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Radiotherapy is an integral part of cancer therapy. Due to the low tissue specificity of radiation, damage to tumor-surrounding healthy tissue remains a major concern. Radio-enhancers based on inorganic nanomaterials have attracted considerable attention in recent years. In addition to widely exploited metal and metal oxides nanoparticles, 2D materials may offer potential advantages due to their intrinsically high specific surface area. Here, we report on the promising radio-enhancement properties of Ti3C2Tx MXenes. We show that Ti3C2Tx MXenes are readily internalized and well-tolerated by mammalian cells. In contrast to MXenes suspended in aqueous buffers which fully oxidize within days (yielding rice-grain shaped rutile nanoparticles), MXenes internalized by cells display slower oxidation rates, in line with cell-free experiments showing slower oxidation in cell media and lysosomal buffers compared to antioxidant-devoid dispersants. The MXenes show potent radio-enhancement properties with dose enhancement factors of up to 2.5 in human soft tissue sarcoma cells and no toxicity towards healthy human fibroblasts. Benchmarking against oxidized MXenes and commercial titanium dioxide nanoparticles indicates superior radio-enhancement properties of the intact 2D titanium carbide flakes. Taken together, this work provides direct evidence for the potent radio-enhancement properties of Ti3C2Tx MXenes rendering them a promising candidate material for radiotherapy enhancement.

Calcific degeneration is the most frequent type of heart valve failure, with rising incidence due to the ageing population. The gold standard treatment to date is valve replacement. Unfortunately, calcification oftentimes re-occurs in bioprosthetic substitutes, with the governing processes remaining poorly understood. Here, we present a multiscale, multimodal analysis of disturbances and extensive mineralisation of the collagen network in failed bioprosthetic bovine pericardium valve explants with full histoanatomical context. In addition to highly abundant mineralized collagen fibres and fibrils, calcified micron-sized particles previously discovered in native valves were also prevalent on the aortic as well as the ventricular surface of bioprosthetic valves. The two mineral types (fibers and particles) were detectable even in early-stage mineralisation, prior to any macroscopic calcification. Based on multiscale multimodal characterisation and high-fidelity simulations, we demonstrate that mineral occurrence coincides with regions exposed to high haemodynamic and biomechanical indicators. These insights obtained by multiscale analysis of failed bioprosthetic valves may serve as groundwork for the evidence-based development of more durable alternatives.Graphical Abstract

Inorganic nanomaterials have gained increasing attention in radiation oncology, owing to their radiation therapy enhancing properties. To accelerate candidate material selection and overcome the disconnect between conventional 2D cell culture and in vivo findings, screening platforms unifying high-throughput with physiologically relevant endpoint analysis based on 3D in vitro models are promising. Here, a 3D tumor spheroid co-culture model based on cancerous and healthy human cells is presented for the concurrent assessment of radio-enhancement efficacy, toxicity, and intratissural biodistribution with full ultrastructural context of radioenhancer candidate materials. Its potential for rapid candidate materials screening is showcased based on the example of nano-sized metal–organic frameworks (nMOFs) and direct benchmarking against gold nanoparticles (the current “gold standard”). Dose enhancement factors (DEFs) ranging between 1.4 and 1.8 are measured for Hf-, Ti-, TiZr-, and Au-based materials in 3D tissues and are overall lower than in 2D cell cultures, where DEF values exceeding 2 are found. In summary, the presented co-cultured tumor spheroid—healthy fibroblast model with tissue-like characteristics may serve as high-throughput platform enabling rapid, cell line-specific endpoint analysis for therapeutic efficacy and toxicity assessment, as well as accelerated radio-enhancer candidate screening., Advanced Biology, ISSN:2701-0198

Osteoderms are hard tissues embedded in the dermis of vertebrates and have been suggested to be formed from several different mineralized regions. However, their nano architecture and micro mechanical properties had not been fully characterized. Here, using electron microscopy, µ-CT, atomic force microscopy and finite element simulation, an in-depth characterization of osteoderms from the lizard Heloderma suspectum, is presented. Results show that osteoderms are made of three different mineralized regions: a dense apex, a fibre-enforced region comprising the majority of the osteoderm, and a bone-like region surrounding the vasculature. The dense apex is stiff, the fibre-enforced region is flexible and the mechanical properties of the bone-like region fall somewhere between the other two regions. Our finite element analyses suggest that when combined into the osteoderm structure, the distinct tissue regions are able to shield the body of the animal by bearing the external forces. These findings reveal the structure-function relationship of the Heloderma suspectum osteoderm in unprecedented detail. STATEMENT OF SIGNIFICANCE: The structures of bone and teeth have been thoroughly investigated. They provide a basis not only for understanding the mechanical properties and functions of these hard tissues, but also for the de novo design of composite materials. Osteoderms, however, are hard tissues that must possess mechanical properties distinct from teeth and bone to function as a protective armour. Here we provide a detailed analysis of the nanostructure of vertebrate osteoderms from Heloderma suspectum, and show that their mechanical properties are determined by their multiscale hierarchical tissue. We believe this study contributes to advance the current knowledge of the structure-function relationship of the hierarchical structures in the Heloderma suspectum osteoderm. This knowledge might in turn provide a source of inspiration for the design of bioinspired and biomimetic materials.

