Stem Cell-Mediated Intervertebral Disc Regeneration

Currently, degenerative disk disease (DDD) and the subsequent chronic lower back pain that results from it represent a significant source of morbidity and mortality worldwide. The available treatment modalities such as pain therapy and surgical interventions aim to provide symptomatic relief; however, they do not address the underlying pathophysiology of DDD. The disease also has high societal health care costs (Chan et al., 2006; Cassinelli et al, 2001). Many modalities exist for symptomatic treatment of this condition, including bed rest, massage, stretching, strengthening exercises, physical therapy, epidural injections and other pain management therapies, and spinal surgery. Most conservative therapies are


Introduction
Currently, degenerative disk disease (DDD) and the subsequent chronic lower back pain that results from it represent a significant source of morbidity and mortality worldwide.The available treatment modalities such as pain therapy and surgical interventions aim to provide symptomatic relief; however, they do not address the underlying pathophysiology of DDD.The disease also has high societal health care costs (Chan et al., 2006;Cassinelli et al, 2001).Many modalities exist for symptomatic treatment of this condition, including bed rest, massage, stretching, strengthening exercises, physical therapy, epidural injections and other pain management therapies, and spinal surgery.Most conservative therapies are  attempted before surgery with the intent to spare patients the possible complications associated with surgical intervention.However, these conservative measures and even surgery itself with its associated risks only address the symptoms with no impact on the disease process in the disc itself.Recent research has given further insight into the pathogenesis of DDD, which has borne out a renewed interest in biologic therapies centered on the nucleus pulposus (NP) and the annulus fibrosus and the potential of stem cells to reverse the disease process at a histological and cellular level.In this chapter, we will systemically review the current literature and the most salient studies regarding biologic therapies in the regeneration of the intervertebral disc (IVD).We go on to describe the direction this field is heading in and the future potential of the therapies being developed using ESCs.

Basic science laboratory studies
Before examining the utility of stem cells in human and animal models, it is important to review several of the basic science benchtop laboratory studies that have provided the rationale for in-vivo testable treatments and hypotheses.These studies examined factors influencing both mesenchymal and embryonic stem cell proliferation and differentiation towards a NP-like phenotype.We will examine how these studies have provided valuable information regarding multiple factors that can stimulate embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs) towards a chondrocytic lineage, as well as factors that can inhibit this differentiation in basic in-vitro models.

Genetic studies
DDD is a condition that rises from a combination of a genetic predisposition (Chan et al., 2006) along with environmental modifiers (Stokes & Iatridis, 2004).Several causes of age-related degeneration of the IVD include loss of biomechanical support by surrounding muscular and ligamentous structures, uneven force loading as the aging spine deforms while trying to compensate for these changes, cell senescence, loss of viable progenitor cells, accumulation of degraded matrix molecules, and fatigue failure of both the disc matrix and surrounding annulus fibrosus.Correlations have been made between DDD and collagen, aggrecan, and matrix metalloproteinase polymorphisms coding for structural proteins (Ala-Kokko, 2002).

Factors influencing stem cell proliferation
In order to further study how these cells would interact in various factor environments, it became crucial to more fully characterize these cells.This point is very important with regard to stem cell research because it is essential to characterize and identify what factors provide the best type of environment to stimulate ESCs and MSCs to differentiate toward a chondrocytic-type cell lineage.

