Development of an integrated environmental monitoring protocol for SARS-CoV-2 contamination. Applications at the IRCSS San Martino Polyclinic Hospital in Genoa, Italy.

SCIENTIFIC BACKGROUND: Environmental sampling of SARS-CoV-2 is a fundamental tool for evaluating the effectiveness of non-specific prophylaxis measures in counteracting virus spread. The purpose of our work was to evaluate the effectiveness of the different sampling methods in the hospital setting to assess their correlation with the structural, functional, and operational situation of the monitored departments and to define the dynamics of the spread of the virus in indoor environments. METHODS: The monitoring (air bubbling sampling, surface wipe test) was carried out at the San Martino Polyclinic Hospital (Genoa, Italy) in the period since April 2020 to June 2021. The presence of viral RNA in the collected samples was evaluated by qPCR. The infection capacity of the samples collected was also evaluated by an in vitro challenge test on cells sensitive to SARS-CoV-2 infection. RESULTS: The percentage of positivity with respect to the number of tests performed (sensitivity) were air bubbler 50%, wipe test 17%, and challenge test 11%. Only 20% of the samples tested positive in the wipe test and 43% of the samples tested positive in the bubbler sampling were also positive in the challenge test. All the positivity obtained was detected at a distance of less than 2 m and height of less than 1.5 from COVID-19 patients. CONCLUSIONS: Environmental contamination from SARS-CoV-2 detected at the San Martino Polyclinic Hospital is found lower than similar assessments performed in other hospitals both in Italy and abroad. Our study predicted that environmental monitoring of SARS-CoV-2 must be carried out in an integrated way by not using a single sampling method, as each individual test has a different biological significance and performance. However, the virus detected by wipe test only is often a degraded viral fragment and not an intact infecting virion.

Coarse-grained elastic network modelling: A fast and stable numerical tool to characterize mesenchymal stem cells subjected to AFM nanoindentation measurements.

The knowledge of the mechanical properties is the starting point to study the mechanobiology of mesenchymal stem cells and to understand the relationships linking biophysical stimuli to the cellular differentiation process. In experimental biology, Atomic Force Microscopy (AFM) is a common technique for measuring these mechanical properties. In this paper we present an alternative approach for extracting common mechanical parameters, such as the Young's modulus of cell components, starting from AFM nanoindentation measurements conducted on human mesenchymal stem cells. In a virtual environment, a geometrical model of a stem cell was converted in a highly deformable Coarse-Grained Elastic Network Model (CG-ENM) to reproduce the real AFM experiment and retrieve the related force-indentation curve. An ad-hoc optimization algorithm perturbed the local stiffness values of the springs, subdivided in several functional regions, until the computed force-indentation curve replicated the experimental one. After this curve matching, the extraction of global Young's moduli was performed for different stem cell samples. The algorithm was capable to distinguish the material properties of different subcellular components such as the cell cortex and the cytoskeleton. The numerical results predicted with the elastic network model were then compared to those obtained from hertzian contact theory and Finite Element Method (FEM) for the same case studies, showing an optimal agreement and a highly reduced computational cost. The proposed simulation flow seems to be an accurate, fast and stable method for understanding the mechanical behavior of soft biological materials, even for subcellular levels of detail. Moreover, the elastic network modelling allows shortening the computational times to approximately 33% of the time required by a traditional FEM simulation performed using elements with size comparable to that of springs.

The biomechanics of the umbilical cord Wharton Jelly: Roles in hemodynamic proficiency and resistance to compression.

