Mechanism Study Of Improving Full-thickness Skin Grafts By TNF-α-activated ADSCs Pretreatment Of Donor Skin

Background and objective:Repair of skin and soft tissue defects has always been a hot topic in plastic surgery.Compared with skin flap transplantation,skin grafting is more convenient,but the early postoperative nutritional supply of grafts depends only on plasma diffusion without blood supply,and requires at least 48 h for the pre-existing blood vessels in the skin graft to anastomose with the recipient vessels to restore blood supply,resulting in disadvantages such as poor texture and high contraction rate.Previous studies have shown that injecting adipose-derived stem cells(ADSCs)into the recipient site of skin grafts can improve the contraction and texture of skin grafts by exerting its pro-angiogenic effects.However,there are still several problems with its application: 1.ADSCs have heterogeneity.The age and pathological status of the donor and recipient can affect the efficacy of stem cells.How to obtain highly functional stem cells to maximize their therapeutic potential still needs further exploration;2.The recipient site of skin grafts is often accompanied by infection,hypoxia,and tumor resection.Direct injection of ADSCs into the wound may result in low survival rate and unknown safety consequences;3.It takes at least 3-5 days for ADSCs injected into the wound to promote the formation of new blood vessels,which cannot shorten the time of blood supply shortage after skin grafting.Based on the above considerations,this study proposes to use TNF-α to pre-activate ADSCs and enhance their cell function.Then,TNF-α-activated ADSCs are used to pretreat the skin in the donor site to increase the blood vessel.The pretreated skin in the donor site is used for full-thickness skin grafts(FTSGs)transplantation,which is expected to accelerate the anastomosis efficiency of pre-existing blood vessels in the skin graft and the recipient vessels,shorten the nutritional deprivation time of the epidermis,dermis,and skin appendages,and improve the survival rate and quality of FTSGs.This study explores the potential mechanisms of TNF-α-activated ADSCs to enhance their pro-angiogenic capabilities,and hopes to obtain the flap-like effects through simple skin grafting.PART 1: Isolation,Culture,and Identification of ADSCsObjective: To obtain high-quality ADSCs from adult adipose tissue.Methods: Adipose tissue was obtained from healthy adult women by abdominal liposuction.The tissue was minced,digested with collagenase,centrifuged,resuspended,and cultured through passage to obtain purified ADSCs.The cells were observed under a light microscope for morphology,surface markers were identified by flow cytometry,and adipogenic and osteogenic differentiation were induced and evaluated by staining.Results:1.ADSCs appeared spindle-or elongated-shaped under a light microscope,similar to fibroblasts.Some cells extended protrusions and,when the density was high,cells grew in a helical or radial colony.2.The cells obtained showed strongly positive surface expression of CD90(98.0%)and negative expression of HLA-DR.3.The cells obtained had multipotent differentiation capacity and could differentiate into adipocytes and osteocytes.Conclusion: We successfully obtained high-quality ADSCs using enzyme digestion and identified them as possessing general characteristics of mesenchymal stem cells with multipotent differentiation capacity.These cells can be used for further experimental research.PART 2: The Effect of TNF-α on ADSCs FunctionObjective: To investigate the effect of TNF-α preactivation on the proliferation,migration,and angiogenic ability of ADSCs in vitro.Methods:1.The effect of different concentrations of TNF-α on ADSCs proliferation was investigated by CCK-8 cell proliferation assay,flow cytometry cell cycle analysis,flow cytometry apoptosis detection,and Ki67 immunofluorescence staining to determine the appropriate concentration of TNF-α for subsequent research.2.The effect of different concentrations of TNF-α on ADSCs migration was investigated by scratch assay.3.HUVECs were co-cultured with ADSCs or TNF-α-activated ADSCs(t-ADSCs)in a Transwell chamber,and then the cell proliferation and angiogenesis ability of HUVECs were detected by Ed U cell proliferation assay and tube formation assay.Results:1.TNF-α preactivation at 5 ng/ml enhanced ADSCs proliferation,increased the proportion of cells in the cell cycle,increased the percentage of Ki67-positive cells,and did not induce significant cell apoptosis.2.TNF-α preactivation at 5 ng/ml enhanced ADSCs migration,with the scratch being covered by cells after 24 hours.3.Coculture of t-ADSCs with HUVECs increased the Ed U staining positivity rate and the number of formed tubes.Conclusion: TNF-α preactivation at 5 ng/ml significantly enhanced the proliferation,migration,and