THE SPINE SCHOLAR VOLUME 1, NUMBER 1, 2017 SEATTLE SCIENCE FOUNDATION

REVIEW

Molecular Biology and Interactions in Intervertebral Disc Development, Homeostasis, and Degeneration, with Emphasis on Future Therapies: A Systematic Review

Loai Aker1, Malik Ghannam1, Muayad A Alzuabi2, Fareed Jumah1, Saja Mohammad Alkhdour1, Shaden Mansour1, Amjad Samara MD1, Katharine Cronk3, Justin Massengale3, James Holsapple3, Nimer Adeeb3, Rod J. Oskouian, R. Shane Tubbs4,5

1An-Najah National University Hospital, Nablus, Palestine 2 Neuroscience Institute, Ain Shams University, Cairo, Egypt 3Department of Neurosurgery, Boston Medical Center, Boston University, Boston, MA, USA 4Department of Anatomical Sciences, St. George’s University, Grenada 5Seattle Science Foundation, Seattle, WA, USA http://thespinescholar.com https://doi.org/10.26632/ss.3.2017.1.1

Keywords: intervertebral disc, molecular, genetics, development, markers, proteoglycans, homeostasis, hypoxia, osmolarity, degeneration, stem cells

ABSTRACT The unique properties of the intervertebral disc (IVD) are evident in its structural complexity and functional importance for spinal support and stability. The contributions of the different cellular and extracellular components to the function of the IVD depend on their distinctive molecular features and pathways. Disruption of these molecular pathways influences the pathological changes involved in IVD degeneration. Therefore, the molecular features of the IVD have been the focus of interest for many researchers seeking to elucidate its normal functioning, potential pathologies, and appropriate therapies. The aim of the present article is to review the molecular aspects of IVD development, specific cellular markers, and the interactions between cellular and extracellular components responsible for homeostasis, degeneration and potential therapies. The literature available via PubMed and Google Scholar was reviewed and the relevant references in review articles were searched manually. Spine Scholar 1:2-20, 2017

INTRODUCTION Owing to its importance for spinal function and its involvement in age-related degeneration, the intervertebral disc (IVD) continues to be a focus of interest for scientists and clinicians. The IVD consists of an inner nucleus pulposus (NP), an outer anulus fibrosus (AF), and cartilaginous endplates. Different cellular and extracellular components interact to ensure the normal development of the IVD and provide adaptive strategies to withstand changes in its environment. These interactions ultimately maintain the necessary support, movement, and stability of the spine. In order to elucidate these interactions thoroughly, various molecular aspects of the IVD have been investigated. Disc degeneration with associated low back pain is widespread, with significant consequences for individuals’ qualities of life. Understanding the cell populations within the IVD and their molecular characteristics is crucial for delineating the pathological changes in disc degeneration and for developing novel therapeutic and preventive modalities. Nevertheless, this field remains challenging because of the complicated and continuously evolving nature of the cell profile and the extracellular matrix (ECM) in relation to development, growth, mechanical and metabolic functioning, interaction with the environment, aging, and degeneration. The limited availability of human cells and intact tissues has necessitated the use of different animal models to study the molecular pathways in IVD cells (Pattappa et al., 2012). These models have helped to solve various biochemical and genetic puzzles regarding the basic molecular and cellular pathways in human IVD cells. However, interspecies differences in cellular development, biomechanical environment, and the rate of cellular changes from notochord-like to mature NP cells should be considered (Pattappa et al., 2012).

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Figure 1: Schematic view of anterior intervertebral disc noting the nucleus pulposus and layered anulus fibrosus.

The molecular biology of intervertebral disc development

The notochordal tissue provides the template for the multi-stage development of mature disc cells (Adams et al., 1990; Hunter et al., 2004; Pattappa et al., 2012; Peacock, 1951). After gastrulation, the rod-like early notochord arising from the mesoderm directs the differentiation of ventral somatic derivatives into sclerotomal cells, which form the peri-notochordal sheath (Aszodi et al., 1998; Pourquie et al., 1993). Many developmental signals such as Sonic Hedgehog (SHH) and brachyury participate in forming the IVD (Choi et al., 2008; Choi and Harfe, 2011). SHH regulates PAX1 activity, which is detected in the developing AF from 14.5 dpc until adulthood. PAX1 is believed to be important for cell proliferation, maintenance of chondrocyte- specific gene expression in the sclerotomal cells, facilitating the response of sclerotome cells to notochord signals, and regulating pattern formation during embryogenesis (DiPaola et al., 2005). SHH also regulates FOXF1 activity to control cell growth and differentiation of different tissues (Pattappa et al., 2012). The hedgehog receptor “patched” protein produced from the NP indicates the presence of SHH in the developing NP. Indian Hedgehog expression has been detected in the early condensation of cartilage and is later restricted to prehypertrophic chondrocytes. Its expression is important for the growth and differentiation of cells within the vertebral endplate. Furthermore, the Noggin gene is crucial for restricting bone morphogenetic protein (BMP) action during development and maintaining IVD shape into adulthood. SHH, like PAX-1, could be involved in inducing Noggin in the anulus to inhibit BMP-4 in somite patterning (DiPaola et al., 2005). In the Pressure mouse model, the developing vertebrae exert pressure on the notochord, leading to cell movement into the region where discs are about to form (Lawson and Harfe, 2015). In support of this inference, a mouse model with no notochordal sheath or condensing vertebrae retained a rod-like notochord (Choi and Harfe, 2011), which also suggested that the vertebrae being formed around regions of the notochord contribute to NP formation. In contrast, according to the Repulsion/Attraction model, the tendency of the notochord to reside in the region of the NP could be due to the influences of certain attractants or repellants. The Eph/ephrin pathway and the Robo/Slit signaling pathway could be essential for consolidating notochord cells in the forming disc (Lawson and Harfe, 2015). The Eph/ephrin family, which is needed for the patterning of numerous tissues during development, involves binding Eph proteins, which are tyrosine kinase receptors with membrane-bound ligands called ephrins. The Eph/ephrin pathway could contribute to attracting notochord cells into regions of disc formation and inhibiting the mixing of notochordal with mesenchymal cells. In support of this model, the EphA4 receptor has been detected in the notochord along with ephrins found in the surrounding mesenchyme (Durbin et al., 1998). However, the Eph/ephrin pathway is not necessarily the only mechanism underpinning the transition of the notochord into the NP because it requires direct cell-cell contact, which cannot be achieved between all cells with surrounding mesenchyme (Rohani et al., 2011). In the other arm of the proposed model, the Robo/Slit pathway, which contributes to both neuron and organ positioning, has been investigated in vivo and in vitro: Slit protein repels cells expressing Robo. Three Slit genes are expressed in the mouse notochord (Domyan et al., 2013), with Robo1 and Robo2 found in mesenchymal cells surrounding the notochord. Therefore, Robo-expressing mesenchymal cells could be repelled from notochordal cells expressing Slit, and this could inhibit the mixing of the two cell types during the transition of the notochord into the NP. The AF develops from a repeated annular condensation derived from mesenchymal cells in the peri-notochordal sheath, while the cartilaginous endplate (CEP) arises from non-condensed areas of the sheath as part of the developing vertebral body (Aszodi et al., 1998; Pattappa et al., 2012). Normally, the notochord also pushes against the surrounding annular condensation, which helps to create an inner and outer AF. After condensation of the sheath the notochord starts to condense and eventually forms the NP (Choi et al., 2008; Choi and Harfe, 2011; Pattappa et al., 2012; Risbud et al., 2010). Interestingly, deletion of transcription factors such as SOX-5 and SOX-6 in the embryonic spine leads to notochord loss, disrupting NP formation (Choi and Harfe, 2011; Smits and Lefebvre, 2003). By late adolescence, human NCs are replaced by chondrocyte- like ‘NP cells’. However, progenitor/stem cells are present in a number of human and non-human NPs and it is likely that these stem/progenitor cells migrate to the NP during development, and perhaps retain the potential to differentiate even in degenerative human IVDs (Erwin and Hood, 2014).

Collagen component

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As early as 42 days of gestation, collagen type IIA mRNA is detected in cells of the pre-chondrogenic area, cells of the notochord, and those surrounding it within the dense zone (pre-IV disc area). Type I and type III collagen mRNAs are mainly expressed in the dense zone. The IVD region shows clearly-defined NP and AF at 54 days of gestation (McAlinden et al., 2002; Zhu et al., 2001). There, type IIA procollagen mRNA is found in the cells of the inner anulus, NP, and pre-chondrogenic area that will form the articular endplate. Type I collagen mRNA can be demonstrated in the cells of the outer anulus and of the NP. Type III collagen mRNA has a more widespread expression than type I at day 54 (McAlinden et al., 2002; Zhu et al., 2001). Type IIA pNprocollagen, where the N-terminal propeptide is attached to the collagen fibrillar domain and the C-terminal domain is absent, can be detected in the inner anulus and vertebral bodies during that time, while the C-terminal domain is always absent in the ECM and is cleaved from the procollagen molecule intracellularly (McAlinden et al., 2002; Zhu et al., 2001). N-proteinase activity cannot be detected at this phase of development, but intracellular C-proteinases are active. On days 72-101 of development, type IIA N-terminal propeptide is cleaved by N-proteinases in the inner anulus cells leading to the secretion of only the major fibrillar domain of type II collagen into the matrix of the inner anulus, suggesting that the N-terminal propeptide could have a growth factor function that is no longer required (McAlinden et al., 2002; Zhu et al., 2001). During chondrogenesis, type IIA procollagen is synthesized by chondroprogenitor cells, which switch to type IIB synthesis during maturation to chondrocytes by alternative splicing of exon 2. In the inner anulus, instead of alternative splicing, the N-terminal propeptide is cleaved from the fibrillar domain by an N-proteinase. Therefore, the N-terminal propeptide in type IIA procollagen can be removed by either pre-mRNA splicing or protein processing (McAlinden et al., 2002; Zhu et al., 2001). It is speculated that the type IIA procollagen N-terminal propeptide regulates BMPs during cell differentiation, and the N-terminal propeptide can be removed by matrix metalloproteinases (MMPs). The cleavage function of MMP can be activated if BMP binds to the type IIA procollagen N-terminal propeptide. This activation could have a role not only in development but also in repair during degeneration, like MMP-3 and MMP-9(McAlinden et al., 2002; Zhu et al., 2001). BMP-2/4 and their receptors have been detected in the degenerated mouse AF. In addition to the catabolic function of MMPs within the disc matrix, an anabolic effect can be achieved after the cleavage of BMP-binding proteins such as type IIA procollagen. This could activate the growth factor to promote anabolic effects (McAlinden et al., 2002; Zhu et al., 2001). Besides collagen, a significant interaction has been reported between Cfm proteins and filamin, which contribute to stabilizing the actin cytoskeleton and maintaining the interactions between actin and transmembrane components. During chondrocyte differentiation, expression of the Cfm2 gene is elevated, particularly in cartilaginous tissues such as the developing IVD (Mizuhashi et al., 2014). Cfm double knockout (DKO) mice demonstrated skeletal abnormalities that resembled those occurring in filamin B (Flnb)-deficient mice, particularly scoliosis, kyphosis, fusion of the vertebrae, and shortening of the distal appendages. It was concluded that Cfm1 and Cfm2 proteins function cooperatively in chondrogenesis and IVD formation. Therefore, loss of the Cfm genes disrupts cartilaginous cell development. The same study reported that Cfm DKO mice had a decreased primary chondrocyte surface area with fewer actin filament bundles and a shortened nuclear long axis, suggesting a correlation between Cfm proteins within chondrocytes and filamin-mediated stabilization of actin filaments. It is also suggested that Flnb deficiency is associated with increased chondrocyte apoptosis, and that Cfm DKO mice have a decreased primary chondrocyte proliferation rate.

