Laser crosslinking for photo-chemical enhancement of collagenous tissues

Abstract

A method for treating collagenous tissue, comprising: illuminating a region of the collagenous tissue with an illumination from an illumination source, the illumination being transmitted through a substrate having a first portion that contacts the collagenous tissue and a second portion that is free of contact with the collagenous tissue, the illumination comprising any one or more of a femtosecond laser illumination or an ultraviolet-A (UVA) illumination, an agent being disposed between the illumination source and the collagenous tissue such that interaction between the agent and the illumination evolves a plasma that gives rise to crosslinking of the collagenous tissue, the agent optionally being biocompatible, and the plasma optionally being a low-density plasma.

Description

CU24195 / 101879.003099 LASER CROSSLINKING FOR PHOTO-CHEMICAL ENHANCEMENT OF COLLAGENOUS TISSUES RELATED APPLICTIONS

[0001] The present application claims priority to and the benefit of United States patent application no.63 / 633,982, “Laser Crosslinking For Photo-Chemical Enhancement Of Collagenous Tissues,” filed April 15, 2024; and United States patent application no. 63 / 625,458, “Femtosecond Laser Crosslinking For Photo-Chemical Enhancement Of Collagenous Tissues,” filed January 26, 2024. All foregoing applications are incorporated herein by reference in their entireties for any and all purposes. GOVERNMENT RIGHTS

[0002] This invention was made with government support under AR073289 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD

[0003] The present disclosure relates to the field of tissue engineering, in particular to the field of crosslinking collagenous tissues. BACKGROUND

[0004] It has been shown that femtosecond laser-based crosslinking of collagenous tissues (cornea and cartilage) in absence of photosensitizers is possible. At that time, we hypothesized that when one restricts the lasing regime below the optical breakdown, under appropriate conditions low-density plasma is formed which ionizes interstitial water in the tissue creating reactive oxygen species, which react with collagen to form crosslinks. There is, however, a need for further methods of crosslinking collagenous tissues. SUMMARY

[0005] In meeting the described needs, the present disclosure provides a method for treating collagenous tissue, comprising: illuminating a region of the collagenous tissue withCU24195 / 101879.003099 an illumination from an illumination source, the illumination being transmitted through a substrate having a first portion that contacts the collagenous tissue and a second portion that is free of contact with the collagenous tissue, the illumination comprising any one or more of a femtosecond laser illumination or an ultraviolet-A (UVA) illumination, an agent being disposed between the illumination source and the collagenous tissue such that interaction between the agent and the illumination evolves a plasma that gives rise to crosslinking of the collagenous tissue, the agent optionally being biocompatible, and the plasma optionally being a low-density plasma.

[0006] Also provided is a system, the system configured to perform the method of the present disclosure, for example the method according to any one of Aspects 1-20.

[0007] Further disclosed is a system for treatment of a collagenous tissue having an initial shape, comprising: an illumination source, the illumination source comprising any one or more of a femtosecond laser illumination or an ultraviolet-A (UVA) illumination; a substrate, the substrate having a first portion arranged for contact with the collagenous tissue and a second portion arranged to be free of contact with the collagenous tissue; and an agent, the agent positioned between the illumination source and the collagenous tissue such that interaction between the agent and illumination from the illumination source evolves a plasma that gives rise to crosslinking of the collagenous tissue.

[0008] Also disclosed is a method for treating collagenous tissue, comprising: applying a femtosecond laser to a particular area of a collagenous tissue so as to induce localized optical breakdown of the collagenous tissue at the particular area; at least one of (i) directing the femtosecond laser away from the particular area so as to initiate low-density plasma formation or (ii) removing the femtosecond laser from the particular area so as to initiate low-density plasma formation; and crosslinking the collagenous tissue by the low- density plasma formation.

[0009] Also provided is a method for treating collagenous tissue, comprising: applying a femtosecond laser to a pigmented area of a collagenous tissue so as to induce localized optical breakdown of the collagenous tissue at the pigmented area; at least one of (i) directing the femtosecond laser away from the pigmented area so as to initiate low-density plasma formation or (ii) removing the femtosecond laser from the pigmented area so as to initiate low-density plasma formation; and crosslinking the collagenous tissue by the low- density plasma formation.CU24195 / 101879.003099

[0010] Also provided is a method for treating collagenous tissue, comprising: illuminating a region of the collagenous tissue with an illumination from an illumination source, the illumination comprising any one or more of a femtosecond laser illumination or an ultraviolet-A (UVA) illumination, the illumination evolving reactive oxygen species that modulate cytokine binding within the collagenous tissue, the method optionally being performed such that the method does not give rise to crosslinking within the collagenous tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In the drawings, which are not necessarily drawn to scale, like numerals may describe similar components in different views. Like numerals having different letter suffixes may represent different instances of similar components. The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed in the present document. In the drawings:

[0012] FIG.1 provides an exemplary depiction of the disclosed technology. As shown, when laser application was applied to a marked region of material, the marker was depleted.

[0013] FIG.2 provides a description of an exemplary setup of the disclosed technology. As shown, the plasma was generated at the location of the dark marker; no sparking was seen while the laser was passed over unmarked parts of the sample.

[0014] FIG.3 provides exemplary multiphoton confocal imaging data related to the disclosed technology.

[0015] FIG.4 provides exemplary optical coherence tomography (OCT) imaging data related to the disclosed technology, showing the differences between an eye treated by the disclosed technology and a control eye.

[0016] FIG.5 provides exemplary diopter data comparing an eye treated according to the disclosed technology and a control eye.

[0017] FIG.6 provides exemplary diopter data comparing an eye treated according to the disclosed technology and a control eye. In particular, FIG.6 provides a time history of the diopter change in treated and controlled eyes, similar to FIG 5. At each time point (horizontal axis), between 5 and 10, topographic measurements using a clinical, hand-held topographer (EyeSys Vista) are taken. Multiple operators perform the measurement at eachCU24195 / 101879.003099 time point to avoid operator’s bias, and from the topographic measurements, one can calculate the diopter. The number 1 in the 1TA refers to the which pair was treated first, T stands for the treated eye; analogously C strands for control eye; two eye pairs were labeled pair A and pair B.

[0018] FIG.7 provides exemplary diopter data comparing an eye treated according to the disclosed technology and a control eye; these data represent the kind of information provided in FIG.5 and FIG.6.

[0019] FIG.8 provides exemplary diopter data comparing an eye treated according to the disclosed technology and a control eye; these data represent the kind of information provided in FIG.5, FIG.6, and FIG.7.

[0020] FIG.9 provides exemplary diopter data comparing an eye treated according to the disclosed technology and a control eye. These data represent the kind of information provided in FIGs. 5-8; each image provides results for a particular eye pair or two.

[0021] FIG.10 provides exemplary treatment descriptions, protocols, and treatment pictures for eyes using the disclosed technology. Exemplary, non-limiting data are shown for eyes treated with 10% and 100% India ink marker.

[0022] FIG.11 provides description of exemplary treatment according to the present disclosure. As shown, a sample was hydrated, marked with black marker, and a laser was focused on the black marker. During laser treatment, so-called sparking was observes near by the boundary of the sample. After laser treatment, the black marker was depleted where the laser was scanning; the sample exhibited a mild shape change. Without being bound to any particular theory, crosslink formation changes the mechanical properties of the sample.

[0023] FIG.12 provides further description of an exemplary arrangement of the disclosed technology. Samples were subjected to mechanical loading, allowed to reach creep equilibrium, and subjected to laser treatment.

[0024] FIG.13 provides exemplary, non-limiting autofluorescence signal comparison. Microscopy shows increased autofluorescence signal in laser crosslinked cartilage, as compared to blank control and thermal control.

[0025] FIG.14 provides exemplary mechanical testing data before and after laser treatment. The results shows that laser crosslinking stiffens the samples.

[0026] FIG.15 provides exemplary data, showing mild morphological changes in samples.CU24195 / 101879.003099

[0027] FIG.16 provides exemplar data for samples before and after laser treatment. Some sparse spark generation was seen at the top of the sample.

[0028] FIG.17 provides exemplary, non-limiting images of cell viability data for a laser-treated sample and an untreated control. As shown, the two samples exhibited similar levels of cell viability.

[0029] FIG.18A provides exemplary Eff.RP changes from corneal topography. This figure provides a time history of the diopter change assessed with corneal topography. As explained above, corneal topography is measured at time intervals before and after the changes; FIG.18A provides a full, statistically significant data set.

[0030] FIG.18B provides exemplary data from samples subjected to the disclosed technology. In particular, this figure provides representative images of the corneal cross- section acquired with OCT, measuring corneal thickness. Because are experiments on ex vivo eyes, the corneas are swelling over time, and it was desired to determine that the laser treatment is responsible for the diopter change, and not swelling.

[0031] FIG.18C provides exemplary data from samples subjected to the disclosed technology. In particular, this figure provides nanoindentation results; nanoindentation was used to measure changes in the mechanical properties. The top image shows stiffening of the cornea; corneal stiffening is one of the hallmarks of the crosslinking process, and as shown, the laser-treated corneas are stiffer than controls. The bottom image shows a difference in the viscoelastic ratio between treated eyes and control eyes. We previously observed that changes in viscoelastic ratio are significant in the eyes subjected to laser treatment (compared to paired controls), whereas there is no significance when traditional crosslinking (UVA light+riboflavin) is applied. Without being bound to any particular theory or embodiment, the nature of crosslinks may be different between laser treated and UVA light+riboflavin crosslinking.

[0032] FIG.19 provides exemplary data from samples subjected to the disclosed technology. In particular, this figure illustrates an articular cartilage experiment for treatment of early osteoarthritis. The objective of this experiment was to further prove that the laser treatment is responsible for cartilage stiffening. Certain samples were immersed in Vitamin C solution, as Vitamin C is known scavenger of reactive oxygen species, and due to the presence of Vitamin C the laser crosslinking treatment was less effective for those samples.CU24195 / 101879.003099

[0033] FIG.20 provides exemplary data from samples subjected to the disclosed technology. In FIG.20, immature bovine cartilage plugs are shown in the images before and after treatment. As seen, each sample stiffened after the laser treatment (103%, 66%, 91%, and so on, as shown). The figure also reports the temperature of the sample during the treatment. The temperature is below the denaturation temperature of the collagen, which signifies that the stiffening is due to the laser treatment only. Further, one can observe that the temperature rise is very modest, which means that the laser treatment will not adversely affect the cells.

[0034] FIG.21 provides exemplary data from samples subjected to the disclosed technology. In particular, FIG.21 shows the same data as FIG 20, but at different processing conditions – the average laser power is 100 mW in FIG.21, and in FIG 20 it is 150 mW; also a different number of treatment layers were applied in the two figures. Lowering the laser power resulted in less stiffening, as shown.

[0035] FIG.22 provides exemplary data from samples subjected to the disclosed technology. This image provides live / dead staining of live immature bovine cartilage samples 4 days after the treatment. As seen, the laser treatment does not kill the cells. This has particular value for cartilage treatment, as cells in cartilage do not regenerate.

[0036] FIG.23 provides exemplary data from samples subjected to the disclosed technology. In particular, FIG.23 provides the same data as FIG.22, but with different processing conditions. Note that the darkened dot on the top images (depicting dead cells) is believed to relate to sample handling and not to laser treatment.

[0037] FIG.24 provides an exemplary overview of the use of low-density plasma to produce reactive oxygen species. Low-density plasma can be understood as referring to plasmas that do not result in tissue disruption through formation of the thermoacoustic and shock waves; LDP likely has lower electron density when compared with a dense plasma.

[0038] FIG.25 provides an exemplary overview of the use of low-density plasma to produce reactive oxygen species. This figures provides reference data, which illustrates that the efficacy of reactive oxygen species production is a function of a few parameters, including the laser repetition rate.

[0039] FIG.26 provides an exemplary overview of the formation of crosslinks using laser pulses. This slide illustrates that melanin increases laser absorption, which results in a more efficient treatment process.CU24195 / 101879.003099

[0040] FIG.27 provides an exemplary description of the use of femtosecond laser- produced reactive oxygen species to crosslink proteins.

[0041] FIG.28 provides an overview of the growth of myopia prevalence in the United States.

[0042] FIG.28 provides an overview of keratoconus.

[0043] FIG.29 provides an exemplary discussion of the effect of UVA-riboflavin crosslinking (CxL) on vison correction.

