Modulation of TRPV4 protects against degeneration induced by sustained loading and promotes matrix synthesis in the intervertebral disc.

While it is well known that mechanical signals can either promote or disrupt intervertebral disc (IVD) homeostasis, the molecular mechanisms for transducing mechanical stimuli are not fully understood. The transient receptor potential vanilloid 4 (TRPV4) ion channel activated in isolated IVD cells initiates extracellular matrix (ECM) gene expression, while TRPV4 ablation reduces cytokine production in response to circumferential stretching. However, the role of TRPV4 on ECM maintenance during tissue-level mechanical loading remains unknown. Using an organ culture model, we modulated TRPV4 function over both short- (hours) and long-term (days) and evaluated the IVDs' response. Activating TRPV4 with the agonist GSK101 resulted in a Ca2+ flux propagating across the cells within the IVD. Nuclear factor (NF)-κB signaling in the IVD peaked at 6 h following TRPV4 activation that subsequently resulted in higher interleukin (IL)-6 production at 7 days. These cellular responses were concomitant with the accumulation of glycosaminoglycans and increased hydration in the nucleus pulposus that culminated in higher stiffness of the IVD. Sustained compressive loading of the IVD resulted in elevated NF-κB activity, IL-6 and vascular endothelial growth factor A (VEGFA) production, and degenerative changes to the ECM. TRPV4 inhibition using GSK205 during loading mitigated the changes in inflammatory cytokines, protected against IVD degeneration, but could not prevent ECM disorganization due to mechanical damage in the annulus fibrosus. These results indicate TRPV4 plays an important role in both short- and long-term adaptations of the IVD to mechanical loading. The modulation of TRPV4 may be a possible therapeutic for preventing load-induced IVD degeneration.

Bullying, harassment, and discrimination of musculoskeletal researchers and the impact of the COVID-19 pandemic: an international study.

PURPOSE: Bullying, harassment, and discrimination (BHD) are prevalent in academic, scientific, and clinical departments, particularly orthopedic surgery, and can have lasting effects on victims. As it is unclear how BHD affects musculoskeletal (MSK) researchers, the following study assessed BHD in the MSK research community and whether the COVID-19 pandemic, which caused hardships in other industries, had an impact. METHODS: A web-based anonymous survey was developed in English by ORS Spine Section members to assess the impact of COVID-19 on MSK researchers in North America, Europe, and Asia, which included questions to evaluate the personal experience of researchers regarding BHD. RESULTS: 116 MSK researchers completed the survey. Of respondents, 34.5% (n = 40) focused on spine, 30.2% (n = 35) had multiple areas of interest, and 35.3% (n = 41) represented other areas of MSK research. BHD was observed by 26.7% (n = 31) of respondents and personally experienced by 11.2% (n = 13), with mid-career faculty both observing and experiencing the most BHD. Most who experienced BHD (53.8%, n = 7) experienced multiple forms. 32.8% (n = 38) of respondents were not able to speak out about BHD without fear of repercussions, with 13.8% (n = 16) being unsure about this. Of those who observed BHD, 54.8% (n = 17) noted that the COVID-19 pandemic had no impact on their observations. CONCLUSIONS: To our knowledge, this is the first study to address the prevalence and determinants of BHD among MSK researchers. MSK researchers experienced and observed BHD, while many were not comfortable reporting and discussing violations to their institution. The COVID-19 pandemic had mixed-effects on BHD. Awareness and proactive policy changes may be warranted to reduce/eliminate the occurrence of BHD in this community.

Dysregulation of STAT3 signaling is associated with endplate-oriented herniations of the intervertebral disc in Adgrg6 mutant mice.

Degenerative changes of the intervertebral disc (IVD) are a leading cause of disability affecting humans worldwide and has been attributed primarily to trauma and the accumulation of pathology during aging. While genetic defects have also been associated with disc degeneration, the precise mechanisms driving the initiation and progression of disease have remained elusive due to a paucity of genetic animal models. Here, we discuss a novel conditional mouse genetic model of endplate-oriented disc herniations in adult mice. Using conditional mouse genetics, we show increased mechanical stiffness and reveal dysregulation of typical gene expression profiles of the IVD in adhesion G-protein coupled receptor G6 (Adgrg6) mutant mice prior to the onset of endplate-oriented disc herniations in adult mice. We observed increased STAT3 activation prior to IVD defects and go on to demonstrate that treatment of Adgrg6 conditional mutant mice with a small molecule inhibitor of STAT3 activation ameliorates endplate-oriented herniations. These findings establish ADGRG6 and STAT3 as novel regulators of IVD endplate and growth plate integrity in the mouse, and implicate ADGRG6/STAT3 signaling as promising therapeutic targets for endplate-oriented disc degeneration.

