Comparative Sample Preparation Using Focused Ion Beam and Ultramicrotomy of Human Dental Enamel and Dentine for Multimicroscopic Imaging at Micro- and Nanoscale.

(1) Background: The aim of this study was to systematically compare TEM sections of mineralized human enamel and dentine prepared by focused ion beam (in situ lift-out) technique and ultramicrotomy through a combination of microscopic examination methods (scanning electron microscopy and transmission electron microscopy). In contrast with published studies, we compared the TEM preparation methods using the same specimen blocks as those for the ultramicrotomy and FIB technique. (2) Methods: A further evaluation of TEM sample preparation was obtained by confocal laser scanning microscopy and atomic force microscopy. In addition, ultramicrotome- and focused ion beam-induced artefacts are illustrated. (3) Results: The FIB technique exposed a major difference between non-decalcified enamel and dentine concerning the ultrastructural morphology compared to ultramicrotome-prepared sections. We found that ultramicrotomy was useful for cutting mineralized dentine, with the possibility of mechanical artefacts, but offers limited options for the preparation of mineralized enamel. FIB preparation produced high-quality TEM sections, showing the anisotropic ultrastructural morphology in detail, with minor structural artefacts. Our results show that the solution of artificial saliva and glutardialdehyde (2.5% by volume) is a very suitable fixative for human mineralized tissue. (4) Conclusions: The protocol that we developed has strong potential for the preparation of mineralized biomaterials for TEM imaging and analysis.

Correlation of ultrasound microscopy and Vickers hardness measurements of human dentin and enamel - A pilot study.

OBJECTIVE: To investigate if Vickers microhardness of dentin and enamel correlated with acoustic velocity c(l) or acoustic reflection from the sample's top (amplitude). METHODS: Eight transversal sections of a sound human tooth were investigated with scanning acoustic microscopy (SAM) and Vickers microhardness measurements. Longitudinal acoustic velocity c(l), amplitude and microhardness MHV were evaluated and for each c(l) test point corresponding amplitude and MHV were linearly interpolated and graphically analyzed. Spearman rank order correlation (rS) was calculated (p<0.05). RESULTS: c(l) was predominantly 6100-7000ms-1 in enamel and 3800-4600ms-1 in dentin and correlated significantly with MHV with 27-420 in enamel and 20-90 in dentin (rs=0,57). Amplitudes significantly correlated with MHV, too, but even better (rs=0,77). SIGNIFICANCE: Acoustic velocity and amplitudes were appropriate to detect microhardness differences of dentin and enamel and certain value ranges of both could be assigned to certain MHV ranges. Further research is needed to differentiate more precisely between the different hard tooth tissues.

Flexible Automation System for Determination of Elemental Composition of Incrustations in Clogged Biliary Endoprostheses Using ICP-MS.

Automation systems are well established in industries and life science laboratories, especially in bioscreening and high-throughput applications. An increasing demand of automation solutions can be seen in the field of analytical measurement in chemical synthesis, quality control, and medical and pharmaceutical fields, as well as research and development. In this study, an automation solution was developed and optimized for the investigation of new biliary endoprostheses (stents), which should reduce clogging after implantation in the human body. The material inside the stents (incrustations) has to be controlled regularly and under identical conditions. The elemental composition is one criterion to be monitored in stent development. The manual procedure was transferred to an automated process including sample preparation, elemental analysis using inductively coupled plasma mass spectrometry (ICP-MS), and data evaluation. Due to safety issues, microwave-assisted acid digestion was executed outside of the automation system. The performance of the automated process was determined and validated. The measurement results and the processing times were compared for both the manual and the automated procedure. Finally, real samples of stent incrustations and pig bile were analyzed using the automation system.

No influence of simultaneous bone-substitute application on the success of immediately loaded dental implants: a retrospective cohort study.

