A raw chicken egg was washed and put into 1% acetic acid. After 24 h of soaking, the egg was washed using running water and the ESM peeled off and washed with distilled water. The ESM was then carefully cut into patches (3 × 1.5 mm) (Figure 1). The patches were examined by scanning electron microscopy (SEM).

The ESM was treated with dopamine (DA) and heparin as we have previously described (Yan et al., 2020). First, a tris(hydroxymethyl) aminomethane solution was prepared (10 mM, pH 8.5) as the buffer to dissolve the dopamine. The heparin solution (1 mg/ml) was prepared using the abovementioned buffer. The ESMs were immersed into the heparin solution at 4°C for 12 h, which was enough time for the heparin to covalently bond. These samples were named HESM.

Platelet adhesion assay was performed as we have previously described (Yan et al., 2020). The original ESM and HESM samples were used in the platelet adhesion tests. Single-donor human whole blood was purchased from Innovative Research. The blood was added into the centrifuge tube and followed by centrifuging at 2000 rpm for 10 min to obtain platelet-rich plasma (PRP). Then, 200 μl of PRP was added to each sample, and it was incubated at 37°C for 2 h. After incubation, the samples were rinsed three times with PBS and the absorbed platelets were fixed with 2.5 wt% glutaraldehyde at 4°C overnight. Then the samples were dehydrated with different concentrations of alcohol (50, 60, 70, 80, 90, and 100%) for 15 min each and sufficiently dried in a freeze dryer, then followed by platinum coating and imaging using SEM.

Experiments were carried out as we have previously described (Yan et al., 2020). The human umbilical vein endothelial cells (HUVECs) were purchased from Lonza. They were used for cell viability testing to assess the feasibility of ESM and HESM. The culture was performed in a thermostatic incubator at 37°C in a 5% CO₂ and 95% air environment. At confluence, HUVECs were dissociated with TrypLE Express enzyme (Gibco) to obtain suspended cells, which were centrifuged and resuspended in medium prior to cell seeding.

The HUVECs on the ESM and HESM patches were evaluated by phalloidin/4′,6-diamidino-2-phenylindole (DAPI) staining and the LIVE/DEAD assay. To further confirm the live and dead cells, fluorescence staining was performed on chosen samples using a LIVE/DEAD Viability/Cytotoxicity Kit (Life Technologies). Cell viability was determined after culturing for 7 days. A kit containing green fluorescent calcein-AM and red fluorescence ethidium homodimer-1 (EthD-1) was used to stain both live and dead cells simultaneously. The samples were immersed in a staining solution (300 μl/cm²) away from light for 20 min at 24°C and then imaged with a Nikon fluorescence microscope.

DAPI (Sigma-Aldrich) and phalloidin fluorescent staining (Biotium) were used for marking the cell nuclei and cytoskeleton f-actin molecules, respectively, to visualize the HUVECs morphology on the samples. The samples with HUVECs were washed three times, then fixed with 4% paraformaldehyde in PBS for 15 min on ice, followed by a PBS rinse, and then permeabilized with 0.5% Triton X-100 in PBS for 10 min. Next, red fluorescent phalloidin solution was used to stain the cell cytoskeleton for 20 min and blue fluorescent DAPI solution used to stain the cell nuclei for 5 min; these were carried out both away from light and at room temperature. The staining processes followed the manufacturer's recommendations. The images were then captured at day 7 using the same fluorescence microscope.

The decellularization procedure was carried out as we have previously described (Bai et al., 2021e). Briefly, the thoracic aorta (TA) was harvested and incubated in 10 ml of sodium dodecyl sulfate buffer (1.8 mM sodium dodecyl sulfate, 1 M NaCl, and 25 mM EDTA in PBS) for 24 h and then rinsed with PBS; the decellularized TA was cut into patches (3 × 1.5 mm).

Male Sprague-Dawley rats (aged 6–8 weeks) were used and anesthetized with an intraperitoneal injection of 10% chloral hydrate (Wu et al., 2021). Adequate anesthesia was confirmed by a lack of response to a toe and tail pinch, ointment was placed on the eyes to prevent dryness while the animals were under anesthesia, and the ventral abdomen hair was removed using a hair remover while wearing sterile gloves. For postoperative analgesia, buprenorphine was given at 0.1 mg/kg intramuscularly, no less than every 12 h for 24 h following the surgical procedures. The status of the animal was checked every day in the animal room, ensuring proper recovery from the perioperative period as well as adequate treatment of postsurgical pain. The aorta patch angioplasty model was performed as we have previously described (Bai et al., 2017b). The microsurgical procedures were performed aseptically using a dissecting microscope (Nikon, Japan). The TA, ESM, and HESM patches (approximately 3 × 1.5 mm²) were implanted to the infrarenal aorta of the rats using continuous 11-0 nylon sutures; the eggshell side of the ESM was facing the aortic lumen. The rats were sacrificed on postoperative day 14, and the patches were explanted for analysis. No immunosuppressive agents, antibiotics, or antiplatelet agents were administered at any time.

The rats were anesthetized with an intraperitoneal injection of 10% chloral hydrate, and the tissues were fixed with transcardial perfusion of PBS, followed by that of 10% formalin. The tissues were removed and fixed overnight in 10% formalin followed by a 24 h immersion in 70% alcohol. They were then embedded in paraffin and sectioned (4-μm thickness). The tissue sections were deparaffinized and stained using the hematoxylin and eosin (H&E) staining kit (Baso, Zhuhai, China) according to the manufacturer's recommendations. Neointimal thickness was the mean of the measurements taken from the edge of the surface to the edge of the patch in three independent areas.

