived purified cell growth
factors for the present invention is disclosed in U.S. Pat. Nos.
5,290,763, 5,371,191 and 5,563,124, all incorporated herein by
reference. Bone-derived cell growth factors produced according to
these patents are hereinafter referred to as "BP."
[0020] Disc regeneration occurs as the cross-linked collagen and
proteoglycan matrix supports living cells (which may include
exogenous cells as well as native disc or other autologous cells)
having inherent capability to synthesize Type II collagen fibrils
and proteoglycans in vivo, among other extracellular matrix
molecules. Where the patient's native disc cells have been removed
or are otherwise insufficient to cause such proliferation, living
cells may be added to the three-dimensional matrix of cross-linked
nucleus pulposus material to further promote disc regeneration.
Accordingly, in another embodiment, the present invention comprises
a three-dimensional matrix of cross-linked nucleus pulposus tissue
to which exogenous arid/or autologous living cells have been added.
The injectable combination of three-dimensional matrix material and
exogenous and/or autologous living cells is termed herein an
injectable cell matrix. Suitable cells for such an injectable cell
matrix may be obtained, for example, from the nucleus pulposus of a
mammalian vertebral disc, from cartilage, from fatty tissue, from
muscle tissue, from bone marrow, or from bone material (i.e.,
mesenchymal stem cells), but are not limited to these tissue types.
These cells are preferably cultured in vitro to confirm their
viability and, optionally, to increase the cells' proliferation and
synthesis responses using cell growth factors.
[0021] Growth factors may optionally be added to cell cultures to
stimulate cellular development and elaboration of Type II collagen
fibrils and proteoglycans suitable for maintaining an effective
disc hydrogel matrix in vivo. An injectable fluid combining
purified cell growth factors and a plurality of living cells is
termed an injectable cell suspension, and is useful in treating
DDD. While an injectable cell matrix alone (i.e., without growth
factors) may substantially regenerate hydrodynamic function in an
intervertebral disc in vivo if sufficient native cell growth
factors are present in the disc, purified (exogenous) cell growth
factors may be added to an injectable cell matrix of the present
invention to form yet another embodiment of the present
invention.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1 is a diagram illustrating components of healthy
nucleus pulposus tissue in a vertebrate.
[0023] FIG. 2 is a diagram illustrating a process for preparation
and use of a cross-linked matrix of porcine nucleus pulposus tissue
in a preferred embodiment of the invention.
[0024] FIG. 3 is a photographic reproduction of an SDS-PAGE (sodium
dodecyl sulfate polyacrylamide gel electrophoresis) analysis
comparing the amount of proteins extracted from a cross-linked
matrix of the present invention with a non-cross-linked
control.
[0025] FIG. 4 is a photographic comparison of an H & E
(hematoxylin and eosin) stained section of fresh porcine nucleus
pulposus tissue with a cross-linked matrix of the present
invention, both at 300.times. magnification.
[0026] FIG. 5 is a photographic reproduction of a stained
nitrocellulose membrane comparing the reactivity of Type II
collagen digested from a cross-linked matrix of the present
invention and a non cross-linked control.
[0027] FIG. 6 is a comparison graph of the hydraulic/swelling
capacity of a cross-linked matrix of the present invention and a
non-crosslinked control.
[0028] FIG. 7 is a diagram of an experimental process used to
demonstrate stimulation of sheep cell ingrowth, proliferation, and
new matrix synthesis in an embodiment of the present invention
comprising a cross-linked matrix combined with bone protein growth
factors (BP).
[0029] FIG. 8 is a graph and a photograph indicating the results of
an Alcian blue assay for matrix production in sheep nucleus
pulposus cells stimulated by growth factors.
[0030] FIG. 9 is a graph indicating the results of immunogenicity
tests for a cross-linked matrix of the present invention in rabbit
immunizations and sheep serum.
[0031] FIG. 10 is a diagram of the protocol for an in vivo study of
a matrix and growth factor combination of the present
invention.
[0032] FIG. 11 is a radiograph of a vertebral column from a sheep
sacrificed at 2 months after an injection of a matrix and growth
factor combination in an in vivo study of an embodiment of the
present invention.
[0033] FIG. 12 is a photographic reproduction of histology slides
of vertebral discs of a sheep sacrificed at 2 months after an
injection of a matrix and growth factor combination of the present
invention.
[0034] FIG. 13 is a radiograph of a vertebral column of a sheep
sacrificed at 4 months after an injection of a matrix and growth
factor combination in an in vivo study of the present
invention.
[0035] FIG. 14 is a photographic reproduction of histology slides
of vertebral discs of a sheep sacrificed at 4 months after an
injection of a matrix and growth factor combination of the present
invention.
[0036] FIG. 15 is a graph representing the results of an ELISA
performed to measure the synthesis of Type II collagen and
Chondroitin-6-sulfate under growth factor stimulation
[0037] FIG. 16a is a graph indicating the results of an Alcian blue
assay for proteoglycan synthesis in human intervertebral disc cells
stimulated by growth factor.
[0038] FIG. 16b is a graph indicating the results of an Alcian blue
assay for proteoglycan synthesis in another human intervertebral
disc cells stimulated by growth factor.
[0039] FIG. 17 is a graph depicting the results of an Alcian blue
assay for proteoglycan synthesis in baboon intervertebral disc
cells stimulated by growth factor.
[0040] FIG. 18 is an SDS-PAGE gel of HPLC fractions 27-16 from a
sample of BP.
[0041] FIG. 19 is an SDS-PAGE gel of HPLC fractions 27-16 with
identified bands indicated according to the legend of FIG. 20.
[0042] FIG. 20 is an SDS-PAGE gel of BP with identified bands
indicated.
[0043] FIG. 21 is a 2-D (two-dimensional) SDS-PAGE gel with
internal standards indicated by arrows.
[0044] FIG. 22 is a 2-D SDS-PAGE gel with circled proteins (growth
factors) identified as in legend.
[0045] FIGS. 23A-23O are Mass Spectrometer results for tryptic
fragments.
[0046] FIG. 24 is a 2-D gel Western blot with anti-phosphotyrosine
antibody.
[0047] FIGS. 25A-25D are 2-D gel Western blots with antibodies for
the indicated proteins. For FIG. 25A, the growth factors are BMP-3
and BMP-2; for FIG. 25B the growth factors are BMP-3 and BMP-7; for
FIG. 25C the growth factors are BMP-7 and BMP-2; and for FIG. 25D
the growth factors are BMP-3 and TGF-.beta.1.
[0048] FIG. 26 is a PAS (periodic acid schiff) stained SDS-PAGE gel
of HPLC fractions.
[0049] FIG. 27 is an anti-BMP-7 stained SDS-PAGE gel of PNGase F
treated BP.
[0050] FIG. 28 is an anti-BMP-2 stained SDS-PAGE gel of PNGase F
treated BP.
[0051] FIGS. 29A-29B are bar charts showing explant mass of
glycosylated BP samples (FIG. 29A) and ALP Score (FIG. 29B) of the
same samples.
[0052] FIG. 30 is a chart showing antibody listing and
reactivity.
