Stratum corneum lipids liposomes for the topical delivery of 5-aminolevulinic acid in photodynamic therapy of skin cancer: preparation and in vitro permeation study

Abstract Background Photodynamic therapy (PDT) using 5-aminolevulinic acid (5-ALA) is a skin cancer therapy that still has limitations due to the low penetration of this drug into the skin. We have proposed in this work a delivery system for 5-ALA based on liposomes having lipid composition similar to the mammalian stratum corneum (SCLLs) in order to optimize its skin delivery in Photodynamic Therapy (PDT) of skin cancers. Methods SCLLs were obtained by reverse phase evaporation technique and size distribution of the vesicles was determinated by photon correlation spectroscopy. In vitro permeation profile was characterized using hairless mouse skin mounted in modified Franz diffusion cell. Results Size exclusion chromatography on gel filtration confirmed vesicle formation. SCLLs obtained by presented a degree of encapsulation of 5-ALA around 5.7%. A distribution of vesicle size centering at around 500 nm and 400 nm respectively for SCLLs and SCLLs containing 5-ALA was found. In vitro 5-ALA permeation study showed that SCLLs preparations presented higher skin retention significantly (p < 0.05) on the epidermis without SC + dermis, with a decreasing of skin permeation compared to aqueous solution. Conclusions The in vitro delivery performance provided by SCLLs lead to consider this systems adequate for the 5-ALA-PDT of skin cancer, since SCLLs have delivered 5-ALA to the target skin layers (viable epidermis + dermis) to be treated by topical PDT of skin cancer.

CCL3 and CXCL12 production in vitro by dental pulp fibroblasts from permanent and deciduous teeth stimulated by Porphyromonas gingivalis LPS

Objective: The aim of this study was to compare the production of the chemokines CCL3 and CXCL12 by cultured dental pulp fibroblasts from permanent (PDPF) and deciduous (DDPF) teeth under stimulation by Porphyromonas gingivalis LPS (PgLPS). Material and Methods: Primary culture of fibroblasts from permanent (n=3) and deciduous (n=2) teeth were established using an explant technique. After the fourth passage, fibroblasts were stimulated by increasing concentrations of PgLPS (0 – 10 µg/mL) at 1, 6 and 24 h. The cells were tested for viability through MTT assay, and production of the chemokines CCL3 and CXCL12 was determined through ELISA. Comparisons among samples were performed using One-way ANOVA for MTT assay and Two-way ANOVA for ELISA results. Results: Cell viability was not affected by the antigen after 24 h of stimulation. PgLPS induced the production of CCL3 by dental pulp fibroblasts at similar levels for both permanent and deciduous pulp fibroblasts. Production of CXCL12, however, was significantly higher for PDPF than DDPF at 1 and 6 h. PgLPS, in turn, downregulated the production of CXCL12 by PDPF but not by DDPF. Conclusion: These data suggest that dental pulp fibroblasts from permanent and deciduous teeth may present a differential behavior under PgLPS stimulation.

Solubility and permeability as in vitro predictors for in vivo performance of fenofibrate IR solid dosage forms

This current work focused on the simulation of in vivo dissolution and permeation in order to be able to predict the in vivo performance of orally administered fenofibrate immediate release formulations. Therefore, the effects of the formulation surfactants on in vivo solubility and permeation of fenofibrate under physiologically relevant excipient concentrations were emphasized.rnIt was shown that the surfactant sodium dodecyl sulfate (SDS) may decrease rather than increase the solubility of fenofibrate in vivo. This was related to the interference of SDS with the vesicular system of the biorelevant medium, FaSSIFmod, and therefore its solubilization capacity. rnMoreover, in vitro permeation studies revealed that SDS concentrations inversely correlated with fenofibrate permeability. Through combination of the observed permeation and solubility data a good in vitro/in vivo correlation regarding Cmax values in humans could be established for five fenofibrate formulations which were based on the same manufacturing technique.rnBesides the experimental part, the major characteristics and their potential implementation in a dissolution/permeation device were discussed based on the promising realization of the in vitro solubility and permeation methods. rn

