Quantifying mechanical forces during vertebrate morphogenesis.

Morphogenesis requires embryonic cells to generate forces and perform mechanical work to shape their tissues. Incorrect functioning of these force fields can lead to congenital malformations. Understanding these dynamic processes requires the quantification and profiling of three-dimensional mechanics during evolving vertebrate morphogenesis. Here we describe elastic spring-like force sensors with micrometre-level resolution, fabricated by intravital three-dimensional bioprinting directly in the closing neural tubes of growing chicken embryos. Integration of calibrated sensor read-outs with computational mechanical modelling allows direct quantification of the forces and work performed by the embryonic tissues. As they displace towards the embryonic midline, the two halves of the closing neural tube reach a compression of over a hundred nano-newtons during neural fold apposition. Pharmacological inhibition of Rho-associated kinase to decrease the pro-closure force shows the existence of active anti-closure forces, which progressively widen the neural tube and must be overcome to achieve neural tube closure. Overall, our approach and findings highlight the intricate interplay between mechanical forces and tissue morphogenesis.

The role of the extracellular matrix in the reduction of lateral force transmission in muscle bundles: A finite element analysis.

BACKGROUND AND OBJECTIVE: Aging is associated with a reduction in muscle performance, but muscle weakness is characterized by a much greater loss of force loss compared to mass loss. The aim of this work is to assess the contribution of the extracellular matrix (ECM) to the lateral transmission of force in humans and the loss of transmitted force due to age-related modifications. METHODS: Finite element models of muscle bundles are developed for young and elderly human subjects, by considering a few fibers connected through an ECM layer. Bundles of young and elderly subjects are assumed to differ in terms of ECM thickness, as observed experimentally. A three-element-based Hill model is adopted to describe the active behavior of muscle fibers, while the ECM is modeled assuming an isotropic hyperelastic neo-Hookean constitutive formulation. Numerical analyses are carried out by mimicking, at the scale of a bundle, two experimental protocols from the literature. RESULTS: When comparing numerical results obtained for bundles of young and elderly subjects, a greater reduction in the total transmitted force is observed in the latter. The loss of transmitted force is 22 % for the elderly subjects, while it is limited to 7.5 % for the young subjects. The result for the elderly subjects is in line with literature studies on animal models, showing a reduction in the range of 20-34 %. This can be explained by an alteration in the mechanism of lateral force transmission due to the lower shear stiffness of the ECM in elderly subjects, related to its higher thickness. CONCLUSIONS: Computational modeling allows to evaluate at the bundle level how the age-related increase of the ECM amount between fibers affects the lateral transmission of force. The results suggest that the observed increase in ECM thickness in aging alone can explain the reduction of the total transmitted force, due to the impaired lateral transmission of force of each fiber.

How does atmospheric pressure cold helium plasma affect the biomechanical behaviour on alkali-lesioned corneas?

BACKGROUND: Melting corneal ulcers are a serious condition that affects a great number of animals and people around the world and it is characterised by a progressive weakening of the tissue leading to possible severe ophthalmic complications, such as visual impairment or blindness. This disease is routinely treated with medical therapy and keratoplasty, and recently also with alternative regenerative therapies, such as cross-linking, amniotic membrane transplant, and laser. Plasma medicine is another recent example of regenerative treatment that showed promising results in reducing the microbial load of corneal tissue together with maintaining its cellular vitality. Since the effect of helium plasma application on corneal mechanical viscoelasticity has not yet been investigated, the aim of this study is first to evaluate it on ex vivo porcine corneas for different exposition times and then to compare the results with previous data on cross-linking treatment. RESULTS: 94 ex vivo porcine corneas divided into 16 populations (healthy or injured, fresh or cultured and treated or not with plasma or cross-linking) were analysed. For each population, a biomechanical analysis was performed by uniaxial stress-relaxation tests, and a statistical analysis was carried out considering the characteristic mechanical parameters. In terms of equilibrium normalised stress, no statistically significant difference resulted when the healthy corneas were compared with lesioned plasma-treated ones, independently of treatment time, contrary to what was obtained about the cross-linking treated corneas which exhibited more intense relaxation phenomena. CONCLUSIONS: In this study, the influence of the Helium plasma treatment was observed on the viscoelasticity of porcine corneas ex vivo, by restoring in lesioned tissue a degree of relaxation similar to the one of the native tissue, even after only 2 min of application. Therefore, the obtained results suggest that plasma treatment is a promising new regenerative ophthalmic therapy for melting corneal ulcers, laying the groundwork for further studies to correlate the mechanical findings with corneal histology and ultrastructural anatomy after plasma treatment.

