Targeted gene-and-host progenitor cell therapy for nonunion bone fracture repair

Abstract

Nonunion fractures present a challenge to orthopedics with no optimal solution. In-vivo DNA electroporation is a gene-delivery technique that can potentially accelerate regenerative processes. We hypothesized that in vivo electroporation of an osteogenic gene in a nonunion radius bone defect site would induce fracture repair. Nonunion fracture was created in the radii of C3H/HeN mice, into which a collagen sponge was placed. To allow for recruitment of host progenitor cells (HPCs) into the implanted sponge, the mice were housed for 10 days before electroporation. Mice were electroporated with either bone morphogenetic protein 9 (BMP-9) plasmid, Luciferase plasmid or injected with BMP-9 plasmid but not electroporated. In vivo bioluminescent imaging indicated that gene expression was localized to the defect site. Microcomputed tomography (µCT) and histological analysis of murine radii electroporated with BMP-9 demonstrated bone formation bridging the bone gap, whereas in the control groups the defect remained unbridged. Population of the implanted collagen sponge by HPCs transfected with the injected plasmid following electroporation was noted. Our data indicate that regeneration of nonunion bone defect can be attained by performing in vivo electroporation with an osteogenic gene combined with recruitment of HPCs. This gene therapy approach may pave the way for regeneration of other skeletal tissues.

Figure 1

Host progenitor cells populate the defect site. A 1.5-mm long defect was made in the mouse radius, and a collagen sponge was placed in the defect site. Ten days after surgery, the radius was harvested and stained using H&E. (a–c) Radial defect 10 days after surgery. Staining shows the presence of HPCs (arrowheads). HPC, host progenitor cells; H&E, hematoxylin and eosin; M, defect margin; U, ulna.

Figure 2

Gene transfer efficiency and localization to HPCs. (a) BLI was used to monitor luciferase activity in bone defects following pLuc injection and electroporation. The x axis displays time postelectroporation; the y axis shows activity in RLUs. *P < 0.05, two-tailed t-test, n = 4. Note the representative pictures of one mouse. (b) Results of another BLI study performed in a representative mouse in order to verify gene delivery to HPCs. After they underwent the BLI study, mice were sacrificed and cells from explanted radii were isolated. (c) photomicrograph shows HPCs isolated from the radial explants. (d) Isolated HPCs were lyzed and mRNA was isolated. Using RT-PCR followed by real-time PCR, the presence of Luc expression in the isolated cells was verified (EP Luc). HPCs isolated from defects in which electroporation was not performed were used as a negative control (no EP). BLI, bioluminescence imaging; HPC, host progenitor cells; RLU, relative light unit; RT-PCR, reverse transcription PCR.

Figure 3

Bone formation in the defect area. (a) Bone volume analysis: a comparison of newly formed bone in the radial defect in animals in the pBMP-9 with electroporation (BMP-9, six mice), pLuc with electroporation (Luc, four mice), and pBMP-9 without electroporation (no EP, four mice) groups and of segments of native radii having the same dimensions (native, six mice). *P < 0.01, two-tailed t-test. Note that bone formation in the defect site was significantly higher than in controls. No statistical significant difference was found between mice electroporated with pLuc and mice with pBMP-9 without electroporation. (b) µCT reconstruction comparison of defects treated with pLuc and electroporation (Luc), defects treated with pBMP-9 and electroporation (BMP-9), and defects treated with BMP-9 without electroporation (no EP). M, defect margin. Arrowhead indicates new bone formation in the defect. In the 3D images, orange regions denote new bone formation. (c) Histological sections containing newly formed bone. 1.5-mm long defects were made in mice radii, and a collagen sponge was placed in the defect site. Ten days postoperation, the radius was injected with either pBMP-9 or pLuc followed by electroporation. As a control, radii were injected with pBMP-9 but electroporation was not performed. Five weeks postelectroporation, the radii were harvested and stained using Masson's trichome. M, defect margin; U, ulna. Arrowheads indicate new bone formation; asterisks indicate soft tissue in the defect site. BMP, bone morphogenetic protein 9; µCT, microcomputed tomography.

Figure 4

Quantitative analysis of structural parameters of induced bone formation in the defect area. (a–f) Newly formed bone in the radial defect in mice in the pBMP with electroporation group (BMP-9) was compared to segments of native radii having the same dimensions (native). *P < 0.05, two-tailed t-test, n = 6 in each group. The structural parameters include: (a) bone thickness (mm); (b) trabecular number (1/mm); (c) bone separation (mm); (d) connectivity density (1/mm³); (e) bone volume density (BV/TV, mm/mm); and (f) bone mineral density (mg HA/cm³). BMP-9, bone morphogenetic protein 9.

Figure 5

Setup of the electroporation system. A 1.5-mm long defect was made in the mouse radius, and a collagen sponge was placed in the defect site. Ten days after surgery pLuc or pBMP-9 was injected into the defect site, and electroporation was immediately performed using needle electrodes. (a) Mouse ready for electroporation. The animal is mounted on the fluoroscope detector and needle electrodes are placed. (b) Locations of the electrodes and the needle used for plasmid injection are verified using the fluoroscope.