Disc cell therapies: critical issues

IntroductionLow back pain is a debilitating disorder affecting a multitude of people and is currently ranked sixth among costliest treatable chronic illnesses.1 Low back pain is often attributed to...

Background Abnormal Smad7 expression can lead to apoptosis in different cell types. Previously, we found high expression of Smad7 in rat degenerative discs. However, the exact role of Smad7 in the apoptosis of disc cells and the possible underlying mechanism remain unclear. Methods Degenerative and nondegenerative human lumbar intervertebral discs were collected from patients during operation. The expressions of SMAD7 mRNA and protein in the different components of these discs were measured with real-time PCR and Western blotting, respectively. Annulus fibrosus (AF) cells were isolated and cultivated from the discs of young healthy rats. Smad7 in the AF cells was overexpressed with adenovirus and knocked down with siRNA. IL-1β was used to induce apoptosis in the AF cells. Loss-and-gain cell function experiments were performed to show the effect of Smad7 on the apoptosis of AF cells. The function recovery experiments were performed to verify whether Smad7 regulates the apoptosis of AF cells through the mitochondria-mediated pathway. Results Both the mRNA and protein expressions of Smad7 were significantly higher in the different components of human degenerative discs than in those of the nondegenerative discs. IL-1β stimulated apoptosis while upregulating the Smad7 expression in the AF cells in vitro. Overexpression of Smad7 in AF cells exaggerated the IL-1β-induced apoptosis in the cells while knockdown of Smad7 expression suppressed this apoptosis. With the exaggerated apoptosis in the AF cells with Smad7 overexpression, both active cleaved caspase-3 and cleaved caspase-9, the ratio of Bax/Bcl-2, and Cyt-c increased significantly. However, the inhibitor of caspase-9, Z-LEHD-FMK, significantly diminished the apoptosis in these cells. ConclusionSmad7 is highly expressed in human degenerative discs and participates in IL-1β-induced apoptosis of rat AF cells via the mitochondria pathway. Smad7 may be a potential target for the prevention and treatment of degenerative disc disease.

