Ion release from titanium particles

Simulated body fluid (SBF) was chosen as the dissolution test solution as we were interested in what happens when the particles become embedded in the soft/hard tissue rather than their interaction with saliva. SBF was prepared using the Kokubo method. Seven hundred millilitres of deionised (DI) water in a 1-L polypropylene beaker was warmed to 37 °C in a water bath. The reagents were slowly added to the DI water in the order given in, while the solution was continuously stirred. The pH was continuously monitored to avoid precipitation due to sudden increase. Once the reagents were mixed, the SBF was filled to 1 L with DI water. SBF was stored at 37 °C and used within 2 days. The pH was adjusted to 7.4 before use at 37 °C.

Grade 4 and 5 titanium (Ti) particles were suspended in SBF at concentrations of 0.75, 1.5 and 3 mg ml⁻¹ in airtight polyethylene containers, which were placed on an orbital shaker rotating at 120 rpm inside a 37 °C incubator. One millilitre of aliquots was taken at day 0, 3 and 10 for the analysis of pH and ionic concentration.

Cell culture and viability assay

Human gingival fibroblasts (HGFs) (PCS-201-018^TM, ATCC®, UK) were culture expanded in basal Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 100 unit ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin and 10% (v/v) foetal bovine serum (FBS) in standard tissue-culture flasks in a humidified atmosphere containing 5% CO₂. Upon confluence, cells were subcultured using 1× trypsin-EDTA (500 μg ml⁻¹ trypsin with 200 μg ml⁻¹ EDTA in Hank’s Balanced Salt Solution).

To evaluate the potential cytotoxic effect of grade 4 and 5 Ti particles on HGFs, two groups of test samples were prepared for cell culture, the particles in media and the dissolution products of the particles. Ti particles were sterilised with 70% ethanol for 1 min before use. Group 1 (dissolution): grade 4 and 5 Ti particles were suspended in DMEM at concentrations of 0.75, 1.5 and 3 mg ml⁻¹ in airtight polyethylene containers, which were placed on an orbital shaker rotating at 120 rpm. inside a 37 °C incubator for 72 h.