U.S. patent application number 12/058193 was filed with the patent office on 2008-12-04 for intervertebral disc repair, methods and devices therefor.
This patent application is currently assigned to ISTO TECHNOLOGIES, INC.. Invention is credited to H. Davis Adkisson, IV, Mitchell S. Seyedin, Robert Spiro.
Application Number | 20080299214 12/058193 |
Document ID | / |
Family ID | 34910797 |
Filed Date | 2008-12-04 |
United States Patent
Application |
20080299214 |
Kind Code |
A1 |
Seyedin; Mitchell S. ; et
al. |
December 4, 2008 |
Intervertebral Disc Repair, Methods and Devices Therefor
Abstract
The present application discloses compositions, methods and
devices for treatment of a degenerative intervertebral disc. A
composition can comprise chondrocytes expressing type II collagen.
These chondrocytes can be obtained from human cadavers up to about
two weeks following death, and can be grown in vitro. The
compositions can further comprise one or more biocompatible
molecules. Treatment of a degenerative disc can comprise injecting
or implanting a composition comprising the chondrocytes into a
degenerative disc.
Inventors: |
Seyedin; Mitchell S.; (Monte
Sereno, CA) ; Spiro; Robert; (Half Moon Bay, CA)
; Adkisson, IV; H. Davis; (St. Louis, MO) |
Correspondence
Address: |
POLSINELLI SHALTON FLANIGAN SUELTHAUS PC
700 W. 47TH STREET, SUITE 1000
KANSAS CITY
MO
64112-1802
US
|
Assignee: |
ISTO TECHNOLOGIES, INC.
St. Louis
MO
|
Family ID: |
34910797 |
Appl. No.: |
12/058193 |
Filed: |
March 28, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11063183 |
Feb 22, 2005 |
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12058193 |
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60546619 |
Feb 20, 2004 |
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Current U.S.
Class: |
424/548 |
Current CPC
Class: |
A61P 19/00 20180101;
A01K 2217/00 20130101; A01K 2227/10 20130101; A61K 35/12 20130101;
A01K 2267/03 20130101; A61K 35/32 20130101; A01K 2207/15 20130101;
A61K 38/4833 20130101; C12N 2533/56 20130101; A61K 38/39 20130101;
C12N 5/0655 20130101 |
Class at
Publication: |
424/548 |
International
Class: |
A61K 35/32 20060101
A61K035/32; A61P 19/00 20060101 A61P019/00 |
Claims
1-35. (canceled)
36. A method for treating intervertebral disc degeneration in a
subject in need thereof, the method comprising: providing a hyaline
cartilage composition comprising non-intervertebral cartilage
tissue and a first biocompatible molecule; combining the hyaline
cartilage composition with a second composition comprising a second
biocompatible molecule to form a third composition; injecting the
third composition into an intervertebral disc of the subject.
37. A method according to claim 36 wherein the first biocompatible
molecule is fibrinogen and the second biocompatible molecule is
thrombin.
38. A method according to claim 36 wherein the first biocompatible
molecule is thrombin and the second biocompatible molecule is
fibrinogen.
39. A method according to claim 36 wherein a treatment provider
combines the hyaline cartilage composition with the second
composition to form the third composition immediately prior to
injecting the third composition into the intervertebral disc of the
subject.
40. A method according to claim 36 wherein a treatment provider
combines the hyaline cartilage composition with the second
composition to form the third composition in conjunction with
injecting the third composition into the intervertebral disc of the
subject.
41. A method according to claim 36 wherein the non-intervertebral
cartilage tissue comprises cadaver cartilage.
42. A method for treating a degenerative intervertebral disc, the
method comprising: injecting into the intervertebral disc: a
cartilage composition comprising non-intervertebral cartilage
tissue; an amount of a first biocompatible molecule; and an amount
of a second biocompatible molecule; wherein when injected into the
intervertebral disc, contact between the amount of the first
biocompatible molecule and the amount of the second biocompatible
molecule is sufficient to form a tissue sealant at the injection
site.
43. A method according to claim 42 wherein the first biocompatible
molecule comprises thrombin, the second biocompatible molecule
comprises fibrinogen, and the tissue sealant comprises fibrin.
44. A method according to claim 42 wherein the first biocompatible
molecule comprises fibrinogen, the second biocompatible molecule
comprises thrombin, and the tissue sealant comprises fibrin.
45. A method according to claim 42 wherein injecting into the
intervertebral disc the cartilage composition and the amount of the
second biocompatible molecule comprises combining the cartilage
composition and the amount of the second biocompatible molecule
immediately prior to injecting.
46. A method according to claim 42 wherein injecting into the
intervertebral disc the cartilage composition and the amount of the
second biocompatible molecule comprises injecting the cartilage
composition in conjunction with injecting the amount of the second
biocompatible molecule.
47. A kit for treating a degenerative intervertebral disc, the kit
comprising: a suspension of non-intervertebral cartilage tissue; an
amount of a first biocompatible molecule packaged separately from
the cartilage tissue suspension; and an amount of a second
biocompatible molecule packaged separately from the cartilage
tissue suspension and the amount of the first biocompatible
molecule.
48. A method according to claim 47 wherein the first biocompatible
molecule is fibrinogen and the second biocompatible molecule is
thrombin.
49. A method according to claim 47 wherein the first biocompatible
molecule is thrombin and the second biocompatible molecule is
fibrinogen.
50. A kit for treating a degenerative intervertebral disc, the kit
comprising: a suspension of non-intervertebral cartilage tissue
comprising an amount of a first biocompatible molecule; and an
amount of a second biocompatible molecule packaged separately from
the cartilage tissue suspension comprising the first biocompatible
molecule.
