Application of endostatin using nonviral gene delivery toward the regeneration of articular cartilage

Abstract

Articular cartilage is avascular, and defects have limited capacity for spontaneous healing. Angiogenesis may interfere with maturation of naturally avascular tissues. Our rationale is that the use of endostatin, a potent angiogenesis inhibitor, will facilitate the formation of hyaline cartilage during regeneration. The objective of this thesis was to develop a system with a novel approach for treating cartilage defects, namely endostatin-producing cartilaginous constructs. The constructs were engineered using nonviral gene therapy, through evaluation of select variables, including regulators (culture media, endostatin plasmid load, method of pEndo lipoplex incorporation, and oxygen tension), scaffold formulation, and cell type. We also investigated select aspects of the in vivo cartilage defect model in which the construct can be implanted, including the post-surgical rehabilitation protocol and the use of osteogenic protein (OP)- 1. The principal achievement was the engineering of endostatin-expressing cartilaginous constructs in vitro using chondrocytes and mesenchymal stem cells, collagen sponge-like scaffolds and hydrogels, and chondrogenic medium. Peaks in endostatin protein were observed during the first few days of culture, followed by decreases. The endostatin levels were comparable to therapeutic levels in vitro and physiological levels in vivo. Most of the endostatin protein was released into the expended medium; little retention was observed, including in scaffolds supplemented with heparan sulfate, chondroitin sulfate, and heparin. In vivo work examining chondral defects in the goat knee demonstrated that long-term post-operative immobilization, even with periodic passive motion exercise, resulted in significant joint degeneration. Cell-seeded scaffolds were observed in the defect 2 months following implantation and short-term immobilization, and yielded results at least as good as historical data obtained using other treatment techniques, including autologous chondrocyte implantation and microfracture, suggesting that a cell-seeded scaffold is a viable option for cartilage repair. There was no significant benefit of multiple treatments of OP-I on chondral defects. Neovascularization was observed in the largely fibrous reparative tissue filling the chondral defects, providing further rationale for the use of endostatin. A notable finding was the observation of laminin and type IV collagen, 2 common basement membrane molecules, in both in vitro engineered cartilaginous constructs and in vivo cartilage repair samples.