Bioreactors for guiding muscle tissue growth and development.

Muscle tissue bioreactors are devices which are employed to guide and monitor the development of engineered muscle tissue. These devices have a modern history that can be traced back more than a century, because the key elements of muscle tissue bioreactors have been studied for a very long time. These include barrier isolation and culture of cells, tissues and organs after isolation from a host organism; the provision of various stimuli intended to promote growth and maintain the muscle, such as electrical and mechanical stimulation; and the provision of a perfusate such as culture media or blood derived substances. An accurate appraisal of our current progress in the development of muscle bioreactors can only be made in the context of the history of this endeavor. Modern efforts tend to focus more upon the use of computer control and the application of mechanical strain as a stimulus, as well as substrate surface modifications to induce cellular organization at the early stages of culture of isolated muscle cells.

Effect of streptomycin on the active force of bioengineered heart muscle in response to controlled stretch.

In this study, we describe a bioreactor system to deliver controlled stretch protocols to bioengineered heart muscle (BEHMs) and test the system when streptomycin (an aminoglycoside antibiotic, which blocks stretch-activated channels) is either added to or excluded from the culture medium. Streptomycin is a very commonly used component of cell culture antibiotic-antimycotic media additives, so its effects on muscle development and functional response to mechanical signals in vitro is worthy of investigation. Our hypothesis is that BEHMs will not adapt to the applied mechanical stretch protocol when streptomycin is present in the culture medium, but will do so when streptomycin is excluded. Bioengineered heart muscles were formed by culturing primary neonatal cardiac myocytes in a fibrin gel using a method previously developed in our laboratory. A custom bioreactor system was designed using SolidWorks and structural components manufactured using fusion deposition modeling. We utilized a stretch protocol of 1 Hz, 10% strain for 7 d. BEHMs were stretched in the presence and absence of streptomycin. As controls, BEHMs were maintained in a cell culture incubator with and without streptomycin. The contractile properties of all BEHMs were evaluated to determine the active force. We were able to demonstrate compatibility of the bioreactor system with BEHMs and were able to stretch 58 constructs with zero incidence of failure. When the BEHMs were stretched in the absence of streptomycin, the active force increased from a mean value of 51.7 +/- 5.6 (N = 10) to 102.4 +/- 16.3 microN (N = 10), with p < 0.05. However, BEHMs that were stretched in the presence of streptomycin did not show any significant increase in active force generation. The average active force of BEHMs increased from a mean value of 57.6 +/- 10.2 (N = 10) to 91.4 +/- 19.8 microN (N = 10) when stretched in the presence of streptomycin. In this study, we demonstrate compatibility of the a bioreactor system with BEHMs, stability of the BEHMs in response to stretch protocols, and significant functional improvement in response to controlled stretch only when streptomycin is excluded from the culture medium, supporting our hypothesis.

Effects of adaptation on the capacity to differentiate simultaneously delivered dual-site vibrotactile stimuli.

The capacity of 20 healthy adult subjects for detecting differences in the amplitude of two simultaneously delivered 25 Hz vibrotactile stimuli was assessed both in the absence and presence of prior exposure to different conditions of adapting stimulation. Results obtained from this study demonstrate that increasing durations of adapting stimulation at one of the two skin sites, in the range of 0.2 to 2.0 s, lead to a systematic and progressive decrease in a subject's ability to accurately discriminate between the two different amplitudes. Delivery of adapting stimuli to both of the sites of skin stimulation prior to simultaneous delivery of the test and standard stimuli, however, leads to an improvement in amplitude discrimination performance--a finding which is consistent with prior published psychophysical studies that demonstrate improvements in discriminatory capacity with much longer durations of adaptation. Striking parallels between the results obtained in this study and those reported in a prior study of the effects of vibrotactile adaptation on the optical response of squirrel monkey contralateral SI cortex to vibrotactile stimulation [Simons, S.B., Chiu, J., Favorov, O.V., Whitsel, B.L., Tommerdahl, M., 2007. Duration-dependent response of SI to vibrotactile stimulation in squirrel monkey. J Neurophysiol. 97, 2121-9, Simons, S.B., Tannan, V., Chiu, J., Favorov, O.V., Whitsel, B.L., Tommerdahl, M., 2005. Amplitude-dependency of response of SI cortex to flutter stimulation. BMC Neurosci. 6, 43] suggest that the perceptual effects detected in this study could be attributable to adaptation-induced alterations of SI response.

