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Musculoskeletal modeling is a well-established method in spine biomechanics and generally employed for investigations concerning both the healthy and the pathological spine. It commonly involves inverse kinematics and optimization of muscle activity and provides detailed insight into joint loading. The aim of the present work was to develop and validate a procedure for the automatized generation of semi-subject-specific multi-rigid body models with an articulated lumbar spine. Individualization of the models was achieved with a novel approach incorporating information from annotated EOS images. The size and alignment of bony structures, as well as specific body weight distribution along the spine segments, were accurately reproduced in the 3D models. To ensure the pipeline's robustness, models based on 145 EOS images of subjects with various weight distributions and spinopelvic parameters were generated. For validation, we performed kinematics-dependent and segment-dependent comparisons of the average joint loads obtained for our cohort with the outcome of various published in vivo and in situ studies. Overall, our results agreed well with literature data. The here described method is a promising tool for studying a variety of clinical questions, ranging from the evaluation of the effects of alignment variation on joint loading to the assessment of possible pathomechanisms involved in adjacent segment disease., Frontiers in Bioengineering and Biotechnology, 9, ISSN:2296-4185

Minimizing the spread of viruses in the environment is the first defense line when fighting outbreaks and pandemics, but the current SARS-CoV-2 pandemic demonstrates how difficult this is on global scale, particularly in a sustainable and environmental-friendly way. Here we introduce and develop a sustainable and bio-degradable anti-viral filtration membrane composed of amyloid nanofibrils made from food-grade milk proteins and iron oxyhydroxide nanoparticles synthesized in-situ from iron salts by simple pH-tuning. Thus, all the membrane components are made of environmentally friendly, non-toxic and widely available materials. The membrane has outstanding efficacy against a broad range of viruses, including enveloped, non-enveloped, airborne and waterborne viruses, such as SARS-CoV-2, H1N1 (the influenza A virus strain responsible for the swine flu pandemic in 2009), and enterovirus 71 (EV71; a non-enveloped virus resistant to harsh conditions such as highly acidic pH), highlighting a possible role in fighting the current and future viral outbreaks and pandemics., Nature Nanotechnology, 16, ISSN:1748-3387, ISSN:1748-3395

When speakers of two or more languages interact, they are likely to influence each other: contact leaves traces in the linguistic record, which in turn can reveal geographic areas of past human interaction and migration. However the complex, multi-dimensional nature of contact has hindered the development of a rigorous methodology for detecting its traces. Specifically, other factors may contribute to similarities between languages. Inheritance (a property is passed from an ancestor to several descendant languages), and universal preference (a property is universally preferred), may both overshadow contact signals. How can we find geographic contact areas in language data, while accounting for the confounding effects of inheritance and universal preference? We presentsBayes, an algorithm for Bayesian clustering in the presence of confounding effects. The algorithm learns which similarities in a set of features are better accounted for by confounders, and which are due to contact effects. Contact areas are free to take any shape or size, but an explicit geographic prior ensures their spatial coherence. We test the clustering method on simulated data and apply it in two case studies to reveal language contact in South America and the Balkans. Our results are supported by —mostly qualitative— findings from previous studies. While we focus on the specific problem of language contact, the method can also be used to uncover other traces of shared history in cultural evolution, and more generally, to reveal latent spatial clusters in the presence of confounders.

BACKGROUND: Avatars in Virtual Reality (VR) can not only represent humans, but also embody intelligent software agents that communicate with humans, thus enabling a new paradigm of human-machine interaction. OBJECTIVE: The research agenda proposed in this paper by an interdisciplinary team is motivated by the premise that a conversation with a smart agent avatar in VR means more than giving a face and body to a chatbot. Using the concrete communication task of patient education, this research agenda is rather intended to explore which patterns and practices must be constructed visually, verbally, para- and nonverbally between humans and embodied machines in a counselling context so that humans can integrate counselling by an embodied VR smart agent into their thinking and acting in one way or another. METHODS: The scientific literature in different bibliographical databases was reviewed. A qualitative narrative approach was applied for analysis. RESULTS: A research agenda is proposed which investigates how recurring consultations of patients with healthcare professionals are currently conducted and how they could be conducted with an embodied smart agent in immersive VR. CONCLUSIONS: Interdisciplinary teams consisting of linguists, computer scientists, visual designers and health care professionals are required which need to go beyond a technology-centric solution design approach. Linguists’ insights from discourse analysis drive the explorative experiments to identify test and discover what capabilities and attributes the smart agent in VR must have, in order to communicate effectively with a human being.

Glioma stem-like cells dynamically transition between a chemoradiation-resistant state (preferentially in the perivascular niche) and a chemoradiation-sensitive state (away from blood vessels). However, physical barriers in the tumour microenvironment restrict the delivery of chemotherapy to tumour compartments distant from blood vessels. Here we show that a computational stochastic model of the spatiotemporal dynamics of the perivascular niche that incorporates glioma stem-like cells, differentiated tumour cells, endothelial cells and stromal cells as well as relevant subcellular, cellular and tissue-level phenomena can be used to optimize the treatment schedules of chemoradiation and of standard radiation fractionation with the administration of temozolomide. In mice with platelet-derived-growth-factor-driven glioblastoma, the model-optimized treatment schedule increased the survival of the animals. For standard radiation fractionation in patients, the model predicts that chemotherapy may be optimally administered about an hour before the application of radiation. Massively parallel simulations of the spatiotemporal dynamics of the tumour microenvironment might predict tumour responses to a broader range of treatments and be used to optimize treatment regimens., Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Nanoscale mapping of the distinct electronic phases characterizing the metal-insulator transition displayed by most of the rare-earth nickelate compounds is fundamental for discovering the true nature of this transition and the possible couplings that are established at the interfaces of nickelate-based heterostructures. Here, we demonstrate that this can be accomplished by using scanning transmission electron microscopy in combination with electron energy-loss spectroscopy. By tracking how the O K and Ni L edge fine structures evolve across two different NdNiO3/SmNiO3 superlattices, displaying either one or two metal- insulator transitions depending on the individual layer thickness, we are able to determine the electronic state of each of the individual constituent materials. We further map the spatial configuration associated with their metallic/insulating regions, reaching unit cell spatial resolution. With this, we estimate the width of the metallic/insulating boundaries at the NdNiO3/SmNiO3 interfaces, which is measured to be on the order of four unit cells.

