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.

BACKGROUND: Lower back pain affects 75%-85% of people at some point in their lives. The detection of biochemical changes with sodium (23Na) MRI has potential to enable an earlier and more accurate diagnosis.
OBJECTIVE: To measure 23Na relaxation times and apparent tissue sodium concentration (aTSC) in ex-vivo intervertebral discs (IVDs), and to investigate the relationship between aTSC and histological Thompson grade.
METHODS: Ex-vivo.
METHODS: Thirty IVDs from the lumbar spines of 11 human body donors (4 female, 7 male, mean age 86 ± 8 years).
UNASSIGNED: 3 T; density-adapted 3D radial sequence (DA-3D-RAD).
RESULTS: IVD 23Na longitudinal (T1), short and long transverse (T2s* and T2l*) relaxation times and the proportion of the short transverse relaxation (ps) were calculated for one IVD per spine sample (11 IVDs). Furthermore, aTSCs were calculated for all IVDs. The degradation of the IVDs was assessed via histological Thompson grading.
METHODS: A Kendall Tau correlation (τ) test was performed between the aTSCs and the Thompson grades. The significance level was set to P < 0.05.
RESULTS: Mean 23Na relaxation parameters of a subset of 11 IVDs were T1 = 9.8 ± 1.3 msec, T2s* = 0.7 ± 0.1 msec, T2l* = 7.3 ± 1.1 msec, and ps = 32.7 ± 4.0%. A total of 30 IVDs were examined, of which 3 had Thompson grade 1, 4 had grade 2, 5 had grade 3, 5 had grade 4, and 13 had grade 5. The aTSC decreased with increasing degradation, being 274.6 ± 18.9 mM for Thompson grade 1 and 190.5 ± 29.5 mM for Thompson grade 5. The correlation between whole IVD aTSC and Thompson grade was significant and strongly negative (τ = -0.56).
CONCLUSIONS: This study showed a significant correlation between aTSC and degenerative IVD changes. Consequently, aTSC has potential to be useful as an indicator of degenerative spinal changes.
METHODS: 2 TECHNICAL EFFICACY: Stage 1.

Diagnostic tests were heralded as crucial during the Coronavirus disease (COVID-19) pandemic with most of the key methods using bioanalytical approaches that detected larger molecules (RNA, protein antigens or antibodies) rather than conventional clinical biochemical techniques. Nucleic Acid Amplification Tests (NAATs), like the Polymerase Chain Reaction (PCR), and other molecular methods, like sequencing (that often work in combination with NAATs), were essential to the diagnosis and management during COVID-19. This was exemplified both early in the pandemic but also later on, following the emergence of new genetic SARS-CoV-2 variants. The 100 day mission to respond to future pandemic threats highlights the need for effective diagnostics, therapeutics and vaccines. Of the three, diagnostics represents the first opportunity to manage infectious diseases while also being the most poorly supported in terms of the infrastructure needed to demonstrate effectiveness. Where performance targets exist, they are not well served by consensus on how to demonstrate they are being met; this includes analytical factors such as limit of detection (LOD) false positive results as well as how to approach clinical evaluation. The selection of gold standards or use of epidemiological factors such as predictive value, reference ranges or clinical thresholds are seldom correctly considered. The attention placed on molecular diagnostic tests during COVID-19 illustrates important considerations and assumptions on the use of these methods for infectious disease diagnosis and beyond. In this manuscript, we discuss state-of-the-art approaches to diagnostic evaluation and explore how they may be better tailored to diagnostic techniques like NAATs to maximise the impact of these highly versatile bioanalytical tools, both generally and during future outbreaks.

Objective. To use automation to facilitate the monitoring of each treatment fraction using an electronic portal imaging device (EPID) basedin vivodosimetry (IVD) system, allowing optimisation of breast radiotherapy delivery for individual patients and cohorts.Approach. A suite of in-house software was developed to reduce the number of manual interactions with the commercial IVD system, dosimetry check. An EPID specific pixel sensitivity map facilitated use of the EPID panel away from the central axis. Point dose difference and the change in standard deviation in dose were identified as useful dose metrics, with standard deviation used in preference to gamma in the presence of a systematic dose offset. Automated IVD was completed for 3261 fractions across 704 patients receiving breast radiotherapy.Main results. Multiple opportunities for treatment optimisation were identified for individual patients and across patient cohorts as a result of successful implementation of automated IVD. 5.1% of analysed fractions were out of tolerance with 27.1% of these considered true positives. True positive results were obtained on any fraction of treatment and if IVD had only been completed on the first fraction, 84.4% of true positive results would have been missed. This was made possible due to the automation that saved over 800 h of manual intervention and stored data in an accessible database.Significance. An improved EPID calibration to allow off-axis measurement maximises the number of patients eligible for IVD (36.8% of patients in this study). We also demonstrate the importance in selecting context-specific assessment metrics and how these can lead to a managable false positive rate. We have shown that the use of fully automated IVD facilitates use on every fraction of treatment. This leads to identification of areas for treatment improvement for both individuals and across a patient cohort, expanding the uses of IVD from simply gross error detection towards treatment optimisation.

