Biocompatible and antibacterial Flammulina velutipes-based natural hybrid cryogel to treat noncompressible hemorrhages and skin defects

Abstract

Hemorrhage, infection, and frequent replacement of dressings bring great clinical challenges to wound healing. In this work, Flammulina velutipes extract (FV) and hydroxyethyl cellulose (HEC) were chemically cross-linked and freeze-dried to obtain novel HFV cryogels (named HFVn, with n = 10, 40, or 70 corresponding to the weight percentage of the FV content), which were constructed for wound hemostasis and full-thickness skin defect repair. Systematic characterization experiments were performed to assess the morphology, mechanical properties, hydrophilic properties, and degradation rate of the cryogels. The results indicated that HFV70 showed a loose interconnected-porous structure and exhibited the highest porosity (95%) and water uptake ratio (over 2,500%) with a desirable degradation rate and shape memory properties. In vitro cell culture and hemocompatibility experiments indicated that HFV70 showed improved cytocompatibility and hemocompatibility. It can effectively mimic the extracellular matrix microenvironment and support the adhesion and proliferation of L929 cells, and its hemolysis rate in vitro was less than 5%. Moreover, HFV70 effectively induced tube formation in HUVEC cells in vitro. The results of the bacteriostatic annulus confirmed that HFV70 significantly inhibited the growth of Gram-negative E. coli and Gram-positive S. aureus. In addition, HFV70 showed ideal antioxidant properties, with the DPPH scavenging rate in vitro reaching 74.55%. In vivo rat liver hemostasis experiments confirmed that HFV70 showed rapid and effective hemostasis, with effects comparable to those of commercial gelatin sponges. Furthermore, when applied to the repair of full-thickness skin defects in a rat model, HFV70 significantly promoted tissue regeneration. Histological analysis further confirmed the improved pro-angiogenic and anti-inflammatory activity of HFV70 in vivo. Collectively, our results demonstrated the potential of HFV70 in the treatment of full-thickness skin defects and rapid hemostasis.

Keywords: Flammulina velutipes polysaccharides; cryogel; hemostasis; hydroxyethyl cellulose; wound healing.

FIGURE 1

Preparation process and biological function of HFVs.

FIGURE 2

Appearance, physical characterization, and mechanical properties of HFVs. (A) Macro and micromorphology of HFVs: photographs (I–III), micro-CT images (IV–VI), and cross-sectional SEM images (VII–IX: low-magnification, 300 ×; X–XII: high magnification, 1500 ×). (B) Average pore size of HFVs. (C) Porosity of HFVs. (D) FTIR spectra. (E) XRD patterns. (F) Compressive stress–strain curve. (G) Tensile stress–strain curve. (H) Elongation at the break. (I) Tensile strength. *p < 0.05; **p < 0.01.

FIGURE 3

Evaluation of cell compatibility and blood compatibility of HFVs in vitro. (A) SEM (I–III) and confocal microscopic images (IV–VI) of L929 cells cultured on HFVs after 3 days. (B) CCK8 results of L929 cells cultured in extracts from HFVs for 1, 2, and 3 days. (C) Macro photographs of the hemolysis test and the corresponding hemolysis percentage. *p < 0.05; **p < 0.01; ****p < 0.0001, in comparison with the control group.

FIGURE 4

Shape memory performance, water absorption and retention test, and hemostasis in the normal rat liver perforation wound model. (A) Macro photographs of the water-triggered shape recovery of HFV70. (B) Water absorption of HFVs. (C) Water retention of HFVs. (D) Dependence of the in vitro degradation ratio of HFVs soaked in PBS on soaking time. (E) Photographs of the hemostatic effect of the gauze, gelatin sponge, and HFV70. (F) Blood loss from the liver incision. (G) Hemostatic time of different treatment groups. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, in comparison with the control group.

FIGURE 5

Evaluation of angiogenesis and antibacterial and antioxidant properties of HFVs in vitro. (A) Images of HUVEC tube formation. (B) Quantified results of the total tube nodes of HUVEC tube formation. (C) Inhibition zone images of E. coli and S. aureus on agar plates after being treated with samples. (D) Areas of ZOI against E. coli by the HFVs. (E) Areas of ZOI against S. aureus by HFVs. (F) Macro photographs of the DPPH scavenging test. (G) DPPH scavenging rates. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, in comparison with the control group.

FIGURE 6

Treatment effect evaluation in the rat full-thickness skin defect model. (A) Images of initial wounds covered with different dressings and the subsequent images of the wounds at different time points (4, 8, and 16 days). (B) Wound area changes in each group. (C) Body temperature changes in each group.

FIGURE 7

H&E and Masson’s staining images of wound areas in each group and detailed images corresponding to the epidermis (black square border), dermis (red square border), and subdermal tissues (blue square border).

FIGURE 8

Immunofluorescence analysis of wound tissues. (A) Representative images of regenerated skin tissues stained with anti-CD31, anti-CK10 (red), and DAPI (blue). (B) Analysis of positive staining ratios of CD31 in the healing wound sections. (C) Analysis of positive staining ratios of CK10 in the healing wound sections. *p < 0.05; **p < 0.01; ***p < 0.001; *comparing to the HFV70 group.