[An in vitro experiment on the stability and irritant of hypochlorous acid in oral cavity].

PURPOSE: To study the stability of physicochemical properties and sterilizing effect about two commercially available hypochlorous acid (HClO) products under simulated clinical conditions, and to evaluate the compatibility of HClO on soft and hard tissues and cells in oral cavity. METHODS: Samples of HClO solution with different production processes were prepared, to detect the changes of physicochemical indexes of each sample over time under simulated clinical conditions (shielded from light at 20-25 ℃, open the cover for 5 minutes every day), including free available chlorine, oxidation-reduction potential and pH. Through suspension quantitative germicidal test, the antibiosis-concentration curve of HClO solution was made, so as to calibrate the change of antibacterial ability of disinfectant with the decrease of available chlorine content during storage. Pulp, tongue and dentine were immersed in PBS, 100 ppm HClO, 200 ppm HClO and 3% NaClO. The influence on soft and hard tissues was evaluated by weighing method and microhardness test. The toxic effects of HClO, NaClO and their 10-fold diluent on human gingival fibroblasts were determined by CCK-8 cytotoxicity assay. GraphPad PRIS 8.0 software was used to analyze the data. RESULTS: Under simulated conditions, the free available chlorine (FAC) of HClO solution decayed with time, and the attenuation degree was less than 20 ppm within 1 month. The bactericidal effect of each HClO sample was still higher than 5log after concentration decay. There was no obvious dissolution and destruction to soft and hard tissues for HClO(P＞0.05). The cell viability of HClO to human gingival fibroblast cells (HGFC) was greater than 80%, which was much higher than 3% NaClO (P＜0.001). CONCLUSIONS: The bactericidal effect and stability of HClO solution can meet clinical needs, which has low cytotoxicity and good histocompatibility. It is expected to become a safe and efficient disinfection product in the field of living pulp preservation and dental pulp regeneration.

Recent Advances in Functional Hydrogels for Treating Dental Hard Tissue and Endodontic Diseases.

Oral health is the basis of human health, and almost everyone has been affected by oral diseases. Among them, endodontic disease is one of the most common oral diseases. Limited by the characteristics of oral biomaterials, clinical methods for endodontic disease treatment still face large challenges in terms of reliability and stability. The hydrogel is a kind of good biomaterial with an adjustable 3D network structure, excellent mechanical properties, and biocompatibility and is widely used in the basic and clinical research of endodontic disease. This Review discusses the recent advances in functional hydrogels for dental hard tissue and endodontic disease treatment. The emphasis is on the working principles and therapeutic effects of treating different diseases with functional hydrogels. Finally, the challenges and opportunities of hydrogels in oral clinical applications are discussed and proposed. Some viewpoints about the possible development direction of functional hydrogels for oral health in the future are also put forward. Through systematic analysis and conclusion of the recent advances in functional hydrogels for dental hard tissue and endodontic disease treatment, this Review may provide significant guidance and inspiration for oral disease and health in the future.

Effect of mesencephalic astrocyte-derived neurotrophic factor on the inflammatory response in human gingival fibroblasts cells.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a unique member of the neurotrophic factor family residing in the endoplasmic reticulum, where it functions as a stress response protein maintaining endoplasmic reticulum homeostasis, in addition to being secreted extracellularly as a neurotrophic factor to bind with receptors to initiate intracellular signal transduction pathways. Interestingly, MANF has shown an important protective role in the inflammatory response of many diseases. In neural stem cells, pancreatic ß cells, and retinal cells, MANF can inhibit the inflammatory response, modulate the immune response, and promote tissue repair. However, the role of MANF in the periodontal inflammatory response remains unclear. In the present study, we used lipopolysaccharide (LPS) from Porphyromonas gingivalis (Pg) to establish a Pg-LPS-stimulated periodontal inflammatory model in human gingival fibroblasts cells (HGF-1) to investigate the role of MANF in vitro. We found that MANF could inhibit pro-inflammatory cytokine secretion, alleviate the endoplasmic reticulum stress response, promote cell survival, and inhibit cell apoptosis. Therefore, MANF might be a novel promising target for the treatment of periodontitis.

