MicroRNA-140-3p alleviates intervertebral disc degeneration via KLF5/N-cadherin/MDM2/Slug axis.

MicroRNAs (miRNAs) are associated with healing or deteriorating degenerated intervertebral disc (IVD) tissues in spinal cord diseases, including intervertebral disc degeneration (IDD). IDD represents a chronic process of extracellular matrix destruction, but the relevant molecular mechanisms implicated in the regenerative effects of miRNAs are unclear. Here, we investigated the regenerative effects of microRNA-140 (miR-140-3p) in an IDD model induced by annulus needle puncture. Bioinformatics analysis was conducted to identify regulatory factors (KLF5/N-cadherin/MDM2/Slug) linked to miR-140-3p effects in IDD. Mesenchymal stem cells (MSCs) were extracted from degenerated IVD nucleus pulposus (NP), and the expression of miR-140-3p/KLF5/N-cadherin/MDM2/Slug was manipulated to explore their effects on cell proliferation, migration, apoptosis and differentiation. The results showed that miR-140-3p was under-expressed in the degenerated IVD NP, whereas its overexpression alleviated IDD. Mechanistic studies suggested that miR-140-3p targeted KLF5 expression, and high KLF5 expression impeded the migration and differentiation of MSCs. In degenerated IVD NP-derived MSCs, MiR-140-3p-mediated KLF5 downregulation simultaneously elevated N-cadherin expression and transcriptionally inhibited MDM2, thus upregulating Slug expression. The experimental data indicated that miR-140-3p enhanced the proliferation, migration and differentiation of degenerated IVD NP-derived MSCs and repressed their apoptosis. The in vivo validation experiment also demonstrated that miR-140-3p inhibited IDD by modulating the KLF5/N-cadherin/MDM2/Slug axis. Collectively, our results uncovered the regenerative role of miR-140-3p in IDD via regulation of the KLF5/N-cadherin/MDM2/Slug axis, which could be a potential therapeutic target for IDD.Abbreviations: miR-140-3p: microRNA-140-3p; IDD: intervertebral disc degeneration; MSCs: Mesenchymal stem cells; IVD: intervertebral disc; MSCs: mesenchymal stem cells; KLF5: Kruppel-like factor 5; MDM2: mouse double minute 2; NC: negative control; DHI: disc height index.

Simultaneous quantification of cefuroxime and clindamycin in human lumbar anulus fibrosus, nucleus pulposus and serum via UPLC-MS/MS.

BACKGROUND: This study aimed to develop a sensitive, accurate method for simultaneously quantifying cefuroxime and clindamycin in human serum, lumbar anulus fibrosus and nucleus pulposus. METHODS: Cefuroxime and clindamycin were quantified using ultra high-performance liquid chromatography-electrospray ionization tandem mass spectrometry in multiple-reaction-monitoring mode on a triple-quadrupole AB Qtrap 5500 system in positive ion mode. Internal standards were D3-cefuroxime and D3,13C-clindamycin. Samples were pretreated by precipitating total protein. RESULTS: The method showed high sensitivity and good linearity over broad calibration ranges from 100 to 100 000 ng/mL for cefuroxime and 10 to 10 000 ng/mL for clindamycin in serum, and from 10 to 10 000 ng/mL for cefuroxime and 1 to 1 000 ng/mL for clindamycin in lumbar nucleus pulposus. In all sample types, correlation coefficients were greater than 0.99, intra- and inter-day precision (relative standard deviation) was less than 15%, and accuracy (relative error) was within 14% for both analytes. This method was effective at quantifying penetration of cefuroxime and clindamycin in patients undergoing oblique lumbar interbody fusion surgery. CONCLUSIONS: A very sensitive, specific method for simultaneous detection of cefuroxime and clindamycin has been developed for human lumbar anulus fibrosus, nucleus pulposus and serum samples.

Simultaneous detection of multiple mRNAs and proteins in bovine IVD cells and tissue with single cell resolution.

