André L. Rossi

Growth of Crystalline Hydroxyapatite Thin Films at Room Temperature by Tuning the Energy of the RF-Magnetron Sputtering Plasma

Right angle radio frequency magnetron sputtering technique (RAMS) was redesigned to favor the production of high-quality hydroxyapatite (HA) thin coatings for biomedical applications. Stoichiometric HA films with controlled crystallinity, thickness varying from 254 to 540 nm, crystallite mean size of 73 nm, and RMS roughness of 1.7 ± 0.9 nm, were obtained at room temperature by tuning the thermodynamic properties of the plasma sheath energy. The plasma energies were adjusted by using a suitable high magnetic field confinement of 143 mT (1430 G) and a substrate floating potential of 2 V at the substrate-to-magnetron distance of Z = 10 mm and by varying the sputtering geometry, substrate-to-magnetron distance from Z = 5 mm to Z = 18 mm, forwarded RF power and reactive gas pressure. Measurements that were taken with a Langmuir probe showed that the adjusted RAMS geometry generated a plasma with an adequate effective temperature of Teff ≈ 11.8 eV and electron density of 2.0 × 10(15) m(-3) to nucleate nanoclusters and to further crystallize the nanodomains of stoichiometric HA. The deposition mechanism in the RAMS geometry was described by the formation of building units of amorphous calcium phosphate clusters (ACP), the conversion into HA nanodomains and the crystallization of the grain domains with a preferential orientation along the HA [002] direction.

Trojan-Like Internalization of Anatase Titanium Dioxide Nanoparticles by Human Osteoblast Cells.

Dentistry and orthopedics are undergoing a revolution in order to provide more reliable, comfortable and long-lasting implants to patients. Titanium (Ti) and titanium alloys have been used in dental implants and total hip arthroplasty due to their excellent biocompatibility. However, Ti-based implants in human body suffer surface degradation (corrosion and wear) resulting in the release of metallic ions and solid wear debris (mainly titanium dioxide) leading to peri-implant inflammatory reactions. Unfortunately, our current understanding of the biological interactions with titanium dioxide nanoparticles is still very limited. Taking this into consideration, this study focuses on the internalization of titanium dioxide nanoparticles on primary bone cells, exploring the events occurring at the nano-bio interface. For the first time, we report the selective binding of calcium (Ca), phosphorous (P) and proteins from cell culture medium to anatase nanoparticles that are extremely important for nanoparticle internalization and bone cells survival. In the intricate biological environment, anatase nanoparticles form bio-complexes (mixture of proteins and ions) which act as a kind of ‘Trojan-horse’ internalization by cells. Furthermore, anatase nanoparticles-induced modifications on cell behavior (viability and internalization) could be understand in detail. The results presented in this report can inspire new strategies for the use of titanium dioxide nanoparticles in several regeneration therapies.

The effects of strontium on bone mineral: A review on current knowledge and microanalytical approaches

The interest in effects of strontium (Sr) on bone has greatly increased in the last decade due to the development of the promising drug strontium ranelate. This drug is used for treating osteoporosis, a major bone disease affecting hundreds of millions of people worldwide, especially postmenopausal women. The novelty of strontium ranelate compared to other treatments for osteoporosis is its unique effect on bone: it simultaneously promotes bone formation by osteoblasts and inhibits bone resorption by osteoclasts. Besides affecting bone cells, treatment with strontium ranelate also has a direct effect on the mineralized bone matrix. Due to the chemical similarities between Sr and Ca, a topic that has long been of particular interest is the incorporation of Sr into bones replacing Ca from the mineral phase, which is composed by carbonated hydroxyapatite nanocrystals. Several groups have analyzed the mineral produced during treatment; however, most analysis were done with relatively large samples containing numerous nanocrystals, resulting thus on data that represents an average of many crystalline domains. The nanoscale analysis of the bone apatite crystals containing Sr has only been described in a few studies. In this study, we review the current knowledge on the effects of Sr on bone mineral and discuss the methodological approaches that have been used in the field. In particular, we focus on the great potential that advanced microscopy and microanalytical techniques may have on the detailed analysis of the nanostructure and composition of bone apatite nanocrystals produced during treatment with strontium ranelate.

