Development, establishment and implementation of molecular biological methods for the detection of enterotoxinogenic Bacillus cereus

Bacillus cereus ist als Sporenbildner mit ubiquitärem Vorkommen sowie ausgeprägten lipo- und proteolytischen Eigenschaften ein Problemkeim in der Lebensmitteltechnologie und -hygiene und verursacht Lebensmittelintoxikationen und -infektionen, die zu den wichtigsten lebensmittelassoziierten Krankheiten gezählt werden. Derzeit besteht in der Routinediagnostik ein Mangel an schnellen und zuverlässigen Methoden zur simultanen Detektion der für diese Erkrankungen kausalen B. cereus Toxine bzw. der zugrunde liegenden Toxin-Gene hbl, nhe, ces und cytK1. Im Rahmen dieser Studie wurden deshalb konventionelle und real-time multiplex PCRs zum simultanen Nachweis der B. cereus Toxin-Gene (hbl, nhe, ces und cytK1) entwickelt und an zuvor bereits immunologisch, zell- und molekularbiologisch charakterisierten B. cereus Stämmen (n = 146) der Stammsammlung des Instituts etabliert: Zur Anwendung in konventionellen Systemen konnten vier multiplex PCRs (PCR 1 – 4) realisiert werden, wobei PCR 1 und 2 dem gleichzeitigen Nachweis der Gene hblC, nheA, ces (PCR1) bzw. hblC, nheA, ces und cytK1 (PCR 2) dienen und mit PCR 3 und 4 die spezifische Detektion der Gene hblC, hblD und hblA bzw. nheA, nheB und nheC ermöglicht wird. Sowohl in den beiden Screening PCRs als auch in den PCRs zur Feindifferenzierung wurde eine Interne Amplifikationskontrolle (IAC) eingesetzt. Für die Applikation in real-time Systemen wurde eine auf SYBR Green I basierende multiplex PCR zur Detektion der B. cereus Toxin-Gene hblD, nheA, ces und cytK1 entwickelt und erfolgreich in den Cyclern LC 2.0 und LC 480 eingesetzt. Die Schwellenwert-Zyklen der multiplex Reaktionen stimmten gut mit denen der singleplex Assays überein und es konnte kein relevanter Sensitivitätsverlust festgestellt werden. In beiden Cyclern wurden für die vier Amplifikate signifikante Unterschiede der Schmelztemperaturen berechnet (p < 0,001). Anschließend wurden die multiplex PCRs zur Charakterisierung von Bacillus cereus Isolaten (n = 208) aus Lebensmittel- und Hygienestatuskontrollen der Bundeswehr eingesetzt. Anhand der detektierten Toxin-Gene konnten die B. cereus Isolate in drei Profile eingeteilt werden (nhe + hbl; nhe + ces; nhe) mit Prävalenzen von 99,5 % für nhe, 62,9 % für hbl sowie 4,3 % für ces. In einem zweiten Schritt wurden die B. cereus Isolate mit validierten, auf monoklonalen Antikörpern basierenden Enzymimmuntests und Zytotoxizitätstests untersucht, wobei eine hohe Übereinstimmung der mit den drei unabhängigen Methoden (PCR, EIA, Zelltest) erhaltenen Ergebnisse verzeichnet wurde. Im Vergleich zu früheren Studien konnte somit eine deutliche Verbesserung der Zuverlässigkeit der molekularbiologischen Ergebnisse erreicht werden.Due to its spore-forming ability combined with ubiquitary occurrence and highly lipolytic and proteolytic properties Bacillus cereus represents a problematic microorganismn in food technology and food hygiene and causes food intoxications and food infections, which are counted among the major food associated diseases. Currently, in routine diagnostics there is a lack of fast and reliable methods for simultaneous detection of the causative toxins and alternatively the underlying toxin genes hbl, nhe, ces and cytK1. Therefore, this study deals with the development of conventional and real-time multiplex PCRs for the simultaneous detection of B. cereus toxin genes (hbl, nhe, ces and cytK1). All methods were established using well characterized B. cereus strains (n = 146) from the strain collection of the institute previously tested with immunological, cellular and molecular biological assays: We developed a total of four conventional multiplex PCRs (PCR 1 – 4) enabling simultaneous detection of genes hblC, nheA, ces in PCR1 and hblC, nheA, ces and cytK1 in PCR 2 as well as the specific analysis of genes hblC, hblD, hblA and nheA, nheB, nheC in PCR 3 and PCR 4, respectively. An internal amplification control (IAC) was implemented in both, the two screening PCRs and the PCRs for fine-typing. For application in real-time systems a multiplex PCR based on SYBR Green I was developed for the detection of B. cereus toxin genes hblD, nheA, ces and cytK1 and successfully applied to cyclers LC 2.0 and LC 480. The threshold cycles in the multiplex assay were in good accordance with the values obtained in the singleplex PCRs and no relevant reduction in sensitivity was observed. In both cyclers significant differences of melting temperatures were calculated for the four PCR products (p < 0.001). Subsequently, the multiplex PCRs were applied for characterization of Bacillus cereus isolates (n = 208) derived from food and hygiene status controls in facilities of the German Federal Armed Forces. The B. cereus isolates could be subdivided into three profiles according to the detected toxin genes (nhe + hbl; nhe + ces; nhe) and prevalences of 99.5 % for nhe, 62.9 % for hbl and 4.3 % for ces were observed. Afterwards the B. cereus isolates were tested with validated enzyme immunoassays based on monoclonal antibodies and cytotoxicity tests. The results obtained with the three independent methods (PCR, EIA, cell test) were in good agreement. Thus, compared to previous studies a considerable improvement was achieved regarding the reliability of molecular biological results

