Research Models in Dentine Development and Regeneration.

Dentine is a major component of teeth and is responsible for many of their functions, such as mastication and neural sensation/transduction. Over the past decades, numerous studies have focused on dentine development and regeneration using a variety of research models, including in vivo, ex vivo and in vitro models. In vivo animal models play a crucial role in the exploration of biochemical factors that are involved in dentine development, whereas ex vivo and in vitro models contribute mainly to the identification of biophysical factors in dentine regeneration, of which mechanical force is most critical. In the present review, research models involved in studies related to dentine development and regeneration were screened from publications released in recent years and summarised comprehensively, particularly in vivo animal models including prokaryotic microinjection, Cre/LoxP, CRISPR/Cas9, ZFN and TALEN, and scaffold-based in vitro and ex vivo models. The latter were further divided by the interactive forces. Summarising these research models will not only benefit the development of future dentine-related studies but also provide hints regarding the evolution of novel dentine regeneration strategies.

[Oral Squamous Cell Carcinoma-Derived Cell-Free DNA Modulates Stemness and Migration of Oral Squamous Cell Carcinoma Cell Line by Inducing M2 Macrophage Polarization].

Objective: To investigate the effect of oral squamous cell carcinoma (OSCC)-derived cell-free DNA (cfDNA) on the polarization of macrophages and the regulatory effect of polarized macrophages on the stemness and migration of OSCC cells. Methods: A total of 30 OSCC tissue samples, 10 dysplastic oral tissue samples, and 10 normal oral tissue samples were collected. The status of all tissue samples was confirmed by pathology analysis. Immunohistochemical (IHC) staining and immunofluorescence (IF) staining were performed to examine the cell count and location of M2 macrophages in different types of oral tissue samples. The conditioned medium (CM) of OSCC cell line CAL-27 from the human tongue was collected and the cfDNA was concentrated and isolated for identification. The macrophages were treated by cfDNA and their morphological characteristics were observed under microscope. The expression levels of polarization-related indicators were determined by RT-qPCR. CAL-27 cell line was treated with macrophage CM induced by cfDNA and the expression levels of stemness-related genes were determined by RT-qPCR. Scratch-wound assay was conducted to verify that the migration ability of CAL-27 was modulated by macrophages induced by cfDNA. Results: There were more M2 macrophages in the deep connective tissue of dysplastic oral epithelium and the stroma of OSCC compared with those in the normal oral tissues ( P<0.05). OSCC cell line CAL-27 could secret cfDNA of 10000-15000 bp in length. cfDNA secreted by CAL-27 could induced in macrophages significantly higher expression of M2-macrophage-related genes ( P<0.05). cfDNA-treated macrophages induced significantly increased expression of stemness-related genes in CAL-27 cell line ( P<0.05) and promoted the migration ability of CAL-27 cell line ( P<0.05). Conclusion: OSCC-derived cfDNA promotes stemness and migration of OSCC cell line by inducing M2 macrophage polarization.

Roles of Immune Cells and Mechanisms of Immune Responses in Periodontitis.

Periodontitis is one of the severe oral diseases that threatens both the oral and general health of humans. It is an inflammatory disease caused by the complex interaction between the plaque microorganisms and the host immune system. The innate immune response is activated when pathogens invade the periodontium. An excessive innate immune response leads to inflammation and the destruction of periodontal tissues, which then activates the adaptive immune response. Although systemic initial therapy and guided tissue regeneration (GTR) can control periodontal inflammation to a certain extent and promote periodontal tissue regeneration, their effects are still limited. Periodontal treatment will be significantly improved if it is possible to screen the potential therapeutic targets and regulate the key molecules involved in periodontal disease; however, relevant research on the prevention and treatment of periodontitis remains limited. Thus, with the aim of assisting the immunoregulation of periodontitis, this article summarises the cells and mechanisms involved in the innate immune response and adaptive immune response caused by pathogens in the periodontium.

[Polarity of ameloblasts and odontoblasts and their related regulators].

The polarity of ameloblasts and odontoblasts is crucial for their differentiation and function. Polarity-related molecules play an important role in this process. This review summarizes the process of polarity formation of ameloblasts and odontoblasts and their related regulators.

[The role of bone morphogenetic protein signaling pathway in tooth root development].

