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Treatment of DDD/Disc Regeneration: Growth Factors and Platelet Rich Plasma
the quantitative assessment of pain reduction using a properly
scaled dose injection produced in a preclinical model is not well
established. Even positive results of imaging analyses, such as
MRI and X-ray, and histological assessments indicating favorable structural changes, do not guarantee that the therapy will
provide pain relief. Some research attempts to target aspects of
pain, addressing the above points, have been made. Kawakami
83
showed that the autologous transplantation of NP tissues
et al
obtained from the rattail disc compression model into the dorsal root ganglion induced significant allodynia and that the
injection of OP-1 into the disc abrogated the pain induction.
84
Olmarker et al
also reported behavioral changes due to pain
from disc degeneration by disc puncture in the rat. These studies may provide additional clinically relevant evidence in animal models for the development of an injection therapy.
12.3.6 Placebo Effects of an Injec tion
Procedure
A recent clinical trial for fibrin sealant85and the preliminary
results of the GDF-5 clinical trial (see ClinicalTrials.gov) indicate
that the placebo effect for pain or spontaneous relief of pain are
substantial. The further understanding of placebo effects and
the selection of a patient population for an injection treatment
are essential.
12.4 Conclusion
Various studies have shown the effectiveness of growth factor
injections in preclinical models. Although some clinical trials
have been performed, the clinical effect of growth factor injection treatment is not conclusive. Although the patient number
was relatively small, a recent double-blinded study for PRP sug-
54
gested short-term efficacy.
Further research on pain aspects
after growth factor administration may fill the gap between
preclinical results and clinical efficacy. One key component is
the careful selection of appropriate patients for the stimulation
of matrix production and to avoid further disease stage
advancement due to depletion of nutrients. Multidisciplinary
efforts by physicians, biologists, and radiologists may be a key
to advance science in this field and to provide clinical benefits
to a billion back pain patients.
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109
Treatment of DDD/Disc Regeneration: Stem Cells, Chondrocytes
13 Treatment of Degenerative Disc Disease/Disc
Regeneration: Stem Cells, Chondrocytes or Other Cells, and
Tissue Engineering
Steven Presciutti and Howard An
Abstract
Intervertebral disc (IVD) degeneration remains a significant
treatment challenge, but the use of stem cells has emerged as
one of the most promising and exciting techniques to restore
the degenerated disc. Many challenges remain. The use of stem
cells in treating disc disease as well as the obstacles that remain
are reviewed in this chapter in an evidence-based manner.
Keywords: intervertebral disc degeneration, mesenchymal stem
cell, MSC, stem cells, tissue engineering
13.1 Historical Perspective
The intervertebral disc (IVD) is composed of the nucleus pulposus (NP), the annulus fibrosus (AF), and the two end plates of
adjacent vertebral bodies. Adult NP primarily contains chondrocyte-like cells capable of synthesizing proteoglycans (PGs) and
type II collagen and is contained within the AF, which principally contains fibroblast-like cells capable of synthesizing type I
collagen.
NP is characterized by attenuation of PG synthesis, development of NP dehydration, and an increase in waste products.
6
ularly implicated in the pathogenesis of IDD.
genic NP cells and their replacement by fibroblast-like cells has
also been implicated in the later stages of IDD.
imbalance in the production and degradation of extracellular
matrix (ECM) components can lead to a perturbed mechanical
balance between the NP and the AF, leading to structural compromise of the disc. This can ultimately result in a variety of
clinically significant spinal disorders including radiculopathy
and spinal stenosis. IDD has also been linked to low back pain
itself; however, the exact relationship between the two remains
unclear.
tive modalities (i.e., physical therapy, medication, epidural steroid injections) as well as eventually surgical options including
spinal fusion or artificial disc replacement if nonoperative
treatment fails.
treatment modalities has been reported, long-term outcomes
have been plagued with high rates of recurrent pain.
tion, none of the aforementioned treatment strategies focus on
halting or reversing the underlying disc degeneration process.
