Chapter 5: Summary and Future prospects
B, GNP-SF conduits with cell (GNP
4.4 Discussions
green. Normal cellular morphology and abundant live cells were observed upto PA treatment concentration of 1000μg/ml through live-dead staining assay (Fig 4.1C-H).
The molecular weight distribution of a polymer is essential for analysing its physical, chemical and biological properties. The molecular weight of synthesised polyaniline has been previously studied using chromatographic techniques (Kolla et al, 2005).
However, solubility of the sample in an organic solvent is an essential prerequisite to obtain accurate results by chromatography. Polyaniline has been reported to be almost insoluble in any solvent and only exists in a dispersion state when mixed with organic solvents (Wessling, 2000). Hence, for obtaining accurate molecular weight distribution of polyaniline, we performed Matrix-assisted laser desorption/ionization (MALDI). Both solvent-less and solvent based MALDI were carried out to study the role of solvents in isolating higher molecular weight oligomers of polyaniline.
Polyaniline primarily produces three types of oligomers based on the presence/absence of terminal amine (-NH2) which differ by a m/z value of 16. (Fig 4.9) (Dolan et al, 2014). Further, the addition of one phenyl group (-C6H4NH-) or 1- mer to the oligomer chain led to an increase of m/z value by 91 (Dolan et al, 2014).
Fig 4.9: Representative structures of different types of aniline oligomers. (i) Aniline oligomers terminated by phenyl group on both ends (lowest molecular weight species), (ii) Oligomers having an amine in one terminal and equal number of six- carbon rings and nitrogen atoms, (iii) Oligomers having amine group in both terminals.
Solvent based MALDI of polyaniline using acetonitrile and THF as solvent produced prominent peaks corresponding to 6-mer (m/z =542,558,574), 7-mer (m/z=634,650,666) and low intensity peaks for 8-mer (m/z=725,741). Polyaniline mixed in THF produces an additional peak for 8-mer around m/z = 757
corresponding to the structure depicted in Fig 4.2(A-B). Solvent based MALDI analysis of polyaniline could detect oligomers upto a maximum of 8 repeat units since only very low molecular weight portions are sparingly soluble in organic solvents like acetonitrile and THF (Fig 4.2A-B).
In order to detect higher molecular weight oligomers we performed solvent less MALDI. The peaks for the oligomers in this case were observed to be of much higher intensity as compared to solvent based MALDI and polymers with upto 12 repeat units were detected (Fig 4.2C-D). The results provide strong indication that solvent less MALDI is an ideal method to study molecular mass spectrometry of insoluble polymers like polyaniline and can also detect higher oligomers more efficiently as compared to conventional solvent based techniques.
Fig 4.10: Wave pattern of compound muscle action potential. Action potential recorded after stimulating at the proximal and distal ends of implanted conduit A) PASF group after 6 months, B) PASF group after 12 months, C) SF group after 6 months, D) SF group after 12 months, E) Cell seeded PASF conduits after 6 months, F) Cell seeded PASF conduits after 12 months, G) Cell seeded SF conduits after 6 months and H) Cell seeded SF conduits after 12 months.
In the present study, aggregates of low molecular weight polyaniline were blended with silk fibroin protein in formic acid to form composite nanofibers through electrospinning. However, since PA is insoluble in formic acid and exists in a dispersed state it was observed through electron microscopy that the polyaniline aggregates were broken down to nano dimensions during electrospinning thereby forming a uniform covering of polyaniline nanoparticles (average size 40-60nm) over the silk nanofibers (Fig 4.3B-C). Consequently, the average diameter of PASF nanofibers (350-450nm) were found to be larger than pristine SF fibers (150-250nm) (Fig 4.3D).
Polyaniline based composite scaffolds have been previously demonstrated to support adhesion and differentiation of neuronal cells (Guarino et al, 2013). In our study, both PASF and SF nanofibers were found to support adhesion of Schwann cells (Fig 4.3E-F). The nanofibrous scaffolds (both PASF and SF) by virtue of their higher surface area were able to support growth and proliferation of cells for a longer time as compared to the control cell culture surface (Fig 4.3G).
The resistance of silk fibroin was observed to decrease by 12 folds upon addition of polyaniline powder in a very low concentration (1% of silk fibroin). Further decrease in resistance could be achieved by increasing the concentration of polyaniline in the polymer blend (data not shown), however, the propensity of PA powder to produce cytotoxic effect at higher concentration lead to its restricted use in the present study in order to balance between electrical conductivity and biocompatibility.
