Proliferative effect of cannabidiol in human gingival fibroblasts via the mitogen-activated extracellular signal-regulated kinase (MEK) 1/2.

BACKGROUND AND OBJECTIVES: Cannabidiol exerts its anti-inflammatory and anti-oxidant activities in various human cells. However, its proliferative effect has not been extrapolated to human gingival fibroblasts (HGFs). This study aimed to determine the proliferative and promigratory effects of cannabidiol in HGFs and to elucidate the signaling mechanism(s). MATERIALS AND METHODS: HGFs, characterized by their CD73, CD90, and CD105 expressions by flow cytometry, were treated with cannabidiol at 0.01-30 µM. The cytotoxicity was determined by the MTT assay, while the proliferative effect was examined by the BrdU assay, immunoblot and immunofluorescence for cyclin D1 and Ki-67 expressions, respectively, and cell cycle analysis. The promigratory effect of cannabidiol was investigated by a wound healing assay. Phosphorylation of the p38 MAPK, JNK, and ERK upon treatment with cannabidiol was explored, and their involvement in cell proliferation and cyclin D1 and Ki-67 expressions was studied using pharmacological inhibitors. RESULTS: No toxicity was found in HGFs treated with any doses of cannabidiol up to 30 µM. The mean percentage of cell proliferation was significantly enhanced by treatment with cannabidiol at 3 or 10 µM (p < .001), consistent with upregulated expressions of cyclin D1 and Ki-67 and increased percentages of HGFs in the S and G2/M phases. Moreover, treatment with cannabidiol significantly induced cell migration (p < .05). The p38 MAPK and ERK1/2 were significantly activated by cannabidiol (p < .05), but only pretreatment with UO126, a MEK1/2 inhibitor, significantly inhibited cell proliferation and cyclin D1 and Ki-67 expressions (p < .05). CONCLUSION: Treatment with cannabidiol at non-toxic doses promotes HGFs' proliferation and migration.

An in vitro anti-inflammatory effect of Thai propolis in human dental pulp cells.

OBJECTIVE: To explore the potential for development of Thai propolis extract as a pulp capping agent to suppress pulpal inflammation from dental pulp infections. This study aimed to examine the anti-inflammatory effect of the propolis extract on the arachidonic acid pathway, activated by interleukin (IL)-1ß, in cultured human dental pulp cells. METHODOLOGY: Dental pulp cells, isolated from three freshly extracted third molars, were first characterized for their mesenchymal origin and treated with 10 ng/ml of IL-1ß in the presence or absence of non-toxic concentrations of the extract from 0.08 to 1.25 mg/ml, as determined by the PrestoBlue cytotoxic assay. Total RNA was harvested and analyzed for mRNA expressions of 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2). Western blot hybridization was performed to investigate COX-2 protein expression. Culture supernatants were assayed for released prostaglandin E2 levels. Immunofluorescence was conducted to determine involvement of nuclear factor-kappaB (NF-kB) in the inhibitory effect of the extract. RESULTS: Stimulation of the pulp cells with IL-1ß resulted in the activation of arachidonic acid metabolism via COX-2, but not 5-LOX. Incubation with various non-toxic concentrations of the propolis extract significantly inhibited upregulated COX-2 mRNA and protein expressions upon treatment with IL-1ß (p<0.05), resulting in a significant decrease in elevated PGE2 levels (p<0.05). Nuclear translocation of the p50 and the p65 subunits of NF-kB upon treatment with IL-1ß was also blocked by incubation with the extract. CONCLUSIONS: Upregulated COX-2 expression and enhanced PGE2 synthesis upon treatment with IL-1ß in human dental pulp cells were suppressed by incubation with non-toxic doses of Thai propolis extract via involvement of the NF-kB activation. This extract could be therapeutically used as a pulp capping material due to its anti-inflammatory properties.

