Antonio Cacchioli

Evaluation of different preparations of plasma-spray hydroxyapatite coating on titanium alloy and duplex stainless steel in the rabbit

Many variables are involved in hydroxyapatite coating of metals by plasma-spray techniques. The authors have investigated the biological response to some of the most relevant variables in a controlled in vivo trial. The bone response in the rabbit towards hydroxyapatite coated cylinders was studied keeping the following variables fixed: (a) crystallinity of coating (greater than 90% and between 70% and 60%); (b) thickness of coating (50 and 100 μm); (c) metallic substrate (titanium alloy and duplex stainless stell). Analysis of the results highlights the importance of defining the crystallinity of the coating to forecast its in vivo behaviour: highly crystalline coating is more stable in time but can give rise to fragmented bulky particles; a less crystalline coating is subject to slow degradation in the long term but facilitates its substitution by newly formed bone. Furthermore, it has been found that no relevant differences can be ascribed to a variation in coating thickness between 50 and 100 μm. It has, also, been observed that there are no differences when duplex stainless steel is used instead of titanium alloy as metallic substrate, confirming that bone responds primarily to the coating.

Flow cytometry detection of serotonin content and release in resting and activated platelets

Summary. Early detection of platelet activation is important for the diagnosis and follow‐up of several pathological conditions that primarily or secondarily involve platelets in their pathogenesis. The golden standard assay to detect thrombocyte activation is represented by the release of serotonin, classically performed by demanding methodologies, such as high‐performance liquid chromatography, 14C‐labelling and enzyme‐linked immunosorbent assay (ELISA). We developed a non‐radioactive method, based on individual cells, for the detection of serotonin content in activated and resting platelets by flow cytometry. The assay was standardized on cells activated by Ca2+ ionophore or by sera from patients with heparin‐induced thrombocytopenia (HIT). Cells were identified by CD41a surface staining and their serotonin content measured by specific antiserotonin intracytoplasmic staining, while their activation was independently shown by annexin V binding. Cellular degranulation was detected by flow cytometry in all the cases that were also positive by standard ELISA. Moreover, multiparametric flow cytometry analysis revealed that, although virtually all activated cells bind annexin V, serotonin was released only by the platelet subset that downmodulates surface CD41a.

Exogenous hydrogen sulfide induces functional inhibition and cell death of cytotoxic lymphocytes subsets

The toxic effects of exogenous hydrogen sulfide on peripheral blood lymphocytes have been investigated in detail. Hydrogen sulfide is now considered as a gasotransmitter with specific functional roles in different cell types, like neurons and vascular smooth muscle. Here we show that exogenous hydrogen sulfide induces a caspase‐independent cell death of peripheral blood lymphocytes that depends on their intracellular glutathion levels, with a physiologically relevant subset specificity for CD8+ T cells and NK cells. Although lymphocyte activation does not modify their sensitivity to HS−, after 24 h exposure to hydrogen sulfide surviving lymphocyte subsets show a dramatically decreased proliferation in response to mitogens and a reduced IL‐2 production. Overall, our data demonstrate that HS− reduces the cellular cytotoxic response of peripheral blood lymphocytes as well as their production of IL‐2, therefore de‐activating the major players of local inflammatory responses, adding new basic knowledge to the clinically well known anti‐inflammatory effects of sulfur compounds. J. Cell. Physiol. 213:826–833.

HIV‐1 matrix protein p17 enhances the proliferative activity of natural killer cells and increases their ability to secrete proinflammatory cytokines

Summary. We investigated the effects of human immunodeficiency type‐1 virus (HIV‐1) matrix protein p17 on freshly isolated and purified human natural killer (NK) cells. HIV‐1 p17 increased the cytokines interleukin (IL) 2, IL‐12 and IL‐15, and induced natural killer cell proliferation, but not cytotoxicity. This effect was specific because it was abrogated by anti‐p17 monoclonal antibody. Moreover, HIV‐1 p17 enhanced the cytokine‐induced production of tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ by NK cells. IL‐4 downregulated IFN‐γ and TNF‐α secretion in IL‐2‐ and IL‐15‐treated NK cells. HIV‐1 p17 restored the ability of NK cells to produce both cytokines when added to the cultures simultaneously with IL‐4. The property of p17 to increase the production of TNF‐α and IFN‐γ might be a mechanism used by HIV‐1 to modulate the immune system to support its replication and spreading.

