Cold-stimulated browning graft fat enhances burn wound recovery through accelerating DAMP-ADSC activation

Abstract

Suboptimal skin regeneration in patients with severe burns leads to significant trauma. Due to their favorable biological properties, fat grafts have been widely used in wound repair. The present study aimed to investigate the regenerative benefits of cold-stimulated fat graft in a contact burn model using C57BL/6J mice. Transplantation of browning fat grafts, with enhanced adipogenic capacity and decreased fibrosis, effectively promoted granulation tissue thickness and re-epithelialization areas. Cold-stimulated fat graft had a significant accumulation of ADSCs, which migrated into wound skin. In adipocyte-deficient mice, the impaired wound-healing phenotype was reversed by browning fat graft. Cold-stimulated fat also exhibited higher levels of damage-associated molecular patterns (DAMPs), which induced the proliferation of ADSCs and promoted ADSC proliferation and differentiation into mature adipocytes. Inhibition of DAMP-related signaling abolished the repair benefits of browning fat graft. In conclusion, transplantation of cold-stimulated fat represents a highly effective strategy for treating burn wounds through promoting DAMP signaling and dermal adipose remodeling.

Graphical Abstract

Browning fat graft improves burn wound healing through regulating DAMP-ADSC axis. Cold-stimulated browning subcutaneous fat graft, exhibited excellent graft feature, accelerated burn skin repair via supplying ADSCs and DAMPs. In wound skin, increased DAMPs further induced the proliferation of ADSCs, and differentiated ADSCs to mature adipocytes.

Introduction

Burns represent a class of injuries that require critical medical attention, often associated with high morbidity and mortality. Severe burns trigger immune-inflammatory responses, metabolic disturbances, and distributive shock, which are difficult to control and may lead to multiple organ failure. ¹ Similar to other types of wounds, burn healing involves multiple highly coordinated and overlapping phases: the inflammatory phase, cellular infiltration, matrix synthesis phase, re-epithelialization phase, and tissue remodeling phase. ² Effective strategies to improve these processes are therefore critical for wound healing.

In recent years, autologous fat grafting has attracted considerable attention in both research and clinical applications. ³ Studies have reported that fat grafting enhances wound angiogenesis and improves burn scar recovery. ⁴ Adipocytes are recognized as key participants in skin repair and regeneration. Previous studies have focused on dermal white adipose tissue (dWAT), with ample evidence supporting a significant correlation between dermal fat and tissue repair efficacy.^{5 –7} In addition, adipose-derived stem cells (ADSCs), recognized for their multipotent differentiation potential and abundant availability, have emerged as promising candidates in grafting and tissue repair. ⁸ However, multiple clinical studies consistently indicate that ADSCs do not persist long-term at the recipient site, and their proliferative and migratory capacities are impaired in the wound microenvironment, significantly limiting therapeutic outcomes.^9,10 Therefore, developing more effective treatment strategies to enhance the therapeutic benefits of ADSCs is of great clinical importance.

Upon injury, innate immune system is immediately activated, leading to local vasodilation and the recruitment of monocytes, macrophages, and neutrophils to the injury site, initiating the inflammatory phase. These cells release inflammatory cytokines, while simultaneously clearing tissue debris and pathogens from the wound. ¹¹ Endogenous damage-associated molecular patterns (DAMPs), serving as key initiators of inflammatory responses, are primarily released by damaged or necrotic cells as well as activated immune cells.^12,13 Several findings highlighted the considerable potential of DAMPs in wound repair.^{14 –16} Nevertheless, the role of graft-derived DAMPs in regulating wound repair and stem cell activity within the burn wound microenvironments remains inadequately investigated.

Recent studies have begun to link adipose browning to the regulation of ADSC stemness. Loss of the tumor suppressor Pdcd4 in mice enhances ADSC stemness, as evidenced by increased expression of CD105, CD90, Nanog, and Oct4, as well as enhanced colony-forming ability, and also promotes a white-to-beige adipocyte conversion program in ADSC-derived adipocytes, indicating that beige-prone ADSCs are intrinsically more stem-like. ¹⁷ In parallel, cold exposure induces beige adipogenesis from adipose progenitors and remodels the adipose stem cell niche by increasing vascularization, sympathetic innervation, and thermogenic adipokine secretion.^{18 –21} Among these secreted factors, CXCL12 has recently been identified as a cold-induced chemokine enriched in brown adipocytes,^{22 –24} while ADSCs express its receptor, CXCR4, and depend on CXCL12/CXCR4 signaling for migration and tissue repair.^25,26 Together, these findings supported the concept that cold-stimulated browning created a thermogenic, CXCL12-rich niche that favors ADSC survival, retention, and maintenance of stemness.

Research has confirmed that both white adipose tissue and browning adipose tissue contain ADSCs with similar multi-differentiation potential. But browning fat-derived ADSCs exhibit a stronger propensity to differentiate into functional brown adipocytes. ²⁷ Therefore, this study aimed to transplant cold-stimulated adipose tissue adjacent to wound skin, and investigate burn wound recovery. By analyzing the morphology and functional changes in both fat graft and wound skin tissues, the present study provided a theoretical basis for the application of browning fat graft in promoting tissue repair.