Quorum sensing system effectively enhances DegU-mediated degradation of pyrethroids by Bacillus subtilis.

The contamination of the natural environment is a growing concern that threatens all life forms, including microorganisms. Bacteria protect themselves by initiating quorum sensing (QS), a bacterial cell-cell communication, to generate adaptive responses to these pollutants. Bacillus subtilis has a typical QS ComQXPA system that regulates the phosphorylation of the transcription factor DegU (DegU-P), and thus can mediate the expression of various downstream genes under different stress conditions. Herein, we found that cesB, a gene of Bacillus subtilis 168, plays a key role in pyrethroid degradation, and cesB-mediated degradation could be enhanced by coordinating with the ComX communication system. Using ß-cypermethrin (ß-CP) as a paradigm, we demonstrated that DegU-P increased upon exposure to ß-CP, thus facilitating ß-CP degradation by binding to the upstream regulatory regions of cesB, leading to the activation of the expression of cesB. Further, we showed that the expression of different levels of phosphorylated DegU in a degU deletion strain resulted in varying degrees of ß-CP degradation efficiency, with phosphorylated DegUH12L achieving 78.39% degradation efficiency on the first day, surpassing the 56.27% degradation efficiency in the wild type strain. Consequently, based on the conserved regulatory mechanism of ComQXPA system, we propose that DegU-P-dependent regulation serves as a conserved defense mechanism owing to its ability to fine-tune the expression of genes involved in the degradation of pollutants upon exposure to different pesticides.

Endodontic treatment of the mandibular first molar with six root canals: A case report and literature review.

The mandibular first molars normally have three or four root canals and rarely have five or more root canals. The present study reported a rare anatomical configuration with six root canals in the mandibular right first molar diagnosed during endodontic treatment using a dental operating microscope and confirmed with the help of cone-beam computed tomography (CBCT) images. The present case report revealed that there is an increasing possibility of detecting additional canals through the magnification of the microscope and the improvement of CBCT diagnostic technology. As more abnormal morphologies in root canals are reported, dentists need to understand this diversity in anatomical structure and improve treatment techniques.

Osteoprotegerin/receptor activator of nuclear factor-κB ligand are involved in periodontitis-promoted vascular calcification.

The present study explored the potential role of osteoprotegerin (OPG)/receptor activator of nuclear factor-κB (RANK)/receptor activator of nuclear factor-κB ligand (RANKL) in promoting vascular calcification by periodontitis. Thirty-six male Wistar rats were randomly assigned to four groups to establish animal models as follows: the sham group (group C), vascular calcification group (group VDN), periodontitis group (group CP), and test group (group CP+VDN). After eight weeks, all the rats were sacrificed. The periodontal and vascular calcification indices were detected. Quantitative polymerase chain reaction (qPCR), immunohistochemistry, western blot analysis, and enzyme-linked immunosorbent assay (ELISA) were used to quantify OPG/RANK/RANKL expression in vascular tissue and serum. Protein expression analyses revealed the expression of OPG and RANKL in the vascular tissues of the four groups. The expression of OPG in group C was the highest, which was similar to group CP+VDN, and the expression of OPG in groups CP and VDN were lower. However, the expression of RANKL was inversely correlated with OPG, and the ratio of RANKL/OPG was also higher in groups CP and VDN than that in groups C and CP+VDN. In conclusion, OPG/RANK/RANKL may play an essential role in the promotion of vascular calcification by periodontitis. However, the expression levels of OPG and RANKL were not simply superimposed when periodontitis and vascular calcification co-existed.

Bactericidal Efficacy of Photon-Induced Photoacoustic Streaming-Erbium:Yttrium-Aluminum-Garnet Laser Combined with Irrigation Solution on Enterococcus faecalis in Curved Root Canals: An In Vitro Study.