Toward a 3D in vitro model based on decellularized thymus to maintain adult thymic ephitelial cells functionality

During my PhD,I have been develop an innovative technique to reproduce in vitro the 3D thymic microenvironment, to be used for growth and differentiation of thymocytes, and possible transplantation replacement in conditions of depressed thymic immune regulation. The work has been developed in the laboratory of Tissue Engineering at the University Hospital in Basel, Switzerland, under the tutorship of Prof.Ivan Martin. Since a number of studies have suggested that the 3D structure of the thymic microenvironment might play a key role in regulating the survival and functional competence of thymocytes, I’ve focused my effort on the isolation and purification of the extracellular matrix of the mouse thymus. Specifically, based on the assumption that TEC can favour the differentiation of pre-T lymphocytes, I’ve developed a specific decellularization protocol to obtain the intact, DNA-free extracellular matrix of the adult mouse thymus. Two different protocols satisfied the main characteristics of a decellularized matrix, according to qualitative and quantitative assays. In particular, the quantity of DNA was less than 10% in absolute value, no positive staining for cells was found and the 3D structure and composition of the ECM were maintained. In addition, I was able to prove that the decellularized matrixes were not cytotoxic for the cells themselves, and were able to increase expression of MHC II antigens compared to control cells grown in standard conditions. I was able to prove that TECs grow and proliferate up to ten days on top the decellularized matrix. After a complete characterization of the culture system, these innovative natural scaffolds could be used to improve the standard culture conditions of TEC, to study in vitro the action of different factors on their differentiation genes, and to test the ability of TECs to induce in vitro maturation of seeded T lymphocytes.

Variabilita' ed evoluzione dei prioni: Mutabilita' in vitro

I prioni, privi di acidi nucleici, esistono come ceppi e possono mutare, in particolare quando attraversano una barriera di specie. Numerosi studi convergono sulla conclusione che le caratteristiche ceppo-specifiche siano inscritte nella conformazione della PrPSc, con la variabilità di ceppo associata a varianti conformazionali della PrPSc. In questo studio ci siamo avvalsi del PMCA, tecnica che riproduce in vitro molti aspetti della biologia dei prioni, per mettere a punto condizioni sperimentali di replicazione che permettessero di osservare fenomeni di mutazione e selezione, onde investigare i meccanismi molecolari e di popolazione alla base della mutabilità dei prioni. In condizioni di replicazione eterologa, che mima la trasmissione tra diverse specie, è stato inizialmente possibile identificare un mutante difettivo della scrapie, caratterizzato da una diversa conformazione della PrPSc e capace di replicare in vitro ma non più in vivo. Le condizioni in cui tale mutante è emerso hanno permesso di sviluppare ulteriori ipotesi di lavoro, basate sul concetto della quasi-specie. Impartendo diversi regimi di replicazione e seguendo l’evoluzione di due ceppi, è stato possibile evidenziare fenomeni di mutazione anche in condizioni di replicazione omologa, in assenza di forti pressioni selettive. In entrambi i ceppi sono emerse varianti conformazionali di PrPSc durante passaggi replicativi ad ampia popolazione, mentre le popolazioni sottoposte a ripetuti colli di bottiglia hanno mostrato un rapido declino del tasso di replicazione. Sono stati infine investigati l’efficacia e il potenziale mutageno di molecole anti-prioniche, ottenendo importanti risultati preliminari sull’efficacia di molecole che legano la PrPC. Questi risultati evidenziano come la mutabilità sia una caratteristica intrinseca dei prioni e supportano l’idea che i prioni siano molto variabili, similmente alle quasi-specie virali, e perciò adattabili e proni a fenomeni di mutazione e selezione. Tali conclusioni hanno impatto su problematiche sanitarie quali lo studio del potenziale zoonotico e i fenomeni di farmaco-resistenza dei prioni.

