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

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.

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

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.

Light-activated sealing of skin wounds.

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.

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.

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.

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.

Light-activated sutureless closure of wounds in thin skin.

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.

A photoactivated nanofiber graft material for augmented Achilles tendon repair.

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.

A light-activated method for repair of corneal surface defects.

BACKGROUND AND OBJECTIVE: Amniotic membrane transplantation (AMT) sealed with sutures has been routinely used to treat ocular surface defects (OSD). However, the sutures used to secure the graft on the cornea cause additional injury, infection, and scarring. A new light-activated technique, called photochemical tissue bonding (PTB), has been developed for securing amniotic membrane (AM) over the corneal surface. The purpose of this study was to compare PTB versus traditional sutures in AMT for repair of OSD. MATERIALS AND METHODS: An OSD was created in the left eye of 40 rabbits. The eyes were randomized into two repair groups: AMT using sutures and AMT using PTB with 0.1% Rose Bengal (RB) and 532 nm laser at 0.4 W/cm(2) for 200 seconds. Eyes were examined for re-epithelialization, inflammation, neovascularization, and scarring histologically and biochemically on postoperative days 1, 3, 14, and 28. RESULTS: PTB strongly bonded AM over corneal defects. Corneal re-epithelialization did not differ significantly between the suture and PTB groups. Histology, immunohistology and Western blotting revealed that the numbers of inflammatory cells and the level of tumor necrosis factor-alpha in the PTB group were dramatically lower than those in the suture group on postoperative day 3. Many fewer neo-vessels were present in the PTB group (2.91 ± 1.00) compared to the suture group (4.33 ± 1.15) at day 28 (P < 0.05). The collagen fibers in the PTB group were well organized and orientated as assessed by second harmonic generation microscopy, suggesting that PTB treatment led to less corneal scarring. CONCLUSIONS: PTB is a superior method for securing AM over OSD with improved wound healing compared to sutures.

Photochemical tissue bonding: a potential strategy for treating limbal stem cell deficiency.

BACKGROUND AND OBJECTIVE: To determine the feasibility of attaching human amniotic membrane (HAM), pre-cultured with limbal stem cells (LSCs), to cornea using a novel, light-activated tissue bonding method. MATERIALS AND METHODS: LSCs were isolated from rabbit eyes, and then cultured on de-epithelialized HAM to create grafts (HAM/LSC). These were then transplanted onto rabbit eyes with surgically created limbal stem cell deficiency (LSCD). The grafts were secured either by sutures or by a light-activated method called photochemical tissue bonding (PTB). Outcomes included corneal opacity, inflammation, neovascularization, and collagen alignment. RESULTS: The isolated and cultured cells were verified to be LSCs based on their K19+/intergrin ß1+/P63+/K3 profile. Securing the HAM/LSC graft with PTB provided better outcomes. At 28 days post-surgery, the corneal opacity scores were significantly lower after securing the graft with PTB compared with suture attachment (0.8 ± 0.5 vs. 1.8 ± 0.5, P < 0.01). Similarly, neovascularization scores were lower after PTB (0.8 ± 0.5 vs. 1.5 ± 0.6, P < 0.01). Quantification of MPO and CD31 levels from immunofluorecent staining indicated that PTB stimulated less neutrophil infiltration (5.3 ± 2.2 vs. 13.3 ± 3.1, P < 0.01) and less new blood vessels formation (2.0 ± 0.8 vs. 6.3 ± 1.3, P < 0.01) at the wound site. The collagen alignment in PTB-treated corneas, as shown by immunofluorescence and second harmonic generation image, was better organized in the PTB-treated group than in the suture group. CONCLUSION: Bonding LSC grafts with PTB produced improved outcomes compared to suture attachment. This light-activated method is a promising modality for treating patients with LSCD.

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.

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.

Phototoxicity is not associated with photochemical tissue bonding of skin.

BACKGROUND AND OBJECTIVE: We have developed a light-activated method called photochemical tissue bonding (PTB) for closing wounds using green light and a photosensitizing dye (Rose Bengal-RB) to initiate photochemical crosslinking of wound surface proteins. These studies were designed to determine whether RB causes phototoxicity during closure of skin incisions with PTB. STUDY DESIGN/MATERIALS AND METHODS: RB phototoxicity was evaluated after sealing incisions in porcine skin ex vivo and rabbit skin in vivo using PTB (1 mM RB, 100 J/cm(2), 532 nm, 0.3 or 0.5 W/cm(2).) Dead cells were identified by pyknotic nuclei and eosinophilic cytoplasm on H&E-stained sections. The influence on RB phototoxicity of penetration of RB into the wound wall (by confocal microscopy), RB concentration in the tissue (by extraction), and fluence of 532 nm reaching depths in skin (calculated from skin optical properties) were investigated. RESULTS: No significant differences were found in the percent dead cells in PTB-treated and control incisions in porcine skin at 24 hours or in rabbit skin at 2 hours and 3 and 7 days after surgery. RB was retained in a approximately 100 microm wide band next to the wound wall. The mean RB concentration within this band was 0.42+/-0.03 mM. Monte Carlo modeling of light distribution indicated that the fluence rate decreased from the subsurface peak to 0.5 W/cm(2) in the mid-dermis (approximately 350 microm.) In vitro RB phototoxicity to dermal fibroblasts yielded an LD(50) of 0.50+/-0.09 J/cm(2) when the cells contained 0.46 mM RB. CONCLUSIONS: PTB does not cause phototoxicity when used to repair skin wounds even though the RB concentration and 532 nm fluence in the mid-dermis during PTB are much greater than the LD(50) for RB phototoxicity in vitro. These results indicate that phototoxicity is not a concern when using PTB for tissue repair.

