Impairment of the glial phagolysosomal system drives prion-like propagation in a Drosophila model of Huntington's disease.

Protein misfolding, aggregation, and spread through the brain are primary drivers of neurodegenerative diseases pathogenesis. Phagocytic glia are responsible for regulating the load of pathogenic protein aggregates in the brain, but emerging evidence suggests that glia may also act as vectors for aggregate spread. Accumulation of protein aggregates could compromise the ability of glia to eliminate toxic materials from the brain by disrupting efficient degradation in the phagolysosomal system. A better understanding of phagocytic glial cell deficiencies in the disease state could help to identify novel therapeutic targets for multiple neurological disorders. Here, we report that mutant huntingtin (mHTT) aggregates impair glial responsiveness to injury and capacity to degrade neuronal debris in male and female adult Drosophila expressing the gene that causes Huntington's disease (HD). mHTT aggregate formation in neurons impairs engulfment and clearance of injured axons and causes accumulation of phagolysosomes in glia. Neuronal mHTT expression induces upregulation of key innate immunity and phagocytic genes, some of which were found to regulate mHTT aggregate burden in the brain. Finally, a forward genetic screen revealed Rab10 as a novel component of Draper-dependent phagocytosis that regulates mHTT aggregate transmission from neurons to glia. These data suggest that glial phagocytic defects enable engulfed mHTT aggregates to evade lysosomal degradation and acquire prion-like characteristics. Together, our findings reveal new mechanisms that enhance our understanding of the beneficial and potentially harmful effects of phagocytic glia in HD and potentially other neurodegenerative diseases.Significance Statement Deposition of amyloid aggregates is strongly associated with neurodegenerative disease progression and neuronal cell loss. Many studies point to glial cells as dynamic mediators of disease, capable of phagocytosing toxic materials, but also promoting chronic inflammation and proteopathic aggregate spread. Thus, glia have emerged as promising therapeutic targets for disease intervention. Here, we demonstrate in a Drosophila model of Huntington's disease that neuronal mHTT aggregates interfere with glial phagocytic engulfment, phagolysosomal processing, and innate immunity transcriptional responses. We also identify Rab10 as a novel modifier of prion-like transmission of mHTT aggregates. Our findings add to a growing narrative of glia as double-edged players in neurodegenerative diseases.

Impairment of the glial phagolysosomal system drives prion-like propagation in a Drosophila model of Huntington's disease.

Protein misfolding, aggregation, and spread through the brain are primary drivers of neurodegenerative diseases pathogenesis. Phagocytic glia are responsible for regulating the load of pathogenic protein aggregates in the brain, but emerging evidence suggests that glia may also act as vectors for aggregate spread. Accumulation of protein aggregates could compromise the ability of glia to eliminate toxic materials from the brain by disrupting efficient degradation in the phagolysosomal system. A better understanding of phagocytic glial cell deficiencies in the disease state could help to identify novel therapeutic targets for multiple neurological disorders. Here, we report that mutant huntingtin (mHTT) aggregates impair glial responsiveness to injury and capacity to degrade neuronal debris in male and female adult Drosophila expressing the gene that causes Huntington's disease (HD). mHTT aggregate formation in neurons impairs engulfment and clearance of injured axons and causes accumulation of phagolysosomes in glia. Neuronal mHTT expression induces upregulation of key innate immunity and phagocytic genes, some of which were found to regulate mHTT aggregate burden in the brain. Finally, a forward genetic screen revealed Rab10 as a novel component of Draper-dependent phagocytosis that regulates mHTT aggregate transmission from neurons to glia. These data suggest that glial phagocytic defects enable engulfed mHTT aggregates to evade lysosomal degradation and acquire prion-like characteristics. Together, our findings reveal new mechanisms that enhance our understanding of the beneficial and potentially harmful effects of phagocytic glia in HD and potentially other neurodegenerative diseases.

Imaging a microfocus X-ray focal spot with a thin coded aperture.

Imaging of the focal spot size in X-ray generators can be achieved by means of a pinhole in a highly attenuating material, such as gold. For microfocus generators with spot sizes of only around 10 microns or less, the material must be very thin to avoid an impractical aspect ratio. With a 90 kV source, only 11% attenuation is attained with 5 µm gold. For a pinhole that is smaller than the focal spot, the signal-to-noise ratio (SNR) may be less than 1. To image the focal spot of a medical X-ray generator, a coded aperture has been used previously to reduce the exposure time required, however the same technique does not appear to have been used to increase the SNR when the attenuation is very low. Such a method is used here, using a no-two-holes-touching variation of a modified uniformly redundant array (MURA). In a prototype sample, with only 5 µm gold having 2.75 µm holes, the focal spot of a microfocus X-ray generator used in a micro-CT system could be clearly visualised and quantified. Directionality of the aberrations made focussing of the X-ray spot more intuitive and reduced the time required to obtain an optimal, quantifiable focus.

