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The Pennsylvania State University
The Graduate School
Department of Engineering Science and Mechanics
FIBROUS PARYLENE-C THIN-FILM SUBSTRATES FOR
IMPLANT INTEGRATION AND PROTEIN ASSAYS
A Dissertation in
Engineering Science and Mechanics
by
Lai Wei
© 2011 Lai Wei
Submitted in Partial Fulfillment
of the Requirements
for the Degree of
Doctor of Philosophy
December 2011
ii
The dissertation of Lai Wei has been reviewed and approved* by the following:
Akhlesh Lakhtakia
Charles Godfrey Binder Professor in Engineering Science and Mechanics
Dissertation Advisor
Chair of Committee
Henry J. Donahue
Michael and Myrtle Baker Professor in Orthopaedics, College of Medicine
Jian Xu
Associate Professor of Engineering Science and Mechanics
MD Amanul Haque
Associate Professor of Mechanical and Nuclear Engineering, Engineering Science
and Mechanics graduate faculty
Judith A. Todd
Professor of Engineering Science and Mechanics
P. B. Breneman Department Head Chair
*Signatures are on file in the Graduate School
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ABSTRACT
Polymeric biomaterials are used in medical devices that can be surgically
implanted in human beings. Long-term bio-compatibility and strong tissue integration are
essential to the longevity of implanted prosthesis. Surface roughness and wettability are
essential for effective cellular attachment and integration. Therefore, materials should be
tailored so that their surface conditions are optimal for excellent integration with selected
proteins and cells.
This dissertation investigates the development of parylene-C thin films with good
control over surface roughness and surface wettability. Based on these qualities, different
degrees of cell and protein adhesion have been achieved, depending on surface properties
and the cell/protein type. In addition, a morphology-composition gradient panel has been
developed with a wide range of surface roughness and wettability, which can be used to
optimize tissue growth with high-throughput screening assays and with gradient surfaces.
The effects of the surface roughness and wettability of parylene-C thin films on the
adhesion of human fibroblast cells and biotinylated serum proteins have been
investigated. In addition, a simple method of fabricating nano-/micro-textured, free-
standing, parylene-C thin-film substrate has been developed, which has been
demonstrated to support cellular attachment and growth.
iv
TABLE OF CONTENTS
LIST OF ACRONYMS………………………………………………………………….vii
LIST OF FIGURES…………………………………………………………………......viii
LIST OF TABLES……………………………………………………………………….xii
ACKNOWLEDGEMENTS…..………………………………………………………...xiii
Chapter 1 Introduction………………………………………………………………….....1
1.1 Overview of the Issues………………………………………………………………...1
1.2 Research Objective……………………………………………………………………4
1.3 Outline of Dissertation……………………...…………………………………………5
Chapter 2 Background…….………...………………………………………………….....6
2.1 General Introduction to STFs……………………………………………………….....6
2.1.1 Growth Mechanics of STFs ……………………………………………………….10
2.1.2 Polymeric STFs…………………………………………………………………….12
2.2 Properties of Parylene-C……………………………………………………………..15
2.2.1 Physical Properties…………………………………………………………………16
2.2.2 Chemical Properties………………………………………………………………..17
2.3 Parylene-C Growth and Biomedical Applications…………………………………...18
2.3.1 Fabrication of Dense Parylene-C Thin Films…………………………..………….18
2.3.2 Biomedical Applications of Dense Parylene-C Thin Films………………………..21
2.3.3 Parylene-C STFs as Bioscaffolds...………………………………………………...21
2.4 Concluding Remarks……..…………………………………………………………..23
Chapter 3 Growth Mechanics and Characterizations of Parylene-C STFs………..……..24
3.1 Physicochemical-Vapor-OAD Method……......……………………………………..24
3.2 Effects of Deposition Conditions…………………………………………………….26
3.2.1 Effect of Substrate Motion…………………………………………………………27
3.2.2 Effect of Vapor Incidence Angle…………………………………………………..29
3.2.3 Effect of Deposition Pressure……………………………………………………...30
3.3 Parylene-C Surface Treatment……………………………………………………….31
3.3.1 Surface Wettability………………………………………………………………...31
3.3.2 Photo-Oxidation…..…….………………………………………………………….32
3.3.3 Oxygen-Plasma Treatment…………………………………………………………33
3.4 Characterization of Fibrous Parylene-C Thin-Film Substrates…….………………...34
v
3.5 Concluding Remarks…..……………………………………………………………..34
Chapter 4 Thickness-Controlled Hydrophobicity of Fibrous Parylene-C Thin-Film
Substrates…………………………………………………………………..36
4.1 Fabrication of Thin-Film Substrates….……………………………………………...36
4.2 Surface Morphology of Fibrous Thin-Film Substrates…...………………………….37
4.3 Surface Wettability and Fibrous Thin Films……………………………….……..….39
4.3.1 Thickness-Controlled Wettability………………………………………………..39
4.3.2 Wenzel and Cassie-Baxter Models………………………………………………...40
4.3.3 Surface Wettability Analysis………………………………………………………41
4.3.4 Oxygen-Plasma-Treated Surface Wettability…..………………………………….41
4.4 Concluding Remarks..………………………………………………………………..42
Chapter 5 Human Fibroblast Attachment on Fibrous Parylene-C Thin-Film Substrates..44
5.1 Introduction…………………………………………………………………………..45
5.2 Thin-Film Substrates and Cellular Attachment Assay Design…………...………….45
5.2.1 Cell Culture………………………………………………………………………...45
5.2.2 Cell Attachment Assays……………………………………………………………46
5.2.3 Oxygen-Plasma Treatment of Parylene-C Thin-Film Substrates…...……………..47
5.2.4 Fibroblast Attachment Characterization…………………………………………...47
5.2.5 Fluorescence Microscopy of Fibroblast Attachment………………………………48
5.3 Results and Discussion………………………………………………………………49
5.3.1 Oxygen-Plasma Treatment and Advancing Contact Angle Measurement……...…49
5.3.2 Influence of Substrate Morphology, Oxygen-Plasma Treatment and Serum Proteins
on Fibroblast Attachment to Parylene-C Thin-Film Substrates……...…………….50
5.3.3 Characterization of Fibroblast Attachment to Flat and Fibrous Parylene-C Thin-
Film Substrates……...……………………………………………………………..54
5.3.4 Fluorescence Microscopy of Oxygen-Plasma and Serum Influence on Fibroblast
Attachment to Parylene-C Thin-Film substrates……….………………………….55
5.4 Concluding Remarks……………………………………………………………........57
Chapter 6 2D Surface Morphology-composition Gradient Panel for Protein-Binding
Assays……………………………………………………………………….59
6.1 Introduction…………………………………………………………………………..60
6.2 Gradient Panels Fabrication and Characterization Method...………………………..61
6.2.1 Fabrication of 1D Surface Morphology Gradient Panels……..…………………...61
6.2.2 Fabrication of 2D Surface Morphology-Composition Gradient Panels ..…………63
6.2.3 Characterization Methods of 2D Wettability Gradient Panels...…………………..64
6.3 2D Surface Morphology-Composition Gradient Panel……………………………....65
vi
6.3.1 Morphology Gradient Along The x Axis………………………………………......65
6.3.2 Wettability Gradient Along The x Axis (before oxygen-plasma treatment)…..…...67
6.3.3 Analysis on 2D Wettability Gradient Panel………………………………………..69
6.4 Protein Binding Test to Gradient Panel……………………………………………...71
6.4.1 Serum Protein-Binding Assay Design………………….…………………….........71
6.4.2 Result of Protein Binding to The 2D Wettability Gradient Panel……………...….73
6.5 Concluding Remarks………………………………………………………………....76
Chapter 7 Free-Standing, Fibrous Thin-Film Substrates of Parylene-C for Cellular
Attachment and Growth……………..…………………………………….....77
7.1 Introduction…………………………………………………………………………..78
7.2 Approach 1: PMMA-Assisted Fabrication of Free-Standing Parylene-C Thin-Film
Substrates………………….………………………………………………………...79
7.2.1 PMMA Sacrificial Layer…………………………………………………………...79
7.2.2 Deposition of Parylene-C Thin-Film Substrate……...…………………….………79
7.2.3 Parylene-C Thin-Film Substrate………...……..…………………………………..80
7.3 Approach 2: Soap Solution-Assisted Fabrication of Free-Standing Parylene-C Thin-
Film Substrates………………………………………………………………………83
7.3.1 Step 1: Soap-Solution-Assisted Surface Treatment………………………………..84
7.3.2 Step 2: Deposition of Parylene-C Thin-Film Substrates…………………………...86
7.3.3 Step 3: Thermal Release of Parylene-C Thin-Film Substrate.……………………..89
7.4 Cellular Attachment and Growth to Free-Standing Parylene-C Thin Films…….…...92
7.4.1 Attachment and Growth Assay Design…………………………………………….92
7.4.2 Attachment and Growth Assay Result and Discussion…………….…………...….94
7.5 Concluding Remarks………..………………………………………………………..96
Chapter 8 Conclusions and Future Research…………………………………………….98
8.1 Conclusions…………………………………………………………………………..98
8.2 Future Research…………………………………………………………………….100
Bibliography……………………………………………………………………………101
vii
LIST OF ACRONYMS
2D: two-dimensional
3D: three-dimensional
ACA: advancing contact angle
AFM: atomic force microscope
B-A: biotinylated albumin
B-IgG: biotinylated IgG
BSA: bovine serum albumin
BTF: biotinylated transferring
CTF: columnar thin-film
DMEM: Dulbecco's modified Eagle's medium
EDTA: ethylene diamine tetra acetic acid
FBS: fetal bovine serum
FDA: food and drug administration
FESEM: field emission scanning electron microscopy
OAD: oblique-angle deposition
PMMA: polymethyl methacrylate
PS: phosphatase substrate
PVD: physical vapor deposition
SAP: streptavidin-alkaline phosphatase
SEM: scanning electron microscopy
SNTFs: sculptured nematic thin films
STF: sculptured thin-film
TBS: tris-buffered saline
TFHBMs: thin-film helicoidal bianisotropic mediums
viii
LIST OF FIGURES
Fig. 2.1. Cross-sectional SEM images of 2D STFs: (a) chevronic STF made of
magnesium fluoride [54], (b) slanted columnar STF made of chromium [56], (c) C-
shaped STF made of magnesium fluoride [51], and (d) S-shaped STF made of silicon
oxide [56]………………………………………………………………………………….7
Fig. 2.2. Cross-sectional SEM images of 3D STFs: (a) helical STF made of magnesium
fluoride [57], and (b) superhelical STF made of magnesium fluoride [54]……………….8
Fig. 2.3. Cross-sectional SEM image of a multi-section STF made of silicon oxide [59]..8
Fig. 2.4. Schematic of the deposition showing the relative angle v of the average vapor
direction with respect to the platform on which the thin-film substrate is deposited.
(Picture regenerated from [61])…………………………………………………………...9
Fig. 2.5. (a) Schematic of the deposition of a CTF. The platform is held fixed at some
oblique angle v with respect to the vapor incidence direction. (b) Schematic of the
deposition of a chiral STF. The platform is rotating about a central normal axis while the
platform is oriented at some oblique angle v . (Pictures regenerated from [51])………10
Fig. 2.6. (a) SiO2 thin film of chiral morphology grown on an Si substrate, (b) void in the
thin film filled with photoresist HPR504, (c) thin layer of photoresist removed from the
top of filled thin-film, and (d) STF of nanopoles with SiO2 etched by hydrofluoric acid
[66]……………………………………………………………………………………….13
Fig. 2.7. Interference in photoresist SU8 of seven beams of 325 nm free-space
wavelength with six beams linearly polarized and one circularly polarized. SEM images
of chiral morphology: (a) overall (scale bar 2 μm), and (b) close-up view (scale bar 1 μm)
[68]……………………………………………………………………………………….14
Fig. 2.8. Cross-sectional SEM image of a parylene-C chiral STF [36]……………….…15
Fig. 2.9. Chemical formula of the parylene-C monomer …………………...…………...16
Fig. 2.10. Process and equipment for deposition of dense parylene thin films on surfaces.
(Picture regenerated from [81])………………………………………………………….18
Fig. 2.11. Schematic diagram of the combined plasma–parylene reactor for deposition on
a surface. (Picture regenerated from [79])……………………………………………….19
Fig. 2.12. Schematic diagram of a reactor for ion-assisted parylene-C deposition on a
surface. (Picture regenerated from [80])…………………………………………………20
Fig. 2.13. (a) AFM image on top surface of a parylene-C chiral STF, and (b) confocal
laser microscope image of COS-7 fibroblast cells grown on a parylene-C chiral STF: Cell
nuclei are shown in red and amines are shown in green [86]……………………………23
ix
Fig. 3.1. Schematic of the physicochemical vapor deposition system developed at the
Pennsylvania State University [36] to deposit parylene-C thin-film substrates of
customizable morphologies on either glass disks or silicon slides………………………25
Fig. 3.2. (a) Photograph of PDS2010 parylene-C deposition chamber. (b) Photograph of
modified deposition chamber. (c) Photograph of facilities inside the chamber…………26
Fig. 3.3. A: Cross-sectional SEM images of four morphological types of parylene-C thin-
film substrates: (a) slanted columnar, (b) chevronic, (c) chiral, and (d) flat. B: Top-
surface SEM images of four types of parylene-C thin-film substrates: (a) slanted
columnar, (b) chevronic, (c) chiral, and (d) flat………………………………………….28
Fig. 3.4. Cross-sectional SEM images of fibrous parylene-C thin-film substrates with
slanted columnar morphologies grown at different vapor incidence angles v : (a) 25º, (b)
20º, (c) 15º, and (d) 10º. Deposition pressure was set at 26 mTorr……………………...30
Fig. 3.5. Cross-sectional SEM images of fibrous thin-film substrates of parylene-C with
slanted columnar morphology ( v =10º) grown at different deposition pressures: (a) 18
mTorr, and (b) 26 mTorr…………………………………………………………………31
Fig. 3.6. SEM images of the top-surface morphology of a parylene-C chiral STF ( v
=10º, 26 mTorr, 30-µm-thick) (a) before and (b) after oxygen-plasma treatment for 60
s…………………………………………………………………………………………..33
Fig. 4.1. Top-surface SEM images of (a-e) CTF substrate and (f) a chiral STF substrate.
Thicknesses are as follows: (a) 0.22 μm, (b) 1.29 μm, (c) 6.0 μm, (d) 35.6 μm, (e) 63.3
μm, and (f) 23.0 μm……………………………………………………………………...38
Fig. 4.2. Measured advancing contact angle θ (black solid curve) and calculated solid
fraction f (red dashed curve) as functions of thickness of a CTF substrate of parylene-C.
