Electrical stimulation facilitates the angiogenesis of human umbilical vein endothelial cells through MAPK/ERK signaling pathway by stimulating FGF2 secretion.

Electrical stimulation (ES) is able to enhance angiogenesis by stimulating fibroblasts. Fibroblast growth factor 2 (FGF2) is an independent angiogenesis inducer. The present study aimed to evaluate the role of ES-induced FGF2 secretion in affecting angiogenesis during wound healing via the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway. Fibroblasts and human umbilical vein endothelial cells (HUVECs) were exposed to ES, and the HUVECs were cocultured with ES-treated fibroblast culture solution. ES exposure showed no toxic effects on fibroblasts or HUVECs. ES led to enhanced growth of fibroblasts and HUVECs as well as FGF2 secretion, which is induced through the NOS pathway. ES-induced FGF2 secretion was shown to increase vascular endothelial growth factor (VEGF) protein and enhance migration, invasion, and angiogenesis of HUVECs. Also, ES-induced FGF2 secretion activated the MAPK/ERK signaling pathway. However, inhibition of the MAPK/ERK signaling pathway reversed the positive effects of ES-induced FGF2 secretion. In vitro experiments showed positive effects of ES on wound healing. Taken together, the findings suggested that ES promoted FGF2 secretion and then activated the MAPK/ERK signaling pathway by facilitating angiogenesis and promoting wound healing.

Cephalometric Analysis of Modifications of the Mandible Due to Reduction Mandibuloplasty in Patients With Prominent Mandibular Angle.

Cephalometry has been used to measure hard and soft facial tissues as well as to conduct the ostectomy to determine the characteristics of the prominent mandibular angles (PMA). The changes produced on the mandibles by reduction mandibuloplasty are not known. The objective of the present study was to identify by cephalometric analysis the anatomical changes of the mandibles that occur in patients with PMA after reduction mandibuloplasty. Forty-two patients with PMA were submitted to cephalometric analysis before, 1 week and 6 months after surgery to evaluate the changes of the mandibles produced by reduction mandibuloplasty. Cephalometry was standardized to obtain descriptive measurements of the dimensions of the mandibles. The modifications of the mandibles due to reduction mandibuloplasty showed a significant change obtained by cephalometry. At 1 week after surgery, the average distance between the gonions decreased 17.70 ±â€Š8.46 mm, the average length of the mandibular ramus reduced 5.84 ±â€Š3.26 mm, the average mandibular body length increased 4.61 ±â€Š2.74 mm, the average gonial angle increased 14.78 ±â€Š6.65°, the average mandibular plane angle increased 10.29 ±â€Š3.82°. At 6 months postoperatively, the first 3 linear measurements increased 3.68 ±â€Š2.91, 1.66 ±â€Š2.51, and 2.10 ±â€Š2.37 mm respectively; however, the last 2 angular measurements reduced 2.86 ±â€Š3.02° and 1.77 ±â€Š2.62° respectively. The results demonstrated that reduction mandibuloplasty can modificate mandibular contouring three-dimensionally. The data of 6 months postoperatively compare to those of the beauty people reported in the literature, there were statistically differences between the linear measurements, but no statistically differences between the angular measurements. Despite bone regeneration result in linear or angular measurements change, postoperative angular shape was predominantly maintained, and the preoperative angular prominence did not recur.

Gene expression of osteogenic factors following gene therapy in mandibular lengthening.

