Research Interests Robert G. Dennis, Ph.D.

Tissue Engineering

Although I have research interests in many areas, the focus of my research since finishing my Ph.D. in 1996 has been Tissue Engineering.  Specifically, I am interested in developing functional skeletal muscle constructs in vitro.  There is a sizable literature on skeletal muscle tissue engineering, but when I began research in this area, the contractility of engineered muscle had been measured and reported only once¹. In this very important paper, avian muscle cells were engineered into a small cylinder, and activated by elevating the extra cellular potassium concentration while maintaining the muscle at 4^oC.  This resulted in a slow contraction of the muscle organoid over a time of 30 to 60 seconds.

I decided to develop a skeletal muscle construct, engineered in vitro, with the following design considerations:
- Self organizing (no scaffold², which could interfere with the cell fusion and force generation)
- Artificial tendons (to allow attachment of force transducers and servo motors for functional evaluation)
- Mammalian tissue (so that the engineering process would have immediate clinical relevance)
- Primary cells (not 'immortalized' cell lines, which typically are isolated from tumors and have abnormalities)
- Electrically excitable (to permit the tissue excitability and contractility to be quantified experimentally)
- Robust (able to be maintained and fully activated at physiologic temperatures, ~37^oC.

My graduate student, Paul Kosnik, and I developed a process for harvesting the primary cells from rats and mice, for inducing the self organization of the cells into a functional engineered tissue, and for evaluating the excitability and contractility of the muscle constructs.  We termed the constructs 'myooids', because they are muscle like in form and function.  Our work in this area has lead to the filing of four patents, the first of which (for the Development Emulator system) was issued in September 2000.

The myooid model system is very useful for the study of muscle function and development.  We have recently submitted three manuscripts for publication, with an additional six manuscripts in preparation for submission in the near future.  Paul Kosnik now works with Herman Vandenburgh at  Cell Based Delivery, Inc., and we have an active collaboration in the development of engineered muscle, culture automation systems, and muscle-tendon interfaces.

My current and future research in tissue engineering will focus on the following topics:
- Automated cell culture systems (for applying controlled interventions and monitoring the resulting function)
- Control of expression of adult muscle phenotype (adult myosin isoforms: fast twitch & slow twitch)
- Compliant tendons (for mechanical impedance matching and the reduction of stress concentrations)
- Motor unit level control of engineered skeletal muscle constructs (a collaboration with CNCT in EECS)
- Nerve-muscle interface (to provide nerve-derived trophic factors and to aid in expression of adult phenotype)
- Angiogenesis and perfusion (to allow large tissue cross sections)

For more detailed technical information, please visit my Current Research Interests page.

Notes:
1  Vandenburgh et al., FASEB J. 5: 2860-2867, 1991.
2  Artificial scaffolds are often employed in engineered tissues.  Typically, these tissues are predominantly extracellular matrix (ECM), such as bone, tendon, ligament, skin, or cartilage.  These tissues generally have 30-90% of their volume occupied by ECM, so the presence of an artificial scaffold presents no theoretical problem.  Muscle, by contrast, has less than 2% by volume ECM, so the pre-existence of a significant ECM in the engineered tissue is, in my opinion, not desirable.  In addition, Muscle is the only structural tissue with multinuclear cells (typically >= 400 nuclei per adult cell, or fiber), and the scaffolds currently in use tend to prevent cell fusion and the formation of long, continuous muscle fibers, which are essential to the adult phenotype of muscle.

In 1991 I began a collaboration with surgeons and scientists in the Department of Surgery at the University of Michigan to do research on the possibility of constructing a left ventricular assist device, using transposed muscle from within the body of the patient.  The Idea was to re-engineer existing skeletal muscle to help a failing heart to continue to pump blood until a heart transplant became available for a patient in end-stage congestive heart failure.  The Latissimus Dorsi muscle was selected, because it was a large sheet of muscle, near the heart, and could be transposed without cutting the nerve or blood supply while resulting in minimal loss of limb function after the transposition.  A small section of the posterior rib cage was removed, and the muscle was transposed through this hole and wrapped around the heart.  For this to work, the muscle had to be re-engineered to express slow twitch characteristics, so that the muscle could provide continuous duty pumping action to assist the remaining heart muscle.  For this reason, I began to design implantable muscle stimulators.

