Non-viral gene delivery of nanostructured-calcium-phosphate (NanoCaP) carriers for bone regeneration

Abstract

OBJECTIVES: Local gene delivery for bone regeneration requires a safe and stable gene delivery system. There is a need to develop safe systems compared to viral gene delivery methods plagued with safety concerns. The objective of this study is to design a non-viral gene delivery for bone regeneration based on the long proven calcium phosphate systems whose biocompatibility and safety is well documented. We have uniquely synthesized a composite comprising fibrin gel and nanosized calcium phosphates (NanoCaPs) as a gene delivery carrier. METHODS: Fibrin gel matrix was synthesized in the presence of NanoCaPs and 100μg of plasmid DNA (pDNA). The complex fibrin/NanocaPs/pDNA was implanted in the hind-leg muscle of C57Bl/6 mice. We initially used the luciferase reporter gene to determine the optimal concentration of NanoCaPs that would provide the best transfection efficiency. We assessed five different concentrations of NanoCaPs (1X, 2X, 3X, 4.5X and 6X). At days 5 and 10 post-implantation, the tissues were collected and luciferase expression was quantified. We then incorporated the BMP-7 gene as the pDNA encoding for an osteoinductive agent. . The implant was assessed at week 1, 2, 4 and 8 following the implantation for bone formation employing radiography, micro-CT scan and histological assessment. RESULTS: At day 5 post-implantation, luciferase assay showed a significant increase in gene transfection when 3X NanoCaPs complexed with pDNA was used compared to the other concentrations of NanoCaPs or controls. The X-ray analysis demonstrated ectopic bone formation at 4 and 8 weeks post-implantation. Micro-CT scan also showed that NanoCaPs complexed with BMP-7 pDNA increased the volume of the new deposited bone at weeks 4 and 8 as compared with BMP-7 without NanoCaPs. CONCLUSION: Nanostructured calcium phosphates can be a safe alternative gene delivery system for bone regeneration.