Date Published: July 19, 2019
Publisher: Public Library of Science
Author(s): Marcel Orth, Claudia Scheuer, Christina Backes, Andreas Keller, Mika F. Rollmann, Benedikt J. Braun, Nicole Ludwig, Eckart Meese, Tim Pohlemann, Matthias W. Laschke, Michael D. Menger, Tina Histing, Xing-Ming Shi.
Although cellular and molecular mechanisms during the course of bone healing have been thoroughly investigated, the regulation of gene expression by microRNA during bone regeneration is still poorly understood. We hypothesized that nonunion formation is associated with different microRNA expression patterns and that target proteins of these microRNAs are differently expressed in callus tissue of nonunions compared to physiologically healing bones. In a well-established femoral osteotomy model in CD-1 mice osteotomies were induced which result either in healing or in nonunion formation. MicroRNA and target protein expression was evaluated by microarray, quantitative real-time polymerase chain reaction (qrt-PCR) and Western blot. Microarray analyses demonstrated 44 microRNAs to be relevant for nonunion formation compared to physiological bone healing. In nonunions qrt-PCR could validate a higher expression of microRNA-140-3p and microRNA-140-5p. This was associated with a reduced expression of Dnpep and stromal cell-derived factor (SDF)-1α, which are both known to be target proteins of microRNA-140 and also to be involved in the process of bone healing. These data suggest that an increased expression of microRNA-140-3p and microRNA-140-5p markedly contributes to the development of nonunions, most probably by affecting bone morphogenetic protein (BMP)-2 function during the early stage of healing due to a reduced SDF-1α expression.
Approximately 5–10% of all fractures show delayed bone healing or nonunion formation . Bone healing is a highly complex and well-orchestrated process, which involves numerous regulating factors. It is dependent on the interaction of bone marrow-derived hematopoietic and immune cells with endothelial and skeletal progenitor cells from the blood stream and the surrounding tissues . By means of in vivo and in vitro experiments, considerable efforts have been made to understand the cellular and molecular mechanisms, which contribute to the process of bone healing [2,3]. Accordingly, the knowledge on distinct cellular and biochemical pathways during bone healing has markedly increased during the last two decades. In contrast, the regulation of gene expression during bone regeneration is still poorly understood.
The present study indicates different expression patterns of miRs in callus tissue of nonunions compared to physiologically healing bones after osteotomy in mice. A total of 44 miRs were identified, which may be relevant for the bone healing process in mice. Nonunions revealed a higher expression level of mmu-miR-140-3p and mmu-miR-140-5p. This was associated with lower expression levels of SDF-1α and Dnpep, which are known to be target proteins of miR-140. These novel data suggest that the expression of mmu-miR-140-3p and mmu-miR-140-5p markedly contributes to the development of nonunions.
In conclusion, the present study demonstrates different miR expression patterns for nonunions compared to physiological bone healing in mice during the early stage of bone healing. The present data further highlight a role of miR-140 in nonunion formation, most likely by inhibiting the inflammatory response and, indirectly, modulating the biological effect of BMP-2. Accordingly, further studies may elucidate whether a manipulation of miR-140 expression is effective in the prevention or treatment of nonunion formation.