Observational pilot study of multi-wavelength wearable light dosimetry for erythropoietic protoporphyria.

BACKGROUND: Erythropoietic protoporphyria (EPP) causes painful light sensitivity, limiting quality of life. Our objective was to develop and validate a wearable light exposure device and correlate measurements with light sensitivity in EPP to predict and prevent symptoms. METHODS: A wearable light dosimeter was developed to capture light doses of UVA, blue, and red wavelengths. A prospective observational pilot study was performed in which five EPP patients wore two light dosimeters for 3 weeks, one as a watch, and one as a shirt clip. RESULTS: Standard deviation (SD) increases from the mean in the daily blue light dose increased the odds ratio (OR) for symptom risk more than the self-reported outdoor time (OR 2.76 vs. 2.38) or other wavelengths, and a one SD increase from the mean in the daily blue light wristband device dose increased the OR for symptom risk more than the daily blue light shirt clip (OR 2.45 vs. 1.62). The area under the receiver operator curve for the blue light wristband dose was 0.78, suggesting 78% predictive accuracy. CONCLUSION: These data demonstrate that wearable blue light dosimetry worn as a wristband is a promising method for measuring light exposure and predicting and preventing symptoms in EPP.

Light-Related Cutaneous Symptoms of Erythropoietic Protoporphyria and Associations With Light Sensitivity Measurements.

Importance: Erythropoietic protoporphyria (EPP) is a rare and underdiagnosed genetic disease characterized by painful sensitivity to light. A better understanding and characterization of its light-induced cutaneous symptoms may aid in the identification of EPP in patients. Objectives: To describe the cutaneous symptoms of erythropoietic protoporphyria (EPP) and to determine if these symptoms are associated with the degree of light sensitivity. Design, Setting, and Participants: This was a cross-sectional study of adolescent and adult (≥15 years) patients with EPP across the US conducted by a single academic hospital via a remotely administered survey, measurements of light sensitivity by light dosimetry and by text message symptom assessments. Data analyses were conducted from November 2020 to April 2022. Exposures: Sunlight exposure. Main Outcomes and Measures: Self-reported symptoms and association with measured light sensitivity. Results: The study sample consisted of 35 patients with EPP (mean [SD] age, 39.1 (15.5) years; 21 [60%] female; 14 [40%] male; 35 [100%] White individuals). The patients' median [range] skin tone was 3.0 (1.0-8.0), based on self-reporting from 1 (lightest) to 12 (darkest). A total of 24 participants completed the light dosimeter measurements. Phototoxic reactions were characterized by pain (97%; 34 patients), burning (97%; 34), tingling (97%; 34), pruritus (83%; 29), allodynia (89%; 31), improvement of symptoms with cold (89%; 31), achiness (24%; 12), fatigue (46%; 16), mild swelling (83%; 29), severe swelling (63%; 22), erythema (51%; 18), petechiae (40%; 14), skin cracking (43%; 15), scabbing (46%; 16), scarring (66%; 23), and other chronic skin changes (40%; 14). Patients with EPP reported that their hands, feet, and face were most sensitive to light and that their shoulders and legs were least sensitive; 25.7% (9 patient) reported no chronic skin changes, and 5.7% (2 patients) reported never having had any visible symptoms. None of these findings varied with the degree of light sensitivity except that lower overall light sensitivity was associated with lower ranked sensitivity of the neck and arms. Conclusions and Relevance: The findings of this cross-sectional study suggest that patients with EPP have distinctive cutaneous symptoms that may aid in identification of this underdiagnosed disease. Characteristic EPP symptoms include light-induced cutaneous burning pain and occasional swelling, particularly over the hands, with a prodrome of pruritus and paresthesias. Minimal skin changes or the absence of visible skin changes during reactions to light, including lack of erythema, do not exclude an EPP diagnosis nor suggest low EPP disease burden.

Nanocaging Rose Bengal to Inhibit Aggregation and Enhance Photo-induced Oxygen Consumption†.

