Patterned Poly(chlorotrifluoroeth ylene) Guides Primary Nerve Cell Adhesion and Neurite Outgrowth

112 Pages·2000·5.26 MB·English

Patterned Poly(chlorotrifluoroethy lene) Guides
Primary Nerve Cell Adhesion and
Neurite Outgrowth
Samar Saneinejad
A thesis submitted in confonity mth the requirements for the degree of
Masten of Applied Science.
Graduate Department of Chemical Engineering and Applied Chemistry
lnstitute of Biomaterials and Biomedical Engineering
in the University of Toronto
@Copyrightb y Samar Saneinejad (1 999)
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Panemed Poly(cMorotrifluoroethylene) Guides Rimary Nerve Ceil Adhesion and Ne* Outgrowtb
MASc., 1999
By Samar Saneinejad
Department of Chernical Engineering and Applied Chemkûy
Institute of Biomaterials and Biomedical Engineering
University of Toronto
Centrai nervous system (CNS) neumns do not regenerate foilowing injury.
Recently, it has been shown that in the developing CNS a combination of ceU-adhesive
and cell-repulsive cues guide growiug axons to their targets. By mimicking the eEects of
these mes, it may be possible to guide nerve cell adhesion and neurite outgrowth in vitro.
The objectives of this research project were to modiQ poly(chloro~uoroethylene)
(PCTFE) surfaces, incorporating patterns of ceil-adhesive (peptide) and non-adhesive
Cpolyethylene glycol (PEG)] regions and to study primary nerve ceii interaction with
these surfaces. PCTFE was modified with PEG-alkoxide, pattemed with gold by sputter-
coating, and PCTFE-Au regions were modifïed with faminin-derived peptides,
CGMGSR or CSIKVAV. On homogeneously modified surfaces, cell adhesion increased
in the order: PE-PCTFE-Au<CSIKVAVcPoly(L-lysine)Aaminin<CGWGSR Neurite
outgrowth studies showed that CSIKVAV nufaces were comparable to positive controls
[(Poly (L-1 y sine)Aaminin] with respect to neurite Length. On patterned surfaces, ceii
arihesion and neurite outgrowth were successfully directed on 180 p PEG/90
peptide patterns.
Acknowledgements
1 would iike to begin by thanking Professor Moily Shoichet for her supervision
and guidance throughout the last two years. Workïng under MoUy's supervision proved
to be a challenging yet wonderful leaming experience and 1 feel fomuiate to have been a
member of her research group. 1 would also iike to thank the other members of the
"S ho ichet Research Group": Xudong Cao, Yen Tong, Dan Lousenberg, Chantal Holy,
Paul Dalton, and Yumin Yuan, for their help and advice and for sharing their expertise! 1
would especially 1&e to thank Xudong for passing on his ceil culture techniques, Dan for
his help in the lab, and Yen for his help with ceil culture and with any cornputer probiems
that were encountered!
1 would also like to acknowledge the assistance of Suzanne Mamiche with the
sputtering machine, Prof. Rana Sodhi with XPS dysis, and Prof. Chris Yip for his help
with AFM analysis and for his numerous helpful suggestions. In addition, 1 would iike to
thank Prof. Sefton for his unique teaching style and for taking time out of his busy
schedule to be on my defense cornmitte.
1 was very fortunate to go have gone through this stage of my academic life
dongside two exceptionai young ladies, Nikki Porter and Dolores Baksh (aka the
Biomaterials Girls). Our morning coffee break conversations where we tackled research
obstacles and discussed fhre plans (4 years!) were mernorable moments. I would like
to thank Nikki and Dolores for their invaluable fnendship. 1 would also like to thank a
special Wend, Anandhi Narayana, for listening to me cornplain and for making me
smile. O
Finally, 1 am grateful to my parents for their love, support, and encouragement
throughout the last 25 years. 1 would like to thank them for allowing me to make my
own decisions and for teaching me so many thhgs about Life that are not taught in school.
1 wouid also like to thank my brother Ali for his support and for the beautifid house he is
going to design for me one day!
List of Tables
List of Figures
List of Abbreviations
1. Introduction
1.1 Axonal Regeneration
1.2 Axonal Guidance
1.3 Primary Nerve Celis
1.4 Hippocampal Cells
1- 5 S dace Modification
1.6 Patternhg Techniques
1.7 Fluoropolymers
1 -8 Poiy(chiototrifluomethylene)
1.9 Protein/CeU-Resistant Surfaces
1-10 Poly(ethy1ene glycol)
1.1 1 CeU-Adhesive Surfaces
1.12 Cell-Adhesive Peptides
1.1 3 Hypothesis
1.14 Objectives
2.1 Materials
2.2 Surface Modifications
2.2.1 PEG Modification
2.2.2 Gold Sputtering
2.2.3 Pattemîng
22.4 Peptide Modification
2.2.5 Peptide Labelhg and Quantscation
2.3 Surfâce Characterhtion
2.3.1 Contact Angie Anaiysis
2.3.2 X-ray Photoelectron Sp ectroscopy
2.3.3 Angle-Resolvcd XPS
2-3.4 EUipsometry
2.3.5 AFM
2.4 Ce11 Culture Experiments
2.4.1 Sterilization of Substrates
2.4.2 Preparation of Positive Control
2.4.3 Ce11 Dissociation and Plating
2.4.4 Competitive CeU Adhesion Assay
2.4.5 CeU Viability Assay
2.4.6 Quantitative and Quaütative Analysis
2.4.7 Statistical Analysis
3.1 PEG Modification
3.1.1.1 The EEect ofR eaction Time
3.1-1.2 The Effect of PEG MW
3.1.1 -3 The Efféct of Solvent System
3.1.2 Contact Angle
3.1 -3 Thickness and Surface Coverage
3.1.4 AFM
3.2 Peptide Modification
3.2.1 XPS
3 -2.2 Contact Angle
3 .2.3 Thickness and Surface Coverage
3.3 Ce11 Culture
3 -3.1 Qualitative Analysis
3.3 -2 CeU Adhesion Analysis
3.3- 3 Competitive Celi Adhesion Assay
3 -3- 4 Neurite Outgrowih Analysis
3.3- 5 Neurite Length Analysis
3.3.6 Pattemed Surfaces
4. Discussion
4.1 PEG Modification
4.1.1 The Proposed Mechanisms
4.1.2 The Effects of PEG MW
4.1.3 The Effects of the Co-solvent Systern
4.2 Peptide Modification
4.3 Cell-Surfacc Interactions on Homogeneous Surfaces
4.4 Cell-Surface Interactions on Pattemed Surfaces
4.5 The Effects of Pattern Dimension on Guiding Cell Adhesion
and Neurite Outgrowth
5. Conclusions and Recommendations for Future Work
5.1 Conclusions
5.2 Recornmendations for Future Work
6. References
Tabie 2.1 : The sputtering system panuneters used in the
deposition of Au ont0 modifieci PCTFE films.
