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In this study, the influence of a plasma electrolytic oxidation (PEO) surface treatment on a medical-grade WE43-based magnesium alloy is examined through an experimental and computational framework that considers the effects of localised corrosion features and mechanical properties throughout the corrosion process. First, a comprehensive in-vitro immersion study was performed on WE43-based tensile specimens with and without PEO surface modification, which included fully automated spatial reconstruction of the phenomenological features of corrosion through micro-CT scanning, followed by uniaxial tensile testing. Then the experimental data of both unmodified and PEO-modified groups were used to calibrate parameters of a finite element-based surface corrosion model. In-vitro, it was found that the WE43-PEO modified group had a significantly lower corrosion rate and maintained significantly higher mechanical properties than the unmodified. While corrosion rates were ∼50% lower in the WE43-PEO modified specimens, the local geometric features of corroding surfaces remained similar to the unmodified WE43 group, however evolving after almost the double amount of time. We were also able to quantitatively demonstrate that the PEO surface treatment on magnesium continued to protect samples from corrosion throughout the entire period tested, and not just in the early stages of corrosion. Using the results from the testing framework, the model parameters of the surface-based corrosion model were identified for both groups. This enabled, for the first time, in-silico prediction of the physical features of corrosion and the mechanical performance of both unmodified and PEO modified magnesium specimens. This simulation framework can enable future in-silico design and optimisation of bioabsorbable magnesium devices for load-bearing medical applications.

Abstract Background Nuclear factor erythroid 2–related factor 2 (Nrf2) is a crucial transcription factor for cellular redox homeostasis. The association of Nrf2 with elderly female osteoporotic has yet to be fully described. The aim was to elucidate a potential age-dependent Nrf2 contribution to female osteoporosis in mice. Methods Eighteen female wild type (WT) and 16 Nrf2-knockout (KO) mice were sacrificed at different ages (12 weeks = young mature adult and 90 weeks = old) to analyze their femurs. The morphological properties (trabecular and cortical) were evaluated by micro-computed tomography (μCT) and compared to gold standard histochemistry analysis. The quasi-static compression tests were performed to calculate the mechanical properties of bones. Additionally, the population of bone resorbing cells and aromatase expression by osteocytes was immunohistochemically evaluated and empty osteocyte lacunae was counted in cortical bone. Results Old Nrf2-KO mice revealed a significantly reduced trabecular bone mineral density (BMD), cortical thickness, cortical area, and bone fraction compared to old WT mice, regardless of no significant difference in skeletally mature young adult mice between WT and KO. Specifically, while all old WT mice showed thin metaphyseal trabeculae, trabecular bone was completely absent in 60% of old KO mice. Additionally, old KO mice showed significantly more osteoclast-like cells and fewer aromatase-positive osteocytes than WT mice, whereas the occurrence of empty osteocyte lacunae did not differ between both groups. Nrf2-KO mice further showed an age-dependently reduced fracture resilience compared to age-matched WT mice. Conclusion Our results suggest that chronic Nrf2 loss can lead to age-dependent progression of female osteoporosis.

Abstract Background The evaluation of bone remodelling and dental root resorption can be performed by histological techniques or micro-computed tomography (micro-CT). The present study aimed to evaluate the relationship between these two procedures in the context of cleft repair in a rat model. Methods The reconstructed maxillae and the orthodontically-moved first molar of 12 rats were analysed for correlations between the histological and radiological findings retrospectively. The alveolar cleft repairs were performed using bone autografts or (human) xenografts. Four weeks after the operation, the intervention of the first molar protraction was initiated and lasted for eight weeks. The newly formed bone and the root resorption lacunae were determined via histology. In the micro-CT analysis, the average change of bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness and trabecular separation of the jaw, as well as the volume of the root resorptions were determined. The Pearson correlation coefficient was applied to study the associations between groups. Results Positive correlations were found only between the newly formed bone (histology) and BMD changes (micro-CT) in the autograft group (r = 0.812, 95% CI: 0.001 to 0.979, p = 0.05). The relationship of newly formed bone and BV/TV was similar but not statistically significant (r = 0.691, 95% CI: −0.274 to 0.963, p = 0.013). Regarding root resorption, no significant correlations were found. Conclusions Due to the lack of correlation between histological and radiological findings of bone remodelling and the development of root resorptions, both methods should be combined in this cleft model in rats for a comprehensive analysis.