Functional dna nanostructures for in vitro biosensing in live cells

DNA is a fascinating biomolecule that is well known for its genetic role in living systems. The emerging area of DNA nanotechnology provides an alternative view that exploits unparallel self-assembly ability of DNA molecules for material use of DNA. Although many reports exist on the results of DNA self-assembling systems, still few of them focus on the in vitro study about the function of such DNA nanostructures in live cells. Due to this, there are still a limited research about the in vitro functionality of such designs. To address an aspect of this issue, we have designed, synthesized and characterized two multifunctional fluorescencent nanobiosensors by DNA self-assembling. Each structure was designed and implemented to be introduced in live cells in order to give information on their functioning in real-time. Computational tools were used in order to design a graphic model of two new DNA motifs and also to obtain the specific sequences to all the ssDNA molecules. By thermal self-assembly techniques we have successfully synthesized the structure and corroborate their formation by the PAGE technique. In addition, we have established the conditions to characterize their structural conformation change when they perform their sensor response. The sensing behavior was also accomplished by fluorescence spectroscopy techniques; FRET evaluation and fluorescence microscopy imaging. Providing the evidence about their adequate sensing performance outside and inside the cells detected in real-time. In a preliminary evaluation we have tried to show the in vitro functionality of our structures in different cancer cell lines with the ability to perform local sensing responses. Our findings suggest that DNA sensor nanostructures could serve as a platform to exploit further therapeutic achievements in live cells.

Functional polymer based nanoparticles : development of defined complex structures as basis for in-vitro and in-vivo applications

In Rahmen der vorliegenden Arbeit wurde ein neuartiger Zugang zu einer Vielzahl von Polymerstrukturen auf Basis des klinisch zugelassenen Polymers Poly(N-(2-Hydroxypropyl)-methacrylamide) (PHPMA) entwickelt. Der synthetische Zugang beruht zum einen auf der Verwendung von Reaktivesterpolymeren und zum anderen auf der Reversible Addition Fragmentation Chain Transfer (RAFT) Polymerisationsmethode. Diese Form einer kontrollierten radikalischen Polymerisation ermöglichte es, neben der Synthese von besser definierten Homopolymeren auch statistische und Blockcopolymere herzustellen. Die Reaktivesterpolymere können durch einfache Aminolyse in HPMA-basierte Systeme überführt werden. Somit können sie als eine vielversprechende Basis zur Synthese von umfangreichen Polymerbibliotheken angesehen werden. Die hergestellten Polymere kombinieren verschiedene Funktionalitäten bei konstantem Polymerisationsgrad. Dies ermöglicht eine Optimierung auf eine gezielte Anwendung hin ohne den Parameter der Kettenlänge zu verändern.rnIm weiteren war es durch Verwendung der RAFT Polymerisation möglich partiell bioabbaubare Blockcopolymere auf Basis von Polylactiden und HPMA herzustellen, in dem ein Kettentransferreagenz (CTA) an ein wohl definiertes Polylactid Homopolymer gekoppelt wurde. Diese Strukturen wurden in ihrer Zusammensetzung variiert und mit Erkennungsstrukturen (Folaten) und markierenden Elementen (Fluoreszenzfarbstoffe und +-emittierenden Radionukleide) versehen und im weiteren in vitro und in vivo evaluiert.rnAuf Grund dieser Errungenschaften war es möglich den Einfluss der Polymermikrostruktur auf das Aggregationsverhalten hin mittel Lichtstreuung und Fluoreszenzkorrelationsspektroskopie zu untersuchen. Es konnte gezeigt werden, dass erst diese Informationen über die Überstrukturbildung die Kinetik der Zellaufnahme erklären können. Somit wurde die wichtige Rolle von Strukturwirkungsbeziehungen nachgewiesen.rnSomit konnte neben der Synthese, Charakterisierung und ersten biologischen Evaluierungen ein Beitrag zum besseres Verständnis zur Interaktion von polymeren Partikeln mit biologischen Systemen geleistet werden.