Result

Cold-stimulated fat graft exhibits improved adipogenic capacity and reduction of fibrosis

Our previous study has already demonstrated cold-stimulated fat graft had higher adipose retention after transplantation in mice for 8-week. ²⁸ However, adipose remodeling in short-term transplantation remained unclear, especially in the burn wound area. To this end, the mice with burn wound were transplanted with fat graft (Figure S1). Compared with normal inguinal white adipose tissue (iWAT), mice with cold challenge exhibited significantly smaller adipocyte with multilocular structure (Figure S2A). The relative expression levels of browning markers, including UCP1, Prdm16, PGC1-α, SCA1, CD81, and PDGFRα were significantly increased in the cold-stimulated iWAT (Figure S2B, p < 0.01). After establishing 6 mm diameter burn wound models using heated copper rods, the recipient mice were transplanted cold-stimulated or normal iWAT adjacent to the wounds. Histological staining indicated the cold-stimulated grafts (COLD-g) exhibited superior structural integrity (Figure 1(a)), with smaller adipocyte size compared to room temperature-treated grafts (RT-g). The proportion of adipocytes with a diameter in the 20–40 μm range was increased, while that in the 60–90 μm range was decreased in COLD-g group (Figure 1(b)). The number of cystic structures with thick fibrous walls which were defined as oil cysts was significantly reduced (Figure 1(c), p < 0.05). Adiponectin is an adipokine exclusively secreted by mature adipocytes, and A-FABP is a key fatty acid-binding protein. ²⁹ The expression levels of adiponectin in the cold-stimulated adipose grafts were significantly upregulated, indicating that the grafts possessed stronger adipogenic capacity and better adipose tissue properties (Figure 1(d), p < 0.05).

Figure 1.

Cold stimulation of iWAT improves graft adipogenic capacity and reduces fibrosis levels. One week after fat transplantation adjacent to the wound, the graft fat was harvested for relevant verification. (a) Representative hematoxylin and eosin staining images of the transplanted adipose tissue harvested from recipients (Scale bar 100 μm; n = 6 mice per group). (b and c) Measurement of cell size in the adipose grafts and calculate the cell percentage of different size interval (b). Calculation of structures with a diameter larger than 90 μm were defined as oil cysts (n = 6 mice per group; (c)). (d) Immunoblot analysis of Adiponectin and A-FABP, along with the statistical analysis of their relative expression levels (n = 6 mice per group). (e) Representative Sirius red-stained images for assessing fibrosis levels, and statistical comparison of relative density levels (Scale bar 100 μm; n = 6 mice per group). (f) Immunoblot analysis of α-SMA and TGF-β1, along with the statistical analysis of their relative expression levels (n = 6 mice per group). (g and h) RNA transcriptional sequencing of adipose grafts (n = 4 mice per group). The heatmap displays the levels of adipose fibrosis markers, characterizing the fibrosis level of the grafts (g). The GSEA ridge plot reveals the expression levels of extracellular matrix and SMAD-related pathways in fat grafts (h). All data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ****p < 0.0001.

Sirius red staining showed that the fibrotic area of transplanted adipose tissue in the COLD-g group was much smaller than that in the RT-g group (Figure 1(e), p < 0.01). Transforming growth factor-β1 (TGF-β1) is a core signaling molecule regulating collagen synthesis, and α-smooth muscle actin (α-SMA) is a marker protein of key cells responsible for collagen secretion. ³⁰ Western blot results demonstrated that the expression levels of TGF-β1 and α-SMA were significantly downregulated (Figure 1(f), p < 0.05). Transcriptional sequencing analysis also showed that the expression of fibrosis-related genes, including members of the Tgf and collagen families, fibronectin, and other fibrotic markers, were remarkably decreased (Figure 1(g)). The GSEA ridge plot visualization results showed that the significant enriched pathways in fibrotic signaling, including collagen-containing extracellular matrix, extracellular matrix, and cell substrate adhesion signaling (Figure 1(h)). Here indicated cold-stimulated fat grafts had the significant improvement of transplantation-induced fibrosis. Collectively, these results indicated that transplantation of cold-stimulated beige adipose tissue results in improved graft quality.

Cold-stimulated fat graft enhances burn wound recovery in mice

The morphological changes in skin tissues were recorded on day 0, 3, 5, and 7, and tissues were harvested on day 7 after establishment of the mouse burn model with fat transplantation. Direct observation of wound photographs showed that the wound area in the COLD-g group was much smaller than that in the Sham group or RT-g group during the wound healing process (Figure 2(a) and (b), p < 0.01). Statistical analysis of histological staining demonstrated that cold-stimulated fat graft increased granulation tissue thickness in the wound skin, with a reduced un-epithelialized area (p < 0.01), showing better repair efficacy under microscopy (Figure 2(c)–(e), p < 0.0001). Sirius red staining and immunoblot analysis confirmed that transplantation of browning fat significantly enhanced the fibrosis level in wound skin, as evidenced by a significantly increased positive-staining area and relative density (Figure 2(f), p < 0.01), and higher expression levels of TGF-β1 and α-SMA (Figure 2(g), p < 0.01). Immunofluorescence staining of TUNEL confirmed browning fat graft decreased the percentage of apoptotic cells in wound skin (Figure 2(h), p < 0.01), compared with Sham group. These results demonstrated that browning fat graft effectively improved the recovery of burn wounds.

Figure 2.

Transplantation of browning iWAT accelerates the rate of wound healing. During the adipose transplantation period, wound photographs of mice were taken at regular intervals. Tissues were harvested on day 7, and the repair quality was evaluated through skin tissue analysis. (a) Direct photographic comparison of wounds at day 0, 3, 5, and 7 after adipose tissue transplantation adjacent to the wound. (b) Calculation of wound area based on direct photographs of each individual mouse. (n = 6 mice per group) (c) Representative hematoxylin and eosin staining images of wound skin tissues harvested on day 7 (Scale bar 500 μm). (d and e) Statistical analysis of the unepithelialized area and granulation tissue thickness of wound skin tissues on day 7 (n = 6 mice per group). (f) Representative Sirius red-stained images for assessing fibrosis levels, and statistical comparison of relative density levels (Scale bar 500 μm; n = 6 mice per group). (g) Immunoblot analysis of α-SMA and TGF-β1, along with the statistical analysis of their relative expression levels (n = 6 mice per group). (h) TUNEL staining characterizes apoptosis levels in wound tissues, with corresponding statistical analysis of relative percentages (Scale bar 200 μm; n = 6 mice per group). All data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ****p < 0.0001.