[0044] FIG.30 provides a brief description of the use of simultaneous CxL and mechanical deformation to effect a change in corneal curvature. In particular, this figure illustrates exemplary difference between clinical data of crosslinking using UVA light and riboflavin and our laser treatment. Without being bound to any particular theory or embodiment, one can argue that both UVA light + riboflavin crosslinking and the disclosed laser-based crosslinking is due to induction of reactive oxygen species that subsequently interact with collagen to form crosslinks. One can see, however, that UVA + riboflavin treatment does not induce diopter changes, whereas the disclosed laser-based crosslinking treatment can correct vision. One can further see that vision correction can be achieved by combining mechanical load and crosslinking.

[0045] FIG.31 provides a further description of the use of simultaneous CxL and mechanical deformation to effect a change in corneal curvature. In particular, this figure supports the implications of FIG 30. As shown, a combination of mechanical load and (traditional) UVA light + riboflavin crosslinking can also be used for vision correction.

[0046] FIG.32 provides a further description of the use of simultaneous CxL and mechanical deformation to effect a change in corneal curvature. This figure uses optical coherence tomography to assess corneal thickness after crosslinking. As seen, the diopter change is due to crosslinking and is not related to swelling of post mortem corneas.

[0047] FIG.33 provides a description of the use of accelerated glycation-mediated simultaneous CxL and deformation to effect a change in corneal curvature. This figure illustrates that crosslinking can suitably be paired with mechanical loading to achieve vision correction. Here, we crosslinked corneas (while they were deformed) via an accelerated glycation process; a ribose solution was applied and the acceleration was achieved by exposing the ribose solution-soaked eyes to UV light; both UV light exposure and femtosecond laser worked. The images on the left show representative topographies of theCU24195 / 101879.003099 crosslinked (top) and control cornea (bottom). The scaled bar indicated diopters. In the middle of the figure, a line graph shows the time history of diopter changes after the treatment. At the top, a 1.5 hrs long treatment line (square markers) is the diopter of treated eye, circles represent control eye; shaded region is standard deviation. The bottom graph shows a 3 hrs long treatment. The bar charts on the right show immediate (right after the treatment)and sustained (after 10 hrs) diopter change. As seen, crosslinking when properly paired with mechanical deformation can be used to correct vision.

[0048] FIG.35 provides description of the use of glycation-mediated CxL and deformation to stiffen corneal tissue. In particular, this figure provides nanoindentation data, from which we can see that crosslinked corneas were stiffer than paired controls. The crosslinking for this example was achieved with accelerated glycation mediated crosslinking. Acceleration of the glycation was achieved with three different ways (IR femtosecond laser with 1069 nm wavelength, UV femtosecond laser with 400 nm wavelength and UV lamp), and compared against paired controls and also positive controls (traditional crosslinking: UVA light+ riboflavin), hence 4 datasets in each graph. The graph on the left shows representative load / unload curve during the indentation process, from which we calculate stiffening, the bar chart in the middle shows stiffening , and the bar chart on the right shows viscoelastic ratio.

[0049] FIG.36 provides description of the use of glycation-mediated CxL and deformation to stiffen corneal tissue. This figure provides further results showing that the treatment imparts crosslinking. The images at the bottom are representative multiphoton microscopy images of treated and control corneas; an increase in fluorescence is being measured, and treated corneas have higher intensity (more fluorescence) than paired controls. Without being bound to any particular theory, it is understood that increased fluorescence corresponds to higher crosslink density, with the crosslinks hence being imparted by the disclosed treatment.

[0050] FIG.37 provides exemplary illustration of the use of a modified melanin- laser protocol to effect a change in corneal curvature.

[0051] FIG.38 provides description of the use of a modified melanin-laser protocol to effect stiffening in in corneal curvature. As seen, the data in FIG.38 are similar to the data provided in FIG.18A.CU24195 / 101879.003099

[0052] FIG.39 provides a summary of the use of simultaneous CxL and mechanical deformation for non-invasive vision correction.

[0053] FIG.40 provides a summary of the use of CxL to repair articular cartilage.

[0054] FIG.41 provides a summary of the use of CxL to stiffen articular cartilage. As seen, the results on the left (bar chart) show an increase in the stiffness of immature bovine cartilage subjected to the femtosecond laser treatment. The increase in stiffness results in improved wear resistance, which is a basis for the disclosed treatment of early osteoarthritis. The photograph in the middle shows the treated and control cartilage plug after wear test. As seen, the laser treated sample resists wear, whereas the control becomes damaged. The figure on the right shows the similar results, using a different imaging technique.

[0055] FIG.42 provides a summary of the use of CxL to stiffen articular cartilage. As seen, these results show stiffening (right bar chart) of human cartilage obtained from the cadaver joint. Two cartilage samples for which results were shown were extracted from the cadaver joint and treated. The stiffness was assessed before and after the treatment, and we see that the laser crosslinking increased the stiffness. The image on the left shows from which region of the joint the samples were extracted. The figure in the middle shows arthritic damage to the cartilage.

[0056] FIG.43 provides exemplary cell death data related to the use of the disclosed technology for arthritis treatment.

[0057] FIG.44 provides an exemplary summary of illustrative results.

[0058] FIG.45 provides an overview of the use of CxL to treat corneal wounds.

[0059] FIG.46 provides a summary of the use of femtosecond (FS )laser generated reactive oxygen species in cultured porcine corneas. As shown, FS laser can initiate low- density plasma to create ROS from water molecule ionization. In particular, FIG.46 illustrates that short bursts of laser-induced reactive oxygen species prevent cell (keratocyte apoptosis). For this illustration, the outermost layer of the cornea (epithelium – single layer of cells) has been scrapped off. Without being bound to any particular theory or embodiment, when the epithelium is damaged it signals to the cells below (keratocytes, located in corneal stroma) to die, so that a potential infection is prevented; this also slows down post-trauma healing. If one modulates cell apoptosis, one can manage inflammation and enhance theCU24195 / 101879.003099 wound healing process. The image on the left shows that cells in the control cornea died at higher rate than those in the treated corneas.

[0060] FIG.47 provides a summary of the use of femtosecond (FS) laser treatment to enhance corneal wound healing. As seen, this figure provides a schematic diagram showing the follow up experiment to results shown in FIG 46. As seen, the experiment included three groups of eyes, epithelium removed and laser treated (epithelium removal simulates scratch injury, a common injury of an eye); epithelium removed without laser treatment (control) and blank controls (no epithelium removal).

[0061] FIG.48 provides a summary of the use of femtosecond (FS) laser treatment to accelerate epithelial recovery. As seen, this figure provides results of the experiment described in FIG 47. The epithelium is more quickly restored in laser treated corneas, when compared to control eyes, which also had their epithelium removed, but no laser treatment. The eyes were stained and exposed to UV light to expose area without epithelium.

[0062] FIG.49 provides a summary of the use of femtosecond (FS) laser mediate ROS treatment to maintain stromal keratocyte viability. As shown, cells (keratocytes) are restored faster in laser treated corneas than in the paired controls.

[0063] FIG.50 provides a summary of the use of femtosecond (FS) laser mediated ROS treatment, showing the lack of adverse effects onto endothelial cells. In particular, this figure provides a control measurement which shows that endothelium is not affected, as expected.

[0064] FIG.51 provides a summary of the use of femtosecond (FS) laser mediated ROS treatment to affect wound healing through cytokine affinity modulation. As seen, one can (without being bound to any particular theory or embodiment) hypothesize that the laser treatment modulated receptors that receive signals from damaged epithelium to start apoptosis. Again without being bound to any particular theory or embodiment, damaged epithelium releases interleukins IL-1a,b which signal to keratocytes to die. The laser generated reactive oxygen species modulate the signal and the receptor, thereby weakening the information.

[0065] FIG.52 provides a summary of the response of IL-1β and IL-1 βR to femtosecond (FS) laser mediated ROS treatment. As seen, FIG.52 provides actual results of the discussion provided in connection with FIG.51.CU24195 / 101879.003099

[0066] FIG.53 provides a summary of the effect of FS laser induced ROS treatment on cytokines and corneal wound healing.

[0067] FIG.54 provides (a) Representative cornea undergoing simultaneous UVA riboflavin crosslinking and mechanical loading for corneal steepening. (b) Representative cornea undergoing simultaneous ROS-Glycation-CxL and mechanical loading for corneal flattening.

[0068] FIG.55 provides Serial dilution ELISA curves of A) HighQ and B) Coherent Fidelity II laser irradiated (treated) and non-irradiated (control) solutions of 1) cytokine IL-1β and 2) IL-1β receptor (n=2 technical repeats at each concentration). Two-way ANOVA tests were performed for each data set to evaluate the interaction effect of laser treatment and protein concentrations (**** p<0.0001). Post-hoc Tukey and one-way ANOVA tests were performed at each concentration. (* p<0.05, ** p<0.01, error bars are standard deviations).

[0069] FIG.56 depicts the corneal wound healing process and the role of inflammatory interleukins. DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0070] The present disclosure may be understood more readily by reference to the following detailed description of desired embodiments and the examples included therein.

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0072] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0073] As used in the specification and in the claims, the term "comprising" can include the embodiments "consisting of" and "consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that requireCU24195 / 101879.003099 the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions or processes as "consisting of" and "consisting essentially of" the enumerated ingredients / steps, which allows the presence of only the named ingredients / steps, along with any impurities that might result therefrom, and excludes other ingredients / steps.

[0074] As used herein, the terms “about” and “at or about” mean that the amount or value in question can be the value designated some other value approximately or about the same. It is generally understood, as used herein, that it is the nominal value indicated ±10% variation unless otherwise indicated or inferred. The term is intended to convey that similar values promote equivalent results or effects recited in the claims. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but can be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is understood that where “about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0075] Unless indicated to the contrary, the numerical values should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

[0076] All ranges disclosed herein are inclusive of the recited endpoint and independently of the endpoints. The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0077] As used herein, approximating language can be applied to modify any quantitative representation that can vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term or terms, such as “about” and “substantially,” may not be limited to the precise value specified, in some cases. In at least some instances, the approximating language can correspond to the precision of an instrument for measuring the value. The modifier “about” should also be considered as disclosing theCU24195 / 101879.003099 range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” can refer to plus or minus 10% of the indicated number. For example, “about 10%” can indicate a range of 9% to 11%, and “about 1” can mean from 0.9-1.1. Other meanings of “about” can be apparent from the context, such as rounding off, so, for example “about 1” can also mean from 0.5 to 1.4.

[0078] Further, the term “comprising” should be understood as having its open- ended meaning of “including,” but the term also includes the closed meaning of the term “consisting.” For example, a composition that comprises components A and B can be a composition that includes A, B, and other components, but can also be a composition made of A and B only. Any documents cited herein are incorporated by reference in their entireties for any and all purposes.

[0079] Any embodiment or aspect provided herein is illustrative only and does not limit the scope of the present disclosure or the appended claims. Any part or parts of any one or more embodiments or aspects can be combined with any part or parts of any one or more other embodiments or aspects.

[0080] Disclosure

[0081] Investigation has shown that irradiating the target tissue is not a sufficient condition for the photochemical reaction to occur. Crosslinking can be initiated with an optical breakdown, which can occur in the vicinity of the area that is to be crosslinked. The femtosecond laser crosslinking may be useful for treatment of early osteoarthritis, non- invasive vision correction, treatment of keratoconus.

[0082] Unlike current clinical practice in which crosslinking is achieved by exposing riboflavin-soaked eyes to UVA light, the disclosed treatment does not require photosensitizer, is not cytotoxic, and does not require debridement of epithelium. Furthermore, there is no treatment for early osteoarthritis. Experiments have shown that articular cartilage cannot be stiffened via application of riboflavin and UV light. This disclosure shows that crosslinking of various collagenous tissues to enhance their mechanical properties or change their shapes and surfaces may be accomplished using femtosecond laser crosslinking.

