Microstructured Click Hydrogels for Cell Contact Guidance in 3d

Abstract

Advances in the design of hydrogels as artificial cellular microenvironments have been leveraged by emergent chemistries, allowing for rapid, bioorthogonal, and cytocompatible reactions. Here, we report the synthesis of clickable alginate (ALG) hydrogels with random hydrophobic microstructures that improve protein retention and cell anchoring and guidance in 3D. Alginate was conjugated with cyclooctyne groups (K) at low (ALG-KL) and high (ALG-KH) modification degrees, which allow biofunctionalization by strain-promoted azide-alkyne cycloaddition (SPAAC), while generating self-associative amphiphilic derivatives. Both derivatives (ALG-KL and ALG-KH) showed the ability to interact with hydrophobic compounds and significantly improved protein retention than unmodified ALG. Remarkably, the spontaneous segregation between hydrophobic/hydrophilic regions of ALG-KH generated microstructured hydrogels with denser/stiffer microdomains inside a looser lattice. Mesenchymal stem/stromal cells (MSCs) cultured within ALG-K hydrogels SPAACclicked with integrin-binding peptides sensed and responded to the random stiffer microdomains. While MSCs adopted round morphology in homogenous ALG-K hydrogels, they were able to spread, produce pericellular matrix and assemble into multicellular clusters within the microstructured ALG-KH hydrogels. These versatile hydrogels can be click-functionalized on-demand in the presence of cells, sequester/release compounds with hydrophobic moieties, and guide 3D cellular organization and endogenous matrix deposition, providing unique and transformative platforms for tissue engineering.

Note:
Funding Declaration: This work was supported by project EndoSWITCH (PTDC/BTM-ORG/5154/2020) funded by FCT (Portuguese Foundation for Science and Technology). The authors thank FCT for CCB’s IF research position (Grant No: IF/00296/2015) and MIN’s scholarship (Grant No: SFRH/BD/129855/2017 and COVID/BD/151886/2022).

Conflict of Interests: The authors declare no conflicts of interest.

Keywords: self-assembling, microtopographical cues, protein sequestration, cell instructive scaffolds, BCN-amine, Regenerative medicine