Human IVD Research Articles

CILP-2 expression in the intervertebral discs of patients with lumbar radiculopathy

BackgroundIntervertebral disc (IVD) degeneration (IVDD) is one of the main causes of low back pain. One of the most important features of IVDD is the loss of extracellular matrix (ECM) with its structural components. Cartilage intermediate layer proteins (CILPs), minor glycoproteins residing in ECM, have been found to be increased in IVD as degeneration and aging progresses. The aim of the present study was to evaluate the expression of CILP-2 in the IVD of patients with lumbar radiculopathy.MethodsThe IVD samples were collected from 25 patients during spinal surgery (interlaminectomy, herniated disc removal). The control IVD samples were obtained from nine patients who underwent lateral corpectomies in the thoracic region. CILP-2 expression was detected by immunohistochemistry. The patients were divided into two groups – aged under or over 50 years. A standardized clinical examination with assessment of radicular signs and deficits was performed. Subjective disability and pain were assessed using the visual analogue scale and Oswestry Disability Index (ODI). The pre-operative MRI was graded for the degree of IVD degeneration by Pfirrmann grading system. IVD samples obtained during operations were subjected to the standardized histopathological analysis applying modified Boos classification. The data were analysed by t-test, Mann-Whitney U-test, and Spearman correlation test.ResultsBoth histopathology scores and Pfirrmann grades did not differ between patients’ groups. Also, no correlations were found between histopathology and Pfirrmann grades, neither were any differences seen when correlating both grades to ODI, back pain or leg pain scores. CILP-2 staining was noted in all studied samples, notably strong staining was seen around large cell clusters. However, no differences in CILP-2 staining were seen between the age groups of patients. No correlations were found between CILP-2 staining and Pfirrmann grades. Grading of CILP-2 immunostaining in nine control patient samples resulted in significantly lower values. The difference is statistically significant (P = 0.002) compared to CILP-2 staining scores of all 25 patients’ samples.ConclusionIn this study, we detected increased CILP-2 expression in the human IVD as compared to the control group patients. CILP-2 can be a possible IVDD marker; however, as knowledge about the role of CILP-2 is limited, further studies are required.

Phospholipase A2 group IVD mediates the transacylation of glycerophospholipids and acylglycerols

In mammalian cells, glycerolipids are mainly synthesized using acyl-CoA-dependent mechanisms. The acyl-CoA-independent transfer of fatty acids between lipids, designated as transacylation reaction, represents an additional mechanism for lipid remodeling and synthesis pathways. Here, we demonstrate that human and mouse phospholipase A2 group IVD (PLA2G4D) catalyzes transacylase reactions using both phospholipids and acylglycerols as substrates. In the presence of mono- and diacylglycerol (MAG and DAG), purified PLA2G4D generates DAG and triacylglycerol (TAG), respectively. The enzyme also transfers fatty acids between phospholipids and from phospholipids to acylglycerols. Overexpression of PLA2G4D in COS7 cells enhances the incorporation of polyunsaturated fatty acids into TAG stores and induces the accumulation of lysophospholipids. In the presence of exogenously added MAG, the enzyme strongly increases cellular DAG formation, while MAG levels are decreased. PLA2G4D is not or poorly detectable in commonly used cell lines. It is expressed in keratinocytes, where it is strongly upregulated by proinflammatory cytokines. Pla2g4d-deficient mouse keratinocytes exhibit complex lipidomic changes in response to cytokine treatment, indicating that PLA2G4D is involved in the remodeling of the lipidome under inflammatory conditions. Transcriptomic analysis revealed that PLA2G4D modulates fundamental biological processes including cell proliferation, differentiation, and signaling. Together, our observations demonstrate that PLA2G4D has broad substrate specificity for fatty acid donor and acceptor lipids, allowing the acyl-CoA-independent synthesis of both phospholipids and acylglycerols. Loss-of-function studies indicate that PLA2G4D affects metabolic and signaling pathways in keratinocytes, which is associated with complex lipidomic and transcriptomic alterations.

GRAM-POSITIVE BACTERIA WITHIN THE INTERVERTEBRAL DISC AND THEIR POTENTIAL INFLUENCE

IntroductionMultiple studies have identified Cutibacterium acnes (C.acnes) and other microbes in intervertebral disc tissue using 16S DNA Sequencing and microbial cultures. However, it remains unclear whether these bacteria are native to the discs or result from perioperative contamination. Our study aimed to detect Gram-positive bacteria in non-herniated human disc samples and explore correlations with Toll-like receptors (TLR) 2, TLR4, NLRP3, and Gasdermin D.MethodsImmunohistochemical staining was conducted on 75 human IVD samples for Gram-positive bacteria, S. aureus, C.acnes, TLR2, TLR4, NLRP3, and Gasdermin D. Cell detection and classification were performed using QuPath. NP cells were treated with Lipopolysaccharide (LPS) and Peptidoglycan (PGN) in monolayer and alginate beads for up to 72 hours, followed by secretome analysis using Luminex. Statistical analysis included Kruskal-Wallis, Dunn's multiple comparison test, and Pearson correlation.ResultsImmunohistochemical staining revealed Gram-positive bacteria exclusively within cells, with C. acnes positivity ranging from 5–99% and correlating with patient age (r=0.41, p= 0.007). TLR2 positivity ranged from 5–99% and TLR4 from 3–72%, showing a strong correlation (r= 0.62, p= 1.5e-006). Females with mid-degenerative grades exhibited significantly decreased TLR2 expression compared to those without degeneration signs. Treatment with LPS and PGN increased catabolic cyto- and chemokines associated with IVD degeneration.ConclusionIn conclusion, this study confirms Gram-positive bacteria presence in non-herniated human disc samples and highlights their role in triggering a catabolic response in disc cells.No conflicts of interest Sources of fundingThis project is part of the Disc4All Training network to advance integrated computational simulations in translational medicine, applies to intervertebral disc degeneration and funded by Horizon 2020 (H2020-MSCA-ITN-ETN-2020 GA: 955735)