Transforming growth factor

In mouse models with deletion of the TGF-h type II receptor gene (Tgfbr2), particularly in Col2a expressing cells, the vertebral bodies were only moderately affected but the IVDs were either missing or incomplete. This suggests that the signaling pathway through the TGF-type II receptor is important for the development of normal dorsal and lateral parts in the lumbar vertebrae. Also, Tgfbr2 is crucial for establishing the boundaries between the vertebral body and IVD as the inner anulus did not form in Col2acre+;Tgfbr2loxP/loxP mice, and mineralization extended from the vertebral body into the region where the IVD cartilage was supposed to be maintained. In contrast to Tgfb2-null mice, Col2acre+;Tgfbr2loxP/loxP mice exhibited vertebral defects that were not limited to the lower thoracic and lumbar vertebrae but found along the entire length of the spine. Interestingly, IVDs were found in Tgfb2-null mice, but were missing or absent in Col2acre+;Tgfbr2loxP/loxP mice (Baffi et al., 2004).

Clinical correlation

Chordomas are known to be of notochordal origin because of molecular similarities and the proximity of the cancer cells to the vertebral column. The expression of CD24 in chordoma cells, thought to be an NP cell marker, supports this view of the origin of chordomas (Fujita et al., 2005). It has been proposed that failure of the notochord cells to reach their destined residence at the NP could increase the number of such cells in the adult vertebral column, which could be linked to an increased risk for chordoma, as these cells are more susceptible to mutation-activating brachyury, which could be linked to chordoma (Lawson and Harfe, 2015). The brachyury transcription factor, which contributes to notochordal development, is only expressed by the embryonic notochord and chordomas. However, in one study, brachyury could not be detected in the NP, arguing against the hypothesis that the NP is a derivative of the notochord and suggesting that brachyury is a potential marker for the notochord and notochord-derived tumors (Vujovic et al., 2006). Copper is essential for notochordal development, and some vascular and neurological defects in the offspring of patients with Menkes disease or copper deficiency have been attributed to the disruption of patterning leading to notochord abnormalities (Mendelsohn et al., 2006). Genetic studies of animal models have shown that phenotype sensitivity to copper deficiency depends on the gene dosage of ATP7a, but transplantation experiments have revealed that this sensitivity is related to the autonomous nature of cells as any cell with adequate ATP7a for a given availability of copper will yield active copper enzymes. These findings suggest that as copper becomes limiting in the developing embryo, higher levels of ATP7a are expressed, which would be beneficial (Mendelsohn et al., 2006).

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The molecular biology of nucleus pulposus components and their interactions

The NP is a heterogeneous structure with large fibrillar spaces within a 3D mesh of collagen fibrils that contain proteoglycans (Pattappa et al., 2012). It expresses more collagen II, proteoglycans and aggrecan, a large proteoglycan capable of forming aggregates with hyaluronan, than other disc cells. Interestingly, the 27:1 proteoglycan-to-collagen ratio within the NP distinguishes it from the CEP, where the ratio is 2:1 (Mwale et al., 2004). However, less decorin and versican are expressed in the NP than the AF. Small leucine-rich proteoglycans (SLRP) contribute to regulating ECM assembly and help to modify the interactions between the matrix and different glycoproteins, retinoic acid, cytokines, and growth factors (Chen et al., 2002). Also, the negatively charged glycosaminoglycan side chains in proteoglycans in the NP can bind electrostatically to polar water molecules, crucial for maintaining the well-hydrated state that enables the disc to undergo reversible deformation under compressive loading (Erwin and Hood, 2014; Pattappa et al., 2012). Within bovine NP cell cytoplasm, F-actin is punctate in its distribution, while actin filaments extend out into cell processes in the outer AF (OAF), correlating with a fibroblast-like morphology. In both the NP and OAF there is an extensive meshwork of β-tubulin throughout the cytoplasm. The β-tubulin mRNA and protein levels are higher in NP than OAF cells, irrespective of age. Vimentin, a cytoskeletal protein crucial for cell rigidity, shape, and mechanical load absorption, traverses the NP cell cytoplasm from the plasma to the nuclear envelope, while vimentin filaments are present throughout the AOF cytoplasm and extend into the cell processes. The OAF has a higher content of vimentin mRNA than the NP cells. The vimentin content of the OAF decreases with age (Li et al., 2008a).

Cellular structure

Notochordal cells of the postnatal NP usually appear in clusters and have intracellular vacuoles occupying at least 25% of the cell area (Pattappa et al., 2012). The hypoxic and avascular region around NP cells with decreased levels of glucose leads to the distinctive metabolic features of these cells as their energy source is provided via anaerobic glycolysis and ATP production (Erwin and Hood, 2014). Cells in the nucleus resemble those in the inner anulus as they are both chondrocyte-like, with rounded appearance and separated by an extensive ECM, and having cytoplasm-filled cellular processes as found in bovine models (Errington et al., 1998). Notochordal cells seem to be key to maintaining NP integrity, regulating the synthesis of neural progenitor cells (NPCs) and probably the turnover of matrix components. BMP, which is produced by the notochordal cells, is thought to be involved in this process, which is mostly dependent on soluble factors produced by notochordal cells rather than cell-cell contact (Aguiar et al., 1999). When substrates of basement membrane extract were used in culturing, NP cell organization in vitro was remarkably influenced by substrate stiffness, clustering behavior being lost when the substrate elastic modulus was increased by 500 Pa. Also, changes in the ECM ligand environment such as a lower abundance of laminin ligands can impair cellular clustering behaviors. This could explain how the increased stiffness of the NP and age-related changes in the abundance of NP tissue ligands could have a role in the dissociation, differentiation, or apoptosis of clustered immature NP cells. Therefore, it is suggested that under specific ECM conditions, NP cells favor cell-cell interactions over cell-substrate interactions. Further research is needed to design future therapies using soft laminin biomaterials (Gilchrist et al., 2011).

Proliferation

Tsirimonaki et al., (2013 reported that NP cells are distinctly plastic, as cell lines tolerated multiple freeze-and-thaw cycles and retained their general growth and molecular phenotypic features. Under monolayer culture conditions with multiple passages, these cells retained the mRNA expression of chondrocytic markers such as SOX9 and receptors for lysophosphatidic acid (LPA), but not Col2A1 (Tsirimonaki et al., 2013). Also, a subpopulation of cells with a notochordal-like morphology was propagated for several passages and proved crucial for supporting proliferation. There was a low but sustained level of ERK1/2 activation, which is known to induce differentiation. In human NP cells, PKCε signaling increased aggrecan expression as part of an PKCε/ERK/CREB/AP-1-dependent transcriptional process with associated increases in ACAN and hsa-miR-377 expression, and decreased expression of the hsa-miR-377 target ADAMTS5 (Tsirimonaki et al., 2013). Lee et al. demonstrated that donor age is the sole predictor of the replicative potential of human NP cells, this potential decreasing with aging. In contrast, initial telomere length and telomerase activity could not predict the replicative potential (Lee et al., 2015). Moreover, both the telomere length and telomerase activity of NP cells declined steadily with increasing population doubling, indicating that telomerase did not significantly prevent telomere shortening during the expansion of NPCs in vitro (Lee et al., 2015). Other studies have suggested that telomerase reverse transcriptase can preserve telomere length and extend the replicative potential (Wu et al., 2014; Wu et al., 2011). Cellular differentiation

To differentiate NP cells from AF or articular chondrocyte (AC) cells, many markers have been investigated, with interspecies differences and similarities (Lv et al., 2014). Although collagen type II is predominant in NP cells, many reports have shown that more collagen type II is produced in AC than NP cells, and the levels of COL2A1 in human NP and AF cells are comparable (Lv et al., 2014). Interestingly, Sive et al. showed a higher aggrecan expression in human NP than AF (Sive et al., 2002), in contrast to another study where aggrecan expression levels in these sources were equal (Minogue et al., 2010b), with a slight downregulation of the aggrecan level in degenerated NP. Those studies disqualify these traditional chondrogenic markers as appropriate NP markers(Lv et al., 2014). A microarray analysis in rats revealed higher CD24 and glypican 3 expression in NP than AF cells, suggesting they are candidate NP markers(Fujita et al., 2005). Subsequent studies showed that annexin A3 (ANXA3), GPC3, KRT19 and pleiotrophin (PTN) are also potential NP/AF markers. Another study on beagles showed that four genes — alpha-2-macroglobulin (A2M), desmocollin 2 (DSC2), keratin 18 (KRT18) and neural cell adhesion molecule 1 (NCAM1) - have a high mRNA and protein level ratio of NP:AF (Lv et al., 2014; Sakai et al., 2009).

