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Dual-Microstructured Porous, Anisotropic Film for Biomimicking of Endothelial Basement Membrane Zuyong Wang, Swee Hin Teoh, Minghui Hong, Fangfang Luo, Erin Yiling Teo, Jerry Kok Yen Chan, and Eng San Thian ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 01 Jun 2015 Downloaded from http://pubs.acs.org on June 3, 2015

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ACS Applied Materials & Interfaces

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Dual-Microstructured Porous, Anisotropic Film for

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Biomimicking of Endothelial Basement Membrane

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Zuyong Wang,† Swee Hin Teoh,‡ Minghui Hong,§ Fangfang Luo,§ Erin Yiling Teo,@ Jerry Kok

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Yen Chan,@,♯,&,* and Eng San Thian†,*

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†

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1, Singapore 117576, Singapore

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‡

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Singapore 637459, Singapore

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§

Department of Mechanical Engineering, National University of Singapore, 9 Engineering Drive

School of Chemical and Biomedical Engineering, Nanyang Technological University,

Department of Electrical and Computer Engineering, National University of Singapore, 2

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Engineering Drive 3, Singapore 117576, Singapore

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@

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Timah Road, Singapore 229899, Singapore

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♯

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University of Singapore, 14 Medical Drive, Singapore 117599, Singapore

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&

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Singapore 169857, Singapore

Department of Reproductive Medicine, KK Women’s and Children’s Hospital, 100 Buikit

Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National

Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, 8 College Road,

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ACS Paragon Plus Environment

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ABSTRACT: Human endothelial basement membrane (BM) plays a pivotal role in vascular

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development and homeostasis. Here, a bio-responsive film with dual-microstructured geometries

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was engineered to mimic the structural roles of the endothelial BM in developing vessels, for

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vascular tissue engineering (TE) application. Flexible poly(ε-caprolactone) (PCL) thin film was

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fabricated with microscale anisotropic ridges/grooves and through-holes using a combination of

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uniaxial thermal stretching and direct laser perforation, respectively. Through optimizing the

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inter-hole distance, human mesenchymal stem cells (MSCs) cultured on the PCL film’s

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ridges/grooves obtained an intact cell alignment efficiency. With prolonged culturing for 8 days,

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these cells formed aligned cell multilayers as found in native tunica media. By co-culturing

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human umbilical vein endothelial cells (HUVECs) on the opposite side of the film, HUVECs

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were observed to build up transmural interdigitation cell-cell contact with MSCs via the through-

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holes, leading to a rapid endothelialization on the PCL film surface. Furthermore, vascular tissue

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construction based on the PCL film showed enhanced bioactivity with an elevated total nitric

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oxide level as compared to single MSCs or HUVECs culturing and indirect MSCs/HUVECs co-

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culturing systems. These results suggested that the dual-microstructured porous and anisotropic

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film could simulate the structural roles of endothelial BM for vascular reconstruction, with

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aligned stromal cell multilayers, rapid endothelialization, and direct cell-cell interaction between

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the engineered stromal and endothelial components. This study has implications of recapitulating

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endothelial BM architecture for the de novo design of vascular TE scaffolds.

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KEYWORDS: endothelial basement membrane, porous micropatterned film, cellular alignment,

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cell-cell interaction, vascular tissue engineering

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INTRODUCTION

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Current therapeutic approaches for cardiovascular diseases the globally leading cause of death

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remain inadequate for many patients.1 Tissue engineering (TE) offers a promising route for the

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improvement or regeneration of diseased vessel function using a combination of biomaterials,

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cells and engineering techniques.2 However, vascular TE grafts presently have limitations in the

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mechanical (e.g. un-matched compliance)3 and biological (e.g. prothrombotic nature)4 functions

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to hinder their clinical translation. To construct proper vascular TE grafts, tissue engineers have

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been looking for the recapitulation of developmental elements, which establish natural vessel

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structure and physiological function. The endothelial basement membrane (BM) located between

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tunica media and intima is such a key targeted evolutionary component, and acts a pivotal role as

