Human ADSC were isolated and cultured as previously described (Song Y. et al., 2023). Extract adipose stem cells from liposuction patients. Rinse with phosphate-buffered saline phosphate-buffered saline (PBS; Beyotime, China) and digest fat tissue with type I collagenase at 2 mg/mL for 45 min (Gibco, United States). Add fetal bovine serum (FBS; Gibco, United States) to stop the digestion. Screen through a 70 μm mesh filter then centrifuge 3,000 rpm for 5 min. Re-suspend precipitates in PBS and reseed into onto 10 cm culture dishes in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, United States), supplemented with 10% fetal bovine serum (FBS; Gibco, United States) and 1% penicillin/streptomycin (Gibco, United States) at 37°C in a 5% CO₂ humidified incubator (Thermo Fisher Scientific, United States). The medium was replenished every 2–3 days. When adherent cells reach about 80%–90% confluence, we passage them at a 1:2 or 1:3 ratio. After three passages, use ADSCs for further experiments. Inspect the cell morphology under the microscope. Lastly, identify ADSC’s surface markers CD90 (11-0909-42, Thermo Fisher Scientific, United States), CD45 (58-0459-42, Thermo Fisher Scientific, United States), CD105 (17-1057-42, Thermo Fisher Scientific, United States), CD31 (17-0319-42, Thermo Fisher Scientific, United States), CD29 (12-0299-42, Thermo Fisher Scientific, United States) and CD11b (12-0118-42, Thermo Fisher Scientific, United States) via flow cytometry.

After attaining 90%–100% confluence, the medium was discarded, and the cells were gently washed twice or thrice with phosphate-buffered saline (PBS; Beyotime, China) and lysed with a solution comprising 0.25% Triton X-100 (Beyotime, China) and 10 mM NH₄OH (Adamas, Switzerland) for 90s at 37°C (Kim et al., 2022). Upon completion of the decellularization process, super nuclease (Beyotime, China) was added and gently lysed at 37°C for 90 min. The solution was collected and frozen overnight at −80°C, and then lyophilized using freeze dryer (Scientz, China).

Quant-iT™ PicoGreen™ dsDNA assay kit (Thermo Fisher Scientific, United States) was used. Dilute the PicoGreen reagent 200-fold with 1X TE buffer. Dilute the 2 μg/mL DNA stock solution to 50, 5, and 500 pg/mL with 1X TE buffer. Then, add 100 μL of DNA dilution and 100 μL of PicoGreen dilution to each well of the 96-well plate. Incubate for 5 min at room temperature, avoiding light. After incubation, the fluorescence of the samples was measured using an enzyme marker (excitation ∼480 nm, emission ∼520 nm). The fluorescence value of each sample was subtracted from the fluorescence value of the reagent blank to make a standard fluorescence versus DNA concentration curve. Prepare 1 mg/mL ADSC-dECM solution. Add 100 μL ADSC-dECM solution and 100 μL PicoGreen dilution to each well of a 96-well plate. Avoid light for 5 min of incubation at room temperature and measure the fluorescence values with the same parameters using an enzyme labeler. Subtract the blank group’s fluorescence value from each sample’s fluorescence value. The DNA concentration of the ADSC-dECM solution was determined from the DNA standard curve.

Samples of ADSC and ADSC-dECM were treated with RIPA lysis buffer (Beyotime, China) containing a protein inhibitor cocktail (Beyotime, China) at 4°C for 30 min. The lysates were centrifuged at 12,000 rpm for 15 min, and the supernatant was collected. The protein concentration was measured with the BCA kit protein assay (Thermo Fisher Scientific, United States) to determine the total protein concentration.

Equal amounts of protein were loaded on 4%–20% Hepes-Tris gels (EpiZyme, China) and run using electrophoresis, and then transferred to a polyvinylidene fluoride (PVDF; EpiZyme, China) membrane. The membrane was blocked with 5% nonfat blocking grade milk and then incubated overnight at 4°C with the following primary antibodies: collagen type I (COL I; 66761-1-Ig, Proteintech, China), collagen type III (COL III; 22734-1-AP, Proteintech, China), collagen type IV (COL IV; AF0510, Affinity, United States), fibronectin (66042-1-Ig, Proteintech, China), laminin (23498-1-AP, Proteintech, China) and elastin (ab307150, Abcam, United States). The appropriate secondary antibodies (1:5,000, Cell Signaling Technology, China) were chosen for 2 h of incubation at room temperature. The density of the bands was measured by ImageJ software, and GAPDH (EpiZyme, China) was selected as the internal reference.

