Administration Of Different Growth Factors Promote Secondary Necrotic Flap Wound Healing In An Extended Animal Model

BackgroundIn reconstructive surgery, flap technology is supposed to be one of the most important skill for plastic and reconstruction surgery. Flap is normally used to cover large open wounds, ulcers, tumor excisions, and large congenital abnormalities. Consequently, flap surgery is one of the most important techniques in reconstructive surgery. Transferred tissue often receives insufficient blood supply, especially in the periphery. In both clinical and laboratory studies, flap necrosis is difficult to avoid absolutely. Once happened, the dead tissue should be removed completely and will bring about serious complications. However, if the recipient site is inappropriate for a new graft or flap, what we can do is only waiting.A great deal of research has been conducted attempting to improve the flap survival rate pharmacologically. Furthermore, no improved intervention for treatment of necrotic wounds has been developed. Although there has been a lot of research on necrosis preservation, there has been little research on the promotion of necrotic wound healing. The currently available pharmacological agents are sympatholytics and vasodilators, some of which will induce a conflict, or are incompatible with flap necrosis preservation. Although flap survival rate is generally high, necrosis is inevitable in many experimental and clinical cases. Little research is available on the treatment of flap necrotic wound. Keratinocyte growth factor, vascular endothelial growth factor (VEGF), and Epidermal growth factor (EGF) are the three hottest growth factors in contemporary research. Quite a lot of papers have been published to proof the special effect on epidermal cells, fibroblast, and vascular endothelial cells. KGF has been found to protect skin, stomach, intestine, lung, bladder, corneal epithelium and oral mucosa from damage, and can reduce some adverse side effects of chemotherapy. KGF specifically targets epithelial cells, and thus has no effects on fibroblasts and other cells. EGF is the most common clinical used growth factor to stimulate epithelial cells to grow and has got the recognition from patients and doctors. VEGF is the best growth factor for promoting angiogenesis and will greatly improve the flap survival rate and prevent flap from necrosis.The gene therapy methods are vast potential for flap necrosis prevention and treatment. Nowadays, most of the gene therapies use the growth factors to conquer the field of gene transfection. Many alternative techniques have been developed to inject therapeutic gene in to objective site and organ. Adenovirus can achieve more efficient transduction and expression than non-virus vectors. After adenovirus enters a cell, it does not integrate with the host cell genome, thus indicating a safety feature for adenovirus usage and the first choice for gene transmission.Adenovirus-mediated KGF (Ad-KGF) gene therapy and co-administration of EGF-VEGF may provide a clinical tool for the promotion of secondary flap necrotic wound healing We set out to validate an advanced animal model of necrosis, and test the hypothesis that local injection of Ad-KG or EGF-VEGF can accelerate healing of necrotic flap wound.Methods1. The plasmid was digested by endonuclease Pac Ⅰ; the product was confirmed by agarose gel electrophoresis and DNA sequencing. Ad-KGF and Ad were generated by plasmid-Ad-KGF and plasmid-Ad digested product in293cells that were used to replicate deficient adenovirus. Ad-KGF and control adenoviruses (Ad) were liberated by three freeze-thaw cycles. Adenoviral titers were tested with a plaque-forming assay on293cells and diluted to1×109/ml. Virus stocks were diluted in PBS and stored at-80℃. The plasmid sequence was confirmed by gene sequencing.2. Ad-KGF gene expression was analyzed by real time PCR. PA317,293cells infected with Ad (293+Ad), and293cells infected with Ad-KGF (293+Ad-KGF), were collected with TRIzol(?) Reagen. cDNA species were synthesized using Promega M-MLV. Real-time PCR was performed using SYBR(?) Premix Ex TaqTM. KGF concentration levels in Ad-KGF infected293cells were determined48hours after infection by ELISA using a human KGF ELISA kit. Transwell plates chamber were used.293+Ad,293+Ad-KGF,293+KGF, or KGF were added to the lower chamber in600μL DMEM.24hours later,1×105sorted HaCaT, HUVEC, and PA317were seeded onto the upper side. The cells were incubated for17hours at37℃. Migrating cells on the bottom of the membrane were stained with0.1%crystal violet. Penetrating cells were stained and counted under a microscope.3. Rat’s back artery and vein distribution were determined. A dorsal skin flap of8cm×2cm was designed on the back of the rat. Arteries that could fertilize the flap were disconnected. Flaps were raised on the dorsum, returned and sutured to the bed with3-0silk sutures. Body weight and pictures were recorded at the same time every other day until the necrosis healed. The first drug injection took place on day5when necrosis started. The rats were sacrificed15,25, and45days postoperatively. The samples from the necrosis area were selected and histochemical staining and image analysis were carried out.4.60rats were randomly divided into four groups according to the treatment they received:1ml PBS,1ml PBS+dexamethasone (DXM),1ml PBS+Ad-KGF (1×109units/ml)+DXM, or lml PBS+Ad (1×109units/ml)+DXM. The same solutions were injected on day10,20, and30after surgery. The flaps were photographed using a digital camera on day5,10,15,25, and35. No additional treatments were performed before the rat