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.

Albumin-genipin solder for laser tissue repair.

BACKGROUND: Laser tissue soldering (LTS) is an alternative technique to suturing for tissue repair that avoids foreign body reaction and provides immediate sealing of the wound. One of the major drawbacks of LTS, however, is the weak tensile strength of the solder welds when compared to sutures. In this study, a crosslinking agent of low cytotoxicity was investigated for its ability to enhance the bond strength of albumin solders with sheep intestine. STUDY DESIGN/MATERIALS AND METHODS: Solder strips were welded onto rectangular sections of sheep small intestine using a diode laser. The laser delivered in continuous mode a power of 170 +/- 10 mW at lambda = 808 nm, through a multimode optical fiber (core size = 200 microm) to achieve a dose of 10.8 +/- 0.5 J/mg. The solder thickness and surface area were kept constant throughout the experiment (thickness = 0.15 +/- 0.01 mm, area = 12 +/- 1.2 mm2). The solder was composed of 62% bovine serum albumin (BSA), 0.38% genipin, 0.25% indocyanin green dye (IG), and water. Tissue welding was also performed with a BSA solder without genipin, as a control group. The repaired tissue was tested for tensile strength by a calibrated tensiometer. Murine fibroblasts were also cultured in extracted media from heat-denatured genipin solder to assess cell growth inhibition in a 48 hours period. RESULTS: The tensile strength of the genipin solder was doubled that of the BSA solder (0.21 +/- 0.04 N and 0.11 +/- 0.04 N, respectively; P = 10(-15) unpaired t-test, N = 30). Media extracted from crosslinked genipin solder showed negligible toxicity to fibroblast cells under the culture conditions examined here. CONCLUSION: Addition of a chemical crosslinking agent, such as genipin, significantly increased the tensile strength of adhesive-tissue bonds. A proposed mechanism for this enhanced bond strength is the synergistic action of mechanical adhesion with chemical crosslinking by genipin.

Impact of solubility on laser tissue-welding with albumin solid solders.

BACKGROUND AND OBJECTIVE: The correlation between the solubility of solid albumin solders and their laser weld strength was investigated. STUDY DESIGN/MATERIALS AND METHODS: Sections of dog intestine were laser welded with soluble or insoluble solid strips of solder. Two different treatments were followed for tissue soldering: "wet weld" and "dry weld." These treatments were chosen to assess the impact of solubility on the repair strength. The laser power and radiation dose were 0.14 W and 14 J/mg, respectively. Calorimetric measures of solders were also performed. RESULTS: The moisture on the tissue partially dissolved the soluble strips at the tissue interface. Hence, the repair strength of the soluble solder was significantly stronger than the repair strength of the insoluble solder (0.22 N and 0.06 N, P < 0.0001). Temperature (approximately 70 degrees C) and enthalpy variation (approximately 1.4 J/g) for denaturing the soluble and insoluble solders were not significantly different (P > 0.05). CONCLUSION: The soluble solid solder behaved like dense liquid solder at the tissue interface. Hence, the interface strength of these two forms of solder should be similar. This correlation made it possible to identify an intrinsic limit for the weld strength of albumin solders.

Carotid artery anastomosis with albumin solder and near infrared lasers: a comparative study.

BACKGROUND AND OBJECTIVE: Laser tissue-welding has been used for anastomosis of carotid arteries. During welding, thermal injury sustained by the vessel walls should be minimized to prevent thrombosis. Two different types of lasers were used and effects on tissue damage were studied in vitro and in vivo. STUDY DESIGN/MATERIALS AND METHODS: End-to-end anastomosis of dog carotid arteries (n = 10) was performed by using a human albumin solder (HAS) in conjunction with Nd:YAG or diode lasers (lambda = 1.32 microm and 1.9 microm, respectively). The arteries were evaluated for patency and evidence of histologic injury after 21 days. Another group of arteries was laser soldered in vitro to measure the intimal and adventitial temperatures by using thermocouples. RESULTS: The arteries repaired with the diode laser sustained significantly less thermal damage than those repaired with Nd:YAG laser, both in vitro and in vivo. In particular, the intimal temperature was significantly lower (P < 0.05) for the diode than for the Nd:YAG repairs (approximately 35 degrees C and approximately 50 degrees C, respectively). In the latter group, the patency rate was 75%, but thrombosis occurred in 75% of the specimens at 21 days. All diode anastomoses were patent and thrombosis developed in only 17% of the arteries. CONCLUSION: Use of the diode laser and albumin solders may provide a means to successfully repair carotid arteries with minimal thermal damage.

Self-expandable chitosan stent: design and preparation.

