Devina Purmessur

Expression and regulation of neurotrophins in the nondegenerate and degenerate human intervertebral disc

IntroductionThe neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) have been identified in the human intervertebral disc (IVD) and have been implicated in the mechanisms associated with nerve ingrowth and nociception in degeneration of the IVD. The aim of the current study was to investigate an association between neurotrophin expression in the IVD and the severity of disc degeneration, including the effect of disc-related proinflammatory cytokines on neurotrophin and neuropeptide expression in cells derived from the human IVD.MethodsImmunohistochemical analysis was performed to examine the expression of NGF, BDNF and their high-affinity receptors Trk-A and Trk-B in human IVD samples, divided into three categories: non-degenerate, moderate degeneration and severe degeneration. In order to study the effect of disc-related cytokines on neurotrophin/neuropeptide gene expression, nucleus pulposus cells derived from non-degenerate and degenerate IVD samples were seeded in alginate and were stimulated with either IL-1β or TNFα for 48 hours. RNA was extracted, cDNA was synthesised and quantitative real-time PCR was performed to examine the expression of NGF, BDNF and substance P.ResultsImmunohistochemistry showed expression of NGF and BDNF in the native chondrocyte-like cells in all regions of the IVD and in all grades of degeneration. Interestingly only BDNF significantly increased with the severity of degeneration (P < 0.05). Similar expression was observed for Trk-A and Trk-B, although no association with disease severity was demonstrated. In cultured human nucleus pulposus cells, stimulation with IL-1β led to significant increases in NGF and BDNF gene expression (P < 0.05). Treatment with TNFα was associated with an upregulation of substance P expression only.ConclusionOur findings show that both the annulus fibrosus and nucleus pulposus cells of the IVD express the neurotrophins NGF and BDNF, factors that may influence and enhance innervation and pain in the degenerate IVD. Expression of Trk-A and Trk-B by cells of the nondegenerate and degenerate IVD suggests an autocrine role for neurotrophins in regulation of disc cell biology. Furthermore, modulation of neurotrophin expression by IL-1β and modulation of substance P expression by TNFα, coupled with their increased expression in the degenerate IVD, highlights novel roles for these cytokines in regulating nerve ingrowth in the degenerate IVD and associated back pain.

Complex loading affects intervertebral disc mechanics and biology

BACKGROUND Complex loading develops in multiple spinal motions and in the case of hyperflexion is known to cause intervertebral disc (IVD) injury. Few studies have examined the interacting biologic and structural alterations associated with potentially injurious complex loading, which may be an important contributor to chronic progressive degeneration. OBJECTIVE This study tested the hypothesis that low magnitudes of axial compression loading applied asymmetrically can induce IVD injury affecting cellular and structural responses in a large animal IVD ex-vivo model. METHODS Bovine caudal IVDs were assigned to either a control or wedge group (15°) and placed in organ culture for 7 days under static 0.2MPa load. IVD tissue and cellular responses were assessed through confined compression, qRT-PCR, histology and structural and compositional measurements, including Western blot for aggrecan degradation products. RESULTS Complex loading via asymmetric compression induced cell death, an increase in caspase-3 staining (apoptosis), a loss of aggrecan and an increase in aggregate modulus in the concave annulus fibrosis. While an up-regulation of MMP-1, ADAMTS4, IL-1β, and IL-6 mRNA, and a reduced aggregate modulus were induced in the convex annulus. CONCLUSION Asymmetric compression had direct deleterious effects on both tissue and cells, suggesting an injurious loading regime that could lead to a degenerative cascade, including cell death, the production of inflammatory mediators, and a shift towards catabolism. This explant model is useful to assess how injurious mechanical loading affects the cellular response which may contribute to the progression of degenerative changes in large animal IVDs, and results suggest that interventions should address inflammation, apoptosis, and lamellar integrity.

