Biology subjects

Müller, R.

Publications and source records attributed to Müller, R..

31 records · Page 2Linked to original sources

bioRxiv2021

Large-scale quantification of human osteocyte lacunar morphological biomarkers as assessed by ultra-high-resolution desktop micro-computed tomography

Ultra-high-resolution imaging of the osteocyte lacuno-canalicular network (LCN) three-dimensionally (3D) in a high-throughput fashion has greatly improved the morphological knowledge about the constituent structures - positioning them as potential biomarkers. Technologies such as serial focused ion beam/scanning electron microscopy (FIB/SEM) and confocal scanning laser microscopy (CLSM) can image in extremely high resolution, yet only capture a small number of lacunae. Synchrotron radiation computed tomography (SR-CT) can image with both high resolution and high throughput but has a limited availability. Desktop micro-computed tomography (micro-CT) provides an attractive balance: high-throughput imaging on the micron level without the restrictions of SR-CT availability. Over the past decade, desktop micro-CT has been used to image osteocyte lacunae in a variety of animals, yet few studies have employed it to image human lacunae using clinical biopsies. In this study, accuracy, reproducibility, and sensitivity of large-scale quantification of human osteocyte lacunar morphometries were assessed by ultra-high-resolution desktop micro-computed tomography. For this purpose, thirty-one transiliac human bone biopsies containing trabecular and cortical regions were imaged using ultra-high-resolution desktop micro-CT at a nominal isotropic voxel resolution of 1.2m. The resulting 3D images were segmented, component labeled, and the following morphometric parameters of 7.71 million lacunae were measured: Lacunar number (Lc.N), density (Lc.N/BV), porosity (Lc.TV/BV), volume (Lc.V), surface area (Lc.S), surface area to volume ratio (Lc.S/Lc.V), stretch (Lc.St), oblateness (Lc.Ob), sphericity (Lc.Sr), equancy (Lc.Eq), and angle (Lc.{theta}). Accuracy was quantified by comparing automated lacunar identification to manual identification. Mean true positive rate (TPR), false positive rate (FPR), and false negative rate (FNR) were 89.0%, 3.4%, and 11.0%, respectively. Regarding the reproducibility of lacunar morphometry from repeated measurements, precision errors were low (0.2 - 3.0%) and intraclass correlation coefficients were high (0.960 - 0.999). Significant differences between cortical and trabecular regions (p<0.001) existed for Lc.N/BV, Lc.TV/BV, local lacunar surface area ( ), and local lacunar volume ( ), all of which demonstrate the sensitivity of the method and are possible biomarker candidates. This study provides the foundation required for future large-scale morphometric studies using ultra-high-resolution desktop micro-CT and high-throughput analysis of millions of osteocyte lacunae in human bone samples. Furthermore, the validation of this technology for imaging of human lacunar properties establishes the quality and reliability required for the accurate, precise, and sensitive assessment of osteocyte morphometry in clinical bone biopsies.

bioengineering↗

bioRxiv2020

Sesbanimide R, a novel cytotoxic polyketide produced by magnetotactic bacteria

Genomic information from various magnetotactic bacteria suggested that besides their common ability to form magnetosomes they potentially also represent a source of bioactive natural products. By using targeted deletion and transcriptional activation, we connected a large biosynthetic gene cluster (BGC) of the trans-AT PKS type to the biosynthesis of a novel polyketide in the alphaproteobacterium Magnetospirillum gryphiswaldense. Structure elucidation by mass spectrometry and NMR revealed that this secondary metabolite resembles sesbanimides which were very recently reported from other taxa. However, sesbanimide R exhibits an additional arginine moiety the presence of which reconciles inconsistencies in the previously proposed sesbanimide biosynthesis pathway when comparing the chemical structure and the potential biochemistry encoded in the BGC. In contrast to sesbanimides D, E and F, we were able to assign the stereocenter of the arginine moiety experimentally and two of the remaining three stereocenters by predictive biosynthetic tools. Sesbanimide R displayed strong cytotoxic activity against several carcinoma cell lines. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY ImportanceThe finding of this study contributes a new secondary metabolite member to the glutarimide-containing polyketides. The determined structure of sesbanimide R correlates with its cytotoxic bioactivity characteristic for members of this family. Sesbanimide R represents the first natural product isolated from magnetotactic bacteria and identifies this highly diverse group as a so far untapped source for the future discovery of novel secondary metabolites.

microbiology↗

bioRxiv2020

The Behavior of Molecular Measures of Natural Selection after a Change in Population Size.