This study develops a three-dimensional automated detection framework (PitScan) that systematically evaluates the severity and phenomenology of pitting corrosion. This framework uses a python-based algorithm to analyse microcomputer-tomography scans (μCT) of cylindrical specimens undergoing corrosion. The approach systematically identifies several surface-based corrosion features, enabling full spatial characterisation of pitting parameters, including pit density, pit size, pit depth as well as pitting factor according to ASTM G46-94. Furthermore, it is used to evaluate pitting formation in tensile specimens of a Rare Earth Magnesium alloy undergoing corrosion, and relationships between key pitting parameters and mechanical performance are established. Results demonstrated that several of the parameters described in ASTM G46-94, including pit number, pit density and pitting factor, showed little correlation to mechanical performance. However, this study did identify that other parameters showed strong correlations with the ultimate tensile strength and these tended to be directly linked to the reduction of the cross-sectional area of the specimen. Specifically, our results indicate, that parameters directly linked to the loss of the cross-sectional area (e.g. minimum material width), are parameters that are most suited to provide an indication of a specimen's mechanical performance. The automated detection framework developed in this study has the potential to provide a basis to standardise measurements of pitting corrosion across a range of metals and future prediction of mechanical strength over degradation time.

Abstract To minimize the postoperative risks posed by grafting autologous transplants for cleft repair, efforts are being made to improve grafting materials for use as potential alternatives. The aim of this study was to compare the bone graft quality of different bone substitutes including the gold standard autografts during the healing processes after cleft repair in the context of orthodontic treatment. In 21 Wistar rats, a complete, continuity-interrupting cleft was created. After 4 weeks, cleft repair was performed using autografts from the hips’ ischial tuberosity, human xenografts, or synthetic bone substitutes [beta-tricalcium phosphate (β-TCP)/hydroxyapatite (HA)]. After another 4 weeks, the first molar movement was initiated in the reconstructed jaw for 8 weeks. The bone remodeling was analyzed in vivo using micro-computed tomography (bone mineral density and bone volume fraction) and histology (new bone formation). All the grafting materials were statistically different in bone morphology, which changed during the treatment period. The β-TCP/HA substitute demonstrated less resorption compared to the autologous and xenogeneic/human bone, and the autografts led to a stronger reaction in the surrounding bone. Histologically, the highest level of new bone formation was found in the human xenografts, and the lowest was found in the β-TCP/HA substitute. The differences between the two bone groups and the synthetic materials were statistically significant. Autografts were confirmed to be the gold standard in cleft repair with regard to graft integration. However, parts of the human xenograft seemed comparable to the autografts. Thus, this substitute could perhaps be used as an alternative after additional tissue-engineered modification.

Small animal micro computed tomography (μCT) is an important tool in cancer research and is used to quantify liver and lung tumors. A type of cancer that is intensively investigated with μCT is hepatocellular carcinoma (HCC). μCT scans acquire projections from different angles of the gantry which rotates X-ray source and detector around the animal. Motion of the animal causes inconsistencies between the projections which lead to artifacts in the resulting image. This is problematic in HCC research, where respiratory motion affects the image quality by causing hypodense intensity at the liver edge and smearing out small structures such as tumors. Dealing with respiratory motion is particularly difficult in a high throughput setting when multiple mice are scanned together and projection removal by retrospective respiratory gating may compromise image quality and dose efficiency. In mice, inhalation anesthesia leads to a regular respiration with short gasps and long phases of negligible motion. Using this effect and an iterative reconstruction which can cope with missing angles, we discard the relatively few projections in which the gasping motion occurs. Moreover, since gated acquisition, i.e., acquiring multiple projections from a single gantry angle is not a requirement, this method can be applied to existing scans. We applied our method in a high throughput setting in which four mice with HCC tumors were scanned simultaneously in a multi-mouse bed. To establish a ground truth, we manually selected projections with visible respiratory motion. Our automated intrinsic breathing projection selection achieved an accordance of 97% with manual selection. We reconstructed volumetric images and demonstrated that our intrinsic gating method significantly reduces the hypodense depiction at the cranial liver edge and improves the detectability of small tumors. Furthermore, we show that projection removal in a four mice scan discards only 7.5% more projections than in a single-mouse setting, i.e., four mouse scanning does not substantially compromise dose efficiency or image quality. To the best of our knowledge, no comparable method that combines multi-mouse scans for high throughput, intrinsic respiratory gating, and an available iterative reconstruction has been described for liver tumor imaging before.

Acetylsalicylic acid (ASA) is a well-established drug for heart attack and stroke prophylaxis. Furthermore, numerous studies have reported an anti-carcinogenic effect, but its exact mechanism is still unknown. Here, we applied VEGFR-2-targeted molecular ultrasound to explore a potential inhibitory effect of ASA on tumor angiogenesis in vivo. Daily ASA or placebo therapy was performed in a 4T1 tumor mouse model. During therapy, ultrasound scans were performed using nonspecific microbubbles (CEUS) to determine the relative intratumoral blood volume (rBV) and VEGFR-2-targeted microbubbles to assess angiogenesis. Finally, vessel density and VEGFR-2 expression were assessed histologically. CEUS indicated a decreasing rBV in both groups over time. VEGFR-2 expression increased in both groups up to Day 7. Towards Day 11, the binding of VEGFR-2-specific microbubbles further increased in controls, but significantly (p = 0.0015) decreased under ASA therapy (2.24 ± 0.46 au vs. 0.54 ± 0.55 au). Immunofluorescence showed a tendency towards lower vessel density under ASA and confirmed the result of molecular ultrasound. Molecular US demonstrated an inhibitory effect of ASA on VEGFR-2 expression accompanied by a tendency towards lower vessel density. Thus, this study suggests the inhibition of angiogenesis via VEGFR-2 downregulation as one of the anti-tumor effects of ASA.

