Engineered Surfaces That Promote Capture of Latent Proteins to Facilitate Integrin-Mediated Mechanical Activation of Growth Factors.

Conventional osteogenic platforms utilize active growth factors to repair bone defects that are extensive in size, but they can adversely affect patient health. Here, an unconventional osteogenic platform is reported that functions by promoting capture of inactive osteogenic growth factor molecules to the site of cell growth for subsequent integrin-mediated activation, using a recombinant fragment of latent transforming growth factor beta-binding protein-1 (rLTBP1). It is shown that rLTBP1 binds to the growth-factor- and integrin-binding domains of fibronectin on poly(ethyl acrylate) surfaces, which immobilizes rLTBP1 and promotes the binding of latency associated peptide (LAP), within which inactive transforming growth factor beta 1 (TGF-ß1) is bound. rLTBP1 facilitates the interaction of LAP with integrin ß1 and the subsequent mechanically driven release of TGF-ß1 to stimulate canonical TGF-ß1 signaling, activating osteogenic marker expression in vitro and complete regeneration of a critical-sized bone defect in vivo.

Investigating multi-material hydrogel three-dimensional printing for in vitro representation of the neo-vasculature of solid tumours: a comprehensive mechanical analysis and assessment of nitric oxide release from human umbilical vein endothelial cells.

Many solid tumours (e.g. sarcoma, carcinoma and lymphoma) form a disorganized neo-vasculature that initiates uncontrolled vessel formation to support tumour growth. The complexity of these environments poses a significant challenge for tumour medicine research. While animal models are commonly used to address some of these challenges, they are time-consuming and raise ethical concerns. In vitro microphysiological systems have been explored as an alternative, but their production typically requires multi-step lithographic processes that limit their production. In this work, a novel approach to rapidly develop multi-material tissue-mimicking, cell-compatible platforms able to represent the complexity of a solid tumour's neo-vasculature is investigated via stereolithography three-dimensional printing. To do so, a series of acrylate resins that yield covalently photo-cross-linked hydrogels with healthy and diseased mechano-acoustic tissue-mimicking properties are designed and characterized. The potential viability of these materials to displace animal testing in preclinical research is assessed by studying the morphology, actin expression, focal adhesions and nitric oxide release of human umbilical vein endothelial cells. These materials are exploited to produce a simplified multi-material three-dimensional printed model of the neo-vasculature of a solid tumour, demonstrating the potential of our approach to replicate the complexity of solid tumours in vitro without the need for animal testing.

Time course changes to structural, mechanical and material properties of bone in rats after complete spinal cord injury.

OBJECTIVE: Characterise the spatiotemporal trabecular and cortical bone responses to complete spinal cord injury (SCI) in young rats. METHODS: 8-week-old male Wistar rats received T9-transection SCI and were euthanised 2-, 6-, 10- or 16-weeks post-surgery. Outcome measures were assessed using micro-computed tomography, mechanical testing, serum markers and Fourier-transform infrared spectroscopy. RESULTS: The trabecular and cortical bone responses to SCI are site-specific. Metaphyseal trabecular BV/TV was 59% lower, characterised by fewer and thinner trabeculae at 2-weeks post-SCI, while epiphyseal BV/TV was 23% lower with maintained connectivity. At later-time points, metaphyseal BV/TV remained unchanged, while epiphyseal BV/TV increased. The total area of metaphyseal and mid-diaphyseal cortical bone were lower from 2-weeks and between 6- and 10-weeks post-SCI, respectively. This suggested that SCI-induced bone changes observed in the rat model were not solely attributable to bone loss, but also to suppressed bone growth. No tissue mineral density differences were observed at any time-point, suggesting that decreased whole-bone mechanical properties were primarily the result of changes to the spatial distribution of bone. CONCLUSION: Young SCI rat trabecular bone changes resemble those observed clinically in adult and paediatric SCI, while cortical bone changes resemble paediatric SCI only.

Spatiotemporal responses of trabecular and cortical bone to complete spinal cord injury in skeletally mature rats.

