Kyounga Cheon

Genetic Diversity of Plaque Mutans Streptococci with rep-PCR

Mutans streptococci (MS) are key organisms associated with the etiology of dental caries. Using probabilities that were tested by oversampling, we designed this study to determine the minimal number of MS isolates from an individual required to evaluate diversity of genotypes. MS isolates were genotyped by repetitive extragenic palindromic-polymerase chain-reaction (rep-PCR). Analysis of 20 isolates from individuals resulted in a mean of 1.6 and 2.4 genotypes in children (N = 12) and adults (N = 10), respectively. In a follow-up study, reducing the number of isolates to 7-10 resulted in a theoretical probability of up to 78% for detecting up to 4 genotypes. A mean of 1.5 genotypes was found in 35 children and 10 adults. These findings provide evidence for the design of studies of MS genotyping that can serve as a model for the analysis of genotypes within individuals.

Characteristics of Streptococcus mutans genotypes and dental caries in children

This longitudinal cohort study evaluated the diversity, commonality, and stability of Streptococcus mutans genotypes associated with dental caries history. Sixty-seven 5- and 6-yr-old children, considered as being at high caries risk, had plaque collected from baseline through 36 months for S. mutans isolation and genotyping using repetitive extragenic palindromic-PCR (4,392 total isolates). Decayed, missing, or filled surfaces (dmfs (primary teeth)/DMFS (secondary teeth)) for each child were recorded at baseline. At baseline, 18 distinct genotypes were found among 911 S. mutans isolates from 67 children (diversity), and 13 genotypes were shared by at least two children (commonality). The number of genotypes per individual was positively associated with the proportion of decayed surfaces (p-ds) at baseline. Twenty-four of the 39 children who were available at follow-up visits maintained a predominant genotype for the follow-up periods (stability) and this was negatively associated with the p-ds. The observed diversity, commonality, and stability of S. mutans genotypes represent a pattern of dental caries epidemiology in this high-caries-risk community, which suggests that fewer decayed surfaces are significantly associated with lower diversity and higher stability of S. mutans genotypes.

Biomimetic microenvironments for regenerative endodontics

Regenerative endodontics has been proposed to replace damaged and underdeveloped tooth structures with normal pulp-dentin tissue by providing a natural extracellular matrix (ECM) mimicking environment; stem cells, signaling molecules, and scaffolds. In addition, clinical success of the regenerative endodontic treatments can be evidenced by absence of signs and symptoms; no bony pathology, a disinfected pulp, and the maturation of root dentin in length and thickness. In spite of the various approaches of regenerative endodontics, there are several major challenges that remain to be improved: a) the endodontic root canal is a strong harbor of the endodontic bacterial biofilm and the fundamental etiologic factors of recurrent endodontic diseases, (b) tooth discolorations are caused by antibiotics and filling materials, (c) cervical root fractures are caused by endodontic medicaments, (d) pulp tissue is not vascularized nor innervated, and (e) the dentin matrix is not developed with adequate root thickness and length. Generally, current clinical protocols and recent studies have shown a limited success of the pulp-dentin tissue regeneration. Throughout the various approaches, the construction of biomimetic microenvironments of pulp-dentin tissue is a key concept of the tissue engineering based regenerative endodontics. The biomimetic microenvironments are composed of a synthetic nano-scaled polymeric fiber structure that mimics native pulp ECM and functions as a scaffold of the pulp-dentin tissue complex. They will provide a framework of the pulp ECM, can deliver selective bioactive molecules, and may recruit pluripotent stem cells from the vicinity of the pulp apex. The polymeric nanofibers are produced by methods of self-assembly, electrospinning, and phase separation. In order to be applied to biomedical use, the polymeric nanofibers require biocompatibility, stability, and biodegradability. Therefore, this review focuses on the development and application of the biomimetic microenvironments of pulp-dentin tissue among the current regenerative endodontics.

Assessment of two multilocus sequence typing (MLST) schemes available for Streptococcus mutans.

