Biomimetics - Biologically Inspired Technologies - Yoseph Bar Cohen Episode 1 Part 10 docx

organ culture and when properly employed are effective for long-term maintenance of livingtissueex vivo.9.6.2.2 Mechanical Failure within the Tissue Intracellular, ECM Also known as cont

organ culture and when properly employed are effective for long-term maintenance of livingtissueex vivo.

9.6.2.2 Mechanical Failure within the Tissue (Intracellular, ECM)

Also known as contraction-induced injury, this mode of failure is prevalent in muscle tissuesubjected to maximal contractions during forced lengthening, and affects all classes of muscleactuators The effective countermeasure involves employing control algorithms that preventrepeated eccentric contraction of fully activated muscle actuators Living muscle can functionallyadapt to tolerate lengthening contractions if the proper maintenance protocols are employed Anattempt can be made to implement such protocols in the muscle actuator bioreactors using feedbackcontrol

9.6.2.3 Mechanical Failure at the Tissue Interface

Less common for musclein vivo, this is a major failure mode for explanted and engineered tissues

in general For whole explanted muscles, the interface typically involves suture or adhesive applied

to the preexisting tendons Lack of process control in this tissue or synthetic junction leads tounpredictable mechanical failures over time In engineered tissues the problem is more serious, astissue failure frequently occurs at the tissue or synthetic interface under relatively mild mechanicalconditions We have extensive experimental data on this failure mode in engineered muscle tissuesubjected to external loading We hypothesize the failure to be due to stress concentration at thetissue or synthetic interface, compounded by inadequate force transduction from the appropriateintracellular force generating machinery to the extracellular synthetic load bearing fixtures, leading

to cell membrane damage at the interface with subsequent rapid tissue degradation and necrosis.The best countermeasure requires the engineering of a muscle–tendon interface (MTJ), which is amajor objective of current research in muscle tissue engineering Tendon tissue is 80 to 90% ECM,composed chiefly of parallel arrays of collagen fibers The tendon-to-synthetic interface, wherebiology meets machine, is a separate and equally important technical challenge

9.6.2.4 Metabolic Failure

This failure mode results most frequently from inadequate delivery of metabolic substrates andinadequate clearance of metabolic byproducts, and is exacerbated at elevated temperatures Thebest countermeasure for this failure mode is to restrict the muscle actuator cross-section to morethan approximately 200 mm diameter, or to provide perfusion through a vascular bed in the case oflarger cross-sections This mode of failure typically initiates at the axial core of cylindrical muscleactuators For this reason, sustained angiogenesis and perfusion is a major technical objective incurrent tissue engineering research

9.6.2.5 Cellular Necrosis and Programmed Cell Death

Several controllable circumstances can lead to this general mode of failure in all classes of muscleactuators Cellular hypercontraction and hyperextension in muscle results in rapid necrosis Thismechanism will occur more or less uniformly across the muscle cross-section, but will theoreticallyoccur more frequently in areas with reduced physiologic cross-sectional area or inhibited sarco-meric function This failure mode can be prevented by control of the internal mechanical compli-ance and stroke of the muscle actuator Muscle maintained at an inappropriate length, either tooshort or too long, will deteriorate, even if the muscle is quiescent In explanted muscles, mainte-nance at lengths greater than the plateau of the length–tension curve appears to be the mostdamaging over time

9.6.2.6 Fatigue (Mechanical and Metabolic)

These failure modes apply to all classes of living muscle actuators For metabolic fatigue thepreferred countermeasures will include genetic engineering of the muscle to promote fatigue-resistant fiber types, the provision of adequate perfusion of the tissue actuator, and the development

of protocols for actuator control that optimize total work output, such as the intermittent locomotorybehavior of both terrestrial and aquatic animals It is in terms of mechanical fatigue that livingactuators have an enormous advantage over fully synthetic actuators By monitoring the state ofhealth of the actuator and modifying the mechanical demands accordingly, it is possible to promotefunctional adaptation of the living component of the actuator as well as the tissue or syntheticinterface It will be necessary to identify biomarkers of mechanical fatigue, such as reduced oraltered contractility, to actively detect these markers, and to respond with appropriate modifications

of the embedded excitation and control algorithms to allow tissue functional adaptation Inprinciple a properly monitored and controlled living muscle actuator will exhibit improved dy-namic performance and structural resilience with use over a period of decades, unlike any syntheticactuator technology currently available

