Artificial Scaffolds for Neural Tissue Engineering

An alternative approach to nerve autotransplantation is the entubulation of nerve clefts using nerve conduits or nerve guides made of synthetic or natural polymers. These studies indicate that the effectiveness of silk fibroin as a biomaterial to facilitate nerve regeneration can be increased by incorporating electrical signals into the nerve channels. The fabrication of novel electroactive composite scaffolds using nanoparticles and conducting polymers for the development of nerve tracts and subsequent in vivo evaluation of neuro-regeneration is also highly desirable.

Review of Literature

Chapter 3: Silk-Gold nanocomposite based scaffolds for neural tissue engineering

The challenges in the repair of nerve injury at the neurotmesis level and the potential of neural tissue engineering strategies in its treatment are discussed. The chapter focuses on explaining the intended problem, developing the rationale behind the thesis and formulating objectives for the research to address some of the existing gaps in the field. The chapter is further divided into separate sections describing the introduction, materials and methods, results, discussions, and conclusions.

Chapter 4: Silk-Polyaniline nanocomposite based scaffolds for neural tissue engineering

Summary and Future prospects

Peripheral Nerve Injury

Physiological events post nerve injury

Such degenerative processes occur after damage to the degree of axonotmesis and neurotmesis and are called Wallerian degeneration. Glial cells of the PNS, i.e. Schwann cells, play a major role in Wallerian degeneration, as well as in axon regeneration. During the late stages of Wallerian degeneration, stacks of Schwann cells form columns called Bands of Bungner, which act as guides for axon sprouting.

Peripheral Nerve Repair Strategies

Conventional methods of nerve repair
Alternative surgical and therapeutic strategies
Enhancing axonal regeneration
Entubulation strategies for nerve repair
Glue based repair of nerve
Delaying onset of Wallerian degeneration
Minimising muscle denervation post nerve injury

Axonal regeneration can be enhanced by the direct application of neurotrophins or growth factors (NGF, GDNF, FGF etc.) to the proximal nerve stump after injury or in combination with nerve conduits (Grinsell et al, 2014; Lee et al, 2000; Konofaos et al .al, 2013). The PTB binding is achieved using a Nd/YAG laser, photoactive dye and a non-immunogenic amnion sheath (O'Neill et al, 2009; O'Neill AC et al, 2009; Johnson et al, 2007; Henry et al. , 2009) in which the collagen present in the amnion covalently binds with the neural epineurium. In vivo studies with such PTB-bound sheaths reported improved axon counts and gait function when tested in rat sciatic nerve as well as rabbit peroneal nerve injury models (O'Neill et al, 2009; O'Neill AC et al, 2009; . Johnson et al, 2007; Henry et al, 2009).

Neural Tissue Engineering and Artificial Nerve Conduits

An ideal conduit
Neural regeneration through a nerve conduit

By combining synthetic and natural polymers with appropriate fabrication techniques, it is possible to fine-tune the flexibility, permeability, swelling, and biodegradation properties of a nerve conduit. Like all scaffolds used in tissue engineering, a neural canal should ideally remain intact until complete axonal regeneration within the gap. This fibrin cable later provides topographic guidance for Schwann cells (SC), endothelial cells and fibroblasts to migrate from the proximal and distal nerve trunks [Mukhatyar et al, 2009; Belkas et al, 2004].

Rationale of the study and Objectives

Long duration studies – Regeneration of nerves following severe neurotmesis grade injury with complete axonal loss and conduction failure is a highly complex, time
Using nanoparticles in Neural Tissue Engineering – There is at present much scope of using nanoparticles in fabricating scaffolds for neural tissue regeneration
Monitoring functional regeneration of muscle – Neurotmesis grade nerve injuries are not only associated with complete loss of a section of a nerve but also
Fabrication method – The human body contains a variety of peripheral nerves which differ from one another in morphology as well as functionality. Electrospining on
Objectives of the study

Electrospinning on a rotating mandrel, injection molding, freeze-drying, and other fabrication methods used so far to develop neural canals only allow the formation of a single canal of a given dimension at a time. Therefore, at present, there is an urgent need to develop fabrication methods for designing multiple neural channels of different dimensions at a single time. Three types of nerve channels were fabricated using silk fibroin nanofibers, silk-gold nanocomposite nanofibers, and silk-polyaniline composite nanofibers.

