BCMs provided by Nameide Biotechnology of Shandong, China, were evaluated through (FT-IR) spectra. The scan was conducted from 4000 cm⁻¹ to 500 cm⁻¹ with a resolution of 0.5 cm⁻¹ for each measurement. To ensure data credibility, we conducted three random sample extraction tests.

The pores of the BCM were evaluated through a porosity analyzer (PSMA-10, China). The BCM was immersed in water for 24 h. Isobutanol and deionized water were mixed at 1:1 for 4–8 h. The upper liquid (alcohol phase) of the mixture was taken to soak the BCM for about 510 min. Finally, the BCM was installed on the aperture detector, the software was opened for testing, and the results were processed and analyzed. To ensure data credibility, we conducted three random sample extraction tests.

The BCM was soaked in water for 24 h, fixed on the test bench of the SEM (Quanta 200, USA), and observed after gold spraying. To ensure data credibility, we conducted three random sample extraction tests.

BMSCs (Cyagen, USA) derived from Balb/c mice were cultured in complete DMEM/low-glucose medium (HyClone, USA) containing 10% fetal bovine serum (Gibco, USA) and 1% 100 U/ml Penicillin-Streptomycin (Gibco). In BMSCs of P2 generation with a degree of integration up to 80% at the logarithmic growth stage, they were labeled by Cell Tracker TM CM-Dil (MKbio, China) according to the manufacturer's instructions. Specifically, CM-Dil was dissolved in 1 mg/ml dimethyl sulfoxide as a working solution and added to the BMSCs (3 ml of working solution per Petri dish). Subsequently, cells were incubated at 37 °C for 3 min and then incubated for another 15 min at 4 °C. Light should be avoided during labeling. Finally, BMSCs were washed with phosphate-buffered solution (PBS) and incubated with complete DMEM/low-glucose medium (Thermo Fisher Scientific, MA, USA). The culture medium was replaced once a day, and the subculture was used when the degree of fusion reached 80%–90%. BMSCs of the fifth–sixth generations were used for further utilization (16).

BCMs (NanoMed Biotech, China) fermented by Acetobacter xylinum (17) were cut into round pieces (1.5 cm in diameter) under sterile conditions and rehydrated in serum-free DMEM/low-glucose medium (containing 1% 100 U/ml Penicillin-Streptomycin) for 24 h. The liquid on the surface of the BCM was dried and spread at the bottom of the 24-well plate (one piece per well). Subsequently, CM-Dil-labeled BMSCs were injected into the BCM by multi-point injection method at a density of 5 × 10⁴ cells/well. Then, 500 μl of serum-free DMEM/low-glucose medium was added to each well. The 24-well plate was incubated in a constant temperature incubator at 37 °C, 5% CO₂ with saturated humidity for 24 h. Finally, 50 μl of Cell Count Kit-8 (CCK-8, Elabscience, China) detection reagent was added to each well. The absorbance at 450 nm (OD₄₅₀) was measured by a microplate reader (Thermo Fisher Scientific) at 0 h, 1 h, and 2 h.

As described in 2.5, the BCMs were cut into round pieces (1.5 cm in diameter) under sterile conditions and rehydrated in serum-free DMEM/low-glucose medium for 24 h. The liquid on the surface of the BCM was dried and transferred to the 6-well plate (one piece per well). Subsequently, CM-Dil-labeled BMSCs were injected into the BCM at a density of 1 × 10⁵ cells/well. Thereafter, 2 ml of serum-free DMEM/low-glucose medium was added to each well. The 6-well plate was incubated for 24 h. The status of CM-Dil-labeled BMSCs on the BCM scaffolds was observed under an inverted fluorescence microscope (Nikon, Japan), and scaffolds with good activity were selected as bioactive wound dressings (BWDs) for further utilization (18).

As described in 2.6, the BWDs were prepared and transferred in 6-well plates, and 2 ml of serum-free DMEM/low-glucose medium was added to each well. The 6-well plate was incubated for 24 h. The culture medium was collected and filtered into a conditioned medium (CM) with a 0.22-μm filter and stored at −80 °C for further utilization.

To assess the effect of BWDs on wound healing, we screened two kinds of cells closely related to wound healing (i.e., human dermal fibroblasts [HDFs] and human vascular endothelial cells [HuVECs]) to detect the proliferation ability in vitro. Briefly, HDFs/HuVECs were seeded into 96-well plates at a density of 1 × 10⁴/well. CM and serum-free DMEM/low-glucose medium were added to the experimental group and control group, respectively, at a density of 100 μl/well. Subsequently, the 96-well plate was incubated for 24 h. Then, 10 μl of the CCK-8 solution was added to each well and incubated at 37 °C. The absorbance at 450 nm at 0 h, 1 h, and 2 h was measured by a microplate reader. Three repeated parallel replicates were used at least every time.