Development and validation of the CHIME simulation model to assess lifetime health outcomes of prediabetes and type 2 diabetes in Chinese populations: A modeling study.

BACKGROUND: Existing predictive outcomes models for type 2 diabetes developed and validated in historical European populations may not be applicable for East Asian populations due to differences in the epidemiology and complications. Despite the continuum of risk across the spectrum of risk factor values, existing models are typically limited to diabetes alone and ignore the progression from prediabetes to diabetes. The objective of this study is to develop and externally validate a patient-level simulation model for prediabetes and type 2 diabetes in the East Asian population for predicting lifetime health outcomes. METHODS AND FINDINGS: We developed a health outcomes model from a population-based cohort of individuals with prediabetes or type 2 diabetes: Hong Kong Clinical Management System (CMS, 97,628 participants) from 2006 to 2017. The Chinese Hong Kong Integrated Modeling and Evaluation (CHIME) simulation model comprises of 13 risk equations to predict mortality, micro- and macrovascular complications, and development of diabetes. Risk equations were derived using parametric proportional hazard models. External validation of the CHIME model was assessed in the China Health and Retirement Longitudinal Study (CHARLS, 4,567 participants) from 2011 to 2018 for mortality, ischemic heart disease, cerebrovascular disease, renal failure, cataract, and development of diabetes; and against 80 observed endpoints from 9 published trials using 100,000 simulated individuals per trial. The CHIME model was compared to United Kingdom Prospective Diabetes Study Outcomes Model 2 (UKPDS-OM2) and Risk Equations for Complications Of type 2 Diabetes (RECODe) by assessing model discrimination (C-statistics), calibration slope/intercept, root mean square percentage error (RMSPE), and R2. CHIME risk equations had C-statistics for discrimination from 0.636 to 0.813 internally and 0.702 to 0.770 externally for diabetes participants. Calibration slopes between deciles of expected and observed risk in CMS ranged from 0.680 to 1.333 for mortality, myocardial infarction, ischemic heart disease, retinopathy, neuropathy, ulcer of the skin, cataract, renal failure, and heart failure; 0.591 for peripheral vascular disease; 1.599 for cerebrovascular disease; and 2.247 for amputation; and in CHARLS outcomes from 0.709 to 1.035. CHIME had better discrimination and calibration than UKPDS-OM2 in CMS (C-statistics 0.548 to 0.772, slopes 0.130 to 3.846) and CHARLS (C-statistics 0.514 to 0.750, slopes -0.589 to 11.411); and small improvements in discrimination and better calibration than RECODe in CMS (C-statistics 0.615 to 0.793, slopes 0.138 to 1.514). Predictive error was smaller for CHIME in CMS (RSMPE 3.53% versus 10.82% for UKPDS-OM2 and 11.16% for RECODe) and CHARLS (RSMPE 4.49% versus 14.80% for UKPDS-OM2). Calibration performance of CHIME was generally better for trials with Asian participants (RMSPE 0.48% to 3.66%) than for non-Asian trials (RMPSE 0.81% to 8.50%). Main limitations include the limited number of outcomes recorded in the CHARLS cohort, and the generalizability of simulated cohorts derived from trial participants. CONCLUSIONS: Our study shows that the CHIME model is a new validated tool for predicting progression of diabetes and its outcomes, particularly among Chinese and East Asian populations that has been lacking thus far. The CHIME model can be used by health service planners and policy makers to develop population-level strategies, for example, setting HbA1c and lipid targets, to optimize health outcomes.

The age-dependent effect of high-dose X-ray radiation on NFκB signaling, structure, and mechanical behavior of the intervertebral disc.