The umbilical cord is a complex structure containing three vessels, one straight vein and two coiled arteries, encased by the Wharton Jelly (WJ) a spongy structure made of collagen and hydrated macromolecules. Fetal blood reaches the placenta through the arteries and flows back to the fetus through the vein. The role of the WJ in maintaining cord circulation proficiency and the ultimate reason for arterial coiling still lack of reasonable mechanistic interpretations. We performed biaxial tension tests and evidenced significant differences in the mechanical properties of the core and peripheral WJ. The core region, located between the arteries and the vein, resulted rather stiffer close to the fetus. Finite element modelling and optimization based inverse method were used to create 2D and 3D models of the cord and to simulate stress distribution in different hemodynamic conditions, compressive loads and arterial coiling. We recorded a facilitated stress transmission from the arteries to the vein through the soft core of periplacental WJ. This condition generates a pressure gradient that boosts the venous backflow circulation towards the fetus. Peripheral WJ allows arteries to act as pressure buffering chambers during the cardiac diastole and helps to dissipate compressive forces away from vessels. Altered WJ biomechanics may represent the structural basis of cord vulnerability in many high-risk clinical conditions.

A hybrid characterization framework to determine the visco-hyperelastic properties of a porcine zona pellucida.

The zona pellucida (ZP) is a specialized extracellular matrix surrounding the developing oocyte. This thick matrix consists of various types of glycoprotein that play different roles in the fertilization process. Nowadays, several techniques are available for assessing ZP's mechanical response. The basic assumption behind these methods is that the ZP behaves like an elastic body: hence, dissipative forces are neglected and Young's modulus remains unaffected by probe dynamics. However, dissipative forces are strongly regulated by the slippage of ZP chains past one another while reaction forces related to elastic deformations (driven by the ability of each chain to stretch) depend on the ZP structure (i.e. number of cross-links and distances between knots). Although viscous reaction forces generated by the ZP are one of the main factors regulating sperm transit, their peculiar behaviour along the ZP structure remains poorly understood and rarely investigated. In order to overcome this limitation, a novel visco-hyperelastic model describing the porcine ZP reaction forces generated by nanoindentations at different probe rates is developed and verified in this study. Visco-hyperelastic parameters of porcine ZP membranes are determined by means of a hybrid characterization framework combining atomic force microscopy nanoindentation measurements, nonlinear finite-element analysis and nonlinear optimization. Remarkably, it is possible to separate the contributions of hyperelastic and viscous terms to ZP mechanical response and evaluate the error made in the determination of ZP mechanical properties if viscous effects were not considered.

A model of tissue differentiation and bone remodelling in fractured vertebrae treated with minimally invasive percutaneous fixation.

In spite of the consolidated clinical use of minimally invasive percutaneous fixation techniques, little is reported in the literature providing a mechanobiological explanation for how the design of fixation devices can affect the healing process within fractured vertebrae. The aim of this study was to develop a multi-scale mechano-regulation model capable of predicting how the patterns of tissue differentiation within a vertebral fracture change in the presence or in the absence of fixation devices and how the dimensions of the device, and the materials it is made from (Ti-6Al-4V alloy and cobalt chrome alloy) can affect the outcome of the healing process. The macro-scale model simulates the spinal segment L3-L4-L5, including the fractured body of the L4 vertebra, while the micro-scale model represents a fractured portion of cancellous bone. The macro-scale model also includes a minimally invasive percutaneous fixation device. The model predicts that fixation devices significantly shorten healing times. Increasing values of the rod diameter D and decreasing values of its radius of curvature R lead to shorter durations of the healing period. Manufacturing the rods in cobalt chrome alloy is also predicted to reduce slightly the healing period by providing greater mechanical stability within the fracture callus.

Finite element method (FEM), mechanobiology and biomimetic scaffolds in bone tissue engineering.

