Identifying chemical changes in subchondral bone taken from murine knee joints using Raman spectroscopy.

Application of Raman spectroscopy to analysis of subchondral bone is described. The effect of cartilage health on subchondral bone has been widely studied using radiological and histological methods; however, there is no method to directly assay mineral components. We present Raman spectra of femur condyles and observe mineral bands that arise from the subchondral bone. In two separate experiments, transgenic mouse models of early-onset osteoarthritis (OA) and lipoatrophy were compared to tissue from wild-type mice. Raman spectroscopy was used to identify chemical changes in the mineral of subchondral bone that may accompany or precede morphological changes that can be observed by histology. The transgenic mice were compared to age-matched wild-type mice. Subtle alterations in the mineral or collagen matrix were observed by Raman spectroscopy using established Raman markers such as the carbonate-to-phosphate ratio, mineral-to-matrix ratio (MTMR), and amide I ratio. The Raman microscope configuration enabled rapid collection of Raman spectra from the mineralized layer that lies under an intact layer of non-mineralized articular cartilage. The effect of the cartilage layer on collection of spectra is discussed. The technique proposed is capable of providing insight into the chemical changes that occur in subchondral bone on a molecular level.

Correlating changes in collagen secondary structure with aging and defective type II collagen by Raman spectroscopy.

A novel application of Raman spectroscopy for monitoring damage to ocular collagen in wild-type mice and Del1 (+/-) transgenic mice, a murine animal model of osteoarthritis, is described. In order to understand the progression of diseases of collagen, it is necessary to use methods that can recognize alterations in affected tissue due to chemical and/or genetic modifications. The heterozygous Del1 (+/-) transgenic mouse is established as a model for early-onset osteoarthritis caused by modifications to the type II collagen gene (COL2A1) that result in a truncated collagen fiber. We expect that abnormal type II collagen is expressed in articular cartilage and eye tissue of the Del1 (+/-) mouse. Eyes excised from a subset of specimens from another study using Del1 (+/-) mice were examined by Raman spectroscopy for evidence of defective collagen. Spectral contributions from the collagen protein were readily observed. The amide III envelope (1220-1280 cm-1) was used to characterize changes in collagen secondary structure. Raman spectra of the sclera component of eyes taken from transgenic and older wild-type mice show an increased collagen disorder, as expected. These preliminary results suggest that Raman is capable of recognizing and measuring abnormality in eye collagen and may have potential as a diagnostic tool for ocular collagen damage.

Automated robotic liquid handling/laser-based nephelometry system for high throughput measurement of kinetic aqueous solubility.

The ability to rapidly and consistently measure aqueous solubility in a preclinical environment is critical to the successful identification of promising discovery compounds. The advantage of an early solubility screen is timely attrition of compounds likely to fail due to poor absorption or low bioavailability before more costly screens are performed. However, due to the large number of compounds and limited sample amounts, thermodynamic solubility measurements are not feasible at this stage. A kinetic solubility measurement is an alternative to thermodynamic measurements at the discovery stage that provides a rank listing of solubility values with minimal sample requirements. A kinetic solubility measurement is attractive from an automation vantage because it features rapid data acquisition and is amenable to multi-well formats. We describe the use of a robotic liquid/plate handler coupled to nephelometry detection for the measurement of kinetic solubility. We highlight the liquid handling validation, serial dilution parameters, and a comparison to the previous method. Experiments to further enhance throughput, or increase confidence in the automation steps, are described and the effects of these experiments are presented. In our integrated nephelometry method, we observe rapid liquid handling with an error of less than 10%, after a series of validation studies, and a sample throughput up to 1800 compounds per week. We compare the nephelometry method with our semi-thermodynamic flow-injection analysis (FIA) method, and find a 75% bin agreement between the methods.