Compositions and methods for intervertebral disc reformation

Methods of reforming degenerated intervertebral discs are provided in accordance with methods of the invention. Hybrid materials useful in methods of the present invention are also provided.

FIELD OF THE INVENTION 
The present invention concerns methods and materials useful for reforming 
degenerated discs of the spine of a vertebrate and in particular the spine 
of a human. 
BACKGROUND OF THE INVENTION 
Back pain is one of the most frequently reported musculoskeletal problems 
in the United States. 80% of the adults will miss work at least three 
times in their career due to back pain. The most common factor causing low 
back pain is the degeneration of the disc. At the ages between 35 to 37, 
approximately a third of the U.S. population will have suffered from a 
herniated disc. 
The main functions of the spine are to allow motion, transmit load and 
protect the neural elements. The vertebrae of the spine articulate with 
each other to allow motion in the frontal, sagittal and transverse planes. 
As the weight of the upper body increases, the vertebral bodies which are 
designed to sustain mainly compressive loads increase in size caudally. 
The intervertebral disc is a major link between the adjacent vertebrae of 
the spine. The intervertebral disc, the surrounding ligaments and muscles 
provide stability to the spine. 
The intervertebral discs make up about 20-33% of the lumbar spine length. 
They are capable of sustaining weight and transferring the load from one 
vertebral body to the next, as well as maintaining a deformable space to 
accommodate normal spine movement. Each disc consists of a gelatinous 
nucleus pulposus surrounded by a laminated, fibrous annulus fibrosus, 
situated between the end plates of the vertebrae above and below. 
The nucleus pulposus contains collagen fibrils and water-binding 
glycosaminoglycans. At birth, the nucleus pulposus contains 88% water, 
however, this percentage decreases with age. This water loss decreases its 
ability to withstand stress. The annulus fibrosus consists of 
fibrocartilaginous tissue and fibrous protein. The collagen fibers are 
arranged in between 10 to 20 lamellae which form concentric rings around 
the nucleus pulposus. The collagen fibers within each lamella are parallel 
to each other and runs at an angle of approximately 60 degrees from 
vertical. The direction of the inclination alternates with each lamellae. 
This crisscross arrangement enables the annulus fibrosus to withstand 
torsional and bending loads. The end-plates are composed of hyaline 
cartilage, and are directly connected to the lamellae which form the inner 
one-third of the annulus. 
When under compressive loads, the nucleus pulposus flattens and bulges out 
radially. The annulus fibrosus stretches, resisting the stress. The 
end-plates of the vertebral body also resist the ability of the nucleus 
pulposus to deform. Thus, pressure is applied against the annulus and 
end-plate, transmitting the compressive loads to the vertebral body. When 
tensile forces are applied, the disc is raised to a certain height 
straining the collagen fibers in the annulus. At bending, one side of the 
disc is in tension while the other side is in compression. The annulus of 
the compressed side bulges out. 
When the disc is subjected to torsion, there are shear stresses which vary 
proportionally to the distance from the axis of rotation, in the 
horizontal and axial plane. The layer of fibers oriented in the angle of 
motion is in tension while the fibers in the preceding or succeeding layer 
are relaxed. Similarly in sliding, the fibers oriented in the sliding 
direction are in tension while the fibers in the other layers relax. 
Repeated rotational loading initiates circumferential tears in the annulus 
fibrosus, which gradually form radial tears into the nucleus pulposus 
until the nucleus degrades within the disc. In addition to the water loss 
which occurs with age, more water is also lost due to nucleus rupture, 
thereby reducing its ability to resist compressive loads. As such, the 
annulus bulges. As the severity of the tear increases, much of the 
contents of the disc is lost, leaving a thin space of fibrous tissue. This 
condition is called disc resorption. 
Increasing disc collapse can cause facet subluxation and stenosis of the 
intervertebral foramen. Subsequently, the degenerative process involves 
the facet joints equally. As the annulus bulges out posteriorly into the 
spinal canal, the nerve root may be compressed causing sciatica. Pain is 
felt from the lower back to the buttocks and the leg. Following the 
rupture of the disc, excessive motions such as excessive extension or 
flexion can occur, resulting in spine segmental instability. The spine is 
thus more vulnerable to trauma. Herniation can occur due to disc 
degeneration or excessive load factors, especially compression. Pain may 
result due to nerve root compression caused by protrusions. 
The unstable phase of the degeneration progress allowing excessive movement 
may result in degenerative spondylolisthesis, which is a breakdown of 
posterior joints. The nerve is trapped between the inferior articular 
facet of the vertebrae above and the body of that below. Thus, sliding of 
a vertebral body on one another damages the posterior joints due to 
fatigue and applies traction on the nerve root causing pain. 
