Extracellular matrix mechanobiology in cancer cell migration.

The extracellular matrix (ECM) is pivotal in modulating tumor progression. Besides chemically stimulating tumor cells, it also offers physical support that orchestrates the sequence of events in the metastatic cascade upon dynamically modulating cell mechanosensation. Understanding this translation between matrix biophysical cues and intracellular signaling has led to rapid growth in the interdisciplinary field of cancer mechanobiology in the last decade. Substantial efforts have been made to develop novel in vitro tumor mimicking platforms to visualize and quantify the mechanical forces within the tissue that dictate tumor cell invasion and metastatic growth. This review highlights recent findings on tumor matrix biophysical cues such as fibrillar arrangement, crosslinking density, confinement, rigidity, topography, and non-linear mechanics and their implications on tumor cell behavior. We also emphasize how perturbations in these cues alter cellular mechanisms of mechanotransduction, consequently enhancing malignancy. Finally, we elucidate engineering techniques to individually emulate the mechanical properties of tumors that could help serve as toolkits for developing and testing ECM-targeted therapeutics on novel bioengineered tumor platforms. STATEMENT OF SIGNIFICANCE: Disrupted ECM mechanics is a driving force for transitioning incipient cells to life-threatening malignant variants. Understanding these ECM changes can be crucial as they may aid in developing several efficacious drugs that not only focus on inducing cytotoxic effects but also target specific matrix mechanical cues that support and enhance tumor invasiveness. Designing and implementing an optimal tumor mimic can allow us to predictively map biophysical cue-modulated cell behaviors and facilitate the design of improved lab-grown tumor models with accurately controlled structural features. This review focuses on the abnormal changes within the ECM during tumorigenesis and its implications on tumor cell-matrix mechanoreciprocity. Additionally, it accentuates engineering approaches to produce ECM features of varying levels of complexity which is critical for improving the efficiency of current engineered tumor tissue models.

Biomimetic Vasculatures by 3D-Printed Porous Molds.

Despite recent advances in biofabrication, recapitulating complex architectures of cell-laden vascular constructs remains challenging. To date, biofabricated vascular models have not yet realized four fundamental attributes of native vasculatures simultaneously: freestanding, branching, multilayered, and perfusable. In this work, a microfluidics-enabled molding technique combined with coaxial bioprinting to fabricate anatomically relevant, cell-laden vascular models consisting of hydrogels is developed. By using 3D porous molds of poly(ethylene glycol) diacrylate as casting templates that gradually release calcium ions as a crosslinking agent, freestanding, and perfusable vascular constructs of complex geometries are fabricated. The bioinks can be tailored to improve the compatibility with specific vascular cells and to tune the mechanical modulus mimicking native blood vessels. Crucially, the integration of relevant vascular cells (such as smooth muscle cells and endothelial cells) in a multilayer and biomimetic configuration is highlighted. It is also demonstrated that the fabricated freestanding vessels are amenable for testing percutaneous coronary interventions (i.e., drug-eluting balloons and stents) under physiological mechanical states such as stretching and bending. Overall, a versatile fabrication technique with multifaceted possibilities of generating biomimetic vascular models that can benefit future research in mechanistic understanding of cardiovascular diseases and the development of therapeutic interventions is introduced.

From qualitative data to correlation using deep generative networks: Demonstrating the relation of nuclear position with the arrangement of actin filaments.

