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A simple, disposable, and integrated electronic-tube cap (E-tube cap) for DNA detection at the point-of-care was designed, fabricated, and tested. The E-tube cap contains a 3D printed electrode substrate for DNA extraction and label-free pH sensing detection. One Flinders Technology Associates (Whatman FTA) membrane was incorporated into the 3D printed electrode substrate for the isolation, concentration, and purification of DNA. The E-tube cap with captured DNA by the membrane was inserted directly into a reaction tube for loop-mediated isothermal amplification (LAMP). The isothermal amplification process was monitored in real-time by a 3D printed electrochemical electrode coated with pH-sensitive material (carbon/iridium oxide layer). The pH sensing electrode showed an excellent linear response within the pH range of 6–9 with a slope of −31.32 ± 0.5 mV/pH at room temperature. The utility of the integrated E-tube cap was demonstrated by detecting the presence of lambda DNA spiked in saliva samples with a sensitivity of 100 copies per mL sample within 30 min. Such a simple, rapid, and affordable diagnostic device is particularly suitable for point-of-care molecular diagnostics of infectious diseases.

Physical forces have long been recognized for their effects on the growth, morphology, locomotion, and survival of eukaryotic organisms1. Recently, mechanical forces have been shown to regulate processes in bacteria, including cell division2, motility3, virulence4, biofilm initiation5,6, and cell shape7,8, although it remains unclear how mechanical forces in the cell envelope lead to changes in molecular processes. In Gram-negative bacteria, multicomponent protein complexes that form rigid links across the cell envelope directly experience physical forces and mechanical stresses applied to the cell. Here we manipulate tensile and shear mechanical stress in the bacterial cell envelope and use single-molecule tracking to show that shear (but not tensile) stress within the cell envelope promotes disassembly of the tripartite efflux complex CusCBA, a system used by E. coli to resist copper and silver toxicity, thereby making bacteria more susceptible to metal toxicity. These findings provide the first demonstration that mechanical forces, such as those generated during colony overcrowding or bacterial motility through submicron pores, can inhibit the contact and function of multicomponent complexes in bacteria. As multicomponent, trans-envelope efflux complexes in bacteria are involved in many processes including antibiotic resistance9, cell division10, and translocation of outer membrane components11, our findings suggest that the mechanical environment may regulate multiple processes required for bacterial growth and survival.

Diffuse matrix damage in rat cortical bone has been observed to self-repair efficiently in two weeks without activating bone remodeling, and unlike the case with linear cracks, the local osteocytes at the sites of diffuse damage remain healthy. However, the reason(s) for such high efficiency of matrix repair remains unclear. We hypothesized that transport of minerals and other compounds essential for damage repair is enhanced at the damaged sites and further increased by the application of tensile loading. To test our hypothesis, diffuse damage was introduced in notched bovine wafers under cyclic tensile loading and unloading. Using the Fluorescence Recovery After Photobleaching (FRAP) approach, we measured the transport of a small fluorescent tracer (sodium fluorescein, 376 Da) in damaged vs. undamaged regions and under varying tensile load magnitudes (0.2 N, 10 N, 20 N, and 30 N), which corresponded to nominal strains of 12.5, 625, 1250, and 1875 microstrains, respectively. We found a 37% increase in transport of fluorescein in damaged regions relative to undamaged regions and a further ∼18% increase in transport under 20N and 30N tension compared to the non-loaded condition, possibly due to the opening of the cracking surfaces. The elevated transport of minerals and other adhesive proteins may, at least partially, account for the highly effective repair of diffuse damage observed in vivo. Clinical Significance: Diffuse damage adversely affects bone's fracture resistance and this study provided quantitative data on elevated transport, which may be involved in repairing diffuse damage in vivo. This article is protected by copyright. All rights reserved

