Micro- and Nanofluidic pH Sensors Based on Electrodiffusioosmosis.

Recently, various kinds of micro- and nanofluidic functional devices have been proposed, where a large surface-to-volume ratio often plays an important role in nanoscale ion transport phenomena. Ionic current analysis methods for ions, molecules, nanoparticles, and biological cells have attracted significant attention. In this study, focusing on ionic current rectification (ICR) caused by the separation of cation and anion transport in nanochannels, we successfully induce electrodiffusioosmosis with concentration differences between protons separated by nanochannels. The proton concentration in sample solutions is quantitatively evaluated in the range from pH 1.68 to 10.01 with a slope of 243 mV/pH at a galvanostatic current of 3 nA. Herein, three types of micro- and nanochannels are proposed to improve the stability and measurement accuracy of the current-voltage characteristics, and the ICR effects on pH analysis are evaluated. It is found that a nanochannel filled with polyethylene glycol exhibits increased impedance and an improved ICR ratio. The present principle is expected to be applicable to various types of ions.

High-throughput mechanical phenotyping and transcriptomics of single cells.

The molecular system regulating cellular mechanical properties remains unexplored at single-cell resolution mainly due to a limited ability to combine mechanophenotyping with unbiased transcriptional screening. Here, we describe an electroporation-based lipid-bilayer assay for cell surface tension and transcriptomics (ELASTomics), a method in which oligonucleotide-labelled macromolecules are imported into cells via nanopore electroporation to assess the mechanical state of the cell surface and are enumerated by sequencing. ELASTomics can be readily integrated with existing single-cell sequencing approaches and enables the joint study of cell surface mechanics and underlying transcriptional regulation at an unprecedented resolution. We validate ELASTomics via analysis of cancer cell lines from various malignancies and show that the method can accurately identify cell types and assess cell surface tension. ELASTomics enables exploration of the relationships between cell surface tension, surface proteins, and transcripts along cell lineages differentiating from the haematopoietic progenitor cells of mice. We study the surface mechanics of cellular senescence and demonstrate that RRAD regulates cell surface tension in senescent TIG-1 cells. ELASTomics provides a unique opportunity to profile the mechanical and molecular phenotypes of single cells and can dissect the interplay among these in a range of biological contexts.

Extracting the gradient component of the gamma index using the Lie derivative method.

Objective. The gamma index (γ) has been extensively investigated in the medical physics and applied in clinical practice. However,γhas a significant limitation when used to evaluate the dose-gradient region, leading to inconveniences, particularly in stereotactic radiotherapy (SRT). This study proposes a novel evaluation method combined withγto extract clinically problematic dose-gradient regions caused by irradiation including certain errors.Approach. A flow-vector field in the dose distribution is obtained when the dose is considered a scalar potential. Using the Lie derivative from differential geometry, we definedL,S, andUto evaluate the intensity, vorticity, and flow amount of deviation between two dose distributions, respectively. These metrics multiplied byγ(γL,γS,γU), along with the threshold valueσ, were verified in the ideal SRT case and in a clinical case of irradiation near the brainstem region using radiochromic films. Moreover, Moran's gradient index (MGI), Bakai's χ factor, and the structural similarity index (SSIM) were investigated for comparisons.Main results. A highL-metric value mainly extracted high-dose-gradient induced deviations, which was supported by highSandUmetrics observed as a robust deviation and an influence of the dose-gradient, respectively. TheS-metric also denotes the measured similarity between the compared dose distributions. In theγdistribution,γLsensitively detected the dose-gradient region in the film measurement, despite the presence of noise. The thresholdσsuccessfully extracted the gradient-error region whereγ> 1 analysis underestimated, andσ= 0.1 (plan) andσ= 0.001 (film measurement) were obtained according to the compared resolutions. However, the MGI, χ, and SSIM failed to detect the clinically interested region.Significance. Although further studies are required to clarify the error details, this study demonstrated that the Lie derivative method provided a novel perspective for the identifying gradient-induced error regions and enabled enhanced and clinically significant evaluations ofγ.

Deep learning-based detection of patients with bone metastasis from Japanese radiology reports.

