Publications by authors named "Irene E Kochevar"

61 Publications

Influence of Rose Bengal Dimerization on Photosensitization.

Photochem Photobiol 2021 Jan 11. Epub 2021 Jan 11.

Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.

Protein crosslinking photosensitized by rose Bengal (RB ) has multiple medical applications and understanding the photosensitization mechanism can improve treatment effectiveness. To this end, we investigated the photochemical efficiencies of monomeric RB (RB ) and dimeric RB (RB ) and the optimal pH for anaerobic RB photosensitization in cornea. Absorption spectra and dynamic light scattering (DLS) measurements were used to estimate the fractions of RB and RB . RB self-photosensitized bleaching was used to evaluate the photoactivity of RB and RB . The pH dependence of anaerobic RB photosensitization was evaluated in ex vivo rabbit corneas. The 549 nm/515 nm absorption ratio indicated that concentrations > 0.10 mm RB contained RB . Results from DLS gave estimated mean diameters for RB and RB of 0.70 ± 0.02 nm and 1.75 ± 0.13 nm, respectively, and indicated that 1 mm RB contained equal fractions of RB and RB . Quantum yields for RB bleaching were not influenced by RB in RB solutions although accounting for RB concentration effects on the reaction kinetics demonstrated that RB 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 RB -photosensitized protein crosslinking in tissues.
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http://dx.doi.org/10.1111/php.13379DOI Listing
January 2021

Arginine as an Enhancer in Rose Bengal Photosensitized Corneal Crosslinking.

Transl Vis Sci Technol 2020 07 14;9(8):24. Epub 2020 Jul 14.

Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, USA.

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/cm. The minimal effective fluence for cornea stiffening was 65.4 J/cm. 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.
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http://dx.doi.org/10.1167/tvst.9.8.24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7422776PMC
July 2020

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

Invest Ophthalmol Vis Sci 2020 03;61(3):28

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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.
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http://dx.doi.org/10.1167/iovs.61.3.28DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401826PMC
March 2020

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

Invest Ophthalmol Vis Sci 2019 06;60(7):2563-2570

Harvard Medical School and Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts, United States.

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.
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http://dx.doi.org/10.1167/iovs.19-26709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6586079PMC
June 2019

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

Photochem Photobiol 2019 09 10;95(5):1097-1115. Epub 2019 Jul 10.

Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA.

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.
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http://dx.doi.org/10.1111/php.13126DOI Listing
September 2019

Enhancing Rose Bengal-Photosensitized Protein Crosslinking in the Cornea.

Invest Ophthalmol Vis Sci 2019 05;60(6):1845-1852

Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States.

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.
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http://dx.doi.org/10.1167/iovs.19-26604DOI Listing
May 2019

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

Invest Ophthalmol Vis Sci 2018 10;59(12):4821-4830

Departamento de Biología Celular, Histología y Farmacología, GIR de Técnicas Ópticas para el Diagnóstico, Universidad de Valladolid, Valladolid, Spain.

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.
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http://dx.doi.org/10.1167/iovs.18-24881DOI Listing
October 2018

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

Invest Ophthalmol Vis Sci 2017 12;58(14):6292-6298

Wellman Center for Photomedicine-Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States.

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.
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http://dx.doi.org/10.1167/iovs.17-22426DOI Listing
December 2017

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

Cornea 2018 Feb;37(2):211-217

San Antonio Uniformed Services Health Education Consortium, San Antonio, TX.

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.
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http://dx.doi.org/10.1097/ICO.0000000000001389DOI Listing
February 2018

Rose Bengal Binding to Collagen and Tissue Photobonding.

ACS Omega 2017 Oct 11;2(10):6646-6657. Epub 2017 Oct 11.

Wellman Center for Photomedicine, Massachusetts General Hospital and Harvard Medical School, 40 Blossom Street, Boston, Massachusetts 02114, United States.

