Pore size estimation in axon-mimicking microfibers with diffusion-relaxation MRI.

PURPOSE: This study aims to evaluate two distinct approaches for fiber radius estimation using diffusion-relaxation MRI data acquired in biomimetic microfiber phantoms that mimic hollow axons. The methods considered are the spherical mean power-law approach and a T2-based pore size estimation technique. THEORY AND METHODS: A general diffusion-relaxation theoretical model for the spherical mean signal from water molecules within a distribution of cylinders with varying radii was introduced, encompassing the evaluated models as particular cases. Additionally, a new numerical approach was presented for estimating effective radii (i.e., MRI-visible mean radii) from the ground truth radii distributions, not reliant on previous theoretical approximations and adaptable to various acquisition sequences. The ground truth radii were obtained from scanning electron microscope images. RESULTS: Both methods show a linear relationship between effective radii estimated from MRI data and ground-truth radii distributions, although some discrepancies were observed. The spherical mean power-law method overestimated fiber radii. Conversely, the T2-based method exhibited higher sensitivity to smaller fiber radii, but faced limitations in accurately estimating the radius in one particular phantom, possibly because of material-specific relaxation changes. CONCLUSION: The study demonstrates the feasibility of both techniques to predict pore sizes of hollow microfibers. The T2-based technique, unlike the spherical mean power-law method, does not demand ultra-high diffusion gradients, but requires calibration with known radius distributions. This research contributes to the ongoing development and evaluation of neuroimaging techniques for fiber radius estimation, highlights the advantages and limitations of both methods, and provides datasets for reproducible research.

Direct jet coaxial electrospinning of axon-mimicking fibers for diffusion tensor imaging.

Hollow polymer microfibers with variable microstructural and hydrophilic properties were proposed as building elements to create axon-mimicking phantoms for validation of diffusion tensor imaging (DTI). The axon-mimicking microfibers were fabricated in a mm-thick 3D anisotropic fiber strip, by direct jet coaxial electrospinning of PCL/polysiloxane-based surfactant (PSi) mixture as shell and polyethylene oxide (PEO) as core. Hydrophilic PCL-PSi fiber strips were first obtained by carefully selecting appropriate solvents for the core and appropriate fiber collector rotating and transverse speeds. The porous cross-section and anisotropic orientation of axon-mimicking fibers were then quantitatively evaluated using two ImageJ plugins-nearest distance (ND) and directionality based on their scanning electron microscopy (SEM) images. Third, axon-mimicking phantom was constructed from PCL-PSi fiber strips with variable porous-section and fiber orientation and tested on a 3T clinical MR scanner. The relationship between DTI measurements (mean diffusivity [MD] and fractional anisotropy [FA]) of phantom samples and their pore size and fiber orientation was investigated. Two key microstructural parameters of axon-mimicking phantoms including normalized pore distance and dispersion of fiber orientation could well interpret the variations in DTI measurements. Two PCL-PSi phantom samples made from different regions of the same fiber strips were found to have similar MD and FA values, indicating that the direct jet coaxial electrospun fiber strips had consistent microstructure. More importantly, the MD and FA values of the developed axon-mimicking phantoms were mostly in the biologically relevant range.

Validating pore size estimates in a complex microfiber environment on a human MRI system.

