Thin Films of Collagen Affect Smooth Muscle Cell Morphology

Abstract

Thin films of the extracellular matrix protein, collagen, were prepared by adsorbing native or heatdenatured type I collagen onto hexadecanethiol self-assembled monolayers. The resulting films were characterized by atomic force microscopy, ellipsometry, and light microscopy. Denatured collagen formed a topographically smooth ∼3.6 nm thick film, consistent with an adsorbed protein monolayer. In contrast, the native collagen thin film consisted of supramolecular collagen fibrils. The density of the large fibrils could be varied by changing the native collagen concentration in the solution from which the films were prepared. The biomimetic nature of the thin collagen films was partially assessed by examining their effects on vascular smooth muscle cells. Automated quantitative analysis indicated that the morphology of smooth muscle cells on the thin films was dependent on whether the collagen was heat-denatured or was in its native fibrillar form. The area of cells on denatured collagen films was significantly larger than that of cells on thin films of native fibrillar collagen. This response closely mimicked the response of these cells to thick collagen gels. Examination of the relationship between collagen fibril density and cell area indicated that large fibrils play a role in determining how cells respond to collagen. Cells assumed a larger morphology on native collagen films with a lower density of large fibrils. In this study, it is clear that cell morphology on these films is determined by micron-scale interactions between cells and the matrix molecules and is not dependent on the bulk materials properties of collagen gels.

Evaluating the performance of fibrillar collagen films formed at polystyrene surfaces as cell culture substrates

“…9 1-Hexadecanethiol ͑Aldrich, St. Louis MO͒, as a 0.5 mM solution in 99.5% ethanol ͑Aldrich͒, was allowed to adsorb to gold films by incubating gold surfaces directly in the thiol solution for 16 h. These substrates were rinsed with ethanol and dried with 0.02 m pore size filtered N 2 prior to incubation with collagen solution.…”

“…9 Phase microscopy images were collected through a 20ϫ objective on a Zeiss Axiovert 100 inverted microscope. Collagen-coated gold coverslip or polystyrene was rinsed with ultra-purified H 2 O ͑Millipore, Billerica, MA͒ and dried with filtered N 2 .…”

“…9 Thiolate monolayers have frequently been used to produce a highly ordered chemically modified surface coating to allow adsorption of ECM proteins such as fibronectin. 10 Our studies with collagen have shown than at a hydrophobic alkanethiol interface, a network of collagen fibrils forms a thin film with an effective medium thickness on the order of 40 nm.…”

“…Larger fibrils 200 nm in diameter and tens of microns in length carpet the surface and appear to grow out of smaller fibrils about 25 nm in diameter that are in intimate contact with the alkanethiolate monolayer. 9 We have shown by ellipsometry a͒ Electronic mail: jelliott@nist.gov and atomic force microscopy ͑AFM͒ that thin films of collagen can be formed reproducibly, 9 and that the supramolecular structure of fibrils can be tuned by altering the solution concentration of collagen 9 or the chemistry of the alkylthiolate surface. 11 We have shown that cells are sensitive to the density of large fibrils 9 and to the mechanical properties 12 and orientation 13 of collagen fibrils.…”

Construction and Biofunction of 3‐Dimensional Self‐assembly Collagen Nanostructure on PLLA Substrate

“…These controllable patterned structures of collagen were verified to be good scaffolds for cell or soft/hard tissue culture. [10][11][12][13] Some of the works have demonstrated that the supramolecular organization of collagen layers has a strong effect on the behavior of cells, 6,14 showing their potential applications in tissue engineering region. However, the biomineralization inductivity of collagen was not studied although their arrangement and assembly state were said to relate directly to the bone regeneration in vivo.…”