The measured values of θ of a flat thin-film substrate (control) and a chiral thin-film
substrate are also marked………………………………………………………………...39
Fig. 4.3. Behavior of a water droplet (blue) on a rough surface. Left: Wenzel model; right:
Cassie-Baxter model [93]………………………………………………………………..40
Fig. 4.4. Left: Photograph of a water droplet on a 35.6-μm-thick CTF substrate showing
θ=165.6°. Right: Photograph of a water spread on the surface of a 22.0-μm-thick CTF
substrate after oxygen-plasma treatment………………………………………………...42
Fig. 5.1. Quantification of fibroblast attachment after 2 h to parylene-C thin-film
substrates of four morphological types. The number of samples of each type is five…...51
Fig. 5.2. Quantification of fibroblast attachment to flat and slanted columnar parylene-C
thin-film substrates under four conditions. +P: with oxygen-plasma treatment; -P: without
oxygen-plasma treatment; +S: with serum proteins; -S: with serum replacement………52
Fig. 5.3. SEM images showing cellular attachment to (a) a fibrous thin-film substrate
with slanted columnar morphology (bar = 20 µm); (b) a fibrous thin-film substrate with
x
chevronic morphology (bar = 25 µm); (c & d) a fibrous thin-film substrate with chiral
morphology (bars = 20 µm, 5 µm; and (e & f) a flat thin-film substrate (bars = 50 µm, 5
µm). Note that (d) is the magnified version of the rectangular region marked in (c);
images (f) and (e) are similarly related…………………………………………………..55
Fig. 5.4. Fluorescence-microscope images of fibroblasts adhering to flat parylene-C thin-
film substrates for 2 h and 24 h, 20X objective. +P: with oxygen-plasma treatment; -P:
without oxygen-plasma treatment; +S: with serum proteins; -S: with serum substitute…56
Fig. 6.1. Schematic of the fabrication method for a panel comprising 3 rows of 16 3mm
3mm plates of glass placed adjacent to each other with virtually no space between
neighbors. The x axis begins at the proximal end………………………………………..62
Fig. 6.2. CTF thickness as a function of the distance x from the proximal end. The
standard deviations are so small that the error bars are not easily discernible…………..65
Fig. 6.3. Optical image of one row of 16 square plates coated with parylene-C. A
thickness gradient is evident as a transparency gradient………………………………...66
Fig. 6.4. Top-view SEM images of the CTF at different distances from proximal end: (a)
x = 4 mm, (b) x = 12 mm, (c) x = 20 mm, (d) x = 28 mm, (e) x = 36 mm, and (f) x = 44
mm. These images were taken before oxygen-plasma treatment, which effects a change in
surface chemical composition but not in surface rugosity……………………………….67
Fig. 6.5. Measured mean values of θ as function of x and t. Data for t = 60 s are close to
those for t = 40 s, and therefore have not been shown. The standard deviation, being less
than 1.1º at any (x,t), has not been shown because of the complexity of the figure. Zones
I-III are discussed in the text……………………………………………………………..69
Fig. 6.6. Normalized absorbance with respect to x of B-IgG bound to a wettability
gradient panel, for t = 0 s (squares) and t = 60 s (circles), where t is the duration of
oxygen-plasma treatment. The measured absorbances were normalized with respect to the
highest value measured…………………………………………………………………..74
Fig. 6.7. Same as Fig. 6.6, but for B-A…………………………………………………..75
Fig. 6.8. Same as Fig. 6.6, but for B-TF …...……………………………………………75
Fig. 7.1. Cross-sectional SEM images of three morphological types of parylene-C thin-
film substrates on PMMA-coated glass slides: (a) chiral, (b) slanted columnar, (c)
chevronic, and (d) magnified version of chevronic……………………………………...81
Fig. 7.2. Photograph of free-standing, fibrous thin-film substrates of parylene-C separated
from glass slides: ―D-film‖ indicates separated thin-film and ―S‖ indicates glass slide...82
Fig. 7.3. Top: Schematic of the three-step process to fabricate nano/micro-textured, free-
standing, fibrous thin-film substrate of parylene-C. Inset: vapor incidence angle v in
Step 2…………………………………………………………………………………….83
xi
Fig. 7.4. Optical microscope images of the top surfaces of unbaked as well as baked
coatings of soap on silicon slide. (a) Without baking; (b-f) baking temperature: (b) 100 ºC,
(c) 150 ºC, (d) 200 ºC, (e) 250 ºC, and (f) 300 ºC………………………………………..85
Fig. 7.5. FESEM images of (a, c) cross-sectional and (b, d) top views of parylene-C thin-
film substrates with slanted columnar morphology and of thickness (a, b) 100 nm and (c,
d) 100 μm. FESEM images of top views of parylene-C thin-film substrates with (e)
chevronic morphology and 75-μm thickness and (f) chiral morphology and 85-μm
thickness………………………………………………………………………………….88
Fig. 7.6. Same as Fig. 7.4, except the images are of the bottom surfaces of the released
parylene-C thin-film substrates of slanted columnar morphology and 500-nm
thickness………………………………………………………………………………….90
Fig. 7.7. (a) Photograph of a parylene-C thin-film with columnar morphology deposited
on an uncoated silicon slide. (b-d) Photographs of fibrous thin-film substrates with
slanted columnar morphology and of thickness (b) 100 nm, (c) 6 μm, and (d) 100 μm...92
Fig. 7.8. Quantification of cell attachment and growth on free-standing fibrous parylene-
C thin-film substrates, with and without oxygen-plasma treatment……………………..95
xii
LIST OF TABLES
Table 5.1. Advancing contact angles on parylene-C thin-film substrates of four different
morphological types before oxygen-plasma treatment…………………………………..50
xiii
ACKNOWLEDGEMENTS
First, this work would not have been possible without funding provided for three
and a half years by Materials Research Institute Fellowship, a seed grant from the
Woodward Research Foundation, and the Charles Godfrey Binder Endowment at
Pennsylvania State University.
Second, I would like to express my sincere gratitude to my advisor, Dr. Akhlesh
Lakhtakia, for providing me the opportunity to complete my Ph.D. dissertation at the
Pennsylvania State University, for his invaluable guidance and encouragement
throughout my Ph.D. research period. His attitude to research, work and life, vision in
exploring new research fields impress me a lot and all that I have learned from him will
be helpful in my future career. I am very grateful to Dr. Timothy M. Ritty for
participating actively as my co-advisor, but his role could not be formally acknowledged
in a befitting manner in the end as he left Pennsylvania State University.
Third, I am indebted to Dr. Erwin A. Vogler and Dr. Purnendu Parhi for helping
me in contact angle measurement, to Lanfranco Leo and Adriana P. Roopnariane for
providing the assistance in the lab. I would like to thank Dr. Henry J. Donahue, Dr. Jian
Xu, and Dr. MD Amanul Haque for serving on my committee and their invaluable
suggestions on future research work.
Fourth, I would like to give my special gratitude to my family for being there for
me. I sincerely thank my parents and my fiancée for their constant love, support and
encouragement. Without their supports and efforts, I could never go this far.
1
Chapter 1
Introduction
1.1 Overview of the Issues
Polymeric biomaterials are essential in making medical devices that can be
surgically implanted in human beings [1, 2]. Long-term bio-compatibility and strong
tissue integration are likewise attributes that biomaterials must possess [1, 3]. Whatever
its claims to biocompatibility, every implanted prosthesis or device elicits a foreign-body
response [4] whereby the surrounding tissue—instead of integrating with the prosthesis
or device— produces a dense, fibrous encapsulation. The result can be a lack of stability,
which, in turn, can shorten the useful life-span of the prosthesis or device. Furthermore,
the tribological issue should also be considered in identifying long-life materials [5, 6].
The accumulation of wear debris encourages the formation of macrophages that cause a
reaction from the body, tending to eliminate the debris as a foreign body. The debris is
usually large in size, and thus the macrophages aggregate and form polynucleate cells,
which results in the long-term failure of a prosthesis.
True integration is not possible if the implant surfaces are smooth. Surface
modifications that improve the interface between cells and biomaterials may promote
physiological healing and thus better integration of the medical device with the tissue [7].
There is growing awareness in the biomedical-materials community that cellular growth
on a substrate is influenced by texturing the latter at submicron- and nano-length scales.
In fact, it is accepted that cellular attachment can be controlled by texturing the substrate
at these scales [8-10]. Both cellular proliferation [11, 12] and cellular differentiation [13,
2
14] can be favorably influenced. Thus, by texturing the polymeric-implant surfaces
appropriately, their performance in a biological system can be improved significantly.
Similarly, another factor that influences cellular interactions with biomaterials is
the degree of hydrophobicity/hydrophilicity or wettability [15, 16]. Wettability of the
surface of a solid material is important for biomedical applications such as tissue-culture
substrates and coatings of implantable prostheses. Mammalian cells often attach to and
spread efficiently on hydrophilic surfaces [17], whereas cell attachment and spreading on
hydrophobic substrata is much less efficient [18]. But the relationship between wettability
and cellular attachment is far from simple, because the cell adhesion mechanism may
vary on surfaces of similar wettability but differing chemistries [19]. Also, different cell
types exhibit different wettability preferences for attachment [20]. To complicate matters
even further, proteins abundant in biological systems that can either facilitate (e.g.,
fibronectin and vitronectin) or inhibit (e.g., serum albumin) cellular attachment to a
surface also vary in adhesion, depending on the hydrophobic/philic nature of the surface
[21].
Protein adsorption on the implant surface is the first step of tissue integration and
the nature of adsorbed proteins in turn determines the cellular response to the biomaterial
[22]. The surface properties of a biomaterial can determine the types, amounts, and
conformations of adsorbed proteins [23]. Furthermore, the influence of nanoscale surface
morphology on protein adsorption has been studied [24-27], and no consistent conclusion
has been reached. Some studies report that nanoscale surface texturing has no effect on
protein adsorption [24, 25], and others demonstrate increased protein adsorption on nano-
3
textured surfaces. In addition, surface wettability, charge, and composition have been
reported to affect protein adsorption [28-32].
For these reasons, it is desirable to be able to tailor the surface of polymeric
materials used for biomedical applications for optimum performance in a specific
application; e.g., the surface hydrophobicity of implantable prostheses promotes efficient
lubrication so that debris does not accumulate and cause inflammation [33]. For good
biological integration, the surface should be textured to control both wettability and
biocompatibility [34, 35]. Moreover, a flexible, free-standing polymeric thin-film
substrate is desirable for implant surgery applications. The tissue grown on a free-
standing flexible substrate could be implanted as an integrated assembly on a nonplanar
surface inside a human body.
Parylene-C is one such candidate polymeric material for biomedical applications.
This material stands out among the available biomaterials, as it has shown to possess
acceptable physical, chemical, and biocompatible properties, which are presented in more
detail in Chapter 2. Fabricated by physicochemical deposition [24, 25], this polymer has
been used to fabricate dense, flat films [36], and it is commonly used to coat implantable
devices [37-40]. Methods of parylene-C growth for rough surfaces and surface wettability
modifications [36, 41] have also been reported. However, there is no report on the ability
to preferentially control surface morphology and wettability of parylene-C in a way that
is helpful for biomedical applications as discussed earlier. It should be noted, too, that no
reports have been published on portability and implantability of the tissue grown using
free-standing parylene-C. Yet, a free-standing flexible substrate is highly desirable for a
material to be implanted as an integrated assembly on a nonplanar surface.
4
1.2 Research Objective
Nanotechnology is playing an important role in designing surface characteristics
that interface optimally with selected proteins and cells. Specifically, a top-down, a
bottom-up approach, or a combination of these, can be taken to the task of engineering
surface roughness. Top-down approaches include lithographic and template-based
techniques [42] and plasma treatment of the surfaces [43, 44]. Bottom-up approaches
involve mostly self-assembly and self-organization [45, 46]. One method that combines
both bottom-up and top-down approaches is casting the polymer solution and phase
separation [47]; another is electro-spinning [48, 49].
However, these methods have certain drawbacks: the electron-beam lithographic
approach, is both expensive and time-consuming; self-assembly requires molecule
engineering; electro-spinning results in the absence of an organized pattern for phase
separation and only simple morphologies are produced [50]. Thus, both new biomaterials
and novel nanotechnologies are required for practical applications.
The aim of the research reported in this dissertation is to develop efficient
parylene-C thin-film substrates as scaffolds for tissue integration that can be used as
implant surfaces. Three key tasks are related to this aim. The first task is that of
developing parylene-C thin-film substrates using the sculptured thin-film (STF)
technology [51], which should have good control over surface roughness and wettability.
Compared with the established nanotechnology techniques described earlier, this
emerging approach should be able to tailor surface conditions efficiently. The second task
is that of developing a morphology-composition gradient panel of parylene-C that will
allow systematic analysis of protein binding in relation to surface properties and also
5
work for high-throughput screening assays and gradient surfaces for optimized growth of
tissue. The third task is to demonstrate a method of fabricating a nano/micro-textured,
free-standing, parylene-C thin-film substrate and test its ability to support the attachment
and growth of human cells.
1.3 Outline of Dissertation
Chapter 2 consists of a literature review on STF technology, the properties of
parylene-C, the growth method, and the biological applications of fibrous and dense
parylene-C thin-film. Chapter 3 describes parylene-C STF fabrication and discusses the
deposition conditions and characterization methods. A discussion of the thickness-
controlled hydrophobicity of fibrous parylene-C STFs is presented in Chapter 4. The
attachment of human fibroblast cells on fibrous parylene-C thin-film substrates is
reported in Chapter 5. The fabrication and characterization of a morphology-composition
gradient parylene-C panel is described in Chapter 6, wherein proteins attachment assays
are studied. Chapter 7 introduces a new method for fabricating nano-/micro-textured,
free-standing, thin-film substrates of parylene-C. It also discusses the cellular attachment
and growth test on this free-standing thin-film substrate. Chapter 8 presents the overall
conclusions and offers several directions for future work.
6
Chapter 2
Background
This chapter provides an overview of STF growth mechanics, particularly for polymeric
STFs. It also presents a physicochemical vapor deposition technique developed for
growing parylene-C STFs as well as dense, flat films of parylene-C. The properties of
both types of materials for biomedical applications are also discussed.
2.1 General Introduction to STFs
STFs are a class of nanostructured thin films comprising columns whose growth
direction can be changed instantaneously during fabrication [51, 52]. They are generally
grown by physical vapor deposition (PVD) or variations thereof [53]. A great variety of
columnar morphologies can be fabricated through a PVD process, by continuously or
discontinuously tilting and/or rotating a substrate (usually a glass/silicon slide or disk)
with respect to the average direction of the incident vapor flux. The morphologies can be
classified as two-dimensional (2D) and three-dimensional (3D), termed sculptured
nematic thin films (SNTFs) [54] and thin-film helicoidal bianisotropic mediums
(TFHBMs) [52, 55], in optics, respectively. For example, the shapes of columns of 2D
STFs include chevrons, zigzags, slanted columns, C’s and S’s, whereas the columns can
be helical in 3D STFs. Cross-sectional images of STFs of different 2D and 3D
morphologies [51, 54, 56, 57] producing by scanning electron microscopy (SEM) are
given in Figs. 2.1 and 2.2. More complex morphologies and multi-section STFs can also
be achieved [58, 59]. The two-section STF shown in Fig. 2.3 comprises a chevronic
section on top of a helical section.
7
Fig. 2.1. Cross-sectional SEM images of 2D STFs: (a) chevronic STF made of
magnesium fluoride [54], (b) slanted columnar STF made of chromium [56], (c) C-
shaped STF made of magnesium fluoride [51], and (d) S-shaped STF made of silicon
oxide [56].
8
Fig. 2.2. Cross-sectional SEM images of 3D STFs: (a) helical STF made of magnesium
fluoride [57], and (b) superhelical STF made of magnesium fluoride [54].
Fig. 2.3. Cross-sectional SEM image of a multi-section STF made of silicon oxide [59].
The basic technique for fabricating STFs employs a typical PVD system with a
substrate holder that can be tilted and/or rotated [51], as shown in Fig. 2.4. A vapor flux
from a solid source is incident obliquely towards a platform, in a low-pressure chamber
under suitable conditions [51, 60]. The adatomic self-shadowing effect creates a thin film
9
with columnar microstructure, as shown in Fig. 2.5 under suitable conditions. This
process is also referred to as the oblique-angle deposition (OAD) method. The column
inclination angle is generally larger than the vapor incidence angle v , unless both
angles are equal to 90º, when the substrate holder is stationary. The exact morphology
depends on many factors such as substrate temperature, deposition rate, angular
distribution of the deposition flux, and deposition pressure [51]. Columnar thin-film
(CTF) is formed by fixing the vapor incidence angle v , as shown in Figure 2.5(a). 2D
STFs with chevronic, C-shaped, and S-shaped columns can be realized by changing v
as a function of time. 3D STFs are fabricated by fixing the vapor incidence angle v ,
followed by rotation of the platform about a central normal axis [55]. Helical columns are
formed with the helix direction perpendicular to the substrate [51], as shown in Fig.
2.5(b), if the deposition occurs at a fixed rate and the rotational speed does not vary. Such
TFHBMs are generally called chiral STFs.
Fig. 2.4. Schematic of the deposition showing the relative angle v of the average vapor
direction with respect to the platform on which the thin-film substrate is deposited.
(Picture regenerated from [61])
10
Fig. 2.5. (a) Schematic of the deposition of a CTF. The platform is held fixed at some
oblique angle v with respect to the vapor incidence direction. (b) Schematic of the
deposition of a chiral STF. The platform is rotating about a central normal axis while the
platform is oriented at some oblique angle v . (Pictures regenerated from [51])
STFs are porous. The void formation in STFs results from a self-shadowing effect,
which means that certain areas of the growing thin film are blocked by features in front of
them along the incident flux direction. The void formation and the tilted columnar
morphology introduce morphological anisotropy. Thus, OAD generates anisotropic
characteristics, which have been regarded as one important feature of STFs. Another
important feature of STFs is porosity, which is greatly enhanced by the self-shadowing
effect. The mass density of an STF is highly dependent upon , and, therefore, v [54,
56].
2.1.1 Growth Mechanics of STFs
For most thin films deposited on non-ionic platforms, there are four stages in the
growth process: nucleation, islanding, channel filling, and continuous film growth [62].
When the vapor flux impinges on the platform, the vapor particles can be either reflected
or adsorbed physically. The adsorbed atoms (also called adatoms) often move some
11
distance before being incorporated into the film. This process is called surface diffusion.
