Vital confocal microscopy in bone

Abstract

Summary:We wished to exploit confocal microscopy for high spatial and temporal resolution vital microscopy in bone. To this end, we evolved implants with glass windows supported in titanium, which were placed in the medial proximal tibial plateau of the rabbit, and special small, self-focussing objectives (dry 10/0.25, water immersion 20/0.45, and oil immersion 45/0.65 and 120/1.0) which mated and matched to the conical window entrance section of the metal components. At intervals of up to 21 months after implant healing, these lenses were used to study live tissue using two genera of confocal microscope: multiple aperture disc, tandem scanning, microscopes for observation in reflection, and video rate confocal laser scanning microscopes for recording, mainly in the fluorescence mode. The latter allowed the study of a variety of intravenously administered substances, including fluorescein, fluorescein-dextrans, fluorescent microspheres, acridine orange, DASPMI, calcein, and tetracycline. We were able to remove blood, stain cells with fluorescent markers, and replace them into the circulation. Calcein and tetracycline bind to the mineral front in bone: this labelling was studied in progress. We observed that both substances partition and remain for long periods (at least days) in adipocytes. Further characterisation of the system used both confocal fluorescence and scanning electron microscopy methods in the study of retrieved implants. These studies showed that the subimplant cortical bone remodelled to a less compact structure with a rich microvasculature extremely close to bone. The points of attachment of bone to glass were found to involve coarse fibres, with the matrix containing large numbers of large cells: some of this tissue was cartilage and some immature bone. An amorphous, mineralised matrix was in immediate contact with glass. The results provide further confirmation of the general utility of high-scan speed confocal methodology in physiology.

Inelastic behaviour of collagen networks in cell–matrix interactions and mechanosensation

“…As the interactions between adherent cells and fibrillar collagen networks are strongly influenced by fibre geometry, physical properties [29] and network organization [30], we imaged collagen matrices prior to cell seeding and determined whether the presence of an underlying rigid foundation affects collagen fibre organization. Confocal reflected light microscopy was used to examine the effect of collagen concentration and the presence of an underlying rigid foundation on collagen gel organization [31,32]. Similar to floating gels, visualization of collagen gels without cells showed a similar distribution and orientation of collagen fibres across the gel width in both (1 mg ml 21 and 3 mg ml 21 ) attached collagen gels (figure 5a,c).…”

Transparent hydroxyapatite ceramics with nanograin structure prepared by high pressure spark plasma sintering at the minimized sintering temperature

“…The IR spectrum was used to estimate the loss of OH in connection with the sintering. The spectrums were scaled with the height of the absorption band of O-P-O bending modes of the PO 4 3− at 600 cm −1 . …”

“…In this regard, transparency and nanostructured feature constitute two important material parameters that will determine the possible application of confocal microscopy allowing direct observation of the bio-interfacial reactions with improved spatial and temporal resolution. 4,5 Optical transparency is readily achievable in HAp ceramics despite HAp possess a non-cubic crystal symmetry. There are already a number of reports that deals with preparation of transparent HAp ceramics.…”

Porous nanoapatite scaffolds synthesized using an approach of interfacial mineralization reaction and their bioactivity

“…32 It is commonly used in cell morphology examination. [32][33][34] The cells were rinsed twice with PBS and fixed in 4% paraformaldehyde solution for 2 h at room temperature. And then the fixed cells were incubated in 0.1 mL PBS containing 1% AO for 15 min at 37 C and rinsed with PBS to remove excess dye.…”