Materials used to fabricate nerve conduits require specific material properties that make them suitable for application in the animal model and eventual clinical trials. A critical feature for nerve conduits is their mechanical properties. The implanted nerve tubes have to be flexible and comply with tensile and bending stress especially when situated in the proximity of joints while at the same time they have to withstand the pulling forces typically exerted by a 9-0 nerve suture. PCLF-PPy scaffolds exhibit excellent suture pullout strengths, and unique thermal transitions that occur near the physiological temperature of 37 °C. By controlling the molecular weight of PCLF the thermal transitions, crystallinity and resulting mechanical properties of cross-linked PCLF can be manipulated as previously reported[45
]. Therefore warming PCLF and the composite PCLF-PPy scaffolds to 37 °C turned them from semi-crystalline to an amorphous state, which significantly increased their flexibility while still providing enough strength to hold a nerve suture.
The biodegradability of the nerve conduit is very important and a balance between providing support, guidance, and degradation is critical. The degradation of the conduit can prevent the regenerating nerve from being compressed and limits chronic foreign body reactions[46
]. Also the conduit should not degrade too quickly, because it has to maintain its mechanical integrity to provide nerve guidance. Additionally, large amounts of small degradation products might increase the osmotic pressure of the conduit causing increased material swelling[7
] which in general can lead to deleterious compression of the nerve[46
]. According to our results, PCLF-PPy scaffolds meet these requirements featuring a lack of swelling when submerged in aqueous solvent and slow but consistent degradation rates, thus being able to provide a strong guidance to outgrowing axons, without leading to compression over time.
The main interest in PCLF-PPy scaffolds is their electrical conductivity and being able to pass electrical current through them to stimulate growing nerve cells. Based on previous results PCLF-PPy networks doped with NSA and DBSA anions[39
] were selected because they demonstrated best material properties and most extensive cell attachment. The Rs
was shown to be as low as 2 kΩ thus exhibiting a similar conductivity as recently reported for other PPy composite materials[33
]. Additionally, the scaffolds proved to be electrically stable when different ES treatment regimens were applied, especially if NSA was the utilized dopant. This combined with the fact that compared to PCLF-PPyDBSA
samples were able to adsorb a higher amount of NGF to their surface led to the conclusion to use this type of scaffold for the cell culture experiments.
PC12 cells were electrically stimulated with 10 μA of current for 1 h per day. The current was either constant direct current or direct current with a frequency of 20 Hz. The amount of current, 10 μA, was chosen because it renders a surface current density of 7.2 μA/cm2
which induced the most favorable cell response from a previous study by Zhang et al.[48
]. The frequency of 20 Hz was chosen because 20 Hz is considered an effective frequency for stimulating nerve regeneration in the rat model[2
]and in human[49
]. Using ES with a frequency of 20 Hz instead of constant current may be advantageous because it resembles average firing frequencies of motor neurons[50
]. Even though the electrical stability of the samples was tested with three different time regimens, the cells were only subject to the 1 h/day ES treatment because it is a commonly used regimen when trying to stimulate neuronal regeneration in vivo[19
Image analysis of PC12 stimulated with a frequency of 20 Hz revealed an impressive 67% increase in the percentage of neurite bearing cells, dramatically higher than the 5% recently achieved in another study with similar PPy-PCL scaffolds[40
]. Using PPy coated PLGA nanofibers to apply ES treatments on PC12 cells, an even higher relative increase of 92% was reported, however, in that case no increase in numbers of neurites per cell was found[33
]. In comparison, when treatments of 1 h/day 10 μA 20 Hz ES were applied on cells seeded on PCLF-PPyNSA
samples the average number of neurites per cell increased by 52%. Considering the length of the extending neurites when electrical stimulation regimens are applied relative increases of 30%[40
], and 90.5%[52
] are reported in the literature. The 33.0% increase achieved when 10 μA 20 Hz ES were applied on PCLF-PPyNSA
scaffolds might therefore seem low at first sight, however it has to be taken into consideration that the increase in cells bearing multiple neurites leads to a shift of the median neurite length to lower values, because the neurites grow longer, but at the same time many new short ones appear. This explanation is corroborated by the observation that constant ES, which had a lower relative increase of the average number of neurites per cell showed a higher relative increase of 41.0% in the median neurite length. As mentioned by Lee et al. the exact mechanism of action of ES on neurons is not fully understood, but might involve increased adsorption of the extracellular matrix protein fibronectin to the scaffold surface[53
], changes in membrane potential[54
], and the vectored accumulation of surface glycoproteins[55
]. According to our results, pulsed ES treatments of 20 Hz led to a significant increase of the number of neurites per cell when compared to constant stimulation. Considering the widely accepted use of pulsed ES treatments of 20 Hz to aid nerve regeneration in vivo[2
] and the finding that PC12 cells show increased viability when stimulated with pulsed rather than constant treatments[37
], the use of pulsed ES in future studies for enhancing neurite extension on electrically conductive scaffolds might lead to improved results.
Since outgrowing axons need to find the desired trajectory towards and into the distal endoneurial tubes[42
], micropatterned surfaces[42
] or aligned nanofibers[58
] have been investigated for their potential to guide outgrowing nerve axons, rendering promising results. When analyzing the orientation of neurite extension in this study, alignment with the current direction was found. Combined with the fact that electrical fields are able to influence the direction of neurite extension in general[55
], this finding indicates an additional method to guide axon outgrowth within nerve conduits by applying electrical current.