1. 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

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

3. 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

4. 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: /jbm.b.33849

5. 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

6. 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.

7. 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.

8. 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.

9. 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.

10. 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

11. 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 +/ µm
3.6 +/- 0.4 µm
Detach, let reach equilibrium in water, then irradiate
0.07 +/ µm
3.6 +/- 0.7 µm
Irradiate, then detach from template
0.3 +/- 0.1 µm
3.6 +/- 0.3 µm

12. 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.

13. 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.

14. 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

15. 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

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

17. Thank you for your attention