carbocyanines and Osteoarthritis

Articular cartilage is the hydrated tissue that lines the ends of long bones in load bearing joints and provides joints with a smooth, nearly frictionless gliding surface. However, the deterioration of articular cartilage occurs in the early stages of osteoarthritis (OA) and is clinically and radiographically silent. Here two cationic near infrared fluorescent (NIRF) dipicolylamine (DPA) probes, Cy5-DPA-Zn and Cy7-DPA-Zn, were prepared for cartilage degeneration imaging and OA early detection through binding to the anionic glycosaminoglycans (GAGs). The feasibility of NIRF dye labeled DPA-Zn probes for cartilage degeneration imaging was examined ex vivo and in vivo. The ex vivo studies showed that Cy5-DPA-Zn and Cy7-DPA-Zn not only showed the high uptake and electrostatic attractive binding to cartilage, but also sensitively reflected the change of GAGs contents. In vivo imaging study further indicated that Cy5-DPA-Zn demonstrated higher uptake and retention in young mice (high GAGs) than old mice (low GAGs) when administrated via local injection in mouse knee joints. More importantly, Cy5-DPA-Zn showed dramatic higher signals in sham joint (high GAGs) than OA side (low GAGs), through sensitive reflecting the change of GAGs in the surgical induced OA models. In summary, Cy5-DPA-Zn provides promising visual detection for early cartilage pathological degeneration in living subjects.

Topics: Amines; Animals; Carbocyanines; Cartilage, Articular; Female; Fluorescent Dyes; Glycosaminoglycans; Humans; Infrared Rays; Knee Joint; Mice; Mice, Nude; Optical Imaging; Osteoarthritis; Picolinic Acids

Among the classical collagenases, matrix metalloproteinase-13 (called MMP-13, collagenase-3) is one of the most important components for cartilage destruction of osteoarthritis (OA) developments. Despite many efforts, the detection methods of MMP-13 activity have been met with limited success in vivo, in part, due to the low sensitivity and low selectivity by homology of MMP family. Previously, we demonstrated the use of strongly dark-quenched fluorogenic probe allowed for the visual detection of MMP-13 in vitro and in OA-induced rat models. In this study, we described the optimization of MMP-13 fluorogenic probe for OA detection in vivo. Three candidate probes demonstrated recovered fluorescent intensity proportional with MMP-13 concentrations, respectively; however, Probe 2 exhibited both high signal amplification and selective recognition for MMP-13, not MMP-2 and MMP-9 in vitro. When Probe 2 was applied to OA-induced rat models, clear visualization of MMP-13 activity in OA-induced cartilage was obtained. Optimized MMP-13 fluorogenic probe can be applied to detect and image OA and have potential for evaluating the in vivo efficacy of MMP-13 inhibitors which are being tested for therapeutic treatment of OA.

Topics: Animals; Carbocyanines; Disease Models, Animal; Fluorescent Dyes; Humans; Kinetics; Matrix Metalloproteinase 13; Molecular Imaging; Molecular Probes; Osteoarthritis; Peptides; Rats; Rats, Sprague-Dawley; Sensitivity and Specificity; Spectroscopy, Near-Infrared

The specific objectives of this study using organ culture were (1) to transplant chondrocytes onto an intact cartilage surface; (2) to genetically modify endogenous and transplanted chondrocytes; and (3) to assess the ability of these cells to continually express a gene product. The specific objective with in vivo experiments was to transplant chondrocytes with intraarticular injections to cartilage.. Fluorescent membrane and intracellular dyes were used in conjunction with confocal microscopy to observe the integration of transplanted chondrocytes into cartilage both in vitro and in vivo. The distribution and duration of binding of rat, canine, and bovine chondrocytes to cartilage explants and the duration of expression of genes transduced into the transplanted chondrocytes were also determined. We used the vector AdlacZ, an E1 and E3 deleted replication defective adenoviral vector that contains the beta-galactosidase gene driven by the beta-actin promoter and the cytomegalovirus enhancer.. The transplanted chondrocytes had a patchy distribution after in vitro or in vivo transplantation and buried themselves within the cartilage over time. Chondrocytes infected with the adenoviral vector AdlacZ soon or well after transplant to cartilage explants were maintained on the cartilage and continued throughout the duration of each trial to produce beta-galactosidase coded by the adenoviral vector. The cartilage plugs were infected with AdlacZ at 2 days or one, 2, 5, or 8 weeks after the chondrocytes were transplanted. The cartilage slices were then cultured from 15 days for chondrocytes infected at 8 weeks to 60 days for chondrocytes infected at 2 days post-transplant before determining the expression of beta-galactosidase.. These results support the possibility of repairing cartilage by intraarticular injections of chondrocytes. Transduction of chondrocytes with genes producing a variety of matrix promoting proteins should further enhance the reconstruction of osteoarthritic cartilage.

Topics: Animals; Carbocyanines; Cartilage; Cattle; Chondrocytes; Dogs; Fluorescent Dyes; Genes, Reporter; Genetic Therapy; Injections, Intra-Articular; Lac Operon; Osteoarthritis; Transfection