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Minogue et al. reported that only KRT19, KRT18 and NCAM1 have high NP:AF expression ratios in bovines. Furthermore, keratin 8 (KRT8), synaptosomal-associated protein (SNAP25), cadherin 2 (CDH2), sclerostin domain containing 1 (SOSTDC1), brain acid soluble protein 1 (BASP1), and fibulin 1 (FBLN1) showed significantly higher expression, while integrin- binding sialoprotein (IBSP), tenomodulin (TNMD), TNF-α-induced protein 6 (TNFAIP6), aquaporin 1 (AQP1), forkhead box F1 (FOXF1) and forkhead box F2 (FOXF2) exhibited significantly lower NP:AF ratios (Lv et al., 2014). Expression of BASP1, TNMD, TNFAIP6, FOXF1, FOXF2 and AQP1 was high in both AF and NP while IBSP and FBLN1 showed low expression in both cell types, indicating that they cannot be appropriate markers for distinguishing NP from AF. To conclude, KRT19, KRT18, NCAM1, KRT8, SNAP25, CDH2 and SOSTDC1 can be considered as NP/AF markers in bovines. On the other hand, Rutges et al. reported that KRT19, NCAM1, A2M and DSC2 are NP+/AF- markers in humans, while cartilage oligomeric matrix protein (COMP) and GPC3 are AF+/NP- markers (Lv et al., 2014). Power et al. identified C-type lectin domain family 2 member B (CLEC2B), carbonic anhydrase 12 (CA12), sarcoglycan gamma (SGCG), and TYRO3 protein tyrosine kinase (TYRO3) as human NP+/AF-genes(Power et al., 2011). Currently, the candidates showing consistently significant levels of expression in most studies of NP cells in the tested species (rat, bovine, and human) are KRT19, NCAM1 and CDH2. However, further validation studies are required (Lv et al., 2014). To distinguish NP from AC, Lee et al. reported that ANXA3, GPC3, KRT19, PTN and vimentin (VIM) are highly expressed in the NP of rats. However, GPC3, VIM, and PTN demonstrated a different trend in dogs, where A2M, KRT18, and NCAM1 were reported to be NP+/AC- markers (Lee et al., 2007). Later, Gilson et al. showed KRT8 to be highly expressed at the protein level in bovine NP cells (Gilson et al., 2010). However, using a microarray technique on bovine cells, Minogue et al. failed to confirm the pattern of NP:AC markers from the previous findings, except for KRT19, KRT18, and NCAM1. The other NP/AC- specific genes demonstrated were SNAP25, CDH2, and versican (VCAN) (Minogue et al., 2010b). Minogue et al. defined paired box 1 (PAX1), FOXF1, hemoglobin beta (HBB), CA12, and ovostatin 2 (OVOS2) as potential NP+/AC- markers(Lv et al., 2014; Minogue et al., 2010a). Power et al. demonstrated high expression of CLEC2B, attractin-like 1 (ATRNL1), choline transporter-like protein 1 (SLC44A1), CA12, transmembrane protein 27 TMEM27), tetraspanin 31 (TSPAN31), SGCG, delta/notch-like epidermal growth factor (DNER), desmoglein 2 (DSG2), cadherin 19 (CDH19), matrix remodeling associated 7 (MXRA7), neuropilin and tolloid-like 2 (NETO2), corticotropin releasing hormone receptor 1(CRHR1) and TYRO3 in human NP vs AC cells(Power et al., 2011). It was concluded in this review that the NP(+)/AC(-) markers include KRT19, KRT18, CDH2, VCAN, BASP1, TNFAIP6, FOXF1, FOXF2 and CA12. On the other hand, COMP, MGP, IBSP and FBLN1 were potential NP(-)/AC(+) markers (Lv et al., 2014). To differentiate mature from immature NP cells, Chen et al. (2006) found lower gene expression of collagen type I, biglycan, and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in immature porcine NP than mature cells, with no difference in the expression of SOX9, collagen type II and aggrecan genes between notochordal and mature porcine NP cells. However, integrins α1 and α6 were more highly expressed in the ‘notochordal-like’ cells, and these molecules seem to interact with collagen and laminins during tissue development (Chen et al., 2006; Pattappa et al., 2012). Also, laminins can help to differentiate between notochordal and mature porcine NP cells (Chen et al., 2009). Another cell population was found in the NP of young human discs (subject age range: 0–17 years) that expressed notochordal cell markers including cytokeratin-8, -18, -19, and also galectin-3 (Weiler et al., 2010), which is known to help regulate cellular differentiation and mediate inflammation. The expression of these markers decreased during aging, but if they are present in mature discs (>18 years), especially galectin-3, this could facilitate disc degeneration. This finding contradicts previous studies, as notochordal cells have a pivotal role in tissue development and regeneration (Henriksson et al., 2009; Minogue et al., 2010b; Pattappa et al., 2012; Risbud et al., 2010; Sakai et al., 2009). Like the cellular response to hydrostatic pressure, in vitro models of NP cells might not reflect in vivo conditions regarding cell–tissue interaction and consequent notochordal cell behavior. Therefore, the presence and function of notochordal cell populations in the mature human NP needs further study. Regarding the derivation of NP cells, one hypothesis argues that the notochord disappears or undergoes apoptosis by early childhood and is then replaced by the cartilaginous NP (Hunter et al., 2004; Vujovic et al., 2006). In support of this hypothesis, cytokeratins and brachyury are not expressed by the NP (Pazzaglia et al., 1989; Vujovic et al., 2006), suggesting there is no molecular evidence that the NP develops directly from the notochord. Furthermore, collagen X has been detected in the NP, which, in addition to its capacity to calcify, makes this tissue more likely to be developmentally unrelated to the notochord (Aigner et al., 1997; Boos et al., 1997; Vujovic et al., 2006). There is increasing evidence from phenotype and surface marker expression and genome-wide microarray studies to support the hypothesis that mature NP cells derive from the notochordal cells present in the disc during embryogenesis (Choi et al., 2008; Fujita et al., 2005; Gilson et al., 2010; Lee et al., 2007; Minogue et al., 2010b; Pattappa et al., 2012; Power et al., 2011; Risbud et al., 2010; Sakai et al., 2009; Sive et al., 2002). Both the NP and NC express cytokeratin-8, -18, -19 and brachyury or T-Box genes (Lee et al., 2007; Minogue et al., 2010a; Pattappa et al., 2012; Sakai et al., 2009). Mature NP cells are smaller than notochordal cells and lack intracellular vacuoles. Risbud et al. supported the hypothesis that mature NP cells derive from notochordal cells as the variations in cell size and morphology correlate with maturation and function. Also, Sonic Hedgehog (Shh) signaling is essential for NP formation, but is not required in the development of AF or CEP (Pattappa et al., 2012; Risbud et al., 2010). Different NP markers have been identified by microarray studies. Although they are not absolutely definitive, they help us to understand the development and metabolism of NP cells and monitor the phenotype of MSC as they differentiate into an NP-like phenotype (Pattappa et al., 2012). In both healthy and degenerative discs, NP cells express chondrocyte markers such as SOX-9 and collagen type II (Clouet et al., 2009; Sive et al., 2002). In rats, COMP and MGP are more highly expressed in aged (24 months) rats than immature (three months) rats. Subsequently, MGP and COMP were found to be potential NP degeneration marker in humans (Rutges et al., 2010), with KRT19 as a healthy NP marker. Minogue et al.(Minogue et al., 2010b) demonstrated the up-regulation of FBLN1 and downregulation of SNAP25, KRT8, KRT18 and N-cadherin (CDH2) in degenerated human NP. Interestingly, Power et al. (2011) found that CA12 mRNA expression, which helps regulate intracellular pH under hypoxia, is negatively correlated with both age and Thompson grade, even though this is not reflected at the protein level.

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On an epigenetic scale of rat samples, there was upregulation of BASP1, NCDN and CD155 in aged NP (21 months) while neuropilin-1 (NRP-1) and CD221 showed decreased levels in aged NPs (Lv et al., 2014; Power et al., 2011). Collectively, high expression of COMP, MGP, FBLN1, BASP1, NCDN, and CD155 was seen in aged/degenerated NP associated with low levels of SNAP25, KRT8, KRT18, CDH2, KRT19, NRP-1, and CD221. Despite having KRT19 downregulated in degenerated NPs, and even in degenerated/aged NPs, KRT19 still had a high NP:AF ratio and a high NP:AC ratio (Lv et al., 2014). Therefore, KRT19 would seem the perfect marker for NP cells. As it is considered a notchordal marker, the decline of its level with age could be due to the replacement of notochordal cells by chondrocyte-like cells in the NP. Owing to the longer life span of humans, the expression level of KRT19 in human NP is not as high as in other animals. Also, it is important to note that NP degeneration starts at 10 years of age with the corresponding downregulation of KRT19, so different results can be found even between normal NP from young healthy human and healthy animal NPs (Lv et al., 2014). Further studies using protein imaging such as microscopic analysis are recommended because the gene expression level is not always correlated with the protein expression level.

Response to compression Different cellular responses to mechanical loading affect the IVD depending on the type of mechanical signal (Iatridis et al., 2006; Iatridis et al., 1998; Li et al., 2011; Pattappa et al., 2012). For example, more β-tubulin and vimentin are expressed in bovine NP than AF cells, probably because NP cells are loaded in compression, while the AF expresses more β-actin to withstand tensile loading (Li et al., 2008b). However, the expression of cytoskeletal elements is influenced by hydrostatic pressure, since tensile loading in bovine NP cells did not induce changes in cytoskeleton remodeling or a gene expression pattern different from AF (Li et al., 2011; Pattappa et al., 2012). NP cells cultured in vitro showed induction of proteoglycan synthesis when the applied hydrostatic pressure was below 3 MPa, but there was increased nitric oxide production and inhibited matrix synthesis when the applied pressure was greater than 3 MPa (Ishihara et al., 1996; Kasra et al., 2003; Le Maitre et al., 2008; Neidlinger-Wilke et al., 2012). Nevertheless, many matrix-associated genes (e.g. type II collagen, aggrecan, and MMPs 3 and 13) and cytokines (e.g. interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α)) were upregulated in NP cells after compressive loading. Load regimes and correlated responses in cellular metabolism could differ between in vitro and in vivo models (Korecki et al., 2009; Pattappa et al., 2012; Wang et al., 2007; Wuertz et al., 2009).

The molecular biology of anulus fibrosus components and their interactions The AF is composed principally of water, collagen, aggregating and non-aggregating proteoglycans and non- collagenous proteins (Pattappa et al., 2012). The aggregating proteoglycans can interact with hyaluronan, unlike non- aggregating ones. Non-aggregating proteoglycans include the proteolytic products of aggrecan with consequent loss of the hyaluronan-binding G1 domain, and small leucine-rich repeat proteoglycans such as decorin and biglycan (Roughley et al., 2014). The proportion of type I collagen increases from the inner part towards the outer anulus, while the proportion of type II collagen follows a diverse distribution (Bron et al., 2009). In an in situ hybridization study, adjacent cells did not necessarily express the same genes or function in a unified way. This puzzling observation could be attributed to either the heterogeneous phenotype of cell populations in the anulus, the presence of senescent cells with altered gene expression patterns and responses to stimuli, or the disruption of cell-to-cell interactions through gap junctions owing to cell loss. Collectively, only 30-57% of the anulus cells expressed aggrecan, types I, II, or VI collagen, or chondroitin-6-sulfotransferase.(Gruber et al., 2008) TGF-β signaling is crucial for proper development of the AF of the IVD since one lesion associated with conditional deletion of Tgfbr2 is loss of the AF without disrupting NP development. E-26 oncogene-related (Erg) is important for maintaining a pool of undifferentiated sclerotomal cells during development and also contributes to AF differentiation, since TGF-β treatment in sclerotome cultures upregulates it. Interestingly, Erg alone does not stimulate the expression of AF markers, evident by upregulation of the pluripotent progenitor cells, Sca1, in cells expressing Erg. However, treatment of Erg-expressing cells with TGF-β caused upregulation of Adamtls2 and Scx, which are markers of AF and tendon, respectively, suggesting interaction between Erg and Smad3. It is also speculated that Erg helps TGF-β to prevent BMP-mediated chondrogenesis and endochondral bone formation in the IVD space (Cox et al., 2014). Proteoglycans occupy a small proportion of the AF and are substituted with negatively charged glycosaminoglycans (GAGs), which are important for hydrating the tissue to help it undergo rapid reversible deformation (Pattappa et al., 2012). The increase in disc size with rapid decline in vascularization during infancy and childhood is associated with glycosaminoglycans changing to more highly sulfated forms in both NP and AF, leading to production of a higher KS:CS ratio during maturation and in adult degenerative discs (Bushell et al., 1977; Scott and Haigh, 1988; Taylor et al., 1992). This is because CS biosynthesis requires oxygen for changing UDP-glucose to UDP-glucuronic acid, while oxygen is not needed for making UDP-galactose during KS production (Taylor et al., 1992). The small proteoglycans, which are characterized by a leucine-rich core protein substituted with GAG side chains, are beneficial as they bind to collagens, growth factors in the matrix, and facilitate the assembly, transport and incorporation of collagen fibrils within the ECM of the IVD for growth, maturation and repair (Melrose et al., 2001). The AF contains the highest amounts of decorin and versican, which are synthesized by cells expressing a fibroblastic phenotype. In contrast, all zones of the fetal IVD contain equally abundant levels of biglycan, which is less abundant in adult AF specimens (Melrose et al., 2001). Versican was also identified and immunolocalized to discrete regions in the ovine IVD; it could have a weight or space-filling role like aggrecan, or help adjacent annular lamellae attach to one another. Fibulin-1, a glycoprotein with a high affinity for the “C-lectin carboxyl terminal G3 domain of versican”, interacts with fibrillin and is also present between adjacent annular lamellae, which could facilitate versican anchorage. Hyaluronan, which has an interlamellar location in human and canine IVDs, is important for lubricating the adjacent lamellae and facilitating movement (Melrose et al., 2001). One study suggested that despite having less pyridinoline cross-linking than articular cartilage, its content did not differ significantly between the AF and NP. There was relatively less hydroxylysine in the AF, where type I collagen predominates, than in the NP, which has a high content of type II collagen. The dorsal part of the AF has more denatured collagen with fewer pyridinoline cross-links than the ventral part, and this could be implicated in disc degeneration(Vonk et al., 2010). Estrogen

