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A. Hubel, R. A. B. Crabb; Macroscopic and Microstructural Properties of a Multilayer Corneal Replacement. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3922. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The native cornea contains keratocytes dispersed between many layers of collagen lamellae. A multilayer corneal replacement was developed to mimic in vitro a structure of the native cornea. The influence of adhesive composition and culture on the properties of the construct was determined.
Collagen films were seeded with stromal fibroblasts, cultured and then covered with either collagen or fibrin gel as an adhesive between the two layers. The modulus and UTS were determined from a single ramp to failure during uniaxial tensile tests and the initial fraction of transmittance was measured. Additional microstructural and immunohistochemical studies were performed to characterize cellular behavior.
When collagen gel was used, the modulus and UTS were initially 14.1 ± 5.4 MPa and 1.1 ± 0.4 MPa respectively, and when fibrin gel was used they were 22.0 ± 1.8 MPa and 1.9 ± 0.3 MPa respectively. Additionally, the fraction of transmittance at 400 and 700 nm of the constructs tested after fabrication were not significantly different between the adhesives; they were 0.58 ± 0.09 and 0.84 ± 0.04 with collagen and 0.53 ± 0.10 and 0.81 ± 0.05 with fibrin at 400 and 700 nm respectively.Confocal imaging of constructs adhered with fibrin gel showed that the stromal fibroblasts had produced a mat of fibronectin and some collagen by one week of culture. Stromal fibroblasts were observed to be both on the collagen film and within multiple layers of the fibrin gel, which showed that the cells were not restricted to the film surface upon which they were seeded. The average new fibril diameter for the multilayered construct was 67 ± 47 nm, and the range was 10-233 nm.
Both collagen and fibrin gels were shown to be capable of adhering two collagen films. The constructs fabricated with fibrin gel also had a greater modulus and ultimate tensile strength. The stromal fibroblasts were also influenced by the presence of a second film and the adhesive with changes in cell behavior. This type of model may be helpful in understanding stromal fibroblast behavior in a three dimensional matrix.
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