MicroRNA-146 inhibits pro-inflammatory cytokine secretion through IL-1 receptor-associated kinase 1 in human gingival fibroblasts.

BACKGROUND: Although various microRNAs (miRNAs) regulate immune and inflammatory responses, the function of miRNAs in periodontitis has not been clearly illuminated. In this study, we measured miRNA-146 (miRNA-146a and miRNA-146b-5p) expression and explored its regulatory function in the inflammatory response in human gingival fibroblasts (HGFs). METHODS: miRNA-146a and miRNA-146b-5p expression was measured by performing real-time polymerase chain reaction in HGFs after Porphyromonas gingivalis (p.g) lipopolysaccharide (LPS) stimulation. After the HGFs were transfected with miRNA-146a and miRNA-146b-5p inhibitor, the expression levels of interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA). Meanwhile, IL-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6) were detected by western blot and quantitative PCR. A luciferase assay was used to detect whether miRNA-146 could directly bind to the 3'-UTR of IRAK1. RESULTS: The expression levels of miRNA-146a and miRNA-146b-5p significantly increased in the P.g LPS-stimulated HGFs compared to the non-stimulated HGFs. The inhibition of miRNA-146a and miRNA-146b-5p resulted in increased IL-1ß, IL-6 and TNF-α secretion. The mRNA and protein levels of IRAK1, but not TRAF6, also increased. We further found that miRNA-146a and miRNA-146b-5p directly bound to the IRAK1 3'-UTR. CONCLUSION: Our data suggest that miRNA-146 inhibits pro-inflammatory cytokine secretion through IRAK1 in HGFs, which indicates that miRNA-146 functions as a negative regulator of periodontal inflammation.

Effect of Porphyromonas gingivalis and Lactobacillus acidophilus on secretion of IL1B, IL6, and IL8 by gingival epithelial cells.

Porphyromonas gingivalis alters cytokine expression in gingival epithelial cells, stimulating inflammatory responses that may lead to periodontal disease. This study explored the effect of Lactobacillus acidophilus on the specific expressions of the interleukins (ILs) IL1B, IL6, and IL8 induced by the pathogen. Human gingival epithelial cells were co-cultured with P. gingivalis, L. acidophilus, or L. acidophilus + P. gingivalis; the control group consisted of the cells alone. Protein and gene expression levels of the ILs were detected using ELISA and qRT-PCR, respectively. The supernatant from the P. gingivalis group held significantly higher protein and mRNA levels of IL1B, IL6, and IL8, compared to the control group. In the mixed bacterial group (L. acidophilus + P. gingivalis), the levels of all three ILs decreased with increasing concentrations of L. acidophilus and were significantly different from the P. gingivalis group. This suggests that in gingival cells, L. acidophilus offsets the P. gingivalis-induced secretion of these ILs in a dose-dependent manner.

[Isolation, cultivation and induced-mineralization of dental pulp cells from goat deciduous teeth in vitro].

PURPOSE: To isolate and cultivate dental cells from goat deciduous teeth ,and explore changes of its biological characters before and after induced-mineralization. METHODS: Pulp cells were cultivated with modified tissue block enzymolytic method, cell lineage in the second passage with SAB methods was checked out. Induced-mineralized cultivation was adopted in the fourth passage, some examinations were used to compare with normal cultivated cells: cell proliferative capality, mineralized ability test, cell morphology change, protein(OCN) expression level, related osteogenic genes(ALP,COL-I,OCN,OPN) expression. RESULTS: Modified tissue block enzymolytic method could culture better pulp cells derived from goat deciduous teeth. Immunohistochemical staining showed that pulp cells were from mesenchyma. MTT method showed that induced-mineralization pulp cells proliferated more slowly than un-induced cells. Compared with uninduced-mineralization cells, induced-mineralization cells had stronger ALP activity and Alizarin red staining rate, its proteins(OCN) and mineralized genes(ALP,OCN) expression were significantly upregulated. CONCLUSIONS: Pulp cells can be cultivated derived from goat exfoliated deciduous teeth with modified tissue block enzymolytic method. Fourteen days after continuous induced-mineralization culture , pulp cells derived from the goat deciduous teeth might own the potential in differentiating to osteoblast and form bone-like tissue.

