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In vitro studies have used protein markers to distinguish between myogenic cells isolated from fast and slow skeletal muscles. The protein markers provide some support for the hypothesis that satellite cells from fast and slow muscles are different, but the data are equivocal. To test this hypothesis directly, three-dimensional skeletal muscle constructs were engineered from myogenic cells isolated from fast tibialis anterior (TA) and slow soleus (SOL) muscles of rats and functionality was tested. Time to peak twitch tension (TPT) and half relaxation time (R[T.sub.1/2]) were ~30% slower in constructs from the SOL. The slower contraction and relaxation times for the SOL constructs resulted in left shift of the force-frequency curve compared with those from the TA. Western blot analysis showed a 60% greater quantity of fast myosin heavy chain in the TA constructs. 14 days of chronic low-frequency electrical stimulation resulted in a 15% slower TPT and a 14% slower R[T.sub.1/2], but no change in absolute force production in the TA constructs. In SOL constructs, slow electrical stimulation resulted in an 80% increase in absolute force production with no change in TPT or R[T.sub.1/2]. The addition of cyclosporine A did not prevent the increase in force in SOL constructs after chronic low-frequency electrical stimulation, suggesting that calcineurin is not responsible for the increase in force. We conclude that myogenic cells associated with a slow muscle are imprinted to produce muscle that contracts and relaxes slowly and that calcineurin activity cannot explain the response to a slow pattern of electrical stimulation. tissue engineering; calcineurin; electrical stimulation; engineered muscle; bioreactors

Fecal incontinence affects people of all ages and social backgrounds and can have devastating psychological and economic consequences. This disorder is largely attributed to decreased mechanical efficiency of the internal anal sphincter (IAS), yet little is known about the pathophysiological mechanisms responsible for the malfunction of sphincteric smooth muscle at the cellular level. The object of this study was to develop a three-dimensional (3-D) physiological model of the IAS bioengineered in vitro from isolated smooth muscle cells. Smooth muscle cells isolated from the IAS of rabbits were seeded in culture on top of a loose fibrin gel, where they migrated and self-assembled in circumferential alignment. As the cells proliferated, the fibrin gel contracted around a 5-mm-diameter SYLGARD mold, resulting in a 3-D cylindrical ring of sphincteric tissue. We found that 1) the bioengineered IAS rings generated a spontaneous basal tone, 2) stimulation with 8-bromo-cAMP (8-Br-cAMP) caused a sustained decrease in the basal tone (relaxation) that was calcium-independent, 3) upon stimulation with ACh, bioengineered IAS rings showed a calcium- and concentration-dependent peak contraction at 30 s that was sustained for 4 min, 4) addition of 8-Br-cAMP induced rapid relaxation of ACh-induced contraction and force generation of IAS rings, and 5) bioengineered sphincter rings show striking functional differences when compared with bioengineered rings made from isolated colonic smooth muscle cells. This is the first report of a 3-D in vitro model of a gastrointestinal smooth muscle IAS. Bioengineered IAS rings demonstrate physiological functionality and may be used in the elucidation of the mechanisms causing sphincter malfunction. extracellular matrix; 8-bromo-adenosine 3',5'-cyclic monophosphate

Skeletal muscles of old rats and elderly humans lose muscle mass and maximum force. Denervation is a major cause of age-related muscle atrophy and weakness, because denervated fibers do not contract, and undergo atrophy. At any age, surgical denervation causes even more dramatic muscle atrophy and loss in force than aging does. Electrical stimulation that generates tetanic contractions of denervated muscles reduces the denervation-induced declines. We investigated whether a stimulation protocol that maintains mass and force of denervated extensor digitorum longus muscles of adult rats would also maintain these properties in denervated muscles of old rats during a 2-month period of age-induced declines in these properties. Contractile activity generated by the electrical stimulation eliminated age-related losses in muscle mass and reduced the deficit in force by 50%. These data provide support for the hypothesis that during aging, lack of contractile activity in fibers contributes to muscle atrophy and weakness.