[0053] FIGS. 31A-31B together comprise a chart showing tryptic
fragment sequencing data.
[0054] FIGS. 32A-32F together comprise a chart showing tryptic
fragment mass spectrometry data.
[0055] FIGS. 33A-33B are an SDS-gel of BP (FIG. 33B) and a scanning
densitometer scan (FIG. 33A).
[0056] FIG. 34 is a chart illustrating the relative mass of major
components of BP.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0057] In a preferred embodiment, the invention comprises a
biodegradable matrix, which is delivered as an incompressible fluid
to induce and/or enhance regeneration or repair of tissues in the
intervertebral disc. The biodegradable matrix comprises hydrophilic
molecules, which will maintain and/or increase the "captured" water
content in intervertebral disc tissues. The biodegradable matrix
may also serve as a carrier substrate for added growth factors
and/or appropriate living cell types.
[0058] Since the biodegradable matrix of the present invention is a
viscous fluid, it furnishes incompressible support when delivered
within a closed, secure disc space. Moreover, because it is
distributed uniformly within a disc, the present fluid matrix has a
force distribution effect, hydraulically transmitting forces evenly
inside the disc. The matrix thus provides resistance against axial
compression and annulus collapse, whereas other matrix materials
(for example, polymer sponges and collagen sponges) will rapidly
collapse under the axial compressive forces within the disc. Solid
matrix materials, in contrast, will concentrate forces from end
plates directly onto implants, leading to rapid deterioration of
implants and/or end plates.
[0059] In a preferred embodiment, the biodegradable matrix of the
present invention is injectable. Clinical application to a patient
can thus be accomplished using minimally invasive techniques,
significantly reducing both the cost of treatment and the
likelihood of complications relative to procedures such as partial
discectomy or vertebral fusion. Similarly, the present invention
avoids the requirement for boring a hole into the annulus to
implant a prosthetic replacement nucleus pulposus device, such as a
relatively solid biodegradable matrix, or to evacuate nucleus
tissue to create space for an implanted biodegradable
substrate.
[0060] The matrix of the present invention is a natural material,
preferably prepared from normal, healthy nucleus tissue of animals
and/or humans. Accordingly, the matrix is comprised of proteins and
matrix molecules especially adapted for efficient hydrodynamic
function in intervertebral discs. Such a matrix remains
biodegradable under normal circumstances in the presence of
specific cellular enzymes, albeit at a slower rate than endogenous
disc matrix. It is an important feature of the invention that
matrix breakdown products associated with the present invention are
digestible by disc cells. In comparison, some matrix materials
previously taught (e.g. polyvinyl alcohol) do not break down by
physiological processes. In addition, some synthetic polymer
substrates create acidic degradation byproducts, in particular PGA
and PLA.
[0061] Immediate (substantially homogeneous) dispersion of cells
within the present matrix is another advantage of the invention.
The viscous fluid formulation preferred for injection can be mixed
directly with cells of the appropriate type(s) and then delivered
immediately to treat an intervertebral disc. In the matrix of the
present invention it is not necessary to culture cells and matrix
together for some days or weeks before implantation, as it is for
certain matrix materials such as PGA and collagen sponges.
[0062] The matrix of the present invention is an appropriate
substrate for cells, uniquely suited to the ingrowth,
proliferation, and residence of intervertebral disc cells.
Intervertebral disc cells preferentially grow into and survive in
the matrix of the present invention, compared to type I collagen
sponges fixed with formalin or glutaraldehyde.
[0063] The following examples illustrate the preparation of
preferred embodiments of the invention and demonstrate its
non-immunogenic and disc regenerative properties.
EXAMPLE 1
Preparation of a Cross-Linked, Fluid Matrix Suitable for Treatment
of Degenerative Disc Disease
[0064] A three-dimensional fluid matrix of cross-linked nucleus
pulposus tissue in accordance with an embodiment of the present
invention may be prepared from donor vertebrates. Although porcine
donors were used in a particularly preferred embodiment, nucleus
pulposus tissues from other vertebrates may also be used, although
mammalian vertebrates are preferred (e.g., human, porcine, bovine,
ovine, etc.).
[0065] Although nucleus pulposus tissues may be harvested by a
variety of methods from many vertebral donors, in a preferred
embodiment nucleus pulposus tissues were dissected aseptically from
spinal intervertebral discs of pigs. In a sterile environment
(i.e., a laminar flow hood), the annulus fibrosus of porcine donors
was sliced radially and the vertebral end plates separated to
expose the nucleus pulposus. The latter material was curetted out
of the central portion of the disc, devoid of annulus and end plate
tissues.
[0066] The nucleus pulposus tissues thus harvested were inserted
into sterile dialysis (filter) tubing having a preferred molecular
weight cutoff of about 3500 Daltons to substantially prevent loss
of low molecular weight proteoglycans from the tissues while
substantially reducing bacterial or other contamination. Other
semipermeable membranes or filtering membrane types may be used to
perform these functions.
[0067] The nucleus pulposus tissues to be cross-linked are also
preferably treated to destroy and remove donor cells and/or cell
fragments. To this end, dialysis tubing containing nucleus pulposus
tissues was submerged in a high-salt, high-sucrose (HSHS) solution
of about 2.2%:8.4% w/v (respectively) for about 48 hours.
Concentration ranges for the HSHS solution may be from 1% to 50%,
but a preferred HSHS solution contains 220 grams NaCl and 837.5
grams of sucrose in 10 L water. Preferred HSHS incubation times are
from about 24 to about 72 hours, although shorter or longer times
may also advantageously be used. Exposure to this HSHS solution
results in osmotic destruction and fragmentation of native
chondrocyte cells (decellularization), and further results in
denaturation of soluble cellular proteins and nucleic acids. The
HSHS solution may also contain other reagents which further degrade
nucleic acids (including but not limited to sulfones and
nucleases), and other reagents which can extract membrane lipids
(including but not limited to alcohol, chloroform, and methanol).
Although native cells of the donor may be retained in other
embodiments of the invention, decellularization and denaturation
are preferred where exogenous (particularly xenogeneic) tissues are
used, so as to reduce the potential for immunogenic responses.
Processes other than exposure to HSHS solutions may be used for his
purpose.
[0068] Cross-linking of the nucleus pulposus tissues is preferably
accomplished by a photo-mediated process in accordance with U.S.
Pat. Nos. 5,147,514, 5,332,475, 5,817,153, and/or 5,854,397. In one
such process, a photoactive dye (methylene blue) was dissolved in
the HSHS solution at a preferred dye concentration of about 20
mg/liter. The photoactive dye was allowed to permeate the nucleus
tissues within the dialysis tubing during the initial
storage/decellularization process in HSHS. A wide range of
photoactive dyes and concentrations, as taught in the foregoing
patents, may be used to obtain cross-linked fluid matrices suitable
for use in regenerating mammalian disc tissues. Preferred dyes
include methylene blue and methylene green at concentrations of
about 0.001% to about 1.0% w/v.
