Angiopoietin-1 derived peptide hydrogel promotes molecular hallmarks of regeneration and wound healing in dermal fibroblasts

Abstract

By providing an ideal environment for healing, biomaterials can be designed to facilitate and encourage wound regeneration. As the wound healing process is complex, there needs to be consideration for the cell types playing major roles, such as fibroblasts. As a major cell type in the dermis, fibroblasts have a large impact on the processes and outcomes of wound healing. Prevopisly, conjugating the angiopoietin-1 derived Q-peptide (QHREDGS) to a collagen-chitosan hydrogel created a biomaterial with in vivo success in accelerating wound healing. This study utilized solvent cast Q-peptide conjugated collagen-chitosan seeded with fibroblast monolayers to investigate the direct impact of the material on this major cell type. After 24 h, fibroblasts had a significant change in release of anti-inflammatory, pro-healing, and ECM deposition cytokines, with demonstrated immunomodulatory effects on macrophages and upregulated expression of critical wound healing genes.

Keywords: Biomaterials; Health sciences; Materials science.

Graphical abstract

Figure 1

Schematic of the experimental design to study impact of Q-peptide, scrambled Q-peptide, and peptide-free hydrogel in comparison to tissue-culture plastic on normal adult human dermal fibroblasts Media is collected at Day 1 and Day 7 for Cytokine analysis via ELISA. This will allow a study of cytokines related to ECM deposition and inflammation. Fluorescent microscopy gives insight into differences in key structural proteins such as Vimentin and α−SMA at Days 1 and 7. Created with Biorender.com.

Figure 2

Fibroblast viability is maintained on all surfaces and proliferation is attenuated on hydrogels (A) Live-Dead staining of HDF cells after 24 h using CFDA-SE and PI (scale bars = 200 μm). Surfaces include: tissue-culture plastic (TCP), peptide-free hydrogel (PF), scrambled Q-peptide hydrogel (SCR), and Q-peptide hydrogel (QP). (B) Quantification of early cell viability on day 1 using the relative counts of CFDA-SE to PI. (C) Representative images of HDF cells stained with Ki-67 (red) and counterstained with DAPI (blue) after 24 h and seven days (scale bars = 200 μm). (D) Quantification of positive Ki-67 staining (%) revealed a significant difference between cells cultured on the Q-peptide hydrogel (QP) and the peptide-free hydrogel (PF) after (i) 24 h but not (ii) seven days. Statistical analysis included one-way ANOVA followed by a Tukey’s post hoc test. n = 3–4. Data presented as mean ±SD ∗ = p < 0.05, ∗∗ = p < 0.01.

Figure 3

Fibroblasts express cell specific marker vimentin on all surfaces and attenuate expression of α-SMA on hydrogels (A) Comparison of HDF cells stained with Vimentin and counterstained with DAPI at days 1 and 7 (scale bars = 200 μm). Surfaces include tissue-culture plastic (TCP), peptide-free hydrogel (PF), scrambled Q-peptide hydrogel (SCR), and Q-peptide hydrogel (QP). (B) Quantification of vimentin staining revealed (i) an increase for QP relative to PF and SCR on day 1 and (ii) decreased staining for all hydrogel groups on day 7 relative to TCP. (C) Aspect Ratio (AR) quantification between the different conditions indicated (i) no significant difference in cell shape on day 1 and ii) increased AR for QP compared to SCR on day 7. (D) Cell count normalized to area (mm²) did not significantly differ between samples on (i) day 1 or (ii) day 7. (E) Comparison of HDF cells stained with Vimentin, α−SMA and DAPI to measure α−SMA positive cells (scale bars = 200 μm). (F) Quantification of α−SMA+ cells as a percentage of the total cell count. (G) Collagen IV staining of HDF cells on day 3 of culture (scale bars = 200 μm). (H) Quantification of collagen IV staining revealed significantly less collagen IV for all hydrogel samples when compared to tissue-culture plastic, with no significant differences between peptide-free, Q-peptide, or scrambled Q-peptide hydrogels. Statistical analysis included one-way ANOVA followed by a Tukey’s post hoc test. n = 3–4. Data presented as mean ±SD ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p<0.001, ∗∗∗∗ = p<0.0001.