51. A method according to claim 50 wherein the first biocompatible
molecule is fibrinogen and the second biocompatible molecule is
thrombin.
52. A method according to claim 50 wherein the first biocompatible
molecule is thrombin and the second biocompatible molecule is
fibrinogen.
53. A method for treating a degenerative intervertebral disc, the
method comprising: providing the treatment provider with at least
two injectable compositions, the injectable compositions
comprising: a cartilage component comprising non-intervertebral
cartilage tissue; an amount of a first biocompatible molecule; and
an amount of a second biocompatible molecule packaged separately
from the injectable composition comprising the first biocompatible
molecule; wherein when injected into the intervertebral disc,
contact between the amount of the first biocompatible molecule and
the amount of the second biocompatible molecule is sufficient to
form a tissue sealant at the injection site.
54. A method according to claim 53 wherein the first biocompatible
molecule comprises thrombin, the second biocompatible molecule
comprises fibrinogen, and the tissue sealant comprises fibrin.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuing application claiming the
benefit of priority from U.S. patent application Ser. No.
11/063,183 filed Feb. 22, 2005, which claims priority from U.S.
Provisional Application Ser. No. 60/706,133 filed on Aug. 4, 2005,
the disclosures of which are incorporated herein by reference in
their entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not Applicable.
BACKGROUND OF THE INVENTION
[0003] Intervertebral disc degeneration is a leading cause of pain
and disability in the adult population. Approximately 80% of the
population experience at least a single episode of significant back
pain in their lifetimes. For many individuals, spinal disorders
become a lifelong affliction. The morbidity associated with disc
degeneration and its spectrum of associated spinal disorders is
responsible for significant health care, economic and social costs.
Furthermore, changes in disc morphology, such as disc compression
associated with aging, can lead to unwanted changes in height or
posture. Current treatments for repairing or ameliorating disc
degeneration, such as spinal fusion, can be expensive, painful, or
lengthy. Alternative treatments are, therefore, needed.
BRIEF SUMMARY OF THE INVENTION
[0004] In view of the need for disc degeneration treatments, the
present inventors have devised compositions, methods and devices
for repair, replacement and/or supplementation of an intervertebral
disc which involve implantation or injection of chondrocytes into a
degenerative disc, as well as compositions and methods for
providing chondrocytes to a treatment provider.
[0005] Some embodiments of the present teachings include methods of
repairing a degenerative intervertebral disc in a human patient in
need of treatment. In these embodiments, a method can comprise
implanting, into the intervertebral disc, chondrocytes obtained
from a cadaver. The cadaver chondrocytes can be from any
cartilaginous tissue of the cadaver, provided the chondrocytes
express type II collagen. Furthermore, the chondrocytes expressing
type II collagen can be chondrocytes expressing high molecular
weight sulfated proteoglycan (HSPG). The chondrocytes can be, for
example, hyaline cartilage chondrocytes. In various configurations,
the chondrocytes can be chondrocytes from one or more
intervertebral discs, or the chondrocytes can be non-intervertebral
disc chondrocytes. Chondrocytes from an intervertebral disc can be
chondrocytes from the annulus of a disc, chondrocytes from the
nucleus pulposus of a disc, or a combination thereof. Non-limiting
examples of non-intervertebral disc tissue which can be sources of
chondrocytes include cartilage of the nose, ears, trachea and
larynx, as well as articular cartilage, costal cartilage, cartilage
of an epiphyseal plate, and combinations thereof. In various
aspects of the present teachings, the chondrocytes can be extracted
from a cadaver at any time following death while the chondrocytes
remain viable. In various configurations, chondrocytes can be
extracted from a cadaver up to about fourteen days following death.
Chondrocytes can be removed from a cadaver from about one hour
following death to about fourteen days following death, from
greater than 24 hours following death to about thirteen days
following death, from about two days following death to about
twelve days following death, from about three days following death
to about twelve days following death, or from about four days
following death to about ten days following death.
[0006] In some embodiments, chondrocytes of the present teachings
can be chondrocytes extracted from a cadaver of any chronological
age at time of death. In various configurations, chondrocytes can
be extracted from a cadaver which is no older than about 40 years
of age at time of death, no older than about 30 years of age at
time of death, no older than about 20 years of age at time of
death, or no older than about 10 years of age at time of death. A
donor cadaver need not be a familial member of a recipient, or be
otherwise matched immunologically.
[0007] In various embodiments, chondrocytes which are extracted
from a cadaver can be grown in vitro prior to their implantation or
injection into a recipient patient or purveyance to a treatment
provider. Growth of chondrocytes in vitro can be used, for example,
to increase the number of chondrocytes available for implantation
or injection. In non-limiting example, chondrocyte numbers can be
increased about two fold or greater, about ten fold or greater, or
about twenty fold or greater. In various configurations, growing
chondrocytes in vitro can comprise placing one or more cartilage
tissue pieces removed from a cadaver into a tissue culture or cell
culture medium which comprises nutrients, buffers, salts, proteins,
vitamins and/or growth factors which promote chondrocyte growth,
and incubating the chondrocytes. In certain configurations, tissue
comprising chondrocytes expressing type II collagen can be
dissociated into single cells or small groups of cells prior to, or
in conjunction with, their introduction into a culture medium. In
addition, in some aspects, in vitro culture of chondrocytes
expressing type II collagen can further comprise removing
non-chondrocyte cells from a cell- or tissue-culture.
