Oral Biology

BMP4 mutations in tooth agenesis and low bone mass Publication date: July 2019 Source: Archives of Oral Biology, Volume 103 Author(s): Miao Yu, Hao Wang, Zhuangzhuang Fan, Chencheng Xie, Haochen Liu, Yang Liu, Dong Han, Sing-Wai Wong, Hailan Feng Abstract Objective To identify an uncommon genetic cause of tooth agenesis (TA) by utilizing whole exome sequencing (WES) and targeted Sanger sequencing in a cohort of 120 patients with isolated TA. Design One deleterious mutation in the gene encoding bone morphogenetic protein 4 (BMP4) was identified in 6 unrelated patients with TA by WES. After that, the coding exons of BMP4 were examined in 114 TA patients using Sanger sequencing. Dual-energy X-ray absorptiometry (DEXA) was used to measure the bone mineral density of patients who carried a BMP4 mutation. Finally, preliminary functional studies of two BMP4 mutants were performed. Results We detected 3 novel missense mutations (c.58 G > A: p.Gly20Ser, c.326 G > T: p.Arg109Leu and c.614 T > C: p.Val205Ala) and 1 reported mutation in the BMP4 gene among 120 TA probands. The previously reported BMP4 mutation (c.751C > T: p.His251Tyr) was associated with urethra and eye anomalies. By extending the pedigrees, we determined that the tooth phenotypes had an autosomal dominant inheritance pattern, as individuals carrying a BMP4 mutation exhibit different types of dental anomalies. Interestingly, we observed that patients harboring a BMP4 mutation manifested early onset osteopenia or osteoporosis. Further in vitrofunctional assays demonstrated that two BMP4 mutants resulted in a decreased activation of Smad signaling. Therefore, a loss-of-function in BMP4 may contribute to the clinical phenotypes seen in this study. Conclusions We identified 4 mutations in the BMP4 gene in 120 TA patients. To our knowledge, this is the first study to describe human skeletal diseases associated with BMP4 mutations.

Interactions among moderate/severe periodontitis, ADIPOQ-rs1501299, and LEPR-rs1137100 polymorphisms on the risk of type 2 diabetes in a Chinese population Publication date: July 2019 Source: Archives of Oral Biology, Volume 103 Author(s): Xiaojing Cao, Pengcheng Huo, Wenjing Li, Peng Li, Lu He, Huanxin Meng Abstract Objective Type 2 diabetes mellitus (T2DM) is a complex disease influenced by genes and the environment. Periodontitis a demonstrated risk factor of T2DM. Previous studies related to gene-environment interactions on the risk of T2DM mainly focused on gene-obesity interactions. However, the impact of gene-periodontitis interaction on the risk of T2DM has not yet been investigated. This study aimed to investigate gene-environment interactions among moderate/severe periodontitis, polymorphisms of adiponectin (ADIPOQ)-rs1501299, and leptin receptor (LEPR)-rs1137100 on T2DM risk in Chinese subjects. Design A case-control study was conducted in 239 Chinese participants from Beijing Hypertension Association Institute (BHAL). After full-mouth periodontal examinations, the participants underwent bilateral buccal swabs for DNA testing. ADIPOQ-rs1501299 and LEPR-rs1137100 were used for genotyping. Generalised multifactor dimensionality reduction (GMDR) and logistic regression were used to examine the interactions among single nucleotide polymorphisms (SNPs) and moderate/severe periodontitis on the risk of T2DM. Results The risk of T2DM was higher in moderate/severe periodontitis [adjusted odds ratio (AOR) = 3.67, 95% confidence interval (95%CI): 1.26–10.71] in ADIPOQ-rs1501299 GG genotype (AOR = 3.42, 95%CI: 1.81–6.46) and LEPR-rs1137100 GG genotype (AOR = 3.16, 95%CI: 1.56–6.39). The GMDR model indicated that there was a significant three-factor model (p = 0.001) involving rs1501299, rs1137100, and moderate/severe periodontitis, demonstrating a potential gene-environment interaction among periodontitis, polymorphisms of rs1501299, and rs1137100 influencing the risk of T2DM. Moderate/severe periodontitis patients with rs1501299-GG and rs1137100-GG have the highest T2DM risk after adjusting for age, gender, BMI, WHR, smoking status, alcohol consumption, economic status, and hypertension (AOR = 20.39, 95%CI: 2.64–157.26). Conclusions Interactions among moderate/severe periodontitis, rs1501299-GG, and rs1137100-GG were associated with an increased risk of T2DM. This study may provide a new insight into the effect of gene-environment interactions on T2DM.

