Architecture and Composition Dictate Viscoelastic Properties of Organ-Derived Extracellular Matrix Hydrogels

Abstract

The proteins and polysaccharides of the extracellular matrix (ECM) provide architectural support as well as biochemical and biophysical instruction to cells. Decellularized, ECM hydrogels replicate in vivo functions. The ECM's elasticity and water retention renders it viscoelastic. In this study, we compared the viscoelastic properties of ECM hydrogels derived from the skin, lung and (cardiac) left ventricle and mathematically modelled these data with a generalized Maxwell model. ECM hydrogels from the skin, lung and cardiac left ventricle (LV) were subjected to a stress relaxation test under uniaxial low-load compression at a 20%/s strain rate and the viscoelasticity determined. Stress relaxation data were modelled according to Maxwell. Physical data were compared with protein and sulfated GAGs composition and ultrastructure SEM. We show that the skin-ECM relaxed faster and had a lower elastic modulus than the lung-ECM and the LV-ECM. The skin-ECM had two Maxwell elements, the lung-ECM and the LV-ECM had three. The skin-ECM had a higher number of sulfated GAGs, and a highly porous surface, while both the LV-ECM and the lung-ECM had homogenous surfaces with localized porous regions. Our results show that the elasticity of ECM hydrogels, but also their viscoelastic relaxation and gelling behavior, was organ dependent. Part of these physical features correlated with their biochemical composition and ultrastructure.

Keywords: ECM hydrogel; Maxwell model; decellularized organs; extracellular matrix; viscoelasticity.

Figure 1

Methods. (a) Decellularization and hydrogel formation of skin-ECM, lung-ECM and LV-ECM (b) Low-load compression testing at 20% strain for 100 s. The data analyzed with a generalized Maxwell model of viscoelasticity. The number of Maxwell elements were determined based on curve-fitting the stress relaxation data (Relaxation modulus; Pa). The figure shows skin-ECM data, where two Maxwell elements were sufficient to explain their viscoelasticity, confirmed by the decrease in Chi². (c) Scanning electron microscopy (SEM). All hydrogels were fixed for 24 h in 2% glutaraldehyde and 2% paraformaldehyde (1:1 ratio) at 4 °C, freeze-dried for 24 h, metal coated and visualized with SEM. (d) Protein and sulphated glycosaminoglycans (sGAGs) quantification with Lowry and DMMB assays. (e) Histology and Immunohistochemistry. Hydrogels were fixed for 24 h in 2% formalin, processed conventionally with a graded ethanol series for dehydration, paraffin embedded and sectioned. Sections (5 µm) were stained with Alcian Blue, Picrosirius Red (PSR) and Masson’s Trichrome (MTC) as well as immune stained for collagen type I (COL1A1) and Elastin and scanned with a Hamamatsu section scanner. (Figure created with BioRender).

Figure 2

Turbidity of skin-ECM, lung-ECM and LV-ECM hydrogels. (a) Turbidity data (mean and S.D.) at every 10 min; (b) Normalized absorbance from the start of gelation. All data derived from a minimum of three independent experiments performed in triplicates. Data are presented as mean with SD.

Figure 3

Viscoelastic properties of skin-ECM, lung-ECM and LV-ECM. (a) Elastic modulus; (b) Time to reach 50% stress relaxation; (c) Average stress relaxation normalized to start from 100%. All data derived from a minimum of three independent experiments performed in triplicates from low-load compression testing at 0.2 strain. Data are presented as mean with SD. Statistical differences according to one-way ANOVA and Dunn’s post hoc test **** p < 0.0001.

Figure 4

Maxwell analysis of skin-ECM, lung-ECM and LV-ECM viscoelasticity. (a) Relative importance of 1st, 2nd and 3rd Maxwell elements; (b) Tau (τ) of 1st, 2nd and 3rd Maxwell elements reported in seconds (s). All data derived from a minimum of three independent experiments performed in triplicates. Data are presented as mean with SD. Statistical differences according to one-way ANOVA and Tukey (1st and 2nd Maxwell Elements) and Student’s t-test (3rd Maxwell Element) * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001.

Figure 5

Quantification of protein and sulfated GAGs (sGAGs) of organ-derived ECM. (a) Protein content (µg/mL) of skin-ECM, lung-ECM and LV-ECM according to Lowry assay; (b) sGAGs content (µg/mL) skin-ECM and LV-ECM and according to DMMB assay. No sGAGs were detected in lung-ECM. All data derived from four independent experiments performed in triplicates. Data are presented as mean with SD. Mean values per experiment (n = 4) are also shown. Lowry data analyzed with one-way ANOVA (p = ns). DMMB data analyzed with Student’s t-test **** p < 0.0001.

Figure 6

Alcian Blue, MTC and PSR, stains in sections of skin-ECM, lung-ECM and LV-ECM. Scale bars: 50 µm.

Figure 7

Immunohistochemistry (red) of COL1A1 and Elastin of skin, lung and LV ECM hydrogels. Scale bars represent 50 µm.

Figure 8

Fluorescent micrographs of PSR stained skin, lung and LV ECM hydrogels. (a) Unstained sections (autofluorescence); (b) PSR-stained sections. Original objective magnification 40×.

Figure 9

Surface microarchitecture of skin-ECM, lung-ECM and LV-ECM. (a) Magnification at 5000×; (b) Magnification, 10,000×. Scale bars: 10 (a) and 2 µm (b).