The cell nucleus is a dynamic structure that changes locales during cellular processes such as proliferation, differentiation, or migration, and its mispositioning is a hallmark of several disorders. As with most mechanobiological activities of adherent cells, the repositioning and anchoring of the nucleus are presumed to be associated with the organization of the cytoskeleton, the network of protein filaments providing structural integrity to the cells. However, demonstrating this correlation between cytoskeleton organization and nuclear position requires the parameterization of the extraordinarily intricate cytoskeletal fiber arrangements. Here, we show that this parameterization and demonstration can be achieved outside the limits of human conceptualization, using generative network and raw microscope images, relying on machine-driven interpretation and selection of parameterizable features. The developed transformer-based architecture was able to generate high-quality, completed images of more than 8,000 cells, using only information on actin filaments, predicting the presence of a nucleus and its exact localization in more than 70 per cent of instances. Our results demonstrate one of the most basic principles of mechanobiology with a remarkable level of significance. They also highlight the role of deep learning as a powerful tool in biology beyond data augmentation and analysis, capable of interpreting-unconstrained by the principles of human reasoning-complex biological systems from qualitative data.

Biomaterials for Mimicking and Modelling Tumor Microenvironment.

This chapter summarizes the current biomaterials and associated technologies used to mimic and characterize the tumor microenvironment (TME) for developing preclinical therapeutics. Research in conventional 2D cancer models systematically fails to provide physiological significance due to their discrepancy with diseased tissue's native complexity and dynamic nature. The recent developments in biomaterials and microfabrication have enabled the popularization of 3D models, displacing the traditional use of Petri dishes and microscope slides to bioprinters or microfluidic devices. These technologies allow us to gather large amounts of time-dependent information on tissue-tissue, tissue-cell, and cell-cell interactions, fluid flows, and biomechanical cues at the cellular level that were inaccessible by traditional methods. In addition, the wave of new tools producing unprecedented amounts of data is also triggering a new revolution in the development and use of new tools for analysis, interpretation, and prediction, fueled by the concurrent development of artificial intelligence. Together, all these advances are crystalizing a new era for biomedical engineering characterized by high-throughput experiments and high-quality data.Furthermore, this new detailed understanding of disease and its multifaceted characteristics is enabling the long searched transition to personalized medicine.Here we outline the various biomaterials used to mimic the extracellular matrix (ECM) and redesign the tumor microenvironment, providing a comprehensive overview of cancer research's state of the art and future.

Hierarchical Colorful Structures by Three-Dimensional Printing of Inverse Opals.

Structural coloration is a recurring solution in biological systems to control visible light. In nature, basic structural coloration results from light interacting with a repetitive nanopattern, but more complex interactions and striking results are achieved by organisms incorporating additional hierarchical structures. Artificial reproduction of single-level structural color has been achieved using repetitive nanostructures, with flat sheets of inverse opals being very popular because of their simple and reliable fabrication process. Here, we control photonic structures at several length scales using a combination of direct laser writing and nanosphere assembly, producing freeform hierarchical constructions of inverse opals with high-intensity structural coloration. We report the first 3D prints of stacked, overhanging and slanted microstructures of inverse opals. Among other characteristics, these hierarchical photonic structures exhibit geometrically tunable colors, focal-plane-dependent patterns, and arbitrary alignment of microstructure facet with self-assembled lattice. Based on those results, novel concepts of multilevel information encoding systems are presented.

Measurements of the swimming speeds of motile microorganisms using object tracking and their correlation with water pollution and rheology levels.

Self-propelled microscopic organisms are ubiquitous in water. Such organisms' motility depends on hydrodynamic and physical factors related to the rheology of the surrounding media and biological factors depending on the organisms' state and well-being. Here we demonstrate that the swimming speed of Paramecium aurelia, a unicellular protozoan, globally found in fresh, brackish, and salt waters, can be used as a measurable frugal indicator of the presence of pollutants in water. This study establishes a significant and consistent relationship between Paramecia's swimming speed and the presence of five different organic and inorganic contaminants at varying concentrations centered around drinking water thresholds. The large size and ubiquity of the targeted microorganism, the avoidance of reagents or specialized tools for the measurement, and the simple data collection based on an object tracking algorithm enable the automatization of the assessment and real-time results using globally available technology.

Martian biolith: A bioinspired regolith composite for closed-loop extraterrestrial manufacturing.