Comparison of microRNA profiles of human periodontal diseased and healthy gingival tissues.

MicroRNAs (miRNAs) have been demonstrated to play an important role in regulation of the immunoinflammatory response; however, the function of miRNAs in periodontal inflammation has not been investigated. The objective of this study was to explore the properties of miRNAs in periodontal inflammation by comparing miRNA profiles of inflamed and healthy gingival tissues. Gingival tissues were obtained from 10 periodontitis patients and 10 healthy subjects. After RNA extraction, miRNA profiles were analyzed by microarray, and expression levels of selected miRNAs were confirmed by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). Analyses using two computational methods, Targetscan and MicroRNA.org, were combined to identify common targets of these miRNAs. Finally, the individual miRNA expression levels of three toll-like receptor (TLR)-related miRNAs from inflamed and healthy gingival tissues were evaluated by RT-PCR. Ninety-one miRNAs were found to be upregulated and thirty-four downregulated over two-fold in inflamed gingival tissue compared with those in healthy gingival tissue. Twelve selected inflammatory-related miRNAs, hsa-miR-126*, hsa-miR-20a, hsa-miR-142-3p, hsa-miR-19a, hsa-let-7f, hsa-miR-203, hsa-miR-17, hsa-miR-223, hsa-miR-146b, hsa-miR-146a, hsa-miR-155, and hsa-miR-205 showed comparable expression levels by microarray and real-time quantitative RT-PCR analyses. In addition, the putative inflammation targets of these miRNAs were predicted, and three that were tested (hsa-miRNA-146a, hsa-miRNA-146b, and hsa-miRNA-155), showed significant differences between inflamed and healthy gingiva. This remarkable difference in miRNA profiles between periodontal diseased and healthy gingiva implicates a probable close relationship between miRNAs and periodontal inflammation. The data also suggest that the regulation of TLRs in periodontal inflammation may involve miRNA pathways.

[Experimental study on stem cells from goat deciduous teeth compounded with porous calcium phosphate cement for ectopic osteogenesis].

PURPOSE: To observe the biocompatibility and ectopic bone-like tissue formation of stem cells from goat deciduous teeth (SGDs) with porous calcium phosphate cement (pCPC). METHODS: The expression of STRO-1 on SGDs was measured with flow cytometry (FCM); the 4th passage SGDs were cultured in induced-mineralization medium in vitro for 7 days. Combined with pCPC, the adhesion and growth of the compounds were observed with scanning electron microscopy (SEM); the ectopic bone-like tissue formation was observed 8 weeks after the compounds implanted subcutaneously into the nude mice. RESULTS: On the third day of SGDs compounded with pCPC, SEM verified that the cells adhered closely and tightly with pCPC, protruded pseudopods and secreted matrix. 8 weeks after the compounds implanted in ectopic sites, HE staining confirmed the formation of bone-like tissue; Immunohistochemistry showed the strongly positive expression of OCN protein in the implanted materials. CONCLUSIONS: SGDs may differentiate into osteoblast and are potential to induce bone matrix formation; combined with pCPC, the compounds may generate bone-like tissue.

[Culture and green fluorescent protein gene transfection of pulp cells from goat deciduous teeth in vitro].

PURPOSE: To isolate and culture pulp cells from goat deciduous teeth, and transfect green fluorescent protein gene. METHODS: Pulp cells from goat deciduous teeth were obtained by tissue culture. Cell growth curve was measured by counting the number of cells, HE and alkaline phophatase(AKP) stain, as well as immunhistochemical stain of vimentin and keratin were performed. Virus supernatant was used to infect cell green fluorescent protein gene. RESULTS: For the pulp cells, the cell group double time was 43.79 hours. AKP stain and immunhistochemical stain of vimentin were both positive, while immunhistochemical stain of keratin was negative. The infected cells expressed green fluorescent. CONCLUSIONS: Pulp cells could be cultured from goat deciduous teeth, and express green fluorescent successfully.