An in vitro anti-inflammatory effect of Thai propolis in human dental pulp cells

Abstract Objective To explore the potential for development of Thai propolis extract as a pulp capping agent to suppress pulpal inflammation from dental pulp infections. This study aimed to examine the anti-inflammatory effect of the propolis extract on the arachidonic acid pathway, activated by interleukin (IL)-1β, in cultured human dental pulp cells. Methodology Dental pulp cells, isolated from three freshly extracted third molars, were first characterized for their mesenchymal origin and treated with 10 ng/ml of IL-1β in the presence or absence of non-toxic concentrations of the extract from 0.08 to 1.25 mg/ml, as determined by the PrestoBlue cytotoxic assay. Total RNA was harvested and analyzed for mRNA expressions of 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2). Western blot hybridization was performed to investigate COX-2 protein expression. Culture supernatants were assayed for released prostaglandin E2 levels. Immunofluorescence was conducted to determine involvement of nuclear factor-kappaB (NF-kB) in the inhibitory effect of the extract. Results Stimulation of the pulp cells with IL-1β resulted in the activation of arachidonic acid metabolism via COX-2, but not 5-LOX. Incubation with various non-toxic concentrations of the propolis extract significantly inhibited upregulated COX-2 mRNA and protein expressions upon treatment with IL-1β (p<0.05), resulting in a significant decrease in elevated PGE2 levels (p<0.05). Nuclear translocation of the p50 and the p65 subunits of NF-kB upon treatment with IL-1β was also blocked by incubation with the extract. Conclusions Upregulated COX-2 expression and enhanced PGE2 synthesis upon treatment with IL-1β in human dental pulp cells were suppressed by incubation with non-toxic doses of Thai propolis extract via involvement of the NF-kB activation. This extract could be therapeutically used as a pulp capping material due to its anti-inflammatory properties.

Expression of human caspase-4 in the gingival epithelium affected with periodontitis: Its involvement in Porphyromonas gingivalis-challenged gingival epithelial cells.

OBJECTIVE: Implication of human caspase-4 in periodontitis and in sensing periodontal pathogens by gingival epithelial cells (GECs) is unclear. This study aimed to determine caspase-4 and interleukin (IL)-18 expressions in gingival tissues affected with periodontitis and to investigate caspase-4 involvement in mediating innate immune responses in GECs. DESIGN: Ex vivo, caspase-4 and IL-18 expressions in gingival biopsies, obtained from healthy participants with periodontitis or clinically healthy gingiva (N = 20 each), were determined by immunohistochemistry. In vitro, caspase-4 activation in cultured GECs stimulated with Porphyromonas gingivalis or Fusobacterium nucleatum was analyzed by immunoblotting. mRNA expressions of human ß-defensin-2 (hBD-2), IL-8, and IL-18 in stimulated GECs in the presence or absence of a caspase-4 inhibitor were assayed by RT-qPCR. RESULTS: Ex vivo, compared with healthy gingival epithelium, the epithelium affected with periodontitis displayed a significant decrease in caspase-4 expression (P = 0.015), whereas IL-18 expression was significantly increased (P = 0.012). Moreover, the expression of caspase-4, but not IL-18, was found to be a predictor of periodontitis (P = 0.007). In vitro, caspase-4 was activated in cultured GECs challenged with P. gingivalis, but not F. nucleatum. mRNA upregulations of hBD-2, IL-8, and IL-18 upon P. gingivalis stimulation were significantly reduced when caspase-4 was inhibited (P < 0.05), whereas the inhibitor failed to suppress those inductions by F. nucleatum. CONCLUSIONS: Caspase-4 expression is diminished in the epithelium affected with periodontitis while that of IL-18 is enhanced. Caspase-4 activation in P. gingivalis-infected GECs upregulates the three innate immune effector molecules, suggesting a possible sensing mechanism of caspase-4 in GECs in periodontal disease pathogenesis.

Overexpression of methyltransferase-like 3 and 14 in oral squamous cell carcinoma.