PURPOSE: Recent advances in diffusion-weighted MRI provide "restricted diffusion signal fraction" and restricting pore size estimates. Materials based on co-electrospun oriented hollow cylinders have been introduced to provide validation for such methods. This study extends this work, exploring accuracy and repeatability using an extended acquisition on a 300 mT/m gradient human MRI scanner, in substrates closely mimicking tissue, that is, non-circular cross-sections, intra-voxel fiber crossing, intra-voxel distributions of pore-sizes, and smaller pore-sizes overall. METHODS: In a single-blind experiment, diffusion-weighted data were collected from a biomimetic phantom on a 3T Connectom system using multiple gradient directions/diffusion times. Repeated scans established short-term and long-term repeatability. The total scan time (54 min) matched similar protocols used in human studies. The number of distinct fiber populations was estimated using spherical deconvolution, and median pore size estimated through the combination of CHARMED and AxCaliber3D framework. Diffusion-based estimates were compared with measurements derived from scanning electron microscopy. RESULTS: The phantom contained substrates with different orientations, fiber configurations, and pore size distributions. Irrespective of one or two populations within the voxel, the pore-size estimates (~5 µm) and orientation-estimates showed excellent agreement with the median values of pore-size derived from scanning electron microscope and phantom configuration. Measurement repeatability depended on substrate complexity, with lower values seen in samples containing crossing-fibers. Sample-level repeatability was found to be good. CONCLUSION: While no phantom mimics tissue completely, this study takes a step closer to validating diffusion microstructure measurements for use in vivo by demonstrating the ability to quantify microgeometry in relatively complex configurations.

Coaxial electrospun biomimetic copolymer fibres for application in diffusion magnetic resonance imaging.

Objective. The use of diffusion magnetic resonance imaging (dMRI) opens the door to characterizing brain microstructure because water diffusion is anisotropic in axonal fibres in brain white matter and is sensitive to tissue microstructural changes. As dMRI becomes more sophisticated and microstructurally informative, it has become increasingly important to use a reference object (usually called an imaging phantom) for validation of dMRI. This study aims to develop axon-mimicking physical phantoms from biocopolymers and assess their feasibility for validating dMRI measurements.Approach. We employed a simple and one-step method-coaxial electrospinning-to prepare axon-mimicking hollow microfibres from polycaprolactone-b-polyethylene glycol (PCL-b-PEG) and poly(D, L-lactide-co-glycolic) acid (PLGA), and used them as building elements to create axon-mimicking phantoms. Electrospinning was firstly conducted using two types of PCL-b-PEG and two types of PLGA with different molecular weights in various solvents, with different polymer concentrations, for determining their spinnability. Polymer/solvent concentration combinations with good fibre spinnability were used as the shell material in the following co-electrospinning process in which the polyethylene oxide polymer was used as the core material. Following the microstructural characterization of both electrospun and co-electrospun fibres using optical and electron microscopy, two prototype phantoms were constructed from co-electrospun anisotropic hollow microfibres after inserting them into water-filled test tubes.Main results. Hollow microfibres that mimic the axon microstructure were successfully prepared from the appropriate core and shell material combinations. dMRI measurements of two phantoms on a 7 tesla (T) pre-clinical scanner revealed that diffusivity and anisotropy measurements are in the range of brain white matter.Significance. This feasibility study showed that co-electrospun PCL-b-PEG and PLGA microfibre-based axon-mimicking phantoms could be used in the validation of dMRI methods which seek to characterize white matter microstructure.

Co-electrospraying of tumour cell mimicking hollow polymeric microspheres for diffusion magnetic resonance imaging.

Diffusion magnetic resonance imaging (dMRI) is considered as a useful tool to study solid tumours. However, the interpretation of dMRI signal and validation of quantitative measurements of is challenging. One way to address these challenges is by using a standard reference material that can mimic tumour cell microstructure. There is a growing interest in using hollow polymeric microspheres, mainly prepared by multiple steps, as mimics of cells in healthy and diseased tissue. The present work reports on tumour cell-mimicking materials composed of hollow microspheres for application as a standard material in dMRI. These microspheres were prepared via one-step co-electrospraying process. The shell material was poly(d,l-lactic-co-glycolic acid) (PLGA) polymers with different molecule weights and/or ratios of glycolic acid-to-lactic, while the core was polyethylene glycol (PEG) or ethylene glycol. The resultant co-electrosprayed products were characterised by optical microscopy, scanning electron microscopy (SEM) and synchrotron X-ray micro-CT. These products were found to have variable structures and morphologies, e.g. from spherical particles with/without surface hole, through beaded fibres to smooth fibres, which mainly depend on PLGA composition and core materials. Only the shell material of PLGA polymer with ester terminated, Mw 50,000-75,000 g mol-1, and lactide:glycolide 85:15 formed hollow microspheres via the co-electrospraying process using the core material of 8 wt% PEG/chloroform as the core. A water-filled test object (or phantom) was designed and constructed from samples of the material generated from co-electrosprayed PLGA microspheres and tested on a 7 T MRI scanner. The preliminary MRI results provide evidence that hollow PLGA microspheres can restrict/hinder water diffusion as cells do in tumour tissue, implying that the phantom may be suitable for use as a quantitative validation and calibration tool for dMRI.

