Distinct membrane mechanical properties of human mesenchymal stem cells determined using laser optical tweezers

The therapeutic efficacy of mesenchymal stem cells (MSCs) in tissue engineering and regenerative medicine is determined by their unique biological, mechanical, and physicochemical characteristics, which are yet to be fully explored. Cell membrane mechanics, for example, has been shown to critically influence MSC differentiation. In this study, we used laser optical tweezers to measure the membrane mechanics of human MSCs and terminally differentiated fibroblasts by extracting tethers from the outer cell membrane. The average tether lengths were 10.6+/-1.1 microm (hMSC) and 3.0+/-0.5 microm (fibroblasts). The tether extraction force did not increase during tether formation, which suggests existence of a membrane reservoir intended to buffer membrane tension fluctuations. Cytoskeleton disruption resulted in a fourfold tether length increase in fibroblasts but had no effect in hMSCs, indicating weak association between the cell membrane and hMSC actin cytoskeleton. Cholesterol depletion, known to decrease lipid bilayer stiffness, caused an increase in the tether length both in fibroblasts and hMSCs, as does the treatment of cells with DMSO. We postulate that whereas fibroblasts use both the membrane rigidity and membrane-cytoskeleton association to regulate their membrane reservoir, hMSC cytoskeleton has only a minor impact on stem cell membrane mechanics.     

Intrinsic mechanical properties of trabecular calcaneus determined by finite-element models using 3D synchrotron microtomography

To determine intrinsic mechanical properties (elastic and failure) of trabecular calcaneus bone, chosen as a good predictor of hip fracture, we looked for the influence of image's size on a numerical simulation. One cubic sample of cancellous bone (9 x 9 x 9 mm(3)) was removed from the body of the calcaneus (6 females, 6 males, 79+/-9 yr). These samples were tested under compressive loading. Before compressive testing, these samples were imaged at 10.13 microm resolution using a 3D microcomputed tomography (muCT) (ESRF, France). The muCT images were converted to finite-element models. Depending on the bone density values (BV/TV), we compared two different finite element models: a linear hexahedral and a linear beam finite element models. Apparent experimental Young's modulus (E(app)(exp)) and maximum apparent experimental compressive stress (sigma(max)(exp)) were significantly correlated with bone density obtained by Archimedes's test (E(app)(exp)=236+/-231 MPa [19-742 MPa], sigma(max)(exp)=2.61+/-1.97 MPa [0.28-5.81 MPa], r>0.80, p<0.001). Under threshold at 40 microm, the size of the numerical samples (5.18(3) and 6.68(3)mm(3)) seems to be an important parameter on the accuracy of the results. The numerical trabecular Young's modulus was widely higher (E(trabecular)(num)=34,182+/-22,830 MPa [9700-87,211 MPa]) for the larger numerical samples and high BV/TV than those found classically by other techniques (4700-15,000 MPa). For rod-like bone samples (BV/TV<12%, n=7), Young's modulus, using linear beam element (E(trabecular)(num-skeleton): 10,305+/-5500 MPa), were closer to the Young's modulus found by other techniques. Those results show the limitation of hexahedral finite elements at 40 microm, mostly used, for thin trabecular structures.     

Carotid artery mechanical properties and stresses quantified using in vivo data from normotensive and hypertensive humans

The goal of this study was to model the in vivo non-linear mechanical behavior of human common carotid arteries (CCAs) and then to compare wall stresses and associated contributions of micro-constituents in normotensive (NT) and treated hypertensive (HT) subjects. We used an established theoretical model of 3D arterial mechanics that assumes a hyperelastic, anisotropic, active–passive, and residually stressed wall. In vivo data were obtained non-invasively from CCAs in 16 NT (21–64 years old) and 25 treated HT (44–69 years old) subjects. The associated quasi-static boundary value problem was solved semi-analytically over a cardiac cycle while accounting for surrounding perivascular tissue. Best-fit values of model parameters, including those describing contributions by intramural elastin, fibrillar collagen, and vascular smooth muscle, were estimated by a non-linear least-squares method. The model (1) captured temporal changes in intraluminal pressure, (2) estimated wall stress fields that appeared to reflect the presence or absence of age and disease, and (3) suggested changes in mechanical characteristics of wall micro-constituents despite medical treatment of hypertension. For example, age was positively correlated with residual stresses and altered fibrillar collagen in NT subjects, which indirectly validated the modeling, and HT subjects had higher levels of stresses, increased smooth muscle tone, and a stiffer elastin-dominated matrix despite treatment. These results are consistent with prior reports on effects of age and hypertension, but provide increased insight into evolving contributions of cell and matrix mechanics to arterial behavior in vivo.     

