Advanced Hollow Cathode Discharge Plasma Treatment of Unique Bilayered Fibrous Nerve Guidance Conduits for Enhanced/Oriented Neurite Outgrowth.

Despite all recent progresses in nerve tissue engineering, critical-sized nerve defects are still extremely challenging to repair. Therefore, this study targets the bridging of critical nerve defects and promoting an oriented neuronal outgrowth by engineering innovative nerve guidance conduits (NGCs) synergistically possessing exclusive topographical, chemical, and mechanical cues. To do so, a mechanically adequate mixture of polycaprolactone (PCL) and polylactic-co-glycolic acid (PLGA) was first carefully selected as base material to electrospin nanofibrous NGCs simulating the extracellular matrix. The electrospinning process was performed using a newly designed 2-pole air gap collector that leads to a one-step deposition of seamless NGCs having a bilayered architecture with an inner wall composed of highly aligned fibers and an outer wall consisting of randomly oriented fibers. This architecture is envisaged to afford guidance cues for the extension of long neurites on the underlying inner fiber alignment and to concurrently provide a sufficient nutrient supply through the pores of the outer random fibers. The surface chemistry of the NGCs was then modified making use of a hollow cathode discharge (HCD) plasma reactor purposely designed to allow an effective penetration of the reactive species into the NGCs to eventually treat their inner wall. X-ray photoelectron spectroscopy (XPS) results have indeed revealed a successful O2 plasma modification of the inner wall that exhibited a significantly increased oxygen content (24 → 28%), which led to an enhanced surface wettability. The treatment increased the surface nanoroughness of the fibers forming the NGCs as a result of an etching effect. This effect reduced the ultimate tensile strength of the NGCs while preserving their high flexibility. Finally, pheochromocytoma (PC12) cells were cultured on the NGCs to monitor their ability to extend neurites which is the base of a good nerve regeneration. In addition to remarkably improved cell adhesion and proliferation on the plasma-treated NGCs, an outstanding neural differentiation occurred. In fact, PC12 cells seeded on the treated samples extended numerous long neurites eventually establishing a neural network-like morphology with an overall neurite direction following the alignment of the underlying fibers. Overall, PCL/PLGA NGCs electrospun using the 2-pole air gap collector and O2 plasma-treated using an HCD reactor are promising candidates toward a full repair of critical nerve damage.

Complete Static Repopulation of Decellularized Porcine Tissues for Heart Valve Engineering: An in vitro Study.

To date, a completely in vitro repopulated tissue-engineered heart valve has not been developed. This study focused on sequentially seeding 2 cell populations onto porcine decellularized heart valve leaflets (HVL) and pericardia (PER) to obtain fully repopulated tissues. For repopulation of the interstitium, porcine valvular interstitial cells (VIC) and bone marrow-derived mesenchymal stem cells (BM-MSC) or adipose tissue-derived stem cells (ADSC) were used. In parallel, the culture medium was supplemented with ascorbic acid 2-phosphate (AA) and its effect on recolonization was investigated. Subsequently and in order to obtain an endothelial surface layer similar to those in native HVL, valvular endothelial cells (VEC) were seeded onto the scaffolds. It was shown that VIC efficiently recolonized HVL and partially also PER. On the other hand, stem cells only demonstrated limited or no subsurface cell infiltration of HVL and PER. Interestingly, the addition of AA increased the migratory capacity of both stem cell populations. However, this was more pronounced for BM-MSC, and recolonization of HVL appeared to be more efficient than that of PER tissue. VEC were demonstrated to generate a new endothelial layer on HVL and PER. However, scanning microscopy revealed that these endothelial cells were not allowed to fully spread onto PER. This study provided a proof of concept for the future generation of a bioactive tissue-engineered heart valve by showing that bioactive HVL could be generated in vitro within 14 days via complete repopulation of the interstitium with BM-MSC or VIC and subsequent generation of an entirely new endothelium.

The role of plasma-induced surface chemistry on polycaprolactone nanofibers to direct chondrogenic differentiation of human mesenchymal stem cells.

