373I.M. Shapiro, M.V. Risbud (eds.), The Intervertebral Disc, DOI 10.1007/978-3-7091-1535-0_23, © Springer-Verlag Wien 2014

23.1 Evolution of Stem Cell Biology in Musculoskeletal Tissues

The ability of a tissue to respond to stress or injury requires the involvement and functions of stem cells resident in tissue-speci fi c microenvironmental niches. Aging has been shown to result in a decrease in stem cell number as well as loss of ability to maintain tissue homeostasis and regenerate lost tis-sue function. Therefore, it is of critical importance to iden-tify stem/progenitor cell populations in different tissues, determine how these cells function in tissue homeostasis, and ascertain their potential utility in tissue engineering.

Classically, stem cells of the musculoskeletal region were de fi ned as undifferentiated cells, found in small numbers in the periosteum or the bone marrow (Caplan 1991 ; Deans and Moseley 2000 ) . Subsequently, these small populations of cells have been found in other stem cell pools, such as the adipose tissues, synovial tissues, perivascular regions in the surrounding tissue environment, and even in the matrix com-ponent of tissues (Bianco et al. 2001 ; Crisan et al. 2008 ) . They were designated “mesenchymal stem cells” because from a developmental perspective, they were thought to be mesenchymal in origin. These cells can undergo sustained proliferation in vitro and potentially give rise to multiple mes-enchymal cell lineages, including osteocytes, chondrocytes, and adipocytes. Since then, many researchers have reported studies of mesenchymal stem cells using different methods of isolation and expansion. With the increasing dif fi culties encountered in comparing and contrasting study outcomes, these cells are now preferentially called “multipotent mesen-chymal stromal cells” (multipotent MSCs). The Tissue Stem Cell Committee of the International Society for Cellular Therapy has proposed minimal criteria to de fi ne human MSC (Dominici et al. 2006 ) . First, MSC must be adherent to plastic when maintained under standard culture conditions. Second, MSC must express the surface molecules CD105, CD73, and CD90, and not express CD45, CD34, CD14, CD11b, CD79 a , CD19, or HLA-DR. Third, MSC must differentiate into osteo-blasts, adipocytes, or chondroblasts in vitro. Despite these

Use of Stem Cells for Regeneration of the Intervertebral Disc

Daisuke Sakai and Joji Mochida

23

D. Sakai , MD, PhD (*) • J. Mochida , MD, PhD Department of Orthopaedic Surgery, Surgical Science , Research Center for Regenerative Medicine, Tokai University School of Medicine , Shimokasuya 143 , Isehara, Kanagawa 259-1193 , Japan e-mail: daisakai@is.icc.u-tokai.ac.jp ; jomo@is.icc.u-tokai.ac.jp

Contents

23.1 Evolution of Stem Cell Biology in Musculoskeletal Tissues ............................................. 373

23.2 Evidence of a Stem Cell System in the Intervertebral Disc ............................................... 374

23.3 Commitment of Stem Cells Toward an Intervertebral Disclike Cell ....................................... 375

23.4 Stem Cell Promotion of Disc Cell Differentiation ........ 376

23.5 Stem Cell Transplantation for Intervertebral Disc Tissue Engineering and Regeneration .................. 377

23.6 Perspectives on the Role of Stem Cell Biology in the Treatment of Intervertebral Disc Disease .......... 379

23.7 Summary of Critical Concepts Discussed in the Chapter .................................................................. 381

References ...................................................................................... 381

374 D. Sakai and J. Mochida

minimal criteria to identify MSC, it is dif fi cult to de fi ne MSC other than by the operational de fi nition of self-renewal and differentiation potential in vitro. Therefore, our knowledge of MSC is based solely on the characterization of cultured cells. We still lack any knowledge of their in vivo characteristics, such as their development, exact tissue localization, and physiological roles.

Despite these dif fi culties, the considerable therapeutic potential of human MSC has generated marked and increas-ing interest within a wide variety of biomedical disciplines, including studies of the intervertebral disc. Published studies involving MSC have increased markedly in this fi eld and may in the long run provide new and useful information on disc biology, the pathogenesis of intervertebral disc degeneration, and possibly offer new therapeutic strategies (Sakai 2011 ) . However, as with any area of study, resear-chers must share similar de fi nitions, terminologies, and methods to ensure that their fi ndings can be compared. It is important for scientists to know the essential features of a stem cell: its capacity for self-renewal and the ability of a single cell to regenerate all the cell types and matrices of the lineage from which the cell was derived (Fig. 23.1 ) (Blanpain et al. 2004 ) . Accordingly, the experimental design and the types of stem cells used in investigational studies and trans-lational cell therapies involving the intervertebral disc must be selected with care.