Regenerative endodontic procedures are an alternative to conventional root-canal treatment and apexification. There are two different tissue engineering approaches that are currently followed, both aiming at the colonisation of the cleaned pulp space by pluripotent cells and subsequent pulp regeneration. Firstly, the transplantation of mesenchymal stem cells (MSCs), and secondly a cell-free strategy that relies on bioactive molecules to trigger the recruitment of the patient’s own cells. The first approach is hampered by costs and regulatory issues. Despite great initial enthusiasm with a clinically used cell-free approach that relies on induced bleeding into the pulp space, results have been revealed to be rather unpredictable, and mere repair rather than regeneration of the pulp-dentin complex is what is typically achieved. Moreover, the extent of further root development is variable, and the concept is limited to immature teeth. This article discusses a third possible way of regenerative endodontics that involves the application of MSC-derived exosomes. These are extracellular vesicles that contain proteins, lipids, and nucleic acids, reflecting the secretome of MSCs. Based on the first in vitro and in vivo studies, exosomes appear to be a potent tool to improve pulp regeneration. This narrative review aims to investigate the therapeutic use of human MSCs or dental pulp-derived exosomes in regenerative endodontics. Furthermore, the focus of this review is on targeting important questions that should be investigated in future in-vivo and clinical studies, such as the choice of scaffold material for exosome delivery into the pulp space.

Additive manufacturing (AM) is an effective tool for accelerating knowledge gain in development processes, as it enables the production of complex prototypes at low cost and with short lead times. In the development of mechanical circulatory support, the use of cheap polymer-based AM techniques for prototype manufacturing allows more design variations to be tested, promoting a better understanding of the respective system and its optimization parameters. Here, we compare four commonly used AM processes for polymers with respect to manufacturing accuracy, surface roughness, and shape fidelity in an aqueous environment. Impeller replicas of the CentriMag blood pump were manufactured with each process and integrated into original pump housings. The assemblies were tested for hydraulic properties and hemolysis in reference to the commercially available pump. Computational fluid dynamic simulations were carried out to support the transfer of the results to other applications. In hydraulic testing, the deviation in pressure head and motor current of all additively manufactured replicas from the reference pump remained below 2% over the entire operating range of the pump. In contrast, significant deviations of up to 620% were observed in hemolysis testing. Only the replicas produced by stereolithography showed a nonsignificant deviation from the reference pump, which we attribute to the low surface roughness of parts manufactured thereby. The results suggest that there is a flow-dependent threshold of roughness above which a surface strongly contributes to cell lysis by promoting a hydraulically rough boundary flow.

Many flowering plants rely on pollinators for their reproductive success. Plant-pollinator interactions usually depend on a complex combination of traits based on a fine-tuned biosynthetic machinery, with many structural and regulatory genes involved. Yet, the physiological mechanisms in plants are the product of evolutionary processes. While evolution has been modifying flowers through millions of years, it is also a rapid process that can change plant traits within few generations. Here we discuss both mechanistic and evolutionary aspects of pollinator attraction. We also propose how latest advances in biotechnology and evolutionary studies, and their combination, will improve the elucidation of molecular mechanisms and evolutionary dynamics of pollinator attraction in changing environments.

Base editors are RNA-programmable deaminases that enable precise single-base conversions in genomic DNA. However, off-target activity is a concern in the potential use of base editors to treat genetic diseases. Here, we report unbiased analyses of transcriptome-wide and genome-wide off-target modifications effected by cytidine base editors in the liver of mice with phenylketonuria. The intravenous delivery of intein-split cytidine base editors by dual adeno-associated viruses led to the repair of the disease-causing mutation without generating off-target mutations in the RNA and DNA of the hepatocytes. Moreover, the transient expression of a cytidine base editor mRNA and a relevant single-guide RNA intravenously delivered by lipid nanoparticles led to ~21% on-target editing and to the reversal of the disease phenotype; there were also no detectable transcriptome-wide and genome-wide off-target edits. Our findings support the feasibility of therapeutic cytidine base editing to treat genetic liver diseases. ISSN:2157-846X

Background: Deficits in interjoint coordination, such as the inability to move out of synergy, are frequent symptoms in stroke subjects with upper limb impairments that hinder them from regaining normal motor function. Kinematic measurements allow a fine-grained assessment of movement pathologies, thereby complementing clinical scales, like the Fugl–Meyer Motor Assessment of the Upper Extremity (FMMA-UE). The study goal was to investigate the effects of the performed task, the tested arm, the dominant affected hand, upper limb function, and age on spatiotemporal parameters of the elbow, shoulder, and trunk. The construct validity of the metrics was examined by relating them with each other, the FMMA-UE, and its arm section.Methods: This is a cross-sectional observational study including chronic stroke patients with mild to moderate upper limb motor impairment. Kinematic measurements were taken using a wearable sensor suit while performing four movements with both upper limbs: (1) isolated shoulder flexion, (2) pointing, (3) reach-to-grasp a glass, and (4) key insertion. The kinematic parameters included the joint ranges of shoulder abduction/adduction, shoulder flexion/extension, and elbow flexion/extension; trunk displacement; shoulder–elbow correlation coefficient; median slope; and curve efficiency. The effects of the task and tested arm on the metrics were investigated using a mixed-model analysis. The validity of metrics compared to clinically measured interjoint coordination (FMMA-UE) was done by correlation analysis.Results: Twenty-six subjects were included in the analysis. The movement task and tested arm showed significant effects (p < 0.05) on all kinematic parameters. Hand dominance resulted in significant effects on shoulder flexion/extension and curve efficiency. The level of upper limb function showed influences on curve efficiency and the factor age on median slope. Relations with the FMMA-UE revealed the strongest and significant correlation for curve efficiency (r = 0.75), followed by shoulder flexion/extension (r = 0.68), elbow flexion/extension (r = 0.53), and shoulder abduction/adduction (r = 0.49). Curve efficiency additionally correlated significantly with the arm subsection, focusing on synergistic control (r = 0.59).Conclusion: The kinematic parameters of the upper limb after stroke were influenced largely by the task. These results underpin the necessity to assess different relevant functional movements close to real-world conditions rather than relying solely on clinical measures.Study Registration: clinicaltrials.gov, identifier NCT03135093 and BASEC-ID 2016-02075.