[0083] Laser treatment relies on low-density plasma induced photo-chemical effect. For example, one can put a black mark with a felt-tip pen around the tissue, or incubate theCU24195 / 101879.003099 hydrated tissue with pigmented solutions such as India ink, Trypan blue, or the biocompatible natural pigment melanin in PBS prior to the laser treatment. The low-density plasma is initiated by an optical breakdown. This optical breakdown occurs on the black marker that encircles the sample. The black marker absorbs the laser beam at significantly higher rate than the sample, resulting in optical breakdown induced dense plasma. We hypothesize that dense plasma produces a cloud of free-electrons that initiate low-density plasma once the laser beam is rasterized onto the cornea, which absorbs the laser at much lower rate. In such a scenario, once transition to low-density plasma occurs, damaging effects of dense plasma are absent, and the treatment is confined to photo-chemical effect that results in crosslinking of the tissue.

[0084] When the laser is focused on the mark or the selected pigments, the high laser beam absorption rate results in localized optical breakdown, visibly observed in the form of a spark. We hypothesize that this event (optical breakdown) generates a localized cloud of free electrons, which are required to initiate low density-plasma formation once the laser focus is moved away from the high absorption area (felt-tip pen mark, or pigment deposition areas). Experiments have shown that absent the triggering event created by the optical breakdown, no crosslinking will occur. We have also demonstrated that with calibrated laser parameters and bio-compatible pigments such as melanin, treated samples have similar cell viability compared to the untreated controls.

[0085] Furthermore, according to our preliminary studies on cornea, the crosslink density is also a function of mechanical load applied onto the target tissue during the laser irradiation, as well as hydration level of the tissue. In corneal crosslinking, simultaneous application of loading and laser irradiation results in change of corneal curvature, required for vision correction (for example, corneal flattening can improve vision of a myopic person).

[0086] Experimental Procedure

[0087] FIGs.1 and 2 show aspects of the experimental procedure. An example procedure is described below.

[0088] 1. Immediately after animal sacrifice, the eyeballs are removed and mounted onto the custom designed holder. IV system is attached to the eye to keep the intraocular pressure in the near physiological range. One eye of the animal was treated and the other served as paired control.CU24195 / 101879.003099

[0089] 2. The epithelium is removed with a scalpel. This step is not necessary for the laser CxL, but taken here for consistency of data acquisition (epithelium of ex vivo eyes sometimes peel off during experiments).

[0090] 3. Pigmented eyes (brown / black) are treated / measured as received. Eyes of albino rabbits have pink / white iris which has insufficient contrast for topography measurements. Thus, for eyes of albino rabbits, Trypan blue dye is applied to capture the refractive power of the cornea (EyeSysclincaltopographer). This step is not necessary for the laser CxL. Trypan blue is not used as a photosensitizer in this wavelength.

[0091] 4. Refractive power of the eyes (Diopter) is measured prior to the experiment and used as a baseline.

[0092] 5. Mechanical loading is applied on both treated and control eyes with the coverslip.

[0093] 6. For the treated eye, black marker is drawn near the laser treatment boundary for the generation of ‘spark’ (optical breakdown)

[0094] 7. Laser treatment is applied.

[0095] 8. Refractive power of both control and treated eyes is measured hourly for 8 hours after the treatment.

[0096] Only PBS (Sigma Aldrich) is used for the hydration of cornea, so the viscosity of hydrating solution does not affect refractive power measurement.

[0097] Optical breakdown on the black mark can be observed by presence of ’sparks.’ IV system ensures stable intraocular pressure.

[0098] Multiphoton Confocal Imaging

[0099] FIG.3 shows aspects of microscopy. Two-photon microscopy has been reported in literature to reveal induced crosslinks. Increased autofluorescence is accepted as indicator of increased crosslink density, induced by external factors. We have confirmed this by exposing samples to glutaraldehyde, a known crosslinker, as well as exposing riboflavin- soaked eyes to UVA light, which is clinically approved crosslinking treatment. Two-photon microscopy shows increased autofluorescence signal in laser crosslinked corneal stroma, compared to control.

[0100] FIG.4 shows aspects of optical coherence tomography. No excessive swelling was observed.

[0101] Corneal Topography (Eyesys)CU24195 / 101879.003099

[0102] FIGs.5-9 provide representative diopter change after laser treatment and mechanical flattening within 8 hours of animal sacrifice (ex vivo). A total of 8 pairs of eyes (8 treated eyes and 8 controls) have been subjected to the proposed treatment. Most controls experience brief drop in diopter (effective refractive power) due to application of mechanical load, followed by recovery to the original diopter over period of 8 hours. In contrast, in all laser treated eyes the change in diopter is sustained, and stable.

[0103] FIG.10 shows additional aspects of femtosecond laser treatment of corneas.

[0104] FIGs.11-23 provide illustrative, non-limiting aspects of femtosecond laser treatment of cartilage tissues.

[0105] Additional Disclosure

[0106] As explained elsewhere herein, low density plasma generated reactive oxygen species (ROS) have been used to crosslink corneal collagen with exacting femtosecond laser pulse parameters. Without being bound to any particular theory or embodiment, one can hypothesize that by increasing the cornea’s optical absorbance through melanin application, ROS can be produced from a denser plasma, leading to robust trans- epithelial corneal crosslinking therapies for keratoconus and vision correction.

[0107] In an example embodiment, fresh rabbit eyes were enucleated, exposed to 1% w / w melanin dissolved in phosphate buffered saline solution, and divided into groups A, B, C, and D (n=3 each). A and C had their epithelium-removed with a scalpel before femtosecond laser irradiation at 1060 nm with 100 mW, whereas B and D maintained intact epithelium before 185 mW irradiation.

[0108] For group A and B rabbit eyes, effective refractive power (Eff.RP) was assessed with corneal topography, and corneal apical thickness was monitored with optical coherence tomography (OCT) on eyeballs kept with 18-20 mm H2O intraocular pressure over a 10-hr period. For group C and D corneas, 5 μm radius nanoindentations were performed on 3 mm diameter corneal buttons after laser treatment. All laser-scanned corneas were mechanically loaded with a coverslip. Paired controls were exposed to melanin but received no light irradiation.

[0109] Results: Over the 10-hr period, the Eff.RP significantly decreased 7.47 diopters for epithelium-removed eyes (A: p<0.01) and 4.67 diopters for epithelium-intact (B: p<0.05) eyes after melanin-assisted laser crosslinking (FIG.18-1). However, there was noCU24195 / 101879.003099 change in the apical corneal thickness compared to the paired control (A: p=0.883; B: P=0.622, FIG.18-2). The equilibrium modulus of crosslinked tissues significantly increased to 80 kPa for epithelium-removed corneas (C: p<0.05) and 33 kPa for epithelium-intact corneas (D: p<0.005), while paired controls were approximately 10 kPa (FIG.18-3).

[0110] Conclusions: Melanin-assisted, femtosecond laser ROS crosslinking generated corneal stiffening and stable refractive power decrease with and without epithelium, proving its utility for transepithelial keratoconus and myopia treatment. The treatment regime at the chosen power levels did not cause notable stromal edema.

[0111] Focused femtosecond laser pulses are widely used in the biomedical field due to their nonlinear multiphoton precision and minimal thermal side effects. Below the threshold of optical breakdown, light energy contributes to photochemical reactions that introduce more chemical bonding in the form of collagen (COL) crosslinking (CxL) in extracellular matrices of transparent tissues such as corneal stroma. Provided here are, inter alia, methods of such treatment.

[0112] Focused femtosecond laser pulses are used in the biomedical field based on their nonlinear multiphoton precision and minimal thermal side effects. Below the threshold of optical breakdown, light energy contributes to photochemical reactions that introduce more chemical bonding in the form of collagen (COL) crosslinking (CxL) in extracellular matrices of transparent tissues such as corneal stroma. Previously, based on the principles of ultrafast laser-tissue interaction, a novel COL CxL method relying on low-density plasma (LDP) generating reactive oxygen species (ROS) was proposed and applied to cornea tissue.

[0113] Relying on the interaction between reactive oxygen species (ROS) created by low-dose photon (LDP) therapy and inflammatory cytokines, epithelium recovery is accelerated on in vivo rabbit corneas. Photochemical reaction-based morphological correction and biomechanical enhancement can be used for corneal diseases such as keratoconus and astigmatism.

[0114] A wavelength-independent, nonenzymatic crosslinking technique based on oxygen-independent, pentose-mediated glycation and ROS acceleration is provided; COL CxL efficiency is tested through collagen autofluorescence microscopy and nanoindentation. Subsequently, the combined effects of simultaneous external mechanical loading and nonenzymatic COL CxL, achieved by both traditional CxL that involves soaking eyes with riboflavin solution, a photosensitizer, and then activating it with ultraviolet A light (UVA-CU24195 / 101879.003099 Riboflavin-CxL) and new ROS catalyzed glycation CxL (ROS-Glycation-CxL) techniques, are investigated on ex vivo rabbit corneas.

[0115] Through x-ray diffraction, permanent adjustments to the ultrastructure of collagen fibril packing are observed, ultimately contributing to refractive power changes in corneal topography. Furthermore, with the addition of melanin application that increases absorption and ionization efficiency, a robust method for generating plasma and reactive oxygen species (ROS) has been proposed and implemented on ex vivo corneas to address ectatic diseases. Discussed here is, inter alia, the effect of plasma-guided laser COL CxL on articular cartilages’ compressive equilibrium modulus and chondrocyte viability. Stemming from the melanin-assisted protocol and ultrafast pulses' high peak power, a plasma spark- mediated laser treatment is hypothesized to biomechanically enhance both bovine and human articular cartilage superficial zone for the treatment of osteoarthritis.

[0116] Self-Focusing

[0117] Under high-intensity laser irradiation, materials with a higher-order susceptibility, a material-dependent parameter due to nonlinear propagation, can generate an intensity-dependent contribution to the index of refraction. This effect, called the optical Kerr nonlinearity, is common in femtosecond laser-tissue interactions even without tightly focusing because the peak pulse power can be significant with nonlinear ultrafast pulses. The nonlinear refractive index, in this case, is defined as: 3^^^ଷ^^^ଶൌ ^ where ^^^ଷ^is materialsusceptibility. The typical values of nonlinear refractive index for dielectric materials far from resonance frequency roughlyranges from 10ି^଼ െ 10ିଶ^ ^^ଶ / ^^. With the addition of nonlinear refractive index, theresulting actual refractive index n is ^^ ൌ ^^^ ^ ^^ଶ^^where the nonlinear refractive index scales with laser intensity, which gives rise to Kerr-effect-related self-focusing. When a collimated laser beam with a Gaussian intensity distribution passes through a material with third-order susceptibility, the refractive index distribution changes from a uniform constant to a curve function matching the laser pulse Gaussian intensity distribution. The materials at the geometric center of the laser beam experience a considerable increase in refractive index at the center due to the higherCU24195 / 101879.003099 centralized intensity compared to the surrounding materials. The new refractive index distribution effectively creates a convex lens and focuses the incident collimated laser beam or Kerr-effect dominated self-focusing to occur, the power of the laser must exceed the self- focusing power threshold expressed as: 3.77^^ଶ^^^^^௧ൌ . 8^^^^^^^ଶ

[0118] Common values which is achievable for a femtosecondlaser with a few hundred nanojoules. The length of the self-focusing ^^^^depends on the ratio between actual laser power and critical power and can be expressed using the Raileigh range: ^^ ^^ோ^^ൌ െ1^^.ହ

[0119] Additionally,length f is used, self-focusing is reinforced by the external focusing. The modified critical power ^^^^^௧ᇱis given as: ^^ ᇱ ^ 0.135^^^^ଶଶ ^^^௧ ^^.ହ ൌ 0.852 ^ ^0.0219 ^ ^^ ^^.ହ^^^^^௧^^ where the wavenumber ^^ ൌ 2^^ / ^^, and a is the input beam radius, and f is the focallength of the lens. The modified self-focusing distance can be calculated using the lens transformation: 1 1 1ᇱ ൌ ^ . ^^ ^^ ^^^^The tendency of a laser beamto contract due to self-focusing would eventually lead to a catastrophic collapse of the incident beam, causing an optical breakdown and subsequent plasma formation, which behaves like a de-focusing lens. It has been observed that laser beams with high power can produce “filaments” in transparent media such as air or glass, with multiple self-focusing and plasma-defocusing sequences hypothesized to be the cause. Some studies suggest that there exists a balance between the effect of self-focusing, ionization plasma, and diffraction, which can be expressed as a “self- guiding” model for filamentation: ^^^^^^ ^1.22^^^^ଶ^CU24195 / 101879.003099 where ^^^^^^ is the electron plasma density. However, the exact cause of filamentation is still unclear, as the “self-guiding model” is theoretically unstable at powers further away from the critical power.

