# Catalyst‐Free Collagen Filament Crosslinking for Engineering Anisotropic and Mechanically Robust Tissue Scaffolds

## TL;DR

A new method creates strong, cell-friendly collagen scaffolds for tissue engineering, supporting muscle regeneration in mice.

## Contribution

A catalyst-free, bioorthogonal crosslinking strategy for collagen scaffolds with mechanical resilience and cell compatibility.

## Key findings

- Collagen hydrogels crosslinked with rhodamine and PEG show enhanced stiffness and mechanical strength.
- Aligned collagen filaments support hASC encapsulation and activate mechanotransductive signaling.
- The method promotes functional muscle regeneration in a murine muscle loss model.

## Abstract

Engineering mechanically resilient hydrogels from naturally derived proteins, such as collagen and gelatin, remains a key challenge in tissue regeneration, particularly when cell compatibility and structural integrity are simultaneously required. Here, a bioorthogonal crosslinking strategy using rhodamine and polyethylene glycol (PEG) is reported to fabricate dense, mechanically reinforced collagen hydrogels. PEG‐mediated dehydration induces spontaneous peptide bond formation between rhodamine and collagen without the need for additional catalysts, yielding fibrous protein networks with enhanced stiffness. To enable anisotropic tissue engineering, this crosslinking method is integrated with wet‐spinning to produce uniaxially aligned collagen filaments. These constructs exhibit high mechanical strength and support human adipose‐derived stem cell (hASC) encapsulation. Mechanotransductive signaling, including cytoskeletal organization and myogenic gene expression, is effectively activated within the aligned filaments. The applicability of cell‐laden filaments in a murine volumetric muscle loss model is demonstrated, which promoted in vitro differentiation and in vivo functional muscle regeneration. This strategy offers a scalable and cytocompatible platform for generating aligned protein‐based scaffolds with tunable mechanical and biological properties, thereby expanding the toolkit for regenerative medicine.

A bioorthogonal rhodamine/PEG crosslinking strategy is introduced to engineer dense collagen hydrogels with high mechanical resilience and cytocompatibility. Integration with wet‐spinning enables the fabrication of uniaxially aligned, cell‐laden collagen filaments that activate mechanotransductive signaling and support functional muscle regeneration in a murine volumetric muscle loss model.