Cellular Therapy for Disc Degeneration

SPINE Volume 30, Number 17S, pp S14 –S19
©2005, Lippincott Williams & Wilkins, Inc.
Cellular Therapy for Disc Degeneration
David Greg Anderson, MD, Todd J. Albert, MD, John K. Fraser, PhD, Makarand Risbud, PhD,
Paul Wuisman, MD, Hans-Jorg Meisel, MD, PhD, Chadi Tannoury, MD, Irving Shapiro, PhD,
and Alexander R. Vaccaro, MD
Study Design. Review article regarding the developing
field of cellular therapies for symptomatic disc degeneration.
Objective. To review the rationale and discuss the results of cellular strategies that have been proposed or
investigated for disc degeneration.
Summary of Background Data. Disc degeneration is a
substantial clinical problem. Disc degeneration begins
with a loss of disc cells and alterations in the extracellular
matrix of the disc. One promising approach for this problem involves the use of cells transplanted to the degenerative disc to achieve functional tissue repair.
Methods. The rationale for using cellular therapy for
disc degeneration is discussed. The basic science studies
involving cellular transplantation to the disc are reviewed
and future directions of this line of research are discussed.
Results. Although substantial work remains, the future
of cellular therapies for symptomatic disc degeneration
appears promising.
Conclusion. Continued research is warranted to further define the optimal cell type, scaffolds, and adjuvants
that will allow successful disc repair in human patients.
Key words: disc degeneration, cell, repair, scaffold.
Spine 2005;30:S14 –S19
Disc degeneration is a universal part of human aging and
in most cases results in minimal or self-limited symptoms. However, the small segment of the population that
experiences disabling spinal pain thought to be in part
attributable to disc degeneration represents a disproportionate medical, societal, and economic challenge. Current treatment options range from medicinal inflammation management, to invasive procedures including spine
fusion and recently spinal arthroplasty. Unfortunately,
all currently available approaches are limited to treating
the symptoms of the degenerative process and not the
underlying biologic alterations of the disc. Recently,
there has been a growing interest in developing strategies
to address the underlying biologic imbalances that lead
to symptomatic disc degeneration.
From the Department of Orthopaedics, Thomas Jefferson University,
Philadelphia, PA.
Acknowledgment date: February 13, 2005. Acceptance date: May 31,
2005.
The manuscript submitted does not contain information about medical
device(s)/drug(s).
No funds were received in support of this work. No benefits in any
form have been or will be received from a commercial party related
directly or indirectly to the subject of this manuscript.
Address correspondence and reprint requests to David Greg Anderson,
MD, Department of Orthopaedics, Thomas Jefferson University, 925
Chestnut Street, 5th Floor, Philadelphia, PA 19107; E-mail: greg.
[email protected]
S14
The normal intervertebral disc is sandwiched between
upper and lower osteochondral endplates and is composed of a tough, outer anulus fibrosus (AF) and gelatinous, proteoglycan-rich inner nucleus pulposus (NP),
which attracts and retains water.1,2 The disc is a specialized biomechanical structure capable of converting axial
spinal loads into tensile hoop stresses in the outer anulus
while allowing motion of the vertebral segment.
With disc degeneration, the NP proteoglycans are
progressively lost, leading to poor hydrodynamic transfer of axial stresses to the outer AF. Simultaneously, the
integrity of the AF is degraded producing radial fissures,
which extend outward toward the periphery of the disc.
The vertebral endplates are also affected by the degenerative process and undergo an ossification process that
may limit the nutritional supply to the disc. Although
progressive disc degeneration results in dramatic changes
to the biomechanical functioning of the disc, biochemical
events probably play a role in producing disabling spinal
pain. Biochemical changes possibly involved in discogenic pain production include the production and release
of inflammatory mediators and cytokines from the disc,
vascular ingrowth into anular fissures, and the stimulation of free nerve endings in the outermost region of
the disc.
As the molecular basis of disc degeneration becomes
increasingly understood, various biologic strategies to
repair or regenerate the degenerative disc have been suggested.3,4 Because the disc has only a limited intrinsic
capacity for regeneration, the therapeutic approaches are
generally geared toward the stimulation of matrix production using growth factors, genes, or transplanting
cells to repair the damaged disc matrix.5–9 This article
will review the cell-based strategies for repair of the degenerated disc and discuss future trends in this rapidly
developing area.
