Functional surface microstructures inspired by nature : From adhesion and wetting principles to sustainable new devices

In the course of evolution nature has arrived at startling materials solutions to ensure survival. Investigations into biological surfaces, ranging from plants, insects and geckos to aquatic animals, have inspired the design of intricate surface patterns to create useful functionalities. This paper reviews the fundamental interaction mechanisms of such micropatterns with liquids, solids, and soft matter such as skin for control of wetting, self-cleaning, anti-fouling, adhesion, skin adherence, and sensing. Compared to conventional chemical strategies, the paradigm of micropatterning enables solutions with superior resource efficiency and sustainability. Associated applications range from water management and robotics to future health monitoring devices. We finally provide an overview of the relevant patterning methods as an appendix

Functional surface microstructures inspired by nature – From adhesion and wetting principles to sustainable new devices

In the course of evolution nature has arrived at startling materials solutions to ensure survival. Investigations into biological surfaces, ranging from plants, insects and geckos to aquatic animals, have inspired the design of intricate surface patterns to create useful functionalities. This paper reviews the fundamental interaction mechanisms of such micropatterns with liquids, solids, and soft matter such as skin for control of wetting, self-cleaning, anti-fouling, adhesion, skin adherence, and sensing. Compared to conventional chemical strategies, the paradigm of micropatterning enables solutions with superior resource efficiency and sustainability. Associated applications range from water management and robotics to future health monitoring devices. We finally provide an overview of the relevant patterning methods as an appendix

The Evolution of Robust Development and Homeostasis in Artificial Organisms

During embryogenesis, multicellular animals are shaped via cell proliferation, cell rearrangement, and apoptosis. At the end of development, tissue architecture is then maintained through balanced rates of cell proliferation and loss. Here, we take an in silico approach to look for generic systems features of morphogenesis in multicellular animals that arise as a consequence of the evolution of development. Using artificial evolution, we evolved cellular automata-based digital organisms that have distinct embryonic and homeostatic phases of development. Although these evolved organisms use a variety of strategies to maintain their form over time, organisms of different types were all found to rapidly recover from environmental damage in the form of wounds. This regenerative response was most robust in an organism with a stratified tissue-like architecture. An evolutionary analysis revealed that evolution itself contributed to the ability of this organism to maintain its form in the face of genetic and environmental perturbation, confirming the results of previous studies. In addition, the exceptional robustness of this organism to surface injury was found to result from an upward flux of cells, driven in part by cell divisions with a stable niche at the tissue base. Given the general nature of the model, our results lead us to suggest that many of the robust systems properties observed in real organisms, including scar-free wound-healing in well-protected embryos and the layered tissue architecture of regenerating epithelial tissues, may be by-products of the evolution of morphogenesis, rather than the direct result of selection

Design of Engineered Biomaterial Architectures Through Natural Silk Proteins

Silk proteins have provided a source of unique and versatile building blocks in the fabrication of biomedical devices for addressing a range of applications. Critical to advancing this field is the ability to establish an understanding of these proteins in their native and engineered states as well as in developing scalable processing strategies, which can fully exploit or enhance the stability, structure, and functionality of the two constituent proteins, silk fibroin and sericin. The research outlined in this dissertation focuses on the evolution in architecture and capability of silks, to effectively position a functionally-diverse, renewable class of silk materials within the rapidly expanding field of smart biomaterials. Study of the process of building macroscopic silk fibers provides insight into the initial steps in the broader picture of silk assembly, yielding biomaterials with greatly improved attributes in the assembled state over those of protein precursors alone. Self-organization processes in silk proteins enable their aggregation into highly organized architectures through simple, physical association processes. In this work, a model is developed for the process of aqueous behavior and aggregation, and subsequent two-dimensional behavior of natural silk sericin, to enable formation of a range of distinct, complex architectures. This model is then translated to an engineered system of fibroin microparticles, demonstrating the role of similar phenomena in creating autonomously-organized structures, providing key insight into future “bottom up” assembly strategies. The aqueous behavior of the water-soluble silk sericin protein was then exploited to create biocomposites capable of enhanced response and biocompatibility, through a novel protein-template strategy. In this work, sericin was added to the biocompatible and biodegradable poly(amino acid), poly(aspartic acid), to improve its pH-dependent swelling response. This work demonstrated the production of a range of porous scaffolds capable providing meaningful response to environmental stimuli, with application in tissue engineering scaffolds and biosensing technologies. Finally, to expand the capabilities of silk proteins beyond process-driven parameters to directly fabricate engineered architectures, a method for silk photopatterning was explored, enabling the direct fabrication of biologically-relevant structures at the micro and nanoscales. Using a facile bioconjugation strategy, native silk proteins could be transformed into proteins with a photoactive capacity. The well-established platform of photolithography could then be incorporated into fabrication strategies to produce a range of architectures capable of addressing spatially-directed material requirements in cell culture and further applications in the use of non-toxic, renewable biological materials

