Complex mechanical loading and inflammation in intervertebral disc degeneration

Introduction Intervertebral disc (IVD) degeneration is the cause of around half of all low back pain cases in young adults, however the initiating and risk factors are poorly understood, limiting development of personalized therapies. [1] Although the effects of pro-inflammatory cytokines and mechanical loading has been investigated within the IVD, [2] it is unknown how IVD response to complex mechanical loading is affected by the presence of cytokines. Thus, we aimed to investigate the combined effects of dynamic compression and torsion with catabolic cytokine interleukin 1 beta (IL-1β) and inhibitory cytokine interleukin 1 receptor antagonist (IL-1Ra), on bovine IVDs using ex vivo culture, magnetic resonance imaging (MRI), and finite element (FE) modeling. Methods Whole bovine IVDs obtained within 3-4 hours portmortem from a local abattoir were isolated and the IVDs cultured in a customized two degrees-of-freedom bioreactor applying diurnal dynamic compression (0.1- 0.5 MPa) and torsion (6°) under normal (HG DMEM), catabolic (10 ng/ml IL-1β) and inhibitory (10 ng/ml IL1Ra) media conditions for one week. Static compression (0.1 MPa) under the same media conditions were used as a control. Before and after culture, the IVDs were imaged using 3T MRI, which was used to create subjectspecific FE models. Downstream analyses included height measurement, qPCR, glycosaminoglycan (GAG) quantification, and cell metabolic activity. For statistical analysis (when n>3), nonparametric distribution was assumed and a Kruskal–Wallis test then Dunn’s multiple comparisons test were performed, and a P < 0.05 considered statistically significant. Results Following one week of culture, IVD height decreased in all conditions (Figure 1a & b), however, this disc height decreases was less pronounced within IVDs stimulated with IL-1Ra. Cellular metabolic activity in the nucleus pulposus (NP) decreased in all conditions, but the difference was only significant in IVDs stimulated with IL-1β and complex dynamic loading (Figure 1c). Similarly, the GAG content decreased in the NP of all conditions, but was not significant. Gene expression of anabolic genes, i.e. collagen type II (COL II) decreased (Figure 1e), while expression of catabolic genes, i.e. matrix metalloproteinase 3 (MMP3) increased in the NP tissue for all conditions except in control static IVDs and in IL-1Ra stimulated IVDs, respectively (Figure 1d). Introduction Intervertebral disc (IVD) degeneration is the cause of around half of all low back pain cases in young adults, however the initiating and risk factors are poorly understood, limiting development of personalized therapies. [1] Although the effects of pro-inflammatory cytokines and mechanical loading has been investigated within the IVD, [2] it is unknown how IVD response to complex mechanical loading is affected by the presence of cytokines. Thus, we aimed to investigate the combined effects of dynamic compression and torsion with catabolic cytokine interleukin 1 beta (IL-1β) and inhibitory cytokine interleukin 1 receptor antagonist (IL-1Ra), on bovine IVDs using ex vivo culture, magnetic resonance imaging (MRI), and finite element (FE) modeling. Methods Whole bovine IVDs obtained within 3-4 hours portmortem from a local abattoir were isolated and the IVDs cultured in a customized two degrees-of-freedom bioreactor applying diurnal dynamic compression (0.1- 0.5 MPa) and torsion (6°) under normal (HG DMEM), catabolic (10 ng/ml IL-1β) and inhibitory (10 ng/ml IL1Ra) media conditions for one week. Static compression (0.1 MPa) under the same media conditions were used as a control. Before and after culture, the IVDs were imaged using 3T MRI, which was used to create subjectspecific FE models. Downstream analyses included height measurement, qPCR, glycosaminoglycan (GAG) quantification, and cell metabolic activity. For statistical analysis (when n>3), nonparametric distribution was assumed and a Kruskal–Wallis test then Dunn’s multiple comparisons test were performed, and a P < 0.05 considered statistically