Replication of a Tissue Microenvironment by Thermal Scanning Probe

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Biological and Medical Applications of Materials and Interfaces

Replication of Tissue Microenvironment by Thermal Scanning Probe Lithography Sze Wing TANG, Md Hemayet Uddin, Wing Yin Tong, Paul Pasic, Wai Yuen, Helmut Thissen, Yun Wah Lam, and Nicolas H. Voelcker ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b05553 • Publication Date (Web): 03 May 2019 Downloaded from http://pubs.acs.org on May 4, 2019

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ACS Applied Materials & Interfaces

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Replication of Tissue Microenvironment by Thermal

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Scanning Probe Lithography

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Sze Wing Tang1, Md Hemayet Uddin4, Wing Yin Tong3, Paul Pasic4, Wai Yuen5, Helmut

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Thissen4, Yun Wah Lam1*, Nicolas H Voelcker2,3,4*

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Kong SAR

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University, 381 Royal Parade, Parkville, Victoria, 3052, Australia

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Department of Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong

Drug Delivery Disposition & Dynamics, Monash Institute of Pharmaceutical Science, Monash

Commonwealth Scientific and Industrial Research Organization (CSIRO), Clayton, Victoria,

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3168, Australia

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Facility, 151 Wellington Road, Clayton, Victoria, 3168, Australia

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5

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Avenue, Hong Kong SAR

Melbourne Centre for Nanofabrication, Victorian Node of the Australian National Fabrication

HealthBaby Biotech (Hong Kong) Co., Ltd, Lakeside 2 West Wing, No. 10 Science Park West

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KEYWORDS: thermal scanning probe lithography, tissue microenvironment, mesenchymal stem

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cells, cell-matrix interactions, cell guidance

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ACS Paragon Plus Environment

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ABSTRACT: Thermal scanning probe lithography (t-SPL) is a nanofabrication technique in

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which an immobilized thermolabile resist, such as polyphthalaldehyde (PPA), is locally

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vaporized by a heated atomic force microscope (AFM) tip. Compared with other nanofabrication

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techniques, such as soft lithography and nanoimprinting lithography, t-SPL is more efficient and

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convenient as it does not involve time-consuming mask productions or complicated etching

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procedures, making it a promising candidate technique for the fast prototyping of nanoscale

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topographies for biological studies. Here, we established the direct use of PPA-coated surfaces as

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a cell culture substrate. We showed that PPA is biocompatible and that the deposition of

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allylamine by plasma polymerization on a silicon wafer before PPA coating can stabilize the

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immobilization of PPA in aqueous solutions. When seeded on PPA coated surfaces, human

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mesenchymal stem cells (MSC) adhered, spread and proliferated in a manner indistinguishable

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from cells cultured on glass surfaces. This allowed us to subsequently use t-SPL to generate

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nanotopographies for cell culture experiments. As a proof of concept, we analyzed the surface

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topography of bovine tendon sections, previously shown to induce morphogenesis and

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differentiation of MSC, by means of AFM, and then “ wrote ” topographical data on PPA by

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means of t-SPL. The resulting substrate, matching the native tissue topography on the nanoscale,

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was directly used for MSC culture. The t-SPL substrate induced similar changes in cell

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morphology and focal adhesion formation in MSC compared to native tendon sections,

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suggesting that t-SPL can rapidly generate cell culture substrates with complex and spatially

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accurate topographical signals. This technique may greatly accelerate the prototyping of models

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for the study of cell-matrix interactions.

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Introduction

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The extracellular matrix (ECM) of biological tissues contains nanoscale topographical features

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that are essential for maintaining and regulating cell functions 1-3. The topography of tissue

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microenvironment influences cell functions through mechanosensing pathways 4. Mechanisms

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underlying this phenomenon are traditionally studied by using in vitro models in which cells are

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seeded on synthetic surfaces that harbor topographical patterns, followed by imaging and

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biochemical characterizations. In recent years, this approach has been accelerated and refined by

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the rapid development of fabrication technologies. For example, submicron grooves fabricated

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on polyimide and polydimethylsiloxane (PDMS) have been shown to guide morphogenesis and

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migration behaviours of induced pluripotent stem cells 5. When cultured in conjunction with

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retinol acid, on surfaces with nanoscale gratings, human mesenchymal stem cells elongate, align

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and express neuronal makers 6. Substrates with 200 nm nanopore-patterns, fabricated by using

