The past, present, and the future of disc nucleus replacement. A systematic review of a large diversity of ideas and experiences.

Disc nucleus replacement (NR) is a challenging surgical technique used as a medical treatment for early-stage disc herniation to restore disc height and the biomechanical function of a motion segment, which may reduce low back pain. The surgical procedure involves the removal and replacement of the degenerated nucleus pulposus with a substitute by accessing the annulus fibrosos via a created hole. Over the decades, nucleus replacement has been an important issue, leading to the development of different substitute alternatives. The first ideas are dated to the 1950s and since then, more than a hundred nucleus replacement concepts can be identified. There were numerous attempts and several clinical trials; however, after more than 70 years of research, no gold standard for nucleus pulposus replacement has been identified. This review aims to collect the different nucleus replacements reported in the literature, thus understanding what failed, what could be improved and what are the opportunities for the future. A systematic review of the literature was performed using a keyword-based search on PubMed, Web of Science, and Scopus databases to detect all nucleus replacements presented in the past by clinicians and engineers. Several studies were extracted from which the main nucleus replacements over the years were investigated, including the ones that received CE mark, FDA approval, or IDE approval and, also those involved in clinical trials. A total of 116 studies were included in this review. The extracted data concern the nucleus replacements proposed over the years to create a historical background as complete as possible, including their mechanical and biomechanical characterization and the clinical trials conducted over the years. Nucleus disc arthroplasty has been explored for many years. Unfortunately, even today there is still nothing safe and definitive in this surgical practice. This review provides an overview of the nucleus replacement history. A breakthrough could be the improvements in technologies for the annulus fibrous closing or sealing and the tissue engineering and medical regenerative techniques which could certainly ensure a higher NR implantation success rate in the future of this clinical treatment. It is not yet clear what is the future of this clinical practice. Only scientific research can answer the question: is the nucleus replacement still a possible clinical solution?

Mitochondrial 'Birth-Death' coordinator: An intelligent hydrogen nanogenerator to enhance intervertebral disc regeneration.

Currently, mitochondrial dysfunction caused by oxidative stress is a growing concern in degenerative diseases, notably intervertebral disc degeneration (IVDD). Dysregulation of the balance of mitochondrial quality control (MQC) has been considered the key contributor, while it's still challenging to effectively harmonize different MQC components in a simple and biologically safe way. Hydrogen gas (H2) is a promising mitochondrial therapeutic molecule due to its bio-reductivity and diffusibility across cellular membranes, yet its relationship with MQC regulation remains unknown. Herein, we propose a mitochondrial 'Birth-Death' coordinator achieved by an intelligent hydrogen nanogenerator (Fe@HP-OD), which can sustainably release H2 in response to the unique microenvironment in degenerated IVDs. Both in vitro and in vivo results prove alleviation of cellular oxidative stress and restoration of nucleus pulposus cells function, thereby facilitating successful IVD regeneration. Significantly, this study for the first time proposes the mitochondrial 'Birth-Death' coordination mechanism: 1) attenuation of overactivated mitochondrial 'Death' process (UPRmt and unselective mitophagy); and 2) activation of Adenosine 5'-monophosphate-activated protein kinase (AMPK) signaling pathway for mitochondrial 'Birth-Death' balance (mitochondrial biogenesis and controlled mitophagy). These pioneering findings can fill in the gaps in molecular mechanisms for H2 regulation on MQC homeostasis, and pave the way for future strategies towards restoring equilibrium of MQC system against degenerative diseases.

Collagen II enrichment through scAAV6-RNAi-mediated inhibition of matrix-metalloproteinases 3 and 13 in degenerative nucleus-pulposus cells degenerative disc disease and biological treatment strategies.