Stability and reproducibility of co-electrospun brain-mimicking phantoms for quality assurance of diffusion MRI sequences.

Grey and white matter mimicking phantoms are important for assessing variations in diffusion MR measures at a single time point and over an extended period of time. This work investigates the stability of brain-mimicking microfibre phantoms and reproducibility of their MR derived diffusion parameters. The microfibres were produced by co-electrospinning and characterized by scanning electron microscopy (SEM). Grey matter and white matter phantoms were constructed from random and aligned microfibres, respectively. MR data were acquired from these phantoms over a period of 33 months. SEM images revealed that only small changes in fibre microstructure occurred over 30 months. The coefficient of variation in MR measurements across all time-points was between 1.6% and 3.4% for MD across all phantoms and FA in white matter phantoms. This was within the limits expected for intra-scanner variability, thereby confirming phantom stability over 33 months. These specialised diffusion phantoms may be used in a clinical environment for intra and inter-site quality assurance purposes, and for validation of quantitative diffusion biomarkers.

Axon mimicking hydrophilic hollow polycaprolactone microfibres for diffusion magnetic resonance imaging.

Highly hydrophilic hollow polycaprolactone (PCL) microfibres were developed as building elements to create tissue-mimicking test objects (phantoms) for validation of diffusion magnetic resonance imaging (MRI). These microfibres were fabricated by the co-electrospinning of PCL-polysiloxane-based surfactant (PSi) mixture as shell and polyethylene oxide as core. The addition of PSi had a significant effect on the size of resultant electrospun fibres and the formation of hollow microfibres. The presence of PSi in both co-electrospun PCL microfibre surface and cross-section, revealed by X-ray energy dispersive spectroscopy (EDX), enabled water to wet these fibres completely (i.e., zero contact angle) and remained active for up to 12 months after immersing in water. PCL and PCL-PSi fibres with uniaxial orientation were constructed into water-filled phantoms. MR measurement revealed that water molecules diffuse anisotropically in the PCL-PSi phantom. Co-electrospun hollow PCL-PSi microfibres have desirable hydrophilic properties for the construction of a new generation of tissue-mimicking dMRI phantoms.

A biomimetic tumor tissue phantom for validating diffusion-weighted MRI measurements.

PURPOSE: To develop a biomimetic tumor tissue phantom which more closely reflects water diffusion in biological tissue than previously used phantoms, and to evaluate the stability of the phantom and its potential as a tool for validating diffusion-weighted (DW) MRI measurements. METHODS: Coaxial-electrospraying was used to generate micron-sized hollow polymer spheres, which mimic cells. The bulk structure was immersed in water, providing a DW-MRI phantom whose apparent diffusion coefficient (ADC) and microstructural properties were evaluated over a period of 10 months. Independent characterization of the phantom's microstructure was performed using scanning electron microscopy (SEM). The repeatability of the construction process was investigated by generating a second phantom, which underwent high resolution synchrotron-CT as well as SEM and MR scans. RESULTS: ADC values were stable (coefficients of variation (CoVs) < 5%), and varied with diffusion time, with average values of 1.44 ± 0.03 µm2 /ms (Δ = 12 ms) and 1.20 ± 0.05 µm2 /ms (Δ = 45 ms). Microstructural parameters showed greater variability (CoVs up to 13%), with evidence of bias in sphere size estimates. Similar trends were observed in the second phantom. CONCLUSION: A novel biomimetic phantom has been developed and shown to be stable over 10 months. It is envisaged that such phantoms will be used for further investigation of microstructural models relevant to characterizing tumor tissue, and may also find application in evaluating acquisition protocols and comparing DW-MRI-derived biomarkers obtained from different scanners at different sites. Magn Reson Med 80:147-158, 2018. © 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Hollow Polycaprolactone Microspheres with/without a Single Surface Hole by Co-Electrospraying.