significant. Results Following one week of culture, IVD height decreased in all conditions (Figure 1a & b), however, this disc height decreases was less pronounced within IVDs stimulated with IL-1Ra. Cellular metabolic activity in the nucleus pulposus (NP) decreased in all conditions, but the difference was only significant in IVDs stimulated with IL-1β and complex dynamic loading (Figure 1c). Similarly, the GAG content decreased in the NP of all conditions, but was not significant. Gene expression of anabolic genes, i.e. collagen type II (COL II) decreased (Figure 1e), while expression of catabolic genes, i.e. matrix metalloproteinase 3 (MMP3) increased in the NP tissue for all conditions except in control static IVDs and in IL-1Ra stimulated IVDs, respectively (Figure 1d). Introduction Intervertebral disc (IVD) degeneration is the cause of around half of all low back pain cases in young adults, however the initiating and risk factors are poorly understood, limiting development of personalized therapies. [1] Although the effects of pro-inflammatory cytokines and mechanical loading has been investigated within the IVD, [2] it is unknown how IVD response to complex mechanical loading is affected by the presence of cytokines. Thus, we aimed to investigate the combined effects of dynamic compression and torsion with catabolic cytokine interleukin 1 beta (IL-1β) and inhibitory cytokine interleukin 1 receptor antagonist (IL-1Ra), on bovine IVDs using ex vivo culture, magnetic resonance imaging (MRI), and finite element (FE) modeling. Methods Whole bovine IVDs obtained within 3-4 hours portmortem from a local abattoir were isolated and the IVDs cultured in a customized two degrees-of-freedom bioreactor applying diurnal dynamic compression (0.1- 0.5 MPa) and torsion (6°) under normal (HG DMEM), catabolic (10 ng/ml IL-1β) and inhibitory (10 ng/ml IL1Ra) media conditions for one week. Static compression (0.1 MPa) under the same media conditions were used as a control. Before and after culture, the IVDs were imaged using 3T MRI, which was used to create subjectspecific FE models. Downstream analyses included height measurement, qPCR, glycosaminoglycan (GAG) quantification, and cell metabolic activity. For statistical analysis (when n>3), nonparametric distribution was assumed and a Kruskal–Wallis test then Dunn’s multiple comparisons test were performed, and a P <0.05 considered statistically significant. Results Following one week of culture, IVD height decreased in all conditions (Figure 1a & b), however, this disc height decreases was less pronounced within IVDs stimulated with IL-1Ra. Cellular metabolic activity in the nucleus pulposus (NP) decreased in all conditions, but the difference was only significant in IVDs stimulated with IL-1β and complex dynamic loading (Figure 1c). Similarly, the GAG content decreased in the NP of all conditions, but was not significant. Gene expression of anabolic genes, i.e. collagen type II (COL II) decreased (Figure 1e), while expression of catabolic genes, i.e. matrix metalloproteinase 3 (MMP3) increased in the NP tissue for all conditions except in control static IVDs and in IL-1Ra stimulated IVDs, respectively (Figure 1d).Discussion We hypothesized that catabolic cytokines, i.e. IL-1β, in the microenvironment of the IVD are sufficient to negatively alter the cellular response to complex loading, leading to further downstream degeneration. However, markers of degeneration were found in all conditions, which could indicate that loading was supraphysiological and catabolic alone. This will be investigated further using subject-specific FE models developed from the MRI images. Additionally, short half-lives of cytokines could have prevented them from diffusing through the IVD effectively, thus limiting the cellular response. This will be further investigated using immunohistochemistry and ELISA. Nevertheless, the results convey that static or complex dynamic loading is sufficient to induce catabolism with or without cytokine stimulation. References 1. Baumgartner L et al., J. Int J Mol Sci. 2021; 22(2):703. 2. Walter BA et al., PLOS ONE. 2015; 10(3): e0118358.5