9.6.2.7 Toxicity

A serious problem for all classes of living muscle actuators, the best countermeasure is barrierexclusion of exogenous toxic agents, the use of biocompatible materials in the fluid-space of thehybrid actuator assembly, and the clearance of toxic metabolic byproducts via a perfusion andfiltration system integrated with the living actuator

9.6.2.8 Electrochemical Tissue Damage

This failure mode affects all classes of living muscle actuators when exposed to chronic electricalstimulation The single best countermeasure is to promote and maintain tissue phenotype exhibitingvery high excitability In addition to vastly improving the excitation efficiency of the tissue, adultmuscle phenotype excitability can yield as much as a 99.9% reduction in electrical pulse energyrequirements for any given level of muscle activation, when compared with chronically denervated

or tissue engineered muscle tissue arrested at early developmental stages For this reason, thedevelopment of electro-mechanical muscle bioreactor systems and maintenance stimulation proto-cols form a core component of all current research on muscle tissue engineering Additionalcountermeasures include the selection of appropriate electrode materials, the use of minimallyenergetic stimulation protocols, the use of pure bipolar stimulation pulses with careful attention tocharge balancing, and the use of high-impedance outputs to the electrodes when not stimulating.9.6.2.9 Damage from Incidental Mechanical Interference

The living actuator will require electrodes to be placed in contact with the tissue, the presence oftubing for perfusion, and other structures required within the hybrid actuator Lateral mechanicalcontact between these synthetic objects and the living muscle tissue can result in a range ofmechanical failures, including abrasion, incision, and chronic pressure atrophy The appropriatecountermeasure for this is careful mechanical design of the hybrid actuator assembly, with theseconsiderations explicitly included in the system Design Specification

9.6.2.10 Retrograde or Arrested Phenotype (Failure to Thrive)

Effective countermeasures for this failure mode have been reported for denervated whole muscles

in vivo, employing a long-term electrical stimulation protocol (Dennis et al., 2003; Dow et al.,

2004) This failure mode is most prevalent in engineered muscle maintained in culture There aretwo approaches to dealing with this in engineered muscle: (1) genetic enhancement and (2)development of electromechanical tissue maintenance protocols In the case of genetic enhance-ment, the approach is to forcibly express desired genes in an attempt to promote the desired tissuephenotype The effectiveness of this approach is the core issue in gene therapy for diseases ofmuscle, but this approach has not yet been demonstrated to be effective for engineered muscleexvivo Optimal tissue maintenance protocols are a much more natural and subtle approach, basedupon the fact that all viable muscle cells contain the necessary genetic machinery to develop anydesired muscle phenotype, if the correct signals and growth conditions prevail In addition togenetic engineering of myocytes to enhance performance of tissue-based actuators, other potentialcountermeasures include: (1) development of appropriate tissue interfaces to permit signal trans-duction to the cellular machinery, (2) development of tissue and organ culture bioreactors to allowthe experimental determination of optimal control and maintenance protocols forex vivo muscletissue, (3) use of these protocols to guide tissue development (cell phenotype and tissue architec-ture), and (4) implementation of this technology into the hybrid actuator system This topic iscurrently an area of very active research Success in terms of counteracting this failure mode inengineered muscle will constitute an extraordinarily significant scientific contribution, as well asproviding the key enabling technology to the further development of practical living actuators.

9.7 SELF-ORGANIZING MUSCLE TISSUESSelf-organization within developing animals gives rise to an enormous array of muscle actuatorarchitectures Each myogenic precursor cell contains the genetic potential to self-organize intomuscle tissue with the desired phenotype and tissue interface The ability to guide the development

of self-organizing muscle tissues in culture will provide the systems engineer with the greatestlevel of design flexibility, since it will in principle be possible to start with a small population ofmuscle progenitor cells and guide them to self-organize into a muscle actuator of any imaginablegeometry It will also be possible to construct hybrid actuators not found in nature, containingregionally organized tissue structures, perhaps even consisting of fundamentally different types

of muscle tissue (skeletal, cardiac, or smooth), depending upon the functional requirements ofthe actuator system It is implicit in most muscle tissue engineering research programs thatskeletal muscle self-organization and development can be guided by the application of the correctexternal cues The general method of guided tissue self-organization in culture (Figure 9.1)briefly is:

. Isolate and coculture the desired cells The cells may be primary or from cell lines

. Engineer a cell culture substrate with controlled adhesion properties for the cells

. Provide permanent anchor points and surfaces to guide tissue architecture formation

. Culture the cells to permit the formation of a cohesive monolayer

. Induce monolayer delamination from the substrate at the appropriate point in cell differentiation(the monolayer remains attached to the anchor points)

. Promote tissue self-organization and further development by applying external signals: chemical,electrical, mechanical

Self-organization of tissues in culture is one effective way to produce small functional tissueconstructs from a range of tissues Examples include:

. Cardiac myocytes cocultured at confluence with fibroblasts will self-organize into long cylindersand tapered cones in culture in 340 to 400 h These constructs are electrically excitable and alsospontaneously contract as a syncytium to continuously generate significant mechanical workcycles Such constructs could be engineered to power cell-scaled implantable pumps, pumps for

stand-alone hybrid tissue actuators, or to engineer cardiac tissue for surgical transplantation incardiac reconstructive surgery.

. Tendon (Ligament) tissue will self-organize in culture under the appropriate conditions The blasts within the tissue produce a prodigious amount of ECM material, with collagen fibers that areoriented along lines of tensile stress, particularly at locations within the tissue where mechanicalinterfaces are present (such as suture anchor materials, metal posts, etc.) Self-organization is driven

fibro-by loss of substrate adhesion and the generation of internal tensile stress fibro-by the action of thefibroblasts on the order of 0 to 6 Pa, which can be experimentally controlled by external factorssuch as the presence of ascorbic acid, serum concentration in the cell culture medium, pH, etc

. Muscle Chimeras: One additional interesting technical possibility is the in vitro fusion of myogenicprecursor cells from different tissue sources to form chimeric self-organized engineered muscles.Preliminary experiments demonstrate that skeletal muscle satellite cells from differing species willfuse to form multinucleated myotubes with desirable contractile function In addition, isolatedcardiac myocytes will fuse into preexisting myotubes in culture, to produce a skeletal–cardiac musclehybrid Such chimeric muscle tissues are not known to exist in nature, but our preliminary dataindicate that they are both stable and functional in culture The contractile function of such chimericcells and tissues could potentially be engineered to produce tissue-based actuators with combinations

of desired characteristics that would be advantageous for use in hybrid bioactuator applications

9.8 ACELLULARIZED–RECELLULARIZED ECM ENGINEERED MUSCLESThe native ECM of muscle tissue occupies approximately less than 5% of the tissue volume, yet itcontains information about the complex architecture of muscle and the corresponding soft tissue

Figure 9.1 (See color insert following page 302) (A) Self-organized skeletal muscle construct after 3 months

in culture, length ~12 mm (B) Rat cardiac myocyteþ fibroblast monolayer in the process of delaminating and

self-organizing into a functional cardiac muscle construct, 340 h in culture (C) Self-organized cardiac muscle construct, attached to laminin-coated suture anchors, 380 h in culture (D) Electrically elicited force trace from the cardiac muscle construct shown in C, stimulation pulses shown below, contractile force trace shown above (raw data, unfiltered).

interfaces The cellular components of muscle can be chemically removed while retaining thedetailed architecture of the muscle ECM Preliminary results indicate the success of the reintro-duction of myogenic cells into these natural ECM scaffolds This approach to engineering muscles

as actuators has several advantages, among these are that heterogenic cells can be introduced intothe preexisting matrix For example, skeletal–cardiac chimeric muscles could be employed ormyogenic precursors from an entirely different species The main advantage of the use ofnatural ECM scaffolds is that the fine architecture of the entire muscle organ is retained by theacellularized ECM scaffold It is possible to perfuse the scaffold using the remnant vascular bedECM to reintroduce cells and later to provide perfusion to the reengineered muscle organ Theacellularized muscle ECM also has matrix architecture specific to the MTJ and tendon, which may

be advantageous in the development of this very critical tissue interface The principal disadvantage

of this approach is that the ECM scaffold architecture is limited to those architectures that areavailable in nature

