Regulation of OPG and RANKL expressed by human dental follicle cells in osteoclastogenesis.

We investigate whether the expression of the receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) in human dental follicle cells (HDFCs) regulated by colony stimulating factor 1 (CSF-1), parathyroid hormone-related protein (PTHrP) and bone morphogenetic protein-2 (BMP-2) contributes to osteoclastogenesis. Adolescent human impacted third mandibular molars were used to separate HDFCs. These cells were incubated with PTHrP (10 ng/ml), CSF-1 (25 ng/ml), or BMP-2 (100 ng/ml) for 0.5, 1, 3, 6 and 12 h. The expression of OPG and RANKL was investigated by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). Two co-culture systems and tartrate-resistant acid phosphatase (TRAP) staining were used to examine osteoclast formation. Scanning electron microscopy was utilized for the resorption pit assay. RANKL and OPG were expressed innately in HDFCs. Exogenous PTHrP, CSF-1 and BMP-2 chronologically regulated the expression of RANKL and OPG in HDFCs. PTHrP and CSF-1 had similar regulative patterns leading to the up-regulated expression of RANKL and the down-regulated expression of OPG and opposite for BMP-2. The number of TRAP-positive peripheral blood mononuclear cells (PBMCs) slightly increased in contacted co-culture of HDFCs and PBMCs, whereas secreted OPG from HDFCs inhibited osteoclastogenesis in the transwell co-culture system. Contacted co-culture of HDFCs and PBMCs exhibited small and shallow resorption pits, whereas in the transwell co-culture system, secreted OPG from HDFCs reduced the resorption pits, reflecting the difference in osteoclast production. Collectively, we found a dual action of HDFCs in osteoclastogenesis; moreover, PTHrP, CSF-1 and BMP-2 might influence osteoclastogenesis by regulating the expression of RANKL and OPG in HDFCs.

Osteogenic-related gene expression profiles of human dental follicle cells induced by dexamethasone.

AIM: Human dental follicle cells (hDFC) have the ability to differentiate into mineralized tissue-forming cells during root and periodontal development or osteogenic induction in vitro. The present study aimed to validate the osteogenic induction of hDFC by dexamethasone (DEX) and to explore the changes of related genes responsible for the osteogenic differentiation process. METHODS: Passage-cultured hDFC were induced by DEX and analyzed for mineralization activity by morphological observation, alkaline phosphatase (ALP) activity, and alizarin red S staining. GEArray Q series human osteogenesis gene array was used to describe large-scale gene expression in treated hDFC compared to the control group. Quantitative real-time RT-PCR was performed to confirm the microarray data by analyzing the expression of 7 critical transcripts. RESULTS: Osteogenic differentiation of hDFC was confirmed by morphological change, elevated ALP activity and calcified nodules. In 96 genes investigated through the microarray analysis, 20 genes were upregulated and 8 genes were downregulated more than 2-fold. The results of the real-time RT-PCR correlated with the microarray analysis. The expression of the transforming growth factor-beta superfamily showed varying degrees of increase, and fibroblast growth factors exhibited a differential changing trend of expression. The expression of most types of collagen genes representative of extracellular matrixes increased under DEX treatment while small mothers against decapentaplegic 6 and 7 expressions significantly decreased. CONCLUSION: Our results demonstrated that hDFC displayed osteoblastic features in both phenotypic and genotypic traits induced by DEX in vitro.

[Anti-tumor immune response in vitro induced by fusion of Tca8113 cells with macrophages].

OBJECTIVE: To evaluate the therapeutic effectiveness of fusion tumor vaccine in tongue cancer treatment. METHODS: Human macrophages fused with human tongue carcinoma cell line Tca8113 cell. The fusion cells were selected by magnetic cell sorting (MACS) and cultured. The biological properties of fusion cells and anti-tumor immune response in vitro induced by fusions were observed. RESULTS: In contrast to Tca8113, the fused cells grew significantly slow in vitro. The expression of MHC I, II antigen of the fusion cells which was detected by flow cytometry (FCM) was higher than that of Tca8113. The fused cells significantly increased the proliferation of mixed lymphocyte and induced their cytotoxicity on parental Tca8113. CONCLUSIONS: The fusion tumor vaccine of macrophages and OSCC cells increase in vitro immunogenicity significantly. This indicates that fusion tumor vaccine could be a new method of anti-tumor immunotherapy, which has important potentials for effective individualized human OSCC vaccine.