To enhance the osteogenic activity of BMP, combination BMP2 and BMP7 gene transfer was performed. This approach led to a significant increase in osteoblastic differentiation of mesenchymal precursors compared with single BMP gene transfer in vitro. When tested in 78 rats, combination gene transfer enhanced mechanically stable spine fusion and bone formation rate versus single BMP gene transfer.



Introduction: Although clinical bone morphogenetic protein (BMP) therapy is effective, required doses are very high. Previous studies have suggested that the co-expression of two different BMP genes can result in the production of heterodimeric BMPs that may be more potent than homodimers. In this study, combined BMP2 and BMP7 gene transfer was performed to test whether this approach improves osteoblastic differentiation and bone formation compared with single BMP gene transfer.



Materials and Methods: A producer cell (A549) was co-transfected with adenovirus vectors encoding BMP2 (AdBMP2) and BMP7 (AdBMP7) or, as controls, each vector alone, AdNull (with no transgene) or no virus. Supernatants were compared for their ability to stimulate osteoblastic differentiation of C2C12 myoblasts and MC3T3-E1 pre-osteoblasts. In a rat posterolateral spine fusion model, co-administration of AdBMP2 and AdBMP7 was compared with treatment with each vector alone, AdNull or no virus in 78 rats. The spines were assessed 8 weeks after surgery for radiographic and mechanical fusion, bone formation, and mineralization.



Results: BMP2 and BMP7 were co-precipitated from supernatants of cells co-transfected with AdBMP2 and AdBMP7, indicating the presence of BMP2/7 heterodimer. Supernatants of co-transfected cells containing relatively low doses (7–140 ng/ml) of BMPs induced osteocalcin expression and alkaline phosphatase activity in both C2C12 and MC3T3-E1 cells, that were up to 6- and 40-fold higher, respectively, than levels induced by maximal doses (200–1000 ng/ml) of either BMP2 or BMP7 alone. In the spine fusion model, co-administration of AdBMP2 and AdBMP7 resulted in a significantly greater number of mechanically stable fusions and also 2-fold higher mineralization rate and bone volume in the fusion mass versus single BMP gene transfer (p < 0.02, all comparisons).



Conclusion: Combined BMP2 and BMP7 gene transfer is significantly more effective in inducing osteoblastic differentiation and spine fusion than individual BMP gene transfer.

Combined bone morphogenetic protein-2 and -7 gene transfer enhances osteoblastic differentiation and spine fusion in a rodent model.

Nanomedicines and gene therapy for the delivery of growth factors to improve perfusion and oxygenation in wound healing.

Background

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. VEGF A also plays an important role in wound healing of the skin by promoting angiogenesis and by stimulating blood vessel growth. Therefore we tested the hypothesis that flap survival could be increased by the preoperative injection of AdVEGF165.



Methods

We studied the effect of AdVEGF165 in an overdimensioned ischemic random-pattern-flap model in the rat (n = 50) with a length-to-width ratio of 4 : 1. VEGF cDNA was administered in two concentrations of 5 × 108 plaque-forming units (pfU) and 1 × 109 pfU using a recombinant adenoviral vector. Recombinant virus was injected subdermally 7, 3 or 0 days prior to flap harvest for the lower concentration and 7 days prior for the higher concentration. Flap survival and necrosis were observed at day 7, the day the animals were sacrificed.



Results

Adenoviral gene transfer with VEGF165 3 and 7 days before flap harvest showed a significantly increased flap survival of 50% together with a significantly reduced necrosis (p < 0.01). Injection using a titer of 1 × 109 pfU 7 days prior to surgery increased flap survival even more, though failing to reach statistical significance compared to the lower concentration. VEGF protein concentration in the injected skin was significantly higher than in controls (p < 0.01). Flap perfusion was increased as well, demonstrated by indocyanine green (ICG) fluoroscopy (p < 0.001).



