Effects of canine bone marrow stromal cells after different stages of mineralization on the differentiation, proliferation of canine periodontal ligament stem cells

To investigate the inductive effects of canine periodontal ligament stem cells (PDLSCs) cocultured with canine disparate differentiating-period iliac bone marrow stromal cells (I-BMSCs).

Purified PDLSCs were isolated by in vitro culture of cBMSCs and flow cytometry. Third-generation PDLSCs were obtained, and conditioned culture medium derived fromiliac bone marrow stromal cells (I-BMSCs-CM) was added as indicated for coculture of PDLSCs. As the control group, pure uninduced PDLSCs were routinely cultured in DMEM culture medium containing 15%FBS. The experimental groups included the I-BMSCs-CM, I-BMSCs-CM-10ds and I-BMSCs-CM-15ds groups. The I-BMSCs-CM group consisted of PDLSCs induced by I-BMSCs, the I-BMSCs-CM-10ds group consisted of PDLSCs induced by I-BMSCs after osteogenic induction for 10 days, and the I-BMSCs-CM-15ds group consisted of PDLSCs induced by I-BMSCs after osteogenic induction for 15 days. The cocultured PDLSCs were examined via the MTT assay. Total mRNA and protein were prepared at 3 and 7 days. The mRNA expression levels of runt-related transcription factor 2(Runx2), special AT-rich sequence binding protein 2(Satb2) and osteocalcin (OCN) were measured by qRT - PCR. The protein expression levels of Satb2, Runx2 and OCN were detected by Western blot.

The PDLSCs showed a spindle-like morphology. While the BMSC-conditioned media increased PDLSCs proliferation, the media conditioned by BMSCs allowed to differentiate for 15 days (I-BMSCs-CM-15days) significantly enhanced PDLSCs proliferation (F＝342.8, P＝0.017). The expression levels of the analyzed genes were upregulated in the coculture groups, and the protein expression levels of Satb2, Runx2 and OCN were higher in the test groups than in the control group at 7 days. At the protein level, I-BMSCs-CM-15days upregulated the expression of Satb2 by 3.04-fold (F_Satb2＝ 24.48, P＝0.014), Runx2 by 5.1-fold (F_Runx2＝12.25, P < 0.001), and OCN by 3.67-fold (F_OCN＝18.35, P＝0.022).

The conditioned medium of I-BMSCs may enhance the proliferation of PDLSCs, and that of terminally differentiated bone cells probably triggered the osteogenesis of PDLSCs, suggesting important implications for periodontal engineering.