Protein-Substrate Adhesion in Microcontact Printing Regulates Cell

Jan 5, 2018 - Microcontact printing (μCP) is widely used to create patterns of biomolecules essential for studies of cell mechanics, migration, and t...
1 downloads 15 Views 2MB Size
Subscriber access provided by University of Florida | Smathers Libraries

Article

Protein-Substrate Adhesion in Microcontact Printing Regulates Cell Behaviors Shuhuan Hu, Ting-Hsuan Chen, Yanhua Zhao, Zuankai Wang, and Raymond H. W. Lam Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02935 • Publication Date (Web): 05 Jan 2018 Downloaded from http://pubs.acs.org on January 6, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1 2 3 4

Protein-Substrate Adhesion in Microcontact Printing

5

Regulates Cell Behaviors

6 7

Shuhuan Hua*, Ting-Hsuan Chenab, Yanhua Zhaoa, Zuankai Wangab and Raymond

8

H.W. Lamab*

9 10

a

11

Kong, Hong Kong;

12

b

Department of Mechanical and Biomedical Engineering, City University of Hong

City University of Hong Kong Shenzhen Research Institute, Shenzhen, China;

13 14 15

* Correspondence should be addressed to S Hu (email:

16

[email protected]; Tel: +852-3442-7174) or RHW Lam (email:

17

[email protected]; Tel: +852-3442-8577; Fax: +852-3442-0172).

18

1 ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Abstract

2

Microcontact printing (μCP) is widely used to create patterns of biomolecules

3

essential for studies of cell mechanics, migration, and tissue engineering. However,

4

different types of μCPs may create micropatterns with varied protein-substrate

5

adhesion, which may change cell behaviors and pose uncertainty in result

6

interpretation. Here, we characterize two μCP methods for coating extracellular

7

matrix (ECM) proteins (stamp-off and covalent-bond) and demonstrate for the first

8

time the important role of protein-substrate adhesion in determining cell behaviors.

9

We found that, as compared to cells with weaker traction force (e.g. endothelial cells),

10

cells with strong traction force (e.g. vascular smooth muscle cells) may delaminate the

11

ECM patterns, which reduced cell viability as a result. Importantly, such ECM

12

delamination was observed on patterns by stamp-off, but not on the patterns by

13

covalent-bond. Further comparisons on the displacement of the ECM patterns

14

between the normal VSMCs and the force-reduced VSMCs suggested that cell

15

traction force plays an essential role in this ECM delamination. Together, our results

16

indicated that μCPs with insufficient adhesion may lead to ECM delamination and

17

cause cell death, providing a new insight for micropatterning in cell-biomaterial

18

interaction on biointerfaces.

19 20 21

Keyword

22

Microcontact printing, protein, substrate, extracellular matrix, adhesion, micropattern,

23

traction force, smooth muscle cell

24

2 ACS Paragon Plus Environment

Page 2 of 30

Page 3 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

Introduction

2

Microcontact printing (μCP) is a widely used biofabrication to coat or self-assemble

3

macromolecules such as deoxyribonucleic acids, extracellular matrix (ECM) proteins

4

and antibodies with precisely defined micropatterns on planar substrates 1-3. Over the

5

past two decades, μCP has become a fundamental tool for cell research, drug

6

screening and tissue engineering 4-7. In particular, μCP has enabled detailed studies on

7

subcellular motility and cytoskeleton mediated by well-defined adhesion sites

8

confined by the ECM proteins. For example, it has been reported that μCP can

9

achieve collagen VI stripes with different spacing distances to regulate chondrocytes

10

behaviors for development of cartilage replacement 8. Recently a new microcontact

11

printing technique called Pattern on Topography (PoT) was developed to examine the

12

synergistic regulating effects of biophysical and biochemical cues to the

13

cardiomyocytes.9

14

The working principle of μCP mostly relies on the cell adhesion to ECM protein,

15

which is essential for many physiological activities. At the cell level, cell adhesion

16

regulates apoptosis, mitogenesis, cell differentiation, and cell migration 10-15. At the

17

tissue/organ level, the adhesive property of ECM is required for tissue integrity 16-18