PURPOSE: Deep learning (DL) is a state-of-the-art technique for developing artificial intelligence in various domains and it improves the performance of natural language processing (NLP). Therefore, we aimed to develop a DL-based NLP model that classifies the status of bone metastasis (BM) in radiology reports to detect patients with BM. MATERIALS AND METHODS: The DL-based NLP model was developed by training long short-term memory using 1,749 free-text radiology reports written in Japanese. We adopted five-fold cross-validation and used 200 reports for testing the five models. The accuracy, sensitivity, specificity, precision, and area under the receiver operating characteristics curve (AUROC) were used for the model evaluation. RESULTS: The developed model demonstrated classification performance with mean ± standard deviation of 0.912 ± 0.012, 0.924 ± 0.029, 0.901 ± 0.014, 0.898 ± 0.012, and 0.968 ± 0.004 for accuracy, sensitivity, specificity, precision, and AUROC, respectively. CONCLUSION: The proposed DL-based NLP model may help in the early and efficient detection of patients with BM.

Detecting hand joint ankylosis and subluxation in radiographic images using deep learning: A step in the development of an automatic radiographic scoring system for joint destruction.

We propose a wrist joint subluxation/ankylosis classification model for an automatic radiographic scoring system for X-ray images. In managing rheumatoid arthritis, the evaluation of joint destruction is important. The modified total Sharp score (mTSS), which is conventionally used to evaluate joint destruction of the hands and feet, should ideally be automated because the required time depends on the skill of the evaluator, and there is variability between evaluators. Since joint subluxation and ankylosis are given a large score in mTSS, we aimed to estimate subluxation and ankylosis using a deep neural network as a first step in developing an automatic radiographic scoring system for joint destruction. We randomly extracted 216 hand X-ray images from an electronic medical record system for the learning experiments. These images were acquired from patients who visited the rheumatology department of Keio University Hospital in 2015. Using our newly developed annotation tool, well-trained rheumatologists and radiologists labeled the mTSS to the wrist, metacarpal phalangeal joints, and proximal interphalangeal joints included in the images. We identified 21 X-ray images containing one or more subluxation joints and 42 X-ray images with ankylosis. To predict subluxation/ankylosis, we conducted five-fold cross-validation with deep neural network models: AlexNet, ResNet, DenseNet, and Vision Transformer. The best performance on wrist subluxation/ankylosis classification was as follows: accuracy, precision, recall, F1 value, and AUC were 0.97±0.01/0.89±0.04, 0.92±0.12/0.77±0.15, 0.77±0.16/0.71±0.13, 0.82±0.11/0.72±0.09, and 0.92±0.08/0.85±0.07, respectively. The classification model based on a deep neural network was trained with a relatively small dataset; however, it showed good accuracy. In conclusion, we provided data collection and model training schemes for mTSS prediction and showed an important contribution to building an automated scoring system.

Periodontal diseases assessed by average bone resorption are associated with microvascular complications in patients with type 2 diabetes.

Periodontal disease often develops in patients with diabetes, and further exacerbated with diabetic complications. It would be clinically important to clarify the relationship between diabetic microvascular diseases and periodontal disease. This study aimed to evaluate the association between periodontal disease and diabetic complications in patients with type 2 diabetes with poor glycemic control. A total of 447 patients with type 2 diabetes hospitalized at Rakuwakai Otowa Hospital, Japan, were initially recruited in this study. After excluding 134 patients who lacked clinical data or were edentulous, 312 were included in our study. The severity of periodontal disease was evaluated based on the average bone resorption rate. Patients with diabetic nephropathy developed severe periodontal disease (multivariate-adjusted odds ratio, 3.00 [95% CI 1.41-5.19]). Diabetic neuropathy was positively associated with the severity of periodontal disease; the multivariate-adjusted odds ratio (95% CI) was 1.62 (0.87‒2.99) for moderate and 4.26 (2.21‒8.20) for severe periodontal disease. In contrast, diabetic retinopathy was linked with moderate periodontal disease (multivariate-adjusted odds ratio 2.23 [95% CI 1.10-4.10]), but not with severe conditions (multivariate-adjusted odds ratio 0.92 [95% CI 0.67-3.07]). In conclusion, periodontal disease, evaluated by average bone resorption rate, was associated with diabetic nephropathy and neuropathy. Supplementary Information: The online version contains supplementary material available at 10.1007/s13340-022-00591-0.

Local Electric Field and Electrical Conductivity Analysis Using a Glass Microelectrode.

