Dose-dependent pro- or anti-scar-preventing responses of freeze-dried amniotic membrane extract in relation to suppression of connective tissue growth factor and α-smooth muscle actin

Abstract

Dose-dependent pro- or anti-scar-preventing effects of amniotic membrane extract (AME) are unclear. This study investigated the dose-dependent effects of freeze-dried AME on connective tissue growth factor (CTGF) and α-smooth muscle action (α-SMA) (key determinants of excessive scarring) expression in an attempt to develop medication for scarless wound healing. The inhibitory effect of freeze-dried AME on α-SMA was visualized through immunofluorescence confocal microscopy. The suppressive effect of 10 different concentrations of AME (0.0025–0.25 µg/mL) on CTGF and α-SMA was determined, and rates of collagen lattice contraction were measured. AME inhibited the trans-differentiation of fibroblasts to myofibroblasts, a hallmark of scar formation. AME downregulated CTGF and α-SMA in a dose-dependent manner; supplementation of 0.0175 µg/mL of AME in cell culture media downregulated CTGF (by 49%; p < 0.05) and α-SMA (by 33%; p < 0.05) relative to control values, the effect saturating with a subsequent increase in AME levels to 0.025 µg/mL. Collagen lattice contraction was maximally inhibited (75 ± 8%) upon treatment with 0.0175 µg/mL AME relative to the control (44 ± 6%). AME downregulates α-SMA and inhibits the trans-differentiation of fibroblasts to myofibroblasts in a dose-dependent manner. Treatment of cells with high AME concentrations retained high α-SMA concentration levels. A delicate balance between the wound healing properties and pro-fibrotic abilities of AME should be considered for selecting an appropriate therapeutic dose.