Stents are largely used in surgical procedures to relieve pathological obstructions. The purpose of the present study was to design and prepare a biocompatible stent with a self-expandable mechanism. Thin films were prepared from deacetylated chitosan (4% w/v) dissolved in acetic acid solution (2% v/v). The chitosan films were tested by a calibrated tensiometer to measure the Young's module (E). The films were used to manufacture stents by pulling and winding them around a cylindrical rod in a helical fashion. Thirteen stents (diameter = 0.5 +/- 0.05 mm, length approximately 4 mm) were inserted into the vas deferens of wistar rats. Upon stent insertion, the vasal anastomosis was achieved with a laser-soldering technique. The animals were sacrificied 8 weeks later. The stress test showed that the chitosan film was elastic (maximum strain = 105% +/- 6%, E = 0.7655 +/- 0.0288 Mpa). The stents self-expanded by releasing their elastic energy. All the stents but one remained open inside the vasa despite high incidence of sperm granuloma. A biocompatible and self-expandable stent with a helical design is proposed.

Bone marrow segmentation in leukemia using diffusion and T (2) weighted echo planar magnetic resonance imaging.

Magnetic resonance images of leukemic bone marrow were acquired over large regions of the pelvis and lower abdomen with minimal interference from overlying tissues using diffusion and T(2) weighted echo planar imaging. Data acquisition times were on the order of 1 min for scanning volumes of up to 25 l at a spatial resolution of 31 microl. A survey of 21 patients with leukemia and eight healthy adult volunteers was undertaken to determine the magnitude of the observed effect and its dependence upon specific pathologies. The acquisition methods yielded high-quality segmentation of leukemic bone marrow prior to therapy in seven of seven patients with acute lymphocytic leukemia, chronic lymphocytic leukemia or chronic myelogenous leukemia, and who had hypercellular (>95%) bone marrow at the time of the study. The quality of the segmentation was sufficient to allow the use of maximum intensity projection images which afforded a convenient evaluation of both intra- and extramedullary disease. The measured signal-to-noise ratios agreed with a theoretical estimate that accounted for the percentage cellularity, T(2) relaxation time of water, and self-diffusion coefficient of water in iliac bone marrow. In addition, the mean signal-to-noise ratios from iliac marrow were strongly dependent upon the time after the initiation of chemotherapeutic regimens, implying that the methods may be useful for therapeutic monitoring.

Laser-assisted demucosalized gastrocystoplasty with autoaugmentation in a canine model.

OBJECTIVES: Laser-assisted autoaugmentation gastrocystoplasty has been performed successfully. Experiments were performed to determine the optimal laser for tissue welding during demucosalized autoaugmentation gastrocystoplasty using both a 1.9-microm diode and a 1.32-microm neodymium:yttrium-aluminum-garnet (Nd:YAG) laser with and without thermal control. METHODS: Autoaugmentation gastrocystoplasty was performed on 18 female mongrel dogs. Anastomoses were performed by either suture or laser welding with a 50% human albumin solution. A 1.9-microm diode laser was compared with a 1.32-microm Nd:YAG laser with and without thermal control. In vivo canine bladder capacity measurements were performed both before gastrocystoplasty and at euthanasia. The animals were studied on days 4 and 14. Samples of the anastomotic area from each group were taken to measure tensile strength. Histologic samples were assessed for tissue damage. RESULTS: There was a significant increase in bladder volume in the 4-day group compared with pregastrocystoplasty values. Both the 1.9-microm diode laser and suture control dogs with the 14-day repairs had significantly more tensile strength than their 4-day counterparts. In contrast, no statistical difference was found between the 4 and 14-day 1.32-microm Nd:YAG groups. The suture control group had evidence of minor tissue devitalization at the anastomosis at both 4 and 14 days. The 1.9 and 1.32-microm laser groups both had evidence of tissue devitalization at 4 and 14 days. The 1.32-microm laser group had primarily severe tissue injury. The laser groups at 14 days demonstrated an inflammatory reaction that was localized to the albumin. CONCLUSIONS: Demucosalized gastrocystoplasty with autoaugmentation can be safely and successfully performed with a 1.9-microm diode laser without significant differences in tensile strength when compared with suture controls. The 1.32-microm Nd:YAG laser can also be successfully used; however, the long-term results appear to be inferior to the 1.9-microm diode laser.

Assessment of the degradation of denatured albumin solder by human urine.

