Combined Effects Of Artificial Dermis And Vascular Endothelial Growth Factor Concentration Gradient On Wound Healing In Diabetic Porcine Model

1.IntroductionWounds in patients with diabetes mellitus(DM)are one of the most prevalent impaired wounds in the world.Both intrinsic factors(peripheral neuropathy or vascular disease,and diabetes severity)and extrinsic(wound infection,callus formation,and excessive pressure to the wound site)effect these kinds of wounds do not follow an orderly and reliable progression of wound healing.Multiple pathogenic abnormalities interact with each other and develop vicious cycles of pathogenicity in diabetic wounds.Thereinto,insufficient blood perfusion and decreased growth factor with the addition of impaired angiogenesis complicates healing process.Vascular endothelial growth factor(VEGF)is one of the most important proangiogenic mediators.It acts as an endothelial cell mitogen,chemotactic agent,and inducer of vascular permeability.Wounds of streptozotocin induced diabetic mice demonstrate diminished synthesis of several growth factors,including VEGF.In diabetic patients,low levels of VEGF reduce vessel density,decrease granulation tissue formation,eventually delay wound closure.Sufficient VEGF levels are believed to stimulate wound healing through multiple activities including deposition of collagen,angiogenesis and re-epithelialization.VEGF alone or combine with other treatment modalities have proved to be an effective treatment for diabetic wounds.Artificial dermal such as Pelnac(?)(Gunze Corp.,Osaka,Japan)has been shown in humans and in animal models a great therapeutic potential in full-thickness skin wounds.It consists of two layers:a porcine tendon-derived atelocollagen sponge layer and silicon film.It can effectively induce the formation of new dermis-like granulation tissue,hereby improves the outcome of wound healing.In our previous vitro study,we have demonstrated that with a certain concentration of VEGF could promote both proliferation and migration of the vascular endothelial cells,and concentration gradient of VEGF improves the migration of vascular endothelial cells.Therefore,in this study,we conducted this study to investigate the effect of a combination therapy with AD and VEGF in wounded diabetic swine models.Take full advantage of the AD and VEGF on improve wound healing process.Utilize the AD as a sustained VEGF delivery system.Meanwhile,compare the efficacy between different constant concentration of VEGF and VEGF concentration gradient.2.Methods2.1.Preparation of artificial dermisIn order to determine the absorptivity and release efficiency of artificial dermis(AD,Pelnac(?)).It was cut into 1cm×1cm square fragment for use(Fig.1a).Reconstitute VEGF(Recombinant Rat VEGF,PEPROTECH(?),USA)in PBS to concentration of 200ng/ml.Completely soak AD piece in 2ml VEGF solution,incubation for 4h at 37?.Remove saturated AD piece and measure the residual volume(R)of the solution.The absorptivity of AD =(2ml-R)/lcm.Saturated AD piece was maintained in 5ml PBS.Take 100?l solution sample after 24h for ELISA to determine the concentration of VEGF,once daily for a week.Add 100?l PBS each time after taking a sample to maintain the total solution volume.Calculate the variation of concentration to determine release efficiency of AD.AD was cut to other two kinds of shapes for following surgical procedures.Type 1 was a circle with diameter of 18mm and prepare for soaking into different concentration of VEGF(Fig.lb).Type 2 were three concentric circles(H,M,L).H:diameter 6mm.M:internal diameter 6mm and external diameter 12mm.L:internal diameter 12mm and external diameter 18mm(Fig.1 c1,c2).Basis on previous study,Type 2 were soaked into three concentration of VEGF.High concentration(H):100ng/ml.Medium concentration(M):25ng/ml.Low concentration(L):5ng/ml.Thereby a concentration gradient of VEGF was established.2.2.Animal modelsSix female domestic swine weighing 20-25 kg were allowed to acclimate for one week.Established diabetic model by intravenous administration of streptozotocin(STZ)at a dose of 125mg/kg.Blood glucose levels were tested once daily using a monitoring meter(HEA-,Omron)during the entire experiment.Subcutaneous injected insulin to maintain blood glucose levels between 250 and 500mg/dl.Two of these pigs are used to evaluate healing rates.The other four are used to subsequent tissue harvesting.2.3.Surgical procedures1 week after stable diabetic induction,animals underwent dorsal wounding surgeries:following anesthesia induction with xylazine,clean and shaving dorsal skin.Under general anesthesia with propofol,surgical area was sterilized and draped.Total 36 paraspinal full-thickness excisional wounds were created(18mm diameter)on each pig(Fig.).These wounds were divided into six groups randomly:blank control group(C,PBS only,n=6),artificial dermis group(AD,AD only,n=6),low concentration group(AD+L,AD+5ng/ml VEGF,n=6),medium concentration group(AD+M,AD+25ng/ml VEGF,n=6),high concentration group(AD+H,AD+100ng/ml VEGF,n=6),concentration gradient group(AD+G,AD+VEGF concentration