Promotion Of Diabetic Wound Healing By Collagen-binding Vascular Endothelial Growth Factor In Diabetic Rat Model

Background and purpose:In recent years, diabetic wounds have become a global health concern with the increase in incidence of diabetes. Diabetic wound is a kind of chronic and refractory ulcer. It generally due to microcirculatory disturbances and reduced levels of endogenous growth factors. Among the angiogenic growth factors,vascular endothelial growth factor (VEGF) plays a pivotalrole in angiogenesis. VEGF can increase the capillary density in diabetic wounds, thereby improving the blood perfusion and metabolism in wound tissue, and then promoting wound healing. VEGF is the main regulating factor in the revascularization and permeability of the wound site, and participates formation of the granulation tissue. In diabetic wounds, the relatively low level of VEGF in the local wound is an important reason.VEGF effectively improves the proliferation and migration of endothelial cells in vitro and triggers a series of events that induce new blood vessel growth in vivo. It has been reported that VEGF can improve angiogenesis and accelerate the blood supply in dibetic wounds, and takes the effect in dose-dependent manner. Although many reports suggest that the effects of VEGF are beneficial, the safety of it in therapy is still a major concern. Low doses of VEGF may lead to retarded angiogenesis but high doses of VEGF may lead to the formation of hemangioma,and the diffusion of VEGF may cause undesirable side effects. Moreover, in practical applications, there are still two problems:One is that the half-life of VEGF is too short and the metabolic degradation is too fast to be sustained at the target site for enough time. The other is that VEGF is susceptible to metabolic loss by the inflammatory exudate's scouring effect, which can not create an effective local concentration of VEGF. Studies have shown that both the capillary density and the content of collagen I are all low in diabetic ulcer wound. This two points lead the ulcer healing delay and the poor healing quality. Therefore, the identification of a specific target of VEGF in injured local wounds may offer an optimal therapy for dibetic wounds. In this study, we used collagen-binding VEGF in a diabetic rat model to investigate the effects of this new method.Methods:MaterialsWe produced the fusion protein and collagen scaffold (CBD-VEGF) consisting of VEGF and a collagen-binding domain (CBD), which allowed VEGF to bind to collagen.Methods of Part 1:1.72 male Sprague-Dawley (SD) rats were randomly divided into 3 groups, baseline of blood glucose and body mass have been measured before operation.2. Group A:PBS menstruum group (n=24), Group B:NAT-VEGF group (n=24), Group C: CBD-VEGF group (n=24).3. The dissolved drugs were evenly sprayed onto the traumatic surface using syringe, and the trauma sites were covered with sterile gauze 5 minutes later.4. All animals were executed by the method of C-spine luxation on the 3rd,7th,14th, and 21 st day postsurgery.5. Photographs were taken on each time point by digital camera, and then were analyzed by computer image analysis system to calculate the healing rates.6. On day 3,7,14,21 after surgery, rats were sacrificed by the method of C-spine luxation and the wound granulation tissues were harvested in each time point postsurgery and take histological analysis.7. The serum was collected from peripheralblood 3 and 6 hours after administration on the 7th day of the experiment and measured with a human VEGF ELISA kit. Methods of Part 2:1.96 male Sprague-Dawley (SD) rats were randomly divided into 4 groups, baseline of blood glucose and body mass have been measured before operation.2. All collagen scaffold received scanning electron microscopy test.3. Group A:blank control (n=24), Group B:collagen scaffold loaded with PBS (n=24), Group C:collagen scaffold loaded with NAT-VEGF (n=24), Group D:collagen scaffold loaded with CBD-VEGF (n=24). The wounds of Group A were covered with gauze and the wounds of Group B,C,D were grafted by corresponding collagen scaffolds respectively.4. All animals were executed by the method of C-spine luxation on the 3rd,7th,14th, and 21 st day postsurgery.5. Photographs were taken on each time point by digital camera, and then were analyzed by computer image analysis system to calculate the healing rates.6. On day 3,7,14,21 after surgery, rats were sacrificed by the method of C-spine luxation and the wound granulation tissues were harvested in each time point postsurgery and take histological analysis.7. The serum was collected from peripheral blood 3d and 7d after administration and measured with a human VEGF ELIS A kit.Results:Results of Part 1:1. After 7 days treatment, group C showed the highest rate of healing (7d 70.3%±5.4,14d 91.9%±1.8,21d 96.5%±1.6) in three groups and showed statistically significant difference(P<0.05, P<0.01).2. Histological analysis:2.1 As show in HE staining,7 days after the experiment, group C had more blood vessels in the granulation tissue.2.2 As show in immunohistochemisty staining,7 days after the experiment, compared with group A (3.67±1.63) and group B (7.50±2.07) respectively, group C (15.33±1.63) had higher blood vessel density in the wound granulation tissue.2.3 As show in immunohistochemisty staining, after 7 days, the level of CBD-VEGF was significantly higher than that of NAT-VEGF in the diabetic wound granulation zone.3. Serum VEGF levels were higher than both control and serum CBD-VEGF levels 3 hours after administration.6 hours after administration, serum VEGF levels were also higher than control and serum CBD-VEGF (B:13.08±4.60pmol/ml 3h,4.25±2.62 pmol/ml 6h; C: 0.64±0.44 pmol/ml 3h,0.60±0.45 pmol/ml 6h; p<0.01 3h; p<0.05 6h).Results of Part 2:1. Scanning electron microscopy shows that the scaffold has porous stereoscopic structure and the pore is 20 to 200μm in size.2. After 7 days treatment, group D showed the highest rate of healing (7d 71.4%±3.4,14d 90.7%±1.2,21d 98.3%±1.4) in four groups and showed statistically significant difference(P<0.05, P<0.01).3. Histological analysis:3.1 As show in HE staining,7 days after the experiment, group D had more cellularization and vascularization in the granulation tissue.3.2 As show in immunohistochemisty staining,7 days after the experiment, compared with group B (5.1±1.37) and group C (10.4±1.43) respectively, group D (18.7±2.01) had higher blood vessel density in the wound granulation tissue.3.3 As show in immunohistochemisty staining, after 7 days, the level of CBD-VEGF was significantly higher than that of NAT-VEGF in the diabetic wound granulation zone.4. Serum NAT-VEGF levels were highest in all groups on the day 3 postoperatively (A: 0.17±0.09pmol/ml, B:0.18±0.09 pmol/ml; C:8.30±2.09 pmol/ml, D:0.25±0.06 pmol/ml; p<0.01,3d) Conclusions:1. CBD-VEGF induced a greater amount of blood vessels formation and cell migration to accelerate the tissue regeneration in the diabetic wound repair.2. Compared with NAT-VEGF, CBD-VEGF have higher binding ability in the collagen existing in diabetic wounds site, and thus improved its therapeutic efficiency.3. CBD-VEGF can promote diabetic wound healing in rat model, which could potentially provide a better therapeutic option for diabetic wounds.