18

and was highlighted by many genetic and autoimmune diseases, such as muscular

19

dystrophies 19. Clinical practices have revealed the effectiveness of using immobilized

20

ECM proteins in the central nervous system to repair nerves 20. However, increasing

21

evidences suggest that cells do not passively attach to ECM. Instead, this cell-ECM

22

adhesion is a two-way mechanical interaction mediated by focal adhesions (FAs) 21-23

23

and cytoskeletal networks 24-27. That is, the mechanical signals may transduce from

24

ECM to cells and reorganize the cytoskeleton networks. As a result, the cellular force

25

is then iteratively changed, causing a disruption of the constitution and structure of

26

ECM 28-31.

27

Early μCP methods involved binding of molecules on a substrate through chemical

28

reactions such as metal-alkanethiolate bonding 32-33, silanization 34 and carbodiimide

29

crosslinking schemes 33. More recently, researchers have implemented μCP using the 3 ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

non-covalent bindings to achieve the more rapid and flexible micropatterns.35-36 These

2

μCP procedures usually include polymer surface activation, either oxygen plasma

3

bombardment 2 or ultraviolet-ozone irradiation 37. However, different μCP methods

4

may create micropatterned ECM protein with varied protein-substrate adhesion. As a

5

result, the cell traction force is resisted in different levels, leading to changes of

6

cytoskeleton organization and regulation of cell behaviors, but it is rarely addressed.

7

In this paper, we analyze cell behaviors growing on ECM protein micropatterns

8

coated with two different µCP methods (stamp-off and covalent-bond). The stamp-off

9

patterning relies on the direct molecular adsorption while the covalent-bond scheme

10

applies intermediating molecules with the higher binding strength by linking both

11

ECM proteins and substrates with covalent bonds. We seed cells onto the substrates

12

and inspect the role of protein-substrate adhesion in the two-way cell-ECM

13

interactions. On the ECM side, we observe any changes in the protein patterns

14

induced by the seeded cells. On the other side, we examine whether the cell

15

characteristics such as morphology, viability, and cytoskeleton formation are different

16

on the micropatterns coated with the different µCP methods.

17

4 ACS Paragon Plus Environment

Page 4 of 30

Page 5 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

Experimental Section

2 3

ECM Patterning Techniques

4

We adopted two μCP techniques (stamp-off and covalent-bond) with different levels

5

of the protein-substrate adhesion. Polydimethylsiloxane (PDMS), the widely-used

6

biocompatible polymeric material (Dow Corning, Midland, MI)38, is used as the

7

substrate. All the PDMS substrates were casted from a

8

trichloro(1H,1H,2H,2H-perfluorooctyl)silane (Sigma-Aldrich, St. Louis, MO) treated

9

flat silicon wafer with a monomer-curing agent weight ratio of 10:1 after baking at 80

10

ºC for >20 hr. In this work, we selected the fibronectin conjugated with fluorescent

11

molecules (Alexa Fluor 488 protein label kit, Thermo Fisher Scientific, Waltham, MA)

12

as the ECM protein for visualizing the coated protein patterns using the selected μCP

13

methods.

14

The first μCP method (stamp-off) utilized a microstructured stamp to create

15

patterns by removing ECM protein from some regions on a fibronectin-adsorbed

16

PDMS substrate 35. As shown in Fig. 1a, we prepared a PDMS stamp (10:1

17

monomer-curing agent ratio) with microstructures of a ‘negative’ pattern based on the

18

traditional soft lithography 39. The PDMS stamp was prepared using photolithography

19

of SU-8 photoresist (Microchem) on a silicon wafer followed by silanization of

20

trichloro(1H,1H,2H,2H-perfluorooctyl)silane. The PDMS stamp was further treated

21

with oxygen plasma (PDC001, Harrick Plasma, Ithaca, NY) to enhance its molecular

22

adhesion to fibronectin. To coat fibronectin on the PDMS substrate, we pipetted 50

23

μg/ml of the fluorescent fibronectin in water to cover the PDMS substrate for 2 hr in

24

order to facilitate the protein adsorption. After blow-drying with compressed air, the

