CD24 Positive Nucleus Pulposus Cells in Adult Human Intervertebral Discs Maintain a More Notochordal Phenotype Than GD2 Positive Cells.

Background: Notochordal cells (NCs) present in the nucleus pulposus (NP) of the developing human intervertebral disc (IVD) disappear during the first decade of life. This loss coincides with the onset of IVD degeneration, therefore these cells are hypothesized to be important in NP homeostasis. Putative NC-derived (CD24) and progenitor (TIE2/GD2) cell sub-populations have previously been identified in the adult human NP, but their characteristics have yet to be compared.

Progenitor-like cells contributing to cellular heterogeneity in the nucleus pulposus are lost in intervertebral disc degeneration.

The nucleus pulposus (NP) in the intervertebral disc (IVD) arises from embryonic notochord. Loss of notochordal-like cells in humans correlates with onset of IVD degeneration, suggesting that they are critical for healthy NP homeostasis and function. Comparative transcriptomic analyses identified expression of progenitor-associated genes (GREM1, KRT18, and TAGLN) in the young mouse and non-degenerated human NP, with TAGLN expression reducing with aging.

Engineered extracellular vesicle-based gene therapy for the treatment of discogenic back pain.

Painful musculoskeletal disorders such as intervertebral disc (IVD) degeneration associated with chronic low back pain (termed "Discogenic back pain", DBP), are a significant socio-economic burden worldwide and contribute to the growing opioid crisis. Yet there are very few if any successful interventions that can restore the tissue's structure and function while also addressing the symptomatic pain. Here we have developed a novel non-viral gene therapy, using engineered extracellular vesicles (eEVs) to deliver the developmental transcription factor FOXF1 to the degenerated IVD in an in vivo model.

Transcriptomic profiling reveals key early response genes during GDF6-mediated differentiation of human adipose-derived stem cells to nucleus pulposus cells.

Background: Stem cell-based therapies show promise as a means of repairing the degenerate intervertebral disc, with growth factors often used alongside cells to help direct differentiation toward a nucleus pulposus (NP)-like phenotype. We previously demonstrated adipose-derived stem cell (ASC) differentiation with GDF6 as optimal for generating NP-like cells through evaluating end-stage differentiation parameters. Here we conducted a time-resolved transcriptomic characterization of ASCs response to GDF6 stimulation to understand the early drivers of differentiation to NP-like cells.

Collagen-like Osteoclast-Associated Receptor (OSCAR)-Binding Motifs Show a Co-Stimulatory Effect on Osteoclastogenesis in a Peptide Hydrogel System.

Osteoclastogenesis, one of the dynamic pathways underlying bone remodelling, is a complex process that includes many stages. This complexity, while offering a wealth of therapeutic opportunities, represents a substantial challenge in unravelling the underlying mechanisms. As such, there is a high demand for robust model systems to understand osteoclastogenesis.

Mechanical loading and hyperosmolarity as a daily resetting cue for skeletal circadian clocks.

Daily rhythms in mammalian behaviour and physiology are generated by a multi-oscillator circadian system entrained through environmental cues (e.g. light and feeding).

Recommendations for intervertebral disc notochordal cell investigation: From isolation to characterization.

Background: Lineage-tracing experiments have established that the central region of the mature intervertebral disc, the nucleus pulposus (NP), develops from the embryonic structure called "the notochord". However, changes in the cells derived from the notochord which form the NP (i.e.

The clock transcription factor BMAL1 is a key regulator of extracellular matrix homeostasis and cell fate in the intervertebral disc.

The circadian clock in mammals temporally coordinates physiological and behavioural processes to anticipate daily rhythmic changes in their environment. Chronic disruption to circadian rhythms (e.g.

Harmonization and standardization of nucleus pulposus cell extraction and culture methods.

Background: In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab-to-lab variability jeopardizes the much-needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources.

Importance of Matrix Cues on Intervertebral Disc Development, Degeneration, and Regeneration.