Purpose: Ionizing radiation damages tissue and provokes inflammatory responses in multiple organ systems. We investigated the effects of high-dose X-ray radiation on the molecular inflammation and mechanical function of the intervertebral disc (IVD).Methods: Functional spine units (FSUs) containing the vertebrae-IVDs-vertebrae structure extracted from 1-month, 6-month, and 16-month-old NFκB-luciferase reporter mice and from 6-month-old myeloid differentiation factor 88 (MyD88)-null mice. After a preconditioning period in culture, the FSUs were subjected a single dose of ionizing X-ray radiation at 20 Gys, and then NFκB expression was monitored. The IVDs were then subjected to mechanical testing using dynamic compression, glycosaminoglycan (GAG) quantification, and histological analyses.Results: In the 1-month-old FSUs, the NFκB-driven luciferase activity was significantly elevated for 1 day following the exposure to radiation. The 6-month-old FSUs showed increased NFκB activity for 3 days, while the 16-month-old FSUs sustained elevated levels of NFκB activity throughout the 10-day culture period. All irradiated groups showed significant loss of disc height, GAG content, mechanical function and changes in structure. Ablation of MyD88 blunted the radiation-mediated NFκB signaling, and preserved GAG content, and the IVDs' structure and mechanical performance.Conclusions: These results suggest that high-dose radiation affects the IVDs' NFκB-dependent inflammatory processes that subsequently lead to functional deterioration. Blocking the transactivation potential of NFκB via MyD88 ablation preserved the structure and mechanical function of the FSUs. The long-term effects of radiation on IVD homeostasis should be considered in individuals susceptible to occupational and medical exposure.

Clinical measurements of bone tissue mechanical behavior using reference point indentation.

Over the last thirty years, it has become increasingly clear the amount of bone (e.g. 'bone quantity') and the quality of the bone matrix (e.g. 'bone quality') both critically contribute to bone's tissue-level mechanical behavior and the subsequent ability of bone to resist fracture. Although determining the tissue-level mechanical behavior of bone through mechanical testing is relatively straightforward in the laboratory, the destructive nature of such testing is unfeasible in humans and in animal models requiring longitudinal observation. Therefore, surrogate measurements are necessary for quantifying tissue-level mechanical behavior for the pre-clinical and clinical evaluation of bone strength and fracture risk in vivo. A specific implementation of indentation known as reference point indentation (RPI) enables the mechanical testing of bone tissue without the need to excise and prepare the bone surface. However, this compromises the ability to carefully control the specimen geometry that is required to define the bone tissue material properties. Yet the versatility of such measurements in clinical populations is provocative, and to date there are a number of promising studies that have utilized this tool to discern bone pathologies and to monitor the effects of therapeutics on bone quality. Concurrently, on-going efforts continue to investigate the aspects of bone material behavior measured by RPI, and the compositional factors that contribute to these measurements. There are currently two variants, cyclic- and impact- RPI, that have been utilized in pre-clinical and clinical studies. This review surveys clinical studies that utilize RPI, with particular emphasis on the clinical instrument, as well as the endeavors to understand the fundamental mechanisms of such measurements. Ultimately, an improved awareness in the tradeoffs and limitations of in vivo RPI is critical towards the effective and successful utilization of this tool for the overall improvement of fragility determination in the clinic.

Novel Augmentation Technique for Patellar Tendon Repair Improves Strength and Decreases Gap Formation: A Cadaveric Study.

