Generation of a Bioengineered Substitute of the Human Sclero-Corneal Limbus Using a Novel Decellularization Method

Area of Science:

• Biomaterials Science
• Ophthalmology
• Regenerative Medicine

Background:

• Severe limbal stem cell deficiency presents a significant therapeutic challenge, particularly when limbal niche structural alterations are present.
• Existing treatments for limbal dysfunction are often limited, especially in cases of structural damage.

Purpose of the Study:

• To evaluate seven decellularization protocols for human sclero-corneal limbus tissue.
• To identify protocols that efficiently remove cellular material while preserving extracellular matrix integrity and supporting cell regeneration.

Main Methods:

• Seven decellularization protocols using various detergents (SDS, Triton X-100, SDC, sulfobetains) and enzymatic treatments were tested on human limbal tissue.
• Decellularization efficiency, DNA removal, extracellular matrix preservation, biocompatibility (macrophage response), and support for corneal epithelial and mesenchymal stem cell attachment and differentiation were assessed.

Main Results:

• Protocols P1-P4 using SDS were inefficient for decellularization.
• Protocols P5, P6, and P7 effectively removed >95% of DNA while preserving 60-100% of extracellular matrix components.
• Protocols P6 and P7 demonstrated biocompatibility, supported cell attachment and differentiation, with P6 showing superior limbal cell marker expression.

Conclusions:

• Protocol P6 is recommended for generating human limbal substitutes via tissue engineering using decellularized human limbi.
• The regenerative biomaterial holds potential for clinical application in patients with structural limbal damage, including those with chemical burns and aniridia.