gradient,n=6)(Fig.2).All AD were secured along wound edges with sutures and covered with tie-over dressings.Animals were bandaged up and separately kept in cages.Thereafter,correspond concentration of VEGF was injected into the collagen sponge layer of the AD once a week.We have been carried out with Chinese Ministry of Public Health(CMPH)guidelines for the care and use of animals.All animals survived in this experiment.2.4.Tissue harvestingTissue samples were harvested from all wounds of four pigs on day 7,14,21 postsurgery under general anesthesia(Fig.3).After the superficial silicone film layer of AD was removed.The incision was made range from the center to edge of the wound,including the underlying subcutaneous tissue.Harvested tissue specimens were fixed in buffered formaldehyde for 48h and embedded in paraffin for histological and immunohistochemical analysis.2.5.Measurement of wound healing ratesWound healing rates were calculated in 7,14,21 days postsurgery.Photographs of each wound were taken by digital camera(DMC-LX2,Panasonic)on each time point,and analyzed by image analysis software(Image J,National Institutes of Health,Bethesda,Md.).Healing rate =[(original size-non-healing area)/original area]×100%.2.6.Histological and immunohistochemical analysisParaffin-embedded tissues were cut into 5?m thick sections,and stained with hematoxylin and eosin(H&E).Microscopic imaging of stained sections was performed.The tissue samples stained with H&E were examined in terms of general morphology.Granulation tissue taken from the middle area of the wounds was analyzed under 100 × magnification.Serial samples were immunolabeled with anti-von Willebrand factor antibody(Abcam,USA)to detect the neovascularization level of the granulation tissues.The number of blood vessels was counted from 6 randomly selected fields in each section by two independent observers under 400 × magnification.Samples from day 21 were immunolabeled with anti-VEGF polyclonal antibody(BIOSS,Beijing,China).Each section was analyzed in 6 randomly areas under 400×magnification.VEGF protein specific staining area was quantified using Image J.Percentage of VEGF protein specific staining area=VEGF protein positive area/total tissue area× 100%.Average value was calculated for each section.2.7.Statistical analysisAll data were presented as mean ± standard deviation(SD).Statistics were calculated with SPSS statistical software(Version 23.0,IBM(?),USA).One-way analysis of variance(ANOVA)followed by Tukey's post hoc test was conducted for multiple comparisons.Values of*P<0.05 were indicative of statistically significant differences.3.Results3.1.Absorptivity and release efficiency of artificial dermisThe absorptivity of AD was 0.3 ml/cm2.Saturated AD continuously released VEGF at high level in first four days(day 1:24.96±2.38 ng/ml,day 2:22.19±1.27 ng/ml,day 3:20.20±1.62 ng/ml,day 4:10.30±1.79 ng/ml).From day 5 the release efficiency significantly decreased(day 5:5.24±0.93 ng/ml,day 6:1.80±0.34 ng/ml),and approached 0 in day 7(Fig.4).3.2.Diabetic swine models were induced successfullyA significant hypoglycemia occurred in first 24 h following intravenous injection of STZ.Animals were fed with high sucrose diet to prevent death.After 24 h,animals maintained a permanent hyperglycemic condition(blood glucose between 300 and 500 mg/dl).Low dose insulin was subcutaneously injected only when blood glucose level is above 500 mg/dl.Hereto,the diabetic swine models were successfully established.3.3.Healing ratePhotographs of each wound were taken and analyzed(Fig.5 I).On day 7 after the operation,the healing rates had no significant difference in all groups(P>0.05).The healing rates of group AD+M,group AD+H and AD+G on day 14 and group AD+H and AD+G on day 21 are significantly higher than the control group(P<0.05).But there was no significant difference between these three groups(P>0.05)(Fig.5?).No infections were developed in all wounds.These results indicated that the artificial dermis combined with VEGF achieved a satisfactory effect on promoting wound healing.3.4.Analysis of Granulation Tissue ThicknessGranulation tissue taken from the middle area of the wounds was analyzed(Fig.6 ?).The formation of granulation tissue was much faster in all treated groups on day 7 after operation compared with control group.A thick granulation tissue response was found in group AD+H and AD+G,with a more than 2-fold increase compared with control group.There was also no significant difference between these two groups(P>0.05).The thickness of granulation in group AD+H and AD+G were significantly different from control group through all the experiments(P<0.05)(Fig.6 ?).But the formation of granulation tissue slowed with the epithelization.3.5.AngiogenesisAfter hematoxylineosin staining,the neovascularization level of granulation tissues was counted(Fig.7 ?).The blood vessel density in group AD+H(21.7±2.0 per field)and AD+G(19.6±1.6 per field)was significantly higher than the control group(4.2±1.0 per field)on day 7(P<0.05).AD group showed a lower number of blood vessels(5.0±1.1 per