Microscopic characterization of collagen modifications induced by low-temperature diode-laser welding of corneal tissue

BACKGROUND AND OBJECTIVE: Laser welding of corneal tissue that employs diode lasers (810 nm) at low power densities (12-20 W/cm(2)) in association with Indocyanine Green staining of the wound is a technique proposed as an alternative to conventional suturing procedures. The aim of this study is to evaluate, by means of light (LM) and transmission electron microscopy (TEM) analyses, the structural modifications induced in laser-welded corneal stroma. MATERIALS AND METHODS: Experiments were carried out in 20 freshly enucleated pig eyes. A 3.5 mm in length full-thickness cut was produced in the cornea, and was then closed by laser welding. Birefringence modifications in samples stained with picrosirius red dye were analyzed by polarized LM to assess heat damage. TEM analysis was performed on ultra-thin slices, contrasted with uranyl acetate and lead citrate, in order to assess organization and size of type I collagen fibrils after laser welding. RESULTS: LM evidenced bridges of collagen bundles between the wound edges, with a loss of regular lamellar organization at the welded site. Polarized LM indicated that birefringence properties were mostly preserved after laser treatment. TEM examinations revealed the presence of quasi-ordered groups of fibrils across the wound edges preserving their interfibrillar spacing. These fibrils appeared morphologically comparable to those in the control tissue, indicating that type I collagen was not denatured during the diode laser corneal welding. CONCLUSIONS: The preservation of substantially intact, undenatured collagen fibrils in laser-welded corneal wounds supported the thermodynamic studies that we carried out recently, which indicated temperatures below 66 degrees C at the weld site under laser irradiation. This observation enabled us to hypothesize that the mechanism, proposed in the literature, of unwinding of collagen triple helixes followed by fibrils "interdigitation" is not likely to occur in the welding process that we set up for the corneal suturing.     

CO2 and argon laser vascular welding: acute histologic and thermodynamic comparison

CO2 and argon lasers have been used successfully for vascular welding in both experimental and clinical settings. This study compared the thermodynamics during CO2 and argon laser welding of 1-cm longitudinal arteriotomies in a canine model. Continuous recordings using an AGA 782 digital thermographic system with spatial and thermal resolution of +/-0.2 mm and +/-0.2 degree C, respectively, were analyzed. A HGM argon laser using a 300-microns optic fiber held at 1 cm from the vessel edges (spot diameter = 2.8 mm) with concomitant room temperature saline irrigation (1 drop/sec) was used for argon welds. Total exposure time was 150 sec/cm. CO2 welds were performed with a Sharplan CO2 laser (spot diameter = 0.22 mm) with no irrigation for total exposure time of 10 sec/cm. Thermodynamic results and laser parameters are summarized as follows: Argon-n = 20; power = 500 mW; energy fluence = 1,400 J/cm2; Tmax = 48.8 degrees C; T mean +/- S.D. = 45.1 +/- 2.7 degrees C; CO2-n = 20; power = 150 mW; energy fluence = 3,000 J/cm2; Tmax 84.0 degrees C; T mean +/- S.D. = 60.7 +/- 9.8 degrees C. There was a significant difference (P less than .05) in thermal measurements between successful CO2 and argon vascular welds. Temperature rise during the argon welds was limited by saline irrigation. In contrast, during CO2 laser welding, the temperature rose quickly to its maximum and was maintained at a relatively high level as the laser progressed (0.1 cm/sec) along the anastomosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chitosan adhesive for laser tissue repair: in vitro characterization

BACKGROUND AND OBJECTIVES: Laser tissue repair usually relies on hemoderivate protein solders, based on serum albumin. These solders have intrinsic limitations that impair their widespread use, such as limited tensile strength of repaired tissue, poor solder solubility, and brittleness prior to laser denaturation. Furthermore, the required activation temperature of albumin solders (between 65 and 70 degrees C) can induce significant thermal damage to tissue. In this study, we report on the design of a new polysaccharide adhesive for tissue repair that overcomes some of the shortcomings of traditional solders. STUDY DESIGN/MATERIALS AND METHODS: Flexible and insoluble strips of chitosan adhesive (elastic modulus approximately 6.8 Mpa, surface area approximately 34 mm2, thickness approximately 20 microm) were bonded onto rectangular sections of sheep intestine using a diode laser (continuous mode, 120 +/- 10 mW, lambda = 808 nm) through a multimode optical fiber with an irradiance of approximately 15 W/cm2. The adhesive was based on chitosan and also included indocyanin green dye (IG). The temperature between tissue and adhesive was measured using a small thermocouple (diameter approximately 0.25 mm) during laser irradiation. The repaired tissue was tested for tensile strength by a calibrated tensiometer. Murine fibroblasts were cultured in extracted media from chitosan adhesive to assess cytotoxicity via cell growth inhibition in a 48 hours period. RESULTS: Chitosan adhesive successfully repaired intestine tissue, achieving a tensile strength of 14.7 +/- 4.7 kPa (mean +/- SD, n = 30) at a temperature of 60-65 degrees C. Media extracted from chitosan adhesive showed negligible toxicity to fibroblast cells under the culture conditions examined here. CONCLUSION: A novel chitosan-based adhesive has been developed, which is insoluble, flexible, and adheres firmly to tissue upon infrared laser activation.     

