Effects of rhBMP-2 gene transfection to periodontal ligament cells on osteogenesis.

The present study aims to investigate the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on the osteogenesis of periodontal ligament (PDL) cells. The expression vector of rhBMP-2 (pcDNA3.1-rhBMP-2) was established. PDL cells were obtained through the enzymatic digestion and tissue explant methods and verified by immunohistochemistry. Cells were classified into experimental (cells transfected with pcDNA3.1/rhBMP-2-EGFP), blank (cells with no transfection) and control group (cells transfected with empty plasmid). rhBMP-2 expression was assessed via Western blotting analysis. The mineralization ability, alkaline phosphatase (ALP) activity and level of related osteogenic biomarkers were detected to evaluate the osteogenic characteristics of PDL cells. The rhBMP-2 expression vector (pcDNA3.1-rhBMP-2) was successfully established. Primary PDL cells displayed a star or long, spindle shape. The cultured cells were long, spindle-shaped, had a plump cell body and homogeneous cytoplasm and the ellipse nucleus contained two or three nucleoli. Cells displayed a radial, sheaf-like or eddy-like arrangement after adherence growth. Immunohistochemical staining confirmed that cells originated from mesenchymal opposed to epithelium. The experimental group exhibited an enhanced mineralization ability, higher ALP activity and increased expression of rhBMP-2 and osteogenic biomarkers (Runx2, collagen type I and osteocalcin) than the blank and control group. The present study demonstrated that rhBMP-2 transfection enhances the osteogenesis of PDL cells and provides a possibility for the application of rhBMP-2 expression products in dental disease treatment.

IL-7 suppresses osteogenic differentiation of periodontal ligament stem cells through inactivation of mitogen-activated protein kinase pathway.

Periodontal ligament stem cells (PDLSCs) are tissue-specific mesenchymal stem cells (MSCs), having an important role in regenerative therapy for teeth loss. Interleukin-7 (IL-7) is a key cytokine produced by stromal cells including MSCs, and exhibits specific roles for B and T cell development and osteoblasts differentiation of multiple myeloma. However, the effect of IL-7 on osteogenic differentiation of PDLSCs remains unclear. Therefore, in the present study we determined whether IL-7 affects the proliferation and osteogenic differentiation of PDLSCs in vitro and explored the associated signaling pathways for IL-7-mediated cell differentiation. The results demonstrated that the isolated human PDLSCs possessed MSCs features, highly expressing CD90, CD44, CD105, CD29 and CD73, and almost did not expressed CD34, CD45, CD11b, CD14 and CD117. IL-7 could not significantly affect the proliferation of PDLSCs, but it decreased their osteogenic differentiation and inhibited alkaline phosphatase (ALP) activity. The results of quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting exhibited that the expression levels of Runx-2, SP7 and osteocalcin (OCN) were significantly reduced by IL-7. Further studies indicated that IL-7 did not significantly change JNK, ERK1/2 and p38 protein production, but markedly suppressed their phosphorylation levels. These data suggest that IL-7 inhibits the osteogenic differentiation of PDLSCs probably via inactivation of mitogen-activated protein kinase (MAPK) signaling pathway.

Tormentic acid inhibits LPS-induced inflammatory response in human gingival fibroblasts via inhibition of TLR4-mediated NF-κB and MAPK signalling pathway.

