Stem Cell Regeneration of Degenerated Intervertebral Discs: Current Status Stephen M. Richardson, PhD, and Judith A. Hoyland, PhD
Corresponding author Stephen M. Richardson, PhD Tissue Injury and Repair Group, School of Clinical and Laboratory Sciences, Faculty of Medical and Human Sciences, Stopford Building, The University of Manchester, Oxford Road, Manchester, M13 9PT, UK. E-mail:
[email protected] Current Pain and Headache Reports 2008, 12:83–88 Current Medicine Group LLC ISSN 1531-3433 Copyright © 2008 by Current Medicine Group LLC
Low back pain (LBP) is one of the most common musculoskeletal conditions, and intervertebral disc (IVD) degeneration is associated with most cases. Although many treatment options are available, they focus on the removal of symptoms rather than repair of the degenerate tissue. However, there is a growing interest in the potential of cell-based tissue engineering strategies for regeneration of the damaged IVD. To achieve this, investigators are now focusing on the use of mesenchymal stem cells (MSCs), which offer several advantages over more mature cell types. A number of problems must be overcome for MSC-based IVD regeneration to be successful, including determining a method for the differentiation of stem cells into nucleus pulposus-like cells. Although this is still a relatively new field, it offers huge potential for the clinical treatment of IVD degeneration and LBP in the future.
Introduction Low back pain (LBP) is one of the most common musculoskeletal disorders [1]; it is estimated that 84% of the population will experience LBP at some point in their lifetime [2], with prevalence increasing with age [3]. Each year, approximately 7% of the adult population consults their general practitioner with symptoms of back pain [4]. A recent UK study has shown an increased prevalence when comparing current data with that of the 1950s [5]. LBP places a huge socioeconomic burden on societies worldwide. In terms of direct costs to health care systems and indirect costs (eg, lost productivity, insurance costs, and disability benefits), LBP is estimated to cost the United Kingdom up to £12 billion per annum [6].
Most people who suffer acute attacks of LBP will recover without intervention or with conservative therapies [7]. However, the small number of patients who fail to recover within 3 months and suffer chronic LBP account for the greatest cost and present a problem in terms of management and treatment. Until recently, little has been known about the pathogenesis of chronic LBP. Although LBP is multifactorial in nature, there have been signs of intervertebral disc (IVD) degeneration in almost every case studied. While affecting the disc, IVD degeneration also affects the surrounding ligaments and musculature, leading to spinal instability, which can accentuate the problem and lead to advancing degeneration, pain, and reduced spinal mobility.
Current Treatments for Chronic LBP Initially, short-term pain relief can be administered, and NSAIDs have been shown to be effective in reducing acute LBP. Combined with this are a range of physiotherapy and exercise therapies designed to improve core stability, strengthen muscles, and improve motion. Alternative therapies, such as massage, acupuncture, and spinal manipulation, or the use of devices such as transcutaneous electrical nerve stimulators, have also been shown to have a positive effect on the treatment of chronic LBP. Alternatively, epidural, facet joint, or local corticosteroid injections have also been used for treatment, although their benefit is unclear [8]. When conservative therapies are ineffective, surgical intervention may be the only option. A wide range of surgical treatments are available, but the most common procedure is spinal fusion. This occurs at the affected level and is aimed at removing the source of pain. However, evidence of its effectiveness is controversial. A 2001 Swedish study showed that 2 years post-surgery, patients who underwent fusion did better than those who were treated conservatively [9], whereas a more recent UK study commissioned by the Medical Research Council demonstrated no significant improvement in patients who underwent surgery compared with the nonsurgical group [10]. Furthermore, there is concern and evidence to show that lumbar fusion may have detrimental effects on spinal biomechanics and may induce degenerative changes in adjacent disc levels [11].
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More recently, a range of disc prostheses have been developed, aimed at replacing the degenerate IVD. Early indications suggest that these devices, including Charite (DePuy Spine Inc., Warsaw, IN) and ProDisc (Synthes Spine Inc., West Chester, PA), provide significant reduction in pain up to 24 months [12,13]. However, it is too early to establish long-term effectiveness, and there is evidence of potential migration, prolapse, and failure of these artificial prostheses [14]. One of the overriding problems with these treatments is their inability to repair the tissue. In each case the aim of the treatment is the removal of symptoms (eg, pain) and, with the exception of spinal fusion, to increase mobility. However, the emergence of tissue- and cellbased engineering in recent years has led to increased interest in the application of regenerative strategies for repair of the degenerate IVD and treatment of the associated pain.