Objective: Characterise the spatiotemporal responses of trabecular and cortical bone to complete spinal cord injury (SCI) in the skeletally mature rat in the acute (4-week) period following injury. Methods: The spinal cord of 5-month old male rats was transected at the T9 level. Outcome measures were assessed using micro-computed tomography, three-point bending and serum markers at 1-, 2-, and 4-weeks post-transection. Comparison was made with time-0 and sham animals. Results: Lower levels of circulating serum bone formation markers and higher bone resorption markers suggested uncoupled bone turnover as early at 1-week post-transection. Micro-computed tomography showed metaphyseal and epiphyseal trabecular bone loss was observed only at 4-weeks post-transection. The bone loss was site-specific with a more severe reduction in trabecular BV/TV observed in the metaphyseal (50%) relative to epiphyseal (19%) region. Metaphyseal trabecular bone exhibited a 54% reduction in connectivity density while the epiphyseal trabecular bone was unaffected. Cortical bone deficits were not seen over the time periods examined. Conclusions: The study demonstrates that the skeletally mature spinal cord transected rat model replicates the biphasic pattern of osteoporotic changes observed in the human SCI population, providing a relevant model for testing the efficacy of interventions against SCI-induced osteoporosis.

Additive Manufacture of Small-Scale Metamaterial Structures for Acoustic and Ultrasonic Applications.

Acoustic metamaterials are large-scale materials with small-scale structures. These structures allow for unusual interaction with propagating sound and endow the large-scale material with exceptional acoustic properties not found in normal materials. However, their multi-scale nature means that the manufacture of these materials is not trivial, often requiring micron-scale resolution over centimetre length scales. In this review, we bring together a variety of acoustic metamaterial designs and separately discuss ways to create them using the latest trends in additive manufacturing. We highlight the advantages and disadvantages of different techniques that act as barriers towards the development of realisable acoustic metamaterials for practical audio and ultrasonic applications and speculate on potential future developments.

Physiological Basis of Noise-Induced Hearing Loss in a Tympanal Ear.

Acoustic overexposure, such as listening to loud music too often, results in noise-induced hearing loss. The pathologies of this prevalent sensory disorder begin within the ear at synapses of the primary auditory receptors, their postsynaptic partners and their supporting cells. The extent of noise-induced damage, however, is determined by overstimulation of primary auditory receptors, upstream of where the pathologies manifest. A systematic characterization of the electrophysiological function of the upstream primary auditory receptors is warranted to understand how noise exposure impacts on downstream targets, where the pathologies of hearing loss begin. Here, we used the experimentally-accessible locust ear (male, Schistocerca gregaria) to characterize a decrease in the auditory receptor's ability to respond to sound after noise exposure. Surprisingly, after noise exposure, the electrophysiological properties of the auditory receptors remain unchanged, despite a decrease in the ability to transduce sound. This auditory deficit stems from changes in a specialized receptor lymph that bathes the auditory receptors, revealing striking parallels with the mammalian auditory system.SIGNIFICANCE STATEMENT Noise exposure is the largest preventable cause of hearing loss. It is the auditory receptors that bear the initial brunt of excessive acoustic stimulation, because they must convert excessive sound-induced movements into electrical signals, but remain functional afterward. Here we use the accessible ear of an invertebrate to, for the first time in any animal, characterize changes in auditory receptors after noise overexposure. We find that their decreased ability to transduce sound into electrical signals is, most probably, due to changes in supporting (scolopale) cells that maintain the ionic composition of the ear. An emerging doctrine in hearing research is that vertebrate primary auditory receptors are surprisingly robust, something that we show rings true for invertebrate ears too.

Global and site-specific analysis of bone in a rat model of spinal cord injury-induced osteoporosis.