We investigated two critical aspects of rose Bengal (RB) photosensitized protein cross-linking that may underlie recently developed medical applications. Our studies focused on the binding of RB to collagen by physical interaction and the effect of this binding and certain amino acids on RB photochemistry. Molecular dynamics simulations and free-energy calculation techniques, complemented with isothermal titration calorimetry, provided insight into the binding between RB and a collagen-like peptide (CLP) at the atomic level. Electrostatic interactions dominated, which is consistent with the finding that RB bound equally well to triple helical and single chain collagen. The binding free energy ranged from -5.7 to -3 kcal/mol and was strongest near the positively charged amino groups at the N-terminus and on lysine side chains. At high RB concentration, a maximum of 16 ± 3 bound dye molecules per peptide was found, which is consistent with spectroscopic evidence for aggregated RB bound to collagen or the CLP. Within a tissue-mimetic collagen matrix, RB photobleached rapidly, probably due to electron transfer to certain protein amino acids, as was demonstrated in solutions of free RB and arginine. In the presence of arginine and low oxygen concentrations, a product absorbing at 510 nm formed, presumably due to dehalogenation after electron transfer to RB. In the collagen matrix without arginine, the dye generated singlet oxygen as well as the 510 nm product. These results provide the first evidence of the effects of a tissue-like environment on the photochemical mechanisms of rose Bengal.
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http://dx.doi.org/10.1021/acsomega.7b00675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6644953PMC
October 2017

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

J Biomed Opt 2017 Sep;22(9):1-7

Massachusetts General Hospital, Wellman Center for Photomedicine, Boston, Massachusetts, United States.

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.
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http://dx.doi.org/10.1117/1.JBO.22.9.095005DOI Listing
September 2017

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

Invest Ophthalmol Vis Sci 2017 07;58(9):3471-3480

Departamento de Biología Celular, Histología y Farmacología, GIR de Técnicas Ópticas para el Diagnóstico, Universidad de Valladolid, Valladolid, Spain.

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.
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http://dx.doi.org/10.1167/iovs.16-21365DOI Listing
July 2017

Flexible Optical Waveguides for Uniform Periscleral Cross-Linking.

Invest Ophthalmol Vis Sci 2017 05;58(5):2596-2602

Harvard Medical School and Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts, United States 2Harvard-MIT Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States.

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.
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http://dx.doi.org/10.1167/iovs.17-21559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5433838PMC
May 2017

An intraluminal stent facilitates light-activated vascular anastomosis.

J Trauma Acute Care Surg 2017 07;83(1 Suppl 1):S43-S49

From the Wellman Center for Photomedicine (P.S.-K., J.H.N.-G., I.E.K., R.W.R.), Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts, Division of Plastic and Reconstructive Surgery, Department of Surgery (P.S.-K., J.H.N.-G., M.A.R., J.M.W.), Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts, and Department of Orthopedic Surgery (H.B.-S., O.K.M.), Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts.

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.
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http://dx.doi.org/10.1097/TA.0000000000001487DOI Listing
July 2017

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

Invest Ophthalmol Vis Sci 2017 03;58(3):1612-1620

Instituto de Optica, Consejo Superior de Investigaciones Cientificas, Madrid, Spain.

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.
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http://dx.doi.org/10.1167/iovs.17-21475DOI Listing
March 2017

Antimicrobial Blue Light Therapy for Infectious Keratitis: Ex Vivo and In Vivo Studies.

Invest Ophthalmol Vis Sci 2017 01;58(1):586-593

Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States.

Purpose: To investigate the effectiveness of antimicrobial blue light (aBL) as an alternative or adjunctive therapeutic for infectious keratitis.

Methods: We developed an ex vivo rabbit model and an in vivo mouse model of infectious keratitis. A bioluminescent strain of Pseudomonas aeruginosa was used as the causative pathogen, allowing noninvasive monitoring of the extent of infection in real time via bioluminescence imaging. Quantitation of bacterial luminescence was correlated to colony-forming units (CFU). Using the ex vivo and in vivo models, the effectiveness of aBL (415 nm) for the treatment of keratitis was evaluated as a function of radiant exposure when aBL was delivered at 6 or 24 hours after bacterial inoculation. The aBL exposures calculated to reach the retina were compared to the American National Standards Institute standards to estimate aBL retinal safety.

Results: Pseudomonas aeruginosa keratitis fully developed in both the ex vivo and in vivo models at 24 hours post inoculation. Bacterial luminescence in the infected corneas correlated linearly to CFU (R2 = 0.921). Bacterial burden in the infected corneas was rapidly and significantly reduced (>2-log10) both ex vivo and in vivo after a single exposure of aBL. Recurrence of infection was observed in the aBL-treated mice at 24 hours after aBL exposure. The aBL toxicity to the retina is largely dependent on the aBL transmission of the cornea.

Conclusions: Antimicrobial blue light is a potential alternative or adjunctive therapeutic for infectious keratitis. Further studies of corneal and retinal safety using large animal models, in which the ocular anatomies are similar to that of humans, are warranted.
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http://dx.doi.org/10.1167/iovs.16-20272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5283079PMC
January 2017

Erratum: Light-Activated Sealing of Acellular Nerve Allografts following Nerve Gap Injury.

