Fig 2: Inhibition of miR-181a-5p suppressed osteogenic differentiation. (A) miR-181a-5p expression in the scramble and inhibitor groups treated with saline or osteogenesis induction medium. (B, C) ALP and ARS staining of the scramble and inhibitor groups treated with saline or osteogenesis induction medium. (D, E) Transcriptional levels of ALP and RUNX2 by polymerase chain reaction (PCR). (F, G) ALP, RUNX2 and beta-tubulin are determined and quantified by densitometric evaluation of western blots, further normalized to beta-tubulin. All data represent mean ± s.e.m. (n = 6). *P<0.05 compared with the scramble group treated with saline. #P<0.05 compared with the scramble group induced for differentiation.

Fig 3: Periostin secreted by IL-4 treated nasal fibroblasts mediates the osteogenesis of MG63 cells. (A) Schematic overview of the conditioned medium preparation and MG63 osteogenic differentiation assay. HNFs were stimulated with IL-4 (5 ng/mL) for 2 days, and the conditioned medium was concentrated using a 30 kDa filter. Periostin concentrations were quantified by ELISA, and the periostin-containing medium was designated as CM-HNF. MG63 cells were cultured in osteogenic medium supplemented with CM-HNF, CM-HNF pretreated with a periostin-neutralizing antibody (CM-HNF + Ab), or recombinant human periostin (rhPOSTN, 200 ng/mL) for 10 days to induce osteogenic differentiation. (B) Representative photomicrographs of ALP staining showing osteoblast differentiation of MG63 cells induced by the osteogenic medium (scale bars = 500 μm). (C) Quantification of ALP-positive cells by counting the number of cells per high-power field in each well (n = 6, pooled from two independent experiments with three wells each). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test. (D) Quantitative RT-PCR analysis of ALP and OCN mRNA expression in MG63 cells under each treatment condition, with values normalized to those of the control group. Experiments were independently repeated three times. Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test. Results represent the mean ± SEM. Exact P values are shown in the graph. Ab, antibody; ALP, alkaline phosphatase; rhPOSTN, recombinant human periostin; ELISA, enzyme-linked immunosorbent assay; HNF, human nasal fibroblast; IL, interleukin; OCN, osteocalcin; SEM, standard error of the mean.