Conclusions

Our results confirm the important role of VEGF165 on angiogenesis in ischemic flaps. Indeed by injecting VEGF165 at 3 to 7 days preoperatively in a concentration of 1 × 109 pfU our data show that length-to-width ratio for random-pattern-flaps could be increased from 2 : 1 to 3 : 1 and therefore may allow a wider range of applications of this simple flap technique. Copyright © 2004 John Wiley & Sons, Ltd.

AdVEGF165 gene transfer increases survival in overdimensioned skin flaps.

A major challenge in reconstructive surgery is flap ischemia, which might benefit from induction of therapeutic angiogenesis. Here we demonstrate the effect of an adeno-associated virus (AAV) vector delivering vascular endothelial growth factor (VEGF)165 in two widely recognized in vivo flap models. For the epigastric flap model, animals were injected subcutaneously with 1.5 × 1011 particles of AAV-VEGF at day 0, 7, or 14 before flap dissection. In the transverse rectus abdominis musculocutaneous flap model, AAV-VEGF was injected intramuscularly. The delivery of AAV-VEGF significantly improved flap survival in both models, reducing necrosis in all treatment groups compared to controls. The most notable results were obtained by administering the vector 14 days before flap dissection. In the transverse rectus abdominis musculocutaneous flap model, AAV-VEGF reduced the necrotic area by >50% at 1 week after surgery, with a highly significant improvement in the healing process throughout the following 2 weeks. The therapeutic effect of AAV-VEGF on flap survival was confirmed by histological evidence of neoangiogenesis in the formation of large numbers of CD31-positive capillaries and α-smooth muscle actin-positive arteriolae, particularly evident at the border between viable and necrotic tissue. These results underscore the efficacy of VEGF-induced neovascularization for the prevention of tissue ischemia and the improvement of flap survival in reconstructive surgery.

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Improved survival of ischemic cutaneous and musculocutaneous flaps after vascular endothelial growth factor gene transfer using adeno-associated virus vectors

Objective: To explore the effects of adenovirus vector-mediated gene transfer of CTLA4Ig fusion protein on CD4+CD25+ T cells in experimental autoimmune myocarditis (EAM).Methods: EAM was induced by porcine cardiac myosin as previously described. Adenovirus vector-mediated CTLA4Ig gene was administrated intravenously in EAM rats on days 1, 4 and 7, with EGFP as control. On day 21, myocardium histopathology was examined and CD4+CD25+ T cells were isolated. Proliferation and suppression assays were used to evaluate the suppressive capacity of CD4+CD25+ T cells in vitro. Relative mRNA level of Foxp3 and TGF-β was determined by quantitative real-time RT-PCR; expression of CTLA-4, B7-1 and B7-2 protein was compared with Western blot in CD4+CD25+ Tregs.Results: Severe inflammatory lesions were observed in the hearts of EGFP-treated EAM rats and the untreated ones, while Ad–CMV–CTLA4Ig alleviated the myocarditis histologically. Adenovirus vector-mediated CTLA4Ig gene transfer up-regulated the proportion of CD4+CD...

Upregulation of CD4+CD25+ T lymphocyte by adenovirus-mediated gene transfer of CTLA4Ig fusion protein in experimental autoimmune myocarditis.

Objective: To evaluate the effect of local application of vascular endothelial growth factor ( VEGF ) via adenovirus-mediated gene transfer on survival of full thickness flaps selected randomly in rats.
Methods: Thirty Sprague-Dawley rats weighing 480-520 g were used in this study. A dorsal flap (8 cm × 2 cm) in full thickness with the pedicle located at the level of the iliac crest was designed. Then the rats received 1 012 pfu replication-deficient recombinant adenovirus carrying VEGF ( AdCMV-VEGF group, n = 10 ), 1012 pfu recombinant β-galactosidase adenovirus ( AdCMV-Gal group, n = 10) and 1 ml saline (saline group, n = 10), respectively, in the distal two thirds of the proposed flap by means of subdermal injection at 8 different locations. Three days after treatment, the flaps were elevated as originally designed and sutured back in situ. The survival rate of the flaps was evaluated on day 7 after operation.
Results: The survival rate of the flaps in the AdCMV-VEGF group increased significantly as compared with those of the AdCMV-Gal group (P 0.01) and the saline group ( P 0.01). Immunohistochemical staining showed that VEGF was expressed in the survival flaps injected with AdCMV-VEGF. Histological analysis showed that more granulation tissues and angiogenesis were observed in the AdCMV-VEGF group than those in the AdCMV-Gal and the saline groups.
Conclusions: Local application of adenovims-mediated VEGF165 cDNA may efficiently improve the survival of ischemic skin flaps.