[0069] To cross-link the collagen within the nucleus tissues, the
dialysis tubing containing, the dye-permeated nucleus tissues was
placed in a photooxidation chamber and exposed to broad-spectrum
visible light for 48 hours. In preferred embodiments of the
invention, the tissues may be cross-linked from about 24 to about
72 hours. A solution of methylene blue in phosphate buffered saline
(PBS) was maintained under controlled temperature at 10.degree. C.
and circulated around the dialysis tubing within the photooxidation
chamber to provide substantially constant temperature regulation of
the nucleus tissues. Precise temperature control is not critical to
the practice of the invention; however, maintaining a relatively
cooler temperature is preferred to avoid damaging the tissues.
Following photo-crosslinking of the collagen, the treated nucleus
tissues were collected, lyophilized in a vacuum under
centrifugation, and finely pulverized in a freezer-mill under
liquid nitrogen. The cross-linked matrix product thus prepared can
be sterilized using gamma radiation, ethylene oxide (or other
sterilants) and stored at -80.degree. C. until rehydrated for use.
A preferred process for preparing a matrix according to the present
invention is illustrated in FIG. 2.
[0070] In addition to preparation of the cross-linked matrix,
control (non-crosslinked) tissues were prepared following the above
procedures, except that they were not exposed to light. These
control, non-crosslinked tissues were used for comparison
purposes.
[0071] To investigate the swelling capacity of cross-linked matrix
versus non-crosslinked control, lyophilized samples of cross-linked
matrix and non-crosslinked control were suspended in water and the
increase in weight due to water absorption was measured at various
times from 0 to 96 hours. As illustrated in FIG. 6, the
cross-linked matrix retained 95% of the hydraulic capacity of the
non-crosslinked control.
EXAMPLE 2
Testing of Fluid Matrix to Evaluate Protein Modification Induced by
the Cross-Linking Process
[0072] One half gram of the matrix material obtained prior to the
lyophilization step of EXAMPLE 1 was placed in 15 mls of a solution
of 4M guanidine hydrochloride and agitated on a shaker for 24 hours
to solubilize proteoglycans. After centrifugation, the supernatant
was discarded and the pellet washed in distilled water 3 times for
5 minutes each. The pelleted matrix material was then removed and
blot-dried on filter paper.
[0073] One hundred mg of the blot-dried matrix was placed in a 1.5
ml microcentrifuge tube with 1000 .mu.l of 1% sodium dodecyl
sulfate (SDS) containing 5% beta-mercaptoethanol (BME). The matrix
in SDS/BME was boiled for one hour to extract proteins (e.g.,
collagens). Samples were then centrifuged at 12000 rpm for 1 hour
and aliquots of the supernatant were subjected to electrophoresis
in gradient polyacrylamide gels.
[0074] Gels were stained with Coomassie blue or silver to visualize
proteins extracted by the SDS/BME and heat treatment. As
illustrated in FIG. 3, collagen bands stained prominently in
control, non-crosslinked tissues but exhibited only faint staining
in cross-linked matrix. These results demonstrated that in the
cross-linked matrix material, collagen proteins were not easily
extracted by the above treatment, indicating that crosslinking had
occurred. In contrast, stained gels of the control tissues
demonstrated that collagen proteins were readily extracted from
non-crosslinked material by the above treatment. See FIG. 3.
EXAMPLE 3
Matrix Histology to Evaluate Cellular Debris and Residual
Membranous Material
[0075] Cross-linked matrix material obtained prior to the
lyophilization step of Example 1 was placed in 4% paraformaldehyde
for tissue fixation. Standard histology techniques of embedding,
sectioning, and staining of sections with hematoxylin & eosin
dyes were performed. Visualization of cross-linked matrix in H
& E-stained sections demonstrated that the matrix preparation
process facilitates destruction of cellular membranes and
intracellular elements, with minimal membrane material remaining as
compared to fresh porcine nucleus pulposus material as well as
non-crosslinked tissue decellularized by HSHS treatment,
freeze-thaw cycles, and HSHS treatment plus freeze-thaw cycles. See
FIG. 4.
EXAMPLE 4
Evaluation of Matrix Antigenic Reactivity Using Monoclonal
Antibodies to Type II Collagen
[0076] Cross-linked matrix material obtained prior to the
lyophilization step of Example 1 was also subjected to pepsin
digestion to cleave Type II collagen proteins. The protein digests
were run on SDS/PAGE and then transferred to a nitrocellulose
membrane. Total protein transferred to the membrane was visualized
using Colloidal Gold.
[0077] The visualized nitrocellulose membranes were incubated with
a mouse monoclonal antibody to Type II collagen and a secondary
antibody (anti-mouse) conjugated with alkaline phosphatase. The
antibody reactivity was visualized through addition of alkaline
phosphatase substrate. As depicted in FIG. 5, the antibodies toward
Type II collagen did not react with pepsin digests of the
cross-linked matrix as much as with the pepsin digests of the
non-crosslinked control tissue. The results indicate that the
matrix of the invention may have reduced antigenic epitopes for
Type II collagen, and thus have less immunogenicity than
non-crosslinked tissues. Sec FIG. 5.
EXAMPLE 5
Evaluation of Matrix Immunogenicity in Rabbit Antisera
Production
[0078] One gram of the lyophilized and pulverized matrix material
prepared according to EXAMPLE 1 was dispersed in PBS (i.e.,
rehydrated) and centrifuged. The protein concentration of the
supernatant was then determined using the. BCA assay and the
supernatant was diluted with PBS to a final concentration of 200
.mu.g of protein per ml of PBS. The diluted supernatant was then
sterilized for injection protocols. Three rabbits were immunized
with 100 .mu.g of protein from the sterilized supernatant. Each
rabbit received 9 immunizations over a 14 week period and sera was
collected from the rabbits on a regular schedule.
[0079] Antisera production against the protein extract was measured
using an enzyme-linked immunosorbent assay (ELISA). Type II
collagen was included as a positive control in the ELISA.
Colorimetric evaluation of antisera directed against the matrix
material demonstrated very low immunogenicity in rabbits. See FIG.
9.
EXAMPLE 6
Matrix Formulation Including Serum and Other Fluids for Injections
and Delivery
[0080] One gram of the lyophilized and pulverized matrix material
prepared according to EXAMPLE 1 was sterilized with 70% ethanol and
the ethanol was removed by successive PBS rinses. The dispersed
matrix was centrifuged and the pellet was suspended in
heat-inactivated sheep serum at a ratio of 0.5 g lyophilized matrix
to 1 ml serum to prepare a viscous fluid matrix which can be loaded
into a standard syringe and delivered via a small gauge needle. In
preferred embodiments of the invention, the serum is collected from
the vertebrate animal or human patient to be treated,
heat-inactivated to destroy unwanted protein components (complement
proteins), and passed through a 0.2 micron sterile filtration unit.
Different matrix/serum ratios may also be advantageously employed.
Ratios ranging from 0.1 g to 2.0 g of lyophilized matrix to 1 ml of
serum are preferred.
[0081] Serum is a preferred fluid for mixture and delivery of the
cross-linked matrix of the present invention because it contains
various intrinsic growth factors that are beneficial to
intervertebral disc cells. Serum also serves as a suitable carrier
for extrinsic protein growth factors and/or small molecules. The
beneficial effects of extrinsic growth factors on intervertebral
disc cells are enhanced by the addition of serum.
