Microenvironment-mediated cancer dormancy: Insights from metastability theory.

Dormancy is an evolutionarily conserved protective mechanism widely observed in nature. A pathological example is found during cancer metastasis, where cancer cells disseminate from the primary tumor, home to secondary organs, and enter a growth-arrested state, which could last for decades. Recent studies have pointed toward the microenvironment being heavily involved in inducing, preserving, or ceasing this dormant state, with a strong focus on identifying specific molecular mechanisms and signaling pathways. Increasing evidence now suggests the existence of an interplay between intracellular as well as extracellular biochemical and mechanical cues in guiding such processes. Despite the inherent complexities associated with dormancy, proliferation, and growth of cancer cells and tumor tissues, viewing these phenomena from a physical perspective allows for a more global description, independent from many details of the systems. Building on the analogies between tissues and fluids and thermodynamic phase separation concepts, we classify a number of proposed mechanisms in terms of a thermodynamic metastability of the tumor with respect to growth. This can be governed by interaction with the microenvironment in the form of adherence (wetting) to a substrate or by mechanical confinement of the surrounding extracellular matrix. By drawing parallels with clinical and experimental data, we advance the notion that the local energy minima, or metastable states, emerging in the tissue droplet growth kinetics can be associated with a dormant state. Despite its simplicity, the provided framework captures several aspects associated with cancer dormancy and tumor growth.

Solvent Cavitation during Ambient Pressure Drying of Silica Aerogels.

Ambient-pressure drying of silica gels stands out as an economical and accessible process for producing monolithic silica aerogels. Gels experience significant deformations during drying due to the capillary pressure generated at the liquid-vapor interface in submicron pores. Proper control of the gel properties and the drying rate is essential to enable reversible drying shrinkage without mechanical failure. Recent in operando microcomputed X-ray tomography (µCT) imaging revealed the kinetics of the phase composition during drying and spring-back. However, to fully explain the underlying mechanisms, spatial resolution is required. Here we show evidence of evaporation by hexane cavitation during the ambient-pressure drying of silylated silica gels by spatially resolved quantitative analysis of µCT data supported by wide-angle X-ray scattering measurements. Cavitation consists of the rupture of the pore liquid put under tension by capillary pressure, creating vapor bubbles within the gels. We found the presence of a homogeneously distributed vapor-air phase in the gels well ahead of the maximum shrinkage. The onset of this vapor/air phase corresponded to a pore volume shrinkage of ca. 50 vol % that was attributed to a critical stiffening of the silica skeleton enabling cavitation. Our results provide new aspects of the relation between the shape changes of silica gels during drying and the evaporation mechanisms. We conclude that stress release by cavitation may be at the origin of the resistance of the silica skeleton to drying stresses. This opens the path toward producing larger monolithic silica aerogels by fine-tuning the drying conditions to exploit cavitation.

Bone Material Properties in Bone Diseases Affecting Children.

PURPOSE OF REVIEW: Metabolic and genetic bone disorders affect not only bone mass but often also the bone material, including degree of mineralization, matrix organization, and lacunar porosity. The quality of juvenile bone is moreover highly influenced by skeletal growth. This review aims to provide a compact summary of the present knowledge on the complex interplay between bone modeling and remodeling during skeletal growth and to alert the reader to the complexity of bone tissue characteristics in children with bone disorders. RECENT FINDINGS: We describe cellular events together with the characteristics of the different tissues and organic matrix organization (cartilage, woven and lamellar bone) occurring during linear growth. Subsequently, we present typical alterations thereof in disorders leading to over-mineralized bone matrix compared to those associated with low or normal mineral content based on bone biopsy studies. Growth spurts or growth retardation might amplify or mask disease-related alterations in bone material, which makes the interpretation of bone tissue findings in children complex and challenging.

Three-dimensional structural interrelations between cells, extracellular matrix, and mineral in normally mineralizing avian leg tendon.

