Enhanced adhesion of osteoblastic cells on polystyrene films by independent control of surface topography and wettability

We independently controlled surface topography and wettability of polystyrene (PS) films by CF₄ and oxygen plasma treatments, respectively, to evaluate the adhesion and proliferation of human fetal osteoblastic (hFOB) cells on the films. Among the CF₄ plasma-treated PS films with the average surface roughness ranging from 0.9 to 70 nm, the highest adhesion of hFOB cells was observed on a PS film with roughness of ~ 11 nm. When this film was additionally treated by oxygen plasma to provide a hydrophilic surface with a contact angle less than 10°, the proliferation of bone-forming cell was further enhanced. Thus, the plasma-based independent modification of PS film into an optimum nanotexture for human osteoblast cells could be appplied to materials used in bone tissue engineering.     

Polymer films with surfaces unmodified and modified by non-thermal plasma as new substrates for cell adhesion

The surface properties of biomaterials, such as wettability, polar group distribution, and topography, play important roles in the behavior of cell adhesion and proliferation. Gaseous plasma discharges are among the most common means to modify the surface of a polymer without affecting its properties. Herein, we describe the surface modification of poly(styrene) (PS) and poly(methyl methacrylate) (PMMA) films using atmospheric pressure plasma processing through exposure to a dielectric barrier discharge (DBD). After treatment the film surface showed significant changes from hydrophobic to hydrophilic as the water contact angle decreasing from 95° to 37°. All plasma-treated films developed more hydrophilic surfaces compared to untreated films, although the reasons for the change in the surface properties of PS and PMMA differed, that is, the PS showed chemical changes and in the case of PMMA they were topographical. Excellent adhesion and cell proliferation were observed in all films. In vitro studies employing flow cytometry showed that the proliferation of L929 cells was higher in the film formed by a 1:1 mixture of PS/PMMA, which is consistent with the results of a previous study. These findings suggest better adhesion of L929 onto the 1:1 PS/PMMA modified film, indicating that this system is a new candidate biomaterial for tissue engineering.     

Facile surface modification of silicone rubber with zwitterionic polymers for improving blood compatibility

A facile approach to modify silicone rubber (SR) membrane for improving the blood compatibility was investigated. The hydrophobic SR surface was firstly activated by air plasma, after which an initiator was immobilized on the activated surface for atom transfer radical polymerization (ATRP). Three zwitterionic polymers were then grafted from SR membrane via surface-initiated atom transfer radical polymerization (SI-ATRP). The surface composition, wettability, and morphology of the membranes before and after modification were characterized by X-ray photoelectron spectroscopy (XPS), static water contact angle (WCA) measurement, and atomic force microscopy (AFM). Results showed that zwitterionic polymers were successfully grafted from SR surfaces, which remarkably improved the wettability of the SR surface. The blood compatibility of the membranes was evaluated by protein adsorption and platelet adhesion tests in vitro. As observed, all the zwitterionic polymer modified surfaces have improved resistance to nonspecific protein adsorption and have excellent resistance to platelet adhesion, showing significantly improved blood compatibility. This work should inspire many creative uses of SR based materials for biomedical applications such as vessel, catheter, and microfluidics.     

Biomaterial surface properties modulate in vitro rat calvaria osteoblasts response: Roughness and or chemistry?

The aim of this study was a better understanding of the regulation mechanisms of in vitro osteoblast activity on biomaterials. Rat osteoblast behaviour on different surfaces was studied. Surfaces with different roughness (and a similar surface chemistry) or with different surface chemistry (and a similar roughness) were compared. Cellular morphology was observed by scanning electron microscopy and cell adhesion was quantified using an image analysis system. Osteoblast proliferation was quantified by a MTT test and total protein content and alkaline phosphatase (ALP) activity were evaluated by spectrophotometry. Data were compared by statistical analysis.

