SHP099-containing multi-targeting hydrogel promotes rapid skin reconstruction through modulating a variety of cells

Abstract

Introduction: Adult wound scarring result in functional skin deficits. However, the development of effective measures to modulate the entire wound healing to encourage the skin function reconstruction is still a clinical challenge, as multiple cells are involved in wound healing hierarchically. Hydrogel scaffolds with long-lasting local release provide new insights into the clinical relevance of entire wound healing.

Methods: Herein, a multi-targeting hydrogel loaded with SHP099 (Gel-SHP) is designed to modulate multiple cells during wound repair.

Results: Our results show that Gel-SHP promotes rapid reconstruction of wound skin by modulating macrophages in the inflammatory stage, fibroblasts in the regeneration stage and smooth muscle cells in the remodelling stage. Gel-SHP could increase M2 macrophage differentiation and remodel the dermal shell of hair follicles through in situ release. Moreover, Gel-SHP may modulate myofibroblasts to promote wound contraction through SHP099-scaffold synergistic interactions.

Discussion: Our results provide new insights into the design of functional hydrogels for tissue regeneration applications. Gel-SHP as a promising tool could provide new clues and new research paradigms for future studies and understanding of the wound healing process and dermal shell formation.

Keywords: SHP099; hair follicle dermal shell; macrophage; multi-targetinghydrogel; myofibroblast; wound healing.

FIGURE 1

Gel loaded with SHP099 (SHP) to prepare multi-targeting hydrogel. The multi-targeting hydrogel is hypothesized to promote the reconstruction of wound skin structures by regulating macrophages, fibroblasts and smooth muscle cells at different stages of wound repair.

FIGURE 2

Gel-SHP promotes wound healing and skin remodeling. (a) Scanning electron microscopy (SEM) morphology characterization of Gel hydrogels (Gel) and Gel hydrogels loaded with the multi-targeting inhibitor SHP099 (Gel-SHP). (b) Cumulative release profile of SHP in Gel-SHP (n = 3). We hypothesized that Gel-SHP would regulate macrophages during the inflammatory stage and fibroblasts during the tissue regeneration stage. (c) Mouse wound model. (d, e) Gel-SHP promotes wound closure. (d) Photographs of wounds treated by Gel and Gel-SHP for day 0, 3 and 7 (1 mm per grid). (e) The wound closure rate between groups Gel and Gel-SHP was not significant on day 3 (n = 3, p = 0.15427), whereas the wound closure rate of Gel-SHP group was significantly higher than Gel group on day 7 (n = 3, p = 0.00453). (f) H&E-stained sections (left, day 7 of treatment) show the epidermal thickness (brown dash-dotted line) was significantly higher in group Gel-SHP than in group Gel (right, arrows, n = 3, p = 0.03237). (g) H&E-stained sections on day 14 treatment show (left) hair follicle number in Gel-SHP group was significantly higher than in Gel group (right, n = 3, p = p = 0.00158). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 3

Gel-SHP regulates macrophages during inflammatory stage and promotes tissue regeneration. (a) Q-PCR results of IL-6 expression in macrophages (RAW264.7; LPS-/SHP099- VS LPS+/SHP099-, n = 3, p = 1.71445E-7; LPS+/SHP099- VS LPS+/SHP099+, n = 3, p = 8.43562E-4). (b, c) Gel-SHP promotes M2 differentiation of macrophages in wounds. (b) Immunohistochemically (IHC) stained sections (day 3 of treatment; CD206, brown). (c) The percentage of macrophages positive for CD206 expression in Gel-SHP group was higher than in the Gel group (n = 5, p = 3.60348E-5). (d–f) Gel-SHP promotes collagen formation and fibroblast regeneration in the wound. (d) Masson stained sections (day 3 of treatment; collagen, blue; fibroblast, red) (e) The blue average optical density (AOD) representing collagen was significantly higher in Gel-SHP group than in Gel group (n = 5, p = 0.00135). (f) The blank area was significantly smaller in Gel-SHP group than in Gel group (n = 5, p = 0.00291). (g) Gel-SHP regulates the differentiation of wound macrophages from M1 to M2, while promoting fibroblast proliferation and facilitating collagen synthesis during the inflammatory stage. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 4

