KneEZ Clear, an Effective Tissue Clearing Protocol to Study Musculoskeletal Tissues in the Mouse

Area of Science:

• Biomedical imaging
• Tissue engineering
• Histology

Background:

• Traditional histological methods for musculoskeletal tissues are time- and labor-intensive.
• Current tissue clearing methods involve solvent-based approaches that alter tissue architecture or expensive hydrogel-based methods.
• There is a need for an accessible, optimized aqueous-based clearing method for musculoskeletal tissues.

Purpose of the Study:

• To develop and present KneEZ Clear, a simple, rapid, and flexible aqueous-based method for optical clearing of mineralized and non-mineralized musculoskeletal tissues.
• To demonstrate the efficacy of KneEZ Clear across various murine musculoskeletal tissues.
• To highlight the method's ability to preserve endogenous fluorescence and allow for subsequent histological analysis.

Main Methods:

• KneEZ Clear is an aqueous-based tissue clearing method adapted from the EZ Clear protocol.
• The method was applied to various murine musculoskeletal tissues, including knee joints, vertebral column, hindlimb, skull, and teeth.
• The protocol was tested for its ability to retain endogenous fluorescence and allow for post-clearing histological processing.

Main Results:

• KneEZ Clear successfully rendered diverse murine musculoskeletal tissues optically transparent.
• The method demonstrated flexibility across multiple tissue types without requiring specialized equipment.
• Endogenous fluorescence signals were retained, and samples could be further processed for 2D histology.
• KneEZ Clear was applied to disease models, revealing alterations in tissue architecture and homeostasis.

Conclusions:

• KneEZ Clear provides a simple, rapid, and versatile solution for optical clearing of musculoskeletal tissues.
• The method overcomes limitations of existing techniques, preserving tissue integrity and endogenous signals.
• KneEZ Clear has the potential to accelerate research on cellular interactions in tissue homeostasis and disease.