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

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

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 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

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

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

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 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.

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) 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.

Passage number change Same working parameters of 4w 9ns 20 passes Speed kept constant at 500mmsec Number of passes changed 20-50 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

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

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.

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

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

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