Effect of electroporation-mediated transfecting recombinant plasmid pIRES-hBMP2-hVEGF165 on mandibular distraction osteogenesis.

Distraction osteogenesis requires a long consolidation period and has a low but real failure rate. Bone morphogenetic proteins (BMPs) accelerate bone deposition in fractures and critical-sized bone defects. Vascular endothelial growth factor (VEGF) is a promising reagent for inducing angiogenesis, and is an essential coordinator of extracellular matrix remodeling, angiogenesis, and bone formation in the growth plate. However, their effects on mandibular distraction osteogenesis are unknown. We investigated the effect of local delivery of plasmid pIRES-hBMP-2-hVEGF165 into a distraction area by electroporation-mediated approach.A New Zealand rabbit model were used. Activation of the device was commenced after 3 days of latency period and proceeded at the rate of 0.8 mm per day for 7 days. After the completion of activation, the rabbits were randomly divided into 5 groups: group A: recombinant plasmid 2 µg (0.1 µg/µL) pIRES-hVEGF165-hBMP2 was injected into the distraction area after the completion of activation; group B: recombinant plasmid pIRES-hBMP2 was injected into the distraction area; group C: recombinant plasmid pIRES-hVEGF165 was injected into the distraction area; group D: pIRES was injected into the distraction area, and group E: normal saline was injected into the distraction area. After injection every group used electroporation. Subsequently, the rabbits were examined by quantitative computed tomography, mechanical testing, and histomorphometric analysis.BMD of newly formed bone of the distraction area in groups A, B, and C were remarkably higher than those of groups D and E at different times (P < 0.001). At 4 and 8 weeks of consolidation, the crushing strength of 3 points of the newly formed bone in group A was remarkably higher than those of groups B, C, D, and E (P < 0.01). The results demonstrated statistically remarkable increase in regenerated bone in the gene-transfected groups.Electroporation-mediated transfecting recombinant plasmid pIRES-hVEGF165-hBMP2 could produce a satisfactory proceeding of osteogenesis and calcification, which surpassed that of the control group. This finding indicates that a combination of VEGF and BMP may make osteogenesis and angiogenesis appear at the same time. Furthermore, it may magnify the effect of single growth factor, and promote growth and reparative process of bone.

[Effect of gene transfection at different time on bone mineral density and strength of newly formed bone in mandibular distraction gap in rabbit].

OBJECTIVE: To investigate the effect of gene transfection at different time on bone mineral density and strength of newly formed bone in mandibular distraction gap in rabbit, so as to explore the optimal time for gene therapy and enhance the therapeutic effect. METHODS: 48 New-Zealand rabbits were employed to receive mandibular osteotomy and implantation of distraction devices bilaterly. Then the rabbits were randomly divided into 4 groups as group A, B and C and D. The animals in group A, B, and C were transfected with recombinant plasmids pIRES-hBMP2-hVEGF165 via electroporation-mediated approach at latency period, distraction period, consolidation period respectively. Group D was used as control group without gene transfection. After 3 days of latency period, the distraction devices were activated at the rate of 0.8 mm per day for 10 days. Three rabbits in each group were sacrificed at 1 wk, 2 wk, 4 wk and 8 wk of consolidation respectively. The mandibles were harvested and the left mandible received X-ray examination for bone healing, and quantitative computed tomography (QCT) dectection for the bone mineral density (BMD) of newly formed bone in the distraction gap. The biomechanical properties of the new generation bone at 4 th and 8 th week of consolidation in each group were detected by three point bending test. RESULTS: The bone mineral density and the biomechanical strength of newly formed bone increased along the length of consolidation in each group. After 1 week of consolidation, there was no significant difference in BMD among group A (83.43 +/- 9.96), group B (92.29 +/- 11.25), group C (89.93 +/- 14.15), P > 0.05. But the BMD of group A, B and C was higher than that of group D (70. 31 +/- 3.30), P < 0.05. After 2wk, 4 wk and 8 wk of consolidation, the BMD of group B (137.54 +/- 7.20,492.93 +/- 17.57, 790.48 +/- 12.19) was significantly higher than those of group A (121.44 +/- 9.27, 396.15 +/- 15.70, 603.39 +/- 16.46), C (125.06 +/- 7.24, 464.15 +/- 15.45, 764.15 +/- 17.28), and D (98.86 +/- 8.13, 336.45 +/- 11.95, 577.89 +/- 18.43), P < 0.05. The biomechanical parameters were also higher in group B than those of group A, C and D after four and eight weeks of consolidation (P < 0.05). CONCLUSION: It is better to transfect gene at the beginning of distraction (distraction period) than at other stages of DO. In this way, more remarkable effect could be obtained on new bone formation. It suggests that the distraction stage is the optimal time for gene therapy.

