Increased reactivity and in vitro cell response of titanium based implant surfaces after anodic oxidation.

In the quest for improved bone growth and attachment around dental implants, chemical surface modifications are one possibility for future developments. The biological properties of titanium based materials can be further enhanced with methods like anodic polarization to produce an active rather than a passive titanium oxide surface. Here we investigate the formation of hydroxide groups on sand blasted and acid etched titanium and titanium-zirconium alloy surfaces after anodic polarization in an alkaline solution. X-ray photoelectron spectroscopy shows that the activated surfaces had increased reactivity. Furthermore the activated surfaces show up to threefold increase in OH(-) concentration in comparison to the original surface. The surface parameters Sa, Sku, Sdr and Ssk were more closely correlated to time and current density for titanium than for titanium-zirconium. Studies with MC3T3-E1 osteoblastic cells showed that OH(-) activated surfaces increased mRNA levels of osteocalcin and collagen-I.

Thin films of TiO2:N for photo-electrochemical applications.

Dc-pulsed magnetron sputtering from Ti target in reactive Ar+O2+N2 atmosphere was used to grow stoichiometric TiO2:N and non-stoichiometric TiO2-x:N thin films. X-ray diffraction at glancing incidence, atomic force microscopy AFM, scanning electron microscopy SEM, X-ray photoelectron spectroscopy XPS, and optical spectrophotometry were applied for sample characterization. Measurements of photocurrent versus voltage and wavelength over the ultraviolet uv and visible vis ranges of the light spectrum were performed in order to assess the performance of nitrogen-doped titanium dioxide thin films as photoanodes for hydrogen generation in photoelectrochemical cells, PEC. Undoped TiO2 and TiO2-x films were found to be composed of anatase and rutile mixture with larger anatase crystallites (25-35 nm) while the growth of smaller rutile crystallites (6-10 nm) predominated at higher nitrogen flow rates etaN2 as measured in standard cubic centimeters, sccm. Nitrogen-to-titanium ratio increased from N/Ti = 0.05 at etaN2 = 0.8 sccm for stoichiometric TiO2:N to N/Ti = 0.11 at etaN2 = 0.8 sccm for nonstoichiometric TiO2-x:N thin films. A red-shift in the optical absorbance was observed with an increase in etaN2. Doping with nitrogen improved photoelectrochemical properties over the visible range of the light spectrum in the case of nonstoichiometric samples.

Nanostructured collagen layers obtained by adsorption and drying.

The supramolecular organization of collagen adsorbed from a 7 microg/ml solution on polystyrene was investigated as a function of the adsorption duration (from 1 min to 24 h) and of the drying conditions (fast drying under a nitrogen flow, slow drying in a water-saturated atmosphere). The morphology of the created surfaces was examined by atomic force microscopy (AFM), while complementary information regarding the adsorbed amount and the organization of the adsorbed layers was obtained using radioassays, X-ray photoelectron spectroscopy (XPS), and wetting measurements. The collagen adsorbed amount increased up to an adsorption duration of 5 h and then leveled off at a value of 0.9 microg/cm2. For samples obtained by fast drying, modeling of the N/C ratios obtained by XPS in terms of thickness and surface coverage, in combination with the adsorbed amount, water contact angle measurements and AFM images, indicated that the adsorbed layer formed a felt starting from 30 min of adsorption, the density and/or the thickness of which increased with the adsorption time. Upon slow drying, the collagen layers formed after adsorption times up to about 2 h underwent a strong reorganization. The obtained nanopatterns were attributed to dewetting, the liquid film being ruptured and adsorbed collagen being displaced by the water meniscus. At higher adsorption times, the organization of the collagen layer was similar to that obtained after fast drying, because the onset of dewetting and/or collagen displacement were prevented by the high density of the collagen felt.

[Studies on development of composite biomaterials for reconstruction of the larynx]. / Badania nad opracowaniem biomaterialu kompozytowego do rekonstrukcji krtani.

The paper presents the results of studies on the development of composite material to be used in laryngology. The composite was manufactured from carbon fibres and polysulfone in the form of multiphase laminate having suitable surface properties. Mechanical properties of three different types of composite materials were analysed. Of the elaborated composites the material possessing the most advantageous mechanical properties was than analysed by means of FTIR and SEM methods. Results obtained permit determination of such composite material in laryngeal and tracheal reconstruction.

Incorporation of sol-gel bioactive glass into PLGA improves mechanical properties and bioactivity of composite scaffolds and results in their osteoinductive properties.