IVD Cell Biology and Pathophysiology The IVD is comprised of three morphologically distinct regions—the central, gelatinous nucleus pulposus (NP), the ligamentous annulus fibrosus (AF; which surrounds the NP circumferentially), and the cartilaginous endplates (which connect the disc to the inferior and superior vertebral bodies). Within each region are populations of phenotypically distinct cells. The NP cells are chondrocyte-like (rounded and within lacunae), whereas the AF cells are more elongated and fibroblastic [15]. These morphologic differences in the cell types are reflected in their phenotypes. NP cells produce a matrix that is rich in proteoglycans (PGs), predominantly aggrecan and type-II collagen, whereas the AF cells produce a matrix that is rich in type-I collagen with little PG or type-II collagen. The structure of the NP is an amorphous mix of PGs and collagens, and although the component molecules are similar to those of articular cartilage, the ratios of PG:collagen are much higher in the NP (27:1 vs 2:1 in cartilage) [16]. PGs have a high positive charge, which gives the NP a high osmotic potential, drawing in water and giving the tissue a more hydrogel-like consistency than cartilage. Although there is no clear demarcation between NP and AF, the amount of PG and type-II collagen decreases outwards from the center of the NP as the amount of type-I collagen increases. In the AF itself the type-I collagen fibers form lamellae that lie at 60° oblique angles and constrain the NP. This coordinated structure allows the IVD to withstand the loads experienced in the spine through deformation and load distribution within the NP and constrained bulging of the AF. There is no consensus on the causes of degeneration, with altered biomechanics and load, genetic predisposition, decreased nutrition, and altered cell physiology all argued to be either cause or effect of degeneration. However, an ever increasing body of evidence has shed light on the
changes that occur at a cellular and tissue level in degeneration, which may help elucidate key events in degeneration and suggest possible routes for regeneration. In degeneration there is a breakdown of the matrix of the NP leading to fissures that eventually extend into the AF. Combined with this is evidence of increased apoptosis [17] and cellular senescence [18] leading eventually to a decrease in cell number. Recent research from our laboratory has demonstrated a key role for the catabolic cytokine interleukin (IL)-1 in stimulating the production of a range of proteolytic enzymes including the matrix metalloproteinases (MMPs) and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs), which play a key role in the extracellular matrix degradation seen in degeneration [19]. Although there is an increase in IL-1 in degenerate discs, there is no increase in its antagonist, IL-1Ra. Overexpressing IL-1Ra in disc cells results in inhibition of proteolytic enzyme activity, suggesting a potential role for IL-1Ra therapy in treatment of IVD degeneration [20]. However, although gene therapy approaches using IL-1Ra, along with molecules and growth factors such as osteogenic protein-1 [21], growth differentiation factor-5 [22], and latent membrane protein-1 [23], have all been suggested as potential candidates for inhibiting degeneration or inducing repair, problems of site-specific delivery and issues with gene therapy for non–life-threatening illnesses make this therapeutic strategy difficult to introduce clinically.
Potential for Cell-based Therapies Cell-based therapies for tissue regeneration offer an attractive solution to current conservative, surgical, pharmaceutical, or gene therapy interventions. Through the introduction of suitable cells, it is possible to repair and produce a tissue with similar characteristics to the original. This has been demonstrated in articular cartilage defects through the use of autologous chondrocyte implantation (ACI), which has been used clinically to successfully repair small, full-thickness cartilage lesions [24]. However, there are limitations in its application to larger lesions and to diseases such as osteoarthritis, in which the loss of cartilage is too extensive and requires large numbers of implanted cells. Similar systems have been tried in animal models of IVD degeneration, with some success [25••], and a human clinical trial is currently underway that shows an increase in disc height and a reduction in pain after autologous disc-chondrocyte reimplantation [26]. However, the IVD provides a number of problems that suggest an ACI-style system may not be successful. Even compared with articular cartilage, which has a cell density of around 15,000 cells/mm3 [27], the number of cells in the normal IVD is relatively low (about 4000 cells/mm3 in the NP) [28]. ACI works by removing cartilage from an unaffected non–load-bearing region,
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extracting cells, and expanding them to sufficient numbers. However, the patient’s cells are not a suitable source of cell. We have shown that a significant number of cells exhibit cellular senescence, which results in an altered phenotype with increased matrix degradation [18]. Furthermore, this also reduces their ability to be manipulated ex vivo (ie, in culture), thus limiting the number of cells that can be obtained for reimplantation. Additionally, although isolation of normal cells from a non-degenerate disc level has been hypothesized, such cell harvesting is dangerous and can itself initiate degeneration. Because of these reasons, our group and others have focused on stem cells as an autologous cell source for such therapy.
