| Popis: |
One of the major obstacles to be addressed by approaches to gene therapy in regenerative medicine remains the attainment of an optimal gene delivery method. Recent indications point out in-vivo electroporation as a safe, simple and effective gene delivery method. Therefore, we hypothesized that direct in vivo electroporation of an osteogenic gene (BMP-9) into a nonunion radial bone defect site would induce complete bone regeneration and fracture repair. Plasmid luciferase (pLuc) and plasmid bone morphogenetic protein|[ndash]|9 (pBMP9) were amplified using standard procedures. A 2.5-mm segmental defect was created in the radius bone of C3H/HeN mice. Three days later, the mice were anesthetized with a 40-ul mixture of xylazine and ketamine, designated electroporation sites were shaven, and pLuc, pBMP9, or PBS was injected into the bone defect. The amount of DNA was determined to be 50 ug based on prior experiments performed in the thigh muscle. Immediately postinjection the skin was covered with 40% glycerol in PBS and transcutaneous electric pulses were applied using two 1-cm cube electrodes placed 2 to 4 mm apart at both sides of the limb. To generate pulses an ECM 830 electroporator was used. To monitor transgene expression in the defect site noninvasively and quantitatively, luciferase activity was detected in vivo in real time by using the Roper Chemiluminescence Imaging System. For a detailed qualitative and quantitative three-dimensional (3D) evaluation of bone formation in the defect, we scanned the electroporated limbs with a high-resolution micro-CT (uCT) system during the experiment. Seven weeks after electroporation the mice were sacrificed, and the dissected limbs were subjected to high-resolution uCT scanning which enabled us to quantitatively analyze the bone formation at the site of the defect. To determine how many copies of plasmid DNA were delivered and expressed in the defect site, DNA and RNA were extracted from the bone defect site 3 days after electroporation with pLuc and pBMP-9. Our results based on the uCT analysis of the radii electroporated with pBMP9 demonstrated massive bone formation surrounding the bone defect. No bone formation was noted in limbs electroporated with pLuc or PBS (1a). Non-invasive, quantitative molecular imaging of electroporated radii indicated that the luciferase gene was expressed in the defect site up to 24 days after electroporation, with peak expression on Day 10 (1b). Our results indicate for the first time that regeneration of bone in a nonhealing bone defect can be attained using in vivo electroporation of an osteogenic gene. Moreover, our method may pave the way for the regeneration of other skeletal tissues such as cartilage and tendon. |