The electrospun mats (SF and PASF) were characterised by FTIR and XRD analysis and compared with polyaniline powder to confirm the formation of composite nanofibers (Fig 4.4A-D). The PASF nanocomposite mat exhibited a FTIR peak around 1614 cm-1 which correlates to 1668 cm-1 absorbance peak in SF for amide I and 1635 cm-1peak present in PA powder corresponding to quinone ring C=N stretching vibration (Zhang et al, 2011; Kim et al, 2003). The lower frequency 1541 cm-1 peak of PASF nanocomposite can be attributed to 1579 cm-1 benzenoid diamine ring stretching of PA as well as amide II region (1514 cm-1) of SF nanofibers (38,39).
Similarly the peak at 1246 cm-1 for PASF nanofibers can be ascribed to the amide III structure of SF at 1240 cm-1 and the characteristic C-N stretching of PA (Zhang et al, 2011; Kim et al, 2003). A small peak was observed around 1146 cm-1 in SF
electrospun mat corresponding to aliphatic amine whereas a broad peak around 1119 cm-1 was exhibited by PASF nanocomposite scaffolds. However no similar peak was found in the polyaniline powder which comprises entirely of aromatic amines. A series of small peaks observed at 931 cm-1,777 cm-1, 725 cm-1,644 cm-1, 517 cm-1 in PA powder can be ascribed to out-of plane deformation of C-H (Zhang et al, 2011; Abdolahi et al, 2012). These were observed to be merged together forming a broad peak around 690 cm-1 in PASF nanocomposite mat.
Fig 4.11: Morphological and electrophysiological assessment in control group. A) No tissue regeneration was observed in the nerve gap region after 12 months, B) No action potential could be detected by nerve conduction studies, C) MUP recorded in the gastrocnemius region.
The crystalline form of polyaniline has been reported to exhibit three characteristic XRD peaks at 2θ=15o, 20o and 25o corresponding to (011), (020) and (200) crystal planes with the one at 25o being the dominant peak (Abdolahi et al, 2012; Lee et al, 2006). However, we observed only a small and broad peak at 2θ=24o for the synthesised PA powder (Fig 4.4D). It indicates the low crystallinity and comparatively higher amorphous nature of the synthesised PA powder which is due to the high monomer (aniline) to APS ratio used during synthesis. The XRD pattern of SF electrospun nanofibrous mat exhibited a broad peak around 2θ=21o characteristic of amorphous silk fibroin protein in β-sheet configuration (Fig 4.4C) (Wang et al, 2014). The PASF nanocomposite sheet showed two peaks around 2θ=20.65o and 2θ=24.8o indicating the formation of a composite comprising amorphous silk fibroin and polyaniline (Fig 4.4B).
Fig 4.12: Sciatic Function Index (SFI) calculated through walking track analysis.
Polyaniline generally exhibits three major weight losses by TGA around 100C, 2000C and 5000C corresponding to water loss, loss of acid molecules and decomposition of polymer respectively (Wei Y et al, 1989). In the present study, PA was thoroughly dried and hence limited weight loss around 100oC was observed. A major weight loss occurred between 2050C-3520C which can be attributed to the loss of hydrochloric acid molecules used as dopant. The polymer was found to steadily decompose to below 45% of its initial weight by 7000C (Fig 4.5). Silk fibroin nanofibers showed a major weight loss around 245oC due to thermal degradation of the protein and continued till 464oC before becoming stable upto 7000C (Fig 4.5) (Wei K et al, Membranes, 2011). PASF composite nanofibers exhibited thermal properties similar to that of pure silk fibroin since PA concentration was restricted to only 1% of the composite. The sharp weight loss around 246oC points to the degradation of silk fibroin. The composite continued to degrade with increase in temperature and less than 45% of initial weight remained upto 7000C (Fig 4.5).
The electrospun sheets (both SF and PASF) were peeled off and rolled over a needle of appropriate diameter. Such sheet rolling method enables fabrication of multiple nerve conduits with varying dimensions (wall thickness, diameter and length) from a single electrospun sheet. Multiple rotations of the electrospun sheet created a lamellar architecture of the nerve conduit which mimics the layer by layer deposition of myelin by Schwann cells over axons (Fig 4.6A-D). Consequently,
Schwann cells when cultured over the conduits (both SF and PASF) were found to adhere and proliferate in the form of clusters (Fig 4.6E-F). The fabricated nerve conduits exhibited no evident signs of cytotoxicity when cultured with Schwann cells over a period of 10 days. Additionally, such stacking and rolling of multiple layers of densely arranged nanofibers contributed to the overall low porosity and swelling tendency of the conduits (Fig 4.7).
