Abstract
Purpose:
In response to injury cornea secretes extracellular matrix (ECM) proteins such as collagens, laminin and fibronectin. Previously we demonstrated the upregulation of corneal epithelial cell cycle protein expression with different ECM proteins. The changes observed with these proteins were comparable to the changes that occurred during the repair of complete rabbit corneal epithelial debridement injury (ARVO 2014, Program 5530/Abstract A0104). In the current study we have evaulated the cell regenerating potential of a preparation containing different ECM proteins (ECMIX).
Methods:
Porcine or rabbit corneal epithelial primary cultures were grown in DMEM/F12. ECMIX containing collagen I, collagen IV, laminin and fibronectin was prepared in DMEM/F12. Cell growth promoting potential of ECMIX was determined by CyQuant proliferation assay. Changes in the expression of different cell cycle progression facilitating cyclins and CDKs as well as Erk and Akt activation that control cell cycle were assessed by immunoblotting. To develop a device for therapeutic applications we constructed pectin (1%)-calcium gel matrix embedded with ECMIX and corneal epithelial cells.
Results:
ECMIX preparation increased the growth of corneal epithelial cells. In the presence of ECMIX cell proliferation (related to DNA content) was increased by 40-45%. However, when cells were treated with collagen or laminin or fibronectin alone no consistent increase in growth was observed. In the presence of ECMIX, activation of Erk and Akt occurred. The expression of cell cycle progressing proteins cyclin A, cyclin E, Cdk 2, Cdk4 and Cdc42 was increased by 40-50%. When corneal epithelial cells were treated with collagen or fibronectin or laminin the upregulation in cell cycle proteins was much more pronounced. ECMIX facilitated in-gel as well as outgrowth of embedded corneal epithelial cells from pectin gel matrix. Epithelial outgrowth from gel matrix was sustained for 7-10 days.
Conclusions:
Collective influence of different ECM proteins on cell cycle progression may be required for the promotion of corneal epithelial cell regeneration. Sustained cell-regenerating potential of ECMIX could be useful for the treatment of difficult-to-heal corneal epithelial injuries.