Objective: This experiment aimed to study the bactericidal effect of photon-induced photoacoustic streaming (PIPS)-erbium:yttrium-aluminum-garnet (Er:YAG) laser on Enterococcus faecalis in curved root canals. Materials and methods: Sixty-two molars with moderately curved roots (10°-20°) and 62 molars with severely curved roots (25°-40°; one root was selected in each tooth) were assigned to group A and group B, respectively. A curved root canal model with E. faecalis infection was established. Four samples were used for sterility test, and 20 samples were used for testing if the modeling was valid. The remaining 100 samples were randomly divided into 5 subgroups (A1/A2/A3/A4/A5 and B1/B2/B3/B4/B5, n = 10) and treated as follows: A1/B1: PIPS-Er:YAG laser +5.25% sodium hypochlorite (NaOCl); A2/B2: passive ultrasonic irrigation +5.25% NaOCl; A3/B3: PIPS-Er:YAG laser+normal saline (NS); A4/B4: two-hole root canal irrigator +5.25% NaOCl; A5/B5: two-hole root canal irrigator+NS. After treatment, bacterial culture counts and scanning electron microscopic (SEM) observations were carried out for each subgroup, and the bacterial clearance rate of each subgroup was calculated. SPSS 23 software package was used for statistical analysis of the data, and a single-factor analysis of variance was used to compare the subgroups. Results: The bacterial clearance rate in group A was higher than that in group B; however, in each group, A or B, there were significant differences between the subgroups (p < 0.001) except for subgroups 1 and 2 (p > 0.05). SEM revealed that the antibacterial and smear layer removal effect of root canal in subgroups 1 and 2 was better than that in subgroups 3, 4, and 5. Conclusions: PIPS-Er:YAG can significantly enhance the bactericidal effect of NaOCl on E. faecalis in moderately and severely curved root canals.

Breast cancer susceptibility gene 1 regulates oxidative damage via nuclear factor erythroid 2-related factor 2 in oral cancer cells.

OBJECTIVE: To determine whether the breast cancer susceptibility gene 1 (BRCA1) regulates oxidative damage in oral cancer cells by interacting with nuclear factor erythroid 2-like 2 (NRF2). DESIGN: The BRCA1 gene was silenced in CAL-27 and DOK cells using specific shRNA, and NRF2 was activated with sulforaphane. The expression levels of BRCA1, NRF2 and its target genes were assessed by quantitative real-time polymerase chain reaction and western blotting. Cell counting kit-8 assay was used to detect cell proliferation, apoptosis was detected by flow cytometry, and 8-OXo-2'-deoxyguanosine level was measured by enzyme-linked immunosorbent assay. The expression of BRCA1 and NRF2 in patients with oral leukoplakia and oral squamous cell carcinoma were evaluated by immunohistochemistry. RESULTS: BRCA1 knockdown downregulated NRF2 and its target genes, increased proliferation rates, reduced apoptosis, and increased 8-OXo-2'-deoxyguanosine levels compared to the control. Activation of NRF2 by sulforaphane significantly upregulated NRF2 levels in the BRCA1-depleted cells, and restored proliferation, apoptosis and 8-OXo-2'-deoxyguanosine level in a dose-dependent manner. Compared with patients with leukoplakia, BRCA1 and NRF2 expression were increased in patients with oral squamous cell carcinoma. CONCLUSIONS: BRCA1 depletion increases oxidative damage and promotes the malignant phenotype, which may eventually promote oral carcinogenesis. The NRF2-activator sulforaphane is a potential chemo-preventive agent for oral cancer.

Effect of endoplasmic reticulum stress-induced apoptosis in the role of periodontitis on vascular calcification in a rat model.