Untersuchung zur endogenen Bildung von oxidativen DNA-Schäden in vivo und in vitro

In dieser Arbeit sollte der Einfluss einer Überproduktion von humaner Superoxiddismutase 1 (hSOD1) auf die Spiegel der DNA-Schäden in verschiedenen Geweben von transgenen Mäusen untersucht werden. Tiere die eine Defizienz des Ogg1- und Csb- Proteins aufweisen und deshalb oxidative Purinmodifikationen nicht oder nur schwer reparieren können, akkumulieren 8-oxoG im Laufe ihres Lebens (Osterod, et al. 2001). Aus diesem Grund sind diese ein gutes Modell, um protektive Eigenschaften von Antioxidantien wie z.B. Substanzen oder Enzymen zu untersuchen. Fusser, et al. 2011 konnten beispielsweise zeigen, dass das pflanzliche Polyphenol Resveratrol die endogenen Spiegel an 8-oxoG sowie die spontanen Mutatiosraten im Lac I - Gen senken kann. Um den Einfluss von hSOD1 in vivo zu untersuchen, wurden in zwei Zuchtschritten 4 Mausgenotypen generiert, nämlich (Csb -/- Ogg1 -/- und Csb +/- Ogg1 +/- Mäuse jeweils mit ohne hSOD1 Überexpression). Diese wurden in verschiedenen Altersstufen auf die Basalspiegel an oxidativen Schäden (Einzelstrangbrüche und Fpg-sensitive Läsionen) in der Leber, der Niere und der Milz untersucht. Die Genotypen wurden zunächst charakterisiert und die hSOD1-Überexpression mittels qRT-PCR, Western Blot und Enzymaktivitätsbestimmung verifiziert. Es konnte an diesen Tieren erstmalig gezeigt werden, dass SOD die Generierung von DNA-Schäden in vivo mit zunehmendem Alter der Tiere senkt und dass deshalb Superoxid eine der reaktiven Sauerstoffspezies ist, die unter physiologischen Bedingungen für die DNA-Schäden verantwortlich ist. Außerdem kann ein möglicher toxischer Effekt der Überproduktion von SOD ausgeschlossen werden. Erhöhte Spiegel an oxidativen DNA-Schäden durch womöglich erhöhte Spiegel an H2O2 konnten in dieser Studie nicht beobachtet werden. Eine Messung der Genexpression anderer antioxidativer Enzyme wie Katalase, SOD2 und SOD3, GPX oder HO1 sind an diesem Effekt nicht beteiligt. Auch konnte kein Einfluss des redoxsensitiven Transkriptionsfaktors Nrf2 gezeigt werden. rnUm mögliche Quellen der für die oxidativ gebildeten DNA-Schäden verantwortlichen ROS zu identifizieren, wurde der Einfluss des Dopaminstoffwechsels untersucht. Während des Dopaminmetabolismus werden intrazellulär Reaktive Sauerstoffspezies (H2O2 und O2.-) gebildet und tragen sehr wahrscheinlich zur Entstehung von neurodegenerativen Erkrankungen wie Parkinson bei. In dem gängigen Parkinson-Zellkulturmodell SH-SY5Y konnte keine Erhöhung von oxidativen Schäden in nukleärer DNA nach Dopaminbehandlung nachgewiesen werden. Eine Überexpression der Dopaminmetabolisierenden Enzyme MAO-A und MAO-B zeigen bei niedrigen Dosen Dopamin eine leichte jedoch nicht signifikante Erhöhung der Fpg-sensitiven Modifikationen. Die Überproduktion des Dopamintransporters zeigte keinen Effekt nach Dopaminzugabe. Es kann geschlussfolgert werden, dass durch erhöhte MAO-A und MAO-B endogen ROS gebildet werden, die die Bildung Fpg-sensitiver Läsionen hervorrufen. Bei hohen Dosen und langer Inkubationszeit steht die Dopaminautoxidation, anschließende Neuromelaninbildung und als Konsequenz Apoptose im Vordergrund.rn

In Vitro and In Vivo Validation of Human and Goat Chondrocyte Labeling by Green Fluorescent Protein Lentivirus Transduction

