Preparation of large biological samples for high-resolution, hierarchical, synchrotron phase-contrast tomography with multimodal imaging compatibility.

Imaging across different scales is essential for understanding healthy organ morphology and pathophysiological changes. The macro- and microscale three-dimensional morphology of large samples, including intact human organs, is possible with X-ray microtomography (using laboratory or synchrotron sources). Preparation of large samples for high-resolution imaging, however, is challenging due to limitations such as sample shrinkage, insufficient contrast, movement of the sample and bubble formation during mounting or scanning. Here, we describe the preparation, stabilization, dehydration and mounting of large soft-tissue samples for X-ray microtomography. We detail the protocol applied to whole human organs and hierarchical phase-contrast tomography at the European Synchrotron Radiation Facility, yet it is applicable to a range of biological samples, including complete organisms. The protocol enhances the contrast when using X-ray imaging, while preventing sample motion during the scan, even with different sample orientations. Bubbles trapped during mounting and those formed during scanning (in the case of synchrotron X-ray imaging) are mitigated by multiple degassing steps. The sample preparation is also compatible with magnetic resonance imaging, computed tomography and histological observation. The sample preparation and mounting require 24-36 d for a large organ such as a whole human brain or heart. The preparation time varies depending on the composition, size and fragility of the tissue. Use of the protocol enables scanning of intact organs with a diameter of 150 mm with a local voxel size of 1 µm. The protocol requires users with expertise in handling human or animal organs, laboratory operation and X-ray imaging.

Altered DNA methylation at age-associated CpG sites in children with growth disorders: impact on age estimation?

Age estimation based on DNA methylation (DNAm) can be applied to children, adolescents and adults, but many CG dinucleotides (CpGs) exhibit different kinetics of age-associated DNAm across these age ranges. Furthermore, it is still unclear how growth disorders impact epigenetic age predictions, and this may be particularly relevant for a forensic application. In this study, we analyzed buccal mucosa samples from 95 healthy children and 104 children with different growth disorders. DNAm was analysed by pyrosequencing for 22 CpGs in the genes PDE4C, ELOVL2, RPA2, EDARADD and DDO. The relationship between DNAm and age in healthy children was tested by Spearman's rank correlation. Differences in DNAm between the groups "healthy children" and the (sub-)groups of children with growth disorders were tested by ANCOVA. Models for age estimation were trained (1) based on the data from 11 CpGs with a close correlation between DNAm and age (R ≥ 0.75) and (2) on five CpGs that also did not present significant differences in DNAm between healthy and diseased children. Statistical analysis revealed significant differences between the healthy group and the group with growth disorders (11 CpGs), the subgroup with a short stature (12 CpGs) and the non-short stature subgroup (three CpGs). The results are in line with the assumption of an epigenetic regulation of height-influencing genes. Age predictors trained on 11 CpGs with high correlations between DNAm and age revealed higher mean absolute errors (MAEs) in the group of growth disorders (mean MAE 2.21 years versus MAE 1.79 in the healthy group) as well as in the short stature (sub-)groups; furthermore, there was a clear tendency for overestimation of ages in all growth disorder groups (mean age deviations: total growth disorder group 1.85 years, short stature group 1.99 years). Age estimates on samples from children with growth disorders were more precise when using a model containing only the five CpGs that did not present significant differences in DNAm between healthy and diseased children (mean age deviations: total growth disorder group 1.45 years, short stature group 1.66 years). The results suggest that CpGs in genes involved in processes relevant for growth and development should be avoided in age prediction models for children since they may be sensitive for alterations in the DNAm pattern in cases of growth disorders.

Blood Vessel Detection Algorithm for Tissue Engineering and Quantitative Histology.

Immunohistochemistry for vascular network analysis plays a fundamental role in basic science, translational research and clinical practice. However, identifying vascularization in histological tissue images is time consuming and markedly depends on the operator's experience. In this study, we present "blood vessel detection-BVD", an automatic algorithm for quantitative analysis of blood vessels in immunohistochemical images. BVD is based on extraction and analysis of low-level image features and spatial filtering techniques, which do not require a training phase. BVD algorithm performance was comparatively evaluated on histological sections from three different in vivo experiments. Collectively, 173 independent images were analyzed, and the algorithm's results were compared to those obtained by human operators. The developed BVD algorithm proved to be a robust and versatile tool, being able to quantify number, area, and spatial distribution of blood vessels within all three considered histologic datasets. BVD is provided as an open-source application working on different operating systems. BVD is supported by a user-friendly graphical interface designed to facilitate large-scale analysis.