Development and preclinical evaluation of bioactive nerve conduits for peripheral nerve regeneration: A comparative study.

In severe peripheral nerve injuries, nerve conduits (NCs) are good alternatives to autografts/allografts; however, the results the available devices guarantee for are still not fully satisfactory. Herein, differently bioactivated NCs based on the new polymer oxidized polyvinyl alcohol (OxPVA) are compared in a rat model of sciatic nerve neurotmesis (gap: 5 mm; end point: 6 weeks). Thirty Sprague Dawley rats are randomized to 6 groups: Reverse Autograft (RA); Reaxon®; OxPVA; OxPVA + EAK (self-assembling peptide, mechanical incorporation); OxPVA + EAK-YIGSR (mechanical incorporation); OxPVA + Nerve Growth Factor (NGF) (adsorption). Preliminarily, all OxPVA-based devices are comparable with Reaxon® in Sciatic Functional Index score and gait analysis; moreover, all conduits sustain nerve regeneration (S100, ß-tubulin) without showing substantial inflammation (CD3, F4/80) evidences. Following morphometric analyses, OxPVA confirms its potential in PNI repair (comparable with Reaxon®) whereas OxPVA + EAK-YIGSR stands out for its myelinated axons total number and density, revealing promising in injury recovery and for future application in clinical practice.

Development and In Vitro/In Vivo Comparative Characterization of Cryopreserved and Decellularized Tracheal Grafts.

Tracheal reconstruction represents a challenge when primary anastomosis is not feasible. Within this scenario, the study aim was to develop a new pig-derived decellularized trachea (DecellT) to be compared with the cryopreserved counterpart (CryoT) for a close predictive analysis. Tracheal segments underwent decellularization by a physical + enzymatic + chemical method (12 cycles); in parallel, cryopreserved samples were also prepared. Once decellularized (histology/DNA quantification), the two groups were characterized for Alpha-Gal epitopes/structural proteins (immunohistochemistry/histology/biochemical assays/second harmonic generation microscopy)/ultrastructure (Scanning Electron Microscopy (SEM))/mechanical behaviour. Cytotoxicity absence was assessed in vitro (extract-test assay/direct seeding, HM1SV40 cell line) while biocompatibility was verified in BALB/c mice, followed by histological/immunohistochemical analyses and SEM (14 days). Decellularization effectively removed Alpha-Gal epitopes; cartilage histoarchitecture was retained in both groups, showing chondrocytes only in the CryoT. Cryopreservation maintained few respiratory epithelium sparse cilia, not detectable in DecellT. Focusing on ECM, preserved structural/ultrastructural organization and collagen content were observed in the cartilage of both; conversely, the GAGs were significantly reduced in DecellT, as confirmed by mechanical study results. No cytotoxicity was highlighted by CryoT/DecellT in vitro, as they were also corroborated by a biocompatibility assay. Despite some limitations (cells presence/GAGs reduction), CryoT/DecellT are both appealing options, which warrant further investigation in comparative in vivo studies.

Compressive Mechanical Behavior of Partially Oxidized Polyvinyl Alcohol Hydrogels for Cartilage Tissue Repair.

