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 17ß-estradiol and insulin and to find a possible model to study cartilage metabolism in vitro, we evaluated the expression of estrogen receptors α and ß (ERα, ERß), 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 17ß-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 ß, AR and IR at mRNA and protein levels. In immortalized C-28/I2 chondrocytes, we showed that increasing concentrations of 17ß-estradiol diminished the 95kDa band of IR. Since 17ß-estradiol suppresses insulin-induced proline incorporation and type II collagen synthesis, as we have previously demonstrated, our findings give the first clue that 17ß-estradiol may have negative effects on cartilage anabolism triggered by insulin during hormonal imbalance. Compared to chondrocytes cultured without hormones, immunostaining for ERα/ß, 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.

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.

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.

Histological and histomorphometric investigations on bone integration of rapidly resorbable calcium phosphate ceramics.

Resorbable ceramics can promote the bony integration of implants. Their rate of degradation should ideally be synchronized with bone regeneration. We report here the results of a histological study of implants with two resorbable calcium phosphate ceramic coatings: Ca(2)KNa(PO(4))(2)-(GB14) and Ca(10)[K/Na](PO(4))(7)-(602020). The results attained with these ceramic-coated implants show the benefits of these materials with regard to bioactive bone-healing stimulation, compared with uncoated implants. The GB14 ceramic coating exhibited greater bone regeneration and differentiation on its surface than the conventional hydroxyapatite coating and helped bone tissue achieve more extensive contact free of connective tissue. Not until the coating disintegrated did the histological features of GB14- and 602020-coated implants converge-both implant types were integrated into bone. Rapid disintegration of the coating material, as with 602020, supports osteoblast proliferation but has negative effects on bone mineralization. Both resorbable ceramics tested, GB14 and 602020, demonstrated bioactivity; even metal surfaces coated with these materials were populated by mature bone tissue without connective tissue after disintegration of their ceramic coating. The less rapidly degrading material, GB14, achieved better results. Degradable calcium phosphate coatings have the potential to stimulate bone regeneration. From the histological viewpoint, the resorbable ceramics examined here can be recommended as coating materials for clinical use.

[Light microscopic examination of rabbit skulls following conventional and Piezosurgery osteotomy]. / Auflichtmikroskopische Untersuchungen an der Kaninchenkalotte nach ultraschallgestützter und konventioneller Osteotomie.

INTRODUCTION: The novel ultrasonic osteotomy technique (Piezosurgery) is an alternative to conventional osteotomy devices. The aim of the present study was to carry out morphological comparison of the bone surface using conventional osteotomy techniques in comparison to the rather new ultrasonic osteotomy technique by means of a reflected-light microscopic examination. MATERIALS AND METHODS: Following the sacrifice of 12 rabbits, 24 standardized bone samples were removed from the skull. The osteotomy devices used were a rotating instrument (Lindemann bur), an oscillating micro-saw, and an ultrasonic osteotomy device (Piezosurgery) with insert tips OT6 and OT7. The times needed for osteotomy were measured. The bone surfaces were examined using a reflected-light microscope with a magnification of 40x and 100x. RESULTS: Osteotomy with Piezosurgery is significantly more time consuming than osteotomy with conventional methods (p<0.05). Following osteotomy with the ultrasonic device, the reflected-light microscopic examinations of the unmodified bone samples revealed typical bone structure of the calvaria, including compacta externa, diploe and compacta interna. On the contrary, following osteotomy with the conventional devices, the diploe structure presented distinct modifications. The cancellous spaces were filled with bone debris, and the cancellous structure was demolished. The samples prepared by the micro-saw technique showed a superficially condensed and grooved surface. CONCLUSION: In the present study, well-defined differences were observed following osteotomy with conventional devices and osteotomy with the ultrasonic device. The integrity of the bony structure observed after the ultrasonic technique could benefit the bone healing process. Further studies dealing with the bone healing process after using different osteotomy techniques are recommended.

Frequency and resolution dependence of the anisotropic impedance estimation in cortical bone using time-resolved scanning acoustic microscopy.

The influences of frequency and spatial resolution on the anisotropic impedance estimation of cortical bone was investigated in the frequency range 25-100 MHz. A set of spherically focused transducers provided a spatial resolution in the range from 150 down to about 20 mum. Four embedded cortical bone samples (two male, two female, two donors aged <30 years, two donors aged >70 years) were cut with different orientations relative to the long axis of the femur (0-90 degrees ). From each section, impedance maps were acquired in the C-scan mode. Histogram evaluations showed a similar angular dependence with a characteristic off-axis maximum of the estimated impedance for all samples and frequencies. The impedance values obtained with the 25-MHz transducer were significantly lower than those obtained with the 50- and 100-MHz transducers. Morphological parameters of the macrostructure, for example, size and distribution of the haversian channels and the resulting porosity, were estimated from the high-resolution acoustic images. These structures appeared to have a significant influence on the measured properties of the bone matrix for the low-frequency and low-aperture measurements.

Investigation of elasto-mechanical properties of alginate microcapsules by scanning acoustic microscopy.

High-frequency scanning acoustic microscopy (SAM) was used for investigation of acoustic impedance and 3D-surface topography of full alginate microspheres that act as model of artificial biological cells. Elasto-mechanical properties of the investigated specimens have been characterized by acoustic impedance. Mean surface impedance of microspheres (diameter: 300 microm) was measured with SAM at 900 MHz with a spatial resolution of 1.5 microm. The sensitivity and reproducibility of SAM had to be increased considerable to receive and quantify signals in the very low impedance region. The multilayer analysis method was used to get quantitative data of acoustic impedance with SAM at a microscopic level. 3D images show details of structure and surface topography. As a reference, bulk measurements were performed on full alginate cylinders. The acoustical impedance and the mechanical stiffness c(11) were obtained from mass density and longitudinal ultrasound velocity at 6 MHz. The impedances received with both methods are in close agreement. The results demonstrate the SAM as a powerful tool for characterizing mechano-elastical parameters as well as surface structure and topography of microspheres with high spatial resolution.