Effect of adenovirus-mediated gene transfection of vascular endothelial growth factor on survival of random flaps in rats

Objective\ The purpose of this study was to determine the effects of local delivery of vascular endothelial growth factor(VEGF)transferred with adenovirus\|mediated gene on the survival of ischemic random skin flap in rats. Methods\ The animals were divided into three groups randomly( n =10).A 2 cm×8 cm dorsal skin flap was designed with the pedicle at the level of the iliac crest.In group A (AdCMV\|VEGF),each animal received 10 12 PFU replication\|deficient recombinant adenovirus(AdCMV\|VEGF)in the distal two\|thirds of the proposed flap by means of the subdermal injection at ten different locations.In group B (AdCMV\|Gal),each received 10 12 PFU AdCMV\|Gal.In Group C (Saline),each received 1 ml saline.Three days after the treatment,the flap was elevated as planed way and re\|sutured back to its donor site.All the animals were evaluated 7 days after the operation. Results\ The mean percentage of surviving flap area was(85.91±2.54)% in group A,(59.56±1 18)% in group B,and (61 48±1 09)% in group C.There was a significant increase in the percentage of the survival area in the flaps of the group A,compared with the group B and group C(Group B vs.Group A, P 0 01;Group C vs.Group A, P 0 01,Group B vs.Group C, P 05).Hybridization in the situ,the immunohistochemical stain showed that the VEGF was expressed in the survival tissue of the flap treated with the AdCMV\|VEGF,but it was not found in the control groups.Histological analysis demonstrated qualitatively greater amount of granulation tissue and angiogenesis was found in the group treated with the AdCMV\|VEGF than the controls. Conclusions\ The results may indicate that Ad vector carrying VEGF cDNA could be useful in enhancing the survival of the skin flap due to the effect of the local delivery of the VEGF.\;

[Effects of hVEGF cDNA on random skin flap via a replication-deficient adenovirus vector].

OBJECTIVE To properly evaluate the benefits of in utero repair of cleft lip, an animal model of cleft lip was developed and the fetal scarless wound healing was observed in SD rat fetus. METHODS Through a maternal celiotomy and hysterotomy, twenty excisional cleft was created in the upper lip of rat fetus. Another group of 20 rat fetus were given full-thickness incisional wounds in the upper lips. The fetus with cleft lip were returned into utero for further development till birth.Wound specimens from the incisional wounds in the other group were removed successively 12, 24, 48, 72h postwounding for histological examination. RESULTS The fetus with excisional wound demonstrated a complete cleft lip after birth. In contrast, the wound in the fetus with incisional wounds regenerated with normal architecture histologically, indistinguishable from the surrounding normal skin. CONCLUSION The results of this preliminary study indicates that the fetal rat cleft lip is technically feasible with an excellent survival rate. Healing occurs without scar formation. This model will be used to document facial growth following in utero repair of a cleft lip.

Fa-cheng Li

Papers

Effect of adenovirus-mediated gene transfection of vascular endothelial growth factor on survival of random flaps in rats

[Effects of hVEGF cDNA on random skin flap via a replication-deficient adenovirus vector].

[Intra-uterine model of cleft lip and scarless wound healing of fetal rats].