This study investigated the effect of gene therapy on the expression of osteogenic mediators in mandibular distraction osteogenesis rabbits. Bilateral mandibular osteotomies were performed in 45 New-Zealand rabbits. After a latency of 3 days, the mandibles were elongated using distractors with a rate of 0.8 mm/d for 7 days. After the completion of distraction, the rabbits were randomly divided into 5 groups: 2 µg (0.1 µg/µL) of recombinant plasmid pIRES-hVEGF165-hBMP-2, recombinant plasmid pIRES-hBMP2, recombinant plasmid pIRES-hVEGF165, pIRES, and the same volume of normal saline were injected into the distraction gap of groups A, B, C, D, and E, respectively, followed by electroporation. Three animals were killed at the 7th, 14th, and 28th day after gene transfected in different groups, respectively. The lengthened mandibles were harvested and processed for immunohistochemical examinations; the mean optic densities (MODs) and integral optical density of bone morphogenetic protein (BMP-2) and transforming growth factor ß1 (TGF-ß1)-positive cells were measured by CMIAS-2001A computerized image analyzer. The data were analyzed with SPSS (SPSS Inc, Chicago, IL). Bone morphogenetic protein 2 and TGF-ß1 staining was mainly located in inflammatory cells, monocytes, fibroblasts, osteoblasts, osteocytes, and chondrocytes in the distraction zones. Their strongest expression reached to the peak at the seventh day and decreased at the 14th day of consolidation stage; at the 28th day, they expressed weakly. Image analysis results show that, at the seventh day, the expression of BMP-2 in group B (0.26 ± 0.03, 0.36 ± 0.02) was the strongest; there was significant difference among them (P < 0.01), whereas the expression of TGF-ß1 in group C (0.38 ± 0.06, 1.05 ± 0.19) is strongest followed by group A (0.34 ± 0.05, 0.95 ± 0.16) and B (0.33 ± 0.07, 0.90 ± 0.19). At every time point, the level of expression of BMP-2 and TGF-ß1 in gene therapy groups (groups A, B, and C) was remarkably higher than those in non-gene therapy groups(groups D and E). There were significant differences between gene therapy groups and non-gene therapy groups (P < 0.05 or P < 0.001). These results indicated that local gene transfection can up-regulate the expression of osteogenic mediators (BMP-2 and TGF-ß1), which may promote cell differentiation and proliferation and stimulate extracellular matrix synthesis and new bone formation in distraction gap.

Effect of gene transfecting at different times on mandibular distraction osteogenesis.

BACKGROUND: Recently, numerous research of gene therapy for mandibular distraction has been published. Based on previous study, the authors used New Zealand rabbits bilateral mandibular distraction model and used electroporation mediate gene therapy at different time, to explore the optimal time for gene therapy and obtain a better effect. METHODS: Forty-eight New-Zealand rabbits were used; after accomplished osteotomy and implant distraction devices on mandible bilaterly, the rabbits were randomly divided into 4 groups: groups A, B, and C were transfected recombinant plasmids pIRES-hBMP2-hVEGF165 via electroporation-mediated approach at latency period, distraction period, and consolidation period, respectively. Group D is a control group, only distracted without gene transfection. After 3 days of latency period, the device was activated at the rate of 1 mm per day for 10 days. Three rabbits of each group were sacrificed at 1, 2, 4, and 8 weeks of consolidation, respectively. The mandibles were harvested; the left was subject to radiograph examination for bone healing, and quantitative computed tomography detect for the bone mineral density (BMD) of newly formed bone in the distraction gap. The biomechanical properties of the new generation bone at the fourth and eighth weeks of consolidation of each group were detected using 3-point bending test. RESULTS: The BMD and the stiffness of newly formed bone increased with the pass of the consolidation time in each group. After 1 week of consolidation, there is no significant difference of BMD among groups A, B, and C. However, the BMD of groups A, B, and C is higher than that of group D. After 2, 4, and 8 weeks of consolidation, the BMD of group B is significantly higher than those of groups A, C, and D. The biomechanical parameters are also higher in group B than those of groups A, C, and D after 4 and 8 weeks of consolidation. CONCLUSIONS: It is better to transfect gene at distraction period than at other stages of DO; in this way, we can obtain more remarkable effect on new bone formation. It suggests that the distraction stage is the optimal time for gene therapy.

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.

[Effect of continuous elastic outside distraction on change of collagen content in female minipig's nipples and their supporting tissues].

