Background: To date, therapeutic method for in vivo gene delivery has not been established on bone engineering though their potential usefulness has been suggested. For clinical applications, an effective condition should be developed to transfer the genes in vivo without any of transfection-reagents or virus-vectors.
Objective: The aim of this study is to have a new understanding on the usefulness of delivering non-viral GAM without cell transplantation or any transfection reagents/kits for facilitating clinical setting of bone engineering.
Methods: In this study, to facilitate the clinical setting of this strategy, we simply investigated whether manufactured gene activated-matrix (GAM) with atelocollagen containing a certain amount of plasmid (p) DNA encoding osteogenic-proteins could augment the cranial bone in rat. GAMs were manufactured by mixing 0.02, 0.1, 0.5 or 1 mg of AcGFP plasmid-vectors harboring cDNA of BMP4 (pBMP4) or Runx2 (pRunx2) with 2% bovine-atelocollagen and β-TCP granules. Before manufactured GAMs, to determine the biological activity of generated pDNAs, we confirmed GFP-expression and increased-level of ALP activities in MC3T3-E1 cells transfected with pBMP4 or pRunx2 during culture. Then, GAMs were lyophilized and transplanted to onlay placement on the cranium.
Results: At 2 weeks of transplantation, GFP-expressing cells could be detectable in only GAMs containing 0.5 and 1 mg of AcGFP plasmid vectors. Then, at 4 weeks, significant bone formation was recognized in GAMs containing 0.5 and 1 mg of pDNAs encoding BMP4 or Runx2 but not in 0.02 or 0.1 mg of GAMs. These newly formed bone tissues surrounded by osteocalcin-stained area were augmented markedly until 8 weeks after transplantation. In contrast, minimal bone formation was observed in GAMs without harboring cDNA of osteogenic-proteins. Meanwhile, when GAMs were transplanted to the cranial bone defect, bone formation was detectable in specimens containing 1 mg of pBmp4 or pRunx2 at 8 weeks as well.
Conclusion: Thus, atelocollagen-based GAM reliably could form the engineered-bone even for the vertical augmentation when contained a certain amount of plasmid-vectors encoding osteogenic-proteins. This study supports facilitating the clinical application of GAM for bone engineering.