The spatial-temporal relationship between cells, extracellular matrices, and mineral deposits is fundamental for an improved understanding of mineralization mechanisms in vertebrate tissues. By utilizing focused ion beam-scanning electron microscopy with serial surface imaging, normally mineralizing avian tendons have been studied with nanometer resolution in three dimensions with volumes exceeding tens of micrometers in range. These parameters are necessary to yield sufficiently fine ultrastructural details while providing a comprehensive overview of the interrelationships between the tissue structural constituents. Investigation reveals a complex lacuno-canalicular network in highly mineralized tendon regions, where ∼100 nm diameter canaliculi emanating from cell (tenocyte) lacunae surround extracellular collagen fibril bundles. Canaliculi are linked to smaller channels of ∼40 nm diameter, occupying spaces between fibrils. Close to the tendon mineralization front, calcium-rich deposits appear between the fibrils and, with time, mineral propagates along and within them. These close associations between tenocytes, tenocyte lacunae, canaliculi, small channels, collagen, and mineral suggest a concept for the mineralization process, where ions and/or mineral precursors may be transported through spaces between fibrils before they crystallize along the surface of and within the fibrils.

The mechanoresponse of bone is closely related to the osteocyte lacunocanalicular network architecture.

Organisms rely on mechanosensing mechanisms to adapt to changes in their mechanical environment. Fluid-filled network structures not only ensure efficient transport but can also be employed for mechanosensation. The lacunocanalicular network (LCN) is a fluid-filled network structure, which pervades our bones and accommodates a cell network of osteocytes. For the mechanism of mechanosensation, it was hypothesized that load-induced fluid flow results in forces that can be sensed by the cells. We use a controlled in vivo loading experiment on murine tibiae to test this hypothesis, whereby the mechanoresponse was quantified experimentally by in vivo micro-computed tomography (µCT) in terms of formed and resorbed bone volume. By imaging the LCN using confocal microscopy in bone volumes covering the entire cross-section of mouse tibiae and by calculating the fluid flow in the three-dimensional (3D) network, we could perform a direct comparison between predictions based on fluid flow velocity and the experimentally measured mechanoresponse. While local strain distributions estimated by finite-element analysis incorrectly predicts preferred bone formation on the periosteal surface, we demonstrate that additional consideration of the LCN architecture not only corrects this erroneous bias in the prediction but also explains observed differences in the mechanosensitivity between the three investigated mice. We also identified the presence of vascular channels as an important mechanism to locally reduce fluid flow. Flow velocities increased for a convergent network structure where all of the flow is channeled into fewer canaliculi. We conclude that, besides mechanical loading, LCN architecture should be considered as a key determinant of bone adaptation.

Quantitative Backscattered Electron Imaging of Bone Using a Thermionic or a Field Emission Electron Source.

Quantitative backscattered electron imaging is an established method to map mineral content distributions in bone and to determine the bone mineralization density distribution (BMDD). The method we applied was initially validated for a scanning electron microscope (SEM) equipped with a tungsten hairpin cathode (thermionic electron emission) under strongly defined settings of SEM parameters. For several reasons, it would be interesting to migrate the technique to a SEM with a field emission electron source (FE-SEM), which, however, would require to work with different SEM parameter settings as have been validated for DSM 962. The FE-SEM has a much better spatial resolution based on an electron source size in the order of several 100 nanometers, corresponding to an about [Formula: see text] to [Formula: see text] times smaller source area compared to thermionic sources. In the present work, we compare BMDD between these two types of instruments in order to further validate the methodology. We show that a transition to higher pixel resolution (1.76, 0.88, and 0.57 µm) results in shifts of the BMDD peak and BMDD width to higher values. Further the inter-device reproducibility of the mean calcium content shows a difference of up to 1 wt% Ca, while the technical variance of each device can be reduced to [Formula: see text] wt% Ca. Bearing in mind that shifts in calcium levels due to diseases, e.g., high turnover osteoporosis, are often in the range of 1 wt% Ca, both the bone samples of the patients as well as the control samples have to be measured on the same SEM device. Therefore, we also constructed new reference BMDD curves for adults to be used for FE-SEM data comparison.

The effect of aging on the nanostructure of murine alveolar bone and dentin.

