T. Clive Lee

Dual responsive chemosensors for anions: the combination of fluorescent PET (Photoinduced Electron Transfer) and colorimetric chemosensors in a single molecule

The design and synthesis of two novel fluorescent PET anion sensors is described, based on the principle of ‘fluorophore-spacer-(anion)receptor’. The sensors 1 and 2 employ simple diaromatic thioureas as anion receptors, and the fluorophore is a naphthalimide moiety that absorbs in the visible part of the spectrum and emits in the green. Upon recognition of anions such as F− and AcO− in DMSO, the fluorescence emission of 1 and 2 was ‘switched off’, with no significant changes in the UV–vis spectra. This recognition shows a 1:1 binding between the receptor and the anions. In the case of F−, further additions of the anion, gave rise to large changes in the UV–vis spectra, where the λmax at 455 nm was shifted to 550 nm. These changes are thought to be due to the deprotonation of the 4-amino moiety of the naphthalimide fluorophore. This was in fact found to be the case, using simple naphthalimide derivatives such as 6. Sensors 1 and 2 can thus display dual sensing action; where at low concentrations, the fluorescence emission is quenched, and at higher concentrations the absorption spectra are modulated.

Microcrack accumulation at different intervals during fatigue testing of compact bone

Fatigue damage in bone occurs in the form of microcracks. This microdamage contributes to the formation of stress fractures and acts as a stimulus for bone remodelling. A technique has been developed, which allows microcrack growth to be monitored during the course of a fatigue test by the application of a series of fluorescent chelating agents. Specimens were taken from bovine tibiae and fatigue tested in cyclic compression at a stress range of 80MPa. The specimens were stained before testing with alizarin and up to three other chelating agents were applied during testing to label microcracks formed at different times. Microcracks initiated in interstitial bone in the early part of a specimens life. Further accumulation of microcracks is then suppressed until the period late in the specimens life. Microcracks were found to be longer in the longitudinal than in the transverse direction. Only a small proportion of cracks are actively propagating; these are longer than non-propagating cracks. These results support the concept of a microstructural barrier effect existing in bone, whereby cracks initiate easily but slow down or stop at barriers such as cement lines.

An improved labelling technique for monitoring microcrack growth in compact bone.

Fatigue-induced damage plays an important role in bone remodelling and in the formation of stress and fragility fractures. Recently, a technique has been developed (Lee, T.C. et al., Sequential labelling of microdamage in bone using chelating agents. Journal of Orthopedic Research, 18 (2000) 322-325) which allows microcrack growth in trabecular bone to be monitored by the application of a series of chelating fluorochromes, however, some limitations were identified with the process. The aims of this study were to refine the method of detection using these agents in order to determine the optimal sequence of application and the optimal concentrations which allowed all the agents to fluoresce equally brightly using UV epifluorescence. A chemical analysis process, ion chromatography, followed by validation tests on bone samples showed that the optimal sequence of application and concentration of each agent was alizarin complexone (0.0005 M) followed by xylenol orange (0.0005 M), calcein (0.0005 M) and calcein blue (0.0001 M). A fifth agent, oxytetracycline was excluded from the study after recurring problems were found with its ability to chelate exposed calcium when applied in sequence with the other agents. This work has developed a sequential labelling technique, which allows for microcrack propagation during fatigue testing of bone specimens to be monitored without the problem of chelating agent substitution occurring.

A highly selective and sensitive fluorescent PET (photoinduced electron transfer) chemosensor for Zn(II)

The naphthalimide derivative 1 was designed as a fluorescence PET sensor for Zn(II); 1 showed excellent selectivity for Zn(II) at pH 7.4, even in the presence of other competitive cations, the emission, being pH independent above pH 3.5, was switched on upon Zn(II) recognition.

Selective Imaging of Damaged Bone Structure (Microcracks) Using a Targeting Supramolecular Eu(III) Complex As a Lanthanide Luminescent Contrast Agent

The synthesis and photophysical evaluation of a new supramolecular lanthanide complex is described which was developed as a luminescent contrast agent for bone structure analysis. We show that the Eu(III) emission of this complex is not pH dependent within the physiological pH range and its steady state emission is not significantly modulated by a series of group I and II as well as D-metal ions and that this agent can be successfully employed to image mechanically formed cracks (scratches) in bone samples after 4 or 24 h, using confocal laser-scanning microscopy.