Bone marrow-derived mesenchymal stromal cells (BMSCs) are extensively being utilized for cartilage regeneration owing to their excellent differentiation potential and availability. However, controlled differentiation of BMSCs towards cartilaginous phenotypes to heal full-thickness cartilage defects remains challenging. This study investigates how different surface properties induced by either coating deposition or biomolecules immobilization onto nanofibers (NFs) could affect BMSCs chondro-inductive behavior. Accordingly, electrospun poly(ε-caprolactone) (PCL) NFs were exposed to two surface modification strategies based on medium-pressure plasma technology. The first strategy is plasma polymerization, in which cyclopropylamine (CPA) or acrylic acid (AcAc) monomers were plasma polymerized to obtain amine- or carboxylic acid-rich NFs, respectively. The second strategy uses a combination of CPA plasma polymerization and a post-chemical technique to immobilize chondroitin sulfate (CS) onto the NFs. These modifications could affect surface roughness, hydrophilicity, and chemical composition while preserving the NFs' nano-morphology. The results of long-term BMSCs culture in both basic and chondrogenic media proved that the surface modifications modulated BMSCs chondrogenic differentiation. Indeed, the incorporation of polar groups by different modification strategies had a positive impact on the cell proliferation rate, production of the glycosaminoglycan matrix, and expression of extracellular matrix proteins (collagen I and collagen II). The chondro-inductive behavior of the samples was highly dependent on the nature of the introduced polar functional groups. Among all samples, carboxylic acid-rich NFs promoted chondrogenesis by higher expression of aggrecan, Sox9, and collagen II with downregulation of hypertrophic markers. Hence, this approach showed an intrinsic potential to have a non-hypertrophic chondrogenic cell phenotype.

Potentials of a Plasma-Aerosol System for Wound Healing Advanced by Drug Introduction: An In Vitro Study.

Cold plasmas have found their application in a wide range of biomedical fields by virtue of their high chemical reactivity. In the past decades, many attempts have been made to use cold plasmas in wound healing, and within this field, many studies have focused on plasma-induced cell proliferation mechanisms. In this work, one step further has been taken to demonstrate the advanced role of plasma in wound healing. To this end, the simultaneous ability of plasma to induce cell proliferation and permeabilize treated cells has been examined in the current study. The driving force was to advance the wound healing effect of plasma with drug delivery. On this subject, we demonstrate in vitro the healing effect of Ar, Ar+N2 plasma, and their aerosol counterparts. A systematic study has been carried out to study the role of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in cell adhesion, signaling, differentiation, and proliferation. An additional investigation was also performed to study the permeabilization of cells and the delivery of the modeled drug carrier fluorescein isothiocyanate (FITC) labeled dextran into cells upon plasma treatment. Short 35 s plasma treatments were found to promote fibroblast adhesion, migration, signaling, proliferation, and differentiation by means of reactive oxygen and nitrogen species (RONS) created by plasma and deposited into the cell environment. The impact of the plasma downstream products NO2- and NO3- on the expressions of the focal adhesion's genes, syndecans, and collagens was observed to be prominent. On the other hand, the differentiation of fibroblasts to myofibroblasts was mainly initiated by ROS produced by the plasma. In addition, the ability of plasma to locally permeabilize fibroblast cells was demonstrated. During proliferative cell treatment, plasma can simultaneously induce cell membrane permeabilization (d ∼ 7.3 nm) by the species OH and H2O2. The choice for a plasma or a plasma-aerosol configuration thus allows the possibility to change the spatial chemistry of drug delivery molecules and thus to locally deliver drugs. Accordingly, this study offers a pivotal step toward plasma-assisted wound healing advanced by drug delivery.

Biological activity and antimicrobial property of Cu/a-C:H nanocomposites and nanolayered coatings on titanium substrates.

Infection associated with titanium based implants remains the most serious problem in implant surgery hence it is important to find optimal strategies to prevent infections. In the present study, we investigated the surface properties, antibacterial activity and biocompatibility of nanocomposite coatings based on an amorphous hydrocarbon (a-C:H) film containing copper nanoparticles (CuNPs) deposited on Ti discs via a gas aggregation cluster source. Three different Cu/a-C:H coatings with approximately the same amount of embedded CuNPs with and without barrier a-C:H layer were fabricated. The obtained results revealed that different structures of the produced coatings have significantly different release rates of Cu ions from the coatings into the aqueous media. This subsequently influences the antibacterial efficiency and osteoblast cell viability of the treated coatings. Coatings with the highest number of CuNPs resulted in excellent antibacterial activity exhibiting approximately 4 log reduction of E.coli and S.aureus after 24 h incubation. The cytotoxicity study revealed that after 7 day cell seeding, even the coating with the highest Cu at.% (4 at.%) showed a cell viability of Ì´90%. Consequently, the coating, formed with a properly tailored number of CuNPs and a-C:H barrier thickness offer a strong antibacterial effect without any harm to osteoblast cells.

