Review Accepted after revision: June 19, 2012 Published online: September 4, 2012
Cells Tissues Organs DOI: 10.1159/000341153
Development and Regeneration Potential of the Mammalian Intervertebral Disc Helena Barreto Henriksson Helena Brisby Department of Orthopedics, Institute of Clinical Sciences, Sahlgrenska University Hospital, Gothenburg University, Gothenburg, Sweden
Key Words Cartilage biology Cell and molecular biology Cell migration Intervertebral disc Mesenchymal stem cells Notochord Notochordal cells Regeneration
Abstract At the present time, the normal cell proliferation rate and regeneration processes in the intervertebral disc (IVD) are not fully known. Historically, the IVD has been considered an organ with little or no regenerative capacity. However, several studies have identified the presence of cells expressing progenitor/stem cell markers in adult cartilage tissue and recent data suggest that adult mammalian IVDs have regenerative capacity, albeit slow. The aim of this review is to give an overview of the present knowledge regarding IVD development, regeneration and repair mechanisms in mammals, with a special focus on human discs. At a time when regenerative medicine is making progress and biological treatment options, such as stem cell therapy, are suggested for patients with degenerated discs causing chronic low back pain, basic knowledge about disc cells and their regenerative capacity form a useful basis for the exploration of new treatment options. Copyright © 2012 S. Karger AG, Basel
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Introduction
The adult mammalian intervertebral disc (IVD) is an avascular and aneural fibrocartilage tissue composed of an inner nucleus pulposus (NP) and an outer zone, the annulus fibrosus (AF) localized adjacent to the endplates [Peacock, 1952; Rufai et al., 1995; Kim et al., 2003]. The NP is hydrated, gelatinous tissue and contains chondrocyte-like cells embedded in extracellular matrix (ECM). The hydrophilic proteoglycans in the ECM retain water and thereby provide strength and spongy
Abbreviations used in this paper AC AF CK ECM EMT IVD LMP1 MMP MSC NP
articular cartilage annulus fibrosus cytokeratin extracellular matrix epithelial-mesenchymal transition intervertebral disc Lim mineralization protein-1 matrix metalloproteinase mesenchymal stem cells nucleus pulposus
Dr. Helena Barreto Henriksson Department of Orthopedics, Sahlgrenska University Hospital Gothenburg University SE–413 45 Gothenburg (Sweden) Tel. +46 31 342 6895, E-Mail helena.barreto.henriksson @ gu.se
features to the IVD [Bibby et al., 2001; Martin et al., 2002]. NP cells are similar but not identical to chondrocytes isolated from articular cartilage (AC), hence they are termed chondrocyte-like cells [Sive et al., 2002]. Specifically, they have a cell morphology similar to AC cells with rounded cells enclosed in lacunae and they express SOX9 (sex-determining region y-box 9), collagen II and aggrecan mRNA. The AF surrounds the NP and is organized in fibrous ring-like structures: the lamellae. The IVD obtain nutrition from the endplates by diffusion mechanisms. The AF is attached to the vertebrae at the superior and interior surfaces. In general, cartilage tissues are considered to no or only poor repair capacity. The avascular structure is believed to be one of the major reasons. In general, adult cartilage has been considered to be a tissue consisting only of terminally differentiated cells such as chondrocyte-like cells, which are present in the NP of the IVD [Hollander et al., 2010]. Cartilage is a type of dense connective tissue derived from the mesoderm [Onyekwelu et al., 2009] (with the exception of craniofacial cartilage, e.g. mandibular cartilage, that originates from neural crest cells deriving from ectoderm) [Noden, 1991; BronnerFraser, 1994; Olsen et al., 2000]. There are three main different types of cartilage in mammals; (1) articular cartilage, e.g. covering the surface of synovial joints; (2) fibrocartilage, e.g. in the meniscus and the IVDs (NP and the inner part of AF), and (3) elastic cartilage, e.g. in the epiglottis [Nielsen and Bytzer, 1979; Rufai et al., 1995; Hunziker et al., 2002]. Frequently presented problems with chronic low-back pain, which are believed to have a strong connection with disc degeneration, promote further studies to elucidate normal development and repair processes of the IVD. The understanding of these processes may be of importance for the use of regenerative medicine (potential biological treatment options) for disc degeneration. At the present time, the available treatment options for degenerated disc disease include symptomatic treatment (analgesics, physiotherapy and cognitive treatment) and/or surgical treatment (spinal fusion and disc replacement with artificial disc prostheses). However, these treatment modalities do not attack the underlying cause of disc degeneration. Biological treatment options of the disc, including cell therapy, have been suggested as complementary/ optional treatment methods for disc degeneration [Leung et al., 2006]. This review aims to examine the current knowledge regarding the regeneration potential of the IVD.