Intervertebral disc (IVD) degeneration damaging the extracellular matrix (ECM) of IVDs is the main cause of spine-associated disorders. Degenerative disc disease (DDD) is a multifaceted disorder, where environmental factors, inflammatory cytokines and catabolic enzymes act together. DDD starts typically due to imbalance between ECM biosynthesis and degradation within IVDs, especially through unbalanced degradation of aggrecan and collagen II in nucleus pulposus (NP). Current treatment approaches are primarily based on conservative or surgical therapies, which are insufficient for biological regeneration. The disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs) and matrix metalloproteinases (MMPs) are the key proteolytic enzymes for degradation of aggrecan and collagens. Previously, high expression levels of ADAMTS4, ADAMTS5, MMP3 and MMP13, which are accompanied with low levels of aggrecan and collagen II, were demonstrated in degenerative human NP cells. Moreover, self-complementary adeno-associated virus type 6 (scAAV6) mediated inhibitions of ADAMTS4 and ADAMTS5 by RNA-interference (RNAi) could specifically enhance aggrecan level. Thus, MMPs are apparently the main degrading enzymes of collagen II in NP. Furthermore, scAAV6-mediated inhibitions of MMP3 and MMP13 have not yet been investigated. Therefore, we attempted to enhance the level of collagen II in degenerative NP cells by scAAV6-RNAi-mediated inhibitions of MMP3 and MMP13. MRI was used to determine preoperative grading of IVD degeneration in patients. After isolation and culturing of NP cells, cells were transduced with scAAV6-shRNAs targeting MMP3 or MMP13; and analysed by fluorescence microscopy, FACS, MTT assay, RT-qPCR, ELISA and western blotting. scAAV6-shRNRs have no impact on cell viability and proliferation, despite high transduction efficiencies (98.6%) and transduction units (1383 TU/Cell). Combined knockdown of MMP3 (92.8%) and MMP13 (90.9%) resulted in highest enhancement of collagen II (143.2%), whereby treatment effects were significant over 56 days (p < 0.001). Conclusively, scAAV6-RNAi-mediated inhibitions of MMP3 and MMP13 help to progress less immunogenic and enduring biological treatments in DDD.

Ultrashort-Echo-Time MRI of the Disco-Vertebral Junction: Modulation of Image Contrast via Echo Subtraction and Echo Times.

INTRODUCTION: The disco-vertebral junction (DVJ) of the lumbar spine contains thin structures with short T2 values, including the cartilaginous endplate (CEP) sandwiched between the bony vertebral endplate (VEP) and the nucleus pulposus (NP). We previously demonstrated that ultrashort-echo-time (UTE) MRI, compared to conventional MRI, is able to depict the tissues at the DVJ with improved contrast. In this study, we sought to further optimize UTE MRI by characterizing the contrast-to-noise ratio (CNR) of these tissues when either single echo or echo subtraction images are used and with varying echo times (TEs). METHODS: In four cadaveric lumbar spines, we acquired 3D Cones (a UTE sequence) images at varying TEs from 0.032 ms to 16 ms. Additionally, spin echo T1- and T2-weighted images were acquired. The CNRs of CEP-NP and CEP-VEP were measured in all source images and 3D Cones echo subtraction images. RESULTS: In the spin echo images, it was challenging to distinguish the CEP from the VEP, as both had low signal intensity. However, the 3D Cones source images at the shortest TE of 0.032 ms provided an excellent contrast between the CEP and the VEP. As the TE increased, the contrast decreased in the source images. In contrast, the 3D Cones echo subtraction images showed increasing CNR values as the second TE increased, reaching statistical significance when the second TE was above 10 ms (p < 0.05). CONCLUSIONS: Our study highlights the feasibility of incorporating UTE MRI for the evaluation of the DVJ and its advantages over conventional spin echo sequences for improving the contrast between the CEP and adjacent tissues. Additionally, modulation of the contrast for the target tissues can be achieved using either source images or subtraction images, as well as by varying the echo times.

HDAC/H3K27ac-mediated transcription of NDUFA3 exerts protective effects on high glucose-treated human nucleus pulposus cells through improving mitochondrial function.

Diabetes mellitus (DM) is a well-documented risk factor of intervertebral disc degeneration (IVDD). The current study was aimed to clarify the effects and mechanisms of NADH: ubiquinone oxidoreductase subunit A3 (NDUFA3) in human nucleus pulposus cells (HNPCs) exposed to high glucose. NDUFA3 was overexpressed in HNPCs via lenti-virus transduction, which were co-treated with high glucose and rotenone (a mitochondrial complex I inhibitor) for 48 h. Cell activities were assessed for cell viability, cell apoptosis, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP) ratio, oxygen consumption rate (OCR) and mitochondrial complexes I activities. High glucose decreased cell viability, increased apoptotic cells, increased ROS production, decreased MMP levels and OCR values in HNPCs in a dose-dependent manner. Rotenone co-treatment augmented the high glucose-induced injuries on cell viability, apoptosis, ROS production and mitochondrial function. NDUFA3 overexpression counteracted the high glucose-induced injuries in HNPCs. HDAC/H3K27ac mechanism was involved in regulating NDUFA3 transcription. NDUFA3 knockdown decreased cell viability and increased apoptotic cells, which were reversed by ROS scavenger N-acetylcysteine. HDAC/H3K27ac-mediated transcription of NDUFA3 protects HNPCs against high glucose-induced injuries through suppressing cell apoptosis, eliminating ROS, improving mitochondrial function and oxidative phosphorylation. This study sheds light on candidate therapeutic targets and deepens the understanding of molecular mechanisms behind DM-induced IVDD.

