Neural Tissue Engineering, by Jonathan Hummel
Nerve Tissue Damage
Damage to nervous tissue in both the central (CNS) and peripheral (PNS) nervous system arises from a wide variety of causes. Direct mechanical trauma, inadequate blood supply, or failure of the cellular machinery of the neurons themselves can result in the severing of axons, damage of neuron cell bodies, or disruption of existing synapses. While all three types of nerve tissue damage are interconnected, the repair of severed axons has historically received the most study, largely due to the fact that the prospects of recovery are much better in the absence of extensive cell body damage or synapse obstruction by aggregates. Regardless of the initial cause, the response to axon damage by the local cells in neural tissues differs dramatically between the CNS and PNS, resulting in different challenges when developing treatments. Glial cells, or more specifically Schwann cells, are the cells responsible for forming the myelin sheath that surrounds, protects, and nourishes the axons of neurons. Shortly after the disruption of an axon in the PNS, macrophages are attracted to the area to swiftly remove the cellular debris of the Schwann cells, as well as any debris from the damaged axon itself. Damage of the axon triggers the cell body associated with that axon to express a variety of genes associated with the production of axon components for elongation. Surviving Schwann cells in the area proliferate and reorganize into a linear supporting structure. They also release growth factors and proteins that guide the direction in which the growth cone of the extending axon moves. This ensures that the extending axon moves toward the appropriate target neuron or tissue to repair communication. In the CNS, the response to the same axon damage results in a different course of events that are much less favorable for tissue repair. The cellular debris are not cleaned up nearly as rapidly as they are in the PNS and persist for weeks, rather than days, which poses obstacles for axon extension and reformation of myelin structures. On top of that, expression of genes associated with axon repair rarely occurs in CNS neurons the way it does in the PNS. When axon extension is initiated by a CNS neuron, proper function of the growth cone is inhibited by factors released by oligodendrocytes, the CNS equivalent of the Schwann cells. Another type of glial cell, called astrocytes, release other factors in response to axon damage that inhibits the extension of the axon. Thus, in the PNS there are mechanisms that actively promote the repair of damaged neural tissues, while in the CNS there are mechanisms in place to actively prevent the repair of neural tissues. The goal of neural tissue engineering is to restore lost neural tissue function by using engineered cells and/or materials. The strategies behind tissue engineering in CNS and PNS tissues are thus very different, and as one can imagine, there has been much more success in the PNS because preexisting repair mechanisms simply need to be aided. It should also be mentioned that other sources of nerve tissue damage, such as degenerative diseases like Alzheimer's, do not result in direct axon damage but a buildup of proteins and aggregates that impede proper neural function. Thus, approaches to treatment of these malfunctions are very different and further understanding of these disorders is required.
Economic and Societal Impact
The exact costs associated with nerve tissue damage are difficult to estimate, largely due to the fact that there are such a wide variety of neurological disorders and traumas, some of which are not fully understood or are under the realm of psychology and/or psychiatry. That being said, the costs both economically and emotionally that stem from nerve tissue damage are surely noteworthy. To get a sense for the economic impacts, one can look at the costs associated with any individual health issue related to nerve tissue damage or malfunction. For example, the overall annual cost of traumatic brain injuries in the United States is estimated to be $48.3 billion and the lifetime cost of caring for a traumatic brain injury survivor is estimated to be between $0.6 and $1.9 million. The costs associated with degenerative diseases are even larger, with a projected $226 billion to be spent due to Alzheimer's in 2015. Especially in the case of nerve tissue damage and disease, the emotional and quality of life impacts are just as important as the economic effects. Damage and degeneration of nerve tissues in the PNS often result in pain, loss of feeling, and loss of control or function in the affected parts of the body. These symptoms result in varying degrees of disability and loss of productivity in those affected, not to mention general discomfort. In nerve tissue damage and degeneration that affects the CNS, the greatest losses are often in cognition and the ability to normally interact with family or friends. In the case of CNS degeneration, even before the more grave effects set in, there is often a level of fear and pain associated with the prospect of losing cognition or control, which also cannot be overlooked. What is often most discouraging for those affected by neurological damage or degeneration is the lack of effective treatment to solve or combat these problems. This is largely due to a massive knowledge gap with regards to the nervous system and the specific causes of disorders and modes of repair. While the field of neural tissue engineering is currently only able to address simple PNS damage, further study of tissue engineering in both the PNS and CNS could someday lead to solutions to even the most complex and detrimental neurological degenerative diseases.
