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Bonding dissimilar materials has been a persistent challenge for decades. This paper presents a method to modify a stainless steel surface (316 L), routinely used in medical applications to enable the significant adhesion of a biopolymer (silk fibroin). The metallic surface was first covalently grafting with polyacrylamide, to enable a hydrogen bonding compatible surface. The polymerisation was initiated via the irreversible electrochemical reduction of a 4-nitrobenzene diazonium salt (20 mM), in the presence of an acrylamide monomer (1 M) at progressively faster scan rates (0.01 V/s to 1 V/s). Examination of the modified samples by FT-IR was consistent with successful surface modification, via observations of the acrylamide carbonyl (1600-1650 cm

In this study we report the extraction and characterization of sericin using water alone, from two non-mulberry silks - Antheraea assamensis (A. assamensis) and Philosamia ricini (P. ricini) compar...

Silk fibroin (SF) membranes are finding widespread use as biomaterial scaffolds in a range of tissue engineering applications. The control over SF scaffold degradation kinetics is usually driven by the proportion of SF crystalline domains in the formulation, but membranes with a high β-sheet content are brittle and still contain amorphous domains, which are highly susceptible to enzymatic degradation. In this work, photo-cross-linking of SF using a ruthenium-based method, and with the addition of glycerol, was used to generate robust and flexible SF membranes for long-term tissue engineering applications requiring slow degradation of the scaffolds. The resulting mechanical properties, protein secondary structure, and degradation rate were investigated. In addition, the cytocompatibility and versatility of porous micropatterning of SF films were assessed. The photo-cross-linking reduced the enzymatic degradation of SF in vitro without interfering with the β-sheet content of the SF material, while adding glycerol to the composition grants flexibility to the membranes. By combining these methods, the membrane resistance to protease degradation was significantly enhanced compared to either method alone, and the SF mechanical properties were not impaired. We hypothesize that photo-cross-linking protects the SF amorphous regions from enzymatic degradation and complements the natural protection offered by β-sheets in the crystalline region. Overall, this approach presents broad utility in tissue engineering applications that require a long-term degradation profile and mechanical support. Postprint

Sustained, local delivery of the antibiotic ciprofloxacin under different formats from porous silk protein-based memory foam systems was studied. Similarly, protease XIV was incorporated during processing to provide control of the degradation kinetics of the silk materials. In vitro antibiotic release studies combined with degradation assessments were utilized to assess the mechanisms and kinetics of release from the silk materials. The sequestered protease XIV affected the degradation profiles of the silk foams yet did not impact the release kinetics of the ciprofloxacin, which was controlled by solubility and diffusion of the drug. The ability to tune the release of ciprofloxacin between 1 and 200 days, combined with the option to modulate the degradation rate up to 80% in 2 weeks via incorporation of a protease, suggests utility for drug release devices. Further, we anticipate that this approach could also be extended to other medical implant needs and other drugs.

Silk nanofibers have been produced and examined in recent years for a range of advanced biomedical and biotechnological applications. Their fabrication involves the canonical approach of dissolving...

Wet spinning of silkworm silk has the potential to overcome the limitations of the natural spinning process, producing fibers with exceptional mechanical properties. However, the complexity of the extraction and spinning processes have meant that this potential has so far not been realized. The choice of silk processing parameters, including fiber degumming, dissolving, and concentration, are critical in producing a sufficiently viscous dope, while avoiding silk's natural tendency to gel via self-assembly. This study utilized recently developed rapid Bayesian optimization to explore the impact of these variables on dope viscosity. By following the dope preparation conditions recommended by the algorithm, a 13% (w/v) silk dope was produced with a viscosity of 0.46 Pa·s, approximately five times higher than the dope obtained using traditional experimental design. The tensile strength, modulus, and toughness of fibers spun from this dope also improved by a factor of 2.20×, 2.16×, and 2.75×, respectively. These results represent the outcome of just five sets of experimental trials focusing on just dope preparation. Given the number of parameters in the spinning and post spinning processes, the use of Bayesian optimization represents an exciting opportunity to explore the multivariate wet spinning process to unlock the potential to produce wet spun fibers with truly exceptional mechanical properties.

