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.
This study proposes a treatment to improve post-trauma corneal wound healing through application of short-lived bursts of reactive oxygen species obtained via ionization of interstitial water by an ultrafast laser-induced low-density plasma. Laser irradiation has been restricted to intensities below the optical breakdown threshold and applied onto interleukin-1β (IL-1β) in aqueous solution, and rabbit corneas in vivo with epithelium removed to simulate external trauma. Quantitative ELISA assays have shown lowered binding affinity of the laser-treated IL-1β to receptor IL1-R1 in aqueous solutions. Laser application on epithelium removed in-vivo rabbit corneas resulted in a preservation of stromal keratocytes and accelerated healing.
Our recent work has demonstrated the application of femtosecond oscillators to crosslink corneal collagen in absence of photosensitizers to correct refractive errors and enhance corneal mechanical properties. Here we propose a new design of the femtosecond laser station enabling significantly reduce the time of the treatment procedure to clinically acceptable duration and report the results of one-dimensional parametric study on the control of the adjustment of corneal curvature. Fresh porcine eyes were subject to the laser irradiation ex vivo. The volumetric exposure to the laser has been executed by treating multiple planar areas at varying depths, measured from the ocular surface. The degree of the refractive error correction was adjusted by varying the number the laser treated planar areas. The eye topography has been monitored within 24-hour window post-treatment to assess degree and short term stability of the induced corrections. Results have shown that the correction of corneal refractive power can be controlled with resolution of approximately 0.75 diopter. Inflation tests were performed post-treatment accordingly to assess the viscoelastic response of crosslinked corneas. Rate dependent hysteresis curves obtained from the pressure-deformation response of treated corneas under physiological conditions showed statistically significant increase in stiffness in contrast to controls, in which no change has been observed.
A new paradigm for strengthening of corneal tissue as well as permanent correction of refractive errors has been proposed. Ultrafast laser irradiation is confined to the levels below optical breakdown such that tissue damage is avoided while creating an ionization field responsible for subsequent photochemical modification of the stroma. The concept was assed using newly developed platform for precise application of a near-IR femtosecond laser irradiation to the cornea in in-vitro experiments. Targeted irradiation with tightly focused ultrafast laser pulses allows spatially resolved crosslinking in the interior of the porcine cornea in the absence of photosensitizers. The formation of intra- or interstromal covalent bonds in collagen matrix locally increases lamellar density. Due to high resolution, treatment is spatially resolved and therefore can be tailored to either enhance structure of corneal stroma or adjust corneal curvature towards correcting refractive errors. As the induced modification is primarily driven by nonlinear absorption, the treatment is essentially wavelength independent, and as such potentially less harmful than current method of choice, joint application of UVA light irradiation in conjunction with riboflavin. Potential applicability of a near-IR femtosecond laser for biomechanical stabilization of cornea and non-invasive refractive eye corrections is discussed.
The onset of osteoarthritis (OA)in articular cartilage is characterized by degradation of extracellular matrix (ECM). Specifically, breakage of cross-links between collagen fibrils in the articular cartilage leads to loss of structural integrity of the bulk tissue. Since there are no broadly accepted, non-invasive, label-free tools for diagnosing OA at its early stage, Raman spectroscopyis therefore proposed in this work as a novel, non-destructive diagnostic tool. In this study, collagen thin films were employed to act as a simplified model system of the cartilage collagen extracellular matrix. Cross-link formation was controlled via exposure to glutaraldehyde (GA), by varying exposure time and concentration levels, and Raman spectral information was collected to quantitatively characterize the cross-link assignments imparted to the collagen thin films during treatment. A novel, quantitative method was developed to analyze the Raman signal obtained from collagen thin films. Segments of Raman signal were decomposed and modeled as the sum of individual bands, providing an optimization function for subsequent curve fitting against experimental findings. Relative changes in the concentration of the GA-induced pyridinium cross-links were extracted from the model, as a function of the exposure to GA. Spatially resolved characterization enabled construction of spectral maps of the collagen thin films, which provided detailed information about the variation of cross-link formation at various locations on the specimen. Results showed that Raman spectral data correlate with glutaraldehyde treatment and therefore may be used as a proxy by which to measure loss of collagen cross-links in vivo. This study proposes a promising system of identifying onset of OA and may enable early intervention treatments that may serve to slow or prevent osteoarthritis progression.
Collagen cross-linking in cornea has the capability of enhancing its mechanical properties and thereby providing an alternative treatment for eye diseases such as keratoconus. Currently, riboflavin assisted UVA light irradiation is a method of choice for cross-link induction in eyes. However, ultrafast pulsed laser interactions may be a powerful alternative enabling in-depth treatment while simultaneously diminishing harmful side effects such as, keratocyte apoptosis. In this study, femtosecond laser is utilized for treatment of bovine cornea slices. It is hypothesized that nonlinear absorption of femtosecond laser pulses plays a major role in the maturation of immature cross-links and the promotion of their growth. Targeted irradiation with tightly focused laser pulses allows for the absence of a photosensitizing agent. Inflation test was conducted on half treated porcine cornea to identify the changes of mechanical properties due to laser treatment. Raman spectroscopy was utilized to study subtle changes in the chemical composition of treated cornea. The effects of treatment are analyzed by observing shifts in Amide I and Amide III bands, which suggest deformation of the collagen structure in cornea due to presence of newly formed cross-links.
Laser assisted corneal surgeries often rely on the nonlinear absorption effect of ultrafast lasers to induce features in the interior of the cornea without affecting the surface. In particular, corneal flap formation in femtosecond assisted Laser- Assisted in situ Keratomileusis (LASIK) is based on the bubble creation. This study focuses on the interaction between the tissue and the femtosecond laser. Interior of cornea is treated with tightly focused femtosecond laser pulses. Due to the nature of the process, heating of the tissue within and around the focal volume is practically instantaneous. The affected region is subject to thermoelastic stress that arises with the steep temperature elevation. To predict the size of the region subject to the morphological changes due to the laser treatment, the temperature field is calculated. Cavitation bubble initiation and expansion process, which acts as precursor to the stress induced tissue trauma, is studied as well. Theoretical findings are compared against experimental results. High-speed camera is utilized to assess the laser treatment process, showing the temporal development of the cavitation bubbles. The results obtained in this study facilitate a better understanding of the effects of femtosecond laser assisted corneal surgeries and help in choosing optimal laser parameters.
In recent years, Raman spectroscopy has emerged as a potentially viable tool for automated cancer diagnostics.
However, due to the complexity of the signal obtained from a tissue, most of the studies have been confined to statistical analysis of the spectra with principal component analysis being most often reported as the analysis of choice. These types of analyses are sensitive to modification of the Raman spectra due to tissue processing. The study presented here addresses the modifications of the Raman spectra obtained from prostate tissue histopathological slides due to the tissue treatment and its influence on the automated cancer diagnostics via Raman spectroscopy.
Nonlinear absorption of femtosecond laser pulses enables the induction of bubble cavities in the interior of eye cornea
without affecting other parts of an eye, a phenomena utilized for flap formation in laser assisted corneal surgery. In the
present study laser pulses were focused in the interior of the sections of bovine cornea. Tight focus of the laser pulses
results in the plasma formation followed by its explosive expansion, which drives cavity formation. The morphology of
the generated features as well as the nature of the physical mechanisms of the phenomenon as a function of process
parameters is discussed. Numerical model is proposed to develop predictive capabilities for the feature size and shape
and the results are compared against the experimental findings.
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