Zirconium Oxide Thin Films Obtained by Atomic Layer Deposition Technology Abolish the Anti-Osteogenic Effect Resulting from miR-21 Inhibition in the Pre-Osteoblastic MC3T3 Cell Line

Abstract

Aleksandra Seweryn,1 Ariadna Pielok,2 Krystyna Lawniczak-Jablonska,1 Rafal Pietruszka,1 Klaudia Marcinkowska,2 Mateusz Sikora,2 Bartlomiej S Witkowski,1 Marek Godlewski,1 Krzysztof Marycz,2,3 Agnieszka Smieszek2 1Institute of Physics, Polish Academy of Sciences, Warsaw PL-02668, Poland; 2Wroclaw University of Environmental and Life Sciences, Department of Experimental Biology, Wroclaw PL-50375, Poland; 3Cardinal Stefan Wyszynski University, Collegium Medicum, Warsaw PL-01938, PolandCorrespondence: Aleksandra SewerynPolish Academy of Sciences, Aleja Lotnikow 32/46, Warsaw PL-02668, PolandTel [phone]Fax [phone]Email [email protected] SmieszekWroclaw University of Environmental and Life Sciences, Norwida St. 27 B, Wroclaw PL-50-375, PolandTel [phone]Fax [phone]Email [email protected]: The development of the field of biomaterials engineering is rapid. Various bioactive coatings are created to improve the biocompatibility of substrates used for bone regeneration, which includes formulation of thin zirconia coatings with pro-osteogenic properties. The aim of this study was to assess the biological properties of ZrO2 thin films grown by Atomic Layer Deposition (ALD) technology (ZrO2ALD).Methodology: The cytocompatibility of the obtained layers was analysed using the mice pre-osteoblastic cell line (MC3T3) characterized by decreased expression of microRNA 21-5p (miR-21-5p) in order to evaluate the potential pro-osteogenic properties of the coatings. The in vitro experiments were designed to determine the effect of ZrO2ALD coatings on cell morphology (confocal microscope), proliferative activity (cell cycle analysis) and metabolism, reflected by mitochondrial membrane potential (cytometric-based measurement). Additionally, the influence of layers on the expression of genes associated with cell survival and osteogenesis was studied using RT-qPCR. The following genes were investigated: B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), p53 and p21, as well as osteogenic markers, i.e. collagen type 1 (Coll-1), osteopontin (Opn), osteocalcin (Ocl) and runt-related transcription factor 2 (Runx-2). The levels of microRNA (miRNA/miR) involved in the regulation of osteogenic genes were determined, including miR-7, miR-21, miR-124 and miR-223.Results: The analysis revealed that the obtained coatings are cytocompatible and may increase the metabolism of pre-osteoblast, which was correlated with increased mitochondrial membrane potential and extensive development of the mitochondrial network. The obtained coatings affected the viability and proliferative status of cells, reducing the population of actively dividing cells. However, in cultures propagated on ZrO2ALD coatings, the up-regulation of genes essential for bone metabolism was noted.Discussion: The data obtained indicate that ZrO2 coatings created using the ALD method may have pro-osteogenic properties and may improve the metabolism of bone precursor cells. Given the above, further development of ZrO2ALD layers is essential in terms of their potential clinical application in bone regenerative medicine.Keywords: ALD, zirconia-based coatings, pro-osteogenic properties, cytocompatibility, osteoblasts precursors

Keywords

• ald
• zirconia-based coatings
• pro-osteogenic properties
• cytocompatibility
• osteoblasts precursors