25

microstructured PDMS stamp was placed onto the flat fibronectin-coated PDMS for

26

20 s. As the plasma-treated PDMS stamp had a stronger molecular adhesion,

27

fibronectin on the contact surfaces between the stamp and the substrate were removed

28

after the stamp was peeled off 35, 40-41. We then applied a 0.1 % surfactant (pluronic

29

F127; Sigma-Aldrich) on the substrate for 15 min to prevent unexpected cell-substrate 5 ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2

attachment in the uncoated regions and rinsed the substrate with distilled water. The second µCP method (covalent-bond) applied intermediate molecules for

3

achieving covalent bonds between the protein coating and the substrate for stronger

4

protein-substrate adhesion (Fig. 1b). An ester based protein immobilization was

5

adapted to bind the fibronectin molecules onto the PDMS substrate through covalent

6

bonding 42 (Fig. 2a). Briefly, we treated the PDMS substrate with oxygen plasma,

7

following by applying 4 % (v/v) 3-mercaptopropyl trimethoxysilane (MPTMS; Gelest,

8

Morrisville, PA) in ethanol to cover the PDMS substrate for 45 min. The substrate was

9

washed with ethanol and then covered by 0.28 % (w/v) N-γ-maleimidobutyryloxy

10

succinimide ester (GMBS; Thermo Fisher Scientific) in ethanol for another 15 min.

11

On the other side, fibronectin was applied onto a ‘positive’ microstructured stamp. We

12

then placed the stamp onto the chemically treated substrate for 20 s. After peeling of

13

the stamp, we applied pluronic F127 and rinsed the substrate to avoid any unexpected

14

cell attachment.

15 16

Surface Characterization

17

X-ray photoelectron spectroscopy (XPS) experiments were conducted using an XPS

18

facility (PHI Model 5802 with a monochromatic Al Kα X-ray source at 1486.6 eV;

19

Physical Electronics, Chanhassen, MN) with a pressure of 10-10 mBar and a resolution

20

of 0.1081 eV. An attenuated total reflectance Fourier transform infrared spectroscopy

21

(ATR-FTIR, Perkin Elmer 1600, Perkin Elmer, Waltham, MA) together with a zinc

22

selenide crystal was used to characterize the covalent bonds between molecules. An

23

ellipsometer with a resolution of 0.1 nm (Jobin Yvon-PZ2000; Horiba Scientific,

24

Edison, NJ) using a He–Ne laser (632.8 nm) was used to measure the thickness of the

25

protein layer. The adhesion force were determined by the Universal Testing Machine

26

(UTM-EZ-LX, Shimadzu Scientific Instruments, Columbia, MD). Immediately before

27

the test, the moving plate (diameter: 8 mm) was treated with underwater glue

28

(JH-5553; Jin Hong Glues, Hangzhou, China). The speed of the moving plate was set

29

to 10 mm•min-1 to obtain the load-displacement curve. The applying load was set as

30

10 N and the contact time between the sample and the moving plate surface was 5 min. 6 ACS Paragon Plus Environment

Page 6 of 30

Page 7 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

In addition, we measured surface hydrophobicity and infrared spectra of the

2

fibronectin-coated substrates in different fabrication stages. The surface

3

hydrophobicity was measured using a drop-shape analyzer (DSA100, Kruss,

4

Hamburg, Germany).

5 6

Cell Culture

7

Primary human vascular smooth muscle cells (VSMCs; Lonza, Walkersville, MD)

8

were cultured in the standard medium kit (SmGM-2™ BulletKit™, Lonza). Primary

9

human umbilical vein endothelial cells (VECs; Lonza) were cultured in the standard

10

medium kit (EGM™-2 BulletKit™, Lonza). NIH/3T3 fibroblasts (3T3; ATCC,

11

Manassas, VA) were cultured in the Dulbecco’s modified Eagle’s medium (DMEM;

12

Life Technologies, Carlsbad, CA) supplemented with 10% Bovine Serum, 100

13

units/mL penicillin and 1 % L-glutamine. The cells were cultured in 5% CO2

14

humidified cell culture incubator at 37 °C. The cells were trypsinized and subcultured

15

once their population reached >80% confluence. Only the cells with a passage number

16