[Experimental study of subcutaneous osteogenesis by bone marrow stromal cells with A-PCPC in Beagle dogs].

PURPOSE: To study the osteogenic capability of the construct combined dog's bone stromal cells with active porous calcium phosphate cement (A-PCPC) in nude mice in vivo. METHODS: Isolated bone marrow stromal cells (BMSCs) were expanded and osteogenically induced in vitro. Their osteogenic phenotype was evaluated by cytochemistry. The tissue engineering complex was constructed with BMSCs/A-PCPC in vitro. After SEM scanning, the complex of BMSCs/A-PCPC was implanted into the subcutaneous tissue of the nude mice as experimental group, and A-PCPC as control group. The engineered bone was harvested 2,4,8weeks post-implantation and processed for HE staining, then evaluated by histology and histomorphometry. RESULTS: Cytochemistry showed alkaline phosphate activity, Von Kossa staining proved the formation of mineralization nodules. Scanning electron microscopy showed the cells adhered to the inner surface of the A-PCPC. HE staining showed a small group of woven bone formation 2 weeks later in the experimental group, while the formation of bone less in the control group. Woven bone turned into trabecular bone gradually at 4 weeks in the experimental group, while the control group showed a large number of bone-like tissue. Histomorphometry showed more mature bone in the experimental group than the control group at 8 weeks. CONCLUSIONS: The A-PCPC/BMSCs composites show good osteogenetic activity and could promote mineralization of the immature bone. It can be used as the bone tissue engineering scaffolds. Supported by Innovation Fund for Science and Technology Development of Pudong New District(Grant No. PKJ2009-Y19).

[Preliminary study on DNA damage of ZrO(2)/LaPO(4) diphase ceramics on human peripheral blood lymphocytes in vitro].

PURPOSE: To detect the genotoxicity of dental machinable ZrO(2)/LaPO(4) diphase ceramics on human peripheral blood lymphocytes in vitro. METHODS: The evaluation of DNA damage on human lymphocytes was performed by comet assay for three groups of ZrO(2)/LaPO(4) diphase ceramics with 30wt% of LaPO(4) (with 3wt% and 5wt% of Y(2)O(3)) and 40wt% of LaPO(4) (with 5wt% of Y(2)O(3)). The results were analyzed with SPSS16.0 software package for one-factor ANOVA and LSD. RESULTS: Three experimental groups with different concentration of LaPO(4) of ZrO(2)/LaPO(4) diphase ceramics, the negative control of IPS Empress II ceramics and the blank behaved little migration of the DNA strands respectively after six-day test, and there was no significant difference in all the groups except the positive control (P>0.05). CONCLUSION: The study indicates little effect of DNA damage of ZrO(2)/LaPO(4) diphase ceramics.

Sequential fluorescent labeling observation of maxillary sinus augmentation by a tissue-engineered bone complex in canine model.

AIM: To evaluate the effects of maxillary sinus floor elevation by a tissue-engineered bone complex of beta-tricalcium phosphate (beta-TCP) and autologous osteoblasts in dogs. METHODOLOGY: Autologous osteoblasts from adult Beagle dogs were cultured in vitro. They were further combined with beta-TCP to construct the tissue-engineered bone complex. 12 cases of maxillary sinus floor elevation surgery were made bilaterally in 6 animals and randomly repaired with the following 3 groups of materials: Group A (osteoblasts/beta-TCP); Group B (beta-TCP); Group C (autogenous bone) (n=4 per group). A polychrome sequential fluorescent labeling was performed post-operatively and the animals were sacrificed 24 weeks after operation for histological observation. RESULTS: Our results showed that autologous osteoblasts were successfully expanded and the osteoblastic phenol-types were confirmed by ALP and Alizarin red staining. The cells could attach and proliferate well on the surface of the beta-TCP scaffold. The fluorescent and histological observation showed that the tissue-engineered bone complex had an earlier mineralization and more bone formation inside the scaffold than beta-TCP along or even autologous bone. It had also maximally maintained the elevated sinus height than both control groups. CONCLUSION: Porous beta-TCP has served as a good scaffold for autologous osteoblasts seeding. The tissue-engineered bone complex with beta-TCP and autologous osteoblasts might be a better alternative to autologous bone for the clinical edentulous maxillary sinus augmentation.