We investigated whether human articular chondrocytes can be labeled efficiently and for long-term with a green fluorescent protein (GFP) lentivirus and whether the viral transduction would influence cell proliferation and tissue-forming capacity. The method was then applied to track goat articular chondrocytes after autologous implantation in cartilage defects. Expression of GFP in transduced chondrocytes was detected cytofluorimetrically and immunohistochemically. Chondrogenic capacity of chondrocytes was assessed by Safranin-O staining, immunostaining for type II collagen, and glycosaminoglycan content. Human articular chondrocytes were efficiently transduced with GFP lentivirus (73.4 +/- 0.5% at passage 1) and maintained the expression of GFP up to 22 weeks of in vitro culture after transduction. Upon implantation in nude mice, 12 weeks after transduction, the percentage of labeled cells (73.6 +/- 3.3%) was similar to the initial one. Importantly, viral transduction of chondrocytes did not affect the cell proliferation rate, chondrogenic differentiation, or tissue-forming capacity, either in vitro or in vivo. Goat articular chondrocytes were also efficiently transduced with GFP lentivirus (78.3 +/- 3.2%) and maintained the expression of GFP in the reparative tissue after orthotopic implantation. This study demonstrates the feasibility of efficient and relatively long-term labeling of human chondrocytes for co-culture on integration studies, and indicates the potential of this stable labeling technique for tracking animal chondrocytes for in cartilage repair studies.

Teeth restored using fiber-reinforced posts: in vitro fracture tests and finite element analysis

In dentistry the restoration of decayed teeth is challenging and makes great demands on both the dentist and the materials. Hence, fiber-reinforced posts have been introduced. The effects of different variables on the ultimate load on teeth restored using fiber-reinforced posts is controversial, maybe because the results are mostly based on non-standardized in vitro tests and, therefore, give inhomogeneous results. This study combines the advantages of in vitro tests and finite element analysis (FEA) to clarify the effects of ferrule height, post length and cementation technique used for restoration. Sixty-four single rooted premolars were decoronated (ferrule height 1 or 2 mm), endodontically treated and restored using fiber posts (length 2 or 7 mm), composite fillings and metal crowns (resin bonded or cemented). After thermocycling and chewing simulation the samples were loaded until fracture, recording first damage events. Using UNIANOVA to analyze recorded fracture loads, ferrule height and cementation technique were found to be significant, i.e. increased ferrule height and resin bonding of the crown resulted in higher fracture loads. Post length had no significant effect. All conventionally cemented crowns with a 1-mm ferrule height failed during artificial ageing, in contrast to resin-bonded crowns (75% survival rate). FEA confirmed these results and provided information about stress and force distribution within the restoration. Based on the findings of in vitro tests and computations we concluded that crowns, especially those with a small ferrule height, should be resin bonded. Finally, centrally positioned fiber-reinforced posts did not contribute to load transfer as long as the bond between the tooth and composite core was intact.

Effect on infection resistance of a local antiseptic and antibiotic coating on osteosynthesis implants: an in vitro and in vivo study

