Mechanical stimulation improves tissue-engineered human skeletal muscle

Human bioartificial muscles (HBAMs) aretissue engineered by suspending muscle cells in collagen/MATRIGEL,casting in a silicone mold containing end attachment sites, andallowing the cells to differentiate for 8 to 16 days. The resultingHBAMs are representative of skeletal muscle in that they containparallel arrays of postmitotic myofibers; however, they differ in manyother morphological characteristics. To engineer improved HBAMs, i.e.,more in vivo-like, we developed Mechanical Cell Stimulator (MCS)hardware to apply in vivo-like forces directly to the engineeredtissue. A sensitive force transducer attached to the HBAM measuredreal-time, internally generated, as well as externally applied, forces.The muscle cells generated increasing internal forces during formationwhich were inhibitable with a cytoskeleton depolymerizer. Repetitivestretch/relaxation for 8 days increased the HBAM elasticity two- tothreefold, mean myofiber diameter 12%, and myofiber area percent 40%.This system allows engineering of improved skeletal muscle analogs aswell as a nondestructive method to determine passive force andviscoelastic properties of the resulting tissue.

     

Buckle muscle tension transducer: what does it measure?

The question is considered whether the strain of a buckle transducer attached to a muscle tendon provides a proportional measure of the force of the muscle acting directly on that tendon. It is shown that if muscle contains elastic and/or viscous elements in parallel with the force generator, the transducer strain may, under certain conditions, reflect other applied forces acting on the load (limb) in addition to the muscle force.     

Biophysical stimuli induced by passive movements compensate for lack of skeletal muscle during embryonic skeletogenesis

In genetically modified mice with abnormal skeletal muscle development, bones and joints are differentially affected by the lack of skeletal muscle. We hypothesise that unequal levels of biophysical stimuli in the developing humerus and femur can explain the differential effects on these rudiments when muscle is absent. We find that the expression patterns of four mechanosensitive genes important for endochondral ossification are differentially affected in muscleless limb mutants, with more extreme changes in the expression in the humerus than in the femur. Using finite element analysis, we show that the biophysical stimuli induced by muscle forces are similar in the humerus and femur, implying that the removal of muscle contractile forces should, in theory, affect the rudiments equally. However, simulations in which a displacement was applied to the end of the limb, such as could be caused in muscleless mice by movements of the mother or normal littermates, predicted higher biophysical stimuli in the femur than in the humerus. Stimuli induced by limb movement were much higher than those induced by the direct application of muscle forces, and we propose that movements of limbs caused by muscle contractions, rather than the direct application of muscle forces, provide the main mechanical stimuli for normal skeletal development. In muscleless mice, passive movement induces unequal biophysical stimuli in the humerus and femur, providing an explanation for the differential effects seen in these mice. The significance of these results is that forces originating external to the embryo may contribute to the initiation and progression of skeletal development when muscle development is abnormal.     

Influence of muscle activity on the forces in the femur: An in vivo study

Experiments were performed on two patients with custom-made instrumented massive proximal femoral prostheses implanted after tumour resection. In vivo axial forces transmitted along the prostheses were telemetered during level walking, single- and double-leg stance, and isometric exercises of the hip muscles. These activities varied the lever arms available to the external loads: minimum for double-leg stance and maximum for hip isometric exercises. Kinematic, force plate, EMG and telemetered force data were recorded simultaneously. The force magnification ration (FMR; the ratio of the telemetered axial force to the external force) was calculated. The FMRs ranged from 1.3 (during double-leg stance) to 29.8 (during abductors test), indicating that a major part of the axial force in the long bones is a response to muscle activity, the strength of which depends on the lever arms available to the external loads. From these results, it was shown that the bulk of the bending moment along limbs is transmitted by a combination of tensile forces in muscles and compressive forces in bones, so moments transmitted by the bones are smaller than the limb moments. It was concluded that appropriate simulation of muscle forces is important in experimental or theoretical studies of load transmission along bones.     