OBJECTIVES: Interactions of cells with their neighbors and influences by the surrounding extracellular matrix (ECM) is reflected in a cells transcriptome and proteome. In tissues comprised of heterogeneous cell populations or cells depending on ECM signalling cues such as those of the intervertebral disc (IVD), this information is obscured or lost when cells are pooled for the commonly used transcript analysis by quantitative PCR or RNA sequencing. Instead, these cells require means to analyse RNA transcript and protein distribution at a single cell or subcellular level to identify different cell types and functions, without removing them from their surrounding signalling cues. RESULTS: We developed a simple, sequential protocol combining RNA is situ hybridisation (RISH) and immunohistochemistry (IHC) for the simultaneous analysis of multiple transcripts alongside proteins. This allows one to characterize heterogeneous cell populations at the single cell level in the natural cell environment and signalling context, both in vivo and in vitro. This protocol is demonstrated on cells of the bovine IVD, for transcripts and proteins involved in mechanotransduction, stemness and cell proliferation. CONCLUSIONS: A simple, sequential protocol combining RISH and IHC is presented that allows for simultaneous information on RNA transcripts and proteins to characterize cells within a heterogeneous cell population and complex signalling environments such as those of the IVD.

A possible injectable tissue engineered nucleus pulposus constructed with platelet-rich plasma and ADSCs in vitro.

BACKGROUND: Injectable tissue engineered nucleus pulposus is a new idea for minimally invasive repair of degenerative intervertebral disc. The platelet-rich plasma (PRP) and adipose-derived stromal cells (ADSCs) could be harvested from autologous tissue easily. PRP contains numerous autologous growth factors and has reticulate fibrous structure which may have the potential to make ADSCs differentiate into nucleus pulposus-like cells. The goal of this study was to explore the feasibility of constructing a possible injectable tissue engineered nucleus pulposus with PRP gel scaffold and ADSCs. METHODS: After identification with flow cytometry, the rabbit ADSCs were seeded into PRP gel and cultured in vitro. At the 2nd, 4th, and 8th week, the PRP gel/ADSCs complex was observed by macroscopy, histological staining, BrdU immunofluorescence, and scanning electron microscopy. The glycosaminoglycans (GAG) in the PRP gel/ADSCs complex were measured by safranin O staining with spectrophotometry. In PRP gel/ADSCs complex, gene expression of HIF-1α, aggrecan, type II collagen were tested by RT-PCR. The injectability of this complex was evaluated. RESULTS: Macroscopically, the complex was solidified into gel with smooth surface and good elasticity. The safranin O dye was almost no positive staining at 2nd week; however, the positive staining of extracellular matrix was enhanced obviously at 4th and 8th week. The HE staining and SEM demonstrated that the cells were well-distributed in the reticulate scaffold. BrdU immunofluorescence showed that ADSCs can survive and proliferate in PRP gel at each time points. The level of GAG at 4th week was higher than those at 2nd week (P < 0.05), and significant difference was also noted between 4th and 8th week (P < 0.05). HIF-1α, aggrecan, type II collagen gene expression at 4th week were much more than those at 2nd week (P < 0.05), and significant differences were also noted between 4th and 8th week (P < 0.05). The flow rate of complex was 0.287 mL/min when passed through the 19-gauge needle with the 100 mmHg injection pressure. CONCLUSIONS: Our preliminary findings suggest that the PRP gel make it possible for rabbit ADSCs differentiated into nucleus pulposus-like cells after coculture in vitro. According to the results, it is a better feasible method for construction of autologous injectable tissue engineered nucleus pulposus.

Thermosensitive injectable decellularized nucleus pulposus hydrogel as an ideal biomaterial for nucleus pulposus regeneration.

Extracellular matrix loss is one of the early manifestations of intervertebral disc degeneration. Stem cell-based tissue engineering creates an appropriate microenvironment for long term cell survival, promising for NP regeneration. We created a decellularized nucleus pulposus hydrogel (DNPH) from fresh bovine nucleus pulposus. Decellularization removed NP cells effectively, while highly preserving their structures and major biochemical components, such as glycosaminoglycan and collagen II. DNPH could be gelled as a uniform grid structure in situ at 37°C for 30 min. Adding adipose marrow-derived mesenchymal stem cells into the hydrogel for three-dimensional culture resulted in good bioactivity and biocompatibility in vitro. Meanwhile, NP-related gene expression significantly increased without the addition of exogenous biological factors. In summary, the thermosensitive and injectable hydrogel, which has low toxicity and inducible differentiation, could serve as a bio-scaffold, bio-carrier, and three-dimensional culture system. Therefore, DNPH has an outstanding potential for intervertebral disc regeneration.