In this study, 3D porous bioactive composite scaffolds were produced and evaluated for their physico-chemical and biological properties. Polymer poly-L-lactide-co-glycolide (PLGA) matrix scaffolds were modified with sol-gel-derived bioactive glasses (SBGs) of CaO-SiO2-P2O5 systems. We hypothesized that SBG incorporation into PLGA matrix would improve the chemical and biological activity of composite materials as well as their mechanical properties. We applied two bioactive glasses, designated as S2 or A2, differing in the content of SiO2 and CaO (i.e. 80 mol% SiO2, 16 mol% CaO for S2 and 40 mol% SiO2, 52 mol% CaO for A2). The composites were characterized for their porosity, bioactivity, microstructure and mechanical properties. The osteoinductive properties of these composites were evaluated in human bone marrow stromal cell (hBMSC) cultures grown in either standard growth medium or treated with recombinant human bone morphogenetic protein-2 (rhBMP-2) or dexamethasone (Dex). After incubation in simulated body fluid, calcium phosphate precipitates formed inside the pores of both A2-PLGA and S2-PLGA scaffolds. The compressive strength of the latter was increased slightly compared to PLGA. Both composites promoted superior hBMSC attachment to the material surface and stimulated the expression of several osteogenic markers in hBMSC compared to cells grown on unmodified PLGA. There were also marked differences in the response of hBMSC to composite scaffolds, depending on chemical compositions of the scaffolds and culture treatments. Compared to silica-rich S2-PLGA, hBMSC grown on calcium-rich A2-PLGA were overall less responsive to rhBMP-2 or Dex and the osteoinductive properties of these A2-PLGA scaffolds seemed partially dependent on their ability to induce BMP signaling in untreated hBMSC. Thus, beyond the ability of currently studied composites to enhance hBMSC osteogenesis, it may become possible to modulate the osteogenic response of hBMSC, depending on the chemistry of SBGs incorporated into polymer matrix.

Enzymatic mineralization of gellan gum hydrogel for bone tissue-engineering applications and its enhancement by polydopamine.

Interest is growing in the use of hydrogels as bone tissue-engineering (TE) scaffolds due to advantages such as injectability and ease of incorporation of active substances such as enzymes. Hydrogels consisting of gellan gum (GG), an inexpensive calcium-crosslinkable polysaccharide, have been applied in cartilage TE. To improve GG suitability as a material for bone TE, alkaline phosphatase (ALP), an enzyme involved in mineralization of bone by cleaving phosphate from organic phosphate, was incorporated into GG hydrogels to induce mineralization with calcium phosphate (CaP). Incorporated ALP induced formation of apatite-like material on the submicron scale within GG gels, as shown by FTIR, SEM, EDS, XRD, ICP-OES, TGA and von Kossa staining. Increasing ALP concentration increased amounts of CaP as well as stiffness. Mineralized GG was able to withstand sterilization by autoclaving, although stiffness decreased. In addition, mineralizability and stiffness of GG was enhanced by the incorporation of polydopamine (PDA). Furthermore, mineralization of GG led to enhanced attachment and vitality of cells in vitro while cytocompatibility of the mineralized gels was comparable to one of the most commonly used bone substitute materials. The results proved that ALP-mediated enzymatic mineralization of GG could be enhanced by functionalization with PDA.

Linseed oil based nanocapsules as delivery system for hydrophobic quantum dots.

In the present work, the CdSe/ZnS hydrophobic quantum dots were embedded within the polyelectrolyte nanocapsules. The core of the capsules, which consists of a mixture of the linseed oil with chloroform, was prepared using the spontaneous emulsification technique. The obtained emulsions were stabilized with lecithin and encapsulated using the layer-by-layer (LbL) adsorption of polyelectrolytes. The pair of biocompatible polyelectrolytes was used: the cationic poly-l-lysine hydrobromide (PLL) together with the anionic poly-d-glutamic acid sodium salt. The saturation LbL method, which is based on the stepwise formation of consecutive layers on the initial emulsion without the intermediate rinsing step, was applied to form the capsule shells. Their growth was evidenced by the capsule size and electrophoretic mobility measurements. The emulsion and the capsules were deposited on a mica surface and the deposit topology was examined by the means of atomic force microscopy (AFM). The presence of quantum dots within the oil cores was confirmed by recording the fluorescent spectra of the samples containing CdSe/ZnS. In order to evaluate cytotoxicity of the capsules, their influence on the viability of mouse embryonic fibroblasts was examined using the MTT test, followed by optical-microscope observation of morphology of the cells after hematoxylin-eosin staining.