Individual muscle force parameters and fiber operating ranges for elbow flexion-extension and forearm pronation-supination

We have quantified individual muscle force and moment contributions to net joint moments and estimated the operating ranges of the individual muscle fibers over the full range of motion for elbow flexion/extension and forearm pronation/supination. A three dimensional computer graphics model was developed in order to estimate individual muscle contributions in each degree of freedom over the full range of motion generated by 17 muscles crossing the elbow and forearm. Optimal fiber length, tendon slack length, and muscle specific tension values were adjusted within the literature range from cadaver studies such that the net isometric joint moments of the model approximated experimental joint moments within one standard deviation. Analysis of the model revealed that the muscles operate on varying portions of the ascending limb, plateau region, and descending limb of the force-length curve. This model can be used to further understand isometric force and moment contributions of individual muscles to net joint moments of the arm and forearm and can serve as a comprehensive reference for the forces and moments generated by 17 major muscles crossing the elbow and wrist.     

Simultaneous prediction of muscle and contact forces in the knee during gait

Musculoskeletal models are currently the primary means for estimating in vivo muscle and contact forces in the knee during gait. These models typically couple a dynamic skeletal model with individual muscle models but rarely include articular contact models due to their high computational cost. This study evaluates a novel method for predicting muscle and contact forces simultaneously in the knee during gait. The method utilizes a 12 degree-of-freedom knee model (femur, tibia, and patella) combining muscle, articular contact, and dynamic skeletal models. Eight static optimization problems were formulated using two cost functions (one based on muscle activations and one based on contact forces) and four constraints sets (each composed of different combinations of inverse dynamic loads). The estimated muscle and contact forces were evaluated using in vivo tibial contact force data collected from a patient with a force-measuring knee implant. When the eight optimization problems were solved with added constraints to match the in vivo contact force measurements, root-mean-square errors in predicted contact forces were less than 10 N. Furthermore, muscle and patellar contact forces predicted by the two cost functions became more similar as more inverse dynamic loads were used as constraints. When the contact force constraints were removed, estimated medial contact forces were similar and lateral contact forces lower in magnitude compared to measured contact forces, with estimated muscle forces being sensitive and estimated patellar contact forces relatively insensitive to the choice of cost function and constraint set. These results suggest that optimization problem formulation coupled with knee model complexity can significantly affect predicted muscle and contact forces in the knee during gait. Further research using a complete lower limb model is needed to assess the importance of this finding to the muscle and contact force estimation process.     

Elbow and wrist joint contact forces during occupational pick and place activities

A three-dimensional, mathematical model of the elbow and wrist joints, including 15 muscle units, 3 ligaments and 4 joint forces, has been developed. A new strain gauge transducer has been developed to measure functional grip forces. The device measures radial forces divided into six components and forces of up to 250N per segment can be measured with an accuracy of +/-1%. Ten normal volunteers were asked to complete four tasks representing occupational activities, during which time their grip force was monitored. Together with kinematic information from the six-camera Vicon data, the moment effect of these loads at the joints was calculated. These external moments are assumed to be balanced by the internal moments, generated by the muscles, passive soft tissue and bone contact. The effectiveness of the body's internal structures in generating joint moments was assessed by studying the geometry of a simplified model of the structures, where information about the lines of action and moment arms of muscles, tendons and ligaments is contained. The assumption of equilibrium between these external and internal joint moments allows formulation of a set of equations from which muscle and joint forces can be calculated. A two stage, linear optimisation routine minimising the overall muscle stress and the sum of the joint forces has been used to overcome the force-sharing problem. Humero-ulnar forces of up to 1600N, humero-radial forces of up to 800N and wrist joint forces of up to 2800N were found for moderate level activity. The model was validated by comparison with other studies.     

