Advisory Committee Chair
Rafael Grytz
Advisory Committee Members
Joel L Berry
Thomas T Norton
Yong Zhou
Document Type
Thesis
Date of Award
2016
Degree Name by School
Master of Biomedical Engineering (MBE) School of Engineering
Abstract
Purpose: Increasing evidence suggests that unknown scleral remodeling mechanisms underlie myopia development. We are proposing a novel organ culture system in combination with two-photon fluorescence imaging to quantity collagen remodeling at the tissue- (macro-level) and lamellar-level (micro-level). Methods: Tree shrew scleral shells were cultured up to 7 days in serum-free media and cellular viability was investigated under: (i) minimal tissue manipulations; (ii) removal of intraocular tissues; (iii) gluing the eye to a stainless steel washer with 50 μL of glue; (iv) same as condition (iii) with 200 μL of glue; (v) supplementing media with Ham's F-12 Nutrient Mixture; and (vi) culturing eyes subjected to 15 mmHg intraocular pressure in a bioreactor. Two different bioreactors were preliminarily tested before a design was finalized. Using our finalized bioreactor, we cultured and fluorescently labeled two scleral shells of normal juvenile tree shrews. Using two-photon microscopy, grid patterns were photobleached into multiple collagen lamellae and across the apex of the scleral shell. These patterns were imaged daily for 3 days, and tissue-/lamella-level strains were calculated from the deformed patterns. Results: No significant reduction in cell viability was observed under condition (i). All tissue manipulations lowered the average viability. Compared to condition (i), cell viability was significantly reduced at day 0 in condition (ii), at day 3 in conditions (ii, iii, iv, vi), and at day 7 in conditions (ii, iii, iv). One bioreactor was determined superior due to its closed design and ability to be used with a multiphoton microscope. Micro- and macro-level normal strains were -0.36±1.51% and 1.22±0.74% at day 2 to -0.19±1.90% and 2.31±1.41% at day 3, respectively. Intralamellar shear angle was 0.63º (IQR = 0.34-1.07) on day 2, and 0.62º (IQR = 0.26-1.12) on day 3. Conclusions: Findings suggest that tree shrew sclera can be cultured in serum-free media for 7 days with no significant reduction in cell viability. Scleral fibroblasts are highly sensitive to mechanical tissue manipulations and tissue gluing, while Ham's F-12 Nutrient Mixture has a protective effect on cell viability. This is the first study to quantify collagen remodeling deformations over a prolonged period in organ culture suggesting that scleral remodeling in juvenile tree shrews involves tissue elongation, lamellar elongation, and intralamellar deformations.
Recommended Citation
Baldivia, Sarah, "Establishing and Organ Culture to Study Scleral Collagen Remodeling in Myopia" (2016). All ETDs from UAB. 1092.
https://digitalcommons.library.uab.edu/etd-collection/1092