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Neural Tissue Engineering

GETTING ON YOUR NERVES:Left: courtesy of Anthony Windebank; right courtesy of Christine SchmidtTo facilitate nerve regrowth, tissue engineers employ biodegradable polymer scaffolds. Shown at left, a micrograph of an actual scaffold used to stimulate spinal cord regeneration in rats. Center, a piece of neural tissue grows on an electrically conductive polymer used primarily for peripheral nerve repair (image colored for effect). At right, stained section of a peripheral nerve showing axons in red

Aileen Constans
<p>GETTING ON YOUR NERVES:</p>

Left: courtesy of Anthony Windebank; right courtesy of Christine Schmidt

To facilitate nerve regrowth, tissue engineers employ biodegradable polymer scaffolds. Shown at left, a micrograph of an actual scaffold used to stimulate spinal cord regeneration in rats. Center, a piece of neural tissue grows on an electrically conductive polymer used primarily for peripheral nerve repair (image colored for effect). At right, stained section of a peripheral nerve showing axons in red and myelin in green.

Despite advances in knowledge about the mechanisms of nerve injury and repair, regeneration strategies for peripheral and central nervous system (PNS and CNS) damage are still in their infancies. "Neuroscientists are very good at finding out, okay, this enzyme would work or this trophic factor would work, but translating that to a controlled application that will help lead to clinical translation is a different story," says Ravi Bellamkonda, a biomedical engineer...

MAKING CONNECTIONS

The bulk of neural tissue engineering efforts center on peripheral nerve repair, for one simple reason: The human body has its own PNS repair pathway. If the injury is small enough (that is, if the nerve is not severed or crushed), a fibrin cable forms at the damaged ends, creating a bridge that eventually becomes lined with nerve growth factor (NGF)-excreting Schwann cells. But this pathway fails if the nerve gap is wider than a centimeter or so.

That's where the tissue engineers come in. Gaps in peripheral nerves are commonly repaired using autologous nerve tissue grafts. The grafted tissue (usually sensory nerves) acts as a template for regrowing nerve fibers, explains tissue engineer Christine Schmidt at the University of Texas, Austin. "It serves as an internal Band-Aid that helps to physically guide the regenerating neurons and provide biochemical cues to the neurons so that they can regrow and find their muscle targets again," she says.

But nerve grafts are invasive, requiring two surgeries, and they cause some sensory impairment. As an alternative, a number of groups are developing materials that can serve as a regrowth scaffold. Collagen-based grafts are currently on the market, but these can provide regeneration only over short distances and are mainly used for small injuries, Schmidt explains.

Strategies involving biologically compatible polymers aim to cover larger distances by recapitulating the nerve structure and/or providing biochemical or physical cues that nurture regrowth. "We're trying to develop materials that will in a sense mimic the nerve as best as possible or enhance the nerve's ability to regenerate," says Schmidt.

EARLY SUCCESSES

These approaches have met with some success in small-animal models. "Across a 10-mm gap, we can match the performance of an autograft in a rat model, and we are now working on extending that to longer nerve gaps," says Bellamkonda, who incorporates laminin and NGF in his synthetic conduits. He notes that clinical applications will require the conduits to bridge 25–80 mm gaps.

Among the candidates for nerve-regrowth materials are poly(lactic-co-glycolic acid) (PLGA), a material used in biodegradable sutures, and polyhydroxybutyrate (PHB), a polymer used for myocardial patching. Tissue engineer Giorgio Terenghi of the Blond McIndoe Centre, Manchester, UK, who employs PHB as a scaffold, notes that PHB fibers align in the direction of nerve regeneration. "So not only does this material offer protection, but it also offers guidance. It's like the fiber runs along a track to reach from one end to the other of the injured nerve," says Terenghi.

Schmidt's variant of the standard synthetic scaffold goes even further. Noting that electric fields have been shown to enhance the regeneration of such tissues as bile duct, skin, and nerve, Schmidt's team builds its materials from electrically conductive polymer, thus providing both a physical pathway for nerve regeneration and a growth-enhancing electrical signal. The material has been further refined to include hyaluronic acid, a sugar molecule that increases the blood vessel supply needed for wound healing.