We characterised fibres and papers of microfibrillated silk from Bombyx mori produced by mechanical and enzymatic process. Milling increased the specific surface area of fibres from 1.5 to 8.5 m2/g and that enzymatic pre-treatment increased it further to 16.5 m2/g. These fibrils produced a uniform, significantly strong (tenacity 55 Nm/g) and stiff (Young's modulus > 2 GPa) papers. Enzymatic pre-treatment did not reduce molecular weight and tensile strength of papers but significantly improved fibrillation. Silk remained highly crystalline throughout the fibrillation process. Protease biodegradation was more rapid after fibrillation. Biodegradation was impacted by structural change due to enzymatic pre-treatment during the fibrillation. Biodegraded silk had much higher thermal degradation temperature. The unique combination of high strength, slow yet predicable degradation and controllable wicking properties make the materials ideally suited to biomedical and healthcare applications.

Hydrogel bioprinting is a major area of focus in the field of tissue engineering. However, 3D printed hydrogel scaffolds often suffer from low printing accuracy and poor mechanical properties because of their soft nature and tendency to shrink. This makes it challenging to process them into structural materials. In this study, natural chitosan hydrogel scaffolds were, for the first time, reinforced with milled silk particles and fabricated by 3D printing. Compared with pure chitosan scaffolds, the addition of silk particles resulted in up to a 5-fold increase in compressive modulus as well as significantly better printing accuracy and improved scaffold stability. The chitosan/silk inks flowed well during printing; loading of up to 300% silk (w/w) resulted in only minor changes in the rheological properties of the ink. Particle loading also enabled tuning of the surface roughness of the scaffolds and improved scaffolds' biodegradability. The printed composite hydrogel scaffolds showed no cytotoxicity and supported adherence and growth of human fibroblast cells.

Low-molecular weight (LMW) silk was utilized as a LMW silk plasticizer for regenerated silk, generating weak physical crosslinks between high-molecular weight (HMW) silk chains in the amorphous regions of a mixed solution of HMW/LMW silk. The plasticization effect of LMW silk was investigated using mechanical testing, Raman spectroscopy, and wide-angle X-ray scattering (WAXS). Small amounts (10%) of LMW silk resulted in a 19.4% enhancement in fiber extensibility and 37.8% increase in toughness. The addition of the LMW silk facilitated the movement of HMW silk chains during drawing, resulting in an increase in molecular chain orientation when compared with silk spun from 100% HMW silk solution. The best regenerated silk fibers produced in this work had an orientation factor of 0.94 and crystallinity of 47.82%, close to the values of natural degummedBombyx mori silk fiber. The approach and mechanism elucidated here can facilitate artificial silk systems with enhanced properties.

Silk fibroin is an excellent biopolymer for application in a variety of areas, such as textiles, medicine, composites and as a novel material for additive manufacturing. In this work, silk membranes were surface modified by in situ polymerization of aqueous acrylic acid, initiated by the reduction of various aryldiazonium salts with vitamin C. Treatment times of 20 min gave membranes which possessed increased tensile strength, tensile modulus, and showed significant increased resistance to needle puncture (+131%), relative to &lsquo, untreated&rsquo, standards. Most interestingly, the treated silk membranes were able to be reversibly formed into various shapes via the hydration and plasticizing of the surface bound poly(acrylic acid), by simply steaming the modified membranes. These membranes and their unique properties have potential applications in advanced textiles, and as medical materials.

Silk fiber is formed by an assembly of fibrils. The fibrils can be isolated by a top-down mechanical process called microfibrillation and the fibrils are known as microfibrillated silk (MFS). The process involves chopping, milling, enzyme treatment and high-pressure homogenization. The milling is an important manufacturing step and to optimize the milling step, a response surface methodology was used in this work where the influence of fiber content in milled suspension, milling time and alkaline concentration were investigated. Output responses for the optimization were diameter distribution of fibrils, size and percentage of different diameter fractions, and the aspect ratio. The main and interaction effects of the milling parameters on these responses were statistically analysed. Milling time was the most significant factor for producing finer fibrils while the fiber content in milling had the maximum impact in reducing the number of large fibrils. Milling time had a positive correlation with the aspect ratio. The optimized milling resulted in MFS with an average diameter of 55.35 nm and 90% of MFS less than 100 nm based on high-magnification SEM image analysis. The aspect ratio of the MFS was 137. The MFS suspension was stable over the pH range 3–11.

Silk fibroin is an excellent biopolymer for application in a variety of areas, such as textiles, medicine, composites and as a novel material for additive manufacturing. In this work, silk membranes were surface modified by in situ polymerization of aqueous acrylic acid, initiated by the reduction of various aryldiazonium salts with vitamin C. Treatment times of 20 min gave membranes which possessed increased tensile strength, tensile modulus, and showed significant increased resistance to needle puncture (+131%), relative to ‘untreated’ standards. Most interestingly, the treated silk membranes were able to be reversibly formed into various shapes via the hydration and plasticizing of the surface bound poly(acrylic acid), by simply steaming the modified membranes. These membranes and their unique properties have potential applications in advanced textiles, and as medical materials.