Surgical treatments for herniated disc include laminectomy, spinal fusion 
and disc replacement with protheses. 
At this time, 150,000 spinal fusion procedures are performed per year in 
the U.S. alone, and the numbers are growing exponentially. However, the 
results of spinal fusions are very varied. Some of the effects include 
non-unions, slow rate of fusion even with autografts, and significant 
frequency of morbidity at the graft donor site. In addition, even if the 
fusion is successful, joint motion is totally eliminated. Adverse effects 
of spinal fusions have also been reported on adjacent unfused segments 
such as disc degeneration, herniation, instability spondylolysis and facet 
joint arthritis. A long-term follow-up of lower lumbar fusions in patients 
from 21 to 52 years of age found that 44% of patients with spinal fusions 
were currently still experiencing low-back pain and 57% had back pain 
within the previous year. 53% of the patients tracked were on medication, 
5% had late sequela secondary surgery, 15% had a repeat lumbar surgery, 
42% had symptoms of spinal stenosis, and 45% had instability proximal to 
their fusion. This clinical data shows that significant long-term 
limitations are associated with spinal fusion. 
An alternative to spinal fusion is the use of an intervertebral disc 
prosthesis. Ideally, a successful disc prosthesis will simulate the 
function of a normal disc. The disc replacement must be capable of 
sustaining weight and transferring load from one vertebral body to the 
next. It should be robust enough not to be injured during movement and 
should maintain a deformable space between the vertebral body to 
accommodate movement. 
Disc protheses should last for the lifetime of the patient, should be able 
to be contained in the normal intervertebral disc space, should have 
sufficient mechanical properties for normal body function, should be able 
to be fixed to the vertebrae adjacent to the disc, should be possible to 
implant, should not cause any damage should the disc fail, and should be 
biocompatible. 
There are at least 56 artificial disc designs which have been patented or 
identified as being investigated, McMillin C. R. and Steffee A. D., 20th 
Annual Meeting of the Society for Biomaterials (abstract) (1994), although 
not all these devices have actually been made or tested. They can be 
divided into two main categories. Lee et al., Spine, Vol. 16, 253-255 
(1991). A first class includes devices for nucleus pulposus replacements 
which includes metal ball bearing, a silicone rubber nucleus, and a 
silicone fluid filled plastic tube. Devices for total or subtotal 
replacement of the disc have also been proposed such as a spring system, 
low-friction sliding surfaces, a fluid filled chamber, elastic disc 
prosthesis and elastic disc encased in a rigid column. 
An example of total disc replacement is described by Urbaniak et al., Bio. 
J. Med. Mater. Res. Sym., Vol. 4, 165-186 (1973) who developed and tested, 
using chimpanzees, an intervertebral disc device made of a central 
silicone layer sandwiched between two layers of Dacron embedded in the 
silicone. The open-mesh Dacron was chosen to allow tissue ingrowth for 
fixation to the adjacent vertebrae. While spinal mobility was restored and 
the device tolerated by the host, due to inexact fit of the device, bone 
resorption and reactive bone formation were observable. Loose fibrous 
tissue also indicated possible movement of the device. 
Hou et al., Chinese Medical Journal, Vol. 104(5), 381-386 (1991), developed 
a disc implant made of silicone rubber which restored normal disc 
function. However, the presence of fibrous tissue surrounding the implant 
indicated possible movement of the device. 
The SB Charite intervertebral disc endoprosthesis, White and Panjabi, 
Clinical biomechanics of the lumbar spine, Churchill Livingstone, London 
(1989), which has been tested clinically, is fabricated from a biconvex 
polyethylene core sandwiched between two concave-molded titanium 
end-plates. However, the endoprosthesis shows insufficient mechanical 
performance and unlikely long-term bone fixation to the device. 
Two types of disc prostheses were developed and evaluated by Lee et al., 
35th Annual Meeting of the Orthopaedic Research Society, Las Vegas, Nev., 
February 6-9 (1989): Dacron fiber-reinforced polyurethane elastomer 
(reinforcement located for the annulus section), and a prosthesis made 
from thermoplastic polymer which is increasingly rigid moving from the 
nucleus out to the end-plates. Yet another design is made of 
cobalt-chromium-molybdenum (Co--Cr--Mo) alloy by Hedman et al., Spine, 
Vol. 16, 256-60 (1991). 
