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.

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.

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.

Co-culture with periodontal ligament stem cells enhanced osteoblastic differentiation of MC3T3-E1 cells and osteoclastic differentiation of RAW264.7 cells.

OBJECTIVES: Periodontal ligament stem cells (PDLSCs) are characterized by having multipotential differentiation and immunoregulatory properties, which are the main mechanisms of PDLSCs-mediated periodontal regeneration. Periodontal or bone regeneration requires coordination of osteoblast and osteoclast, however, very little is known about the interactions between PDLSCs and osteoblast-like cells or osteoclast precursors. In this study, the indirect co-culture approach was introduced to preliminarily elucidate the effects of PDLSCs on differentiation of osteoblast-like cells and osteoclast precursors in vitro. MATERIALS AND METHODS: Human PDLSCs were obtained from premolars extracted and their stemness was identified in terms of their colony-forming ability, proliferative capacity, cell surface epitopes and multi-lineage differentiation potentials. A noncontact co-culture system of PDLSCs and preosteoblastic cell line MC3T3-E1 or osteoclast precursor cell line RAW264.7 was established, and osteoblastic differentiation of MC3T3-E1 and osteoclastic differentiation of RAW264.7 were evaluated. RESULTS: PDLSCs exhibited features of mesenchymal stem cells. Further investigation through indirect co-culture system showed that PDLSCs enhanced ALP activity, expressions of ALP, Runx2, BSP, OPN mRNA and BSP, OPN proteins and mineralization matrix deposition in MC3T3-E1. Meanwhile, they improved maturation of osteoclasts and expressions of TRAP, CSTK, TRAF6 mRNA and TRAP, TRAF6 proteins in RAW264.7. CONCLUSIONS: PDLSCs stimulates osteoblastic differentiation of osteoblast precursors and osteoclastic differentiation of osteoclast precursors, at least partially, in a paracrine fasion.

Expression of Osterix in mechanical stress-induced osteogenic differentiation of periodontal ligament cells in vitro.

Osterix (Osx) is an osteoblast-specific transcription factor required for the differentiation of pre-osteoblasts into functional osteoblasts. This study sought to examine the changes of Osx expression in periodontal ligament cells (PDLC) subjected to mechanical force, and to investigate whether Osx is involved in the mechanical stress-induced differentiation of PDLC. Human PDLC were exposed to centrifugal force for 1-12 h. Real-time polymerase chain reaction (PCR), western blot, and immunofluorescence assays were used to examine the mRNA and protein expression of Osx and its subcellular localization. Furthermore, PDLC were transfected with the expression vector pcDNA3.1 flag-Osx and subjected to mechanical force for 6 h. The changes in alkaline phosphatase (ALP) activity and in the expression of core-binding factor alpha1 (Cbfa1), ALP, osteopontin, bone sialoprotein, osteocalcin, and collagen I were measured. After the application of mechanical force, Osx was upregulated in a time-dependent manner at both mRNA and protein levels, and Osx protein was translocated from the cytosol into the cell nuclei. Overexpression of Osx did not affect the expression of Cbfa1, but it significantly enhanced the ALP activity and the mRNA expression of all the aforementioned osteogenic marker genes, all of which increased further under mechanical stress. These results suggest that Osx might play an important role in the mechanical stress-induced osteogenic differentiation of PDLC and therefore be involved in alveolar bone remodeling during orthodontic therapy.

[Expression of mineral-associated factors in the mineral stage tooth germ of mice].

PURPOSE: To study the distribution and expression of core binding factor alpha 1 (Cbf alpha1), bone sialoprotein (BSP), osteopontin (OPN), osteocalcin (OC) and alkaline phosphatase (ALP) in mineral stage tooth germ of mice, so as to understand their roles in the development of the mineral stage tooth germ. METHODS: Thirty BALB/c mice in different days were killed, and their bilateral mandibular first molar germs with surrounding alveolar bone were taken out, then the tissues were fixed with 4% paraformaldehyde at 4 degrees C overnight, dehydrated, embedded in paraffin and serially sectioned at 5 microm. Immunohistochemical assay was adopted to determine the tissue distribution and cellular localization of Cbf alpha1, OPN, BSP, ALP and OC in the mineral stage tooth germ by these sections of BALB/c mice. RESULTS: There were Cbf alpha1, BSP and OPN expressing in the alveolar bone, and Cbf alpha1, ALP, OC in predentin, while in other tissues such as dental follicle, pulp cells, stratum intermedium, stellate reticulum, only ALP and OC were detected, but none of them were observed in the dentin. CONCLUSIONS: The formation mechanisms of hard tooth tissues are different. They are controlled by different factors. Cbf alpha1, ALP and OC are involved in the early formation of dentin, while Cbf alpha1, OPN and BSP involved in the early formation of alveolar bone, ALP involved in the stratum intermedium. They all play an important role in the start of mineralization.

[Expression of epidermal growth factor receptor in human periodontal ligament cells during their mineralization in vitro].

OBJECTIVE: To investigate the expression of epidermal growth factor receptor (EGFR) during the mineralization of human periodontal ligament cells (hPDLC) in vitro. METHODS: Studies using specific antibodies to immunolocalize EGFR in the mineral differentiating hPDLC were undertaken to investigate the different expression during the inducing process. In situ hybridization and RT-PCR technique were used to investigate the transcripts encoding the protein of EGFR. RESULTS: The results showed that immunocytochemical labeling gradually decreased following the elong of the induce time, downing to nearly negative at the 4th week and the signal of EGFR transcripts was weaker in the induced hPDLC than that in uninduced. CONCLUSION: EGFR has a negative regulation function during the mineralization of hPDLC.

[Expression of core binding factor a1, bone morphogenetic proteins and osteopontin in the developing periodontal tissues of mice].

OBJECTIVE: To study the expression and interaction of core binding factor a1 (Cbfa1), bone morphogenetic proteins (BMPs) and osteopontin (OPN) in the developing periodontal tissues of mice. METHODS: A mice developing periodontal tissues study model was created histologically by 5-27 day postnatal BALB/c mice, then the immunohistochemical localization of Cbfa1, BMPs and OPN in different developing stages were undertaken. RESULTS: In early stage of postnatal mice periodontal tissues development, only BMPs expressed in dental follicle cells, though the signal was weak. When root was forming, all of them were expressed in periodontal ligament cells and cementoblasts, while only OPN in acellular cementum, cellular cementum and the surface of alveolar bone, Cbfa1 only in cellular cementum and BMPs was seen in neither acellular cementum nor cellular cementum. CONCLUSION: Cbfa1, BMPs and OPN all involve in the development of periodontal tissues, while OPN is crucial for cementum.

[Expression of Runx2/Cbfa1 in the developing pulpo-dentinal complex of postnatal mice].

OBJECTIVE: To study the expression of Runx2/Cbfa1 in the developing dentin and differentiating odontoblasts. METHODS: A postnatal mice teeth developing model was built histologically. Immunohistochemical technique was adopted to determine the expression of Runx2/Cbfa1 in the developing pulpo-dentinal complex in mice. RESULTS: Runx2/Cbfa1 was merely present in predentin in the exact and before the 11th day's postnatal stages. Meanwhile, it was positively located in odontoblasts and dental pulp cells in root region, but negatively in coral part after the 11th day's stages. CONCLUSION: Runx2/Cbfa1 may play an important role in the deposing of tooth dentin and in the differentiating of odontoblasts and pulp cells.