The present study aimed to investigate the mechanism(s) through which endoplasmic reticulum stress (ERS)-induced apoptosis, in the role of periodontitis, affects vascular calcification. Rat models of periodontitis, vascular calcification, periodontitis-vascular calcification, and a normal group were established. Cardiovascular tissues were obtained, and hematoxylin-eosin staining was applied to demonstrate the morphological changes in vascular tissues. Immunohistochemical staining was applied to analyze apoptosis in cardiovascular tissues. The expression levels of apoptotic factor cysteinyl aspartate specific proteinase 3 (Caspase-3), ERS-induced apoptotic factors glucose-regulated protein 78 (GRP78), 94 (GRP94), and ERS-induced apoptosis pathways Caspase-12, C/EBP homologous protein (CHOP), and c-Jun N-terminal kinase (JNK) were analyzed and compared. Hematoxylin-eosin staining revealed that the arterial layers in the normal group were structurally intact. The structural damage to the aortic wall gradually aggravated from the periodontitis group to the vascular calcification group to the combined group. The immunohistochemistry results showed Caspase-3, GRP78, GRP94, and ERS-induced apoptosis pathways in the cardiovascular tissues cells in the periodontitis group, vascular calcification group, and combined group. The Caspase-3, GRP78, GRP94, and CHOP expression levels in the combined group were significantly higher than that in the normal group (P < 0.05); however, the Capase-12 and JNK expression levels in the four groups exhibited no significant differences (P > 0.05). Apoptosis induced by ERS is involved in the effect of periodontitis on vascular calcification and might be mainly achieved through the activation of the CHOP transcription pathway.

Effect of Porphyromonas gingivalis lipopolysaccharide on calcification of human umbilical artery smooth muscle cells co-cultured with human periodontal ligament cells.

Periodontitis is an independent risk factor for coronary heart disease. Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) was considered to be one of the main virulence factors. In addition, vascular smooth muscle cells transform into osteoblast-like cells in an arterial calcification process under chronic inflammatory conditions. The present study aimed to determine the calcification induced by Pg-LPS in human umbilical artery smooth muscle cells (HUASMCs) co-cultured with human periodontal ligament cells (HPDLCs). An in vitro co-culture system was established using Transwell inserts. HUASMC proliferation and alkaline phosphatase (ALP) activity were measured with a Cell Counting Kit-8 and an ALP kit, respectively. Calcium nodule formation was detected using alizarin red S staining. The effects of Pg-LPS on the mRNA expression of the calcification genes of ALP, core-binding factor α1 (Runx2) and bone sialoprotein (BSP) were assessed using reverse transcription-quantitative PCR. The results indicated that Pg-LPS increased HUASMC proliferation and ALP activity. Furthermore, among all of the groups, calcium nodule formation was most extensive in co-cultured cells in the mineralization-inducing medium containing Pg-LPS. In addition, the expression of specific osteogenic genes (Runx2, ALP and BSP) significantly increased in the presence of Pg-LPS and mineralization-inducing medium, which was further enhanced in co-culture with HPDLCs. In conclusion, co-culture with HPDLCs increased the effect of Pg-LPS to stimulate the calcification of HUASMCs. It was suggested that besides the inflammation, periodontitis may promote the occurrence of vascular calcification. The study indicated that periodontal treatment of subgingival scaling to reduce and/or control Porphyromonas gingivalis may decrease the occurrence or severity of vascular calcification.

In-vivo imaging revealed antigen-directed gingival B10 infiltration in experimental periodontitis.

Our previous study demonstrated that IL-10 secreting B (B10) cells alleviate inflammation and bone loss in experimental periodontitis. The purpose of this study is to determine whether antigen-specificity is required for the local infiltration of B10 cells. Experimental periodontitis was induced in the recipient mice by placement of silk ligature with or without the presence of live Porphyromonas gingivalis (P. gingivalis). Donor mice were pre-immunized by intraperitoneal (IP) injection of formalin-fixed P. gingivalis, or PBS as non-immunized control. Spleen B cells were purified and treated with LPS and CpG for 48 h to expand the B10 population in vitro. Fluorescence-labelled B10 cells were transferred into the recipient mice by tail vein injection and were tracked on day 0, 3, 5 and 10 using IVIS Spectrum in vivo imaging system. The number of B10 cells and P. gingivalis-binding B cells were significantly increased after in vitro treatment of LPS and CpG. On day 5, the fluorescence intensity in gingival tissues was the highest in mice transferred with B10 cells from pre-immunized donor mice. Gingival expression of IL-6, TNF-α, RANKL/OPG ratio and periodontal bone loss in recipient mice were significantly reduced, and the expression of IL-10 and the number of CD19+ B cells were significantly increased after pre-immunized B10 cell transfer in the presence of antigen, compared to those with non-immunized B10 cell transfer or no antigen presence. This study suggests that antigen specificity dictate the local infiltration of B10 cells into periodontal tissue and these antigen-specific B10 cells promote anti-inflammatory responses.

