Contactless mechanical stimulation of tissue engineered constructs: Development and validation of an air-pulse device.

OBJECTIVE: Tissue engineering (TE) is the study and development of biological substitutes to restore, maintain or improve tissue function. Tissue engineered constructs (TECs) still present differences in mechanical and biological properties compared to native tissue. Mechanotransduction is the process through which mechanical stimulation triggers proliferation, apoptosis, and extracellular matrix synthesis, among other cell activities. Regarding that aspect, the effect of in vitro stimulations such as compression, stretching, bending or fluid shear stress loading modalities have been extensively studied. A fluid flow used to produce contactless mechanical stimulation induced by an air pulse could be easily achieved in vivo without altering the tissue integrity. METHODS: A new air-pulse device for contactless and controlled mechanical simulation of a TECs was developed and validated in this study conducted in the following three phases: 1) conception of the controlled air-pulse device combined with a 3D printed bioreactor; 2) experimental and numerical mechanical characterization of the air-pulse impact by digital image correlation; and 3) achieving sterility and noncytotoxicity of the air-pulse and of the 3D printed bioreactor using a novel dedicated sterilization process. RESULTS: We demonstrated that the treated PLA (polylactic acid) was noncytotoxic and did not influence cell proliferation. An ethanol/autoclaved sterilization protocol for 3D printed objects in PLA has been developed in this study, enabling the use of 3D printing in cell culture. A numerical twin of the device was developed and experimentally characterized by digital image correlation. It showed a coefficient of determination R2 = 0.98 between the numerical and averaged experimental surface displacement profiles of the TEC substitute. CONCLUSION: The results of the study assessed the noncytotoxicity of PLA for prototyping by 3D printing the homemade bioreactor. A novel sterilization process for PLA was developed in this study based on a thermochemical process. A numerical twin using fluid-structure interaction method has been developed to investigate the micromechanical effects of air pulses inside the TEC, which cannot all be measured experimentally, for instance, wave propagation generated during the air-pulse impact. The device could be used to study the cell response to contactless cyclic mechanical stimulation, particularly in TEC with fibroblasts, stromal cells and mesenchymal stem cells, which have been shown to be sensitive to the frequency and strain level at the air-liquid interface.

Strain rate and anisotropy effects on the tensile failure characteristics of human skin.

The anisotropic failure characteristics of human skin are relatively unknown at strain rates typical in impact biomechanics. This study reports the results of an experimental protocol to quantify the effect of dynamic strain rates and the effect of sample orientation with respect to the Langer lines. Uniaxial tensile tests were carried out at three strain rates (0.06s(-1), 53s(-1), and 167s(-1)) on 33 test samples excised from the back of a fresh cadaver. The mean ultimate tensile stress, mean elastic modulus and mean strain energy increased with increasing strain rates. While the stretch ratio at ultimate tensile stress was not affected by the strain rate, it was influenced by the orientation of the samples (parallel and perpendicular to the Langer lines. The orientation of the sample also had a strong influence on the ultimate tensile stress, with a mean value of 28.0 ± 5.7 MPa for parallel samples, and 15.6 ± 5.2 MPa for perpendicular samples, and on the elastic modulus, with corresponding mean values of 160.8 MPa ± 53.2 MPa and 70.6 MPa ± 59.5 MPa. The study also pointed out the difficulties in controlling the effective applied strain rate in dynamic characterization of soft tissue and the resulting abnormal stress-strain relationships. Finally, data collected in this study can be used to develop constitutive models where high loading rates are of primary interest.

Geometrical and material parameters to assess the macroscopic mechanical behaviour of fresh cranial bone samples.

The present study aims at providing quantitative data for the personalisation of geometrical and mechanical characteristics of the adult cranial bone to be applied to head FE models. A set of 351 cranial bone samples, harvested from 21 human skulls, were submitted to three-point bending tests at 10 mm/min. For each of them, an apparent elastic modulus was calculated using the beam's theory and a density-dependant beam inertia. Thicknesses, apparent densities and percentage of ash weight were also measured. Distributions of characteristics among the different skull bones show their symmetry and their significant differences between skull areas. A data analysis was performed to analyse potential relationship between thicknesses, densities and the apparent elastic modulus. A specific regression was pointed out to estimate apparent elastic modulus from the product of thickness by apparent density. These results offer quantitative tools in view of personalising head FE models and thus improve definition of local injury criteria for this body part.

In-vivo analysis of thoracic mechanical response under belt loading: the role of body mass index in thorax stiffness.