OBJECTIVE Two multilocus sequencing typing (MLST) schemes are currently available for Streptococcus mutans. The first, introduced by Nakano et al. in 2007, consists of 8 conserved housekeeping genes. The second, introduced in 2010 by Do et al., includes 6 housekeeping genes and 2 putative virulence genes. The purpose of the current study was to compare the two MLST schemes for use in validating repetitive extragenic palindromic polymerase chain reaction (rep-PCR) genotypes. DESIGN Thirty-three S. mutans isolates, representing the 11 most commonly occurring rep-PCR genotype groups, were selected for MLST. MLST was performed with SYBR Green™ PCR with published primers for both MLST schemes. Amplicons were purified, sequenced, and data checked against the www.PubMLST.org database for allelic and sequence type (ST) assignment. Discriminatory power, congruence, and convenience criteria were evaluated. Concatenated sequences for each scheme were analyzed using MEGA to generate phylogenetic trees using minimum evolution with bootstrap. RESULTS No significant difference in discriminatory power was observed between the two MLST schemes for S. mutans. Clonal clusters were consistent for both schemes. Overall, MLST demonstrated marginally greater discriminatory power than rep-PCR; however all methods were found to be congruent. New alleles and ST are reported for each scheme and added to the PubMLST database. CONCLUSIONS Clonality, supported by both methods and rep-PCR, indicates S. mutans genotypes are shared between unrelated subjects. Both Nakano and Do schemes demonstrates similar genotype discrimination for S. mutans isolates suggesting each are well designed and may be used to verify rep-PCR genotypes.

Assessment of clonality and serotypes of Streptococcus mutans among children by multilocus sequence typing

Studies using multilocus sequence typing (MLST) have demonstrated that Streptococcus mutans isolates are genetically diverse. Our laboratory previously demonstrated clonality of S. mutans using MLST but could not discount the possibility of sampling bias. In this study, the clonality of randomly selected S. mutans plaque isolates from African-American children was examined using MLST. Serotype and the presence of collagen-binding proteins (CBPs) encoded by cnm/cbm were also assessed. One-hundred S. mutans isolates were randomly selected for MLST analysis. Sequence analysis was performed and phylogenetic trees were generated using start2 and mega. Thirty-four sequence types were identified, of which 27 were unique to this population. Seventy-five per cent of the isolates clustered into 16 clonal groups. The serotypes observed were c (n = 84), e (n = 3), and k (n = 11). The prevalence of S. mutans isolates of serotype k was notably high, at 17.5%. All isolates were cnm/cbm negative. The clonality of S. mutans demonstrated in this study illustrates the importance of localized population studies and are consistent with transmission. The prevalence of serotype k, a recently proposed systemic pathogen, observed in this study, is higher than reported in most populations and is the first report of S. mutans serotype k in a United States population.

Genetic Diversity and Evidence for Transmission of Streptococcus mutans by DiversiLab rep-PCR

This two-part study investigated the genetic diversity and transmission of Streptococcus mutans using the DiversiLab repetitive extragenic palindromic PCR (rep-PCR) approach. For children with S. mutans and participating household members, analysis for evidence of unrelated child-to-child as well as intra-familial transmission was evaluated based on commonality of genotypes. A total of 169 index children and 425 household family members from Uniontown, Alabama were evaluated for genetic diversity using rep-PCR. Thirty-four unique rep-PCR genotypes were observed for 13,906 S. mutans isolates. For transmission, 117 child and household isolates were evaluated for shared genotype (by child and by genotype cases, multiple matches possible for each child). Overall, children had 1-9 genotypes and those with multiple genotypes were 2.3 times more likely to have caries experience (decayed, missing and filled teeth/surfaces>0). Only 28% of children shared all genotypes within the household, while 72% had at least 1 genotype not shared with anyone in the household. Children had genotype(s) not shared with any household members in 157 cases. In 158 cases children and household members shared a genotype in which 55% (87/158 cases) were shared with more than one family member. Children most frequently shared genotypes with their mothers (54%; 85/158), siblings (46%; 72/158) and cousins (23%; 37/158). A reference library for S. mutans for epidemiological surveillance using the DiversiLab rep-PCR approach is detailed. The genetic diversity of S. mutans in this population demonstrated frequent commonality of genotypes. Evidence for both child-to-child and intra-familial transmission of S. mutans was observed by rep-PCR.