Evidence for differences in DNA methylation between Germans and Japanese.

As a contribution to the discussion about the possible effects of ethnicity/ancestry on age estimation based on DNA methylation (DNAm) patterns, we directly compared age-associated DNAm in German and Japanese donors in one laboratory under identical conditions. DNAm was analyzed by pyrosequencing for 22 CpG sites (CpGs) in the genes PDE4C, RPA2, ELOVL2, DDO, and EDARADD in buccal mucosa samples from German and Japanese donors (N = 368 and N = 89, respectively).Twenty of these CpGs revealed a very high correlation with age and were subsequently tested for differences between German and Japanese donors aged between 10 and 65 years (N = 287 and N = 83, respectively). ANCOVA was performed by testing the Japanese samples against age- and sex-matched German subsamples (N = 83 each; extracted 500 times from the German total sample). The median p values suggest a strong evidence for significant differences (p < 0.05) at least for two CpGs (EDARADD, CpG 2, and PDE4C, CpG 2) and no differences for 11 CpGs (p > 0.3).Age prediction models based on DNAm data from all 20 CpGs from German training data did not reveal relevant differences between the Japanese test samples and German subsamples. Obviously, the high number of included "robust CpGs" prevented relevant effects of differences in DNAm at two CpGs.Nevertheless, the presented data demonstrates the need for further research regarding the impact of confounding factors on DNAm in the context of ethnicity/ancestry to ensure a high quality of age estimation. One approach may be the search for "robust" CpG markers-which requires the targeted investigation of different populations, at best by collaborative research with coordinated research strategies.

Imaging intact human organs with local resolution of cellular structures using hierarchical phase-contrast tomography.

Imaging intact human organs from the organ to the cellular scale in three dimensions is a goal of biomedical imaging. To meet this challenge, we developed hierarchical phase-contrast tomography (HiP-CT), an X-ray phase propagation technique using the European Synchrotron Radiation Facility (ESRF)'s Extremely Brilliant Source (EBS). The spatial coherence of the ESRF-EBS combined with our beamline equipment, sample preparation and scanning developments enabled us to perform non-destructive, three-dimensional (3D) scans with hierarchically increasing resolution at any location in whole human organs. We applied HiP-CT to image five intact human organ types: brain, lung, heart, kidney and spleen. HiP-CT provided a structural overview of each whole organ followed by multiple higher-resolution volumes of interest, capturing organotypic functional units and certain individual specialized cells within intact human organs. We demonstrate the potential applications of HiP-CT through quantification and morphometry of glomeruli in an intact human kidney and identification of regional changes in the tissue architecture in a lung from a deceased donor with coronavirus disease 2019 (COVID-19).

[COVID-19-induced coagulopathy and thrombosis manifestations]. / COVID-19-induzierte Koagulopathien und thromboembolische Manifestationen.

CLINICAL ISSUE: Clinically, COVID-19 (coronavirus disease 2019) is increasingly seen as a systemic disease associated with multiorgan involvement through a hypercoagulatory condition in the sense of vasculopathy. STANDARD TREATMENT: Treatment with antiplatelet drugs or heparins appears to be indicated. The current evidence, at least for acetylsalicylic acid (ASA), is lacking. DIAGNOSTIC WORK-UP: Corresponding to the significant proportion of primarily microstructural vascular changes, the radiological diagnosis showed not only macrovascular pathologies, but also diffuse perfusion disorders. PERFORMANCE: Regional hypoperfusion in the lungs can be detected with and without pulmonary arterial embolism. Similar findings can be found in almost all organ systems. PRACTICAL RECOMMENDATIONS: A therapeutic intervention using low molecular weight heparins in hospitalized patients in situation-adapted dosage is indicated and is discussed in detail. In the detection of micro- and macrovascular thrombosis in the context of COVID-19, extended radiological diagnostics play a central role and are the basis of adapted therapy and secondary prevention.