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Cells Tissues Organs
Embryonic Development of the Vertebrae and the IVDs
Knowledge of the embryonic development phases and processes of the IVD may help to understand the adult regeneration of the IVD. In the development of the IVD, in early human embryogenesis at the end of the 4th week, cells from several somite compartments come together to form the vertebrae. Cells from the somitocoel together with ventral cells form the IVDs and the vertebral joint surfaces. Fundamental steps that take place in the formation of the vertebrae are: (1) mesenchymal cells differentiate and migrate from the sclerotome to the region of the future vertebrae and ribs; (2) mesenchymal cells form a precartilage mass; (3) the shape of the cartilage forms, and (4) bone formation occurs via replacement of the cartilaginous tissue with bone tissue [Christ and Wilting, 1992; Langman, 2000; Carlson, 2009]. Cell migration is important in the embryo and the failure of migration to specific areas can result in malfunction of limb development. The development of the vertebrae is controlled at different levels and during several developmental periods of the embryo by signaling systems, e.g. regulation by the HOX gene products (homeobox genes) [Alberts et al., 1998; Mallo et al., 2010]. The development of the individual vertebrae begins with a Shh (sonic hedgehog)-mediated induction by the notochord cells on the somite in order to form the sclerotome. Other signaling pathways that participate in the formation of the vertebrae include the BMP- and the WNT signaling systems [Hoang et al., 1998; Day et al., 2005; Mundy et al., 2011]. Mesodermal chondrogenic lineage markers include: GDF5 (growth and differentiation factor-5, synonym CDMP1) and SOX9, SOX5 and SOX6, which are expressed in prechondrocytic cells (progenitor cells) during the formation of cartilage and Brachyury [Langman, 2000; Smits et al., 2004; DiPaola et al., 2005; Carlson, 2009; Dy et al., 2010]. In mammals, Pax-1 (paired box 1) is continuously expressed during the development of the IVD during embryogenesis. The Pax-1 gene encodes transcriptional factors that regulate and take part in vertebrae formation. It is believed that Pax-1 and the subsequent formation of the IVD is an important factor in maintaining the segmental character of the vertebral column [Langman, 2000; DiPaola et al., 2005; Carlson, 2009; Risbud et al., 2010] (see also the description of markers in table 1). When IVD are formed during the human embryonic development, the notochord disappears from the vertebral bodies and forms the condensed mesenchymal primordia (the earliest cells) of the IVD. The tissue will form Henriksson/Brisby
Table 1. Summary of selected genes involved in embryonic development of bone and cartilage tissues
Abbreviation
Full name/gene family
Description
References
BMP-1 BMP-2 BMP-7 BMP-14
name is an abbreviation of bone morphogenic proteins; BMPs are a subfamily of the TGF- gene superfamily
BMP signaling pathway involved in the formation of bone and cartilage tissues in the embryo
Langman, 2000; Smits et al., 2004; DiPaola et al., 2005; Carlson, 2009; Dy et al., 2010
DELTA4
DELTA4 belongs to the NOTCH gene family
involved in NOTCH signaling pathway, ligand to NOTCH
Artavanis-Tsakonas et al., 1999; Henriksson et al., 2009; Tao et al., 2010
GDF5
growth and differentiation factor 5; syn. BMP14, CDMP1; member of the TGF- family
GDF5 has a role in skeletal and joint development; regulator of cell growth and differentiation in both embryonic and adult tissues
Langman, 2000; Henriksson et al., 2009
HOX-genes
subsets of homeobox genes
HOX genes are involved in embryonic development of e.g. the patterning formation along the anteroposterior body axis
Langman, 2000; Carlson, 2009
JAGGED1
JAGGED1 belongs to the NOTCH gene family
involved in NOTCH signaling pathway ligand to NOTCH
Artavanis-Tsakonas et al., 1999; Henriksson et al., 2009; Tao et al., 2010
LMP-1
gene encode the Lim protein-1
involved in bone formation
Yoon et al., 2004
NOTCH1
NOTCH homolog1 belongs to the NOTCH gene family
NOTCH signaling controls cell proliferation/differentiation processes during embryonic and adult life; functions as a receptor for membranebound ligands, e.g. Jagged1
Artavanis-Tsakonas et al., 1999; Henriksson et al., 2009; Tao et al., 2010
Shh