The first generation of implantable stimulators was designed simply to apply a series of continuous pulses to normally-innervated skeletal muscle, to cause the muscle fiber type to convert from fast twitch to slow twitch.  Over the years, I have developed a series of implantable devices for a wide range of research projects, including maintenance of the mass and contractility of long-term denervated muscles, gene expression in denervated muscles, glucose metabolism in chronically active muscles, and contraction-induced injury in dystrophic mouse muscles.  I have expanded the capabilities of the current system to allow multiple stimulation outputs, transcutaneous optical and magnetic control of the devices, and even the addition of internal sensors, so that the devices can act as implantable data acquisition systems.

For more detailed technical information, please visit my Current Research Interests page.

In 1995, I began a collaboration with Tom Goodwin at NASA Johnson Space Center to investigate the effects of low level electro-magnetic fields on tissues in culture.  Based on Tom's experimental requirements, I developed the instrumentation and protocols to subject normal human neural progenitor (NHNP) cells to low level fields in both 2- and 3-dimensional tissue culture systems.  At NASA JSC, Tom used the instrumentation to apply low-level (<100 mGauss) magnetic fields with continuous (DC), and bipolar sine, square, narrow pulse (delta), and triangle waveforms.  For comparison, the Earth's magnetic field strength at 45^o latitude is approximately 500 mGauss, so the fields that we applied were quite low when compared with the fields typically generated by common electronic devices.

The results were quite surprising. We found that these low level fields could exert a very significant influence over cells in culture under certain conditions.  The effect was greatest for the most rapid magnetic (B) field slew rate (square and delta waves), less for slower B field changes (sine and triangle), and no effect for continuous (DC) B fields.  The results for the square and delta functions were indistinguishable, and we therefore conclude that the effect results from the rapid variation of the B field, rather than the DC component of the field itself.

For more detailed technical information, please visit my Current Research Interests page.

   The Biomechatronics Group at the MIT Artificial Intelligence Laboratory has recently been funded by DARPA to develop muscle based actuators for robotic and prosthetic applications.  With my colleague, Hugh Herr, Ph.D., I am developing the technology to employ both engineered skeletal muscle as well as re-engineered native whole muscles as actuators within robotic and prosthetic devices.  The ultimate vision is to integrate muscle actuators into prosthetic limbs.  The prosthetic devices will evolve in complexity by inclusion of greater and greater biological content, until the entire device is biologic rather than synthetic.  The technology will be developed to utilize cells from the prosthetic user themselves, so that eventually the re-engineered limb will be entirely compatible with the person for whom it has been engineered.  Tissue interfaces will also be developed, to allow innervation, blood circulation, and mechanical connection with the muscle actuators.  at MIT, I have designed and tested a biomechatronic device, a surface swimming fish, that employs a pair of muscle actuators, an embedded microcontroller, and an infra red optical link to demonstrate that muscle-powered robots are feasible.

For more detailed technical information, please visit my Current Research Interests page.

Force Transducers
     I have a general interest in instrumentation development, both for basic and applied research.  I have developed three different types of force transducers for research applications.  The Interferometric Force Transducer uses laser interferometry to permit extremely low compliance load detection.  This device has been patented (U.S. patent # 5555470).  A second force transducer device, also with very high stiffness, is based on total internal reflection.  This device works only in compression mode, unlike the other two designs, and also has a highly non-linear output.  The third design is a robust, low cost, simple optical force transducer, specifically designed to measure forces in the range generated by single muscle fibers and engineered muscle tissue.

