Influence of bisphosphonates on endothelial cells, fibroblasts, and osteogenic cells.

Bisphosphonate-associated osteonecrosis of the jaws (BP-ONJ) is a side effect primarily in patients receiving highly potent nitrogen-containing bisphosphonates. The exact etiopathology is unknown. In addition to reduced bone remodeling, there may also be an impact on soft tissues. The impact of nitrogen- (ibandronate, pamidronate, zoledronate) and non-nitrogen-containing bisphosphonates (clodronate) on human umbilicord vein endothelial cells (HUVEC), fibroblasts and osteogenic cells was analyzed employing cell viability testing and a scratch wound assay. The impact on the cell morphology of vital-stained osteogenic cells was investigated by cell visualization (confocal laser scanning microscopy). Pamidronate and zoledronate had the greatest negative impact on all cell lines, whereas the impact of ibandronate and clodronate was less distinct. The effect of clodronate on HUVEC and fibroblasts was particularly marginal. BP-ONJ could be a multifactorial event with multicellular impairments. This might result in altered wound healing. The increased impact of the highly potent bisphosphonates, particularly on non-bone cells, may explain the higher occurrence of BP-ONJ.

Influence of apple juice on human enamel surfaces of the first and second dentition - an in vitro study.

Dental erosion caused by acidic beverages is common and occurs with increasing tendency. The aim of this in vitro study was to analyse the erosive potential of apple juice on human enamel samples from the first and second dentition. Apple-juice-containing beverages (n = 23) were selected, and pH and buffering capacity were determined. Enamel samples were prepared from impacted, surgically removed wisdom teeth (20 mm superset2) and from deciduous teeth (16 mm superset2). Prepared enamel slices were incubated with a selected apple juice (pH = 3.5) for up to 24 h; the amounts of released calcium were determined colorimetrically, and mean surface roughness (Ra) of the enamel was measured using an optical profilometric device (perthometer, Mahr, Göttingen, Germany). Controls were incubated with a 0.9 % sodium chloride solution under the same conditions (37 degrees C, humidified atmosphere of 5% CO subset2 and 95 % air). The surfaces of the enamel samples were visually examined by CLSM (Leica TCS SP2). The pH-values of the apple juices ranged from 3.3 to 4.2. Incubating the enamel slices (from both dentitions) with a selected apple juice caused a time dependent release of calcium. After 24 h, the primary dentition showed Ca-release values of 0.61 +/- 0.035 mg/ 20 mm superset2 and the second dentition of 0.41 +/- 0.085 mg/ 20 mm superset2; the surface roughness for the primary teeth was 6.8 +/- 1.09 microm and for the second dentition 6.2 +/- 0.41 microm. CLSM show structural changes on all surfaces when compared to the controls. In this in vitro study, the erosive potential of apple juice on teeth of the first and second dentition could be demonstrated. However, it must be considered that numerous modifying factors influence the human enamel surface in vivo; therefore, a direct translation from in-vitro conditions can only be done with caution.

Turned, machined versus double-etched dental implants in vivo.

BACKGROUND: Positive effects on the clinical outcome of moderately rough implant surfaces are described. Intercomparison of clinical data, however, is rarely found. PURPOSE: The aim of this study was to compare the clinical results of two macroscopically identical implants, the one with a turned, machined and the other with an etched surface. MATERIALS AND METHODS: In a retrospective cohort study, the included implants followed the criteria: standard surgical protocol, >12 months in situ; minimally rough self-threading implants with a turned, machined surface (Mk II Nobel Biocare AB, Göteborg, Sweden], n=210); etched implants of the same macrodesign (3i Implant Innovations Inc., Palm Beach Gardens, FL, USA], n=151), length > or = 10 mm. Clinical data and implant success were rated. Resonance frequency analysis (RFA) and Periotest (Siemens AG, Bensheim, Germany) were measured and related to the corresponding implant survival rate in the respective group. RESULTS: The total number of implants was 361, of which 264 (73%) were subject to clinical reexamination. RFA and Periotest could be recorded in 25% of the implants. Neither clinically relevant nor statistically significant differences between the surface designs were found in the RFA (64 +/- 8.6 vs 63 +/- 9.7), in Periotest (-2 +/- 3.3 vs -1 +/- 5.1), and in mean survival periods (49 months, 95% confidence interval CI]: 46-51 months, for the turned vs 46 months, 95% CI: 43-49 months, for the double-etched implant). After osteoplastic procedures, a significantly higher rate of implant losses in the turned, machined implant group was observed (17 vs 1) with a mean survival period of 43 (40-46) months for the turned and 46 (45-48) months for the double-etched implants. CONCLUSION: No difference between implants with two different minimally rough surfaces was found. A positive effect of surface roughness is observed in poor quality bone, but the pivotal proof of this effect is still lacking.