[Comparison of the attachment and growth characteristics between human junctional epithelium and oral epithelium cells].

OBJECTIVE: To compare the attachment and growth characteristics between human junctional epithelium (JE) and oral epithelium cells. METHODS: The healthy JE biopsies were derived from the human teeth extracted due to impaction or orthodontic purpose. Enzyme digestion was used to isolate JE cells, which were then cultured in DKGM. The co-culture model of JE cell-tooth slice was built up by adding 3 decalcification cementum slices (5 mm x 3 mm x 1 mm) into sterilized plate containing 1 ml of JE cells (5 x 10(8)/L), 21 slices all together,and incubated in an atmosphere containing 5% CO2 at 37 degrees C for 1-14 days. The attachment structure was observed under transmission electron microscope, and the OE cells was used as control. RESULTS: The human JE cells were polymorphous in shape and CK19 positive, while OE cells were consisted of equal and closely packed epithelial-like cells in a paving stone arrangement, and CK19 was only strained in a few cells. There were a few cells in JE-slice when co-cultured for 1-3 days, and electron dense plaques on the JE cell surface of the attached slice were observed at 9 days, and 2-3 layer of JE cells and hemidesmosome-like structure formed within 11-14 days. There were more OE cells within 1-3 days, electron dense plaques appeared at 7 days, and stratified epithelium and hemidesmosome-like structure formed in OE-slice at 9 days. CONCLUSIONS: The cultured JE cells were immature and lower differentiated epithelial cells which were different from OE cells. Under the same condition the growth and attachment of JE cells on the cementum slice surface were slower than that of OE cells. Their attachment strength needs further study.

[An experimental study on biocompatibility of Bio-oss collagen with cultured bone marrow stromal cells].

PURPOSE: To study the biocompatibility of Bio-oss collagen with cultured bone marrow stromal cells. METHODS: The bone marrow stromal cells (BMSCs) of rhesus monkey were cultured with Bio-oss collagen in vitro. Cell attachment was observed with laser confocal microscope. Cell proliferation rates were assessed with MTT assay. ALP activity was also detected. The data was statistically analyzed with SAS6.12 software package for Student's t test. RESULTS: The rhesus BMSCs could attach to the surface of Bio-oss collagen. In cell proliferation rates, there was no significant difference between the control group and experimental group at 2-day and 5-day(P>0.05). However, at 8-day, the difference was significant (P<0.05). For ALP activity, there was no significant difference among different time point (P>0.05). CONCLUSIONS: The Bio-oss collagen has good biocompatibility with rhesus BMSCs, and could be used to repair periodontal bone defects as a biomaterial in tissue engineering.

[Effect of enamel matrix protein on periodontal cells in an in vitro wound healing model].

PURPOSE: The aim of this study was to evaluate the influence of enamel matrix protein (EMP) on wound filling of periodontal ligament cells (PDLC) and gingival fibroblasts (GF), using an in vitro model of wound healing. METHODS: In vitro wound models were mechanically created in subconfluent cultures of human GF and PDLC, by removing a 7mm wide band of the cell layer respectively. Wounded cultures were then incubated for a time periods up to 2,6,9 days in a media containing 10% fetal bovine serum (FBS), stimulated with EMP (100microg/ml) simultaneously, negative controls were those cultured only with media containing 10% FBS. Slides were fixed, stained with crystal violet and cell filling area within the wound boundaries was quantified by computer assisted histomorphometry. Statistical analysis was performed by SAS6.12 software package to determine the differences between the time points and groups. RESULTS: in the control group, there was difference between GF and PDLC in filling wound in vitro over 9 days of healing period. The difference was significant (P<0.05) at the 9th day after wound creating, with GF filling the wound faster than PDLC. In contrast, under the stimulation of EMP (100microg/ml), there was no significant difference (P>0.05) between the filling rate of two types of periodontal cells at the 6th, 9th day after wound creating. CONCLUSION: GF has a significantly greater ability to fill a wound than PDLC. However, EMP appears to exert an influence on cells that is compatible with improved wound healing, especially in that of PDLC.