The Disc: A Challenging Environment for Cells
The normal NP has an acidic pH, low oxygen tension,
and paucity of basic nutrients and thus is one of the most
hostile biologic environments in the body. Mechanical
loading of the disc subjects this environment to high
pressures and complex shear stresses. The gel-like substance of the NP is normally devoid of blood vessels,
making anaerobic metabolism a primary means of generating energy. Because the disc must rely on simple diffusion to exchange metabolites and waste products
across the barrier of the vertebral endplates, the supply
of nutrients is severely limited.10 –12 Although disc cells
can survive in an environment devoid of oxygen, they are
Cellular Therapy for Disc Degeneration • Anderson et al S15
highly dependent on the availability of glucose.13 The
normal disc pH is acidic in the range of 6.9 to 7.2, as the
result of lactate production; however, metabolic stress
may cause the pH to fall as low as 6.1.14 Under highly
acidic conditions, the production of matrix proteins by
disc cells is suppressed.15
To survive in this harsh environment, disc cells are
highly specialized. These cells function quite well in a
mildly acidic environment with a lowered oxygen tension.15 Disc cells use specific signaling pathway activity,
which facilitates survival in the specialized, low-oxygen
microenvironment.16 Within the disc, the cell concentration is relatively sparse, making up only about 1% of the
disc volume. This low concentration of cells may be
adaptive to the environment, which has a limited supply
of nutrients to support cell proliferation.
At least two morphologically distinct cells types populate the NP region of the disc in young animals. One cell
type is small and round, similar to a chondrocyte. The
second cell type is much larger, with a vacuolated appearance and prominent intracellular glycogen deposits.17 The larger cells are thought to be a remnant of the
primitive notochord.18 At the ultrastructural level, the
large NP cells contain multiple intracellular vacuoles and
prominent Golgi apparatus, suggesting that these cells
are actively involved in the synthesis of proteins. However, few mitochondria are seen, probably because of the
relatively poor supply of oxygen and a reliance on anaerobic energy pathways (Figure 1).
In humans, the population of notochordal cells diminishes rapidly during childhood, leading to a paucity of
these cells in the adult disc. Until recently, no reliable
distinguishing cell markers were available to definitely
identify the NP or notochordal cell phenotype.11,19 Recently, however, certain cell markers, used in combina-
tion, have been suggested to define NP phenotype.20
Rajpurohit et al found that differential expression of the
two HIF-1 (hypoxia inducible factor) isoforms, HIF-1␣
and HIF-1␤, along with the expression of GLUT-1 and
MMP-2 markers, can provide a phenotypic signature
that permits NP cells to be distinguished from neighboring tissues.20
Whole Disc and Disc Tissue Transplantation
Frick et al attempted to address the degenerative process
by transplanting whole discs along with the endplates to
the spine of mature dogs.21 Although technically feasible, the biologic outcome of this study was disappointing
in that proteoglycan synthesis and NP morphology were
substantially degraded by the 4-month time point.
Katsuura and Hukuda implanted cryopreserved intact allogeneic discs to the intervertebral disc space of
dogs following discectomy.22 As with the Frisk et al
study,21 the authors observed modest preservation of
disc architecture over the short-term, but progressive degeneration with longer follow-up. These findings may be
due in part to the difficulty in maintaining adequate viably of the cryopreserved disc cells as suggested by the
work of Nishimura and Mochida.23 These researchers
compared cryopreserved NP to fresh tissue transplants in
a rat nucleotomy model.23 Although both fresh and
cryopreserved disc tissue decelerated degeneration compared with an artificial substitute, the fresh tissue transplants significantly outperformed the cryopreserved tissue in maintaining disc height.
Luk et al transplanted whole disc/endplate constructs
to the spines of 14 Rhesus monkeys and demonstrated
successful healing of the implants but noted early loss of
disc height.24 However, with additional follow-up, there
was partial reconstitution of disc height and only minimal disc degeneration noted by the 12-month time
point.24
Transplantation of Cultured Disc Cells
Figure 1. Electron micrograph of nucleus pulposus (NP) cell from
adult rat disc. Note that cell is surrounded by dense matrix (M) and
has many vesicles (V) filled with a ground substance. Cell shows
extensive Golgi (GA) network but very few mitochondria suggesting its special metabolic status (original magnification ⫻10,000)
Okuma et al documented slowing of the degenerative
process in rabbit discs following the insertion of cocultured NP and AF cells.25 In another study, this group
compared intact NP transplants with cultured NP cells in
a degenerative, young rabbit model that had previously
undergone NP aspiration.26 Two weeks after the induction of degeneration, intact NP tissue or cultured NP
cells (up to 50,000 cells) from an allogeneic donor were
injected into the central region of the rabbit discs. At the
14-week time point, the discs treated with intact NP tissue demonstrated less degeneration than discs treated
with cultured NP cells or untreated controls. Unfortunately, data on the survival of cultured donor cells were
not included, making it impossible to determine if the
relatively poor performance of the transplanted cells was
due to a lack of cell survival or was related to the efficacy
of cell transplantation strategy. Cell viability has been
shown to affect the outcome of other related tissue engi-
S16 Spine • Volume 30 • Number 17S • 2005
neering strategies, making analysis of cell survival an
important step in cell research design.27
Gruber et al analyzed the survival and function of
cultured autologous disc cells in the sand rat (Psammomys obesus), an animal that undergoes spontaneous disc
degeneration.28,29 In this study, an average of 10,000
cultured disc cells embedded in a 2-mm3 collagen matrix
were implanted into a defect in the lumbar disc. The cells
were labeled before implantation to allow postimplantation tracking. Labeled cells were seen in the discs of animals as late as 8 months postimplantation, but the efficacy of disc repair was not reported.