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nanoimprinting and nanoinjection, can guide human embryonic stem cells and iPSC cells into

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endoderm differentiation 7. Furthermore, the nano- and micro-scale topography of cell culture

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surface can influence cell morphology and adhesion dynamics 8-9. Although a large variety of

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surface topographies, such as pillars 10-12, grooves 13 and pits 14-15 and pores 16-17, have

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been fabricated and studied, most of these shapes and dimensions are empirically designed and

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their simplistic geometries do not necessarily reflect the complexity of native tissue

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microenvironment. As a result, the physiological relevance of this reductionist approach remains

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uncertain. For example, we have previously shown that surface topography of tendon sections,

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when replicated on PDMS by mold casting, can induce profound changes in cell morphology and

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differentiation 18. Ultrastructural analysis revealed that tendon sections have highly complex


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surfaces, and subtle changes in the topographical patterns leads to dramatic changes in cell

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morphology 19.

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To recapitulate the functional roles of the tissue microenvironment on a cell culture substrate, the

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fabrication process must be accurate enough to copy the topographical information in the tissue

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microenvironment with nanometer accuracy, while efficient enough to produce such complex

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pattern without tedious procedures. Furthermore, the resulting substrate must be biocompatible.

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Some patterning methods, such as soft lithography 20-21, do not have sufficient spatial

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resolution. Whereas photolithography allows the production of nanoscale structures, it is a long

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and complicated process that involves the use of toxic photoresists and hazardous etching

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procedure. Nanoimprinting lithography is able to create three dimensional scaffolds 22 but its

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spatial resolution is limiting. Although multiscale structures are achievable with next generation

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nanoimprinting 21, 23, the level of complexity is still far from that of tissue microenvironment.

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While electron beam lithography (EBL) can create sub 10 nm scale patterns, the accuracy of

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patterning is limited by the proximity effect 24, which is caused by scattering of the electron

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beam into non-target area of the resist 25. Although proximity effect corrections have greatly

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improved the writing accuracy 26, the application of EBL in producing physiological relevant

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complex designs is still relatively limited.

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Thermal scanning probe lithography (t-SPL) is a maskless 27-28 close-loop technology 29-30 in

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which a heated atomic force microscope cantilever is used to precisely engrave a pattern on a

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layer of thermal sensitive resist adhered to a silicon wafer 31-32. The spatial resolution of t-SPL


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is within a few nanometers. The most popular thermal sensitive resist in t-SPL, polypthalamide

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(PPA) is biocompatible, as it is commonly used as medical catheters 33-34. However, the use of

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t-SPL in the fabrication of topographical features on cell culture substrates has not been

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explored. This is, at least in part, due to the detachment of PPA from the silicon wafer in aqueous

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solutions. In this study, we overcame this problem by the use of plasma polymerization to

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deposit an adhesive layer of allylamine that stabilizes the binding of PPA to the silicon wafer.

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Using this approach, we demonstrated the use of PPA as a cell culture substrate, and further

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explored the use of t-SPL in producing complex, biomimetic tissue topographies for cell culture

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experiments.

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Materials and methods

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Substrate Preparation

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Oxidized silicon wafers (Silicon Quest Intl. Ltd.) were cleaned by ultrasonication for 20 min in

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1% RBS-35 detergent (Pierce, USA) /10% ethanol in water and then rinsed with Milli-Q water.

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The wafers were then subjected to 15 min UV/ozone oxidation treatment for the removal of trace

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organic contaminants. Plasma polymerization was conducted in a custom-built reactor as

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previously described 35. In brief, the upper and lower circular electrodes were placed 150 mm

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apart within a cylindrical reactor chamber. Samples were placed on the lower grounded

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electrode. Allylamine (Sigma-Aldrich, 145831) plasma polymer (ALApp) depositions were


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performed under the following conditions: radio frequency of 200kHz, a load power of 20 W and

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an initial monomer pressure of 0.200 mbar for 1 s, 2 s and 5 s. Air was pumped out from the

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reactor chamber down to 0.02 mbar before ALApp deposition to ensure initial monomer pressure

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is accurate. The resist films were prepared by spin coating of PPA (Allresist, AR-P 8100.07). To

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achieve 100 nm thick films, PPA were spun at 4,000 rpm for 60 s at ambient conditions followed

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by incubation at 90oC for 3 min to remove the residual solvent.

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Atomic force microscopy (AFM)

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Measurements were performed using an atomic force microscope (Bruker Dimension Icon).