23.2 Evidence of a Stem Cell System in the Intervertebral Disc

As mentioned above, investigation of the tissue-speci fi c stem cell niche is a key factor in understanding the degenerative processes and tissue regeneration. Although research into the

stem cell system in the intervertebral disc is still in its infancy, recent studies have shown evidence of a potential stem cell niche in the intervertebral disc region. Henriksson et al. ( 2009 ) attempted to detect cell proliferation zones and label-retaining cells with in vivo 5-bromo-2 ¢ -deoxyuridine (BrdU) labeling in rabbit discs. They also investigated the localization of pro-genitor cell markers (Notch1, Delta4, Jagged1, C-KIT, KI67, and Stro-1) with immunohistochemistry in the degenerated intervertebral discs of rabbits, rats, mini pigs, and humans. Although slow, ongoing proliferation was detectable in both the nucleus pulposus and annulus fi brosus regions. They also found a high number of BrdU-positive cells at the annulus fi brosus borders with the ligament zone and the perichon-drium region at early time points, whereas only a few label-retaining cells were observed at later time points, identifying a stem cell niche-like pattern. This may support the fi ndings of Melrose et al. ( 2007 ) , who demonstrated spatial annular remodeling in experimentally injured ovine discs.

The recruitment of cells from the surrounding environ-ment is another phenomenon often seen in the regenerative process. Using the BrdU in vivo labeling technique described above, Henriksson et al. ( 2011 ) observed BrdU-labeled cells in the intervertebral disc niche, adjacent to the epiphyseal plate at early time points, but at later time points, these cells were mainly in the outer region of the annulus fi brosus, sug-gesting possible migratory pathway. Tzaan and Chen ( 2011 ) have extended this concept by enhancing bone marrow cell migration to the disc by stimulation with granulocyte colony-stimulating factor. However, their results only demonstrated increased bone marrow cells in the endplate. Cell migration processes are thus underde fi ned and require substantial investigation.

There is increasing evidence that stem/progenitor cells are present in the intervertebral disc. Risbud et al. ( 2007 ) reported that cells isolated from degenerative human disc tis-sues expressed CD105, CD166, CD63, CD49a, CD90, CD73, p75 low-af fi nity nerve growth factor receptor, and CD133/1, proteins that are characteristic of MSC and repre-sent the differentiation capacity of these cells toward osteo-genesis, adipogenesis, and chondrogenesis. Feng et al. ( 2010 ) have demonstrated that the human degenerative annulus con-tains cells that express the MSC markers CD29, CD49e, CD51, CD73, CD90, CD105, CD166, CD184, and Stro-1 and two neuronal stem cell markers, nestin and neuron-speci fi c enolase. In an in vitro assay, they differentiated annulus fi brosus-derived cells into adipocytes, osteoblasts, chondrocytes, neurons, and endothelial cells. Blanco et al. ( 2010 ) investigated MSC markers in nucleus pulposus cells isolated from degenerated discs and compared their differen-tiation capacities with those of bone marrow-derived MSC from the same patient. They found that nucleus pulposus-derived MSC ful fi lled almost all the morphological, immu-nophenotypical, and differentiation criteria for MSC

Definition of a stem/progenitor cell

Clonogenicity

High proliferation

Self-renewal

Multipotency

Fig. 23.1 De fi nition of a stem/progenitor cell. Criteria of stem/pro-genitor cell include clonogenicity, high proliferative capacity, self-renewal capability (from a single cell to reconstitute a tissue in which the cell was derived), and multipotency

37523 Use of Stem Cells for Regeneration of the Intervertebral Disc

described by the International Society of Cell Therapy, except that nucleus-derived MSCs were unable to differenti-ate into adipocytes. Likewise, Liu et al. ( 2011 ) investigated the characteristics of MSC derived from degenerated human disc cartilage endplates and reported that the morphology, proliferation rate, cell cycle, immunophenotype, and expres-sion of stem cell genes were similar to those isolated from the bone marrow. These research fi ndings suggest that the stimulation of endogenous stem cell populations may be an effective strategy for treating intervertebral disc degenera-tion or to provide cells for the allogeneic transplantation of somatic-tissue-speci fi c stem cells.

23.3 Commitment of Stem Cells Toward an Intervertebral Disclike Cell

In contrast to the small steps yet taken in investigating the endogenous stem cell system in the intervertebral disc, a number of studies have reported the utilization of MSC in new, targeted therapeutic strategies (Fig. 23.2 ). MSC utiliza-tion can be subdivided into three main types of studies. The fi rst approach utilizes the multipotent differentiation capac-ity of MSC to induce stem cells to commit toward an inter-vertebral disclike cell. A considerable number of studies in the literature describe this strategy (Risbud et al. 2004 ; Li et al. 2005 ; Steck et al. 2005 ; Richardson et al. 2006 ; Sobajima et al. 2008 ; Vadalà et al. 2008 ; Wuertz et al. 2008 ; Chen et al. 2009 ; Kim et al. 2009 ; Le Maitre et al. 2009 ; Wei et al. 2009a ; Korecki et al. 2010 ; Strassburg et al. 2010 ; Bertolo et al. 2012 ; Choi et al. 2011 ; Feng et al. 2011a ; Luo