Ultrastructure of regenerated bone mineral surrounding hydroxyapatite–alginate composite and sintered hydroxyapatite

We report the ultrastructure of regenerated bone surrounding two types of biomaterials: hydroxyapatite-alginate composite and sintered hydroxyapatite. Critical defects in the calvaria of Wistar rats were filled with micrometer-sized spherical biomaterials and analyzed after 90 and 120 days of implantation by high-resolution transmission electron microscopy and Fourier transform infrared attenuated total reflectance microscopy, respectively. Infrared spectroscopy showed that hydroxyapatite of both biomaterials became more disordered after implantation in the rat calvaria, indicating that the biological environment induced modifications in biomaterials structure. We observed that the regenerated bone surrounding both biomaterials had a lamellar structure with type I collagen fibers alternating in adjacent lamella with angles of approximately 90°. In each lamella, plate-like apatite crystals were aligned in the c-axis direction, although a rotation around the c-axis could be present. Bone plate-like crystal dimensions were similar in regenerated bone around biomaterials and pre-existing bone in the rat calvaria. No epitaxial growth was observed around any of the biomaterials. A distinct mineralized layer was observed between new bone and hydroxyapatite-alginate biomaterial. This region presented a particular ultrastructure with crystallites smaller than those of the bulk of the biomaterial, and was possibly formed during the synthesis of alginate-containing composite or in the biological environment after implantation. Round nanoparticles were observed in regions of newly formed bone. The findings of this work contribute to a better understanding of the role of hydroxyapatite based biomaterials in bone regeneration processes at the nanoscale.

Synthesis of hydroxyapatite nanotubes for biomedical applications

Single phase, stoichiometrically pure, hollow nanotubes of hydroxyapatite have been synthesized and single-particle analysis has been performed to successfully prove the sole formation of Ca10(PO4)6(OH)2 phase. The facile synthesis involves a sol-gel process under neutral conditions in the presence of a sacrifical anodic alumina template. The structures formed are hollow nanotubes that have been characterized by XRD, SEM, TEM, SAED, EELS, EDS and BET measurements. The diameter of the resulting tubes is in the range of 140-350 nm, length is on the order of a few microns and the wall thickness of the tubes was found to be ca. 30 nm. Moreover these tubes had a large BET surface area of 115 m(2)/g and were found to be biocompatible. They displayed inertness in the presence of NIH 3T3 mouse fibroblast cells as dictated by an MTT assay.

Effect of strontium ranelate on bone mineral: Analysis of nanoscale compositional changes.

Strontium ranelate has been used to prevent bone loss and stimulate bone regeneration. Although strontium may integrate into the bone crystal lattice, the chemical and structural modifications of the bone when strontium interacts with the mineral phase are not completely understood. The objective of this study was to evaluate apatite from the mandibles of rats treated with strontium ranelate in the drinking water and compare its characteristics with those from untreated rats and synthetic apatites with and without strontium. Electron energy loss near edge structures from phosphorus, carbon, calcium and strontium were obtained by electron energy loss spectroscopy in a transmission electron microscope. The strontium signal was detected in the biological and synthetic samples containing strontium. The relative quantification of carbon by analyzing the CK edge at an energy loss of ΔE = 284 eV showed an increase in the number of carbonate groups in the bone mineral of treated rats. A synthetic strontium-containing sample used as control did not exhibit a carbon signal. This study showed physicochemical modifications in the bone mineral at the nanoscale caused by the systemic administration of strontium ranelate.

Calcareous sponges from São Paulo State, Brazil (Porifera: Calcarea: Calcinea) with the description of two new species

Calcareous sponges (Calcarea: Calcinea) are being described, for the first time, from Sao Paulo State, Brazil. A total of five species are described, two of which are new to science (Clathrina alcatraziensis sp. nov. and Leucascus roseus sp. nov.). The remaining three species have their distribution widened (Clathrina aurea, C. conifera and C. tetractina).