9.9 TISSUE INTERFACES: TENDON, NERVE, AND VASCULAR

For any type of muscle actuator, it will be essential to provide appropriate tissue interfaces In somecases, the tissue interfaces are already in place and specific measures must be taken to maintainthem properly In other cases, their formation must be guided and facilitated Based upon ourin vivowork, we have demonstrated that muscle phenotype can be controlled and maintained in theabsence of innervation via electrical stimulation A considerable volume of published researchhas been directed toward the promotion of adult phenotype in muscle tissue in culture directly

by electrical stimulation, in the absence of nerve-derived trophic factors or depolarization viathe neuromuscular junction and related synaptic structures It remains to be demonstrated,however, that muscle can be guided through the necessary developmental stages in the absence

of innervation to achieve adult phenotype Adequate and functional vascular and tendon interfaces

to muscle engineered in vitro are also yet to be demonstrated, although they are the topic ofintensive research

9.9.1 Vascular Tissue Interface

Nutrition and oxygen delivery in static culture conditions always limit the cross-sectional area,particularly for tissues with high metabolic demand, such as muscle Therefore, a 3-D organ culturesystem with perfusion of a vascular bed within the muscle tissue is a core objective of currentresearch Cell types associated with angiogenesis, such as endothelial cells, are also crucial players

in organ development (Bahary and Zon, 2001) Endothelial progenitor cells from peripheral bloodare readily isolated, and have been shown to incorporate into neovessels (Asahara et al , 1997) andalso have potential to expand to more than 1019-fold in vitro (Lin et al., 2000) Furthermore,functional small-diameter neovessels can be created in culture by using endothelial progenitor cells(Kaushal et al., 2001)

9.9.2 Strategies for Engineering Functional Vascularized Muscle Tissue

There are three strategies for generating vascularized muscle constructs:

(1) Recellularization of an acellular muscle construct

(2) Coculture of myoblasts with endothelial cells and growth factor stimulation for induction of theendothelial cells to form capillary like structures

(3) Induction of sprouting of microvessels into temporarily implanted tissues or from vascularized andperfused tissue explants (such as adipose) cultured adjacent to the engineered muscle

The strategies for generating functional muscle tissue can be broadly divided intoin vitro and in vivostrategies, the ultimate outcome of which would be a vascularized muscle construct In any case,once a vascular bed is established, the constructs need to be maintained in a bioreactor to providefurther electrical, mechanical, and chemical stimulation, thus guiding both the phenotype andresulting in the development of a fully function muscle construct.

9.9.2.1 Recellularization of an Acellular Muscle Construct

This experimental approach involves harvesting muscle tissue from any natural source andusing chemical acellularization to remove myoblasts and fibroblasts leaving behind an intactECM The ECM should be evaluated for structural integrity and immunogenic behavior andits ability to support myoblast growth and differentiation The ECM should then be used asscaffolding material for seeding primary myoblast and the construct will be placed in a perfusionbioreactor allowing formation of functional skeletal muscle tissue (Hall, 1997) Immunohis-tochemical studies should be performed to determine which ECM components are present inthe acellular construct, such as collagen types I and IV, fibronectin, laminin, vitronectin,entactin, heparin sulfate, proteoglycan, and elastin The acellular muscle can be repopulated

by obtaining a purified sample of myogenic precursor cells, which may be injected or perfusedinto the acellular muscle Although some initial success has been reported with thisgeneral approach, it has not yet been possible to maintain perfusion of the tissue samples in culturefor a period sufficiently long to promote and maintain full cellular infiltration into the acellularscaffold

9.9.2.2 Coculture Systems

Since the early 1990s, there have been reports of the use of various coculture systems to study cell–cell interactions and the formation of tissue interfaces For vasculogenesis, the cells in question arepresumed to be myoblast and endothelial cells Although promising initial reports have beenpublished, a truly successful demonstration of a vascular bed self-organizing within a tissueconstruct has yet to be demonstrated The design of bioreactors for such a technology muststimulate the myoblasts to form functional muscle tissue and simultaneously guide the endothelialcells to form capillary-like structures within the newly forming muscle tissue, while providingperfusion during development The environment, which the bioreactor provides together withsoluble growth factor stimulation, will presumably allow formation of a functional muscle con-struct (Vernon, 1999)