J Reconstr Microsurg 2016 11 24;32(9):e1. Epub 2016 Jun 24.

Wellman Centre for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts.

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http://dx.doi.org/10.1055/s-0036-1584882DOI Listing
November 2016

Corneal Crosslinking With Rose Bengal and Green Light: Efficacy and Safety Evaluation.

Cornea 2016 Sep;35(9):1234-41

*Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA; †Department of Ophthalmology, Shanghai First People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; and ‡Boston Eye Group, Boston, MA.

Purpose: To evaluate crosslinking of cornea in vivo using green light activation of Rose Bengal (RGX) and assess potential damaging effects of the green light on retina and iris.

Methods: Corneas of Dutch belted rabbits were de-epithelialized, then stained with Rose Bengal and exposed to green light, or not further treated. Corneal stiffness was measured by uniaxial tensiometry. Re-epithelialization was assessed by fluorescein fluorescence. Keratocytes were counted on hematoxylin and eosin (H&E)-stained sections, and iris cell damage was assessed by lactate dehydrogenase staining. Thermal effects on the blood-retinal barrier (BRB) were assessed by fluorescein angiography and those on photoreceptors, retinal pigment epithelium (RPE), and choriocapillaris by light microscopy and transmission electron microscopy.

Results: RGX (10-min irradiation; 150 J/cm) increased corneal stiffness 1.9-fold on day 1 (1.25 ± 0.21 vs. 2.38 ± 0.59 N/mm; P = 0.036) and 2.8-fold compared with controls on day 28 (1.70 ± 0.74 vs. 4.95 ± 1.86 N/mm; P = 0.003). Keratocytes decreased only in the anterior stroma on day 1 (24.0 ± 3.0 vs. 3.67 ± 4.73, P = 0.003) and recovered by day 28 (37.7 ± 8.9 vs. 34.5 ± 2.4, P = 0.51). Iris cells were not thermally damaged. No evidence of BRB breakdown was detected on days 1 or 28. Retina from RGX-treated eyes seemed normal with RPE cells showing intact nuclei shielded apically by melanosomes, morphologically intact photoreceptor outer segments, normal outer nuclear layer thickness, and choriocapillaris containing intact erythrocytes.

Conclusions: The substantial corneal stiffening produced by RGX together with the lack of significant effects on keratocytes and no evidence for retina or iris damage suggest that RGX-initiated corneal crosslinking may be a safe, rapid, and effective treatment.
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http://dx.doi.org/10.1097/ICO.0000000000000916DOI Listing
September 2016

UV-A Irradiation Activates Nrf2-Regulated Antioxidant Defense and Induces p53/Caspase3-Dependent Apoptosis in Corneal Endothelial Cells.

Invest Ophthalmol Vis Sci 2016 Apr;57(4):2319-27

Schepens Eye Research Institute Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.

Purpose: To examine whether Nrf2-regulated antioxidant defense and p53 are activated in human corneal endothelial cells (CEnCs) by environmental levels of ultraviolet A (UV-A), a known stimulator of oxidative stress.

Methods: Immortalized human CEnCs (HCEnCi) were exposed to UV-A fluences of 2.5, 5, 10, or 25 J/cm2, then allowed to recover for 3 to 24 hours. Control HCEnCi did not receive UV-A. Reactive oxygen species (ROS) were measured using H2DCFDA. Cell cytotoxicity was evaluated by lactate dehydrogenase (LDH) release. Levels of Nrf2, HO-1, NQO-1, p53, and caspase3 were detected by immunnoblotting or real-time PCR. Activated caspase3 was measured by immunoblotting and a fluorescence assay.

Results: Exposure of HCEnCi to 5, 10, and 25 J/cm2 UV-A increased ROS levels compared with controls. Nrf2, HO-1, and NQO-1 mRNA increased 1.7- to 3.2-fold at 3 and 6 hours after irradiation with 2.5 and 5 J/cm2 UV-A. At 6 hours post irradiation, UV-A (5 J/cm2) enhanced nuclear Nrf2 translocation. At 24 hours post treatment, UV-A (5, 10, and 25 J/cm2) produced a 1.8- to 2.8-fold increase in phospho-p53 and a 2.6- to 6.0-fold increase in activated caspase3 compared with controls, resulting in 20% to 42% cell death.

