U.S. patent application number 10/312655 was filed with the patent office on 2004-04-29 for intervertebral disc.
Invention is credited to Kandel, Rita.
Application Number | 20040083001 10/312655 |
Document ID | / |
Family ID | 22801316 |
Filed Date | 2004-04-29 |
United States Patent
Application |
20040083001 |
Kind Code |
A1 |
Kandel, Rita |
April 29, 2004 |
Intervertebral disc
Abstract
The present invention relates to an engineered biological
material comprising or enriched for tissue of intervertebral disc;
tissue derived from an engineered biological material; constructs
comprising one or more tissues from an engineered biological
material; methods for producing the engineered biological materials
and constructs; and methods of using the engineered biological
materials and constructs.
Inventors: |
Kandel, Rita; (Toronto,
CA) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Family ID: |
22801316 |
Appl. No.: |
10/312655 |
Filed: |
August 4, 2003 |
PCT Filed: |
June 29, 2001 |
PCT NO: |
PCT/CA01/00972 |
Current U.S.
Class: |
623/17.16 ;
623/914 |
Current CPC
Class: |
A61F 2002/445 20130101;
A61L 2430/38 20130101; A61F 2/28 20130101; A61F 2002/30677
20130101; A61P 17/00 20180101; A61F 2002/4445 20130101; A61F
2310/00359 20130101; A61L 27/3817 20130101; A61F 2002/4435
20130101; A61L 27/3633 20130101; A61L 27/3612 20130101; A61L
27/3856 20130101 |
Class at
Publication: |
623/017.16 ;
623/914 |
International
Class: |
A61F 002/44 |
Claims
We claim:
1. An engineered biological material comprising one or more tissues
of intervertebral disc.
2. An engineered biological material as claimed in claim 1
characterized by a continuous layer of nucleus pulposus tissue.
3. An engineered biological material as claimed in claim 1
characterized by a continuous layer of annulus fibrosus tissue.
4. An engineered biological material as claimed in claim 1, 2, or 3
further characterized by the presence of extracellular matrix,
large proteoglycans and collagen.
5. An engineered biological material as claimed in claim 2 or 4
wherein the continuous layer of nucleus pulposus tissue is on a
substrate.
6. An engineered biological material as claimed in claim 3 or 4
wherein the continuous layer of annulus fibrosus tissue is on a
substrate.
7. An engineered biological material as claimed in claim 5 or 6
wherein the substrate is selected from the group consisting of
bone, an engineered biomaterial, preferably an engineered bone
substitute, and porous tissue culture inserts.
8. Nucleus pulposus tissue obtained from an engineered biological
material of claim 2 or 5.
9. Annulus fibrosus tissue obtained from an engineered biological
material of claim 3 or 6.
10. An intervertebral disc construct comprising nucleus pulposus
tissue as claimed in claim 8 or annulus fibrosus tissue as claimed
in claim 9 fused to a substrate.
11. An intervertebral disc construct comprising nucleus pulposus
tissue as claimed in claim 8 fused to a continuous layer of
cartilage tissue on a substrate.
12. An intervertebral disc construct as claimed in claim 11 wherein
the nucleus pulposus tissue is surrounded by a continuous layer of
annulus fibrosus tissue.
13. A process for producing an engineered biological material as
claimed in claim 1 comprising: (a) isolating nucleus pulposus cells
or annulus fibrosus cells from intervertebral disc; (b) forming a
layer of the nucleus pulposus cells or annulus fibrosus cells on a
substrate, and; (c) culturing the nucleus pulposus cells or annulus
fibrosus cells in growth media under suitable conditions so that
the nucleus pulposus cells or annulus fibrosus cells accumulate
extracellular matrix and form a continuous layer of nucleus
pulposus tissue or annulus fibrosus tissue.
14. A process for producing an engineered biological material as
claimed in claim 1 comprising: (a) isolating nucleus pulposus cells
and annulus fibrosus cells from intervertebral disc; (b) forming a
layer of the nucleus pulposus cells surrounded by the annulus
fibrosus cells on a substrate; and (c) culturing the nucleus
pulposus cells and annulus fibrosus cells in growth media under
suitable conditions so that the nucleus pulposus cells and annulus
fibrosus cells accumulate extracellular matrix and form nucleus
pulposus tissue surrounded by annulus fibrosus tissue.
15. A process as claimed in claim 13 or 14 wherein the substrate is
selected from the group consisting of bone, an engineered
biomaterial preferably an engineered bone substitute, and porous
tissue culture inserts.
16. An engineered biological material or intervertebral disc
construct as claimed in any preceding claim wherein cells of the
nucleus pulposus tissue or annulus fibrosus tissue are transformed
with recombinant vectors containing an exogenous gene encoding a
biologically active protein that corrects or compensates for a
genetic deficiency, or stimulates cell growth or stimulates
extracellular matrix production by cells, or encodes a drug.
17. A system for testing a substance that affects nucleus pulposus
cells or annulus fibrosus cells comprising: (a) generating or
culturing an engineered biological material, or construct as
claimed in any preceding claim in the presence of a substance which
is suspected of affecting nucleus pulposus tissue or annulus
fibrous tissue; and (b) determining the biochemical composition
and/or physiological organization of tissue generated or cultured
with the biochemical composition and/or physiological organization
of the biological material or construct generated or cultured in
the absence of the substance.
18. A method of using an engineered biological material or
construct as claimed in any preceding claim to test pharmaceutical
preparations for efficacy in the treatment of diseases of
intervertebral disc.
19. A method of replacing or repairing damaged or deficient
intervertebral discs or portions thereof of a patient comprising
implanting an engineered biological material, nucleus pulposus
tissue, annulus fibrosus tissue, or construct as claimed in any
preceding claim into the site of the damaged or deficient
intervertebral disc or portions thereof, of the patient.
20. A method for enhancing healing of an intervertebral disc in a
patient which comprises inserting an engineered biological
material, nucleus pulposus tissue, annulus fibrosus tissue, or
construct as claimed in any preceding claim into the site of a
damaged intervertebral disc.
21. A method for repairing damaged or degenerated intervertebral
discs comprising evacuating tissue from a nucleus pulposus portion
of a degenerated intervertebral disc space, preparing an engineered
biological material in accordance with a process as claimed in
claim 13 using nucleus pulposus cells from the evacuated tissue,
and implanting the engineered biological material in the space
where the tissue was evacuated.
22. A method for investigating the metabolism or degeneration of
nucleus pulposus or annulus fibrosus cells or tissue using an
engineered biological material or construct as claimed in any
preceding claim as an in vitro model.
23. A gene therapy method comprising using an engineered biological
material or construct as claimed in any preceding claim.
24. Use of an engineered biological material, nucleus pulposus
tissue, annulus fibrosus tissue, or construct as claimed in any
preceding claim as an implant to replace or repair damaged or
deficient intervertebral disc.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to an engineered biological
material comprising tissue of intervertebral disc; constructs
comprising one or more engineered biological materials; methods for
producing the biological materials and constructs; and methods of
using the biological materials or constructs.