et al. 2011 ; Purmessur et al. 2011 ; Ruan et al. 2012 ; Stoyanov et al. 2011 ) . Various methods to induce MSC differentiation have been evaluated such as stimulation with growth factors under speci fi c culture conditions, coculture with terminally differentiated intervertebral disc cells, or seeding cells onto a microenvironment-mimicking scaffold. Steck et al. ( 2005 ) compared the molecular phenotypes of human disc cells and articular chondrocytes to determine whether MSC can dif-ferentiate toward both cell types after transforming growth factor- b (TGF b )-mediated induction in vitro. Their results demonstrated that the gene expression pro fi le adopted by MSC resembled that of native disc tissue more closely than that of native joint cartilage. As well as TGF b 3, insulin-like growth factor 1 (IGF1), fi broblast growth factor 2, and plate-let-derived growth factor BB have been shown to induce MSC differentiation toward nucleus pulposus-like cells (Ehlicke et al. 2010 ) . Richardson et al. ( 2006 ) assessed the value of a coculture system with or without cell–cell contact to induce MSC differentiation toward a nucleus pulposus-like phenotype. Real-time quantitative polymerase chain reaction (RT-PCR) demonstrated a signi fi cant increase in the expression of nucleus pulposus marker genes in stem cells when the cells were cocultured with contact for 7 days, and this change was regulated by the cell ratio. However, no signi fi cant changes in marker gene expression was observed when the cells were cultured with either the nucleus pulpo-sus cells or stem cells without contact, indicating a require-ment for cell–cell contact in this induction process.

In a follow-up study, Strassburg et al. ( 2010 ) used human nucleus pulposus cells from degenerate and nondegenerate intervertebral discs to show that MSC differentiated into a

Activation

Culture insert

IVD cell

MSC

IVD cell-like cell

Growth factorCoculture etc.

MSC

Induction of MSCtowards IVD cell-like cells IVD cell activation by MSC Direct transplanation

MSC

Activated IVD cell

Utilization of MSC for IVD regeneration Fig. 23.2 Utilization of multipotent mesenchymal stromal cells for disc regenera-tion. Mesenchymal stem cells ( MSCs ) can be directly induced to differentiate toward an intervertebral disc cell ( IVD ) using growth factor and coculture techniques ( left ). In addition, activation can be achieved using coculture systems in which there is direct cell–cell contact ( middle ). Alternatively, MSC can be directly transplanted into the disc and commitment enhanced by the local conditions and the presence of resident host cells ( right )

376 D. Sakai and J. Mochida

nucleus pulposus-like phenotype following direct coculture with either of the tissues with signi fi cantly upregulated expression of the genes encoding SOX9, collagen VI, aggre-can, and versican, together with the simultaneous upregula-tion of growth differentiation factor 5 (GDF5), TGF b 1, IGF1, and connective tissue growth factor expression. The direct coculture of normal nucleus pulposus cells with MSC had no effect on the phenotype of the nucleus cells, whereas coculture with degenerated nucleus pulposus cells resulted in enhanced matrix gene expression in the degenerate cells, accompanied by increases in both TGFB1 and GDF5 gene expressions. These results suggest that cellular interactions between MSC and degenerated nucleus pulposus cells both stimulate MSC differentiation to a nucleus pulposus-like phenotype and promote the endogenous nucleus pulposus cell population to regain a nondegenerated phenotype, con-sequently enhancing matrix synthesis for self-repair. Similarly, when human nucleus pulposus cells were main-tained in a pellet coculture with human MSC in different ratios, the 75:25 and 50:50 NP:MSC ratios yielded the great-est increases in extracellular matrix (ECM) production (Sobajima et al. 2008 ) . Vadalà et al. ( 2008 ) also reported a reduction in the expression of collagen I and an increase in the expression of collagen II and aggrecan in MSCs after coculture with nucleus pulposus cells in alginate hydrogels, which allowed short-distance paracrine cell interactions. Stoyanov et al. ( 2011 ) have shown the important role of hypoxia, together with the addition of GDF5, in enhancing the expression of nucleus pulposus markers in a bone mar-row-derived MSC coculture.

MSCs from sources other than the bone marrow have also been examined. Lu et al. ( 2007 ) studied the interac-tions between human nucleus pulposus cells and adipose- tissue-derived stem cells in Transwell coculture, using both monolayer and micromass con fi gurations. A similar cocul-ture study was performed by Chen et al. ( 2009 ) utilizing syn-ovium-derived MSC. Vadalà et al. ( 2008 ) investigated mus-cle-derived MSC, and Ruan et al. ( 2012 ) assessed whether Wharton’s jelly cells could be induced to differentiate toward nucleus pulposus-like cells in similar coculture studies.

