Muestra N° 01 (En relación a la Población N° 01)

Peripheral nerve injuries can be broadly classifi ed into two categories, large gaps (greater than 4 mm) and small gaps (less than 4 mm).^28,29 Th ere are several treatment options that currently exist, with some of them showing great promise in the clinic trails. For the regeneration of peripheral nerve injuries, the gold standard is an autograft , i n w hich t he i njured ner ve gap i s fi lled w ith a pat ient’s own ner ve.²⁹ However, the autograft is adopted only when coaptation (approximation and suture of the 2 ends) of the nerve is not possible owing to t he length of the gap c reated by the injury.³⁰ Specifi cally, in the case of large nerve defects, an autograft is usually extracted from the sural nerve, located near the ankle, and is placed at the site of injury. However, in cases of trauma or tumor, where large nerve sections and several nerve sections are required, an autograft was not suffi cient to repair all the injuries.

In most cases, the scaff old is a g uidance approach that allows for guiding severed axons across the gap. Th is is the most vital component in the regeneration of nerve injuries. A few decades ago, several guidance st rategies were attempted to r epair la rge nerve defects.³¹ One of t he earliest approaches to guide axons across nerve injuries was the application of tube-like structures usually made of autologous tissues, a f at sheath,³² tendons,³³ veins,^34,35 or sub-mucosa³⁶ in most cases. However, inherent to these techniques was the fact that these nerve graft s did not m imic the native nerve microstructure of the actual ner ve. Also, optimal growth factors and ECM proteins that are needed for guiding the a xons across the ends were not suffi cient.³⁷

In o rder to a lleviate d rawbacks a ssociated w ith t he a utograft s, s everal re searchers h ave adopte d allograft -based techniques to repair and regenerate large nerve defects.³⁸ However, the allograft s possess

the risk of disease transmission, unfavorable immune response, and more importantly lack of donors.

In order to m itigate drawbacks associated with these techniques, several tissue engineering strategies were adopted to study the regeneration process in the nervous system.

Tissue engineering is composed of three major components: the scaff olds, proteins (in the form of growth factors and surface tethered ECM proteins), and most importantly the cells, which are composed of the glial support cells and the main neurons in the case of nerve tissue engineering. Th e se critical parameters for peripheral nerve regeneration are summarized in Figure 10.1 and are discussed below in detail.

10.2.1 Optimal Physical Properties of Scaffolds

Th e conventional approach in the fabrication of functional tissue engineered graft s is creation of biode-gradable graft s with optimal physical properties, that support and promote axonal growth from the prox-imal end to the distal ends, and cause functional recovery. Porosity plays a major role in the regeneration process. First, it allows the migration of cells, specifi cally the glial cells, and more importantly the neu-ral processes or axon growth across the scaff old. Second, porosity allows medium or blood vessel infl ux into the scaff old. Th e blood circulation is a vital source of nutrients for the survival of the nerves as well as a major source of debris clearance when the scar tissues, polymer graft , and sutures are cleared from the site of injury.³⁹ However, the porosity, degradability, and mechanical properties are all interrelated and changes in one of the parameters alternatively aff ect the other.

Mechanical properties play a v ital role i n the success of the peripheral nerve regeneration process.

Ideally, the engineered graft s should match a nat ive acellular nerve graft , which has to b e a f actor of consideration, during scaff old design and material selection. An acellular nerve graft has tensile stress of about 0.2–0.5 MPa and a modulus of 1–5 MPa.⁴⁰

Physiological properties Scaffolds

• Biodegradable • Nonbiodegradable • Natural polymers

• Biodegradability

• Bone marrow stromal cells (BMSCs)

FIGURE 1 0.1 Critical p arameters for ne rve re generation: s caff olds, ph ysiological prop erties, c ells, g rowth factors, and extracellular matrix proteins.