At this stage, the adatoms can desorb or collide with each other and with the surface
thereby forming the bonds of the film material already on the surface. The initial
aggregation of the film material is known as nucleation, and is a random process. The
adatoms become less mobile as the nuclei grow larger; as this occurs, the adatoms tend
to bond to the surface where they serve as anchors on which other adatoms can
congregate when the nuclei reach a certain size. Islands form as these aggregations
continuously grow and the gaps between the islands eventually decrease in size and
become channels and voids. At this stage, the self-shadowing effect has a strong impact
on void formation, as the conglomerations of some adatoms can effectively shadow areas
from the incoming flux. The islanding-and-channel-formation process leads to the
creation of columnar structures and voids in the growing thin-film.
The fabrication of STFs takes advantage of the self-shadowing effect, which is
associated with various deposition conditions. According to the structure zone model [63,
64], the emergence of columnar morphology depends on the ratio of the platform
temperature Ts during deposition, and the melting temperature Tm of the deposition
material. The higher the ratio Ts/Tm, the more mobile the adatoms are and the more
dominant the surface diffusion becomes. Thus, a surface with low adatom mobility
clearly provides conditions that support the formation of STFs.
Another factor related to STF deposition is the degree of collimation in the vapor
flux. The vapor flux leaves the source with a wide angular distribution. As the distance
increases from the source to the substrate, the angle of the vapor flux across the platform
becomes smaller, which results in more collimated flux towards the platform.
12
Furthermore, lower pressure also leads to better collimation at the platform because under
these conditions the mean free path becomes longer.
2.1.2 Polymeric STFs
Attempts to fabricate polymeric STFs were made in the early days of STF
research. It is difficult to fabricate polymeric STFs through PVD. Experimental
approaches for polymeric STFs include the replamineform method and the holographic
lithography and mixed-vapor deposition technique.
The replamineform route was implemented [65, 66] to realize polymeric STFs or
STFs of nanoholes. For the latter structure, there are three stages, an example of which is
shown in Fig. 2.6. First, grow an STF from some inorganic material by PVD; second, fill
the void regions with a polymer; third, etch out the inorganic skeleton. For an STF with
polymeric nanowire, the route has five stages. The first three stages are the same as those
for fabricating an STF of nanoholes. In the fourth stage, the nanoholes are filled with the
desired polymer, followed by the fifth stage in which the first polymer is removed.
13
Fig. 2.6. (a) SiO2 thin film of chiral morphology grown on an Si substrate, (b) void in the
thin film filled with photoresist HPR504, (c) thin layer of photoresist removed from the
top of filled thin-film, and (d) STF of nanopoles with SiO2 etched by hydrofluoric acid
[66].
Another way to fabricate a polymeric STF is through holographic lithography. In
this technique, several optical beams with the same free-space wavelength are
simultaneously launched in a photoresist, and the interference pattern is recorded in the
photoresist. After exposed photoresist has developed, a complicated morphology is
obtained [67]. The chiral morphology is achieved if at least one of the beams is
elliptically polarized, as shown in Fig. 2.7 [68].
14
Fig. 2.7. Interference in photoresist SU8 of seven beams of 325 nm free-space
wavelength with six beams linearly polarized and one circularly polarized. SEM images
of chiral morphology: (a) overall (scale bar 2 μm), and (b) close-up view (scale bar 1 μm)
[68].
A single-step mixed-vapor deposition for chiral STFs of parylene-C was reported
by Penn State researchers [36]. The route combined chemical vapor deposition and
physical vapor deposition is the OAD format. The raw material was parylene-C, a
polymer widely used for coating biomedical devices. Parylene-C in its dimer form was
first vaporized and then pyrolized into a monomer form. Then the monomer flux was
directed to a rotating platform to form chiral STFs, with the cross-sectional SEM image
presented in Fig. 2.8. Compared with the replamineform and holographic lithography
routes, this method is much easier to implement. Further, it should be noted that
parylene-C has excellent biocompatibility [69]. Therefore, the physicochemical-vapor-
15
OAD method was selected for fabricating parylene-C STF substrates for the research
reported in this dissertation.
Fig. 2.8. Cross-sectional SEM image of a parylene-C chiral STF [36].
2.2 Properties of Parylene-C
Parylene is the generic name for a set of chemicals, including parylene-N,
parylene-C, and parylene-D. The parylene monomer (also termed parylene-N) is
composed of an aromatic ring with methylene groups attached at the para positions.
Parylene-C has one chlorine atom on the aromatic ring replacing the hydrogen bond, as
shown in Fig. 2.9, and parylene-D has two chlorine atoms on the aromatic ring [70].
Compared with the other two derivatives, parylene-C is more popular due to its useful
combination of physical and electrical properties, such as its ability to provide an
16
efficient barrier to solvents and its ability to conformally coat a surface without forming
pinholes [71].
Fig. 2.9. Chemical formula of the parylene-C monomer.
2.2.1 Physical Properties
Conventional parylene-C deposition (as discussed later in Sec. 2.3) has the ability
to make the vapor surround and penetrate closely spaced components. Thin parylene-C
coatings with high density, conformality, and thickness uniformity can be achieved on
non-planar surfaces [72]. Thus, parylene-C coatings form a good moisture barrier and
function as a good dielectric insulator [71].
Parylene-C deposition is a polymerization process that induces no thermal or
mechanical stress in the coating layer and takes place at room temperature. Thus, the
original physical attributes of the coated surface are unaffected [72]. Parylene-C also
possesses high tensile strength [70] and high yield strength [73], which can increase the
pull strength of the coated surface. Additionally, parylene-C coatings have good wear
resistance and abrasion resistance compared with fluoropolymers [72].
With a melting point of 290 ºC, parylene-C has good thermal stability. Its thin-
film coatings can remain stable below 125 ºC in air and can survive below 350 ºC in
17
nitrogen [70]. It has good mechanical properties from -200 °C to 275 ºC [72]. Annealing
at proper temperature increases its hardness and abrasion resistance [71].
Parylene-C thin films in general do not adhere well to surfaces. The chemical
composition of parylene-C makes it difficult to bond to various materials [74]. However,
there are techniques capable of increasing its bond strength, including silane-based
coupling agents [75] and the plasma treatment of the surface to be coated [76].
2.2.2 Chemical Properties
Parylene-C is chemically very inert, and it can work as an effective barrier to all
known organic solvents and most acids and alkali reagents at temperatures below its
melting point. Due to its excellent chemical resistance, parylene-C thin-film coatings can
survive sterilization processes like autoclaving and radiation, which is a necessary ability
in biomedical applications [72].
However, the chemical composition of parylene-C surfaces can be modified with
either an oxygen-plasma treatment [41] or by photo-oxidation [77]. These two techniques
are presented in more detail in Sec. 3.3. The induced composition change can provide a
range of surface wettability.
18
2.3 Parylene-C Growth and Biomedical Applications
2.3.1 Fabrication of Dense Parylene-C Thin Films
Methods for dense parylene thin-film fabrication have been reported [78] [79]
[80]. The basic growth mechanism is physicochemical deposition composed of three
steps: sublimation, pyrolysis, and polymerization. Compared with the physicochemical-
vapor-OAD method for fibrous parylene STFs, there is no issue of platform orientation
with respect to incident vapor flux, which comes to the platform from all directions. As
shown in Fig. 2.10 [81], the parylene dimer is sublimated in the vaporizer at 150 ºC, after
which it is dissociated into two monomers at 680 ºC through pyrolysis. Finally, parylene
monomers are condensed and polymerized on the platform in a deposition chamber at
room temperature. A continuous, dense, thin-film of parylene is formed.
Fig. 2.10. Process and equipment for deposition of dense parylene thin films on surfaces.
(Picture regenerated from [81])
19
The adhesion of parylene-C to most surfaces is inherently weak [74]. To improve
adhesion, two modified physicochemical vapor deposition methods have been reported.
The first is plasma-polymerization-assisted chemical vapor deposition [79], by which a
plasma-polymerized film is deposited before parylene polymerization takes place. The
plasma-polymerized film works as an adhesion primer layer and provides a covalent bond
to enhance the adhesion of parylene-C. As shown in Fig. 2.11, it is a combined plasma
and parylene-C reactor. The parylene-C reactor consists of sublimation and pyrolysis
furnaces. In the plasma reactor, a gas, such as methane used in the preparation of the
primer layer, is introduced through an inlet valve, after which deposition occurs on the
substrate with plasma generated by radiofrequency power.
Fig. 2.11. Schematic diagram of the combined plasma–parylene reactor for deposition on
a surface. (Picture regenerated from [79])
20
The other method is ion-assisted parylene-C physicochemical vapor deposition
[80]. The schematic diagram is shown in Fig. 2.12. Parylene-C monomer is formed
through evaporator and pyrolysis filaments, and then is fractionally ionized by ionizer
filament. The ionized parylene-C monomer is then accelerated towards the surface at
high energy by accelerating electrodes. The accelerated monomer penetrates the surface,
thereby improving the material’s adhesion to the surface.
Fig. 2.12. Schematic diagram of a reactor for ion-assisted parylene-C deposition
on a surface. (Picture regenerated from [80])
21
2.3.2 Biomedical Applications of Dense Parylene-C Thin Films
A foreign-body response [4] would be elicited for an implanted prosthesis or
device in which the surrounding tissue produces a dense, fibrous encapsulation but does
not integrate with the tissue. For this reason, a biocompatible coating material is needed.
Parylene-C, an FDA-approved (Food and Drug Administration) biopolymer [69], is
extensively used as a biocompatible electrical insulator for sensors [39, 82], electronic
circuits [37, 38], and other implantable biomedical devices [83, 84]. For example,
parylene-C has been examined for use as insulator coating on microelectrode arrays for
recording electromyographic signals [82]. It has also been demonstrated that parylene-C
is the choice for conformal and corrosion prevention coating of high-reliability
implantable electronic circuits [37]. Another example is the use of parylene-C as a
pacemaker coating to prevent muscle twitch [84].
2.3.3 Parylene-C STFs as Bioscaffolds
As discussed in Sec. 1.1, in addition to long-term biocompatibility, strong tissue
integration is also important for successful biomaterials [1, 3]. True integration is not
possible if the implant surfaces are smooth [7]. Thus, an investigation into how fibrous
parylene-C STFs interact with cells and proteins is crucial if solid implantation
applications are to be realized.
In addition to biocompatibility, parylene-C STFs are being considered as
bioscaffolds for cell–surface interactions tests for two reasons [85]. First, they have
features similar to those of the native extra-cellular matrix in biological tissue: clusters of
22
1–3 nm diameter coalesce to form columns or nanowires of 30–50 nm cross-sectional
diameter which, in turn, form bundles of 300–500 nm cross-sectional diameter. Second,
STFs have increased surface area as well as multi-scale and engineerable porosity for cell
attachment. Thus, parylene-C STFs can also serve as simulative tools for studying the
interactions, transport, and synthesis of biomolecules in confined environments and on
tissue-culture substrates [85].
The results of preliminary tests on the ability of parylene-C STFs to support
fibroblast cell attachment and proliferation [86], suggest the possible use of parylene-C
STF as substrates for growing implantable tissue. A parylene-C chiral STF approximately
50-µm-thick was fabricated by physicochemical-vapor-OAD method. The top-surface
atomic force microscope (AFM) image is shown in Fig. 2.13 (a) [86]. The figure shows
that parylene-C chiral STF consists of assemblies of nanowires and 200-nm-diameter
columns comprising 50 nm nanowires. COS-7 fibroblast cells were seeded on parylene-C
STF and cultured in Petri dishes. After 72 h of growth, the fixed cells were labeled with
dyes in order to locate the amines and nuclei. As shown in Fig. 2.13 (b), the fluorescent
localization of amines and nuclei reveals the cell growth on the chiral STF. The nuclei
(red) undergoing division indicates continuing cell growth. Cells adhered well to
neighbors, and 2D biofilm was formed on the STF.
23
Fig. 2.13. (a) AFM image on top surface of a parylene-C chiral STF, and (b) confocal
laser microscope image of COS-7 fibroblast cells grown on a parylene-C chiral STF: Cell
nuclei are shown in red and amines are shown in green [86].
2.4 Concluding Remarks
This chapter began with an introduction to STF growth mechanics. An overview
of polymeric STF growth methods was then provided. A physicochemical vapor
deposition technique was adapted for growing parylene-C STFs and dense thin films for
this dissertation. Modified dense parylene-C thin-film deposition approaches were
discussed, which can enhance the thin-film adhesion to a surface, but maybe unnecessary
for some biomedical applications. The properties of parylene-C and the biomedical
applications of dense thin-film and STFs were also discussed. Fibrous parylene-C STFs
can work as bioscaffolds supporting fibroblast cell attachment and proliferation. Surface
properties have a significant impact on tissue integration. The modifications made to the
surface properties and the corresponding reactions of cells and proteins are discussed in
the following chapters.
24
Chapter 3
Growth Mechanics and Characterizations of Parylene-C STFs
This chapter presents the fabrication details of dense and fibrous parylene-C thin-
film substrates and the effects of deposition conditions on thin-film morphology. It also
presents the surface treatments that can be used to modify the surface wettability of
parylene-C thin-film substrates. The characterization methods for fibrous parylene-C
thin-film substrates are also discussed.
3.1 Physicochemical-Vapor-OAD Method
A physicochemical vapor deposition method has been used for several decades to
make dense, flat, thin films of parylene-C [78]. This technique was modified in order to
allow fibrous thin films with specific morphology to be deposited using the
physicochemical-vapor-OAD method [36]. The modified technique was adopted for this
dissertation.
Figure 3.1 shows the diagram of the deposition system based on PDS2010
equipment (Specialty Coating Systems, Inc., Indianapolis, IN). The parylene-C dimer, the
raw material, is first vaporized at 150 °C and then pyrolyzed at 650 °C. Pyrolysis cleaves
the dimer into two reactive monomers. Then the reactive-monomer flux is directed
through a nozzle onto a glass/silicon slide mounted on a platform whose orientation can
be manipulated dynamically. Room-temperature polymerization of the monomer flux
occurs on the slide.
Two motors are used to dynamically adjust the orientation of the platform holding
the glass/silicon slide. The rotation motor rotates the flat platform about a central axis
25
passing normally through it, and the rocking motor tilts the flat platform about an axis
tangential to it. Computer-controlled dynamic adjustment of the platform orientation is
used to fabricate the parylene-C STFs with different morphologies. A cold trap in the
vacuum system catches the excess monomer before it enters the mechanical pump.
Fig. 3.1. Schematic of the physicochemical vapor deposition system developed at the
Pennsylvania State University [36] to deposit parylene-C thin-film substrates of
customizable morphologies on either glass disks or silicon slides.
26
The photographs of PDS2010 equipment and modified deposition system are
shown in Fig. 3.2. The modified deposition chamber is larger than the one in the
PDS2010 system, in which the nozzle, two motors and the platform are placed.
Fig. 3.2. (a) Photograph of PDS2010 parylene-C deposition chamber. (b) Photograph of
modified deposition chamber. (c) Photograph of facilities inside the chamber.
3.2 Effects of Deposition Conditions
As discussed in Chapter 2, the morphology of STFs is related to deposition
parameters such as substrate motion, vapor incidence angle, and deposition pressure. The
relationship between thin-film morphology and deposition parameters is presented in
more detail in the next section.
27
3.2.1 Effect of Substrate Motion
At the fixed vapor incidence angle v and 26mTorr pressure, fibrous parylene-C
STFs of three different morphologies were fabricated: (i) slanted columnar, (ii) chevronic,
and (iii) chiral. The motion of the platform holding a glass/silicon slide determined the
morphology of the thin-film substrate deposited. For all three morphologies, the rocking
motor was manipulated to ensure that the direction of the reactive-monomer flux was
fixed at v = 10º with respect to the platform plane. The rotating motor was not used for
engineering the slanted columnar morphology, but that motor was abruptly rotated by
180º every 60 s for the chevronic morphology, and continuously rotated at 0.012 rps for
the chiral morphology. Fibrous thin-film substrates of approximately 70 μm thickness
were made. Cross-sectional SEM images of thin-film substrates of all three
morphological types are presented in Fig. 3.3A(a)–(c). These images confirm the fibrous
morphology and the associated porosity, with fibers of cross-sectional diameter ~5 μm.
The fibrous thin-film substrates with the chevronic and the chiral morphologies are
periodic on the ~12 μm scale in the thickness direction. As the fibers are all distinct, these
STFs have porosity on two scales: (i) one on the large scale (~5 μm) in between
neighboring fibers and (ii) the other on the small scale (~50 nm), as previously
demonstrated with AFM images [86]. Top-surface SEM images of the three
morphological types of fibrous thin-film substrates, shown in Figs. 3.3B(a)–(c), reveal
similar top-surface morphologies.