A common model to describe natural selection at the molecular level is the nearly neutral theory, which emphasizes the importance of mutations with slightly deleterious fitness effects as they have a chance to get fixed due to genetic drift. Since genetic drift is stronger in smaller than in larger populations, a negative relationship between molecular measures of selection and population size is expected within the nearly neutral theory. Originally, this hypothesis was formulated under equilibrium conditions. A change in population size, however, pushes the selection-drift balance off equilibrium leading to alterations in the efficacy of selection. To investigate the nonequilibrium behavior, we relate measures of natural selection and genetic drift to each other, considering both, measures of micro- and macroevolution. Specifically, we use a Poisson random field framework to model{pi} N/{pi}S and{omega} as time-dependent measures of selection and assess genetic drift by an effective population size. This analysis reveals a clear deviation from the expected equilibrium selection-drift balance during nonequilibrium periods. Moreover, we find that microevolutionary measures quickly react to a change in population size and reflect a recent change well, at the same time as they quickly lose the knowledge about it. Macroevolutionary measures, on the other hand, react more slowly to a change in population size but instead capture the influence of ancient changes longer. We therefore conclude that it is important to be aware of the different behaviors of micro- and macroevo- lutionary measures when making inference in empirical studies, in particular when comparing results between studies.

evolutionary biology↗

bioRxiv2020

The local and global geometry of trabecular bone

AO_SCPLOWBSTRACTC_SCPLOWThe organization and shape of the microstructural elements of trabecular bone govern its physical properties, are implicated in bone disease, and can serve as blueprints for biomaterial design. To devise fundamental structure-property relationships, it is essential to characterize trabecular bone from the perspective of geometry, the mathematical study of shape. Here, we used the micro-computed tomography images of 70 donors at five different sites to characterize the local and global geometry of human trabecular bone, respectively quantified by surface curvatures and Minkowski functionals. We find that curvature density maps provide sensitive shape fingerprints for bone from different sites. Contrary to a common assumption, these curvature maps also show that bone morphology does not approximate a minimal surface but exhibits a much more intricate curvature landscape. At the global (or integral) perspective, our Minkowski analysis illustrates that trabecular bone exhibits other types of anisotropy/ellipticity beyond interfacial orientation, and that anisotropy varies substantially within the trabecular structure. Moreover, we show that the Minkowski functionals unify several traditional morphometric indices. Our geometric approach to trabecular morphometry provides a fundamental language of shape that could be useful for bone failure prediction, understanding geometry-driven tissue growth, and the design of complex tissue engineering scaffolds.

bioinformatics↗

bioRxiv2020

Automated Segmentation of Fractured Distal Radii by 3D Geodesic Active Contouring of in vivo HR-pQCT Images

Radius fractures are among the most common fracture types; however, there is limited consensus on the standard of care. A better understanding of the fracture healing process could help to shape future treatment protocols and thus improve functional outcomes of patients. High-resolution peripheral quantitative computed tomography (HR-pQCT) allows monitoring and evaluation of the radius on the micro-structural level, which is crucial to our understanding of fracture healing. However, current radius fracture studies using HR-pQCT are limited by the lack of automated contouring routines, hence only including small number of patients due to the prohibitively time-consuming task of manually contouring HR-pQCT images. In the present study, a new method to automatically contour images of distal radius fractures based on 3D morphological geodesic active contours (3D-GAC) is presented. Contours of 60 HR-pQCT images of fractured and conservatively treated radii spanning the healing process up to one year post-fracture are compared to the current gold standard, hand-drawn 2D contours, to assess the accuracy of the algorithm. Furthermore, robustness was established by applying the algorithm to HR-pQCT images of intact radii of 73 patients and comparing the resulting morphometric indices to the gold standard patient evaluation including a threshold- and dilation-based contouring approach. Reproducibility was evaluated using repeat scans of intact radii of 19 patients. The new 3D-GAC approach offers contours within inter-operator variability for images of fractured distal radii (mean Dice score of 0.992 {+/-} 0.004 versus median operator Dice score of 0.993 {+/-} 0.006). The generated contours for images of intact radii yielded morphometric indices within the in vivo reproducibility limits compared to the current gold standard. Additionally, the 3D-GAC approach shows an improved robustness against failure (n = 4) when dealing with cortical interruptions, fracture fragments, etc. compared with the automatic, default manufacturer pipeline (n = 40). Using the 3D-GAC approach assures consistent results, while reducing the need for time-consuming hand-contouring.

