Fabio Bianchi

Probing multi-scale mechanical damage in connective tissues using X-ray diffraction

The accumulation of microstructural collagen damage following repetitive loading is linked to painful and debilitating tendon injuries. As a hierarchical, semi-crystalline material, collagen mechanics can be studied using X-ray diffraction. The aim of the study was to describe multi-structural changes in tendon collagen following controlled plastic damage (5% permanent strain). We used small angle X-ray scattering (SAXS) to interrogate the spacing of collagen molecules within a fibril, and wide angle X-ray scattering (WAXS) to measure molecular strains under macroscopic loading. Simultaneous recordings of SAXS and WAXS patterns, together with whole-tissue strain in physiologically hydrated rat-tail tendons were made during increments of in situ tensile loading. Results showed that while tissue level modulus was unchanged, fibril modulus decreased significantly, and molecular modulus significantly increased. Further, analysis of higher order SAXS peaks suggested structural changes in the gap and overlap regions, possibly localising the damage to molecular cross-links. Our results provide new insight into the fundamental damage processes at work in collagenous tissues and point to new directions for their mitigation and repair.nnnSTATEMENT OF SIGNIFICANCEnThis article reports the first in situ loading synchrotron studies on mechanical damage in collagenous tissues. We provide new insight into the nano- and micro-structural mechanisms of damage processes. Pre-damaged tendons showed differential alteration of moduli at macro, micro and nano-scales as measured using X-ray scattering techniques. Detailed analysis of higher order diffraction peaks suggested damage is localised to molecular cross-links. The results are consistent with previous X-ray scattering studies of tendons and also with recent thermal stability studies on damaged material. Detailed understanding of damage mechanisms is essential in the development of new therapies promoting tissue repair.

Adaptive internal impedance control for stable walking on uncertain visco-elastic terrains

This paper investigates how a walker could maintain the variability of an arbitrary set of state variables within desired margins while walking on an uncertain soft terrain. The state variables are dynamically related to the visco-elastic impedance parameters of the body on a given set of uncertain soft terrains using internal memory primitives. A rimless wheel, a walker in its simplest form, is used to perform numerical simulations based on analytical dynamic models and hardware experiments to test a novel algorithm. The rimless wheel model is widely used by the legged locomotion research community to understand basic collision and energetics during passive dynamic walking. Very often, variability of punctuated force perturbations across collisions between the legs and the ground cause uncertain steady state dynamics of walking. This leads to the existence of a finite probability that certain state variables can reach unstable regions. Such phenomenon is known as metastability of walking. In this case, we actuate the rimless wheel with a constant torque leaving it to develop any speed profile for a given visco-elastic impedance distribution of the ground and its own vertical visco-elastic impedance that pushes the rimless wheel against the ground. Here we measure the robustness of the novel algorithm by its ability to shift the distribution of collision forces to a safer region in order to minimize the probability of reaching a given critical force threshold. Our analysis shows that the generalization of the variability of walking in different regions of the internal and external visco-elastic impedance spaces can simplify the computational challenges of robust walking on uncertain visco-elastic terrains.

Rapid and efficient differentiation of functional motor neurons from human iPSC for neural injury modelling

Primary rodent neurons and immortalised cell lines have overwhelmingly been used for in vitro studies of traumatic injury to peripheral and central neurons, but have some limitations of physiological accuracy. Motor neurons (MN) derived from human induced pluripotent stem cells (iPSCs) enable the generation of cell models with features relevant to human physiology. To facilitate this, it is desirable that MN protocols both rapidly and efficiently differentiate human iPSCs into electrophysiologically active MNs. In this study, we present a simple, rapid protocol for differentiation of human iPSCs into functional spinal (lower) MNs, involving only adherent culture and use of small molecules for directed differentiation, with the ultimate aim of rapid production of electrophysiologically functional cells for short-term neural injury experiments. We show successful differentiation in two unrelated iPSC lines, by quantifying neural-specific marker expression, and by evaluating cell functionality at different maturation stages by calcium imaging and patch clamping. Differentiated neurons were shown to be electrophysiologically altered by uniaxial mechanical deformation. Spontaneous network activity decreased with applied stretch, indicating aberrant network connectivity. These results demonstrate the feasibility of this rapid, simple protocol for differentiating iPSC-derived MNs, suitable for in vitro neural injury studies focussing on electrophysiological alterations caused by mechanical deformation or trauma.