Conditioned medium of intervertebral disc cells inhibits osteogenesis on autologous bone-marrow-derived mesenchymal stromal cells and osteoblasts

INTRODUCTION: Low back pain (LBP) is a significant global burden and is associated with the degeneration of the spine and human intervertebral discs (hIVD). Current surgical treatment for hIVD degeneration is the removal of the affected tissue with a cage to promote spinal fusion and relieve discomfort. Despite progress in the treatment of LBP, however, in ~30% of all cases this procedure ends in non-fusion and painful pseudo-athrosis after operation. Previous research from our laboratory showed morphogenetic protein (BMP) antagonists secreted by the intervertebral disc have been potentially associated with inhibiting the process of osteogenesis. However, the co-cultured cells did not come from the same donor. In this study, we investigated the hypothesis that IVD cells secrete BMP inhibitors that inhibit osteogenesis in autologous osteoblast (hOB) and bone marrow mesenchymal stem cell (hMSC). METHODS: Conditioned Medium (CM) collected from primary hIVD cells in 3D alginate culture was co-cultured with seven donor-matched hOB and hMSC in 2D culture. Osteogenesis after 10 days was then quantified at the transcript level using qPCR to measure the expression of bone makers and BMP antagonists, and at the protein level by alkaline phosphatase (ALP) activity. Additionally, they were evaluated histologically by alizarin red (ALZR) staining on Day 21. The relationship between ALP activity, osteogenesis and Noggin expression in hOB or hMSC or hIVD was investigated to uncover the potential causes. RESULTS: ALP activity significantly decreased and the formation of calcium deposits in alizarin red staining was inhibited after culture with CM derived from hIVD. Interestingly, less changes of bone makers and BMP inhibitors’ expression was found in hOB or hMSC on Day 10. Noggin was relatively higher expressed (Average fold change: AF, 6.9; CEP, 10.0; NP, 6.3; relative to autologous hOB. AF, 2.3; CEP, 3.4; NP, 3.2; relative to autologous hMSC.) in hIVD compared to hOB or hMSC. DISCUSSION: The up-regulation of Noggin mRNA (and possibly other BMP inhibitors) in residual hIVD tissue after spinal fusion surgery is potentially a potent reason for prevention of successful osteogenesis. Similar results were found previously with allogenic co-cultures. However, in previous study there were merely trends of inhibition. Here we show a significant decrease with autologous donor-matched samples

Cartilaginous endplates: A comprehensive review on a neglected structure in intervertebral disc research

The cartilaginous endplates (CEP) are key components of the intervertebral disc (IVD) necessary for sustaining the nutrition of the disc while distributing mechanical loads and preventing the disc from bulging into the adjacent vertebral body. The size, shape, and composition of the CEP are essential in maintaining its function, and degeneration of the CEP is considered a contributor to early IVD degeneration. In addition, the CEP is implicated in Modic changes, which are often associated with low back pain. This review aims to tackle the current knowledge of the CEP regarding its structure, composition, permeability, and mechanical role in a healthy disc, how they change with degeneration, and how they connect to IVD degeneration and low back pain. Additionally, the authors suggest a standardized naming convention regarding the CEP and bony endplate and suggest avoiding the term vertebral endplate. Currently, there is limited data on the CEP itself as reported data is often a combination of CEP and bony endplate, or the CEP is considered as articular cartilage. However, it is clear the CEP is a unique tissue type that differs from articular cartilage, bony endplate, and other IVD tissues. Thus, future research should investigate the CEP separately to fully understand its role in healthy and degenerated IVDs. Further, most IVD regeneration therapies in development failed to address, or even considered the CEP, despite its key role in nutrition and mechanical stability within the IVD. Thus, the CEP should be considered and potentially targeted for future sustainable treatments

Immuno-modulatory effects of intervertebral disc cells

Low back pain is a highly prevalent, chronic, and costly medical condition predominantly triggered by intervertebral disc degeneration (IDD). IDD is often caused by structural and biochemical changes in intervertebral discs (IVD) that prompt a pathologic shift from an anabolic to catabolic state, affecting extracellular matrix (ECM) production, enzyme generation, cytokine and chemokine production, neurotrophic and angiogenic factor production. The IVD is an immune-privileged organ. However, during degeneration immune cells and inflammatory factors can infiltrate through defects in the cartilage endplate and annulus fibrosus fissures, further accelerating the catabolic environment. Remarkably, though, catabolic ECM disruption also occurs in the absence of immune cell infiltration, largely due to native disc cell production of catabolic enzymes and cytokines. An unbalanced metabolism could be induced by many different factors, including a harsh microenvironment, biomechanical cues, genetics, and infection. The complex, multifactorial nature of IDD brings the challenge of identifying key factors which initiate the degenerative cascade, eventually leading to back pain. These factors are often investigated through methods including animal models, 3D cell culture, bioreactors, and computational models. However, the crosstalk between the IVD, immune system, and shifted metabolism is frequently misconstrued, often with the assumption that the presence of cytokines and chemokines is synonymous to inflammation or an immune response, which is not true for the intact disc. Therefore, this review will tackle immunomodulatory and IVD cell roles in IDD, clarifying the differences between cellular involvements and implications for therapeutic development and assessing models used to explore inflammatory or catabolic IVD environments