sonic hedgehog belongs to the hedgehog gene family
sonic hedgehog plays a role during embryogenesis in cell growth, limb formation and shaping (patterning) of the body
Langman, 2000; DiPaola et al., 2005; Carlson, 2009
SOX5-2 SOX-6 SOX-9
sex-determining region, y-box members of SOX gene family
SOX genes are involved in the development of e.g. cartilage tissues
Smits et al., 2004; Dy et al., 2010
PAX-1
name is an abbreviation of paired box 1; belongs to the group of paired box genes
PAX-1 is expressed during embryonic development in the formation of the vertebrae and IVD
Langman, 2000; DiPaola et al., 2005; Carlson, 2009; Risbud et al., 2010
WNT
name derived from a combination of wingless and INT genes
WNT signaling pathway, a highly conserved signaling pathway that has a central role in embryonic development and tissue regeneration
Hoang et al., 1998; Langman, 2000; Day et al., 2005; Carlson, 2009; Mundy et al., 2011
around the degraded notochord between the developing vertebrae. The notochord becomes discontinuous, persisting only inside the primitive AF, where it is believed that the entrapped notochord cells are involved in the formation of the primitive immature NP in the full-term fetus [Peacock, 1951; Rufai et al., 1995; Christopherson et al., 1999; Langman, 2000; Hayes et al., 2001; Carlson, 2009]. During embryogenesis, the notochord is gradually
surrounded and replaced by immigrating mesenchymal cells, which synthesize the fibrocartilaginous AF and the vertebral bodies [Peacock, 1951; Babic, 1991] (fig. 1). The remains of the notochord can be found in the center of the IVD: the NP. The cells of the adult NP more closely resemble AC and are therefore termed chondrocyte-like cells [Walmsley, 1953; Errington et al., 1998] (fig. 1).
Development and Potential Regeneration of the Mammalian IVD
Cells Tissues Organs
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Notochord
Notochord vestige
Bone trabeculae
Fibrous tissue
‘Immature’ NP
Notochord cells
AF
Vertebral body
Mesenchymal tissue
Fibrocartilage Cartilage endplate
a
b
Fig. 1. Schematic simplified image of stages in the normal devel-
opment of the human IVD with the notochord and notochordal cells illustrated (image based on Peacock [1951] and Christopherson et al. [1999]). a In the 4th week of gestation, mesenchymal cells occur in the peripheral region of the disc and more rounded irregularly distributed cells are present around the notochord with capsules of immature cartilage (15-mm embryo). b In the 5th week of gestation, fibers at the periphery of the AF are now present and more mature fibrocartilage is developed adjacent to im-
Origin of Chondrocyte-Like Cells
The origin of human chondrocyte-like cells of the IVD is not fully understood, but two hypotheses have been put forward. It is unclear at present whether the alteration in the IVD cell population from notochordal cells to chondrocyte-like cells is due to the continued and terminal differentiation of the notochordal cells into a chondrocyte-like phenotype (hypothesis 1) or programmed apoptosis of the resident IVD cells followed by invasion of progenitor cells destined to be NP cells, derived from the cartilaginous endplates, AF or possibly from another location (hypothesis 2) [Walmsley, 1953; Rufai et al., 1995; Hunter et al., 2003; Kim et al., 2003; Risbud et al., 2010].
c
mature cartilage. Notochordal cells are present in the center surrounded by a mucoid matrix (29-mm embryo). c Full-term fetus: bony trabeculae and intertrabecular marrow have developed within the vertebral body. No vestige of the notochord is present. Small groups of notochordal cells can be found in the center of the notochordal region and at the circumference of this region small areas of fibrocartilage (‘immature’ NP) which are surrounded by AF fibers.
et al., 1991; Chelberg et al., 1995]. The cells are distributed as solitary cells or in cell clusters. The ECM which is produced by chondrocyte-like cells is composed mainly of type II collagen, but type VI and XI collagens are also present in small amounts [Bibby et al., 2001; Roberts et al., 2006; Singh et al., 2009]. Further, proteoglycans such as aggrecan and versican (most common components) as well as decorin, biglycan and lumican (more sparsely distributed) are present in the ECM of the NP [Roberts et al., 1994, 2006; Singh et al., 2009].