The bone morphogenetic protein (BMP) family is an important factor in the regulation of cell ular life activities and in the development of almost all tissues. BMP-mediated signaling plays an important role in tooth root development, which is a part of tooth development. Epithelial and mesenchymal interactions are involved in tooth root development, but the BMP signaling pathway has a different effect on tooth root development in epithelial and mesenchymal. This review summarizes the advances of BMP signaling in tooth root development.

Effects of human bone morphogenetic protein 2 (hBMP2) on tertiary dentin formation.

Formation of tertiary dentin to maintain pulp vitality is a major odontoblastic response to dental pulp injury. Human bone morphogenetic protein 2 (hBMP2) can promote proliferation and differentiation of odontoblasts. Current study is interested in evaluating if the hBMP2 can promote the regeneration of tertiary dentin and cure dental pulp injury using the adenoviral vector to deliver hBMP2 cDNA into the pulp. Primary culture of dental pulp cells of exfoliated deciduous teeth (hDPCs) was established. Human serotype 5 adenoviral vector, AdCMV-hBMP2, was created. AdCMV-hBMP2 was used to transduce hDPCs in vitro and dental pulp cells in animal model in vivo. Data clearly demonstrated that hBMP2 increased ALP and mineralization. Reverse transcription-real time quantitative PCR (RT-QPCR) data showed that hBMP2 dramatically increased gene expressions of Runx2 (Runt-related transcription factor 2), ALP, Col Iα (Collagen 1a1), SP7 (Osterix), DMP1 (dentin matrix acidic phosphoprotein 1), DSPP (dentin sialophosphoprotein), and BSP (bonesialoprotein), which are normally involved in osteogenesis/odontogenesis. Data from in vivo assays demonstrated that hBMP2 promoted pulp cell proliferation and increased formation of tertiary dentin in dental pulp. Our in vitro and in vivo data suggest that hBMP2 gene can efficiently be delivered into the dental pulp cells by adenovirus, and show potential clinical application for the treatment of dental pulp damage.

The Influence of Surface Modification on the Photoluminescence of CdTe Quantum Dots: Realization of Bio-Imaging via Cost-Effective Polymer.

To impart biocompatibility, stability, and specificity to quantum dots (QDs)-and to reduce their toxicity-it is essential to carry out surface modification. However, most surface-modification processes are costly, complicated, and time-consuming. In addition, the modified QDs often have a large size, which leads to easy aggregation in biological environments, making it difficult to excrete them from in vivo systems. To solve these problems, three kinds of conventional polymers, namely, polyvinyl alcohol (PVA, neutral), sodium polystyrene sulfonate (PSS, negative charged), and poly(diallyl dimethyl ammonium chloride) (PDDA, positive charged) were selected to modify the surface of QDs at low cost via a simple process in which the size of the QDs was kept small after modification. The effect of polymer modification on the photoluminescence (PL) properties of the QDs was systematically investigated. High quantum yields (QYs) of 65 % were reached, which is important for the realization of bio-imaging. Then, the cytotoxicity of CdTe QD-polymer composites was systematically investigated via MTT assay using the Cal27 and HeLa cell lines, especially for high concentrations of QD-polymer composites in vitro. The experimental results showed that the cytotoxicity decreased in the order CdTe-PDDA>CdTe>CdTe-PSS>CdTe-PVA, indicating that PSS and PVA can reduce the toxicity of the QDs. An obvious cytotoxicity of CdTe-PVA and CdTe-PSS was present until 120 h for the Cal27 cell line and until 168 h for the HeLa cell line. At last, the Cal27 cell line was selected to realize bio-imaging using CdTe-PSS and CdTe-PVA composites with different emission colors under one excitation wavelength.

[Effects of lactoferrin on vascular endothelial growth factor and basic fibroblast growth factor expression in human Tca8113 cell line].