The mechanisms underlying the detection of critically loaded or micro-damaged regions of bone by bone cells are still a matter of debate. Our previous studies showed that calcium efflux originates from pre-failure regions of bone matrix and MC3T3-E1 osteoblasts respond to such efflux by an increase in the intracellular calcium concentration. The mechanisms by which the intracellular calcium concentration increases in response to an increase in the pericellular calcium concentration are unknown. Elevation of the intracellular calcium may occur via release from the internal calcium stores of the cell and/or via the membrane bound channels. The current study applied a wide range of pharmaceutical inhibitors to identify the calcium entry pathways involved in the process: internal calcium release from endoplasmic reticulum (ER, inhibited by thapsigargin and TMB-8), calcium receptor (CaSR, inhibited by calhex), stretch-activated calcium channel (SACC, inhibited by gadolinium), voltage-gated calcium channels (VGCC, inhibited by nifedipine, verapamil, neomycin, and ω-conotoxin), and calcium-induced-calcium-release channel (CICRC, inhibited by ryanodine and dantrolene). These inhibitors were screened for their effectiveness to block intracellular calcium increase by using a concentration gradient induced calcium efflux model which mimics calcium diffusion from the basal aspect of cells. The inhibitor(s) which reduced the intracellular calcium response was further tested on osteoblasts seeded on mechanically loaded notched cortical bone wafers undergoing damage. The results showed that only neomycin reduced the intracellular calcium response in osteoblasts, by 27%, upon extracellular calcium stimulus induced by concentration gradient. The inhibitory effect of neomycin was more pronounced (75% reduction in maximum fluorescence) for osteoblasts seeded on notched cortical bone wafers loaded mechanically to damaging load levels. These results imply that the increase in intracellular calcium occurs by the entry of extracellular calcium ions through VGCCs which are sensitive to neomycin. N-type and P-type VGCCs are potential candidates because they are observed in osteoblasts and they are sensitive to neomycin. The calcium channels identified in this study provide new insight into mechanisms underlying the targeted repair process which is essential to bone adaptation.

The aim of this study was to understand the polymer degradation and drug release mechanism from PLGA microspheres embedded in a PVA hydrogel. Two types of microspheres were prepared with different molecular weight PLGA polymers (approximately 25 and 7 kDa) to achieve different drug release profiles, with a 9-day lag phase and without a lag phase, respectively. The kinetics of water uptake into the microspheres coincided with the drug release profiles for both formulations. For the 25 kDa microspheres, minimal water uptake was observed in the early part of the lag phase followed by substantial water uptake at the later stages and in the drug release phase. For the 7 kDa microspheres, water uptake occurred simultaneously with drug release. Water uptake was approximately 2-3 times that of the initial microsphere weight for both formulations. The internal structure of the PLGA microspheres was evaluated using low temperature scanning electron microscopy (cryo-SEM). Burst drug release occurred followed by pore forming from the exterior to the core of both microspheres. A well-defined hydrogel/microsphere interface was observed. For the 25 kDa microspheres, internal pore formation and swelling occurred before the second drug release phase. The surface layer of the microspheres remained intact whereas swelling, and degradation of the core continued throughout the drug release period. In addition, microsphere swelling reduced glucose transport through the coatings in PBS media and this was considered to be a as a consequence of the increased thickness of the coatings. The combination of the swelling and microdialysis results provides a fresh understanding on the competing processes affecting molecular transport of bioanalytes (i.e. glucose) through these composite coatings during prolonged exposure in PBS.