Other groups design conduits containing directional information. Biomedical engineer Bob Tranquillo at the University of Minnesota, Minneapolis, uses a magnetic field to direct the growth of biopolymer fibers along the axis of the tube. " As [axons] grow out, they are able to perceive the aligned fibers in the axial direction of the tube, ane that serves to guide them more efficiently from the proximal to the distal nerve stump," says Tranquillo. The fibers can be used to entrap small beads containing nerve growth factor to further stimulate axonal growth. "It's the one-two punch; we both stimulate them to elongate and as they're elongating we guide them with the aligned fibers," Tranquillo explains.

Tranquillo has not yet tested the bead strategy in animal models and notes that the current design releases growth factor simultaneously from all the particles entrapped in the tube. "All the axons of course start at one end. If the beads at the other end are already releasing the factor, it may well diffuse out of the tube and have no benefit in terms of stimulating the axons that are all the way at the other end," Tranquillo explains. His lab is developing methods for spatially controlled release to overcome this problem.

Surya Mallapragada, a chemical engineer at Iowa State University in Ames, inserts a thin biodegradable film etched with micropatterned grooves containing extracellular matrix proteins and Schwann cells into nerve guidance conduits to help align, direct, and accelerate axonal growth. In designing the patterns, Mallapragada's group takes its cues from nature. "We did lots of in vitro studies to see what sort of pattern sizes and dimensions would work best in aligning both the Schwann cells and the neurons. And it turns out that they prefer groove widths about the size of the cells," or around 10 μm, she explains. In studies of rats with sciatic nerve injury, Mallapragada found that her micropatterned conduits elicited functional recovery faster than empty tubes. "We see significant improvement in the sciatic function index at six weeks, compared to just using conduits without the micropatterning," she says.

Artificial scaffolds mimic the actual physical structure of nerves as closely as possible. Schmidt explains that this is a difficult task and that a better approach might be to improve what already works, by using natural nerve tissue. To this end, Schmidt and colleagues take tissue from donors (cadavers or animals) and try to eliminate chemically those components that give rise to immune response and rejection. "We've been able to take out the cells that cause the immune response and leave behind the physical structure," says Schmidt. In the future she plans to test whether adding components back to the graft – for example, the patient's own Schwann cells – will further enhance regeneration.

CREATING THE RIGHT ENVIRONMENT

While evidence exists that damaged tracts in the spinal cord can branch out and regrow after injury, functional recovery does not occur; after the initial trauma, secondary damage caused by inflammation and scarring occurs in the tissue surrounding the injury site. This secondary damage ultimately forms a barrier, or inhibitive environment, to regrowth. Complicating matters, healthy nerve tissue in the area surrounding the injury site will eventually atrophy and die.

Strategies to mitigate spinal cord injury thus focus on halting secondary damage to the injured site (including scarring), preventing further atrophy of healthy tissue surrounding the injury, reconnecting the surviving axons across the injured site, and ensuring they reach the appropriate target.

All these strategies rely on an understanding of how neurons respond to the spinal cord injury environment. "In my group we want to take a step back and try to sort out a bit more how the neuron takes in that combination of inhibitory and permissive information," says biomedical engineer Diane Hoffman-Kim of Brown University.

Hoffman-Kim's group uses micropatterning techniques to create polymer molds in which cells can be injected and positioned, generating in vitro models of neurons interacting with permissive and inhibitory glial cells or molecular factors. "We're really trying to understand in a quantitative manner ... how much permissive information needs to be there in order for neurons to overcome the inhibition that they're faced with," she explains. Using chromophore-assisted laser inactivation, Hoffman-Kim can transiently inactivate the function of a protein in a growing neuron or glial cell to further probe the cell's response to changes in its environment.

MAKING STRIDES

<p>NERVE MIMIC:</p>

©2004 Mayo Clinic

A trellis-like, biodegradable polymer scaffold that is designed to encourage nerve cells to grow in a predetermined direction after injury. The scaffold is engineered to anchor nerve cells, deliver drugs that promote nerve regeneration, and dissolve after a predetermined time to make room for more nerve growth.