To further determine whether the early beneficial effects observed within 7 days could be maintained during the later stage of wound repair, we extended the observation period to 14 days after burn injury and fat transplantation. The morphological changes in skin tissues were recorded on days 0, 3, 5, 7, and 14, and tissues were harvested on day 14 after establishment of the mouse burn model with fat transplantation. Direct observation of wound photographs showed that the wound area in the COLD-g group was much smaller than that in the Sham group or RT-g group during the wound-healing process (Figures S3A and S3B, p < 0.05). Statistical analysis of histological staining demonstrated that the cold-stimulated fat graft increased granulation tissue thickness in the wound skin, with an increased re-epithelialized area (p < 0.05), showing better repair efficacy under microscopy (Figures S3C–S3E, p < 0.01). Sirius red staining and immunoblot analysis confirmed that transplantation of browning fat significantly enhanced the fibrosis level in wound skin, as evidenced by a significantly increased positive-staining area and relative density (Figure S3F, p < 0.01). These results demonstrated that browning fat graft effectively improved burn wound recovery. Together, these findings demonstrate that browning fat grafts exert sustained pro-healing effects in burn wounds, promoting wound closure, epithelial regeneration, granulation tissue formation, and collagen-rich matrix remodeling at 14 days post-injury.

Dermal adipose tissue contributes to wound repair in browning graft fat-treated mice

Utilizing the public dataset (GSE8056), we found that adipose tissue-related metabolic pathways, including adipogenesis, estrogen response, and myogenesis, were significantly correlated with wound repair efficacy (Figure 3(a)). The expression of adipogenesis genes, such as adipoQ, Leptin, and Plin2, were significantly downregulated in postburn skin tissues (Figure 3(b)). Measuring histological changes on day 3, 5, and 7 of wound healing, we observed a decrease level of adipocyte number and perilipin expression during the healing process (Figure 3(c)–(e)).

Figure 3.

Cold-stimulated fat grafts promote dermal adipose remodeling and restore the structure of dermal adipose tissue beneath wounds. (a and b) The public human post-burn skin transcriptomic dataset GSE8056 was reanalyzed. GSEA curves display the enrichment of downregulated pathways in the HALLMARK gene set, involving adipogenesis pathway (a). Heatmap shows the expression levels of adipogenic differentiation and lipid metabolism-related genes in Control and Postburn groups (b). (c–e) Representative wound area, H&E-stained wound sections, and immunofluorescence staining of perilipin on day 0, 3, 5, and 7 ((c) Scale bar 200 μm). Quantitative analysis of wound area (d) and relative fluorescence density of perilipin (e) (n = 6 mice per group). (f and g) One week after adipose tissue transplantation adjacent to the wound, the wound tissues were collected. Representative H&E-staining of dermal sections (Scale bar 100 μm, (f)) and quantification of dermal adipocyte area (g). (h) Immunoblot analysis of Adiponectin and A-FABP, along with statistical analysis of relative expression levels. (n = 6 mice per group). (i) Representative images of perilipin-stained wound skin tissue and relative intensity analysis. (scale bar 100 μm; n = 6 mice per group). All data are presented as mean ± SEM; **p < 0.01, ****p < 0.0001.

To this end, we verified the dermal fat remodeling in wound skin after fat transplantation. The results showed that transplantation of cold-stimulated fat promoted the accumulation of adipocytes in wound skin (Figure 3(f) and (g), p < 0.0001). Meanwhile, the expression levels of adiponectin and A-FABP in COLD-g group were higher than those in the sham and RT-g groups (Figure 3(h), p < 0.0001)). Immunofluorescence staining further confirmed COLD-g treated wound skin had higher level of perilipin-positive density (Figure 3(i), p < 0.0001). During wound healing process, accumulation of stem cells is one of key contributors to wound regeneration. As shown in the public dataset (GSE241297), most gene levels of adipose-derived stem cell (ADSC) were increased during wound repair, accompanied with obvious gene reduction of mature adipocyte markers (Figure 4(a)). The immunofluorescence staining showed COLD-g-treated wound skin had higher CD34 expression (Figure 4(b), p < 0.01), and higher mRNA level of stem cell markers, including KLF4, C-Myc, OCT4, and Sox2 (Figure S4A).

Figure 4.

Browning fat grafts promote the generation of ADSCs and reverse the impaired wound healing caused by adipocyte deficiency. (a) The public datasheet of mouse wound skin tissue (GSE241297) is re-analyzed. Heatmap displaying temporal dynamics of ASPCs and mature adipocyte markers during the wound healing process. (b) One week after adipose tissue transplantation adjacent to the wound, the wound tissues were collected. Representative immunofluorescence images of CD34, a key stemness marker of ADSCs, with statistical analysis of relative fluorescence intensity. (scale bar 200 μm; n = 6 mice per group). (c and d) Wound images of wild-type (WT), Bscl2⁻/⁻ (KO), WT mice transplanted with Cold-g (WT-g), and KO mice transplanted with Cold-g (KO-g) on day 0, 3, 5, and 7 (c), with quantification of wound area (n = 6 mice per group; (d)). (e–g) Representative images of H&E staining and Sirius red staining on day 7 after wound (scale bar 500 μm; E), with statistical analysis of re-epithelialized area (f) and relative density of collagen deposit (n = 6 mice per group; (g)). (h) Immunoblot analysis of TGF-β1 and α-SMA expression with statistical analysis of relative expression levels. (n = 6 mice per group). All data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