Methods of Neural Tissue Repair
As described earlier, the mechanisms of response to damage in the CNS and PNS are dramatically different and thus the challenges are different when designing repair methods in each part of the nervous system. While there is a long way to go in expanding the treatment options for both the CNS and PNS, there are currently many more methods of repair that have been developed to treat damage in the PNS:
In the Peripheral Nervous System
Historically, the main way to treat damage of nerves in the PNS has been via various forms of nerve connecting surgery. These procedures started out with the direct suturing of larger disconnected nerves over short distances. In the mid 20th century, the concept of nerve tissue grafting was introduced which greatly improved the recovery process, largely due to the fact that direct suturing causes tensions in nerve tissues that stunt the healing process. Today, serious PNS damage is treated primarily with nerve tissue grafts and this method is generally accepted as the most reliable. This is sure to change, however, as improvements in nerve grafting have stagnated and the technique does not work in many situations (i.e. certain sized nerves or over certain distances). In the early 2000's and on there has been an explosion of research into the use of decellularized and artificial nerve conduits to guide nerve tissue regeneration. Many of these engineered nerve conduits have shown increased or equal efficacy to traditional nerve grafts and some have even gained FDA approval; however their use in patients is still not widespread.
While direct suturing of severed nerves is still used in cases where the nerve bundle is sufficiently large, the distance to span is sufficiently small, and tension enforced on the bundle is low, the most common method used is currently grafting. Nerve grafting involves taking a nerve from another source that matches the damaged nerve in terms of size and structure and stitching the appropriate ends together to bridge the gap. While one outcome of a nerve graft is that the transplanted nerve provides a functional rout of neural communication between severed ends, the most important outcome is that the transplanted nerve provides a guide for the axonal end of the damaged nerve to repair itself in the right direction. There are three main types of nerve grafts and each have their advantages and disadvantages:
- Autografts - These are nerve grafts using a transplant nerve taken from elsewhere within the patient being treated. This is the most commonly used type of nerve graft, the most positive feature being the low risk of immune response to the transplanted nerve. This comes at a price, however, as the site where the nerve was taken from loses nerve function locally, and two surgeries are needed which puts more of a load on the patient during recovery. In autografts, nerves are most often taken from locations where loss of function will be least problematic, such as sensory nerves just below the skin. The fact that nerves need to be taken from the patient with minimal loss of important function highly limits the size and type of nerve damage that autographs can successfully repair.
- Allografts - These are nerve grafts taken from another donor of the same species. While this type of graft has the advantage of taking a load off of the patient's body, it introduces a higher risk of immune response to the foreign tissue. There is also the issue that allografts, much like organ transplants, are highly limited by the amount of compatible donors available.
- Xenografts - These are grafts taken from a donor of another species. The major advantage of this method is that it eliminates some of the intrinsic limitations of the other two, such as the availability of donors or adequately sized transplant nerves. One study in the late 90's showed that xenografts to repair sciatic nerves in rabbits yielded recoveries as successful as those attained by allograft. The main limitation of this method is that as the donor tissue is less and less genetically similar to the target tissue, the risk of immune response increases. Unlike the other two methods, xenograft has not yet been used in human treatment 
The shortcomings of autografts, along with the fact that the potential gains of allografts and xenografts are overshadowed by immune response, lead researchers to begin exploring decellularized nerve conduits in the early to mid 2000's. To create the decellularized nerve conduits, allograft or xenograft nerve tissues with the appropriate features are exposed to conditions that lead to the destruction and removal of all cells and cellular debris. All that is ideally left after the decellularization process are the components of the extracellular matrix (ECM) and basal lamina from the original nerve tissue. There are many methods for deceullarizing tissues but some methods used on nerve tissues include lyophilization, exposure to high pressure, sonication, agitation, freeze-thaw, acid/base exposure, detergents, and exposure to hypertonic or hypotonic solutions. A successfully decellularized nerve tissue is use to treat damage in the PNS much the way a nerve graft is. The nerve conduit is surgically implanted across the site of severance and it aids the extending axon(s) in the right direction. The primary difference from nerve graft is that ECM components are the same in nerve conduits across species and thus the risk of immune response is dramatically lower. Also, the hollowness of the conduit and micro-environment provided by the ECM components of the decellularized tissue potentially provide better conditions for successful axon extension. The FDA has given approval to a brand of decellularized nerve tissue obtained from allograft called Avance®, which was designed by AxoGen Inc. Avance® has been compared to the performance of a prominent synthetic conduit called NeuraGen®, which is made from Type I Collagen, and was found to be just as effective after 12 weeks. In 2009, it was also tested in clinical trials in the digital and dorsal sensory nerves of 5 men and women, resulting in restored sensation after 9 months. Positive results using decellularized conduits seeded with stem cells have also been shown, which are discussed under the stem cells section below.
