Our laboratory facilities

Repetitive loading weakens components over time, leading to catastrophic failure at loads lower than the component maximum load bearing capacity if fatigue properties are not considered. Small cracks or crystal defects invisible to the naked eye, grow or compile as the component is loaded repeatedly below its yield strength. This leads to a gradual decrease in the component’s load bearing capacity, followed by permanent deformation or catastrophic failure. Testing implant systems according to ISO 14801 increases patient safety by helping mitigate the need for revision surgery caused by inadequate implant designs.

The DYNA5dent produced by DYNA-MESS Prüfsysteme GmbH is especially built for testing according to ISO 14801. Each machine can run five parallel and fully independent tests. The machines are also supplied with a temperature control unit which circulates water or saline in the around the test specimen. This means tests also take into consideration important defect nucleation and growth mechanisms such as corrosion fatigue, which is an important factor in the harsh oral environment.

With two DYNA5dent machines in our laboratory, NIOM can run ten parallel in vitro tests with high precision. This means we can provide representative test results in accordance with ISO14801 in a time efficient manner. NIOMs aim is to use this capability to ensure implant systems on the Nordic market are safe and to enhance their research on related material properties.

Flow cytometry is a technology that simultaneously measures and then analyzes multiple characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light.

The technology is based upon detection of fluorescence and light scatter from a high number (usually several thousand) of single cells in suspension (Figure 1). Cells can be labeled with fluorescent molecules (fluorochromes) that bind to specific components in the cells, such as DNA and protein. The cells are sent through an intense laser light causing light emission from the fluorochrome. This light is collected by a special type of optics that focuses the light in a fluorescence detector (photomultiplier). The photomultiplier converts light to electronic spikes that are proportional to the amount of fluorescent compound in the cell.

Flow cytometers have a wide range of applications. They are used in routine clinical laboratories and in many research laboratories.

NIOM has flow cytometers with the ability to analyze cells with the use of UV, 488 nm and 640 nm lasers. One example of measuring DNA in cells is seen in Figure 2. A cell line (BEAS 2b) was exposed to 2-hydroxyethyl methacrylate (HEMA) for 24 hours, and differences in growth pattern are recorded. Other examples are measuring glutathione and reactive oxygen content in cells.

The hardness tester at NIOM is capable of measuring the hardness of many different materials (Figure 1). The instrument is currently used in a range of different research projects. One example is the effect of abrasion and erosion on surface hardness of different dental materials.

Two visiting researchers have previously investigated the results of different treatment methods to improve the surface of molar-incisor-hypomineralisation (MIH)-affected teeth (Figure 2). Hardness is an important mechanical property of dental materials and is defined as the resistance to permanent surface indentation. Sufficient hardness ensures that the placed restorations are resistant to in-service scratching, from both mastication and abrasion.

The instrument is configured to carry out two types of microhardness testing; Vickers and Knoop. In both methods a standardized load is applied to a fixed point on the sample. This creates a symmetrically shaped indentation, which can be measured and the hardness of the material calculated. In the Vickers hardness test, a diamond in the shape of a pyramid is used to make the indentation with a force load ranging from 100g to 30kg. In contrast, the diamond indenting tool used in the Knoop method is more narrow and elongated and a lower load is applied (20g to 2kg) (Figure 3).

Traditionally, Vickers is used to measure the hardness of very hard and brittle materials, such as cast dental alloys, but is also applicable to softer materials. Knoop has been used for a wider range of materials, from amalgam and ceramics to resin-based composites, but is also useful for materials that vary in hardness over an area of interest, such as enamel and dentin

NIOM has a high sensitivity ultra-high performance liquid chromatography mass spectrometry (UHPLC-MS) instrument.

The UHPLC allows separation of components in a short time and the triple quadrupole MS allows quantification of substances present in very small amounts (picogram-femtogram range).

