Photodynamically tunable ROS-generating hydrogels for accelerated tissue regeneration

Abstract

Wound healing progresses through many key cellular activities, including fibroblast and keratinocyte proliferation and angiogenesis. This study explored the wound-healing potential of reactive oxygen species (ROS)-generating hyaluronic acid (HA) hydrogels. We fabricated a chlorin e6-conjugated HA (Ce6-HA) hydrogel that generates ROS when subjected to irradiation from an LED light source. In vitro studies revealed that the ROS generated by the Ce6-HA hydrogels enhanced the proliferation of fibroblasts and keratinocytes. Further, the fibroblasts were found to have high levels of intracellular ROS, elevated expression of p-ERK1/2, p-p38 MAPK, p-Akt, and cyclin D1 proteins, and enhanced collagen deposition. Moreover, the Ce6-HA hydrogel also promoted endothelial angiogenesis in vitro. In vivo studies demonstrated the ROS-generating HA hydrogels significantly improved wound closure and tissue regeneration compared to control groups. The Ce6-HA hydrogel-treated group exhibited accelerated wound healing, with enhanced fibroblast proliferation, increased keratinocyte proliferation, and better angiogenesis. Histopathological and immunohistochemical analyses showed elevated levels of key growth factors and signaling molecules, which are critical to wound healing. The controlled ROS generation from the Ce6-HA hydrogels activated broader molecular pathways necessary for effective skin tissue repair. Therefore, ROS-triggering HA hydrogels could be a viable approach to accelerate recovery and reduce scarring in clinical settings.

Keywords: Angiogenesis; Hyaluronic acid; Hydrogel; Proliferation; Reactive oxygen species; Wound healing.

This study investigates a hyaluronic acid hydrogel conjugated with chlorin e6 (Ce6-HA), which generates ROS upon exposure to LED light. In vitro experiments were conducted using fibroblasts, keratinocytes, and endothelial cells to evaluate the cellular responses. In vivo studies demonstrated that the Ce6-HA hydrogel significantly enhanced wound closure and tissue regeneration. Histological analysis further confirmed elevated expression of growth factors and wound healing related markers.

Scheme 1

This study investigates a hyaluronic acid hydrogel conjugated with chlorin e6 (Ce6-HA), which generates ROS upon exposure to LED light. In vitro experiments were conducted using fibroblasts, keratinocytes, and endothelial cells to evaluate the cellular responses. In vivo studies demonstrated that the Ce6-HA hydrogel significantly enhanced wound closure and tissue regeneration. Histological analysis further confirmed elevated expression of growth factors and wound healing-related markers.

Fig. 1

Animal experiment group classification and schedule (A) The animals were divided into four groups; only wound (CON), HA treatment on the wound (HA), LED irradiation on HA-treated wound (HAL), and LED irradiation on Ce6-HA treated wound (CHL). (B) ROS-induced wound healing model of balb/c nude mouse. (C) Every 3–4 d, macroscopic examination was carried out to assess the size of the wound and its body weights. Every week, tissue was excision for histology analysis.

Fig. 2

Preparation of Ce6-HA hydrogels, LED irradiation device setup, and ROS detection (A) Schematic illustration of the preparation of crosslinked Ce6-bonded HA hydrogel. (B) Blue print of LED equipment. (C) Photograph of the LED equipment emitting light. (D) Degradation of DPBF measured upon LED irradiation at 50, 100, 250, 500, 750 μW/cm² and 1 mW/cm² at 10 min intervals. Data are presented as mean ± SD (n = 5).