In this regard, regenerative cellular therapy has attracted significant attention because of its potential to directly address the
degenerative process. Cell-based therapies are part icularly
attractive in treatment of more advanced IDD, such as Pfirrmann grades IV or V (▶ Fig. 13.1).
medicine, tissue engineering, and stem cell therapies with the
aim of treating and reversing IDD as well as augmenting and
1,2
Intervertebral disc degeneration (IDD) affecting the
3,4,5,
Alterations in the cell population of the IVD have been partic-
9,10
Conventional treatment options include multiple nonopera-
11
Although short-term pain relief with these
Substantial progress has occurred in the fields of regenerative
7
Loss of chondro-
8
Over time, an
12
In a ddi-
enhancing current treatments. A stem cell can renew itself
through cell division and can exert beneficial effects on other
cell types through a variety of mechanisms (i.e., paracrine
effects, cell–cell interactions). These characteristics make stem
cells worthy of further investigation as a source of cells for
treating IDD. Although not currently routinely employed in the
management of IDD, these therapeutic characteristics of mesenchymal stem cells (MSCs) have led to an increasing number
of preclinical and clinical studies attempting to demonstrate
the efficacy and safety of progenitor cells for the treatment of
IDD. The first reported use of cell-based therapies to treat IDD
in humans was in 2005 by Haufe et al
tive results brought about intense interest among researchers
and have resulted in many more preclinical and clinical studies
investigating the role of cell-based therapies to treat IDD.
This chapter reviews the biology of stem cells, the clinical evidence concerning the use of MSCs, the hindrances to clinical
use, and the potential risks associated with the application of
MSCs for the treatment of IDD. Due to the fact that this specific
subject matter uses terminology that can often times be confusing, a list of definitions is provided to the reader in ▶ Table 13.1.
13
and those initial posi-
13.2 Determining the Ideal Cell
Type
Although considerable research has been dedicated to strategies that boost the ability of native disc cells to synthesize and
maintain the ECM, the introduction of exogenous cells such as
stem cells is gaining popularity as a potential treatment for
7
The goal of stem cell therapy is to replace or replenish dis-
IDD.
eased tissue through the localized differentiation of transplanted stem cells into native cells, which advance the healing
process or directly restore the tissue physically. In addition,
therapeutic stem cells also release trophic factors that have
been shown to stimulate the metabolism of IVD cells as well as
suppress inflammatory reactions.
cell source, however, is vital for the successful outcome of cell
15
therapy.
to obtain, and the capacity to differentiate into a desired cell
type. The cells of choice also need to be viable in the hypoxic
and the hypoglycemic environment found within the disc, with
minimal or no immune response. They should have low or no
risk for tumor growth.
Adult stem cells, like all stem cells, share at least two characteristics. First, they can make identical copies of themselves for
long periods of time; this ability to proliferate is referred to as
long-term self-renewal. Second, they can give rise to mature cell
types that have characteristic morphologies (shapes) and specialized functions. The product of a stem cell undergoing division
is at least one additional stem cell that has the same capabilities
of the originating cell. Typically, stem cells generate an
The choice of cells is based on their abundance, ease
14
Choosing the appropriate
110
Treatment of DDD/Disc Regeneration: Stem Cells, Chondrocytes
Fig. 13.1 Grading system for the assessment of
lumbar disc degeneration. (a) Grade I: The
structure of the disc is homogeneous, with a
bright hyperintense white signal intensity and a
normal disc height. (b) Grade II: The structure of
the disc is inhomogeneous, with a hyperintense
white signal. (c) Grade III: The structure of the
disc is inhomogeneous, with an intermediate
gray signal intensity. (d) Grade IV: The structure
of the disc is inhomogeneous, with a hypointense
dark gray signal intensity. (e) Grade V: The
structure of the disc is inhomogeneous, with a
hypointense black signal intensity. Grading is
performed on T2-weighted mid-sagittal images.
(Reprinted with permission from Pfirrmann et al.
Magnetic resonance classification of lumbar
intervertebral disc degeneration. Spine 2001;26
(17):1873–1878.)