Scanning electron microscopy (SEM) is widely used for materials characterization. It has also been successfully applied to the imaging of biological samples, providing invaluable insights into the topography, morphology and composition of biological structures, including pathological minerals, in diseases affecting cardiovascular, kidney and ocular tissues. Here we provide a comprehensive and detailed guide on how to use colored SEM to aid the visualization and characterization of pathological calcification, and identify the effects of different sample preparation protocols for the visualisation of these minerals.

Millions of patients every year undergo gastrointestinal surgery. While often lifesaving, sutured and stapled reconnections leak in around 10% of the cases. Penetration of digestive fluids into the peritoneal cavity may lead to dreadful complications, including sepsis and premature death. Modern suture supports and tissue adhesives only insufficiently address the issue. Due to the scarcity of alternatives, surgeons rely on monitoring surrogate markers and clinical symptoms, which oftentimes lack sensitivity and specificity, hence only offering late-stage detection of already fully developed leaks.Here, a first-of-its-kind, modular, intelligent suture support patch capable of sealing and monitoring leaks under harsh gastrointestinal conditions is presented. The smart adhesive layered hydrogel patch provides, in addition to unprecedented tissue sealing under most demanding conditions, unique leak-detection capabilities based on pH and/or enzyme-responsive sensing elements, which can be read out by non-invasive point-of-need ultrasound imaging. Reliable detection of the breaching of sutures in as little as 3 hours in intestinal leak and 15 minutes in gastric leak conditions, and before an actual leak develops, is demonstrated. This technology paves the way for next-generation suture support materials that offer disambiguation in cases of anastomotic leaks based on point-of-need monitoring, without reliance on complex electronics or bulky (bio)electronic implantables.SummaryElectronic-free smart surgical hydrogel sealants leveraging tissue-penetrating polymer networks and trigger-responsive echogenic entities to enable point-of-need monitoring and early anastomotic leak detection using a hand-held ultrasound transducer and a smartphone.

Physiological calcification plays an essential part in the development of the skeleton and teeth; however, the occurrence of calcification in soft tissues such as the brain, heart, and kidneys associates with health impacts, creating a massive social and economic burden. The current paradigm for pathological calcification focuses on the biological factors responsible for bone‐like mineralization, including osteoblast‐like cells and proteins inducing nucleation and crystal growth. However, the exact mechanism responsible for calcification remains unknown. Toward this goal, this review dissects the current understanding of structure–function relationships and physico‐chemical properties of pathologic calcification from a materials science point of view. We will discuss a range of potential mechanisms of pathological calcification, with the purpose of identifying universal mechanistic pathways that occur across multiple organs/tissues at multiple length scales. The possible effect of extracellular components in signaling and templating mineralization, as well as the role of intrinsically disordered proteins in calcification, is reviewed. The state‐of‐the‐art in vitro models and strategies that can recreate the highly dynamic environment of calcification are identified.

Protein-mediated tethering of vesicles in the extracellular matrix contributes to microcalcification formation., Extracellular vesicles (EVs) including plasma membrane–derived microvesicles and endosomal-derived exosomes aggregate by unknown mechanisms, forming microcalcifications that promote cardiovascular disease, the leading cause of death worldwide. Here, we show a framework for assessing cell-independent EV mechanisms in disease by suggesting that annexin A1 (ANXA1)–dependent tethering induces EV aggregation and microcalcification. We present single-EV microarray, a method to distinguish microvesicles from exosomes and assess heterogeneity at a single-EV level. Single-EV microarray and proteomics revealed increased ANXA1 primarily on aggregating and calcifying microvesicles. ANXA1 vesicle aggregation was suppressed by calcium chelation, altering pH, or ANXA1 neutralizing antibody. ANXA1 knockdown attenuated EV aggregation and microcalcification formation in human cardiovascular cells and acellular three-dimensional collagen hydrogels. Our findings explain why microcalcifications are more prone to form in vulnerable regions of plaque, regulating critical cardiovascular pathology, and likely extend to other EV-associated diseases, including autoimmune and neurodegenerative diseases and cancer.