Table 2.2: Grid dimensions used to create micron scaîe pattenis
on modifieci PCTFE.
Table 3.1 :X PS results for PEG MW5000 modification of
PCTFE (90' take-off angle).
Table 3.2: XPS results for PEG MW5000 modification of
PCTFE (1 S0 take-off angle).
Table 3.3 : XPS results cornparhg dinerent MW PEûs in toluene
vs. toluene/THF solvents (90° take-offa ngle).
Table 3.4: XPS redts cornparhg dinerent MW PEGs in toluene
vs. toluene/THF soivents (lSo take-offa ngle).
Table 3.5: XPS redts for peptide modiiidon rractions (90°
take-off angle).
Table 3 -6:W S r edts for peptide modification reactions (1 S0
take-off angle).
vii
Figure 1.1 : In vivo, growing axons are guided by a combination
of adhesive and repulsïve cues.
Figure 1.2: An image of a typicai hippocarnpai ceu in culture.
Figure 1 -3: The molecular fondas of PTFE and PCTFE.
Figure 1.4: The molecular formula of PEG.
Figure 1 .S: Surface bound PEG molecules are highiy hydraîed
and their excluded volume is believed to play a key
role in resisting protein adsorption and cell adhesion.
Figure 1.6: The structure of laminin, showing the locations of
YIGSR, IKVAV, and RGD peptide sequences.
Figure 2.1 : The experimental scheme showing the thme d a c e
modification steps: 1) FE0 modification, 2) Au
sputtering, and 3) Peptide modification.
Figure 2.2: The preparation of PEG-atkoxide and its reaction with
PCTFE films.
Figure 2.3: A mask in combinaiion with EM grids is used to
create micron sale patterns on the d a c eo f the
films.
Figure 2.4: Peptide modification of PCTFE-Au films.
Figure 3. L : High resolution XPS sptms howing the Cls regions
of a) PCTFE, b) PCTFE-PEG at a take-off angle of
90°, and c) PCTFE-PEG at a take-off angle of 15'.
Figure 3.2: For the same depth of modification @) and the same
depth of anaiysis (d), an incraw in surfiacc
percentages of carbon and oxygen with decreasing
MW PEG suggests that the surfâce coverage is greater
on the PEG MW2000-modified SUrf8ce.
Figure 3.3: Advancing and receding contact angie data for the
PEG modification reactions.
Figure 3.4: AFM height images of A) PCTFE and B) PCTFE-
PEG surfaces showing surfàce topography @an size
ofSpX5pm).
Figure 3.5: Advancing and receding contact angle data for the
peptide modification reactions.
Figure 3.6: Representative images of hippocampai celis on
PCTFE on a) day 1, b) day 4, and c) day 7 after ceIl
plating.
Figure 3.7: Representative images of hippocampal ceUs on
a) PCTFE-PEG- day 1 after ceil plating (100X
-
magdication), b) PCTFE-PEG &y 1 a f kc ell
plating (2OOX magriincation). and c) PCTFE-PEG-
-
Au day 1 a hce li plathg (2ûûX maguification).
Figure 3.8: Representative images of hippocampai celison
CGYIGSR-modified mrfaces on a) &y 1 after celi
plating, b) day 4 aftrr c dp lating. c) &y 7 derc e11
plating.
Figure 3.9: Representative images of hippocampal celis on
CSIKVAV-modified SUrf8ces on a) &y 1 derc ell
plating, b) &y 4 after ce11 platin& and c) day 7 after
cell plating.
Figure 3.10: Representative images of hippocampai ceils on
PLLAaminin (positive control) SUTfaces on a) day 1
after celi plating, b) &y 4 a f kc ell plating, c) day 7
after celi plating.
Figure 3.1 1: Average number of ceiis pet field (1 field = 25 mm2)
as deterrnined for 10 random fields per de.
Figure 3.12: Resuits of the competitive assay showing a
signincant rcduction in the number of cells pet field
if the ceus werc prc-incubated with peptides.
Figure 3.1 3 : Average numùer of neurites pci celi as determined
for 50 mdom celis per dace.
Figure 3.14: Average neurite length on each surfe as
deterxnined for 50 random cells per dace.