BACKGROUND: Patellar tendon ruptures commonly are repaired using transosseous patellar drill tunnels with modified-Krackow sutures in the patellar tendon. This simple suture technique has been associated with failure rates and poor clinical outcomes in a modest proportion of patients. Failure of this repair technique can result from gap formation during loading or a single catastrophic event. Several augmentation techniques have been described to improve the integrity of the repair, but standardized biomechanical evaluation of repair strength among different techniques is lacking. QUESTIONS/PURPOSES: The purpose of this study was to describe a novel figure-of-eight suture technique to augment traditional fixation and evaluate its biomechanical performance. We hypothesized that the augmentation technique would (1) reduce gap formation during cyclic loading and (2) increase the maximum load to failure. METHODS: Ten pairs (two male, eight female) of fresh-frozen cadaveric knees free of overt disorders or patellar tendon damage were used (average donor age, 76 years; range, 65-87 years). For each pair, one specimen underwent the standard transosseous tunnel suture repair with a modified-Krackow suture technique and the second underwent the standard repair with our experimental augmentation method. Nine pairs were suitable for testing. Each specimen underwent cyclic loading while continuously measuring gap formation across the repair. At the completion of cyclic loading, load to failure testing was performed. RESULTS: A difference in gap formation and mean load to failure was seen in favor of the augmentation technique. At 250 cycles, a 68% increase in gap formation was seen for the control group (control: 5.96 ± 0.86 mm [95% CI, 5.30-6.62 mm]; augmentation: 3.55 ± 0.56 mm [95% CI, 3.12-3.98 mm]; p = 0.02). The mean load to failure was 13% greater in the augmentation group (control: 899.57 ± 96.94 N [95% CI, 825.06-974.09 N]; augmentation: 1030.70 ± 122.41 N [95% CI, 936.61-1124.79 N]; p = 0.01). CONCLUSIONS: This biomechanical study showed improved performance of a novel augmentation technique compared with the standard repair, in terms of reduced gap formation during cyclic loading and increased maximum load to failure. CLINICAL RELEVANCE: Decreased gap formation and higher load to failure may improve healing potential and minimize failure risk. This study shows a potential biomechanical advantage of the augmentation technique, providing support for future clinical investigations comparing this technique with other repair methods that are in common use such as transosseous suture repair.

Age-related changes in the plasticity and toughness of human cortical bone at multiple length scales.

The structure of human cortical bone evolves over multiple length scales from its basic constituents of collagen and hydroxyapatite at the nanoscale to osteonal structures at near-millimeter dimensions, which all provide the basis for its mechanical properties. To resist fracture, bone's toughness is derived intrinsically through plasticity (e.g., fibrillar sliding) at structural scales typically below a micrometer and extrinsically (i.e., during crack growth) through mechanisms (e.g., crack deflection/bridging) generated at larger structural scales. Biological factors such as aging lead to a markedly increased fracture risk, which is often associated with an age-related loss in bone mass (bone quantity). However, we find that age-related structural changes can significantly degrade the fracture resistance (bone quality) over multiple length scales. Using in situ small-angle X-ray scattering and wide-angle X-ray diffraction to characterize submicrometer structural changes and synchrotron X-ray computed tomography and in situ fracture-toughness measurements in the scanning electron microscope to characterize effects at micrometer scales, we show how these age-related structural changes at differing size scales degrade both the intrinsic and extrinsic toughness of bone. Specifically, we attribute the loss in toughness to increased nonenzymatic collagen cross-linking, which suppresses plasticity at nanoscale dimensions, and to an increased osteonal density, which limits the potency of crack-bridging mechanisms at micrometer scales. The link between these processes is that the increased stiffness of the cross-linked collagen requires energy to be absorbed by "plastic" deformation at higher structural levels, which occurs by the process of microcracking.

Comparison of different intubation techniques performed inside a moving ambulance: a manikin study.

OBJECTIVE. Airway management and endotracheal intubation may be required urgently when a patient deteriorates in an ambulance or aircraft during interhospital transfer or in a prehospital setting. The objectives of this study were: (1) to compare the effectiveness of conventional intubation by Macintosh laryngoscope in a moving ambulance versus that in a static ambulance; and (2) to compare the effectiveness of inverse intubation and GlideScope laryngoscopy with conventional intubation inside a moving ambulance. DESIGN. Comparative experimental study. SETTING. The experiment was conducted in an ambulance provided by the Auxiliary Medical Service in Hong Kong. PARTICIPANTS. A group of 22 doctors performed endotracheal intubation on manikins with Macintosh laryngoscope in a static and moving ambulance. In addition, they performed conventional Macintosh intubation, inverse intubation with Macintosh laryngoscope, and GlideScope intubation in a moving ambulance in both normal and simulated difficult airways. MAIN OUTCOME MEASURES. The primary outcome was the rate of successful intubation. The secondary outcomes were time taken for intubation, subjective glottis visualisation grading, and eventful intubation (oesophageal intubation, intubation time >60 seconds, and incisor breakage) with different techniques or devices. RESULTS. In normal airways, conventional Macintosh intubation in a static ambulance (95.5%), conventional intubation in a moving ambulance (95.5%), as well as GlideScope intubation in a moving ambulance (95.5%) were associated with high success rates; the success rate of inverse intubation was comparatively low (54.5%; P=0.004). In difficult airways, conventional Macintosh intubation in a static ambulance (86.4%), conventional intubation in a moving ambulance (90.9%), and GlideScope intubation in a moving ambulance (100%) were associated with high success rates; the success rate of inverse intubation was comparatively lower (40.9%; P=0.034). CONCLUSIONS. En-route intubation in an ambulance by conventional Macintosh laryngoscopy is superior to inverse intubation unless the cephalad access is impossible. GlideScope laryngoscopy appears to be associated with lower rates of eventful intubation in difficult airways and has better laryngoscopic view versus inverse intubation.