Given plans to revisit the lunar surface by the late 2020s and to take a crewed mission to Mars by the late 2030s, critical technologies must mature. In missions of extended duration, in situ resource utilization is necessary to both maximize scientific returns and minimize costs. While this present a significantly more complex challenge in the resource-starved environment of Mars, it is similar to the increasing need to develop resource-efficient and zero-waste ecosystems on Earth. Here, we make use of recent advances in the field of bioinspired chitinous manufacturing to develop a manufacturing technology to be used within the context of a minimal, artificial ecosystem that supports humans in a Martian environment.

Cellulose Nanofibers for Encapsulation and Pluripotency Preservation in the Early Development of Embryonic Stem Cells.

Materials for three-dimensional cultures aim to reproduce the function of the extracellular matrix, enabling cell adhesion and growth by remodeling the environment. However, embryonic stem cells (ESCs) must develop in environments that prevent adhesion and preserve their pluripotency. In this study, we used cellulose nanofiber hydrogels to mimic the developing conditions required for ESCs. These plant-based hydrogels are simultaneously biocompatible and exogenous to mammalian cells, preventing remodeling and attachment. The storage modulus of these hydrogels could be fine-tuned by varying the degree of oxidation to enable selective degradation. The ESCs proliferated in the artificial environment, forming increasingly large embryoid bodies for 15 days. Unlike traditional cultures in which ESCs begin differentiating upon the removal of the chemical inhibition, the expression of pluripotency markers in the ESC population remained high for the entire two weeks. Cellulase from Trichoderma reesei was used to retrieve the ESC cultures selectively. The proposed unique system is a prospective model with which to study the early development of embryonic cells, as well as a nonchemical method of preserving undifferentiated populations of ESCs.

Tough and Strong: Cross-Lamella Design Imparts Multifunctionality to Biomimetic Nacre.

The creation of structural composites with combined strength, toughness, low density, and biocompatibility remains a long-standing challenge. On the other hand, bivalve marine shells-Clinocardiumspp.-exhibit strength, stiffness, and toughness that surpass even that of the nacre that is the most widely mimicked model for structural composites. The superior mechanical properties of Clinocardiumspp. shells originate from their cross-lamella design, comprising CaCO3 mineral platelets arranged in an "interlocked" herringbone fashion. Reproduction of such hierarchical designs could offer multifunctionality, potentially combining strength and toughness at low densities, and the capability for seamless integration with biological systems. Here, we demonstrate manufacturing of the cross-lamella design by biomineralizing aragonite films with sawtooth patterns and assembling them in a chitosan/fibroin matrix to generate a composite with interlocked mineral layers. The resultant composite, with a similar constitution to that of the biological counterpart, nearly doubles the strength of previous nacre-mimetic composites while improving the tensile toughness and simultaneously exhibiting stiffness and biocompatibility.

Circular manufacturing of chitinous bio-composites via bioconversion of urban refuse.

Bioinspired manufacturing, in the sense of replicating the way nature fabricates, may hold great potential for supporting a socioeconomic transformation towards a sustainable society. Use of unmodified ubiquitous biological components suggests for a fundamentally sustainable manufacturing paradigm where materials are produced, transformed into products and degraded in closed regional systems with limited requirements for transport. However, adoption is currently limited by the fact that despite their ubiquitous nature, these biopolymers are predominantly harvested as industrial and agricultural products. In this study, we overcome this limitation by developing a link between bioinspired manufacturing and urban waste bioconversion. This result is paramount for the development of circular economic models, effectively connecting the organic by-products of civilization to locally decentralized, general-purpose manufacturing.

Stimuli-responsive injectable cellulose thixogel for cell encapsulation.