28
Fig. 3.3. A: Cross-sectional SEM images of four morphological types of parylene-C thin-
film substrates: (a) slanted columnar, (b) chevronic, (c) chiral, and (d) flat. B: Top-
surface SEM images of four types of parylene-C thin-film substrates: (a) slanted
columnar, (b) chevronic, (c) chiral, and (d) flat.
29
3.2.2 Effect of Vapor Incidence Angle
The rotating motor was not used and the rocking motor was manipulated for
different vapor incidence angles. The deposition pressure was fixed at 26 mTorr.
A dense thin-film substrate with flat surface was deposited by directing the
reactive-monomer flux normally ( v = 90º) towards the glass/silicon disk. The cross-
sectional and top views are shown in Fig. 3.3. In contrast to the fibrous thin-film
substrates, the flat thin-film substrates are dense and have very little porosity. These flat
substrates were used as controls to assess the efficacy of the fibrous thin-film substrates
of the three different morphologies.
The slanted columnar morphology forms for v ≤ 30º. At lower values of v , the
column inclination angle is lower and the porosity of the thin-film substrate is higher,
which is evident from cross-sectional SEM images shown in Fig. 3.4. At large values of
v , the self-shadowing effect is of minor importance compared with the surface diffusion
in thin-film morphology, and under these conditions, too, it is difficult for columnar
morphology to emerge.
30
Fig. 3.4. Cross-sectional SEM images of fibrous parylene-C thin-film substrates with
slanted columnar morphologies grown at different vapor incidence angles v : (a) 25º, (b)
20º, (c) 15º, and (d) 10º. Deposition pressure was set at 26 mTorr.
3.2.3 Effect of Deposition Pressure
The morphology of a thin-film substrate in relation to deposition pressure was
investigated under vapor incidence angle v = 10º and without substrate motion. As
shown in Fig. 3.5, at a low deposition pressure of 18 mTorr, the slanted columnar
morphology is obscure compared with the morphology at the high deposition pressure of
26 mTorr. At low pressure, the monomer flow rate is lower than at high pressure, as is the
deposition rate. This is because the adatoms have more time to diffuse at low pressure
before more adatoms arrive. Thus, the self-shadowing effect is weak and slanted
columnar morphology is hard to form. The deposited film is thin and dense. Conversely,
31
the monomer flow rate is higher at the high deposition pressure, and the self-shadowing
effect dominates, thereby yielding the slanted columnar morphology.
Fig. 3.5. Cross-sectional SEM images of fibrous thin-film substrates of parylene-C with
slanted columnar morphology ( v =10º) grown at different deposition pressures: (a) 18
mTorr, and (b) 26 mTorr.
3.3 Parylene-C Surface Treatment
As stated in Sec. 1.1, the surface wettability of a solid material influences its
interactions with the cells and tissues. Thus, it is desirable for polymeric materials used in
biomedical applications to have the ability to be tailored to the preferred surface
wettability without changing surface morphology. For this purpose, photo-oxidation and
an oxygen-plasma treatment are most often used in such applications [41, 77].
3.3.1 Surface Wettability
A direct quantification of surface wettability is by the advancing contact angle
(ACA) θ of a water droplet on the surface, which is defined as the maximum angle
32
allowable without increasing the liquid–solid interfacial area by adding volume
dynamically [87]. A hydrophilic surface has the advancing contact angle θ < 65°, and a
surface with θ > 65° is considered hydrophobic [88]. The wettability of a surface is
directly related to surface energy. Materials containing functional groups such as –CH
have a low surface energy, which results in a hydrophobic surface. Conversely, high
surface energy with chemical composition involving C-O, for instance, leads to a
hydrophilic surface.
3.3.2 Photo-Oxidation
Photo-oxidation is conducted by exposing the parylene-C surface to ultraviolet
(UV) light in the presence of oxygen. The oxygen molecules attacks the radical sites
created by UV photoinitiation [77]. Photo-oxidation, however, does not take place on the
parylene-C surface until the UV intensity is higher than a certain value, and different
functional groups are introduced at different UV intensities. At an intensity of 4000
mJ/cm2 with a wavelength of 254 nm, C-O or C=O groups dominate on the surface. At
the higher intensity of 10,000 mJ/cm2 with the same wavelength, O-C=O groups have
been found to appear [77]. The parylene-C surface becomes more hydrophilic at a higher
UV intensity [77]. As the light intensity of lab UV radiometers at 254 nm is around
several mW/cm2 [77, 89], the photo-oxidation process takes up to several hours before the
desired exposure dose is administered. Therefore, this method was not used for the results
reported in this dissertation.
33
3.3.3 Oxygen-Plasma Treatment
The oxygen-plasma treatment was conducted on parylene-C surfaces to acquire
the desired wettability. The oxygen-plasma treatment system Metroline M4L Plasma
Etcher (PVATePla, Corona, CA) was used with the radiofrequency power set at 50 W.
Oxygen flowing at a rate of 50 sccm was applied and the working pressure was 400
mTorr. Surface wettability is expected to increase into hydrophilic with treatment,
introducing functional groups (such as C=O, C-O, and O-C=O) to the surface [41].
Oxygen-plasma treatment is a nondestructive process. The top-surface SEM
images of a 30-µm-thick parylene-C thin film with and without oxygen-plasma treatment
are shown in Fig. 3.6. There is little significant difference in overall surface morphology
and roughness between the results of the two procedures. Compared with photo-oxidation,
oxygen-plasma treatment is much faster and normally takes less than 60 s to achieve the
desired surface wettability [90].
Fig. 3.6. SEM images of the top-surface morphology of a parylene-C chiral STF ( v
=10º, 26 mTorr, 30-µm-thick) (a) before and (b) after oxygen-plasma treatment for 60 s.
34
3.4 Characterization of Fibrous Parylene-C Thin-Film Substrates
A Hitachi S3500N SEM and LEO 1530 field-emission SEM (FESEM) were used
to image top surfaces and cross-sections of the deposited thin-film substrates. For cross-
sectional images, the samples were soaked in liquid nitrogen for 2 min and then cracked
to create a sharp interface. A thin layer of gold was sputtered on the interface before
visualization at 5 kV by SEM/FESEM. Substrates with morphology on the micrometer
scale were also imaged with a Nikon L200ND optical microscope.
Film thickness was initially measured using a cross-sectional SEM/FESEM image.
A KLA-Tencor Alphastep 500 profilometer (KLA-Tencor, Milpitas, CA) was used to
measure the film thickness precisely.
In order to quantify surface wettability, measurements of the ACA [87] were
carried out using the FTA1000 automated contact angle goniometer (First Ten Angstroms,
Portsmouth, VA), which employs the captive water-drop method [91]. Deionized water
drops (~10 μl in volume) were expanded using a motor-driven syringe. The shape of each
drop was recorded by a camera built into the instrument. The advancing contact angle is
defined as the maximum angle allowable without increasing the liquid–solid interfacial
area by adding volume dynamically. The accuracy of this method for measuring the
advancing contact angle has been confirmed as precise to within ±0.5° [92].
3.5 Concluding Remarks
In this chapter, the fabrication details of dense and fibrous parylene-C thin-film
substrates and the effects of deposition conditions on thin-film morphology were
discussed. For fibrous thin-film deposition, the deposition pressure should not be too low
35
and the vapor incidence angle should not be too high. The in-situ control of substrate
rotation and rocking can control the film morphology and porosity.
The surface treatments that can be used to modify the surface wettability of
parylene-C thin-film substrates were also considered. Compared with photo-oxidation,
oxygen-plasma treatment is much faster and for this reason it was selected for this
research. Finally, this chapter also presented characterization methods for fibrous
parylene-C thin-film substrates.
36
Chapter 4
Thickness-Controlled Hydrophobicity of Fibrous Parylene-C Thin-Film Substrates*
This chapter demonstrates a thickness-controlled method to tailor the surface
wettability of a parylene-C thin-film substrate for a specific demand in a biomedical
application. Fibrous parylene-C thin film substrates with slanted columnar morphology
were deposited using the physicochemical-vapor-OAD method. Advancing contact
angles increased from 103.9° (smooth hydrophobic surface) to 168.5° (rough
superhydrophobic surface) as the thickness of the thin-film substrate increased from ~100
nm to ~60 μm, reflecting changes in the top-surface morphology. Oxygen-plasma
treatment can render these fibrous thin-film substrates completely water wettable.
4.1 Fabrication of Thin-Film Substrates
The basic fabrication process for the thin-film substrates is same as described in
Chap. 3. Slanted columnar thin-film (CTF) substrates with different thicknesses were
fabricated on glass slides with vapor incidence angle v = 10° and the deposition
pressure approximately set at 26 mTorr. With the same vapor incidence angle and
pressure, a parylene-C chiral thin-film substrate of 23.0-μm thickness was fabricated with
the rotating motor working at 0.0125 rps. A highly dense and flat parylene-C thin-film
substrate of 15.0-μm thickness was also deposited by setting v = 90° and pressure at 26
mTorr, as a control for comparison with fibrous thin-film substrates.
*This chapter is based in part on the following paper: L. Wei, P. Parhi, E.A. Vogler, T.M.
Ritty, and A. Lakhtakia, ―Thickness-controlled hydrophobicity of fibrous parylene-C
films,‖ Mater. Lett., vol. 64, 2010, pp. 1063–1065.
37
4.2 Surface Morphology of Fibrous Thin-Film Substrates
Top-view SEM images were taken of all fibrous thin-film substrates. The images
of CTF substrates of thicknesses 0.22 μm and 1.29 μm are presented in Figs. 4.1(a) and
(b), respectively. Due to the shadowing effect [51] during OAD, parylene-C nuclei
initially present on the slide and nanoparticles with diameters ranging from 50 nm to 200
nm were formed, as shown in Fig. 4.1(a). The particles are more densely distributed and
even clusters of nanoparticles can be observed in the thicker CTF substrate, as presented
in Fig. 4.1(b). As the thickness increases to 6.0 μm, the columnar morphology emerges
due to self-shadowing, as is evident in Fig. 4.1(c). The columns are separated from each
other and each column is built up of nanoparticles with diameters ranging from 50 nm to
200 nm. As the CTF substrate keeps on growing, both the columnar cross-sectional
dimensions and the inter-columnar spacing continue to increase. In Fig. 4.1(d), the 35.6-
μm-thick CTF substrate comprises nanowires and nanoparticles on average of diameter
100 nm, which are finer than of the thinner CTF substrate. The 63.3-μm-thick CTF
substrate in Fig. 4.1(e) does not have top-surface features significantly different from the
previous CTF substrate, but there is a slight reduction in the columnar diameter leading to
slightly increased roughness.
It is clearly demonstrated that, as the CTF substrate grows thicker, the surface
gets rougher. However, surface roughness increases very slowly beyond a certain
thickness. The CTF substrates possess morphology at two or three length scales, which
enhances surface roughness.
In order to assess the effect of the shape of the column on surface wettability, a
chiral parylene-C thin-film substrate of 23.0-μm thickness was also fabricated, with a
38
top-surface SEM image shown in Fig. 4.1(f). The helical columns are separated from
each other, and the top-surface morphology is multi-scale.
Fig. 4.1. Top-surface SEM images of (a-e) CTF substrate and (f) a chiral STF substrate.
Thicknesses are as follows: (a) 0.22 μm, (b) 1.29 μm, (c) 6.0 μm, (d) 35.6 μm, (e) 63.3
μm, and (f) 23.0 μm.
39
4.3 Surface Wettability of Fibrous Thin Films
4.3.1 Thickness-Controlled Wettability
The advancing contact angle θdense
for the dense and flat control thin-film
substrate was measured as 103.4°, which indicates that bulk parylene-C is a hydrophobic
material with low surface free energy [93]. Values of the ACA θ were then measured for
CTF substrates of different thicknesses. The data presented in Fig. 4.2 show that θ first
increases rapidly with thickness but then begins to level off. This angle is 103.9° for a
100-nm-thick slanted columnar thin-film (CTF) substrate, almost the same value as that
of the control film; then it rises to 159.0° for a 22.0-μm-thick CTF substrate and almost
levels off to 168.5° when the thickness is 63.3 μm.
Fig. 4.2. Measured advancing contact angle θ (black solid curve) and calculated solid
fraction f (red dashed curve) as functions of thickness of a CTF substrate of parylene-C.
The measured values of θ of a flat thin-film substrate (control) and a chiral thin-film
substrate are also marked.
40
4.3.2 Wenzel and Cassie-Baxter models
The behavior of a water droplet on a rough surface is schematically shown in Fig.
4.3. Water can either penetrate into the asperities or be suspended over the asperities.
These two situations have been named as Wenzel [94] and Cassie-Baxter [87] models,
respectively.
Fig. 4.3. Behavior of a water droplet (blue) on a rough surface. Left: Wenzel model; right:
Cassie-Baxter model [93].
Suppose that a solid material exhibits an intrinsic ACA θi and rough surface
having an apparent ACA θ. Also suppose the rough surface has a roughness factor γ and
solid area fraction ƒ (0≤ƒ≤1). Thus for the Wenzel model, the apparent ACA obeys the
relationship: cosθ = γ cos θi [94]. For the Cassie-Baxter model, the relationship is: cosθ =
ƒ(1+cosθi)−1 [87].
41
4.3.3 Surface Wettability Analysis
Since the advancing contact angle on a CTF substrate increases with surface
roughness, the Cassie-Baxter model should be applied. Figure 4.2 contains a plot of ƒ
against the CTF substrate thickness calculated after assuming θi = θdense
. As the CTF
substrate grows thicker, ƒ reduces quickly from unity to a remarkably low value of 0.03.
This shows that the hydrophobic asperities on the topmost surface entrap air and prevent
water wicking into the bulk of the film. Note that the solid area fraction ƒ cannot drop
significantly below 0.03 with increase of thickness.
The advancing contact angle of water on the chiral thin-film substrate of 23.0-μm
thickness is 156.0°, not very different from 159.0° measured for a 22.0-μm-thick CTF
substrate. Thus, the chirality of the columns does not strongly influence overall
wettability, possibly because superhydrophobicity is controlled only by ƒ and θi.
4.3.4 Oxygen-Plasma-Treated Surface Wettability
It was found that, just like a flat thin-film substrate of parylene-C [95], CTF
substrates of the same material can be made hydrophilic by oxygen-plasma treatment. A
22.0-μm-thick CTF substrate was treated with oxygen plasma. During the 30-s treatment,
radiofrequency power was maintained at 50 W, while oxygen flowed at 50 sccm at 400
mTorr pressure. The advancing contact angle on plasma-treated CTF substrates was
evanescent—as shown in the right panel of Fig. 4.4—indicating that brief oxidation
introduced surface functional groups that interacted with water in a way that defeated the
superhydrophobic effect and made the oxygen-plasma-treated CTF substrates completely
42
water wettable. This is the first example of oxygen-plasma-treated fibrous Parylene-C
thin-film.
Fig. 4.4. Left: Photograph of a water droplet on a 35.6-μm-thick CTF substrate showing
θ=165.6°. Right: Photograph of a water spread on the surface of a 22.0-μm-thick CTF
substrate after oxygen-plasma treatment.
4.4 Concluding Remarks
This chapter presented a method of fabricating fibrous thin-film substrates of
parylene-C, using a physicochemical version of the OAD technique. The surface
wettability—as evaluated by advancing contact angle measurements—decreases as a
function of the thickness of the thin-film substrate to an asymptotic value near 170°. The
columnar morphology does not seem to have a significant effect on wettability. Parylene-
C, a polymer proven useful in numerous biomedical application in its dense form, can
thus be made to exhibit a range of wettability characteristics from hydrophobic to
superhydrophobic, merely by changing the thickness of a fibrous thin-film substrate
43
deposited by the OAD technique. Hydrophilicity in these fibrous thin-film substrates can
be induced by oxygen-plasma treatment.
The ability of surface morphology and wettability modification by the described
methods opens up several possibilities. One is that different degrees of cell adhesion and
protein attachment can be achieved, depending on surface properties and the cell/protein
type. Details are presented in Chaps. 5 and 6. The second is the development of a
morphology-composition gradient panel that could be used for high-throughput screening
assays as well as gradient surfaces for optimized growth of tissue, which is presented in
Chap. 6. The third is the fabrication of free-standing thin-film substrates for portability
and implantability of tissue grown ex vivo, as discussed in Chap. 7.