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the basolateral of stromal and endothelial cells, for both vascular development5 and maintenance

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of the adult vessel function.6

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Human endothelial BM comprises of a three-dimensional (3D) architecture with highly porous

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and organized laminin/collage IV networks.6-7 The importance of such architecture for designing

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vascular TE grafts has been revealed by how substrates biomimicking endothelial BM such as

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the roughness,8 anisotropy9-10 and porosity11 can influence enhanced cell adhesion, spreading,

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proliferation, and specific gene expressions in smooth muscle cells (SMCs) and endothelial cells

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(ECs). Such architectural simulation on TiO substrate has also been shown to impact ECs

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mobility and differentiation over surface chemistry.12 However, these substrates are designed

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with singular structure, and cannot mimic the architectural complexity of endothelial BM such as

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co-existence of the anisotropy7 and holes.13-16 For example, the endothelial BM of developing

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vessel is not entirely continuous, and has gaps to allow direct interaction between the ECs and

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perivascular stromal cells such as pericytes14-15 and SMCs,13,16-17 importantly for preventing


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nascent vessel regression. Furthermore, the 3D architecture of endothelial BM guides both SMCs

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and ECs with specific localization, to form the unique vessel structure and function such as the

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aligned SMCs multilayers in tunica media for mechanical strength and compliance support.2-3

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The current engineered solutions of endothelial BM have not addressed simulation of the

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architectural complexity. Here, we report a flexible dual-microstructured thin film to mimic the

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structural roles of the endothelial BM in developing vessels, for promoting vascular

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reconstruction. This bio-responsive film will be able to facilitate vascular reconstruction through

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guiding stromal cells to form aligned cellular multilayers on the film’s tunica media surface,

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while promoting endothelialization on the film’s tunica intima side via direct transmural cell-cell

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interaction.

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EXPERIMENTAL SECTION

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Materials. Poly(ε-caprolactone) (PCL, MW = 80,000), phosphate buffer saline (PBS),

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penicillin-streptomycin (PS), TRITC-conjugated phalloidin, paraformaldehyde (PFA), triton-X

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100, bovine serum albumin (BSA), 4',6-diamidino-2-phenylindole (DAPI) and PHK cell linker

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dye kits were purchased from Sigma-Aldrich (Singapore). Fetal bovine serum (FBS), Trypsin-

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EDTA and dulbecco's modified eagle medium (DMEM) were purchased from Life Technologies

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(Singapore), endothelial cell growth medium (EGM) from Lonza (Singapore), and NO assay kits

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from Cell Biolabs, Inc. (USA). Tissue culture plates (TCP) and flasks, and low-adhesion cell

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culture plates were purchased from Thermo Fisher Scientific (USA), Nunc (Singapore), and

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Corning (Costar, Singapore), respectively.

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Preparation of Dual-Microstructured PCL Films. Flexible PCL thin films were engineered

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with dual-microstructured geometries through a combination of uniaxial thermal stretching and

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direct femtosecond laser perforation (Figure 1). PCL solid mass after two-roll milling was heat


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pressed at 80 oC and 300 MPa into a thick film named HP-PCL. The film was then subjected to

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uniaxial stretching at 54 oC with a draw ratio of 4. The uniaxial-stretched PCL film was thinner

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and named as UXHP-PCL. To perforate the PCL films, a Ti:Sapphire femtosecond laser was used

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with a wavelength of 800 nm, pulse duration of 110 fs and repetition rate of 1 kHz (Spectra-

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Physics, USA). Laser beam scanning was controlled using a U500 MMI software (Aerotech,

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UK). HP-PCL and UXHP-PCL after femtosecond laser perforation were named as PHP-PCL and

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PUXHP-PCL, respectively.

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Morphological Characterization of PCL films. PCL film samples were sputter-gold coating

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at 10 mA for 30 s, and imaged using a field emission scanning electron microscope (SEM; S-

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4300, Hitachi, Japan) at different magnifications with an accelerating voltage of 15 kV.

