1. Development and characterisation of laser-patterned polymer substrates for controlling cell growth
Michael Irving

2. Breakdown 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

3. 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

4. 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

5. Applications Issues - 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

6. 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

7. 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.

8. 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.  

9. 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

10. Preliminary Results 1
Investigating the Effects of Processing Speed & Pass Number using SPI Laser

11. Characterization of SEM images – Change in Speed
200mm/sec
300mm/sec
Power 4w
Pulse duration 9ns
Frequency 500khz.
Number of passes 15
Parameters being investigated - speed and pass number.
50um hatch spacing
400mm/sec
500mm/sec
Observations suggest by increasing speed you can decrease the number of micro pits.
Faster speeds seem preferable due to lower numbers

12. 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)

13. Characterizing AFM Images
200mm/sec
300mm/sec
400mm/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.

14. Micro Pit
Width ~5um
Depth ~800nm

15. AFM height profile of 400mm/sec processing

16. AFM height profile of 500 mm/sec processing

17. Surface roughness from Wyko analysis
White light interferometer used to determine surface roughness of processed areas
Surface Roughness (Nm)
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.

18. Passage number change Same working parameters of 4w 9ns 20 passes
Speed kept constant at 500mmsec
Number of passes changed passes
50um hatch spacing
20 passes
30 passes
40 passes
50 passes
Initial observations suggest lower pass numbers preferable, improved definition seen with SEM images

19. 20p
30p
40p
50p
Images show a decrease in edge definition as pass number increases.
Suggests use of lower pass number for use in testing cell behaviour

20. Surface roughness from Wyko analysis
Shows general increase in Ra value as number of passes increases
Surface Roughness (Nm)
Number of passes
Clear difference in surface roughness between 20/30 and 40/50 pass number. Potentially due to decrease in surface definition.

21. 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 protocols for biocompatibility.
Will be characterised to determine accuracy of cast

22. 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

23. Preliminary Results 2
Investigating the Effects of Processing Speed & Pass Number Using Green laser

24. Green Laser (532 nm) 1w 6p speed change 1200 mm/sec 200mm/sec

25. 500mmsec 6p power change 2W
0.5W
2w 500mmsec passage number change 1p
10p

26. Thank you for Listening