Silicon Nitride Bioceramics Induce Chemically Driven Lysis in Porphyromonas gingivalis.

Organisms of Gram-negative phylum bacteroidetes, Porphyromonas gingivalis, underwent lysis on polished surfaces of silicon nitride (Si3N4) bioceramics. The antibacterial activity of Si3N4 was mainly the result of chemically driven principles. The lytic activity, although not osmotic in nature, was related to the peculiar pH-dependent surface chemistry of Si3N4. A buffering effect via the formation of ammonium ions (NH4(+)) (and their modifications) was experimentally observed by pH microscopy. Lysis was confirmed by conventional fluorescence spectroscopy, and the bacteria's metabolism was traced with the aid of in situ Raman microprobe spectroscopy. This latter technique revealed the formation of peroxynitrite within the bacterium itself. Degradation of the bacteria's nucleic acid, drastic reduction in phenilalanine, and reduction of lipid concentration were observed due to short-term exposure (6 days) to Si3N4. Altering the surface chemistry of Si3N4 by either chemical etching or thermal oxidation influenced peroxynitrite formation and affected bacteria metabolism in different ways. Exploiting the peculiar surface chemistry of Si3N4 bioceramics could be helpful in counteracting Porphyromonas gingivalis in an alkaline pH environment.

Analysis of defect luminescence in Ga-doped ZnO nanoparticles.

We applied cathodoluminescence (CL) spectroscopy to evaluate the defect-induced luminescence within ZnO and Ga-doped ZnO (GZO) nanoparticles. The observed emissions from defect sites present in the GZO lattice exhibited a strong dependence on both dopant content and synthesis methods. The strong and broad defect-induced emissions and inhomogeneous population of intrinsic defects in nano-sized ZnO particles could effectively be suppressed by Ga doping, although large dopant amounts caused the generation of negatively-charged defects, VZn and Oi, with a subsequent increase of the luminescence. Upon deconvolution of the retrieved CL spectra into individual sub-bands, the physical origin of all the sub-bands could be clarified, and related to sample composition and synthesis protocol. This study lays the foundation of quantitative CL evaluation of defects to assess the quality of GZO optoelectronic devices.

Vibrational algorithms for quantitative crystallographic analyses of hydroxyapatite-based biomaterials: I, theoretical foundations.

The Raman spectroscopic method has quantitatively been applied to the analysis of local crystallographic orientation in both single-crystal hydroxyapatite and human teeth. Raman selection rules for all the vibrational modes of the hexagonal structure were expanded into explicit functions of Euler angles in space and six Raman tensor elements (RTE). A theoretical treatment has also been put forward according to the orientation distribution function (ODF) formalism, which allows one to resolve the statistical orientation patterns of the nm-sized hydroxyapatite crystallite comprised in the Raman microprobe. Close-form solutions could be obtained for the Euler angles and their statistical distributions resolved with respect to the direction of the average texture axis. Polarized Raman spectra from single-crystalline hydroxyapatite and textured polycrystalline (teeth enamel) samples were compared, and a validation of the proposed Raman method could be obtained through confirming the agreement between RTE values obtained from different samples.

Vibrational algorithms for quantitative crystallographic analyses of hydroxyapatite-based biomaterials: II, application to decayed human teeth.

A systematic investigation, based on highly spectrally resolved Raman spectroscopy, was undertaken to research the efficacy of vibrational assessments in locating chemical and crystallographic fingerprints for the characterization of dental caries and the early detection of non-cavitated carious lesions. Raman results published by other authors have indicated possible approaches for this method. However, they conspicuously lacked physical insight at the molecular scale and, thus, the rigor necessary to prove the efficacy of this spectroscopy method. After solving basic physical challenges in a companion paper, we apply them here in the form of newly developed Raman algorithms for practical dental research. Relevant differences in mineral crystallite (average) orientation and texture distribution were revealed for diseased enamel at different stages compared with healthy mineralized enamel. Clear spectroscopy features could be directly translated in terms of a rigorous and quantitative classification of crystallography and chemical characteristics of diseased enamel structures. The Raman procedure enabled us to trace back otherwise invisible characteristics in early caries, in the translucent zone (i.e., the advancing front of the disease) and in the body of lesion of cavitated caries.

Chemistry-driven structural alterations in short-term retrieved ceramic-on-metal hip implants: Evidence for in vivo incompatibility between ceramic and metal counterparts.