Identification of significant gene biomarkers of low back pain caused by changes in the osmotic pressure of nucleus pulposus cells.

The incidence of intervertebral disc (IVD) degeneration disease, caused by changes in the osmotic pressure of nucleus pulposus (NP) cells, increases with age. In general, low back pain is associated with IVD degeneration. However, the mechanism and molecular target of low back pain have not been elucidated, and there are no data suggesting specific biomarkers of low back pain. Therefore, the research aims to identify and verify the significant gene biomarkers of low back pain. The differentially expressed genes (DEGs) were screened in the Gene Expression Omnibus (GEO) database, and the identification and analysis of significant gene biomarkers were also performed with various bioinformatics programs. A total of 120 patients with low back pain were recruited. Before surgery, the degree of pain was measured by the numeric rating scale (NRS), which enables comparison of the pain scores from individuals. After surgery, IVD tissues were obtained, and NP cells were isolated. The NP cells were cultured in two various osmotic media, including iso-osmotic media (293 mOsm/kg H2O) to account for the morbid environment of NP cells in IVD degeneration disease and hyper-osmotic media (450 mOsm/kg H2O) to account for the normal condition of NP cells in healthy individuals. The relative mRNA expression levels of CCL5, OPRL1, CXCL13, and SST were measured by quantitative real-time PCR in the in vitro analysis of the osmotic pressure experiments. Finally, correlation analysis and a neural network module were employed to explore the linkage between significant gene biomarkers and pain. A total of 371 DEGs were identified, including 128 downregulated genes and 243 upregulated genes. Furthermore, the four genes (CCL5, OPRL1, SST, and CXCL13) were identified as significant gene biomarkers of low back pain (P < 0.001) based on univariate linear regression, and CCL5 (odds ratio, 34.667; P = 0.003) and OPRL1 (odds ratio, 19.875; P < 0.001) were significantly related to low back pain through multivariate logistic regression. The expression of CCL5 and OPRL1 might be correlated with low back pain in patients with IVD degeneration disease caused by changes in the osmotic pressure of NP cells.

Thixotropic PVA hydrogel enclosing a hydrophilic PVP core as nucleus pulposus substitute.

A thixotropic polyvinyl alcohol (PVA) hydrogel, containing a hydrophilic poly-vinyl pyrrolidone (PVP) core, was obtained in order to develop a preformed 3D network able to maintain injectability. PVA was mixed with PVP in two different molar ratios (1:1 and 1:3) and chemically cross-linked using trisodium trimetaphosphate (STMP), which is able to react only with PVA component. A combination of Time of Flight- Secondary Ion Mass Spectrometry (ToF-SIMS), elemental analysis and UV spectroscopy permitted to determine both the cross-linking arm length and the crosslinking degree. Hydrogels were characterized in terms of swelling pressurization, rheological and mechanical behaviour. In particular, the viscoelastic behaviour of the hydrogel was analysed in shear and compression stress under dynamic conditions and compared with the performance of healthy human nucleus pulposus. In conclusion, the study demonstrated that the scaffold obtained mixing PVA and PVP in a molar ratio 1:1 can be considered a promising material to be utilised in the replacement of nucleus pulposus.

Ethanol-mediated compaction and cross-linking enhance mechanical properties and degradation resistance while maintaining cytocompatibility of a nucleus pulposus scaffold.