OBJECTIVES: This study aimed to determine expressions of methyltransferase-like 3 (METTL3) and METTL14, two enzymes essential for mRNA methylation at the adenosine (m6 A), in oral squamous cell carcinoma (OSCC) and to investigate in vitro aggressiveness of their aberrant expressions. METHODS: METTL3 and METTL14 expressions in 50 OSCC and 11 normal oral tissues were examined by immunohistochemistry. METTL3 and METTL14 expressions and m6 A amounts were determined in three OSCC cell lines, including HN5, HN6, and HN15. Cell proliferation, migration, and invasion were studied by BrdU, wound healing, and Transwell chamber assays, after silencing of METTL3, METTL14, or both by siRNA transfection. RESULTS: Immunostaining of METTL3 and METTL14 was localized in cancer cell nuclei. The mean percentages of METTL3- and METTL14-positive cells were significantly increased in OSCC tissues (p < 0.001). The percentages of METTL3- and METTL14-positive cells were correlated with the advanced pTNM stages (p < 0.05) and with the degrees of histopathological differentiation in OSCC (r = 0.564 and r = 0.316, respectively; p < 0.001). By the COX multivariate analysis, both overexpressed METTL3 and METTL14 were significantly associated with short overall survival (p < 0.05). Both METTL3 and METTL14 expressions and the m6 A amounts were significantly increased in HN6 (p < 0.05). Silencing of METTL3 and METTL14 in HN6 significantly inhibited cell proliferation (p < 0.01), but it failed to mitigate cell migration or invasion. CONCLUSIONS: METTL3 and METTL14 are overexpressed in OSCC tissues and in the HN6 OSCC cell line that promotes cell proliferation. Overexpressed METTL3 or METTL14 is found to be an independent prognostic factor for short overall survival in patients with OSCC.

Relationship Between O-GlcNAcase Expression and Prognosis of Patients With Osteosarcoma.

Several studies have demonstrated a role of O-GlcNAcylation (O-GlcNAc) in tumorigenesis of various carcinomas by modification of tumor-associated proteins. However, its implication in the pathogenesis of osteosarcoma remains unclear. This study aimed to investigate the levels of O-GlcNAc and the expressions of O-linked N-acetylglucosamine transferase (OGT) and O-GlcNAcase (OGA) in human osteosarcoma tissues, by using immunohistochemistry; and to find correlations between the levels or expressions and several clinicopathologic parameters. There were 109 first diagnosed osteosarcoma patients, including Enneking stage IIB (n=70) and III (n=39). Correlations between the immunoreactive score (IRS) and clinicopathologic parameters, overall survival, and metastasis-free survival were evaluated. A positive correlation was found between the IRS of OGA and the percentage of postchemotherapeutic tumor necrosis (r=0.308; P=0.017). Univariate analysis revealed significantly lower OGA IRS in metastatic patients (P=0.020) and poor chemotherapeutic-responder patients (P=0.001). By multivariate analysis, presence of tumor metastasis (P=0.002) and lower OGA IRS (P=0.004) was significantly associated with shorter overall survival. Subgroup analysis in stage IIB osteosarcoma (n=70) demonstrated that male sex (P=0.019), presence of tumor recurrence (P=0.026), poor chemotherapeutic responder (P=0.022), and lower OGA IRS (P=0.019) were significantly correlated with short metastasis-free survival. But, lower OGA IRS was the only independent predictor for short metastasis-free survival (P=0.006). Our findings suggested that O-GlcNAc pathway, especially OGA, may involve in pathogenesis and aggressiveness of osteosarcoma. Low level of OGA expression may be used as a poor prognostic indicator.

Anticancer Activities of Hesperidin via Suppression of Up-Regulated Programmed Death-Ligand 1 Expression in Oral Cancer Cells.

Up-regulated expression of programmed death-ligand 1 (PD-L1) by interferon-gamma (IFN-γ) has been associated with promotion of cancer cell survival and tumor cell escape from anti-tumor immunity. Therefore, a blockade of PD-L1 expression can potentially be used as a molecular target for cancer therapy. The aim of this study was to investigate whether suppression of IFN-γ induced PD-L1 expression in two oral cancer cell lines, HN6 and HN15, by hesperidin effectively decreased cell proliferation and migration. Further, our objective was to elucidate the involvement of the signal transducer and activator of transcription 1 (STAT1) and STAT3 in the inhibition of induced PD-L1 expression by hesperidin. Our findings indicate that IFN-γ induced expression of PD-L1 protein in HN6 and HN15 via phosphorylation of STAT1 and STAT3 and that hesperidin significantly reduced that induction through suppression of phosphorylated STAT1 and STAT3 in both cell lines. Moreover, hesperidin also significantly decreased the viability, proliferation, migration, and invasion of both cell lines. In conclusion, hesperidin exerted anticancer effects against oral cancer cells through the suppression of PD-L1 expression via inactivation of the STAT1 and STAT3 signaling molecules. The findings of this study support the use of hesperidin as a potential adjunctive treatment for oral cancer.