Techniques of bone reconstructive surgery are largely based on conventional, non-cell-based therapies that rely on the use of durable materials from outside the patient's body. In contrast to conventional materials, bone tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences towards the development of biological substitutes that restore, maintain, or improve bone tissue function. Bone tissue engineering has led to great expectations for clinical surgery or various diseases that cannot be solved with traditional devices. For example, critical-sized defects in bone, whether induced by primary tumor resection, trauma, or selective surgery have in many cases presented insurmountable challenges to the current gold standard treatment for bone repair. The primary purpose of bone tissue engineering is to apply engineering principles to incite and promote the natural healing process of bone which does not occur in critical-sized defects. The total market for bone tissue regeneration and repair was valued at $1.1 billion in 2007 and is projected to increase to nearly $1.6 billion by 2014.Usually, temporary biomimetic scaffolds are utilized for accommodating cell growth and bone tissue genesis. The scaffold has to promote biological processes such as the production of extra-cellular matrix and vascularisation, furthermore the scaffold has to withstand the mechanical loads acting on it and to transfer them to the natural tissues located in the vicinity. The design of a scaffold for the guided regeneration of a bony tissue requires a multidisciplinary approach. Finite element method and mechanobiology can be used in an integrated approach to find the optimal parameters governing bone scaffold performance.In this paper, a review of the studies that through a combined use of finite element method and mechano-regulation algorithms described the possible patterns of tissue differentiation in biomimetic scaffolds for bone tissue engineering is given. Firstly, the generalities of the finite element method of structural analysis are outlined; second, the issues related to the generation of a finite element model of a given anatomical site or of a bone scaffold are discussed; thirdly, the principles on which mechanobiology is based, the principal theories as well as the main applications of mechano-regulation models in bone tissue engineering are described; finally, the limitations of the mechanobiological models and the future perspectives are indicated.

Analysis of the performance of different orthodontic devices for mandibular symphyseal distraction osteogenesis.

The aim of this study was to investigate the performance of different orthodontic devices for mandibular symphyseal distraction osteogenesis (MSDO). Two performance parameters were analysed, the first of which concerned the stability guaranteed by a distractor in the fracture gap under mastication loads and the second the level of reliability with which a distractor transfers a given expansion to the mandibular bone, inasmuch as the more reliable the device the smaller the difference between the degree of expansion provided to the device and the displacement achieved on the mandibular arch. Hence, a non-linear finite element (FE) model of a human mandible with different devices (tooth-borne, bone-borne, and hybrid) was constructed and then utilized to assess the structural behaviour of the mandibular bone under distraction and mastication loads. An ad hoc algorithm was developed to simulate progressive expansion of the devices; a distraction protocol comprising a 10 day latency period and a 6 day distraction period was hypothesized. The first hypothetical expansion given to the device was 2 mm, and the five subsequent expansions were 1 mm. The results showed that the hybrid device was the most stable appliance under mastication loads, followed by the tooth- and bone-borne devices. However, parasitic rotations of the mandibular arms caused by mastication might counteract the benefits of distraction. The tooth-borne device was found to have the highest reliability in transferring expansion to the mandibular bone. For this device, mandibular expansion was less than the nominal aperture of the distractor by no more than 15 per cent. Lower values of reliability were achieved with the bone-borne device. As the values of the aperture of the appliances increased, the stability guaranteed in the fracture gap increased while the reliability in transferring expansion to the mandibular arch decreased.

Numerical/experimental analysis of the stress field around miniscrews for orthodontic anchorage.

The aims of this study were to analyse the stress distribution developing around an orthodontic miniscrew (OM) inserted into the maxilla and to determine the stress field changes for different screw lengths and for different levels of osseointegration occurring at the bone/screw interface. An integrated experimental/numerical approach was adopted. Using the photoelastic technique, the stress field arising in the bone after screw insertion and the application of the initial orthodontic load was assessed. The finite element (FE) method was used to determine the stress acting in the bony tissue after a given time following screw application, when, for the viscoelastic relaxation effects, the only stress field remaining was that due to the application of the orthodontic load. Different levels of osseointegration were hypothesized. Photoelastic analyses showed that stress distribution does not change significantly for moderate initial orthodontic loads. From the FE simulations, it was found that critical conditions occur for screws 14 mm long with an orthodontic load of 2 N. The optimal screw length seems to be 9 mm. For such a dimension, small stress values were found as well as low risk of lesion to the anatomical structures.

Finite element analysis of cancellous bone failure in the vertebral body of healthy and osteoporotic subjects.

