Electron beam structuring of Ti6Al4V: New insights on the metal surface properties influencing the bacterial adhesion

Soft tissue adhesion and infection prevention are currently challenging for dental transmucosal or percutaneous orthopedic implants. It has previously been shown that aligned micro-grooves obtained by Electron Beam (EB) can drive fibroblast alignment for improved soft tissue adhesion. In this work, evidence is presented that the same technique can also be effective for a reduction of the infection risk. Grooves 10-30 \u3bcm wide and around 0.2 \u3bcm deep were obtained on Ti6Al4V by EB. EB treatment changes the crystalline structure and microstructure in a surface layer that is thicker than the groove depth. Unexpectedly, a significant bacterial reduction was observed. The surfaces were characterized by field emission scanning electron microscopy, X-ray diffraction, confocal microscopy, contact profilometry, wettability and bacterial adhesion tests. The influence of surface topography, microstructure and crystallography on bacterial adhesion was systematically investigated: it was evidenced that the bacterial reduction after EB surface treatment is not correlated with the grooves, but with the microstructure induced by the EB treatment, with a significant bacterial reduction when the surface microstructure has a high density of grain boundaries. This correlation between microstructure and bacterial adhesion was reported for the first time for Ti alloys

Generation of cytocompatible superhydrophobic Zr–Cu–Ag metallic glass coatings with antifouling properties for medical textiles

Zirconium–Copper-based metallic glass thin films represent promising coatings in the biomedical sector for their combination of antibacterial property and wear resistance. However, finding a Zr–Cu metallic glass composition with desirable cytocompatibility and antibacterial property is extremely challenging. In this work, we have created a cytocompatible and (super-)hydrophobic Zr–Cu–Ag metallic glass coating with ≈95% antifouling properties. First, a range of different chemical compositions were prepared via Physical Vapor Deposition magnetron by co-sputtering Zr, Cu, and Ag onto a Polybutylene terephthalate (PBT) substrate among which Zr93·5Cu6·2Ag0.2, Zr76·7Cu22·7Ag0.5, and Zr69·3Cu30·1Ag0.6 were selected to be further investigate for their surface properties, antibacterial activity, and cytocompatibility. Scanning electron microscopy (SEM) images revealed a micro-roughness fibrous structure holding superhydrophobic properties demonstrated by specimens' static and dynamic contact angle measurements ranging from 130° to 150°. The dynamic contact angle measurements have shown hysteresis below 10° for all coated samples which indicated the superhydrophobicity of the samples. To distinguish between antifouling and bactericidal effect of the coating, ions release from coatings into Luria Bertani Broth (LB), and Dulbecco's Modified Eagle Medium (DMEM) solutions were evaluated by inductively coupled plasma mass spectrometry (ICP-MS) measurements after 24 ​h and 5 days. Antifouling properties were evaluated by infecting the specimens' surface with the Gram-positive Staphylococcus aureus and the Gram-negative Escherichia coli strain reporting a ≈95% reduction of bacteria adhesion as visually confirmed by FESEM and fluorescent live/dead staining. Human mesenchymal stem cells (hMSC) were used for direct cytocompatibility evaluation of coated samples and their metabolic activity was evaluated via relative fluorescence unit after 24 ​h and 5 days confirming that it was comparable to the controls (>97% viable cells). The results were further visualized by FESEM, fluorescent staining by Live/Dead Viability/Cytotoxicity Kit and confirmed the cytocompatibility of all coated samples. Finally, hMSC′ cytoplasm was stained by May Grunwald and Giemsa after 5days to detect and visualize the released ions which have diffused through the cells' membrane

Surface Properties and Antioxidant Activity of Silicate and Borosilicate Bioactive Glasses

Herein, silicate and borosilicate bioactive glasses are synthetized and characterized. The antioxidant activity, in the presence and absence of human osteoblasts' progenitor cells, of the different glass compositions, is correlated to the surface properties: wettability, zeta potential, hydroxylation degree, reactivity in simulated body fluid (SBF), and Tris buffer. An enhancing effect of boron in glass reactivity and a stabilizing role of Sr and Mg are evidenced. The scavenging potential of the analyzed bioactive glasses toward reactive oxygen species (ROS) is clearly proved. Moreover, cellular tests confirm the protective effect of the bioactive glasses toward viable cells acting as ROS/RNS species scavenger. The obtained results represent an original improvement of the knowledge concerning the intrinsic antioxidant ability of bioactive glasses with different compositions and the mechanisms involved

Electrophoretic deposition of composite coatings based on alginate matrix/45S5 bioactive glass particles doped with B, Zn or Sr

