[The Roles of RUNX1 in the Proliferation and Osteogenic and Adipogenic Differentiation of Dental Pulp Stem Cells].

OBJECTIVE: To investigate the influence of Runt-related transcription factor 1 (RUNX1) on the proliferation, osteogenic differentiation and adipogenic differentiation of dental pulp stem cells (DPSC) in vitro. METHODS: DPSCs were transfected through lentiviral vector carrying the target gene RUNX1 and green fluorescent protein (GFP). After 48 h, transfection efficiency was determined with the fluorescent marking of GFP and Western blot. The effect of the overexpression of RUNX1 on DPSC proliferation and colony formation was determined with CCK-8 and colony formation assay; cell cycle of DPSC was detected by flow cytometry. RUNX1 siRNA was transfected into the DPSCs. After mineralized induction, the effect of RUNX1 overexpression/silencing on the osteogenetic differentiation of DPSC was tested by alkaline phosphatase (ALP) staining and alizarin red staining. After adipogenic induction, oil red O staining was done in order to observe the effect of overexpression/silencing of RUNX1 on the adipogenic differentiation of DPSC. RESULTS: RUNX1 protein was overexpressed in DPSC after lentiviral transfection. Fluorescent test showed successful transfection of lentiviral transfection and over 70% of the cells showed stable expression of GFP protein. The proliferation and colony-formation efficiency of DPSC was enhanced significantly and the proportion of DPSCs in the S phase was significantly increased in the RUNX1-overexpessed group ( P<0.05). ALP activity and mineralized nodule formation ability increased, while lipid droplets decreased in the RUNX1-overexpessed group ( P<0.05). ALP activity and mineralized nodule formation ability decreased, while lipid droplets increased in the RUNX1 knockdown group ( P<0.05) . CONCLUSION: RUNX1 promotes DPSC proliferation and osteogenic differentiation while it inhibits DPSC adipogenic differentiation.

Commensal bacteria-dependent select expression of CXCL2 contributes to periodontal tissue homeostasis.

The oral and intestinal host tissues both carry a heavy microbial burden. Although commensal bacteria contribute to healthy intestinal tissue structure and function, their contribution to oral health is poorly understood. A crucial component of periodontal health is the recruitment of neutrophils to periodontal tissue. To elucidate this process, gingival tissues of specific-pathogen-free and germ-free wild-type mice and CXCR2KO and MyD88KO mice were examined for quantitative analysis of neutrophils and CXCR2 chemoattractants (CXCL1, CXCL2). We show that the recruitment of neutrophils to the gingival tissue does not require commensal bacterial colonization but is entirely dependent on CXCR2 expression. Strikingly, however, commensal bacteria selectively upregulate the expression of CXCL2, but not CXCL1, in a MyD88-dependent way that correlates with increased neutrophil recruitment as compared with germ-free conditions. This is the first evidence that the selective use of chemokine receptor ligands contributes to neutrophil homing to healthy periodontal tissue.

Laser capture microdissection enables cellular and molecular studies of tooth root development.

Epithelial-mesenchymal interactions (EMIs) are critical for tooth development. Molecular mechanisms mediating these interactions in root formation is not well understood. Laser capture microdissection (LCM) and subsequent microarray analyses enable large scale in situ molecular and cellular studies of root formation but to date have been hindered by technical challenges of gaining intact histological sections of non-decalcified mineralized teeth or jaws with well-preserved RNA. Here,we describe a new method to overcome this obstacle that permits LCM of dental epithelia,adjacent mesenchyme,odontoblasts and cementoblasts from mouse incisors and molars during root development. Using this method,we obtained RNA samples of high quality and successfully performed microarray analyses. Robust differences in gene expression,as well as genes not previously associated with root formation,were identified. Comparison of gene expression data from microarray with real-time reverse transcriptase polymerase chain reaction (RT-PCR) supported our findings. These genes include known markers of dental epithelia,mesenchyme,cementoblasts and odontoblasts,as well as novel genes such as those in the fibulin family. In conclusion,our new approach in tissue preparation enables LCM collection of intact cells with well-preserved RNA allowing subsequent gene expression analyses using microarray and RT-PCR to define key regulators of tooth root development.