Figure 4

Unique secretion profile of pro- and anti-inflammatory cytokines by dermal fibroblasts cultivated on Q-peptide hydrogel Cytokine secretion of pro-inflammatory cytokines. (A–E) TNF-α, (B) IL-2, (C) IL-6, (D) IL-8, and anti-inflammatory cytokines (E) IL-10, and (F) IL-13 at (i) 1 day and (ii) 7 days of culture. Surfaces include tissue-culture plastic (TCP), peptide-free hydrogel (PF), scrambled Q-peptide hydrogel (SCR), and Q-peptide hydrogel (QP). Concentration (pg/mL) expressed after subtraction of baseline media control and normalized to cell number (10,000 cells). Statistical Analysis included One-way ANOVA followed by a Tukey’s post hoc test. n = 4. Data presented as mean ±SD ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p<0.001, ∗∗∗∗ = p<0.0001.

Figure 5

Fibroblasts cultivated on Q-peptide gel exhibit early enhanced secretion of cytokines implicated in ECM deposition (A–F) Cytokine release of (A) GM-CSF, (B) IL-1RA, (C) IL-4, (D) IL-5, (E) IL-12 P40, and (F) MCP-1 at (i) 1 day and (ii) 7 days of culture. Surfaces include tissue-culture plastic (TCP), peptide-free hydrogel (PF), scrambled Q-peptide hydrogel (SCR), and Q-peptide hydrogel (QP). Concentration (pg/mL) expressed after subtraction of baseline media control and normalized to cell number (10,000 cells). Statistical Analysis included One-way ANOVA followed by a Tukey’s post hoc test. n = 4. Data presented as mean ± SD ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, ∗∗∗∗ = p < 0.0001.

Figure 6

Fibroblasts cultivated on Q-peptide gel exhibit enhanced secretion of anti-fibrotic cytokines (A–F) Cytokine release of anti-fibrotic cytokines (A) IL-1β, (B) IFN-γ, and TGF-β isoforms (C) TGF-β1, (D) TGF-β2, (E) TGF-β3, and (F) TGF-β3/TGF-β1 ratio at (i) 1 day and (ii) 7 days of culture. Surfaces include tissue-culture plastic (TCP), peptide-free hydrogel (PF), scrambled Q-peptide hydrogel (SCR), and Q-peptide hydrogel (QP). Concentration (pg/mL) for IL-1β, and IFN-γ expressed after subtraction of baseline media control and normalized to cell number (10,000 cells). Concentration (pg/mL) for TGF-β1-3 normalized to cell number (10,000 cells). Blue and yellow lines indicate the range of concentrations of TGF-β family cytokines measured in the starting culture media (or just blue line when consistent amongst replicates). Statistical analysis included One-way ANOVA followed by a Tukey’s post hoc test. n = 4. Data presented as mean ± SD ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, ∗∗∗∗ = p < 0.0001.

Figure 7

Fibroblasts cultured on Q-peptide hydrogels show enhanced expression profile of wound healing-related genes (A) Clustergram analysis of a wound healing qPCR array comparing the expression profile of fibroblasts grown on TCP to that of Q-and scrambled Q-peptide hydrogels. (B and C) Volcano plots highlighting significantly upregulated and downregulated genes in fibroblasts grown on (B) QP versus TCP and (C) QP versus SCR. (D and E) Wound healing array heatmaps depicting gene symbols and fold changes for (D) QP versus TCP and (E) QP versus SCR. Bold and underlined symbols indicate statistically significant differences. For all plots: n = 3 or 4, p < 0.05 is considered significant.