PURPOSE: To examine the influence of bone-substitute application during implantation on the success of immediately placed and loaded dental implants. MATERIALS AND METHODS: A total of 147 consecutive patients (age, 16.5-80.4 years) were provided with 696 immediately loaded implants. The mean follow-up time was 34.1 months. Of these implants, 50.4% (n=351) were immediately placed into extraction sockets. A total of 119 implants were added by simultaneous bone-substitute application (NanoBone, Artoss GmbH, Rostock Germany), whereas the other implants were placed in healed bone. Univariate and multivariate analysis was performed using IBM SPSS V.20. RESULTS: The overall implant success rate was 96.1%. Implants with simultaneous bone replacement had a hazard ratio of 0.877 (p=0.837); 95% CI, 0.253-3.04). Factors found to be statistically significant modifiers of success on multivariate analysis (p<0.05) included type of superstructure (p<0.001), implant-abutment connection (p<0.001), membrane use (p=0.010), and jaw (p=0.026). None of the other factors investigated were significant modifiers. CONCLUSIONS: The present study demonstrates high success rates for immediately loaded implants and their superstructures independent of the simultaneous application of bone substitute. The declared aim of socket preservation, the prevention avoiding bone loss, is achieved in the immediate implant placement scenario under immediate-loading conditions.

Development of a biodegradable tissue adhesive based on functionalized 1,2-ethylene glycol bis(dilactic acid). II.

In body regions where damage and bleeding must be avoided, a substitute for mechanical tissue fixation by sutures or staplers is needed. Since tissue adhesives provide easy and fast handling they are a promising alternative. The present study reports the development and analysis of a tissue adhesive that consists of two adhesive components: hexamethylene diisocyanate (HDI) functionalized 1,2-ethylene glycol bis(dilactic acid) (ELA-NCO) and chitosan chloride. This composition was chosen based on preliminary studies on several chain elongation agents. The present study evaluates this adhesive system by IR-spectroscopy, tensile tests, and gel point measurements in comparison to fibrin glue. The system's in vitro biocompatibility was tested with mouse fibroblasts (L929) according to ISO 10993-5. Furthermore, an implantation study was performed in SPF-Wistar rats. The adhesive strength of manually applied mixtures or mixtures applied by double chamber syringes with a mixing extruder was determined to be significantly higher than that of fibrin glue on bovine muscle tissue at 37°C. Tensile strength increased further when exposure time of the adhesive was increased from 10 min to 48 h. The rheological gel point determination showed that the mixture of ELA-NCO/DMSO and chitosan chloride offers a time window large enough to readjust the fused joint during surgery, as opposed to fibrin glue. Additionally, the in vitro and in vivo biocompatibility studies of the adhesive system revealed no toxic effects on the surrounding tissue.

[A novel cryo-grinding technique for evaluating new cochlear implant electrodes after ex vivo insertions in temporal bone specimens]. / Entwicklung einer Kryo-Schlifftechnik für Felsenbeinpräparate: eine neue Möglichkeit zur Beurteilung ex vivo implantierter CI-Test-Elektroden.

BACKGROUND: Prior to clinical application, newly developed prototypes of cochlear implant electrode arrays must prove their suitability with the smallest possible tissue damage in ex vivo temporal bones. So far, after insertion of the electrodes the temporal bone specimens have to be processed in a rather intricate technique, including embedding, sectioning or grinding prior to histological evaluation. The question remains whether for special indications this time-consuming method, which even causes artifacts, can be replaced by a new technique based on cryo-grinding. MATERIALS AND METHODS: The main principle of the method described is to grind the temporal bone with the inserted electrode in a frozen state, provided by a fixation device filled with dry ice. After creating a plane surface and staining it (still in a frozen state), the specimen can be examined and photographed with a projection microscope. This procedure is continued by subsequently grinding and examining new surfaces in defined distances. RESULTS: In numerous trial runs the method proved feasible, saving much time and manpower. After grinding, each plane could be examined sufficiently; the site of the electrodes and the corresponding tissue damage could be documented properly. DISCUSSION: The new concept of cryo-grinding provides relatively easy and fast examinations of temporal bones after inserting test electrodes. The examiner is enabled to correlate his "sensations" during the insertion (e.g., smoothness, resistances) almost directly with the morphologic findings, without having to wait a long time while the temporal bone specimens are being processed conventionally. Furthermore, this method avoids artifacts due to soft tissue shrinking during drying. In further steps of development, the grinding device will be optimized for standard use.

Development of a biodegradable tissue adhesive based on functionalized 1,2-ethylene glycol bis(dilactic acid). I.