Efficacy of double antibiotics in hydroxypropyl methylcellulose for bactericidal activity against Enterococcus faecalis and Streptococcus gordonii in biofilm.

OBJECTIVE: This study aimed to investigate the efficacy of double antibiotics, including ciprofloxacin and metronidazole, in a new vehicle, hydroxypropyl methylcellulose (HPMC), against Enterococcus faecalis and Streptococcus gordonii grown in biofilm. DESIGN: Human mandibular premolars were prepared and divided into four groups: (i) negative control, (ii) positive control, (iii) infected with E. faecalis and S. gordonii for 21 days and intracanally medicated with double antibiotics in HPMC, and (iv) infected with E. faecalis and S. gordonii for 21 days and intracanally medicated with calcium hydroxide (UltraCal™). The efficacy of medication for 14 or 28 days was determined by bacterial cultures and RT-qPCR for absolute quantities of E. faecalis and S. gordonii cDNA and for relative mRNA expressions of pbp5 and gtfG genes. RESULTS: There were significant decreases in the mean colony forming units and mean cDNA amounts of E. faecalis and S. gordonii in group (iii) on days 14 and 28 compared to those in group (ii) (p < 0.01). However, the mean cDNA amounts of E. faecalis and S. gordonii in group (iv) were found to be significantly increased on day 28 (p < 0.05). The mRNA expression of gtfG was significantly decreased in groups (iii) and (iv) on days 14 and 28, whereas that of pbp5 was significantly increased in group (iv) on days 14 and 28 (p < 0.01). CONCLUSION: Double antibiotics in HPMC gel showed an in vitro efficacy against E. faecalis and S. gordonii grown in biofilm, suggesting its clinical application as an intracanal medicament for both primary and persistent infections.

Involvement of the A disintegrin and metalloproteinase 9 in oral cancer cell invasion.

The aims of this study were to determine the functional roles of the transmembrane glycoprotein, Disintegrin and metalloproteinase domain-containing protein 9 (ADAM 9), in the phosphorylation of epidermal growth factor receptor (EGFR) and AKT and in the aggressiveness of oral cancer cells. Immunohistochemistry and immunoblotting were conducted to determine expression of ADAM 9 and the levels of EGFR phosphorylated at the tyrosine 1173 residue (p-EGFRtyr1173 ) and AKT phosphorylated at the serine 473 residue (p-AKTser473 ) in oral cancer tissues and in the oral cancer cell lines HN5, HN6, HN15, and HN008. Small interfering RNA (siRNA) was used to inhibit expression of ADAM9 mRNA, and thus production of ADAM9 protein, in oral cancer cells. ADAM9-knockdown cells were examined for p-EGFRtyr1173 and p-AKTser473 levels and used for cell proliferation and invasion assays. A positive correlation among overexpression of ADAM 9, p-EGFRtyr1173 , and p-AKTser473 was found in oral cancer tissues. These biomolecules were also overexpressed in HN6 and HN15 cell lines. Expression of ADAM9 in HN6 and HN15 cells was statistically significantly inhibited by siRNA against ADAM9 mRNA (siADAM9) compared with the negative-control siRNA (scramble). The levels of p-AKTser473 , but not those of p-EGFRtyr1173 , were statistically significantly blocked by siADAM9. Although the proliferation rates of ADAM9 knocked-down HN6 and HN15 cells did not differ from those of cells exposed to scramble, a statistically significant decrease in cell invasion was found in these ADAM9-silenced cells. These results suggest a functional role of the ADAM 9/AKT signaling pathway in oral cancer cell invasion, which may be beneficial as a therapeutic target of oral cancer.

Juberg-Hayward syndrome is a cohesinopathy, caused by mutation in ESCO2.

