Helena Barreto Henriksson
Intervertebral disc regeneration
Studies on stem cell niches and cell transplantation
Department of Orthopaedics Institute of Clinical Sciences
at Sahlgrenska Academy University of Gothenburg
Göteborg, Sweden 2010
ISBN 978-91-628-8147-4
This thesis is a collaboration project between the Institution of Clinical Sciences, Department of Orthopaedics and the Institute of Biomedicine, Department of Clinical Chemistry and Transfusion Medicine, the
Sahlgrenska Academy, University of Gothenburg. Sweden
© Helena Barreto Henriksson Department of Orthopaedics
Institute of Clinical Sciences at Sahlgrenska Academy University of Gothenburg
Sahlgrenska University Hospital SE-413 45 Gothenburg
Sweden
helena.barreto.henriksson@gu.se
Printed by Intellecta Infolog AB, 2010 Illustrations by Pontusartproduction
Cover illustration: Annulus fibrosus outer region with BrdU positive cells. © Helena Barreto Henriksson
ISBN 978-91-628-8147-4
The copyright of the original papers and images belongs to the journals, which have give permission for reprints in this thesis.
To my family
“Impossible is nothing”
Abstract
Intervertebral disc regeneration
Studies on stem cell niches and cell transplantation
Helena Barreto Henriksson Gothenburg 2010
Low back pain is a common condition in the Western world and disc degeneration (DD) is considered a major cause. DD is characterized by dysfunctional cells and decreased matrix production. The aim of this thesis was to explore normal growth and regeneration in the intervertebral disc (IVD). Further, to test possibilities of cell therapy treatment for DDs.
The methods used include in vitro- and in vivo experiments. In vitro methods were:
monolayer, 3D cell cultures and explants models with human mesenchymal stem cells (hMSCs), articular chondrocytes and IVD cells. Cells/ tissues were analyzed for cell proliferation markers; BrdU, KI67, migration markers: β1-INTEGRIN, SNAIL-homolog-1 (SNAI1), SNAIL-homolog-2 (SLUG), progenitor/stem cell markers: STRO1, C-KIT, Notch1, CD105 and chondrogenic lineage markers:
GDF5 and SOX9, matrix markers: COLLAGEN I and II, glycosaminoglycans, AGGRECAN by biochemical methods, flowcytometry, Real-time PCR and microscopy. Disc appearance was evaluated with MRI.
Results from normal regeneration studies: a potential stem cell niche was identified in the IVD region lateral to the epihyseal plate and in the annulus fibrosus outer region, based on findings of label-retaining cells and presence of cells expressing stem cell/progenitor markers, in young and mature animals. Migrating cells expressing SNAI1, SLUG, β1-integrin and GDF5 and SOX9 around niches were observed. Results from the cell therapy experiments; In vitro analyses; 3D co- culture system of hMSC and IVD cells showed an increased COLLAGEN II production. In vivo: Xenotransplanted cells survived in vivo 6 months (porcine IVDs) and produced matrix in hydrogel/MSCs injected IVDs. Taken together, these findings illustrate a normal slow regeneration of the IVD, and that growth and regeneration is presumably supported by progenitor cells deriving from niches adjacent to the IVD. Further, that human IVD cells and MSCs interact positively on matrix production when co-cultured and the survival of transplanted cells in vivo support the possibility for cell therapy treatment of DD. These results encourage further studies to arrest IVD degeneration, by stimulation of regenerative mechanisms in situ or by cell therapy.
ISBN 978-91-8147-4
Populärvetenskaplig sammanfattning
Ländryggsmärta är ett vanligt sjukdomstillstånd i västvärlden, och en vanlig bakomliggande orsak anses vara disk degeneration. I en degenerativ disk fungerar diskcellerna inte längre normalt och bildar mindre mängd omkringliggande matrix, vilket medför en uttorkning av disken och försämrad funktion hos denna. Det anses i allmänhet att broskvävnad har liten eller ingen läkningsförmåga och kunskapen om den normala regenerationsförmågan hos ryggdisk är begränsad. Studier av den normala regenerationsförmågan hos ryggdisken kan öka förståelsen av hur olika sjukdomstillstånd i ryggdisken utvecklas samt ligga till grund för att påverka dessa i positiv riktning.
I dessa studier har tecken på en normal omsättning av den friska ryggdisken identifierats och progenitor celler (omogna celler) från stamcell nischer som är lokaliserade till områden i ryggdiskens utkant har visats bidra till denna. Vidare studerades möjligheter till att transplantera celler till en skadad ryggdisk i djurmodeller och där konstaterades cellöverlevnad och en viss läkning av den skadade disken.
De erhållna resultaten uppmuntrar till vidare studier avseende att återställa eller stoppa upp degeneration av en ryggdisk antingen genom att stimulera befintliga lokala stam-/progenitor celler eller genom utveckling av metoder för celltransplantation, som ett alternativ till
dagens behandlingsformer.
List of publications
The thesis is based on the following papers: (I-IV)
I. Identification of cell proliferation zones, progenitor cells and a potential stem cell niche in the intervertebral disc region. A study in four species
Henriksson HB, Thornemo M, Karlsson C, Hägg O, Junevik K, Lindahl A, Brisby H. Spine (Phila Pa 1976). 2009 Oct 1;34 (21):2278-87.
II. Migrating prechondrocytic cells from stem cell niches supports growth and regeneration of the mammal intervertebral disc. A study in three species
Henriksson HB, Svala E, Skioldebrand E, Junevik K, Lindahl A, Brisby H. Submitted.
III. Human disc cells from degenerated discs and mesenchymal stem cells in co-culture result in increased matrix production
Svanvik T, Henriksson HB, Karlsson C, Hagman M, Lindahl A, Brisby H. Cells Tissues and Organs. 2010; 191(1): 2-11
IV. Transplantation of human mesenchymal stems cells into intervertebral discs in a xenogeneic porcine model
Henriksson HB, Svanvik T, Jonsson M, Hagman M, Horn M, Lindahl A, Brisby H. Spine (Phila Pa 1976). 2009 Jan 15;34 (2):141-8.