An assessment of mineral concentration of dental enamel neighbouring hypothetical orthodontic brackets using X-ray microtomography.

OBJECTIVES: Differences in the mineral concentration (MC) level of dental enamel may represent a precursor of white spot lesions adjacent to fixed orthodontic brackets. The aim of the current in vitro study was to compare the MC level central, occlusal and cervical to orthodontic attachments. METHODS AND MATERIALS: A total of 16 enamel blocks were obtained from sound human premolar samples extracted for orthodontic reasons. The buccal portion of the dental enamel blocks was divided into central, occlusal, and cervical regions and then imaged and measured to calculate the level of MC using quantitative X-ray microtomography methods (XMT) at each site. RESULTS: There was a substantial variation in the mineral concentration with the lowest level being detected in the cervical region when compared with other regions. The MC of the gingival zone was significantly lower than that of the middle zone (P<0.05) and was insignificantly lower than that of the occlusal zone. CONCLUSION: Within the limitations of the current study, it can be concluded that the cervical region of the permanent enamel had the lowest mineral concentration using XMT. The cervical region may therefore be more vulnerable to the development of white spot lesions (WSLs) adjacent to a fixed orthodontic appliance during orthodontic treatment. CLINICAL SIGNIFICANCE: Using X-ray microtomography lower mineral concentration in the cervical region of the enamel was observed. This may make these areas particularly susceptible to demineralisation during fixed appliance-based orthodontic treatment and may influence the bond strength of fixed orthodontic attachments.

Development of two-dimensional beam hardening correction for X-ray micro-CT.

BACKGROUND: Beam-hardening in tomography with polychromatic X-ray sources results from the nonlinear relationship between the amount of substance in the X-ray beam and attenuation. Simple linearisation curves can be derived with the use of an appropriate step wedge, however, this does not yield good results when different materials are present whose relationships between X-ray attenuation and energy are very different. OBJECTIVE: To develop a more accurate method of beam-hardening correction for two-phase samples, particularly immersed or embedded biological hard tissue. METHODS: Use of a two-dimensional step wedge is proposed in this study. This is not created physically but is derived from published X-ray attenuation coefficients in conjunction with a modelled X-ray spectrum, optimised from X-ray attenuation measurements of a calibration carousel. To test this method, a hydroxyapatite disk was scanned twice; first dry, and then immersed in 70% ethanol solution (commonly used to preserve biological specimens). RESULTS: With simple linearisation the immersed disk reconstruction exhibited considerable residual beam hardening, with edges appearing approximately 10% more attenuating. With 2-dimensional correction, the attenuation coefficient showed only around 0.5% deviation from the dry case. CONCLUSION: Two-dimensional beam-hardening correction yielded accurate results and does not require segmentation of the two phases individually.

Clinical and Radiographic Outcome of Non-Surgical Endodontic Treatment Using Calcium Silicate-Based Versus Resin-Based Sealers-A Systematic Review and Meta-Analysis of Clinical Studies.

The aim of this paper is to systematically analyse the effect of calcium silicate-based sealers in comparison to resin-based sealers on clinical and radiographic outcomes of non-surgical endodontic treatment in permanent teeth. Methods: The study was conducted according to the guidelines of the Cochrane Handbook for Systematic Reviews of Interventions and Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) statement. The literature search was performed using PubMed/MEDLINE, Cochrane Central Register of Controlled Trials, Web of Science, DOAJ and OpenGrey with no language restrictions. Two reviewers critically assessed the studies for eligibility. Grading of Recommendations, Assessment, Development and Evaluations (GRADE) was carried out to assess the evidence. Meta-analysis of the pooled data with subgroups was carried out using the RevMan software (p < 0.05). Results: Results from the included studies showed that there were no significant differences between the groups in the 24 h post-obturation pain levels (mean difference (MD), −0.19, 95% CI = −0.43−0.06, p = 0.14, I2 = 0%), but at 48 h (MD, −0.35, 95% CI = −0.64−0.05, p = 0.02, I2 = 0%), a significant difference was observed in favour of calcium silicate sealers. Furthermore, there were no significant differences between the two sealers due to risk of onset or intensity of postoperative pain, need for analgesic and extrusion of the sealer. The heterogeneity assessed using Q test between the included studies was 97% (I2). Conclusions: Within the limitations of this review, the paper shows that calcium silicate-based sealers exhibited optimal performance with similar results to resin-based sealers in terms of average level of post-obturation pain, risk of onset and pain intensity at 24 and 48 h. The observations from the included studies are informative in the clinical evaluation of calcium silicate-based sealers and provide evidence for the conduction of well-designed, controlled randomised clinical trials for a period of at least four years in the future.