Reported is a fast and versatile protocol to surface modify pre-cast silk membranes targeting tyrosine residues. Enriched alkyne silk membranes were prepared using this method and azides possessing a range of functional groups were tethered to the membrane surface using click chemistry to give a range of water contact angles from 85 ± 3° to 34 ± 6°.

Two-photon lithography (2 PL) has been proposed as an additive manufacturing method to fabricate structures at the microscale, including curing protein-based bioinks. In this study, we explored the use of 2 PL for biomedical applications to photo-cross-link silk fibroin solution into a solid hydrogel with directly tuneable physico-chemical properties. 2 PL allowed control of the cross-linking degree and the porosity of the resulting hydrogel at the micrometric scale by regulating the intensity of the IR laser exposure, tested between 800 and 2400 mW, during the 3D printing process. The regulation of the cross-linking degree in this way allowed for the production of hydrogels with significantly different degradation rates, ranging from 0.5 to 2 h during accelerated enzymatic degradation assay, while their β-sheet content, assessed by Fourier-transform infra-red spectroscopy (FTIR), did not show significant changes. The hydrogels Young’s modulus, assessed by quantitative microelastography, could also be controlled within a 9.6 to 47.2 kPa range and was found to increase with the rate of cross-linking. When human dermal fibroblasts were included in the silk fibroin solution for bioprinting testing at different laser exposures, cell viability was assessed over 95% during 21 days of in vitro culture. Moreover, cells showed a higher proliferation rate in less cross-linked hydrogels and were able to align and migrate to areas of increased stiffness printed in gradient hydrogels. Overall, this versatile additive manufacturing method represents an advancement of silk fibroin biomaterials for soft tissue engineering, enabling control of the microstructure and degradation rate of the products and the proliferation of the engrafted cells.

Objective To evaluate the recent developments in optical coherence tomography (OCT) for tympanic membrane (TM) and middle ear (ME) imaging and to identify what further development is required for the technology to be integrated into common clinical use. Data Sources PubMed, Embase, Google Scholar, Scopus, and Web of Science. Review Methods A comprehensive literature search was performed for English language articles published from January 1966 to January 2018 with the keywords "tympanic membrane or middle ear,""optical coherence tomography," and "imaging." Conclusion Conventional imaging techniques cannot adequately resolve the microscale features of TM and ME, sometimes necessitating diagnostic exploratory surgery in challenging otologic pathology. As a high-resolution noninvasive imaging technique, OCT offers promise as a diagnostic aid for otologic conditions, such as otitis media, cholesteatoma, and conductive hearing loss. Using OCT vibrometry to image the nanoscale vibrations of the TM and ME as they conduct acoustic waves may detect the location of ossicular chain dysfunction and differentiate between stapes fixation and incus-stapes discontinuity. The capacity of OCT to image depth and thickness at high resolution allows 3-dimensional volumetric reconstruction of the ME and has potential use for reconstructive tympanoplasty planning and the follow-up of ossicular prostheses. Implications for Practice To achieve common clinical use beyond these initial discoveries, future in vivo imaging devices must feature low-cost probe or endoscopic designs and faster imaging speeds and demonstrate superior diagnostic utility to computed tomography and magnetic resonance imaging. While such technology has been available for OCT, its translation requires focused development through a close collaboration between engineers and clinicians.

This study aimed to sequence and identify a glycosyl hydrolase family 9 (GHF9) endo-β-1,4-glucanase expressed in the midgut gland of the herbivorous gecarcinid land crab, Gecarcoidea natalis. Hence this would explain the gene responsible for the production of previously purified and characterised endo-β-1,4-glucanases. Three different transcripts, two complete and one partial were sequenced from cDNA and an open reading frame of 1383bp was produced. Translated, this would produce a putative protein of 460 amino acid residues, including a 16 amino acid residue signal peptide. The mature protein (without signal peptide) is predicted to have a molecular mass of 47.6-47.7kDa; this closely matches the molecular mass (47.4kDa) of one of the three endo-β-1,4-glucanase/lichenase enzymes purified previously from G. natalis. It is therefore proposed that the gene described here encodes one of the previously characterised enzymes. The presence of multiple transcripts suggests gene duplication. To confirm that the gene is widely expressed within the Crustacea, cDNA encoding a GHF9 endo-β-1,4-glucanase was also sequenced in diverse crustaceans, the deposit feeding soldier crab, Mictyris platycheles and the terrestrial hermit crabs, Coenobita purlatus and C. brevimanus. An open reading frame of 1356bp was sequence from M. platycheles, while an incomplete open reading frames of 1384 and 1523bp were respectively sequenced from Coenobita brevimanus and C. perlatus. The midgut gland of M. platycheles contained activity (0.704±0.218μmol reducing sugars produced. min-1·mg-1 tissue wet weight) of a 26.3±0.3(5) endo-β-1,4-glucanase isozyme (determined from activity staining). These species, particularly M. platycheles does not consume and digest significant amounts of plant cellulose. This implies that the ancestral enzyme is not a cellulase, but rather it may be involved in hydrolysing cellulose like polysaccharides within other organisms such as algae.