BACKGROUND: Juberg-Hayward syndrome (JHS; MIM 216100) is a rare autosomal recessive malformation syndrome, characterized by cleft lip/palate, microcephaly, ptosis, short stature, hypoplasia or aplasia of thumbs, and dislocation of radial head and fusion of humerus and radius leading to elbow restriction. OBJECTIVE: To report for the first time the molecular aetiology of JHS. PATIENT AND METHODS: Clinical and radiographic examination, whole exome sequencing, Sanger sequencing, mutant protein model construction, and in situ hybridization of Esco2 expression in mouse embryos were performed. RESULTS: Clinical findings of the patient consisted of repaired cleft lip/palate, microcephaly, ptosis, short stature, delayed bone age, hypoplastic fingers and thumbs, clinodactyly of the fifth fingers, and humeroradial synostosis leading to elbow restriction. Intelligence is normal. Whole exome sequencing of the whole family showed a novel homozygous base substitution c.1654C>T in ESCO2 of the proband. The sister was homozygous for the wildtype variant. Parents were heterozygous for the mutation. The mutation is predicted to cause premature stop codon p.Arg552Ter. Mutations in ESCO2, a gene involved in cohesin complex formation, are known to cause Roberts/SC phocomelia syndrome. Roberts/SC phocomelia syndrome and JHS share similar clinical findings, including autosomal recessive inheritance, short stature, cleft lip/palate, severe upper limb anomalies, and hypoplastic digits. Esco2 expression during the early development of lip, palate, eyelid, digits, upper limb, and lower limb and truncated protein model are consistent with the defect. CONCLUSIONS: Our study showed that Roberts/SC phocomelia syndrome and JHS are allelic and distinct entities. This is the first report demonstrating that mutation in ESCO2 causes JHS, a cohesinopathy.

Significant elevation of salivary human neutrophil peptides 1-3 levels by probiotic milk in preschool children with severe early childhood caries: a randomized controlled trial.

OBJECTIVES: To determine salivary human neutrophil peptides 1-3 (HNP1-3) levels in caries-free preschool children and in those with early childhood caries (ECC) or severe-ECC, in a daily probiotic group, receiving reconstituted milk with the probiotic Lactobacillus paracasei SD1 once daily; a triweekly probiotic group, receiving the probiotic milk 3 days a week; and a placebo group. MATERIALS AND METHODS: Oral examination and unstimulated whole saliva collection were conducted in 354 children at baseline, 6 months after intervention (T6), and after probiotic discontinuation (T12). Of the 354, adequate volume of saliva samples from 268 children were simultaneously analyzed for Streptococcus mutans and total lactobacilli levels using qPCR and for HNP1-3 levels using ELISA. RESULTS: In the severe-ECC status, significant increases in the median HNP1-3 levels at T12 were found in both daily and triweekly probiotic groups (p < 0.001). The median S. mutans levels in the daily group were significantly decreased at T6 and T12 (p < 0.01), whereas the median total lactobacilli levels were significantly increased at T6 (p < 0.001). Significantly inverse correlations between altered HNP1-3 and S. mutans levels and significant decreases in caries progression were found in both probiotic groups (p < 0.05). CONCLUSIONS: In the severe-ECC status, daily or triweekly consumption of L. paracasei SD1 significantly enhanced salivary HNP1-3 levels, but reduced S. mutans levels, possibly resulting in reduction of caries progression. CLINICAL RELEVANCE: Significant enhancement of salivary HNP1-3 levels by probiotic consumption is associated with reduction in S. mutans levels, consistent with diminished caries progression in children with severe-ECC.

Efficacy of Probiotic Milk for Caries Regression in Preschool Children: A Multicenter Randomized Controlled Trial.

This multicenter double-blind randomized controlled trial aimed to determine the efficacy of daily or triweekly consumption of reconstituted milk powder, containing Lactobacillus paracaseiSD1, in preschool children for caries prevention. A 6-month intervention was conducted in 487 children (aged 37.6 ± 9.2 months) with ≤4 decayed teeth from 8 childcare centers. Using stratified block randomization, participants were randomly allocated into 3 groups, comprising: (1) daily probiotic, receiving probiotic milk once daily, (2) triweekly probiotic, randomly receiving probiotic milk 3 days a week and the placebo milk for the remaining 4 days, and (3) placebo, receiving milk without probiotics. Each tooth surface was assessed for caries status using the modified Nyvad criteria at baseline (T0), 6 (T6) and 12 (T12) months after T0. Study outcomes were caries transition, including caries progression and regression during the T0-T6, T6-T12 and T0-T12 periods. Negative binomial regression with a generalized linear model was used to estimate the caries outcomes, which were reported as incidence rate ratios (IRRs) and 95% confidence intervals (CIs). At baseline, caries prevalence and mean decayed surfaces were 81.9% and 7.29 ± 7.60, respectively. Three groups were balanced at baseline with an overall dropout rate of 25.2%. After adjusting for age, sex, and the number of noncavitated and cavitated caries at baseline, a decreased caries risk during T0-T6 was shown in both the daily and triweekly probiotic groups (adjusted IRR 0.83, 95% CI 0.72-0.96 in both groups) and during T0-T12 (adjusted IRR 0.84, 95% CI 0.76-0.94, and adjusted IRR 0.86, 95% CI 0.76-0.96, respectively) compared to the placebo group. In contrast, significantly increased regressive surfaces were demonstrated during T0-T6 by adjusted IRR 1.76 (95% CI 1.25-2.48) and 2.01 (95% CI 1.42-2.85) and during T0-T12 by adjusted IRR 1.98 (95% CI 1.50-2.61) and 1.92 (95% CI 1.45-2.55) for the daily and triweekly groups, respectively. In conclusion, probiotic milk consumption either daily or triweekly can modestly prevent new caries, but considerably reverse carious lesions, suggesting that a daily or triweekly dose interval is sufficient to reverse carious lesions.

