J. Barbara Nebe

Osteoblast response to biomimetically altered titanium surfaces

Bioinert titanium (Ti) materials are generally encapsulated by fibrous tissue after implantation into the living body. To improve the bone-bonding ability of Ti implants, we activated commercially pure titanium (cpTi) by a simple chemical pre-treatment in HCl and NaOH. Subsequently, we exposed the treated samples to simulated body fluid (SBF) for 2 (TiCT) and 14 days (TiHCA), respectively, to mimic the early stages of bone bonding and to investigate the in vitro response of osteoblasts on thus altered biomimetic surfaces. Sample surfaces were characterized by scanning electron microscopy, energy-dispersive X-ray analysis, cross-sectional transmission electron microscopy analyses, Fourier transform infrared and Raman spectroscopy. It was shown that the efflorescence consisting of sodium titanate that is present on pre-treated cpTi surfaces transformed to calcium titanate after 2 days in SBF. After 14 days in SBF a homogeneous biomimetic apatite layer precipitated. Human osteoblasts (MG-63) revealed a well spread morphology on both functionalized Ti surfaces. On TiCT, the gene expression of the differentiation proteins alkaline phosphatase (ALP) and bone sialo protein was increased after 2 days. On both TiCT and TiHCA, the collagen I and ALP expression on the protein level was enhanced at 7 and 14 days. The TiCT and the TiHCA surfaces reveal the tendency to increase the differentiated cell function of MG-63 osteoblasts. Thus, chemical pre-treatment of titanium seems to be a promising method to generate osteoconductive surfaces.

Cold atmospheric plasma in combination with mechanical treatment improves osteoblast growth on biofilm covered titanium discs

Treatment of implants with peri-implantitis is often unsuccessful, because an instrumented implant surface and residual microbial biofilm impedes re-osseointegration. The application of cold atmospheric plasma (CAP) could be a simple and effective strategy to overcome the inherent problems of peri-implantitis treatment. CAP is able to destroy and eliminate bacterial biofilms. Additionally, it increases the wettability of titanium, which supports cellular attachment. In this study, the behaviour of osteoblasts on titanium discs was analysed after treatment of bacterial biofilms with CAP, brushing, or a combination of both. A human plaque biofilm was cultured on titanium discs. Treatment with a brush (BR), 1% oxygen/argon CAP (PL), or brushing combined with CAP (BR+PL) was used to eliminate the biofilm. Discs without biofilm (C), autoclaved biofilm (AUTO) and untreated biofilm (BIO) served as controls. Subsequently, human osteoblastic cell growth (MG-63) was observed after 1 and 24 h. Biofilm remnants on BR and PL impaired osteoblastic cell development, whereas the BR+PL provided an increased area of osteoblastic cells. A five-day cell growth was only detectable on BR+PL treated discs. The combination of established brushing and CAP application may be a promising strategy to treat peri-implantitis.

Impact of plasma chemistry versus titanium surface topography on osteoblast orientation.

Topographical and chemical modifications of biomaterial surfaces both influence tissue physiology, but unfortunately little knowledge exists as to their combined effect. There are many indications that rough surfaces positively influence osteoblast behavior. Having determined previously that a positively charged, smooth titanium surface boosts osteoblast adhesion, we wanted to investigate the combined effects of topography and chemistry and elucidate which of these properties is dominant. Polished, machined and corundum-blasted titanium of increasing microroughness was additionally coated with plasma-polymerized allylamine (PPAAm). Collagen I was then immobilized using polyethylene glycol diacid and glutar dialdehyde. On all PPAAm-modified surfaces (i) adhesion of human MG-63 osteoblastic cells increased significantly in combination with roughness, (ii) cells resemble the underlying structure and melt with the surface, and (iii) cells overcome the restrictions of a grooved surface and spread out over a large area as indicated by actin staining. Interestingly, the cellular effects of the plasma-chemical surface modification are predominant over surface topography, especially in the initial phase. Collagen I, although it is the gold standard, does not improve surface adhesion features comparably.