Human IVDs NP Cells The NP (human) has a cell distribution of 4 ! 103cells/ mm3 [Roberts et al., 2006; Liebscher et al., 2011]. The chondrocyte-like cells are the dominating cell type in the NP of the mature human IVD [Hunter et al., 2004; Cappello et al., 2006]. The chondrocyte-like cells present in the NP are rounded cells with a spheroid nucleus [Roberts
Notochordal Cells In human childhood, the two major populations of cells in the NP are the notochordal and chondrocyte-like cells. The notochordal cells are typically physaliferous or vacuole-containing cells and have a large cellular size (25–85 m). They contain ‘immature’ mitochondria associated with the rough endoplasmatic reticulum, cytoplasmic inclusions (e.g. glycogen) and filaments [Trout et al., 1982; Hunter et al., 2003; Guehring et al., 2008]. The exact function of these different cellular structures is still unclear. The notochordal cells are believed to gradually disappear and be replaced by chondrocyte-like cells, the cells that make up the center (NP) of the IVD by about 10 years of age in humans. However, Weiler et al. [2010] reported data supporting that a remaining population of notochordal cells is present in the adult human IVD and
Cells Tissues Organs
Henriksson/Brisby
Cell Types and Matrix of the IVD
4
identified cells positive for the notochordal markers: cytokeratin (CK) proteins (CK8, CK18 and CK19) and galectin-3, which were detected in different age groups ranging from 1–86 years. The expression of these markers declined with age but their presence was found in both the young and elder populations. Further, expression of the markers CK8, CK18 and CK19 was recently reported by Minogue et al. [2010] in adult humans. Similar results were obtained in a bovine study on small populations of residual notochordal cells expressing CK8 [Gilson et al., 2010]. These markers are described in table 2. A specialized function of notochordal cells was proposed by Hunter et al. [2003], where the notochordal cell could be seen either as a stem cell itself or an ‘organizer cell’ that directs the migration of stem cells from the surrounding mesenchyme during growth and undergoes apoptosis when the formation of the IVD is complete. The persistence of notochordal cells in human adulthood is supported by the occurrence of rare tumors, both non-malignant [Haasper et al., 2007] and malignant (e.g. chordoma tumors), originating from adult notochordal cells/tissues [Le Charpentier et al., 1988; Yamaguchi et al., 2002; Riopel and Michot, 2007]. AF Cells The AF has a cellular distribution of 9 ! 103 cells/ mm3 and is composed of 15–25 fibrous lamellae enclosing the NP. The cells of the inner AF consist of chondrocyte-like cells while the cells of the outer AF are more elongated and have a fibroblast-like morphology [Roberts et al., 1991; Errington et al., 1998; Roberts, 2002]. The outer lamellae are rich predominantly in type I collagen which decreases toward the NP; the inner lamellae consist mainly of collagen II. Other minor types of collagen in the AF include type III, V, VI and IX [Buckwalter and Mankin, 1998; Eyre et al., 2002]. Non-Human IVDs In many mammalian species, cellular IVD density is higher and the notochordal cells are still present in the adult animal, e.g. in the rat, pig and rabbit [Hunter et al., 2003, 2004]. Further, the collagen ratio in the IVD also differs between species that are of dipod or tetrapod origin. A limitation with animal models in research is that they are not always comparable to the human situation due to differences in anatomy (e.g. dipod vs. tetrapod construction) which creates differences in the load imposed on the IVD. In addition, the differences in cellular composition can influence and make the interpretation of results more complicated [Lotz, 2004]. Development and Potential Regeneration of the Mammalian IVD
The Degenerated Disc The degenerated disc is characterized by increased cell clustering, reduced disc height and cell death as well as a decreased fluid-binding ability causing concomitant dehydration of the tissue. Changes in the AF include fibrocartilage formation, fissures and disorganization of the annular architecture and increase in collagen II [Adam and Deyl, 1984]. In the NP, the expression of collagen II decreases and is replaced by other collagens, e.g. collagen types I, III, V, VI and X [Nerlich et al., 1998; Rutges et al., 2010]. These alterations precede the morphological reorganization associated with disc degeneration: loss of disc height, disc bulge, disc herniation and endplate defects. Furthermore, the ECM composition of the IVD is altered: a decrease in glycosaminoglycan production could be seen as well as an increase in the matrix degrading enzymes [e.g. matrix metalloproteinases (MMPs): MMP1, MMP3, MMP9 and MMP13) and cytokines (e.g. interleukins 1 and 1) [Le Maitre et al., 2005; Goupille et al., 2007; Ulrich et al., 2007].
