Antonietta M. Gatti

Biocompatibility of micro- and nano-particles in the colon. Part II.

Pathological colonic tissues were investigated with an Environmental Scanning Electron Microscope technique to verify the presence of inorganic, non-biodegradable pollutants, i.e. micro- and nano-debris of exogenous origin, after debris in liver and kidney had been discovered. In all, 18 samples of colon tissues affected by cancer and Crohns disease were evaluated and found in all the cases to contain micro- and nano-particles. Their chemistry, detected with an X-ray microprobe, indicated a heterogeneous nature, whereas the size of the particles was homogeneous. Three control samples of healthy, young, cadavers were analysed and showed the absence of debris within the normal, healthy colon mucosa. The study reveals the presence of particulate debris, generally considered as biocompatible, in pathological specimens of human colon. The findings suggest a possible link between the presence of such particles and the underlying pathology in the cases analysed.

Behaviour of tricalcium phosphate and hydroxyapatite granules in sheep bone defects

Granules of hydroxyapatite (HA) and tricalcium phosphate (TCP) were implanted in separate holes drilled in mandibular bone of sheep to check the bone growth and in vivo behaviour of the materials. The experiment was performed in three sheep, killed respectively at 4, 8, 12 month. Samples of bone with the materials were explanted, microradiographed and sectioned to evaluate the interface under optical and electron scanning electron microscope. The hole, left empty as a reference, showed no full repair; whereas 4 month after implantation the TCP granules induce total repair of the hole. HA granules crumbled and no new bone induction was seen even 12 month after implantation.

Toxicity of antimony trioxide nanoparticles on human hematopoietic progenitor cells and comparison to cell lines.

Nanoparticles (NPs) are materials with one dimension in the range of 1-100 nm. The toxicity of NPs remains widely unknown and still poses concerns, due to the peculiar characteristics of materials in the nano-size range. We analyze the toxicity of seven NPs ((Fe2O3, Fe3O4, Sb2O3, Au, TiO2, Co, and Ag) on primary cultures of human hematopoietic progenitor cells from the bone marrow of healthy donors with CFU assays, and show that antimony oxide (Sb2O3) NPs and cobalt (Co) NPs have a toxic effect, while the other NPs have no effect at the tested concentrations (5, 25 and 100 microg/ml). While Co NPs suspension is toxic to both erythroid and granulocytic-monocytic precursors, Sb2O3 NPs at 5 microg/ml are specifically toxic to erythroid colony development, suggesting a highly selective type of toxicity. With liquid culture assays we show that Sb2O3 NPs impair the proliferation of erythroid progenitors, while no toxic effect is observed when Sb2O3 NPs are added during erythroid differentiation. CFU assays and liquid culture assays on seven human cell lines of hematopoietic origin (K562, HL-60, CEM, CEM-R, Thp-1, Jurkat, and Molt-4) show that, contrary to what observed on primary cultures of bone marrow progenitors, Sb2O3 NPs have no toxic effect on proliferation of any of the cell lines, raising concerns about the use of immortalized cell lines for nanotoxicology tests.

Analysis of the in vivo reactions of a bioactive glass in soft and hard tissue

A bioactive glass, S53P4, was implanted as granules subcutaneously in muscles and connective tissue of rabbits, as well as in the mandibular bone of a sheep. After the implantation period of 2-3 months, cross-sections were prepared and studied by scanning electron microscopy and energy dispersive X-ray analysis. The glass reacted essentially in the same way in all types of tissue. The granules consisted of an unreacted core and a reacted layer with a silica-rich and calcium phosphate-rich zone. Large hydroxyapatite crystals were occasionally found on top of the calcium phosphate surface of the granules implanted in soft tissue. On the basis of elemental analysis of the reaction layers it was found that the release of calcium from inside the glass is sufficient to account for the formation of the calcium phosphate surface layer, whereas the release of phosphate from the glass is not sufficient.

Biological tolerance of different materials in bulk and nanoparticulate form in a rat model: sarcoma development by nanoparticles

In order to study the pathobiological impact of the nanometre-scale of materials, we evaluated the effects of five different materials as nanoparticulate biomaterials in comparison with bulk samples in contact with living tissues. Five groups out of 10 rats were implanted bilaterally for up to 12 months with materials of the same type, namely TiO2, SiO2, Ni, Co and polyvinyl chloride (PVC), subcutaneously with bulk material on one side of the vertebral column and intramuscularly with nanoparticulate material on the contralateral side. At the end of each implantation time, the site was macroscopically examined, followed by histological processing according to standard techniques. Malignant mesenchymal tumours (pleomorphic sarcomas) were obtained in five out of six cases of implanted Co nanoparticle sites, while a preneoplastic lesion was observed in an animal implanted with Co in bulk form. In the Ni group, all animals rapidly developed visible nodules at the implanted sites between 4 and 6 months, which were diagnosed as rhabdomyosarcomas. Since the ratio of surface area to volume did not show significant differences between the Ni/Co group and the TiO2/SiO2/PVC group, we suggested that the induction of neoplasia was not mediated by physical effects, but was mediated by the well-known carcinogenic impact of Ni and Co. The data from the Co group show that the physical properties (particulate versus bulk form) could have a significant influence on the acceleration of the neoplastic process.