To verify the roles of adipocyte and ADSC in burn wound repair, an adipocyte-specific Bscl2-deficient mouse model was established. The gross observation of wound remodeling revealed that the wound recovery effect in knockout (Bscl2 KO) mice was poorer than that in wild-type (WT) mice, with larger wound areas on day 5 and day 7 (Figure 4(c) and (d), p < 0.01). Transplantation of browning fat obviously accelerated wound recovery in both WT and KO mice (Figure 4(c) and (d)), and increased re-epithelialized area and fibrotic formation (Figure 4(e)–(g)). The fibrosis level of burn wounds in KO mice was weakened, as shown by decreased collagen accumulation (Figure 4(e) and (g), p < 0.0001), TGF-β1 ((Figure 4(h), p < 0.01) and α-SMA (Figure 4(h), p < 0.01). Browning graft fat enhanced fibrotic substance and TGF signaling activity in both WT and KO mice (Figure 4(e)–(h)). More importantly, the CD34-positive cell number was decreased in wound skin from KO mice, but browning graft fat induced ADSC accumulation (Figures S5A and S5B). The proliferative signaling, such as AKT and ERK activity, was consistently upregulated in browning graft fat-treated mice (Figure S5C). These results identified dermal adipose homeostasis was one of critical contributors to burn wound repair, and browning graft fat might support the accumulation of ADSC in wound skin tissue.

Browning graft fat-derived ADSC migrates into wound skin

To disclose the stemness activity in graft fat, transcriptional sequencing of ADSC markers was conducted in RT-g and COLD-g. The expression of key stem markers, such as Cd44, Cd34, Itga5, and Pdgfrα, were upregulated in COLD-g-treated wound mice (Figure 5(a)). Browning graft fat had higher number of CD34-positive stem cell (Figure 5(b), p < 0.05) and mRNA level of stem cell markers (Figure 5(c)). Then, the ZsGreen-labeled ADSCs were injected into the browning graft fat prior to transplantation. Prior to this, fluorescence microscopy revealed that ZsGreen-labeled ADSCs exhibited significant intrinsic fluorescence compared to the PBS control group, indicating successful labeling (Figure 5(d), p < 0.0001). At day 7 post-transplantation, there was a remarkable accumulation of ZsGreen-positive ADSCs in the wound skin, which was further supported by the microscopic detection of green fluorescence in wound skin sections (Figure 5(e) and (f), p < 0.0001).

Figure 5.

Migration of fat graft-derived ADSCs to skin wound. (a) One week after adipose tissue transplantation adjacent to the wound, adipose grafts were harvested for relevant verification. Heatmap displaying the markers of ASPCs in transplanted fat. (b) Representative immunofluorescence images of CD34 with statistical analysis of relative fluorescence intensity (scale bar 100 μm; n = 6 mice per group). (c) Expression levels of stemness factors KLF4, SOX2, c-Myc, and OCT4 (n = 6 mice per group). (d) Representative fluorescence images and quantification of relative fluorescence intensity of ZsGreen-labeled ADSCs prior to injection (scale bar 400 μm; n = 6). (e and f) Fluorescence images (e) and quantification of relative fluorescence intensity (f) of ZsGreen-labeled ADSCs in frozen sections of harvested wound tissues at day 7 post-transplantation (scale bar 400 μm; n = 6 mice per group). (g). Immunoblot analysis of proliferative signaling, including p-AKT and p-ERK expression with statistical analysis of relative expression levels (n = 6 mice per group). (h). Relative mRNA levels of stemness markers. (n = 6 mice per group). All data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Immunoblot analysis of wound skin confirmed the upregulated activities of proliferative AKT and ERK signaling in browning graft fat (Figure 5(g), p < 0.01). The mRNA levels of stemness markers were also significantly increased in COLD-g group (Figure 5(h), p < 0.01). The migration of graft fat-derived ADSC into burn wound tissue enhanced proliferative activation and wound recovery.

Elevation of DAMP-ADSC activation confers to burn wound repair

Although graft fat-derived ADSC contributed skin regeneration, the upstream signaling regulating ADSC homeostasis remained unclear. Volcano plot analysis showed many factors were upregulated in COLD-g group, especially labeled as upregulated secretory proteins (Figure 6(a)). KEGG pathway analysis consistently showed enrichment of inflammatory signaling pathways, including IL-17, TNF, and NF-kappa B signaling, which also contributed to damage-associated molecular patterns (DAMPs) formation (Figure 6(b)). In detail, genes involved in DAMP signaling were obviously upregulated in browning graft fat (Figure 6(c)). The immune microenvironment alterations were further analyzed by xCell. The COLD-g group had higher scores of ImmuneScore, StromaScore, and MicroenvironmentScore values compared with the RT-g group (Figure S6A). Analysis of immune cell types indicated several pro-inflammatory cells, such as neutrophils, DCs, and CD4-positive cells, enriched in COLD-g (Figure S6B). Real-time PCR further identified browning graft fat had higher expression levels of DAMP-related genes, especially increased mRNA of S100A8 and Cxcl1 (Figure 6(d), p < 0.0001). Correlation analysis between cell-type abundance and DAMPs showed both S100A8 and Cxcl1 expression were closely associated with the abundance of neutrophils and monocytes (Figure S6C). COLD-g also secreted higher levels of S100A8 and Cxcl1 in ex vivo graft fat culture compared with RT-g (Figure 6(e)).

Figure 6.

