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MA Watsky, CJ Doillon, M Hakim, J Wang, R Munger, N Laycock, M Griffith; A Collagen-based Scaffold for a Tissue Engineered Human Cornea: Physical and Physiological Properties . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2996.
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Purpose: To develop and characterize a stabilized collagen-glycosaminoglycans (GAG) based matrix that serves as a scaffold for a tissue engineered human corneal equivalent. Methods: Immortalized human corneal cells similar to low passage primary cells were used. Hydrated matrices were constructed by blending neutralized type I collagen (bovine dermis or rat tail tendon) with one of the following: chondroitin sulphates A, B, C or collagen type III, and then stabilizing by glutaraldehyde crosslinking. Glycine was added to deactivate excess glutaraldehyde, and stromal cells were mixed into the matrix. For some experiments, epithelial and endothelial cells were added. Constructs were supplemented with growth media, with or without ascorbic acid. Some were treated with chemicals to cause different degrees of wounding to evaluate their ability to recover. Results: Native, non-crosslinked gels were soft and exhibited limited longevity. Glutaraldehyde cross-linking strengthened the matrix while still permitting cell growth. FTIR spectra indicated that bovine collagen was more easily cross-linked by glutaraldehyde than rat tail collagen. Chondroitin sulphate addition increased transparency. Keratocytes grown within the matrix had a higher frequency of K+ channel expression than identical keratocytes grown on a culture dish. Ascorbic acid addition increased degradation rate of the uncrosslinked matrix in the presence of keratocytes, but enhanced keratocyte proliferation and endogenous collagen synthesis. Wounded constructs showed recovery from chemical exposure. Conclusion: Presence of keratocytes and ascorbic acid influences the scaffold properties. A very low concentration of glutaraldehyde carries out most of the biophysical and biological characteristics required for a reliable stroma substitute.
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