Transport phenomena in microfluidic chips are induced by electric fields and electrolyte concentrations. Liquid flows are often affected by ionic currents driven by electric fields in narrow channels, which are applied in microelectromechanical systems, microreactors, lab-on-a-chip, and so forth. Even though numerical studies to evaluate those local fields have been reported, measurement methods seem to be under construction. To deeply understand the dynamics of ions at the microscale, measurement techniques are necessary to be developed. In this study, we propose a novel method to directly measure electrical potential differences in liquids, local electric fields, and electrical conductivities, using a glass microelectrode. Scanning an electrolyte solution, for example, KCl solutions, with a 1 µm tip under constant ionic current conditions, a potential difference in liquids is locally measured with a micrometer-scale resolution. The conductivity of KCl solutions ranging from 0.56 to 100 mM is evaluated from electric fields locally measured, and errors are within 5% compared with the reference values. It is found that the present method enables us to directly measure local electric fields under constant current and that the electrical conductivity is quantitatively evaluated. Furthermore, it is suggested that the present method is available for various electrical analyses without calibration procedures before measurements.

Visualization of Optical Vortex Forces Acting on Au Nanoparticles Transported in Nanofluidic Channels.

The optical manipulation of nanoscale objects via structured light has attracted significant attention for its various applications, as well as for its fundamental physics. In such cases, the detailed behavior of nano-objects driven by optical forces must be precisely predicted and controlled, despite the thermal fluctuation of small particles in liquids. In this study, the optical forces of an optical vortex acting on gold nanoparticles (Au NPs) are visualized using dark-field microscopic observations in a nanofluidic channel with strictly suppressed forced convection. Manipulating Au NPs with an optical vortex allows the evaluation of the three optical force components, namely, gradient, scattering, and absorption forces, from the in-plane trajectory. We develop a Langevin dynamics simulation model coupled with Rayleigh scattering theory and compare the theoretical results with the experimental ones. Experimental results using Au NPs with diameters of 80-150 nm indicate that our experimental method can determine the radial trapping stiffness and tangential force with accuracies on the order of 0.1 fN/nm and 1 fN, respectively. Our experimental method will contribute to broadening not only applications of the optical-vortex manipulation of nano-objects, but also investigations of optical properties on unknown nanoscale materials via optical force analyses.

Synchronized resistive-pulse analysis with flow visualization for single micro- and nanoscale objects driven by optical vortex in double orifice.

Resistive-pulse analysis is a powerful tool for identifying micro- and nanoscale objects. For low-concentration specimens, the pulse responses are rare, and it is difficult to obtain a sufficient number of electrical waveforms to clearly characterize the targets and reduce noise. In this study, we conducted a periodic resistive-pulse analysis using an optical vortex and a double orifice, which repetitively senses a single micro- or nanoscale target particle with a diameter ranging from 700 nm to 2 [Formula: see text]m. The periodic motion results in the accumulation of a sufficient number of waveforms within a short period. Acquired pulses show periodic ionic-current drops associated with the translocation events through each orifice. Furthermore, a transparent fluidic device allows us to synchronously average the waveforms by the microscopic observation of the translocation events and improve the signal-to-noise ratio. By this method, we succeed in distinguishing single particle diameters. Additionally, the results of measured signals and the simultaneous high-speed observations are used to quantitatively and systematically discuss the effect of the complex fluid flow in the orifices on the amplitude of the resistive pulse. The synchronized resistive-pulse analysis by the optical vortex with the flow visualization improves the pulse-acquisition rate for a single specific particle and accuracy of the analysis, refining the micro- and nanoscale object identification.

Mathematical model of the auditory nerve response to stimulation by a micro-machined cochlea.