Passive transverse mechanical properties of skeletal muscle under in vivo compression

The objective of the present study is to determine the passive transverse mechanical properties of skeletal muscle. Compression experiments were performed on four rat tibialis anterior muscles. To assess the stress- and strain-distributions in the muscle during the experiment, a plane stress model of the cross section was developed for each muscle. The incompressible viscoelastic Ogden model was used to describe the passive muscle behaviour. The four material parameters were determined by fitting calculated indentation forces on measured indentation forces. The elastic parameters, mu and alpha, were 15.6+/-5.4 kPa and 21.4+/-5.7, respectively. The viscoelastic parameters, delta and tau, were 0.549+/-0.056 and 6.01+/-0.42 s. When applying the estimated material parameters in a three-dimensional finite element model, the measured behaviour can be accurately simulated.     

In vivo characterization of mechanical tissue properties of internal organs using endoscopic microscopy and inverse finite element analysis

Knowledge about mechanical tissue properties is required for functional modelling and simulating of tissue and organ responses to external mechanical stress. To get the right properties especially for functional modelling of organs, tissue properties have to be determined in vivo. There are only few described methods for characterization of internal organ's tissue mechanics that can be applied in vivo. We introduce and evaluate a method to determine mechanical tissue properties, especially those of lung tissue, endoscopically. Inverse finite element analysis (utilizing a Neo-Hookean model for hyperelastic materials) and image processing algorithms are used to determine the shear modulus of a soft tissue. The resulting values for shear moduli were normally distributed. The shear modulus of the artificial tissue sample was determined with a relative error of 0.47% compared to the value obtained by uniaxial tensile test.     

Ex vivo stretch reveals altered mechanical properties of isolated dystrophin-deficient hearts

Duchenne muscular dystrophy (DMD) is a progressive and fatal disease of muscle wasting caused by loss of the cytoskeletal protein dystrophin. In the heart, DMD results in progressive cardiomyopathy and dilation of the left ventricle through mechanisms that are not fully understood. Previous reports have shown that loss of dystrophin causes sarcolemmal instability and reduced mechanical compliance of isolated cardiac myocytes. To expand upon these findings, here we have subjected the left ventricles of dystrophin-deficient mdx hearts to mechanical stretch. Unexpectedly, isolated mdx hearts showed increased left ventricular (LV) compliance compared to controls during stretch as LV volume was increased above normal end diastolic volume. During LV chamber distention, sarcomere lengths increased similarly in mdx and WT hearts despite greater excursions in volume of mdx hearts. This suggests that the mechanical properties of the intact heart cannot be modeled as a simple extrapolation of findings in single cardiac myocytes. To explain these findings, a model is proposed in which disruption of the dystrophin-glycoprotein complex perturbs cell-extracellular matrix contacts and promotes the apparent slippage of myocytes past each other during LV distension. In comparison, similar increases in LV compliance were obtained in isolated hearts from β-sarcoglycan-null and laminin-α(2) mutant mice, but not in dysferlin-null mice, suggesting that increased whole-organ compliance in mdx mice is a specific effect of disrupted cell-extracellular matrix contacts and not a general consequence of cardiomyopathy via membrane defect processes. Collectively, these findings suggest a novel and cell-death independent mechanism for the progressive pathological LV dilation that occurs in DMD.     

