Karen M. McNally-Heintzelman

Optimal parameters for laser tissue soldering

Variations in laser irradiance, exposure time, solder composition, chromophore type and concentration have led to inconsistencies in published results of laser-solder repair of tissue. To determine optimal parameters for laser tissue soldering, an in vitro study was performed using an 808-nm diode laser in conjunction with an indocyanine green (ICG)- doped albumin protein solder to weld bovine aorta specimens. Liquid and solid protein solders prepared from 25% and 60% bovine serum albumin (BSA), respectively, were compared. The effects of laser irradiance and exposure time on tensile strength of the weld and temperature rise as well as the effect of hydration on bond stability were investigated. Optimum irradiance and exposure times were identified for each solder type. Increasing the BSA concentration from 25% to 60% greatly increased the tensile strength of the weld. A reduction in dye concentration from 2.5 mg/ml to 0.25 mg/ml was also found to result in an increase in tensile strength. The strongest welds were produced with an irradiance of 6.4 W/cm2 for 50 s using a solid protein solder composed of 60% BSA and 0.25 mg/ml ICG. Steady-state solder surface temperatures were observed to reach 85 plus or minus 5 degrees Celsius with a temperature gradient across the solid protein solder strips of between 15 and 20 degrees Celsius. Finally, tensile strength was observed to decrease significantly (20 to 25%) after the first hour of hydration in phosphate-buffered saline. No appreciable change was observed in the strength of the tissue bonds with further hydration.

In vivo tissue repair using light-activated surgical adhesive in a porcine model

An in vivo study was conducted to investigate the feasibility, mechanical function, and chronic biocompatibility of a new light-activated surgical adhesive for achieving rapid hemostasis of the puncture site following diagnostic catheterization and interventional cardiac procedures. Porcine carotid arteries (nequals6) and femoral arteries (nequals6) were exposed, and an incision was made in the arterial walls using a 16G needle. The surgical adhesive, composed of a poly(L-lactic-co-glycolic acid) scaffold doped with the traditional protein solder mix of serum albumin and indocyanine green dye, was used to close the incisions in conjunction with an 805-nm diode laser. Blood flow was restored to the vessels immediately after the procedure and the incision sites were checked for patency. The strength and hemostatic abilities of the new surgical adhesive were evaluated in the context of arterial pressure, persistence of hemostatis and presence of any inflammatory reaction after 3 days. After this evaluation period, the surgical procedure was repeated on the carotid arteries (nequals6) and femoral arteries (nequals6) of three additional animals that had been heparinized prior to surgery to closer approximate the conditions seen in a typical vascular surgical setting.

ICG-doped albumin protein solders for improved tissue repair

An in vitro study was performed using an 808nm-diode laser in conjunction with indocyanine green-doped albumin protein solders to repair bovine aorta specimens. Investigations were conducted to determine optimal solder and laser parameters for tissue repair in terms of tensile strength, temperature rise and damage and the microscopic nature of the bonds formed. Liquid and solid protein solders prepared from 25% and 60% bovine serum albumin (BSA), respectively, were compared. The tensile strengths of the repairs were greatly improved with an increase in BSA concentration from 25% to 60% and a reduction in ICG dye concentration from 2.5 mg/ml to 0.25 mg/ml. Increasing the later irradiance and thus surface temperature resulted in an increased severity of histological injury. Thermal denaturation of the tissue substrate increased laterally and in depth with higher temperatures. Optimal repairs in terms of bond strength and thermal damage were achieved by denaturing a solid protein solder composed of 60% BSA and .025mg/ml ICG with an irradiance of 6.4 W/cm2. Using this combination of solder and laser parameters, surface temperatures were observed to reach 85±5°C with an average temperature difference across the solder strips of 15°C across a thickness of 150 μm. Histological examination of the repairs formed using these parameters showed negligible evidence of collateral thermal damage to the underlying tissue. Scanning electron microscopy suggested albumin intertwining within the itssue collagen matrix and subsequent fusion with the collagen as the mechanism for laser tissue soldering.

