On the Potential of Bulk Metallic Glasses for Dental Implantology: Case Study on Ti40Zr10Cu36Pd14.

Ti40Zr10Cu36Pd14 Bulk Metallic Glass (BMG) appears very attractive for future biomedical applications thanks to its high glass forming ability, the absence of toxic elements such as Ni, Al or Be and its good mechanical properties. For the first time, a complete and exhaustive characterization of a unique batch of this glassy alloy was performed, together with ISO standard mechanical tests on machined implant-abutment assemblies. The results were compared to the benchmark Ti-6Al-4V ELI (Extra-Low-Interstitial) to assess its potential in dental implantology. The thermal stability, corrosion and sterilization resistance, cytocompatibility and mechanical properties were measured on samples with a simple geometry, but also on implant-abutment assemblies' prototypes. Results show that the glassy alloy exhibits a quite high thermal stability, with a temperature range of 38 °C between the glass transition and crystallization, a compressive strength of 2 GPa, a certain plastic deformation (0.7%), a hardness of 5.5 GPa and a toughness of 56 MPa.√m. Moreover, the alloy shows a relatively lower Young's modulus (96 GPa) than the Ti-6Al-4V alloy (110-115 GPa), which is beneficial to limit bone stress shielding. The BMG shows a satisfactory cytocompatibility, a high resistance to sterilization and a good corrosion resistance (corrosion potential of -0.07 V/SCE and corrosion current density of 6.0 nA/cm²), which may ensure its use as a biomaterial. Tests on dental implants reveal a load to failure 1.5-times higher than that of Ti-6Al-4V and a comparable fatigue limit. Moreover, implants could be machined and sandblasted by methods usually conducted for titanium implants, without significant degradation of their amorphous nature. All these properties place this metallic glass among a promising class of materials for mechanically-challenging applications such as dental implants.

In vitro and in vivo evaluation of a polylactic acid-bioactive glass composite for bone fixation devices.

Poly(lactic acid) is nowadays among the most used bioabsorbable materials for medical devices. To promote bone growth on the material surface and increase the degradation rate of the polymer, research is currently focused on organic-inorganic composites by adding a bioactive mineral to the polymer matrix. The purpose of this study was to investigate the ability of a poly(L,DL-lactide)-Bioglass® (P(L,DL)LA-Bioglass(®) 45S5) composite to be used as a bone fixation device. In vitro cell viability testing of P(l,dl)LA based composites containing different amounts of Bioglass(®) 45S5 particles was investigated. According to the degradation rate of the P(L,DL)LA matrix and the cytocompatibility experiments, the composite with 30 wt % of Bioglass® particles seemed to be the best candidate for further investigation. To study its behavior after immersion in simulated physiological conditions, the degradation of the composite was analyzed by measuring its weight loss and mechanical properties and by proceeding with X-ray tomography. We demonstrated that the presence of the bioactive glass significantly accelerated the in vitro degradation of the polymer. A preliminary in vivo investigation on rabbits shows that the addition of 30 wt % of Bioglass(®) in the P(L,DL)LA matrix seems to trigger bone osseointegration especially during the first month of implantation. This composite has thus strong potential interest for health applications.

Staphylococcus epidermidis biofilm formation and structural organization on different types of intraocular lenses under in vitro flow conditions.

AIM: To compare the adherence and structural organization of Staphylococcus epidermidis biofilm on intraocular lenses (IOLs). METHODS: IOLs made of 3 different biomaterials [polymethyl methacrylate (PMMA), hydrophilic acrylic or hydrophobic acrylic] were incubated into an S. epidermidis bacterial solution. Scanning electron microscopy was used to count the bound bacteria and to analyze the structural biofilm architecture. RESULTS: After 4-6 h of incubation, adherence was statistically weakest on the hydrophilic acrylic polymer. On the hydrophobic acrylic material, the bacterial cells tended to cover the substratum in a horizontal spread in a continuous monolayer. On the hydrophilic acrylic material or on the PMMA material bacterial cells tended to form only few, small scattered cell clusters. CONCLUSIONS: The data suggest that the pattern of S. epidermidis adhesion varies with the IOL biomaterial. Hydrophobic IOLs seem to be more permissive to S. epidermidis adhesion.

The ratio 1660/1690 cm(-1) measured by infrared microspectroscopy is not specific of enzymatic collagen cross-links in bone tissue.

