Salvianolic-Acid-B-Loaded HA Self-Healing Hydrogel Promotes Diabetic Wound Healing through Promotion of Anti-Inflammation and Angiogenesis

Abstract

Inflammatory dysfunction and angiogenesis inhibition are two main factors leading to the delayed healing of diabetic wounds. Hydrogels with anti-inflammatory and angiogenesis-promoting effects have been considered as promising wound care materials. Herein, a salvianolic acid B (SAB)-loaded hyaluronic acid (HA) self-healing hydrogel (HA/SAB) with anti-inflammatory and pro-angiogenesis capacities for diabetic wound healing is reported. The HA hydrogel was prepared via the covalent cross-linking of aldehyde groups in oxidized HA (OHA) and hydrazide groups in adipic dihydrazide (ADH)-modified HA (HA-ADH) with the formation of reversible acylhydrazone bonds. The obtained HA hydrogel exhibited multiple favorable properties such as porous structures, excellent self-healing properties, a sustainable release capacity of SAB, as well as excellent cytocompatibility. In addition, the effects of the SAB-loaded HA self-healing hydrogel were investigated via a full-thickness skin defect model using diabetic rats. The HA/SAB hydrogel showed enhanced skin regeneration effects with accelerated wound closure, shorter remaining dermal space length, thicker granulation tissue formation, and more collagen deposition. Furthermore, reduced inflammatory response and enhanced vascularization were found with HA/SAB2.5 hydrogel-treated wounds, indicating that the hydrogel promotes diabetic wound healing through the promotion of anti-inflammation and angiogenesis. Our results suggest that the fabricated SAB-loaded HA self-healing hydrogel is promising as a wound dressing for the treatment of diabetic wounds.

Keywords: angiogenesis; anti-inflammation; diabetic wound healing; salvianolic acid B; self-healing hydrogel.

Figure 1

Characterization of the basic properties of the HA hydrogel. (A) Synthesis of adipic dihydrazide (ADH)-modified HA (HA-ADH) and oxidized HA (OHA). (B) FT-IR spectra of HA, ADH, OHA, and HA/SAB2.5 hydrogel. (C) ¹H NMR spectrum of HA, ADH, and HA-ADH. (D) Photographs of the injectable (i), moldable (ii), and self-healing (iii) properties of the HA hydrogel. (E) Schematic illustration of the HA hydrogel formation and self-healing properties.

Figure 2

(A) SEM images of the blank HA and salvianolic acid B (SAB)-loaded HA hydrogels (HA/SAB1, HA/SAB2.5, and HA/SAB5) (Scale bar = 100 μm). (B) Pore size distribution of the blank HA, HA/SAB1, HA/SAB2.5, and HA/SAB5 hydrogels (n = 20). (C) Storage modulus (G’) and loss modulus (G’’) of the HA hydrogel cross-linked by adipic dihydrazide (ADH)-modified HA (HA-ADH) and oxidized HA (OHA) at different times at 1% strain and 10 rad/s angular frequency. (D,E) Strain sweep of the HA hydrogel with strain from 0.1% to 1000% at an angular frequency of 10 rad/s. (F) Angular frequency sweep of the HA hydrogel from 0.1 to 10 rad/s at a constant strain of 1%. (G) Self-healing property of the blank HA and HA/SAB2.5 hydrogels at continuous step strain test of 1% and 300% strain every 200 s at an angular frequency of 10 rad/s. (H) Swelling and degradation behaviors of the blank HA, HA/SAB1, HA/SAB2.5, and HA/SAB5 hydrogels in PBS at 37 °C. (I) SAB release profile of the HA/SAB hydrogels in PBS at 37 °C. Data represent mean ± SD, n = 3.

Figure 3

Cytocompatibility of the blank HA and salvianolic acid B (SAB)-loaded HA hydrogels (HA/SAB1, HA/SAB2.5, and HA/SAB5) in vitro. Cytotoxicity analysis of the HA hydrogels with CCK-8 assay (A) and LIVE/DEAD staining assay (B) using NIH/3T3 cells on day 1 and 3. (Scale bar = 200 μm). Data represent mean ± SD, n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 4

The mRNA expression level of (A) IL-1β and (B) ccr7 in (LPS + IFN-γ)-stimulated THP-1-derived macrophages following treatment of salvianolic acid B (SAB, 100 μM). (C) The mRNA expression level of CD206 in IL-4-stimulated THP-1-derived macrophages following treatment of SAB (100 μM). Data represent mean ± SD, n = 3; * p < 0.05; *** p < 0.001.

Figure 5

(A) Schematic of the establishment, treatment, and assessment of diabetic wound model. (B) Representative photographs of diabetic wounds on day 0, 3, 7, and 14 for control, blank HA, and salvianolic acid B (SAB)-loaded HA hydrogel (HA/SAB2.5). (C) Schematic diagram of the wound healed by different treatments during 14 days. (D) Quantitative data of wound healing rate on day 3, 7, and 14 following treatment of blank HA hydrogel and HA/SAB2.5 hydrogel, n = 6. (E,F) The mRNA expression level of IL-1β and IL-10 in wound sites on day 7 following treatment of blank HA hydrogel and HA/SAB2.5 hydrogel, n = 3. Data represent mean ± SD; * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 6

Histological evaluation of regenerated skin. (A) Representative images of H&E staining (A) (scale bar = 1 mm; red arrow: wound site length; black arrow: granulation tissue) and Masson trichrome staining (B) (scale bar = 1 mm/200 μm) for control, blank HA, and salvianolic acid B (SAB)-loaded HA hydrogel (HA/SAB2.5) on day 14. (C–E) Quantification of the wound site length, granulation tissue thickness, and collagen deposition of wound sites on day 14. Data represent mean ± SD, n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 7

HA hydrogels promoted angiogenesis during wound healing process. (A) Immunofluorescence staining images of α-SMA (green), CD31 (red) and DAPI (blue) on day 14 post wounding for control, blank HA, and salvianolic acid B (SAB)-loaded HA hydrogel (HA/SAB2.5) (scale bar = 100 μm). (B) Mean intensity of CD31-positive cells in wounds. (C) Quantitative graph of blood vessel density on day 14 corresponding to α-SMA-positive staining in diabetic wounds. Data represent mean ± SD, n = 3; *** p < 0.001.

Figure 8

Salvianolic acid B-loaded HA self-healing hydrogel promotes diabetic wound healing through promotion of anti-inflammation and angiogenesis. Red arrow: HA/SAB hydrogel increased the expression of IL-10, CD31 and α-SMA. Blue arrow: HA/SAB hydrogel decreased the expression of IL-1β and ccr7. Red circular: wound sites established on the back of diabetic rats. Blue circular: wound sites treated with hydrogels.