Bone Regeneration by pDNA Encoding MicroRNA-200c in Rat Critical-Size Defect

Objectives: Defects in oral and craniofacial bone tissues resulting from trauma, congenital abnormalities, or cancer resection are major medical concerns. Gene therapy approach to bone and periodontal tissue engineering has become one of the most promising therapeutic technologies for bone regeneration. Our previous studies have reported that plasmid DNA encoding microRNA (miR)-200c delivered by polyethylenimine (PEI) enhanced osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs).
The objective of this study is to evaluate the effect of naked plasmid DNA encoding miR-200c in promoting osteogenic differentiation of hBMSCs and bone regeneration in critical-size calvarial defects of rats.
Methods: We evaluated the expression level of miR-200c, viability, and proliferation of hBMSCs after plasmid DNA encoding miR-200c was transfected. The biomarkers of osteogenic differentiation in the hBMSCs were quantitated using qPCR and ELISA up to 28 days. In addition, we implanted collagen matrix incorporating plasmid DNA encoding miR-200c (10 μg/implant) into the created nonhealing calvarial defects (8 mm-in-diameter) of 12 week-old rats. The bone regeneration in calvarial defects was analyzed using µCT and histology after 6 weeks.
Results: miR-200c expression was significantly increased in transfected hBMSCs in a dose-dependent manner. The mRNA of RUNX-2, ALP, and OCN were up-regulated in the hBMSCs with miR-200c overexpression. miR-200c significantly increased RUNX-2 and OCN secretion and calcium deposition in hBMSCs after 4 weeks. Additionally, the in vivo transfection of miR-200c by the naked plasmid DNA was evidenced by miR-200c overexpression in the scaffolds 1 week after implantation. The µCT and histology analysis revealed that miR-200c-loaded collagen scaffolds had significantly stronger osteogenic capability than controls.

Conclusions: Plasmid DNA encoding miR-200c promoted the osteogenic differentiation of hBMSCs and bone regeneration in a rat model, which indicating it may potentially be used to restore craniofacial and periodontal bone defects.