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.

The progression of neurovascular features and chemokine signatures of the intervertebral disc with degeneration.

Inflammatory cytokine production and de novo neurovascularization have been identified in painful, degenerated intervertebral discs (IVDs). However, the temporal trajectories of these key pathoanatomical features, including the cascade of inflammatory chemokines and neo- vessel and neurite infiltration, and their associations with IVD degeneration, remain relatively unknown. Investigating this process in the caudal mouse IVD enables the opportunity to study the tissue-specific response without confounding inflammatory signaling from neighboring structures. Thus this study aims to define the progression of chemokine production and neurovascular invasion during the IVD degeneration initiated by injury in the caudal spine 3-month-old C57BL6/J mice. Forty-nine IVD-secreted chemokines and matrix metalloproteinases (MMPs) was measured using multiplex ELISA, and the intradiscal infiltrating vessels (endomucin) and nerves (protein-gene-product 9.5) was quantified in the tissue volume using immunohistochemistry. Injury provoked the increase secretion of IL6, CCL2, CCL12, CCL17, CCL20, CCL21, CCL22, CXCL2 and MMP2 proteins. The centrality and structure of inflammatory networks in IVDs evolved over the 12 post-injury weeks, highlighting distinct responses between the acute and chronic phases. Neurites propagated rapidly within 2-weeks post-injury and remained relatively constant until 12-weeks. Vascular vessel length was observed to peak at 4-weeks post-injury and it regressed by 12-weeks. These findings identified the temporal flux of inflammatory chemokines and pain-associated pathoanatomy in a model of IVD degeneration using the mouse caudal spine.

Assessment of bovine cortical bone fracture behavior using impact microindentation as a surrogate of fracture toughness.

The fracture behavior of bone is critically important for evaluating its mechanical competence and ability to resist fractures. Fracture toughness is an intrinsic material property that quantifies a material's ability to withstand crack propagation under controlled conditions. However, properly conducting fracture toughness testing requires the access to calibrated mechanical load frames and the destructive testing of bone samples, and therefore fracture toughness tests are clinically impractical. Impact microindentation mimicks certain aspects of fracture toughness measurements, but its relationship with fracture toughness remains unknown. In this study, we aimed to compare measurements of notched fracture toughness and impact microindentation in fresh and boiled bovine bone. Skeletally mature bovine bone specimens (n = 48) were prepared, and half of them were boiled to denature the organic matrix, while the other half remained preserved in frozen conditions. All samples underwent a notched fracture toughness test to determine their resistance to crack initiation (KIC) and an impact microindentation test using the OsteoProbe to obtain the Bone Material Strength index (BMSi). Boiling the bone samples increased the denatured collagen content, while mineral density and porosity remained unaffected. The boiled bones also showed significant reduction in both KIC (P < .0001) and the average BMSi (P < .0001), leading to impaired resistance of bone to crack propagation. Remarkably, the average BMSi exhibited a high correlation with KIC (r = 0.86; P < .001). A ranked order difference analysis confirmed the excellent agreement between the 2 measures. This study provides the first evidence that impact microindentation could serve as a surrogate measure for bone fracture behavior. The potential of impact microindentation to assess bone fracture resistance with minimal sample disruption could offer valuable insights into bone health without the need for cumbersome testing equipment and sample destruction.

Bone canonical Wnt signaling is downregulated in type 2 diabetes and associates with higher advanced glycation end-products (AGEs) content and reduced bone strength.