The area of neural tissue engineering that has expanded the most in the last decade has been the development of nerve conduits from either biologically derived or artificially synthesized materials. Manufactured nerve conduits eliminate the risk of tissue death or rejection around the graft site, which is always a potential problem with both normal and decellularized grafts. General themes of nerve conduit design can be seen in the diagram to the left, where a variety of physical structures and the use of seeded cells, growth factors, and neurotrophic factors are all options to consider. Regardless of the materials used or components seeded onto the conduit, the goal is to design a conduit that has is biocompatible, biodegradable, flexible, highly porous, neuroinductivite, neuroconductivite on the inner surfaces, mechanically resilient, and complies with FDA standards. As can be seen, these nerve conduits are surgically implanted into the area of damage much the way nerve grafts are. All of the following are materials that have been used in artificial nerve conduits:
- P3HB - this is a type of biological polyester from bacteria that has been studied for use in nerve conduits for over two decades.
- PHBV - another type of biological polyester that is commonly used in nerve conduit research.
- Collagen - a family of proteins that make up much of the ECM. Particularly, Type I Collagen has been used extensively in nerve conduits both alone and in conjunction with many other materials. Several collagen-based nerve conduits have been given FDA approval, one of which is NeuraGen®, mentioned earlier.
- Gelatin - this is collagen that has been denatured and cross-linked with other chemicals. It has been successfully used as a conduit material in nerve repair.
- Fibronectin - another major component of the ECM that is extremely important for cell adhesion, which in-turn effects migration, differentiation, and morphology. Fibronectin has been successfully used in many cell conduit designs, primarily to guide the direction of cell growth within the conduit.
- Keratin - a type of protein produced by skin cells that has been used to fill the insides of nerve conduits, creating a complex scaffold network.
- Silk fibroin - a protein polymer produced by Arachnids that has been used as a successful conduit material. The major strength of using silk is the reduced occurrence of immune response and the resilience of the material.
- Laminin - another type of ECM protein that has often been used to coat various forms of collagen, which are then used to fill or line a conduit formed by a synthetic material.
- Chitosan - a linear polysaccharide that comes from the exoskeletons of insects and crustaceans that has been used alone and in combination with synthetic polymers to create conduits that are highly porous and exhibit good mechanical resilience.
- Hyaluronic acid - a polysaccharide that is found in the human body and does not activate any immune response. It has been used clinically over the last three decades and more recently has been used to make hydrogels and electrospun fibers for use as nerve conduit material.
- PLA - polylactic acid is a type of synthetic polyester. Several types of PLAs have been used to make effective conduits in conjunction with biologically derived materials.
- PGA - polyglycolic acid is another type of polyester used in conduits which is notable for its mechanical durability and biodegradability. The first biodegradable synthetic conduit design to gain FDA approval was made using PGA.
- PLGA - a copolyester that is notable for the fact that it does not induce much inflammatory response and is easy to use for fabrication. It has FDA approval and has been used in many nerve conduit schemes.
- PCL - polycaprolacetone is another polyester that has been tested extensively as a nerve conduit material and has often seeded with stem cells and neural growth factors.
- PU - polyurethane is a polymer that is frequently used in medical devices and has shown promising results when used to make conduits.
- PVA - polyvinyl alcohol is a polymer that has been shown to be a successful conduit material, especially when combined with chitosan. The fact that it is not biodegradable makes it weaker than other synthetic or biological candidates.