Regenerated silk fibers typically fall short of silkworm cocoon fibers in mechanical properties due to reduced fiber crystal structure and alignment. One approach to address this has been to employ inorganic materials as reinforcing agents. The present study avoids the need for synthetic additives, demonstrating the first use of exfoliated silk nanofibers to control silk solution crystallization, resulting in all-silk pseudocomposite fibers with remarkable mechanical properties. Incorporating only 0.06 wt% silk nanofibers led to a ≈44% increase in tensile strength (over 600 MPa) and ≈33% increase in toughness (over 200 kJ kg

Organic powders are polymers with organic origin in a powdered form. They are biocompatible, biodegradable, and possess positive biological attributes, and can form constructs with high mechanical properties due to their powder form. However, there are various constraints that limit organic powders to be solely used with current 3D printers. Many organic powders cannot be fused by heat and light exposure and not easy to dissolve by the printing ink. Moreover, binding solutions for silk and other organic powders are mostly acidic in nature which cause damage to current 3D powder printers as they use neutral or slight low pH inks. This work aims to address this problem by developing a 3D printer that enables the printing of an organic powder, silk. This paper presents the design and implementation of the first prototype of a novel organic powder printer. The printer incorporates a novel compression mechanism that enables the compression of the powder during the printing process. It also includes a non-corrosive binder supply system consisting of silicone peroxide tubing, a custom-built peristaltic pump, and a nozzle built from a 33-gauge needle. The powder management system consists of a powder canister apparatus, depositing mechanism, and a powder bed designed to be compatible with low flowability powders, such as silk powder. The implementation of each component and the whole printer is presented. Evaluating the functionality of the organic powder printer found that several silk powder scaffolds were able to be printed with a varying magnitude of control over architecture. Future work is required to further advance the printable constructs up to a standard found in commercial 3D printers. This work demonstrated that a 3D printer system can be developed to fabricate constructs solely out of an organic powder. The paper highlights the limitations of the current design and suggests future improvements.

Regenerated silk fibroin membranes tend to be brittle when dry. The use of plasticisers such as glycerol improve membrane ductility, but, when combined with aqueous processing, can lead to a higher degradation rate than solvent-annealed membranes. This study investigated the use of formic acid as the solvent with glycerol to make deformable yet degradation-resistant silk membranes. Here we show that membranes cast using formic acid had low light scattering, with a diffuse transmittance of less than 5% over the visible wavelengths, significantly lower than the 20% transmittance of aqueous derived silk/glycerol membranes. They had 64% β-sheet content and lost just 30% of the initial silk weight over 6h when tested with an accelerated enzymatic degradation assay, in comparison the aqueous membranes completely degraded within this timeframe. The addition of glycerol also improved the maximum elongation of formic acid derived membranes from under 3% to over 100%. They also showed good cytocompatibility and supported the adhesion and migration of human tympanic membrane keratinocytes. Formic acid based, silk/glycerol membranes may be of great use in medical applications such as repair of tympanic membrane perforation or ocular applications where transparency and resistance to enzymatic degradation are important.

This study describes for the first time the development of a silk powder-based 3D printing formulation that is compatible with Binder Jetting, a commercial additive manufacturing (AM) technique. The dynamic and bulk properties of the precursor powder were measured, including particle sizing, shape, flow energy, and compressibility, and the relationships between these properties, particle flow and printability were investigated. We used two different types of silk powder, super fine silk powder (SFSP) with an average particle size of 5 µm and fine silk powder (FSP), average particle size of 20 µm, and found that FSP provided good flow, spreadability and printability with polyvinyl alcohol (PVA) used as the solid binder. An optimized SP/PVA powder formulation was developed and successfully printed into intricate structures with a resolution as high as 200 µm. The printed green samples were analysed thoroughly to determine the printing performance, resolution, porosity, and mechanical strength. The compressive modulus of the printed SP/PVA parts reached 3 MPa, which was comparable to that of some ceramic printed parts. An effective infusing and immersion post-crosslinking method was developed and found to enhance the water stability of the printed constructs, making the printed parts suitable for potential load-bearing biomedical applications.

