Effective Gene-activated Matrix with Self-assembly Nano-devices for Bone Engineering

Objectives: Therapeutic method for in vivo gene delivery has not been established on bone engineering. To deliver the plasmid (p) DNAs effectively, we have recently developed a novel biocompatible self-assembly nano-device “Nanoball” based on electrostatic interaction which arranges the anionic macromolecules to polymeric nanomaterials for nucleic-acid carriers. This study aims to investigate whether GAM comprised of atelocollagen and nanoballs containing pDNAs encoding BMP4 and GFP (Nano-BMP4) can promote the bone regeneration.
Methods: To determine the biological function after gene transfection of cells, which migrate to injured sites of bone, pDNAs encoding BMP4 (pBMP4) was transferred to rat macrophages, fibroblasts and bone-marrow mesenchymal stem cells (BMMSCs) in culture by nanoball-devices. Then, GAMs were manufactured by mixing 25-200μg of Nano-BMP4 with 100μl of 2% bovine atelocollagen and 20mg β-TCP granules. After that, GAMs were lyophilized and transplanted to the rat cranial bone surfaces.
Results: When cultured the macrophages, fibroblasts and BMMSCs with Nano-BMP4 for 48-hours, up-regulation of its mRNA expression and subsequent production of BMP4 were observed in these cells. Then, when transplanted the GAMs containing Nano-BMP4, newly formed bone tissues were markedly observed after 4-weeks of transplantation, compared with GAMs containing Nano-GFP without encoding any osteogenic protein. Particularly, enough volume of new bones was recognized even though only 25μg of Nano-BMP4 was contained in GAMs. In contrast, more than 500μg of pBMP4 was required to induce the same level of new bone formation when naked-pDNAs were directly incorporated to GAMs.
Conclusions: Nanoball may facilitate the clinical setting of GAMs for bone engineering. We are currently carrying out the additional experiments, such as developing an appropriate matrix and verifying the minimal concentration of pDNAs, for clarifying the actual usefulness of nanoball-based GAMs.