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Height information was obtained using tapping mode. The film thickness was obtained by

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subtracting the value of lower uncoated surface from the upper coated surfaces.

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Thermal scanning probe lithography (t-SPL) patterning

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All t-SPL were performed by NanoFrazor Explore (SwissLitho), following manufacturer ’ s

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instructions. Pattern dimension was 12 nm pixel size in y-axis and 12 nm in x-axis. The

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generated patterns were clean room packed and transported to the tissue culture room for cell

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culture experiments. The samples were sterilized by rinsing in 70% ethanol for 5 min followed

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by rinsing with 1 x phosphate buffered saline (PBS) before cell seeding.

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Cell culture


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Mesenchymal stem cells (MSC) were derived from human umbilical cord (UC) obtained from

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healthy human subjects and processed within 24 h after collection, in a biological safety cabinet

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(BSC) in a class 10,000 cleanroom area. A length of the UC (roughly 8 cm) was disinfected by

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70% alcohol for 30 s and washed with Dulbecco PBS (Gibco: 14190) for 3 times. Then, the UC

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was minced and digested by 0.05% trypsin/EDTA (Gibco: 15400) at 37 ° C for 30 min. The

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resulting tissues were subsequently cultured in low glucose (1g/L) Dulbecco’s Modified Eagle

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Medium (DMEM, Gibco: 11885) with 20% fetal bovine serum (FBS, Gibco: 10438) and 1%

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Penicillin-streptomycin (Gibco: 15140) for 7 d in humidified atmosphere of 95% air and 5%

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CO2 at 37°C. When the cell colonies were formed from tissue, cells were harvested using 0.05%

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trypsin/EDTA and defined at the primary culture (P0). Immunophenotyping by flow cytometry

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showed that the isolated cells met the defining criteria for MSC, i.e., positive for CD29-PE0Cy5,

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CD44-PE, CD73-PE, CD90-PE-Cy5, CD105-PE and negative for CD31-FITC, CD34-PE,

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CD45-PE-Cy5, HLA-DR-FITC (all antibodies from BD Biosciences). The culture media were

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refreshed every three days and cell confluence was always kept under 80%.

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Resist peel-off resistant assay

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Oxidized silicon wafers coated with allylamine plasma polymer and PPA were submersed in 1x

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PBS. The time for visible coating lift off under 50x light microscopy (Nikon C-HGFI) is

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recorded.

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Cell adhesion assay


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A suspension of MSC (2x105 in 5 ml of medium) was applied on the PPA substrate. At different

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intervals (30 min and 60 min) after cell seeding, the substrates were washed with PBS (4 x 30 s).

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The attached cells were fixed with 4% paraformaldehyde (Sigma, 158127) and then stained with

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Hoechst 33342 (1:20000, Sigma, 14533). The stained samples were imaged with an

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epifluorescence microscope (Nikon C-HGFI). The number of cells in five randomly detected

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fields from each sample was counted.

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Histology

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Freshly dissected Achilles tendon was obtained from a local wet market and stored at -80oC. The

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tissues were trimmed into blocks (~ 1 cm3) before cryosectioning (Jung CM 1500, Leica

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Instruments). Longitudinal cryosections (50 μ m thickness) were thawed onto glass slides

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(Menzel-Glaser Superfrost Ultra Plus). The sections as well as adherent cells cultured on glass

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coverslips were rehydrated with phosphate buffered saline (PBS) and then fixed with 4%

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paraformaldehyde (PFA) (Sigma-Aldrich, 30525-89-4) in PBS for one hour at room temperature.

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The slides were then washed with PBS twice (10 min each).

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Scanning electron microscopy (SEM)

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SEM was performed as previously described 36. In brief, the sample were fixed with

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glutaraldehyde and dehydrated in a series of ethanol, methanol and acetone, followed by a 30 s

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CO2 critical point drying (BAL-TEC CPD 030 Critical Point Dryer). The samples were then


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mounted on an aluminium stub tape and then coated with carbon. The carbon-coated section was

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imaged using an environmental scanning electron microscope (FEI/Philips XL30 Esem-FEG).