Mutual promotions between periodontitis and vascular calcification by rat animal model.

OBJECTIVE AND BACKGROUND: To study the relationship between periodontitis and vascular calcification by establishing rat model of chronic periodontitis and vascular calcification. METHODS: Forty male Wistar rats were divided into four groups randomly: control group, periodontitis group, vascular calcification group, and compound periodontitis and calcification group. Each group rats accepted the corresponding manages to establish the animal model. Clinical examinations and hematoxylin and eosin staining of periodontal tissue were taken to test the periodontal model; calcium assay, alkaline phosphatase activity, expression of mineral-related factors including osteopontin, alkaline phosphatase, core-binding factor-α1 and bone sialoprotein, hematoxylin and eosin staining and von Kossa staining of vascular tissue were taken to test the vascular calcification model; inflammatory factors including C-reactive protein, interleukin-1ß, tumor necrosis factor-α, interleukin-6, prostaglandin E2, and serum lipid in serum were also detected at the same time. RESULTS: The rat model was established. Inflammation of periodontal tissue and alveolar bone resorption in compound group and periodontitis group were more obvious than those in control group and vascular calcification group (P < .05). However, the calcium assay, alkaline phosphatase activity, and mineralized deposition in vascular calcification group and compound group were higher than those in control group and periodontitis group (P < .05), and compound group were the highest (P < .05); as for serum lipid, the level of total cholesterol and low-density lipoprotein-cholesterol in compound group and vascular calcification group were higher than that in control group and periodontitis group (P < .05), and compound group was the highest (P <.05); but the level of high-density lipoprotein cholesterol was higher in control group and periodontitis group. Inflammatory factors expression in serum were higher in compound group and periodontitis group, while mineral-related factors expression were higher in compoundgroup and vascular calcification group. CONCLUSION: There are some mutual promotions between periodontitis and vascular calcification, which might be related to the increasing inflammatory factors, lipids level, and mineral-related factors.

RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling.

BACKGROUND: The present study was to determine the effect of local anti-RANKL antibody administration in the presence or absence of microRNA-146a on ligature-induced peri-implant bone resorption, and the potential role of TLR2/4 signaling in such effect. RESULTS: Titanium implants were placed in the left maxilla alveolar bone 6 weeks after extraction of first and second molars in C57/BL6 wild-type (WT) and TLR2-/- TLR4-/- (TLR2/4 KO) mice. Silk ligatures were tied around the implants 4 weeks after implantation. Anti-RANKL antibody (500 µg/mL) with or without microRNA 146a (miR-146a) (100 nM) was injected into palatal gingiva around implant on days 3, 6, and 9 during 2 weeks of ligation period. Bone resorption around the implants was assessed by 2D imaging using area measurement and 3D imaging using micro-computed tomography (µCT). Real-time quantitative PCR (RT-qPCR) was used to determine the peri-implant gingival mRNA expression levels of pro-inflammatory cytokines (TNF-α) and osteoclastogenesis-related cytokines (RANKL). In both WT and TLR2/4 KO mice, the bone resorption around implants was significantly increased in the ligation only group when compared to the non-ligation group, but TLR2/4 KO mice showed significantly less bone loss compared to WT mice after ligation. As expected, gingival injection of anti-RANKL antibody significantly reduced bone loss compared with the ligation only group in both WT and TLR2/4 KO mice. Moreover, injection of miR-146a in addition to anti-RANKL antibody significantly enhanced the inhibition of bone loss in WT mice but not in TLR2/4 KO mice. Gingival mRNA expressions of RANKL were significantly reduced by anti-RANKL antibody treatment in both WT and TLR2/4 KO mice but were not affected by the additional miR-146a treatment. Gingival mRNA expression of TNF-α was significantly reduced by miR-146a treatment in WT mice but not in TLR2/4 KO mice. The number of gingival inflammatory cell infiltration and peri-implant TRAP-positive cell formation was significantly reduced by the additional miR-146a treatment in WT mice but not in TLR2/4 KO mice. CONCLUSIONS: This study suggests that anti-inflammatory miR-146a enhance anti-RANKL-induced inhibition of peri-implant bone resorption through the regulation of TLR2/4 signaling and inhibition of TNF-α expression.