INTRODUCTION: Alveolar bone, dentin, and cementum provide a striking example of structurally different collagen-based mineralized tissues separated only by periodontal ligament. While alveolar bone is strongly remodeled, this does not hold for dentin and cementum. However, additional dentin can be deposited on the inner surface of the pulp chamber also in older age. By investigating alveolar bone and molar of mice, the aim of our study is to detect changes in the mineral nanostructure with aging. MATERIALS AND METHODS: Buccal-lingual sections of the mandible and first molar from C57BL/6 mice of three different age groups (young 5 weeks, adult 22 weeks and old 23 months) were characterized using synchrotron small and wide-angle X-ray scattering. Local average thickness and length of the apatite particles were mapped with several line scans covering the alveolar bone and the tooth. RESULTS: In alveolar bone, a spatial gradient was seen to develop with age with the thickest and longest particles in the distal part of the bone. The mineral particles in dentin were found to be become thicker, but then decrease of average length from adult to old animals. The mineral particle characteristics of dentin close to the pulp chamber were not only different to the rest of the tooth, but also when comparing the different age groups and even between individual animals in the same age group. CONCLUSIONS: These results indicated that mineral particle characteristics were found to evolve differently between molar and alveolar bone as a function of age.

The spider cuticle: a remarkable material toolbox for functional diversity.

Engineered systems are typically based on a large variety of materials differing in composition and processing to provide the desired functionality. Nature, however, has evolved materials that are used for a wide range of functional challenges with minimal compositional changes. The exoskeletal cuticle of spiders, as well as of other arthropods such as insects and crustaceans, is based on a combination of chitin, protein, water and small amounts of organic cross-linkers or minerals. Spiders use it to obtain mechanical support structures and lever systems for locomotion, protection from adverse environmental influences, tools for piercing, cutting and interlocking, auxiliary structures for the transmission and filtering of sensory information, structural colours, transparent lenses for light manipulation and more. This paper illustrates the 'design space' of a single type of composite with varying internal architecture and its remarkable capability to serve a diversity of functions. This article is part of the theme issue 'Bio-derived and bioinspired sustainable advanced materials for emerging technologies (part 1)'.

Advanced materials design based on waste wood and bark.

Trees belong to the largest living organisms on Earth and plants in general are one of our main renewable resources. Wood as a material has been used since the beginning of humankind. Today, forestry still provides raw materials for a variety of applications, for example in the building industry, in paper manufacturing and for various wood products. However, many parts of the tree, such as reaction wood, branches and bark are often discarded as forestry residues and waste wood, used as additives in composite materials or burned for energy production. More advanced uses of bark include the extraction of chemical substances for glues, food additives or healthcare, as well as the transformation to advanced carbon materials. Here, we argue that a proper understanding of the internal fibrous structure and the resulting mechanical behaviour of these forest residues allows for the design of materials with greatly varying properties and applications. We show that simple and cheap treatments can give tree bark a leather-like appearance that can be used for the construction of shelters and even the fabrication of woven textiles. This article is part of the theme issue 'Bio-derived and bioinspired sustainable advanced materials for emerging technologies (part 1)'.

Spatiotemporal Measurement of Osmotic Pressures by FRET Imaging.

Osmotic pressures (OPs) play essential roles in biological processes and numerous technological applications. However, the measurement of OP in situ with spatiotemporal resolution has not been achieved so far. Herein, we introduce a novel kind of OP sensor based on liposomes loaded with water-soluble fluorescent dyes exhibiting resonance energy transfer (FRET). The liposomes experience volume changes in response to OP due to water outflux. The FRET efficiency depends on the average distance between the entrapped dyes and thus provides a direct measure of the OP surrounding each liposome. The sensors exhibit high sensitivity to OP in the biologically relevant range of 0-0.3 MPa in aqueous solutions of salt, small organic molecules, and macromolecules. With the help of FRET microscopy, we demonstrate the feasibility of spatiotemporal OP imaging, which can be a promising new tool to investigate phenomena involving OPs and their dynamics in biology and technology.

Globular structure of the hypermineralized tissue in human femoral neck.

Bone becomes more fragile with ageing. Among many structural changes, a thin layer of highly mineralized and brittle tissue covers part of the external surface of the thin femoral neck cortex in older people and has been proposed to increase hip fragility. However, there have been very limited reports on this hypermineralized tissue in the femoral neck, especially on its ultrastructure. Such information is critical to understanding both the mineralization process and its contributions to hip fracture. Here, we use multiple advanced techniques to characterize the ultrastructure of the hypermineralized tissue in the neck across various length scales. Synchrotron radiation micro-CT found larger but less densely distributed cellular lacunae in hypermineralized tissue than in lamellar bone. When examined under FIB-SEM, the hypermineralized tissue was mainly composed of mineral globules with sizes varying from submicron to a few microns. Nano-sized channels were present within the mineral globules and oriented with the surrounding organic matrix. Transmission electron microscopy showed the apatite inside globules were poorly crystalline, while those at the boundaries between the globules had well-defined lattice structure with crystallinity similar to the apatite mineral in lamellar bone. No preferred mineral orientation was observed both inside each globule and at the boundaries. Collectively, we conclude based on these new observations that the hypermineralized tissue is non-lamellar and has less organized mineral, which may contribute to the high brittleness of the tissue.