Table 13.1 Cell-based therapy terminology
Adult stem cell An undifferentiated cell found in a
Allogenic Two or more individuals (or cell lines) are stated
Autologous transplant
Bone marrow The soft, living tissue that fills most bone
Bone marrow stem
cell (BMSC)
Chondrocytes Cartilage cells
Differentiation The process whereby an unspecialized early
differentiated tissue that can renew itself and
(with certain limitations) differentiate to yield
all the specialized cell types of the tissue from
which it originated
to be allogenic to one another when the genes
at one or more loci are not identical in
sequence in each organism.
Transplanted tissue derived from the intended
recipient of the transplant. Such a transplant
helps avoid complications of immune rejection.
cavities and contains hematopoietic stem cells,
from which all red and white blood cells evolve.
The bone marrow also contains mesenchymal
stem cells that a number of cell types come
from, including chondrocytes, which produce
cartilage.
One of at least two types of multipotent stem
cells: hematopoietic stem cell and mesenchymal stem cell
embryonic cell acquires the features of a
specialized cell such as a heart, liver, or muscle
cell
Ectoderm The upper, outermost of the three primitive
Embryonic stem (ES)
cells
Endoderm Lower layer of a group of cells derived from the
Hematopoietic stem
cell (HSC)
Mesenchymal stem
cells (MSCs)
Mesoderm The middle layer of the embryonic disk, which
Multipotent stem
cells
Pluripotent stem cell
(PSC)
Precursor cells In fetal or adult tissues, these are partly differ-
germ layers of the embryo; it gives rise to skin,
nerves, and brain.
Primitive (undifferentiated) cells from the
embryo that have the potential to become a
wide variety of specialized cell types
inner cell mass of the blastocyst; it later
becomes the lungs and digestive organs.
A stem cell from which all red and white blood
cells evolve
Cells from the immature embryonic connective
tissue. A number of cell types come from
mesenchymal stem cells, including
chondrocytes, which produce cartilage.
consists of a group of cells derivedfrom the inner
cellmass of theblastocyst. Thismiddle germlayer
is known as gastrulation and is the precursor to
bone, muscle, and connective tissue.
Stem cells that have the capability of
developing cells of multiple germ layers
A single stem cell that has the capability of
developing cells of all germ layers (endoderm,
ectoderm, and mesoderm)
entiated cells that divide and give rise to differentiated cells. Also known as progenitor cells.
111
Treatment of DDD/Disc Regeneration: Stem Cells, Chondrocytes
Primary germ layers The three initial embryonic germ
layers—endoderm, mesoderm, and
ectoderm—from which all other somatic
tissue-types develop
Somatic cell Any cell of a plant or animal other than a germ
cell or germ cell precursor
Stem cell A cell that has the ability to divide for indefinite
periods in culture and to give rise to specialized
cells
Totipotent Having unlimited capability. The totipotent
cells of the very early embryo have the capacity
to differentiate into extra embryonic
membranes and tissues, the embryo, and all
postembryonic tissues and organs.
intermediate cell type or types before they achieve their fully
differentiated state. The intermediate cell is called a precursor or
progenitor cell. Progenitor or precursor cells are partly differentiated cells that divide and give rise to differentiated cells. Such
cells are usually regarded as “committed” to differentiating along
a particular cellular development pathway (▶ Fig. 13.2).
Most scientists use the term pluripotent to describe stem cells
that can give rise to cells derived from all three embryonic germ
layers—mesoderm, endoderm, and ectoderm. These three germ
layers are the embryonic source of all cells of the body. All of
the many different kinds of specialized cells that make up the
body are derived from one of these germ layers (▶ Table 13.2).
Pluripotent cells have the potential to give rise to any type of
cell, a property observed in the natural course of embryonic
development and under certain laboratory conditions.