Mutans streptococci enumeration and genotype selection using different bacitracin-containing media

The primary etiological agents associated with dental caries include the mutans streptococci (MS) comprised of Streptococcus mutans and Streptococcus sobrinus. The effective cultivation and isolation of MS are necessary for the study of MS, including their proper clinical assessment in the epidemiological study of dental caries. Several selective media have been developed for the isolation, enumeration, and characterization of MS. However, inhibition of MS may occur, reducing counts and perhaps limiting selection of some strains. The purpose of this study was to compare five culture media containing bacitracin recommended for the isolation of MS. Five commonly used bacitracin-containing media (MSB, MSKB, GTSB, TYS20B, and TYCSB) used for MS isolation were quantitatively evaluated. Standard plate counts were performed in duplicate for 2 prototype MS strains (S. mutans UA159 and S. sobrinus 6715) and for MS isolates from clinical saliva samples obtained from 16 children (approximate age 5years) to determine total plate counts, and total S. mutans counts. Selected isolates (n=249) from all five media for 5 saliva samples were further confirmed as S. mutans with real-time PCR then subsequently evaluated qualitatively with rep-PCR for genotype determination. All media resulted in variable enumeration with no significant difference in MS counts. MS prototype strains grew well on all five media; clinical isolates demonstrated more variability in counts but no overall significant differences were found. MSB demonstrated comparable ability to grow S. mutans but allowed for more non-S. mutans growth. All 5 media identified a consistent predominant genotype by rep-PCR. Recovery of minor genotypes was not inhibited by media type.

Angiogenic and Osteogenic Synergy of Human Mesenchymal Stem Cells and Human Umbilical Vein Endothelial Cells Cocultured on a Nanomatrix

To date, bone tissue regeneration strategies lack an approach that effectively provides an osteogenic and angiogenic environment conducive to bone growth. In the current study, we evaluated the osteogenic and angiogenic response of human mesenchymal stem cells (hMSCs) and green fluorescent protein-expressing human umbilical vein endothelial cells (GFP-HUVECs) cocultured on a self-assembled, peptide amphiphile nanomatrix functionalized with the cell adhesive ligand RGDS (PA-RGDS). Analysis of alkaline phosphatase activity, von Kossa staining, Alizarin Red quantification, and osteogenic gene expression, indicates a significant synergistic effect between the PA-RGDS nanomatrix and coculture that promoted hMSC osteogenesis. In addition, coculturing on PA-RGDS resulted in enhanced HUVEC network formation and upregulated vascular endothelial growth factor gene and protein expression. Though PA-RGDS and coculturing hMSCs with HUVECs were each previously reported to individually enhance hMSC osteogenesis, this study is the first to demonstrate a synergistic promotion of HUVEC angiogenesis and hMSC osteogenesis by integrating coculturing with the PA-RGDS nanomatrix. We believe that using the combination of hMSC/HUVEC coculture and PA-RGDS substrate is an efficient method for promoting osteogenesis and angiogenesis, which has immense potential as an efficacious, engineered platform for bone tissue regeneration.

Effects of the nitric oxide releasing biomimetic nanomatrix gel on pulp-dentin regeneration: Pilot study

Successful disinfection alongside complete endodontic tissue regeneration and revascularization are the most desired clinical outcomes of regenerative endodontics. Despite reported clinical successes, significant limitations to the current regenerative endodontic procedure (REP) have been elucidated. To improve the current REP, an antibiotics and nitric oxide (NO) releasing biomimetic nanomatrix gel was developed. The study evaluates antibacterial effects of an antibiotics and NO releasing biomimetic nanomatrix gel on multispecies endodontic bacteria. Antibiotics, ciprofloxacin (CF) and metronidazole (MN) were mixed and encapsulated within the NO releasing biomimetic nanomatrix gel. The gel was synthesized and self-assembled from peptide amphiphiles containing various functional groups. Antibacterial effects of the antibiotics and NO releasing biomimetic nanomatrix gel were evaluated using bacterial viability assays involving endodontic microorganisms including clinical samples. Pulp-dentin regeneration was evaluated via animal-model experiments. The antibiotics and NO releasing biomimetic nanomatrix gel demonstrated a concentration dependent antibacterial effect. In addition, NO alone demonstrated a concentration dependent antibacterial effect on endodontic microorganism. An in vivo analysis demonstrated the antibiotics and NO releasing biomimetic nanomatrix gel promoted tooth revascularization with maturation of root canals. An optimal concentration of and NO releasing nanomatrix gel is suggested for its potential as a root treatment material for REP and an appropriate protocol for human trials. Further investigation is required to obtain a larger sample size and decide upon ideal growth factor incorporation.