Raman Spectroscopy Detection of Molecular Changes Associated with Osteoarthritis.

Abstract

Vibrational spectroscopic methods are minimally invasive, and are appropriate for use in clinical contexts. Methods were developed in this dissertation for evaluating joint damage and disease using Raman spectroscopy. Subtle changes in the molecular structure of joint tissue and synovial fluid precede morphological changes in the joint. The goal of this research is to develop Raman spectroscopic methods for the examination of joint tissue and biological fluids, for monitoring and detecting molecular alterations associated with osteoarthritis. We identified Raman spectroscopic markers of altered molecular structure in subchondral bone and relevant biological fluids. Using Raman spectroscopy the molecular structure of joint tissues was measured, and the results were compared to the results from micro computed tomographic and histopathologic analysis. Raman spectra of subchondral bone collected from Del1 (+/-) transgenic mice, a mouse model for early-onset osteoarthritis, indicated lower bone mineralization in transgenic mice (5.73 ± 0.28 vs. 6.87 ± 0.225 in wild-type mice, p=0.003). A fiber-optic Raman probe for arthroscopic measurements was developed to demonstrate the feasibility of measuring the molecular structure of joint tissue with clinically-relevant instrumentation. The carbonate-to-phosphate ratio, a Raman spectroscopic measurement of bone mineral composition, was measured from subchondral bone under an intact layer of cartilage. Our initial work on a human proximal radius specimen indicated that an arthroscope configuration is capable of providing similar carbonate-to-phosphate values as spectra collected on a Raman microscope (0.25 vs. 0.24). In addition to cartilage and subchondral bone, the chemical structure of synovial fluid molecules is a key factor in maintaining healthy joint function. Synovial fluid from normal and diseased joints was examined using a novel drop deposition/Raman spectroscopic method. Raman spectra of synovial fluid from patients with radiographic evidence of osteoarthritis showed evidence of altered protein structure, as shown by increased Raman band intensity ratios at 1080 cm-1/1002 cm-1 (0.054 ± 0.07 vs. 0.038 ± 0.003, p<0.01) and 1670 cm-1/1655 cm-1 (0.68 ± 0.06 vs. 0.51 ± 0.05, p<0.01). These studies show that Raman spectroscopic measurements of joint tissue and synovial fluid correlate with established techniques for osteoarthritis detection and Raman spectroscopy may potentially provide early detection of joint damage.