Automatic Actin Filament Quantification of Osteoblasts and Their Morphometric Analysis on Microtextured Silicon-Titanium Arrays

Microtexturing of implant surfaces is of major relevance in the endeavor to improve biorelevant implant designs. In order to elucidate the role of biomaterial’s topography on cell physiology, obtaining quantitative correlations between cellular behavior and distinct microarchitectural properties is in great demand. Until now, the microscopically observed reorganization of the cytoskeleton on structured biomaterials has been difficult to convert into data. We used geometrically microtextured silicon-titanium arrays as a model system. Samples were prepared by deep reactive-ion etching of silicon wafers, resulting in rectangular grooves (width and height: 2 µm) and cubic pillars (pillar dimensions: 2 × 2 × 5 and 5 × 5 × 5 µm); finally sputter-coated with 100 nm titanium. We focused on the morphometric analysis of MG-63 osteoblasts, including a quantification of the actin cytoskeleton. By means of our novel software FilaQuant, especially developed for automatic actin filament recognition, we were first able to quantify the alterations of the actin network dependent on the microtexture of a material surface. The cells’ actin fibers were significantly reduced in length on the pillared surfaces versus the grooved array (4–5 fold) and completely reorganized on the micropillars, but without altering the orientation of cells. Our morpho-functional approach opens new possibilities for the data correlation of cell-material interactions.

Evaluation of Osseointegration of Titanium Alloyed Implants Modified by Plasma Polymerization

By means of plasma polymerization, positively charged, nanometre-thin coatings can be applied to implant surfaces. The aim of the present study was to quantify the adhesion of human bone cells in vitro and to evaluate the bone ongrowth in vivo, on titanium surfaces modified by plasma polymer coatings. Different implant surface configurations were examined: titanium alloy (Ti6Al4V) coated with plasma-polymerized allylamine (PPAAm) and plasma-polymerized ethylenediamine (PPEDA) versus uncoated. Shear stress on human osteoblast-like MG-63 cells was investigated in vitro using a spinning disc device. Furthermore, bone-to-implant contact (BIC) was evaluated in vivo. Custom-made conical titanium implants were inserted at the medial tibia of female Sprague-Dawley rats. After a follow-up of six weeks, the BIC was determined by means of histomorphometry. The quantification of cell adhesion showed a significantly higher shear stress for MG-63 cells on PPAAm and PPEDA compared to uncoated Ti6Al4V. Uncoated titanium alloyed implants showed the lowest BIC (40.4%). Implants with PPAAm coating revealed a clear but not significant increase of the BIC (58.5%) and implants with PPEDA a significantly increased BIC (63.7%). In conclusion, plasma polymer coatings demonstrate enhanced cell adhesion and bone ongrowth compared to uncoated titanium surfaces.

Preparation, microstructures, mechanical properties, and cytocompatibility of TiMn alloys for biomedical applications

The titanium-manganese (TiMn) alloys have been extensively used in aerospace and hydrogen storage. In this study, the TiMn alloys with various manganese contents ranging from 2 to 12 wt % were prepared by using mechanical alloying and spark plasma sintering (SPS) techniques. The microstructures, mechanical properties including hardness, elastic modulus and ductility, cytotoxicity and cell proliferation properties of the TiMn alloys were investigated to explore their biomedical applications. The addition of manganese to the titanium reduced the alpha to beta transformation temperature and was confirmed as a beta stabilizer element. The manganese increased the relative density of the alloy and thus high density TiMn alloys with alpha+beta structure were prepared by using SPS at 700 degrees C. The hardness increased significantly ranging from 2.4 GPa (Ti2Mn) to 5.28 GPa (Ti12Mn) and the elastic modulus ranging from 83.3 GPa (Ti2Mn) to 122 GPa (Ti12Mn), the ductility decreased ranging from 21.3% (Ti2Mn) to 11.7% (Ti12Mn) with increasing manganese content in the Ti. Concentrations of Mn below 8 wt % in titanium reveal negligible effects on the metabolic activity and the cell proliferation of human osteoblasts. The Mn could be used in lower concentrations as an alloying element for biomedical titanium. The Ti2Mn, Ti5Mn, and Ti8Mn alloys with supervisor mechanical properties and acceptable cytocompatibility have a potential for use as bone substitutes and dental implants.