Micro-Computed Tomography bone analysis is the gold standard method for assessing trabecular and cortical bone microarchitecture in small animal bones. This technique reports morphometric parameters as averages over selected volumes of interest (VOIs). This study proposes the introduction of an additional global 2D morphometric step into the analysis process, that provides a survey of the underlying morphometric variation present throughout both trabecular and cortical bone. The visualisation of these morphometric distributions provides a systematic approach to VOI selection that provides rationale and adds confidence to subsequent 3D morphometric analysis. To test the applicability and value of this methodological addition it was applied to the distal femur of a rat model of spinal cord injury (SCI)-induced osteoporosis. The 2D morphometric variation of both trabecular and cortical bone was quantified as a function of bone length. SCI-induced osteoporosis was localised in i) trabecular bone, where metaphyseal bone was more severely affected than epiphyseal bone, and there was a significant reduction in Distal Femoral Trabecular Extent, a new parameter defined here that quantifies how far trabecular bone penetrates in to the marrow cavity, ii) cortical bone, where diaphyseal bone underwent significant lowering of both cortical area and thickness, while distal-metaphyseal bone did not. Theses site-specific changes were validated, further elucidated and compared with follow-up conventional 3D analysis. The techniques applied here are equally applicable to other long bones (tibia, humerus, radius, ulna), other types of imaging modality and other types of experimental design including the effects of rehabilitation, aging, loading, gene knockout and pharmacological intervention.

Correction to: Transmission of the frequency components of the vibrational signal of the glassy-winged sharpshooter, Homalodisca vitripennis, within and between grapevines.

Unfortunately, Fig. 3 was incorrectly published in the original publication. The correct version of Fig. 3 is updated here.

Transmission of the frequency components of the vibrational signal of the glassy-winged sharpshooter, Homalodisca vitripennis, within and between grapevines.

The agricultural pest, Homalodisca vitripennis, relies on vibrational communication through plants for species identification, location, and courtship. Their vibrational signal exhibits a dominant frequency between 80 and 120 Hz, with higher frequency, lower intensity harmonics occurring approximately every 100 Hz. However, previous research revealed that not all harmonics are recorded in every signal. Therefore, how the female H. vitripennis vibrational signal changes as it travels through the plant was investigated. Results confirmed that transmission was a bending wave, with decreased signal intensity for increasing distance from the source; moreover, at distances of 50 cm, higher frequencies traveled faster than lower frequencies, suggesting that dispersion of H. vitripennis signal components may enable signaling partners to encode distance. Finally, H. vitripennis generates no detectable airborne signal (pressure wave), yet their low vibrational frequency components are detectable in neighboring plants as a result of leaf-to-air-to-leaf propagation. For instance, with isolated key female signal frequencies, 100 Hz was detected at a 10 cm gap between leaves, whereas 600 Hz was detectable only with a 0.1 cm gap. Together, these results highlight the complexity of vibration propagation in plants and suggest the possibility of the animals using the harmonic content to determine distance to the signaling H. vitripennis source.

Porpoise click classifier (PorCC): A high-accuracy classifier to study harbour porpoises (Phocoena phocoena) in the wild.

Harbour porpoises are well-suited for passive acoustic monitoring (PAM) as they produce highly stereotyped narrow-band high-frequency (NBHF) echolocation clicks. PAM systems must be coupled with a classification algorithm to identify the signals of interest. Here, the authors present a harbour porpoise click classifier (PorCC) developed in matlab, which uses the coefficients of two logistic regression models in a decision-making pathway to assign candidate signals to one of three categories: high-quality clicks (HQ), low-quality clicks (LQ), or high-frequency noise. The receiver operating characteristics of PorCC was compared to that of PAMGuard's Porpoise Click Detector/Classifier Module. PorCC outperformed PAMGuard's classifier achieving higher hit rates (correctly classified clicks) and lower false alarm levels (noise classified as HQ or LQ clicks). Additionally, the detectability index (d') for HQ clicks for PAMGuard was 2.2 (overall d' = 2.0) versus 4.1 for PorCC (overall d' = 3.4). PorCC classification algorithm is a rapid and highly accurate method to classify NBHF clicks, which could be applied for real time monitoring, as well as to study harbour porpoises, and potentially other NBHF species, throughout their distribution range from data collected using towed hydrophones or static recorders. Moreover, PorCC is suitable for studies of acoustic communication of porpoises.

