Facile fabrication of nano-bioactive glass functionalized blended hydrogel with nucleus pulposus-derived MSCs to improve regeneration potential in treatment of disc degeneration by in vivo rat model

Background: Intervertebral disc (IVD) degeneration is a common degenerative disease that can lead to collapse or herniation of the nucleus pulposus (NP) and result in radiculopathy in patients.Methods: NP tissue and cells were isolated from patients and mice, and the expression profile of cortistatin (CST) was analysed. In addition, ageing of the NP was compared between 6-month-old WT and CST-knockout (CST-/-) mice. Furthermore, NP tissues and cells were cultured to validate the role of CST in TNF-α-induced IVD degeneration. Moreover, in vitro and in vivo experiments were performed to identify the potential role of CST in mitochondrial dysfunction, mitochondrial ROS generation and activation of the NLRP3 inflammasome during IVD degeneration. In addition, NF-κB signalling pathway activity was tested in NP tissues and cells from CST-/- mice.Results: The expression of CST in NP cells was diminished in the ageing- and TNF-α-induced IVD degeneration process. In addition, compared with WT mice, aged CST-/- mice displayed accelerated metabolic imbalance and enhanced apoptosis, and these mice showed a disorganized NP tissue structure. Moreover, TNF-α-mediated catabolism and apoptosis were alleviated by exogenous CST treatment. Furthermore, CST inhibited mitochondrial dysfunction in NP cells through IVD degeneration and suppressed activation of the NLRP3 inflammasome. In vitro and ex vivo experiments indicated that increased NF-κB pathway activity might have been associated with the IVD degeneration observed in CST-/- mice.Conclusion: This study suggests the role of CST in mitochondrial ROS and activation of the NLRP3 inflammasome in IVD degeneration, which might shed light on therapeutic targets for IVD degeneration.

Intervertebral disc generation tissue engineering treatment platform: gelatin-methacryloyl hydrogel composite system.

Accumulating evidence demonstrates that estrogen receptor α (ERα) and microRNAs (miRNAs) play crucial roles in intervertebral disc degeneration (IDD). However, the specific miRNA that related with ERα during IDD development remains unknown. Therefore, we aimed to explore the role of ERα-related miRNA in the IDD model. Nucleus pulposus (NP) cells were isolated from IDD patients. ERα-related miRNAs were selected and verified in NP tissues from IDD patients using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Also, the related cytokine mRNA levels were detected by qRT-PCR. Protein levels were determined by Western blot. The concentrations of inflammatory cytokines in culture supernatants were detected by enzyme-linked immunosorbent assay. MiR-203-3p was found to be upregulated in NP tissues of high-grade IDD patients compared with low-grade IDD patients, and negatively associated with ERα expression. MiR-203-3p directly targeted ERα in NP cells of IDD patients. After lipopolysaccharides (LPS) stimulation, miR-203-3p expression increased, while ERα expression decreased in NP cells. MiR-203-3p inhibition suppressed the effect of LPS on ERα expression and IDD related genes, while ERα downregulation rescued the effect of LPS. In conclusion, suppression the expression of miR-203-3p could inhibit LPS-induced human intervertebral disc inflammation and degeneration through upregulating ERα.

Decision letter: DIPPER, a spatiotemporal proteomics atlas of human intervertebral discs for exploring ageing and degeneration dynamics

The spatiotemporal proteome of the intervertebral disc (IVD) underpins its integrity and function. We present DIPPER, a deep and comprehensive IVD proteomic resource comprising 94 genome-wide profiles from 17 individuals. To begin with, protein modules defining key directional trends spanning the lateral and anteroposterior axes were derived from high-resolution spatial proteomes of intact young cadaveric lumbar IVDs. They revealed novel region-specific profiles of regulatory activities and displayed potential paths of deconstruction in the level- and location-matched aged cadaveric discs. Machine learning methods predicted a ‘hydration matrisome’ that connects extracellular matrix with MRI intensity. Importantly, the static proteome used as point-references can be integrated with dynamic proteome (SILAC/degradome) and transcriptome data from multiple clinical samples, enhancing robustness and clinical relevance. The data, findings, and methodology, available on a web interface (http://www.sbms.hku.hk/dclab/DIPPER/), will be valuable references in the field of IVD biology and proteomic analytics.

Eugenol attenuates intervertebral disc degeneration by inhibiting nucleus pulposus cell ferroptosis via the Nrf2/GPX4 pathway.

