Microstructural Characteristics of Extracellular Matrix Produced by Stromal Fibroblasts

Abstract

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 microm 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.

Morphologic Characterization of Organized Extracellular Matrix Deposition by Ascorbic Acid–Stimulated Human Corneal Fibroblasts

“…16,17,22 The resultant matrix, which is intended to replace the disorganized degrading scaffolding, does not exhibit organized arrays of collagen fibrils. 78 Although our model is not a perfect analog for stromal development, some strong similarities suggest that the same mechanisms may be involved in the syntheses of the organized arrays of collagen fibrils. It is, therefore, a potentially powerful tool in which to study how fibroblasts produce organized matrix.…”

Effect of Substrate Composition and Alignment on Corneal Cell Phenotype

“…HCKs were found to secrete more mesh-like structures after 2 weeks of culture time. 31 SEM analysis of our samples has indicated different matrix structures produced by RCFs on TCP and RCFs on fibrous, collagen scaffolds (Fig. 8).…”

“…HCKs cultured on collagen films promoted HCKs to deposit smaller-diameter fibers than HCKs cultured on 1-2 mm microgrooved films. 31 In addition, they showed that the length of culture time can affect ECM morphology. HCKs were found to secrete more mesh-like structures after 2 weeks of culture time.…”

“…Crabb et al identified similar mesh-like ECM structures in their cultures and confirmed these to be fibronectin, which is a common ECM protein found in wound-healing phenotypes. 31 Additionally, other research groups have attributed loss of transparency to a decreased organization of the ECM of their tissue constructs. 25 The differences in ECM morphology may explain why RCFs cultured on TCP scatter less light than those on unaligned samples despite the fact that RCFs express more a-SMA when cultured on TCP.…”

The Development of a Tissue-Engineered Cornea: Biomaterials and Culture Methods

“…Collagen films have also been investigated as potential scaffolds for corneal replacement (38). Collagen films were prepared from dispersions of bovine type I collagen.…”