Acacia honey is a natural product which has proven to have therapeutic effects on skin wound healing, but its potential healing effects in corneal wound healing have not been studied. This study aimed to explore the effects of Acacia honey (AH) on corneal keratocytes morphology, proliferative capacity, cell cycle, gene and protein analyses. Keratocytes from the corneal stroma of six New Zealand white rabbits were isolated and cultured until passage 1. The optimal dose of AH in the basal medium (FD) and medium containing serum (FDS) for keratocytes proliferation was identified using MTT assay. The morphological changes, gene and protein expressions of aldehyde dehydrogenase (ALDH), marker for quiescent keratocytes and vimentin, marker for fibroblasts were detected using q-RTPCR and immunocytochemistry respectively. Flowcytometry was performed to evaluate the cell cycle analysis of corneal keratocytes.
There has been no effective treatment or agent that is available for corneal injury in promoting corneal wound healing. Previous studies on edible bird's nest extract (EBN) had reported the presence of hormone-like substance; avian epidermal growth factor that could stimulate cell division and enhance regeneration. This study aimed to investigate the effects of EBN on corneal keratocytes proliferative capacity and phenotypical changes.
The in vivo quiescent corneal stroma keratocytes need to be transformed to activated state in order to obtain sufficient number of cells either for monolayer evaluation or corneal stroma reconstruction. This study aimed to investigate the phenotypic characterization of corneal stromal cells during culture expansion from the limbal region of the cornea. Isolated corneal keratocytes from limbal tissue of New Zealand White Strain rabbits' corneas (n = 6) were culture expanded until three passages. Keratocytes morphology was examined daily with viability, growth rate, number of cell doubling and population doubling time were recorded at each passage. The expression of collagen type 1, aldehyde dehydrogenase (ALDH), lumican and alpha smooth muscle actin (α-SMA) were detected by RT-PCR. Immunocytochemistry was also used to detect ALDH, α-SMA, collagen type I and Cytokeratin-3 (CK3). Growth kinetic study revealed that the growth rate was low at the initial passage but increase to about two folds with concomitant reduction in population doubling time in later passages. Freshly isolated and cultured keratocytes expressed collagen type 1, ALDH and lumican but α-SMA expression was absent. However, α-SMA was expressed along with the other genes during culture expansion. Keratocytes at P1 expressed all the proteins except CK3. These results suggest that cultured keratocytes maintained most of the gene expression profile of native keratocytes while the emergence of α-SMA in serial passages showed a mix population of various phenotypes. The phenotypic characterization of monolayer keratocytes provides useful information before reconstruction of bioengineered tissue or in vitro pharmaceutical applications.