Abstract Cancer nanomedicines rely on the enhanced permeability and retention (EPR) effect for efficient target site accumulation. The EPR effect, however, is highly heterogeneous among different tumor types and cancer patients and its extent is expected to dynamically change during the course of nanochemotherapy. Here the authors set out to longitudinally study the dynamics of the EPR effect upon single‐ and double‐dose nanotherapy with fluorophore‐labeled and paclitaxel‐loaded polymeric micelles. Using computed tomography‐fluorescence molecular tomography imaging, it is shown that the extent of nanomedicine tumor accumulation is predictive for therapy outcome. It is also shown that the interindividual heterogeneity in EPR‐based tumor accumulation significantly increases during treatment, especially for more efficient double‐dose nanotaxane therapy. Furthermore, for double‐dose micelle therapy, tumor accumulation significantly increased over time, from 7% injected dose per gram (ID g–1) upon the first administration to 15% ID g–1 upon the fifth administration, contributing to more efficient inhibition of tumor growth. These findings shed light on the dynamics of the EPR effect during nanomedicine treatment and they exemplify the importance of using imaging in nanomedicine treatment prediction and clinical translation.

We provide a well-defined accurate brute force implementation without approximations as a reference to support future development of correct and fast algorithms to measure the trabecular thickness and spacing. Using artificial ellipsoid examples, the systematic error is shown to be related to the voxelization, which can be reduced by upsampling. Furthermore, we collected a database of 40 three-dimensional micro-computed tomography images of trabecular bone regions of a sheep femur. With the coverage of a broad range of trabecular thickness and spacing, the database is suitable for assessing the correctness of algorithms for the measurements of trabecular thickness and spacing. The bone data is shared publicly in the online repository ‘Figshare’ and the brute force code is freely available in Code Ocean. Scientists are encouraged to implement faster algorithms, e.g., graphic processing unit-accelerated, with accurate or controlled approximate behaviour to compute trabecular thickness and spacing. Especially, the provided data can be re-employed together with the reference algorithm to evaluate their accuracies.

Obesity and metabolic syndrome are of increasing global concern. In order to understand the basic biology and etiology of obesity, research has turned to animals across the vertebrate spectrum including zebrafish. Here, we carefully characterize zebrafish in a long-term obesogenic environment as well as zebrafish that went through early lifetime caloric restriction. We found that long-term obesity in zebrafish leads to metabolic endpoints comparable to mammals including increased adiposity, weight, hepatic steatosis and hepatic lesions but not signs of glucose dysregulation or differences in metabolic rate or mitochondrial function. Malnutrition in early life has been linked to an increased likelihood to develop and an exacerbation of metabolic syndrome, however fish that were calorically restricted from five days after fertilization until three to nine months of age did not show signs of an exacerbated phenotype. In contrast, the groups that were shifted later in life from caloric restriction to the obesogenic environment did not completely catch up to the long-term obesity group by the end of our experiment. This dataset provides insight into a slowly exacerbating time-course of obesity phenotypes.

Abstract Background Cancer cachexia represents a central obstacle in medical oncology as it is associated with poor therapy response and reduced overall survival. Systemic inflammation is considered to be a key driver of cancer cachexia; however, clinical studies with anti‐inflammatory drugs failed to show distinct cachexia‐inhibiting effects. To address this contradiction, we investigated the functional importance of innate immune cells for hepatocellular carcinoma (HCC)‐associated cachexia. Methods A transgenic HCC mouse model was intercrossed with mice harbouring a defect in myeloid cell‐mediated inflammation. Body composition of mice was analysed via nuclear magnetic resonance spectroscopy and microcomputed tomography. Quantitative PCR was used to determine adipose tissue browning and polarization of adipose tissue macrophages. The activation state of distinct areas of the hypothalamus was analysed via immunofluorescence. Multispectral immunofluorescence imaging and immunoblot were applied to characterize sympathetic neurons and macrophages in visceral adipose tissue. Quantification of pro‐inflammatory cytokines in mouse serum was performed with a multiplex immunoassay. Visceral adipose tissue of HCC patients was quantified via the L3 index of computed tomography scans obtained during routine clinical care. Results We identified robust cachexia in the HCC mouse model as evidenced by a marked loss of visceral fat and lean mass. Computed tomography‐based analyses demonstrated that a subgroup of human HCC patients displays reduced visceral fat mass, complementing the murine data. While the myeloid cell‐mediated inflammation defect resulted in reduced expression of pro‐inflammatory cytokines in the serum of HCC‐bearing mice, this unexpectedly did not translate into diminished but rather enhanced cachexia‐associated fat loss. Defective myeloid cell‐mediated inflammation was associated with decreased macrophage abundance in visceral adipose tissue, suggesting a role for local macrophages in the regulation of cancer‐induced fat loss. Conclusions Myeloid cell‐mediated inflammation displays a rather unexpected beneficial function in a murine HCC model. These results demonstrate that immune cells are capable of protecting the host against cancer‐induced tissue wasting, adding a further layer of complexity to the pathogenesis of cachexia and providing a potential explanation for the contradictory results of clinical studies with anti‐inflammatory drugs.