Ganey et al transplanted autologous cells to the disc of
skeletally mature dogs following a posterolateral aspiration injury (⬃100 mg of AF and ⬃100 mg of NP were
removed).30 Twelve weeks later, approximately 6 million autologous cells cultured from tissue aspiration were
injected into the NP region of the injured discs. The animals were followed for 12 months and disc height was
measured with high resolution radiography. Significant
improvements in disc height were noted at the 12-month
time point in the cell-treated discs compared with control
discs, although prior time points failed to show a significant difference. This delay may reflect the slow biosynthetic rate of NP cells in the disc. Histologic evaluation at
the 6-, 9-, and 12-month time points indicated less scarring and more proteoglycan content in the cell-treated
discs compared with controls. The labeled cells survived
in vivo at least 6 months. Despite the improvements in
the cell-treated discs, none of the treated specimens regained normal architecture by the 12-month time point.
Using a similar approach, Ganey and Meisel conducted a human pilot safety study and have initiated a
pivotal study using autologous NP cells derived from
therapeutic discectomy that were cultured under Good
Manufacturing Practices and delivered 12 weeks following discectomy.31 In this nonrandomized and unblinded
study, a preliminary data analysis has demonstrated
some examples of MR imaging improvements consistent
with increased proteoglycan matrix within the NP.31 Although encouraging, these findings require validation
from prospective, randomized clinical studies before this
approach can be widely recommended in humans.
Implantation of Disc Cell/Scaffold Constructs
Many different types of scaffolds have been used for the
growth of disc cells. The purpose of a cellular scaffold is
to provide an optimal microenvironment for cellular migration and proliferation that allows the cells to maintain the appropriate phenotype. It is well known that NP
cells will alter their phenotype depending on the culture
technique used; therefore, selection of an appropriate
scaffold and culture technique is crucial.
Alini et al successfully proliferated bovine coccygeal
disc cells using a collagen/hyaluronate and chitosanbased scaffold.32,33 Se´guin et al grew bovine caudal NP
cells using a sintered calcium polyphosphate scaffold and
reported successful production of proteoglycans and col-
lagen but noted that the engineered tissue fell short of
reaching levels of proteoglycan production found in the
native disc.34
Sato et al used a honeycomb-shaped scaffold of modified Type II collagen to grow AF cells and later reported
placing these tissue constructs into laser-mediated defects in rabbit discs.35,36 Using a lipophilic dye to track
the cells, survival of the implants was documented out to
the 12-week time point along with robust proteoglycan
production and disc height maintenance.
Mizuno et al used a composite scaffold composed of
an outer region of polyglycolic acid polymer and an inner
region of alginate/calcium sulfate to grow AF and NP
cells, respectively.37 After a 24-hour culture period, the
constructs were implanted to the subcutaneous space of
athymic mice. Cell survival and matrix protein production were documented over the 12-week follow-up period; however, the amount of proteoglycans produced
fell well short of the levels expected in the native intervertebral disc.