Induction of graft-derived DAMPs promotes the proliferation and differentiation of ADSCs. (a) RNA transcriptional sequencing of adipose grafts (n = 4 mice per group). Volcano plot displays upregulated secretory proteins in the COLD-g group based on HPA secretome data after human-mouse homolog mapping. The DAMP-related proteins are highlighted in red. (b) KEGG GSEA bubble plot shows enrichment of DAMP-related recognition and activation signaling pathways. (c) Heatmap of DAMP signaling-related gene expression levels in fat grafts. (d) Relative mRNA levels of representative DAMPs, including S100A8, S100A9, IL-1β, IL-6, and Cxcl1 in fat grafts. (e) Concentrations of S100A8 and Cxcl1 in conditioned medium from culturing fat grafts. (f–i) ADSCs are incubated with conditional medium from culturing fat grafts, and specific antibodies were utilized for blocking S100A8 and Cxcl1. Measurement of cell viability (f). (g) EdU staining of proliferative ADSCs and quantification of EdU+ cell percentage (scale bar 100 μm). (h) The ADSCs are differentiated into mature adipocytes with conditional medium. Oil Red O staining of mature adipocytes, and quantitative analysis of relative density (scale bar 100 μm). (i) Relative mRNA levels of mature adipocyte markers, including Fasn, Adipoq, and A-fabp. (n = 5 individual experiments per group). All data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Then, we collected the conditional medium (CM) from culturing RT-g and COLD-g, and incubated the CM with primary ADSC. COLD-g remarkably stimulated the proliferative activity, as measured by cell viability and EdU staining (Figure 6(f) and (g), p < 0.0001). However, blockage of S100A8 and Cxcl1 inhibited the proliferation of cultured ADSC, as compared with COLD-g group (Figure 6(f) and (g), p < 0.001). The differentiation activity of mature adipocyte was also elevated in COLD-g-treated ADSC, but antibodies of S100A8 and Cxcl1 synergically inhibited adipocyte differentiation, as measured by lipid staining and adipogenesis gene levels (Figure 6(h) and (i)). These findings indicated browning graft fat-derived DAMP might enhance the proliferation and adipocyte differentiation of ADSC.

To specifically target DAMP signaling in the fat grafts, we employed Paquinimod, a specific inhibitor of S100A8/S100A9.^31,32 Following local administration of Paquinimod in the browning graft fat, the expression levels of Cxcl1, IL-6, and IL-1β in the grafts were significantly reduced (Figure S7A). This indicates that the drug attenuated the effector function of DAMPs mediated by S100A8/9. Furthermore, ADSC accumulation (Figure 7(a), p < 0.05) and stemness gene levels (Figure S7B) in the wound skin were suppressed. The proliferative pathways, including AKT and ERK signaling, were also obviously inhibited in Paq-treated COLD-g (Figure 7(b) and (c)). The protein levels of skin adipogenesis markers, such as adiponectin and A-FABP, were downregulated (Figure 7(b) and (c)), while mature adipocyte differentiation was also inhibited in the COLD-g + Paq group (Figure S7C, p < 0.05). Consequently, the burn wound recovery effect of browning graft fat was remarkably weakened after Paq intervention (Figure 7(d) and (e)). Both re-epithelialization and granulation tissue thickness were impaired in the COLD-g + Paq group (Figure 7(f)–(h)). Sirius red staining further identified the inhibitory effects of Paq on fibrotic formation in wound skin (Figure 7(i), p < 0.05), whereas the protein levels of TGF-β1 and α-SMA were downregulated (Figure 7(j)). Suppression of inflammatory DAMPs blocked the wound repair effects of browning graft fat, and activation of DAMP-ADSC signaling was one of the critical mediators in skin regeneration.

Figure 7.

Administration of Paquinimod abolishes the wound repair benefits of browning adipose graft in mice. (a) Immunofluorescence staining of CD34 and quantitative analysis of relative fluorescence intensity. (scale bar 100 μm; n = 6 mice per group). (b and c) Immunoblot analysis of stemness and adipogenesis markers (b), along with the statistical analysis of their relative expression levels (n = 6 mice per group; (c)). (d and e) Representative wound images at different healing stages (d) and corresponding wound area quantification (e) (n = 6 mice per group). (f–h) Representative H&E-stained images of wound tissues on day 7 (scale bar 500 μm; (f)), with statistical analysis of re-epithelialization rate (g) and granulation tissue thickness (n = 6 mice per group; (h)). (i) Representative Sirius red-stained images for fibrosis assessment with relative density quantification (scale bar 500 μm n = 6 mice per group). (j) Immunoblot analysis of α-SMA and TGF-β1, along with the statistical analysis of their relative expression levels. (n = 6 mice per group). All data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Discussion

Severe burns are among the most traumatic and physically debilitating injuries, affecting nearly every organ system. ² Early burn wound excision and skin grafting are common clinical practices that significantly reduce mortality and shorten hospital stays, thereby improving outcomes for patients with severe burns. ³³ However, delayed wound healing and poor quality of healed skin remain major challenges in burn management. Fat transplantation has been widely used as a modality for burn wound repair, with notable benefits such as promoting wound angiogenesis and improving scar healing.^34,35 To overcome the adverse effects of traditional fat transplantation, we adopted transplantation of cold-stimulated beige fat to enhance burn wound healing. The results showed that cold-stimulated browning iWAT markedly accelerated burn wound healing (including reducing wound area and increasing granulation tissue thickness) and improved repair quality (promoting collagen deposition and dermal fat remodeling). Mechanistic analysis found that graft fat-derived inflammatory DAMPs induced ADSC migration, proliferation, and differentiation into mature adipocytes, which supported dermal fat homeostasis and wound skin recovery.