was sacrificed for the sake of avoiding unbalanced and incomparable treatments. The rats were sacrificed15,25, and35days postoperatively. The samples from the necrosis area were selected and histochemical staining and image analysis were carried out. Real-time PCR and blood serum ELISA were performed to determine whether the Ad-KGF was expressed in vivo.5. The migration ability of the keratinocyte cell line HaCaT treated with, EGF, EGF+VEGF or PA317were tested first though transwell cell migration chambers.40rats were randomly divided into four groups according to the treatment they received:1ml PBS,1ml PBS+EGF (150mg/kg),1ml PBS+VEGF(10μg), or1ml PBS+EGF (150mg/kg)+VEGF (10μg). Five days after the operation, drugs were injected subdermally around the edge of the flaps and wounds. The same solutions were injected every two days postoperatively. The flaps were photographed using a digital camera on day5,10,15,20, and25. Body weight was recorded every other day until the necrosis healed. The samples from the necrosis area were selected and histochemical staining and image analysis were carried out. Blood serum ELISA was performed to determine whether the Ad-KGF was expressed in vivo.Results1. The digested product showed the plasmid was the right adenoviral plasmid. The sequence of Ad-KGF was consistent with the previously published KGF data. The transcription initial site was185bp, ATG; the transcription end site was678bp, TAA. There was no gene mutation in the plasmid.24hours after the virus transfected into293cells, the cells turned round, and started to express GFP;96hours later, all293cells showed a cytopathic effect and expressed strong GFP signal. The Ad-KGF were amplified in293cells and collected. The final Ad-KGF titer we got were2×1011PFU/ml,and3×1012PFU/ml for Ad-GFP(Ad).2. Semi-quantitative PCR confirmed that PA317and293cells+Ad-KGF highly expressed KGF mRNA. The level of KGF expression varied with Ad-KGF titer. The migration ability of the keratinocyte cell line HaCaT treated with Ad-KGF or KGF significantly increased in comparison to293cells and293cells+Ad KGF did not stimulate proliferation of PA317or HUVEC.3. There were more than forty arteries and seven veins fertilize the back of the rat. It is clear that necrosis peaked on day9. From days9to47, the necrosis decreased gradually and was almost cleared by day47. Throughout the47days, rats in the flap necrotic wound group had a relatively stable weight profile compared with normal rats. However, the two groups’weight has no detectable statistical significance. The histochemical staining showed that, at the edge of the wound the skin had epithelial hyperplasia with little fiber at15days postoperatively.35days after the surgery, the epithelial hyperplasia seems compromised.4. The Ad-KGF group had the smallest necrotic area and fastest wound healing speed from day10to day35. Thirty-five days after the operation, the necrotic area was relatively small in the Ad-KGF group. Epidermal sections were stained with Masson Trichrome. The epidermis showed greater active proliferation in the Ad-KGF group compared with the other three groups10days postoperatively. After35days, the Ad-KGF group did not have any unhealed areas, while the others still had necrotic lesions. All groups had similar collagen fibers in the healing area. The Ad-KGF group had thickest epidermis on day15and25. Immunohistochemical staining shows that in the Ad-KGF group, the epidermis and subcutaneous tissues could express KGF. The KGF level in the blood serum increased dramatically10and15days after the operation. KGF mRNA levels in vivo were measured in the four different groups. The Ad-KGF group had the highest expression level when compared with the other three groups.5. The migration ability of the fibroblast cell line PA317treated with EGF or EGF+VEGF did not significantly increased. The migration ability of the human umbilical vein endothelial cell line HUVEC treated with EGF, VEGF, EGF+VEGF or PA317significantly increased in comparison to blank control. The EGF, VEGF, EGF-VEGF groups had faster wound healing speed from day10to day25. The EGF-VEGF group had the fastest healing speed. On the25th day, the necrosis was almost healed in EGF-VEGF group. Throughout the25days, rats from both groups had relatively stable weight profile. EGF-VEGF group showed greater active proliferation compared with the PBS control groups15days postoperatively. After25days, EGF and EGF-VEGF groups had more collagen fibers in the healing area than the control group. EGF and EGF-VEGF groups had thicker epidermis on day15and25. Immunohistochemical staining shows that in the EGF, VEGF, and EGF-VEGF groups had better vascularization. The EGF-VEGF group had the most vessels number in the necrotic wound area. Both the EGF and VEGF level did not show a significant difference in the blood5,10,15,20, and25, days postoperatively.Conclusions1. We had successful built Adenovirus vector though pAd-KGF digested product. The virus got the ability of transfecting cells. The final virus titer we had were2×10uPFU/ml, and3×1012PFU/mlforAd,2. Ad-KGF and Ad could highly transfected PA317, HUVEC and HaCaT cells. KGF had the potential to stimulate HaCaT cells to grow and migration and did not promote PA317and HUVEC cells to grow.3. We had modified the classical rat’s back flap model, and made it suitable for flap necrosis studies which was simple and convince.4. Ad-KGF benefited necrotic areas at the early stage of necrosis in an extended rat dorsal necrotic wound model.5. We injected EGF-VEGF directly into rat flap necrotic wound and found it helpful for wound healing without any side effect.