Evidences of the Effect of GO and rGO in PCL Membranes on the Differentiation and Maturation of Human Neural Progenitor Cells

The effect of doping graphene oxide (GO) and reduced graphene oxide (rGO) into poly(ε-caprolactone) (PCL) membranes prepared by solvent induced phase separation is evaluated in terms of nanomaterial distribution and compatibility with neural stem cell growth and functional differentiation. Raman spectra analyses demonstrate the homogeneous distribution of GO on the membrane surface while rGO concentration increases gradually toward the center of the membrane thickness. This behavior is associated with electrostatic repulsion that PCL exerted toward the polar GO and its affinity for the non-polar rGO. In vitro cell studies using human induced pluripotent cell derived neural progenitor cells (NPCs) show that rGO increases marker expression of NPCs differentiation with respect to GO (significantly to tissue culture plate (TCP)). Moreover, the distinctive nanomaterials distribution defines the cell-to-nanomaterial interaction on the PCL membranes: GO nanomaterials on the membrane surface favor higher number of active matured neurons, while PCL/rGO membranes present cells with significantly higher magnitude of neural activity compared to TCP and PCL/GO despite there being no direct contact of rGO with the cells on the membrane surface. Overall, this work evidences the important role of rGO electrical properties on the stimulation of neural cell electro-activity on PCL membrane scaffolds.

Probing multi-scale mechanics of peripheral nerve collagen and myelin by X-ray diffraction

Peripheral nerves are continuously subjected to mechanical forces, both during everyday movement and as a result of traumatic events. Current mechanical models focus on explaining the macroscopic behaviour of the tissue, but do not investigate how tissue strain translates to deformations at the microstructural level. Predicting the effect of macro-scale loading can help explain changes in nerve function and suggest new strategies for prevention and therapy. The aim of this study was to determine the relationship between macroscopic tensile loading and micro scale deformation in structures thought to be mechanically active in peripheral nerves: the myelin sheath enveloping axons, and axially aligned epineurial collagen fibrils. The microstructure was probed using X-ray diffraction during in situ tensile loading, measuring the micro-scale deformation in collagen and myelin, combined with high definition macroscopic video extensiometry. At a tissue level, tensile loading elongates nerves axially, whilst simultaneously compressing circumferentially. The non-linear behaviour observed in both directions is evidence, circumferentially, that the nerve core components have the ability to rearrange before bearing load and axially, of a recruitment process in epineurial collagen. At the molecular level, axially aligned epineurial collagen fibrils are strained, whilst the myelin sheath enveloping axons is compressed circumferentially. During induced compression, the myelin sheath shows high circumferential stiffness, indicating a possible role in mechanical protection of axons. The myelin sheath is deformed from low loads, despite the non-linearity of whole tissue compression, indicating more than one mechanism contributing to myelin compression. Epineurial collagen shows similar load-bearing characteristics to those of other collagenous connective tissues. This new microstructural knowledge is key to understand peripheral nerve mechanical behaviour, and will support new regenerative strategies for traumatic and repetitive injury.

Strain partitioning between nerves and axons: Estimating axonal strain using sodium channel staining in intact peripheral nerves

BACKGROUNDnPeripheral nerves carry afferent and efferent signals between the central nervous system and the periphery of the body. When nerves are strained above physiological levels, conduction blocks occur, resulting in debilitating loss of motor and sensory function. Understanding the effects of strain on nerve function requires knowledge of the multi-scale mechanical behaviour of the tissue, and how this is transferred to the cellular environment.nnnNEW METHODnThe aim of this work was to establish a technique to measure the partitioning of strain between tissue and axons in axially loaded peripheral nerves. This was achieved by staining extracellular domains of sodium channels clustered at nodes of Ranvier, without altering tissue mechanical properties by fixation or permeabilisation.nnnRESULTSnStained nerves were imaged by multi-photon microscopy during in situ tensile straining, and digital image correlation was used to measure axonal strain with increasing tissue strain. Strain was partitioned between tissue and axon scales by an average factor of 0.55.nnnCOMPARISONS WITH EXISTING METHODSnThis technique allows non-invasive probing of cell-level strain within the physiological tissue environment.nnnCONCLUSIONSnThis technique can help understand the mechanisms behind the onset of conduction blocks in injured peripheral nerves, as well as to evaluate changes in multi-scale mechanical properties in diseased nerves.

