Dynamic Studies On Effects Of Adipose Tissue In The Repair And Rebuilding Process Of Burned Wound Healing

Objective: ① To investigate the regularity of microcirculiation change of adipoise tissue under the burned wound(ATUW), and to detect the effect of the interval between thermal injury and escharectomy on ATUW; ② To explore the relationship between adipose tissue preservation and healing speed, wound construction rate, some kinds of healing-related index respectively; the effect of adipose tissue preservation on bacterium content in the wounds; the effect of the interval between thermal injury and escharectomy on the function of ATUW; as well as the relationship between the change of microcirculiation change of ATUW and the changes of healing-related index; ③ Compared with the traditional skingrafting technique on deep fascia of wound, to evaluate the survival rate and the survival quality of skingrafting on ATUW; ④ To explore a part of mechanism of the capability of adipose tissue on improving wound healing by observing the effect of the homogenate of adipose tissue on the biological properties and generation ability of porcine MSCs in vitro; ⑤ To elucidate the effect of adipose tissue preservation on maturity and proliferation of burn scar by studying on the reparation and regeneration quality of human scars which had been healed on various base structure of wound.Methods: Seventy-two full thickness skin thermal wounds were produced on the back in 6 male minipigs. ① LDF was used to detect the regulation of microcirculation changes of both deep fascia and adipose tissue under the wounds. ② we preserved various thickness of adipose tissue in escharectomy which undertook 24 hours after the burn injury. On day 3, 7, 14 and 21 after injury, we compare the healing speeds and the rate of wound contraction in wounds on the whole subcutaneous fat (n = 24), half subcutaneous fat (n = 24) and deep fascia (n = 24) in burned pigs after excision of eschar by using sketchy observation . light microscope , electron microscope , HE staining ,VG staining and immunohistochemistry, RT-PCR. ELISA. Western blotting methods and bacterial culture were performed to detect thedifference of genes or proteins of -SMA, F-VDIRA, PCNA, bFGF, bFGf-R, TGF-β1, TGF-β3, Lep, Lep-R, ADRP, and the changes of bacteria content in wounds. ③ 24 wounds on minipig were undertook excision of eschar and autografting on various thickness of adipose tissue 24 hours after burn injury, We compare the survival rate and survival quality of grafting on the whole subcutaneous fat (n = 8), half subcutaneous fat (n = 8) and deep fascia (n = 8) by using sketchy observation, light microscope, electron microscope and immunohistochemistry methods to detect the difference of proteins of F-ⅧRA and bFGF. ④ We use homogenate of both normal adipose tissue and adipose tissue under the wounds to intervene the biological behavior and generation ability of MSCs. Grouth curve of MSCs was made, the population doubling time of MSCs was culculated and cell cycle, DNA content ,apoptosis rate of MSCs was detected by Flow Cytometry. ⑤ 26 scar samples obtained from 18 patients who had suffered burn injury and the wounds had healed with scar. All scar samples were divided into 3 groups according to the thickness of adipose tissue under the scar: Scar healed on deep fascia group(n=15), Scar healed on adipose tissue group(n=ll) and normal tissue control group(n=26).We use histological examination, Western-bloting, PCR method to detect protein level changes of TGF-β1,TGF-β3 in samples.Results: ①The volume of microcirculation(VM)in adipose tissue and in deep fascia tissue had no statistic difference before injury; But the VM both in AUW and DUW reduced rapidly after burn injury, the VM in AUW decreased more than that of DUW 1 ～60 hours after the thermal injury. the AUW's VM in E72h were lower than that in E24h within 60 hours after escharectomy,and the AUW's VM in E7d remained lower than that in E24h and in E72h 14 days after escharectomy. ② The healing time of the three groups had no difference. The wounds' construction occurred earlier and the wounds' construction rate (WCR) which had relationship with the thickness of preserved adipose tissue in A group remains more than that in other groups to the end .The totle amount of cytes in wounds of B and C group were more than that of A group 7 days and 14 days after escharectomy, But when 3 months passed , the changes of totle amount of cytes in wounds was reverse,the group A contained morecytes component than the other two groups. The MFB appeared ealier and remained more functional for longer time in group A than that in the other groups. The a-SMA positive cytes in group A were more than that in B and C group. The granulation tissue in B and C gtoup generate earlier and become more abundant, cell multiplication was more active in earlier phase of healing than that in group A, which was manifested by more F-ⅧRA