Enhanced analysis of equine plasma for the presence of recombinant human erythropoietin - Implementation of an improved workflow.

An improved screening workflow and a robust capillary flow LC-MS confirmatory method for the detection of recombinant human erythropoietin (rHuEPO) has been implemented to increase the sensitivity of rHuEPO detection and to reduce the number of suspect samples committed to confirmatory testing. The influence of repeated dosing of epoetin-β on the detection window of rHuEPO in equine plasma was assessed using the optimised method. Samples were initially assessed using an economical R&D Human EPO Duo-Set ELISA Development System. Samples indicating a result greater than the batch baseline were analysed using the complementary R&D Human EPO Quantikine IVD ELISA kit. All samples recording an abnormal screening result were subjected to confirmatory analysis. Confirmation of rHuEPO in plasma (≥2.5ml) in the range of 4-13 mIU/ml (n = 6) was achieved using immunoaffinity enrichment, tryptic digestion, and capillary flow LC-MS/MS. Four horses were administered a single dose of epoetin-β (10,000 IU) via the subcutaneous and intravenous routes, on two occasions, seven days apart. The excretion profile was rapid with epoetin-β detection times of 48 to 72 h following each administration, with no appreciable difference observed between the two routes of administration. This workflow has been shown as an effective anti-doping strategy related to rHuEPO misuse and supports the use of out-of-competition testing of horses in the 2 to 3-day period prior to race-day.

Expression of Glial-Cell-Line-Derived Neurotrophic Factor Family Ligands in Human Intervertebral Discs.

Glial-cell-line-derived neurotrophic factor (GDNF) family ligands (GFLs) contribute to the sensitization of primary afferents and are involved in the pathogenesis of inflammatory pain. The purpose of this preliminary study was to examine the expression of other GFLs (neurturin (NRTN), artemin (ARTN), persephin (PSPN)) and receptors in human IVD cells and tissues exhibiting early and advanced stages of degeneration. Human IVD cells were cultured as a monolayer after isolation from the nucleus pulposus (NP) and anulus fibrosus (AF) tissues. The mRNA expression of NRTN, ARTN, PSPN, and their receptors (GFRA2-GFRA4) was quantified using real-time PCR. Protein expression was evaluated using immunohistochemistry and Western blotting. The expression of NRTN, ARTN, PSPN, and their co-receptors (GFRA2-GFRA4) was identified in human IVD cells at both mRNA and protein levels. A trend was noted wherein the mRNA expression of ARTN, PSPN, and GFRA2 was upregulated by IL-1β treatment in a dose-dependent manner. The percentages of immunopositive cells in the advanced degenerate stage of ARTN, PSPN, and GFRA2 were significantly higher than those in the early degenerate stage. Their expression was enhanced in advanced tissue degeneration, which suggests that GFLs (ARTN and PSPN) may be involved in the pathogenesis of discogenic pain.

Correlation between Adrenoceptor Expression and Clinical Parameters in Degenerated Lumbar Intervertebral Discs

Despite advanced knowledge of the cellular and biomechanical processes of intervertebral disc degeneration (IVDD), the trigger and underlying mechanisms remain unclear. Since the sympathetic nervous system (SNS) has been shown to exhibit catabolic effects in osteoarthritis pathogenesis, it is attractive to speculate that it also influences IVDD. Therefore, we explored the adrenoceptor (AR) expression profile in human IVDs and correlated it with clinical parameters of patients. IVD samples were collected from n = 43 patients undergoing lumbar spinal fusion surgery. AR gene expression was analyzed by semi-quantitative polymerase chain reaction. Clinical parameters as well as radiological Pfirrmann and Modic classification were collected and correlated with AR expression levels. In total human IVD homogenates α1A-, α1B-, α2A-, α2B-, α2C-, β1- and β2-AR genes were expressed. Expression of α1A- (r = 0.439), α2A- (r = 0.346) and β2-AR (r = 0.409) showed a positive and significant correlation with Pfirrmann grade. α1A-AR expression was significantly decreased in IVD tissue of patients with adjacent segment disease (p = 0.041). The results of this study indicate that a relationship between IVDD and AR expression exists. Thus, the SNS and its neurotransmitters might play a role in IVDD pathogenesis. The knowledge of differential AR expression in different etiologies could contribute to the development of new therapeutic approaches for IVDD.

Role of the miR-133a-5p/FBXO6 axis in the regulation of intervertebral disc degeneration.