Gel-SHP regulates fibroblasts during regeneration stage and promotes angiogenesis. (a, b) Transcriptomic sequencing reveals Gel-SHP regulated genes and gene functions in the regeneration stage. (a) Volcano map of differentially expressed genes (day 7 of treatment; Gel-SHP vs Gel, n = 3). P < 0.05, log₂ (Fold change) > 2 was defined as significant difference. (b) Gene Ontology (GO) enrichment analysis of significantly upregulated genes in biological progress (BP). Significant enrichment was defined as p value < 0.05 (Top 5 in BP). (c, d) Gel-SHP promotes fibroblast alpha-smooth muscle actin (alpha-SMA) expression in wounds. (c) IHC stained sections of alpha-SMA (day 7 of treatment; red). (d) The AOD of alpha-SMA in fibroblasts of Gel-SHP group is significantly higher than Gel group (n = 5, p = 0.00125). (e, f) Gel-SHP promotes angiogenesis of the wound. (e) H&E-stained sections (7 day of treatment). (f) Statistical results show that the number of blood vessels at the Gel-SHP group wound was significantly higher than that in Gel group (n = 5, p = 3.8429E-4). (g–i) Gel-SHP promotes alpha-SMA expression in neo-regenerated hair follicles. (g) IHC stained sections of alpha-SMA (day 7 of treatment; red). (h) The alpha-SMA positive hair follicle rate was significantly higher in Gel-SHP group than Gel group (n = 5, p = 0.00499). (i) The hair follicle alpha-SMA AOD in Gel-SHP group was significantly higher than that Gel group (n = 5, p = 2.88749E-6). (j, k) Gel-SHP did not significantly increase the number of hair follicles after day 7 of treatment. (j) H&E-stained sections (day 7 of treatment). (k) The difference in the number of hair follicles regenerated in the wound (Regeneration area: red box = 1 mm²) between Gel-SHP group and Gel group was not significant (n = 3, p = 0.34). (l) Gel-SHP modulates the differentiation of fibroblasts into myofibroblasts, promotes angiogenesis and promotes alpha-SMA expression during the regeneration stage. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 5

Gel-SHP promotes hair follicle regeneration via modulating Cald1 expression during the remodeling stage. (a) Gel-SHP promotes Ki67 expression in neonatal hair follicle matrix cells. Immunofluorescence (IF) stained sections (left; 7 day of treatment). The Ki67-positive follicles (follicles with Ki67-positive cells were defined as positive follicles) ratio (n = 5, p = 0.01081) and the number of positive cells in positive follicles (15 randomly selected hair follicles:3 follicles close to the wound per section, n = 15, p = 1.058E-4) were significantly higher in Gel-SHP group than in Gel group. (b) Western blot results of L929 showed that SHP099 promoted Cald1 expression in fibroblasts. (c) IF results (left, day 3 incubated) showed that SHP099 altered the morphology of fibroblasts and significantly decreased the length/width ratio of fibroblasts (n = 10, p = 1.17055E-9). (d, e) Gel-SHP promotes Cald1 expression in wound fibroblasts. (d) IHC stained sections of Cald1 (day7 of treatment; red). (e) Cald1 AOD in wound fibroblasts in Gel-SHP group was significantly higher than that in Gel group (n = 5, p = 2.62218E-4). (f–h) Gel-SHP promotes Cald1 expression in neo-regenerated hair follicles. (f) IHC stained sections of Cald1 (day 7 of treatment; red). (g) Cald1 positive neo-regenerated hair follicle percentage in Gel-SHP group was significantly higher than that in Gel group (n = 5, p = 6.04142E-5). (h) Cald1 AOD in neo-regenerated hair follicles in Gel-SHP group was significantly higher than that in Gel group (10 randomly selected hair follicles:2 follicles close to the wound per section, n = 10, p = 2.86295E-8). (i) Gel-SHP maintains the dermal shell of hair follicles to promote follicular regeneration by promoting Cald1 expression during the remodeling stage. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.