BACKGROUND OF THE INVENTION
[0002] The human spine consists of thirty-three vertebral bodies of
which the distal nine are fused to form the sacrum and coccyx
(Simon, S R, et al 1994; Bogduk, N., 1997). The 24 vertebrae, with
the exception of C1 and C2, are each separated by an intervertebral
disc (IVD). The IVD anchors adjacent vertebral bodies and by doing
so allows for spinal stabilization, load bearing, and movement. The
intervertebral disc is a specialized structure consisting of two
interdependent tissues, the annulus fibrosus (AF) and the nucleus
pulposus (NP) which merge with the the cartilage endplate (Bogduk,
N., 1997; Eyre, D R, 1979). The composition of the AF and nucleus
pulposus varies with anatomical site in the tissue and the age of
the individual (Eyre, D R, 1979; Buckwalter, 1995). The normal
function of the disc is dependent on maintenance of the
composition, organization, and integrity of the different
components (Chiba, 1998). The annulus fibrosus is responsible for
withstanding circumferential tensile forces while the nucleus
pulposus resists compressive forces during normal activity (Simon,
1994; Bogduk, 1997; Eyre, 1979; Buckwalter, 1995). The disc is
relatively avascular as only the outer portion of the annulus
contains blood vessels in adults (Buckwalter, 1995). The disc cells
rely on diffusion of nutrients from these vessels and from blood
vessels in the vertebral body.
[0003] The annulus fibrosus surrounds the nucleus pulposus and
consists of approximately 10-20 lamellar sheets each composed of
collagen fibres oriented parallel to each other and about
65.degree. from the vertical. Although the angle is the same, the
direction of the inclination alternates with each sheet such that
the fibres in one lamella are 65.degree. to the right, while in the
next lamella they are 65.degree. to the left. Every second lamella
has the same orientation (Bogduk, N., 1997). This very specific
collagen organization allows the disc to rotate and flex. Collagen
makes up about 70% of the dry weight of the annulus (Buckwalter,
1995). Type I collagen is the predominate collagen but types II,
III, V, VI and type IX collagen are also present in lesser amounts
(Bogduk, N., 1997; Buckwalter, 1995; Nerlich, 1998). The average
diameter of the collagen fibril is 50-60 nm as determined by
transmission electron microscopy (Eyre, 1979). The annulus also
contains a small amount of proteoglycans and these also have a
specific distribution. The proteoglycan content in the tissue is
lowest in the periphery of the annulus fibrosus and increases in
amount towards the nucleus pulposus (Bogduk, N., 1997). The major
proteoglycan is aggrecan (Bogduk, N., 1997; Inerot, 1991; Roberts,
1994; Antoniou, 1996; and Sztrolovics, 1997). Small proteoglycans
such as decorin, biglycan, fibromodulin are also present (Gotz,
1997; Sztrolovics, 1999). Elastin and other non-collagenous
proteins are detected in the disc (Bogduk, 1997). The cellularity
across the annulus varies, as it is more cellular in the outer
third (0.7 ug DNA/gm dry weight) when compared to the inner
two-thirds (0.1 ug DNA/gm dry weight) of the annulus (Bayliss,
1998).
[0004] The nucleus pulposus (NP) is gelatinous type tissue, which
is surrounded by the annulus fibrosus and confined by the
cartilaginous endplates of the vertebral bodies (Bogduk, 1997). It
consists of proteoglycans within a loose network of collagen and
does not show the same degree of collagen organization in the
matrix as the annulus fibrosus (Eyre, 1979; Aguiar, 1999).
Proteoglycans comprise approximately up to 65% of the dry weight of
the nucleus. Aggrecan is the major proteoglycan present in the
nucleus pulposus and about 60% of it is present in a form that does
not aggregate. Other proteoglycans, such as decorin, biglycan, and
fibromodulin, are also present (Buckwalter, 1995; Gotz, 1997;
Sztrolovics, 1999; and Oegema, 1993). The nucleus pulposus contains
predominantly type II collagen but there are other collagen types
present, such as III, VI, IX and XI (Eyre, 1979; Buckwalter, 1995;
Aulisa, 1998). Type I collagen has been detected in small amounts
in the nucleus pulposus of humans (Eyre, 1979) and rats (Rufai,
1995). The average diameter of the collagen fibrils is around 30 nm
as determined by transmission electron microscopy (Eyre, 1979). In
childhood, the nucleus contains notochordal cells but these
disappear with age. It has been postulated that these cells
contribute to the maintenance of the nucleus pulposus and their
absence in the adult disc explains the high prevalence of disc
degeneration (Aguiar, 1999).
[0005] The other component of the disc is the cartilage endplate, a
thin layer of articular cartilage that is integrated with the
underlying bone of the vertebral body (Bogduk, 1997). As the
endplate covers a portion of the vertebral body it confines the
nucleus pulposus entirely but only a portion of the annulus
fibrosus. The peripheral portion of the annulus fibrosus inserts
directly into the bone. The endplate is considered part of the disc
as it can easily be separated from the vertebral body (Bogduk,
1997). Like articular cartilage, the endplate consists
predominantly of water, proteoglycans and collagen (Bogduk, 1997;
Antoniou, 1996). The zone of tissue closer to the bone is richer in
collagen as compared to the zone closer to the nucleus pulposus,
which contains less collagen and more proteoglycans and water
(Bogduk, 1997).
[0006] Intervertebral disc prolapse is a very common problem and
currently there is no optimal treatment for persistent disease. In
an autopsy study, 97% of individuals 50 years or older showed disc
degeneration (Miller, 1988). It is not known why it is so common,
but may be due in part to the relative avascularity of the tissue
until there is prolapse (Ozaki, 1999), mechanical factors
(Hadjipavlou, 1999), the absence of notochordal cells (Aguiar, 199)
or genetic factors (Kawaguchi, 1999). The back pain that can
develop as a result of this disease is often self-limited, but a
percentage of affected individuals require surgery (Kraemer, 1994;
Borenstein, 1999). Although the surgical intervention may relieve
pain faster, this procedure does not restore disc height or its
original load bearing capacity. Post-discotomy syndrome, which is
characterized by persistent pain and occurs after disc surgery, may
be treated by spinal fusion. This is a less than optimal treatment
as it is not always successful and results in limited flexibility
and degenerative changes in adjacent vertebrae (Javedan, 1999).
[0007] IVD replacement, with allografts or prosthetic devices, has
been attempted but met with limited success (Leivseth, 1999; Hou,
1991; Enker 1993; Bao, 1996; and Kostiuk, 1997). Alternative
treatments such as laser treatment are now being studied (Zeegers,
1999). Intraspinal injection of chymopapain has been used; however,
a recent study of 51 patients showed that this treatment had no
effect (Choy, 1998). Although not used currently, gene therapy may
be another way to treat this disease (Nishida, 1998; Nishida, 1999;
and Evans & Robbins, 1999). There is clearly a need to develop
novel approaches to the treatment f disc disease.
SUMMARY OF THE INVENTION
[0008] The present inventor has produced an engineered biological
material comprising or enriched for one or more tissues of
intervertebral disc.
[0009] In particular, the present inventor has produced an
engineered biological material comprising a continuous layer of
nucleus pulposus tissue. The tissue formed in vitro was
characterized and compared to in vivo nucleus pulposus tissue and
it was found to mimic the organization of nucleus pulposus tissue
in vivo. In particular, the accumulation of sulfated proteoglycans
in the nucleus pulposus tissue continued up to 10 weeks and this
was paralleled by an increase in tissue thickness and dry weight.
DNA content decreased over time. The amount of DNA and
proteoglycans per mg dry weight of the tissue generated in 10 weeks
old cultures were substantially the same as for the in vivo tissue.
There was no significant difference between in vitro and in vivo
tissues. The cells in culture synthesized large proteoglycans which
were similar in size to those synthesized by cells in nucleus
pulposus tissue explant culture as determined by Sepharaose CL-2B
column chromatography. The cells also synthesized type II
collagen.
