Evaluation of bacterial nanocellulose-based uniform wound dressing for large area skin transplantation

Bacterial nanocellulose (BNC) was biosynthesized by Gluconacetobacter xylinus. The surface area, physicochemical structure and morphology of the materials were characterized. Here provides a method for an efficient production of uniform BNC, which is beneficial for the fast characterization and evaluation of BNC. In vitro cytotoxicity of the materials was evaluated by the proliferation, the adhesion, the viability and the morphology of NIH/3T3 cells. Low cytotoxicity of the BNC was observed, and micrographs demonstrate a good proliferation and adhesion of NIH/3T3 cells on BNC. Large area full-thickness skin defects were made on the back of C57BL/6 mice in animal surgery. The wounds were transplanted with BNC films and the results compared to those in a control group. The rehabilitation of the wound surfaces and the pathological sections of mice were investigated and are discussed. Histological examinations demonstrated faster and better healing effect and lower inflammatory response in the BNC group than those in the control group. Preliminary results on wound dressings from BNC show a curative effect promoting the healing of epithelial tissue. BNC is a promising natural polymer with medical applications in wound dressings.     

Critical fluid shear stress analysis for cell–polymer adhesion

A simple methodology to assess cell adhesion on materials was developed. We demonstrated that the cell adhesion strength could be quantified. Using this method, we were able to compare the NIH/3T3 Swiss mouse fibroblasts adhesion strength to poly(methyl methacrylate) and polycarbonate. A controlled fluid shear stress was applied to cells using a parallel plate rotational system. Cells detached from the surface in the radial direction. Results showed that there was a critical radius where the shear stress experienced by the cells equaled the cell adhesion strength. The cells outside this radius were removed while those inside maintained initial confluency. The quantitative evaluation of cell adhesion is beneficial for development of biomaterials.     

Plasma-modified and polyethylene glycol-grafted polymers for potential tissue engineering applications

Modified and grafted polymers may serve as building blocks for creating artificial bioinspired nanostructured surfaces for tissue engineering. Polyethylene (PE) and polystyrene (PS) were modified by Ar plasma and the surface of the plasma activated polymers was grafted with polyethylene glycol (PEG). The changes in the surface wettability (contact angle) of the modified polymers were examined by goniometry. Atomic Force Microscopy (AFM) was used to determine the surface roughness and morphology and electrokinetical analysis (Zeta potential) characterized surface chemistry of the modified polymers. Plasma treatment and subsequent PEG grafting lead to dramatic changes in the polymer surface morphology, roughness and wettability. The plasma treated and PEG grafted polymers were seeded with rat vascular smooth muscle cells (VSMCs) and their adhesion and proliferation were studied. Biological tests, performed in vitro, show increased adhesion and proliferation of cells on modified polymers. Grafting with PEG increases cell proliferation, especially on PS. The cell proliferation was shown to be an increasing function of PEG molecular weight.     

Poly-l -lactic acid modified by etching and grafting with gold nanoparticles

This work is focused on characterization of plasma treated and consequently etched and grafted biocompatible polymer poly(l-lactide acid) (PLLA). The interaction of biodegradable polymers with cold plasma is of a great importance in a tissue engineering and surface science. Cold plasma exposure, grafting with gold nanoparticles and etching processes were successfully applied to biopolymer substrate. A method for biopolymer nanostructuring as combination of cold plasma treatment and Au nanoparticle grafting for biocompatibility improvement is introduced. Surface roughness, morphology and surface chemistry was determined. The plasma modification leads to significant increase in surface roughness of PLLA and appearance of sharp spikes and ridges on the PLLA surface. Modification by grafting and etching leads to significant changes in PLLA surface morphology and chemistry. The surface ablation of PLLA has been proved to be significant. In etching of plasma-modified PLLA, methanol proves to be stronger etching agent than water. The grafting of PLLA with gold nanoparticles improved mouse embryonic fibroblasts (NIH 3T3) adhesion and proliferation significantly.     

