Abstract Skin grafts often suffer from contracture, complicating recovery. SVF-gel shows promise in addressing scar contracture, but its therapeutic effects and mechanisms are not fully understood. This study evaluates the efficacy of SVF-gel in full-thickness skin grafting. Full-thickness skin grafts were harvested from mice dorsal skin, rotated and sutured. SVF-gel or saline was injected beneath the muscle fascia. Immunohistochemistry assessed SVF-gel’s effects on angiogenesis, collagen deposition, fibrosis, and dermal adipocytes. Keloid-related genes from [40]GSE92566 and [41]GSE158395 were analyzed for functional enrichment and protein-protein interactions, with hub genes validated using [42]GSE190626. SVF-gel significantly increased the grafted area, thickness of the epidermal and dermal adipose layers, and hair follicles compared to the control group. SVF-gel enhanced CD31 and perilipin expression, decreased α-SMA expression, and identified HLA^+ cells around CD31^+ cells in the dermal microvessels and adipose tissue of the graft. Sixty commonly downregulated keloid-related genes were identified, with KEGG pathway analysis indicating enrichment in the PPAR signaling pathway and lipolysis regulation. Five hub genes (ADIPOQ, FABP4, KRT7, LEP, and PIP) were validated. SVF-gel shows promise as a stem cell therapy for skin grafts, improving outcomes by enhancing revascularization, increasing collagen fiber density and regularity, accelerating myofibroblast turnover, promoting adipogenesis, and increasing hair follicles. Keywords: SVF-gel, Full-thickness skin graft, Skin contracture, Angiogenesis, Adipogenesis Subject terms: Trauma, Mesenchymal stem cells Introduction Skin grafts are commonly used to repair large skin defects resulting from trauma, tumor excision, or congenital abnormalities^[43]1. While these procedures are effective in restoring skin barrier function and reducing infection risk, they may suffer from undesirable complications such as local pigmentation changes, decreased friction tolerance, and notably, scarring that may result in persistent grafted skin contracture^[44]2. The incidence of contracture following skin grafting can reach 20-30%, particularly in areas like joints, necessitating additional interventions such as contracturelysis, skin grafting, and skin flap transplantation to address functional and aesthetic concerns^[45]3,[46]4. Techniques like splints, pressure garments, and other methods have been employed to improve outcomes^[47]5,[48]6, but effective therapies for skin contracture are still lacking, highlighting the need for innovative treatments. The healing process of skin grafts involves three distinct phases: serum imbibition, revascularization, and organization^[49]7,[50]8. Tissue remodeling begins once the graft integrates with the recipient bed, followed by secondary contraction driven by myofibroblasts in the host bed, leading to hyperfibrosis and scar formation^[51]9. Myofibroblast density and collagen concentration are critical factors during skin contracture^[52]10. Therefore, techniques that limit collagen deposition and myofibroblast activity may help alleviate skin contracture, potentially increasing the efficacy and clinical applicability of full-thickness skin grafts . Adipose-derived stem cells (ADSCs) and their products have shown therapeutic benefits in various fibrotic conditions, such as liver cirrhosis, pulmonary fibrosis, and keloids^[53]11. Stromal vascular fraction (SVF) and ADSCs can secrete various cytokines and differentiate into multiple cell types, benefiting applications like skin grafts, hypertrophic scar treatment, and wound healing^[54]12–[55]16. However, isolating stem cells involves in vitro cell expansion, which is time-consuming, complex, expensive, and raises biosecurity concerns^[56]17. Human adipose-derived stem cells extracellular matrix/stromal vascular fraction-gel (SVF-gel), a newly developed injectable derived from adipose tissue, shows promise for clinical use in stem cell therapy^[57]18. Research indicates that SVF-gel is effective in treating hypertrophic scars and chronic wounds^[58]19,[59]20. However, its impact on skin graft outcomes remains unexplored. This study aims to establish a new method to improve the contracture and texture of full-thickness skin grafts. We investigated the transplantation of SVF-gel in combination with skin grafts to determine its potential in enhancing skin graft outcomes. Methods Ethical statement Approval for the study was obtained from the Animal Ethics Committee of Fujian Medical University (Approval No. IACUC FJMU 2018-CX-26) and the Human Ethics Committee of the First Affiliated Hospital of Fujian Medical University (Approval No. [2017]078). All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines ([60]https://arriveguidelines.org). Informed consent was obtained from all subjects. Harvesting fat and preparation of SVF-gel Under general anesthesia, human abdominal or femoral lipoaspirates were obtained via liposuction. The SVF-gel was prepared as previously described^[61]18. Briefly, the extracted fat was kept in ice water for 10 min, after which the liquid portion was discarded, and the fat layer was retained for further processing. The fat was