[Effect of electroporation-mediated gene transfect on the expression of cyclins during mandible distraction in rabbit].

OBJECTIVE: To investigate the effect of electroporation-mediated gene transfect on the expression of cyclins during mandible distraction in rabbit. METHODS: Bilateral mandibular osteotomy was performed in 45 New-Zeland rabbits. After a latency of 3 days, the mandibles were elongated using distractors with a rate of 0.8 mm/day for 7 days. After the completion of distraction, the rabbits were randomly divided into 5 groups. 2 microg (0.1 microg/microl) of pIRES-hVEGF165-hBMP2, recombinant plasmid pIRES-hBMP2, recombinant plasmid pIRES-hVEGF165, pIRES and the same volume of normal saline (NS) was injected into the distraction area in each group, respectively. After injection, electroporation was performed in every group. Three animals in each group were sacrificed at 7, 14, and 28 days after completion of distraction, respectively. The lengthened mandibles were harvested and processed for immunohistochemical examinations. The expression of cyclins A, D1 ,E in positive cells were measured by CMIAS-2001A computerized image analyzer. The data were analyzed with the single factor analysis of variance and q test. RESULTS: Cyclins A, D1, E staining was mainly located in inflammatory cells, granulation tissue monocyte, fibroblast, osteoblasts, osteocyte and the connective tissues around the new bone. The expression reached to the peak at 7th day of consolidation, and decreased at 14th day, and weak at 28th day. Image analysis results showed that, at 7th day, the expression absorbance A in group C (0.59 +/- 0.14) was the strongest, compared to group A (0.41 +/- 0.13), B (0.38 +/- 0.14), D (0.34 +/- 0.12) and E (0.31 +/- 0.10), showing a significant difference (P < 0.05, P < 0.01). There was no significance difference between group A and B (P > 0.05), but the difference between group A/B and group D/E (P < 0.05). At 14th and 28th day, there was no significant difference among group A (0.39 +/- 0.11), B (0.34 +/- 0.10) and C (0.33 +/- 0.09) (P > 0.05), but there was significant difference between group A/B/C and group D (0.19 +/- 0.12) or E (0.14 +/- 0.04) (P < 0.05 or P < 0.01). CONCLUSIONS: Electroporation-mediated gene transfection can promote cyclins A, D1, E expression effectively, which may promote cell differentiation and proliferation, stimulate extracellular matrix synthesis and new bone formation in distraction gap.

[The effect of electroporation mediated gene therapy on bone mineral density and strength of new-formed bone in mandibular distraction gap in rabbit].

OBJECTIVE: To explore the effect of electroporation mediated gene therapy on bone mineral density and strength of new-formed bone in mandibular distraction gap, so as to enhance the osteogenesis and shorten the distraction term. METHODS: New-Zealand rabbits were employed. The distraction began after 3 days of latency period at the rate of 0. 8 mm per day for 7 days. After distraction, the rabbits were randomly divided into 5 groups to receive injection in the distraction gap with recombinant plasmid 2 microg (0.1 microg/microl) pIRES-hVEGF165-hBMP2 in group A, with recombinant plasmid pIRES-hBMP2 in group B, with recombinant plasmid pIRES-hVEGF165 in group C, with pIRES in group D, and with normal saline (NS) in group E. After injection, electroporation was performed in all the groups. After 1 week, 2 weeks, 4 weeks and 8 weeks of consolidation, all the animals underwent X-ray and quantitative computed tomography (QCT). The new-formed bone in distraction gap was selected as regions of interest (ROI) to measure the bone mineral density(BMD). Then the rabbits were sacrificed and the new-formed bone samples were harvested to detect 3-point crushing strength. RESULTS: BMD of newly formed bone in group A, B and C was markedly higher than that in group D and E (P < 0.01). After 2 weeks of consolidation, BMD in group A was much higher than that in the other groups, but there was no difference between group B and C. After 4 weeks of consolidation, BMD in group A and B was markedly higher than that in group C, D and E (P < 0.01). After 8 weeks of consolidation, BMD in group A was markedly higher than that in the other groups. While the BMD was not significantly different between group B and C, but the BMD in group B and C was higher than that in group D and E (P < 0.01). After 4 weeks of consolidation, the 3-point crushing strength of newly formed bone in group A was markedly higher than that in group B,C, D and E (P < 0.01), which was still the same after 8 weeks of consolidation. And the crushing strength in group B was higher than that in group C, D and E (P < 0. 05). CONCLUSIONS: Electroporation-mediated transfection of recombinant plasmid pIRES-hVEGF165-hBMP2 could greatly enhance osteogenesis and calcification. A combination of VEGF and BMP may promote osteogenesis and angiogenesis simultaneously, so as to magnify the effect of each growth factor, resulting a synergetic effect.