Differences in Marker Profiles between Chondrocyte-Like Cells (NP) and Chondrocytes (AC)
It is of importance to understand the cell biology (e.g. cell surface marker profiles) in order to be able to develop new biological cell therapy strategies (e.g. for degenerated discs). Transplantation of a suitable cell type in order to achieve a good cell adaptation to the environment and high ECM production would be preferable. However, gene expression analysis of ECM components showed that IVD cells exhibit different collagen 2A/collagen 1A and collagen 1A/aggrecan 1 ratios compared with AC in a rabbit model [Clouet et al., 2009]. In the same study, collagen 5A1 was predominantly expressed in AF cells and barely detectible in cells from AC or NP [Clouet et al., 2009]. In a bovine model, differences in gene expression, i.e. between NP chondrocyte-like cells and AC, were observed. In that study, chondrocytes but not NP cells expressed MMP12 and MMP27; this difference was suggested to be a potential marker to distinguish NP cells from chondrocytes [Cui et al., 2010]. Until now, there has been little evidence of a single unique marker(s) for chondrocytelike cells. However, recently, Minogue et al. [2010b] identified potential IVD markers (bovine and human studies) after comparing NP and AC cell gene expression from bovine microarray analyses, e.g. TNMD (tenomodulin), TNFAIP6 (tumor necrosis factor -induced protein 6), FOXF1 Cells Tissues Organs
5
Table 2. Overview of cellular markers detected in the IVD region (gene and protein level) Abbreviation
Full name
Potential markers for notochordal cells CK8 cytokeratin CK18 CK19 Galectin-3
galectin-3 member of the lectin family
Potential markers for IVD cells (NP and AF) AQP1 Aquaporin 1, member of the membrane intrinsic protein gene family
Description/function
References
cytokeratin proteins are part of the cytoskeleton; expressed in different types e.g. epithelial cells
Minogue et al., 2010; Weiler et al., 2010
-galactoside binding protein, involved in e.g. cell adhesion/differentiation; expressed on epithelial/ immunoreactive cells
Weiler et al., 2010
AQP1 function as water-selective channels in many water-transporting cells/tissues
Minogue et al., 2010b
FOFXF1
forkhead box f1, belongs to the forkhead box genes
FOXF1 proteins are important in the embryonic development of the mesenchyme
Minogue et al., 2010b
FOFXF2
forkhead box f2 belongs to the forkhead box genes
FOXF2 proteins are important in the embryonic development in e.g. cellular differentiation/survival
Minogue et al., 2010b
TNFA1P6
TNF--induced protein 6, member of the hyaluronan binding family
TNFA1P6 contains a hyaluronan binding domain and is involved in ECM stability/cell migration and inflammatory processes
Minogue et al., 2010b
TNMD
tenomodulin, member of the type II transmembrane glycoprotein family
tenomodulin is predominantly expressed in tendons, ligaments and take part in e.g. cell proliferation
Minogue et al., 2010b
cell membrane-bound receptor for collagen, integrin subunit, e.g. expressed on MSCs
Risbud et al., 2007
Stem cell markers (detected in the IVD) CD49a cluster of differentiation 49a CD73
cluster of differentiation 73; syn. ecto-5-nucleotidase
cell membrane-bound protein expressed on e.g. MSCs, involved in inflammatory processes and immunoreactions
Blanco et al., 2010
CD90
cluster of differentiation 90; syn. THY, thymocyte differentiation antigen 1
cell membrane-bound protein expressed on MSCs; function not fully known
Henriksson et al., 2009; Blanco et al., 2010
CD105
Cluster of differentiation 105; syn. endoglin
cell membrane-bound protein expressed on e.g. MSCs; transmembrane glycoprotein, takes part in TGF- signaling
Henriksson et al., 2009; Blanco et al., 2010
CD166
cluster of differentiation 166; syn. activated leukocyte cell adhesion molecule (ALCAM)
cell membrane-bound protein expressed on e.g. MSCs, involved in adhesion interactions/immunoreactions
Henriksson et al., 2009; Blanco et al., 2010
STRO-1
stromal cell antigen-1
cell membrane-bound protein expressed on MSCs. STRO1+ cells can differentiate into the e.g. osteogenic, chondrogenic lineages
Henriksson et al., 2009
CD117
cluster of differentiation 117 syn. C-KIT
primarily, a hemapoetic stem cell marker (also expressed in non-hemapoetic subpopulations), cell membrane-bound protein, tyrosine kinase receptor
Henriksson et al., 2009
CD133
cluster of differentiation 117 syn. prominin-1
cell membrane-bound glycoprotein expressed on e.g. hemapoetic stem cell and endothelial progenitor cells; function unknown
Risbud et al., 2007
Markers involved in cellular migration and EMT (detected in the IVD) protein involved in cellular adhesion, cell signaling and 1-integrin 1-Integrin ECM interactions, widely expressed in many cell/tissue syn. fibronectin receptor subunit , CD29 types
Henriksson et al., 2012
SNAI1
SNAIL homolog 1 member of the SNAIL gene family
cellular protein expressed during EMT, down-regulates E-cadherin (cell-to-cell adhesion molecule), SNAI1 is expressed in e.g. migrating tumor cells
Nieto, 2002; Thiery and Sleeman, 2006; BarralloGimeno and Nieto, 2009
SLUG
SNAIL homolog 2 SNAI2 member of the SNAIL gene family
cellular protein expressed during EMTfunction as repressor of E-cadherin transcription, SLUG is expressed in e.g. migrating tumor cells
Nieto, 2002; Thiery and Sleeman, 2006; BarralloGimeno and Nieto, 2009
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Cells Tissues Organs
Henriksson/Brisby
(forkhead box f1), FOXF2 (forkhead box f2) and AQP1 (aquaporin 1). These markers showed significantly higher expression in NP and AF cells than in AC cells, and these results were more pronounced in the bovine model than in the human samples [Minogue et al., 2010b] (table 2).