[0010] Therefore, in one aspect, the invention relates to an
engineered biological material comprising a continuous layer of
nucleus pulposus tissue. In an embodiment, an engineered biological
material is provided comprising in combination a substrate and a
continuous layer of nucleus pulposus tissue on the substrate.
[0011] In an embodiment, the invention provides an engineered
biological material comprising in combination nucleus pulposus
tissue and a substrate for the nucleus pulposus tissue, the nucleus
pulposus tissue being reconstituted on the substrate in vitro from
isolated nucleus pulposus cells and being a continuous layer
comprising nucleus pulposus cells and an extracellular matric
containing sulfated proteoglycans and type II collagen.
[0012] In another aspect, the invention relates to an engineered
biological material comprising a continuous layer of annulus
fibrosus tissue. In an embodiment the biological material comprises
in combination a substrate and a continuous layer of annulus
fibrosus tissue on the substrate.
[0013] In an embodiment, the invention provides an engineered
biological material comprising in combination annulus fibrosus
tissue and a substrate for the annulus fibrosus tissue, the annulus
fibrosus tissue being reconstituted on the substrate in vitro from
isolated annulus fibrosus cells and being a continuous layer
comprising annulus fibrosus cells and extracellular matrix.
[0014] In another aspect of the invention an engineered biological
material is provided comprising a continuous layer of nucleus
pulposus tissue surrounded by annulus fibrosus tissue.
[0015] The invention also relates to nucleus pulposus tissue and/or
annulus fibrosus tissue derived from the engineered biological
materials of the invention.
[0016] Still further the invention contemplates an intervertebral
disc construct comprising nucleus pulposus or annulus fibrosus
tissue derived from a biological material of the invention fused to
a substrate (e.g. gone substitute). A construct is also provided
comprising nucleus pulposus tissue derived from a biological
material of the invention fused to a continuous layer of cartilage
tissue on a substrate. The nucleus pulposus tissue may be
surrounded by a continuous layer of annulus fibrosus tissue.
[0017] The invention also relates to a process for producing an
engineered biological material of the invention comprising
isolating nucleus pulposus cells or annulus fibrosus cells from
intervertebral disc; forming a layer of the nucleus pulposus cells
or annulus fibrosus cells on a substrate, and; culturing the
nucleus pulposus cells or annulus fibrosus cells in growth media
under suitable conditions so that the nucleus pulposus cells or
annulus fibrosus cells accumulate extracellular matrix and form
nucleus pulposus tissue or annulus fibrosus tissue,
respectively.
[0018] The invention also provides a process for producing an
engineered biological material of the invention comprising
isolating nucleus pulposus cells and annulus fibrosus cells from
intervertebral disc; forming a layer of the nucleus pulposus cells
surrounded by the annulus fibrosus cells on a substrate, and;
culturing the nucleus pulposus cells and annulus fibrosus cells in
growth media under suitable conditions so that the nucleus pulposus
cells and annulus fibrosus cells accumulate extracellular matrix
and form nucleus pulposus tissue surrounded by annulus fibrosus
tissue.
[0019] In an embodiment the substrate is selected from the group
consisting of bone, an engineered biomaterial preferably an
engineered bone substitute, and porous tissue culture inserts.
[0020] The nucleus pulposus cells or annulus fibrosus cells in the
engineered biological materials or constructs of the invention may
be transformed with recombinant vectors containing an exogenous
gene encoding a biologically active protein that corrects or
compensates for a genetic deficiency, or stimulates cell growth or
stimulates extracellular matrix production by cells, or
alternatively, encoding a drug. Therefore, the invention also
contemplates an engineered biological material or construct of the
invention wherein nucleus pulposus cells or annulus fibrosus cells
in the biological material or construct are transformed with
recombinant vectors containing an exogenous gene encoding a
biologically active protein which can correct or compensate for a
genetic deficiency or have a stimulatory effect, or encoding a
drug.
[0021] The invention still further relates to a system for testing
a substance or agent that affects nucleus pulposus tissue or
annulus fibrosus tissue comprising: generating or culturing a
biolgoical material, or construct of the invention in the presence
of a substance or agent which is suspected of affecting nucleus
pulposus tissue or annulus fibrosus tissue, and determining the
biochemical composition and/or physiological organization of tissue
generated or cultured, with the biochemical composition and/or
physiological organization of the biological material or construct
generated or cultured in the absence of the substance or agent.
[0022] The invention still further relates to a method of using the
biological materials and constructs of the invention to test
pharmaceutical preparations for efficacy in the treatment of
diseases of intervertebral disc.
[0023] Still another aspect of the present invention provides a
method of conducting a drug discovery business comprising:
[0024] (a) identifying agents that affect the biochemical
composition and/or physiological organization of an engineered
biological material of the invention;
[0025] (b) conducting therapeutic profiling of agents identified in
step (a), or further analogs thereof, for efficacy and toxicity in
animals; and
[0026] (c) formulating a pharmaceutical preparation including one
or more agents identified in step (b) as having an acceptable
therapeutic profile.
[0027] In certain embodiments, the subject method can also include
a step of establishing a distribution system for distributing the
pharmaceutical preparation for sale, and may optionally include
establishing a sales group for marketing the pharmaceutical
preparation.
[0028] Yet another aspect of the invention provides a method of
conducting a target discovery business comprising:
[0029] (a) providing one or more engineered biological materials
for identifying agents by their ability to affect the biochemical
composition and/or physiological organization of the engineered
biological material;
[0030] (b) (optionally) conducting therapeutic profiling of agents
identified in step (a) for efficacy and toxicity in animals;
and
[0031] (c) licensing, to a third party, the rights for further drug
development and/or sales for agents identified in step (a), or
analogs thereof.
[0032] The invention provides methods of using an engineered
biological material or tissues obtained therefrom or construct of
the present invention as an implant to replace or repair damaged or
deficient intervertebral disc, and methods for repairing damaged or
degenerated intervertebral discs. Methods of the invention may be
used to treat vertebrates suffering from degenerated intervertebral
disc conditions, and in particular to treat humans with such
conditions.
[0033] Therefore, the invention contemplates a method of replacing
or repairing damaged or deficient intervertebral discs or portions
thereof (preferably nucleus pulposus) of a patient comprising
implanting an engineered biological material (or tissue therefrom)
or construct of the invention into the site of the damaged or
deficient intervertebral disc of the patient. Methods for enhancing
healing of an intervertebral disc in a patient are contemplated
which comprise inserting a biological material (or tissue
therefrom) or construct of the invention into the site of a damaged
intervertebral disc.
[0034] In an embodiment, the invention provides a method for
replacing or repairing a degenerated or damaged nucleus pulposus
tissue of an intervertebral disc comprising implanting in the disc
space, after the removal of the degenerated or damaged nucleus
pulposus tissue, an engineered biological material of the invention
comprising a continuous layer of nucleus pulposus tissue or nucleus
pulposus tissue obtained therefrom.
[0035] In an embodiment, the invention provides a method for
replacing or repairing a degenerated or damaged annulus fibrosus
tissue of an intervertebral disc comprising implanting in the disc
space, after the removal of the degenerated or damaged annulus
fibrosus tissue, an engineered biological material of the invention
comprising a continuous layer of annulus fibrosus tissue or annulus
fibrosus tissue obtained therefrom.
[0036] In an aspect of the invention, a method for repairing
damaged or degenerated intervertebral disc is provided comprising
evacuating tissue from the nucleus pulposus portion of a
degenerated intervertebral disc space, preparing a biological
material of the invention using nucleus pulposus cells from the
vacuated tissue, and implanting the biological material in the
evacuated nucleus pulposus space.
