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.

Three-Dimensional Bioprinting of Naturally Derived Hydrogels for the Production of Biomimetic Living Tissues: Benefits and Challenges.

Three-dimensional bioprinting is the process of manipulating cell-laden bioinks to fabricate living structures. Three-dimensional bioprinting techniques have brought considerable innovation in biomedicine, especially in the field of tissue engineering, allowing the production of 3D organ and tissue models for in vivo transplantation purposes or for in-depth and precise in vitro analyses. Naturally derived hydrogels, especially those obtained from the decellularization of biological tissues, are promising bioinks for 3D printing purposes, as they present the best biocompatibility characteristics. Despite this, many natural hydrogels do not possess the necessary mechanical properties to allow a simple and immediate application in the 3D printing process. In this review, we focus on the bioactive and mechanical characteristics that natural hydrogels may possess to allow efficient production of organs and tissues for biomedical applications, emphasizing the reinforcement techniques to improve their biomechanical properties.

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.

Development of detailed finite element models for in silico analyses of brain impact dynamics.

BACKGROUND AND OBJECTIVE: In the last few decades, several studies have been performed to investigate traumatic brain injuries (TBIs) and to understand the biomechanical response of brain tissues, by using experimental and computational approaches. As part of computational approaches, human head finite element (FE) models show to be important tools in the analysis of TBIs, making it possible to estimate local mechanical effects on brain tissue for different accident scenarios. The present study aims to contribute to the computational approach by means of the development of three advanced FE head models for accurately describing the head tissue dynamics, the first step to predict TBIs. METHODS: We have developed three detailed FE models of human heads from magnetic resonance images of three volunteers: an adult female (32 yrs), an adult male (35 yrs), and a young male (16 yrs). These models have been validated against experimental data of post mortem human subjects (PMHS) tests available in the literature. Brain tissue displacements relative to the skull, hydrostatic intracranial pressure, and head acceleration have been used as the parameters to compare the model response with the experimental response for validation. The software CORAplus (CORrelation and Analysis) has been adopted to evaluate the bio-fidelity level of FE models. RESULTS: Numerical results from the three models agree with experimental data. FE models presented in this study show a good bio-fidelity for hydrostatic pressure (CORA score of 0.776) and a fair bio-fidelity brain tissue displacements relative to the skull (CORA score of 0.443 and 0.535). The comparison among numerical simulations carried out with the three models shows negligible differences in the mechanical state of brain tissue due to the different morphometry of the heads, when the same acceleration history is considered. CONCLUSIONS: The three FE models, thanks to their accurate description of anatomical morphology and to their bio-fidelity, can be useful tools to investigate brain mechanics due to different impact scenarios. Therefore, they can be used for different purposes, such as the investigation of the correlation between head acceleration and tissue damage, or the effectiveness of helmet designs. This work does not address the issue to define injury thresholds for the proposed models.

Customized bioreactor enables the production of 3D diaphragmatic constructs influencing matrix remodeling and fibroblast overgrowth.

The production of skeletal muscle constructs useful for replacing large defects in vivo, such as in congenital diaphragmatic hernia (CDH), is still considered a challenge. The standard application of prosthetic material presents major limitations, such as hernia recurrences in a remarkable number of CDH patients. With this work, we developed a tissue engineering approach based on decellularized diaphragmatic muscle and human cells for the in vitro generation of diaphragmatic-like tissues as a proof-of-concept of a new option for the surgical treatment of large diaphragm defects. A customized bioreactor for diaphragmatic muscle was designed to control mechanical stimulation and promote radial stretching during the construct engineering. In vitro tests demonstrated that both ECM remodeling and fibroblast overgrowth were positively influenced by the bioreactor culture. Mechanically stimulated constructs also increased tissue maturation, with the formation of new oriented and aligned muscle fibers. Moreover, after in vivo orthotopic implantation in a surgical CDH mouse model, mechanically stimulated muscles maintained the presence of human cells within myofibers and hernia recurrence did not occur, suggesting the value of this approach for treating diaphragm defects.

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.

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.

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.

Fibre and extracellular matrix contributions to passive forces in human skeletal muscles: An experimental based constitutive law for numerical modelling of the passive element in the classical Hill-type three element model.

The forces that allow body movement can be divided into active (generated by sarcomeric contractile proteins) and passive (sustained by intra-sarcomeric proteins, fibre cytoskeleton and extracellular matrix (ECM)). These are needed to transmit the active forces to the tendon and the skeleton. However, the relative contribution of the intra- and extra- sarcomeric components in transmitting the passive forces is still under debate. There is limited data in the literature about human muscle and so it is difficult to make predictions using multiscale models, imposing a purely phenomenological description for passive forces. In this paper, we apply a method for the experimental characterization of the passive properties of fibres and ECM to human biopsy and propose their clear separation in a Finite Element Model. Experimental data were collected on human single muscle fibres and bundles, taken from vastus lateralis muscle of elderly subjects. Both were progressively elongated to obtain two stress-strain curves which were fitted to exponential equations. The mechanical properties of the extracellular passive components in a bundle of fibres were deduced by the subtraction of the passive tension observed in single fibres from the passive tension observed in the bundle itself. Our results showed that modulus and tensile load bearing capability of ECM are higher than those of fibres and defined their quantitative characterization that can be used in macroscopic models to study their role in the transmission of forces in physiological and pathophysiological conditions.

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.

Mechanical properties variation and constitutive modelling of biomedical polymers after sterilization.

