Clinical application of multiphoton microscopy and Raman spectroscopy : Focus on Osteoarthritis
Bio-mechanical investigation of osteoarthritic human articular cartilage
The investigation of stress relaxation behavior of osteoarthritic articular cartilage revealed that there is a significant reduction in stiffness and change in stress relaxation behavior found in advance stage of osteoarthritc human cartilage while there is no statistical significant difference observed between moderate and early stage.
In a synovial joint the bone ends are covered with articular cartilage. The main functions of articular cartilage are to decrease the contact stress in the joint and facilitate the smooth movement of joint during the motion. Despite of superficial similarity, animal and human articular cartilage differ significantly in their material properties (Athanasiou KA et. al., J Orthop Res. 1991). Viscoelastic behavior and function of cartilage in synovial joint are highly dependent on its mechanical properties. The mechanical properties of the cartilage tissues are strongly related of osteoarthritic stage of the articular cartilage. Therefore, from the clinical point of view biomechanical investigation of human osteoarthritic cartilage is essential. The experimental investigation of stress relaxation behavior of osteoarthritic human cartilage revealed that there is statistical significant reduction in stiffness exist between ICRS grade I and III and ICRS grade II and III while there is no significant difference observed between ICRS grade I and II of osteoarthritic cartilage. It was also observed that stress relaxation proceeds faster in advance stage of osteoarthritic cartilage. Investigations have already shown that mechanical properties of articular cartilage depend on its biochemical compositions (Athanasiou KA et. al., J Orthop Res. 1991). Based on our current biomechanical characterizations (Rajesh Kumar et. al., manuscript ready for submission, 2017) and earlier Raman spectroscopic studies on osteoarthritic cartilage (Rajesh Kumar et. al., Anal and Bioanal Chem. 2015), we hypothesized that the decrease in proteoglycan content in extracellular matrix of cartilage is responsible for reduction in stiffness and relaxation time in advance stage of osteoarthritic cartilage. More detailed information about the mechanical properties of articular cartilage, including the changes that might be triggered by physical and chemical stimuli, may be of practical importance for a better understanding of both cartilage mechanics and cartilage disease progression. A detail biomechanical characterization of osteoarthritic cartilage is essential, as alteration in biomechanical properties is observed before any gross morphological change visible in the standard clinical imaging systems. Accurate characterization of osteoarthritic cartilage would not only enhance our understanding about the progression of osteoarthritis but, can also further aid in prognosis and therapy. Such study may also be useful for orthopaedic surgeons because, stiffness values of articular cartilage is often measured in situ by indentation testing using clinical millimeter-scale indentation device during arthroscopic surgery. In our forthcoming investigation, the correlation studies between biomechanical properties and the multipoton microscopic (~morphological) and Raman spectroscopic (~biochemical) studies can reveal the other hidden features of the progression of osteoarthrits, which can't be done by standard clinical imaging modalities due to its technical limitations. The active collaboration and guidance of St. Olavs Hospital-Norway and Massachusetts Institute of Technology-USA is highly appreciated and acknowledged.
Analysis of Osteoathritis using advance optical tools
Advance optical techniques can provide high resolution image and biochemical information of articular cartilage without the need of external tag. This post-doctoral project is exploring the novel features obtained from human osteoarthritic samples in order to understand the underlying mechanism behind progression of disease.
Osteoarthritis (OA) is a complex musculoskeletal disorder whose origin is not exactly clear and as time progresses, the views about this disorder are continuously evolving. Advance technique like two-photon microscopy can provide high resolution image (higher than existing clinical modalities e.g., CT, US, MRI and arthroscope) and, the technique of Raman spectroscopy can provide bio-chemical specific information of articular cartilage without any need of external label. In this post-doctoral research project, we are characterizing different grades of osteoarthritic human samples to find novel morphological, biochemical and mechanical features and any correlation between those features, if exist. Exploring the novel features associated with OA in a systematic way is an important step forward towards understanding the underlying mechanism of the disorder, which eventually may lead towards early diagnosis and better treatment of disease. This project has several sub-projects. (1) In pilot project, an investigation relevant to inherent biochemical analysis in different grades of osteoarthritic cartilage by Raman spectroscopy is completed and is published (PMID: 26319282). The obtained results are highly encouraging and we are proceeding towards further investigations. (2) Due to recent new regulations, in order to obtain control tissue samples, the modified version of previously approved ethical application (2013/265 REK, Norway) is re-submitted, and is in progress now. The scientific idea is to characterize normal cartilage and to compare with the results already obtained by different grades of osteoarthritic cartilage. E.g., as seen by two-photon excited fluorescence microscopy, we hypothesized that collagen VI might be an early biomarker of OA. However, the obtained result needs to verify by other valid techniques/methodologies. (3) The new experimental data associated with subchondral bone is acquired in collaboration with Nofima, Oslo. Now, advance data analysis on acquired data is in progress under collaboration with MIT, USA, and results are not clear yet. However, the goal is to figure out early biochemical changes in subchondral bone and cartilage and any correlation between those changes in different grades of osteoarthritic samples. (4) The stress relaxation data obtained by mechanical indentation from three different ICRS grades of osteoarthritic cartilage are being analyzed. We are investigating the change in visco-elastic behavior of osteoarthritic cartilage and any correlation between change in visco-elastic behavior and histological evaluation of osteoarthritic cartilage. In the long term, the analysis of osteoarthritic samples with Raman probe can be included that may help in feasibility analysis for the development of Raman arthroscopic tool. However, initially it is necessary to understand the novel features of OA which are being obtained using two-photon microscopy and Raman spectroscopy. The project has been underway for less than a year and we are moving ahead in collaboration with mainly Magnus B. Lilledahl (Professor, NTNU), Jon O. Drogset, (Orthopaedic Surgeon, St. Olav's Hospital) Catharina de Lange Davies (Professor NTNU), and Gajendra P. Singh (Scientist, MIT, USA). In October 2015, the project has received a citation under the title 'Høyoppløste bilder avslører tegn på slitasjegikt' (High resolution pictures reveal signs of osteoarthritis), which is published in http://forskning.no/ and www.gemini.no
Optical investigation of osteoarthritic human cartilage (ICRS grade) by confocal Raman spectroscopy: a pilot study.
Anal Bioanal Chem 2015 Oct;407(26):8067-77. Epub 2015 aug 29
Compressive stress relaxation investigation of ICRS grade osteoarthritic human articular cartilage.
Journal of the Mechanical Behavior of Biomedical Materials, February-2017 (manuscript ready for submission).