Polycaprolactone (PCL) scaffolds have been widely investigated for tissue engineering applications, however, they exhibit poor cell adhesion and mechanical properties. Subsequently, PCL composites have been produced to improve the material properties. This study utilises a natural material, Bombyx mori silk microparticles (SMP) prepared by milling silk fibre, to produce a composite to enhance the scaffolds properties. Silk is biocompatible and biodegradable with excellent mechanical properties. However, there are no studies using SMPs as a reinforcing agent in a 3D printed thermoplastic polymer scaffold. PCL/SMP (10, 20, 30 wt%) composites were prepared by melt blending. Rheological analysis showed that SMP loading increased the shear thinning and storage modulus of the material. Scaffolds were fabricated using a screw-assisted extrusion-based additive manufacturing system. Scanning electron microscopy and X-ray microtomography was used to determine scaffold morphology. The scaffolds had high interconnectivity with regular printed fibres and pore morphologies within the designed parameters. Compressive mechanical testing showed that the addition of SMP significantly improved the compressive Young's modulus of the scaffolds. The scaffolds were more hydrophobic with the inclusion of SMP which was linked to a decrease in total protein adsorption. Cell behaviour was assessed using human adipose derived mesenchymal stem cells. A cytotoxic effect was observed at higher particle loading (30 wt%) after 7 days of culture. By day 21, 10 wt% loading showed significantly higher cell metabolic activity and proliferation, high cell viability, and cell migration throughout the scaffold. Calcium mineral deposition was observed on the scaffolds during cell culture. Large calcium mineral deposits were observed at 30 wt% and smaller calcium deposits were observed at 10 wt%. This study demonstrates that SMPs incorporated into a PCL scaffold provided effective mechanical reinforcement, improved the rate of degradation, and increased cell proliferation, demonstrating potential suitability for bone tissue engineering applications.

Silver nanoparticles (AgNPs) are in situ synthesized for the first time on microfibrillated silk (MFS) exfoliated from domesticated Philosamia cynthia ricini (eri) and Bombyx mori (mulberry) silkworm silk fibers. The process is rapid (hours time), does not rely on harmful chemicals, and produces robust and flexible AgNPs coated MFS (MFS-AgNPs) protein papers with excellent handling properties. None of these can be achieved by approaches used in the past to fabricate AgNPs silk systems. MFS bonds the AgNPs strongly, providing good support and stabilization for the NPs, leading to strong wash fastness. The mechanical properties of the MFS-AgNPs papers largely do not change compared to the MFS papers without nanoparticles, except for some higher concentration of AgNPs in the case of mulberry silk. The improved tensile properties of eri silk papers with or without AgNPs compared to mulberry silk papers can be attributed to the higher degree of fibrillation achieved in eri silk and its inherent higher ductility. MFS-AgNPs from eri silk also exhibit strong antibacterial activity. This study provides the basis for the development of smart protein papers based on silk fiber and functional nanomaterials.

Silk, with highly crystalline structure and well-documented biocompatibility, is promising to be used as reinforcing material and build functionalized composite scaffolds. In the present study, we developed chitosan/silk composite scaffolds using silk particles, silk microfibres and nanofibres via 3D printing method. The three forms of silk fillers with varied shapes and dimensions were obtained via different processing methods and evaluated of their morphology, crystalline structure and thermal property. All silk fillers showed different degrees of improvement on printability in terms of ink rheology and printing shape fidelity. Different silk fillers led to different scaffold surface morphology and different roughness, while all reduced the contact angle compared to pure chitosan. Similar reinforcements were observed on compressive modulus, while oscillatory gel strength reinforcement was found to be positively correlated to the filler aspect ratio. Addition of silk introduced no cytotoxicity for that all scaffolds supported a steady cell growth using human fibroblasts. Meanwhile different cellular behaviours were observed on different scaffold surfaces, which can possibly intriguer specific application on soft tissue engineering.