How to build a biological machine using engineering materials and methods

We present work in 3D printing electric motors from basic materials as the key to building a self-replicating machine to colonise the Moon. First, we explore the nature of the biological realm to ascertain its essence, particularly in relation to the origin of life when the inanimate became animate. We take an expansive view of this to ascertain parallels between the biological and the manufactured worlds. Life must have emerged from the available raw material on Earth and, similarly, a self-replicating machine must exploit and leverage the available resources on the Moon. We then examine these lessons to explore the construction of a self-replicating machine using a universal constructor. It is through the universal constructor that the actuator emerges as critical. We propose that 3D printing constitutes an analogue of the biological ribosome and that 3D printing may constitute a universal construction mechanism. Following a description of our progress in 3D printing motors, we suggest that this engineering effort can inform biology, that motors are a key facet of living organisms and illustrate the importance of motors in biology viewed from the perspective of engineering (in the Feynman spirit of "what I cannot create, I cannot understand")

Pathways of biomineralization and microstructure evolution in brachiopod shells

Biominerals of shells, bones and teeth are composits of minerals and organic tissue components precipitated by organisms. Accordingly, it is very important to understand (1) the relation between the soft and hard tissues in composite materials of living organisms, (2) the resulting micro- and nanostructure of the constituting biominerals (3) and the function of the minerals of the biomineralization epithelial cells in producing these materials. Brachiopod shells were selected to be the principal subject of this work as they are major geochemical archives for paleo-environmental reconstruction of sea water conditions. The shell of modern brachiopods is secreted by the outer mantle epithelium (OME) of the animal. Despite several decades of research, it is still unknown how the mineral is transported from OME cells to the site of mineralization. For brachiopod shells the biomineralization process was not yet described and often biomineralization of mollusc shells was used as a reference. In order to understand mineral transport and shell secretion, we investigated the ultrastructure of OME cells and their spatial relation to the growing shell for the terebratulide brachiopod Magellania venosa (Chapters 2.1 and 2.2). The animals were chemically fixed and high pressure frozen. We worked with high resolution panorama images formed of up to 350 TEM images. This ensured a general overview as well as a detailed description of the ultrastructure of the OME. We found and described the specific differences between (1) the OME ultrastructure at the commissure and that at central shell regions as well as (2) differences between areas in the central region where active secretion takes place and those areas where secretion is finished. The OME at the commissure consists of several cell layers, while at central shell regions it is single-layered. It is significantly thinner at the central shell region in comparison to the commissure. Especially at sites of actively forming calcite fibres, OME cells are only a few tens of nanometre thin. Where the mineral deposition takes place, the apical membrane of OME cells is in direct contact with the calcite of the forming fibre. At these sites the extracellular organic membrane at the proximal convex surface of the fibre is absent. When mineral secretion is finished the cells form an extracellular organic membrane which lines the proximal surface of fibres. The extracellular organic membrane is attached to the apical cell membrane via apical hemidesmosomes. Tonofilaments cross the cell, connect apical to basal hemidesmosomes, stabilize the contact between epithelium and fibres and keep the mantle attached to the shell. Furthermore, communication and cooperation of neighbouring OME cells could be proved in this work as individual fibres are secreted by several cooperating cells at the same time (Chapters 2.1 and 2.2). The extracellular space, the space between the epithelium and the growing fibres, is either absent or very narrow. Quantitative analysis demonstrated that there are no significant differences in the volume fraction of vesicles between secreting and non-secreting regions of the OME. The latter and the extreme reduction in cell thickness at sites of mineral secretion suggest that for Magellania venosa shells mineral transport to the sites of mineralization does not occur by transport with organelles such as vesicles but via ion transport mechanisms through the cell membrane. For the central shell region the previously discussed data was complemented with atomic force microscopy (AFM) and electron backscatter diffraction (EBSD) measurements. In the central region of the shell the fibrous layer is secreted. The fibrous layer of modern terebratulide brachiopod shells has an overall plywood-like organization