Notochordal conditioned media from tissue increases proteoglycan accumulation and promotes a healthy nucleus pulposus phenotype in human mesenchymal stem cells

IntroductionNotochordal cells (NCs) are influential in development of the intervertebral disc (IVD) and species that retain NCs do not degenerate. IVD repair using bone marrow derived mesenchymal stem cells (MSCs) is an attractive approach and the harsh microenvironment of the IVD suggests pre-differentiation is a necessary first step. The goal of this study was to use soluble factors from NCs in alginate and NCs in their native tissue to differentiate human MSCs to a young nucleus pulposus (NP) phenotype.MethodsHuman MSCs (cultured under micromass conditions for 21 days in hypoxia) were differentiated with conditioned medium derived from porcine notochordal cells in native tissue (NCT) or in alginate beads (NCA), and compared with chondrogenic (TGFβ-3) or basal medium. A PCR array of 42 genes was utilized to screen a large number of genes known to be associated with the healthy NP phenotype and pellet cultures were also evaluated for glycosaminoglycan content, histology and viability. Proteomic analysis was used to assess candidate soluble factors in NCA and NCT.ResultsNotochordal cell conditioned media had diverse effects on MSC phenotype. NCT resulted in the highest levels of glycosaminoglycan (GAG), as well as up-regulation of SOX9 and Collagen II gene expression. NCA demonstrated effects that were catabolic yet also anti-fibrotic and minimally hypertrophic with down-regulation of Collagens I and III and low levels of Collagen X, respectively. Micromass culture and hypoxic conditions were sufficient to promote chondrogenesis demonstrating that both basal and chondrogenic media produced similar phenotypes. Candidate matricellular proteins, clusterin and tenascin were identified by proteomics in the NCA group.ConclusionsNCs secreted important soluble factors capable of differentiating MSCs to a NP phenotype synthesizing high levels of proteoglycan while also resisting collagen fiber expression and hypertrophy, yet results were sensitive to the conditions in which media was generated (cells in alginate versus cells in their native tissue) so that further mechanistic studies optimizing culture conditions and defining important NC secreted factors are required. Matricellular proteins, such as clusterin and tenascin, are likely to be important to optimize differentiation of MSCs for maximum GAG production and reduced collagen fiber expression.

A role for TNFα in intervertebral disc degeneration: A non-recoverable catabolic shift

This study examines the effect of TNFα on whole bovine intervertebral discs in organ culture and its association with changes characteristic of intervertebral disc degeneration (IDD) in order to inform future treatments to mitigate the chronic inflammatory state commonly found with painful IDD. Pro-inflammatory cytokines such as TNFα contribute to disc pathology and are implicated in the catabolic phenotype associated with painful IDD. Whole bovine discs were cultured to examine cellular (anabolic/catabolic gene expression, cell viability and senescence using β-galactosidase) and structural (histology and aggrecan degradation) changes in response to TNFα treatment. Control or TNFα cultures were assessed at 7 and 21 days; the 21 day group also included a recovery group with 7 days TNFα followed by 14 days in basal media. TNFα induced catabolic and anti-anabolic shifts in the nucleus pulposus (NP) and annulus fibrosus (AF) at 7 days and this persisted until 21 days however cell viability was not affected. Data indicates that TNFα increased aggrecan degradation products and suggests increased β-galactosidase staining at 21 days without any recovery. TNFα treatment of whole bovine discs for 7 days induced changes similar to the degeneration processes that occur in human IDD: aggrecan degradation, increased catabolism, pro-inflammatory cytokines and nerve growth factor expression. TNFα significantly reduced anabolism in cultured IVDs and a possible mechanism may be associated with cell senescence. Results therefore suggest that successful treatments must promote anabolism and cell proliferation in addition to limiting inflammation.

