The anti-scar effects of basic fibroblast growth factor on the wound repair in vitro and in vivo

Abstract

Hypertrophic scars (HTS) and keloids are challenging problems. Their pathogenesis results from an overproduction of fibroblasts and excessive deposition of collagen. Studies suggest a possible anti-scarring effect of basic fibroblast growth factor (bFGF) during wound healing, but the precise mechanisms of bFGF are still unclear. In view of this, we investigated the therapeutic effects of bFGF on HTS animal model as well as human scar fibroblasts (HSF) model. We show that bFGF promoted wound healing and reduced the area of flattened non-pathological scars in rat skin wounds and HTS in the rabbit ear. We provide evidence of a new therapeutic strategy: bFGF administration for the treatment of HTS. The scar elevation index (SEI) and epidermal thickness index (ETI) was also significantly reduced. Histological reveal that bFGF exhibited significant amelioration of the collagen tissue. bFGF regulated extracellular matrix (ECM) synthesis and degradation via interference in the collagen distribution, the α-smooth muscle actin (α-SMA) and transforming growth factor-1 (TGF-β1) expression. In addition, bFGF reduced scarring and promoted wound healing by inhibiting TGFβ1/SMAD-dependent pathway. The levels of fibronectin (FN), tissue inhibitor of metalloproteinase-1 (TIMP-1) collagen I, and collagen III were evidently decreased, and matrix metalloproteinase-1 (MMP-1) and apoptosis cells were markedly increased. These results suggest that bFGF possesses favorable therapeutic effects on hypertrophic scars in vitro and in vivo, which may be an effective cure for human hypertrophic scars.

Figure 1. Wound closure and histopathological characteristics of bFGF treated wound healing in rat.

(A) Representative photographs of full-thickness skin wounds at various time points after treatment with or without 1 µg/ml bFGF. (B) The wound healing rates of bFGF. *P<0.05 compared to control group, n = 8. (C) Histopathological observation and masson staining of collagen in wound healing at day 14 post-wounding (×200).

Figure 2. The expression of PCNA, CD68 and TGF-β1 after bFGF treatment.

Immunohistochemistry of (A) PCNA, (B) CD68 and (C) TGF-β1 was performed on the indicated day (×200); the histogram represents the positive cells and optical density of the immunohistochemistry results. *P<0.05 and **P<0.01 compared to control group, n = 8.

Figure 3. bFGF alleviated the scar formation in rabbit ear model.

(A) The averaged Epidermal Thickness Index (ETI) of the scars. Epidermal hypertrophy was displayed by ETI. ETI >1 depicts a hypertrophic epidermis. (B) The averaged scar elevation index (SEI) of the scars. Dermal hypertrophy is displayed by the SEI, where SEI >1 depicts a hypertrophic scar. *P<0.05, **P<0.01 compared to control group, n = 6. (C) The microscopic histology of wounds that control or bFGF at day 40, HE stain.

Figure 4. bFGF decreased collagen I and collagen III synthesis.

The levels of expression of (A) collagen Ι and (B) collagen III in scars treated with saline or bFGF, *P<0.05, **P<0.01 compared to control group, n = 6. (C) Immunohistochemistry of the expression of collagen III in scars treated with saline or bFGF (×200). (D) Analysis of relative density collagen III, *P<0.05, **P<0.01 compared to control group, n = 6.

Figure 5. bFGF decreased α-SMA and TGF-β1 expression in scars.

Immunohistochemistry of the expression of (A) α-SMA and (B) TGF-β1 in scars treated with saline or bFGF in scars. (C) The levels of α-SMA and TGF-β1 in scars treated with saline or bFGF by Western blot. Analysis of the relative protein of (E) α-SMA and (F) TGF-β1 was performed. **P<0.01 compared to control group.

Figure 6. Effects of bFGF on mRNA levels in HSF.

RT-PCR analysis the mRNA levels of type collagen Ι and (B) collagen III, (C) CTGF, (D) HGF, (E) FN, (F) MMP-1, (G) TIMP-1, (H) SMAD-2, (I) SMAD-7, (J) α-SMA, (K) Lysine hydroxylases and (L) Prolyo hydroxylase in HSF treated with bFGF or saline for 5 days. *P<0.05 compared to control group.

Figure 7. bFGF induced cells apoptosis and inhibited TGF-β1/SMAD signaling pathway.

(A and B) TUNEL staining analysis of the apoptosis cells in rat skin by bFGF or saline in day 14 after wounding. The nuclear was labeled by Hoechst (blue), the myofibroblast was labeled by α-SMA (red), the apoptosis cells was labeled by TUNEL (green). Colocalization of α-SMA and TUNEL indicate apoptosis myofibroblast (white arrow) (×200). *P<0.05 compared to control. (C and D) Western blot analysis of the phosphorylation of SMAD2/3 (Ser 423/425) in HSF incubated with TGF-β1 (5 ng/ml). (E and F) Western blot analysis of the phosphorylation of SMAD2/3 in HSF incubated with TGF-β1 and bFGF (10 ng/ml). *P<0.05, **P<0.01 compared to control group.