Results showed that NiTi surface roughness did not influence osteoblasts morphology, adhesion, total protein content and ALP activity whereas it modulated cell proliferation. Roughness was shown to stimulate cell proliferation. For smooth surfaces exhibiting two different chemical compositions, adhesion rate was found to be higher on Thermanox® than on NiTi whereas proliferation was shown to be smaller. ALP activity was also modulated by surface chemistry. Thus, cell adhesion and ALP activity were found to be more governed by surface chemistry than by roughness whereas cell proliferation was shown to be modulated by roughness (this effect increasing during cell culture) and by chemistry (this effect remaining stable in time) together. Total protein content and cell morphology were found to be independent of both parameters (roughness and chemistry). Effects of surface chemistry were discussed in terms of wettability and electron acceptor/donor properties of the surfaces of interest. Immunofluorescence images of adhesion proteins could not demonstrate differences between the three surfaces.     

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.     

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.     

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.     

Endothelial cells on plasma-treated segmented-polyurethane: Adhesion strength, antithrombogenicity and cultivation in tubes

When the surface of segmented-polyurethane (SPU), where endothelial cells are not capable of proliferating, is modified by plasma treatment, the adhesion and proliferation of bovine aortic endothelial cells (BAECs) can be drastically improved. The cells were capable of proliferating on the inner surface of a plasma-treated SPU-coated tube (length: 50 mm; inner diameter: 1.5 mm). When a steady flow shear stress of 9 Pa was applied to the cells proliferated on the modified SPU surface for 90 min, most cells did not detach from the surface. From an in vitro evaluation test of antithrombogenicity, the cell surface can be considered to provide an inert surface against thrombus formation and blood coagulation. From analyses of the plasma-treated SPU surface, it was suggested that the improvements in BAEC proliferation and adhesion after plasma treatment were due to the change in wettability of the surface. Data suggest that the plasma treatment would be useful for developing a small-calibre hybrid vascular graft.     

碳/碳复合材料表面粗糙度对成骨细胞生长行为的影响

采用化学气相沉积工艺制备了碳/碳(C/C)复合材料, 用表面轮廓仪检测了表面粗糙度, 用MG-63成骨细胞进行了细胞试验, 研究了C/C复合材料表面粗糙度对成骨细胞形貌、粘附和增殖的影响规律. 结果表明: C/C复合材料表面粗糙度越高则成骨细胞在其表面的粘附率越高, 增殖率越低; C/C复合材料表面粗糙度对成骨细胞的生长方向和形貌具有诱导作用, 粗糙度越高则方向诱导作用越明显, 且细胞附着形貌呈梭形或长条状, 立体感强, 反之成骨细胞则呈现片状, 铺展状态好.     

Surface modification of polymers by ion-assisted reaction

This paper deals with a new surface modification technique of polymers, the so-called ion-assisted reaction (IAR) to improve the surface properties of polymers and provides outstanding experimental results regarding wettability and adhesion of various polymers. In the IAR, polymer surfaces were subjected to low energy ion irradiation at different dosage in reactive gas environment. Dramatic improvements in wettability and surface energy are observed for the IAR-treated polymer surfaces and can be explained by the addition of functional groups, responsible for the increase of polar component in surface energy. The formation of functional groups results from the interaction among ion, reactive gas and polymer chain involved in IAR treatment, depending on the reactive ion species, the flow rate of the reactive gas and the irradiating ion fluence. The improvement in adhesion between the IAR-treated polymers and coating materials was explained in terms of the increased surface energy as well as surface roughness in the polymers modified by the IAR and possible adhesion enhancement mechanism is to be discussed.     

Surface modification of implant materials and its effect on attachment and proliferation of bone cells

Osteoblast-like cell response in variation with the air plasma sprayed (APS) TiO2 coating process parameters correlated with coating properties were investigated to evaluate the durability and biocompatibility of the surface-modified implant. The Taguchi technique was used to determine the coating properties affected by plasma spraying parameters on Ti-6AI-4V alloy substrate. The coating properties were characterized by porosity and surface roughness using an image analyzer and surf analyzer, respectively. The MG-63 osteoblast like cell morphology and proliferation data on TiO2 coated substrate were measured by SEM observation and direct cell counting. It was demonstrated that surface roughness increased as spray distance decreased but gas flow rates and spray distance were major factors in the case of porosity. The osteoblast adhesion morphology and proliferation data indicated that osteoblast-like cell morphology was not influenced by process parameters, but cell proliferation was affected to some extent by surface roughness and porosity among TiO2 coated specimens. Specifically, the difference between those of substrate and coating layer was relatively more visible.     