9.9.2.3 Induced Microvessel Sprouting

This approach can be attempted eitherin vivo or ex vivo using small vascularized tissue explantswhich are cannulated and perfused while adjacent to an avascular tissue such as engineered skeletalmuscle This is an active area of current research For the in vivo approach, it is necessary tomechanically support the muscle tissue while implanted to prevent hypercontraction and subse-quent tissue damage It is also necessary to take measures to prevent tissue rejection to implantationinto syngenic animals, or the use of immune-suppressive agents, is required Otherwise, this method

is relatively quite simple and often yields satisfactory results In addition to vascularization of theimplanted muscle tissue, there are collateral effects, as yet not fully understood, that also tend todrive the muscle phenotype toward an adult phenotype, with enhanced contractility For this reason,

it is likely that the future of tissue engineering will see increasingly common application of theapproach where the intended recipient is used as a ready-made bioreactor vessel The engineeredtissues would be implanted within the person, presumably along with means to enhance tissuedevelopment and to prevent tissue degeneration or resorption while implanted The tissue need not

be implanted at the ultimate site for which it is intended, however, it is essential to consider themorbidity of the site at which the disuse will be initially developed.

9.9.3 Engineered Tissue Interface: Tendon

The MTJ is critical for the ability of muscle tissue to transduce force to and from the externalenvironment, and to produce maximal power without subsequent injury to the muscle cells inthe contractile tissue The MTJ contains specialized structures at the cell membrane whichfacilitate transmembrane transmission of force from the contractile proteins (biomolecularmotors) within the cell to the surrounding collagen fibrils in the ECM (Trotter, 1993) Thesestructures include a large number of infoldings of the muscle cell membrane at the MTJ, increasingthe membrane surface area and acting to transfer stress from the cytoskeleton to the ECM inthe tendon These structures have also been demonstrated to occur when myotubes are coculturedwith fibroblasts concentrated near the ends of the muscle constructs in vitro (Swasdison andMayne, 1991) In the case of whole explanted muscle actuators, the MTJ already exists, and it

is necessary to maintain this structure in vitro In all other classes of muscle actuator it isnecessary to generate or regenerate the MTJ and tendon structures Currently, attempts to engineertendon-like structures and muscle–tendon junctions in culture follow one of three distinctapproaches:

(1) Scaffold-based tendon, used as an anchor material for engineered muscle

(2) Self-organizing tendon and muscle-tendon structures in co-culture

(3) Direct laser transfer of muscle and tendon cells into defined 3-D structures

9.9.4 Nerve–Muscle Interfaces

Skeletal muscle phenotype is defined largely by the motor nerve which innervates each musclefiber Adult muscles may be either fast- or slow-twitch, but in general in humans muscles aremixed, containing significant populations of both fast- and slow-twitch fibers Denervated musclerapidly loses tissue mass and the adult phenotype, with contractility eventually dropping toessentially zero Although it is possible to maintain adult phenotype of adult skeletal muscle inthe absence of innervation, it is not yet clear whether it is possible to guide skeletal muscletissue development to an adult phenotype in an entirely aneural culture environment For thatreason, nerve–muscle synaptogenesis in culture is an area of active research in tissue engineering.Putative synaptic structuresin vitro have been reported for decades (Ecob et al., 1983; Ecob, 1983,1984; Ecob and Whalen, 1985), in some cases axon sprouting from nerves to muscle tissue

in culture is clearly visible (Figure 9.2) and verified upon histologic examination; however,functional nerve–musclein vitro systems that result in advanced tissue development have yet to

be demonstrated

9.9.5 Tissue–Synthetic Interfaces

Another key challenge is to develop means to mechanically interface living muscle cells and tissues

to synthetic fixtures in such a way that the tissue development and function will not be inhibited.The technical challenge is to provide a transition of mechanical stiffness and cell density in theregion between the contractile tissue and the synthetic fixture, to reduce stress concentrations at thetissue interface and provide mechanical impedance matching Several approaches are currentlyunder investigation, including the chemical functionalization of synthetic surfaces to bind collagen,and the use of porous scaffolds to promote tissue in-growth at the desired tissue or syntheticinterface