[0120] Aside from the Kerr effect, recent studies have also found the possibility of self-focusing in plasma. Self-focusing in plasma can occur through thermal, relativistic, and ponderomotive effects. Thermal self-focusing occurs when plasma, heated through collisions with electromagnetic radiation, undergoes temperature escalation. This results in hydrodynamic expansion, elevating the refractive index and amplifying heating. Relativistic self-focusing arises from electron mass augmentation as they approach light speed, altering the plasma's refractive index.

[0121] Ponderomotive self-focusing stems from the ponderomotive force, which pushes electrons away from laser beam regions with the highest intensity, thus effectively elevating the refractive index and inducing self-focusing. Specifically, the critical power for plasma-mediated self-focusing ^^^^^ି^^is proportional to the ratio of incident laser field frequency and plasma frequency ^^^: ^^ ଶ ^^ଶ∝^^^^^^^ ^ ^^^^

[0122] Theorders of giga watts (10ଽ^^^ for comparable incident laser and plasma frequency. However, if the intensity- dependent plasma electron density ^^^^^^ is high enough to reachఠ ଶఠ^~10 , the required laser power could be significantly reduced.

[0123] The above describes a self-focusing mechanism made possible by a single laser pulse. However, multi-pulse accumulation may also contribute to self-focusing, which can be induced by a permanent refractive index change of the target material resulting from consecutive pulse exposure. For example, it has been observed that silica glass, under prolonged UV laser irradiation with pulse fluence of 0.1 to 10 ^^^^ / ^^^^ଶpulse fluence and 24ns pulse width, undergoes structural changes in density, refractive index, and optical absorption. The accumulated self-focusing manifests as a wave-guiding effect with a microchannel created by plasma spark captured with in-situ microscopy. The size of the self- focusing length and filamentation may depend on the damage threshold of the glass medium.

[0124] Additional DisclosureCU24195 / 101879.003099

[0125] One feature of exiting approaches is the use of external chemical agents such as riboflavin and melanin. Both UVA or 760nm activation of riboflavin photochemical crosslinking have been reported to cause stromal cell death in corneas. Cytotoxicity originating from the interaction of laser pulses and melanin has also been observed. Because of chondrocytes’ highly specialized function to create ECM, low population density, and limited capacity to regenerate, loss of cell viability is highly detrimental to OA treatment and recovery. Development of a stable, cartilage SPZ (superficial zone) crosslinking method without the need of external photosensitizer or agent may be useful.

[0126] In response to advancing and adapting the laser generated plasma crosslinking method, spark-mediated laser treatment was developed. Instead of the cartilage tissue’s direct exposure to plasma and absorption-enhancing pigment such as melanin, plasma in the form of a spark is generated on top of the coverslip through the application of an alcohol-containing pigment. Due to the optical Kerr effect, one can hypothesize that the high peak laser irradiance causes the refractive index of the coverslip to increase, effectively making a local focusing lens. After focusing, the high intensity at the second focal point leads to the second plasma generation, which can act as a defocusing lens. This phenomenon is widely studied as self-focusing and filamentation in transparent bulk media by femtosecond lasers.

[0127] The threshold of self-focusing is dependent on a critical power. With a typical soda-lime glass type coverslip, the refractive index is 1.5 and the nonlinear refractiveindex at ~3∗ 10ିଶ^^^ଶ / ^^, which yields the threshold for self-focusing to be 3.8MW. For ourcurrent laser treatment using the Fidelity II laser and an average power of 200mW, the peak pulse energy can be calculated as 2.857nJ, and the maximum peak power is 70.34 kW or 0.70 MW with a 40.6fs pulse duration. (For the previous HighQ laser with an average power of 65mW, the peak pulse energy would be 1.25nJ, and the maximum peak power is 12.61 kW or 0.12MW with a 99fs pulse duration). It is obvious that self-focusing would not occur if we only consider the Kerr effect and a standard glass coverslip.

[0128] Interestingly, self-focusing may also occur in generated plasma for ultra- high intensity laser pulses as a result of the relative mass increase of electrons close to the speed of light and the reduction of electron density from the pondermotive force at the laser focal volume. The threshold of plasma-mediated self-focusing is quadratically proportional to the ratio of laser frequency and plasma frequency, which is dependent on the plasmaCU24195 / 101879.003099 electron density. Without being bound to any particular theory or embodiment, it has been hypothesized that if the plasma frequency is high enough, the critical power can be brought down to the order of 10kW.

[0129] Additionally – but without being bound to any particular theory or embodiment, the alcohol containing pigment may also contribute to plasma generation robustness. Plasma-spark was generated at the intersection of alcohol-rich pigment and coverslip glass. In the field of laser material processing, this form of plasma generation was proposed as “laser-induced backside wet etching”. Compared to a transparent bulk solid material such as soda-glass or fused silica, the ablation or plasma generation threshold in the auxiliary liquid is smaller; alcohol such as ethanol is reported to have the lowest ionization threshold compared to water and glass. Plasma generated in the auxiliary liquid then subsequently interacts with the bulk material. With accumulated exposure to laser pulses, it is possible that plasma-spark-initiated ionization of the coverslip surface can lead to the formation of microchannels or micron-size waveguides throughout the coverslip thickness, which has been reported with pulsed excimer lasers on silica glass. Compared to direct laser- tissue interaction, the plasma spark-mediated laser treatment requires a much lower power threshold and increases the repeatability and robustness of multiphoton ionization of water if self-focusing filamentation is successful.

[0130] Spark-mediated laser treatment may generate a thermal effect due to objectively creating more plasma near the sample surroundings. To minimize thermal shock damage to the chondrocytes, we provided a flowing PBS environment to promote heat transfer through convection.

[0131] This addition can be clinically relevant, as cartilage orthopedic surgeries can be performed with the SPZ submerged underneath saline or synovial fluid. The thermocouple probe measured the temperature of the sample-surrounding environment under the additional PBS flow, stayed at approximately 30 ºC during treatment, and dropped back down to room temperature in minutes without laser exposure.

[0132] In a further study, spark-mediated laser bovine treatment led to an average of 50% of modulus change. Laser treatment with VC (Vitamin C) significantly decreased the magnitude of stiffening, which confirmed that ROS is a major component in the study, as ascorbic acid is a major ROS scavenger. Furthermore, increased VC concentration increases osmolarity, which has proven to help with crosslinking efficiency; however, bovine samplesCU24195 / 101879.003099 treated in a hyperosmolar environment due to VC supplementation instead had a decreased crosslinking efficiency. These results further suggest the mitigating effect of VC and ROS dependency on collagen crosslinking. In the VC-supplemented control group, there were trends of softening of the actual equilibrium modulus before and after treatment. Without being bound to any particular theory or embodiment, one may hypothesize that VC might be interfering with protease inhibitor function, leading to more collagen degradation.

[0133] In a further study, laser treatment yielded significant stiffening in human OA cartilages. However, there were variances in the stiffening amount for the treated samples, which had more surface irregularities and uneven features compared to healthy bovine samples used in other studies. Without being bound to any particular theory or embodiment, it is possible that uneven surfaces lead to irregularity during laser focusing, which may decrease the amount of filamentation delivered plasma exposure to the sample in the spark-mediated laser treatment process.

[0134] In a further study, to further validate the safety of the spark-mediated laser treatment, chondrocyte cell viability was tested using a live / dead cell staining kit. The results demonstrated favorable cell viability. Small areas of decreased cell viability may come from small physical scratches and uneven marker distribution. Cell deaths were more commonly found around the edge because the hexapod motor system used decelerates at the edge, leading to extended laser exposure at the sample peripherical. Laser focal point dwelling in one position leads to more severe cell death, so focusing procedure can be assisted with a CCD camera to find the “spark” for the Z position away from the sample. The observed cell death zone a particular sample might – again without being bound to any particular theory or embodiment – be due to the uneven thickness of the sample: part of the sample (higher end) gets compressed more or might be exposed to more spark and excessive laser irradiation due to uneven focusing. When the lower half of the focal volume coincides with the higher part of the sample, the starting Z position may not in all cases be accurate.

[0135] Here is provided a plasma spark-mediated laser treatment for the multiphoton ionization of interstitial water and subsequent crosslinking of cartilage SPZ collagen. The compressive bulk equilibrium after crosslinking can be increased while also preserving chondrocyte viability.

[0136] Additional DisclosureCU24195 / 101879.003099

[0137] 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. Short bursts of ROS were imparted into 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 absence of tissue damaging shock and thermoacoustic waves, and 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's ability to modulate molecular interactions.

[0138] About 55 million eye injuries occur annually worldwide, 1.6 million resulting in blindness and 2.3 million in low bilateral vision. Most common eye injuries include corneal abrasion (scratched cornea), blunt trauma, and penetration by a foreign object. The cornea, the eye's outermost layer, gets injured the most. Cornea has a highly organized structure, and its architecture plays an integral role in light refraction onto the retina while simultaneously acting as a barrier that protects intraocular content from external factors. Scar-driven fibrosis healing fails to fully restore the corneal transparency because of the disruption of carefully maintained periodic spatial ECM structures, and thus often leads to aberrant light refraction into the retina . In this study, a low-powered femtosecond laser (oscillator) has been employed to produce controlled short bursts of reactive oxygen species (ROS) to manage the inflammation and enhance the wound healing process.

[0139] Disruption of the corneal healing process, such as misregulated molecular pathways or excessive collagen deposition, can lead to the loss of structural integrity and transparency of the stromal ECM, resulting in scarring and corneal haze, corneal neovascularization, and even blindness. The cornea needs to remain transparent and able to properly refract light onto the retina. Existing therapies have not yet led to substantial progress in scar-free, regenerative wound recovery: single-agent therapies, such as theCU24195 / 101879.003099 administration of a particular growth factor, have a limited impact due to considerable complexity and rapid activity of the wound site components.

[0140] Wound repair and healing include multiple stages such as inflammation, new tissue formation, and remodeling. Upon injury, various intra- and intercellular pathways are activated in the immune and inflammatory systems through molecular agents such as cytokines and growth factors.

[0141] Such pathways regulate and coordinate metabolism, proliferation, differentiation, and migration of cells near the wound site. Problems with wound healing can be roughly divided into two categories. The first is delayed healing, which is a characteristic of chronic or non-healing wounds such as pressure or diabetic ulcers. The second is excessive healing, characterized by the deposition of large amounts of extracellular matrix and alterations in local vascularization. Many such excessive healings produce large masses of tissue that can physically distort surface structures. Wound healing in the ocular system is particularly sensitive to excessive fibrosis healing because it interferes with specialized tissue function and characteristics for light interaction.

[0142] Post-traumatic corneal wound repair includes re-epithelization, cell proliferation, as well as synthesis and remodeling of stromal extracellular matrix (ECM). All steps in the corneal wound healing process are mainly regulated by cytokines and growth factors (FIG.56). Injury to the epithelium triggers the release of proinflammatory cytokines such as interleukin-1 (IL-1a, β) and tumor necrosis factor-a (TNF-a), which attract inflammatory cells but also initiate keratocyte apoptosis. IL-1a, β signal keratocyte apoptosis in the vicinity of the wound, resulting in the formation of an acellular area. Keratocytes surrounding the acellular region get activated and turn into fibroblasts and, along with inflammatory cells, migrate into the acellular region. Fibroblasts secrete proteases, chemokines, and growth factors such as transforming growth factors (TGFβ1, TGFβ2) that stimulate fibroblasts differentiation into myofibroblasts, which are seen as instrumental in wound contraction and scarring. For example, IL-1 and TNF-a levels are transiently elevated in routine wound healing; however, if their concentrations remain persistently high, the inflammation is prolonged, and epithelium repair is impeded, which can result in the repair defect. Further, a disbalance between elevated levels of IL-1 and receptor antagonist (IL-1R) results in delayed wound healing, which is particularly important in cases where IL-1R is suppressed.CU24195 / 101879.003099

[0143] Reactive oxygen species (ROS) lead to the oxidation of amino acid residue side chains, formation of protein crosslinks, and oxidation of the protein backbones, resulting in protein fragmentation since ROS moieties, by their nature, have high reactivity with various molecules. Specifically, the biological activity of IL-33 at its receptor ST2 was shown to be regulated by an oxidation-driven conformational change involving the formation of two di-sulfate bridges, resulting in a structural change of the binding site and affecting the binding affinity of the cytokine. Other IL-1 family cytokines are also susceptible to cysteine oxidation changes that could regulate their biological activity. However, the effect of ROS on biological processes is a function of content, strength, and duration of exposure. While ROS-related signaling pathways were established, ROS' cellular influence varies from anabolic to cytotoxic. Excessive oxidative stress can potentially cause various deleterious events such as damaged protein structure, interfered activation of signaling cascades, and even cell apoptosis. In contrast, low doses of oxidative stress possess an adaptive stimulatory effect on cells and activate pathways leading to migration and collagen synthesis. ROS- production enhancing modalities such as anti-bacterial H2O2 containing topical solutions, galvanic zinc-copper microparticles, Galectin-1, hyperbaric O2therapy, and photodynamic therapy are increasingly utilized to promote skin wound healing and manage inflammation. Nevertheless, the optimal delivery system of ROS is still elusive; to the best of authors’ knowledge, there are no marketed or in-pipeline ROS-based therapies for ophthalmic wound recovery at the epithelial-stromal level.