Measuring the shape and size of microcracks in bone

Microcracks in bone are normally measured from two-dimensional, transverse sections but this provides incomplete information about their three-dimensional shape and size. The methods of stereology can usefully be applied in such a case. This problem has been addressed using a theoretical model and a numerical simulation. Results show that the data are consistent with a crack shape which is elliptical, with axis ratio approximately 4.5:1 and with size variation (expressed as the ratio of standard deviation to mean length) of at least 0.1. Measured lengths will, on average, be smaller and have more scatter than true lengths. Errors caused by omitting small cracks are relatively unimportant. Knowledge of the true crack dimensions, and their variability, is important for the analysis of damage, stress fracture, remodelling and adaptation.

The nature of fatigue damage in bone

Abstract Bone is unusual among structural materials as it is alive and capable of self-repair. Fatigue-induced microdamage is repaired by bone remodelling, but if damage accumulates too quickly, or remodelling is deficient, fatigue failure may result. Fatigue is thought to contribute to both stress and fragility fractures which are of major clinical importance. Despite this, we do not fully understand the nature of fatigue damage in bone. Human rib sections, containing microcracks stained with basic fuchsin, were serially sectioned and microcracks identified and reconstructed in three dimensions using computer software. Microcracks were elliptical in shape, 400 μm long and 100 μm wide, typical of a transversely isotropic material. Chelating agents which bind Ca 2+ were found to label microcracks in rib, as well as mineralising bone surfaces and resorption sites, suggesting that microcracks are Ca 2+ ion-lined discontinuities in the hydroxyapatite matrix. Ca 2+ ions were exposed by scratching the surface of bovine bone specimens and labelled with chelating agents in sequence. The optimal four agent sequence was: alizarin, xylenol orange, calcein and calcein blue. Two dye sequences were used to differentiate between pre-existing and test-induced microdamage in bovine samples fatigue tested in compression and longer sequences labelled microcrack growth. Microcrack dimensions can be used to calculate stress intensity values and, together with fatigue test data, can aid theoretical models to predict fatigue failure in bone.

RANKL and OPG activity is regulated by injury size in networks of osteocyte-like cells.

Bone remodelling is an intricate process encompassing numerous paracrine and autocrine biochemical pathways and mechanical mechanisms. It is responsible for maintaining bone homeostasis, structural integrity and function. The RANKL-RANK-OPG cytokine system is one of the principal mediators in the maintenance of bone cell function and activation of bone remodelling by the Basic Multicellular Unit (BMU) which carries out remodelling. Theories surrounding the initiation of bone remodelling include mechanical loading, fluid flow and microdamage as potential stimuli. This study focused on microdamage. In an in vitro simulated bone environment, gel embedded MLO-Y4 cell networks were subjected to damage in the form of planar, crack-like defects of constant area and varying thickness. The biochemical response was determined by ELISA and luciferase assay. The results showed that RANKL release increased and OPG decreased in a manner which depended on injury size (i.e. thickness) and time following application of injury. The effect of microdamage on cell viability and apoptosis was also evaluated. This work demonstrates that injury alone, in the absence of imposed strain or fluid flow, is sufficient to initiate changes in cytokine concentrations of the type which are known to stimulate bone remodeling.

The role of osteocytes and bone microstructure in preventing osteoporotic fractures

The skeleton alters its geometry following trauma, the introduction of artificial defects and of fatigue-induced microcracks. The precise mechanism by which the skeleton adapts remains unclear. Microcracks might directly affect the cell by damaging the osteocyte cell network or causing apoptosis. Bone microstructure may play an important role in these processes by diverting and arresting propagating microcracks and so prevent fracture failure. This paper discusses the effects of microstructure on propagating cracks, how microdamage may act as a stimulus for bone adaptation and its potential effects on bone biochemistry.

Compression data on bovine bone confirms that a “stressed volume” principle explains the variability of fatigue strength results

The literature contains many measurements of the fatigue properties of compact bone, but these experimental results have been difficult to interpret and use due to a large amount of apparent scatter: variation in the number of cycles to failure for a given cyclic stress or strain range. Recently Taylor (1998a, Journal of Orthopaedic Research, 16, 163-169) showed that much of this scatter could be explained using a statistical model which took into account specimen size, or more specifically stressed volume. The present paper describes an attempt to test this model by using it to predict some new data, for bovine bone tested in compressive loading at room temperature at physiological loading rates. Twenty specimens were tested at the same applied load range (100 MPa). The theory was able to predict the mean behaviour of the specimens very well, with an accuracy (expressed in terms of stress) of 2%. It was also able to predict the degree of scatter (i.e. the variation of Nf), which was shown to be similar to that measured by other workers.