Conclusions: Lower fluences of UV-A induce Nrf2-regulated antioxidant defense and higher fluences activate p53 and caspase3, indicating that even near-environmental levels of UV-A may affect normal CEnCs. This data suggest that UV-A may especially damage cells deficient in antioxidant defense, and thus may be involved in the etiology of Fuchs' endothelial corneal dystrophy (FECD).
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http://dx.doi.org/10.1167/iovs.16-19097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4855825PMC
April 2016

A light-activated amnion wrap strengthens colonic anastomosis and reduces peri-anastomotic adhesions.

Lasers Surg Med 2016 07 21;48(5):530-7. Epub 2016 Mar 21.

Wellman Center for Photomedicine, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts, 02114.

Background And Objective: Colonic anastomotic failure is a dreaded complication, and multiple surgical techniques have failed to eliminate it. Photochemical tissue bonding (PTB) is a method of sealing tissue surfaces by light-activated crosslinking. We evaluated if a human amniotic membrane (HAM), sealed over the anastomotic line by PTB, increases the anastomotic strength.

Study Design: Sprague-Dawley rats underwent midline laparotomy followed by surgical transection of the left colon. Animals were randomized to colonic anastomosis by one of the following methods (20 per group): single-layer continuous circumferential suture repair (SR); SR with a HAM wrap attached by suture (SR+ HAM-S); SR with HAM bonded photochemically over the anastomotic site using 532 nm light (SR+ HAM-PTB); approximation of the bowel ends with only three sutures and sealing with HAM-PTB (3+ HAM-PTB). A control group underwent laparotomy alone with no colon resection (NR). Sub-groups (n = 10) were sacrificed at days 3 and 7 post-operatively and adhesions were evaluated. A 6 cm section of colon was then removed and strength of anastomosis evaluated by burst pressure (BP) measurement.

Results: A fourfold increase in BP was observed in the SR+ HAM-PTB group compared to suture repair alone (94 ± 3 vs. 25 ± 8 mm Hg, P < 0.0001) at day 3. At day 7 the burst pressures were 165 ± 40 and 145 ± 31 mm Hg (P = 1), respectively. A significant decrease in peri-anastomotic adhesions was observed in the SR+ HAM-PTB group compared to the SR group at both time points (P < 0.001).

Conclusion: Sealing sutured colonic anastomotic lines with HAM-PTB increases the early strength of the repair and reduces peri-anastomotic adhesions. Lasers Surg. Med. 48:530-537, 2016. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/lsm.22507DOI Listing
July 2016

Corneal Biomechanical Response Following Collagen Cross-Linking With Rose Bengal-Green Light and Riboflavin-UVA.

Invest Ophthalmol Vis Sci 2016 Mar;57(3):992-1001

Instituto de Optica Consejo Superior de Investigaciones Cientificas, Madrid, Spain.

Purpose: To compare the biomechanical corneal response of two different corneal cross-linking (CXL) treatments, rose bengal-green light (RGX) and riboflavin-UVA (UVX), using noninvasive imaging.

Methods: A total of 12 enucleated rabbit eyes were treated with RGX and 12 with UVX. Corneal dynamic deformation to an air puff was measured by high speed Scheimpflug imaging (Corvis ST) before and after treatment. The spatial and temporal deformation profiles were evaluated at constant intraocular pressure of 15 mm Hg, and several deformation parameters were estimated. The deformation profiles were modeled numerically using finite element analysis, and the hyperelastic corneal material parameters were obtained by inverse modeling technique.

Results: The corneal deformation amplitude decreased significantly after both CXL methods. The material parameters obtained from inverse modeling were consistent with corneal stiffening after both RGX and UVX. Within the treated corneal volume, we found that the elasticity decreased by a factor of 11 after RGX and by a factor of 6.25 after UVX.

Conclusions: The deformation of UVX-treated corneas was smaller than the RGX-treated corneas. However, the reconstructed corneal mechanical parameters reveal that RGX produced in fact larger stiffening of the treated region (100-μm depth) than UVX (137-μm depth). Rose bengal-green light stiffens the cornea effectively, with shorter treatment times and shallower treated areas. Dynamic air puff deformation imaging coupled with mechanical simulations is a useful tool to characterize corneal biomechanical properties, assess different treatments, and possibly help optimize the treatment protocols.
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http://dx.doi.org/10.1167/iovs.15-18689DOI Listing
March 2016

Light-Activated Sealing of Acellular Nerve Allografts following Nerve Gap Injury.

J Reconstr Microsurg 2016 Jul 15;32(6):421-30. Epub 2016 Feb 15.

Wellman Centre for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts.