UHPLC-MS is used in a range of scientific disciplines, such as the pharmaceutical industry, forensics, food and materials science, to detect and quantify substances in solution. This combined analytical technique works by separating the components, e.g. according to polarity, in the liquid phase (UHPLC) before they are injected in the mass spectrometer (MS). In the MS the components of interest are ionized to charged particles and are separated in the gas phase according to their mass-to-charge (m/z) ratio.

Assessing the extent of leaching from different materials is an essential part of NIOM’s activities and UHPLC-MS is therefore an important tool for improving patient safety.

The curing of resin-based dental materials is never complete. These complex materials may leach unreacted components, as well as additives, degradation products and contaminants into the oral environment. The concentrations of the leaching components are often very low, but the high sensitivity of the new MS will allow detection and quantification of the leaching components, eg. methacrylate monomers and bisphenol A (Figure 2), at very low concentrations. Assessing the extent of leaching from different materials is an essential part of NIOM’s activities and UHPLC-MS is therefore an important tool for improving patient safety.

The methacrylate-based monomers in dental materials have been shown to affect cells in vitro by reducing cell growth. However, the mechanisms through which this process occurs are not fully understood. Research projects at NIOM are currently focused on elucidating these mechanisms through the use of mass spectrometry to study interactions between the dental monomers and proteins in cells. In the long run, the new UHPLC-MS instrument will thus provide information that can be used to improve the biocompatibility of resin-based dental materials.

The analytical method GC-MS is used to identify and quantify volatile substances within a test sample.

It combines the features of gas-chromatography which separates substances in a mixture primarily based on the boiling point but also on the affinity of the column used for the separation. The mass spectrometer breaks each molecule into ionized fragments and sorts the ions based on their mass-to-charge ratio.

Using both GC and MS gives more certainty when identifying substances. Using a column coated with an appropriate stationary phase causes the substances within the sample to elute at different times, known as the retention time. In some cases, substances may travel through the column using the same amount of time, which results in two or more substances that co-elute. When using MS as the detector, these substances may be identified by the mass spectra. Two different substances may also have a similar pattern of ionized fragments when using a mass spectrometer, as shown in the mass spectra of Diethyleneglycol dimethacrylate and Triethyleneglycoldimethacrylate, but often have different retention times.

With the use of GC-MS we are able to identify substances often used in dental materials e.g. composites, sealants and denture base materials.

NIOM has extensive experience with the use of gas chromatography and have made our own library of substances over the years, which helps us to identify unknowns based on retention time. For identification we also use Safety data sheets, manufacturer’s instruction, a NIST MS Search program, and reference solutions.

When substances are identified they may also be quantified using GC-MS. The detection/quantification limits are set for each substance.

NIOM is experienced in quantification of residual methyl methacrylate according to the two standards of ISO 20795 — Dentistry — Base polymers Part 1 and 2.

In terms of dental materials, thermal properties are of high importance, especially when different materials are to be joined together. Dental crowns or bridges consist of metal alloy or ceramic cores covered with veneering ceramics. These materials undergo changes during heating and cooling.

Thermomechanical analysis, TMA, can provide valuable information on materials that influence on production conditions for dental restorations, which is difficult to obtain by other analytical techniques.

NIOM can perform measurements of coefficients of linear thermal expansion for dental metals, alloys and crystalline ceramics, as well as of glass transition temperatures for glass-containing ceramics. NIOM also offers accredited testing of theses parameters using a TMA 402 F3 Hyperion.

Thermomechanical analysis is also used in research projects. Glass transition is typical for amorphous materials, like glass and polymer materials. Therefore, this instrument is also suitable for determining the same parameters for polymer materials.

TMA provides valuable insight into structural changes or phase transformations. Coefficients of thermal expansion for the core material and the veneering material will always be different and stresses will build up between the two materials by repeated heating and cooling during veneering.

Metal alloys and crystalline ceramics have almost a linear thermal expansion from low temperatures up to a possible phase transformation or melting point while veneering ceramics consisting of a glass matrix filled with different crystals have a less well-defines change in the thermal expansion at the glass transition temperature (Tg).

