Objective：To study the expression patterns of bone morphogenetic proteins 7 (BMP7) during deciduous molar tooth development in miniature pig. Methods： The staged miniature pig embryos at embryonic day (E) 40th day, 50th day, 60th day were collected. Mandibles were fixed, decalcified and embedded before section. HE staining, immunohistochemistry, in situ hybridization using specific radionuclide labeled cRNA probes and real-time PCR were used to detect BMPs expression patterns during morphogenesis of the third deciduous molar tooth of miniature pig. Results：HE staining showed that the deciduous third molar at (E) 40th day, 50th day, 60th day represent the cap stage, bell stage and late bell stage (or secretory phase), respectively. Immunohistochemical staining showed BMP7 were expressed in both enamel organ and dental mesenchyme at E40 day and E50 day. However, at E60 day, expression of BMP7 was mainly located in pre-odontoblasts and pre-ameloblasts. Conclusions：BMP7 were temporal and spatial expressed during swine deciduous molar development, suggesting these molecules are associated with tooth morphogenesis and cell differentiation.

Objective：To gain insight into the molecular mechanisms associated with mouse tongue myogenesis. Methods：Different genes in the tongue at mouse embryonic day 13.25 (E13.25) and 15.5 was investigated using Affymetrix Mouse GeneChip. Using the twice significance of difference as the standard, the molecular mechanisms of tongue development were studied and several molecules related were identified by DAVID functional annotation clustering analysis. Results：Genes of higher expression level at E13.25 were related to cell cycle and cell adhesion, of whom Exo1, Gsk3B, Kif20b, Skp2 ( cell cycle related factors) and Neo1 and lama1 (cell adhesion factors) were activated. While genes of higher expression level at E15.5 were related to cytoskeleton, such as titin and Hspb7. Conclusions：The proliferation and determination of tongue were related with gene clusters of cell cycle and cell adhesion, and, differentiation and maturation of tongue were relevant to gene cluster of cytoskeleton. It had highlighted potential cascades and important candidates for further investigation on the genetic mechanism and clinical therapy of tongue related diseases.

Objective：To study the in vitro biologic characteristics of bone marrow stromal cells with cleidocranial dysplasia (BMSCs-CCD), including osteogenesis, proliferation ability, stemness and senescence. Methods：MTT and cell cycle detection for proliferation ability, Western blot and alizarin red staining for osteogenesis ability, Western blot and clony-formation for stemness ability, Western blot and senescence staining for senescence characteristics. Results：The optical density value was lower in BMSCs-CCD in MTT experiment and lower BMSCs-CCD were under S or G2 stage while more under G1 stage in cell cycle detection. BMSCs-CCD expressed lower levels of Runx2, Opn, Osterix and weaker alizarin red staining. Moreover, BMSCs-CCD also exhibited weaker stemness associate proteins (Oct4, Nanog, and Sox2) and clony-formation rates. However, stronger senescence associated proteins (p16, p21) and more senescenced cells were found in BMSCs-CCD. Conclusions：BMSCs-CCD exhibited weaker proliferation ability, osteogenesis and stemness while stronger senescence ability as compared to BMSCs. These results are helpful for us to understand pathological mechanism of CCD bone disease.

Objective：To investigate the influence of bone morphogenetic protein-2(BMP-2) on osteogenic differentiation of human inflammatory dental pulp stem cells(DPSCs-IPs) in vitro. Methods：Passage 3 cells dental pulp stem cells from inflamed teeth in medium with or without osteogenic matter. According to whether 100 μg/L BMP-2 was added they were divided into four groups: group 1，DPSCs-IPs culturing with 100 μg/L BMP-2 and osteogenic medium containing; group 2, DPSCs-IPs culturing with 100 μg/L BMP-2 but without osteogenic medium containing; group 3, DPSCs-IPs culturing without 100 μg/L BMP-2 but with osteogenic medium containing; group 4, DPSCs-IPs culturing without 100 μg/L BMP-2 and osteogenic medium containing. After one week of their culture in nonosteogenic condition, we used Trichrome to stain the secretion of collagen matrix, and compared the difference of osteogenic differentiation between them. Real-time RT-PCR was used to test the expression of COL-ⅠmRNA. Three weeks after their culture in osteogenic condition, the cell differentiation was examined by von Kossa staining. Real-time RT-PCR was used to test nuclear transcription factor Nanog, Oct4, SOX2 and osteogenesis related molecules ALP, OCN, BSP, CEMP-1 and COL-Ⅰ gene expression. Results：One week later, Trichrome staining matrix secretion of DPSCs-IPs with BMP-2 group is more obvious than the control group in the nonosteogenic medium. Real-time RT-PCR tests showed that expression of COL-ⅠmRNA increased (P<0.05). The results suggested that BMP-2 could induce DPSCs-IPs deposit more collagen matrix; 3 weeks later, more mineralized matrix secretion of DPSCs-IPs can be observed in the osteogenic medium. In addition, real-time RT-PCR tests showed that experimental group cells express more Nanog, Oct4 and SOX2. And the osteogenesis related molecules ALP, OCN, BSP, CEMP-1 and COL-Ⅰ expression increased significantly compared with the control group (P<0.05). Conclusions：BMP-2 can promote osteogenic differentiation of human inflammatory dental pulp stem cells in vitro in osteogenic condition.