Back pain is one of the leading causes of disability worldwide and is frequently caused by degeneration of the intervertebral discs. The discs' development, homeostasis, and degeneration are driven by a complex series of biochemical and physical extracellular matrix cues produced by and transmitted to native cells. Thus, understanding the roles of different cues is essential for designing effective cellular and regenerative therapies.

Incorporation of Natural and Recombinant Collagen Proteins within Fmoc-Based Self-Assembling Peptide Hydrogels.

Hydrogel biomaterials mimic the natural extracellular matrix through their nanofibrous ultrastructure and composition and provide an appropriate environment for cell-matrix and cell-cell interactions within their polymeric network. Hydrogels can be modified with different proteins, cytokines, or cell-adhesion motifs to control cell behavior and cell differentiation. Collagens are desirable and versatile proteins for hydrogel modification due to their abundance in the vertebrate extracellular matrix and their interactions with cell-surface receptors.

Peptide location fingerprinting identifies species- and tissue-conserved structural remodelling of proteins as a consequence of ageing and disease.

Extracellular matrices (ECMs) in the intervertebral disc (IVD), lung and artery are thought to undergo age-dependant accumulation of damage by chronic exposure to mechanisms such as reactive oxygen species, proteases and glycation. It is unknown whether this damage accumulation is species-dependant (via differing lifespans and hence cumulative exposures) or whether it can influence the progression of age-related diseases such as atherosclerosis. Peptide location fingerprinting (PLF) is a new proteomic analysis method, capable of the non-targeted identification of structure-associated changes within proteins.

Self-Assembling Peptide Hydrogels as Functional Tools to Tackle Intervertebral Disc Degeneration.

Low back pain (LBP), caused by intervertebral disc (IVD) degeneration, is a major contributor to global disability. In its healthy state, the IVD is a tough and well-hydrated tissue, able to act as a shock absorber along the spine. During degeneration, the IVD is hit by a cell-driven cascade of events, which progressively lead to extracellular matrix (ECM) degradation, chronic inflammation, and pain.

Restoring the dampened expression of the core clock molecule BMAL1 protects against compression-induced intervertebral disc degeneration.

The circadian clock participates in maintaining homeostasis in peripheral tissues, including intervertebral discs (IVDs). Abnormal mechanical loading is a known risk factor for intervertebral disc degeneration (IDD). Based on the rhythmic daily loading pattern of rest and activity, we hypothesized that abnormal mechanical loading could dampen the IVD clock, contributing to IDD.

Lumbar Intervertebral Disc Herniation: Annular Closure Devices and Key Design Requirements.

Lumbar disc herniation is one of the most common degenerative spinal conditions resulting in lower back pain and sciatica. Surgical treatment options include microdiscectomy, lumbar fusion, total disc replacement, and other minimally invasive approaches. At present, microdiscectomy procedures are the most used technique; however, the annulus fibrosus is left with a defect that without treatment may contribute to high reherniation rates and changes in the biomechanics of the lumbar spine.

Acidic and basic self-assembling peptide and peptide-graphene oxide hydrogels: characterisation and effect on encapsulated nucleus pulposus cells.

Extracellular pH can have a profound effect on cell metabolism, gene and protein expression. Nucleus pulposus (NP) cells, for example, under acidic conditions accelerate the production of degradative enzymes and pro-inflammatory cytokines, leading ultimately to intervertebral disc degeneration, a major cause of back pain. Self-assembling peptide hydrogels constitute a well-established class of biomaterials that could be exploited as pH-tunable platform to investigate cell behaviour under normal and non-physiological pH.

Hydroxyapatite-decorated Fmoc-hydrogel as a bone-mimicking substrate for osteoclast differentiation and culture.

Hydrogels are water-swollen networks with great potential for tissue engineering applications. However, their use in bone regeneration is often hampered due to a lack of materials' mineralization and poor mechanical properties. Moreover, most studies are focused on osteoblasts (OBs) for bone formation, while osteoclasts (OCs), cells involved in bone resorption, are often overlooked.

Regional variations in discrete collagen fibre mechanics within intact intervertebral disc resolved using synchrotron computed tomography and digital volume correlation.

