Biological behavior of sol-gel coated dental implants.

The biocompatibility of dental implants coated with titania/hydroxyapatite (HA) and titania/bioactive glass (BG) composites obtained via sol-gel process was investigated using an in vitro and in vivo model. A device for the in vitro testing of screw-shaped dental implants was developed, in order to well compare the two experimental models studying the behavior of human MG63 osteoblast-like cells seeded onto a particular geometry. The expression of some biochemical parameters of osteoblastic phenotype (alkaline phosphatase specific activity, collagen and osteocalcin production) and some indications on cells morphology obtained by scanning electron microscopy were evaluated. The in vitro and in vivo models were compared after implants insertion in rabbit tibia and femur. The removal torque and histomorphometric parameters (percentage of bone in contact with implant surface and the amount of bone inside the threaded area) were examined. A good agreement was found between the in vitro and in vivo models. These experiments showed better performances of HA and BG sol-gel coated dental implants with respect to uncoated titanium; in particular, it was found that in vitro the HA coating stimulates osteoblastic cells in producing higher level of ALP and collagen, whereas in vivo this surface modification resulted in a higher removal torque and a larger bone-implant contact area. This behavior could be ascribed to the morphology and the chemical composition of the implants with rough and bioactive surfaces.

Three-dimensional reconstruction of confocal laser microscopy images to study the behaviour of osteoblastic cells grown on biomaterials.

The adhesion, spreading and cytoskeletal organization of osteoblastic cells seeded onto titanium and titania/hydroxyapatite composite coating (TiO2/HA) were studied using images acquired by confocal laser scanning microscopy. The fluorescence staining technique was employed to visualize actin cytoskeletal organization of cells, 2-D images were exhaustive when the cells were seeded at low density (in the first 24 h of incubation), but they were less clear when the cells proliferated and appeared stacked. Since the shareware software were not satisfactory, a new 3-D image reconstruction was developed using ordinary software and a model was obtained directly from the optical section set, in order to achieve a more realistic and faithful vision of morphological structures and to evaluate the behaviour of bone cells grown on materials. The results showed that the cells grown on titanium conform to the irregular substrate surfaces maximizing the contact between the cell membrane and the substrate and proliferate disposing close to each other. On the contrary, the osteoblasts seeded onto TiO2/HA coating develop clusters where the cells aggregated extending processes in order to establish intercellular connections. Cell aggregation is an early and critical event leading to cell differentiation and mineralization process and could be a first signal of the tendency of TiO2/HA coating to stimulate cell differentiation.

Physico-chemical properties and degradability of non-woven hyaluronan benzylic esters as tissue engineering scaffolds.

The development of biocompatible materials which can be processed into three-dimensional scaffolds and the design of appropriate configurations in order to enable the cellular infiltration and proliferation is a major issue in the tissue engineering. The hyaluronan total benzyl ester (Hyaff 11) has been found to be suitable substrate to grow a variety of cell types. Since structural, physical, chemical and biological data can help for tailoring appropriate scaffold for tissue engineering, information on chemicophysical properties on degradability of hyaluronan total benzyl ester non-woven has been obtained. The thermal analysis, the evaluation of the surface chemical composition, the morphology, the mechanical behaviour and the swelling tests were carried out on these materials. The hyaluronan total benzyl ester non-woven showed a thermal stability up to 220 degrees C and the surface composition differed from that of the bulk for C-O and C-C contribution. No contaminant were detected. The non-woven swelled in culture medium. Moreover the mechanical tests showed that when submitted to a press treatment, the samples have best mechanical properties. The pressed Hyaff 11 non-woven undergoes degradation when exposed to DMEM. The frying and breaking of the fibres, a decrease of the mechanical properties and a molecular weight loss have been observed. First, the ester bond of the Hyaff 11 non-woven is hydrolysed and the benzylic alcohol is released and the low molecular weight values indicate that a cleavage of the polymer is promoted by the components of the culture medium. After 11 days, some fragments, constituted by hyaluronic acid with a molecular weight of 23,000 Da became soluble in the medium. No oligomer was detected.

Biocompatibility of poly(vinyl alcohol)-hyaluronic acid and poly(vinyl alcohol)-gellan membranes crosslinked by glutaraldehyde vapors.