Flame aerosol technology is a versatile method for scalable synthesis of nanoparticles. Since particles are produced and collected in a dry state, dispersibility and further functionalization could pose hurdles to their biomedical use. We report on a one-pot, scalable and robust procedure for the PEGylation of flame-made yttria and silica nanoparticles. We demonstrate improved colloidal stability, attenuated activation of blood coagulation and decreased uptake into phagocytic cells, all of which pave the way for facilitated biomedical use of flame-made oxide nanoparticles.

In this chapter, the basic principles of electron microscopy and its application to the study of cardiovascular calcification will be presented, with the main objective of introducing a technique that could be routinely used in most laboratories working with pathological calcification. With this chapter, we hope the reader will achieve a basic understanding on how to prepare and image pathological minerals in situ. The chapter will discuss the most suitable sample preparation protocols for precise visualisation and characterization of pathological minerals using conventional scanning and transmission electron microscopy.

Pathological mineralization has been reported countless times in the literature and is a well-known phenomenon in the medical field for its connections to a wide range of diseases, including cancer, cardiovascular, and neurodegenerative diseases. The minerals involved in calcification, however, have not been directly studied as extensively as the organic components of each of the pathologies. These have been studied in isolation and, for most of them, physicochemical properties are hitherto not fully known. In a parallel development, materials science methods such as electron microscopy, spectroscopy, thermal analysis, and others have been used in biology mainly for the study of hard tissues and biomaterials and have only recently been incorporated in the study of other biological systems. This review connects a range of soft tissue diseases, including breast cancer, age-related macular degeneration, aortic valve stenosis, kidney stone diseases, and Fahr’s syndrome, all of which have been associated with mineralization processes. Furthermore, it describes how physicochemical material characterization methods have been used to provide new information on such pathologies. Here, we focus on diseases that are associated with calcium-composed minerals to discuss how understanding the properties of these minerals can provide new insights on their origins, considering that different conditions and biological features are required for each type of mineral to be formed. We show that mineralomics, or the study of the properties and roles of minerals, can provide information which will help to improve prevention methods against pathological mineral build-up, which in the cases of most of the diseases mentioned in this review, will ultimately lead to new prevention or treatment methods for the diseases. Importantly, this review aims to highlight that chemical composition alone cannot fully support conclusions drawn on the nature of these minerals.

Despite its crucial role, the placenta is the least understood human organ. Recent clinical studies indicate a direct association between placental calcification and maternal and offspring health. This study reveals distinct characteristics of minerals formed during gestational ageing using cutting-edge nano-analytical characterization and paves the way for investigations focused on the identification of potential markers for disease risks in a clinical setting based on atypical placental mineral fingerprints.

Drusen are lipid-, mineral-, and protein-containing extracellular deposits that accumulate between the basal lamina of the retinal pigment epithelium (RPE) and Bruch's membrane (BrM) of the human eye. They are a defining feature of age-related macular degeneration (AMD), a common sight-threatening disease of older adults. The appearance of heterogeneous internal reflectivity within drusen (HIRD) on optical coherence tomography (OCT) images has been suggested to indicate an increased risk of progression to advanced AMD. Here, in a cohort of patients with AMD and drusen, we show that HIRD indicated an increased risk of developing advanced AMD within 1 year. Using multimodal imaging in an independent cohort, we demonstrate that progression to AMD was associated with increasing degeneration of the RPE overlying HIRD. Morphological analysis of clinically imaged cadaveric human eye samples revealed that HIRD was formed by multilobular nodules. Nanoanalytical methods showed that nodules were composed of hydroxyapatite and that they differed from spherules and BrM plaques, other refractile features also found in the retinas of patients with AMD. These findings suggest that hydroxyapatite nodules may be indicators of progression to advanced AMD and that using multimodal clinical imaging to determine the composition of macular calcifications may help to direct therapeutic strategies and outcome measures in AMD.

Cellular function depends on the maintenance of protein homeostasis (proteostasis) by regulated protein degradation. Chronic dysregulation of proteostasis is associated with neurodegenerative and age-related diseases, and drugs targeting components of the protein degradation apparatus are increasingly used in cancer therapies. However, as chronic imbalances rather than loss of function mediate their pathogenesis, research models that allow for the study of the complex effects of drugs on tissue properties in proteostasis-associated diseases are almost completely lacking. Here, to determine the functional effects of impaired proteostatic fine-tuning, we applied a combination of materials science characterisation techniques to a cell-derived, in vitro model of bone-like tissue formation in which we pharmacologically perturbed protein degradation. We show that low-level inhibition of VCP/p97 and the proteasome, two major components of the degradation machinery, have remarkably different effects on the bone-like material that human bone-marrow derived mesenchymal stromal cells (hMSC) form in vitro. Specifically, whilst proteasome inhibition mildly enhances tissue formation, Raman spectroscopic, atomic force microscopy-based indentation, and electron microscopy imaging reveal that VCP/p97 inhibition induces the formation of bone-like tissue that is softer, contains less protein, appears to have more crystalline mineral, and may involve aberrant micro- and ultra-structural tissue organisation. These observations contrast with findings from conventional osteogenic assays that failed to identify any effect on mineralisation. Taken together, these data suggest that mild proteostatic impairment in hMSC alters the bone-like material they form in ways that could explain some pathologies associated with VCP/p97-related diseases. They also demonstrate the utility of quantitative materials science approaches for tackling long-standing questions in biology and medicine, and could form the basis for preclinical drug testing platforms to develop therapies for diseases stemming from perturbed proteostasis or for cancer therapies targeting protein degradation. Our findings may also have important implications for the field of tissue engineering, as the manufacture of cell-derived biomaterial scaffolds may need to consider proteostasis to effectively replicate native tissues.