The effects of scaffold properties on the differentiation of MSC toward intervertebral disc cells have also been investi-gated. Bertolo et al. ( 2012 ) evaluated four types of matrices as scaffolds, approved as medical devices for other applica-tions: two made of equine or porcine collagen, one of gela-tin, and one of chitosan. They showed that although the collagen scaffolds induced better chondrogenic differentia-tion than the other scaffolds, the phenotype of the MSC was not fully equivalent to that of nucleus pulposus cells. Feng et al. ( 2011a ) investigated the use of three-dimensional (3D) nano fi brous poly( l -lactide) scaffolds seeded with rabbit MSCs which were induced to differentiate along the nucleus pulposus pathway in a hypoxic chamber (2 % O

2 ) in the

presence of TGF b 1. The nano fi brous scaffold supported the differentiation of rabbit MSC toward a nucleus pulposus-like phenotype in vitro, with the upregulated expression of a few important nucleus-associated genes (encoding aggrecan, col-lagen II, and Sox-9), the abundant deposition of ECM (gly-cosaminoglycan and collagen II), and the continuous expression of the nucleus pulposus-speci fi c marker, hypoxia-inducible factor 1- a (HIF1- a ).

The effect of physiological stimulation on MSC differen-tiation toward intervertebral disclike cells has also been stud-ied. Luo et al. ( 2011 ) cultured MSC under simulated microgravity in a chemically de fi ned medium supplemented with TGF b 1 (positive control group). The results showed that MSC independently and spontaneously differentiated toward a nucleus pulposus-like phenotype under simulated microgravity without the addition of any external bioactive stimulant, such as factors from the TGF b family, which were previously considered necessary.

Recently, another unique approach to inducing MSC was reported by Korecki et al. ( 2010 ) . Because notochord-derived cells play a major role in nucleus development, they tested whether the culture medium from porcine notochordal cells could direct the differentiation of MSCs. Human MSC pel-lets were cultured fi rst in serum-free medium for 4 days and then in notochordal cell-conditioned medium for 7 days. It was found that there was glycosaminoglycan accumulation and increased gene expression toward a nucleus pulposus-like phenotype, together suggesting that there was differen-tiation toward that of the intervertebral disc phenotype.

The results of these studies suggest that MSC from any source can be forced to express some of the molecular mark-ers of intervertebral disc cells in vitro and many external stimuli can accelerate differentiation. However, at the end of the day, they do not develop beyond nucleus pulposus-like cells because the default differentiation pathway is deter-mined by the localization of the MSC. The most important factor in studying the induction of MSC toward interverte-bral disclike cells is to demonstrate their functional capacity in vivo.

23.4 Stem Cell Promotion of Disc Cell Differentiation

Another approach to the utilization of stem cells is to exploit their capacity to nourish other cells. Stem cells can act as feeder cells to stimulate target cells directly through cell–cell contact or indirectly through the secretion of various factors. In a rabbit disc cell culture, Yamamoto et al. ( 2004 ) showed that direct cell–cell contact between nucleus pulposus cells and MSC occurred across a membrane with 0.45 mm pores, which allowed only the cell processes to adhere to each other, without any more extensive contact between the cultured

37723 Use of Stem Cells for Regeneration of the Intervertebral Disc

cells. Compared with the culture systems with no cell–cell contact, a coculture system that allowed intercellular adhe-sion between nucleus pulposus cells and MSC yielded a marked increase in target cell proliferation, DNA synthesis, and proteoglycan synthesis. This is possibly attributable to the increased secretion of cytokines found in the culture system.

An interesting study by He and Pei ( 2012 ) tested the effects of the ECM deposited by synovium-derived MSC on the rejuvenation of nucleus pulposus cells. The nucleus pul-posus cells that were expanded on ECM grew much faster, were smaller, and had a more fi broblastic shape than those expanded in plastic fl asks. ECM-treated nucleus pulposus cells acquired enhanced CD90 expression and higher mRNA levels of collagen I, collagen II, and collagen X and aggrecan as well as a robust redifferentiation capacity for up to six pas-sages. The authors concluded that the ECM provides a tissue-speci fi c microenvironment for the rejuvenation of nucleus pulposus cells with a higher proliferation rate and greater redifferentiation capacity. These characteristics may improve any future autologous disc cell-based minimally invasive therapeutic approach to the physiological reconstruction of a biologically functional disc in the clinical setting.

Another use of MSC is in the delivery of bioactive factors to resident disc cells. Meyerrose et al. ( 2010 ) suggested the use of MSC for the sustained in vivo production of supra-physiological levels of cytokines to support co-transplanted stem cells and resident cells in intervertebral disc therapies.

23.5 Stem Cell Transplantation for Intervertebral Disc Tissue Engineering and Regeneration

The fi nal approach to MSC utilization in intervertebral disc tissue engineering and regeneration involves the construc-tion of a disclike tissue in vitro and its transplantation or the direct delivery of stem cells into the intervertebral disc. Gaetani et al. ( 2008 ) , Nesti et al. ( 2008 ) , Driscoll et al. ( 2011 ) , and See et al. ( 2011 ) have all attempted to construct this type of tissue in vitro. To improve the quality of in vitro-reconstructed tissue, Gaetani et al. ( 2008 ) constructed a nucleus pulposus-like tissue using a 3D culture of nucleus cells and adipose-derived MSC. To develop a biphasic con-struct, Nesti et al. ( 2008 ) seeded MSC onto a hyaluronic acid–nano fi brous scaffold that consisted of an electrospun, biodegradable nano fi brous scaffold enveloping a hyaluronic acid hydrogel center. The seeded MSCs were induced to undergo chondrogenesis in vitro in the presence of TGF b for up to 28 days. The cartilaginous hyaluronic acid–nano fi brous scaffold construct resembled a native disc architecturally, with an outer annulus fi brosus-like region and an inner nucleus-like region. Histological and biochemical analyses,