Knowledge of rates of protein turnover is important for a quantitative understanding of tissue synthesis and catabolism. In this work, we have used the racemization of aspartic acid as a marker for the turnover of collagen obtained from healthy and pathological human intervertebral disc matrices. We measured the ratio of the d- and l-isomers in collagen extracted from these tissues as a function of age between 16 and 77 years. For collagen taken from healthy discs, the fractional increase of d-Asp was found to be 6.74 x 10(-4)/year; for degenerate discs, the corresponding rate was 5.18 x 10(-4)/year. Using the racemization rate found previously for the stable population of collagen molecules in dentin, we found that the rate of collagen turnover (k(T)) in discs is not constant but rather a decreasing function of age. The average turnover rate in normal disc between the ages of 20 and 40 is 0.00728 +/- 0.00275/year, and that between the ages of 50 and 80 is 0.00323 +/- 0.000947/year, which correspond to average half-lives of 95 and 215 years, respectively. Turnover of collagen from degenerate discs may be more rapid than that found for normal discs; however, statistical analysis leaves this point uncertain. The finding of a similar correlation between the accumulation of d-Asp and that of pentosidine for three normal collagenous tissues further supports the idea that the accumulation of pentosidine in a particular tissue can, along with the racemization of aspartic acid, be used as a reliable measure of protein turnover.

Intervertebral disc degeneration (IDD) is a key element resulting in low back pain, but the mechanisms underlying IDD remain largely unknown. The purpose of the study was to investigate the influence of microRNA-155-3p (miR-155-3p) on proliferation and autophagy of nucleus pulposus (NP) cells in IDD with the involvement of hypoxia-inducible factor 1 α (HIF1α)/histone lysine demethylase 3A (KDM3A) axis. IDD NP tissues of patients with lumbar disc herniation and traumatic intervertebral disc NP tissues from patients with traumatic lumbar fracture were collected. Apoptosis in NP tissues was observed, and autophagy marker proteins in NP tissues were detected. NP cells in IDD were transfected with miR-155-3p mimic or KDM3A-siRNA to explore their roles in cell proliferation, autophagy and apoptosis. MiR-155-3p, KDM3A and HIF1α expression in NP tissues and cells were detected. Decreased miR-155-3p, and elevated HIF1α and KDM3A were presented in NP tissues and cells of IDD. Elevated miR-155-3p or silenced KDM3A promoted the proliferation and autophagy, and inhibited the apoptosis of NP cells of IDD. Moreover, elevated miR-155-3p decreased KDM3A and HIF1α expression, while silenced KDM3A decreased HIF1α expression in NP cells with IDD. The study concludes that up-regulated miR-155-3p or silenced KDM3A promotes the proliferation, autophagy, and restrains the apoptosis of NP cells of IDD via inhibition of HIF1α, which may be a promising approach for the treatment of IDD.

Aim: Intervertebral disc (IVD) degeneration (IDD) is one of the main causes for spinal degenerative diseases, such as disk herniation, spinal canal stenosis, and spinal deformities. Growing evidence has highlighted the contribution of oxidative stress in pathogenesis of IDD, and antioxidant treatment is thus considered to be a promising therapeutic strategy for IDD. The aim of this study was to investigate whether N-tert-butyl-α-phenylnitrone (PBN), a free radical scavenger, could attenuate the pathological changes of IDD by alleviating oxidative stress. Materials and Methods: Nucleus pulposus (NP) cells were isolated from rabbit lumbar disks. MTT assay, real-time PCR and western blotting were employed to evaluate the effects of PBN on oxidative damages induced by 2,2ʹ-azobis (2-amidinopropane) dihydrochloride (AAPH) in NP cells. Results: AAPH induced oxidative stress and the subsequent degenerative changes in NP cells via the ERK/MAPK pathway. On the contrary, the oxidative stress induced by AAPH was significantly ameliorated by PBN. Moreover, PBN also attenuated AAPH-induced expression of matrix degradation proteases and apoptosis. PBN suppresses AAPH-induced activation of ERK/MAPK pathway, which may be the underlying mechanism for the protective effects of PBN. Conclusions: Our study for the first time identified a novel role and mechanism for PBN in protecting the IVD against oxidative stress, matrix catabolism and apoptosis, which may have implications for its further application in combating IVD degenerative diseases. Abbreviations: AAPH: 2,2ʹ-azobis(2-methylpropanimidamidine) dihydrochloride; ADAMTS: a disintegrin and metalloproteinase with thrombospondin motifs; AF: annulus fibrosus; CEP: cartilage endplate; DCF: 2ʹ7’-dichlorofluorescein; IDD: intervertebral disc degeneration; IVD: intervertebral disc; LPS: lipopolysaccharide; MMP: matrix metalloproteinase; MTT: methyl-thiazolyl-tetrazolium; NP: nucleus pulposus; PBN: N-tert-butyl-alfa-phenylnitrone; PGs: proteoglycans; ROS: reactive oxygen species; SDS: sodium dodecyl sulfate

PPAR-γ agonist pioglitazone protects against IL-17 induced intervertebral disc inflammation and degeneration via suppression of NF-κB signaling pathway

