Multilayered Cryogel Enriched with Exosomes Regenerates and Maintains Cartilage Architecture and Phenotype in Goat Osteochondral Injuries

Treatment of critical-size osteochondral (OC) injuries at load-bearing sites has remained a major clinical challenge in orthopedic surgery. This is due to the anisotropic characteristics of OC tissue and the stratified structure of the cartilage. Here, we developed a multilayered OC scaffold by employing cryogelation technology. Gelatin, chitosan, and chondroitin sulfate were utilized for designing three distinct, 2425 ± 120 μm thick layers of cartilage having different alignments, while nanohydroxyapatite and gelatin were used for the subchondral bone layer. Exosomes derived from articular chondrocytes in the range of 60–110 nm were used to promote chondrogenesis. The biocompatibility and cartilage formation potential of the scaffold and exosomes were initially evaluated in rat OC defects. The application of exosome-loaded scaffolds was then investigated in a critical-size OC injury (8 × 10 mm) created in the goat knee. Artificial synovial fluid was designed and utilized as a carrier for exosomes for a booster dose administered as an intra-articular injection. X-ray imaging and micro-CT analysis revealed that the treatment resulted in improved subchondral bone regeneration. The defect region exhibited healthy hyaline cartilage formation, as detected by MRI imaging. Moreover, histological examination revealed that the treatment group showed augmented cell proliferation, matrix deposition, secretion of proteoglycans, and the formation of stratified hyaline cartilage over a long-term (6 and 12 months), whereas the control group demonstrated the formation of fibrocartilage. Treatment-induced upregulation of collagen II, aggrecan, and SOX 9 genes (∼10 fold) further provided evidence that the cartilage phenotype was well preserved. Hence, the proposed treatment has significant translational potential for treating adverse OC clinical injuries.