with the basic mineral units, the calcite fibres, being assembled with a microstructure resembling an ‘anvil-type’ arrangement (Chapter 2.2). The observations on the TEM images and on etched sample surfaces under AFM lead us to develop a model for calcite fibre secretion and fibre shape formation for Magellania venosa is described as a dynamic process coordinated by outer mantle epithelium cells (OME). The secretion process consists of the following steps: (i) local detachment of epithelial cell membrane from the organic membrane of previously formed fibres, (ii) onset of secretion of calcite at these sites, (iii) organic membrane formation along the proximal, convex side of the forming fibre during achievement of the full width of the fibre, (iv) start of membrane secretion at the corners of fibres progressing towards the centre of the fibre, (v) attachment of the cells via apical hemidesmosomes to newly formed organic membrane, and (vi) suspension of calcite secretion at sites where the proximal, organic membrane of the calcite fibre is fully developed and the apical cell membrane is attached to the latter with apical hemidesmosomes. Thecideide brachiopods are an anomalous group of invertebrates. Their position within the phylogeny of the Brachiopoda and the identification of their origin is still not fully resolved. Studies of morphological features such as shell structure and body size aimed to shed more light on thecideide evolution. However, none of these did provide a definitive answer, possibly because of their complex and diverse evolutionary track. In this thesis (Chapter 2.3) we attempt to trace thecideide shell evolution from a microstructure and texture point of view. We describe for this group of brachiopods the appearance and disappearance of a variety of calcite biocrystals that form the shells and trace these from Late Triassic to Recent times. The results and conclusions are based on EBSD measurements that form the basis of a phylogenetic tree. With this thesis we present a new phylogenetic hypothesis for the evolution of Thecideida. This is the first study that links microstructure and texture results gained from EBSD measurements with phylogenetic analysis and implications derived from phylogenetic evolution. BSD measurements demonstrated the presence of a large variety of mineral units within thecideide shells throughout the geological record. With geologic time there is a progressive loss of the fibrous layer in favour of highly disordered acicular and granular microstructures. This loss can be seen as a paedomorphic pattern in the complex mosaic of evolutionary changes characterizing thecideide brachiopods. The Upper Jurassic species has transitional forms. The shells are composed of stacks of acicles on the external part of the shell. The fibrous layer is kept only in some regions next to the soft tissue of the animal. The regularity of biocrystal shape, mineral unit size, and the strength of calcite co-orientation decreases from the Late Triassic to Recent species. Even though, since the Upper Jurassic the thecideide shell microstructure shows the same type of mineral unit morphologies made of (i) nanometric to small granules, (ii) acicles, (iii) fibres, (iv) polygonal crystals, (v) large roundish crystals. I deduce from my studies that the change in microstructure and texture of thecideide brachiopods may be related to the ecological strategy to exploit distinct niches and life styles, in particular attachment to hard substrates. The clear and well defined microstructure of this brachiopod group is well distinguishable and can help to unravel the phylogenetic relationships between different taxa. Brachiopods are one of the very few marine organism groups which have a complete fossil record. First species appeared in early Cambrian. The end-Permian extinction erased the majority of Paleozoic brachiopod taxa and reset taxonomic, morphological, functional and ecological brachiopod diversity. A few groups survived end-Permian extinction, diversified and occupied new ecological niches. Representatives of these form today the extant orders of the Lingulida, Craniida, Rhynchonellida, and Terebratulida. The Thecideida appeared after the end-Permian crisis, in the Triassic. The geological record shows that brachiopods were and are able to adopt to many marine environments. Accordingly, a large diversity in body plans as well as morphological, structural and chemical features of their shell became developed. With this thesis I highlight structural features of the shells of selected terebratulide, rhynchonellide, thecideide and craniide taxa. Chapter 2.4 describes the difference in shell structure for brachiopods with different life-styles, highlights the distinctness between the structure of the primary shell layer of Terebratulida, Rhynchonellida and the shell structure of Thecideida. I detail the nanometer scale calcite organization of Rhynchonellide and Terebratulide fibers, describe some advantages of a hierarchical composite hard tissue, address possible determinants for primary, fibrous and columnar shell calcite of Terebratullida and discuss a possible usage of thecideide shell for paleoenvironment reconstruction

