[Effect of circRASA2 targeting miR-543/TRAF6 axis on LPS-induced periodontal ligament cell proliferation and osteogenic differentiation].

PURPOSE: To investigate the possible role of circRASA2 in periodontitis and its potential regulatory mechanism. METHODS: Periodontitis cell model was established by lipopolysaccharide(LPS)-induced periodontal ligament cells(PDLCs). Cell proliferation activity was detected by CCK-8 assay, cell migration ability was detected by Transwell chamber assay, and the expression of osteogenic differentiation-related proteins in cells was detected by Western blot. The target miRNA of circRASA2 and its downstream target genes were predicted using the databases circinteractome and starBase, respectively, and the targeting relationship between the target genes was verified by dual-luciferase reporter gene experiment. GraphPad Prism 8.0 software package was used to analyze the data. RESULTS: circRASA2 was highly expressed in LPS-treated PDLCs cells. LPS-induced PDLCs cell proliferation activity, migration ability and osteogenic differentiation ability decreased, while knockdown of circRASA2 promoted proliferation, migration and osteogenic differentiation ability of PDLCs under LPS treatment. circRASA2 targeted and negatively regulated the expression of miR-543, and overexpression of miR-543 promoted proliferation, migration and osteogenic differentiation of PDLCs under LPS treatment. TRAF6 was a downstream target gene of miR-543, knockdown of circRASA2 down-regulated the expression of TRAF6 through the sponge action of miR-543. Overexpression of TRAF6 reversed the promotion of circRASA2 knockdown on proliferation, migration and osteogenic differentiation of PDLCs. CONCLUSIONS: circRASA2 accelerated the pathological process of periodontitis in vitro through miR-543/TRAF6 axis, and might improve periodontitis by targeting down the expression of circRASA2.

[An analysis of the wall width before and after root canal therapy in mandibular second molars with C-shaped canals].

PURPOSE: To measure the wall width in mandibular second molars with C-shaped canals before and after root canal therapy using cone-beam CT(CBCT). METHODS: A total of 55 mandibular second molars from 38 patients which met the criteria for inclusion at Nanjing Stomatological Hospital, Affiliated Stomatological Hospital of Medical School of Nanjing University from January 2020 to July 2021 were analyzed．From this sample, ten teeth had been treated, while another 45 of them not. CT images of the teeth were reestablished by Mimics software 20.0. Then we made a section every 1mm perpendicular to the long axis of the teeth from apex to pulp floor. The first slice from apex to pulp chamber was named the slice 1. Along the slice 1 to crown 1 mm was called slice 1, and so forth. The wall thickness at different locations of all the slices was measured. The data was entered into SPSS 20.0 software package for analysis. RESULTS: Regardless of whether the teeth were treated or not, both the mesial and distal canal walls' average width were thicker than 1mm in all slices. At the same time, the mesial and distal canal walls' width were thicker than the width of buccal and lingual canal walls in all the slices from C-shaped root canal, except slices which were near pulp chamber(P＜0.05). As for the C-shaped root canals without root canal therapy, the width of lingual wall in the slice 1 to 4, as well as apex third root, was thinner than 1 mm. The width of buccal canal wall was thicker than the width of lingual canal wall in all slices except slice 11 and 12. As for the C-shaped root canals with root canal therapy, the width of buccal canal wall in slice 1 to 5, equivalent of apex half root, and the width of lingual wall in the slice 1 to 7, amount to apex two-thirds of root, was thinner than 1 mm. The width of buccal canal wall was thicker than the width of lingual wall in all slices except slice 1 and 9. There was no significant difference between the distal canal walls' width of C-shaped canals with and without root canal therapy(P＞0.05) . There was significant difference between the buccal canal walls' width of C-shaped canals with and without root canal therapy, as same as the mesial canal walls' width and the width of lingual canal wall (P＜0.05). CONCLUSIONS: The lingual canal walls' width in apex third root of C-shaped root canal were thin before canal preparation. The buccal walls' width in apex half root and the lingual canal walls' in apex two-thirds of root of C-shaped root canal were thin after canal preparation.

[Guided endodontic access of calcified root canal by laser melting templates].