Atomic force microscopy and transmission electron microscopy analyses of low-temperature laser welding of the cornea

Low-temperature laser welding of the cornea is a technique used to facilitate the closure of corneal cuts. The procedure consists of staining the wound with a chromophore (indocyanine green), followed by continuous wave irradiation with an 810 nm diode laser operated at low power densities (12–16 W/cm²), which induces local heating in the 55–65 °C range. In this study, we aimed to investigate the ultrastructural modifications in the extracellular matrix following laser welding of corneal wounds by means of atomic force microscopy and transmission electron microscopy. The results evidenced marked disorganization of the normal fibrillar assembly, although collagen appeared not to be denatured under the operating conditions we employed. The mechanism of low-temperature laser welding may be related to some structural modifications of the nonfibrillar extracellular components of the corneal stroma.     

Microvascular anastomosis using a photochemical tissue bonding technique

BACKGROUND AND OBJECTIVES: Photochemical tissue bonding (PTB) combines photoactive dyes with visible light to create fluid-tight seals between tissue surfaces without causing collateral thermal damage. The potential of PTB to improve outcomes over standard of care microsurgical reanastomoses of blood vessels in ex vivo and in vivo models was evaluated. STUDY DESIGN: The mechanical strength and integrity of PTB and standard microsurgical suture repairs in ex vivo porcine brachial arteries (n = 10) were compared using hydrostatic testing of leak point pressure (LPP). Femoral artery repair in vivo was measured in Sprague-Dawley rats using either standard microvascular sutures (n = 20) or PTB (n = 20). Patency was evaluated at 6 hours (n = 10) and 8 weeks post-repair (n = 10) for each group. RESULTS: PTB produced significantly higher LPPs (1,100+/- 150 mmHg) than suture repair (350+/-40 mmHg, P<0.001) in an ex vivo study. In an in vivo study all femoral arteries in both suture and PTB repair groups were patent at 6 hours post-repair. At 8 weeks post-repair the patency rate was 80% for both groups. No evidence of aneurysm formation was seen in either group and bleeding was absent from the repair site in the PTB-treated vessels, in contrast to the suture repair group. CONCLUSION: PTB is a feasible microvascular repair technique that results in an immediate, mechanically robust bond with short- and long-term patency rates equal to those for standard suture repair.     

In-vivo response to free electron laser incision of the rabbit cornea

BACKGROUND AND OBJECTIVE: We analyzed the in vivo ocular response to corneal incisions made by Medical Free Electron Laser (MFEL) as a function of scan rate and incision depth. Additionally, we compared biomicroscopy, optical coherence tomography (OCT), and light microscopy as ocular response diagnostic tools. STUDY DESIGN/MATERIALS AND METHODS: Rabbit corneas were incised with pulsed MFEL radiation at 2.94 microm wavelength, scalpel incisions or focal cautery treatment were used as controls. The MFEL beam scan rate ranged from 0.2 to 1.0 mm/second. Ocular effects were monitored for 2 hours postoperatively using OCT and slit lamp examination. Ocular tissue was fixed for light microscopic examination. RESULTS: Anterior chamber fibrin formation correlated with MFEL incision depth. Slower scan rates resulted in deeper incisions and greater fibrin formation. OCT was better than slit lamp biomicroscopy at identifying fibrin attachments. OCT and light microscopy proved to be excellent companion technologies. CONCLUSIONS: Deep corneal incisions in the rabbit produced by the MFEL resulted in fibrin formation in the anterior chamber.     

Solid state ultraviolet laser (213 nm) ablation of the cornea and synthetic collagen lenticules.

