Single cell RNA sequencing reveals a shift in cell function and maturation of endogenous and infiltrating cell types in response to acute intervertebral disc injury.

Intervertebral disc (IVD) degeneration contributes to disabling back pain. Degeneration can be initiated by injury and progressively leads to irreversible cell loss and loss of IVD function. Attempts to restore IVD function through cell replacement therapies have had limited success due to knowledge gaps in critical cell populations and molecular crosstalk after injury. Here, we used single cell RNA sequencing to identify the transcriptional changes of endogenous and infiltrating IVD cell populations, as well as the potential of resident mesenchymal stem cells (MSCs) for tissue repair. Control and Injured (needle puncture) tail IVDs were extracted from 12 week old female C57BL/6 mice 7 days post injury and clustering analyses, gene ontology, and pseudotime trajectory analyses were used to determine transcriptomic divergences in the cells of the injured IVD, while immunofluorescence was utilized to determine mesenchymal stem cell (MSC) localization. Clustering analysis revealed 11 distinct cell populations that were IVD tissue specific, immune, or vascular cells. Differential gene expression analysis determined that Outer Annulus Fibrosus, Neutrophils, Saa2-High MSCs, Macrophages, and Krt18+ Nucleus Pulposus (NP) cells were the major drivers of transcriptomic differences between Control and Injured cells. Gene ontology of DEGs suggested that the most upregulated biological pathways were angiogenesis and T cell related while wound healing and ECM regulation categories were downregulated. Pseudotime trajectory analyses revealed that cells were driven towards increased cell differentiation due to IVD injury in all IVD tissue clusters except for Krt18+ NP which remained in a less mature cell state. Saa2-High and Grem1-High MSCs populations drifted towards more IVD differentiated cells profiles with injury and localized distinctly within the IVD. This study strengthens the understanding of heterogeneous IVD cell populations response to injury and identifies targetable MSC populations for future IVD repair studies.

Secreted chemokines reveal diverse inflammatory and degenerative processes in the intervertebral disc of the STZ-HFD mouse model of Type 2 diabetes.

The chronic inflammation present in type 2 diabetes causes many chronic inflammatory comorbidities, including cardiovascular, renal, and neuropathic complications. Type 2 diabetes is also associated with a number of spinal pathologies, including intervertebral disc (IVD) degeneration and chronic neck and back pain. Although confounding factors such as obesity are thought to increase the loads to the musculoskeletal system and subsequent degeneration, studies have shown that even after adjusting age, body mass index, and genetics (e.g. twins), patients with diabetes suffer from disproportionately more IVD degeneration and back pain. Yet the tissue-specific responses of the IVD during diabetes remains relatively unknown. We hypothesize that chronic diabetes fosters a proinflammatory microenvironment within the IVD that accelerates degeneration and increases susceptibility to painful disorders. To test this hypothesis, we evaluated two commonly used mouse models of diabetes - the leptin-receptor deficient mouse (db/db) and the chronic high-fat diet in mice with impaired beta-cell function (STZ-HFD). The db/db is a genetic model that spontaneous develop diabetes through hyperphagia, while the STZ-HFD mouse first exhibits rapid obesity development under HFD and pronounced insulin resistance following streptozotocin administration. Both animal models were allowed to develop sustained diabetes for at least twelve weeks, as defined by elevated hemoglobin A1C, hyperglycemia, and glucose intolerance. Following the twelve-week period, the IVDs were extracted in quantified in several measures including tissue-specific secreted cytokines, viscoelastic mechanical behavior, structural composition, and histopathologic degeneration. Although there were no differences in mechanical function or the overall structure of the IVD, the STZ-HFD IVDs were more degenerated. More notably, the STZ-HFD model shows a significantly higher fold increase for eight cytokines: CXCL2, CCL2, CCL3, CCL4, CCL12 (monocyte/macrophage associated), IL-2, CXCL9 (T-cell associated), and CCL5 (pleiotropic). Correlative network analyses revealed that the expression of cytokines differentially regulated between the db/db and the STZ-HFD models. Moreover, the STZ-HFD contained a fragmented and modular cytokine network, indicating greater complexities in the regulatory network. Taken together, the STZ-HFD model of type 2 diabetes may better recapitulate the complexities of the chronic inflammatory processes in the IVD during diabetes.

Analysis of Infiltrating Immune Cells Following Intervertebral Disc Injury Reveals Recruitment of Gamma-Delta (γδ) T cells in Female Mice.

Inadequate repair of injured intervertebral discs (IVD) leads to degeneration and contributes to low back pain. Infiltrating immune cells into damaged musculoskeletal tissues are critical mediators of repair, yet little is known about their identities, roles, and temporal regulation following IVD injury. By analyzing longitudinal changes in gene expression, tissue morphology, and the dynamics of infiltrating immune cells following injury, we characterize sex-specific differences in immune cell populations and identify the involvement of previously unreported immune cell types, γδ and NKT cells. Cd3+Cd4-Cd8- T cells are the largest infiltrating lymphocyte population with injury, and we identified the presence of γδ T cells in this population in female mice specifically, and NKT cells in males. Injury-mediated IVD degeneration was prevalent in both sexes, but more severe in males. Sex-specific degeneration may be associated with the differential immune response since γδ T cells have potent anti-inflammatory roles and may mediate IVD repair.

Post or perish? Social media strategies for disseminating orthopedic research.