NIOM posesses a Varian model 670 FT-IR (Agilent Technologies) equipped with ATR, DRIFT, and transmission accessories. The instrument is used among others to find the degree of conversion of polymer-based dental materials.

Molecules interact with light over a wide wavelength range, and the interaction between light and molecules will form a pattern that can give us detailed information about the structure of the molecule.

Infrared spectroscopy is one of the most widely used techniques for identification of chemical compounds and materials, including liquids, solid substances and gases, through their ability to characterize absorption of infrared radiation.

There are four most used sampling techniques in FTIR depending on what type of samples and consistency it is:

• Transmission
• Attenuated Total Reflection (ATR)
• Specular Reflection
• Diffuse Reflectance

Degree of conversion
The degree of conversion (DC) is a measurement of the curing of a resin-based material. FT-IR may be used to determine the DC. The method calculates the conversion of reactive methacrylate groups in the material. The calculation is based on ratios of area or height of specific peaks in the FTIR-spectrum of the material.

A typical FTIR spectrum with a characteristic peaks.
The ATR technique may be used to measure DC at different depths of a material, for instance a cylinder may be used to prepare samples of varying thickness. This method is particularly relevant for light-curing composite materials, where the layering of the material in the restoration is an issue. The material may then be cured from the top surface using an ordinary curing unit in a setup resembling the in situ curing. The method can be used for evaluation of bulk-fill materials for which thicker layers (e.g. 4 mm) are used, compared to conventional composites.

NIOM may perform measurements of degree of conversion of restorative materials and other polymer-based biomaterials using our FTIR instrument. We have equipment for measuring transmission and reflection (ATR) as well as a DRIFT accessory (diffuse reflectance) for powder samples.

NIOM has a Shimadzu UV-2550 UV/Vis spectrophotometer with double monochromator design that provides a high-energy throughput optical system with ultra-low stray light. While featuring low stray light, excellent energy characteristics are provided over an extremely wide wavelength range.

Qualitative analysis can be used to identify certain classes of compounds both as pure samples and in biological mixtures. This type of spectroscopy is most commonly used for quantification of biological samples either directly or via colorimetric assays.

UV-visible spectra will not give an absolute identification of an unknown compound, but the spectra are frequently used to confirm the identity of a substance through comparison of the measured spectrum with a reference spectrum.

NIOM has several microscopes for research of different topics:

For biological science (cells and bacteria):

Confocal Laser scanning microscope, CLSM, system FV1200, Olympus BX61WI, equipped with two lasers, a diode laser emitting at 473 nm and a helium-neon laser emitting at 543 nm, 10X and 60X with oil or water (see NIOM Newsletter August 2016).
Fluorescence microscope: Olympus BX51 with 10X, 40X/oil and 100X/oil objectives, filters for UV excitation, blue excitation and green excitation and a DP70 camera.
Inverted microscope with phase contrast: Olympus CX41 with 4X, 10X and 20X objectives and Olympus C-7070 camera.

For material science (polymers, metals, alloys and ceramics):

• Inverted microscope, Nikon Epiphot with 5X, 10X, 20X, 40X, and 100X objectives and accessories like analyzer, polarizer, first order red compensator with λ-filter and dark-field, Numarski prisms, scale bar and a SPOT RT digital camera.
• Profile projector, Nikon with 10X mirror, 50X and 100X objectives and the accessories: Two illumination systems – surface and contour, micrometer, X- and Y-measurements and condenser lever with anti-glare filter.
• Photo macroscope, Wild M400, Wild-Leitz with Macrozoom lens 6,3X – 32 X and two adaptors, ½X and 2X, two illuminations, Intralux 250 HL, Volpi and EasyLED SP, Schott; a SPOT digital photo adapter for the SPOT RT camera.
• Euromex Nexus Zoom EVO stereo microscope with 10x widefield, 0,5 – 5,5X zoom + 1,5X objective, 3W LED, polarizer and analyzer for transmitted illumination and Ring light with 144 LEDs of up to 23.000 LUX.