then centrifuged at 1200 g for 3 min to produce Coleman fat. After centrifugation and removal of liquid components, the fat layer was mechanically emulsified by alternating between two 10-ml syringes connected by a female-to-female Luer-Lock connector. The nanofat was then combined with 0.5 ml of oil until flocculation was observed within the emulsion. Finally, the product was centrifuged at 2000 g for 3 min, and the substance remaining beneath the oil layer was defined as SVF-gel. Characterization of SVF-gel 0.1% type I collagenase was incorporated into the SVF-gel, followed by incubation at 37℃ for 40–60 min. Connective tissue was removed through filtration, and the resulting filtrate was collected in a centrifuge tube. PBS was then added to halt the digestion process. The cell suspension was obtained through successive centrifugation and resuspension steps. Subsequently, cells were labeled with antibodies (CD31-PE, CD34-FITC, CD45-APC) and analyzed using multi-color flow cytometry with a flow cytometer. Skin grafting model in mice Specific pathogen-free grade, 6-8-week-old, 22–25 g BALB/c nude mice were purchased from the Experimental Animals Centre of Fujian Medical University. The animals were housed in a controlled environment (12 h light/dark cycles at 23 °C) with unrestricted access to water and diet. The animals were randomly separated into two groups of thirty mice each. The mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (40 mg/kg), the dorsal skin was disinfected with 75% alcohol. Full-thickness skin excision wounds of 1.5 cm × 1.5 cm were created by removing the whole dorsal skin in the midline of the mice. The excised skin was placed in saline for subsequent grafting. 0.2 ml of SVF-gel (SVF-gel group) or 0.2 ml of saline (NS group) was injected into the deep fascia of the dorsal musculature at equal points. After rotation 180 degree in situ, the skin was sutured with 5 − 0 nylon single stitches at 0.5 cm intervals. Wounds were treated with chlortetracycline and then covered with a sterile dressing post-operation. The surgical technique was well tolerated by the animals, with no anesthesia-related deaths. Postoperative mice were housed individually to prevent mutual interference. Digital photographs were taken at the time of surgery and 7, 14, 30, 60, and 90 days later (Fig. [62]1). The graft area was assessed as a ratio of contracture using ImageJ software. Fig. 1. [63]Fig. 1 [64]Open in a new tab Timeline for the experimental design.Timeline of the experimental design. Saline or SVF-gel was injected into the skin graft area, and the skin graft size was measured and digitally photographed at the time of surgery and at 7, 14, 30, 60, and 90 days. Histological observation Mice in all groups were anesthetized, and full-thickness skin was collected from the survival area. Skin samples were fixed in 4% formalin, embedded in paraffin, and cut into 8–10 μm sections. While some sections were laid on slides and stained, others were frozen at − 80 °C. The slices were stained with hematoxylin and eosin (H&E) for histological analysis according to the manufacturer’s instructions. Additionally, collagen accumulation was evaluated by Masson staining. All images were captured using a microscope and then analyzed using ImageJ software. Immunohistochemistry and Immunofluorescence The sections were prepared as described above. For immunohistochemistry analysis, dehydrated sections were antigen retrieved with sodium citrate in a pressure cooker and blocked with 5% bovine serum albumin at 37 °C for 1 h. The sections were then incubated with the anti-α-SMA antibody (1:200 dilution, Abcam, Cambridge, MA) overnight at 4 °C. After rinsing three times with PBS, the sections were incubated with a secondary antibody for 1 h at room temperature. Signals were observed using an avidin-biotin-horseradish peroxidase detection system. Slices were reviewed using an Olympus CX21 microscope. For immunofluorescence, the prepared sections were placed on glass slides and rinsed with PBS. After incubation with mouse anti-human leukocyte antigen antibody (HLA, 1:200 dilution, Abcam, Cambridge, MA) in a wet box for 1 h, the slices were incubated with rabbit anti-mouse secondary antibody for 1 h. Additionally, the same method was used to stain adipocytes and nuclei with mouse anti-human Perilipin antibody (1:200 dilution, Abcam, Cambridge, MA) and 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, Carlsbad, CA). The samples were observed under a Zeiss LSM 700 confocal fluorescence microscope. Collection of keloid genes Microarray expression data and high throughput sequencing data of keloid patients were obtained from the GEO database. [65]GSE92566 and [66]GSE158395 were selected as training cohorts. [67]GSE92566 consisted of 4 keloid samples and 3 normal samples. [68]GSE158395 contained 6 control samples and 7 keloid samples. The training datasets were analyzed using the limma package, with an adjusted p-value < 0.05 and |log2FoldChange| > 1 as the screening criteria for important differentially expressed genes (DEGs). Gene enrichment analysis To investigate biological activities and pathways, we utilized Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The functional characteristics