Effect of analogues of cationic peptides on dentin mineralization markers in odontoblast-like cells Publication date: July 2019 Source: Archives of Oral Biology, Volume 103 Author(s): Karina S. Caiaffa, Fernanda G. Basso, Norival A. Santos-Filho, Carlos Alberto de Souza-Costa, Vivien T. Sakai, Eduardo M. Cilli, Cristiane Duque Abstract Objectives To evaluate the effect of analogues of cationic peptides on the viability and the expression of phenotypic and genotypic markers of dentin mineralization in MDPC-23 odontoblast-like cells. Materials and methods Cells were exposed to serial dilutions of analogues of cationic peptides hBD-3-1CV and KR-12-a5 compared to peptide LL-37 and their viability was assessed by methyltetrazolium assay. Next, peptides (0.78–62.5 μg/mL) were applied on the MDPC-23 cells for evaluating the total protein (TP) production, alkaline phosphatase (ALP) activity and mineralized nodule deposition. Gene expression of mineralization markers (DSPP and DMP-1) was also determined by quantitative PCR. Results LL-37 and hBD-3-1CV treatment did not affect cellular viability at concentrations below 62.5 μg/mL. KR-12-a5 reduced cell viability above 31.25 μg/mL. TP production was similar for all groups compared with the control group, except by hBD-3-1CV (at 15.62 μg/mL). LL-37 (at 62.5 μg/mL) induced higher ALP activity than control and other experimental groups. LL-37 and hBD-3-1CV, at 62.5 μg/mL and KR-12-a5 at 31.25 μg/mL stimulated the highest deposition of mineralized nodule. Overall, no statistical differences were observed between the groups for DSPP-1 and DMP-1 expressions. Conclusions LL-37 was the only peptide that induced both ALP activity and mineralized nodules deposition, without affecting cell viability. None of peptides tested induced the expression of DSPP or DMP-1, genes commonly involved in active dentin mineralization.

Tooth agenesis-related GLI2 and GLI3 genes may contribute to craniofacial skeletal morphology in humans Publication date: July 2019 Source: Archives of Oral Biology, Volume 103 Author(s): Guido Artemio Marañón-Vásquez, Beatriz Dantas, Christian Kirschneck, Juliana Arid, Arthur Cunha, Alice Gomes de Carvalho Ramos, Marjorie Ayumi Omori, Amanda Silva Rodrigues, Ellen Cardoso Teixeira, Simone Carvalho Levy, Agnes Schroeder, Mírian Aiko Nakane Matsumoto, Peter Proff, Lívia Azeredo A. Antunes, Alexandre R. Vieira, Leonardo Santos Antunes, Erika Calvano Küchler Abstract Objective The present cross-sectional, multi-centre, genetic study aimed to determine, whether single nucleotide polymorphisms (SNPs) in tooth agenesis (TA)-associated GLI2 and GLI3 genes contribute to the development of craniofacial skeletal morphology in humans. Design Orthodontic patients from an ethnically heterogeneous population were selected for the present study (n = 594). The presence or absence of TA was determined by analysis of panoramic radiography and dental records. The subjects were classified according to their skeletal malocclusion and facial growth pattern by means of digital cephalometric analysis. Genomic DNA was extracted from squamous epithelial cells of the buccal mucosa and SNPs in GLI2 (rs3738880, rs2278741) and GLI3 (rs929387, rs846266) were analysed by polymerase chain reaction using TaqMan chemistry and end-point analysis. Results Class II skeletal malocclusion presented a significantly lower frequency of TA (P < 0.05). Subjects without TA showed significantly higher ANB angles (P < 0.05). Genotype and/or allele distributions of the SNPs in GLI2(rs3738880, rs2278741) and GLI3 (rs846266) were associated with the presence of TA (P < 0.05). The SNPs rs3738880, rs2278741 and rs929387 were also associated with some type of skeletal malocclusion (P < 0.05), but not with the facial growth pattern (P > 0.05). The G allele for TA-related GLI2 rs3738880 was strongly linked to the presence of Class III skeletal malocclusion (OR = 2.03; 95% CI = 1.37–3.03; P<3125 × 10−6). GLI2rs2278741 C allele was overrepresented in individuals without TA, suggesting it as a protective factor for this dental phenotype (OR = 0.43; 95% CI = 0.24−0.78; P<625 × 10−5). Conclusion The present study suggests that SNPs in TA-associated GLI2 and GLI3 genes may also play a role in the development of skeletal malocclusions. rs3738880 and rs2278741 in GLI2 seems to contribute to the genetic background for skeletal Class III and TA, respectively. TA could be an additional predictor of craniofacial morphology in some cases. Further research replicating the reported associations should be performed.