Mesenchymal Stem Cells
Transforming growth factor-β3 (TGF-β3) is one factor that has been shown in multiple studies (Steck et al., 2005;Risbud et al., 2004;Shen, 2009) to stimulate cells to differentiate into chondrocytes.Several studies have shown that after TGF-β3 stimulation, MSCs turned positive for collagen type II protein and expressed a large panel of genes characteristic for chondrocytes, such as aggrecan, decorin, fibromodulin, and cartilage oligomeric matrix protein (Steck et al., 2005;Risbud et al., 2004).Shen et al. have shown that bone morphogenic protein-2 (BMP-2) can help to enhance TGF-β3-mediated chondrogenesis in MSCs (Shen, 2009).The combination of BMP-2 and TGF-β3 in alginate culture was found to be superior to the standard differentiation method using TGF-β3 alone as evinced by increased mRNA expression of aggrecan, type II collagen, Sox-9, BMP-2, and BMP-7, all of which are chondrocyte markers.This effect was even more pronounced when TGF-β3 and rhBMP-2 were both added (Kuh et al., 2008).This synergistic effect was consistently found in the study, providing further support as to an as yet unknown pathway towards chondrocytic differentiation.

Embryonic Stem Cells
Hoben et al performed a similar characterization study using human ESCs (Hoben et al., 2009).Growth factors were studied with a coculture method for 3 weeks and evaluated for collagen and glycosaminoglycan (GAG) synthesis.The growth factors studied were TGF-β3, BMP-2, BMP-4, BMP-6, and sonic hedgehog protein.The investigators found that the combination of BMP-4 and TGF-β3 within the fibrochondrocyte coculture led to an increase in cell proliferation and GAG production compared to either treatment alone.Koay et al had similar results with BMP-2 and TGF-β3 leading human ESCs down a differentiation path that produced an end product with high type I collagen content (Koay et al., 2007).However, they also found that human ESCs treated with TGF-β3 followed by TGF-β1 and IGF-1 produced constructs with no collagen I, showing that different growth factor application in different temporal sequences can have a marked impact on end-product composition and biomechanical properties.The importance of temporal sequences cannot be understated with regard to stem cell development and has important implications pertaining to harvesting and large-scale production of these cells for future potential therapeutic uses.

Stem cell growth in the native IVD microenvironment
Several groups have conducted well-designed in-vitro studies that have gone one step beyond identifying environmental factors that affect differentiation of stem cells into NPlike cells, and have actually studied how these factors may correlate to the current in-vivo microenvironment of the IVD.This was done in order to obtain a clear picture of what would happen if these stem cells were implanted into these native biological conditions.Culturing under IVD-like glucose conditions (1.0 mg/mL glucose) stimulated aggrecan and collagen I expression and deposition.IVD-like osmolarity (485 mOsm) and pH (pH = 6.8) conditions, on the other hand, strongly decreased proliferation and expression of matrix proteins.Combining these conditions resulted in decreased proliferation and gene expression of matrix proteins, demonstrating that, in this case, osmolarity and pH play a larger impact in inhibiting differentiation than glucose does in stimulating it (Wuertz et al., 2008).
Another study by the same group showed that acidity caused an inhibition of aggrecan and collagen I expression, as well as a decrease in proliferation and cell viability.This demonstrates that pH may be the major limitation for stem cell-based IVD repair (Wuertz, 2009).This also illustrates the importance of early intervention and the role of predifferentiation when planning to use stem cells for reparative treatments.However, some studies have shown that implantation of stem cells at a later stage in the DDD process may result in a greater increase in disc height when compared to implantation at an earlier stage (Ho et al., 2008).This finding highlights the importance of studies involving stem cellbased intervertebral disc regeneration being carefully controlled in the context of stage of disc degeneration.Again, this point highlights the importance of temporal sequence when examining therapeutics with stem cells.Additionally, inflammatory processes have been shown to inhibit the chondrogenic differentiation of stem cells, whereas hypoxic conditions exert beneficial effects on chondrogenesis and phenotype stability of transplanted stem cells (Felka et al., 2009).

