Comparative analysis of biofilm models to determine the efficacy of antimicrobials.

Biofilms are one of the greatest challenges in today's treatment of chronic wounds. While antimicrobials kill platonic bacteria within seconds, they are rarely able to harm biofilms. In order to identify effective substances for antibacterial therapy, cost-efficient, standardized and reproducible models that aim to mimic the clinical situation are required. In this study, two 3D biofilm models based on human plasma with immune cells (lhBIOM) or based on sheep blood (sbBIOM) containing S. aureus or P. aeruginosa, are compared with the human biofilm model hpBIOM regarding their microscopic structure (scanning electron microscopy; SEM) and their bacterial resistance to octenidine hydrochloride (OCT) and a sodium hypochlorite (NaOCl) wound-irrigation solution. The three analyzed biofilm models show little to no reaction to treatment with the hypochlorous solution while planktonic S. aureus and P. aeruginosa cells are reduced within minutes. After 48 h, octenidine hydrochloride manages to erode the biofilm matrix and significantly reduce the bacterial load. The determined effects are qualitatively reflected by SEM. Our results show that both ethically acceptable human and sheep blood based biofilm models can be used as a standard for in vitro testing of new antimicrobial substances. Due to their composition, both fulfill the criteria of a reality-reflecting model and therefore should be used in the approval for new antimicrobial agents.

Platysma and the cervical superficial musculoaponeurotic system - Comparative analysis of facial crease and platysmal band development.

OBJECTIVE: The superficial musculoaponeurotic system connects the mimic muscles to the skin, allowing mimic expressions with regional morphological architectural differences. The aim of this study was to perform an architectural analysis of the cervical platysma-skin interaction, determine the morphological implications in platysmal band development and compare the findings to the facial SMAS architectural types. METHOD: Full-thickness blocks of skin, SMAS and platysma from seven hemifaces (three male and two female) and full-thickness blocks of skin, SMAS and mimic muscles of the periorbital, perioral, forehead and midfacial regions from six hemifaces (three male and three female) of donor bodies were collected postmortem. Serial histological sections were cut and stained with Azan. After the morphological analysis, three-dimensional reconstruction of the tissue block was performed with AutoCAD. The morphological and mechanical properties of the different facial SMAS types were compared with those of the cervical SMAS. RESULTS: The architecture of the cervical SMAS (type V) consists of parallel, aligned septum fibrosus profundus and septum fibrosus superficialis tissue connected by vertical, aligned septa fibrotica commisurales tissue delimiting fatty tissue compartments transferring platysmal contractions to the skin. The facial morphological dynamic mimic pattern (SMAS types I, II and III) describes the point-by-point transfer of mimic muscle bundle contractions to the skin, explaining facial crease formation. The cervical morphological dynamic mimic pattern (SMAS type V) can be explained by dual traction force collimation over the septum fibrosus superficialis and profundus in platysmal band development. CONCLUSIONS: The cervical SMAS (type V) description supports the hypothesis that the SMAS and platysma have different morphological origins. The two different facial and cervical morphological dynamic mimic patterns support the phenotypical difference between facial fold and platysmal band development.

Impact of probiotics on pathogen survival in an innovative human plasma biofilm model (hpBIOM).