In postmenopausal osteoporosis, an impairment in enzymatic cross-links (ECL) occurs, leading in part to a decline in bone biomechanical properties. Biochemical methods by high performance liquid chromatography (HPLC) are currently used to measure ECL. Another method has been proposed, by Fourier Transform InfraRed Imaging (FTIRI), to measure a mature PYD/immature DHLNL cross-links ratio, using the 1660/1690 cm(-1) area ratio in the amide I band. However, in bone, the amide I band composition is complex (collagens, non-collagenous proteins, water vibrations) and the 1660/1690 cm(-1) by FTIRI has never been directly correlated with the PYD/DHLNL by HPLC. A study design using lathyritic rats, characterized by a decrease in the formation of ECL due to the inhibition of lysyl oxidase, was used in order to determine the evolution of 1660/1690 cm(-1) by FTIR Microspectroscopy in bone tissue and compare to the ECL quantified by HPLC. The actual amount of ECL was quantified by HPLC on cortical bone from control and lathyritic rats. The lathyritic group exhibited a decrease of 78% of pyridinoline content compared to the control group. The 1660/1690 cm(-1) area ratio was increased within center bone compared to inner bone, and this was also correlated with an increase in both mineral maturity and mineralization index. However, no difference in the 1660/1690 cm(-1) ratio was found between control and lathyritic rats. Those results were confirmed by principal component analysis performed on multispectral infrared images. In bovine bone, in which PYD was physically destructed by UV-photolysis, the PYD/DHLNL (measured by HPLC) was strongly decreased, whereas the 1660/1690 cm(-1) was unmodified. In conclusion, the 1660/1690 cm(-1) is not related to the PYD/DHLNL ratio, but increased with age of bone mineral, suggesting that a modification of this ratio could be mainly due to a modification of the collagen secondary structure related to the mineralization process.

Crystallization processes at the surface of polylactic acid-bioactive glass composites during immersion in simulated body fluid.

We report on the crystallization processes occurring at the surface of PDLLA-Bioglass® composites immersed in simulated body fluid. Composites manufactured by injection molding and containing different amounts (0, 20, 30, and 50 wt %) of 45S5 Bioglass® particles were tested for durations up to 56 days and compared with Bioglass® particles alone. Crystallization processes were followed by visual inspection, X-ray diffraction (with Rietveld analysis) and scanning electron microscopy. Both calcite and hydroxyapatite were formed at the surface of all materials, but their relative ratio was dependent on the Bioglass® content and immersion time. Hydroxyapatite was always the major phase after sufficient immersion time, insuring bioactivity of such composites especially for Bioglass® content higher than 30 wt %. A scenario of crystallization is proposed. Rapid degradation of the composites with 50 wt % was also observed during immersion. Therefore, composites with 30 wt % of Bioglass® particles seem to exhibit the best balance between bioactivity and stability at least during the first weeks of immersion in contact with body fluids.

Transglutaminase-2: a new endostatin partner in the extracellular matrix of endothelial cells.

Endostatin, a C-terminal fragment of collagen XVIII, binds to TG-2 (transglutaminase-2) in a cation-dependent manner. Recombinant human endostatin binds to TG-2 with an affinity in the nanomolar range (Kd=6.8 nM). Enzymatic assays indicated that, in contrast with other extracellular matrix proteins, endostatin is not a glutaminyl substrate of TG-2 and is not cross-linked to itself by the enzyme. Two arginine residues of endostatin, Arg27 and Arg139, are crucial for its binding to TG-2. They are also involved in the binding to heparin [Sasaki, Larsson, Kreuger, Salmivirta, Claesson-Welsh, Lindahl, Hohenester and Timpl (1999) EMBO J. 18, 6240-6248], and to alpha5beta1 and alphavbeta3 integrins [Faye, Moreau, Chautard, Jetne, Fukai, Ruggiero, Humphries, Olsen and Ricard-Blum (2009) J. Biol. Chem. 284, 22029-22040], suggesting that endostatin is not able to interact simultaneously with TG-2 and heparan sulfate, or with TG-2 and integrins. Inhibition experiments support the hypothesis that the GTP-binding site of TG-2 is a potential binding site for endostatin. Endostatin and TG-2 are co-localized in the extracellular matrix secreted by endothelial cells under hypoxia, which stimulates angiogenesis. This interaction, occurring in a cellular context, might participate in the concerted regulation of angiogenesis and tumorigenesis by the two proteins.

In vitro and in vivo evaluation of an alumina-zirconia composite for arthroplasty applications.

