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.

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.

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.

Painful connections: densification versus fibrosis of fascia.

Deep fascia has long been considered a source of pain, secondary to nerve pain receptors becoming enmeshed within the pathological changes to which fascia are subject. Densification and fibrosis are among such changes. They can modify the mechanical properties of deep fasciae and damage the function of underlying muscles or organs. Distinguishing between these two different changes in fascia, and understanding the connective tissue matrix within fascia, together with the mechanical forces involved, will make it possible to assign more specific treatment modalities to relieve chronic pain syndromes. This review provides an overall description of deep fasciae and the mechanical properties in order to identify the various alterations that can lead to pain. Diet, exercise, and overuse syndromes are able to modify the viscosity of loose connective tissue within fascia, causing densification, an alteration that is easily reversible. Trauma, surgery, diabetes, and aging alter the fibrous layers of fasciae, leading to fascial fibrosis.

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.

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.

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.

A constitutive model for the mechanical characterization of the plantar fascia.

A constitutive model is proposed to describe the mechanical behavior of the plantar fascia. The mechanical characterization of the plantar fascia regards the role in the foot biomechanics and it is involved in many alterations of its functional behavior, both of mechanical and nonmechanical origin. The structural conformation of the plantar fascia in its middle part is characterized by the presence of collagen fibers reinforcing the tissue along a preferential orientation, which is that supporting the major loading. According to this anatomical evidence, the tissue is described by developing an isotropic fiber-reinforced constitutive model and since the elastic response of the fascia is here considered, the constitutive model is based on the theory of hyperelasticity. The model is consistent with a kinematical description of large strains mechanical behavior, which is typical of soft tissues. A fitting procedure of the constitutive model is implemented making use of experimental curves taken from the literature and referring to specimens of human plantar fascia. A satisfactory fitting of the tensile behavior of the plantar fascia has been performed, showing that the model correctly interprets the mechanical behavior of the tissue in the light of comparison to experimental data at disposal. A critical analysis of the model with respect to the problem of the identification of the constitutive parameters is proposed as the basis for planning a future experimental investigation of mechanical behavior of the plantar fascia.

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.

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.

Mechanics of crural fascia: from anatomy to constitutive modelling.

Ten dissections of inferior limbs and histological studies were performed to describe the structural conformation of the muscular fascia of the leg (crural fascia) and to propose a constitutive model to be adopted for the analysis of its biomechanical behaviour. The crural fascia had a mean thickness of 924 microm and was composed of three layers (mean thickness 277.6 microm) of parallel, collagen fibre bundles separated by a thin layer of loose connective tissue (mean thickness 43 microm). Only a few elastic fibres were highlighted. The disposition of the collagen fibres gives the crural fascia anisotropic characteristics. In addition, their crimped conformation is the cause of the non-linear elastic behaviour of the tissue. Both these aspects are included in the constitutive model. The constitutive modelling of the crural fascia represents a useful tool to rationally interpret the correlation between functional behaviour and structural conformation.

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.

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.

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.

Investigation of bone inelastic response in interaction phenomena with dental implants.

OBJECTIVES: The aim of the paper is to analyze the effects of misfits in multi-implant oral prostheses caused by defects in manufacturing of bar connecting implants. The consequent stress-strain state on peri-implant bone tissue must be carefully considered because of the significant effects induced. MATERIALS AND METHODS: The case of a two-implant prosthesis connected by a titanium cast bar in the pre-molar mandible region is investigated. The complex geometry requires the use of refined finite element model. In consideration of the action induced on the bone tissue, a specific constitutive model that includes inelastic behavior is implemented. The linear misfits considered, estimated with reference to experimental works already present in the literature, induce relevant strains in the peri-implant bone tissue up to a post-elastic phase. Relaxation phenomena are also evaluated. The interaction between the bone and implant is modeled by using contact elements to represent possible detachments at the bone-implant interface. RESULTS: The response of the bone material is reported with regard to the stress/strain field induced, evaluating the inelastic behavior in terms of plastic and relaxation responses. The effects on the peri-implant bone tissue at the interface are evaluated. SIGNIFICANCE: The analysis confirms that the interaction phenomena between the multi-implant oral prostheses and bone induced by the misfit defects of normal intensity induce significant strain in the bone tissue and inelastic phenomena must be taken into account. Local permanent strains of bone tissue and relaxation phenomena represent short-term tissue response for a correct interpretation of the real biomechanical behavior of multiple implant frames.

Constitutive modelling of inelastic behaviour of cortical bone.

A visco-elasto-plastic constitutive model is formulated for investigating the mechanics of cortical bone tissue, accounting for an anisotropic configuration and post-elastic and time-dependent phenomena. The constitutive model is developed with reference to experimental data obtained from literature on the behaviour of cortical bone taken from multiple samples. Regarding the constitutive model, a specific procedure based on a coupled deterministic and stochastic method is applied in order to determine the values of the constitutive parameters with regard to human samples. The procedure entails processing of data deduced from mechanical tests to achieve relationships between permanent and total strain, elastic modulus and strain rate, and creep elastic modulus and time. Numerical results obtained by using a finite element model are compared with tensile experimental data on cortical bone including the post-elastic range and creep phenomena. The model shows an excellent capability to describe the tensile behaviour of the cortical bone for the specific mechanical condition analysed.