Tracking the degradation of polysaccharide hydrogels by non-invasive near-infrared fluorescence imaging

Purpose: Hydrogels can mimic hydrated native cartilage tissue and are considered suitable scaffolds for cartilage tissue engineering. Compared with preformed hydrogels, in situ forming hydrogels offer various advantages in cartilage repair. An essential step in the development of in-situ forming biodegradable hydrogels for cartilage regeneration is the monitoring of their in vivo fate. Fluorescence imaging techniques in this respect are preferred for these avoid the need to sacrifice large numbers of animals. Meanwhile, this approach has the potential to provide a non-invasive tool for locating, monitoring and tracking the in vivo status of implanted biomaterials. In this study, the degradation behavior of near infrared (NIR) fluorescence labeled polysaccharide-tyramine conjugate hydrogels was followed by monitoring the changes in signal intensity of subcutaneously implanted hydrogels in mice. Methods: The NIR fluorescent label was covalently linked to a dextran co-conjugated with both tyramine and free amine groups (Figure 1). Hydrogels implanted were prepared by the enzymatic crosslinking of either dextran-tyramine or dextran-tyramine/hyaluronic acid-tyramine (50/50: wt/wt) conjugates with the addition of the near infrared dye labeled conjugate using Horseradish peroxidase in the presence of hydrogen peroxide. The presence of tyramine groups in the NIR labeled conjugate ensures a covalent coupling of the conjugate in the hydrogel matrix. Human bone marrow derived mesenchymal stem cells (hMSCs) and bovine primary chondrocytes (bPCs) were isolated as previously reported. Both hydrogel constructs with and without hMSCs/bPCs in an 80/20 ratio were studied. The gels were implanted subcutaneous in mice for 10 weeks. Results: The free amine groups or the NIR label had no influence on the gelation time, swelling and mechanical properties of the enzymatically crosslinked hydrogels prepared. In general gelation times are within 10–20 seconds and can be regulated by the concentration of Horseradish peroxidase or hydrogen peroxide. In previous research it was proven that these concentrations do not invoke any cytotoxic reactions. In vitro fluorescence imaging was performed to determine the necessary concentration for imaging prior to hydrogel implantation. A decrease in fluorescence upon enzymatic crosslinking of the conjugate hydrogels was observed likely due to reaction of the radical formed in the process with the fluorescent dye. The concentration of the NIR dye was set at 100 nM and at this concentration an appropriate signal for imaging was obtained. As an example an image of the signal and mouse and superposition is presented in Figure 2. The total fluorescence decreased to 60% for the dextran-tyramine hydrogels and to 40% for the 50/50 dextran-tyramine hyaluronic acid-tyramine conjugates during the 10 weeks implantation period indicating hydrogels degradation. Results were similar for hydrogel/cell constructs. Currently histologic analysis of explants is performed which will be reported soon. Conclusions: This research revealed that in-situ forming NIR fluorescence labeled polysaccharide hydrogels can be traced by a non-invasive procedure and provides new strategies to study the fate of injectable hydrogels in vivo.