Oral Biology

The effect of iron deficiency anemia on experimental dental caries in mice Publication date: September 2019 Source: Archives of Oral Biology, Volume 105 Author(s): Dania Bahdila, Kenneth Markowitz, Siddhi Pawar, Krupa Chavan, Daniel H. Fine, Kabilan Velliyagounder Abstract Objective The objective of this study was to examine the relationship between iron deficiency and caries susceptibility in a mouse model. Materials and methods Three-week-old C57BL/J6 mice were fed a cariogenic diet containing either standard iron (48 ppm Fe) or low iron (4 ppm Fe) levels. Concurrently, groups of mice with both diets were orally inoculated with Streptococcus mutans (1 × 108) cells on three consecutive days. At the end of the 5th week after infection, mice were sacrificed and jaws were collected for caries scoring, rating the number and severity of lesions using a modified Keyes method applicable to mice. Results Blood analysis by the end of the 5th week revealed marked reduction in the hemoglobin and hematocrit levels of the mice fed the iron deficient diet (IDA and IDA-S. mutans). Anemic mice in both groups lacked the incisor enamel pigmentation observed in mice fed an iron deficient diet. Anemic infected mice had the highest caries severity scores reflecting extensive deep lesions (P < 0.05). S. mutans infected mice fed a standard iron diet had similar numbers of lesions and severity scores as un-infected IDA animals (p < 0.05). IDA did not alter S. mutans CFU counts in infected animals (P < 0.05). Conclusion These results demonstrated that IDA mice are at a higher risk of developing deep dental caries compared to non-anemic mice; highlighting the protective role of iron against dental caries.

Long non-coding RNAs mortal obligate RNA transcript regulates the proliferation of human periodontal ligament stem cells and affects the recurrence of periodontitis Publication date: September 2019 Source: Archives of Oral Biology, Volume 105 Author(s): Yanhua Wang, Yingying Sun, Peng Zheng, Chunyu Cai, Yu Jiang, Huiyan Zhang, Zuntai Li, Qing Cai Abstract Objective The aim of this study is to investigate the role of long non-coding RNAs (lncRNA), mortal obligate RNA transcript (MORT), in human periodontal ligament stem cells. Design Periodontal ligament tissues were collected from 48 periodontitis patients underwent tooth extraction and 38 people in similar age and gender distributions who experienced orthodontic treatment. After treatment, periodontitis patients were followed up for 2 years. MORT in periodontal ligament stem cells (PDLSCs) was detected by qRT-PCR. Proliferation of PDLSCs was detected by CCK-8 assay. Results The proliferation rate of PDLSCs isolated from periodontitis-affected teeth was significantly higher than PDLSCs from healthy teeth. Overexpression of MORT inhibited the proliferation of both types of periodontal ligament stem cells. After treatment, periodontitis patients were followed up for 2 years and patients with high level of MORT expression showed relatively lower recurrence rate comparing to low expression group. Conclusion MORT is involved in the proliferation of human PDLSCs and affects the recurrence of periodontitis.

Salivary biological biomarkers for Alzheimer's disease Publication date: September 2019 Source: Archives of Oral Biology, Volume 105 Author(s): Dan Liang, Hao Lu Abstract Alzheimer's disease (AD) is becoming a threat to aging population all over the world. The pathogenic process of AD is likely initiated many years before clinical onset, thus biomarkers for AD diagnosis are critical for the prevention and therapy for the disease at the early stage in order to reduce the global burden brought by the disease. Saliva is treated as a potential alternative and universal diagnostic fluid that can be collected noninvasively by participants with moderate training and without side effects. Several potential salivary biomarkers, which might prove to be significant diagnostic tools in AD, have been researched. We address here the present and the future of these salivary biological biomarkers for AD.