Glutaraldehyde (GTA) solutions are commonly used to crosslink biomolecules and artificial polymers in order to reduce the degradation rate and to avoid the rapid dissolution in biological fluids. The toxicity of these materials is often due to the presence of GTA residuals unremoved by washing procedures. In this study membranes of PVA-hyaluronic acid and PVA-gellan with different composition have been obtained by solution casting technique and crosslinked by exposure to vapors of GTA in acid environment. The harmful effects of GTA residuals released from the membranes have been evaluated by the cytotoxicity and cytocompatibility in vitro tests, based on the cell culture method. The results showed that these materials have no toxic effects: they do not affect cell viability and proliferation, nor exert damages on mithocondrial and lysosomal functions. The poor adhesion of cells seeded directly onto membranes is due to the surface properties of these materials which are completely refractory at cell adhesion and proliferation. The use of GTA in vapor phase as crosslinking agent of natural and artificial polymer blends is demonstrated to be an efficacious procedure that avoids the presence of toxic residuals into materials.

In vitro response of primary rat osteoblasts to titania/hydroxyapatite coatings compared with transformed human osteoblast-like cells.

The biocompatibility of titania/hydroxyapatite (TiO(2)HA) composite coatings, at different ratio obtained by sol-gel process, was investigated studying the behavior of primary cultures of rat osteoblastic cells, isolated by femoral trabecular bone tissue. Moreover, the results have been compared with the response of human osteoblast-like MG63 cell line. Cytotoxicity of coatings was assessed by lactate dehydrogenase activity (LDH). The cellular behavior was analyzed by the cell proliferation (MTT test), cell morphology (SEM) and the biochemical markers evaluation of osteoblastic phenotype, such as alkaline phosphatase activity (ALP) and osteocalcin production. The results showed that TiO(2)/HA coatings have no toxic effects and seemed to be a good support for cell adhesion and proliferation. Moreover, these materials allowed the differentiation of osteoblasts, stimulating the expression of alkaline phosphatase activity. The responses of the primary rat osteoblasts and human osteoblast-like MG63 cell line grown onto these coatings were similar in terms of proliferation and ALP activity. Differences were found considering the osteocalcin production. The results show that these coatings, thanks to their chemical composition and the deposition technique, are very promising for the potential orthopedic and dental applications.

Comparative investigation of the surface properties of commercial titanium dental implants. Part I: chemical composition.

The surfaces of five commercially available titanium implants (Brånemark Nobel Biocare, 3i ICE, 3i OSSEOTITE, ITI-TPS, and ITI-SLA) were compared by scanning electron microscopy, X-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectroscopy. All five implant types were screw-shaped and fabricated from commercially pure (cp) titanium, but their surface properties differed both as regards surface morphology and surface chemical composition. The macro- and microstructure of the implant surfaces were investigated by scanning electron microscopy. The surfaces chemical composition was determined using the surface-sensitive analytical techniques of X-ray photoelectron spectroscopy and time-of-flight secondary ion spectrometry. Surface topographies were found to reflect the type of mechanical/chemical fabrication procedures applied by the manufacturers. The titanium oxide (passive) layer thickness was similar (5-6 nm) and typical for oxide films grown at or near room temperature. A variety of elements and chemical compounds not related to the metal composition were found on some implant types. They ranged from inorganic material such as sodium chloride to specific organic compounds believed to be due to contamination during fabrication or storage. The experimental findings are believed to make a contribution to a better understanding of the interplay between industrial fabrication procedure and physico-chemical implant surface properties.

Surface and biological evaluation of hydroxyapatite-based coatings on titanium deposited by different techniques.

Hydroxyapatite coatings have been deposited on titanium cp by plasma spray, sol-gel, and sputtering techniques for dental implant applications. The latter two techniques are of current interest, as they allow coatings of micrometer dimensions to be deposited. Coating morphology, composition, and structure have been investigated by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD). All coatings were homogeneous and exhibited a rough morphology suitable for implant applications. The sputtered (after annealing), plasma spray, and sol-gel coatings all showed diffraction peaks corresponding to hydroxyapatite. The surface contaminants were observed to be different for the different coating types. The sputtered coatings were found to have a composition most similar to hydroxyapatite; the sol-gel deposits also showed a high concentration of hydroxyl ions. A discrepancy in the Ca/P ratio was observed for the plasma spray coatings, and a small concentration of carbonate ions was found in the sputter-deposited coatings. The in vitro cell-culture studies using MG63 osteoblast-like cells demonstrated the ability of cells to proliferate on the materials tested. The sol-gel coating promotes higher cell growth, greater alkaline phosphatase activity, and greater osteocalcin production compared to the sputtered and plasma-sprayed coatings.

Poly(L-lactide)acid/alginate composite membranes for guided tissue regeneration.

