Fibrin-mediated endothelial cell adhesion to vascular biomaterials resists shear stress due to flow

In vitro endothelial cell (EC) seeding onto biomaterials for blood-contacting applications can improve the blood compatibility of materials. Adhesive proteins adsorbed from serum that is supplemented with the culture medium intercede the initial cell adhesion and subsequent spreading on material surface during culture. Nevertheless, physical and chemical properties of vascular biomaterial surface fluctuate widely between materials resulting in dissimilarity in protein adsorption characteristics. Thus, a variation is expected in cell adhesion, growth and the ability of cell to resist shear stress when tissue engineering on to vascular biomaterials is attempted. This study was carried out with an objective to determine the significance of a matrix coating on cell adhesion and shear stress resistance when cells are cultured on materials such as polytetrafluoroethylene (PTFE, Teflon) and polyethyleneterephthalate (Dacron), ultra high molecular weight polyethylene (UHMWPE) and titanium (Ti), that are used for prosthetic devices. The study illustrates the distinction of EC attachment and proliferation between uncoated and matrix-coated surfaces. The cell attachment and proliferation on uncoated UHMWPE and titanium surfaces were not significantly different from matrix-coated surfaces. However, shear stress resistance of the cells grown on composite coated surfaces appeared superior compared to the cells grown on uncoated surface. On uncoated vascular graft materials, the cell adhesion was not supported by serum alone and proliferation was scanty as compared to matrix-coated surface. Therefore, coating of implant devices with a composite of adhesive proteins and growth factors can improve EC attachment and resistance of the cells to the forces of flow.     

The impact of the RGD peptide on osteoblast adhesion and spreading on zinc-substituted hydroxyapatite surface

The incorporation of zinc into the hydroxyapatite structure (ZnHA) has been proposed to stimulate osteoblast proliferation and differentiation. Another approach to improve cell adhesion and hydroxyapatite (HA) performance is coating HA with adhesive proteins or peptides such as RGD (arginine–glycine–aspartic acid). The present study investigated the adhesion of murine osteoblastic cells to non-sintered zinc-substituted HA disks before and after the adsorption of RGD. The incorporation of zinc into the HA structure simultaneously changed the topography of disk’s surface on the nanoscale and the disk’s surface chemistry. Fluorescence microscopy analyses using RGD conjugated to a fluorescein derivative demonstrated that ZnHA adsorbed higher amounts of RGD than non-substituted HA. Zinc incorporation into HA promoted cell adhesion and spreading, but no differences in the cell density, adhesion and spreading were detected when RGD was adsorbed onto ZnHA. The pre-treatment of disks with fetal bovine serum (FBS) greatly increased the cell density and cell surface area for all RGD-free groups, overcoming the positive contribution of zinc to cell adhesion. The presence of RGD on the ZnHA surface impaired the effects of FBS pre-treatment possibly due to competition between FBS proteins and RGD for surface binding sites.     

Osteoblast cell behavior on the new beta-type Ti–25Ta–25Nb alloy

Among metallic materials used as bone substitutes, β titanium alloys gain an increasing importance because of their low modulus, high corrosion resistance and good biocompatibility. In this work, an investigation of the in vitro cytocompatibility of a recently new developed β-type Ti–25Ta–25Nb alloy was carried out by evaluating the behavior of human osteoblasts. The metallic Ti–6Al–4V biomaterial, which is one of representative α + β type titanium alloys for biomedical applications, and Tissue Culture Polystyrene (TCPS), were also investigated as reference Ti-based material and control substrate, respectively. Both metallic surfaces were analyzed by X-ray diffraction, atomic force microscopy and X-ray photoelectron spectroscopy. The cellular response was quantified by assessments of viability, cell attachment and spreading, cell morphology, production and extracellular organization of fibronectin and cell proliferation. Polished surfaces from both materials having an equiaxed grain microstructure and nanometre scale surface roughness elicited an essentially identical osteoblast response in terms of all analyzed cellular parameters. Thus, on both surfaces the cells displayed high survival rates, good cell adhesion and spreading, a dense and randomly dispersed fibronectin matrix and increasing cell proliferation rates over the incubation time. Furhermore, the enhanced biological performance of Ti–25Ta–25Nb was highly supported by the results obtained in comparison with TCPS. These findings, together with previously shown superelastic behavior, low Young's modulus and high corrosion resistance, recommend Ti–25Ta–25Nb as good candidate for applications in bone implantology.     