Herein, we present the synthesis of surface-oxidized cellulose nanofiber (CNF) hydrogel and characterization with various physicochemical analyses and spectroscopic tools as well as its suitability for cellular encapsulation and delivery. The structure-property relationship as shear thinning, thixotropy, creep-recovery and stimuli responsiveness are explored. The CNF hydrogel is capable to inject possessing shear thinning behavior at shear rate (~10 s-1) range in the normal injecting process. In time-dependent thixotropy, the hydrogel showed rapid transform from flowable fluid back to structured hydrogel fully recovering in less than 60 s. The presence of cell-culture media did not alter shear thinning behavior of CNF hydrogel and showed increased thixotropicity with respect to the control gel. The CNF hydrogel forms 3D structures, without any crosslinker, with a wide range of tunable moduli (~36-1000 Pa) based on concentration and external stimuli. The biological characteristics of the thixotropic gels are studied for human breast cancer cells and mouse embryonic stem cells and indicated high cell viability, long-term survival, and spherical morphology.

Large-scale additive manufacturing with bioinspired cellulosic materials.

Cellulose is the most abundant and broadly distributed organic compound and industrial by-product on Earth. However, despite decades of extensive research, the bottom-up use of cellulose to fabricate 3D objects is still plagued with problems that restrict its practical applications: derivatives with vast polluting effects, use in combination with plastics, lack of scalability and high production cost. Here we demonstrate the general use of cellulose to manufacture large 3D objects. Our approach diverges from the common association of cellulose with green plants and it is inspired by the wall of the fungus-like oomycetes, which is reproduced introducing small amounts of chitin between cellulose fibers. The resulting fungal-like adhesive material(s) (FLAM) are strong, lightweight and inexpensive, and can be molded or processed using woodworking techniques. We believe this first large-scale additive manufacture with ubiquitous biological polymers will be the catalyst for the transition to environmentally benign and circular manufacturing models.

Direct Bonding of Chitosan Biomaterials to Tissues Using Transglutaminase for Surgical Repair or Device Implantation.

Natural biomaterials, such as chitosan and collagen, are useful for biomedical applications because they are biocompatible, mechanically robust, and biodegradable, but it is difficult to rapidly and tightly bond them to living tissues. In this study, we demonstrate that the microbial transglutaminase (mTG), can be used to rapidly (<5 min) bond chitosan and collagen biomaterials to the surfaces of hepatic, cardiac, and dermal tissues, as well as to functionalized polydimethylsiloxane (PDMS) materials that are used in medical products. The mTG-bonded chitosan patches effectively sealed intestinal perforations, and a newly developed two-component mTG-bonded chitosan spray effectively repaired ruptures in a breathing lung when tested ex vivo. The mechanical strength of mTG-catalyzed chitosan adhesive bonds were comparable to those generated by commonly used surgical glues. These results suggest that mTG preparations may be broadly employed to bond various types of organic materials, including polysaccharides, proteins, and functionalized inorganic polymers to living tissues, which may open new avenues for biomedical engineering, medical device integration, and tissue repair.

Unexpected strength and toughness in chitosan-fibroin laminates inspired by insect cuticle.

A material inspired by natural insect cuticle and composed of chitosan and fibroin is created. The material exhibits the strength of an aluminum alloy at half its weight, while being clear, biocompatible, biodegradable, and micromoldable. The bioinspired laminate exhibits strength and toughness that are ten times greater than the unstructured component blend and twice that of its strongest constituent.

Simultaneous biochemical and topographical patterning on curved surfaces using biocompatible sacrificial molds.

A method for the simultaneous (bio)chemical and topographical patterning of enclosed structures in poly(dimethyl siloxane) (PDMS) is presented. The simultaneous chemical and topography transference uses a water-soluble chitosan sacrificial mold to impart a predefined pattern with micrometric accuracy to a PDMS replica. The method is compared to conventional soft-lithography techniques on planar surfaces. Its functionality is demonstrated by the transference of streptavidin directly to the surface of the three-dimensional PDMS structures as well as indirectly using streptavidin-loaded latex nanoparticles. The streptavidin immobilized on the PDMS is tested for bioactivity by coupling with fluorescently labeled biotin. This proves that the streptavidin is immobilized on the PDMS surface, not in the bulk of the polymer, and is therefore accessible for use as signaling/binding element in micro and bioengineering. The use of a biocompatible polymer and processes enables the technique to be used for the chemical patterning of tissue constructions.

