Study On The Effect Of Decellularized Adipose Matrix Combined With Vacuum Sealing Drainage On Skin Wound Healing

ObjectiveThe aim of this study was to investigate the effects of decellularized adipose matrix(DAM)combined with vacuum sealing drainage(VSD)on wound healing rate,vascularization and inflammatory response,as well as the performances in the treatment of exposed skull wounds.At the same time,explain the effect and mechanism of this treatment.Methods(1)Human decellularized adipose matrix scaffold material were prepared.Examination of its structure and decellularizing effect were examed by HE staining.12 Bama miniature pigs were recruited.Four skin wounds(4 cm in diameter each)were made on the back of each pig.Four wounds were randomly divided into 4 groups for different treatments:DAM/VSD group(decellularized adipose matrix combined with vacuum sealing drainage),DAM group(decellularized adipose matrix alone),VSD group(vacuum sealing drainage alone)and control group(CON,sterile gauze dressing).The wounds were photographed and the remaining wound area was measured on the day of the wound established and on day 7,10,14,17,21 post treatment.Meanwhile,HE staining,vWF immunohistochemical staining and microvessel count was performed on day 7,14 and 21 post treatment.Moreover,the expression of vascularization-related factors was detected by fluorescence quantitative PCR.(2)The animal wound model as described above was used in this part.HE staining was performed on the wound tissues at 3,7,10,and 14 days to observe the neutrophil infiltration of the wound tissue.The number of neutrophils from wound tissues was counted,and the inflammatory response-related factors IL-1?,IL-6,TNF-?,as well as macrophage isoforms iNOS and ARG-1 were detected by fluorescence qRT-PCR.(3)Create cranial bone-exposed wounds model on Bama miniature pigs.Twelve smallBama pigs were recruited.A 2cm diameter,periosteum-stripped skull exposed wound was made in the forehead of each miniature pig.They were randomly divided into four treatment groups:DAM/VSD group(DAM combined with VSD),DAM group(DAM alone),VSD group(VSD alone),blank control CON group(sterile gauze dressing change).On the day of the wound established and on day 7,10,14,17,21 post treatment,the wound were photographed and the remaining wound area was measured.On day 7,14,21 post treatment,the wound tissue samples were taken,and CD31,a-SMA(a-smooth muscle actin)double-stained immunofluorescence was performed.Mean while,fluorescence qRT-PCR was used to detect vascularization related factor CD31 and VEGF expression.Results(1)Decellularized adipose matrix obtained in this study had a loose porous structure,with a proximately complete decellularization.The remaining wound area of the DAM/VSD group was significantly smaller than that in the other three groups on day 14,17 and 21 after treatment(P<0.05).The healing time span was less than the other three groups(P<0.01).The microvessel counts in the DAM/VSD group were higher than those in the other three groups on day 14 and 21(P<0.01).Similarly,the expression of vascularization related factors was higher than the other three groups on day 7 and 14(P<0.05).(2)Compared with sterile gauze change treatment,DAM or VSD alone,DAM/VSD group had less neutrophil infiltration and lower expression of pro-inflammatory factors IL-1?,IL-6,TNF-a and M1 macrophage marker iNOS,while the expression of the M2 macrophage marker ARG-1 which associated with tissue repair was higher.(3)In the cranial bone-exposed wounds,the wounds in the DAM/VSD group also showed faster healing than the other three groups,and the complete epithelialization occurred earlier.On day 21,CD31 and a-SMA double staining immunofluorescence showed that the positive fluorescence intensity and the number of mature blood vessels in the DAM/VSD group were higher than those in the other three groups.The results of fluorescence qRT-PCR were consistent with the results.The expression of CD31 and VEGF in the wounds of DAM/VSD group was also significantly higher than that of the other three groups at each observation time.Conclusions(1)As an ideal natural scaffold material,DAM can promote the healing of skin soft tissue wounds in combination with VSD.Compared with the single use of each,DAM combined with VSD treatment is more effective,which accelerates the healing rate,promotes the establishment of wound neovascularization,and regulates the expression of inflammatory factors as well as macrophage phenotype,reducing the tissue damage secondary to inflammatory responses.(2)DAM combined with VSD can heal the exposed skull wounds with periosteum removed and accelerate the healing speed by promoting the formation and maturation of new blood vessels.This indicates that this method has the ability to make the wound heal across the area lacking blood supply,and may be applied to the non-surgical treatment of various refractory wounds in the future.