Engineering microvasculature by 3D bioprinting of prevascularized spheroids in photo-crosslinkable gelatin.

To engineer tissues with clinically relevant dimensions by three-dimensional bioprinting, an extended vascular network with diameters ranging from the macro- to micro-scale needs to be integrated. Extrusion-based bioprinting is the most commonly applied bioprinting technique but due to the limited resolution of conventional bioprinters, the establishment of a microvascular network for the transfer of oxygen, nutrients and metabolic waste products remains challenging. To answer this need, this study assessed the potential and processability of spheroids, containing a capillary-like network, to be used as micron-sized prevascularized units for incorporation throughout the bioprinted construct. Prevascularized spheroids were generated by combining endothelial cells with fibroblasts and adipose tissue-derived mesenchymal stem cells as supporting cells. To serve as a viscous medium for the bioink-based deposition by extrusion printing, spheroids were combined with a photo-crosslinkable methacrylamide-modified gelatin (gelMA) and Irgacure 2959. The influence of gelMA encapsulation, the printing process and photo-crosslinking conditions on spheroid viability, proliferation and vascularization were analyzed by live/dead staining, immunohistochemistry, gene expression analysis and sprouting analysis. Stable spheroid-laden constructs, allowing spheroid outgrowth, were achieved by applying 10 min UV-A photo-curing (365 nm, 4 mW cm-2), while the construct was incubated in an additional Irgacure 2959 immersion solution. Following implantationin ovoonto a chick chorioallantoic membrane, the prevascular engineered constructs showed anastomosis with the host vasculature. This study demonstrated (a) the potential of triculture prevascularized spheroids for application as multicellular building blocks, (b) the processability of the spheroid-laden gelMA bioink by extrusion bioprinting and (c) the importance of photo-crosslinking parameters post printing, as prolonged photo-curing intervals showed to be detrimental for the angiogenic potential and complete vascularization of the construct post printing.

Combinatorial effects of coral addition and plasma treatment on the properties of chitosan/polyethylene oxide nanofibers intended for bone tissue engineering.

Given the complex calcified nature of the fibrous bone tissue, a combinatorial approach merging specific topographical/biochemical cues was adopted to design bone tissue-engineered scaffolds. Coral having a Ca-enriched structure was added to electrospun chitosan (CS)/polyethylene oxide (PEO) nanofibers that were subjected to plasma surface modifications using a medium pressure Ar, air or N2 dielectric barrier discharge. Plasma incorporated oxygen- and nitrogen-containing functionalities onto the nanofibers surface thus enhancing their wettability. Plasma treatment enhanced the performance of osteoblasts and the interplay between plasma treatment and coral was shown to boost initial cell adhesion. The fibers capacity to trigger calcium phosphate growth was predicted via immersion in simulated body fluid. Globular carbonate apatite nanocrystals were deposited on plasma-treated CS/PEO NFs while thicker layers of flake-like nanocrystals were covering plasma-treated Coral/CS/PEO fibers without blocking the interfibrous pores. Overall, the exclusive multifaceted plasma-treated Coral/CS/PEO nanofibers are believed to revolutionize the bone tissue engineering field.

Hybrid Bioprinting of Chondrogenically Induced Human Mesenchymal Stem Cell Spheroids.