In recent years, the regulatory effects of cold stimulation have garnered widespread attention. Numerous studies have demonstrated that cold exposure induces the browning of WAT, leading to the formation of beige adipose tissue characterized by multilocular lipid droplets and high expression of uncoupling protein 1 (UCP-1) .^{36 –38} BAT and browned WAT can improve metabolic health in obese patients or accelerate cachexia in cancer patients and burn survivors via thermogenesis.^{39 –41} Notably, skin injury triggers the browning of sWAT, and this transformation effectively enhances wound repair outcomes. ⁴² Our previous study also identified that browning graft fat had higher fat retention, and improved mouse metabolism after 8-week transplantation. ²⁸ Based on this, we verified the burn wound model treated with browning fat. During the 7-day burn wound healing process, browning graft fat effectively accelerated skin re-epithelialization and granulation tissue formation. Interestingly, transplantation of browning fat also improved burn wound recovery in adipocyte-deficient mice, which indicating that the accumulated ADSCs might migrate from graft fat. Using a cell-labeling approach, our results supported the migration of graft fat-derived ADSCs into wound skin.

Cold stimulation promotes β-hydroxybutyrate secretion from beige adipose tissue, which inhibits adipose progenitor cells from differentiating into myofibroblasts, thereby reducing graft fibrosis. ⁴³ However, in the inflammatory milieu of burn wounds, cold-stimulated grafts and migrating stem cells may release factors that activate fibroblasts and elevate TGF-β1 mediated collagen production, augmenting granulation tissue thickness.^{44 –46} These findings suggest that the enhanced repair efficacy is closely linked to the biological behavior of the transplanted tissue and its interaction with the wound environment.

Dermal WAT exhibits dedifferentiation and redifferentiation potential under specific conditions, ⁴⁷ suggesting that its regenerative abilities are closely linked to stem cell properties. ADSCs show great advantages and potential in promoting healing: they can accelerate the repair process, regulate the wound microenvironment, and directly differentiate into fibroblasts, keratinocytes, and other cell types to restore wound morphology and function.^{48 –51} Furthermore, numerous studies have shown that the maintenance and enhancement of ADSC stemness are important research contents topics in regenerative medicine and tissue repair, which can effectively promote the therapeutic effects of ADSCs.^{52 –54} Therefore, effective treatment methods to enhance the stemness of adipose stem cells in wounds are highly beneficial for tissue repair.

Skin wound healing is a complex biological process involving four phases: hemorrhage and exudation, inflammatory response, tissue proliferation, and wound remodeling. ⁵⁵ Inflammation plays a fundamental role in wound healing, serving as the primary defense mechanism against microorganisms. ⁵⁶ DAMPs are recognized by pattern recognition receptors, such as toll-like receptors (TLRs) and NOD-like receptors (NLRs). ¹ Ligand binding to TLRs and NLRs activates downstream inflammatory pathways, leading to the release of various inflammatory mediators. S100A8, a member of the DAMP family, has been shown to regulate the production of pro-inflammatory mediators. However, its role in wound healing remains relatively underexplored.^57,58 Previous studies have confirmed that S100A8 enhances wound healing efficiency in inflammatory environments. ¹⁵ DAMPs also exert significant regulatory effects on adipose tissue, modulating the release of inflammatory factors and directly influencing adipokine secretion levels. ⁴¹ The present study found that DAMPs, such as S100A8 and Cxcl1, induced the proliferation of ADSCs and promoted their differentiation into mature adipocytes. The migration of ADSCs might be attributed to browning graft fat-derived DAMPs, which exhibited higher levels of chemokines, including Cxcl1, Sdf-1α, Cxcl4, and Mcp-1.

Currently, clinical approaches for burn wound recovery include drug therapy, negative pressure wound therapy, and skin transplantation.^59,60 However, drug therapy carries the risks of adverse reactions, negative pressure therapy damages deep tissues, and skin transplantation faces challenges such as donor scarcity and immune rejection.^59,60 In contrast, beige fat transplantation offers the advantages of abundant sources, simple preparation, and low cost. Cold challenge, as the optimal an effective physiological strategy to initiate WAT browning, activates sympathetic nerves in a norepinephrine-dependent manner. ⁶¹ Additionally, our previous study found that cold-induced beige fat grafts exhibit higher retention rates and better structural integrity, facilitating improved graft survival and function. ²⁸ Moreover, unlike cell therapy, tissue-level adipose transplantation preserves a favorable microenvironment for adipocytes, better mimics natural components and reduces artificial drug or reagent intervention.

However, our study has limitations. For instance, despite standardized procedures to ensure consistent graft size during fat injection, there were slight deviations in injection sites and graft quality. There was a higher level of inflammatory response in wound skin at 7-day, indicating the critical role of immune response in skin regeneration. In the contrast to our previous study on fat retention at 8 weeks, which had lower level of inflammation in graft, it is necessary to identify the graft fat profiles in wound skin after long-term intervention.

In conclusion, the present study provided a novel approach for promoting wound recovery in burned skin. Our results showed that transplantation of cold-stimulated browning fat significantly enhanced DAMP-ADSC interaction and promoted dermal fat remodeling. These findings offered a new strategy for facilitating burn wound healing and contributed to the advancement of skin tissue engineering.

Materials and methods

Animal experiments

All animal operations have been reviewed and approved by the Shenzhen University ethics committee (approval No. IACUC-202200070). All surgeries were performed on male C57 mice (aged 10 week, 24–26 g), the mice were euthanized by using 3% pentobarbital sodium (0.03 g/ml, intraperitoneal injection). Afterward, hair removal cream to shave the backs of the mice. The metal column (copper column; diameter 0.6 cm; height 4 cm; weight 9.5 g) was heated in a metal bath (100℃) for 15 min, and then applied to the marked site on the back of the mice without pressure. Use a copper rod to iron for 15 s to make two deep second degree burn wounds (6 mm in diameter). Wound mice were divided into three groups: No intervention group (Sham), transplanted with normal fat group (RT-g), and transplanted with cold-stimulated fat group (Cold-g). In the fat graft mice, inguinal adipose tissue was transplanted adjacent to the wound, with a volume of 0.03 ml/wound. Digital photographs were taken on days 0, 3, 5, 7, and 14. On day 7 and 14, the mice were euthanized, and the samples were collected for further analysis. The detail procedures were illustrated in Figure S1.