A portable tactile sensory diagnostic device.

Current methods for applying multi-site vibratory stimuli to the skin typically involve the use of two separate vibrotactile stimulators, which can lead to difficulty with positioning of stimuli and in ensuring that stimuli are delivered perfectly in phase at the same amplitude and frequency. Previously, we reported a two-point stimulator (TPS) that was developed in order to solve the problem of delivering two-point stimuli to the skin at variable distances between the sites of stimulation. Because of the success of the TPS, we designed and fabricated a new stimulator with four significant improvements over our original device. First, the device is portable, lightweight and can be used in a variety of non-laboratory settings. Second, the device consists of two independently controlled stimulators which allow delivery of stimuli simultaneously to two distinct skin sites with different amplitude, frequency and/or phase. Third, the device automatically detects the skin surface and thus allows for much better automated control of stimulus delivery. Fourth, the device is designed for rapid manufacture and, therefore, can be made readily available to other research (non-laboratory) settings. To demonstrate the device, a modified Bekesy tracking method was used to evaluate the simultaneous amplitude discrimination capacity of 20 subjects.

Development of a novel bioreactor for the mechanical loading of tissue-engineered heart muscle.

OBJECTIVE: In this study, we describe a novel bioreactor system to deliver controlled stretch protocols to bioengineered heart muscle (BEHM) constructs. Our primary objective was to evaluate the effect of mechanical stretch on the contractile properties of three-dimensional cardiac constructs in vitro. METHODS: BEHMs were formed by culturing primary neonatal cardiac myocytes in a fibrin gel using a method previously developed in our laboratory. A custom bioreactor system was designed using SolidWorks (Concord, MA) and structural components were manufactured using fusion deposition modeling. We utilized the bioreactor to evaluate the effect of 2-, 6-, and 24-hour stretch protocols on the stretch-induced changes in contractile function of BEHMs. RESULTS: We were able to demonstrate compatibility of the bioreactor system with BEHMs and were able to stretch all the constructs with zero incidence of failure. We found that loading the constructs for 2, 6, and 24 hours during a 24-hour period using a stretch protocol of 1 Hz, 10% stretch did not result in any significant change in the active force, specific force, pacing characteristics, and morphological features. CONCLUSIONS: In this study, we demonstrate compatibility of a novel bioreactor system with BEHMs and the stability of the BEHMs in response to stretch protocols.

Stimulus-dependent effects on tactile spatial acuity.

