| Objective:(1) In order to investigate the property of rabbit corneal keratocytes cultured in physiological status, keratocyte alignment cultured on microgrooved patterns of polystyrene plates and cellular biology were studied. (2) To explore the proper density and the cellular biological characteristics when corneal epithelial cells and oral mucosal epithelial cells were seeded on temperature-responsive Nunc UpCell Surface. The carrier-free cell sheets fabricated on the temperature-responsive culture surfaces. (3) To investigate the influence of recombinant maxadilan to keratin CK3 expression of oral mucosal epithelial cells when co-culturing keratocytes. The proliferation effects of different concentration maxadilan to keratocytes were also studied.Methods:(1) The alignment patterns were designed as alternating grooves and ridges, including 0.25mm lineation space (narrow groove group), lmm lineation space (wide groove group), and no alignment pattern worked as control group. The suspension of rabbit P2 keratocytes was inoculated on the culture plates at a density of 1×105 cells/ml. Morphology of cultured cells was observed under the phase contrast microscope daily. The cellular growth and contact guidance property were investigated with HE staining and immunofluorescence staining of vimentin. Cells in narrow groove group was incubated at 37℃in a closed dry 5% CO2 environment and the processes of cellular adherence, contact guidance proliferation, and apoptosis of 24 hour time-lapse were observed under real-time microscope. (2) Different densities of primary corneal epithelial cells and oral mucosal epithelial cells were seeded on Nunc UpCell surface and tissue culture plate (TCP) surface of 6 well plates and incubated. Phase contrast microscope examination was performed daily to observe morphological differences in different cellular densities. Acridine orange (AO) staining was used to evaluated the cell viability. HE staining was carried to observe the paraffin longitudinal section of the cell sheets. SEM examination was carried out to observe the cell junction and the structure. (3) RT-PCR assay was used to detect the expression of PAC1 receptor in keratocytes. Western-Blot assay was used to detect the expression of PAC1 receptor in corneal epithelial cells, keratocytes and oral mucosa epithelial cells. The proliferation effects of keratocytes in 1nM～500nM maxadilan using DMEM medium with 0.3% FBS or 10% FBS were assessed by CCK-8 method. Real-Time PCR assay was established for detection of CK3 gene expression of four groups as bellow:rabbit mucosal epithelial cells co-culture with keratocytes through transwell culture (group one), group one +100nM recombinant maxadilan (group two), rabbit mucosal epithelial cells (group three) and corneal epithelial cells (group four) as control group.Results:(1) Keratocytes in groove groups grew along the grooves, but cells in control group showed no alignment pattern. Histological evaluation of HE staining and immunofluorescence staining showed that keratocytes in groove groups had contact guidance feature and elongated on the surface with microgrooved patterns and aligned along that patterns, the cells of three groups presented the positive response for Vimentin. Comparison to the wide group, the cells in narrow group showed more obvious growing characteristics of alignment. The images of cells in narrow group under the real-time microscope confirmed that cells proliferated along the arranged grooves to abide by the contact guidance. The cellular adherence was firstly observed in cell body, but its apoptosis occurred firstly in cell foot processes. (2) There were not obvious discrimination in different densities of primary corneal epithelial cells and oral mucosal epithelial cells seeded on Nunc UpCell surface and TCP surface during the early stage. With the time prolongs, the cells of 5×104/ml density presented retardation, the cells of 2×105/ml density displayed apoptosis state, while the cells of 1×105/ml density were easy to form cobble-like morphology and colony formation. There were multilayer cells and cells reached confluence on day 8. Cell sheets were harvested by reducing the culture temperature to 20℃-25℃. HE staining showed that the cell sheets were carrier-free. SEM showed tight junction of the cells. (3)RT-PCR assay demonstrated the expression of PAC1 receptor in keratocytes. Western-Blot detected the expression of PAC1 receptor in corneal epithelial cells, keratocytes and oral mucosa epithelial cells at protein level. The CCK8 assay revealed that maxadilan promoted the proliferation of keratocytes. Real-Time PCR demonstrated that maxadilan promoted the expression of CK3 in oral mucosal epithelial cells when co-culture keratocytes.Conclusions:(1) Keratocytes cultured on microgrooved surfaces have alignment pattern, especially in the pattern of 0.25mm lineation space. Keratocytes show contact guidance feature in alignment growth and close to the physiological status, which is suitable to the three-dimensional corneal stroma reconstruction in vitro. (2) Cell sheets obtained from Nunc UpCell surface show carrier-free without exogenous materials, which corresponds the requirements of reconstructing physiological condition of cornea cellular layers. (3) There is the expression of PAC1 receptor in corneal epithelial cells, keratocytes and oral mucosa epithelial cells. Maxadilan, an agonist of PAC1 receptor, can promote the proliferation of keratocytes and the expression of CK3 of oral mucosa epithelial cells. So Maxadilan is to the benefit of seeding cells of corneal tissue engineering.
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