3D printed microneedle patches using stereolithography (SLA) for intradermal insulin delivery.

3D printed microneedle arrays were fabricated using a biocompatible resin through stereolithography (SLA) for transdermal insulin delivery. Microneedles were built by polymerising consecutive layers of a photopolymeric resin. Thin layers of insulin and sugar alcohol or disaccharide carriers were formed on the needle surface by inkjet printing. The optimization of the printing process resulted in superior skin penetration capacity of the 3D printed microneedles compared to metal arrays with minimum applied forces varying within the range of 2 to 5 N. Micro-CT analysis showed strong adhesion of the coated films on the microneedle surface even after penetration to the skin. In vivo animal trials revealed fast insulin action with excellent hypoglycaemia control and lower glucose levels achieved within 60 min, combined with steady state plasma glucose over 4 h compared to subcutaneous injections.

Nanoscale Coatings for Ultralow Dose BMP-2-Driven Regeneration of Critical-Sized Bone Defects.

While new biomaterials for regenerative therapies are being reported in the literature, clinical translation is slow. Some existing regenerative approaches rely on high doses of growth factors, such as bone morphogenetic protein-2 (BMP-2) in bone regeneration, which can cause serious side effects. An ultralow-dose growth factor technology is described yielding high bioactivity based on a simple polymer, poly(ethyl acrylate) (PEA), and mechanisms to drive stem cell differentiation and bone regeneration in a critical-sized murine defect model with translation to a clinical veterinary setting are reported. This material-based technology triggers spontaneous fibronectin organization and stimulates growth factor signalling, enabling synergistic integrin and BMP-2 receptor activation in mesenchymal stem cells. To translate this technology, plasma-polymerized PEA is used on 2D and 3D substrates to enhance cell signalling in vitro, showing the complete healing of a critical-sized bone injury in mice in vivo. Efficacy is demonstrated in a Münsterländer dog with a nonhealing humerus fracture, establishing the clinical translation of advanced ultralow-dose growth factor treatment.

"Pipe Organ" Inspired Air-Coupled Ultrasonic Transducers With Broader Bandwidth.

Piezoelectric micromachined ultrasonic transducers (PMUTs) are used to receive and transmit ultrasonic signals in industrial and biomedical applications. This type of transducer can be miniaturized and integrated with electronic systems since each element is small and the power requirements are low. The bandwidth of the PMUT may be narrow in some conventional designs; however, it is possible to apply modified structures to enhance this. This paper presents a methodology for improving the bandwidth of air-coupled PMUTs without sensitivity loss by connecting a number of resonating pipes of various lengths to a cavity. A prototype piezoelectric diaphragm ultrasonic transducer is presented to prove the theory. This novel device was fabricated by additive manufacturing (3-D printing), and consists of a polyvinylidene fluoride thin film over a stereolithography designed backplate. The backplate design is inspired by a pipe organ musical instrument, where the resonant frequency (pitch) of each pipe is mainly determined by its length. The -6-dB bandwidth of the "pipe organ" air-coupled transducer is 55.7% and 58.5% in transmitting and receiving modes, respectively, which is ~5 times wider than a custom-built standard device.

Active Hearing Mechanisms Inspire Adaptive Amplification in an Acoustic Sensor System.

Over many millions of years of evolution, nature has developed some of the most adaptable sensors and sensory systems possible, capable of sensing, conditioning and processing signals in a very power- and size-effective manner. By looking into biological sensors and systems as a source of inspiration, this paper presents the study of a bioinspired concept of signal processing at the sensor level. By exploiting a feedback control mechanism between a front-end acoustic receiver and back-end neuronal based computation, a nonlinear amplification with hysteretic behavior is created. Moreover, the transient response of the front-end acoustic receiver can also be controlled and enhanced. A theoretical model is proposed and the concept is prototyped experimentally through an embedded system setup that can provide dynamic adaptations of a sensory system comprising a MEMS microphone placed in a closed-loop feedback system. It faithfully mimics the mosquito's active hearing response as a function of the input sound intensity. This is an adaptive acoustic sensor system concept that can be exploited by sensor and system designers within acoustics and ultrasonic engineering fields.