Commercially available ELISA-KITs (Boster, China) containing hVEGF, hPDGF-BB, hPDGF-AA, hEGF, hFGF-1, and hFGF-7 were employed to measure the levels of these six growth factors. According to the manufacturer’s instructions, the OD values of the supernatant from each well were determined using an enzyme label reader (Thermo Fisher Scientific, United States) at a wavelength of 450 nm. Standard solutions of known concentration were employed to construct the standard curve. The regression equation for the standard curve was calculated and the concentrations of the samples could be determined based on their OD values by the standard curve.

The protein solution was firstly digested into a peptide mixture with protease (Ragelle et al., 2017). Then, LC-MS/MS was performed using a Q Exactive mass spectrometer coupled with an Easy nLC (Thermo Fisher Scientific, United States). The peptide sample was loaded onto the C18-reversed phase analytical column (Thermo Fisher Scientific, United States) in buffer A (0.1% formic acid in HPLC grade water), and separated with a linear gradient of buffer B (80% acetonitrile and 0.1% formic acid) with a flow rate at 300 nL/min. The linear chromatographic gradient was achieved with linear increase of buffer B percentage. After that, the peptide entered into the Q Exactive mass spectrometer (Thermo Fisher Scientific, United States). The MS analysis was set for 60 min in a positive ion mode. MS data was acquired using a data-dependent top10 method dynamically choosing the most abundant precursor ions from the full scan (350–1,800 m/z) for HCD fragmentation. The raw data obtained was then imported into Proteome Discoverer 2.2 (Thermo Fisher Scientific, United States) for protein identification, then embedded Mascot 2.6 engines was used for database searches. Protein identification was performed using reviewed database.

Biologically active scaffolds were prepared by freeze-drying within a mold utilizing CMC powder and ADSC-dECM powder. Briefly, 2.5% of CMC was dissolved in PBS, and ADSC-dECM was added to a concentration of 0.1% w/v (1 mg/mL) with the inclusion of 2.5% CMC. Subsequently, the prepared solution comprising either CMC or ADSC-dECM-CMC was transferred into a 10cm mold, subsequently underwent freezing at −80°C for an extended period. After that, the freeze-drying process was initiated to yield the CMC patch or the CMC patch supplemented with ADSC-dECM.

The SEM (Hitachi, Japan) was used to examine the microstructure of the patch. Before observation, coat the freeze-dried patch with gold sputtering.

To estimate the effect of ADSC-dECM on cell proliferation, human keratinocytes cells (HaCaT), human fibroblasts (HFB) and human umbilical vein endothelia cells (HUVEC) were treated with ADSC-dECM at different concentrations (0, 10, 1, 100, 10, 1 ng/mL). The culture medium without ADSC-dECM was set as the control group. Cells were counted and seeded at a density of 2,000 cells per well in a 96-well plate and cultured in a 37°C, 5% CO₂ incubator. After 24 h, the cells adhered to the wall and then treated with ADSC-dECM. Following cultivation for 0, 6, 12, and 24 h, cell counting assay reagent (CCK-8; Solabio, Japan) was incorporated into the cell culture medium, post which it was incubated for an additional 2 h. Subsequently, optical density (OD) value of each well was measured at a wavelength of 450 nm using a microplate reader (Thermo Fisher Scientific, United States). The following formula was employed to assess the viability of cells: cell viability (%) = [(OD of the ADSC-dECM group - OD of the negative control group)/(OD of the control group - OD of the negative control group)] × 100%.

Cell migration was estimated using a scratch assay. HaCaT, HFB and HUVEC were cultured at 1 × 10⁵ cells per well (3 wells per group) until 100% fusion was achieved. Subsequently, scratch treatment was applied using a 200 μm micropipette tip. The scratched cells were washed with PBS and cultured in serum-free medium containing different concentrations of ADSC-dECM (0, 1, 100, 10 and 1 ng/mL). The culture medium without ADSC-dECM was set as the control group. The width of the scratch was visualized with a light microscope, and the wound closure rate was measured by ImageJ software at various timepoints extending from 0, 12, 24, 36, to 48 h.