ObjectiveLow back pain is a leading cause of disabilities worldwide, and intervertebral disc degeneration (IVDD)-related disorders have been recognised as one of the main contributors. Nevertheless, the underlying mechanism has not yet been fully understood. The aim of this study was to investigate the role of the miR-133a-5p/FBXO6 axis in the regulation of IVDD.MethodsRT-qPCR, WB and IHC were performed to assess the expression of FBXO6 in human IVD tissues. Nucleus pulposus (NP) cells were treated with IL-1β to induce IVDD cellular model. Silence of FBXO6 was achieved using specific siRNAs. CCK-8 assay, flow cytometry, TUNEL assay, RT-qPCR and WB were used to evaluate the role and mechanism of FBXO6 in the process of IVDD. Online tools, GSE datasets and RT-qPCR were used to search the candidate miRNAs targeting FBXO6. The direct binding sites between FBXO6 and miR-133a-5p were further verified by a dual luciferase assay. RT-qPCR, WB and rescue experiments were conducted to identify the regulatory function of miR-133a-5p on the expression of aggrecan, collagen Ⅱ, MMP3, ADAMTS5, IL-6 and COX2. In addition, the role of the NF-κB pathway in regulating miR-133a-5p was studied using lentiviral shRNA, WB and RT-qPCR.ResultsResults showed that FBXO6 mainly expressed in the NP tissue of IVD and the expression of FBXO6 decreased with the process of IVDD as well as under IL-1β stimulation. The silence of FBXO6 led to the decreased expression of aggrecan and collagen Ⅱ and the increased expression of MMP3, ADAMTS5, IL-6 and COX2, which further induced the degeneration of NP cells. The bioinformatic analysis showed that miR-133a-5p was the candidate miRNA targeting FBXO6. miR-133a-5p was upregulated in IVDD tissues and significantly inhibited the expression of FBXO6. The inhibition of miR-133a-5p ameliorated the acceleration of IVDD induced by the silence of FBXO6 in vitro. Moreover, it was demonstrated that IL-1β regulated the expression of the miR-133a-5p/FBXO6 axis via the NF-κB pathway in NP cells.ConclusionmiR-133a-5p was upregulated by IL-1β to aggravate intervertebral disc degeneration via sponging FBXO6. Inhibiting miR-133a-5p expression or rescuing FBXO6 expression may be promising strategies for the treatment of IVDD.The translational potential of this articleThis study suggests that the miR-133a-5p/FBXO6 axis could regulate NP cells proliferation, apoptosis, synthesis and degradation of extracellular matrix, which provides a promising therapeutic target and strategy for the treatment of IVDD.

The effects of rivaroxaban, an oral anticoagulant, on human IVD primary cultures.

IntroductionThe present study aimed to investigate the potential effects of rivaroxaban, an oral anticoagulant that inhibits the effects of factor Xa, on intact intervertebral disc tissue cells and the extracellular matrix (ECM).Material and methodsRivaroxaban was applied to primary human cell cultures prepared from tissues of the intervertebral disc. Comparative molecular analyses were performed on non-drug-treated control group samples. Descriptive statistics were presented as the mean ± standard deviation. An analysis of variance test was performed to determine whether there were significant differences in the mean across the groups. When differences across groups were observed, Tukey’s honestly significant difference post-hoc test was used for multiple pairwise comparisons. The significance of the obtained data was determined statistically. The α significance value was < 0.05.ResultsThe cells in the control group and in the rivaroxaban-treated group were viable, healthy, and proliferated (p < 0.05). However, the expression levels of the chondroadherin gene (CHAD), cartilage oligo matrix protein (COMP), matrix metalloproteinase (MMP)-13, and MMP-19 genes were changed (p < 0.05).ConclusionsAlthough rivaroxaban does not suppress cell proliferation due to morphological, biological, and biochemical changes in the intervertebral disc tissue, it may change the expression of genes that are related to ECM maintenance.

88. Cell stiffness decreases with severity of disc degeneration and inflammatory stimulation

88. Cell stiffness decreases with severity of disc degeneration and inflammatory stimulation

Inhibiting IL-1 signaling pathways to inhibit catabolic processes in disc degeneration.

Intervertebral disc degeneration is characterized by an imbalance between catabolic and anabolic signaling, with an increase in catabolic cytokines particularly IL-1β, a key regulator of IVD degeneration. This study aimed to investigate intracellular signaling pathways activated by IL-1β, and GDF-5 in the degenerate IVD to identify potential new therapeutic targets. Human NP cells were cultured in alginate beads to regain in vivo phenotype prior to stimulation with IL-1β or GDF-5 for 30 min, a proteasome profiler array was initially utilized to screen activation status of 46 signaling proteins. Immunoflourescence was used to investigate activation of the NFκB pathway. Cell-based ELISAs were then deployed to confirm results for ERK1/2, p38 MAPK, c-jun, and IκB signaling. IHC was utilized to investigate native activation status within human IVD tissue between grades of degeneration. Finally, cells were stimulated with IL-1β in the absence or presence of p38 MAPK, c-jun, JNK, and NFκB inhibitors to investigate effects on MMP3, MMP13, IL-1β, IL-6, and IL-8 mRNA expression. This study demonstrated three key signaling pathways which were differentially activated by IL-1β but not GDF-5; namely p38 MAPK, c-jun, and NFκB. While ERK 1/2 was activated by both GDF-5 and IL-1. Immunohistochemistry demonstrated p38 MAPK, c-jun, and NFκB were activated during human IVD degeneration and inhibition of these pathways reduced or abrogated the catabolic effects of IL-1β, with inhibition of NFκB signaling demonstrating most widespread inhibition of IL-1β catabolic effects. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:74-85, 2017.

