Francesco Boschetto

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 bacterias 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 bacterias 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.

Bioactive silicon nitride: A new therapeutic material for osteoarthropathy

While the reciprocity between bioceramics and living cells is complex, it is principally governed by the implant’s surface chemistry. Consequently, a deeper understanding of the chemical interactions of bioceramics with living tissue could ultimately lead to new therapeutic strategies. However, the physical and chemical principles that govern these interactions remain unclear. The intricacies of this biological synergy are explored within this paper by examining the peculiar surface chemistry of a relatively new bioceramic, silicon nitride (Si3N4). Building upon prior research, this paper aims at obtaining new insights into the biological interactions between Si3N4 and living cells, as a consequence of the off-stoichiometric chemical nature of its surface at the nanometer scale. We show here yet unveiled details of surface chemistry and, based on these new data, formulate a model on how, ultimately, Si3N4 influences cellular signal transduction functions and differentiation mechanisms. In other words, we interpret its reciprocity with living cells in chemical terms. These new findings suggest that Si3N4 might provide unique new medicinal therapies and effective remedies for various bone or joint maladies and diseases.

Human osteoblasts grow transitional Si/N apatite in quickly osteointegrated Si3N4 cervical insert

Silicon nitride (Si3N4) ceramics possesses surface chemistry that accelerates bone repair, as previously established by in vitro experiments using both osteosarcoma and mesenchymal cells. The release of silicic acid and nitrogen compounds from the surface Si3N4 enhanced in vitro cellular activity. The results of this study demonstrate for the first time that the osseointegration behavior previously observed is operative with a peculiar chemistry within the human milieu. Si and N elements stimulated progenitor cell differentiation and osteoblastic activity, which ultimately resulted in accelerated bone ingrowth. At the molecular scale, insight into the effect of silicon and nitrogen ions released from the Si3N4 surface was obtained through combined histomorphometric analyses, Raman, Fourier-transform-infrared, and X-ray photoelectron spectroscopies. Identical analyses conducted on a polyetheretherketone (PEEK) spinal explant showed no chemical changes and a lower propensity for osteogenic activity. Silicon and nitrogen are key elements in stimulating cells to generate bony apatite with crystallographic imperfections, leading to enhanced bioactivity of Si3N4 biomedical devices. STATEMENT OF SIGNIFICANCE This research studies osseointegration processes comparing results from explanted PEEK and Si3N4 spinal spacers. Data show that the formation of hydroxyapatite on silicon nitride bio-ceramic surfaces happens with a peculiar mechanism inside the human body. Silicon and nitrogen were incorporated inside the bony tissue structure allowing the developing of off-stoichiometric bony apatite and stimulating progenitor cell differentiation/osteoblastic activity. Silicon and nitrogen ions released from the Si3N4 surface were detected through combined histologic analyses, Raman microspectroscopy, Fourier-transform-infrared, and X-ray photoelectron spectroscopies.

Development of a SiYAlON glaze for improved osteoconductivity of implantable medical devices

The application of bioactive coatings onto orthopaedic appliances is commonly performed to compensate for the otherwise bioinert nature of medical devices and to improve their osseointegration. Calcium phosphates, hydroxyapatite (HAp), and bioglasses are commercially available for this purpose. Until recently, few other inorganic compounds have been identified with similar biofunctionality. However, silicon nitride (Si3 N4 ) has emerged as a new orthopaedic material whose unique surface chemistry also enhances osteoconductivity. Recent research has confirmed that its minority intergranular phase, consisting of silicon yttrium aluminum oxynitride (SiYAlON), is principally responsible for this improvement. As a result, it was hypothesized that SiYAlON itself might serve as an effective osteoconductive coating or glaze for medical devices. To test this hypothesis, a process inspired by traditional ceramic whiteware glazing was developed. A slurry containing ingredients similar to the intergranular SiYAlON composition was applied to a Si3 N4 surface, which was then subjected to a heat treatment to form a glaze. Various analytical tools were employed to assess its chemistry and morphology. It was found that the glaze was comprised predominately of Y5 Si3 O12 N, a compound commonly referred to as N-apatite, which is isostructural to native HAp. Subsequent exposure of the glazed surface to acellular simulated body fluid led to increased deposition of biomimetic HAp-like crystals, while exposure to Saos-2 osteosarcoma cells in vitro resulted in greater HAp deposition relative to control samples. The observation that SiYAlON exhibits enhanced osteoconductivity portends its potential as a therapeutic aid in bone and tissue repair.

Incorporating Si3N4 into PEEK to Produce Antibacterial, Osteocondutive, and Radiolucent Spinal Implants

Polyetheretherketone (PEEK) is a popular polymeric biomaterial which is primarily used as an intervertebral spacer in spinal fusion surgery; but it is developed for trauma, prosthodontics, maxillofacial, and cranial implants. It has the purported advantages of an elastic modulus which is similar to native bone and it can be easily formed into custom 3D shapes. Nevertheless, PEEKs disadvantages include its poor antibacterial resistance, lack of bioactivity, and radiographic transparency. This study presents a simple approach to correcting these three shortcomings while preserving the base polymers biocompatibility, chemical stability, and elastic modulus. The proposed strategy consists of preparing a PEEK composite by dispersing a minor fraction (i.e., 15 vol%) of a silicon nitride (Si3 N4 ) powder within its matrix. In vitro tests of PEEK composites with three Si3 N4 variants-β-Si3 N4 , α-Si3 N4 , and β-SiYAlON-demonstrate significant improvements in the polymers osteoconductive versus SaOS-2 cells and bacteriostatic properties versus gram-positive Staphylococcus epidermidis bacteria. These properties are clearly a consequence of adding the bioceramic dispersoids, according to chemistry similar to that previously demonstrated for bulk Si3 N4 ceramics in terms of osteogenic behavior (vs both osteosarcoma and mesenchymal progenitor cells) and antibacterial properties (vs both gram-positive and gram-negative bacteria).