OBJECTIVE: Periodontal disease is one of the most prevalent oral diseases, which is associated with inflammation of the tooth-supporting tissues. Tormentic acid (TA), a triterpene isolated from Rosa rugosa, has been reported to exert anti-inflammatory effects. The aim of this study was to investigate the anti-inflammatory effects of TA on lipopolysaccharide (LPS)-stimulated human gingival fibroblasts (HGFs). METHODS: The levels of inflammatory cytokines such as interleukin (IL)-6 and chemokines such as IL-8 were detected by enzyme-linked immunosorbent assay (ELISA). The expression of Toll-like receptor 4 (TLR4), nuclear factor kappa B (NF-κB), IκBα, p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) was determined by Western blotting. RESULTS: The results showed that Porphyromonas gingivalis LPS significantly upregulated the expression of IL-6 and IL-8. TA inhibited the LPS-induced production of IL-6 and IL-8 in a dose-dependent manner. Furthermore, TA inhibited LPS-induced TLR4 expression; NF-κB activation; IκBα degradation; and phosphorylation of ERK, JNK, and P38. CONCLUSION: TA inhibits the LPS-induced inflammatory response in HGFs by suppressing the TLR4-mediated NF-κB and mitogen-activated protein kinase (MAPK) signalling pathway.

Effects of hypoxia on the proliferation, mineralization and ultrastructure of human periodontal ligament fibroblasts in vitro.

This study aimed to investigate the effects of hypoxia on the proliferation, mineralization and ultrastructure of human periodontal ligament fibroblasts (HPLFs) at various times in vitro in order to further study plateau-hypoxia-induced periodontal disease. HPLFs (fifth passage) cultured by the tissue culture method were assigned to the slight (5% O2), middle (2% O2), and severe hypoxia (1% O2) groups and the control (21% O2) group, respectively. At 12, 24, 48 and 72 h, the proliferation and alkaline phosphatase (ALP) activities were detected. The ultrastructure of the severe hypoxia group was observed. HPLFs grew more rapidly with an increase in the degree of hypoxia at 12 and 24 h, and significant levels of proliferation (P<0.05) were observed in the severe hypoxia group at 24 h. Cell growth was restrained with an increase in the degree of hypoxia at 48 and 72 h, and the restrictions were clear (P<0.05) in the middle and severe hypoxia groups. ALP activity was restrained with increasing hypoxia at each time point. The restrictions were marked (P<0.05) in the severe hypoxia group at 24 h and in the middle and severe hypoxia groups at 48 and 72 h. However, the restriction was more marked (P<0.05) in the severe hypoxia group at 72 h. An increase was observed in the number of mitochondria and rough endoplasmic reticula (RER), with slightly expanded but complete membrane structures, in the severe hypoxia group at 24 h. At 48 h, the number of mitochondria and RER decreased as the mitochondria increased in size. Furthermore, mitochondrial cristae appeared to be vague, and a RER structural disorder was observed. At 72 h, the number of mitochondria and RER decreased further when the mitochondrial cristae were broken, vacuolar degeneration occurred, and the RER particles were reduced while the number of lysosomes increased. HPLF proliferation and mineralization was restrained. Additionally, HPLF structure was broken for a relatively long period of time in the middle and severe hypoxia groups. This finding demonstrated that hypoxia was capable of damaging the metabolism, reconstruction and recovery of HPLFs. The poor state of HPLFs under hypoxic conditions may therefore initiate or aggravate periodontal disease.

20-Hydroxyecdysone-induced bone morphogenetic protein-2-dependent osteogenic differentiation through the ERK pathway in human periodontal ligament stem cells.