Morphological changes can affect distribution of dose in patients. Determination of the dose distribution changes for each fraction radiotherapy can be done by relativein vivodosimetry (IVD). This study analysed the distribution of doses per fraction based on the fluence map recorded by the electronic portal imaging device (EPID) of the patient\'s transit dose. This research examined cases involving the cervix, breast, and nasopharynx. Transit dose analysis was performed by calculating the gamma index (GI) with composite and field-by-field methods. The gamma passing rate (GPR) value was assessed for its correlation with the subject\'s body weight. In the case of the nasopharynx, breast, and cervix, the GPR value decreased as the fraction increased. In the case of the nasopharynx, the correlation between the GPR and fraction radiotherapy showed no difference when using either composite or field-by-field methods. However, in cases involving the cervix and breast, there was a difference in the correlation values between the composite and field-by-field methods, where the subject had a significant correlation (p< 0.05) when it was done using a field-by-field method. In addition, the nasopharynx had the highest number of subjects with significant correlation (p< 0.05) between GPR and body weight, followed by the cervix and breast. In the nasopharynx, breast, and cervix, the reproducibility of the dose distribution decreased. This decreased reproducibility was associated with changes in body weight.

According to the World Health Organization, blood must be screened for major transmitted infections before transfusion to prevent the possibility of passing an infection to the recipient. For accurate detection of infectious disease pathogens in the blood of donors, in-vitro diagnostic medical devices (IVDs) of high specificity and sensitivity should be used. In mature healthcare systems, the regulatory authorities authorize the usage of devices with the highest performance capabilities, which are also controlled through active market oversight. However, in Sub-Saharan African countries, the regulation of IVDs is often poorly developed. With the lack of stringent regulatory oversight, IVDs of poor quality can be put on the market and used for blood donor screening, which, ultimately, poses a great public health threat. The BloodTrain is a humanitarian project from the Germany Federal Ministry of Health that aims to help strengthen the regulatory authorities in Sub-Saharan partner countries. Here, we present the status of IVD regulation in the partner countries and the objectives that the BloodTrain project aims to achieve in the region toward regulating IVDs.

BACKGROUND: Dachshunds have a high prevalence of intervertebral disc disease (IVDD) to which they are predisposed due to early intervertebral disc (IVD) degeneration and calcification. Moreover, the recently found 12-FGF4 retrogene (RG) is associated with calcified discs visible on radiographs (CDVR) and IVDD. Earlier studies suggest that all IVDs of one-year-old Dachshunds show signs of degeneration. This prospective, analytical, blinded study aimed to investigate the extent and distribution of IVD degeneration in young adult (24-31 months) asymptomatic Dachshunds (n = 21) hypothesizing that not all IVDs of two-year-old Dachshunds are degenerated. Another aim was to explore the correlations between IVD degeneration evaluated with magnetic resonance imaging (MRI), the number of CDVR, and the dog\'s 12-FGF4RG status. The study protocol included grading the CDVR on spinal radiographs, grading the IVD degeneration on T2-weighted sagittal and transverse high-field MR images of all IVDs (n = 546), and 12-FGF4RG variant genotyping.
RESULTS: Of all IVDs evaluated, 2% (n = 11) were normal based on MRI grading. Despite the study population having moderately degenerated IVDs (median MRI grade 3), there was also variation in the degree of IVD degeneration between individuals and in the distribution of IVD degeneration between different vertebral regions. The number of CDVR correlated significantly with the magnitude of IVD degeneration based on MRI evaluation and with the 12-FGF4RG genotype. The odds for being 12-FGF4RG homozygous were higher for Dachshunds with CDVR. However, the 12-FGF4RG variant did not alone explain the phenotypic variation in IVD degeneration.
CONCLUSIONS: The number of CDVR is a valid indicator of overall IVD degeneration, as it correlates with MRI-based IVD grading. Also, as the extent and distribution of IVD degeneration varies between individual Dachshunds, selective breeding against IVDD using radiographic screening and 12-FGF4RG variant genotyping is possible.

