Plasma activation of electrospun scaffolds for neural tissue engineering

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

Acrylic acid plasma coated 3D Scaffolds for Cartilage tissue engineering applications

Abstract The current generation of tissue engineered additive manufactured scaffolds for cartilage repair shows high potential for growing adult cartilage tissue. This study proposes two surface modification strategies based on non-thermal plasma technology for the modification of poly(ethylene oxide terephthalate/poly(butylene terephthalate) additive manufactured scaffolds to enhance their cell-material interactions. The first, plasma activation in a helium discharge, introduced non-specific polar functionalities. In the second approach, a carboxylic acid plasma polymer coating, using acrylic acid as precursor, was deposited throughout the scaffolds. Both surface modifications were characterized by significant changes in wettability, linked to the incorporation of new oxygen-containing functional groups. Their capacity for chondrogenesis was studied using ATDC5 chondroblasts as a model cell-line. The results demonstrate that the carboxylic acid-rich plasma coating had a positive effect on the generation of the glucoaminoglycans (GAG) matrix and stimulated the migration of cells throughout the scaffold. He plasma activation stimulated the formation of GAGs but did not stimulate the migration of chondroblasts throughout the scaffolds. Both plasma treatments spurred chondrogenesis by favoring GAG deposition. This leads to the overall conclusion that acrylic acid based plasma coatings exhibit potential as a surface modification technique for cartilage tissue engineering applications

Plasma Polymerization for Tissue Engineering Purposes

The ability of non-equilibrium plasmas to modify surfaces has been known for many years. And a promising way to perform surface modifications without altering the bulk properties is plasma polymerization since this technique is versatile and can be applied to a wide range of materials. Plasma polymer films usually show good biocompatibility when compared to classical biomaterials. The possible biomedical use of plasma polymers motivates the study of their behavior during storage and in aqueous environment. Therefore, it is of major importance to understand the change of properties of these plasma polymers over time and when in contact with certain fluids. Recently, plasma polymer gradients (surfaces that display a change in at least one physicochemical property over distance) have attracted significant attention from the biomedical filed where the interaction of cells with a material surface is of major interest. This chapter discusses biomaterial functionalization via plasma polymerization focusing on their use in the biomedical field as well as their aging and stability behaviors. Plasma polymer gradients as valuable tools to investigate cell-surface interactions will also be reviewed

Non-thermal Plasma Technology for the Improvement of Scaffolds for Tissue Engineering and Regenerative Medicine - A Review

Non-thermal plasma technology is one of those techniques that suffer relatively little from diffusion limits, slow kinetics, and complex geometries compared to more traditional liquid-based chemical surface modification techniques. Combined with a lack of solvents, preservation of the bulk properties, and fast treatment times; it is a well-liked technique for the treatment of materials for biomedical applications. In this book chapter, a review will be given on what the scientific community determined to be essential to obtain appropriate scaffolds for tissue engineering and how plasma scientists have used non-thermal plasma technology to accomplish this. A distinction will be made depending on the scaffold fabrication technique, as each technique has its own set of specific problems that need to be tackled. Fabrication techniques will include traditional fabrication methods, rapid prototyping, and electrospinning. As for the different plasma techniques, both plasma activation and grafting/polymerization will be included in the review and linked to the in-vitro/in-vivo response to these treatments. The literature review itself is preceded by a more general overview on cell communication, giving useful insights on how surface modification strategies should be developed

Random and aligned plasma-activated nanofibers of different diameters : impact on cell morphology and proliferation

INTRODUCTION: Poly-ε-caprolactone (PCL) is an interesting polymer for tissue engineering purposes due to its good mechanical properties and tunable degradation rate. However, its hydrophobicity raises a big challenge for cell adhesion and proliferation. To overcome this problem, plasma surface treatment could be used. Moreover, surface topography is also described to play an important role in cellular interaction. Therefore, in this study, PCL nanofibers of different diameters and orientations were produced and cellular interaction was evaluated for both untreated and plasma-activated nanofibers. MATERIALS AND METHODS: Random and aligned PCL nanofibers of three different diameters were produced by electrospinning and subsequently plasma-activated. Furthermore, cellular interaction was evaluated by seeding adipose-derived stem cells onto the nanofibers. Cell morphology was evaluated by means of scanning electron microscopy, while cell survival and proliferation was evaluated with live/dead staining and MTT assays. RESULTS AND DISCUSSION: Fiber orientation had a clear influence on cell morphology, with irregular-shaped cells on the random nanofibers and more elongated cells on the aligned nanofibers. Cell proliferation, on the other hand, was similar for random and aligned nanofibers. Due to the incorporation of functional groups, plasma treatment of the PCL nanofibers resulted in higher proliferation rates as compared to untreated samples, both for random and aligned nanofibers. In case of the untreated samples, higher nanofiber diameters seemed to have a positive influence on cell proliferation. For the plasma-treated samples, however, this could not be observed. CONCLUSIONS: To improve cellular interaction with PCL nanofibers, either plasma-treatment or an increase of surface roughness can be employed, but a combination of both has no synergistic effect