The clinical treatment of large, full-thickness skin injuries with tissue-engineered autologous dermo–epidermal skin substitutes is an emerging alternative to split-thickness skin grafting. However, their production requires about one month of in vitro cell and tissue culture, which is a significant drawback for the treatment of patients with severe skin defects. With the aim to reduce the production time, we developed a new dynamic bioreactor setup that applies cyclic biaxial tension to collagen hydrogels for skin tissue engineering. By reliably controlling the time history of mechanical loading, the dynamic culturing results in a three-fold increase in collagen hydrogel stiffness and stimulates the embedded fibroblasts to enter the cell cycle. As a result, the number of fibroblasts is increased by 75% compared to under corresponding static culturing. Enhanced fibroblast proliferation promotes expression of dermal extracellular matrix proteins, keratinocyte proliferation, and the early establishment of the epidermis. The time required for early tissue maturation can therefore be reduced by one week. Analysis of the separate effects of cyclic loading, matrix stiffening, and interstitial fluid flow indicates that cyclic deformation is the dominant biophysical factor determining fibroblast proliferation, while tissue stiffening plays a lesser role. Local differences in the direction of deformation (in-plane equibiaxial vs. uniaxial strain) influence fibroblast orientation but not proliferation, nor the resulting tissue properties. Importantly, dynamic culturing does not activate fibroblast differentiation into myofibroblasts. The present work demonstrates that control of mechanobiological cues can be very effective in driving cell response toward a shorter production time for human skin substitutes., Biomaterials, 273

SERS tags are a class of nanoparticles with great potential in advanced imaging experiments. The preparation of SERS tags however is complex, as they suffer from the high variability of the SERS signals observed even at the slightest sign of aggregation. Here, we developed a method for the preparation of SERS tags based on the use of gold nanostars conjugated with neutravidin. The SERS tags here obtained are extremely stable in all biological buffers commonly employed and can be prepared at a relatively large scale in very mild conditions. The obtained SERS tags have been used to monitor the expression of fibroblast activation protein alpha (FAP) on the membrane of primary fibroblasts obtained from patients affected by Crohn’s disease. The SERS tags allowed the unambiguous identification of FAP on the surface of cells thus suggesting the feasibility of semi-quantitative analysis of the target protein. Moreover, the use of the neutravidin–biotin system allows to apply the SERS tags for any other marker detection, for example, different cancer cell types, simply by changing the biotinylated antibody chosen in the analysis.

State-of-the-art preoperative biomechanical analysis for the planning of spinal surgery not only requires the generation of three-dimensional patient-specific models but also the accurate biomechanical representation of vertebral joints. The benefits offered by computational models suitable for such purposes are still outweighed by the time and effort required for their generation, thus compromising their applicability in a clinical environment. In this work, we aim to ease the integration of computerized methods into patient-specific planning of spinal surgery. We present the first pipeline combining deep learning and finite element methods that allows a completely automated model generation of functional spine units (FSUs) of the lumbar spine for patient-specific FE simulations (FEBio). The pipeline consists of three steps: (a) multiclass segmentation of cropped 3D CT images containing lumbar vertebrae using the DenseVNet network, (b) automatic landmark-based mesh fitting of statistical shape models onto 3D semantic segmented meshes of the vertebral models, and (c) automatic generation of patient-specific FE models of lumbar segments for the simulation of flexion-extension, lateral bending, and axial rotation movements. The automatic segmentation of FSUs was evaluated against the gold standard (manual segmentation) using 10-fold cross-validation. The obtained Dice coefficient was 93.7% on average, with a mean surface distance of 0.88 mm and a mean Hausdorff distance of 11.16 mm (N = 150). Automatic generation of finite element models to simulate the range of motion (ROM) was successfully performed for five healthy and five pathological FSUs. The results of the simulations were evaluated against the literature and showed comparable ROMs in both healthy and pathological cases, including the alteration of ROM typically observed in severely degenerated FSUs. The major intent of this work is to automate the creation of anatomically accurate patient-specific models by a single pipeline allowing functional modeling of spinal motion in healthy and pathological FSUs. Our approach reduces manual efforts to a minimum and the execution of the entire pipeline including simulations takes approximately 2 h. The automation, time-efficiency and robustness level of the pipeline represents a first step toward its clinical integration., Frontiers in Bioengineering and Biotechnology, 9, ISSN:2296-4185

Lifting up objects from the floor has been identified as a risk factor for low back pain, whereby a flexed spine during lifting is often associated with producing higher loads in the lumbar spine. Even though recent biomechanical studies challenge these assumptions, conclusive evidence is still lacking. This study therefore aimed at comparing lumbar loads among different lifting styles using a comprehensive state-of-the-art motion capture-driven musculoskeletal modeling approach. Thirty healthy pain-free individuals were enrolled in this study and asked to repetitively lift a 15 kg-box by applying 1) a freestyle, 2) a squat and 3) a stoop lifting technique. Whole-body kinematics were recorded using a 16-camera optical motion capture system and used to drive a full-body musculoskeletal model including a detailed thoracolumbar spine. Continuous as well as peak compressive, anterior-posterior shear and total loads (resultant load vector of the compressive and shear load vectors) were calculated based on a static optimization approach and expressed as factor body weight (BW). In addition, lumbar lordosis angles and total lifting time were calculated. All parameters were compared among the lifting styles using a repeated measures design. For each lifting style, loads increased towards the caudal end of the lumbar spine. For all lumbar segments, stoop lifting showed significantly lower compressive and total loads (−0.3 to −1.0BW) when compared to freestyle and squat lifting. Stoop lifting produced higher shear loads (+0.1 to +0.8BW) in the segments T12/L1 to L4/L5, but lower loads in L5/S1 (−0.2 to −0.4BW). Peak compressive and total loads during squat lifting occurred approximately 30% earlier in the lifting cycle compared to stoop lifting. Stoop lifting showed larger lumbar lordosis range of motion (35.9 ± 10.1°) than freestyle (24.2 ± 7.3°) and squat (25.1 ± 8.2°) lifting. Lifting time differed significantly with freestyle being executed the fastest (4.6 ± 0.7 s), followed by squat (4.9 ± 0.7 s) and stoop (5.9 ± 1.1 s). Stoop lifting produced lower total and compressive lumbar loads than squat lifting. Shear loads were generally higher during stoop lifting, except for the L5/S1 segment, where anterior shear loads were higher during squat lifting. Lifting time was identified as another important factor, considering that slower speeds seem to result in lower loads., Frontiers in Bioengineering and Biotechnology, 9, ISSN:2296-4185