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.

Photochemical sealing improves outcome following peripheral neurorrhaphy.

INTRODUCTION: Peripheral nerve transection initiates a complex molecular response in the severed nerve endings, resulting in the release of neurotrophic and neurotropic factors that are central to axonal survival and regeneration. In this study we tested the hypothesis that sealing the neurorrhaphy site from the surrounding environment using a photochemically bonded nerve wrap would optimize the endoneural environment and enhance regeneration and nerve function recovery. MATERIALS AND METHODS: Adult rats underwent unilateral sciatic nerve transection and standard epineural nerve repair. The repair site was wrapped with amniotic membrane or autologous vein and then was either sealed using photochemical tissue bonding (PTB) or secured with sutures. Photochemical sealing without a wrap was also carried out. Functional recovery was assessed at 2-wk intervals using walking track analysis and nerve histomorphometry was assessed at 12 wk. RESULTS: Treating nerves with PTB-sealed amnion significantly improved functional recovery and increased distal axon and fiber diameters and myelin thickness compared to nerves treated with standard neurorrhaphy alone. Direct PTB sealing of the repair site also improved function. Neither amnion secured with sutures nor vein wraps exhibited improved functional or histological recovery compared to standard neurorrhaphy. CONCLUSIONS: These results suggest that sealing the peripheral nerve repair site with amnion using a photochemical technique may lead to earlier restoration of neural homeostasis and consequent enhanced repair of nerve injury.

Low level light effects on inflammatory cytokine production by rheumatoid arthritis synoviocytes.

BACKGROUND AND OBJECTIVE: Low level light therapy (LLLT) is being evaluated for treating chronic and acute pain associated with rheumatoid arthritis (RA) and other inflammatory diseases. The mechanisms underlying the effectiveness of LLLT for pain relief in RA are not clear. The objectives of this study were to determine whether LLLT decreased production of pro-inflammatory cytokines by cells from RA joints, and, if so, to identify cellular mechanisms. STUDY DESIGN/MATERIALS AND METHODS: Synoviocytes from RA patients were treated with 810 nm radiation before or after addition of tumor necrosis factor-alpha (TNF-alpha). mRNA for TNF-alpha, interleukin (IL)-1beta, IL-6, and IL-8 was measured after 30, 60, and 180 minutes using RT-PCR. Intracellular and extracellular protein levels for 12 cytokines/chemokines were measured at 4, 8, and 24 hours using multiplexed ELISA. NF-kappaB activation was detected using Western blotting to follow degradation of IkappaBalpha and nuclear localization of the p65 subunit of NF-kappaB. RESULTS: Radiation at 810 nm (5 J/cm(2)) given before or after TNF-alpha decreases the mRNA level of TNF-alpha and IL-1beta in RA synoviocytes. This treatment using 25 J/cm(2) also decreases the intracellular levels of TNF-alpha, IL-1beta, and IL-8 protein but did not affect the levels of seven other cytokines/chemokines. TNF-alpha-induced activation of NF-kappaB is not altered by 810 nm radiation using 25 J/cm(2). CONCLUSIONS: The mechanism for relieving joint pain in RA by LLLT may involve reducing the level of pro-inflammatory cytokines/chemokines produced by synoviocytes. This mechanism may be more general and underlie the beneficial effects of LLLT on other inflammatory conditions.

Engineering cartilage in a photochemically crosslinked collagen gel.

This study's purpose was to investigate whether photochemically crosslinking collagen gel to encapsulate chondrocytes (articular, auricular, costal) would permit new cartilage formation in vivo, and to determine whether this neocartilage had the ability to integrate with existing native cartilage. Chondrocytes from swine were embedded in collagen gel that was photochemically crosslinked using riboflavin and visible light. Controls were collagen gels containing cells that were not crosslinked. Cylindrical implants (0.1 cc) were placed in athymic mice for 4 and 8 weeks. To study integration, the constructs were crosslinked within articular cartilage rings and implanted in the mice. Samples were analyzed in terms of macroscopic, microscopic, and biochemical aspects. Photocrosslinking did not affect the amount of glycosaminoglycan and type II collagen produced by the cells. We found that photochemical crosslinking collagen gel enhances the physical parameters of the gel and permits new cartilage formation that can integrate with existing native cartilage.