Stimulation of a calcified cartilage connecting zone by GDF-5-augmented fibrin hydrogel in a novel layered ectopic in vivo model.

Tissue engineering approaches for reconstructing full-depth cartilage defects need to comprise a zone of calcified cartilage to tightly anchor cartilage constructs into the subchondral bone. Here, we investigated whether growth and differentiation factor-5-(GDF-5)-augmented fibrin hydrogel can induce a calcified cartilage-layer in vitro that seamlessly connects cartilage-relevant biomaterials with bone tissue. Human bone marrow stromal cells (BMSCs) were embedded in fibrin hydrogel and subjected to chondrogenesis with TGF-β with or without GDF-5 before constructs were implanted subcutaneously into SCID mice. A novel layered ectopic in vivo model was developed and GDF-5-augmented fibrin with BMSCs was used to glue hydrogel and collagen constructs onto bone disks to investigate formation of a calcified cartilage connecting zone. GDF-5 significantly enhanced ALP activity during in vitro chondrogenesis while ACAN and COL2A1 mRNA, proteoglycan-, collagen-type-II- and collagen-type-X-deposition remained similar to controls. Pellets pretreated with GDF-5 mineralized faster in vivo and formed more ectopic bone. In the novel layered ectopic model, GDF-5 strongly supported calcified cartilage formation that seamlessly connected with the bone. Pro-chondrogenic and pro-hypertrophic activity makes GDF-5-augmented fibrin an attractive bioactive hydrogel with high potential to stimulate a calcified cartilage connecting zone in situ that might promote integration of cartilage scaffolds with bone. Thus, GDF-5-augmented fibrin hydrogel promises to overcome poor fixation of biomaterials in cartilage defects facilitating their long-term regeneration.