[0144] Aside from wound healing therapies, ROS based collagen crosslinking has been adapted prevalently for the treatment of keratoconus. Specifically, activation of riboflavin molecule (riboflavin-5-phosphate, or R5P) with ultraviolet A (UVA) light at 365nm generates ROS, leading to the crosslinking of stromal collagen crosslinking. The main drawback of traditional UVA activation includes a lack of spatial specificity and endothelium cytotoxicity.

[0145] In some embodiments, laser intensity is restricted such that the optical breakdown is never reached (~1013W / cm2). and thus, the process is limited to photochemical reactions in the absence of damaging effects of dense plasma such as ablation, as well as shock or thermoacoustic waves. Induced LDP ionizes and dissociates interstitial water in the corneas, resulting in a cascade of reactions that produce ROS such as H2O2, hydrogen ion (H3O+), hydroxyl ion (OH-), hydroxy radical (OH*). Further, because the number of laser-CU24195 / 101879.003099 generated free electrons produced during a single pulse is a function of irradiance, lasing parameters can be tuned to control the amount of ROS delivered to the target region of the ocular system. The proposed treatment has a high degree of spatial accuracy due to the nature of multiphoton ionization at NIR wavelength. In vivo results presented here show that the targeted introduction of ROS enhances the wound healing process in rabbit models subjected to corneal abrasion. Follow-up experiments on isolated cytokines and corresponding receptors confirm that ROS indeed modulated the binding affinity of targeted molecules.

[0146] Epithelium Recovery

[0147] To simulate corneal surface abrasion (scratch wound – most common eye injury), apical corneal epithelia were removed in treated eyes and paired controls, but not in positive control eyes. Eyes were divided into three groups: Group A: intact, positive control, epithelia not debrided and not subject to lasing; Group B control eyes: epithelium removed, and subject to the same treatment conditions except laser irradiation (sham controls), and Group C treated eyes: epithelium removed and subsequently laser treated.

[0148] Top row in FIG.48 shows eyes before the treatment. The eye on the top left is a pristine (positive) control, and the other two eyes have their epithelium removed. Size of the injury, and the healing process after the laser treatment was monitored via staining with topically applied fluorescein eyedrops and exposing to a light source. The size of the initial injury is approximately the same in both eyes. Injured corneas with exposed stromata appear fluorescent green under cobalt-blue inspection illumination, whereas intact or recovered central epithelium surfaces did not show artificial colors.

[0149] On the 3rd to 4th day after the laser irradiation, non-laser treated cornea surface showed partial recovery of epithelium in Group B in Figure.2 (2nd row), whereas treated corneal surface in Group C (bottom right) showed complete recovery of the epithelium layer, suggesting laser treatment’s acceleration effect on the overall wound healing process. The ratio of unhealed wound area to the overall area of the cornea at the 3rd- 4th day timepoint was summarized in Figure.2D through image segregation and pixel counting for epithelium removed eyes. The average wound to cornea ratio for laser treated eyes was 12.5±9.10%, whereas the average wound to cornea ratio for non-laser treated eyes was higher at 26.9±12.1%. However, a one-tailed t-test showed no statistical significance between the two groups(p=0.163).

[0150] Keratocyte Cell ViabilityCU24195 / 101879.003099

[0151] Keratocyte count was performed to assess the injury induced cell loss, and whether the laser treatment can mitigate it. In vivo rabbit stromal keratocyte densities were calculated from CLSM images of the laser-treated (epithelium removed, laser-treated), control (epithelium removed, no laser treatment) and positive control (no epithelium removed) within the 200um thick region below the anterior stromal surface at three time- points: before-treatment, 3 to 4-day and 14-day after the treatment. A mixed repeated measures ANOVA was performed to analyze the effect of time and treatments on keratocyte density. The variations in keratocyte density across time points are statistically different for each laser treatment protocol (F=4, P<0.05). Post-hoc tests were performed to evaluate the simple main effects within the same treatment group: Tukey’s multiple comparisons test showed a significant decrease of keratocyte density from the before-treatment to the 3 to 4- day timepoints for epithelium removed corneas without laser treatment (p<0.05). No other significance was found across 3 time-points within 3 different treatment groups (Figure 3A). In essence, there is no statistically significant change in keratocyte cell density in positive controls and the laser-treated eyes before the injury and at day 3-4 after the injury, whereas there is a significant drop in keratocyte count between day 0 and day 3-4 in injured eyes that did not receive the laser treatment. The latter observation is consistent with prior reports that epithelium injury triggers keratocyte apoptosis in the anterior segment of the corneas

[0027] . In contrast, the laser treatment reduces the injury-mediated cell apoptosis.

[0152] Endothelium Cell Viability

[0153] In healthy corneas, endothelium cells maintain hydration and transparency. Since the endothelium cells cannot replicate, their sufficient loss can result in endothelium barrier dysfunction and vision loss. In vivo rabbit endothelium cell densities were calculated from CLSM images at the before-treatment, 3rd to 4th day and 14-day time points. A mixed repeated measures ANOVA was performed to analyze the effect of time and treatments on epithelial cell density. The effect of time on endothelium cell viability did not depend on laser treatment (F= 0.6630, p=0.6257). There was no significant main effect on endothelium cell density among different treatment groups (F=0.7897, p=0.4831). Time did have a significant main effect on endothelium cell density (F=4.187, p=0.0321). Post-hoc Tukey’s multiple comparisons tests showed that there was a significant decrease of endothelium cell density at the 3rd to 4th day timepoint from the before-treatment baseline for all samples (p<0.05) (Figure 3B).CU24195 / 101879.003099

[0154] Binding Affinity of IL-1β and IL-1β-receptor

[0155] Elevated pro-inflammatory cytokine and receptor interaction potentially leads to cellular behavior induced prolonged and irregular wound healing. To further investigate the mechanism of laser treatment’s effect on epithelial healing and keratocyte viability, changes to binding interaction between IL-1β and IL-1β-receptor after the Nd:Glass and fiber laser irradiation were assessed through ligand-receptor interaction quantitative ELISA, where optical density (OD) was measured as an indication of binding affinity. Ordinary two-way ANOVA tests were performed to analyze the effect of protein concentrations and laser treatments on OD levels.

[0156] Figure.55A1 shows Nd:Glass laser treated IL-1β cytokine’s binding activity with unmodified IL-1β-R on ELISA plate. The effect of IL-1β concentration on binding affinity significantly depended on Nd:Glass laser treatments, and vice versa (F=12.91, p<0.0001). Post-hoc Tukey test showed a significant OD difference between the laser treated and control cytokines at 10pg / ml (p<0.05) and 20pg / ml (p<0.0001) IL-1β concentrations.

[0157] Figure.55A2 shows Nd:Glass laser treated IL-1β-receptor’s binding activity to unmodified IL-1β cytokines on ELISA plate. The effect of IL-1β-R concentration on binding affinity did not depend on Nd:Glass laser treatment (F=1.311, p=0.3152). Laser treatment did not have a significant main effect on the OD levels (F=0.3248, p=0.5777). Post- hoc Tukey test showed no significant OD difference between the laser treated and control cytokines at any IL-1β-receptor concentrations.

[0158] FIG.55B1 shows fiber laser treated IL-1β cytokine’s binding activity to unmodified IL-1β-R on ELISA plate. The effect of IL-1β concentration on binding affinity significantly depended on fiber laser treatments, and vice versa (F=11.49, p<0.0001). One way ANOVA test at each protein concentration identified significant differences of laser treatment powers on OD at 8pg / ml (p<0.01), 16pg / ml (p<0.01), 32pg / ml (p<0.01), 128pg / ml (p<0.01), and 256pg / ml (p<0.01) IL-1β concentrations.

[0159] FIG.55B2 shows fiber laser treated IL-1β-receptor’s binding activity to unmodified IL-1β cytokines on ELISA plate. The effect of IL-1β-R’s concentration on binding affinity significantly depended on fiber laser treatments, and vice versa (F=13.66, p<0.0001). One way ANOVA test at each protein concentration identified significant effectCU24195 / 101879.003099 of different laser treatment powers on OD at 128pg / ml (p<0.05), 512pg / ml (p<0.01), 1024pg / ml (p<0.05), and 2048pg / ml (p<0.01) IL-1β-receptor concentrations.

[0160] Discussion

[0161] This study has demonstrated that NIR femtosecond laser irradiation enhanced the corneal wound healing process after the induced abrasion. After the scratch injury, the laser treatment targets the anterior part of exposed stroma since the rabbits do not have a Bowman’s layer. The laser irradiation pattern takes advantage of the effective size of the multiphoton focal volume which is 50um in depth (25um in Rayleigh range) and covers the 200um depth of the anterior stroma. This spatial positioning of the laser treatment allows for the investigation of the epithelial-stroma interaction during wound healing.

[0162] The relative size of the corneal wound was quantitatively assessed and compared between the laser-treated and control groups at the midpoint of the study (3rdto 4thday post-treatment). All corneas were subjected to an identical injury, and complete epithelial healing was observed in all specimens by two weeks following treatment. Fluorescein was administered topically to the corneas, which were subsequently exposed to ultraviolet (UV) light to visualize areas lacking epithelial coverage. A total of three laser-treated corneas and two control corneas were evaluated; the third control cornea could not be imaged due to logistical constraints. At the 3rdto 4thday assessment, one laser-treated cornea exhibited complete recovery, while the other two had 16.26% and 21.33% of the corneal surface without epithelial coverage, respectively. In contrast, the control corneas displayed 39.01% and 14.81% of their surfaces uncovered by epithelium at the same time point.

[0163] Delayed healing of the cornea and epithelium is generally considered detrimental, as an exacerbated and prolonged inflammatory response may result in persistent corneal defects, leading to scarring, ulceration, and opacification . To mitigate unnecessary delays in epithelial healing, therapeutic modulation of the immune response may be implicated. Our quantitative enzyme-linked immunosorbent assay (qELISA) and cell counting data indicate that near-infrared femtosecond laser treatment significantly influenced the immune response during the healing process by reducing cytokine binding affinity and decreasing stromal keratocyte apoptosis. This potential downregulation of the immune system may have contributed to the observed trend of accelerated healing.

[0164] Wounding of the corneal epithelium initiates the rapid loss of stromal keratocytes in rabbits and primates. Our in vivo CLSM images within the 0-200um regionCU24195 / 101879.003099 confirmed that at the 3rdto 4thday timepoint; epithelium-removed corneas of the control Group B had a significant decrease in stromal keratocyte density. In contrast, epithelium- removed and laser-treated corneas of Group C had a similar time-dependent response to the epithelium-intact corneas of the positive control Group A and showed no significant keratocyte density difference (FIG.49, FIG.50). This similarity suggests that NIR femtosecond laser treatment altered the post-injury keratocytes response. First, the laser irradiation has reduced keratocyte apoptosis near the wound. Second, keratocyte differentiation into fibroblasts and myofibroblasts could also have been mediated since the transformed cells in general are larger in volume. Lastly, the keratocyte response sequence might have been accelerated by the laser treatment such that at the 3rdto 4thday timepoint the stromal keratocytes network has restored to the baseline structure. We observed a combination of the above potential effects in stromal keratocyte CLSM images within the laser treatment volume. Keratocyte density below the treated region showed no significant difference across all three groups at all three time points, suggesting an anterior stroma- confined effect of the laser treatment; however, this might also be due to the distance from the induced wound at the surface of the cornea.