angiogenic ability of ADSCs without inducing significant cell apoptosis.TNF-α preactivation can also enhance ADSCs’ angiogenic ability significantly.PART 3: Mechanisms of TNF-α Enhanced ADSCs-Mediated AngiogenesisObjective: To explore and validate potential mechanisms by which TNF-α enhances the angiogenic capacity of ADSCs in vitro.Methods:1.m RNA sequencing was performed to detect differentially expressed genes(DEGs)between t-ADSCs and ADSCs,and data mining was conducted to screen signal pathways and cytokines that may contribute to the enhanced angiogenic ability of ADSCs.2.ELISA was used to detect the secretion levels and patterns of angiogenic factors IL-8 and VEGF by t-ADSCs,and the effect of NF-κB signaling pathway inhibitor anacardic acid on IL-8 and VEGF expression was verified.3.Western blotting was performed to detect the expression of key proteins p-p65,p65,and IκBα on the NF-κB signaling pathway in ADSCs after TNF-α treatment,reflecting the activation of the NF-κB signaling pathway.Results:1.m RNA sequencing showed that compared to ADSCs,t-ADSCs had 1539 significantly DEGs,including 722 up-regulated genes and 817 down-regulated genes.GO annotation,KEGG enrichment,protein interaction network analysis,and Hub gene screening revealed that the NF-κB signaling pathway and IL-8 were most likely to be associated with TNF-α-enhanced ADSCs cell function.2.ELISA results showed that TNF-α pre-activation enhanced ADSCs’ expression of IL-8 and VEGF,which could be inhibited by anacardic acid,and was dose-dependent.After TNF-α stimulation was removed,ADSCs could maintain high IL-8 secretion for a long time.3.Western blotting showed that after TNF-α treatment,the key protein p-p65/p65 of the NF-κB signaling pathway increased and the content of IκBα increased,indicating that the activation of the NF-κB signaling pathway was enhanced.Conclusion: Pre-activation of ADSCs with TNF-α may enhance ADSCs’ paracrine IL-8 secretion ability by activating the intracellular NF-κB signaling pathway,thereby exerting their angiogenic capacity.PART 4: T-ADSCs promote the generation of blood vessels in the donor skin toimprove the outcome of full-thickness skin grafts(FTSGs)transplantationObjective: To establish an animal model of full-thickness skin defects in the back of rats,pre-treat the donor skin with t-ADSCs for 7 days,and then perform FTSGs transplantation.The effect of t-ADSCs on blood vessel generation in the donor skin and the outcome of FTSGs were observed.Methods:1.Rats were randomly divided into control group,PBS group,ADSCs group,and t-ADSCs group for corresponding intradermal injection pretreatment.After 7 days,the difference in blood vessel content in the donor skin was detected by HE staining and CD31 immunofluorescence staining.2.After the donor skin was pretreated for 7 days,rats were divided into 5 groups according to different pretreatment methods: control group(no injection),pre-PBS group,pre-ADSCs group,pre-t-ADSCs group,and post-ADSCs group for FTSGs transplantation.3.48 hours after transplantation,a thermal imaging camera was used to evaluate blood perfusion to assess the degree of anastomosis between the graft and recipient vessels.The graft area was photographed and recorded at 2,3,5,7,14,21,and 28 days after transplantation to calculate graft survival and contraction rates.On the 28 th day after skin grafting,the grafts were harvested for HE and Masson staining to evaluate histological changes.Results:1.After t-ADSCs pretreatment of the donor skin for 7 days,HE staining and CD31 immunofluorescence staining showed significantly higher blood vessel content than other groups.2.48 hours after transplantation,the thermal imaging measurement showed that the pre-t-ADSCs group had the fastest temperature recovery.3.Seven days after transplantation,the order of graft necrosis rates was as follows:control group > pre-PBS group > pre-ADSCs group > post-ADSCs group > pre-t-ADSCs group;28 days after transplantation,the order of graft contraction rates was as follows:control group > pre-PBS group > pre-ADSCs group > post-ADSCs group > pre-t-ADSCs group.4.On the 28 th day after transplantation,HE staining showed that the t-ADSCs pretreatment group had the thickest epidermis,the lowest epidermal detachment rate,and the most hair follicles retained.Masson staining showed that the collagen arrangement in the t-ADSCs pretreatment group was more regular.Conclusion: Pre-treatment of the donor skin with t-ADSCs can effectively promote the generation of blood vessels in the donor skin,increase the pre-existing blood vessel content,and improve the outcome of FTSGs transplantation by advancing the restoration of blood supply to the graft,increasing graft survival,and reducing graft contraction rate,as well as enhancing the transplantation effect at the epidermis,dermis,and skin appendage levels.