THE SPINE SCHOLAR 7 VOLUME 1, NUMBER 1, 2017 receptor-β is expressed in the anulus cells of the human IVD. Exposing anulus cells to 17-β-estradiol led to significantly greater proliferation than in low serum controls. The effect of estrogen on the disc cells could be related to the anti-apoptotic effect of IGF-1 on them (Gruber et al., 2002). The AF is made of a series of lamellae, collagen fibers paralleling each lamella and a network of elastic fibers between them (Pattappa et al., 2012). One study demonstrated that following elastase treatment, the respective falls in modulus and increases in extensibility indicate that elastic fibers are crucial for guiding and restraining the deformation of the collagen matrix, and their removal causes the collagenous elements to separate and rearrange more easily. Extensibility appeared significantly higher for posterolateral specimens, both before and after elastase treatment, which could be attributed to differences in collagenous architecture rather than elastic fibers or glycosaminoglycans (Smith et al., 2008). The complexity of the mechanical behavior of the AF – it is nonlinear, anisotropic (direction-dependent) and viscoelastic (rate-dependent) - is determined by the ECM around its cells. Replacing collagen II by collagen type I in the inner anulus during aging leads to deterioration of this mechanical behavior (Pattappa et al., 2012). The elastic modulus increased while collagen II decreased in degenerated human AF (Antoniou et al., 1996b), while the outer anulus was kept under tension, eventually leading to annular rupture. The cells of the AF range morphologically from ‘fibroblast-like' in the outer AF to `chondrocyte-like' in the inner AF, while NP cells are more typically `chondrocytic', which could be attributed to the type of collagen produced: type II collagens are made in the NP, switching to type I in the outer AF (Melrose et al., 2001). Also, the cellular processes are shorter and thinner in the inner anulus than in the nucleus or outer anulus. Processes in the outer anulus cells extend from and in the direction of the long axis of the cell, or into the matrix, perpendicular to the long axis. It is speculated that these processes act as mechanotransducers (Errington et al., 1998). Although definitive phenotypic markers could not be identified, Clouet et al. (2009) reported higher expression of type V collagen in rabbit AF than NP cells or articular chondrocytes. However, there are interspecies differences in gene expression (Pattappa et al., 2012). Tenomodulin, a small proteoglycan, could be a potential AF marker as demonstrated in both human and bovine species (Minogue et al., 2010a). In addition, the tenomodulin gene is more highly expressed in degenerated human AF cells than normal ones. Microarray analysis (Gruber et al., 2010; Pattappa et al., 2012) has demonstrated that mitogen-activated protein kinase p38, growth arrest and DNA-damage-inducible β, retinoblastoma (Rb)-associated KRAB repressor gene, discoidin CuB and LCCL domain-containing protein 2, gene inhibitor of growth family member 5, somatostatin receptor 3, interferon-induced transmembrane protein 1, sphingosine-1-phosphate receptor 2, nitric oxidase synthase 1, and heat shock 70 kDa protein 6 are related to cell senescence in AF. In addition, pleiotrophin mRNA levels in AF were correlated with aging, which could be linked to stimulated vascular in-growth in degenerative AF (Pattappa et al., 2012). Also, orosomucoid 1 was positively correlated, and spondin 2 and tubulin polymerization promoting protein family member three gene expressions were negatively correlated, with age and degeneration in human AF cells. Glypican 3, cytokeratin-19, matrix gla protein, and pleiotrophin mRNA were more highly expressed in aged rat AF than young tissue (Lee et al., 2007).

The molecular biology of the cartilaginous endplate The healthy endplate has osseous and cartilaginous parts, the latter referred to as the CEP. The main component of the CEP is water, followed by type II collagen and proteoglycans. Smaller types of proteoglycans have been detected in the CEP including decorin and biglycan (Hayes et al., 2011b; Pattappa et al., 2012). The orientation of collagen fibrils changes across the CEP, the fibrils being parallel with vertebral bodies in the CEP center but becoming curved adjacent to the inner AF region. The CEP separates the IVD from adjacent vertebrae in addition to containing the NP. It also provides the main route for solutes to diffuse into the avascular NP. Small molecules diffuse most rapidly in the central zone of the end-plate. Although the outer AF permits some small molecules to diffuse, the inner AF is impermeable (Pattappa et al., 2012). In addition to CEP permeability, molecule size and ionic charge affect transport. The CEP also mediates the equal distribution of compressive loading from the IVD on to the vertebral body, a feature depending on balanced collagen, water, and proteoglycan contents, and the integrity of the matrix. During IVD degeneration, as the cartilage endplate becomes thinner, fissures develop with associated sclerosis of the subchondral bone(Roberts et al., 2006). The disc height decreases and eventually cartilage and new bone form around the prolapsed region. It has been speculated that highly vascularized regions are sites of defects in the absence in trauma because of scar formation and weakening in that area in comparison to the rest of the CEP matrix. Interestingly, CEP permeability decreases with IVD degeneration. Concomitant with the onset of NP breakdown during the second decade, the CEP becomes less vascular as it becomes more calcified. Later, calcified regions are entirely replaced by bone with occlusion of nutrient canals (Pattappa et al., 2012). Benneker et al. (2005) reported that the density of openings in the range 20–50 lm in the human endplate was significantly correlated with the degree of disc degeneration. However, magnetic resonance imaging (MRI) and X-ray microtomography demonstrated an increase in endplate permeability and porosity with aging, suggesting that the primary reason for disc degeneration is the alteration in cell function and capillary density rather than the inhibition of disc nutrition via the endplate (Pattappa et al., 2012). CEP cells from herniated discs showed greater senescence and increased matrix metalloproteinase production. Ariga et al. found that mouse cell apoptosis increased with aging. Even though apoptotic cells were detected in the NP, AF and CEP, most of them were found in the CEP. After apoptotic processes, the endplate was ossified and this preceded IVD degeneration (Ariga et al., 2001; Pattappa et al., 2012). CEP cells resemble articular chondrocytes in their rounded morphology, yet they differ: the CEP shows slight local variations in the distribution of the cells while articular cartilage has no distinct layers. The CEP has a higher cell density than the AF and NP (Pattappa et al., 2012). Antoniou et al. (1996a) reported that the first phase in ECM turnover involves active production of matrix molecules with active denaturation of type II collagen. During the second phases, referred to as aging and maturation, the synthetic and denaturation activities declined. In the third phase, degeneration entails type II collagen denaturation with associated type 1 procollagen production in progress.

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Although aggrecan and collagen make up both the CEP and the other disc tissues, their synthetic profiles differ significantly. Types I and II procollagen epitope levels in younger groups (< 5 years of age) are significantly lower in the CEP than the NP and AF. On the other hand, there is a higher level of the aggrecan chondroitin sulphate 846 epitope (CS-846), indicating aggrecan synthesis in the CEP (Antoniou et al., 1996a).

Proteoglycans Aggrecan, a proteoglycan with numerous glycosaminoglycan (GAG) chains attached to a core protein, represents the major non-collagenous part of the IVD. Its unique ability to form aggregates with hyaluronan defines aggregan as a large chondroitin sulfate (CS)/keratan sulfate (KS) aggregating proteoglycan. It also varies with species owing to differences in GAG synthesis, KS and CS chain length and sulfation pattern. Aggrecan molecules consist of three globular regions termed G1, G2 and G3, with a short interglobular domain (IGD) separating G1 from G2 and a long GAG-attachment region separating G2 from G3 (Sivan et al., 2014). Regarding spatial distribution, the water and GAG contents are highest in the nucleus and lowest in the outer anulus. The water and GAG contents near the posterior nucleus/transition zone are slightly greater than in the anterior nucleus/transition zone. In the axial direction, the GAG and water contents are greatest in the central region, with a small dip in the central nucleus region, which could reflect degenerative injury (Iatridis et al., 2007). Three-dimensional cell culturing revealed that cultured chondrocytes or nucleus cells accumulated more glycosaminoglycan when the cell density increased. Although GAG production can be increased by increasing cell densities, there would be a steep fall in concentration of oxygen and nutrients from the periphery of the construct to its center, with consequent slowing of metabolism and apoptosis and cell death (Kobayashi et al., 2008). Many functional aspects of these components remain to be delineated, but G1 is required for the interaction with hyaluronic acid, while the function of G2 is not yet well understood. G3 is crucial for “normal trafficking of the molecule through the cell and subsequent secretion” (Sivan et al., 2014). Even though human G3 has a unique feature of variant exon splicing, it is not known whether this feature has any functional significance. IGD is susceptible to cleavage by MMPs and aggrecanases. The KS-rich domain contains multiple 6-residue amino acid repeats that contain proline–serine (Sivan et al., 2014). The serine residues are susceptible to substitution with O-linked oligosaccharides or KS chains; the type of substitution and the structure of KS chains change with aging, but with unknown significance (Sivan et al., 2014). There are two CS-rich domains in aggrecan, CS1 and CS2, which have variations in their amino acid compositions allowing for different susceptibilities to proteolysis. The CS1 domain in humans is distinguished by variations in repeat number among individuals, ranging from 13 to 33. Interestingly, aggrecan function is negatively affected when there are few repeats, which can be a risk factor for early disc degeneration. The CS2 domain also shows more variation in its amino acid structure, conferring different susceptibility to proteolysis on the two domains. Unlike the CS1 domain, CS2 can be cleaved by aggrecanases. The CS chain structure also changes with age (Sivan et al., 2014). The core protein in aggrecan allows it to aggregate, giving the molecule its bulk and stable location within the tissue. The aggrecan molecule interacts well with HA only when it is within the ECM so it can reach more remote areas after secretion. Because the CS and KS chains are highly sulfated, the GAG chains are crucial for establishing the osmotic properties of aggrecan, which allow it to withstand compressive loads by swelling in the NP where its content is highest. This osmotic tendency is balanced by the collagenous framework within the surrounding AF. This process helps maintain adequate waste removal and nutrition of the disc, and explains the diurnal discrepancy in disc height in humans due to differences in gravitational forces on discs. A tear in the AF compromises its ability to resist NP swelling, leading to disc herniation (Sivan et al., 2014). The abundance of aggrecan from the NF prevents blood vessel and nerve growth in normal discs owing to its avascular and aneural nature, and inhibits the calcification of the region as sulfate is a counterion to calcium, so free calcium ions do not precipitate. In disc degeneration, the aggregan content declines, which explains the vessel and nerve ingrowth along with the concomitant calcification (Sivan et al., 2014). GAG chains undergo various changes during growth that seem to be functionally significant, including a decrease in CS length, a change in its sulfation positions, and an increase in KS length, which seems to increase the avascular nature of adult discs (Sivan et al., 2014). Aggrecan is susceptible to cleavage by most proteinases, especially in the IGD and the domains of the GAG attachment region. Nevertheless, only a few of these proteinases can be functional in vivo, especially MMPs and aggrecanases. These can be secreted after mechanical loading or certain inflammatory mediators such as interleukin 1(IL1) and tumor necrosis factor α (TNF-α) (Sivan et al., 2014). In vitro, the collagenases (MMP1, 8 and 13) and gelatinases (MMP2 and 9) have the lowest activities while MMP3, 7 and 12 are the most active. Aggrecanases, which belong to the ADAMTS family, have an exclusive cleavage activity within the IGD and CS2 domain, and the most active ones are ADAMTS 4 and 5 (aggrecanase 1 and 2, respectively)(Sivan et al., 2014). Regardless of which proteinase cleaves aggrecan, a C-terminal fragment is produced that is no longer attached to HA and can diffuse more freely within the tissue. However, these fragments accumulate as movement from the NP is limited by the surrounding AF and adjacent vertebra. Proteolysis results in loss of the G3 domain along with truncation of the core protein, while only the G1 domain remains bound to HA. These events can be considered detrimental, predisposing to disc degeneration (Sivan et al., 2014). Also, the size of aggrecan aggregates is reduced by extracellular degradation of HA. This can be achieved by hyaluronidases activated by cytokines such as oncostatin M, or by free radicals. This degradation can be partially prevented by the incorporation of link proteins. Reducing sugars such as ribose can mediate glycation of aggrecan, leading to modification of lysine residues and formation of advanced glycation end-products (AGEs) such as pentosidine. This modification can make aggrecan unable to interact with HA and eventually destabilizes the aggregates. If this holds true in vivo, this process could explain why diabetes is related to disc degeneration (Illien-Junger et al., 2013; Robinson et al., 1998; Roughley et al., 2014; Sivan et al., 2014).