The ability to resist mechanical forces is necessary for the survival and division of bacteria and has traditionally been probed using specialized, low-throughput techniques such as atomic force microscopy and optical tweezers. Here we demonstrate a microfluidic technique to profile the stiffness of individual bacteria and populations of bacteria. The approach is similar to micropipette aspiration used to characterize the biomechanical performance of eukaryotic cells. However, the small size and greater stiffness of bacteria relative to eukaryotic cells prevents the use of micropipettes. Here we present devices with sub-micron features capable of applying loads to bacteria in a controlled fashion. Inside the device, individual bacteria are flowed and trapped in tapered channels. Less stiff bacteria undergo greater deformation and therefore travel further into the tapered channel. Hence, the distance traversed by bacteria into a tapered channel is inversely related to cell stiffness. We demonstrate the ability of the device to characterize hundreds of bacteria at a time, measuring stiffness at 12 different applied loads at a time. The device is shown to differentiate between two bacterial species, E. coli (less stiff) and B. subtilis (more stiff), and detect differences between E. coli submitted to antibiotic treatment from untreated cells of the same species/strain. The microfluidic device is advantageous in that it requires only minimal sample preparation, no permanent cell immobilization, no staining/labeling and maintains cell viability. Our device adds detection of biomechanical phenotypes of bacteria to the list of other bacterial phenotypes currently detectable using microchip-based methods and suggests the feasibility of separating/selecting bacteria based on differences in cell stiffness.

Topographical cues from the extracellular microenvironment can influence cellular activity including proliferation and differentiation. Information on the effects of material topography on tenogenic differentiation of human mesenchymal stem cells (human MSCs) is limited. A methodology using the principles of isoelectric focusing has previously been developed in our laboratory to synthesize electrochemically aligned collagen (ELAC) threads that mimics the packing density, alignment and strength of collagen dense connective tissues. In the current study, human MSCs were cultured on ELAC and randomly-oriented collagen threads and the effect of collagen orientation on cell morphology, proliferation and tenogenic differentiation was investigated. The results indicate that higher rates of proliferation were observed on randomly oriented collagen threads compared to ELAC threads. On the other hand, tendon specific markers such as scleraxis, tenomodulin, tenascin-C and collagen-III were significantly increased on ELAC threads compared to randomly oriented collagen threads. Additionally, osteocalcin, a specific marker of bone differentiation was suppressed on ELAC threads. Previous studies have reported that BMP-12 is a key growth factor to induce tenogenic differentiation of human MSCs. To evaluate the synergistic effect of BMP-12 and collagen orientation, human MSCs were cultured on ELAC threads in culture medium supplemented with and without BMP-12. The results revealed that BMP-12 did not have an additional effect on the tenogenic differentiation of human MSCs on ELAC threads. Together, these results suggest that ELAC induces tenogenic differentiation of human MSCs by presenting an aligned and dense collagen substrate, akin to the tendon itself. In conclusion, ELAC has a significant potential to be used as a tendon replacement and in the development of an osteotendinous construct towards the regeneration of bone-tendon interfaces.

The mechanisms by which bone cells sense critically loaded regions of bone are still a matter of ongoing debate. Animal models to investigate response to microdamage involve post mortem immunohistological analysis and do not allow real-time monitoring of cellular response during the emergence of the damage in bone. Most in vitro mechanical stimulation studies are conducted on non-bone substrates, neglecting the damage-related alterations in the pericellular niche and their potential effects on bone cells. The current study reports spontaneous efflux of calcium ions (Ca2+) (1.924 ± 0.742 pmol cm− 2 s− 1) from regions of devitalized bone matrix undergoing post-yield strains, induced by a stress concentrator. When these samples are seeded with MC3T3-E1 osteoblasts, the strain-induced Ca2+ efflux from bone elicits cell response at the stress concentration site as manifested by activation of intracellular calcium signaling (increase in fluorescence by 52% ± 27%). This activity is associated with extracellular calcium because the intracellular calcium signaling in response to mechanical loading subsides when experiments are repeated using demineralized bone substrates (increase in fluorescence by 6% ± 10%). These results imply a novel perspective where bone matrix acts as an intermediary mechanochemical transducer by converting mechanical strain into a chemical signal (pericellular calcium) to which cells respond. Such a mechanism may be responsible for triggering repair at locations of bone matrix undergoing critical deformation levels.

