Evaluating potential of tissue-engineered cryogels and chondrocyte derived exosomes in articular cartilage repair.

Treatment of articular cartilage injuries especially osteochondral tissue requires intervention of bioengineered scaffold. In this study, we investigated the potential of the tissue-engineered cryogel scaffold fabricated using cryogelation technology. Two types of cryogels viz. chitosan-gelatin-chondroitin sulfate (CGC) for articular cartilage and nano-hydroxyapatite-gelatin (HG) for subchondral bone were fabricated. Further, novel bilayer cryogel designed using single process fabrication of two layers (CGC as top layer and HG as the lower layer) was designed to mimic osteochondral unit. CGC cryogel was tested for their biocompatibility using the enzymatically isolated chondrcoytes from goat articular cartilage while HG cryogel was tested using pre-osteoblast cell line. Extracellular vesicles, specifically exosomes were isolated from the spent media of chondrocytes to validate their effect over cell proliferation and migration which are required for defect healing and infiltration respectively. These isolated exosomes were characterized and analyzed for confirming their size distribution profile and visualized morphologically using advanced microscopy techniques. For cartilage part, CGC cryogels were examined as delivery system for delivering exosomes at defect site, where 80% of release was observed in 72 h. Release of 18.7 µg chondroitin sulfate/mg cryogel was obtained in a period of one week from CGC cryogel (termed cryogel extract) which has chondroprotective effect. Further, effect of exosome concentration (10 and 20 µg/ml), CGC extract and combination of exosome and CGC extract (Exo-Ex) were assessed over the chondrocytes. In addition, in vitro scratch wound assay was performed to analyse the migration capacity over the micro-injury when treated with exosomes, cryogel extract and Exo-Ex. The overall results thus answer key questions of therapeutic potential of chondrocyte exosomes, cryogel extract in addition to potential of CGC and HG cryogel for osteochondral repair.