Translating Dental, Oral, and Craniofacial Regenerative Medicine Innovations to the Clinic through Interdisciplinary Commercial Translation Architecture.

Few university-based regenerative medicine innovations in the dental, oral, and craniofacial (DOC) space have been commercialized and affected clinical practice in the United States. An analysis of the commercial translation literature and National Institute for Dental and Craniofacial Research's (NIDCR's) portfolio identified barriers to commercial translation of university-based DOC innovations. To overcome these barriers, the NIDCR established the Dental Oral Craniofacial Tissue Regeneration Consortium. We provide generalized strategies to inform readers how to bridge the "valley of death" and more effectively translate DOC technologies from the research laboratory or early stage company environment to clinical trials and bring needed innovations to the clinic. Three valleys of death are covered: 1) from basic science to translational development, 2) from translational technology validation to new company formation (or licensing to an existing company), and 3) from new company formation to scaling toward commercialization. An adapted phase-gate model is presented to inform DOC regenerative medicine teams how to involve regulatory, manufacturability, intellectual property, competitive assessments, business models, and commercially oriented funding mechanisms earlier in the translational development process. An Industrial Partners Program describes how to conduct market assessments, industry maps, business development processes, and industry relationship management methods to sustain commercial translation through the later-stage valley of death. Paramount to successfully implementing these methods is the coordination and collaboration of interdisciplinary teams around specific commercial translation goals and objectives. We also provide several case studies for translational projects with an emphasis on how they addressed DOC biomaterials for tissue regeneration within a rigorous commercial translation development environment. These generalized strategies and methods support innovations within a university-based and early stage company-based translational development process, traversing the many funding gaps in dental, oral, and craniofacial regenerative medicine innovations. Although the focus is on shepherding technologies through the US Food and Drug Administration, the approaches are applicable worldwide.

Photon-counting detectors in computed tomography: from quantum physics to clinical practice.

Over the last decade, a fundamentally new type of computed tomography (CT) detectors has proved its superior capabilities in both physical and preclinical evaluations and is now approaching the stage of clinical practice. These detectors are able to discriminate single photons and quantify their energy and are hence called photon-counting detectors. Among the promising benefits of this technology are improved radiation dose efficiency, increased contrast-to-noise ratio, reduced metal artifacts, improved spatial resolution, simultaneous multi-energy acquisitions, and the prospect of multi-phase imaging within a single acquisition using multiple contrast agents. Taking the conventional energy-integrating detectors as a reference, the authors demonstrate the technical principles of this new technology and provide phantom and patient images acquired by a whole-body photon-counting CT. These images serve as a basis for discussing the potential future of clinical CT.

[Microvascular changes in COVID-19]. / Mikrovaskuläre Veränderungen bei COVID-19.

BACKGROUND: Clinically, coronavirus disease 2019 (COVID-19) is associated with a wide range of symptoms, which can range from mild complaints of an upper respiratory infection to life-threatening hypoxic respiratory insufficiency and multiorgan failure. OBJECTIVE: The initially identified pulmonary damage patterns, such as diffuse alveolar damage in acute lung failure, are accompanied by new findings that draw a more complex scenario. These include microvascular involvement and a wide range of associated pathologies of multiple organ systems. A back-scaling of microstructural vascular changes is possible via targeted correlation of pathological autopsy results with radiological imaging. MATERIAL AND METHODS: Radiological and pathological correlation as well as microradiological imaging to investigate microvascular involvement in fatal COVID-19. RESULTS: The cases of two COVID-19 patients are presented. Patient 1 showed a relative hypoperfusion in lung regions that did not have typical COVID-19 infiltrates; the targeted post-mortem correlation also showed subtle signs of microvascular damage even in these lung sections. Patient 2 showed both radiologically and pathologically advanced typical COVID-19 destruction of lung structures and the case illustrates the damage patterns of the blood-air barrier. The perfusion deficit of the intestinal wall shown in computed tomography of patient 2 could not ultimately clearly be microscopically attributed to intestinal microvascular damage. CONCLUSION: In addition to microvascular thrombosis, our results indicate a functional pulmonary vasodysregulation as part of the pathophysiology during the vascular phase of COVID-19. The clinical relevance of autopsies and the integration of radiological imaging findings into histopathological injury patterns must be emphasized for a better understanding of COVID-19.