Are There Tissue-Specific Populations of Mesenchymal Stem Cells within the IVD?
Human mesenchymal stem cells (MSCs) isolated from IVDs have been demonstrated to be capable of differentiation into osteocytes and chondrocytes by Risbud et al. [2007] and Blanco et al. [2010]. Regarding the differentiation capability of these cells into adipocytes, contrasting findings were obtained. Risbud et al. [2007] found that isolated IVD-MSCs were able to differentiate along the adipogenic lineage while Blanco et al. [2010] did not. Furthermore, Blanco et al. [2010] compared cells that expressed MSC cell surface markers which were isolated from human degenerated discs (NP) [Blanco et al., 2010] and bone marrow-derived MSCs. The cells isolated from the degenerated discs were positive when assayed by flow-cytometric methods (FACS) for: CD90, CD73, CD105 and CD166, and negative for CD45, CD34, CD14 and HLA-DR, which, according to the International Society for Cell Therapy, is the expression profile for MSCs (from bone marrow). Risbud et al. [2007] isolated immature cells from human degenerated whole discs and demonstrated the expression of the same markers with the addition of the markers CD63, CD49a and CD133. In addition, an in vivo study has recently demonstrated cells expressing stem/progenitor cell markers (e.g. STRO-1, NOTCH1 and CD117) in normal mammalian IVD (rabbit, porcine and rat IVD) and expression of CD105, CD166, STRO1, NOTCH1 and CD117 was further detected in human degenerated IVD tissue biopsies [Henriksson et al., 2009]. Some of the markers described above were also identified at gene level by PCR methods [Risbud et al., 2007; Blanco et al., 2010]. The full profile of tissuederived IVD stem cells remains to be completed but there are several indications of the presence of stem/progenitor cells in the IVD (see description of markers in table 2).
Potential Stem Cell Niches in the IVD Region
In their niches, stem cells can divide according to a hypothetical asymmetric cell division pattern where one daughter cell gives rise to a more differentiated progeniDevelopment and Potential Regeneration of the Mammalian IVD
tor cell while the other cell maintains the original genome [Karpowicz et al., 2005; Mitsiadis et al., 2007; Knoblich, 2008]. Stem cell niches located adjacent to the epiphyseal plate and in the outer AF border have been proposed [Henriksson et al., 2009]. Similar observations in a corresponding region adjacent to the epiphyseal plate in the knee joint have also been reported [Karlsson et al., 2009] as well as the presence of progenitor cells in AC of the knee joint [Dowthwaite et al., 2004; Williams et al., 2010]. The stem cell niche is a specific anatomical localization which harbors stem cells [Ohlstein et al., 2004; Li and Xie, 2005; Moore and Lemischka, 2006; Mitsiadis et al., 2007], and the location and nature of these niches can vary depending on the tissue type [Ohlstein et al., 2004; Mitsiadis et al., 2007; Voog and Jones, 2010]. The epithelial-mesenchymal transition (EMT) is an evolutionary well-conserved process that is present in many organisms. For example, EMT take part in the process of migration of immunoreactive cells, e.g. macrophages and tumor cells during the development of tumor metastases in cancer [Brabletz et al., 2005; Vasiliev, 2008]. During EMT, the cell cytoskeleton is rearranged to a more flattened migratory phenotype [Doherty and McMahon, 2008]. Members of the snail super family proteins that trigger EMT are involved in cytoskeleton rearrangement processes and have been suggested to be involved in many developmental processes, e.g. neural differentiation, normal migration, maintenance of adult stem cell phenotype and cell survival [Nieto, 2002; Barrallo-Gimeno and Nieto, 2009]. Snail genes have a central role in the regulation of EMT, for example enhanced levels of Snai1 have a downregulatory effect upon E-cadherin (a cell-to-cell adhesion molecule) [Thiery and Sleeman, 2006]. EMT involves a series of events in which cell-to-cell and cell-to-ECM interactions are altered to release epithelial cells from the surrounding tissue where the cytoskeleton is reorganized to provide the ability to move through the surrounding tissue [Thiery, 2003; Hotz et al., 2010]. From the niches, migrating progenitor cells are believed to participate in normal growth and repair mechanisms, for example to react to distant signals from a site of injury and migrate to that location. Homeostasis of many tissues such as the skin, blood and gastrointestinal epithelium is regulated through a balance of proliferation, differentiation and migration of adult stem cells [Potten and Loeffler, 1990; Moore and Lemischka, 2006]. Different hypotheses have been proposed for the existence of adult stem cells. One hypothesis is that stem cells occur during embryonic development and thereafter persist at different locations in specific microenvironments. Cells Tissues Organs
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IVD Regeneration