To date, the treatment of articular cartilage lesions remains challenging. A promising strategy for the development of new regenerative therapies is hybrid bioprinting, combining the principles of developmental biology, biomaterial science, and 3D bioprinting. In this approach, scaffold-free cartilage microtissues with small diameters are used as building blocks, combined with a photo-crosslinkable hydrogel and subsequently bioprinted. Spheroids of human bone marrow-derived mesenchymal stem cells (hBM-MSC) are created using a high-throughput microwell system and chondrogenic differentiation is induced during 42 days by applying chondrogenic culture medium and low oxygen tension (5%). Stable and homogeneous cartilage spheroids with a mean diameter of 116 ± 2.80 µm, which is compatible with bioprinting, were created after 14 days of culture and a glycosaminoglycans (GAG)- and collagen II-positive extracellular matrix (ECM) was observed. Spheroids were able to assemble at random into a macrotissue, driven by developmental biology tissue fusion processes, and after 72 h of culture, a compact macrotissue was formed. In a directed assembly approach, spheroids were assembled with high spatial control using the bio-ink based extrusion bioprinting approach. Therefore, 14-day spheroids were combined with a photo-crosslinkable methacrylamide-modified gelatin (gelMA) as viscous printing medium to ensure shape fidelity of the printed construct. The photo-initiators Irgacure 2959 and Li-TPO-L were evaluated by assessing their effect on bio-ink properties and the chondrogenic phenotype. The encapsulation in gelMA resulted in further chondrogenic maturation observed by an increased production of GAG and a reduction of collagen I. Moreover, the use of Li-TPO-L lead to constructs with lower stiffness which induced a decrease of collagen I and an increase in GAG and collagen II production. After 3D bioprinting, spheroids remained viable and the cartilage phenotype was maintained. Our findings demonstrate that hBM-MSC spheroids are able to differentiate into cartilage microtissues and display a geometry compatible with 3D bioprinting. Furthermore, for hybrid bioprinting of these spheroids, gelMA is a promising material as it exhibits favorable properties in terms of printability and it supports the viability and chondrogenic phenotype of hBM-MSC microtissues. Moreover, it was shown that a lower hydrogel stiffness enhances further chondrogenic maturation after bioprinting.

Fabrication and Plasma Modification of Nanofibrous Tissue Engineering Scaffolds.

This paper provides a comprehensive overview of nanofibrous structures for tissue engineering purposes and the role of non-thermal plasma technology (NTP) within this field. Special attention is first given to nanofiber fabrication strategies, including thermally-induced phase separation, molecular self-assembly, and electrospinning, highlighting their strengths, weaknesses, and potentials. The review then continues to discuss the biodegradable polyesters typically employed for nanofiber fabrication, while the primary focus lies on their applicability and limitations. From thereon, the reader is introduced to the concept of NTP and its application in plasma-assisted surface modification of nanofibrous scaffolds. The final part of the review discusses the available literature on NTP-modified nanofibers looking at the impact of plasma activation and polymerization treatments on nanofiber wettability, surface chemistry, cell adhesion/proliferation and protein grafting. As such, this review provides a complete introduction into NTP-modified nanofibers, while aiming to address the current unexplored potentials left within the field.

Water-Stable Plasma-Polymerized N,N-Dimethylacrylamide Coatings to Control Cellular Adhesion.

The plasma polymerization of amide-based precursors is a nearly unexplored research area, which is in contrast with the abundance of reports focusing on amide-based surface modification using wet chemistry. Therefore, this study aims to profoundly investigate the near-atmospheric pressure plasma polymerization of N,N-dimethylacrylamide (DMAM) to obtain stable coatings. In contrast to the unstable coatings obtained at lower discharge powers, the stable coatings that were obtained at higher powers showed a lower hydrophilicity as assessed by water contact angle (WCA). This decrease in hydrophilicity with increasing plasma power was found to be related to a reduced preservation of the monomer structure, as observed by Fourier transform infrared (FTIR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), and XPS C60 depth profiling, a rarely used but effective combination of techniques. Furthermore, the chemical composition of the coating was found to be in good agreement with the plasma active species observed by optical emission spectroscopy. Additionally, XPS C60 depth profiling indicated a difference between the top layer and bulk of the plasma polymer due to spontaneous oxidation and/or postplasma coating deposition. Finally, the stable coatings were also found to have cell-interactive behavior toward MC3T3 as studied by in vitro live/dead fluorescence imaging and (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assays. With the latter technique, a cell viability of up to 89% as compared with tissue culture plates after 1 day of cell culture was observed, indicating the potential of these coatings for tissue engineering purposes.

Investigation of Ag/a-C:H Nanocomposite Coatings on Titanium for Orthopedic Applications.