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Aggrecan turnover varies throughout life but is consistently higher for degenerative IVDs than normal ones. The mean turnover rate for normal aggrecan is 0.126 per year and the mean half-life is 5.7 years. Aggrecan has a shorter mean half-life than collagen since it is more susceptible to proteolysis. In the fetal and neonatal periods, most of the aggrecan is located in the gelatinous NP of the human disc, where its abundance increases gradually until it reaches a maximum in the young adult. Surprisingly, much of the aggrecan during this period is fragmented and cannot interact with HA. Later, the aggrecan content of the NP gradually decreases because of degradation in the ECM, but it increases in the inner AF until it becomes the major deposit in the IVD in mature adults(Sivan et al., 2014). Fragmentation is slow and the fixed charge density of the remaining fragments within the disc can mean that decades pass before functionally significant loss occurs. Future therapy should aim toward promoting aggrecan synthesis and preventing its degradation. BMP7 and transforming growth factor β (TGFβ) proved successful in animal models with potential adverse effects (Sivan et al., 2014). Link N peptide, the N-terminal peptide of link protein produced in vivo during tissue turnover, upregulates the expression of types I and II collagen in human NP cells with an associated decrease in p-p38 MAPK. While the results suggest that Link N upregulates type II collagen expression in both low- and high-expression cells, it seems more capable of stimulating type I collagen expression in a tissue where such expression is low, via a signaling pathway involving p38 (Petit et al., 2011). Also, the full length of LPP is needed to increase the expression of aggrecan and collagen II and inhibit expression of the catabolic regulators IL-lß and MMPI, which are linked to disc degeneration. In vitro studies of bone morphogenetic proteins 2, 7, 14 showed a regenerative potential in IVD pathology with upregulation of proteoglycans and decreasing MMP expression (Belykh et al., 2015). However, they were associated with ectopic ossification. Lpp had a lower osteoinductive effect than BMP2 and BMP7 (Wang et al., 2013). A synthetic peptide (Link N) not only stimulated both NP and AF cells to produce aggrecan, but also inhibited the expression of many MMPs. Although it is cheaper than recombinant growth factors, its disadvantage is that it can only be administered by intradiscal injection (Sivan et al., 2014). CS is composed of repeating disaccharide units in which hydroxyl groups can be differentially sulfated, allowing for structural heterogeneity within CS chains and a strong tendency to attract positively-charged matrix molecules, which are fundamental for the hydrodynamic properties of the disc in withstanding biomechanical load (Hayes et al., 2011a). Using rat models to study chondrotin sulfation epitopes, Hayes et al. found that these epitopes had complex, dynamic and specific distributions in the disc and vertebral tissues during their differentiation, growth and aging. During initial disc differentiation, the presence of sulfation variants and the absence of C-4-S and C-6-S stubs in the disc anlagen could lead to a lower swelling pressure than in the vertebral bodies, helping the notochord to bulge between the vertebral bodies into these “weak” points. The alterations in the expression patterns of CS sulfation motifs during fetal development are expected to occur on a range of matrix and cell-associated PGs. Also, the composition of cellular GAG chains in their PGs seems to be modified in a specific and spatially unique manner through differential regulation of sulfotransferase enzymes. Postnatally, CS sulfation motifs demonstrate more uniform epitope expression labeling in the inner anulus matrix, and later in the outer anulus matrix with full differentiation of the fibrocartilage. Versican is considered a splice variant of a larger proteoglycan (PG) called PG-Mhersican (PG-M). The long central chondroitin sulfate-attachment region of the PG0/m core protein is encoded by exons 7 and 8. At all ages in normal IVD the versican isoform with exon 8 but not exon 7 (V1) is present, while those possessing neither exon 7 nor 8 (V3) or expressing both of these exons (V0) can only be identified in the fetus, and the isoform with only exon 7 (V2) cannot be detected at all (Sztrolovics et al., 2002). While multiple core protein sizes of more than 200 kDa were detected in articular cartilage samples from the fetus to the mature adult, extracts from adult cartilage showed a higher proportion of smaller core proteins. Interestingly, osteoarthritic cartilage exhibited comparable core protein sizes to the normal adult, indicating that osteoarthritis does not affect the splicing pattern, though it has been reported that degeneration involves increased versican expression. The reported increased synthesis could suggest a reparative response in the degenerated tissue, masking any evidence of degradation (Cs-Szabo et al., 1997; Sztrolovics et al., 2002). However, at all postnatal ages, the IVD demonstrated greater size heterogeneity, with an abundant component of about 50 kDa that increased when the sample was treated with keratanase, indicating that the GI region of versican in the disc is susceptible to substitution with KS (Sztrolovics et al., 2002). In the IVD, the versican G1 region accumulated after the versican core protein degraded. Aggrecan shows a similar pattern with aging as its G1 region accumulates owing to the effects of both aggrecanases and matrix MMPs. However, versican and aggrecan do not share the same proteolytic cleavage mechanisms. Alternatively, the inability to detect the versican GI region in cartilage could relate to the lower abundance of the parent proteoglycan in cartilage than disc, or to the preferential loss of the G1 regions from the articular cartilage, which is thin relative to the thick entrapped disc (Sztrolovics et al., 2002).

Different molecular signals and interactions in normal intervertebral disc

The response of gene expression to environmental stimuli differs between cells in the AF and NP, so ECM composition differs between the AF and NP regions. This could be attributed to integrins, which have a role in presenting matrix signals that facilitate cellular responses in the IVD. Human AF tissue showed a higher incidence of staining for the β3, β4 and β6 integrin subunits than human articular cartilage, while staining for the αV subunit was slightly lower in human AF tissue. Importantly, the staining profile (α5, β1, β3 and β5) was similar in porcine and human AF tissues, but human tissue expression of α1 and β4 was lower than porcine. While human AF tissue stained for αv and β6 subunits, porcine AF did not. In contrast, porcine AF tissue stained for the α6 subunit exclusively. Expression of α6a and α6b is necessary for mesenchymal cells to differentiate through the chondrogenic pathway and express types II and X collagen (Nettles et al., 2004; Segat et al., 2002). Differences in expression of the α6 integrin could explain the differences between the NP and AF in biological responses to environmental stimuli (Nettles et al., 2004).

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Immunohistochemical examination of S-100 protein to study mechanoreceptors in the lower IVDs (L4-L5, L5-S1) revealed that the most abundant receptor types were Ruffini type receptors, which contribute to maintaining muscle tone (low threshold, slow adaptation), followed by Golgi endings, which are stimulated at extremes of joint motion (high threshold, slow adaptation). Free nerve fibers were frequent, having either avasomotor, nociceptive or proprioceptive roles. The frequency of encapsulated receptors was significantly greater in the anterior part of the L5–S1 disc than the other parts. This difference can be attributed to the high shear forces affecting the lumbosacral junction, or regional variations in the tensile properties of the disc (Dimitroulias et al., 2010). E-cadherin expression is elevated in rabbit IVD cells with a subsequent increase in the expression of the matrix macromolecules aggrecan and collagen II, stimulation of the expression of BMP-4 and BMP-7 genes, and enhancement of Smad1/5 phosphorylation (Wang et al., 2012). Also, of the two subtypes of sodium-dependent vitamin C transporter (SVCT), RT-PCR demonstrated SVCT2 expression only in the IVDs of rabbits, with higher levels in NF than AF cells. There was a significant upregulation of SVCT2 mRNA expression by IGF-1 and dexamethasone in the IVD, and as ascorbic acid is crucial for collagen and proteoglycan biosynthesis, future therapies implementing IGF-1 and dexamethasone could stimulate AA reuptake and perhaps help degenerated discs regain some of their normal functions (Chothe et al., 2013). The expression of xylosyltransferase-1 (XT-1), a key enzyme in the initial step of GAG synthesis, is not influenced by the activities of the inflammatory cytokines TNF-α and IL-1β. Also, XT-1 expression is similar during the early and late stages of the degenerative process. Jun/AP-1 and complementary action Sp1 and Sp3 transcription factors are essential for maintaining XT-1 expression in NP cells, which is achieved by interacting with the respective cognate binding sites in the XT-1 promoter. XT-1 expression in human NP cells is positively correlated with Jun, Fos, and Sp1 expression (Ye et al., 2015). Treating human NP cells with TNF-α induces the expression of microsomal prostaglandin E2 synthase 1 (PTGES) and prostaglandin F2α synthase (PTGFS), while prostacyclin synthase (PTGIS) is decreased(Vo et al., 2010). PGE2 decreases the levels of aggrecan mRNA and proteoglycan synthesis significantly. PGF2α increases aggrecan levels while decreasing versican expression. The messenger for collagen XV, a nonfibrillar collagen with many sites for GAG chain attachment, is significantly influenced by PGF2α. Both PGE2 and PGF2α caused a significant decline in the IGF-1 messenger, which prevents anulus disc cell senescence and stimulates disc matrix synthesis. Therefore, chronic exposure of PG has a negative effect on the disc (Vo et al., 2010). Further studies are still needed to determine the effect of prostaglandins on MMPs and/or aggrecanase to establish which PG leads to the greatest net matrix degradation. Connective tissue growth factor (CTGF/CCN2) acts as a signal transducer or modifier. From early disc development to maturation, many studies have reported an anabolic effect of CCN2 in maintaining the matrix components of the disc (Chiou et al., 2006; Hall-Glenn and Lyons, 2011; Huang et al., 2010; Tran et al., 2013a; Tran et al., 2013b; Tran et al., 2011). In the degenerative disc, several recent studies have demonstrated a positive correlation between CCN2 levels and the severity of degeneration (Tran et al., 2010) and the presence of pain (Peng et al., 2009). Despite the anabolic effect of CCN2 on matrix synthesis in NP cells in vivo and in vitro, it is speculated that CCN2 has a limited reparative response in degenerative discs, similar to the mechanism illustrated in articular cartilage. Nishida and colleagues (Nishida et al., 2004; Tran et al., 2013a; Tran et al., 2010; Tran et al., 2011) reported that the change in cell response to CCN2 is due to alterations in the surrounding microenvironment during degeneration(Abbott et al., 2013). Interestingly, the increase in CCN2 and decrease in CCN3 in degenerate discs could also be attributed to the increased level of TGF-β, a positive regulator of CCN2 in NP cells (Tran et al., 2011). CCN3 is expressed in NP cells, basal level expression being regulated by TGFβ. CCN3 suppression is partially mediated by the p38 and ERK signaling pathways. Treating NP cells with CCN3 leads to a decrease in cell number and in the expression of versican, aggrecan, type I collagen, and CCN2. Also, TGF-β signaling stimulates CCN3 promoter activity via Smad3 and facilitates the repression of CCN3 via MAPK signaling. The repression can override the stimulation mediated by Smads through a mechanism that limits their access to the promoter (Tran et al., 2011). Also, CCN3 expression decreases with aging in both NP and AF. The anti-proliferative effects of CCN3 are mediated by its interaction with notch and notch ligands in addition to the connexin43 signaling pathways (Fu et al., 2004; Sakamoto et al., 2002; Tran et al., 2011). TGF-β also stimulates cellular proliferation in the NP (Tran et al., 2011; Zhang et al., 2006) even after the addition of CCN3, suggesting that it overrides the antiproliferative effects of CCN3. A regulatory triad has been proposed comprising the opposing effects of TGF-β on the expression of CCN2 and CCN3 and the downregulation of CCN2 by CCN3 (Kawaki et al., 2008; Tran et al., 2011).