44
Chapter 5
Human Fibroblast Attachment on Fibrous Parylene-C Thin-Film Substrates*
This chapter examines the effects of (i) surface morphology, (ii) surface
wettability, and (iii) the presence of serum proteins on fibroblast cell attachment to
fibrous parylene-C thin-film substrates. The physicochemical-vapor-OAD method
described in Sec. 3.1 was implemented to deposit dense, flat thin-film as well as fibrous
thin-film substrates of three different morphologies: slanted columnar, chevronic, and
chiral. The flat parylene-C thin-film substrate was moderately hydrophobic while the
fibrous ones were superhydrophobic. Oxygen-plasma treatment changed a thin-film
substrate from hydrophobic to superhydrophilic. The attachment efficiency of human
fibroblast cells to the flat and three fibrous thin-film substrates of parylene-C was
investigated. Fibroblast attachment was better on fibrous thin-film substrates than on flat
thin-film substrates, and oxygen-plasma treatment was found to facilitate fibroblast
attachment on all four morphologies. Serum proteins also facilitated cell attachment on
all substrates. The combination of oxygen-plasma treatment and serum proteins increased
fibroblast adhesion in an additive manner on flat, but not on fibrous parylene-C substrates.
The morphology of cells differed between fibrous and flat parylene-C thin-film substrates.
*This chapter is based in part on the following paper: L. Wei, A. Lakhtakia, A.P.
Roopnariane, and T.M. Ritty, ―Human fibroblast attachment on fibrous parylene-C thin-
film substrates,‖ Mater. Sci. Eng. C, vol. 30, 2010, pp. 1252–1259.
45
5.1 Introduction
As discussed in Chap. 1, texturing of polymeric-implant surfaces is known to
significantly influence performance in a biological system. Besides, surface wettability
can also have an influence on cellular interactions with biomaterials. For example, there
is growing awareness in the biomedical-materials community that cellular behavior on a
substrate is influenced by texturing the latter at submicron- and nanometer-length scales
[8-14]. Thus, a desirable biomaterial would be amenable to surface modification for
desired wettability and texturing by specific, controllable design parameters in order to
perform specific biological tasks.
For the research reported in this chapter, fibrous parylene-C thin-film substrates
were fabricated, as discussed in Chap. 4, and the attachment of human fibroblast cells to
the fibrous thin-film substrates of three different morphologies was investigated in
relation to their dense, flat counterparts. Besides, this chapter describes the effects of
altering the surface wettability of parylene-C thin-film substrates on cell adhesion, and
the supplemental role of adhesion-mediating serum proteins coupled with high or low
wettability on fibroblast attachment.
5.2 Thin-film Substrates and Cellular Attachment Assay Design
5.2.1 Cell Culture
Human foreskin fibroblasts, immortalized with hTERT and designated ―BJ-5ta‖,
were purchased (ATCC, Manassas, VA). The cells were cultured in Dulbecco's Modified
Eagle's Medium (DMEM) containing 4mM L-glutamine, 4.5g/L glucose, antibiotics, and
46
10% fetal bovine serum (FBS) at 37 °C in a humidified incubator with 5% CO2. For
experimental use, the fibroblasts were lifted with a 0.25% trypsin / 0.5mM ethylene
diamine tetra acetic acid (EDTA) solution (Gibco/Invitrogen, Grand Island, NY), washed
by centrifugation in DMEM, resuspended in the appropriate cell-culture medium
supplemented either with 10% FBS or with 10% KnockOut SR (Invitrogen, Carlsbad, CA)
serum replacement, and plated on the thin-film substrates.
5.2.2 Cell Attachment Assays
Before being used for cell culture, parylene-C thin-film substrates of all four
morphological types (i.e., three fibrous and one flat of 20-μm-thick fabricated under v =
10º at 26 mTorr) were sterilized by immersion for 1 h in absolute ethanol, and dried in a
sterile laminar-flow hood. Two types of attachment assays were performed. The first type
of attachment assay was designed to assess the effect of fibrous morphology on cellular
attachment. Glass disks, each coated with a parylene-C thin-film substrate of one of four
different morphologies, were placed in 1.5-cm cell-culture wells (24-well plate), and 50K
cells in 500 μl of DMEM with 10% KnockOut SR serum replacement were seeded per
well in non-tissue-culture-treated polystyrene plates that do not facilitate cell adhesion
and are intended for suspension cultures (Thermo Fisher Scientific, Waltham, MA).
KnockOut SR is an FBS substitute that does not contain cellular attachment-facilitating
proteins such as fibronectin and vitronectin. The fibroblasts were allowed to attach for
2 h at 37 °C. Then the medium was removed, unattached fibroblasts were removed by
washing, and the remaining attached cells were quantified by CellTiter-Glo assay
47
(Promega, Madison, WI) according to the manufacturer's protocol, and luminescence was
measured on a Synergy HT (BioTek, Winooski, VT) plate reader with KC4 analysis.
The second type of attachment assay was designed to test the effect of oxygen-
plasma treatment, with or without serum proteins, on cellular attachment to fibrous and
flat parylene-C thin-film substrates. Two hours after 50K cells were seeded in 500 μl of
DMEM containing either 10% FBS or KnockOut SR, the thin-film substrates were
washed to remove unattached cells. The attached cells were quantified by CellTiter-Glo
luminescence assay (Promega, Madison, WI). For all attachment assays, five replicate
samples of each type of parylene-C thin-film substrate were used and the results were
compared against a standard curve. Assays were repeated twice with comparable results.
5.2.3 Oxygen-plasma Treatment of Parylene-C Thin-Film Substrates
A set of parylene-C thin-film substrates of all four morphological types were
treated for surface modification from hydrophobic to hydrophilic. Treatment with oxygen
plasma is a nondestructive process and oxygen-plasma-treated thin films of parylene-C
have hydrophilic surfaces [91, 95]. The oxygen-plasma treatment was applied for 30 s.
Measurements of the advancing contact angle [87] were carried out to quantify surface
wettability.
5.2.4 Fibroblast Attachment Characterization
In order to later acquire SEM images of attached fibroblasts, cells were seeded
onto each of the four morphological types of parylene-C thin-film substrates, grown in
DMEM with 10% KnockOut SR for 24 hr, and then washed 3X in 0.1M Na cacodylate
48
buffer. The thin-film substrates were not treated with oxygen plasma. Fibroblasts were
fixed in 3% gluteraldehyde and 1% paraformaldehyde in 0.1M NaCac for 1 h at room
temperature, and then 2.5 h at 4 °C. The samples were washed extensively in 0.1M
cacodylate buffer and treated with 1% osmium tetroxide in 0.1M cacodylate buffer at
room temperature for 1.5 h. The samples were taken through graded-ethanol dehydration,
critical point dried, sputter-coated with a thin layer of gold, and imaged with SEM.
5.2.5 Fluorescence Microscopy of Fibroblast Attachment
To determine the effects of oxygen-plasma treatment and attachment-mediating
serum proteins on cell adhesion and spreading with time, focus was put only on the dense,
flat thin-film substrates. This choice had to be made because the fibrous thin-film
substrates have a frosty white color due to multiple scattering [36], which makes
fluorescent microscopy of cells impractical. In contrast, flat thin-film substrates are
transparent and do not pose problems due to light scattering.
50K cells were plated onto the flat parylene-C thin-film substrates under one of
the following four conditions for either 2 h or 24 h: (i) no oxygen-plasma treatment and
seeding in DMEM with 10% KnockOut SR (-P-S); (ii) no oxygen-plasma treatment and
seeding in DMEM with 10% FBS (-P+S); (iii) oxygen-plasma treatment and seeding in
DMEM with 10% KnockOut SR (+P-S); (iv) oxygen-plasma treatment and seeding in
DMEM with 10% FBS (+P+S). To fluorescently label the attached cells, the culture
medium was replaced by phosphate-buffered saline containing 10 μm CFDA-SE
(Molecular Probes, Carlsbad, CA) for 10 min at 37 °C. The fibroblasts were then washed
49
and photographed with an UTB190 microscope (Olympus, Center Valley, PA) mounted
with a Spot RT SE digital camera (Diagnostic Instruments, Sterling Heights, MI).
5.3 Results and Discussion
5.3.1 Oxygen-Plasma Treatment and Advancing Contact Angle Measurement
As stated in Sec. 3.3.3, oxygen-plasma treatment was applied to convert the
hydrophobic surfaces of the thin-film substrates into hydrophilic ones. The advancing
contact angle for the flat thin-film substrate was measured as 103.4º, which indicates that
the surface of bulk parylene-C is a moderately hydrophobic material with low free energy.
The advancing contact angle after oxygen-plasma treatment dropped to 30º, which
indicates that the surface became moderately hydrophilic.
Table 5.1 shows that the advancing contact angles for all three types of fibrous
thin-film substrates exceeded 150º before oxygen-plasma treatment; hence, their surfaces
must be considered as superhydrophobic [91]. After oxygen-plasma treatment, the ACA
on any of the fibrous thin-film substrates was evanescent; in other words, these substrates
became completely water wettable with superhydrophilic surfaces and high surface
energy [91].
Thus, the effect of treatment by oxygen plasma is much less on the flat thin-film
substrates than on the fibrous thin-film substrates. This may be due to the larger surface
areas of the fibrous thin-film substrates getting more functional groups during oxygen-
plasma treatment than the much smaller surface areas of the flat thin-film substrates.
50
Table 5.1. Advancing contact angles on parylene-C thin-film substrates of four different
morphological types before oxygen-plasma treatment.
Type of substrate Thickness (µm) ACA
Flat (dense) 18 103.4º
Fibrous: Slanted columnar 22 159º
Fibrous: Chevronic 22 157º
Fibrous: Chiral 22 155º
5.3.2 Influence of Substrate Morphology, Oxygen-plasma Treatment and Serum
Proteins on Fibroblast Attachment to Parylene-C Thin-Film Substrates
The attachment of fibroblast cell to the fibrous thin-film substrates of three
morphological types was assessed against attachment to flat thin-film substrates. The
mean and the standard deviation for five replicate samples of each type is presented in
Fig. 5.1. One factor analysis of variance (ANOVA) was performed to determine the
significance of differences between the numbers of cells attached to any two different
types of substrates. OriginPro 8 (OriginLab, Northampton, MA) was used to perform the
analysis, with the probability p of the difference occurring by statistical accident being <
0.05. A significant difference between fibrous thin-film substrates of any morphological
type on the one hand and flat thin-film substrates on the other hand exists. In the absence
of oxygen-plasma treatment, fibroblasts adhered better to fibrous thin-film substrates of
all three types than to the flat thin-film substrates. As the surfaces of the fibrous thin-film
substrates are considerably rougher than of the flat thin-film substrates, the former
provide more surface area for fibroblast attachment. There is no significant difference in
51
attachment among the three types of fibrous substrates, likely because the surface
morphologies are similar to each other (see Fig. 3.3B). These data are consistent with
previous studies of other materials that show surface roughness to influence cell
attachment [96, 97].
Fig. 5.1. Quantification of fibroblast attachment after 2 h to parylene-C thin-film
substrates of four morphological types. The number of samples of each type is five.
The effects of oxygen-plasma treatment and attachment-mediating serum proteins
on the attachment of fibroblasts to the thin-film substrates were assessed, with results
shown in Fig. 5.2. ANOVA was also performed to statistically evaluate the numbers of
attached cells under all four combinations:
(-P-S) without oxygen-plasma treatment and without serum proteins,
52
(+P-S) with oxygen-plasma treatment and without serum proteins,
(-P+S) without oxygen-plasma treatment and with serum proteins, and
(+P+S) with oxygen-plasma treatment and with serum proteins,
Fig. 5.2. Quantification of fibroblast attachment to flat and slanted columnar parylene-C
thin-film substrates under four conditions. +P: with oxygen-plasma treatment; -P: without
oxygen-plasma treatment; +S: with serum proteins; -S: with serum replacement.
For each type of thin-film substrate, treatment with oxygen plasma significantly
increased cell adhesion (p < 0.05). As discussed in Sec. 5.3.1, oxygen-plasma treatment
alters the surfaces of the flat parylene-C substrates to become moderately hydrophilic,
and the surfaces of fibrous parylene-C substrates to become superhydrophilic. After
53
oxygen-plasma treatment, the net effect for the flat and the fibrous thin-film substrates
was to increase cell adhesion 5.7 and 3.5 fold, respectively. These results are in
agreement with previous studies of cell adhesion to flat, oxygen-plasma-pretreated
parylene-C and poly(dimethylsiloxane) substrates [98, 99]. Similarly, for flat and fibrous
thin-film substrates that had not been pretreated with oxygen plasma (hydrophobic), the
addition of serum proteins during cell adhesion enhanced cell adhesion by factors of 8.0
and 4.1, respectively.
For flat thin-film substrates, the addition of serum proteins significantly increased
cell adhesion on the same substrate; i.e. –P-S versus -P+S and +P-S versus +P+S
(p<0.05). However, among treatments on fibrous thin-film substrates, serum only
increased cell adhesion on non-oxygen-plasma-treated fibrous thin-film substrates. The
use of both serum proteins and oxygen-plasma treatment resulted in a 13.5 fold increase
in cell attachment on flat substrates as compared to flat –P-S substrate conditions. On
fibrous thin-film substrates, cell attachment in the presence of serum proteins to oxygen-
plasma-pretreated substrates increased only 2.3 fold over untreated fibrous substrates in
the absence of serum proteins. Thus, the combination of serum proteins and oxygen-
plasma treatment had a synergistic effect on flat thin-film substrates, but not on fibrous
thin-film substrates. This discrepancy may be indicative of mechanistic differences
between cell attachment to flat versus rough, fibrous substrates.
54
5.3.3 Characterization of Fibroblast Attachment to Flat and Fibrous Thin-Film
Substrates
In order to determine if there are morphological differences in the ways cells
attach to flat and fibrous parylene-C thin-film substrates, the nature of the interactions
was imaged by SEM. Representative images are presented in Fig. 5.3. On each of the
three types of fibrous thin-film substrates, fibroblasts spanned the top contours of the
surfaces and attached by making small point contacts with the fibers, as exemplified by
Figs. 5.3(a)-(d). The attached fibroblasts adopted an elongated, spindle-shaped
morphology. Surprisingly, on fibrous parylene-C thin-film substrates, fibroblasts did not
appear to take advantage of the increased surface per unit area available to them for
attachment or spreading. It may be that the cells were unable to organize their
cytoskeletal elements due to the undulating surface disrupting the geometrical linearity
required for spreading in many directions. In contrast, on the dense and flat parylene-C
thin-film substrates, fibroblasts took on a very thin, flat shape with cell processes
extending in many directions and several cell-cell contacts apparent; see Figs. 5.3(e) and
(f). Under these conditions, many lamellipodiums were visible and extensive filopodium
formation was evident indicating a high degree of cytoskeletal organization.
55
Fig. 5.3. SEM images showing cellular attachment to (a) a fibrous thin-film substrate
with slanted columnar morphology (bar = 20 µm); (b) a fibrous thin-film substrate with
chevronic morphology (bar = 25 µm); (c & d) a fibrous thin-film substrate with chiral
morphology (bars = 20 µm, 5 µm; and (e & f) a flat thin-film substrate (bars = 50 µm, 5
µm). Note that (d) is the magnified version of the rectangular region marked in (c);
images (f) and (e) are similarly related.
5.3.4 Fluorescence Microscopy of Oxygen-plasma and Serum Influence on
Fibroblast Attachment to Parylene-C Thin-Film substrates
To determine how the progression of fibroblast attachment and spreading is
influenced by oxygen-plasma treatment and serum proteins over time, cells were plated
on flat parylene-C thin-film substrates under four experimental conditions and imaged by
fluorescent microscopy. Typical images acquired are presented in Fig. 5.4. Without
oxygen-plasma treatment and in the absence of serum proteins, fibroblast attachment was
56
numerically low after 2 h, and the cells were rounded. After 24 h, a small number of cells
attached and spread, but most remained rounded and not securely attached. The inclusion
of 10% FBS in the cell-culture medium greatly aided attachment (Fig. 5.2) but only
slightly increased spreading on non-oxygen-plasma-pretreated substrates gauged visually
(Fig. 5.4). Oxygen-plasma treatment enhanced attachment and greatly enhanced
fibroblast spreading into elongated spindle shapes by 24 h. The greatest spreading was
observed after 2 h as well as after 24 h by the combination of oxygen-plasma pretreated
substrates and serum proteins in the cell-culture medium.
Fig. 5.4. Fluorescence-microscope images of fibroblasts adhering to flat parylene-C thin-
film substrates for 2 h and 24 h, 20X objective. +P: with oxygen-plasma treatment; -P:
without oxygen-plasma treatment; +S: with serum proteins; -S: with serum substitute.
57
The relationship between surface wettability and cell attachment is not a simple
one. Hydrophilic surfaces can provide better adhesion than hydrophobic ones [100], but
this appears to be specific to the material, as the opposite relationship has also been
demonstrated [16]. Additionally, different cell types can vary in the degree of
hydrophobicity preferred for attachment [20].