It should be noted that many, if not all, of the materials listed above have been combined and hybridized in a diverse array of schemes yielding successful nerve conduits. While some conduits are entirely made of ECM components like collagen, others are made of synthetic polymers and incorporate ECM components to yield more compatible environments for seeded or recovering cells. There are also many instances where biological polymers are chemically linked and combined with synthetic ones for further gains in cell compatibility. Virtually all types of conduits, regardless of the base material, have been either infused with neural growth factors or seeded with Schwann cells to improve the axon growth and guidance process. Various types of stem cells, such as mesenchymal stem cells, bone marrow stem cells, and adipose derived stem cells, have also been seeded into nerve conduits with successful results.
The Role of Stem Cells
One of the greatest weaknesses of the grafts and conduits discussed thus far is that all of these methods are not able to treat nerve damage that is much longer than 2cm. As was discussed previously, the use of various types of stem cells has been shown to greatly improve the performance of these treatments. Another line of research, which parallels nerve conduit design, attempts to produce a better understanding of stem cells and their potential role in repair. This could someday lead to the use of stem cells to dramatically improve the length of injury that grafts and conduits can effectively treat. There has been extensive research into the use of embryonic stem cells (ESC), neural stem cells (NSC), induced pluripotent stem cells (iPSC), and adult mesenchymal stem cells (MSC) in cell therapies to treat nerve damage. ESCs have been shown to differentiate into the Schwann cells and aid in reformation of the myelin sheath in mouse models. It has been shown that NSCs can be genetically engineered to overexpress a neural growth factor called GDNF to speed up nerve regeneration when seeded into conduits. Another study has shown that iPSCs can be used to generate neural crest cells, which are multipotent stem cells that can differentiate into key nerve tissue cells like glia. The use of MSCs has been the most widely accepted due to lowered ethical concerns and less risk of undesirable differentiation or teratoma formation. Bone marrow derived MSCs in particular have been shown to differentiate into Schwann-like cells that aid in nerve regeneration. Adipose derived MSCs have also been shown to differentiate into Schwann-like cells and are more practical to obtain as no marrow harvesting procedure is needed. Stem cells are already being applied to nerve conduit designs, but further research on the role of various types of stem cells in nerve regeneration is needed before their full potential can be reached.
In the Central Nervous System
Overall, there has been far less success in developing methods of repair to treat damage in the CNS. As was mentioned in earlier sections, this is largely due to the fact that the local response to damage differs greatly between the CNS and PNS. In the PNS, there are a myriad of repair mechanisms and signals in place that contribute to axon extension and reformation of the myelin sheath, as well as other neural connective tissues. In the CNS, these repair mechanisms still exist in the genomes of the local neurons and glial cells, however, the local glial cells also express an array of factors that inhibit repair activity. This response to damage in the CNS has long caused frustration among biologists and neuroscientists, because in some ways the CNS is normally dynamic in its ability to change neural connections (aka neural plasticity). Some believe that this type of response favors stability over the risk of potentially losing information in an overly dynamic CNS. Regardless of the purpose for the CNS inhibitory pathways, recent work has begun to hone in on the specific mechanisms by which various glial cells of the CNS, such as oligodendrocytes and astrocytes, prevent axon extension. As a result of this research, better understanding has been gained of how these glial cells compromise the integrity of the axon growth cone and withdraw the structural and chemical support that they normally supply to axons. On top of that, it has been shown that both injection of macrophages to the damaged area and inhibition of the receptors for Neurite Growth Inhibitory Factors, such as Nogo-A, result in much better axon sprouting and extension. This is not entirely surprising, and it has been hypothesized for some time that to see better healing the CNS, PNS-like conditions need to be forced locally. It is not until very recently, however, that understanding of the CNS has reached a point that allows researchers to successfully counteract the mechanisms that inhibit repair in the CNS.
Neural tissue engineering is one of the sub-fields of tissue engineering that will require the most work before widespread benefits will be felt by the general population. While various forms of nerve graft have been successfully used on patients for decades, and a plethora of nerve guiding conduits are being designed, these methods can still only effectively treat injuries that are around 2cm or less in length. Thus, the primary goal of neural tissue engineering in the PNS will be to increase the maximum treatment length by finding the optimal combinations materials, growth factors, and stem cells for axon extension and myelination. The primary goal in repairing CNS damage will be to further fill in gaps in knowledge in order to develop ways to efficiently work against the inhibitory mechanisms in place. Better understanding is also needed of the wide variety of neurological degenerative diseases that exist, as many of these disorders cause different types of damage than those currently addressed by tissue engineering.
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