A novel spherical GelMA-HAMA hydrogel encapsulating APET×2 polypeptide and CFIm25-targeting sgRNA for immune microenvironment modulation and nucleus pulposus regeneration in intervertebral discs.

METHODS: Single-cell transcriptomics and high-throughput transcriptomics were used to screen factors significantly correlated with intervertebral disc degeneration (IDD). Expression changes of CFIm25 were determined via RT-qPCR and Western blot. NP cells were isolated from mouse intervertebral discs and induced to degrade with TNF-α and IL-1ß. CFIm25 was knocked out using CRISPR-Cas9, and CFIm25 knockout and overexpressing nucleus pulposus (NP) cell lines were generated through lentiviral transfection. Proteoglycan expression, protein expression, inflammatory factor expression, cell viability, proliferation, migration, gene expression, and protein expression were analyzed using various assays (alcian blue staining, immunofluorescence, ELISA, CCK-8, EDU labeling, transwell migration, scratch assay, RT-qPCR, Western blot). The GelMA-HAMA hydrogel loaded with APET×2 polypeptide and sgRNA was designed, and its effects on NP regeneration were assessed through in vitro and mouse model experiments. The progression of IDD in mice was evaluated using X-ray, H&E staining, and Safranin O-Fast Green staining. Immunohistochemistry was performed to determine protein expression in NP tissue. Proteomic analysis combined with in vitro and in vivo experiments was conducted to elucidate the mechanisms of hydrogel action. RESULTS: CFIm25 was upregulated in IDD NP tissue and significantly correlated with disease progression. Inhibition of CFIm25 improved NP cell degeneration, enhanced cell proliferation, and migration. The hydrogel effectively knocked down CFIm25 expression, improved NP cell degeneration, promoted cell proliferation and migration, and mitigated IDD progression in a mouse model. The hydrogel inhibited inflammatory factor expression (IL-6, iNOS, IL-1ß, TNF-α) by targeting the p38/NF-κB signaling pathway, increased collagen COLII and proteoglycan Aggrecan expression, and suppressed NP degeneration-related factors (COX-2, MMP-3). CONCLUSION: The study highlighted the crucial role of CFIm25 in IDD and introduced a promising therapeutic strategy using a porous spherical GelMA-HAMA hydrogel loaded with APET×2 polypeptide and sgRNA. This innovative approach offers new possibilities for treating degenerated intervertebral discs.

Glucose deprivation-induced disulfidptosis in human nucleus pulposus cells: a novel pathological mechanism of intervertebral disc degeneration.

BACKGROUND: Limited supply of certain nutrients and deregulation of nucleus pulposus (NP) plays a key role in the pathogenesis of intervertebral disc degeneration (IVDD). However, whether nutrient deprivation-induced cell death, particularly disulfidptosis, contributes to the depletion of NP cells and the development of IVDD, is unknown. METHODS: RNA-seq, single-cell RNA-seq, and Genome-wide DNA methylation datasets of nucleus pulposus tissue were collected for bioinformatic analysis. Predictive models of disulfidptosis related genes in IVDD were constructed by machine learning and their differential expression was analyzed. In addition, we performed cell subsets identification analysis, cell-cell communications analysis, and functional enrichment analysis of key genes in the core subset based on single-cell RNA-seq data of NP tissues isolated from one normal sample and one IVDD sample. Finally, glucose deprivation-induced disulfidptosis in human NP cells (HNPCs) was verified by various cell death inhibitors and disulfidptosis-related molecular markers. RESULTS: We found the disulfidptosis signal was significantly activated in the IVDD group. Using single-cell RNA-seq analysis, we focused on the chondrocytes and found that disulfidptosis-related genes significantly highly expressed in the IVDD C4 chondrocyte subset, which was identified as a new disulfidptosis-associated cell subset. Correlation analysis revealed the negative correlation between SLC7A11 (driving gene of disulfidptosis) and the glucose transporter GLUTs (SLC2A1-4) family genes (suppressing genes of disulfidptosis) in the IVDD group. We also found obvious cell death in HNPC upon glucose starvation, while employment of various cell death inhibitors could not inhibit glucose starvation-induced death in HNPCs. Moreover, the accumulation of disulfide bonds in cytoskeletal proteins was indicated by slowed migration in non-reducible protein blotting experiments. 2-DG, a key disulfidptosis inhibitor, significantly rescued cell death caused by glucose starvation through lowering the NADP+/NADPH ratio. CONCLUSIONS: We validated the occurrence of disulfidptosis in HPNCs and identified a novel disulfidptosis-associated cell subset, followed by experimental verification of disulfidptosis in a glucose-limited context to mimic a fall in nutrient supply during the development disc degeneration. These findings provided new insights into the pathological mechanisms of IVDD and encourage us to explore potential therapeutic targets involved in the regulation of disulfidptosis for the prevention of intervertebral disc degeneration.

