1 Caries prophylaxis: next generation nanotechnologyJagiellonian University Medical College Caries prophylaxis: next generation nanotechnology Palina Vyhouskaya Supervisor: Wirginia Krzyściak PhD Good afternoon, ladies and gentlemen. My name is PV and I am very grateful for the opportunity to present the results of the study, which was carried out in the Medical College of the Jagiellonian University. Scientific Association at the Department of Medical Diagnostics, Jagiellonian University, Medical College, Pharmacy Faculty, Medyczna 9, Krakow, Poland 2 – 5 June 2015 ISCOMS 2015
2 The objective of the presentationThe objective of the presentation is to determine the role of next generation nanotechnology in caries prophylaxis. During the presentation I will focus on the results of research on the prevention of dental caries based on nanosolutions of the XXI century.
3 Agenda Dental caries: epidemiologyDental caries: etiology and pathogenesis Nanotechnology products The study: methods I will focus on the epidemiology of dental caries, its’ etiology and pathogenesis; later I will tell about nanocompounds that can be used in dental practice, about the research and discuss its’ results. The study: results and conclusions
4 Dental caries prevalence in 2-3 year old childrenDental caries is the most common disease of childhood. Results show that the prevalence of dental caries in from Poland is about 70%. Compared to other countries, it is situated on the top of the list. Those results were the subject of publication of my department.
5 Etiology of the disease: earlierEtiology of the disease: now Etiology of the disease: earlier Dental caries Host Microbes Host Environment Agent Enamel crystal structure Plaque quantity Firmicutes Diet Time Enamel minerals Plaque quality Proteobacteria Saliva quality Enzymes Actinobacteria Saliva quantity Minerals Bacteroidetes Dental caries In spite of a huge number of reports on the etiology of caries, researchers still did not reach the consensus. Till recently, S. mutans, has been established as a main risk factor for dental caries, in addition to diet, time and host factors. However, nowadays more attention has been paid to the fact, that several thousands of other bacterial species are also involved in this process, and altogether they form biofilm structure. Moreover, dental caries is also a consequence of the host and environmental factors. Immune response Bacterial substrate Fusobacteria Host behavior Socio economics Other bacteria Host attitudes
6 Ecological plaque hypothesisSucrose Neutral pH S.oralis, S.sanguinis Remineralization Environmental and biofilm exchange Ecological and biofilm shift Acids Currently, the main hypothesis of caries is the ecological plaque hypothesis. It assumes that changes in the oral cavity conditions initiate a change in the balance of bacterial flora, resulting in the development of more kariogenic species, what leads to the demineralization of enamel. Dietary carbohydrates are considered to be one of the most important factors responsible for those changes in the biofilm. Acidic pH S. mutans Demineralization Eps Lactobacilli ↑ porosiry
7 Microbiology techniquesFirmicutes Microbiology techniques Streptococcus Veillonella Lactobacillus Gemella Paenibacillus Proteobacteria Neisseria Kingella Derxia Classical microbiology methods Actinobacteria Molecular biology methods Actinomyces Corynebacterium Rothia Kineococcus Angustibacter Bacteroidetes Prevotella Capnocytophaga Fusobacteria Leptotrichia Fusobacterium Other bacteria Determination of etiologic factors now depends on analytical methods, among which are methods of classical microbiology and molecular biology methods. It was estimated that about 35% of bacterial species identified with genetic methods have not been grown on conventional media. Studies on metagenomes sequencing revealed five dominant bacteria phyla. The participation of those phyla in the etiology of dental caries is estimated on the level of 80-95%, wherein the contribution of Streptococcus is above 50%. Spirochaetes Tenericutes Synergistetes Chloroflexi Cyanobacteria Planctomycetes Deferribacteres Gemmatimonadetes Verrucomicrobia etc…
8 SUCROSE Biofilm formation GlycolysisGlucosyltransferases secreted by bacteria Exopolysaccharides Lactic acid Acid pH SUCROSE LACTIC ACID SUCROSE H+ GLYCOLYSIS H+ As was mentioned earlier, the very important role in caries pathogenicity is played by the ability of the bacteria to create a complex structure called biofilm. During the biofilm maturation EPS matrix is being built and bacterias are being integrated. Their mutual influence on each other as well as microbe-host interactions are major determinants in the developiing biofilm structure. During the adaptation to stress conditions it comes to the change in the activity of numerous cellular proteins, f.ex. the amount of synthesized glycolytic enzymes is increasing. That’s why S. mutans is more resistant to glycolytic enzyme inactivation in response to environmental pH decrease. S. mutans can continue its’ metabolism, even when the external pH decreases below 4, what stops metabolism of most microorganisms which don’t have ATR mechanisms. ADP GENE EXPRESSION Initial adhesion Initial adhesion Exopoli- saccharide matrix Exopoli- saccharide matrix Biofilm maturation Biofilm maturation Microorganism-host interactions Microorganism-host interactions Own pictures