BACKGROUND: Despite of medical advances, the number of patients suffering on non-healing chronic wounds is still increasing. This fact is attended by physical and emotional distress and an economic load. The majority of chronic wounds are infected of harmful microbials in a protecting extracellular matrix. These biofilms inhibit wound healing. Biofilm-growing bacteria developed unique survival properties, which still challenge the appropriate wound therapy. The present in-vitro biofilm models are not suitable for translational research. By means of a novel in-vivo like human plasma biofilm model (hpBIOM), this study systematically analysed the influence of 3 probiotics on the survival of five clinically relevant pathogenic microorganisms. METHODS: Human plasma was used to produce the innovate biofilm. Pathogenic microorganisms were administered to the plasma. By stimulating the production of a fibrin scaffold, stable coagula-like discs with integrated pathogens were produced. The five clinically relevant pathogens P. aeruginosa, S. aureus, S. epidermidis, E. faecium and C. albicans were challenged to the probiotics L. plantarum, B. lactis and S. cerevisiae. The probiotics were administered on top of the biofilm and the survival was quantified after 4 h and 24 h of incubation. For statistics, two-way ANOVA with post-hoc Tukey's HSD test was applied. P-value > 0.05 was considered to be significant. RESULTS: SEM micrographs depicted the pathogens on the surface of the fibrin scaffold, arranged in close proximity and produced the glycocalyx. The application of probiotics induced different growth-reducing capacities towards the pathogens. B. lactis and S. cerevisiae showed slight bacteria-reducing properties. The survival of C. albicans was not affected at all. The most antimicrobial activity was detected after the treatment with L. plantarum. CONCLUSIONS: This study successfully reproduced a novel human biofilm model, which provides a human wound milieu and individual immune competence. The success of bacteriotherapy is dependent on the strain combination, the number of probiotics and the activity of the immune cells. The eradicating effect of L. plantarum on P. aeruginosa should be emphasized.

Histological, SEM and three-dimensional analysis of the midfacial SMAS - New morphological insights.

INTRODUCTION: The superficial musculoaponeurotic system (SMAS) of the midface has a complex morphological architecture, and a multitude of controversial opinions exist regarding its in vitro appearance and clinical relevance. The aim of this study was to investigate the three-dimensional architecture of the midfacial SMAS. METHOD: Histological and SEM analyses were performed on tissue blocks of the skin, subcutaneous tissue and mimic musculature of the midfacial region between the anterior parotid gland pole and lateral to the nasolabial fold and tissue blocks of the skin, subcutaneous tissue and parotid fascia. Blocks were collected postmortem from six formalin-fixed donor bodies. Serial histological sections were made, stained with Azan and digitized. Three-dimensional reconstructions and visualization of the tissue blocks were performed using AutoCAD. RESULTS: Two different SMAS architectures were found in the midfacial region: parotideal (type IV) and preparotideal (type I) SMAS. Type I SMAS showed three-dimensional interconnecting fibrous chambers embracing fat tissue lobules that cushioned the space between the skin and mimic musculature. Fibrous septa divided the mimic musculature surrounding the muscular bundles. Beneath the mimic muscular level, SMAS septa were oriented parallel to the muscular plane. Above the mimic muscular plane, SMAS septa were oriented perpendicularly, inserted into the skin. Type IV SMAS showed a parallel alignment of the fibrous septa to the skin level, anchoring the skin to the parotid fascia, presenting lymphatic nodes in the fat tissue compartments. The fat cells of the SMAS were enveloped in a fibrotic membrane at the border of the fibro-muscular septa. The SMAS blood supply comprised two subcutaneously epimuscularly spreading anastomosing vascular systems. CONCLUSIONS: Midfacial SMAS represents a functional unit with physical and immunological tasks appearing in two different morphological architecture types. A well-defined nomenclature is needed to prevent controversy.

Bacterial viability in oral biofilm after tooth brushing with amine fluoride or sodium fluoride.

OBJECTIVE: The aim of this study was to investigate the effects of sodium fluoride (NaF) and amine fluoride (AmF) on bacterial viability in the oral cavity. MATERIAL AND METHODS: Healthy subjects brushed their teeth with either fluoride free toothpaste, NaF- or AmF-containing toothpaste. Biofilm smears from different locations were collected before and immediately and 30 and 120 min after tooth brushing. The smears were stained with live/dead bacterial staining, and the number of the respective bacteria was counted. The data were statistically analyzed by comparing the numbers of bacteria before and after the application of no fluoride, NaF and AmF. RESULTS: The highest numbers of bacteria were found in the tongue biofilm, followed by the palatal and cheek biofilm. The lowest numbers were found in the mouth floor biofilm. After the application of AmF, no changes in the numbers of bacteria were found in the biofilms, except for the cheek, where they were reduced. After the application of NaF, the number of bacteria decreased significantly in all biofilms. After 120 min, bacterial regrowth was complete. CONCLUSIONS: AmF has only little effect on the bacterial viability of oral biofilms. NaF application reduces the number of living bacteria in the oral biofilms. This effect lasts not longer than 120 min.

