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Chi-Chin Sun, Jui-Yang Lai, Kuo-Ying Lee; Effects of chitosan membrane deacetylation on sphere formation of corneal keratocytes. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5150.
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To investigate the physical and morphological effects of degree of deacetylation on in vitro chitosan membrane-cultivated keratocyte spheroids.
Alkaline treatment method was applied to the preparation of a more highly deacetylated chitosan. To isolate stromal keratocytes, corneas of New Zealand white rabbits stripped of both endothelium and epithelium were placed in a solution of collagenase overnight at 37°C. The keratocytes were washed and resuspended in culture medium consisting of DMEM-F12/HAM basal medium supplemented with 10% FBS. Confluent cell layers were treated with trypsin-EDTA and seeded onto the chitosan coatings(C74, C84 and C94, larger number indicating higher deacetylation) or plastic dishes. The cultures were subjected to different analyses including tension strength, contact angle, surface potential, cell adhesion, light microscopy and scanning electron microscopy(SEM) examinations.
The results show that Young's modulus in C74, C84, C94 groups are 58.1 ± 2.5 MPa, 78.6 ± 5.0 MPa, and 94.0 ± 3.6 MPa(P <0.05) respectively. The contact angle on plastic dish(60.4 ± 1.9°) is significantly less than those on C74 (93.3 ± 2.64°), C84 (85.1 ± 2.3°) and C94(75.1 ± 2.2°)(P <0.05). This result is associated with the chemical composition of the surface material, indication a hydrophilic property on chitosan membrane. The zeta potential are TCPS(-10.6 ± 0.9mV), C74(15.7 ± 1.1mV), C84(20.2 ± 1.8 mV) and C94(27.5 ± 1.4 mV), respectively. As degree of deacetylation increases, the positive potential also rises up accordingly. MTT assay shows that the number of cell attachment increases with the higher chitosan membrane deacetylation degree. SEM observations demonstrate that cell adhesion on plastic dishes is the most smoothness followed by C94, C84 and C74. Cells on C74 show the highest bulging contour. These results echo the findings of MTT assay. Spheroid formation observed by the light microscopy demonstrates that the lower the degree of deacetylation of the chitosan membrane(C74), the greater number of the sphere formation, probably due to the different cell adhesion effect.
The less deacetylation degree of chitosan membrane(C74) is less favorable for corneal keratocyte adhesion, but promotes spheroid formation in vitro.
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