Development and characterisation of laser-patterned
polymer substrates for controlling cell growth
Michael Irving
Breakdown
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Project Background
Aims
Methodology
Preliminary results 1 - SPI Laser
- Characterisation of SEM images
- Characterisation of AFM images
- Wyko Analysis
- Continued Work
• Preliminary results 2 - Green Laser
• The Challenge
Project background
• Our body is made of billions of cells that pack
together tightly to form tissues
• In the body the cells attach within a 3D scaffold
support known as the extracellular matrix (ECM)
• The interaction of cells with the external environment
(i.e. ECM) is essential for for many cellular functions
Project background
• There is great interest in trying to develop micro
and nano-patterned materials which promote
cellular interaction and thus can be used to control
aspects of cellular behaviour e.g.
• Growth Rate
• Adhesion
• Cell Migration Direction
• Applications
- Stents
- Hip Implants
- Dental Implants
- Ocular implants
- wound healing
• Issues
- surfaces need to be characterised
- need to be reproducible
- if surface effects cell behaviour important to know why
- if altering specific parameters e.g. speed, power alters
effect surface has
What methods are currently used to
develop patterned surfaces?
Much of the research in this area has focussed on using the following techniques
• Electron beam Lithography
• Photolithography
• Silicon shadow mask
Laser Processing to develop Micro-patterned
Surfaces
Laser processing has emerged as an effective method for structure
manufacturing as it has been shown that laser processing is an excellent tool
for micro-patterning due to it being a rapid, direct-write and flexible process
while also capable of processing on a large scale.
Aims
The aims of this work is to:
1. Produce nano/micro scale structures on polymer substrates
2. To characterise the 3D surface parameters using Atomic
Force Microscopy (AFM), Laser Scanning Confocal Microscopy
(LSM), Scanning Electron Microscopy (SEM) and white-light
interferometry (Wyko)
3. To carry out a range of biological assays in order to
determine how the 3D surfaces influence important aspects of
cellular behaviour.
Methodology
Developing the surfaces
• Involves changing parameters to determine optimum settings
• Looking for minimum debris, defined features
• Surfaces need to be reproducible
Parameters using SPI laser
• Previous work showed that settings of 4w 9ns and 500khz
produced workable features
Parameters for Green Laser
• Range of parameters, speed, power and pass number all
changed
Preliminary Results 1
Investigating the Effects of Processing
Speed & Pass Number using SPI Laser
300mm/sec
200mm/sec
Characterization of SEM images – Change in Speed
• Power 4w
• Pulse duration 9ns
• Frequency 500khz.
500mm/sec
400mm/sec
• Number of passes 15
• Parameters being
investigated - speed
and pass number.
• 50um hatch spacing
Observations suggest by increasing speed you can decrease the number of
micro pits.
Faster speeds seem preferable due to lower numbers
Characterising the surfaces with SEM
Using SEM imaging measured width of processed area and non processed area,
30 individual measurements were taken for statistical analysis.
SPSS software was used for analysis
When compared the width of the processed areas were not significantly
different apart from between the 400 and 500mm/sec processing (p>0.005)
Characterizing AFM Images
200mm/sec
400mm/sec
300mm/sec
500mm/sec
Visual inspection suggests an increase in surface definition as the speed is increased
There seems to be a decrease in roughness due to reduction in debris.
Micro Pit
Width ~5um
Depth ~800nm
AFM height profile of 400mm/sec processing
AFM height profile of 500 mm/sec processing
Surface roughness from Wyko analysis
White light interferometer used to determine surface roughness of
processed areas
Surface Roughness (Nm)
How Change in Speed Effects Surface Roughness of
SS (Ra)
380
360
340
320
300
280
260
240
220
200
200
300
400
500
Speed of processing (mm/sec)
Clear difference in surface roughness between 200/300 and 400/500mm/sec
processing, maybe due to the improved definition of surface features.
30 passes
20 passes
Passage number change
Same working parameters of
4w 9ns
Speed kept constant at
500mmsec
50 passes
40 passes
Number of passes changed
20-50 passes
50um hatch spacing
Initial observations suggest lower pass numbers preferable, improved definition seen with
SEM images
20p
40p
30p
50p
Images show a decrease in edge definition as pass number increases.
Suggests use of lower pass number for use in testing cell behaviour
Surface roughness from Wyko analysis
Shows general increase in Ra value as number of passes increases
Surface Roughness (Nm)
How Change in Passage number Effects
Surface Roughness
380
360
340
320
300
280
260
240
220
200
20
30
40
50
Number of passes
Clear difference in surface roughness between 20/30 and 40/50 pass number.
Potentially due to decrease in surface definition.
Continued Work with SPI laser
Increased number of passes seems to reduce definition between processed areas
Look at using lower pass numbers for their effect
If improve definition can decrease width between processed areas
Casting of surfaces
using SS mould polyurethane cast will be taken
b9 Series A polyurethanes have been independently tested and are compliant with USP
Class VI, and ISO 10993 protocols for biocompatibility.
Will be characterised to determine accuracy of cast
Testing Cell Behaviour
Testing of cell behaviour will be done
• Adhesion test
- Seed cells for 2/3hrs, remove medium and wash surface. Determine how
many cells remain on surface
• Migration test
- Seed cells on surface image cells over extended period of time
determine direction of cell movement and distance moved.
• Staining
- Seed cells on surface, stain structural proteins (actin/ vinculin). Can
compare with flat surface for changes in the arrangement of proteins
LL24 fibroblast cells stained for Actin
Preliminary Results 2
Investigating the Effects of Processing
Speed & Pass Number Using Green laser
Green Laser (532 nm)
1w 6p speed change
1200 mm/sec
200mm/sec
2w 6p speed change
200mm/sec
1200 mm/sec
500mmsec 6p power change
2W
0.5W
2w 500mmsec passage number change
1p
10p
Thank you for Listening