Tissue adhesives are a valuable alternative for mechanical tissue fixation by sutures or staples. Adhesives are desirable in body regions where damage and bleeding must be avoided. Tissue adhesives provide easy and fast handling. This study reports the development of a tissue adhesive based on 1,2-ethylene glycol bis(dilactic acid) (ELA) functionalized with hexamethylene diisocyanate (HDI) to produce isocyanate terminated ELA-NCO which was characterized by NMR and FTIR spectroscopy. ELA-NCO together with chain elongation agents forms an adhesive system suitable for tissue fixation. Several biodegradable polymers, such as hyaluronic acid, gelatin, chitosan acetate, and chitosan chloride were tested as chain elongation agents to obtain an adhesive system and studied on bovine muscle tissue to evaluate their adhesive strength and compared to fibrin glue. Tensile strength of glued joints was determined by a Zwick universal testing machine at ambient temperature. Mixtures of ELA-NCO and chitosan acetate or chloride, showed significantly higher adhesive strength than fibrin glue. Reaction between ELA-NCO and chitosan chloride produced polyurethane was traced by FTIR spectroscopy. NMR, FTIR, and rheological measurements demonstrated that ELA-NCO and chitosan chloride can be sterilized by gamma-rays or superheated water vapor without alterations, respectively. A mixture of ELA-NCO and chitosan chloride can be useful as medical tissue adhesive.

[The influence of macro- and microstructure on the surface wettability and retention properties of endodontic posts in vitro]. / Der Einfluss der Makro- und Mikrostruktur auf die Benetzungs- und Retentionseigenschaften von Wurzelkanalstiften in vitro.

Aesthetical and biomechanical requirements on fiber-reinforced endodontic posts play an important role in clinical application. Leading to a long-term clinical success, post systems must ensure an adequate force transmission and optimal retention behavior into the root channel. The aim of this study was to determine the microstructure and morphology of commonly used fiber reinforced endodontic posts in vitro. Post-specific fiber diameter, fiber arrangement over the cross-sections and length, and fiber-matrix ratio could be associated with mechanical properties, such as three-point bending strength and structure modulus. Furthermore, our investigation focused on the quantification of the wettability of the post surface. By using a modified contact-angle measurement, the meniscus on the post surface was characterized. In additional investigations, posts were inserted in artificial root channels and the extraction force was measured. The results show a strong correlation between the extraction force and the wettability of the post surface.

[Drug release of coated dental implant neck region to improve tissue integration]. / Pharmakafreisetzende biodegradierbare Polymerbeschichtung von dentalen Titanimplantaten zur Verbesserung der Weichgewebsintegration.

In order to improve tissue integration, the neck region of dental implants was coated with the biodegradable polymer poly (L-lactide) incorporating tetracycline, ibuprofen and the combination of both drugs using a solvent dip-coating process. Metallographic analysis, light microscopy and electron microscopy were used to detect the thickness range and the surface characteristics of the coatings. Cytotoxicity was evaluated using the tetrazolium colorimetric method with the fibroblast cell line L929. The in vitro drug release was measured in isotonic sodium chloride solution by UV spectroscopy. To explore if drug release is concentration-dependent, the total amount of drug was varied in the coating (20% wt, 30% wt and 40% wt). The results showed a continuous release of the embedded drugs in relevant dosage over a period of 6 months. In contrast to high tetracycline concentrations, high ibuprofen concentrations resulted in a decreased metabolic activity of the L929 fibroblasts.

Electromyographical recording of the electrically elicited stapedius reflex via a bipolar hook electrode.

OBJECTIVE: To prove the feasibility of recording stapedius reflexes by bipolar electromyography intra-operatively during cochlea implant via a bipolar hook needle electrode. STUDY DESIGN: Prospective study. SETTING: Tertiary referral center. PATIENTS: Adults receiving a MED-EL cochlear implant. INTERVENTION: Diagnostic. MAIN OUTCOME MEASURES: Stapedius reflex responses can be recorded by electromyography. RESULTS: A double hook electrode could be inserted into the muscle tissue via the natural opening of the pyramid process along with the stapedius tendon. In 7 out of 10 patients supplied with MED-EL CIs (PULSARCI100 or SONATATI100), electrically elicited stapedius reflex potentials were recorded after eliminating stimulation artifacts caused by electromagnetic fields during electrical stimulation by low pass filtering. CONCLUSION: Intraoperative stapedius reflex recording by bipolar electromyography via a hook electrode is feasible. Further research will show whether these signals may be used for establishing a self-adjusting speech processor.