[0008] In various embodiments of the present teachings,
chondrocytes expressing type II collagen can be comprised by a
composition which can be implanted or injected into an
intervertebral disc of a patient in need of treatment. Accordingly,
in certain embodiments, the present teachings also include
compositions comprising cadaver chondrocytes expressing type II
collagen for use in implantation or injection into a degenerative
intervertebral disc of a patient in need of treatment. In some
configurations of these embodiments, the chondrocytes of these
compositions can comprise chondrocytes expressing high molecular
weight sulfated proteoglycan. In some configurations, a composition
comprising chondrocytes expressing type II collagen can further
comprise at least one biocompatible molecule. Non-limiting examples
of biocompatible molecules which can be comprised by a composition
of the present teachings include fibrinogen, fibrin, thrombin, type
I collagen, type II collagen, type III collagen, fibronectin,
laminin, hyaluronic acid (HA), hydrogel, pegylated hydrogel,
chitosan, and combinations thereof.
[0009] In various embodiments, the present teachings include
methods of forming a composition comprising cadaver chondrocytes. A
composition formed by these methods can further comprise one or
more biocompatible molecules such as those described supra.
Accordingly, methods of these embodiments can comprise contacting
cadaver chondrocytes expressing type II collagen with one or more
biocompatible molecules, such as, for example, fibrinogen, fibrin,
thrombin, type I collagen, type II collagen, type III collagen,
fibronectin, laminin, hyaluronic acid, hydrogel, pegylated
hydrogel, chitosan and combinations thereof. The cadaver
chondrocytes expressing type II collagen can be, in some
configurations, chondrocytes which also express high molecular
weight sulfated proteoglycan. In certain aspects, the chondrocytes
can be incubated in vitro in a culture medium prior to the
contacting with one or more biocompatible molecules.
[0010] In various embodiments, the present teachings include
methods of forming a composition comprising cadaver tissue
comprising chondrocytes. A composition formed by these methods can
further comprise one or more biocompatible molecules such as those
described supra. Accordingly, methods of these embodiments can
comprise contacting cadaver tissue comprising chondrocytes
expressing type II collagen with one or more biocompatible
molecules, such as, for example, fibrinogen, fibrin, thrombin, type
I collagen, type II collagen, type III collagen, fibronectin,
laminin, hyaluronic acid, hydrogel, pegylated hydrogel, chitosan
and combinations thereof. The cadaver chondrocytes expressing type
II collagen can be, in some configurations of these embodiments,
chondrocytes which also express high molecular weight sulfated
proteoglycan. In certain aspects of these embodiments, cadaver
tissue comprising chondrocytes expressing type II collagen can be
incubated in vitro in a culture medium prior to the contacting with
one or more biocompatible molecules.
[0011] In various aspects of the present teachings, a composition
comprising both cadaver chondrocytes expressing type II collagen
and one or more biocompatible molecules can be implanted or
injected into a degenerative intervertebral disc in a patient in
need of treatment. In various aspects, implantation or injection of
a composition into a disc can comprise implantation or injection of
the composition into the annulus of the disc, implantation or
injection of the composition into the nucleus pulposus of the disc,
implantation or injection of the composition into one or both
endplates of the disc, or a combination thereof. In some
configurations, an aperture can be formed in an annulus of a
degenerative disc, and a composition can be introduced into the
disc through the aperture. In some configurations, surgical
techniques such as vertebroplasty and kyphoplasty (Garfin, S. R.,
et al., Spine 26: 1511-1515, 2001) can be adapted or modified for
introducing chondrocytes into a degenerative disc of a patient.
[0012] In various embodiments, the present teachings include an
apparatus configured for injection of chondrocytes expressing type
II collagen to an intervertebral disc of a patient in need of
treatment. An apparatus configured for injection of chondrocytes
expressing type II collagen into an intervertebral disc can
comprise chondrocytes expressing type II collagen. Chondrocytes of
these embodiments can comprise chondrocytes expressing high
molecular weight sulfated proteoglycan. In various configurations,
the apparatus can comprise a composition which comprises the
chondrocytes and at least one biocompatible molecule, such as, for
example, a biocompatible molecule described supra. In certain
embodiments, the chondrocytes expressing type II collagen comprised
by the apparatus can be cadaver chondrocytes. The cadaver
chondrocytes in these embodiments can be intervertebral disc
chondrocytes, or non-intervertebral disc chondrocytes, such as
those described supra. In some configurations of these embodiments,
the chondrocytes can be comprised by cadaver tissue. An apparatus
of the present teachings can further comprise, in some
configurations, a syringe, a double syringe, a hollow tube, such as
a hollow needle (for example, a Jamshidi needle), a cannula, a
catheter, a trocar, a stylet, an obturator, or other instruments,
needles or probes for cell or tissue injection, injection, or
transfer known to skilled artisans. In certain configurations, the
apparatus can be configured for injection of chondrocytes
expressing type II collagen into a nucleus pulposus of an
intervertebral disc, an annulus of an intervertebral disc, an
endplate of an intervertebral disc or a combination thereof.
[0013] In various embodiments of the present teachings, methods are
provided for purveying to a treatment provider chondrocytes for
repairing a degenerative intervertebral disc in a patient in need
thereof. In various aspects, a method of these embodiments can
comprise growing cadaver chondrocytes expressing type II collagen
in vitro, and delivering the chondrocytes expressing type II
collagen to the treatment provider. Chondrocytes expressing type II
collagen of these embodiments can be, in some configurations,
chondrocytes which also express high molecular weight sulfated
proteoglycan. Methods of these embodiments can further comprise
obtaining chondrocytes expressing type II collagen from a cadaver.
In these embodiments, the cadaver chondrocytes expressing type II
collagen can be obtained at various time intervals following death
of the donor as described supra. Furthermore, a donor cadaver of
chondrocytes expressing type II collagen can be of an age at time
of death as described supra. The chondrocytes of these embodiments
can be chondrocytes of tissue sources such as those described
supra.
