Technical strategies to improve tissue engineering of cartilage-carrier-constructs.

Technical aspects play an important role in tissue engineering. Especially an improved design of bioreactors is crucial for cultivation of artificial three-dimensional tissues in vitro. Here formation of cartilage-carrier-constructs is used to demonstrate that the quality of the tissue can be significantly improved by using optimized culture conditions (oxygen concentration, growth factor combination) as well as special bioreactor techniques to induce fluid-dynamic, hydrostatic or mechanical load during generation of cartilage.

Biomimetic implant coatings.

Biomaterials and tissue engineering technologies are becoming increasingly important in biomedical practice, particularly as the population ages. Cellular responses depend on topographical properties of the biomaterial at the nanometer scale. Structures on biomaterial surfaces are used as powerful tools to influence or even control interactions between implants and the biological system [; ]. The influence of nanometer sized surface structures on osteoblastlike cell interactions was tested with niobium oxide coatings on polished titanium slices (cp-Ti grade 2). The aim of the study was to investigate the influence of nanoscopic surface structures on osteoblast interactions in order to support collagen I production and cell adhesion. The coatings were done by means of the sol-gel process. The surface structure was adjusted by annealing of the metaloxide ceramic coatings due to temperature depended crystal growth. The applied annealing temperatures were 450, 550 and 700 degrees C for 1 h, corresponding to Ra-numbers of 7, 15 and 40 nm. The surfaces were characterized by means of AFM, DTA/TG, diffractometry and white light interferometry. The cell reactions were investigated concerning adhesion kinetics, migration, spreading, cell adhesion, and collagen I synthesis. The smooth surface (Ra=7 nm) resulted in the fastest cell anchorage and cell migration. The closest cell adhesion was reached with the surface structure of Ra=15 nm. The roughest surface (Ra=40 nm) impedes the cell migration as well as a proper spreading of the cells. The best results concerning cell adhesion and spreading was reached with an intermediate surface roughness of Ra=15 nm of the niobium oxide coating on cp-titanium slices.

Nanostructured niobium oxide coatings influence osteoblast adhesion.

The interaction of osteoblasts was correlated to the roughness of nanosized surface structures of Nb(2)O(5) coatings on polished CP titanium grade 2. Nb(2)O(5) sol-gel coatings were selected as a model surface to study the interaction of osteoblasts with nanosized surface structures. The surface roughness was quantified by determination of the average surface finish (Ra number) by means of atomic force microscopy. Surface topographies with Ra = 7, 15, and 40 nm were adjusted by means of the annealing process parameters (time and temperature) within a sol-gel coating procedure. The observed osteoblast migration was fastest on smooth surfaces with Ra = 7 nm. The adhesion strength, spreading area, and collagen-I synthesis showed the best results on an intermediate roughness of Ra = 15 nm. The surface roughness of Ra = 40 nm was rather peaked and reduced the speed of cell reactions belonging to the adhesion process.

[Corrosion behaviour, metal release and biocompatibility of implant materials coated by TiO2-sol gel chemistry]. / Einfluss einer TiO2-beschichtung auf Biokompatibilität, Korrosions- und Auslaugverhalten verschiedener Implantatlegierungen.

Alloys based on titanium or cobalt have been used as implant materials for decades with good success. Because of their natural oxide layer these alloys reveal good corrosion behaviour. In contact with physiological solution metal release takes place, which can cause inflammation. Coatings can improve the corrosion behaviour. In this study Ti6Al4V and Co28Cr6Mo alloys, which are frequently used as implant materials, were tested. Polished discs of these alloys and polished discs, which were coated with TiO2-layers by sol-gel chemistry, were compared regarding their corrosion behaviour and metal ion releasing. The releasing of Al, V, Ti, Co, Cr and Mo was quantified by ICP-MS analysis. The TiO2-coating reduced the release of all ions except of the Al-ion. Both alloys showed a deviating kinetic of ion releasing. In addition, cell response (cell vitality, cell proliferation, endothelial marker CD31 and actin allocation) of osteoblasts and endothelial cells were investigated.

Biocompatible Nb2O5 thin films prepared by means of the sol-gel process.

Thin biocompatible oxide films with an optimised composition and structure on the surface of titanium and its alloys can improve the implant integration. The preparation of these thin oxide layers with the intended improvement of the surface properties can be realised by means of the sol-gel process. Nb2O5 is a promising coating material for this application because of its extremely high corrosion resistance and thermodynamic stability. In this study, thin Nb2O5 layers ( < 200 nm) were prepared by spin coating of polished discs of cp-titanium with a sol consisting of a mixture of niobium ethoxide, butanol and acetylacetone. The thickness, phase composition, corrosion resistance and the wettability of the oxide layers were determined after an optimisation of the processing parameters for deposition of oxide without any organic impurities. The purity of the oxide layer is an important aspect in order to avoid a negative response to the cell adhesion. The biocompatibility of the oxide layers which was investigated by in vitro tests (morphology, proliferation rate, WST-1, cell spreading) is improved as compared to uncoated and TiO2 sol-gel coated cp-titanium concerning the spreading of cells, collagen I synthesis and wettability.

Biocompatibility of beta-stabilizing elements of titanium alloys.

