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.

Healing of osteoporotic defects is challenging and requires innovative approaches to elicit molecular mechanisms promoting osteoblasts-osteoclasts coupling and bone homeostasis. Cytocompatibility and biocompatibility of previously characterised nanocomposites, i.e Ca5(PO4)3OH/Fe3O4 (later called nHAp/IO) functionalised with microRNAs (nHAp/IO@miR-21/124) was tested. In vitro studies were performed using a direct co-culture system of MC3T3-E1 pre-osteoblast and 4B12 pre-osteoclasts. The analysis included determination of nanocomposite influence on cultures morphology (confocal imaging), viability and metabolic activity (Alamar Blue assay). Pro-osteogenic signals were identified at mRNA, miRNA and protein level with RT-qPCR, Western blotting and immunocytochemistry. Biocompatibility of biomaterials was tested using bilateral cranial defect performed on a senescence-accelerated mouse model, ie SAM/P6 and Balb/c. The effect of biomaterial on the process of bone healing was monitored using microcomputed tomography. The nanocomposites promoted survival and metabolism of bone cells, as well as enhanced functional differentiation of pre-osteoblasts MC3T3-E1 in co-cultures with pre-osteoclasts. Differentiation of MC3T3-E1 driven by nHAp/IO@miR-21/124 nanocomposite was manifested by improved extracellular matrix differentiation and up-regulation of pro-osteogenic transcripts, ie late osteogenesis markers. The nanocomposite triggered bone healing in a cranial defect model in SAM/P6 mice and was replaced by functional bone in Balb/c mice. This study demonstrates that the novel nanocomposite nHAp/IO can serve as a platform for therapeutic miRNA delivery. Obtained nanocomposite elicit pro-osteogenic signals, decreasing osteoclasts differentiation, simultaneously improving osteoblasts metabolism and their transition toward pre-osteocytes and bone mineralisation. The proposed scaffold can be an effective interface for in situ regeneration of osteoporotic bone, especially in elderly patients.