Polyvinyl alcohol (PVA) hydrogels are extensively used as scaffolds for tissue engineering, although their biodegradation properties have not been optimized yet. To overcome this limitation, partially oxidized PVA has been developed by means of different oxidizing agents, obtaining scaffolds with improved biodegradability. The oxidation reaction also allows tuning the mechanical properties, which are essential for effective use in vivo. In this work, the compressive mechanical behavior of native and partially oxidized PVA hydrogels is investigated, to evaluate the effect of different oxidizing agents, i.e., potassium permanganate, bromine, and iodine. For this purpose, PVA hydrogels are tested by means of indentation tests, also considering the time-dependent mechanical response. Indentation results show that the oxidation reduces the compressive stiffness from about 2.3 N/mm for native PVA to 1.1 ÷ 1.4 N/mm for oxidized PVA. During the consolidation, PVA hydrogels exhibit a force reduction of about 40% and this behavior is unaffected by the oxidizing treatment. A poroviscoelastic constitutive model is developed to describe the time-dependent mechanical response, accounting for the viscoelastic polymer matrix properties and the flow of water molecules within the matrix during long-term compression. This model allows to estimate the long-term Young's modulus of PVA hydrogels in drained conditions (66 kPa for native PVA and 34-42 kPa for oxidized PVA) and can be exploited to evaluate their performances under compressive stress in vivo, as in the case of cartilage tissue engineering.

Preclinical Development of Bioengineered Allografts Derived from Decellularized Human Diaphragm.

Volumetric muscle loss (VML) is the traumatic/surgical loss of skeletal muscle, causing aesthetic damage and functional impairment. Suboptimal current surgical treatments are driving research towards the development of optimised regenerative therapies. The grafting of bioengineered scaffolds derived from decellularized skeletal muscle may be a valid option to promote structural and functional healing. In this work, a cellular human diaphragm was considered as a scaffold material for VML treatment. Decellularization occurred through four detergent-enzymatic protocols involving (1) sodium dodecyl sulfate (SDS), (2) SDS + TergitolTM, (3) sodium deoxycholate, and (4) TergitolTM. After decellularization, cells, DNA (≤50 ng/mg of tissue), and muscle fibres were efficiently removed, with the preservation of collagen/elastin and 60%-70% of the glycosaminoglycan component. The detergent-enzymatic treatments did not affect the expression of specific extracellular matrix markers (Collagen I and IV, Laminin), while causing the loss of HLA-DR expression to produce non-immunogenic grafts. Adipose-derived stem cells grown by indirect co-culture with decellularized samples maintained 80%-90% viability, demonstrating the biosafety of the scaffolds. Overall, the tested protocols were quite equivalent, with the patches treated by SDS + TergitolTM showing better collagen preservation. After subcutaneous implant in Balb/c mice, these acellular diaphragmatic grafts did not elicit a severe immune reaction, integrating with the host tissue.

Time-dependent mechanical behavior of partially oxidized polyvinyl alcohol hydrogels for tissue engineering.

Polyvinyl alcohol (PVA) hydrogels are synthetic polymers which can be used as scaffolds for tissue engineering due to their biocompatibility and large water content. To improve their biodegradation properties, partial oxidation of PVA is achieved by means of different oxidizing agents, such as potassium permanganate, bromine and iodine. The effect of this process on hydrogels mechanical performance has not been fully investigated in view of tissue engineering applications. In this work, the time-dependent mechanical behavior of unmodified and partially oxidized PVA hydrogels is evaluated by means of uniaxial tensile and stress relaxation tests, to evaluate the effect of different oxidizing agents on the viscoelastic response. Tensile tests show an isotropic and almost-incompressible behavior, with a stiffness reduction after PVA oxidation. The time-dependent response of oxidized PVA is comparable to the one of unmodified PVA and is modeled as a quasi-linear viscoelastic behavior. Finite Element (FE) models of PVA samples are developed and numerical analyses are used to evaluate the effect of different strain rates on the mechanical response under uniaxial tension. This model can be exploited to predict the time-dependent mechanical behavior of partially oxidized PVA in tissue engineering application under tensile loading.

Mechanical behaviour of healthy versus alkali-lesioned corneas by a porcine organ culture model.