Introduction Photochemical tissue bonding (PTB) uses visible light to create sutureless, watertight bonds between two apposed tissue surfaces stained with photoactive dye. In phase 1 of this two-phase study, nerve gaps repaired with bonded isografts were superior to sutured isografts. When autograft demand exceeds supply, acellular nerve allograft (ANA) is an alternative although outcomes are typically inferior. This study assesses the efficacy of PTB when used with ANA. Methods Overall 20 male Lewis rats had 15-mm left sciatic nerve gaps repaired using ANA. ANAs were secured using epineurial suture (group 1) or PTB (group 2). Outcomes were assessed using sciatic function index (SFI), gastrocnemius muscle mass retention, and nerve histomorphometry. Historical controls from phase 1 were used to compare the performance of ANA with isograft. Statistical analysis was performed using analysis of variance and Bonferroni all-pairs comparison. Results All ANAs had signs of successful regeneration. Mean values for SFI, muscle mass retention, nerve fiber diameter, axon diameter, and myelin thickness were not significantly different between ANA + suture and ANA + PTB. On comparative analysis, ANA + suture performed significantly worse than isograft + suture from phase 1. However, ANA + PTB was statistically comparable to isograft + suture, the current standard of care. Conclusion Previously reported advantages of PTB versus suture appear to be reduced when applied to ANA. The lack of Schwann cells and neurotrophic factors may be responsible. PTB may improve ANA performance to an extent, where they are equivalent to autograft. This may have important clinical implications when injuries preclude the use of autograft.
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http://dx.doi.org/10.1055/s-0035-1571247DOI Listing
July 2016

Toward New Engagement Paradigms For Intraocular Lenses: Light-Initiated Bonding of Capsular Bag to Lens Materials.

Invest Ophthalmol Vis Sci 2015 Jul;56(8):4249-56

Wellman Center for Photomedicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, United States.

Purpose: Successful intraocular lens procedures, that is, implantation of accommodating intraocular lenses (A-IOL), require firm engagement of the IOL haptics to the capsular bag. We evaluated the use of photochemical bonding to engage IOL materials to the capsular bag.

Methods: Freshly enucleated eyes of New Zealand rabbits were used in two types of photobonding experiments using Rose Bengal (RB) photoinitiation and green light (532-nm) irradiation. First, RB-stained capsular bag strips were photobonded ex vivo to IOL polymer [poly(2-hydroxyethyl methacrylate) pHEMA] strips in an atmosphere of air and of nitrogen. Second, IOLs were implanted intracapsularly and photobonded intraocularly. Irradiation times were between 30 and 180 seconds, and laser irradiance was between 0.25 and 0.65 W/cm(2). The strength of the bonding was tested using a custom-developed uniaxial extensiometry system and the breakage load (the load that caused breakage per bonded area) was calculated.

Results: The breakage load of ex vivo capsule-pHEMA bonds increased exponentially with irradiation time, using 0.45 W/cm(2). In air, the average breakage load across all conditions was 1 g/mm(2) and 1.6 times lower than that in a nitrogen atmosphere. Intraocularly, RB-stained IOLs were strongly photobonded to the capsule bag with breakage loads > 0.8 g/mm(2).

Conclusions: Breakage of the photobonded linkage between IOL material and capsular bag required loads substantially greater than the maximum force of ciliary muscle, suggesting that this technology may introduce a new paradigm for engagement of A-IOLs. The bonding produced in air was stronger than that in nitrogen atmosphere, suggesting that oxygen is involved in the chemical mechanism for photobonding.
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http://dx.doi.org/10.1167/iovs.15-17070DOI Listing
July 2015

Light-activated sealing of skin wounds.

Lasers Surg Med 2015 Jan 23;47(1):17-29. Epub 2014 Nov 23.

Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts; Dermatology Department, Shanghai East Hospital, Tongji University, Shanghai, China.

Background And Objectives: We have developed a light-activated technology for rapidly sealing skin surgical wounds called photochemical tissue bonding (PTB). The goals of this study were to evaluate parameters influencing PTB in order to optimize its clinical efficacy and to determine whether PTB can be used to seal wounds in moderately to highly pigmented skin.

Study Design/materials And Methods: Application of Rose Bengal (RB) followed by exposure to 532 nm was used to seal linear incisions (1.5 mm deep, 2 cm long) in lightly pigmented (Yorkshire) and darkly pigmented (Yucatan) swine skin. The force required to open the seal (the bonding strength) was measured by in situ tensiometry. Reflectance spectra, epidermal transmission spectra, and histology were used to characterize the skin. The relationships of RB concentration and fluence to bonding strength were established in Yorkshire skin. Surface temperature was measured during irradiations and cooling was used while sealing incisions in Yucatan skin. Monte Carlo simulations were carried out to estimate the effect of epidermal melanin on the power absorbed in the dermis at the incision interface.