Bone cells are responsible for sensing and converting the mechanical signals into cellular signals to drive bone adaptation and damage repair [1]. Cell-mediated repair of bone is reported to be in preferential association with regions filled with microdamage [2]. Although different theories have been proposed for mechanisms involved in those processes (such as substrate deformation, fluid flow shear, and hydrostatic pressure in mechanotransduction [3], or microcrack and osteocyte apoptosis in damage detection [4]), knowledge on the exact form of physical stimuli which trigger bone cells, especially in critically loaded regions of bone, is still elusive.Copyright © 2011 by ASME

Owing to the low-loss and high refractive index variations derived from the basic building block of bone structure, we, for the first time to our knowledge, demonstrate coherent random lasing action originated from the bone structure infiltrated with laser dye, revealing that bone tissue is an ideal biological material for random lasing. Our numerical simulation shows that random lasers are extremely sensitive to subtle structural changes even at nanoscales and can potentially be an excellent tool for probing nanoscale structural alterations in real time as a novel spectroscopic modality. (C) 2010 Optical Society of America

Image subtraction has been an extremely useful tool for capturing subtle changes in pixel intensity with extremely high temporal resolution, and has been used for decades in the astronomy and metal corrosion fields. However, to date, image subtraction has not been used as a mainstream technique for investigating morphological changes in cells, tissues, or whole organisms. We introduce a user-friendly differential imaging technique for monitoring real time (~msec) changes in morphology within the micrometer to millimeter spatial scale. The technique is demonstrated by measuring morphological changes morphology for biomedical (bone stress), agricultural (crop root elongation), and environmental (zooplankton ecotoxicology) applications. Subtle changes in growth that would typically only be observed by highly skilled experts are easily resolved via image subtraction and the use of convolution kernels. When coupled with techniques characterizing real time biochemical transport (e.g., respiration, ion/substrate transport), physiology can be directly quantified with a high temporal and spatial resolution. Because of the ease of use, this technique can be readily applied to any field of science concerned with bridging the gap between form and function.

We, for the first time, demonstrate coherent random lasing action in bone tissue infused with laser dye. This could potentially be used to probe structural alterations at nanoscales in real-time as a novel spectroscopic modality.

A prominent opacity is evident in the process zone of notched thin wafers of bone loaded in tension. Being recoverable upon unloading, this opaque zone can be stained only when the sample is under load, unlike the classically reported forms of damage which take up the stain in the unloaded state. Furthermore, despite the stain uptake, microcracks are absent in the stained area examined by high magnification optical microscopy and atomic force microscopy (AFM). Therefore, the size scale and the electric charge of the features involved in the process zone were probed at the submicron level by using a wide range of fluorescent dyes of different molecular weights and charges. It was observed that negatively charged dyes penetrate the process zone and that dyes greater than 10 kDa (about 10-20 nm in size) were unable to label the process zone. Digital image correlation (DIC) measurements indicated that the opacity initiates at about 1% principal strain and the strain accumulates up to 14%. While the opacity was largely recoverable upon unloading, the core regions which experienced large strains had permanent residual strains up to 2%, indicating that the observed deformation phenomenon can be interlocked within bone matrix without the formation of microcracks. Based on the similarity of size and their known affinity for negatively charged species, exposure of mineral nanoplatelets is proposed as prime candidates. Therefore, the deformation process reported here may be associated with debonding of mineral crystals from the neighboring collagen molecules. Overall, post-yield deformation of bone at the micron scale takes place by large strain events which are accommodated in bone matrix by the generation of nanoscale positively charged interfaces.

We demonstrate that the unique characteristics of random lasing in bone can be used to assess nanoscale structural alterations as a mechanical or structural biosensor, given that bone is a partially disordered biological nanostructure. In this proof-of-concept study, we conduct photoluminescence experiments on cortical bone specimens that are loaded in tension under mechanical testing. The ultra-high sensitivity, the large detection area, and the simple detection scheme of random lasers allow us to detect prefailure damage in bone at very small strains before any microscale damage occurs. Random laser-based biosensors could potentially open a new possibility for highly sensitive detection of nanoscale structural and mechanical alterations prior to overt microscale changes in hard tissue and biomaterials.