Objective: To research the mechanisms of the muscle-specific microRNAs in the tongue’s differentiation of the retinoic acid induced tongue dysplasia by measuring the expression of MyomiRs, MRFs and Pax3/Pax7. Methods: A retinoic acid induced tongue dysplasia mouse model was established. The tongues were collected at E13.5, E14.5 and E15.5. The expression levels of MyomiRs, MRFs and Pax3/Pax7 were detected by real-time quantitative PCR. Results: During the tongue development, the expression of miR-1 and miR-206 was keeping rising both in the tongues of RA-treated mice and normal ones. While the expression of miR-1 and miR-206 in the RA-treated mice’s tongues was less than in the normal ones. The results of miR-1 at E14.5 and E15.5 had statistical significance (P<0.01) and the results of miR-206 at E13.5 had statistical significance (P<0.05). The expression of MyoD and Myf5 in the normal and RA-treated mice’s tongues reached the peak at E14.5 then reduced. At E13.5 and E14.5, the expression of MyoD in the RA-treated mice’s tongues was less than the normal ones. But at E15.5, the expression of MyoD in the RA-treated mice’s tongues was higher (P<0.01). And at E14.5 and E15.5, the expression of Myf5 in the RA-treated mice’s tongues was lesser compared to the normal ones. During the tongue myogenesis of normal and RA-treated mice the expression of Pax3 in the tongue reached the peak at E14.5, and the expression of Pax7 in both of them reached the peak at E15.5. Compared to the normal mice, the expression of Pax3 was higher at E14.5 (P<0.05), and the expression of Pax7 was higher at E13.5 (P<0.01) in the RA-treated mice’s tongues. Conclusions: During the tongue myogenesis, miR1/miR-206, Pax3/Pax7 and Myf5/MyoD were associated. In the retinoic acid induced tongue dysplasia, the correlation still existed. RA may down-regulate miR-1/miR-206 which targeted on Pax3/Pax7, following down-regulate the expression of MyoD/Myf5 to inhibit the myogenesis in tongues of RA-treated mice, resulted in tongue dysplasia.

Objective：To analyze different protein extraction methods from tooth germ, and find a suitable protein extraction method for high through-put proteome analysis of tooth development. Methods：The deciduous third molars were collected from miniature pig on embryonic 50 day and embryonic 60 day. The matrix samples of partial calcification tooth germ were fractionated with 50 mM carbonate buffer (pH 10.8). The protein extraction effect was observed by sodium dodecylsulphate–polyacrylamide gelelectrophoresis (SDS-PAGE), pulverized methods under liquid nitrogen into fine powder or with an electro-grinding machine were compared. Then proteins were quantified with the method of bicinchoninic acid (BCA), RIPA lysis buffer and 8 mol/L urea were compared. Results：SDS-PAGE showed that electro-grinding got higher quantity of tooth germ proteins than pulverizing under liquid nitrogen. At developing stages of tooth germ in miniature pigs, the extraction quantity of tooth germ proteins raised obviously when 8 mol/L urea replaced RIPA lysis buffer. Conclusions：The electro-grinding method was better for tissue utilization and 8 mol/L urea solution was suitable for extracting proteins from tooth germ with higher value of protein concentrations. The successful establishment of the extraction technique lays the foundation for the analysis methods in down- stream proteomics study.

Objective：To investigate the effect of cryopreservation on proliferation and differentiation of periodontal ligament stem cells (PDLSCs) sheet. Methods：We compared cryopreserved PDLSCs sheet with freshly PDLSCs sheet in terms of cell proliferation rate and multilineage differentiation potential by MTT, von Kossa staining and Oil Red staining. Results：Cryopreserved PDLSCs sheet showed a similar cell proliferation rate and multilineage differentiation potential as fresh PDLSCs sheet. Conclusions：This study demonstrates that cryopreservation does not affect the proliferation and differentiation of PDLSCs sheet, supporting the feasibility of PDLSCs sheet cryopreservation in nitrogen.

Objective: The odontogenic epithelial cells largely disappear after tooth eruption. It is proved that the non-odontogenic epithelial cells can be used as alternative seed cells of tooth regeneration. This study will characterizes the role of conditioned culture media on expansion of non-odontogenic epithelial cells in vitro for potential seed cells of further tissue engineering tooth. Method: The oral mucosa epithelial cells were harvested from 1day postnatal mouse and cultured to confluency in conditioned media supplemented with Y27632, then passaged and plated onto 3T3 feeder cells in conditioned media with Y27632 for continuous passage. Histology and immunohistochemistry were performed for biological characteristics analysis of cells. Results: Compared with the traditional ways of epithelial cell culture, the oral mucosa epithelial cells plated onto 3T3 feeder cells in conditioned media supplemented with Y27632 grew rapidly, which showed a cuboidal, polygonal or pavement-like shape and expressed CK14, SOX2 and LGR5. After continuous passage, the cell morphology, growth curve and cell markers have no obvious changes. Conclusion: The conditioned culture media supplemented with Y27632 have potential to effectively expand non-odontogenic epithelial cells in vitro, and maintain the characteristics of stem cells, which is expected for tissue engineering tooth as seed cells.

Objective：To explore effects of viability on human umbilical artery smooth muscle cells affected by two different fimAgenetypes of Porphyromonas gingivalis. Methods：The tissue-block attached method was used for primary culture of human umbilical artery smooth muscle cells. Porphyromonas gingivalis was cultured in the anaerobic conditions. Human umbilical artery smooth muscle cells were invaded by fimA genotype Ⅰ and Ⅳstrains respectively for 2, 8, 24 and 48 h. The CCK-8 assay was applied in testing viability of smooth muscle cells. Results： After fimA typeⅠstrains were cultured with vascular smooth muscle cells, smooth muscle cells were significantly increasing at 8 h, but obviously, cytotoxicity appeared at 24 h and 48 h (P<0.05). Cells invaded by fimA type Ⅳ Porphyromonas gingivalis did not have any significant change as compared with that of control group. Conclusions：There are diversities between the two different strains. FimA type Ⅰ strain plays a role in the processes of facilitating proliferation and cytotoxicity of vascular smooth muscle cells.