V. Investigations of different cell types and gel carriers for cell based intervertebral disc therapy - in vitro and in vivo studies
Henriksson HB, Hagman M, Horn M, Lindahl A and Brisby H.
Submitted.
Abbreviations
ACI Auotologous Chondrocyte Implantation AF Annulus Fibrosus
AFo Annulus Fibrosus outer region AFb Annulus Fibrosus border region bFGF basic Fibroblast Growth Factor BMP Bone Morphogenic Protein BrdU 5-Bromo-2-deoxy-Uridine cDNA complementary DNA
CDMP1 Cartilage Derived Morphogenetic Protein 1 C-KIT stem cell factor receptor
COX2 CytOchrome C Oxidase subunit 2 CXCL12 Chemokine (C-X-C motif) Ligand 12 Delta4 notch ligand protein
DD Degenerated Disc cDNA complementary DNA dsDNA double stranded DNA
DMB DiMethylmethylene Blue method DNA deoxy-riboNucleic Acid
EDTA EthyleneDiamineTetraacetic Acid
EMT Epithelial- and Mesenchymal Transitions ES cells Embryonic Stem cells
FACS Fluorescent Activated Cell Sorting FSC Forward SCatter
GAGs GlycosAminoGlycans GvHd Graft Versus Host Disease
GDF5 Growth-and Differentiation Factor 5 IL InterLeukin
iPS induced Pluripotent cellS HA Hyaluronic Acid
hC human Chondrocytes
hDC human intervertebral Disc Cells hES human Embryonic Stem cells HNA anti Human Nuclei Antibody
HOX HOX genes are a subgroup of home box genes HP HydroxyProline
HvGd Host versus Graft disease KI67 nuclear protein
IHC ImmunoHistoChemistry IVD InterVertebral Disc
Jagged Notch ligand protein expressed during proliferation MR1 Migration Route 1
MR2 Migration Route 2
MRI Magnetic Resonance Imaging MSC Mesenchymal Stem Cell
NO Nitric Oxide
Notch1 Notch homolog 1 NP Nucleus Pulposus
MAPK Mitogen Activated Protein Kinase
MIAMI Marrow Isolated Adult Multilineage Inducible cells P Perichondrium
PCR Polymerase chain reaction RNA RiboNucleic Acid
RAs a small GTPase, name derived from RAt sarcoma RGD aRginin-Glycin-aspartate sequence Domain
roi1 region of interest 1 roi2 region of interest 2
SCID Severe Combined ImmunoDefiency SOX Sex determining region Y-box SSC Side SCatter
STRO1 membran bound protein on bone marrow STROmal cells 1 Taq Thermous aquatious
TGFβ Transforming Growth Factor β TZ Transitional Zone
WNT Name derived from combination of Wingless and INT genes Wntch Wnt and Notch signaling
Table of contents
Abstract ___________________________________________ 5 Populärvetenskaplig sammanfattning __________________ 6 List of publications __________________________________ 7 Abbreviations ______________________________________ 8 Table of contents ___________________________________ 11 1. Introduction _____________________________________ 15 1.1 General background ___________________________________ 15 1.2 Anatomy _____________________________________________ 15
1.2.1 Anatomy of the spine _________________________________________ 15 1.2.2 Anatomy of the vertebrae ______________________________________ 16 1.2.3 Anatomy of the IVD___________________________________________ 16 1.2.4 The nucleus pulposus _________________________________________ 17 1.2.5 Annulus fibrosus _____________________________________________ 18 1.2.6 Embryonic development of the vertebrae and the IVD ________________ 19
1.3 Cartilage characteristics ________________________________ 21
1.3.1 Matrix composition ___________________________________________ 21 1.3.2 Regeneration of cartilage ______________________________________ 22 1.3.3 The epiphyseal plate__________________________________________ 23 1.3.4 Overview of cell signaling in normal growth and regeneration of cartilage _ 25 1.3.5 The chondrogenic lineage markers- GDF5 and SOX9 ________________ 25 1.3.6 BMP signaling _______________________________________________ 26
1.4 Stem cells ____________________________________________ 27
1.4.1 Stem cells characteristics ______________________________________ 27 1.4.2 The stem cell niche ___________________________________________ 29 1.4.3 Cellular migration mechanisms __________________________________ 31 1.4.4 Stem cell marker used in the studies- CD105 _______________________ 32 1.4.5 Stem cell marker used in the studies- STRO-1 ______________________ 32 1.4.6 Stem cell marker used in the studies- C-KIT _______________________ 33 1.4.7 Progenitor marker used in studies- Notch __________________________ 33
1.5 The degenerated IVD ___________________________________ 35
1.5.1 Tissue morphology and cellular characteristics _____________________ 35 1.5.2 Treatment options for the degenerated disc ________________________ 37 1.5.3 Cell sources for cell therapy ____________________________________ 37 1.5.4 Carriers for transplantation of cells _______________________________ 39 1.5.5 Ethical considerations _________________________________________ 39
2. Aim of the thesis _________________________________ 42 2.1. Overall aim of the thesis _______________________________ 42
2.1.2 Specific aims _______________________________________________ 42
3. Methods ________________________________________ 43 3.1 Overview of methods: paper I-V __________________________ 43
3.1.2 Ethical approvals ____________________________________________ 44
3.2 Human tissues and cells________________________________ 44
3.2.1 Human IVD cell isolation and culture _____________________________ 44 3.2.2 Human chondrocytes isolation and culture _________________________ 44 3.2.3 Human MSC isolation and culture _______________________________ 45
3.3 In vitro models ________________________________________ 45
3.3.1 In vitro model – monolayer culture _______________________________ 45 3.3.2 In vitro model-chondrogenic differentiation _________________________ 45 3.3.3 In vitro model – co-culture systems ______________________________ 46 3.3.4 Preparation of conditioned media for co-cultures ____________________ 46 3.3.5 Iron labeling of MSC for co-cultures ______________________________ 47 3.3.6 Cell carriers ________________________________________________ 47 3.3.7 In vitro– test of cell carriers _____________________________________ 48
3.4 In vivo models-animals _________________________________ 48
3.4.1 The Sprague Dawley rat _______________________________________ 48 3.4.2 The New Zealand white rabbit __________________________________ 49 3.4.3 The Göttinger mini pig ________________________________________ 49
3.5 Animal tissues and cells ________________________________ 49