Thoracic injuries are a major cause of mortality in frontal collisions, especially for elderly and obese people. Car occupant individual characteristics like BMI are known to influence human vulnerability in crashes. In the present study, thoracic mechanical response of volunteers quantified by optical method was linked to individual characteristics. 13 relaxed volunteers of different anthropometries, genders and age were submitted to non-injurious sled tests (4 g, 8 km/h) with a sled buck representing the environment of a front passenger restrained by a 3-point belt. A resulting shoulder belt force was computed using the external and internal shoulder belt loads and considering shoulder belt geometry. The mid sternal deflection was calculated as the distance variation between markers placed at mid-sternum and the 7th vertebra spinous process of the subject. Force-deflection curves were constructed using resulting shoulder belt force and midsternal deflection. Average maximum chest compression was 7.9±2.3% and no significant difference was observed between overweight subjects (BMI≥25 kg/m²) and normal subject (BMI<25 kg/m²). The overweight subjects exhibited significantly greater resultant belt forces than normal subjects (715±132 N vs. 527±111 N, p<0.05), higher effective stiffness (30.9±10.6N/mm vs. 19.6±8.9 N/mm, p<0.05) and lower dynamic stiffness (42.7±8.71 N/mm vs. 61.7±15.5 N/mm, p<0.05).

In vivo analysis of thoracic mechanical response variability under belt loading: specific behavior and relationship to age, gender and body mass index.

Thoracic injuries are a major cause of mortality in frontal collisions, especially for elderly female and obese people. Car occupant individual characteristics like age, gender and Body Mass Index (BMI) are known to influence human vulnerability tolerance in crashes. The objective of the this study was to perform in vivo test experiments to quantify the influence of subject characteristics in terms of age, gender and anthropometry and on thorax mechanical response variability under belt loading. Thirty-nine relaxed volunteers of different anthropometries, genders and age were submitted to non-injurious sled tests (4 g, 8 km/h) with a sled buck representing the environment of a front passenger restrained by a 3-point belt. A resulting shoulder belt force FRes was computed using the external and internal shoulder belt loads and considering shoulder belt geometry. The mid sternal deflection D was calculated as the distance variation between markers placed at mid-sternum and at the 7th vertebra spinous process of the subject. Linear stiffness (K) and damping coefficient (µ) of a spring-dashpot model were identified from the FRes-D curves of each test. The analysis suggests that among subjects over 40 years old, thinness leads to higher K-values.

Automated estimation of collagen fibre dispersion in the dermis and its contribution to the anisotropic behaviour of skin.

Collagen fibres play an important role in the mechanical behaviour of many soft tissues. Modelling of such tissues now often incorporates a collagen fibre distribution. However, the availability of accurate structural data has so far lagged behind the progress of anisotropic constitutive modelling. Here, an automated process is developed to identify the orientation of collagen fibres using inexpensive and relatively simple techniques. The method uses established histological techniques and an algorithm implemented in the MATLAB image processing toolbox. It takes an average of 15 s to evaluate one image, compared to several hours if assessed visually. The technique was applied to histological sections of human skin with different Langer line orientations and a definite correlation between the orientation of Langer lines and the preferred orientation of collagen fibres in the dermis (p < 0.001, R(2) = 0.95) was observed. The structural parameters of the Gasser-Ogden-Holzapfel (GOH) model were all successfully evaluated. The mean dispersion factor for the dermis was κ = 0.1404±0.0028. The constitutive parameters µ, k(1) and k(2) were evaluated through physically-based, least squares curve-fitting of experimental test data. The values found for µ, k(1) and k(2) were 0.2014 MPa, 243.6 and 0.1327, respectively. Finally, the above model was implemented in ABAQUS/Standard and a finite element (FE) computation was performed of uniaxial extension tests on human skin. It is expected that the results of this study will assist those wishing to model skin, and that the algorithm described will be of benefit to those who wish to evaluate the collagen dispersion of other soft tissues.

Characterization of the anisotropic mechanical properties of excised human skin.

The mechanical properties of skin are important for a number of applications including surgery, dermatology, impact biomechanics and forensic science. In this study, we have investigated the influence of location and orientation on the deformation characteristics of 56 samples of excised human skin. Uniaxial tensile tests were carried out at a strain rate of 0.012 s(-1) on skin from the back. Digital Image Correlation was used for 2D strain measurement and a histological examination of the dermis was also performed. The mean ultimate tensile strength (UTS) was 21.6±8.4 MPa, the mean failure strain 54%±17%, the mean initial slope 1.18±0.88 MPa, the mean elastic modulus 83.3±34.9 MPa and the mean strain energy was 3.6±1.6 MJ/m(3). A multivariate analysis of variance has shown that these mechanical properties of skin are dependent upon the orientation of the Langer lines (P<0.0001-P=0.046). The location of specimens on the back was also found to have a significant effect on the UTS (P=0.0002), the elastic modulus (P=0.001) and the strain energy (P=0.0052). The histological investigation concluded that there is a definite correlation between the orientation of the Langer lines and the preferred orientation of collagen fibres in the dermis (P<0.001). The data obtained in this study will provide essential information for those wishing to model the skin using a structural constitutive model.