Background: Biological organ engineering is a novel experimental approach to generate functional liver grafts by decellularization and repopulation. Currently, healthy organs of small or large animals and human organs with preexisting liver diseases are used to optimize decellularization and repopulation.However, the effects of morphological changes on allo- and xenogeneic cell-scaffold interactions during repopulation procedure, e.g., using scaffold-sections, are unknown. We present a sequential morphological workflow to identify murine liver scaffold-sections with well-preserved microarchitecture. Methods: Native livers (CONT, n = 9) and livers with experimentally induced pathologies (hepatics steatosis: STEA, n = 7; hepatic fibrosis induced by bile duct ligation: BDL, n = 9; nodular regenerative hyperplasia induced by 90% partial hepatectomy: PH, n = 8) were decellularized using SDS and Triton X-100 to generate cell-free scaffolds. Scaffold-sections were assessed using a sequential morphological workflow consisting of macroscopic, microscopic and morphological evaluation: (1) The scaffold was evaluated by a macroscopic decellularization score. (2) Regions without visible tissue remnants were localized for sampling and histological processing. Subsequent microscopical examination served to identify tissue samples without cell remnants. (3) Only cell-free tissue sections were subjected to detailed liver-specific morphological assessment using a histological and immunohistochemical decellularization score. Results: Decellularization was feasible in 33 livers, which were subjected to the sequential morphological workflow. In 11 of 33 scaffolds we achieved a good macroscopic decellularization result (CONT: 3 scaffolds; STEA: 3 scaffolds; BDL: 3 scaffolds; PH: 2 scaffolds). The microscopic assessment resulted in the selection of 88 cell-free tissue sections (CONT: 15 sections; STEA: 38 sections; BDL: 30 sections; PH: 5 sections). In 27 of those sections we obtained a good histological decellularization result (CONT: 3 sections; STEA: 6 sections; BDL: 17 sections; PH: 1 section). All experimental groups contained sections with a good immunohistochemical decellularization result (CONT: 6 sections; STEA: 5 sections; BDL: 4 sections; PH: 1 section). Discussion: Decellularization was possible in all experimental groups, irrespectively of the underlying morphological alteration. Furthermore, our proposed sequential morphological workflow was suitable to detect tissue sections with well-preserved hepatic microarchitecture, as needed for further repopulation experiments.

The plasma protein fetuin-A mediates the formation of protein-mineral colloids known as calciprotein particles (CPP)-rapid clearance of these CPP by the reticuloendothelial system prevents errant mineral precipitation and therefore pathological mineralization (calcification). The mutant mouse strain D2,Ahsg-/- combines fetuin-A deficiency with the calcification-prone DBA/2 genetic background, having a particularly severe compound phenotype of microvascular and soft tissue calcification. Here we studied mechanisms leading to soft tissue calcification, organ damage and death in these mice. We analyzed mice longitudinally by echocardiography, X-ray-computed tomography, analytical electron microscopy, histology, mass spectrometry proteomics, and genome-wide microarray-based expression analyses of D2 wildtype and Ahsg-/- mice. Fetuin-A-deficient mice had calcified lesions in myocardium, lung, brown adipose tissue, reproductive organs, spleen, pancreas, kidney and the skin, associated with reduced growth, cardiac output and premature death. Importantly, early-stage calcified lesions presented in the lumen of the microvasculature suggesting precipitation of mineral containing complexes from the fluid phase of blood. Genome-wide expression analysis of calcified lesions and surrounding (not calcified) tissue, together with morphological observations, indicated that the calcification was not associated with osteochondrogenic cell differentiation, but rather with thrombosis and fibrosis. Collectively, these results demonstrate that soft tissue calcification can start by intravascular mineral deposition causing microvasculopathy, which impacts on growth, organ function and survival. Our study underscores the importance of fetuin-A and related systemic regulators of calcified matrix metabolism to prevent cardiovascular disease, especially in dysregulated mineral homeostasis.