Interactions between cells and titanium surfaces.

The interaction between cells and implant materials is determined by the surface structure and/or surface composition of the material. In the past years, titanium and titanium alloys have proved their superiority over other implant materials in many clinical applications. This predominant behaviour is caused by a dense passive oxide layer which forms within milliseconds in oxidizing media. Titanium dioxide layers of 100 nm thickness were produced on the surface of cp-titanium grade 2, and on an experimental alloy of high vanadium content (Ti1.5Al25V) as a harmful control. The layers were produced by thermal and anodic oxidation and by coating by means of the sol-gel process. The resulting oxide layers were characterized with respect of their structure and chemical composition. In cell tests (proliferation, MTT, morphology, actin staining), the reaction of the cells was examined. It was shown that the sol-gel-produced titanium oxide layer is able to shield the cells from toxic alloying elements, with the result that the cell reaction is influenced only by the thin titanium oxide surface layer and not by the composition of the bulk material.

Electron spectroscopic studies of interactions between superficially-applied fluorides and surface enamel.

Despite comparable acidities and fluoride concentrations, distinctly different characteristics of fluoride deposition and enamel degradation were observed. In the sequence of the amine hydrofluorides, this may possibly be due to the increasing cation polarity. Thus, by the concomitant rise in the dissociation constants of the compounds, the increasing effectiveness of fluoride accumulation is due to the presence of more free fluoride in solution. On the other hand, sodium fluoride shows behavior comparable to that of the unsaturated amine hydrofluoride. Presumably, specific interactions between the cations and surface apatite may favor fluoride deposition. Under present conditions of treatment, however, neither sodium nor quaternary ammonium cations are deposited in significant amounts on tooth surfaces. Thus, the behavior of fluoridizing agents cannot be understood only by their properties in bulk solution. Obviously, specific reaction mechanisms between surface-adjacent solutions and enamel apatite must be considered. Similar reactions have been studied extensively in connection with corrosion problems. The investigations clearly demonstrated that, for the interpretation of mechanisms, all relevant parameters must be considered: the ionic composition of solutions, the temperature, the time of treatment, etc. Thus, a series of additional experiments will be necessary for more insight into the reaction mechanisms of fluoride on superficial dental enamel. The consequence of the present studies is that the cariostatic effect of superficially applied fluoridizing agents cannot be discussed in terms of fluoride accumulation only.

Validation of three-dimensional surface characterising methods: scanning electron microscopy and confocal laser scanning microscopy.

Surface characteristics of enosseous titanium implants have been known to influence the quality of osseointegration. Parameters recommended for metrical analysis should be supplemented by a topographical description. In this study, Ra values obtained by established tactile and optical profilometric methods are correlated with those obtained by stereo scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). As test specimens, standardised CVD diamond-coated titanium alloys with different microwave coating power ranging from 2,000 to 3,000 W were used. A non-CVD-coated corund grit-blasted specimen (NC) was used as reference. After coating with a CVD, power of 2,000 W Ra ranges from 4.33 to 5.69 microm depending on the method used. With increasing power of the coating process, the amplitude of the surface roughness is significantly increased to 4.53 to 6.89 microm. Ra values of the same sample obtained by different methods are also significantly different (p = 0.001). Compared with the established methods of tactile and optical profilometry, SEM and CLSM offer valid data on the surface roughness accompanied by a topographical imaging. In future studies, the underlying method should be specified to interpret roughness values correctly, as not every method is suitable for each specimen and values obtained by different methods vary extremely.

Changes in the extracellular matrix of the vein wall--the cause of primary varicosis?