One of the leading causes of failure for any bone implant is implant-associated infections. The implant-bone interface is in fact the crucial site of infection where both the microorganisms and cells compete to populate the newly introduced implant surface. Most of the work dealing with this issue has focused on the design of implant coatings capable of preventing infection while ignoring cell proliferation or vice versa. The present study is therefore focused on investigating the antibacterial and biological properties of nanocomposite coatings based on an amorphous hydrocarbon (a-C:H) matrix containing silver nanoparticles (AgNPs). a-C:H coatings with varying silver concentrations were generated directly on medical grade titanium substrates using a combination of a gas aggregation source (GAS) and a plasma-enhanced chemical vapor deposition (PE-CVD) process. The obtained results revealed that the surface silver content increased from 1.3 at % to 5.3 at % by increasing the used DC magnetron current in the GAS from 200 to 500 mA. The in vitro antibacterial assays revealed that the nanocomposites with the highest number of silver content exhibited excellent antibacterial activities resulting in a 6-log reduction of Escherichia coli and a 4-log reduction of Staphylococcus aureus after 24 h of incubation. An MTT assay, fluorescence live/dead staining, and SEM microscopy observations of MC3T3 cells seeded on the uncoated and coated Ti substrates also showed that increasing the amount of AgNPs in the nanocomposites had no notable impact on their cytocompatibility, while improved cell proliferation was especially observed for the nanocomposites possessing a low amount of AgNPs. These controllable Ag/a-C:H nanocomposites on Ti substrates, which simultaneously provide an excellent antibacterial performance and good biocompatibility, could thus have promising applications in orthopedics and other biomedical implants.

Fabrication of Microporous Coatings on Titanium Implants with Improved Mechanical, Antibacterial, and Cell-Interactive Properties.

The success of an orthopedic implant therapy depends on successful bone integration and the prevention of microbial infections. In this work, plasma electrolytic oxidation (PEO) was performed to deposit TiO2 coatings enriched with Ca, P, and Ag on titanium to improve its surface properties and antibacterial efficacy while maintaining normal biological functions and thus to enhance the performance of orthopedic implants. After PEO treatment, the surface of Ti was converted to anatase and rutile TiO2, hydroxyapatite, and calcium titanate phases. The presence of these crystalline phases was further increased with an increased Ag content in the coatings. The developed coatings also exhibited a more porous morphology with an improved surface wettability, roughness, microhardness, and frictional coefficient. In vitro antibacterial assays indicated that the Ag-doped coatings can significantly prevent the growth of both Staphylococcus aureus and Escherichia coli by releasing Ag+ ions, and the ability to prevent these bacteria was enhanced by increasing the Ag content in the coatings, resulting in a maximal 6-log reduction of E. coli and a maximal 5-log reduction of S. aureus after 24 h of incubation. Moreover, the in vitro cytocompatibility evaluation of the coatings showed that the osteoblast (MC3T3) cell integration on the PEO-based coatings was greatly improved compared to untreated Ti and no notable impact on their cytocompatibility was observed on increasing the amount of Ag in the coating. In conclusion, the coating with favorable physicochemical and mechanical properties along with controlled silver ion release can offer an excellent antibacterial performance and osteocompatibility and can thus become a prospective coating strategy to face current challenges in orthopedics.

Biocompatibility of Cyclopropylamine-Based Plasma Polymers Deposited at Sub-Atmospheric Pressure on Poly (ε-caprolactone) Nanofiber Meshes.

In this work, cyclopropylamine (CPA) monomer was plasma-polymerized on poly (ε-caprolactone) nanofiber meshes using various deposition durations to obtain amine-rich surfaces in an effort to improve the cellular response of the meshes. Scanning electron microscopy and X-ray photoelectron spectroscopy (XPS) were used to investigate the surface morphology and surface chemical composition of the PCL samples, respectively. The measured coating thickness was found to linearly increase with deposition duration at a deposition rate of 0.465 nm/s. XPS analysis revealed that plasma exposure time had a considerable effect on the surface N/C and O/C ratio as well as on amino grafting efficiency and amino selectivity. In addition, cell studies showed that cell adhesion and proliferation significantly improved for all coated samples.

Micropatterning of beta tricalcium phosphate bioceramic surfaces, by femtosecond laser, for bone marrow stem cells behavior assessment.

