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 – Clinocardium exhibit strength, stiffness and toughness that surpass even that of the nacre which is the most widely mimicked model for structural composites. The superior mechanical properties of Clinocardium 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 combing strength and toughness at low densities and capability for seamless integration with biological systems. Here, we demonstrate manufacturing of the cross-lamella design by biomineralizing aragonite films with saw-tooth patterns and assembling them in a chitosan/fibroin matrix to generate a composite with interlocked mineral layers. The resultant composite —with a similar composition to the biological counterpart, nearly doubles the strength of previous nacre-mimetic composites while improving the tensile toughness and simultaneously exhibiting stiffness and biocompatibility.
A dramatic transformation is necessary to reach a sustainable society revolving around the control and use of biological materials and designs. This biomaterial age ushers a completely new technological paradigm favoring the development of circular economic models and sustainable societies.
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 presents 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.
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.erein, 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
Manipulating microorganisms with inherent motility is a challenging yet significant aim with implications in many biological, environmental, and technological applications. Many microorganisms that are broadly available in nature can be used as self‐powered systems that can be directed with external stimuli. Paramecium is a unicellular protozoan that exhibits a negative galvanotaxis where the cell follows the direction of weak electric fields. Here, the galvanotactic behavior of Paramecia is studied to achieve the precise manipulation of these organisms. Using a specially devised microfluidic chip and computer vision, unprecedented levels of manipulation and isolation of Paramecia are demonstrated, enabling their integration, use, and study in micro‐electromechanical systems.
The mechanics of the extracellular matrix (ECM) have long been known to have important implications for cancer metastasis and cell migration. An atypical increase in tumor ECM stiffness occurs because of the heightened deposition of ECM proteins and increased crosslinking density of fibrillar collagen. This tissue stiffening is an essential contributor to disease progression; however, its precise role remains mostly unidentified. Recent advances in synthetic ECM analogs have enabled the concurrent exploration of the effects of crosslinking density, ligand concentrations, matrix stiffness, and pore sizes on tumor cell invasion. However, this convolution of parameters prevents an understanding of the independent contribution of each separate parameter to tumorigenesis. Here, the use of a precisely adjusted degree of methacryloyl substitution in gelatin‐based hydrogel to capture the heterogeneity in cancer cell behavior in response to matrix stiffness is characterized and demonstrated. The proposed ECM model and biomimetic stiffening mechanism are used to produce complex 3D environments with physiological characteristics and independently tunable stiffness. Two populations of invasive and noninvasive human breast adenocarcinoma are embe dded in these matrices and monitored by computer vision, enabling the reproduction and characterization of distinct cell migratory patterns as a result of differences in matrix stiffness and cell metastatic potential.
Embryonic stem cells act as a valuable and promising resource in the field of regenerative cell-based therapies and in studying various developmental models. These derived cells can be induced in vitro to differentiate into numerous distinct cell lineages by the formation of Embryoid Bodies (EBs), in a process strongly conditioned by the geometrical characteristics of the EB. This artificial conditioning of cellular differentiation is performed using tools prioritizing geometrical definition but lacking versatility for the adaptation of the geometry to different conditions. Here we demonstrate the production of cardiomyocytes by using high definition direct writing technologies to influence EB aggregation from stem cells. We gauged the impact of this technology over the standard known methods in terms of size dispersion, cell packing density, cardiac tissue health, and the number of cardiomyocytes produced. The feasibility to create small variations of a geometry enabled optimizing EB formation for cardiogenesis and its threefold increase respect traditional techniques. This result highlights how the fast-paced improvement of geometrical control in additive manufacture might hold the key to unprecedented control of stem cell differentiation for regenerative medicine.
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.