The mechanics of the in vivo infant and toddler trunk during respiratory physiotherapy.

BACKGROUND: The purpose of this study is to quantify the in vivo mechanical response of the child trunk under loading during physiotherapy treatments. METHODS: Twenty-six children aged 45 days to 7 years (14 girls and 12 boys) took part in this study. The forces applied by the physiotherapist were recorded using a force-plate embedded in the manipulation table supporting the child. Two synchronized cameras filmed the scene in a calibrated environment. The displacement of reflective targets glued on the physiotherapist's hands was calculated using an automatic tracking procedure and the 3D reconstruction "Direct Linear Transformation" algorithm. The progression of physical parameters was evaluated according to the age of the child. They included force, displacement, normalized displacement, loading speed, displacement and normalized displacement at the maximum force, force at the maximum displacement, viscous criterion and effective stiffness. FINDINGS: For all patients, the mean maximum displacement and load were 22 mm (SD 9 mm) and 240 N (SD 46 N) respectively. The force-displacement curves had shown the complexity of the in vivo behavior: four phases have been distinguished with cycles in respect with the respiratory phases. The increase in force always occurred before the increase in displacement. INTERPRETATION: This study helps to understand the in vivo behavior of the child trunk subjected to repetitive non-injurious mechanical loading. Further analysis in other populations and with different therapeutic maneuvers would refine the results.

Clavicle fracture prediction: simulation of shoulder lateral impacts with geometrically personalized finite elements models.

BACKGROUND: Human body numerical models can help to develop protection devices against effects of road crashes. In the context of a side impact, a shoulder model able to predict shoulder injuries and more especially clavicle fracture would be helpful. METHODS: A shoulder model derived from an existing finite element model of the human body representing an average male (50th percentile), HUMOS1, has been upgraded. An isolated clavicle model was assessed thanks to experimental corridors derived from dynamic tests up to failure. Then, the whole upgraded shoulder model was evaluated by comparison with results from experimental side impact tests on the shoulder. Eventually, the upgraded model was geometrically personalized toward the anthropometry of the subjects and its ability to simulate fractures was assessed. RESULTS: The isolated clavicle model was assessed as validated. The upgraded 50th percentile shoulder model provided accurate results in the subinjurious domain. At higher velocities, the personalized models produced realistic shoulder injuries: clavicle fracture was accurately predicted in four cases of six, the model was conservative for the two other cases. CONCLUSION: The upgraded shoulder model presented here was successfully submitted to a rigorous assessment process. Once geometrically personalized, it provided positive results for clavicle fracture prediction. As clavicle fracture is the major shoulder injury, this model could help the design of safety devices for shoulder protection. Furthermore, this study enhances the need for geometrical personalization methods when using finite element model for injury risk prediction.

Influence of geometrical personalization on the simulation of clavicle fractures.

Finite element body models enable the evaluation of car occupant protection. In general, these models represent average males and inter-individual geometry variability is not taken into account. As the most frequent shoulder injury during car lateral accidents is a clavicle fracture, the purpose of this study is to investigate whether clavicle geometry has an influence on bone response until failure, and whether geometrical personalization of clavicle models is required. Eighteen clavicles from 9 subjects (5 males and 4 females, mean age: 76 +/- 12 years) were harvested. Six clavicles were scanned, enabling the development of subject-specific models and the quantification of geometrical features defining shape and cortical thickness. Bone mineral densities (BMD) were measured through double X-ray absorptiometry. Then, the general clavicle responses to dynamic compression until failure were studied. Simulations of the compression tests were carried out with the subject-specific models to assess the sensitivity of force-deflection clavicle responses to geometrical features. Clavicle fractures occurred at an average velocity of 1.41 +/- 0.4 ms(-1), with a fracture force of 1.48 +/- 0.46 kN and a deflection of 5.4 +/- 1.1 mm. A significant difference was found between male and female clavicle force values at rupture although their BMDs were not significantly different. Simulations with subject-specific models led to the conclusion that cortical bone thickness and bone shape have large effects on bone responses until failure and on fracture location. This study highlights the need for a geometrical personalization of clavicle models in order to take into account both gender discrepancies concerning clavicle shape and aging effects affecting cortical thickness.