Intervertebral disc degeneration is a complex, cell-mediated process originating in the nucleus pulposus (NP) and is associated with extracellular matrix catabolism leading to disc height loss and impaired spine kinematics. Previously, we developed an acellular bovine NP (ABNP) for NP replacement that emulated human NP matrix composition and supported cell seeding; however, its mechanical properties were lower than those reported for human NP. To address this, we investigated ethanol-mediated compaction and cross-linking to enhance the ABNP's dynamic mechanical properties and degradation resistance while maintaining its cytocompatibility. First, volumetric and mechanical effects of compaction only were confirmed by evaluating scaffolds after various immersion times in buffered 28% ethanol. It was found that compaction reached equilibrium at ~30% compaction after 45 min, and dynamic mechanical properties significantly increased 2-6× after 120 min of submersion. This was incorporated into a cross-linking treatment, through which scaffolds were subjected to 120 min precompaction in buffered 28% ethanol prior to carbodiimide cross-linking. Their dynamic mechanical properties were evaluated before and after accelerated degradation by ADAMTS-5 or MMP-13. Cytocompatibility was determined by seeding stem cells onto scaffolds and evaluating viability through metabolic activity and fluorescent staining. Compacted and cross-linked scaffolds showed significant increases in DMA properties without detrimentally altering their cytocompatibility, and these mechanical gains were maintained following enzymatic exposure. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2488-2499, 2019.

Comparison of B0 versus B0 and B1 field inhomogeneity correction for glycosaminoglycan chemical exchange saturation transfer imaging.

PURPOSE: The study compares glycosaminoglycan chemical exchange saturation transfer (gagCEST) imaging of intervertebral discs corrected for solely B0 inhomogeneities or both B0 and B1 inhomogeneities. METHODS: Lumbar intervertebral discs of 20 volunteers were examined with T2-weighted and gagCEST imaging. Field inhomogeneity correction was performed with B0 correction only and with correction of both B0 and B1. GagCEST effects measured by the asymmetric magnetization transfer ratio (MTRasym) and signal-to-noise ratio (SNR) were compared between both methods. RESULTS: Significant higher MTRasym and SNR values were obtained in the nucleus pulposus using B0 and B1 correction compared with B0-corrected gagCEST. The GagCEST effect was significantly different in the nucleus pulposus compared with the annulus fibrosus for both methods. CONCLUSION: The B0 and B1 field inhomogeneity correction method leads to an improved quality of gagCEST imaging in IVDs compared with only B0 correction.

Proteomic Analysis of Nucleus Pulposus Cell-derived Extracellular Matrix Niche and Its Effect on Phenotypic Alteration of Dermal Fibroblasts.

Reconstituting biomimetic matrix niche in vitro and culturing cells at the cell niche interface is necessary to understand the effect and function of the specific matrix niche. Here we attempted to reconstitute a biomimetic extracellular matrix (ECM) niche by culturing nucleus pulposus cells (NPCs) in a collagen microsphere system previously established and allowing them to remodel the template matrix. The reconstituted NPC-derived complex ECM was obtained after decellularization and the composition of such niche was evaluated by proteomic analysis. Results showed that a complex acellular matrix niche consisting of ECM proteins and cytoskeletal proteins by comparing with the template collagen matrix starting material. In order to study the significance of the NPC-derived matrix niche, dermal fibroblasts were repopulated in such niche and the phenotypes of these cells were changed, gene expression of collagen type II and CA12 increased significantly. A biomimetic NPC-derived cell niche consisting of complex ECM can be reconstituted in vitro, and repopulating such matrix niche with fibroblasts resulted in changes in phenotypic markers. This work reports a 3D in vitro model to study cell niche factors, contributing to future understanding of cellular interactions at the cell-niche interface and rationalized scaffold design for tissue engineering.

Reverse Reconstruction and Bioprinting of Bacterial Cellulose-Based Functional Total Intervertebral Disc for Therapeutic Implantation.

The degradation of intervertebral discs (IVD), a typical hierarchical structured tissue, causes serious neck and back pain. The current methods cannot fully reconstitute the unique structure and function of native IVD. In this study, by reverse reconstruction of the structure of native IVD and bioprinting bacterial cellulose (BC) nanofibers with a high-throughput optimized micropattern screening microchip, a total IVD is created that contained type II collagen-based nucleus pulposus (NP) and hierarchically organized and micropatterned BC-based annulus fibrosus (AF), mimicking native IVD tissue. The artificial NP contains rat NP cells, whereas the AF contains concentrically arranged BC layers with aligned micropatterns and attached AF cells in +/-30° alternate directions between adjacent layers. Long-term (3 months) implantation experiments on rats demonstrate the excellent structural (shape maintenance, hydration, tissue integration) and functional (mechanical support and flexibility) performance of the artificial IVD. This study provides a novel strategy for creating highly sophisticated artificial tissues.