Various fates of neuronal progenitor cells observed on several different chemical functional groups

Neuronal progenitor cells cultured on gold-coated glass surfaces modified by different chemical functional groups, including hydroxyl (-OH), carboxyl (-COOH), amino (-NH₂), bromo (-Br), mercapto (-SH), -Phenyl and methyl (-CH₃), were studied here to investigate the influence of surface chemistry on the cells’ adhesion, morphology, proliferation and functional gene expression. Focal adhesion staining indicated in the initial culture stage cells exhibited morphological changes in response to different chemical functional groups. Cells cultured on -NH₂ grafted surface displayed focal adhesion plaque and flattened morphology and had the largest contact area. However, their counter parts on -CH₃ grafted surface displayed no focal adhesion and rounded morphology and had the smallest contact area. After 6 days culture, the proliferation trend was as follows: -NH₂>-SH>-COOH>-Phenyl>-Br>-OH>-CH₃. To determine the neural functional properties of the cells affected by surface chemistry, the expression of glutamate decarboxylase (GAD67), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were characterized. An increase of GAD67 expression was observed on -NH₂, -COOH and -SH grafted surfaces, while no increase in NGF and BDNF expression was observed on any chemical surfaces. These results highlight the importance of surface chemistry in the fate determination of neuronal progenitor cells, and suggest that surface chemistry must be considered in the design of biomaterials for neural tissue engineering.     

PTFE surface modification by Ar plasma and its characterization

Polytetrafluoroethylene (PTFE) samples were exposed to argon plasma discharge and the changes of the PTFE surface properties were studied by different methods. Surface wettability and aging of the plasma modified PTFE were derived from the contact angle measured by standard goniometry. Electrokinetical potential (ζ-potential) was determined by SurPASS Instrument. The ζ-potential and the contact angle characterize “chemistry” of the PTFE surface and also make possible to follow the aging of the PTFE surface after the plasma treatment. The surface morphology and roughness were examined by Atomic Force Microscopy (AFM). Surface concentrations of elements were determined by X-ray photoelectron spectroscopy (ARXPS). The contact angle of the modified PTFE decreases with increasing time of the plasma treatment. During the aging of the plasma modified PTFE the contact angle increases. After the plasma treatment dramatic changes in the PTFE surface morphology and roughness were observed. Also a significant increase of ζ-potential is observed, which indicates more hydrophilic surface of the modified PTFE in comparison with pristine one. With increasing time of the plasma treatment the total oxygen content in the polymer surface layer increases.     

Surface modified electrospun nanofibrous scaffolds for nerve tissue engineering

The development of biodegradable polymeric scaffolds with surface properties that dominate interactions between the material and biological environment is of great interest in biomedical applications. In this regard, poly-ε-caprolactone (PCL) nanofibrous scaffolds were fabricated by an electrospinning process and surface modified by a simple plasma treatment process for enhancing the Schwann cell adhesion, proliferation and interactions with nanofibers necessary for nerve tissue formation. The hydrophilicity of surface modified PCL nanofibrous scaffolds (p-PCL) was evaluated by contact angle and x-ray photoelectron spectroscopy studies. Naturally derived polymers such as collagen are frequently used for the fabrication of biocomposite PCL/collagen scaffolds, though the feasibility of procuring large amounts of natural materials for clinical applications remains a concern, along with their cost and mechanical stability. The proliferation of Schwann cells on p-PCL nanofibrous scaffolds showed a 17% increase in cell proliferation compared to those on PCL/collagen nanofibrous scaffolds after 8 days of cell culture. Schwann cells were found to attach and proliferate on surface modified PCL nanofibrous scaffolds expressing bipolar elongations, retaining their normal morphology. The results of our study showed that plasma treated PCL nanofibrous scaffolds are a cost-effective material compared to PCL/collagen scaffolds, and can potentially serve as an ideal tissue engineered scaffold, especially for peripheral nerve regeneration.     