[0082] Other fluids are also suitable for mixture and delivery of
the viscous fluid matrix. For example, sterile saline or sterile
water may also be used. The examples herein are not meant to be
limiting as to the variety of carrier fluids which may be used to
mix and deliver the matrix in the present invention.
EXAMPLE 7
Injection of Matrix Formulation to Intervertebral Discs Using
Pressure-Mediated Syringe
[0083] Matrix material was prepared according to EXAMPLE 6 (mixed
with serum) to form a viscous fluid and loaded into a standard
syringe having a small gauge needle (e.g., 18-31 gauge) attached.
Syringe injection pressure can be controlled simply by the fingers
of the hand. In other embodiments of the invention, pressure to
inject the viscous fluid can be controlled by an external device
which concomitantly measures (e.g., via a pressure transducer) and
delivers (e.g., by compressed air) a predetermined force to the
syringe plunger.
[0084] In one preferred embodiment of this device, a thermal
element is included in the needle. By providing a needle having a
thermal element, it is possible to deliver heat to the outer layers
of the annulus fibrosus at the end of the treatment and during
removal of the syringe needle in order to shrink collagen fibers
around the needle and effectively seal the site of needle
penetration.
[0085] It is further contemplated that the matrix of the present
invention can be delivered to the disc space of a patient
transpedicularly (i.e., through the pedicle of the vertebrae). In
particular, the cross-linked matrix can be administered
percutaneously via a biopsy cannula inserted through a channel in
the pedicle. After delivery of the matrix, the channel can then be
filled with bone cement or other like material to seal the
channel.
EXAMPLE 8
Isolation of Human, Sheep, and Baboon Intervertebral Disc Nucleus
Pulposus Cells
[0086] Human intervertebral nucleus pulposus tissues were collected
during surgery, suspended in Dulbecco's Modified Eagle Medium/
Nutrient Mixture F-12 (DMEM/F-12) in a 1:1 v/v mixture supplemented
with antibiotics. The tissues were kept on ice until dissection, at
which time they were rinsed 2-3 times in sterile Dulbecco's
Phosphate Buffer Saline (DPBS) to remove any blood. In a laminar
flow hood, the nucleus tissues were isolated and diced into small
(2 mm) cubes, and then placed in a Tissue Culture Medium
(hereinafter referred to as "TCM") comprising DMEM/F-12 culture
media supplemented with 10% heat inactivated fetal bovine serum,
0.25% penicillin, 0.4% streptomycin, 0.001% amphotericin B, and 50
.mu.g/ml ascorbic acid. Only tissues clear of blood and other
anomalous elements were used. Placed on a shaker at 37.degree. C.,
the tissues were digested with 0.01% hyaluronidase (Calbiochem) in
TCM for 2 hours, 0.01% protease (Sigma) in TCM for 1 hour, and 0.1%
collagenase Type II (Sigma) in TCM overnight to obtain a suspension
of human intervertebral disc nucleus pulposus cells.
[0087] The foregoing procedure was also applied to sheep and baboon
intervertebral disc nucleus pulposus tissues to obtain suspensions
of sheep and baboon intervertebral disc nucleus pulposus cells,
respectively.
EXAMPLE 9
Primary Culture and Expansion of Human, Sheep, and Baboon
Intervertebral Disc Nucleus Pulposus Cells
[0088] Human intervertebral disc nucleus pulposus cells from
EXAMPLE 8 were expanded by culturing in TCM at 37.degree. C. in 5%
CO.sub.2 atmosphere and 95% relative humidity. The TCM was changed
every 2-3 days and the cells were passaged with trypsin to another
container, when 80-90% confluent, for continued expansion.
[0089] The foregoing procedure was also applied to sheep and baboon
intervertebral disc nucleus pulposus tissues to obtain an expanded
supply of sheep and baboon intervertebral disc nucleus pulposus
cells.
EXAMPLE 10
Alcian Blue Assay of Disc Cell Matrix Production in Human, Sheep,
and Baboon Intervertebral Disc Nucleus Pulposus Cells
[0090] Human intervertebral disc cells from EXAMPLE 9 were seeded
and grown in 24 well plates in TCM in the presence or absence of
exogenous growth factors. At various time points, TCM was aspirated
out from the wells and the wells washed 3 times with PBS. The cells
were then fixed with 4% paraformaldehyde (pH 7.4) for 10 min. The
fixed cells were washed 2 times with PBS and then stained overnight
with 0.5% Alcian blue in 0.1N hydrochloric acid (pH 1.5). After
overnight staining, excess stain was rinsed out with 3 rinses of
PBS. The remaining Alcian blue stain (bound to proteoglycans) was
dissolved overnight into 6M guanidine hydrochloride and the
absorbance at 630 nm was measured using a spectrophotometer,
providing an indication of the induction of matrix production by
exogenous growth factors in human nucleus pulposus cells.
[0091] The foregoing procedure was also applied to sheep and baboon
intervertebral nucleus pulposus cells from EXAMPLE 9 to obtain an
indication of the induction of matrix production by exogenous
growth factors in sheep and baboon nucleus pulposus cells.
EXAMPLE 11
Enzyme Linked Immunosorbent Assay (ELISA) on Ovine Intervertebral
Disc Nucleus Pulposus Cells
[0092] To detect specific antigenic epitopes in the synthesized
matrix, sheep intervertebral nucleus pulposus cells from EXAMPLE 9,
seeded and grown in monolayer, were fixed in 2% glutaraldehyde for
1 hour at room temperature. The fixed cells were washed 3 times
with TBS for 5 min. each. To block non-specific antibody binding,
the cells were incubated in a solution of Tris buffered saline
(TBS) containing 1 mM ethylene-diamine-tetraacetic acid (EDTA),
0.05% Tween-20, and 0.25% bovine serum albumin for 1 hour. The
blocking step was followed by 3 washes with TBS for 5 min. each.
The cells were incubated with the primary antibody at room
temperature for 2.5 hours, and the excess primary antibody was
removed by 3 washes with TBS for 5 min. each. A second incubation
with blocking buffer was performed for 10 min., followed by 3
washes with TBS. The cells were then incubated with the secondary
antibody, which was conjugated with alkaline phosphatase enzyme,
for 3 hours at room temperature. The unbound secondary antibodies
were removed by 3 washes of TBS for 5 min each. The bound primary
and secondary antibodies were detected by addition of an
enzyme-specific substrate which produced a colored reaction. The
colorimetric measurement was performed using a spectrophotometer,
providing a quantitative measure of the presence of the bound
antibodies.
EXAMPLE 12
Effect of Exogenous Growth Factors on Proteoglcan Synthesis in
Ovine Intervertebral Disc Nucleus Pulposus Cells
[0093] Transforming growth factor-.beta.1 (TGF.beta.1) and a
mixture of bone-derived protein growth factors (BP) produced
according to U.S. Pat. Nos. 5,290,763, 5,371,191 and 5,563,124,
were tested for their effects on stimulation of proteoglycan
synthesis in ovine nucleus pulposus cells. Sheep intervertebral
disc nucleus cells were collected and cultured as described in
EXAMPLES 8 and 9. Sheep cells were seeded in micromass (200,000)
into the wells of a 24 well plate. Growth factor dilutions were
prepared in TCM supplemented with 0.5% heat-inactivated fetal
bovine serum. TGF.beta.1 and BP were both tested at 10 ng/ml; BP
was also tested at a concentration of 10 .mu.g/ml. Control wells
without growth factors contained TCM supplemented with 0.5% and 10%
heat-inactivated fetal bovine serum. The cells were incubated in
continuous exposure to the various growth factors for 7 and 10
days. At these time points, the cells were fixed and the amount of
proteoglycan synthesis was measured by the Alcian blue assay as
described in EXAMPLE 10.
[0094] At both 7 and 10 day time points, proteoglycan synthesis was
significantly greater in the 10% fetal bovine serum control
cultures than in the 0.5% fetal bovine serum control cultures. At
the 7 day time point, BP at the higher 10 .mu.g/ml concentration
produced a significant (93%) increase in proteoglycan synthesis
above the level in 10% serum control culture and a greater (197%)
increase above the 0.5% serum control. Slight increases in
proteoglycan synthesis above the 0.5% serum control were observed
in the 10 ng/ml TGF.beta.1 and BP cultures, but these increases
were not significant.
[0095] At the 10 day time point (FIG. 8), 10 .mu.g/ml BP produced a
significant increase (132%) in proteoglycan synthesis over the 10%
serum control, while 10 ng/ml TGF.beta.1 produced a significant
increase (52%) above the 0.5% serum control. At 10 ng/ml, BP
exhibited a modest 20% increase in proteoglycan synthesis over the
0.5% serum control, while at the 10 .mu.g/ml concentration, BP
produced an 890% increase above the 0.5% serum control.
EXAMPLE 13
Effect of Exogenous Growth Factors on Type II Collagen and
Chondroitin-6-Sulfate Produced by Ovine Intervertebral Disc Nucleus
Pulposus Cells
[0096] TGF.beta.1 and BP were tested for their effects on
stimulation of Type II collagen and chondroitin-6-sulfate synthesis
in sheep intervertebral disc nucleus pulposus cells. The cells were
obtained and cultured as described in EXAMPLES 8 and 9 and seeded
into tissue culture dishes. The TGF.beta.1 and BP growth factors
were prepared in TCM supplemented with 0.5% heat inactivated fetal
bovine serum. TGF.beta.1 was tested at a concentration of 10 ng/ml;
BP was tested at a concentration of 10 .mu.g/ml. Control cultures
were incubated in TCM supplemented with 0.5% serum alone.
[0097] After incubation with growth factors for 7 days, cell
cultures were fixed in 2% glutaraldehyde and the quantity of Type
II collagen and chondroitin-6-sulfate produced in the cell cultures
was detected by ELISA according to the procedure described in
EXAMPLE 11. The primary antibodies used were mouse anti-human Type
II collagen and mouse anti-human chondroitin-6-sulfate.
[0098] At 7 days, cell cultures incubated with 10 .mu.g/ml BP
produced 324% more Type II collagen and 1780% more
chondroitin-6-sulfate than control cultures. 10 ng/ml TGF.beta.1
increased production of Type II collagen by 115% and
chondroitin-6-sulfate by 800% over controls. See FIG. 15.
EXAMPLE 14
Effect of Exogenous Growth Factors on Proteoglycan Synthesis in
Human Intervertebral Disc Nucleus Pulposus Cells
[0099] TGF.beta.1 and BP were tested for their effects on
stimulation of proteoglycan synthesis in human nucleus cells. Human
intervertebral disc nucleus pulposus cells obtained from Disc L5-S1
of a 40 yr old female patient were cultured as described in
EXAMPLES 8 and 9 and seeded into 24 well plates. After the cells
adhered to the well surface, multiple dilutions of different growth
factors were added. The concentrations of growth factors tested
were 10 ng/ml TGF.beta.1, and 10 and 20 .mu.g/ml of BP. The
dilutions were prepared in TCM. The cells were fixed after 5 and 8
days of continuous exposure to growth factors and proteoglycans
synthesized were detected by the Alcian blue assay as described in
EXAMPLE 10.
[0100] At 5 days only BP produced a significant increase in Alcian
blue staining over controls. At 10 .mu.g/ml BP there was a 34%
increase over the control while at 20 .mu.g/ml there was a 23%
increase over the control. The difference between the averages of
10 and 20 .mu.g/ml BP was not significant.
[0101] At 8 days (FIG. 16a), both growth factors exhibited a
significant increase in Alcian blue staining over the control.
TGF.beta.1 at 10 ng/ml had a 42% increase over the control. BP had
a 60% increase at 10 .mu.g/ml and 66% increase at 20 .mu.g/ml over
the control.
EXAMPLE 15
Effect of Exogenous Growth Factors on Proteoglycan Synthesis in
Human Intervertebral Disc Nucleus Pulposus Cells
[0102] A second experiment to test the effects of TGF.beta.1 and BP
on proteoglycan synthesis was performed on a different human
patient from that described in EXAMPLE 14. Human intervertebral
disc cells obtained from another 40-year-old female patient were
cultured as described in EXAMPLES 8 and 9 and seeded into 24 well
plates. Growth factors were added after the cells were allowed to
adhere overnight. TGF.beta.1 was tested at a concentration of 10
ng/ml; BP was tested at 10 .mu.g/ml. After 6 and 9 days the cells
were fixed and the amount of proteoglycans synthesized was measured
by the Alcian blue assay as described in EXAMPLE 10.
[0103] At 6 days cells stimulated with 10 ng/ml TGF.beta.1 produced
54% more proteoglycans than control, and 10 .mu.g/ml BP increased
production by 104% over the control. At 9 days (FIG. 16b), 10 ng/ml
TGF.beta.1 increased production by 74% over controls, and 10
.mu.g/ml BP increased production by 171% over the control.
EXAMPLE 16
Effect of Exogenous Growth Factors on Proteoglycan Synthesis in
Baboon Intervertebral Disc Nucleus Pulposus Cells
[0104] TGF.beta.1 and BP were tested for their effects on
stimulation of proteoglycan synthesis in baboon nucleus cells.
Baboon intervertebral disc nucleus pulposus cells were obtained
from a 7 year old male baboon, cultured as described in EXAMPLES 8
and 9, and seeded into a 24 well plate. The cells were allowed to
adhere to the well surface before the addition of growth factors.
The concentrations of growth factors tested were 10 .mu.g/ml BP and
10 .mu.g/ml TGF.beta.1. The dilutions were prepared in TCM. The
cells were fixed after 4 and 8 days of continuous exposure to
growth factors, and proteoglycan synthesis was detected by the
Alcian blue assay as described in EXAMPLE 10.