Fig. 13.2 Distinguishing features of progenitor/
precursor cells and stem cells. The product of a
stem cell undergoing division is at least one
additional stem cell that has the same capabilities
of the originating cell. Shown here is an example
of a hematopoietic stem cell (HSC) producing a
second generation stem cell and a neuron. This is
in contrast to a progenitor cell, which can yield
two specialized cells upon cell division. Shown
here is an example of a myeloid progenitor
undergoing cell division to yield two specialized
cells (a neutrophil and a red blood cell).
112
Treatment of DDD/Disc Regeneration: Stem Cells, Chondrocytes
Table 13.2 Embryonic germ layers from which differentiated tissues de-
velop
Embryonic germ
layer
Endoderm Thymus
Mesoderm Bone marrow (blood)
Ectoderm Skin
Differentiated tissue
Thyroid, parathyroid glands
Larynx, trachea, lung
Urinary bladder, vagina, urethra
Gastrointestinal (GI) organs
Lining of GI tract
Lining of respiratory tract
Adrenal cortex
Lymphatic tissue
Skeletal, smooth, and cardiac muscle
Connective tissues (including bone, cartilage)
Urogenital system
Heart and blood vessels
Neural tissue (neuroectoderm)
Adrenal medulla
Pituitary gland
Connective tissue of the head and face
Eyes, ears
Because NP cells share similarities with chondrocytes in
both phenotype and molecular markers, cells with the
capacity of differentiating toward chondrocytes have largely
been considered ideal in the treatment of IDD. Given this,
autologous chondrocyte implantation has been attempted as
a treatm ent strategy for IDD . Articular chondrocytes, such as
NP cells, prod uce aggrecan and type II collagen and can be
surgically obtained from the non–weight-bearing parts of
16
the knee. In a study by Gorensek and colleagues,
the NP of
rabbit IVDs was r emoved and a utologous chondrocytes were
transferred to the defect site. Discs injected with chondrocytes produced a hyaline-like cartilage in the center without
any elastic fibers, but with a higher collagen to aggrecan
ratiothaninNPECM.Inaporcinedenucleationmodel,
autologous chondrocytes injected with a fibrin carrier
showed dense cartilage formation by 3 months and cell via-
17
bility up to 12 months after t ransplantation.
In vitro coculture experiments have also suggested cartilage tissue
stimulates NP cell anabolism through moderation of inflam-
18
mation.
lagen ratio than NP cells,
However, chondrocytes produce a lower PG to col-
19
so further work needs to be
done to establish restoration of disc biochemical and
biomechanical properties. Chondrocyte transplantation also
suffers from some of the sam e limitations as NP cells (inva-
sive second surgery and low supply of donor tissue), so
chondrocytes themselves may not be an ideal source for cell
therapy.
Given this, multiple sources of stem cells that have the ability
to become chrondrocyte-like cells (i.e., NP cells) have also been
explored as potential candidates and are discussed below. A list
of the completed human clinical trials using cell-based therapies to treat IDD is provided in ▶ Table 13.3.
13,20,21,22,23,24
Simi-
larly, ▶ Table 13.4 provides a list of the current ongoing clinical
25,26,27,28,29,30
trials.
13.2.1 Embryonic Stem Cells and
Induced Pluripotent Stem Cells
Embryonic stem (ES cells) are pluripotent stem cells derived
from discarded early-stage frozen embryos.
back is that there are only a limited number of cell lines available at present for study. ES cells are able to differentiate
toward all der ivatives of the three primary germ layers, includ-
32
ing chondrocytelike cells.
Sheikh et al were able to show that
the subsequent transplantation of these cells into the IVD of
rabbits resulted in maintenance of the native cell’s viability as
well as their appropriate morphology.
Induced pluripotent stem cells (iPSCs) are similar to ES cells
in both phenotype and potential for differentiation, and have
also been reported to have the capacity of differentiating
Table 13.3 Clinical studies of cell therapy for intervertebral disc degen-
a
eration
Location Mode Cell type Indication N Outcome
United
States
Germa-
20,21
ny
Japan
Spain
United
States
Abbreviations: IDD, intervertebral disc degeneration; IVD, intervertebral
disc; MRI, magnetic resonance imaging; MSCs, mesenchymal stem cells.
a
Published as of December 2015.
