Bone Morphogenetic Protein-9 Enhances Osteogenic Differentiation of Human Periodontal Ligament Stem Cells via the JNK Pathway


Bone morphogenetic protein-9 (BMP9) shows great osteoinductive potential in bone regeneration. Periodontal ligament stem cells (PDLSCs) with multi-differentiation capability and low immunogenicity are increasingly used as seed cells for periodontal regenerative therapies. In the present study, we investigated the potent osteogenic activity of BMP9 on human PDLSCs (hPDLSCs), in which the c-Jun N-terminal kinase (JNK) pathway is possibly involved. Our results showed that JNK inhibition by the specific inhibitor SP600125 or adenovirus expressing small interfering RNA (siRNA) targeting JNK (AdR-si-JNK) significantly decreased BMP9-induced gene and protein expression of early and late osteogenic markers, such as runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN), in hPDLSCs. We also confirmed the in-vivo positive effect of JNKs on ectopic bone formation induced by hPDLSCs injected into the musculature of athymic nude mice and BMP9 ex vivo gene delivery. For the cellular mechanism, we found that BMP9 activated the phosphorylation of JNKs and Smad2/3, and that JNKs may engage in cross-talk with the Smad2/3 pathway in BMP9-mediated osteogenesis.This study protocol was approved by the Ethical Review Committee for Research on Human Subjects of the Second Military Medical University. In this study, all isolated human premolars for orthodontic purpose were collected with written informed consent from the children’s parents/guardians prior to study participation. This study is in strict accordance with approved guidelines set by the National Institute of Health Office of Human Subjects Research, regulations on the management of medical waste, and the principles expressed in the Declaration of Helsinki.PDLSCs were isolated from a human PDL cell population using CD146-conjugated immunomagnetic microbeads (Fig 1A). The isolated cells formed colonies, which showed radiating or whirlpool-like morphology (Fig 1B). Most cells exhibited a fibroblastic spindle, and a small round or triangular shape, and most cells had abundant cytoplasm and a large nucleus (Fig 1C). IF staining showed that the isolated PDLCs primarily expressed vimentin rather than cytokeratin, which suggests their mesodermal origin (Fig 1D). The stem cell STRO-1+/CD146+ immunophenotype which are expressed by hPDLSCs, was determined by flow cytometry. The mean percentage of the STRO-1+/CD146+ cells in PDLCs populations after immunomagnetic isolation was 27.9% (Fig 1E). In addition, Alizarin red S staining after three weeks of osteo-induction showed that some mineralized nodules were formed in the isolated PDLSCs (Fig 1F). Similarly, Oil Red O-positive lipid droplets were observed in PDLSCs after three weeks of adipogenic induction (Fig 1G). These results confirm the multi-differentiation potential of the isolated PDLSCs, which could then be used for subsequent experiments.(XLSX)This work was supported by grants from the Science and Technology Commission of Shanghai Municipality (No.13411951202 and NO. 09ZR1400200), and the Natural Science Foundation of China (NO. 81170987).
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