The IVD has often been described as an organ with degeneration continuously with age or initiated by an injury event [Bibby et al., 2001; Adams and Roughley, 2006]. Normal proliferation and spontaneous regeneration processes in the IVD have only been discussed sparsely. In various human studies, indications of a normal cell turnover and minor regeneration processes have been observed within the IVD, especially in the outer rings of the AF [Humzah and Soames, 1988; Videman et al., 2006]. In the inner parts of the AF and in the NP, no clear regeneration capacity has been reported. In vivo labeling with BrdU (5-bromo-2-deoxy-uridine) has been performed in a rabbit model where cell proliferation zones were detected with a stem cell nichelike pattern [Henriksson et al., 2009]. Using in vivo labeling in rabbits and additional stem cell markers, a low number of proliferating cells were detected in NP and the inner parts of the AF. Further potential stem cell niches were identified in the outer AF regions and adjacent to the epiphyseal plate in different species of mammals. The concept of local stem cell niches that take part in normal regeneration of different organs is a theme that runs through the whole animal kingdom. In the niche, stem cells can be in a resting state (so called ‘slow-cycling’- or ‘label-retaining’ cells which can be detected by BrdU using in vivo labeling methods) or acting on distant signaling when the stem cells can emigrate from the niche to support tissue regeneration [Ohlstein et al., 2004; Mitsiadis et al., 2007; Voog and Jones, 2010]. Such a local reservoir of immature cells that undergo EMT and migrate from niches could possibly contribute to the development and support of normal regeneration of the adult mammal IVD (fig. 2). In the in vivo labeling rabbit model, the, migration markers SLUG, SNAI1, B1 and integrin, and the chondrogenic lineage markers GDF5 and SOX9 were also investigated in both young and adult rabbits [Henriksson et al., 2012]. These markers were found in the niche region along the migration route and more sparsely towards the inner regions of the IVD. Based on these findings, it seems possible that progenitor cells are recruited and migrate from the niche into the IVD, and together with solitary stationed progenitor cells maintain the regeneration processes of the IVD, possibly in cooperation with MSCs from the bone marrow niche, the endplates or together with remnant pools of notochordal cells. The presence of the same progenitor and stem cell markers in degenerative human IVD tissue (detected by 8
Cells Tissues Organs
immunohistochemistry and flow cytometry analyses) as found in the non-degenerated IVDs from other investigated species [Henriksson et al., 2009] is of interest since the cell population within the IVD, especially in the NP, largely differs between species [Hunter et al., 2004]. NOTCH1 signaling is known to play a decisive role in cell-to-cell interactions and as a cell fate determinant in different stem cell niche structures [Artavanis-Tsakonas et al., 1999; Tao et al., 2010]. It can be speculated that cells expressing MSC markers could also be recruited from the vascular system to the IVD since the ingrowth of blood vessels in severely degenerated discs has been described [Lee et al., 2011]. However, the finding of cells expressing MSC markers in normal IVDs in several different mammals rather supports the presence of local tissue-specific stem cells within the IVD.
Do Different Cell Types Interact to Maintain IVD Regeneration?
Stem cells in general are small unspecialized cells with few phenotypic characteristics in contrast to terminally differentiated cells that display many distinct morphological characteristics in their phenotype, depending on the cell type. The notochordal cell displays a more characteristic phenotype compared to stem cells, e.g. with glycogen stored within the cytoplasm, which indicates a specialized function. The notochordal cell has been suggested to function as an ‘organizer cell’ during IVD development [Hunter et al., 2003; Kim et al., 2009]. In vitro studies have demonstrated that IVD cells have positive effects on matrix formation in cell cultures when cultured in conditioned media from notochordal cells [Korecki et al., 2010; Purmessur et al., 2011; Abbott et al., 2012]. The fact that notochordal cell conditioned medium (derived from porcine normal control cultures) is capable of stimulating bone marrow-derived human MSC differentiation towards a young NP phenotype in an in vitro environment is interesting and is supported by studies in co-culture systems between notochordal and NP cells. In these studies, proteoglycan accumulation was increased and soluble factors produced by notochordal cells were suggested to be influencing the increased proteoglycan accumulation [Aguiar et al., 1999; Korecki et al., 2010] (fig. 3). In another in vitro study by Richardsson et al. [2006], it was shown that cell-to-cell contact is important for increased matrix accumulation when co-culturing bone marrow-derived MSCs and IVD cells. In addition, SvanHenriksson/Brisby
Fig. 2. Schematic picture that illustrates the hypothesis of cellular movements, directions from niches (above)
and EMT cellular changes (below) to a flattened migratory cellular phenotype within the IVD region. The presence of stem/progenitor cells in the IVD niche and in the hypothetized migration route (MR) and the border of the outer AF (AFb) are indicated with colored dots (image-based studies performed in rabbit models) [Henriksson et al., 2012]. TZ = Transition zone. Reproduced with permission from SPINE (Phila).