OBJECTIVE: To investigate the effect of lactoferrin on vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) expression. METHODS: Lactoferrin at concentration of 0.006, 0.013, 0.025, 0.050 g/L and blank control groups were included in this study. The gene and protein expression of VEGF, bFGF were examined by RT-PCR and Western blotting. RESULTS: The RT-PCR and Western blotting assay showed that VEGF mRNA(0.31 +/- 0.08) and protein (0.68 +/- 0.11) in lactoferrin (0.050 g/L) group were significantly lower than in the control group (P < 0.05), and the bFGFmRNA (0.27 +/- 0.10) and protein (0.68 +/- 0.07) in lactoferrin (0.050 g/L) group were also significantly lower than in the control group (P < 0.05). CONCLUSIONS: Lactoferrin could inhibit the expression of VEGF, bFGFmRNA and protein in Tca8113 cells. This effect might be one of the mechanisms for anticancer function of lactoferrin.

[Effect of simvastatin on the function of MG63 cell line].

PURPOSE: To investigate the effect of simvastatin on the function of MG63 cell line. METHODS: The function of proliferation, osteoprotegerin (OPG) and osteocalcin (OC) gene expression, and migration of MG63 cells were detected with MTT, fluorescent real-time PCR and micro chemotaxis, respectively. The data was analyzed using ANOVA followed by Tukey test with SPSS14.0 software package. RESULTS: 10-9mol/L and 10-8mol/L concentrations of simvastatin slightly promoted MG63 cell proliferation; 10-7mol/L and 10-6mol/L concentrations of simvastatin greatly enhanced the OPG and OC mRNA expression; All concentrations of simvastatin had an inhibitory effect on MG63 cell migration. CONCLUSION: Simvastatin could promote bone defect regeneration by enhancing the bone-related genes expression.

The effect of simvastatin on mRNA expression of transforming growth factor-beta1, bone morphogenetic protein-2 and vascular endothelial growth factor in tooth extraction socket.

AIM: To determine the effect of local simvastatin application on the mRNA expression level of transforming growth factor-beta1 (TGF-beta1), bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) in the tooth sockets of rat. METHODOLOGY: Forty-eight male Wistar rats were randomly divided into experimental and control groups (n = 24). Polylactic acid/polyglycolic acid copolymer carriers, with or without simvastatin, were implanted into extraction sockets of right mandibular incisors. The expression of TGF-beta1, BMP-2 and VEGF mRNA was determined by in situ hybridization in the tooth extraction socket at five days, one week, two weeks and four weeks after implantation. RESULTS: The fusiform stroma cells in the tooth extraction socket began to express TGF-beta1, BMP-2 and VEGF mRNA in both experimental and control groups from one week after tooth extraction until the end of experiment. The expression of TGF-beta1 and BMP-2 mRNA in the experimental group was significantly up-regulated after one, two and four weeks, and expression of VEGF mRNA was significantly increased after one and two weeks compared with that in the control group. CONCLUSION: The findings indicate that local administration of simvastatin can influence alveolar bone remodeling by regulating the expression of a school of growth factors which are crucial to osteogenesis in the tooth extraction socket.

[Targeting transgene effect of adTERT-TRAIL on salivary adenoid cystic carcinoma cell line SACC-83].

OBJECTIVE: To investigate the targeting expression of TRAIL gene driven by human telomerase reverse transcriptase (hTERT) promoter in SACC-83 cell with telomerase activity. METHODS: Adenovirus vector AdTERT-TRAIL was constructed by homologus recombination. After transfecting AdTERT-TRAIL into SACC-83 cell and HEL cell, its effect on these cells in vitro was investigated using RT-PCR technique, MTT method and flow cytometry. RESULTS: After transfection of AdTERT-TRAIL, expression of extrinsic TRAIL gene driven was detected in SACC-83, the proliferation of SACC-83 cell showed significant inhibitory effect (the relative cell viability was 49.70%) and its apoptotic rate was promoted (30.49%), whereas no TRAIL gene was detected in HEL cell, also no inhibitory effect was observed in HEL cell and its apoptotic rate showed little change. CONCLUSION: Adenovirus vector AdTERT-TRAIL was successfully constructed, which can be used to induce expression of TRAIL gene in SACC-83 cell with targeting effect.

[Study of methods of decalcification for making united slices of tooth and affiliated periodontic tissues].