Regenerative Potential of TGFβ3 + Dex and Notochordal Cell Conditioned Media on Degenerated Human Intervertebral Disc Cells

Injection of soluble cell signaling factors into degenerated intervertebral discs (IVDs) offers a minimally invasive treatment that could limit the processes of degeneration by stimulating native matrix repair. This study evaluated the regenerative capacity of degenerated nucleus pulposus (NP) cells obtained from patients undergoing anterior interbody fusions by measuring metabolic activity, DNA content, glycosaminoglycan (GAG) content, and cellular phenotype using qRT‐PCR profiling with a custom array of 42 genes. NP cells were cultured in alginate for 7 days with 4 treatment groups: transforming growth factor beta 3 (TGFβ3) + dexamethasone (Dex), soluble factors released from notochordal cells (NCs) cultured in alginate (NCA), soluble factors released from NCs in their native tissue environment (NCT), and basal media. TGFβ3 + Dex stimulated degenerated human NP cells to proliferate and exhibit an anti‐catabolic gene expression profile (with a decrease in ADAMTS5 and MMP1 compared to basal, and an increase in SOX9, decrease in ADAMTS5, MMP1, collagen I and collagen III compared to day 0), while NCA stimulated the greatest GAG per cell. We conclude that degenerated human NP cells exhibit regenerative potential, and that an optimal treatment will likely require treatments, such as TGFβ3 + Dex, which were able to increase cell metabolism and reduce catabolism, as well as treatments with factors found in NC conditioned medium, that were able to produce high amounts of GAG per cell. Additional studies to optimize NC culture conditions are required to determine if NC conditioned medium can be made with the capacity to enhance NP cell proliferation and metabolism.

Degenerate human nucleus pulposus cells promote neurite outgrowth in neural cells.

Innervation of nociceptive nerve fibres into the normally aneural nucleus pulposus (NP) of the intervertebral disc (IVD) occurs during degeneration resulting in discogenic back pain. The neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), which are associated with stimulation of axonal outgrowth and nociception by neuronal cells, are both expressed by NP cells, with BDNF levels increasing with disease severity. However the mechanism of interaction between human NP cells and neural cells has yet to be fully elucidated. Therefore the aim of this study was to determine whether non-degenerate or degenerate human NP cells inhibit or stimulate neural outgrowth and whether any outgrowth is mediated by NGF or BDNF. Human NP cells from non-degenerate and degenerate IVD were cultured in alginate beads then co-cultured for 48 hours with human SH-SY5Y neuroblastoma cells. Co-culture of non-degenerate NP cells with neural cells resulted in both an inhibition of neurite outgrowth and reduction in percentage of neurite expressing cells. Conversely co-culture with degenerate NP cells resulted in an increase in both neurite length and percentage of neurite expressing cells. Addition of anti-NGF to the co-culture with degenerate cells resulted in a decrease in percentage of neurite expressing cells, while addition of anti-BDNF resulted in a decrease in both neurite length and percentage of neurite expressing cells. Our findings show that while non-degenerate NP cells are capable of inhibiting neurite outgrowth from human neural cells, degenerate NP cells stimulate outgrowth. Neurotrophin blocking studies demonstrated that both NGF and BDNF, secreted by degenerate NP cells, may play a role in this stimulation with BDNF potentially playing the predominant role. These findings suggest that NP cells are capable of regulating nerve ingrowth and that neoinnervation occurring during IVD degeneration may be stimulated by the NP cells themselves.

Soluble factors from the notochordal-rich intervertebral disc inhibit endothelial cell invasion and vessel formation in the presence and absence of pro-inflammatory cytokines