The aim of this work is to assess the fracture risk prediction of the cancellous bone in the body of a lumbar vertebra when the mechanical parameters of the bone, i.e. stiffness, porosity, and strength anisotropy, of elderly and osteoporotic subjects are considered. For this purpose, a non-linear three-dimensional continuum-based finite element model of the lumbar functional spinal unit L4-L5 was created and strength analyses of the spongy tissue of the vertebral body were carried out. A fabric-dependent strength criterion, which accounts for the micro-architecture of the cancellous bone, based on histomorphometric analyses was used. The strength analyses have shown that the cancellous bone of none of the subject types undergoes failure under loading applied during normal daily life like axial compression; however, bone failure occurs for the osteoporotic segment, subjected to a combination of the compression preloading and moments in the sagittal or in the frontal plane, which are conditions that may not be considered to occur 'daily'. In particular, critical stress conditions are met because of the high porosity values in the horizontal direction within the cancellous bone. The computational approach presented in the paper can potentially predict the material fracture risk of the cancellous bone in the vertebral body and it may be usefully employed to draw failure maps representing, for a given micro-architecture of the spongy tissue, the critical loading conditions (forces and moments) that may lead to such a risk. This approach could be further developed in order to assess the effectiveness of biomedical devices within an engineering approach to the clinical problem of the spinal diseases.

Tissue differentiation and bone regeneration in an osteotomized mandible: a computational analysis of the latency period.

Mandibular symphyseal distraction osteogenesis is a common clinical procedure to modify the geometrical shape of the mandible for correcting problems of dental overcrowding and arch shrinkage. In spite of consolidated clinical use, questions remain concerning the optimal latency period and the influence of mastication loading on osteogenesis within the callus prior to the first distraction of the mandible. This work utilized a mechano-regulation model to assess bone regeneration within the callus of an osteotomized mandible. A 3D model of the mandible was reconstructed from CT scan data and meshed using poroelastic finite elements (FE). The stimulus regulating tissue differentiation within the callus was hypothesized to be a function of the strain and fluid flow computed by the FE model. This model was then used to analyse tissue differentiation during a 15-day latency period, defined as the time between the day of the osteotomy and the day when the first distraction is given to the device. The following predictions are made: (1) the mastication forces generated during the latency period support osteogenesis in certain regions of the callus, and that during the latency period the percentage of progenitor cells differentiating into osteoblasts increases; (2) reducing the mastication load by 70% during the latency period increases the number of progenitor cells differentiating into osteoblasts; (3) the stiffness of new tissue increases at a slower rate on the side of bone callus next to the occlusion of the mandibular ramus which could cause asymmetries in the bone tissue formation with respect to the middle sagittal plane. Although the model predicts that the mastication loading generates such asymmetries, their effects on the spatial distribution of callus mechanical properties are insignificant for typical latency periods used clinically. It is also predicted that a latency period of longer than a week will increase the risk of premature bone union across the callus.

The influence of expansion rates on mandibular distraction osteogenesis: a computational analysis.

Mandibular distraction osteogenesis is a clinical procedure used for modifying the mandibular geometry when problems of dental overcrowding and arch shrinkage occur. The objective of this study is to use a computational model of tissue differentiation to examine the influence of the rate of distraction on bone re-growth within the fracture callus of a human mandible submitted to symphyseal distraction osteogenesis. A 3D model of the mandible is reconstructed from CT scan data and meshed into finite elements. Two different mastication loadings have been investigated: a 'full' mastication load and a 'reduced' mastication load where the action of each muscle was reduced by 70%. Four different distraction rates were analyzed: 0.6, 1.2, 2, and 3 mm/day, allowing a total displacement of 6 mm. In the early stages of the distraction process it is predicted that there is a decrease in the amount of bone tissue forming within the center of the fracture gap for all distraction rates. After the initial phases of expansion, the bone tissue within the callus increases for the slower rate of distraction or continues to decrease at the faster rates of distraction. At the end of the simulated maturation period, 47% of the distracted callus was predicted to consist of bone tissue for a distraction rate of 0.6 mm/day, decreasing to 22% for a distraction rate of 3 mm/day. Significantly higher amounts of bone formation were predicted for all distraction rates for the case of reduced mastication loading. Disparities between the model predictions and what is observed in vivo were found. For instance, during the latency period, the distraction period and beyond, the model is predicting larger than expected amounts of cartilage tissue formation within the callus. This and other limitations of the proposed model are discussed and possible specific explanations for these disparities are provided in the paper. The model predicts a distraction rate of around 1.2 mm/day to be optimal as higher rates produce less bone tissue while the risk of a premature bone union is greater at slower rates of distraction because in the latter stages of the distraction process bone tissue is predicted to form between the left and right side of the bone callus.

