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Study Design.Porcine intervertebral discs (IVDs) were excised and then drilled to simulate degeneration before being electrically stimulated for 21 days while undergoing mechanical loading. The discs were then analyzed for gene expression and morphology to assess regeneration. Objective.The purpose of this study was to investigate the effectiveness of the electrical stimulation of IVD treatment as an early intervention method in halting the progression of degenerative disc disease using an ex-vivo porcine model. Summary of Background Data.Treatments for degenerative disc disease are limited in their efficacy and tend to treat the symptoms of the disease rather than repairing the degenerated disc itself. There is a dire need for an early intervention treatment that not only halts the progression of the disease but contributes to reviving the degenerated disc. Methods.Lumbar IVDs were extracted from a mature pig within 1 hour of death and were drilled with a 1.5 mm bit to simulate degenerative disc disease. Four IVDs at a time were then cultured in a dynamic bioreactor system under mechanical loading for 21 days, two with and two without the electrical stimulation treatment. The IVDs were assessed using histological analysis, magnetic resonance imaging, and quantitative reverse transcriptase polymerase chain reaction to quantify the effectiveness of the treatment on the degenerated discs. Results.IVDs with electrical stimulation treatment exhibited extensive annular regeneration and prevented herniation of the nucleus pulposus (NP). In contrast, the untreated group of IVDs were unable to maintain tissue integrity and exhibited NP herniation through multiple layers of the annulus fibrosus. Gene expression showed an increase of extracellular matrix markers and antiinflammatory cytokine interleukin-4 (IL-4), while decreasing in pro-inflammatory markers and pain markers in electrically stimulated IVDs when compared to the untreated group. Conclusion.The direct electrical stimulation application in NP of damaged IVDs can be a viable option to regenerate damaged NP and annulus fibrosus tissues.
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Enzymatic Digestion of the Intervertebral Disc Alters Intradiscal Injection and Leakage Mechanics
Abstract 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.
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- Award ID(s):
- 2138342
- PAR ID:
- 10629295
- Publisher / Repository:
- ASME
- Date Published:
- Journal Name:
- Journal of Biomechanical Engineering
- Volume:
- 146
- Issue:
- 11
- ISSN:
- 0148-0731
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract BackgroundLow back pain is a leading cause of disability worldwide and is frequently attributed to intervertebral disc (IVD) degeneration. Though the contributions of the adjacent cartilage endplates (CEP) to IVD degeneration are well documented, the phenotype and functions of the resident CEP cells are critically understudied. To better characterize CEP cell phenotype and possible mechanisms of CEP degeneration, bulk and single-cell RNA sequencing of non-degenerated and degenerated CEP cells were performed. MethodsHuman lumbar CEP cells from degenerated (Thompson grade ≥ 4) and non-degenerated (Thompson grade ≤ 2) discs were expanded for bulk (N=4 non-degenerated,N=4 degenerated) and single-cell (N=1 non-degenerated,N=1 degenerated) RNA sequencing. Genes identified from bulk RNA sequencing were categorized by function and their expression in non-degenerated and degenerated CEP cells were compared. A PubMed literature review was also performed to determine which genes were previously identified and studied in the CEP, IVD, and other cartilaginous tissues. For single-cell RNA sequencing, different cell clusters were resolved using unsupervised clustering and functional annotation. Differential gene expression analysis and Gene Ontology, respectively, were used to compare gene expression and functional enrichment between cell clusters, as well as between non-degenerated and degenerated CEP samples. ResultsBulk RNA sequencing revealed 38 genes were significantly upregulated and 15 genes were significantly downregulated in degenerated CEP cells relative to non-degenerated cells (|fold change| ≥ 1.5). Of these, only 2 genes were previously studied in CEP cells, and 31 were previously studied in the IVD and other cartilaginous tissues. Single-cell RNA sequencing revealed 11 unique cell clusters, including multiple chondrocyte and progenitor subpopulations with distinct gene expression and functional profiles. Analysis of genes in the bulk RNA sequencing dataset showed that progenitor cell clusters from both samples were enriched in “non-degenerated” genes but not “degenerated” genes. For both bulk- and single-cell analyses, gene expression and pathway enrichment analyses highlighted several pathways that may regulate CEP degeneration, including transcriptional regulation, translational regulation, intracellular transport, and mitochondrial dysfunction. ConclusionsThis thorough analysis using RNA sequencing methods highlighted numerous differences between non-degenerated and degenerated CEP cells, the phenotypic heterogeneity of CEP cells, and several pathways of interest that may be relevant in CEP degeneration.more » « less