[0105] At 4 days there was no significant increase in proteoglycan
synthesis between the different growth factors and the control. At
8 days (FIG. 17), TGF.beta.1 and BP significantly increased
proteoglycan synthesis over the control, but the increase was only
marginal. In particular, TGF.beta.1 produced a 21% increase over
the control while BP produced a 22% increase over the control.
EXAMPLE 17
Staining of Seeded Matrix Material with Phalloidin
[0106] Cross-linked matrix seeded with living cells was stained
with phalloidin to indicate the growth and proliferation of living
cells into the matrix. The media was rinsed from the matrix with 3
PBS washes of 5 min each. The matrix was fixed for 1 hour at room
temperature with 4% paraformaldehyde. The 4% paraformaldehyde was
washed off with 3 PBS rinses. The matrix was treated with 0.1%
Triton-X 100 for 3 min and then washed with 3 PBS rinses. The
matrix was then stained with phalloidin-conjugated rhodamine, made
up in PBS, for 45 min. Excess phalloidin was washed off with PBS.
The matrix was mounted on slides and viewed under fluorescence with
filter of .lamda. range 530-550 nm.
EXAMPLE 18
Growth and Proliferation of Sheep Intervertebral Disc Nucleus
Pulposus Cells into. Non-Homogenized Matrix with BP Growth
Factor
[0107] Ingrowth and proliferation of growth factor stimulated sheep
intervertebral disc nucleus pulposus cells into the matrix of the
present invention was investigated. Cross-linked matrix material
obtained prior to the lyophilization step of Example 1 was cut into
square pieces 75 mm on each side and sterilized in 70% ethanol for
3 hours. Remaining steps in the protocol were performed under
aseptic conditions.
[0108] Ethanol was removed from the matrix with two 1-hour washes
in sterile PBS, followed by a one hour wash in TCM. The matrix
pieces were then suspended overnight in TCM having BP
concentrations of 20 ng/ml and 20 .mu.g/ml. The control was
cross-linked matrix suspended in 20 .mu.g/ml BSA (bovine serum
albumin). Each matrix piece was then placed in a well of a 24 well
plate and seeded with TCM containing sheep intervertebral disc
nucleus cells at 40,000 cells/ml. The cells were allowed to grow
into the matrix and the TCM was changed every 2-3 days. Sample
matrix pieces were fixed at 3, 6 and 9 days and stained with
phalloidin as described in EXAMPLE 17. The process is illustrated
in FIG. 7.
[0109] Infiltration of sheep nucleus pulposus cells into the matrix
was observed at all of the 3, 6 and 9 day timepoints, indicating
that the matrix is biocompatible. The number of cells observed per
field was higher at 6 and 9 days, indicating that the cells were
proliferating into the matrix. More cells were observed in matrix
pieces that had been suspended in TCM containing BP than in
controls having no growth factor. BP at 20 .mu.g/ml produced the
greatest infiltration and proliferation of cells into the
matrix.
EXAMPLE 19
Growth and Proliferation of Sheep Intervertebral Disc Nucleus
Pulposus Cells into Homogenized Matrix with BP Growth Factor
[0110] A further investigation of the ingrowth and proliferation of
growth factor stimulated sheep intervertebral disc nucleus pulposus
cells into the matrix of the present invention was made using
homogenized matrix, as opposed to the non-homogenized matrix in
EXAMPLE 18. Cross-linked matrix material obtained prior to the
lyophilization step of Example 1 was homogenized using a tissue
homogenizer, and sterilized in 70% ethanol for 3 hours. All
subsequent steps in the protocol were under aseptic conditions.
[0111] The homogenized matrix was centrifuged at 3200 rpm for 10
min and the supernatant was discarded. The pelleted matrix was
rinsed with two 1-hour PBS washes, followed by a 1-hour TCM wash.
Between each wash the matrix was centrifuged, and the supernatant
was discarded. The pelleted matrix was then suspended overnight in
TCM having BP concentrations of 20 ng/ml and 20 .mu.g/ml. The
control was cross-linked matrix suspended in 20 .mu.g/ml BSA
[0112] The TCM/matrix mixture was then centrifuged and the
supernatant was discarded. The matrix pellet was resuspended in TCM
containing sheep intervertebral disc nucleus cells, obtained
according to the procedure in EXAMPLES 8 and 9. The matrix/cell
suspension was pipetted into wells of a 24 well plate. The TCM was
changed every 2-3 days. The homogenized matrix seeded with cells
was fixed at 4 days and stained with phalloidin as described in
EXAMPLE 17. The process is illustrated in FIG. 7.
[0113] After 4 days, the layer of cross-linked matrix soaked in 20
.mu.g/ml BP and seeded with cells had contracted to form a rounded
clump of compact tissue. This tissue was comprised of both the
original cross-linked matrix and the newly synthesized matrix
produced by the infiltrated cells. There were very few cells
adherent to the well surface, indicating that most cells had
infiltrated the matrix. This conclusion was reinforced by the dense
infiltration of cells into the matrix as visualized by phalloidin
staining. The cells had assumed a rounded morphology which is
characteristic of nucleus chondrocytic cells, indicating reversion
to their original morphology. Cells had also grown into matrix
soaked in 20 ng/ml BP by 4 days, but cell ingrowth was not as dense
as in the matrix soaked in 20 .mu.g/ml BP.
[0114] The control matrix suspended in BSA also had cells
infiltrating into it, but it was the least populated among the
different dilutions.
EXAMPLE 20
In Vivo Evaluation of Cross-Linked Matrix and Bone Protein (BP)
Growth Factor for Nucleus Pulposus Regeneration in an Ovine Lumbar
Spine Model
[0115] Pilot studies were conducted to evaluate preparative and
surgical methods for the implantation of the cross-linked matrix
containing BP growth factors into the intervertebral disc space of
the sheep lumbar spine, to evaluate whether implantation of the
matrix with growth factors arrests degeneration and/or stimulates
regeneration of nucleus pulposus in a sheep disc degeneration model
over a period of six months, and to assess the antibody- and
cell-mediated immune response in sheep to the matrix/BP
combination.
[0116] Study #1
[0117] One-half gram (0.5 g) of cross-linked, lyophilized and
pulverized matrix prepared as described in EXAMPLE 1 was rehydrated
and sterilized by two 4 hour rinses in 70% isopropanol. The matrix
was centrifuged and pelleted, and then rinsed in sterile PBS three
times for 2 hours each to remove the isopropanol. The rehydrated
matrix was again centrifuged and pelleted.
[0118] Bone Protein (BP) prepared according to U.S. Pat. Nos.
5,290,763 and 5,371,191 was obtained from Sulzer Biologics, Inc.
(Wheat Ridge, Colo.) in a lyophilized form. Two milligrams (2 mg)
of BP was suspended in 100 .mu.l dilute 0.01M hydrochloric acid to
produce a 20 mg/ml BP stock solution. The BP stock solution was
diluted to 100 .mu.g/ml in sheep serum and the BP/serum suspension
was sterile-filtered through a 0.2 micron filter. Next, 1.0 ml of
the sterile BP/serum suspension was added to 1.0 ml of the
rehydrated matrix described above to obtain a final concentration
of 50 .mu.g BP per ml of cross-linked, rehydrated matrix/serum
suspension. At the time of surgery, one aliquot (0.5 ml) of the
rehydrated matrix/BP/serum suspension was loaded into a sterile 3
ml pressure control syringe with an 18 or 20 gauge needle for
injection.