BACKGROUND Chronic low back pain can be associated with the pathological ingrowth of blood vessels and nerves into intervertebral discs (IVDs). The notochord patterns the IVD during development and is a source of anti-angiogenic soluble factors such as Noggin and Chondroitin sulfate (CS). These factors may form the basis for a new minimally invasive strategy to target angiogenesis in the IVD. OBJECTIVE To examine the anti-angiogenic potential of soluble factors from notochordal cells (NCs) and candidates Noggin and CS under healthy culture conditions and in the presence of pro-inflammatory mediators. DESIGN NC conditioned media (NCCM) was generated from porcine NC-rich nucleus pulposus tissue. To assess the effects of NCCM, CS and Noggin on angiogenesis, cell invasion and tubular formation assays were performed using human umbilical vein endothelial cells (HUVECs) ± tumor necrosis factor alpha (TNFα [10 ng/ml]). vascular endothelial growth factor (VEGF)-A, MMP-7, interleukin-6 (IL-6) and IL-8 mRNA levels were assessed using qRT-PCR. RESULTS NCCM (10 & 100%), CS (10 and 100 μg) and Noggin (10 and 100 ng) significantly decreased cell invasion of HUVECs with and without TNFα. NCCM 10% and Noggin 10 ng inhibited tubular formation with and without TNFα and CS 100 μg inhibited tubules in Basal conditions whereas CS 10 μg inhibited tubules with TNFα. NCCM significantly decreased VEGF-A, MMP-7 and IL-6 mRNA levels in HUVECs with and without TNFα. CS and Noggin had no effects on gene expression. CONCLUSIONS We provide the first evidence that soluble factors from NCs can inhibit angiogenesis by suppressing VEGF signaling. Notochordal-derived ligands are a promising minimally invasive strategy targeting neurovascular ingrowth and pain in the degenerated IVD.

Inflammatory Kinetics and Efficacy of Anti-inflammatory Treatments on Human Nucleus Pulposus Cells.

Study Design. Human nucleus pulposus (NP) cell culture study investigating response to tumor necrosis factor-&agr; (TNF&agr;), effectiveness of clinically available anti-inflammatory drugs, and interactions between proinflammatory cytokines. Objective. To characterize the kinetic response of proinflammatory cytokines released by human NP cells to TNF&agr; stimulation and the effectiveness of multiple anti-inflammatories with 3 substudies: Timecourse, Same-time blocking, Delayed blocking. Summary of Background Data. Chronic inflammation is a key component of painful intervertebral disc degeneration. Improved efficacy of anti-inflammatories requires better understanding of how quickly NP cells produce proinflammatory cytokines and which proinflammatory mediators are most therapeutically advantageous to target. Methods. Degenerated human NP cells (n = 10) were cultured in alginate with or without TNF&agr; (10 ng/mL). Cells were incubated with 1 of 4 anti-inflammatories (anti-IL-6 receptor/atlizumab, IL-1 receptor anatagonist, anti-TNF&agr;/infliximab and sodium pentosan polysulfate/PPS) in 2 blocking-studies designed to determine how intervention timing influences drug efficacy. Cell viability, protein, and gene expression for IL-1&bgr;, IL-6, and IL-8 were assessed. Results. Timecourse: TNF&agr; substantially increased the amount of IL-6, IL-8, and IL-1&bgr;, with IL-1&bgr; and IL-8 reaching equilibrium within ∼72 hours (IL-1&bgr;: 111 ± 40 pg/mL, IL-8: 8478 ± 957 pg/mL), and IL-6 not reaching steady state after 144 hours (1570 ± 435 pg/mL). Anti-TNF&agr; treatment was most effective at reducing the expression of all cytokines measured when added at the same time as TNF&agr; stimulation. Similar trends were observed when drugs were added 72 hours after TNF&agr; stimulation, however, no anti-inflammatories significantly reduced cytokine levels compared with TNF control. Conclusion. IL-1&bgr;, IL-6, and IL-8 were expressed at different rates and magnitudes suggesting different roles for these cytokines in disease. Autocrine signaling of IL-6 or IL-1&bgr; did not contribute to the expression of any proinflammatory cytokines measured in this study. Anti-inflammatory treatments were most effective when applied early in the inflammatory process, when targeting the source of the inflammation. Level of Evidence: N/A

