Biomechanical and Morphologic Effects of Collagen Cross-Linking in Human Tarsus

Abstract

Purpose: To investigate the feasibility of increasing the stiffness of human tarsal tissue following treatment with riboflavin and ultraviolet A (UVA) to induce cross-linking of collagen fibers.

Methods: In this case control study, 18 right and left upper eyelids were excised en bloc from 18 fresh-frozen cadavers. One side served as the control while the samples from the opposite side were cross-linked. Four 2 × 6-mm vertical strips of central tarsus were cut from the superior to inferior border of each tarsal plate. Sample tissue was irradiated with UVA at 6 mW/cm² for 18 minutes. A microtensile load cell and an optical coherence tomography scanner allowed calculation of stiffness (Young's modulus). Six cross-linked samples and corresponding controls were stained with hematoxylin and eosin (H&E) and Masson trichrome stains. Four controls and four cross-linked samples were also reviewed with a transmission electron microscope.

Results: Mean Young's modulus in the linear region for controls was 28 ± 9 MPa and was much higher at 138 ± 8 MPa for cross-linked samples (P < 0.001), yielding a 493% mean stiffness increase. Staining with H&E and Masson did not reveal any histologic changes. Transmission electron microscopy showed a decrease in average diameter of 50 randomly selected collagen fibers from 47.2 ± 1.9 nm prior to cross-linking to 34.2 ± 1.1 nm post cross-linking (P < 0.001). Qualitatively, the collagen fibers appeared more closely packed following cross-linking.

Conclusions: The findings of this study suggest that collagen cross-linking is a viable and effective modality for increasing the stiffness of human tarsal plates.

Translational relevance: This work provides proof that collagen cross-linking produces stiffening of the human tarsal plate and may be used in disorders that cause eyelid laxity.

Keywords: cross-linking; floppy eyelids; tarsus.

Figure 1

Microtensile load cell. A strain gauge (A) attached to a linear motor (B) was connected in the temperature- and humidity-controlled chamber to the specimen clamp (E) that was supported by a frictionless air bearing (C). An OCT scanner (D) was directed at the specimen.

Figure 2

Intensity of UV light that has passed through a human tarsal plate at different illumination intensities.

Figure 3

Stress-strain plot averaging 18 cross-linked and 18 control human tarsal specimens. The slope of the curves in the linear region (dotted lines) represents Young's modulus in the high-strain region.

Figure 4

Masson trichrome staining of control (A) and cross-linked (B) tissue. Meibomian glands appear red (yellow arrows). Collagen appears blue. Blood vessels (BV) also appear red.

Figure 5

Transmission electron microscopy of the tarsal plates at 100,000× magnification, revealing a change in collagen organization and narrowing of collagen fibers.