[0037] In another aspect of the invention, a method for repairing
damaged or degenerated intervertebral discs is provided comprising
evacuating tissue from the annulus fibrosus portion of a
degenerated intervertebral disc space, preparing a biological
material of the invention using annulus fibrosus cells from the
evacuated tissue, and implanting the biological material in the
evacuated annulus fibrosus space.
[0038] The invention also contemplates methods for using the
biological materials and constructs of the invention in gene
therapy.
[0039] A biological material or construct of the invention can be
used as an in vitro model for investigating the metabolism and
degeneration of nucleus pulposus or annulus fibrosus cells and
tissues.
[0040] These and other aspects of the present invention will become
evident upon reference to the following detailed description and
attached drawings.
DESCRIPTION OF THE DRAWINGS
[0041] The invention will be better understood with reference to
the drawings in which:
[0042] FIG. 1A is a photomicrograph of toluidine blue-stained
section of 10 wk old formalin-fixed, paraffin-embedded nucleus
pulposus cell cultures showing the formation of nucleus pulposus
tissue. The filter is still present.
[0043] FIG. 1B is a photomicrograph of in vitro formed 8 week old
nucleus pulposus tissue (toluidine blue, magnification
.times.600).
[0044] FIG. 2 shows the determination of culture tissue thickness
over time. The cultures were harvested, paraffin-embedded and
histological sections were used to measure the nucleus pulposus
tissue thickness. The results are expressed as mean.+-.SEM of three
separate experiments and each time point was done in
triplicate.
[0045] FIG. 3 shows the determination of culture tissue dry weight
over time. The cultures were harvested, lyophilized and weighed.
The results are expressed as mean.+-.SEM of three separate
experiments and each time point was done in triplicate.
[0046] FIG. 4 shows the measurement of DNA content of the nucleus
pulposus tissue in culture over time. The results are expressed as
mean.+-.SEM of three separate experiments and each time point was
done in triplicate.
[0047] FIG. 5 shows the proteoglycan content of the nucleus
pulposus tissue in culture over time which was determined by
measuring glycosaminoglycan content. The results are expressed as
mean.+-.SEM of three separate experiments and each time point was
done in triplicate.
[0048] FIG. 6 shows proteoglycan elution profiles of newly
synthesized proteoglycans extracted from 10 week old nucleus
pulposus cultures (.cndot.-.cndot.) and of proteoglycans extracted
from nucleus pulposus ex-vivo cultures (o-o). The size (K.sub.av)
of newly synthesized proteoglycan monomers was determined by
gel
[0049]
[0050] The present invention relates to an engineered biological
material comprising a continuous layer f nucleus pulposus tissue.
The tissue is characterized by nucleus pulposus cells that
synthesize large sulfated proteoglycans and type II collagen
characteristic of nucleus pulposus cells in vivo. The tissue in the
biological material is also characterized by becoming less cellular
with age. The nucleus pulposus tissue is further characterized by
having a three dimensional organization that is characteristic of
nucleus pulposus tissue in vivo.
[0051] In an embodiment, the engineered biological material
comprises a continuous layer of nucleus pulposus tissue with a
thickness of between about 100 to 500 .mu.m after histological
processing.
[0052] The present invention also relates to an engineered
biological material comprising a substrate and a continuous layer
of nucleus pulposus tissue on the substrate.
[0053] The invention also relates to an engineered biological
material comprising a substrate and a continuous layer of annulus
fibrosus tissue on the substrate. The annulus fibrosus cells are
characterized by being capable of synthesizing types I and II
collagen and proteoglycans similar in size to those synthesized by
annulus fibrosus cells in vivo. The annulus fibrosus tissue is
further characterized by having a three dimensional organization
that is characteristic of annulus fibrosus tissue in vivo.
[0054] The invention also relates to a method for producing an
engineered biolgoical material of the invention comprising
isolating nucleus pulposus cells or annulus fibrosus cells of
intervertebral disc; forming a layer of the cells on a substrate;
culturing the cells in growth media under suitable conditions so
that the cells accumulate intracellular matrix and form a
continuous layer of nucleus pulposus or annulus fibrosus
tissue.
[0055] The cells used in the method of the invention may be
isolated from intervertebral discs (lumbar discs, thoracic discs,
or cervical discs) from animals, preferably humans, bovines,
ovines, rabbits, most preferably humans. The tissue may be isolated
from adult or fetal tissue. In one embodiment of the invention, the
cells are isolated from intervertebral disc of the lumbar spine of
sheep. Intervertebral disc tissue may be extracted from a patient
being treated, or alternatively from a donor, using known surgical
techniques.
[0056] The nucleus pulposus or annulus fibrosus cells may be
isolated from intervertebral disc tissue by sequential enzyme
digestion techniques, such as those described in Boyle et al,
Osteoarthritis and Cartilage 3, 117-125, 1995. For example, the
cells may be treated with 0.5% protease followed by 0.1% bacterial
collagenase.
[0057] In accordance with the method of the invention a continuous
layer of cells is placed on a substrate. Suitable substrates
include bone, engineered biomaterials, and porous tissue culture
inserts, for example filter inserts.
[0058] The substrate is optionally coated with an attachment
factor. Attachment factors are known in the art, see for example,
Streuli and Bissell, J. Cell. Biol. 110:1405, 1990 and Buck and
Horwitz, Ann, Rev. Cell Biol. 3:179, 1987. Examples f attachment
factors include type I collagen, type II collagen, type IV
collagen, a synthetic peptide of a segment of collagen, (e.g. a
fifteen amino acid sequence 766GTPGPQGIAGQRGVV780 which is present
in the .alpha.1 chain of collagen) (Bhatnagar and Qian, 38th Annual
Meeting of the Orthopedic Research Society 17:106, 1992),
fibronectin, gelatin, laminin, polylysine, vitronectin, cytotactin,
echinonectin, entactin, tenascin, thrombospondin, uvomorulin,
biglycan, chondroitin sulfate, decorin, dermatan sulfate, and
heparin. A preferred attachment factor that may be used in the
method of the invention is collagen, most preferably type II
collagen. When the substrate is coated it may be air dried and
sterilized.
[0059] In a preferred embodiment of the invention the substrate is
a tissue culture insert known as Millicell CM.RTM., (Millipore
Corp., Bedford, Mass., U.S.A.), pore size 0.4 .mu.m, coated with an
attachment factor, preferably type II collagen (Sigma Chemical Co.,
St. Louis, Mo., U.S.A.).
[0060] The substrate may be a bone substitute, in particular an
engineered bone substitute such as coral derivatives (Interpore
International Inc., CA), deproteinized bovine bone (Bio-oss.RTM.,
Geistlich Biomaterials, Switzerland), or a porous biodegradable
biomaterial which is formed from sintered calcium polyphosphate
(CPP) as described in U.S. Ser. No. 6,077,989 to Kandel et al. and
PCT Application No. PCT/CA97/00331 (WO97/45147 published Dec. 4,
1997). A sintered porous CPP can be formed with a preferred pore
size and percent porosity through selection of sintering parameters
(time, temperature, starting particle size). Thus, structures can
be formed that more closely mimic the structure of the bone into
which a composite construct of the invention may be placed.
