Although long noncoding RNAs (lncRNAs) have been emerging as critical regulators in various tissuesand biological processes, little is known about their expression and regulation during the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in inflammatory microenvironment. In this study, we have identified 63 lncRNAs that are not annotated in previous database. These novel lncRNAs were not randomly located in the genome but preferentially located near protein-coding genes related to particular functions and diseases, such as stem cell maintenance and differentiation, development disorders and inflammatory diseases. Moreover, we have identified 650 differentially expressed lncRNAs among different subsets of PDLSCs. Pathway enrichment analysis for neighboring proteincoding genes of these differentially expressed lncRNAs revealed stem cell differentiation related functions. Many of these differentially expressed lncRNAs function as competing endogenous RNAs that regulate protein-coding transcripts through competing shared miRNAs.Periodontal diseases, such as periodontitis, are the most common causes of tooth loss 1 . Periodontal regeneration via the application of stem cells is the future direction of therapy for periodontal diseases. To date, several types of stem cells, including mesenchymal stem cells (MSCs), embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have been investigated for periodontal regeneration. MSCs are gaining more appreciation for their application in periodontal regeneration because they are not subject to ethical issues 2-5 . MSCs have the potential to be involved in multiple types of differentiation, including neurogenic, cardiomyogenic, chondrogenic and osteogenic differentiation. Periodontal ligament stem cells (PDLSCs) are a type of MSC. PDLSCs are more suited for maintenance and regeneration of periodontal tissues, compared to other mesenchymal stem cells, such as bone marrow stromal cells (BMSCs) 3,6 . Several factors have been shown to regulate the potency of PDLSCs, including tissue origin 7 , age of the donor 8 , inflammatory condition 9 and growth factors 10 . Increasing evidence shows that inflammation hampers the osteogenic differentiation potency of PDLSCs 9,11 . Therefore, determining the molecular mechanism of osteogenic differentiation of PDLSCs in the inflammatory microenvironment is critical for tooth regeneration.

Proinflammatory responses are important aspects of the immune response to biomaterials, which may cause peri-implantitis and implant shedding. The purpose of this study was to test the cytotoxicity and proinflammatory effects of dicalcium silicate particles on RAW 264.7 macrophages and to investigate the proinflammatory response mechanism induced by C2S and tricalcium phosphate (TCP). C2S and TCP particles were characterized using scanning electron microscopy (SEM), energy spectrum analysis (EDS) and X-ray diffraction (XRD). Cytotoxicity and apoptosis assays with C2S and TCP in the murine RAW 264.7 cell line were tested using the cell counting kit-8 (CCK-8) assay and flow cytometry (FCM). The detection results showed that C2S and TCP particles had no obvious toxicity in RAW 264.7 cells and did not cause obvious apoptosis, although they both caused an oxidative stress response by producing ROS when the concentrations were at 100 μg/mL. C2S particles are likely to induce a proinflammatory response by inducing high TLR2, TNF-α mRNA, TNF-α proinflammatory cytokine, p-IκB, and p-JNK1 + JNK2 + JNK3 expression levels. When we added siRNA-TLR2-1, a significant reduction was observed. These findings support the theory that C2S particles induce proinflammatory responses through the TLR2-mediated NF-κB and JNK pathways in the murine RAW 264.7 macrophage cell line.