3D Bioprinting of Human Tissues: Biofabrication, Bioinks, and Bioreactors

The field of tissue engineering has progressed tremendously over the past few decades in its ability to fabricate functional tissue substitutes for regenerative medicine and pharmaceutical research. Conventional scaffold-based approaches are limited in their capacity to produce constructs with the functionality and complexity of native tissue. Three-dimensional (3D) bioprinting offers exciting prospects for scaffolds fabrication, as it allows precise placement of cells, biochemical factors, and biomaterials in a layer-by-layer process. Compared with traditional scaffold fabrication approaches, 3D bioprinting is better to mimic the complex microstructures of biological tissues and accurately control the distribution of cells. Here, we describe recent technological advances in bio-fabrication focusing on 3D bioprinting processes for tissue engineering from data processing to bioprinting, mainly inkjet, laser, and extrusion-based technique. We then review the associated bioink formulation for 3D bioprinting of human tissues, including biomaterials, cells, and growth factors selection. The key bioink properties for successful bioprinting of human tissue were summarized. After bioprinting, the cells are generally devoid of any exposure to fluid mechanical cues, such as fluid shear stress, tension, and compression, which are crucial for tissue development and function in health and disease. The bioreactor can serve as a simulator to aid in the development of engineering human tissues from in vitro maturation of 3D cell-laden scaffolds. We then describe some of the most common bioreactors found in the engineering of several functional tissues, such as bone, cartilage, and cardiovascular applications. In the end, we conclude with a brief insight into present limitations and future developments on the application of 3D bioprinting and bioreactor systems for engineering human tissue