Gas-Discharge Plasma-Assisted Functionalization of Titanium Implant Surfaces

A crucial factor for in-growth of metallic implants in the bone stock is the rapid cellular acceptance whilst prevention of bacterial adhesion on the surface. Such contradictorily adhesion events could be triggered by surface properties. There already exists fundamental knowledge about the influence of physicochemical surface properties like roughness, titanium dioxide modifications, cleanness, and (mainly ceramic) coatings on cell and microbial behavior in vitro and in vivo. The titanium surface can be equipped with antimicrobial properties by plasma-based copper implantation, which allows the release and generation of small concentrations of copper ions during contact with water-based biological liquids. Additionally, the titanium surface was equipped with amino groups by the deposition of an ultrathin plasma polymer. This coating on the one hand does not significantly reduce the generation of copper ions, and on the other hand improves the adhesion and spreading of osteoblast cells. The process development was accompanied by physicochemical surface analyses like XPS, FTIR, contact angle, SEM, and AFM. Very thin modified layers were created, which are resistant to hydrolysis and delamination. These titanium surface functionalizations were found to have either an antimicrobial activity or cell-adhesive properties. Intramuscular implantation of titanium samples coated with the cell-adhesive plasma polymer in rats revealed a reduced inflammation reaction compared to uncoated titanium.

Time-dependent metabolic activity and adhesion of human osteoblast-like cells on sensor chips with a plasma polymer nanolayer.

Purpose To improve orthopedic implant ingrowth, knowledge of the effect of chemical surface modifications on vital cell function in vitro is of importance. Early in our investigations we recognized that amino groups, positively charged via plasma polymerized allylamine, increased cell growth and the actin-filament formation in the initial cell-material contact phase. To gain insight into continuous vital cell behavior on this plasma polymer layer, here we present the metabolic activity of osteoblasts and their time-dependent adhesion using the sensor chip technology. Methods We demonstrate a new method for continuous 24 hour-measurements with vital human osteoblast-like cells (MG-63, ATCC) on sensor chips (Bionas® SC 1000) modified with plasma polymerized allylamine (PPAAm). The PPAAm film deposited on the chip is a cross-linked, strongly fixed plasma polymer with relatively high amino functionality and well defined chemical surface composition. We assessed continuous cell adhesion and the metabolic activity, i.e., oxygen consumption and acidification. Results We determined that adhesion of vital cells on PPAAm is not only enhanced shortly (1 h) after cell seeding but remained continuously higher for 24 h, which is significant. This nanometer-thin PPAAm layer did not change the overall metabolic activity of MG-63 cells during 24 h. Conclusion This tool – using adhesion and metabolic sensor chips – appears to be a suitable method for the recognition of vital cell physiology in biocompatibility measurements of plasma chemical treated surfaces.

Aging of Plasma-Polymerized Allylamine Nanofilms and the Maintenance of Their Cell Adhesion Capacity

The long-term stability and γ-sterilisability of bioactive layers is the precondition for the application of implants. Thus, aging processes of a microwave deposited, plasma polymerized allylamine nanofilm (PPAAm) with positively charged amino groups were evaluated concerning physicochemical characteristics and cell adhesion capacity over the course of one year. XPS, FT-IR, surface free energy, and water contact angle measurements elucidated not only the oxidation of the PPAAm film due to atmospheric oxygen reacting with surface free radicals but also the influence of atmospheric moisture during sample storage in ambient air. Surprisingly, within 7 days 70% of the primary amino groups are lost and mostly converted into amides. A positive zeta-potential was verified for half a year and longer. Increasing polar surface groups and a water contact angle shift from 60° to 40° are further indications of altered surface properties. Nevertheless, MG-63 human osteoblastic cells adhered and spread out considerably on aged and additionally γ-sterilized PPAAm layers deposited on polished titanium alloys (Ti-6Al-4V_P). These cell-relevant characteristics were highly significant over the whole period of one year and may not be related to the existence of primary amino groups. Rather, the oxidation products, the chemical amide group, that is, seem to support the attachment of osteoblasts at all times up to one year.

Cellular Activity and Biomaterial's Surface Topography

The contact of a cell on the biomaterial’s surface is mediated by its adhesion components. The topography of titanium surfaces influences these adhesion components of osteoblasts, e.g. the integrins, the adapter proteins and the actin cytoskeleton. In our current experiments we were interested in why osteoblasts were strongly aligned to the grooves of a structured pure titanium surface (grade 2). The titanium was characterized by EIS to get insights in the electro-chemically active surface. We used MG-63 human bone cells, cultured in DMEM with 10% FCS at 37°C. For protein adsorption the titanium discs were incubated for 24h with complete medium containing soluble fibronectin at 37°C. Interestingly, only in the grooves cells adhered and were aligned and this is not dependent on the gravitation. The cell adhesion seems to depend on the protein adsorption of fibronectin which we could find to be adsorbed exclusively in the valleys. We speculate that there are local differences in electro-chemical characteristics of this structured titanium surface.