[0165] Importantly, spatially resolved laser treatment did not affect endothelium cell density, although we observed that endothelial cell density decreased in all corneas regardless of treatment groups from the baseline at the 3rdto 4thday timepoint after treatment but recovered at the 2-weeks timepoint (FIGs.49, 50). One can hypothesize that perhaps a procedure in animal handling unrelated to the principal laser treatment application, such as transportation from the animal facility to the laser operating room, might be the potential cause. H&E-stained histological sections of epithelium removed rabbit corneas obtained at 3rdto 4thday and 0-day timepoints showed no stromal collagen disorganization and no endothelium detachment after laser treatment.

[0166] Further, one might suspect that the drop in the endothelium cell density might be due to the coverslip mechanical loading being applied on top of the cornea, as external strain leads to an increase of intraocular pressure (IOP), which may have affected the size of the endothelial cell and the area density reported through cell counting. Additionally, even though endothelial cells in humans are considered to lack regenerative capacity, it has been shown that rabbit endothelium have some capability of regeneration,CU24195 / 101879.003099 which might be relevant to how the rabbit endothelium density increased slightly form the 2ndtime point to the 3rdtimepoint.

[0167] We further hypothesize that different stromal keratocytes and epithelium recovery behavior results between the laser treated and control corneas is due to the laser- initiated LDP-produced ROS. Produced from the interstitial water in the stromal ECM, short lived ROS interact with cytokines secreted by regenerating epithelium and activated keratocytes during the wound healing process. For example, earlier studies defined the important role of pro-inflammatory cytokine IL-1α and β and their receptor IL-1R1 as modulators of the epithelial-stromal cell interactions. Through quantitative ELISA, we showed that the identical Nd:Glass laser treatment applied to in vivo rabbit corneas significantly decreased the binding affinity of the irradiated IL-1β to unmodulated IL-1R1. However, the same laser treatment did not affect the biding affinity of the irradiated IL-1R1 to unmodulated IL-1. We only observed an increase in the magnitude of binding affinity change as IL-1β concentration increases, while the IL-1R1 concentration’s effect on OD levels did not depend on laser treatment (FIG.55A). Because we speculated that ROS-led, oxidation-driven conformational change of the amino acids is the primary reason for the binding affinity difference, it is possible – without being bound to theory – the binding site structure of the bulkier IL-1R1 at ~50kDa could be less susceptible to the ROS oxidation effect compared to the smaller IL-1β at ~17.5kD.

[0168] The interaction of femtosecond laser pulses with IL-1 and IL-1R1 was further investigated with a different optical setup using the fiber laser that can provide significantly higher out of objective average power at 80mW, 160mW, and 320mW. We demonstrated that the fiber laser treatment significantly decreased the binding affinity of both the irradiated IL-1β and IL-1R1 to their respective unmodulated counterparts. Binding affinity changes increase with both protein concentrations and laser powers in a nonlinear fashion. One can attribute this non-linearity to the multiphoton nature of the laser treatment in transparent aqueous media, as the energy absorption process depends on multiple photons interacting with the target nearly simultaneously (10ି^^s), leading to a non-linear, higher order dependence on light intensity. The binding affinity dependency on laser powers were not significant for all concentrations (FIG.55B). It is worth noting that at the 80mW fiber laser were approximated to have the same ~3.7e10 W / cm2focal area intensity and at ~1.2nJ pulse energy as the Nd:Glass laser treatment at 60mW. However, the IL-1R1 had a differentCU24195 / 101879.003099 response to the two lasers as the binding affinity only decreased for the fiber laser. The fiber laser has a slightly higher wavelength, shorter pulse duration and higher repetition rate compared to the Nd:Glass laser treatment. These variable lasing parameters might have increased the efficiency of the LDP to produce more ROS that altered the 50kDa IL-1R1 protein structure. Overall, the differential response of IL-1β and IL-1R1 to different lasing parameters shows the availability of target-specific laser treatment.

[0169] The concentration ladder chosen here was close to the reported IL-1β concentration in serum. Interestingly, it has been reported that the concentration of pro- inflammatory cytokines such as IL-1β increases not only in routine wound healing, but also in diabetic complications. Inhibiting the IL-1β pathways in wounds of diabetic host induced a switch from proinflammatory to healing-associated macrophage phenotypes and improved healing of persistent diabetic wounds. Since the laser treatment produced higher magnitude of binding affinity changes with higher cytokine concentrations, this treatment is increasingly effective for severe wounds with high pro-inflammatory cytokine concentrations.

[0170] Corneal wound healing depends on many intertwining factors, such as stromal cells, epithelial and endothelial cells, ECM, secreted chemokines, proteinases, and biophysical properties. Current therapeutic approaches to modulate the corneal wound healing processes, such as gene therapy, nano-particles, and stem cell transplantation, might have multiple mechanisms of action but rarely investigate the cumulative effects of multiple factors. When NIR ultrashort pulses carrying nano-joule energy below the optical breakdown are relatively loosely focused onto collagen-rich biological media, ROS produced by LDP in the focal volume interacts with stromal ECM to form collagenous crosslinks. The LDP is differentiated from a dense plasma by its inability to generate thermoacoustic or shock waves. A femtosecond laser induced dense plasma is a product of an optical breakdown and its freeelectron density is about 10^଼ െ 10ଶ^ cmିଷ. On the other hand, LDP can occur even whenlasing parameters do not reach the optical breakdown threshold and have lower free electron density than the dense plasma.

[0171] When applied to the corneal stroma, this technique enhances the biomechanical properties of the cornea with spatial precision and has therapeutic potential for keratoconus and vision correction. Notably, the laser parameters used in corneal stroma crosslinking overlap with this study's wound healing lasing condition. This study showed the first pieces of evidence of possible NIR femtosecond laser-induced alteration of epithelial-CU24195 / 101879.003099 stromal interactions, affecting not only epithelial cells and stromal keratocyte network but also potentially the ECM structure due to the collagen crosslinking introduced by the laser treatment.

[0172] Methods

[0173] Animals and Epithelium Recovery Treatment Groups

[0174] In vivo experiments were performed on young adult Dutch belted rabbits delivered to the Columbia University Institute of Comparative Medicine (ICM) animal house. The Institutional Animal Care and Use Committee (IACUC) of Columbia University provided review and approval to the experimental protocols (AC-AAAV2456 and AAAT6466) for all pre- and post-treatment animal-handling procedures. All in vivo experiments were conducted in compliance of IACUC regulations and reported in accordance with the ARRIVE 2.0 Essential 10 guidelines. A total of 6 Dutch Belt rabbits were used to study the epithelium wound healing process. For all rabbits, one eye was randomly chosen to be kept intact and the paired eye was subjected to epithelium debriding at the central cornea by brush. Out of the 6 apical epithelium removed eyes, the laser treatment was applied on 3 corneas; the remaining 3 corneas were left to heal naturally. Fluorescein and cobalt-blue inspection illumination was used to evaluate the epithelium wound state at two time points: before treatment (BT) and 3rdto 4thday after treatment. Due to the noticeable physical difference from epithelium removal and recovery, the experimenter could not be blinded to whether the animal was laser treated.

[0175] Corneal Epithelial Debridement Protocol: Vacuum fixated corneal well was placed on the central 6 mm of the cornea (Barron Vacuum Punch; Barron Precision Instruments) filled with proparacaine solution and kept for 30 seconds to loosen the epithelial cells while providing additional analgesia. Proparacaine solution was then aspirated before removing the vacuum well. The corneal epithelium (6 mm) was scraped off using either blunt spatula or Amoils epithelial scrubber (Innovative excimer solutions) for 10 seconds.

[0176] Laser Treatment: Two low pulse energy, high repetition rate femtosecond lasers – laser oscillators, were used in the study: (1) 1059.6nm wavelength Nd:Glass femtosecond oscillator (High Q, Spectra-Physics, Austria) with 1059.6 nm central wavelength and 99 femtosecond temporal pulse width, and (2) fiber-based femtosecond oscillator (Fidelity II, Coherent, Santa Clara, CA) with central wavelength at 1069 nm and 52 femtosecond pulse duration. The first laser (Nd:Glass) was focused with a Zeiss Plan-CU24195 / 101879.003099 Neofluar 40x Zeiss objective to produce 65mW of average power, measured after the objective. The effective numerical aperture was estimated to be 0.098 due to the out-of-cavity 0.2mm beam diameter; The fiber oscillator (Fidelity II) was focused with the same objective at 0.1463NA to produce 80, 160, and 320mW average power, also measured after the objective. Out-of-cavity powers were adjusted with continuous neutral density filters (NDC- 50C-2M-B, Thorlabs, Newton, NJ). Treatments on interleukins solutions (please see below) and rabbit corneas were accomplished with a raster scan regime, with a 50um increment zig- zag pattern in a 25mm2square parallel to the cornea or solution surface and 5 layers of consecutive treatment planes into the depth of the sample. A 0.15mm thick coverslip was put on top of the sample to capture the focal position of the laser beam after the objective. The laser beam was rasterized by a custom assembled 3-axis motorized linear stage (Z825B and PT1, Thorlabs, Newton) with a speed of 2mm / s and a maximum acceleration of 1.5mm / s2. In vivo rabbit handling, and anaesthetization procedures were described in a previous study

[0037] . Briefly, the rabbits were anaesthetized with an intramuscular injection of ketamine (3.5mg kg-1) and xylazine (5 mg kg-1) before treatment; the depth of anesthesia was confirmed by the absence of ear pinch and pedal reflexes. All relevant parameters are reported in Supplementary Table 1.

[0177] Image Analysis for Epithelium Recovery Pictures: Digital pictures taken at the 3rdto 4thday after treatment timepoints for epithelium removed corneas were segmented based on contrast of color from intact cornea (black) and unhealed, exposed stroma (green). Pixel counting using custom process in Python and area percentage calculation were performed to represent the wound area to total corneal area ratio.

[0178] Confocal laser scanning microscopy (CLSM) and Cell Counting: CLSM was performed with an HRT3-RCM 670nm laser scanning system (Heidelberg Engineering, Dossenheim, Germany) and a 63x / 0.95NA water immersion objective (Zeiss, Germany) on the same set of 6 rabbits at three time points: before the treatment, 3rdto 4thday, and 2 weeks after the treatment. GenTeal water-based gel was placed between the cornea and the objective as a coupling medium. For stromal keratocytes, the cell per unit volume (100*100*70um3) was acquired and then converted to cell per millimeter cubed. Cell counting was performed with Image J software. The representative keratocyte counts for each cornea at each time point were taken from the average of three unit-volumes in the treatment region (0 to 200umCU24195 / 101879.003099 anterior stroma). For endothelium cells, the cell per unit area (100*100um2) was acquired and then converted to cell per millimeter squared.

[0179] Quantitative ELISA Assay: ELISA plate protocols were used to evaluate the possible alteration effect of the NIR laser irradiation on the binding affinity of IL-1b and IL-1b-receptor (IL1R1) molecules. Recombinant human IL-1b protein (Thermo Fisher Scientific, PHC0815) was reconstituted to 5 ug / mL with dH2O according to the manufacturer’s recommendations. Then protein solution was sampled into shallow (2 mm depth) dishes, covered with microscope cover slides and divided in two groups. One group was treated with the Nd:glass laser, and the other group was left as untreated control. Serial dilutions of the control and Nd:glass laser treated samples were assayed using IL-1b US ELISA kit (Invitrogen, KHC0014). Effects of Nd:glass laser treatment on recombinant human IL1R1 receptor protein (Thermo Fisher Scientific, 10126H08H5) were also assayed following the above procedure using Human IL-1R1 (IL-1RA) ELISA Kit (Thermo Fisher scientific, EHIL1R1). The experiments were repeated by using the fiber laser for the irradiation at various average power. The binding affinity of IL-1b (Thermo Fisher Scientific, PHC0815) and IL-1b-receptor (Sinobiological, 10126-hcch) subject to the fiber laser treatment was similarly investigated through corresponding ELISA assays (Thermo Fisher Scientific, BMS224-2 and EHIL1R1). The optical density at 450nm was acquired with kinetic microplate spectrophotometers for Nd:Glass (Benchmark Microplate Reader, Bio-Rad Laboratories, Hercules, CA) and fiber laser (POLARstar Omega, BMG LABTECH, Cary, NC) treatments. Only readings of optical density on the same plate between the treated and control groups were compared.