vik et al. [2010] obtained similar results in a 3D in vitro co-culture study and in this study when using conditioned media cultures, no evidence for a soluble factor was found. These findings suggest that there are probably several different cell types co-existing in adult mammal IVD during their life cycle that interact to maintain normal regeneration of the IVD.
Stimulation of IVD Regeneration
Different approaches to stimulate regeneration have been suggested that include molecular factors, environmental factors such as oxygen, mechanical loading or physical exercise. Development and Potential Regeneration of the Mammalian IVD
One possibility to stimulate the regenerative capacity within the disc would be to inject small molecule compounds with stimulatory effects on cells present in the disc, e.g. growth factors. Following the injection of growth and differentiation factor 5 (GDF5 synonym BMP14, CDPMP1) into the bovine disc, some positive effects were observed on disc height, improvement in magnetic resonance imaging scores [Chujo et al., 2006] and matrix formation [Le Maitre et al., 2009]. Additionally, in vitro studies involving BMP-2 and TGF- gene therapy models have also shown some positive effects on matrix synthesis [Yoon et al., 2004; Levicoff et al., 2005; Moon et al., 2008]. Approaches to influence regeneration of degenerated discs also include cell therapy strategies which in the last years have been explored in many different animal models where Cells Tissues Organs
9
Soluble factors BMPs? MSC
Notochordal cells WNTs?
– Differentiation – Cell-to-cell contact
Cell signaling
NP
GAGs
AF
Chondrocyte Mobile ions Collagen Proteoglycan
Chondrocyte-like cells
Link protein Hyaluronan IVD
Fig. 3. Schematic image illustrating the hypothesis of paracrine interactions in vivo within the NP that may take
part in the regeneration of the IVD and ECM components in the NP. Paracrine interactions between remnant pools of notochordal cells and MSC/progenitor cells may be one type of cellular interactions that support differentiation of MSC/progenitor cells into a chondrocyte-like matrix-producing phenotype.
various cell types have been transplanted into the discs in combination with a variety of cell carriers [Sakai, 2008; Svanvik et al., 2010; Henriksson et al., 2011; Hilborn, 2011]. Recently, in an in vitro study, Stoyanov et al. [2011] reported indications that hypoxia culture conditions in combination with stimulation with GDF5 could contribute to the differentiation of MSCs into a chondrocyte-like phenotype. Further, overexpression of the protein Lim mineralization protein-1 (LMP1), which takes part in bone formation, resulting in increased levels of BMP2 and BMP7 with positive effects on matrix formation in the IVD, has been reported by Yoon et al. [2004] based on in vitro and in vivo investigations. In addition, in vivo studies performed in rats running on treadmill in combination with in vivo labeling with BrdU showed that physical exercise was ob-
served to influence matrix production and the total cell proliferation in the IVD, which may contribute to local regeneration processes [Brisby et al., 2010; Sasaki et al., 2012]. Possible limitations of the outcome of cell stimulation within a degenerated IVD could be that this degenerated tissue has low water content and that these dysfunctional cells may not respond normally to cell stimuli.
Cells Tissues Organs
Henriksson/Brisby
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Concluding Remarks
Accumulating data suggest that there exist immature cell populations that could represent mesenchymal tissue-specific stem cells within the IVD. However, their precise nature, cell type and cellular marker profile re-
mains to be further investigated. In addition, it may be reasonable to hypothesize that notochordal cells interact and function as ‘organizer cells’ and cooperate with tissue-specific stem cells in normal growth and regeneration of the IVD. In order to increase the knowledge on the pathophysiology of disc degeneration, further studies elucidating the different cell types and cellular processes involved in normal growth and regeneration within the IVD are of importance. Further, from a clinical and tissue engineering
perspective, this increased knowledge is of importance in order to design and explore new treatment methods (e.g. cell stimulation with growth factors or cell therapy) for degenerated discs causing chronic low-back pain. Acknowledgements This study was supported with grants from the Swedish research council, Dr. Felix Neubergh foundation and ALF Västra Götaland. All illustrations by Pontusartproductions.
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