Mechanical behavior of an osteotomized mandible with distraction orthodontic devices.

This work analyzes the mechanical behavior of a human mandible when distraction orthodontic devices are used for correcting problems of dental overcrowding and/or arch shrinkage. The mandible 3D model is reconstructed from CT scan data and meshed into finite elements. The distractor is also modeled. FEM analysis included geometric non-linearity. Displacement field of healthy and osteotomized mandibles are compared. Progressive expansion of the distractor and effects of mastication are also analyzed. Finally, we compare two distraction protocols PROT1 and PROT2 where device is, respectively, expanded by 0.6 or 1.2mm/day. The global displacement is 6mm according to clinical recommendations. It came out that mastication forces generate displacements compatible with bone remodeling. However, parasitic rotations of the mandible arms due to mastication may counteract arch expansion induced by the device. Stress concentrations occurred where the device is fixed: stress peaks stay however below yield limit. Finally, PROT2 reduced by about 10% stresses in mandible and reproduces better than PROT1 the displacement field imposed by the device.

Biomechanical effects of prosthesis neck geometries to contrast limb lengthening after hip replacement.

Hip prostheses should meet the anatomical and physiological characteristics of patients; this is the rationale for designing modular implants of different sizes. To optimize implant geometry, it is necessary to consider, not only the prosthesis component design, but also the final configuration of the implanted leg. This means the necessity to consider the specific morphological and functional condition of ""that"" patient and not only of ""that"" hip to restore, at best, limb functions. Variations in the length of the implanted limb are frequent; therefore, the variations in the three geometrical features of the hip prosthesis neck, which can affect the restoration of the anatomical symmetry of the limbs, were investigated: (i) neck lengths (Ln ), between 50.5 and 64.5 mm; (ii) cervico-diaphyseal (CD) angle ( ã ), between 135 and 125 degrees and; (iii) anteversion (AV) angle ( â ), between 0 and 15 degrees . Adopting a three-dimensional (3D) simplified biomechanical model, the resultant load acting on the hip was estimated for each different design solution; corresponding stress distributions and contact pressures at the interface between the prosthesis head and the ultra high molecular weight polyethylene (UHMWPE) layer were evaluated by 3D finite element (FE) analyses and using the Strozzi approach. The following values have been assumed as physiological values: ã = ã p = 125 degrees , â = â p = 15 degrees and Ln = 57 mm; it was found that to contrast limb lengthening, if the CD angle varies from 135-125 degrees (with neck length Ln = 64.5 mm and AV = 0 degrees ), the joint resultant load decreases by 8.8% (7.2% if AV = 15 degrees ); the contact pressure de-creases by 5.8%, (5% if AV = 15 degrees ); the bending moment in the stem neck increases by 10.9% (13.8% if AV = 15 degrees ) and the torque increases by 1% (12.8% if AV = 15 degrees ).

Tonic and phasic tetrodotoxin block of sodium channels with point mutations in the outer pore region.