WNT10A mutations causing oligodontia Publication date: July 2019 Source: Archives of Oral Biology, Volume 103 Author(s): Haemin Park, Ji-Soo Song, Teo Jeon Shin, Hong-Keun Hyun, Young-Jae Kim, Jung-Wook Kim Abstract Objectives To identify the molecular genetic etiology of the families with non-syndromic multiple missing permanent teeth (oligodontia). Materials and methods Genomic DNA was isolated and measured, and whole-exome sequencing was performed. The obtained sequencing reads were aligned to the human reference genome and subsequently processed by a series of bioinformatics programs. Finally, short insertions/deletions and single nucleotide variations were annotated with dbSNP build 138. Results The proband of family 1 was missing 14 permanent teeth, and the mutational analysis revealed compound heterozygousWNT10A mutations (c.364A > T and c.511C > T). Two affected individuals in family 2 were missing 20 and 12 permanent teeth, respectively, and compound heterozygous WNT10A mutations (c.364A > T and c.637G > A) were also identified. Conclusions This study reveals compound heterozygousWNT10A missense mutations in two families with non-syndromic oligodontia which will improve the understanding of odontogenesis and the pathogenesis related to WNT10Amutations.

The effect of vanillic acid on ligature-induced periodontal disease in Wistar rats Publication date: July 2019 Source: Archives of Oral Biology, Volume 103 Author(s): Ozkan Karatas, Hatice Balci Yuce, Mehmet Murat Taskan, Fikret Gevrek, Fatma Ucan Yarkac, Aslı Keskin, Sukruye Firuze Ocak Karatas, Hulya Toker Abstract Objective Vanillic acid, also known as 4-hydroxy-3-methoxy benzoic acid has a potent effect on bone metabolism. The purpose of the present study was to specify the effects of vanillic acid (VA) on preventing inflammation and bone destruction in experimental periodontitis as inflammatory bone disease. To evaluate the effects of VA, osteoblast, osteoclast and inflammatory cell counts, iNOS, CD68, MMP-1, and TIMP-1 levels were determined. Methods 32 female Wistar rats were divided into four experimental groups as; Group 1: healthy control (C, n = 8), group 2: Periodontitis (P, n = 8), group 3: periodontitis and 50 mg/kg VA administered group (P + VA-50, n = 8) and group 4: periodontitis and 100 mg/kg VA delivered group (P + VA-100, n = 8). Ligature-induced experimental periodontitis was carried out at mandibular first molar teeth of the right quadrant by placing submarginal 4-0 silk ligatures. VA was administered by oral gavage for 14 days beginning from the first day. Rats were euthanized on the 15th day. Morphological changes in alveolar bone were evaluated via a stereomicroscope. Mandibles were subjected to histological procedures. Osteoblasts, tartrate-resistant acid phosphatase synthesizing osteoclasts and inflammatory cells were counted. Inducible nitric oxide synthase (iNOS), cluster of differentiation (CD)-68, Matrix metalloproteinase (MMP)-1, tissue inhibitor of MMP-1, runt-related x factor-2 (RUNX2), and cyclooxygenase (COX)-2 expressions were determined by immunohistochemistry. Results The rats in the periodontitis group had the highest alveolar bone loss compared to the other groups. Both doses of VA significantly decreased alveolar bone loss but not the control levels. TRAP-positive osteoclast and inflammatory cell counts were also highest in the P group, and both 50 and 100 mg/kg VA reduced these counts. Control rats had the lowest osteoclast and inflammatory cell counts compared to the other groups. Similar to osteoclast counts, MMP-1, iNOS, CD68, and COX-2 expressions were the highest in the P group compared to the other groups. Both doses of VA significantly decreased these levels. Osteoblast cells were higher in the VA groups compared to the control and periodontitis groups. RUNX2 levels were lower in the periodontitis group compared to the control group. A slight increase was also observed in VA groups. However, the difference in the TIMP-1 levels was significant only between P and VA100 groups. Conclusion VA administration successfully ameliorated periodontitis symptoms by decreasing alveolar bone and collagen destruction, periodontal inflammation, and increasing osteoblastic activity.