Comparison of mesenchymal stem cell and osteosarcoma cell adhesion to hydroxyapatite

Immortalized cells are often used to model the behavior of osteogenic cells on orthopaedic and dental biomaterials. In the current study we compared the adhesive behavior of two osteosarcoma cell lines, MG-63 and Saos-2, with that of mesenchymal stem cells (MSCs) on hydroxyapatite (HA). It was found that osteosarcoma cells demonstrated maximal binding to fibronectin-coated HA, while MSCs alternately preferred HA coated with collagen-I. Interesting, the binding of MG-63 and Saos-2 cells to fibronectin was mediated by both α5 and αv-containing integrin heterodimers, whereas only αv integrins were used by MSCs. Cell spreading was also markedly different for the three cell types. Osteosarcoma cells exhibited optimal spreading on fibronectin, but poor spreading on HA disks coated with fetal bovine serum. In contrast, MSCs spread very well on serum-coated surfaces, but less extensively on fibronectin. Finally, we evaluated integrin expression and found that MSCs have higher levels of α2 integrin subunits relative to MG-63 or Saos-2 cells, which may explain the enhanced adhesion of MSCs on collagen-coated HA. Collectively our results suggest that osteosarcoma cells utilize different mechanisms than MSCs during initial attachment to protein-coated HA, thereby calling into question the suitability of these cell lines as in vitro models for cell/biomaterial interactions.     

Appearance of cell-adhesion factor in osteoblast proliferation and differentiation of apatite coating titanium by blast coating method

We have already reported that the apatite coating of titanium by the blast coating (BC) method could show a higher rate of bone contact from the early stages in vivo, when compared to the pure titanium (Ti) and the apatite coating of titanium by the flame spraying (FS) method. However, the detailed mechanism by which BC resulted in satisfactory bone contact is still unknown. In the present study, we investigated the importance of various factors including cell adhesion factor in osteoblast proliferation and differentiation that could affect the osteoconductivity of the BC disks. Cell proliferation assay revealed that Saos-2 could grow fastest on BC disks, and that a spectrophotometric method using a LabAssay^TM ALP kit showed that ALP activity was increased in cells on BC disks compared to Ti disks and FS disks. In addition, higher expression of E-cadherin and Fibronectin was observed in cells on BC disks than Ti disks and FS disks by relative qPCR as well as Western blotting. These results suggested that the expression of cell-adhesion factors, proliferation and differentiation of osteoblast might be enhanced on BC disks, which might result higher osteoconductivity.     

The effects of loading waveform and microstructure on the fatigue response of titanium aero-engine compressor disk alloys

Microstructural variations produced from manufacturing processes and their influence on fatigue crack growth in titanium disks were investigated. Charpy‐tests on titanium disk material were performed and materials with fracture energy values in the range of 3.8–19.1 J/cm² were selected for tests under cyclic loads. Results of Charpy‐tests were compared with fractographic features related to fatigue crack growth in Ti?6Al?3Mo?0.4Si and Ti?6Al?3Mo?2Cr alloys with a two‐phase (α + β) lamellar structure under various cyclic waveforms using specimens made from compressor disks. The material sensitivity to cyclic load waveform can be seen for in‐service disks using a criteria based on fracture energy values determined in Charpy‐tests. A difference in fatigue crack growth periods of 2.5 times was discovered for specimens made from the disk with a filament type microstructure and the mainly globular two‐phase structure of the Ti?6Al?3Mo?0.4Si alloy. The shorter crack growth period correlated with the mainly facetted pattern formation with local zones of fatigue striations when fatigue crack growth is along the planes of the filaments. Fatigue striations are the major fracture surface relief when crack growth occurs in the perpendicular direction to the plane of the filaments. A quantitative fractographic method for estimating the crack growth period for in‐service failed disks was performed for the case of crack development along planes of such microstructural filaments created during the manufacturing process. Specimen tests involving a hold‐time in the cyclic loads are recommended for in‐service accepted titanium disks using a criteria based on the fracture energy value. Selection of disks based on these criteria can indicate a material sensitivity to cyclic load waveforms.     