The bioactivity of synthetic bone implants is highly impacted by their surface topography, especially by the presence of micro-patterns likely to generate cells growth guidance. In this study, laser machining technology was employed in order to produce controlled regular micro-patterns on dense calcium phosphate surfaces, without any contamination. The choice of the source was directed towards a femtosecond pulsed laser in order to limit the thermal impact of such a process and thus to avoid the unwanted phase transformations potentially induced by the temperature elevation. Beta tricalcium phosphate substrates with perfectly controlled micro-patterning and without any secondary phase were obtained by optimization of the process parameters (laser power, scanning speed, pulse frequency). The microstructural characteristics were investigated by microscopy (optical, confocal, scanning electron) and the phase identification was performed by X-ray diffraction. This work allowed highlighting the effects of the process parameters on the patterning. The high benefits of the laser treatment on wettability were shown by contact angle assays. Finally, the influence of surface micro-patterning on cell behavior was highlighted in vitro. This technique seems to provide an interesting alternative to conventional surface treatments of calcium phosphates.

Influence of the Aliphatic Side Chain on the Near Atmospheric Pressure Plasma Polymerization of 2-Alkyl-2-oxazolines for Biomedical Applications.

Plasma polymerization is gaining popularity as a technique for coating surfaces due to the low cost, ease of operation, and substrate-independent nature. Recently, the plasma polymerization (or deposition) of 2-oxazoline monomers was reported resulting in coatings that have potential applications in regenerative medicine. Despite the structural versatility of 2-oxazolines, only a few monomers have been subjected to plasma polymerization. Within this study, however, we explore the near atmospheric pressure plasma polymerization of a range of 2-oxazoline monomers, focusing on the influence of the aliphatic side-chain length (methyl to butyl) on the plasma polymerization process conditions as well as the properties of the obtained coatings. While side-chain length had only a minor influence on the chemical composition, clear effects on the plasma polymerization conditions were observed, thus gaining valuable insights in the plasma polymerization process as a function of monomer structure. Additionally, cytocompatibility and cell attachment on the coatings obtained by 2-oxazoline plasma polymerization was assessed. The coatings displayed strong cell interactive properties, whereby cytocompatibility increased with increasing aliphatic side-chain length of the monomer, reaching up to 93% cell viability after 1 day of cell culture compared to tissue culture plates. As this is in stark contrast to the antifouling behavior of the parent polymers, we compared the properties and composition of the plasma-polymerized coatings to the parent polymers revealing that a significantly different coating structure was obtained by plasma polymerization.

Surface Treatment of PEOT/PBT (55/45) with a Dielectric Barrier Discharge in Air, Helium, Argon and Nitrogen at Medium Pressure.

This work describes the surface modification of 300PEO-PEOT/PBT 55/45 thin films using a medium pressure dielectric barrier discharge system operated in argon, helium, nitrogen or dry air to improve cell-surface interactions of this established biomaterial. The first part of the paper describes the optimization of the plasma processing parameters using water contact angle goniometry. The optimized samples are then characterized for changes in surface topography and surface chemical composition using atomic force microscopy (AFM) and X-ray fluorescence spectroscopy (XPS) respectively. For all plasma treatments, a pronounced increase in surface wettability was observed, of which the extent is dependent on the used plasma discharge gas. Except for dry air, only minor changes in surface topography were noted, while XPS confirmed that the changes in wettability were mainly chemical in nature with the incorporation of 5-10% of extra oxygen as a variety of polar groups. Similarly, for the nitrogen plasma, 3.8% of nitrogen polar groups were additionally incorporated. Human foreskin fibroblast (HFF) in vitro analysis showed that within the first 24 h after cell seeding, the effects on cell-surface interactivity were highly dependent on the used discharge gas, nitrogen plasma treatment being the most efficient. Differences between untreated and plasma-treated samples were less pronounced compared to other biodegradable materials, but a positive influence on cell adhesion and proliferation was still observed.

Plasma Functionalization of Polycaprolactone Nanofibers Changes Protein Interactions with Cells, Resulting in Increased Cell Viability.

