Introduction: Macroporous cryogel scaffolds are being developed for tissue engineering applications as 3D analogs of the extracellular matrix. Compared to bulk hydrogels without macropores, the increased precursor-concentration in the non-frozen liquid microphase of cryogels results in denser polymer networks surrounding the macropores, which is designated as cryo-concentration effect. Due to the cryo-concentration effect, the struts of the sponge-like cryogels can be assumed to exhibit a higher local stiffness than the corresponding bulk hydrogel materials obtained from the same reaction mixture at room temperature. However, this was not demonstrated experimentally, yet. Thus, the aim of this study was to develop, test, and apply a method for the direct determination of the microscale Young’s modulus of cryogel struts
Material and Methods: Gels were formed via cross-linking of amino-end-functionalized starPEG with EDC/ sulfo-NHS-activated carboxylic acid groups of heparin as described[1]. Sub-zero
temperature treatment of the gel forming reaction mixtures
and subsequent lyophilization of the incompletely frozen gels
resulted in macroporous biohybrid cryogels showing rapid swelling, porosity of up to 92% with interconnected large pores (30− 180 μm), low bulk stiffness, and high mechanical stability upon compression[2]. Reference bulk hydrogels were formed from the same reaction mixture at room temperature. A microtome cryostat was utilized to cut cryogel sections of high uniform thickness (Figure 1) that enabled the visualization of indivudual cryogel struts. The Young’s moduli of cryogel struts and corresponding bulk hydrogels were determined by AFM-based nanoindentation experiments (Figure 1) in the PBS swollen state.
Results and Discussion: Resulting from the higher crosslinking degree, the Young’s moduli of bulk hydrogels and the cryogel struts increased with increasing molar starPEG to heparin ratio. For a given ratio, the cryogel struts exhibited a significantly higher Young’s modulus than the respective bulk hydrogels (Figure 1). These results are the first experimental proof of an increased stiffness of the cryogel struts compared to the corresponding bulk hydrogel material. Besides analyzing the impact of cryogelation on the Young’s modulus of the cryogel strut material, the interplay between local strut Young’s modulus, and integral modulus of the cryogels (determined by uniaxial stress–strain compression experiments) was also examined. It was found that the intrinsic mechanical properties of the struts for different cryogel samples cannot simply be deduced from their integral mechanical properties.
Conclusion: Local mechanical properties of macroporous starPEG–heparin cryogel scaffolds can be directly measured by AFM-based nanoindentation methodology. The results provide valuable insights about the effect of cryogelation parameters on local scaffold mechanics allowing for a far-going customization of the cryogels to match particular tissue elasticities and to induce desired cellular responses.
Figure 1: Schematic representation of the starPEG-heparin network (right). Confocal microscopy image of the sponge-like microstructure of the PBS swollen cryogel scaffold (left). Arrows indicate where AFM-based nanoindentation was performed. Comparison of Young’s moduli of the cryogel struts and the corresponding bulk hydrogels (bottom).
References:
[1] U. Freudenberg, A. Hermann, P.B. Welzel, K. Stirl, S.C. Schwarz, M. Grimmer, et al., A star-PEG–heparin hydrogel platform to aid cell replacement therapies for neurodegenerative diseases, Biomaterials. 30 (2009) 5049–5060.
[2] P.B. Welzel, M. Grimmer, C. Renneberg, L. Naujox, S. Zschoche, U. Freudenberg, et al., Macroporous StarPEG-Heparin Cryogels, Biomacromolecules. 13 (2012) 2349–2358.