of the common keloid-related genes from [69]GSE92566 and [70]GSE158395 were examined using these analyses with R’s cluster profile package, setting a p-value < 0.05 as the threshold. Protein–protein interaction (PPI) network analysis and screening for common Hub genes A PPI network was constructed using the STRING database ([71]https://string-db.org/) with a medium confidence score > 0.4. Visualization was done via Cytoscape (Version 3.10.2)^[72]21. Hub genes were identified through five algorithms (MCC, Degree, BottleNeck, Closeness, Betweenness) in the cytoHubba plugin of Cytoscape. Common hub genes for [73]GSE92566 and [74]GSE158395 were determined by intersecting the top 10 genes from each algorithm. Validation of hub genes in an external dataset To minimize false positives, hub genes were validated using another keloid dataset from the GEO database, specifically [75]GSE190626, which includes samples from 3 keloid and 3 non-keloid individuals. The GEOquery package was used to obtain expression data for the keloid-related hub genes. Statistical analysis Statistical analysis was performed with SPSS 19.0 software, and all data were expressed as mean ± SD. Comparisons between each group were analyzed with independent T or paired T-tests, as appropriate. A value of p < 0.05 was considered statistically significant. Results Preparation and characterization of SVF-gel The SVF-gel was prepared according to established methodology, resulting in a final volume representing less than 15% of the initial volume and suitable for injection through a 27-gauge needle (Fig. [76]2A). Flow cytometry was employed to assess the quality of the SVF-gel, with cell surface expression profiles used to quantify the components present. The results, depicted in Fig. [77]2B, indicated that in 1 ml of SVF-gel, the concentration of ADSCs (CD45^− / CD31^− / CD34^+) exceeded 4.5 × 10^5 cells/ml, while ECs (CD45^− / CD31^+ / CD34^+) were present at levels greater than 0.75 × 10^5 cells/ml, and other cells (CD45^− / CD31^− / CD34^−) were observed at 2 × 10^4 cells/ml. The total number of SVF cells in the sample was calculated to be 13.0 × 10^5 cells/ml, exceeding the minimum threshold of 4.0 × 10^5 cells/ml required for SVF-gel standards^[78]18. These findings demonstrated that the SVF-gel met the criteria for the subsequent experimental. Fig. 2. [79]Fig. 2 [80]Open in a new tab Preparation and Characterization of SVF-gel. (A) A schematic flowchart represents the preparation of the SVF-gel. (B). CD45(+) cells were hematopoietic cells, whereas CD45(-) cells were adipose-derived cells and were subjected to further analyses. CD45(-)/CD31(-)/CD34(-) cells were regarded as other cells, CD45(-)/CD31(+)/CD34(+) cells were regarded as vascular endothelial cells, and CD45(-)/CD34(+)/CD31(-) cells were regarded as ADSCs. Full-thickness skin graft in mice model and histology of skin transplants To assess the impact of skin contracture post-grafting, SVF-gel was administered to the grafts on the dorsal area of nude mice. The condition of the skin grafts was monitored at various time points post-operation (7, 14, 30, 60, and 90 days), with normal saline serving as the control group. Within the initial 7-day period, grafts in the control group exhibited paleness and blackening at the edges, along with areas of scab formation indicating compromised survival or necrosis. In contrast, grafts in the SVF-gel group exhibited a healthy, ruddy, and soft appearance with no signs of necrosis. By the 14th day, necrosis was observed at the edges of the skin in the NS group, whereas the SVF-gel group continued to show no signs of necrosis. A significant difference in grafted skin area between the two groups persisted, with the SVF-gel group demonstrating a significantly larger area compared to the NS group (p < 0.05). This trend continued on the 30th, 60th, and 90th days, with the grafted skin area of the SVF-gel group consistently larger than that of the NS group, showing statistically significant differences (p < 0.05) (Fig. [81]3A -B). Fig. 3. [82]Fig. 3 [83]Open in a new tab Full-thickness skin graft in mice model and histology of skin transplants. (A). Representative images of the full-thickness skin graft in mice model demonstrate that SVF-gel treatment alleviated transplanted skin contractures more effectively than saline. (B). Following surgery, the grafted skin region was assessed. The skin area acquired for transplantation was greater in the SVF-gel group than in the saline group. (C). Hematoxylin and eosin-stained skin transplants at 7, 14, 30, 60, and 90 days after surgery. The epidermis is labeled as E, and the dermis as D. The black dashed line represents the boundary between the epidermis and dermis, while the green dashed line demarcates the dermal adipose tissue. Hair follicles are marked with yellow asterisks (*), and the pink fibers between the black and green dashed lines represent collagen fibers. (× 40, scale bars = 100 μm; significance was set to *p < 0.05, **p < 0.01, and ***p < 0.001, n = 5 animals per time point/group). (D). HE staining of transplanted skin at 7, 14, 30, 60, and 90 days post-surgery was used to measure the thickness of the epidermis, dermis, and dermal adipose tissue. Histological