Nonlinear optical microscopy and Raman spectroscopy analysis of Osteoarthritis

Can Osteoarthritis be diagnosed at an early stage? Osteoarthritis (OA) is a highly prevalent, disabling, complex joint disorder, and a leading cause of individual and socioeconomic burden. The tissue that contributes the most extraordinary functional capacities and forms the bearing surface of all synovial (e.g., knee) joints is the articular cartilage. Degeneration of the articular cartilage is directly associated with the progression of OA. Due to the lack of resolution and sensitivity, currently used clinical imaging modalities (e.g., X-ray, MRI, Ultrasound) are not efficient in the assessment of early cartilage disorder. Nonlinear optical imaging and Raman spectroscopy are evolving, advanced techniques that may be used for biomedical applications. By using such techniques in the analysis of different grades of osteoarthritic cartilage in humans rather than an animal model is more relevant to demomstrate clinical potential. The goal of this thesis is to demonstrate the capability of nonlinear optical microscopy (NLOM) and Raman spectroscopy for morphological and biochemical characterization of human articular cartilage obtained from the femoral condyle of the knee. Novel morphological features (like microsplits and wrinkles) in early stage of osteoarthritic cartilage were observed by second harmonic generation (SHG) microscopy, structures that would otherwise not be visible in existing clinical imaging modalities. Within the group of ICRS Grade-I cartilage, possibly distinct phases of OA were observed. In order to perform polarization-SHG (p-SHG) microscopy, a portable polarization optical module was developed and integrated in a commercial microscope. The presence of fibrocartilage in early stage (ICRS Grade-I) of OA was observed by p-SHG microscopy. Furthermore, it was demonstrated that alteration of the collagen molecules pitch angle can be quantified by p-SHG microscopy. By using Raman spectroscopy a relative assessment of proteoglycan and amide (ordered vs. disordered protein coil) content, in different grade of osteoarthritic cartilage (ICRS Grade-I, II, III), were performed and their respective indication were discussed. Additionally, a label free investigation of single cells (chondrocytes) isolated from osteoarthritic cartilage was performed, which demonstrated that Raman micro-spectroscopy may reveal changes in biochemical compositions at the cellular level. In summary, our proof-of-principle investigation present NLOM and Raman spectroscopy as potential tools, which can assess the quality of the articular cartilage and therefore may be able to identify the stage of OA more precisely than other existing clinical modalities. An essential feature of NLOM and Raman method is that these techniques are minimally invasive and label free optical techniques. By the use of a miniaturized fiber based probe head, these techniques can be integrated with modern clinical arthroscopes and thus may potentially be used for in vivo analysis. Our proof-of-concept study encourages development of a NLOM and Raman arthroscope as a potential diagnostic tool for the use in orthopaedics