Influence of charge densities of randomly sulfonated polystyrene surfaces on cell attachment and proliferation

Attachment and proliferation of NIH-3T3 fibroblast cells on random polymer surfaces, polystyrene sulfonated acid (PSSAx) with five different degrees of sulfonation (x = 0%, 5%, 10%, 15% and 33%) and on a tissue culture polystyrene (TCPS) surface were studied. The surface properties, wettability and roughness were measured by water-contact angle and atomic force microscopy measurement. The wettability and surface roughness increased with increasing the content of sulfonic acid groups on the surfaces. The number of cells attached on the surface after seeding increased with increasing x and reached to the maximum value on PSSA15. The cell proliferation also increased with increasing x. However, cell proliferation was slow down on PSSA33 in comparison to PSSA10 and PSSA15 surfaces after 48 h culture.     

Effects of LP-MOCVD prepared TiO2 thin films on the in vitro behavior of gingival fibroblasts

We report on the in vitro response of human gingival fibroblasts (HGF-1 cell line) to various thin films of titanium dioxide (TiO₂) deposited on titanium (Ti) substrates by low pressure metal-organic chemical vapor deposition (LP-MOCVD). The aim was to study the influence of film structural parameters on the cell behavior comparatively with a native-oxide covered titanium specimen, this objective being topical and interesting for materials applications in implantology. HGF-1 cells were cultured on three LP-MOCVD prepared thin films of TiO₂ differentiated by their thickness, roughness, transversal morphology, allotropic composition and wettability, and on a native-oxide covered Ti substrate. Besides traditional tests of cell viability and morphology, the biocompatibility of these materials was evaluated by fibronectin immunostaining, assessment of cell proliferation status and the zymographic evaluation of gelatinolytic activities specific to matrix metalloproteinases secreted by cells grown in contact with studied specimens. The analyzed surfaces proved to influence fibronectin fibril assembly, cell proliferation and capacity to degrade extracellular matrix without considerably affecting cell viability and morphology. The MOCVD of TiO₂ proved effective in positively modifying titanium surface for medical applications. Surface properties playing a crucial role for cell behavior were the wettability and, secondarily, the roughness, HGF-1 cells preferring a moderately rough and wettable TiO₂ coating.     

Influence of unsaturated carbonic acids on hemocompatibility and cytotoxicity of poly-vinylacetate based co-polymers

The aim of this study was to investigate hemocompatibility and cytotoxicity properties of synthetic polymer coatings containing various unsaturated carbonic acids with vinylacetate. Co-polymers of vinylacetate and crotonic acid (CA), maleic acid (MA), and itaconic acid (IA) were made. The materials were characterized in terms of their adhesion to metal supports (titanium and stainless steel) as well as hemocompatibility (% hemolysis, wettability, erythrocyte aggregation, hemoglobin content, thrombocyte count and lipid peroxidation levels) and cytotoxicity (human endothelial cell activity in vitro and chromosome aberrations, bone marrow proliferation and cell ploidy in rats). Co-polymers of unsaturated carbonic acids with vinylacetate exhibited good hemocompatibility properties, as opposed to vinylacetate homopolymer for which substantial levels of hemolysis were observed. By coating the metal supports with co-polymers the cytotoxic effects associated with the bare metal samples were markedly reduced. MA samples showed excellent hemocompatibility and no cytotoxicity, yet they lacked good adhesion properties to metal substrate, probably due to high water content. CA samples, having the highest density of carboxylic groups among the samples under investigation, showed increased bone marrow proliferation activity and cell ploidy in rats, as compared to controls. The most promising results in the present study were obtained for the samples with IA, which showed good adhesion to metal substrates, good hemocompatibility and low cytotoxicity. Thus, co-polymers of vinylacetate and IA rich in carboxylic groups are promising materials for the design of novel drug-eluting stents.     