Stem Cell Biology and Identification Stem cells are populations of unspecialized cells that are capable of self-renewal and differentiation into a number of specialized cell types. Although much discussion surrounds the use of embryonic stem cells, adult mesenchymal stem cells (MSCs) have reached prominence because of their ready availability from a number of sites (bone marrow and fat), their proliferative capacity, and their ability to differentiate into several cell types, including chondrocytes. However, at present there is still speculation over the exact definition of these cells. Although there is a growing range of cell surface (including CD44, CD71, CD90, CD105, CD120A, CD124, and CD166) and other markers (including ILs, leukemia inhibitory factor, and granulocyte-macrophage colony-stimulating factor) available to aid identification and separation from contaminating cell types, there is still no consensus opinion or unambiguous definition to what a stem cell is or how it should be identified and cultured [29•]. Therefore, these aspects may require clarification before clinical application is a possibility, although this has not prevented numerous studies investigating the use of progenitor cells in tissue regeneration.
The Importance of Cellular Scaffolds The use of cellular scaffolds is potentially a key determinant for the success or failure of any stem cell–based regenerative therapy for the IVD for several reasons. The human IVD is the largest avascular organ in the human body, with cells as far as 8 mm from the nearest blood supply [30]. Therefore, nutrition within the disc is limited and in the degenerate disc, where the endplates may be calcified, there is an increase in waste products and a decrease in nutrition, creating an acidic environment. The tissue is also hypoxic and under constant mechanical load, which creates an environment not suited to cell survival. The implantation of a large number of stem cells into this environment is likely to result
in large-scale cell death, rather than differentiation and matrix formation. Furthermore, after discectomy to remove degenerate tissue, implanted cells are unlikely to differentiate and produce new tissue quickly enough for a successful outcome; a more likely scenario is the formation of scar tissue, which is unacceptable. Application of a cellular scaffold is also important for both cell–cell and cell–matrix interactions in directing cellular differentiation and matrix formation. Cell–cell communication is instrumental in MSC differentiation during development, and our studies have shown that coculture of MSCs with NP cells causes MSC differentiation only in cocultures conducted with direct cell–cell contact [31]. Cells within the NP exist in a three-dimensional matrix and interact with it through a pericellular matrix to control homeostasis. NP cells in vitro have also been shown to prefer a three-dimensional environment and, like chondrocytes, can only retain their phenotype during culture in three-dimensional rather than twodimensional systems [32]. The most important factor in the use of stem cells is the ability to successfully differentiate the cells into NP-like cells before implantation or to ensure that implanted cells are capable of differentiating to the correct phenotype in vivo. This requires a scaffold that permits or enhances stem cell differentiation to NP-like cells, is non-immunogenic and biodegradable, and can withstand the loaded environment within the IVD. Studies in vitro have demonstrated differentiation of MSCs to NP-like cells in alginate beads [33]; however, alginate is mechanically unstable and therefore unsuitable for implantation. Studies have also investigated the use of polymeric scaffolds, and we have demonstrated MSC differentiation on poly-L-lactic acid (PLLA) scaffolds [34]. These scaffolds degrade slowly and are mechanically stable, and therefore act to support the spine mechanically while the seeded cells differentiate and synthesize a new matrix. Other studies have used calcium polyphosphate scaffolds [35] and type-I collagen/hyaluronan scaffolds [36] and achieved good differentiation and matrix formation in vitro. An increasing area of interest is the use of hydrogels, such as chitosan, collagen, and hyaluronan gels, which can be injected into the disc or implanted using a minimally invasive approach. This is attractive clinically because the surgery required is less invasive (through a needle or arthroscope) and, therefore, faster and cheaper than conventional implantation techniques required for rigid scaffolds and causes less damage to the AF. These hydrogels also offer a three-dimensional environment that more closely mimics the microenvironment present within the NP and allows cells (including stem cells) to more easily obtain a rounded morphology. We recently demonstrated MSC differentiation in a chitosan-glycerophosphate hydrogel in vitro without the need for exogenous factors [37•]. This gel’s advantage is that it is thermoreversible (a liquid at room temperature and a gel at around 37° C), meaning cells can
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be encapsulated, cultured in vitro, and then cooled before injection into the IVD. An in vivo study in rabbits using a thermosensitive atellocollagen gel seeded with MSCs demonstrated a 91% increase in disc height by 24 weeks compared with sham-operated controls, as well as evidence of cellular differentiation and the production of a PG-rich extracellular matrix [38••]. We are currently focusing on the use of thermosensitive hydrogels for regeneration of the NP and are testing a range of molecules aimed at modifying the biological and biomechanical properties of these gels to improve differentiation, matrix formation, and stability after in vivo implantation.