9.10 MUSCLE BIOREACTOR DESIGN FOR THE IDENTIFICATION,

CONTROL, AND MAINTENANCE OF MUSCLE TISSUEThe engineering of complex functional tissues such as skeletal muscle is by definition a systemsengineering problem Functional muscles are composed of a number of highly integrated tissuesystems, none of which is known to function in isolation for any significant period of time withoutmassive deterioration in performance Any attempt to engineer a functional muscle tissue system

ex vivo, and to employ that muscle system as a source of motility in robots or prostheses, will bynecessity require the development of bioreactor technologies to (1) guide the tissue development tothe desired phenotypeex vivo, (2) maintain the tissue at the desired phenotype while it is performingits function, and (3) control the mechanical output of the tissue through electrical stimulation.Critical to these three objectives are bioreactor technologies that are capable of monitoring andcontrolling a muscle’s mechanical and electrical environment

In Figure 9.3, a muscle bioreactor is shown that can implement muscle identification, control,and maintenance protocols under generalized boundary conditions while also providing flexiblefeedback control of electrical stimulation parameters (Farahat and Herr, 2005) These features areaccomplished by having two real-time control loops running in parallel The first loop, or themechanical boundary condition (MBC) control loop, ensures that the mechanical response ofthe servo simulates the dynamics of the associated muscle boundary condition For example,

if the desired boundary condition is a second order, mass–spring–damper system, the MBC controlloop controls the motion of the end points of the muscle–tendon unit as if the muscle–tendon wereactually pulling against physical mass–spring–damper mechanical elements The MBC controlloop allows for a whole host of boundary conditions, from finite (but nonzero) to infinite impedanceconditions Clearly, to understand muscle tissue performance, muscle dynamics, and the dynamics

of the load for which the muscle acts upon must be taken into consideration Examples of impedance boundary conditions include loads such as springs, dampers, masses, viscous friction,coulomb friction, or a combination thereof Such loads prescribe boundary conditions that aregenerally defined in terms of dynamic relationships between force and displacement Under theseloading conditions, it would be expected that the dynamics of the load will interact with thecontraction dynamics of the muscle, leading to a behavior that is a resultant of both This is

finite-Figure 9.2 (See color insert following page 302) Left: axonal sprouting (A) from an explanted motor neuron cell cluster (V) toward a target tissue (T), in this case, an aneural cultured skeletal muscle ‘‘myooid.’’ Right: a simple cell culture system demonstrating axonal sprouting between neural (PC–12) and myogenic (C2C12) cell lines This co-culture system allows the study of synaptogenesis in culture (Photographs taken by members of the Functional Tissue Engineering Laboratory at the University of Michigan: Calderon, Dow, Borschel, Dennis.)

primarily because the force generated by muscle is dependent on its mechanical state, namely itslength and velocity.

The second control loop for the bioreactor design of Figure 9.3 implements the electricalstimulus (ES) control based on measurements of the muscle’s mechanical response This loop,referred to as the ES control loop, offers simultaneous real-time modulation of pulse width,amplitude, frequency, and the number of pulses per cycle There is increasing experimental interest

in real-time control of muscles, primarily in the context of functional electrical stimulation (FES)(Chizeck et al., 1988; Veltink et al., 1992; Eser et al., 2003; Jezernik et al., 2004) In theseinvestigations, attempts were made to control the response of muscle(s) and associated loads to adesired trajectory by varying electrical stimulation parameters as a function of time Electricalstimulation patterns are typically square pulses characterized by frequency, amplitude, pulse width,and number of pulses per trigger (considering the cases of doublets, triplets, or more generallyN-lets) For testing a variety of FES algorithms, the ES control loop is designed for real-timemodulation of these stimulation parameters as a function of a muscle’s mechanical response,including tissue length, contraction velocity, and borne muscular force

9.11 CASE STUDY IN BIOMECHATRONICS:

A MUSCLE ACTUATED SWIMMING ROBOTBiomechatronics is the integration of biological materials with artificial devices, in which thebiological component enhances the functional capability of the system, and the artificial componentprovides specific environmental signals that promote the maintenance and functional adaptation ofthe biological component Recent investigations have begun to examine the feasibility of usinganimal-derived muscle as an actuator for artificial devices in the millimeter to centimeter size scale