The degumming process to remove sericin decreases silk fiber strength; however, the impact of degumming on the mechanical properties of regenerated silk biomaterials has not been established. This study investigated the effect of degumming temperature, time, alkaline component and alkaline concentration on the mechanical properties of silk fibroin films. Sericin removal was estimated using weight loss; 10 samples with 12.2–29.4% weight loss were then further characterized in terms of fiber mechanical properties, fiber surface morphology, molecular weight distribution and film tensile strength. A negative correlation was found between weight loss and fiber tensile strength. This loss of fiber strength under harsher degumming conditions had a direct impact on the tensile strength of regenerated films. Mild degumming conditions (weight loss of 12.2%) led to higher film strength (8.9 MPa), whereas aggressive degumming conditions (with 29.4% weight loss) resulted in significantly weaker films (4.3 MPa). The presence of some residual sericin, after mild degumming, is likely to affect the mechanical properties of the regenerated silk films. These results will assist in the development of materials with mechanical and biocompatibility properties tuned to specific biomedical applications.

3D printing technologies have enabled advances in biomedical research and development, including rapid fabrication of complex, customised constructs for personalised treatment of patients. However, there are still limitations with the 3D printing technologies in relation to the diversity of biomaterials, and the versatility of the printing methods to print such diverse materials. While many materials have been used for biomedical applications involving high load bearing forces, e.g. ceramics and metals, they struggle in areas of biodegradability, and supporting tissue adhesion. An alternative are natural polymers, which are currently being investigated in the biomedical industry and are generally biocompatible, biodegradable and bioactive. However, they lack optimal mechanical properties necessary for high load bearing applications. Natural polymers in a powdered form, also referred to as organic powders, can allow for much denser and higher integrity constructs compared to their more common bioink counterparts, whilst maintaining many of the desired biological attributes. However, many organic powders are either do not respond to light and degrade in heat, preventing their use in many light and thermal based 3D printing processes. Powder inkjet printing is a 3D printing technology that utilizes an alternative means of solidifying the powdered material. These include physical or chemical bonding, instead of photopolymerisation, melting or sintering via the exposure of heat or light. Nevertheless, the binders or solutions that are commonly used with organic powders are high in acidity which can damage the printer components, further limiting this materials printability. This paper investigates the current 3D printing technologies that can print with biomaterials. Several elements are investigated, including current biomedical applications, fusion technology, material limitations and useable biomaterials. In addition to the printing processes, a study on available biomaterials, in particular organic powders, is elaborately discussed. The origins of the biomaterials are explored, in addition to the acceptable 3D printers, current applications, and printable forms. The limitations found with the current available printing processes is also discussed, in relation to their compatibility with organic powders, and their required binder solutions.

The acoustic and mechanical properties of silk membranes of different thicknesses were tested to determine their suitability as a repair material for tympanic membrane perforations. Membranes of different thickness (10-100μm) were tested to determine their frequency response and their resistance to pressure loads in a simulated ear canal model. Their mechanical rigidity to pressure loads was confirmed by tensile testing. These membranes were tested alongside animal cartilage, currently the strongest available myringoplasty graft as well as paper, which is commonly used for simpler procedures. Silk membranes showed resonant frequencies within the human hearing range and a higher vibrational amplitude than cartilage, suggesting that silk may offer good acoustic energy transfer characteristics. Silk membranes were also highly resistant to simulated pressure changes in the middle ear, suggesting they can resist retraction, a common cause of graft failure resulting from chronic negative pressures in the middle ear. Part of this strength can be explained by the substantially higher modulus of silk films compared with cartilage. This allows for the production of films that are much thinner than cartilage, with superior acoustic properties, but that still provide the same level of mechanical support as thicker cartilage. Together, these in vitro results suggest that silk membranes may provide good hearing outcomes while offering similar levels of mechanical support to the reconstructed middle ear.

SUMMARY A 160 kDa enzyme with β-glucosidase activity was purified from the midgut gland of the land crab Gecarcoidea natalis. The enzyme was capable of releasing glucose progressively from cellobiose, cellotriose or cellotetraose. Although β-glucosidases (EC 3.2.1.21) have some activity towards substrates longer than cellobiose, the enzyme was classified as a glucohydrolase (EC 3.2.1.74) as it had a preference for larger substrates (cellobiose

