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Dongyul Chai, Tibor Juhasz, Donald J. Brown, James V. Jester; Non-Linear Optical (NLO) Collagen Crosslinking. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1116.
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Non-linear optical, femtosecond laser-induced, two photon excitation provides for activation of photosensitizers within femtoliter volumes, thus allowing for precise control of photoactivated tissue reactions. The purpose of this study was to determine if NLO based excitation of riboflavin could induce crosslinking (CXL) and mechanical stiffening of collagen hydrogels.
Six compressed collagen hydrogels composed of rat-tail collagen type-1, averaging 179.33 + 73.2 μm in thickness, were divided into two groups (3 gels each), NLO (group 1) and conventional UVA crosslinking (group 2). NLO CXL of riboflavin soaked (0.5% w/v in PBS) hydrogels was performed by focusing 87 mW, 760 nm femtosecond laser light into the gels using a Zeiss 510 Meta CLSM and a 20x Zeiss Apochromat objective lens (NA=0.75). Gels were then scanned over a 5.2 mm x 5.2 mm area through the gel thickness at 2 um steps. Conventional CXL was achieved by irradiating a similar area with 3mW/cm2, 370 nm UVA light for 30 min. Mechanical stiffness was determined by measuring the indentation force through 1 mm displacement at 20 µm/sec velocity using a 250 μm diameter, round tip probe fixed to a force transducer after preloading. Hydrogel thickness was measured using second harmonic generation imaging. Hydrogel thickness and stiffness was measured at baseline and after crosslinking. Indentation modulus, E, was calculated using the following equation,E = f(v)3 a2 P / (d3h), f(v) ≈ 1.049 - 0.146v - 0.158v2E: indentation modulus, d: indentation displacement, v: 0.5 (Poisson’s ratio), a: radius of window holding gel, h: gel thickness, P: indenting force
Mechanical stiffness of hydrogels before CXL averaged 0.45 (± 0.14) MPa. Following CXL there was a 3.35 (± 1.80) fold increase using NLO and a 4.38 (± 4.61) fold increase using UVA CXL. There was no difference between NLO and UVA CXL.
This is the first study to demonstrate that non-linear optical photodynamic therapy (NLO-PT) can be used to increase the mechanical stiffness of collagen hydrogels through crosslinking. Translation of this technology to the cornea may provide for a more precise, safe and effective therapeutic strategy for treating Keratoconus and other ectatic disorders compared to conventional UVA crosslinking.
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