evaluation of skin grafts was conducted through HE staining at various time points post-transplantation. The results revealed that the skin morphology in the SVF-gel group closely resembled that of normal skin tissue (Fig. [84]3C). Furthermore, during the process of skin graft tissue remodeling, the SVF-gel group exhibited increased thickness in both the epidermal and dermal adipose tissue layers compared to the saline group (Fig. [85]3D). SVF-gel lessens transplanted skin contracture and enhances angiogenesis Early revascularization is a crucial process in the success of skin grafts. To assess vascular reconstruction post-operation, endothelial cells were labeled with the endothelial cell marker CD31 at various time intervals, and microvessel density was quantified through microscopic observation (Fig. [86]4A-B). The microvessel density in the SVF-gel group was found to be significantly higher than that in the NS group at 7 and 14 days post-operation. However, by the 30th day, there was no significant disparity in microvessel density between the two groups. These data indicated that SVF-gel may enhance early revascularization following skin grafting and positively impact early skin graft survival. Human leukocyte antigen (HLA) serves as a marker for human-derived cells and was utilized in staining to assess the cellular source within the grafts. The findings revealed the presence of local human-derived cells in the SVF-gel group, whereas no human-derived cells were observed in the NS group. Above all, the presence of HLA-positive cells surrounding CD31-positive endothelial cells suggests that a portion of the neovascularization within the grafts originated from the host bed (Fig. [87]4C). Fig. 4. [88]Fig. 4 [89]Open in a new tab SVF-gel lessens transplanted skin contracture and enhances angiogenesis.(A). Representative images of skin transplant tissue slices immunostained for CD31, along with a measurement of (B) the number of microvessels in each skin transplant at 7, 14, and 30 days after surgery. (C, E). Tissue sections from skin transplants immunostained for HLA and α-SMA at 60 and 90 days post-surgery are shown in these representative pictures. (D) Masson’s trichrome staining and microscopy were used to visualize dermis collagen deposition at 14 days after surgery. The epidermis is labeled as E, and the dermis as D. The black dashed line represents the boundary between the epidermis and dermis, while the orange dashed line demarcates the dermal adipose tissue. Hair follicles are marked with green asterisks (*), and the blue fibers between the black and orange dashed lines represent collagen fibers. (× 200, scale bars = 50 μm; *p < 0.05; n = 5 animals per group/time point). (F) Sirius Red staining and microscopy were used to visualize the types of collagen deposition in the dermis at 14 and 30 days after surgery (× 200, scale bars = 100 μm). Masson staining was employed to assess collagen deposition post skin grafting. The results demonstrated a notable disparity in collagen morphology between the two groups at the 30-day post transplantation. Specifically, the collagen density in the SVF-gel group exceeded that of the NS group, with a more organized arrangement observed (Fig. [90]4D). These findings indicated that SVF-gel can improve collagen deposition and arrangement after skin grafting. Meanwhile, the presence of myofibroblasts identified by α-SMA is indicative of their role in wound contraction. By the 60th day, α-SMA-positive myofibroblasts were significantly higher in the NS group than in the SVF-gel group, suggesting that SVF-gel may decrease the number of myofibroblasts and alleviate skin contracture (Fig. [91]4E). Additionally, Sirius Red staining at Day 14 and 30 showed that the SVF-gel group had more organized and denser collagen fibers compared to the saline group, supporting the findings from Masson staining that SVF-gel enhances collagen deposition and tissue remodeling during skin grafting (Fig. [92]4F). SVF-gel’s inherent mechanism for alleviating transplanted skin contracture Adipocytes play a crucial role in intercellular communication that influences myofibroblast activity during skin wound healing^[93]22. Perilipin was utilized as a marker for the detection of adipocytes in this study. The findings revealed a temporal pattern in the number of dermal adipocytes following skin grafting, with an initial increase followed by a subsequent decrease. Specifically, within the first 30 days post-transplantation, the number of dermal adipocytes was significantly higher in the SVF-gel group than in the NS group. However, between 60 and 90 days post-transplantation, the number of dermal adipocytes in the NS group surpassed that of the SVF-gel group. Notably, the peak number of dermal adipocytes in the SVF-gel group was significantly higher than that of the NS group (Fig. [94]5A-C). The strong correlation between hair follicle cycling and the spatiotemporal behavior of dermal white adipose tissue was investigated in this study. Over time, the number of hair follicles gradually decreased, with the SVF-gel group showing a higher number compared to the NS group (Fig. [95]5D). Fig. 5. [96]Fig. 5 [97]Open in a new tab SVF-gel’s inherent mechanism for alleviating