Terrestrial decapods consume a wide variety of plant and animal material. The potential adaptations of carnivorous, omnivorous, and herbivorous terrestrial crustaceans were studied by examining the functional morphology of the gastric mill. Two closely related species from each feeding preference group were examined to identify which features of the mill were due to phylogeny and which were due to adaptation. The morphology of the gastric mill matched the diet well; the gastric mills of the carnivorous species (Geograpsus grayi and Geograpsus crinipes) possessed a blunt, rounded medial tooth and flattened lateral teeth with a longitudinal grinding groove. These features make them well suited to a carnivorous diet of soft animal tissue as well as hard material, such as arthropod exoskeleton. In contrast, the mill of the herbivorous gecarcinids (Gecarcoidea natalis and Discoplax hirtipes) consisted of a medial tooth with sharp transverse ridges and lateral teeth with sharp interlocking cusps and ridges and no grinding surface. These features would efficiently shred fibrous plant material. The morphology of the mill of the omnivorous coenobitids (Coenobita perlatus and Birgus latro) was more generalized toward a mixed diet. However, the mill of B. latro was more adapted to deal with highly nutritious food items, such as nuts and heavily calcified decapods. Its mill possessed lateral teeth with extended ridges, which sat close to the calcified cardiopyloric valve to form a flattened floor. Hard items trapped in the mill would be crushed against this surface by the medial tooth.

To identify the gene responsible for the production of a β-1,3-glucanase (laminarinase) within crustacea, a glycosyl hydrolase family 16 (GHF16) gene was sequenced from the midgut glands of the gecarcinid land crab, Gecarcoidea natalis and the freshwater crayfish, Cherax destructor. An open reading frame of 1098 bp for G. natalis and 1095 bp for C. destructor was sequenced from cDNA. For G. natalis and C. destructor respectively, this encoded putative proteins of 365 and 364 amino acids with molecular masses of 41.4 and 41.5 kDa. mRNA for an identical GHF16 protein was also expressed in the haemolymph of C. destructor. These putative proteins contained binding and catalytic domains that are characteristic of a β-1,3-glucanase from glycosyl hydrolase family 16. The amino acid sequences of two short 8–9 amino acid residue peptides from a previously purified β-1,3-glucanase from G. natalis matched exactly that of the putative protein sequence. This plus the molecular masses of the putative proteins matching that of the purified proteins strongly suggests that the sequences obtained encode for a catalytically active β-1,3-glucanase. A glycosyl hydrolase family 16 cDNA was also partially sequenced from the midgut glands of other amphibious (Mictyris platycheles and Paragrapsus laevis) and terrestrial decapod species (Coenobita rugosus, Coenobita perlatus, Coenobita brevimanus and Birgus latro) to confirm that the gene is widely expressed within this group. There are three possible hypothesised functions and thus evolutionary routes for the β-1,3-glucanase: 1) a digestive enzyme which hydrolyses β-1,3-glucans, 2) an enzyme which cleaves β-1,3-glycosidic bonds within cell walls to release cell contents or 3) an immune protein which can hydrolyse the cell walls of potentially pathogenic micro-organisms.

Both Engaeus sericatus and Cherax destructor are omnivorous crayfishes consuming a variety of food items. Materials identified in the faeces of both E. sericatus and C. destructor consisted of mainly plant material with minor amounts of arthropod animals, algae and fungi. The morphology of the gastric mill of C. destructor suggests that it is mainly involved in crushing of food material while the gastric mill of E. sericatus appears to be better suited to cutting of food material. Given this, the gastric mill of E. sericatus may be better able to cut the cellulose and hemicellulose fibres associated with fibrous plant material. In contrast, the gastric mill of C. destructor appears to be more efficient in grinding soft materials such as animal protein and algae. Both species accumulated high amounts of lipids in their midgut glands (about 60% of the dry mass) which were dominated by triacylglycerols (81-82% of total lipids). The dominating fatty acids were 16:0, 16:1(n-7), 18:1(n-9), 18:2(n-6), and 18:3(n-3). The two latter fatty acids can only be synthesised by plants, and are thus indicative of the consumption of terrestrial plants by the crayfishes. The similarity analysis of the fatty acid patterns showed three distinct clusters of plants and each of the crayfish species. The complement of digestive enzymes, proteinases, total cellulase, endo-beta-1,4-glucanase, beta-glucosidase, laminarinase and xylanase within midgut gland suggests that both C. destructor and E. sericatus are capable of hydrolysing a variety of substrates associated with an omnivorous diet. Higher activities of total cellulase, endo-beta-1,4-glucanase and beta-glucosidase indicate that E. sericatus is better able to hydrolyse cellulose within plant material than C. destructor. In contrast to E. sericatus, higher total protease and N-acetyl-beta-D-glucosaminidase activity in the midgut gland of C. destructor suggests that this species is better able to digest animal materials in the form of arthropods. Differences in total cellulase and gastric mill morphology suggest that E. sericatus is more efficient at digesting plant material than C. destructor. However, the contents of faecal pellets and the fatty acid compositions seem to indicate that both species opportunistically feed on the most abundant and easily accessible food items.