Meniscus, articular cartilage and nucleus pulposus: a comparative review of cartilage-like tissues in anatomy, development and function.

The degradation of cartilage in the human body is impacted by aging, disease, genetic predisposition and continued insults resulting from daily activity. The burden of cartilage defects (osteoarthritis, rheumatoid arthritis, intervertebral disc damage, knee replacement surgeries, etc.) is daunting in light of substantial economic and social stresses. This review strives to broaden the scope of regenerative medicine and tissue engineering approaches used for cartilage repair by comparing and contrasting the anatomical and functional nature of the meniscus, articular cartilage (AC) and nucleus pulposus (NP). Many review papers have provided detailed evaluations of these cartilages and cartilage-like tissues individually but none have comprehensively examined the parallels and inconsistencies in signaling, genetic expression and extracellular matrix composition between tissues. For the first time, this review outlines the importance of understanding these three tissues as unique entities, providing a comparative analysis of anatomy, ultrastructure, biochemistry and function for each tissue. This novel approach highlights the similarities and differences between tissues, progressing research toward an understanding of what defines each tissue as distinctive. The goal of this paper is to provide researchers with the fundamental knowledge to correctly engineer the meniscus, AC and NP without inadvertently developing the wrong tissue function or biochemistry.

The intervertebral disc contains intrinsic circadian clocks that are regulated by age and cytokines and linked to degeneration.

OBJECTIVES: The circadian clocks are internal timing mechanisms that drive ∼24-hour rhythms in a tissue-specific manner. Many aspects of the physiology of the intervertebral disc (IVD) show clear diurnal rhythms. However, it is unknown whether IVD tissue contains functional circadian clocks and if so, how their dysregulation is implicated in IVD degeneration. METHODS: Clock gene dynamics in ex vivo IVD explants (from PER2:: luciferase (LUC) reporter mice) and human disc cells (transduced with lentivirus containing Per2::luc reporters) were monitored in real time by bioluminescence photon counting and imaging. Temporal gene expression changes were studied by RNAseq and quantitative reverse transcription (qRT)-PCR. IVD pathology was evaluated by histology in a mouse model with tissue-specific deletion of the core clock gene Bmal1. RESULTS: Here we show the existence of the circadian rhythm in mouse IVD tissue and human disc cells. This rhythm is dampened with ageing in mice and can be abolished by treatment with interleukin-1ß but not tumour necrosis factor α. Time-series RNAseq revealed 607 genes with 24-hour patterns of expression representing several essential pathways in IVD physiology. Mice with conditional knockout of Bmal1 in their disc cells demonstrated age-related degeneration of IVDs. CONCLUSIONS: We have established autonomous circadian clocks in mouse and human IVD cells which respond to age and cytokines, and control key pathways involved in the homeostasis of IVDs. Genetic disruption to the mouse IVD molecular clock predisposes to IVD degeneration. These results support the concept that disruptions to circadian rhythms may be a risk factor for degenerative IVD disease and low back pain.

Interleukin-17 upregulates vascular endothelial growth factor by activating the JAK/STAT pathway in nucleus pulposus cells.

