Effect of compressive force combined with vibration on CCL2 and CCL5 in human periodontal ligament cells.

Purpose: To investigate the effect of compressive force combined with vibration on expression of CC-chemokine ligand 2 (CCL2) and 5 (CCL5) in human periodontal ligament (hPDL) cells. Methods: Human PDL cells were cultured and assigned into four groups: control (Con), compressive force 2.0 g/cm2 for 24 h and 48 h (C), vibration 0.3 g 30 Hz for 20 min every 24 h (V), and compressive force combined with vibration (VC). At 24 h and 48 h, mRNA and protein levels of CCL2 and CCL5 were examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Results: At 24 h and 48 h, CCL2 mRNA and protein levels in C and VC were significantly higher than Con. At 24 h, VC showed significantly higher CCL2 mRNA expression than C. However, there was no significant difference between CCL2 protein in C and VC at both time points. At 24 h and 48 h, CCL5 mRNA expression was significantly down-regulated in V and VC, whereas CCL5 protein was undetectable in all groups. Conclusions: Application of compressive force combined with vibration resulted in the upregulation of CCL2 mRNA and protein levels, whereas CCL5 mRNA expression was down-regulated.

Biocompatibility and mineralization activity of modified glass ionomer cement in human dental pulp stem cells.

Background/purpose: Fortilin is a multi-functional protein involved in several cellular processes. It has been shown promising potential to be a bioactive molecule that can be incorporated in the dental materials. This study aimed to compare the biocompatibility and mineralization activities of modified glass ionomer cement (Bio-GIC) and Biodentine by direct and indirect method on human dental pulp stem cells (hDPSCs). Materials and methods: Conventional glass ionomer cement (GIC), Bio-GIC (GIC supplemented with chitosan, tricalcium phosphate, and recombinant fortilin from Fenneropenaeus merguiensis), and Biodentine were examined in this study. Recombinant fortilin was purified and tested for its cytotoxicity by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay. Human DPSCs were treated with different material eluate for particular time intervals. At given time points, viability of hDPSCs was examined using MTT assay and calcium deposition was assessed by Alizarin red staining assay. Comparisons of the data among groups were analyzed by analysis of variance and Tukey's multiple comparisons. Results: All test materials demonstrated no cytotoxicity. In addition, Bio-GIC promoted cell proliferation at 72 h. For direct and indirect method, cells treated with Bio-GIC demonstrated significantly higher calcium deposition than other groups (P < 0.05). Conclusion: Bio-GIC and Biodentine are not cytotoxic to hDPSCs. Bio-GIC demonstrates enhanced calcium deposition comparable to Biodentine. Bio-GIC may be further developed as a bioactive material for dentin regeneration.

Biological Activities of Glass Ionomer Cement Supplemented with Fortilin on Human Dental Pulp Stem Cells.

This study aimed to determine the most suitable recombinant fortilin and evaluate the biological activities of glass ionomer cement (GIC) incorporated with fortilin on human dental pulp stem cells (hDPSCs). Full-length and three fragments of Penaeus merguiensis fortilin were cloned and examined for their proliferative and cytoprotective effects on hDPSCs by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. Human DPSCs were cultured with GIC supplemented with fortilin, tricalcium phosphate, or a combination of tricalcium phosphate and fortilin, designated as GIC + FL, GIC + TCP, and GIC + TCP + FL, respectively (n = 4 for each group). At given time points, hDPSCs were harvested and analyzed by MTT, quantitative reverse transcription polymerase chain reaction, alkaline phosphatase activity, and Alizarin Red assays. The full-length fortilin promoted cell proliferation and significantly increased cell survival. This protein was subsequently added into the GIC along with tricalcium phosphate to investigate the biological activities. All experimental groups showed reduced cell viability after treatment with modified GICs on days 1 and 3. The GIC + TCP + FL group significantly promoted odontoblastic differentiation at particular time points. In addition, alkaline phosphatase activity and calcium phosphate deposit were markedly increased in the GIC + TCP + FL group. Among all experimental groups, the GIC incorporated with fortilin and tricalcium phosphate demonstrated the best results on odontogenic differentiation and mineral deposition in hDPSCs.

Long-Term Effect of Modified Glass Ionomer Cement with Mimicked Biological Property of Recombinant Translationally Controlled Protein.

This study modified glass ionomer cement (GIC) by adding mimicked biological molecules to reduce cell death. GIC was modified to BIOGIC by adding chitosan and bovine serum albumin for enhancing protein release. The BIOGIC was supplemented with tricalcium phosphate (TCP) and recombinant translationally controlled tumor protein (TCTP) to improve its biological properties. Four groups of materials, GIC, BIOGIC, BIOGIC+TCP, and BIOGIC + TCP + TCTP, were examined by XRD and SEM-EDX. TCTP released from the specimens was determined by an ELISA method. Human dental pulp stem cells (hDPSCs) were harvested and analyzed by MTT assay, apoptosis, gene expression, and cell differentiation. All groups had the same crystallization characteristic peaks of La2O3. The elemental compositions composed of La, Si, and Al are the main inorganic components. The results show that BIOGIC + TCP + TCTP presented significantly higher percentages of cell viability than other groups on day 1 to day 23 (p < 0.05), but were not different after day 24 to day 41 and had reduced cell apoptosis including BAX, TPT1, BCL-2, and Caspase-3. The BIOGIC + TCP + TCTP demonstrated higher odontoblast mineralization and differentiation markers including ALP activity, DSPP, DMP-1, ALP, BMP-2, and OPN. It enhanced cell proliferation and differentiation as well as mineralization with down-regulation of genes related to apoptosis compared with other groups.