<Emphasis Type="Italic">Streptococcus sanguinis</Emphasis> adhesion on titanium rough surfaces: effect of shot-blasting particles

Dental implant failure is commonly associated to dental plaque formation. This problem starts with bacterial colonization on implant surface upon implantation. Early colonizers (such as Streptococcus sanguinis) play a key role on that process, because they attach directly to the surface and facilitate adhesion of later colonizers. Surface treatments have been focused to improve osseointegration, where shot-blasting is one of the most used. However the effects on bacterial adhesion on that sort of surfaces have not been elucidated at all. A methodological procedure to test bacterial adherence to titanium shot-blasted surfaces (alumina and silicon carbide) by quantifying bacterial detached cells per area unit, was performed. In parallel, the surface properties of samples (i.e., roughness and surface energy), were analyzed in order to assess the relationship between surface treatment and bacterial adhesion. Rather than roughness, surface energy correlated to physicochemical properties of shot-blasted particles appears as critical factors for S. sanguinis adherence to titanium surfaces.     

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.     

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.     

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.     

Statistical demonstration of the relative effect of surface chemistry and roughness on human osteoblast short-term adhesion

The effects of material composition, surface chemistry or surface topography on cell attachment (short-term adhesion) have been largely studied on bone-derived cells. However, no statistical demonstration of these effects has been performed until now. With this objective, we quantified the attachment after 24 hours of human osteoblasts on pure titanium, titanium alloy and stainless steel substrates presenting 6 different surface morphologies and 2 different roughness amplitude obtained by sand-blasting, electro-erosion, acid etching, polishing and machine-tooling. The coating by a gold-palladium layer of these surfaces allowed determining the relative effect of the surface roughness and of the surface chemistry. By multiple analysis of variance, we demonstrated that neither material composition nor surface roughness amplitude influenced cell attachment except on sandblasted pure titanium substrates. On the contrary, a high significant influence of the process used to produce the surface was observed meaning that the main influent factor on cell attachment could be either the surface morphology or the surface chemistry induced by the process. As the coating of surfaces by a gold-palladium layer decreased significantly the attachment of cells on the majority of substrates, we concluded that attachment is rather influenced by surface chemistry than by surface topography.     

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.     

Studies of calcium-precipitating oral bacterial adhesion on TiN,TiO2 single layer,and TiN/TiO2 multilayer-coated 316L SS

Titanium nitride (TiN), titanium oxide (TiO₂) single layer, and TiN/TiO₂ multilayer coatings were deposited on a 316L stainless steel substrate using reactive magnetron sputtering process with the aim of preventing bacterial adhesion. The crystal structures of as-prepared coatings were evaluated using X-ray diffraction analysis. The cubic structure of TiN, anatase, and rutile structure of TiO₂ was noticed. Atomic force microscopy images exhibited a relatively smooth surface for all coatings. The surface wettability studies confirmed that the coatings were hydrophilic in nature. The rate of bacterial adhesion was evaluated using scanning electron microscopy and epifluorescence microscopy. These results demonstrated that the coated substrates could help to effectively reduce the bacterial adhesion and biofilm formations.     

Evaluation of the attachment,proliferation, and differentiation of osteoblast on a calcium carbonate coating on titanium surface

Titanium has been reported to have some limitations in dental and orthopaedic clinical application. This study described a coating process using a simple chemical method to prepare calcium carbonate coatings on smooth titanium (STi) and sandblasted and acid-etched titanium (SATi), and evaluated the biological response of the materials in vitro. The surfaces of STi, SATi, calcium carbonate coated STi (CC-STi) and calcium carbonate coated SATi (CC-SATi) were characterized for surface roughness, contact angles, surface morphology and surface chemistry. The morphology of MG63 cells cultured on the surfaces was observed by SEM and Immuno-fluorescence staining. Cell attachment/proliferation was assessed by MTT assay, and cell differentiation was evaluated by alkaline phosphatase (ALP) activity. MG63 was found to attach favorably to calcium carbonate crystals with longer cytoplasmic extensions on CC-STi and CC-SATi, resulting in lower cell proliferation but higher ALP activity when compared to STi and SATi respectively. Moreover, CC-SATi is more favorable than CC-STi in terms of biological response. In conclusion, the calcium carbonate coatings on titanium were supposed to improve the osteointegration process and stimulate osteoblast differentiation, especially in early stage. And this method could possibly be a feasible alternative option for future clinical application.     