Many soft tissues, such as the intervertebral disc (IVD), have a hierarchical fibrous composite structure which suffers from regional damage. We hypothesise that these tissue regions have distinct, inherent fibre structure and structural response upon loading. Here we used synchrotron computed tomography (sCT) to resolve collagen fibre bundles (∼5μm width) in 3D throughout an intact native rat lumbar IVD under increasing compressive load.

Peptide Location Fingerprinting Reveals Tissue Region-Specific Differences in Protein Structures in an Ageing Human Organ.

In ageing tissues, long-lived extracellular matrix (ECM) proteins are susceptible to the accumulation of structural damage due to diverse mechanisms including glycation, oxidation and protease cleavage. Peptide location fingerprinting (PLF) is a new mass spectrometry (MS) analysis technique capable of identifying proteins exhibiting structural differences in complex proteomes. PLF applied to published young and aged intervertebral disc (IVD) MS datasets (posterior, lateral and anterior regions of the annulus fibrosus) identified 268 proteins with age-associated structural differences.

Role of OSCAR Signaling in Osteoclastogenesis and Bone Disease.

Formation of mature bone-resorbing cells through osteoclastogenesis is required for the continuous remodeling and repair of bone tissue. In aging and disease this process may become aberrant, resulting in excessive bone degradation and fragility fractures. Interaction of receptor-activator of nuclear factor-κB (RANK) with its ligand RANKL activates the main signaling pathway for osteoclastogenesis.

TGF-β3-loaded graphene oxide - self-assembling peptide hybrid hydrogels as functional 3D scaffolds for the regeneration of the nucleus pulposus.

Intervertebral disc (IVD) degeneration is a process that starts in the central nucleus pulposus (NP) and leads to inflammation, extracellular matrix (ECM) degradation, and progressive loss of disc height. Early treatment of IVD degeneration is critical to the reduction of low back pain and related disability. As such, minimally invasive therapeutic approaches that can halt and reverse NP degeneration at the early stages of the disease are needed.

Circadian time series proteomics reveals daily dynamics in cartilage physiology.

Objective: Cartilage in joints such as the hip and knee experiences repeated phases of heavy loading and low load recovery during the 24-h day/night cycle. Our previous work has shown 24 h rhythmic changes in gene expression at transcript level between night and day in wild type mouse cartilage which is lost in a circadian clock knock-out mouse model. However, it remains unknown to what extent circadian rhythms also regulate protein level gene expression in this matrix rich tissue.

Site-Directed Differentiation of Human Adipose-Derived Mesenchymal Stem Cells to Nucleus Pulposus Cells Using an Injectable Hydroxyl-Functional Diblock Copolymer Worm Gel.

Adipose-derived mesenchymal stem cells (ASCs) have been identified for their promising therapeutic potential to regenerate and repopulate the degenerate intervertebral disk (IVD), which is a major cause of lower back pain. The optimal cell delivery system remains elusive but encapsulation of cells within scaffolds is likely to offer a decisive advantage over the delivery of cells in solution by ensuring successful retention within the tissue. Herein, we evaluate the use of a fully synthetic, thermoresponsive poly(glycerol monomethacrylate)-poly(2-hydroxypropyl methacrylate) (PGMA-PHPMA) diblock copolymer worm gel that mimics the structure of hydrophilic glycosaminoglycans.

Non-viral reprogramming of human nucleus pulposus cells with FOXF1 via extracellular vesicle delivery: an in vitro and in vivo study.

Intervertebral disc (IVD) degeneration is characterized by decreased cellularity and proteoglycan synthesis and increased inflammation, catabolism, and neural/vascular ingrowth. Regenerative methods for IVD degeneration are largely cell-therapy-based or involve viral vectors, which are associated with mutagenesis and undesired immune responses. The present study used bulk electroporation and engineered extracellular vesicles (EVs) to deliver forkhead-box F1 (FOXF1) mRNA to degenerate human nucleus pulposus (NP) cells as a minimally invasive therapeutic strategy for IVD regeneration.