Frequency doubling by active in vivo motility of mechanosensory neurons in the mosquito ear.

Across vertebrate and invertebrate species, nonlinear active mechanisms are employed to increase the sensitivity and acuity of hearing. In mosquitoes, the antennal hearing organs are known to use active force feedback to enhance auditory acuity to female generated sounds. This sophisticated form of signal processing involves active nonlinear events that are proposed to rely on the motile properties of mechanoreceptor neurons. The fundamental physical mechanism for active auditory mechanics is theorized to rely on a synchronization of motile neurons, with a characteristic frequency doubling of the force generated by an ensemble of motile mechanoreceptors. There is however no direct biomechanical evidence at the mechanoreceptor level, hindering further understanding of the fundamental mechanisms of sensitive hearing. Here, using in situ and in vivo atomic force microscopy, we measure and characterize the mechanical response of mechanosensory neuron units during forced oscillations of the hearing organ. Mechanoreceptor responses exhibit the hallmark of nonlinear feedback for force generation, with movements at twice the stimulus frequency, associated with auditory amplification. Simultaneous electrophysiological recordings exhibit similar response features, notably a frequency doubling of the firing rate. This evidence points to the nature of the mechanism, whereby active hearing in mosquitoes emerges from the double-frequency response of the auditory neurons. These results open up the opportunity to directly investigate active cellular mechanics in auditory systems, and they also reveal a pathway to study the nanoscale biomechanics and its dynamics of cells beyond the sense of hearing.

Extreme call amplitude from near-field acoustic wave coupling in the stridulating water insect Micronecta scholtzi (Micronectinae).

Underwater acoustic transducers, particularly at low frequencies, are beset by problems of scale and inefficiency due to the large wavelengths of sound in water. In insect mating calls, a high call volume is usually desirable, increasing the range of signal transmission and providing a form of advertisement of the signaller's quality to a potential mate; however, the strength of the call is constrained by body size and by the need to avoid predators who may be listening in. Male crickets and water boatmen avoid some of the limitations of body size by exploiting resonant structures, which produce sharply tuned species-specific songs, but call frequency and volume remain linked to body size. Recently, the water boatman Micronecta scholtzi was found to circumvent this rule, producing a louder mating call than that of similar, but much larger, Corixa The resonant structure in Corixidae and Micronectinae is believed to be the trapped air reserves around the insect as it dives, driven by a stridulatory apparatus. However, the method by which energy is transferred from the striated area to the bubble is unknown. Here, we present modelling of a system of near-field coupling of acoustic sources to bubbles showing an exponential increase in sound power gain with decreasing distance that provides a simple solution to the stimulus of the air bubbles in Corixidae and Micronectinae and explains the discrepancy of M. scholtzi's extreme call volume. The findings suggest a possible route to engineered systems using near-field coupling to overcome size constraints in low-frequency (less than 500 Hz) underwater transducers, where the input efficiency of a piezoelectric device can be coupled through the hydrodynamic field to the high radiative efficiency of a near-ideal monopole emitter.

Hearing on the fly: the effects of wing position on noctuid moth hearing.

The ear of the noctuid moth has only two auditory neurons, A1 and A2, which function in detecting predatory bats. However, the noctuid's ears are located on the thorax behind the wings. Therefore, as these moths need to hear during flight, it was hypothesized that wing position may affect their hearing. The wing was fixed in three different positions: up, flat and down. An additional subset of animals was measured with freely moving wings. In order to negate any possible acoustic shadowing or diffractive effects, all wings were snipped, leaving the proximal-most portion and the wing hinge intact. Results revealed that wing position plays a factor in threshold sensitivity of the less sensitive auditory neuron A2, but not in the more sensitive neuron A1. Furthermore, when the wing was set in the down position, fewer A1 action potentials were generated prior to the initiation of A2 activity. Analyzing the motion of the tympanal membrane did not reveal differences in movement due to wing position. Therefore, these neural differences arising from wing position are proposed to be due to other factors within the animal such as different muscle tensions.