Green Tea Polyphenols Coupled with a Bioactive Titanium Alloy Surface: In Vitro Characterization of Osteoinductive Behavior through a KUSA A1 Cell Study

A chemically-treated titanium alloy (Ti6Al4V) surface, able to induce hydroxyapatite precipitation from body fluids (inorganic mineralization activity), was functionalized with a polyphenolic extract from green tea (tea polyphenols, TPH). Considering that green tea polyphenols have stimulating effects on bone forming cells (biological mineralization), the aim was to test their osteoinductive behavior due to co-operation of inorganic and biological mineralization on mesenchymal stem cells KUSA A1. The functionalized surfaces were characterized by using the Folin–Ciocalteu method and X-ray photoelectron spectroscopy to confirm the successful outcome of the functionalization process. Two cell cultures of mesenchymal stem cells, KUSA A1 were performed, with or without osteoinductive factors. The cells and surfaces were characterized for monitoring cell viability and hydroxyapatite production: Fourier Transform Infrared Spectroscopy and Raman spectroscopy analyses showed deposition of hydroxyapatite and collagen due to the cell activity, highlighting differentiation of KUSA A1 into osteoblasts. A higher production of extracellular matrix was highlighted on the functionalized samples by laser microscope and the fluorescence images showed higher viability of cells and greater presence of osteocalcin in these samples. These results highlight the ability of polyphenols to improve cell differentiation and to stimulate biological mineralization, showing that surface functionalization of metal implants could be a promising way to improve osteointegrability.

Raman Imaging of Individual Membrane Lipids and Deoxynucleoside Triphosphates in Living Neuronal Cells during Neurite Outgrowth

Recent developments in Raman imaging at the microscopic scale were exploited here with the specific purpose of locating spectral fingerprints of individual membrane lipids and deoxynucleoside triphosphates during neuronal cell networking and separation. After carefully screening the Raman spectra of isolated lipid components, we located an in situ mapped specific Raman fingerprints from individual phospholipids at the micrometric level in comparison with the total lipid distribution within single living cells. We concurrently examined silent zones of lipid emissions and exploited those peculiar spectral ranges for mapping both abundance and localization of individual DNA nucleoside triphosphates. This work represents a first step toward label-free/molecular-selective Raman patterning with high spectral resolution of the relevant chemical species involved with the functionality of neuronal cells.

Understanding Silicon Nitride’s Biological Properties: From Inert to Bioactive Ceramic

Due to the favourable combination of mechanical strength and fracture toughness, silicon nitride has been applied as a load-bearing bioceramic, in particular for implants used in spinal fusion surgery. Only recently it has been observed that the supposedly inert surface of silicon nitride is actually bioactive: a slow, but not negligible, pH controlled, ionic exchange between nitrogen and oxygen leads to the formation and elution of silicic acid and ammonia groups, also resulting in an effective protection against bacteria colonization. These properties could be further modulated by chemical and mechanical treatments.

Monitoring metabolic reactions in Staphylococcus epidermidis exposed to silicon nitride using in situ time-lapse Raman spectroscopy

Abstract. Staphylococcus epidermidis (S. epidermidis) is one of the leading nosocomial pathogens, particularly associated with periprosthetic infections of biomedical implants. Silicon nitride (Si3N4), a nonoxide biomaterial widely used in spinal implants, has shown bacteriostatic effects against both gram-positive and gram-negative bacteria; however, the physicochemical interactions between Si3N4 and bacteria yet remain conspicuously unexplored. In situ time-lapse Raman spectroscopic experiments were conducted by exposing S. epidermidis for 12, 24, and 48 h to Si3N4 substrates to understand the evolution of bacterial metabolism and to elucidate the ceramics antimicrobial behavior. The Raman probe captured an initial metabolic response of the bacteria to the adverse chemistry of the Si3N4 surface, which included peroxidation of membrane phospholipids and protein structural modifications to adjust for survivorship. However, beyond 24 h of exposure, the Raman signals representing DNA, lipids, proteins, and carbohydrates showed clear fingerprints of bacterial lysis. Bands related to biofilm formation completely disappeared or underwent drastically reduced intensity. Bacterial lysis was confirmed by conventional fluorescence microscopy methods. Spectroscopic experiments suggested that a pH change at the Si3N4’s surface induced variations in the membrane structure and D-alanylation of teichoic acids in its peptidoglycan layer. Concurrent stimulation of peptidoglycan hydrolase (i.e., an enzyme involved with autolysis) ultimately led to membrane degradation and cellular death. An additional finding was that modulating the Si3N4 surface by increasing the population of amine groups improved the efficiency of the substrate against S. epidermidis, thus suggesting that optimization of the near-surface (alkaline) conditions may be a viable approach to bacterial reduction.