The role of miR-155-5p in inflammation and mechanical loading during intervertebral disc degeneration.

BACKGROUND: Intervertebral disc (IVD) degeneration is a multifactorial pathological process resulting in the dysregulation of IVD cell activity. The catabolic shift observed in IVD cells during degeneration leads to increased inflammation, extracellular matrix (ECM) degradation, aberrant intracellular signaling and cell loss. Importantly, these pathological processes are known to be interconnected and to collectively contribute to the progression of the disease. MicroRNAs (miRNAs) are known as strong post-transcriptional regulators, targeting multiple genes simultaneously and regulating numerous intracellular pathways. Specifically, miR-155-5p has been of particular interest since it is known as a pro-inflammatory mediator and contributing factor to diseases like cancer and osteoarthritis. This study investigated the role of miR-155-5p in IVD degeneration with a specific focus on inflammation and mechanosensing. METHODS: Gain- and loss-of-function studies were performed through transfection of human Nucleus pulposus (NP) and Annulus fibrosus (AF) cells isolated from degenerated IVDs with miR-155-5p mimics, inhibitors or their corresponding non-targeting control. Transfected cells were then subjected to an inflammatory environment or mechanical loading. Conditioned media and cell lysates were collected for phosphorylation and cytokine secretion arrays as well as gene expression analysis. RESULTS: Increased expression of miR-155-5p in AF cells resulted in significant upregulation of interleukin (IL)-8 cytokine secretion during cyclic stretching and a similar trend in IL-6 secretion during inflammation. Furthermore, miR-155-5p mimics increased the expression of the brain-derived neurotrophic factor (BDNF) in AF cells undergoing cyclic stretching. In NP cells, miR-155-5p gain-of-function resulted in the activation of the mitogen-activated protein kinase (MAPK) signaling pathway through increased phosphorylation of p38 and p53. Lastly, miR-155-5p inhibition caused a significant increase in the anti-inflammatory cytokine IL-10 in AF cells and the tissue inhibitor of metalloproteinases (TIMP)-4 in NP cells respectively. CONCLUSION: Overall, these results show that miR-155-5p contributes to IVD degeneration by enhancing inflammation through pro-inflammatory cytokines and MAPK signaling, as well as by promoting the catabolic shift of AF cells during mechanical loading. The inhibition of miR-155-5p may constitute a potential therapeutic approach for IVD degeneration and low back pain.

A2AR regulate inflammation through PKA/NF-κB signaling pathways in intervertebral disc degeneration.

BACKGROUND: Reduction of inflammatory damage and inhibition of nucleus pulposus (NP) apoptosis are considered to be the main effective therapy idea to reverse the intervertebral disc degeneration (IDD) and alleviate the chronic low back pain. The adenosine A2A receptor (A2AR), as a member of G protein-coupled receptor families, plays an important role in the anti-inflammation and relieving pain. So far, the impact of A2AR on IDD therapy is unclear. The aim of this study was to explore the role of Adenosine A2A receptor (A2AR) in the intervertebral disc degeneration (IDD) and clarify potential mechanism. MATERIALS AND METHODS: IL-1ß and acupuncture was used to establish IDD model rats. A2AR agonist CGS-21680 and A2AR antagonist SCH442416 were used to investigate the therapeutical effects for IDD. Histological examination, western blotting analysis and RT-PCR were employed to evaluate the the association between A2AR and cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway. RESULTS: A2AR activity of the intervertebral disc tissues was up-regulated in feedback way, and cAMP, PKA and CREB expression were also increased. But in general, IL-1ß-induced IDD promoted the significant up-regulation the expression of inflammatory factors. The nucleus pulposus (NP) inflammation was exacerbated in result of MMP3 and Col-II decline through activating NF-κB signaling pathway. A2AR agonist CGS-21680 exhibited a disc protective effect through significantly increasing A2AR activity, then further activated cAMP/PKA signaling pathway with attenuating the release of TNF-α and IL-6 via down-regulating NF-κB. In contrast, SCH442416 inhibited A2AR activation, consistent with lower expression levels of cAMP and PKA, further leading to the acceleration of IDD. CONCLUSIONS: The activation of A2AR can prevent inflammatory responses and mitigates degradation of IDD thus suggest a potential novel therapeutic strategy of IDD.