Low intensity pulsed ultrasound increases mandibular height and Col-II and VEGF expression in arthritic mice Publication date: August 2019 Source: Archives of Oral Biology, Volume 104 Author(s): Jacqueline Crossman, Nadia Alzaheri, Mohamed-Nur Abdallah, Faleh Tamimi, Patrick Flood, Hatem Alhadainy, Tarek El-Bialy Abstract Objective Rheumatoid arthritis (RA) is a chronic inflammatory disease involving persistent inflammation resulting in cartilage and bone damage. RA can affect the temporomandibular joint (TMJ), and damage to the TMJ condyle can lead to craniofacial developmental disturbances, causing micrognathia, malocclusion, retrognathia, and increased overjet. Current treatments of TMJ arthritis are unsatisfactory. This pilot study aimed to investigate the effect of low intensity pulsed ultrasound (LIPUS) on the mandible and TMJ condyles in an RA mouse model using micro-computed tomography (Micro-CT), histologic, and immunohistochemical analyses. Methods MRL-lpr/lpr mice received LIPUS application to their TMJs for 20 min/day for 2 and 4 weeks. Micro-CT analysis measured condylar length and width, posterior mandibular height (P.M.H), mandibular ramus length (M.R.L), effective mandibular length (Ef.M.L), angular process length (A.P.L), mandibular plane (M.P), mandibular axis (M.Ax), and lower incisor height (L.I.H). Condylar cartilage thickness was histologically measured, and type II collagen (Col-II), vascular endothelial growth factor (VEGF), nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) expression was analyzed using immunohistochemistry. Results Comparing the LIPUS-treated group with the control, P.M.H, M.R.L, and M.P were significantly greater in the LIPUS-treated group. Immunostaining for Col-II and VEGF was stronger in the LIPUS-treated group after 4 weeks. OPG showed slightly more expression in the LIPUS group. Conclusions LIPUS may enhance mandibular and TMJ condylar bone formation in this RA mouse model by preventing any growth disturbances involved in inflammation. Further studies are recommended to analyze the effect of LIPUS on TMJ of RA in other animal models.

Low-intensity pulsed ultrasound repair in mandibular condylar cartilage injury rabbit model Publication date: August 2019 Source: Archives of Oral Biology, Volume 104 Author(s): Hang-yu Zhou, Qiang Li, Jian-xiong Wang, Yu-jie Xie, Shi-qi Wang, Lei Lei, Ya-qian Gao, Mao-Mao Huang, Yue Hu, Fang-yuan Xu, Chi Zhang Abstract Objectives Beneficial effects of low-intensity pulsed ultrasound(US) have been reported for knee articular cartilage injury. It is unclear whether the same effect could be observed on mandibular condylar cartilage. This study was designed to explore the efficacy of ultrasound cartilage repair via autophagy regulation. Methods A total of 18 adult rabbits were divided into a sham operation group (exposure to condylar articular surface only), operation without US group (only cartilage surgery), and operation with US group (received ultrasonic therapy daily on day 4 after cartilage surgery). The rabbits were then sacrificed to construct a temporomandibular joint (TMJ) cartilage injury model and HE staining was conducted to observe pathological changes of cartilage in each group. Expression of FGF18, FGFR4, beclin1, ATG3 and ATG7 in rabbit TMJ cartilage were detected using RT-PCR and western blotting. Finally, protein-protein interaction (PPI) analysis was used to observe the interaction among the network of important biomarkers in this injury model. Results Compared to the operation without US group, the severity of cartilage injury was decreased in the operation with US group according to HE staining. The expression of autophagy biomarkers, beclin1, ATG3, ATG7, FGF18 and FGFR4, in operation with US group were up-regulated compared with those in sham operation group and operation without US group p < 0.05). In PPI analysis, ATG3, ATG7, PIK3C3, PIK3R4, BECN1 were identified as hub nodes connecting with most proteins network. Conclusions Our results suggest US has therapeutic potential for the treatment of mandibular condylar cartilage injury, and may affect chondrocyte autophagy.