field).On day 14 and day 21,all groups' neovascularization level continuously increased.Group AD+H and AD+G induced angiogenesis most obviously in the experiments(Fig.7 ?).VEGF increased the blood vessel formation in diabetic wounds.Thus,improved the blood perfusion and metabolism in wound granulation tissue to promote wound healing process.3.6.Expression of VEGF in woundsThe protein level VEGF of samples from day 21 were detected(Fig.8 ?).All treated groups showed a larger percentage of VEGF protein specific staining area,especially group AD+H and AD+G with a 3-fold and a 2-flod content compared with control group respectively(P<0.05).AD shows a relatively stable slow-release feature and sustainability(Fig.8 II).Much more VEGF was retained at the wound granulation zone.4.DiscussionRecent date from the World Health Organization(WHO)shows that an estimated 422 million adults were living with DM globally in 2014,compared to 108 million in 1980.Prevalence of DM has nearly doubled since 1980,rising from 4.7%to 8.5%in the worldwide adult population.With the dramatic increase of incidence,more patients with DM have the potential to develop a diabetic wound.Especially in lower limb,such as diabetic foot ulcer(DFU)is an autonomic neuropathy and microvascular disease,which can lead to infection,gangrene,and eventual amputation.Once the ulceration develops,the main characteristic of DFUs is the inability to self-repair timely and orderly.For an efficient diabetic wound healing,a proper angiogenesis and thereby enhance blood perfusion hold tremendous potential to address the shortage of current diabetic wound care.Long-term followed-up studies of the use of Pelnac(?)in clinical treatment of full-thickness skin defects showed good results.In this study,we observed that its inner sponge layer not only serves as a scaffold for vascularization and colonization by dermal fibroblasts,and that exhibits definite capacity of drug absorption.AD has a relatively stable slow-release feature.It released a high level VEGF in first four days and continuously released until day 7(Fig.4).We injected correspond concentration of VEGF into the collagen sponge on day 7 and day 14 after operation.Hereby maintain the VEGF levels of the wound bed.In our previous study,we have demonstrated that with a certain concentration of VEGF could promote both proliferation and migration of the vascular endothelial cells,and concentration gradient of VEGF improves the migration of vascular endothelial cells in vitro.In this study,we have found that AD with or without VEGF increased granulation tissue formation after operation.AD combine with high concentration of VEGF or concentration gradient of VEGF had the most obvious effect.Along with the re-epithelialization,the formation of granulation tissue slowed in all experiment groups.AD's structural characteristics and adequate VEGF levels can contribute to accelerate the granulation tissue formation,eventually promote the wound healing process.Neovascularization is a committed step in the complicated wound healing.VEGF has been shown to play an important in neovascularization.According to the absorptivity and release efficiency of AD,we used it as a sustained VEGF delivery system.Result showed that,all treated groups still expressed a sufficient level of VEGF at the end of this experiment,especially group AD+H and AD+G.AD effectively maintained the VEGF concentration levels of the wound.All group using VEGF increased the blood vessel density through the whole experiments particularly AD+H and AD+G groups(Fig.7 ?).Demonstrate that sufficient VEGF levels can stimulate wound healing through angiogenesis and re-epithelialization.The healing rates had no significant difference in all groups on day 7.Because of AD's structural and slowly release feature,granulation tissue and blood vessel density gradually increased.The healing rates of group AD+M,group AD+H and AD+G on day 14 and group AD+H and AD+G on day 21 are significantly higher than the control group.All treated groups' healing rates were higher than the control group at the end of the experiments.Artificial dermis combined with VEGF achieved a satisfactory effect on promoting wound healing.AD combine with sufficient concentration of VEGF and concentration gradient of VEGF had similar effect on granulation tissue formation,neovascularization.But,concentration gradient of VEGF induced migration and proliferation of the vascular endothelial cells with less additive dosage of VEGF.This combination may be more economic and efficacious to treat diabetic wound.5.ConclusionsPelnac(?)combine with sufficient concentration of VEGF had the significantly capability to improve the diabetic wound healing.Thereinto,the combination of Pelnac(?)and certain concentration gradient of VEGF used less dosage but achieved similar effect.This combination could provide an appropriate angiogenesis,increase epithelization,eventually accelerate the direct healing of diabetic wounds or make good preparation for secondary skin graft.This idea could be a novelty method for diabetic wound treatment.