This experiment involved the procurement of 15 six-week-old male C57BL/6J mice from GemPharmatech Company. All animal experiments were conducted in strict accordance with the guidelines for experimental animals and were approved by the ethics committee. The mice were housed under controlled conditions, with a temperature of 23°C ± 2°C, humidity of 50% ± 5%, and a light-dark cycle of 12/12 h.

Prior to the experiment, mice were anesthetized with isoflurane and depilated of their dorsal hair using depilatory cream. Iodophor was used to disinfect the skin of mice, and a 10 mm diameter skin punch was applied to the back area of the mice to form a circular wound. A retractor, needle holder, and sutures were used to suture an anti-contracture ring to the wound. The CMC patches and ADSC-dECM-CMC patches were cut into circular discs with a diameter of 12 mm and sterilized under UV light with a wavelength of 254 nm for 30 min. The mice were randomly divided into three groups (n = 5 per group) including control group treated with PBS, CMC group treated with CMC patches, and ADSC-dECM-CMC group treated with ADSC-dECM-CMC patches. After patch treatment, all mice received a dressing of an oily gauze and sterile gauze to maintain humidity and prevent infection. Then, an aseptic tape dressing was applied to prevent the mice from biting the skin defect area. The treatment was repeated for three consecutive days. The detailed evaluation of the wound’s morphology captured photographically on days 7, 10, 14, and 21, and were assessed by ImageJ software. Wound closure rate (%) = (S₀- S_t)/S₀*100% (S₀: wound area on day 0; S_t: wound area on day T).

Skin tissue specimens were collected from mice aged 7, 14, and 21 days and subjected to fixation with a 4% paraformaldehyde (PFA) solution (pH 7.40, 4°C) for 24 h, subsequently embodied in paraffin, and serially sectioned. The sections were then stained with hematoxylin-eosin (HE) and Congo red to assess the recovery phase of the skin tissue.

Immunohistochemistry was performed on the skin tissue sections. Simply put, the sections were rehydrated with an ethanol gradient and treated with 0.3% hydrogen peroxide and 0.3% Triton X-100 for 30 min, followed by three washes with PBS. The sections were then incubated with anti-CD31 (ab28364, Abcam, United States) and anti-Ki67 (ab15580, Abcam, United States) at 4°C overnight. On the subsequent day, the sections were washed three times with PBS and incubated with appropriate secondary antibody (1:500, ThermoFisher Scientific, United States) at 37°C for 30 min. And then, the sections were washed again for three times with PBS and stained with hematoxylin for 2 min. Lastly, the sections were dehydrated and analyzed under a fluorescence microscope.

Statistical analyses were conducted using SPSS 21.0 and Graphpad Prism 9. All experiments were replicated at least three times to ensure reliable results. Single-factor analysis of variance (ANOVA), parametric measurement of Bonferroni multiple comparison post hoc test, nonparametric measurement of Kruskal–Wallis test, Dunn post hoc test, and linear regression for construction of standard curves wereapplied appropriately. Statistical significance was determined by a p-value less than 0.05.

We used flow cytometry to identify ADSCs, and the analysis results showed that ADSCs highly expressed surface markers CD90, CD105, and CD29, while low expressed CD45, CD31, and CD11b (Supplementary Figure S1).

The fluorescence value of 1 mg/mL dECM solution was tested and compared with the standard curve to obtain a DNA residue of 1.57 ± 0.49 ng/mg (n = 5) for dECM, which is much lower than 50 ng/mg (Crapo et al., 2011) (Supplementary Figure S2).

The isolated ADSC were employed for the preparation of ADSC-dECM. Once the cells reached 100%, the DNA and nuclei were eliminated using the optimal decellularization method documented in literature (Kim et al., 2022). And we also optimized the preservation of the ECM content. A single 10 cm culture dish of cells yielded a substantial quantity of 8 mg dECM following the extraction of ECM and subsequent freeze-drying. To verify the preservation of the active ingredients, the protein contents of cells was analyzed before and after decellularization through the application of three methods, including WB, ELISA, and MS. As a result, the WB analysis revealed negligible changes in the expression of COL Ⅲ, laminin, and elastin, while the content of COL Ⅰ, COL Ⅳ and fibronectin decreased by 40%, 20%, and 55%, respectively, following decellularization (Figures 1A, B). The ELISA assay demonstrated an increase in the content of PDGF-BB and a corresponding decrease in VEGF, PDGF-AA, FGF-1 and FGF-7 after decellularization (Figure 1C). LC-MS/MS was further used to evaluate the composition and quantitative analysis and showed both ADSC and ADSC-dECM encompassed a multitude of collagen proteins, which indicating that the collagen components after decellularization were well preserved (Table 1).