Figure 9.3 (See color insert following page 302) Muscle Bioreactor Technology Muscle identification, control, and maintenance apparatus is shown with the primary sensors and actuators noted The coarse positioning stage is adjusted at the beginning of the experiment to accommodate different tissue lengths, but is typically kept at a constant position during a particular contraction The primary stage provides the motion that simulates the boundary condition force law with which the muscle specimen pulls against The vertical syringe has a suction electrode at its tip that is connected to the stimulation electronics in the background The encoder and load cell measure muscle displacement and force, respectively, and are employed as sensory control inputs during FES control experimen- tation Silicone tubing recirculates solution via a peristaltic pump, while oxygen is injected in the loop.

(Herr and Dennis, 2004) Although a great deal of research has been conducted to develop anactuator technology with muscle-like properties, engineering science has not yet produced a motorsystem that can mimic the contractility, energetics, scalability, and plasticity of muscle tissue(Hollerbach et al., 1991; Meijer et al., 2003) As a demonstratory proof of concept, Herr and Dennis(2004) designed, built, and characterized a swimming robot actuated by two explanted frogsemitendinosus muscles and controlled by an embedded microcontroller (Figure 9.4) Using openloop stimulation protocols, their robot performed basic swimming maneuvers such as starting,stopping, turning (turning radius ~ 400 mm), and straight-line swimming (max speed > 1/3 bodylengths/sec) A broad-spectrum antibiotic or antimycotic ringer solution surrounded the muscleactuators for long-term maintenance,ex vivo The robot swam for a total of 4 h over a 42-h lifespan(10% duty cycle) before its velocity degraded below 75% of its maximum The mechanicalswimming efficiency of the biomechatronic robot, as determined by a slip value of 0.32, waswithin the biological efficiency range Slip values increase with swimming speed in biologicalswimming, ranging from 0.2 to 0.7 in most fish (Gillis, 1997, 1998).

The development of functional biomechatronic prototypes with integrated musculoskeletaltissues is the first critical step toward the long-term objective of controllable, adaptive, and robustbiomechatronic robots and prostheses The results of the swimming robot study of Herr and Dennis(2004), although preliminary, suggest that some degree ofex vivo robustness and controllability ispossible for natural muscle actuators if adequate chemical and electromechanical interventions aresupplied from a host robotic system An important area of future research will be to establishprocesses by which optimal intervention strategies are defined to maximize tissue longevity for agiven hybrid-machine task objective Another important research area is tissue control It is wellestablished that natural muscle changes in size and strength depending on environmental work-load, and when supplied with appropriate signals, changes frequency characteristic or fiber type(Green et al., 1983, 1984; Delp and Pette, 1994) Hence, an important area of future work will be toput forth strategies by which muscle tissue plasticity can be monitored and controlled Still further,

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Figure 9.4 (See color insert following page 302) Biomechatronic swimming robot To power robotic swimming, two frog semitendinosus muscles (1), attached to either side of elastomeric tail (2), alternately contract to move the tail back and forth through a surrounding fluid medium Two electrodes per muscle (3), attached near the myotendonous junction, are used to stimulate the tissues and to elicit contractions To depolarize the muscle actuators, two lithium ion batteries (4) are attached to the robot’s frame (5) During performance evaluations, the robot swam through a glucose-filled ringer solution to fuel muscle contractions.

strategies must also be devised to control the force and power output of muscle, in the context ofrobotic systems, through the modulation of electrical pulses to the muscle cell Also, for thedevelopment of controllable, adaptive and robust biomechatronic systems, feedback control sys-tems that monitor and adapt the mechanical, electrical, and chemical environment of muscleactuators are of critical importance.

