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

Abstract

PurposeTo 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.MethodsIn 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.ResultsMean 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.ConclusionsThe findings of this study suggest that collagen cross-linking is a viable and effective modality for increasing the stiffness of human tarsal plates.Translational RelevanceThis work provides proof that collagen cross-linking produces stiffening of the human tarsal plate and may be used in disorders that cause eyelid laxity.

Collagen Structural Changes in Rat Tarsus After Crosslinking

“…Tarsal tissue from larger animals or human tissue are therefore needed before we can say with certainty that crosslinking at these energy parameters is effective in human tarsus. Additionally, our histopathology studies are mainly qualitative, although they do agree with the more quantitative histology work done by Ugradar et al 5 …”

“…Although others [3][4][5][6] have previously described methodologies for crosslinking tarsus, we are, to the best of our knowledge, the first to use SHG microscopy to study collagen fiber changes after CXL in the tarsus. This also has the advantage of not requiring delicate tissues to be stained or fixed, processes that are known to alter the cellular nanostructure.…”

“…Ugradar et al . 5 crosslinked thawed cadaveric human eyelids with 1% hypertonic riboflavin solution and a 365-nm LED power source of 6 mW/cm 2 over 18 minutes (total energy, 6.48 J/cm 2 ). Finally, DeParis et al .…”

“…Similarly, Ugradar et al . 5 achieved CXL with the second-lowest energy output (6.48 J/cm 2 ) and used 1% hypertonic riboflavin. Finally, it may also be true that crosslinking can be achieved with either combination, meaning low energy and high-concentration riboflavin are effective, as well as high energy and lower concentration riboflavin.…”

“…Recently, tarsal crosslinking (CXL), or the process of forming strong covalent bonds between neighboring collagen fibrils, has been explored in animal and cadaver models as an alternative treatment to stiffen tarsus in FES. [3][4][5][6] Crosslinking is already well known to most ophthalmologists as a treatment for keratoconic corneas. In the US Food and Drug Administrationapproved Dresden protocol, corneas incubated with riboflavin are exposed to ultraviolet A (UV-A) radiation.…”