Optimizing conditions to promote proliferation
There is currently an avid interest in using our accumulated data and knowledge of the factors influencing stem cell proliferation and the exact conditions in the native IVD microenvironment to optimize the chances for stem cell proliferation.
Multiple studies have investigated culturing MSCs with NP cells in a co-culture system, allowing for cell-to-cell contact (Yang et al., 2009;Le Maitre et al., 2009;Vadalà et al., 2009;Richardson et al., 2006;Richardson et al., 2008).This contact has been shown to stimulate these MSCs to differentiate toward a chondrocytic lineage, therefore removing the need for pre-differentiation in-vitro (Watanabe et al., 2010;Svanvik et al., 2010;Niu et al., 2009;Wei et al., 2009;Tao et al., 2008;Le Visage et al., 2006;Richardson et al., 2006).This was evidenced by mRNA expression levels of Type II collagen and aggrecan being elevated in co-cultured cells and cells undergoing morphological changes to form three-dimensional micromasses expressing collagen-2, aggrecan, and Sox-9 at RNA and protein levels after 14 days of coculture.These changes were unique and not detected in the samples of stem cells cultured alone (Svanvik et al., 2010;Niu et al., 2009;Wei et al., 2009).Furthermore, MSCs from older individuals differentiate spontaneously into chondrocyte-like NP cells upon insertion into NP tissue in-vitro, and thus may not require additional stimulation to induce differentiation.This is a key finding, as such a strategy would minimize the level of external manipulation required prior to insertion of these cells into the patient, thus simplifying the treatment strategy and reducing costs (Le Maitre et al., 2009).
Adipose-Derived Stem Cells (ADSCs) have also been shown to be able to differentiate into NP cells in multiple in-vitro studies (Xie et al., 2009;Tapp et al., 2008;Lu et al., 2007;Lu et al., 2008;Li et al., 2005).Soluble factors released by NP cells direct chondrogenic differentiation of ADSCs in collagen hydrogels, and combination with a nucleus-mimicking collagen type II microenvironment enhances differentiation towards a more pronounced cartilaginous lineage (Lu et al., 2007;Lu et al., 2008).
Studies using annulus fibrosus cells isolated from nondegenerated intervertebral discs have shown that these cells have the capability of differentiating into adipocytes, osteoblasts, chondrocytes, neurons, and endothelial cells in-vitro.These cells may also be induced to become more plastic, allowing them to differentiate along more mesenchymal lineages (Li et al., 2005;Feng et al., 2010;Saraiya et al., 2010).However, when annulus cells are differentiated into a chondrocyte micromass, it was not as rounded or compact as that which occurs with stem cells induced into chondrocyte differentiation (Saraiya et al., 2010).TGF-β stimulation of fetal cells cultured in high cell density led to the production of aggrecan, type I and II collagens and variable levels of type X collagen, although fetal cells had lower adipogenic and osteogenic differentiation capacity than MSCs and variability in matrix synthesis was observed between specific donors (Quintin et al., 2009;Quintin et al., 2010).

