Abstract:

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.

Abstract:

We present research work on a manufacturing process deploying natural composite materials. The objective of the project is to create a sustainable manufacturing process integrating materials, hardware, software and fabrication logic from the ground up. We deploy a bioinspired natural composite comprised by renewable, widely available, biodegradable and low-cost natural components. Material properties closely resemble those of high-density foams or low-density timbers and it is produced without any petrochemical or harmful solvents associated with adverse environmental effects. We designed a mobile material deposition system using the Direct Ink Writing method, with work envelope of over 3 m vertically and indefinite horizontal range, comprised of industrial robotic hardware and purpose-built mechanical mobile platform. We performed testing in characterizing material properties with and without the introduction of the printing process, tightly integrated material behavior with manufacturing and developed design software for direct transition from design to production. To address scaling, we approached the fabrication process from the perspective of fusing the best principles from both additive and subtractive manufacturing, offering geometric freedom and material efficiency of additive manufacturing while targeting production and quality efficiencies of subtractive and forming processes. We believe this process has the potential of significant impact on general manufacturing as well as the building industry.

Abstract:

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.

Abstract:

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:

• Environmental Impact: Redefining Our Understanding (Video)
  Wyss Institute, 2015
• Surprise Finding Heightens Concern Over Tiny Bits Of Plastic Polluting Our Oceans (Article)
  The Huffington Post, 2015

Abstract:

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.

 In the Media:

• Discovery Channel: Can We Make Plastic from Shrimp? (Article)
  Carin Bondar, 2014
• Science/AAAS: Making Plastic Out of Shrimp Shells (Article)
  Kristen Kusek, 2014
• Huffington Post: Nature Has A Promising Replacement For Plastics (Article)
  Lynne Peeples, 2014
• Business Insider: Biodegradable Plastic (Article)
  Douglas Main, 2014

Abstract:

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.

Abstract:

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.

In the Media:

• The Guardian: Five wonder materials that could change the world(Article)
  Ian Sample, 2014
• Scientific American: 9 Materials That Will Change Manufacturing (Article)
  Steven Ashley and Larry Greenemeier, 2013
• National Geographic Magazine: Super Materials (Article)
  Ann Williams, 2013
• The Daily Mail: Shrilk’ is a lab-grown version of insect armour which could replace plastics (Article)
  Rob Waugh, 2011
• Science Daily: Inspired by Insect Cuticle, Scientists Develop Material That’s Tough and Strong (Article)
  Dan Hogan, 2011
• Fast Company: Insect-Inspired Material That Could Solve Our Plastic Problem (Article)
  Nidhi Subbaraman, 2011
• Scientific American: Insect Cuticle Inspires New Material (Article)
  Cynthia Graber, 2011
• PhysOrg: ‘Shrilk’: Inspired by insect cuticle, researchers develop low-cost material with exceptional strength” (Article)
  John Benson, 2011
• Science Business: Natural Lightweight Material Exhibits Strength, Toughness (Article)
  Alan Kotok, 2011

Abstract:

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.

Abstract:

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.

In the Media:

• CNET: Breakthrough in tissue engineering: ‘Bio-Legos’” (Article)
  Elizabeth Armstrong Moore, 2010
•  New Scientist: ‘Human Lego’ may one day build artificial organs (Article)
  Wendy Wolfson, 2010
•  The New York Times: Micromasonry & Biological Lego (Article)
  Ben Schott, 2010
•  MIT News: Building organs block by block (Article)
  Anne Trafton, 2010

Abstract:

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).

Abstract:

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.

Abstract:

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.

Abstract:

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.

Abstract:

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.

Abstract:

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.

 Abstract:

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.