From visible light to X-ray microscopy: major steps in the evolution of developmental models for calcification of invertebrate skeletons

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Deciphering mollusc shell production: the roles of genetic mechanisms through to ecology, aquaculture and biomimetics

Most molluscs possess shells, constructed from a vast array of microstructures and architectures. The fully formed shell is composed of calcite or aragonite. These CaCO3 crystals form complex biocomposites with proteins, which although typically less than 5% of total shell mass, play significant roles in determining shell microstructure. Despite much research effort, large knowledge gaps remain in how molluscs construct and maintain their shells, and how they produce such a great diversity of forms. Here we synthesize results on how shell shape, microstructure, composition and organic content vary among, and within, species in response to numerous biotic and abiotic factors. At the local level, temperature, food supply and predation cues significantly affect shell morphology, whilst salinity has a much stronger influence across latitudes. Moreover, we emphasize how advances in genomic technologies [e.g. restriction site-associated DNA sequencing (RAD-Seq) and epigenetics] allow detailed examinations of whether morphological changes result from phenotypic plasticity or genetic adaptation, or a combination of these. RAD-Seq has already identified single nucleotide polymorphisms associated with temperature and aquaculture practices, whilst epigenetic processes have been shown significantly to modify shell construction to local conditions in, for example, Antarctica and New Zealand. We also synthesize results on the costs of shell construction and explore how these affect energetic trade-offs in animal metabolism. The cellular costs are still debated, with CaCO3 precipitation estimates ranging from 1-2 J/mg to 17-55 J/mg depending on experimental and environmental conditions. However, organic components are more expensive (~29 J/mg) and recent data indicate transmembrane calcium ion transporters can involve considerable costs. This review emphasizes the role that molecular analyses have played in demonstrating multiple evolutionary origins of biomineralization genes. Although these are characterized by lineage-specific proteins and unique combinations of co-opted genes, a small set of protein domains have been identified as a conserved biomineralization tool box. We further highlight the use of sequence data sets in providing candidate genes for in situ localization and protein function studies. The former has elucidated gene expression modularity in mantle tissue, improving understanding of the diversity of shell morphology synthesis. RNA interference (RNAi) and clustered regularly interspersed short palindromic repeats - CRISPR-associated protein 9 (CRISPR-Cas9) experiments have provided proof of concept for use in the functional investigation of mollusc gene sequences, showing for example that Pif (aragonite-binding) protein plays a significant role in structured nacre crystal growth and that the Lsdia1 gene sets shell chirality in Lymnaea stagnalis. Much research has focused on the impacts of ocean acidification on molluscs. Initial studies were predominantly pessimistic for future molluscan biodiversity. However, more sophisticated experiments incorporating selective breeding and multiple generations are identifying subtle effects and that variability within mollusc genomes has potential for adaption to future conditions. Furthermore, we highlight recent historical studies based on museum collections that demonstrate a greater resilience of molluscs to climate change compared with experimental data. The future of mollusc research lies not solely with ecological investigations into biodiversity, and this review synthesizes knowledge across disciplines to understand biomineralization. It spans research ranging from evolution and development, through predictions of biodiversity prospects and future-proofing of aquaculture to identifying new biomimetic opportunities and societal benefits from recycling shell products.FCT: UID/Multi/04326/2019; European Marine Biological Research Infrastructure Cluster-EMBRIC (EU H2020 research and innovation program) 654008; European Union Seventh Framework Programme [FP7] ITN project 'CACHE: Calcium in a Changing Environment' under REA 60505; NERC Natural Environment Research Council NE/J500173/1info:eu-repo/semantics/publishedVersio

From visible light to X-ray microscopy: major steps in the evolution of developmental models for calcification of invertebrate skeletons

The calcareous skeletons built by invertebrate organisms share a paradoxical property. Although growing outside the mineralizing cell layers the crystal-like skeleton units exhibit morphologies and three-dimensional arrangements that imply an efficient link between crystallization process and taxonomy. Almost two centuries of investigation led to a series of developmental models in which biological and physical or chemical influences are variously balanced. Recent innovative methods allow for their re-examination. From control of the overall shape of the shell to photo-spectroscopic evidence at the atomic level, influence of the biological processes on mineral properties may be a widely shared specificity of the calcareous biomineralization mechanism