Differentiation of human mesenchymal stem cells on plasma-treated polyetheretherketone

Polyetheretherketone (PEEK) generally exhibits physical and chemical characteristics that prevent osseointegration. To activate the PEEK surface, we applied oxygen and ammonia plasma treatments. These treatments resulted in surface modifications, leading to changes in nanostructure, contact angle, electrochemical properties and protein adhesion in a plasma power and process gas dependent way. To evaluate the effect of the plasma-induced PEEK modifications on stem cell adhesion and differentiation, adipose tissue-derived mesenchymal stem cells (adMSC) were seeded on PEEK specimens. We demonstrated an increased adhesion, proliferation, and osteogenic differentiation of adMSC in contact to plasma-treated PEEK. In dependency on the process gas (oxygen or ammonia) and plasma power (between 10 and 200 W for 5 min), varying degrees of osteogenic differentiation were induced. When adMSC were grown on 10 and 50 W oxygen and ammonia plasma-treated PEEK substrates they exhibited a doubled mineralization degree relative to the original PEEK. Thus plasma treatment of PEEK specimens induced changes in surface chemistry and topography and supported osteogenic differentiation of adMSC in vitro. Therefore plasma treated PEEK holds perspective for contributing to osseointegration of dental and orthopedic load-bearing PEEK implants in vivo.     

Chemical activation and changes in surface morphology of poly(ε-caprolactone) modulate VEGF responsiveness of human endothelial cells

The high degree of clinical routine in percutaneous transluminal coronary angioplasty (PTCA) with and without stenting has not changed the fact that a large number of coronary heart disease patients are still affected by post-operative complications such as restenosis and thrombosis. Because re-endothelialization is the crucial aspect of wound healing after cardiovascular implant surgery, there is a need for modern biomaterials to aid endothelial cells in their adhesion and functional recovery post-stenting. This study systematically examines the potential of numerous chemical polymer modifications with regard to endothelialization. Poly(ε-caprolactone) (PCL) and its chemically activated forms are investigated in detail, as well as the impact of polymer surface morphology and precoating with matrix protein. Human umbilical vein endothelial cells (HUVECs) are used to characterize endothelial cell responses in terms of in vitro viability and adhesion. As a potential component in drug eluting implants, VEGF is applied as stimulus to boost endothelial cell proliferation on the polymer. In conclusion, plasma chemical activation of PCL combined with VEGF stimulation best enhances in vitro endothelialization. Examining the impact of morphological, chemical and biological modifications of PCL, this study makes an important new contribution towards the existing body of work on polymer endothelialization.     

Cell adhesion on modified polyethylene

Polyethylene (PE) was irradiated with 10 and 63 keV Ar⁺ ions to fluences of 1 × 10¹⁷ to 3 × 10¹⁹ m^–2 and then it was grafted with aminoacid (alanine). The changes of surface polarity, electrical conductivity, and oxygen concentration were examined on pristine, as-irradiated, and irradiated-grafted PE. The in vitro adhesion of mice fibroblasts on the modified PE was evaluated 24 hours after inoculation. It was proved that for the PE irradiated at 10 keV ion energy, the presence of chemically bound alanine increases cell adhesion and its homogenity. For PE irradiated with 63 keV ions, however, the alanine grafting leads to a reduction of the number of adhering cells. It was found that a rising surface polarity increases cell adhesion, but when its value is too high the cell adhesion starts to decrease. No correlation between electrical conductivity and cell adhesion was observed. In general, higher cell adhesion is observed on modified PE in comparison with pristine one.     

Cells cultured on microgrooves with or without surface coating: Correlation between cell alignment,spreading and local membrane deformation

The behaviors of cells cultured on patterned substrates vary with the material stiffness, the geometry and the biochemical properties of the pattern. By using a reversed cell imprinting (RCI) technique, together with phase contrast microscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM), we have exploited reversed side cellular morphology on patterned microgrooves of different geometries with or without surface coating of adhesion molecules. We have shown a close correlation between the effect of contact guidance and penetration of cellular membrane. Without surface coating, roughly 80% of HeLa cells were aligned along the groove direction regardless of the groove spacing. When the microgrooves were coated with fibronectin, the area of cell spreading was increased but the percentage of aligned cells was significantly decreased. In both cases, the deformation of cell membrane at the cell-pattern interfaces could be measured. We found that the local penetration of the cellular membrane into the grooves was correlated to the cellular alignment for both HeLa and NIH 3T3 cells, and that such a correlation was cell-type dependent.     