Additionally, degradation rates can be controlled to some extent
through control of percent porosity and pore size (i.e. total free
surface area. In an embodiment, the substrate is an
appropriately-sized porous disc (e.g. 4 mm diameter and 3 mm thick)
that may be encased with an inert tubing to help cell retention by
preventing overflow.
[0061] Cartilage can be formed on, and anchored to, an engineered
bone substitute, preferably a porous CPP substrate. Articular
chondrocytes may be cultured on a porous CPP disc to form a
continuous layer of cartilagneous tissue using the method described
in U.S. Pat. No. 5,326,357.
[0062] Nucleus pulposus or annulus fibrosus cells may be seeded on
a selected substrate at a cell density of about to 1.times.10.sup.5
to 8.times.10.sup.6 cells/cm.sup.2, preferably 2-4.times.10.sup.6
cells/cm.sup.2, more preferably 3-3.5.times.10.sup.6
cells/cm.sup.2, most preferably 3.3.times.10.sup.6 cells/cm.sup.2.
The cells seeded on a coated or uncoated substrate are grown in
suitable culture conditions. Examples of suitable culture media are
known in the art, such as Ham's F12 and/or Dulbecco's modified
Eagle's medium (DMEM). Preferably DMEM is used. The culture medium
may contain serum, for example, heat inactivated fetal bovine serum
in a concentration range of about 2-20%, preferably 10-20%, and may
further contain growth factors, and optionally ascorbic acid. The
culture media is applied above and below the substrate. The cells
may be cultured at 37.degree. C. in a humidified atmosphere
supplemented with CO.sub.2.
[0063] In a preferred embodiment of the invention, the isolated
cells are grown in DMEM containing 10% fetal bovine serum for about
5 days, the medium is then changed to DMEM containing 20% fetal
bovine serum, and ascorbic acid (100 .mu.g/ml, final concentration)
at about 7 days.
[0064] The cells are cultured for an additional 5 weeks to obtain
the engineered biological material described herein. The cells may
be cultured for less than 5 weeks, or greater than 5 weeks, to
obtain a product which may be suitable for some uses such as
transplantation or gene therapy.
[0065] Mechanical force(s) may be administered during in vitro
formation of the engineered biological material in order to enhance
the development of tissues that are highly suited for implantation
and physiol gical weight bearing. Torsion, compression, and/or
shear forces may be applied during tissue formati n. Forces,
together or alone, may be applied, consecutively, simultaneously,
or cyclically. The mechanical forces may be applied through the use
f a mechanical stimulation system that all ws for loading cell
cultures under sterile conditions. For example, the Mach-1.TM.
system (Biosyntech, Montreal) is capable of supplying simultaneous
compressive and linear shear forces, and can include the
application of torsional shear forces. For each type of force
application, a skilled artisan can determine the optimal conditions
to induce tissue growth and organization (i.e. force amplitude,
frequency and duration of stimulation).
[0066] In an embodiment of the invention, either sinusoidal
compressive or torsional forces are applied to the developing
tissue. Compressive forces may be applied at about day 3, in a
range of unconstrained loading between 0.1 to 10 N (approximately
corresponding to compressive stresses of 0.01 to 1 MPa), through a
compliant, biocompatible, autoclavable elastomer (e.g. medical
grade silicone or polyurethane) placed on the actuator to avoid
direct contact with the cells. The duration of loading may range
from 100 to 1200 cycles/day and may be applied at a frequency of
1.0 Hz. (1 Hz approximates normal gait frequency of disc loading).
Minimal numbers of loading cycles may be preferred to stimulate
organization of IVD tissues. For example, 20 sec. of 1 MPa of
hydrostatic pressure may be sufficient to stimulate proteoglycan
synthesis by inner annulus cells.
[0067] Torsional shear force application may consist of a
compressive preload followed by varying degrees of cyclic torsional
shear. Angular deformation amplitudes ranging from 0.005 rad to
0.05 rad at a frequency of 1 rad/sec, may be used (approximately
corresponding to a maximal torque of 0.5N.mm). Cyclic compressive
and torsional shear forces may be simultaneously applied.
[0068] The invention also contemplates an intervertebral disc
construct. The construct may comprise one or both of annulus
fibrosus and nucleus pulposus tissue, with cartilagenous tissue
and/or a substrate (e.g. bone substitute). In one embodiment, the
construct comprises an engineered bone substitute with
cartilagenous tissue formed thereon, and nucleus pulposus tissue
derived from an engineered biological material of the invention
fused to the bone substitute-cartilagenous tissue. This construct
may be prepared by culturing articular chondrocytes on porous CPP
discs for about 3 weeks using the methods described in U.S. Pat.
No. 5,326,357. Simultaneously, nucleus pulposus cells may be grown
on a substrate, preferably a filter insert, more preferably a
Millipore CM.RTM. filter as described herein. At about 3 weeks, a
piece of nucleus pulposus tissue formed in vitro may be punched out
from the substrate (e.g. Millipore CM.RTM. filter), and placed on
the CPP-cartilagenous tissue construct. The tissue components may
be held together using fibrin glue, or other suitable adhesive, and
maintained in culture for about 3 weeks. The composite may be
harvested to form the construct.
[0069] In another embodiment of the invention, the construct
resembles a natural disc. Thus, a substrate (e.g. bone substitute)
may be made with a central depression, and articular cartilage
tissue may be cultured in the depression. Articular cartilage
tissue may be cultured in the depression using the methods
described in U.S. Pat. No. 5,326,357. Annulus fibrosus tissue
derived from an engineered biological material of the invention (or
other source) may be grown on the cartilagenous tissue formed on
the substrate. After fusion of the annulus fibrosus and
cartilagenous tissues, a plug of annulus fibrosus tissue may be
removed from the centre f the annulus fibrosus tissue and replaced
with nucleus pulposus tissue derived from an engineered biomaterial
of the invention. The resulting composite comprising annulus
fibrosus, nucleus pulposus, cartilage endplate, and substrate is
grown in culture to produce a construct comprising fused annulus
fibrosus tissue, nucleus pulposus tissue, and cartilage tissue,
with a substrate.
[0070] In a specific embodiment of the invention a calcium
polyphosphate porous cylinder may be generated that has a central
depression on its surface. Articular chondrocytes may be isolated
(e.g. from sheep knee joint) (Boyle et al, Osteoarthritis and
Cartilage 3, 117-125, 1995) and plated in the depression. The cells
may be grown under the tissue culture conditions described in U.S.
Pat. No. 5,326,357 for about 3 weeks during which time they will
form cartilagenous tissue. Annulus fibrosus and nucleus pulposus
cells may be isolated from sheep lumbar spine. The nucleus pulposus
cells may be plated on a substrate, preferably filter inserts (e.g.
Millicell CM.RTM., Millipore Corp) and grown as described herein
for about 4 weeks. At about 4 weeks, the nucleus pulposus tissue
formed in vitro has sufficient strength to be handled. The annulus
fibrosus cells are plated on top of the cartilagenous tissue which
has formed on the CPP porous cylinder. This composite may be grown
in culture for about 4 weeks under the optimal loading conditions
as described herein. At about 4 weeks, a plug of annulus fibrosus
tissue (e.g. 1-3 mm, preferably 2 mm diameter) is punched out from
the centre and a plug of nucleus pulposus tissue (obtained from the
nucleus pulposus tissue culture) is placed in the resulting defect.
This composite consisting of annulus fibrosus, nucleus pulposus,
cartilage endplate and CPP may be be grown in culture for an
additional 4 to 6 weeks as described herein.