U.S. Pat. No. 4,911,718 (Lee et al.), U.S. Pat. No. 5,002,576 (Fuhrmann et 
al.), U.S. Pat. No. 4,911,718 (Lee et al.) and U.S. Pat. No. 5,458,642 
(Beer et al.) also teach permanent intervertebral disc endoprostheses for 
total disc replacement. 
All of the foregoing intervertebral disc prostheses, however, merely 
replace all or a part of the disc with synthetic materials which must 
remain in place ad infinitum. These prostheses are generally permanent 
implants which require observation of long term biologic responses 
throughout the life of the prothesis. Furthermore, discs that are not 
comprised of biocompatible material may be rejected by the patient. 
Procedures by which the tissues of the intervertebral disc are made to 
reform or replace the degenerated tissue of the intervertebral disc would 
be highly desirable and a significant improvement over the current state 
of the art which presently use such permanent implants. Although efforts 
in tissue-engineering have been reported, no one has, until now, 
accomplished reformation of intervertebral disc tissue. 
Repair of skin tissue has been achieved. For instance, skin deficiencies 
which arise in severely burnt patients or in decubitus wounds of diabetic 
patients have been so treated. Sabolinski, Biomaterials, Vol. 17, 311-320 
(1996). Cells are seeded onto templates of either resorbable or 
non-resorbable material. Once tissue begins to form the templates are 
dressed onto the site in need of treatment. Tissue engineering of the 
skin, however, is significantly different from tissue engineering of the 
intervertebral disc because tissue compositions differ significantly. In 
addition, the mechanical requirements of engineered skin tissue are 
significantly different from those of intervertebral disc tissue. 
Some intervertebral disc prostheses provide for regrowth of the 
intervertebral disc and concurrent resorption of the prothesis. For 
example, U.S. Pat. Nos. 4,772,287 and 4,904,260 (Ray et al.) teach 
prosthetic discs having an outer layer of strong, inert fibers 
intermingled with bioresorbable materials which attract tissue ingrowth. 
However, this prosthesis is purely a synthetic material at the time of 
implantation and does not include any cells or developing tissue. In 
addition, it provides only partial resorption and the problems associated 
with permanent implants remain. 
U.S. Pat. Nos. 5,108,438 and 5,258,043 (Stone) teach a porous matrix of 
biocompatible and bioresorbable fibers which may be interspersed with 
glycosaminoglycan molecules. The matrix serves as a scaffold for 
regenerating disc tissue and replaces both the annulus fibrosus and 
nucleus pulposus. However, replacement of this much tissue is a relatively 
invasive procedure which requires lengthy recovery time. Furthermore, 
these matrices do not use any cells to stimulate tissue recovery nor is 
there any incipient tissue formation in this device at the time of 
implantation. 
Various materials have been seeded with cells in order to facilitate cell 
function including proliferation and extracellular matrix synthesis. For 
instance, El-Ghannam, et al., Journal of Biomedical Materials Research, 
Vol. 29, 359-370 (1974), teaches in vitro synthesis of bone-like tissue 
using bioactive glass templates. Schepers, et al., J. Oral Rehab., Vol. 
18, 439-452 (1991), analyzed the use of bioactive glass as fillers for 
bone lesions. Also, porous polymeric matrices have been used. The polymers 
include poly(lactic acid), poly(glycolic acid) and their co-polymers. 
However, these polymers have not been taught to be appropriate substrates 
for intervertebral disc cells which until now have not been used to seed 
implants of any sort. 
Ideally, intervertebral disc treatment would guide and possibly stimulate 
the reformation of the tissue of affected intervertebral disc, especially 
nucleus pulposus and annulus fibrosus tissue. It could also biodegrade 
while allowing concurrent nucleus pulposus and annulus fibrosus tissue 
ingrowth, thereby providing for disc regeneration. Such an intervertebral 
disc material which is biodegradable while still satisfying the mechanical 
requirements of an intervertebral disc, has not been available until now. 
OBJECTS OF THE INVENTION 
An object of the present invention is to provide a method of inducing 
and/or guiding intervertebral disc reformation using biodegradable support 
substrates. 
Yet another object of the present invention is provided biodegradable 
substrates useful for intervertebral disc tissue reformation. 
Still another object of the invention is to provide material useful for 
guiding and/or stimulating intervertebral disc tissue reformation. 
Another object of the invention is to provide methods of culturing 
intervertebral disc cells.

SUMMARY OF THE INVENTION 
In accordance with methods of the present invention there are provided 
methods for repairing damaged or degenerated intervertebral discs. These 
methods comprise evacuating tissue from the nucleus pulposus portion of a 
degenerated intervertebral disc space, preparing hybrid material by 
combining isolated intervertebral disc cells with a biodegradable 
substrate, and implanting the hybrid material in the evacuated nucleus 
pulposus space. In accordance with methods of the invention intervertebral 
disc cell growth is guided and/or stimulated and intervertebral disc 
tissue is reformed. 
