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In this work we present a standardised quantitative ultrasound imaging (SQUI) approach for the non-destructive three-dimensional imaging and quantification of cartilage formation in hydrogel based bioscaffolds. The standardised concept involves the processing of ultrasound backscatter data with respect to an acellular phantom in combination with the compensation of sound speed mismatch diffraction effects between the bioscaffold and the phantom. As a proof-of-concept, the SQUI approach was tested on a variety of bioscaffolds with varying degree of neocartilage formation. These were composed of Gelatine Methacryloyl (GelMA) hydrogels laden with human adipose-derived stem cells (hADSCs). These were cultured under chondrogenic stimulation following a previously established protocol, where the degree of the neocartilage formation was modulated using different GelMA network densities (6, 8, 10 % w/v) and culture time (0, 14, 28 days). Using the SQUI approach we were able to detect marked acoustic and morphological changes occurring in the bioscaffolds a result of their different chondrogenic outcome. We defined an acoustic neocartilage indicator, the sonomarker, for the selective imaging and quantification of neocartilage formation. The sonomarker, of backscatter intensity logIBC -2.4, was found to correlate with data obtained via standard destructive bioassays. The ultrasonic evaluation of human specimens confirmed the sonomarker as a relevant intensity, although it was found to shift to higher intensity values in proportion to the cartilage condition as inferred from sound speed measurements. This study demonstrates the potential of the SQUI approach for the realization of non-destructive analysis of cartilage regeneration over-time. STATEMENT OF SIGNIFICANCE: As tissue engineering strategies for neocartilage regeneration evolve towards clinical implementation, alternative characterisation approaches that allow the non-destructive monitoring of extracellular matrix formation in implantable hydrogel based bioscaffolds are needed. In this work we present an innovative standardized quantitative ultrasound imaging (SQUI) approach that allows the non-destructive, volumetric, and quantitative evaluation of neocartilage formation in hydrogel based bioscaffolds. The standardised concept aims to provide a robust approach that accounts for the dynamic changes occurring during the conversion from a cellular bioscaffold towards the formation of a neocartilage construct. We believe that the SQUI approach will be of great benefit for the evaluation of constructs developing neocartilage, not only for in-vitro applications but also potentially applicable to in-vivo applications.
Assuntos
Condrogênese , Hidrogéis , Cartilagem/diagnóstico por imagem , Cartilagem/fisiologia , Humanos , Hidrogéis/farmacologia , Engenharia Tecidual/métodos , UltrassonografiaRESUMO
Point of care testing using micro-total-analysis systems (µTAS) is critical to emergent healthcare devices with rapid and robust responses. However, two major barriers to the success of this approach are the prohibitive cost of microchip fabrication and poor sensitivity due to small sample volumes in a microfluidic format. Here, we aimed to replace the complex microchip format with a low-cost textile substrate with inherently built microchannels using the fibers' spaces. Secondly, by integrating this textile-based microfluidics with electrophoresis and wireless bipolar electrochemistry, we can significantly improve solute detection by focusing and concentrating the analytes of interest. Herein, we demonstrated that an in situ metal electrode simply inserted inside the textile-based electrophoretic system can act as a wireless bipolar electrode (BPE) that generates localized electric field and pH gradients adjacent to the BPE and extended along the length of the textile construct. As a result, charged analytes were not only separated electrophoretically but also focused where their electrophoretic migration and counter flow (EOF) balances due to redox reactions proceeding at the BPE edges. The developed wireless redox focusing technique on textile constructs was shown to achieve a 242-fold enrichment of anionically charged solute over an extended time of 3000 s. These findings suggest a simple route that achieves separation and analyte focusing on low-cost surface-accessible inverted substrates, which is far simpler than the more complex ITP on conventional closed and inaccessible capillary channels.