Photosensitized crosslinking of proteins in tissues has many medical applications including sealing wounds, strengthening tissues, and beneficially altering tissue properties. Rose Bengal (RB) is used most frequently as the photosensitizer but is not as efficient as would be desired for broad utilization in medicine. Aggregation of RB, at the high concentrations used for medical treatments, decreases the yield of singlet oxygen, which mediates protein crosslinking. We hypothesized that nanocages that sequester RB would inhibit self-association, increasing photosensitization efficiency. We tested cucurbituril and cyclodextrin nanocages, demonstrating that hydroxypropyl-functionalized cyclodextrins are most effective in inhibiting RB aggregation. For these RB/cyclodextrin solutions, we investigated the effect of nanocaging on the photobleaching and oxygen consumption kinetics under 530 nm LED light in aqueous phosphate-buffered solutions. At 100 µm RB, the initial oxygen consumption rates increased by 58% and 80% compared with uncaged RB for the ß and γ (2-hydroxypropyl) cyclodextrins, respectively. For 1 mm RB, the enhancement in these rates was much greater, about 200% and 300%, respectively. In addition, at 1 mm RB these two cyclodextrins increased the RB photobleaching rate by ~20% and ~75%. These results suggest that nanocages can minimize RB aggregation and may lead to higher-efficiency photo-medical therapies.

Detection of singlet oxygen luminescence for experimental corneal rose bengal photodynamic antimicrobial therapy.

Rose bengal photodynamic antimicrobial therapy (RB-PDAT) treats corneal infection by activating rose bengal (RB) with green light to produce singlet oxygen (1O2). Singlet oxygen dosimetry can help optimize treatment parameters. We present a 1O2 dosimeter for detection of 1O2 generated during experimental RB-PDAT. The system uses a 520 nm laser and an InGaAs photoreceiver with bandpass filters to detect 1O2 luminescence during irradiation. The system was validated in RB solutions and ex vivo in human donor eyes. The results demonstrate the feasibility of 1O2 dosimetry in an experimental model of RB-PDAT in the cornea.

Influence of Rose Bengal Dimerization on Photosensitization.

Protein crosslinking photosensitized by rose Bengal (RB2- ) has multiple medical applications and understanding the photosensitization mechanism can improve treatment effectiveness. To this end, we investigated the photochemical efficiencies of monomeric RB2- (RBM 2- ) and dimeric RB2- (RBD 2- ) and the optimal pH for anaerobic RB2- photosensitization in cornea. Absorption spectra and dynamic light scattering (DLS) measurements were used to estimate the fractions of RBM 2- and RBD 2- . RB2- self-photosensitized bleaching was used to evaluate the photoactivity of RBM 2- and RBD 2- . The pH dependence of anaerobic RB2- photosensitization was evaluated in ex vivo rabbit corneas. The 549 nm/515 nm absorption ratio indicated that concentrations > 0.10 mm RB contained RBD 2- . Results from DLS gave estimated mean diameters for RBM 2- and RBD 2- of 0.70 ± 0.02 nm and 1.75 ± 0.13 nm, respectively, and indicated that 1 mm RB2- contained equal fractions of RBM 2- and RBD 2- . Quantum yields for RB2- bleaching were not influenced by RBD 2- in RB2- solutions although accounting for RB2- concentration effects on the reaction kinetics demonstrated that RBD 2- is not a photosensitizer. Optimal anaerobic photosensitization occurred at pH 8.5 for solutions containing 200 mm Arg. These results suggest potential approaches to optimizing RBM 2- -photosensitized protein crosslinking in tissues.

Arginine as an Enhancer in Rose Bengal Photosensitized Corneal Crosslinking.