Interleukin-17 plays a role in pulp inflammation partly by WNT5A protein induction Publication date: July 2019 Source: Archives of Oral Biology, Volume 103 Author(s): Mengyu Liu, Yuan Zhao, Chenglin Wang, Haiyun Luo, Peng A, Ling Ye Abstract Objective Our study aimed to investigate the role of interleukin (IL)-17 in dental pulp inflammation and the relationship between WNT5A and IL-17. Methods Immunohistochemical staining was used to detect the expression of tumor necrosis factor-α (TNF-α), WNT5A and IL-17 in pulp tissues. Anti-IL-17 neutralizing antibody was used in rat pulpitis model and to study the role of IL-17 in pulpitis. TNF-α, WNT5A or IL-17 recombinant protein were used to treat human dental pulp cells. RT-PCR, Western blot, and Enzyme linked immunosorbent assay were used to detect the expression of mRNA and protein. Transwell assay was used to measure the migration of THP-1 cells, which is a human monocytic cell line. Results IL-17 and WNT5A are co-expressed in TNF-α high-expressed region in human and rat pulpitis tissue. IL-17 mainly contributes to its positive regulatory role in inflammation through up regulate cytokines and mediated macrophages migration. Anti-IL-17 neutralizing antibody can suppress the inflammatory cell infiltration and TNF-α expression in dental pulpitis. TNF-α promotes the expression of IL-17 partly through WNT5A and WNT5A regulates IL-17 expression by mitogen-activated protein kinase (MAPK)-(P38 and ERK) pathway. Conclusions IL-17 acts as an inflammatory mediator in dental pulp inflammation. The expression of IL-17 can be partially regulated by WNT5A.

Effects of 7S globulin 3 derived from the adzuki bean [Vigna angularis] on the CSP- and eDNA- dependent biofilm formation of Streptococcus mutans Publication date: June 2019 Source: Archives of Oral Biology, Volume 102 Author(s): Hidenobu Senpuku, Shota Mohri, Mamiko Mihara, Toshiaki Arai, Yusuke Suzuki, Yoji Saeki Abstract Objective Streptococcus mutans is a principal bacterium that forms pathogenic biofilm involved in the development of dental caries. S. mutans possesses a quorum sensing system (QS) stimulated by competence stimulating peptide (CSP), which is associated with bacteriocin production, genetic competency and biofilm formation. Inhibiting CSP-dependent QS is one of the aims leading to the inhibition of biofilm formation and is useful for establishing new prevention systems for dental caries. Design In this study, we selected adzuki bean [Vigna angularis] extract as a candidate component to inhibit CSP-dependent biofilm formation among various foods. To purify an inhibitory component from the adzuki extracts, we performed the salting-out method, two rounds of ion-exchange chromatography, and SDS and native PAGE. Results A primary protein band that inhibits CSP-dependent biofilm formation appeared at approximately 50 kDa and was identified as 7S globulin 3 (7S3), a major seed storage protein in adzuki bean. To determine the characteristics of 7S3 as an inhibitory component, aggregated proteins were extracted from the adzuki crude extracts at pH values lower than 6. The aggregated proteins inhibited CSP- and eDNA-dependent biofilm formation and showed 50 kDa band, which is identical with 7S3 in the purified sample. Moreover, 7S globulin 3 in the adzuki bean extract directly interacted with CSP at low pH conditions but not at neutral conditions, and inhibited CSP-dependent bacteriocin production. Conclusion It was suggested that 7S3 might be a safe and useful material to prevent pathogenic activities in the biofilm formation of S. mutans.

