Plasma activation of electrospun scaffolds for neural tissue engineering

Subtitle:Plasma-activatie van elektrogesponnen draagstructuren voor regeneratie van zenuwweefsel

An alerting high incidence of peripheral nerve injuries (PNI) reaching over one million cases worldwide is still lingering over the centuries. This is due to the fact that peripheral nerves are not protected like the brain and the spinal cord by a bone tissue and a nerve-blood barrier, making them susceptible to physical, chemical, thermal and/or ischemic damages at any anatomic site. Luckily, unlike the central nervous system having a very limited to no ability of regeneration, the peripheral nervous system is marked by its regenerative machinery initiating nerve repair up to a certain extent. However, the complex pathophysiology involved in PNI makes the spontaneous regeneration not always successful. Therefore, interventions to achieve an effective repair are in most cases unavoidable. Direct nerve repair is the most efficient therapeutic approach but is limited to extremely short nerve gaps where a tensionless suturing can still be performed. In the frequent cases involving bigger nerve gaps, the interposition of a supportive structure is necessary to span the injured site. The use of autologous nerve graft is currently considered the gold standard and has stayed so all along the previous 50 years. However, the autograft is associated with some weighty drawbacks such as donor site morbidity, use of sensory-only nerve, size and fascicular pattern mismatching, extra surgical step, possible neuroma formation and a success rate of only 50%. In the last few decades, the advancements in the multidisciplinary tissue engineering (TE) field have led to the development of nerve guidance conduits (NGCs) as alternative. Just as most scaffolds used in different TE applications, NGCs typically consist of a physical scaffolding made up of natural or synthetic polymeric material possibly amalgamated with biomolecular components and/or support cells. Nerve gaps of maximum 4.0 cm in humans and 1.5 cm in rats have been successfully bridged. However, nerve regeneration becomes very limited or completely absent in bigger gaps and functional repair remains deficient across all gap sizes. Therefore, several inventive strategies focused on adding neurotrophic factors, Schwann cells (SCs), stem cells, intraluminal fillers, wall guidance structure or changing the whole conduit design were implemented. Moreover, combinatorial strategies merging the advantages of previous NGCs and adopting additional levels of complexities are investigated. However, all approaches are still failing in outperforming the regeneration levels of autograft or even in attaining similar outcomes especially in critical nerve gaps. Therefore, the main goal of this thesis is to tackle large nerve gaps by designing a novel NGC possessing the ideal topographical, mechanical, chemical and cellular cues triggering a robust regenerative capacity. From a topographical point of view, one elemental factor that can guaranty the implant success is the mimicry of the fibrillary architecture of the extracellular matrix (ECM) that is known to govern most of the cellular activities in the body. Different physical, chemical and electrostatic techniques have already been developed for the recreation of ECM fibers. Of those available, electrospinning is by far the most widely used due to its simplicity, versatility and affordability. Moreover, its capacity to align the fibers and adapt their diameter down to the nanometer size renders it a powerful technique as it can recapitulate the in vivo tissue-specific properties in terms of orderliness and scale. In fact, the fiber diameter plays a decisive role in modulating cellular adhesion, gene expression, proliferation and differentiation. Moreover, fiber alignment provides directional cues triggering cell elongation, directed migration and regeneration enhancement of ordered tissues such as nerves. In this sense, NGCs made up of random and aligned electrospun fibers of different diameters have shown moderate successes in nerve regeneration over the other topographical designs. In this dissertation, the right electrospinning parameters leading to the finest fiber diameter and alignment are vigilantly picked to optimize the previous generation of electrospun NGCs. From a mechanical point of view, the base material should be meticulously selected to match the strength and elasticity of the innate environment and support cell growth. Biodegradable aliphatic polyesters constitute the most eminent polymer group for NGC fabrication owing to their proper mechanical properties, biocompatibility and FDA approval for clinical suitability as nerve conduits. A remarkable supremacy of polycaprolactone (PCL) is observed in the literature dealing with electrospun NGCs. PCL fibers are non-toxic as they do not lead to the formation of high concentrations of organic acid degradation products, which minimizes the risk inflammatory responses. In vivo performances of PCL NGCs showed good results in bridging nerve gaps of 1 cm by exhibiting large number of myelinated axons. Recently, the copolymer poly(ethylene oxide terephthalate)-poly(buylene terephthalate) (PEOT-PBT) commercially known as Polyactive® (PA) was shown to support the regeneration in longer gaps because of its in vivo slower degradation rate coinciding with the longer regeneration time. Another advantage of PA over the conventional polymers is the possibility to tune more adequately its mechanical properties by tackling the composition of the two polymers. Therefore, both PCL and PA are used in this thesis dissertation. From a biochemical point of view, adding free neurotrophic factors to NGCs is not enough to support cellular activities. A critical limitation of most NGCs