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Immunostaining

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MSC were cultured on the substrate for 48 h, and were fixed in freshly prepared 4% PFA for 15

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min at room temperature. The cells were washed twice with PBS (5 min each time) and

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permeabilized in 0.1% Triton X-100 in PBS for 2 min at room temperature. The cells were

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washed with PBS twice (5 min each time) and blocked with 1% bovine serum albumin (BSA) in

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PBS for 30 min at room temperature. The cells were incubated with mouse anti-vinculin

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antibody (1:200, Thermofisher, MA5-11690) overnight followed by three PBS washings (5 min

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each time) before incubation with Alexa 647 goat anti-mouse antibody (1:200, Invitrogen,

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A21235) for 60 min. The cells were then washed twice with PBS (5 min on rocker). The

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immunolabelled samples were imaged with a laser scanning confocal microscope (Leica SP8).

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Viability assay

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MSC were cultured on silicon wafer with ALA/PPA and coverglass for 24 hours before

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performing the essay. Culture medium was replaced with fresh culture medium with Hoechst

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33342 (1:20000, Sigma, 14533) for 30 min. At 15 min mark, propidium iodide (PI) was added to

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the culture at a final concentration of 2 µg/ml for a further 15 min. Afterwards, the medium was

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removed and the cells were washed with 1X PBS twice and fixed with 4% paraformaldehye


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(PFA). Four randomly chosen fields on each sample were imaged with an epifluorescence

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microscopy (Nikon C-HGFI) for data analysis.

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Statistical analysis

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Statistical difference between each treated group was determined by Analysis of variance

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(ANOVA). The hypothesis was accepted at a 95% significant level (p < .05). All data were

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presented as mean± standard deviation (SD).

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Results & Discussion

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Plasma polymerization of allylamine enhances the adhesion of PPA resist to the substrate

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Thermal scanning probe lithography is a nanofabrication technique that involves the accurate

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removal of thermolabile resist. Traditionally, t-SPL samples are prepared by the spin coating of a

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layer of PPA resist on to a silicon wafer followed by soft baking. However, upon contact with

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aqueous solutions such as PBS, regions of the resist film were observed to peel off from the

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silicon wafer within the first 24 h of incubation (Fig 1A & C). The instability of the PPA layer

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makes this substrate unsuitable for cell culture experiments involving prolonged incubation in

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culture media.

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Amine plasma polymerization is widely used to modify diverse types of material surfaces by

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introducing a pinhole-free layer of amine functional groups 37-38. The resulting amine-

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containing layer creates an electrostatic environment which allows further functionalization with

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the electrostatic absorption of another polymer 39-40, and biomolecules such as DNA 41.

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Allylamine (ALA) was chosen as the precursor for plasma polymerization to enhance the

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adhesion of PPA to silicon wafer because ALA is a hydrophilic polymer 40 and is known to be

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non-toxic to cells 42. Fig 1B illustrates the difference between traditional t-SPL sample and

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plasma polymer modified t-SPL sample. After allylamine plasma polymerization (ALApp) onto

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a silicon wafer, the PPA layer stayed intact even after 14 d of incubation. We then optimized the

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thickness of the plasma-polymer coating, by varying the time of deposition. As shown in Fig 1D,

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the thickness of ALApp could be adjusted to 2 - 10 nm (Fig. 1D) by changing the deposition

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time from 2 to 10 s. Fig 1C shows that the stability of the PPA layer increased as the thickness of

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the ALApp coating increased. For 5 s and 10 s ALApp deposition samples, PPA stayed intact

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over 2 weeks of incubation. Deposition of ALApp for over 10 s, however, appeared to affect the

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accuracy of the thermal lithography probe (data not shown). The 5 s ALApp was hence chosen

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for all further experiments.

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The ALApp/PPA coated surface is compatible with cell culture

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The ALApp/PPA coated surface was then tested for its bioocompatibility. First, the adhesion of

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human MSC to this surface was comparable to that in standard polystyrene tissue culture dishes

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(Fig 2A). There was also no significant toxicity of 5s ALApp/PPA sample to MSC as shown in

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Fig 2B. MSC cultured on the ALApp/PPA substrate exhibited no observable difference in


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morphology and number of focal adhesions (FAs) from cells on glass coverslips (Fig. 2D). These

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results indicate that ALApp/PPA coating enabled the use of thermolabile resist as cell culture

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substrates. Topographical patterns engraved by t-SPL can be directly exposed to cells, without

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the need of transferring the patterns into a separate, bioocompatible material. This t-SPL

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workflow, demonstrated here for the first time, allows the direct prototyping of surface

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topography, with nanoscale resolution, and assessment of cellular response to the topography

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within one day. This not only streamlines the procedure but also dramatically increases the

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spatial accuracy of the resulting pattern.