Nerves are more abundant than blood vessels in the degenerate human intervertebral disc

BackgroundChronic low back pain (LBP) is the most common cause of disability worldwide. New ideas surrounding LBP are emerging that are based on interactions between mechanical, biological and chemical influences on the human IVD. The degenerate IVD is proposed to be innervated by sensory nerve fibres and vascularised by blood vessels, and it is speculated to contribute to pain sensation. However, the incidence of nerve and blood vessel ingrowth, as well as whether these features are always associated, is unknown. We investigated the presence of nerves and blood vessels in the nucleus pulposus (NP) of the IVD in a large population of human discs.MethodsImmunohistochemistry was performed with 61 human IVD samples, to identify and localise nerves (neurofilament 200 [NF200]/protein gene product 9.5) and blood vessels (CD31) within different regions of the IVD.ResultsImmunopositivity for NF200 was identified within all regions of the IVD within post-mortem tissues. Nerves were seen to protrude across lamellar ridges and through matrix towards NP cells. Nerves were identified deep within the NP and were in many cases, but not always, seen in close proximity to fissures or in areas where decreased matrix was seen. Fifteen percent of samples were degenerate and negative for nerves and blood vessels, whilst 16 % of all samples were degenerate with nerves and blood vessels. We identified 52 % of samples that were degenerate with nerves but no blood vessels. Interestingly, only 4 % ofall samples were degenerate with no nerves but positive for blood vessels. Of the 85 samples investigated, only 6 % of samples were non-degenerate without nerves and blood vessels and 7 % had nerves but no blood vessels.ConclusionsThis study addresses the controversial topic of nerve and blood vessel ingrowth into the IVD in a large number of human samples. Our findings demonstrate that nerves are present within a large proportion of NP samples from degenerate IVDs. This study shows a possible link between nerve ingrowth and degeneration of the IVD and suggests that nerves can migrate in the absence of blood vessels.

Identification and characterization of human nucleus pulposus cell specific serotypes of adeno-associated virus for gene therapeutic approaches of intervertebral disc disorders.

BackgroundIntervertebral disc (IVD) disorders are often accompanied by painful inflammatory and immunopathological processes. Nucleus pulposus (NP) cells play a pivotal role in maintenance of IVD by organizing the expression of anabolic, catabolic, anti-catabolic and inflammatory cytokines. Human NP cells have been targeted by gene therapeutic approaches using lentiviral or adenoviral systems that could be critical due to genome incorporation or immunological side effects. Adeno-associated viruses (AAVs), which do not express any viral gene and are not linked with any known disease in humans, are attractive gene delivery vectors. However, their lack of specific tissue tropism and preexisting immune response are main problems for therapeutic applications. Heretofore, AAVs have not been studied in human IVD research. Therefore, we attempted to identify NP cell specific AAV serotype by targeting human NP cells with different self-complementary AAV (scAAV) serotypes.Identification and characterization of the proper serotype is crucial to establish less immunogenic and safer gene therapeutic approaches of IVD disorders.MethodsPreoperative magnetic resonance imaging (MRI) was used for grading of IVD degeneration. NP cells were isolated, cultured with low-glucose and transduced with green fluorescent protein (GFP) packing scAAV serotypes (scAAV1-8) in a dose-dependent manner. scAAV titers were determined by quantitative polymerase chain reaction (qPCR). Transduction efficiencies were determined by fluorescence microscopy and fluorescence-activated cell sorting within 48 days of post-transduction. The 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine NP cell viability. Three-dimensional (3D) cell culture and enzyme-linked immunosorbant assay (ELISA) were performed to examine the expression levels of inflammatory, catabolic and matrix proteins in NP cells.ResultsscAAV6, scAAV2 and scAAV3 showed high and prolonged transgene GFP expressions with transdution efficiencies of 98.6 %, 91.5 % and 89.6 % respectively (p ≤ 0.002). Unlike scAAV6, the serotypes scAAV2 and scAAV3 declined the viability of NP cells by about 25 % and 10 % respectively (p ≤ 0.001). Moreover, scAAV6 did not affect the expression of the inflammatory, catabolic and matrix proteins.ConclusionsAs original primary research evaluating AAVs in degenerative human IVDs, this study identified scAAV6 as a proper serotype for high, stable and non-immunogenic target gene expression in human NP cells. The data could be very important to design efficient and safer gene therapeutic approaches of IVD disorders.

Substance P Receptor Antagonist Suppresses Inflammatory Cytokine Expression in Human Disc Cells.