bioengineering↗

bioRxiv2020

Longitudinal in vivo micro-CT-based approach allows spatio-temporal characterization of fracture healing patterns and assessment of biomaterials in mouse femur defect models

Thorough preclinical evaluation of functionalized biomaterials for treatment of large bone defects is essential prior to clinical application. Using in vivo micro-computed tomography (micro-CT) and mouse femoral defect models with different defect sizes, we were able to detect spatio-temporal healing patterns indicative of physiological and impaired healing in three defect sub-volumes and the adjacent cortex. The time-lapsed in vivo micro-CT-based approach was then applied to evaluate the bone regeneration potential of functionalized biomaterials using collagen and BMP-2. Both collagen and BMP-2 treatment led to distinct changes in bone turnover in the different healing phases. Despite increased periosteal bone formation, 87.5% of the defects treated with collagen scaffolds resulted in non-unions. Additional BMP-2 application significantly accelerated the healing process and increased the union rate to 100%. This study further shows potential of time-lapsed in vivo micro-CT for capturing spatio-temporal deviations preceding non-union formation and how this can be prevented by application of functionalized biomaterials. This study therefore supports the application of longitudinal in vivo micro-CT for discrimination of normal and disturbed healing patterns and for the spatio-temporal characterization of the bone regeneration capacity of functionalized biomaterials.

bioengineering↗

bioRxiv2020

Individualized cyclic mechanical loading improves callus properties during the remodelling phase of fracture healing in mice as assessed from time-lapsed in vivo imaging

Fracture healing is regulated by mechanical loading. Understanding the underlying mechanisms during the different healing phases is required for targeted mechanical intervention therapies. Here, the influence of individualized cyclic mechanical loading on the remodelling phase of fracture healing was assessed in a mouse femur defect model. After bridging of the defect, a loading group (n=10) received individualized cyclic mechanical loading (8-16 N, 10 Hz, 5 min, 3x/week) based on computed strain distribution in the callus using animal-specific real-time micro-finite element analysis. Controls (n=10) received 0 N treatment at the same post-operative time-points. By registration of consecutive scans, structural and dynamic callus morphometric parameters were followed in three callus sub-volumes and the adjacent cortex showing that the remodelling phase of fracture healing is highly responsive to cyclic mechanical loading with changes in dynamic parameters leading to significantly larger callus formation and mineralization. Loading-mediated maintenance of callus remodelling was associated with distinct effects on Wnt-signalling-associated molecular targets Sclerostin and RANKL in callus sub-regions and the adjacent cortex. Given these distinct local protein expression patterns induced by cyclic mechanical loading, the femur defect loading model with individualized load application seems suitable to understand the local spatio-temporal mechano-molecular regulation of the different fracture healing phases.

bioengineering↗

bioRxiv2020

Subject-specific mechanical loading of bones: Improved healing of mouse vertebral defects using adaptive loading regimes.