Assuntos
Eletroforese Capilar , Microfluídica , Eletroquímica , Eletrodos , TêxteisRESUMO
Quantitative ultrasound has a great potential for the non-destructive evaluation of tissue engineered constructs, where the local attenuation and the integrated backscatter coefficient (IBC) can be used for monitoring the development of biological processes. The local determination of both parameters can be achieved using the reference phantom method (RPM). However, its accuracy can be affected when evaluating constructs of evolving sound speed, attenuation and thickness, for example, when evaluating biodegradable hydrogels developing neocartilage. To assess the feasibility of using the RPM under such dynamic conditions while employing a 50-MHz transducer, we conducted a series of experiments on 3-mm-thick acellular hydrogels laden with microspheres. The ultrasonic evaluation procedure used was validated by detecting and compensating for large attenuation variations occurring in the construct, up to 20-fold with respect to the reference phantom, with estimations errors below 1%. We found that sound speed mismatch does not affect the local attenuation estimation, but causes a strong diffraction effect by reducing the backscatter intensity. Such intensity reduction was compensated by determining the IBC percentage change (IBCΔ) as function of sound speed mismatch with respect to the reference phantom (ΔSS), with the equation IBCΔâ¯=â¯(0.63 ± 0.07) ΔSSâ¯+â¯(8.54 ± 0.76) 10-3 ΔSS2. The investigated ΔSS interval was up to 120 m/s and using two different concentrations of microspheres, with estimation errors below 7% relative to the construct's actual IBC. Finally, we found that the spectral difference method is sufficient to measure within a few millimetres in depth mismatch, and when combining with sound speed mismatch, we found negligible additional effects. These results pave the way for the use of a generic reference phantom for the evaluation of thin dynamic constructs, thus simplifying the need for using different phantoms depending on the construct's properties.
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Imagens de Fantasmas , Ultrassonografia/métodos , Estudos de Viabilidade , Hidrogéis , MicroesferasRESUMO
In the present work we have revisited the application of quantitative ultrasound imaging (QUI) to cellular hydrogels, by using the reference phantom method (RPM) in combination with a local attenuation compensation algorithm. The investigated biological samples consisted of cell-laden collagen hydrogels with PC12 neural cells. These cell-laden hydrogels were used to calibrate the integrated backscattering coefficient (IBC) as a function of cell density, which was then used to generate parametric images of local cell density. The image resolution used for QUI and its impact on the relative IBC error was also investigated. Another important contribution of our work was the monitoring of PC12 cell proliferation. The cell number estimates obtained via the calibrated IBC compared well with data obtained using a conventional quantitative method, the MTS assay. Evaluation of spectral changes as a function of culture time also provided additional information on the cell cluster size, which was found to be in close agreement with that observed by microscopy. Last but not least, we also applied QUI on a 3D printed cellular construct in order to illustrate its capabilities for the evaluation of bioprinted structures. STATEMENT OF SIGNIFICANCE: While there is intensive research in the areas of polymer science, biology, and 3D bio-printing, there exists a gap in available characterisation tools for the non-destructive inspection of biological constructs in the three-dimensional domain, on the macroscopic scale, and with fast data acquisition times. Quantitative ultrasound imaging is a suitable characterization technique for providing essential information on the development of tissue engineered constructs. These results provide a detailed and comprehensive guide on the capabilities and limitations of the technique.
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Hidrogéis/química , Processamento de Imagem Assistida por Computador , Impressão Tridimensional , Alicerces Teciduais/química , Animais , Células PC12 , Ratos , UltrassonografiaRESUMO
Using a customized ultrasound setup we investigate the feasibility of using a contactless approach to study the bulk mechanical properties of swollen hydrogels. The study involved two different hydrogels, gelatin methacrylate (GelMa) and green algae extract methacrylate (GAEM), which were prepared to provide materials with varying modulus and different swelling properties. Two approaches have been developed. In the first case, ultrasound was compared to Young's modulus measured by indentation. It was found that can be linearly related to indentation modulus values only when the hydrogel swelling ratio is taken into account. In the second approach, an exponential dependency between swelled thickness and indentation modulus was found. This is representative for each hydrogel and purification method in addition to being independent of the conditions used within the toughness range explored. The results of this study indicate that a simple thickness measurement via the proposed approach can provide a direct relationship to Young's modulus upon calibration.