OBJECTIVE: To observe the effect of continuous elastic outside distraction on the change of collagen content in female minipig's nipples and their supporting tissues, and to investigate the mechanism of continuous elastic outside distraction correcting inverted nipples. METHODS: Three 3-month-old female minipigs (weighing 18.5-22.0 kg), which had 12 nipples, were employed. Four nipples of each minipig were not treated as control group (n = 12), and the other nipples were continuously distracted with inverted nipple correction instruments as experimental group (n = 24). The nipple specimens were harvested at 2, 4, 8, and 12 weeks after distraction and HE staining was performed to observe the change of their tissue structure. And saturated picric acid sirius red staining was used to observe the distribution and content of collagen types I and III, image analysis software for quantitative analysis. RESULTS: The control group had normal structure of epidermis at all time points. In experimental group, the epidermis thickened; basal cells, fibroblasts, and capillary significantly proliferated along with the times; and the content and the density of collagen types I and III increased gradually. There were significant differences in collagen type I at 4, 8, and 12 weeks, and in collagen type III at 2, 4, 8, and 12 weeks between 2 groups (P < 0.01). There were significant differences in the ratio of collagen type I to III at 2 and 4 weeks between 2 groups (P < 0.05). CONCLUSION: Continuous elastic outside distraction can increase the quantity of collagen types I and III in the tissue, the thickness of the dermis, and the height of the nipple, which may be one of key mechanisms of correction the inverted nipple by continuous elastic outside distraction.

[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.

Effects of gene transfection at different time on the expression of bone morphogenetic protein-2 during mandibular distraction / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investgate the expression patterns of bone morphogenetic protein-2 (BMP-2) in the distraction area following plRES-hBMP2-VEGFI65 gene transfection at different time during mandibular distraction osteogenesis in a rabbit model.</p><p><b>METHODS</b>48 New-Zeland rabbits were employed to underwent osteotomy and distraction devices implantation on mandible bilaterly. After 3 days of latency period, the devices were activated at the rate of 0. 8 mm per day for 10 days. The rabbits were randomly divided into 4 groups (group A, B, C and D). Group A, B and C were transfected 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. Three rabbits in each group were sacrificed at 1, 2, 4 weeks of consolidation respectively. The mandibles were harvested for immunohistochemical staining detection of BMP-2 expression respectively, which were analyzed by CMIAS series multifunction color quantitative analysis of pathological image analysis system.</p><p><b>RESULTS</b>BMP-2 expression was found to be mainly located in the monocyte, fibroblast of the granulation tissue, the osteoblasts, osteocyte on the surface of new formed trabecular, and the connective tissues surrounding the new formed bone. The expression in group B was superior to other groups. Image analysis showed that, at the first week and second week of consolidation, the expression abosordbance A in group B (0. 58 ± 0. 03 and 0. 34 ± 0. 02) was relatively higher, when compared with that in group A (0. 42 ± 0. 02 and. 31 ±0.01), C(0.32 ±0.01 and 0.30 ±0.01)and D(0.27 ±0.01 and 0.23 ±0.02), showing a significant difference(P <0. 05). It was also relatively higher in group A(0. 42 ± 0. 02 and 0. 31 ± 0. 01) and C(0. 32 ± 0.01 and 0.30 ± 0.01), when compared with that in group D(0. 27 ±0.01 and 0.23 ± 0. 02), showing a significant difference( P < 0. 05) , but there was no significant difference ( P > 0. 05) between group A and group C. At the fourth week of consolidation, the expression decreased and there was no significant difference among group A, B, C, D.</p><p><b>CONCLUSIONS</b>The electroporation-mediated gene transfection which is transfected at the beginning of traction can promote BMP-2 expression effectively, stimulate bone marrix synthesis and induce proliferation and differentiation of fibroblasts, osteoblasts, endothelial cells, which further effectively promote the new bone formation. It suggests that the distraction stage is the optimal time for gene therapy.</p>