The purpose of this study was to acquire information about the effect of an antibacterial and biodegradable poly-L-lactide (PLLA) coated titanium plate osteosynthesis on local infection resistance. For our in vitro and in vivo experiments, we used six-hole AO DC minifragment titanium plates. The implants were coated with biodegradable, semiamorphous PLLA (coating about 30 microm thick). This acted as a carrier substance to which either antibiotics or antiseptics were added. The antibiotic we applied was a combination of Rifampicin and fusidic acid; the antiseptic was a combination of Octenidin and Irgasan. This produced the following groups: Group I: six-hole AO DC minifragment titanium plate without PLLA; Group II: six-hole AO DC minifragment titanium plate with PLLA without antibiotics/antiseptics; Group III: six-hole AO DC minifragment titanium plate with PLLA + 3% Rifampicin and 7% fusidic acid; Group IV: six-hole AO DC minifragment titanium plate with PLLA + 2% Octenidin and 8% Irgasan. In vitro, we investigated the degradation and the release of the PLLA coating over a period of 6 weeks, the bactericidal efficacy of antibiotics/antiseptics after their release from the coating and the bacterial adhesion of Staphylococcus aureus to the implants. In vivo, we compared the infection rates in white New Zealand rabbits after titanium plate osteosynthesis of the tibia with or without antibacterial coating after local percutaneous bacterial inoculations at different concentrations (2 x 10(5)-2 x 10(8)): The plate, the contaminated soft tissues and the underlying bone were removed under sterile conditions after 28 days and quantitatively evaluated for bacterial growth. A stepwise experimental design with an "up-and-down" dosage technique was used to adjust the bacterial challenge in the area of the ID50 (50% infection dose). Statistical evaluation of the differences between the infection rates of both groups was performed using the two-sided Fisher exact test (p < 0.05). Over a period of 6 weeks, a continuous degradation of the PLLA coating of 13%, on average, was seen in vitro in 0.9% NaCl solution. The elution tests on titanium implants with antibiotic or antiseptic coatings produced average release values of 60% of the incorporated antibiotic or 62% of the incorporated antiseptic within the first 60 min. This was followed by a much slower, but nevertheless continuous, release of the incorporated antibiotic and antiseptic over days and weeks. At the end of the test period of 42 days, 20% of the incorporated antibiotic and 15% of the incorporated antiseptic had not yet been released from the coating. The antibacterial effect of the antibiotic/antiseptic is not lost by integrating it into the PLLA coating. The overall infection rate in the in vivo investigation was 50%. For Groups I and II the infection rate was both 83% (10 of 12 animals). In Groups III and IV with antibacterial coating, the infection rate was both 17% (2 of 12 animals). The ID50 in the antibacterial coated Groups III and IV was recorded as 1 x 10(8) CFU, whereas the ID50 values in the Groups I and II without antibacterial coating were a hundred times lower at 1 x 10(6) CFU, respectively. The difference between the groups with and without antibacterial coating was statistically significant (p = 0.033). Using an antibacterial biodegradable PLLA coating on titanium plates, a significant reduction of infection rate in an in vitro and in vivo investigation could be demonstrated. For the first time, to our knowledge, we were able to show, under standardized and reproducible conditions, that an antiseptic coating leads to the same reduction in infection rate as an antibiotic coating. Taking the problem of antibiotic-induced bacterial resistance into consideration, we thus regard the antiseptic coating, which shows the same level of effectiveness, as advantageous.

Clinical investigations of polymethylmethacrylate cement viscosity during vertebroplasty and related in vitro measurements

Percutaneous vertebroplasty, comprising an injection of polymethylmethacrylate (PMMA) into vertebral bodies, is a practical procedure for the stabilization of osteoporotic compression fractures as well as other weakening lesions. Cement leakage is considered to be one of the major and most severe complications during percutaneous vertebroplasty. The viscosity of the material plays a key role in this context. In order to enhance the safety for the patient, a rheometer system was developed to measure the cement viscosity intraoperatively. For this development, it is of great importance to know the proper viscosity to start the procedure determined by experienced surgeons and the relation between the time period when different injection devices are used and the cement viscosity. The purpose of the study was to investigate the viscosity ranges for different injection systems during conventional vertebroplasty. Clinically observed viscosity values and related time periods showed high scattering. In order to get a better understanding of the clinical observations, cement viscosity during hardening at different ambient temperatures and by simulation of the body temperature was investigated in vitro. It could be concluded, that the direct viscosity assessment with a rheometer during vertebroplasty can help clinicians to define a lower threshold viscosity and thereby decrease the risk of leakage and make adjustments to their injection technique in real time. Secondly, the acceleration in hardening of PMMA-based cements at body temperature can be useful in minimizing leakages by addressing them with a short injection break.