[0071] In an embodiment, nucleus pulposus cells are plated in the
center of a substrate (e.g. disc) with annulus cells surrounding
them, and grown in culture together in the presence or absence of
mechanical stimulation.
[0072] The engineered biological material and constructs of the
present invention can be used as model systems for in vitro studies
of intervertebral disc (or components thereof i.e. annulus fibrosus
and nucleus pulposus tissue) function and development.
[0073] In accordance with one embodiment of the invention, an
engineered biological material, may be used to test substances
which affect intervertebral disc or components thereof (e.g.
nucleus pulposus or annulus fibrosus tissue). A system for testing
a substance that affects intervertebral disc in accordance with the
invention comprises generating or culturing an engineered
biological material or construct of the invention in the presence
of a substance which is suspected of affecting intervertebral disc
or components thereof, and determining the biochemical composition
and/or physiological organization of tissue generated or cultured,
and comparing with the biochemical composition and/or physiological
organization of the engineered biological material in the absence
of the substance.
[0074] The substance may be added to the culture or the cells in
the engineered biological materials (nucleus pulposus or annulus
fibrosus cells) may be genetically engineered to express the
substance i.e. the cells may serve as an endogenous source of the
substance. Cells may be engineered by viral or retroviral-mediated
gene transfer using methods known in the art to produce a specific
substance. The engineered cells are constructed and maintained such
that they release the substance into the medium for the desired
period of time for the culture.
[0075] The system may be used to analyze the effects of
substance(s) on different stages of intervertebral disc
development. Effects on cells at very early, intermediate, and late
stages of development may be evaluated by assessing the biochemical
composition and/or physi logical organization of the tissue
generated in the cultures at various times such as 2, 4, 6 and 8
weeks.
[0076] The biochemical composition and/or physiological
organization of the tissue generated in the cultures may be
assessed using the methods described herein. (See, for example, the
methods described in Example 1)
[0077] In a preferred embodiment of the invention, the biological
materials of the present invention may be used in the testing of
pharmaceutical preparations useful in the treatment of diseases of
intervertebral disc.
[0078] The biological materials of the invention may also be
implanted into patients to replace or repair damaged or deficient
intervertebral disc. In particular, the biological materials of the
invention may be implanted into individuals with idiopathic
scoliosis, herniated disc, degenerative disc disease, recurrent
disc herniation, or spinal stenosis.
[0079] It is also contemplated that the biological materials of the
present invention can be used to enhance healing of damaged or
deficient intervertebral discs when inserted into the site of the
disc.
[0080] The invention also contemplates using the biological
materials of the invention in gene therapy. Therefore, recombinant
vectors containing an exogenous gene encoding a biologically active
protein that is selected to modify the genotype and/or phenotype of
a cell to be infected may be introduced into cells in the
biological materials of the invention. An exogenous gene coding for
a biologically active protein which corrects or compensates for a
genetic deficiency or a drug may be introduced into cells in the
biological materials. For example, IGF-I could be introduced into
the cells so that the cells secrete this protein and stimulate
production of proteoglycans resulting in disc regeneration. The
expression of the exogenous gene may be quantitated by measuring
the expression levels of a selectable marker encoded by a selection
gene contained in the recombinant vector.
[0081] The following non-limiting examples are illustrative of the
present invention:
EXAMPLE 1
[0082] The following materials and methods were utilized in the
investigations outlined in the examples:
Materials and Methods
[0083] Cell cultures
[0084] The lumber spines from up to 9 month-old sheep were removed.
The muscle tissue was cleared from the ventral portion of the spine
and then the ligamentous tissue surrounding the disc was carefully
excised aseptically and discarded. The annulus fibrosus (AF) and
nucleus pulposus (NP) were identified and the nucleus pulposus was
dissected out with blunt forceps and placed in Ham's F12. The
accuracy of the dissection was determined by submitting random
tissue fragments for paraffin embedding and histological assessment
at each harvesting.
[0085] The tissue underwent sequential enzyme digestion consisting
of 0.5% protease (Sigma Chemical Co., St. Louis, Mo., USA) for 1 hr
at 37.degree. C., followed by 0.1% collagenase (Boehringer Mannheim
GmbH, Indianapolis, Ind., USA) overnight at 37.degree. C. The cell
suspension was then filtered through a sterile mesh and plated at a
cell density of 3.3.times.10.sup.6/cm.sup.2 on filter inserts
(Millicell CMR.RTM. Millipore Corp., Bedford, Mass.) precoated with
type II collagen (Sigma Chemical Co., St. Louis, Mo., USA) as
described previously (Kandel 1997, Yu 1997). The cells were grown
in Dulbecco's Modified Eagles medium (DMEM) supplemented with 10%
fetal bovine serum (FBS). After 5 days the FBS was increased to 20%
and asorbic acid (100 .mu.g/ml, final concentration) was added to
the medium at day 7. The medium was changed every 2-3 days and
fresh ascorbic acid was added each time. The cultures were
harvested at 2, 4, 6, 8, and 10 weeks for histologic assessment and
biochemical analysis.
[0086] Histology
[0087] The nucleus pulposus cell cultures were harvested at
selected intervals and fixed in 10% formalin. They were
paraffin-embedded and 5 .mu.m sections were cut and stained with
either hematoxylin and eosin or toluidine blue. Toluidine blue
stains sulphated proteoglycans.
[0088] The culture tissue thickness was measured morphometrically
using light microscopy and a digitized board connected to an IBM
computer equipped with the Bioquant Image Analysis program. Ten
separate points in each section were examined and three sections
per culture were quantified. Tissue thickness was calculated by
determining the mean value of three separate experiments.
[0089] Determination of dry weight of culture tissue
[0090] The cultures, harvested at various times, and representative
fragments of the in vivo nucleus pulposus were lyophilized
overnight. The lyophilized tissues were weighed on an electrical
balance (Mettler Instruments, AG, Greifensee-Zurich,
Switzerland).
[0091] DNA Quantification
[0092] The cultures at different time points and representative
fragments of the in vivo nucleus pulposus tissue were digested with
papain [40 ug/ml in buffer consisting of 20 mM ammonium acetate, 1
mM ethylenediaminetetraacetic acid (EDTA) and 2 mM dithiotreitol]
for 48 hours at 65.degree. C. The DNA content was measured using
Hoescht 33258 dye (Polysciences, Inc., Warrington, Pa.) and
fluorometry (emission wavelength 365 nm and excitation wavelength
458 mm) as described by Kim et al. (1988). Calf thymus DNA (Sigma
Aldrich Co., St. Louis, Mo. USA) was used to generate the standard
curve.
[0093] Quantification of Proteoglycans and Collagen
[0094] Proteoglycan content in the in vitro formed tissue and the
in vivo tissue was determined by measuring the amount of
glycosaminoglycans in the papain digest using the dimethylmethylene
blue dye binding assssay (Polysciences Inc., Warrington, Pa.) and
spectrophotometry as described by Farndale et al. (1986) and
modified by Goldberg et al (1990). Chondroitin sulphate (Sigma
Aldrich Co., St. Louis, Mo., USA) was used to generate the standard
curve.
[0095] The collagen content was determined by measuring the amount
of hydroxyproline in the papain digest of the cultures. An aliquot
was hydrolyzed overnight at 110.degree. using 12N hydrochloric
acid. The hydroxproline was measured by high pressure liquid
chromatography using a C18 reverse column and a Waters PicoTag
amino acid analysis system. The amount of collagen was calculated
from the hydroxyproline content which comprises about 10% of the
weight of collagen (Berg, R A, 1982, Determination of 3 and 4
hydroxyproline. In Methods of Enzymology pp. 393-94, Ed. by L. W.
