Graft-Host Interaction and Its Effect on Wound Repair Using Mouse Models

Abstract

Autologous skin grafting has been commonly used in clinics for decades to close large wounds, yet the cellular and molecular interactions between the wound bed and the graft that mediates the wound repair are not fully understood. The aim of this study was to better understand the molecular changes in the wound triggered by autologous and synthetic grafting. Defining the wound changes at the molecular level during grafting sets the basis to test other engineered skin grafts by design. In this study, a full-thickness skin graft (SKH-1 hairless) mouse model was established. An autologous full-thickness skin graft (FTSG) or an acellular fully synthetic Biodegradable Temporising Matrix (BTM) was grafted. The wound bed/grafts were analysed at histological, RNA, and protein levels during the inflammation (day 1), proliferation (day 5), and remodelling (day 21) phases of wound repair. The results showed that in this mouse model, similar to others, inflammatory marker levels, including Il-6, Cxcl-1, and Cxcl-5/6, were raised within a day post-wounding. Autologous grafting reduced the expression of these inflammatory markers. This was different from the wounds grafted with synthetic dermal grafts, in which Cxcl-1 and Cxcl-5/6 remained significantly high up to 21 days post-grafting. Autologous skin grafting reduced wound contraction compared to wounds that were left to spontaneously repair. Synthetic grafts contracted significantly more than FTSG by day 21. The observed wound contraction in synthetic grafts was most likely mediated at least partly by myofibroblasts. It is possible that high TGF-β1 levels in days 1-21 were the driving force behind myofibroblast abundance in synthetic grafts, although no evidence of TGF-β1-mediated Connective Tissue Growth Factor (CTGF) upregulation was observed.

Keywords: IL-6; TGF-β1; myofibroblast; skin grafting; wound repair.

Figure 1

Wound epithelisation/contraction. Representative images of (A) ungrafted full-thickness wounds and wounds grafted with (B) FTSG or (C) BTM on day of surgery (D0), day 5 (D5), day 10 (D10), day 15 (D15), and day 21 (D21). (D) The wound surface area (SA) was measured at D0, D5, D10, D15, and D21 using ImageJ version 1.54f software. Data analysed using two-way ANOVA. Values represent mean +/− SEM in each group (n = 4 mice per group, ns = not significant, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001).

Figure 2

Effect of skin grafting on inflammation and proliferation markers. Graft tissue was harvested from ungrafted full-thickness wounds or wounds grafted with FTSG and BTM. Dermal tissue was separated at days 1, 5, and 21 post-operation. (A) inflammatory and (B) proliferative cytokine/growth factors were measured using RT-qPCR. Data were analysed as expression fold changes of the targets against the average of the C_t values of three housekeeping genes (Polr2a, Eef1a1, Tuba1a), and normalised against the C_t values of the same genes of the “tail” skin collected on the same day. (C) Protein isolated from ungrafted and FTSG/BTM-grafted wounds were analysed for IL-6, TNF-α, and IL-10 secretion using ELISA. Statistical analysis was performed using two-way ANOVA. Values represent mean +/− SEM in each group (n = 3 mice per group, ns = not significant, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001).

Figure 3

Histological analysis of grafts. Representative images of (A) haematoxylin and eosin and (B) Masson’s trichrome staining of the native skin prior to wounding, full-thickness ungrafted wounds, or wounds grafted with FTSG and BTM. Red arrows on haematoxylin and eosin images indicate the abundance of purple stained cells likely to be immune cells in BTM grafts. (C) Quantification of the total collagen area (area per µm²) that is stained blue with Masson’s trichrome at day 1, 5, and 21 post-grafting using ImageJ version 1.54f software. Statistical analysis was performed using one-way ANOVA. Values represent mean values +/− SEM in each group (n = 4–5 mice per group, * = p ≤ 0.05, ** = p ≤ 0.01).

Figure 4

Abundance of myofibroblasts in grafts. Native skin prior to wounding, ungrafted full-thickness wounds, and wounds grafted with FTSG and BTM were co-stained for alpha-smooth muscle actin (α-SMA) in green and vimentin (VIM, a fibroblast marker) in red. Representative images of (A) α-SMA and VIM co-staining on day 5 and day 21 of FTSG and BTM grafts compared with native skin. Total number of cells expressing (B) VIM, α-SMA, and both (C) α-SMA⁺/VIM⁺ myofibroblast compared with the percentage of α-SMA⁺ cells and (D) α-SMA⁺/VIM⁺ myofibroblast compared with the percentage of VIM⁺ fibroblast were measured using NIS-elements version 5.21.00 software and normalised against no primary antibody negative control staining for each section. Data analysed using one-way ANOVA in (B) or an unpaired t-test in (C,D). Values represent mean +/− SEM in each group (n = 4 mice per group, ns = not significant, * = p ≤ 0.05).

Figure 5

Myofibroblast differentiation in BTM grafts is likely to be mediated at least partly by transforming growth factor beta 1 (TGF-β1). (A) TGF-β1 mRNA was measured by RT-qPCR from the tissue of ungrafted full-thickness wounds or wounds grafted with FTSG and BTM on day 1, day 5, and day 21. Data were analysed as expression fold changes of the targets against the average of the C_t values of three housekeeping genes (Polr2a, Eef1a1, Tuba1a), and normalised against the C_t values of the same genes of the “tail” skin. Proteins isolated from ungrafted and FTSG/BTM-grafted wounds were analysed for (B) TGF-β1 and its downstream mediator (C) connective tissue growth factor (CTGF) secretion using ELISA. Data analysed using two-way ANOVA. Values represent mean +/− SEM in each group (n = 3–4 mice per group, * = p ≤ 0.05, *** = p ≤ 0.001).