The purpose of the research work, an overview thereof presented here, is to create a new digital manufacturing technology with an emphasis on its sustainability characteristics. We designed a family of natural composite materials comprised of exclusively renewable, widely available, biodegradable, and low-cost components. Their physical and mechanical properties closely resemble those of high-density synthetic foams and low-density natural timbers. They are produced without inclusion of petrochemical products or harmful solvents and adhesives, often associated with adverse human and environmental effects. We designed a material extrusion system based on additive manufacturing principles similar to the Fused Deposition Modeling and the Direct Ink Writing methods. The mechanical system used is comprised of a mobile industrial robotic unit, a viscous liquid transport, and dispensing sub-system and programmable control logic. We performed extensive modeling and testing of material properties with the objective of tightly integrating material behavior with manufacturing. We developed design software for direct transition from design to production, including support scaffold generation for accelerated curing by evaporation and predictive models for process parameter control. To address the challenge of scale, we approached the fabrication process from a hybrid perspective including additive, net-zero-change, and subtractive operations. Early proof of concept demonstrators offers encouraging results towards manufacturing with two of the most abundant and widely distributed natural materials on earth. We believe that, with persistent effort of controlling the innate variability of natural materials and tighter integration with contemporary fabrication methods through predictive computational modeling, this process has very strong potential for a significant impact on product design, general manufacturing, and the building construction industry.
We present a system for 3D printing large-scale objects us-ing natural bio-composite materials which comprises of a preci-sion extruder mounted on an industrial six-axis robot. This paper highlights work on controlling process settings to print filaments of desired dimensions while constraining the operating point to a region of maximum tensile strength and minimum shrinkage. Response surface models relating the process settings to geomet-ric and physical properties of extruded filaments, are obtained through Face-Centered Central Composite Designed experi-ments. Unlike traditional applications of this technique which identify a fixed operating point, the models are used to uncover dimensions of filaments obtainable within operating boundaries of our system. Process setting predictions are then made through multi-objective optimization of the models. An interesting out-come of this study is the ability to produce filaments of different shrinkage and tensile strength properties, by solely changing pro-cess settings. As a follow up, we identify optimal lateral overlap and inter-layer spacing parameters to define toolpaths to print structures. If unoptimized, the material’s anisotropic shrinkage and non-linear compression characteristics cause severe delami-nation, cross-sectional tapering and warpage. Lastly, we show the linear scalability of the shrinkage model in 3D space which allows for suitable toolpath compensation to improve dimen-sional accuracy of printed artefacts. We believe this first ever study on the parametrization of large-scale additive manufacture technique with bio-composites will serve as reference for future sustainable developments in manufacturing.
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.
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. Here, we demonstrate that the microbial enzyme 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 Shrilk patches composed of a chitosan-fibroin laminate 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.
Chitin is found in abundance in invertebrates, fungi and microalgae, and is the second most prevalent biopolymer in the biosphere next to cellulose. There has been a longstanding belief that vertebrates lack endogenous chitin. Moreover, the targeted inhibition of chitin synthesis is used as a strategy to control invertebrate pests and parasites. A finding, therefore, of the endogenous production of chitin in the vertebrates has broad ranging ramifications in the biological sciences.
We present compelling evidence demonstrating that chitin is endogenously produced in fishes and amphibians, collectively which comprise over half of the vertebrates on the earth. First we report that chitin synthase (CHS) genes are present in the genomes of fishes and amphibians, and show that these genes are actively transcribed. Next, using a sensitive affinity histochemistry assay to detect chitin in situ, we demonstrate that it is found throughout the lumen of the developing zebrafish gut as well as in cell populations within and adjacent to the larval gut, and in scale epithelia of both zebrafish and salmon. We also detected chitin in at least three different cell types in larval salamander appendages. Knockdown of an embryonically expressed zebrafish chitin synthase gene resulted in marked diminution of chitin staining in the developing gut, whereas chitinase treatment of whole zebrafish larvae or scale epithelial sections resulted in concomitant reduction of chitin staining. Finally, using chemical means, we extracted a polysaccharide from the adult scales of salmon that exhibits all the chemical hallmarks of chitin. Our data and analyses demonstrate the existence of endogenous chitin in the vertebrates and suggest that it may serve multiple and hitherto unknown roles in vertebrate biology.