Assessment of the Cytocompatibility of Poly‐(N‐hexylvinylpyridinium) Used as an Antibacterial Implant Coating

Medical implants made of titanium have a wide variety of applications, ranging from replacement of a single tooth to extraoral maxillofacial prosthetic rehabilitation or hip endoprosthesis. The long‐term success of such osseointegrated titanium implants is endangered by inflammation of periimplant hard or soft tissues caused by a bacterial infection. Therefore, implants should ideally inhibit bacterial adhesion and growth, but allows strong attachment of connective tissues or epithelium at the same time. Antimicrobial polymers like poly(vinyl‐N‐hexylpyridinium bromide) (hexyl‐PVP) are a promising approach as implant coatings to inhibit bacterial adhesion, but little is known about the biocompatibility of these polymers. The aim of the present study was to develop a method for evaluation of the cell acceptance of hexyl‐PVP or copolymers of vinyl‐N‐hexylpyridinium bromide and (4‐vinylbenzyl)phosphonic acid diethylester (poly((hexyl‐VP)‐co‐VBP)) as coating on titanium disks. Primary human gingival fibroblasts were used and biocompatibility was assessed by cell adhesion and proliferation. The cell morphology of the fibroblasts on these surfaces was analyzed by scanning electron microscopy (SEM) and was used as additional criterion. The results indicate no significant differences in adhesion or proliferation rate between primary human gingival fibroblasts seeded on polymer‐coated titanium disks and uncoated titanium disks as a control. Although SEM micrographs displayed moderate differences in cell morphology between the two groups, application of hexyl‐PVP or the corresponding copolymers as antibacterial coatings for medical implants or devices appears to be promising.     

Nanostructured titanium-45S5 Bioglass scaffold composites for medical applications

Titanium–10 wt.% 45S5 Bioglass scaffold nanocomposites were synthesized by the combination of mechanical alloying and by a “space-holder” sintering process. The porous structure and corrosion properties were investigated. In vitro biocompatibility of these materials was evaluated and compared with a conventional microcrystalline titanium, where normal human osteoblast (NHOst) cells from Cambrex (CC-2538) were cultured on the disks of the materials and cell growth was examined. The morphology of the cell cultures obtained on Ti–10 wt.% 45S5 Bioglass nanocomposite was similar to those obtained on the microcrystalline titanium. On the other hand, on porous scaffold, the cells adhered with their whole surface to the insert penetrating the porous structure, while on the polished surface, more spherical cells were observed with a smaller surface of adhesion. The present study has demonstrated that titanium–10 wt.% 45S5 Bioglass scaffold nanocomposite is a promising biomaterial for bone tissue engineering.     

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.     

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.     

Kinetics of Rutile Formation via Oxidation of Titanium in Air at 850°C

The kinetics of rutile growth in the form of disks up to 5 mm in thickness and 60 mm in diameter during direct oxidation of titanium preforms in air at 850°C are studied by thermogravimetry. The materials thus prepared are close in dielectric and elastic properties to single-crystal rutile. The rate and extent of rutile formation are shown to depend on the surface curvature of the titanium preform. A thermogravimetric system for kinetic studies and procedures of dielectric and mechanical measurements are described.     

羟基磷灰石/钛合金骨替代材料体外培养成骨细胞的实验研究

实验选用小鼠头盖骨成骨细胞,采用体外细胞培养技术对具有羟基磷灰石涂层的钛合金(HA/Ti)与未经过表面改性的钛合金两种骨替代材料进行细胞相容性评价,动态观察两种骨替代材料对成骨细胞生长、附着的影响。结果表明两种骨替代材料对成骨细胞生长无抑制作用,未发生细胞毒性反应,细胞在两种材料表面均能正常粘附、生长、增殖,均具有良好的细胞附着形态和细胞增殖率,而HA/钛合金材料具有更好的成骨性,是一种骨细胞相容性良好的骨替代材料。     