Climate Change Meets the Law of the Horse

The climate change policy debate has only recently turned its full attention to adaptation - how to address the impacts of climate change we have already begun to experience and that will likely increase over time. Legal scholars have in turn begun to explore how the many different fields of law will and should respond. During this nascent period, one overarching question has gone unexamined: how will the legal system as a whole organize around climate change adaptation? Will a new distinct field of climate change adaptation law and policy emerge, or will legal institutions simply work away at the problem through unrelated, duly self-contained fields, as in the famous Law of the Horse? This Article is the first to examine that question comprehensively, to move beyond thinking about the law and climate change adaptation to consider the law of climate change adaptation. Part I of the Article lays out our methodological premises and approach. Using a model we call Stationarity Assessment, Part I explores how legal fields are structured and sustained based on assumptions about the variability of natural, social, and economic conditions, and how disruptions to that regime of variability can lead to the emergence of new fields of law and policy. Case studies of environmental law and environmental justice demonstrate the model’s predictive power for the formation of new distinct legal regimes. Part II applies the Stationarity Assessment model to the topic of climate change adaptation, using a case study of a hypothetical coastal region and the potential for climate change impacts to disrupt relevant legal doctrines and institutions. We find that most fields of law appear capable of adapting effectively to climate change. In other words, without some active intervention, we expect the law and policy of climate change adaptation to follow the path of the Law of the Horse - a collection of fields independently adapting to climate change - rather than organically coalescing into a new distinct field. Part III explores why, notwithstanding this conclusion, it may still be desirable to seek a different trajectory. Focusing on the likelihood of systemic adaptation decisions with perverse, harmful results, we identify the potential benefits offered by intervening to shape a new and distinct field of climate change adaptation law and policy. Part IV then identifies the contours of such a field, exploring the distinct purposes of reducing vulnerability, ensuring resiliency, and safeguarding equity. These features provide the normative policy components for a law of climate change adaptation that would be more than just a Law of the Horse. This new field would not replace or supplant any existing field, however, as environmental law did with regard to nuisance law, and it would not be dominated by substantive doctrine. Rather, like the field of environmental justice, this new legal regime would serve as a holistic overlay across other fields to ensure more efficient, effective, and just climate change adaptation solutions

Global and national Burden of diseases and injuries among children and adolescents between 1990 and 2013

