Effects of low-dose of TGF-β1 on maintaining bovine corneal stromal cell growth and retarding extra cellular matrix fibrosis in a three-dimensional culture model

Background

Transforming growth factor-β₁ (TGF-β₁) plays an important role in corneal wound healing.The effects of TGF-β₁ on the synthesis of extra cellular matrix (ECM) vary upon different concentrations.Previous studies focused on the effects of high concentration of TGF-β₁ on keratocytes under the two-dimensional culture condition, and the effect of low concentration of TGF-β₁ on the synthesis of ECM in keratocytes remains unclear.

Objective

This study was to investigate the growth of Pellet, a three-dimensional model of corneal stroma cells in vitro, and its ECM synthesis under a low concentration of TGF-β₁.

Methods

Bovine corneal stromal cells were isolated from fresh bovine eyeballs by two-step digestion by collagenase and cultured using DMEM/F12 medium with 10% fetal bovine serum (FBS). Pellets derived fresh bovine keratocytes with culture medium containing 0.25 ng/ml TGF-β₁+ 5% FBS and 0.50 ng/ml TGF-β₁+ 5% FBS were established, respectively.The morphology of Pellets was observed under the natural light at 48 hours, 1 week, 2 weeks and 3 weeks after culture.In 3 weeks after culture, the cell structures was observed by hematoxylin-eosin staining, and Calcein-AM/propidium (Calcein-AM/PI) staining was used to assay the cell viability.Real-time fluorescence quantitative PCR and immunofluorescence technology were applied to analyze the expressions of α-smooth muscle actin (α-SMA), fibronectin (FN), type Ⅰ collagen (ColⅠ) and type Ⅲ collagen (Col Ⅲ) mRNA and proteins.RT-PCR was employed to detect the expressions of lumican (LUM) mRNA and keratocan (KERA) mRNA in the cells.

Results

Cells in Pellet clustered throughout the culture duration.Hematoxylin-eosin staining showed the mass red-dyed collagen fibers in both 0.25 ng/ml TGF-β₁+ 5% FBS group and 0.50 ng/ml TGF-β₁+ 5% FBS group, and most cells possessed complete structures.The death rate of the cells was (33.60±1.65)% in the 0.25 ng/ml TGF-β₁+ 5% FBS group and (30.90±0.78)% in the 0.50 ng/ml TGF-β₁+ 5% FBS group, showing an insignificant difference between them (t=0.144, P=0.887). The expressions of α-SMA, FN and Col Ⅲ proteins in 0.25 ng/ml TGF-β₁+ 5% FBS group were lower than those in the 0.50 ng/ml TGF-β₁+ 5% FBS group (t_α-SMA=4.622, P=0.010; t_FN =2.973, P=0.040; t _{Col Ⅲ}=7.845, P<0.001), but the expression of Col Ⅰ in 0.25 ng/ml TGF-β₁+ 5% FBS group was higher than that in 0.50 ng/ml TGF-β₁+ 5% FBS group (t_ColⅠ=4.022, P=0.016). The ratio of Col Ⅲ/ColⅠ in 0.25 ng/ml TGF-β₁+ 5% FBS group was lower than that in the 0.50 ng/ml TGF-β₁+ 5% FBS group in both mRNA and protein level (t_mRNA =-3.039, P=0.038; t_protein=3.215, P=0.032). The expression of LUM mRNA and KERA mRNA were detected in Pellet at different time points.The expression of LUM mRNA in 0.25 ng/ml TGF-β₁+ 5% FBS group increased over time.While in 0.50 ng/ml TGF-β₁+ 5% FBS group, the expression of LUM mRNA peaked at 1 week but declined at 2 weeks.The expression of KERA mRNA in two groups were all peaked at 1 week but declined at 2 weeks.

Conclusions

Low-dose TGF-β₁ in Pellet can maintain the normal growth of keratocytes and synthesize ECM.The expression of ECM tends to the normal condition after reducing the concentration of TGF-β₁, implying a scarless expression.