Assessment of Tie2-Rejuvenated Nucleus Pulposus Cell Transplants from Young and Old Patient Sources Demonstrates That Age Still Matters.

Cell transplantation is being actively explored as a regenerative therapy for discogenic back pain. This study explored the regenerative potential of Tie2+ nucleus pulposus progenitor cells (NPPCs) from intervertebral disc (IVD) tissues derived from young (<25 years of age) and old (>60 years of age) patient donors. We employed an optimized culture method to maintain Tie2 expression in NP cells from both donor categories. Our study revealed similar Tie2 positivity rates regardless of donor types following cell culture. Nevertheless, clear differences were also found, such as the emergence of significantly higher (3.6-fold) GD2 positivity and reduced (2.7-fold) proliferation potential for older donors compared to young sources. Our results suggest that, despite obtaining a high fraction of Tie2+ NP cells, cells from older donors were already committed to a more mature phenotype. These disparities translated into functional differences, influencing colony formation, extracellular matrix production, and in vivo regenerative potential. This study underscores the importance of considering age-related factors in NPPC-based therapies for disc degeneration. Further investigation into the genetic and epigenetic alterations of Tie2+ NP cells from older donors is crucial for refining regenerative strategies. These findings shed light on Tie2+ NPPCs as a promising cell source for IVD regeneration while emphasizing the need for comprehensive understanding and scalability considerations in culture methods for broader clinical applicability.

IL-32 aggravates metabolic disturbance in human nucleus pulposus cells by activating FAT4-mediated Hippo/YAP signaling.

Extracellular matrix (ECM) metabolism disorders in the inflammatory microenvironment play a key role in the pathogenesis of intervertebral disc degeneration (IDD). Interleukin-32 (IL-32) has been reported to be involved in the progression of various inflammatory diseases; however, it remains unclear whether it participates in the matrix metabolism of nucleus pulposus (NP) cells. Therefore, this study aimed to investigate the mechanism of IL-32 on regulating the ECM metabolism in the inflammatory microenvironment. RNA-seq was used to identify aberrantly expressed genes in NP cells in the inflammatory microenvironment. Western blotting, real-time quantitative PCR, immunohistochemistry and immunofluorescence analysis were performed to measure the expression of IL-32 and metabolic markers in human NP tissues or NP cells treated with or without tumor necrosis factor-α (TNF-α). In vivo, an adeno-associated virus overexpressing IL-32 was injected into the caudal intervertebral discs of rats to assess its effect on IDD. Proteins interacting with IL-32 were identified via immunoprecipitation and mass spectrometry. Lentivirus overexpressing IL-32 or knocking down Fat atypical cadherin 4 (FAT4), yes-associated protein (YAP) inhibitor-Verteporfin (VP) were used to treat human NP cells, to explore the pathogenesis of IL-32. Hippo/YAP signaling activity was verified in human NP tissues. IL-32 expression was significantly upregulated in degenerative NP tissues, as indicated in the clinical samples. Furthermore, IL-32 was remarkably overexpressed in TNF-α-induced degenerative NP cells. IL-32 overexpression induced IDD progression in the rat model. Mechanistically, the elevation of IL-32 in the inflammatory microenvironment enhanced its interactions with FAT4 and mammalian sterile 20-like kinase1/2 (MST1/2) proteins, prompting MST1/2 phosphorylation, and activating the Hippo/YAP signaling pathway, causing matrix metabolism disorder in NP cells. Our results suggest that IL-32 mediates matrix metabolism disorders in NP cells in the inflammatory micro-environment via the FAT4/MST/YAP axis, providing a theoretical basis for the precise treatment of IDD.

A structured biomimetic nanoparticle as inflammatory factor sponge and autophagy-regulatory agent against intervertebral disc degeneration and discogenic pain.