Effects of tannin-fluoride and milk-fluoride mixture on human enamel erosion from inappropriately chlorinated pool water.

This in vitro study aimed to investigate the efficacy of tannin-fluoride and milk-fluoride mixtures on human enamel erosion after exposure to inappropriately chlorinated pool water. Enamel specimens were immersed in swimming pool water (pH 2.7) for 30 min and in each test reagent for 4 min once a day for 60 consecutive days (group I: control, group II: tannin-fluoride, group III: milk-fluoride, group IV: tannin-fluoride before and milk-fluoride after erosive challenge, and group V: milk containing tannin-fluoride before and after erosive exposure). Surface microhardness was assessed on days 0, 30, and 60. Scanning electron microscopy (SEM) and electron probe microanalysis (EPMA) were performed after treatment of samples for 60 days. Surface microhardness of experimental groups was ranked as follows: group III > group IV-group V > group II > group I (P < 0.05). Moreover, SEM images revealed deposition of substances on erosive enamel surface after treatment with tannin-fluoride and milk-fluoride mixtures. Furthermore, EPMA profiles showed decrease of phosphorus and increase of fluoride content in groups II and IV. In conclusion, we demonstrated that treatment with fluoridated milk with or without tannin-fluoride has protective effects against enamel erosion caused by low-pH swimming pool water.

Expression of translationally controlled tumor protein in heat-stressed human dental pulp cells.

OBJECTIVE: The aim of this study was to investigate the effects of heat stress on cell viability, translationally controlled tumor protein (TCTP) expression, and the effects of recombinant TCTP on heat-stressed human dental pulp cells (HDPCs). METHODS: HDPCs were isolated from human teeth and cultured at 37°C. For heat stress, HPDCs were incubated at 43°C for 45min. After heat stress, recombinant TCTP were added to HDPCs and cultured for various periods of time at 37°C. Heat-treated cells were then analyzed by DNA staining with Hoechst 33258, MTT, and caspase 3 activity assays. TCTP expression level was assessed by real-time PCR and western blot analysis. RESULTS: Heat-treated cells displayed lower cell density and nuclear morphology resembling apoptotic body. Heat stress significantly decreased cell viability and induced activity of caspase 3. The effect of recombinant TCTP on pulp cell death from heat stress varied depending on each subject and TCTP concentration. Heat stress up-regulated TCTP mRNA expression level. In contrast, TCTP protein level remained unchanged. Recombinant TCTP did not affect TCTP mRNA expression but down-regulated TCTP protein in heat-treated cells. CONCLUSIONS: Heat stress induces caspase 3 activation and up-regulates TCTP mRNA expression in HDPCs. TCTP did not play a key role on pulp cell recovery from heat stress.

Identification of a novel nuclear localization signal and speckle-targeting sequence of tuftelin-interacting protein 11, a splicing factor involved in spliceosome disassembly.

Tuftelin-interacting protein 11 (TFIP11) is a protein component of the spliceosome complex that promotes the release of the lariat-intron during late-stage splicing through a direct recruitment and interaction with DHX15/PRP43. Expression of TFIP11 is essential for cell and organismal survival. TFIP11 contains a G-patch domain, a signature motif of RNA-processing proteins that is responsible for TFIP11-DHX15 interactions. No other functional domains within TFIP11 have been described. TFIP11 is localized to distinct speckled regions within the cell nucleus, although excluded from the nucleolus. In this study sequential C-terminal deletions and mutational analyses have identified two novel protein elements in mouse TFIP11. The first domain covers amino acids 701-706 (VKDKFN) and is an atypical nuclear localization signal (NLS). The second domain is contained within amino acids 711-735 and defines TFIP11's distinct speckled nuclear localization. The identification of a novel TFIP11 nuclear speckle-targeting sequence (TFIP11-STS) suggests that this domain directly interacts with additional spliceosomal components. These data help define the mechanism of nuclear/nuclear speckle localization of the splicing factor TFIP11, with implications for it's function.

TFIP11 interacts with mDEAH9, an RNA helicase involved in spliceosome disassembly.