The scratch-wound assay was used to detect basic cell migration parameters of CM on wound healing-related cells. HDFs/HuVECs were seeded into 6-well plates at a density of 1 × 10⁵/well, and 2 ml of complete DMEM/low-glucose medium was added to each well. When cells reached 90% confluence, a thin “wound” was introduced by scratching with a 10–200-ml pipette tip. The cells shed due to streaking and were washed by PBS, and 2 ml CM or blank medium (as a vehicle) was added to each well. At 0, 6, 12, and 24 h, cells on the microscope were selected on field or fields of view to observe cell migration, and data were analyzed by ImageJ. Each condition was performed in triplicate (19).

Total RNAs were extracted by animal tissue/cell total RNA extraction kit (Tiangen, China), and 500 ng total RNAs were used as templates of reverse-transcription with the oligo-dT primer for the qRT-PCR. The primers were synthesized by Sangon Biotech (China), and the sequences are listed in Table 1. GAPDH was used as the reference gene for calculations. The relative expression value was calculated with the following formula: ΔCt = Ct (target gene)−Ct (reference gene), and the relative = 2^−ΔΔCt.

Six-week-old Balb/C male mice were randomly divided into three groups: control, BCM, and BWDs. Full-thickness skin defect models were made using a tissue punch on the back of the mice. Briefly, the mice were anesthetized by isoflurane inhalation, and the hair on the back was removed. A full-thickness skin defect model with a diameter of 15 mm was made using a tissue punch on the back of the mice. Then, the wound was covered with an aseptic dressing (control, saline gauze; BCM, and BWD). The radiation-sterilized anti-shrinkage aseptic ring (20 mm in outer diameter, 15 mm in inner diameter, and 2 mm in thickness) was sewn to the edge of each defective skin with a 4–0 mousse thread. Each silicone ring was sutured with eight sites for fixation (Figure 6A). At the end of the operation, the wound was covered with a 3 M transparent film.

The activities of mice and wound dressings were observed daily, and the dressing was removed on day 7. Wound healing was observed on days 0, 7, and 14 after the operation, and the wound healing rate was calculated and analyzed by ImageJ. The percentage wound closure was calculated as [(original wound area−wound area) / original wound area] × 100%. On days 7 and 14, the animals were killed with excessive anesthesia, and the wound samples were taken for further utilization. Each group contained five mice.

γ-Secretase inhibitor DAPT was purchased from AbMole (USA) and diluted with dimethyl sulfoxide (DMSO). In an inhibiting assay, HDFs were incubated with BWD-CM and DMSO or DAPT (5 μM) for 72 h. Then, the cells were harvested for qRT-PCR. In the in vivo experiments, full-thickness skin wounds in mice were treated with BWD and subcutaneous injection of 100 μl of DMSO or DAPT (10 μM).

Data were statistically analyzed using SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). All data are presented as the mean ± SEM (X¯ ± SX¯). Significance was determined by the analysis of variance or standard t-tests. P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, and NS indicated the absence of statistical difference.

When CM-Dil-labeled BMSCs were observed under a fluorescence microscope, red fluorescence was observed in some labeled cells at 0 h (Figure 2A). The number and brightness of labeled cells were significantly increased at 24 h (Figure 2B), the labeled cells had >80% of confluence at 48 h, and the fluorescence brightness was weakened (Figure 2C). Three visual fields were randomly selected under 10 × microscope for cell counting, and the cells labeled by CM-Dil had biological activity and could proliferate stably (Figure 2D). However, the fluorescence intensity of CM-Dil labeling decreased gradually with time. The above results provided a basis for follow-up short-term tracking of BMSCs on the BCM.

BMSCs were injected into the BCM by the multi-point injection method to prepare BWDs. BWDs were cultured in vitro for 3 days and then observed under a microscope. Interestingly, in this side of the graph, BMSCs grew radially around the puncture point and adhered to the BCM (Figure 3A). On the surface and inside the BCM, adherent cells, colony cells, and apoptotic non-adherent cells were observed. In Figure 3B, quantitative analysis of CCK8 showed a significant difference (P < 0.001) between the two groups. These results suggested that the BCM had biocompatibility and could promote the proliferation of BMSCs; thus, it could be used to prepare bioactive dressings.

Wound repair is closely related to the proliferation and migration of fibroblasts and angiogenesis. The BCM and BMSCs may play an important role in the proliferation and migration of fibroblasts and endothelial cells. To confirm this hypothesis, CM was used to treat HDFs or HuVECs. We conducted a CCK8 assay to detect cell proliferation and scratch-wound assay for migration. The BCM and BWD promoted the proliferation (Figure 4A) and migration (Figures 4C,E) of HDF at 24 h. BWD had a more significant effect on proliferation, but no difference was found in promoting cell migration. In the experimental evidence on HuVECs, the BCM and BWD had no significant effect on proliferation at 24 h (Figure 4B). However, the BCM and BWD promoted migration (Figures 4D,F) of HuVECs at 12 h, and the effect of BWD was even more significant. Taken together, these results suggested that BCMs and BWDs had good biocompatibility and could accelerate wound healing by promoting the proliferation and migration of fibroblasts and endothelial cells.

