<p>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</p>

Abstract

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 2 thin films grown by Atomic Layer Deposition (ALD) technology (ZrO 2 ALD). Methodology: The cytocompatibility of the obtained layers was analysed using the mice preosteoblastic 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 2 ALD 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 ZrO 2 ALD coatings, the up-regulation of genes essential for bone metabolism was noted. Discussion: The data obtained indicate that ZrO 2 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 2 ALD layers is essential in terms of their potential clinical application in bone regenerative medicine.

Novel Nanohydroxyapatite (nHAp)-Based Scaffold Doped with Iron Oxide Nanoparticles (IO), Functionalized with Small Non-Coding RNA (miR-21/124) Modulates Expression of Runt-Related Transcriptional Factor 2 and Osteopontin, Promoting Regeneration of Osteoporotic Bone in Bilateral Cranial Defects in a Senescence-Accelerated Mouse Model (SAM/P6). PART 2

“…Detailed protocols for trypsinisation of pre-osteoblasts and osteoclast precursors were established and published previously. 7,25,26…”

Bone marrow stromal cells (BMSCs CD45‐/CD44+/CD73+/CD90+) isolated from osteoporotic mice SAM/P6 as a novel model for osteoporosis investigation

“…The gating procedure of cells’ populations was based on the positive and negative controls. 32 , 33 , 34 …”

Investigations of Structural and Electrical Properties of ALD Films Formed with the Ozone Precursor

“…According to the type of films, the used oxidants were deionized water (Millipore Milli-Q) or ozone received from the generator mentioned above. As previously reported, all processes were performed under vacuum, below 0.5 Torr [ 24 , 26 ]. Several ALD cycles were optimized to deposition of 10 nm and 20 nm films thick in the case of alumina and 20 nm and 50 nm films thick in the case of hafnia.…”