1114. Bone Regeneration by Osteogenic Gene Delivery without Cell Transplantation Using Supramolecular Nanocarrier

Abstract

In the fields of tissue engineering, the exotic gene introduction for regulating the cell function and differentiation is one of the most decisive and important issues. For this purpose, a novel type of supramolecular nanocarrier was developed. This system is based on the self-assembly of poly(ethylene glycol) (PEG)-polycation block copolymers and pDNA, forming polyplex micelles through an electrostatic interaction. In this study, we introduced a newly-developed PEG-polycation copolymers, PEG-PAsp(DET), possessing plural cationic groups with distinctive pKa in the side chain. By the unique feature of the regulated location of primary and secondary amino groups in the side chain, this block copolymer enabled both sufficient complexation with DNA and the endosome buffering capacity (proton sponge effect). By the nanocarrier, the excellent gene expressions were obtained in various primary cells, comparable to LPEI and Fugene6. Worth noting is that the nanocarrier showed almost no cytotoxicity for any cells. Owing to this low cytotoxicity, the prolonged incubation in the presence of the nanocarrier could be carried out. Without changing medium, the cells could survive and showed high gene expression for up to 5 days. Interestingly, the gene expression was low at the beginning, and subsequently it continuously increased for several days in both protein and mRNA levels. This tendency was only observed with this nanocarrier. Then, the experiments of inducing cell differentiation by delivering genes encoding osteogenic factors with this nanocarrier were carried out. The mouse-derived calvarial cells were used and the differentiation was evaluated by the expression of osteocalcin mRNA. On Day 5 after transfection, the increase in osteocalcin expression was confirmed, and on Day 10, the expression was drastically increased. To our surprise, the induction was not observed by Fugene6, even though a comparable gene expression of the reporter gene was observed on the second day, suggesting the importance of gene expression manner for inducing the cell differentiation. Finally, we applied this system for in vivo local delivery. After admixed with calcium phosphate paste, the nanocarrier containing pDNA of osteogenic factor were implanted to mouse calvaria bone defects. After 4w, it transfected adjacent recipient cells and induced bone regeneration. To worth noting is that the bone regeneration was achieved without cell transplantation. Thus, this system may be a promising candidate of intelligent gene delivery system for constructing the innovative method of tissue engineering.