immunohistochemistry, and gene expression pro fi ling revealed the time-dependent development of the chondro-cytic phenotype of the seeded cells. The cells also maintained the microarchitecture of the native disc. An electrospun nano fi brous scaffold has also been used to examine the native biomechanics of the annulus fi brosus (Driscoll et al. 2011 ) . Previous studies have shown that the tensile and shear prop-erties of the native tissue are dependent on the fi ber angle and the sample aspect ratio, respectively, so the effects of changing the fi ber angle and the sample aspect ratio on the shear properties of aligned electrospun poly( e -caprolactone) scaffolds were evaluated. How ECM deposition by the resi-dent MSC modulates the measured shear response was also determined. This team showed that the fi ber orientation and sample aspect ratio signi fi cantly in fl uenced the response of scaffolds in shear; indeed, the shear properties of both cel-lular and cell-seeded formulations can match or even exceed the native tissue.

A different approach to tissue engineering the annulus fi brosus was reported by See et al. ( 2011 ) . They constructed cell sheets from bone marrow MSC and incorporated them onto silk scaffolds to simulate the native lamellae of the annulus fi brosus. They then wrapped the construct around silicone nucleus pulposus, mimicking an intervertebral disc construct, and used a bioreactor to provide compressive mechanical stimulation to the silicone disc. Under static con-ditions, the MSC sheets remained viable, with no signi fi cant change in cell numbers for 4 weeks. A histological analysis showed that the MSC sheets adhered well to the silk scaf-folds and glycosaminoglycans were detected within the ECM. The ratio of collagen I to collagen II within the ECM of the MSC sheets also decreased signi fi cantly over the period of culture. These results suggest that extensive remod-eling of the ECM occurred within the simulated disclike assembly and that this assembly is suitable for the regenera-tion of the inner annulus fi brosus.

Finally, there have been several attempts to supplement viable cells in the disc by direct stem cell transplantation. The thought behind this concept is the fi nding that degenera-tion is initiated by reductions in the numbers, viability, and functions of disc cells, especially those of the nucleus pulpo-sus. Basic in vitro studies have shown that disc cells have a low proliferative capacity and that most cells in the adult human disc are in a senescent state. These facts have led researchers to focus on the idea of transplanting stem cells, which may show transplanted site-dependent differentiation and function to produce a functional ECM.

Sakai et al. ( 2003 ) reported the fi rst MSC transplantation study in a rabbit model of disc degeneration. In follow-up studies (Sakai et al. 2005, 2006 ) , they showed that trans-planted MSCs survive, proliferate, and differentiate into cells expressing chondroitin sulfate; keratan sulfate; collagen I, collagen II, and collagen IV; HIF1- a and HIF1- b ; HIF2- a

378 D. Sakai and J. Mochida

and HIF2- b ; glucose transporter type 1 (GLUT1) and GLUT3; and matrix metalloproteinase 2 (MMP2), proteins that characterize the native nucleus pulposus cells. They also used RT-PCR to quantify the expression of the genes encod-ing aggrecan, versican, collagen I and II, interleukin 1 b (IL1 b ), IL6, tumor necrosis factor- a , MMP9, and MMP13, thus demonstrating the increased expression of nucleus pul-posus cell markers and the downregulated expression of in fl ammatory genes. Magnetic resonance imaging (MRI) and radiography con fi rmed the regenerative effects of the procedure, showing that MSC transplanted into degenerating discs in vivo can survive, proliferate, and differentiate into cells that express the phenotype of nucleus pulposus cells, with suppressed in fl ammatory gene expression. Since then, numerous similar studies have used different animal models and cell carriers and MSC from various sources (Table 23.1 ) (Crevensten et al. 2004 ; Zhang et al. 2005 ; Hiyama et al. 2008 ; Hoogendoorn et al. 2008 ; Ganey et al. 2009 ; Wei et al. 2009a ) .