BACKGROUND: Previous studies have shown that spatio-tactile acuity is influenced by the clarity of the cortical response in primary somatosensory cortex (SI). Stimulus characteristics such as frequency, amplitude, and location of tactile stimuli presented to the skin have been shown to have a significant effect on the response in SI. The present study observes the effect of changing stimulus parameters of 25 Hz sinusoidal vertical skin displacement stimulation ("flutter") on a human subject's ability to discriminate between two adjacent or near-adjacent skin sites. Based on results obtained from recent neurophysiological studies of the SI response to different conditions of vibrotactile stimulation, we predicted that the addition of 200 Hz vibration to the same site that a two-point flutter stimulus was delivered on the skin would improve a subject's spatio-tactile acuity over that measured with flutter alone. Additionally, similar neurophysiological studies predict that the presence of either a 25 Hz flutter or 200 Hz vibration stimulus on the unattended hand (on the opposite side of the body from the site of two-point limen testing - the condition of bilateral stimulation - which has been shown to evoke less SI cortical activity than the contralateral-only stimulus condition) would decrease a subject's ability to discriminate between two points on the skin. RESULTS: A Bekesy tracking method was employed to track a subject's ability to discriminate between two-point stimuli delivered to the skin. The distance between the two points of stimulation was varied on a trial-by-trial basis, and several different stimulus conditions were examined: (1) The "control" condition, in which 25 Hz flutter stimuli were delivered simultaneously to the two points on the skin of the attended hand, (2) the "complex" condition, in which a combination of 25 Hz flutter and 200 Hz vibration stimuli were delivered to the two points on the attended hand, and (3) a "bilateral" condition, in which 25 Hz flutter was delivered to the two points on the attended hand and a second stimulus (either flutter or vibration) was delivered to the unattended hand. The two-point limen was reduced (i.e., spatial acuity was improved) under the complex stimulus condition when compared to the control stimulus condition. Specifically, whereas adding vibration to the unilateral two-point flutter stimulus improved spatial acuity by 20 to 25%, the two-point limen was not significantly affected by substantial changes in stimulus amplitude (between 100 - 200 microm). In contrast, simultaneous stimulation of the unattended hand (contralateral to the attended site), impaired spatial acuity by 20% with flutter stimulation and by 30% with vibration stimulation. CONCLUSION: It was found that the addition of 200 Hz vibration to a two-point 25 Hz flutter stimulus significantly improved a subject's ability to discriminate between two points on the skin. Since previous studies showed that 200 Hz vibration preferentially evokes activity in cortical area SII and reduces or inhibits the spatial extent of activity in SI in the same hemisphere, the findings in this paper raise the possibility that although SI activity plays a major role in two-point discrimination on the skin, influences relayed to SI from SII in the same hemisphere may contribute importantly to SI's ability to differentially respond to stimuli applied to closely spaced skin points on the same side of the body midline.

Functional development of engineered skeletal muscle from adult and neonatal rats.

A myooid is a three-dimensional skeletal muscle construct cultured from mammalian myoblasts and fibroblasts. The purpose was to compare over several weeks in culture the morphology, excitability, and contractility of myooids developed from neonatal and adult rat cells. The hypotheses tested were as follows: (1) baseline forces of myooids correlate with the cross-sectional area (CSA) of the myooids composed of fibroblasts, and (2) peak isometric tetanic forces normalized by total CSA (specific P(o)) of neonatal and adult rat myooids are not different. Electrical field stimulation was used to measure the excitability and peak tetanic forces. The proportion of the CSA composed of fibroblasts was greater for neonatal (40%) than adult (17%) myooids. For all myooids the baseline passive force normalized by fibroblast CSA (mean = 5.5 kPa) correlated with the fibroblast CSA (r(2) = 0.74). A two-element cylindrical model was analyzed to determine the contributions of fibroblasts and myotubes to the baseline force. At each measurement period, the specific P(o) of the adult myooids was greater than that of the neonatal myooids. The specific P(o) of the adult myooids was approximately 1% of the control value for adult muscles and did not change with time in culture, while that of neonatal myooids increased.

Excitability and contractility of skeletal muscle engineered from primary cultures and cell lines.

The purpose of this study was to compare the excitability and contractility of three-dimensional skeletal muscle constructs, termed myooids, engineered from C2C12 myoblast and 10T1/2 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 approximately 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 C2C12-10T1/2 cells exhibited greater rheobase, time to peak tension, and one-half relaxation time than myooids engineered from adult rodent cultures, and myooids from C2C12-10T1/2 and neonatal rat cells had greater resting baseline forces than myooids from adult rodent cultures.

Excitability and isometric contractile properties of mammalian skeletal muscle constructs engineered in vitro.