Association of individual surgeon volume and postoperative outcome in esophagomyotomy for achalasia.

BACKGROUND: Many surgical disciplines have demonstrated superior outcomes when procedures are performed at "high-volume". Esophagomyotomy is commonly performed for achalasia, however it's unclear what constitutes "high-volume" for this procedure, and if individual procedure volume and outcome are related. We identified physicians performing esophagomyotomy, stratified them by individual case volume, and examined their outcomes with the hypothesis that high-volume surgeons will be associated with improved outcomes as compared to low-volume surgeons. METHODS: The 2015-2019 Florida Agency for Health Care Administration (AHCA) inpatient dataset was queried for esophagomyotomy. Surgeons who performed ≥ 10 procedures during the study period were placed into the high-volume cohort, and those performing < 10 into the low-volume cohort. Groups were compared by length of stay, discharge disposition, and postoperative complications. Patient demographics were evaluated using student's t test and chi square test, p < 0.05 considered significant. RESULTS: Six hundred and sixty-two procedures performed by 135 surgeons were identified. The mean number of esophagomyotomies per surgeon was 4.9 (Range 1-147). The high-volume group (n = 12) performed 362 of the 662 procedures (55%), while the low-volume group (n = 123) performed the remaining 300 (45%). Patients of high-volume physicians had decreased length of stay (1.4 ± 0.8 days vs 4.9 ± 6.7 days, p = 0.01) and were more likely to be discharged to home following surgery (92.8% vs 86.0, p = 0.04). High volume physicians also had statistically significant differences in rates of urinary tract infection (1.4% vs 4.0%, p = 0.034), postoperative malnutrition (5.8% vs 11.0%, p = 0.015), and postoperative fluid and electrolyte disorders (5.5% vs 13.3%, p < 0.0001). CONCLUSION: Surgeons who perform higher volumes of esophagomyotomies are associated with decreased length of stay, higher likelihood of patient discharge to home, and decreased rates of some postoperative complications. This research should prompt further inquiry into defining what constitutes a high-volume center in foregut surgery and their role in improving patient outcomes.

Unlocking history through automated virtual unfolding of sealed documents imaged by X-ray microtomography.

Computational flattening algorithms have been successfully applied to X-ray microtomography scans of damaged historical documents, but have so far been limited to scrolls, books, and documents with one or two folds. The challenge tackled here is to reconstruct the intricate folds, tucks, and slits of unopened letters secured shut with "letterlocking," a practice-systematized in this paper-which underpinned global communications security for centuries before modern envelopes. We present a fully automatic computational approach for reconstructing and virtually unfolding volumetric scans of a locked letter with complex internal folding, producing legible images of the letter's contents and crease pattern while preserving letterlocking evidence. We demonstrate our method on four letterpackets from Renaissance Europe, reading the contents of one unopened letter for the first time. Using the results of virtual unfolding, we situate our findings within a novel letterlocking categorization chart based on our study of 250,000 historical letters.

Physico-Chemical Properties of Calcium-Silicate vs. Resin Based Sealers-A Systematic Review and Meta-Analysis of Laboratory-Based Studies.

INTRODUCTION: The aim of this systematic review is to analyse the effect of physico-chemical properties of calcium silicate-based sealers in comparison to epoxy resin sealers in permanent teeth using a single-cone obturation technique. METHODS: The study was conducted according to the guidelines of Cochrane Handbook for Systematic Reviews of Interventions and Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) statement. Literature search was performed using the PubMed/MEDLINE, Cochrane Central Register of Controlled Trials, Web of Science, DOAJ, Open Gray with no language restrictions until October 2020. Two reviewers assessed the studies for eligibility. Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) was carried out to assess the evidence. Meta-analysis of the pooled data with subgroups was performed using the RevMan software (p < 0.05). RESULTS: Results from the 28 included studies showed that the mean difference in adaptation to root canal walls (marginal adaptation, interfacial gaps and void volume) for both sealers were non-significant. However, void volume values showed a significant mean difference (p < 0.00001) favouring the calcium silicate-based sealers. The pooled meta-analysis reported statistically significant differences for apical microleakage (p = 0.0007) whilst there were non-significant mean differences for fracture resistance (p = 0.09) and push-out bond strength (p = 0.63). The heterogeneity among the included studies was 97% (I2). CONCLUSIONS: Within the limitations of this review, calcium silicate-based sealers demonstrated a similar or superior performance in comparison to resin-based sealers in terms of the physico-chemical properties.