In the Media:
Despite the urgent need for sustainable materials for mass-produced commercial products, and the incredible diversity of naturally biodegradable materials with desired structural properties, the use of regenerated biomaterials in modern engineering remains extremely limited. Chitin is a prime example: although it is responsible for some of the most remarkable mechanical properties exhibited by natural materials, including nacre, insect cuticle, and crustacean shells, and it is the most abundant organic compound on earth after cellulose, it has not been utilized in manufacturing strategies for commercial applications. Here we describe how analysis of differences in the molecular arrangement and mechanical properties of chitosan polymer that result from different processing methods led to development of a scalable manufacturing strategy for production of large three-dimensional (3D) objects of chitosan. This chitosan fabrication method offers a new pathway for large-scale production of fully compostable engineered components with complex forms, and establishes chitosan as a viable bioplastic that could potentially be used in place of existing non-degradable plastics for commercial manufacturing.
Chitin—the second most abundant organic material on earth—is a polysaccharide that combines with proteinaceous materials to form composites that provide the structural backbone of insect cuticles, crustacean exoskeletons, cephalopod shells and covering surfaces of many other living organisms. Although chitin and its related chitosan materials have been used in various industrial and medical applications based on their chemical properties, their unique mechanical functions have not date been leveraged for commercial applications. The use of chitinous materials for structural applications has been limited by our inability to reproduce, or even fully understand, the complex hierarchical designs behind naturally occurring chitin composites. In this article, an example of engineered chitinous materials is used to introduce the reader to the potential value that bioinspired materials offer for engineering of synthetic and biological materials. The nature of chitin and its general characteristics are first reviewed, and examples of chitinous structures are presented that are designed to perform very different functions, such as nacre and the insect cuticle. Investigation of the structural organization of these materials leads to understanding of the principles of natural materials design that are beginning to be harnessed to fabricate biologically-inspired composites for materials engineering with tunable properties that mimic living materials, which might provide useful for environmental challenges, as well as medical applications.
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.
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.
A biocompatible method for general construction of 3D structures by aggregation of micrometric polymeric subunits is presented. Shape-controlled microgels are forced to self-assemble, in a structure similar to a brick wall, in different shapes by limiting their movement onto a surface. Scaffolds with high spatial resolution in the aggregation and composed by the addition of multiple layers are produced.
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).
Polymers with high glass transition temperatures, fluorinated ethylene propylene copolymer (FEP) and poly(ethylene naphthalate) (PEN), have been used in imprint lithography as a protective support layer and as a secondary mould, to imprint superficial structures into a polymer with a lower glass transition temperature, namely poly(methyl methacrylate) (PMMA). As a support layer, FEP replaces fragile silicon based supports for the production of freestanding, structured sheets of PMMA, useful, for example, in biomedical applications where transmittance optical microscopy is required. Secondary PEN moulds, produced by imprinting using silicon-based primary moulds, have been used to transfer sub-micrometer tall structures to a freestanding PMMA sheet. Similarly, hole structures, with different dimensions, have been embossed in both sides of a PMMA sheet simultaneously.
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 sim60 nm tall and sim500 × 500 nm2 to be produced on freestanding sim0.5 mm thick sheets of the polymer when cured at 293 K, and structures sim400 nm tall and 5 × 5 mum2 to be produced when cured at 283 K. Nonstructured chitosan thin sheets (sim200 mum 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- 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 ?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.
A “forced” soft lithography (FSL) technique is described for production of micro- and nanostructures into the surface of polymers at room temperature. The technique can be used with polymer/mould combinations that are unsuitable for conventional soft lithography, and has been used to structure the surface of the Chitosan biopolymer. Theoretical descriptions of the filling of the mould cavities and the possible formation of bubbles in the polymer are given.
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°, 30°, and 45° 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.
The design and method for the production of an all-polymer microfluidic particle sorter, for use in biomedical applications, is described. The sorter is made from biocompatible materials with properties, such as high optical transparency, that make it useful in a biological laboratory. The method of sorting is designed to be gentle on biological species, using a method of guiding the particles towards the filter, and has been successfully used to separate latex beads depending on their diameters. Preliminary qualitative experiments have been able to separate beads of 45 and 90 μm in diameter from a mixture of the two. These dimensions are on the same scale as those of some eukaryotic cells.