We report a novel mathematical model of an artificial auditory system consisting of a micro-machined cochlea and the auditory nerve response it evokes. The modeled micro-machined cochlea is one previously realized experimentally by mimicking functions of the cochlea [Shintaku et al, Sens. Actuat. 158 (2010) 183-192; Inaoka et al, Proc. Natl. Acad. Sci. USA 108 (2011) 18390-18395]. First, from the viewpoint of mechanical engineering, the frequency characteristics of a model device were experimentally investigated to develop an artificial basilar membrane based on a spring-mass-damper system. In addition, a nonlinear feedback controller mimicking the function of the outer hair cells was incorporated in this experimental system. That is, the developed device reproduces the proportional relationship between the oscillation amplitude of the basilar membrane and the cube root of the sound pressure observed in the mammalian auditory system, which is what enables it to have a wide dynamic range, and the characteristics of the control performance were evaluated numerically and experimentally. Furthermore, the stimulation of the auditory nerve by the micro-machined cochlea was investigated using the present mathematical model, and the simulation results were compared with our previous experimental results from animal testing [Shintaku et al, J. Biomech. Sci. Eng. 8 (2013) 198-208]. The simulation results were found to be in reasonably good agreement with those from the previous animal test; namely, there exists a threshold at which the excitation of the nerve starts and a saturation value for the firing rate under a large input. The proposed numerical model was able to qualitatively reproduce the results of the animal test with the micro-machined cochlea and is thus expected to guide the evaluation of micro-machined cochleae for future animal experiments.

Characterisation of the static offset in the travelling wave in the cochlear basal turn.

In mammals, audition is triggered by travelling waves that are evoked by acoustic stimuli in the cochlear partition, a structure containing sensory hair cells and a basilar membrane. When the cochlea is stimulated by a pure tone of low frequency, a static offset occurs in the vibration in the apical turn. In the high-frequency region at the cochlear base, multi-tone stimuli induce a quadratic distortion product in the vibrations that suggests the presence of an offset. However, vibrations below 100 Hz, including a static offset, have not been directly measured there. We therefore constructed an interferometer for detecting motion at low frequencies including 0 Hz. We applied the interferometer to record vibrations from the cochlear base of guinea pigs in response to pure tones. When the animals were exposed to sound at an intensity of 70 dB or higher, we recorded a static offset of the sinusoidally vibrating cochlear partition by more than 1 nm towards the scala vestibuli. The offset's magnitude grew monotonically as the stimuli intensified. When stimulus frequency was varied, the response peaked around the best frequency, the frequency that maximised the vibration amplitude at threshold sound pressure. These characteristics are consistent with those found in the low-frequency region and are therefore likely common across the cochlea. The offset diminished markedly when the somatic motility of mechanosensitive outer hair cells, the force-generating machinery that amplifies the sinusoidal vibrations, was pharmacologically blocked. Therefore, the partition offset appears to be linked to the electromotile contraction of outer hair cells.

Development of glass micro-electrodes for local electric field, electrical conductivity, and pH measurements.

In micro- and nanofluidic devices, liquid flows are often influenced by ionic currents generated by electric fields in narrow channels, which is an electrokinetic phenomenon. Various technologies have been developed that are analogous to semiconductor devices, such as diodes and field effect transistors. On the other hand, measurement techniques for local electric fields in such narrow channels have not yet been established. In the present study, electric fields in liquids are locally measured using glass micro-electrodes with 1-µm diameter tips, which are constructed by pulling a glass tube. By scanning a liquid poured into a channel by glass micro-electrodes, the potential difference in a liquid can be determined with a spatial resolution of the size of the glass tip. As a result, the electrical conductivity of sample solutions can be quantitatively evaluated. Furthermore, combining two glass capillaries filled with buffer solutions of different concentrations, an ionic diode that rectifies the proton conduction direction is constructed, and the possibility of pH measurement is also demonstrated. Under constant-current conditions, pH values ranging from 1.68 to 9.18 can be determined more quickly and stably than with conventional methods that depend on the proton selectivity of glass electrodes under equilibrium conditions.

Effect of hydrodynamic inter-particle interaction on the orbital motion of dielectric nanoparticles driven by an optical vortex.

We experimentally and theoretically characterize dielectric nano- and microparticle orbital motion induced by an optical vortex of the Laguerre-Gaussian beam. The key to stable orbiting of dielectric nanoparticles is hydrodynamic inter-particle interaction and microscale confinement of slit-like fluidic channels. As the number of particles in the orbit increases, the hydrodynamic inter-particle interaction accelerates orbital motion to overcome the inherent thermal fluctuation. The microscale confinement in the beam propagation direction helps to increase the number of trapped particles by reducing their probability of escape from the optical trap. The diameter of the orbit increases as the azimuthal mode of the optical vortex increases, but the orbital speed is shown to be insensitive to the azimuthal mode, provided that the number density of the particles in the orbit is same. We use experiments, simulation, and theory to quantify and compare the contributions of thermal fluctuation such as diffusion coefficients, optical forces, and hydrodynamic inter-particle interaction, and show that the hydrodynamic effect is significant for circumferential motion. The optical vortex beam with hydrodynamic inter-particle interaction and microscale confinement will contribute to biosciences and nanotechnology by aiding in developing new methods of manipulating dielectric and nanoscale biological samples in optical trapping communities.

