Human bone marrow derived mesenchymal stem cells modulation by microanostructured ATZ surfaces treated with femtosecond laser

摘要

Introduction: ATZ (Alumina toughened Zirconia) has been recently studied for orthopedic and dental applications, as a replacement for metals, considering its well documented properties. It is widely accepted that further improvement in implants can only be achieved by endowing biomaterials with improved surface functionality. In this work, we used a femtosecond laser ablation technique to modify ATZ, developing surfaces structured at the micro and nanoscale levels, in a controlled and reproducible manner. The aim was to characterize these newly developed materials and evaluate the cell-surface interaction between Human bone marrow derived mesenchymal stem cells (hMSCs) and these microanostructured surfaces (μATZ) and an untreated control (ATZ). Materials and Methods: A commercial powder of ATZ was used to prepare discs using a uniaxial press. The discs were pre-sintered, polished and sintered at 1500°C. The laser treatments were performed in ambient atmosphere using an Yb:KYW chirped-pulse-regenerative amplification laser system with a radiation wavelength of 1030 nm and a pulse duration of 500 fs. The patterns were produced by the direct writing method, using a beam delivery system based on a Michelson interferometer, as described in Oliveira et all3'. The obtained surfaces were characterized by SEM, EDS and XRD. hMSCs were cultured on both materials for 14 days. Cells metabolic activity, proliferation and morphology were evaluated at days 1,7 and 14 of culture. Focal adhesions were observed at day 1 by CLSM. Results and Discussion: SEM images of the μATZ and ATZ surfaces are displayed in figure 1. EDS analysis detected the same elements in both surfaces - Zr, Al, O and C. The peaks obtained by XRD correspond all to alumina and the tetragonal phase of zirconia, meaning that the laser treatment didn't cause any change to the ZrO2 monodinic phase. In terms of biological characterization, cells metabolic activity increased with time of culture for both surfaces, and at day 14 the μATZ samples exhibited a statistically significant increase. The same results were obtained for cell proliferation by DMA quantification. Cells focal adhesions on the patterned substrate were topographically organized, mainly with the microstructure, and were larger and more mature than on the control, showing a stronger bond to the respective surface. Cells morphology at days 1 and 7 are shown in figure 2. Cells on ATZ were randomly attached while μATZ modulated hMSCs adhesion and proliferation, mainly along the microtopographlc features with some cells oriented according to the nanostmctures. Thus, indicating that the microtextured cues had a higher influence on cells behaviour. Conclusion: While some methods for texturing ceramics have been described, few are capable of modifying hard ceramics surface and pattern it in a completely precise and reproducible manner. Currently, there is a lack of studies evaluating the biological potential of these laser treated ceramic surfaces. With this study we showed that the microanostructures modulate cell attachment and proliferation and improve cell behaviour, in relation to an untreated ATZ surface. The micropattems showed to have a higher impact on cells behaviour. This laser technique allows for surface modifications of implants with complex shapes and with expected long shelf life, showing potential to ensure long term lifespan.