Heterogeneity of the osteocyte lacuno-canalicular network architecture and material characteristics across different tissue types in healing bone.

Various tissue types, including fibrous connective tissue, bone marrow, cartilage, woven and lamellar bone, coexist in healing bone. Similar to most bone tissue type, healing bone contains a lacuno-canalicular network (LCN) housing osteocytes. These cells are known to orchestrate bone remodeling in healthy bone by sensing mechanical strains and translating them into biochemical signals. The structure of the LCN is hypothesized to influence mineralization processes. Hence, the aim of the present study was to visualize and match spatial variations in the LCN topology with mineral characteristics, within and at the interfaces of the different tissue types that comprise healing bone. We applied a correlative multi-method approach to visualize the LCN architecture and quantify mineral particle size and orientation within healing femoral bone in a mouse osteotomy model (26 weeks old C57BL/6 mice). This approach revealed structural differences across several length scales during endochondral ossification within the following regions: calcified cartilage, bony callus, cortical bone and a transition zone between the cortical and callus region analyzed 21 days after the osteotomy. In this transition zone, we observed a continuous convergence of mineral characteristics and osteocyte lacunae shape as well as discontinuities in the lacunae volume and LCN connectivity. The bony callus exhibits a 34% higher lacunae number density and 40% larger lacunar volume compared to cortical bone. The presented correlations between LCN architecture and mineral characteristics improves our understanding of how bone develops during healing and may indicate a contribution of osteocytes to bone (re)modeling.

Alterations of bone material properties in adult patients with X-linked hypophosphatemia (XLH).

X-linked hypophosphatemia (XLH) caused by PHEX mutations results in elevated serum FGF23 levels, renal phosphate wasting and low 1,25-dihydroxyvitamin D. The glycophosphoprotein osteopontin, a potent inhibitor of mineralization normally degraded by PHEX, accumulates within the bone matrix. Conventional therapy consisting of supplementation with phosphate and vitamin D analogs is burdensome and the effects on bone material poorly characterized. We analyzed transiliac bone biopsies from four adult patients, two of them severely affected due to no diagnosis and no treatment until adulthood. We used light microscopy, qBEI and FTIRI to study histology, histomorphometry, bone mineralization density distribution, properties of the organic matrix and size of hypomineralized periosteocytic lesions. Non-treatment resulted in severe osteomalacia, twice the amount of mineralized trabecular volume, multiple osteon-like perforations, continuity of lamellae from mineralized to unmineralized areas and distinctive patches of woven bone. Periosteocytic lesions were larger than in treated patients. The latter had nearly normal osteoid thicknesses, although surface was still elevated. The median calcium content of the matrix was always within normal range, although the percentage of lowly mineralized bone areas was highly increased in non-treated patients, resulting in a marked heterogeneity in mineralization. Divalent collagen cross-links were evident independently of the mineral content of the matrix. Broad osteoid seams lacked measurable pyridinoline, a mature trivalent cross-link and exhibited considerable acidic lipid content, typically found in matrix vesicles. Based on our results, we propose a model that possibly integrates the relationship between the observed mineralization disturbances, FGF23 secretion and the known osteopontin accumulation in XLH.

Multiscale characterization of the mineral phase at skeletal sites of breast cancer metastasis.