Purpose: Oxygen-independent cornea crosslinking (CXL) using rose bengal (RB) and green light may have unique clinical applications. These studies were designed to gain insight into the arginine (arg)-enhanced anaerobic crosslinking process, to maximize crosslinking efficiency, and to test a clinically feasible method for oxygen-free CXL. Methods: Rabbit corneas were treated ex vivo using 1 mM RB and 532 nm light. RB photodecomposition, monitored by absorption spectrophotometry, was used to optimize arg concentration and to develop an irradiation and re-dying protocol. The minimal effective green light fluence was identified by linear tensile strength measurements. RB penetration into the stroma was determined by fluorescence microscopy. To favor the anaerobic pathway, a contact lens was used to minimize stromal oxygen level during irradiation. Stromal cell toxicity was evaluated by a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Results: RB photodecomposition reached 75% of its maximal effect at 200 mM arg and the optimal fluence increment was 32.7 J/cm2. The minimal effective fluence for cornea stiffening was 65.4 J/cm2. Placement of a contact lens promoted oxygen-independent cornea stiffening, similar to that obtained on isolated, oxygen-deprived cornea. RB penetration into the stroma with arg present was limited to ∼120 µm, about 25% deeper than without arg. Stromal cell toxicity was limited to the depth of RB and arg penetration. Conclusions: An oxygen-independent pathway in cornea for RB-CXL was characterized and optimized, including a possible clinical protocol for its use. Translational Relevance: Oxygen-independent RB-CXL is an efficient and effective process that can be developed further for unique clinical applications.

Corneal Collagen Ordering After In Vivo Rose Bengal and Riboflavin Cross-Linking.

Purpose: Photoactivated cornea collagen cross-linking (CXL) increases corneal stiffness by initiating formation of covalent bonds between stromal proteins. Because CXL depends on diffusion to distribute the photoinitiator, a gradient of CXL efficiency with depth is expected that may affect the degree of stromal collagen organization. We used second harmonic generation (SHG) microscopy to investigate the differences in stromal collagen organization in rabbit eyes after corneal CXL in vivo as a function of depth and time after surgery. Methods: Rabbit corneas were treated in vivo with either riboflavin/UV radiation (UVX) or Rose Bengal/green light (RGX) and evaluated 1 and 2 months after CXL. Collagen fibers were imaged with a custom-built SHG scanning microscope through the central cornea (350 µm depth, 225 × 225 µm en face images). The order coefficient (OC), a metric for collagen organization, and total SHG signal were computed for each depth and compared between treatments. Results: OC values of CXL-treated corneas were larger than untreated corneas by 27% and 20% after 1 month and 38% and 33% after 2 months for the RGX and UVX, respectively. RGX OC values were larger than UVX OC values by 3% and 5% at 1 and 2 months. The SHG signal was higher in CXL corneas than untreated corneas, both at 1 and 2 months after surgery, by 18% and 26% and 1% and 10% for RGX and UVX, respectively. Conclusions: Increased OC corresponded with increased collagen fiber organization in CXL corneas. Changes in collagen organization parallel reported temporal changes in cornea stiffness after CXL and also, surprisingly, are detected deeper in the stroma than the regions stiffened by collagen cross-links.

Selective Equatorial Sclera Crosslinking in the Orbit Using a Metal-Coated Polymer Waveguide.

Purpose: Photochemical crosslinking of the sclera is an emerging technique that may prevent excessive eye elongation in pathologic myopia by stiffening the scleral tissue. To overcome the challenge of uniform light delivery in an anatomically restricted space, we previously introduced the use of flexible polymer waveguides. We presently demonstrate advanced waveguides that are optimized to deliver light selectively to equatorial sclera in the intact orbit. Methods: Our waveguides consist of a polydimethylsiloxane cladding and a polyurethane core, coupled to an optical fiber. A reflective silver coating deposited on the top and side surfaces of the waveguide prevents light leakage to nontarget, periorbital tissue. Postmortem rabbits were used to test the feasibility of in situ equatorial sclera crosslinking. Tensometry measurements were performed on ex vivo rabbit eyes to confirm a biomechanical stiffening effect. Results: Metal-coated waveguides enabled efficient light delivery to the entire circumference of the equatorial sclera with minimal light leakage to the periorbital tissues. Blue light was delivered to the intact orbit with a coefficient of variation in intensity of 22%, resulting in a 45 ± 11% bleaching of riboflavin fluorescence. A 2-fold increase in the Young's modulus at 5% strain (increase of 92% P < 0.05, at 25 J/cm2) was achieved for ex vivo crosslinked eyes. Conclusions: Flexible polymer waveguides with reflective, biocompatible surfaces are useful for sclera crosslinking to achieve targeted light delivery. We anticipate that our demonstrated procedure will be applicable to sclera crosslinking in live animal models and, potentially, humans in vivo.

