Myelination and node of Ranvier formation on sensory neurons in a defined in vitro system.

One of the most important developmental modifications of the nervous system is Schwann cell myelination of axons. Schwann cells ensheath axons to create myelin segments to provide protection to the axon as well as increase the conduction of action potentials. In vitro neuronal systems provide a unique modality to study a variety of factors influencing myelination as well as diseases associated with myelin sheath degradation. This work details the development of a patterned in vitro myelinating dorsal root ganglion culture. This defined system utilized a serum-free medium in combination with a patterned substrate, utilizing the cytophobic and cytophilic molecules (poly)ethylene glycol (PEG) and N-1[3 (trimethoxysilyl) propyl] diethylenetriamine (DETA), respectively. Directional outgrowth of the neurites and subsequent myelination was controlled by surface modifications, and conformity to the pattern was measured over the duration of the experiments. The myelinated segments and nodal proteins were visualized and quantified using confocal microscopy. This tissue-engineered system provides a highly controlled, reproducible model for studying Schwann cell interactions with sensory neurons, as well as the myelination process, and its effect on neuronal plasticity and peripheral nerve regeneration. It is also compatible for use in bio-hybrid constructs to reproduce the stretch reflex arc on a chip because the media combination used is the same that we have used previously for motoneurons, muscle, and for neuromuscular junction (NMJ) formation. This work could have application for the study of demyelinating diseases such as diabetes induced peripheral neuropathy and could rapidly translate to a role in the discovery of drugs promoting enhanced peripheral nervous system (PNS) remyelination.

Tissue engineering the mechanosensory circuit of the stretch reflex arc: sensory neuron innervation of intrafusal muscle fibers.

The sensory circuit of the stretch reflex arc, composed of specialized intrafusal muscle fibers and type Ia proprioceptive sensory neurons, converts mechanical information regarding muscle length and stretch to electrical action potentials and relays them to the central nervous system. Utilizing a non-biological substrate, surface patterning photolithography and a serum-free medium formulation a co-culture system was developed that facilitated functional interactions between intrafusal muscle fibers and sensory neurons. The presence of annulospiral wrappings (ASWs) and flower-spray endings (FSEs), both physiologically relevant morphologies in sensory neuron-intrafusal fiber interactions, were demonstrated and quantified using immunocytochemistry. Furthermore, two proposed components of the mammalian mechanosensory transduction system, BNaC1 and PICK1, were both identified at the ASWs and FSEs. To verify functionality of the mechanoreceptor elements the system was integrated with a MEMS cantilever device, and Ca(2+) currents were imaged along the length of an axon innervating an intrafusal fiber when stretched by cantilever deflection. This system provides a platform for examining the role of this mechanosensory complex in the pathology of myotonic and muscular dystrophies, peripheral neuropathy, and spasticity inducing diseases like Parkinson's. These studies will also assist in engineering fine motor control for prosthetic devices by improving our understanding of mechanosensitive feedback.

Engineering a titanium and polycaprolactone construct for a biocompatible interface between the body and artificial limb.

Intraosseous transcutaneous amputation prostheses may be able to overcome the problems that stem from the nonuniform distribution of pressure seen in the conventional stump-socket prosthetic replacement devices. Transcutaneous devices have had limited success in amputees. By optimizing the attachment of the skin to the prosthetic, intraosseous transcutaneous amputation prostheses may become clinically viable options. This report details studies evaluating the development of a modified titanium construct with a specially machined surface to increase the adherence of tissue as well as scaffold. A computer-aided biology tool was used to fabricate polycaprolactone (PCL) scaffolds with a specific three-dimensional architecture. To extrude the PCL, it was dissolved in acetic acid to produce a 70% PCL liquid. A scaffold with a porosity of >50% was fabricated to have a tensile strength similar to skin. The presence of a specially machined surface greatly increased the adhesion of the PCL scaffold to the titanium constructs. When the 70% PCL was properly neutralized by heating at 55 degrees C and washing in 90% ethanol (EtOH), there was only a decrease (10%) in the viability of cells seeded onto the PCL constructs when compared with the cells in culture. The antibacterial properties of titanium dioxide anatase, silver nanoparticles, and chlorhexidine diacetate mixed in either type I collagen or hyaluronic acid (HA) were assessed. The addition of 1% (w/w) chlorhexidine diacetate in HA resulted in a 71% decrease in bacteria seen in nontreated HA. These results show promise in developing a novel engineered titanium and PCL construct that promotes effective adhesion between the titanium-skin interface.

Temporal neurotransmitter conditioning restores the functional activity of adult spinal cord neurons in long-term culture.

The ability to culture functional adult mammalian spinal cord neurons represents an important step in the understanding and treatment of a spectrum of neurological disorders including spinal cord injury. Previously, the limited functional recovery of these cells, as characterized by a diminished ability to initiate action potentials and to exhibit repetitive firing patterns, has arisen as a major impediment to their physiological relevance. In this report, we demonstrate that single temporal doses of the neurotransmitters serotonin, glutamate (N-acetyl-DL-glutamic acid) and acetylcholine-chloride lead to the full electrophysiological functional recovery of adult mammalian spinal cord neurons, when they are cultured under defined serum-free conditions. Approximately 60% of the neurons treated regained their electrophysiological signature, often firing single, double and, most importantly, multiple action potentials.