Nonoperative Management of Discogenic Back Pain: A Systematic Review

Study Design. Systematic review of the literature. Objective. A systematic evaluation of the literature was performed to investigate current nonoperative management of the treatment of discogenic low back pain. Summary of Background Data. Back pain is a major health care concern with up to 39% being discogenic in origin according to one study. Nonoperative therapy is likely to be the initial treatment strategy for discogenic low back pain. Methods. PubMed, EMBASE, and Cochrane Central Register of Controlled Trials were searched for clinical studies evaluating nonoperative methods of treating discogenic back pain that were published between 2000 and 2012. Only prospective randomized controlled studies that compared a nonsurgical intervention with sham or placebo therapy were included. After removal of duplicate citations, a total of 226 articles were initially identified from the search terms. From these, we identified 11 randomized controlled trials (RCTs) from which data analysis was performed. Results. The 11 RCTs investigated traction therapy, injections, and ablative techniques. Results from 5 RCTs investigating methylene blue injection, steroid injection, ramus communicans ablation, intradiscal electrothermal therapy, and biacuplasty favored intervention over sham therapy. However, results from the study on methylene blue injections have not been replicated in other RCTs. Evaluation of the selection criteria used in the studies on ramus communicans ablation and intradiscal biacuplasty and a stratified analysis of results from the RCTs on intradiscal electrothermal therapy casts doubt on whether the conclusions from these RCTs can be applied to the general patient population with discogenic pain. Conclusion. There are few high-quality studies evaluating nonoperative treatments for reducing discogenic low back pain. Although conclusions from several studies favor intervention over sham, it is unclear whether these interventions confer stable long-term benefit. There is some promise in newer modalities such as biacuplasty; however, more inclusive studies need to be performed. Level of Evidence: 2

Degenerative grade affects the responses of human nucleus pulposus cells to link-N, CTGF, and TGFβ3.

Study Design: Cells isolated from moderately and severely degenerated human intervertebral disks (IVDs) cultured in an alginate scaffold. Objective: To compare the regenerative potential of moderately versus severely degenerated cells using 3 proanabolic stimulants. Summary of Background Data: Injection of soluble cell signaling factors has potential to slow the progression of IVD degeneration. Although degenerative grade is thought to be an important factor in targeting therapeutic interventions it remains unknown whether cells in severely degenerated IVDs have impaired metabolic functions compared to lesser degenerative levels or if they are primarily influenced by the altered microenvironment. Methods: Nucleus pulposus (NP) cells were cultured in alginate for 21 days and treated with 3 different proanabolic stimulants: a growth factor/anti-inflammatory combination of transforming growth factor &bgr;3 (TGF&bgr;3)+dexamethasone (Dex), or matricellular proteins connective tissue growth factor (CTGF) or Link-N. They were assayed for metabolic activity, DNA content, glycosaminoglycan, and qRT-PCR gene profiling. Results: Moderately degenerated cells responded to stimulation with increased proliferation, decreased IL-1&bgr;, MMP9, and COL1A1 expression, and upregulated HAS1 as compared with severely degenerated cells. TGF&bgr;R1 (ALK5) receptors were expressed at greater levels in moderately than severely degenerated cells. TGF&bgr;3+Dex had a notable stimulatory effect on moderately degenerated NP cells with increased anabolic gene expression and decreased COL1A1 and ADAMTS5 gene expression. Link-N and CTGF had similar responses in all assays, and both treatments upregulated IL-1&bgr; expression and had a more catabolic response than TGF&bgr;3+Dex, particularly in the more severely degenerated group. All groups, including different degenerative grades, produced similar amounts of glycosaminoglycan. Conclusions: Proanabolic stimulants alone had limited capacity to overcome the catabolic and proinflammatory cytokine expression of severely degenerated NP cells and likely require additional anti-inflammatory treatments. Moderately degenerated NP cells had greater TGF&bgr; receptor 1 expression and better responded to anabolic stimulation.