Introduction

Limitations of Neuroregeneration among mammals and potential of neural tissue engineering
Components of an artificial nerve conduit

As the nervous system matures after birth, myelination is finalized with oligodendrocytes ensheathing axons to prevent aberrant sprouting and astrocytes secreting chondroitin sulfate proteoglycans (CSPGs) to further limit structural plasticity in the adult. As similar mechanisms appear short. plasticity in the mature and long-distance axon repair after injury, alleviating these inhibitory influences may not only improve the regeneration of severed axons, but may also promote compensatory sprouting. However, the glial cells in the peripheral nervous system (PNS), ie the Schwann cells, play a much more constructive role in nerve regeneration than their CNS counterparts.

Scaffolds in Neural Tissue Engineering

Natural Polymers in Neural Tissue Engineering
Synthetic Polymers in Neural Tissue Engineering
Electrical stimulation and electrically conductive scaffolds in Neural Tissue Engineering

Several studies have shown that neural stem cells (NSCs) can develop on PLLA scaffold and this supports neurite outgrowth Vasita et al, 2006; . Tierney et al, 2009). Soft lithography and molding Wang DY et al, 2008 Braiding and molding Wang A et al, 2007 Chitosan/PLA. Collagen Dip coating and crosslinking Alluin O et al, 2009 Injection molding and crosslinking Chamberlain et al, 2000.

Support cells in Neural Tissue Engineering

Schwann cells/Glial cells and macrophages
Olfactory Ensheathing Cells (OEC)
Stem Cells
Genetically Modified Cells

Neural stem cells have been isolated from rodent brain (Kocsis et al, 2002) or spinal cord (Kalyani et al, 1998) and have shown promising results when cultured on electrospun scaffolds (Christopherson et al, 2009). Mesenchymal stem cells derived from bone marrow samples have been shown to differentiate into neural cells under appropriate induction factors. For example, fibroblasts have been engineered to produce NGF, BDNF, bFGF (Nakahara et al, 1996) and GDNF (Blesch et al, 2001).

Growth factors in Neural Tissue Engineering

However, the use of NGF is not without disadvantages: the application of exogenous NGF to spinal cord injuries has been associated with significant sprouting of uninjured sensory axons (Romero et al ,2001). This sprout has been linked to serious side effects, including chronic pain (Romero et al ,2001; . Priestley et al, 2002) and inappropriate neuronal reflexes (Romero et al ,2001). However, research examining the effects of BDNF on nerve regeneration has produced inconclusive results in both the PNS (Utley et al, 1996) and the spinal cord (Nakahara et al, 1996, Oudega et al, 1996).

In vivo, NT-3 plays an important role in the regeneration of peripheral nerves (Sterne et al, 1997) and spinal cord (Oudega et al, 1999, Bradbury et al, 1999) NT-3 has also been associated with the increased ability of sensory axons to grow from the dorsal root ganglia, across the PNS-CNS transition zone, and into the spinal cord (Ramer et al, 2000; Priestley et al, 2002; Bradbury et al, 1999). The application of exogenous CNTF has been associated with increased levels of regeneration after injury in both the spinal cord and peripheral nerve (Newman et al, 1996). However, a disadvantage is that CNTF has been demonstrated to play a role in glial scarring (Winter et al, 1995).

The fibroblast growth factors are strong promoters of angiogenesis (Friesel et al., 1995) and may therefore directly and indirectly aid the healing of injured nerves. Although neurotrophic factors promote regeneration in the PNS and CNS, in vivo responses may vary due to the method of growth factor delivery.