Background Studying bacterial adhesion and early biofilm development is crucial for understanding the physiology of sessile bacteria and forms the basis for the development of novel antimicrobial biomaterials. Microfluidics technologies can be applied in such studies since they permit dynamic real-time analysis and a more precise control of relevant parameters compared to traditional static and flow chamber assays. In this work, we aimed to establish a microfluidic platform that permits real-time observation of bacterial adhesion and biofilm formation under precisely controlled homogeneous laminar flow conditions. Results Using Escherichia coli as the model bacterial strain, a microfluidic platform was developed to overcome several limitations of conventional microfluidics such as the lack of spatial control over bacterial colonization and allow label-free observation of bacterial proliferation at single-cell resolution. This platform was applied to demonstrate the influence of culture media on bacterial colonization and the consequent eradication of sessile bacteria by antibiotic. As expected, the nutrient-poor medium (modified M9 medium containing 1 g/l of organic nitrogen) was found to promote bacterial adhesion and to enable a higher adhesion rate compared to the nutrient-rich medium (tryptic soy broth containing 20 g/l of organic nitrogen). However, in rich medium the adhered cells colonized the glass surface faster than those in poor medium under otherwise identical conditions. For the first time, this effect was demonstrated to be caused by a higher retention of newly generated bacteria in the rich medium, rather than faster growth especially during the initial adhesion phase. These results also indicate that higher adhesion rate does not necessarily lead to faster biofilm formation. Antibiotic treatment of sessile bacteria with colistin was further monitored by fluorescence microscopy at single-cell resolution, allowing in situ analysis of killing efficacy of antimicrobials.Conclusion The platform established here represents a powerful and versatile tool for studying environmental effects such as medium composition on bacterial adhesion and biofilm formation. Our microfluidic setup shows great potential for the in vitro assessment of new antimicrobials and antifouling agents under flow conditions.

In recent years, cell-based assays have been frequently used in molecular interaction analysis. Cell-based assays complement traditional biochemical and biophysical methods, as they allow for molecular interaction analysis, mode of action studies and even drug screening processes to be performed under physiologically relevant conditions. In most cellular assays, biomolecules are usually labeled to achieve specificity. In order to overcome some of the drawbacks associated with label-based assays, we have recently introduced cell-based molography as a biosensor for the analysis of specific molecular interactions involving native membrane receptors in living cells. Here, we expand this assay to cytosolic protein-protein interactions. First, we created a biomimetic membrane receptor by tethering one cytosolic interaction partner to the plasma membrane. The artificial construct is then coherently arranged into a two-dimensional pattern within the cytosol of living cells. Thanks to the molographic sensor, the specific interactions between the coherently arranged protein and its endogenous interaction partners become visible in real-time without the use of a fluorescent label. This method turns out to be an important extension of cell-based molography because it expands the range of interactions that can be analyzed by molography to those in the cytosol of living cells., 22 pages, 4 figures

In the field of reproductive biology, there is a strong need for a suitable tool capable of non-destructive evaluation of oocyte viability and function. We studied the application of brilliant cresyl blue (BCB) as an intra-vital exogenous contrast agent using multispectral optoacoustic tomography (MSOT) for visualization of porcine ovarian follicles. The technique provided excellent molecular sensitivity, enabling the selection of competent oocytes without disrupting the follicles. We further conducted in vitro embryo culture, molecular analysis (real-time and reverse transcriptase polymerase chain reaction) and DNA fragmentation analysis to comprehensively establish the safety of BCB-enhanced MSOT imaging in monitoring oocyte viability. Overall, the experimental results suggest that the method offers a significant advance in the use of contrast agents and molecular imaging for reproductive studies. Our technique improves the accurate prediction of ovarian reserve significantly and, once standardized for in vivo imaging, could provide an effective tool for clinical infertility management.

An alternative intracranial aneurysm embolic agent is emerging in the form of hydrogels due to their ability to be injected in liquid phase and solidify in situ. Hydrogels have the ability to fill an aneurysm sac more completely compared to solid implants such as those used in coil embolization. Recently, the feasibility to implement photopolymerizable poly(ethylene glycol) dimethacrylate (PEGDMA) hydrogels in vitro has been demonstrated for aneurysm application. Nonetheless, the physical and mechanical properties of such hydrogels require further characterization to evaluate their long-term integrity and stability to avoid implant compaction and aneurysm recurrence over time. To that end, molecular weight and polymer content of the hydrogels were tuned to match the elastic modulus and compliance of aneurysmal tissue while minimizing the swelling volume and pressure. The hydrogel precursor was injected and photopolymerized in an in vitro aneurysm model, designed by casting polydimethylsiloxane (PDMS) around 3D printed water-soluble sacrificial molds. The hydrogels were then exposed to a fatigue test under physiological pulsatile flow, inducing a combination of circumferential and shear stresses. The hydrogels withstood 5.5 million cycles and no significant weight loss of the implant was observed nor did the polymerized hydrogel protrude or migrate into the parent artery. Slight surface erosion defects of 2–10 μm in depth were observed after loading compared to 2 μm maximum for non-loaded hydrogels. These results show that our fine-tuned photopolymerized hydrogel is expected to withstand the physiological conditions of an in vivo implant study., Frontiers in Bioengineering and Biotechnology, 8, ISSN:2296-4185