Adipocyte-deficient mice (Bscl2 KO) and wild-type mice (WT) were generated by Cyagen Biosciences (Guangzhou). Male mice, aged 10-week, were underwent the same procedures for wound creation, transplantation of graft fat and tissue harvesting.

For Paquinimod intervention, burn wound mice were transplanted with cold-stimulated fat. On day 1, 3, and 5 after fat transplantation, Paquinimod (6 mg/kg, MCE, Cat#HY-100442) was injected into graft fat. Digital photographs were taken on days 0, 3, 5, and 7. On day 7, the mice were euthanized, and the samples were collected for further analysis.

Preparation of browning fat graft

The cage, food and water were pre cooled overnight at 4℃. Then, the donor mice, with two mice per cage, were housed in a 4℃ environment. After 48 h of cold stimulation, the mice were immediately euthanized for collecting the inguinal fat for the fat grafting surgery. The collected inguinal fat was placed in a culture dish, and the fat was minced with ophthalmic scissors for 10 min to prepare the fat graft material. After that, the minced fat was loaded into a 2 ml syringe and then injected into the burn wound.

Histology, hematoxylin-eosin, and sirius red

The harvested samples were fixed in 4% paraformaldehyde fix Solution overnight, dehydrated in graded ethanol (100%, 90%, 80%, 70%), transparent in xylene, and finally embedded in paraffin. About 5 μm-thick section were used for staining with hematoxylin (Sigma, #51275) and eosin solution (Sigma, #318906) and Sirius red staining (direct red 80, Sigma, #195251). H&E staining was performed to visualize the wound structure. Sirius Red staining to observe the deposition of collagen beneath the wound, and the positively stained areas were quantified using ImageJ. Cell diameter and the number of oil cysts were quantified in section stained with H&E solution. In a double-blind manner, 50 adipocytes were selected from each region of each sample using ImageJ for diameter measurement, with more than five regions chosen from each sample. Oil cysts were defined as cystic structures with a diameter greater than 90 μm and thick fibrous walls, and their quantification was performed.

Immunofluorescent staining

To further evaluate the specific structure of the sample, the sections near the wound center were blocked with 3% bovine serum albumin (BSA) for 1 h at room temperature, and then incubated with primary anti-f4/80 (Abcam, #ab6640), anti-CD34 (Cell Signaling Technology, #26233), anti-perilipin (Santa Cruz Biotechnology, #sc-390169) antibody at 4°C overnight. Subsequently, the sections were incubated with Alexa fluor 488- and Alexa fluor 633 conjugated secondary antibodies of the corresponding species (Invitrogen, #a11008 or a21052) for 1 h at room temperature. Finally, nuclei were stained with DAPI (Sigma, #f6057). The fluorescence density was quantified in images captured by fluorescence microscope.

Western blot analysis

Proteins were extracted from tissues by RIPA buffer (Thermo Fisher, #89901) containing complete protease inhibitor cocktail (Beyotime, # P1005). PVDF membrane (Millipore, #IPVH00010) was probed with anti-adiponectin (Abcam, #ab22554), anti-A-FABP (Santa Cruz Biotechnology, #sc-271529), anti-p-IκBα (Cell Signaling Technology, #2859), anti-IκBα (Cell Signaling Technology, #9242), anti-TGF-β1 (Abcam, #ab215715), anti-α-SMA (Abcam, #ab7817), anti-IL-1β (Cell Signaling Technology, #12242), and anti-β-tubulin (Cell Signaling Technology, #2146) antibodies. Then, the membranes were incubated with HRP-conjugated secondary antibodies of the same species (Cell Signaling Technology, #7074 or #7076). The protein bands were visualized with HRP substrate peroxide solution (Millipore) and quantified using ImageJ.

Quantitative real-time polymerase chain reaction

Total RNA was extracted by Trizol (Invitrogen, #15596018) and reverse transcribed into cDNA by using HiScript III RT SuperMix (Vazyme, #AQ602). Real-time PCR reactions were performed using QuantStudio 3 (Applied Biosystems). The relative levels of gene expression were calculated by the 2^−ΔΔCT method, after normalization with the abundance of 18s RNA. The primer sequences were listed in Table S1.

In vitro culture of ADSCs

The stromal vascular fraction (SVF) was isolated from mouse inguinal adipose tissue as described in our previous study, ⁶² with minor modifications. Briefly, male C57BL/6J mouse was euthanized, and inguinal fat pads were digested in 0.1% (w/v) collagenase type I (Sigma, #C0130) for 30 min at 37°C with gentle shaking. The digestion mixture was passed through a 100 μm cell strainer (BD Biosciences, #352360) and centrifuged at 800g for 10 min. The pellets were collected for culturing in DMEM medium with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin Solution. ADSCs were incubated with conditional medium from culturing fat grafts, and specific antibodies were utilized for blocking S100A8 and Cxcl1. For EdU incorporation, 5 μM EdU (Invitrogen, #C10337) was added to the ADSCs along with conditional medium. The ADSC was differentiated to mature adipocyte as described in our previous study, ⁶³ and the conditional medium was added in day 3 after starting differentiation.

In vivo ADSC tracking

ADSCs were transduced in vitro with recombinant AAV2/9 viral vectors encoding the ZsGreen fluorescent reporter gene to achieve stable genetic labeling. Prior to transplantation, the transduction efficiency and fluorescence intensity were confirmed by fluorescence microscopy. Following expansion, 5 × 10⁶ ZsGreen-positive ADSCs were injected into the adipose grafts. Additionally, 5 μm tissue sections were prepared and examined under a fluorescence microscope to evaluate the spatial distribution and density of the grafted ZsGreen⁺ ADSCs within the host tissue.

