The overall objective of this investigation was to characterize the extracellular matrix deposited by the stromal fibroblasts as a function of time in culture and matrix microstructure. Stromal fibroblasts were seeded onto collagen matrices and cultured for up to 5 weeks. The collagen matrices contained collagen fibrils with an average diameter of 215±20 nm. When cultured on a collagen film, an average fibril diameter of 62±39 nm was observed for single layer films with only slight variations with time in culture, and after 1 week of culture between two film layers 67±47 nm fibrils were observed after 1 week. When the film surface was molded into 1 and 2 lm microgrooves, the initial average fibril diameter of the extracellular matrix was 73±21 and 73±31 nm respectively. When cultured on a collagen sponge, an average fibril diameter of 107±20 nm was initially observed and decreased to 47.5±17 nm after 1 week in culture. For cells cultured on a collagen sponge, Western blotting showed an increase in myofibroblast phenotype expression with time in culture. Shifts in phenotype were less distinct for cells cultured on collagen films. The microstructure, rather than geometry, of the matrix substrate appeared to influence the newly synthesized extracellular matrix and cell phenotype.
Bibliographical noteFunding Information:
The authors would like to thank the Fight for Sight Foundation and the American Society of Biomechanics for their fellowship support of RABC and the support of NSF for the Research Experience for Undergraduates Program in Mechanical Engineering at the University of Minnesota for DMD. Human corneal tissue for cell isolation was provided by the Minnesota Lions Eye Bank. The authors would also like to thank Audrey Bernstein and Sandra Masur for their discussions about corneal cell phenotype and Ching Yuan for his guidance with Western blotting. Lastly, the authors would like to thank the laboratory of Dr. William R. Kennedy, particularly Gwen Wendelschafer-Crabb, for the use of the confocal microscope and the gifts of Zamboni’s fixative, DAPI, and secondary antibodies.
Copyright 2012 Elsevier B.V., All rights reserved.