Muscle tissue as a mechanical actuator has great, though as-yet unrealized potential for use

in engineered systems Synthetic technologies such as electroactive polymers are rapidly emerging

as quantitatively functional equivalents to muscle tissue, and it is likely that the technologicalevolution of EAP muscles will soon out-pace the natural functional evolution of living muscletissue This means that the quantitative performance advantages that muscle tissue has over someforms of synthetic actuators in terms of efficiency, power density, and so forth are not likely toremain the case for very much longer One then invariably must ask why it is advantageous to evenconsider the use of living muscle tissue as a mechanical actuator It is easy to point out that themany disadvantages of muscle outweigh the few performance advantages it may have The answerlies chiefly in the qualitative differences between muscle and competing synthetic actuator tech-nologies, among these are those qualities that arise from muscle being a living tissue: its ability tofunctionally adapt and to potentially integrate seamlessly with other living structures So it is likelythat living muscle actuators will only be employed in practical systems where their qualitativeadvantages as living tissue can be exploited to maximum benefit, such as in hybrid biomechatronicprosthetic systems and implants, and perhaps in bioreactors where their biological products (such asedible proteins) are of primary importance Certainly though, living muscle tissue serves as theexplicit benchmark against which the performance of synthetic actuator technologies will beevaluated for many decades to come

FURTHER READING

The following list of papers and book chapters comprises a set of useful references for further work in this area.These were not referenced directly in the text, but have been included because the authors have found them to

be useful during the course of the development of the technology discussed in this chapter

Agoram, B and Barocas, V.H Coupled macroscopic and microscopic scale modeling of fibrillar tissues andtissue equivalents.J Biomech Eng 2001, 123(4): 362–369

Askew, G.N., Marsh, R.L et al The mechanical power output of the flight muscles of blue-breasted quail(Coturnix chinensis) during take-off J Exp Biol 2001, 204: 3601–3619

Barrett, S Propulsive Efficiency of a Flexible Hull Underwater Vehicle PhD Thesis, Massachusetts Institute

of Technology, Cambridge, Massachusetts, 1996

Biewener, A.A., Dial, K.P et al Pectoralis muscle force and power output during flight in the starling.J Exp.Biol 1992, 164: 1–18

Broadie, K.S Development of electrical properities and synaptic transmission at the embryonic neuromuscularjunction.Neuromuscular Junctions Drosophila 1999, 43: 45–67

Brown, K.J et al A novelin vitro assay for human angiogenesis Lab Invest 1996, 75(4): 539–555.Calve, S., Arruda, E., Dennis, R.G., and Grosh, K Influence of mechanics on tendon and muscle development.WCCM Abstracts, 2002

Campbell, P.G., Durham, S.K., Hayes, J.D., Suwanichkul, A., and Powell, D.R Insulin-like growth binding protein–3 binds fibrinogen and fibrin.J Biol Chem 1999, 274(42): 30215–30221

factor-Cederna, P.S., Kalliainen, L.K., Urbanchek, M.G., Rovak, J.M., and Kuzon, W.M ‘‘Donor’’ muscle structureand function following end-to-side neurorrhaphy.Plast Reconstr Surg 2001, 107: 789–796

Close, R Effects of cross-union of motor nerves to fast and slow skeletal muscles Nature 1965, 206:831.

Close, R Dynamic properties of fast and slow skeletal muscles of rat after nerve cross-union.J Physiol Lond

Conrad, G.W et al Differencesin vitro between fibroblast-like cells from cornea, heart, and skin of embryonicchicks.J Cell Sci 1977, 26: 119–137

Dennis, R.G Bipolar implantable stimulator for long-term denervated muscle experiments.Med Biol Eng.Comput Med Biol Eng Comput 1998 March, 36: 225–228

Dennis, R.G Engineered skeletal muscle: nerve and tendon tissue interfaces, contractility, excitability, andarchitecture In:Functional Tissue Engineering, Guilak, F., Butler, D., Mooney, D., and Goldstein, S.(eds) Springer-Verlag, New York, 2003

Dennis, R.G and Kosnik, P Excitability and isometric contractile properties of mammalian skeletal muscleconstructs engineeredin vitro In Vitro Cell Dev Biol Anim 2000, 36(5): 327–335

Dennis, R.G., Dow, D.E., Hsueh, A., and Faulkner, J.A Excitability of engineered muscle constructs,denervated and denervated-stimulated muscles of rats, and control skeletal muscles in neonatal,young, adult, and old mice and rats.Biophys J 2002, 82: 364A

Dial, K.P and Biewener, A.A Pectoralis muscle force and power output during different modes of flight inpigeons (Columba livia) J Exp Biol 1993, 176: 31–54

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