Yeast proteins Ntr1, Ntr2 and Prp43 function in spliceosome disassembly. An Ntr1-Ntr2 protein complex recruits Prp43 to allow the removal of the lariat-intron in late-stage RNA splicing activity. Based on amino-acid sequence similarities across species, TFIP11 and mDEAH9/Dhx15 have been identified as homologues of yeast Ntr1 and Prp43, respectively. The N-terminal region of TFIP11 contains a G-patch, which is a highly conserved domain of many RNA-processing proteins. TFIP11 displays a unique and characteristic subnuclear localization pattern, in close proximity to SC35 nuclear speckles. Transfected GFP-tagged mDEAH9 displays an evenly distributed nuclear localization and is excluded from the nucleoli; however when TFIP11 and mDEAH9 are co-transfected, both proteins colocalize to distinct nuclear speckles. These data show that TFIP11 recruits mDEAH9 suggesting that these two proteins have similar biological activities to their yeast counterparts.

TFIP11, CCNL1 and EWSR1 Protein-protein Interactions, and Their Nuclear Localization.

Previous studies using the yeast two-hybrid assay (Y2H) have identified cyclin L1 (CCNL1) and Ewing sarcoma breakpoint region 1 protein (EWSR1) as being interacting partners of tuftelin-interacting protein 11 (TFIP11). All three proteins are functionally related to the spliceosome and involved in pre-mRNA splicing activities. The spliceosome is a dynamic ribonucleoprotein complex responsible for pre-mRNA splicing of intronic regions, and is composed of five small nuclear RNAs (snRNAs) and µ140 proteins. TFIP11 appears to play a role in spliceosome disassembly allowing for the release of the bound lariat-intron. The roles of CCNL1 and EWSR1 in the spliceosome are poorly understood. Using fluorescently-tagged proteins and confocal microscopy we show that TFIP11, CCNL1 and EWSR1 frequently co-localize to speckled nuclear domains. These data would suggest that all three proteins participate in a common cellular activity related to RNA splicing events.

Using the yeast two-hybrid assay to discover protein partners for the leucine-rich amelogenin peptide and for tuftelin-interacting protein 11.

The established structural proteins of the enamel matrix are amelogenin, ameloblastin, and enamelin. Historically, tuftelin and tuftelin-interacting protein 11 (TFIP11) have also been discussed as possible enamel proteins. Protein complexes are achieved by protein-protein interactions, and it is protein complexes that control biomineralization. The purpose of our recent studies was to catalog protein partners for these proteins that are, or have been, implicated in tooth formation. We used the sensitive yeast two-hybrid assay to identify proteins that interact directly with amelogenin, ameloblastin, enamelin, the leucine-rich amelogenin peptide (LRAP) and TFIP11. In this manuscript we refer to, or document, potential protein partners for the proteins listed above. The yeast two-hybrid assay may ultimately prove to be a valuable proteomics methodology for using to decipher molecular events that ultimately result in enamel biomineralization.

An amelogenin minigene to study alternative splicing.

Diversity in gene expression is commonly observed as a result of alternative splicing of RNA transcripts. This is true in the case of amelogenin, one of the enamel matrix proteins. Our hypothesis is that additional amelogenin mRNA transcripts are generated in vivo, but these transcripts have yet to be observed because of the limitations of currently used detection methodologies. For this study our objective was to create an amelogenin minigene to study amelogenin RNA splicing events in cell lines of diverse character. Mouse genomic DNA was used as a PCR template to amplify the amelogenin DNA sequence spanning exons 2-7. The resulting PCR-generated DNA was subcloned in an expression vector. This resulting amelogenin minigene was shown to be functionally active by transfection into multiple cell lines. We have successfully cloned an amelogenin minigene, and as a result we describe and discuss novel amelogenin alternatively spliced transcripts.

Enamel matrix protein interactions.

UNLABELLED: The recognized structural proteins of the enamel matrix are amelogenin, ameloblastin, and enamelin. While a large volume of data exists showing that amelogenin self-assembles into multimeric units referred to as nanospheres, other reports of enamel matrix protein-protein interactions are scant. We believe that each of these enamel matrix proteins must interact with other organic components of ameloblasts and the enamel matrix. Likely protein partners would include integral membrane proteins and additional secreted proteins. INTRODUCTION: The purpose of this study was to identify and catalog additional proteins that play a significant role in enamel formation. MATERIALS AND METHODS: We used the yeast two-hybrid assay to identify protein partners for amelogenin, ameloblastin, and enamelin. Once identified, RT-PCR was used to assess gene transcription of these newly identified and potential "enamel" proteins in ameloblast-like LS8 cells. RESULTS: In the context of this yeast assay, we identified a number of secreted proteins and integral membrane proteins that interact with amelogenin, ameloblastin, and enamelin. Additionally, proteins whose functions range from the inhibition of soft tissue mineralization, calcium ion transport, and phosphorylation events have been identified as protein partners to these enamel matrix proteins. For each protein identified using this screening strategy, future studies are planned to confirm this physiological relationship to biomineralization in vivo. CONCLUSION: Identifying integral membrane proteins of the secretory surface of ameloblast cells (Tomes' processes) and additional enamel matrix proteins, based on their abilities to interact with the most abundant enamel matrix proteins, will better define the molecular mechanisms of enamel formation at its most rudimentary level.