Our purpose was to engineer three-dimensional skeletal muscle tissue constructs from primary cultures of adult rat myogenic precursor cells, and to measure their excitability and isometric contractile properties. The constructs, termed myooids, were muscle-like in appearance, excitability, and contractile function. The myooids were 12 mm long and ranged in diameter from 0.1 to 1 mm. The myooids were engineered with synthetic tendons at each end to permit the measurement of isometric contractile properties. Within each myooid the myotubes and fibroblasts were supported by an extracellular matrix generated by the cells themselves, and did not require a preexisting scaffold to define the size, shape, and general mechanical properties of the resulting structure. Once formed, the myooids contracted spontaneously at approximately 1 Hz, with peak-to-peak force amplitudes ranging from 3 to 30 microN. When stimulated electrically the myooids contracted to produce force. The myooids (n = 14) had the following mean values: diameter of 0.49 mm, rheobase of 1.0 V/mm, chronaxie of 0.45 ms, twitch force of 215 microN, maximum isometric force of 440 microN, resting baseline force of 181 microN, and specific force of 2.9 kN/m2. The mean specific force was approximately 1% of the specific force generated by control adult rat muscle. Based on the functional data, the myotubes in the myooids appear to remain arrested in an early developmental state due to the absence of signals to promote expression of adult myosin isoforms.

Measurement of recovery of function following whole muscle transfer, myoblast transfer, and gene therapy.

For a skeletal muscle tissue engineer, the most important issue following an experimental intervention is the evaluation of the recovery of the functional capabilities of the tissue, relative to those of the control tissue. Whether investigators perform whole muscle transfers with spontaneous (1) or surgical (2) vascular and nerve repair, myoblast transfers (3), or manipulations of muscle-specific genes (4,5), the question remains the same: Has the intervention impaired, maintained, or enhanced the functional capabilities of the skeletal muscles involved? In each case, determining structure-function relationships is of vital importance because structure-function relationships are frequently disrupted following an intervention, so that muscle mass and total muscle fiber cross-sectional area (CSA) are not different from control values, but function is impaired, or both are impaired, but with different magnitudes of impairment.

Microtubule assembly is regulated by externally applied strain in cultured smooth muscle cells.

Mechanical forces clearly regulate the development and phenotype of a variety of tissues and cultured cells. However, it is not clear how mechanical information is transduced intracellularly to alter cellular function. Thermodynamic modeling predicts that mechanical forces influence microtubule assembly, and hence suggest microtubules as one potential cytoskeletal target for mechanical signals. In this study, the assembly of microtubules was analyzed in rat aortic smooth muscle cells cultured on silicon rubber substrates exposed to step increases in applied strain. Cytoskeletal and total cellular protein fractions were extracted from the cells following application of the external strain, and tubulin levels were quantified biochemically via a competitive ELISA and western blotting using bovine brain tubulin as a standard. In the first set of experiments, smooth muscle cells were subjected to a step-increase in strain and the distribution of tubulin between monomeric, polymeric, and total cellular pools was followed with time. Microtubule mass increased rapidly following application of the strain, with a statistically significant increase (P<0.05) in microtubule mass from 373+/-32 pg/cell (t=0) to 514+/-30 pg/cell (t=15 minutes). In parallel, the amount of soluble tubulin decreased approximately fivefold. The microtubule mass decreased after 1 hour to a value of 437+/-24 pg/cell. In the second set of experiments, smooth muscle cells were subjected to increasing doses of externally applied strain using a custom-built strain device. Monomeric, polymeric, and total tubulin fractions were extracted after 15 minutes of applied strain and quantified as for the earlier experiments. Microtubule mass increased with increasing strain while total cellular tubulin levels remained essentially constant at all strain levels. These findings are consistent with a thermodynamic model which predicts that microtubule assembly is promoted as a cell is stretched and compressional loads on the microtubules are presumably relieved. Furthermore, these data suggest microtubules are a potential target for translating changes in externally applied mechanical stimuli to alterations in cellular phenotype.

Bipolar implantable stimulator for long-term denervated-muscle experiments.

A micropower bipolar implantable stimulator has been developed and tested for long-term (four weeks-six months) use in experiments involving the stimulation of denervated skeletal muscle. Implantable stimulators are typically operated from a single lithium battery at 3 V. After the first week of denervation, stimulation of denervated muscles of rats requires voltages in the range of 6-12 V. The stimulator described can deliver voltages up to 15 V, with variable pulsewidth, frequency and duty cycle. All stimulation parameters are set prior to implantation by selection of appropriate resistors and capacitors. Each primary failure mode for implantable stimulators is addressed. Long-term reliability rates in excess of 95% are achievable if the construction details are followed closely. Methods for battery power management, circuit component selection, electrode construction and encapsulation are described in detail. This device is not intended for use in humans.