Confocal laser scanning microscope (CLSM) system FV1200 from Olympus opens new possibilities for high-quality imaging of biological and material samples at NIOM.

The confocal microscope works principally by using laser light to excite emission of fluorescence from dyes or molecules in a sample. The CLSM focuses light to a spot and scans it across the sample to build an image, in contrast to a traditional microscope that excite the whole field at once. The confocal microscope has a pinhole that excludes out-of-focus fluorescence. This feature enables the operator to take a set of images at different levels of depth and reconstruct a three- dimensional image of the object of interest. The CLSM at NIOM is equipped with two lasers, a diode laser emitting at 473 nanometres (nm) and a helium-neon laser emitting at 543 nm. The microscope is also equipped with a motorized stage, enabling automatic mosaic imaging defined by the user.

In addition, the microscope can be used as a traditional fluorescence microscope with a mercury lamp as excitation source. Specifications of the available objectives are listed in the table below. CLSM is widely used in many biological science disciplines, such as microbiology and eukaryotic cell biology. Biofilms, a surface-associated bacterial community embedded in an extracellular matrix, may have a complex structure.

The CLSM is ideal for imaging biofilms on material surfaces and enables investigation of the spatial organization of bacteria within a biofilm. In addition, CLSM may aid in discriminating between live and dead bacterial cells in a biofilm by using dyes for cell viability. In eukaryotic biology, the CLSM is used to investigate e.g. localization of cellular organelles, the cytoskeleton and other cellular components.

Magnification

10x
60x
60x

Numerical aperture

0.3
1.35
1.0

Working distance (mm)

10.00
0.15
2.00

Immersion

Dry
Oil
Water

Real-time polymerase chain reaction detection system.

Polymerase chain reaction (PCR) is a common technique in modern molecular biology for amplifying DNA in order to get sufficient DNA for qualitative and quantitative detection. Conventional PCR is an end-point analysis where the amplified DNA is detected as bands on an agarose gel, while real-time PCR detects DNA as fluorescence from each amplification cycle as it occurs (real time). This enables measurement of fluorescence in the exponential phase of amplification, where ideally there is a doubling of product for each cycle, and thus gives a more accurate quantification compared to the end point PCR.

NIOM’s laboratory possess a CFX96 Touch™ Real Time PCR Detection System from Bio-Rad which has the possibility to discriminate up to five different targets in one reaction well. The instrument has several applications, such as investigating gene expression in mammalian cells and bacteria, identification of single nucleotide polymorphism, and identification and quantifying microorganism from different samples.

Western blotting is a common method to identify and quantify level-changes of proteins in tissues and cells exposed to possible toxic compounds.

Proteins in a sample are separated based on their size by gel electrophoresis. Subsequently, the proteins are transferred to a solid membrane. The membrane is incubated with different antibodies that specifically bind proteins of interest. Fluorochromes attached to the antibodies make it possible to quantify a bound antibody based on fluorescence intensity.

The amount of bound antibody depends on the amount of the specific protein in each sample, and comparing fluorescence intensity from the samples yields relative protein levels. At NIOM, we use the Odyssey CLx Infrared Imaging System to develop a digital image and quantify fluorescence from each sample. This technique delivers fast and reproducible results, and the possibility to analyze these right away, without the troubles of film and darkroom.

Synergy™ H1 is a multi-mode microplate reader with monochromator-base optics for top and bottom read of fluorescence and UV-visible absorbance. For data collection and analysis the instrument uses Gen5 software.

Example of use of Synergy™ H1 at NIOM:

• Metabolic cell proliferation like MTT and XTT
• Enzyme-linked immunosorbent assay, ELISA
• Glutathione
  Reactive oxygen species, ROS
• Resazurin cell viability in biofilm
• Bacteria growth rate

Microplate types: 6- to 384-well plates
Read methods: Endpoint, kinetic, spectral scanning, well area scanning
Wavelength range absorbance: 230 – 999 nm
Wavelength range fluorescence: 250 – 700 nm

Casting machines

NIOM has two casting machines for casting different dental alloys. A vacuum press machine, Heraeus Combilabor CL-G2002, for the casting of precious and low-precious alloys, and a vacuum pressure induction casting machine, Heracast iQ, for casting of base metals. The casting machines are primarily used to cast samples for testing according to ISO 22674 and ISO 9693. These are samples used for measuring mechanical properties, corrosion, and tarnish testing and tests for metal and porcelain bonding. The casting machines are also used to make samples for different research projects where alloys are involved. It might be elucidation of corrosion properties, both in static and dynamic situations, or different tests of mechanical properties, including elastic-modules, and debonding of metal-ceramic restorations.