Laboratory study. To evaluate whether blockade of the Substance P (SP) NK1R attenuates its proinflammatory effect on human intervertebral disc cells (IVD), and to evaluate the signaling pathways associated with SP. SP and its receptors are expressed in human IVD cells, and cause upregulation of inflammatory mediators; however, the effects of blocking these receptors have not been studied in human IVD cells. Human annulus fibrosus (AF) and nucleus pulposus (NP) cells were expanded in monolayer, and then suspended in alginate beads. The alginate beads were treated with culture medium first containing a high affinity NK1R antagonist (L-760735) at different concentrations, and then with medium containing both NK1R antagonist and SP at 2 concentrations. Ribonucleic acid was isolated and transcribed into cDNA. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to evaluate expression of interleukin (IL)-1β, IL-6, and IL-8. Western blot analysis was performed to examine levels of the phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal regulated kinase 1/2 (ERK1/2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB p65). The cells were pretreated with specific inhibitors of p38 (SB203580), ERK1/2 (PD98059), and p65 (SM7368) and then stimulated with SP. We detected expression of NK1R, neurokinin receptor 2 (NK2R), and neurokinin receptor 3 (NK3R) in AF and NP cells. Treatment of disc cells with the NK1R antagonist was able to suppress expression of IL-1β, IL-6, and IL-8 in a dose-dependent manner. SP stimulation increased phosphorylation of p38-MAPK and ERK1/2, but not of NFκB p65. This indicates that p38-MAPK and ERK1/2 control SP-induced cytokine expression independently from NF-kB p65. Inhibition of p38 and ERK1/2 activation reduced SP-induced IL-6 production in human disc cells. NK1R is responsible for the proinflammatory effect of SP on IVD cells and this effect can be blocked by preventing binding of SP to NK1R. This study shows for the first time that SP mediates signaling in disc cells through NK1R and that SP activates the proinflammatory p38-MAPK and ERK1/2 pathways. 4.

Progranulin knockout accelerates intervertebral disc degeneration in aging mice.

Intervertebral disc (IVD) degeneration is a common degenerative disease, yet much is unknown about the mechanisms during its pathogenesis. Herein we investigated whether progranulin (PGRN), a chondroprotective growth factor, is associated with IVD degeneration. PGRN was detectable in both human and murine IVD. The levels of PGRN were upregulated in murine IVD tissue during aging process. Loss of PGRN resulted in an early onset of degenerative changes in the IVD tissue and altered expressions of the degeneration-associated molecules in the mouse IVD tissue. Moreover, PGRN knockout mice exhibited accelerated IVD matrix degeneration, abnormal bone formation and exaggerated bone resorption in vertebra with aging. The acceleration of IVD degeneration observed in PGRN null mice was probably due to the enhanced activation of NF-κB signaling and β-catenin signaling. Taken together, PGRN may play a critical role in homeostasis of IVD, and may serve as a potential molecular target for prevention and treatment of disc degenerative diseases.

SIRT1 protects against apoptosis by promoting autophagy in degenerative human disc nucleus pulposus cells.

SIRT1 could protect degenerative human NP cells against apoptosis, and there were extensive and intimate connection between apoptosis and autophagy. Up to now, the role of autophagy in the process of human IVD degeneration is unclear. We sought to explore the relationship between autophagy and human IVD degeneration and to understand whether autophagy is involved in the protective effect of SIRT1 against apoptosis in NP cells. Our results showed that the autophagosomes number, the mRNA level of LC3 and Beclin-1, the protein expression of LC3-II/I and Beclin-1, decreased in NP from DDD. Resveratrol could increase the protein expression of LC3-II/I and Beclin-1, and reduce apoptosis in degenerative NP cells. In contrast, the protein levels of LC3-II/I and Beclin-1 were down-regulated and apoptosis level was significantly up-regulated in treatment with nicotinamide or SIRT1-siRNA transfection. Further analysis identified that the expression of cleaved Caspase3 and apoptosis incidence significantly increased with the pretreatment of bafilomycin A, whether resveratrol was added or not. These suggested that autophagy may play an important role in IVD degeneration, and SIRT1 protected degenerative human NP cells against apoptosis via promoting autophagy. These findings would aid in the development of novel therapeutic approaches for degenerative disc disease treatment.

Intervertebral disc and stem cells cocultured in biomimetic extracellular matrix stimulated by cyclic compression in perfusion bioreactor