OBJECTIVE: The aim of this study is to treat calcified root canal and assess the accuracy of guided endodontics using laser melting templates. METHODS: Two cases with calcified anterior teeth were treated with laser melting templates. Cone beam computed tomography (CBCT) was used to scan the maxillary teeth of patients before surgery to establish the root canal system model. The maxillary impression of the patient was made and it was scanned by a 3D scanner. The CBCT scans were matched with surface scans of plaster model. Mimics 19.0 and 3-matic 11.0 software were used to design the virtual planning to access cavities. The templates were produced by laser melting 3D printing. Access cavity was prepared under the guidance of laser melting template. Then the deviations of planned and prepared cavities in three dimensions and angle were measured. RESULTS: The two teeth obtained satisfactory results. The first case had a angle deviation of 1.77°, a drilling base deviation of 0.403-0.497 mm, and a tip of 0.433-0.537 mm. The second case had a angle deviation of 3.26°, a drill base deviation of 0.18-0.347 mm, and a tip of 0.310-0.463 mm. CONCLUSIONS: Laser melting template-guided endodontics is an effective technique for the treatment of calcified root canal and can be used as a new strategy for the treatment of calcified canal.

DSP-PP precursor protein cleavage by tolloid-related-1 protein and by bone morphogenetic protein-1.

Dentin sialoprotein (DSP) and phosphophoryn (PP), acidic proteins critical to dentin mineralization, are translated from a single transcript as a DSP-PP precursor that undergoes specific proteolytic processing to generate DSP and PP. The cleavage mechanism continues to be controversial, in part because of the difficulty of obtaining DSP-PP from mammalian cells and dentin matrix. We have infected Sf9 cells with a recombinant baculovirus to produce large amounts of secreted DSP-PP(240), a variant form of rat DSP-PP. Mass spectrometric analysis shows that DSP-PP(240) secreted by Sf9 cells undergoes specific cleavage at the site predicted from the N-terminal sequence of PP extracted from dentin matrix: SMQG(447)↓D(448)DPN. DSP-PP(240) is cleaved after secretion by a zinc-dependent activity secreted by Sf9 cells, generating DSP(430) and PP(240) products that are stable in the medium. DSP-PP processing activity is constitutively secreted by Sf9 cells, but secretion is diminished 3 days after infection. Using primers corresponding to the highly conserved catalytic domain of Drosophila melanogaster tolloid (a mammalian BMP1 homolog), we isolated a partial cDNA for a Spodopotera frugiperda tolloid-related-1 protein (TLR1) that is 78% identical to Drosophila TLR1 but only 65% identical to Drosophila tolloid. Tlr1 mRNA decreased rapidly in Sf9 cells after baculovirus infection and was undetectable 4d after infection, paralleling the observed decrease in secretion of the DSP-PP(240) processing activity after infection. Human BMP1 is more similar to Sf9 and Drosophila TLR1 than to tolloid, and Sf9 TLR1 is more similar to BMP1 than to other mammalian homologs. Recombinant human BMP1 correctly processed baculovirus-expressed DSP-PP(240) in a dose-dependent manner. Together, these data suggest that the physiologically accurate cleavage of mammalian DSP-PP(240) in the Sf9 cell system represents the action of a conserved processing enzyme and support the proposed role of BMP1 in processing DSP-PP in dentin matrix.

[Effect of longterm lower Porphyromonas gingivalis infection on the progression of atherosclerosis in apolipoprotein E-knocked out mice].

OBJECTIVE: To assess the effect of longterm and lower oral inoculation with Porphyromonas gingivalis (Pg) on the progression of atherosclerosis in apolipoprotein E-knocked out (ApoE(-/-)) mice. METHODS: Six-week-old male ApoE(-/-) mice were inoculated orally with 0.1 ml live Pg(10(13)/L) or bouillon culture-medium quintic per week for 15 consecutive weeks, altogether 75 times of inoculations. The lesion area of atherosclerosis in the aortic tree was measured by en face quantification by red oil O staining method. The atherosclerotic lesion was examined by histopathology. The levels of total cholesterol and triglycerides were compared. RESULTS: At 22 weeks after inoculation, the mean atherosclerotic lesion area in inoculated mice was (98 363.68 ± 12 043.00) µm(2), which was significantly greater than that in noninoculated mice, which was (62 985.06 ± 7419.64) µm(2) (P = 0.035). CONCLUSIONS: Longterm lower oral inoculation of Pg can accelerate the progression of atherosclerosis in apolipoprotein E-knocked out mice.