OBJECTIVE: To study the methods of decalcification for making united slices of tooth and affiliated periodontic tissues. METHODS: Twenty-one samples containing dog molars and affiliated periodontic tissues were divided into seven mean groups. The pH value of solution, time of decalcification, weight and volume of samples, and content of decalcified calcium were detected. The slices were observed by HE, specific, and immunohistochemical stain. RESULTS: The velocity of decalcification increased with decrease of solution pH. The weight of samples lightened by 37.61%, the volume reduced by 25.97% on average, and calcium decalcified was 174.49 mg per gram humid samples. The EDTA decalcification was slowest, but it was best. Decalcification was fast in Plank-Rycho solution while the section was worst, and faster in the formyl solution containing aluminium chloride than in EDTA, and the section was better. CONCLUSIONS: The 50% formyl solution containing aluminium chloride is an ideal decalcifying solution.

[Inhibition of proliferation of salivary adenoid cystic carcinoma by targeting gene transfer].

OBJECTIVE: To investigate the expression of gene TRAIL driven by human telomerase reverse transcriptase (hTERT) promoter in SACC-83 cell tumor necrosis factor related apoptosis in dncing ligand. METHODS: After pACTERT-TRAIL plasmid transfected was into SACC-83 and HEL cells through liposome, the expression of TRAIL was examined using RT-PCR technique, the cells' survival rate by methyl thiazolyl tetrazolium (MTT) method and apoptosis rate by flow cytometry. RESULTS: Expression of extrinsic TRAIL gene driven by hTERT promoter was detected in SACC-83 cells, and not detected in human embryonic lung fibroblast (HEL) cells. After transfection of pACTERT-TRAIL, the proliferation of SACC-83 cells was significantly inhibited, and its apoptotic rate was promoted, whereas no inhibited effect was observed on HEL cells and its apoptotic rate showed little change. CONCLUSIONS: hTERT promoter can be used to induce tumor-specific expression of TRAIL gene and apoptosis in SACC-83 cells.

[Effect of nano-hydroxyapatite to glass ionomer cement].

OBJECTIVE: To investigate the mechanical character, microleakage and mineralizing potential of nano-hydroxyapatite (nano-HAP)-added glass ionomer cement(GIC). METHODS: 8% nano-HAP were incorporated into GIC as composite, and pure GIC as control. Both types of material were used to make 20 cylinders respectively in order to detect three-point flexural strength and compressive strength. Class V cavities were prepared in 120 molars extracted for orthodontic treatment, then were filled by two kinds of material. The microleakage at the composite-dentine interface was observed with confocal laser scanning microscope (CLSM) after stained with 1% rhodamin-B-isothiocyanate for 24 hours. Class V cavities were prepared in the molars of 4 healthy dogs, filled with composite, and the same molars in the other side were filled with GIC as control. The teeth were extracted to observe the mineralizing property with polarimetric microscope in 8 weeks after filling. RESULTS: Three-point flexural strength and compressive of nano-HAP-added GIC were increased compared with pure GIC (P < 0.001, P < 0.05). The nanoleakages and microleakages appeared at the material-dentine interface in the two groups, but there were more microleakages in control group than in experiment group (P = 0.004). New crystals of hydroxyapatite were formed into a new mineralizing zone at the interface of tooth and nano-HAP-added GIC, while there was no hydroxyapatite crystals formed at the interface of tooth and pure GIC. CONCLUSION: 8% nano-HAP-added GIC can tightly fill tooth and have mineralizing potential, and can be used as liner or filling material for prevention.

[Effect of TRAIL gene in human squamous cell carcinoma cell line induced by adenovirus].

OBJECTIVE: To study the apoptotic effect on the squamous cell carcinoma cell line TCa83 induced by recombined adenovirus vector containing TRAIL gene and CMV promoter. METHODS: The TCa83 cell line was firstly infected with different titre of AdCMV-EGFP containing enhanced green fluorescence protein gene (EGFP) as control, and investigated the transducing rate through fluorescence to obtain the definite titre. Then TCa83 cell line was infected with AdCMV-TRAIL in proper titre, and TRAIL gene was detected by means of RT-PCR. After TCa83 cell line was infected with AdCMV-TRAIL and AdCMV-EGFP at day 1, 3, 5, 7, the activity of TCa83 cell line were evaluated by MIT and the apoptosis were detected by flow cytometer. RESULTS: Proper titre was of 1,000 particles/cell, and TCa83 cell line could be infected 100% in this titre. TRAIL gene was detected by RT-PCR after infected with AdCMV-TRAIL. The activity of TCa83 decreased in both groups, but the AdCMV-TRAIL group decreased more sharply than AdCMV-EGFP group (P < 0.001). Both AdCMV-TRAIL and AdCMV-EGFP could lead to apoptosis of TCa83 cells, but the AdCMV-TRAIL, function stronger than AdCMV-EGFP. Especially there was remarkable statistic difference between two groups (P < 0.0001). CONCLUSION: AdCMV-TRAIL could effectively decrease the activity of TCa83 cell line and induce apoptosis.

