PDLSCs exhibit ability of self-renew and show the potentiality to differentiate into ipocytes, osteo/cementoblast-like cells and collagen-forming cells. However, differentiation of the stem cells may be triggered by specific signals provided by the local environment [
17]. During tooth development, after the completion of crown formation, the apical mesenchyme develops the periodontium as well as the inner and outer enamel epithelia fuse under the level of the crown cervical margin to produce a bilayered epithelial sheath termed Hertwig's epithelial root sheath (HERS).It has been reported that epithelial–mesenchymal interactions which take place between Hertwig’s sepithelial root sheath and underlying root mesenchyme seem to play an important role in root/periodontal tissue development [
18]. Primary APTGs were heterogeneous, containing both cobblestone-like epithelial and spindle-shaped mesenchymal cell types, which provide multiple molecular signals or growth factors necessary for PDLSCs proliferation and differentiation. In this study, primary cultures of human PDLSCs from 4 individual donors was established and their osteo-differentiation potential were confirmed by co-culture with APTG-CM. And then we determined Osteogenic differentiation by mineralization in vitro, osteoblast marker gene expression, and alkaline phosphatase activity. And it was evidenced that APTG-CM-induced PDLSCs exhibited significantly increased calcified nodule formation and enhanced osteogenic gene expression and high ALP activity. The expression of osteogenic differentiation marker genes ALP and BSP was significantly up-regulated, and the expression of PLAP1, which is a negative regulator of periodontal ligament mineralization, was remarkably down-regulated. In contrast, non-induced PDLSCs exhibited low ALP activity and reduced mineralization formation. The results mentioned above showed that PDLSCs had the potentiality to differentiate into osteoblast with APTG-CM induction and exhibited stem cell characteristics similar to that of normal human osteoblast-like cells.
MicroRNAs (miRNAs) are an integral part of this regulatory network with essential roles in pluripotent maintenance, proliferation and differentiation. The role of miRNA in osteogenic differentiation of MSCs has been indicated by several studies [
19,
20]. However, few studies investigate the mechanism of miRNA in osteoblast differentiation of PDLSCs. It has been reported that ibandronate promote the proliferation of PDLSCs and enhance the expression of alkaline phosphatase (ALP), type I collagen (COL-1), osteoprotegerin (OPG), osteocalcin (OCN), and Runx2. The expression of miRNAs, including miR-18a, miR-133a, miR-141 and miR-19a, was significantly altered in the PDLSCs cultured with ibandronate [
21]. Some results indicate that the 3D granules, in contact with hPDLSCs, showed not only osteoconductive properties, evaluated through the adhesion and proliferation process, but also the ability to stimulate VEGF secretion in hPDLSCs via miR-210 involvement. And miR- 2861, are involved in osteogenic differentiation and open a new scenario in the study of biomaterial performance [
22,
23]. In this study, we applied miRNA sequencing to investigate the profile of miRNA expression in APTG-CM-induced and non-induced PDLSCs from 3 individual donors. Eleven miRNAs were significantly expressed in PDLSCs co-cultured with APTG-CM, and then miR-146a-5p was identified by computational miRNA target prediction analyses, which was up-regulated during miRNA expression profile. Using RT-qPCR, we validated the expression of miR-146a-5p, and the results of the expression level of miR-146a-5p were highly consistent with the results of miRNA sequencing. MiR-146a-5p differed from those highly expressed in human multipotent mesenchymal stromal cells (MSCs), such as hsa-miR-30c, hsa-miR-15b, and hsa-miR-130b, and the pattern of its expression in PDLSCs also differed from MSCs during osteogenic differentiation [
24,
25]. Several previous studies have showed that miR-146a play a key regulatory role in MSC differentiation. They showed that miR-146a were significantly up-regulated in neuronally differentiated bone marrow-derived mesenchymal stem cells (BMSCs) [
26]. Expression levels of miR-146a were dynamically changed during differentiation of hESCs to CD34
+ hematopoietic cells, and in subsequent differentiation of the CD34
+ cells into the erythroid lineage [
27]. They also showed that miR-146a was up-regulated in differentiation of PDL cells with the help of ascorbic acid treatment and promoted the differentiation in PDL cells through the down-regulation of NF-kβ signaling [
14]. The present study showed that the expression of miR-146a-5p was up-regulated in PDLSCs co-cultured with APTG-CM, and miRNA sequencing results were validated by RT-qPCR, which demonstrated that miR-146a-5p was involved in the promotion effect of APTG-CM on osteoblastic differentiation of PDLSCs. Our results suggested that miR-146a-5p might be a novel way in deciding the direction of PDLSCs differentiation.