International audience; With the current COVID-19 outbreak, it has become essential to develop efficient methods for the treatment and detection of this virus. Among the new approaches that could be tested, that relying on nanotechnology finds one of its main grounds in the similarity between nanoparticle (NP) and coronavirus (COV) sizes, which promotes NP−COV interactions. Since COVID-19 is very recent, most studies in this field have focused on other types of coronavirus than COVID-19, such as those involved in MERS or SARS diseases. Although their number is limited, they have led to promising results on various COV using a wide range of different types of nanosystems, e.g., nanoparticles, quantum dos, or nanoassemblies of polymers/proteins. Additional efforts deserve to be spent in this field to consolidate these findings. Here, I first summarize the different nanotechnology-based methods used for COV detection, i.e., optical, electrical, or PCR ones, whose sensitivity was improved by the presence of nanoparticles. Furthermore, I present vaccination methods, which comprise nano-particles used either as adjuvants or as active principles. They often yield a better-controlled immune response, possibly due to an improved antigen presentation/processing than in non-nanoformulated vaccines. Certain antiviral approaches also took advantage of nanoparticle uses, leading to specific mechanisms such as the blocking of virus replication at the cellular level or the reduction of a COV induced apoptotic cellular death.

Appropriate macrophage response to an implanted biomaterial is crucial for successful tissue healing outcomes. In this work we investigated how intrinsic topological cues from electrospun biomaterials and extrinsic mechanical loads cooperate to guide macrophage activation and macrophage-tendon fibroblast cross-talk. We performed a series of in vitro and in vivo experiments using aligned or randomly oriented polycaprolactone nanofiber substrates in both mechanically loaded and unloaded conditions. Across all experiments a disorganized biomaterial fiber topography was alone sufficient to promote a pro-inflammatory signature in macrophages, tendon fibroblasts, and tendon tissue. Extrinsic mechanical loading was found to strongly regulate the character of this signature by reducing pro-inflammatory markers both in vitro and in vivo. We observed that macrophages generally displayed a stronger response to biophysical cues than tendon fibroblasts, with dominant effects of cross-talk between these cell types observed in mechanical co-culture models. Collectively our data suggest that macrophages play a potentially important role as mechanosensory cells in tendon repair, and provide insight into how biological response might be therapeutically modulated by rational biomaterial designs that address the biomechanical niche of recruited cells., BIOMATERIALS, 249

Athletic performance relies on tendons, which enable movement by transferring forces from muscles to the skeleton. Yet, how load-bearing structures in tendons sense and adapt to physical demands is not understood. Here, by performing calcium (Ca2+) imaging in mechanically loaded tendon explants from rats and in primary tendon cells from rats and humans, we show that tenocytes detect mechanical forces through the mechanosensitive ion channel PIEZO1, which senses shear stresses induced by collagen-fibre sliding. Through tenocyte-targeted loss-of-function and gain-of-function experiments in rodents, we show that reduced PIEZO1 activity decreased tendon stiffness and that elevated PIEZO1 mechanosignalling increased tendon stiffness and strength, seemingly through upregulated collagen cross-linking. We also show that humans carrying the PIEZO1 E756del gain-of-function mutation display a 13.2% average increase in normalized jumping height, presumably due to a higher rate of force generation or to the release of a larger amount of stored elastic energy. Further understanding of the PIEZO1-mediated mechanoregulation of tendon stiffness should aid research on musculoskeletal medicine and on sports performance.

Vascularization of tissue engineered implants is crucial for their survival and integration in the recipient’s body. Pre-vascularized, fibrin-based implants offer a solution since low concentration fibrin hydrogels (1 mg/mL) have been shown to promote tube formation of endothelial cells in co-culture with adipogenic stem cells. However, higher fibrinogen concentrations (> 20 mg/mL) enabling the fabrication of stable implants are necessary. We here characterized fibrin gels of 1–30 mg/mL for their rheological properties and whether they support tube formation of endothelial cell–adipogenic stem cell co-cultures for up to 7 days. Moreover, 20 mg/mL gels containing preformed channels and endothelial cell–adipogenic stem cell co-culture were perfused continuously in a customized flow chamber with 3.9 dyn/cm2 for 12 days and analyzed for capillary formation. Rheology of fibrin gels showed increasing stability proportional to fibrinogen concentration with 20 mg/mL gels having a storage module of 465 Pa. Complex tube networks stable for 7 days were observed at 1–5 mg/mL gels whereas higher concentrations showed initial sprouting only. However, perfusion of 20 mg/mL fibrin gels resulted in endothelialized pore formation in several layers of the gel with endothelial cell–adipogenic stem cell co-culture. Thus, perfusion supports the formation of capillary-like structures in fibrin gels that are too dense for spontaneous tube formation under static conditions. Future studies are necessary to further increase pore density and to investigate proper nutrition of tissue-specific target cells in the scaffold.

Efforts to scale neuroimaging towards the direct visualization of mammalian brain-wide neuronal activity have faced major challenges. Although high-resolution optical imaging of the whole brain in small animals has been achieved ex vivo, the real-time and direct monitoring of large-scale neuronal activity remains difficult, owing to the performance gap between localized, largely invasive, optical microscopy of rapid, cellular-resolved neuronal activity and whole-brain macroscopy of slow haemodynamics and metabolism. Here, we demonstrate both ex vivo and non-invasive in vivo functional optoacoustic (OA) neuroimaging of mice expressing the genetically encoded calcium indicator GCaMP6f. The approach offers rapid, high-resolution three-dimensional snapshots of whole-brain neuronal activity maps using single OA excitations, and of stimulus-evoked slow haemodynamics and fast calcium activity in the presence of strong haemoglobin background absorption. By providing direct neuroimaging at depths and spatiotemporal resolutions superior to optical fluorescence imaging, functional OA neuroimaging bridges the gap between functional microscopy and whole-brain macroscopy.