MMP9 protects against LPS-induced inflammation in osteoblasts.

The matrix metalloproteinase (MMP) family is widely involved in the destruction of the pulp and apical tissues in the inflammatory process. MMP9 is closely related to oral inflammation. Nevertheless, the specific function of MMP9 during oral inflammation, as well as its mechanism, is not well understood. Our previous studies found that in experimentally induced apical periodontitis, more severe inflammation occurred in MMP9 knockout mice compared with the wild type mice. Moreover, the pathology phenomenon of alveolar bone destruction was even more evident in MMP9 knockout mice compared with the wild type mice. We proposed that MMP9 has "anti-inflammatory" properties. We aimed to study the effects of MMP9 on inflammatory response as well as on bone formation and bone destruction. We found a specific relationship between MMP9 and inflammation. qRT-PCR and Western blot revealed that the production of IL-1ß, TNF-α, RANK, RANKL, TLR2, and TLR4 was reduced by MMP9 in LPS-stimulated MC3T3-E1 cells. Meanwhile, the expressions of OPG and OCN were increased by MMP9 in LPS-stimulated cells. MMP9 plays a protective role in LPS-induced inflammation, thereby providing new clues to the prevention and treatment of apical periodontitis.

[Intramuscular type of nodular fasciitis: a report of 3 cases and review of literature].

PURPOSE: To retrospectively analyze the clinical characteristics of impacted supernumerary teeth in 115 patients. METHODS: One hundred and fifteen patients with im-pacted supernumerary teeth who were admitted to the Department of Oral and Max-illofacial Surgery of Hefei Stomatological Hospital were selected randomly. The age, sex, number of teeth, location, direction, clinical manifestation, anaes-thesia method and operation time were analyzed retrospectively, T test and Chi-square test were used to determine the statistical differences with SPSS 19.0 software package. RESULTS: Among 115 patients, there were 176 impacted supernu-merary, most of them were in mixed dentition period (66.96%), the sex ratio was 2.29:1, and Most patients (59.1%) had one supernumerary tooth, followed by two supernumerary teeth(33.9%). Most supernumerary teeth were located in the middle of the maxilla (68.2%). Inverted ones were the most common (52.8%). The most common symptoms were delayed eruption, displacement, crowding, torsion and space of the adjacent teeth. 92.2% of patients underwent general anesthesia. The dee-per the locations of impacted supernumerary were, the longer the operation time was. CONCLUSIONS: There are regional characteristics of supernumerary teeth in Hefei City, which can provide a reference for clinical diagnosis and treatment.

[Cytotoxicity of carboxymethyl chitosan zinc and peptide to human periodontal ligament cells].