Cannabidiol, cannabinol and their combinations act as peripheral analgesics in a rat model of myofascial pain Publication date: August 2019 Source: Archives of Oral Biology, Volume 104 Author(s): Hayes Wong, Brian E. Cairns Abstract Objective This study investigated whether local intramuscular injection of non-psychoactive cannabinoids, cannabidiol (CBD), cannabinol (CBN), cannabichromene (CBC) and their combinations can decrease nerve growth factor (NGF)-induced masticatory muscle sensitization in female rats. Design In awake rats, changes in mechanical sensitivity induced by intramuscular injection of NGF and cannabinoids were measured by applying an electronic von Frey hair over the masseter muscle to measure the withdrawal response. The effect of CBD (5 mg/ml) and CBN (1 mg/ml) or their combinations CBD/CBN (1:1 mg/ml or 5:1 mg/ml) were assessed. To confirm a peripheral action, electrophysiological experiments were undertaken in anesthetized rats to examine whether intramuscular injections of CBD (5 mg/ml) and CBN (1 mg/ml) altered the mechanical threshold of masticatory muscle mechanoreceptors. Results In behavioral experiments, CBD (5 mg/ml) or CBN (1 mg/ml) decreased NGF-induced mechanical sensitization. Combinations of CBD/CBN induced a longer-lasting reduction of mechanical sensitization than either compound alone. No significant change in mechanical withdrawal threshold was observed in the contralateral masseter muscles and no impairment of motor function was found with the inverted screen test after any of the treatments. Consistent with behavioral results, CBD (5 mg/ml), CBN (1 mg/ml) and the combination of CBD/CBN (1:1 mg/ml) increased the mechanical threshold of masseter muscle mechanoreceptors. However, combining CBD/CBN (5:1 mg/ml) at a higher ratio reduced the duration of this effect. This may indicate an inhibitory effect of higher concentrations of CBD on CBN. Conclusions These results suggest that peripheral application of these non-psychoactive cannabinoids may provide analgesic relief for chronic muscle pain disorders such as temporomandibular disorders and fibromyalgia without central side effects.

Salivary and serum cystatin SA levels in patients with type 2 diabetes mellitus or diabetic nephropathy Publication date: August 2019 Source: Archives of Oral Biology, Volume 104 Author(s): Suteera Techatanawat, Rudee Surarit, Kongthawat Chairatvit, Sittiruk Roytrakul, Weerapan Khovidhunkit, Supanee Thanakun, Yuichi Izumi, Siribang-on Piboonniyom Khovidhunkit Abstract Objective To investigate putative salivary biomarkers for screening and diagnosis of type 2 diabetes mellitus and diabetic nephropathy. Design Saliva and serum samples were collected from 29 patients with type 2 diabetes, 20 patients with diabetic nephropathy, eight patients with non-diabetic induced nephropathy, and 25 healthy subjects. Initially, pooled unstimulated saliva samples from six sex- and age-matched healthy subjects and six patients with type 2 diabetes were subjected to two-dimensional gel electrophoresis, followed by mass spectrometry. Protein expression of cystatin SA in the saliva of patients with type 2 diabetes was further examined in saliva and serum using enzyme-linked immunosorbent assay (ELISA). Results Two-dimensional gel electrophoresis revealed upregulation of salivary cystatin SA in patients with type 2 diabetes. ELISA showed a weak trend of increasing salivary cystatin SA levels in patients with type 2 diabetes, compared with those levels in healthy subjects. When patients were stratified according to periodontal status, linear regression analyses revealed that salivary cystatin SA levels were associated with Periodontal Screening and Recording (PSR) index (β = 0.297, p <  0.05) when the analysis was adjusted for age, sex, HbA1C, estimated glomerular filtration rate (eGFR), and number of teeth. Serum cystatin SA levels were negatively associated with eGFR (β = −0.534, p <  0.0001) when the analysis was adjusted for age, sex, HbA1C, number of teeth, and PSR index. Conclusions Salivary cystatin SA was associated with periodontal disease severity; moreover, serum cystatin SA levels could reflect kidney function.

Salivary gland metabolism in an animal model of chronic kidney disease Publication date: August 2019 Source: Archives of Oral Biology, Volume 104 Author(s): R.A. Carvalho, A.C. Romero, F.K. Ibuki, F.N. Nogueira Abstract Objective The aim of this study was to determine the effects of experimental CKD into the metabolism of parotid and submandibular glands of rats. CKD was induced by 5/6 nephrectomy. Design Serum analyses of BUN (Blood Urea Nitrogen) and creatinine concentrations were performed. Major salivary glands metabolism was investigated in vivo, both at rest and during salivary stimulation conditions by NMR isotopomer analysis, using [U-13C]glucose as metabolic tracer. Results CKD increases BUN and serum creatinine concentrations (p < 0.001). Multiple metabolic alterations were detected in the parotid glands of this animal model, including decreased concentrations of alanine (p < 0.05) and creatine (p < 0.05) and increased lactate/alanine ratios (p < 0.05). The salivary stimulus fostered accumulations of acetate at both analyzed glands of the CKD model (p < 0.05), indicative of disruption of the oxidative metabolic process. Conclusions Experimental CKD induced by 5/6 nephrectomy altered the parotid salivary gland function, since glucose metabolism is clearly affected after stimulation for salivation in this gland.