Social media usage, particularly Twitter, among scientists in academia has increased in recent years. However, Twitter's use in scholarly post-publication dissemination of orthopaedic research and musculoskeletal advocacy remains low. To enhance usage of Twitter among musculoskeletal researchers, this article reviews data supporting the professional benefits of using the platform to disseminate scholarly works. Next, we provide a linear workflow for Tweet curation, discuss the importance of data-driven decision making behind tweet curation and posting, and propose new guidelines for professional Twitter usage. Since this workflow may not eliminate all the identified barriers and new institutionalized shifts in policies regarding curation and consumption of social media on Twitter, we also briefly introduce and explore using other social media platforms. We hope this information will be persuasive and compelling to those in the orthopedic research field and be broadly applicable to others in related scientific fields who wish to disseminate findings and engage a public audience on social media. In addition, we encourage the Orthopedic Research Society (ORS) and Journal of Orthopedic Research (JOR) communities to take advantage of the many tools curated by the Wiley editorial office and the ORS social media committee to increase dissemination of their scholarly works online. Twitter and social media can assist in accomplishing our mission of creating a world without musculoskeletal limitations via the timely dissemination of orthopedic information. However, this can only be accomplished if the orthopedic research community has a unified and strong online presence actively engaged in orthopaedic research findings and news.

Single Cell RNA-Sequence Analyses Reveal Uniquely Expressed Genes and Heterogeneous Immune Cell Involvement in the Rat Model of Intervertebral Disc Degeneration.

Intervertebral disc (IVD) degeneration is characterized by a loss of cellularity, and changes in cell-mediated activity that drives anatomic changes to IVD structure. In this study, we used single-cell RNA-sequencing analysis of degenerating tissues of the rat IVD following lumbar disc puncture. Two control, uninjured IVDs (L2-3, L3-4) and two degenerated, injured IVDs (L4-5, L5-6) from each animal were examined either at the two- or eight-week post-operative time points. The cells from these IVDs were extracted and transcriptionally profiled at the single-cell resolution. Unsupervised cluster analysis revealed the presence of four known cell types in both non-degenerative and degenerated IVDs based on previously established gene markers: IVD cells, endothelial cells, myeloid cells, and lymphoid cells. As a majority of cells were associated with the IVD cell cluster, sub-clustering was used to further identify the cell populations of the nucleus pulposus, inner and outer annulus fibrosus. The most notable difference between control and degenerated IVDs was the increase of myeloid and lymphoid cells in degenerated samples at two- and eight-weeks post-surgery. Differential gene expression analysis revealed multiple distinct cell types from the myeloid and lymphoid lineages, most notably macrophages and B lymphocytes, and demonstrated a high degree of immune specificity during degeneration. In addition to the heterogenous infiltrating immune cell populations in the degenerating IVD, the increased number of cells in the AF sub-cluster expressing Ngf and Ngfr, encoding for p75NTR, suggest that NGF signaling may be one of the key mediators of the IVD crosstalk between immune and neuronal cell populations. These findings provide the basis for future work to understand the involvement of select subsets of non-resident cells in IVD degeneration.

TGFß signaling is required for sclerotome resegmentation during development of the spinal column in Gallus gallus.

We previously showed the importance of TGFß signaling in development of the mouse axial skeleton. Here, we provide the first direct evidence that TGFß signaling is required for resegmentation of the sclerotome using chick embryos. Lipophilic fluorescent tracers, DiO and DiD, were microinjected into adjacent somites of embryos treated with or without TGFßRI inhibitors, SB431542, SB525334 or SD208, at developmental day E2.5 (HH16). Lineage tracing of labeled cells was observed over the course of 4 days until the completion of resegmentation at E6.5 (HH32). Vertebrae were malformed and intervertebral discs were small and misshapen in inhibitor injected embryos. Hypaxial myofibers were also increased in thickness after treatment with the inhibitor. Inhibition of TGFß signaling resulted in alterations in resegmentation that ranged between full, partial, and slanted shifts in distribution of DiO or DiD labeled cells within vertebrae. Patterning of rostro-caudal markers within sclerotome was disrupted at E3.5 after treatment with TGFßRI inhibitor with rostral domains expressing both rostral and caudal markers. We propose that TGFß signaling regulates rostro-caudal polarity and subsequent resegmentation in sclerotome during spinal column development.

Canonical and noncanonical TGF-ß signaling regulate fibrous tissue differentiation in the axial skeleton.

Previously, we showed that embryonic deletion of TGF-ß type 2 receptor in mouse sclerotome resulted in defects in fibrous connective tissues in the spine. Here we investigated how TGF-ß regulates expression of fibrous markers: Scleraxis, Fibromodulin and Adamtsl2. We showed that TGF-ß stimulated expression of Scleraxis mRNA by 2 h and Fibromodulin and Adamtsl2 mRNAs by 8 h of treatment. Regulation of Scleraxis by TGF-ß did not require new protein synthesis; however, protein synthesis was required for expression of Fibromodulin and Adamtsl2 indicating the necessity of an intermediate. We subsequently showed Scleraxis was a potential intermediate for TGF-ß-regulated expression of Fibromodulin and Adamtsl2. The canonical effector Smad3 was not necessary for TGF-ß-mediated regulation of Scleraxis. Smad3 was necessary for regulation of Fibromodulin and Adamtsl2, but not sufficient to super-induce expression with TGF-ß treatment. Next, the role of several noncanonical TGF-ß pathways were tested. We found that ERK1/2 was activated by TGF-ß and required to regulate expression of Scleraxis, Fibromodulin, and Adamtsl2. Based on these results, we propose a model in which TGF-ß regulates Scleraxis via ERK1/2 and then Scleraxis and Smad3 cooperate to regulate Fibromodulin and Adamtsl2. These results define a novel signaling mechanism for TGFß-mediated fibrous differentiation in sclerotome.