[Effects of enamel matrix proteins on the proliferation of human gingival epithelial cells in vitro].

PURPOSE: To evaluate the effect of enamel matrix proteins (EMPs) on the proliferation of human gingival epithelial cells in vitro. METHODS: EMPs were extracted from pig tooth germ by acetic acid. The gingival tissues cut off during gingivectomy were separated into two parts through Dispase II digestion and the epithelium part was cultured to acquire the gingival epithelial cells. The first passage epithelial cells were inoculated into 96-well plate, 22500 cells per well, and exposed to different concentrations of EMPs (50, 100, 200 microg/ml respectively). The control was epithelial cells cultured in the same medium except without EMPs. The proliferation rates were carried out over a 5-day period and assessed by an MTT assay and the data were analysed by one-way variance. RESULTS: It was shown that gingival epithelial cells well attached and spread on EMPs-coated substrata. There were no significant differences between the control group and various concentrations of EMPs groups at the initial stage, however, EMPs at a concentration of 200 microg/ml significantly inhibited gingival epithelial cells proliferation from day 3 over the experiment. CONCLUSIONS: The proliferation of gingival epithelial cells was significantly inhibited by EMPs in a dose- and time-dependent manner, which provides some evidence for the mechanism of EMPs in promoting the periodontal tissue regeneration.

[Preliminary study on adult odontoblast culture in situ].

PURPOSE: To establish an in situ culture model of adult odontoblasts. METHODS: Thirty intact and healthy third molars freshly prepared from 20-30 year old individuals were randomly divided into three groups. Each group had 10 molars. Group 1 was pulp tissue extraction group. Group 2 was serum-containing culture group. Group 3 was serum-free culture group. The root was dissected from the crown and the pulp was pulled out to make odontoblasts remaining in the crown. The odontoblasts were cultured in situ either in medium containing serum or serum-free medium for up to 7 days. The growth status of the cells was examined by light microscopy and cell morphology and distribution was analyzed by scanning electron microscopy. Cell viability was determined by trypan blue staining. RESULTS: After pulp removal at room temperature, odontoblasts remained in the wall of the pulp chamber, and kept viable and good morphology during the 7-day culture. CONCLUSION: We have successfully established an in situ culture model of adult primary odontoblasts in either serum-containing or serum-free medium.

[Culture and comparison of the biological characteristics of human junctional epithelium and gingival epithelium].

PURPOSE: To study the different biological characteristics of human junctional epithelium (JE) and gingival epithelium (GE). METHODS: Human JE cells were cultured and identified by the cell culture and immunohistochemistry, and the biological characteristics of JE cells and GE cells were compared. RESULTS: The morphology of cultured JE cells was various and unequal, the arrangement of the cells was loose and mitosis was common, while GE colony was consisted of equal and closely packed epithelial-like cells in a paving stone arrangement. CK-Pan staining was positive in all JE and GE cells. CK19 was strongly stained in all JE cells but only moderately stained in some GE cells, and most GE cells were negative which was obviously different from JE cells. JE cells had longer latent period (7 days) than GE cells (4 days) during the cell growth period, and then the cells proliferated rapidly (4 days) to attain the maximum and descended rapidly in the declining period. While GE cells ascended evenly (7 days) to attain the maximum and descended slowly in the declining period. Proliferation study demonstrated the doubling time of JE cells was 48 to 60 hours and that of GE cells was 72 to 96 hours. It was possible to subculture JE cells up to 5 times serially, and that of GE cells was up to 7 times. CONCLUSIONS: The human JE cells are a kind of unique non-differentiated epithelial cells different from GE cells. In this experimental culture condition the subculture times of JE cells were less than GE cells, which affects JE cells, so the culture methods and conditions should be improved.