[Research of the surface oxide film on anodizing Ni-Cr porcelain alloy].

OBJECTIVE: To study the shape, thickness and oxide percentage of major metal element of oxide film on Ni-Cr porcelain alloy after anodizing pretreatment. METHODS: 10 samples were made and divided into 2 groups at random. Then after surface pretreatment, the oxide films of two samples of each group were analyzed using electronic scanning microscope. The rest 3 samples were measured by X-ray photoelectron spectroscopy (XPS) and Auger electron spectroscopy (AES). RESULTS: Lightly selective solution appeared because the different component parts of the alloy have dissimilar electrode, whose dissolve velocity were quite unlike. The sample's metal surface expanded, so the mechanical interlocking of porcelain and metal increased bond strength. The thickness of oxide film was 1.72 times of the control samples. The oxide percentage of major metal elements such as Cr, Ni and Mo were higher, especially Cr. It initially involved the formation of a thin oxide bound to the alloy and second, the ability of the formed oxide to saturate the porcelain, completing the chemical bond of porcelain to metal. CONCLUSION: The method of anodizing Ni-Cr porcelain alloy can easily control the forming of oxide film which was thin and its surface pattern was uniform. It is repeated and a good method of surface pretreatment before firing cycle.

[Effect of simvastatin on residual ridge resorption after tooth extraction].

OBJECTIVE: To observe the effect of simvastatin carried by poly (lactide-co-glycolide) (PLGA) on residual ridge resorption following tooth extraction. METHODS: Sixty male Wistar rats were divided into experimental groups and control groups (30 rats/group). PLGA was immediately implanted with or without simvastatin into extraction sockets of the mandibular incisors. Soft X-ray photography, bone mineral density (BMD) and histopathologic study were conducted at 7, 14, 28, 56, and 84 days after implantation. RESULTS: The relative length values of residual alveolar ridge of the experimental groups were greater than those of the controls at 14, 28, 56, and 84 days after implantation, and there was a significant difference between the experimental and control groups (P < 0.05). The BMD of the specific region was higher in the experimental groups [(7.101 +/- 0.025), (7.178 +/- 0.039), and (7.162 +/- 0.052) g/cm(2)] than that in the control groups [(7.074 +/- 0.014), (7.117 +/- 0.012), and (7.059 +/- 0.037) g/cm(2)] (P < 0.05) after 28, 56, and 84 days. Light microscopy showed that bone formation rate and quality of the experimental group were better than those in the control group at the same time. CONCLUSIONS: Simvastatin carried by PLGA could induce bone formation of tooth socket. Local application of simvastatin would be potential to preserve the length and bone volume of alveolar ridge after tooth extraction.

[Effect of K2O addition on the crystallization property of dental glass-ceramics].

OBJECTIVE: To evaluate the effect of K2O addition on the crystallization property of dental glass-ceramics in the Li2O-SiO2-Al2O3-P2O5-ZnO system. METHODS: Different content of K2O was added into Li2O-SiO2-Al2O3-P2O5-ZnO glass system. The heat-treated system of the glass-ceramics was determined by differential thermal analyses (DTA), then the crystallization components and the microstmcture of the glass-ceramics with different content of K2O were investigated from X-ray diffraction (XRD) analyses and scanning electron microscopy (SEM). RESULTS: Addition of K2O helped to reduce the viscosity of the glass system and improved crystallization. More lithium disilicate crystals appeared after heated-treatment of the glass system which contained 5.3 wt% addition of K2O, and the homogeneously lath-shaped crystals were 4 gm in length. CONCLUSION: Certain content of K2O can improve the crystallization property of dental glass-ceramics in the Li2O-SiO2-Al2O3-P2O5-ZnO system.