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Cell Isolation and In Vitro Culture. Human tissue collection for research purposes was

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approved by the Domain Specific Review Board of National Healthcare Group, in compliance

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with international guidelines regarding to the use of fetal tissues for research.18 In all cases,

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patients gave separate written consent for the use of their collected tissues.

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Mesenchymal stem cells (MSCs) were isolated from human fetal bone marrow as previously

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described.2 Single-cell suspension from the marrow components of femurs was seeded in a tissue

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culture flask at a density of 106 cells/cm2. Non-adherent cells were removed after 3 days of

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culturing in D10 medium (DMEM + 10% FBS + 1% PS). The adherent cells were further

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cultured for one week, recovered and characterized with stem cell phenotype.2 GFP-labeled

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human umbilical vein endothelial cells (HUVECs) were derived as previously reported,19 and

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provided by Mr Sandikin Dedy (National University of Singapore, Singapore).

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Cell Morphology and Organization. Cell morphology was characterized through a fixed-cell

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cytoskeletonal staining as previously described.20 MSCs (5,000 cells/cm2) were seeded on UXHP-


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PCL (control group) and PUXHP-PCL, and cultured in D10 medium for 3 days. After PBS

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washing, the cells were fixed and permeabilized by PFA (3.7% in PBS; for 15 min) and Triton-X

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100 (0.1% in PBS; for 5 min), respectively. Cells were then washed with PBS thrice, blocked by

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BSA (2% in PBS; for 30 min) and incubated with TRITC-conjugated phalloidin (1:200 in PBS;

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for 60 min) for F-actin labeling. After PBS washing thrice, the cells were further incubated with

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DAPI (1:1000 in PBS; for 5 min) for nucleus DNA labeling. Cells were imaged using a CLSM

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(FV1000, Olympus, Japan) under identified parameters for all samples.

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Cell organization was characterized through a live-cell cytoplasmic staining as previously

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described.2,18 MSCs were seeded on PCL films and cultured in D10 medium for predetermined

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periods. HP-PCL and UXHP-PCL were used as negative and positive control, respectively. After

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PBS washing thrice, the cells were fluoresence-labeled using FDA (5 µg/L; for 10 min) at room

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temperature. Cells after further PBS washing were immediately imaged using CLSM under a low

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powered magnification of x10. Image analysis was performed using the built-in function of NIH

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ImageJ software (USA), with fluorescence from the hole scattering and the cells that were in

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contact with other cells and image edges being manually removed from the data sets.18 Cellular

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angle was determined as an orientation of the major elliptic axis of individual cell. A preferential

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cell orientation has been determined and set as 0o. All cellular angles were then normalized to the

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preferential cell orientation. The number of cells within each degree from -90o to +89o was

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calculated and normalized such that the total sum was unity. Cells with angles falling into ±10o

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was considered to be aligned.18 For comparison, the cell alignment efficiency of each group was

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finally normalized to that of positive control, which has a value of 1. Three film samples were

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used for each experimental group.


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Direct Cell-Cell Interaction. Fluorescence labeling was performed for different cells for the

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visualization of direct cell-cell interaction across PUXHP-PCL. MSCs were labeled with red

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fluorescence using a PHK-26 kit. Cells collected from culture expanding were re-suspended in

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Diluent (2x final concentration, 0.5 ml). PHK26 dye was diluted into a 2x solution (0.5 ml). The

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cell suspension and PHK26 dilution were then mixed, and incubated at 21 oC for 5 min.

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Fluorescence-labeling reaction was stopped by adding 1 ml FBS. Lenti-virally infected GFP

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HUVECs was identified with green fluorescence.