We used a Q-switched Nd:YAG laser with non-linear optical crystals to produce the 5th (213 nm) and the 4th (266 nm) harmonic frequencies. Using these two wavelengths, we ablated fresh porcine corneas and type I collagen synthetic epikeratoplasty lenticules. For the 213-nm ablation, radiant exposure was 1.3 J/cm2. The ablation rate was 0.23 micron per pulse for the epikeratoplasty lenticules. We examined all tissues with light microscopy, transmission electron microscopy, and scanning electron microscopy. Histology for the 213-nm ablation showed a clean ablation crater with minimal collagen lamellae disruption and a damage zone less than 1 micron. In comparison, the 266 nm radiation showed more charring at the edges with a damage zone approximately 25 microns deep with disruption of the stromal lamella. Our results show that this solid state UV laser is a potential alternative to the excimer laser for cornea surgery.     

In vitro and in vivo tissue repair with laser-activated chitosan adhesive

BACKGROUND AND OBJECTIVES: Sutures are currently the gold standard for wound closure but they are still unable to seal tissue and may induce scarring or inflammation. Biocompatible glues, based on polysaccharides such as chitosan, are a possible alternative to conventional wound closure. In this study, the adhesion of laser-activated chitosan films is investigated in vitro and in vivo. In particular we examine the effect of varying the laser power, as well as adding a natural cross-linker (genipin) to the adhesive composition. STUDY DESIGN/MATERIALS AND METHODS: Flexible and insoluble strips of chitosan films (surface area approximately 34 mm(2), thickness approximately 20 microm) were bonded to sheep intestine using several laser powers (0, 80, 120, and 160 mW) at 808-nm wavelength. The strength of repaired tissue was tested by a calibrated tensiometer to select the best power. A natural cross-linker (genipin) was also added to the film and the tissue repair strength compared with the strength of plain films. The adhesive was also bonded in vivo to the sciatic nerve of rats and the thermal damage induced by the laser assessed 4 days post-operatively. RESULTS: Chitosan adhesives successfully repaired intestine tissue, attaining a maximum repair strength of 14.7+/-4.3 kPa (n = 30) at the laser power of 120 mW. The chitosan-genipin films achieved lower repair strength (9.1+/-2.9 kPa). The laser caused partial demyelination of axons at the site of operation, but the myelinated axons retained a normal morphology proximally and distally. CONCLUSIONS: The chitosan adhesive effectively bonded to tissue causing only localized thermal damage in vivo, when the appropriate laser parameters were selected.     

Cell vitality in cartilage tissue culture following excimer laser radiation: An in vitro examination

Excimer laser is used for cartilage debridement, although the resulting cell damage is yet unclear. For examination of cartilage survival after treatment, we used short-term tissue cultures of human joint cartilage. Specimens were treated with a XeCl-Excimer laser using different laser parameters, pulse energies, and repetition rates. Following treatment, discs were cultured for 8 days prior to examination. In contrast to the 20 μm damage zone as instant visible effect in histomorphologic examinations, we found a 0.3 mm zone in which ～ 50% of cartilage cells had morphological signs of damage on light microscopic examinations. Autoradiography revealed that cartilage cells in an 0.5–0.7 mm area surrounding the laser craters had no collagen synthesis. This examination indicates that cell damage of excimer laser is higher than expected from prior studies. © 1993 Wiley-Liss, Inc.     

In vitro comparison of thulium-holmium-chromium: YAG and argon ion lasers for welding of biliary tissue

Laser welding offers several potential advantages over suture closure, including improved healing, lack of a nidus for stone formation, and greater speed and ease. We examined in vitro gallbladder cystic duct welds created by two different systems, the thulmium-holmium-chromium (THC):YAG (2,150 nm) and argon ion (488-514 nm) lasers, in an effort to define suitable parameters for tissue fusion. Mean bursting pressures for argon welds were 95 mm Hg at 1.5 W CW and 26 mm Hg at 1.5 W, 50 msec chopped delivery. For the THC:YAG laser, the mean bursting pressure for welds created with 300 mJ pulses was 45 mm Hg. Full-thickness tissue fusion and limited collateral thermal damage were observed histologically for both the CW argon and pulsed THC:YAG welds. Examination of the suggested mechanisms of tissue fusion for these photothermal lasers suggests that increased duration of tissue heating at the appropriate temperature results in more extensive collagen crosslinking and a stronger weld.