20-Hydroxyecdysone, an ecdysteroid hormone, can induce osteogenic differentiation in mesenchymal stem cells. Periodontal ligament stem cells (PDLS cells) have mesenchymal-stem-cell-like qualities and are considered as one of the candidates of future clinical application in periodontitis treatment. However, there are no studies describing the effect of 20-Hydroxyecdysone on PDLS cells. In this paper, we report a detailed study on the effect of 20-Hydroxyecdysone on PDLS cell proliferation in vitro. PDLS cells were developed from human PDL cells and were treated with 20-Hydroxyecdysone to understand different aspects of its effects. 20-Hydroxyecdysone promoted PDLS cell proliferation; significantly increased the gene expression levels of runt-related transcription factor 2, alkaline phosphatase (ALP), type I collagen, and osteocalcin. Moreover, 20-Hydroxyecdysone enhanced bone formation by PDLS cells and significantly increased bone morphogenetic protein-2 (BMP-2) mRNA and protein expression. However, 20-Hydroxyecdysonemediated increase in ALP activity was blocked with a BMP-2-specific neutralizing antibody or with the antagonist noggin; and20-Hydroxyecdysone mediated induction of BMP-2 expression and increase of ALP activity were abolished by the extracellular regulated protein kinase (ERK) MAPK pathway inhibitor PD98059. 20-Hydroxyecdysone also increased the phosphorylation of ERK. These findings provide evidence to state that 20-Hydroxyecdysone stimulates cell proliferation and induces osteogenic differentiation through the induction of BMP-2 expression in PDLS cells. It also shows that the ERK pathway is involved in 20-Hydroxyecdysone induced BMP-2 expression and osteogenic differentiation. These results are suggesting its potential as a drug for periodontal regenerative therapy.

Ectopically expressed IBP promotes cell proliferation in oral squamous cell carcinoma.

IFN regulatory factor 4 binding protein (IBP) has been shown to play an important role in the progression of malignant tumors such as breast cancer cells, but its function in oral squamous cell carcinoma (OSCC) remains unclear. We found that IBP ectopically expressed in some OSCC specimens but not in normal oral mucosa epithelium tissues. IBP expression was significantly correlated with tumor size, differentiation, clinical stage, and distant metastasis. Furthermore, IBP markedly promoted OSCC cell proliferation, shortened the G1 interval in the cell cycle, and increased cyclin D1 expression. These findings suggest that IBP may be a potential therapeutic target for OSCC.

[Isolation of aerobic degrading strains for TBBPA and the properties of biodegradation].

In order to investigate the characteristics of the microbial degradation of tetrabromobisphenol A (TBBPA), four aerobic strains were isolated from the phenol granular sludge using selective medium with TBBPA as the sole carbon source. Among them, strain H could accommodate the TBBPA culture medium and degrade TBBPA with high efficiency, which was identified as Rhodococcus sp. via DNA sequencing test. The optimal conditions for TBBPA degradation by strain H was pH 6.5, 30 degrees C, 150 r x min(-1), the concentration of humic acid being 200 mg x L(-1) and the concentration of K+ being 1 000 mg x L(-1). Under the optimal conditions, the debromination rate of TBBPA reached 20.75% after 21 d. The results of LC-MS showed that the major degradation product was monobromophenol, which was formed by the removal of isopropyl from the benzene ring in TBBPA molecule. The results of protein gel electrophoresis (SDS-PAGE) showed that strain H had a protein band between (90-117) x 10(3) kDa, which might be the enzyme responsible for TBBPA degradation.

IL-33 is induced by amyloid-ß stimulation and regulates inflammatory cytokine production in retinal pigment epithelium cells.

Age-related macular degeneration (AMD) is the predominant cause of irreversible blindness in the elderly population. Despite intensive basic and clinical research, its pathogenesis remains unclear. However, evidence suggests that immunological and inflammatory factors contribute to the pathogenesis of AMD. A newly identified cytokine, IL-33, appears to be an important pro-inflammatory cytokine promoting tissue inflammation. In this study, IL-33 was increased through amyloid-beta(1-40) (Aß(1-40)) stimulation and regulated inflammatory cytokines including IL-6, IL-8, IL-1ß, and TNF-α secretion using different signaling pathways in retinal pigment epithelium (RPE) cells. Furthermore, ST2L, the important component of the IL-33 receptor, was significantly increased following recombinant human IL-33 stimulation in RPE cells. These findings suggest that IL-33-mediated inflammatory responses in RPE cells are involved in the pathogenesis of AMD. Greater understanding of the inflammatory effect of IL-33 and its role in RPE cells should aid the development of future clinical therapeutics and enable novel pharmacological approaches towards the prevention of AMD.