Influence of Nanopillar Arrays on Fibroblast Motility,Adhesion, and Migration Mechanisms

Surfaces decorated with high aspect ratio nanostructures are a promising tool to study cellular processes and design novel devices to control cellular behavior. However, little is known about the dynamics of cellular phenomenon such as adhesion, spreading, and migration on such surfaces. In particular, how these are influenced by the surface properties. In this work, fibroblast behavior is investigated on regular arrays of 1 µm high polymer nanopillars with varying pillar to pillar distance. Embryonic mouse fibroblasts (NIH‐3T3) spread on all arrays, and on contact with the substrate engulf nanopillars independently of the array pitch. As the cells start to spread, different behavior is observed. On dense arrays which have a pitch equal or below 1 µm, cells are suspended on top of the nanopillars, making only sporadic contact with the glass support. Cells stay attached to the glass support and fully engulf nanopillars during spreading and migration on the sparse arrays which have a pitch of 2 µm and above. These alternate states have a profound effect on cell migration rates. Dynamic F‐actin puncta colocalize with nanopillars during cell spreading and migration. Strong membrane association with engulfed nanopillars might explain the reduced migration rates on sparse arrays.     

The effect of plasma polymer coating using atmospheric-pressure glow discharge on the shear bond strength of composite resin to ceramic

If plasma technology can come out of the vacuum chamber and plasma can be extruded through a small pencil-type torch, it can be applied widely to dental practices. For this study, we designed a small pencil-type non-thermal atmospheric-pressure glow discharge plasma torch. The purpose of this study was to determine the effect of plasma polymer coating on the adhesion of composite resin to feldspathic porcelain. The effect of plasma polymer coating was evaluated using shear bond strength (SBS) test. Contact angle measurements and fracture mode analysis were also performed. Among the groups treated with plasma polymer coating, the SBS of the adhesive (Adper Scotchbond Multi-Purpose, 3M ESPE) to the ceramic surface pre-treated sequentially with water plasma and triethyleneglycol dimethacrylate (TEGDMA) plasma in helium gas was significantly higher than that of the adhesive to the untreated surface (p < 0.05). In this group, the predominant fracture mode was mixed fracture, where small cohesively fractured fragments of ceramic were dispersed on the adhesively fractured flat adhesive surface. However, the SBS values of all the plasma polymer-coated groups were lower than those obtained through a routine porcelain bonding procedure with HF acid and silane coupling agent (p < 0.05). The non-thermal atmospheric-pressure plasma polymer coating technique was found to have a potential promoting adhesion to dental materials.     

Increased response of Vero cells to PHBV matrices treated by plasma

The copolymers poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) are being intensely studied as a tissue engineering substrate. It is known that poly 3-hydroxybutyric acids (PHBs) and their copolymers are quite hydrophobic polyesters. Plasma-surface modification is an effective and economical surface treatment technique for many materials and of growing interest in biomedical engineering. In this study we investigate the advantages of oxygen and nitrogen plasma treatment to modify the PHBV surface to enable the acceleration of Vero cell adhesion and proliferation. PHBV was dissolved in methylene chloride at room temperature. The PHBV membranes were modified by oxygen or nitrogen-plasma treatments using a plasma generator. The membranes were sterilized by UV irradiation for 30 min and placed in 96-well plates. Vero cells were seeded onto the membranes and their proliferation onto the matrices was also determined by cytotoxicity and cell adhesion assay. After 2, 24, 48 and 120 h of incubation, growth of fibroblasts on matrices was observed by scanning electron microscopy (SEM). The analyses of the membranes indicated that the plasma treatment decreased the contact angle and increased the surface roughness; it also changed surface morphology, and consequently, enhanced the hydrophilic behavior of PHBV polymers. SEM analysis of Vero cells adhered to PHBV treated by plasma showed that the modified surface had allowed better cell attachment, spreading and growth than the untreated membrane. This combination of surface treatment and polymer chemistry is a valuable guide to prepare an appropriate surface for tissue engineering application.     

