Differential regulation of stiffness, topography, and dimension of substrates in rat mesenchymal stem cells

Abstract

a b s t r a c tThe physiological microenvironment of the stem cell niche, including the three factors of stiffness, topography, and dimension, is crucial to stem cell proliferation and differentiation. Although a growing body of evidence is present to elucidate the importance of these factors individually, the interaction of the biophysical parameters of the factors remains insufficiently characterized, particularly for stem cells. To address this issue fully, we applied a micro-fabricated polyacrylamide hydrogel substrate with two elasticities, two topographies, and three dimensions to systematically test proliferation, morphology and spreading, differentiation, and cytoskeletal re-organization of rat bone marrow mesenchymal stem cells (rBMSCs) on twelve cases. An isolated but not combinatory impact of the factors was found regarding the specific functions. Substrate stiffness or dimension is predominant in regulating cell proliferation by fostering cell growth on stiff, unevenly dimensioned substrate. Topography is a key factor for manipulating cell morphology and spreading via the formation of a large spherical shape in a pillar substrate but not in a grooved substrate. Although stiffness leads to osteogenic or neuronal differentiation of rBMSCs on a stiff or soft substrate, respectively, topography or dimension also plays a lesser role in directing cell differentiation. Neither an isolated effect nor a combinatory effect was found for actin or tubulin expression, whereas a seemingly combinatory effect of topography and dimension was found in manipulating vimentin expression. These results further the understandings of stem cell proliferation, morphology, and differentiation in a physiologically mimicking microenvironment.

Differential regulation of morphology and stemness of mouse embryonic stem cells by substrate stiffness and topography

“…Topographical substrates were constructed using PA hydrogel via a soft-contact lithography technique described in our previous work [29]. Briefly, 10 ml of 40% acrylamide and 2% bis-acrylamide were mixed in water to form a solution with a constant concentration of 10% acrylamide and two with concentrations of 0.03 and 0.30% bis-acrylamide.…”

“…Finally, the PA hydrogel was prepared in the form of discs (20 mm in diameter and 0.1 mm in thickness) with the following three topographies: groove (ridge width/ditch width ¼ 5/15 mm), hexagonal (ridge width/ side-length ¼ 5/15 mm), and square pillar (side-length/inter-pillar gap size ¼ 10/ 10 mm) configurations with the same depths of 5 mm; a planar PA hydrogel of the same diameter and thickness was used as the control. Collagen I (1 ml of 200 mg/ml) was cross-linked securely onto the PA hydrogel surface prior to seeding the cells [29,30].…”

“…After adding 0.2 mg/ml sulfo-SANPAH solution (cross-linking agent) in an adequate volume, the mixture was irradiated with ultra-violet light for 5 min. The planar PA hydrogel was cut into rectangular strips to estimate its Young's modulus using a selfweighing assay, which yielded E ¼ 6.1 and 46.7 kPa at the two bis-acrylamide concentrations, respectively [29]. Finally, the PA hydrogel was prepared in the form of discs (20 mm in diameter and 0.1 mm in thickness) with the following three topographies: groove (ridge width/ditch width ¼ 5/15 mm), hexagonal (ridge width/ side-length ¼ 5/15 mm), and square pillar (side-length/inter-pillar gap size ¼ 10/ 10 mm) configurations with the same depths of 5 mm; a planar PA hydrogel of the same diameter and thickness was used as the control.…”

Bioactive calcium silicate extracts regulate the morphology and stemness of human embryonic stem cells at the initial stage

“…On one hand, CS bioceramics and their analogs are able to direct the differentiation of hMSCs into osteoblastic cells 22,23,25,27 . On the other hand, substrate stiffness, microtopography, and geometry are able to synergistically regulate the differentiation of ESCs 43 or even MSCs 44 . In this work, our data suggest that the 1+3 days culture of hESC colonies in the presence of CS extracts maintains cell stemness (Figs.…”

“…In some cases, TRITC-labeled phalloidin was used at a concentration of 5 μg/ml to stain actin. The samples were incubated with Hoechst 33342 for 10 min at room temperature (RT), washed 3-5 times with PBS, and stored at 4°C for examination with confocal laser scanning microscopy (Zeiss L710, Germany) 44 .…”