In order to improve the reliability and the mechanical properties of orthopaedic hip prosthesis, new ceramic composites starting with nanosized powders of alumina and zirconia have been recently developed. The aim of the present study was to investigate the biological tolerance of one of these sintered ceramics and of its alumina and zirconia constitutive nanosized powders with both in vitro and in vivo approaches. At first, osteoblasts and fibroblasts were cultured either upon sintered ceramic discs with polished or rough surfaces or in the presence of the corresponding alumina or zirconia powders at various concentrations. Thereafter, we chronically injected these powders in the knee articulation of rats. In vitro, the materials showed no deleterious effect on cell proliferation, extra-cellular matrix production (human type I collagen and fibronectin) or on cell morphology. In vivo, the histological examination showed only a very moderate and non-specific granulomatous response of the synovial membrane but no major inflammation as clinically described with metals or polyethylene wear debris. Besides its improved physical properties, this recently developed alumina-zirconia composite showed satisfactory biocompatibility.

Preparation and development of anti-chitosan antibodies.

Polyclonal antibodies directed against chitosan were produced using several immunogens, prepared by binding the polymer according to two ways (covalent and electrostatic) with a protein (bovine serum albumin or hemocyanin). It appeared that the presence of a carrier protein linked to chitosan was necessary to enhance the immune response and to obtain antibodies in a stable and reproducible way. Direct and inhibition enzyme-linked immunosorbent assay experiments were performed to assess the affinity and the specificity of the antibodies. The interactions of these antibodies with modified chitosans showed no influence of the degree of polymerization of the polymer in the range studied (from 24 to 2261), by contrast with the degree of acetylation. The affinity decreased when the degree of acetylation increased. Absence of cross-reactivity with glycosaminoglycans was observed whatever the antibody. The cationicity of the amine function along the polymer chains may have a role in the immunological recognition of the chitosan structure.

Long-term remodeling of a bilayered living human skin equivalent (Apligraf) grafted onto nude mice: immunolocalization of human cells and characterization of extracellular matrix.

Type I collagen is a clinically approved biomaterial largely used in tissue engineering. It acts as a regenerative template in which the implanted collagen is progressively degraded and replaced by new cell-synthesized tissue. Apligraf, a bioengineered living skin, is composed of a bovine collagen lattice containing living human fibroblasts overlaid with a fully differentiated epithelium made of human keratinocytes. To investigate its progressive remodeling, athymic mice were grafted and the cellular and the extracellular matrix components were studied from 0 to 365 days after grafting. Biopsies were analyzed using immunohistochemistry with species-specific antibodies and electron microscopy techniques. We observed that this bioengineered tissue provided living and bioactive cells to the wound site up to 1 year after grafting. The graft was rapidly incorporated within the host tissue and the bovine collagen present in the graft was progressively replaced by human and mouse collagens. A normal healing process was observed, i.e., type III collagen appeared transiently with type I collagen, the major collagen isoform present at later stages. New molecules, such as elastin, were produced by the living human cells contained within the graft. This animal model combined with species-specific immunohistochemistry tools is thus very useful for studying long-term tissue remodeling of bioengineered living tissues.

Immunofluorescent localization of collagen types I, III, IV, fibronectin and laminin during morphogenesis of scales and scaleless skin in the chick embryo.

Collagen types I and III were purified from the skin of 3-or 7-week-old chickens, collagen type IV from bovine skin or EHS mouse tumour, fibronectin from human serum, and laminin from EHS mouse tumour. Antibodies were produced in rabbits or sheep, and used in indirect immunofluorescence on frozen sections of 9-to 16-day-old normal or mutant (scaleless) chick-embryo foot skin. In normal scale-forming skin and inscaleless skin, the distribution of anti-laminin and anti-type IV collagen label was uniform along the dermal-epidermal junction and showed no stage-related variations, except for fluorescent granules located in the dermis of early scale rudiments. By contrast, in normal scale-forming skin, the density of anti-types I and III label decreased in the dermis within scale rudiments, whereas it gradually increased in interscale skin. Conversely, anti-fibronectin label accumulated at a higher density within scale rudiments than in interscale skin. In the dermis of thescaleless mutant, anti-types I and III label and antifibronectin label were distributed evenly: the density of anti-collagen label increased with age, while that of antifibronectin decreased and almost completely vanished in 16-day-old skin, except around blood vessels. The microheterogeneous distribution of some extracellular matrix components, namely interstitial collagen types I and III and fibronectin, is interpreted as part of the morphogenetic message that the dermis is known to transmit to the epidermis during the formation of scales. The even distribution of these components in mutantscaleless skin is in agreement with this view. Basement membrane constituents laminin and type-IV collagen do not appear to be part of the dermal morphogenetic message.