Methods of culturing intervertebral disc cells are also provided in some 
aspects of the invention whereby intervertebral disc tissue is digested 
with collagenase and incubated in medium supplemented with hyaluronidase. 
In still other aspects of the invention biodegradable substrates are 
provided comprising polymer foam coated with bioactive materials, which 
substrates are useful for intervertebral disc tissue reformation. 
In yet another aspect of the invention are provided hybrid materials for 
reforming degenerate intervertebral disc tissue. The hybrid materials can 
be made in the form of shaped bodies comprising biodegradable substrate 
and intervertebral disc cells. 
DETAILED DESCRIPTION OF THE INVENTION 
The present invention is directed to methods of inducing intervertebral 
disc repair by reformation of intervertebral disc tissue. By implanting a 
hybrid material comprising intervertebral disc cells and a biodegradable 
support substrate into the intervertebral disc space, ingrowth of 
intervertebral disc cells is induced. Thus, the present invention provides 
methods of inducing self-regeneration of viable tissue and functional 
joints. 
Methods of the present invention are useful to treat vertebrates suffering 
from degenerated intervertebral disc conditions, and in particular may be 
used to treat humans with such conditions. 
A degenerated intervertebral disc has lost or damaged some or all of its 
intervertebral disc tissue, primarily including its nucleus pulposus 
tissue, due to any number of factors discussed herein, including age and 
stress due to rotational loading. Degenerated discs vary in severity from 
bulging discs to herniated or ruptured discs. Patients suffering from a 
degenerated disc experience a number of symptoms which include pain of the 
lower back, buttocks and legs and may also include sciatica and 
degenerative spondylolysis. In accordance with methods of the present 
invention reformation or regeneration of intervertebral disc tissue occurs 
in situ, replacing lost or damaged tissue and resulting in amelioration or 
elimination of the conditions associated with the degenerated disc. 
In accordance with the present invention hybrid materials used to induce 
and/or guide reformation of intervertebral disc tissue comprise 
biodegradable substrates. Biodegradable means that the substrate degrades 
into natural, biocompatible byproducts over time until the substrate is 
substantially eliminated from the implantation site and, ultimately, the 
body. Preferably in accordance with methods of the present invention, the 
rate of biodegradation of the substrate is less than or equal to the rate 
of intervertebral disc tissue formation such that the rate of tissue 
formation is sufficient to replace the support material which has 
biodegraded. 
In some aspects of the present invention the biodegradable substrate may be 
bioactive. Bioactive, as used herein, is meant to refer to substrates 
which enhance cell function as compared to cell function of the same cell 
type in the absence of the substrate. For instance, bioactive glass 
granules have been shown to enhance cell growth of typical bone cells, 
Schepers et al., U.S. Pat. No. 5,204,106. In addition, dense bioactive 
glass discs have been found to enhance osteoprogenitor cell 
differentiation beyond even those levels of enhanced differentiation 
elicited by bone morphogenic protein, H. Baldick, et al., Transactions 5th 
World Biomaterials Conference, Toronto, II-114 (June, 1996). 
The biodegradable substrate must also have sufficient mechanical strength 
to act as a load bearing spacer until intervertebral disc tissue is 
regenerated. In addition, the biodegradable substrate must be 
biocompatible such that it does not elicit an autoimmune or inflammatory 
response which might result in rejection of the implanted hybrid material. 
Biodegradable support substrates useful in methods of the present invention 
include bioactive glass, polymer foam, and polymer foam coated with sol 
gel bioactive material. 
In accordance with some methods of the present invention bioactive glass is 
employed as a substrate. Bioactive glass is described in U.S. Pat. No. 
5,204,106, incorporated by reference herein in its entirety. The bioactive 
glass contains oxides of silicon, sodium, calcium and phosphorous in the 
following percentages by weight: about 40 to about 58% SiO.sub.2, about 10 
to about 30% Na.sub.2 O, about 10 to about 30% CaO, and 0 to about 10% 
P.sub.2 O.sub.5. In preferred embodiments of the invention the nominal 
composition of bioactive glass by weight is 45% SiO.sub.2, 24.5% Na.sub.2 
O, 24.5% CaO and 6% P.sub.2 O.sub.5 and is known as 45S5 bioactive glass. 
Bioactive glass may be obtained from commercial sources such as Orthovita, 
Inc. (Malvern, Pa.). 
