Enhancing osteogenic differentiation of mouse embryonic stem cells by nanofibers

Abstract

Controlled differentiation of embryonic stem cells (ESC) is necessary to their use as a cell source for tissue engineering or regeneration. To date, most studies have concentrated on chemical cues to direct ESC differentiation. However, during normal embryonic development, multiple factors beyond chemical cues play a role, including the extracellular matrix (ECM) in bone development. In this study, we use nanofibrous (NF) matrices to mimic the morphology of the ECM to examine the contribution of the ECM morphology to the differentiation of mouse ESC. After 12 h of differentiation culture, mouse ESC form protrusions interacting with NF matrices, while they appear not to interact with flat films. Immunofluorescence staining after 26 days of differentiation culture indicates a greater degree of differentiation for mouse ESC on NF matrices compared to flat films. Polymerase chain reaction results, also, show greater degree of osteogenic differentiation on NF matrices compared to flat films when osteogenic supplements are added to the culture. Overall, these results demonstrate that NF morphology contributes to the controlled differentiation of mouse ESC.

FIG. 1.

SEM micrographs of (A) nanofibrous matrix, scale bar = 10 μm; (B) solid films, scale bar = 10 μm; and (C) D3 cells after 12 h under differentiation conditions on nanofibrous matrix (Nano), solid films (Solid), and gelatin-coated tissue culture plastic (Control), scale bar = 5 μm.

FIG. 2.

Expression of neuronal, mesodermal, and early osteogenic markers and integrins after 12 days of culture under osteogenic conditions. (A) PCR on NF matrix (N), solid films (S), and gelatin-coated tissue culture plastic (C). (B) Quantification of brachyury expression on nanofibrous matrix (Nano), solid films (Solid), and gelatin-coated tissue culture plastic (Control); *p < 0.05. (C) Quantification of nestin expression on nanofibrous matrix (Nano), solid films (Solid), and gelatin-coated tissue culture plastic (Control); *p < 0.05.

FIG. 3.

Effects of integrin blocking on mesodermal and osteogenic differentiation after 12 days of differentiation culture. (A) PCR of α2 integrin RNA expression over time on nanofibrous matrix (N) and flat (solid) films (S). (B) Quantitative PCR of brachyury RNAs isolated from cells grown on nanofibrous matrix (Nano), on nanofibrous matrix with control IGG isotype (IGG nano), on nanofibrous matrix with CD49b antibody (α2 blocking nano), on flat (solid) films (Solid), on flat (solid) films with control IGG isotype (IGG solid), and on flat (solid) films with CD49b antibody (α2 blocking solid); **p < 0.01. (C) Quantitative PCR of Runx2 RNAs isolated from cells grown on nanofibrous matrix (Nano), on nanofibrous matrix with control IGG isotype (IGG nano), on nanofibrous matrix with CD49b antibody (α2 blocking nano), on solid-walled matrix (Solid), on flat (solid) films with control IGG isotype (IGG solid), and on flat (solid) films with CD49b antibody (α2 blocking Solid); **p < 0.01. (D) Quantitative PCR of brachyury RNAs isolated from cells grown on nanofibrous matrix (Nano), on nanofibrous matrix with control IGG isotype (IGG nano), on nanofibrous matrix with CD49e antibody (α5 blocking nano), on flat (solid) films (Solid), on flat (solid) films with control IGG isotype (IGG solid), and on solid-walled matrix with CD49e antibody (α5 blocking solid); *p < 0.05. (E) Quantitative PCR of Runx2 RNAs isolated from cells grown on nanofibrous matrix (Nano), on nanofibrous matrix with control IGG isotype (IGG nano), on nanofibrous matrix with CD49e antibody (α5 blocking nano), on solid-walled matrix (Solid), on flat (solid) films with control IGG isotype (IGG solid), and on flat (solid) films with CD49e antibody (α5 blocking solid); *p < 0.05.

FIG. 4.

Protein adsorption to materials after exposure to differentiation media containing 20% bovine serum protein or purified bovine fibronectin (100 μg/mL) for 1 h: (A) 4–12% polyacrylamide gels stained with Coomassie blue from protein extracts from nanofibrous matrix (N) and flat (solid) films (S) treated with media; (B) Western blot of fibronectin extracted from nanofibrous matrix (Nano) and flat (solid) films (Solid) treated with media; (C) Western blot of fibronectin extracted from nanofibrous matrix (Nano) and flat (solid) films (Solid) treated with purified bovine fibronectin.

FIG. 5.

Expression of osteogenic markers after 26 days of culture under osteogenic differentiation conditions. (A) PCR of RNAs isolated from cells grown on nanofibrous matrix (N), flat (solid) films (S), and gelatin-coated tissue culture plastic (C). (B) Quantitative PCR of osteocalcin RNAs isolated from cells grown on nanofibrous matrix (Nano), flat (solid) films (Solid), and gelatin-coated tissue culture plastic (Control); **p < 0.01. (C) Quantitative PCR of bone sialoprotein RNAs isolated from cells grown on nanofibrous matrix (Nano), flat (solid) films (Solid), and gelatin-coated tissue culture plastic (Control); *p < 0.05 and **p < 0.01.

FIG. 6.

Mineralization and extracellular characterization after 26 days of culture under osteogenic differentiation conditions. (A) Calcium staining after 26 days under osteogenic differentiation conditions on nanofibrous matrix (Nano), solid films (Solid), and gelatin-coated tissue culture plastic (Control). (B) Calcium staining after 26 days under osteogenic differentiation conditions on nano-fibrous matrix without ESC. (C) Immunofluorescence localization neuronal (TUJ1) and late bone differentiation (Osteocalcin) marker expression after 26 days under osteogenic differentiation conditions on nanofibrous matrix (Nano), solid films (Solid), and gelatin-coated tissue culture plastic (Control). Scale bar = 50 μm. Color images available online at www.liebertonline.com/ten.