Morphological analysis and three-dimensional reconstruction of the SMAS surrounding the nasolabial fold.

BACKGROUND: The superficial musculoaponeurotic system (SMAS), a structure that has been discussed with some controversy, has a complex morphological architecture. MATERIAL AND METHODS: Histological analysis was performed on tissue blocks of the nasolabial fold (NLF) collected postmortem from formalin-fixed bodies of one male and one female donor. Serial histological sections were made, stained and digitized. Three-dimensional reconstructions of the histological structures were performed. Specimen- and location-specific differences were determined. SEM analysis of the NLF tissue block was performed. RESULTS: The NLF SMAS is a fibro-muscular, three-dimensional meshwork bolstered with fat cells. Two SMAS structure types were identified adjacent to the NLF. The cheek SMAS structure showed a regular, vertical and parallel alignment of the fibrous septa, building a three-dimensional meshwork of intercommunicating compartments. It changed its morphology, condensing while transiting the NLF and passing over to form an irregular structure in the upper lip region. SEM analysis demonstrated the connection between the fibrous meshwork and the fat cells. SMAS blood circulation expanded subcutaneously without perforating the fibro-muscular septa. CONCLUSIONS: The NLF has a recognizable condensed cheek SMAS structure and represents the transition zone between the two SMAS types. Specimen-specific morphological differences necessitate individual planning and area-specific surgical procedures.

Three-dimensional reconstruction of the suborbicularis oculi fat and the infraorbital soft tissue.

The aim of this study was to reveal the histomorphological connections among the suborbicularis oculi fat (SOOF), the orbicularis oculi muscle (OOM), the superficial musculoaponeurotic system (SMAS), the infraorbital fat and the skin. Full graft tissue blocks of the infraorbital region with the skin, SMAS, OOM and SOOF were collected post mortem from one female and two male formalin-fixed body donors. Serial histological sections were made, stained and digitized. Digitalization and three-dimensional (3D) reconstruction of the histological meshwork were performed. SOOF was revealed as a fibro-adipose tissue underlying the OOM, which was strictly separated from the intraorbital fat pad by the orbital septum. SOOF, OOM and SMAS were connected by fibrous septa derived from the SOOF, traversing the OOM with division into multiple muscular bundles, continuing above the muscular plane by forming the SMAS and ending with skin insertion. In the infraorbital region, two different types of SMAS bordering the infraorbital fold have been recognized. Muscle cells have been demonstrated in the SMAS fibrous septa of both SMAS types. Together with the OOM, the SMAS and the skin, SOOF forms an anatomical functional unit. Muscular contraction of the OOM could be transferred by the SMAS to the skin level, producing periorbital mimic expression. The 3D reconstruction facilitates the comprehension of the morphological structure, its connections and space correlations in the infraorbital area. The morphological and topographical peculiarities of the infraorbital structures make it possible to conclude that surgical interventions in this area need to be elaborated and individualized.

Parallel study about the effects of psychotherapy on patients with dental phobia determined by anxiety scores and saliva secretion and composition.

BACKGROUND: The aim of this study was to determine the success of psychotherapeutic treatment for dental phobia by measurement of anxiety using the dental anxiety score (DAS), the state trait anxiety score (STAI state), salivary cortisol and protein concentrations and the salivary secretion rate. Primary endpoint of the study was the comparison of the data before and after psychotherapeutic treatment. METHODS: Forty patients were included into the study. Twenty-four were allocated to the phobic group, 16 to the control group. Saliva was collected upon entering the dental clinic and again after three weeks of psychotherapy. The results were compared with those of a control group. The DAS and STAI questionnaires were completed at each visit. RESULTS: A reduction in DAS values was found after psychotherapy. However, the values remained significantly higher in the phobic group than in the controls. Similar results were found for STAI scores. A slightly higher salivary cortisol level was found in the phobic group. No changes occurred in cortisol or protein concentrations. The salivary secretion rate increased in the phobic patients after psychotherapy. CONCLUSIONS: It could be concluded that psychotherapy is effective in the treatment of dental phobic patients. TRIAL REGISTRATION: This study has been retrospectively registered in the German Clinical Trials Register (# DRKS00009552 ) on 10/19/15.