Tonic and use-dependent block by tetrodotoxin (TTX) has been studied in cRNA-injected Xenopus oocytes expressing mutants W386Y, E945Q, D1426K, and D1717Q, of the outer-pore region of the rat brain IIA alpha-subunit of sodium channels. The various phenotypes are tonically half-blocked at TTX concentrations, IC50(t), that span a range of more than three orders of magnitude, from 4 nM in mutant D1426K to 11 microM in mutant D1717Q. When stimulated with repetitive depolarizing pulses at saturating frequencies, all channels showed a monoexponential increase in their TTX-binding affinity with time constants that span an equally wide range of values ([TTX] approximately IC50(t), from approximately 60 s for D1426K to approximately 30 ms for D1717Q) and are in most phenotypes roughly inversely proportional to IC50(t). In contrast, all phenotypes show the same approximately threefold increase in their TTX affinity under stimulation. The invariance of the free-energy difference between tonic and phasic configurations of the toxin-receptor complex, together with the extreme variability of phasic block kinetics, is fully consistent with the trapped-ion mechanism of use dependence suggested by and developed by. Using this model, we estimated for each phenotype both the second-order association rate constant, kon, and the first-order dissociation rate constant, koff, for TTX binding. Except for mutant E945Q, all phenotypes have roughly the same value of kon approximately 2 microM-1 s-1 and owe their large differences in IC50(t) to different koff values. However, a 60-fold reduction in kon is the main determinant of the low TTX sensitivity of mutant E945Q. This suggests that the carboxyl group of E945 occupies a much more external position in the pore vestibule than that of the homologous residue D1717.

The block of Shaker K+ channels by kappa-conotoxin PVIIA is state dependent.

kappa-conotoxin PVIIA is the first conotoxin known to interact with voltage-gated potassium channels by inhibiting Shaker-mediated currents. We studied the mechanism of inhibition and concluded that PVIIA blocks the ion pore with a 1:1 stoichiometry and that binding to open or closed channels is very different. Open-channel properties are revealed by relaxations of partial block during step depolarizations, whereas double-pulse protocols characterize the slower reequilibration of closed-channel binding. In 2.5 mM-[K+]o, the IC50 rises from a tonic value of approximately 50 to approximately 200 nM during openings at 0 mV, and it increases e-fold for about every 40-mV increase in voltage. The change involves mainly the voltage dependence and a 20-fold increase at 0 mV of the rate of PVIIA dissociation, but also a fivefold increase of the association rate. PVIIA binding to Shaker Delta6-46 channels lacking N-type inactivation or to wild phenotypes appears similar, but inactivation partially protects the latter from open-channel unblock. Raising [K+]o to 115 mM has little effect on open-channel binding, but increases almost 10-fold the tonic IC50 of PVIIA due to a decrease by the same factor of the toxin rate of association to closed channels. In analogy with charybdotoxin block, we attribute the acceleration of PVIIA dissociation from open channels to the voltage-dependent occupancy by K+ ions of a site at the outer end of the conducting pore. We also argue that the occupancy of this site by external cations antagonizes on binding to closed channels, whereas the apparent competition disappears in open channels if the competing cation can move along the pore. It is concluded that PVIIA can also be a valuable tool for probing the state of ion permeation inside the pore.

Calcium dependent shifts of Na+ channel activation correlated with the state dependence of calcium-binding to the pore.

Calcium ions block the open configuration and antagonise the tonic binding of TTX to the closed state of sodium channels in very different ranges of extracellular concentration, [Ca]o. We measured the open-state block in channels expressed in Xenopus oocytes by alpha-subunits from rat brain (rBIIa) or adult rat skeletal muscle (rSkM1). Recordings of instantaneous tail-currents from cell-attached macro patches show that the binding of Ca2+ to the blocking site has a dissociation constant of about 20 mM at 0 mV and senses about 30% of the membrane potential drop, whereas the concentration of half-inhibition of TTX-binding is less than 1 mM and voltage-insensitive. Assuming that both effects involve a single binding site, a simple model predicts that the state-dependency of the dissociation constant entails positive shifts of activation and faster kinetics of deactivation at increasing [Ca]o. The shifts of activation measured for rBIIA and rSkM1 channels are comparable in size to those predicted by the model, which accounts also for the observed larger shifts of the rBIIA-mutant K226Q as a consequence of its reduced voltage-sensitivity. Shifts attributable to surface-charge screening effects seem smaller in the oocyte than in native cell-membranes. The experimental [Ca]o-dependence of deactivation kinetics is also consistent with the model and with the idea that Ca(2+)-binding changes to the same extent, but in opposite directions, the activation free-energies of both opening and closing transitions.