Aims. This study was performed to explore the effect of melatonin on pyroptosis in nucleus pulposus cells (NPCs) and the underlying mechanism of that effect. Methods. This experiment included three patients diagnosed with lumbar disc herniation who failed conservative treatment. Nucleus pulposus tissue was isolated from these patients when they underwent surgical intervention, and primary NPCs were isolated and cultured. Western blotting, reverse transcription polymerase chain reaction, fluorescence staining, and other methods were used to detect changes in related signalling pathways and the ability of cells to resist pyroptosis. Results. Western blot analysis confirmed the expression of cleaved CASP-1 and melatonin receptor (MT-1A-R) in NPCs. The cultured NPCs were identified by detecting the expression of CD24,
Background. Current clinical treatment for spinal instability requires invasive spinal fusion with cages and screw instrumentation. We previously reported a novel injectable hydrogel (Bgel), which supports the delivery and differentiation of mesenchymal stem cells (MSCs) to bone forming cells and supports bone formation in vivo. Here, we investigated whether this system could be utilised to induce bone formation within intervertebral disc tissue as a potential injectable spinal fusion approach. Methodology. Bovine and Human Nucleus pulpous tissue explants were injected with Bgel with and without MSCs. Tissue samples were cultured under hypoxia (5%) in standard culture media for 4 weeks. Cell viability, histological assessment of matrix deposition, calcium formation, and cell phenotype analysis using immunohistochemistry for NP matrix and bone markers. Results. Following injection of B-gel into tissue explants following culture for 4 weeks, cells were visualized within the regions of the B-gel. Demonstrating that native cells were able to migrate into regions of B-gel. Increased collagen deposition was seen in tissue explants injected with Bgel, with increased collagen type I and X but decreased
Purpose of study and background. We have previously reported the development of injectable hydrogels for potential disc regeneration (NPgel) or bone formation which could be utilized in spinal fusion (Bgel). As there are multiple sources of mesenchymal stem cells (MSCs), this study investigated the incorporation of patient matched hMSCs derived from adipose tissue (AD) and bone marrow (BM) to determine their ability to differentiate within both hydrogel systems under different culture conditions. Methods and Results. Human fat pad and bone marrow derived MSCs were isolated from femoral heads of patients undergoing hip replacement surgery for osteoarthritis with informed consent. MSCs were encapsulated into either NPgel or Bgel and cultured for up to 6 weeks in 5% (NPgel) or 21% (Bgel) O. 2. Histology and immunohistochemistry was utilized to determine phenotype. Both fat and bone marrow derived MSCs, were able to differentiate into both cell lineages. NPgel culture conditions increased expression of matrix components such as
Background. Intervertebral disc (IVD) degeneration is a major cause of Low back pain (LBP). We have reported an injectable hydrogel (NPgel), which following injection into bovine NP explants, integrates with NP tissue and promotes NP cell differentiation of delivered mesenchymal stem cells (MSCs) without growth factors. Here we investigated the injection of NPgel+MSCs into bovine NP explants under degenerate culture conditions to mimic the in vivo environment of the degenerate IVD. Methods. hMSCs were incorporated within liquid NPgel and injected into bovine NP explants alongside controls. Explants were cultured for 6 weeks under hypoxia (5%) with ± calcium 5.0mM CaCl. 2. or IL-1β individually or in combination to mimic the degenerate microenvironment. Cell viability was assessed by caspase 3 immunohistochemistry. Histological and immunohistochemical analysis was performed to investigate altered matrix synthesis and matrix degrading enzyme expression. Results. CFSe positive hMSCs were identified in all NPgel injected explants and cell viability was maintained. The NPgel integrated with NP tissue and hMSCs produced matrix components: aggrecan,