3.5.1 Animal tissues and cells _______________________________________ 49 3.5.2 In vitro model – organ culture ___________________________________ 50
3.6 In vivo labeling of cells _________________________________ 52
3.6.1 In vivo labeling with BrdU ______________________________________ 52
3.7 Immunohistochemistry and histology staining methods _____ 53
3.7.1 Preanalytical treatment of tissues and cell pellets ___________________ 53 3.7.2 Immunohistochemistry ________________________________________ 53 3.7.3 Histology -Alcian blue van Gieson staining _________________________ 54 3.7.4 Histology-Von Kossa staining ___________________________________ 54 3.7.5 Staining for ferric iron deposits __________________________________ 54
3.8 Classification of characteristics in cartilage cell pellets ______ 55
3.8.1 Bern score classification _______________________________________ 55
3.9 Biochemical analyses __________________________________ 55
3.9.1 Sulphated glycosaminoglycans (GAGs) ___________________________ 55 3.9.2 DNA ______________________________________________________ 56 3.9.3 Collagens __________________________________________________ 56
3.10 Characterization of cells _______________________________ 56
3.10.1 Flow cytometry _____________________________________________ 56
3.11 Gene expression methods _____________________________ 57
3.11.1 Isolation of RNA ____________________________________________ 57 3.11.2 cDNA synthesis ____________________________________________ 58 3.11.3 Quantitative Real-time PCR analysis ____________________________ 58
3.12 Radiology imaging methods ___________________________ 59
3.12.1 Magnetic resonance imaging __________________________________ 59
3.13 Statistical methods ___________________________________ 60
3.13.1 Statistical methods in paper II __________________________________ 60 3.13.2 Statistical methods in paper III _________________________________ 60
4. Results _________________________________________ 61 4.1 Paper I ______________________________________________ 61 4.2 Paper II ______________________________________________ 67 4.3 Paper III _____________________________________________ 79 4.4 Paper IV _____________________________________________ 84 4.5 Paper V ______________________________________________ 87 5. Discussion ______________________________________ 92 5.1 General discussion ____________________________________ 92 5.2 Normal growth and regeneration of the IVD ________________ 92
5.2.1 Progenitor markers in the normal IVD region _______________________ 94 5.2.2 Progenitor markers in the degenerated IVD ________________________ 95
5.3 Stem cell niches ______________________________________ 96
5.3.1 Cellular activity ______________________________________________ 96 5.3.2 Cellular migration ____________________________________________ 97
5.4 Cell therapy __________________________________________ 98
5.4.1 Cell therapy-in vitro observations ________________________________ 98 5.4.2 Cell therapy – in vivo observations ______________________________ 100
5.5 Evaluation aspects of animal models ____________________ 104 5.6 Clinical relevance ____________________________________ 104 6. Summary and conclusions ________________________ 105 7. Future directions ________________________________ 106 8. Acknowledgement ______________________________ 107 9. Financial support _______________________________ 110 10. References ____________________________________ 111 Papers I-IV _______________________________________ 126
1. Introduction
1.1 General background
Low back pain is a common condition in the Western world. The costs for the patient and the society are large in terms of the great load on healthcare systems and a reduced capacity for labour in affected individuals. [1] About 80 % of the adult population will experience low back pain during their life time [2] and 5% will experience chronic spinal disease. [3] The annual costs for the US healthcare industry are about 200 billion dollars. [4] This is an escalating problem since the elderly population in the Western world is increasing in numbers. There are many underlying causes, injuries and diseases that can result in low back pain. Disc degeneration (DD) is believed to be one of the major factors for back pain and this thesis will focus on this condition. Intervertebral discs (IVDs) with declined cell number and reduced water content and matrix component turnover are associated with DD. [5] [6] [7] Little is known about the normal regeneration capability of the IVD and until recently, the IVD has been considered to be a tissue with none or very poor self repair just as cartilaginous tissue in other locations.
1.2 Anatomy
1.2.1 Anatomy of the spine
The human spine columna vertebralis is columnar shaped and consists of 33 bony vertebrae with 23 cartilageous intervertebral disc (IVD), disci intervertebrales situated between each vertebrae. The spine is divided in five regions: the neck part, pars cervicalis, the chest part, pars thoracica, the low back part, pars lumbalis, the pelvic part, pars pelvina and the remnant of tail part, pars caudalis.
1.2.2 Anatomy of the vertebrae
There are 7 cervical -, 12 thorical -, 5 lumbar -, 5 sacral -, and 4 caudal vertebrae which vary in size with the lumbar vertebrae being the largest.
The vertebrae protect and support the spinal cord and are the main bearers of weight load exposed on the spine. The vertebrae consist of a large bone called the vertebral body and laminae which extend from the body and enclose the spinal cord. The laminae include the bony spinous processes; pedicles, articular- and transverse processes. Facet joints and ligaments link the vertebrae together and when stacked on top of each other they form a hollow tube; the vertebral foramen. The spinal cord runs through this tube along the vertebral column. Between each pair of vertebrae there are intervertebral fibrocartilageous discs (IVDs), which assist in load bearing and prevent friction between bones. [8]
1.2.3 Anatomy of the IVD
The shape of the IVDs is flattened and cylindrical. It supports movement by having a load bearing and absorbing function. Each IVD is attached to the longitudinal ligaments of the spine and axially to the adjacent vertebral body.
The IVD is an avascular structure composed of an inner gelatinous nucleus pulposus (NP) and an outer fibrous zone, the annulus fibrosus (AF). These two are separated by the transitional zone (TZ), a thin membrane layer. The AF is attached to the vertebrae at the superior and interior surfaces. Figure 1.
1.2.4 The nucleus pulposus
The central NP consists of fibrocartilage and is a hydrated gelatinous structure composed of water, extra cellular matrix (ECM), and cellular elements. The NP has a cell distribution of 4x103/mm3. [9] In healthy and young IVDs, the water content of the nucleus is 80 – 90 %. The water content decreases with age, especially after the fourth decade of life. [10] The ECM is composed of mainly type II collagen but also type VI and XI collagens are present in small amounts.
Figure 1. Anatomical picture with description of the location and components of the intervertebral disc.
Further, proteoglycans such as aggrecan, versican (the major fractions), decorin, biglycan and lumican (minor fractions) are present in the ECM.
[11] [12] [13] [14] The hydrophilic proteoglycans retain water in the IVD, which gives the nucleus its strength and spongy features. [15] [16]
In childhood, the two major populations of cells in the NP are the notochordal- and chondrocyte-like cells. In the developing spine, the cells of the notochord take part in tissue formation and development of the NP during embryogenesis of the IVD [17] [18] and it is believed that the notochordal cells are entrapped within the IVD and gradually replaced by chondrocyte-like cells during adolescence. In human, the notochordal cells are believed to disappear at about 10 years of age. In some species including rats, pigs, rabbits and mice, notochordal cells remain in adulthood. [17, 19] The chondrocyte-like cells are the dominating cell type in the NP of the mature human IVD.
1.2.5 Annulus fibrosus
The annulus fibrosus (AF) is a fibrous structure containing 65 % water with cells, collagen fibers, microfibrilles and proteoglycans organized in ring like structures, the lamellae. [20] [21]. A network of elastic fibers is present between and in bridge formations across the lamellae. [22] The AF has a cell distribution of 9x103/mm3 and is composed of 15 to 25 lamellae enclosing the NP. The outer lamellae are anchored to the edges of the adjacent vertebral bodies and the inner lamellae are attached to the vertebral endplates. The outer lamellae are rich in predominantly type I collagen which decreases toward the NP, the inner lamellae consists mainly of type II collagen. Other minor types of collagen in the AF include type III, V, VI and IX. [23] [24] The cell types of AF are mainly fibroblast like cells in the outer lamellae and chondrocyte- like cells in the inner lamellae. [11, 25] The only innervated part of the IVD is the outer 1-3 mm region of AF (border zone). [15] The transition zone (TZ) is a thin layer which separates the NP from the AF. Figure 2.
1.2.6 Embryonic development of the vertebrae and the IVD
In early human embryogenesis at the end of the fourth week, cells from several somite compartments come together to form the vertebrae. Cells deriving from the somitocoel form with ventral cells the IVDs and the vertebral joint surfaces. The fundamentally 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 is formed and 4) the formation of the bone is carried out by the replacement of the cartilaginous tissue with bone tissue. [26] Migration of cells is important in the embryo and the failure of migration to specific areas can result in malfunction of e.g. limb development. The development of the vertebrae is controlled on different levels and during several development periods of the embryo and signaling systems by e.g.
Figure 2. An anatomical image of a rabbit intervertebral disc:
AF= annulus fibrosus, NP = nucleus pulposus, TZ= transitional zone and EP=epihyseal plate. (Image constructed of 10 separate photos)
HOX genes. The development of individual vertebrae begins with a Sonic Hedgehog (Shh) mediated induction by the notochord cells on the somite in order to form the sclerotome. Other signaling pathways that participate in formation of the vertebrae include the BMP-signaling and the WNT systems. Mesodermal chondrogenic lineage markers include:
Brachyury, GDF5, SOX9, SOX5 and SOX6 which are expressed in prechondrocytic cells (progenitor cells) during formation of cartilage.
[8] [27] [28] [26] When the IVDs are formed around the fourth week of the human embryonic development, the notochord disappears from the vertebral bodies and the notochord forms the condensed mesenchymal primordia (the earliest cells) of the IVDs. Between the developing vertebrae the tissue will form around the degraded notochord. The tissue around the notochord will form the AF part of the IVD. The remains of the notochord will be found in the center of the IVD, the NP. [8, 29] In the adult human, the notochordal cells are believed to gradually disappear and be replaced by NP cells, the cells that make up the center of the IVD. However, the origin of the chondrocyte like cells in the adult human NP is at the present time not totally clarified. [30] [31] The notochordal cells are gradually disappearing in humans while in other species they remain in adulthood e.g. rat, rabbit. [17, 19] [8]
1.3 Cartilage characteristics
Cartilage is a type of dense connective tissue derived from mesoderm (with the exception of craniofacial cartilage e.g. mandibular cartilage which originates from the neural crest cells deriving from the ectoderm).
Cartilage is a white compact but still flexible tissue and is found on e.g.
on the surfaces of joints or in the rib cage. The main purpose of cartilage is to support movements of the body by providing a low friction surface that covers e.g. the long bones. Cartilage is classified in four types: 1) fibrocartilage, 2) articular cartilage (hyaline cartilage), 3) elastic cartilage and 4) epiphyseal cartilage.