Introduction Back pain is a fairly common problem which affects a large portion of the population across all ages and has an impact on quality of life. Intervertebral disk (IVD) degeneration is the single most common implicated cause of back pain. Presently, there is no medical treatment or therapeutic agent to address this problem and surgery is the only offered option. Link-N peptide represents the 16 amino acid sequence from the N-terminus of the link protein that stabilizes the proteoglycan aggregates present in cartilage and disk. Link-N peptide is released from the link protein as a result of proteolysis, and has been suggested to play a role in matrix homeostasis by promoting new matrix synthesis. We evaluated its regenerative potential in intact human IVD. Materials and Methods Lumbar IVDs were obtained through organ donations via Transplant Quebec. Disks from seven individuals, five disks per spine, were harvested. Cells were isolated from nucleus pulposus (NP) and inner annulus fibrosus regions of the disks. Single cells were beaded in 1.2% alginate and cultured in DMEM containing 1 g/L glucose and 10% FBS. Alginate beads were exposed to 10–10000 ng/mL Link-N peptide for 48 hours. Intact disks were prepared for organ culture by parallel cuts through the adjacent vertebral bodies close to the end plates, and the remaining bone and the calcified part of the cartilage endplates were removed using a high-speed bone burr. Disks were maintained and cultured with no external load applied in DMEM containing 1 g/L glucose and supplemented with 1% FBS. Link-N was conjugated with 5-TAMRA dye then injected into the center of the disk. The distribution of Link-N in the medium and within the disk was studied to determine whether Link-N is retained in the disk. Disks from adjacent levels were matched for the degree of degeneration and were injected in their NP region with 50µCi 35SO4 along with 0.1 mg or 1 mg of Link-N in 100 µL of medium per disk and harvested after 48 hours. Sustained effect of Link-N was evaluated by injecting the disk with Link-N and injecting 35SO4 1 week later. Proteoglycan synthesis was evaluated by measuring 35SO4 incorporation. Results When human lumbar disk cells from NP and iAF regions beaded in alginate were exposed to Link-N peptide for 48 hours, proteoglycan synthesis was observed to increase in a dose-dependent manner with the maximal response at 1000 ng/mL Link-N. Fluorescently labelled Link-N peptide was injected into the disks to determine if Link-N is retained in the disks matrix or freely diffuses throughout the tissue and equilibrates with surrounding medium. Samples were taken continually from the surrounding medium and from the disk tissue at the termination of the experiment. Fluorescent-Link-N was detectable in the medium at 24 hours and reached equilibrium after 48 hours. The fluorescent peptide was found in the NP and NP/iAF junction but not in the remaining AF. Thus loss of Link-N appears to occur by diffusion through the endplates (Fig. 1). Cell viability was maintained in the NP, at >96%, after injection of 1 mg of Link-N/disk. Disks injected with Link-N showed increased proteoglycan synthesis in the NP and iAF compared to adjacent level control disks matched for grade of degeneration. To evaluate the duration of the effect, disks were injected with 35SO4 1 week after the injection of Link-N. Proteoglycan synthesis remained elevated in Link-N injected disks compared to adjacent level control disks suggesting a sustained effect. Conclusion Link-N peptide has previously been shown to promote matrix protein synthesis by bovine disk cells in monolayer and pellet cultures. In this work, we show that Link-N can promote proteoglycan synthesis not only in human disk cells cultured in 3D constructs, but also in intact adult human disks where the cells are in their native environment. Recently, an increase in disk height measured by MRI was shown in an in vivo rabbit model, where degenerated disks were injected with Link-N. If a similar restoration of disk function could be achieved in the human disks, then Link-N could be a promising candidate for biologically induced disk repair, and could provide an alternative to surgical intervention for early stage disk degeneration. Link-N has a significant cost advantage over growth factors, such as BMP7, TGFβ and, GDF5. Based on prior in vivo studies in the rabbit, Link-N is over 100 times less expensive than recombinant growth factors that have a similar repair response. Thus, Link-N peptide injection could be both effective and cost-efficient therapy for retarding the ongoing degenerative process in early stage disk disease and help relieve back pain. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Intervertebral disc (IVD) degeneration is a major cause of low back pain. The transcription factor c-Fos/Activator Protein-1 (AP-1) controls the expression of inflammatory cytokines and matrix metalloproteinases (MMPs) that contribute to the pathogenesis IVD degeneration. We investigated the effects of inhibition of c-Fos/AP-1 on IVD degeneration and associated pain. A selective inhibitor, T-5224, significantly suppressed the interleukin-1β-induced up-regulation of Mmp-3, Mmp-13 and Adamts-5 transcription in human nucleus pulposus cells and in a mouse explant culture model of IVD degeneration. We used a tail disc percutaneous needle puncture method to further assess the effects of oral administration of T-5224 on IVD degeneration. Analysis of disc height, T2-magnetic resonance imaging (MRI) findings, and histology revealed that IVD degeneration was significantly mitigated by T-5224. Further, oral administration of T-5224 ameliorated pain as indicated by the extended tail-flick latency in response to heat stimulation of rats with needle-puncture-induced IVD degeneration. These findings suggest that the inhibition of c-Fos/AP-1 prevents disc degeneration and its associated pain and that T-5224 may serve as a drug for the prevention of IVD degeneration.

Patterns of lumbar disc degeneration are different in degenerative disc disease and disc prolapse magnetic resonance imaging analysis of 224 patients

Back pain is a leading cause of global disability and is strongly associated with intervertebral disc (IVD) degeneration (IDD). Hallmarks of IDD include progressive cell loss and matrix degradation. The Akt signaling pathway regulates cellularity and matrix production in IVDs and its inactivation is known to contribute to a catabolic shift and increased cell loss via apoptosis. The PH domain leucine-rich repeat protein phosphatase (Phlpp1) directly regulates Akt signaling and therefore may play a role in regulating IDD, yet this has not been investigated. The aim of this study was to investigate if Phlpp1 has a role in Akt dysregulation during IDD. In human IVDs, Phlpp1 expression was positively correlated with IDD and the apoptosis marker cleaved Caspase-3, suggesting a key role of Phlpp1 in the progression of IDD. In mice, 3 days after IVD needle puncture injury, Phlpp1 knockout (KO) promoted Akt phosphorylation and cell proliferation, with less apoptosis. At 2 and 8 months after injury, Phlpp1 deficiency also had protective effects on IVD cellularity, matrix production, and collagen structure as measured with histological and immunohistochemical analyses. Specifically, Phlpp1-deletion resulted in enhanced nucleus pulposus matrix production and more chondrocytic cells at 2 months, and increased IVD height, nucleus pulposus cellularity, and extracellular matrix deposition 8 months after injury. In conclusion, Phlpp1 has a role in limiting cell survival and matrix degradation in IDD and research targeting its suppression could identify a potential therapeutic target for IDD.