Allogenic Juvenile
13
Allogenic Adult
22
Allogenic Bone
23
Autologous
Autolo-
24
gous
chondrocytes
mesenchymal
precursor
cells
marrow
MSCs
IVD cells IVD her-
Adipose
MSCs
IDD with
low back
pain
IDD with
low back
pain
IDD with
low back
pain
niation
with back
and / or
leg pain
IDD with
low back
pain
31
One major draw-
10 No improve-
ment
112 Improvement
of low back
pain and on
radiograph
2 Improvement
on radiograph
and on MRI
10 Improvement
of low back
pain and on
MRI
15 Improvement
of low back
pain and on
MRI
113
Treatment of DDD/Disc Regeneration: Stem Cells, Chondrocytes
Table 13.4 ongoing clinical trials of cell therapy for intervertebral disc
degeneration
Location Mode Cell type Indica-
United
States
United
States
Spain
Austria,
Germa-
28
ny
Korea
United
States
Abbreviations: IDD, intervertebral disc degeneration; IVD, intervertebral
disc; MSCs, mesenchymal stem cells.
Allogenic Juvenile
25
Allogenic Adult
26
27
Allogenic Bone
Autologous
29
Autologous
Autolo-
30
gous
chondrocytes
mesenchymal
precursor
cells
marrow
MSCs
IVD cells IVD her-
Adipose
MSCs
Adipose
MSCs
tion
IDD with
low back
pain
IDD with
low back
pain
IDD with
low back
pain
niation
with back
and / or
leg pain
IDD with
low back
pain
IDD with
low back
pain
ClinicalTrials.gov
identifier
NCT01771471
NCT01290367
NCT01860417
NCT01640457
NCT01643681
NCT02097862
Status
Active,
but not
recruiting
Active,
but not
recruiting
Active,
but not
recruiting
Recruiting
Recruiting
Recruiting
Bone Marrow Stromal Cells
Bone marrow is an excellent source of MSCs for most orthopaedic applications including chondrogenic differentiation for the
diseased IVD. Yamamoto and colleagues
marrow–derived stromal cells in a direct contact co-culture system with native NP cells. The authors found significantly
increased cell proliferation, deoxyribonucleic acid (DNA) synthesis, and PG synthesis in the NP cells. This was most pronounced in the NP cells that had direct cell-to-cell contact with
the bone marrow MSCs. These in vitro findings have also been
extended to an in vivo rat model by Allon and colleagues.
After injecting bone marrow–derived MSCs into the discs or
rabbits, the authors were able to demonstrate an increase in PG
content as well as partial restoration of both disc height and
48,49
disc hydration.
Similarly in the ovine model, the injection of
MSCs directly into the IVD has been shown to restore disc ECM,
increase disc height, and reduce both radiological and histological grading scores 6 months following injection.
Early clinical research has also been promising. Yoshikawa
22
reported two cases of autologous MSC implantation in
et al
markedly degenerative IVDs. Symptomatic and radiological
improvement was demonstrated without significant adverse
effects. In addition, a larger pilot study was subsequently conducted in which 10 patients suffering from low back pain associated with IDD were injected with autologous bone marrow–
23
derived MSCs.
The authors found that improvements in patient-reported outcomes were similar to those observed following spinal fusion. These small human studies, along with
numerous animal studies, suggest that MSC implantation could
indeed be a useful tool for the treatment of IDD.
46
investigated bone
50,51
47
toward chondrocytelike cells.33iPSCs are derived from somatic
cells by inducing the expression of ectopic reprogramming
transcriptional factors. Unfortunately, some of these factors are
known oncogenes. Although some studies showed that these
transcriptional factors can be replaced by small molecules,
tumorigenesis is still a major concern that limits the clinical
utility of both ES cells and iPSCs.