Occlusal interference induces oxidative stress and increases the expression of UCP3 in the masseter muscle: A rat model Publication date: June 2019 Source: Archives of Oral Biology, Volume 102 Author(s): Donglei Wu, Jing Liu Abstract Objective To determine whether occlusal alteration contributes to masticatory muscle damage by inducing oxidative stress. Design Thirty Sprague-Dawley rats were randomly divided into six groups, including occlusal interference groups (3 days, 7 days, 14 days, 21 days, and removal for 3 days) and a sham group. A rat experimental model of occlusal interference was generated by a 0.6-mm unilateral bite-raise. The rats were euthanised for evaluation of histologic changes in the masseter muscles using haematoxylin-eosin staining. To further investigate the role of oxidative stress and uncoupling protein (UCP3) in the development of occlusal dysfunction-induced masseter damage, levels of UCP3 protein were measured by western blot analysis. Results Compared with the sham group, the connective tissue of the masseter muscle was extended partially and inflammatory cells appeared following the induction of malocclusion. With respect to the oxidative stress markers, there were increases in malondialdehyde (MDA) content but decreases in superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities; furthermore, the expression of UCP3 was upregulated. After eliminating the occlusal interference for 3 days, the degree of inflammation was substantially alleviated, the MDA content decreased, and SOD and GPX activities increased. The expression of UCP3 decreased. Conclusions Occlusal interference induces oxidative stress in the masseter muscle, regulated by UCP3. Overall, these findings have significant implications for the understanding of how occlusal dysfunction causes muscle fatigue and pain.

Effect of dioxin-related compounds on oral pigmentation in patients affected by the Yusho incident Publication date: June 2019 Source: Archives of Oral Biology, Volume 102 Author(s): Goro Kawasaki, Izumi Yoshitomi Abstract Background/aim Toxins such as polychlorinated biphenyls (PCBs) and dioxins dramatically affect patients even decades after exposure. Patients with Yusho, a condition caused by exposure to PCBs and dioxins, have diverse mental and physical complaints, even though it is almost 50 years since the Yusho incident. Oral pigmentation is one of the major symptoms in Yusho patients. Patients and methods A total of 183 participants in the Yusho health study were examined. Oral examinations, including recording the prevalence of oral pigmentation, were performed by two oral surgeons. Demographic and clinical characteristics, including blood concentration of PCB and 2,3,4,7,8-pentachlorodibezofuran (2,3,4,7,8-PeCDF), which are the major causes of Yusho, were obtained from the results of recent surveys conducted by the Yusho Study Group. Results The mean serum PCB and 2,3,4,7,8-PeCDF levels of the 183 Yusho patients were 1.59 ± 1.25 ppb and 29.0 ± 42.9 pg/g lipid, respectively. There was a significant correlation between the levels of PCB and 2,3,4,7,8-PeCDF (r = 0.64, p < 0.01). The rate of oral pigmentation in Yusho patients (25.7%) was significant higher than among potential victims of Yusho (13 of 183, 7.1%) (p < 0.05). Conclusion The prevalence of oral pigmentation was still significantly higher in Yusho patients, even 50 years after exposure, although blood PCB levels have decreased in that time.

Alexandros Sfakianakis
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alsfakia@gmail.com