Periodontal ligament enhancement in mesio-angulated impaction of third molars after orthodontic tooth movement: A prospective cohort study.

INTRODUCTION: The mesio-angulated impaction of mandibular third molars makes them unsuitable as donor teeth for tooth autotransplantation. However, uprighted molars may be applicable for autotransplantation. This study aimed to determine the amount of periodontal ligament (PDL) on the root surfaces of extracted third molars after an application of uprighting force and to examine the amount of PDL at the tension and compression sites. METHODS: In this prospective cohort study, 15 mesio-angulated mandibular third molars (iM8s) from 15 patients planned for orthodontic extraction, were uprighted using springs connected to miniscrews, whereas 15 nonopposing and fully erupted mandibular third molars from 15 other patients served as controls. The altered angulation was monitored and assessed from panoramic radiographs. All 30 molars, removed by simple extraction, were stained with 0.04% (w/v) toluidine blue to analyze the percentages of stained PDL on the root surfaces. RESULTS: An average period of 3.4 months was necessary to upright the iM8s at a mean rate of 8.3° per month. The mean percentage of stained PDL on the loaded iM8s was significantly greater than that on the unloaded molars (P <0.05). The mean percentages of stained PDL were significantly increased at the cervical and middle thirds and the buccal, mesial, and distal surfaces of the loaded iM8s compared with those of the unloaded molars (P <0.05), whereas the apical third and the lingual surface, corresponding to the compression sites, showed no significant increases. CONCLUSIONS: Orthodontic uprighting leads to significantly increased proliferative PDL on certain radicular portions and surfaces of iM8s, which might be useful for tooth autotransplantation.

Periodontal ligament proliferation and expressions of bone biomolecules upon orthodontic preloading: Clinical implications for tooth autotransplantation.

OBJECTIVE: Preservation of the periodontal ligament (PDL) is vital to the success of tooth autotransplantation (TAT). Increased PDL volumes and facilitated tooth extraction have been observed upon orthodontic preloading. However, it is unclear whether any changes occur in the expressions of bone biomolecules in the increased PDL volumes. This study aimed to determine the expressions of runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) in PDL upon preloading. METHODS: Seventy-two premolars from 18 patients were randomly assigned to experimental groups that received a leveling force for 1, 2, or 4 weeks or to a control unloaded group. Following extraction, PDL volumes from 32 premolars of eight patients (21.0 ± 3.8 years) were evaluated using toluidine blue staining. The expressions of the biomolecules in the PDL from 40 premolars of ten patients (21.4 ± 4.0 years) were analyzed via immunoblotting. RESULTS: The median percentage of stained PDL was significantly higher at 2 and 4 weeks after preloading than in the unloaded condition (p < 0.05). The median RUNX2 and ALP expression levels were significantly higher at 2 and 4 weeks after preloading than in the unloaded condition (p < 0.05), whereas the median RANKL/OPG ratios were significantly higher at 1 and 4 weeks after preloading (p < 0.05). CONCLUSIONS: Orthodontic preloading for 4 weeks enhances PDL volumes as well as the expressions of RUNX2, ALP and the RANKL/OPG ratio in the PDL, suggesting this loading period is suitable for successful TAT.

Enhancement of human periodontal ligament by preapplication of orthodontic loading.

