Influence of micropattern width on differentiation of human mesenchymal stem cells to vascular smooth muscle cells

doi:10.1016/j.colsurfb.2014.06.013

Colloids and Surfaces B: Biointerfaces

Volume 122, 1 October 2014, Pages 316-323

https://doi.org/10.1016/j.colsurfb.2014.06.013 Get rights and content

Highlights

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A gradient micropattern of different stripe width was prepared.
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The width of micropattern stripes showed obvious effect on hMSCs orientation and differentiation.
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Narrow stripes allowed hMSCs to spread along the stripes and limited spreading in perpendicular direction.
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Cells cultured on the narrow stripes showed a significantly higher expression of VSMC marker protein.

Abstract

In recent years, various approaches have been taken to generate functional muscle tissue by tissue engineering. However, in vitro methods to generate smooth muscle with physiologically aligned structure remains limited. In order to mimic the in vivo highly organized structure of smooth muscle cells, we used micropatterning technology for engineering parallel aligned cells. In this study, a gradient micropattern of different width of cell-adhesive polystyrene stripes (5, 10, 20, 40, 60, 80, 100, 200, 400, 600, 800 and 1000 μm) was prepared and the effects of micropattern width on human mesenchymal stem cells (hMSCs) orientation, morphology and smooth muscle cell differentiation were investigated. The width of micropattern stripes showed obvious effect on cell orientation, morphology and smooth muscle cell differentiation. The cells showed higher degree of orientation when the micropattern stripes became narrower. Higher expression of calponin and smooth muscle actin was observed among the narrow micropatterns ranging from 200 μm to 20 μm, compared to the non-patterned area and wide micropattern areas which showed similar levels of expression.

Graphical abstract

Introduction

Vascular smooth muscle cells (VSMCs) perform a crucial function in angiogenesis, mechanical support of vessels and blood pressure control [1]. To successfully construct tissue-engineered blood vessels, regeneration of functional VSMC layer is required. Since the lifespan of autologous VSMCs derived from elder donors who are the majority of potential recipients of vascular grafts for the treatment of cardiovascular diseases [2], [3] is limited, increasing the lifespan of autologous VSMCs from elder donors is still a big challenge. Human mesenchymal stem cells (hMSCs) can differentiate into a variety of cell types including myocytes [4] and they are considered to be nonimmunogenic [5]. Their potential as a source of smooth muscle progenitor cells for vascular engineering approach has received widespread attention.

To direct hMSCs differentiation to a VSMCs lineage, an appropriate microenvironment that mimics the in vivo physicochemical and biological cues is desirable. One approach using micropatterning technique to regulate cell alignment is considered effective on mimicking the structure, composition and function of muscular tissue. Huang et al. [6] have reported that myoblasts on micropatterned polymer surfaces had well organized F-actin assembly to the direction of microgrooves, together with enhanced level of myotube formation at early time point. In another report, Tay et al. [7] have developed a biomimetic PLGA micropattern to modulate hMSCs response. The cells were well aligned along the micropattern and the myogenic activity of hMSCs was promoted. It has also been reported to use micropatterning network structure to spatially control MSCs fate within 3-dimensional hydrogels [8]. These researches highlight the importance of physical cues in creating aligned muscle for tissue engineering and muscle regeneration applications. However, systemic comparison of cell alignment influence on stem cells behavior and differentiation requires the cells to be controlled at different degree of alignment on a single culture plate. Kim et al. have used anisotropic micro- and nanotopographic pattern arrays with variable groove widths to compare the their influence on cell shape, orientation and migration [9].

In our previous studies, a photolithographic method was developed to prepare poly(vinyl alcohol) (PVA)-micropatterned surfaces to investigate the influence of cell spreading area, cell geometry and surface composition on osteogenic and adipogenic differentiation of hMSCs [10], [11], [12] at a single cell level. The method has also been used to prepare gradient micropatterns to control cell density for investigation of cell density influence on osteogenic and adipogenic differentiation of hMSCs [13], [14]. This method has the advantage over other micropatterning methods for creation of stable micropatterns for long period cell culture. In this research, the method was used to prepare a micropatterned polymer surface with cell-adhesive polystyrene lines of different widths (5, 10, 20, 40, 60, 80, 100, 200, 400, 600, 800 and 1000 μm). The effects of micropattern width on cell orientation, morphology and smooth muscle cell differentiation of hMSCs were compared on a single micropattern surface.

Section snippets

Preparation of PVA-micropatterned surface

Azidophenyl-derivatized poly (vinyl alcohol) conjugate (AzPhPVA) was synthesized by coupling poly (vinyl alcohol) with 4-azidobenzoic acid as previously described [14]. Briefly, dimethyl sulfoxide solution (DMSO, 2 mL) containing dicyclohexyl carbodiimide (234.09 mg, 1.13 mmol, Watanabe Chemical Industries, Ltd) was added dropwise to another DMSO solution (5 mL) containing 4-azidobenzoic acid (185.37 mg, 1.13 mmol, Tokyo Chemical Industry Co., Ltd) at room temperature in the dark. Subsequently, 2 mL

PVA-micropatterned surface characterization

Phase contrast micrograph of the pre-designed photomask consisting of UV-non-transparent dark stripe areas and UV-transparent light stripe areas with different widths (5, 10, 20, 40, 60, 80, 100, 200, 400, 600, 800 and 1000 μm) is shown in Fig. 2(a). The UV-non-transparent dark stripes and UV-transparent stripes had the same width. After preparation of PVA-micropatterned surface, formation of micropatterns on the polystyrene plate was observed with a phase contrast microscope (Fig. 2(b)). The

Conclusion

A gradient PVA micropattern with different stripe widths ranging from 5 to 1000 μm was prepared to investigate the effect of stripe width on the differentiation of hMSCs to VSMCs. Micropattern stripe width had an obvious effect on orientation and VSMC differentiation of hMSCs. Narrow stripes limited cell spreading in perpendicular direction and allowed cell to spread in the direction of stripes. As a result, smaller alignment angles were observed among the narrow micropattern stripes. Compared

Acknowledgements

The authors would like to acknowledge the financial support from World Premier International (WPI) Research Center Initiative, Ministry of Education, Culture, Sports, Science and Technology, Japan.

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Influence of micropattern width on differentiation of human mesenchymal stem cells to vascular smooth muscle cells

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Preparation of PVA-micropatterned surface

PVA-micropatterned surface characterization

Conclusion

Acknowledgements

Lancet

Biomaterials

Biomaterials

Biochem. Biophys. Res. Commun.

Colloids Surf., B

J. Biol. Chem.

J. Biol. Chem.

Cell Stem Cell

Curr. Opin. Cell Biol.

Physiol. Rev.

EMBO Rep.

Science

Stem Cells