Circulating miRNAs are associated with successful bone regeneration

Julia Frank ^1,2 Carina Kampleitner ^3,4,5 Patrick Heimel ^2,3,4 Gabriele Leinfellner ^2,4 Dominik Hanetseder ^2,4 Simon Sperger ^2,4 Amelie Frischer ^2,4 Barbara Schädl ^2,4 Stefan Tangl ^3,4 Claudia Lindner ^4,6,7 Johanna Gamauf ^4,6 Regina Grillari-Voglauer ^4,6 Heinz Redl ^2,4 Matthias Hackl ^4,8 Johannes Grillari ^2,4,9 Darja Marolt Presen ^10,2,4*

• ¹ Herz Jesu Krankenhaus, Baumgasse, Vienna, Austria
• ² Ludwig Boltzmann Institute for Traumatology The research center in cooperation with AUVA, Vienna, Austria
• ³ University Clinic of Dentistry, Medical University of Vienna, Vienna, Vienna, Austria
• ⁴ Austrian Cluster for Tissue Regeneration, Vienna, Austria
• ⁵ Ludwig Boltzmann Institute for Traumatology, The Research Centre in Cooperation with AUVA, Vienna, Austria
• ⁶ Evercyte (Austria), Vienna, Vienna, Austria
• ⁷ Phoenestra, Vienna, Austria
• ⁸ Tamirna GmbH, Vienna, Austria
• ⁹ Institute of Molecular Biotechnology , Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria
• ¹⁰ Centre for the Technologies of Gene and Cell Therapy, The National Institute of Chemistry, Ljubljana, Slovenia

Introduction: Bone healing is a well-orchestrated process involving various bone cells and signaling pathways, where disruptions can result in delayed or incomplete healing. MicroRNAs (miRNAs) are small non-coding RNAs capable of influencing various cellular processes, including bone remodeling. Due to their biological relevance and stable presence in biofluids, miRNAs may serve as candidates for diagnosis and prognosis of delayed bone healing. The aim of the study was to investigate changes in miRNAs circulating in the blood during the healing of rat calvaria defects as biomarkers of successful bone regeneration. Methods: Standardized calvaria defects were created in 36 Wistar rats with a trephine drill and treated with collagen hydroxyapatite (CHA) scaffolds. The treatment groups included CHA scaffolds only, CHA scaffolds containing a plasmid coding for bone morphogenetic protein 2 (BMP2) and miR-590-5p, CHA scaffolds containing mesenchymal stromal cell-derived extracellular vesicles, and empty defects as a control group. After 1, 4 and 8 weeks of healing, the animals were evaluated by microcomputed tomography (microCT), as well as subjected to histological analyses. Blood was sampled from the tail vein prior to surgeries and after 1, 4, and 8 weeks of healing. miRNAs circulating in the plasma were determined using next generation sequencing. Results: Variability of bone regeneration within the four groups was unexpectedly high and did not result in significant differences between the groups, as indicated by the microCT and histological analyses of the newly formed bone tissue. However, irrespective of the treatment group and regenerative activity, we identified miRNAs with distinct expression patterns of up- and downregulation at different time points. Furthermore, rats with high and low regenerative activity were characterized by distinct circulating miRNA profiles. miR-133-3p was identified as the top up-regulated miRNA and miR-375-3p was identified as the top down- regulated miRNA in animals exhibiting strong regeneration over all time points evaluated. Conclusion: Our study indicates that regardless of the treatment group, success or lack of bone regeneration is associated with a distinct expression pattern of circulating microRNAs. Further research is needed to determine whether their levels in the blood can be used as predictive factors of successful bone regeneration.