The barrier membranes for guided tissue regeneration (GTR) to treat bone defects have to satisfy the criteria of biocompatibility, cell-occlusiveness, space-making, tissue integration and clinical manageability. In this study a system constituted of a poly(L-lactide) acid (PLLA) asymmetric membrane combined with an alginate film was prepared. The PLLA membrane functions to both support the alginate film and separate the soft tissue; the alginate film is intended to act as potential vehicle for the growth factors to promote osteogenesis. The structural, morphological, and mechanical properties of the bilamellar membrane and its stability in culture medium were evaluated. Moreover, the feasibility of using the alginate membranes as controlled-release delivery vehicles of TGF-beta was monitored. Finally, the bacterial adhesion and permeability of Streptococcus mutans, selected for the high adhesive affinity, were monitored. The results showed that the surfaces of the alginate side, to be used in contact with the bone defect, were rougher than PLLA ones. When in contact with complete culture medium, the PLLA-alginate membrane retained its mechanical and structural properties for more than 100 days. Then, the degradation processes occurred but the membrane continued to be stable and manageable for 6 months. Growth factors such as TGF-beta can be incorporated into alginate membranes functioning as drug delivery vehicle, and retain the biological activity when tested in an in vitro model system. The obtained membrane acted as a barrier to the passage of S. mutans bacteria and showed to promote a lower bacterial adhesion with respect to commercial GTR membranes.

Physicochemical, mechanical, and biological properties of commercial membranes for GTR.

Barrier membranes for guided tissue regeneration (GTR) to treat bone defects have to satisfy criteria of biocompatibility, cell-occlusiveness, spacemaking, tissue integration, and clinical manageability. In this study, the morphological and mechanical properties of two commercial biodegradable membranes (Resolut LT and Biofix) as a function of the incubation time have been compared. Moreover, their permeability to both fluids and epithelial cells as well as the bacteria adhesion have been evaluated. The membranes are asymmetric and composed of a dense polymeric layer coupled with nonwoven (Resolut LT) or woven (Biofix) fibers. Both of the membranes, when incubated in complete culture medium, completely lose the structural and mechanical properties within 30 days. Moreover the results of solute permeability show that Resolut LT and Biofix membranes cannot be considered selective membranes to the solute crossing. On the contrary, they act as a barrier to the passage of the gingivial cells and to S. mutans bacteria.

Preparation and characterisation of titania/hydroxyapatite composite coatings obtained by sol-gel process.

In the present work a titania network encapsulating a hydroxyapatite particulate phase is proposed as a bioceramic composite coating. The coating on a titanium substrate was produced starting from a sol containing a mixture of titania colloidal particles and hydroxyapatite submicron particles using the dip-coating technique. The microstructure, the morphology and the surface chemical composition of the coating were characterised using X-ray diffraction (XRD), scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS), respectively. Adhesion tests were also performed. These analyses showed that the obtained coating was chemically clean, homogeneous, rough, porous, with a low thickness and well-defined phase composition as well as a good adhesion to the substrate.

The influence of titania/hydroxyapatite composite coatings on in vitro osteoblasts behaviour.

The biocompatibility of titania/hydroxyapatite (TiO2 /HA) composite coatings, at different ratio obtained by sol-gel process, were investigated studying the behaviour of human MG63 osteoblast-like cells. The biocompatibility was evaluated by means of cytotoxicity and cytocompatibility tests. Cytotoxicity tests, i.e., neutral red (NR), MTT and kenacid blue (KB) assays, were performed to assess the influence of the material extracts on lysosomes, mitochondria and cell proliferation, respectively. Cell proliferation, some preliminary indications of cell morphology, alkaline phosphatase activity, collagen and osteocalcin production of MG63 cells, cultured directly onto TiO2/HA substrates, were evaluated. The results showed that these materials have no toxic effects. Cell growth and morphology were similar on all the materials tested: on the contrary, alkaline-phosphatase-specific activity and collagen production of osteoblasts cultured on TiO2/HA coatings were significantly higher than uncoated titanium and polystyrene of culture plate and were influenced by chemical composition of the coatings. In particular, TiO2/HA coating at 1:1 ratio (w/w) seems to stimulate more than others the expression of some differentiation markers of osteoblastic phenotype. TiO2/HA coatings resulted to be bioactive owing to the presence of hydroxyl groups detected on their surface that promote the calcium and phosphate precipitation and improve the interactions with osteoblastic cells.

Distribution of fluoride in glass ionomer cement determined using SIMS.