PURPOSE: In this work, the mechanical behavior of two block copolymers for biomedical applications is studied with particular regard to the effects induced by a steam sterilization treatment that biomedical devices usually undergo in healthcare facilities. This investigation is aimed at describing the elasto-plastic behavior of the stress-strain response, determining a functional dependence between material constitutive parameters, to obtain an optimal constitutive model. METHODS: The mechanical properties of these polymers are analyzed through uniaxial tensile tests, before and after the sterilization process. The effect of sterilization on the mechanical behavior is evaluated. The Ramberg-Osgood model is used to describe the elasto-plastic behavior of the stress-strain response. RESULTS: Data from uniaxial tensile tests are discussed in the light of previous data on the same polymeric materials, in order to highlight the correlation between physicochemical and mechanical properties variation. The material constitutive parameters are determined and the functional dependence between them is found, thus enabling an optimal constitutive model to be obtained. CONCLUSIONS: The effect of sterilization on the material constitutive parameters is studied, to evaluate the suitability of the model in describing the mechanical response of biomedical polymer before and after sterilization treatment. The same approach can be applied to other biomaterials, under various tensile tests, and for several processes that induce variation in mechanical properties.

From single muscle fiber to whole muscle mechanics: a finite element model of a muscle bundle with fast and slow fibers.

Muscles exhibit highly complex, multi-scale architecture with thousands of muscle fibers, each with different properties, interacting with each other and surrounding connective structures. Consequently, the results of single-fiber experiments are scarcely linked to the macroscopic or whole muscle behavior. This is especially true for human muscles where it would be important to understand of how skeletal muscles disorders affect patients' life. In this work, we developed a mathematical model to study how fast and slow muscle fibers, well characterized in single-fiber experiments, work and generate together force and displacement in muscle bundles. We characterized the parameters of a Hill-type model, using experimental data on fast and slow single human muscle fibers, and comparing experimental data with numerical simulations obtained from finite element (FE) models of single fibers. Then, we developed a FE model of a bundle of 19 fibers, based on an immunohistochemically stained cross section of human diaphragm and including the corresponding properties of each slow or fast fiber. Simulations of isotonic contractions of the bundle model allowed the generation of its apparent force-velocity relationship. Although close to the average of the force-velocity curves of fast and slow fibers, the bundle curve deviates substantially toward the fast fibers at low loads. We believe that the present model and the characterization of the force-velocity curve of a fiber bundle represents the starting point to link the single-fiber properties to those of whole muscle with FE application in phenomenological models of human muscles.

Investigation of interaction phenomena between crural fascia and muscles by using a three-dimensional numerical model.

The focus of this work is the numerical modeling of the anterior compartment of the human leg with particular attention to crural fascia. Interaction phenomena between fascia and muscles are of clinical interest to explain some pathologies, as the compartment syndrome. A first step to enhance knowledge on this topic consists in the investigation of fascia biomechanical role and its interaction with muscles in physiological conditions. A three-dimensional finite element model of the anterior compartment is developed based on anatomical data, detailing the structural conformation of crural fascia, composed of three layers, and modeling the muscles as a unique structure. Different constitutive models are implemented to describe the mechanical response of tissues. Crural fascia is modeled as a hyperelastic fiber-reinforced material, while muscle tissue via a three-element Hill's model. The numerical analysis of isotonic contraction of muscles is performed, allowing the evaluation of pressure induced within muscles and consequent stress and strain fields arising on the crural fascia. Numerical results are compared with experimental measurements of the compartment radial deformation and intracompartmental pressure during concentric contraction, to validate the model. The numerical model provides a suitable description of muscles contraction of the anterior compartment and the consequent mechanical interaction with the crural fascia.

Decellularized Human Skeletal Muscle as Biologic Scaffold for Reconstructive Surgery.

Engineered skeletal muscle tissues have been proposed as potential solutions for volumetric muscle losses, and biologic scaffolds have been obtained by decellularization of animal skeletal muscles. The aim of the present work was to analyse the characteristics of a biologic scaffold obtained by decellularization of human skeletal muscles (also through comparison with rats and rabbits) and to evaluate its integration capability in a rabbit model with an abdominal wall defect. Rat, rabbit and human muscle samples were alternatively decellularized with two protocols: n.1, involving sodium deoxycholate and DNase I; n.2, trypsin-EDTA and Triton X-NH4OH. Protocol 2 proved more effective, removing all cellular material and maintaining the three-dimensional networks of collagen and elastic fibers. Ultrastructural analyses with transmission and scanning electron microscopy confirmed the preservation of collagen, elastic fibres, glycosaminoglycans and proteoglycans. Implantation of human scaffolds in rabbits gave good results in terms of integration, although recellularization by muscle cells was not completely achieved. In conclusion, human skeletal muscles may be effectively decellularized to obtain scaffolds preserving the architecture of the extracellular matrix and showing mechanical properties suitable for implantation/integration. Further analyses will be necessary to verify the suitability of these scaffolds for in vitro recolonization by autologous cells before in vivo implantation.

Biomechanical behavior of human crural fascia in anterior and posterior regions of the lower limb.

The present work focuses on the numerical modeling of the mechanical behavior of the crural fascia, the deep fascia enwrapping the lower limb muscles. This fascia has an important biomechanical role, due to its interaction with muscles during contraction and its association with pathological events, such as compartment syndrome. The mechanical response of the crural fascia is described by assuming a hyperelastic fiber-reinforced constitutive model, with families of fibers disposed according to the spatial disposition of the collagen network, as shown in histological analyses. A two-dimensional finite element model of a lower limb transversal section has been developed to analyze deformational behavior, with particular attention on interaction phenomena between crural fascia and enwrapped muscles. The constitutive model adopted for the crural fascia well fits experimental data taken along the proximal-distal and medial-lateral directions. The finite element analysis allows for interpreting the relation between change in volume and pressure of muscle compartments and the crural fascia deformation.