Scaffold Of Transglutaminase Cross-linked Collagen Gel For Three-dimensional Corneal Stroma Reconstruction

Objective:(1) To research the characteristics of rabbit corneal keratocytes which were cultured in vitro, and to lay a foundation for tissue engineering of cornea. (2) To study the status of rabbit keratocytes growing on collagen gel and investigate the feasibility of collagen matrix for cornea stroma. (3)To investigate the property of scaffold of transglutaminase cross-linked collagen gel and the changes of seeded keratocytes in order to provide a new way to enhance the mechanical strength of tissue engineering corneal stroma.Methods:(1) Rabbit keratocytes were cultured by tissue inoculation or digestion methods and the cells were subcultured after they became confluent. The growing characteristics were observed every day. (2) The suspension of the third passage of keratocytes were cultured on collagen gel at a density of 5×104cells/ml. The cells were incubated for approximately 1h at 37℃to allow the cells to facilitate attachment to the surfaces before medium was added. Medium was replaced every 2 days and the surfaces were examined daily by phase contrast microscopy. (3) The suspension of the third passage of keratocytes was mixed with collagen solution on an ice bath at a density of 5×104/ml cells, and three-dimensional corneal stroma was reconstructed. The collagen gel was prepared with transglutaminase in experimental group, and collagen gel without transglutaminase was used as control group. Phase contrast microscope examination was performed to observe the state of cell-seeded scaffolds daily. The proliferation of keratocytes was assessed at 1,2,3,5 days separately by Alamar-Blue reduction assay. Immunohistochemistry was carried out to examine the cellular response to vimentin antibody under the confocal microscope. Transmittance of the collagen samples was evaluated at 2 and 3 weeks. The cross-linked statue of cell-seeded collagen substrates was observed to assess the digestion procedure.Results:(1) Rabbit keratocytes grew out from the rim of tissue and formed a confluent monolayer in 2-3 weeks. The cells cultured by digestion adhered within 4 hours and formed a confluent monolayer in 5-6 days. The subcultured cells separated by the two methods still maintained the characteristics of the primary cells. (2) Keratocytes cultured on collagen gel adhered to the surfaces within 3 hours and attachmented tightly with gel after 24 hours. Intercellular junctions were obvious, and pseudopods penetrated into the matrix in 4-5 days. At a later stage, collagen matrix showed obvious degradation and contraction. (3) Keratocytes elongated and showed triangle or dendritic shape with a stronger ability of forming network in cross-linked collagen compared with spindle-shape cells in the native collagen without transglutaminase. Proliferation ability of cell was increased in both groups after culture. The keratocytes showed the positive response for vimentin after 14-day culture in both groups, and the cellular pseudopods were abundant in experimental group. Light transmission of transglutaminase crosslinked cell-seeded scaffold was lower than that of native collagen cell-seeded scaffold, but the stability was superior to control group. The transglutaminase-treated collagen substrates showed a stronger resistance to collagenase in comparison with untransglutaminase-treated collagen substrates.Conclusions:(1) This research explores the characteristics of rabbit corneal keratocytes which are cultured in vitro, and establishes a simple and efficient method for culture of rabbit corneal keratocytes in vitro. (2) Collagen is well compatible with keratocytes, which can construct tissue engineered cornea stroma, so as to further reconstruct. (3) The cellular morphology and activity of keratocytes within transglutaminase crosslinked collagen are similar to natural cornea. The scaffold of transglutaminase cross-linked collagen gel is favorable for the three-dimensional corneal stroma reconstruction.