3d bioprinting of human tissues: Biofabrication, bioinks and bioreactors

The field of tissue engineering has progressed tremendously over the past few decades in its ability to fabricate functional tissue substitutes for regenerative medicine and pharmaceutical research. Conventional scaffold‐based approaches are limited in their capacity to produce constructs with the functionality and complexity of native tissue. Three‐dimensional (3D) bioprinting offers exciting prospects for scaffolds fabrication, as it allows precise placement of cells, biochemical factors, and biomaterials in a layer‐by‐layer process. Compared with traditional scaffold fabrication approaches, 3D bioprinting is better to mimic the complex microstructures of biological tissues and accurately control the distribution of cells. Here, we describe recent technological advances in bio‐fabrication focusing on 3D bioprinting processes for tissue engineering from data processing to bioprinting, mainly inkjet, laser, and extrusion‐based technique. We then review the associated bioink formulation for 3D bioprinting of human tissues, including biomaterials, cells, and growth factors selection. The key bioink properties for successful bioprinting of human tissue were summarized. After bioprinting, the cells are generally devoid of any exposure to fluid mechanical cues, such as fluid shear stress, tension, and compression, which are crucial for tissue development and function in health and disease. The bioreactor can serve as a simulator to aid in the development of engineering human tissues from in vitro maturation of 3D cell‐laden scaffolds. We then describe some of the most common bioreactors found in the engineering of several functional tissues, such as bone, cartilage, and cardiovascular applications. In the end, we conclude with a brief insight into present limitations and future developments on the application of 3D bioprinting and bioreactor systems for engineering human tissue.ISSN:1422-006

Protocol for preparing formalin-fixed paraffin-embedded musculoskeletal tissue samples from mice for spatial transcriptomics

Here, we present a protocol for using spatial transcriptomics in bone and multi-tissue musculoskeletal formalin-fixed paraffin-embedded (FFPE) samples from mice. We describe steps for tissue harvesting, sample preparation, paraffin embedding, and FFPE sample selection. We detail procedures for sectioning and placement on spatial slides prior to imaging, decrosslinking, library preparation, and final analyses of the sequencing data. The complete protocol takes ca. 18 days for mouse femora with adjacent muscle; of this time, >50% is required for mineralized tissue decalcification. For complete details on the use and execution of this protocol, please refer to Wehrle et al.1 and Mathavan et al.2ISSN:2666-166

Mouse models of accelerated aging in musculoskeletal research for assessing frailty, sarcopenia, and osteoporosis – A review

Musculoskeletal aging encompasses the decline in bone and muscle function, leading to conditions such as frailty, osteoporosis, and sarcopenia. Unraveling the underlying molecular mechanisms and developing effective treatments are crucial for improving the quality of life for those affected. In this context, accelerated aging models offer valuable insights into these conditions by displaying the hallmarks of human aging. Herein, this review focuses on relevant mouse models of musculoskeletal aging with particular emphasis on frailty, osteoporosis, and sarcopenia. Among the discussed models, PolgA mice in particular exhibit hallmarks of musculoskeletal aging, presenting early-onset frailty, as well as reduced bone and muscle mass that closely resemble human musculoskeletal aging. Ultimately, findings from these models hold promise for advancing interventions targeted at age-related musculoskeletal disorders, effectively addressing the challenges posed by musculoskeletal aging and associated conditions in humans.ISSN:1568-1637ISSN:1872-964

The impact of low-magnitude high-frequency vibration on fracture healing is profoundly influenced by the oestrogen status in mice

Fracture healing is impaired in aged and osteoporotic individuals. Because adequate mechanical stimuli are able to increase bone formation, one therapeutical approach to treat poorly healing fractures could be the application of whole-body vibration, including low-magnitude high-frequency vibration (LMHFV). We investigated the effects of LMHFV on fracture healing in aged osteoporotic mice. Female C57BL/6NCrl mice (n=96) were either ovariectomised (OVX) or sham operated (non-OVX) at age 41 weeks. When aged to 49 weeks, all mice received a femur osteotomy that was stabilised using an external fixator. The mice received whole-body vibrations (20 minutes/day) with 0.3 G: peak-to-peak acceleration and a frequency of 45 Hz. After 10 and 21 days, the osteotomised femurs and intact bones (contra-lateral femurs, lumbar spine) were evaluated using bending-testing, micro-computed tomography (μCT), histology and gene expression analyses. LMHFV disturbed fracture healing in aged non-OVX mice, with significantly reduced flexural rigidity (-81%) and bone formation (-80%) in the callus. Gene expression analyses demonstrated increased oestrogen receptor β (ERβ, encoded by Esr2) and Sost expression in the callus of the vibrated animals, but decreased β-catenin, suggesting that ERβ might mediate these negative effects through inhibition of osteoanabolic Wnt/β-catenin signalling. In contrast, in OVX mice, LMHFV significantly improved callus properties, with increased flexural rigidity (+1398%) and bone formation (+637%), which could be abolished by subcutaneous oestrogen application (0.025 mg oestrogen administered in a 90-day-release pellet). On a molecular level, we found an upregulation of ERα in the callus of the vibrated OVX mice, whereas ERβ was unaffected, indicating that ERα might mediate the osteoanabolic response. Our results indicate a major role for oestrogen in the mechanostimulation of fracture healing and imply that LMHFV might only be safe and effective in confined target populations