Lower back pain (LBP) is a common condition closely associated with intervertebral disc degeneration (IDD), causing a significant socioeconomic burden. Inflammatory activation in degenerated discs involves pro-inflammatory cytokines, dysregulated regulatory cytokines, and increased levels of nerve growth factor (NGF), leading to further intervertebral disc destruction and pain sensitization. Macrophage polarization is closely related to autophagy. Based on these pathological features, a structured biomimetic nanoparticle coated with TrkA-overexpressing macrophage membranes (TMNP@SR) with a rapamycin-loaded mesoporous silica core is developed. TMNP@SR acted like sponges to adsorbe inflammatory cytokines and NGF and delivers the autophagy regulator rapamycin (RAPA) into macrophages through homologous targeting effects of the outer engineered cell membrane. By regulating autophagy activation, TMNP@SR promoted the M1-to-M2 switch of macrophages to avoid continuous activation of inflammation within the degenerated disc, which prevented the apoptosis of nucleus pulposus cells. In addition, TMNP@SR relieved mechanical and thermal hyperalgesia, reduced calcitonin gene-related peptide (CGRP) and substance P (SP) expression in the dorsal root ganglion, and downregulated GFAP and c-FOS signaling in the spinal cord in the rat IDD model. In summary, TMNP@SR spontaneously inhibits the aggravation of disc inflammation to alleviate disc degeneration and reduce the ingress of sensory nerves, presenting a promising treatment strategy for LBP induced by disc degeneration.

Needle-scalpel therapy inhibits the apoptosis of nucleus pulposus cells via the SDF-1/CXCR4 axis in a rat degenerative cervical intervertebral disc model.

As a common disease, cervical spondylosis (CS) results from the degeneration of the cervical intervertebral disc. However, there are still no effective clinical strategies for the treatment of this disease. Needle-scalpel (Ns), a therapy guided by traditional Chinese medicine theory, alleviates intervertebral disc degradation and is widely used in the clinic to treat CS. Stromal cell-derived factor-1 (SDF-1) and its receptor CXC receptor 4 (CXCR4) in nucleus pulposus cells play an important role in CS onset and development. This study aimed to explore whether Ns can relieve pain and regulate the SDF-1/CXCR4 axis in nucleus pulposus cells to inhibit apoptosis, thereby delaying cervical intervertebral disc degradation in a rat model of CS. It was found that the Ns-treated groups exhibited higher mechanical allodynia scores than the model group, and H&E staining, MRI, and scanning electron microscopy revealed that Ns therapy inhibited intervertebral disc degeneration. Additionally, Ns therapy significantly inhibited increases in the RNA and protein expression levels of SDF-1 and CXCR4. Furthermore, these treatments alleviated the apoptosis of nucleus pulposus cells, which manifested as a decline in the proportion of apoptotic nucleus pulposus cells and inhibition of the decrease in the levels of Bcl-2/Bax. These findings indicated that Ns mitigated CS-induced pain, inhibited the apoptosis of nucleus pulposus cells, and alleviated intervertebral disc degeneration in CS rats. These effects may be mediated by specifically regulating the SDF-1/CXCR4 signaling axis. Based on these findings, we conclude that Ns might serve as a promising therapy for the treatment of CS.

Isorhapontigenin delays senescence and matrix degradation of nucleus pulposus cells via PI3K/AKT/mTOR-mediated autophagy pathway in vitro and alleviates intervertebral disc degeneration in vivo.

Intervertebral disc degeneration (IVDD), a common degenerative disc disease, is a major etiological factor for back pain, affecting a significant number of middle-aged and elderly individuals worldwide. Thus, IVDD is a major socio-economic burden. The factors contributing to the complex IVDD etiology, which has not been elucidated, include inflammation, oxidative stress, and natural aging. In particular, inflammation and aging of nucleus pulposus cells are considered primary pathogenic factors. Isorhapontigenin (ISO) is a polyphenolic compound commonly found in traditional Chinese herbs and grapes. We have demonstrated that ISO exerts anti-inflammatory and anti-aging effects and mitigates extracellular matrix (ECM) degradation. In this study, in vitro experiments revealed that, ISO delays aging and ECM degradation by promoting PI3K/AKT/mTOR-mediated autophagy. Meanwhile, in vivo experiments affirmed that ISO delays the progression of IVDD.

Enzymatic Digestion of the Intervertebral Disc Alters Intradiscal Injection and Leakage Mechanics.