Granule size of the bioactive glass is selected based upon the degree of 
vascularity of the affected tissue and generally will be less than about 
1000 .mu.m in diameter. In some embodiments of the present invention it is 
preferred that the bioactive glass granules be from about 200 .mu.m to 
about 300 .mu.m in diameter. In still other embodiments of the present 
invention granule size is from about 50 .mu.m to about 100 .mu.m. 
In some embodiments of the present invention bioactive glass has pores. 
Percent density (100%-percent porosity) of less than about 80% may be used 
in some aspects of the invention. Percent density of about 10% to about 
68% can be used for other aspects of the invention. In some aspects of the 
present invention the pore size should be less than about 850 .mu.m in 
diameter while about 150 .mu.m to about 600 .mu.m pore diameter is 
preferred. 
One method of preparing porous bioactive glass is by mixing bioactive glass 
granules of a desired size with sieved sacrificial agent camphor particles 
of a desired amount and size. The camphor sublimates during sintering 
leaving pores in the sintered glass. Thus, the average particle size and 
weight percent of the camphor particles is chosen to optimize the pore 
size and percent porosity, respectively, of the glass. In some aspects of 
the invention particle size may be less than about 850 .mu.m in diameter 
while about 150 .mu.m to about 600 .mu.m is preferred. Thereafter the 
glass may be treated with any aqueous buffer solution containing ions, the 
identity and concentration of which is found in interstitial fluid. Said 
treatments result in the formation of a calcium phosphate rich layer at 
the glass surface. Typical buffers include those prepared as described by 
Healy and Ducheyne, Biomaterials, Vol. 13, 553-561 (1992), the subject 
matter of which is incorporated herein by reference in its entirety. 
In still other aspects of the present invention the support substrate 
comprises polymer foam. Polymer foam useful in these aspects of the 
invention are biocompatible and include polyglycolide (PGA), 
poly(D,L-lactide)(D,L-PLA), poly(L-lactide) (L-PLA), 
poly(D,L-lactide-co-glycolide),(D,L-PLGA), poly(L-lactide-co-glycolide) 
(L-PLGA), polycaprolatone(PCL), polydioxanone, polyesteramides, 
copolyoxalates, and polycarbonates. D,L-PLGA, which is preferred in some 
embodiments of the invention, may comprise 50% polylactide and 50% 
polyglycolide. About 75% polylactide and about 25% polyglycolide is still 
more preferred although it is anticipated that ratios may be varied to 
optimize particular features of the individual polymers. For instance, the 
mechanical strength of a polymer may be adjusted by varying the percentage 
of PLA and the percentage of PGA may be adjusted to optimize cell growth. 
In some aspects of the invention polymer foam is coated with sol gel 
bioactive material. Sol gel materials include glasses and ceramics. Such 
bioactive compound are prepared by mixing a desired polymer foam with NaCl 
to create the desired porosity and pore size. Thereafter, the polymer, 
including the pores and interstices, is coated with sol gel material. 
Sol gel glass is prepared by combining a metal alkoxide precursor with 
water and an acid catalyst to produce a gel. A typical process is 
described in U.S. Ser. No. 08/477,585 (U.S. Pat. No. 5,591,453) which is 
incorporated by reference herein in its entirety. Once dried the gel 
consists mostly of metal oxide with a glass consistency. Sol gel bioactive 
material may be comprised of from about 60 to about 100% silicon dioxide, 
up to about 40% calcium oxide and up to about 10% diphosphorous pentoxide. 
A final product of 70% SiO.sub.2, 25% CaO and 5% P.sub.2 O.sub.5 is 
preferred in some methods of the present invention although the 
concentration of each may be adjusted to optimize critical features of the 
sol gel. Other sol gel materials may be prepared by methods known in the 
art. For instance, Qui, Q., et al., Cells and Materials, Vol. 3, 351-60 
(1993), incorporated by reference herein in its entirety, teaches methods 
of preparing calcium phosphate sol gel bioactive material. 
To coat the polymer, the polymer foam is dipped into the sol during the sol 
gelation phase. The sol-filled foam is then placed in a syringe filter and 
the sol is pulled through the foam by creating a vacuum below using the 
syringe. Thus, the polymer is substantially coated with sol gel, with 
residual sol gel being evacuated from the polymer matrices. While it is 
preferred that most or all of the polymer surfaces, including the surfaces 
of the pores and interstices, be coated with sol gel bioactive glass, 
polymers which are only partially coated with sol gel bioactive glass may 
also be useful in some aspects of the present invention. It is desired in 
some embodiments of the invention that greater than about 50% of the 
polymer surface be coated. 