Evaluation of poly(vinyl alcohol) hydrogels as a component of hybrid artificial tissues

Hydrogels are three-dimensional polymeric networks very similar to biological tissues. Many synthetic polymers can be used in preparing hydrogels. Among them poly(vinyl alcohol) (PVA), physically crosslinked by repeated freeze-thawing cycles of polymer aqueous solutions, is widely employed to make hydrogels for biomedical applications. To increase the similarity between hydrogels and natural tissues and to obtain polymeric hybrid tissues, we attempted to incorporate 3T3 cells, from a mouse fibroblast cell line, into PVA hydrogels obtained by one freeze-thawing cycle using as a solvent complete culture medium. Hydrogels were also made using eight freeze-thawing cycles from PVA solutions prepared using as a solvent either complete culture medium or water. Cell adhesion experiments were performed by seeding 3T3 and human umbilical vein endothelial cells (HUVEC) on to the hydrogel surface. The effect of the solvent and of the different number of freeze-thawing cycles on the mechanical characteristics of the PVA hydrogels were investigated by dynamic-mechanical techniques. A scanning force microscope analysis of the hydrogel surface viscoelastic properties was also carried out. Our results show that PVA is not cytotoxic. Although PVA hydrogel surface characteristics do not seem to favour the adhesion of substrate-dependent cells, encouraging results were obtained with the 3T3 cells incorporation. DMA analysis indicates that the networks prepared by eight freeze-thawing cycles possess a mechanical consistency comparable, even slightly better, than the ones prepared by only one freeze-thawing cycle and used for the cell incorporation studies.     

Influence of polymer type, composition, and interface on the structural and mechanical properties of core/sheath type bicomponent nonwoven fibers

In this study, we investigated the effect of polymer type, composition, and interface on the structural and mechanical properties of core–sheath type bicomponent nonwoven fibers. These fibers were produced using poly(ethylene terephthalate)/polyethylene (PET/PE), polyamide 6/polyethylene (PA6/PE), polyamide 6/polypropylene (PA6/PP), polypropylene/polyethylene (PP/PE) polymer configurations at varying compositions. The crystallinity, crystalline structure, and thermal behavior of each component in bicomponent fibers were studied and compared with their homocomponent counterparts. We found that the fiber structure of the core component was enhanced in PET/PE, PA6/PE, and PA6/PP whereas that of the sheath component was degraded in all polymer combinations compared to corresponding single component fibers. The degrees of these changes were also shown to be composition dependent. These results were attributed to the mutual interaction between two components and its effect on the thermal and stress histories experienced by polymers during bicomponent fiber spinning. For the interface study, the polymer–polymer compatibility and the interfacial adhesion for the laminates of corresponding polymeric films were determined. It was shown that PP/PE was the most compatible polymer pairing with the highest interfacial adhesion value. On the other hand, PET/PE was found to be the most incompatible polymer pairings followed by PA6/PP and PA6/PE. Accordingly, the tensile strength values of the bicomponent fibers deviated from the theoretically estimated values depending on core–sheath compatibility. Thus, while PP/PE yielded a higher tensile strength value than estimated, other polymer combinations showed lower values in accordance with their degree of incompatibility and interfacial adhesion. These results unveiled the direct relation between interface and tensile response of the bicomponent fiber.     

Adhesion and proliferation of fibroblasts on cluster-assembled nanostructured carbon films: the role of surface morphology

We have investigated the influence on adhesion and proliferation of NIH 3T3 fibroblasts of the surface morphology of cluster assembled carbon films deposited by Supersonic Cluster Beam Deposition. Nanostructured carbon films exhibit a multi-scale morphology, which resembles the surface structure of the extracellular matrix, and possess a high specific area, while being relatively smooth at all scales. Correlations between measured morphological parameters and adaptive cell response have been brought out. High specific area and smoothness appear to conceivably favour both the early attachment of plated cells and the long-term survival of adherent cells. Moreover, nano-structured carbon films affect the cells morphology as well as the extension and the number of the focal contacts.     