transplanted skin contracture. (A). Representative images of skin transplant tissue slices immunostained for perilipin at 7, 14, 30, 60, and 90 days post-surgery. (B-D). The thickness of the epidermis, dermis, and dermal adipose tissue as well as the percentage of hair follicles were measured using HE staining and perilipin immunostaining of transplanted skin at 7, 14, 30, 60, and 90 days post-surgery. (× 200, scale bars = 50 μm; *p < 0.05; n = 5 animals per group/time point). (E). Immunofluorescence staining of CD206 (red) and CD86 (green) at 14 and 30 days post-surgery. Nuclei are stained with DAPI (blue) (× 400, scale bars = 20 μm). (F) Immunofluorescence staining of HLA (red), CD31 (green), and CD34 (magenta) at 14, 30, 60, and 90 days post-surgery. Nuclei are stained with DAPI (blue) (× 400, scale bars = 20 μm). The immunomodulatory and pro-regenerative properties of SVF-gel were further elucidated by analyzing macrophage polarization and vascular remodeling dynamics. Immunofluorescence staining revealed distinct temporal patterns in macrophage phenotypes between the SVF-gel and NS groups (Fig. [98]5E). At postoperative day 14, the SVF-gel group exhibited a predominant infiltration of CD206⁺ anti-inflammatory macrophages, whereas the NS group showed a higher abundance of CD86⁺ pro-inflammatory macrophages. This phenotypic disparity persisted through day 30, with SVF-gel maintaining a sustained anti-inflammatory milieu characterized by elevated CD206⁺ cell populations, in contrast to the NS group’s persistent pro-inflammatory dominance. These findings suggest that SVF-gel actively modulates the immune microenvironment by promoting macrophage polarization toward a reparative phenotype, thereby ameliorating excessive inflammatory responses and creating favorable conditions for tissue regeneration. Concurrently, vascularization and graft integration were significantly enhanced in the SVF-gel group (Fig. [99]5F). At day 14, robust angiogenesis was evidenced by increased CD31⁺ vascular endothelial cells and CD34⁺ adipose-derived stem cells within SVF-gel-treated grafts. By day 30, this group demonstrated advanced vascular maturation and persistent stem cell recruitment, with CD31⁺ vessel density surpassing that of the NS group. Strikingly, SVF-gel facilitated long-term graft survival, as evidenced by HLA⁺ human-derived cell persistence and structurally mature vascular networks at days 60–90. In contrast, NS-treated grafts displayed sparse vasculature and absent HLA⁺ cells. These observations underscore SVF-gel’s dual capacity to orchestrate immunoregulation and sustain neovascularization, which synergistically mitigate fibrotic contracture by suppressing myofibroblast activation through cytokine equilibrium and mechanotransduction pathways. Collection of keloid genes and gene enrichment analysis We obtained 1149 keloid-related DEGs from [100]GSE92566 and [101]GSE158395. By taking the intersection of 844 DEGs from [102]GSE92566 with 305 DEGs from [103]GSE158395, we identified 196 genes common to both datasets, including 136 upregulated and 60 downregulated keloid-related genes (Fig. [104]6A). The top GO terms and KEGG pathways according to the 196 common keloid-related genes are summarized in Fig. [105]6B. GO annotation includes three categories: biological process, cell component, and molecular function. For biological processes, the common genes were enriched in extracellular matrix organization, collagen metabolic process, and fat cell differentiation. Cell component items were mainly enriched in the collagen-containing extracellular matrix, endoplasmic reticulum lumen, and collagen trimer. The molecular function category was mainly enriched in extracellular matrix structural constituent, glycosaminoglycan binding, and receptor ligand activity. KEGG pathway enrichment analysis included protein digestion and absorption, PI3K-Akt signaling pathway, focal adhesion, ECM-receptor interaction, and PPAR signaling pathway (Fig. [106]6C). Based on the 60 common downregulated keloid-related genes, GO analysis revealed enrichment in apical part of cell, basal part of cell, and lipid droplet in cell component items, while the molecular function category was primarily enriched in phosphatase inhibitor activity, protein phosphatase inhibitor activity, and cadherin binding involved in cell-cell adhesion (Fig. [107]6D-E). KEGG pathways of the 60 common downregulated keloid-related genes were mostly enriched in PPAR signaling pathway, regulation of lipolysis in adipocytes, and vibrio cholerae infection (Fig. [108]6D-E). Fig. 6. [109]Fig. 6 [110]Open in a new tab Collection of keloid genes and gene enrichment analysis. (A). Common gene representation through a Venn diagram. 196 genes from among 844 DEGs in [111]GSE92566 and 305 DEGs in [112]GSE158395 were found to be common genes. (B-C). GO and KEGG pathway enrichment analysis of 196 common genes between [113]GSE92566 and [114]GSE158395.