Mineral exchange within restorative materials following incomplete carious lesion removal using 3D non-destructive XMT subtraction methodology.

OBJECTIVES: The objective of this study was to quantify the changes in mineral and selected element concentrations within residual carious dentine and restorative materials following incomplete carious lesion removal (ICLR) using different cavity liners, with non-destructive subtraction 3D-X-ray Microtomography (XMT, QMUL, London, UK). MATERIALS AND METHODS: A total of 126 extracted teeth with deep dental caries were assessed using International Caries Risk and Assessment (ICDAS). Eight teeth were subsequently selected after radiographic evaluation. Each lesion was removed, leaving a thin layer of leathery dentine at the deepest part of cavity. Different cavity lining materials were placed; Mineral Trioxide Aggregate (MTA), calcium hydroxide, (Ca(OH)2), resin-based material (RBM). For each, the restorative material was an encapsulated glass ionomer (GIC) and the control group had a GIC restoration alone. Each tooth was immediately placed in Simulated Body Fluid (SBF). All samples were then imaged using XMT at baseline, and three weeks after treament. The XMT images were then subtracted to show the mineral concentration changes three weeks after treatment. RESULTS: There were significant increases in mineral concentrations within the residual demineralised dentine in individual teeth treated with Ca(OH)2, MTA, RBM, and GIC following immersion in SBF for three weeks. GIC group without any liners showed the greatest increase in mineral concentration, followed by MTA and Ca(OH)2. CONCLUSION: Mineral changes in demineralised dentine and within restorative materials are quantifiable using non-destructive 3D-XMT subtraction methodology. This laboratory study suggested that calcium, phosphate and strontium ion-exchange occurs with GIC, MTA and Ca(OH)2 in deep dentinal lesions following ICLR. CLINICAL RELEVANCE: In clinical practice, incomplete carious lesion removal could be performed to avoid the dental pulp exposure. 3D non-destructive XMT subtraction methodology in a laboratory setting is advantageous to provide evidence for different restorative materials on deep carious lesions prior to clinical investigations.

Fabrication of Porous Bone Scaffolds Using Alginate and Bioactive Glass.

Porous composite scaffold using an alginate and bioactive glass ICIE16M was synthesized by a simple freeze-drying technique. The scaffold was characterized using compression testing, Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), X-ray diffraction (XRD), X-ray microtomography (XMT) and scanning electron microscopy (SEM). The bioactivity of the scaffold was evaluated by its ability to form apatite on its surface in simulated body fluid (SBF). The data collected showed evidence that the novel material produced had an appropriate pore size for osteoconduction, with an average pore size of 110 µm and maximum pore size of 309 µm. Statistical analysis confirmed that the glass filler significantly (P < 0.05) increased the collapse yield of the scaffolds compared with pure alginate scaffolds. The ICIE16M glass had an amorphous structure, favorable for bioactivity.

Crystallographic texture and mineral concentration quantification of developing and mature human incisal enamel.

For human dental enamel, what is the precise mineralization progression spatially and the precise timing of mineralization? This is an important question in the fundamental understanding of matrix-mediated biomineralization events, but in particular because we can use our understanding of this natural tissue growth in humans to develop biomimetic approaches to repair and replace lost enamel tissue. It is important to understand human tissues in particular since different species have quite distinct spatial and temporal progression of mineralization. In this study, five human central incisors at different stages of enamel maturation/mineralization were spatially mapped using synchrotron X-ray diffraction and X-ray microtomography techniques. From the earliest developmental stage, two crystallite-orientation populations coexist with angular separations between the crystallite populations of approximately 40° varying as a function of position within the tooth crown. In general, one population had significantly lower texture magnitude and contributed a higher percentage to the overall crystalline structure, compared to the other population which contributed only 20-30% but had significantly higher texture magnitude. This quantitative analysis allows us to understand the complex and co-operative structure-function relationship between two populations of crystallites within human enamel. There was an increase in the mineral concentration from the enamel-dentin junction peripherally and from the incisal tip cervically as a function of maturation time. Quantitative backscattered-electron analyses showed that mineralization of prism cores precedes that of prism boundaries. These results provide new insights into the precise understanding of the natural growth of human enamel.

