Editorial: Immunological imbalance: What is its role in intervertebral disc degeneration?

Low back pain (LBP) is a major cause of disability worldwide (GBD, 2017 Disease and Injury Incidence and Prevalence Collaborators, 2018) and belongs to the most urgent priorities to identify novel therapies, especially for the elderly (Teichtahl et al., 2015; Chen et al., 2020; Lee et al., 2021). Global health costs are generally rising but in the field of orthopedics and mostly for spine, the costs are exploding in recent years compared to other diseases (Wieser et al., 2011; Martin et al., 2019). Surgical options to treat LBP efficiently are very limited; clinical outcome is often non-satisfactory for the patients with very high re-operation rates (Nachemson et al., 1996; Knezevic et al., 2021).

The evolutionary importance of cell ratio between notochordal and nucleus pulposus cells: an experimental 3-D co-culture study

Introduction: Notochordal cells and nucleus pulposus cells are co-existing in the intervertebral disc at various ratios among different mammalians. This fact rises the question about the interactions and the evolutionary relevance of this phenomenon. It has been described that these relatively large notochordal cells are mainly dominant in early lifetime of all vertebrates and then differences occur with ageing. Human, cattle, sheep, and goat lose the cells with age, whereas rodents and lagomorphs maintain these throughout their lifetime. Materials and methods: Here, we addressed the importance of cell ratio using alginate bead 3-D co-culture of bovine nucleus pulposus cells (bNPC) and porcine notochordal cells (pNCs) for 14days using culture inserts. Result: We found a significant stimulation of bNPC in the presence of pNC in terms of cell activity and glycosaminoglycan production, but not for proliferation (DNA content). Relative gene expression was significantly stimulated for collagen type 2 and aggrecan. Conclusion: The stimulating effect of NC was confirmed and the ideal ratio of NPC: NC was found to be ~50:50. This has direct implications for tissue-engineering approaches, which aim to repopulate discs with NP-like precursor cell

Erratum to: The effects of dynamic loading on the intervertebral disc

Loading is important to maintain the balance of matrix turnover in the intervertebral disc (IVD). Daily cyclic diurnal assists in the transport of large soluble factors across the IVD and its surrounding circulation and applies direct and indirect stimulus to disc cells. Acute mechanical injury and accumulated overloading, however, could induce disc degeneration. Recently, there is more information available on how cyclic loading, especially axial compression and hydrostatic pressure, affects IVD cell biology. This review summarises recent studies on the response of the IVD and stem cells to applied cyclic compression and hydrostatic pressure. These studies investigate the possible role of loading in the initiation and progression of disc degeneration as well as quantifying a physiological loading condition for the study of disc degeneration biological therapy. Subsequently, a possible physiological/beneficial loading range is proposed. This physiological/beneficial loading could provide insight into how to design loading regimes in specific system for the testing of various biological therapies such as cell therapy, chemical therapy or tissue engineering constructs to achieve a better final outcome. In addition, the parameter space of ‘physiological' loading may also be an important factor for the differentiation of stem cells towards most ideally ‘discogenic' cells for tissue engineering purpos

Exploring a Novel Spheroid 3D Cell Culture System for Tie2+ Nucleus Pulposus Cells of the Intervertebral Disc