Physical and biological characterizations of a novel multiphase anodic spark deposition coating to enhance implant osseointegration

The present study assessed in vitro the short-term cellular response to surface physico-chemical properties of a new, purposed bioactive surface treatment called BioSpark^TM performed on simply machined and on sand-blasted titanium. Material characterisation was carried out using scanning electron microscopy, energy dispersion spectroscopy, laser profilometry, and thin film X-ray diffraction. The in vitro biological study showed a suitable cellular response with adhesion and spreading level comparable for all the tested specimens. The proliferation analysis demonstrated that all the surfaces successfully supported cellular colonisation; in particular, higher cellular proliferation activity was observed on the BioSpark^TM-treated materials, with values higher than machined titanium. The results suggest that the BioSpark^TM treatment represents a smart way to enhance osteoblastic cellular colonisation and thus improve osteointegration processes of machined and sandblasted titanium for orthopaedic and dental implants.     

Promoting Bone Mesenchymal Stem Cells and Inhibiting Bacterial Adhesion of Acid-Etched Nanostructured Titanium by Ultraviolet Functionalization

Titanium(Ti) and its alloys are used extensively in orthopedic implants because of their excellent biocompatibility,mechanical properties and corrosion resistance. However,titanium-based implant materials face many severe complications,such as implant loosening due to poor osseointegration and bacterial infections,which may lead to implant failure. Hence,preparing a biomaterial surface,which enhances the interactions with host cells and inhibits bacterial adhesion,may be an optimal strategy to reduce the incidence of implant failure. This study aims to improve osseointegration and confer antibacterial properties on Ti through a combination of two surface modifications including nanostructuring generated by acid etching and ultraviolet(UV) light treatment.Our results showed that without UV treatment,the acid etching treatment of Ti surface was effective at both improving the adhesion of bone mesenchymal stem cells(BMSCs) and increasing bacterial adhesion. A further UV treatment of the acid-etched surface however,not only significantly improved the cell adhesion but also inhibited bacterial adhesion. The acid-etched nanostructured titanium with UV treatment also showed a significant enhancement on cell proliferation,alkaline phosphatase(ALP) activity and mineralization. These results suggest that such nanostructured materials with UV treatment can be expected to have a good potential in orthopedic applications.     

In vitro Response of Human Mesenchymal Stromal Cells to Titanium Coated Peek Films and Their Suitability for Magnetic Resonance Imaging

Medical imaging is an important tool for the post-operative checkup of an accurate position of an implant as well as for monitoring the integration in the adjacent tissue that may influence the success of a medical device.Unfortunately,the possibility to use imaging methods is associated with the implant material and all the established metallic materials for surgery do not show a proper "imaging compatibility".The present study is a combined investigation of the in vitro response to human mesenchymal stromal cells(hMSC) and magnetic resonance imaging(MRI) compatibility of the potential material combination polyetheretherketone/titanium(PEEK/Ti) for medical devices.Because of the advantageous imaging properties and the mechanical and chemical stability,PEEK becomes more and more an alternative to common metallic implant materials like titanium or cobalt-chrome.However,PEEK is a bioinert material having a limited ability for direct bone incorporation.Due to its excellent biocompatibility,Ti was chosen as coating material to enhance the cellular response.The result is a combination with advantageous properties:the magnetic susceptibility and elastic modulus close to bone,corrosion resistance and mechanical flexibility of PEEK and the excellent biocompatibility of titanium.The appearance of metal-related artifact was discussed in electrical resistivity and magnetic susceptibility.Therefore,two titanium coatings have been investigated:a complete coating and a structured surface avoiding surface conductivity.To determine the in vitro biocompatibility,the cell responses were assessed in terms of the overall morphology of the hMSC and their cell area distribution,proliferation,osteogenic differentiation and mineral deposition.The cellular stress was evaluated by the prostaglandin E_2 level.The bonded materials both produced no disturbing artifacts in magnetic resonance imaging.Compared to the pure PEEK material,the titanium coated specimens showed an enhanced biocompatibility,which is indicated by a higher cell number,larger activity of the enzyme tissue non-specific alkaline phosphatase and therefore a greater amount of deposited calcium and phosphate.The results on bare PEEK are accompanied with a higher cellular stress level,which is indicated by prostaglandin E_2.     