[Temporary hyposmia in COVID-19 patients]. / Temporäre Hyposmie bei COVID-19-Patienten.

BACKGROUND: This is a report on the high incidence of olfactory dysfunction in COVID-19 patients in the first cohort of COVID-19 patients in Germany (Webasto cluster). METHODS: Loss of sense of smell and/or taste was reported by 26 of 63 COVID-19 patients (41%), whereas only 31% of the patients experiencing hyposmia had simultaneous symptoms of rhinitis. Smell tests were performed in 14 of these patients and taste tests in 10. The measurements were conducted in a patient care setting in an early COVID-19 cohort. RESULTS: An olfactory disorder was present in 10/14 patients, before as well as after nasal decongestion. In 2 of these patients, hyposmia was the leading or only symptom of SARS-CoV­2 infection. All tested patients reported recovery of smell and/or taste within 8 to 23 days. CONCLUSION: The data imply that a) COVID-19 can lead to hyposmia in a relevant number of patients, the incidence was approximately 30% in this cohort; b) in most cases, the olfactory disturbance was not associated with nasal obstruction, thus indicating a possible neurogenic origin; and c) the olfactory disorder largely resolved within 1-3 weeks after the onset of COVID-19 symptoms. There were no indications of an increased incidence of dysgeusia. These early data may help in the interpretation of COVID-19-associated hyposmia as well as in the counseling of patients, given the temporary nature of hyposmia observed in this study. Furthermore, according to the current experience, hyposmia without rhinitic obstruction can be the leading or even the only symptom of a SARS-CoV­2 infection.

Scanning Magnetic Microscope Using a Gradiometric Configuration for Characterization of Rock Samples.

Scanning magnetic microscopy is a tool that has been used to map magnetic fields with good spatial resolution and field sensitivity. This technology has great advantages over other instruments; for example, its operation does not require cryogenic technology, which reduces its operational cost and complexity. Here, we presented a spatial domain technique based on an equivalent layer approach for processing the data set produced by magnetic microscopy. This approach estimated a magnetic moment distribution over a fictitious layer composed by a set of dipoles located below the observation plane. For this purpose, we formulated a linear inverse problem for calculating the magnetic vector and its amplitude. Vector field maps are valuable tools for the magnetic interpretation of samples with a high spatial variability of magnetization. These maps could provide comprehensive information regarding the spatial distribution of magnetic carriers. In addition, this approach might be useful for characterizing isolated areas over samples or investigating the spatial magnetization distribution of bulk samples at the micro and millimeter scales. This technique could be useful for many applications that require samples that need to be mapped without a magnetic field at room temperature, including rock magnetism.

Head Loss in Thin-Walled Drip Tapes with Continuous Labyrinth.

Thin-walled drip tapes with continuous labyrinth have been used for irrigation of vegetables and other short-cycle crops, especially due to their low cost. The continuous labyrinths welded into the pipe inner wall affect the head loss along such emitting pipes. In addition, the flow cross section of thin-walled pipes may change due to the effects of the operating pressure, which also has consequences for the head loss. The objective of this work was to investigate experimentally the friction factor and the head loss on thin-walled drip tapes with continuous labyrinths operated under various pressures. Two models of commercial thin-walled drip tapes with continuous labyrinths were evaluated. Nonperforated samples were used to determine the head-loss equations. The equations were adjusted as a function of flow rate and pressure head at the pipe inlet. Alternatively, the diameter in the Darcy-Weisbach equation was adjusted as a function of the pressure head by a power-law model. The possibility of using a mean diameter in the Darcy-Weisbach equation was also analyzed. Experimental investigation indicated that the friction factor in the Darcy-Weisbach equation can be accurately described using a power-law model, like the Blasius equation, but characterized by a coefficient a=0.3442 for the Turbo Tape and a=0.3225 for the Silver Tape. The obtained values of a are larger than those generally used and available in the literature. The influence of the operating pressure on the pipe diameter can be neglected for the purpose of calculating the head loss. The two approaches, considering the variation of the diameter with the pressure head and considering an optimum average diameter for the calculation of head loss by the Darcy-Weisbach equation, produce similar results, allowing accurate prediction of head loss. Evaluating the proposed mathematical models, 95% of predictions presented relative errors of head loss smaller than 5%. For the Turbo Tape, the optimum diameter for the purpose of calculating the head loss is 16.01 mm, which is very close to the value indicated by its manufacturer (15.9 mm). For the Silver Drip, the optimum diameter is 15.71 mm, while the manufacturer gives a value of 16.22 mm, which produces considerable error in the calculation of head loss.