Fetal ventral mesencephalon of human and rat origin maintained in vitro and transplanted to 6-hydroxydopamine-lesioned rats gives rise to grafts rich in dopaminergic neurons

Free-floating roller tube cultures of human fetal (embryonic age 6-10 weeks post-conception) and rat fetal (embryonic day 13) ventral mesencephalon were prepared. After 7-15 days in vitro, the mesencephalic tissue cultures were transplanted into the striatum of adult rats that had received unilateral injections of 6-hydroxydopamine into the nigrostriatal bundle 3-5 weeks prior to transplantation. Graft survival was assessed in tyrosine hydroxylase (TH)-immunostained serial sections of the grafted brains up to post-transplantation week 4 for the human fetal xenografts and post-transplantation week 11 for the rat fetal allografts. D-amphetamine-induced rotation was monitored up to 10 weeks after transplantation in the allografted animals and compared with that of lesioned-only control animals. All transplanted animals showed large, viable grafts containing TH-immunoreactive (ir) neurons. The density of TH-ir neurons in the human fetal xenografts and in rat fetal allografts was similar. A significant amelioration of the amphetamine-induced rotation was observed in the animals that received cultured tissue allografts. These results promote the feasibility of in vitro maintenance of fetal human and rat nigral tissue prior to transplantation using the free-floating roller tube technique.

Quantitative comparison of 3 enamel-stripping devices in vitro: how precisely can we strip teeth?

INTRODUCTION In this in-vitro study, we aimed to investigate the predictability of the expected amount of stripping using 3 common stripping devices on premolars. METHODS One hundred eighty extracted premolars were mounted and aligned in silicone. Tooth mobility was tested with Periotest (Medizintechnik Gulden, Modautal, Germany) (8.3 ± 2.8 units). The selected methods for interproximal enamel reduction were hand-pulled strips (Horico, Hapf Ringleb & Company, Berlin, Germany), oscillating segmental disks (O-drive-OD 30; KaVo Dental, Biberach, Germany), and motor-driven abrasive strips (Orthofile; SDC Switzerland, Lugano-Grancia, Switzerland). With each device, the operator intended to strip 0.1, 0.2, 0.3, or 0.4 mm on the mesial side of 15 teeth. The teeth were scanned before and after stripping with a 3-dimensional laser scanner. Superposition and measurement of stripped enamel on the most mesial point of the tooth were conducted with Viewbox software (dHal Software, Kifissia, Greece). The Wilcoxon signed rank test and the Kruskal-Wallis test were applied; statistical significance was set at alpha ≤ 0.05. RESULTS Large variations between the intended and the actual amounts of stripped enamel, and between stripping procedures, were observed. Significant differences were found at 0.1 mm of intended stripping (P ≤ 0.05) for the hand-pulled method and at 0.4 mm of intended stripping (P ≤ 0.001 to P = 0.05) for all methods. For all scenarios of enamel reduction, the actual amount of stripping was less than the predetermined and expected amount of stripping. The Kruskal-Wallis analysis showed no significant differences between the 3 methods. CONCLUSIONS There were variations in the stripped amounts of enamel, and the stripping technique did not appear to be a significant predictor of the actual amount of enamel reduction. In most cases, actual stripping was less than the intended amount of enamel reduction.

A new optical detection method to assess the erosion inhibition by in vitro salivary pellicle layer

OBJECTIVES Application of the recently developed optical method based on the monitoring of the specular reflection intensity to study the protective potential of the salivary pellicle layer against early enamel erosion. METHODS The erosion progression was compared between two treatment groups: enamel samples coated by the 15 h-in vitro-formed salivary pellicle layer (group P, n=90) and the non-coated enamel surfaces (control group C, n=90). Different severity of the erosive impact was modelled by the enamel incubation in 1% citric acid (pH=3.6) for 2, 4, 8, 10 or 15 min. Erosion quantification was performed by the optical method as well as by the microhardness and calcium release analyses. RESULTS Optical assessment of the erosion progression showed erosion inhibition by the in vitro salivary pellicle in short term acidic treatments (≤ 4 min) which was also confirmed by microhardness measurements proving significantly less (p<0.05) enamel softening in the group P at 2 and 4 min of erosion compared to the group C. SEM images demonstrated less etched enamel interfaces in the group P at short erosion durations as well. CONCLUSIONS Monitoring of the specular reflection intensity can be successfully applied to quantify early erosion progression in comparative studies. In vitro salivary pellicle (2h) provides erosion inhibition but only in short term acidic exposures. CLINICAL SIGNIFICANCE The proposed optical technique is a promising tool for the fast and non-invasive erosion quantification in clinical studies.