(D-E). GO and KEGG pathway enrichment analysis of 60 common downregulated genes between [115]GSE92566 and [116]GSE158395. GO gene ontology, KEGG kyoto encyclopedia of genes and genomes, BP biological process, CC cellular component, MF molecular function. PPI network analysis and validation of hub genes in external dataset To explore protein interactions, we constructed a PPI network of 60 downregulated keloid-related genes using the STRING database. The network contained 38 nodes and 62 edges, which was visualized by Cytoscape (Fig. [117]7A). We screened the top 10 hub genes with five algorithms (MCC, Degree, BottleNeck, Closeness, Betweenness) from the cytoHubba plugin. Five hub genes (ADIPOQ, FABP4, KRT7, LEP, and PIP) were identified by taking the intersection of the top 10 genes in the five algorithms (Fig. [118]7B). ADIPOQ, FABP4, KRT7, LEP, and PIP were significantly downregulated in [119]GSE92566 and [120]GSE158395 (Fig. [121]7C-D). To validate the reliability of the five hub genes, we verified their expression in the [122]GSE190626 dataset. ADIPOQ, FABP4, KRT7, LEP, and PIP were significantly downregulated in the external dataset (Fig. [123]7E). Fig. 7. [124]Fig. 7 [125]Open in a new tab PPI network analysis and validation of hub genes in external dataset. (A). The PPI network of 60 common downregulated genes. (B). Five common hub genes were identified by five algorithms(MCC, Degree, BottleNeck, Closeness, Betweenness) of the cytoHubba plugin. (C-D). The expression of ADIPOQ, FABP4, KRT7, LEP, and PIP in the testing databases([126]GSE92566 and [127]GSE158395). (E). The expression of ADIPOQ, FABP4, KRT7, LEP, and PIP in the external database([128]GSE190626).(Abbreviations: PPI, Protein–protein interaction). (F) Immunofluorescence staining of SMA (red), FABP4 (green), and Caspase3 (magenta) at 14, 30, 60, and 90 days post-surgery. Nuclei are stained with DAPI (blue) (× 400, scale bars = 20 μm). Immunofluorescence analysis of SMA, FABP4, and Caspase3 in full-thickness skin grafts further elucidated the mechanistic role of SVF-gel in modulating adipocyte metabolism and myofibroblast apoptosis (Fig. [129]7F). At day 14 post-surgery, the saline (NS) group exhibited a higher proportion of FABP4⁺/Caspase3⁺ co-stained apoptotic adipocytes and SMA⁺ myofibroblasts compared to the SVF-gel group, which displayed sparse SMA⁺ and Caspase3⁺ signals. By day 30, the SVF-gel group maintained a lower apoptotic cell ratio alongside elevated FABP4⁺ adipocyte populations, whereas the NS group showed a significant increase in apoptotic adipocytes, indicative of progressive dermal adipocyte loss. Notably, at day 60, SVF-gel-treated grafts demonstrated a marked rise in SMA⁺/Caspase3⁺ co-localized cells, reflecting enhanced adipogenic potential and tissue remodeling capacity. In contrast, the NS group retained elevated SMA⁺ myofibroblast activity, consistent with unresolved fibrosis. The results align with the protein-protein interaction (PPI) network analysis, which identified FABP4 as a central regulatory hub gene. These findings reinforce the dual role of SVF-gel in promoting adipocyte differentiation and inducing myofibroblast apoptosis, both of which contribute to improved tissue regeneration and reduced fibrosis. Discussion The integumentary system, the body’s largest organ, functions as the primary barrier against external threats, rendering it vulnerable to harm. Clinical intervention often involves skin grafting to address significant skin injuries. Nonetheless, the disparity in texture between grafted skin and native tissue, along with the potential for scarring and graft contraction, can have detrimental implications for patient well-being. At present, there are no pharmacological interventions available to effectively prevent fibrotic scar formation, contraction, and associated functional complications subsequent to skin grafting^[130]23,[131]24. In an effort to tackle the challenge of fibrotic contraction post-skin grafting, we implemented SVF-gel treatment concurrently with the grafting process. The group receiving SVF-gel treatment demonstrated enhanced neovascularization and superior dermal remodeling, thereby alleviating graft contraction. SVF-gel, a substance abundant in ADSCs and ECM acquired through physical means, offers an improved extracellular milieu for cells^[132]18. SVF-gel has been shown to promote angiogenesis through the secretion of angiogenic factors, decrease dermal macrophage infiltration, reduce myofibroblast levels and collagen deposition, ultimately leading to a reduction in scar dermis thickness and accelerated wound healing^[133]25,[134]26. Our study has demonstrated the efficacy of SVF-gel in skin grafts. The healing process of skin grafts involves three distinct phases: serum imbibition, revascularization marked by neovascularization and inosculation, and organization^[135]27,[136]28. The period of serum imbibition following graft placement onto the recipient bed typically spans 48 hours, during which fibrin plays a crucial role in securing the graft to its new location and sustains the graft through plasma exudate from the wound bed. Nutrient acquisition occurs through passive diffusion. Subsequently, the fibrin ‘glue’ is replaced by granulation tissue, providing a more enduring attachment of the graft to the recipient bed^[137]28. The revascularization phase commonly includes two mechanisms: anastomosis between the graft and host vessels, and the ingrowth of host bed blood vessels into the graft. The latter is recognized as the principal