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‘Reading and writing” of tissue microenvironment with AFM and t-SPL

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As a proof of concept, we applied this workflow to fabricate a complex and physiologically

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relevant topographical pattern. We have previously shown that cells exposed to the tissue

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microenvironment revealed on a histological section of the Achilles tendon exhibited changes in

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morphology and function 18. Here, we tested the feasibility of “ reading ” the nanoscale

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topography of this microenvironment, and then “writing” the pattern on a ALApp/PPA coated

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surface. The tendon microenvironment consists of the aligned arrangement of triple-helical

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collagen fibrils 43, with regular bandings on each fibril (Fig 3). To replicate this characteristic

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topography by t-SPL, we first scanned a representative region of a tendon section by AFM (Fig

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3), and then converted the three-dimensional data into an image in which the depth information

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was encoded by grey-scale. Consecutive windows of AFM fields were stitched into a composite

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image of 3 µm x 3 µm. This image was used as the writing template for t-SPL. Fig 3 shows that

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this approach could accurately reproduce the topography of the tendon microenvironment. To


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our knowledge, this is the first demonstration of the direct copying of a biological surface on to a

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synthetic material. This method allows the accurate extraction of the topographical information

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of this surface, while removing other factors, such as the biochemical signals embedded in the

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ECM.

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Generalized tendon ECM

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The AFM image of the tendon section for t-SPL in Fig 3 was extremely data-rich, as it contained

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all the detailed topographical information in the selected region. Some of the information in this

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snapshot might represent localized damages of tissue introduced during dissection and

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sectioning, or local variations of collagen bundle orientation (Fig. 4A). This redundant spatial

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data may not be generally represented in the tendon tissue, and it significantly slowed down the

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t-SPL writing. We have previously shown, by varying the sectioning angle relative to the main

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axis of the tendon, that the spacing between the bands on the collagen fibrils is more important to

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MSC morphogenesis and differentiation than other topographical features such as bundle

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thickness 36. To understand the general architecture of tendon, we quantitated the geometry of

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collagen fibrils and banding by conducting AFM analysis of sections obtained from different

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regions of the tendon (Fig. 4A and 4B).

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topography of tendon was created (Fig. 4C). Numerous studies have reported the tendon collagen

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fibril structure 19, 44-45. The spacing between bands on collagen fibrils was reportedly in a

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range of 64 – 67 nm and the fibril diameters between 20 and 280 nm depending on state of

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lateral growth of the particular growth 46-47. Our measurements by means of AFM showed the

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band size to be 68.7 +/- 5.5 nm and 195.4 +/- 30 nm for fibril diameter. The consistency of our

Based on these measurements, a “ generalized ”


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data to the published literature indicated the stringency of tendon structure and the spatial

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accuracy of the replication. To optimize the spatial accuracy of t-SPL, we further converted the

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generalized topography into a 2-depth bit map template (Fig 4D) that utilizes the cantilever

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properties and tip geometry to reproduce the cylindrical shape of the fibrils. Fig 4D shows a

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comparison of AFM image of native tendon, generalized tendon, optimized template and the

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resulting t-SPL product. The result indicates that the t-SPL-generated pattern accurately matched

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the generalized tendon (Fig. 4C), and was similar to the native tendon at the nano- to microscale.

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The use of a 2-depth bit map also eliminated unnecessary depth information, thus increasing the

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writing efficiency so that arrays of multiple patterns could be generated on the same substrate in

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one session.

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Tendon microenvironment generated by t-SPL mimics the effect of native tendon on

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human mesenchymal stem cells

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Next, we examined the effect of the t-SPL-generated tendon topography on human MSC. MSC

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were seeded on the t-SPL pattern, on a native tendon section, a flat poly-allylamine/PPA surface

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and standard glass coverslip. MSC displayed similar morphology on the t-SPL pattern and the

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native tendon section. To examine cell-matrix interactions, the cells were fixed and probed with

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anti-vinculin antibody to visualize focal adhesions (FA). As shown in Figure 5A, MSC exposed

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to the t-SPL tendon pattern produced fewer FA (Fig. 5A, Fig. 2C & Fig. 5B) compared to flat

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poly-allylamine/PPA surface. The number of FA on the t-SPL pattern and native tendon section

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was similar (Fig. 5B). This implies that the tissue microenvironment induced by t-SPL generated

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nanostructure reflected the biological activity of the native tissue.