INTRODUCTION: The quantity of remaining periodontal ligament (PDL) on the root surface of donor teeth is essential for the success of tooth autotransplantation. Preapplication of orthodontic loading increases this quantity on rat tooth root surfaces. However, little is known about the effects of preloading on human PDL or the ease of tooth extraction. This study aimed to determine the optimal duration of preloading for enhanced PDL on the root surface of extracted human premolars and for facilitating extraction. METHODS: Sixty patients received orthodontic preloading with a bracket connected to an archwire on one of their maxillary first premolars for 2, 4, 6, or 8 weeks, whereas the contralateral premolar was not loaded as a control. Premolar extractions were performed with a record of the duration and difficulty of extraction. The extracted premolars were fixed and stained with toluidine blue. Digitized images were recorded under a stereomicroscope, and the percentage of stained PDL was analyzed. RESULTS: Orthodontic preloading for 4, 6, and 8 weeks significantly increased the percentage of stained PDL on the root surface compared with the control (P <0.05). The duration and difficulty of extraction were significantly less in preloaded than that of unloaded teeth after 4, 6, and 8 weeks (P <0.05). CONCLUSIONS: A 4-week duration of orthodontic preloading is suggested as the shortest duration to adequately enhance PDL and ease tooth extraction; both outcomes may be beneficial for tooth autotransplantation.

Effects of low-dose irradiation on human osteoblasts and periodontal ligament cells.

OBJECTIVE: To investigate the effects of dental x-ray on proliferation and mineralization in human primary osteoblasts as well as on proliferation and apoptotic potential in human periodontal ligament (PDL) cells. DESIGN: Primary osteoblasts and PDL cells were irradiated with various doses of periapical radiography by repeated exposures and further incubated for 1, 3 or 7 days. Cell proliferation was assayed by BrdU incorporation. The effect of dental x-ray on mineralization in osteoblasts either before or after x-ray exposures was determined by Alizarin red staining. Both mRNA and protein expressions of BCL-2, an anti-apoptotic gene, and BAX, a pro-apoptotic gene, in PDL cells were analyzed by RT-qPCR and immunoblotting analysis, respectively. RESULTS: Neither the proliferative nor the mineralization ability of irradiated osteoblasts was different from that of non-irradiated osteoblasts at any doses or time points. By contrast, there was a significant decrease in the proliferation of PDL cells on day 3 after repeated exposures to dental x-ray for 20 times (P < 0.05), whereas the ratio of BCL-2 to BAX mRNA and protein expressions in these irradiated PDL cells was significantly increased (P < 0.05). CONCLUSIONS: Upon multiple exposures to dental x-ray used in intraoral radiography up to 20 times, there is no effect on the proliferation or the mineralization of osteoblasts, whereas the proliferative and apoptotic potentials of PDL cells are transiently decreased.

Anesthetic efficacies of intrapapillary injection in comparison to inferior alveolar nerve block for mandibular premolar extraction: a randomized clinical trial.

OBJECTIVE: Intrapapillary injection (IPI) has been suggested to improve pulpal anesthesia of mandibular teeth and to avoid complications from inferior alveolar nerve block (IANB). This study aimed to determine and compare clinical efficacies and prostaglandin E2 (PGE2) levels between IPI and IANB. MATERIALS AND METHODS: IANB was randomly selected for mandibular premolar anesthesia on one side of 40 patients, whereas IPI was locally administered to the contralateral premolar. Pulpal anesthesia, pain during injection and extraction, patients' satisfaction, and complications were assessed from 30 patients. Gingival crevicular fluid from ten patients was collected for PGE2 quantification by ELISA. RESULTS: Of 30 patients, 18 preferred IPI after injection due to significantly faster mean onset of pulpal anesthesia (p < 0.001) and lower mean score of injection pain (p = 0.017) than IANB, but 21 preferred IANB instead after extraction due to less postoperative pain, consistent with the significantly lower median PGE2 level on the IANB side than that on the IPI at 30 min (p = 0.047). However, there was no difference in the mean satisfaction score between the two techniques. Ulcerated epithelium and sloughing tissues were found at the IPI site in some patients with complete healing within 2 weeks. CONCLUSIONS: The anesthetic efficacies of IPI for mandibular premolar extraction are comparable to those of IANB. However, postoperative pain and local complications at the IPI site should be considered. CLINICAL RELEVANCE: IPI may be used for dental procedures that require only a short anesthetic duration to avoid failure of pulpal anesthesia, complications, and discomfort from IANB.