method of revascularization^[138]8. Our research documented the existence of nearby human cellular structures enveloping CD31 + endothelial cells, providing evidence for the concept of blood vessel infiltration into the graft within the recipient bed. Maintaining sufficient revascularization is essential for the viability of the graft in the early stages of transplantation. Our study revealed a significant increase in the number of blood vessels in the SVF-gel group during the first and second weeks post skin grafting compared to the control group, followed by a gradual decrease after two weeks. In contrast, the control group exhibited a slow increase in the number of blood vessels after two weeks. Additionally, immunohistochemistry using HLA demonstrated that the injected SVF-gel had migrated to the interstitium of the dermal microvessels and dermal adipose tissue of the skin graft. The findings indicate that SVF-gel has the potential to enhance the revascularization and maturation of skin grafts, expedite the tissue remodeling phase, and hasten the overall repair process. Furthermore, both SVF-gel and SVF have been shown to stimulate angiogenesis^[139]15,[140]19, thereby enhancing graft survival. SVF-gel also demonstrates efficacy in reducing levels of macrophages and myofibroblasts, leading to improved treatment of hypertrophic scarring and fibrosis^[141]29,[142]30. Skin grafting contracture can be categorized into primary and secondary contracture. Primary contracture is characterized by the contraction of elastic fibers in the dermis following skin separation. On the other hand, secondary contractures occur when the skin is transplanted to the recipient area and are influenced by the pulling force exerted by myofibroblasts at the edge of the receptor bed. The extent of secondary contracture is positively associated with the lifespan of myofibroblasts in the wound, with longer survival leading to more pronounced contracture^[143]28,[144]31,[145]32. Myofibroblasts play a crucial role in the synthesis and deposition of extracellular matrix (ECM) components, leading to the replacement of temporary matrix. The disordered and dense arrangement of collagen fibers in keloids contributes to the rough and uneven appearance of the skin^[146]33,[147]34. Our study revealed that the utilization of SVF-gel in skin transplantation has demonstrated enhancements in collagen fiber density and regularity within the dermis, accelerated turnover of myofibroblasts, and restructuring of dermal tissue, ultimately leading to a substantially larger skin area in the treatment group as opposed to the control group. Furthermore, our study observed alterations in the adipocyte population within the dermis. Adipocytes have the ability to secrete BMP-4 and activate PPARɣ signaling, leading to the induction of myofibroblast reprogramming, a process that is crucial in anti-fibrotic mechanisms^[148]35. Additionally, recent research has shown that SVF-gel can enhance the proliferation of dermal papilla cells and cells in the bulge, promoting hair growth^[149]36. In accordance with our research findings, histological analysis indicated a higher quantity of hair follicles in the SVF-gel group compared to the NS group. Furthermore, dermal white adipose tissue has been identified as a potential target for modulating hair growth, suggesting a possible mechanism by which SVF-gel facilitates hair growth^[150]37. The phenomenon of skin graft contracture is intricately linked to scar fibrosis, the underlying pathophysiology of which remains incompletely elucidated^[151]38. Skin grafting contracture is a multifactorial process influenced by mechanical stress, infection, inflammatory responses, and patient-specific factors such as age, comorbidities, and anatomical site of grafting. Mechanical tension at the graft-recipient interface has been shown to activate mechanotransduction YAP/TAZ signaling pathways, promoting myofibroblast differentiation and collagen overproduction independent of fibrosis^[152]39,[153]40. Similarly, prolonged inflammation due to bacterial colonization or immune dysregulation can exacerbate ECM disorganization and graft shrinkage^[154]41. Our study focused on fibrotic contraction due to its dominant role in long-term functional and cosmetic outcomes, particularly in full-thickness grafts. Furthermore, current clinical strategies for mitigating mechanical stress (e.g., splinting) and infection (e.g., antimicrobial dressings) are well-established, whereas pharmacological or bioengineered solutions targeting fibrosis remain limited^[155]5,[156]6. To address this gap in knowledge, our study employed genomic analysis of keloids to uncover the pathogenesis of skin fibrosis. We identified 60 downregulated keloid-related genes that were common to both datasets, with the KEGG pathway showing enrichment in the PPAR signaling pathway, regulation of lipolysis in adipocytes, and vibrio cholerae infection. Subsequently, a comprehensive analysis was conducted to construct the protein-protein interaction (PPI) network, which revealed ADIPOQ, FABP4, KRT7, LEP, and PIP as hub genes. These findings were further validated by external datasets. ADIPOQ, FABP4, and LEP are all adipokines, and KEGG analysis also