Establishment of a three-dimensional corneal stroma extracellular matrix fibrosis model induced by transforming growth factor-β1 in vitro

Extracellular matrix (ECM) fibrosis leads to corneal scaring during the process of cornea wound healing.Transforming growth factor-β₁ (TGF-β₁) is known to mediate overproduce of ECM components.Our previous study developed a three-dimensional model for corneal stromal cells culture in vitro.

The hypothesis of this study was to apply TGF-β₁ in the three-dimensional culture system to establish a corneal stroma ECM fibrosis model.

Fresh bovine corneas were extracted for the culture of bovine keratocytes in constructed three-dimension culture system.The Pellets were cultured in the DMEM/F12+ 10% fetal bovine serum(FBS) medium with 0.5 ng/ml or 1.0 ng/ml TGF-β₁ or without TGF-β₁, respectively.Calcein AM/(propidium iodide) PI staining was employed to assay the cell viability 2 weeks after culture.The expressions of α-smooth muscle actin (α-SMA), type Ⅰ collagen (Col Ⅰ) and Col Ⅲ mRNA and protein in the cells were detected by real-time PCR and Western blot respectively 48 hours, 1 week and 2 weeks after cultured.The results were statistically analyzed.

Cultured for 48 hours in the Pellet system, corneal stromal cells clustered and was identified alive by Calcein-AM/PI staining in 2 weeks.The relative expression levels of α-SMA, Col Ⅰ and Col Ⅲ mRNA were elevated in both the 0.5 ng/ml and 1.0 ng/ml TGF-β₁ supplement groups in comparison with the only DMEM/F12+ 10%FBS group, with marked difference among the three groups (F_group=696.745, P<0.001; F_group=35.166, P<0.001; F_group=33.677, P<0.001), and the expression levels increased with the lapse of culture time(F_time=5.863, P<0.05; F_time=298.614, P<0.001; F_time=607.472, P<0.001). The synthetic rate of Col Ⅲ mRNA was obviously faster than that of Col Ⅰ mRNA.Western blot showed that only a trace of α-SMA, Col Ⅰ and Col Ⅲ were detected 48 hours and 1 week after culture.The expression levels of α-SMA, Col Ⅰ and Col Ⅲ in Pellet system in 0.5 ng/ml TGF-β₁ medium were 0.395±0.208, 1.060±0.175 and 0.629±0.382, and in 1.0 ng/ml TGF-β₁ medium were 0.758±0.228, 1.201±0.187 and 0.753±0.468, respectively 2 weeks after culture, significant differences were shown among the three groups (α-SMA: F=10.691, P<0.05; Col Ⅰ: F=14.094, P<0.05; Col Ⅲ: F=10.995, P<0.05).

Addition of TGF-β₁ and serum enhance the assembly and fibrosis of ECM, showing the higher expressions of specific fibrotic markers in bovine keratocytes Pellet.This culture systerm can be used as a candidate three-dimensional model for corneal stroma ECM fibrosis.

Corneal keratocyte; Wound healing; Cell culture techniques/methods; Transforming growth factor-β₁; Extracellular matrix; Fibrosis; Collagen type Ⅰ; Collagen type Ⅲ