In order to investigate the impact of ADSC-dECM on the behaviors of HaCaT, HUVEC and HFB, after coculture with ADSC-dECM with varying concentrations, cell proliferation and migration were evaluated. The CCK-8 assay was employed to detect the proliferation of these cells at measurement time points. The results showed ADSC-dECM could significantly promote cell proliferative rates in HaCaT, HUVEC and HFB; and the effect of promoting cell proliferation became more significant with increasing ADSC-dECM concentration from 1 to 100 ng/mL (Figure 2). However, with the further increase of concentration in these cells, the effect of promoting proliferation of ADSC-dECM was weakened (Figure 2). In addition, the cell migration results confirmed that ADSC-dECM treatment could significantly promote the migration of both HaCaT and HFB; whereas its effect on HUVEC exhibited a relative attenuation (Figure 3). These results demonstrated that ADSC-dECM treatment could significantly enhance the proliferation and migration of HaCaT, HUVEC and HFB.

The gross observations of ADSC-dECM powder and ADSC-dECM-CMC patch after undergoing lyophilization were shown in Figure 4. Microporous structures could be seen in SEM. The wound healing capacity of the ADSC-dECM was examined using the full-thickness skin wound model. In the normal wound model, 10-mm full-thickness skin wounds were produced on the dorsum of C57BL/6 mice, and these mice were randomly grouped and treated with the CMC patch, ADSC-dECM-CMC patch, or PBS. Images of the wound were obtained at days 0, 1, 3, 5, 7, 10, 14 and 21 (Figure 5A). After 10 days of treatment, the wound area of each group decreased, with the ADSC-dECM-CMC patch group having the highest wound closure rate and a smooth and glossy wound surface, indicating complete epithelialization and wound healing. However, the control and CMC patch groups exhibited small amounts of exudate and bleeding, with their healing rates being notably lower than that of the ADSC-dECM CMC patch group (p < 0.05). On day 14, both control and experimental groups achieved almost complete epithelialization.

The HE staining was further performed on the sample tissue on day 21, at which point the proliferation in the skin stopped and remodeling was active, with the aim of exploring the long-term effect of ADSC-dECM on wound healing (Figure 5C). The HE staining results showed that the skin of the control group was composed of immature skin tissue, forming scar tissue with only a small amount of skin appendages. However, the ADSC-dECM-CMC patch group showed more hair follicles on the wound skin, notably surpassing both the control and CMC groups. Furthermore, there was no notable disparity of epidermal and dermal thickness in the control and CMC groups; however, both were thinner than that of the control group. These findings collectively implied that ADSC-dECM-derived scaffolds exhibited regenerative potency and could elicit angiogenesis and wound retraction. Meanwhile, a reduction in the epidermal tissue thickness and an augmentation in hair follicles may facilitate wound remodeling.

Angiogenesis represents a pivotal component of the wound healing process. Vessels are capable of delivering progenitor cells, oxygen, and nutrients, thereby fostering cellular proliferation and supporting matrix restoration. CD31 is a specific marker for endothelial cells and can be used to detect neovascularization. As demonstrated in Figure 6A, the ADSC-dECM-CMC patch group exhibited a substantial augmentation in vascular volume detected via the CD31-positive vessel area. The ADSC-dECM-CMC group demonstrated a significant enhancement in vascularization at the 7- and 14-day marks relative to the control and CMC groups, as indicated by an increased vessel area and count. Notably, after 14 days, there was an observable augmentation in vessel size within the ADSC-dECM-CMC and Control groups, suggestive of progressive vessel maturation (Figures 6B–D). IHC staining with cell proliferation marker Ki67 was carried out on days 7 and 14 to further evaluate the proliferation ability. As shown in Figure 6F, we found that in comparison to other groups, the ADSC-dECM-CMC patch group exhibited a notable enhancement in Ki67 expression, while the control group had relatively fewer Ki67-positive cells. These data confirmed that ADSC-dECM-CMC patch could effectively promote cell proliferation and angiogenesis.