Long-term stability of structured gelatin hydrogels Die Angaben in der Fußziele auf der Folie eintragen. Long-term stability of structured gelatin hydrogels Meike Tadsen Materialwissenschaftliches Seminar 23.06.2017

Outline Motivation Methods Sample production Sample analysis Results Non-irradiated samples Drying and re-hydrating samples Irradiated samples Problems and outlook

Motivation Motivation Gelatin is a promising biomimetic material that could be used as a cell carrier to repair soft tissue, e.g. blood vessels However, pure gelatin liquefies at below body temperature Crosslinking can be achieved chemically, but leaves toxic residues Electron irradiation is a good alternative Cells align themselves to microstructures on their substrate

10wt% gelatin, left to dissolve for 1h, then heated until liquid Methods: Sample production 10wt% gelatin, left to dissolve for 1h, then heated until liquid Liquid gelatin poured onto preheated silicon templates with grooves of 3.5µm width and 800nm depth Stored overnight at 4°C to solidify If sample was irradiated: 40kGy in 5kGy steps Gelatin was detached from template Long-term storage: in the fridge at 4°C in the incubator at 37°C Some samples were dried at room temperature, then re-hydrated Heyart et al., Micropatterning of reagent-free, high energy crosslinked gelatin hydrogels for bioapplications, 2017. doi: 10.1002/jbm.b.33849

The samples were taken out of storage, dried with pressured nitrogen, Methods: Sample analysis The samples were taken out of storage, dried with pressured nitrogen, then analysed under a NanoFocus µsurf explorer confocal microscope, using 100x magnification. The microscope uses a rotating multi-pinhole disc to filter out-of focus light and enable seamless scanning over the sample size. A 1st order polynomial correction was applied to all pictures to account for the slanted sample holder. The pictures were then exported in ASCII format. Picture: NanoFocus AG

A python script was used to collect heights and widths: Methods: Sample analysis A python script was used to collect heights and widths: Samples are positioned with vertical structures Every line of the 2D data file is one height profile A baseline correction is applied peakutils is used for peak detection Widths are measured at the mean Heights and widths are collected over an entire picture Lines with >30% standard deviation in heights or widths are discarded.

A comparison of AFM and confocal measurements Methods: Sample analysis A comparison of AFM and confocal measurements of the same sample on the same day shows that the confocal microscope is likely underestimating the height of the ridges, but measuring the width correctly.

Results: Non-irradiated samples Development of heights and widths over time for several samples of 10wt% gelatin, structured on a template with 800nm x 3.75µm ridges. Stored in water at 4°C.

Results: Non-irradiated samples Development of heights and widths for different storage methods: in water at 4°C (red), airtight at 4°C (blue), in air at 4°C (green). 10wt% gelatin, structured on a template with 800nm x 3.75µm ridges.

Drying and then re-hydrating a sample preserves the structure. Results: Drying and re-hydrating Drying and then re-hydrating a sample preserves the structure. Top left: Surface of the sample after preparation Top middle: Surface after 7 days of drying at room temperature Top right: Surface after another 7 days of re-hydrating in water at 4°C Bottom left: Height profiles of each of these surfaces

Different preparation methods for irradiated samples: Height Width Results: Irradiated samples Different preparation methods for irradiated samples: Height Width Detach from template, then irradiate 0.06 +/- 0.01 µm 3.6 +/- 0.4 µm Detach, let reach equilibrium in water, then irradiate 0.07 +/- 0.05 µm 3.6 +/- 0.7 µm Irradiate, then detach from template 0.3 +/- 0.1 µm 3.6 +/- 0.3 µm

Top row: day 1, day 5, day 19 and day 29, respectively. Results: Irradiated samples 10wt% gelatin, irradiated with 40kGy on the template. Stored in cell culture medium at 37°C. Top row: day 1, day 5, day 19 and day 29, respectively. Bottom right: heights (red) and widths (blue) of the structures over time.

left: 40 kGy vs. unirradiated sample. Top: heights, bottom: widths. Results: Irradiated samples Comparisons: left: 40 kGy vs. unirradiated sample. Top: heights, bottom: widths. right: 40kGy vs. a sample crosslinked with 1% glutaraldehyde for 24h. Top: heights, bottom: widths.

Problems Problems: Samples need to be completely dry to produce meaningful results in the confocal microscope. Top left: same region, pictures taken about a minute apart. Top right: profiles for the two pictures on the left. Red: before, blue: after Bottom left: profile with jagged bottoms due to residual water. Other problems: dirt, bacteria, defects from handling the sample, difficulties taking AFM pictures

Next steps higher radiation dosages different concentrations Outlook Next steps higher radiation dosages different concentrations reproduce results from the sample in the incubator collagen on templates better AFM pictures cell cultures

Special thanks to Stefanie Riedel Katharina Bela Robert Konieczny Toni Liebeskind Prof. Mayr

Thank you for your attention