Variation of roughness and adhesion strength of deposited apatite layers on titanium dental implants

It is known that surface roughness and chemical composition of the titanium surface influence the osseointegration of titanium implants. Most commercial dental implants offer a shot-blasted rough surface. It is also known that apatite layers coating the surface of titanium implants improve bone response, but the adhesion of the layer to the substrate poses some problems.In this study the roughness and adhesion strength to a titanium dental implant surface of an apatite layer deposited via wet chemistry after a thermochemical treatment were compared with those of plasma-sprayed apatite layers and machined titanium surfaces. Different surface conditions have been studied: (a) as-received machined dental implant surface; (b) grit-blasted titanium surface; (c) grit-blasted and thermochemically-treated titanium surface; (d) titanium surfaces coated with plasma-sprayed apatite. The morphology and roughness of the samples were measured and compared. The adhesion of the apatite layers to the titanium was compared by means of a scratch test.Measured roughness showed that the deposition of an apatite layer did not affect roughness but plasma-sprayed apatite produced a decrease on roughness values when compared to control samples. Both roughness and adhesion strength of the deposited apatite layer to the titanium substrate were higher than those of the plasma-sprayed apatite.     

Surface modification of titanium by hydrothermal treatment in Mg-containing solution and early osteoblast responses

Surface modification on titanium was carried out in order to improve its bioactivity. Pure titanium was hydrothermally treated in distilled water and 0.1 M MgCl₂ solutions at 200°C for 24 h. Surface morphology, roughness, wettability and chemical composition were characterized before and after treatment. Bovine serum albumin was used as model to study protein adsorption. MC3T3-E1 cells were cultured and initial cell attachment, morphology, proliferation were evaluated. After hydrothermal treatment, nano-sized precipitations were observed and samples showed superhydrophilicity. Magnesium (Mg) was immobilized into titanium surface by hydrothermal treatment. Protein adsorption was significantly increased on Mg-containing samples. Cell attachment was improved and cell spreading was enhanced on Mg-containing samples compared with untreated or those treated in distilled water. Increased early cellular attachment on the MgTi surface resulted in subsequent increase of number of proliferated cells. Hydrothermal treatment in MgCl₂ solution was expected to be an effective method to fabricate titanium implant with good bioactivity.     

In vitro evaluation of a multispecies oral biofilm over antibacterial coated titanium surfaces

Peri-implantitis is an infectious disease that affects the supporting soft and hard tissues around dental implants and its prevalence is increasing considerably. The development of antibacterial strategies, such as titanium antibacterial-coated surfaces, may be a promising strategy to prevent the onset and progression of peri-implantitis. The aim of this study was to quantify the biofilm adhesion and bacterial cell viability over titanium disc with or without antibacterial surface treatment. Five bacterial strains were used to develop a multispecies oral biofilm. The selected species represent initial (Streptococcus oralis and Actinomyces viscosus), early (Veillonella parvula), secondary (Fusobacterium nucleatum) and late (Porphyromonas gingivalis) colonizers. Bacteria were sequentially inoculated over seven different types of titanium surfaces, combining different roughness level and antibacterial coatings: silver nanoparticles and TESPSA silanization. Biofilm formation, cellular viability and bacterial quantification over each surface were analyzed using scanning electron microscopy, confocal microscopy and real time PCR. Biofilm formation over titanium surfaces with different bacterial morphologies could be observed. TESPSA was able to significantly reduce the cellular viability when compared to all the surfaces (p?<?0.05). Silver deposition on titanium surface did not show improved results in terms of biofilm adhesion and cellular viability when compared to its corresponding non-coated surface. The total amount of bacterial biofilm did not significantly differ between groups (p?>?0.05). TESPSA was able to reduce biofilm adhesion and cellular viability. However, silver deposition on titanium surface seemed not to confer these antibacterial properties.

