Diminished type III collagen promotes myofibroblast differentiation and increases scar deposition in cutaneous wound healing

Abstract

The repair of cutaneous wounds in the postnatal animal is associated with the development of scar tissue. Directing cell activities to efficiently heal wounds while minimizing the development of scar tissue is a major goal of wound management and the focus of intensive research efforts. Type III collagen (Col3), expressed in early granulation tissue, has been proposed to play a prominent role in cutaneous wound repair, although little is known about its role in this process. To establish the role of Col3 in cutaneous wound repair, we examined the healing of excisional wounds in a previously described murine model of Col3 deficiency. Col3 deficiency (Col3+/-) in aged mice resulted in accelerated wound closure with increased wound contraction. In addition, Col3-deficient mice had increased myofibroblast density in the wound granulation tissue as evidenced by an increased expression of the myofibroblast marker, α-smooth muscle actin. In vitro, dermal fibroblasts obtained from Col3-deficient embryos (Col3+/- and -/-) were more efficient at collagen gel contraction and also displayed increased myofibroblast differentiation compared to those harvested from wild-type (Col3+/+) embryos. Finally, wounds from Col3-deficient mice also had significantly more scar tissue area on day 21 post-wounding compared to wild-type mice. The effect of Col3 expression on myofibroblast differentiation and scar formation in this model suggests a previously undefined role for this ECM protein in tissue regeneration and repair.

Fig. 1

Wound size is reduced in aged Col3-deficient (Col3+/–) mice relative to wild-type (Col3+/+) mice at day 7 post-excisional wounding. a Graph showing gross WA measurements of skin wounds 7 days postoperatively in young (6–11-week) and aged (>1-year) mice of both genotypes. Each bar represents the mean ± SEM. ** p < 0.001. b Gross appearance of excisional wounds on the dorsum and head of aged Col3+/+ (left) and Col3+/– (right) mice at 7 days. Wounds of a representative pair of mice from each genotype are shown.

Fig. 2

Col3-deficient mice exhibit accelerated wound closure. Punch biopsy wounds (6 mm in diameter) were created on the dorsum of wild-type (Col3+/+) or Col3-deficient (Col3+/–) mice. Wound healing was expressed as a percent change in the WA relative to the original wound size (mean ± SD). Col3+/– values are significantly different from Col3+/+ values at corresponding time points at * p < 0.05 and ** p < 0.01.

Fig. 3

Wound contraction is accelerated in aged Col3-deficient (Col3+/–) mice compared to aged wild-type (Col3+/+) mice. a Representative H&E-stained sections through the central wounds from aged Col3+/+ (top) and Col3+/– (bottom) mice. Wounds were harvested at 7 days postoperatively. Arrows indicate the lateral margins of hyperproliferative epithelium (lateral edge of the reepithelialized wound). The scale bar beneath the histologic sections is equal to the original wound diameter of 6 mm. b Quantitative comparison of the percentage of wound closure due to contraction (mean ± SD). At 7 days postwounding, the Col3+/– wounds exhibited significantly more contraction than those in Col3+/+ mice (* p < 0.001, n = 10 mice per group).

Fig. 4

Wounds in aged Col3-deficient mice show increased wound contraction and GT α-SMA expression. Immunohistochemical staining for α-SMA (brown) in representative wounds from Col3+/+ (a) and Col3+/– (b) mice 7 days after wounding by excisional punch biopsy. Detailed views of the GT and overlying neoepithelium in wounds from Col3+/+ (c) and Col3+/– (d) mice reveal increased α-SMA immunoreactivity in wounds from Col3-deficient mice (Col3+/–) compared to wild-type mice. Corresponding negative controls from adjacent serial sections are shown (e and f, respectively). Scale bars = 500 μm (a, b) and 50 μm in high-magnification views (c–f).

Fig. 5

Collagen gel contraction by wild-type and Col3-deficient embryonic (∼E18.5) dermal fibroblasts. Wild-type and Col3-deficient fibroblasts were grown in attached collagen gels for 5 days. Stressed lattices were then released and the percentage of contraction calculated at 30, 60, 120, and 240 min post-release. Values represent means ± SEM for individual cell isolates from at least 3 embryos for each genotype with 3 lattices per individual cell isolate. Col3+/– and Col3–/– values were found to be significantly different from Col3+/+ values at all time points (** p < 0.01; * p < 0.05).

Fig. 6

Increased α-SMA expression by Col3-deficient (Col3+/– and Col3–/–) cells compared to Col3 wild-type (Col3+/+) cells. Cells from all 3 genotypes, utilized in the attached fibroblast-populated collagen gel assay, were immunostained for α-SMA (green) and counterstained with DAPI (blue). Collagen lattices seeded with cell isolates from at least 3 embryos per genotype were analyzed. a Myofibroblasts within collagen lattices were identified by α-SMA expression. α-SMA incorporation into stress fibers (arrows) could be visualized in cells from all 3 genotypes. b–d Representative images showing the percentage of myofibroblasts in cultures of Col3+/+ (b), Col3+/– (c), and Col3–/– (d) cells are presented. e Graphic representation of the percentage of α-SMA-positive cells quantitated as described in Materials and Methods, expressed as means ± SD. The percentages of α-SMA-positive Col3-deficient (Col3+/– and Col3–/–) fibroblasts were significantly increased relative to that of Col3+/+ fibroblasts. ** p < 0.001; * p < 0.05. Scale bars = 20 μm (a) and 100 μm (b–e).

Fig. 7

Scar formation is increased in wounds of Col3-deficient (Col3+/–) mice relative to wild-type (Col3+/+) mice. a Representative Masson's trichrome-stained sections of bisected wounds from Col3+/+ (top) and Col3+/– (bottom) mice at 21 days postwounding. b Histomorphometric analysis reveals a significantly increased scar tissue area associated with Col3 deficiency (* p < 0.05). Scale bars = 500 μm.