10.2.2 Materials for Scaffold Fabrication

Many biodegradable and biocompatible materials have been developed and tested for applications in the regeneration o f t he p eripheral ner ve i njuries. Th ere h ave b een s everal nat ural m aterials a s w ell a s synthetic materials that have been applied for fi lling the nerve guidance channel (NGC) as well as fabri-cation of the whole graft by various techniques. Some of the most common natural materials that have been used for nerve tissue engineering include collagen (described above),^41,42 chitosan,^43,44 alginate,⁴⁵ dextran,⁴⁶ ge latin,⁴⁷ fi brin,⁴⁸ a nd fi bronectin.⁴⁹ Th ese m aterials p ossess s everal p roperties t hat m ake them attractive for use as nerve graft s, especially in fi lling guidance channels. Th ey form a hydrogel-like matrix, which mimics the soft -tissue architecture of the nerve, and a l arge amount of critical growth factors such as NGF, neurophin-3 (NT-3), or BDNF can be encapsulated into the systems. Th ere are also some nonbiodegradable systems that have been used extensively for nerve tissue engineering. Agarose is one of the most common nonbiodegradable hydrogel that has been evaluated for PNS and CNS regen-eration.⁵⁰ Another versatile platform, which is biocompatible but nonbiodegradable is poly(hydroxyethyl methacrylate) (PHEMA). PHEMA has shown great promise as a substitute for regeneration.⁵¹

However, inherent to the hydrogel-based system is the fact that it is mechanically very weak and therefore in most cases needs a housing system like the silicone tube. Th erefore, several synthetic materials have been de veloped t hat have ideal mechanical properties, degradation r ates a s well a s do not need an external system, like silicone to form the NGCs. Some of the most common materials used f or t he f abrication o f ner ve g raft s i nclude p olycaprolactone ( PCL),⁵² po ly la ctide ( PLLA),⁵³ polyglycolide (PGA), and their copolymers.⁵⁴ Th ese materials have a stable degradation period in vivo and produce by- products t hat c an b e e asily c leared by s ystem. A lso, by c hanging t he r atio of t he polymers (PGA and PLLA) the degradation rates can be precisely controlled. Th ere are several other materials that have been used for the fabrication of scaff olds for tissue engineering, including electro-conductive materials such as polypyrrole (ppy).^55,56 Several review articles^7,57,58 have dwelled on the materials a spect o f ner ve t issue eng ineering, a nd w ould g ive a mo re de tailed p erspective o n t he materials used.

10.2.3 Protein Factors for Regeneration 10.2.3.1 Extracellular Matrix Proteins

During the development and repair of the nervous system, glial cells, especially Schwann cells, play a key role.⁵⁹ Th ey have been regarded as a potential guide towards the regeneration process, and have been attributed to the success of autograft s as compared to other NGCs.⁶⁰ Th ey provide vital molecules for the regeneration process, either as diff usible cues (growth factors) or as substrate bound proteins (ECM proteins), such as collagen, fi bronectin, and laminin.⁶¹ Th ey are also a major source of signaling mole-cules that enhance the migration of Schwann cells into the injury site, macrophages for debris clearance, and more importantly axons towards the proximal end.

Collagen: Collagen is the most prominent protein in the PNS and the CNS. Collagen exists in the form of nanofi bers that are oriented in the scaff olds and aid in the guidance of the cells and axons across the nerve. Th ey also have several motifs that allow for cell surface interactions and also house several growth factors that are produced by the cells.⁶²

Laminin: Th e second most prominent ECM protein in the nervous system is laminin. Th ey are usu-ally present in the basement membrane of cells and play vital roles in cell migration, adhesion, prolifera-tion, d iff erentiaprolifera-tion, a nd neu rite o utgrowth i n t he ner vous s ystem d uring de velopment a nd regeneration.^63,64 Th ere is considerable evidence t hat laminin is a v ital component i n t he embryonic development stages as well during the process of regeneration. Laminin at optimal concentrations pro-motes axonal and glial cell attachment, migration, diff erentiation, survival, and overall development.

However, at larger concentrations, it tends to aff ect the normal development processes and aid in the formation of glial scars that might retard neurite growth.⁶⁵

Other surface bound proteins that are vital to t he regeneration processes are, fi bronectin, which is over-expressed immediately postinjury and plays a major role in the regeneration and wound healing process; NCAM and N-cadherin, have signifi cant impact in the signaling processes.⁶⁶

10.2.3.2 Neurotrophic Factors

Neurotrophic f actors, a lso k nown a s g rowth f actors, a re s oluble a gents f ound i n t he e xtracellular fl uid surrounding most cells and account for diff erentiation, proliferation, gene expression, proper net-working, as well as survival of neurons.^67,68 Th ere are several neurotrophic factors whose eff ects have been extensively studied in neural tissue regeneration; NGF, BDNF, CNTF, glial cell line-derived growth factor (GDNF), NT-3, neurophin-4/5 (NT-4/5), acidic and basic fi broblast growth factor (aFGF, bFGF) are all important neurotrophic factors.^69,70 Local delivery of growth factors through use of nanoparti-cles, scaff olds, or gels is highly desirable in order to c ompete with the inhibitory agents present aft er trauma to the nervous system. Th e following part gives a more detailed insight into the critical growth factors that aff ect the nerve regeneration process.