Type 2 diabetes (T2D) is associated with higher fracture risk, despite normal or high bone mineral density. We reported that bone formation genes (SOST and RUNX2) and advanced glycation end-products (AGEs) were impaired in T2D. We investigated Wnt signaling regulation and its association with AGEs accumulation and bone strength in T2D from bone tissue of 15 T2D and 21 non-diabetic postmenopausal women undergoing hip arthroplasty. Bone histomorphometry revealed a trend of low mineralized volume in T2D (T2D 0.249% [0.156-0.366]) vs non-diabetic subjects 0.352% [0.269-0.454]; p=0.053, as well as reduced bone strength (T2D 21.60 MPa [13.46-30.10] vs non-diabetic subjects 76.24 MPa [26.81-132.9]; p=0.002). We also showed that gene expression of Wnt agonists LEF-1 (p=0.0136) and WNT10B (p=0.0302) were lower in T2D. Conversely, gene expression of WNT5A (p=0.0232), SOST (p<0.0001), and GSK3B (p=0.0456) were higher, while collagen (COL1A1) was lower in T2D (p=0.0482). AGEs content was associated with SOST and WNT5A (r=0.9231, p<0.0001; r=0.6751, p=0.0322), but inversely correlated with LEF-1 and COL1A1 (r=-0.7500, p=0.0255; r=-0.9762, p=0.0004). SOST was associated with glycemic control and disease duration (r=0.4846, p=0.0043; r=0.7107, p=0.00174), whereas WNT5A and GSK3B were only correlated with glycemic control (r=0.5589, p=0.0037; r=0.4901, p=0.0051). Finally, Young's modulus was negatively correlated with SOST (r=-0.5675, p=0.0011), AXIN2 (r=-0.5523, p=0.0042), and SFRP5 (r=-0.4442, p=0.0437), while positively correlated with LEF-1 (r=0.4116, p=0.0295) and WNT10B (r=0.6697, p=0.0001). These findings suggest that Wnt signaling and AGEs could be the main determinants of bone fragility in T2D.

Type 2 diabetes is a long-term metabolic disease characterised by chronic high blood sugar levels. This in turn has a negative impact on the health of other tissues and organs, including bones. Type 2 diabetes patients have an increased risk of fracturing bones compared to non-diabetics. This is particularly true for fragility fractures, which are fractures caused by falls from a short height (i.e., standing height or less), often affecting hips or wrists. Usually, a lower bone density is associated with higher risk of fractures. However, patients with type 2 diabetes have increased bone fragility despite normal or higher bone density. One reason for this could be the chronically high levels of blood sugar in type 2 diabetes, which alter the properties of proteins in the body. It has been shown that the excess sugar molecules effectively 'react' with many different proteins, producing harmful compounds in the process, called Advanced Glycation End-products, or AGEs. AGEs are ­ in turn ­thought to affect the structure of collagen proteins, which help hold our tissues together and decrease bone strength. However, the signalling pathways underlying this process are still unclear. To find out more, Leanza et al. studied a signalling molecule, called sclerostin, which inhibits a signalling pathway that regulates bone formation, known as Wnt signaling. The researchers compared bone samples from both diabetic and non-diabetic patients, who had undergone hip replacement surgery. Analyses of the samples, using a technique called real-time-PCR, revealed that gene expression of sclerostin was increased in samples of type 2 diabetes patients, which led to a downregulation of Wnt signaling related genes. Moreover, the downregulation of Wnt genes was correlated with lower bone strength (which was measured by compressing the bone tissue). Further biochemical analysis of the samples revealed that higher sclerostin activity was also associated with higher levels of AGEs. These results provide a clearer understanding of the biological mechanisms behind compromised bone strength in diabetes. In the future, Leanza et al. hope that this knowledge will help us develop treatments to reduce the risk of bone complications for type 2 diabetes patients.

Impact of the COVID-19 pandemic on the productivity and career prospects of musculoskeletal researchers.