Fibrocartilage is found in the IVD, articular cartilage on the surface of synovial joints, elastic cartilage in the epiglottis and epiphyseal cartilage in growing long bones. Cartilage tissue is avascular, aneural and consists of 70-80 % water, extracellular matrix (ECM), consisting of collagen fibers and proteoglycans, with chondrocytes embedded in the ECM. The ECM composition, which is produced by the chondrocytes, consists of collagen fibers that form a cross linked net work, and proteoglycans. [32- 33] In the nucleus pulposus of the IVD the ECM producing cells are called chondrocyte-like cells.
1.3.1 Matrix composition
Collagen II is the most common type of collagen found in cartilage (e.g.
articular cartilage and in the NP) and can be found on genetic level in two different splicing variants; Collagen type IIA and Collagen type IIB.
Collagen IIA is expressed by prechondrocytes and Collagen IIB is expressed by differentiated chondrocytes or chondrocyte-like cells. [34]
Collagen X is produced by prehypertrophic and hypertrophic chondrocytes in the calcified layer in epihyseal cartilage and participates in the mineralization process during bone formation. [23] Other collagen types present in minor fractions in cartilage are e.g. collagen type I, III,
V, VI and IX. [35-36] Proteoglycans e.g. aggrecan are macromolecules which have a central part consisting of hyaluronan (HA) with glycos- aminoglycans (GAGs) bound to them, the complex is being stabilizated by link proteins.
Figure 3.
The GAG, most
commonly found in cartilage tissue is chondroitin sulphate.
Others found in lesser extent are keratan- and heparan sulphate.
The GAGs are
negatively charged, attracts and bind water which leads to osmotic swelling, and thus contribute to the features of cartilage.
[37-38]
1.3.2 Regeneration of cartilage
Cartilage tissue in general is considered to lack or have poor repair capacity. The avascular structure is believed to be one of the major reasons. Further, the chondrocytes or chondrocyte-like cells of the IVD have a slow cellular turnover which contributes to less repopulation and repair of an injury. In general, cartilage has been considered to be a tissue consisting of one cell type, terminally differentiated chondrocytes or chondrocyte-like cells. Several studies have however identified the Figure 3. Schematic picture of a chondrocyte and the
extracellular matrix components of cartilage tissue.
presence of cells expressing progenitor/stem cell markers in adult cartilage tissue [39] [40] [41] and cartilage cells with clone formation capacity has been demonstrated in vitro. [42-43] The normal cell proliferation rate and regeneration processes in the IVD are at the present time not fully known. Small injuries in knee joint articular cartilage have in young animals been demonstrated to heal, whereas larger injuries exhibit a poor self-regeneration capacity. [44-45] Also within the IVD, observations of minor self-repair processes have been seen, especially in the outer rings of the annulus fibrosus (AF). [46] [47] In the inner part of the AF and in the NP no clear regeneration capacity has been reported.
Further, in vitro, it has been suggested that differentiated chondrocytes can dedifferentiate into a more primitive cell type, with the capacity to give rise to other cell types e.g. osteocytes. [48]
1.3.3 The epiphyseal plate
The epiphyseal plate supports longitudinal growth of the skeleton. [49]
The epiphyseal plate is located between the epiphysis and metaphysis in the growing long bones, and in each end of the vertebrae in the vertebral column, the epiphyseal plates are located beneath the cartilaginous endplate in caudal and rostral direction of the IVD. Figure 4. The fusion of the epiphysis and metaphysis in puberty (humans) is known as closure of the physis. In some species the closure is incomplete in the adult animal. The different layers of the epiphyseal plate are the germinal-, proliferative- and the hypertrophic layers. The germinal layer (synonymous names: reserve zone, stem cell layer) contains cells next to the epiphysis which are none organized distributed and embedded in ECM. It has been demonstrated that these cells of the germinal layer have stem cell properties (knee joint). [50] [51] [50] The next layer after the germinal layer is the proliferative layer where the cells are organized in parallel columnar formations of cells. Then follows the hypertrophic layer were the hypertrophic chondrocyte die through programmed cell
death, the so called chondroptosis. These events attract vascular ingrowth, calcification and invasion of bone cells. This process is also called endochondral bone formation. [50] [52] The zone of Ranvier´s groove is an anatomical structure first identified in the knee joint by Ranvier 1873. [53] The zone of Ranvier is a clearly defined anatomical structure in the knee joint of growing mammals and is located in the periphery of the epiphyseal plate. It consists of the zone of Ranvier´s groove and the ring of LaCroix. Figure 4.
Cells of different morphology have been observed within this structure and in the center of the groove the cells are more densely packed. The area of densely packed cells has been demonstrated to harbor progenitor cells. [53-54] The ring of LaCroix, a fibrous layer surrounding the groove has also been suggested to contain precartilageinous cells and serves as a suppliant reservoir of cells for the germinal zone. [55] The epiphyseal plate and an intact perichondrial zone are crucial for normal growth of the long bones. [49] Injuries in childhood in the zone of Ranvier´s groove and in the ephiphyseal plate e.g. Salter-Harris type IV fractures results in severe skeletal growth disturbances. [56-57]
Figure 4. Images of the zone of Ranvier´s groove to the left with densely packed cells and the epihyseal plate with cells in characteristic columnar formation to the right, in a rabbit IVD, age 3 months. The locations are indicated by squares.