Introduction Low back pain (LBP) is a big problem in today's Western European society1. LBP impacts the patient's quality of life and places an immense burden on the healthcare system worldwide2,3. Degeneration of the intervertebral disc (IVD) is one of the common causes of LBP4. Recently, nucleus pulposus (NP) progenitor cells (NPPC) were discovered, which are positive for Angiopoietin-1 receptor (aka. Tie2/CD202b). These NPPCs are a promising cell source for IVD regeneration5,6. NPPCs are rare (2-10% of all IVD cells) in human IVDs and diminish in number with increasing age6. It has been demonstrated that 3D culture is superior to classic 2D culture to maintain the pluripotent phenotype of the NPPCs7-9. The goal of this research project is to test the expansion and culture of bovine NPPC in a novel spheroid plate. Methods NP tissue was isolated from bovine tails (aged 10-14 months). NP cells were isolated by using a mild two-step digestion protocol. Then, the primary NP cells were sorted by fluorescence activated cell sorting (FACS). NP cells were expanded for two weeks under hypoxia (2% O2) and were supplemented with 2.5 ng/ml basic fibroblast growth factor 2(bFGF2) (fig. 1). Tie2+ and Tie2- cells were then seeded in the functionalized and the 2D control wells of the Sphericalplate 5D (SP5D) of Kugelmeier, Ltd (fig. 2). The cells were cultured for two weeks under hypoxia and with bFGF2. A colony forming unit-assay was performed on day 0. Cell activity, DNA/GAG content and gene expression of selected genes were measured on day one, eight, and 15. Results Cell isolation from NP tissue yielded on average 15.7 million primary cells, of which ~10 k cells (0.063%) were positive for Tie2. Assessing the colony-forming ability of expanded cells revealed no significant differences between Tie2+ and Tie2- cells. Cells in 2D plastic control wells proliferated significantly more compared to the spheroids (p = 0.0002). Analyzing gene expression after expansion revealed a five-fold increase of KRT19 and a four-fold increase of SOX2 in Tie2+ cells in relation to Tie2- cells. Gene expression of KRT19 and SOX2 was found to be significantly downregulated in Tie2+ cells in spheroid and 2D culture after 15 days (fig. 3). Discussion The results indicate that sorted NPPSs differentiated during expansion, resulting in a low share of Tie2+ cells. Unexpectedly, successive culture in the SP5D revealed that such expanded Tie2+ cells could not be kept in a non-differentiated stem-cell like state although such speriod-like cultures have been promising for many stem cells from various tissues and also in Tie2+ cells7,8. The biological nature of the NPPCs are undeniably a promising approach for therapy of the degenerated IVDs. Acknowledgments This research was supported by project a Swiss National Science Bridge Discovery Project # 40B2-0_211510/1 (https://data.snf.ch/grants/grant/211510)

A comparative analysis of tie2-positive IVD progenitor cells in single-cell and bulk transcriptomics.

Introduction: Back pain and disability are often attributed to intervertebral disc (IVD) degeneration1, where current treatments are limited by an incomplete understanding of IVD biology. This study focuses on the Angiopoietin-1 receptor Tie2, which marks a progenitor cell subset in the nucleus pulposus (NP)2, crucial for repair and regeneration3,4. We aim to elucidate the transcriptomic profile of these Tie2-positive NP progenitor cells (NPPCs) to understand their role in IVD homeostasis and repair. Methods: We utilized single-cell and bulk RNA sequencing to characterize the transcriptomic profiles of Tie2-positive NPPCs from bovine and human IVDs. For single-cell sequencing, reanalysis was conducted on the dataset published by Caliò et al. (2021)5, which included samples comprising pooled cells from three adjacent discs of either distal or proximal region of the bovine coccygeal tail of two biological replicates. Single-cell suspension was obtained through enzymatic dissociation using Collagenase Type II. Library preparation followed the single-cell 3’-version-3-protocol6 and sequencing was performed on the Illumina NovaSeq platform. The sequencing data were aligned to the Bos taurus reference genome (UMD-v3.1 Release-92) from Ensembl. Post-sequencing analysis involved cell cluster identification using the Seurat package. In parallel, bulk RNA sequencing was conducted on human IVD samples. Cells were isolated, and Tie2-positive cells were obtained by subsequent fluorescent activated cell sorting (FACS). These cells were subsequently expanded for one week. We compared Tie2-enriched samples from four healthy individuals, to Tie2-negative samples from three healthy individuals using the NovaSeq system. Differential expression analysis was carried out using DESeq2 to pinpoint marker genes specific to Tie2-enrichment. Furthermore, we performed Pearson correlation analysis contrasting the expression profiles of each identified single-cell cluster against the average profile of the Tie2-enriched samples, which facilitated the delineation of potential NPPC clusters. Results: We identified 14 distinct cell clusters (Fig. 1), underscoring NP cellular heterogeneity. Although not expressing a significant higher expression of the TEK gene, the gene for Tie2, Tie2-positive NPPCs exhibited a unique gene expression profile when compared to Tie2-negative cells, with specific marker genes identified. Through correlation analysis, these NPPCs were associated with specific clusters, suggesting a specialized “niche” in the IVD. Enrichment analysis indicated their involvement in tissue homeostasis and regeneration

The first molecular phylogeny of Buthidae (Scorpiones)