[0119] Three sheep were anesthetized and the dorsolateral lumbar
area prepared for surgery. Blood was drawn from each sheep
pre-operatively, centrifuged, and serum collected for immunology
studies. A ventrolateral, retroperitoneal approach was made through
the oblique abdominal muscles to the plane ventral to the
transverse processes of the lumbar spine. The annuli fibrosi of
intervertebral discs L3-4, L4-5, and L5-6 were located, soft
tissues retracted, and a discrete 5 mm deep by 5 mm long incision
was made into both L3-4 and L5-6 discs. The intervening, middle
L4-5 disc remained intact to serve as an intra-operative control.
Following annulus stab procedures, the musculature and subcutaneous
tissues were closed with absorbable suture. After postoperative
recovery, sheep were allowed free range in the pasture.
[0120] Two months after the annulus stab surgical procedures, the
sheep were operated upon a second time. After anesthesia and
preparation for surgery, the three operated lumbar spine levels
were again exposed. Two hundred microliters (200 .mu.l) of the
prepared test material (i.e., rehydrated matrix/BP/serum
suspension) was injected into the intradiscal space of one (L5-6)
of the experimentally-damaged discs. The second operated disc
(L3-4) served as a sham-treated degenerative disc; the syringe
needle punctured the annulus but no material was injected. After
disc treatments, the musculature and subcutaneous tissues were
closed with absorbable suture. Following postoperative recovery,
sheep were allowed free range of movement. The study design is
diagrammatically represented in FIG. 10.
[0121] The sheep were sacrificed at 2, 4, and 6 months after the
second surgery. The radiograph from the 2 month sheep showed a
degenerative appearance of the untreated disc but a normal
appearance in the control and treated discs (FIG. 11). Histological
analysis of the 2 month sheep as illustrated in FIG. 12 confirmed
extensive degeneration within the sham-treated, stab-induced
degenerative disc. In both the control disc and the
matrix/BP-treated disc, a normal sized gelatinous nucleus and
regular, compact annulus were observed. In the 4 month and 6 month
sheep, no obvious changes were seen in the radiograph of the three
discs. A radiograph of the 4 month sheep is shown in FIG. 13.
However, on gross dissection in the 4 month sheep, the sham-treated
disc exhibited obvious gross degeneration while the control and
treated discs were normal in appearance (FIG. 14). In the 6 month
sheep, there were no gross differences between the sham-treated,
control, and treated discs.
[0122] Although there was some variation in the rate of
degeneration using the annulus stab technique (i.e., the absence of
clear degeneration in the 6 month sheep), these results suggest
that the cross-linked matrix/BP treatment may protect against or
impede the progress of stab-induced degeneration in sheep
intervertebral discs.
[0123] Study #2
[0124] For the second study, matrix material was rehydrated and
combined with BP and serum to produce a matrix/BP/serum suspension
as described in Study #1.
[0125] Twelve sheep were anesthetized and the dorsolateral lumbar
area prepared for surgery. Blood was drawn from each sheep
pre-operatively, centrifuged, and serum collected for immunology
studies. A ventrolateral, retroperitoneal approach was made through
the oblique abdominal muscles to the plane ventral to the
transverse processes of the lumbar spine. The annuli fibrosi of
intervertebral discs L1-2, L2-3, L3-4, L4-5, and L5-6 were located,
soft tissues retracted, and a small diameter hole punched through
the annulus using a syringe needle in 4 of the 5 discs. A small
curette was then placed through the hole into the intradiscal space
to remove a discrete portion of nucleus pulposus from each of the
four discs in each sheep. In 2 of the 4 damaged discs, 0.5 ml of
the matrix/BP/serum suspension was injected into the intradiscal
spaces and the needle punctures were sealed off with ligament
sutured over them. The immediate injection of this suspension was
considered an "acute" treatment protocol. The 2 other damaged discs
were left untreated at that time but were sealed off with ligament
sutured over the needle punctures. The intervening, middle L3-4
disc remained intact in all sheep spines to serve as an
intra-operative control. Following these procedures, the
musculature and subcutaneous tissues were closed with absorbable
suture. After postoperative recovery, sheep were allowed free
range.
[0126] Six weeks after the first surgery to remove portions of the
nucleus pulposus, the sheep were operated upon a second time. After
anesthesia and preparation for surgery, the five operated lumbar
spine levels were again exposed. In one of the two remaining
nontreated discs which had been damaged six weeks before, 0.5
milliliters of the prepared test material (i.e., rehydrated
matrix/BP/serum suspension) was injected into the intradiscal space
of the disc. The injection of this suspension six weeks later into
a damaged disc was considered a "delayed" treatment protocol. The
second nontreated damaged disc served as a sham-treated
degenerative disc; the syringe needle punctured the annulus but no
material was injected. The treatment method used in each of the
four experimentally-damaged discs was randomized for location
within the spines. That is, except for the intact control disc
(L3-4), the locations of an "acute" treatment disc, a "delayed"
treatment disc, or a nontreated, damaged disc, were randomly
assigned to one of the four different lumbar disc levels. After
disc treatments, the musculature and subcutaneous tissues were
closed with absorbable suture. Following postoperative recovery,
sheep were allowed free range.
[0127] The sheep were sacrificed at 2, 4, and 6 months after
matrix/BP/serum injections and the spines were fixed for histology
in formalin. Cross-sections were taken from plastic-embedded discs,
stained with H & E and Saffranin-O, and evaluated for
chondrocyte proliferation (cloning), proteoglycan staining
intensity, level of fibrosis, and level of ossification. An
evaluation of the "acute" treatment discs, "delayed" treatment
discs, sham-treated, and control was made in a blinded fashion and
ranked +1, +2, or +3 (low, medium, or high) for each parameter
listed above. Semiquantitative evaluation of the histological
results was compared in 2 month, 4 month, and 6 month sheep for
both the "acute" and "delayed" (6 week) treatments.
[0128] The results demonstrated overall that injected matrix+BP
stimulated chondrocyte cloning and accumulation of Saffranin-O
staining of glycosaminoglycans in the nucleus matrix of damaged
discs. In particular, the extent of regenerative repair was much
greater in both "acute" treatment discs and "delayed" treatment
discs, compared to that observed in non-treated, damaged discs.
This greater level of repair in matrix/BP-treated discs was
statistically significant at the 0.01 level of confidence. There
was also less fibrosis and ossification seen in the acute and
delayed treatment discs compared to the non-treated discs.
[0129] A significant difference was also noted between the
"delayed" treatment discs and the "acute" treatment discs in the
level of proteoglycan staining. For example, Saffranin-O staining
as an index to proteoglycan synthesis and content in the nucleus
matrix was greater in the "delayed" matrix/BP-treatment discs than
in the "acute" matrix/BP-treatment discs. Additional benefits
apparent in the histological evaluation, which were associated with
"delayed" treatment with matrix/BP, were an overall lack of bony
transformation (ossification) or fibrous tissue accumulation
(fibrosis) within the treated discs compared to the non-treated,
damaged discs. In general, the results in Study #2 support and
elaborate earlier indications from Study #1 that treatment of
damaged discs with the cross-linked matrix/BP may protect against
or impede the progress of degeneration in experimentally-damaged
sheep intervertebral discs.