The anatase phase of nanotopography titania plays an important role on osteoblast cell morphology and proliferation

The surface properties of biomaterials play a vital role in cell morphology and behaviors such as cell adhesion, migration, proliferation and differentiation. Three different crystal phases of titania film (rutile, anatase and amorphous titania) with similar roughness were successfully synthesized by DC reactive magnetron sputtering. The surface roughness of each film was about 8-10 nm. Primary rat osteoblasts were used to observe changes in morphology and to evaluate cell behavior at the film surface. The number of the osteoblasts on anatase film was significantly higher than rutile and amorphous films after 36 and 72 h incubation. More importantly, synthesis of alkaline phosphatase was significantly greater by osteoblasts cultured on anatase film than on rutile and amorphous films after 7 and 14 days. In addition, the cells grown on the anatase phase film had the largest spreading area; the actin filaments in cells with regular directions were well defined and fully spreaded. The results indicate that the anatase phase of titania with nanoscale topography yield the best biological effects for cell adhesion, spreading, proliferation and differentiation. There are strong therapeutic prospects for this biomaterial film for osteoblast proliferation, with possible applications for orthopedic and dental implant.     

Reduced fibroblast adhesion and proliferation on plasma-modified titanium surfaces

Soft tissue complications are clinically relevant problems after osteosynthesis of fractures. The goal is to develop a method for reduction of fibroblast adhesion and proliferation on titanium implant surfaces by plasma polymerisation of the organo-silicon monomer hexamethyldisiloxane (HMDSO). HMDSO was deposited under continuous wave conditions in excess oxygen (ppHMDSO surface) and selected samples were further modified with an additional oxygen plasma (ppHMDSO + O₂ surface). Surface characterization was performed by scanning electron microscopy, profilometry, water contact angle measurements, infrared reflection absorption spectroscopy and X-ray photoelectron spectroscopy. In our experimental setup the mechanical properties, roughness and topography of the titanium were preserved, while surface chemistry was drastically changed. Fibroblast proliferation was assessed by alamarBlue assay, cell morphology by confocal microscopy visualization of eGFP-transducted fibroblasts, and cell viability by Annexine V/propidium iodide assay. Both modified surfaces, non-activated hydrophobic ppHMDSO and activated hydrophilic ppHMDSO + O₂ were able to dramatically reduce fibroblast colonization and proliferation compared to standard titanium. However, this effect was more strongly pronounced on the hydrophobic ppHMDSO surface, which caused reduced cell adhesion and prevented proliferation of fibroblasts. The results demonstrate that plasma modifications of titanium using HMDSO are valuable candidates for future developments in anti-adhesive and anti-proliferative coatings for titanium fracture implants.     

Improved proliferation of human osteosarcoma cells on oxygen plasma treated polystyrene

Polystyrene (PS) foils were exposed to oxygen plasma in order to improve the ability of human osteosarcoma cells (HOS) proliferation. Plasma was created in an electrodeless radio-frequency (RF) discharge in pure oxygen. Plasma treatment time of 30 s allowed for saturation of the surface with oxygen rich functional groups. Incubation of proteins from cell culture media was studied versus incubation time for untreated and plasma treated PS by X-ray photoelectron spectroscopy (XPS). Then HOS cells were cultivated according to a standard procedure, applied on the PS substrates and incubated. The proliferation was studied qualitatively by optical microscopy, scanning electron microscopy (SEM) and quantitatively by MTT assay. All techniques revealed significant differences in HOS cells proliferation on untreated and oxygen plasma treated PS. The differences were most pronounced after 24 h of incubation when the cells have already stared to proliferate on plasma treated PS, while they were still in quiescence on untreated PS. The confluency was achieved till the 6th day on plasma treated PS, while on untreated PS such confluency has never been achieved. The cell viability assay revealed that HOS cells grew better on plasma treated PS for various incubation times. Similar trend was observed regarding the adhesion of proteins. They adhered better on plasma treated PS causing better environment for further cell proliferation. The results clearly show that oxygen plasma treatment allows for much better proliferation of HOS cells on plasma treated PS as compared to the untreated PS substrates.     