Animal studies
Many studies using stem cells for disc regeneration have been performed in a wide array of animal models with promising results.Two recent studies were conducted utilizing ADSCs in a murine (Jeong et al., 2010) and a canine model (Ganey et al., 2009).Staining in both studies demonstrated increased Type II collagen and aggrecan in the transplantation group.Additionally, at 6 weeks after transplantation, discs exhibited a restoration of disc hydration and MRI T2 signal intensity and more closely resembled the healthy controls as evidenced by matrix translucency, compartmentalization of the annulus, and increased cell density within the nucleus pulposus.Discs also showed a significantly smaller reduction in disc height when compared with controls.
Multiple studies have shown that MSCs are able to proliferate and survive inside the IVD, with assessments being made as far out as six months post-transplant (Tan et Sakai et al., 2005).Cells were also shown to exhibit NP phenotypic markers (Sakai et al., 2005).The injected discs had a central NP-like region which had a close similarity to the normal biconvex structure of the IVD and contained viable chondrocytes forming a matrix like that of the normal disc (Sakai et al., 2003;Revell et al., 2007).Omlor et al. studied the practical phenomenon of transplanted stem cell loss through the actual annular puncture which was used to not only simulate disc damage and herniation but also to inject the stem cells themselves.They made a logical conclusion that IVD regeneration strategies should increasingly focus on annulus reconstruction in order to reduce implant loss due to annular failure (Omlor et al., 2010).Most studies focusing on this point are still ongoing.Many of the stem cells in these studies were xenografted from other species and the recipient animals were not treated with immunosuppressive agents.In spite of this, there was a lack of immune response suggesting an unrecognized immune-privileged site within the intervertebral disc space (Wei et al., 2009;Sheikh et al., 2009).On top of this, there has been some study with MSC showing that transplantation contributes to this immunosuppressive phenomenon by the differentiation of these cells into cells expressing FasL, which has been shown to be an immunosuppressive factor (Hiyama et al., 2008).(Sheikh et al., 2009).This study used a needle puncture model with appropriate controls to simulate disc injury.The effects of implanted murine ESCs were measured at 8 weeks using imaging, histological, and immunohistochemical analyses.In-vivo new notochordal cell populations were seen in ESC-injected discs, providing convincing evidence for stem-cell mediated regeneration of the IVD.Another study established the utility of stem cells implanted at 12 weeks post-injury in regenerating the IVD and maintaining perfusion to the endplate and subchondral bone in a porcine model (Bendtsen et al., 2010).Sobajima et al used a rabbit model to show that IVD cells harvested 48 weeks post-implantation revealed a restoration of both glycoprotein content and matrix characteristics (Sobajima et al., 2008).These analyses all provide further evidence that ESC transplantation does have strong potential for clinical use in regenerating the IVD and reversing the cascade of degeneration that occurs with time.

Human studies
To date, there have been only two studies where stem cells were injected into the IVD in humans to stimulate regeneration of the disc.Yoshikawa et al percutaneously grafted MSCs into degenerated IVDs in two women aged 67 and 70 years.After two years, both individuals had alleviation of symptoms and radiographic changes that included improvement of vacuum phenomenon on X-ray and increased signal intensity of IVDs on T2-weighted MRI (Yoshikawa et al., 2010).Another study involved intradiscal injection of hematopoietic stem cells into ten patients that had confirmed disc pain and these patients' pain was assessed at 6-month and 12-month intervals.In contrast to previous study, none of these individuals had any relief of symptoms (Haufe et al., 2006).These trials suggest that stem cells have the potential to relieve symptoms of DDD and restore normal IVD anatomy; however, more human studies are needed to truly establish this.

Future potential of ESCs
Although many laboratory and animal studies have been performed utilizing stem cells for the purposes of cell characterization and inducing chondrocyte formation, much further study is needed before human trials are undertaken on a larger scale.Several studies have already showcased the ability of ESCs to differentiate towards a chondrocytic lineage invitro and also to improve DDD in in-vivo animal and human trials, using a combination of imaging and histological analyses.Several benchtop lab studies have been performed to show that ESCs can be successfully stimulated to differentiate into chondrocyte-like cells (Hoben et (Kramer et al., 2000).Injection of ESCs in a DDDinduced rabbit model led to viable notochordal-type cells within the discs (Sheikh et al., 2009).These animal studies demonstrate the ability of ESCs to differentiate into a chondrocytic lineage in-vitro and in-vivo.
Our group is currently developing chondroprogenitor stem cell lines that can restore the functional capability of the IVD (Sheikh et al., 2009).Our rationale stemmed from the idea that currently there is no biologic therapy for repairing a degenerated IVD and that ESCs have a potential to fill this role based on their regenerative potential.Studies have shown that ESCs can be induced to differentiate into specific cell lineages by using selective culture media and growth environments (Kawaguchi et al., 2005).
Relying on the significant strides made by these basic science groups with regard to cell and factor characterization, our lab proceeded for further refine these methods and develop a protocol for both stem cell differentiation along a chondrocytic lineage and also for examining the utility of transplantation of these cells in a rabbit model of DDD.We initially developed a novel percutaneous animal model of disc degeneration using New Zealand white rabbits (Figure 1) and used this model to explore the possibility of ESC implantation for both structural regeneration and for the growth and continued presence of notochordal stem cells in the disc space (Sheikh et al., 2009).
Previous research transplanting MSCs into degenerated rabbit discs has shown consistent biochemical and radiographic (MRI) evidence of IVD restoration (Sakai et al., 2005).Human A. MSCs have also been investigated for their bone-forming capabilities with good results (Jaiswal et al., 1997).Stem cells are already being used in therapeutic applications with placement of cells directly at the site of intended spinal fusion during open surgical procedures.