[0180] Aspects

[0181] The following Aspects are illustrative only and do not limit the scope of the present disclosure or the appended claims. Any part or parts of any one or more Aspects can be combined with any part or parts of any one or more other Aspects.

[0182] Aspect 1. A method for treating collagenous tissue, comprising: illuminating a region of the collagenous tissue with an illumination from an illumination source, the illumination being transmitted through a substrate having a first portion that contacts the collagenous tissue and a second portion that is free of contact with the collagenous tissue, the illumination comprising any one or more of a femtosecond laser illumination or an ultraviolet-A (UVA) illumination, an agent being disposed between theCU24195 / 101879.003099 illumination source and the collagenous tissue such that interaction between the agent and the illumination evolves a plasma that gives rise to crosslinking of the collagenous tissue, the agent optionally being biocompatible, and the plasma optionally being a low-density plasma.

[0183] As described, illumination can be any one or more of a femtosecond laser illumination or an ultraviolet-A (UVA) illumination. Suitable femtosecond lasers and UVA sources are known in the field. In the non-limiting example of using a femtosecond laser, the femtosecond laser induces a low-density plasma that generates an ionization field resulting in the generation of reactive oxygen species (ROS) in and around the collagenous tissue. Treatment can be achieved over a broad range of wavelengths, for example, including 1060 nm. An example pulse duration can be about 80-90 femtoseconds, the repetition rate can be around 52 MHz, and average power after focusing objective can be between about 10 mW and about 100 mW, e.g., about 60 mW. A beam can be focused, for example with a 40× / 0.6 objective. Without being bound by theory, the illumination and / or the resulting ROS interact with collagen fibrils in the extracellular matrix (ECM). Biochemical reactions with ROS can result in crosslink (CxL) formation.

[0184] Again without being bound by theory, the illumination and / or the resulting ROS controls tissue remodeling. Such tissue remodeling including changing the stiffness in the corneal tissue, resulting in changes in the optical characteristics, such as refractive power. In some embodiments, the tissue remodeling including applying a mechanical load to the tissue, e.g., the steepening or flattening procedures described herein.

[0185] As discussed herein, an ultrafast laser-tissue interaction can occur. When lasing is restricted below the optical breakdown, it can ionize and dissociate interstitial water in collagenous tissues. The ionization results in production of refractive oxygen species, which in turn interact with collagen and form crosslinking. There are numerous potential applications of this process in translational medicine, including, for example, ophthalmology and orthopedics. Ultrafast laser-based corneal crosslinking is as an attractive choice due to absence of photosensitizers, and no need for epithelial debriding. Crosslinking of corneal tissue can be used to treat keratoconus, and for noninvasive vision correction. The Latter application includes the combination of crosslinking, which can be done with an ultrafast laser or with more traditional methods (riboflavin / UVA light), and application of mechanical load. Without being bound to a particular theory, it is believed that incoming photons can be in resonance with interstitial water in collagenous tissue to trigger ionization. There may beCU24195 / 101879.003099 more than one resonance peak, and one of the peaks may be Fano resonance. Further, the laser pulse duration is below 100 femtoseconds to produce the resonance.

[0186] Suitable agents for the disclosed technology include, without limitation, agents that comprise a pigment. Without being bound to any particular theory or embodiment, a pigment can promote the formation of a plasma – which can be a low-density plasma – that in turns gives rise to crosslinking of the collagenous tissue. The pigment can be, for example, melanin or an ink. Black, blue, green, brown, and other colors are suitable for use as pigments.

[0187] Aspect 2. The method of Aspect 1, wherein the plasma is a low-density plasma.

[0188] Aspect 3. The method of Aspect 2, wherein the low-density plasma self- focuses. Additional disclosure related to self-focusing is provided elsewhere herein.

[0189] Aspect 4. The method of Aspect 1, wherein the illumination comprises femtosecond laser illumination.

[0190] Aspect 5. The method of Aspect 1, wherein the illumination comprises UVA illumination.

[0191] Aspect 6. The method of any one of Aspects 1-5, wherein the agent comprises a pigment.

[0192] Aspect 7. The method of Aspect 6, wherein the pigment comprises any one or more of melanin or India ink.

[0193] Aspect 8. The method of any one of Aspects 1-7, wherein the agent is disposed so as to contact the collagenous tissue during the illuminating. As one example, the agent can be placed directly on the collagenous tissue. This can be accomplished by, for example, writing, spraying, dipping, tapping, dispensing, and the like. The agent can also be placed on the first portion of the substrate such that when the substrate is contacted to the collagenous tissue, the agent then contacts the tissue. In some embodiments, the agent is a biocompatible agent. This can be particularly suitable when the agent contacts the collagenous tissue. The agent can be present as a single mark, but can also be present as multiple marks. The agent can be present in a pattern, shape, line, or other non-random configuration.

[0194] Aspect 9. The method of any one of Aspects 1-8, wherein the agent is disposed on the second portion of the substrate such that the agent is free of contact with theCU24195 / 101879.003099 collagenous tissue during the illuminating. In such embodiments, the agent does not directly contact the collagenous tissue. The agent can, for example, be written, sprayed, brushed, dripped, tapped, or otherwise applied to the substrate. The agent can be present as a single mark, but can also be present as multiple marks. The agent can be present in a pattern, shape, line, or other non-random configuration.

[0195] Aspect 10. The method of any one of Aspects 1-8, wherein the agent is disposed within the substrate. As but one example, the substrate can include a pigment, tint, or other colorant therein. For example, a colored glass – such as a brown or other colored glass – is considered to have an agent within the substrate.

[0196] Aspect 11. The method of any one of Aspects 9-10, wherein illumination gives rise to filamentation within the substrate. As described elsewhere herein, it has been observed that laser beams with high power can produce “filaments” in media such as air or glass, with multiple self-focusing and plasma-defocusing sequences hypothesized to be the cause. In this way, plasma in the form of a spark is generated on top of the substrate through the application of a pigment, which pigment can include alcohol. Due to the optical Kerr effect, one can hypothesize that the high peak laser irradiance causes the refractive index of the coverslip to increase, effectively making a local focusing lens. After focusing, the high intensity at the second focal point leads to the second plasma generation, which can act as a defocusing lens. This phenomenon is widely studied as self-focusing and filamentation in transparent bulk media by femtosecond lasers.

[0197] Aspect 12. The method of any one of Aspects 1-11, wherein the substrate comprises glass. The substrate can be, for example, a glass slide or coverslip. In some embodiments, the substrate is transparent, but this is not a requirement.

[0198] Aspect 13. The method of any one of Aspects 1-12, further comprising contacting the substrate to a region of the collagenous tissue during the illumination.

[0199] Aspect 14. The method of claim 13, wherein the contacting comprises exerting the substrate against the region of the collagenous tissue such that the region of the collagenous tissue attains a modified shape. The exertion can be performed to flatten or otherwise applanate the region of the collagenous tissue. In this way, the user can change the shape of the collagenous tissue to arrive at a desired shape. As but one example, a user may seek to change the shape of a subject’s cornea.CU24195 / 101879.003099

[0200] Aspect 15. The method of Aspect 14, wherein the crosslinking causes the region to retain the modified shape.

[0201] Aspect 16. The method of any one of Aspects 1-15, wherein the collagenous tissue is comprised in at least one of a cornea and cartilage. Application of the disclosed technology to a subject’s cornea is particularly advantageous.

[0202] Aspect 17. The method of any one of Aspects 1-16, further comprising effecting glycation of the collagenous tissue. Example, non-limiting glycations are described elsewhere herein. Without being bound to any particular theory, glycation can effect acceleration of the crosslinking.

[0203] Aspect 18. The method of Aspect 17, further comprising contacting the collagenous with a solution that comprises a sugar, the sugar optionally comprising ribose. Other sugars – such as glucose and fructose – are also suitable.

[0204] Aspect 19. The method of any one of Aspects 1-18, wherein the method is performed to effect vision correction in a subject.

[0205] Aspect 20. The method of any one of claims 1-19, wherein the method is performed to effect stiffening of a joint cartilage in a subject.

[0206] Aspect 21. A system, the system configured to perform the method of any one of Aspects 1-20.

[0207] Aspect 22. A system for treatment of a collagenous tissue having an initial shape, comprising: an illumination source, the illumination source comprising any one or more of a femtosecond laser illumination or an ultraviolet-A (UVA) illumination; a substrate, the substrate having a first portion arranged for contact with the collagenous tissue and a second portion arranged to be free of contact with the collagenous tissue; and an agent, the agent positioned between the illumination source and the collagenous tissue such that interaction between the agent and illumination from the illumination source evolves a plasma that gives rise to crosslinking of the collagenous tissue.

[0208] Example femtosecond laser and UVA illumination sources will be known to those of ordinary skill in the art. Example agents are described elsewhere herein, and include, for example, inks, melanin, and the like.

[0209] Aspect 23. The system of Aspect 22, wherein the agent is disposed on the second portion of the substrate. As described, however, the agent can also contact the collagenous tissue. In some embodiments, the agent is placed directly on the collagenousCU24195 / 101879.003099 tissue. In other embodiments, the agent is placed on the first portion of the substrate, and the agent then contacts the collagenous tissue when the first portion of the substrate is contacted to the collagenous tissue.

[0210] Aspect 24. The system of Aspect 23, further comprising an actuator configured to contact the substrate against a region of the collagenous tissue. The actuator can be, without limitation, a servo, a piston, a motor, and the like. The actuator can be manually controllable, but can also be automated.

[0211] Aspect 25. The system of Aspect 24, wherein the actuator is configured to contact the substrate against the region of the collagenous tissue such that the region of the collagenous tissue attains a modified shape.

[0212] Aspect 26. A method for treating collagenous tissue, comprising: applying a femtosecond laser to a particular area of a collagenous tissue so as to induce localized optical breakdown of the collagenous tissue at the particular area; at least one of (i) directing the femtosecond laser away from the particular area so as to initiate low-density plasma formation or (ii) removing the femtosecond laser from the particular area so as to initiate low- density plasma formation; and crosslinking the collagenous tissue by the low-density plasma formation. As described, the disclosed methods can be useful in, as but some examples, vision treatment, osteoarthritis treatment, and the link. The disclosed methods can be applied to applications in which the user modifies one or more of the mechanical properties or the shape of collagenous tissue. Such tissue can be, for example, cartilage and / or corneal tissue.

[0213] Aspect 27. The method of Aspect 26, wherein the method is performed such that the femtosecond laser has a power that exceeds the self-focusing power threshold of the laser.

[0214] Aspect 28. The method of Aspect 27, wherein the self-focusing power threshold is expressed as 3.77^^ଶ^^^^^௧ൌ . 8^^^^^^^ଶ

[0215] Aspect 29. The method of Aspect 26, wherein the method is performed such that the femtosecond laser has a power below the self-focusing power threshold of the laser.

[0216] Aspect 30. The method of any one of Aspects 26-29, wherein the method is performed such that the low-density plasma self-focuses.CU24195 / 101879.003099

[0217] Aspect 31. The method of any one of Aspects 26-30, wherein the particular area comprises a an agent present thereon, the agent optionally comprising a pigment.

[0218] Aspect 32. The method of any one of Aspects 26-31, wherein a substrate between the laser and the collagenous tissue has an agent disposed thereon, the agent optionally comprising a pigment, and the substrate optionally comprising a cover slip.

[0219] Aspect 33. The method of Aspect 32, wherein the pigment comprises an alcohol.

[0220] Aspect 34. The method of any one of Aspects 32-33, wherein application of the laser causes a refractive index of the substrate to increase.

[0221] Aspect 35. The method of any one of Aspects 26-34, further comprising removal of heat from the collagenous tissue.

[0222] Aspect 36. The method of Aspect 35, wherein the removal comprises effecting heat transfer from at least one of conduction and convection.

[0223] Aspect 37. The method of Aspect 36, wherein the removal comprises collecting heat from the collagenous tissue with a moving heat-absorbing medium.

[0224] Aspect 38. The method of any one of Aspects 26-37, wherein an amount of Vitamin C contacts the collagenous tissue during application of the laser.

[0225] Aspect 39. The method of Aspect 38, further comprising contacting the collagenous tissue with the amount of Vitamin C.

