P625 Platelet derivatives stimulate cartilage repair activating progenitor cells and maintaining human hyaline cartilage phenotype by Chondromodulin-I induction

Objective: Regeneration of damaged hyaline cartilage still remains a tremendous clinical challenge; new trends in cartilage regeneration often contemplate the use of platelet derivatives (PRP or PL) for the effect on the proliferation of human articular chondrocytes preserving their somatic differentiation potential and inducing the cartilage repair. Our study identify new pathway induced by platelet derivatives that are able to regulate articular cartilagehomeostasis. Materials and Methods: From human articular cartilage biopsy adult human articular chondrocytes (HAC) and chondro-progenitor cells (CPC) were isolated and expanded “in vitro”. The CPC population was obtained by PL (platelet lyset) treatment of human cartilage chips: after 10 days in culture the cells migrate and proliferate. Both cell populations were tested “in vitro” for clonogenic capability, for phenotype and gene expression of typical chondrogenic markers and for differentiation potential. Results: Our data strongly suggested that fully differentiated chondrocytes possess “reserved stemness,” which are reactivated during in vitro expansion only in presence of PL, gradually displaying multipotent stem/progenitor cell characteristics. At the same time PRP is able to activate and recruit CPCs positive for nestin, gremlin-1 and sox-9 genes normally expressed in dividing cells during the early stage of development. “In vivo”, cartilage matrix formation was assessed by histology after subcutaneous transplantation of HAC/CPC isolated cell culture; cells were seeded on PGA-HA scaffolds with or without PL and implanted into immunocompromised mice. The addition of platelet derivatives to cartilage grafts resulted in robustly formation of hyaline-like cartilage that showed the expression of type II collagen and Chondromodulin-1 (ChM1), a specific anti-angiogenic factor strongly present in avascular cartilage. Moreover, type X collagen and VEGF expression were not observed in the tissue graft, supporting the maintenance of hyaline cartilage phenotype and no hypertrophy tissue transition. Conclusions: The combination of articular chondrocytes or precursors, platelet derivatives and PGA-HA scaffold can synergistically promote cartilage formation. At the same time PL is able to drive cartilage regenerative response by activating and recruiting CPCs positive for nestin and other early genes. Nestin is an intermediate filament protein expressed in dividing cells during the early stage of development and it is normally used as a marker of proliferating and migrating adult stem cell in multiple tissues. This protein is up regulated in response to injury and pathological processes. The newly tissue formed maintains the cartilage phenotype by the overexpression of ChM1 mediated by PL treatment. ChM1 maintains cartilage homeostasis and inhibits endochondral ossification, suggesting the importance of ChM1 in governing stable chondrocyte phenotype. More studies are in progress to investigate the role of platelet derivatives on hypoxia environment and hypoxia-inducible factors (HIFs) modulation as critical markers on the maintaining of the articular chondrocyte phenotype.