B.
Our lab has developed chondroprogenitor cells lines that can restore the functional capacity of the IVD, with these cells differentiating into chondrocytes.Using our novel percutaneous model of disc degeneration in a rabbit model, we obtained MRIs preoperatively and at 2, 4, and 8 weeks postoperatively (Figure 2).Before implantation, ESCs were cultured with cisretinoic acid, TGF-beta, ascorbic acid, and insulin-like growth factor to induce differentiation along a chondrocyte lineage.After MRI confirmation of disc degeneration, the discs were then injected with murine ESCs that were labeled with mutant green fluoroscent protein (GFP).At 8 weeks post-implantation, IVDs were harvested and analyzed with hematoxylin and eosin staining along with immunohistochemical analyses (Figure 3).Three groups were analyzed: group A consisted of control animals with nonpunctured discs; group B consisted of control animals with experimentally punctured discs; and group C consisted of animals with experimentally punctured discs that were subsequently implanted with ESCs.Gel electrophoresis was used to analyze ESCs for cartilaginous tissue formation.MRI confirmed IVD degeneration after needle puncture starting at 2 weeks postoperatively.Postmortem histological analysis of group A IVDs showed chondrocytes, but no notochordal cells.Group B disc displayed intact annulus fibrosus but disorganized A. fibrous tissue in the NP.Group C discs showed new notochordal cell growth, indicating survival and proper differentiation of the injected ESCs.Fluorescent microscopic analysis was positive in group C tissue, confirming the viability of GFP-labeled ESCs within the injected IVD.In addition, the notochordal cells in group C stained positive for cytokeratin and vimentin, providing further evidence of their chondrocyte origin.There was no inflammatory response in group C discs, indicating no cell-mediated immune response.

B.
Our study provides a novel, reproducible model for the study of disc degeneration.New notochordal cell populations were seen in discs injected with ESCs.The lack of an immune response to xenograft-implanted mouse stem cells in an immune-competent rabbit suggests an immunoprivileged site within the IVD.Although preliminary, this study highlights the possible use of stem cells to promote IVD regeneration.Further ongoing studies are in the process of fully elucidating the processes involved with ESC differentiation along chondrogenic cell lines and how they may be used for new disc formation in the future.These studies will provide a good deal of evidence with regard to the future potential of ESCs for use in restoring the IVD in humans.

