gms | German Medical Science

Objectives: Regeneration of large bone defects is a major problem in trauma surgery and orthopedics. The gold standard for their treatment is autologous bone, taken from the iliac crest. However, this procedure is fraught with donor site complications. Tissue engineering approaches using cells with osteoinductive potential might circumvent those limitations. Bone marrow mononuclear cells (BMC) can be harvested and reintroduced to the patient within hours and were effective in animal models of bone healing. MicroRNAs (MiR) comprise a class of small noncoding RNAs with a mean length of 19-25 nucleotides. MiRs regulate gene expression by binding to their target mRNAs resulting in translational repression or mRNA degradation. MiR-92A influences key angiogenic processes negatively and MiR-335 impairs osteogenic differentiation. Hence, the aim is to evaluate, if the neutralization of MiR-92A (vascularization) and MiR-335 (osteogenic differentiation) in BMC using specific antagoMiRs leads to a further improvement of the BMC supported therapy of large bone defects.

Methods: A total of 96 male SD-rats, 250g weight, were allocated to 8 treatment groups. Syngenic BMC from donor rats were transiently transfected by lipofection with antagoMiR-92A, -335, -92A and -355 or control antagoMiR and placed on a beta-TCP scaffold into the femoral bone defect (5 mm) of male SD-rats. Animals were sacrificed after 1 week for assessment of target gene expression (VEGF, ITGA5, RUNX2) and after 8 weeks for assessment of bone mineral density (BMD) and biomechanical stability. Kruskal-Wallis-test with Bonferroni-Holm post hoc analysis was used for statististical evaluation.

Results and Conclusion: A transfection rate between 32% - 37% of total BMC was gained. Differences of target gene expression (VEGF, ITGA5, RUNX2) between treatment groups were not seen at one week after surgery. Significantly increased BMD were found in antagoMiR-92A group in comparison to the antagoMiR-335 group at eight weeks after surgery. New bone formation was seen in all groups but osseus integration of the beta-TCP-scaffolds as well as occurrence of bony bridges were most prominent in the antagoMiR-335 group respectively in the combination group (antagoMiR-92A and -335). Concomitantly we found a significantly increased biomechanical stability of the bone defect in animals treated with BMC transfected with antagoMiR-335 and in trend in animals treated with BMC transfected with antagomir-92A and -335 in comparison to control.

Discussion: We demonstrated that the targeted inhibition of impairing vascularisation and osteogenic differentiation MiRs is a new approach that improves BMC supported therapy of large bone defects. Further improvement might be gained through neutralisation of additional MiRs that were involved in key processes of bone healing.