Academic researchers faced a multitude of challenges posed by the COVID-19 pandemic, including widespread shelter-in-place orders, workplace closures, and cessation of in-person meetings and laboratory activities. The extent to which these challenges impacted musculoskeletal researchers, specifically, is unknown. We developed an anonymous web-based survey to determine the pandemic's impact on research productivity and career prospects among musculoskeletal research trainees and faculty. There were 116 musculoskeletal (MSK) researchers with varying demographic backgrounds who completed the survey. Of respondents, 48.3% (n = 56) believed that musculoskeletal funding opportunities decreased because of COVID-19, with faculty members more likely to hold this belief compared to nonfaculty researchers (p = 0.008). Amongst MSK researchers, 88.8% (n = 103) reported research activity was limited by COVID-19, and 92.2% (n = 107) of researchers reported their research was not able to be refocused on COVID-19-related topics, with basic science researchers less likely to be able to refocus their research compared to clinical researchers (p = 0.030). Additionally, 47.4% (n = 55) reported a decrease in manuscript submissions since the onset of the pandemic. Amongst 51 trainee researchers, 62.8% (n = 32) reported a decrease in job satisfaction directly attributable to the COVID-19 pandemic. In summary, study findings indicated that MSK researchers struggled to overcome challenges imposed by the pandemic, reporting declines in funding opportunities, research productivity, and manuscript submission. Trainee researchers experienced significant disruptions to critical research activities and worsening job satisfaction. Our findings motivate future efforts to support trainees in developing their careers and target the recovery of MSK research from the pandemic stall.

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. Lay Summary: The intervertebral disc (IVD) is a spinal joint that accumulates damage with age but has limited tissue repair capabilities. IVD damage progresses into degeneration, and IVD degeneration is a leading cause of lower back pain. There are no effective therapies to treat IVD degeneration, but understanding the cell populations that change and respond to injury will uncover targets to restore IVD function. Mesenchymal stem cells (MSCs) are cells within the IVD that can potentially replenish the cells lost after IVD damage. To identify the cell populations of the IVD and how they change with injury, we performed single cell RNA sequencing of IVD tissue 7 days post injury and analyzed the differences in gene regulation. We identified diverse cells populations such as IVD specific tissues, immune cells, vascular cells, and MSCs. We discovered the presence of Saa2 and Grem1 expressing MSCs that become less stem cell-like and express higher levels of IVD gene markers after injury. We also determined that Saa2 and Grem1 have slightly different expression patterns in IVD tissues, and this expression becomes reduced after injury. These MSCs could be used in future stem cell therapies to prevent IVD degeneration.

Comparing shades of darkness: trolling victims' experiences on social media vs. online gaming.

Although there is ample literature available on toxicity in games, as there is regarding trolling on social media, there are few to no cross-platform studies on toxicity and trolling. In other words, the extant literature focuses on one platform at a time instead of comparing and contrasting them. The present work aims to rectify this gap by analyzing interviews from a larger study of 22 self-proclaimed victims of in-game trolling to not only determine whether social media or gaming communities are considered more toxic but also to explore how definitions of the word 'trolling' change depending on the platform in question. We found that while definitions of in-game trolling behavior focused on behavioral styles of trolling (e.g., throwing one's avatar into enemy fire to disadvantage one's team, and blocking other players' avatars' movement), social media trolling is defined by more sinister actions such as misinformation spreading and 'canceling' other users. We also found that gaming is perceived as generally more toxic than social media, often due to company policies or lack thereof. Practical and theoretical implications for the study of toxicity in all online communities - gaming or social-media based - are discussed.

Relative Effects of Radiation-Induced Changes in Bone Mass, Structure, and Tissue Material on Vertebral Strength in a Rat Model.

The observed increased risk of fracture after cancer radiation therapy is presumably due to a radiation-induced reduction in whole-bone strength. However, the mechanisms for impaired strength remain unclear, as the increased fracture risk is not fully explained by changes in bone mass. To provide insight, a small animal model was used to determine how much of this whole-bone weakening effect for the spine is attributable to changes in bone mass, structure, and material properties of the bone tissue and their relative effects. Further, because women have a greater risk of fracture after radiation therapy than men, we investigated if sex had a significant influence on bone's response to irradiation. Fractionated in vivo irradiation (10 × 3 Gy) or sham irradiation (0 Gy) was administered daily to the lumbar spine in twenty-seven 17-week-old Sprague-Dawley rats (n = 6-7/sex/group). Twelve weeks after final treatment, animals were euthanized, and lumbar vertebrae (L4 and L5 ) were isolated. Using a combination of biomechanical testing, micro-CT-based finite element analysis, and statistical regression analysis, we separated out the effect of mass, structural, and tissue material changes on vertebral strength. Compared with the sham group (mean ± SD strength = 420 ± 88 N), the mean strength of the irradiated group was lower by 28% (117 N/420 N, p < 0.0001). Overall, the response of treatment did not differ with sex. By combining results from both general linear regression and finite element analyses, we calculated that mean changes in bone mass, structure, and material properties of the bone tissue accounted for 56% (66 N/117 N), 20% (23 N/117 N), and 24% (28 N/117 N), respectively, of the overall change in strength. As such, these results provide insight into why an elevated clinical fracture risk for patients undergoing radiation therapy is not well explained by changes in bone mass alone. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