PURPOSE: The purpose of this investigation was to study the cytotoxicity of carboxymethyl chitosan zinc and peptide to human periodontal ligament cells in vitro, in order to provide a reference for understanding materials' safety. METHODS: The primary cells were obtained from human periodontal tissues and cultured. The cultured cells were identified by observing the shape under microscope and by immunocytochemical method. HPDLCs were cultured in different concentrations of the tested materials extract and the activity of cells was evaluated using CCK-8 assay. The cytotoxicity grade was determined in term of the cell relative growth rates. The concentration of the tested materials extract was 100%, 75%, 50% and 25% in group 1-4, respectively. The fifth group had no materials. All statistical analysis was performed using SPSS 17.0 software package. RESULTS: The primary cells owned fibroblasts' shape. Immunocytochemical analysis showed the cells were stained positively to antibodies against vimentin, and negatively to antibodies against cytokeratin, which indicated that they were external embryo mesenchymal cell. The cell relative growth rates were more than 100%, no matter the concentrations of materials extract were. The cytotoxicity grade of CMC-Zn+-P was 0. CONCLUSIONS: CMC-Zn+-P exhibits no cytotoxicity to HPDLCs in vitro, which meets the requirements of the national standard.

[Expression of OPG/RANK/RANKL in the rat dental pulp tissue of periodontitis combined with vascular calcification and its clinical significance].

PURPOSE: To study the expression and possible role of OPG/RANK/RANKLin the rat dental pulp of periodontitis combined with vascular calcification. METHODS: Thirty-six male Wister rats were randomly divided into 4 groups: control group(group C), periodontitis group(group CP), vascular calcification group(group VDN) and compound group(group CP+VDN). Each group underwent corresponding management to establish animal model. When the model was successful, the maxillae including molars were sectioned, pulp tissue was examined by H-E staining; Immunohistochemical staining method was used to evaluate the expression and ratio of OPG and RANKL in pulp tissues. Statistical analysis was carried out using SPSS 19.0 software package. RESULTS: The pulp tissue of group CP, VDN, CP+VDN showed varied degrees of damage, neutrophil infiltration, pulp vascular congestion, odontoblasts vacuolar changes, pulp necrosis by H-E staining, and the changes in CP+VDN group was the most significant, followed by CP group, VDN group. Immunohistochemistry showed OPG in pulp tissues in group CP, VDN, CP+VDN were significantly lower than that in normal group (P<0.05), and the expression in group CP+VDN was the least;Expression of RANKL in pulp tissues in group CP, VDN, CP+VDN were significantly higher than that in normal group(P<0.05)，and the expression in group CP+VDN was the highest. The ratio of OPG/RANKL in normal group was the highest, and the ratio in CP+VDN group was the lowest. CONCLUSIONS: Periodontitis and vascular calcification can damage the pulp tissue, periodontitis compound with vascular calcification may aggravate the injury; OPG/RANKL/RANK system may play an important role in pulp tissue injury.

[The effect of carboxymethyl chitosan zinc and peptide on IL-1,TNF-α and PGE-2 in gingival crevicular fluid of miniature pigs].

PURPOSE: This experiment was aimed at exploring whether carboxymethyl chitosan zinc and peptide （CMC-Zn(+)-P） can reduce the occurrence and development of periodontal tissue inflammation effectively by observing the change of IL-1,TNF-α and PGE-2 level in gingival crevicular fluid （GCF） before and after brushing, so as to find a new effective material in preventing and treating periodontal diseases. METHODS: Miniature pigs were selected as experimental subjects and divided into 4 groups randomly: the control group; CMC-Zn(+)-P group (material group);brushing group; brushing + CMC-Zn(+)-P group (composite group). Gingival crevicular fluid before and one month after the experiment was collected. The levels of IL-1, TNF-α and PGE-2 were examined by enzyme-linked immune-sorbent assay, while the clinical periodontal index was recorded. SPSS 18.0 software package was used for statistical analysis. RESULTS: There was no significant difference in levels of IL-1, TNF-α and PGE-2 and clinical periodontal index between the 4 groups before experiment. After one month, the levels of IL-1, TNF-α, PGE-2 in GCF had significant difference between 4 groups. The levels of IL-1, TNF-α, PGE-2 in composite group were significant lower than that of the other three groups (P<0.008).The levels of IL-1, TNF-α and PGE-2 in the material group and brushing group were significantly lower than that of the control group (P<0.008). Compared with materials group, the brushing group had significantly lower level of IL-1,significantly higher level of PGE-2 ,but no difference in the level of TNF-α.In addition, the teeth calculus index of composite group was significantly lower than that of other groups (P<0.05). CONCLUSIONS: CMC-Zn(+)-P can effectively reduce periodontal tissue inflammation and cut down the speed of deposition of dental calculus. If used cooperatively with brushing, the effect will be better.