revealed their involvement in adipogenic signaling pathways such as PPAR, suggesting that adipogenesis plays a crucial role in the treatment of skin fibrosis diseases. The NCoR/PPAR-γ pathway has been shown to be dysregulated in systemic sclerosis (SSc), with a mouse model of adipocyte-specific NCoR deletion demonstrating PPAR-γ activation and resistance to skin fibrosis. Pharmacological inhibition of PPAR-γ in this model reverses the antifibrotic phenotype^[157]42. Additionally, PPAR-γ plays a critical role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) by regulating liver adipogenesis, improving insulin resistance, reducing liver inflammation, mitigating oxidative stress, alleviating endoplasmic reticulum stress, and inhibiting liver fibrosis^[158]43. This suggests that PPAR-γ is a key regulator in fibrotic conditions and may serve as a potential therapeutic target for both skin fibrosis and liver fibrosis. Our findings revealed that SVF-gel treatment enhanced FABP4 expression in regenerating skin tissues, indicating increased adipogenic activity, and promoted a shift in macrophage polarization from the pro-inflammatory M1 phenotype (CD86-positive) toward the anti-inflammatory M2 phenotype (CD206-positive). These results are consistent with previous studies showing that increased FABP4 expression is associated with elevated secretion of adipokines, including adiponectin and leptin, which play important roles in modulating fibrosis and promoting tissue repair^[159]44. In addition, the observed macrophage polarization aligns with reports that an M2-dominant microenvironment supports improved wound healing and reduced fibrosis^[160]45. Additionally, the introduction of chyle fat into hypertrophic scar tissue resulted in a decrease in fibroblast density and quantity, as well as normalization of the arrangement, quantity, and morphology of type III collagen^[161]46. Adipocytes have been identified as a therapeutic target for anti-fibrosis, with adiponectin secretion being a key factor in this process. Studies in mice have demonstrated that the absence of adiponectin leads to an exaggerated dermal fibrotic response, while overexpression of adiponectin results in selective dermal white adipose tissue expansion and protection from skin and peritoneal fibrosis^[162]47. These findings underscore the beneficial role of adipocytes in anti-fibrosis and their significant contribution to preventing skin contracture. In summary, the utilization of SVF-gel in conjunction with skin grafts has shown improvements in early revascularization, collagen fiber density and regularity in the dermis, hastening the turnover of myofibroblasts and promoting adipogenesis, restructuring of dermal tissue, and resulting in a significantly increased grafted skin surface area (Fig. [163]8). The use of a nude mouse skin graft model was chosen to assess the effect of human-derived SVF-gel in improving skin graft contraction, as our intention is to prepare SVF-gel from autologous adipose tissue during clinical translation. Therefore, immunogenicity is unlikely to be a significant concern, and the graft-versus-host response is minimal. Fig. 8. [164]Fig. 8 [165]Open in a new tab Graphical abstract. Graph showing the role of SVF-gel in alleviating full-thickness skin graft contraction. This study provides promising insights into the potential of SVF-gel for reducing skin graft contraction, but several limitations should be considered. First, while the use of a nude mice model is common in research, it may not fully replicate the complexities of human skin. Differences in skin structure and disease progression between humans and rodents may limit the direct applicability of our findings to clinical settings. Second, the duration of the study may not adequately capture the long-term effectiveness and safety of SVF-gel treatment. Extended follow-up is necessary to assess the sustainability of the observed effects. Third, accurately characterizing the composition of SVF-gel is challenging. The difficulty in quantifying the cellular components, extracellular matrix, and soluble factors within the gel can introduce variability, which may impact clinical trial outcomes. This variability could complicate the ability to draw definitive conclusions. To address these issues, future research should focus on establishing standardized measurement methods for SVF-gel, which will be critical for its clinical application in treating skin graft contraction. These limitations underline key areas for future investigation into the therapeutic use of SVF-gel. Conclusion The SVF-gel represents an innovative approach to stem cell therapy and shows potential as an effective treatment for skin grafts. Its mechanisms of action involve enhancing early revascularization, improving collagen fiber density and regularity in the dermis, accelerating the turnover of myofibroblasts, promoting adipogenesis, and restructuring dermal tissue. Furthermore, it has the potential to increase the number of hair follicles in the dermis, leading to improved aesthetic outcomes post-transplantation. Future research efforts will be directed towards elucidating the role of adipokines in the treatment of skin grafting complications. Acknowledgements