The effects of muscle stretching and shortening on isometric forces on the descending limb of the force-length relationship

The purpose of this study was to examine the effects of stretching and shortening on the isometric forces at different lengths on the descending limb of the force-length relationship. Cat soleus (N = 10) was stretched and shortened by various amounts on the descending limb of the force-length relationship, and the steady-state forces following these dynamic contractions were compared to the isometric forces at the corresponding muscle lengths. We found a shift of the force-length relationship to greater force values following muscle stretching, and to smaller force values following muscle shortening. Shifts in both directions critically depended on the magnitude of stretching/shortening and the final muscle length. We confirm recent findings that the steady-state isometric force following some stretch conditions clearly exceeded the maximal isometric forces at optimum muscle length, and that force enhancement was associated with an increase in the passive force, i.e., a passive force enhancement. When the passive force enhancement was subtracted from the total force enhancement, forces following stretch were always equal to or smaller than the isometric force at optimum muscle length. Together, these findings led to the conclusions: (a). that force enhancement is composed of an "active and a "passive" component; (b). that the "passive" component of force enhancement allows for forces greater than the maximal isometric forces at the muscle's optimum length; and (c). that force enhancement and force depression are critically affected by muscle length and stretch/shortening amplitude.     

Estimating the muscle forces generated in the human lower extremity when walking: a physiological solution

A model capable of estimating individual muscle activity during human lower extremity activity has been developed. This model was implemented and applied to normal gait. Both 3D cinematography and force platform records were collected to define the kinematics of the lower limb segments and the ground reaction forces. From these data the lengths, velocities, and moment arms of the muscles and the net muscle moments were calculated. These data were then used to estimate the output from a set of pattern generators which specified the neural input to and hence the force generated in each muscle. Results were obtained which demonstrate that it is feasible to construct a hierarchical physiological model which will estimate individual muscle forces. This statement must be tempered somewhat in that better anatomical and neurophysiological data must be made available and that this model, and all others attempting to estimate individual muscle forces, must be rigorously tested before being applied in a research, sports, or clinical setting.     

Barrels XII - Proceedings

Spinalized frogs were microstimulated in the intermediate grey layers of the lumbar spinal cord; the forces evoked in the hindlimb were measured at several limb positions. The data were expressed as force fields. After the collection of many force fields, the dorsal roots were cut with the stimulating electrode in place, and the position-dependent stimulation-evoked forces were again measured repeatedly. We found that the position-dependent pattern of evoked forces—the force fields—did not change after the dorsal roots were cut. In other words, the postcut evoked forces pointed in the same direction as the precut evoked forces. This result was predicted and confirmed by the muscle activations (EMGs): Before and after the dorsal roots were cut, the same muscles were activated in the same proportions. In all limb positions, the rank ordering of the muscle activations remained fixed. The stimulation needed to evoke forces was increased by deafferentation, and there were subtle changes in the force magnitudes that were consistent with a linearization of the muscle stiffness by the afferents. We conclude that the microstimulation activated specific muscle synergies that resulted in limb forces pointing toward a particular posture. The patterns of evoked forces were predominantly attributable to feedforward activation of these muscle synergies.     

A comparison of the triceps surae and residual muscle moments at the ankle during cycling

The rigid linked system model and principles of inverse dynamics have been widely used to calculate residual muscle moments during various activities. EMG driven models and optimization algorithms have also been presented in the literature in efforts to estimate skeletal muscle forces and evaluate their possible contribution to the residual muscle moment. Additionally, skeletal muscle-tendon forces have been measured, directly, in both animals and humans. The purpose of this investigation was to calculate the moment produced by the triceps surae muscles and compare it to the residual muscle moment at the ankle during cycling at three power outputs (90, 180 and 270 W). Inferences were made regarding the potential contribution made by each triceps surae component to the tendon force using EMG and muscle-tendon length changes. A buckle-type transducer was surgically implanted on the right Achilles tendon of one male subject. Achilles tendon forces measured in vivo were multiplied by their corresponding moment arms to yield the triceps surae moment during the three working conditions. Moment arm lengths were obtained in a separate experiment using magnetic resonance imaging (MRI). Pedal reaction forces, body segment accelerations (determined from high speed film), and appropriate mass parameters served as input to the inverse solution. The triceps surae moment was temporally in phase with and consistently represented approximately 65% of the residual muscle moment at the ankle. These data demonstrate the feasibility of using implanted transducers in human subjects and provide a greater understanding of musculoskeletal mechanics during normal human movements.     