To further investigate the molecular biological evidence that the BCM and BWD regulate the biological behaviors of HDFs and HuVECs, we detected the expressions of COL-1 and VEGF-A by qRT-PCR. The figures show that the BCM and BWD could upregulate the expressions of COL-1 in HDFs (Figure 5A) and VEGF-A in HuVECs (Figure 5B). The effect of BWD was more obvious (Figures 5A,B). The results suggested that the BCM and BWD promoted extracellular matrix synthesis and angiogenesis to promote wound healing by regulating the behaviors of HDFs and HuVECs.

Gauze soaked with physiological saline, BCM, and BWD were used to interfere with full-thickness skin defect wounds in mice. The results showed that the wounds treated with BCM and BWD healed basically on day 14, and the effect of BWD was more significant (Figures 6A,E). The full-thickness skin of the wound was removed, fixed, dehydrated, and embedded into paraffin sections on day 7. Hematoxylin and eosin staining and Masson staining were performed to observe the angiogenesis and collagen formation. As shown in Figure 6C, compared with the control and BCM groups, the BWD group showed numerous closely arranged fibroblasts, abundant and orderly arranged collagen fibers, and more new capillaries and hair follicles. The re-epithelialization rates of all groups are presented in Figure 6F. The epidermal gaps were smaller in the BWD group on days 7 and 14 after wounding, which suggested more rapid re-epithelialization. The results showed that BWD could significantly improve wound healing.

To determine this conclusion from the molecular level, the wound samples of mice were taken on days 7 and 14, and the expressions of COL-1 and VEGF-A were detected by qRT-PCR. As shown in Figures 6G,H, both BCM and BWD upregulated the expression of COL-1 in the wound on days 7 and 14, and the effect of BWD was more obvious. Regarding VEGF-A, the BCM downregulated its expression on day 7, but the expression of VEGF-A was reversed and upregulated significantly on day 14. By contrast, the expression of VEGF-A in BWD-treated wounds was always at a high level. Overall, these results indicate that both the BCM and BWD could promote the healing of acute full-thickness skin defects in mice, and the effect of BWD was more significant, which was confirmed at the molecular level.

The Notch signaling pathway is involved in ontogeny and tissue regeneration. We speculated that the promotion of wound healing by the BCM and BWD was related to the activation of the Notch signaling pathway. Jagged-1 is an important ligand of the Notch signaling pathway, and Hes-1 is the downstream protein. To further determine this hypothesis, wound samples of mice were taken on days 7 and 14, and the expressions of genes related to the Notch signaling pathway were detected by qRT-PCR. The expressions of Notch-1, Jagged-1, and Hes-1 in the BCM group increased significantly on day 7 and decreased sharply on day 14. By contrast, the gene expression in the BWD group was stable at high levels (Figures 7A,B), which provided important insights that BWDs may promote wound healing by activating the Notch signaling pathway.

To determine whether BWD can promote wound healing in a Notch-dependent manner, we treated HDFs with DAPT, the γ-secretase inhibitor, to block the Notch receptor cleavage at the cell surface. HDFs were treated with DMSO or DAPT (5 μM), and the expression of genes related to the Notch signaling pathway and COL-1 were detected by qRT-PCR. We found that DAPT partly abolished the positive regulating effect on COL-1, genes related to the Notch signaling pathway (Notch-1 and Jagged-1), and expression of BWD on HDFs (Figure 7C).

Correspondingly, we performed DAPT-inhibition experiments in mice. Full-thickness skin wounds in mice were treated with BWD and DMSO or DAPT (10 μM). Pictures of wounds were collected on days 0, 7, and 14 after the operation, which also showed that DAPT partly abolished the positive regulating effect on wound healing of BWD (Figure 6B). HE staining and Masson staining were performed to observe the angiogenesis and collagen formation on day 7. BWD with DAPT showed a significant reduction of collagen fibers and angiogenesis (Figure 6D). We further verify this conclusion at the molecular level. The levels of Notch-1, Jagged-1, and Hes-1 decreased after adding the Notch inhibitor, DAPT, indicating that DAPT successfully inhibited the Notch signaling pathway. In addition, the expressions of COL-1 and VEGF-A were reduced after DAPT was added (Figure 7D). These results indicate that BWD can activate the wound healing capacity via the Notch signaling pathway, and these effects can be inhibited when DAPT was used, illustrating the role of the Notch signaling pathway in wound healing.