Silk based

Silk based scaffolds in Tissue Engineering

Evolution of nano-scale materials and concept of nanotechnology
Nanotools for Neuro-imaging

Magnetic nanoparticles in Neuro-imaging
Liposomes in Neuro-imaging

Silk fibroin has been found to promote mineralization in the presence of solutions containing calcium or phosphate. The compatibility of silk fibroin with endothelial cells prompted the development of silk fibroin-based prosthetic devices for use as vascular grafts. Electrospun silk fibroin nanofibrous scaffolds provided higher surface area for adhesion and proliferation of human aortic endothelial cells (HAEC) and human coronary artery smooth muscle cells (HCASMC) [Zhang et al, 2008].

Electrospun composite scaffolds have been reported with silk fibroin-gelatin [Wang et al, 2010], silk fibroin-collagen [Zhou et al, 2010], silk fibroin-poly (L-lactic acid-co-epsilon-caprolactone (PLLA- CL ) [Zhang et al, 2010], polydioxanone-polycaprolactone-silk fibroin [McClure et al, 2009], polylactide/silk fibroin-gelatin [Wang et al, 2009]. Formic acid-linked 3D silk fibroin was also reported because they have better biocompatibility in skin fibroblasts and keratinocytes [Dal Pra et al, 2006] Silk fibroin coated with synthetic polymers such as poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) was found to increase biocompatibility its and increases the growth of human smooth muscle cells [Zhang et al, 2007].

It mainly consists of three layers of tissue - the inner mucosa (made up of transepithelial cells called urothelial cells), the middle submucosa layer and the detrusor muscle which helps to expel urine from the bladder by contraction. Attempts to replicate the complex architecture in vitro using the retinal pigment epithelial cell line ARPE-19 cultured on porous silk fibroin membranes resulted in the expression of F-actin and ZO1 markers [Shadforth et al, 2012]. Furthermore, olfactory ensheathing cells grown on silk fibroin nanofibers were also found to secrete neurotrophic factors emerging as a potential construct for the treatment of spinal cord injury.

For the first time, silk fibroinpoly (L-lactide-coglycolide (PLGA) films were shown to promote Schwann cell adhesion and proliferation in vitro [Kim et al, 2011].

The ways in which lipoproteins may be modified to act as contrast agents. (Adapted with permission from Cormode et al, 2010)

Quamtum dots in Neuro-imaging
Gold nanoparticles in Neuro-imaging
Nanomaterial based assay platforms for Neuro-diagnostics

Gold nanoparticle based assays for early detection of Alzheimer’s disease
Nanomaterial based assays for early detection of Parkinson’s disease Like AD, another neurodegenerative disease primarily affecting the aged called

Nanotools for Neurotherapy

Nanotherapeutics in Glioblastoma multiforme
Nanotherapeutics in Alzheimer’s disease
Nanotherapeutics in Parkinson’s disease

Nanotechnology in Neuroregeneration
Sciatic Nerve Injury Model in Neuro-regenerative research

Sciatic Nerve Injury Model in compression lesions
Sciatic Nerve Injury Model in chronic denervation
Sciatic Nerve Injury Model in autograft studies
Sciatic Nerve Injury Model in nerve conduit studies
Surgical considerations of using Sciatic Nerve Injury Model
Functional assessment in Sciatic Nerve Injury Model
Electrophysiological assessment in Sciatic Nerve Injury Model
In vivo imaging in Sciatic Nerve Injury Model
Ethical issues in Sciatic Nerve Injury Model

Commercially available artificial nerve conduits

Polyvinyl alcohol hydrogel (Salubridge TM ; SaluTunnel TM )
Polyglycolic acid (Neurotube)
Poly D,L lactide-co-e-carprolactone (Neurolac)
Conclusions

Introduction
Materials and Methods

Cytotoxicity of synthesized GNPs Cytotoxic effect of Centella
Cytotoxicity of synthesized GNPs

Cytotoxicity of GNPs. A, MTT assay of GNPs on rat Schwann cell line SCTM41

Fabrication and characterization of nanocomposite mat
Culture of Schwann cells over nanofibrous scaffolds
Fabrication of nerve conduit and pre-seeding with Schwann cells
Porosity and Swelling ratio of Nerve conduits
In vivo intra dermal test
Surgical implantation of nerve conduits Imaging (MRI)
Functional analysis of regenerated sciatic nerve
Morphological analysis of regenerated sciatic nerve