Investigation of the Presence of Inorganic Micro- and Nanosized Contaminants in Bread and Biscuits by Environmental Scanning Electron Microscopy

A European project called “Nanopathology” allowed to develop a new diagnostic tool through which the presence of inorganic particulate matter in pathological human tissues of the digestive tract could be shown. This unexpected evidence induced the authors to put forward the hypothesis that that sort of contamination was present in ingested food. In order to demonstrate this hypothesis, 86 samples of wheat bread and 49 of wheat biscuits from 14 different countries were analyzed by means of an Environmental Scanning Electron Microscopy to detect inorganic, micro-, and nano-scaled contaminants. The X-ray microprobe of an Energy Dispersion Spectroscope was employed to identify their chemical composition. The results indicate that 40% of the samples analyzed contained foreign bodies as ceramic and metallic debris of probable environmental or industrial origin. Because of the great variety of chemical composition of the particulate matter, those contaminants were listed according to the most expressed element. The majority of these debris are not biodegradable, some are chemically toxic, and none of them have any nutritive value. The work discusses the possible origin of such a pollution and the role that it can play on human life.

Bone Augmentation with Bioactive Glass in Three Cases of Dental Implant Placement

This study reports the clinical use of a bioactive bone graft material, PerioGlas®, in the treatment of dental extraction sites before dental implant placement, to effect bone regeneration and to give early fixation to the implant. PerioGlas®, granules, ranging from 90 to 710 mm, are implanted after tooth extraction in three patients; after 6 months bone biopsies were performed in the site of the glass implantation and observed under Electron Scanning Microscopy. All the granules showed a biodegradation involving precipitation of calcium phosphate that worked as a scaffold for osteoblasts colonization. All cases examined showed the bioactivity of PerioGlas® granules resulting in new bone formation and biodegradation of the glass. After a two-year clinical follow-up all the implants were successfully loaded and appeared stable.

Long-term behaviour of active glasses in sheep mandibular bone

Granules of a glass (A) prepared according to Henchs formula and a new vitreous material for biological applications (AKRA 15) were used for repair of bone defects in the dental field. The behaviour of these materials implanted in holes drilled in sheeps mandibular bone was examined 4, 8, 12 month after implantation. Microradiographic analyses, optical and scanning electron microscopic observations, and X-ray microprobe evaluations were carried out using undecalcified, methacrylate-embedded sections of the jaw containing the granules. After one year the granules of A disappeared, but not important bone growth was observed also in the holes containing AKRA 15. SEM and microprobe showed: disappearance of Na and Si ions at different stages; increase of P and Ca up to 4 month and then decrease, but in different ways in the two glasses; unexpected appearance of K ions after 4 month only in AKRA 15.

Mg-Substituted Tricalcium Phosphates: Formation and Properties

This study aimed to investigate the formation and properties of mag nesium (Mg)substituted tricalcium phosphate, β-TCMP, its properties and potential as biomaterial for bone repair. β-TCMPs were prepared and characterized using x-ray diffraction, FT-IR and SEM. Dissolution properties were determined in acidic buffer. β-TCMP discs were implanted in surgically created holes in femoral and tibial diaphyses of rabbits. Results demonstrated that the formation of β-TCMP and Mg incorporation in β-TCMP were dependent on reaction pH, temperature and solution Mg/Ca ratios. Sintere d β-TCMP was significantly less soluble than β-TCP. Implanted unsintered β-TCMP showed osteoconductive properties associated with new bone formation. This study suggests that β-TCMP (sintered or unsintered), alone or in combination with other calcium phosphates, may be useful as biomaterials for bone repair and maybe useful in cases where s low r biodegradation than that of β-TCP is desired.

Toxic effects of colloidal nanosilver in zebrafish embryos

A variety of consumer products containing silver nanoparticles (Ag NPs) are currently marketed. However, their safety for humans and for the environment has not yet been established and no standard method to assess their toxicity is currently available. The objective of this work was to develop an effective method to test Ag NP toxicity and to evaluate the effects of ion release and Ag NP size on a vertebrate model. To this aim, the zebrafish animal model was exposed to a solution of commercial nanosilver. While the exposure of embryos still surrounded by the chorion did not allow a definite estimation of the toxic effects exerted by the compound, the exposure for 48 h of 3‐day‐old zebrafish hatched embryos afforded a reliable evaluation of the effects of Ag NPs. The effects of the exposure were detected especially at molecular level; in fact, some selected genes expressed differentially after the exposure. The Ag NP toxic performance was due to the combined effect of Ag+ ion release and Ag NP size. However, the effect of NP size was particularly detectable at the lowest concentration of nanosilver tested (0.01 mg l–1) and depended on the solubilization media. The results obtained indicate that in vivo toxicity studies of nanosilver should be performed with ad hoc methods (in this case using hatched embryos) that might be different depending on the type of nanosilver. Moreover, the addition of this compound to commercial products should take into consideration the Ag NP solubilization media. Copyright