To investigate the osteogenic properties of maxillary sinus membrane stem cells (MSMSCs).

Beagle maxillary sinus mucosa was collected, immunomagnetic bead method was applied for isolation of CD146⁺ cells, and MSMSCs were harvested and cultured from the canine maxillary sinus floor mucosa. The levels of the cell surface antigens CD44, CD146, and CD34 were determined at passage one by flow cytometry. Cells at passage one were cultured in basal medium and osteogenic inductive medium. Real-time PCR, immunohistochemical staining, alkaline phosphatase activity, alizarin red staining and Von Kossa staining were used to investigate the osteogenic properties in vitro.

The canine MSMSCs were cultured successfully. The results of flow cytometry were positive for CD146 and CD44 expression but negative for CD34 expression. The relative mRNA expression of runt-related transcription factor 2 (RUNX2) (t = 14.44, P < 0.001), osteopontin (OPN) (t = 7.85, P = 0.001) and alkaline phosphatase alkaline phosphatase (t = 14.27, P < 0.001) was apparently higher in the osteoinductive medium group than in the basal medium group, the differences in relative mRNA expression between the groups were significant. The protein levels of RUNX2 and OPN increased in the osteoinductive medium group. The alkaline phosphatase activity of the MSMSCs increased when the cells were cultured in osteoinductive medium; the activity increased to a level that was significantly higher than that in basal medium, particularly at days 3 (t = 8.79, P < 0.001), 7 (t = 9.75, P < 0.001), 14 (t = 12.14, P < 0.001), 21 (t = 19.62, P < 0.001) and 28 (t = 17.53, P < 0.001). Obvious mineralized nodules were observed by alizarin red staining or Von Kossa staining.

Maxillary sinus membrane stem cells exhibit osteogenic ability.

To detect the expression level of miR-27a during the osteogenic differentiation of beagle maxillary sinus membrane stem cells (MSMSCs) and explore the role of miR- 27a in the osteogenic differentiation of MSMSCs.

Beagle MSMSCs were cultured in vitro. The expression level of miR-27a was detected via RT-PCR after an osteogenic inductive culture was prepared. The mRNA expression levels of Runx2 and OPN were examined via RT-PCR, and the protein expression levels of Runx2 and OPN were examined via Western blot after the cells were transfected with pre-miR-27a or anti-miR-27a. Finally, osteoprogenitor cells transfected with pre-miR-27a were composited with Bio-Oss particles and subcutaneously implanted into nude mice to form ectopic bone formation models, and then the inhibition of bone formation from miR-27a was observed in vivo.

The expression level of miR- 27a in the beagle MSMSCs decreased after osteogenic inductive culturing. The relative miR-27a levels were significantly decreased at day 1 (t＝3.795, P＝0.023), day 3 (t＝4.493, P＝0.011), day 7 (t＝11.591, P < 0.001), day 14 (t＝ 12.542, P < 0.001), and day 21 (t＝5.621, P＝0.008) compared with day 0. In addition, the expression levels of Runx2 mRNA (t＝4.923, P＝0.007) and protein (t＝4.425, P＝0.008) were reduced after the cells were transfected with premiR-27a. The expression levels OPN mRNA (t＝5.253, P＝0.006) and protein (t＝5.132, P＝0.006) were also reduced. In contrast, the mRNA expression levels of Runx2 (t＝3.925, P＝0.013) and OPN (t＝3.712, P＝0.019) were increased after the cells were transfected with anti-miR-27a, and bone formation was observed after the subcutaneous implantation of beagle MSMSCs composited with Bio-Oss in nude mice. Nevertheless, ectopic bone formation was inhibited by pre-miR-27a-transfected beagle MSMSCs composited with Bio-Oss (t＝7.219, P＝0.0020).

MiR- 27a negatively regulates the osteogenic differentiation of MSMSCs.