Sarcomere dynamics and contraction-induced injury to maximally activated single muscle fibres from soleus muscles of rats.

1. The focal nature of contraction-induced injury to skeletal muscle fibres may arise from heterogeneities in sarcomere length that develop during contractions. We tested the hypothesis that when a maximally activated single permeabilized fibre segment is stretched and a deficit in maximum isometric force (force deficit) is produced, the regions of sarcomeres with the longest lengths of prior to the stretch contain the majority of the damaged sarcomeres when the fibre is returned to optimum length (Lo) after the stretch. 2. Single fibre segments (n = 16) were obtained from soleus muscles of rats. Average sarcomere length at five discrete positions along the length of each fibre was determined by lateral deflection of a diode laser spot. Diffraction patterns were obtained while fibres were relaxed and immediately before, during and after a single stretch of 40% strain relative to Lo. Following the stretch, the regions of each fibre that potentially contained damaged sarcomeres were identified by an increased scatter of the first-order diffraction patterns. The damage was confirmed by light and electron microscopy. 3. While single fibre segments were in relaxing solution, the mean value for all of the average sarcomere lengths sampled (n = 80) was 2.53 +/- 0.01 microns (range, 2.40-2.68 microns). During the maximum isometric contraction before each stretch, the mean sarcomere length decreased to 2.42 +/- 0.02 microns and the range increased to 2.12-3.01 microns. 4. During the stretch of 40% strain, all regions of sarcomeres were stretched onto the descending limb of the length-force curve, but sarcomere lengthening was non-uniform. After the stretch, when the maximally activated fibres were returned to Lo, the force deficit was 10 +/- 1%. Microscopic evaluation confirmed that the regions with the longest sarcomere lengths before the stretch contained the majority of the damaged sarcomeres after the stretch. We conclude that when heterogeneities in sarcomere length develop in single permeabilized fibre segments during a maximum isometric contraction, the sarcomeres in the regions with the longest lengths are the most susceptible to contraction-induced injury.

A parametric model of muscle moment arm as a function of joint angle: application to the dorsiflexor muscle group in mice.

A parametric model was developed to describe the relationship between muscle moment arm and joint angle. The model was applied to the dorsiflexor muscle group in mice, for which the moment arm was determined as a function of ankle angle. The moment arm was calculated from the torque measured about the ankle upon application of a known force along the line of action of the dorsiflexor muscle group. The dependence of the dorsiflexor moment arm on ankle angle was modeled as r = R sin(a + delta), where r is the moment arm calculated from the measured torque and a is the joint angle. A least-squares curve fit yielded values for R, the maximum moment arm, and delta, the angle at which the maximum moment arm occurs as offset from 90 degrees. Parametric models were developed for two strains of mice, and no differences were found between the moment arms determined for each strain. Values for the maximum moment arm, R, for the two different strains were 0.99 and 1.14 mm, in agreement with the limited data available from the literature. While in some cases moment arm data may be better fitted by a polynomial, use of the parametric model provides a moment arm relationship with meaningful anatomical constants, allowing for the direct comparison of moment arm characteristics between different strains and species.

Action of inflammatory mediators on middle ear mucosa. A method for measuring permeability and swelling.

We describe a technique for measuring changes of permeability and vasodilation-edema in middle ear and Eustachian tube mucosa when this tissue is challenged with an inflammatory mediator. A ten-minute exposure to histamine, bradykinin, and prostaglandin E1 or E2 causes marked increases in permeability and a vasodilation-edema that lasts from 30 minutes to two hours. The passage across the mucosa of a small ion-pertechnetate-is increased by these mediators. Protein molecules, normally prevented from permeating the blood-mucosal barrier, permeate in substantial amounts after the middle ear mucosa is challenged with inflammatory mediators. Thus, the mediators seem capable of producing two of the cardinal signs of middle ear inflammation. They may be the proximal cause of middle ear effusions and their continued presence may be responsible for the chronic nature of this disease.