Background contextIntervertebral disc (IVD) degeneration often causes back pain. Current treatments for disc degeneration, including both surgical and nonsurgical approaches, tend to compromise the disc movement and cannot fully restore functions of the IVD. Instead, cell-based IVD tissue engineering seems promising as an ultimate therapy for IVD degeneration. PurposeTo tissue-engineer an IVD ex vivo as a biological substitute to replace degenerative IVD. Study designAn extracellular matrix (ECM) structure-mimetic scaffold, cocultured human IVD cells and human mesenchymal stem cells (hMSCs), and mechanical stimulation were used to biofabricate a tissue-engineered IVD. MethodsAn optimal ratio of human annulus fibrosus (hAF) cells to hMSCs for AF generation within aligned nanofibers, and that of human nucleus pulposus (hNP) cells to hMSCs for NP generation within hydrogels were first determined after comparing different coculture ratios of hAF or hNP cells to hMSCs. Nanofibrous strips seeded with cocultured hAF cells/hMSCs were constructed into multilayer concentric rings, enclosing an inner core of hydrogel seeded with hNP cells/hMSCs. A piece of nonwoven nanofibrous mat seeded with hMSC-derived osteoblasts was assembled on the top of the cellular nanofiber/hydrogel assembly, as an interface layer between the cartilagenous end plate and vertebral body. The final assembled construct was then maintained in an osteochondral cocktail medium and stimulated with compressive loading to further enhance the hAF and hNP cells differentiation and increase the IVD ECM production. ResultsAmong all cocultured groups, hAF cells and hMSCs in the ratio of 2:1 cultured in nanofibers showed the closest mRNA expression levels of AF-related markers to positive control hAF cells, whereas hNP cells and hMSCs in the ratio of 1:2 cultured in hydrogels showed the closest expression levels of NP-related markers to positive control hNP cells. The effects of compressive loading on chondrogenesis of hAF or hNP cell and hMSC coculture were dependent on the scaffold structure; the expression of cartilage-related markers in AF nanofibers was downregulated, whereas that in NP hydrogel was upregulated. Interestingly, we found that hMSC-derived osteogenic cells in the interface layer were turned into chondrogenic lineage cells, with decreased expression of osteogenic markers and increased expression of chondrogenic markers. ConclusionsWe demonstrate a unique approach using a biomimetic scaffold, IVD and stem cell coculture, and mechanical stimulation to tissue-engineer a biological IVD substitute. The results show that our approach provides both favorable physical and chemical cues through cell-matrix and cell-cell interactions and mechanobiological induction to enhance IVD generation ex vivo. Our findings may lead to viable tissue engineering applications of generating a functional biological IVD for the treatment of disc degeneration.

Novel Molecular Phenotype of Notochordal-Like Cells in Nucleus Pulposus of Human Intervertebral Disk

Introduction Cells of the immature nucleus pulposus (NP) are believed to originate from the notochord with many unique morphologic features,1–3 including larger cell size and vacuolated appearance. NP cells of immature IVD tissues are heterogeneous with a population of cells that are notochordal in appearance. While these morphological characteristics of notochordal cells are absent in NP cells of adult human, it is unknown if a distinct molecular phenotype for notochordal-like cells exists in the cells of human IVD. Previously, we discovered a set of novel cell surface proteins (CD24, CD54, CD155, CD166, CD221), one transcriptional factor (brachyury, T), and three neuronal-related proteins (brain abundant membrane attached signal protein 1, Basp1; Neurochondrin, Ncdn, and Neuropilin, Nrp1) in cells from immature rat NP that is enriched in notochordal-like cells.4,5 The goal of this study is to evaluate whether these proteins can serve as markers of notochordal-like NP cells in the human IVD and the change of these markers during aging by both flow cytometry and immunohistochemistry analysis. Materials and Methods Human IVD cells were isolated by a nonenzymatic protocol from NP tissues of patients undergoing surgery for scoliosis (juvenile group, n = 4, 6 to 17yo) and degenerative disk disease (adult group, n = 3, 43 to 71yo). Cells were allowed to become confluent before removal via trypsin and incubated with antibodies against human CD24, CD54, CD155, CD166, and CD221 (with isotype controls and secondary fluorescent antibodies). To evaluate brachyury expression, cells were first fixed and permeabilized before incubation with antibodies. The percentage of cells with positive protein and mean fluorescence intensity (MFI) was analyzed by flow cytometry (Accuri C6). Analyses were performed to detect differences in percentage of ( + ) cells between the two age groups (one-factor ANOVA). Frozen tissue sections were fixed and permeabilized, then incubated with specific antihuman antibodies for selected NP markers (CDs, brachyury T, Basp1, Ncdn, Nrp) and secondary antibodies, counterstained with propidium iodide and imaged using confocal microscopy. Results NP cells from the juvenile group stained positively for all studied cell surface antigens and brachyury ( Table 1 ). A significant lower percent of adult NP cells stained positively for CD24, CD54, and brachyury ( p &lt; 0.05), as compared to the juvenile NP cells; in contrast, a higher percentage of adult NP cells stained positively for CD155 and CD166 ( p &lt; 0.05). No differences in CD221 expression was detected between the two age groups (TABLE). All CD surface proteins, brachyury T, Basp1, Ncdn, and Nrp1 were detected in juvenile NP tissues via immunostaining, however only Basp1 and CD24 stained intensively positive (Fig. 1C) and others were only moderately positive. In contrast, immunostaining only detected moderate expression for CD54 and CD166, but not brachyury T, CD24, CD155, CD221, Basp1, Ncdn, and Nrp1 in adult NP tissues (Fig. 1D). [Table: see text][Figure: see text] Conclusion The results of this study suggest the utility of a new set of NP cell markers (CD24, CD54, CD155, CD166, CD221, brachyury T, Basp1, Ncdn, and Nrp1) for identifying notochordal-like NP cells in the human IVD. These findings are consistent with our previous findings for notochordal-like cells in the rat NP5. Our data confirm consistent expression of notochordal-like NP molecular phenotypes across species and in cases where the notochordal-like cell morphology is not preserved. Acknowledgments Supported by NIH AR057410, AR047442 and EB002263 I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Rufai et al. Anatomy and Embryology (Berl) 1996;192:53 Trout et al. Tiss Cell 1982;14:359 Urban and Roberts. Molecular Medicine Today 1995;1:329 Chen et al. Trans ORS 2007;32:1104 Tang X et al. Trans ORS 2011;36:470

Regulation of Chemokine Expression by Interleukin-1 in Human Intervertebral Disk Degeneration

