ASSESSMENT OF NEURAL TISSUE REGENERATION BY COMBINING BIOENGINEERING APPROACHES AND BIOACTIVE COMPOUNDS

Abstract

Much effort has been taken in devising and fabricating biocompatible and functional scaffolds for nervous system regeneration. Considering the very demanding nature of neurons, it becomes very challenging to tackle issues, like, biocompatibility, mechanical stability and electrical cues. Apart from this, it is needed to consider geometrical cues. At present, mainly polymeric blends are being used for synthesizing the scaffolds. Out of these, some polymers are conducting in nature but their conductivity is not optimum to excite the neuronal cells. So, for electrical cues, we need conductive nanofillers. Additionally, polymers are also having quite low mechanical strength. Carbon nanofillers are a good choice to enhance the conductivity and mechanical properties of polymers. Till now, most of the studies focused on growing and patterning carbon nanofillers on the top surfaces of hard substrates, like, glass, mica and silicon, for growing cells of neural tissue. But such substrates do not offer enough adhesion strength of carbon nanofillers to the scaffold surface, posing a high risk of in vivo release of the nanostructures, when implanted. Once the carbon nanofillers are uprooted from the surface of the scaffolds and taken away by the circulatory system, the scaffolds are not able to offer topographical or electrical cues to the regenerating tissues. Apart from this issue, there is another serious issue with using such carbon nanofillers grown structures as neural scaffolds. Carbon nanofillers’ growth through chemical routes on substrates requires very high temperatures, which polymers cannot withstand. Therefore, most of the studies have used silicon or metallic materials as substrates. However, these hard and stiff substrates cannot be used in vivo as scaffolds, considering the soft nature of the neural tissues. As it shows, these scaffolds are not a real solution, which can be used in neural tissue engineering. Thus, the ideal solution at this point is to have a soft base material, like, polymers, for neural scaffold, with carbon nanofillers reinforced in the structure, following some patterns, like, alignment in a particular direction. This can only be achieved by synthesizing a polymer-based composite, in which the carbon nanofillers can be aligned as per the requirement of the nerve tissue, it is going to replace. However, it is not easy to arrange carbon nanofillers in a particular pattern inside any polymer matrix. Thus, it is very important to identify and select a simple and effective fabrication method to align carbon nanofillers inside any polymer matrix in a particular direction, suitable for neural tissue regeneration. Upon successful alignment of the nanofillers inside the polymer matrix, the whole structure should be able to provide effective mechanical, electrical and topographical cues, a pre-requisite to support the regeneration of the neural tissues. Apart from the patterning of the nanofillers, another important factor is the determination of the optimum concentration of the nanofillers to be reinforced inside the polymer matrix. Because a lesser amount than the optimum concentration of nanofillers may not able to provide the ideal physical properties to the scaffolds, whereas, more amount than the optimum concentration may cause systemic toxicity. Thorough studies regarding the determination of the optimum concentration of carbon nanofillers, in the context of neural tissue engineering are majorly lacking. Apart from adding nanofillers to the polymer matrix, different groups tried to increase the biological activity of the scaffolds by incorporating cells or growth factors in the scaffolds. But the limitations of these scaffolds are their non-cost effectivity, storage issue and stability. So, some alternative strategies must be taken into account to resolve these issues. Thus, the overarching theme of this research was to prepare scaffolds that can provide biological, biochemical, mechanical, electrical and topographical cues simultaneously to the regenerating neural tissues. In this study, the scaffolds were synthesized through two methods, namely, solution casting and electrospinning and the prepared scaffolds were characterized for their morphology, physical (mechanical, electrical and topographical) properties, degradation kinetics, biocompatibility with neuronal cells and in vivo nerve regeneration efficacy. The main objective, as mentioned above, was achieved through three sub-objectives. The first sub-objective dealt with concentration optimization and alignment of carbon nanofillers to be reinforced in the chitosan polymer matrix. An electric field was used to align the carbon nanofillers inside the matrix. It was found that 0.5 wt% of multi-walled carbon nanotubes (MWCNTs) and 2 wt% graphene nanoplatelets were the optimum concentration for the reinforcement, as these concentrations maximally improved the mechanical and electrical properties of the scaffolds. However, all the nanofiller compositions used to prepare the scaffolds were found biocompatible. Apart from that, the shape of the nanofillers was found to influence the cellular morphology. On MWCNT-reinforced scaffolds, cells become spindle-shaped, having bidirectional neurite outgrowth. But the cells cultured on GNP-reinforced scaffolds took astral morphology. Moreover, on MWCNT-reinforced aligned scaffolds cells got aligned in the direction parallel to the alignment direction of the MWCNTs, whereas, no such alignment of cells was observed on GNP-reinforced scaffolds. This portion delineates the enormous potential offered by the chitosan-GNP/MWCNT scaffolds for the regeneration of the central nervous system (CNS),, as well as the peripheral nervous system (PNS). In the next sub-objective, the optimized concentrations of the carbon nanofillers were reinforced in the Polycaprolactone (PCL)-collagen peptide scaffolds and used these scaffolds as platforms to provide electrical stimulation to differentiate normal neuronal cells and mouse mesenchymal stem cells (mMSCs). It was observed that both the nanofillers reinforced aligned scaffolds efficiently conducted the provided electrical field and promoted differentiation of both normal neuronal, as well as, mesenchymal stem cells. Overall, it can be said that when needed, the carbon nanofillers reinforced aligned scaffolds can be used as platforms to provide localized electrical stimulation for the regeneration of both the nervous system. The last chapter of this study dealt with the synthesis of an extracellular matrix, mimicking electrospun nanofibrous scaffold reinforced with 0.5wt% MWCNTs and its biofortification. In this portion of the study, initially, a biocompatible, 0.5wt% MWCNT reinforced, anisotropically conductive, electrospun, aligned nanofibrous scaffold was prepared. This scaffold was observed to efficiently regenerate the injured sciatic nerve of the rats, as the electrically conductive aligned fibers helped in propagating electrical impulses across the lesion site. After fabricating this basic structure, this study aimed at upscaling the biological property of this scaffold. Instead of using any growth factors, this study tried to incorporate a plant extract (Bacopa monnieri) that has neurogenic properties to make the scaffold cost-effective. Thus, in the next portion of this chapter, the neurogenic property of this extract was established in terms of its neuroprotective and neurodifferentiation properties. It was observed that this extract efficiently protects neuronal cells from extreme oxidative stress and promoted differentiation of the neuronal cells. After establishing these properties, this extract was incorporated in MWCNT reinforced electrospun, aligned nanofibrous scaffold and checked for improvements in its neuro regeneration efficiency in vivo in a rat sciatic nerve injury model. It was observed that this biofortified scaffold indeed release the incorporated plant extract in the injury site and expedited the regeneration process of the injured sciatic nerve, when compared to the only MWCNT-reinforced aligned scaffold. This chapter notes the efficacy of the ECM mimicking biofortified conductive aligned nanofibrous scaffolds, with the capability of establishing remarkable advancement in the field of peripheral neural regeneration.