In comparison to the presently used alpha + beta titanium alloys for biomedical applications, beta-titanium alloys have many advantageous mechanical properties, such as an improved wear resistance, a high elasticity and an excellent cold and hot formability. This will promote their future increased application as materials for orthopaedic joint replacements. Not all elements with beta-stabilizing properties in titanium alloys are suitable for biomaterial applications-corrosion and wear processes cause a release of these alloying elements to the surrounding tissue. In this investigation, the biocompability of alloying elements for beta- and near beta-titanium alloys was tested in order to estimate their suitability for biomaterial components. Titanium (grade 2) and the implant steel X2CrNiMo18153 (AISI 316 L) were tested as reference materials. The investigation included the corrosion properties of the elements, proliferation, mitochondrial activity, cell morphology and the size of MC3T3-E1 cells and GM7373 cells after 7 days incubation in direct contact with polished slices of the metals. The statistical significance was considered by Weir-test and Lord-test (alpha = 0.05). The biocompatibility range of the investigated metals is (decreasing biocompatibility): niobium-tantalum, titanium, zirconium-aluminium-316 L-molybdenum.

Cell orientation and cytoskeleton organisation on ground titanium surfaces.

A stable connection between the biomaterial surface and the surrounding tissue is one of the most important prerequisites for the long-term success of implants. Therefore, a strong adhesion of the cells on the biomaterial surface is required. Beside the surface composition the surface topography influences the properties of the adherent cells. The quality of the connection between the cell and the biomaterial is-among other factors-determined by the dimensions of the surface topography. Osteoblasts and fibroblast-like cells in contact with a ground biomaterial surface spread in the direction of the surface structures. These aligned cells provide a more favourable adhesion behaviour than a spherically shaped cell. To determine the influence of the surface structure on the cell alignment and cytoskeleton organisation or arrangement, substrate discs of cp-titanium were ground, producing different roughness of the substrates. The oriented cells had a higher density of focal contacts when they were in contact with the edges of the grooves and showed a better organisation of the cytoskeleton and stronger actin fibres. These changes of the aligned cells depend on the peak to valley height of the surface structures.

Interactions between cells and titanium surfaces.

The interaction between cells and implant materials is determined by the surface structure and/or surface composition of the material. In the past years, titanium and titanium alloys have proved their superiority over other implant materials in many clinical applications. This predominant behaviour is caused by a dense passive oxide layer which forms within milliseconds in oxidizing media. Titanium dioxide layers of 100 nm thickness were produced on the surface of cp-titanium grade 2, and on an experimental alloy of high vanadium content (Ti1.5Al25V) as a harmful control. The layers were produced by thermal and anodic oxidation and by coating by means of the sol-gel process. The resulting oxide layers were characterized with respect of their structure and chemical composition. In cell tests (proliferation, MTT, morphology, actin staining), the reaction of the cells was examined. It was shown that the sol-gel-produced titanium oxide layer is able to shield the cells from toxic alloying elements, with the result that the cell reaction is influenced only by the thin titanium oxide surface layer and not by the composition of the bulk material.

[The in vitro biological behavior of TiNb30 alloy treated with hydroxyapatite and tricalcium phosphates]. / Le comportement biologique in vitro d'un alliage TiNb30 traité avec hydroxyapatite et phosphates tricalciques.

An in vitro study has been carried out in different cell systems to determine the biological response of TiNb30 alloy before and after a surface treatment with hydroxyapatite (HA) and tricalcium phosphate (TCP) by the sol-gel method. TiNb30 pure Ti induce favorable cell viability with respect to pure Ni showing a high cytotoxic effect. After surface treatment with HA or HA-TCP mixtures, no difference in cell proliferation can be observed between amorphous and cristalline forms. However, HA decreases (75 +/- 15%) and HA-TCP mixtures increase (133 +/- 11%) significantly cell proliferation compared with controls.

Influence of the surface structure of titanium materials on the adhesion of fibroblasts.

Of utmost importance for the successful use of an implant is a good adhesion of the surrounding tissue to the biomaterial. In addition to the surface composition of the implant, the surface topography also influences the properties of the adherent cells. The aim of this investigation was thus to study the influence of the surface structure of the substrate on the formation of focal contacts and on the orientation of cultivated gingival fibroblasts by means of fluorescence microscopy. A further goal was to determine the effect of the material composition on the cell shape, on the assumption that in each case a lengthening of the cells can be expected to provide a more favourable adhesion behaviour than a spherical cell shape. In order to describe the shape of the cell, a shape factor was defined which was calculated from the area covered by the cells and from their circumference. To determine the influence of the surface structure, substrate platelets of cp-titanium, TiAl6V4 and TiTa30 were ground. Onto these specimens human gingival fibroblasts of the 5th to 7th passages were cultivated. After a culture time of two days the cells were fixed and stained. The number of orientated cells was determined as a function of the surface roughness of the substrate. The number of orientated cells was shown to increase---independent of the material---with increasing roughness of the ground substrate. On a polished surface the number of orientated cells was 11% (average peak-to-valley height 0.04 microns); at a peak-to-valley height of 1.36 microns the number of orientated cells increased to 72%. It could be observed that the orientated cells had a higher density of focal contacts where they were in contact with the edges of the grinding grooves. In order to determine the effect of the surface composition, gingival fibroblasts were cultured for 14 d on polished substrate specimens of cp-titanium, TiAl6V4 and TiTa30 and examined for differences in shape. The cells grown on cp-titanium and on TiTa30 had shape factors of 1.76 and 1.58 respectively, whereas those grown on TiAl6V4 had a shape factor of 0.93. The unfavourable spherical shape of the fibroblasts (resulting in a poor adhesion) grown on TiAl6V4 after a culture period of 14 d may be the result of a locally increased vanadium concentration in the substrate, with an accompanying increase in the release of toxic vanadium ions.