Ceramic-on-metal (CoM) hip implants were reported to experience lower wear rates in vitro as compared to metal-on-metal (MoM) bearings, thus hinting metal-ion release at lower levels in vivo. In this article, we show a spectroscopic study of two short-term retrieval cases of zirconia-toughened alumina (ZTA) femoral heads belonging to CoM hip prostheses, which instead showed poor wear performances in vivo. Metal contamination and abnormally high fractions of tetragonal-to-monoclinic (t→m) polymorphic transformation of the zirconia phase could be found on both ZTA heads, which contrasted with the optimistic predictions of in vitro experiments. At the molecular scale, incorporation of metal ions into the ceramic lattices could be recognized as due to frictionally assisted phenomena occurring at the ceramic surface. Driven by abnormal friction, diffusion of metal ions induced lattice shrinkage in the zirconia phases, while residual stress fields became stored at the surface of the femoral head. Diffusional alterations destabilized the chemistry of the ceramic surface and resulted in an abnormal increase in t→m phase transformation in vivo. Frictionally driven metal transfer to the ceramic lattice thus hinders the in vivo performance of CoM prostheses. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1469-1480, 2017.

Vibrational monitor of early demineralization in tooth enamel after in vitro exposure to phosphoridic liquid.

The Raman spectroscopic method has been applied to quantitatively assess the in vitro degree of demineralization in healthy human teeth. Based on previous evaluations of Raman selection rules (empowered by an orientation distribution function (ODF) statistical algorithm) and on a newly proposed analysis of phonon density of states (PDOS) for selected vibrational modes of the hexagonal structure of hydroxyapatite, a molecular-scale evaluation of the demineralization process upon in vitro exposure to a highly acidic beverage (i.e., CocaCola™ Classic, pH=2.5) could be obtained. The Raman method proved quite sensitive and spectroscopic features could be directly related to an increase in off-stoichiometry of the enamel surface structure since the very early stage of the demineralization process (i.e., when yet invisible to other conventional analytical techniques). The proposed Raman spectroscopic algorithm might possess some generality for caries risk assessment, allowing a prompt non-contact diagnostic practice in dentistry.

Surface toughness of silicon nitride bioceramics: II, Comparison with commercial oxide materials.

Raman microprobe-assisted indentation, a micromechanics method validated in a companion paper, was used to compare the surface toughening behaviors of silicon nitride (Si3N4) and alumina-based bioceramics employed in joint arthroplasty (i.e., monolithic alumina, Al2O3, and yttria-stabilized zirconia (ZrO2)-toughened alumina, ZTA). Quantitative assessments of microscopic stress fields both ahead and behind the tip of Vickers indentation cracks propagated under increasing indentation loads were systematically made using a Raman microprobe with spatial resolution on the order of a single micrometer. Concurrently, crack opening displacement (COD) profiles were monitored on the same microcracks screened by Raman spectroscopy. The Raman eye clearly visualized different mechanisms operative in toughening Si3N4 and ZTA bioceramics (i.e., crack-face bridging and ZrO2 polymorphic transformation, respectively) as compared to the brittle behavior of monolithic Al2O3. Moreover, emphasis was placed on assessing not only the effectiveness but also the durability of such toughening effects when the biomaterials were aged in a hydrothermal environment. A significant degree of embrittlement at the biomaterial surface was recorded in the transformation-toughened ZTA, with the surface toughness reduced by exposure to the hydrothermal environment. Conversely, the Si3N4 biomaterial experienced a surface toughness value independent of hydrothermal attack. Crack-face bridging thus appears to be a durable surface toughening mechanism for biomaterials in joint arthroplasty.

Surface toughness of silicon nitride bioceramics: I, Raman spectroscopy-assisted micromechanics.

Indentation micro-fracture is revisited as a tool for evaluating the surface toughness of silicon nitride (Si3N4) bioceramics for artificial joint applications. Despite being unique and practical from an experimental perspective, a quantitative assessment of surface fracture toughness using this method is challenging. An improved method has been developed, consisting of coupling indentation with confocal (spatially resolved) Raman piezo-spectroscopy. Empowered by the Raman microprobe, the indentation micro-fracture method was found to be capable of providing reliable surface toughness measurements in silicon nitride biomaterials. In designing the microstructures of bioceramic bearing couples for improved tribological performance, surface toughness must be considered as a fundamentally different and distinct parameter from bulk toughness. The coupling of indention crack opening displacements (COD) with local stress field assessments by spectroscopy paves the way to reliably compare the structural properties of bioceramics and to quantitatively monitor their evolution during environmental exposure.

