Selective two-photon collagen crosslinking: Why more is better

Selective two-photon collagen crosslinking has been shown to stiffen any three-dimensional region inside corneal tissue without damaging surrounding tissues.

Reviewed by Seok Hyun Yun, PhD, and Sheldon J.J. Kwok, BA

Cambridge, MA—Investigators at five top research centers have developed a new light-based polymerization, two-photon crosslinking (2P-CXL) that mechanically strengthens corneal tissue in situ without damaging surrounding untreated tissue.

CXL is used extensively in Europe and Canada for treating patients with keratoconus and has recently received FDA approval in the United States.

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The currently used single-photon CXL process typically uses ultraviolet light to treat the cornea. 2P-CXL differs from standard CXL in that it uses two-photon absorption-mediated processes using near infrared light supplied by a femtosecond laser, according to Seok Hyun Yun, PhD, who explained that this technologic advance offers the benefit of “far superior spatial resolution that is confined to the focal volume.”

This technology itself, while not entirely new, has been used for a liquid-based, three-dimensional printing process, in the fabrication of micro-optic components, and hydrogel-based cellular scaffolds.

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However, its newest application is for treatment of soft biologic tissue.

To date, results have been reported with artificial cardiac tissue and collagen hydrogels, and a previous study reported using 2P-CXL in corneal tissue in which changes in collagen second harmonic generation were seen but no evidence of tissue stiffening.

Dr. Yun and colleagues have moved another step forward in a study in which they showed spatially selective 2P-CXL of intact tissue for the first time. The investigators reported their findings in the journal Optica (2016;3:469-472).

Brillouin microscopy


In this animal study, they tested the ability to control the technology’s spatial selectivity in tissue and pinpoint its clinical effects more precisely. They used femtosecond laser pulses and riboflavin for 2P-CXL in the corneas of the animals.

Following treatment, confocal Brillouin microscopy was performed to validate their results. Brillouin microscopy is a technique developed recently by Dr. Yun and colleagues that uses light to probe mechanical properties non-invasively in cells and tissues.

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Currently, Brillouin microscopy is used to study the cornea in normal subjects and patients with keratoconus.

“While conventional CXL crosslinks the entire stromal depth of the cornea (over 500 μm), 2P-CXL enables crosslinking of any arbitrary three-dimensional region within the cornea,” explained Sheldon Kwok, BA, lead author on the paper. Kwok is an MD/PhD candidate, Harvard-Massachusetts Institute of Technology Health Sciences and Technology, Harvard Medical School, and a member of the Yun Bio-Optics Lab, Wellman Center for Photomedicine, Massachusetts General Hospital, Boston.

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The investigators used their technique to demonstrate crosslinking of a rectangular region 50 μm below the cornea surface. Using Brillouin microscopy, they found the crosslinked region was significantly stiffer than surrounding regions.

Importantly, the tissue stiffening induced by 2P-CXL was found to be comparable to that achieved with conventional CXL.

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The investigators concluded, “…we have demonstrated three-dimensional selective CXL in corneal tissue. Our technique involves in situ 2P-CXL of an arbitrary three-dimensional structure, and verification and characterization with confocal Brillouin microscopy.”

Compare with conventional procedures


Compared with conventional procedures for evaluating 2P-CXL-induced stiffening, Brillouin microscopy does not require perturbation of the surrounding non-crosslinked regions, they noted.

The investigators commented, “Our results demonstrate the ability to alter the microstructure and mechanical modulus locally in the cornea by 2P-CXL with microscopic resolution and illustrate the unique advantage of Brillouin microscopy in evaluating 2P-CXL-induced changes nondestructively.”

They also pointed out other uses for 2P-CXL in ophthalmology in addition to treating thin ectatic corneas (<400 μm), such as corneal flap bonding post-LASIK, and the selective modulation of corneal curvature for refractive error correction.

“Since 2P-CXL has such high spatial selectivity in three-dimensions, and the fact that it is a non-surgical and non-invasive technique,” Kwok said, “it could have unique advantages for correcting refractive errors in patients.”

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Seok Hyun Yun, PhD


Dr. Yun and Mr. Kwok have a patent pending for this technology. The institutions involved in this research were Harvard-MIT Division of Health Sciences and Technology and Wellman Center for Photomedicine at Massachusetts General Hospital, both in Boston; and the Departments of Biomedical Engineering at Johns Hopkins University, Sungkyunkwan University, and University of Maryland, in Baltimore, Suwon, South Korea, and College Park, MD, respectively.

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