Fluorescence-based confocal laser endomicroscopy for imaging osteoarthritis

Abstract

Purpose: Osteoarthritis (OA) is the most common type of joint disease characterized by slow occurring cartilage degeneration, cartilage loss and pain. This process frequently occurs in the knee joint and becomes a chronic disease over time. It is a degenerative disorder that results from the biochemical breakdown of articular cartilage in the synovial joints. Accumulated mechanical stress and chemical factors which are produced from joint tissues are involved in the progression of OA. Conventional radiographs remain the current standard for the imaging diagnosis of OA. The diagnosis can be made when joint narrowing and morphological changes in subchondral bone structure and osteophyte formation are seen. One important characteristic of OA is that different abnormalities are found in the pressure (i.e, contact) and non-pressure areas of the affected joint. Radiographic findings may be incorrectly found to be ‘normal’ in the early stages of the disease because the cartilage is not directly visualized. In this study we performed experiments with a confocal microprobe in the OA knee joint to track the delivery of therapeutically targeted compounds encapsulated in nanoparticles. This procedure allows for high quality images of a very thin focusing field. Methods: 10 weeks old male C57/Bl6 mice were used to induce OA. OA was induced by two consecutive intra articular injections of 10 μl highly purified bacterial type VII collagenase (10 U) into the right knee. As analgesic, the mice received also a subcutaneous injection of Temgesic (0.01 mg/kg body weight). To track the delivery of therapeutic compounds, PLGA based nanoparticles (NP) loaded with drug and Near Infra-Red (NIR) 800 dye were prepared with a water-in-oil-in-water double emulsion and solvent evaporation method. After characterizing with imaging techniques, 100 μl NP was injected in the mouse tail vein (iv). The NP delivery and distribution were followed by for several weeks with the Pearl Impulse imaging system (Li-Cor, Nebraska, USA) and Cellvizio 785 nm prototype fluorescence-based Confocal Laser Endomicroscopy (CLE) system (Mauna Kea, Paris, France). The Pearl Impulse is specifically optimized for whole small animal imaging while the CLE 785 nm prototype system is made to image the tissue of interest and provides real-time in vivo video sequences at cellular resolution. This system allows us to monitor in real time the delivery of a drug to a target site compound of interest within its anatomical environment in the living animal by simply to bring the flexible microprobe in contact with the tissue of interest to record real-time images of the tissue at cellular resolution. The knee joint was exposed by a superficial skin incision and a S300B with 3.3 μm resolution confocal microprobe was directly positioned onto the surface to scan the NIR 800 dye. During the scans all the mice were anesthetized with a 2% isoflurane/N2O/O2 mixture. Results: CLE imaging in vivo, after induction of OA using collagenase, was performed according to the results of the whole animal fluorescent imaging and knee joint. No significant fluorescent signal was observed in the controls. The results from ex vivo fluorescent imaging of knee joint cryosections were consistent with in vivo CLE findings. Conclusions: Our study shows that targeted imaging using a fluorescently labeled NP is possible with CLE. These findings indicate that we can follow the NP loaded with drug and NIR dye in the OA knee joint at different stages of the disease. This will impact upon future clinical approaches for OA, whereby diagnosis can be determined by optical imaging.