Separation of Nano- and Microparticle Flows Using Thermophoresis in Branched Microfluidic Channels.

Particle flow separation is a useful technique in lab-on-a-chip applications to selectively transport dispersed phases to a desired branch in microfluidic devices. The present study aims to demonstrate both nano- and microparticle flow separation using microscale thermophoresis at a Y-shaped branch in microfluidic channels. Microscale thermophoresis is the transport of tiny particles induced by a temperature gradient in fluids where the temperature variation is localized in the region of micrometer order. Localized temperature increases near the branch are achieved using the Joule heat from a thin-film micro electrode embedded in the bottom wall of the microfluidic channel. The inlet flow of the particle dispersion is divided into two outlet flows which are controlled to possess the same flow rates at the symmetric branches. The particle flow into one of the outlets is blocked by microscale thermophoresis since the particles are repelled from the hot region in the experimental conditions used here. As a result, only the solvent at one of outlets and the residual particle dispersion at the other outlet are obtained, i.e., the separation of particles flows is achieved. A simple model to explain the dynamic behavior of the nanoparticle distribution near the electrode is proposed, and a qualitative agreement with the experimental results is obtained. The proposed method can be easily combined with standard microfluidic devices and is expected to facilitate the development of novel particle separation and filtration technologies.

Uncovering dehydration in cytochrome c refolding from urea- and guanidine hydrochloride-denatured unfolded state by high pressure spectroscopy.

To investigate the dehydration associated with protein folding, the partial molar volume changes for protein unfolding (ΔV u) in cytochrome c (Cyt c) were determined using high pressure absorption spectroscopy. ΔV u values for the unfolding to urea- and guanidine hydrochloride (GdnHCl)-denatured Cyt c were estimated to be 56±5 and 29±1 mL mol-1, respectively. Considering that the volume change for hydration of hydrophobic groups is positive and that Cyt c has a covalently bonded heme, a positive ΔV u reflects the primary contribution of the hydration of heme. Because of the marked tendency of guanidium ions to interact with hydrophobic groups, a smaller number of water molecules were hydrated with hydrophobic groups in GdnHCl-denatured Cyt c than in urea-denatured Cyt c, resulting in the smaller positive ΔV u. On the other hand, urea is a relatively weak denaturant and urea-denatured Cyt c is not completely hydrated, which retains the partially folded structures. To unfold such partial structures, we introduced a mutation near the heme binding site, His26, to Gln, resulting in a negatively shifted ΔV u (4±2 mL mol-1) in urea-denatured Cyt c. The formation of the more solvated and less structured state in the urea-denatured mutant enhanced hydration to the hydrophilic groups in the unfolding process. Therefore, we confirmed the hydration of amino acid residues in the protein unfolding of Cyt c by estimating ΔV u, which allows us to discuss the hydrated structures in the denatured states of proteins.

Generation and Control of Electrohydrodynamic Flows in Aqueous Electrolyte Solutions.

To drive electrohydrodynamic (EHD) flows in aqueous solutions, the separation of cation and anion transport pathways is essential because a directed electric body force has to be induced by ionic motions in liquid. On the other hand, positive and negative charges attract each other, and electroneutrality is maintained everywhere in equilibrium conditions. Furthermore, an increase in an applied voltage has to be suppressed to avoid water electrolysis, which causes the solutions to become unstable. Usually, EHD flows can be induced in non-aqueous solutions by applying extremely high voltages, such as tens of kV, to inject electrical charges. In this study, two methods are introduced to generate EHD flows induced by electrical charge separations in aqueous solutions, where two liquid phases are separated by an ion-exchange membrane. Due to a difference in the ionic mobility in the membrane, ion concentration polarization is induced between both sides of the membrane. In this study, we demonstrate two methods. (i) The relaxation of ion concentration gradients occurs via a flow channel that penetrates an ion-exchange membrane, where the transport of the slower species in the membrane selectively becomes dominant in the flow channel. This is a driving force to generate an EHD flow in the liquid. (ii) A long waiting time for the diffusion of ions passing through the ion-exchange membrane enables the generation of an ion-dragged flow by externally applying an electric field. Ions concentrated in a flow channel of a 1 x 1 mm2 cross-section determine the direction of the liquid flow, corresponding to the electrophoretic transport pathways. In both methods, the electric voltage difference required for an EHD flow generation is drastically reduced to near 2 V by rectifying the ion transport pathways.