All Universal Chimeric Antigen Receptor T-cells (UniCAR T-cells) are T-cells which have been engineered to recognize a haptenated ligand. Due to this feature, UniCAR T-cells have the potential to mediate a potent and selective tumor killing only in the presence of a haptenated tumor ligand, thus avoiding the long-lasting biocidal effects of conventional CAR T-cells. We have used fluorescein-labeled versions of small organic ligands and different antibody formats specific to carbonic anhydrase IX (a tumor-associated antigen) in order to assess whether the killing potential of UniCAR T-cells depended on the molecular features of the haptenated molecule. Both small molecule ligands and larger antibody fragments were potent in mediating tumor cell killing over a broad concentration range. Antibodies could be conveniently used both in IgG format and as smaller diabody fragments. Importantly, the use of site-specific chemical modification strategies for the antibody coupling to fluorescein led to a substantial improvement of tumor cell killing performance, compared to the random modification of primary amino groups on the antibody surface.

Stem cells are essential for tissue homeostasis and regeneration throughout the lifespan of multicellular rganisms. The decline in stem cell function during advanced age is associated with a reduced regenerative potential of tissues that leads to an increased frequency of diseases. Age-related changes also occur in the dental pulp tissue that represents a reliable model tissue, with high regenerative capability, for studying senescence mechanisms. However, little information is available concerning the effects of ageing on dental stem-cell function. In this mini-review, recent data on how the molecular and functional alterations that accumulate in stem cell populations during ageing result in modifications of dental pulp physiology are discussed. Changes that accumulate during ageing such as how reduction of pulp chamber volume, decreased vascular supply and modifications to the stem cell niches affect stem cell functions and, therefore, dental pulp regenerative potential in response to various stressful agents. Dental pulp cells from aged individuals are still metabolically active and secrete pro-inflammatory and matrix-degrading molecules. Furthermore, miRNAs and exosomes derived from dental pulp stem cells constitute an attractive source of nanovesicles for the treatment of age-related dental pathologies. Further investigation of the epigenetic alterations in dental pulp stem cells, accumulating during ageing, might reveal crucial information for potential stem cell-based therapeutic approaches in the elderly.

ACS Sensors, 4 (2), ISSN:2379-3694

Intracranial aneurysms are increasingly being treated with endovascular therapy, namely coil embolization. Despite being minimally invasive, partial occlusion and recurrence are more frequent compared to open surgical clipping. Therefore, an alternative treatment is needed, ideally combining minimal invasiveness and long-term efficiency. Herein, we propose such an alternative treatment based on an injectable, radiopaque and photopolymerizable polyethylene glycol dimethacrylate hydrogel. The rheological measurements demonstrated a viscosity of 4.86 ± 1.70 mPa.s, which was significantly lower than contrast agent currently used in endovascular treatment (p = 0.42), allowing the hydrogel to be injected through 430 μm inner diameter microcatheters. Photorheology revealed fast hydrogel solidification in 8 min due to the use of a new visible photoinitiator. The addition of an iodinated contrast agent in the precursor contributed to the visibility of the precursor injection under fluoroscopy. Using a customized light-conducting microcatheter and illumination module, the hydrogel was implanted in an in vitro silicone aneurysm model. Specifically, in situ fast and controllable injection and photopolymerization of the developed hydrogel is shown to be feasible in this work. Finally, the precursor and the polymerized hydrogel exhibit no toxicity for the endothelial cells. Photopolymerizable hydrogels are expected to be promising candidates for future intracranial aneurysm treatments., Frontiers in Bioengineering and Biotechnology, 8, ISSN:2296-4185

Ventricular assist devices (VADs), among which the HeartMate 3 (HM3) is the latest clinically approved representative, are often the therapy of choice for patients with end-stage heart failure. Despite advances in the prevention of pump thrombosis, rates of stroke and bleeding remain high. These complications are attributed to the flow field within the VAD, among other factors. One of the HM3's characteristic features is an artificial pulse that changes the rotor speed periodically by 4000 rpm, which is meant to reduce zones of recirculation and stasis. In this study, we investigated the effect of this speed modulation on the flow fields and stresses using high-resolution computational fluid dynamics. To this end, we compared Eulerian and Lagrangian features of the flow fields during constant pump operation, during operation with the artificial pulse feature, and with the effect of the residual native cardiac cycle. We observed good washout in all investigated situations, which may explain the low incidence rates of pump thrombosis. The artificial pulse had no additional benefit on scalar washout performance, but it induced rapid variations in the flow velocity and its gradients. This may be relevant for the removal of deposits in the pump. Overall, we found that viscous stresses in the HM3 were lower than in other current VADs. However, the artificial pulse substantially increased turbulence, and thereby also total stresses, which may contribute to clinically observed issues related to hemocompatibility.

Additive manufacturing of bone tissue engineering scaffolds will become a key element for personalized bone tissue engineering in the near future. Several additive manufacturing processes are based on extrusion where the deposition of the filament will result in a three-dimensional lattice structure. Recently, we studied diverse lattice structures for bone tissue engineering realized by laser sintering of titanium. In this work, we used lithography-based ceramic manufacturing of lattice structures to produce scaffolds from tricalcium phosphates (TCP) and compared them in vivo to congruent titanium scaffolds manufactured with the identical computer-aided design data to look for material-based differences in bony healing. The results show that, during a 4-week period in a noncritical-size defect in a rabbit calvarium, both scaffolds with the identical microarchitecture performed equally well in terms of bony regeneration and bony bridging of the defect. A significant increase in both parameters could only be achieved when the TCP-based scaffolds were doped with bone morphogenetic protein-2. In a critical-size defect in the calvarial bone of rabbits, however, the titanium scaffold performed significantly better than the TCP-based scaffold, most likely due to its higher mechanical stability. We conclude that titanium and TCP-based scaffolds of the same microarchitecture perform equally well in terms of bone regeneration, provided the microarchitecture meets the mechanical demand at the site of implantation.

