Cultures and co-cultures of human blood mononuclear cells and endothelial cells for the biocompatibility assessment of surface modified AISI 316L austenitic stainless steel.

Samples of AISI 316L austenitic stainless steel were subjected either to grinding and polishing procedure, or to grinding and then low temperature glow-discharge nitriding treatment, or to grinding, nitriding and subsequently coating with collagen-I. Nitrided samples, even if only ground, show a higher corrosion resistance in PBS solution, in comparison with ground and polished AISI 316L. Biocompatibility was evaluated in vitro by incubating the samples with either peripheral blood mononuclear cells (PBMC) or human umbilical vein endothelial cells (HUVEC), tested separately or in co-culture. HUVEC-PBMC co-culture and co-incubation of HUVEC with PBMC culture medium, after the previous incubation of PBMC with metallic samples, allowed to determine whether the incubation of PBMC with the different samples might affect HUVEC behaviour. Many biological parameters were considered: cell proliferation, release of cytokines, matrix metalloproteinases (MMPs) and sICAM-1, gelatinolytic activity of MMPs, and ICAM-1 protein expression. Nitriding treatment, with or without collagen coating of the samples, is able to ameliorate some of the biological parameters taken into account. The obtained results point out that biocompatibility may be successfully tested in vitro, using cultures of normal human cells, as blood and endothelial cells, but more than one cell line should be used, separately or in co-culture, and different parameters should be determined, in particular those correlated with inflammatory phenomena.

In vitro response of human peripheral blood mononuclear cells to AISI 316L austenitic stainless steel subjected to nitriding and collagen coating treatments.

Surface modification treatments can be used to improve the biocompatibility of austenitic stainless steels. In the present research two different modifications of AISI 316L stainless steel were considered, low temperature nitriding and collagen-I coating, applied as single treatment or in conjunction. Low temperature nitriding produced modified surface layers consisting mainly of S phase, which enhanced corrosion resistance in PBS solution. Biocompatibility was assessed using human peripheral blood mononuclear cells (PBMC) in culture. Proliferation, lactate dehydrogenase (LDH) levels, release of cytokines (TNF-α, IL-1ß, IL-12, IL-10), secretion of metalloproteinase (MMP)-9 and its inhibitor TIMP-1, and the gelatinolytic activity of MMP-9 were determined. While the 48-h incubation of PBMC with all the sample types did not negatively influence cell proliferation, LDH and MMP-9 levels, suggesting therefore a good biocompatibility, the release of the pro-inflammatory cytokines was always remarkable when compared to that of control cells. However, in the presence of the nitrided and collagen coated samples, the release of the pro-inflammatory cytokine IL-1ß decreased, while that of the anti-inflammatory cytokine IL-10 increased, in comparison with the untreated AISI 316L samples. Our results suggest that some biological parameters were ameliorated by these surface treatments of AISI 316L.

In vitro interaction between surface-treated Ti-6Al-4V titanium alloy and human peripheral blood mononuclear cells.

The Ti-6Al-4V titanium alloy is widely employed as an implant material. The effects of Ti-6Al-4V samples, tested in both an untreated state and one in which the samples were subjected to a glow-discharge treatment performed with the use of air, on human peripheral blood mononuclear cells (PBMC) were studied. Apoptosis, undetectable after 24-h contact of PBMC with the two sample types, is induced after 48 h by treated samples, and, after 48 h, but in the presence of 1.5 microg/mL PHA, by both sample types. The expression of intercellular adhesion molecule-1 (ICAM-1) always increases, in comparison with control, in PBMC put in contact with the two sample types. In the same way, a remarkable increase in tumor necrosis-alpha (TNF-alpha) release in the culture medium is registered, when PBMC are put in contact with the two sample types for 24 and 48 h. Human umbilical-vein endothelial cells (HUVEC) cocultured for 48 h with PBMC, previously incubated with the two sample types for 24 h, show an increase in ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) protein expression in comparison with control (HUVEC cocultured with control PBMC), indicating that inflammatory phenomena might occur. Taken together, these results suggest that, although plasma-treated titanium alloy shows a better biocompatibility in comparison with the untreated one, attention must be paid to the careful control of the first signs of inflammation.

Effects of surface treatment of Ti-6Al-4V titanium alloy on biocompatibility in cultured human umbilical vein endothelial cells.

Among the titanium alloys employed as implant materials, the Ti-6Al-4V alloy is still widely used. Ti-6Al-4V titanium alloy samples, in untreated state and subjected to treatments in air by furnace or glow-discharge processes, were put in contact with human umbilical vein endothelial cells (HUVEC) in order to evaluate their effects on biocompatibility. In HUVEC kept for 48 h in the presence of the three sample types neither cell proliferation nor protein content nor lactate dehydrogenase release in the culture medium are affected, while apoptosis is induced after 48- and 96-h contact of the cells with the untreated sample type, and after 96-h contact with the plasma treated one, the furnace treated sample type being ineffective. The expression of two adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was also studied. The incubation of HUVEC with the three sample types for 48 or 96 h induces a significant increase in ICAM-1 protein levels, in comparison with control cells, while VCAM-1 expression is not detectable. In the same way, TNF-alpha release in the culture medium, assayed after 48- and 96-h contact of the cells with the three sample types, is significantly higher, in comparison with control, even if the highest values are registered in the presence of the untreated samples. Taken together, these data indicate that, although Ti-6Al-4V alloy samples, and in particular the treated ones, show a good biocompatibility, attention must be given to the first signs of inflammation.