Poly(3-hydroxybutyrate) (PHB) patches for covering anterior skull base defects - an animal study with minipigs.

CONCLUSION: We conclude that PHB patch material may fulfil the specific requirements that are necessary for a dural substitute, including defect closure, stability and biocompatibility. Our results support the assumed positive influence of PHB on bone regeneration. OBJECTIVES: Although many experimental and clinical studies have been performed to identify a suitable material to repair defects of the dura mater, no ideal dural substitute is currently available. PHB is a biodegradable and biocompatible polymer that might serve as dural substitute and osteosynthesis material in cranial bone defects. MATERIALS AND METHODS: Different standardized PHB patches were used in six minipigs for covering defined bone defects in the anterior skull base including a dura mater lesion as well as in the frontal sinus front wall. After a defined time of implantation of 3, 6, and 9 months PHB patches were explanted and examined for clinical findings, biodegradation, presence of microorganisms, histological findings, and electron microscopy. RESULTS: The examinations revealed an increasing closure of bone defect corresponding with time. The anterior skull base bone defect was completely closed after 9 months. The histological findings revealed a connective tissue and callus formation around the PHB patches with fibroblasts and foreign body/giant cell reaction growing through PHB membrane pores. There were no reactions or adhesions between brain and PHB or dura mater and PHB, respectively. Investigations of biodegradation and electron microscopy revealed a continuous breakdown of PHB in the course of time with variations due to different PHB structures. Microbiological investigations could not detect any florid intracranial infection.

Optical coherence tomography as an orientation guide in cochlear implant surgery?

CONCLUSION: With optical coherence tomography (OCT) it is basically possible to reveal parts of the cochlear morphology without opening its enveloping membranes. Thus, it may serve as a helpful guide for the surgeon to localize the scala tympani precisely before opening the fluid-filled inner ear to insert the electrode array. OBJECTIVE: To improve anatomical orientation in cochlear implant surgery before definitively opening the fluid-filled inner ear. The question was whether a new imaging technique, OCT, might provide information about the site of the underlying inner ear structures (scala tympani, scala vestibuli) and could, consequently, guide the surgeon towards the scala tympani. MATERIALS AND METHODS: In a preliminary study, OCT was carried out on human temporal bone preparations, in which a cochleostomy ('fenestration') was performed leaving the endosteum and the fluid-filled inner ear intact. OCT was applied via a prototype of a specially equipped operating microscope. The mode of OCT used in this context was spectral-domain (SD)-OCT. RESULTS: On scans, which can be read analogous to B-mode sonography, OCT provides information about structures on the inner surface of the partly exposed but still intact membranous cochlear lining - such as scala tympani or scala vestibuli.

Severe damage of the femoral head after dislocation and difficult reduction maneuvers after total hip arthroplasty.

INTRODUCTION: Aseptic loosening associated with osteolysis and release of wear particles is the main reason for revision of total hip arthroplasty (THA). Damage of femoral heads is well known to increase the wear rate at the articulating surface. Instability and dislocation are serious complications and are the second most frequent reason for revision surgery after THA. In the present work the possible damaging of the femoral head as a result of difficult reduction maneuvers of dislocated THA should be investigated. PATIENTS AND METHODS: In three cases of total hip dislocation an open reduction was performed after dislocation of primary THA. Thereby one metal and two ceramic femoral heads were retrieved. Each head was analyzed macroscopically and by scanning electron microscopy (SEM) subsequently. RESULTS: In the first case the SEM analysis revealed severe titanium deposits on the retrieved ceramic head in the case of repeated unsuccessful closed reduction maneuvers. In the second case the retrieved ceramic head showed multiple scratches and a spalling of the surface structure. In the third case the retrieved metal head presented a roughened surface and severe scratches at the articulating area. DISCUSSION: Difficult reduction maneuvers following total hip dislocation can lead to severe damaging of the femoral head associated with an increase of the surface roughness. While repeated reduction maneuvers can lead to metallic deposits and spalling of the surface of ceramic heads, severe scratches can occur at metal heads. The damaging of the femoral head at the articulating surface can lead to increased wear and subsequent aseptic implant loosening. Therefore, open joint reduction has to be considered in cases of difficult or unsuccessful closed reduction maneuvers to prevent severe femoral head damage which may cause an increased release of wear particles.