Calcifications can disrupt organ function in the cardiovascular system and the kidney, and are particularly common in patients with chronic kidney disease (CKD). Fetuin-A deficient mice maintained against the genetic background DBA/2 exhibit particularly severe soft tissue calcifications, while fetuin-A deficient C57BL/6 mice remain healthy. We employed molecular genetic analysis to identify risk factors of calcification in fetuin-A deficient mice. We sought to identify pharmaceutical therapeutic targets that could be influenced by dietary of parenteral supplementation. We studied the progeny of an intercross of fetuin-A deficient DBA/2 and C57BL/6 mice to identify candidate risk genes involved in calcification. We determined that a hypomorphic mutation of the Abcc6 gene, a liver ATP transporter supplying systemic pyrophosphate, and failure to regulate the Trpm6 magnesium transporter in kidney were associated with severity of calcification. Calcification prone fetuin-A deficient mice were alternatively treated with parenteral administration of fetuin-A dietary magnesium supplementation, phosphate restriction, or by or parenteral pyrophosphate. All treatments markedly reduced soft tissue calcification, demonstrated by computed tomography, histology and tissue calcium measurement. We show that pathological ectopic calcification in fetuin-A deficient DBA/2 mice is caused by a compound deficiency of three major extracellular and systemic inhibitors of calcification, namely fetuin-A, magnesium, and pyrophosphate. All three of these are individually known to contribute to stabilize protein-mineral complexes and thus inhibit mineral precipitation from extracellular fluid. We show for the first time a compound triple deficiency that can be treated by simple dietary or parenteral supplementation. This is of special importance in patients with advanced CKD, who commonly exhibit reduced serum fetuin-A, magnesium and pyrophosphate levels.

Abstract Background Alveolar cleft repair is performed via bone grafting procedure to restore the dental arch continuity. A suitable bone substitute materials should possess osteoinductive and osteoconductive properties, to promote new bone formation, along with a slowly resorbable scaffold that is subsequently replaced with functionally viable bone. Calcium phosphate biomaterials have long proved their efficacy as bone replacement materials. Dentin in several forms has also demonstrated its possibility to be used as bone graft replacement material in several studies. The purpose of this study was to evaluate bone regeneration pattern and quantify bone formation after grafting pre-established experimental alveolar clefts defects model in rabbits using composite xenogenic dentin and β-TCP in comparison to β-TCP alone. Methods Unilateral alveolar cleft defects were created in 16 New Zealand rabbits according to previously described methodology. Alveolar clefts were allowed 8 weeks healing period. 8 defects were filled with β-TCP, whereas 8 defects filled with composite xenogenic dentin with β-TCP. Bone regeneration of the healed defects was compared at the 8 weeks after intervention. Quantification of bone formation was analyzed using micro-computed tomography (µCT) and histomorphometric analysis. Results µCT and histomorphometric analysis revealed that defects filled with composite dentin/β-TCP showed statistically higher bone volume fraction, bone mineral density and percentage residual graft volume when compared to β-TCP alone. An improved surgical handling of the composite dentin/β-TCP graft was also noted. Conclusions Composite xenogenic dentin/β-TCP putty expresses enhanced bone regeneration compared to β-TCP alone in the reconstruction of rabbit alveolar clefts defects.

The liver has a unique regenerative capability upon injury or partial resection. The regeneration process comprises a complex interplay between parenchymal and non-parenchymal cells and is tightly regulated at different scales. Thus, we investigated liver regeneration using multi-scale methods by combining non-invasive imaging with immunohistochemical analyses. In this context, non-invasive imaging can provide quantitative data of processes involved in liver regeneration at organ and body scale. We quantitatively measured liver volume recovery after 70% partial hepatectomy (PHx) by micro computed tomography (μCT) and investigated changes in the density of CD68+ macrophages by fluorescence-mediated tomography (FMT) combined with μCT using a newly developed near-infrared fluorescent probe. In addition, angiogenesis and tissue-resident macrophages were analyzed by immunohistochemistry. Based on the results, a model describing liver regeneration and the interactions between different cell types was established. In vivo analysis of liver volume regeneration over 21 days after PHx by μCT imaging demonstrated that the liver volume rapidly increased after PHx reaching a maximum at day 14 and normalizing until day 21. An increase in CD68+ macrophage density in the liver was detected from day 4 to day 8 by combined FMT-μCT imaging, followed by a decline towards control levels between day 14 and day 21. Immunohistochemistry revealed the highest angiogenic activity at day 4 after PHx that continuously declined thereafter, whereas the density of tissue-resident CD169+ macrophages was not altered. The simulated time courses for volume recovery, angiogenesis and macrophage density reflect the experimental data describing liver regeneration after PHx at organ and tissue scale. In this context, our study highlights the importance of non-invasive imaging for acquiring quantitative organ scale data that enable modeling of liver regeneration.