To prepare the hybrid material, intervertebral disc cells are combined with 
biodegradable substrate material. Intervertebral disc cells may be 
isolated from tissue extracted from any accessible intervertebral disc of 
the spine. For instance, tissue may be extracted from the nucleus pulposus 
of lumbar discs, sacral discs or cervical discs. Preferably, 
intervertebral disc cells are primarily nucleus pulposus cells. In some 
embodiments it is preferred that disc cells are at least 50% nucleus 
pulposus cells while 90% nucleus pulposus cells is still more preferred. 
Cells may be obtained from the patient being treated, or alternatively 
cells may be extracted from donor tissue. 
The present invention provides advantages over prior art methods in that 
the entire degenerated disc need not be removed to treat a degenerated 
disc. Rather, only the nucleus pulposus tissue need be evacuated from the 
degenerated intervertebral disc. Degenerated nucleus pulposus refers to a 
region of the intervertebral disc where the tissue has severely reduced 
mechanical properties or which has lost some or most of the nucleus 
pulposus tissue. The present invention thus provides a less invasive 
procedure than that of the prior art. In addition, the methods and hybrid 
materials of the present invention prompt biological repair of normal 
tissue in the disc which will result in better long term results than that 
obtained with synthetic prostheses. 
Evacuation of the degenerated intervertebral disc tissue, and primarily the 
nucleus pulposus tissue, is performed using known surgical tools with 
procedures developed to meet the needs of the present invention. Generally 
an incision or bore is made at the lateral edge in the annulus fibrosus 
and the intervertebral disc tissue is extracted from the nucleus pulposus 
via, for example, the guillotine cutting approach. The tissue may be 
extracted using a scalpel, bore, or curette. Alternatively, tissue may be 
aspirated. Ideally, the annulus fibrosus, or significant portions thereof, 
are left intact. It is preferred, for instance, that at least 50% of the 
annulus fibrosus remain intact. It is still more preferred that at least 
85% of the annulus fibrosis remain intact. Arthroscopic techniques are 
most preferred in accordance with methods of the present invention. 
Similar surgical techniques are utilized to extract intervertebral disc 
tissue from other, non-degenerate intervertebral discs of the spine of the 
patient or donor. For instance, similar techniques may be used to obtain 
intervertebral tissue from sacral discs. Minor modifications necessary to 
tailor the procedure to a particular region of the spine would be 
appreciated by those skilled in the art. 
Where there is lag time between tissue evacuation and implantation of the 
hybrid material, the evacuated space may be temporarily filled with gel 
foam or other load bearing spacers known in the art. 
Intervertebral disc cells are isolated from extracted tissue. Generally, 
tissue is fragmented and treated with enzymes such as collagenase to 
disaggregate the cells into individual cells. Preferably isolated cells 
are primarily nucleus pulposus cells with 50% nucleus pulposus cells being 
preferred and 90% nucleus pulposus cells being more preferred. The cells 
are isolated using centrifugation. Cells may then be combined with a 
biodegradable substrate and implanted into the evacuated nucleus pulposus. 
Alternatively, isolated intervertebral disc cells may be cultured alone or 
seeded onto a biodegradable substrate and cultured together with the 
biodegradable substrate for later implantation. 
In some aspects of the present invention the hybrid material may also 
include factors to enhance cell growth. For instance, TGF-.beta. and EGF 
may be added to the hybrid material to enhance cell growth. Cells may be 
incubated alone or seeded on a substrate in a tissue culture medium such 
as Dulbecco's Modified Eagle Medium (DMEM) (pH 7.0), which may be 
supplemented with serum such as heat-inactivate fetal bovine serum. 
Antifungal and antibacterial agents may also be added. In preferred 
methods of the present invention cells are incubated with about 0.5% to 
about 1.5% hyaluronidase. 
In some aspects of the present invention the end plate may be partially 
decorticated to enhance vascularization. Thereafter, cells may be 
implanted or alternatively, after cell attachment, hyaluronidase is 
removed and incubation is resumed with a medium supplemented with 0.001% 
ascorbic acid in the absence of hyaluronidase. Medium supplemented with 
0.0025% ascorbic acid is used to replenish the cell solutions. 
Hybrids of intervertebral disc cells and biodegradable substrate may then 
be implanted into the evacuated intervertebral disc space using surgical 
procedure such as described above. 