We describe the co-electrospraying of hollow microspheres from a polycaprolactone (PCL) shell solution and various core solutions including water, cyclohexane, poly(ethylene oxide) (PEO), and polyethylene glycol (PEG), using different collectors. The morphologies of the resultant microspheres were characterized by scanning electron microscopy (SEM), confocal microscopy, and nano-X-ray computed tomography (nano-XCT). The core/shell solution miscibility played an important role in the co-electrospraying process and the formation of microsphere structures. Spherical particles were more likely to be produced from miscible combinations of core/shell solutions than from immiscible ones. Hollow PCL microspheres with a single hole in their surfaces were produced when an ethanol bath was used as the collector. The mechanism by which the core/shell structure is transformed into single-hole hollow microspheres is proposed to be primarily based on the evaporation through the shell and extraction by ethanol of the core solution and is described in detail. Additionally, we present a 3D macroscopic tubular structure composed of hollow PCL microspheres, directly assembled on a copper wire collector during co-electrospraying. SEM and nano-XCT confirm that microspheres in the 3D bulk structure remain hollow.

Preparation and characterization of polycaprolactone microspheres by electrospraying.

The ability to reproducibly produce and effectively collect electrosprayed polymeric microspheres with controlled morphology and size in bulk form is challenging. In this study, microparticles were produced by electrospraying polycaprolactone (PCL) of various molecular weights and solution concentrations in chloroform, and by collecting materials on different substrates. The resultant PCL microparticles were characterized by optical and electron microscopy to investigate the effect of molecular weight, solution concentration, applied voltage, working distance, and flow rate on their morphology and size. The work demonstrates the key role of a moderate molecular weight and/or solution concentration in the formation of spherical PCL particles via an electrospraying process. Increasing the applied voltage was found to produce smaller and more uniform PCL microparticles. There was a relatively low increase in the particle average size with an increase in the working distance and flow rate. Four types of substrates were adopted to collect electrosprayed PCL particles: a glass slide, aluminium foil, liquid bath, and copper wire. Unlike 2D bulk structures collected on the other substrates, a 3D tubular structure of microspheres was formed on the copper wire which could find application in the construction of 3D tumor mimics.

Biomimetic phantom for cardiac diffusion MRI.

PURPOSE: Diffusion magnetic resonance imaging (MRI) is increasingly used to characterize cardiac tissue microstructure, necessitating the use of physiologically relevant phantoms for methods development. Existing phantoms are generally simplistic and mostly simulate diffusion in the brain. Thus, there is a need for phantoms mimicking diffusion in cardiac tissue. MATERIALS AND METHODS: A biomimetic phantom composed of hollow microfibers generated using co-electrospinning was developed to mimic myocardial diffusion properties and fiber and sheet orientations. Diffusion tensor imaging was carried out at monthly intervals over 4 months at 9.4T. 3D fiber tracking was performed using the phantom and compared with fiber tracking in an ex vivo rat heart. RESULTS: The mean apparent diffusion coefficient and fractional anisotropy of the phantom remained stable over the 4-month period, with mean values of 7.53 ± 0.16 × 10(-4) mm(2) /s and 0.388 ± 0.007, respectively. Fiber tracking of the 1st and 3rd eigenvectors generated analogous results to the fiber and sheet-normal direction respectively, found in the left ventricular myocardium. CONCLUSION: A biomimetic phantom simulating diffusion in the heart was designed and built. This could aid development and validation of novel diffusion MRI methods for investigating cardiac microstructure, decrease the number of animals and patients needed for methods development, and improve quality control in longitudinal and multicenter cardiac diffusion MRI studies.