In vivo passive mechanical properties of the human gastrocnemius muscle belly

The purpose of the present study was to determine the in vivo passive mechanical properties, including the length below the slack length, of the gastrocnemius muscle (GAS) belly in humans. Transverse ultrasound images of the medial head of the GAS were taken in 11 subjects during passive knee extension from 80 degrees to 5 degrees with a constant ankle joint angle of 10 degrees (0 degrees is the neutral ankle position: positive values for dorsiflexion). The change in passive ankle joint moment (Mp), which is produced only by the GAS length change, was also measured during passive knee extension. The onset of Mp during passive knee extension was found to be 43+/-8 degrees (mean+/-SD) when the baseline of the Mp was set at the average Mp in the range of 55-60 degrees where the Mp was almost constant (SD<0.03 Nm). At this onset, the muscle fascicle length of the GAS (Lf) was 46+/-7 mm (slack length; Lfs). Lf at 80 degrees was 6+/-4 mm (13+/-6%) less than the Lfs, and Lf at 5 degrees was 12+/-5 mm (27+/-11%) greater than the Lfs. The passive force-resisting compression of the GAS did not produce a dorsiflexion moment in the joint angle range adopted. The passive ankle joint moment increased linearly with Lf (coefficient of determination (R2)=0.85-0.96), and the slopes of the relationships between Lf and Mp, and between the relative Lf to Lfs and Mp were 0.093+/-0.038 Nm/mm and 0.043+/-0.021 Nm/%Lfs. The findings of the present study can be implemented in musculoskeletal modeling, which would provide a more accurate evaluation of the passive mechanical properties of muscle during movement.     

Oxygen binding properties of backswimmer (Notonectidae, Anisops) haemoglobin, determined in vivo

Aquatic backswimmers (Anisops spp.) collect oxygen from the atmosphere in order to breathe underwater, carrying it within a bubble of air on the ventral surface of their body and bound within haemoglobin-filled cells inside their abdomen. These oxygen stores are interconnected via the abdominal spiracles and the tracheal system. Fibre optic oxygen probes were used to measure PO₂ changes within the air bubbles of submerged backswimmers (Anisops deanei) and these measurements were transformed into in vivo haemoglobin–oxygen equilibrium curves (OECs) using a biotonometric approach. The haemoglobin displayed a triphasic, highly sigmoid OEC with a P₅₀ of 3.90 kPa. Comparisons made with a previous in vitro analysis of Anisops haemoglobin demonstrate that while the apparent cooperativity and oxygen affinity are considerably higher in vivo, both measurements share unusual Hb–O₂ binding characteristics. The affinity and cooperativity of the backswimmers’ haemoglobin appears adaptive as it lengthens dives and promotes neutral buoyancy. While there are limitations associated with biotonometry, the in vivo OEC accurately represents the loading and unloading of biologically available oxygen within the backswimmers’ haemoglobin cells. Potential errors associated with determining the OEC are small, as evaluated with sensitivity analyses in numerical models.     

In vivo human tendon mechanical properties: effect of resistance training in old age

Recent advances in ultrasound scanning have made it possible to obtain the mechanical properties of human tendons in vivo. Application of the in vivo method in elderly individuals showed that their patellar tendons stiffened in response to a 14-week resistance training program by approximately 65% both structurally and materially. The rate of muscle torque development increased by approximately 27%, indicating faster contractile force transmission to the skeleton. The present findings suggest that strength training in old age can, at least partly, reverse the deteriorating effect of ageing on tendon properties and function.     

Mechanical properties of coronary stents determined by using finite element analysis.

The mechanical function of a stent deployed in a damaged artery is to provide a metallic tubular mesh structure. The purpose of this study was to determine the exact mechanical characteristics of stents. In order to achieve this, we have used finite-element analysis to model two different type of stents: tubular stents (TS) and coil stents (CS). The two stents chosen for this modeling present the most extreme mechanical characteristics of the respective types. Seven mechanical properties were studied by mathematical modeling with determination of: (1) stent deployment pressure, (2) the intrinsic elastic recoil of the material used, (3) the resistance of the stent to external compressive forces, (4) the stent foreshortening, (5) the stent coverage area, (6) the stent flexibility, and (7) the stress maps. The pressure required for deployment of CS was significantly lower than that required for TS, over 2.8 times greater pressure was required for the tubular model. The elastic recoil of TS is higher than CS (5.4% and 2.6%, respectively). TS could be deformed by 10% at compressive pressures of between 0.7 and 1.3 atm whereas CS was only deformed at 0.2 and 0.7 atm. The degree of shortening observed increases with deployment diameter for TS. CS lengthen during deployment. The metal coverage area is two times greater for TS than for CS. The ratio between the stiffness of TS and that of CS varies from 2060 to 2858 depending on the direction in which the force is applied. TS are very rigid and CS are significantly more flexible. Stress mapping shows stress to be localized at link nodes. This series of finite-element analyses illustrates and quantifies the main mechanical characteristics of two different commonly used stents. In interventional cardiology, we need to understand their mechanisms of implantation and action.     