Crevensten et al. ( 2004 ) used a 15 % hyaluronan gel as the carrier and injected fl uorescently labeled MSC into rat coccygeal discs. Although the number of MSCs retained decreased signi fi cantly during the fi rst 2 weeks after injec-tion, the initial cell number was restored after 4 weeks, and cell viability and disc height were maintained. These results indicate that the injected cells had started to proliferate within the rat disc. Zhang et al. ( 2005 ) implanted allogeneic MSC containing the marker gene lacZ from young rabbits into rab-bit intervertebral discs to determine the potential of this cell-based approach. As the transplanted allogeneic MSC survived and increased the proteoglycan content within the disc, it lent strong support to the notion that these cells could be used as a potential treatment for intervertebral disc degenera-tion. Along the same lines, Hiyama et al. ( 2008 ) injected one million autologous MSCs into lumbar discs 4 weeks after

nucleus pulposus aspiration in mature beagle dogs (a chon-drodystrophoid breed), and the animals were followed for another 8 weeks. Radiological, histological, biochemical, immunohistochemical, and gene expression analyses dem-onstrated the clear regenerative effects of the transplanted cells. Importantly, MSC found in the nucleus 8 weeks after transplantation expressed Fas-ligand protein, which has not been detected in MSCs before transplantation. Fas-ligand, which is present in tissues with isolated immune privilege, including the nucleus pulposus, plays an important role in nucleus maintenance. The expression of Fas-ligand indicates that MSC transplantation may also contribute to the preser-vation of the immune privilege of the disc environment. In a follow-up study using the same model, Serigano et al. ( 2010 ) investigated the effects of the numbers of MSC transplanted. To determine the optimal number of MSCs for transplanta-tion, 10 5 , 10 6 , and 10 7 MSCs were transplanted, and their sur-vival and apoptosis after transplantation were compared, together with their regenerative effects, which were assessed with histology, radiography, and MRI images. The transplan-tation of 10 6 MSCs, rather than 10 5 or 10 7 cells, best main-tained the structure of the intervertebral disc and optimally inhibited degeneration. This study demonstrates that the number of cells transplanted affects the regenerative capacity of MSC transplants in animal models of disc degeneration and addresses the importance of cell number in the clinical application of MSC transplantation.

Leung et al. ( 2006 ) investigated the allogeneic transplan-tation of MSC and reported many advantages of such trans-plantations in treating disc disease. They reasoned that if the nucleus pulposus is an immunoprivileged environment, then by presenting fewer antigens, MSC should escape alloanti-gen recognition. Wei et al. ( 2009b ) have also reported the xenogeneic transplantation of bone marrow-derived MSC in rats, and Henriksson et al. ( 2009 ) transplanted human bone

Table 23.1 Summary of in vivo animal experimental studies on stem cell transplantation therapy for intervertebral disc degeneration

Stem cell type Mode Animal model Author Year

Bone marrow MSCs (Autologous MSCs expanded) Rabbit (nucleus aspiration) Sakai et al. 2003, 2005, 2006 Bone marrow MSCs (MSCs expanded) Rat (no injury) Crevensten et al. 2004 Bone marrow MSCs (Allogeneic MSCs expanded) Rabbit no injury Zhang et al. 2005 Bone marrow MSCs (Allogeneic MSCs expanded) Rabbit (nucleus puncture) Leung et al. 2006 Bone marrow MSCs (Autologous MSCs expanded) Canine (nucleotomy) Hiyama et al. 2008 Adipose MSCs (Autologous MSCs expanded) Goat (ABC chondroitinase) Hoogendoorn et al. 2008 Adipose MSCs (Autologous MSCs expanded) Canine (nucleotomy) Ganey et al. 2009 Bone marrow human MSCs (Xenogeneic MSC expanded) Rat (no injury) Wei et al. 2009a and 2009b Bone marrow human MSCs (Xenogeneic MSC expanded) Porcine(nucleus aspiration) Henriksson et al. 2009 Synovial MSCs (Allogeneic MSCs expanded) Rabbit (nucleus puncture) Miyamoto et al. 2010 Bone marrow MSCs (Autologous MSCs expanded) Rabbit (nucleus aspiration) Yang et al. 2010 Bone marrow human MSCs (Xenogeneic MSC expanded) Rat (nucleotomy) Allon et al. 2010 Bone marrow MSC + autologous NP cells

(Autologous MSCs expanded) Rabbit (nucleus aspiration) Feng et al. 2010

Bone marrow MSCs (Autologous MSCs expanded) Mini pig (annulus incision) Bendtsen et al. 2011 Adipose human MSCs (Xenogeneic MSCs expanded) Rabbit (nucleus puncture) Chun et al. 2012

37923 Use of Stem Cells for Regeneration of the Intervertebral Disc

marrow MSC into a porcine model of disc degeneration, pro-ducing a regenerative effect.

Various stem cells have already been tested as cell sources. Hoogendoorn et al. ( 2008 ) reported that adipose-derived MSC may be bene fi cial in cell-based therapies for interverte-bral disc disease because they are isolated more easily than are bone marrow MSC. Ganey et al. ( 2009 ) studied the ef fi cacy of autologous adipose-tissue-derived stem cells in promoting disc regeneration in a canine model of disc injury and found enhanced disc matrix production and improved overall disc morphology. A recent study by Chun et al. ( 2012 ) in a rabbit trauma model has also shown that the transplanta-tion of adipose-derived MSC increased ECM secretion, with little ossi fi cation of the damaged cartilage in the nucleus pul-posus compared with degenerated control discs. MSC puri fi ed from the synovial tissue of knee joints demonstrated superior proliferative ability and greater chondrogenic differentiation capacity than those of bone marrow MSC. Miyamoto et al. ( 2010 ) transplanted allogeneic synovial MSC into rabbits and found that synovial MSC injected into the nucleus pulposus space promoted the synthesis of collagen II in the remaining nucleus pulposus cells and inhibited the expressions of degra-dative enzymes and in fl ammatory cytokines. This allowed the structure of the intervertebral disc to be maintained.