BACKGROUND: Conflicting theories on the development of primary varicosis have led to the molecular biological investigation of the vein wall or, more accurately, of the extracellular matrix. It was the aim of this study to quantify matrix expression and to compare pathological changes in the vein wall with valve-orientated staging of varicosis, in order to determine indicators of the primary cause of varicosis. MATERIALS AND METHODS: Three hundred seventy-two tissue specimens of greater saphenous veins were obtained from 17 patients with varicosities and categorised according to Hach stage and procurement site. The specimens were compared with 36 specimens collected from six patients without varicosities, incubated with fluorescence-stained antibodies for collagen 4, laminin, fibronectin and tenascin prior to being assessed with confocal laser scan microscopy. In addition, 22 vein specimens (16 varicose, 6 normal veins) serving as negative controls were investigated. RESULTS: Image analysis and statistical evaluation showed that compared with normal veins, varicose veins are associated with a significant increase in matrix protein expression for collagen 4, laminin and tenascin. A trend towards an increase in matrix expression was further observed for fibronectin. There was, however, no difference between varicose veins and clinically healthy vein segments inferior to a varicose segment. CONCLUSION: If the findings of the present investigation can be confirmed by other studies, alterations in the vein wall may be regarded as the primary cause of varicosis and valvular insufficiency as the result of these changes.

Nanoleakage at the composite-dentin interface: a review.

The term "nanoleakage" was introduced to describe a specific type of leakage within the dentin margins of restorations. Nanoleakage appears as a consequence of the acid etching procedure allowing the penetration of oral and pulpal liquids such as acids into porosities within or adjacent to the hybrid layer. Nanoleakage is independent from microleakage. The amount of penetration depends on the type of bonding agent and on different parameters of the application technique (e.g. etching time, dentin moisture). Nanoleakage is much less extensive than microleakage and has probably no short-term clinical relevance. The long-term stability of the adhesive bond between dentin and restorative material, however, might be adversely affected. Nevertheless, based on the knowledge to date, acid etching prior to dentin bonding should be performed.

Influence of different etching times on hybrid layer formation and tensile bond strength.

PURPOSE: To correlate the thickness of the dentin/resin hybrid layer and the influence of different etching times to the composite resin tensile bond strength. MATERIALS AND METHODS: 775 human molars were prepared by removing the occlusal cusps to expose middle dentin using a microtome saw. Five commercially available dentin bonding agents were applied according to the manufacturers' instructions. Only the etching times were varied. For confocal laser scanning microscopy (CLSM) measurements, 325 teeth were used and the etching times were varied (not etched, 15, 30, 60 or 120 seconds). The primer components or self-priming adhesive, respectively, were fluorescence-labeled by rhodamine B isothiocyanate. In each case, the prepared surfaces were then covered by a layer of composite resin, sectioned and examined using a CLSM in fluorescent mode. The extension of hybrid layers was quantified by measuring the distances between the dentin-composite junction to a visible boundary. For the tensile bond strength measurements (Zwick testing machine), 450 molars were prepared, using different dentin etching times (not etched, 15, 30, 60, 120 and 180 seconds). RESULTS: A hybrid layer was found in all etched teeth. The CLSM data gave evidence for an exponent-function relationship between the periods of etching and the thickness of the hybrid layer. The highest tensile bond strengths were achieved after 15 s of etching, followed by 30 s and 60 s. Under these etching conditions and irrespective of the bonding agent, bond strengths were significantly higher (P < or = 0.05) than without etching or after 120 and 180 s of etching. There was no linear correlation between the thickness of the hybrid layer and the bond strength.

Effect of intrapulpal pressure simulation in vitro on shear bond strengths and hybrid layer formation.

PURPOSE: To test the hypothesis that simulation of an intrapulpal pressure during the application of dentin bonding systems influences hybrid layer formation in vitro. MATERIALS AND METHODS: 180 teeth and three different bonding agents were used for shear bond strength measurements and for confocal laser scanning microscopy studies on the dentin-composite interface. The bonding agents (30 teeth each material) were applied without (90 teeth, Group A) and with intrapulpal pressure simulation of 34 cm H20 with water (90 teeth, Group B). 45 specimens from each group were sheared in a testing machine, and 45 teeth from each group were used for CLSM studies after labeling the primer components of the bonding agents with a fluorescent dye. RESULTS: After simulation of pulpal pressure the bond strengths decreased significantly in all groups (Syntac [without/with]: 16.0 +/- 4.5/8.0 +/- 3.7, P= 0.001; Gluma: 13.3 +/- 4.6/8.4 +/- 4.4, P= 0.008; Prime & Bond NT: 14.8 +/- 5.8/8.7 +/- 5.7, P= 0.007; n = 15 each; U-tests). There were no significant differences between the hybrid layer thickness without/with simulation of pulpal pressure (Syntac: 3.6 +/- 0.6/4.1 + 1.2, P= 0.35; Gluma: 3.5 +/- 0.6/3.7 + 0.9, P= 0.49; Prime & Bond NT: 3.5 +/- 0.9/3.8 + 0.8, P= 0.44; n = 15 each; U-tests). In the case of simulation of an intrapulpal pressure, CLSM examination revealed a distinctly shallower penetration of the adhesives into dentin compared to the samples treated without intrapulpal pressure.