NGF: Amongst all the neurotrophic factors, NGF is possibly the best-characterized growth factor as it has shown i mportance i n peripheral ner ve regeneration. NGF i s a d imeric (27 kDa) neu rotrophic factor that is produced in the target organs of the sympathetic and sensory nerves.⁷¹ Normally, sympa-thetic a nd sensory ner ves contain a lo w concentration of NGF as compared to t heir target organs.²⁴ However, following axotomy, Schwann cells in the distal stump start producing NGF, which has been shown to stimulate and promote the survival of sensory ganglia and nerves.^72,73 Two distinct receptors for NGF exist, including a h igh affi nity trkA receptor and a lo w affi nity p75 receptor.⁷⁴ Activation of NGF receptors initiates a cascade of signaling events and leads to neuronal diff erentiation.⁷⁵ Although NGF has shown success in the growth and development of sensory neurons, its eff ects are limited with respect to moto r neuron regeneration.^76–80 Nonetheless, the delivery of NGF to neuronal injuries has been studied extensively.^81,82

BDNF: BDNF is another neurotrophic factor that promotes the axonal growth of sensory neurons.⁸³ Additionally, BDNF supports survival a nd a xonal g rowth of motor neurons.^84,85 Although research investigating the eff ects of BDNF on nerve regeneration has led to some inconclusive results in the PNS, it has been noted that BDNF must be delivered locally at high concentrations to have an eff ect on nerve regeneration.^78,86

NT-3 and NT-4/5: Similar to BDNF, NT-3 promotes motor neurons survival and outgrowth, as well as sensory nerve axonal growth.^86,87 It has shown promising results in vivo with respect to the regeneration of peripheral nerves.^88,89 NT-4/5 can promote the survival of motor neurons^86,90 and sensory neurons.⁹¹ Th is factor aids in growth of sensory axons from dorsal root ganglia into the spinal cord,^92,93 and also promotes axonal outgrowth in motor neurons^86,91 and the regeneration of peripheral nerves.⁹⁴

CNTF: Similar to t he other growth factors, CNTF accounts for survival and outgrowth of motor neurons.^95–97 Its increased mRNA levels near the spinal cord injury indicate that CNTF might be involved in a healing response mechanism. It has also shown promise in the regeneration of periph-eral nerves⁹⁸ and spinal cord. However, some literature indicates that the introduction of CNTF into spinal c ord i njuries le ads to g lial s carring, w hich i nhibits t he re generation process i n s pinal c ord injuries.⁹⁹

GDNF: GDNF supports sensory,^100,101 autonomic,¹⁰² and motor¹⁰³ neurons’ survival, it has been shown as most eff ective survival and tropic factor for motor neurons.^86,104,105 It can also increase peripheral nerve re generation.^106–108 It s e ff ects o n s ensory neu ral g rowth a re g reater t han t hose o f N GF a nd NT-3.^93,94

aFGF a nd b FGF: aFGF and bFGF have been shown to increase the regeneration of peripheral nerve.^109,110 Like several other neurotrophic factors, these fi broblast growth factors signifi cantly increase angiogenesis levels which in turn helps heal injury to a nerve.¹¹¹ bFGF has also been linked to increased sensory neural outgrowth. However, FGF has limited eff ectiveness at regenerating axons over a prolonged period of time.¹¹²

In summary, neurotrophic factors encourage a variety of neural responses that include the survival of motor and sensory nerves in the PNS amongst others. Due to diff erent methods of delivering growth factors, in vivo responses are not always consistent. As a result, the development of highly controllable delivery devices is required and currently part of ongoing research.

10.3 Nanotechnology Approaches to Peripheral