Enhancing Rose Bengal-Photosensitized Protein Crosslinking in the Cornea.

Purpose: Rose bengal (RB)-photosensitized protein crosslinking has been proposed for several applications in the eye. This study identifies oxygen-dependent and oxygen-independent mechanistic pathways in cornea for RB-photosensitized crosslinking to enhance its efficiency for ocular treatments. Methods: Rabbit corneas ex vivo were stained with 1 mM RB and irradiated at 532 nm. RB photobleaching, measured by spectrophotometry and linear tensile strength testing, were performed with and without oxygen present. The effects of sodium azide, D2O, arginine, and ascorbate were used to discriminate between mechanisms involving energy transfer (forming singlet oxygen) and electron transfer (forming radical ions). The influence of corneal depth on RB photobleaching was determined using inclined corneal incisions. Results: RB photobleaching was greater in the presence than the absence of oxygen, enhanced by D2O and partially inhibited by azide, indicating a singlet oxygen pathway. Photobleaching without oxygen was enhanced by arginine and ascorbate and accompanied by a shift in the absorption to shorter wavelengths, suggesting that electron transfer initiates RB photodecomposition. The RB-photosensitized tensile strength increase in air was enhanced by D2O and inhibited by azide. In an O2-free environment, arginine was required for an increase in tensile strength, which matched that attained by irradiation in air without arginine, suggesting an efficient electron transfer pathway. Rapid photobleaching was observed below 80 to 120 µm only when arginine was present. Conclusions: These results indicate that RB photosensitizes crosslinking in cornea by both singlet oxygen and electron transfer mechanisms and that adding enhancers may increase the efficiency of this treatment.

Medical Applications of Rose Bengal- and Riboflavin-Photosensitized Protein Crosslinking.

This review summarizes research on many of the potential applications of photosensitized crosslinking of tissue proteins in surgery and current knowledge of the photochemical mechanisms underlying formation of the covalent protein-protein crosslinks involved. Initially developed to close wounds or reattach tissues, protein photocrosslinking has also been demonstrated to stiffen and strengthen tissues, decrease inflammatory responses and facilitate tissue bioengineering. These treatments appear to result largely from crosslinks within and between collagen molecules in tissue that typically form by an oxygen-dependent mechanism. Surgical applications discussed include sealing wounds in skin, cornea and bowel; reattaching severed nerves, blood vessels and tendons; strengthening cornea and vein; reducing capsular contracture after breast implants; and regenerating joint cartilage.

Rose Bengal and Green Light Versus Riboflavin-UVA Cross-Linking: Corneal Wound Repair Response.

Purpose: To study corneal wound healing after two cross-linking techniques using either rose bengal and green light (RGX) or the conventional treatment using riboflavin and UVA radiation (UVX). Methods: Corneas of New Zealand rabbits were monolaterally treated with UVX (21 eyes) or RGX (25 eyes). Treatments involved corneal de-epithelialization (8-mm diameter), soaking with photosensitizer (0.1% riboflavin in 20% dextran for 30 minutes for UVX; 0.1% rose bengal for 2 minutes for RGX), and light irradiation (370 nm, 3 mW/cm2, 30 minutes for UVX; 532 nm, 0.25 W/cm2, 7 minutes for RGX). Contralateral eyes were used as controls. Clinical follow-up included fluorescein staining, haze measurement, and pachymetry. Healing events analyzed after euthanasia at 2, 30, and 60 days included cell death (TUNEL assay), cell proliferation (BrdU [bromodeoxyuridine] immunofluorescence), and differentiation to myofibroblasts (α-SMA [alpha smooth muscle actin] immunohistochemistry). Results: Re-epithelialization and pachymetries were similar after RGX and UVX. The haze from day 1 to 15 was greater after UVX. Cell death was deeper after UVX, being localized in the anterior and middle stroma, and was superficial (anterior third) after RGX. Cell proliferation appeared after 2 days and was localized in the middle and posterior stroma in the UVX group but was superficial in the RGX group. After 60 days the number of stromal cells had not returned to the control number in either group. Conclusions: The deeper and longer-lasting cell damage caused by UVX compared to RGX may underlie the slower cell repopulation after UVX and other differences in healing. Shallower damage and a shorter treatment time suggest that RGX may be appropriate for stiffening thin corneas.