Methods to repair bone defects arising from trauma, resection, or disease, continue to be sought after. Cyclic mechanical loading is well established to influence bone (re)modelling activity, in which bone formation and resorption are correlated to micro-scale strain. Based on this, the application of mechanical stimulation across a bone defect could improve healing. However, if ignoring the mechanical integrity of defected bone, loading regimes have a high potential to either cause damage or be ineffective. This study explores real-time finite element (rtFE) methods that use three-dimensional structural analyses from micro-computed tomography images to estimate effective peak cyclic loads in a subject-specific and time-dependent manner. It demonstrates the concept in a cyclically loaded mouse caudal vertebral bone defect model. Using rtFE analysis combined with adaptive mechanical loading, mouse bone healing was significantly improved over non-loaded controls, with no incidence of vertebral fractures. Such rtFE-driven adaptive loading regimes demonstrated here could be relevant to clinical bone defect healing scenarios, where mechanical loading can become patient-specific and more efficacious. This is achieved by accounting for initial bone defect conditions and spatio-temporal healing, both being factors that are always unique to the patient.

bioengineering↗

bioRxiv2020

Development of a micro-CT mouse holder for a femur defect mouse model

Correct fixation of an object is essential for accurate micro computed tomography scanning. In this document, we provide a motivation for, description of, and use cases for a mouse holder appropriate for holding an externally fixated mouse in femur fracture/defect healing experiments. In addition to rigid fixation, the holder provides heating and anaesthetic gas to ensure correct anaesthetic conditions for the animal. We provide the description and design files for a Scanco viva40 scanner, but with small changes, the holder can be used with other scanners.

systems biology↗

bioRxiv2020

Reducing variance in a mouse defect healing model: Real-time Finite Element Analysis allows homogenization of tissue scale strains

Mechanical loading allows both investigation into the mechano-regulation of fracture healing as well as interventions to improve fracture-healing outcomes such as delayed healing or non-unions. However, loading is seldom individualised or even targeted to an effective mechanical stimulus level within the bone tissue. In this study, we use micro-finite element analysis to demonstrate the result of using a constant loading assumption for all mouse femurs in a given group. We then contrast this with the application of an adaptive loading approach, denoted real time Finite Element (rtFE) adaptation, in which micro-computed tomography images provide the basis for micro-FE based simulations and the resulting strains are manipulated and targeted to a reference distribution. Using this approach, we demonstrate that individualised femoral loading leads to a better-specified strain distribution and lower variance in tissue mechanical stimulus across all mice, both longitudinally and cross-sectionally, while making sure that no overloading is occurring leading to refracture of the femur bones.

systems biology↗

bioRxiv2020

Supersampling Enables Accurate Microstructural Bone Adaptation Simulations in Human in vivo HR-pQCT Images

In silico trials of treatments in a virtual physiological human (VPH) would revolutionize research in the biomedical field. Hallmarks of bone disease and treatments can already be simulated in pre-clinical models and in ex vivo data of humans using microstructural bone adaptation simulations. The increasing availability of in vivo high resolution peripheral quantitative computed tomography (HR-pQCT) images provides novel opportunities to validate and ultimately utilize microstructural bone adaptation simulations to improve our understanding of bone diseases and move towards in silico VPH decision support systems for clinicians. In the present study, we investigated if microstructural bone adaptation simulations of in vivo human HR-pQCT images yielded accurate results. Since high-resolution ground truth images cannot be obtained in vivo, we applied an ex vivo approach to study resolution dependence and the effect of upscaling on morphometric accuracy. To address simulation initialisation issues, we developed an input regularisation approach to reduce initialisation shocks observed in microstructural bone adaptation simulations and evaluated upscaling as a way to improve the accuracy of model inputs. Finally, we compared our ex vivo results to simulations run on in vivo images to investigate whether in vivo image artefacts further affect simulation outcomes.

bioinformatics↗

bioRxiv2020

Effects of long-term in vivo micro-CT imaging on hallmarks of osteopenia and frailty in aging mice