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钛金属表面活化处理及表面细胞响应特性

采用不同比例的氢氟酸(1％(质量分数)HF)与双氧水(30％(质量分数)H2O2)以及二者不同配比(1∶1、5∶1和1∶5)的混合溶液对钛金属表面进行处理,制备出具有不同拓扑结构的钛活性表面.用扫描电镜(SEM)、原子力显微镜(AFM)、接触角测试和X射线光电子能谱(XPS)等手段分别对各组样品的表面形貌、粗糙度、亲疏水性及表面化学组成变化等进行分析和表征.将各组样品分别与骨肉瘤细胞株(MG63)共培养,用SEM观察细胞形态变化,利用MTT比色法测定细胞增殖能力,通过体外细胞培养实验考察处理后的钛金属表面对MG63细胞形貌及增殖分化特性的影响.结果表明,经HF和H2O2混合溶液处理后的样品表面粗糙,并含有丰富的F-和OH-基团,促进了细胞的粘附、铺展、生长和增殖,大幅改善了钛表面的生物活性.其中,氢氟酸与双氧水按1∶5配比的混合溶液处理后的样品在细胞培养前期显示出更加优良的细胞相容性,这对促进钛种植体与周围骨组织间的快速整合具有积极意义.     

Suppressed proliferation of mouse osteoblast-like cells by a rough-surfaced substrate leads to low differentiation and mineralization

The cellular responses of mouse osteoblast-like MC3T3-E1 cells to the surface roughness were examined in the sequential events of cell adhesion, proliferation, differentiation, and mineralization. The cells were plated and cultured on sandblasted borosilicate glass slideslips with different surface roughnesses. DNA synthesis at day 1 after plating and the cell number at day 5 significantly decreased as the surface roughness increased. The suppressed cell proliferation on the rough-surfaced substrates, closely related to the round cell morphology, caused underdeveloped intercellular contacts via the gap junction due to the low population of neighboring cells. Expressions of the representative osteoblastic genes at day 14, alkaline phosphatase activity at day 21, and mineralization at day 28 were markedly reduced on the rough-surfaced substrates. These results clearly indicated that the reduced cell differentiation and mineralization resulted from the early cellular responses of the suppressed cell proliferation depending on the surface roughness and the consequent poor intercellular communication. The specific changes in the early gene expression profiles at day 1, depending on the surface roughness, were examined by a large-scale analysis of the gene expression using a mouse DNA chip. The ribosomal protein S6 kinase polypeptide 1 gene, which is a cell growth-related gene involved in the PI3-kinase/Akt pathway, was found to be the most down-regulated among the 4277 screened genes.     

S. sanguinis adhesion on rough titanium surfaces: Effect of culture media

Bacterial colonization plays a key role in dental implant failure, because they attach directly on implant surface upon implantation. Between different types of bacteria associated with the oral environment, Streptococcus sanguinis is essential in this process since it is an early colonizer. In this work the relationship between titanium surfaces modified by shot blasting treatment and S. sanguinis adhesion; have been studied in approached human mouth environment. Bacteria pre-inoculated with routinary solution were put in contact with titanium samples, shot-blasted with alumina and silicon carbide, and adhesion results were compared with those obtained when bacteria were pre-inoculated with modified artificial saliva medium and on saliva pre-coated titanium samples. Our results showed that bacterial adhesion on titanium samples was influenced by culture conditions. When S. sanguinis was inoculated in routinary culture media, colonies forming unities per square millimeter presented an increment correlated with roughness and surface energy, but separated by the type of particle used during shot-blasting treatment; whereas in modified artificial saliva only a relationship between bacteria adhered and the increment in both roughness and surface energy were observed, regardless of the particle type. Finally, on human saliva pre-coated samples no significant differences were observed among roughness, surface energy or particle.