The results show that human fibroblasts spread best on oxygen-plasma-pretreated
parylene-C substrates, and little spreading was evident on substrates without oxygen-
plasma treatment. The effects of attachment-mediating serum proteins on cell spreading
were greatest on the hydrophilic oxygen-plasma pretreated parylene-C substrates, and
had little effect on the hydrophobic non-oxygen-plasma pretreated substrates. This
observation is consistent with the fact that fibronectin is absorbed significantly more on
hydrophilic than on hydrophobic surfaces [101, 102].
5.4 Concluding Remarks
For the research reported in this chapter, fibrous parylene-C thin-film substrates
of three different morphological types, along with flat thin-film substrates were fabricated
by physicochemical vapor depositions. Whereas the fibrous thin-film substrates are
porous and their surfaces are superhydrophobic, the flat thin-film substrates are dense and
have moderately hydrophobic surfaces. Treatment with oxygen plasma made the surfaces
of fibrous substrates completely wettable with water, and the surfaces of the flat thin-film
substrates became moderately hydrophilic.
The attachment of human fibroblasts on four morphological types of thin-film
parylene-C substrates was investigated. Cell attachment was significantly better on
58
fibrous than on flat parylene-C thin-film substrates. SEM images showed that the
mechanisms of attachment to fibrous and flat parylene-C substrates differ, as cells formed
small point contacts with the surfaces of the fibrous substrates, but extended broad
pseudopodia on the surfaces of the flat substrates. Fibroblast attachment to both flat and
fibrous parylene-C thin-film substrates was greatly enhanced by oxygen-plasma
treatment. The presence of serum proteins improved fibroblast attachment to all four
morphological types of substrates, but only resulted in enhanced cell spreading on
parylene-C thin-film substrates that had been pretreated with oxygen plasma.
59
Chapter 6
2D Surface Morphology-Composition Gradient Panel for Protein-Binding Assays*
This chapter describes a 2D morphology-composition gradient panel, which has a
gradient in surface morphology at constant chemical composition along its length and a
gradient in surface chemical composition without change of surface morphology in the
orthogonal direction. The panels, which exhibit a 2D gradient in surface wettability, were
fabricated by thermolysis of parylene-C followed by oxygen-plasma treatment for various
times. As a demonstration study, the binding of three different biotinylated serum
proteins (B-IgG, B-A, and BTF) to the 2D panel was investigated. The 2D gradient
panels will facilitate development of optimized binding surfaces for various
biotechnological applications.
*This chapter is based in part on the following paper: L. Wei, E.A. Vogler, T.M. Ritty,
and A. Lakhtakia, ―A 2D surface morphology-composition gradient panel for protein-
binding assays,‖ Mater. Sci. Eng. C, vol. 31, 2011, pp. 1861–1866.
60
6.1 Introduction
Gradient panels are experimental vehicles that multiplex investigations of surface
interactions by exposing a panel of surface properties that vary systematically with
position on the panel to the phenomenon under study. In this way, a multiplicity of
surface interactions are captured simultaneously in a manner that is much more efficient
than sequential preparation of single-test units with a single specific surface property for
one-variable-at-a-time experimentation—which is tedious and time-consuming because
many different samples, each with a different value of the surface variable of interest, are
required.
Gradient panels have been widely used in biotechnology to study protein
adsorption [103] and cell adhesion [104] and are of use in various kinds of high-
throughput screening assays [105]. The technology was introduced by Elwing et al. [106]
who created a gradient in surface energy (water wettability) on glass by exposure to a
concentration gradient of imethyldichlorosilane in xylene. Pitt employed a moving
radiofrequency discharge to fabricate a wettability-gradient panel by increasing oxidation
along the length of a polymer strip [107]. Ueda-Yukoshi et al. engineered a gradient on
poly(vinylene carbonate) by successive wet chemical reactions [108]. Roth et al.
developed a colloidal-silver-nanoparticle gradient panel by drying the colloidal solution
between two walls of different solvent wettability [109]. In all of these efforts, a
continuous change of chemical composition in one direction on the gradient panel was
realized.
Morphology, chemical composition and wettability are important properties of
surfaces. In particular, surface rugosity and chemical composition can combine to
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produce technologically useful properties such as superhydrophobicity [93] or
superoleophobicity [110]. The surface properties of a biomaterial determine the types,
amounts, and conformations of adsorbed proteins [23]. The nature of adsorbed proteins in
turn determines the cellular response to the biomaterial [22], thereby underscoring the
essential role of surface properties in the bioactivity of biomaterials. This chapter reports
herein the design, fabrication, and testing of a 2D gradient panel with mutually
orthogonal gradients in surface morphology and surface chemical composition that can
be used to identify optimal combinations of surface chemical and morphological
characteristics. Parylene-C is thermolyzed onto an array of glass plates in such a way that
a columnar thin-film (CTF) is deposited with surface density and commensurate rugosity
that varies along the length of the panel, thereby creating a wettability gradient that varies
from hydrophobic to superhydrophobic [91]. Subsequent oxygen-plasma treatment of the
surface-morphology gradient changes the wetting characteristics in relation to the
treatment time in a way that permits systematic variation of the CTF wettability gradient
over a 120-deg range of the advancing water contact angle. As a demonstration study,
this 2D wettability-gradient panel was deployed in the study of protein binding using
immunoglobulin G (B-IgG, 160 kDa), albumin (B-A, 66 kDa), and transferrin (B-TF, 76
kDa).
6.2 Gradient Panels Fabrication and Characterization Method
6.2.1 Fabrication of 1D Surface Morphology Gradient Panels
A physicochemical vapor deposition technique, used for several decades to make
flat, dense thin-film substrates of parylene-C for implantable devices [20, 37, 40] was
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modified to deposit fibrous thin-film substrates with specific volumetric morphology [36,
91, 111]. In this technique, as discussed in detail in Sec. 3.1, the raw material is parylene-
C dimer (SCS Coatings, Indianapolis, IN). This material is first vaporized at 150 ºC and
then thermolyzed at ~650 ºC so that each dimer is cleaved into two reactive monomers. A
vapor comprising reactive monomers is then directed through a nozzle onto a flat
platform on which a specially prepared array of glass plates is mounted to fabricate the
gradient panel. Room-temperature polymerization of the collimated reactive-monomer
flux occurs on the exposed surface.
Fig. 6.1. Schematic of the fabrication method for a panel comprising 3 rows of 16 3mm
3mm plates of glass placed adjacent to each other with virtually no space between
neighbors. The x axis begins at the proximal end.
The platform must be held stationary for a CTF to be deposited. As shown in Fig.
6.1, the reactive-monomer flux was directed at an angle v = 10 deg with respect to the
platform plane. 3mm 3mm square plates of glass were assembled together on a 16 3
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lattice as a 48mm 9mm rectangular array. This configuration was chosen mainly for
ease of conducting protein-binding experiments. The rectangular array was mounted on
the platform with its long axis, identified as the x axis in Fig. 6.1, parallel to the
projection of the direction of the reactive-monomer flux on the platform plane, and
deposition took place at a pressure of approximately 26 mTorr for 10 min. As a result,
CTFs of parylene-C [91, 111] were formed on each of the 48 square plates of glass, the
plates close to the proximal end (x = 0 mm) of the rectangular array having thicker CTFs
than those close to the distal end (x = 48 mm).
The CTF-thickness gradient along the x axis on the rectangular panel yielded a
lengthwise gradient in surface morphology [91], as discussed later in Sec. 6.3.1, which
therefore exhibits a lengthwise gradient in surface hydrophobicity [91], as discussed later
in Sec. 6.3.2.
6.2.2 Fabrication of 2D Surface Morphology-Composition Gradient Panels
Oxygen-plasma treatment is a nondestructive process to introduce functional
groups that interact with water to overcome the inherent hydrophobicity of many
polymers. Oxygen-plasma-treated dense thin-film substrates of parylene-C have
hydrophilic surfaces [41], and so do their CTF counterparts [91].
As shown in Fig. 6.1, each morphology-gradient panel consists of three rows of
16 square plates of glass. Two such panels (six rows altogether) were fabricated. One row
was not treated with oxygen plasma, but each of the remaining five rows was separately
treated with oxygen plasma for a specific duration t. The chosen values of t are 0s, 5s, 10s,
20s, 40s and 60s. The oxygen-plasma-treatment system Metroline M4L Plasma Etcher
64
(PVATePla, Corona, CA) was used with the radiofrequency power set at 50 W, while
oxygen flow was maintained at 50 sccm with the chamber pressure set at 400 mTorr.
Surface wettability is expected to increase with t, as longer treatment introduces
more functional groups (such as C=O, C-O, and O-C=O) to the surface [41]. Thus, a 2D
surface morphology-composition panel comprising six rows of 16 square plates each was
assembled to function as a 2D wettability gradient panel.
6.2.3 Characterization Methods of 2D Wettability Gradient Panels
A Hitachi S3500 SEM was used to qualitatively evaluate the surface of a 1D
surface morphology gradient panel. A thin layer of gold was sputtered on the surface
before visualization at 5 kV by the SEM. The CTF thickness was measured by a Tencor
500 profilometer.
For surface wettability measurements, linear arrays of 3mm 3mm square pieces
from the proximal to the distal were re-assembled without intervening spaces. The
surface wettability was quantified with respect to surface morphology by measuring the
advancing contact angle θ [87], which is defined as the maximum angle allowable
without increasing the water-solid interfacial area by adding volume dynamically, at
fixed distances x from the proximal end. As different linear arrays had been subjected to
oxygen-plasma treatment for different durations t, the surface wettability was thus
additionally quantified with respect to surface chemical composition. Measurements of θ
were carried out using the TechDirect FTA1000 automated contact-angle goniometer that
employs the captive water-drop method [91]. For each set of experimental conditions,
measurement of the ACA was repeated three times and the mean value was calculated.
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6.3 2D Surface Morphology-Composition Gradient Panel
6.3.1 Morphology Gradient Along The x Axis
In order to create a 2D wettability gradient panel, thermolyzed parylene-C is
made to flow along the length of a rectangular array of 3mm 3mm glass plates
illustrated in Fig. 6.1. Because the flow rate of the reactive-monomer flux decreases
along the x axis from the proximal end to the distal end, a gradient of CTF thickness is
created along the length of the rectangular panel, as quantified in Fig. 6.2.
Fig. 6.2. CTF thickness as a function of the distance x from the proximal end. The
standard deviations are so small that the error bars are not easily discernible.
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At higher flow rates, close to the proximal end, the deposition rates are higher and
a self-shadowing effect results in a columnar morphology [91]. Further from the proximal
end, the flow rate lessens and so does the deposition rate, self-shadowing becomes less
important, and the CTF becomes progressively thinner and smoother. In this manner, the
gradient in CTF thickness results in a gradient in surface morphology. It has been shown
in Chap. 4 [91] that thicker CTFs have rougher surfaces, meaning that the rectangular
array has a rougher coating of parylene-C at its proximal end compared to its distal end,
resulting in a visually apparent gradient in thickness and roughness. As shown in Fig. 6.3,
the CTF appearance changes gradually from frosty white at the proximal end to
completely transparent at the distal end. SEM images shown in Fig. 6.4 confirms that the
frosty white coating is rougher than the transparent coating with a variation along the
length of the rectangular panel.
Fig. 6.3. Optical image of one row of 16 square plates coated with parylene-C. A
thickness gradient is evident as a transparency gradient.
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Fig. 6.4. Top-view SEM images of the CTF at different distances from proximal end: (a)
x = 4 mm, (b) x = 12 mm, (c) x = 20 mm, (d) x = 28 mm, (e) x = 36 mm, and (f) x = 44
mm. These images were taken before oxygen-plasma treatment, which effects a change in
surface chemical composition but not in surface rugosity.
6.3.2 Wettability Gradient Along The x Axis (before oxygen-plasma treatment)
A dense smooth thin-film of parylene-C exhibits an advancing contact angle θ =
103.4º, consistent with the surface chemistry of a saturated hydrocarbon [91]. Rough
hydrophobic materials can exhibit the phenomenon of superhydrophobicity wherein the
observed advancing contact angle rises as the surface becomes rougher due to air trapped
68
within the interstices of the rough surface [87]. The water droplet tends to suspend over
the interstices, as shown in Fig. 4.3, according to the Cassie-Baxter model [87]. The
composite ACA of water on air and water on the hydrophobic surface can be much
higher than ordinarily observed on smooth hydrophobic surfaces due to this effect, with
observed advancing water contact angles exceeding 160º [112-114]. As a consequence of
these factors, the gradient in thickness and rugosity along the x axis results in a gradient
in water wettability, ranging from superhydrophobic at the proximal end to
conventionally hydrophobic at the distal end of the rectangular panel.
Figure 6.5 is a summary graphic that shows the combined effects of surface
rugosity and surface chemical composition (described in Sec. 6.3.3) on water wettability
by plotting the advancing contact angle θ against position x in the panel (Fig. 6.1) and the
oxygen-plasma treatment duration t. Measured value of θ for different values of x and t
are organized as iso-x contours (labeled X1-X1 to X9-X9) and as iso-t contours (labeled
as T1-T1 to T5-T5).
The effect of surface rugosity in the absence of oxygen-plasma treatment is
quantified on the back wall of the graphic in Fig. 6.5 as the iso-t contour T1-T1 (t = 0 s),
with annotations X1 (x ≈ 0 mm) to X9 (x ≈ 48 mm) marking positions along the length of
the rectangular panel. Close to the proximal end, θ = 160º (X1) is consistent with the
superhydrophobic effect. Close to the distal end, θ = 110º (X9) indicates conventional
hydrophobicity. Advancing water contact angles increased linearly between X1 and X9
on the iso-t contour T1-T1, smoothly spanning the conventional hydrophobic to the
superhydrophobic wetting regimes.
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Fig. 6.5. Measured mean values of θ as function of x and t. Data for t = 60 s are close to
those for t = 40 s, and therefore have not been shown. The standard deviation, being less
than 1.1º at any (x,t), has not been shown because of the complexity of the figure. Zones
I-III are discussed in the text.
6.3.3 Analysis on 2D Wettability Gradient Panel
Brief oxygen-plasma treatment increased the hydrophilicity of the CTF surfaces
described above. The iso-x contour X9-X9 along the θ-wall of Fig. 6.5 corresponds to the
wetting of a conventionally hydrophobic CTF at different levels of surface oxidation.
Interestingly, the effect of surface oxidation was most pronounced for the roughest
surfaces at the superhydrophobic proximal end of the rectangular panel (iso-x contours
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X1-X1 to X6-X6) compared to the relatively smoother surfaces at the distal end of the
rectangular panel (X7-X7 to X9-X9). In fact, the superhydrophobic effect was effectively
destroyed by even the briefest plasma treatment (t = 5 s), causing the ACA θ on the iso-t
contour T2-T2 to fall below the 120º threshold typically associated with conventional
hydrophobicity. The profile of the iso-t contour T5-T5 (t = 40 s) indicates that nearly
complete wetting (θ → 20º) was obtained after a sufficiently long oxygen-plasma
treatment for the roughest surfaces denoted by X1 and X2, with increasing θ at any
particular plasma treatment time as surfaces became smoother from the proximal end to
the distal end; furthermore, data for t = 60 s is indistinguishable from that for t = 40 s,
indicating a self-limiting mechanism in surface oxidation. These effects resulted in a
ridge that goes from the location (X6, T2) to the location (X8, T5) on the hypothetical 2D
gradient surface formed by the iso-x and the iso-t contours. The hypothetical surface is
partitioned into three regions identified as I, II, and III, and also shaded differently for
easy identification, in Fig. 6.5.
It is suspected that this interesting trend in water-wetting properties is due to
water penetration (wicking) into the rough CTF surface. Oxygen-plasma treatment causes
oxidation at every level of morphology, penetrating deep into the interstices between the
parylene-C columns shown in Fig. 6.4. Water wicks into these interstices, displacing air
that would normally give rise to the superhydrophobic effect, inducing enhanced wetting
for the roughest CTF surfaces (X1-X1 to X6-X6). This wicking effect decreases as the
CTF surfaces become smoother along the x axis, resulting in a peak where the wetting
regime changes from wicking-dominated to conventional wetting (X6-X6 to X9-X9).
Region I in Fig. 6.5 corresponds to a decreasing wicking effect as the CTF surface
71
becomes smoother, region II corresponds to an increasing wicking effect highly
influenced by surface rugosity, and region III corresponds to wicking-dominated wetting
regime (see axes annotations in Fig. 6.5).