1.3.4 Overview of cell signaling in normal growth and regeneration of cartilage
The formation of cartilage is a complex process involving several signaling systems such as e.g. Notch, and BMP signaling. (Hedgehog- and WNT signaling are two additional important signaling systems which are not described in this thesis). Brachyury is one of the earliest proteins expressed in chondrogenesis. Brachyury functions as a transcription factor in the mesodermal T-box and is involved in mesoderm development e.g. involved in formation of the notochord in the embryo. Brachyury expression can be found in adulthood e.g. in chordoma tumours derived from notochordal cells. [58]
1.3.5 The chondrogenic lineage markers- GDF5 and SOX9
Two of the key molecules in the chondrogenic developing program are the important protein lineage markers known as the Growth and differentiation factor 5 (GDF5) (also called Cartilage derived morphogenetic protein 1 (CDMP1)) [59] and SOX9. [60] GDF5 binds preferably to the membrane bound receptor Bone morphogenic protein receptor 1B (BMPR1B). GDF5 is a member of the bone morphogenic protein family and the TGFbeta super family. These proteins are regulators of cell-growth, migration and differentiation in both embryonic and adult tissues. [61-62] As an example, the formation of the synovial joint cavity (the interzone) in the embryo is under regulation of GDF5 signaling cells which migrate towards the center of the joint and takes part in the formation of the interzone, the condensed zone which will finally form the space between joint surfaces. [28] WNT signalling e.g. WNT14 is believed to play a major role in these events, as a regulator of the formation of the joint cavity. [63] In the adult organism GDF5 takes part in normal growth and regeneration of cartilage, e.g. the
GDF5 deficient mice exhibits disc degeneration features with loss of proteoglycan content. A mutation in the GDF5 gene causes several severe malfunctions in joints e.g. Hunter Thompsson syndrome;
brachydactyly (deformed limbs e.g. shortness of digits). [64] [65] One important key regulator in formation of cartilage is the transcription factor; Sex determining region Y-box 9 (SOX9). SOX9 is expressed in during the chondrogenic program and under the influence of SOX9 the prechondrocytes start to produce collagen type II and form ECM. [61]
Genetic defects involving the gene SOX9 gives rise to Campomelic dysplasia which is a congenital syndrome with bowing of the long bones.
[66]
1.3.6 BMP signaling
TGFβ family members are structurally related secreted cytokines, which include TGF isoforms and bone morphogenetic proteins (BMPs). BMP signaling takes part in development and growth of many organs. BMP proteins bind to a transmembrane receptor complex: Bone morphogenic receptors BMPR1 or BMPRII. Activated receptors phosphorylate intracellular Smads 1, 5, 8 which form complexes with Smad4. Activated Smad complexes regulate gene expression of several target genes with widely different functions. The inhibitory Smads (Smad6, Smad7) antagonize signaling. [67] [68] TGFβ and BMPs receptor signaling are required not only for early development and creation of multiple tissues, but also for ongoing maintenance of articular cartilage after birth. [69]
They are strong inducers of cartilage ECM synthesis and inhibit cartilage degradation. Noggin, is a soluble protein, and has an inhibitory effect on BMP signaling, especially BMP-2, -4 and -7. [70] During formation of the synovial joint, Noggin is involved in formation of the joint cavity.
[61]
1.4 Stem cells
1.4.1 Stem cells characteristics
Stem cells are cells that give rise to different cell types and tissues within an organism and they are theoretically capable of an endless amount of cell divisions. Stem cells are unspecialized cells that can differentiate to mature cells and at the same time replicate themselves; (“the immortal strand” hypothesis). [71-72] [73] The stem cells divide according to this hypothetical asymmetric cell division pattern were one daughter cell gives rise to a more differentiated progenitor cell while the other cell maintains the original genome. [73] [74] [75] Figure 5.
In the embryo during the first cell divisions the embryonic stem cells (ES-cells) have pluripotent capability and can hereby differentiate into every cell type of the body. At later stages the stem cells are multipotent,
Figure 5. Schematic picture of asymmetric cell division to the left and to the right normal cell division
capable to differentiate within their lineages of endoderm, ectoderm and mesoderm origin in the organism growth and development stages. [8]
[26] In the adult organism (mammals) there are multipotent e.g.
mesenchymal stem cells and unipotent tissue specific stem cells that take part in normal regeneration and repair of injured tissues. The unipotent stem cells are destined to be differentiated cells within their lineage.
Figure 6.
Further, stem cells can home into injured tissues by transportation through the blood stream by stimulation of certain homing factors e.g.
chemokine (C-X-C motif) ligand 12 (CXCL12). [76]
MSCs are further known to also be inducible to differentiate beyond their lineage to e.g. neurons or pancreatic cells. These cells are called marrow isolated adult multilineage inducible cells, (MIAMI cells) [77] [78] and Figure 6. Schematic overview over multipotent bone marrow derived MSCs and their differentiation capability into different cell types.
are a subpopulation of the total MSC population. Stem cell activity has been demonstrated in many adult tissues but their locations are not always clear. This is due to the current lack of well defined organ/tissue specific stem cell classification system for determination of e.g.
“stemness” properties of adult tissue specific stem cells. There is no ultimate method to identify bone marrow derived hMSCs, since no single cell surface marker exists. Typically, a set of accepted markers are used for characterization and control of the purity of a hMSCs cell population.
According to the International Society of Cellular Therapy (ISCT) human MSCs must be positive for the cell surface proteins CD105, CD73, C90 and be negative for CD45, CD34, CD14, CD11b, CD79a or CD19 and HLA-DR , and must be able to differentiate into osteoblasts, chondrocytes and adipocytes under differentiation standard conditions.
[79] [80] Other bone marrow stem cell markers include STRO-1 [81]
[82] which is expressed on stromal cells and C-KIT which is primarily a hemapoetic stem cell marker but also present in other stem cell populations. [83] [84]
1.4.2 The stem cell niche
The term stem cell niche arose about nearly four decades ago and could be described as ” a specific location where stem cells can reside for an indefinite period of time and produce cells with progenitor properties while self-renewing”. [85] [86] [87] The stem cell niche is an ancient evolutionary structure which is thought to conserve features across diverse organisms. [85] Many of these niches seem to be relatively similar in structures and use common mechanisms for operating but there are probably differences in cell signalling systems. Typically, the stem cell niche area is delineated by a special ECM composition which also encloses the stem cells. [88] [89] [53] Stem cell niches have been identified in e.g. skin, intestine, gonads, brain and bone marrow. [74]
[90] [91] [92-93] [94] [95] The stem cell niches can be divided into:
simple-, complex- and storage niches. Simple niches contains stem cells associated with partner cells by an adherent junction where the stem cells divides asymmetrically and give rise to one differentiating daughter cell and one stem cell. A complex niche contains two or more different types of stem cells which are supported by one or more partner cells and these stem cells give rise to multiple coordinated cells by regulatory signals.