BACKGROUND: Cornea is a composite tissue exhibiting nonlinear and time-dependent mechanical properties. Corneal ulcers are one of the main pathologies that affect this tissue, disrupting its structural integrity and leading to impaired functions. In this study, uniaxial tensile and stress-relaxation tests are developed to evaluate stress-strain and time-dependent mechanical behaviour of porcine corneas. RESULTS: The samples are split in two groups: some corneas are analysed in an unaltered state (healthy samples), while others are injured with alkaline solution to create an experimental ulcer (lesioned samples). Furthermore, within each group, corneas are examined in two conditions: few hours after the enucleation (fresh samples) or after 7 days in a specific culture medium for the tissue (cultured samples). Finally, another condition is added: corneas from all the groups undergo or not a cross-linking treatment. In both stress-strain and stress-relaxation tests, a weakening of the tissue is observed due to the imposed conditions (lesion, culture and treatment), represented by a lower stiffness and increased stress-relaxation. CONCLUSIONS: Alkali-induced corneal stromal melting determines changes in the mechanical response that can be related to a damage at microstructural level. The results of the present study represent the basis for the investigation of traditional and innovative corneal therapies.

Porcine Decellularized Diaphragm Hydrogel: A New Option for Skeletal Muscle Malformations.

Hydrogels are biomaterials that, thanks to their unique hydrophilic and biomimetic characteristics, are used to support cell growth and attachment and promote tissue regeneration. The use of decellularized extracellular matrix (dECM) from different tissues or organs significantly demonstrated to be far superior to other types of hydrogel since it recapitulates the native tissue's ECM composition and bioactivity. Different muscle injuries and malformations require the application of patches or fillers to replenish the defect and boost tissue regeneration. Herein, we develop, produce, and characterize a porcine diaphragmatic dECM-derived hydrogel for diaphragmatic applications. We obtain a tissue-specific biomaterial able to mimic the complex structure of skeletal muscle ECM; we characterize hydrogel properties in terms of biomechanical properties, biocompatibility, and adaptability for in vivo applications. Lastly, we demonstrate that dECM-derived hydrogel obtained from porcine diaphragms can represent a useful biological product for diaphragmatic muscle defect repair when used as relevant acellular stand-alone patch.

Conformation and mechanics of the polymeric cuff of artificial urinary sphincter.

The surgical treatment of urinary incontinence is often performed by adopting an Artificial Urinary Sphincter (AUS). AUS cuff represents a fundamental component of the device, providing the mechanical action addressed to urethral occlusion, which can be investigated by computational approach. In this work, AUS cuff is studied with reference to both materials and structure, to develop a finite element model. Materials behavior is investigated using physicochemical and mechanical characterization, leading to the formulation of a constitutive model. Materials analysis shows that AUS cuff is composed by a silicone blister joined with a PET fiber-reinforced layer. A nonlinear mechanical behavior is found, with a higher stiffness in the outer layer due to fiber-reinforcement. The cuff conformation is acquired by Computer Tomography (CT) both in deflated and inflated conditions, for an accurate definition of the geometrical characteristics. Based on these data, the numerical model of AUS cuff is defined. CT images of the inflated cuff are compared with results of numerical analysis of the inflation process, for model validation. A relative error below 2.5% was found. This study is the first step for the comprehension of AUS mechanical behavior and allows the development of computational tools for the analysis of lumen occlusion process. The proposed approach could be adapted to further fluid-filled cuffs of artificial sphincters.

Biomechanics of stomach tissues and structure in patients with obesity.

Even though bariatric surgery is one of the most effective treatment option of obesity, post-surgical weight loss is not always ensured, especially in the long term, when many patients experience weight regain. Bariatric procedures are largely based on surgeon's expertise and intra-operative decisions, while an integrated in-silico approach could support surgical activity. The effects of bariatric surgery on gastric distension, which activates the neural circuitry promoting satiety, can be considered one of the main factors in the operation success. This aspect can be investigated trough computational modelling based on the mechanical properties of stomach tissues and structure. Mechanical tests on gastric tissues and structure from people with obesity are carried out, as basis for the development of a computational model. The samples are obtained from stomach residuals explanted during laparoscopic sleeve gastrectomy interventions. Uniaxial tensile and stress relaxation tests are performed in different directions and inflation tests are carried out on the entire stomach residual. Experimental results show anisotropic, non-linear elastic and time-dependent behavior. In addition, the mechanical properties demonstrate to be dependent on the sample location within the stomach. Inflation tests confirm the characteristics of time-dependence and non-linear elasticity of the stomach wall. Experimental activities developed provide a unique set of data about the mechanical behavior of the stomach of patients with obesity, considering both tissues and structure. This data set can be adopted for the development of computational models of the stomach, as support to the rational investigation of biomechanical aspects of bariatric surgery.