Complex microstructured 3D surfaces using chitosan biopolymer.

A technique for producing micrometer-scale structures over large, nonplanar chitosan surfaces is described. The technique makes use of the rheological characteristics (deformability) of the chitosan to create freestanding, three-dimensional scaffolds with controlled shapes, incorporating defined microtopography. The results of an investigation into the technical limits of molding different combinations of shapes and microtopographies are presented, highlighting the versatility of the technique when used irrespectively with inorganic or delicate organic moulds. The final, replicated scaffolds presented here are patterned with arrays of one-micrometer-tall microstructures over large areas. Structural integrity is characterized by the measurement of structural degradation. Human umbilical vein endothelial cells cultured on a tubular scaffold show that early cell growth is conditioned by the microtopography and indicate possible uses for the structures in biomedical applications. For those applications requiring improved chemical and mechanical resistance, the structures can be replicated in poly(dimethyl siloxane).

Micro- and nanostructuring of freestanding, biodegradable, thin sheets of chitosan via soft lithography.

A technique for imparting micro- and nanostructured topography into the surface of freestanding thin sheets of chitosan is described. Both micro- and nanometric surface structures have been produced using soft lithography. The soft lithography method, based on solvent evaporation, has allowed structures approximately 60 nm tall and approximately 500 x 500 nm(2) to be produced on freestanding approximately 0.5 mm thick sheets of the polymer when cured at 293 K, and structures approximately 400 nm tall and 5 x 5 microm(2) to be produced when cured at 283 K. Nonstructured chitosan thin sheets (approximately 200 microm thick) show excellent optical transmission properties in the visible portion of the electromagnetic spectrum. The structured sheets can be used for applications where optical microscopic analysis is required, such as cell interaction experiments and tissue engineering.

Micro/nanopatterning of proteins via contact printing using high aspect ratio PMMA stamps and nanoimprint apparatus.

Micro- and nanoscale protein patterns have been produced via a new contact printing method using a nanoimprint lithography apparatus. The main novelty of the technique is the use of poly(methyl methacrylate) (PMMA) instead of the commonly used poly(dimethylsiloxane) (PDMS) stamps. This avoids printing problems due to roof collapse, which limits the usable aspect ratio in microcontact printing to 10:1. The rigidity of the PMMA allows protein patterning using stamps with very high aspect ratios, up to 300 in this case. Conformal contact between the stamp and the substrate is achieved because of the homogeneous pressure applied via the nanoimprint lithography instrument, and it has allowed us to print lines of protein approximately 150 nm wide, at a 400 nm period. This technique, therefore, provides an excellent method for the direct printing of high-density sub-micrometer scale patterns, or, alternatively, micro-/nanopatterns spaced at large distances. The controlled production of these protein patterns is a key factor in biomedical applications such as cell-surface interaction experiments and tissue engineering.

Directional alignment of MG63 cells on polymer surfaces containing point microstructures.

MG63 cells cultured on regular arrays of point microstructures (posts and holes) are shown to preferentially align at certain angles to the pattern of the structures, at 0 degrees, 30 degrees, and 45 degrees in particular. The effect is found to be more pronounced for post rather than hole structures (although no significant difference is found for the angles the cells make to the holes or posts) and is thought to be due to the fact that the cells use the posts as anchorage points to hold themselves to the surface. It is also shown that cells preferentially align with the structures depending on the dimensions of the structures and the distance between neighboring structures. This is important when designing structured surfaces for cell-surface interaction studies for materials to be used in, for example, drug delivery or tissue engineering.