Dentinal adhesion and histomorphology of two dentinal bonding agents under the influence of eugenol.

This study examined the influence of eugenol on the dentinal adhesion of an enamel-dentin bonding agent that removes the smear layer (total-etch technique) and a system that partially dissolves the smear layer (self-conditioning adhesive system). The tensile bond strength was measured on flat, dentinal surfaces after application and removal of pure eugenol for 15 minutes and a eugenol-containing temporary luting agent for 24 hours. In addition, the resin-dentin interface was evaluated with confocal laser scanning microscopy. Dentinal adhesion was found to be significantly reduced after the application of eugenol when a dentin bonding agent that dissolves the smear layer was used. Under confocal laser scanning microscopy, the resin-dentin interdiffusion zone of both adhesive was found to be changed, not only after application of eugenol but also after application of a eugenol-containing temporary luting agent.

Structural changes of acid etched enamel examined under confocal laser scanning microscope.

The aim of the present investigation was to elucidate structural alternations in enamel subjected to acid etch technique and treatment under remineralizing conditions by means of a new microscopy technique known as confocal laser scanning microscopy (CLSM). Blocks of enamel were treated with 37% phosphoric acid and exposed for 3 weeks to an oral environment. Tomographic CLSM images were subsequently obtained and compared with controls. CLSM proved to be a reliable, highly reproducible and simple method of qualitative assessment of structural changes occurring on the surface of enamel and in areas below the surface as deep as 100 microns or more. Structural alterations associated with acid application were observed to occur up to 100 microns below the surface; these largely remained after exposure to an oral environment. Changes manifested as differential light reflection were detected in experimental samples in the form of a hyper-reflexible zone extending as a 10 to 15 microns wide band below the etched surface. This finding indicates that deposition of salivary material occurs on and below the surface of etched enamel exposed to an oral environment. Once qualitative results of CLSM are correlated with quantitative measurements, this technique may facilitate large scale remineralization studies.

Cariostatic effect of a light-cured, resin-reinforced glass-ionomer for bonding orthodontic brackets in vivo. A combined study using microradiography and confocal laser scanning microscopy.

The aim of this study was to evaluate in vivo the cariostatic potential of the resin-reinforced glass-ionomer (Vitremer core build-up restorative; 3M Dental Product Division) when used as a bonding agent for orthodontic brackets. The mineral distribution and topography of the enamel surface adjacent to the bracket base was determined by quantitative microradiography (TMR) and confocal laser scanning microscopy (CLSM). The study was designed in split-mouth technique using 9 pairs of premolars to be extracted for orthodontic reasons. One tooth of each pair was bonded with the resin-reinforced glass-ionomer, and the control contralateral premolar with the non-fluoridated composite (Concise, 3M Dental Products Division). After 4 weeks all teeth were extracted and stored until analysis. The lesion depths and mineral loss values in enamel adjacent to brackets bonded with Vitremer were significantly lower than in teeth bonded with the composite, indicating that the resin-reinforced glass-ionomers significantly reduced caries lesion development in vivo. CLSM images show a severe cariogenic challenge around orthodontic brackets and support TMR measurements.

Dental implantation: ultrasound transmission velocity to evaluate critical bone quality--an animal model.

