Multilayered Cryogel\nEnriched with Exosomes Regenerates\nand Maintains Cartilage Architecture and Phenotype in Goat Osteochondral\nInjuries

Treatment of critical-size osteochondral (OC) injuries\nat load-bearing\nsites has remained a major clinical challenge in orthopedic surgery.\nThis is due to the anisotropic characteristics of OC tissue and the\nstratified structure of the cartilage. Here, we developed a multilayered\nOC scaffold by employing cryogelation technology. Gelatin, chitosan,\nand chondroitin sulfate were utilized for designing three distinct,\n2425 ± 120 μm thick layers of cartilage having different\nalignments, while nanohydroxyapatite and gelatin were used for the\nsubchondral bone layer. Exosomes derived from articular chondrocytes\nin the range of 60–110 nm were used to promote chondrogenesis.\nThe biocompatibility and cartilage formation potential of the scaffold\nand exosomes were initially evaluated in rat OC defects. The application\nof exosome-loaded scaffolds was then investigated in a critical-size\nOC injury (8 × 10 mm) created in the goat knee. Artificial synovial\nfluid was designed and utilized as a carrier for exosomes for a booster\ndose administered as an intra-articular injection. X-ray imaging and\nmicro-CT analysis revealed that the treatment resulted in improved\nsubchondral bone regeneration. The defect region exhibited healthy\nhyaline cartilage formation, as detected by MRI imaging. Moreover,\nhistological examination revealed that the treatment group showed\naugmented cell proliferation, matrix deposition, secretion of proteoglycans,\nand the formation of stratified hyaline cartilage over a long-term\n(6 and 12 months), whereas the control group demonstrated the formation\nof fibrocartilage. Treatment-induced upregulation of collagen II,\naggrecan, and SOX 9 genes (∼10 fold) further provided evidence\nthat the cartilage phenotype was well preserved. Hence, the proposed\ntreatment has significant translational potential for treating adverse\nOC clinical injuries.