The size of polyaniline particles have been found to influence the cytotoxicity and particularly its nanoform has been reported to be highly toxic at concentrations above 10μg/ml causing cell death, increase cellular reactive oxygen species and disturbed mitochondrial membrane potential (Li et al, 2014). Since the PASF nanocomposite fibers comprised of polyaniline nanoparticles it was essential to confirm their biocompatibility through intracutaneous skin irritation and toxicity studies. However no adverse immunogeneic or toxic response was noted among the animals (both PASF and SF groups) which may be attributed to the strong interaction between PA nanoparticles and SF protein which prevents any washout of nanoparticles into the saline extract (Fig 4.8A).
Fig 4.13: Gross and histological examination. Nerve conduits were harvested for gross observation and histological examination by hematoxylin-eosin staining after (i) 6months and (ii) 12 months of implantation.
The electrophysiological studies carried out to evaluate functional regeneration of the sciatic nerve can be broadly divided into a) nerve conduction velocity and muscle action potential measurements and b) motor unit potential recorded from the surrounding gastrocnemius muscle. The compound muscle action potential (CMAP) waveforms for all the experimental groups are depicted in Fig 4.10 whereas the quantitative analysis of CMAP and NCV are represented in Table 4.1. The animals implanted with PASF conduits (both with and without cells) showed appreciable improvement in wave pattern with enhanced amplitude and higher area under the curve with time from 6 months to 12 months. A similar trend was also observed with animals having SF conduits (both with and without cells) after 6 and 12 months.
However, the nanocomposite group with PASF conduits (both with and without cells)
clearly outperformed the animals with plain SF conduits (both with and without cells) with higher NCV and CMAP values after 12 months indicating better functional regeneration of the transacted nerve. The animals implanted with Schwann cell seeded PASF conduits exhibited recovery as high as 86.2% of NCV and 80.0% of CMAP as compared to normal rat thereby suggesting the positive role of Schwann cell pre-seeding in enhancing nerve regeneration outcomes. The animals that were left untreated (control group) did not exhibit any action potential even after applying stimulation up to 18.4mA at the proximal and distal end of the nerve gap over a period of 12 months indicating the absence of regenerated neural tissue across the gap (Fig 4.11B).
Nerve conduction studies and CMAP analysis form only a part of complete peripheral neurophysiological examination and are generally accompanied by monitoring motor unit potentials (MUP) recorded from the surrounding muscle area through needle EMG. A motor unit is the basic functional element of skeletal muscle comprising of the anterior horn cell, its axon and all the muscle fibers it innervates including the neuromuscular junctions. The action potential generated when a motor unit fires upon voluntary contraction of muscle is recorded as MUP. Since sciatic nerve is the major mixed nerve innervating the gastrocnemius region we conducted MUP measurements through needle EMG to evaluate the neuromuscular regeneration in the region. MUP recorded immediately post surgery exhibited minute potential of 27μV which could be attributed to smaller nerves other than the sciatic nerve which also innervate the gastrocnemius muscle fibers. The animals implanted with PASF conduits pre-seeded with Schwann cells exhibited highest MUP after 12 months corresponding to 76.07% of MUP of normal rat (Table 4.1). The result indicates enhanced re-innervations of the gastrocnemius muscle fibers with regenerated axons. MUP recorded from the untreated animals (control group) exhibited minute potential of 30μV which could be attributed to smaller nerves other than the sciatic nerve which also innervate the gastrocnemius muscle fibers (Fig 4.11C).
Fig 4.14: Ultra structure of regenerated tissue under TEM. A) Normal sciatic nerve, B) Regenerated tissue through PASF conduits 1year post implantation, C) Regenerated tissue through Schwann cell seeded PASF conduits 1year post implantation, D) Higher magnification image of myelin sheath in a single axon of cell seeded PASF conduits 1year post implantation, E-F) Regenerated tissue through SF conduits 1year post implantation, G- H) Regenerated tissue through cell seeded SF conduits 1year post implantation.