Though CNS repair remains elusive, some groups are making progress. The tissue engineering research team at the Mayo Clinic, headed by Windebank and orthopedic tissue engineer Mike Yaszemski, has used computer-aided design and microfabrication techniques to construct PLGA scaffolds containing parallel channels loaded with Schwann cells. They found that axons grow successfully through these tubes when implanted in severed rat spinal cord.

Materials scientist Samuel Stupp's group at Northwestern University, Evanston, Ill., developed a self-assembling bioactive scaffold that can direct the differentiation of neural progenitor cells into neurons rather than a combination of neurons and astrocytes. (Astrocytes synthesize the glial scar that blocks regrowth after spinal cord injury.) The material is composed of peptide amphiphile molecules in solution that form an ordered gel-like substance in the presence of cultured neural progenitor cells.

"When cells are added to this liquid in their own culture medium, the electrolytes in the culture medium cause the molecules to assemble into nanofibers," Stupp explains. The surface of the resulting scaffold contains, in high density, a five-amino-acid-long laminin epitope that has been shown to promote neuron growth. "We have an unprecedented differentiation response in neural stem cells," says Stupp, who points out that most studies report mixed-cell populations.

Stem cell-based approaches such as Stupp's are gaining momentum in neural tissue engineering. Georgia Tech biomedical engineer Michelle LaPlaca develops injectable methylcellulose-laminin hydrogels that serve as a scaffold for neural stem cells. The gels are designed to be minimally invasive. "In traumatic injury and other acute [CNS] injuries, you often have very irregularly shaped defects in the tissue, whether it be a cyst in the spinal cord or in an infarct in the brain. A minimally invasive gel would let you fill the space without opening up the brain or the spinal cord," says LaPlaca.

Polymer chemist Molly Shoichet of the University of Toronto has achieved some success treating spinal cord injury in rats using porous tubes filled with growth factor-containing matrices. In model studies, her laboratory has shown that axons can be guided through scaffolds modified with immobilized concentration gradients of growth factors. "Scaffolds are like a mesh or a fence that gives the cells something to grow along. By creating a gradient – you can imagine just like a slope of growth factors – the cells are guided, similar to what is observed in development," and grow along the scaffold in a spatially-directed manner. Similar guidance has recently been observed in three-dimensional biochemical adhesive channels separated by nonadhesive regions.

Another strategy pursued by the Shoichet lab involves a polymer matrix that gels upon injection and delivers drugs directly to the injured site; it is a less invasive strategy intended for use when the spinal cord is crushed but not severed. Shoichet has demonstrated safety of this new technique and has seen moderate functional improvement in rats using the delivery method. But so far, her lab has been able to mitigate only some cell damage and promote growth of neural progenitor cells. "We have further to go in terms of promoting regeneration and seeing functional improvements," says Shoichet.

As in the peripheral nervous system, a major problem with CNS drug delivery is controlling spatial and temporal patterning. Yale University biomedical engineer Erin Lavik explains that growth factors are upregulated in the neonatal spinal cord and non-neural tissue after injury. "But it doesn't just all happen at once. It happens over time and in different particular areas," she says.

Lavik's group seeds regenerative scaffolds with microspheres that degrade in the body over time, releasing growth factors at a rate that depends on the polymer makeup of the spheres. By placing specific beads at different points in the scaffold, the team can control spatial issues as well. Lavik's ultimate goal is to mimic the cascade of events leading to repair of the neonatal spinal cord, which heals itself to some degree after injury. "We need to figure out exactly which growth factors are important at which time and in which place," says Lavik. Whether a successful combination can be found remains to be seen, she adds. "People have been investigating spinal cord injury for an awfully long time, and there hasn't been a big breakthrough."

Nevertheless, Bellamkonda remains optimistic. "Neural tissue engineering in a way is a new part of tissue engineering," he says. "If you look at biomaterials used clinically, if you look at orthopedic or cardiovascular, there are a lot of biomaterials that are already used. In the brain, there are not as many and there aren't as many labs doing it. That's the challenge and the opportunity, I think."

Aileen Constans can be contacted at aconstans@the-scientist.com.

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