The net effect of superhydrophobicity and wicking creates the hypothetical
surface illustrated in Fig. 6.5 that varies these two phenomenons orthogonally along the x
and the t axes, respectively. This hypothetical surface was physically realized by
assembling glass plates shown in Fig. 6.3 side-by-side in progressive order of oxygen-
plasma treatment time. Even higher resolution is possibly with larger numbers of smaller
square plates. Alternatively, the moving discharge method invented by Pitt [107] could be
used to impart the gradient in surface oxidation, or different mask technologies might be
used to generate a similar effect.
6.4 Protein-Binding Test to Gradient Panel
6.4.1 Serum Protein-Binding Assay Design
B-IgG (Sigma, St. Louis, MO), B-A (Sigma, St. Louis, MO), and B-TF (Sigma,
St. Louis, MO) were chosen for a study to demonstrate the efficacy of the 2D wettability
gradient panels for protein binding, because the non-biotinylated versions of these three
proteins are present in very high concentrations in human serum.
In a manner paralleling that described for surface wettability measurements, rows
of 16 square plates aligned along the x axis were coated with parylene-C. For each of the
three proteins tested, a total of ten replicate rows were fabricated. Five rows underwent
oxygen-plasma treatment for 60 s, and the other five rows were untreated. Each square
plate with specific surface chemical composition and surface topography had five
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replicates. Thus, measurement of protein binding was carried out for each protein using
five replicate surfaces for each combination of surface morphology and surface chemical
composition (oxygen-plasma treatment). Since proteins bind easily with glass, the back
surfaces of all square plates were coated three times with silane [115], and this step
reduced protein binding to negligible amounts.
Tests were carried out in 96-well plates that had been pretreated with 200 μl of
1mg/ml of bovine serum albumin (BSA) (Sigma, St. Louis, MO) in tris-buffered saline
(TBS) (Sigma, St. Louis, MO) for 30 min at room temperature to block binding of the
protein under study to the walls of the wells. Each parylene-C-coated square plate was
placed in a separate well, 100 μl/well of a 200 μg/ml protein solution (B-IgG/B-A/B-TF)
was added, and the square plate was incubated in a humidified environment at 37 ºC for
60 min. Fluid was aspirated from the well and washed thrice with 1mg/ml BSA in TBS to
remove unbound proteins. Serum proteins bound to the square plate were detected by the
addition of 100 μl/well 2.0u/ml streptavidin-alkaline phosphatase (SAP) (Pierce,
Rockford, IL) reporter molecule and incubation at 37 ºC for 30 min. All square plates
were washed as before to remove unbound protein, and then transferred to new 96-well
plates where 200 μl of 1.0 mg/ml phosphatase substrate (PS) (Sigma, St. Louis, MO) was
added and incubated at 37 ºC for 30 min. PS was cleaved by alkaline phosphatase to
produce a soluble end product that was quantified spectrophotometrically at 405 nm
wavelength on a MRX Revelation Microplate Reader (Dynex, Chantilly, VA).
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6.4.2 Result of Protein Binding to The 2D Wettability Gradient Panel
The relative amounts of three biotinylated serum proteins bound to the 2D
wettability gradient panel described above, were measured using the spectrophotometric
assay described in Sec. 6.4.1. This assay measures color development (or spectral
absorbance) that is proportional to the amount of protein bound to a square plate shown
in Fig. 6.1 after rinsing away any weakly bound protein. It should be noticed that this
method was not intended to measure total adsorption from solution but rather only the
amount of protein that becomes bound to the surface through any number of possible
mechanisms including entrapment and chemical interactions dependent upon the amount
of charge imparted on the parylene-C by the oxygen-plasma treatment and the unique
surface charge profile of each protein as determined by its amino-acid composition.
Normalized absorbance—shown with respect to x in Figs. 6.6, 6.7, and 6.8 for B-IgG, B-
A, and B-TF, respectively, for both t = 0 s and 60 s—was not calibrated to moles or mass
of protein bound per unit area and absolute adsorbent capacity was not measured.
Furthermore, normalized absorbance for different proteins cannot be quantitatively
compared because each protein exhibits different levels of biotinylation which affects
rate and intensity of color development in solution. Instead, the assay quantifies the
relative amount of a particular protein bound to the 2D gradient panel as a function of
surface morphology (rugosity) and surface chemical composition (oxidation) in a way
that permits general trends for a particular protein and among different proteins to be
compared.
Inspection of Figs. 6.6-6.8 reveals that more protein was bound to the rougher
surfaces on the gradient panel (x → 0 mm) than to the smoother surfaces (x → 48 mm).
74
This trend became less pronounced when the gradient panel was treated with oxygen
plasma for t = 60 s. On focusing on Fig. 6.6 corresponding to B-IgG for which the most
data was collected, it can be seen that the half-maximum of the sigmoid-like protein-
binding trend occurred near the X4 position of Fig. 6.5, rising sharply as the surfaces
became rougher and maximizing at the maximum in the superhydrophobicity trend (X1-
X1). Half as much protein was bound at surfaces smoother than on the iso-x contour X6-
X6. More protein was bound to oxygen-plasma-treated surfaces with a minimum that was
nearly twice greater than untreated CTF. Qualitatively similar trends can be gleaned from
Figs. 6.7 and 6.8 for B-A and B-TF, respectively.
Fig. 6.6. Normalized absorbance with respect to x of B-IgG bound to a wettability
gradient panel, for t = 0 s (squares) and t = 60 s (circles), where t is the duration of
oxygen-plasma treatment. The measured absorbances were normalized with respect to the
highest value measured.
75
Fig. 6.7. Same as Fig. 6.6, but for B-A.
Fig. 6.8. Same as Fig. 6.6, but for B-TF.
76
The protein-binding trend observed is consistent with a physical entrapment
mechanism in which the protein intercalates into the porous structure of rough surfaces,
with more protein becoming entrapped in the pores of the roughest surfaces. Oxygen-
plasma treatment enhances this effect by wicking the protein solution into the pores
where it is prevented from being washed away in surface-rinsing steps.
6.5 Concluding Remarks
This chapter described a facile technique devised to fabricate a 2D surface
morphology-composition gradient panel using the physicochemical-vapor-OAD method
and subsequent oxygen-plasma treatment that takes advantage of three wetting regimes:
conventional hydrophobicity, superhydrophobicity, and wicking. A protein-binding
demonstration study showed that surface rugosity and surface chemical composition
control the amount of protein that can be bound to the surface. It is expected that these
2D wettability gradient panels shall be useful in various biotechnical applications,
especially high-throughput screening assays.
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Chapter 7
Free-Standing, Fibrous Thin-Film Substrates of Parylene-C for Cellular
Attachment and Growth*
Two approaches for fabricating free-standing, fibrous thin-film substrates of
parylene-C using physicochemical-vapor-OAD of parylene-C on glass/silicon slides were
devised for this research. Compared with the first approach that is based on using PMMA
as a sacrificial layer, the second approach, using soap solution to replace PMMA layer, is
preferable as it does not require the use of potentially harmful chemicals. The thin-film
substrates are engineered with different volumetric fibrous morphologies, range from 100
nm to 100 μm in thickness, and have top-surface texture on nanometer- to micrometer-
length scales. In the soap-solution approach, the bottom surfaces of the thin-film
substrates are morphological negatives of the top surfaces of the soap coatings, which can
assist in integration with implant surfaces. The attachment and growth of human
osteosarcoma cells on the top surfaces of thin-film substrates is demonstrated in this
chapter.
*This chapter is based in part on the following two papers:
1. A. Lakhtakia, L. Wei, A. Kumar, and J. Kumar, ―Facile fabrication of free standing
submicron textured films of Parylene-C,‖ Mater. Res. Innovat., vol. 15, 2011, pp. 1–3.
2. L. Wei, L. Leo, T.M. Ritty, and A. Lakhtakia, ―Nano/Micro-textured, free-standing,
thin-film substrates of parylene-C for cellular attachment and growth,‖ Mater. Res.
Innovat.,(Accepted)
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7.1 Introduction
Compared with fibrous thin-film substrates attached to rigid slides (such as glass),
sufficiently flexible, free-standing, fibrous thin-film substrates are very desirable to
conformally cover implant surfaces. Also, tissue grown ex vivo on a free-standing flexible
substrate could be implanted as an integrated assembly on a nonplanar surface inside a
human body. These thoughts initiated the physicochemical-vapor-OAD of parylene-C
thin-film substrates with different fibrous volumetric morphologies on slides with special
surface treatments, with separation being carried afterwards [116, 117]. Two approaches
were devised.
In the first approach, a glass slide is coated with polymethyl methacrylate
(PMMA) as a sacrificial layer and a parylene-C thin-film substrate is deposited thereon,
with separation being carried later by immersion in chloroform [116]. This procedure
yields free-standing, thin-film substrates of parylene-C that are less than 100 μm in
thickness and have surface texture on at least the submicron length scale. However, as
residual chloroform can be potentially harmful, an alternative approach is definitely
desirable.
In the second approach, soap solution is spin-coated on a flat, polished silicon
slide; the soap-solution-coated slide is then baked; a thermal-release frame is applied; and
a parylene-C thin-film substrate of fibrous volumetric morphology and thickness between
100 nm and 100 μm is deposited thereon. A free-standing thin-film substrate of parylene-
C with thermal-release frame is peeled off the soap-solution-coated silicon slide.
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7.2 Approach 1: PMMA-Assisted Fabrication of Free-Standing Parylene-C Thin-
Film Substrates
7.2.1 PMMA Sacrificial Layer
In the first approach, a glass slide used to deposit a submicron-textured, fibrous
thin-film substrate of parylene-C is specially prepared as follows. PMMA of molecular
weight 15,000 dalton is dissolved in chloroform and the solution is spin coated on a
microscope glass slide, after the slide has been first cleaned with acetone and then dried
with nitrogen. The thickness of the PMMA sacrificial layers thus produced were
measured on different spots as 850±250 nm, using a DekTak surface profiler (Veeco Inc.,
Plainview, NY).
7.2.2 Deposition of Parylene-C Thin-Film Substrate
The physicochemical-vapor-OAD method [36] was applied for depositing the
submicron-textured, fibrous thin-film substrate of parylene-C on the PMMA-coated glass
slide. Three different morphological types of submicron-textured parylene-C thin film
substrates were fabricated: chiral, slanted columnar and chevronic. For all three types, the
direction of the reactive-monomer flux was fixed at v = 10º with respect to the platform
plane [111].
Parylene-C thin-film substrates between 10 μm and 100 μm in thickness were
produced. An SEM was used to image the cross-sections of the deposited films. Finally,
the parylene-C thin-film substrates were separated from the PMMA-coated glass slides
by immersion for 12 h in chloroform, which dissolves PMMA but not parylene-C. If
80
desired, faster dissolution of the release layer is facilitated by choosing polymers of lower
molecular weight and solvents. Finally, the thin-film substrates were repeatedly washed
with deionized water to remove residual chloroform.
7.2.3 Parylene-C Thin-film Substrate
Cross-sectional SEM images of the three morphological types of parylene-C thin-
film substrates deposited on the PMMA-coated glass slides are provided in Fig. 7.1. The
lowest layer in each image is the glass slide and the top layer is the textured, fibrous thin-
film substrate of parylene-C. The middle layer is the PMMA sacrificial layer. The image
of the thin-film substrate with chiral morphology in Fig. 7.1(a) shows that the helixes of
parylene-C are highly intertwined. The slanted columnar morphology is clearly evident in
Fig. 7.1(b), whereas the chevronic morphology in Fig. 7.1(c) has a period of ~12 μm in
the thickness direction. The texture is on at least the submicron scale, as is evident from
the high magnification image presented in Fig. 7.1(d). These images also make it clear
that the parylene-C thin-film substrates produced are not dense solid films; instead, they
are porous and possess a fibrous volumetric morphology.
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Fig. 7.1. Cross-sectional SEM images of three morphological types of parylene-C thin-
film substrates on PMMA-coated glass slides: (a) chiral, (b) slanted columnar, (c)
chevronic, and (d) magnified version of chevronic.
All three types of submicron-textured, fibrous thin-film substrates of parylene-C
on PMMA-coated glass slides were soaked in chloroform. Complete delamination
occurred in 12 h. The submicron-textured thin-film substrates separated from the glass
slides are shown floating in acetone in the optical image presented in Fig. 7.2. The chiral
and the columnar thin-film substrates are opaque and white in color, whereas the
chevronic thin-film substrate is quite transparent.
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Fig. 7.2. Photograph of free-standing, fibrous thin-film substrates of parylene-C separated
from glass slides: ―D-film‖ indicates separated thin-film and ―S‖ indicates glass slide.
Figure 7.1 also shows that a submicron-textured, fibrous thin-film substrate of
parylene-C has a transitional layer above the PMMA sacrificial layer. Lacking the texture
that emerges later with increased thickness, the transitional layer keeps the parylene-C
thin-film substrate robust after separation from the glass slide. Between 10 μm and 100
μm in thickness, the separated thin-film can be handled easily.
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7.3 Approach 2: Soap Solution-Assisted Fabrication of Free-Standing Parylene-C
Thin-Film Substrates
The devised approach is simple and easy to implement. However, as the free-
standing thin-film substrate is to be used to grow cells, any residual chloroform can be
potentially harmful. Therefore, an alternative approach that does not require the use of
potentially harmful chemicals was devised.
The second approach is a simple and inexpensive three-step process. As
schematically presented in Fig. 7.3, in the first step, a polished silicon slide is prepared in
a specific way; the second step is the physicochemical-vapor-OAD of the thin-film
substrate; in the third step, the thin-film substrate is released from the specially prepared
silicon slide.
Fig. 7.3. Top: Schematic of the three-step process to fabricate nano/micro-textured, free-
standing, fibrous thin-film substrate of parylene-C. Inset: vapor incidence angle v in
Step 2.
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7.3.1 Step 1: Soap-Solution-Assisted Surface Treatment
Soap solution has shown the ability to serve as a release agent for dense thin films
of parylene from solid surfaces [118, 119]. The same treatment could be suitable for
fibrous parylene-C thin-film substrates.
The silicon slides used to deposit parylene-C thin-film substrates were specially
prepared as follows. A 15mm 15mm slide of polished silicon was first cleaned with
acetone for 10 min and then with isopropyl alcohol for 10 min. Both operations were
carried out in a 3510 Branson ultrasonic cleaner (American Airworks, Sophia, WV).
After dehydration on a hot plate at 220 ºC for 5 min, the silicon slide was spin-coated
with 2% Micro-90 soap solution (VWR International, Radnor, PA) at 4000 rpm for 45 s.
Thereafter, the coating of soap solution was baked for 2 min at 100 ºC, 150 ºC, 200 ºC,
250 ºC or 300 ºC; for comparison, unbaked samples were also prepared.
Representative optical images of the top surfaces of unbaked as well as baked
coatings of soap on silicon are presented in Fig. 7.4.
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Fig. 7.4. Optical microscope images of the top surfaces of unbaked as well as baked
coatings of soap on silicon slide. (a) Without baking; (b-f) baking temperature: (b) 100 ºC,
(c) 150 ºC, (d) 200 ºC, (e) 250 ºC, and (f) 300 ºC.
The variation in colors represents the variation in thickness of the soap coatings.
Without baking, the soap coating has a connected-filamentary texture, as shown in Fig.
7.4(a). As the boiling temperature of soap solution is around 100 ºC [120], the solvent in
the soap-solution layer is partially driven out on baking for a short duration. The leftover
86
coating of soap solution has a texture that depends on the baking temperature. When the
baking temperature ranges between 100 ºC and 150 ºC, a discotic texture with disk
diameters from 50 μm to 100 μm shows up, as presented in Figs. 7.4(b) and (c). Smaller
disks diminish in number as the baking temperature is raised to 150 ºC, but increase again
when the baking temperature is raised to 200 ºC, as is evident in Fig. 7.4(d). At higher
baking temperatures, the soap coating becomes discontinuous, as shown in Figs. 7.4(e)
and (f). The leftover solvent is minimal when the baking temperature is 300 ºC.
7.3.2 Step 2: Deposition of Parylene-C Thin-Film Substrates
After the silicon slide was coated with soap solution and baking treatment was
applied, a square frame of thermal-release tape (Semiconductor Equipment, Moorpark,
CA) was attached on the edge of the silicon slide (inside square 10 mm 10 mm/outside
square 20mm 20mm). The thermal-release tape adheres firmly to the slide at room
temperature and easily peels off when heated to 95 ºC.
A fibrous thin-film substrate of parylene-C was deposited on the silicon slide
treated with soap solution and then framed with thermal-release tape. The fabrication
process used is the physicochemical-vapor-OAD method presented in Sec. 3.1 [36].
The direction of the reactive-monomer flux was fixed at v = 10º with respect to
the platform plane. Three different morphological types of fibrous thin-film substrates of
parylene-C were deposited: (i) slanted columnar, (ii) chevronic, and (iii) chiral [111]. Flat
thin-film substrate of parylene-C were deposited by directing the reactive-monomer flux
normally ( v = 90º) towards the platform plane [111].