Although evidence of the regenerative effects of MSC transplantation has accrued, whether MSCs perform better than transplanted nucleus pulposus cells is as yet unknown. Feng et al. ( 2011b ) compared the regenerative effects of nucleus pulposus cell and MSC transplantation in a rabbit model. No signi fi cant differences in gene expression were observed between the groups transplanted with nucleus cells or MSCs, supporting the role of MSC as a useful cell therapy substitute for nucleus pulposus cells for intervertebral disc degeneration. Furthermore, recent studies have explored vari-ous modi fi cations to this method to enhance its effect. In vitro studies have shown that the coculture of nucleus pulposus cells and MSC promotes ECM production. The use of bilami-nar coculture pellets (BCPs) of MSC and nucleus pulposus cells has also been explored by Allon et al. ( 2010 ) . BCPs, in which MSCs are enclosed in a shell of nucleus pulposus cells, were transplanted into denucleated rat-tail discs with a fi brin-sealant carrier. Cell retention was higher in the BCP-treated group than in the control group. The disc height and disc grade increased over time only in the BCP-treated group. Only after transplantation with BCPs was proteoglycan staining evident in the nuclear space after 35 days. This approach has been combined with growth factor therapy (Yang et al. 2010 ) . MSC transplanted with pure fi brinous gelatin-TGF b 1 resulted in less degeneration and a reduction in apoptotic cells in rabbits.

The modalities used to evaluate disc degeneration have advanced markedly with the evolution of MRI technology. Bendtsen et al. ( 2011 ) investigated the regenerative effects of MSC transplantation with or without hydrogel in mini pigs, using dynamic contrast-enhanced MRI to focus on endplate

function. They showed that MSC transplantation partly regenerated the degenerative disc and maintained the perfu-sion and permeability characteristics of the vertebral end-plate and subchondral bone.

With increasing evidence of the ef fi cacy of MSC trans-plantation therapy in promoting intervertebral disc regenera-tion, three reports of stem cell transplantation in humans have been published. Haufe and Mork ( 2006 ) injected hematopoi-etic stem cells from the bone marrow into ten patients with discogenic back pain, diagnosed with provocative discogram. There was no improvement in any patient after 1 year. This study cautions that the appropriate selection of patients, cells, and methods must be made with care before the clinical application of this technique. Yoshikawa et al. ( 2010 ) trans-planted autologous bone marrow MSC into discs showing the vacuum phenomenon and instability in two patients undergoing decompression surgery for spinal stenosis. The MSCs were cultured in medium containing autologous serum. During surgery, the stenosed spinal canal was fenes-trated, and pieces of collagen sponge containing autologous MSCs were then grafted percutaneously onto the degener-ated disc. Two years after surgery, radiography and computed tomography showed improvements in the vacuum phenome-non in both patients. T2-weighted MRI showed high signal intensity in the intervertebral discs treated with cell grafts, indicating a high moisture content, and dynamic radiography showed less lumbar disc instability. Orozco et al. ( 2011 ) also transplanted autologous bone marrow MSC into ten patients with chronic back pain, diagnosed with lumbar disc degen-eration with an intact annulus fi brosus. The feasibility and safety of the treatment were con fi rmed and strong indications of its clinical ef fi cacy identi fi ed. The patients displayed rapid improvement in their pain and disability (to 85 % of maxi-mum in 3 months), which approached 71 % of optimal ef fi cacy, comparing favorably with the results for other pro-cedures, such as spinal fusion and total disc replacement. MRI evaluations 1 year after transplantation showed that although the disc height was not restored, the water content was signi fi cantly elevated. They concluded that MSC therapy might be a valid alternative treatment for chronic back pain caused by degenerative disc disease. Its advantages over the current gold standard treatments include that it is a simpler and more conservative intervention, without surgery, it pre-serves the normal biomechanics of the intervertebral disc, and it offers the same or better pain relief.