9 Chlorhexidine (Eludril)Antimicrobial, biofilm Inhibitors Antibacterial alternative substances Delmopinol Chlorhexidine (Eludril) Phenols (Triklosan) Vomiting, diarrhea Tooth discoloration, altered taste, epithelial desquamation Addiction The growing problem of antibiotic resistance of strains makes us to seek other methods of dealing with them. Today we know a number of substances which have effective antibacterial activity and inhibit biofilm development. Unfortunately, most of them cause side effects such as vomiting, diarrhea, addiction or discoloration of the teeth. That’s why, alternative substances having antibacterial activity which would be safe for users are still sought. SAFE FOR USERS
10 Nanotechnology products in dentistryPAST TODAY ACP Amorphous Calcium Phosphate NACP Nanoparticles of Amorphous Calcium Phosphate CRACKING RESISTANCE (>200 µm) (1-100 nm) PLASTICITY For years, a fundamental role in the fight against caries plays a calcium phosphate having remineralization characteristics. Very soon was invented a special resin, which was about to release large amounts of elements gradually in places that require reconstruction. Later this compound has gained the form of nanoparticle composite (NACP). The advantages of NACP are: improved mechanical possibilities … DEFORMATION RESISTANCE
11 Nanotechnology products in dentistryPAST TODAY ACP Amorphous Calcium Phosphate NACP Nanoparticles of Amorphous Calcium Phosphate pH ≈ 6 ↓ pH Good conditions for caries formation DEGRADATION ION RELEASE NEUTRALIZATION an increase in the ions release in an acidic environment fast neutralization of low pH to a safe value, while ACP quits its’ activity. pH NORMALIZATION
12 NACP QAS Modification of NACP Remineralizational propertiesAntibacterial domains Amphiphilicity The loss of membrane properties Remineralizational properties QAS NACP QAS The change of membrane enzymes activity Antibacterial properties The change of the metabolic activity of the cell In search of a substance that in addition to remineralization properties would have antibacterial properties as well, NACP was enriched with quaternary ammonium salts (QAS), such as quaternary ammonium dimethacrylate (QADM). It has two active antibacterial domains, and it is easily mixed with other dental materials. Its use results in decrease in the viability and metabolic activity of microbes and reduction in the acid production.
13 Electron transport damageModification of NACP NAg QAS Lipoteichoic acid Peptidoglycan Cell wall damage Cytoplasmic membrane The diagram shows the effects of the usage of nanoparticle of silver (NAg), another important antibacterial compound, and QAS, whose mechanism involves the absorption of polymer particles by peptidoglycan of a membrane surface. This process is responsible for the malfunction of microbial cell membranes. Oxidative damage Electron transport damage
14 Cetylpyridinium chloride, CPCLethal properties of QAS are also used in antibacterial nanoemulsions like cetylpyridinium chloride (CPC), which destabilizes lipids integration in bacteria’s cell membrane. CPC is also capable of inhibiting enzymes. NANOEMULSION Surfactant Water Lipid
15 Earlier studies have shown that the combination of some nanocompounds give much better results than the use of these substances separately. On the photographs from scanning fluorescence microscope living cells are in green, in red are dead cells. So the data relative to the antibacterial / anticariogenic action of these combinations is intriguing.
16 Materials & Methods STREPTOtest 24 Identification 24h, 37 ºC+ quantifcation In order to examine the effect of combination of Nag, CPC and QADM in different concentrations on single- and mixed biofilm, scrapings from patients with caries were collected, and then immediately inoculated on a selective medium (HLR-S). Standard strains of Streptococcus mutans, Lactobacillus rhamnosus, Streptococcus sanguinis, Streptococcus mitis were grown on BHI medium. After growth, the strains were id-ed and quant-ed. BA 48h, 37 ºC, 10% CO2 HLR-S 48h, 37 ºC, 10% CO2 Material collection
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19 Conclusions Bonding agent including a combination QADM, NAg and CPC is promising to eradicate bacteria in tooth cavity and inhibit caries. This study showed that bonding agent including QADM, NAg and nanoemulsion is promising to eradicate bacteria in tooth cavity and inhibit caries. Additionally, this components may have broad applicability for adhesives, cements, sealants and composites.
20 Thank you for your attention…Thank you for the attention, do you have any questions?