Intradiscal injection is required to deliver therapeutic agents to the intervertebral disc (IVD) nucleus pulposus (NP). However, injectate leakage following needle retraction may result in decreased treatment efficacy and adverse side effects. While enzymatic digestion is a common research approach for simulating degeneration in healthy animal IVDs, contributions to the leakage phenomenon are unknown. In this study, bovine caudal discs were treated with injection into the NP of either a tris buffer control, collagenase (to primarily target collagen), or trypsin (to primarily target proteoglycans) and then injected with fluorescent saline using a through-puncture defect protocol. Pressure-volume records during injection were used to determine volume and pressure at leakage. Discs were then frozen, transected, and photographed to visualize injectate dispersion. Collagenase treatment resulted in a large increase in injectate dispersion, along with a decrease in injection pressure relative to control. Trypsin treatment resulted in a moderate increase in dispersion, with no associated effect on pressure. This study concludes that care should be taken when employing enzymatic digestion to simulate IVD degeneration, as NP tissue disruption may affect both retention and dispersion of subsequent therapeutic injections.

Biomimetic kartogenin containing κ-carrageenan hydrogel for nucleus pulposus regeneration.

Intervertebral disc degeneration is a clinical disease that reduces the quality of patient's life. The degeneration usually initiates in the nucleus pulposus (NP), hence the use of hydrogels represents a promising therapeutic approach. However, the viscoelastic nature of hydrogel and its ability to provide biomimetic architecture and biochemical cues influence the regeneration capability. This study focused on tuning the physical nature of a glycosaminoglycan hydrogel (κ-carrageenan) as well as the release kinetics of a chondrogenic factor (kartogenin - KGN) through physical cross-linking. For this, κ-carrageenan was cross linked with 2.5 % and 5 % potassium chloride (KCl) for 15 and 30 min and loaded with KGN molecule at 50 µM and 100 µM. The tight network structure with low water retention and degradation property was seen in hydrogel cross-linked with increased KCl concentration and time. However, optimal degradation along with NP mimicking viscoelastic nature was exhibited by 5 wt% KCl treated hydrogel (H3 hydrogel). All hydrogel groups exhibited burst KGN release at 24 h followed by a sustained release for 5 days. However, hydrogel cross-linked with 5 wt% KCl enhanced chondrogenic differentiation, mainly at lower KGN dose. In summary, this study shows the potential application of biomimetic KGN laden carrageenan hydrogel in NP regeneration.

IL-1ra loaded chondroitin sulfate-functionalized microspheres for minimally invasive treatment of intervertebral disc degeneration.

Presently, the clinical treatment of intervertebral disc degeneration (IVDD) remains challenging, but the strategy of simultaneously overcoming the overactive inflammation and restoring the anabolic/catabolic balance of the extracellular matrix (ECM) in the nucleus pulposus (NP) has become an effective way to alleviate IVDD. IL-1ra, a natural antagonist against IL-1ß, can mitigate inflammation and promote regeneration in IVDD. Chondroitin sulfate (CS), an important component of the NP, can promote ECM synthesis and delay IVDD. Thus, these were chosen and integrated into functionalized microspheres to achieve their synergistic effects. First, CS-functionalized microspheres (GelMA-CS) with porous microstructure, good monodispersion, and about 200 µm diameter were efficiently and productively fabricated using microfluidic technology. After lyophilization, the microspheres with good local injection and tissue retention served as the loading platform for IL-1ra and achieved sustained release. In in vitro experiments, the IL-1ra-loaded microspheres exhibited good cytocompatibility and efficacy in inhibiting the inflammatory response of NP cells induced by lipopolysaccharide (LPS) and promoting the secretion of ECM. In in vivo experiments, the microspheres showed good histocompatibility, and local, minimally invasive injection of the IL-1ra-loaded microspheres could reduce inflammation, maintain the height of the intervertebral disc (IVD) and the water content of NP close to about 70 % in the sham group, and retain the integrated IVD structure. In summary, the GelMA-CS microspheres served as an effective loading platform for IL-1ra, eliminated inflammation through the controlled release of IL-1ra, and promoted ECM synthesis via CS to delay IVDD, thereby providing a promising intervention strategy for IVDD. STATEMENT OF SIGNIFICANCE: The strategy of simultaneously overcoming the overactive inflammation and restoring the anabolic/catabolic balance of the extracellular matrix (ECM) in nucleus pulposus (NP) has shown great potential prospects for alleviating intervertebral disc degeneration (IVDD). From the perspective of clinical translation, this study developed chondroitin sulfate functionalized microspheres to act as the effective delivery platform of IL-1ra, a natural antagonist of interleukin-1ß. The IL-1ra loading microspheres (GelMA-CS-IL-1ra) showed good biocompatibility, good injection with tissue retention, and synergistic effects of inhibiting the inflammatory response induced by lipopolysaccharide and promoting the secretion of ECM in NPCs. In vivo, they also showed the beneficial effect of reducing the inflammatory response, maintaining the height of the intervertebral disc and the water content of the NP, and preserving the integrity of the intervertebral disc structure after only one injection. All demonstrated that the GelMA-CS-IL-1ra microspheres would have great promise for the minimally invasive treatment of IVDD.