Despite the various reported approaches to generate osteochondral composites by combination of different cell types and materials, engineering of templates with the capacity to autonomously and orderly develop into cartilage-bone bi-layered structures remains an open challenge. Here, we hypothesized that the embedding of cells inducible to endochondral ossification (i.e. bone marrow derived mesenchymal stromal cells, BMSCs) and of cells capable of robust and stable chondrogenesis (i.e. nasal chondrocytes, NCs) adjacent to each other in bi-layered hydrogels would develop directly in vivo into osteochondral tissues. Poly(ethylene glycol) (PEG) hydrogels were functionalized with TGFβ3 or BMP-2, enzymatically polymerized encapsulating human BMSCs, combined with a hydrogel layer containing human NCs and ectopically implanted in nude mice without pre-culture. The BMSC-loaded layers reproducibly underwent endochondral ossification and generated ossicles containing bone and marrow. The NC-loaded layers formed cartilage tissues, which (under the influence of BMP-2 but not of TGFβ3 from the neighbouring layer) remained phenotypically stable. The proposed strategy, resulting in orderly connected osteochondral composites, should be further assessed for the repair of osteoarticular defects and will be useful to model developmental processes leading to cartilage-bone interfaces.

Journal of Nanobiotechnology, 16 (1), ISSN:1477-3155

Wounds in the fetus can heal without scarring. Consequently, biomaterials that attempt to recapitulate the biophysical and biochemical properties of fetal skin have emerged as promising pro-regenerative strategies. The extracellular matrix (ECM) protein fibronectin (Fn) in particular is believed to play a crucial role in directing this regenerative phenotype. Accordingly, Fn has been implicated in numerous wound healing studies, yet remains untested in its fibrillar conformation as found in fetal skin. Here, we show that high extensional (~1.2 × 10(5) s(−1)) and shear (~3 × 10(5) s(−1)) strain rates in rotary jet spinning (RJS) can drive high throughput Fn fibrillogenesis (~10 mL/min), thus producing nano fiber scaffolds that are used to effectively enhance wound healing. When tested on a full-thickness wound mouse model, Fn nanofiber dressings not only accelerated wound closure, but also significantly improved tissue restoration, recovering dermal and epidermal structures as well as skin appendages and adipose tissue. Together, these results suggest that bioprotein nanofiber fabrication via RJS could set a new paradigm for enhancing wound healing and may thus find use in a variety of regenerative medicine applications.

Live-imaging of C. elegans is essential for the study of conserved cellular pathways (e.g. EGFR/Wnt signaling) and morphogenesis in vivo. However, the usefulness of live imaging as a research tool has been severely limited by the need to immobilize worms prior to and during imaging. Conventionally, immobilization is achieved by employing both physical and chemical interventions. These are known to significantly affect many physiological processes, and thus limit our understanding of dynamic developmental processes. Herein we present a novel, easy-to-use microfluidic platform for the long-term immobilization of viable, normally developing C. elegans, compatible with image acquisition at high resolution, thereby overcoming the limitations associated with conventional worm immobilization. The capabilities of the platform are demonstrated through the continuous assessment of anchor cell (AC) invasion and distal tip cell (DTC) migration in larval C. elegans and germ cell apoptosis in adult C. elegans in vivo for the first time. ispartof: LAB ON A CHIP vol:18 issue:9 pages:1359-1368 ispartof: location:England status: published

Fracture-related infection (FRI) is a major complication in surgically fixed fractures. Instability of the fracture after fixation is considered a risk factor for infection; however, few experimental data are available confirming this belief. To study whether stable fractures led to higher infection clearance, mouse femoral osteotomies were fixed with either stable or unstable fixation and the surgical site was contaminated with either Staphylococcus epidermidis (S. epidermidis) or Staphylococcus aureus (S. aureus) clinical isolates. Infection progression was assessed at different time points by quantitative bacteriology, total cell counts in spleen and lymph node and histological analysis. Operated, non-inoculated mice were used as controls. Two inbred mouse strains (C57BL/6 and BALB/c) were included in the study to determine the influence of different host background in the outcome. Stable fixation allowed a higher proportion of C57BL/6 mice to clear S. epidermidis inoculation in comparison to unstable fixation. No difference associated with fixation type was observed for BALB/c mice. Inoculation with S. aureus resulted in a more severe infection for both stable and unstable fractures in both mouse strains; however, significant osteolysis around the screws rendered the stable group functionally unstable. Our results suggested that fracture stability could have an influence on S. epidermidis infection, although host factors also played a role. No differences were observed when using S. aureus, due to a more severe infection, leading to osteolysis and loss of stability in both groups. Further studies are required in order to address the biological features underlying the differences observed.

Finding the appropriate cues to trigger the desired differentiation is a challenge in tissue engineering when stem cells are involved. In this regard, three-dimensional environments are often compared to cells' two-dimensional culture behaviour (plastic culture dish). Here, we compared the gene expression pattern of human adipose-derived stem cells (ASC) seeded in a three-dimensional (3D) electrospun mesh and on a two-dimensional (2D) film - both of exactly the same material. Additionally, we conducted experiments with a scaffold floating above a film to investigate two-way paracrine effects (co-system). Electrospun meshes (3D scaffolds) and films (2D), consisting either of pristine poly-lactic-co-glycolic acid (PLGA) or of PLGA-containing dispersed amorphous calcium phosphate nanoparticles (PLGA/aCaP), were seeded with ASCs and cultured either in Dulbecco Minimum Essential Medium (DMEM) or in osteogenic medium. After two weeks, minimum stem cell criteria markers as well as typical markers for osteogenesis, endothelial cell differentiation, adipogenesis and chondrogenesis were analysed by quantitative real-time PCR. Interestingly, mostly osteogenic genes of cells seeded on 3D meshes were upregulated compared to those on 2D films, while stem cell markers seemed to be only slightly affected. Runx2 and osteocalcin showed an especially strong upregulation under all conditions, while most other factors analysed for 2D/3D changes were highly dependent on the material composition, the culture medium and on paracrine signalling effects. The beneficial 3D environment for stem cells found in many studies has therefore not to be attributed to the third dimension alone and should carefully be compared to 2D films fabricated of the same material. Furthermore, paracrine interactions triggering differentiation are not negligible.