Results: The lowest fluence, 25 J/cm(2), delivered at an irradiance of 0.5 W/cm(2) substantially increased the bonding strength (∼ 10-fold) compared to controls in Yorkshire swine skin. Increasing the fluence to 100 J/cm(2) enhanced bonding strength by a further 1.5-fold. Application of 0.1% RB for 2 minutes produced the greatest bonding strength using 100 J/cm(2) and limited the penetration of RB to an ∼ 50 μm band on the dermal incision wall. Reflectance spectra indicated that Yorkshire skin had minimal melanin and that Yucatan skin was a good model for highly pigmented human skin. In Yucatan skin, the bonding strength increased 1.7-fold using 0.1% RB and 200 J/cm(2) at 1.5 W/cm(2) with cooling and epinephrine. Monte Carlo simulation indicated that absorption of 532 nm light by epidermal melanin in dark skin decreased the power absorbed along the incision in the dermis by a factor of 2.7.

Conclusions: These results suggest that in lightly pigmented skin the PTB treatment time can be shortened without compromising the bonding strength. Sealing incisions using PTB in moderately and highly pigmented skin will require a careful balance of irradiance and cooling.
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http://dx.doi.org/10.1002/lsm.22308DOI Listing
January 2015

Decreased DJ-1 leads to impaired Nrf2-regulated antioxidant defense and increased UV-A-induced apoptosis in corneal endothelial cells.

Invest Ophthalmol Vis Sci 2014 Jul 31;55(9):5551-60. Epub 2014 Jul 31.

Schepens Eye Research Institute, Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.

Purpose: To investigate the role of DJ-1 in Nrf2-regulated antioxidant defense in corneal endothelial cells (CECs) at baseline and in response to ultraviolet A (UV-A)-induced oxidative stress.

Methods: DJ-1-deficient CECs were obtained by transfection of an immortalized normal human corneal endothelial cell line (HCECi) with DJ-1 small interfering RNA (siRNA) or by isolation of CECs from ex vivo corneas of DJ-1 knockout mice. Levels of reactive oxygen species (ROS), protein carbonyls, Nrf2 subcellular localization, Nrf2 target genes, and protein interaction between Keap1/Nrf2 and Cul3/Nrf2 were compared between normal and DJ-1-deficient CECs. Oxidative stress was induced by irradiating HCECi cells with UV-A, and cell death and levels of activated caspase3 and phospho-p53 were determined.

Results: DJ-1 siRNA-treated cells exhibited increased levels of ROS production and protein carbonyls as well as a 2.2-fold decrease in nuclear Nrf2 protein when compared to controls. DJ-1 downregulation led to attenuated gene expression of Nrf2 and its target genes HO-1 and NQO1. Similar levels of Nrf2 inhibitor, Keap1, and Cul3/Nrf2 and Keap1/Nrf2 were observed in DJ-1 siRNA-treated cells as compared to controls. Ultraviolet A irradiation resulted in a 3.0-fold increase in cell death and elevated levels of activated caspase3 and phospho-p53 in DJ-1 siRNA-treated cells compared to controls.

Conclusions: Downregulation of DJ-1 impairs nuclear translocation of Nrf2, causing decreased antioxidant gene expression and increased oxidative damage. The decline in DJ-1 levels leads to heightened CEC susceptibility to UV-A light by activating p53-dependent apoptosis. Targeting the DJ-1-Nrf2 axis may provide a potential therapeutic approach for enhancing antioxidant defense in corneal endothelial disorders.
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http://dx.doi.org/10.1167/iovs.14-14580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4160071PMC
July 2014

Why is rose bengal more phototoxic to fibroblasts in vitro than in vivo?

Photochem Photobiol 2014 Mar-Apr;90(2):297-305. Epub 2013 Dec 16.

Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA.