Intervertebral disc (IVD) degeneration and associated spinal disorders are leading sources of morbidity, and they can be responsible for chronic low back pain. Treatments for degenerative disc diseases continue to be a challenge. Intensive research is now focusing on promoting regeneration of degenerated discs by stimulating production of the disc matrix. Link protein N-terminal peptide (LPP) is a proteolytic fragment of link protein, an important cross-linker and stabilizer of the major structural components of cartilage, aggrecan and hyaluronan. In this study we investigated LPP action in rabbit primary intervertebral disc cells cultured ex vivo in a three-dimensional alginate matrix. Our data reveal that LPP promotes disc matrix production, which was evidenced by increased expression of the chondrocyte-specific transcription factor SOX9 and the extracellular matrix macromolecules aggrecan and collagen II. Using colocalization and pulldown studies we further document a noggin-insensitive direct peptide-protein association between LPP and BMP-RII. This association mediated Smad signaling that converges on BMP genes leading to expression of BMP-4 and BMP-7. Furthermore, through a cell-autonomous loop BMP-4 and BMP-7 intensified Smad1/5 signaling though a feedforward circuit involving BMP-RI, ultimately promoting expression of SOX9 and downstream aggrecan and collagen II genes. Our data define a complex regulatory signaling cascade initiated by LPP and suggest that LPP may be a useful therapeutic substitute for direct BMP administration to treat IVD degeneration and to ameliorate IVD-associated chronic low back pain.

The present study aimed to explore the underlying mechanisms of bone morphogenetic protein 2 (BMP2) in alleviating intervertebral disc degeneration (IDD). A rat puncture IDD model was constructed, and the rats were randomly divided into six groups: Control; IDD (model); IDD+PBS [containing 1010 adeno‑associated virus serotype 2 (AAV)]; and IDD + AAV2‑BMP2 (106, 108 and 1010). IL‑1β was used to treat primary nucleus pulposus (NP) cells to mimic IDD in vitro. The effects of BMP2 in IDD were determined by magnetic resonance imaging (MRI), hematoxylin and eosin staining and Alcian Blue staining in vivo. The levels of collagen II, aggrecan, transcription factor SOX9 (SOX9) and matrix metalloproteinase 13 (MMP‑13) were examined using western blot analysis and reverse transcription quantitative polymerase chain reaction (RT‑qPCR) in NP tissues and cells. The expression of C‑telopeptide of type II collagen (CTX‑II) in the sera or cell supernatants was determined by ELISA. In addition, the levels of phosphorylation of phosphoinositide 3‑kinase (PI3K) and protein kinase B (Akt), and the levels of apoptosis‑associated proteins and apoptosis ratio of NP cells were also determined by western blot analysis and flow cytometry, respectively. LY29400, an inhibitor of PI3K, was used to additionally confirm the signal pathway mechanism of BMP2 treatment in IDD. BMP2 significantly extended the interval between discs and alleviated the fibrous ring rupture and the decrease in the levels of glycoproteins in IDD rats, as determined by MRI and histological staining. Additionally, BMP2 treatment significantly upregulated the levels of collagen II, aggrecan and SOX9, but downregulated the levels of MMP‑13 and CTX‑II in IDD rats and NP cells in a dose‑dependent manner. Concurrently, recombinant human (rh)BMP2 pretreatment also significantly decreased the apoptosis ratio of interleukin (IL)‑1β‑treated NP cells via downregulating the level of cleaved caspase‑3 and upregulating the level of uncleaved poly (adenosine 5'‑diphosphate‑ribose) polymerase. It was demonstrated that rhBMP2 also significantly decreased the inflammatory response in NP tissues and cells, based on levels of IL‑6, TNF‑α and IL‑10. In addition, rhBMP2 inhibited cell apoptosis via upregulating the phosphorylation levels of the PI3K/Akt signaling pathway, and LY29400 pretreatment inhibited the effects of BMP2 in IL‑1β treated NP cells. BMP2 alleviated IDD via the PI3K/Akt signaling pathway by inhibiting NP cell apoptosis and decreasing the levels of matrix proteins.

Targeting oxidative stress with amobarbital to prevent intervertebral disc degeneration: Part I. in vitro and ex vivo studies

P53 is an apoptosis marker which is involved in determining nucleus pulposus (NP) cell fate. Little is known about P53 interaction with N-Myc downstream-regulated gene 2 (NDRG2) in intervertebral disc degeneration (IVDD). Here, we studied the role of the P53-NDRG2 axis in IVDD. We found that NDRG2 was expressed in NP tissue obtained from patients with IVDD. The level of NDRG2 was positively related to the severity of IVDD, as determined by Pfirrmann grading. Subsequently, we overexpressed NDRG2 in human NP cells by adenoviral transfection and studied the effects of increased levels of NDRG2 on the viability and apoptosis of these cells. NDRG2 overexpression induced NP cell apoptosis and reduced viability in NP cells obtained from patient with IVDD. We also found that the level of P53 was elevated in NP cells from patients with IVDD and treatment with exogenous P53 upregulated NDRG2 in NP cells. Last, IVDD model was established in P53 knockout mice and the pathological changes in the intervertebral discs and NDRG2 expression were examined. P53 knockout can reduce the damage of NP tissues after IVDD surgery to some extent. Restoration of NDRG2 antagonized the effect of P53 knockout on IVDD. Collectively, this study suggests that elevated P53 in NP cells stimulates apoptosis of the cells by upregulating NDRG2 expression, thereby exacerbating IVDD.

MINK1 deficiency stimulates nucleus pulposus cell pyroptosis and exacerbates intervertebral disc degeneration