Predifferentiation Versus In Vivo Differentiation The successful differentiation of stem cells and formation of an NP-like matrix are reliant on biological and biomechanical cues that the cells receive. Growth factors are instrumental in the differentiation process, in particular members of the transforming growth factor (TGF)-β superfamily [39], as well as insulin-like growth factor-1 [40] and the cartilage-derived morphogenetic proteins [41]. We have demonstrated that SOX-9 overexpression and culture in a differentiating media containing TGF-β can induce MSC differentiation and matrix formation on PLLA scaffolds, as well as in monolayer [34]. Reduction in oxygen tension has also been shown to induce an NPlike phenotype in MSCs, and studies using application of hypoxia or compressive load have been demonstrated to induce a chondrocyte-like phenotype in MSCs [39,42]. Therefore, although Sakai et al. [38••] demonstrated good differentiation and matrix formation by MSC implantation into rabbit IVD, by pre-conditioning cells and exposing them to an environment similar to that found in vivo, it may be possible to induce differentiation before implantation and therefore accelerate matrix formation in vivo.
Future Directions For all its apparent simplicity, the IVD is a complex environment that provides numerous clinical and scientific challenges to successful regenerative treatment for degeneration. A number of factors must be considered to achieve long-term success—most notably the choice of cells. Adult stem cells offer a seemingly ideal choice due to their ease of acquisition, rapid proliferation, and differentiation capacity. Although the evidence presented here suggests the ability of MSCs to differentiate to NP-like cells, the markers used in these studies are not unique to NP cells. In fact, most marker genes are shared with articular chondrocytes and although studies have suggested marker gene profiles, such as expression of MMP-2, glucose transporter-1, and hypoxia-inducible factor-1α [43], these are not definitive and are not unique to NP cells. Microarray studies have been used
to investigate NP cell gene signatures but to date have only been conducted in animals such as the rat [44••], which retains the presence of notochordal cells that may influence the expression profiles. If a unique marker or profile cannot be found then the differentiation state of the stem cells will only be known through examination of the tissue formed after in vivo implantation experiments. However, the unique bipedal stance of humans cannot be replicated in animal models; therefore, model systems are of limited relevance. Although our group has developed a novel in vitro loading system capable of mimicking the loads experienced in the human disc over extended time periods [45], it relies on an in vitro culture environment that is not truly representative. Therefore, although there is convincing evidence of the ability of stem cells to differentiate and produce a matrix similar to that found in the native human IVD, it is only through clinical application in humans that the true differentiation state can be ascertained. Clinical application itself also presents a problem. Hydrogels, unlike rigid scaffolds, offer the versatility of minimally invasive implantation. However, their structural integrity under the loaded, but constrained, conditions of the human NP is yet to be elucidated. For implantation or minimally invasive injection/insertion into the NP through the AF, the problem remains of how to seal the AF and prevent extrusion of the gel/ scaffold or prolapse of the disc. A number of systems have been used in animal models, including application of fibrin glue and/or suturing; however, these methods may not be applicable in humans due to the increased loads applied.
Conclusions Cell-based tissue engineering and regenerative medicine for repair of the human IVD are in their infancy, especially with regard to the application of stem cells. However, the increased understanding of IVD biology and pathophysiology, combined with increased knowledge surrounding stem cell biology and the experience learned from other more established fields such as cartilage repair, has allowed a rapid advancement of the science over the past few years. Although a clearer picture of the requirements for a successful strategy is emerging, the optimal choice of scaffold, culture conditions, and implantation technique must still be identified and will require the continued collaboration of scientists and clinicians. However, a stem cell–based therapy for regeneration of the IVD and treatment of LBP is still the goal of many focused teams around the world and will no doubt one day be a reality.
Disclosures No potential conflicts of interest relevant to this article were reported.
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