ELISA measurement of S100A8 and Cxcl1 in conditional medium

About 50 mg graft fat was collected and cut into smaller pieces, then incubated in DMEM medium with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin Solution for 24-h. The concentrations of S100A8 (Invitrogen, #EM67RB) and Cxcl1 (Invitrogen, #EMCXCL1) in conditioned medium were measured by commercial kits.

Publicly available cohort datasets and preprocessing

Bulk RNA-sequencing data were obtained from adipose graft tissues. Raw sequencing reads were first assessed for quality using FastQC, and adapter sequences and low-quality bases were removed by Trimmomatic. Clean reads were then aligned to the Mus musculus reference genome (GRCm39) using STAR (v2.7.10a), and gene-level counts were summarized using feature Counts (Subread v2.0.3). Gene expression levels were normalized as fragments per kilobase of exon model per million mapped fragments (FPKM). Genes with minimal expression (FPKM < 1 in all samples) were excluded from downstream analysis. Differentially expressed genes (DEGs) were identified using the DESeq2 R package (v1.38.3), with an adjusted p-value (FDR) < 0.05 and |log₂FC| > 0.5 as the significance threshold. A volcano plot was generated to visualize global transcriptional differences, highlighting upregulated secreted proteins. Secreted protein information was retrieved from the Human Protein Atlas (HPA) secretome dataset, and corresponding mouse orthologs were obtained using the biomaRt package (v2.58.0). Expression patterns of upregulated secreted proteins were visualized through heatmaps generated by pheatmap (v1.0.12). To explore the mechanisms related to fibrosis regulation, Gene Set Enrichment Analysis (GSEA, v4.3.2) was performed using KEGG and Hallmark gene sets, based on ranked gene lists from differential expression analysis. Enrichment results were visualized with ridge plots and bubble plots.

To further investigate adipogenic and regenerative transcriptional dynamics during wound healing, publicly available RNA-sequencing datasets were analyzed. The GEO dataset GSE241297, which profiles murine skin wound tissues across different healing stages, was downloaded from the NCBI Gene Expression Omnibus. Raw data were normalized and grouped by healing stage: 0d and 3d samples were merged as the Early stage, 6d as the Middle stage, and 9d as the Late stage. Expression levels of adipose stem/progenitor cell (ASPC) markers (e.g. Pdgfra, Cd34, Ly6a) and mature adipocyte markers (e.g. Adipoq, Plin1, Fabp4) were extracted, and their temporal expression patterns were visualized using heatmaps generated by the pheatmap (v1.0.12) R package. To validate the observed transcriptional trends in the wound model, bulk RNA-seq data from the current study (RT vs COLD groups) were processed in parallel, and ASPC marker expression patterns were analyzed using identical normalization and visualization pipelines. Additionally, the human dataset GSE8056, which contains transcriptomic profiles of skin after thermal injury, was reanalyzed to characterize gene expression associated with adipocyte metabolism. Normalized expression matrices were used for heatmap visualization of adipocyte-related genes, and GSEA (v4.3.2) was performed based on the HALLMARK gene sets from the MSigDB database to assess global pathway alterations. Enrichment plots were generated to illustrate pathway activity changes related to adipogenesis and metabolic regulation.

Given the hypothesis that browning graft fat released damage-associated molecular patterns (DAMPs), a curated list of DAMP-related genes was constructed. Expression of these genes was compared between groups and presented as clustered heatmaps. Cluster Profiler (v4.8.1) was used to analyze KEGG pathway enrichment of DAMP-related gene signatures. To evaluate immune microenvironment alterations, xCell (v1.1.0) was applied to the normalized bulk transcriptome data to infer the relative abundance of 64 immune and stromal cell types. Immune Score, Stroma Score, and Micro-environment Score were computed and compared between groups. Additionally, correlations between key cytokines (e.g. S100a8, Cxcl1) and immune cell subsets were evaluated via Spearman’s correlation and displayed as heatmaps. All bioinformatic analyses were conducted using R (v4.3.2) unless otherwise specified.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 9.5.0. All quantitative analyses of staining were conducted by investigators blinded to the experimental groups. Before applying parametric tests, data normality was assessed using the Shapiro-Wilk test to confirm that the data followed a normal distribution. Statistical significance was primarily evaluated using one-way or two-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons test for post hoc analysis. For comparisons between two groups, unpaired two-tailed Student’s t tests were used where appropriate. All data are presented as mean ± SEM, with specific sample sizes provided in each figure legend. p < 0.05 was considered statistically significant.

Supplemental Material

sj-docx-1-tej-10.1177_20417314261451461 – Supplemental material for Cold-stimulated browning graft fat enhances burn wound recovery through accelerating DAMP-ADSC activation

Supplemental material, sj-docx-1-tej-10.1177_20417314261451461 for Cold-stimulated browning graft fat enhances burn wound recovery through accelerating DAMP-ADSC activation by Wenhui Ma, Zhenyu Quan, Li Jiang, Pengya An, Qi Zhao, Yucheng Luo, Yueqi Zhang, Xiaohua Feng and Yong Pan in Journal of Tissue Engineering

Supplemental Material

sj-docx-2-tej-10.1177_20417314261451461 – Supplemental material for Cold-stimulated browning graft fat enhances burn wound recovery through accelerating DAMP-ADSC activation

Supplemental material, sj-docx-2-tej-10.1177_20417314261451461 for Cold-stimulated browning graft fat enhances burn wound recovery through accelerating DAMP-ADSC activation by Wenhui Ma, Zhenyu Quan, Li Jiang, Pengya An, Qi Zhao, Yucheng Luo, Yueqi Zhang, Xiaohua Feng and Yong Pan in Journal of Tissue Engineering