EXAMPLE 21
Characterization of BP
[0130] Specific growth factors present in the mixture of growth
factors produced according to U.S. Pat. Nos. 5,290,763, 5,371,191,
and 5,563,124 (i.e., BP) have been identified. BP has been
partially characterized as follows: HPLC fractions have been
denatured, reduced with DTT (dithiothreitol), and separated by
sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE). One minute high performance liquid chromatography
(HPLC) fractions taken at from 27 to 36 minutes are shown in FIG.
18. Size standards (ST) of 14, 21, 31, 45, 68 and 97 kDa were
obtained as Low Range size standards from BIORAD.TM. and are shown
at either end of the Coomassie blue stained gel (FIGS. 18 and 19).
In the usual protocol, HPLC fractions 29 through 34 are pooled to
produce BP (see box in FIGS. 18 and 19), as shown in a similarly
prepared SDS-PAGE gel in FIG. 33B.
[0131] An SDS-PAGE gel of BP was also analyzed by Western
immunoblot with a series of antibodies, as listed in FIG. 30.
Visualization of antibody reactivity was by horse radish peroxidase
conjugated to a second antibody and using a chemiluminescent
substrate. The reactivities are as indicated in FIG. 30.
[0132] The BP was further characterized by 2-D (two dimensional)
gel electrophoresis, as shown in FIGS. 21 and 22. The proteins are
separated in horizontal direction according to charge (pI) and in
the vertical direction by size according to the method of O'Farrell
et al. (Cell, 12:1133-1142, 1977). Internal standards, specifically
tropomyosin (33 kDa, pI 5.2) and lysozyme (14.4 kDa, pI 10.5-11.0),
are included and the 2-D gel was visualized by Coomassie blue
staining. FIG. 21 shows the stained 2-D gel with size standards
indicated on the left. Tropomyosin (left arrow) and lysozyme (right
arrow) are also indicated.
[0133] The same gel is shown in FIG. 22 with several identified
proteins indicated by numbered circles. The proteins were
identified by mass spectrometry and amino acid sequencing of
tryptic peptides, as described below. The identity of each of the
labeled circles is provided in the legend of FIG. 22.
[0134] The various components of the BP were characterized by mass
spectrometry and amino acid sequencing of tryptic fragments where
there were sufficient levels of protein for analysis. The major
bands in the 1-D (one dimensional) gels were excised, eluted,
subjected to tryptic digestion, purified by HPLC and sequenced by
methods known in the art. The major bands are identified by band
number, as shown in FIGS. 19 and 20. The sequence data was compared
against known sequences, and the fragments are identified as shown
in FIG. 31. In some cases, the identification is tentative due to
possible variation between the human and bovine sequences and/or
possible post translational modifications, as discussed below.
[0135] The same tryptic protein fragments were analyzed by mass
spectrometry and the mass spectrograms are shown in FIGS. 23A-23O.
The tabulated results are shown in the Table depicted in FIGS.
32A-32F, which provides identification information for each of the
indicated bands, as identified in FIGS. 19 and 20. As above,
assignment of band identity may be tentative based on species
differences and post translational modifications.
[0136] The identified components of BP were quantified as shown in
FIGS. 33A and 33B. FIG. 33B is a stained SDS-PAGE gel of BP and
FIG. 33A represents a scanning densitometer trace of the same gel.
The identified proteins were labeled and quantified by measuring
the area under the curve. These results are presented in FIG. 34 as
a percentage of the total peak area.
[0137] As FIG. 34 indicates, there are 11 major bands in the BP
SDS-PAGE gel representing about 60% of the protein in BP. Further,
TGF-.beta.1 was quantified using commercially pure TGF-.beta.1 as a
standard, and was determined to represent less than 1% of the BP
protein. The identified proteins fall roughly into three
categories: the ribosomal proteins, the histones, and growth
factors, including active growth factors comprising members of the
TGF-.beta. superfamily of growth factors, which includes the bone
morphogenic proteins (BMPs). It is believed that the ribosomal
proteins and histone proteins may be removed from the BP without
loss of activity, and the specific activity is expected to increase
correspondingly.
[0138] Because several of the proteins migrated at more than one
size (e.g., BMP-3 migrating as 5 bands) investigations were
undertaken to investigate the extent of post-translational
modification of the BP components. Phosphorylation was measured by
anti-phosphotyrosine immunoblot and by phosphatase studies. FIG. 24
shows a 2-D gel, electroblotted onto filter paper and probed with a
phosphotyrosine mouse monoclonal antibody by SIGMA (#A-5964).
Several proteins were thus shown to be phosphorylated at one or
more tyrosine residues.
[0139] Similar 2-D electroblots were probed with BP component
specific antibodies, as shown in FIGS. 25A-D. The filters were
probed with BMP2, BMP-3 (FIG. 25A), BMP-3, BMP-7 (FIG. 25B), BMP-7,
BMP-2 (FIG. 25C), and BMP-3 and TGF-.beta.1 (FIG. 25D). Each shows
the characteristic, single-size band migrating at varying pI, as is
typical of a protein existing in various phosphorylation
states.
[0140] Native and phosphatase treated BP samples were also assayed
for morphogenic activity by explant mass and ALP (alkaline
phosphatase) score. The results showed that AcP treatment reduces
the explant mass and ALP score from 100% to about 60%.
[0141] The BP was also analyzed for glycosylation. FIG. 26 shows an
SDS-PAGE gel stained with periodic acid schiff (PAS)--a
non-specific carbohydrate stain, indicating that several of the BP
components are glycosylated (starred protein identified as BMP-3).
FIGS. 27 and 28 show two specific proteins (BMP-7, FIG. 27 and
BMP-2, FIG. 28) treated with increasing levels of PNGase F
(Peptide-N-Glycosidase F), and immunostained with the appropriate
antibody. Both BMP-2 and BMP-7 show some degree of glycoslyation,
but appear to have some level of protein that is resistant to
PNGase F, as well (plus signs indicate increasing levels of
enzyme). Functional activity of PNGase F and sialadase treated
samples were assayed by explant mass and by ALP score, as shown in
FIG. 29A and 29B, indicating that glycosylation is required for
full activity.
[0142] In summary, BMPs 2, 3 and 7 are modified by phosphorylation
(.sup.-33%) and glycosylation (50%). These post-translation
modifications do affect protein morphogenic activity.
[0143] Matrix compositions useful in treating intervertebral disc
impairment in vertebrates, including humans, may be prepared
according to the foregoing descriptions and examples. While various
embodiments of the inventions have been described in detail,
modifications and adaptations of those embodiments will be apparent
to those of skill in the art in view of the present disclosure.
However, such modifications and adaptations are within the spirit
and scope of the present inventions, as set forth in the following
claims.
* * * * *