To promote and support tendon healing, one viable strategy is the use or administration of growth factors at the wound/rupture site. Platelet derived growth factor-BB (PDGF-BB), together with other growth factors, is secreted by platelets after injury. PDGF-BB promotes mitogenesis and angiogenesis, which could accelerate tendon healing. Therefore, in vitro studies with PDGF-BB have been performed to determine its effect on tenocytes and tenoblasts. Moreover, accurate and sophisticated drug delivery devices, aiming for a sustained release of PDGF-BB, have been developed, either by using heparin-binding and fibrin-based matrices or different electrospinning techniques. In this review, the structure and composition, as well as the healing process of tendons, are described. Part A deals with in vitro studies. They focus on the multiple effects evoked by PDGF-BB on the cellular level. Moreover, they address strategies for the sustained delivery of PDGF-BB. Part B focuses on animal models used to test different delivery strategies for PDGF-BB, in the context of tendon reconstruction. These studies showed that dosage and timing of PDGF-BB application are the most important factors for deciding which delivery device should be applied for a specific tendon laceration.

Accurate segmentation of connective soft tissues is still a challenging task, which hinders the generation of corresponding geometric models for biomechanical computations. Alternatively, one could predict ligament insertion sites and then approximate the shapes, based on anatomical knowledge and morphological studies. Here, we describe a corresponding integrated framework for the automatic modelling of human musculoskeletal ligaments. We combine statistical shape modelling with geometric algorithms to automatically identify insertion sites, based on which geometric surface and volume meshes are created. For demonstrating a clinical use case, the framework has been applied to generate models of the interosseous membrane in the forearm. For the adoption to the forearm anatomy, ligament insertion sites in the statistical model were defined according to anatomical predictions following an approach proposed in prior work. For evaluation we compared the generated sites, as well as the ligament shapes, to data obtained from a cadaveric study, involving five forearms with a total of 15 ligaments. Our framework permitted the creation of 3D models approximating ligaments' shapes with good fidelity. However, we found that the statistical model trained with the state-of-the-art prediction of the insertion sites was not always reliable. Using that model, average mean square errors as well as Hausdorff distances of the meshes increased by more than one order of magnitude, as compared to employing the known insertion locations of the cadaveric study. Using the latter an average mean square error of 0.59 mm and an average Hausdorff distance of less than 7 mm resulted, for the complete set of ligaments. In conclusion, the presented approach for generating ligament shapes from insertion points appears to be feasible but the detection of the insertion sites with a SSM is too inaccurate., Comment: 16 pages, 8 figures

Chromatin modifications regulate genome function by recruiting proteins to the genome. However, the protein composition at distinct chromatin modifications has yet to be fully characterized. In this study, we used natural protein domains as modular building blocks to develop engineered chromatin readers (eCRs) selective for DNA methylation and histone tri-methylation at H3K4, H3K9 and H3K27 residues. We first demonstrated their utility as selective chromatin binders in living cells by stably expressing eCRs in mouse embryonic stem cells and measuring their subnuclear localization, genomic distribution and histone-modification-binding preference. By fusing eCRs to the biotin ligase BASU, we established ChromID, a method for identifying the chromatin-dependent protein interactome on the basis of proximity biotinylation, and applied it to distinct chromatin modifications in mouse stem cells. Using a synthetic dual-modification reader, we also uncovered the protein composition at bivalently modified promoters marked by H3K4me3 and H3K27me3. These results highlight the ability of ChromID to obtain a detailed view of protein interaction networks on chromatin. The protein complexes associated with specific chromatin marks in living cells are identified using engineered binding proteins.

Flow fields in rotary blood pumps (RBPs) have a significant influence on hemocompatibility. Because flow characteristics vary with flow rate, different operating conditions play a role. Furthermore, turbulence is crucial in the evaluation of blood damage potential, but the level of turbulence in implantable RBPs is still unknown. In this study, we addressed both research aspects and for the first time measured turbulent flow fields in the HeartMate 3 (HM3) at different operating flows. The averaged, three-dimensional velocity field including fluctuating velocity components in a HM3 with a transparent lower housing was measured using three-dimensional particle tracking velocimetry (3D-PTV). In vitro results were compared with computational fluid dynamic (CFD) simulations for two flow cases, representing the lower and upper physiologic flow range (2.7 and 5.7 L/min), using two different turbulence models that account for fluctuating velocity fields: the k-ω shear stress transport and the Reynolds stress model (RSM). The measurements revealed higher mean and turbulent kinetic energies (TKEs) for the low-flow condition especially within the gap beneath the impeller. Computed mean fields agree well with 3D-PTV for both models, but the RSM predicts the TKE levels better than the k-ω model. Computational fluid dynamic results further show wall shear stresses higher than 150 Pa, a commonly used damage threshold, in the bottom gap for the lower flow condition. In conclusion, the low-flow condition was found to be more prone to blood damage. Furthermore, CFD predictions for turbulence must be carefully experimentally validated.

The location of the terminal hinge axis of the temporomandibular joint is still a very wide-spread procedure in dentistry in order to replicate the movement in various articulator devices. Especially pantographic methods are claimed to provide accurate measurements and, additionally, are seen to be able to separate a pure rotation of the joint from a movement with an arbitrary combined shift and rotation. In the latter application, these methods were used in a lot of studies as a reference standard. The aim of this study was to analyze, whether common pantographic methods in general are able to distinguish between a pure rotation and a movement with rotational and translational portions. The mathematical proof of this analysis was done with theoretical kinematic considerations and compared with computer simulations. The results show for the first time that there exist combinations of rotational and translational movements of the temporomandibular joint which cannot be separated from pure rotational movements using actual pantographic methods. Even more, the consequence is a shifted location of the (combined) finite center (axis) of rotation in comparison to the true center (axis) of rotation: in case of a translational portion of only 1 mm, this is a displacement of around ±6 mm and, in case of 2 mm translation, a displacement of ±12 mm. This finding necessitates a critical reinterpretation of former studies using pantographic methods as a reference standard. Further, under some circumstances it may also affect the applicability of articulator concepts and the interpretation of functional signs.