35,36
13.2.2 Mesenchymal Stem Cells
MSCs are multipotent cells derived from mesodermal tissues
and they have the capacity to differentiate toward some or all
of the specialized cell types of that original tissue. MSCs are
reported to be nonimmunogenic and, in contrast to ES cells, do
not as easily undergo malignant transformation.
MSCs have the capacity for self-renewal, enabling maintenance
of an undifferentiated phenotype in multiple subcultures.
However, in the appropriate environment, MSCs are capable of
differentiating into multiple cell types including osteoblasts,
adipocytes, chondrocytes, and neuronal cells.
tiation of MSCs toward chondrocytelike cells has been documented both in vitro and in vivo.
42,43,44,45
differentiated into chondrocytelike cells similar to those found
in the NP, whether those cells are truly NP cells remains to be
7
We next review the various tissues that have been
seen.
explored as a source for MSCs.
37,38
Moreover,
40,41
The differen-
Although MSCs can be
Adipose-Derived Stem Cells
Given the larger number of MSCs that can be obtained compared with bone marrow or other sources as well as the relative
ease of procurement, adipose-derived stem cells (ADSCs) have
gained significant attention in recent years. Multiple in vitro
34
studies have been conducted that have found these MSCs to be
a viable option for IVD regeneration.
52,53
Choi and colleagues
reported that chondrogenic differentiation of ADSCs was possible by co-cult uring them with normal, healthy NP cells in
monolayer. ADSCs cultured alone or with NP cells from degenerative discs were shown to have inferior results.
More recent studies have focused on fine-tuning the chon-
drogenic differentiation of these ADSCs in order to be more
54
similar to actual NP cells. Lu and colleagues
reported that coculture of human ADSCs and NP cells in a three-dimensional
(3D) micromass culture resulted in differentiation of ADSCs into
an NP cell–like phenotype. Likewise, Gaetani et al
39
a 3D culture technique resulted in significantly increased
55
matrix production and a 3D cell organization similar to IVD.
Other Mesenchymal Stem Cell Sources
Other stem cell sources have also been investigated for the
treatment of IDD, although most of these other cell types are
just beginning to be explored. Their true translational promise
for the treatment of IDD remains to be seen. Vadala et al
investigated the use of adult muscle tissue as a stem cell source.
40
showed that
56
114
Treatment of DDD/Disc Regeneration: Stem Cells, Chondrocytes
They used a co-culture in vitro model with NP cells and were
able to show increased matrix production and DNA synthesis of
the NP cells. Recent in vitro studies have also revealed the
57
potential for human AF cells
and NP cells58to serve as a source
of MSCs. The morbidity of harvesting one’s own disc cells is a
concern for the induction of further degeneration, however.
Others studies have recently investigated the use of synovial
cells as a stem cell source for IVD tissue engineering.
59,60,61
Sakai and colleagues62demonstrated a population of progenitor cells within the actual NP itself. Although they were able to
indentify such cells, the proportion of these cells within the disc
was markedly reduced with age and degeneration. Other studies have also found highly proliferative cells expressing stem
cell markers (i.e., Notch1, Delta4, Jagged1, C-KIT) within the
63,64
disc.
These cells have been successfully isolated and have
been shown to be capable of differentiating down the osteo-
65
genic, chondrogenic, and adipogenic lineage.
Although these
endogenous stem cells offer huge potential, it remains to be
seen whether they can be harnessed for regenerative purposes.
Human umbilical cord MSCs are multipotent and have been
reported to differentiate into chondrogenic, adipogenic, and
66
osteogenic lineages.
But the safety of allogenic transplantation
in MSCs has not yet been established. Until further studies
establish the immunologic safety of allogenic human umbilical
cord MSC transplantation in humans, the utility of human
umbilical cord MSCs is limited for the purpose of IDD treatment.
13.3 Understanding the Target
Tissue
13.3.1 Inf luence of the Intervertebral
Disc Microenvironment
To produce matrix, disc cells require an extracellular environment with sufficient oxygen (> 3%) and glucose (1 mM) and the
pH must preferably not be acidic (optimally, ~7.0–7.2). In addition, the osmolarity must be high enough to stimulate matrix
production as well as retain it locally (ideally, 350 mOsm).