SUMMARY Laminarinase and endo-β-1,4-glucanase were purified and characterised from the midgut gland of the herbivorous land crab Gecarcoidea natalis and the crayfish Cherax destructor. The laminarinase isolated from G. natalis was estimated to have a molecular mass of 41 kDa by SDS-PAGE and 71 kDa by gel filtration chromatography. A similar discrepancy was noted for C. destructor. Possible reasons for this are discussed. Laminarinase (EC 3.2.1.6) from G. natalis had a Vmax of 42.0 μmol reducing sugars produced min–1 mg protein–1, a Kmof 0.126% (w/v) and an optimum pH range of 5.5–7, and hydrolysed mainlyβ-1,3-glycosidic bonds. In addition to the hydrolysis ofβ-1,3-glycosidic bonds, laminarinase (EC 3.2.1.39) from C. destructor was capable of significant hydrolysis of β-1,4-glycosidic bonds. It had a Vmax of 19.6 μmol reducing sugars produced min–1 mg protein–1, a Km of 0.059% (w/v) and an optimum pH of 5.5. Laminarinase from both species produced glucose and other short oligomers from the hydrolysis of laminarin. Endo-β-1,4-glucanase (EC 3.2.1.4) from G. natalis had a molecular mass of 52 kDa and an optimum pH of 4–7. It mainly hydrolysed β-1,4-glycosidic bonds, but was also capable of significant hydrolysis of β-1,3-glycosidic bonds. Two endo-β-1,4-glucanases, termed 1 and 2, with respective molecular masses of 53±3 and 52 kDa, were purified from C. destructor. Endo-β-1,4-glucanase 1 was only capable of hydrolysingβ-1,4-glycosidic bonds and had an optimum pH of 5.5. Endo-β-1,4-glucanases from both species produced some glucose, cellobiose and other short oligomers from the hydrolysis of carboxymethyl cellulose.

This study investigated synergism between endo-β-1,4-glucanase and β-glucohydrolase enzymes from Gecarcoidea natalis. Together, these enzymes efficiently hydrolyse the cellulose-like polymer, carboxymethyl cellulose, to glucose. Endo-β-1,4-glucanase and β-glucohydrolase, isolated previously from G. natalis, were incubated in vitro using a ratio of the measured activities that matches that found in their digestive juice (5.4 : 1). Their combined activity, measured as the release of glucose from carboxymethyl cellulose, was greater than the sum of their separate activities. Hence they synergistically released glucose from carboxymethyl cellulose (degree of synergy: 1.27). This may be due to the complementary nature of the products of endo-β-1,4-glucanase activity and the preferred substrates of the β-glucohydrolase. β-glucohydrolase may also enhance cellulose hydrolysis by removing cellobiose, a potential competitive inhibitor of endo-β-1,4-glucanase. The synergistic interaction of these two enzymes further supports the previous suggestion that this species possesses a novel two-enzyme cellulase system that differs from the traditional three-enzyme fungal model.

This article reviews the current knowledge of cellulose and hemicellulose digestion by herbivorous land crabs using the gecarcinid Gecarcoidea natalis as a model species for this group. Cellulose digestion in the gecarcinids is hypothesised to require mechanical fragmentation and enzymatic hydrolysis. Mechanical fragmentation is achieved by the chelae, mandibles and gastric mill, which reduce the material to particles less than 53 µm. The gastric mill shows adaptations towards a plant diet; in particular, there are transverse ridges on the medial and lateral teeth and ventral cusps on the lateral teeth that complement and interlock to provide efficient cutting surfaces. Enzymatic hydrolysis of cellulose and hemicellulose is achieved through cellulase and hemicellulase enzymes. In the gecarcinids, 2–3 endo-β-1,4-glucanases, one β-glucohydrolase and a laminarinase have been identified. The endo-β-1,4-glucanases are multifunctional, with both endo-β-1,4-glucanase and lichenase activity. Complete cellulose hydrolysis is achieved through the synergistic action of the endo-β-1,4-glucanase and β-glucohydrolase. The evidence for the endogenous production of the cellulase and hemicellulase enzymes, their evolutionary origin and possible evolution in invertebrates as they colonised land is also discussed.