Inhibition of miR-222-3p activity promoted osteogenic differentiation of hBMSCs by regulating Smad5-RUNX2 signal axis.

miRNAs are recently found playing important roles in osteogenesis. In this study, we identified that miR-222-3p decreased during osteogenic differentiation of human mesenchymal stem cells (hBMSCs) using Quantitative Real-Time Reverse Transcription PCR (qRT-PCR). Furthermore, we investigated the effect of miR-222-3p on osteogenic differentiation of hBMSCs. Inhibition of miR-222-3p function in hBMSCs using infection of lentiviruses carrying miR-222-3p specific inhibitor promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Whereas, overexpression of miR-222-3p inhibited osteoblast differentiation of hBMSCs in vitro. Moreover, Smad5 and RUNX2, which are the critical transcription factors in osteogenic differentiation, were predicted to be targets of miR-222-3p by bioinformatic analysis. Overexpression of miR-222-3p in hBMSCs significantly suppressed the protein levels of Smad5 and RUNX2, while inhibition of miR-222-3p increased their protein levels. Furthermore, inhibition of miR-222-3p increased phosphorylation of Smad1/5/8, which regulated the expression of osteogenic genes. Our findings suggest that suppression of miR-222-3p activity promoted osteogenic differentiation hBMSCs through regulating Smad5-RUNX2 signaling axis.

Effects of releasing recombinant human growth and differentiation factor-5 from poly(lactic-co-glycolic acid) microspheres for repair of the rat degenerated intervertebral disc.

PURPOSE: The objective of this study was to investigate the therapeutic potential of poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with recombinant human growth and differentiation factor-5 (rhGDF-5) on the disc degeneration induced by needle puncture in a rat caudal disc model. METHODS: The rhGDF-5-loaded PLGA microspheres were prepared by the water-oil-water double-emulsion solvent evaporation method, and release kinetics was determined over 42 days. Rats that underwent 21-G needle puncture at rat tail discs were injected with rhGDF-5/PLGA microspheres at four weeks after needle injury. At eight weeks after the injection, disc height, glycosaminoglycans content, and DNA content of the discs were evaluated. In addition, gene expression analysis of aggrecan, collagen type I, and collagen type II in the rat nucleus pulposus was measured by real-time polymerase chain reaction. Rat discs were also assessed by histology using hematoxylin and eosin stain. RESULTS: Encapsulation of rhGDF-5 in PLGA microspheres guaranteed a sustained release of active rhGDF-5 for more than 42 days. The injection of GDF-5/PLGA microspheres resulted in a statistically significant restoration of disc height (p < 0.01), improvement of sulfated glycosaminoglycan (p < 0.05), DNA content (p < 0.05), and significantly increased mRNA levels of collagen type II (p < 0.01), and the differentiation index (the ratio of collagen type II to collagen type I, p < 0.01). In addition, rhGDF-5/PLGA microspheres treatment also improved histological changes induced by needle puncture. CONCLUSIONS: The results of this study suggest that injection of rhGDF-5 loaded in PLGA microspheres into rat tail discs may be as a promising therapy strategy to regenerate or repair the degenerative disc.