Even though animal trials are a controversial topic, they provide knowledge about diseases and the course of infections in a medical context. To refine the detection of abnormalities that can cause pain and stress to the animal as early as possible, new processes must be developed. Due to its noninvasive nature, thermal imaging is increasingly used for severity assessment in animal-based research. Within a multimodal approach, thermal images combined with anatomical information could be used to simulate the inner temperature profile, thereby allowing the detection of deep-seated infections. This paper presents the generation of anatomical thermal 3D models, forming the underlying multimodal model in this simulation. These models combine anatomical 3D information based on computed tomography (CT) data with a registered thermal shell measured with infrared thermography. The process of generating these models consists of data acquisition (both thermal images and CT), camera calibration, image processing methods, and structure from motion (SfM), among others. Anatomical thermal 3D models were successfully generated using three anesthetized mice. Due to the image processing improvement, the process was also realized for areas with few features, which increases the transferability of the process. The result of this multimodal registration in 3D space can be viewed and analyzed within a visualization tool. Individual CT slices can be analyzed axially, sagittally, and coronally with the corresponding superficial skin temperature distribution. This is an important and successfully implemented milestone on the way to simulating the internal temperature profile. Using this temperature profile, deep-seated infections and inflammation can be detected in order to reduce animal suffering.

The kinase RIPK3 initiates necroptosis, which has been reported to promote inflammation in various pathological conditions. Here, the authors show that genetic ablation of Ripk3results in adipocyte apoptosis and white adipose tissue inflammation in obese mice, which promotes glucose intolerance.

It was hypothesized that strontium (Sr)-doped β-tricalcium phosphate (TCP)-based scaffolds have a positive effect on the regeneration of large bone defects (LBD). Readouts in our mice models were nuclear factor-kappa beta (NF-κB) activity and vascular endothelial growth factor receptor-2 (VEGFR-2) promoter activity during the healing process. A 2-mm critical-size femoral fracture was performed in transgenic NF-κB- and VEGFR-2-luciferase reporter mice. The fracture was filled with a 3D-printed β-TCP scaffold with or without Sr. A bioluminescence in-vivo imaging system was used to sequentially investigate NF-κB and VEGFR-2 expression for two months. After sacrifice, soft and osseous tissue formation in the fracture sites was histologically examined. NF-κB activity increased in the β-TCP + Sr group in the latter stage (day 40–60). VEGFR-2 activity increased in the + Sr group from days 0–15 but decreased and showed significantly less activity than the β-TCP and non-scaffold groups from days 40–60. The new bone formation and soft tissue formation in the + Sr group were significantly higher than in the β-TCP group, whereas the percentage of osseous tissue formation in the β-TCP group was significantly higher than in the β-TCP + Sr group. We analyzed longitudinal VEGFR-2 promoter activity and NF-κB activity profiles, as respective agents of angiogenesis and inflammation, during LBD healing. The extended inflammation phase and eventually more rapid resorption of scaffold caused by the addition of strontium accelerates temporary bridging of the fracture gaps. This finding has the potential to inform an improved treatment strategy for patients who suffer from osteoporosis.

CD62L (L-Selectin) dependent lymphocyte infiltration is known to induce inflammatory bowel disease (IBD), while its function in the liver, especially in non-alcoholic steatohepatitis (NASH), remains unclear. We here investigated the functional role of CD62L in NASH in humans as well as in two mouse models of steatohepatitis. Hepatic expression of a soluble form of CD62L (sCD62L) was measured in patients with steatosis and NASH. Furthermore, CD62L−/− mice were fed with a methionine and choline deficient (MCD) diet for 4 weeks or with a high fat diet (HFD) for 24 weeks. Patients with NASH displayed increased serum levels of sCD62L. Hepatic CD62L expression was higher in patients with steatosis and increased dramatically in NASH patients. Interestingly, compared to wild type (WT) mice, MCD and HFD-treated CD62L−/− mice were protected from diet-induced steatohepatitis. This was reflected by less fat accumulation in hepatocytes and a dampened manifestation of the metabolic syndrome with an improved insulin resistance and decreased cholesterol and triglyceride levels. Consistent with ameliorated disease, CD62L−/− animals exhibited an enhanced hepatic infiltration of Treg cells and a strong activation of an anti-oxidative stress response. Those changes finally resulted in less fibrosis in CD62L−/− mice. Additionally, this effect could be reproduced in a therapeutic setting by administrating an anti-CD62L blocking antibody. CD62L expression in humans and mice correlates with disease activity of steatohepatitis. CD62L knockout and anti-CD62L-treated mice are protected from diet-induced steatohepatitis suggesting that CD62L is a promising target for therapeutic interventions in NASH.