The influence of implant surface properties on cell adhesion and proliferation

Interactions of the foreign material of implant and the living tissue on the cell level can cause prolonged healing or, worse, loss of the implant. The cell response to the presence of some implant materials was studied under in vitro conditions. The influence of physicochemical surface parameters on the response of the cells in the immediate vicinity of implants, namely on adhesion, proliferation and synthetic activity of fibroblasts, and on the blood coagulation were compared. The direct contact of tested materials (titanium and Ti6Al4V alloy with various surface treatments, Cr Co Mo alloy, hydroxyapatite-coated titanium, zirconium oxide ceramics, polyethylene and carbon composite) on cell spreading was monitored and the presence of TNF-α and IL-8 was evaluated in the cultivation medium. The formation of blood clots was investigated on samples immersed in a well with freshly drawn whole rabbit blood using a scanning electron microscope. The surface free energy was estimated using the measurement of static contact angle. Both the advancing and receding contact angles were measured by the dynamic Wilhemy plate method. Two main groups with extremes in cell viability were established. In the first group the increased polar component of surface free energy, the highest cell density, the lowest inflammatory cytokine production, but no fibres in the clotting blood were found. On the contrary, the second group of materials with a very low polar component of the surface free energy showed distinctly higher expression of inflammatory mediators, low cell proliferation, but faster formation of fibres in the blood coagulum.     

Material-selective surface chemistry for nanoplasmonic sensors: optimizing sensitivity and controlling binding to local hot spots

Optical sensors utilizing the principle of localized surface plasmon resonance (LSPR) offer the advantage of a simple label-free mode of operation, but the sensitivity is typically limited to a very thin region close to the surface. In bioanalytical sensing applications, this can be a significant drawback, in particular since the surface needs to be coated with a recognition layer in order to ensure specific detection of target molecules. We show that the signal upon protein binding decreases dramatically with increasing thickness of the recognition layer, highlighting the need for thin high quality recognition layers compatible with LSPR sensors. The effect is particularly strong for structures that provide local hot spots with highly confined fields, such as in the gap between pairs of gold disks. While our results show a significant improvement in sensor response for pairs over single gold disks upon binding directly to the gold surface, disk pairs did not provide larger signal upon binding of proteins to a recognition layer (already for around 3 nm thin layers) located on the gold. Local plasmonic hot spots are however shown advantageous in combination with directed binding to the hot spots. This was demonstrated using a structure consisting of three surface materials (gold, titanium dioxide, and silicon dioxide) and a new protocol for material-selective surface chemistry of these three materials, which allows for controlled binding only in the gap between pairs of disks. Such a design increased the signal obtained per bound molecule by a factor of around four compared to binding to single disks.     

Biofunctionalization strategies on tantalum-based materials for osseointegrative applications

The use of tantalum as biomaterial for orthopedic applications is gaining considerable attention in the clinical practice because it presents an excellent chemical stability, body fluid resistance, biocompatibility, and it is more osteoconductive than titanium or cobalt-chromium alloys. Nonetheless, metallic biomaterials are commonly bioinert and may not provide fast and long-lasting interactions with surrounding tissues. The use of short cell adhesive peptides derived from the extracellular matrix has shown to improve cell adhesion and accelerate the implant’s biointegration in vivo. However, this strategy has been rarely applied to tantalum materials. In this work, we have studied two immobilization strategies (physical adsorption and covalent binding via silanization) to functionalize tantalum surfaces with a cell adhesive RGD peptide. Surfaces were used untreated or activated with either HNO₃ or UV/ozone treatments. The process of biofunctionalization was characterized by means of physicochemical and biological methods. Physisorption of the RGD peptide on control and HNO₃-treated tantalum surfaces significantly enhanced the attachment and spreading of osteoblast-like cells; however, no effect on cell adhesion was observed in ozone-treated samples. This effect was attributed to the inefficient binding of the peptide on these highly hydrophilic surfaces, as evidenced by contact angle measurements and X-ray photoelectron spectroscopy. In contrast, activation of tantalum with UV/ozone proved to be the most efficient method to support silanization and subsequent peptide attachment, displaying the highest values of cell adhesion. This study demonstrates that both physical adsorption and silanization are feasible methods to immobilize peptides onto tantalum-based materials, providing them with superior bioactivity.     

