Nanohydroxyapatite (nHAp) Doped with Iron Oxide Nanoparticles (IO), miR-21 and miR-124 Under Magnetic Field Conditions Modulates Osteoblast Viability, Reduces Inflammation and Inhibits the Growth of Osteoclast – A Novel Concept for Osteoporosis Treatment: Part 1
Krzysztof Marycz, Agnieszka Śmieszek, Klaudia Marcinkowska, Mateusz Sikora, Eliza Turlej, Paulina Sobierajska, Adrian Patej, Alina Bieńko, Rafał J. Wiglusz
Abstract
Purpose: Osteoporosis results in a severe decrease in the life quality of many people worldwide. The latest data shows that the number of osteoporotic fractures is becoming an increasing international health service problem. Therefore, a new kind of controllable treatment methods for osteoporotic fractures is extensively desired. For that reason, we have manufactured and evaluated nanohydroxyapatite (nHAp)-based composite co-doped with iron oxide (IO) nanoparticles. The biomaterial was used as a matrix for the controlled delivery of miR-21-5p and miR-124-3p, which have a proven impact on bone cell metabolism. Methods: The nanocomposite Ca 5 (PO 4 ) 3 OH/Fe 3 O 4 (later called nHAp/IO) was obtained by the wet chemistry method and functionalised with microRNAs (nHAp/ [email protected] /124). Its physicochemical characterization was performed using XRPD, FT-IR, SEM-EDS and HRTEM and SAED methods. The modulatory effect of the composite was tested in vitro using murine pre-osteoblasts MC3T3-E1 and pre-osteoclasts 4B12. Moreover, the anti-inflammatory effects of biomaterial were analysed using a model of LPS-treated murine macrophages RAW 264.7. We have analysed the cells’ viability, mitochondria membrane potential and oxidative stress under magnetic field (MF+) and without (MF-). Moreover, the results were supplemented with RT-qPCR and Western blot assays to evaluate the expression profile for master regulators of bone metabolism. Results: The results indicated pro-osteogenic effects of nHAp/ [email protected] /124 composite enhanced by exposure to MF. The enhanced osteogenesis guided by nHAp/ [email protected] /124 presence was associated with increased metabolism of progenitor cells and activation of osteogenic markers ( Runx-2, Opn, Coll-1 ). Simultaneously, nanocomposite decreased metabolism and differentiation of pre-osteoclastic 4B12 cells accompanied by reduced expression of CaII and Ctsk . Obtained composite regulated viability of bone progenitor cells and showed immunomodulatory properties inhibiting the expression of inflammatory markers, ie, TNF-α, iNOs or IL-1β , in LPS-stimulated RAW 264.7 cells. Conclusion: We have described for the first time a new concept of osteoporosis treatment based on nHAp/ [email protected] /124 application. Obtained results indicated that fabricated nanocomposite might impact proper regeneration of osteoporotic bone, restoring the balance between osteoblasts and osteoclast. Keywords: nanocomposites, hydroxyapatite, osteoblasts, osteoclasts, osteoporosis