Investigation of cartilage modification for controlling immune reaction against xenogeneic cartilage

Introduction: Allogeneic cartilages are decellularized when they are transplanted subcutaneously to avoid the immunoreaction in clinical practice. However, transplanting cartilage with chondrocytes would be beneficial because chondrocytes are essential for the maintenance of the cartilage. In this study, we investigated the effect of modifications to cartilage before transplantation for the suppression of immunoreactions. We utilized xenogeneic models because they are often employed in preclinical safety evaluations prior to clinical trials. If xenogeneic immunoreactions can be controlled, allogeneic rejection should be easier to manage. Methods: Cartilaginous particles differentiated from human iPS cells were modified in two ways. For coverage with syngeneic chondrocytes, cartilaginous particles were cocultured with murine chondrocytes for 7 days. For decellularization, particles were undergone freeze & thaw with or without DNase cycles of either 5 or 15. After these modifications, cartilaginous particles were filled in meshed tubular PLA scaffolds and subcutaneously transplanted into C57BL/6-Tg (CAG-EGFP) for 8 weeks. Immunoreactions were histologically evaluated by immunostaining against CD3, CD4 and Foxp3. Results: In vitro studies showed that chondrocytes successfully covered the cartilaginous particle irrespective of decellularization. In addition, decellularization of cartilage was possible without apparent breakdown of the matrix structure. In transplantation experiments, severe immune reactions were not observed histologically. However, CD4 (+) cells and CD3 (+) cells increased when cartilaginous particles received both chondrocyte coverage and decellularization, while Foxp3 (+) cells were not observed. Conclusions: Contrary to our expectations, combination of decellularization and surface coverage of cartilaginous transplants increased T cells, suggesting the complexity of the immunoreaction against xenogeneic cartilages.