Fig. 3

Assessment of fibroblast response to ROS-induced effects (A) Cellular proliferation assay using NHDF cells subjected to LED irradiation (100 μW/cm²) for different durations. (i) Non-irradiation, (ii) 20 min, (iii) 30 min, (iv) 40 min, (v) 50 min, (vi) 60 min LED irradiation. ∗p < 0.05 vs. HA (n = 5). (B) Estimated intracellular ROS levels at different irradiation time points under 100 μW/cm² intensity. #p < 0.01 vs. con (n = 3). (C) Cells were harvested after 24, 48, and 72 h after LED irradiation. p-ERK1/2, ERK1/2, p-p38 MAPK, p38 MAPK, p-AKT, AKT, cyclin D1 and β-actin proteins levels were measured using Western blot. The graphs show quantified proteins levels; (i) relative p-ERK1/2 level, (ii) p-ERK1/2/ERK1/2, (iii) relative p-p38 MAPK level, (iv) p-p38 MAPK/p38 MAPK, (v) relative p-AKT level, (vi) p-AKT/AKT (vii) relative cyclin D1 level. Data are expressed as means ± SD (n = 3). ∗p < 0.05 vs. 24 h HA (D) (i) Total collagen estimated using the Sircol soluble collagen assay kit. (ii) Collagen amount normalized to total protein content showing increased collagen level due to the higher fibroblast number. ∗p < 0.05 vs. HA (n = 3). Throughout all figures and graphs in the in vitro experiments, HA represents the group using HA hydrogel, while CH denotes the group using Ce6-HA hydrogel. This labeling is consistently applied across all in the in vitro data representations in the manuscript.

Fig. 4

Evaluation of the effects of ROS on keratinocytes (A) HaCaT cells were treated with HA or Ce6-HA (CH) under 100 μW/cm² LED irradiation for 20–60 min; (i) non-irradiation, (ii) 20 min, (iii) 30 min, (iv) 40 min, (v) 50 min, (vi) 60 min ∗ p < 0.05 vs. HA (n = 5). (B) Intracellular ROS levels induced in keratinocytes upon Ce6-HA treatment and LED irradiation. #p < 0.01 vs. con (n = 3).

Fig. 5

Effect of ROS on tube formation analyzed using fluorescence staining (A) Confirmation of angiogenesis using the tube formation assay. Fluorescence image of tube formation. (B) (i) The junction number and (ii) total tube length was analyzed using image J. ∗p < 0.05 vs. ECM + HA (n = 5).

Fig. 6

Wound healing assessment in vivo (A) Representative images of wound healing progression in Balb/c mice. Full-thickness wounds were created using a biopsy punch, and wound size was measured from photographs taken on days 0, 1, 3, 5, 7, 14, 17, and 21. Scale bar = 5 mm. (B) Wound area reduction analysis. Wound sizes were quantified from day 0 to day 21, and statistical comparisons were made. ∗p < 0.05 vs. HA (n = 3). (C) Body weight monitoring of Balb/c nude mice during the observation period (n = 3).

Fig. 7

Histological analysis of tissue at various wound healing stages (A) Histological evaluation of wound healing using H&E staining. (B) Collagen deposition analysis using MT staining. (C) Epidermal thickness measurement at post-surgery day 21 (n = 5).

Fig. 8

Immunostaining analysis for PCNA, CD31 and vimentin (A) Representative IHC staining for PCNA, CD31, and vimentin. PCNA staining was performed on day 7 samples, while CD31 and vimentin staining were performed on day 14 samples. Scale bar = 100 μm. (B) Quantification of (i) PCNA, (ii) CD31, and (iii) vimentin expression in tissue. Data are presented as mean ± SD. ∗p < 0.05 vs. control, #p < 0.05 vs. HA (n = 3).

Fig. 9

Immunohistochemical analysis of CD68 (A) Representative IHC staining images for CD68 in different groups (CON, HA, HAL, CHL). Scale bar = 50 μm. (B) Quantification of CD68-positive staining in tissue. Data are presented as mean ± SD. ∗p < 0.05 vs. CON (n = 3).

Fig. 10

Analysis of cytokines levels in skin tissue (A) EGF and (B) VEGF quantified using ELISA. ∗p < 0.05 vs. con, #p < 0.05 vs. HA (n = 3).