Results

Cytotoxicity of synthesized GNPs
Fabrication and Characterization of nanocomposite mat
Culture of Schwann cells over nanofibrous scaffolds
Morphology of nerve condui proliferation
Porosity and Swelling ratio of Nerve conduits
Surgical implantation of nerve conduits

Discussions

Chronic denervation can be reproduced by complete transection of the sciatic nerve, not followed by its surgical reconstruction. To study electrical resistance, a 1 cm x 1 cm section of electrospun silk fibroin (SF) was in. Cells were found to adhere and proliferate on the outer (left) as well as the inner surface (right) of the nerve canals. Reprinted with permission from Das et al, Biomaterials, 2015).

The porosity of the channels was determined by a previously described ethanol displacement method (Tang et al, 2013). Briefly, the dimensions of the wires (n=4) were measured precisely using a caliper and the volume (V) calculated according to the following formula. Briefly, 24 h saline extract of silk fibroin (n=3) and GNP-incorporated silk fibroin cord (n=3) were taken and injected intradermally.

An overview of the surgical procedure performed is depicted in the data article (Das et al, 2015). In other groups, the wire was sutured to both the proximal and distal ends of the nerve stumps. The dimensions of the leads were found to be suitable for implantation in a rat sciatic nerve.

Immediately after implantation of the tubes, a minute MUP of 32μV was observed in all the groups. The locomotor activities of the animals implanted with nanocomposite tubes after 10 months are presented in Fig 3.11 A-D. The hind limbs of the animals were stained with methylene blue for obtaining footprints (inset).

GNP-SF conduits with cell (GNP

Conclusions
Introduction
Materials and Methods

Synthesis and cytotoxicity of polyaniline
Molecular mass characterization of synthesised polyaniline
Fabrication and characterization of electrospun SF and PASF nanofibrous mat
Fabrication and characterization of pre-seeded and un-seeded nerve conduits

Results

Synthesis and cytotoxicity of polyaniline
Molecular mass characterization of synthesised polyaniline
Fabrication and characterization of electrospun SF and PASF nanofibrous mat
Fabrication of pre-seeded and un-seeded nerve conduit and implantation in rats

Discussions
Conclusions
Summary

Silk-Gold nanocomposite based scaffolds for neural tissue engineering

Future Prospects

However, in the silk fibroin group (SF and SF cell) after 18 months, the gap along the tube was found to be filled with tissue that is fibrous in nature (Fig 3.11 G-H). On gross examination, the silk fibroin ducts (SF and . SF cell) were associated with inflammation at the distal end (Fig 3.12C-D). Histology of the SF tubules after 9 months showed little recruitment of neuronal cells with most of the lumen empty (Fig 3.12C i).

The porosity of the conduits (n=4) was determined using the previously described ethanol displacement method according to the following formula (Tang et al, 2013). Rat Schwann cells (SCTM41) were observed to adhere to both electrospun mats (SF and PASF) (Figure 4.3E-F). Rat Schwann cells (SCTM41) were also found to adhere and proliferate on PASF and SF channels (Figure 4.6E-F).

Histological analysis of the regenerated nerve showed cell recruitment within the PASF nanocomposite channels (with and without cell clusters) (Figure 4.13). In our study, PASF and SF nanofibers were found to support Schwann cell adhesion (Figure 4.3E-F). Multiple rotations of the electrospun plate produced a lamellar architecture of the nerve conduit that mimics the layer-by-layer deposition of myelin by Schwann cells over the axons (Figure 4.6A-D).

Schwann cells when cultured over channels (both SF and PASF) were found to adhere and proliferate in the form of clusters (Fig 4.6E-F). Nerve conductors were collected for gross observation and histological examination with hematoxylin-eosin staining after (i) 6 months and (ii) 12 months after implantation. Electrophysiological studies performed to assess functional regeneration of the sciatic nerve can be broadly divided into a) measurement of nerve conduction velocity and muscle action potential and b) motor unit potential recorded from the surrounding gastrocnemius muscle. The black coating observed around the axons after staining the samples with osmium tetroxide under TEM indicates the deposition of myelin by Schwann cells (Fig 4.14).