Modulating smooth muscle cell response by the release of TGFß2 from tubular scaffolds for vascular tissue engineering.

Tissue engineering has gained considerable attention in the development of small diameter tissue engineered vascular grafts (TEVGs) for treating coronary heart disease. A properly designed acellular and biodegradable TEVG must encourage the infiltration and growth of vascular smooth muscle cells (SMCs). Our group has previously shown that increasing levels of TGFß2 can differentially modulate SMC migration and proliferation. In this study, tubular electrospun scaffolds loaded with TGFß2 were fabricated using various ratios of gelatin/polycaprolactone (PCL), resulting in scaffolds with porous nano-woven architecture suitable for tissue ingrowth. Scaffold morphology, degradation rate, TGß2 release kinetics, and bioactivity were assessed. TGFß2 was successfully integrated into the electrospun biomaterial that resulted in a differential release profile depending on the gelatin/PCL ratio over the course of 42 days. Higher TGFß2 elution was obtained in scaffolds with higher gelatin content, which may be related to the biodegradation of gelatin in culture media. The biological activity of the released TGFß2 was evaluated by its ability to affect SMC proliferation as a function of its concentration. SMCs seeded on TGFß2-loaded scaffolds also showed higher densities and infiltration after 5 days in culture as compared to scaffolds without TGFß2. Our results demonstrate that the ratio of synthetic and natural polymers in electrospun blends can be used to tune the release of TGFß2. This method can be used to intelligently modulate the SMC response in gelatin/PCL scaffolds making the TGFß2-loaded conduits attractive for cardiovascular tissue engineering applications.

The lactate receptor (HCAR1/GPR81) contributes to doxorubicin chemoresistance via ABCB1 transporter up-regulation in human cervical cancer HeLa cells.

The lactate receptor, also known as hydroxycarboxylic acid receptor 1 (HCAR1/GPR81), plays a vital role in cancer biology. Recently, HCAR1 was reported to enhance metastasis, cell growth, and survival of pancreatic, breast, and cervical cancer cells. This study showed, for the first time, the mechanism of HCAR1-mediated chemoresistance to doxorubicin through regulation of ABCB1 transporter. We observed the HCAR1 agonists L-lactate, D-lactate and 3,5-dihydroxybenzoic acid (DHBA) induced up-regulation of ABCB1. HCAR1 silencing decreased ABCB1 mRNA and protein by 80% and 40%, respectively. Moreover, cellular doxorubicin accumulation decreased by 30% after DHBA treatment, while HCAR1 silencing increased accumulation of ABCB1 substrates by nearly 2-fold. Based on growth inhibition assays, cell cycle analysis, and annexin V staining assays, we demonstrated that HCAR1 enhances cell survival and doxorubicin resistance. Finally, DHBA-stimulated up-regulation of ABCB1 functionality was suppressed by pharmacological inhibition of the PKC pathway. Taken together, our study shows the novel role of HCAR1 in development of chemoresistance in cervical carcinoma HeLa cells via ABCB1 transporter up-regulation.

Six-implant-supported immediate fixed rehabilitation of atrophic edentulous maxillae with tilted distal implants.

