Research Area: Nerve Tissue Co-culture Robert G. Dennis, Ph.D.

My research in nerve tissue engineering involves four general areas:

1- Nerve-Muscle co-culture to promote synaptogenesis in engineered skeletal muscle.
    [Collaboration with Marlene Calderon, M.D.]

2- Gene expression and synaptogenesis.
    [Collaboration with Dan Goldman, Ph.D., and Peter Macpherson, Ph.D.]

3- Monitoring and depolarization of cultured nerve axons using sieve electrodes.
    [Collaboration with Dave Anderson, Ph.D. and Jamie Hetke of the Center for Neural Communication Technology]

4- Acellularized nerve tissue ECM for use as a nerve conduit.
    [Collaboration with Bill Kuzon, M.D., Ph.D., and Paul Cederna, M.D.]

These four areas of research in nerve tissue engineering all relate to different aspects of the technology development and ultimate clinical application of engineered functional skeletal muscle tissue.

    In a collaboration with Marlene Calderon, M.D. we were able to develop a nerve-muscle co-culture system in which axonal projections from a ventral horn explant projected to the muscle construct in vitro, as shown in the image below.

Purpose:  to promote enhanced excitability and contractility of the engineered muscle constructs by providing nerve-derived trophic factors and synaptic stimulation at the neuromuscular junction.
 
 

Nerve-muscle co-culture system.  We were able to get axonal projections from the ventral horn spinal explants from embryonic rats to project to the myooid muscle constructs in culture.  This photo is representative of many successful nerve-muscle co-culture systems.  When tested for changes in contractility or tissue excitability, there were no differences between muscles in co-culture and muscles cultured entirely aneurally.  We also were not able to detect synaptogenesis by cholinesterase staining at the putative motor end plates.  We therefore hypothesize that the muscle constructs are not receptive to synaptogenesis.  Our working hypothesis is that the muscle construct must be pre-conditioned by electrical stimulation to promote the correct expression of nicotinic acetylcholine receptors for the muscle tissue to be receptive to reinnervation (see Dan Goldman collaboration below). The scale running parallel to the myooid muscle construct is 1 mm long, with 100 divisions.  The myooid is approximately 300 microns in diameter.  Photo by Marlene Calderon.

    In a collaboration with Dan Goldman and Peter Macpherson, our objective is to control the relative expression of different subunits (g and e) of the nicotinic acetylcholine receptor (nAChR) expressed by the muscle, to allow re-innervation of the skeletal muscle after long periods of denervation.  Prolonged denervation of skeletal muscle is typified by a reversion to a state where the muscle expresses subunits that are not receptive to synaptogenesis.  Peter has used the implantable stimulator system that I have designed and has demonstrated that, by controlled intermittent muscle cell membrane depolarization, he can control and reverse the relative levels of expression.  He is currently executing experiments to demonstrate an increased potential for reinnervation of the long-term denervated skeletal muscles in vivo (rats).  Marlene Calderon is also working on a doctoral project which uses my implantable stimulators to maintain muscle mass and contractility during long-term denervation.  For my part in this collaboration, in addition to designing and providing the electrical stimulators, I will test this hypothesis in vitro in a nerve-muscle co-culture system.  The current engineered muscle model is cultured entirely aneurally (no innervation), and therefore may be refractory to innervation unless properly preconditioned by depolarization activity.

Objectives:  (1) Permit depolarization of individual motor axons in culture to achieve motor unit level control of engineered muscle tissue, (2) exert control over the phenotype of muscle fibers in each motor unit, and (3) monitor spontaneous depolarization of individual motor axons during development in vitro.

Experimental Plan:  Sieve electrodes coated with laminin and other surface adhesion molecules will be interposed between the nerve tissue and the muscle construct in culture.  The iridium metal electrode surfaces form an oxide layer, and allow a pseudo-capacitive electrode coupling to the tissue.  Due to the many iridium oxide transitions, the electrodes can provide an adequate stimulus to discharge a motor axon.  The sieve electrodes can be designed to accommodate any desired geometry, with an arbitrary number of instrumented holes, each hole of arbitrary dimensions.  A representative photograph of a currently available sieve electrode is shown in the photograph below.  Nerve tissue source will be either primary ventral horn explants (as shown above) or PC12 cells, which are known to form synapses with muscle cells in culture.
 

The image to the right is of a sieve electrode specifically designed for in vivo implantation at the site of a nerve transsection.  We are currently collaborating to develop a sieve electrode specifically for the nerve-muscle co-culture configuration shown above.  The probes are fabricated by the University of Michigan Center for Neural Communication Technology, sponsored by NIH NCRR grant P41 RR09754, for Dr. Steven Highstein of Washington University.  (see: Mensinger, et al., J. Neurophys 83: 611-615, 2000).

    In a collaboration with Bill Kuzon and Paul Cederna, I have developed a protocol for complete acellularization of tissues with tubular cell components, such as muscle and nerve.  The purpose for the acellularization process is to provide a scaffold of extracellular matrix proteins for cell ingrowth to guide nerve axon regeneration over long gaps (> 2 cm).  The candidate tissues for the neural conduits are muscle and nerve.  The acellularization process that I have developed works equally well with both tissue types, entirely removing all cellular components while leaving the ECM intact.  A representative photograph showing the effectiveness of this procedure is shown below, using a full section thickness of mouse EDL muscle, which is approximately 3 mm thick.  Before the acellularization process, the tissue is opaque, after the process it is transparent because only the ECM remains after all cellular components have been removed.  When segments of nerve tissue are subjected to the same process, an acellularized structure results that has the correct architecture to support Schwann cell repopulation and axonal regeneration.  In preliminary studies we have found that the acellularized nerve conduits are entirely non-immunogenic when transplanted between animals with major histocompatibility mismatches.  The acellularized tissue elicits a less severely immune response than autograft tissue (due probably to the presence of necrosing cells in the autograft, even though there is no rejection of xenogenetic material in the autograft tissue, per se.)  Our preliminary data also indicate that the acellularized nerve conduits will support Schwann cell repopulation and axonal regeneration.
 

Mouse EDL muscle before acellularization	Mouse EDL muscle after acellularization

Significance:  The ability to create nonimmunogenic acellular nerve conduits that support axonal regeneration over large gaps has direct clinical significance because it then becomes possible to use cadaveric nerve tissue (in plentiful supply) to produce the graft material for a patient, which can then be repopulated by the patient's own Schwann cells prior to reimplantation, so axonal regeneration will be supported and there will be no tissue rejection.