Flame aerosol technology is a versatile method for scalable synthesis of nanoparticles. Since particles are produced and collected in a dry state, dispersibility and further functionalization could pose hurdles to their biomedical use. We report on a one-pot, scalable and robust procedure for the PEGylation of flame-made yttria and silica nanoparticles. We demonstrate improved colloidal stability, attenuated activation of blood coagulation and decreased uptake into phagocytic cells, all of which pave the way for facilitated biomedical use of flame-made oxide nanoparticles.

Background Micro-crystals of calcium phosphate have been detected on the aortic valve of patients with aortic stenosis using scanning electron microscopy. It is not known whether crystalisation is specific to heart valve tissue or a general blood-derived process. Methods To this end we modified the method to determine whether calcium phosphate micro-crystals were present in the blood of patients with aortic stenosis. The method was first validated by adding synthetic calcium phosphate hydroxyapatite micro-crystals to healthy volunteer blood samples and determining the lower limit of detection. Then the method was used to examine the blood of 63 patients with echocardiographically confirmed aortic stenosis and 69 unaffected controls undergoing echocardiography for other reasons. Serum calcium and phosphate were measured and the calcium phosphate product compared in cases and controls. Results In the validation study, synthetic hydroxyapatite micro-crystals were identified down to a lower concentration limit of 0.008mg/mL. In the experimental study no particles were identified in any patient, with or without aortic stenosis, even though serum calcium phosphate was higher in cases compared with controls 2.6mmol/L (2.58–2.77) versus 2.47mmol/L (2.36–2.57), p = 0.005 for the difference. Conclusion The results of our study confirm a positive association between serum calcium phosphate and aortic stenosis, but indicate that the calcium phosphate particles found in valve tissue do not precipitate freely in the blood.

The magnetic separation of pathogenic compounds from body fluids is an appealing therapeutic concept. Recently, removal of a diverse array of pathogens has been demonstrated using extracorporeal dialysis-type devices. The contact time between the fluid and the magnetic beads in such devices is limited to a few minutes. This poses challenges, particularly if large compounds such as bacteria or cells need to be removed. Here, we report on the feasibility to remove cells from body fluids in a continuous dialysis type of setting. We assessed tumor cell removal efficiencies from physiological fluids with or without white blood cells using a range of different magnetic bead sizes (50–4000 nm), concentrations, and contact times. We show that tumor cells can be quantitatively removed from body fluids within acceptable times (1– 2 min) and bead concentrations (0.2 mg per mL). We further present a mathematical model to describe the minimal bead number concentration needed to remove a certain number of cells, in the presence of competing nonspecific uptake. The present study paves the way for investigational studies to assess the therapeutic potential of cell removal by magnetic blood purification in a dialysis-like setting.

Polydimethylsiloxane (PDMS) is widely used in biomedical science and can form composites that have broad applicability. One promising application where PDMS composites offer several advantages is optical ultrasound generation via the photoacoustic effect. Here, methods to create these PDMS composites are reviewed and classified. It is highlighted how the composites can be applied to a range of substrates, from micrometer‐scale, temperature‐sensitive optical fibers to centimeter‐scale curved and planar surfaces. The resulting composites have enabled all‐optical ultrasound imaging of biological tissues both ex vivo and in vivo, with high spatial resolution and with clinically relevant contrast. In addition, the first 3D all‐optical pulse‐echo ultrasound imaging of ex vivo human tissue, using a PDMS‐multiwalled carbon nanotube composite and a fiber‐optic ultrasound receiver, is presented. Gold nanoparticle‐PDMS and crystal violet‐PDMS composites with prominent absorption at one wavelength range for pulse‐echo ultrasound imaging and transmission at a second wavelength range for photoacoustic imaging are also presented. Using these devices, images of diseased human vascular tissue with both structural and molecular contrast are obtained. With a broader perspective, literature on recent advances in PDMS microfabrication from different fields is highlighted, and methods for incorporating them into new generations of optical ultrasound generators are suggested.