TRPV4 differentially controls inflammatory cytokine networks during static and dynamic compression of the intervertebral disc.

Background: The ion channel transient receptor potential vanilloid 4 (TRPV4) critically transduces mechanical forces in the IVD, and its inhibition can prevent IVD degeneration due to static overloading. However, it remains unknown whether different modes of loading signals through TRPV4 to regulate the expression of inflammatory cytokines. We hypothesized that TRPV4 signaling is essential during static and dynamic loading to mediate homeostasis and mechanotransduction. Methods: Mouse functional spine units were isolated and either cyclically compressed for 5 days (1 Hz, 1 h, 10% strain) or statically compressed (24 h, 0.2 MPa). Conditioned media were monitored at 6 h, 24 h, 2 days, and 5 days, with and without TRPV4 inhibition. Effects of TRPV4 activation was also evaluated without loading. The media was analyzed for a panel of 44 cytokines using a microbead array and then a correlative network was constructed to explore the regulatory relationships during loading and TRPV4 inhibition. After the loading regimen, the IVDs were evaluated histologically for degeneration. Results: Activation of TRPV4 led to an increase interleukin-6 (IL-6) family of cytokines (IL-6, IL-11, IL-16, and leukemia inhibitory factor [LIF]) and decreased the T-cell (CCL3, CCL4, CCL17, CCL20, CCL22, and CXCL10) and monocyte (CCL2 and CCL12) recruiting chemokines by the IVD. Dynamic and static loading each provoked unique chemokine correlation networks. The inhibition of TRPV4 during dynamic loading dysregulated the relationship between LIF and other cytokines, while the inhibition of TRPV4 during static loading disrupted the connectivity of IL-16 and VEGFA. Conclusions: We demonstrated that TRPV4 critically mediates the cytokine production following dynamic and static loading. The activation of TRPV4 upregulated a diverse set of cytokines that may suppress the chemotaxis of T-cells and monocytes, implicating the role of TRPV4 in maintaining the immune privilege of healthy IVD.

Quantative MRI predicts tendon mechanical behavior, collagen composition, and organization.

Quantitative magnetic resonance imaging (qMRI) measures have provided insights into the composition, quality, and structure-function of musculoskeletal tissues. Low signal-to-noise ratio has limited application to tendon. Advances in scanning sequences and sample positioning have improved signal from tendon allowing for evaluation of structure and function. The purpose of this study was to elucidate relationships between tendon qMRI metrics (T1, T2, T1ρ and diffusion tensor imaging [DTI] metrics) with tendon tissue mechanics, collagen concentration and organization. Sixteen human Achilles tendon specimens were collected, imaged with qMRI, and subjected to mechanical testing with quantitative polarized light imaging. T2 values were related to tendon mechanics [peak stress (rsp = 0.51, p = 0.044), equilibrium stress (rsp = 0.54, p = 0.033), percent relaxation (rsp = -0.55, p = 0.027), hysteresis (rsp = -0.64, p = 0.007), linear modulus (rsp = 0.67, p = 0.009)]. T1ρ had a statistically significant relationship with percent relaxation (r = 0.50, p = 0.048). Collagen content was significantly related to DTI measures (range of r = 0.56-0.62). T2 values from a single slice of the midportion of human Achilles tendons were strongest predictors of tendon tensile mechanical metrics. DTI diffusivity indices (mean diffusivity, axial diffusivity, radial diffusivity) were strongly correlated with collagen content. These findings build on a growing body of literature supporting the feasibility of qMRI to characterize tendon tissue and noninvasively measure tendon structure and function. Statement of Clinical Significance: Quantitative MRI can be applied to characterize tendon tissue and is a noninvasive measure that relates to tendon composition and mechanical behavior.