[0226] Aspect 40. The method of any one of Aspects 26-39, further comprising use of a camera to assist in setting a location of the plasma. As described, the plasma can be a low-density plasma.

[0227] Aspect 41. The method of any one of Aspects 26-40, wherein the plasma is formed at a distance from the collagenous tissue. As described, the plasma can be a low- density plasma.

[0228] Aspect 42. The method of Aspect 41, wherein the plasma is formed at a distance measured perpendicular from a surface of the collagenous tissue. As described, the plasma can be a low-density plasma.

[0229] Aspect 43. The method of any one of Aspects 26-42, wherein the collagenous tissue is a cornea.

[0230] Aspect 44. The method of any one of Aspects 26-43, wherein the method is performed to effect any one or more of non-invasive vision correction, treatment ofCU24195 / 101879.003099 keratoconus, wound treatment, cartilage stiffening, or cartilage shape change. As described elsewhere herein, the disclosed technology is useful in a broad range of applications, and the foregoing list is not exclusive.

[0231] Aspect 45. The method of any one of Aspects 26-44, wherein the collagenous tissue is cartilage.

[0232] Aspect 46. A method for treating collagenous tissue, comprising: applying a femtosecond laser to a pigmented area of a collagenous tissue so as to induce localized optical breakdown of the collagenous tissue at the pigmented area; at least one of (i) directing the femtosecond laser away from the pigmented area so as to initiate low-density plasma formation or (ii) removing the femtosecond laser from the pigmented area so as to initiate low-density plasma formation; and crosslinking the collagenous tissue by the low-density plasma formation.

[0233] Aspect 47. The method of Aspect 46, wherein the method further comprises applying a pigment to the collagenous tissue so as to give rise to the pigmented area. Pigment can be applied by, for example, a pen, a brush, or other applicator.

[0234] Aspect 48. The method of any one of Aspects 46-47, wherein the collagenous tissue is a cornea.

[0235] Aspect 49. The method of any one of Aspects 46-48, therein the is performed to effect non-invasive vision correction or treatment of keratoconus.

[0236] Aspect 50. The method of any one of Aspects 46-49, wherein the collagenous tissue is cartilage.

[0237] Aspect 51. The method of Aspect 50, wherein the method is performed to treat osteoarthritis.

[0238] Aspect 52. The method of any one of Aspects 46-51, wherein the pigment comprises melanin.

[0239] Aspect 53. The method of any one of Aspects 46-52, wherein the pigment comprises ink.

[0240] Aspect 54. The method of any one of Aspects 46-53, wherein the method further comprises applying a mechanical load to the collagenous tissue during application of the femtosecond laser. The mechanical load can be applied to, for example, give rise to a desired curvature or other shape of the collagenous tissue. Without being bound to any particular theory, the crosslinking can then fix the collagenous tissue in the curvature or otherCU24195 / 101879.003099 shape effected by application of the mechanical load. Again without being bound to any particular theory, the method can be performed to conform the collagenous tissue to at least a part of a form, at least a part of a model, at least a part of a target shape, and the like. As but one example, a user may identify a shape to which the user may desire the collagenous tissue to conform – such as a lens having a particular curvature – and then applying the mechanical load to the collagenous tissue to conform the collagenous tissue to that shape. In this way, the disclosed methods can be applied to effect collagenous tissue having virtually any shape desired.

[0241] Aspect 55. The method of any one of Aspects 46-54, wherein performance of the method gives rise to a persistent change in a curvature of the collagenous tissue. Such persistent change can, as one non-limiting example, last for a period of 4 hours after the mechanical load is removed.

[0242] Aspect 56. A method for treating collagenous tissue, comprising: illuminating a region of the collagenous tissue with an illumination from an illumination source, the illumination comprising any one or more of a femtosecond laser illumination or an ultraviolet-A (UVA) illumination, the illumination evolving reactive oxygen species that modulate cytokine binding within the collagenous tissue, the method optionally being performed such that the method does not give rise to crosslinking within the collagenous tissue. The method can, however, be performed so as to give rise to at least some crosslinking within the collagenous tissue.

[0243] Aspect 57. The method of Aspect 56, wherein the collagenous tissue is comprised in a cornea or in cartilage.

[0244] Aspect 58. The method of any one of Aspects 56-57, wherein the method is performed to effect wound healing in a subject.

Claims

CU24195 / 101879.003099 What is Claimed:

1. A method for treating collagenous tissue, comprising: illuminating a region of the collagenous tissue with an illumination from an illumination source, the illumination being transmitted through a substrate having a first portion that contacts the collagenous tissue and a second portion that is free of contact with the collagenous tissue, the illumination comprising any one or more of a femtosecond laser illumination or an ultraviolet-A (UVA) illumination, an agent being disposed between the illumination source and the collagenous tissue such that interaction between the agent and the illumination evolves a plasma that gives rise to crosslinking of the collagenous tissue, the agent optionally being biocompatible, and the plasma optionally being a low-density plasma.

2. The method of claim 1, wherein the plasma is a low-density plasma.

3. The method of claim 2, wherein the low-density plasma self-focuses.

4. The method of claim 1, wherein the illumination comprises femtosecond laser illumination.

5. The method of claim 1, wherein the illumination comprises UVA illumination.

6. The method of any one of claims 1-5, wherein the agent comprises a pigment.

7. The method of claim 6, wherein the pigment comprises any one or more of melanin or India ink.

8. The method of any one of claims 1-5, wherein the agent is disposed so as to contact the collagenous tissue during the illuminating.CU24195 / 101879.003099 9. The method of any one of claims 1-5, wherein the agent is disposed on the second portion of the substrate such that the agent is free of contact with the collagenous tissue during the illuminating.

10. The method of any one of claims 1-5, wherein the agent is disposed within the substrate.

11. The method of any one of claims 1-5, wherein (1) the agent is disposed on the second portion of the substrate such that the agent is free of contact with the collagenous tissue during the illuminating and wherein illumination gives rise to filamentation within the substrate or (2) wherein the agent is disposed on the second portion of the substrate such that the agent is free of contact with the collagenous tissue during the illuminating and wherein illumination gives rise to filamentation within the substrate.

12. The method of any one of claims 1-5, wherein the substrate comprises glass.

13. The method of any one of claims 1-5, further comprising contacting the substrate to a region of the collagenous tissue during the illumination.

14. The method of claim 13, wherein the contacting comprises exerting the substrate against the region of the collagenous tissue such that the region of the collagenous tissue attains a modified shape.

15. The method of claim 14, wherein the crosslinking causes the region to retain the modified shape.

16. The method of any one of claims 1-5, wherein the collagenous tissue is comprised in any one or more of a cornea and joint cartilage.

17. The method of any one of claims 1-5, further comprising effecting glycation of the collagenous tissue.

18. The method of claim 17, further comprising contacting the collagenous tissue with a sugar, the sugar optionally comprising ribose.

19. The method of any one of claims 1-5, wherein the method is performed to effect vision correction in a subject.CU24195 / 101879.003099 20. The method of any one of claim 1-5, wherein the method is performed to effect stiffening of a joint cartilage in a subject.

21. A system, the system configured to perform the method of any one of claims 1-5.

22. A system for treatment of a collagenous tissue, comprising: an illumination source, the illumination source comprising any one or more of a femtosecond laser illumination or an ultraviolet-A (UVA) illumination; a substrate, the substrate having a first portion arranged for contact with the collagenous tissue and a second portion arranged to be free of contact with the collagenous tissue; and an agent, the agent positioned between the illumination source and the collagenous tissue such that interaction between the agent and illumination from the illumination source evolves a plasma that gives rise to crosslinking of the collagenous tissue.

23. The system of claim 22, wherein the agent is disposed on the second portion of the substrate.

24. The system of claim 23, further comprising an actuator configured to contact the substrate against a region of the collagenous tissue.

25. The system of claim 24, wherein the actuator is configured to contact the substrate against the region of the collagenous tissue such that the region of the collagenous tissue attains a modified shape.

26. A method for treating collagenous tissue, comprising: applying a femtosecond laser to a particular area of a collagenous tissue so as to induce localized optical breakdown of the collagenous tissue at the particular area; at least one of (i) directing the femtosecond laser away from the particular area so as to initiate low-density plasma formation or (ii) removing the femtosecond laser from the particular area so as to initiate low-density plasma formation; andCU24195 / 101879.003099 crosslinking the collagenous tissue by the low-density plasma formation.

27. The method of claim 26, wherein the method is performed such that the femtosecond laser has a power that exceeds the self-focusing power threshold of the laser.

28. The method of claim 27, wherein the self-focusing power threshold is expressed as 3.77^^ଶ^^^^^௧ൌ . 8^^^^^^^ଶ29. The method of claim 26,such that the femtosecond laser has a power below the self-focusing power threshold.

30. The method of any one of claims 26-28, wherein the method is performed such that the low-density plasma self-focuses.

31. The method of any one of claims 26-29, wherein the particular area comprises a an agent present thereon, the agent optionally comprising a pigment.

32. The method of any one of claims 26-29, wherein a substrate between the laser and the collagenous tissue has an agent disposed thereon, the agent optionally comprising a pigment, and the substrate optionally comprising a cover slip.

33. The method of claim 32, wherein the pigment comprises an alcohol.

34. The method of claim 32, wherein application of the laser causes a refractive index of the substrate to increase.

35. The method of any one of claims 26-29, further comprising removal of heat from the collagenous tissue.

36. The method of claim 35, wherein the removal comprises effecting heat transfer from at least one of conduction and convection.

37. The method of claim 36, wherein the removal comprises collecting heat from the collagenous tissue with a moving heat-absorbing medium.CU24195 / 101879.003099 38. The method of any one of claims 26-29, wherein an amount of Vitamin C contacts the collagenous tissue during application of the laser.

39. The method of claim 38, further comprising contacting the collagenous tissue with the amount of Vitamin C.

40. The method of any one of claims 26-29, further comprising use of a camera to assist in setting a location of the plasma.

41. The method of any one of claims 26-29, wherein the plasma is formed at a distance from the collagenous tissue.

42. The method of claim 41, wherein the plasma is formed at a distance measured perpendicular to a surface of the collagenous tissue.

43. The method of any one of claims 26-29, wherein the collagenous tissue is a cornea.

44. The method of any one of claims 26-29, wherein the method is performed to effect any one or more of non-invasive vision correction, treatment of keratoconus, wound treatment, cartilage stiffening, or cartilage shape change.

45. The method of any one of claims 26-29, wherein the collagenous tissue is cartilage.

46. A method for treating collagenous tissue, comprising: applying a femtosecond laser to a pigmented area of a collagenous tissue so as to induce localized optical breakdown of the collagenous tissue at the pigmented area; at least one of (i) directing the femtosecond laser away from the pigmented area so as to initiate low-density plasma formation or (ii) removing the femtosecond laser from the pigmented area so as to initiate low-density plasma formation; and crosslinking the collagenous tissue by the low-density plasma formation.

47. The method of claim 46, further comprising applying a pigment to the collagenous tissue so as to give rise to the pigmented area.

48. The method of any one of claims 46-47, wherein the collagenous tissue is a cornea.CU24195 / 101879.003099 49. The method of any one of claims 46-47, wherein the method is performed to effect non-invasive vision correction or treatment of keratoconus.

50. The method of any one of claims 46-47, wherein the collagenous tissue is cartilage.

51. The method of claim 50, wherein the method is performed to treat osteoarthritis.

52. The method of any one of claims 46-47, wherein the pigment comprises melanin.

53. The method of any one of claims 46-47, wherein the pigment comprises ink.

54. The method of any one of claims 46-47, further comprising applying a mechanical load to the collagenous tissue during application of the femtosecond laser.

55. The method of claim 54, wherein performance of the method gives rise to a persistent change in a curvature of the collagenous tissue.

56. A method for treating collagenous tissue, comprising: illuminating a region of the collagenous tissue with an illumination from an illumination source, the illumination comprising any one or more of a femtosecond laser illumination or an ultraviolet-A (UVA) illumination, the illumination evolving reactive oxygen species that modulate cytokine binding within the collagenous tissue, the method optionally being performed such that the method does not give rise to crosslinking within the collagenous tissue.

57. The method of claim 56, wherein the collagenous tissue is comprised in a cornea or in cartilage.

58. The method of any one of claims 56-57, wherein the method is performed to effect wound healing in a subject.

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