Polyetheretherketone and titanium surface treatments to modify roughness and wettability-Improvement of bioactivity and antibacterial properties

Among the materials available for implant production,titanium is the most used while polyetherether-ketone (PEEK) is emerging thanks to its stability and to the mechanical properties similar to the ones of the bone tissue.Material surface properties like roughness and wettability play a paramount role in cell adhesion,cell proliferation,osteointegration and implant stability.Moreover,the bacterial adhesion to the biomaterial and the biofilm formation depend on surface smoothness and hydrophobicity.In this work,two different treatments,sandblasting and air plasma,were used to increase respectively roughness and wettability of two materials:titanium and PEEK.Their effects were analyzed with profilometry and contact angle measurements.The biological properties of the material surfaces were also investigated in terms of cell adhesion and proliferation of NIH-3T3 cells,MG63 cells and human Dental Pulp Stem Cells.Moreover,the ability of Staphylococcus aureus to adhere and form a viable biofilm on the samples was evaluated.The biological properties of both treatments and both materials were compared with samples of Synthegra(R) titanium,which underwent laser ablation to obtain a porous micropatterning,character-ized by a smooth surface to discourage bacterial adhesion.All cell types used were able to adhere and proliferate on samples of the tested materials.Cell adhesion was higher on sandblasted PEEK samples for both MG63 and NIH-3T3 cell lines,on the contrary,the highest proliferation rate was observed on sandblasted titanium and was only slightly dependent on wettability;hDPSCs were able to proliferate similarly on sandblasted samples of both tested materials.The highest osteoblast differentiation was ob-served on laser micropatterned titanium samples,but similar effects,even if limited,were also observed on both sandblasted materials and air plasma treated titanium.The lowest bacterial adhesion and biofilm formation was observed on micropatterned titanium samples whereas,the highest biofilm formation was detected on sandblasted PEEK samples,and in particular on samples not treated with air-plasma,which displayed the highest hydrophobicity.The results of this work showed that all the tested materials were able to sustain osteoblast adhesion and promote cell proliferation;moreover,this work highlights the fea-sible PEEK treatments which allow to obtain surface properties similar to those of titanium.The results here reported,clearly show that cell behavior depends on a complex combination of surface properties like wettability and roughness and material nature,and while a rough surface is optimal for cell adhesion,a smooth and less hydrophilic surface is the best choice to limit bacterial adhesion and biofilm formation.     

Alumina coatings on PMMA: optimization of adherence

Alumina is deposited on polymethyl methacrylate (PMMA) using the RF magnetron sputtering method. The adhesion is characterized by means of a 180° peeling test and a fragmentation test. The surface energy of the materials is determined by measuring the contact angles using the two liquids method. The plasma surface treatment of the polymer help to smooth it and increase its surface energy. The modifications of the dispersive and polar components of this energy depend on the type of the plasma-gas used. Oxygen rich plasmas, such as air or carbon dioxide, allow the surface polarity of the polymer to be increased by a superficial oxidation whilst limiting the reticulation. They give better adhesion values. The analysis by ATR-FTIR displays the reticulation phenomenon for the extended application of argon plasma at low power. The RBS analysis shows that the quantity of argon incorporated into the alumina deposit is dependent on the operating conditions, particularly the pressure in the sputtering chamber. Different values of surface energy are due to the variations in composition. The alumina films with the lowest percentage of argon have the highest polar component. Their adhesion to the PMMA is also the highest. The best adhesion of alumina on PMMA is obtained for a polymer activation during a short time (10 s), by means of an oxygen rich plasma (CO₂) and for alumina coatings which have the lowest argon content (p = 1 Pa).     