Reduction of Streptococcus mutans by probiotic milk: a multicenter randomized controlled trial.

OBJECTIVE: To investigate the effects of probiotics, Lactobacillus paracasei SD1, on the quantities of Streptococcus mutans in saliva and plaque samples of preschool children. DESIGN: This randomized trial recruited 487 preschool children from eight childcare centers. Participants were assigned to receive a 6-month course of placebo milk daily (group I), probiotic milk either daily (group II) or three days a week (triweekly, group III). The absolute quantities of S. mutans and total lactobacilli in the saliva and plaque samples at baseline (T0), after intervention (T6), and 6 months after discontinuation (T12) were assessed by qPCR. RESULTS: Of 487 children, 354 completed all follow-up periods. However, only 268 children (3.2 ± 0.8 years old; groups I = 86, II = 89, and III = 93) provided adequate saliva for qPCR. Whereas the quantities of S. mutans were significantly decreased in groups II and III compared to group I in the saliva and plaque samples at T6 and T12, those of total lactobacilli were significantly increased (p < 0.0167). There was no difference in the quantities of S. mutans or total lactobacilli between groups II and III at any period. Significant changes in the quantities of S. mutans and total lactobacilli lasted until T12 compared to T0 (p < 0.0167). CONCLUSIONS: Probiotic administration daily or triweekly reduces S. mutans quantities, whereas it increases total lactobacilli quantities that persists at least 6 months after discontinuation in the saliva and plaque samples of preschool children. CLINICAL RELEVANCE: Daily or triweekly consumption of L. paracasei SD1 supplemented in milk may help prevent dental caries in preschool children.

Reduced ADAM8 levels upon non-surgical periodontal therapy in patients with chronic periodontitis.

OBJECTIVE: To determine effect of non-surgical periodontal treatment on a disintegrin and metalloproteinase 8 (ADAM8) levels in gingival crevicular fluid (GCF) of patients with chronic periodontitis (CP) in comparison with those of patients with gingivitis and to find correlations between ADAM8 levels and clinical parameters. DESIGN: Twenty-two and eleven patients with CP and gingivitis, respectively, were examined for four clinical parameters, probing depth, clinical attachment level, gingival and plaque indices. GCF from the selected gingivitis or periodontitis sites with distinct severities was sampled by Periopaper strips. The non-surgical treatments, including scaling and/or root planing and oral hygiene instruction, were provided for all patients. Clinical measurements and GCF sampling were repeated at three months after the treatments. ADAM8 concentrations were analyzed by ELISA and normalized by GCF volumes or total protein amounts. RESULTS: All patients exhibited significant improvement of almost every clinical parameter after treatment, whereas the median ADAM8 concentrations were significantly decreased at the moderate and severe periodontitis sites of patients with CP (p < 0.05). Moreover, the significantly positive correlations between ADAM8 concentrations and four clinical parameters were found in both moderate and severe groups (p < 0.05). CONCLUSION: ADAM8 concentrations were decreased by non-surgical periodontal therapy in patients with chronic periodontitis at the moderate and severe sites and were correlated with four clinical parameters, implying that GCF ADAM8 levels reflect inflammatory and bone-resorbing activities in the periodontal pocket.

Promotion of Dental Pulp Wound Healing in New Zealand White Rabbits' Teeth by Thai Propolis Product.

This study examined and compared wound healing between Thai propolis product and calcium hydroxide paste as pulp-capping agents after partial pulpotomy in New Zealand white rabbits. Forty incisor teeth from 10 rabbits were treated. Thirty-six teeth received class V cavity preparations with partial pulpotomy and application of either propolis or calcium hydroxide paste. Similar cavity preparations were performed in 2 teeth without any capping material as a positive control, whereas 2 teeth without the cavity preparation served as a negative control. Histological evaluation showed that both groups had dentin bridge formation. Dentinal tubules in the dentin bridge were more orderly arranged in the Thai propolis group than in the calcium hydroxide group. Wound healing and the median number of hyperemic blood vessels were not statistically significant different between the 2 groups. Thai propolis product may be used as a pulp-capping agent.