[0014] In some configurations of these methods, the chondrocytes
expressing type II collagen can be purveyed to a treatment provider
along with one or more biocompatible molecules, such as those
described supra. In some configurations, a composition comprising
the chondrocytes and one or more biocompatible molecules can be
purveyed to a treatment provider. In other configurations, the
chondrocytes and the one or more biocompatible molecules can be
purveyed separately to a treatment provider (either simultaneously
or at different times), and the treatment provider can form a
composition comprising the chondrocytes and the one or more
biocompatible molecules prior to, or in conjunction with,
implanting the composition in a patient in need thereof.
[0015] In various embodiments, the teachings of the present
application also disclose use of cadaver chondrocytes expressing
type II collagen for the production of a composition for repairing
a degenerative intervertebral disc in a patient in need thereof. In
some configurations of these embodiments, the chondrocytes can also
express high molecular weight sulfated proteoglycan. In certain
configurations of these embodiments, the chondrocytes can be
cadaver chondrocytes which are grown in vitro, as described supra.
A composition of these embodiments can comprise a composition
comprising cadaver chondrocytes expressing type II collagen and one
or more biocompatible molecules such as those described supra. In
addition, the time interval following death at which the
chondrocytes can be removed from a donor can be a time interval as
described supra, and the age of a donor cadaver at time of death
can be an age as described supra. In some aspects of these
embodiments, the chondrocytes can include chondrocytes removed from
an annulus, chondrocytes removed from a nucleus pulposus,
chondrocytes removed from an endplate of an intervertebral disc of
a donor cadaver, or a combination thereof. In some other aspects of
these embodiments, the chondrocytes can be chondrocytes removed
from other cartilaginous, non-intervertebral disc tissue of a
cadaver, such as, for example, hyaline cartilage from the nose,
ears, trachea or larynx, as well as articular cartilage, costal
cartilage, cartilage of an epiphyseal plate, or combinations
thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] These and other features, aspects, and advantages of the
present invention will become better understood with regard to the
following description, appended claims and accompanying figures
where:
[0017] FIG. 1 illustrates a normal intervertebral disc (left) and a
herniated disc (right),
[0018] FIG. 2 illustrates freshly isolated, juvenile cartilage
tissue that has been dissected to small cubes and implanted into a
damaged nucleus pulposus region of an intervertebral disc in a
composition which can also comprise a biocompatible molecule.
[0019] FIG. 3 illustrates isolated juvenile chondrocytes, freshly
isolated or harvested from expanded in vitro cultures which can be
implanted into the nucleus pulposus region of an intervertebral
disc in a composition which can also comprise a biocompatible
molecule.
[0020] FIG. 4 illustrates the gross appearance of an intact
unoperated disc harvested from the lumbar region of an adult
canine.
[0021] FIG. 5 illustrates an intact nucleus pulposus and the
cartilaginous endplate of the disc shown in FIG. 4.
[0022] FIG. 6 illustrates a section of an intervertebral disk that
was treated with human chondrocytes 12 weeks post-injection.
[0023] FIG. 7 represents the highlighted region of FIG. 6,
illustrating the newly synthesized matrix that has replaced the
native nucleus 12 weeks after chondrocyte injection.
[0024] FIG. 8 illustrates the gross appearance of discs 12 weeks
after chondrocyte injection.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present teachings describe compositions, methods and
devices for repair, replacement and/or supplementation of a
degenerative intervertebral disc. These methods can involve
implantation or injection of chondrocytes into a degenerative disc.
In addition, the present teachings also describe methods for
providing chondrocytes to a treatment provider.
[0026] As used herein, the terms "degenerative intervertebral disc"
and "degenerative disc" refer to an intervertebral disc exhibiting
disease symptoms, abnormalities or malformations, including but not
limited to herniations, disruptions, traumatic injuries, and
morphological changes associated with or attributed to aging.
Indications of a degenerative intervertebral disc can include, but
are not limited to, brittleness of an annulus, tearing of an
annulus, and shrinking of a nucleus pulposus.
[0027] In various embodiments, the present teaching include methods
of repairing a degenerative disc in a human patient in need of
treatment. Methods of these embodiments can comprise implanting or
injecting into the intervertebral disc a composition comprising
cadaver chondrocytes. As used herein, the term "cadaver
chondrocytes" refers to viable chondrocytes originally comprised by
a human cadaver, as well as clonal descendants of such
chondrocytes, such as chondrocytes grown in vitro. Cadaver
chondrocytes for use in the various aspects of the present
teachings can be obtained from tissues comprising chondrocytes from
a cadaver, such as cartilage tissue. Such tissues can be dissected
from a cadaver using standard dissection methods well known to
skilled artisans. The cartilage tissue utilized in the present
teachings can comprise hyaline cartilage, such as cartilage of the
nose, ears, trachea and larynx, articular cartilage, costal
cartilage, cartilage of an epiphyseal plate, and combinations
thereof. In various aspects, the cartilage tissue or chondrocytes
can be intervertebral disc cartilage or chondrocytes, or can be
cartilage or chondrocytes originating from cartilaginous tissues
other than intervertebral disc tissue (herein referred to as
"non-intervertebral disc chondrocytes"). Viable chondrocytes can be
comprised by cartilaginous tissues in a donor cadaver for up to
about two weeks after death of the donor. Accordingly, in some
configurations, the time interval from the time of death of a donor
(as determined, for example, by a physician or a coroner) to the
time of dissection of cartilage tissue from the donor can be any
time from about immediately following a pronouncement of death, to
about two weeks following death, such as, without limitation, about
one hour, greater than 24 hours, about two days, about three days,
about four days, about five days, about six days, about seven days,
about eight days, about nine days about ten days, about eleven
days, about twelve days, about thirteen days, or about fourteen
days after death. In addition, a donor cadaver can be of any
chronological age at time of death. For example, a donor cadaver
can be, at time of death, ten years old or younger, twenty years
old or younger, thirty years old or younger, or forty years old or
younger. A donor cadaver need not be a familial member of a
recipient, or be otherwise matched immunologically. Without being
limited by theory, it is believed that intervertebral cartilage
comprises an "immunologically privileged" tissue, so that
chondrocytes transplanted to an intervertebral disk are not subject
to rejection by the recipient's immune system.