Poly(L-lactide-co-glycolide) scaffolds coated with collagen and glycosaminoglycans: impact on proliferation and osteogenic differentiation of human mesenchymal stem cells.

In this study, we analyzed poly(L-lactide-co-glycolide) (PLGA) scaffolds modified with artificial extracellular matrices (aECM) consisting of collagen type I, chondroitin sulphate, and sulphated hyaluronan (sHya). We investigated the effect of these aECM coatings on proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSC) in vitro. We found that scaffolds were homogeneously coated, and cross-linking of aECM did not significantly influence the amount of collagen immobilized. Cell proliferation was significantly increased on cross-linked surfaces in expansion medium (EM), but was retarded on cross-linked and non-cross-linked collagen/sHya coatings. The alkaline phosphatase activity was increased on sHya-containing coatings in EM even without the presence of differentiation supplements, but was six to ten times higher in differentiation medium (DM) and comparable for cross-linked and non-cross-linked collagen/sHya. The highest amount of calcium phosphate mineral was deposited on day 28 on cross-linked collagen/sHya. Therefore, coatings of PLGA scaffolds with collagen/sHya promoted the osteogenic differentiation of hMSCs in vitro and might be an interesting candidate for the modification of PLGA for bone reconstruction in vivo.

Effects of aliphatic polyesters on activation of the immune system: studies on macrophages.

There is a constant search for biodegradable polymers with biocompatible characteristics. However, the reported materials are rarely tested for their immunostimulatory properties, which is an important issue as immune cells activated by the polymers might cause their rejection and lead to further injury to the host tissues. Therefore, the aim of the present study was to determine if biodegradable polymers are able to activate RAW 264.7 macrophages. Aliphatic polyesters, poly(L-lactide) (PLLA), poly(L-lactide-co-trimethylene carbonate) (PLTMC), poly(glycolide-co-L-lactide) (PGLA), poly(glycolide-co-L-lactide-co-ε-caprolactone) (PGLCap) and poly(glycolide-co-ε-caprolactone) (PGCap), processed into foils by slip-casting, were characterized in terms of their structure ((1)H-NMR, GPC, DSC) and surface properties (chemical composition, water contact angle, surface free energy, topography and roughness). RAW 264.7 cells were cultured on the materials for 3 or 5 days and their adherence, numbers of apoptotic/necrotic cells, as well as production of several cytokines/chemokines and other inflammation-related molecules (matrix metalloproteinases, nitric oxide) was evaluated. The study demonstrated that PLLA and PGLA did not influence macrophage activation and survival. In contrast, PLTMC, PGLCap and PGCap significantly decreased macrophage adherence, increased ratio of apoptosis and up-regulated synthesis/release of numerous inflammatory mediators. Thus, the latter materials might initiate an undesired inflammatory reaction. The above effects of the polymers were attributed to their high hydrophobicity and low polarity due to the presence of ε-caproyl blocks (PGLCap and PGCap), and/or high flexibility and susceptibility to mechanical deformation due to low glasstransition temperature (PLTMC, PGLCap and PGCap). In conclusion, while PLLA and PGLA do not affect macrophage functioning, the other materials (PLTMC, PGLCap, PGCap) up-regulate macrophage activity.

Controlling the supramolecular organisation of adsorbed collagen layers.

The supramolecular organisation of collagen adsorbed on polymer substrates was investigated as a function of properties of the substrates (chemical nature, roughness) and of characteristics of the collagen solution (concentration, state of aggregation) as well as details of the preparation procedure (adsorption time, drying rate). Elongated structures are formed at the interface by assembly of collagen molecular segments protruding into the solution. This is favoured by using a hydrophobic and smooth substrate, by increasing the adsorbed amount and by increasing the adsorption time, even beyond stages at which the adsorbed amount does no longer vary. Collagen adsorbed at low amount on hydrophobic substrates strongly reorganises into a net-like pattern if drying is performed at low rate. This is due to dewetting and collagen displacement by the water meniscus. Applications derived from the control of collagen organisation are presented. Nanostructured polymer surfaces were created starting from a collagen template. The attachment and the cytoskeletal organisation of mammalian cells (MCF-7/6) were also shown to depend on collagen organisation.