Decellularization Induces Hierarchical Disorganization in Ligament Scaffolds
Ligament injuries, particularly those affecting the Anterior Cruciate Ligament (ACL) or Medial Collateral Ligament (MCL), present a significant orthopedic challenge due to the tissue's poor vascularization and limited regenerative capacity. Decellularized extracellular matrix (dECM) scaffolds have emerged as a promising solution, offering a natural framework that minimizes immune rejection while theoretically preserving the native architecture necessary for mechanical function.
However, there is a persistent "blind spot" in tissue engineering: scaffolds that appear structurally sound on a macroscopic level often fail mechanically in vivo. A new study published in European Cells and Materials investigates this discrepancy, utilizing a multi-scale analysis to reveal how standard decellularization protocols can compromise the hierarchical integrity of ligament tissue.
Researchers Sun et al. utilized rabbit medial collateral ligaments (MCL) to evaluate a combined decellularization protocol involving freeze-thaw cycles, guanidine, SDS, and trypsin. On the surface, the results were successful: the protocol removed over 96% of DNA and the gross morphology of the tissue appeared intact. Standard biochemical assays based on hydroxyproline indicated that the total collagen content remained statistically unchanged.
Despite this, mechanical testing revealed a significant drop in the tensile modulus and energy dissipation capacity. Under extreme tensile conditions, the decellularized ligaments exhibited pronounced fiber tearing and misalignment compared to native tissue. To understand why a scaffold with "normal" collagen content was mechanically weaker, the researchers had to look deeper than standard histology allows—specifically at the molecular unfolding of the collagen triple helix.
How did CHPs Uncover Hidden Collagen Damage?
To detect damage that traditional methods missed, the researchers employed Fluorescent Collagen Hybridizing Peptides (F-CHP).
While standard dyes like Fast Green showed that collagen fibers were present, they could not discern the molecular quality of those fibers. The study found that while native ligaments showed almost no CHP signal (indicating intact triple helices), the decellularized tissue exhibited distinct, enhanced green fluorescence.
"The F-CHP staining results revealed that... distinct enhanced green fluorescence was observed in the decellularized tissue. This indicates that the decellularization process induced collagen helix unwinding in the ligament tissue, leading to collagen fiber denaturation and modification."
This CHP analysis provided the "smoking gun" for the mechanical loss. The chemical agents (particularly SDS and Guanidine), while effective at removing cells, disrupted the non-covalent interactions and hydrogen bonding that stabilize the collagen triple helix. This molecular unfolding—undetectable by standard hydroxyproline assays—correlated directly with the loss of micromechanical integrity, increased porosity, and the disruption of D-banding periodicity observed via Atomic Force Microscopy (AFM).
This study highlights a critical consideration for the field of regenerative medicine: collagen quantity does not equal collagen quality.
By utilizing Collagen Hybridizing Peptides, the researchers demonstrated that decellularization can induce hierarchical disorganization at the nanometer scale, compromising the scaffold's ability to bear loads. As the authors suggest, future optimization of decellularization strategies must focus not just on removing cellular material, but on preserving the molecular folding and D-band periodicity of the collagen matrix to ensure successful in vivo application.
Citation:
Sun, J. L., Yang, M. Y., Zhou, J. H., Jiang, N., & Chen, H. Z. (2025). Decellularization induces hierarchical disorganization of ligaments matrix at both micrometer and nanometer scales to change their mechanical behavior. European Cells and Materials, 53, 28–40. DOI: 10.22203/eCM.v053a03