Fabrication of PEOT/PBT nanofibers by atmospheric pressure plasma jet treatment of electrospinning solutions for tissue engineering

This study focuses on the enhanced electrospinning of 300-Polyethylene oxide-polyethylene oxide terephthalate/polybutylene terephthalate (PEOT/PBT). An atmospheric pressure plasma jet for liquid treatment is applied to a solution with 9 w/v% PEOT/PBT dissolved in either chloroform (CHCl3), CHCl3 + N,N-dimethylformamide (DMF), CHCl3 + methanol (MeOH), or CHCl3 + hexafluoroisopropanol (HFIP). For all conditions, the plasma-treated samples present better-quality fibers: less or no-beads and uniform fiber diameter distribution. Except for CHCl3 + DMF, no significant changes to the material bulk are detected, as shown with size exclusion chromatography (SEC). X-ray photoelectron spectroscopy (XPS) spectra performed on nanofibers record an increase in C-C bonds for the CHCl3 + DMF combination upon plasma modification, while a shift and slight increase in oxygen-containing bonds is found for the CHCl3 + HFIP and CHCl3 + MeOH mixtures. MTT assay shows no-cytotoxic effects for CHCl3 + DMF, while a better cellular adhesion is found on nanofibers from CHCl3 + MeOH and CHCl3 + HFIP. Among the examined additives, MeOH is preferable as it produces beadless electrospun nanofibers with an average diameter of 290 +/- 100 nm without causing significant changes to the final nanofiber surface properties

Probing polydopamine adhesion to protein and polymer films: microscopic and spectroscopic evaluation

Polydopamine has been found to be a biocompatible polymer capable of supporting cell growth and attachment, and to have antibacterial and antifouling properties. Together with its ease of manufacture and application, it ought to make an ideal biomaterial and function well as a coating for implants. In this paper, atomic force microscopy was used to measure the adhesive forces between polymer-, protein- or polydopamine-coated surfaces and a silicon nitride or polydopamine-functionalised probes. Surfaces were further characterised by contact angle goniometry, and solutions by circular dichroism. Polydopamine was further characterised with infrared spectroscopy and Raman spectroscopy. It was found that polydopamine functionalisation of the atomic force microscope probe significantly reduced adhesion to all tested surfaces. For example, adhesion to mica fell from 0.27 ± 0.7 nN nm-1 to 0.05 ± 0.01 nN nm-1. The results suggest that polydopamine coatings are suitable to be used for a variety of biomedical applications

Lessons learned: A comparative study of the integration experiences of Armenian and Palestinian refugees in Lebanon

This thesis analyzes the factors contributing to the differing degrees of integration achieved by the Armenian and Palestinian refugees in Lebanon. The two case studies presented include a historical overview of these refugees in Lebanon, the international response to the refugee crisis, the legal status of the discussed refugees in Lebanon, and the evolution of refugee identity. The analysis of the case studies uses the conceptual framework of Alastair Ager and Alison Strang, who wrote Understanding Integration, which appeared in the June 2008 Journal of Refugee Studies. The thesis also reviews the available literature written from a Western perspective, which rarely considers the role of religion in the integration process of refugees. In the Lebanese dilemma, where demographic balance is a major factor in domestic stability, religious affiliation emerges as a main influence in the contrasting treatment of Armenian and Palestinian refugees. With the continued conflict in Syria, Lebanon and other countries face similar challenges in addressing the refugee crisis. Thus, successful integration methods must include not only policies that address the humanitarian side of refugee flows but also the burden on host communities. Based on the case studies, the thesis concludes with recommendations for successfully integrating Syrian refugees in Lebanon, and policies that other host countries could adapt.http://archive.org/details/lessonslearnedac1094555600Captain, Lebanese ArmyApproved for public release; distribution is unlimited