1

Section information

Replicating diseased aorta mechanical

properties via enzymatic digestion and

glutaraldehyde treatment

Noble C.1, Green N.2, Smulders N.3, Matt Carrè4, Sheila

MacNeil2, Lewis R.4, Taylor Z.A.1

1. CISTIB Centre for Computational Imaging and Simulation Technologies in Biomedicine, INSIGNEO Institute for in silico Medicine, Department of Mechanical Engineering, The University of Sheffield, Sheffield, UK

2. Department of Materials Science and Engineering, The Kroto Research Institute, North Campus, University of Sheffield, Broad Lane, Sheffield, UK

3. Philips Research, Eindhoven, The Netherlands

4 Department of Mechanical Engineering, The University of Sheffield, Sheffield, UK

2

Section information

Artery Composition

Composed of three layers:

Intima Endothelial layer

Elastic lamina

Media Smooth muscle cells

Elastin fibres with some directionality

Collagen fibres with large directionality

Adventitia Collagen fibres with less directionality

Gasser, T. C., Ogden, R. W., & Holzapfel, G. a. (2006). Hyperelastic modelling of arterial layers with distributed collagen fibre orientations. Journal of the Royal Society, Interface / the Royal Society, 3(6), 15–35. http://doi.org/10.1098/rsif.2005.0073

3

Section information

Clinical Background

Improving medical device design requires testing on healthy and diseased tissue

Difficult to access human diseased tissue in large quantities necessary

Healthy porcine aorta relatively easy to source

4

Section information

CAN WE USE TAKE HEALTHY TISSUE AND MAKE MODELS OF DISEASED AORTA FOR FUTURE STUDY?

5

Section information

Enzyme and Glutaraldehyde Treatment

Collagenase treatment is expected to be most effective in circumferential direction

Stiffer collagen fibres are predominantly orientation in this direction

Elastase treatment should be most effective in axial direction

Collagen fibres don’t contribute as greatly to mechanical properties in this direction

Glutaraldehyde acts to cross link protein fibres and is used widely in fixation for histology

Should make samples less compliant and more brittle

Used with permission from Microsoft

6

Section information

Sample Preparation

The model chosen was pig aorta

The arteries are transported from a local butcher to the laboratory (~30min)

The thoracic aorta (~10-20cm length) was dissected carefully at room temperature

Samples opened flat along the axial direction

Cut into dog-bone shapes in both axial and circumferential directions

15mm

40mm

10mm

Schriefl, A. J., Zeindlinger, G., Pierce, D. M., Regitnig, P., & Holzapfel, G. A. (2012). Determination of the layer-specific distributed collagen fibre orientations in human thoracic and abdominal aortas and common iliac arteries. Journal of the Royal Society, Interface / the Royal Society, (December 2011), 1275–1286.

7

Section information

Sample Treatment

Enzyme treated samples incubated at 37˚C for 20hours Elastase treatment at 0.2 U/ml

Collagenase at 0.05 U/ml

Glutaraldehyde at 0.1% concentration for 20 hours at 4˚C

Controls were fresh tissue and tissue incubated for 20 hours at 37˚C with no treatment

All samples stored in PBS solution with antibiotics and fungicides

All washed thoroughly after treatment

8

Section information

Mechanical Testing

Performed on Tinius Olsen tensile testing machine at room temperature

Samples moistened in PBS solution immediately prior to testing

Preconditioned with 10 loading cycles of 15mm displacement at 0.1Hz

Sample then pulled to complete failure

Strain measured via tracking motion of markers placed on samples using trackmate plugin within Fiji

9

Section informationMechanical Testing – axial tensile results

Fracture stresses are significant vs controls for collagenase (p<0.05)

Fracture stretches are significant vs controls for collagenase and elastase (p<0.05)

Largest stretches and stresses seen in elastase treated samples

10

Section informationMechanical Testing – circumferential tensile results Fracture stresses are significant vs controls for elastase (p<0.05)

Fracture stretches are significant vs controls for collagenase and elastase (p<0.05)

Largest stresses again seen in elastase treated samples and largest stretches seen in collagenase samples

11

Section information

Diseased Tissue Comparison

Biaxial testing of femoropopliteal arteries of various ages shows loss of compliance with age

Elastin loss also observed

Other enzyme digestion studies show increase in compliance with partial elastin digestion implyingfurther mechanisms at work in vivo(Chow et al. 2013)

Additional collagen synthesised and cross-linkedin vivo to compensate

Kamenskiy, A. V, Pipinos, I. I., Dzenis, Y. a, Phillips, N. Y., Desyatova, A. S., Kitson, J., … MacTaggart, J. N. (2015). Effects of age on the physiological and mechanical characteristics of human femoropopliteal arteries. Acta Biomaterialia, 11, 304–13. http://doi.org/10.1016/j.actbio.2014.09.050

Chow, M.-J., Mondonedo, J. R., Johnson, V. M., & Zhang, Y. (2013). Progressive structural and biomechanical changes in elastin degraded aorta. Biomechanics and Modeling in Mechanobiology, 12(2), 361–72. http://doi.org/10.1007/s10237-012-0404-9

12

Section information

Confocal Multiphoton Imaging

Upright Zeiss LSM510 META confocal microscope

With Plan-Neofluar objective: 40xmagnification/1.3, Oil immersion, DIC, Numerical aperture: 1.3

Collagen visualised via SHG with 950nm excitation wavelength (filter: 447-500nm)

Elastin visualised via TPF with 800nm excitation wavelength (filter: 394-405nm)

Non-stretched samples imaged at depth of 19.5μm

13

Section information

Confocal Multiphoton Imaging

14

Section information

Summary

Partial breakdown of aorta achieved through enzyme digestion

Collagenase treatment made samples weaker and more compliant

Elastase treatment made samples stronger and more compliant

Glutaraldehyde treatment made curve profile more discontinuous but there was no significant changes in fracture properties

15

Section information

Conclusions and Further Work

Can we use take healthy tissue and make models of diseases aorta for future study?

Yes… but further work is necessary

Further characterisation of damage such as peel testing

Combination of elastase digestion and glutaraldehyde treatment may aid in capturing collagen cross-linking compensatory behaviour with elastin loss

Acknowledgements

We gratefully acknowledge support for this research from the

European Commission FP7

UNITISS, FP7-PEOPLE-2011-IAPP/286174

17

Section information

Thank you for listening