In vitro cellular response and in vivo primary osteointegration of electrochemically modified titanium

Anodic spark deposition (ASD) is an attractive technique for improving the implant-bone interface that can be applied to titanium and titanium alloys. This technique produces a surface with microporous morphology and an oxide layer enriched with calcium and phosphorus. The aim of the present study was to investigate the biological response in vitro using primary human osteoblasts as a cellular model and the osteogenic primary response in vivo within a short experimental time frame (2 and 4 weeks) in an animal model (rabbit). Responses were assessed by comparing the new electrochemical biomimetic treatments to an acid-etching treatment as control. The in vitro biological response was characterized by cell morphology, adhesion, proliferation activity and cell metabolic activity. A complete assessment of osteogenic activity in vivo was achieved by estimating static and dynamic histomorphometric parameters at several time points within the considered time frame. The in vitro study showed enhanced osteoblast adhesion and higher metabolic activity for the ASD-treated surfaces during the first days after seeding compared to the control titanium. For the ASD surfaces, the histomorphometry indicated a higher mineral apposition rate within 2 weeks and a more extended bone activation within the first week after surgery, leading to more extensive bone-implant contact after 2 weeks. In conclusion, the ASD surface treatments enhanced the biological response in vitro, promoting an early osteoblast adhesion, and the osteointegrative properties in vivo, accelerating the primary osteogenic response.

Attachment, proliferation and osteogenic response of osteoblast-like cells cultured on titanium treated by a novel multiphase anodic spark deposition process.

A new bioactive titanium surface treatment, labeled Ti-ASD, was developed using the electrochemical anodic spark deposition (ASD) technique and results in a thickened titanium oxide layer with higher levels of calcium and phosphorus typical of newly deposited mineral phase. This study was aimed at extending the knowledge on Ti-ASD treatment, by means of evaluation of the attachment, morphology, proliferation, metabolic activity, differentiation, and mineralization of osteoblast-like cells (SaOS-2) after growth on Ti-ASD treated titanium compared with nontreated titanium (Ti) and with chemically etched titanium (Ti-ETC). This novel type of titanium coating supported cell attachment, cell proliferation, and mineralization, revealing no cytotoxicity effects. The expression of differentiation markers on Ti-ASD treated titanium shows that genes related to the proliferation phase (Collagen type I, Coll I; Cbfa-1) were early expressed, whereas genes related to the mineralization phase (alkaline phosphatase, osteopontin, bone sialo protein) increased in a time-related way. Mineralization occurred on all analyzed surfaces, but on Ti-ASD the number of bone-like nodules and the amount of mineralized area was higher. In conclusion, Ti-ASD resulted to be a good surface for osteoblast attachment and proliferation, also promoting the maintenance of cell differentiation and matrix mineralization, a fundamental requirement for sustain the osseointegration and the clinical success of dental implants.

The Effects of Er:YAG Laser Treatment on Titanium Surface Profile and Osteoblastic Cell Activity: An In Vitro Study

BACKGROUND Laser light has been proposed as a tool to decontaminate the surface of endosseous implants. The effects of this maneuver on the interactions between cells and surface, however, are poorly known. The goal of the present study is to investigate osteoblast growth and differentiation on three commercially available surfaces untreated or after irradiation by erbium-doped:yttrium, aluminum, and garnet (Er:YAG) laser at two levels: 150 and 200 mJ/pulse at 10 Hz. METHODS Human osteoblastic Saos-2 cells were plated on machined, sandblasted and acid-etched titanium, or titanium plasma-sprayed disks. The effects of lasing were observed with a scanning electronic microscope, and cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Moreover, we measured the production of the osteoblast-specific protein osteocalcin and of osteoprotegerin in the supernatants by immunoenzymatic assays. RESULTS Although no visible changes were observed on machined or titanium plasma-sprayed disk samples at the tested levels, titanium peaks on sandblasted and acid-etched titanium disks appeared fused as a consequence of laser irradiation. Interestingly, cell proliferation was slower on irradiated titanium at both intensities on all the surfaces. Cell differentiation, as assessed by osteocalcin production, was generally unaffected by laser treatment, whereas the production of osteoprotegerin was decreased on all the surfaces irradiated at the intensity of 200 mJ/10Hz. CONCLUSIONS These results indicate that Er:YAG laser at energy levels used in this study can alter the surface profile of titanium implants and these changes may negatively affect the viability and the activity of osteoblastic cells. Therefore, Er:YAG lasers should be used with caution on titanium surfaces.

Innate pro-inflammatory and adaptive immune cytokines in PBMC of vaccinated and unvaccinated pigs naturally exposed to porcine circovirus type 2 (PCV2) infection vary with the occurrence of the disease and the viral burden.