Sealing of Corneal Lacerations Using Photoactivated Rose Bengal Dye and Amniotic Membrane.

PURPOSE: Watertight closure of perforating corneoscleral lacerations is necessary to prevent epithelial ingrowth, infection, and potential loss of the eye. Complex lacerations can be difficult to treat, and repair with sutures alone is often inadequate. In this study, we evaluated a potentially sutureless technology for sealing complex corneal and scleral lacerations that bonds the amniotic membrane (AM) to the wound using only green light and rose bengal dye. METHODS: The AM was impregnated with rose bengal and then sealed over lacerations using green light to bond the AM to the deepithelialized corneal surface. This process was compared with suture repair of 3 laceration configurations in New Zealand White rabbits in 3 arms of the study. A fourth study arm assessed the side effect profile including viability of cells in the iris, damage to the blood-retinal barrier, retinal photoreceptors, retinal pigment epithelium, and choriocapillaris in Dutch Belted rabbits. RESULTS: Analyses of the first 3 arms revealed a clinically insignificant increase in polymorphonuclear inflammation. In the fourth arm, iris cells appeared unaffected and no evidence of breakdown of the blood-retinal barrier was detected. The retina from green light laser-treated eyes showed normal retinal pigment epithelium, intact outer segments, and normal outer nuclear layer thickness. CONCLUSIONS: The results of these studies established that a light-activated method to cross-link AM to the cornea can be used for sealing complex penetrating wounds in the cornea and sclera with minimal inflammation or secondary effects.

Interface Bonding With Corneal Crosslinking (CXL) After LASIK Ex Vivo.

Purpose: Interface bonding with corneal crosslinking (CXL) after LASIK using two different photosensitizers was studied ex vivo. Methods: A LASIK flap was created in enucleated rabbit eyes using a femtosecond laser. After the dissection, CXL was performed to seal the interface. In one group interface CXL was performed using rose bengal and green light, whereas in a second group riboflavin and UV-A light was used. In both groups irradiance, radiant exposure, dye concentration, and imbibition time was varied. In a control group, LASIK only was performed. After the procedures, the maximal shear-force required to separate the flap from the stroma was measured. Additionally, corneal transmission spectra were recorded. Results: Optimized parameters for rose bengal/green-light bonding lead to a 2.1-fold increase in shear-force compared with untreated control eyes (P < 0.01). The optimal parameter combination was: irradiance of 180 mW/cm2 for 14 minutes (total radiant exposure 150 J/cm2), rose bengal concentration 0.1%, and an imbibition time of 2 minutes. Optimized riboflavin/UV-A light parameters were 0.5% for 2 minutes with a radiant exposure of 8.1 J/cm2 obtained by an irradiance of 30 mW/cm2 for 4.5 minutes. These optimized parameters lead to a 2-fold increase compared with untreated control eyes (P < 0.01). Optical transmission experiments suggest safety for more posterior structures. Conclusions: Based on ex-vivo results, interface bonding after LASIK using crosslinking with either rose bengal or riboflavin increases the adhesion between flap and stromal bed. In vivo trials are needed to evaluate the temporal evolution of the effect.

Variations in the endogenous fluorescence of rabbit corneas after mechanical property alterations.