The storage niche is a type of niche where quiescent stem cells are maintained until activated by external signals which trigger cell division and migration. [85] These cells are the” slow cycling cells” or label retaining cells and these can be identified by BrdU labeling experiments.
[91, 96-97] Empty niches are niches from which stem cells have migrated out, these can be occupied again by returning stem cells. The stem cell niche consists of a morphological structure where E-cadherin, β-catenin and β1-INTEGRINS are typical anchorage proteins as well as participants in cell signaling [98] [99] [100] and ECM which surrounds the cells. The niche is also maintained by short range signals produced from a localized “source”. The “sources” which produces signaling proteins are often partner cells which can be located several cell layers away. When the stem cells divide some of the daughter cells are located outside the reach of the signal and these begin to differentiate. The effective range of the signal is limited by the “borders” of the stem cell niche.[75]Figure 7.
Figure 7. Schematic picture of the cellular signalling range that surrounds a stem cell
A complex cellular signalling orchestra takes place within and around the niche and triggers the stem cells to e.g. differentiation, regeneration, growth or migration e.g. to a wound in a distant tissue. Primitive signalling systems including WNT-, Notch-, BMP- and Hedgehog- signalling systems are involved in signalling patterns around and within the niche. [59, 101-104] Several metalloproteinases are suggested to be involved both in loosening of anchorage from the niche and in migration processes. [105-107]
1.4.3 Cellular migration mechanisms
Cells require adhesive interactions with either each other and/or an extracellular substrate in order to actively migrate. Cell-to-cell contact, cellular protrusions and formation of invadopodia assists in the mobility of the migrating cell. During cell migration, cells first flatten and spread out to maximize their adhesions through so called epithelial- mesenchymal transitions (EMT). During these events the cell´s cytoskeleton is rearranged in order to form a flattened, more migratory phenotype. EMT is a well conserved evolutionary process that is present in many organisms e.g. non-vertebrate chordates and in vertebrates.
[108-109] EMT means that a cell can dissociate from a certain tissue region by the loss of cell adhesion and migrate to different locations.
EMT is essential for numerous developmental processes; activity of immunoreactive cells e.g. macrophage motility, cellular migration and contributes to development of tumour metastases in cancer. [110] [107, 111]
Stem cells are known to have a good migration capacity and EMT operates when stem cells invade an injured tissue area. [111] [109]
Members of the Snail super family proteins triggers EMT and have been suggested to be involved in many development processes e.g. neural differentiation, normal cell migration e.g. immunoreactive cells or stem cells, maintaining adult stem cell phenotype, cell fate and survival
decisions. [112] Transforming growth factor beta receptor (TGFβ) is a potent inducer of EMT. [113] [114] Members of the Snail super family encode transcription factors of the zinc finger type and includes e.g.
Snail homolog1 (SNAI1) and Snail homolog2 (SLUG), among others.
[115]
Another mechanism involved during migration is the capability of cells to adhere to surfaces which include the INTEGRIN protein family.
INTEGRINS are a family of heterodimeric transmembrane receptors consisting of an α and a β unit. 18 α and 8 β units are known in mammals which can combine to 24 different INTEGRIN receptors which can bind to different molecules. All β-INTEGRINS bind to ECM molecules e.g.
α1β1, α2β1binds to collagen while other integrin combinations bind to other molecules e.g. laminin, fibronectin. [116-118] INTEGRINS, also take part in cell proliferation and differentiation processes. [99] [119]
1.4.4 Stem cell marker used in the studies- CD105
CD105 (syn. endoglin) is a transmembrane cell surface protein associated with e.g. human bone marrow stem cells, endothelia cells and smooth muscle cells. This protein is a component of the TGFβ complex (papers I, IV and V). [120] [79] [121]
1.4.5 Stem cell marker used in the studies- STRO-1
The membrane bound protein on bone marrow stromal cells 1
(STRO-1) is a cell surface protein expressed by human bone marrow stromal cells and erythroid precursors. The frequency of colony forming units fibroblasts (CFU) is high in the STRO-1 population, about a 100 fold yield in the STRO-1+/glycophorin population. STRO-1+ cells is capable of differentiating along the mesenchymal lineages e.g. to osteoblasts, adipocytes, vascular smooth muscle cells and chondrocytes (paper I). [122] [123] [82]
1.4.6 Stem cell marker used in the studies- C-KIT
C-KIT (syn. CD117 or C-KIT receptor is a transmembrane receptor expressed on the surface of human stem cells, (primarily on hemapoetic MSCs, but also on subpopulations of non hemapoetic MSCs) but is also expressed on mast cells. When the stem cell factor (SCF) binds to the C- KIT receptor a signaling cascade is activated that works through the Ras and Mapk kinase pathway and leads to onset of a set of transcription factors that mediates e.g. proliferation, migration and apoptosis.
Mutations involving the C-KIT receptor can lead to development of cancer e.g. gastrointestinal tumors (paper I).