Production and cross-sectional characterization of aligned co-electrospun hollow microfibrous bulk assemblies.

The development of co-electrospun (co-ES) hollow microfibrous assemblies of an appreciable thickness is critical for many practical applications, including filtration membranes and tissue-mimicking scaffolds. In this study, thick uniaxially aligned hollow microfibrous assemblies forming fiber bundles and strips were prepared by co-ES of polycaprolactone (PCL) and polyethylene oxide (PEO) as shell and core materials, respectively. Hollow microfiber bundles were deposited on a fixed rotating disc, which resulted in non-controllable cross-sectional shapes on a macroscopic scale. In comparison, fiber strips were produced with tuneable thickness and width by additionally employing an x-y translation stage in co-ES. Scanning electron microscopy (SEM) images of cross-sections of fiber assemblies were analyzed to investigate the effects of production time (from 0.5 h to 12 h), core flow rate (from 0.8 mL/h to 2.0 mL/h) and/or translation speed (from 0.2 mm/s to 5 mm/s) on the pores and porosity. We observed significant changes in pore size and shape with core flow rate but the influence of production time varied; five strips produced under the same conditions had reasonably good size and porosity reproducibility; pore sizes didn't vary significantly from strip bottom to surface, although the porosity gradually decreased and then returned to the initial level.

Multistep microsatellite mutation leading to father-child mismatch of FGA locus in a case of non-exclusion parentage.

A non-exclusion paternity case with a mismatch in the autosomal short tandem repeats (STR) locus FGA is reported. The genotypes of the suspected father, the mother and the questioned child in FGA locus were 18/25, 20/26 and 20/22, respectively. Examination of 38 autosomal STR loci revealed no mismatches, and the paternity index is up to 1.3618×10(6). The haplotype of 16 Y chromosomal STR in the child matched completely with that of the father. These results suggested that the suspected father is the biological father of the child and that a rare three- or four-step microsatellite mutation had occurred in the paternal allele of FGA.

Multistep microsatellite mutation in a case of non-exclusion parentage.

A non-exclusion paternity with multistep mutation in the locus D5S818 was reported. Examination of 39 autosomal short tandem repeats (STR) loci revealed a mismatch of the maternally or paternally transmitted allele in the locus D5S818 in the questioned child. The composition of the alleles of this locus in the mother, the questioned child and the alleged father are 11/13, 7/13 and 13, respectively. The sequence analysis of the regions flanking the locus D5S818 of the mother, the questioned child and the alleged father excluded the possibility of null allele as a cause of the allelic mismatch in the child. The combined paternity index of 39 autosomal STRs is up to 2.461×10(9). Genotyping of sixteen Y-STR loci in the questioned child matched completely with the alleged father. The results prove that the alleged father is the biological father of the questioned child with four-step or six-step microsatellite mutation in the locus D5S818.

Biomimetic phantom for the validation of diffusion magnetic resonance imaging.

PURPOSE: A range of advanced diffusion MRI (dMRI) techniques are currently in development which characterize the orientation of white matter fibers using diffusion tensor imaging (DTI). There is a need for a physical phantom with microstructural features of the brain's white matter to help validate these methods. METHODS: Hollow, co-electrospun, aligned fibers with a tuneable size distribution have been produced in bulk and with an MR visible solvent infused into the pores. The morphology and size of the phantoms was assessed using scanning electron microscopy (SEM) and compared with DTI results obtained on both a clinical and preclinical scanner. RESULTS: By varying inner diameter of the phantom fibers (from SEM: 9.5 µm, 11.9 µm, 13.4 µm) the radial diffusivity and fractional anisotropy, calculated from DTI, vary between 0.38 ± 0.05 × 10(3) and 0.61 ± 0.06 × 10(3) cm s(-1) and between 0.45 ± 0.05 and 0.33 ± 0.04, respectively. CONCLUSION: We envisage that these materials will be used for the validation of novel and established methods within the field of diffusion MRI, as well as for routine quality assurance purposes and for establishing scanner performance in multicenter trials.