[0028] Cartilage tissue can be removed from a cadaver using any
surgical or dissecting techniques and tools known to skilled
artisans. Following cartilage removal from a cadaver, the cartilage
tissue can be minced, dissociated into single cells or small groups
of cells, and/or placed into tissue or cell culture using standard
techniques and apparatuses well known to skilled artisans, such as
techniques and apparatuses described in the these references.
Non-limiting descriptions of methods of cartilage and chondrocyte
removal and culture can be found in references such as, for
example, Feder, J. et al. in: Tissue Engineering in Musculoskeletal
Clinical Practice. American Academy of Orthopaedic Surgeons, 2004;
Adkisson, H. D. et al., Clin. Orthop. 391S:S280-S294, 2001; and
U.S. Pat. Nos. 6,235,316 and 6,645,316 to Adkisson.
[0029] Cadaver chondrocytes used in the various embodiments of the
present teachings are all cadaver chondrocytes which express type
II collagen. In addition, in some aspects, cadaver chondrocytes can
comprise chondrocytes expressing other molecular markers such as a
high molecular weight sulfated proteoglycan, such as, for example,
chondroitin sulfate (Kato, Y., and Gospodarowicz, D., J. Cell Biol.
100: 477-485. 1985). Presence of such markers can be determined
using materials and methods well known to skilled artisans, such
as, for example, antibody detection and histological staining.
[0030] In some configurations, cadaver chondrocytes or cartilage,
including cartilage tissue as well as cells, either directly
extracted from a cadaver grown in vitro, can be harvested prior to
implantation or injection into a patient using cell culture
techniques and apparatuses well known to skilled artisans, such as
culture methods for neocartilage described in U.S. Pat. Nos.
6,235,316 and 6,645,316 to Adkisson, and other general laboratory
manuals on cell culture such as Sambrook, J. et al., Molecular
Cloning: a Laboratory Manual (Third Edition), Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 2001; and Spector, D.
L., et al., Culture and Biochemical Analysis of Cells, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1998. In vitro
culture of cadaver chondrocytes can be used to increase numbers of
chondrocytes which can be implanted into a patient. In addition,
routine laboratory measures known to skilled artisans can be used
to detect and remove non-chondrocyte cells from a cell culture, or
to test a culture for the presence of biological contaminants such
as microorganisms and viruses. Primary cultures established
starting from cadaver chondrocytes can be grown as long as the
chondrocytes remain viable and maintain their normal in vitro
histological properties.
[0031] Various configurations of the present teachings include
compositions comprising chondrocytes and one or more biocompatible
molecules. These biocompatible molecules can include molecules that
enhance survival an/or integration of implanted chondrocytes or
cartilaginous tissues into an intervertebral disc. Examples of such
molecules include, without limitation, fibrinogen, fibrin,
thrombin, type I collagen, type II collagen, type III collagen,
fibronectin, laminin, hyaluronic acid, hydrogel, pegylated
hydrogel, chitosan, and combinations thereof. Various commercial
formulations comprising such molecules, such as, for example,
Tisseel.RTM. fibrin glue (Baxter Healthcare Corporation, Westlake
Village, Calif.) can comprise a composition of the present
teachings. Accordingly, a composition of the present teachings can
comprise, in non-limiting example, chondrocytes grown in culture
and Tisseel.RTM. fibrin glue.
[0032] In various methods of the present teachings, cadaver
chondrocytes, including but not limited to cadaver chondrocytes
grown in vitro and cartilage tissue maintained in tissue culture in
vitro, can be implanted or injected into an intervertebral disc of
a recipient patient using surgical methods and apparatuses known to
skilled artisans but adapted for such use. In various
configurations, chondrocytes or cartilage of the present teachings
can be implanted or injected into an annulus of a degenerative
intervertebral disc, a nucleus pulposus of an intervertebral disc,
one or both endplates of a degenerative intervertebral disc, or a
combination thereof. In certain aspects, an aperture can be
introduced into the annulus of an intervertebral disc. The aperture
can provide a path for introducing chondrocytes or cartilage tissue
into a disc.
[0033] In various configurations, cells or tissue can be placed
into an apparatus or device configured for transfer of chondrocytes
to or from an intervertebral disc patient, such as, in non-limiting
example a biopsy instrument or transplantation instrument
comprising a hollow tube or needle, a syringe, a double syringe, a
hollow tube, a hollow needle such as a Jamshidi needle, a Cook
needle (Cook incorporated, Bloomington, Ind. USA), a cannula, a
catheter, a trocar, a stylet, an obturator, or other instruments,
needles or probes for cell or tissue injection, injection, or
transfer known to skilled artisans. Accordingly, an apparatus of
the present teachings can comprise cadaver chondrocytes as
described above, as well as at least one hollow needle or tube
through which the chondrocytes can be introduced into an
intervertebral disc of a patient. In some configurations, the
apparatus comprises a composition comprising the chondrocytes as
well as at least one biocompatible molecule as described supra.