In this study, we investigate the use of multifunctional smart radiotherapy biomaterials (SRBs) loaded with immunoadjuvants for boosting the abscopal effect of local radiotherapy (RT). SRBs were designed similar to currently used inert RT biomaterials, incorporating a biodegradable polymer with reservoir for loading payloads of the immunoadjuvant anti-CD40 monoclonal antibody. Lung (LLC1) tumors were generated both on the right and left flank of each mouse, with the left tumor representing metastasis. The mice were randomized and divided into eight cohorts with four cohorts receiving image-guided RT (IGRT) at 5 Gy and another similar four cohorts at 0 Gy. IGRT and Computed Tomography (CT) imaging were performed using a small animal radiation research platform (SARRP). Tumor volume measurements for both flank tumors and animal survival was assessed over 25 weeks. Tumor volume measurements showed significantly enhanced inhibition in growth for the right flank tumors of mice in the cohort treated with SRBs loaded with CD40 mAbs and IGRT. Results also suggest that the use of polymeric SRBs with CD40 mAbs without RT could generate an immune response, consistent with previous studies showing such response when using anti-CD40. Overall, 60% of mice treated with SRBs showed complete tumor regression during the observation period, compared to 10% for cohorts administered with anti-CD40 mAbs, but no SRB. Complete tumor regression was not observed in any other cohorts. The findings justify more studies varying RT doses and quantifying the immune-cell populations involved when using SRBs. Such SRBs could be developed to replace currently used RT biomaterials, allowing not only for geometric accuracy during RT, but also for extending RT to the treatment of metastatic lesions.

BACKGROUND:Liver regeneration consists of cellular proliferation leading to parenchymal and vascular growth. This study complements previous studies on cellular proliferation and weight recovery by (1) quantitatively describing parenchymal and vascular regeneration, and (2) determining their relationship. Both together are needed to (3) characterize the underlying growth pattern. METHODS:Specimens were created by injecting a polymerizing contrast agent in either portal or hepatic vein in normal or regenerating livers after 70% partial hepatectomy. 3D image data were obtained through micro-CT scanning. Parenchymal growth was assessed by determining weight and volume of the regenerating liver. Vascular growth was described by manually determined circumscribed parameters (maximal vessel length and radius of right inferior portal/hepatic vein), automatically determined cumulative parameters (total edge length and total vascular volume), and parameters describing vascular density (total edge length/volume, vascular volume fraction). The growth pattern was explored by comparing the relative increase of these parameters to the increase expected in case of isotropic expansion. RESULTS:Liver volume recovery paralleled weight recovery and reached 90% of the original liver volume within 7 days. Comparing radius-related vascular parameters immediately after surgical resection and after virtual resection in-silico revealed a slight increase, possibly reflecting the effect of resection-induced portal hyperperfusion. Comparing length-related parameters between post-operative day 7 and after virtual resection showed similar vascular growth in both vascular systems investigated. In contrast, radius-related parameters increased slightly more in the portal vein. Despite the seemingly homogeneous 3D growth, the observed vascular parameters were not compatible with the hypothesis of isotropic expansion of liver parenchyma and vascular structures. CONCLUSION:We present an approach for the quantitative analysis of the vascular systems of regenerating mouse livers. We applied this technique for assessing the hepatic growth pattern. Prospectively, this approach can be used to investigate hepatic vascular regeneration under different conditions.

The intra-hepatic vascular anatomy in rodents, its variations and corresponding supplying and draining territories in respect to the lobar structure of the liver have not been described. We performed a detailed anatomical imaging study in rats and mice to allow for further refinement of experimental surgical approaches.LEWIS-Rats and C57Bl/6N-Mice were subjected to ex-vivo imaging using μCT. The image data were used for semi-automated segmentation to extract the hepatic vascular tree as prerequisite for 3D visualization. The underlying vascular anatomy was reconstructed, analysed and used for determining hepatic vascular territories.The four major liver lobes have their own lobar portal supply and hepatic drainage territories. In contrast, the paracaval liver is supplied by various small branches from right and caudate portal veins and drains directly into the vena cava. Variations in hepatic vascular anatomy were observed in terms of branching pattern and distance of branches to each other. The portal vein anatomy is more variable than the hepatic vein anatomy. Surgically relevant variations were primarily observed in portal venous supply.For the first time the key variations of intrahepatic vascular anatomy in mice and rats and their surgical implications were described. We showed that lobar borders of the liver do not always match vascular territorial borders. These findings are of importance for the design of new surgical procedures and for understanding eventual complications following hepatic surgery.

The pathomechanisms of atherosclerosis and vascular remodelling are under intense research. Only a few in vivo tools to study these processes longitudinally in animal experiments are available. Here, we evaluated the potential of micro-CT technology.Lumen areas of the common carotid arteries (CCA) in the ApoE-/- partial carotid artery ligation mouse model were compared between in vivo and ex vivo micro-CT technique and serial histology in a total of 28 animals. AuroVist-15 nm nanoparticles were used as in vivo blood pool contrast agent in a Skyscan 1176 micro-CT at resolution of 18 μmeter voxel size and a mean x-ray dose of 0.5 Gy. For ex vivo imaging, animals were perfused with MicroFil and imaged at 9 μmeter voxel size. Lumen area was evaluated at postoperative days 7, 14, and 28 first by micro-CT followed by histology.In vivo micro-CT and histology revealed lumen loss starting at day 14. The lumen profile highly correlated (r = 0.79, P