Dentin abrasivity of various desensitizing toothpastes.

BACKGROUND: The aim of this study was to compare the abrasivity of various commercially available toothpastes that claim to reduce dentin hypersensitivity. METHODS: Dentin discs were prepared from 70 human extracted molars. The discs were etched with lemon juice for 5 min, and one half of the discs were covered with aluminum tape. Following this, they were brushed with 6 different toothpastes, simulating a total brushing time of 6 months. As a negative control, discs were brushed with tap water only. The toothpastes contained pro-arginine and calcium carbonate, strontium acetate, stannous fluoride, zinc carbonate and hydroxyapatite, new silica, or tetrapotassium pyrophosphate and hydroxyapatite. After brushing, the height differences between the control halves and the brushed halves were determined with a profilometer and statistically compared using a Mann-Whitney U test for independent variables. RESULTS: A significant difference (p < 0.001) in height difference between the controls and the toothpaste-treated samples was found in all cases, except for the stannous fluoride-containing toothpaste (p = 0.583). The highest abrasion was found in the toothpaste containing zinc carbonate and hydroxyapatite, and the lowest was found in the toothpaste containing pro-arginine and calcium carbonate. CONCLUSIONS: Desensitizing toothpastes with different desensitizing ingredients have different levels of abrasivity, which may have a negative effect on their desensitizing abilities over a long period of time.

Effectiveness of various toothpastes on dentine tubule occlusion.

OBJECTIVE: Dentine hypersensitivity is an increasing problem in dentistry. Several products are available that claim to occlude open dentine tubules and to reduce dentine hypersensitivity. The aim of this study was to investigate the effectiveness of several different products on dentine tubule occlusion using qualitative and quantitative methods. MATERIALS AND METHODS: Dentine discs were prepared from extracted human premolars and molars. The dentine discs were brushed with 6 different experimental toothpastes, 1 positive control toothpaste and 1 negative control without toothpaste; the brushing simulated a total brushing time of 1 year. Half of the discs were etched with lemon juice after toothpaste application. Standardized scanning electron microphotographs were taken and converted into binary black and white images. The black pixels, which represented the open dentine tubules, were counted and statistically evaluated. Then, half of the dentine discs were broken, and the occlusion of the dentine tubules was investigated using energy dispersive X-ray spectroscopy (EDS). RESULTS: The number of open dentine tubules decreased significantly after brushing with 5 of the 6 tested toothpastes. A significant effect was observed after acid erosion for 3 of the 6 tested toothpastes. EDS revealed partly closed dentine tubules after brushing with 3 toothpastes; however, no partly closed dentine tubules were observed after acid erosion. CONCLUSIONS: Some toothpastes are capable of partial dentine tubule occlusion. This occlusion is unstable and can be removed with acid erosion. CLINICAL SIGNIFICANCE: Desensitizing toothpastes are the most common products that are used against dentine hypersensitivity, and these toothpastes affect dentine tubule occlusion.

Effects of different amine fluoride concentrations on enamel remineralization.

OBJECTIVES: The aim of this study was to investigate the effects of decreasing fluoride concentrations on repeated demineralizing challenges on human enamel. MATERIALS AND METHODS: In 24 teeth, 3mm×3mm windows were prepared on the buccal and lingual sides and treated in a cycling demineralization-remineralization model. Remineralization was achieved with 100, 10 and 0.1 ppm fluoride from anime fluoride. Coronal sections were cut through the artificial lesions, and three sections per tooth were investigated using polarized light microscopy and scanning electron microscopy with quantitative element analysis. RESULTS: The morphology of the lesions was studied, and the extensions of the superficial layer and the body of the lesion were measured. Using element analysis, the Ca, P and F content were determined. The body of the lesion appeared remineralized after application of 100 ppm fluoride, while remineralization of the lesion was less successful after application of 10 and 0.1 ppm fluoride. The thickness of the superficial layer increased with decreasing fluoride concentrations, and also the extension of the body of the lesion increased. Ca and P content increased with increasing fluoride concentrations. CONCLUSIONS: The effectiveness of fluoride in enamel remineralization increased with increasing fluoride concentration. CLINICAL RELEVANCE: A consistently higher level of fluoride in saliva should be a goal in caries prevention.