Virtual Recovery of Content from X-Ray Micro-Tomography Scans of Damaged Historic Scrolls.

There is a large body of historical documents that are too fragile to be opened or unrolled, making their contents inaccessible. Recent improvements in X-ray scanning technology and computer vision techniques make it possible to perform a "virtual" unrolling of such documents. We describe a novel technique to process a stack of 3D X-ray images to identify the surface of parchment scrolls, unroll them, and create a visualization of their written contents. Unlike existing techniques, we can handle even challenging cases with minimal manual interaction. Our novel approach was deployed on two 15th and 16th century damaged historic scrolls from the manors of Bressingham and Diss Heywood. The former has become fused, probably due to exposure to moisture, and cannot be fully unrolled. The latter was severely burnt several hundred years ago, becoming thoroughly charred, heat-shrunken, and distorted, with all the sheets now brittle and fused together. Our virtual unrolling revealed text that has been hidden for centuries.

Estimating age using permanent molars and third cervical vertebrae shape with a novel semi-automated method.

Estimating chronological age accurately in young adults is difficult and additional methods are required to increase the accuracy. This study explored a new semi-automated method to assess shape change of third cervical vertebra (C3) with age in the living; comparing this as a method to determine whether individuals could be categorised into being less than 18 years of age (<18), or at least 18 years of age (≥18) with tooth formation of the second and third mandibular molars (M2 and M3). The sample was panoramic and lateral skull radiographs of 174 dental patients (78 males, 96 females aged 15-22 years). Twelve variables were compared in two age categories: younger than 18 and at least 18 years of age in males and females separately using a t-test. Tooth formation of M2 and M3 was assessed. Mean values of eight variables of C3 in males and one variable in females were significantly different between the two age categories (p < 0.05). Results for males showed that the best age indicator for age ≥18 was the ratio between height and width of C3 and for females, the ratio between diagonals. Results for molars showed that M2 was mature in 69% of males and 83% of females, within the expected age range of 14-16 years. M3 was highly variable ranging from stages 6-14 for both; M3 was missing in 24% of males and 28% of females and mature in 14% of males and 15% of females. The conclusion was that shape change of C3 has potential as an additional method to group individuals <18 and ≥ 18 years of age.

Comparison of efficacy of dental varnish containing fluoride either with CPP-ACP or bioglass on root caries: Ex vivo study.

OBJECTIVES: To compare the efficacy of fluoride varnishes either casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) or bioglass particles on the severity index of root caries. METHODS: Visual-tactile assessments including lesion hardness was carried out to categorise the severity index of 80 extracted teeth with primary root caries. These teeth were randomly divided into four groups; CPP-ACP and fluoride, bioglass and fluoride, fluoride only, and no treatment. Standardised toothbrushing using a dentifrice containing 1450 ppm fluoride was carried out twice a day for all groups. All samples were stored in remineralising solution at 37°C for 45 days. Visual-tactile assessments were carried out at baseline, and after 45 days. Surface roughness measurements (Ra) were performed at baseline and after 7, 14, 30 and 45 days. X-ray Microtomography was performed at the baseline and after 45 days for three samples from each group to quantify the change in mineral contents in the lesion area. RESULTS: The Visual-tactile assessment results showed a reduction in the severity index of root caries, being 20% in CPP-ACP and fluoride, 100% in bioglass and fluoride, 80% in fluoride only, and 60% in non-varnish (toothbrushing only). Non-significant change in surface roughness was observed in all groups. X-ray Microtomography assessment showed a highly significant increase in the mineral deposition in all cases (p > .001). CONCLUSION: The combination of bioglass with fluoride has a potentially superior effect than either CPP-ACP with fluoride or fluoride only to reverse and arrest the root caries in a laboratory setting. CLINICAL SIGNIFICANCE: The combination of bioglass particles and fluoride formulation is likely to have a significant impact in reversing and arresting root caries in a minimally invasive approach. However, randomised controlled double-blinded clinical trials are required to translate these results into clinical practice.

In vitro and in vivo study of commercial calcium phosphate cement HydroSet™.