Silicon Nitride: A Synthetic Mineral for Vertebrate Biology.

The remarkable stoichiometric flexibility of hydroxyapatite (HAp) enables the formation of a variety of charged structural sites at the material's surface which facilitates bone remodeling due to binding of biomolecule moieties in zwitterionic fashion. In this paper, we report for the first time that an optimized biomedical grade silicon nitride (Si3N4) demonstrated cell adhesion and improved osteoconductivity comparable to highly defective, non-stoichiometric natural hydroxyapatite. Si3N4's zwitterionic-like behavior is a function of the dualism between positive and negative charged off-stoichiometric sites (i.e., N-vacancies versus silanols groups, respectively). Lattice defects at the biomaterial's surface greatly promote interaction with positively- and negatively-charged functional groups in biomolecules, and result in the biologically effective characteristics of silicon nitride. These findings are anticipated to be a starting point for further discoveries of therapeutic bone-graft substitute materials.

Surface modulation of silicon nitride ceramics for orthopaedic applications.

Silicon nitride (Si3N4) has a distinctive combination of material properties such as high strength and fracture toughness, inherent phase stability, scratch resistance, low wear, biocompatibility, hydrophilic behavior, excellent radiographic imaging and resistance to bacterial adhesion, all of which make it an attractive choice for orthopaedic implants. Unlike oxide ceramics, the surface chemistry and topography of Si3N4 can be engineered to address potential in vivo needs. Morphologically, it can be manufactured to have an ultra-smooth or highly fibrous surface structure. Its chemistry can be varied from that of a silica-like surface to one which is predominately comprised of silicon-amines. In the present study, a Si3N4 bioceramic was subjected to thermal, chemical, and mechanical treatments in order to induce changes in surface composition and features. The treatments included grinding and polishing, etching in aqueous hydrofluoric acid, and heating in nitrogen or air. The treated surfaces were characterized using a variety of microscopy techniques to assess morphology. Surface chemistry and phase composition were determined using X-ray photoelectron and Raman spectroscopy, respectively. Streaming potential measurements evaluated surface charging, and sessile water drop techniques assessed wetting behavior. These treatments yielded significant differences in surface properties with isoelectric points ranging from 2 to 5.6, and moderate to extremely hydrophilic water contact angles from ∼65° to ∼8°. This work provides a basis for future in vitro and in vivo studies which will examine the effects of these treatments on important orthopaedic properties such as friction, wear, protein adsorption, bacteriostasis and osseointegration. STATEMENT OF SIGNIFICANCE: Silicon nitride (Si3N4) exhibits a unique combination of bulk mechanical and surface chemical properties that make it an ideal biomaterial for orthopaedic implants. It is already being used for interbody spinal fusion cages and is being developed for total joint arthroplasty. Its surface texture and chemistry are both highly tunable, yielding physicochemical combinations that may lead to enhanced osseointegration and bacterial resistance without compromising bulk mechanical properties. This study demonstrates the ease with which significant changes to Si3N4's surface phase composition, charging, and wetting behavior can be induced, and represents an initial step towards a mechanistic understanding of the interaction between implant surfaces and the biologic environment.

Raman spectroscopy of human skin: looking for a quantitative algorithm to reliably estimate human age.

The possibility of examining soft tissues by Raman spectroscopy is challenged in an attempt to probe human age for the changes in biochemical composition of skin that accompany aging. We present a proof-of-concept report for explicating the biophysical links between vibrational characteristics and the specific compositional and chemical changes associated with aging. The actual existence of such links is then phenomenologically proved. In an attempt to foster the basics for a quantitative use of Raman spectroscopy in assessing aging from human skin samples, a precise spectral deconvolution is performed as a function of donors' ages on five cadaveric samples, which emphasizes the physical significance and the morphological modifications of the Raman bands. The outputs suggest the presence of spectral markers for age identification from skin samples. Some of them appeared as authentic "biological clocks" for the apparent exactness with which they are related to age. Our spectroscopic approach yields clear compositional information of protein folding and crystallization of lipid structures, which can lead to a precise identification of age from infants to adults. Once statistically validated, these parameters might be used to link vibrational aspects at the molecular scale for practical forensic purposes.