Use of Non–Disc-Derived Cells for Disc
Tissue Engineering
Because autologous disc cells cannot be obtained without causing morbidity to the disc and because disc cells
from a degenerative disc may be functionally impaired,
alternative cell types have been considered for disc tissue
engineering. Candidate cell populations must be capable
of producing large amounts of proteoglycans and Type II
collagen (chondrogenic) when placed under suitable conditions. Two good cellular candidates for disc tissue engineering include chondrocytes and mesenchymal stem
cells (MSCs), which may be rendered chondrogenic depending on the microenvironment and/or culture conditions used. MSCs may be obtained from several sources,
but bone marrow38,39 and adipose tissue40 – 43 (adiposederived stem cells) appear to be the most promising
clinically. Although MSCs from different sources have
distinct cell culture requirements42,43 and differ in the
expression of certain cell surface markers,44,45 they share
the ability to generate chondrogenic cells.40,46 Some data
suggest that adipose tissue may be a richer source of
progenitor and stem cells than marrow.44,45,47
Recently, Risbud et al have demonstrated that bone
marrow-derived MSCs can be differentiated into NP-like
cells using microenvironmental conditions similar to
those found in the intact disc.48 Using a combination of
low oxygen tension and high osmolarity, the MSCs took
on the morphologic appearance of NP cells and formed
clusters similar to that seen in the central region of the
disc. In addition, the expression profile of the engineered
MSCs was noted to be similar to NP cells from the disc
(Figure 2). In theory, these engineered cells might be used
to repopulate the NP region of the disc after degeneration has ensued.
Sakai et al cultured autologous, bone marrow-derived
MSCs on a honeycomb-shaped scaffold of modified Type
II collagen.49 The constructs were implanted into rabbit
Cellular Therapy for Disc Degeneration • Anderson et al S17
Figure 2. Differentiation of mesenchymal stem cells (MSC) into
NP-like cells in vitro. MSC were cultured in alginate beads in
hypoxia (Hx) or normoxia (Nx) for 7 days and assessed for their
morphologic and phenotypic characters. I, Celltracker Green
staining of MSC. A, Untreated cells after 24 hours in alginate. B,
Cells treated with TGF-␤1 in normoxia after 7 days in culture. C,
Cells treated with TGF-␤1 in hypoxia after 7 days in culture. Note
that after 7 days in hypoxia, cells forms large aggregates resembling NP cell clusters (arrow). II, Western blot analysis of hypoxia
responsive gene products expressed by MSC. MSC express
HIF-1␣ and MMP-2 in hypoxia. Note that all these genes are
expressed by NP cells in vivo and therefore indicate differentiation
of MSC toward an NP-like phenotype. III, RT-PCR analysis of
genes expressed by MSC cultured in alginate beads in hypoxia
and normoxia. After 1 week in culture, in hypoxia, there is a
significant increase in the level of expression of aggrecan, Type II
and Type XI collagen, and Sox-9.
discs 2 weeks following aspiration of the NP and then
followed for an 8-week period. Using the lac Z reporter
gene, cell viability was documented for at least 4 weeks,
and disc height maintenance and proteoglycan production were observed.
Crevensten et al used MSCs grown in a hyaluronate
gel to populate the coccygeal discs of Sprague-Dawley
rats.50 Using cell labeling, viable cells were detected over
the 28-day study period. The cell numbers initially decreased and then increased, suggesting proliferation of
the cells within the disc. Although normal disc height
was not maintained, this study was limited by the small
size of the rat discs and the relatively small volume of
injected cells.
Leo et al labeled fat and bone marrow-derived MSCs
with the luciferase marker gene and encapsulated the
cells in alginate beads before placing the cell containing
beads into a small defect created in the anterior region of
the intervertebral disc.51 Using a specialized, noninvasive
detection system, the fate of the implanted cells was able
to be followed in the live animals over a 14-day period
(Figure 3). Although cell survival and retention in the
disc region were documented, imaging also detected cells
in the lungs and liver suggesting leakage or migration of
some of the labeled cells. Given the large defect in the
disc used in this study, it is not particularly surprising
that some of the implanted cells were not retained within
the disc.
In addition to the direct production of matrix proteins, MSCs have been shown to stimulate proteoglycan
production by native disc cells. In one experiment,
Yamamoto et al cocultured MSCs in contact with autologous NP cells, using a 0.4-␮m mesh to separate the two
cell types.52 The cocultured NP cells up-regulated the
production of both DNA and proteoglycan synthesis,
suggesting that soluble factors released by the MSCs
stimulated the metabolic output from the NP cells.
Enhancement of Cell Function with Growth Factors
and/or Genes
Multiple growth factors including transforming growth
factor-beta (TGF-␤), epidermal growth factor (EGF),
bone morphogenetic protein-2 (BMP-2), and bone
morphogenetic protein-7 (BMP-7, also referred to as
OP-1)53–58 have also been shown to stimulate matrix
production by disc cells. For instance, Kim et al found a
200% increase in proteoglycan synthesis and 450% increase in aggrecan mRNA in human disc cells exposed to
high doses (up to 2,000 ng/mL) of recombinant human
BMP-2.58 These data suggest that using growth factors
or growth factor genes may improve the success of a
cellular approach to disc tissue engineering.