OBJECTIVES: Intervertebral disc (IVD) related diseases and age-related IVD degeneration are responsible for significant morbidity. Inflammatory mediators and pro-inflammatory cytokines, including interleukin (IL)-17, show elevated expression in degenerated disc tissue. IL-17 is reported to transduce signals across the cell membrane predominantly via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signal transduction pathway, leading to transcriptional activation of target genes. METHODS: In this study, we investigated whether the JAK/STAT pathway plays a role in IL-17-mediated signaling in the nucleus pulposus (NP) cells of IVDs. Vascular endothelial growth factor (VEGF) and IL-17 were found to be highly expressed in human degenerated NP tissue. In isolated rat NP cells, IL-17-induced VEGF expression in a time- and dose-dependent manner. Rat NP cells were co-transfected with VEGF promoter plasmid along with constitutively active STAT1, STAT3 or JAK2 plasmid. VEGF promoter activity was found to be increased by STAT1, STAT3 and JAK2 in IL-17-treated cells. Transfection of cultured rat NP cells with STAT1 or STAT3 lentiviral short hairpin RNAs or treatment with the JAK2 inhibitor AG490 significantly reduced IL-17-stimulated VEGF expression. CONCLUSIONS: IL-17 upregulated VEGF expression in rat NP cells mediated by the JAK/STAT pathway, and elevated levels of IL-17 and VEGF are present in human degenerated NP tissue. These findings provide new insight into the pathology of IVD degeneration.

Biomimetic nucleus pulposus scaffold created from bovine caudal intervertebral disc tissue utilizing an optimal decellularization procedure.

Intervertebral disc (IVD) degeneration (IDD) and herniation (IDH) can result in low back pain and impart significant socioeconomic burden. These pathologies involve detrimental alteration to the nucleus pulposus (NP) either via biochemical degradation or extrusion from the IVD, respectively. Thus, engineering living NP tissue utilizing biomaterial scaffolds that recapitulate native NP microarchitecture, biochemistry, mechanical properties, and which support cell viability represents an approach to aiding patients with IDD and IDH. To date, an ideal biomaterial to support NP regeneration has yet to be developed; however, one promising approach to generating biomimetic materials is to employ the decellularization (decell) of xenogeneic NP tissue to remove host DNA while maintaining critical native extracellular matrix (ECM) components. Herein, 13 different procedures were evaluated in an attempt to decell bovine caudal IVD NP tissue. An optimal method was identified which was confirmed to effectively remove bovine DNA, while maintaining physiologically relevant amounts of glycosaminoglycan (GAG) and type II collagen. Unconfined static and dynamic compressive mechanical properties of scaffolds approached values reported for human NP and viability of human amniotic stem cells (hAMSCs) was maintained on noncrosslinked and EDC/NHS treated scaffolds for up to 14 days in culture. Taken together, NP tissue obtained from bovine caudal IVDs can be successfully decelled in order to generate a biomimetic scaffold for NP tissue regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3093-3106, 2016.

Inhibition of microRNA-34a prevents IL-1ß-induced extracellular matrix degradation in nucleus pulposus by increasing GDF5 expression.

Accumulating evidence indicates that miRNAs, a class of small non-coding RNAs, are implicated in the pathogenesis of various diseases such as cancer and intervertebral disc degeneration. The aim of this study was to investigate the expression and the biological function of microRNA-34a in intervertebral disc degeneration. In this study, microRNA-34a expression was assessed in nucleus pulposus specimens and in IL-1ß-stimulated nucleus pulposus cells by real-time polymerase chain reaction. microRNA-34a functions were investigated by using gain and loss of function experiments in nucleus pulposus cells and a dual luciferase reporter assay in 293T cells. microRNA-34a was dramatically up-regulated in degenerative nucleus pulposus tissues and in IL-1ß-stimulated nucleus pulposus cells when compared with controls. Furthermore, growth differentiation factor 5 was identified as a target of microRNA-34a. Aberrant expression of microRNA-34a inhibited growth differentiation factor 5 expression by direct binding to its 3'-untranslated region. This inhibition was abolished by mutation of the microRNA-34a binding sites. In addition, microRNA-34a silencing reversed IL-1ß-induced decrease in type II collagen and aggrecan expression in nucleus pulposus cells. This effect was substantially suppressed by growth differentiation factor 5 silencing. Our results suggested that microRNA-34a inhibition prevents IL-1ß-induced extracellular matrix degradation in human nucleus pulposus by increasing growth differentiation factor 5 expression. microRNA-34a inhibition may be a novel molecular target for intervertebral disc degeneration treatment through the prevention of nucleus pulposus extracellular matrix degradation.