Hybrid materials are provided by the present invention. Such hybrids can 
then be shaped for insertion into the intervertebral disc space of a 
patient. Exemplary FIG. 1 shows a shaped hybrid material comprising 
biodegradable substrate and intervertebral cells. Intervertebral cells are 
located on the outer surface 10 and on the surfaces of the pores and 
interstices 12 of the shaped substrate. It should be noted that FIG. 1 
depicts one possible embodiment and should not be construed as limiting 
the invention in any way. 
The substrate should generally have a rectangular shape. A cylindrical pad 
shape is also envisioned. 
The following examples are illustrative but are not meant to be limiting of 
the present invention. 
EXAMPLES 
Example 1 
Evacuation of Nucleus Pulposus 
Mature New Zealand rabbits weighing 4-5 kg are used. For each rabbit, L4-L5 
or, when possible L4-L5 and L5-L6 disc spaces are accessed as those are 
the biggest sections. The anesthetics Ketamine, HCl 30 mg/kg, and Xylazine 
6 mg/kg, are administered intramuscularly. Using a paraspinal 
posterolateral splitting approach, the large cephalad-facing transverse 
process of the lumbar spine is identified and removed with a rongeur. The 
intervertebral disc can then be seen. An incision is made in the annulus 
fibrosus. Using a high-power surgical microscope, the nucleus pulposus 
tissue is scraped out carefully with a curette. The space is then packed 
with gel foam. The rabbit is closed provisionally. 
Example 2 
Isolation of Intervertebral Disc Cells 
Intervertebral disc tissue is obtained as described in Example 1 or from an 
amputated tail section. Under aseptic condition, the intervertebral disc 
tissue is diced with a scalpel and placed in a T25 tissue culture flask 
with Dulbecco's Modified Eagle Medium (DMEM) adjusted to pH 7.0, 
supplemented with 10% heat inactivated fetal bovine serum and 1% 
penicillin/streptomycin (TCM). The tissue is then treated with 0.25% 
collagenase for two hours at 37.degree. C. An equal amount of TCM to 
collagenase is added to stop treatment. The mixture is centrifuged at 1000 
r/min for 10 minutes and supernatant is discarded. TCM is added and the 
mixture is filtered to remove debris. The mixture is again centrifuged and 
supernatant discarded. Cells are resuspended in TCM supplemented with 1% 
hyaluronidase (400 u/ml). 
Example 3 
Culture of Intervertebral Disc Cells 
Cells are cultured in TCM supplemented with 1% hyaluronidase (400 u/ml) at 
37.degree. C. in 5% CO2/95% air. Once cells attach medium is changed to 
TCM supplemented with 0.001% ascorbic acid in the absence of 
hyaluronidase. Cells are resuspended in fresh medium supplemented with 
0.0025% ascorbic acid every 3 days. 
Example 4 
Preparation of Bioactive Glass 
Bioactive glass granules (45S5) having diameters of 40 .mu.m to 71 .mu.m 
can be obtained from Orthovita, Inc. (Malvern, Pa.). Prior to implantation 
or addition to cell culture, the specimens are sterilized in ethylene 
oxide. 
Example 5 
Preparation of Sintered Porous Bioactive Glass 
Bioactive glass granules having diameters of 40 .mu.m to 71.mu.m can be 
obtained from Orthovita, Inc. (Malvern, Pa.). The glass granules are mixed 
with 20.2 weight % sieved sacrificial agent camphor C.sub.10 H.sub.16 O 
with grain size of 300 .mu.m to 500 .mu.m. The mixture is mechanically 
mixed overnight, and cold pressed at 350 MPa. The disc obtained is heat 
treated at 575.degree. C. for 45 minutes. The heating rate is 10.degree. 
C./min. It is then left to cool at room temperature. The disc is immersed 
in acetone for 30 minutes and dried at 37.degree. C. The disc is cut to 
the desired dimensions using a diamond-wheel saw. The disc is washed in 
acetone for 15 minutes. The specimen is then conditioned in tris buffer 
with electrolytes added (TE) (El-Ghannam, et al., Journal of Biomedical 
Materials Research, Vol. 29, 359-370 (1974)), for 2 days to obtain the 
desired formation of calcium phosphate-rich layer at the glass surface. 
The specimen is rinsed with methanol and dried at 37.degree. C. The 
specimen is analyzed using scanning electron microscopy (SEM), Fourier 
Transform Infrared (FTIR) spectroscopy and X-ray diffraction (XRD). Prior 
to implantation or introduction to cell culture, the specimen is 
sterilized in ethylene oxide. 