BACKGROUND: The aim of this retrospective study was to evaluate the treatment outcome of six Bredent blueSky™ implants (Bredent GmbH, Senden, Germany) immediately loaded with a fixed full-arch prosthesis (two tilted posterior and four axial frontal and premolar implants). METHODS: All 10 patients with atrophic edentulous maxillae being treated with a standardized procedure from 09/2009 to 01/2013, who had a follow-up of at least 3 years, were included. Sixty implants were placed to support 10 screwed prostheses. Twenty-one of them were inserted in fresh extraction sockets. Lab-side-prepared provisional fixed prostheses were placed at the day of implantation. Periotest (PT) values and implant stability quotient (ISQ) were measured after implant surgery and after 3 months of healing in all patients. RESULTS: The analyzed implants were in function in mean 64 ± 13 months (range 42 to 84 months). One axial and two tilted implants failed in three patients. The mean PT values decreased, and ISQ increased significantly after the first 3 months at the osseointegrated tilted and axial implants. With an area under the curve of 0.503 and 0.506 in the receiver operating characteristic, the PT values and the ISQ were unspecific parameters and unsuitable as a predictor for the risk of non-osseointegration. CONCLUSIONS: Within the limits of this small group (n = 10 patients/60 implants), the failure rate of the analyzed implant system (n = 3 respective 5% implant loss) seems to be comparable with other immediate-loading protocols. The failure rate of tilted implants in the atrophic upper jaw was quite high, but the aimed treatment concept could be achieved in every patient. The rehabilitation of the posterior region in edentulous maxilla remains a challenge.

Clinical outcome of alveolar ridge augmentation with individualized CAD-CAM-produced titanium mesh.

BACKGROUND: The augmentation of the jaw has been and continues to be a sophisticated therapy in implantology. Modern CAD-CAM technologies lead to revival of old and established augmentation techniques such as the use of titanium mesh (TM) for bone augmentation. The aim of this retrospective study was to evaluate the clinical outcome of an individualized CAD-CAM-produced TM based on the CT/DVT-DICOM data of the patients for the first time. METHODS: In 17 patients, 21 different regions were augmented with an individualized CAD-CAM-produced TM (Yxoss CBR®, Filderstadt, Germany). For the augmentation, a mixture of autologous bone and deproteinized bovine bone mineral (DBBM) or autologous bone alone was used. Reentry with explantation of the TM and simultaneous implantation of 44 implants were performed after 6 months. Preoperative and 6-month postoperative cone beam computed tomographies (CBCT) were performed to measure the gained bone height. RESULTS: The success rate for the bone grafting procedure was 100%. Thirty-three percent of cases presented an exposure of the TM during the healing period. However, premature removal of these exposed meshes was not necessary. Exposure rate in augmentations performed with mid-crestal incisions was higher than in augmentations performed with a modified poncho incision (45.5 vs. 20%, p = 0.221). In addition, exposure rates in the maxilla were significantly higher than in the mandible (66.7 vs. 8.3%, p = 0.009). Gender, smoking, periodontal disease, gingiva type, used augmentation material, and used membrane had no significant influence on the exposure rate (p > 0.05). The mean vertical augmentation was 6.5 ± 1.7 mm, and the mean horizontal augmentation was 5.5 ± 1.9 mm. Implant survival rate after a mean follow-up of 12 ± 6 months after reentry was 100%. CONCLUSION: Within the limits of the retrospective character of this study, this study shows for the first time that individualized CAD-CAM TM provide a sufficient and safe augmentation technique, especially for vertical and combined defects. However, the soft tissue handling for sufficient mesh covering remains one of the most critical steps using this technique.

Dephasing and diffusion on the alveolar surface.

We propose a surface model of spin dephasing in lung tissue that includes both susceptibility and diffusion effects to provide a closed-form solution of the Bloch-Torrey equation on the alveolar surface. The nonlocal susceptibility effects of the model are validated against numerical simulations of spin dephasing in a realistic lung tissue geometry acquired from synchotron-based µCT data sets of mouse lung tissue, and against simulations in the well-known Wigner-Seitz model geometry. The free induction decay is obtained in dependence on microscopic tissue parameters and agrees very well with in vivo lung measurements at 1.5 Tesla to allow a quantification of the local mean alveolar radius. Our results are therefore potentially relevant for the clinical diagnosis and therapy of pulmonary diseases.