The anti-bat strategy of ultrasound absorption: the wings of nocturnal moths (Bombycoidea: Saturniidae) absorb more ultrasound than the wings of diurnal moths (Chalcosiinae: Zygaenoidea: Zygaenidae).

The selection pressure from echolocating bats has driven the development of a diverse range of anti-bat strategies in insects. For instance, several studies have proposed that the wings of some moths absorb a large portion of the sound energy contained in a bat's ultrasonic cry; as a result, the bat receives a dampened echo, and the moth becomes invisible to the bat. To test the hypothesis that greater exposure to bat predation drives the development of higher ultrasound absorbance, we used a small reverberation chamber to measure the ultrasound absorbance of the wings of nocturnal (Bombycoidea: Saturniidae) and diurnal moths (Chalcosiinae: Zygaenoidea: Zygaenidae). The absorption factor of the nocturnal saturniids peaks significantly higher than the absorption factor of the diurnal chalcosiines. However, the wings of the chalcosiines absorb more ultrasound than the wings of some diurnal butterflies. Following a phylogenetic analysis on the character state of diurnality/ nocturnality in the Zygaenidae, we propose that diurnality in the Chalcosiinae is plesiomorphic (retained); hence, the absorbance of their wings is probably not a vestigial trait from an ancestral, nocturnal form but an adaptation to bat activity that overlaps their own. On a within-species level, females of the saturniids Argema mittrei and Samia cynthia ricini have significantly higher absorption factors than the males. In the female S. c. ricini, the higher absorption factor corresponds to a detection distance by bats that is at best 20-30% shorter than that of the male.

Evolution of directional hearing in moths via conversion of bat detection devices to asymmetric pressure gradient receivers.

Small animals typically localize sound sources by means of complex internal connections and baffles that effectively increase time or intensity differences between the two ears. However, some miniature acoustic species achieve directional hearing without such devices, indicating that other mechanisms have evolved. Using 3D laser vibrometry to measure tympanum deflection, we show that female lesser waxmoths (Achroia grisella) can orient toward the 100-kHz male song, because each ear functions independently as an asymmetric pressure gradient receiver that responds sharply to high-frequency sound arriving from an azimuth angle 30° contralateral to the animal's midline. We found that females presented with a song stimulus while running on a locomotion compensation sphere follow a trajectory 20°-40° to the left or right of the stimulus heading but not directly toward it, movement consistent with the tympanum deflections and suggestive of a monaural mechanism of auditory tracking. Moreover, females losing their track typically regain it by auditory scanning-sudden, wide deviations in their heading-and females initially facing away from the stimulus quickly change their general heading toward it, orientation indicating superior ability to resolve the front-rear ambiguity in source location. X-ray computer-aided tomography (CT) scans of the moths did not reveal any internal coupling between the two ears, confirming that an acoustic insect can localize a sound source based solely on the distinct features of each ear.

Unpicking the signal thread of the sector web spider Zygiella x-notata.

Remote sensing allows an animal to extend its morphology with appropriate conductive materials and sensors providing environmental feedback from spatially removed locations. For example, the sector web spider Zygiella x-notata uses a specialized thread as both a structural bridge and signal transmitter to monitor web vibrations from its retreat at the web perimeter. To unravel this model multifunctional system, we investigated Zygiella's signal thread structure with a range of techniques, including tensile testing, laser vibrometry, electron microscopy and behavioural analysis. We found that signal threads varied significantly in the number of filaments; a result of the spider adding a lifeline each time it runs along the bridge. Our mechanical property analysis suggests that while the structure varies, its normalized load does not. We propose that the signal thread represents a complex and fully integrated multifunctional structure where filaments can be added, thus increasing absolute load-bearing capacity while maintaining signal fidelity. We conclude that such structures may serve as inspiration for remote sensing design strategies.