Characterization of the in situ pellicle ultrastructure formed under the influence of bovine milk and milk protein isolates Publication date: August 2019 Source: Archives of Oral Biology, Volume 104 Author(s): A. Kensche, A. Dürasch, B. König, T. Henle, C. Hannig, M. Hannig Abstract Objectives The present study aimed to investigate if bovine milk or milk protein isolates, respectively, alter the ultrastructure of thein situ pellicle and might therefore have an influence on oral health. Methods In situ pellicle samples were formed on bovine enamel slabs exposed in the oral cavity of three subjects for 6, 30, 60 or 120 min. After 3 min of pellicle formation, mouthrinses were performed for 3 min with (non-)homogenized UHT- or fresh milk (0.3% or 3.8% fat), 30% UHT-treated cream or different types of casein- or milk protein isolates containing preparations. The specimens were removed after the exposure times and transmission electron microscopy (TEM) was performed. Native pellicle samples served as controls. Results Topical ultrastructural pellicle modifications were detected after mouthrinses with all types of homogenized UHT- or fresh milk and after the application of a 3% native casein micelles containing experimental solution. Atypical globular protein structures, identified as casein micelles, were temporarily adsorbed onto the pellicle. They were closely associated with lipid droplets. Furthermore, the mouthrinses occasionally affected the morphology of salivary bacteria. However, no notable ultrastructural alterations remained after 120 min of pellicle formation. Conclusion For the first time, bovine milk- and micellar casein-induced pellicle modifications were revealed by TEM. The adsorption of micellar casein is possibly due to its molecular interactions. Clinical significance Bovine milk or micellar caseins provide some potential for the development of preventive strategies against bacterial biofilm formation or erosive processes at the tooth surface.

Activity and distribution pattern of enzymes in the in-situ pellicle of children Publication date: August 2019 Source: Archives of Oral Biology, Volume 104 Author(s): Susann Hertel, Annika Schulz, Roman Lang, Thomas Hofmann, Belinda König, Matthias Hannig, Christian Hannig Abstract Objective This study investigated, for the first time, pellicle enzymes with respect to their activity, distribution and fluorescence pattern in children with different caries experience. Design In-situ pellicles were collected from 41 children (aged 4–6 years) with different caries status; 17 of them were caries-free (dmf = 0), 12 had dental restorations but no current caries (dmf ≥ 2) and 12 had at least two carious lesions (dmf ≥ 2). Bovine enamel samples were fixed on individual upper jaw braces for pellicle formation. After 30 min of intraoral exposure, the pellicle and saliva samples were analysed for the activities of amylase, lysozyme, peroxidase and glucosyltransferase (GTF). The distribution of these enzymes, including GTF-isoforms B, C and D, and the pellicle ultrastructure were examined by gold-immunolabelling and transmission electron microscopy (TEM). Furthermore, interactions between pellicle enzymes and adherent bacteria were visualised using combined fluorescence and immunofluorescence labelling. Results There were no significant differences in the pellicle enzyme activities between the study groups. TEM analysis revealed the absence of GTF C and D in the pellicle of caries-active children. Amylase, peroxidase and GTF-isoforms showed a random distribution within the pellicle layer; lysozyme was found in the form of clusters. A similar ultrastructural pattern was observed for all subjects. Fluorescence labelling technique enabled visualisation of all enzymes, except for GTF B. Conclusion Pellicle enzyme activities and ultrastructure are not associated with children's caries status. Further investigation is needed to assess the influence of individual GTF-isoforms on caries susceptibility in children.

Alexandros Sfakianakis
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alsfakia@gmail.com