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.

Introduction: 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 ZrO thin films grown by Atomic Layer Deposition (ALD) technology (ZrO ).

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 ZrO 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-associated X protein (), and , as well as osteogenic markers, i.e. collagen type 1 (), osteopontin (), osteocalcin () and runt-related transcription factor 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 ZrO coatings, the up-regulation of genes essential for bone metabolism was noted.

Discussion: The data obtained indicate that ZrO 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 ZrO layers is essential in terms of their potential clinical application in bone regenerative medicine.