Non-destructive assessment of human ribs mechanical properties using quantitative ultrasound

Advanced finite element models of the thorax have been developed to study, for example, the effects of car crashes. While there is a need for material properties to parameterize such models, specific properties are largely missing. Non-destructive techniques applicable in vivo would, therefore, be of interest to support further development of thorax models. The only non-destructive technique available today to derive rib bone properties would be based on quantitative computed tomography that measures bone mineral density. However, this approach is limited by the radiation dose. Bidirectional ultrasound axial transmission was developed on long bones ex vivo and used to assess in vivo health status of the radius. However, it is currently unknown if the ribs are good candidates for such a measurement. Therefore, the goal of this study is to evaluate the relationship between ex vivo ultrasonic measurements (axial transmission) and the mechanical properties of human ribs to determine if the mechanical properties of the ribs can be quantified non-destructively. The results show statistically significant relationships between the ultrasonic measurements and mechanical properties of the ribs. These results are promising with respect to a non-destructive and non-ionizing assessment of rib mechanical properties.     

The effects of bone and pore volume fraction on the mechanical properties of PMMA/bone biopsies extracted from augmented vertebrae

Vertebroplasty forms a porous PMMA/bone composite which was shown to be weaker and less stiff than pure PMMA. It is not known what determines the mechanical properties of such composites in detail. This study investigated the effects of bone volume fraction (BV/TV), cement porosity (PV/(TV-BV), PV…pore volume) and cement stiffness. Nine human vertebral bodies were augmented with either standard or low-modulus PMMA cement and scanned with a HR-pQCT system before and after augmentation. Fourteen cylindrical PMMA/bone biopsies were extracted from the augmented region, scanned with a micro-CT system and tested in compression until failure. Micro-finite element (FE) models of the complete biopsies, of the trabecular bone alone as well as of the porous cement alone were generated from CT images to gain more insight into the role of bone and pores. PV/(TV-BV) and experimental moduli of standard/low-modulus cement (R(2)=0.91/0.98) as well as PV/(TV-BV) and yield stresses (R(2)=0.92/0.83) were highly correlated. No correlation between BV/TV (ranging from 0.057 to 0.138) and elastic moduli was observed (R(2)< 0.05). Interestingly, the micro-FE models of the porous cement alone reproduced the experimental elastic moduli of the standard/low-modulus cement biopsies (R(2)=0.75/0.76) more accurately than the models with bone (R(2)=0.58/0.31). In conclusion, the mechanical properties of the biopsies were mainly determined by the cement porosity and the cement material properties. The study showed that bone tissue inside the biopsies was mechanically "switched off" such that load was carried essentially by the porous PMMA.     

An experimental study on in vivo dynamic mechanical properties of the knees of cats in valgus loading

In these experiments an attempt was made to investigate in vivo dynamic mechanical properties of the knee in valgus loading. Fifteen adult cats were used. Sinusoidal bending oscillation of 0.3, 1.0, 1.6, 3.3, 5.0, 8.6 and 10.0 Hz was transmitted to the knee-leg complex under each pre-bending strain in the valgus direction, and the bending moment was measured. Dynamic mechanical properties of the knee-leg complex were given as a complex bending elastic coefficient. Bending elastic and bending damping coefficient of the knee could be calculated by the least square method. As a first approximation, the knee was represented as a non-linear Kelvin model. Both bending elastic and bending damping coefficient of the knee increased as pre-bending moment was increased, and decreased as amplitude of bending oscillation was increased. Mechanical loss tangent of the knee-leg complex decreased within the range of pre-bending moment larger than 1.0 X 10(6) dyn cm as bending moment was increased, and decreased as amplitude of bending oscillation was increased.     

