Experimental Study Of The Effect Of EGF Sustained Release Nano-particles On Skin Regeneration In Diabetic Rat Ulcer

Objective:1To investigate the preservation of biological activity of epidermal growth factor (EGF) which EGF-loaded PLGA nano-particles contains, and observe the toxic effects of PLGA on the fibroblast.2To observe whether EGF nano-particles can promote the regeneration of dermal collagen in diabetic ulcer through sustained-release of the EGF, whether it can promote the proliferation of newborn skin through inducing the proliferation of epidermal stem cells, and whether its role in wound repair is more better than free-EGF.Method:1EGF nano-particles was provided by Nanobiotechno-logy Institue of the College of Material Engineering Tianjin University.2Different concentrations of EGF nano-particles and hollow particles and10μg/L of different formulations of EGF (EGF nano-particles, EGF, hollow particles, control group) were added to L929cell line of mouse fibroblast, in order to evaluate the biological activity of EGF nano-particles and the toxicity of PLGA through optical density value of the MTT method.388Sprague-Dawley rats were applied injection of streptozotocin (STZ,45mg/kg) via tail vein to make diabetic model. One week later, diabetic model was established. All rats were created a2.542area full-thickness skin defects on the back, which were treated as wound hard healing model. Then88rats were randomly divided into EGF nano-particles group (group A, n=26), free-EGF group (group B, n=26), hollow particles group (group C, n=20), PBS control group (group D, n=16). All wounds received drug every day, At the3rd,7th,14th and21st day after trauma, the wounds were shoted by digital camera to calculating wound healing rate. At the same time, skin specimens from the wound edge were harvested partially for the observation of hydroxyproline (HYP) contents, and partially for the investigtion of epidermal thickness, histological characteristics and immunohistoch-emistry changes through the paraffin sections, HE staining, integrin β1and keratin19(K19) immunohistochemistry.4The statistical analysis was executed by SPSS16.0software.Results:1Experiment in vitro①Effects of different concentrations of EGF nano-particles on the proliferation of fibroblast:The OD value of different concentrations of EGF nano-particles is higher than the control group.10μg/L is optimal concentration. The variance of OD value between EGF nano-particles groups and control group has statistical significance (P<0.05).②Effects of10μg/L of different formulations of EGF on the proliferation of fibroblast:EGF nano-particles is the highest, and there’s significant difference between EGF nano-particles group and other groups (P<0.05).③ffects of different concentrations of hollow particles on the proliferation of fibroblast:There is no significant difference between all groups (P>0.05).2Experiment in vivo①Wound healing rate:Since the7th day, group A is always the highest, group B follows. There is significant difference between group A and other groups at the14th and21st day (P<0.05).②YP content:Group A is always the highest at the3rd,7th,14th day. At the7th day, there is significant difference between group B and C, group B and D (P<0.05). At the14th day, there is significant difference between group A and other groups (P<0.05).③Epidermis thickness: Group B is the highest at the3rd day. Since the7th day, group A is always the highest, group B follows. At the7th,14th and21st day, there is significant difference between group A and C, group A and D (P<0.05). At the14th day, there is significant difference between group B and C, group B and D (P<0.05).④Positive area ratio of integrin (31:Group B is the highest at the3rd day. Since the7th day, group A is always the highest. At the7th and21st day, there is significant difference between group A and other groups (P<0.05). At the14th day, there is significant difference between group B and C, group B and D (P<0.05).⑤ositive area ratio of K19:Group B is the highest at the3rd day. Since the7th day, group A is always the highest. At the14th day, there is statistically significant difference between group A and other groups (P<0.05).Conclusion:1The biological activity of EGF which EGF nano-particles contains is well preserved. The particulate carrier PLGA is safe for fibroblast.2Through a long period of controlled release of EGF, EGF nano-particles can promote regeneration of dermal collagen and induce the proliferation of epidermal stem cells to promote renascence of epidermis in diabetic ulcer. EGF nano-particles is superior to free-EGF in promoting healing of diabetic ulcer.