In this research work composite coatings made of alginate and 45S5 bioactive glass particles doped with B, Zn or Sr were synthesized by means of electrophoretic deposition and characterized from morphological, compositional, thermogravimetric, mechanical and electrochemical points of view. The developed coatings were also subjected to in vitro test in SBF solution to evaluate their ability to induce hydroxyapatite precipitation and cytocompatibility evaluation using human primary fibroblasts. The obtained results demonstrated a good homogeneity of the coatings, high adhesion and a protective behavior towards the substrate. The thermogravimetric analysis proved that the glass amount was constant before and after the deposition and all the investigated coatings promoted the deposition of hydroxyapatite but with different kinetics. Since the Zn containing coating showed the best bioactive behavior it was subjected to cytocompatibility test, which demonstrated, after an initial reduction of cell viability, a good cell proliferation and the production of collagen from the ECM. These findings suggest that the obtained coatings are promising materials to coat metallic prosthetic devices

Biological evaluation of a new sodium-potassium silico-phosphate glass for bone regeneration: In vitro and in vivo studies

In vitro and in vivo studies are fundamental steps in the characterization of new im-plantable materials to preliminarily assess their biological response. The present study reports the in vitro and in vivo characterizations of a novel experimental silicate bioactive glass (BG) (47.5 B, 47.5 SiO2-10 Na2O-10 K2O-10 MgO-20 CaO-2.5 P2O5 mol.%). Cytocompatibility tests were perfor-med using human mature osteoblasts (U2OS), human mesenchymal stem cells (hMSCs) and human endothelial cells (EA.hy926). The release of the early osteogenic alkaline phosphatase (ALP) marker suggested strong pro-osteogenic properties, as the amount was comparable between hMSCs cultivated onto BG surface and cells cultivated onto polystyrene control. Similarly, real-time PCR revealed that the osteogenic collagen I gene was overexpressed in cells cultivated onto BG surface without biochemical induction. Acute toxicity tests for the determination of the median lethal dose (LD50 ) al-lowed classifying the analyzed material as a slightly toxic substance with LD50 = 4522 ± 248 mg/kg. A statistically significant difference in bone formation was observed in vivo through comparing the control (untreated) group and the experimental one, proving a clear osteogenic effect induced by the implantation at the defect site. Complete resorption of 47.5 B powder was observed after only 3 months in favor of newly formed tissue, thus confirming the high osteostimulatory potential of 47.5 B glass

Bioreactor mechanically guided 3D mesenchymal stem cell chondrogenesis using a biocompatible novel thermo-reversible methylcellulose-based hydrogel

Autologous chondrocyte implantation for cartilage repair represents a challenge because strongly limited by chondrocytes' poor expansion capacity in vitro. Mesenchymal stem cells (MSCs) can differentiate into chondrocytes, while mechanical loading has been proposed as alternative strategy to induce chondrogenesis excluding the use of exogenous factors. Moreover, MSC supporting material selection is fundamental to allow for an active interaction with cells. Here, we tested a novel thermo-reversible hydrogel composed of 8% w/v methylcellulose (MC) in a 0.05 M Na 2 SO 4 solution. MC hydrogel was obtained by dispersion technique and its thermo-reversibility, mechanical properties, degradation and swelling were investigated, demonstrating a solution-gelation transition between 34 and 37 °C and a low bulk degradation (<20%) after 1 month. The lack of any hydrogel-derived immunoreaction was demonstrated in vivo by mice subcutaneous implantation. To induce in vitro chondrogenesis, MSCs were seeded into MC solution retained within a porous polyurethane (PU) matrix. PU-MC composites were subjected to a combination of compression and shear forces for 21 days in a custom made bioreactor. Mechanical stimulation led to a significant increase in chondrogenic gene expression, while histological analysis detected sulphated glycosaminoglycans and collagen II only in loaded specimens, confirming MC hydrogel suitability to support load induced MSCs chondrogenesis

Bioreactor mechanically guided 3D mesenchymal stem cell chondrogenesis using a biocompatible novel thermo-reversible methylcellulose-based hydrogel

Autologous chondrocyte implantation for cartilage repair represents a challenge because strongly limited by chondrocytes' poor expansion capacity in vitro. Mesenchymal stem cells (MSCs) can differentiate into chondrocytes, while mechanical loading has been proposed as alternative strategy to induce chondrogenesis excluding the use of exogenous factors. Moreover, MSC supporting material selection is fundamental to allow for an active interaction with cells. Here, we tested a novel thermo-reversible hydrogel composed of 8% w/v methylcellulose (MC) in a 0.05 M Na 2 SO 4 solution. MC hydrogel was obtained by dispersion technique and its thermo-reversibility, mechanical properties, degradation and swelling were investigated, demonstrating a solution-gelation transition between 34 and 37 °C and a low bulk degradation (<20%) after 1 month. The lack of any hydrogel-derived immunoreaction was demonstrated in vivo by mice subcutaneous implantation. To induce in vitro chondrogenesis, MSCs were seeded into MC solution retained within a porous polyurethane (PU) matrix. PU-MC composites were subjected to a combination of compression and shear forces for 21 days in a custom made bioreactor. Mechanical stimulation led to a significant increase in chondrogenic gene expression, while histological analysis detected sulphated glycosaminoglycans and collagen II only in loaded specimens, confirming MC hydrogel suitability to support load induced MSCs chondrogenesis