     The series of force transducers for physiological measurements was first described in my doctoral dissertation (1996).  They are a simple differential optical displacement transducer, with a total compliance of +/- 30 mm from the neutral position (linear range).  The unamplified output voltage is +/- 0.5 V, so only very low levels of amplification are required.  The mechanical load element is configured to allow the load range to be adjusted in the range of 100 mN to 10 N full scale, with a resolution of approximately 1/2000 of full scale.  My force transducer design is currently in use for muscle physiological measurements, especially engineered skeletal muscle, in several labs across the country.  I am working with a small instrumentation company in Providence, RI to develop these transducers as a standard product for more general use.
 

Laser diffraction
     I have an interest in developing systems for measuring the mechanical strain of small regions of sarcomeres within individual muscle fibers.  For my doctoral dissertation, I developed a system to measure longitudinal pulse propagation in permeabilized muscle fiber segments.  Since I graduated, I have been providing guidance to students in the muscle mechanics laboratory at the University of Michigan who are interested in developing this technology.  We are currently working to expand the system capabilities to allow a focused visible laser beam to be swept along a single muscle fiber.  The position of the laser spot on the fiber is controlled by an acusto-optic modulator (AOM), employed as a beam deflector.  This allows random access of any point along the fiber with an access time of ~2 ms.  By placing surface markers on the muscle fiber (a regularly used technique, published over the last decade or so), and by recording the diffraction angle of the laser beam, it will be possible to measure the differential strain between the cell membrane and the internal contractile apparatus within the muscle fiber.  We plan to use this capability to study the mechanical consequences of genetic modifications that result in disruption of the cytoskeleton.  Examples include the dystrophic mouse model, as well as a- sarcoglycan knockout mice.

I still have a strong interest in this area of research, but I have set it aside to allow room for a highly-creative graduate student to develop the technology with a free hand.  For more information on this topic, and a movie of a laser diffraction signal through skeletal muscle, please visit my Past Projects page and the research topics section of the web site for the Muscle Mechanics Laboratory at the University of Michigan.

Implantable data loggers/stimulators
     Based on my series of implantable stimulators described above, I am developing a series of data loggers/tissue stimulators for use in a wide range of research applications.  This is a long term project that I have been working on for approximately 9 years.  The new devices will be useful for measuring temperature, nerve impulses and forces generated within living organisms, recording the output from sonomicrometers to measure displacements, and for providing any desired stimulus pattern to targeted tissues, including nerve, muscle, skin, bone, tendon, or ligament.  These devices will also interface with the implantable nerve electrodes from the Center for Neural Communication Technology at the University of Michigan.  I will make the design of these devices readily available on this web site for general research use as the technology develops.  These devices will be critical for the re-engineering of native muscle tissues for use in the muscle actuator research currently in progress at the MIT Biomechatronics Group.

Bioreactors
     I am actively engaged in bioreactor design for engineered tissues.  I work with Thomas Goodwin at NASA JSC to develop 2-D and 3-D bioreactor systems to test the effects of electromagnetic fields on tissues in culture.  The 3-D system includes a commercially available microgravity simulator, which allows large tissue masses to be cultured.  I am also developing fully automated cell culture systems to provide controlled electrical, chemical, and mechanical interventions to engineered skeletal muscle constructs.

For more detailed technical information, please visit my Current Research Interests page.

     I am currently using the tissue engineered skeletal muscle model (myooid) to study the regenerative potential of satellite cells from aged mammals, including humans.  The importance of the aging aspect of this basic research can not be overstated.  For most clinical applications, tissue from adult and aged humans must be used, so it is important to include this at an early stage in the technology development.  At the Muscle Mechanics Laboratory at the University of Michigan, I have carried out research on the functional plasticity and capacity of skeletal muscle stem cells isolated from rats of all ages, from 1-day neonates to 36 month old rats (roughly equivalent to a 90 year old human).

For more detailed technical information, please visit my Current Research Interests page.