Photosensitized protein cross-linking has been recently developed to seal wounds and strengthen tissue. Although the photosensitizing dye, Rose Bengal (RB), is phototoxic to cultured cells, cytotoxicity does not accompany RB-photosensitized tissue repair in vivo. We investigated whether the environment surrounding cells in tissue or the high irradiances used for photo-cross-linking inhibited RB phototoxicity. Fibroblasts (FB) grown within collagen gels to mimic a tissue environment and monolayer cultured FB were treated with RB (0.01-1 mm) and the high 532 nm laser irradiances used in vivo for tissue repair (0.10-0.50 W cm(-2)). Monolayer FB were substantially more sensitive to RB photosensitization: the LD50 was >200-fold lower than that in collagen gels. Collagen gel protection was associated with increased Akt phosphorylation, a prosurvival pathway. RB phototoxicity in collagen gels was 25-fold greater at low (0.030 W cm(-2)) that at high (0.50 W cm(-2)) irradiances. Oxygen depletion at high irradiance only partially accounted for the irradiance dependence of phototoxicity as replacing air with nitrogen only increased the LD50 by four-fold in monolayers. These results indicate that the lack of RB phototoxicity during in vivo tissue repair results from upregulation of prosurvival pathways in tissue cells, oxygen depletion and irradiance-dependent RB photochemistry.
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http://dx.doi.org/10.1111/php.12215DOI Listing
June 2015

Collagen cross-linking using rose bengal and green light to increase corneal stiffness.

Invest Ophthalmol Vis Sci 2013 May 13;54(5):3426-33. Epub 2013 May 13.

Boston University Medical Sciences Program, Boston, MA, USA.

Purpose: Photochemical cross-linking of corneal collagen is an evolving treatment for keratoconus and other ectatic disorders. We evaluated collagen cross-linking by rose bengal plus green light (RGX) in rabbit eyes and investigated factors important for clinical application.

Methods: Rose bengal (RB, 0.1%) was applied to deepithelialized corneas of enucleated rabbit eyes for 2 minutes. The diffusion distance of RB into the stroma was measured by fluorescence microscopy on frozen sections. RB-stained corneas were exposed to green (532-nm) light for 3.3 to 9.9 minutes (50-150 J/cm(2)). Changes in the absorption spectrum during the irradiation were recorded. Corneal stiffness was measured by uniaxial tensiometry. The spatial distribution of the stromal elastic modulus was assessed by Brillouin microscopy. Viable keratocytes were counted on H&E-stained sections 24 hours posttreatment.

Results: RB penetrated approximately 100 μm into the corneal stroma and absorbed >90% of the incident green light. RGX (150 J/cm(2)) increased stromal stiffness by 3.8-fold. The elastic modulus increased in the anterior approximately 120 μm of stroma. RB was partially photobleached during the 2-minute irradiation, but reapplication of RB blocked light transmission by >70%. Spectral measurements suggested that RGX initiated cross-linking by an oxygen-dependent mechanism. RGX did not decrease keratocyte viability.

Conclusions: RGX significantly increases cornea stiffness in a rapid treatment (≅12 minutes total time), does not cause toxicity to keratocytes and may be used to stiffen corneas thinner than 400 μm. Thus, RGX may provide an attractive approach to inhibit progression of keratoconus and other ectatic disorders.
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http://dx.doi.org/10.1167/iovs.12-11509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4597485PMC
May 2013

A photoactivated nanofiber graft material for augmented Achilles tendon repair.

Lasers Surg Med 2012 Oct 21;44(8):645-52. Epub 2012 Aug 21.

Department of Burns and Plastic Surgery, No. 3 People's Hospital, and Institute of Traumatic Medicine; School of Medicine, Shanghai Jiao Tong University, Shanghai 201900, P.R. China.

Background And Objective: Suture repair of Achilles tendon rupture can cause infection, inflammation and scarring, while prolonged immobilization promotes adhesions to surrounding tissues and joint stiffness. Early mobilization can reduce complications provided the repair is strong enough to resist re-rupture. We have developed a biocompatible, photoactivated tendon wrap from electrospun silk (ES) to provide additional strength to the repair that could permit early mobilization, and act as a barrier to adhesion formation.

Study Design/material And Methods: ES nanofiber mats were prepared by electrospinning. New Zealand white rabbits underwent surgical transection of the Achilles tendon and repair by: (a) SR: standard Kessler suture + epitendinous suture (5-0 vicryl). (b) ES/PTB: a single stay suture and a section of ES mat, stained with 0.1% Rose Bengal (RB), wrapped around the tendon and bonded with 532 nm light (0.3 W/cm(2) , 125 J/cm(2) ). (c) SR + ES/PTB: a combination of (a) and (b). Gross appearance, extent of adhesion formation and biomechanical properties of the repaired tendon were evaluated at Days 7, 14, or 28 post-operatively (n = 8 per group at each time point).