is their deprivation of immobilized proteins because of their hydrophobic base material surface lacking protein-binding functional groups. In fact, topographical and biochemical cues are concurrently recognized by cells at the cell-scaffold interface that plays a primordial role in the initiation of vital cellular processes such as adhesion and proliferation. However, given the narrow dimensions of the porous conduits, reaching and modifying the inner wall surface without altering the nanofibers delicate structure remain challenging tasks. To solve this issue, this thesis focuses on the application of non-thermal plasmas as a route to bio-activate NGC surface. Plasma treatments are nowadays gaining a great interest in TE over other traditional surface modification techniques. It is a solvent-free method that can be highly controlled to incorporate specific functional groups (plasma activation) or deposit thin polymer coatings (plasma polymerization) on biomaterials thus creating adequate surfaces for subsequent protein immobilization. Moreover, it is a gas-based technique that can reach and treat the overall surface of complex and porous scaffolds, hence NGCs. Despite the considerable improvements offered by plasma-functionalized scaffolds in several TE applications, plasma treatment was never, to the best of our knowledge, applied to NGCs. Yet, tailoring their surface properties using fine-tuned plasma parameters is believed to play a pivotal role in the enhancement of glial and neural cell activities thus activating nerve regeneration across critical nerve gaps. In the body, the directional guidance of neurites that ensures a successful nerve regeneration is mediated by spatial concentration gradients of biomolecules. Inspired by the theory “Nature knows the best”, plasma treatments creating a chemistry gradient along the conduit surface are also performed in this thesis. Gradient plasma treatments were previously applied only on 2D sheets using complex redesigned plasma reactors. For instance, automated stepper motors moving the samples during the treatment, complex gas flow systems, reshaped electrodes and shielding covers were employed for this purpose. Relatively simple dielectric barrier discharge (DBD) and plasma jet (PJ) reactors are used in this thesis to plasma-treat the more complex 3D NGCs in a gradient way. From a cellular point of view, NGCs cultured with SCs prior to in vivo implantation were shown to considerably enhance nerve regeneration compared to their acellular counterparts. If autograft is the gold standard for PNI repair, adding autologous SCs to NGCs is similarly judged as the present gold standard for cellular-based approaches. In addition to their active secretion of growth factors, SCs express cell adhesion molecules, build their own basal lamina and intensely assist, at a later stage, in the remyelination of growing nerve fibers. However, SC cultures are difficult and time-consuming and SC extraction from demands the sacrifice of a nerve tissue. As alternative stem cells are cultured on the NGCs, of which the frequently used adipose derived stem cells (ADSCs). In fact, ADSCs are abundant and easily accessible by a simple liposuction, proliferate rapidly in culture, have a low cost and can differentiate into glial and neural cell lineages. Both, undifferentiated and differentiated ADSCs were shown to enhance the functional recovery when injected in NGCs. Therefore, ADSCs and SCs are cell types studied in this thesis. An evolutionary methodological strategy is adopted in this dissertation gradually paving the way towards the generation of an ideal NCG: In a first step, the effects of different sterilization methods on the physico-chemical and bioresponsive properties of plasma-treated PCL are studied. In fact, the material sterility is a prerequisite for the use of NGCs in vitro and in vivo. This crucial step is often neglected as tissue engineers are mainly focusing on implementing complex scaffold topographies and advanced biofunctionalization. However, given the high sensitivity of biodegradable polymers, the harsh sterilization methods normally used are associated with big risks of compromising the physical and chemical properties of the scaffolds, thereby altering the cell-material interactions. Therefore, the sterilization should be considered early in the scaffold designing process especially when it comes to fine structures subjected to a previous surface modification. This prevents from the risk of damaging the NGCs at advanced stages after the whole optimization processes of biofabrication and plasma treatment. The first experimental chapter is thus dedicated to sterilize PCL films pre-subjected to a plasma treatment using a medium pressure DBD. The second experimental chapter further advances in complexity and transfer from 2D films to 3D electrospun fibers. In this way, an examination of different polymeric topographies is completed to check if the nanofibrous scaffolds are more or equally prone to sterilization-induced damages compared to films. Results show that air and argon plasmas significantly increase the films and fibers wettability due to the incorporation of oxygen-containing functionalities onto PCL surface. Besides surface modification, the plasma potential to sterilize PCL is studied in function of appropriate treatment times, but sterility is not achieved so far. Therefore, plasma-modified samples are subjected to UV, H2O2 plasma (HP) and ethylene oxide (EtO) sterilizations. EtO decreases the wettability of plasma-treated films and fibers and changes the morphology of plasma-treated fibers mainly due to reactions between EtO molecules and the grafted functional groups. Moreover, HP modifies the surface morphology of PCL films and provokes a total loss of the fibrillary architecture of fibers because of the complex