The liver is the central organ for detoxification of xenobiotics in the body. In pharmacokinetic modeling, hepatic metabolization capacity is typically quantified as hepatic clearance computed as degradation in well-stirred compartments. This is an accurate mechanistic description once a quasi-equilibrium between blood and surrounding tissue is established. However, this model structure cannot be used to simulate spatio-temporal distribution during the first instants after drug injection. In this paper, we introduce a new spatially resolved model to simulate first pass perfusion of compounds within the naive liver. The model is based on vascular structures obtained from computed tomography as well as physiologically based mass transfer descriptions obtained from pharmacokinetic modeling. The physiological architecture of hepatic tissue in our model is governed by both vascular geometry and the composition of the connecting hepatic tissue. In particular, we here consider locally distributed mass flow in liver tissue instead of considering well-stirred compartments. Experimentally, the model structure corresponds to an isolated perfused liver and provides an ideal platform to address first pass effects and questions of hepatic heterogeneity. The model was evaluated for three exemplary compounds covering key aspects of perfusion, distribution and metabolization within the liver. As pathophysiological states we considered the influence of steatosis and carbon tetrachloride-induced liver necrosis on total hepatic distribution and metabolic capacity. Notably, we found that our computational predictions are in qualitative agreement with previously published experimental data. The simulation results provide an unprecedented level of detail in compound concentration profiles during first pass perfusion, both spatio-temporally in liver tissue itself and temporally in the outflowing blood. We expect our model to be the foundation of further spatially resolved models of the liver in the future.

For improved tumor staging and therapy control, imaging biomarkers are of great interest allowing a noninvasive characterization of invasiveness. In squamous epithelial skin and cervix lesions, transition to invasive stages is associated with enhanced matrix metalloproteinase (MMP) activity, increased angiogenesis, and worsened prognosis. Thus, we investigated MMP activity as imaging biomarker of invasiveness and the potential of optical tomography in characterizing the angiogenic and invasive behavior of skin squamous cell carcinoma (SCC) xenografts. MMP activity was measured in vivo in HaCaT-ras A-5RT3 tumors at different angiogenic and invasive stages (onset of angiogenesis, intermediate and highly angiogenic, invasive stage) and after 1 week of sunitinib treatment by fluorescence molecular tomography–microcomputed tomography imaging using an activatable probe. Treatment response was additionally assessed morphologically by optical coherence tomography (OCT). In vivo MMP activity significantly differed between the groups, revealing highest levels in the highly angiogenic, invasive tumors that were confirmed by immunohistochemistry. At the onset of angiogenesis with lowest MMP activity, fibroblasts were detected in the MMP-positive areas, whereas macrophages were absent. Accumulation of both cell types occurred in both invasive groups, again to a significantly higher degree at the most invasive and angiogenic stage. Sunitinib treatment significantly reduced the MMP activity and accumulation of fibroblasts and macrophages and blocked tumor invasion that was additionally visualized by OCT. Human cervical SCCs also showed high MMP activity and a similar stromal composition as the HaCaT xenografts, whereas normal tissue was negative. This study strongly suggests MMP activity as imaging biomarker and demonstrates the high sensitivity of optical tomography in determining tumor invasiveness that can morphologically be supported by OCT.

BACKGROUND: Lead-free hunting bullets are an alternative to lead-containing bullets which cause health risks for humans and endangered scavenging raptors through lead ingestion. However, doubts concerning the effectiveness of lead-free hunting bullets hinder the wide-spread acceptance in the hunting and wildlife management community. METHODS: We performed terminal ballistic experiments under standardized conditions with ballistic soap as surrogate for game animal tissue to characterize dimensionally stable, partially fragmenting, and deforming lead-free bullets and one commonly used lead-containing bullet. The permanent cavities created in soap blocks are used as a measure for the potential wound damage. The soap blocks were imaged using computed tomography to assess the volume and shape of the cavity and the number of fragments. Shots were performed at different impact speeds, covering a realistic shooting range. Using 3D image segmentation, cavity volume, metal fragment count, deflection angle, and depth of maximum damage were determined. Shots were repeated to investigate the reproducibility of ballistic soap experiments. RESULTS: All bullets showed an increasing cavity volume with increasing deposited energy. The dimensionally stable and fragmenting lead-free bullets achieved a constant conversion ratio while the deforming copper and lead-containing bullets showed a ratio, which increases linearly with the total deposited energy. The lead-containing bullet created hundreds of fragments and significantly more fragments than the lead-free bullets. The deflection angle was significantly higher for the dimensionally stable bullet due to its tumbling behavior and was similarly low for the other bullets. The deforming bullets achieved higher reproducibility than the fragmenting and dimensionally stable bullets. CONCLUSION: The deforming lead-free bullet closely resembled the deforming lead-containing bullet in terms of energy conversion, deflection angle, cavity shape, and reproducibility, showing that similar terminal ballistic behavior can be achieved. Furthermore, the volumetric image processing allowed superior analysis compared to methods that involve cutting of the soap blocks.