67
These conditions are present in a normal, healthy disc
but in
degenerative discs, osmolarity falls as aggrecan is lost, oxygen
68
levels are variable, and the pH tends to become acidic.
These
microenvironmental conditions are very specific, quite harsh,
and differ from the native environments of the cells being considered as possible sources of MSCs (▶ Fig. 13.3). This could
impair the functioning of any such implanted cells or even
result in their death.
69
For example, Wuertz and colleagues70reported that the
acidic conditions often found in the degenerated disc caused an
inhibition of aggrecan, collagen-1, and tissue inhibitor of metalloproteinase 3 (TIMP-3) expression. They also found a decrease
in cell proliferation and viability as well as altered cell morphology of bone marrow–derived MSCs. Likewise, ADSCs were also
shown to respond negatively to the environmental conditions
found within degenerative IVDs.
52
These difficult physiological conditions found in the healthy
disc phenotype, let alone the degenerative one, poses a significant challenge. These conditions could theoretically be overcome with the use of growth factors as well as with the
preconditioning of cultured cells prior to implantation to
achieve better survival in the disc environment.
71
13.3.2 The Target Cell
To successfully develop an MSC-based therapy for the treatment of IDD it is necessary to understand the phenotype of the
target cell and ensure correct differentiation and synthesis of an
appropriately functioning tissue. NP cells have routinely been
described as chondrocytelike, with traditional chondrogenic
gene markers (collagen type II alpha 1, aggrecan, SRY (sex
determining region Y) box 9 [SOX9]) being used to assess MSC
differentiation toward an NP cell phenotype.
whereas the NP and articular cartilage share a similar matrix
composition, the ratio of PGs and collagens differs between the
two tissues. In fact, the PG to collagen ratio in NP tissue is 27:1
as compared with only 2:1 in articular cartilage tissue.
array studies have also identified differences in the phenotypic
gene profile between NP and articular cartilage cells. Whereas
72,73
However,
74
Micro-
Fig. 13.3 The intravertebral disc (IVD)‐specific
niche is an obstacle to successful IVD regeneration using cell therapy. The specialized microenvironment of the IVD is characterized by low
oxygen level, low pH, and poor nutrient supply,
factors that present a major challenge to the
survival and function of injected cells.
115
Treatment of DDD/Disc Regeneration: Stem Cells, Chondrocytes
the functional significance of these molecules in the IVD is
largely unknown, their use as phenotypic markers is vitally
75
important to help define the NP cell phenotype.
Indeed, these
markers (particularly CA12 and KRT-8, -18, and -19) are being
used in more recent studies to better demonstrate MSC differentiation toward NP-like cells and not just toward more general
chondrocytelike cells. This improvement in our understanding
of the cells that are being generated from MSCs should provide
better results moving forward.
13.4 Induction of Mesenchymal
Stem Cell Discogenic
Differentiation
13.4.1 Growth Factors
Disc cell metabolism is modulated by a variety of growth factors
acting in both paracrine and autocrine roles.
function to modulate the precise homeostasis of ECM production
versus breakdown. In addition, growth factors have been shown
to be very important in coaxing MSCs to differentiate into the
desired cell line. In a 2005 study, Steck et al
ability of adult bone marrow–deriv ed MSCs to differentiate
toward the molecular phenotype of human IVD cells with the use
of transf orming growth factor (TGF)-β. This was further expanded
78
upon by Tapp and colleagues.
In their study, they revealed that
the treatment of TGF- β could significantly stimulate expression of
PG and type I collagen in 3D-cultured sand rat ADSCs.
79
McCanless and colleagues
investigated the effects of bone
morphogenetic protein (BMP)-2 and synthetic peptide B2A on
cell proliferation and ECM synthesis by NP-like differentiated
bone marrow–derived MSCs. The authors found that B2A
induced proliferation, aggrecan synthesis, and stabilized collagen
accumulation consistent with cells of the young, healthy NP.