Long-range crystalline order in spicules from the calcareous sponge Paraleucilla magna (Porifera, Calcarea)

We investigated the ultrastructure and crystallographic orientation of spicules from the calcareous sponge Paraleucilla magna (subclass Calcaronea) by transmission and scanning electron microscopy using two different methods of sample preparation: ultramicrotomy and focused ion beam (FIB). It was found that the unpaired actine from the spicules was oriented in the [211] zone axis. The plane that contains the unpaired actine and divides symmetrically the paired actines is the (-120). This plane is a mirror plane of the hexagonal lattice system. All the spicule types analyzed presented the same crystallographic orientation. Electron nanodiffraction maps from 4μm×4μm regions prepared by FIB showed disorientation of <2° between diffraction patterns obtained from neighbor regions, indicating the presence of a unique, highly aligned calcite crystalline phase. Among the eight FIB sections obtained, four presented high pore density. In one section perpendicular to the actine axis pores were observed only in the center of the spicule aligned in a circular pattern and surrounded by a faint circular contour with a larger radius. The presence of amorphous carbon representative of organic molecules detected by electron energy loss spectroscopy was correlated neither with porosity nor with specific lattice planes.

Improved tribocorrosion performance of bio-functionalized TiO₂ nanotubes under two-cycle sliding actions in artificial saliva

After insertion into bone, dental implants may be subjected to tribocorrosive conditions resulting in the release of metallic ions and solid wear debris, which can induce to peri-implant inflammatory reactions accompanied by bone loss, and ultimately implant loosening. Despite the promising ability of TiO2 nanotubes (NTs) to improve osseointegration and avoid infection-related failures, the understanding of their degradation under the simultaneous action of wear and corrosion (tribocorrosion) is still very limited. This study aims, for the first time, to study the tribocorrosion behavior of bio-functionalized TiO2 NTs submitted to two-cycle sliding actions, and compare it with conventional TiO2 NTs. TiO2 NTs grown by anodization were doped with bioactive elements, namely calcium (Ca), phosphorous (P), and zinc (Zn), through reverse polarization anodization treatments. Characterization techniques such as scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS) and scanning transmission electron microscopy (STEM), were used to characterize the films. Tribocorrosion tests were carried out in artificial saliva (AS) by applying two cycles of reciprocating sliding actions. The open circuit potential (OCP) was monitored before, during, and after both cycles of sliding, during which the coefficient of friction (COF) was calculated. The resulting wear scars were analyzed by SEM and EDS, and wear volume measurements were performed by 2D profilometry. Finally, the mechanical features of TiO2 NTs were accessed by nanoindentation. The results show that bio-functionalized TiO2 NTs display an enhanced tribocorrosion performance, ascribed to the growth of a nano-thick oxide film at Ti/TiO2 NTs interface, which significantly increased their adhesion strength to the substrate and consequently their hardness. Furthermore, it was discovered that during tribo-electrochemical solicitations, the formation of a P-rich tribofilm takes place, which grants both electrochemical protection and resistance to mechanical wear. This study provides fundamental and new insights for the development of multifunctional TiO2 NTs with long-term biomechanical stability and improved clinical outcomes.

Crystallographic orientation and concentric layers in spicules of calcareous sponges.

In this work, the crystallography of calcareous sponges (Porifera) spicules and the organization pattern of the concentric layers present in their inner structure were investigated in 10 species of the subclass Calcaronea and three species of the subclass Calcinea. Polished spicules had specific concentric patterns that varied depending on the plane in which the spicules were sectioned. A 3D model of the concentric layers was created to interpret these patterns and the biomineralization process of the triactine spicules. The morphology of the spicules was compared with the crystallographic orientation of the calcite crystals by analyzing the Kikuchi diffraction patterns using a scanning electron microscope. Triactine spicules from the subclass Calcinea had actines (rays) elongated in the 〈210〉 direction, which is perpendicular to the c-axis. The scale spicules of the hypercalcified species Murrayona phanolepis presented the c-axis perpendicular to the plane of the scale, which is in accordance with the crystallography of all other Calcinea. The triactine spicules of the calcaronean species had approximately the same crystallographic orientation with the unpaired actine elongated in the ∼[211] direction. Only one Calcaronea species, whose triactine was regular, had a different orientation. Three different crystallographic orientations were found in diactines. Spicules with different morphologies, dimensions and positions in the sponge body had similar crystallographic directions suggesting that the crystallographic orientation of spicules in calcareous sponges is conserved through evolution.