Given that bone is an intriguing nanostructured dielectric as a partially disordered complex structure, we apply an elastic light scattering-based approach to image prefailure deformation and damage of bovine cortical bone under mechanical testing. We demonstrate that our imaging method can capture nanoscale deformation in a relatively large area. The unique structure, the high anisotropic property of bone, and the system configuration further allow us to use the transfer matrix method to study possible spectroscopic manifestations of prefailure deformation. Our sensitive yet simple imaging method could potentially be used to detect nanoscale structural and mechanical alterations of hard tissue and biomaterials in a fairly large field of view.

Bone is a biocomposite of collagen and apatite crystals which together constitute a striking hierarchical organization, though it can still become structurally compromised when external load exceeds its threshold. Mechanisms of bone damage have been proposed on different length scales corresponding to its hierarchical structure. However, the damage process was still not completely understood due to the complexity of bone’s hierarchy. We previously reported an opaque process zone feature in bone under tensile loading, which could be stained only when samples were kept in loaded condition, in contrast to the classical damage, which could be stained after the removal of loading. In this study, Digital Image Correlation (DIC) methods have been used to quantify the local strain value at the micro-scale upon dark zone emergence. The process zones observed under transmission illumination was found to colocalize with the high-strain (up to 14%) regions calculated by DIC, and overlap best with the shape of principal strain (e1). The average strain value recorded at the edges of the process zones was about 1.1%, around the proposed threshold for collagen interfibrillar sliding. Thus, we speculate that collagen interfibrillar sliding might be among the causes for this dark zone phenomenon.

To reduce bone fracture incidence, gaining knowledge on the underlying bone damage mechanisms is essential. Although several mechanisms have been proposed on different length scales, the pre-failure deformation processes of bone are still not completely understood. We previously reported an opaque process zone feature in bone under tensile loading which colocalizes with high-strain regions. Unlike the classical damage which can be stained in the unloaded state, the process zone can be stained only when the sample is loaded. In this study, a wide range of fluorescent dyes with different molecular weights (MW) and charges were used to probe the size and charge properties of the structural features leading to process zone emergence. We found none of these fluorescent dyes tested were able to stain the process zone once the loading was removed. All of the negativelycharged dyes with MW less than 70 kDa stained the process zone, while the positively- or neutrally-charged dyes did not, except for the one with smallest MW tested (380 Da) in group of the positively charged dyes. Therefore, we proposed that certain molecular groups with positive charge in bone were exposed during process zone emergence under loading.

Although crack mechanisms in bone have been intensively studied to have a better understanding of bone fracture, exact prefailure damage mechanisms about how cracks or deformations at nanoscales occur still remain unexplored due to technical limitations. In this pilot study, we apply back-directional gated spectroscopic imaging (BGSI) to examine the exact spatial extent of such damage in in-situ mechanical testing of bovine cortical bone. Our imaging approach provides a relatively large field of view, while the wavelength dependence of light elastically backscattered from bone at each pixel can capture structural alterations in a few tens of nanometers. Thus, our imaging method can simultaneously examine various length scales. Using a notched bovine cortical bone wafer, we report that an altered field of a couple of square millimeters forms at the tip of the notch during tensile loading in the transverse orientation, and this field disappears upon unloading. We conducted simple pilot simulations of optical waves in one-dimensional layered media to gain an understanding of the potential mechanisms about the spectral dependence on nanostructure alterations. Our results imply that the bone nanostructure may allow the formation of nanoscale deformation over a relatively large area to prevent microcrack formation or fracture as an energy dissipating mechanism. We further envision that BGSI may facilitate understanding how the nanostructure of bone controls bone characteristics and properties.