Surface wettability of plasma SiOx:H nanocoating-induced endothelial cells' migration and the associated FAK-Rho GTPases signalling pathways

Vascular endothelial cell (EC) adhesion and migration are essential processes in re-endothelialization of implanted biomaterials. There is no clear relationship and mechanism between EC adhesion and migration behaviour on surfaces with varying wettabilities. As model substrates, plasma SiO_x:H nanocoatings with well-controlled surface wettability (with water contact angles in the range of 98.5 ± 2.3° to 26.3 ± 4.0°) were used in this study to investigate the effects of surface wettability on cell adhesion/migration and associated protein expressions in FAK-Rho GTPases signalling pathways. It was found that EC adhesion/migration showed opposite behaviour on the hydrophilic and hydrophobic surfaces (i.e. hydrophobic surfaces promoted EC migration but were anti-adhesions). The number of adherent ECs showed a maximum on hydrophilic surfaces, while cells adhered to hydrophobic surfaces exhibited a tendency for cell migration. The focal adhesion kinase (FAK) inhibitor targeting the Y-397 site of FAK could significantly inhibit cell adhesion/migration, suggesting that EC adhesion and migration on surfaces with different wettabilities involve (p)FAK and its downstream signalling pathways. Western blot results suggested that the FAK-Rho GTPases signalling pathways were correlative to EC migration on hydrophobic plasma SiO_x:H surfaces, but uncertain to hydrophilic surfaces. This work demonstrated that surface wettability could induce cellular behaviours that were associated with different cellular signalling events.     

The method of surface PEGylation influences leukocyte adhesion and activation

The influence of different surface modifications with poly(ethyleneglycol) (PEG) layers on the adsorption of fibrinogen and the adhesion and activation of macrophage-like human leukocytes was investigated. Poly(ethylene terephthalate) (PET) was modified using pulsed AC plasma polymerization with two types of starting monomers to generate: 1) a reactive acid surface using maleic anhydride (MAH) as monomer, and 2) a PEG-like surface using diethyleneglycol methyl vinyl ether (DEGVE) as monomer. The MAH surface was used as a reactive platform to graft linear chains of non-fouling mPEG via an intermediate layer of poly(ethyleneimine) (PEI) under lower critical solution temperature (LCST) conditions of the mPEG. The DEGVE monomer is used to create PEG-like layers by use of low power plasma conditions. The ability of the surfaces to resist protein adsorption was investigated quantitatively using ¹²⁵I-radiolabeled human fibrinogen, and the conformation of the adsorbed protein was tested using an anti-fibrinogen monoclonal antibody in an enzyme-linked immunosorbent assay. The results showed that PEGylated surfaces adsorbed significantly less (up to 90% less) fibrinogen, and that unfolding of adsorbed fibrinogen was more pronounced on the linear mPEG layers than on the PEG-like plasma polymer surfaces. Adhesion of in-vitro differentiated macrophage-like U937 cells was reduced on both the PEG-like plasma polymer surfaces and the linear mPEG layers compared to the unmodified PET surface, but cells adhering to the PEG-like plasma polymer surfaces secreted less tumor necrosis factor-α (TNF- α) than cells adhering to the linear mPEG layers. In conclusion, the method for preparing non-fouling surfaces for long-term implanted devices influence surface-induced cellular responses of the host.     

Dynamin II involves in cell migration and actin formation of NIH3T3 cells

It was previously reported that in Ras transformed NIH3T3 cells, dynamin II acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration. However, these results do not provide sufficient evidence of a relationship between dynamin II and the Ras signal transduction pathway leading to membrane ruffling and cell migration. The results showed that a dynamin II association with myosin II as a signaling molecule is involved in NIH3T3 cell migration through the Ras/PI3K signaling pathway, and is associated with the p85 subunit of PI3K. Confocal microscopy also revealed co-localization between dynamin II and paxillin after PDGF stimulation. In addition, immunofluorescence results showed that dynamin II was colocalized with the actin filament. After stimulating the NIH3T3 cells with PDGF and treating them with an actin inhibitor, such as Cytochalasin D, it was observed that dynamin II with the myosin II complex inhibited binding to the actin. Therefore, dynamin II is localized in focal adhesion when cell migration is triggered and binds to the actin filament component, suggesting that it is a good candidate nanomolecule to regulate the cell attachment and migration to the materials such as implants etc.