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An in-house designed ring was used for co-culturing MSCs and HUVECs on the two surfaces

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of PUXHP-PCL, respectively. PHK26-labeled MSCs (10,000 cells/cm2) were firstly seeded on

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PUXHP-PCL for 1 day of culturing in D10 medium. The film was then flipped over to transfer to

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the ring. GFP-labeled HUVECs (20,000 cells/cm2) were seeded on the film opposite side of

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MSCs. MSCs/HUVECs co-culturing was performed in EGM10 medium, which has been shown

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to support the growth and maintenance of MSCs and HUVECs. The cells after 3 days of co-

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culturing were imaged with CLSM using a z-scanning model. Images were reconstructed using

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the built-in function of Imaris software (Bitplane, Switzerland). The PHK26-labeled MSCs and

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GFP-labeled HUVECs were identified with red and green fluorescences, respectively. The

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MSCs-HUVECs contact was characterized as the co-localization of the red and green colors,

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representing as yellow color.

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Cell Adhesion and Proliferation. To investigate endothelialization on PUXHP-PCL, the cell

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number of HUVECs was assessed by enumerating nucleus number as previously reported.21

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GFP-labeled HUVECs after seeding on the film opposite side of MSCs were co-cultured for 24

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hrs to allow fully cellular adhesion, and co-cultured for another 4 days for proliferation. Cells

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were then fixed, permeabilized, and blocked. Cells were then incubated with DAPI (1:1000 in


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PBS; for 5 min) for cell nucleus labeling. The nucleus number of HUVECs was counted using

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the built-in function of NIH ImageJ software, with cells that have either green- or blue-

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fluorescence alone being manually removed from the data sets. Cell nucleus number enumerated

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at day 1 and 5 represented the adhesion and proliferation of HUVECs on the PCL film surface,

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respectively. Single-culture of HUVECs alone on UXHP-PCL and PUXHP-PCL, and co-culture of

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HUVECs and MSCs on UXHP-PCL via indirect cell-cell interaction were also investigated as

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comparison groups. Three samples were used for each experimental group.

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Total NO Level. To evaluate the bioactivity of vascular tissue construct, system total NO level

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was investigated using a NO assay kit. MSCs and HUVECs were seeded on PCL films in 12-

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well low adhesion plates, and cultured in EGM10 medium (2.5 mL/well) for 8 days. At day 3

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and 5, 0.5 mL new EGM10 medium was added for nutrition supplement. Experimental groups of

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(G1) EGM10 medium alone, (G2) MSCs cultured on PUXHP-PCL in EGM10 medium, (G3)

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HUVECs cultured on PUXHP-PCL in EGM10 medium, (G4) co-culturing of HUVECs/MSCs

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using UXHP-PCL in EGM10 medium and (G5) co-culturing of HUVECs/MSCs using PUXHP-

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PCL in EGM10 medium were investigated. The total nitrate/nitrite (NO3-/NO2-) level in culture

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medium was examined following the procedures of NO assay kit. Briefly, cell culture medium

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(10 µL) was added to a 96-well plate and adjusted to 80 µL using assay buffer. 10 µL enzyme

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cofactors and 10 µL nitrate reducer were added in sequence, and incubated at 21 oC for 30 min

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for reducing nitrate into nitrite. After adding 2,3-diaminonaphthalene (10 µL) and incubating for

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10 min, NaOH solution (20 µL) was added to stop the reaction with enhanced fluorescence

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intensity. To prepare the standard curve, 10 µL EGM10 medium with 70 µL assay buffer was

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used as the blank, and 10 µL EGM10 medium with 20 µL assay buffer and 50 µL nitrate

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standards (0.156 to 10 mol/L) was used as gradient. Fluorescence intensity was detected using


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a microplate reader at an excitation wavelength of 360 nm and emission wavelength of 430 nm.

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Basal fluorescence intensity of EGM10 medium was deducted from all groups. Three samples

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were used for each experimental group.

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Statistical Analysis. Data analysis was performed on Prism 5 software. Results were reported

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as mean ± SD. A value of p 0.05). Such observation agreed with the previous

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report that holes with dimensions in the order of several micrometers could not affect cellular

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proliferation as compared to the cells on non-hole surfaces.32 However, the prolonged culturing

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resulted in considerable increase in the cell proliferation of HUVECs in G-IV, which was higher

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than those of both the single-culturing in G-I and II (16.7-19.6% increase, p

Dual-Microstructured Porous, Anisotropic Film for ... - ACS Publications

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