Porphyromonas gingivalis Lipopolysaccharide Stimulation of Vascular Smooth Muscle Cells Activates Proliferation and Calcification.

BACKGROUND: Porphyromonas gingivalis (Pg) is a major etiologic agent of periodontitis, whose virulence has been attributed to different factors, including lipopolysaccharide (LPS). Vascular ectopic calcification as a well-known major risk factor for adverse cardiovascular diseases is a highly prevalent vascular pathophenotype, and vascular smooth muscle cells (VSMCs) play an important role in mediating vascular calcification. It was hypothesized that Pg-LPS may stimulate vascular calcification through a direct effect on VSMC function. To test this hypothesis, the effect of Pg-LPS on VSMC calcification was determined. METHODS: Primary cultures of VSMCs were obtained and identified by immunochemistry in vitro. The proliferation and alkaline phosphatase (ALP) activity of VSMCs were measured using a cell counting kit and an ALP activity test. Mineral deposition was examined using alizarin red staining. Gene (e.g. ALP, core binding factor α1 [Cbfα1], bone sialoprotein [BSP], and osteopontin [OPN]) expression levels altered by Pg-LPS were determined by reverse transcription-polymerase chain reaction array. RESULTS: Pg-LPS could increase the proliferation of VSMCs at different times and enhance ALP activity of VSMCs after 1 day. Alizarin red staining and quantification showed that, with Pg-LPS treatment, VSMCs displayed more obvious calcification nodules. When stimulated with Pg-LPS, the expression of specific osteogenic genes (e.g., ALP, Cbfα1, BSP, and OPN) was significantly promoted in the presence or absence of mineralization-inducing medium, whereas the expression of the OPN gene was inhibited in the mineralization induction group at day 7. CONCLUSION: Pg-LPS can stimulate VSMC calcification, which results in vascular calcification, further proving the precise relationship between periodontitis and vascular calcification.

[Study of antimicrobial activity of carboxymethyl chitosan zinc complex material against cariogenic bacteria].

PUPPOSE: To evaluate the antimicrobial activity of carboxymethyl chitosan zinc (CMCSZ) and carboxymethyl chitosan zinc-active peptide complex material (CMCSZP) on oral cariogenic bacteria. METHODS: Agar dilution method and K-B disk diffusion susceptibility agents were used to measure the antimicrobial activity of two agents against S.mutans, Lactobacillus, S.sanguinis and Actinomyces viscosus. The former method was used to measure the minimal inhibitory concentration (MIC) and latter was used to measure the inhibitory zone. The effects of pH value, temperature, light, ultraviolet and storage temperature on the active substances were investigated to determine the stability of CMCSZ and CMCSZP. SPSS17.0 software package was used for statistical analysis. RESULTS: All the bacteria were susceptible to active peptide, CMCSZ and CMCSZP with the MIC of CMCSZ/CMCSZP being 625, 1250, 1250 and 2500 mg/L, respectively. At the same concentration, the inhibitory zone of CMCSZP was significantly bigger than that of CMCSZ. Acidic conditions were conducive to increase the antimicrobial activity of CMCSZ, while the effect on CMCSZP was not significant. CMCSZ and CMCSZP exhibited good stability against light, but their antimicrobial activity gradually weakened as the bath temperature rising. In the temperature of 85 degrees centigrade, their antibacterial activity disappeared. CONCLUSIONS: CMCSZ have CMCSZP had strong antimicrobial activity against 4 kinds of cariogenic bacteria. They have good stability against light, but poor thermal stability. This study provides theoretical foundation for the application of CMCSZ/CMCSZP in prevention of cariogenic diseases.