Cunningham and D. W. Frederiksen, New York, Acadmic Press,
1982).
[0096] Analysis of newly synthesized proteoglycans
[0097] To analyze proteoglycan biosynthesis, 8 week old cultures
were incubated with [.sup.35S]-sulphate (4 .mu.Ci/per well) for 24
hours prior to harvesting. Matrix proteoglycans were extracted with
4M guanidine hydrochloride in 50 mM sodium acetate, pH 5.8
containing protease inhibitors (0.1M 6-amino-hexanoic acid, 50 mM
benzamidine HCl, 10 mM EDTA and 5 mM N-ethylmaleimide) for 24 hours
at 4.degree. C. The proteoglycans were precipitated by the addition
of three volumes of ice cold ethanol. After 24 hours at 4.degree.
C. the precipitate was collected, washed with 70% ethanol, and
resuspended in 4 M guanidium HCl with protease inhibitors.
Proteoglycan synthesis was determined by quantitating
[.sup.35S]-sulphate incorporation by a .beta.-scintillation
counter. The proteoglycan size was determined by Sepharose CL-2B
column chromatography (1.times.100 cm) under dissociative
conditions, as described previously (Boyle 1995). Fractions (2 ml)
were collected using a flow rate of 6 ml/hour at 4.degree. C. The
elution profile was analyzed for its partition coefficient
[Kav=(Ve-Vo)/Vt-Vo), where Vo=void volume, Vt=total volume,
Ve=elution volume]. Vt was determined using [.sup.35S]-sulphate and
Vo was determined using dextran blue 2000.
[0098] Sheep ex vivo nucleus pulposus explant cultures were
established as controls. Immediately after being placed in culture
the cultures were labelled for 24 hours with [.sup.35S]-sulphate
and proteoglycans were extracted identically as described for the
filter cultures.
[0099] Analysis of collagens
[0100] The 8 week old cultures were harvested and digected with
pepsin (200 .mu.g/ml in 0.1 N acetic acid; Sigma Aldrich Co., St.
Louis, Mo., USA) for 72 hours at 4.degree. C. The pepsin extracts
were separated on a 5% sodium dodecyl sulfate-polyacrylamide gel
(SDS-PAGE) and either silver stained (Silver Stain Plus Kit,
Bio-Rad, Hercules, Calif.) or transferred to nitrocellulose
membranes (Schleicher & Schuell, Keene, N.H., USA) for Western
blot analysis. The presence of type II collagen was determined by
Western blot using antibody reactive with type II collagen (4
.mu.g/ml. monoclonal clone 6B3; NeoMarkers, Union City, Calif.,
USA). Immunoreactive bands were visualized by a chemiluminescence
using peroxidase-labelled secondary antibodies (Western blotting
kit, Boehringer Mannheim GmbH, Indianapolis, Ind., USA).
[0101] Statistical Analysis
[0102] Student's t-test was used to analyse the data and
significance assigned at a p value <0.05.
Results
[0103] Histologic appearance of cultures
[0104] The nucleus pulposus cultures were examined histologically
at 2, 4, 6, 8, and 10 weeks after initiation of the cultures to
assess matrix accumulation and tissue organization. By 2 weeks in
cultures, the nucleus pulposus cells accumulated extracellular
matrix to form a continuous layer of tissue which contained
sulfated proteoglycans as demonstrated by toluidine blue staining.
Histologically, the extracellular matrix tissue appeared more
abundant over time in culture (FIG. 1A). The cells were in lacunae
similar to the in vivo nucleus pulposus tissue (FIGS. 1B). The
tissue thickness was measured in the histological sections, and it
increased in thickness up to 10 weeks in culture. The nucleus
pulposus tissue which had been dehydrated and paraffin embedded,
attained a thickness of 308.8.+-.29 .mu.m (mean.+-.SEM) at 10 wk.
(FIG. 2).
[0105] Tissue dry weight
[0106] The dry weight of the in vitro nucleus pulposus tissue
increased over time up to 10 weeks. The dry weight of the tissue,
which was 0.87.+-.0.1 mg per culture (mean.+-.SEM) at 2 weeks,
weighed 2.16.+-.0.2 mg per culture (mean.+-.SEM) by 10 weeks (FIG.
3).
[0107] DNA content f the nucleus pulposus tissue
[0108] The DNA content of the nucleus pulposus tissue formed in
culture was stable during the first 4 weeks in culture but then
decreased ver the following 6 weeks of culture. By 10 weeks the
tissue cellularity had plateaued. (FIG. 4). The tissue formed in
vitro at 10 weeks contained 1.25.+-.0.02 .mu.g DNA/mg dry weight
(mean.+-.SEM), whereas the in vivo nucleus pulposus contained
1.04.+-.0.08 .mu.g DNA/mg dry weight (mean.+-.SEM). There was no
significant difference between the in vitro formed tissue and the
in vivo tissue.
[0109] Quantification of proteoglycan and collagen content
[0110] Proteoglycans and collagen are the major macromolecules of
the nucleus pulposus tissue. The proteoglycan and collagen contents
in the extracellular matrix of the nucleus pulposus tissue in
culture were quantified in order to examine matrix accumulation.
The proteoglycan content, as determined by measuring
glycosaminoglycan content, increased up to 10 weeks (FIG. 5). The
tissue formed in vitro at 10 weeks contained 301.6.+-.27.7 .mu.g
GAG/mg dry weight (mean.+-.SEM), and 411.+-.65 .mu.g collagen/mg
dry weight (mean.+-.SEM), whereas the in vivo nucleus pulposus
contained 320.6.+-.21.2 .mu.g GAG/mg dry weight (mean.+-.SEM) and
399.+-.44 .mu.g collagen/mg dry weight (mean.+-.SEM) (Table 1).
There was no significant difference between the in vitro formed
tissue and the in vivo tissue.
[0111] Analysis of the proteoglycans and collagens
[0112] As large proteoglycans and type II collagen are the main
macromolecules present in the matrix of nucleus pulposus tissue,
the proteoglycans and collagens present in the nucleus pulposus
tissue formed in vitro were analyzed to determined whether the
phenotype of these cells was retained under these culture
conditions. To determine the size of the proteoglycans retained in
the matrix, the [.sup.35S]-sulphate labeled proteoglycans were
guanidinium extracted from 8 week old culture. As shown in FIG. 6,
analysis by column chromatography under dissociative conditions
demonstrated that the proteoglycan monomers have a large
hydrodynamic size (K.sub.av=0.26.+-.0.03, mean.+-.SD), which was
similar in size to those synthesized by cells in the ex vivo tissue
cultures (K.sub.av=0.22.+-.0.02, mean.+-.SD, p>0.05). Nucleus
pulposus cells in vitro and in vivo were also synthesizing a
smaller population of proteoglycan with a K.sub.av around 0.7.
[0113] Pepsin extracts of 10 week old tissue formed in vitro were
analyzed by SDS-PAGE and autoradiography. A band similar in size to
the .alpha.1(II) chain of type II collagen was seen. Western blot
analysis of these extracts confirmed the presence of type II
collagen (FIG. 7).
[0114] Discussion
[0115] A cell culture system is described herein in which nucleus
pulposus cells isolated from sheep lumbar spines and grown on
Millipore CM.RTM. filter inserts accumulate extracellular matrix
and form a continuous layer of nucleus pulposus tissue. The nucleus
pulposus cells in these cultures maintained their phenotype as they
synthesized large sulfated PGs and type II collagen which are
characteristic of nucleus pulposus cells. Cells isolated from
rabbit nucleus pulposus also generated tissue in culture under
similar conditions.