23.6 Perspectives on the Role of Stem Cell Biology in the Treatment of Intervertebral Disc Disease

Recent advances in stem cell biology offer a number of entic-ing possible avenues for intervertebral disc research, with therapeutic potential. Undoubtedly, studies of the applications

380 D. Sakai and J. Mochida

of stem cell biology will increase; however, as already dis-cussed, it is important that investigators share a common stem cell terminology. There is an increasing interest in this fi eld and evidence of endogenous stem/progenitor cells within the intervertebral disc. Identi fi cation of these stem/progenitor cells in their niche and the fates of these cells should provide insight into the key elements in the pathogenesis of disc degeneration and the endogenous repair system of the inter-vertebral disc. This insight is required for potential therapeu-tic interventions. For instance, molecules that enhance the

recruitment and function of disc stem/progenitor cells may enhance the longevity of the tissue itself. This area of research is still underde fi ned and awaits rigorous investigation. Supporting this insight, an epoch making fi nding that nucleus pulposus progenitor cell exhaustion relates to aging and degeneration has been recently reported (Sakai et al. 2012 ) . In this study, the authors sorted human and mouse nucleus pul-posus cells using various surface markers and then scored their ability to form colonies. They identi fi ed nucleus pulpo-sus progenitor cells that formed spheroid colonies in Tie2 + and disialoganglioside GD2 + population. These spheroid col-onies demonstrated superior aggrecan and collagen II produc-tion capability compared to other colonies (Fig. 23.3 ). Clonal analyses of Tie2 + GD2 + human nucleus pulposus cell demon-strated that these cells are highly proliferative and clonally capable of differentiation into multiple mesenchymal and nucleus pulposus lineages. Moreover, this multipotent ability is sustained through long-term engraftment and was demon-strated to include the ability for self-renewal. Analyses in clinically obtained tissue provided new concept in the underde fi ned pathology of disc degeneration by showing a gradual exhaustion of Tie2 + cells in the disc correlating with aging and degeneration in humans. Moreover, they de fi ned the importance of Tie2/angiopoietin-1 niche (Fig. 23.4 ) in maintenance of Tie2 + nucleus pulposus progenitor cells, which may lead to new treatment targets. To induce MSC to differentiate into intervertebral disc cells, an obstacle that must be overcome is that induced MSCs never proceed beyond intervertebral disclike cells. This problem is exacerbated by the lack of a speci fi c marker or assays that de fi nes a cell as an intervertebral disc cell. Fundamental studies that can de fi ne a cell as a disc cell that can function in vitro will be most useful in this fi eld. The use of MSC as an activation tool for drug delivery systems may still offer a novel strategy for tissue repair. In the developmental and maturation stages of the disc, the mesenchymal cells surrounding the nucleus pulposus are

Fig. 23.3 Spheroid colony derived from nucleus pulposus progenitor cells. These cells express abundant aggrecan ( red ), the major extracel-lular matrix component of the nucleus pulposus ( blue indicates nuclear staining with diamidino-2-phenylindole) (Reproduced from Sakai et al. 2012 )

Fig. 23.4 Tie2/angiopoietin-1 signaling maintains progenitor cells in the nucleus pulposus of the intervertebral disc. Both Tie2 ( red ) and Ang-1 ( green ) are expressed in cluster of cells found in the nucleus pulposus ( blue indicates nuclear staining with diamidino-2-phenylindole) (Reproduced from Sakai et al. 2012 )

38123 Use of Stem Cells for Regeneration of the Intervertebral Disc

thought to be in fl uenced by notochord-derived cells. Knowledge of the interactions between the components that participate in disc development will certainly provide new information on tissue regeneration and tissue engineering. Finally, more clinical trials of actual MSC transplantation for intervertebral disc repair will also undoubtedly be reported in the future, using various techniques and stem cells from vari-ous sources. However, caution is strongly warranted. For example, using a rabbit model, Vadalà et al. ( 2012 ) reported that cell leakage after MSC injection into the intervertebral disc can cause osteophyte formation. They suggest that cell carriers or annulus-sealing techniques should be assessed or perhaps postsurgical rehabilitation protocols investigated, to minimize leakage. Unintended differentiation and tumorigen-esis are also potential risks usually faced in stem cell thera-pies. Although many animal studies and preliminary human trials have supported the promising future of stem cell therapy for disc disease, careful application of cells and techniques, with the selection of the appropriate types of patients in a strictly controlled manner, are key elements in ensuring the success of these therapies.

23.7 Summary of Critical Concepts Discussed in the Chapter

While de fi nition of stem cells in the mesenchymal tissues • has changed with time, there is still a lack of knowledge of their in vivo characteristics, and therefore, selection of stem cells for regeneration of the intervertebral disc needs to be carefully evaluated. It is important that investigators share a common stem • cell terminology in designing experiments to evaluate the use of stem cells for intervertebral disc research. Laboratory investigations have revealed that a stem cell • system is present in the intervertebral disc and that its exhaustion may be related to aging and degeneration. Stem cells can be induced to express some of the charac-• teristics of intervertebral disc cells. Stem cells are capable of stimulating intervertebral disc • cell metabolism. Stem cells can facilitate intervertebral disc tissue engi-• neering and repair. Investigation of intervertebral disc stem/progenitor cells • in their niche and the fates of these cells should provide insight into key events in the pathogenesis of disc degen-eration and the endogenous repair system. This informa-tion should be of great use in the design of new biological therapies.

Acknowledgments The authors are grateful to the staff of the Education and Research Support Center, Tokai University. This work was supported in part by a Grant-in-Aid for Scienti fi c Research from

the Ministry of Education, Culture, Sports, Science and Technology of Japan and a grant from AOSpine International and AO Foundation to D.S.

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