Introduction It is established that Interleukin-1 (IL-1) is expressed by native nucleus pulposus (NP) cells and that disregulated expression is associated with the pathogenesis of IVD degeneration1. IL-1 has been shown to regulate diverse cellular functions within the IVD including those related to catabolic and anabolic activities2. Here the regulatory potential of IL-1 on chemokines that we have previously identified within the human IVD is investigated to address the hypothesis that regulatory interrelationships exist between cytokines and chemokines in the IVD. Materials and Methods Primary human NP cells cultured in alginate beads were subjected to 48 hours treatment with IL-1. RNA extracted from these cultures was used in qRT-PCR gene expression analysis to determine the effects of treatment on the expression of selected target chemokine genes. Cell culture supernatants from these cultures were used to determine the effects of treatment on protein production of selected chemokines. Results Constitutive expression of CXCL8, CCL2, and CCL7 was observed in alginate bead cultured primary human NP cells. Following 48 hours IL-1 treatment expression of CXCL8 and CCL2 was upregulated (Fig. 1A). Constitutive expression of IL-1 and CCL3 was not observed in alginate cultured primary human NP cells however, expression was induced following 48 hours IL-1 treatment (Fig. 1B). Conclusion This study demonstrates the regulatory potential of IL-1 in relation to a number of chemokines some of which have not been shown previously and adds to the body of evidence implicating IL-1 as a key regulator of NP cell biology. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Le Maitre et al. Arthritis Research and Therapy 2007;7:R732-R745 Le Maitre et al. Arthritis Research and Therapy 2007;9:R77

In Vitro Evaluation of a Thermoreversible Hyaluronan-Based Hydrogel Loaded with Cell-Seeded Polyurethane Particles for Nucleus Pulposus Repair

Introduction Thermoreversible hyaluronan (HA) based hydrogels represent a promising carrier for IVD repair because of the mild gel formation allowing for encapsulation of viable cells and biological stimulation of cells by HA.1,2 Additionally, polyurethane (PU) foams can provide elasticity, which is of key importance for the restoration of IVD mechanical function, as the latter experiences repeated loading during the daily human activities. Finally, the addition of relevant cells may promote a faster restoration of the IVD function. Therefore, the aim of this study was to compare the single and combined effect of PU particles and HA hydrogel on the proliferation and differentiation of human IVD and mesenchymal stem cells. Materials and Methods Human bone marrow stromal cells (hMSCs) or human disk cells (hIVD cells) (P2-P4) were seeded on PU particles; after 12 hours, the seeded particles were suspended in either a 12% w/v thermoreversible hyaluronan-poly(N-isopropylacrylamide) hydrogel (HA-pNIPAM)2 or in a fibrinogen/thrombin solution.3 Each sample contained 50 µL hydrogel, 2.5 mg PU particles, and 2.5 × 105 cells. Controls included hMSC (or hIVD cells) seeded HA-pNIPAM, hMSC (or hIVD cells) seeded fibrin, and hMSC (or hIVD cells) seeded PU. After gelling for 1 hour at 37°C, samples were cultured in chondrogenic medium (DMEM high glucose, 1% ITS, 130 µM ascorbic acid, 100 nM dexamethasone and 10 ng/mL TGF-β1) for one week and analyzed for DNA (by Picogreen), sulfated glycosaminoglycan (GAG) (by 1,9 dimethylmethylene blue), mRNA expression, and histology (toluidine blue) at day 3 and 7. Three independent experiments were performed in triplicates for each group and cell type. After confirming the normality of data distribution and exclusion of the outliers, data were analyzed with a univariate general linear model and post hoc Bonferroni test ( p &lt; 0.05). Results In the MSC cultures, a slight DNA increase was observed between day 3 and 7 for both PU/HA-pNIPAM and PU/fibrin samples. At day 7, the highest amounts of DNA were found for the PU/fibrin group, followed by fibrin and PU groups ( Fig. 1a ). The amount of DNA was higher in the PU group than PU/HA-pNIPAM and HA-pNIPAM, suggesting a lower cell proliferation in the presence of the thermoreversible hydrogel. A similar DNA profile was obtained when hIVD cells were used instead of hMSCs. A significantly increased amount of GAG was observed in hMSC-seeded PU/HA-pNIPAM samples (Fig.1B) and there was an increase of GAG from day 3 to day 7. All other groups had a much lower amount of glycosaminoglycans and there was only a slight increase of GAG from day 3 to day 7. The GAG retention (GAG in sample divided by the sum of GAG in the sample and GAG released in the medium) at day 7 was comprised between 50% and 80% for all groups. Disk cells produced significantly less GAG in the PU/HA-pNIPAM system compared to hMSC, indicating that MSCs were more responsive to the combination of these two biomaterials ( Fig. 1B ). Moreover, the GAG retention at day 7 was lower with human IVD cells (20–60%) compared to hMSCs in all groups. This suggests that an early and higher GAG accumulation is associated to a better GAG retention in the hydrogel. This finding may be related to the fact that while MSCs were obtained from patients undergoing various types of surgeries, IVD cells were obtained from (degenerate) disks of patients undergoing total IVD replacement. The gene expression profile confirmed MSC chondrogenic differentiation with a strong upregulation of collagen type II and, to a lower extent, of aggrecan and SOX9 (compared to cells collected on the day of seeding) without significant differences between the two gels. Histological sections revealed cell migration from PU particles to the hydrogels. [Figure: see text] Conclusion Human disk cells from degenerating disks synthesized less GAG than hMSCs, indicating that MSCs may represent a suitable cell type for the biological repair of IVDs. A synergic effect for MSC differentiation was observed when a thermoreversible hyaluronan-based hydrogel was combined with polyurethane particles. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Mortisen D, et al. Biomacromolecules 2010;11:1261–1272 Peroglio M, et al. European Spine Journal Epub ahead of print Mauth C, et al. European Cells and Material 2009;18:27–38