Consequences of hypoxia at the molecular level in the intervertebral disc

A study on rat models revealed that ANK, a transmembrane channel that controls the transport of inorganic pyrophosphate, is more highly expressed in mature animals than neonates. As ANK expression is sensitive to oxygen tension, the observed high expression levels were surprising since the NP is avascular. High ANK expression was expected in the calcified zone of the endplate, which has a rich vascular supply (Skubutyte et al., 2010). Hypoxia Inducible Factors HIF-1 and HIF-2 have a role in regulating energy metabolism and matrix synthesis in the NP. Generally, their degradation is thought to be regulated by prolyl-4-hydroxylase domain (PHD) proteins, the activity of which depends on tissue oxygen tension. However, in the NP, protein levels of HIF-1α and HIF-2α are similar in both hypoxia and normoxia. The stability of the HIF-1α-ODD (oxygen- dependent degradation domain) and HIF-2α-ODD in NP cells is independent of oxemic tension. Even though PHD2 is highly expressed in the NP, it seems to have a limited role in controlling HIF-1α degradation and thus its turnover. Interestingly, HIF- 1α levels seem to be regulated mainly by oxygen-independent proteasomal and lysosomal processes. In contrast, HIF-2α turnover by the 26S proteasome is largely independent of PHD, and the lysosomal pathway has limited involvement. These findings illustrate the adaptation of NP cells to the avascular microenvironment: the HIF-1α and HIF-2α levels are regulated mostly by oxygen-independent pathways (Fujita et al., 2012a). Once a certain threshold of PHD2 activity is reached in NP cells, it is important for cell survival and function that a steady state level of HIF-1α is attained by increasing either the rate of HIF-1α production or its resistance to further degradation (Fujita et al., 2012a).

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Just as high HIF expression in the NP is independent of oxygen tension, so ANK expression is not affected by pO2. After HIF-1α or HIF-2α expression in the cells is silenced, ANK expression is stimulated by hypoxia, indicating that its expression is regulated in a HIF-dependent manner. Also, within the hypoxic IVD, HIF-1 and HIF-2 expression is important for maintaining basal ANK expression. It is proposed that under normal physiological conditions, control of mineralization is facilitated by Ank, which helps transport the mineralization inhibitor PPi (Skubutyte et al., 2010). In addition to HIF proteins, TGF-β seems to influence ANK expression in NP cells, which increases in response to TGF-β treatment. This could explain how dysregulation of HIF and elevated TGF-β levels in degeneration contribute to increased ANK levels. In this case, high ANK expression raises PPi and alkaline phosphatase levels, which contribute to hydroxyapatite deposition. An aim of future studies will be to test this hypothesis (Skubutyte et al., 2010). Although hypoxic regulation of PHD3 seems to depend on HIF activity (D'Angelo et al., 2003; Fujita et al., 2012b; Fujita et al., 2012c; Marxsen et al., 2004; Metzen et al., 2005), Fujita et al. found that the expression of PHD3 prolyl hydroxylase in NP cells is controlled by TNF-α and IL-1. Furthermore, PHD is upregulated after stimulation by cytokines, particularly NF-κB, and p65, independently of HIF activity. Also, PHD3 contributes to a cytokine-induced NF-B/p65 signaling process that is correlated with disc degeneration. Moreover, the regulation of PHD3 expression in NP cells is context-dependent since PHD3 has a role as a transcriptional co-activator of HIF-1 signaling under hypoxic conditions, but does not contribute to the oxygen-dependent degradation of HIF- 1α (Fujita et al., 2012a; Fujita et al., 2012b; Fujita et al., 2012c). Suyama reported that BMAL1, a circadian regulator, and RORα, which is a regulator of BMAL1, could influence HIF-1 function in NP cells and have a role in the adaptation of the NP to its hypoxic environment (Suyama et al., 2016).

Extracellular osmolarity and matrix homeostasis in the intervertebral disc

The percentages of water in the NP and AF are approximately 77% and 70%, respectively. The IVD is hyperosmolar in comparison to other tissues, with approximate values between 430 and 496 mOsm (Johnson et al., 2014), which influences cell function and matrix synthesis. When the osmolarity of a bovine NP culture medium was increased by adding sodium salts, the rate of sulfate incorporation increased, suggesting an overall increase in proteoglycan synthesis (Johnson et al., 2014). Sucrose had a similar effect to sodium salts, suggesting that the enhancement of proteoglycan synthesis depended on the tonicity of the medium. In human NP and AF cells under hyperosmotic conditions (500 mOsm), more aggrecan and collagen II and less collagen 1 are expressed than in controls (Wuertz et al., 2007). A study on pig models demonstrated that in a hyperosmotic medium, NP and TZ (transition zone) cells showed, respectively, increased and decreased expression of the small proteoglycans biglycan and decorin (Chen et al., 2002). On the other hand, the TZ cells showed increased expression of small proteoglycans, aggrecan, and type II collagen under hypoosmotic conditions, while the gene expression of lumican and tubulin was decreased in the NP cells. Different cells in the IVD respond differently to osmolarity, and this can be attributed to differences in the cytoskeletal components such as those between the fibro-chondrocytic TZ cells and the NP cells of notochordal origin. Also, these responses could differ between species, since there are differences in the number of cells of notochordal origin retained in the NP (Chen et al., 2002). Increasing the medium osmolarity to 500 mOsm in bovine NP cells also increased aggrecan expression and downregulated the levels of MMP-3 mRNA (Neidlinger-Wilke et al., 2012). Mechanical loading had minimal effects on the molecular level within the disc cells, but these loading forces partly depend on osmotic conditions. Aggrecan and collagen II expression was increased in human and bovine NP cells when hydrostatic pressure was applied under hyposmotic conditions, yet expression was inhibited when the same pressure was applied in a hyperosmotic environment. In contrast, hydrostatic pressure had no effect on the expression of collagen I regardless of the osmotic environment (Wuertz et al., 2007). Spillekom et al. reported recently that using a culture medium adjusted to 400 mOsm/L led to optimal levels of brachyury, aggrecan and GAG (Spillekom et al., 2014). AQP-1, an archetypal water channel, is expressed in the NP and inner anulus within the human IVD, but there is zero AQP-1 expression in the outer anulus, which could be correlated with its minimal water content and its minimal role in resistance to compressive loading. While the NP has to deform to resist compression, and the inner anulus should form a transitional region between the NP and outer anulus, AQP-1 expression could help the cells tolerate osmotic stress by regulating water transport across them. As AQP-1 and -3 have similar expression patterns, overlapping function seems plausible. AQP-3 is distinctive in transporting small solutes such as glycerol and urea, so it could have a role in nutrient supply to IVD cells and in waste removal. As the IVD is avascular, this vital function could explain why AQP-3 is present throughout it (Richardson et al., 2008). However, more studies are needed to investigate any relationship between IVD degeneration and dehydration and differences in AQP expression.

General osmotic response of TonEBP

As described above, changes in the extracellular osmotic condition of a tissue can elicit an organized molecular response. A hypertonic challenge induces membrane electrolyte transporters to prevent large volume swings through establishing balanced intercellular and extracellular solute concentrations. This process should not last for long periods since the change in osmotic pressure can have adverse effects on cellular components leading to autophagy, senescence or apoptosis (Johnson et al., 2014). The transcription factor TonEBP can help prevent these destructive events by upregulating genes that help to exchange the accumulated ions for small organic non-ionic osmolytes. Among these TonEBP targets are aldose reductase (AR), betaine- γ-amino butyric acid transporter (BGT1), sodium myoinositol transporter (SMIT), and taurine transporter (TauT), which contribute respectively to regulating the transport of sorbitol, betaine, myo-inositol, and taurine through the plasma membrane (Johnson et al., 2014). TonEBP is robustly expressed in the NP cells of both neonatal and adult rat discs. The protein is also detected in AF cells, which would be expected as the anulus is rich in proteoglycans and hydrodynamically stressed, like the nucleus. TonEBP