Engineered method for directional growth of muscle sheets on electrospun fibers: Engineered method for directional growth

Research on the neuromuscular junction (NMJ) and its function and development spans over a century. However, researchers are limited in their ability to conduct experimentation on this highly specialized synapse between motor neurons and muscle fibers, as NMJs are not easily accessible outside the body. The aim of this work is to provide a reliable and reproducible muscle sheet model for in vitro NMJ study. A novel culture system was designed by engineering a method for the directional growth of myofiber sheets, using muscle progenitor cells cultured on electrospun fiber networks. Myoblastic C2C12 cells cultured on suspended aligned fibers were found to maintain directionality, with alignment angle standard deviations approximately two-thirds lower on fibers than on regular culture surfaces. Morphological studies found nuclei and cytoskeleton aspect ratios to be elongated by 20 and 150%, respectively. Furthermore, neurons were shown to form innervation patterns parallel to suspended fibers when co-cultured on developed muscle sheets, with alignment angle standard deviations three times lower compared with those on typical surfaces. The effect of agrin on samples was quantified through the slow release of agrin medium, encapsulated in alginate pellets and imbedded within culture chambers. Samples exposed to agrin showed significantly increased percentage of AChR-covered area. The developed model has potential to serve as the basis for synaptogenesis and NMJ studies, providing a novel approach to bio-artificial muscle alignment and setting the groundwork for further investigations in innervation.

Aligned electrospun fibers for neural patterning.

ObjectivesTo test a 3D approach for neural network formation, alignment, and patterning that is reproducible and sufficiently stable to allow for easy manipulation.ResultsA novel cell culture system was designed by engineering a method for the directional growth of neurons. This uses NG108-15 neuroblastoma x glioma hybrid cells cultured on suspended and aligned electrospun fibers. These fiber networks improved cellular directionality, with alignment angle standard deviations significantly lower on fibers than on regular culture surfaces. Morphological studies found nuclear aspect ratios and cell projection lengths to be unchanged, indicating that cells maintained neural morphology while growing on fibers and forming a 3D network. Furthermore, fibronectin-coated fibers enhanced neurite extensions for all investigated time points. Differentiated neurons exhibited significant increases in average neurite lengths 96xa0h post plating, and formed neurite extensions parallel to suspended fibers, as visualized through scanning electron microscopy.ConclusionsThe developed model has the potential to serve as the basis for advanced 3D studies, providing an original approach to neural network patterning and setting the groundwork for further investigations into functionality.

Stable walking on variable visco-elastic terrains using meta-parameters for passive state migration

This paper investigates how a walker could estimate the variability of an arbitrary set of state variables when migrating on visco-elastic grounds. The state variables are a function of both the visco-elastic settings of the walking body and soft terrain parameters. A rimless wheel model was developed using a Lagrangian approach in order to obtain analytical solutions for migration across ground conditions. An algorithm was then developed to determine the steady value of the variables as a function of the difference in ground and hub parameters involved in the migration. A generalised migration metaparameter, Δg, function of this difference, was then extrapolated using polynomial approximation. Δg can be used to estimate the expected variability at a state given information on actual and previous ground parameters. A second parameter, Δh, describing local variability of a given state on a given terrain, is used to generate a predictive algorithm capable of stabilising the rimless wheel setup when subject to an abrupt change in ground parameters. We actuate the rimless wheel with a constant torque leaving it to develop any speed profile for a given visco-elastic impedance distribution of the ground and its own vertical visco-elastic impedance. The ground is altered depending on the two migration meta-parameters (Δg and Δr), ensuring both local and migration stability.