In Vitro Evaluation of a Thermoreversible Hyaluronan-Based Hydrogel Loaded with Cell-Seeded Polyurethane Particles for Nucleus Pulposus Repair

IntroductionThermoreversible hyaluronan (HA) based hydrogels represent a promising carrier for IVD repair because of the mild gel formation allowing for encapsulation of viable cells and biological stimulation of cells by HA.1,2 Additionally, polyurethane (PU) foams can provide elasticity, which is of key importance for the restoration of IVD mechanical function, as the latter experiences repeated loading during the daily human activities. Finally, the addition of relevant cells may promote a faster restoration of the IVD function. Therefore, the aim of this study was to compare the single and combined effect of PU particles and HA hydrogel on the proliferation and differentiation of human IVD and mesenchymal stem cells. Materials and MethodsHuman bone marrow stromal cells (hMSCs) or human disk cells (hIVD cells) (P2-P4) were seeded on PU particles; after 12 hours, the seeded particles were suspended in either a 12% w/v thermoreversible hyaluronan-poly(N-isopropylacrylamide) hydrogel (HA-pNIPAM)2 or in a fibrinogen/thrombin solution.3 Each sample contained 50 µL hydrogel, 2.5 mg PU particles, and 2.5 × 105 cells. Controls included hMSC (or hIVD cells) seeded HA-pNIPAM, hMSC (or hIVD cells) seeded fibrin, and hMSC (or hIVD cells) seeded PU. After gelling for 1 hour at 37°C, samples were cultured in chondrogenic medium (DMEM high glucose, 1% ITS, 130 µM ascorbic acid, 100 nM dexamethasone and 10 ng/mL TGF-β1) for one week and analyzed for DNA (by Picogreen), sulfated glycosaminoglycan (GAG) (by 1,9 dimethylmethylene blue), mRNA expression, and histology (toluidine blue) at day 3 and 7. Three independent experiments were performed in triplicates for each group and cell type. After confirming the normality of data distribution and exclusion of the outliers, data were analyzed with a univariate general linear model and post hoc Bonferroni test (p < 0.05). ResultsIn the MSC cultures, a slight DNA increase was observed between day 3 and 7 for both PU/HA-pNIPAM and PU/fibrin samples. At day 7, the highest amounts of DNA were found for the PU/fibrin group, followed by fibrin and PU groups (Fig. 1a). The amount of DNA was higher in the PU group than PU/HA-pNIPAM and HA-pNIPAM, suggesting a lower cell proliferation in the presence of the thermoreversible hydrogel. A similar DNA profile was obtained when hIVD cells were used instead of hMSCs. A significantly increased amount of GAG was observed in hMSC-seeded PU/HA-pNIPAM samples (Fig.1B) and there was an increase of GAG from day 3 to day 7. All other groups had a much lower amount of glycosaminoglycans and there was only a slight increase of GAG from day 3 to day 7. The GAG retention (GAG in sample divided by the sum of GAG in the sample and GAG released in the medium) at day 7 was comprised between 50% and 80% for all groups. Disk cells produced significantly less GAG in the PU/HA-pNIPAM system compared to hMSC, indicating that MSCs were more responsive to the combination of these two biomaterials (Fig. 1B). Moreover, the GAG retention at day 7 was lower with human IVD cells (20–60%) compared to hMSCs in all groups. This suggests that an early and higher GAG accumulation is associated to a better GAG retention in the hydrogel. This finding may be related to the fact that while MSCs were obtained from patients undergoing various types of surgeries, IVD cells were obtained from (degenerate) disks of patients undergoing total IVD replacement. The gene expression profile confirmed MSC chondrogenic differentiation with a strong upregulation of collagen type II and, to a lower extent, of aggrecan and SOX9 (compared to cells collected on the day of seeding) without significant differences between the two gels. Histological sections revealed cell migration from PU particles to the hydrogels.Figure 1 Mean and standard deviation of DNA (a) and sulfated glycosaminoglycans (GAG) (b) in HA-pNIPAM and fibrin, with and without the addition of PU particles after 1 week of culture; * p < 0.05 relative to PU/Fibrin, o p < 0.05 relative to fibrin. ConclusionHuman disk cells from degenerating disks synthesized less GAG than hMSCs, indicating that MSCs may represent a suitable cell type for the biological repair of IVDs. A synergic effect for MSC differentiation was observed when a thermoreversible hyaluronan-based hydrogel was combined with polyurethane particles.I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declaredMortisen D, et al. Biomacromolecules 2010;11:1261–1272Peroglio M, et al. European Spine Journal Epub ahead of printMauth C, et al. European Cells and Material 2009;18:27–38