Paul et al performed adenovirus-mediated transduction of rabbit NP cells with the Sox 9 gene and injected
the gene vector directly into the intervertebral disc of
skeletally mature New Zealand white rabbits immedi-
Figure 3. In vivo bioluminescent
imaging of a rat on postoperative
day 7 following implantation of
fat stem cells transduced ex vivo
with Ad-luc virus carrying the luciferase marker gene.
S18 Spine • Volume 30 • Number 17S • 2005
ately following a stab injury.59 The transduced cells exhibited increased production of collagen II, while the
injected discs exhibited less scarring and improved retention of matrix at the 5-week time point.
Discussion
Clearly, the biology of the intervertebral disc is complex
and represents a significant challenge to those seeking to
achieve successful tissue repair in this relatively hostile
environment. At the current time, the science of disc cell
transplantation is in its infancy. Issues that need to be
worked out include defining the optimal cell type for disc
repair, determining whether cellular scaffolds are necessary or beneficial, determining the optimal mechanobiologic environment for cell proliferation and proteoglycan
production, and understanding which growth factors or
genes may be used to enhance the biologic performance
of the implanted constructs.
Many questions remain. For instance, it is not yet
known whether MSCs are truly capable of forming mature disc cells or whether these engineered cells only
mimic NP cells. Also, long-term survival of MSCs transplanted to the disc has not yet been shown. Because both
autologous and allograft MSCs are readily available, the
characterization of MSCs as a potential source of cells to
repair the disc is an important issue. As better (more
specific) cellular markers of the NP phenotype become
known and longer-term studies using MSC cell transplants to the disc are performed, it should be possible to
make this determination.
The lack of an ideal animal model of human disc degeneration also represents a challenge to the field of cellular disc repair. Virtually all available animal models
have significant differences from the human degenerative
condition. Although much useful information has been
obtained through animal research, most of the existing
studies have been performed on relatively young rodents
or rabbits with normal or recently injured discs. Compared to humans, these models may be more readily able
to achieve tissue repair. Studies using larger animals and
involving discs with more advanced stages of degeneration are necessary as researchers move toward the goal of
achieving meaningful disc tissue repair in humans.
Another limitation of the current approach to regeneration of the degenerated disc is the relatively poor link
between disc degeneration and spinal symptoms. In humans, the real clinical problem is spinal pain and not disc
degeneration per se. The assumption has been that many
patients have pain from disc degeneration and, therefore,
disc repair is a reasonable goal to alleviate the clinical
symptoms of spinal pain. However, this assumption remains unproven. Because it is not possible to reliably
measure pain in animal models, researchers have been
forced to use surrogate methods to evaluate the success
of disc repair experiments by quantifying the histologic
or biochemical status of the disc.
Another issue that must be defined before tissue repair
can be widely adopted in the clinical arena is the optimal
stage at which to treat a patient with painful disc degeneration. It is theoretically possible that intervention will
work best if applied at a very early stage of degeneration,
but often the presence of symptoms is minimal in early
disc degeneration. Alternatively, different disc repair
strategies may have to be developed for different stages of
disc degeneration. Clearly, substantial work remains to
be done in the area of disc tissue engineering.
Fortunately, the field of tissue engineering is rapidly
evolving, and successes thought impossible only a few
years ago have already been achieved. Therefore, continued research into the area of disc repair is clearly warranted. There is reason for optimism as we continue to
search for a successful, biologically sound method of
addressing the problems of human disc degeneration.
Conclusion
Given our current state of knowledge, it appears that
several cell types have the ability to survive, proliferate,
and participate in matrix production within the disc for
at least short periods of time. Some studies suggest that
cell implantation to the disc may help to maintain disc
height and improve biomechanical properties of the disc
following early induced degeneration. Currently, however, the optimal cell therapy and the use of adjuvants
such as cell scaffolds, genes, or growth factors remain to
be defined. Further research is necessary to define the
optimal combination of cells, scaffolds, and growth factors that will provide meaningful, long-term improvement in the biologic integrity of the intervertebral disc.
Key Points
● Disc degeneration is a ubiquitous process that
involves failure of the intrinsic disc cells to maintain normal matrix architecture within the disc.
● Implantation of cells to the disc is a promising
approach that may be useful in repairing the degenerative disc.
● Various cell types, including disc cells, chondrocytes, and stem cells, have been investigated for
disc repair.
● Cellular scaffolds, which provide a microenvironment for the implanted disc cells may be useful
in the repair strategy.
● Growth factors and genes may also be used to
modulate the repair process within the disc.
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