The commercial calcium phosphate cement, HydroSet™, was investigated in vitro, studying phase formation, compressive strength and setting time, followed by an ovine in vivo study to measure osseointegration, bone apposition and bone-to-graft contact. The X-ray diffraction and 31 P Magic Angle Spinning Nuclear Magnetic Resonance (MAS NMR) results showed the initial formation of octacalcium phosphate and hydroxyapatite at one hour. Over 7 days the octacalcium phosphate transformed to apatite, which was the only crystalline phase of the cement at 28 days. This apatite phase is thought to be a calcium deficient apatite. In the scanning electron microscopy, histological images of 12-week ovine in vivo results showed a high degree of osseointegration, 92.5%. Compressive strength comparisons between in vitro and in vivo measurements showed a dramatic difference between the in vitro measurements (highest 25.4 MPa) and in vivo (95 MPa), attributed to bone ingrowth into the cement in vivo. To the best of our knowledge this is the first time phase evolution of HydroSet™ and the properties studied in vitro complement the in vivo evaluation of the cement in a publication. The significance of the new finding of initial formation of octacalcium phosphate in this cement is discussed. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 21-30, 2018.

Real-time observations of tooth demineralization in 3 dimensions using X-ray microtomography.

OBJECTIVES: The immediate aim of this study was to develop and test a method to record, visualize and quantify real-time demineralization (or remineralization) of teeth in vitro using X-ray microtomography (XMT or micro-CT). The longer term objective is to improve understanding of demineralization processes, allowing the creation of better artificial models of dental caries and better quantification of the efficacy of caries treatment and prevention regimes. METHODS: For demineralization studies, the tooth is mounted in a plastic container through which the demineralizing solution is circulated during simultaneous repeated scanning. Key features of the method are safe circulation of the demineralizing solution, periodic interruption to adjust X-ray filament current and re-focus, accurate beam-hardening correction and calibration, alignment of reconstructed scans, and normalization of grey-levels to compensate for changes in the X-ray spectrum. The method was tested by scanning an extracted third molar during 8days of demineralization. RESULTS: From the reconstructed 3D images, the progression of an artificial carious lesion could be visualized and quantified. The lesion progressed at approximately 0.1mm per day and appeared to be more erosive in nature. CONCLUSIONS: A successful method has been developed to monitor real-time demineralization in 3 dimensions. Further work is now needed to create better models of true carious lesions. CLINICAL SIGNIFICANCE: Observation of the dynamics of demineralization and remineralization will aid in the development of therapies to treat and prevent dental caries.

Body wall structure in the starfish Asterias rubens.

The body wall of starfish is composed of magnesium calcite ossicles connected by collagenous tissue and muscles and it exhibits remarkable variability in stiffness, which is attributed to the mechanical mutability of the collagenous component. Using the common European starfish Asterias rubens as an experimental animal, here we have employed a variety of techniques to gain new insights into the structure of the starfish body wall. The structure and organisation of muscular and collagenous components of the body wall were analysed using trichrome staining. The muscle system comprises interossicular muscles as well as muscle strands that connect ossicles with the circular muscle layer of the coelomic lining. The collagenous tissue surrounding the ossicle network contains collagen fibres that form loop-shaped straps that wrap around calcite struts near to the surface of ossicles. The 3D architecture of the calcareous endoskeleton was visualised for the first time using X-ray microtomography, revealing the shapes and interactions of different ossicle types. Furthermore, analysis of the anatomical organisation of the ossicles indicates how changes in body shape may be achieved by local contraction/relaxation of interossicular muscles. Scanning synchrotron small-angle X-ray diffraction (SAXD) scans of the starfish aboral body wall and ambulacrum were used to study the collagenous tissue component at the fibrillar level. Collagen fibrils in aboral body wall were found to exhibit variable degrees of alignment, with high levels of alignment probably corresponding to regions where collagenous tissue is under tension. Collagen fibrils in the ambulacrum had a uniformly low degree of orientation, attributed to macrocrimp of the fibrils and the presence of slanted as well as horizontal fibrils connecting antimeric ambulacral ossicles. Body wall collagen fibril D-period lengths were similar to previously reported mammalian D-periods, but were significantly different between the aboral and ambulacral samples. The overlap/D-period length ratio within fibrils was higher than reported for mammalian tissues. Collectively, the data reported here provide new insights into the anatomy of the body wall in A. rubens and a foundation for further studies investigating the structural basis of the mechanical properties of echinoderm body wall tissue composites.