In vivo micro-CT has already been used to monitor microstructural changes of bone in mice of different ages and in models of age-related diseases such as osteoporosis. However, as aging is accompanied by frailty and subsequent increased sensitivity to external stimuli such as handling and anesthesia, the extent to which longitudinal imaging can be applied in aging studies remains unclear. Consequently, the potential of monitoring individual mice during the entire aging process - from healthy to frail status - has not yet been exploited. In this study, we assessed the effects of long-term in vivo micro-CT imaging - consisting of 11 imaging sessions over 20 weeks - on hallmarks of aging both on a local (i.e., static and dynamic bone morphometry) and systemic (i.e., frailty index (FI) and body weight) level at various stages of the aging process. Furthermore, using a premature aging model (PolgA(D257A/D257A)), we assessed whether these effects differ between genotypes. The 6th caudal vertebrae of 4 groups of mice (PolgA(D257A/D257A) and PolgA(+/+)) were monitored by in vivo micro-CT every 2 weeks. One group was subjected to 11 scans between weeks 20 and 40 of age, whereas the other groups were subjected to 5 scans between weeks 26-34, 32-40 and 40-46, respectively. The long-term monitoring approach showed small but significant changes in the static bone morphometric parameters compared to the other groups. However, no interaction effect between groups and genotype was found, suggesting that PolgA mutation does not render bone more or less susceptible to long-term micro-CT imaging. The differences between groups observed in the static morphometric parameters were less pronounced in the dynamic morphometric parameters. Moreover, the body weight and FI were not affected by more frequent imaging sessions. Finally, we observed that longitudinal designs including baseline measurements at young adult age are more powerful at detecting effects of in vivo micro-CT imaging on hallmarks of aging than cross-sectional comparisons between multiple groups of aged mice subjected to fewer imaging sessions.

physiology↗

bioRxiv2019

Hallmarks of frailty and osteosarcopenia in prematurely aged PolgAD257A/D257A mice: a preclinical model to test anabolic interventions

BackgroundFrailty is a geriatric syndrome characterized by increased susceptibility to adverse health outcomes. One major determinant thereof is the gradual weakening of the musculoskeletal system and the associated osteosarcopenia. To improve our understanding of the underlying pathophysiology and, more importantly, to test potential interventions aimed at counteracting frailty suitable animal models are needed. MethodsTo evaluate the relevance of prematurely aged PolgA(D257A/D257A) mice as a model for frailty and osteosarcopenia, we quantified the clinical mouse frailty index in PolgA(D257A/D257A) and wild type littermates (PolgA(+/+), WT) with age and concertedly assessed the quantity and quality of bone and muscle tissue. Lastly, the anabolic responsiveness of skeletal muscle, muscle progenitors and bone was assessed. ResultsPolgA(D257A/D257A) accumulated health deficits at a higher rate compared to WT, resulting in a higher frailty index at 40 and 46 weeks of age (+166%, +278%, p<0.0001), respectively, with no differences between genotypes at 34 weeks. Concomitantly, PolgA(D257A/D257A) displayed progressive musculoskeletal deterioration such as reduced bone and muscle mass as well as impaired functionality thereof. In addition to lower muscle weights (-14%, p<0.05, -23%, p<0.0001) and fiber area (-20%, p<0.05, -22%, p<0.0001) at 40 and 46 weeks, respectively, PolgA(D257A/D257A) showed impairments in grip-strength and concentric muscle forces (p<0.05). PolgA(D257A/D257A) mutation altered the acute response to various anabolic stimuli in skeletal muscle and muscle progenitors. While PolgA(D257A/D257A) muscles were hypersensitive to eccentric contractions as well as leucine administration, shown by larger downstream signaling response of the mechanistic target of rapamycin complex 1 (mTORC1), myogenic progenitors cultured in vitro showed severe anabolic resistance to leucine and robust impairments in cell proliferation. Longitudinal micro-CT analysis of the 6th caudal vertebrae showed that PolgA(D257A/D257A) had lower bone morphometric parameters (e.g. bone volume fraction, trabecular and cortical thickness, p<0.05) as well as reduced remodeling activities (e.g. bone formation and resorption rate, p<0.05) compared to WT. When subjected to 4 weeks of cyclic loading, young but not aged PolgA(D257A/D257A) caudal vertebrae showed load-induced bone adaptation suggesting reduced mechanosensitivity with age. ConclusionsPolgA(D257A/D257A) mutation leads to hallmarks of age-related frailty and osteosarcopenia and provides a powerful model to better understand the relationship between frailty and the aging musculoskeletal system.

physiology↗