Background. Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP). We have developed an injectable hydrogel (NPgel), which following injection into bovine IVD explants, integrates with IVD tissue and promotes disc cell differentiation of delivered mesenchymal stem cells (MSCs) without growth factors. Here, we investigated the injection of NPgel+MSCs into IVD explants under degenerate culture conditions. Methods and Results. The NPgel integrated with bovine and human degenerate Nucleus Pulposus (NP) tissue and hMSCs produced matrix components: aggrecan,
This study aimed, through bioinformatics analysis and in vitro experiment validation, to identify the key extracellular proteins of intervertebral disc degeneration (IDD). The gene expression profile of GSE23130 was downloaded from the Gene Expression Omnibus (GEO) database. Extracellular protein-differentially expressed genes (EP-DEGs) were screened by protein annotation databases, and we used Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) to analyze the functions and pathways of EP-DEGs. STRING and Cytoscape were used to construct protein-protein interaction (PPI) networks and identify hub EP-DEGs. NetworkAnalyst was used to analyze transcription factors (TFs) and microRNAs (miRNAs) that regulate hub EP-DEGs. A search of the Drug Signatures Database (DSigDB) for hub EP-DEGs revealed multiple drug molecules and drug-target interactions.Aims
Methods
Background. Degeneration of the intervertebral disc (IVD) is a major cause of Low back pain. We have recently reported a novel, injectable liquid L-pNIPAM-co-DMAc hydrogel (NPgel), which promote differentiation of MSCs to nucleus pulposus (NP) cells without the need for additional growth factors. Here, we investigated the behaviour of hMSCs incorporated within the hydrogel injected into NP tissue. Methods. hMSCs were injected either alone or within NPgel, into bovine NP tissue explants and maintained at 5% O. 2. for up to 6wks. Media alone and acellular NPgel were also injected into NP explants to serve as controls. Cell viability was assessed by Caspase 3 immunohistochemistry and the phenotype of injected hMSC was assessed by histology and immunohistochemistry. Mechanical properties were also assessed via dynamic mechanical analysis (DMA). Results. No significant difference in the elastic modulus was observed between NPgel injected NP tissue and media injected controls. CFSe positive hMSCs were identified in all injected tissue samples and cell viability was maintained. Where hMSCs were delivered via NPgel, the hydrogel integrated with native NP tissue and cells producing NP matrix components: aggrecan;
Inflammatory response plays a pivotal role in the pathophysiological process of intervertebral disc degeneration (IDD). A20 (also known as tumour necrosis factor alpha-induced protein 3 (TNFAIP3)) is a ubiquitin-editing enzyme that restricts nuclear factor-kappa B (NF-κB) signalling. A20 prevents the occurrence of multiple inflammatory diseases. However, the role of A20 in the initiation of IDD has not been elucidated. The aim of the study was to investigate the effect of A20 in senescence of TNF alpha (TNF-α)-induced nucleus pulposus cells (NPCs). Immunohistochemical staining was performed to observe the expression of A20 in normal and degenerated human intervertebral discs. The NPCs were dissected from the tail vertebrae of healthy male Sprague-Dawley rats and were cultured in the incubator. In the experiment, TNF-α was used to mimic the inflammatory environment of IDD. The cell viability and senescence were examined to investigate the effect of A20 on TNF-α-treated NPCs. The expression of messenger RNA (mRNA)-encoding proteins related to matrix macromolecules (collagen II, aggrecan) and senescence markers (p53, p16). Additionally, NF-κB/p65 activity of NPCs was detected within different test compounds.Aims
Methods
Mesenchymal stem-cell based therapies have been
proposed as novel treatments for intervertebral disc degeneration,
a prevalent and disabling condition associated with back pain. The
development of these treatment strategies, however, has been hindered
by the incomplete understanding of the human nucleus pulposus phenotype
and by an inaccurate interpretation and translation of animal to
human research. This review summarises recent work characterising
the nucleus pulposus phenotype in different animal models and in
humans and integrates their findings with the anatomical and physiological
differences between these species. Understanding this phenotype
is paramount to guarantee that implanted cells restore the native
functions of the intervertebral disc. Cite this article:
