Introduction: There are many approaches for classification of skeletal asymmetry using either PA cephalograms or SMV radiographs; however, there is no universally accepted one. We developed a new classification system to remove much of the previous diagnostic uncertainty that is also useful in genotyping single nucleotide polymorphisms (SNPs) associated with asymmetry. Also, we investigated whether ACTN3, ENPP1, ESR1, PITX1, and PITX2 genes which contribute to sagittal and vertical malocclusions also contribute to facial asymmetries and temporomandibular disorders (TMD) before and after orthodontic and orthognathic surgery treatment. Methods: One hundred seventy-four patients with a dentofacial deformity were diagnosed as symmetric or subdivided into 4 asymmetric groups according to posteroanterior cephalometric measurements. 13 SNPs in ACTN3, ENPP1, ESR1, PITX,1 and PITX2 were selected for genotyping to determine whether specific allelic variants were overrepresented in subjects with malocclusion subclassifications. TMD examination diagnosis and jaw pain and function (JPF) questionnaires assessed the presence and severity of TMD. Results: Fifty-two percent of the patients were symmetric, and 48% were asymmetric. The asymmetry classification demonstrated significant cephalometric differences between the symmetric and asymmetric groups, and across the 4 asymmetric subtypes: group 1, mandibular body asymmetry; group 2, ramus asymmetry; group 3, atypical asymmetry; and group 4, C-shaped asymmetry. ENPP1 SNP-rs6569759 was associated with group 1 (p=0.004), and rs858339 was associated with group 3 (p=0.002). ESR1 SNP-rs164321 was associated with group 4 (p=0.019). Diagnoses of disc displacement with reduction, masticatory muscle myalgia, and arthralgia were highly prevalent in the asymmetry groups, and all had strong statistical associations with ENPP1 rs858339. The average JPF scores for asymmetric subjects before surgery (JPF, 7) were significantly higher than for symmetric subjects (JPF, 2). Patients in group 3 had the highest preoperative JPF scores, and groups 2 and 3 were most likely to be cured of TMD 1 year after treatment. Conclusions: 1. A new posteroanterior cephalometric analysis using 6 measurements to detect differences in facial sides has been developed to distinguish 4 main classifications of asymmetry that are common in patients with dentofacial deformity. 2. TMD prevalence is much higher in patients with asymmetry compared with patients with dentofacial deformity without asymmetry. a. The most common TMD presentations were disc displacement with reduction, masticatory muscle myalgia, and arthralgia. b. Two of the 4 asymmetry groups had both high positive diagnoses for TMD and subjective patient reporting of symptoms. 3. SNP genotype rs6569759 in ENPP1 was associated with asymmetry group 1, and rs858339 was associated with asymmetry group 3. 4. SNP genotype rs1643821 in ESR1 was associated with asymmetry group 4. rs3020318 in ESR1 was associated with PC1 and PC2, which relate to maxillary canting and menton deviation. 5. SNP genotype rs858339 in ENPP1 was associated with disc displacement with reduction, masticatory muscle myalgia, and arthralgia. 6. Orthodontic and orthognathic treatment of asymmetry alleviates TMD symptoms for at least 1 year into retention in most patients. / Oral Biology

The presence of dentofacial deformities in humans is prevalent, with distortions in jaw growth affecting about 20% of people worldwide. Least is known about the genetic etiology of malocclusions, so it is the purpose of this study to identify genetic factors which influence jaw growth and examine how their expression correlates with vertical and sagittal malocclusions. Myostatin (MSTN) is a negative growth regulator which functions to inhibit excessive growth of muscle. Mice in which this gene was absent exhibited increased muscle mass and altered skeletal form, indicating the role of genetic control on muscle mass and skeletal phenotype. IGF-1 is an anabolic growth factor which acts in coordination with growth hormone to promote myofiber regeneration and hypertrophy. A third gene of interest, myosin 1C is a class I myosin which functions to regulate glucose uptake via facilitated glucose transporter 4 (GLUT 4) in insulin and contraction stimulated pathways. Given its role in muscle metabolism in addition to its association to MYO1H, a paralogous protein which has been associated with Class III malocclusions, the goal of this study was to elucidate the possible role of MYO1C in mediating the metabolic effects of growth factors on fiber size and phenotype and subsequently skeletal form. The aims of this study are as follows: quantify MYO1C expression in masseter muscle from individuals of different occlusal groups; compare MYO1C expression to myosin heavy chain gene expression and fiber percent occupancy by sagittal and vertical malocclusion classes; compare expression of MSTN and IGF-1 to MYO1C to evaluate if a correlation exists; evaluate the expression of MYO1C and MYO1H to identify differences in proportions among malocclusion types. Human masseter muscle samples were provided by oral surgeons at the University of Lille, France from subjects undergoing bilateral sagittal split osteotomy surgery for treatment of malocclusion. Muscle RNA was isolated with TRIzolTM reagent, digested with DNase I, re-isolated with RNAqueous® and quantified in 42 samples by triplicate assays of TaqMan® real time PCR using RNA-to-CTTM 1-Step reagent and an Applied Biosystems Step One Plus instrument. A 25ng amount of skeletal muscle standard was selected as a reference calibrator and relative expression quantities of MYO1C were determined by the comparative threshold cycle (CT) method. The relative quantity (RQ) of expressed RNA is calculated from the CT value. Fiber type, area and percent occupancy had been determined previously for 39 of 42 masseter muscle samples used in this study. Expression of the genes for MyHC-I/β, IIA, IIX, perinatal (neonatal) and α (atrial) in another 39 of the 42 masseter muscle samples had also been previously quantified by RT-PCR for use in correlation analyses with MYO1C expression. Based on the results collected, the final conclusions were drawn: * MYO1C expression is greater in open and normal versus deep bites and Class III versus Class II malocclusions. The highest expression is seen with Class III open bites and the lowest with Class II normal malocclusions. * Class II deep and normal bites showed high correlation between MYO1C expression and atrial and neonatal/atrial MHC gene expression, which require increased MYO1C for oxidative metabolism. They exhibited a negative correlation to type I MHC gene expression and percent occupancy, as deep bites have fewer type I fibers. * Class III open bites had high correlation between MYO1C and neonatal MHC gene expression and low correlation to type II MHC gene expression due to increased percentage of high oxidative type I fibers in open bites and diminished type II fibers. * Correlations between MYO1C and hybrid I/II fiber percent occupancy was unpredictable by occlusal group due to transitional nature of fibers. * MYO1C expression is correlated to growth factor expression in Class III but not in Class II malocclusions, indicating its potential interactive role in masseter metabolism in the Class III group. * Class I myosins are highly expressed in Class III open bites. * Class II deep bites exhibited the lowest expression of MYO1H, indicating the masseters are less regulated by class I myosins. * MYO1H is closely linked with type II MHC gene expression, while MYO1C has a close association with types I and neonatal MHC gene expression. * An association exists between class I myosins and both type I and neonatal/atrial fiber percent occupancy. * A greater sample size of approximately 102 would permit an accurate test for significant differences in future studies. / Oral Biology

The use of mini-screws (MSs) as temporary anchorage devices (TADs) is becoming more common in orthodontic treatment. With the increased use of TADs and the numerous manufactures producing them, research is needed to aid orthodontists in their selection of MSs. One of the major advantages of using mini-screw implants is that they can be loaded immediately and do not require osseoitegration.1 For this to be successful, the mini-screw needs to have adequate primary stability to retain itself in the bone. The mini-screw bone system relies on the stability of the mini-screw, the stability of the bone, and the stability of their interface.19,20 In the literature, this stability if usually quantified by measuring the pullout force, insertion torque and bone-to-implant contact.25,26 To date, much of the research has demonstrated that the geometric configuration of the mini-screw plays a significant role in its primary stability. Features such as pitch (length between threads), thread body design, screw length and diameter factor into primary stability.5,21,23 In order to observe the bone-to-implant contact traditionally, researchers had to perform histologic section. This method is destructive to the sample and does not allow it to be used for other analyses such as pullout force. With micro-CT technology, it is now possible to study the bone-to-implant relationship without destroying the sample and with great accuracy.6 Currently, it is unclear as to the optimal scanning specification to choose when using a micro-CT to measure bone-to-implant contact. The optimal scan is one, which provides the most accurate measurement within the least amount of time. The highest quality scans increase both time and costs of acquisition, while lower quality scans have the potential of introducing inaccuracies. This study aims to determine the optimal specifications needed to scan a mini-screw in bone using a SkyScan 1172 micro-CT, to measure the bone-to-implant contact. A total of three orthodontic mini-screws from Aarhus (American Orthodontics, Sheboygan, Wisc), 1.4 mm in diameter and 8 mm long were inserted into an adult pig mandible. All three mini-screws were inserted into the lingual area in the molar region. Each mini-screw was inserted until all the threads were buried into the bone. After placement, the blocks of bone containing the individual mini-screw were cut out and shaped to facilitate scanning by micro-CT. Each sample was be positioned and scanned individually using micro-CT (SkyScan 1172; SkyScan, Aartselaar, Belgium) under 5 different specifications, see table 1. SkyScan software was used to process the scans and calculate qualitative and quantitative data. For each sample bone-to-implant contact was measured. The software measured the TS, which is the area of the mini-screws surface and the IS, which is where the bone interfaces with the mini-screws. The IS/TSx100 was determined to be the percentage of the mini-screws surface that is in contact with bone or the bone-to-implant contact. Qualitative and quantitative analyses were performed to determine statically significant differences between the various bone-to-implant contact measurements of the samples. BIC varied greatly between the scanning specifications and samples, from 0% to 70.35%. Quantitative and qualitative analysis was performed to compare the differences in BIC(%) values between the 5 specifications. Two of the three samples displayed an accuracy of greater than 95% for specification 3, thus providing latitude in adjustment/reduction of scanning times with minimal variance in data accuracy for BIC(%). The results show for measuring BIC(%), scanning specifications can be modified/optimized to reduce scanning time while maintaining acceptable accuracy when scanning of large sample sizes are needed. It is recommended that for studies where absolute BIC(%) is needed, specification 5 is recommended since it will provide the most accurate measurement. For studies that are comparing changes in BIC(%), specification 3 is recommended as it will provide an acceptable level of accuracy in a reasonable amount time. However, due to limited sample size, more data is needed. / Oral Biology

Objectives: The presence of subgingival dental calculus on tooth root surfaces, an important risk factor in the pathogenesis of human periodontitis, is clinically challenging to reliably detect with existing tactile-based, manual forms of dental instrumentation. In 2003, the United States Food and Drug Administration granted approval for marketing in the United States of a differential reflectometry-based device (DetecTar, NEKS Technologies, Laval, Quebec, Canada) for detection of subgingival dental calculus in humans. The instrument employs a light-emitting diode to deliver red light from the visible light region of the electromagnetic spectrum, with a 635 nm-specific wavelength, onto tooth root surfaces through an optical fiber extending to the tip of a periodontal probe-like handpiece. The optical fiber also collects light reflected back from oral surfaces, from which the optical signature of dental calculus is identified by matching the spectra of the reflected light to an internal computer software database containing red light spectra characteristic of dental calculus in its reference library. To date, only a limited amount of in vitro and in vivo research has been conducted on the DetecTar differential reflectometry device. As a result, the purpose of this study was to to assess, with an in vitro typodont model system, the ability of the DetecTar differential reflectometry device to reliably identify subgingival dental calculus on tooth root surfaces. Methods: A total of 108 subgingival sites on mandibular posterior plastic teeth, of which 73 (67.6%) exhibited artificial dental calculus deposits, were mounted within typodont models of the human oral cavity, comprised of white plastic teeth emerging from and surrounded by anatomically-accurate pink silicone gingival and palatal soft tissues. Each typodont was attached to a phantom head with simulated soft tissue mouth shrouds. Sheep blood was irrigated into subgingival and interproximal areas around typodont teeth to simulate gingival tissue inflammation, and artificial saliva applied onto supragingival typodont tooth surfaces to further simulate typical oral cavity conditions in humans. The 108 test subgingival surfaces were then evaluated with the DetecTar differential reflectometry device in duplicate readings performed by a single periodontist examiner blinded to the typodont distribution of subgingival dental calculus. Emission of a sustained audible signal tone from the DetecTar differential reflectometry device upon entry of its optical fiber tip into typodont periodontal pockets indicated detection of subgingival dental calculus. The diagnostic performance of the DetecTar differential reflectometry device, relative to in vitro detection of subgingival dental calculus, was assessed among all test root surfaces, as well as among proximal and non-proximal root surfaces, with calculations of sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood value, negative likelihood value, diagnostic odds ratio, accuracy (diagnostic effectiveness), and Youden’s Index. Results: Among all root surfaces, the DetecTar differential reflectometry device revealed a sensitivity of 75.4%, specificity of 86.3%, positive predictive value of 86.0%, negative predictive value of 75.9%, positive likelihood value of 5.5, negative likelihood value of 0.3, diagnostic odds ratio of 19.6, accuracy (diagnostic effectiveness) of 80.6%, and Youden’s index value of 0.62, for in vitro detection of subgingival dental calculus. More favorable diagnostic test findings for the device were found on non-proximal (buccal and lingual) than proximal (mesial and distal) root surfaces, with accuracy (diagnostic effectiveness) values 22.7% lower at proximal sites, indicating a poorer performance capability of differential reflectometry within interproximal periodontal pockets. Only a fair level (kappa = 0.42) of reproducibility was found in duplicate scoring of tooth root surfaces for subgingival dental calculus by the DetecTar differential reflectometry device. Conclusions: These study findings suggest marked limitations in the potential clinical utility of the DetecTar differential reflectometry device for detection of subgingival dental calculus. The device demonstrated markedly decreased in vitro accuracy on mesial and distal typodont tooth root surfaces, as compared to non-proximal tooth sites, and exhibited only a fair level of reproducibility in duplicate assessments. The overall performance of the DetecTar differential reflectometry device appears to be inferior to similar assessments of typodont tooth root surfaces conducted by other investigators with more conventional tactile-based, manual instrumentation. Based on these in vitro findings, routine clinical utilization of the DetecTar differential reflectometry device in dental practice is not recommended. / Oral Biology

Edgewise orthodontic brackets are designed with a geometric three-dimensional prescription that allows for specific angulation, inclination, and in-out positioning of individual teeth when a rectangular archwire is engaged and ligated into the bracket’s slot. A precise archwire-bracket slot fit is necessary to produce these prescribed tooth movements. As a result, any discrepancy in bracket slot size, archwire dimension, or bracket configuration diminishes tooth movement position accuracy. The objective is to develop a novel microcomputed tomography approach to examine and more accurately measure the interdimensional fit of archwire-bracket slot engagement. .022x028 inch self-ligating Empower® orthodontic brackets were obtained from American Orthodontics. Self-ligating refers to the manner with which the archwire is retained by the bracket. The Empower® system incorporates a clip into the bracket construction that, when closed, retains an archwire. Using TransbondTM XT light cure adhesive by 3M Unitek, these brackets were bonded to a stone model representing the maxillary left quadrant. A .017x.025 inch nickel-titanium archwire was engaged in the bracket slots. The sample was scanned using microcomputed tomography. Three-dimensional reconstruction, reorientation, and volumetric analyses were completed using the accompanying microcomputed tomography software. The archwire-bracket slot interdimensional fit measurements showed poor interaction between the archwire and bracket slot. All archwire-bracket slot pairs were significantly different from the standard (P<0.05) with excessive remaining space after engagement. The majority of archwire-bracket slot pairs were significantly different from each other (P<0.05). The potential clinical significance of these findings involves incomplete expression of the prescribed tooth movements incorporated into orthodontic brackets. The poor interdimensional fit of the archwire in the bracket slot inhibits the attainment of appropriate tooth angulation, inclination, and in-out positioning. The improved understanding of archwire-bracket slot interaction provided by this study can lead to better, more efficient treatment outcomes. / Oral Biology

Facial esthetics affect an individual's quality of life, self-image, social behavior, and public perception. The oro-facial region, in particular, plays a pivotal role in facial esthetics as dento-facial deformities can cause an individual to be perceived as less attractive, less successful, and less socially acceptable. The ability of orthodontic treatment to alter the soft-tissue facial form makes it a powerful tool to improve facial esthetics. Orthodontic treatment exhibits the most control over the soft-tissue in the lower third of the face, specifically the profile, position of the lips, and lower-third convexity. The position of the soft-tissue in this region plays an important role in the perception of facial attractiveness. Due to the importance of the oro-facial region in facial esthetics, it is of the utmost importance that orthodontists better understand the effects of their treatment on the soft-tissue. The majority of literature evaluates soft tissue changes using 2-D imaging, such as a profile photo or lateral cephalogram; however, the evaluation of soft-tissue changes requires more than just what is seen in a 2-D sagittal image. It is essential that this knowledge is obtained in the most accurate and realistic way possible; to understand changes in three-dimensions instead of two. Using 3dMD technology, pre- and post- three-dimensional soft tissue images of dental Class II patients with high or low mandibular plane angle, treated with or without extraction therapy, were compared. The objective was to determine the effect of extraction treatment on the soft-tissue of different facial types. Subjects were sampled from a database of patients treated by orthodontic residents under the supervision of faculty at the graduate orthodontic clinic of the Maurice H. Kornberg School of Dentistry at Temple University in Philadelphia, PA. Pre- and post-treatment 3dMD images were obtained of 42 dental Class II, high-angle (FMA ≥ 28) and low-angle (FMA < 28) subjects treated with extraction or non-extraction therapy. The 3dMDface system (3dMD, Atlanta, GA) is a stereophotogrammetric system used to capture 3D soft tissue images. Pre- and post-treatment images were superimposed to evaluate soft-tissue changes both quantitatively and qualitatively. Quantitative Analysis: The average linear (mm) changes between the pre- and post-images for each group were recorded for the following areas: right/left commissures and cheeks, upper/lower lips at the midline, chin (pogonion), and tip of the nose. In addition, sectional volumetric (cc) changes of the peri-oral area were recorded. The mean volumetric and linear changes were compared between groups. Qualitative Analysis: Color histograms were created from the superimpositions, representing a color-coded map of the direction and magnitude of soft-tissue changes. The magnitude and direction correspond to color, with warmer colors, such as pink and purple, representing positive changes in the soft-tissue (anterior movement) and cooler colors, like green, indicating a negative change (posterior movement). To allow comparison, the degree of change was coded using symbols, ranging from (+++) to (0) to (---), which correspond to the colors observed in each histogram. Coding was performed for the following areas: upper/lower lip, right/left commissures, right/left cheek, tip of the nose and chin (Horizontal (AP) and Vertical movement), the naso-labial fold, and the mento-labial fold. The coding for each facial area was placed in a table and color coded to aid visual analysis. The color-coded tables were analyzed for patterns within and between groups. While the quantitative analysis found no statistically significant linear and volumetric differences between each group, the qualitative analysis of the 3-D histograms revealed noticeable changes in the soft-tissue. Similar changes resulting from treatment type (extraction vs. non-extraction) were seen in both the high and low angle groups. The changes exhibited by the upper lip were independent of treatment and facial type: retraction was seen in all groups, likely due to Class II mechanics. In contrast, the changes exhibited by the lower lip, nasolabial fold, and mentolabial fold were more dependent on treatment type, becoming fuller in the non-extraction groups, while exhibiting more negative changes in the extraction groups. The commissures and cheeks exhibited unpredictable changes regardless of treatment and facial type. The nose and chin exhibited similar changes in all groups. These data suggest that orthodontic treatment may in fact induce predictable qualitative responses from specific areas of the soft-tissue face. We also conclude that qualitative assessment of soft-tissue facial change is more appropriate than quantitative assessment. / Oral Biology

Growth of the mandible affects the outcome of orthodontic treatment in children. Traditionally, evaluation of mandibular growth uses 2D cephalometric analyses. While still used by orthodontists today, these 2D analyses are fraught with errors in measurement associated with interpreting 2D projections of 3D structures. Cone Beam Computer Tomography (CBCT) provides a means to study craniofacial structures in 3D. The objective of this study is to identify 3D topographical changes of mandible during the period of growth and development in orthodontic patients. Ten subjects with serial CBCT images were selected from the Bolton-Brush collection. The age of the subjects ranged from 8 to 14 years-old. Mandibular growth is evaluated using serial pairs of CBCT images which are segmented, superimposed using regional voxel-based mutual information, and quantitatively evaluated in AmiraXImage®. Surface topography changes over time in these 19 subjects are reported as follows: 56% of surface remodeling at a threshold of 0.5mm, median remodeling of 0.6mm, 0.8mm 0.6mm SD. Intra- and inter-rater ICC reliability for for surface generation (0.82 and 0.90, respectively) and superimpositions (0.94 and 0.93, respectively) demonstrate the reliability of the technique. The findings of this study support theories of bone remodeling reported in histological and radiographic studies of mandibular growth. / Oral Biology

Objectives: Bacterial biofilms are ubiquitous, found in a variety of sites within the human body, and are associated with the pathogenesis of dental caries. They are formed when free-floating microorganisms attach to a surface. One such organism, Streptococcus mutans, has adhesins allowing its attachment to tooth surfaces. S. mutans is associated with all forms of human dental caries. S. mutans can rapidly metabolize dietary sugars to acid, locally creating a low pH on tooth surfaces, where it can optimally grow and become more competitive in dental plaque biofilms, in contrast to acid-sensitive bacterial species associated with sound non-carious tooth surfaces. Commensal and natural biofilm aggregates contain multiple microbial species that are believed to co-exist, interact, and form families with high bacteria and niche diversity. In contrast, most biofilms that are chronically infectious tend to have low bacterial diversity with sovereign mono-species such as S. mutans. Hence, it is advantageous to study mono-species biofilms of dental caries-associated bacteria. Although dental biofilms cannot be completely eliminated, their pathogenicity can be lessened through effective oral hygiene measures. Continuous and regular disruption of dental biofilms is imperative for prevention and management of oral infectious diseases. Mechanical methods, most notably tooth brushing and flossing, are required to regularly and effectively disrupt dental plaque biofilms in the human oral cavity. Antiseptics, such as mouth rinses, can also help control dental plaque biofilms, and may gain access to oral sites inaccessible by mechanical methods. Among available antiseptics, sodium hypochlorite is a particularly potent agent against bacteria, fungi, and viruses. Sodium hypochlorite occurs naturally within phagocytic cells (neutrophils, monocytes, and macrophages) participating in the human innate immune response to microbial infection. Several randomized controlled clinical trials support the efficacy of rinsing twice weekly with 0.25 % sodium hypochlorite to improve periodontal health. To date, no studies have been reported concerning the effect of sodium hypochlorite rinsing on dental caries incidence or progression. Several in vitro studies have addressed the effect of dilute sodium hypochlorite on various oral bacteria species. However, there are no literature reports addressing the effect of sodium hypochlorite on bacteria attached to solid surfaces in biofilms. Hence, the objective of this study is to compare the in vitro antimicrobial effect of sodium hypochlorite to another antiseptic agent, chlorhexidine, on the acidigenic and aciduric bacterial species, S. mutans and Lactobacillus casei, both in a free-form (planktonic) state, and as a biofilm attached to two hard surfaces (glass and hydroxyapatite). Methods: The effect of sodium hypochlorite on S. mutans and L. casei was determined by finding the minimum inhibitory concentration (MIC) by broth dilution assays. The MIC was considered to be the lowest concentration of the agent that prevented bacterial growth, resulting in a clear test tube in a broth dilution assay. Experiments with sodium hypochlorite were repeated twice against each bacterial species, and compared to chlorhexidine in similar assays. The effect of a 20-second exposure of MIC concentration (1.0%) of sodium hypochlorite on the growth of S. mutans planktonic cells was measured. Finally, the effect of a 20-second exposure of 1% hypochlorite on S. mutans single-species biofilms was assayed using sterile microplates and the MBEC Biofilm Inoculator (Innovatech). Results: Values for inhibition of growth of planktonic cells were as follows: sodium hypochlorite (MIC = 0.1%; MBC = 0.1%); chlorhexidine (MIC = 0.0015%; MBC = 0.0025%). A 20-second exposure to either solution at the MBC inhibited growth of planktonic cells in refreshed media. Cells adherent to glass or hydroxyapatite pegs were growth-inhibited, but not detached, in refreshed media by a 20-second exposure to 0.2% and 0.4% sodium hypochlorite, and 0.01% and 0.12 % chlorhexidine, but not by more dilute solutions. Conclusions: Chlorhexidine is more potent than sodium hypochlorite, but dilute solutions of both antimicrobial agents inhibited growth of S. mutans mono-species biofilm cells without detachment of cells. / Oral Biology

Objectives: The oral cavity may be an important and overlooked reservoir of systemic infection, and athletic appliances may constitute a potentially important and under-recognized risk factor for infection. Athletic mouthguards are difficult to clean and provide retentive sites for microorganisms. These microorganisms are organized as biofilms and can adhere to the acrylic, which has porosities on its outer and inner surfaces that create favorable conditions for bacterial colonization. Athletic appliances are not disinfected routinely at home and there is no specific clinical protocol for the control of bacterial biofilm on these appliances. Although adequate brushing or scrubbing with a dentrifice is an effective means of controlling the biofilm, inappropriate home-care quality and frequency are factors that compromise the efficacy of the mechanical control of biofilm. In addition, many athletic mouthguards are often left in the locker-room, and while in use at the athletic site, are often transferred from the mouth to hands, and temporarily stored or carried at distant sites (eg helmuts, shorts, sleeves,socks etc). The two most frequently gram-positive cocci isolated from athletic mouthguards were Staphylococcus aureus and Staphylococcus epidermidis, prominent skin pathogens that are also associated with endocarditis, pericarditis, pneumonia, osteomyelitis, food intoxication, and athletic equipment contamination. Methicillin-resistant S. aureus is an important nosocomial pathogen, which is often transmitted from the colonized site to the site of subsequent infection, and is responsible for considerable morbidity and mortality. iii Athletic mouthguards may be a reservoir for these bacteria to hibernate and thus be a mechanism of transmittal from one site to another. An increase in community associated MRSA (aka close-quarter MRSA) outbreaks has been noted in basketball, football, rugby, volleyball, and wrestling athletes. and some strains of S. aureus are the most common cause of cultured skin abscesses in athletes. Good personal hygiene is the key to prevention and control of community associated MRSA outbreaks. Such practices among athletes include frequent hand washing, covering abrasions or seeping wounds, disallowing athletes with open wounds in whirlpools or saunas, discouraging shared personal items, requiring showers after all practices and games, wearing sandals in showers, isolating athletes who have infections, and washing protective gear after each use. Recommended infection control measures include regular and thorough cleaning of equipment, however when antimicrobial treatment is recommended, specific products are not identified. Therefore , the purpose of this study was to assess alternative methods of disinfecting samples of acrylic from surface and subsurface inoculates of specific bacteria, having in mind methods that maybe used at the athletic site and in the locker-room. Methods: In this study, we tested the efficacy of the Nature/ZoneTm ozone sanitizing chamber and PurellTm hand sanitizer in disinfecting surfaces of acrylic specimens prepared from mouthguards. Staphylococcus epidermidis (aka S. aureus), Lactobacillus casei, and Escherichia coli were purchased from the American Type Culture Collection, Manasas, Va. All experiments were conducted in a laminar flow hood and all equipment was autoclaved prior to use. Growing cells were placed on the surface of sterile acrylic discs and then either Purell was added to the disc for three iv minutes, or the inoculated discs were subjected to ozone in the chamber for three minutes. Alternatively, growing cells were placed in Eppendorf tubes to simulate porosities in the acrylic. All specimens (discs or inoculates) were then transferred to 10 ml of culture broth, grown overnight, and compared to positive and negative controls. After overnight incubation, each working solution was diluted in ten-fold steps for the purpose of estimating bacterial cell numbers. From the dilutions , 0.1 ml was spread evenly across the surface of their respective agars. All agar plates were incubated for 24 hrs. at 37 C, after which all visible colonies were counted. The number of colony forming units (CFU) were determined by counting segments of the plates. All experiments were performed in triplicate, and experimental samples were compared to positive and negative controls. One way analysis of variance was used to compare colony counts between positive control samples , experimental ozone chamber samples, and experimental PurellTM samples. Results: Three trials indicated that there was no inhibition of growth of the organisms when sterile discs or eppendorf tubes were incubated in the ozone chamber for 3 minutes. As compared to positive control and the ozone chamber experimental samples, surface application of hand sanitizer (PurellTm) for 3 minutes significantly inhibited growth of all three bacteria (p = 0.05, one way ANOVA). Conclusions: These findings indicate that ozone produced by the Nature/Zone UV sanitizer is ineffective in reducing bacterial counts of the three organisms used in these in vitro experiments. On the other hand, Purell hand sanitizer, significantly reduced the number of bacterial counts of all three organisms. Thus, whereas the hand sanitizer is an effective disinfectant, the ozone chamber is not an effective disinfectant. / Oral Biology

Objectives: Prevotella intermedia and Prevotella nigrescens are dark-pigmented, non-motile, anaerobic rods regarded as important bacterial pathogens in the etiology and progression of human chronic periodontitis. Because the bacterial species may not be adequately suppressed in the subgingival microbiota of chronic periodontitis lesions by conventional mechanical root debridement when present in high cultivable proportions, systemic and/or local periodontal chemotherapy is often employed to reduce their subgingival numbers to levels compatible with periodontal health. However, many clinical isolates of P. intermedia and P. nigrescens may exhibit resistance to β-lactam antibiotics prescribed in periodontal therapy via expression of β-lactamase enzymes. Conventional methods of in vitro antibiotic susceptibility testing entail cultivation of targeted bacterial species, with evaluations of whether or not the organism is capable of growing in the presence of critical thresholds of test antibiotics, such as β-lactam antibiotics. Because such conventional in vitro antibiotic testing is time-consuming, expensive, and labor-intensive, there is an urgent clinical need for more rapid assays to determine the susceptibility or resistance of important periodontal pathogens, such as P. intermedia and P. nigrescens, to contemplated periodontal antimicrobial chemotherapy. In this regard, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, and associated analytic software for the definitive identification P. intermedia and P. nigrescens in clinical specimens, has been approved for clinical microbiology diagnostic use in the United States by the Food and Drug Administration. This methodology relies upon evaluations of bacterial protein profiles for microbial species identification. Since β-lactamase enzymes conferring bacterial resistance to β-lactam antibiotics are proteins, it is possible that MALDI-TOF mass spectrometry may be able to reliably detect characteristic differences in protein profiles of microorganisms elaborating and not elaborating β-lactamase enzymes. Thus, the potential exists that protein profiles generated by MALDI-TOF mass spectrometry may be used to rapidly distinguish between antibiotic-resistant versus susceptible strains of bacterial species without employing time-consuming, expensive, and labor-intensive culture and in vitro drug testing. As a result, this study aimed carry out a proteomic comparison of amoxicillin-resistant and susceptible periodontal P. intermedia and P. nigrescens clinical isolates with MALDI-TOF mass spectrometry. Methods: A total of 10 amoxicillin-resistant and two amoxicillin-susceptible fresh clinical subgingival isolates of P. intermedia, and 10 amoxicillin-resistant and one amoxicillin-susceptible clinical subgingival isolates of P. nigrescens, were recovered by culture and identified to species level with MALDI-TOF mass spectrometry from the subgingival dental plaque biofilms of adults with severe chronic periodontitis. The amoxicillin-resistant clinical isolates of P. intermedia and P. nigrescens exhibited growth on anaerobically-incubated enriched Brucella blood agar primary isolation culture plates supplemented with amoxicillin at 8 μg/ml, whereas no growth on these plates was found with amoxicillin-susceptible strains. Normalized raw mass spectra, as well as normalized peak list spectrum representations, generated by MALDI-TOF mass spectrometry within a routine operating range of 2-20 kDa were visually compared for the amoxicillin-resistant and amoxicillinsusceptible P. intermedia and P. nigrescens clinical isolates, to see if there were consistently reproducible differences in their distribution of mass spectra peaks. Results: Visual comparisons of normalized raw mass spectra, and normalized peak list spectrum, for amoxicillin-susceptible and amoxicillin-resistant subgingival clinical isolates of both P. intermedia and P. nigrescens failed to reveal consistently reproducible differences in their distribution of mass spectra peaks within a routine MALDI-TOF mass spectrometry operating range of 2-20 kDa. Conclusions: Visual examination of raw mass spectra and mass spectra peaks generated by MALDI-TOF mass spectrometry within a routine operating range of 2-20 kDa failed to reveal noteworthy differences between amoxicillin-resistant and amoxicillin-susceptible subgingival clinical isolates of P. intermedia and P. nigrescens. These findings indicate that use of MALDI-TOF mass spectrometry employed within a routine operating range of 2-20 kDa may not provide sufficient differentiation in protein profiles between amoxicillin-resistant and amoxicillin-susceptible Prevotella species to be of rapid diagnostic use for assessing the species in vitro antibiotic susceptibility to β- lactam antibiotics. Additional evaluation of MALDI-TOF mass spectrometry employing higher kDa ranges beyond a routine upper operating range of 20 kDa is warranted to further evaluate its potential to accurately identify antibiotic-resistant versus susceptible strains of pathogenic microorganisms. / Oral Biology