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Conclusion

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This study established a novel protocol for t-SPL sample preparation that involves the use of

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plasma polymerization of allylamine to improve the immobilization of PPA thermolabile resist

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on the substrate. This expands the application of t-SPL in fabrication of nanostructured materials

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in cell contact. Utilizing the superior spatial accuracy of t-SPL, we demonstrated that complex

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biological surfaces can be copied onto the Biocompatible PPA layer with nanoscale accuracy.

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The cellular response to the copied microenvironment could be immediately assessed by cell

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culture and imaging. Our approach opens up many new biological applications of the t-SPL

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technology. For example, the dimensions of the various geometrical components of the

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biological surface can now be independently modified, allowing the interrogation and the

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optimization of the functional activity of the topography in systematic, hypothesis-driven

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experiments. In future, this method can be applied to the study of other bioactive surfaces. We

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believe this new technique will accelerate the screening and development of biomimetic

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materials.

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FIGURES

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Figure 1. Modification of t-SPL substrate by plasma polymerization. (A) PPA coating peeled off from silicon wafer upon contact with PBS while allylamine plasma polymerization (ALApp) on the silicon wafer increased the immobilization of the PPA layer. (Green arrow: spin coating edge bead; Yellow arrow: PPA residues) (B) Schematic diagram showing ALApp between PPA and silicon wafer prevent peeling off. (C) PPA adhesion to silicon wafer increased with the duration of plasma polymer ALApp deposition. (D) Thickness of ALApp against polymer deposition time.

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Figure 2. Biocompatibility of the ALApp/PPA coated surface. (A) The adhesion efficiency of MSC seeded on PPA immobilized with coatings of different ALApp coatings. (B) The viability of MSC on ALApp modified t-SPL sample and on glass control. (C-D) The numbers of focal adhesions in MSC on ALApp modified t-SPL sample and on glass slide (red arrows: focal adhesions).

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Figure 3. High resolution tissue topography writing by means of t-SPL. t-SPL reproduced the nanoscale features of native tendon by using an AFM image directly as the writing template (red arrow: nanoscale features of collagen fiber).

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Figure 4. The generalized tendon tissue topography. (A) SEM of tendon tissue showing the different orientation of fibrils exposed (red arrows: different fibrils orientation). (B-C) The


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generalized tendon according to AFM measurements of native tendon fibrils. (D) Fabrication of the generalized tendon topography by t-SPL using the 2-depth bitmap ( “Nanofrazor template ”) based on the depth-encoded grayscale image of the generalized tendon.

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Figure 5. Cell culture on ALA modified t-SPL sample (silicon wafer with ALApp/PPA). (A) MSC exposed to generalized tendon pattern and native tendon tissue have significantly reduced focal adhesions as compared to MSC seeded on control glass slide and silicon wafer with ALApp/PPA without pattern (yellow arrows: focal adhesions). (B) MSC on t-SPL have similar numbers of FAs.

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AUTHOR INFORMATION

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Corresponding Author

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*Yun Wah Lam

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[email protected]


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* Nicolas H Voelcker [email protected]

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Author Contributions

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The manuscript was written through contributions of all authors. All authors have given approval

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to the final version of the manuscript.

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ACKNOWLEDGMENT

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This work was supported by Collaborative Research Grant (project number C1013-15G) from

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the Hong Kong Research Grant Council. Additionally, Sze Wing TANG was supported by a PhD

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fellowship (ID: 000618) funded by Hong Kong University Grants Committee (UGC). We thank

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Dr Rosa Chan (Department of Electrical Engineering, City University of Hong Kong) for her

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help in generating the data file for the generalized tendon topography. This work was performed

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in part at the Melbourne Centre for Nanofabrication (MCN) in the Victorian Node of the

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Australian National Fabrication Facility (ANFF).

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ABBREVIATIONS

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ALApp, Allylamine plasma polymer; AFM, Atomic force microscope; BSA, Bovine serum

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albumin; EBL, Electron beam lithography; ECM, Extracellular matrix; FA, Focal adhesions;

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MSC, Human mesenchymal stem cells; α-MEM, Minimum essential medium- alpha medium;

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PFA, Paraformalaldehyde; PBS, Phosphate buffered saline; PDMS, Polydimethylsiloxane; PPA,

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Polyphthaladehyde; PI, Propidium iodide; SEM, Scanning electron microscopy; t-SPL, Thermal

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scanning probe lithography; UC, Umbilical cord


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Replication of a Tissue Microenvironment by Thermal Scanning Probe

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