Jörg Brandt

Nondestructive three-dimensional evaluation of biocompatible materials by microtomography using synchrotron radiation

Microtomography based on synchrotron radiation sources is a unique technique for the 3D characterization of different materials with a spatial resolution down to about 1 micrometers . The interface between implant materials (metals, ceramics and polymers) and biological matter is nondestructively accessible, i.e. without preparation artifacts. Since the materials exhibit different x-ray absorption, it can become impossible to visualize implant material and tissue, simultaneously. Here, we show that coating of polymer implants, which are invisible in bone tissue, does not only improve the interfacial properties but also allows the imaging of the interface in detail. Titanium implants, on the other hand, absorb the x-rays stronger than bone tissue. The difference, however, is small enough to quantify the bone formation near interface. Another advantage of microtomography with respect to classical histology is the capability to examine samples in a hydrated state. We demonstrate that ceramic hollow spheres can be imaged before sintering and fibroblasts marked by OsO4 are visible on polymer textiles. Consequently, scaffolds of different materials designed for tissue engineering and implant coatings can be optimized on the basis of the tomograms.

Multilayer analysis: quantitative scanning acoustic microscopy for tissue characterization at a microscopic scale

An in vitro acoustic microscopy method for the quantitative characterization of biological hard tissues at a microscopic scale is described. At a frequency of 900 MHz, the acoustic impedance is measured as a tissue parameter, which is closely related to its elastomechanical properties. Contrast influences caused by defocus, edges, and surface inclinations, respectively, are either compensated or excluded from the measurement by a special data acquisition and analysis concept. A raster grid was used to validate the capabilities and limitations of the method, and results obtained from human cortical bone are shown. The comparison of different evaluation methods demonstrate the significance of a sophisticated analysis under consideration of topographical and system parameters. Cortical bone impedance maps showed a strong dependence on the anatomical structures, and the mean values were found to be in the range from 3.5 to 6.5 Mrayl within one single osteon.

Trefoil factor 3 is induced during degenerative and inflammatory joint disease, activates matrix metalloproteinases, and enhances apoptosis of articular cartilage chondrocytes

OBJECTIVE Trefoil factor 3 (TFF3, also known as intestinal trefoil factor) is a member of a family of protease-resistant peptides containing a highly conserved motif with 6 cysteine residues. Recent studies have shown that TFF3 is expressed in injured cornea, where it plays a role in corneal wound healing, but not in healthy cornea. Since cartilage and cornea have similar matrix properties, we undertook the present study to investigate whether TFF3 could induce anabolic functions in diseased articular cartilage. METHODS We used reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry to measure the expression of TFF3 in healthy articular cartilage, osteoarthritis (OA)-affected articular cartilage, and septic arthritis-affected articular cartilage and to assess the effects of cytokines, bacterial products, and bacterial supernatants on TFF3 production. The effects of TFF3 on matrix metalloproteinase (MMP) production were measured by enzyme-linked immunosorbent assay, and effects on chondrocyte apoptosis were studied by caspase assay and annexin V assay. RESULTS Trefoil factors were not expressed in healthy human articular cartilage, but expression of TFF3 was highly up-regulated in the cartilage of patients with OA. These findings were confirmed in animal models of OA and septic arthritis, as well as in tumor necrosis factor alpha- and interleukin-1beta-treated primary human articular chondrocytes, revealing induction of Tff3/TFF3 under inflammatory conditions. Application of the recombinant TFF3 protein to cultured chondrocytes resulted in increased production of cartilage-degrading MMPs and increased chondrocyte apoptosis. CONCLUSION In this study using articular cartilage as a model, we demonstrated that TFF3 supports catabolic functions in diseased articular cartilage. These findings widen our knowledge of the functional spectrum of TFF peptides and demonstrate that TFF3 is a multifunctional trefoil factor with the ability to link inflammation with tissue remodeling processes in articular cartilage. Moreover, our data suggest that TFF3 is a factor in the pathogenesis of OA and septic arthritis.