87
Cross-sectional FESEM images of the fibrous parylene-C thin-film substrates on
soap-coated silicon slides are presented in Fig. 7.5. The spin-coated soap was not baked
for these samples. The thinnest thin film had a slanted columnar morphology and 100-nm
thickness, as shown in Fig. 7.5(a). The bottom layer in the image is the silicon slide, the
middle layer (brighter) is the soap coating of 40 nm thickness (as measured from the
FESEM image), and the top layer (darker) is the parylene-C thin-film substrate. This
thin-film substrate is continuous and quite dense, resulting from the diffusion of the
reactive monomer flux during initial deposition. This diffused deposition makes the free-
standing, fibrous thin-film substrates robust for mechanical handling. The top-view image
in Fig. 7.5(b) of the same thin-film substrate shows that the top surface is textured at the
nanometer length scale.
As the parylene-C thin-film substrate grows thicker, the surface roughness
increases to the micrometer scale, as has been shown in Chap. 4 [17]. The thickest
substrate deposited was 100-μm thick and engineered with a slanted columnar
morphology, as is evident from the cross-sectional image in Fig. 7.5(c). The
corresponding top-view image in Fig. 7.5(d) shows that the top surface is rougher with
texture on the micrometer length scale.
Top-view images of parylene-C thin-film substrates with chevronic and chiral
morphologies are shown in Figs. 7.5(e) and (f), respectively. Their thicknesses are 75 μm
and 85 μm, respectively, and their top surfaces exhibit similar roughness as the 100-μm-
thick thin-film-substrate shown in Fig. 7.5(d). Clearly, the fibrous volumetric
morphology of the parylene-C thin-film substrate is not influenced by whether or not the
soap coating is baked.
88
Fig. 7.5. FESEM images of (a, c) cross-sectional and (b, d) top views of parylene-C thin-
film substrates with slanted columnar morphology and of thickness (a, b) 100 nm and (c,
d) 100 μm. FESEM images of top views of parylene-C thin-film substrates with (e)
chevronic morphology and 75-μm thickness and (f) chiral morphology and 85-μm
thickness.
Some framed thin-film substrates were subjected to oxygen-plasma treatment for
30 s to enable top-surface modification from hydrophobic to hydrophilic.
89
7.3.3 Step 3: Thermal Release of Parylene-C Thin-Film Substrate
Each specially prepared slide with the parylene-C thin-film substrate deposited
thereon was placed on a hot plate at 95 ºC, at which temperature the thermal-release
frame peeled off. Then the parylene-C thin-film substrate was separated from the soap-
coated slide by simply lifting off the frame. The framed thin-film substrate was
repeatedly washed with deionized water to remove any residual soap.
Optical microscope images of the bottom surfaces of the thin-film substrates are
shown in Fig. 7.6. Clearly, these images are morphological negatives of the images in Fig.
7.4. This conformal relationship has been previously observed with inorganic thin films
of columnar morphology deposited on nonplanar substrates [121], and has been exploited
for bioreplication [122] as well as solid-state acquisition of fingerprints [123].
Caution is necessary when lifting off a framed thin-film substrate from the soap-
coated slide that has been baked at a high temperature. Since very little solvent is left
after high-temperature baking, the release is non-uniform which creates mechanical stress
in the thin-film substrate, as shown in Figs. 7.6(e) and (f) by black spots indicating
wrinkles.
90
Fig. 7.6. Same as Fig. 7.4, except the images are of the bottom surfaces of the released
parylene-C thin-film substrates of slanted columnar morphology and 500-nm thickness.
Baking of the soap-solution coating allows engineering of the bottom-surface
morphology of the thin-film substrate. If the aim is to glue the thin-film substrate to an
implant surface, the back-surface texture would enhance the contact area, thereby
91
improving adhesion and preventing delamination. If the aim is to spread the thin-film
substrate on healthy tissue in vivo, the back-surface texture would promote integration
with the healthy tissue because surface texture on micrometer- and submicron-length
scales is known to influence cellular growth [10, 11]. Thus, the thin-film substrates can
be better integrated with the engineered surfaces of implants as well as the tissue in vivo.
Figure 7.7(a) presents a photograph of a parylene-C thin-film on a framed but not
soap-coated slide. The thin-film has a dense morphology ( v = 90º) and is 500-nm thick.
Clearly, such a film cannot be peeled off into a free-standing thin-film substrate of
acceptable quality for biomedical use.
Photographs of 100-nm-thick, 6-μm-thick, and 100-μm-thick thin-film substrates
with slanted columnar morphology are presented in Figs. 7.7(b)-(d). These thin-film
substrates were produced with framed and soap-coated slides that had not been baked.
In each of these three photographs, the white frame is the thermal-release tape and
the central square is the nano/micro-textured, free-standing, thin-film substrate of
parylene-C. The thin-film substrate is strongly bonded to the frame, which makes it easy
to handle. As the thickness increases, the thin-film substrate changes from completely
transparent to frosty white and gets dark yellow at the thickest. Such changes in the
appearance of submicron-textured films of parylene-C have been observed for the 2D
gradient panel in Chap. 4 [91, 111]. A thin slice near the frame edge is more transparent,
which results from the self-shadowing effect during the physicochemical-vapor-OAD
step [91]. The frame edge shadows the incident reactive-monomer flux from forming
columnar morphology in this area and instead a denser thin film is formed by diffusion.
92
Fig. 7.7. (a) Photograph of a parylene-C thin-film with columnar morphology deposited
on an uncoated silicon slide. (b-d) Photographs of fibrous thin-film substrates with
slanted columnar morphology and of thickness (b) 100 nm, (c) 6 μm, and (d) 100 μm.
7.4 Cellular Attachment and Growth to Free-Standing Parylene-C Thin Films
7.4.1 Attachment and Growth Assay Design
An assay was designed primarily to ascertain that the freestanding, fibrous thin-
film substrates of parylene-C fabricated with soap-solution approach would allow cellular
attachment and growth. The effects of oxygen-plasma treatment and the duration of
93
incubation on cellular attachment and growth were also evaluated. Six replicates of a (5
mm 5 mm) thin-film substrate with slanted columnar morphology but without a frame
were prepared under each four conditions: (a) with plasma treatment for 24 h, (b) without
plasma treatment for 24 h, (c) with plasma treatment for 168 h, and (d) without plasma
treatment for 168 h.
The free-standing thin-film substrates were anchored to the bottom of non-tissue-
culture-treated polystyrene 24-well plates using ethyl cyanoacrylate glue. The thin-film
substrates were sterilized by immersion in 100% ethanol overnight, followed by
aspiration and drying in a sterile cell-culture hood, and three washes with sterile distilled
water. The human osteosarcoma cell line SAOS-2 (American Type Culture Collection,
Manassas, VA) was cultured under standard conditions in DMEM, with 10% FBS.
For cell attachment and growth assays, each well was seeded with 7.5 104
SAOS-2 cells as determined in duplicate by a TC10 automated cell counter (BioRad,
Hercules, CA). After 24 h, the wells were washed to remove any floating cells or debris.
Any loosely adherent cells on the non-tissue-culture-treated polystyrene perimeter around
the parylene-C thin-film substrate were removed by vacuum aspiration. For the 168-h
time point, the medium was changed every two days and loosely adherent cells on the
non-tissue-culture-treated polystyrene perimeter around the thin-film substrate were
removed by vacuum aspiration on day 3 and immediately before the trypsinization and
counting on day 7. The remaining cells attached to the thin-film substrate were gently
removed by incubation with 0.25% trypsin-EDTA solution (Gibco/Invitrogen, Grand
Island, NY). The trypsin was blocked by adding 1 ml of fresh media with 10% FBS, and
the cells pelleted by centrifugation at 1000g at 4 ºC for 8 min. The pellet was re-
94
suspended in 100 μl of fresh culture medium, and cell numbers were determined by an
automated cell counter after diluting 10 μl of cell suspension in a 1:1 ratio with trypan
blue stain (Gibco/Invitrogen, Grand Island, NY) so that only living cells were counted.
Cell numbers for each well were measured four times twice, and then averaged. Finally,
cell numbers were averaged for all six wells for plasma-treated and non-plasma-treated
thin-film substrates.
7.4.2 Attachment and Growth Assay Result and Discussion
The attachment (24 h) and growth (168 h) of SAOS-2 cells on free-standing
parylene-C thin-film substrates of slanted columnar morphology and 40-μm thickness
were investigated under four experimental conditions. The mean and the standard
deviation for six replicates per experimental condition are presented in Fig. 7.8.
One factor ANOVA (analysis of variance) was performed to determine the
significance of differences between cell numbers under different experimental conditions.
OriginPro 8.0 was used to perform the analysis, with the probability p of the difference
occurring by statistical accident set being < 0.05.
There was a significant difference between cell attachment after 24 h to plasma-
treated thin-film substrates and non-plasma-treated ones. Due to oxygen-plasma
treatment, cell attachment to the fibrous thin-film substrates was higher with cell numbers
1.6 time larger than to ones without treatment. The top surface of a thin-film substrate
changes from hydrophobic to hydrophilic after oxygen-plasma treatment [111]. It seems
that the SAOS-2 cells have attachment preference for hydrophilic surfaces at 24 h. This
may be because the attachment-mediating serum proteins of SAOS-2 bind more to the
95
hydrophilic substrates than to hydrophobic ones. There is no significant difference in cell
numbers after 168 h between plasma-treated and non-plasma-treated thin-film substrates.
Significance of differences on numbers of cells between two time points was detected for
both surface conditions. After 168h, the net effect for plasma-treated and non-plasma-
treated thin-film substrates was to increase cell numbers 2.9 and 4.0 fold, respectively.
Fig. 7.8. Quantification of cell attachment and growth on free-standing fibrous parylene-
C thin-film substrates, with and without oxygen-plasma treatment.
Thus, it was demonstrated that SAOS-2 cells do grow onto free-standing, fibrous
thin-film substrates of parylene-C and the growth rate is higher with non-plasma-treated
hydrophobic surfaces than with plasma-treated hydrophilic ones.
96
7.5 Concluding Remarks
In this chapter, the successful fabrication of free-standing thin-film substrates
with fibrous volumetric morphology and nano/micro-textured top surfaces was
successfully fabricated. Two approaches were devised. In the first approach, a glass slide
is first coated with PMMA, then a thin-film substrate of parylene-C is deposited on it
using the physicochemical-vapor-OAD process, and finally the entire assemblage is
immersed in chloroform. This procedure yields free-standing thin-film substrates of
parylene-C that are less than 100 μm in thickness and have surface texture on at least the
submicron length scale. As the residual chloroform can be potentially harmful, another
approach was devised. In the second approach, a silicon slide is spin-coated with soap
solution and decorated with a frame made of thermal-release tape. Baking of the soap-
coated wafer at some temperature below 250 ºC for 2 min is recommended in order to
engineer the back-surface texture of a thin-film substrate with either an implant surface or
healthy tissue in vivo. The physicochemical-vapor-OAD process is used to deposit on this
specially prepared slide a thin-film substrate of parylene-C with fibrous volumetric
morphology. The free-standing, fibrous thin-film substrate can then be simply produced
by elevating the temperature to release the thermal-release tape. Oxygen-plasma
treatment may be carried out if the biomedical application requires hydrophilicity.
The free-standing thin-film substrates produced by the second approach were
shown viable for cell attachment and growth. The devised technique using the soap
solution is simple. It is also environmentally friendly because the silicon slide can be a
reject from the electronics industry and can be reused numerous times, and soap can also
97
be recycled. It is also scalable, the physicochemical-vapor-OAD of parylene-C being
widely used in industry.
98
Chapter 8
Conclusions and Future Research
8.1 Conclusions
The central goal of the research reported in this dissertation was to efficiently
develop parylene-C thin-film substrates for implant integration and protein assays. To
achieve this goal, in the first step a commercially available parylene-C evaporator was
modified to grow fibrous thin-film substrates with different volumetric morphologies.
Two vacuum stepper motors were mounted on a set of mechanical frames, so that the
substrate-holding platform’s orientation could be dynamically controlled using computer
software and a custom-made circuit board. Physicochemical-vapor-OAD of parylene-C
thin-film was implemented, and the fabricated thin-film substrates were then
characterized.
Control of the thickness of the fibrous thin-film substrate allows surface
morphology and wettability to be controlled. Based on this finding, a 2D morphology-
composition gradient panel with continuous changes in surface roughness and surface
wettability was prepared. The abilities of fibroblast cells to fibrous thin-film substrates
and of biotinylated serum proteins to attach to a morphology-composition gradient panel
were found to be closely related to surface wettability. Different degrees of cell adhesion
and protein binding were achieved, depending on surface properties and cell/protein type.
Finally, free-standing fibrous parylene-C thin-film substrates were fabricated with control
over both surface wettability and surface morphology on both sides. These free-standing,
99
fibrous thin-film substrates were demonstrated to be capable of supporting the attachment
and growth of human osteosarcoma cells.
The scientific consequences of these results can be summarized as follows.
1. Fibrous parylene-C thin-film substrates can work as anti-/adhesion media for
specific proteins or cells, as demonstrated by the different degrees of cell
adhesion and protein attachment found in this dissertation. With controlled
surface roughness, composition, and wettability, the preferred proteins and cells
can be attached, and the adhesion of undesired ones can be deterred.
2. The 2D morphology-composition gradient panel has great promise as an
application for high-throughput screening assays. The panel has a gradient in
surface morphology with constant chemical composition along its length and a
gradient in surface chemical composition without change in its surface
morphology in the orthogonal direction. The panel, therefore, provides a wide
range of surface wettability. Thus a protein of a certain type could be
comprehensively differentiated by its preferential attachment to different locations
on the panel.
3. Free-standing, fibrous thin-film substrates of parylene-C permit the ex vivo
growth of tissue that is portable and implantable. Thus, such thin-film substrates
have potential for promoting tissue implants.
100
8.2 Future Research
One important direction for future work is that of developing fibrous parylene-C
thin-film substrates with large millimeter-scale voids. Cells would be able to penetrate
such voids and interact with the material instead of attaching to the top surface of the
thin-film substrate. Of the voids are on the micrometer-scale, proteins would be able to
do the same. Thus, under such conditions, the contact area for proteins or cells would
become much larger for implant integration to improve significantly. To realize such
substrates, lithographically decorated glass slides will have to be used.
Another way of extending this research would be to comprehensively characterize
2D morphology-composition gradient panels for assorting proteins. Such characterization
should cover a large variety of proteins.
Additional future work could focus on the implantation of tissue of grown on
free-standing, fibrous thin-film substrates. Likewise, the use of such a substrate to
conformally cover nonplanar implant surface is also desirable. As the strong attachment
of the thin films to the implant surface is crucial for the longevity of an implanted
prosthesis, the pattern on the back side of the thin-film should be investigated in relation
to its impact on the degree of attachment.
101
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VITA
Lai Wei
EDUCATION
Ph.D. in Engineering Science and Mechanics, Department of Engineering Science
and Mechanics, Pennsylvania State University, University Park, PA, USA,
August 2008-December 2011
M.S. in Optics, Department of Optical Science and Engineering, Fudan
University, Shanghai, China, September 2005-June 2008
B.E. in Electronics Science and Technology, School of Information Science and
Engineering, Shandong University, Jinan, Shandong, China, September 2001-
May 2005
JOURNAL PUBLICATIONS
L. Wei, L. Leo, T.M. Ritty, and A. Lakhtakia, ―Nano/micro-textured, free-
standing, flexible, thin-film substrates of parylene-C for cellular attachment and
growth,‖ (Accepted for publication in Mater. Res. Innovat.)
L. Wei, E.A. Vogler, T.M. Ritty, and A. Lakhtakia, ―A 2D surface morphology-
composition gradient panel for protein-binding assays,‖ Mater. Sci. Eng. C, vol.
31, 2011, pp. 1861-1866.
A. Lakhtakia, L. Wei, A. Kumar, and J. Kumar, ―Facile fabrication of free
standing submicron-textured films of Parylene-C,‖ Mater. Res. Innovat., vol. 15,
2011, pp. 1-3.
L. Wei, A. Lakhtakia, A.P. Roopnariane, and T.M. Ritty, ―Human fibroblast
attachment on fibrous parylene-C thin-film substrates,‖ Mater. Sci. Eng. C, vol.
30, 2010, pp. 1252-1259.
L. Wei, P. Parhi, E.A. Vogler, T.M. Ritty, and A. Lakhtakia, ―Thickness-
controlled hydrophobicity of fibrous parylene-C films,‖ Mater. Lett., vol. 64,
2010, pp. 1063-1065.