Three-dimensional ultrasound biomicroscopy, environmental and conventional scanning electron microscopy investigations of the human zonula ciliaris for numerical modelling of accommodation.

PURPOSE: Biomechanical modelling of the accommodation process is a useful tool for studying the mechanism of accommodation and presbyopia and can aid in the development of accommodative lens-replacing materials. Existing biomechanical models, however, use a very simplified zonula structure. The aim of this study was to use three-dimensional ultrasonic imaging and scanning electron microscopy to provide a more detailed, three-dimensional description of the structure of the human zonula to improve the modelling of accommodation. METHODS: Five human eyes were examined without invasive manipulation using a custom-made three-dimensional ultrasonic imaging technique that allows scanning of features with a spatial resolution of 30 microm. Environmental and conventional scanning electron microscopy (SEM) provided information to complement the ultrasonic images for use in development of a more anatomically correct finite-element model of the zonula structures. These data along with the material properties of the ocular tissue structures were used to construct an advanced geometric model for finite-element simulation of the accommodation process. RESULTS: Images were obtained through three-dimensional ultrabiomicroscopy (3D-UBM) of anatomical features heretofore not directly imagable in their native state. Ciliary processes and zonula structures were clearly separated by both the 3D-UBM and the SEM methods. It was found that fibres inserting on the anterior and posterior lens capsule emerge anteriorly at the ciliary body. Fibres emerging near the pars plana insert on the lens and the ciliary body. No X-shaped crossing fibres were found. Modelling of the accommodation process with both the simple and the more complex geometric models produced refractive power changes comparable with in vivo findings. CONCLUSIONS: The 3D-UBM allowed examination of zonula structures in their native state with minimized preparation artefacts. While these data were incorporated into a complex and more anatomically correct finite-element simulation of intraocular features including lens, zonular system and ciliary body it was found that a simplified zonular model is sufficient for the numerical simulation of the accommodation process.

An "endosteal electrode" for cochlear implantation in cases with residual hearing? Feasibility study: preliminary temporal bone experiments.

BACKGROUND: Over the years, an increasing number of patients with some degree of residual hearing have received cochlear implants. In these cases, the marginal benefit provided by hearing aids alone is not sufficient; however, as experience has already shown, when hearing aids are used in combination with a cochlear implant, more benefit may be obtained. As a prerequisite, this requires that residual hair cell function must remain intact postoperatively. One of the European pioneers of cochlear implants, Ernst Lehnhardt, questioned whether residual hearing might better be preserved if the implanted electrode permits the fluid-filled inner ear space to remain intact. Subsequently, he proposed insertion of a very flat electrode array design into the extraluminal space between the spiral ligament and the bony cochlear wall (endosteum). OBJECTIVE: Our study aimed to determine whether it is feasible to insert an endosteal electrode model intracochlearly but extraluminarily, anatomically, and ultimately surgically and to determine the impact on surrounding intracochlear structures. METHODS: Insertion of two silicon models of an endosteal electrode were carried out in 15 human temporal bones. Histologic examination of the temporal bones after electrode insertion was performed on both fresh and fixed specimens to determine whether the desired anatomic site of insertion was achieved. In combination with light reflected and electron microscopic techniques, the extent to which the surrounding structures were impacted was also examined. RESULTS: Successful insertion of the prototype silicon endosteal electrodes was performed intracochlearly and extraluminarly in 11 of the 15 temporal bone specimens, confirming the anatomic feasibility of insertion into the crevice between the spiral ligament and endosteum. CONCLUSIONS: On the basis of the anatomy of the human temporal bone, insertion of an "endosteal electrode" is feasible. Subsequently, in vivo animal studies are needed to determine the physical effects of insertion of an endosteal electrode design prototype upon the functionality of the surrounding intracochlear structures and in particularly the ability to preserve hearing function.