C-28/I2 and T/C-28a2 chondrocytes as well as human primary articular chondrocytes express sex hormone and insulin receptors--Useful cells in study of cartilage metabolism.

Sex hormones and insulin have been implicated in articular cartilage metabolism. To supplement previous findings on the regulation of matrix synthesis with 17β-estradiol and insulin and to find a possible model to study cartilage metabolism in vitro, we evaluated the expression of estrogen receptors α and β (ERα, ERβ), androgen receptor (AR) and insulin receptor (IR), in immortalized C-28/I2 and T/C-28a2 chondrocytes and in human primary articular cartilage cells. Chondrocytes were treated with increasing concentrations of 17β-estradiol, dihydrotestosterone or insulin and analyzed by means of RT-PCR and Western blotting. Both cell lines as well as human articular chondrocytes expressed ER α and β, AR and IR at mRNA and protein levels. In immortalized C-28/I2 chondrocytes, we showed that increasing concentrations of 17β-estradiol diminished the 95kDa band of IR. Since 17β-estradiol suppresses insulin-induced proline incorporation and type II collagen synthesis, as we have previously demonstrated, our findings give the first clue that 17β-estradiol may have negative effects on cartilage anabolism triggered by insulin during hormonal imbalance. Compared to chondrocytes cultured without hormones, immunostaining for ERα/β, AR and IR was decreased in both cell lines after incubation of cells with the receptor-specific hormones. It can be assumed that C-28/I2 and T/C-28a2 chondrocytes interact with the respective hormones. Our findings provide a reproducible model for investigating sex hormone and insulin receptors, which are present in low concentrations in articular chondrocytes, in the tissue-specific context of cartilage metabolism.

Variations in the microstructural acousto-mechanical properties of cortical bone revealed by a quantitative acoustic microscopy study

A previously described Multi Layer Analysis (MLA) technique was used to examine the anisotropy, age and gender dependence of lamellar osteon ensembles of human cortical bone, obtained from 17 male and 9 female human cadaver femora. The samples were embedded in polymethylmethacrylate (PMMA) and cut in several directions with respect to the long axis of the femur. For each cut six measurements were made under standardized conditions using a scanning acoustic microscope at 900 MHz. The mean impedance of 998 MLAs was 3.38 Mrayl (std. 0.37 Mrayl), which is significantly lower than published low frequency values. The angular dependence of impedance exhibited a similar behavior as derived for the Youngs modulus from an alternating lamellae model. Statistical analysis of age- and gender specific subgroups showed a general increase of impedance with age and a reversal for the oldest male age group only.

Quantitative Ultraschallrastermikroskopie zur Bestimmung der akustischen Impedanz von kortikalem Knochengewebe

Zusammenfassung Ein In-vitro-Mesverfahren der akustischen Rastermikroskopie wird beschrieben, mit dem elastomechanische Eigenschaften von humanem Knochengewebe in der Grosenordnung zellularer und uberzellularer Organisationsstrukturen erfast werden konnen. Das vorgestellte Verjahren erlaubt die quantitative Messung der akustischen Impedanz als materialbeschreibenden Parameter. Kontrasteinflusse, die durch Defokussierung, Kanten und Oberflachenneigung entstehen, werden durch eine spezielle Datenakquisitions- und Signalverarbeitung separiert oder eliminiert und die mikroskopische akustische Impedanz der verbleibenden Datenpunkte durch eine dynamische Kalibrierungsmethode bestimmt. In Polymethylmethacrylat (PMMA) eingebetteter kortikaler Knochen aus Praparaten der menschlichen Femurkortikalis wurde mit dieser Methode bei einer Frequenz von 900 MHz untersucht. Die gemessenen Impedanzwerte zeigen eine starke strukturelle Abhangigkeit und lagen in einem Bereich von 2 bis 6 Mrayl. Dies ist deutlich niedriger, als die mit niederfrequenten Methoden ermittelten makroskopischen Werte des Knochenverbundes.