The surface properties of electrospun scaffolds can greatly influence protein adsorption and, thus, strongly dictate cell-material interactions. In this study, we aim to investigate possible correlations between the surface properties of argon, nitrogen, and ammonia and helium plasma-functionalized polycaprolactone (PCL) nanofibers (NFs) and their cellular interactions by examining the protein corona patterns of the plasma-treated NFs as well as the cell membrane proteins involved in cell proliferation. As a result of the performed plasma treatments, PCL NFs morphology was preserved, while wettability was improved profoundly after all treatments because of the incorporation of polar surface groups. Depending on the discharge gas, different types of groups are incorporated, which influenced the resultant cell-material interactions. Argon plasma-functionalized PCL NFs, only enriched by oxygen-containing functional groups, were found to show the best cell-material interactions, followed by N2 and He/NH3 plasma-treated samples. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and liquid chromatography-mass spectrometry clearly indicated an increased protein retention compared with non-treated PCL NFs. The nine proteins retained best on plasma-treated NF are important mediators of extracellular matrix interaction, illustrating the importance thereof for cell proliferation and the viability of cells. Finally, 92 proteins that can be used to differentiate how the different plasma treatments are clustered and subjected to a gene ontology study, illustrating the importance of keratinization and extracellular matrix organization.

Effects of a dielectric barrier discharge (DBD) treatment on chitosan/polyethylene oxide nanofibers and their cellular interactions.

In this study, chitosan (CS)/polyethylene oxide (PEO) nanofibrous mats (Ø: 166 ± 43 nm) were fabricated by electrospinning and subsequently surface-modified by a dielectric barrier discharge (DBD) sustained in argon, ammonia/helium or nitrogen. The surface properties of the CS/PEO nanofibers (NFs) before and after plasma treatment were characterized using contact angle measurements, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). Additionally, the mechanical properties and PEO leaching in aqueous conditions of the different NFs under study were examined by tensile tests and nuclear magnetic resonance (1H NMR) spectroscopy respectively. Finally, cell behavior and cell morphology of human foreskin fibroblasts (HFFs) on the CS/PEO NFs were evaluated via live/dead fluorescence microscopy, MTT assays and SEM. The obtained results revealed that the surface free energy of the CS/PEO NFs was significantly increased after plasma modification, which was correlated to an enrichment in surface oxygen (Ar, N2, NH3/He) and nitrogen (N2, NH3/He) functional groups. All performed plasma treatments also led to an increase in ultimate tensile strength, most likely due to an increased fiber-to-fiber friction. Additionally, it was also observed that N2 plasma treatment resulted in a decrease in PEO release, which could be attributed to more pronounced surface cross-linking. Cellular interactions on the CS/PEO NFs also significantly increased due to the performed plasma treatments. The best cellular response was noted for the Ar plasma modification although the surface hydrophilicity was the lowest in this case. These observations thus suggest that not only the wettability characteristics but also the presence of distinct functional groups on plasma-treated CS/PEO NFs have a significant influence on the observed enhanced cellular interactions.

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.

Atmospheric Pressure Plasma Jet Treatment of Poly-ε-caprolactone Polymer Solutions To Improve Electrospinning.

An atmospheric pressure plasma jet (APPJ) specifically designed for liquid treatment has been used in this work to improve the electrospinnability of a 5 w/v % solution of poly-ε-caprolactone (PCL) in a mixture of chloroform and N,N-dimethylformamide. Untreated PCL solutions were found to result in nonuniform fibers containing a large number of beads, whereas plasma-treated solutions (exposure time of 2-5 min) enabled the generation of beadless, uniform nanofibers with an average diameter of 450 nm. This enhanced electrospinnability was found to be mainly due to the highly increased conductivity of the plasma-modified PCL solutions. Consequently, more stretching of the polymer jet occurred during electrospinning, leading to the generation of bead-free fibers. Plasma treatment also results in an increased viscosity and decreased pH values. To explain these observed changes, optical emission spectroscopy (OES) has been used to examine the excited species present in the APPJ in contact with the PCL solution. This study revealed that the peaks attributed to H, CH, CH2, and C2 species could be responsible for the degradation of solvent molecules and/or PCL structures during the plasma treatment. Size exclusion chromatography and X-ray photoelectron spectroscopy results showed that the molecular weight and the chemical composition of PCL were not significantly affected by the APPJ treatment. Plasma exposure mainly results in the degradation of the solvent molecules instead of modifying the PCL macromolecules, preserving the original polymer as much as possible. A hypothesis for the observed macroscopic changes in viscosity and pH values could be the generation of new chemical species such as HCl and/or HNO3. These species are characterized by their high conductivity, low pH values, and strong polarity and could enhance the solvent quality for PCL, leading to the expansion of the polymer coil, which could in turn explain the observed enhanced viscosity after plasma modification.