Air-Coupled Ultrasonic Optical Coherence Elastography Reveals Protocol-Dependent Corneal Stiffening in Epi-On and Epi-Off Riboflavin Crosslinking

Purpose: To evaluate the biomechanical impact of corneal crosslinking (CXL) when combining three different ultraviolet A (UV-A) riboflavin (RB) photosensitizers and protocols (Dresden protocol [DP] and accelerated protocol [AP]) using air-coupled ultrasonic optical coherence elastography in ex vivo rabbit corneas. Methods: An air-coupled ultrasound excitation optical coherence elastography system was used to excite the corneal apex and generate Lamb wave propagation along 16 corneal cross-sectional meridians. Measurements were conducted on ex vivo rabbit eyes (n = 45) during three treatment phases: Virgin, after 30 minutes of RB soaking, and after UV-A irradiation. The protocols used include the DP (3 mW/cm2, 30 minutes) and two accelerated protocols (AP-1: 9 mW/cm2, 10 minutes; and AP-2: 30 mW/cm2, 3 minutes). Each protocol was tested with three photosensitizers: epithelium-on (TE), and epithelium-off with hyperosmolar (M) and hypo-osmolar solutions (D). Lamb wave speed and average corneal thickness were calculated for each meridian to estimate meridian-dependent corneal shear modulus. Differences were statistically analyzed using linear mixed-effects regression. Results: The DP-D produced the strongest significant corneal stiffness increase (480.5 kPa; P < 0.001) during the UV irradiation phase, and the most pronounced corneal thinning (186 µm; P < 0.001). DP-TE achieved the next strongest stiffening (309.5 kPa) during UV irradiation, with no significant thickness change. All other protocol-photosensitizer combinations did not achieve significant corneal stiffening except for AP-1-D and AP-2-D during the RB soaking phase. Conclusions: Accelerated protocols did not produce significant corneal stiffening for any RB photosensitizer except when using dextran-based D. DP-D and DP-TE produced the greatest balance between shear modulus increase and corneal thickness decrease, suggesting DP-TE as a potential compromise between biomechanical impact, corneal integrity, and faster postoperative recovery. Translational Relevance: This work quantified the impact of clinically used crosslinking photosensitizers and protocols on corneal stiffening using an air-coupled ultrasound excitation optical coherence elastography, which holds promise for clinical assessments in patients.