Fibroblast migration correlates with matrix softness. A study in knob-hole engineered fibrin.

Christopher Y Leon-Valdivieso,Roberto Donno,Nicola Tirelli,Matilde Ghibaudi,Enrique Lallana,Iwan Roberts,Annalisa Tirella,Jennifer Wedgwood

doi:10.1063/1.5022841

Christopher Y Leon-Valdivieso, Roberto Donno + Show 6 more

Open Access

https://doi.org/10.1063/1.5022841

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Abstract

The invasion of a matrix by migrating cells is a key step in its remodelling. At least in 2D migration models, cells tend to localize in stiffer areas (durotaxis). Here, we show that mechanical properties affect differently the 3D migration rate: non-proteolytic 3D cell migration is facilitated in softer matrices. In these gels, the modulus was varied by introducing defects in fibres, leaving largely intact the nanostructure. The matrices derive from fibrin via functionalization with a bioinert polymer [poly(ethylene glycol), PEG] through an affinity mechanism identical to that presiding to fibrin own self-assembly. Peptidic end groups on PEG were used to bind fibrinogen globular D regions [GPRP (glycine-proline-arginine-proline) for a holes, GHRP (glycine-histidine-arginine-proline) for b holes; Kd evaluated via isothermal titration calorimetry or fluorescence anisotropy]. In a dose-dependent manner, both PEGylated peptides decreased gel stiffness, but most other properties at a macroscopic [e.g., overall elastic character, strain hardening, and high (>0.5) Poisson ratio] or nano/micro level (fibre dimension and pore size) were largely unaffected, suggesting that the softening effect was due to the introduction of defects within fibres, rather than to differences in the network architecture. In these matrices, the key determinant of fibroblast migration was found to be the elastic modulus, rather than the identity or the dose of the PEGylated peptide; softer materials allowed a faster invasion, even if this meant a higher content of non-adhesive PEG. This does not conflict with fibroblast durotaxis (where stiffness controls accumulation but not necessarily the speed of migration) and indicates a way to fine tune the speed of cell colonization.

Highlights

Remodelling: rapid degradation is accompanied by macroscopic, cell-mediated contraction, leading to non-functional constructs.5 Protease inhibitors such as aminocaproic acid, aprotinin, or matrix metalloproteinase (MMP) inhibitors6 are typically used to ease these problems; this issue can be tackled upstream, by controlling the rate of cell invasion, e.g., via engineering the matrix structure
poly(ethylene glycol) (PEG) was modified with a terminal vinyl sulfone (PEG-VS) to react with the cysteine residues through Michael-type addition; the reaction partners were selected for their reactivity, the absence of side products and the stability of the reaction product: sulfones, unlike, e.g., acrylates, are not hydrolysable, and there is little risk of retro-Michael reaction, as for maleimides
PEG-VS was prepared from 2 kDa PEG-OH and an excess of divinylsulfone (DVS) according to a literature procedure,29 which minimizes the possible side reactions of alcoholates by using a catalytic amount of NaH as a nonnucleophilic base

Summary

Introduction

Remodelling: rapid degradation is accompanied by macroscopic, cell-mediated contraction, leading to non-functional constructs. Protease inhibitors such as aminocaproic acid, aprotinin, or matrix metalloproteinase (MMP) inhibitors are typically used to ease these problems; this issue can be tackled upstream, by controlling the rate of cell invasion, e.g., via engineering the matrix structure. Remodelling: rapid degradation is accompanied by macroscopic, cell-mediated contraction, leading to non-functional constructs.5 Protease inhibitors such as aminocaproic acid, aprotinin, or matrix metalloproteinase (MMP) inhibitors are typically used to ease these problems; this issue can be tackled upstream, by controlling the rate of cell invasion, e.g., via engineering the matrix structure. The fibrinogen-fibrin conversion operated by thrombin has two main steps2,14,15 [Scheme 1(a)]; in the first, the cleavage of fibrinopeptides A (Fp A) exposes N-terminal glycine-proline-arginine (GPR) amino acid. Once grown to 600–800 nm, protofibrils aggregate laterally; the thrombinmediated cleavage of fibrinopeptides B (Fp B) exposes glycine-histidine-arginine (GHR) amino acid sequences (B-knobs) at the N-termini of Bb-polypeptide chains, allowing this second fibrin structure (fibrin II, desAABB fibrin or ab-fibrin16) to bind b-holes in other fibrin molecules within the protofibril.. Once grown to 600–800 nm, protofibrils aggregate laterally; the thrombinmediated cleavage of fibrinopeptides B (Fp B) exposes glycine-histidine-arginine (GHR) amino acid sequences (B-knobs) at the N-termini of Bb-polypeptide chains, allowing this second fibrin structure (fibrin II, desAABB fibrin or ab-fibrin16) to bind b-holes in other fibrin molecules within the protofibril. The fibrillar network is stabilized by further lateral aggregation (intermolecular interactions between aC-domains of different fibrin molecules) and Ca2þ-dependent covalent cross-linking by factor XIIIa (a plasma transglutaminase).

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