Effect of Mg particles on MC3T3-E1 and J774 cellular cycle. Influence of fluoride treatment

ALVAREZ F; R. LOZANO PUERTO; B.T. PEREZ MACEDA; C.A. GRILLO; M. FERNÁNDEZ LORENZO

Maratea

Simposio; 6th Simposium on Biodegradable Metals; 2014

INTRODUCTION: Mg-based materials are promising for orthopedic, dental, and cardiovascular applications [1]. After Mg implantation its high degradation rate causes the release of microscopic debris particles (MgP) and metal ions. Protective treatments involving the generation of magnesium fluoride conversion coatings (Mg-F-C) have been proposed [2]. The aim of this work was to evaluate the possible cytotoxic effects of MgP, and with and without F-C on macrophage (J774) and osteoblast (MC3T3-E1) cell lines. METHODS: MgP (99.8%, 325 mesh, 58.9 µm ± 20.7 µm.) were supplied by Alfa Aesar and were used to simulate Mg debris. Fluoride layers (Mg-F-C) were generated by immersion of 1000 µg/ml of MgP into 0.1M KF (MgP01) or 0.2M KF (MgP02) for 1h at room temperature. After the immersion period, KF-treated MgP were suspended in DMEM and subsequently added to cell cultures. The J774 and the MC3T3-E1 both from Mus musculus origin, were used to test the immune cell responses to the particles. The effect of MgP, MgP01 and MgP02 and their degradation products on cell cycle and cellular viability were analyzed by flow cytometry (FC, Cell Sorter FACS Vantage Becton Dickinson) using propidium iodide and NP40. The presence of F-L on the MgP01 and MgP02 was detected by Maldi-TOF analysis. RESULTS: Cellular viability rate and percentages included in the different cell cycle phases were evaluated in presence of 1000 µg/ml of MgP, MgP01 and MgP02 by FC (Table 1). Machophages were more affected than MC3T3-E1 cells by the three types of particles. J774 viability decreased considerably in all cases. The percentage of SubG1 phase, which is apoptosis-related, increased 5.1% and 11.6% in presence of MgP01 and MgP02, respectively while synthesis phase (S) decreased 18.3% and 12.4% with respect to MgP value. Viability of MC3T3-E1 cells was not affected by MgP but by MgP01 and by MgP02. Table I. Viability and percentages of MC3T3-E1 and J774 cells included in the different cell cycle phases. DISCUSSION  CONCLUSIONS: Mg-F-C temporally decrease corrosion rate of Mg [2]. Reduction of corrosion rate implies the decrease in the release of ions and may increase biocompatibility. Notwithstanding this, in a previous report with J774 cells, higher effect on LDH assays was detected for MgP01 than for MgP for ≥1000 µg/ml. Coincidently, present results showed that Mg-F-C affect viability of both cell lines assayed (MC3T3-E1 and J774). Macrophages seem to be more sensitive to MgP, MgP01 and MgP02 (1000 µg/ml) than MC3T3-E1. The presence of a Mg-F-C on MgP01 and MgP02 also affects the phases of the J774 cell cycle severally. REFERENCES: 1 F. Witte, N. Hort, C.Vogt, S Cohen, K. Ulrich, R. Willurmeit, F. Feyerabend (2008) Degradable biomaterials based on magnesium corrosion, Current Opinion in Solid State and Mat. Science 12:63-72.2 D. Pereda, C. Alonso, L. Burgos, J.A del Valle, O. Ruano, P. Perez, M. Férnandez Lorenzo (2009) Corrosion inhibition of powder metallurgy Mg by fluoride treatments, Acta Biomaterialia 6:1772-1782. ACKNOWLEDGEMENTS:Authors acknowledge the financial support of UNLP, CONICET and ANPCyT.