The uptake by glass ionomer cement of ions (particularly fluoride) from solutions in which the cements have been immersed has been extensively reported. The concentrations within the cement often greatly exceed those in the immersing solution. The distribution of these ions has not been determined. The aim of this study is to use SIMS to investigate the levels of ions within the cement at different depths below the immersed surface of the cement. K+ and F were the ions studied and uptake was into a cement containing neither K nor F (LG30) and one containing F (AH2). The surface was analysed using a Cameca ims4f instrument employing a 14.5 keV Cs+ primary ion beam. This was calibrated on cements made from a series of glasses in which fluorine content was systematically substituted for oxygen (without other elemental changes). XPS, which is very much a surface technique, was used in confirmatory role with respect to the SIMS analysis. Cement discs were made from LG30- and AH2-based cements. After maturation for 72 h these were immersed in 0.275% KF solution for 24 h. SIMS analysis indicated appreciable surface F concentration on LG30 and on AH2 an enhanced F concentration. In contrast, K was not detected on the LG30 surface and only at a low level on AH2. These results were confirmed by XPS. Using the ion beam of the SIMS to sputter away cement enabled the F depth profile on LG30 to be measured to 10 microm. Over this distance the F content drops from 6.2 mmol/g at 0.2 microm from the surface to 0.2 mmol/g at 10 microm. No K was detected down to 13 microm from the surface. From the results of this study, it can be concluded that SIMS is an appropriate tool for further investigation of the distribution of ions uptaken by glass ionomer cements.

Chemico-physical properties of hyaluronan-based sponges.

The aim of this study was to obtain information on the chemico-physical and surface properties of the hyaluronan total benzylic ester sponges to evaluate their stability, surface "cleanliness" and handling for the applications in the tissue engineering. The thermal analysis, the characterization of surface chemical composition and the swelling test were performed on these materials. Moreover, the morphological changes, the rheological behavior, and the molecular weight loss in function of the time were monitored when the sponges were incubated in cell culture medium. The results showed that the sponges were thermally stable up to 220 degrees C and the surface composition was different from that of the bulk for C-O contribution. No contaminants were detected. In culture medium, the samples swelled assuming the rheological properties of biopolymer gel. When the sponges were in contact with the culture medium, their molecular weight remained stable for the first day and a loss of 11% and 31% was recorded for samples removed from culture medium after 3 and 7 days, respectively. With the scanning electron microscopy analysis, the spongy structure appeared with open interconnecting pores. The micrographs related to the samples after incubation in culture medium showed that the degradation was evident on the surface after 1 day. The deterioration of the pore walls and the presence of craters increased with time and, after 3 days, the phenomena were present also in the section.

Gas sensing properties of Langmuir-Blodgett polypyrrole film investigated by surface acoustic waves.

The gas sensing properties of organic polypyrrole (PPS) film, deposited onto LiNbO(3) substrate by Langmuir-Blodgett (LB) technique, have been monitored by surface acoustic wave (SAW) delay lines and studied with respect to sensitivity, selectivity, response time, stability, repeatability, and aging. The SAW PPy elements demonstrate high sensitivity toward NH(3) gas with high selectivity against CH(4), CO, H(2), and O(2). The detectable threshold concentration has been estimated as 20 ppm NH(3) in air; the response time is in the 10s range, and the recovery time is about 15 min; the repeatability of the SAW response toward eight sequential NH(3) gas exposures is within 6%; the aging of the PPy film is within 4% over a month; and the effect of humidity on SAW NH(3) gas response is negligible for the typical conditions at room ambient air. Partially reversible SAW response recognizing NH(3) gas as one component of an interfering gases-mixture has been observed. Simultaneous chemoresponses of SAW phase and insertion loss have been performed in order to investigate the sensing mechanisms. By merging with electrical conductivity gas response, the dominant SAW sensing effects for NH(3 ) gas detection are defined as elastic loading.

Non-destructive characterization of hydrogels.

Hydrogels have been prepared by a freezing-thawing procedure and investigations made of the effect of both number of freezing-thawing cycles and different content of hyaluronic acid (HA) on the mechanical properties of the PVA-HA hydrogels using non-destructive testing. The bulk elastic modulus K of hydrogels has been determined by pulse-echo measurements. It is noted that hydrogel elastic properties improve with the number of the cycles in PVA-HA 100/0; on the other hand samples with a high HA (1,000,000 molecular weight) content, beyond the third cycle, seem to be unaffected by the number of cycles. A bulk elastic modulus fall-off is then observed in samples submitted to an additional overnight freezing between two subsequent cycles. K increases in hydrogels with the highest HA content, when samples undergo pulse-echo measurements soon after their preparation. When hydrogels reach equilibrium, after having been kept in deionized water for 12 h, K values are lower, showing a nearly constant behaviour with different PVA-HA ratios and cycles. Furthermore, by means of scanning laser acoustic microscopy (SLAM) defects have been detected in the hydrogels. In samples which have reached equilibrium, SLAM images show that these defects disappear in PVA-HA hydrogels.