The sections were heated in a citric acid buffer (pH 6.0, Beyotime, Shanghai, China) at 100°C for 10 min for antigen retrieval. They were then treated with 0.3% hydrogen peroxide for 30 min and incubated overnight at 4°C with primary antibodies (Table 1). After overnight incubation, the sections were incubated with appropriate secondary antibodies (Table 1) for 1 h at room temperature and treated with the 3,3N-diaminobenzidine tetrahydrochloride horseradish peroxidase Color Development Kit (Beyotime, Shanghai, China) to detect the reaction products. Finally, the sections were counterstained with hematoxylin (Baso, Zhuhai, China). The positive cells were counted and expressed as n/mm².

Tissue sections were deparaffinized and then incubated with primary antibodies (Table 1) overnight at 4°C. The sections were incubated with secondary antibodies (Table 1) for 1 h at room temperature; subsequently, the sections were stained with the fluorescent dye DAPI (Solarbio, Beijing, China) to stain cellular nuclei. The positive cells were counted and expressed as n/mm².

Data are expressed as mean ± standard errors of mean. Statistical significance for these analyses was determined using t tests (Prism 6; GraphPad Software, La Jolla, CA, United States). p values <0.05 were considered significant.

A chicken egg was immersed in 1% acetic acid solution; after 24 h of soaking, bubbles were observed from the egg shell, and the outside part of the egg shell dissolved; the egg became soft and smooth (Figure 1). Then, the ESM was cut into patches and implanted to the rat abdominal aorta, and decellularized rat TA patch was used as the control. After 14 days, the ESM was incorporated to the native aorta and capsuled by newly formed tissue (Figure 1). H&E staining showed neointima formed on the luminal side of the TA and ESM patches (Figure 2A), and the neointimal thickness was similar in both groups (Figure 2B). In the TA patch, there were few cells infiltrated into the TA patch, but there were more cells infiltrated into the ESM patch compared to the TA patch (Figures 2A,C). A thick adventitia was observed in both the groups, and there was no difference in the number of newly formed capillaries in both the patches (Figures 2A,D).

We then examined the neointimal cells composition in the TA and ESM patches. CD31, alpha smooth muscle actin- (α-SMA), CD3-, and CD68-positive cells in the neointima in both groups were observed (Figure 3A). Immunofluorescence showed that there was a layer of von Willebrand factor (vWF)-positive cells on the luminal side, with several layers of α-SMA-positive cells in the neointima in both patches (Figure 3B). Similar re-endothelialization of the neointima was observed in both the TA and ESM patches; compared to the TA patch, the α-SMA-positive smooth muscle cells were not aligned evenly in the ESM patch (Figure 3C). CD34- and nestin-positive progenitor cells also participated in the neointimal formation, and there were a similar number of CD34-positive cells on the luminal side in the neointima of the TA and ESM patches (Figures 3B,C). We then examined the neointimal endothelial cell identity; Ephrin-B2 and dll-4 are arterial endothelial cell markers (Bai et al., 2018b). In both TA and ESM patch groups, the endothelial cells expressed Ephrin-B2 and dll-4 markers, and there was also a similar number of Ephrin-B2- and dll-4-positive cells in the neointima (Figure 3C). These results mean that the neointimal endothelial cells acquire arterial identity in the arterial microenvironment (Figure 3).

Cell proliferation plays a role in neointimal hyperplasia. We also examined the cell turnover in the neointima, and there were proliferating cell nuclear antigen (PCNA) and α-SMA dual-positive cells in the neointima in both the groups (Figure 4A). There was a smaller number of PCNA-positive cells, and PCNA and α-SMA dual-positive cells in the ESM patch when compared to the TA patch (Figures 4A,B). There were very few cleaved caspase-3-positive cells in both the groups (Figures 4A,B).

A coating of heparin to the biomaterials is commonly used; we have shown heparin coating in our previous researches (Bai et al., 2020; Yan et al., 2020; Wei et al., 2022). We then explored the influence of heparin conjugation to the ESM, and the ESM and HESM were examined by SEM; since heparin is a complex polysaccharide, a specific marker is not always used to detect it. There were heparin particles conjugated to the surface of the EMS (Figures 5A,B). The endothelial cells on the luminal side of the vessel also have an anticoagulation function, and these were always formed several days later, after the formation of neointima, when a prosthetic vascular graft is implanted in the animal's body (Bai et al., 2017a; Bai et al., 2020). Neointimal endothelial cells and heparin play the same role in preventing thrombus formation, so we only tested the platelet accumulation in this experiment. The platelets were found on the ESM surface, while no platelets were found on the HESM (Figures 5C,D). The HUVECs were cultured on the ESM and HESM patches. Fluorescence images of the HUVECs cultured on the ESM and HESM for 7 days showed fewer dead cells in the HESM patch (Figures 5C,D); there were also more cells on the HESM patch at day 7 (Figures 5C,D).

The ESM and HESM were also implanted in the aorta. There was a smaller mural thrombus area in the HESM patch when compared to the ESM patch at day 14 (Figures 6A,B); cells can also migrate into the HESM patch (Figure 6A). We also examined M1 and M2 macrophages in the neointima; there were fewer macrophages, fewer IL-10, and CD68 dual-positive cells (M1), and fewer CD68 and TNF-α dual-positive cells (M2) in the HESM neointima than in the ESM neointima (Figures 6A,B).