PURPOSE: Several studies have been undertaken to evaluate the efficacy of human serum albumin (HSA) as a solder in urologic procedures. The purpose of this study was to evaluate whether albumin solder undergoes significant degradation in urine. MATERIALS AND METHODS: Laser denatured 25% HSA pellets were incubated at 37C for varying times with 1 ml. of either pooled human urine or control diluent solution adjusted to the same pH and osmolality as urine. To assess the contribution of enzymatic degradation, aliquots of urine were boiled and compared with non-boiled urine and diluent. The amount of solubilized HSA in solution was measured using the Bradford assay, while the degradation of albumin was detected by SDS-PAGE electrophoresis. RESULTS: Approximately 5% of the albumin was degraded over a period of 7 days following incubation at 37C, regardless of treatment. SDS-PAGE revealed only minor traces of degradation in urine and controls. The very slight degradation of denatured HSA appears to be non-enzymatic, as it was observed in both urine and diluent samples. CONCLUSIONS: HSA solder appears to be appropriate for use in urologic reconstructive surgery since it is not appreciably degraded in the presence of urine.

Effect of laser welding with human serum albumin on the expression of P-selectin on platelets.

BACKGROUND AND OBJECTIVE: Artery repair by means of laser energy induces activation of platelets with a risk of thrombosis and local inflammatory reactions. The aim of this study was to investigate the effect of human serum albumin, the most common solder in laser surgery, on platelet activation. STUDY DESIGN/MATERIALS AND METHODS: Platelet activation was evaluated in canine blood by using two-color flow cytometry with a phycoerythrin-labeled antibody to a common platelet marker, glycoprotein IIb/IIIa and fluorescein isothiocyanate-labeled antibody to a platelet activation molecule, P-selectin. Human serum albumin was applied in vitro and in vivo, as a solder during laser reconstruction of canine arteries. RESULTS: In vitro, albumin significantly (P < 0.01) reduces the expression of P-selectin on platelets. This is most likely related to the blockage of P-selectin by albumin, which binds to the platelet surface, as confirmed by flow cytometry with fluorescein isothiocyanate-labeled albumin. In vivo, application of albumin solder tended to result in a lower percentage of P-selectin-expressing platelets in laser-repaired arteries compared to suture-repaired arteries. CONCLUSION: Albumin decreases the percentage of P-selectin-expressing platelets in vitro. Further research may allow the platelet activation inhibiting properties of albumin to be further optimized in vivo.

Two-layer film as a laser soldering biomaterial.

BACKGROUND AND OBJECTIVES: A two-layer solder was developed to weld at low laser intensity and to provide a new method of measuring solder-tissue temperature. STUDY DESIGN/MATERIALS AND METHODS: A film solder consisted of a white layer (bovine serum albumin (BSA) and distilled water) and a black layer (BSA, carbon black (CB), and distilled water). This two-layer solder was used with a diode laser to weld sections of dog small intestine (lambda = 810 nm, power = 200 +/- 20 mW, radiation dose = 18 +/- 1 J/mg). Sections of intestine were welded only with one-layer black solders as control group. The temperature difference between the external solder surface and the tissue-solder interface was evaluated during welding. RESULTS: The two-layer solder performed welds as strong as the one-layer solder ( approximately 0.12 N) but with less laser intensity on the black layer. The temperature difference between the external surface of the solder and the solder-tissue interface was significantly less for the two-layer solder than for the one-layer solder ( approximately 6 degrees C and approximately 15 degrees C, respectively; P < 0.05). CONCLUSIONS: The two-layer solder appeared to be more efficient at soldering biomaterials than the one-layer solder. Furthermore, the heat diffusion from the black midplane of the two-layer solder decreased the difference in temperature recorded on the solder external surface and on the solder-tissue interface.

Preliminary study of microsurgical repairs of the inferior alveolar nerve in rats using primary suturing and laser weld techniques.

A comparison of primary suturing and a new laser weld technique is described for the microsurgical repair of the inferior alveolar nerve in Wistar rats. A reliable method of exposure of the inferior alveolar nerve has been developed in order to allow intraosseous repairs of the nerve involving suturing with 10:0 nylon and a laser weld technique using an albumin-based solder, containing indocynine cardiogreen, plus an infrared (810 nm wavelength) diode laser. Seven cases of microsuture and laser weld repairs were performed with a 29.4% reduction in total operating time in the laser weld group. Histochemical analysis showed comparable mean neuron counts and mean tracer uptake by neurons for the microsuture and laser weld groups. Giant cell reactions were identified in two of the primary suture cases and axon deflection in three cases demonstrating possible advantages of the laser weld technique which showed no adverse reactions by axons or epineurium to the coagulative repair with the solder. The technique of laser weld repair, on initial analysis, therefore appears comparable with traditional suture repairs and indeed may possess several advantages. Further studies are recommended.

Laser nerve repair by solid protein band technique. I: identification of optimal laser dose, power, and solder surface area.