Abstract Background The traditional two-point discrimination (TPD) test, a widely used tactile spatial acuity measure, has been criticized as being imprecise because it is based on subjective criteria and involves a number of non-spatial cues. The results of a recent study showed that as two stimuli were delivered simultaneously, vibrotactile amplitude discrimination became worse when the two stimuli were positioned relatively close together and was significantly degraded when the probes were within a subject's two-point limen. The impairment of amplitude discrimination with decreasing inter-probe distance suggested that the metric of amplitude discrimination could possibly provide a means of objective and quantitative measurement of spatial discrimination capacity. Methods A two alternative forced-choice (2AFC) tracking procedure was used to assess a subject's ability to discriminate the amplitude difference between two stimuli positioned at near-adjacent skin sites. Two 25 Hz flutter stimuli, identical except for a constant difference in amplitude, were delivered simultaneously to the hand dorsum. The stimuli were initially spaced 30 mm apart, and the inter-stimulus distance was modified on a trial-by-trial basis based on the subject's performance of discriminating the stimulus with higher intensity. The experiment was repeated via sequential, rather than simultaneous, delivery of the same vibrotactile stimuli. Results Results obtained from this study showed that the performance of the amplitude discrimination task was significantly degraded when the stimuli were delivered simultaneously and were near a subject's two-point limen. In contrast, subjects were able to correctly discriminate between the amplitudes of the two stimuli when they were sequentially delivered at all inter-probe distances (including those within the two-point limen), and improved when an adapting stimulus was delivered prior to simultaneously delivered stimuli. Conclusion Subjects' capacity to discriminate the amplitude difference between two vibrotactile stimulations was degraded as the inter-stimulus distance approached the limit of their two-point spatial discriminative capacity. This degradation of spatial discriminative capacity lessened when an adapting stimulus was used. Performance of the task, as well as improvement on the task with adaptation, would most likely be impaired if the cortical information processing capacity of a subject or subject population were systemically altered, and thus, the methods described could be effective measures for use in clinical or clinical research applications.

Abstract Biomechatronics is the integration of biological components with artificial devices, in which the biological component confers a significant functional capability to the system, and the artificial component provides specific cellular and tissue interfaces that promote the maintenance and functional adaptation of the biological component. Based upon functional performance, muscle is potentially an excellent mechanical actuator, but the larger challenge of developing muscle-actuated, biomechatronic devices poses many scientific and engineering challenges. As a demonstratory proof of concept, we designed, built, and characterized a swimming robot actuated by two explanted frog semitendinosus muscles and controlled by an embedded microcontroller. Using open loop stimulation protocols, the robot performed basic swimming maneuvers such as starting, stopping, turning (turning radius ~400 mm) and straight-line swimming (max speed >1/3 body lengths/second). A broad spectrum antibiotic/antimycotic ringer solution surrounded the muscle actuators for long term maintenance, ex vivo. The robot swam for a total of 4 hours over a 42 hour lifespan (10% duty cycle) before its velocity degraded below 75% of its maximum. The development of functional biomechatronic prototypes with integrated musculoskeletal tissues is the first critical step toward the long term objective of controllable, adaptive and robust biomechatronic robots and prostheses.

Excitability and contractility of skeletal muscle engineered from primary cultures and cell lines. Am J Physiol Cell Physiol 280: C288-C295, 2001. The purpose of this study was to compare the excitability and contractility of three-dimensional skeletal muscle constructs, termed myooids, engineered from [C.sub.2][C.sub.12] myoblast and 10T 1/2 fibroblast cell lines, primary muscle cultures from adult C3H mice, and neonatal and adult Sprague-Dawley rats. Myooids were 12 mm long, with diameters of 0.1-1 mm, were excitable by transverse electrical stimulation, and contracted to produce force. After ~30 days in culture, myooid cross-sectional area, rheobase, chronaxie, resting baseline force, twitch force, time to peak tension, one-half relaxation time, and peak isometric force were measured. Specific force was calculated by dividing peak isometric force by cross-sectional area. The specific force generated by the myooids was 2-8% of that generated by skeletal muscles of control adult rodents. Myooids engineered from [C.sub.2][C.sub.12]-10T1/2 cells exhibited greater rheobase, time to peak tension, and one-half relaxation time than myooids engineered from adult rodent cultures, and myooids from [C.sub.2][C.sub.12]-10T1/2 and neonatal rat cells had greater resting baseline forces than myooids from adult rodent cultures. tissue engineering; myooid; myogenesis; isometric force; rodent tissue culture

Flanders et al is commended for their ingenious and novel approach to the problem of coordinate transformations in motor control, however, exception is taken on the formulation of the proposed model's experimental methods, results and data interpretation. It is suggested that a blocked presentation of targets would have greatly reduced range effects. Also, errors of interpretation give rise to Flanders' statements on shoulder- versus head-centered representations of target parameters.