Delivery of microRNA-302a-3p by APTES modified hydroxyapatite nanoparticles to promote osteogenic differentiation in vitro

To demonstrate the miRNA delivery by hydroxyapatite nanoparticles modified with APTES (HA-NPs-APTES) and promote osteogenic gene expression. Materials and methods Osteosarcoma cells (HOS, MG-63) and primary human mandibular osteoblasts (HmOBs) were co-cultured with HA-NPs-APTES conjugated with miRNA-302a-3p. Resazurin reduction assay was performed to evaluate HA-NPs-APTES biocompatibility. Intracellular uptake was demonstrated by confocal fluorescent and scanning electron microscopy. The miRNA-302a-3p and its mRNA targets expression levels including COUP-TFII and other osteogenic genes were assessed by qPCR on day1 or day5 post-delivery. Calcium deposition induced by the osteogenic gene upregulation was shown by alizarin red staining on day7 and 14 post-delivery. Results Proliferation of HOS cells treated with HA-NPs-APTES was similar to that of untreated cells. HA-NPs-APTES was visualized in cell cytoplasm within 24 hours. MiRNA-302a-3p level was upregulated in HOS, MG-63 and HmOBs as compared to untreated cells. As a result, COUP-TFII mRNA expression was reduced, followed by an increase of RUNX2 and other osteogenic genes mRNA expression. Calcium deposition induced by HA-NPs-APTES-miR-302a-3p in HmOBs was significantly higher than in untreated cells. Conclusion HA-NPs-APTES may support the delivery of miRNA-302a-3p into bone cells, as assessed by osteogenic gene expression and differentiation improvement once this combination is used on osteoblast cultures.</p

3D-printed TCP-HA scaffolds delivering MicroRNA-302a-3p improve bone regeneration in a mouse calvarial model

Objective To demonstrate hydroxyapatite nanoparticles modified with cationic functional molecules. 3-aminopropyltriethoxysilane (HA-NPs-APTES) carrying microRNA-302a-3p (miR) in the 3D-printed tricalcium phosphate/Hydroxyapatite (TCP/HA) scaffold can increase healing of the critical-sized bone defect. Materials and methods 3D-printed TCP/HA were modified with HA-NPs-APTES by two methods (M1, M2). The dispersion of particles was visualized by fluorescent microscopy. Biocompatibility of the scaffolds was tested by alizarin assay. Delivery of miR to the cells and osteogenic gene expression were evaluated by qPCR. After selecting best method (M2), scaffolds, scaffolds+HA-NPs-APTES with or without miR were implanted in 4 mm mouse calvarium defect ( n = 4 per group). After 2,4 and 6 weeks, bone regeneration were evaluated by microCT and histology sections. Results Both M1 and M2 scaffolds were biocompatible with cell adhesion on its surface. M2 scaffold showed significant increase of miR, suggesting successful delivery, resulted in downregulation of its target mRNA COUP-TFII, and upregulation of RUNX2 mRNA. Calvarium defect with M2 scaffold also showed significantly higher BV/TV and higher number of filled spaces at all time points. Histomorphometry demonstrated new bone formed at the center of the HA-NPs-APTES-miR scaffold earlier than controls. Conclusion TCP/HA scaffold modified with HA-NPs-APTES facilitated delivery of miR and enhanced bone regeneration.</p