Furnaces

NIOM also has several furnaces for different purposes such as firing of casting molds and heat treatment of alloys, Jelenko accu-therm II 2000 and Mihm-Vogt M3. For ceramic veneering of metal or ceramic core materials, Jelenko Commodore 100 and Ivolclar EP 5000/G2 are used. The latter are also for pressureable glass ceramics for cores or for veneering of ceramic cores.

Cutting, grinding, polishing and other equipment

NIOM possesses different equipment for cutting, grinding and polishing: An automatic precision machine for serial cutting, Metkon Micracut 201, and a cutting machine for manual single cutting, with accessories; SiC-paper and diamond discs, polishing cloths for diamond and alumina for all kind of materials; sand-blasting.

Workshop

Our laboratory include an instrument maker’s workshop where homemade equipment, jigs and fixtures and accessories are prepared, both for mechanical and electronic devices. We may prepare custom design samples and equipment for research and testing purposes.

The ability to prepare experimental materials is mandatory for a research and test institute of dental materials. Using experimental materials assures that all the components of the material will be carefully controlled and experiments can be set up with variations only in the desired substance(s).

This allows for the study of the effects of the different components on the material properties in a controlled manner. However, mixing experimental composite materials by hand is a time-consuming and difficult process. In addition, hand-mixing may often lead to non-homogenous distribution of the filler particles into the resin matrix. In the next step, when studying either physical or chemical properties of the material, the results may be influenced by this inhomogeneity, giving unstable or incorrect values. On the other hand, using large-scale mixers consumes too much material in the initial testing phase of a new product.

NIOM has acquired a mixing machine for preparing experimental materials for testing and research purposes. The mixing machine is a SpeedMixer™ from Synergy Devices Ltd, model DAC 150.1 FVZ-K. This machine allows us to mix composites, and other materials, in small amounts for research and testing purposes. It mixes well amounts down to 10 grams, which may be useful for initial try-outs of new experimental materials. For more comprehensive studies, material mixes of up to 100 grams may be prepared, and series of different experiments may thus be performed on the same batch of material. Tests have shown that the machine gives excellent mixes of composites with both low and high filler content.

NIOM offers testing of experimental materials, prepared under controlled and repeatable mixing conditions. In combination with our laboratory and test facilities for physical, chemical and biological analyses of biomaterials and our in-house knowledge and experience, we are now capable of performing a complete evaluation of experimental materials.

The delightful combination of a coffee and an ice cream sets the parameters for one of the most used test protocols for dental materials. The international standard for testing dental adhesives stipulates an ageing procedure in which test specimens are held repeatedly first in 5 °C cold water and then in 55 °C hot water for a large number of cycles. The result of subsequent testing invariably shows degradation in adhesive strength. It is important that this loss of bonding is limited.

Thermocycling requires moderately sophisticated equipment to ensure constant temperatures in the water baths and properly timed transfer of the specimens. NIOM designed its first thermocycling apparatus 20 years ago, and has since supplied numerous laboratories with the equipment, each incorporating developments in temperature and mechanical control. While international standards specify immersion times and temperatures for established tests, new materials and test methods create new situations for which thermocycling is a useful method of ageing the materials. In each case, however, the physical process requires analysis. Thermocycling is based on the diffusion of heat, and in porous materials also of moisture, in and out of the test specimen. A diffusion process always acts to even out differences.