THE SPINE SCHOLAR 12 VOLUME 1, NUMBER 1, 2017 enhances aggrecan gene expression, which usually binds water molecules in the NP and helps it withstand mechanical loading (Tsai et al., 2006). Interestingly, mechanical deformation is correlated with TonEBP activity, as integrins α1β1 and α6β4 and stretching, as well as hypertonic stress, induce TonEBP signaling(Johnson et al., 2014). TonEBP homodimers bind to the tonicity responsive enhancer element (TonE) in target gene promoters needed for osmotolerance (Lopez-Rodriguez et al., 2001; Stroud et al., 2002; Tsai et al., 2006). The nuclear-to-cytoplasmic abundance ratio of TonEBP depends on the osmolarity of the cellular environment (Johnson et al., 2014). After silencing of TonEBP in hypertonic medium the survival of NP cells decreased significantly, indicating the importance of this transcription factor for NP cell viability in the hypertonic environment of the disc. Furthermore, the mRNA levels of SMIT, BGT1, and TauT, which are TonEBP target genes, were upregulated after a hyperosmotic challenge, along with the water channel protein AQP-2 (Johnson et al., 2014). In hypertonic stress the activities of both TauT and HSP-70 reporter were increased, which is crucial for cellular function under hypertonic stress, and TonEBP bound to the TonE motif in the DNA, suggesting that TonEBP is transcriptionally active in the NP in a hypertonic environment. The application of DN-TonEBP transfected cells or partially silencing TonEBP expression by siRNA showed forced suppression of aggrecan promoter activity and decreased cell survival especially under osmotic stress (Tsai et al., 2006). Esensten et al. demonstrated that TonEBP increased the expression of TNF-α and IL-6 under hypertonic conditions, which facilitated degeneration by inducing the chemotaxis of immune cells (Esensten et al., 2005; Tew et al., 2011; Wang et al., 2011b). There was an associated increase in MCP-1(CCL2); a functional TonE element is present in the 5′ flanking region of the MCP-1 gene. Deleting TonE resulted in ablation of the hypertonicity-dependent increase in MCP-1 promoter activity (Johnson et al., 2014). The correlation between the daily osmotic pressure and loading swings in human discs and the mentioned effects of TonEBP on proinflammatory targets and osmoregulatory genes could have important consequences in tissues such as the NP (Johnson et al., 2014). In addition to osmolarity-related effects, TonEBP facilitates the effects of Toll-like receptors (TLRs), which are involved in macrophage responses against pathogenic molecules by inducing Nos2, TNF and Il-6 genes (Buxade et al., 2012). Multiple TLRs are expressed in NP cells and chondrocytes (Liu-Bryan and Terkeltaub, 2010; Rajan et al., 2013). Moreover, the expression levels of Vcan, Has1, Fn1, Timp1, Tnc, and Mmp13 are affected by TonEBP and are correlated with matrix homeostasis as a response to lipopolysaccharide (LPS)-mediated TLR stimulation. LPS treatment also induces enhancement of the NF-κB pathway by TonEBF (Johnson et al., 2014). Therefore, two distinct mechanisms can be involved in activating TonEBP. The first is the canonical pathway, which is influenced by changes in the osmotic status of the extracellular milieu. The second is the noncanonical pathway through TLR activation. While the latter pathways have not yet been verified in the IVD, Kim et al. (2013b) demonstrated that the response of osmotic-response genes (AR, BGT1, SMIT) was lessened when macrophages were pretreated with LPS. Nevertheless, cells pretreated with hyperosmotic conditions showed a suppressed response to TLR stimuli (Johnson et al., 2014; Kim et al., 2013b). If these findings can be extended to the osmodependent TonEBP targets in the IVD such as aggrecan, Sox9, and GlcAT-I, their regulation could be negatively affected under inflammatory conditions such as disc degeneration. Finally, in hypertonic environments, TonEBP influences the expression of target genes affecting the synthesis or transport of small polyhydric alcohols (myoinositol and sorbitol), methylamines (betaine), and amino acids (taurine). This response is adventitious as these compounds do not affect the activities of enzymes and other macromolecules within the cytoplasm (Johnson et al., 2014).

Molecular aspects of intervertebral disc degeneration

The process of disc degeneration involves a variety of pathological changes that stem from a multitude of genetic and environmental factors. Although aging and degenerative disc disease have been used interchangeably, they do not seem to reflect the same biological processes. Disc height is well preserved in aging, if there is no disc collapse, while disc degeneration involves significant underlying pathobiological alterations (Erwin and Hood, 2014). In disc degeneration, the IVD experiences a decline in the ability of the NP to retain water, causing compression forces to affect the inner anulus more, along with declining numbers of viable cells and a significant change in the expression of many ECM molecules (Erwin and Hood, 2014; Pattappa et al., 2012). During disc degeneration, the decrease in cell number is significant not only because fewer cells remain to maintain the disc, but also because the coordination facilitated by gap junction communication is disrupted. Intercellular communication via gap junctions in disc cells was significantly lower in human anulus disc cells from older individuals. Connexins Cx43 and Cx45 were highly expressed in the youngest individuals, while the oldest subject demonstrated the least expression (Gruber et al., 2001). Signaling through TGF-β1 is crucial for the expression of both collagen type II and aggrecan, which are involved in IVD maintenance. Impaired TGF-β1 signaling can be related to the development of osteoarthritis and lumbar disc herniation caused by reduced collagen type II and aggrecan expression, respectively, in asporin or cartilage intermediate layer protein mutants (Fujita et al., 2005; Kizawa et al., 2005). In the degenerating disc, the expression of both decorin and biglycan is increased (Chen et al., 2002). There is increased expression of both cathepsin K, a cysteine protease that cleaves the triple helical domains of types I and II collagen, and RANKL in degenerated human IVDs, suggesting that cathepsin K is crucial for the remodeling and degradation of the matrix of a degenerating disc. There could be a role for cathepsin K inhibitors in future therapies for human disc degeneration (Gruber et al., 2011a). It has been speculated that the source of pain in disc degeneration is related to inflammatory pain-related mediators, after annular tears attract and facilitate the ingrowth of nerves mediating nociceptive signals (Erwin and Hood, 2014). In addition, pain-related molecules such as nerve growth factor (NGF) and its receptor (TrkA) are upregulated. Shamji et al. demonstrated increased IL-4, IL-6 and IL-12 in surgical samples from degenerative and herniated discs, which are related to developing disc- related back pain (Erwin and Hood, 2014). In 3D cultures, anulus cells exposed to IL-1ß showed significantly increased expression of NGF, which is important for regulating the growth of sensory neurons and related to the vascular ingrowth into

THE SPINE SCHOLAR 13 VOLUME 1, NUMBER 1, 2017 degenerating discs. Neurotrophin 3, which is important for neurite survival and growth induction, brain-derived neurotrophic factor, and neuropilin 2, was also elevated. These findings could explain the relationship between proinflammatory cytokines in degenerating discs and the associated low back pain. They could be precursors for future novel analgesic therapies based on developing molecular antagonists to neurotrophins(Gruber et al., 2012a). Koerner et al. reported that IL-4, which seemed to limit inflammatory hyperalgesia, IL-5, IL-6, M-CSF, MDC, TNF-β (lymphotoxin), EGF, IGF-1, angiogenin, and leptin showed significantly higher expression in the posterior degenerated AF than the anterior or normal one. This can be attributed to the high levels of strain on the posterior lumbar AF (Koerner et al., 2014). In the aging NP ECM there is an increase in collagen cross-linking and non-enzymatic glycosylation, which impairs the viscoelastic properties of the disc (Erwin and Hood, 2014). Molecular degradation associated with annular fissures and progressive cell death makes the disc more susceptible to mechanical injury. Bovine cultured IVD models demonstrated that transient treatment with TNF-α can lead to permanent biomechanical changes regardless of the continuing presence of inflammatory cytokines since TNF-α-mediated aggrecan degradation led to increased tissue stiffening correlating with permanent changes in the biomechanical behavior of the IVDs. Also, it is speculated that during dynamic loading, most of the TNF-α passes into the NP through the CEP, not the AF of the IVD. This contradicts a study that reported greater hydraulic permeability in the AF than the CEP. Nevertheless, other factors should be considered in assessing TNF-α transport such as the larger exposed surface area of the CEP and the shorter transport distances between the CEP and the NP. Overall, dynamic loading with convective transport facilitated the penetration of TNF-α through bovine IVDs, with subsequent transient elevation of proinflammatory cytokine synthesis (Walter et al., 2015). Immunohistochemical and microarray gene expression analyses demonstrated the presence of a newly-discovered MMP, MMP-26, in human IVD. MMP-26 expression was significantly downregulated in degenerated cells, and TGF-β expression was remarkably greater than in healthier discs. Future studies are needed to investigate the role of MMP-26 in disc degeneration and its relationships with proinflammatory cytokines(Gruber et al., 2012b). Regarding mitochondrial gene expression, there was significant upregulation of p53 in anulus cells in degenerated discs, which is crucial for initiating cell cycle arrest and cellular senescence. Also, proapoptotic genes were upregulated such as BCL2-like 11, caspase 7, apoptosis-related cysteine peptidase, proteasome 26S subunit non-ATPase 10, reticulon 3, programmed cell death 6, apoptosis-inducing factor, and mitochondrion- associated 1 (AIF or AIFM1), which translocates to the nucleus and initiates chromatin condensation and DNA disruption. Membrane-associated ring finger (C3HC4) 5, residing in the mitochondrial outer membrane, was also significantly upregulated. Notably, nitric oxide synthase 3 and interferon were downregulated in degenerated anulus cells. These findings could be exploited in cell-based biological therapy for IVD degeneration to block cellular apoptosis (Gruber et al., 2011b). Although NP cell apoptosis could seem beneficial in minimizing the effects of aberrant NP cell proliferation during degeneration (Ha et al., 2011; Li et al., 2015), apoptosis can be catastrophic owing to the decreased synthesis of interstitial collagen fibers, which are vital for tensile strength. The dominant effect of apoptosis depends on the extent of the cell death (Jiang et al., 2013; Li et al., 2015). MiR-155, which inhibits apoptotic pathways, is downregulated in degenerative NP cells (Li et al., 2015; Wang et al., 2011a). This downregulation is associated with decreased expression of collagens 1 and III in ligamentum flavum fibroblasts. In contrast, MiR-27α is significantly upregulated during NP cell degeneration, which leads to a decrease in phosphoinositide-3 kinase (PI3K) expression and facilitates subsequent NP cell apoptosis. Similar to its activity in cancer cells, miR-10β is overexpressed in degenerative NP cells leading to increased NP cell proliferation by directly targeting HOXD10 (Li et al., 2015). However, MiR-10β stimulates Akt phosphorylation. Also, MiR-21 is significantly upregulated in degenerative NP cells, increasing proliferation and Akt phosphorylation (Li et al., 2015). Ex vivo studies of bovine NP cells demonstrated a role for miR-146 in suppressing IVD degeneration by downregulating the IL-1-mediated catabolism of ECM and COX2, IL-6, iNOS and TNF-α. Moreover, genetic loss of miR146α function is correlated with a higher catabolic response to IL-1 and an increased number of cells expressing MMP-13 and ADAMTS-5 (Gu et al., 2015). Carbonic anhydrase II (CAII), reported by Power et al. (2011) to be a potential NP marker, is highly expressed in degenerated cells. It could counteract acidic cellular conditions in the hypoxic NP and degenerated tissues following the accumulation of lactic acid from glucose metabolism in addition to inflammatory or catabolic byproducts (Power et al., 2011). A bioinformatic analysis of disc cells after treatment with TNF-α demonstrated significant upregulation of matrix metalloproteinase-1 (MMP-1) along with NF-κB and ADAMTS6. There was also a significant increase in the expression of CCL3 and its related genes downstream of the MAPK, NF-κB and C/EBPβ signaling pathways, which are correlated with inflammatory responses. It is speculated that CCL3 recruits macrophages with subsequent inflammatory effects during degeneration. ANKH, which is involved in transporting intracellular inorganic pyrophosphate (PPi) to the extracellular region and regulating tissue calcification, was also downregulated. Therefore, decreased ANKH gene expression could facilitate calcification of the cartilage endplate during the degeneration process (Liu et al., 2015). One study revealed that in degenerative endplate cells, ADAMTS- 7 and ADAMTS-12 mRNA levels were significantly increased (Zhang et al., 2012). Another study using microarray and protein- protein interaction analyses showed that the upregulation of fibronectin, COL2A1 and β-catenin genes could be related to degeneration of the IVD. However, the application of unsupervised clustering in that study showed that the widely-used Thompson grading system demonstrated a marginal association with the molecular classification of IVD degeneration (Chen et al., 2013). Fibulin, a regulator of ADAMTS-1, is upregulated in degenerative human NP (Pattappa et al., 2012). Matrix degrading enzymes such as aggrecanases and collagenases are also upregulated, with associated elevation of inflammatory mediators such as IL-1β and TNF-α during degeneration, culminating in the degradation of aggrecan and collagen II (Pattappa et al., 2012). Simultaneously, senescence markers such as p16 are expressed on NP cells, mediating further degeneration. While the expression of sFasL (soluble Fas ligand) is significantly lower in degenerate NP cells, the expression of sFas (soluble Fas) is increased. These findings can be attributed to the action of sFas in reducing the ability of sFasL to induce apoptosis of infiltrating immune cells. Immune cell infiltration will therefore be facilitated, allowing for destruction of the immune balance of the NP(Sun et al., 2013). In degenerative human NP cells, Tsai et al. demonstrated a significant upregulation of sulfatase 1 (SULF1), an extracellular enzyme that removes the 6-O-sulfates of heparan sulfate, and a subsequent decrease in

THE SPINE SCHOLAR 14 VOLUME 1, NUMBER 1, 2017 the binding affinity of heparan sulfate and Wnt. This is expected to induce further WNT signaling, which is involved in disc degeneration (Tsai et al., 2015).