[124] [125]
1.4.7 Progenitor marker used in studies- Notch
Members of the Notch family proteins are important in many developmental processes by regulating cell fate decisions. Notch positive cells are often partner cells to stem cells and take part in regulating stem cell behavior in the niche region. [91] [74] [85] [93] Notch signaling is an evolutionary conserved intercellular pathway that regulates interactions between physically adjacent cells (lateral inhibition). [126]
[103] [126] Mammals have four transmembrane receptors for Notch proteins; Notch 1-4 which interact with ligands e.g. Delta or Jagged. The Notch signaling cascade is triggered by cell-to cell-contact since the ligands are also transmembrane bound. An intracellular part of the Notch receptor (NCID) when cleaved off, takes part in activating e.g. HES genes. [127] Mutations involving Notch ligands e.g. Jagged -1 can cause abnormal vertebrae segmentation and “butterfly vertebrae” (remaining notochord in vertebrae, one of the abnormalities of the Alagille syndrom) in mammals. [128] [129] [8]
The role of the Notch signaling pathway in cartilage is not fully investigated at the present time. The presence of Notch 1 and Notch 1
the adult organism as well as during chondrogenesis in the embryo. [130]
[131] [103] Further, cross talk between Notch and WNT signaling (Wntch signaling) has been proposed to interact in cell fate decisions [132] [127, 133] as well as interactions with the BMP signaling system (paper I). [134] [132]
1.5 The degenerated IVD
1.5.1 Tissue morphology and cellular characteristics
Primarily, the degenerated disc is characterized by increased cell clustering, reduced disc height and cell death as well as a decrease in fluid binding ability causing tissue concomitant dehydration. Figure 8.
Secondary changes in the AF include fibrocartilage formation, fissures and disorganization of the annular architecture and increase in Collagen type II. [135]
Figure 8. Set of images of a A) normal (non degenerated) human annulus fibrosus, B) normal (non degenerated) human nucleus pulposus, (both samples A-B from a female scoliotic patient, age 14) C) degenerated disc tissue with disorganized morphology with the separate regions of AF and NP no longer distinguishable and D) typical cell clusters of a degenerated disc, magnified image of image C. (sample C-D degenerative disc from a male, age 44)
A B
C D
In the NP, the expression of Collagen II decreases and is replaced by other collagen types e.g. collagen type I, III, V ,VI and X. [136] [136]
[137] These alterations precede the morphological reorganization associated with DD; loss of disc height, disc bulge (also termed disc protrusion), and disc herniation (also termed prolapsed disc) and end plate defects. Further, a decrease in GAG production is seen as well as an increase in the matrix degrading enzymes e.g. MMPs 1, 3, 9 and 13 and cytokines e.g. IL1α and IL1β. [138-141]
DD may cause pain by the secretion of inflammatory factors from the IVD cells. [141] [5] These factors can diffuse and sensitize surrounding innervated tissues. [142-143] The pain is suggested to be derived from surrounding tissue and registered through nocireceptive nerve fibers that are sparsely present in the outer AF and more extensively distributed in the facet joint capsule and the posterior longitudinal ligament. [144] The combination of atypical changes in the IVD, thickening of surrounding ligaments and development of bony spurs can contribute to degenerative arthritis of the spine. [145]
The pathological changes of a degenerated disc can be graded according to the Thompson scale ranging from 1-5 were 1 is the lowest grade of degeneration and 5 the highest score. The Thompson scale is based on IVD fluid content where disc height and contour are visualized by magnetic resonance imaging (MRI). [146] It has been suggested that there is genetic factor involved in the onset of DD, although the specific genes responsible for disc DD have not been identified. However, studies have demonstrated that some of the genetic risk factors associated with DD include polymorphisms in genes encoding for ECM proteins, the vitamin D receptor gene and cartilage intermediate layer protein. [147] A recent study has identified genes for ECM components that were down regulated in degenerated disc samples (AF tissue): e.g. biglycan, fibulin - 2. An up regulation of the interleukins; IL1α, IL7 and IL26 was observed in the same study. [148] Earlier studies has reported increased levels of certain proinflammatory and signaling substances, such as IL-1, IL-6, IL-
8, TNF-α, nitric oxide (NO) and cytochrome C oxidase subunit 2 (COX2), in degenerative discs. [5, 145, 149] Mutations involving the genes encoding for IL-1 have been shown to be related with an increased risk of disc degenerative changes. [141] [138] Other factors that can contribute to DD include obesity, [150] occupational physical loading on the spine and back injury. [151] [152] [153] [141, 154] [6] Further, the presence of Schmorl´s nodes has been suggested to be a contributing risk factor for the development of disc degeneration. [155-156]
1.5.2 Treatment options for the degenerated disc
The current treatment options for degenerated IVD disease are symptomatic treatment; analgesics, physiotherapy, and cognitive treatment or surgical treatment; spinal fusion or total disc replacement using an artificial disc replacement. None of these treatment methods attack the underlying problem. A disadvantage of spinal fusion treatment is that it can affect the biomechanical properties of the nearby vertebrae and may contribute to a future onset of IVD degeneration in other locus along the vertebral column. Biologic treatment options of the IVD, including cell therapy, have been suggested as complementary or optional treatment methods for DD. [157-158] [159]
Further, NP cells have been reported to express the protein FAS ligand (FAS-L, syn. CD95) which are present in immune privileged organs [160] [161] [162] and this is an advantage for cell therapy treatment.
Furthermore, gene therapy for treatment of degenerated discs is under investigation. [163]
1.5.3 Cell sources for cell therapy
The usage of embryonic stem (ES) cells has been suggested for cell therapy, but their drawback is that there are ethical considerations, [164]
[165] difficulties with controlling the ES cells in vitro and a high risk of
development of teratomas in in vivo experiments. [166] More safe alternatives are the MSCs which are already used for e.g. bone marrow transplantations (hemapoetic mesenchymal stem cells) for treatment of certain types of leukemia. [167] [168] The transplantation can be autologous or allogenic where the cells are taken of the bone marrow of the patient or from a suitable donor, such as a relative. There is also evidence for the presence of a MSC population in the umbilical cord that may be a suitable source for cell therapy in the future. Figure 9. [169]
[170] [171] [172]
Many countries, including Sweden, are at present time collecting blood/cells from umbilical cords of newborns, in order to create a future biobank system. Further, adipose derived MSCs are currently
Figure 9. Schematic overview of possible cell sources that can be used for cell therapy treatment for the degenerated disc in the future.