Stretch-induced,steady-state force enhancement in single skeletal muscle fibers exceeds the isometric force at optimum fiber length

Stretch-induced force enhancement has been observed in a variety of muscle preparations and on structural levels ranging from single fibers to in vivo human muscles. It is a well-accepted property of skeletal muscle. However, the mechanism causing force enhancement has not been elucidated, although the sarcomere-length non-uniformity theory has received wide support. The purpose of this paper was to re-investigate stretch-induced force enhancement in frog single fibers by testing specific hypotheses arising from the sarcomere-length non-uniformity theory. Single fibers dissected from frog tibialis anterior (TA) and lumbricals (n=12 and 22, respectively) were mounted in an experimental chamber with physiological Ringer's solution (pH=7.5) between a force transducer and a servomotor length controller. The tetantic force-length relationship was determined. Isometric reference forces were determined at optimum length (corresponding to the maximal, active, isometric force), and at the initial and final lengths of the stretch experiments. Stretch experiments were performed on the descending limb of the force-length relationship after maximal tetanic force was reached. Stretches of 2.5-10% (TA) and 5-15% lumbricals of fiber length were performed at 0.1-1.5 fiber lengths/s. The stretch-induced, steady-state, active isometric force was always equal or greater than the purely isometric force at the muscle length from which the stretch was initiated. Moreover, for stretches of 5% fiber length or greater, and initiated near the optimum length of the fiber, the stretch-enhanced active force always exceeded the maximal active isometric force at optimum length. Finally, we observed a stretch-induced enhancement of passive force. We conclude from these results that the sarcomere length non-uniformity theory alone cannot explain the observed force enhancement, and that part of the force enhancement is associated with a passive force that is substantially greater after active compared to passive muscle stretch.     

A force transducer to measure individual finger loads during activities of daily living.

A new six-degree-of-freedom force transducer has been manufactured, with the sensitivity to measure forces in the range +/-100 N and moments of up to +/-5 Nm. The transducer incorporates two mechanical components: shear forces and bending moments are measured via a strain-gauged tubular section whilst axial forces are transmitted to a cantilevered load cell. Both components are instrumented with 350 ohms strain gauge full bridge circuits and are temperature compensated. After calibration, measurement errors are less than +/-0.3 N for direct forces and +/-0.03 Nm for applied moments. In order to measure sub-maximal finger loads during activities of daily living, the transducer has been incorporated into several housings representing objects in domestic use: a jar, a tap, a key in a lock and a jug kettle.     

History-dependent properties of skeletal muscle myofibrils contracting along the ascending limb of the force–length relationship

There is a history dependence of skeletal muscle contraction: stretching activated muscles induces a long-lasting force enhancement, while shortening activated muscles induces a long-lasting force depression. These history-dependent properties cannot be explained by the current model of muscle contraction, and its mechanism is unknown. The purposes of this study were (i) to evaluate if force enhancement and force depression are present at short lengths (the ascending limb of the force–length (FL) relationship), (ii) to evaluate if the history-dependent properties are associated with sarcomere length (SL) non-uniformity and (iii) to determine the effects of cross-bridge (de)activation on force depression. Rabbit psoas myofibrils were isolated and attached between two microneedles for force measurements. Images of the myofibrils were projected onto a linear photodiode array for measurements of SL. Myofibrils were activated by either Ca²⁺ or MgADP; the latter induces cross-bridge attachment to actin independently of Ca²⁺. Activated myofibrils were subjected to three stretches or shortenings (approx. 4% SL at approx. 0.07 µm s⁻¹ sarcomere⁻¹) along the ascending limb of the FL relationship separated by periods (approx. 5 s) of isometric contraction. Force after stretch was higher than force after shortening at similar SLs. The differences in force could not be explained by SL non-uniformity. The FL relationship produced by Ca²⁺- and MgADP-activated myofibrils were similar in stretch experiments, but after shortening MgADP activation produced forces that were higher than Ca²⁺ activation. Since MgADP induces the formation of strongly bound cross-bridges, this result suggests that force depression following shortening is associated with cross-bridge deactivation.     