Keratoconus is an eye disease in which the cornea progressively deforms due to loss of cornea mechanical rigidity, and thus causes deterioration of visual acuity. Techniques to characterize the mechanical characteristics of the cornea are important to better monitor changes and response to treatments. To investigate the feasibility of using the endogenous fluorescence of cornea for monitoring alterations of its mechanical rigidity, linear tensiometry was used to quantitate stiffness and Young's modulus (YM) after treatments that increase cornea stiffness (collagen photocross-linking) or decrease stiffness (enzymatic digestion). The endogenous ultraviolet fluorescence of cornea was also measured before and after these treatments. The fluorescence excitation/emission spectral ranges were 280 to 430/390 to 520 nm, respectively. A correlation analysis was carried out to identify fluorescence excitation/emission pairs whose intensity changes correlated with the stiffness. A positive correlation was found between variations in fluorescence intensity of the 415-/485-nm excitation/emission pair and YM of photocross-linked corneas. After treatment of corneas with pepsin, the YM decreased as the fluorescence intensity at 290-/390-nm wavelengths decreased. For weakening of corneas with collagenase, only qualitative changes in the fluorescence spectrum were observed. Changes in the concentration of native or newly created fluorescent molecular species contain information that may be directly or indirectly related to the mechanical structure of the cornea.

Corneal Wound Repair After Rose Bengal and Green Light Crosslinking: Clinical and Histologic Study.

Purpose: To evaluate corneal wound healing after treatment with a new collagen crosslinking protocol using rose bengal dye and green light (RGX). Methods: One cornea of 20 New Zealand rabbits was de-epithelialized (DE) in an 8-mm diameter circle and, in another group (n = 25), the DE corneas were then stained with 0.1% rose bengal for 2 minutes and exposed to green light (532 nm) for 7 minutes (RGX). The contralateral eyes without treatment acted as controls. The animals were clinically followed including fluorescein staining and pachymetry. Healing events were analyzed after euthanasia at 2, 30, and 60 days. Cell death (TUNEL assay), cell proliferation (5-bromo-2'-deoxyuridine incorporation), and cell differentiation to myofibroblasts (α-SMA labeling) were carried out. In addition, loss of keratocytes and subsequent repopulation of the corneal stroma were quantified on hematoxylin-eosin-stained sections. Results: Wound closure was slower after RGX (4.4 days) then after DE (3.3 days). Cell death was restricted to the anterior central stroma, and the cellular decrease did not differ significantly between RGX and DE corneas. Cell proliferation in the epithelium and stroma appeared at 2 days. In both DE and RGX corneas, recovery of the epithelium was complete at day 30, although cell repopulation of the stroma was not complete at 60 days. Conclusions: The healing response in corneas after RGX is very similar to that observed after DE alone, suggesting that, along with its short treatment time and limited effect on keratocytes, RGX displays good potential for clinical cornea stiffening.

Flexible Optical Waveguides for Uniform Periscleral Cross-Linking.

Purpose: Scleral cross-linking (SXL) with a photosensitizer and light is a potential strategy to mechanically reinforce the sclera and prevent progressive axial elongation responsible for severe myopia. Current approaches for light delivery to the sclera are cumbersome, do not provide uniform illumination, and only treat a limited area of sclera. To overcome these challenges, we developed flexible optical waveguides optimized for efficient, homogeneous light delivery. Methods: Waveguides were fabricated from polydimethylsiloxane elastomer. Blue light (445 nm) is coupled into the waveguide with an input fiber. Light delivery efficiency from the waveguide to scleral tissue was measured and fit to a theoretical model. SXL was performed on fresh porcine eyes stained with 0.5% riboflavin, using irradiances of 0, 25, and 50 mW/cm2 around the entire equator of the eye. Stiffness of scleral strips was characterized with tensiometry. Results: Light delivery with a waveguide of tapered thickness (1.4-0.5 mm) enhanced the uniformity of light delivery, compared to a flat waveguide, achieving a coefficient of variation of less than 10%. At 8% strain, sclera cross-linked with the waveguides at 50 mW/cm2 for 30 minutes had a Young's modulus of 10.7 ± 1.0 MPa, compared to 5.9 ± 0.5 MPa for no irradiation, with no difference in stiffness between proximally and distally treated halves. The stiffness of waveguide-irradiated samples did not differ from direct irradiation at the same irradiance. Conclusions: We developed flexible waveguides for periscleral cross-linking. We demonstrated efficient and uniform stiffening of a 5-mm-wide equatorial band of scleral tissue.

