Stiffer is better: Influence o fsubstrate stiffness on endothelial colony forming cells

Stiffer is better: Influence o fsubstrate stiffness on endothelial colony forming cells

Citation for published version (APA):

Fioretta, E. S., Bouten, C. V. C., & Baaijens, F. P. T. (2011). Stiffer is better: Influence o fsubstrate stiffness on endothelial colony forming cells. Poster session presented at Mate Poster Award 2011 : 16th Annual Poster Contest.

Document status and date: Published: 01/01/2011

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Proliferation, instead, is comparable on the two substrates (Fig. 2B).

On 21kPa gels, ECFCs express higher level of αSMA compared to glass substrates (Fig. 3B), deposit collagen type III in oriented fiber-like structures, and produce a great amount of collagen type IV. Finally, ECFCs on 21kPa gels develop transient tubular structure that recall the EC vasculogenesis (Fig. 4).

We hypothesized that the likely signaling pathway involved in stiffness-related EndoMT is the TGFβ1-pathway.[5] Despite differences in ECFC behavior when cultured on physiological-like substrates, we cannot confirm EndoMT because cells maintain the endothelial phenotype (Fig. 3A) and they do not express αSMA stress fibers – developed instead by transdifferentiated cells stimulated with TGFβ1 (Fig. 3C).

Conclusion

Since substrate stiffness can influence ECFC phenotype within limits, stiff polymeric materials can be safely used for scaffold for in situ TE to improve cell adhesion and proliferation without inducing changing in cell proliferation, phenotype, or matrix production.

Introduction

In situ vascular tissue engineering (TE) aims at capturing and

differentiating cells from the blood by mimicking the endogenous vascular repair (Fig. 1)[1]. Circulating Endothelial Colony Forming Cells (ECFCs) are a suitable cell source for this approach because they can differentiate into endothelial cells (ECs)[2] and transdifferentiate (EndoMT) into smooth muscle cells (SMCs) using TGFβ1[3]. ECFCs recruited from the blood will perceive a change in mechanical environment because of the adhesion on a stiff (polymeric) scaffold instead of the elastic lamina. The aim of this

study is to evaluate the influence of physiological vessel wall

stiffness (21 kPa)[4] on ECFCs behavior and to compare it with stiff glass substrates (>GPa) to identify if material stiffness can induces changes in lineage specification.

Materials and Methods

Human ECFCs were seeded on fibronectin-coated polyacrylamide gels with a measured stiffness of 21.6±2.9kPa. As control group for stiff polymeric material, cells were seeded on coated glass. To compare the results with EndoMT, cells were also cultured with the addition of TGFβ1. Analysis were performed on proliferation (BrdU), focal adhesion (FAs, vinculin), ECs (CD31) and SMCs (αSMA) markers, cytoskeleton structure (phalloidin) and collagen production (type III and IV).

Results

ECFCs adhere better on glass substrate compared to 21kPa gels (Fig. 2A), as quantified by the amount of FAs expressed per cell.

/ Biomechanics & Tissue Engineering

Fioretta ES et al., Macromol. Biosci. (2011). [1] Obi et al. J Appl. Physiol. (2009). [2] Moonen JR et al. Biomaterials (2010). [3] Discher et al. Science (2009). [4] Fioretta ES et al., J Biomechanics (2011). [5]

Stiffer is Better

Influence of Substrate Stiffness on

Endothelial Colony Forming Cells

Department of Biomedical Engineering, Eindhoven University of Technology, The Netherlands

Figure 1: In situ vascular TE is a straight-forward approach where cells (e.g.: ECFCs) are

selectively captured from the blood inside a functionalized scaffold. The different biomolecules applied to the scaffold will trigger the recruited cells towards (a) differentiation into ECs; (b) transdifferentiation into SMCs, to restore vessel integrity.

Figure 3: Mean fluorescent intensity (MFI) measured by FACS for CD31 (A) and αSMA (B) expressed by ECFCs cultured on substrates with different stiffness. * indicates a

difference (p<0.05) between the two groups. (C) Immunofluorescent images for CD31 (red) and αSMA (green) of ECFCs cultured on (a) 21kPa gels; (b) on glass; (c) on glass with the addition of TGFβ1 to induce EndoMT. Cell nuclei Is stained by DAPI (blue). Scale bar is 50 µm.

Figure 2: (A) Quantification of cell adhesion as number of FAs expressed per cell

(FA/cell). * indicates a difference (p<0.05) between the two groups. (B) ECFCs proliferation on two samples (light and dark bars) measured as the ratio between BrdU positive cells and the total number of cells, in percentage.

Figure 4: Immunofluorescent pictures of ECFCs. On 21kPa gels cells express oriented

(arrow)collagen type III (a) and abundant collagen type IV (b). Phalloidin staining reveals tubular structures (c). ECFCs cultured on glass produced collagen type III (green) and IV (yellow) (d). Cell nuclei is stained by DAPI (blue). Scale bar is 50 µm.

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