The first partial phylogeny of family Buthidae (17 genera) is presented, based on molecular data (16S rRNA mitochondrial DNA). The strong support for a monophyletic Old World group of 13 genera (mainly Palearctic desert forms) is demonstrated, while representative genera from Madagascar (Grosphus) and Southeast Asia (Lychas) group outside, as well as New World genera Centruroides and Rhopalurus. A very strong support is observed for the first time for three groups of Old World genera: (a) Compsobuthus, Mesobuthus, Liobuthus, Kraepelinia; (b) Hottentotta, Buthacus; (c) Orthochirus, Anomalobuthus. Phylogenetic hypotheses are discussed

The effects of dynamic loading on the intervertebral disc

Loading is important to maintain the balance of matrix turnover in the intervertebral disc (IVD). Daily cyclic diurnal assists in the transport of large soluble factors across the IVD and its surrounding circulation and applies direct and indirect stimulus to disc cells. Acute mechanical injury and accumulated overloading, however, could induce disc degeneration. Recently, there is more information available on how cyclic loading, especially axial compression and hydrostatic pressure, affects IVD cell biology. This review summarises recent studies on the response of the IVD and stem cells to applied cyclic compression and hydrostatic pressure. These studies investigate the possible role of loading in the initiation and progression of disc degeneration as well as quantifying a physiological loading condition for the study of disc degeneration biological therapy. Subsequently, a possible physiological/beneficial loading range is proposed. This physiological/beneficial loading could provide insight into how to design loading regimes in specific system for the testing of various biological therapies such as cell therapy, chemical therapy or tissue engineering constructs to achieve a better final outcome. In addition, the parameter space of 'physiological' loading may also be an important factor for the differentiation of stem cells towards most ideally 'discogenic' cells for tissue engineering purpose

The first molecular phylogeny of Buthidae (Scorpiones)

The first partial phylogeny of family Buthidae (17 genera) is presented, based on molecular data (16S rRNA mitochondrial DNA). The strong support for a monophyletic Old World group of 13 genera (mainly Palearctic desert forms) is demonstrated, while representative genera from Madagascar (Grosphus) and Southeast Asia (Lychas) group outside, as well as New World genera Centruroides and Rhopalurus. A very strong support is observed for the first time for three groups of Old World genera: (a) Compsobuthus, Mesobuthus, Liobuthus, Kraepelinia; (b) Hottentotta, Buthacus; (c) Orthochirus, Anomalobuthus. Phylogenetic hypotheses are discussed

The interplay between biochemical mediators and mechanotransduction in chondrocytes: Unravelling the differential responses in primary knee osteoarthritis.

In primary or idiopathic osteoarthritis (OA), it is unclear which factors trigger the shift of articular chondrocyte activity from pro-anabolic to pro-catabolic. In fact, there is a controversy about the aetiology of primary OA, either mechanical or inflammatory. Chondrocytes are mechanosensitive cells, that integrate mechanical stimuli into cellular responses in a process known as mechanotransduction. Mechanotransduction occurs thanks to the activation of mechanosensors, a set of specialized proteins that convert physical cues into intracellular signalling cascades. Moderate levels of mechanical loads maintain normal tissue function and have anti-inflammatory effects. In contrast, mechanical over- or under-loading might lead to cartilage destruction and increased expression of pro-inflammatory cytokines. Simultaneously, mechanotransduction processes can regulate and be regulated by pro- and anti-inflammatory soluble mediators, both local (cells of the same joint, i.e., the chondrocytes themselves, infiltrating macrophages, fibroblasts or osteoclasts) and systemic (from other tissues, e.g., adipokines). Thus, the complex process of mechanotransduction might be altered in OA, so that cartilage-preserving chondrocytes adopt a different sensitivity to mechanical signals, and mechanic stimuli positively transduced in the healthy cartilage may become deleterious under OA conditions. This review aims to provide an overview of how the biochemical exposome of chondrocytes can alter important mechanotransduction processes in these cells. Four principal mechanosensors, i.e., integrins, Ca2+ channels, primary cilium and Wnt signalling (canonical and non-canonical) were targeted. For each of these mechanosensors, a brief summary of the response to mechanical loads under healthy or OA conditions is followed by a concise overview of published works that focus on the further regulation of the mechanotransduction pathways by biochemical factors. In conclusion, this paper discusses and explores how biological mediators influence the differential behaviour of chondrocytes under mechanical loads in healthy and primary OA