Future molecular therapies for intervertebral disc degeneration

A study investigating the active component of Tripterygium wilfordii Hook, triptolide (TPL), showed that in IL-1β-prestimulated human IVD cells, TPL elicited a significant reduction in the mRNA levels of major inflammatory mediators (IL-6, IL-8, PGES2) and matrix degrading enzymes (MMP1, MMP2, MMP3, MMP13), with maximum effects after 18 hours. TPL also decreased the expression of TLR2 and TLR4. These effects were due to the influence of TPL on pathways involving the MAP kinases p38 and ERK. Although TPL seems to be very promising for preventing further disc degeneration, there was a remarkable upregulation of expression of TNF-α mRNA, necessitating further research into this finding in vivo (Klawitter et al., 2012). Bovine lactoferricin (LfcinB) has a promising potential therapeutic effect for disc degeneration as in vitro and ex vivo analyses showed antagonism against both IL-1 and LPS-mediated depletion of proteoglycan, and production of matrix-degrading enzymes including MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5. LfcinB also significantly enhances PG deposition and aggrecan expression through upregulation of biglycan and decorin (Kim et al., 2013a). Biomimetics are being developed that function similarly to aggrecan but cannot be degraded by proteases. One such biomimetic is a GAG analog formed by polymerizing sulfonated monomers of acrylamido-2-methylpropane sulfonic acid (AMPS) and sulfopropyl acrylate (SPA) in the presence of a crosslinking agent. The sulfonate groups on the GAG analog provide the molecule with osmotic properties like those of aggrecan. However, safe administration of this agent is challenging, since direct intradiscal injection is not favorable (Sivan et al., 2014). Soluble factors in porcine notochord cell cultured media (NCCM) inhibit angiogenesis in vitro even in the presence of TNFα, indicating a promising future treatment of vascular ingrowth during degeneration. The effect could be mediated by the suppression of cell invasion and tubule formation by inhibiting the activities of proangiogenic factors such as VEGF, which is a crucial regulator. It is speculated that cadherins or ligand–matrix interactions influence the activity of VEGF in NCCM (Cornejo et al., 2015). The presence of progenitor cells in the tissues of the IVD has been confirmed by several studies (Blanco et al., 2010; Henriksson et al., 2009; Pattappa et al., 2012; Risbud et al., 2007). A slow-cycling stem cell population within the perichondrium or outer anulus region of the human disc could be activated after disc degeneration. A stem cell niche environment was detected by primitive stem cell markers (STRO-1, Ki67). Risbud et al. and Blanco et al. isolated a stem cell-like population from degenerated human IVDs (Blanco et al., 2010; Pattappa et al., 2012; Risbud et al., 2007), while Feng et al. (2010) succeeded in isolating a progenitor cell population from the AF of non-degenerative human discs in which CD marker expression was similar to that in bone marrow-derived mesenchymal stem cells (MSCs). These cells were capable of differentiating towards each of the mesodermal lineages. A progenitor population showing similar characteristics to MSCs was also detected in the degenerative CEP (Liu et al., 2011; Pattappa et al., 2012). These findings of notochordal-like cells suggest that natural repair mechanisms could still be available for activation and regeneration. Yamamoto et al. suggested that coculture with cell-to-cell contact can stimulate the secretion of growth factors such as insulin-like growth factor-1 (IGF-1) to induce matrix production by NP cells (Yamamoto et al., 2004). Studies coculturing human MSC and NP cells with cell-cell contact revealed that the NP cells stimulate specific differentiation and expression of matrix components by MSCs (Richardson et al., 2006). Gene expressions of SOX-9, matrix aggrecan, and type II collagen were particularly upregulated. A ratio of 75:25 NP cells:MSCs was optimal for inducing MSC differentiation, which could be attributed to the high cellular signals mediated from the greater NP cell population. Types I and VI collagen exhibited a minor but significant increase in expression by both the NP cells and MSCs, and the short form of collagen IX was detected. Coculture without contact elicited no significant alterations in matrix gene expression (Richardson et al., 2006). Other studies investigating various cell types cocultured with MSCs without cell-cell contact have demonstrated effects, but none comparable to those elicited by coculturing with cell-cell contact (Rasmusson et al., 2003). Immunohistological staining of cells in animal models confirms the presence of progenitor cells and stem cell niches in the IVD region (Henriksson et al., 2009). Few cells in both the AF and NP areas of the IVD expressed 5-bromo-2-deoxyuridine (BrdU), which demonstrates a continuous but low rate of cellular proliferation. However, the BrdU-positive cells in the perichondrium region (roi 1) and the AF ligament anchoring site (AFo) exhibited a pattern similar to a stem cell niche with a high cell proliferation rate during early stages and only a few slow cycling cells later. A corresponding Jagged 1 expression pattern was noted, especially in the perichondrial densely packed zone (roi 1), which contains progenitor cells. Areas with slow cycle cells had the most abundant proliferation and progenitor markers in the disc (Henriksson et al., 2009). As there are more BrdU- positive cells in the epiphyseal plate close to the perichondrium region than in the middle part of the plate, it is suggested that cells migrate from there to the upper zone of the epiphysial region and probably also toward the AF and/or the NP. The expression of Ki67 along with the progenitor marker Notch1, a crucial cell fate determinant in various stem cell niches, Stro-1, and C-KIT (CD117) demonstrates continuous regeneration in normal discs. These findings could help in developing future biological treatment options for disc degeneration (Henriksson et al., 2009). A distinct immortalization strategy has been reported to generate NP cell lines (Liu et al., 2014; van den Akker et al., 2014). Clonal subtypes can be distinguished according to morphological characteristics, expression of marker genes, and responses to differentiation signals. In one study (van den Akker et al., 2014), an immortal NP cell clone was identified on the basis of positive KRT19 and CD24 expression. TGFβ3 caused downregulation of novel NP markers in primary cells and cell clones (Stoyanov et al., 2011; van den Akker et al., 2014). Matrigel or ACAN mediated a stronger stimulation of conventional chondrogenic markers in the NP cell lines studied, since they led to upregulation of COMP and COL2A1, respectively. Also, there was little or no T expression in the immortal clones, indicating that more mature NP cells were immortalized. Furthermore, it was hypothesized that these immortalized lines can be classified into more differentiated cells (NP-nR), which are GD2+ /CD24+, and more progenitor-like

THE SPINE SCHOLAR 15 VOLUME 1, NUMBER 1, 2017 cells (NP-R), which are GD2− /CD24− with the spheroid-forming capacity (van den Akker et al., 2014). Guan et al. used CD24 as the mature NP cell marker to identify the MSCs in NPs differentiating into mature NP cells in vitro (Guan et al., 2014). Adding TGF-β1 to the NP-MSC culture resulted in positive promotion of CD24 expression. Interestingly, after interacting with inflammatory mediators, the immediate early response gene (EGR1) was stimulated both cell types, associated with NF-κB signaling. However, NP-nR cells illustrated a more mature catabolic response manifested by a higher IL-1β-mediated MMP3 upregulation. Nevertheless, NP-R cells showed a higher anabolic response after treatment with IL-1β and TNF-α than the NP- nR cells. It is concluded that EGR1 mediates cell-specific responses to growth or inflammatory stimulants, and cells subsequently respond according to the epigenetic constitution of their lineage. It is speculated that epigenetic programming occurs during the maturation of NP-R to NP-nR cells (van den Akker et al., 2016). It has been suggested that normal AF tissue contains multipotent progenitor cells, since the human AF contains many cell surface antigens of mesenchymal stem cells, namely CD29, CD49e, CD51, CD73, CD90, CD105, CD166, CD184, and Stro- 1, and two neuronal stem cell markers, nestin and neuron-specific enolase. These multipotent progenitor cells in the AF can differentiate into various cell types that take part in innervating and vascularizing the degenerative IVD (Feng et al., 2010). Wang et al. showed that adding TGF-β1 to a medium containing chitosan/glycerophosphate scaffolds under hypoxic conditions helped immortalized human precartilaginous stem cells to differentiate into NP-like cells, associated with increased expression of type II collagen and aggrecan and elevated protein expression of Sox9 and β-catenin (Wang et al., 2015). An aim of tissue engineering is to develop biodegradable and biocompatible substances for tissue restoration. Polymers are classified into synthetic and natural. The latter include Alginate, Chitosan, and hyaluronic acid (Renani et al., 2012). NP cell proliferation was elevated in association with secretion of ECM on an alginate scaffold. Chitosan, with the advantages of biocompatibility and dissolubility, comprises a glycosamine and N-acetyl glycosamine polymer (Renani et al., 2012). Several studies have demonstrated the efficacy of chitosan scaffolds in elevating both proliferation and secretion processes (Lahiji et al., 2000; Lee et al., 2002). The addition of gelatin to a scaffold enhances cell adhesion and migration (Dang et al., 2006). In a comparison between chitosan–gelatin and alginate scaffolds, NP cell proliferation and viability percentage proved significantly higher in both scaffolds on day 3 than day 1. However, proliferation and viability in both scaffolds declined from day 3 to day 21(Renani et al., 2012). Similar results were reported by Bertolo et al. (2012). The alginate scaffold also supported higher cellular proliferation and viability than the chitosan–gelatin scaffold (Renani et al., 2012). These differences could be attributed to the toxic effect of glutaraldehyde, which is used for cross-linking in chitosan–gelatin scaffolds(Miranda et al., 2011), and the micro-diameter of surface pores in some areas of the chitosan–gelatin scaffold; these eventually become blocked, deceasing nutrition exchange and causing subsequent cell death (Griffon et al., 2006). Hydrogel in alginate scaffolds enhanced cell connection, nutrition and oxygen transport. Furthermore, gelatin could cause wetting and lead to hydrolysis of a chitosan–gelatin scaffold. Therefore, alginate seems more appropriate than chitosan (Renani et al., 2012).

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Correspondence to R. Shane Tubbs, [email protected]

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