Kinetics of fluoride bioavailability in supernatant saliva and salivary sediment.

UNLABELLED: The assessment of the fluoride kinetics in whole saliva as well as in the different salivary phases (supernatant saliva and sediment) is essential for the understanding of fluoride bioavailability. OBJECTIVES: To assess the fluoride content, provided by sodium fluoride and amine fluoride, in the supernatant saliva and in salivary sediment. METHODS: Seven trained volunteers were randomly attributed to 2 groups in a cross-over design and brushed their teeth in the morning for 3 min with a product containing either sodium fluoride or amine fluoride. Saliva was collected before, immediately after tooth brushing and 30, 120, and 360 min later and measured. The samples were centrifuged 10 min at 3024 × g. Fluoride content of the supernatant saliva and of the sediment was analysed using a fluoride sensitive electrode. All subjects repeated the study cycles 2 times, and statistical analyses were made using the nonparametric sign test for related samples, the Wilcoxon-Mann-Whitney-test for independent samples. RESULTS: There was a significant increase in fluoride immediately after tooth brushing in both groups in saliva and sediment. The distribution of fluoride between salivary sediment and supernatant saliva (ratio) varied considerably at the different collection times: decreased from 17.87 in baseline samples of saliva to 0.07 immediately and to 0.86 half an hour after tooth brushing in the sodium fluoride group and from 14.33 to 2.85 and to 3.09 in the amine fluoride group. Furthermore after 120 min and after 360 min after tooth brushing the ratio increased from 17.6 to 31.6 in the sodium fluoride group and from 20.5 to 25.76 in the amine fluoride group. No difference was found in the sediment-supernatant saliva ratio between the sodium fluoride and the amine fluoride groups 360 min after tooth brushing. CONCLUSION: For the assessment of fluoride kinetics in whole saliva it is necessary to pay attention to at least four factors: fluoride formulation, time after fluoride application, fluoride concentration in supernatant saliva and fluoride concentration in salivary sediment. This study was approved by the Ethical Committee of the University of Witten/Herdecke permission 21/2008.

Influence of individual saliva secretion on fluoride bioavailability.

UNLABELLED: The aim of this preliminary investigation was to compare the individual saliva secretion rate with the fluoride bioavailability in saliva after using sodium fluoride and amine fluoride. METHODS: To assess oral fluoride kinetics 10 highly trained volunteers brushed their teeth with one of the formulations and saliva was collected. The amount of saliva was measured, and the fluoride content was determined. Data underwent statistical analysis using the Mann-Whitney-U test and Pearson correlation. The ex vivo experiment I included individual saliva collection of the same volunteers. Then the oral hygiene products were solved in equal amounts of whole saliva (ex-vivo experiment II), and the fluoride content was measured. Finally, both products were dispersed in distilled water (ex-vivo experiment III) to calculate the dissociation of both products in water. RESULTS: In vivo results of fluoride content after 3 min. tooth brushing demonstrated a negative correlation with saliva secretion: for NaF r = -0.695 (p<0.01) and for amine fluoride r = -0.446 (p<0.01). The in-vitro experiment I resulted for NaF in 251.7±22.4 µg/g fluoride and for amine fluoride in 171.7±14.4 µg/g. CONCLUSIONS: Fluoride bioavailability of saliva after exposure to NaF was higher compared to amine fluoride. The individual secretion rate changes the fluoride content and normal secretors keep the fluoride availability longer.

[Periodic medical examinations: problem of concordance].

From bioethics and contemporary medical legislation viewpoints, the authors consider a topic of periodic medical examinations and possibility of concordance between doctor and patient. Opinion is that in order to gain concordance and higher efficiency of the examinations, the doctor should separate occupational examination function from medical function.