Expression of mineralized tissue associated proteins: dentin sialoprotein and phosphophoryn in rodent hair follicles.

BACKGROUND: Mammalian hair development and tooth development are controlled by a series of reciprocal epithelial-mesenchymal interactions. Similar growth factors and transcription factors, such as fibroblast growth factor (FGF), sonic hedgehog homolog (SHH), bone morphogenetic proteins (BMPs) and Wnt10a, were reported to be involved in both of these interactions. Dentin sialoprotein (DSP) and phosphophoryn (PP) are the two major non-collagenous proteins secreted by odontoblasts that participate in dentin mineralization during tooth development. Because of striking similarities between tooth development and hair follicle development, we investigated whether DSP and/or PP proteins may also play a role in hair follicle development. OBJECTIVE: In this study, we examined the presence and location of DSP/PP proteins during hair follicle development. METHODS: Rat PP proteins were detected using immunohistochemical/immunofluorescent staining. DSP-PP mRNAs were detected by in situ hybridization with riboprobes. LacZ expression was detected in mouse tissues using a DSP-PP promoter-driven LUC in transgenic mice. RESULTS: We found that PP proteins and DSP-PP mRNAs are present in rat hair follicles. We also demonstrate that an 8 kb DSP-PP promoter is able to drive lacZ expression in hair follicles. CONCLUSION: We have firmly established the presence of DSP/PP in mouse and rat hair follicles by immunohistochemical/immunofluorescent staining, in situ hybridization with riboprobes and transgenic mice studies. The expression of DSP/PP in hair follicles is the first demonstration that major mineralization proteins likely may also contribute to soft tissue development. This finding opens a new avenue for future investigations into the molecular-genetic management of soft tissue development.

[Effect of static magnetic field on bone morphogenetic protein 2 and collagen type I of osteoblast cell].

PURPOSE: To study the effect of static magnetic field on osteoblast,and to explore the possibility of osteogenesis of static magnetic field. METHODS: Rat calvarial osteoblasts were seeded on the 96-well culture plate, and exposed to 0.062T magnetic intensity field for 24 hours, 48 hours and 72 hours, respectively. Changes of BMP-2 and collagen type I in osteoblast were examined by Western blot. Immunohistochemical staining was carried out at different time periods. The data obtained were analyzed for ANOVA using SPSS 11.5 software package. RESULTS: The results of Western blot showed that magnetic static field affected expression of BMP-2 and collagen type I in osteoblast. The expression of BMP-2 in 48-hour group increased 2.1 fold and the expression of collagen type I in 72-hour group increased 1.6 fold compared to the control group (P<0.01). Moreover, the staining color of the collagen type I in cell of magnetic treated group was deeper than that of the control. CONCLUSIONS: Static magnetic field induced the expression of BMP-2 and stimulated secretion of collagen type I. The results indicate that osteoblasts are sensitive to 0.062T static magnetic field stimulation.

[Effect of static magnetic field on proliferation and cell cycle of osteoblast cell.].

Rat calvarias osteoblasts were cultured and put in the static magnetic field of 0 Gs, 400 Gs, 620 Gs, 830 Gs and 1080 Gs for 24 h, 48 h and 72 h, respectively. MTT method was used for detecting osteoblast proliferation and flow cytometry for detecting cell cycle. When the magnetic exposure time extended to 48 h or 72 h, the corresponding OD value of osteoblast with magnetic treatment of 400 Gs or 620 Gs increased obviously with significant difference as compared with 0 Gs group (P<0.05), but such proliferative effect was not shown in the 1080 Gs group (P>0.05). After magnetic exposure with intensity of 620 Gs, G0/G1 phase percentage decreased, but S phase and G2/M phase percentage was significantly higher as compared with control. Proliferation index (PI) increased as well and there was significant difference in contrast to control (P<0.05). Within a certain range of magnetic intensity, magnetic field enhances cell growth in a dose-dependent pattern. And static magnetic field can activate static G1 phase into S phase, increase DNA synthesis, and accelerate cell proliferation.