71
Stoyanov and colleagues
also compared standard chondrogenic differentiation protocols using TGF- β with a novel
technique utilizing hypoxia, growth and differentiation factor
(GDF)-5, and co-culture with NP cells. The authors found that
both hypoxia and GDF-5 were suitable for directing bone marrow–derived MSCs toward an NP-like phenotype. Feng et al
also examined the effects of GDF-5 on ADSCs. The authors
found GDF-5 to be a potent inducer of chondrogenesis in
ADSCs, and that ADSCs can be genetically engineered to express
prochondrogenic growth factors as a promising therapeutic cell
source for IVD tissue engineering.
In another example of genetic engineering, Yang and col-
81
leagues
engineered ADSCs to continuously express SOX9. The
authors reported improved chondrogenic differentiation in the
engineered cells, indicating a potential application in the treatment of IDD.
13.4.2 Scaffolds
Simply implanting a large number of stem cells into a target tissue can lead to a substantial amount of cell death
rather than cell differentiation and matrix production.
5,76
These factors
77
demonstrated the
Additionally, after the removal of degenerated disc tissue,
the implanted cells may need too much time to differentiate and generate new disc tissue and a more probable scenario might be t hat undesired scar tissue is formed
82
instead.
The goal t hen is to improve the viability of the
cells and to accelerate the process of matrix prod u c tion,
whichhasbeenshowntobeachievedwiththeuseof
extracellular scaffolds. These scaffolds aid in cell delivery
and provide a suitable microenvironment while the
implanted cells generate new tissue.
Thechoiceofscaffold is critical because it can directly
affect the type of cell line that forms from MSCs. Scaffold
fiber diameter and stiffness can influence cell function, proliferation, and orientation. They act as a cellular attachment
site and can provide a privileged mechanical advantage for
15
cell growth.
Agoodscaffold should also aid in the intro-
duction of chemical stimuli by immobilizing growth factors
and keeping them at a higher local concentration than in
the surrounding liquid phase. The scaffold is also an important tool for tissue engineers because it can function as a
delivery vehicle, not only allowing the implantation of cells
into the intended site but also increasing their level of local
retention the reafter. Numerous biomaterials have been
described as scaffolds in the setting of stem cell delivery
includi ng polyactic glycolic acid (PLGA), poly-d-lactide, chitosan, alginate, collagens, gels, calcium polyphosphate, and
demineralized bone matrix.
83
Bertoloet al84compared the performance of four commercially available matrixes in supporting cell differentiation into
NP-like cells. Bone marrow–derived human MSCs were seeded
in these scaffolds or embedded in alginate as a 3D control for 5
weeks. MSCs in collagen matrices and gelatin produced more
messenger ribonucleic acid (mRNA) and proteins of chondrogenic markers when compared with cells embedded in alginate
or chitosan. PG accumulation and cell survival were also higher.
Gene expression studies have shown that the phenotype of the
differentiated MSCs was similar but not completely equivalent
to that of NP cells, however.
85
Gruber and colleagues
developed a small animal model
for autologous disc cell implantation. Cells were either
placed onto a bioresorbable carrier or injected into the
80
recipient disc of the sand rat alone. Immunocytologic identification of the engrafted cells showed that they integrated
into the disc and were surrounded by normal matrix at time
points u p to 8 months after being placed. Another study has
shown the ability of hyaluronan-based hydrogels to drive
differentiation of MSCs when implanted into a nucleated
86
bovine caudal disc.
The authors found that predifferentiation of the MSCs within the hydrogel prior to implantation
was not necessary and in fact led to inferior results when
compared with direct implantation.
87
Sato and colleagues
also investigated the regeneration of
IVDs in a rabbit model. MSCs that had been cultured in a scaffold
for 7 days were allografted into the IVDs of the rabbits whose
NP had been vaporized with the use of a laser. The allografted
cells not only survived but also produced hyalinelike cartilage,
and narrowing of the disc spaces of the cell-containing scaffold
group were found to be significantly inhibited after 12 weeks.
116