Pro-inflammatory (IL-8, TNF-α, IL-1β) and immune (IFN-γ, IL-10) cytokines were evaluated in PCV2-vaccinated and unvaccinated pigs exposed to natural PCV2 infection retrospectively selected according to the time of the onset of viremia and the viral burden, and the presence of PMWS clinical signs. In a farrow-to-finish herd with a history of PMWS in animals aged older than 15 weeks, at weaning (21 ± 3 days of age), vaccinated pigs were intramuscularly inoculated with one dose of Porcilis(®) PCV vaccine+adjuvant whereas the adjuvant alone was administered to the control animals. Thirty animals bled at 16 weeks of age (before the occurrence of the natural infection and the onset of the disease) and then at 19, 20, 22 and 26 weeks of age, were categorized as: (a) vaccinated non-infected and non-PMWS-affected (PCV2-vac), (b) unvaccinated spontaneously infected/non-PMWS-affected (Ctrl) and (c) unvaccinated spontaneously infected/PMWS-affected (Ctrl-PMWS+) pigs. A major evidence of this study is that PMWS-affected animals were not able to mount an efficient innate pro-inflammatory response to cope with PCV2 infection as demonstrated by the low levels of pro-inflammatory cytokines, namely IL-8, TNF-α and IL-1β, and IFN-γ. Conversely, significantly increased gene expression levels of IL-8, TNF-α and IL-1β were detected especially in the PCV2-vac group at the early phase of the infection. Moreover, in PMWS diseased animals, a significant increase of IL-10 occurred at the early phase of infection, while, vaccinated pigs, in addition to the low viremia burden and its frequency and the absence of PMWS disease, showed a more stable IFN-γ response.

Bone response to polymers based on poly-lactic acid and having different degradation times

Authors studied two degradable and resorbable polymers derived from lactic acid: poly-L-Lactic acid (PLLA), with a relatively long time of degradation (longer than 6 months, PL10 Purac NL); poly-DL-Lactic acid (PDLLA), with a relatively short time of degradation (shorter than 6 months, PDL Purac NL). The animal species was the young adult New Zealand White rabbit. The in-vivo study was performed by implantation of small cylinders of 10 × 3 mm in size (length × diameter) in the distal metaepiphysis of the femur; 34 cylinders have been implanted. Retrievals of PLLA specimens took place at 3, 6, 9, 12 and 24 months; for PDLLA specimens at 1, 2, 4 months. Polarized light microscopy of undecalcified tissue sections was performed. The analysis for PLLA and PDLLA has shown a favorable response of bone tissue: alterations in the bone repair, growth and remodeling have not been observed. PLLA is persistent at the times studied; there is never a tight apposition between bone and PLLA implant and an intervening fibrous layer has often been observed. PDLLA is not persistent at the times studied and it degrades quite fast; bone repair of the empty implantations hole occurs by bony growth from the endosteal trabeculae. The newly formed bone covers the holes walls with an elongation parallel to them. For both polymers, whether the degradation is fast or slow, the materials substitution by newly formed bone never starts from the walls of the implantation hole. Only after the complete disappearance of the polymeric material newly formed bone begins to fill the hole.

Titanium dioxide aggregating nanoparticles induce autophagy and under-expression of microRNA 21 and 30a in A549 cell line: A comparative study with cobalt(II, III) oxide nanoparticles

The toxicity of TiO2 nanoparticles (NPs) is controversial, while it is widely accepted for Co3O4 NPs. We present a comparative study concerning the uptake of these NPs and their effect on cytoplasmic organelles and autophagy in a human lung carcinoma cell line (A549), including assays on the expression of autophagy-related microRNAs. The NP accumulation caused a fast dose- and time-dependent change of flow cytometry physical parameters particularly after TiO2 NP exposure. The intracellular levels of metals confirmed it, but the Co concentration was ten times higher than that of Ti. Both NPs caused neither necrosis nor apoptosis, but cytotoxicity was mainly evident for Co3O4 NPs in the first 72h. TiO2 NPs caused autophagy, contrarily to Co3O4 NPs. Furthermore, a significant and persistent downregulation of miRNA-21 and miRNA-30a was observed only in TiO2 NPs-treated cultures. The expression of miRNA-155 was similar for both NPs. Oxidative stress was evident only for Co3O4 NPs, while both NPs perturbed endoplasmic reticulum and p-53 expression. In conclusion, the oxidative stress caused by Co3O4 NPs can influence energy homeostasis and hamper the ability to detoxify and to repair the resulting damage, thus preventing the induction of autophagy, while TiO2 NPs elicit autophagy also under sub-toxic conditions.