An intraluminal stent facilitates light-activated vascular anastomosis.

BACKGROUND: Photochemical tissue bonding (PTB) is a sutureless, light-activated technique that produces a watertight, microvascular repair with minimal inflammation compared to standard microsurgery. However, it is practically limited by the need for a clinically viable luminal support system. The aim of this study was to evaluate a hollow biocompatible stent to provide adequate luminal support to facilitate vascular anastomosis using the PTB technique. METHODS: Forty rats underwent unilateral femoral artery transection. Five rats were used to optimize the stent delivery method, and the remaining 35 rats were randomized into three groups: (1) standard suture repair with 10-0 nylon microsuture (SR), (2) standard suture repair over the stent (SR + S), or (3) PTB repair over stent (PTB + S). For the PTB group, a 2-mm overlapping cuff was painted with 0.1% (wt/vol) Rose Bengal then illuminated for 30 seconds on each side (532 nm, 0.5 W/cm, 30 J/cm). Anastomotic leak and vessel patency (immediate, 1 hour, and 1 week postoperatively) were assessed. RESULTS: Vessels in all three groups were patent immediately and at 1 hour postoperatively. After 1 week, all animals displayed patency in the SR group, while only 5 of 14 and 2 of 8 surviving animals had patent vessels in the PTB + S and SR + S groups, respectively. CONCLUSIONS: This study demonstrated successful use of an intraluminal stent for acute microvascular anastomosis using the PTB technique. However, the longer-term presence of the stent at the anastomotic site led to thrombosis in multiple cases. A rapidly dissolvable stent should facilitate a light-activated microvascular anastomosis with excellent long-term patency.

Biomechanical Changes After In Vivo Collagen Cross-Linking With Rose Bengal-Green Light and Riboflavin-UVA.

Purpose: To compare corneal biomechanical properties after in vivo and ex vivo cross-linking (CXL) using rose bengal-green light (RGX) or riboflavin-UVA (UVX). Methods: Corneas of 30 rabbits were treated in vivo by the two CXL modalities monolaterally (Group 1) or bilaterally (Group 2). Rabbits in Group 1 were euthanized 1 month after treatments and in Group 2 two months after treatment. Ex vivo CXL was also performed. Eyes were measured by Scheimpflug air puff corneal deformation imaging (Corvis ST) under constant IOP. Corneal deformation parameters were assessed. Inherent corneal biomechanical properties were estimated using inverse finite element modeling. Results: Peak to peak distance decreased 16% 2 months after RGX, and 4% and 20% 1 and 2 months after UVX, respectively. The equivalent Young's modulus (Eeq) increased relative to the control during the post treatment period for both RGX and UVX. The Eeq increased by factors of 3.4 (RGX) and 1.7 (UVX) 1 month and by factors of 10.7 (RGX) and 7.3 (UVX) 2 months after treatment. However, the Eeq values for ex vivo CXL were much greater than produced in vivo. The ex vivo Eeq was greater than the 1-month in vivo values by factors of 8.1 (RGX) and 9.1 (UVX) and compared with 2 month by factors of 2.5 (RGX) and 2.1 (UVX). Conclusions: These results indicate that corneal stiffness increases after CXL, and further increases as a function of time after both RGX and UVX. Also, while biomechanical properties determined after ex vivo CXL are indicative of corneal stiffening, they may not provide entirely accurate information about the responses to CXL in vivo.

Collagen-Based Photoactive Agent for Tissue Bonding.

Using a combination of methacrylated collagen and the photosensitizer rose Bengal, a new light-activated biomimetic material for tissue sutureless bonding was developed. This formulation was cross-linked using green light. In vivo tests in mice demonstrate the suitability of the material for sutureless wound closure.