Investigation of effects of Argenine–Glycine–Aspartate (RGD) and nano-scale titanium coatings on cell spreading and adhesion

This paper examines the effects of physical and chemical surface modifications on the biocompatibility of silicon surfaces that are relevant to implantable silicon Bio-micro-electro-mechanical systems (BioMEMS). Two types of surface modifications were explored. The first involved the deposition of nano-scale biocompatible layers of pure titanium on silicon, while the second explored the covalent attachment of the binding peptide Argenine–Glycine–Aspartic acid (RGD) for improved cell adhesion. Improvements in biocompatibility were assessed through examination of cell areas after culture, as well as the measurements of adhesion strengths, as determined by shear assay techniques. The titanium nanolayers and the RGD coating resulted in improvements in biocompatibility. Increased cell spreading areas and improved adhesion strength were obtained from short and long-term studies of Human Osteosarcoma (HOS) cells cultured on the coated surfaces. RGD functionalization resulted in the greatest improvement in cell spreading area and adhesion strength for short culture times. The effects of the titanium, while less than those of RGD for short culture times, appeared to be greater after 48 h of culture.     

Dextran‐based coating system for the immobilization of cell adhesion promoting molecules on titanium surfaces

Immobilization of adhesive peptides interacting with cellular integrin receptors onto metallic implant surfaces represents a promising approach to improve osseointegration of implants into the surrounding tissue. In the present study, a functional dextran‐based coating system consisting of an amino titanate adhesion promoter with dendritic structure and a carboxymethyl dextran was established to bind an RGD‐containing adhesive peptide via a selective coupling methodology onto titanium surfaces. The three‐step reaction procedure was characterized by X‐ray photoelectron spectroscopy. In cell adhesion experiments it could be demonstrated that dextran coatings containing immobilized RGD promote attachment and spreading of fibroblast and pre‐osteoblastic cells compared to native as well as CMD‐coated titanium surfaces without RGD. The direct attachment of the RGD sequence to the metal surface via the amino titanate adhesion promoter did not increase pre‐osteoblastic cell spreading, whereas coupling of RGD to the polymeric carboxy­methyl dextran layer slightly enhanced spreading of the cells.     

Effects of alloying elements on the cytotoxic response of titanium alloys

Titanium alloys, especially β-type alloys containing β-stabilizing elements, constitute a highly versatile category of metallic materials that have been under constant development for application in orthopedics and dentistry. This type of alloy generally presents a high mechanical strength-to-weight ratio, excellent corrosion resistance and low elastic modulus. The purpose of this study is to evaluate the cytotoxicity and adhesion of fibroblast cells on titanium alloy substrates containing Nb, Ta, Zr, Cu, Sn and Mo alloying elements. Cells cultured on polystyrene were used as controls. In vitro results with Vero cells demonstrated that the tested materials, except Cu-based alloy, presented high viability in short-term testing. Adhesion of cells cultured on disks showed no differences between the materials and reference except for the Ti–Cu alloy, which showed reduced adhesion attributed to poor metabolic activity. Titanium alloys with the addition of Nb, Ta, Zr, Sn and Mo elements show a promising potential for biomedical applications.     

The effects of micro arc oxidation of gamma titanium aluminide surfaces on osteoblast adhesion and differentiation

The adhesion and proliferation of human fetal osteoblasts, hFOB 1.19, on micro arc oxidized (MAO) gamma titanium aluminide (γTiAl) surfaces were examined in vitro. Cells were seeded on MAO treated γTiAl disks and incubated for 3 days at 33.5 °C and subsequently for 7 days at 39.5 °C. Samples were then analyzed by scanning electron microscopy (SEM) and alkaline phosphatase assay (ALP) to evaluate cell adhesion and differentiation, respectively. Similar Ti-6Al-4V alloy samples were used for comparison. Untreated γTiAl and Ti-6Al-4V disks to study the effect of micro arc oxidation and glass coverslips as cell growth controls were also incubated concurrently. The ALP Assay results, at 10 days post seeding, showed significant differences in cell differentiation, with P values <0.05 between MAO γTiAl and MAO Ti-6Al-4V with respect to the corresponding untreated alloys. While SEM images showed that hFOB 1.19 cells adhered and proliferated on all MAO and untreated surfaces, as well as on glass coverslips at 10 days post seeding, cell differentiation, determined by the ALP assay, was significantly higher for the MAO alloys.