These apparatuses can be configured for implanting or injecting
chondrocytes into an annulus, a nucleus pulposus, and/or an
endplate of a degenerative disc. Furthermore, surgical techniques
such as vertebroplasty and kyphoplasty (Garfin, S. R., et al.,
Spine 26: 1511-1515, 2001) can be adapted for introduction of
chondrocytes into a degenerative disc of a patient. In non-limiting
example, an instrument for such as a bone tamp/balloon can be
inserted into a degenerative intervertebral disc, and used to
create or expand a space or cavity within a degenerative disc, for
example in the nucleus pulposus of the disc. The balloon can be
removed, and chondrocytes expressing type II collagen can then be
injected into the expanded space, for example through a
catheter.
[0034] In any embodiment the one or more biocompatible molecules
may form a chondro-conductive/inductive matrix, carrier, or milieu
that comprises, for example, fibrinogen. The fibrinogen may be from
any suitable source. For example, one skilled in the art will
recognize that fibrinogen may be derived from blood bank
products--either heterologous (pooled or single-donor) or
autologous cryoprecipitate or fresh frozen plasma. Fibrinogen can
also be derived from autologous fresh or platelet-rich plasma,
obtained using cell-saver or other techniques. U.S. Pat. No.
5,834,420 discloses a method for obtaining fibrinogen.
[0035] In any embodiment the one or more biocompatible molecules
may comprise thrombin. The thrombin may be from any suitable
source. One skilled in the art will recognize that thrombin can be
isolated by well known means or purchased commercially. See U.S.
Pat. No. 4,965,203 and Berliner, J L (Ed), THROMBIN: STRUCTURE AND
FUNCTION, Plenum Pub Corp. (1992) for exemplary methods of
isolation and/or purification.
[0036] In any embodiment the one or more biocompatible molecules
may comprise collagen, hyaluronic acid, fibrinogen, thrombin or any
combination thereof. The biocompatible molecules in combination may
contain equal proportions of each constituent molecule or more of
any one or group of the constituents than other(s). When used in
combination the collagen, hyaluronic acid, fibrinogen and thrombin
may be in any proportion, ranging from one part of one, or one part
of a combination of two or more of them compared to the amount of
the other(s), up to equal proportions of each constituent.
[0037] When practicing any embodiment of the invention that
comprises collagen, hyaluronic acid, fibrinogen, thrombin or any
combination thereof, the fibrinogen and thrombin components should
be kept separate prior to the time of use. A common type of
applicator that may be used for this purpose consists of a double
syringe, joined by a Y-connector where the components mix as they
emerge. This type of applicator, used with a blunt cannula, is
useful for combining of the collagen, hyaluronic acid, thrombin and
the fibrinogen and also useful in the methods of the invention for
disposing or transplanting the inventive compositions to a site
wherein repair, replacement or supplementation of an intervertebral
disc is desired.
[0038] In various embodiments the one or more biocompatible
molecules may comprise one or more of collagen, hyaluronic acid,
fibrinogen, thrombin, fibrinogen/thrombin (Tisseel), Albumin,
in-situ forming PEG hydrogel, fibrin, hyaluronate,
fibrin/hyaluronate, collagen/hyaluronate,
fibrin/collagen/hyaluronate, PEG/hyaluronate, PEG/collagen, PEG
base sealants (CoSeal) or other plasma and protein-based adhesives
and/or sealants other natural adhesives and/or sealants and
combinations thereof that are biocompatible with regard to the
intervertebral nucleus pulposus repair, replacement or
supplementation.
[0039] In use in the method embodiments of the invention, the
composition may be disposed in an intervertebral disc in need of
repair, replacement or supplementation by being injected through
the disc annulus into the center of the damaged disc nucleus. As
the delivery needle, trochanter or portal is withdrawn, additional
material may usefully be injected into the track created in the
annulus. In all cases it is expected that the hyaline cartilage
completely fills the nucleus space and fully integrates with the
annulus. It is further presumed that the hyaline cartilage
comprises of neocartilage or juvenile cartilage and/or cells will
also be useful in repairing damaged annulus.
[0040] In various embodiments and configurations, the present
teachings also disclose methods of purveying to a treatment
provider chondrocytes for repairing a degenerative intervertebral
disc in a patient in need thereof. These methods can comprise
obtaining chondrocytes from a cadaver, growing the chondrocytes in
vitro, then delivering the chondrocytes to the treatment provider.
The chondrocytes can be obtained from a cadaver using methods
described supra, and can be chondrocytes which are adapted for
injection into a degenerative intervertebral disc in a patient. The
adaptation can comprise, in various configurations, expanding the
numbers of chondrocytes through growth in vitro. Chondrocytes
adapted for injection can also comprise, in certain aspects,
chondrocytes which can be loosely connected or unattached to each
other, and can be chondrocytes not comprised by cartilage tissue.
The cadaver chondrocytes of these embodiments can be chondrocytes
expressing type II collagen, as described supra, and can also be
chondrocytes expressing high molecular weight sulfated
proteoglycan, also as described supra. The chondrocytes can be
delivered to a treatment provider, as either a chondrocytes grown
in vitro and/or as cartilage tissue pieces as described supra. The
treatment provider can be, in non-limiting example, a physician
such as an orthopedic surgeon, or an agent or employee of the
physician or a health care institution such as a hospital or
outpatient clinic. Accordingly, in non-limiting example, cadaver
chondrocytes can be grown in vitro, and delivered to the treatment
provider via a delivery service such as, for example, a courier or
an overnight shipper. Cadaver chondrocytes and/or cartilage tissue
can be prepared for delivering by methods well known to skilled
artisans. In some configurations, cadaver chondrocytes and/or
cartilage tissue can be provided in a composition further
comprising at least one biocompatible molecule as described supra.