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Hidrogéis , Teste de Materiais/métodos , Fenômenos Mecânicos , Metacrilatos/química , Ondas UltrassônicasRESUMO
This work presents a study on a capacitively coupled contactless conductivity detector (C4D) for micron-sized fibers. Following a previous report on the qualitative application of C4D for fibers, the present study provides a thorough analysis of the signal response to fiber conductivity. Using reduced graphene oxide (RGO) fibers, the detector response as a function of fiber length, cross-sectional area and resistance has been investigated. To study the effect of insulating coatings, Parylene-coated RGO fibers were also investigated. In addition, measurements were performed in different coupling environments, such as in a capillary tube or air. The analysis of the measured data allowed the determination of the C4D conductivity of various RGO fibers, and the correlation with contact methods through empirical relationships to be determined. It was found that the detection limit and sensitivity of resistance measurements are mainly dependent on the sensor design, and also on the fiber properties. The detection threshold can be defined as the ratio of the coupling impedance to fiber resistance. In our case, the detection limit was found for impedance ratios equal to 14. This limit sets a functioning mode in C4D for fibers, which may be used as an area or resistance detector for the impedance ratio above or below the detection threshold. A semi-log linear response of the fiber resistance to the voltage output was found for impedance ratios between 2.66 and 0.63. These impedance ratios may serve as a reference for designing C4D, depending on the fibers to be tested and the analytical information needed. In summary, we suggest that C4D has the capacity to emerge as a new characterisation tool for micron-sized fibers, due to its applicability to any conductive material, ease of use, and the contactless nature of the measurement.
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The use of capacitively coupled contactless conductivity detection (C(4)D) for the characterisation of thin conductive graphene fibres, graphene composite fibres, and graphene coated fibrous materials is demonstrated for the first time. Within a few seconds, the non-destructive C(4)D detector provides a profile of the longetudinal physical homogeneity of the fibre, as well as extra information regarding fibre mophology and composition. In addition to the theoretical considerations related to the factors affect the output signal, this work evaluates the properties of graphene fibres using scanning C(4)D following the manufacturing process of wet-spinning. Furthermore, conductive graphene-coated fibrous materials and the effectiveness of the coating and reduction procedures applied could be investigated. Apart from the application of C(4)D in the monitoring of such processes, the feasibility of this small, highly sensitive and rapidly-responsive detector to monitor strain and elasticity responses of conductive and elastomeric composite fibres for applications in motion sensing, biomedical monitoring, and stretchable electronics was also demonstrated.
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We present a series of non-stoichiometric cadmium sulfide quantum-dot (QD) models. Using density functional theory (DFT) and semi-empirical molecular orbital (MO) calculations, we explore the ligand binding and exchange chemistry of these models. Their surface morphology allows for these processes to be rationalized on the atomic scale. This is corroborated by ultraviolet-visible (UV-vis), infrared (IR), and inductively coupled plasma-optical emission spectroscopy (ICP-OES).
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Pontos Quânticos , Semicondutores , Espectrofotometria AtômicaRESUMO
We have investigated the role of linker molecules in quantum-dot-sensitized solar cells (QDSSCs) using density-functional theory (DFT) and experiments. Linkers not only govern the number of attached QDs but also influence charge separation, recombination, and transport. Understanding their behavior is therefore not straightforward. DFT calculations show that mercaptopropionic acid (MPA) and cysteine (Cys) exhibit characteristic binding configurations on TiO(2) surfaces. This information is used to optimize the cell assembly process, yielding Cys-based cells that significantly outperform MPA cells, and reach power conversion efficiencies (PCE) as high as 2.7% under AM 1.5 illumination. Importantly, the structural information from theory also helps understand the cause for this improved performance.