Functional muscle synergies constrain force production during postural tasks

We recently demonstrated that a set of five functional muscle synergies were sufficient to characterize both hindlimb muscle activity and active forces during automatic postural responses in cats standing at multiple postural configurations. This characterization depended critically upon the assumption that the endpoint force vector (synergy force vector) produced by the activation of each muscle synergy rotated with the limb axis as the hindlimb posture varied in the sagittal plane. Here, we used a detailed, 3D static model of the hindlimb to confirm that this assumption is biomechanically plausible: as we varied the model posture, simulated synergy force vectors rotated monotonically with the limb axis in the parasagittal plane (r2=0.94+/-0.08). We then tested whether a neural strategy of using these five functional muscle synergies provides the same force-generating capability as controlling each of the 31 muscles individually. We compared feasible force sets (FFSs) from the model with and without a muscle synergy organization. FFS volumes were significantly reduced with the muscle synergy organization (F=1556.01, p<0.01), and as posture varied, the synergy-limited FFSs changed in shape, consistent with changes in experimentally measured active forces. In contrast, nominal FFS shapes were invariant with posture, reinforcing prior findings that postural forces cannot be predicted by hindlimb biomechanics alone. We propose that an internal model for postural force generation may coordinate functional muscle synergies that are invariant in intrinsic limb coordinates, and this reduced-dimension control scheme reduces the set of forces available for postural control.     

Muscle force production during bent-knee,bent-hip walking in humans

Researchers have long debated the locomotor posture used by the earliest bipeds. While many agree that by 3–4 Ma (millions of years ago), hominins walked with an extended-limb human style of bipedalism, researchers are still divided over whether the earliest bipeds walked like modern humans, or walked with a more bent-knee, bent-hip (BKBH) ape-like form of locomotion. Since more flexed postures are associated with higher energy costs, reconstructing early bipedal mechanics has implications for the selection pressures that led to upright walking. The purpose of this study is to determine how modern human anatomy functions in BKBH walking to clarify the links between morphology and energy costs in different mechanical regimes. Using inverse dynamics, we calculated muscle force production at the major limb joints in humans walking in two modes, both with extended limbs and BKBH. We found that in BKBH walking, humans must produce large muscle forces at the knee to support body weight, leading to higher estimated energy costs. However, muscle forces at the hip remained similar in BKBH and extended limb walking, suggesting that anatomical adaptations for hip extension in humans do not necessarily diminish the effective mechanical advantage at the hip in more flexed postures. We conclude that the key adaptations for economical walking, regardless of joint posture, seem to center on maintaining low muscle forces at the hip, primarily by keeping low external moments at the hip. We explore the implications of these results for interpreting locomotor energetics in early hominins, including australopithecines and Ardipithecus ramidus.     

Force depression in human quadriceps femoris following voluntary shortening contractions.

The purpose of this study was to investigate whether the isometric muscle force, redeveloped following maximal-effort voluntary shortening contractions in human skeletal muscle, is smaller than the purely isometric muscle force at the corresponding length. Isometric knee extensor moments, surface electromyographic (EMG) signals of quadriceps femoris, and interpolated twitch moments (ITMs) were measured while 10 subjects performed purely isometric knee extensor contractions at a 60 degrees knee angle and isometric knee extensor contractions at a 60 degrees knee angle preceded by maximal-effort voluntary shortening of the quadriceps muscles. It was found that the knee extensor moments were significantly decreased for the isometric-shortening-isometric contractions compared with the isometric contractions for the group as a whole, whereas the corresponding EMG and ITM values were the same. This study is the first to demonstrate force depression following muscle shortening for voluntary contractions. We concluded that force depression following muscle shortening is an actual property of skeletal muscle rather than a stimulation artifact and that force depression during voluntary contraction is not accompanied by systematic changes in muscle activation as evaluated by EMG and ITM.     