Although several advanced electrophysiological and behavioural tests are available to conduct peripheral neurophysiological examination, walking track analysis of rats by calculating Sciatic Function Index (SFI) is still one of the most preferred non- invasive ways to objectively assess recovery of motor function in peripheral nerve injury models (Varejão et al, 2001). A SFI score of -100 indicates complete inability to walk and severely damaged gait whereas a normal walking pattern gives a SFI score of 0. The untreated animals (with a nerve gap and without any nerve conduit) exhibited constant SFI of around -100 for 12 months (Fig 4.12). Sequential improvement of SFI was observed in all experimental groups with a saturation point after 32 weeks (Fig 4.12). Animals having SF conduits with and without cells showed SFI improvement upto a maximum of -70 and -72 respectively. In comparison Schwann cell seeded PASF conduits exhibited best gait pattern with a SFI of -41 which was much improved than the animals with PASF conduit having SFI of -51.
The conduits were harvested from the animals 6 and 12 months post implantation and the tissue within the nerve gap was histo-morphometricaly analysed for evaluating the extent of neuro-regeneration through the conduits (Fig 4.13). Low
amount of cellular growth was observed across the lumen of SF and cell seeded SF conduits after 6 months. However, after 12 months, accumulation of inflammatory cells inside the conduit was observed in SF and SF cell groups. Culture of Schwann cells over the nanocomposite PASF conduits evidently led to higher amount of normal neuronal cells inside the lumen facilitating regeneration of nervous tissue across the gap. The structural stability of the nerve conduits even after 12 months implantation can be attributed to the stacking of multiple layers of nanofibers leading to minimal swelling ratio of the conduits. In case of animals where no conduit was implanted (control group) the nerve gap increased considerably on both ends and no signs of neural regeneration was observed even after 12 months (Fig 4.11A).
Although some portion of the proximal end remained the distal nerve stump was found to have completely degenerated. This could be due to long duration Wallerian degeneration occurring in neurotmesis grade nerve injury which causes the nerve stumps to degenerate and recede from the distal end.
The process of myelination is an important indicator of accurate neuron-glia interaction which is essential for complete neural regeneration. In the peripheral nervous system myelin is deposited around an axon by rotating Schwann cells in a layer by layer fashion (Garbay et al, 2000). The high lipid to protein ratio (75% lipid and 25% protein) of the myelin sheath confers it with electrically insulating property required for maintaining proper saltatory conduction of nerve impulse (Garbay et al, 2000). This high lipid content is exploited to stain the myelin sheath with lipid binding dyes like osmium tetra oxide, toluidene blue etc. The black coating observed around axons after osmium tetraoxide staining of the samples under TEM indicates deposition of myelin by Schwann cells (Fig 4.14). A closer look at the ultrastructure of the tissue harvested from the lumen of cell seeded PASF conduits revealed thick deposition of myelin in a lamellar fashion indicating proper myelination of the regenerating axons (Fig 4.14 C-D). In comparison, the thin, discontinuous and granular type deposition of myelin observed in the regenerating tissue of SF conduits (with and without cells) (Fig 4.14 4.E-H) points to incomplete and improper myelination which explains the low NCV, CMAP and MUP values observed in animals belonging to SF groups (with and without cells).
Although conductive polymer based scaffolds have been extensively researched for potential use as nerve conduits, their in vivo studies for evaluating nerve
regeneration in animal models have been very limited (Ahn et al, 2015; George et al, 2009, Mottaghitalab et al, 2013; Yu et al, 2014). Further these in vivo studies have been for a short duration (maximum 16 weeks) and monitored neuro-regeneration primarily based on histopathological/immuno-histochemical outcomes without complete electrophysiological analyses. In the present study, we observe the effects of synthetic conducting polymer like polyaniline in vivo over a long duration (1 year) and evaluate functional nerve regeneration through several electrophysiological tests like NCV, CMAP and MUP including walking track analysis. The animals implanted with Schwann cell pre-seeded nanocomposite PASF conduits exhibited excellent NCV and CMAP patterns along with high MUP values which are better than previous reports of nerve regeneration using similar electrically conducting nerve conduits (Ahn et al, 2015;; Mottaghitalab et al, 2013). These electrophysiological results along with thick myelination in a lamellar fashion strongly indicate superior functional and morphological regeneration of nerves in the PASF group as compared to previously reported conducting polymer based nerve conduits.