Diffusion tensor MRI phantom exhibits anomalous diffusion.

This paper reports diffusion weighted MRI measurements of cyclohexane in a novel diffusion tensor MRI phantom composed of hollow coaxial electrospun fibers (average diameter 10.2 µm). Recent studies of the phantom demonstrated its potential as a calibration standard at low b values (less than 1000 s/mm<;sup>2<;/sup>) for mean diffusivity and fractional anisotropy. In this paper, we extend the characterization of cyclohexane diffusion in this heterogeneous, anisotropic material to high b values (up to 5000 s/mm<;sup>2<;/sup>), where the apparent diffusive motion of the cyclohexane exhibits anomalous behavior (i.e., the molecular mean squared displacement increases with time raised to the fractional power 2α/ß). Diffusion tensor MRI was performed at 9.4 T using an Agilent imaging scanner and the data fit to a fractional order Mittag-Leffler (generalized exponential) decay model. Diffusion along the fibers was found to be Gaussian (2α/ß=l), while diffusion across the fibers was sub-diffusive (2α/ß<;l). Fiber tract reconstruction of the data was consistent with scanning electron micrograph images of the material. These studies suggest that this phantom material may be used to calibrate MR systems in both the normal (Gaussian) and anomalous diffusion regimes.

The CONNECT project: Combining macro- and micro-structure.

In recent years, diffusion MRI has become an extremely important tool for studying the morphology of living brain tissue, as it provides unique insights into both its macrostructure and microstructure. Recent applications of diffusion MRI aimed to characterize the structural connectome using tractography to infer connectivity between brain regions. In parallel to the development of tractography, additional diffusion MRI based frameworks (CHARMED, AxCaliber, ActiveAx) were developed enabling the extraction of a multitude of micro-structural parameters (axon diameter distribution, mean axonal diameter and axonal density). This unique insight into both tissue microstructure and connectivity has enormous potential value in understanding the structure and organization of the brain as well as providing unique insights to abnormalities that underpin disease states. The CONNECT (Consortium Of Neuroimagers for the Non-invasive Exploration of brain Connectivity and Tracts) project aimed to combine tractography and micro-structural measures of the living human brain in order to obtain a better estimate of the connectome, while also striving to extend validation of these measurements. This paper summarizes the project and describes the perspective of using micro-structural measures to study the connectome.

Coaxially electrospun axon-mimicking fibers for diffusion magnetic resonance imaging.

The study of brain structure and connectivity using diffusion magnetic resonance imaging (dMRI) has recently gained substantial interest. However, the use of dMRI still faces major challenges because of the lack of standard materials for validation. The present work reports on brain tissue-mimetic materials composed of hollow microfibers for application as a standard material in dMRI. These hollow fibers were fabricated via a simple and one-step coaxial electrospining (co-ES) process. Poly(ε-caprolactone) (PCL) and polyethylene oxide (PEO) were employed as shell and core materials, respectively, to achieve the most stable co-ES process. These co-ES hollow PCL fibers have different inner diameters, which mainly depend on the flow rate of the core solution and have the potential to cover the size range of the brain tissue we aimed to mimic. Co-ES aligned hollow PCL fibers were characterized using optical and electron microscopy and tested as brain white matter mimics on a high-field magnetic resonance imaging (MRI) scanner. To the best of our knowledge, this is the first time that co-ES hollow fibers have been successfully used as a tissue mimic or phantom in diffusion MRI. The results of the present study provide evidence that this phantom can mimic the dMRI behavior of cellular barriers imposed by axonal cell membranes and myelin; the measured diffusivity is compatible with that of in vivo biological tissues. Together these results suggest the potential use of co-ES hollow microfibers as tissue-mimicking phantoms in the field of medical imaging.