Siniša Vukelić

Background: The pentose-initiated Maillard reaction may serve as an oxygen-independent, non-enzymatic crosslinking (CxL) mechanism, but its slow kinetics limit clinical applicability. We hypothesize that glycation-mediated CxL can be accelerated by reactive oxygen species (ROS), potentially enabling its use in the treatment of keratoconus. Methods: was used in combination with either ribose or riboflavin-5-phosphate to generate comparison and control groups. ROS-Glycation-CxLed corneas were flattened with a glass coverslip to minimize oxygen replenishment. Crosslinking efficacy was assessed using 5 µm micro-indentation to measure the equilibrium modulus and viscoelastic ratio post-treatment. Results: Corneal tissues subjected to ROS-glycation-CxL exhibited a significant increase in equilibrium modulus. The modulus change in ribose-exposed tissues treated with UVA light was comparable to that observed with the conventional UVA-riboflavin CxL pathway. In contrast, infrared and blue laser-treated samples showed less stiffening, likely due to the limited tissue volume affected by the laser's focal region. Notably, only the UVA-riboflavin-treated tissues demonstrated a pronounced elastic load-relaxation response. Conclusion: ROS-Glycation-CxL offers a potential alternative to existing crosslinking techniques, with crucial advantages such as reduced oxygen reliance, light-source agnostic mechanism, and customizable treatment patterns with laser light sources. Further investigation is needed to evaluate the clinical viability of ROS-glycation CxL through optimization of sugar concentration, treatment duration, and assessment of stromal extracellular matrix ultrastructure.

Eye injuries are a prevalent global health concern, with millions of cases annually resulting in blindness and vision impairment. Injuries to the cornea, the transparent outer layers of the eye, may lead to scar-driven fibrosis that decreases visual clarity and light refraction to the retina. Traditional wound healing therapies have shown limited success in achieving scar-free wound recovery due to the complex nature of the corneal wound healing process, which involves multiple stages regulated by cytokines and growth factors. In this study, we have utilized rabbit models to investigate whether femtosecond laser-generated reactive oxygen species (ROS) can enhance the wound healing process. A low-power femtosecond laser induced ROS-producing low-density plasma in the injured rabbit cornea, aiming to mitigate inflammation and avoid scar formation. Ultrafast laser-generated low-density plasma ionizes and dissociates interstitial water in the corneal stroma, resulting in ROS production in the absence of tissue-damaging shock and thermoacoustic waves, thus allowing for precise treatment delivery. The proposed treatment has enhanced the wound healing process for corneal abrasion through binding affinity modulation of post-trauma released cytokines. Further experiments on isolated cytokines and their receptors validated ROS' ability to modulate molecular interactions.

Interstitial fluid load support (FLS) is a dominant mechanism of lubrication in cartilage, producing a low friction coefficient while enhancing the tissue’s load bearing capabilities. Due to its viscosity, synovial fluid (SF) may retard loss of FLS by slowing the exudation of interstitial fluid from the cartilage. This study tested this hypothesis by comparing the stress-relaxation (SRL) response of immature bovine articular cartilage immersed either in phosphate buffered saline (PBS) or in healthy mature bovine SF, under unconfined compression (fluid exudation across cut lateral tissue boundary) and indentation testing (fluid exudation across articular surface). To investigate the influence of diffusion of SF molecular constituents into cartilage, the effect of incubation time in SF on SRL was also investigated. The SRL response in unconfined compression was not significantly different in PBS versus SF when compared directly (p=0.98) and had a slope of m=1.00 ± 0.04 (R2=0.989 ± 0.007). Samples tested in PBS exhibited characteristic relaxation times, τPBS=42.6 ± 5.3 s and τSF=40.8 ± 4.7 s, that were not significantly different (p=0.40). Incubation time of 24 hours in SF resulted in no significant difference in the SRL response (p=0.39, m=1.03 ± 0.12; R2=0.983 ± 0.011, and τPBS=43.4 ± 10.7 s versus τSF=41.5 ± 4.8 s, p=0.59). Indentation testing showed some statistically significant, but functionally insignificant, difference in SRL responses in PBS versus SF with a slope of m=0.958 ± 0.060 (R2=0.957 ± 0.020, p=0.029, and τPBS=16.9 ± 2.6 s versus τSF =19.4 ± 3.3 s, p=0.073). Based on these results, we reject the hypothesis that healthy SF can retard the loss of FLS in cartilage due to its viscosity.

Objective: Based on our recent study, which showed that cartilage fatigue failure in reciprocating sliding contact results from cyclical compressive forces, not from cyclical frictional forces, we hypothesize that a major functional role for synovial fluid (SF) is to reduce the rate of articular cartilage fatigue failure from cyclical compressive loading. Design: The rate of cartilage fatigue failure due to repetitive compressive loading was measured by sliding a glass lens against an immature bovine cartilage tibial plateau strip immersed in mature bovine SF, phosphate-buffered saline (PBS), or SF/PBS dilutions (50% SF and 25% SF; n=8 for all four bath conditions). After 24 hours of reciprocating sliding (5,400 cycles), samples were visually assessed and if damage was observed, the test was terminated; otherwise, testing was continued for 72 hours (16,200 cycles), with solution refreshed daily. Results: All eight samples in the PBS group exhibited physical damage after 24 hours, with an average final surface roughness of Rq=0.210±0.067mm. The SF group showed no damage after 24 hours; however, two of eight samples became damaged after 72 hours, producing a significantly lower average surface roughness than the PBS group Rq=0.059±0.030mm;p<10−4). For the remaining groups, at 72 hours one of eight samples damaged in the 50% SF group, and five of eight samples damaged in the 25% SF group. Conclusions: The results strongly support our hypothesis, showing that decreased amounts of SF in the testing bath produces increased rates of fatigue failure in cartilage that was subjected to reciprocating sliding contact.

Reactive Oxygen Species (ROS) affect biological processes in many ways, and their effect is a function of strength, content and the exposure duration. Whereas excessive oxidative stress has potential to cause various deleterious events such as damaged protein structure, interfered activation of signaling cascades, and even cell apoptosis, we argue that targeted and carefully dosed application of ROS can be used as a therapeutic modality. In appropriate dosage ROS can induce stimulatory effects on cells and activate pathways leading to migration and collagen synthesis. At the tissue level ROS are utilized in photochemical crosslinking. Thus, we propose use of femtosecond oscillators as a therapeutic platform that can be utilized for ocular wound healing, non-invasive vision correction, as well as treatment of keratoconus and early osteoarthritis.