thermo-oxidative reactions occurring during the process. UV does not affect the physico-chemical properties of all samples which shows a significantly higher adhesion and proliferation of ADSCs compared to EtO and HP sterilized samples. Overall, it can be concluded that plasma-treated NGCs and other TE scaffolds should be sterilized by UV to maintain their beneficial surface properties induced by non-thermal plasma. After the selection of a suitable sterilization, the second step of this dissertation focuses more on PCL electrospinning process itself. Several highly toxic solvent systems providing a good PCL solubility-spinability are recurrently applied in the overwhelming majority of studies. One of the current major focuses revolves around the challenging generation of aligned fibers that are very desirable in numerous TE applications of which peripheral nerve TE. Moreover, the critical influence of fiber size on cellular performance has led to the use of different solvent systems for the production of specific nano- or micro-sized PCL fibers while neglecting the solvents toxicity. Therefore, the goal of the third experimental chapter is to use the unconventional and non-toxic solvent system acetic acid/formic acid, recently defined as the system producing ultra-thin PCL fibers, in a trial to outspread the size range and to tackle fiber alignment. After profound analysis of the effect of varying collector motion and collector design on fiber alignment, a novel collector producing highly aligned PCL fibers based on synchronic mechanical and electrical effects is designed. In a subsequent step, the fiber diameter is manipulated by analyzing 3 influential parameters: polymer concentration, tip-to-collector distance and the frequently overlooked parameter humidity. The parameters fine-tuning study has resulted in very broad PCL fiber diameter ranges of 94 to 1548 nm and 114 to 1408 nm for random and aligned fibers respectively. The generated fibers are then used in the fourth experimental chapter to investigate the synergistic influence of PCL fiber size, orientation and plasma-modified surface chemistry on ADSC behavior. Despite the incorporation of approximately the same oxygen amount on all samples post-plasma treatment, the hydrophilicity significantly differ between the different fiber sizes and orientations. This highlights the outstanding influence of the fibrous mesh topography on the liquid-solid interface. Extended plasma exposure starts damaging the fibers with a growing risk of drastic alterations on thicker and random fibers compared to thinner and aligned fibers. The diverse responses to plasma stem from the distinct molecular chain arrangement and crystallinity of different fiber diameters and orientations. Plasma treatment strikingly enhances the cell metabolic activity, adhesion, proliferation and cytoplasmic remodeling on all samples. ADSCs adhere multi-directionally on random fibers with a gradual change from a more circular to a more elongated shape on increasing diameters. In contrast, ADSCs overextend in a bipolar and aligned fashion on aligned fibers with a tendency to attach on fewer fibers with increasing fiber diameter. A mimicry of the natural bands of Büngner structure guiding axon extension during nerve regeneration is thus gradually observed, making from the aligned plasma-treated fibers promising candidates in the design of NGCs. Coming to the last experimental chapter of this dissertation, NGCs are electrospun using fine-tuned process parameters engendering an innovative bi-layered architecture. An inner wall composed of aligned fiber bundles with random fibers in between is obtained, thus guiding neurite extension and SC elongation while still allowing nutrient supply through the random fiber pores. In contrast, randomly deposited nanofibers entirely compose the outer wall thus further supplying nutrients and consolidating the whole NGC structure. Medium pressure argon DBD treatment homogeneously increased the inner surface oxygen content from 17 % to 28 % thus highlighting the plasma ability to penetrate through the porous wall. Atmospheric pressure argon PJ treatment created a gradient chemistry throughout the inner wall with an oxygen content gradually increasing from 21% to 30%. A significantly enhanced SC adhesion is observed on plasma-treated NGCs compared to untreated NGCs. A uniform cell distribution is perceived along the homogeneously plasma-treated NGCs. However, cell gradients towards increased surface oxygen contents are interestingly detected on the NGCs subjected to a gradient plasma treatment. With time, the cell gradient becomes steeper and more prominent owing to the better cell proliferation on the oxygen-rich end and to a directed cell migration along the NGCs. A gradual change from a more circular shape to a more elongated SC shape is visualized along the oxygen gradient thus forming SC columns mimicking the natural bands of Büngner structure. Finally, when PC12 cells are cultured on SC pre-seeded scaffolds, neurite outgrowth is only seen on plasma-treated NGCs: DBD treated NGCs display relatively short neurites extending in multiple directions, while PJ treated NGCs show gradually longer neurites mainly directed towards higher oxygen contents. Overall, it can be concluded that the joint use of the electrospinning technique and the non-thermal plasma technology in the engineering of NGCs has a great potential in enhancing peripheral nerve regeneration. Particularly, plasma-induced chemistry gradient along the inner NGC wall offers high promises in bridging critical nerve defects and ensuring a complete functional recovery. A future translation from in vitro to in vivo will hopefully constitute a big step towards the clinical use of plasma-treated NGCs.

ISBN:9789463552325

Publication year:2019

Accessibility:Open