Cation-induced electrohydrodynamic flow in aqueous solutions.

Recently, single-molecule manipulation techniques in micro- and nanofluidic channels have attracted significant attention. To precisely control the transport velocity, the dynamics of the surrounding liquid must be understood in addition to the behavior of the target particles. Some unknowns about interactions between electrolyte ions and solvents remain to be clarified from a microscopic viewpoint. Herein, we propose a technique to generate a liquid flow driven by ion transport phenomena, the so-called electrohydrodynamic (EHD) flow, where electrolyte ions are dialyzed using a cation-exchange membrane. With this method, it is possible to apply an electric body force in liquids, which is different from electroosmotic flows that are limited to ion transport in electric double layers, and is expected to be a good candidate for detailed control of liquid flows in micro- and nanofluidic channels. To collect basic design data based on the knowledge of microscopic fluid dynamics of the present technique, a mathematical model of an EHD flow dragged by electrical carriers in an ionic current is developed and results are compared with experimental data. In our experiments, EHD flows are efficiently driven by applied electric fields in a cation dominant current. To induce such an EHD flow, the externally applied electric potential can be drastically reduced to 2.0 V in comparison with previous methods because we do not need an excessively high voltage to inject electrical charges into liquids. This method enables us to induce EHD flows in aqueous solutions and is expected to open the door to low-voltage driven liquid flow control.

Bullous pemphigoid associated with dipeptidyl peptidase-4 inhibitors: A report of five cases.

Bullous pemphigoid (BP) is an autoimmune blistering skin disorder. Recently, BP induced by dipeptidyl peptidase-4 (DPP-4) inhibitors has been a concern. Although DPP-4 inhibitors are commonly used in the Asian population because of their safety and efficacy, BP associated with DPP-4 inhibitors is sometimes seen in clinical settings. Here, we report five Japanese cases of BP associated with the agents. In the present cases, BP occurred in older adults using four different DPP-4 inhibitors, which showed various clinical manifestations in terms of latency period for BP, sex, glycemic control and diabetes duration. Withdrawal of DPP-4 inhibitors was effective in improving BP, and achieved remission even in cases requiring oral steroid administration and intravenous immunoglobulin therapy. Clinicians should note the importance of early diagnosis of this clinical condition and initiate prompt withdrawal of DPP-4 inhibitors.

An injectable non-cross-linked hyaluronic-acid gel containing therapeutic spheroids of human adipose-derived stem cells.

For chronic wounds, the delivery of stem cells in spheroidal structures can enhance graft survival and stem cell potency. We describe an easy method for the 3D culture of adipose-derived stem/stromal cells (ASCs) to prepare a ready-to-use injectable. We transferred suspensions of monolayer-cultured ASCs to a syringe containing hyaluronic acid (HA) gel, and then incubated the syringe as a 3D culture vessel. Spheroids of cells formed after 12 h. We found that 6 × 106 ASCs/ml in 3% HA gel achieved the highest spheroid density with appropriate spheroid sizes (20-100 µm). Immunocytology revealed that the stem cell markers, NANOG, OCT3/4, SOX-2, and SSEA-3 were up-regulated in the ASC spheroids compared with those in nonadherent-dish spheroids or in monolayer cultured ASCs. In delayed wound healing mice models, diabetic ulcers treated with ASC spheroids demonstrated faster wound epithelialization with thicker dermis than those treated with vehicle alone or monolayer cultured ASCs. In irradiated skin ulcers in immunodeficient mice, ASC spheroids exhibited faster healing and outstanding angiogenic potential partly by direct differentiation into α-SMA+ pericytes. Our method of 3D in-syringe HA gel culture produced clinically relevant amounts of ready-to-inject human ASC microspheroids that exhibited superior stemness in vitro and therapeutic efficacy in pathological wound repair in vivo.