[0116] The composition of the in vitro formed tissue was similar to
the in vivo nucleus pulposus tissue. The cellularity and
proteoglycan content at 10 weeks was comparable to the in vivo
tissue. The predominate proteoglycan in the nucleus pulposus is
aggrecan (Melrose 1994, Oegema 1979), and the cells in filter
culture synthesized large proteoglycans in keeping with this type
of proteoglycan. The size of proteoglycans synthesized by the
nucleus pulposus cells in vitro was similar to that synthesized by
the nucleus pulposus cells in ex-vivo culture as determined by
column chromatography. As well a smaller amount of proteoglycans
with a K.sub.av of approximately 0.7 were also detected.
Proteoglycans of this size may represent degradation products or
the presence of the small proteoglycans, such as decorin,
fibromodulin, which have been shown to be present in the nucleus
pulposus (Melrose 1994; Inkinen 1998; Jahnke 1988; Sztrolovics
1999; Gotz, 1997).
[0117] The cellularity of the in vitro tissue decreased between 4
and 10 weeks of culture and in doing so more closely approximates
the cellularity of the in vivo tissue. The nucleus pulposus in vivo
has been shown to become less cellular with age (Trout 1982;
Buckwalter 1995). Other studies have shown that DNA content
increases with time when nucleus pulposus cells were cultured in
either monolayer or alginate gel. In these culture systems the
cells do not synthesize sufficient matrix to form a continuous
layer of tissue, so it is possible that the nucleus pulposus cells
are in a different microenvironment than those present in the in
vivo tissue (Chiba 1997, Sato 1999, Ichimura 1991).
[0118] This new culture system has several advantages over other
culture systems in that the nucleus pulposus cells do not
dedifferentiate, as indicated by the maintenance of their phenotype
during the time period studied, and form a continuous layer of
tissue which is amenable to histological and biochemical
assessment. In addition the nucleus pulposus cells under these
culture conditions maintain their phenotype. Other methods to
culture nucleus pulposus cells have been described and include
growing cells in monolayer (Ichimura 1991) or encapsulated within
alginate (Chelberg 1995; Chiba 1997, Sato 1999 Maldonado 1992).
However, these do not result in tissue formation and so do not
mimic the organization of the nucleus pulposus tissue i.e. they do
not accumulate sufficient extracellular matrix to form a continuous
layer of tissue and so do not form a three dimensional structure.
For example, nucleus pulposus cells grown in monolayer or
suspension culture do not accummulate sufficient extracellular
matrix to form a continuous layer of tissue (Ichimura 1991, Osada
1996). Nucleus pulposus cells grown in alginate or agarose beads
reamin spherical and accumulate mainly type II collagen and large
proteoglycans but still do not form tissue (Chiba 1997, 1998,
Maldonado 1992, Chelberg 1995). Furthermore, it has been shown that
molecules of up to 200,000 molecular weight are able to diffuse out
of alginate and this may influence what is retained within the
matrix of these cultures (Kupchik 1983). Kusior L J et al (1999)
has reported that nucleus pulposus cells embedded in biocompatible
polymers, such as polyglycolic acid and calcium alginate, will form
tissue. However, the cell seeded scaffolds had to be implanted in
nude mice for tissue formation to occur. Although organ culture of
the intervertebral disc has been used to study metabolism of the
whole tissue it can not been maintained at a comparable metabolism
level to the in vivo tissue. (Urban 1981, Thompson 1991). Recently
an alternative approach was described by Chiba et al. (1998) in
which an intact disc was surrounded with alginate, however this
method did not entirely inhibit the matrix loss that occurs during
the first few days of culture.
[0119] In conclusion, these studies demonstrate that the nucleus
pulposus cells grown on filters generate tissue similar to the in
vivo tissue for the features examined.
EXAMPLE 2
[0120] Formation of disc construct
[0121] The possible formation of a composite of nucleus pulposus
tissue fused to cartilage which is anchored to the porous CPP was
investigated. Articular chondrocytes were plated on the porous CPP
discs and allowed to grow for 3 weeks. Simultaneously nucleus
pulposus cells were grown on Millipore CM.RTM. filters as described
above. At 3 weeks, a piece of nucleus pulposus tissue formed in
vitro was punched out from the Millipore CM.RTM. filter and placed
on the CPP-cartilagenous tissue construct that had been prepared.
The tissue components were held together using fibrin glue and
maintained in culture for 3 weeks. The composite was harvested and
light microscopical examination of the processed constructs showed
that substantial fusion of nucleus pulposus tissue with the
underlying cartilagenous tissue occurred.
EXAMPLE 3
[0122] Generation of intervertebral disc--CCP biomaterial
construct
[0123] One half of a spinal unit consists of an intervertebral disc
fused to a cartilage endplate integrated with subchondral bone. To
more closely mimic a natural disc, CPP will be generated with a
central depression on its surface, the diameter of which will
depend on the diameter of the CPP cyclinder and with a depth of 0.5
mm. Articular chondrocytes can be isolated from sheep knee joint as
described previously (Boyle et al, 1995) and plated in this
depression. The cells will be grown under standard tissue culture
conditions for 3 weeks during which time they will form
cartilagenous tissue. Annulus fibrosus and nucleus pulposus cells
will be isolated from sheep lumbar spine. The nucleus pulposus
cells will be plated on filter inserts (Millicell CM.RTM.,
Millipore Corp) and grown as described above for 4 wks (see Example
1). At 4 weeks the nucleus pulposus tissue formed in vitro has
sufficient strength to be handled. The annulus fibrosus cells will
be plated on top of the cartilagenous tissue which has formed on
the CPP porous cylinder. This composite will be grown in culture
for 4 weeks under the optimal loading conditions described herein.
At 4 weeks a 2 mm diameter plug of annulus fibrosus tissue will be
punched out from the centre and a plug of nucleus pulposus tissue
(obtained from the nucleus pulposus tissue culture) will be placed
in the resulting defect. In preliminary studies, no dead cells were
seen at the edge of the punched out nucleus pulposus tissue
suggesting that this manipulation does not damage the tissue. This
composite consisting of annulus fibrosus, nucleus pulposus,
cartilage endplate and CPP will then be grown in culture for an
additional 4 to 6 wks under loading as defined above.
[0124] Having illustrated and described the principles of the
invention in a preferred embodiment, it should be appreciated to
those skilled in the art that the invention can be modified in
arrangement and detail without departure from such principles. All
modifications coming within the scope of the following claims are
claimed.
[0125] All publications, patents and patent applications referred
to herein are incorporated by reference in their entirety to the
same extent as if each individual publication, patent or patent
application was specifically and individually indicated to be
incorporated by reference in its entirety.
1TABLE 1 Comparison of 10 week olf in vitro generated nucleus
pulposus tissue with in vivo nucleus pulposus tissue (.mu.g/mg dry
weight) in vitro in vivo DNA 1.25 .+-. 0.02 1.04 .+-. 0.08 GAG
301.6 .+-. 27.7 320.6 .+-. 21.2 COLLAGEN 411 .+-. 65 399 .+-.
44
[0126] The dry weight, and DNA, glycosaminoglycans (GAG) and
collagen (hydroxyproline) contents were determined as described in
Example 1. The analyses were performed on three different samples
from each of three separate experiments. The data are expressed as
mean.+-.SEM of the three experiments.
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