Numerical modelling of abdominal wall mechanics: The role of muscular contraction and intra-abdominal pressure.

The biomechanics of the abdominal wall depends on muscular activation, tissue mechanical behavior and Intra-Abdominal Pressure (IAP). In this work, a numerical model of a human abdomen is presented, based on abdominal wall geometry from medical images. Specific constitutive formulations describe tissues mechanical behavior. Connective tissues are modelled as hyperelastic fiber-reinforced materials, while muscular tissues are described by means of a three-element Hill's model. The abdominal cavity is represented by a volume region interacting with the abdominal wall. Numerical analyses are developed by applying a muscular contraction, inducing a volume reduction of the abdominal cavity and a simultaneous IAP increase. Numerical results of abdomen displacement at IAP corresponding to an abdominal crunch are compared with experimental results acquired via 3D laser scanning on a healthy subject. Numerical and experimental results are mutually consistent and show that muscular activation induces a raising in the region adjacent to linea alba along the posterior-anterior direction and a lowering along lateral-medial direction of the abdominal wall sides. The numerical model developed in this work allows a coherent representation of the abdominal wall mechanics.

Halogen-Mediated Partial Oxidation of Polyvinyl Alcohol for Tissue Engineering Purposes.

Partial oxidation of polyvinyl alcohol (PVA) with potassium permanganate turned out to be an efficient method to fabricate smart scaffolds for tissue engineering, endowed with biodegradation and protein delivery capacity. This work considered for the first time the use of halogens (bromine, chlorine and iodine) as less aggressive agents than potassium permanganate to perform controlled PVA oxidation, in order to prevent degradation of polymer molecular size upon chemical modification. Oxidized PVA solutions were chemically characterized (i.e., dinitrophenylhydrazine assay, viscosity measurements, molecular size distribution) before preparing physically cross-linked hydrogels. Scaffolds were assessed for their mechanical properties and cell/tissue biocompatibiliy through cytotoxic extract test on IMR-90 fibroblasts and subcutaneous implantation into BALB/c mice. According to chemical investigations, bromine and iodine allowed for minor alteration of polymer molecular weight. Uniaxial tensile tests demonstrated that oxidized scaffolds had decreased mechanical resistance to deformation, suggesting tunable hydrogel stiffness. Finally, oxidized hydrogels exhibited high biocompatibility both in vitro and in vivo, resulting neither to be cytotoxic nor to elicit severe immunitary host reaction in comparison with atoxic PVA. In conclusion, PVA hydrogels oxidized by halogens were successfully fabricated in the effort of adapting polymer characteristics to specific tissue engineering applications.

Inter-rater reliability and variability of ultrasound measurements of abdominal muscles and fasciae thickness.

Ultrasound (US) imaging is being increasingly used by Physical and Rehabilitation Medicine (PRM) specialists to measure the thickness of abdominal muscles. The current study set out to assess the inter-rater reliability of US measurements of the thickness of the abdominal muscles/fasciae. Three raters (1 = orthopedic specialist, expert on fasciae; 2 = PRM resident; 3 = PRM specialist) with different levels of US training examined the abdominal muscles and fasciae of a healthy volunteer under supine resting and dynamic conditions following a standard US protocol. The probe was positioned along the right lateral abdominal wall at the height of the 12th rib: (1) above the umbilicus at the linea alba, (2) to the side of and approximately 2 cm from the umbilicus, (3) along the mammillary line, and (4) along the anterior axillary line. Each rater measured 17 anatomical structures six times during two sessions. The relative error of the measurements (intra-rater variability) was slightly higher for the fasciae than for the muscles, and during the dynamic condition than the resting condition. Inter-rater reliability was good under both conditions for the fasciae (Intraclass Correlation Coefficient = ICC = 0.83) and excellent for the muscles (ICC = 0.99). Knowledge of the fascial anatomy of the abdominal wall is essential for accurate ultrasound examinations and for improving reliability. These findings confirm that US imaging is a reliable, non-invasive, cost-effective instrument for evaluating the abdominal muscles/fasciae. Clin. Anat. 32:948-960, 2019. © 2019 Wiley Periodicals, Inc.