Kinetic properties of bovine brain proteinl-isoaspartyl methyltransferase determined using a synthetic isoaspartyl peptide substrate

Proteinl-isoaspartyl methyltransferase, an enzyme enriched in brain, is implicated in the repair of age-damaged proteins containing atypical, isoaspartyl peptide bonds. We have investigated the kinetics of methylation using a synthetic peptide substrate having the structure Trp-Ala-Gly-Gly-isoAsp-Ala-Ser-Gly-Glu. Double-reciprocal plots of initial velocity versus concentration of S-adenosylmethionine (AdoMet) at different fixed concentrations of peptide gave straight lines converging at a positive 1/v value and a negative 1/AdoMet value. The product S-adenosylhomocysteine (AdoHcy) was a competitive inhibitor towards AdoMet and a linear mixed-type inhibitor towards peptide. These results are consistent with the rapid-equilibrium random sequential bi-bi mechanism previously proposed for the enzyme, but they also reveal the formation of the deadend, enzyme-peptide-AdoHcy, complex. The rate constants were:V _max=32–34 nmol/min/mg,K ^peptide=7.6–9.4 M,K ^AdoMet=1.9–2.2 M, =0.43–0.53,K ^AdoHcy=0.08 M, =2.9. The interaction factors and indicate that binding of enzyme to peptide increases its affinity for AdoMet and decreases its affinity for AdoHcy. Methylation was linear with time throughout the transfer of 2 mol of methyl groups/mol of enzyme. This absence of burst kinetics suggests that slow release of products cannot explain the low turnover number.Special issue dedicated to Dr. Paul Greengard     

Study of the mechanical properties of myomesin proteins using dynamic force spectroscopy

Myomesin is the most prominent structural component of the sarcomeric M-Band that is expressed in mammalian heart and skeletal muscles. Like titin, this protein is an intracellular member of the Ig-fibronectin superfamily, which has a flexible filamentous structure and which is largely composed of two types of domain that are similar to immunoglobulin (Ig)-like and fibronectin type III (FNIII) domains. Several myomesin isoforms have been identified, and their expression patterns are highly regulated both spatially and temporally. Particularly, alternative splicing in the central part of the molecule gives rise to an isoform, EH (embryonic heart)-myomesin, containing a serine and proline-rich insertion with no well-defined secondary structure, the EH segment. EH-myomesin represents the major myomesin isoform at embryonic stages of mammalian heart and is rapidly down-regulated around birth, but it is re-expressed in the heart of patients suffering from dilated cardio-myopathy. Here, in order to facilitate a better understanding of the physiological, and possibly pathological, functions of myomesin proteins, we explore the mechanical stability, elasticity and force-driven structural changes of human myomesin's sub-molecular segments using single-molecule force spectroscopy and protein engineering. We find that human myomesin molecules are composed of modules (Ig and FNIII), that are designed to withstand force and we demonstrate that the human cardiac EH segment functions like an additional elastic stretch in the middle part of the EH-myomesin and behaves like a random coil. Consequently myomesin isoforms (proteins with or without the EH segment) have different elastic properties, the EH-myomesin being the more compliant one. These findings imply that the compliance of the M-band increases with the amount of EH-myomesin it contains. So, we provide the evidence that not only titin but also other sarcomeric proteins have complicated visco-elastic properties depending on the contractile parameters in different muscle types.     

Evaluation of frontal radiographs of scoliotic spines—Part II. Relations between lateral deviation, lateral tilt and axial rotation of vertebrae

The shape of scoliotic spines as measured from frontal radiographs (see Part I of this paper) is analysed with respect to interrelations between lateral deviation, lateral tilt and axial rotation of the vertebrae. These parameters are represented by sinusoidal functions of the longitudinal coordinate. The interrelations can, therefore, be expressed in terms of amplitude and phase relations. Two additional functions—‘spinal tilt’ and (local) curvature—are calculated from the first and second derivatives of lateral deviation. The method has been applied to three patient groups with different aetiology: 113 patients with idiopathic scoliosis (478 radiographs, partially follow-up examinations), 23 patients with scoliosis secondary to Wilms' tumour irradiation and 18 patients with scoliosis secondary to poliomyelitis. The amplitude and phase relations of all functions reveal a characteristic pattern which is apparently independent of the specific aetiology. The results show that the available biomechanic explanations of coupling of vertebral motions are questionable.     

Ex vivo mechanical loading of tendon

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37 degrees C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.