Isoforms Confer Characteristic Force Generation and Mechanosensation by Myosin II Filaments

Myosin II isoforms with varying mechanochemistry and filament size interact with filamentous actin (F-actin) arrays to generate contractile forces in muscle and nonmuscle cells. How myosin II force production is shaped by isoform-specific motor properties and environmental stiffness remains poorly understood. Here, we used computer simulations to analyze force production by an ensemble of myosin motors against an elastically tethered actin filament. We found that force output depends on two timescales: the duration of F-actin attachment, which varies sharply with the ensemble size, motor duty ratio, and external load; and the time to build force, which scales with the ensemble stall force, gliding speed, and environmental stiffness. Although force-dependent kinetics were not required to sense changes in stiffness, the myosin catch bond produced positive feedback between the attachment time and force to trigger switch-like transitions from transient attachments, generating small forces, to high-force-generating runs. Using parameters representative of skeletal muscle myosin, nonmuscle myosin IIB, and nonmuscle myosin IIA revealed three distinct regimes of behavior, respectively: 1) large assemblies of fast, low-duty ratio motors rapidly build stable forces over a large range of environmental stiffness; 2) ensembles of slow, high-duty ratio motors serve as high-affinity cross-links with force buildup times that exceed physiological timescales; and 3) small assemblies of low-duty ratio motors operating at intermediate speeds are poised to respond sharply to changes in mechanical context—at low force or stiffness, they serve as low-affinity cross-links, but they can transition to force production via the positive-feedback mechanism described above. Together, these results reveal how myosin isoform properties may be tuned to produce force and respond to mechanical cues in their environment.     

The pressure-flow relation for plasma in whole organ skeletal muscle and its experimental verification.

The whole-organ pressure-flow relation in resting rat skeletal muscle is examined for the flow of plasma. Due to the small size of the blood vessels in this organ, inertia and convective forces in the blood are negligible and viscous forces dominate. Direct measurements in the past have shown that skeletal muscle blood vessels are distensible. Theoretical formulations based on these measurements lead to a third order polynomial model for the pressure-flow relation. The purpose of the current study is to examine this relation experimentally in an isolated muscle organ. A high precision feedback controlled pump is used to perfuse artificial plasma into the vasodilated rat gracilis muscle. The results indicate that the pressure-flow curve in this tissue is nonlinear in the low flow region and almost linear at physiological flow rates, following closely the third order polynomial function. Vessel fixation with glutaraldehyde causes the curves to become linear at all pressures, indicating that vessel distention is the primary mechanism causing the nonlinearity. Furthermore, the resistance of the post-fixed tissue is determined by the pressure at which the fixative is perfused. At fixation pressures below 10 mmHg, the resistance is three times higher than in vessels fixed at normal physiological pressures. Dextran (229,000 Dalton) is used to obtain Newtonian perfusates at different viscosities. The pressure-flow relation is found to be linearly dependent on viscosity for all flow rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Length dependence of active force production in skeletal muscle.

The sliding filament and cross-bridge theories of muscle contraction provide discrete predictions of the tetanic force-length relationship of skeletal muscle that have been tested experimentally. The active force generated by a maximally activated single fiber (with sarcomere length control) is maximal when the filament overlap is optimized and is proportionally decreased when overlap is diminished. The force-length relationship is a static property of skeletal muscle and, therefore, it does not predict the consequences of dynamic contractions. Changes in sarcomere length during muscle contraction result in modulation of the active force that is not necessarily predicted by the cross-bridge theory. The results of in vivo studies of the force-length relationship suggest that muscles that operate on the ascending limb of the force-length relationship typically function in stretch-shortening cycle contractions, and muscles that operate on the descending limb typically function in shorten-stretch cycle contractions. The joint moments produced by a muscle depend on the moment arm and the sarcomere length of the muscle. Moment arm magnitude also affects the excursion (length change) of a muscle for a given change in joint angle, and the number of sarcomeres arranged in series within a muscle fiber determines the sarcomere length change associated with a given excursion.