This study aimed to comprehensively characterize the relationship between glycated hemoglobin (HbA1c) and intervertebral disc degeneration (IVDD) by integrating population-level epidemiological profiling, Mendelian randomization (MR) analysis, multi-omics data, and single-cell transcriptomics. The objectives included clarifying the causal effect of HbA1c on IVDD, identifying key HbA1c-related genes, elucidating their cellular and molecular roles in disc degeneration, and exploring potential therapeutic targets. Global Burden of Disease (GBD) 2021 database was used to evaluate the incidence rate of diabetes mellitus (DM) and low back pain (LBP) across the entire global population. To assess the causal effect of HbA1c on IVDD, MR approache was conducted using summary statistics from large genome-wide association studies. Subsequently, integrating cis-eQTL information, bulk RNA sequencing, and single-cell RNA sequencing were performed to identify HbA1c-related genes involved in disc degeneration, and explore their roles along pseudotime differentiation trajectories. Potential associations between hub genes and IVDD, as well as candidate therapeutic compounds targeting these genes, were further evaluated through database screening and molecular docking. The findings from GBD indicated a correlation between diabetes and low back pain in both 1990 and 2021. The results of MR analysis revealed a causal relationship between HbA1c levels and IVDD. After integrated analysis of the eQTLGen database and RNA-seq data, we identified five hub HRGs, including STAT5A, CDKN1C, TRPS1, NME4, and OASL, which have been implicated in multiple biological processes and pathways associated with IVDD. Moreover, the scRNA-seq analysis showed that hub HRGs were associated with specific cell subpopulations in human nucleus pulposus (NP) tissues, such as inflamed chondrocytes, fibrochondrocytes, chondrocytes, and calcifying chondrocytes. Additionally, hub HbA1c related genes (HRGs) have been found to be associated with several DDDs such as back pain, osteoporosis, and spinal stenosis. Meanwhile, 12 drugs, including resveratrol, dexamethasone, and simvastatin, were identified that have the potential to target hub HRGs. This study revealed a causal effect of HbA1c on IVDD and identified five key HbA1c-related genes (STAT5A, CDKN1C, TRPS1, NME4, and OASL) involved in disc degeneration, providing potential targets for future research.

World Forum for Spine Research: The intervertebral disc

BackgroundLow back pain (LBP) is a leading cause of disability in elderly individuals, and intervertebral disc degeneration (IDD) is the major contributor to LBP. Aging is an important factor contributing to IDD. Research has indicated that the incidence of lumbar intervertebral disc protrusion is significantly greater in high-altitude areas. However, the effects of a high-altitude environment on IDD and the underlying mechanism remain unclear.MethodsWe identified changes in the intervertebral discs of rats of different ages in a simulated high-altitude, low-pressure, and hypoxic environment. Furthermore, through transcriptome analysis, we investigated genes that are differentially expressed during intervertebral disc aging and degeneration at different altitudes.ResultsGene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that biological processes and cellular senescence are important molecular events and regulatory pathways for the development of IDD in individuals who live in high-altitude environments. In the transcriptome sequencing of rat intervertebral discs with aging and degeneration at different altitudes, the results of the GO and KEGG analyses revealed that cell cycle arrest is a key factor in aging and degeneration. In addition, through overlap analysis, we observed changes in SFN expression in high-altitude environments, and this change may be meaningful.ConclusionOur findings clarify the effects of high altitude on disc degeneration and predict potential molecular targets that can be used to identify or intervene in disc degeneration caused by high altitude by mining the common features of NP cells associated with the aging process in HA environments through transcriptome sequencing.

12. Assessment of age-related intervertebral disc degenerations of the cervical spine in different age groups: a kinematic MRI study