Example 6 
Preparation of Polymer Foam 
3 g of NaCl with particle sizes 300 .mu.m to 500 .mu.m, and 2 g of D,L-PLGA 
75/25 (75% polylactide/25% polyglycolide) polymer foam were mixed. The 
dispersion is vortexed and cast in a 5 cm petri dish. The solvent is 
allowed to evaporate from the covered petri dish for 48 hrs. To remove 
residual amounts of chloroform, the petri dish is vacuum-dried at 13 Pa 
for 24 hrs. The material is then immersed in 250 ml distilled deionized 
water at 37.degree. C. for 96 hrs. The water is changed every 12 hrs to 
leach out the salt. The salt-free membrane is airdried for 24 hrs, 
followed by vacuum-drying at 13 Pa for 48 hrs. The material is then cut to 
the desired geometry with a razor blade. The membrane is stored in a 
desiccator under vacuum. The specimens are analyzed using SEM. The 
specimen will have 60% pore density with pore sizes 300 to 500 .mu.m. 
Prior to implantation or introduction into cell culture, the specimens are 
sterilized in ethylene oxide. 
Example 7 
Preparation of Polymer Foam Coated with Sol Gel Bioactive Glass 
Tetramethylorthosilane (TMOS), calcium methoxyethoxide and triethyl 
phosphate are mixed for 5 minutes in an argon atmosphere using a magnetic 
stirrer. Respective amounts of each are chosen such that the resulting 
product is 70% SiO.sub.2 -25% CaO-5% P.sub.2 O.sub.2 (upon drying). They 
are mixed using a magnetic stirrer for 5 min. The PLGA polymer foam 
prepared according to Example 5 is dipped into the sol approximately 
halfway to gelation. The foam is dipped 2 to 3 times to make sure that the 
sol completely fills the polymer foam. The sol-filled foam is then placed 
in a syringe filter with appropriate filter pore size which only allows 
the sol to flow through. This syringe filter is attached to a syringe. The 
sol is pulled through the foam by creating a vacuum below using the 
syringe. EDAX and SEM are used to analyze pore size, porosity and the 
thickness/uniformity of the sol gel bioactive glass coating. Prior to 
implantation or introduction to cell culture, the specimens are sterilized 
in ethylene oxide. 
Example 8 
Cell Phenotype 
Cell phenotype of cells cultured in accordance with the method of Example 3 
is examined. Immunofluorescent staining of cells shows positive staining 
for proteoglycan and collagen type II, markers of intervertebral disc cell 
phenotype. Substantially negative staining for collagen I, a annulus 
fibrosus marker, was also observed. 
Example 9 
Cell Reversion 
Intervertebral disc cells cultured as described in Example 3 are tested for 
reversion. Cells are placed in Eppendorf tube with TCM and spun down to 
form a pellet. Cell histology is examined after 4, 8 and 12 days by 
washing the pellet and fixing it with 70% ethanol. The cells are 
dehydrated, embedded and cut. The sample is stained with hematoxylin-eosin 
and toluidine blue. 
The histology of cultured cells is compared to the histology of nucleus 
pulposus tissue prepared immediately upon retrieval. Histology of the 
cells evidences a reversion to the original morphology of the cells. 
Example 10 
Implantation of Biodegradable Substrate 
Cells are prepared in accordance with Examples 2 and 3. Cells are counted. 
Biodegradable substrates prepared as described in Examples 4-7 are each 
placed in a tissue culture dish and immersed in TCM for 1 hour. The cells 
are seeded onto each of the sterile biodegradable substrates prepared as 
described in Examples 4-7 in TCM with hyaluronidase and left to attach for 
at least one hour before flooding the dish with TCM. Cells are incubated 
overnight. Attachment is detected using SEM. 
The rabbit treated as described in Example 1 is reopened per surgical 
technique described in Example 1, and the intervertebral disc space 
accessed. The gel foam is retrieved and the cell-biodegradable substrate 
hybrid material inserted in place. The wound is closed. 
Example 11 
Effect on Neurological Function 
Regular post-operative neurological functions are evaluated to examine the 
subject for any spinal injury such as lameness. The effect of the hybrid 
material on the behavior of the disc can be observed and generally 
compared by taking radiographs of the spine immediately pre-operation, 
post-operation and at 1 month time periods until the animal is sacrificed. 
Example 12 
Histological Analysis 
Histological analysis is performed to determine cell ingrowth, cell types, 
tissue morphology, and absence of inflammation. To this end, the retrieved 
disc is fixed in 70% ethanol and dehydrated. After embedding in methyl 
methacrylate, sections are cut with a diamond saw, ground, polished with 
silicon carbide paper and diamond paste, and stained. Histology is done on 
normal discs and discs retrieved at the various time periods. Analysis 
will show ingrowth of cells with concurrent degradation of implanted 
hybrid material with little to no inflammation.