In alternative configurations, the chondrocytes and/or cartilage
tissue can be packaged and sent separately from any biomolecule(s).
The treatment provider can then form the composition by mixing the
cells with the one or more biomolecules. In some aspects, the
mixing can be done immediately prior to implanting the cells into a
recipient patient.
EXAMPLE
[0041] The following example illustrates transplantation and
survival of human chondrocytes transplanted into canine
intervertebral disc tissue. In this example, a pilot animal study
was conducted to determine whether human articular chondrocytes
survive injection to produce cartilaginous matrices in experimental
defects created in the intervertebral disk of adult canines. Gross
morphologic and histological results obtained from this short-term
pilot study (12 weeks) demonstrate that implanted chondrocytes can
survive to produce cartilaginous matrices which integrate with
surrounding host tissues.
[0042] Surgical Procedure: Prior to induction of anesthesia, six
adult female dogs were sedated by the attending veterinarian or the
veterinary technician/anesthetist using one of the following
combinations: Atropine 0.05 mg/kg IM with or without Acepromizine
0.05-0.2 mg/kg IM. An 18 or 20 gauge 11/4 to 2 inch angio-catheter
was placed in the cephalic saphenous or auricular vein for venous
access. General anesthesia was induced with Pentothal (10-20 mg/kg
IV to effect). Animals were intubated with a 7.0 mm-9.0 mm hi-low
pressure cuff endotracheal tube. Anesthesia was maintained with
isoflurane 2.5-4% in an air-oxygen mixture of 40-60%. The tube was
connected to low-pressure continuous suction, and mechanical
ventilation was initiated and maintained at 10 ml/kg tidal volume
and at a rate of 8-10/minute. Crystalloids were provided at a rate
of 7-10 mg/kg/hr.
[0043] Surgical exposure consisted of a 10 cm incision along the
abdominal midline, followed by soft tissue dissection to permit
transperitoneal exposure of the anterior lumbar spine.
[0044] Blunt dissection using a Cobb elevator and electrocautery
was performed as needed to expose the anterior aspects of the
L3-L4, L5-L6, and L7-S1 intervertebral disc space. Surgical defects
(1.times.3 mm) were created through the annulus into the disc
nucleus using a 16 gauge biopsy needle (Jamshidi needle) and
aspiration. A significant volume of the nucleus was removed in
concert with the annulus.
[0045] Human Neocartilage produced at ISTO Technologies according
to U.S. Pat. Nos. 6,235,316 and 6,645,764 were enzymatically
dissociated in HL-1 Serum-free Medium (Cambrex Bio Science,
Walkersville, Md.) containing 60 units/ml CLS4 collagenase
(Worthington, Lakewood, N.J.) and 50 units/mL hyaluronidase (Sigma,
St. Louis, Mo.). The dissociated chondrocytes (derived from the
articular cartilage of a six year old individual) were washed in
fresh HL-1 medium and briefly exposed to 0.25% EDTA before
pelleting at 500.times.g for 7 minutes. The cells were counted and
stored in sterile cryovials until use. Chondrocyte viability was
estimated to be greater than 90% by trypan blue exclusion. Six
tubes were prepared each containing 2 million chondrocytes. The
cells were then pelleted and the supernate removed. These samples
were hand carried to the operating room on wet ice. Once defects
were created, a chondrocyte suspension was prepared using 100
microliters of thrombin solution (Tisseel.RTM., Baxter Healthcare
Corporation, Westlake Village, Calif.). This step was completed
immediately before mixing with an equivalent volume of the fibrin
component (Tisseel.RTM.) using the Tisseel.RTM. injection device.
150-200 microliters of the cell suspension was injected into the
intervertebral disk closest to the dog's tail (L7-S1 and L5-L6),
whereas the highest vertebral level to be treated (L3-L4 or L4-L5)
was filled with 100-150 microliters of cells or cell carrier. The
cell suspension was injected at the base of the defect through a
needle and withdrawn during expulsion until it began to spill out
of the injection site, forming a solid gel. Two thirds of the
control defects were left untreated (33%) or received fibrin
carrier alone (33%). The final one-third of operated defects was
treated with cells suspended in fibrin carrier as described above.
Treatment at each of the levels was randomized to control for
variability in disc size and location.
[0046] Following the surgical procedure, the fascia and underlying
muscles were closed in an interrupted fashion using -0- Prolene and
the skin approximated using Vicryl.RTM. (Ethicon, Inc. Somerville,
N.J. USA) and Vetbond.TM. tissue adhesive (3M, St. Paul, Minn.
USA). Blood loss, operative times and both intra- and
peri-operative complications were recorded. Observations of
ambulatory activities and wound healing were monitored daily, and
all animals received analgesics after surgery.
[0047] Post-operative Care: After recovery from anesthesia, each
dog was returned to its cage and housed singly for observation
(daily) by veterinary technicians for any sign of adverse events
related to surgery. Buprinorphine (0.01-0.02 mg/k IM or SC) was
administered for relief of pain every 12 hours for the first 24
hours and prn thereafter. In general, the animals were pain free
after 24 hrs.
[0048] Animal Harvest and Sample Collection: Dogs were sacrificed
12 weeks after surgery by overdose with euthanasia solution. Spines
were removed, keeping the upper lumbar and sacral region intact.