positive cytes and more PCNA positive cytes in wounds of B and C group. And the array of collagen fibers trend to be thick, long and regulative earlier in group B and C than that in group A. The overall degree of maturity of repairing tissue in group B and C was higher than that in group A 21 days after escharectomy. The immunohistochemistry staining showed that the bFGF positive cytes increased just after the inury and incraesed more in group C and B than that in group A,but the results of RT-PCR showed that the gene expression of bFGF was not increase until 7 days after the injury, and got to the top at 14 days after injury, the gene expression of bFGF in group B and C was higher than that in group A. And moreover, bFGF level in group C and B increase in small extent 3 months after the escharectomy after a stable decrease, and this phenomena didn't appear in group A. The protein expression of bFGF-R, Lep, Lep-R and ADRP all increased in various time points , and in group B and C these levels were higher than those in group A. 3 months after injury , the TGF-(31, TGF-β3 protein level especially TGF-β3 protein level in the three groups were higher than normal tissue, there was no difference amang the three groups in TGF-β1 level, but there was obvious difference in TGF-β3 level: group C≈group B>group A. The bFGF positive cytes appeared earlier and persist longer in E24h than that in E72h. The Lep,Lep-R,and ADRP level in adipose tissue under the wound were higher than those in normal adipose tissue, and the interval time between escharectomy and injury may affect the Lep,Lep-R,and ADRP levelin wound:the earlier escharectomy was undertaken, the higher level of these indix may present. The cell density of neogenesis vascular endothelial cell were closely correlated with the cell density of endothelial cell in the base tissue of wound, but not the volune of microcirculation in the base tissue of wound 3 days after injury. The bacteria content of wounds were seriously affected by the interval time betweenescharectomy and injury: The earlier escharectomy was made, the fewer bacteria may existed and the preservation of adipose tissue under the scar which was remained longer than 3 days may cause more bacteria content in wounds. ③ The skin graft in the three groups all survived, but the skin grafts in group A contract more than that in the other groups. The healing quality of graft in group C was superior to group B and group A. The interval time for blood vessel hyperplasia and rebuilting and for collagenic deposition and restructure in group A was longer than that in group B and C, and the maturation degree of rebuilt tissue in group A was worse than that in the other groups.And in group A, the expression of bFGF and PCNA were lower than that in group B and C, and the ultramicrostructure change of fibroblast showed that the process of proliferation, function enhancement,and apoptosis, function decline were delayed than group B and C, the maturate degree of collagenous fibers were poor, myofibroblast exist longer time and its founction attenuated much slower. ⑤Compare with normal tissue , in group A,the constructure of scar was more disorder, layer of tissue was blur, the cell component was more, and the occurence of Hyperplastic scar was more frequent than those in group B. the frequent of group A and B was 83.7% and 62.4%,respectively. The TGF-β1 and TGF-β3 protein level in both groups were higher than that of normal tissue, there was no difference in TGF-β1 between A and B group , but the TGF-β3 level in group A was higher than that in group B. Conclusion: ① Adipose tissue is not a kind tissue lacking of blood supply compare with deep fascia tissue.The microcirculation of AUW was obviously decreased,and reach the same level with DUW 60 hours after injury .Early escarectomy may improve the recover of microcirculation . ② Preservation of adipose tissue in burned wound may accelerate healing process, reduce wound contraction rate, enhance healing quality, escharectomy in early phase may promote the recover of adipose tissue' s function, and reduced the infected chance of adipose tissue remained wounds. In early phase of healing process, the generation of vascular endothelial cell and Angiopoiesis were closely correlated with the cell density of endothelial cell in the base tissue of wound, but not the volune of microcirculation in the base tissue ofwound. ③Grafting on adipose tissue may obtain the same survival rate and better survival quality, compared with the traditional grafting technique on deep fascia; ④The perserved adipose tissue in the wound improve the healing quality and promote the grafting survival rate, which may be attributed to the ability of fat to promote the stem cells' generation and differentiation. ⑤The perserved adipose tissue in human burned wound had apotential to improve the quality of healing, to accelerate the maturation of scar, to reduce the occur of hyperplastic scar.