Importance The literature focuses on mortality among children younger than 5 years. Comparable information on nonfatal health outcomes among these children and the fatal and nonfatal burden of diseases and injuries among older children and adolescents is scarce. Objective To determine levels and trends in the fatal and nonfatal burden of diseases and injuries among younger children (aged <5 years), older children (aged 5-9 years), and adolescents (aged 10-19 years) between 1990 and 2013 in 188 countries from the Global Burden of Disease (GBD) 2013 study. Evidence Review Data from vital registration, verbal autopsy studies, maternal and child death surveillance, and other sources covering 14 244 site-years (ie, years of cause of death data by geography) from 1980 through 2013 were used to estimate cause-specific mortality. Data from 35 620 epidemiological sources were used to estimate the prevalence of the diseases and sequelae in the GBD 2013 study. Cause-specific mortality for most causes was estimated using the Cause of Death Ensemble Model strategy. For some infectious diseases (eg, HIV infection/AIDS, measles, hepatitis B) where the disease process is complex or the cause of death data were insufficient or unavailable, we used natural history models. For most nonfatal health outcomes, DisMod-MR 2.0, a Bayesian metaregression tool, was used to meta-analyze the epidemiological data to generate prevalence estimates. Findings Of the 7.7 (95% uncertainty interval [UI], 7.4-8.1) million deaths among children and adolescents globally in 2013, 6.28 million occurred among younger children, 0.48 million among older children, and 0.97 million among adolescents. In 2013, the leading causes of death were lower respiratory tract infections among younger children (905 059 deaths; 95% UI, 810 304-998 125), diarrheal diseases among older children (38 325 deaths; 95% UI, 30 365-47 678), and road injuries among adolescents (115 186 deaths; 95% UI, 105 185-124 870). Iron deficiency anemia was the leading cause of years lived with disability among children and adolescents, affecting 619 (95% UI, 618-621) million in 2013. Large between-country variations exist in mortality from leading causes among children and adolescents. Countries with rapid declines in all-cause mortality between 1990 and 2013 also experienced large declines in most leading causes of death, whereas countries with the slowest declines had stagnant or increasing trends in the leading causes of death. In 2013, Nigeria had a 12% global share of deaths from lower respiratory tract infections and a 38% global share of deaths from malaria. India had 33% of the world’s deaths from neonatal encephalopathy. Half of the world’s diarrheal deaths among children and adolescents occurred in just 5 countries: India, Democratic Republic of the Congo, Pakistan, Nigeria, and Ethiopia. Conclusions and Relevance Understanding the levels and trends of the leading causes of death and disability among children and adolescents is critical to guide investment and inform policies. Monitoring these trends over time is also key to understanding where interventions are having an impact. Proven interventions exist to prevent or treat the leading causes of unnecessary death and disability among children and adolescents. The findings presented here show that these are underused and give guidance to policy makers in countries where more attention is needed

A communal catalogue reveals Earth’s multiscale microbial diversity

Our growing awareness of the microbial world’s importance and diversity contrasts starkly with our limited understanding of its fundamental structure. Despite recent advances in DNA sequencing, a lack of standardized protocols and common analytical frameworks impedes comparisons among studies, hindering the development of global inferences about microbial life on Earth. Here we present a meta-analysis of microbial community samples collected by hundreds of researchers for the Earth Microbiome Project. Coordinated protocols and new analytical methods, particularly the use of exact sequences instead of clustered operational taxonomic units, enable bacterial and archaeal ribosomal RNA gene sequences to be followed across multiple studies and allow us to explore patterns of diversity at an unprecedented scale. The result is both a reference database giving global context to DNA sequence data and a framework for incorporating data from future studies, fostering increasingly complete characterization of Earth’s microbial diversity

A communal catalogue reveals Earth's multiscale microbial diversity

Our growing awareness of the microbial world's importance and diversity contrasts starkly with our limited understanding of its fundamental structure. Despite recent advances in DNA sequencing, a lack of standardized protocols and common analytical frameworks impedes comparisons among studies, hindering the development of global inferences about microbial life on Earth. Here we present a meta-analysis of microbial community samples collected by hundreds of researchers for the Earth Microbiome Project. Coordinated protocols and new analytical methods, particularly the use of exact sequences instead of clustered operational taxonomic units, enable bacterial and archaeal ribosomal RNA gene sequences to be followed across multiple studies and allow us to explore patterns of diversity at an unprecedented scale. The result is both a reference database giving global context to DNA sequence data and a framework for incorporating data from future studies, fostering increasingly complete characterization of Earth's microbial diversity.Peer reviewe

Measuring progress and projecting attainment on the basis of past trends of the health-related Sustainable Development Goals in 188 countries: an analysis from the Global Burden of Disease Study 2016

The UN’s Sustainable Development Goals (SDGs) are grounded in the global ambition of “leaving no one behind”. Understanding today’s gains and gaps for the health-related SDGs is essential for decision makers as they aim to improve the health of populations. As part of the Global Burden of Diseases, Injuries, and Risk Factors Study 2016 (GBD 2016), we measured 37 of the 50 health-related SDG indicators over the period 1990–2016 for 188 countries, and then on the basis of these past trends, we projected indicators to 2030

Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016

As mortality rates decline, life expectancy increases, and populations age, non-fatal outcomes of diseases and injuries are becoming a larger component of the global burden of disease. The Global Burden of Diseases, Injuries, and Risk Factors Study 2016 (GBD 2016) provides a comprehensive assessment of prevalence, incidence, and years lived with disability (YLDs) for 328 causes in 195 countries and territories from 1990 to 2016