Lower back pain is a common concern, and 40% of all cases involve the degeneration of the intervertebral disc (IVD). However, the excessive apoptosis of disc cells plays an important role in IVD degeneration, particularly in the nucleus pulposus (NP). Thus, anti-apoptotic gene therapy to attenuate or reverse the degenerative process within the NP is being developed. Survivin is a unique inhibitor of apoptosis (IAP) and has been extensively investigated in cancer cells. However, little is known of the effects of survivin transfection on NP cells derived from degenerative human disc. In this study, we aimed to investigate the effects of lentivirus (LV)-mediated survivin transfection on the morphology and apoptosis of NP cells derived from degenerative human disc in vitro. NP cells were transfected with LV-mediated survivin. Subsequently, cell morphology was observed and the survivin mRNA expression levels were measured by RT-qPCR. Apoptosis was analyzed by flow cytometry and by measuring caspase-3 activity. The results revealed that the morphology of the NP cells derived from degenerative human disc transfected with LV-mediated survivin was significantly altered as evidenced by cytomorphosis, the reduction of the cytoplasm and cell shrinkage. Following transfection, survivin gene expression significantly increased in the transfected cells and subsequent generation cells; however, no significant differences in the cell apoptotic rate and caspase-3 activity were observed. We found that transfection of the survivin gene into NP cells led to the stable expression of survivin and induced marked changes in cell morphology. Furthermore, no significant anti-apoptotic effects were observed following LV-mediated survivin transfection. Overall, our findings demonstrate that LV carrying surviving may be used to successfully enforce the expression of survivin in NP cells. However, cell morphology was evidently altered, whereas the apoptotic rate did not decrease. Comprehensive studies on the feasibility of using survivin in gene therapy in an aim to attenuate disc degeneration are warranted. Further research on the mechanisms responsible for the changes in cell morphology and cell function are also required.

Back pain is linked to intervertebral disc (IVD) degeneration, but clinical studies show the relationship is complex. This study assessed whether males and females have distinct relationships between IVD degeneration and pain using an in vivo rat model. Forty-eight male and female Sprague–Dawley rats had lumbar IVD puncture or sham surgery. Six weeks after surgery, IVDs were evaluated by radiologic IVD height, histological grading, and biomechanical testing. Pain was assessed by von Frey assay and dorsal root ganglia (DRG) expression of Calca and Tac1 genes. Network analysis visualized which measures of IVD degeneration most related to pain by sex. In both females and males, annular puncture induced structural IVD degeneration, but functional biomechanical properties were similar to sham. Females and males had distinct differences in mechanical allodynia and DRG gene expression, even though sex differences in IVD measurements were limited. Network analysis also differed by sex, with more associations between annular puncture injury and pain in the male network. Sex differences exist in the interactions between IVD degeneration and pain. Limited correlation between measures of pain and IVD degeneration highlights the need to evaluate pain or nociception in IVD degeneration models to better understand nervous system involvement in discogenic pain.

Intervertebral disc (IVD) degeneration (IDD) is a common musculoskeletal disease and its treatment remains a clinical challenge. It is characterised by reduced cell numbers and degeneration of the extracellular matrix (ECM). Nucleus pulposus (NP) cells play a crucial role in this process. The purpose of this study is to explore the role of bevacizumab, a vascular endothelial growth factor (VEGF) inhibitor, in the treatment of IDD through local drug delivery. High expression of VEGF was observed in degenerating human and rat IVDs. We demonstrated that MMP3 expression was decreased and COL II synthesis was promoted, when VEGF expression was inhibited by bevacizumab, thereby improving the degree of disc degeneration. Thus, these findings provide strong evidence that inhibition of VEGF expression by local delivery of bevacizumab is safe and effective in ameliorating disc degeneration in rats. The injectable thermosensitive PLGA-PEG-PLGA hydrogels loaded with bevacizumab is a potential therapeutic option for disc degeneration.

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

Prospective, repeated measures obtained under treatment and control conditions. The purposes of this study were to provide preliminary evidence regarding the immediate change in the diffusion of water in the nuclear region of normal and degenerative lumbar intervertebral discs (IVDs) following a single session of lumbar joint mobilization, and to compare these findings to the immediate change in the diffusion of water following a 10-minute session of prone lying. There is conflicting evidence regarding the effectiveness and efficacy of lumbar joint mobilization. Increased knowledge of the physiologic effects of lumbar joint mobilization can lead to refinement of its clinical application. A total of 24 people (15 males and 9 females), ranging in age from 22 to 58 years, participated in this study. All subjects had a history of activity-limiting low back pain. Diffusion-weighted magnetic resonance images (DW-MRIs) were obtained immediately before and after a 10-minute session of lumbar joint mobilization. At least 1 month later, a second session was performed in which DW-MRIs were obtained immediately before and after a 10-minute session of prone lying. Following lumbar joint mobilization, a significant increase (P = .002) in the mean values for diffusion of water was observed within degenerative IVDs at L5-S1 (22.2% increase; effect size, 0.97). Degenerative IVDs at L1-2 to L4-5 and normal IVDs at L1-2 to L5-S1 did not demonstrate a change in diffusion following joint mobilization. Prone lying was not associated with a change in diffusion for normal or degenerative IVDs. The stimulus provided by lumbar joint mobilization may influence the diffusion of water in degenerative IVDs at L5-S1; however, these are preliminary findings and the relationship of these findings to pain and function needs further investigation.