Results: Ultimate stress (US) and Young's modulus (E) in the SR group were not significantly different from the ES/PTB group at Days 7 (US, P = 0.85; E, P = 1), 14 (US, P = 0.054; E, P = 1), and 28 (US, P = 0.198; E, P = 0.12) post-operatively. Adhesions were considerably greater in the SR group compared to the ES/PTB group at Days 7 (P = 0.002), 14 (P < 0.0001), and 28 (P < 0.0001). The combination approach of SR + ES/PTB gave the best outcomes in terms of E at 7 (P < 0.016) and 14 days (P < 0.016) and reduced adhesions compared to SR at 7 (P < 0.0001) and 14 days (P < 0.0001), the latter suggesting a barrier function for the photobonded ES wrap.

Conclusion: Photochemical sealing of a ES mat around the tendon repair site provides considerable benefit in Achilles tendon repair. Lasers Surg. Med. 44: 645-652, 2012. © 2012 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/lsm.22066DOI Listing
October 2012

Light-activated sutureless closure of wounds in thin skin.

Lasers Surg Med 2012 Feb 13;44(2):163-7. Epub 2011 Dec 13.

Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.

Background And Objectives: Closing lacerations in thin eyelid and periorbital skin is time consuming and requires high skill for optimal results. In this study we evaluate the outcomes after single layer closure of wounds in thin skin with a sutureless, light-activated photochemical technique called PTB.

Study Design/materials And Methods: Dorsal skin of the SKH-1 hairless mouse was used as a model for eyelid skin. Incisions (1.2 cm) were treated with 0.1% Rose Bengal dye followed by exposure to 532 nm radiation (25, 50, or 100 J/cm(2); 0.25 W/cm(2)) for PTB. Other incisions were sutured (five 10-0 monofilament), exposed only to 532 nm (100 J/cm(2)), or not treated. Outcomes were immediate seal strength (pressure causing leakage through incision of saline infused under wound), skin strength at 1, 3, and 7 days (measured by tensiometry), inflammatory infiltrate at 1, 3, and 7 days (histological assessment), and procedure time.

Results: The immediate seal strength, as measured by leak pressure, was equivalent for all PTB fluences and for sutures (27-32 mmHg); these pressures were significantly greater than for the controls (untreated incisions or laser only treatment; P < 0.001). The ultimate strength of PTB-sealed incisions was greater than the controls at day 1 (P < 0.05) and day 3 (P < 0.025) and all groups were equivalent at day 7. Sutures produced greater inflammatory infiltrate at day 1 than observed in other groups (P = 0.019). The average procedure time for sutured closure (311 seconds) was longer than for the PTB group treated with 25 J/cm(2) (160 seconds) but shorter than the group treated with 100 J/cm(2) (460 seconds).

Conclusion: PTB produces an immediate seal of incisions in thin, delicate skin that heals well, is more rapid than suturing, does not require painful suture removal and is easy to apply.
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http://dx.doi.org/10.1002/lsm.21137DOI Listing
February 2012

Light-initiated bonding of amniotic membrane to cornea.

Invest Ophthalmol Vis Sci 2011 Dec 9;52(13):9470-7. Epub 2011 Dec 9.

Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.

Purpose: Suturing amniotic membrane to cornea during surgery is time consuming, and sutures may further damage the eye. The authors introduce a novel sutureless, light-activated technique that securely attaches amnion to cornea through protein-protein crosslinks.

Methods: Cryopreserved human amniotic membrane, stained with Rose Bengal (RB), was placed over a full-thickness wound in deepithelialized rabbit cornea and was treated with green laser. The intraocular pressure that broke the seal (IOP(L)) was measured, and adhesion was measured with a peel test. The influences on bonding strength of fluence, irradiance, RB concentration, and amnion surface bonded were measured. Epithelial cell migration on treated amnion and keratocyte viability after bonding were also measured. The involvement in the bonding mechanism of oxygen, singlet oxygen, and association of RB with stromal collagen was investigated.

Results: Sealing amniotic membrane over cornea using 0.1% RB and 150 J/cm(2) at 532 nm produced an IOP(L) of 261 ± 77 mm Hg ex vivo and 448 mm ± 212 mm Hg in vivo. The ex vivo IOP(L) increased with increasing fluence (50-150 J/cm(2)). Equivalent IOP(L) was produced for bonding basement membrane or stromal amnion surfaces. The bonding treatment was not toxic to keratocytes but slightly reduced the migration of corneal epithelial cells on amnion ex vivo. Mechanism studies indicated that RB forms two complexes with amnion stromal collagen, that bonding requires oxygen, and that singlet oxygen mediates protein crosslinking.

Conclusions: A rapid, light-activated technique produces strong, immediate bonding between amnion and cornea and merits further evaluation for ocular surface surgeries.
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http://dx.doi.org/10.1167/iovs.11-7248DOI Listing
December 2011