The Importance of Effective Ligand Concentration to Direct Epithelial Cell Polarity in Dynamic Hydrogels

Abstract

Epithelial cysts and organoids are multicellular hollow structures formed by correctly polarized epithelial cells. Important in steering these cysts from single cells is the dynamic regulation of extracellular matrix presented ligands, and matrix dynamics. Here, control over the effective ligand concentration is introduced, decoupled from bulk and local mechanical properties, in synthetic dynamic supramolecular hydrogels formed through noncovalent crosslinking of supramolecular fibers. Control over the effective ligand concentration is realized by 1) keeping the ligand concentration constant, but changing the concentration of nonfunctionalized molecules or by 2) varying the ligand concentration, while keeping the concentration of non‐functionalized molecules constant. The results show that in 2D, the effective ligand concentration within the supramolecular fibers rather than gel stiffness (from 0.1 to 8 kPa) regulates epithelial polarity. In 3D, increasing the effective ligand concentration from 0.5 × 10−3 to 2 × 10−3 m strengthens the effect of increased gel stiffness from 0.1 to 2 kPa, to synergistically yield more correctly polarized cysts. Through integrin manipulation, it is shown that epithelial polarity is regulated by tension‐based homeostasis between cells and matrix. The results reveal the effective ligand concentration as influential factor in regulating epithelial polarity and provide insights on engineering of synthetic biomaterials for cell and organoid culture.

Using Chemistry To Recreate the Complexity of the Extracellular Matrix: Guidelines for Supramolecular Hydrogel–Cell Interactions

“…Importantly, the local stiffness of the supramolecular hydrogels did not alter upon changing the hydrogel bulk stiffness. However, local inhomogeneities with areas of bundled fibers may still exist within the supramolecular gels, which were indeed confirmed to exist upon further indentation of the sample . The bulk mechanical properties required for optimal cell performance depend on culture dimensionality.…”

“… Regarding (1) gel mechanics, a base level of bulk stiffness ( G ′ ≈ 1 kPa) is required to offer mechanical support to cells, especially in 3D, to achieve cell adhesion. , However, stiffer gels (with inherent increased network density and smaller pores) might lead to mechanical constriction, hampering tissue growth. However, soft hydrogels will fulfill the needs of cells that do not rely on cell–matrix interactions as much. Lessons learned for (2) the bioactivity are (i) the ligand type needs to match with the expression of the receptor of interest, , (ii) supraphysiological ligand concentrations are required in synthetic gels , (most likely due to steric hindrance/inefficient ligand orientation toward receptors), and (iii) when C eff ≥ 5 mol %, multivalent effects can be observed through facilitating ligand recruitment. For (3) the gel dynamics, slow molecular dynamics (∼10% in 1 h) are required to achieve cell adhesion to withstand cell-pulling forces . The bulk dynamics is heavily dependent on culture dimensionality: in 2D, slow stress–relaxation (τ 1/2 ≈ 1000 s) is crucial to achieve cell spreading and prevent the relaxation of cell traction forces, while in 3D, fast-relaxing gels (τ 1/2 ≈ 50 s) promote the growth of single cells into multicellular organoids by dissipating tissue forces. …”

“…Based on some recent, important findings, guidelines underlying supramolecular hydrogel–cell interactions are proposed (Figure ). Regarding (1) gel mechanics, a base level of bulk stiffness ( G ′ ≈ 1 kPa) is required to offer mechanical support to cells, especially in 3D, to achieve cell adhesion. , However, stiffer gels (with inherent increased network density and smaller pores) might lead to mechanical constriction, hampering tissue growth. However, soft hydrogels will fulfill the needs of cells that do not rely on cell–matrix interactions as much. Lessons learned for (2) the bioactivity are (i) the ligand type needs to match with the expression of the receptor of interest, , (ii) supraphysiological ligand concentrations are required in synthetic gels , (most likely due to steric hindrance/inefficient ligand orientation toward receptors), and (iii) when C eff ≥ 5 mol %, multivalent effects can be observed through facilitating ligand recruitment. For (3) the gel dynamics, slow molecular dynamics (∼10% in 1 h) are required to achieve cell adhesion to withstand cell-pulling forces .…”

Importance of Molecular and Bulk Dynamics in Supramolecular Hydrogels in Dictating Cellular Spreading

“…Nature uses a variety of supramolecular interactions to create functional materials with different mechanical, dynamic, and bio­(chemical) properties, i.e., soft tissues, such as those in the brain, and stiff tissue, such as those in bone . Inspired by biology, synthetic mimics of the environment around cells, better known as the extracellular matrix (ECM), hold great promise in many fields including regenerative medicine and wearable electronics. – Using the chemistry of materials, flexible electronic materials are being developed that can interact with living tissue, with pioneering contributions by the Reichmanis lab and Bao laboratories. ,,– Moving toward the field of regenerative medicine, chemistry of materials is also being employed here to accurately mimic the native ECM to grow cells into more complex living tissues, of which hydrogels are an important and emerging class. ,– As accurate ECM mimics, such hydrogels must possess similar and preferably independently controllable mechanical, dynamic, and bioactive properties as those of the native ECM. – …”

“…Therefore, to achieve design rules for supramolecular hydrogel–cell interactions, we here investigate two different hydrogel systems with similar molecular designs employing slow-exchanging ureidopyrimidinone (UPy) monomers and fast-exchanging benzene-1,3,5-tricarboxamide (BTA) monomers. ,,,, Both hydrogel systems consist of 3 different molecular building blocks: monofunctional (M), bifunctional (B), and bioactive RGD (arginine−glycine−aspartic acid)-functionalized UPy or BTA monomers. , Herein, the M monomers form one-dimensional fibers. The B molecules intercalate in the monofunctional fibers to form cross-linked networks.…”