Real-time finite element analysis allows homogenization of tissue scale strains and reduces variance in a mouse defect healing model

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 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.ISSN:2045-232

Quantification of mechanical stimuli and bone formation in fracture healing using In vivo time-lapsed imaging

During bone regeneration, mechanical loading is believed to be responsible for provoking bone formation, however previous investigations into tissue level loading have been limited to crosssectional\u3cbr/\u3estudies and relied upon idealized models for mechanics.\u3cbr/\u3eBy applying in vivo time-lapse micro-computed tomography (microCT) in concert with imaged based micro-finite element (microFE) analysis we have overcome these limitations and have identified an association between tissue loading and bone formation during fracture healing.\u3cbr/\u3eA femoral defect of 1.24[SD = 0.13] mm was created in five female mice (C57BL/6); the femur was first stabilized with an external fixator (MouseExFix, RISystem, Switzerland). Weekly scans were\u3cbr/\u3eperformed using microCT imaging (vivaCT 40, Scanco Medical, Switzerland) over a period of 6 weeks, resulting in a series of timelapsed images. We determined sites of mineralization by registering\u3cbr/\u3eand overlaying images from the second and third week. Combining this with microFE (Parosol) simulations based upon images of the second week, we separated strains in volumes where mineralization occurred, from volumes where no change occurred.\u3cbr/\u3eTo assess the efficacy of strain as a predictor of mineralization, receiver operating characteristic analysis was used. The optimum strain level correctly predicted 60[SD= 9] % of the mineralization which occurred,\u3cbr/\u3eand the final state for 86[SD= 4] % of the entire volume.\u3cbr/\u3eWe have for the first time, quantitatively demonstrated that an association exists between local tissue strain and bone formation during fracture healing. This could be used to determine the optimal\u3cbr/\u3estiffness for biomaterials intended to promote bone healing

Perspectives on in Silico Bone Mechanobiology: Computational Modelling of Multicellular Systems

Bone mechanobiology is the study of the physical, biological and mechanical processes that continuously affect the multiscale multicellular system of the bone from the organ to the molecular scale. Current knowledge derives from experimental studies, which are often limited to gathering qualitative data in a cross-sectional manner, up to a restricted number of time points. Moreover, the simultaneous collection of information about 3D bone microarchitecture, cell activity as well as protein distribution and level is still a challenge. In silico models can expand qualitative information with hypothetical quantitative systems, which allow quantification, testing and comparison to existing quantifiable experimental data. An overview of multiscale, multiphysics, agent-based and hybrid techniques and their applications to bone mechanobiology is provided in the present review. The study analysed how mechanical signals, cells and proteins can be modelled in silico to represent bone remodelling and adaptation. Hybrid modelling of bone mechanobiology could combine the methods used in multiscale, multiphysics and agent-based models into a single model, leading to a unified and comprehensive understanding of bone mechanobiology. Numerical simulations of in vivo multicellular systems aided in hypothesis testing of such in silico models. Recently, in silico trials have been used to illustrate the mechanobiology of cells and signalling pathways in clinical biopsies and animal bones, including the effects of drugs on single cells and signalling pathways up to the organ level. This improved understanding may lead to the identification of novel therapies for degenerative diseases such as osteoporosis.ISSN:1473-226