Thirty-four tibial nerves in 17 adult male wistar rats were repaired by applying protein bands longitudinally across the nerve join. The bands were then irradiated with a fibre-coupled diode laser (lambda = 810 nm). The relations among the laser weld breaking force, the power, and the solder surface area were investigated, while maintaining a consistent ratio between the total mass of protein solder in a band and total laser energy delivered (the laser energy dose). When this laser energy dose was held constant, the average breaking force of the laser welds irradiated by 72 mW laser output power was weaker than that reached after 90 mW laser radiation. There is a linear relation between the solder breaking force and the solder surface area when band thickness, laser power, and laser dose are unvaried.

Laser nerve repair by solid protein band technique. II: assessment of long-term nerve regeneration.

A total of 18 adult male Wistar rats had left tibial nerve repaired by either the laser-solder technique or a more conventional microsuture technique. The diode laser power was 90 mW and the radiation dose 16 J/mg. Three months postoperatively electrophysiology showed that the average compound muscle action potential (CMAP) of the laser repair group was not significantly different from the CMAP of the sutured nerves. Light microscopy confirmed regeneration of myelinated axons in both groups of animals. The laser-solder technique, when used with such parameters, proved to be a reliable method to achieve satisfactory peripheral nerve anastomosis and nerve regeneration.

Repair strength dependence on solder protein concentration: a study in laser tissue-welding.

BACKGROUND AND OBJECTIVE: A novel laser-activated solid solder has been coupled with a diode laser to investigate the dependence of the solder protein concentration on the tensile strength of the soldered tissues. The uncertainty of laser welding, due to the fluid glue, was overcome using the solid solder. STUDY DESIGN/MATERIALS AND METHODS: Sixty-two severed rat tibial nerves and vas deferens were repaired using rectangular protein bands with two different albumin concentrations (58% and 68% by weight). The laser power (90 mW and 140 mW), dose (12.9 +/- 0.7 J/mg, mean +/- s.d.), and solder dimensions (thickness = 0.15 +/- 0.01 mm, surface area = 7.8 +/- 0.4 mm2) were kept constant during the operations. RESULTS: The laser welds with high protein solder concentration were significantly (P < 0.05) stronger (28 +/- 3.5 g) than the welds with low protein solder concentration (23 +/- 5 g). CONCLUSIONS: The average tensile strength of the laser soldered tissues increased as the protein solder concentration increased.

Solubility study of albumin solders for laser tissue-welding.

BACKGROUND/OBJECTIVE: Current albumin solders for tissue-welding are soluble in physiological fluids, prior to laser irradiation. These solders are therefore subjected to mechanical alterations, which can weaken the solder-tissue repair. In this study, an albumin solder (laser activated) was developed with low solubility and with the ability to retain (partially) its mechanical characteristics in saline solution. STUDY DESIGN/MATERIALS AND METHODS: Gauged protein samples of solder were immersed into 0.5 ml saline solution for fixed intervals of time. The solder samples contained four bovine serum albumin (BSA) concentrations: 56%, 66%, 70%, and 75% (by weight). A Bradford protein assay measured the BSA solubility of the solders. The 70% and 75% BSA solders were also used to weld in vitro Wistar rat intestine sections with a diode laser (lambda = 810 nm, power = 270 mW). RESULTS: The solubility of the 75% BSA solder was significantly decreased with respect to the other solders (Anova, P < 0.05). This solder also showed comparable weld strength (13 gm) to the 70% BSA solder. CONCLUSION: The 75% BSA solder strongly reduced the albumin solubility in saline solution, without affecting its tissue-welding properties.

Laser-activated solid protein bands for peripheral nerve repair: an vivo study.

BACKGROUND AND OBJECTIVE: Severed tibial nerves in rats were repaired using a novel technique, utilizing a semiconductor diode-laser-activated protein solder applied longitudinally across the join. Welding was produced by selective laser denaturation of solid solder bands containing the dye indocyanine green. STUDY DESIGN/MATERIALS AND METHODS: An in vivo study, using 48 adult male Wistar rats, compared conventional microsuture-repaired tibial nerves with laser solder-repaired nerves. Nerve repairs were characterised immediately after surgery and after 3 months. RESULTS: Successful regeneration with average compound muscle action potentials of 2.5 +/- 0.5 mV and 2.7 +/- 0.3 mV (mean and standard deviation) was demonstrated for the laser-soldered nerves and the sutured nerves, respectively. Histopathology confirmed comparable regeneration of axons in laser- and suture-operated nerves. CONCLUSION: The laser-based nerve repair technique was easier and faster than microsuture repair, minimising manipulation damage to the nerve.