The questions that need to be answered are:

• How quickly do gradients in temperature or moisture content fade?
• How large are the transient mechanical and chemical stresses that are imposed?

Heat transfer is a classic field of engineering that gives us the necessary equations. Once the thermal parameters and diffusion properties of a material are available, the temperature throughout the test specimen may be calculated for times after immersion in each bath. NIOM has written software to calculate temperature profiles and the resulting mechanical stresses for both round and square specimens. The flow rate of water past the specimen determines how quickly the specimen achieves the temperature of its bath, and thereby the period required for each thermocycle.

NIOM has a new scanning electron microscope (SEM) TM4000Plus from Hitachi for surface imaging and element analysis within materials science.

SEM is a staple within the materials research community and is a crucial tool for surface analysis within a broad array of disciplines such as chemistry, physics, biology, and engineering. Contrary to a light microscope, which typically uses light in the visible range to investigate the surface of the sample, SEM uses electrons. This has several advantages.

The electrons make it possible to achieve a much higher level of magnification. Our instrument can acquire images in the magnification range from ~20 to 100 000x. The backscatter electrons (BSE) also give us a contrast between different elements, for example, making it possible to identify different composition domains on the surface. We can also collect the secondary electrons (SE), which, because of their elastic interaction with the surface, allows for topographic contrast.

By taking advantage of the characteristic X-rays emitted, because of electrons in-elastically interacting with the surface, we can do quantitative energy-dispersive X-ray spectroscopy (EDX). With EDX, we can get quantitative element composition, achieving an accuracy of approximately 0.1 % (sample dependent) of the sample.

Furthermore, the SEM has a 4-segment BSE detector, which allows for 3D visualization of the surface. By acquiring a segmented image, a 4-quadrant reconstruction is performed (Hitachi 3D Map software), resulting in a 3D model of the surface. This adds another dimension to our SEM imaging, making it possible to acquire metrological data of the surface, including contour analysis. This is beneficial in our work with dental materials when analyzing surface treatments and abrasion effects.

A variety of samples can be analyzed, with the instrument being able to accommodate samples up to 5 cm in thickness and 8 cm in diameter. The current SEM can measure at three different voltages, 5, 10, and 15 eV, with four different beam currents at each voltage.

We can also alter the vacuum of the measurement chamber to reduce charging effects for low conducting samples. Beyond this, we can manipulate samples for optimal imaging, including surface coating (e.g., gold and carbon) for improved surface conductivity, and dehydrate and fixate biological samples.

NIIOM has a digital dental x-ray system using phosphor plates and a digital scanner. The system is used for measuring radio-opacity of dental materials according to ISO standards, such as ISO 6876 (Endodontic materials) and ISO 4049 (Polymer-based restorative materials). By making a specimen of the specific material and place it beside an aluminium step wedge, the x-ray picture is used to calculate the radio-opacity of the material.

The radio-opacity is found by recording the grey values of the material compared to the grey values from the aluminium step wedge. The radio-opacity of the material is expressed in equivalent aluminum thickness.

NIOM possesses equipment for mechanical testing of dental materials. Whether it concerns polymer-based materials, metals, alloys, cements or ceramics we are able to offer testing of the mechanical elastic, plastic and fracture properties of dental materials.

Two testing machines are widely used for research projects and testing of dental materials according to different ISO standards. The materials are exposed to both destructive and non-destructive tests depending on the investigated properties.

The mechanical tests are performed by two different universal testing machines; Zwick 10 kN machine with macro extensometer and Zwickline 5 kN table top machine. Both machines are classified as class 0.5. This means that the accuracy of the testing machines is better than ± 0.5 % and ± 1 %, respectively.

The testing for clients is performed in accordance with a range of test methods for mechanical properties, for the most part from ISO-standards within the field of dentistry/dental materials.

NIOM is accredited for several of these tests, such as biaxial or 3-point flexure tests, debonding strength of metals or high strength ceramics and veneering ceramics, bonding of artificial teeth to denture base materials, adhesion of bonding materials to dentin or enamel, in addition to traditional tensile and compression measurements.