Multiomics analysis reveals the potential of LPCAT1-PC axis as a therapeutic target for human intervertebral disc degeneration.

Intervertebral disc degeneration (IDD) is a highly prevalent musculoskeletal disorder that is associated with considerable morbidity. However, there is currently no drug available that has a definitive therapeutic effect on IDD. In this study, we aimed to identify the molecular features and potential therapeutic targets of IDD through a comprehensive multiomics profiling approach. By integrating transcriptomics, proteomics, and ultrastructural analyses, we discovered dysfunctions in various organelles, including mitochondria, the endoplasmic reticulum, the Golgi apparatus, and lysosomes. Metabolomics analysis revealed a reduction in total phosphatidylcholine (PC) content in IDD. Through integration of multiple omics techniques with disease phenotypes, a pivotal pathway regulated by the lysophosphatidylcholine acyltransferase 1 (LPCAT1)-PC axis was identified. LPCAT1 exhibited low expression levels and exhibited a positive correlation with PC content in IDD. Suppression of LPCAT1 resulted in inhibition of PC synthesis in nucleus pulposus cells, leading to a notable increase in nucleus pulposus cell senescence and damage to cellular organelles. Consequently, PC exhibits potential as a therapeutic agent, as it facilitates the repair of the biomembrane system and alleviates senescence in nucleus pulposus cells via reversal of downregulation of the LPCAT1-PC axis.

Metformin prevents the onset and progression of intervertebral disc degeneration: New insights and potential mechanisms (Review).

Metformin has been the go­to medical treatment for addressing type 2 diabetes mellitus (T2DM) as a frontline oral antidiabetic. Obesity, cancer and bone deterioration are linked to T2DM, which is considered a metabolic illness. Numerous diseases associated with T2DM, such as tumours, cardiovascular disease and bone deterioration, may be treated with metformin. Intervertebral disc degeneration (IVDD) is distinguished by degeneration of the spinal disc, accompanied by the gradual depletion of proteoglycans and water in the nucleus pulposus (NP) of the IVD, resulting in lower back pain. The therapeutic effect of metformin on IVDD has also attracted much attention. By stimulating AMP­activated kinase, metformin could enhance autophagy and suppress cell senescence, apoptosis and inflammation, thus effectively delaying IVDD. The present review aimed to systematically explain the development of IVDD and mechanism of metformin in the treatment and prevention of IVDD to provide a reference for the clinical application of metformin as adjuvant therapy in the treatment of IVDD.

Self-powered triboelectric-responsive microneedles with controllable release of optogenetically engineered extracellular vesicles for intervertebral disc degeneration repair.

Excessive exercise is an etiological factor of intervertebral disc degeneration (IVDD). Engineered extracellular vesicles (EVs) exhibit excellent therapeutic potential for disease-modifying treatments. Herein, we fabricate an exercise self-powered triboelectric-responsive microneedle (MN) assay with the sustainable release of optogenetically engineered EVs for IVDD repair. Mechanically, exercise promotes cytosolic DNA sensing-mediated inflammatory activation in senescent nucleus pulposus (NP) cells (the master cell population for IVD homeostasis maintenance), which accelerates IVDD. TREX1 serves as a crucial nuclease, and disassembly of TRAM1-TREX1 complex disrupts the subcellular localization of TREX1, triggering TREX1-dependent genomic DNA damage during NP cell senescence. Optogenetically engineered EVs deliver TRAM1 protein into senescent NP cells, which effectively reconstructs the elimination function of TREX1. Triboelectric nanogenerator (TENG) harvests mechanical energy and triggers the controllable release of engineered EVs. Notably, an optogenetically engineered EV-based targeting treatment strategy is used for the treatment of IVDD, showing promising clinical potential for the treatment of degeneration-associated disorders.