3D surface imaging of abdominal wall muscular contraction.

BACKGROUND AND OBJECTIVE: The biomechanical analysis of the abdominal wall should take into account muscle activation and related phenomena, such as intra-abdominal pressure variation and abdomen surface deformation. The geometry of abdominal surface and its deformation during contraction have not been extensively characterized, while represent a key issue to be investigated. METHODS: In this work, the antero-lateral abdominal wall surface of ten healthy volunteers in supine position is acquired via laser scanning in relaxed conditions and during abdominal muscles contraction, repeating each acquisition six times. The average relaxed and contracted abdominal surfaces are compared for each subject and displacements measured. RESULTS: Muscular activation induces raising in the region adjacent to linea alba along the posterior-anterior direction and a simultaneous lowering along lateral-medial direction of the abdominal wall sides. Displacements reach a maximum value of 12.5 mm for the involved subjects. The coefficient of variation associated to the abdomen surface measurements in the same configuration (relaxed or contracted) is below 0.75%. Non-parametric Mann-Whitney U test highlights that the differences between relaxed and contracted abdominal wall surfaces are significant (p < 0.01). CONCLUSIONS: Laser scanning is an accurate and reliable method to evaluate surface changes on the abdominal wall during muscular contraction. The results of this experimental activity can be useful to validate numerical models aimed at describing abdominal wall biomechanics.

The effects of the muscular contraction on the abdominal biomechanics: a numerical investigation.

Abdominal wall biomechanics is strongly affected by muscular contraction and intra-abdominal pressure (IAP) which characterize different physiological functions and daily tasks. However, the active muscular behavior is generally not considered in current computational models of the abdominal wall. The aim of this study is to develop a numerical model mimicking muscular activation and IAP. A three dimensional Finite Element model of a healthy abdominal wall is developed detailing the principal abdomen components reconstructed upon anatomical data and medical images. Fascial tissues, aponeuroses and linea alba are modelled as hyperelastic fiber-reinforced materials, while a three-element Hill's model is assumed for muscles. Numerical analyses are performed increasing the IAP up to 100 mmHg and simultaneously activating the muscular structures. The obtained abdominal behavior is compared to a similar model with same IAPs, but passive muscles conditions. Abdomen stiffness and strength are computed in regions in which hernias can potentially occur. A global stiffening of the abdominal wall is found corresponding to a low abdomen deformation and the membrane force on fascial structures is reduced by muscular contraction. Representing active muscular contraction leads to advanced findings, otherwise membrane force results overestimated considering a purely passive behavior for the abdominal wall.

Computational modeling of abdominal hernia laparoscopic repair with a surgical mesh.

PURPOSE: Although new techniques and prostheses have been introduced in ventral hernia surgery, abdominal hernia repair still presents complications, such as recurrence, pain, and discomfort. Thus, this work implements a computational method aimed at evaluating biomechanical aspects of the abdominal hernia laparoscopic repair, which can support clinical research tailored to hernia surgery. METHODS: A virtual solid model of the abdominal wall is obtained from MRI scans of a healthy subject. The mechanical behavior of muscular and fascial tissues is described by constitutive formulations with specific parameters. A defect is introduced to reproduce an incisional hernia. Laparoscopic repair is mimicked via intraperitoneal positioning of a surgical mesh. Numerical analyses are performed to evaluate the mechanical response of the abdominal wall in healthy, herniated and post-surgery configurations, considering physiological intra-abdominal pressures. RESULTS: During the deformation of the abdominal wall at increasing pressures, a percentage displacement increment up to 6% is found in the herniated condition, while the mechanical behavior of the repaired abdomen is similar to the healthy one. In the pressure range between 8 mmHg and 55 mmHg, the herniated abdomen shows an incremental stiffness differing of 7% with respect to the healthy condition, while the post-surgery condition shows an increase of the incremental stiffness up to 58%. CONCLUSIONS: This computational approach may be exploited to investigate different aspects of abdominal wall surgical repair, including mesh mechanical characteristics and positioning. Numerical modeling offers a helpful support for selecting the best-fitting prosthesis for customize pre-surgery planning.