Inhibition of Mineralization by a Calcium Zirconium Phosphate Coating

Bioactive ceramics used as coating materials combine the conductive properties of a bioceramic with the mechanical stability of the metal implant. We studied a calcium zirconium phosphate-containing coating material, FA-CZP [Ca(5)(PO(4))(3)F, CaZr(4)(PO(4))(6)], that is relatively insoluble in the biological milieu. The reaction of bone to this material was investigated histologically and histomorphometrically in an animal trial. Cylindrical Ti6Al4V specimens that had been coated with FA-CZP by plasma spraying were implanted in the femoral condyles of rabbits. The implants were left in place for 2, 4, 6, 12, and 14 weeks. FA-CZP led to impaired mineralization of the newly formed bone at the interface. Noncalcified osteoid was found throughout the whole study period. The layer seemed to become thicker with time. The mineralization disorder is evidently caused by zirconium ions. The presence of zirconium in the osteoid in contact with the implant was demonstrated by means of two different staining methods.

High frequency acoustic dispersion of surface waves using time-resolved broadband microscopy

Broadband time-resolved microscopy was used in order to extend spatial frequency domain analysis of the V(z)-technique from a single frequency to the bandwidth of the transducer. With a special frequency dependent SAW speed imaging mode the occurrence and characteristics of the different SAW waves can be visualized. Phase velocities were estimated using multi-narrow-band spatial frequency analysis. A number of different plastics, metals, quartz glasses, piezo ceramics, tooth enamel, dentin and cortical bone was investigated. The observed velocities were in the range from 2300 to more than 6000 m/s. Recently reported multiple SAW reflections were only observed in low attenuating materials, e.g. titanium and aluminum. This methodology allows to study the frequency dependence of acoustic properties with a high spatial resolution and minor sample preparation, which is of particular interest for the investigation of biological materials.

Quantitative scanning acoustic microscopy investigation of cortical bone using a multilayer analysis method

An in vitro scanning acoustic microscopy measurement technique is described to assess the acoustical properties of human bone tissue at the level of individual lamellae. Contrast influences caused by defocus, surface edges and inclinations, respectively are separated or eliminated. The microscopic acoustic impedances of the remaining data points are determined by a dynamic calibration method. Cortical bone samples of the human femur embedded in polymethyl-methacrylate (PMMA) were investigated at a frequency of 900 MHz. The resulting impedance maps showed a strong structural dependence and the mean values were found to be in the range from 2 to 6 Mrayl, which is significantly lower than macroscopic bulk values of the bone compound measured by acoustic low frequency methods.

Experimental Arthritis in the Rat Induced by the Superantigen Staphylococcal Enterotoxin A.

The pathogenesis of rheumatoid arthritis (RA) is incompletely understood. Human endogenous retroviruses (HERVs) and their superantigenic envelope protein (env) have been implicated in the pathogenesis of RA. In the present investigation, the arthritogenic potential of the superantigen staphylococcal enterotoxin A (SEA) has been investigated. In the present investigation, the bacterial superantigen staphylococcal enterotoxin A (SEA) was injected into the right knee joint of 15 Lewis rats. Further nine animals received saline. Animals were sacrificed one, five and 10 days after the injection, respectively. The antigens CD3, CD4, CD8, MHC class I, MHC class II, Pax5 and CD138 were investigated by immunohistochemistry on cryo‐sections. After intra‐articular SEA injection, the inflammation was initially dominated by CD8+ T cells. In the course of the investigation, the numbers of CD4+, Pax5+, CD138+ and MHC class II+ cells increased. CD3 was expressed in low numbers as compared to CD8. After saline injection, no similar inflammatory response has been detected. The arthritis induced by the superantigen SEA may be a novel model for inflammatory joint diseases, that is rheumatoid arthritis or juvenile idiopathic arthritis.