PURPOSE: In dental implantology, preoperative evaluation of bone quality is an important aspect for the long-term success of the treatment. The insertion of implants into regions with a great portion of poorly mineralised cancellous bone, in particular, increases the risk of subsequent implant failure. The measurement of Ultrasound Transmission Velocity (UTV) proved to be a non-invasive and valid method for the assessment of mechanical properties of bone. The aim of this study was to correlate conventional histomorphometric bone properties with UTV-values from mandibular and iliac crest bone specimens in an animal model. MATERIALS AND METHODS: 12 native (no sample preparation) porcine specimens from mandibular bone and 14 from iliac crest bone, respectively, were studied both by UTV measurement and by conventional histomorphometry. UTV-values were measured bicortically by three different investigators four times at each site. Bone samples from identical sites were obtained and ground down to a thickness of approx. 50 microm. Transmission light microscopy was used to assess the overall percentage of mineralised bone. Mineral bone density less than 40% was defined as "critical". RESULTS: The median bone density was 43 % for mandibular bone and 23% for iliac crest bone, respectively. The median UTV values of the mandible (1756 m/sec) were significantly higher than those of the iliac crest specimens (1613 m/sec). Intra-individual correlation testing illustrates a positive, statistically significant correlation between presurgical UTV measurement and the grade of mineralisation (r=0.54). ROC analysis defined a UTV breakpoint of 1767 m/sec to identify critical bone with a sensitivity of 75%. CONCLUSION: In this ex-vivo model, UTV measurements have proven to be a reliable method for identifying critical bone quality prior to implantation.

Comparative histomorphometry and resonance frequency analysis of implants with moderately rough surfaces in a loaded animal model.

OBJECTIVES: Test of favourable conditions for osseointegration with respect to optimum bone-implant contact (BIC) in a loaded animal model. The varied parameters were surface roughness and surface topography of commercially available dental implants. METHOD: Thirty-two implants of six types of macro and microstructure were included in the study (total 196). The different types were: minimally rough control: Branemark machined Mk III; oxidized surface: TiUnite MkIII and MkIV; ZL Ticer; blasted and etched surface: Straumann SLA; rough control: titanium plasma sprayed (TPS). Sixteen beagle dogs were implanted with the whole set of the above implants. After a healing period of 8 weeks, implants were loaded for 3 months. For the evaluation of the BIC areas, adequately sectioned biopsies were visualized by subsurface scans with confocal laser scanning microscopy (CLSM). RESULTS: The primary statistical analysis testing BIC of the moderately rough implants (mean 56.1+/-13.0%) vs. the minimally rough and the rough controls (mean 53.9+/-11.2%) does not reveal a significant difference (P=0.57). Mean values of 50-70% BIC were found for all implant types. Moderately rough oxidized implants show a median BIC, which is 8% higher than their minimally rough turned counterpart. The intraindividual difference between the TPS and the blasted and etched counterparts revealed no significant difference. The turned and the oxidized implants show median values of the resonance frequency [implant stability quotients (ISQ)] over 60; the nonself-tapping blasted and etched and TPS implants show median values below 60. DISCUSSION: In conclusion, the benefit of rough surfaces relative to minimally rough ones in this loaded animal model was confirmed histologically. The comparison of different surface treatment modalities revealed no significant differences between the modern moderately rough surfaces. Resonance frequency analysis seems to be influenced in a major part by the transducer used, thus prohibiting the comparison of different implant systems.

[Systematic qualitative histology of enossal implants with anodically oxidised surfaces]. / Systematische qualitative Histologie von enossalen Implantaten mit anodisch oxidierten Oberflächen.

Histological analysis of dental implants is often quantified by calculating the bone-implant contact rate (bone volume/total volume), whereas qualitative aspects like osteoconduction are underrepresented. The aim of this study was to focus on the micro-architectural properties of the bone-implant contact under physiologic loading using a systematic analysis of these characteristics. In 16 Beagle dogs we inserted 6 different types of dental implants in the hard bone of the mandible and the soft bone of the maxilla. After a healing period of two months the implants were loaded for three months and then histologically analysed. For the metric evaluation of qualitative histological aspects 12 examiners answered pivotal questions: 1. Is the implant functional sufficient? 2. Do you see close contact to bone, were bone is present? 3. Is the amount of bone at the implant at least similar to the peripheral bone? 4. Is the bone to implant contact homogenous? 5. Does the bone show a functional architecture? 6. Do you find osseoconductive bone apposition at the crestal and apical border? A superiority of anodic oxidized surfaces can be seen in questions 1, 2 and 3 mainly for the comparison of identical macrodesigns (MkIII). The potential for osseoconductivity (questions 4 and 5) shows a tendency for significant differences for the ZL Ticer implant. Homogenous bone to implant contact is rarely found, in contrast to rather positive implant function ratings. This suggests the existence of an optimum in the bone to implant contact rate. Bone to implant contact rate as an isolated quantitative parameter should in future be completed by a systematic, standardised and blinded analysis of qualitative properties.