Musculoskeletal tissue was removed by dissection to expose the
vertebral bodies for further sectioning using a band saw. Gross
observation of the defects was performed using digital photography
and the samples were immediately fixed in 10% neutral buffered
formalin (Fisher Scientific, Fairlawn, N.J.) for 48 hrs. Samples
were subsequently decalcified in 10% disodium EDTA (Sigma-Aldrich
Co., St. Louis, Mo.) after four washes in PBS to remove formalin.
Samples were then dehydrated in a graded alcohol series and
processed using standard paraffin embedding.
[0049] Five micron sections were cut and stained with hematoxylin
and eosin as well as safraninO for microscopic evaluation of the
cartilaginous tissue present in control and operated intervertebral
disks. Discs that were not exposed to the surgical procedures were
used to establish normal histological features of the canine
intervertebral disk.
[0050] Results: In general, the dogs handled the surgical procedure
well, and all of the abdominal wounds healed rapidly without
infection. There appeared to be no detrimental effect of multiple
surgical procedures (operation at three vertebral levels in each
animal) on the activity level of all dogs.
[0051] Gross macroscopic observation of the dissected vertebrae
revealed normal disc structure in those discs that were not
subjected to surgical intervention (FIG. 4). A glistening
gelatinous center, corresponding to the nucleus pulposus, was
identifiable in every case. Histological analysis revealed normal
disc morphology in which the concentric rings of the annulus were
observed to contain lower sulfated glycosaminoglycan content
(fibrocartilaginous tissue) than the nucleus pulposus (NP) and the
cartilage end plates (hyaline tissue), suggesting that surgical
intervention at an adjacent level did not alter the morphological
features of a disc that was not part of the procedure (FIG. 5).
[0052] Those discs receiving neocartilage chondrocytes in fibrin
glue were observed to contain viable chondrocytes in the disc
space, and the injected chondrocytes had synthesized a hyaline
matrix enriched in sulfated proteoglycan (FIGS. 6 and 7). Gross
macroscopic observation of treated discs show viable cartilaginous
tissue occupying the disc space (FIGS. 8A and B).
[0053] These results indicate that fibrin delivery to the disc
space of chondrocytes derived from juvenile articular was
successful and that the nature of newly synthesized tissue produced
by the implanted chondrocytes appeared to be cartilaginous as
determined by SafraninO staining. Most importantly, there was no
histological evidence of lymphocytic infiltration into the
operative site 12 weeks post-injection, suggesting that there was
no immunologic rejection.
[0054] FIG. 4 illustrates the gross appearance of an intact
unoperated disc harvested from the lumbar region of an adult
canine. The disc is split in half to show the morphology of a
normal intervertebral disk. The annulus fibrosus is the outer
fibrocartilaginous structure surrounding the inner jelly-like
structure or nucleus pulposus (NP). The cartilage endplate covers
the surface of the upper and lower vertebral body.
[0055] FIG. 5 illustrates an intact nucleus pulposus and the
cartilaginous endplate of the disc shown in FIG. 4. The section was
stained with Safranin O to identify sulfated glycosaminoglycans in
the NP and in the cartilage end plate. Notice that the NP
chondrocytes are significantly larger than chondrocytes of the
cartilage endplate and that the endplate contains greater levels of
sulfated proteoglycan. Original magnification 100.times.
[0056] FIG. 6 illustrates a Safranin O-stained section of an
intervertebral disk that was treated with human chondrocytes 12
weeks post-injection. The chondrocytes are viable and have
synthesized a cartilaginous matrix that is highly enriched in
sulfated glycosaminoglycans. The injected chondrocytes are much
smaller than native NP chondrocytes identified in FIG. 5. The white
square identifies the region shown in FIG. 7. Original
magnification 40.times..
[0057] FIG. 7 represents the highlighted region of FIG. 6,
illustrating the newly synthesized matrix that has replaced the
native nucleus 12 weeks after chondrocyte injection. The new matrix
appears to be integrated well with the surrounding native tissues.
Chondrocytes in this newly synthesized matrix (identified with
white dotted circles) appear to be randomly distributed and of
similar size to chondrocytes of the cartilaginous endplate.
Original magnification 100.times.
[0058] FIG. 8 is in two parts. Panel A illustrates the gross
appearance of a disc 12 weeks after chondrocyte injection. The
native nucleus is no longer present and is replaced by newly
synthesized cartilaginous tissue. Panel B illustrates the gross
appearance of another disc treated in the same manner. The
histological features of this disc are shown in FIGS. 6 and 7. The
newly synthesized cartilaginous material produced after chondrocyte
injection is expected to remodel and take on morphological features
that are more characteristic of the native annulus and nucleus
within 1 year after treatment.
[0059] It is to be understood that the present invention has been
described in detail by way of illustration and example in order to
acquaint others skilled in the art with the invention, its
principles, and its practical application. Particular formulations
and processes of the present invention are not limited to the
descriptions of the specific embodiments presented, but rather the
descriptions and examples should be viewed in terms of the claims
that follow and their equivalents. While some of the examples and
descriptions above include some conclusions about the way the
invention may function, the inventors do not intend to be bound by
those conclusions and functions, but put them forth only as
possible explanations.
[0060] It is to be further understood that the specific embodiments
of the present invention as set forth are not intended as being
exhaustive or limiting of the invention, and that many
alternatives, modifications, and variations will be apparent to
those of ordinary skill in the art in light of the foregoing
examples and detailed description. Accordingly, this invention is
intended to embrace all such alternatives, modifications, and
variations that fall within the spirit and scope of the following
claims.
[0061] All publications, patents, patent applications and other
references cited in this application are herein incorporated by
reference in their entirety as if each individual publication,
patent, patent application or other reference were specifically and
individually indicated to be incorporated by reference.
* * * * *