The circadian clock in mammals temporally coordinates physiological and behavioural processes to anticipate daily rhythmic changes in their environment. Chronic disruption to circadian rhythms (e.g., through ageing or shift work) is thought to contribute to a multitude of diseases, including degeneration of the musculoskeletal system. The intervertebral disc (IVD) in the spine contains circadian clocks which control ∼6% of the transcriptome in a rhythmic manner, including key genes involved in extracellular matrix (ECM) homeostasis. However, it remains largely unknown to what extent the local IVD molecular clock is required to drive rhythmic gene transcription and IVD physiology. In this work, we identified profound age-related changes of ECM microarchitecture and an endochondral ossification-like phenotype in the annulus fibrosus (AF) region of the IVD in the Col2a1-Bmal1 knockout mice. Circadian time series RNA-Seq of the whole IVD in Bmal1 knockout revealed loss of circadian patterns in gene expression, with an unexpected emergence of 12 h ultradian rhythms, including FOXO transcription factors. Further RNA sequencing of the AF tissue identified region-specific changes in gene expression, evidencing a loss of AF phenotype markers and a dysregulation of ECM and FOXO pathways in Bmal1 knockout mice. Consistent with an up-regulation of FOXO1 mRNA and protein levels in Bmal1 knockout IVDs, inhibition of FOXO1 in AF cells suppressed their osteogenic differentiation. Collectively, these data highlight the importance of the local molecular clock mechanism in the maintenance of the cell fate and ECM homeostasis of the IVD. Further studies may identify potential new molecular targets for alleviating IVD degeneration.

The early bovine embryo is supported by histotroph molecules secreted by endometrial epithelial (EPI) and stroma fibroblast (SF) cells in response to luteal progesterone (P4). We hypothesized that specific histotroph molecule transcript abundance depends on cell type and P4 concentration and that endometrial cell conditioned media (CM) could improve in vitro produced (IVP) embryo development in culture. Primary bovine EPI and SF cells from seven uteri were incubated for 12 h with RPMI medium containing 0 (Control), 1, 15, or 50 ng of P4. RPMI was also incubated without cells (N-CM) and CM from EPI or SF cultures (EPI- or SF-CM) or a combination of the two (1:1; EPI/SF-CM) was used to culture IVP embryos from days 4-8 of development (n = 117). There was an effect of cell type (SLC1A1, SLC5A6, SLC7A1, FGF-2, FGF-7, CTGF, PRSS23 and NID2) and/or P4 concentration (FGF-7 and NID2) on endometrial cell histotroph molecule mRNA (P < 0.05). Compared to N-CM, blastocyst development on day 7 was greater in the EPI or SF-CM (P ≤ 0.05) and tended to be greater in the EPI/SF-CM (P = 0.07). On day 8, blastocyst development was greater only in the EPI-CM (P < 0.05). Further, culturing embryos with endometrial cell CM reduced day 8 blastocyst transcript abundance of cell adhesion molecule LGALS1 (P < 0.01). In conclusion, endometrial cell CM or histotroph molecules may be used to improve IVP embryo development in cattle.

Various implant treatments, including total disc replacements, have been tried to treat lumbar intervertebral disc (IVD) degeneration, which is claimed to be the main contributor of lower back pain. The treatments, however, come with peripheral issues. This study proposes a novel approach that complies with the anatomical features of IVD, the so-called monolithic total disc replacement (MTDR). As the name suggests, the MTDR is a one-part device that consists of lattice and rigid structures to mimic the nucleus pulposus and annulus fibrosus, respectively. The MTDR can be made of two types of thermoplastic polyurethane (TPU 87A and TPU 95A) and fabricated using a 3D printing approach: fused filament fabrication. The MTDR design involves two configurations-the full lattice (FLC) and anatomy-based (ABC) configurations. The MTDR is evaluated in terms of its physical, mechanical, and cytotoxicity properties. The physical characterization includes the geometrical evaluations, wettability measurements, degradability tests, and swelling tests. The mechanical characterization comprises compressive tests of the materials, an analytical approach using the Voigt model of composite, and a finite element analysis. The cytotoxicity assays include the direct assay using hemocytometry and the indirect assay using a tetrazolium-based colorimetric (MTS) assay. The geometrical evaluation shows that the fabrication results are tolerable, and the two materials have good wettability and low degradation rates. The mechanical characterization shows that the ABC-MTDR has more similar mechanical properties to an IVD than the FLC-MTDR. The cytotoxicity assays prove that the materials are non-cytotoxic, allowing cells to grow on the surfaces of the materials.