Surface modification of starch based blends using potassium permanganate-nitric acid system and its effect on the adhesion and proliferation of osteoblast-like cells

The surface modification of three starch based polymeric biomaterials, using a KMnO₄/NHO₃ oxidizing system, and the effect of that modification on the osteoblastic cell adhesion has been investigated. The rationale of this work is as follows—starch based polymers have been proposed for use as tissue engineering scaffolds in several publications. It is known that in biodegradable systems it is quite difficult to have both cell adhesion and proliferation. Starch based polymers have shown to perform better than poly-lactic acid based materials but there is still room for improvement. This particular work is aimed at enhancing cell adhesion and proliferation on the surface of several starch based polymer blends that are being proposed as tissue engineering scaffolds.The surface of the polymeric biomaterials was chemically modified using a KMnO₄/HNO₃ system. This treatment resulted in more hydrophilic surfaces, which was confirmed by contact angle measurements. The effect of the treatment on the bioactivity of the surface modified biomaterials was also studied. The bioactivity tests, performed in simulated body fluid after biomimetic coating, showed that a dense film of calcium phosphate was formed after 30 days. Finally, human osteoblast-like cells were cultured on unmodified (control) and modified materials in order to observe the effect of the presence of higher numbers of polar groups on the adhesion and proliferation of those cells. Two of the modified polymers presented changes in the adhesion behavior and a significant increase in the proliferation rate kinetics when compared to the unmodified controls.     

Surface properties of thin gold layers sputtered on polymers

Thin gold layers were sputtered on the foils of polypropylene-PP, polyethyleneterephthalate-PET, polystyrene-PS, polyethylene-PE and polytetrafluoroethylene-PTFE modified by Ar+ plasma. Surface properties of pristine, plasma treated and gold coated polymers were characterized by two-points method (sheet electrical resistance), electrokinetical analysis (zeta-potential, surface chemistry), goniometry (contact angle), electron paramagnetic resonance (concentration of radicals), atomic force microscopy (AFM, surface morphology and roughness) and scratch test (mechanical properties). Zeta potential and contact angle, as assumed, differ dramatically for plasma treated polymers and for the polymers deposited by Au layers. AFM images indicate that after gold deposition on polymers the surface roughness and the surface morphology change depending on pristine polymer surfaces (roughness and morphology) and sputtering time. Electrical measurements resulted in fact that with increasing layer thickness, the sheet resistance of the gold layer decreases for all polymers with increasing sputtering time. Lower adhesive destruction is observed on the gold layer deposited on plasma treated PE in comparison with pristine.     

Surface tailoring of poly(ethylene terephthalate) via ligand-tethered comb-like PEG to enhance endothelialization

The comb-like PEG (CPEG) end-tethered with l-lysine was explored to surface modification of PET to enhance endothelialization. The hydroxyl end groups of CPEG were oxygenated into aldehyde groups. The CPEG-CHO was grafted onto the aminolysized PET. The l-lysine was then end-tethered onto surface via the residual aldehyde groups. The surface modification was confirmed by ATR-FTIR, contact angle and XPS measurements. The endothelial cell adhesion, proliferation and viability results indicated that the PET-CPEG resisted cell adhesion and growth, where as PET-CPEG-lysine promoted cell adhesion and growth. The MTT assay and total cell protein tests indicated that the endothelial cells on PET-CPEG-lysine had high viability. Cell spread uniformly and covered completely on the PET-CPEG-lysine. The CPEG end tethered with l-lysine could regulate cell adhesion and growth and enhance surface endotheliazation.