Summary
DDD is a high-morbidity condition with many modalities of treatment including surgery and more conservative measures such as pain injections, which only provide symptomatic treatment.No therapy has been developed that targets DDD at the cellular level.Recently, many biologic therapies have emerged that may be able to restore the NP and the normal cellular structure of the IVD.This restoration may in turn alleviate the symptoms of DDD through restoration of foraminal height, removing the compression of nerves.In-vitro studies have been performed to identify what cells are capable of differentiating towards a chondrocytic lineage and to best define parameters and factors that influence this differentiation.Multiple laboratory studies have been performed showing that MSCs, ADSCs, fetal cartilaginous cells, and annulus fibrosus cells all have the ability to differentiate towards a chondrocytic pathway.Factors that can induce these cells to differentiate toward a chondrocytic lineage have been identified and include TGF-β3 and BMP-2, which have a synergistic effect when used together.Other factors that may be beneficial include hypoxia, IVD-like glucose conditions (1.0 mg/mL glucose), and cell-tocell contact with NP cells; the latter negating the need for other soluble factors (i.e.TGF-β3).A major limiting factor may be the acidic pH (6.8) of the IVD, one that may be especially important as acidic pH levels are typical of increasingly degenerated discs.These studies yielded encouraging results with cells in the IVD being positive for markers of chondrocytic differentiation such as collagen type II and aggrecan.Additionally, cells exhibited NP phenotypic markers and had a close similarity to the normal biconvex structure of the NP.In-vitro studies have clearly established that ESCs are capable of differentiating into a chondrocytic lineage and have delineated some of the factors that affect this.The optimal microenvironment needs to be more accurately characterized at this time.
Animal studies of cell implantation have been performed in DDD-induction models.Weeks after injury, stem cells have been implanted and outcomes followed.These outcomes which have included radiographic analyses along with histological and immunohistochemical analyses have provided preliminary data that stem cell therapies are a viable option with regard to IVD regeneration (Sheikh et al., 2009).Human studies have further provided some preliminary evidence that stem cell therapy may be of clinical value (Haufe et al., 2006).The use of ESCs in regenerating IVD shows exciting new possibilities and further studies are needed in humans to establish its efficacy.
ESC-based regeneration of the human IVD is still in its infancy.Much progress has been made regarding laboratory research identifying the correct factors and microenvironment, and initial results from animal studies using stem cells remain promising.ESCs may be useful for repairing DDD as evidenced by their ability to differentiate into a chondrocytic lineage and yield notochordal-type cells in DDD models.ESCs need to be further studied and characterized with respect to safety, and larger human trials with appropriate clinical outcomes such as pain and disability reduction are needed to definitively establish its clinical efficacy.

Conclusions
The last half-century has seen an exponential rate of progress with regard to elucidating the mechanisms of degeneration of the IVD and how targeted therapies can help to alleviate this common condition.These studies have provided us with an improved understanding of the IVD and how it behaves under typical biomechanical forces and loads experienced in invivo conditions.Novel therapies are being studied, including stem cells with their potential regenerative capabilities in the spine.The development and action of these stem cells can be further modified through gene therapy and microenvironment manipulation.Immunologic markers are being used for more efficient targeting of these cells.With enhanced cell delivery and an improved understanding of the cell differentiation process, true regeneration of the IVD and surrounding supportive structures of the spine will become a reality that can be applied to treat patients with this common, debilitating condition.

Fig. 1 .
Fig. 1.Anatomy of the spine with the compartmentalization of the IVD.

Fig. 2 .
Fig. 2. Axial slice model of the intervertebral disc with an image of a disc herniation

Fig. 5 .
Fig. 5. Hematoxylin and eosin staining of the rabbit IVD, showing healthy notochordal cell rests Several xenotransplant studies involving ESCs have been conducted with promising results.Jeong et al have shown that rats receiving human ESCs showed relative restoration of the inner annulus structure compared to a control group (Jeong et al., 2010).This finding may help to address the concern of loss of implanted material through the needle puncture.

Fig. 6 .
Fig. 6.Photographs of our group's rabbit model for IVD degeneration.The rabbit is positioned prone, its back is shaved and prepared for surgery (A), with a corresponding fluoroscopic view (B).

Fig. 8 .
Fig. 8. Photomicrographs of tissue obtained preimplanation for histological analysis of ESCs grown in-vitro with Alcian blue staining showing 86% viability (A) and high power magnification showed adequate GFP cell labeling (B).

Table 1 .
Animal StudiesOur group recently reported seminal work with regard to ESC implantation in a rabbit model www.intechopen.comwww.intechopen.com To date, there have been no human ESC implantation studies into the IVD in humans.Further study is needed to verify safety before such work is undertaken.