Skeletal metastases, the leading cause of death in advanced breast cancer patients, depend on tumor cell interactions with the mineralized bone extracellular matrix. Bone mineral is largely composed of hydroxyapatite (HA) nanocrystals with physicochemical properties that vary significantly by anatomical location, age, and pathology. However, it remains unclear whether bone regions typically targeted by metastatic breast cancer feature distinct HA materials properties. Here we combined high-resolution X-ray scattering analysis with large-area Raman imaging, backscattered electron microscopy, histopathology, and microcomputed tomography to characterize HA in mouse models of advanced breast cancer in relevant skeletal locations. The proximal tibial metaphysis served as a common metastatic site in our studies; we identified that in disease-free bones this skeletal region contained smaller and less-oriented HA nanocrystals relative to ones that constitute the diaphysis. We further observed that osteolytic bone metastasis led to a decrease in HA nanocrystal size and perfection in remnant metaphyseal trabecular bone. Interestingly, in a model of localized breast cancer, metaphyseal HA nanocrystals were also smaller and less perfect than in corresponding bone in disease-free controls. Collectively, these results suggest that skeletal sites prone to tumor cell dissemination contain less-mature HA (i.e., smaller, less-perfect, and less-oriented crystals) and that primary tumors can further increase HA immaturity even before secondary tumor formation, mimicking alterations present during tibial metastasis. Engineered tumor models recapitulating these spatiotemporal dynamics will permit assessing the functional relevance of the detected changes to the progression and treatment of breast cancer bone metastasis.

Unraveling the Rapid Assembly Process of Stiff Cellulosic Fibers from Mistletoe Berries.

The mucilaginous viscin tissue within mistletoe berries possesses an extraordinary ability to be rapidly processed under ambient conditions into stiff cellulosic fibers (>14 GPa) through simple mechanical drawing. This rapid and extreme transformation process is hydration-dependent and involves an astonishing >200-fold increase in length, providing a relevant role model for efforts to produce advanced composites from cellulose-based structures such as cellulose nanocrystals or cellulose nanofibrils. Using a combination of in situ polarized light microscopy, synchrotron X-ray scattering, and humidity-controlled mechanical analysis, we examine here the dynamic transition of a viscin cell bundle from hydrogel-like tissues to high-performance fibers. Our findings indicate a massive phase transition in which cellulose microfibrils containing high-aspect-ratio crystalline domains undergo dramatic reorganization, facilitated by a water-responsive noncellulosic matrix. Transition from an aligned, yet flowing state to a stiff fiber is likely triggered by rapid water loss below 45% relative humidity. These findings not only help understanding the adaptive success of mistletoe but may also be relevant for the development of new facile processing methods for next-generation cellulosic composites.

Self-healing silk from the sea: role of helical hierarchical structure in Pinna nobilis byssus mechanics.

The byssus fibers of Mytilus mussel species have become an important role model in bioinspired materials research due to their impressive properties (e.g. high toughness, self-healing); however, Mytilids represent only a small subset of all byssus-producing bivalves. Recent studies have revealed that byssus from other species possess completely different protein composition and hierarchical structure. In this regard, Pinna nobilis byssus is especially interesting due to its very different morphology, function and its historical use for weaving lightweight golden fabrics, known as sea silk. P. nobilis byssus was recently discovered to be comprised of globular proteins organized into a helical protein superstructure. In this work, we investigate the relationships between this hierarchical structure and the mechanical properties of P. nobilis byssus threads, including energy dissipation and self-healing capacity. To achieve this, we performed in-depth mechanical characterization, as well as tensile testing coupled with in situ X-ray scattering. Our findings reveal that P. nobilis byssus, like Mytilus, possesses self-healing and energy damping behavior and that the initial elastic behavior of P. nobilis byssus is due to stretching and unraveling of the previously observed helical building blocks comprising the byssus. These findings have biological relevance for understanding the convergent evolution of mussel byssus for different species, and also for the field of bio-inspired materials.

Towards a Connectomic Description of the Osteocyte Lacunocanalicular Network in Bone.

PURPOSE OF REVIEW: Osteocytes are the most abundant bone cells. They are completely encased in mineralized tissue, sitting inside lacunae that are connected by a multitude of canaliculi. In recent years, the osteocyte network has been shown to fulfill endocrine functions and to communicate with a number of other organs. This review addresses emerging knowledge on the connectome of the lacunocanalicular network in different types of bone tissue. RECENT FINDINGS: Recent advances in three-dimensional imaging technology started to reveal parameters that are well known from general theory to characterize the function of networks, such as network density, degree of nodes, or shortest path length through the network. The connectome of the lacunocanalicular network differs in some aspects between lamellar and woven bone and seems to change with age. More research is needed to relate network structure to function, such as intercellular transport or communication and its role in mechanosensation, as well as to understand the effect of diseases.