Alternative chromophores for use in light-activated surgical adhesives: optimization of parameters for tensile strength and thermal damage profile

The use of indocyanine green-doped albumin protein solders has been shown to vastly improve the anastomotic strength that can be achieved by laser tissue repair techniques, while at the same time minimizing collateral thermal tissue damage. However, the safety of the degradation products of the chromophore following laser irradiation is uncertain. Therefore, we studied the feasibility of using alternative chromophores in terms of temperature rise at the solder/tissue interface, the extent of thermal damage in the sourrounding tissue, and the tensile strength of repairs. Biodegradable polymer scaffolds of controlled porosity were fabricated with poly(L-lactic-co-glycolic acid), using a solvent-casting and particulate-leaching technique. The porous scaffold acted as a carrier to the traditional protein solder composition of serum albumin and an absorbing chromophore mixed in deionized water. Two commonly used chromophores, indocyanine green and methylene blue were investigated, as well as blue and green food colorings. Temperature rise at the solder surface and at the interface between the solder and tissue were monitored by an IR temperature monitoring system and a type-K thermocouple, respectively, and the extent of thermal damage in the underlying tissue was determined using light microscopy. As expected, temperature rise at the solder/tissue interface, and consequently the degree of collateral thermal tissue damage, was directly related to the penetration depth of the laser light in the protein solder. Optimal tensile strength of repairs was achieved by selecting a chromophore concentration that resulted in a temperature of 66 ± 3°C at the solder/tissue interface.

Mechanisms of laser-induced thermal coagulation of whole blood in vitro

Quantitative data regarding photothermal and damage processes during pulsed laser irradiation of blood are necessary to achieve a better understanding of laser treatment of cutaneous vascular lesions and improve numerical models. In this study, multiple experimental techniques were employed to quantify the effects os single- pulse KTP laser (λ = 532 nm, τp= 10 ms) irradiation of whole blood in vitro: high-speed temperature measurement with a thermal camera in line-scan mode (8 kHz); optical coherence tomography; and transmission measurement with a co-aligned laser beam (λ=635 nm). Threshold radiant exposures for coagulation (4.4-5.0 J/cm2) and ablation (~ 12 J/cm2) were identified. Thermal camera measurements indicated threshold coagulation temperatures of 90-100°C, and peak temperatures of up to 145°C for sub-ablation radiant exposures. Significant changes in coagulum thickness and consistency, and a corresponding decrease in transmission, were observed with increasing radiant exposure. The Arrhenius equation was shown to produce accurate predictions of coagulation onset. The significant of dynamic effects such as evaporative loss and dynamic changes in optical properties was indicated. Implications for numerical modeling are discussed. Most importantly, the threshold temperatures typically quoted in the literature for pulsed laser coagulation (60-70 °C) and ablation (100 °C) of blood do not match the result of this study.

Laser-solder repair technique for nerve anastomosis: temperatures required for optimal tensile strength

Laser-assisted repair of nerves is often unsatisfactory and has a high failure rate. Two disadvantages of laser assisted procedures are low initial strength of the resulting anastomosis and thermal damage of tissue by laser heating. Temporary or permanent stay sutures are used and fluid solders have been proposed to increase the strength of the repair. These techniques, however, have their own disadvantages including foreign body reaction and difficulty of application. To address these problems solid protein solder strips have been developed for use in conjunction with a diode laser for nerve anastomosis. The protein helps to supplement the bond, especially in the acute healing phase up to five days post- operative. Indocyanine green dye is added to the protein solder to absorb a laser wavelength (approximately 800 nm) that is poorly absorbed by water and other bodily tissues. This reduces the collateral thermal damage typically associated with other laser techniques. An investigation of the feasibility of the laser-solder repair technique in terms of required laser irradiance, tensile strength of the repair, and solder and tissue temperature is reported here. The tensile strength of repaired nerves rose steadily with laser irradiance reaching a maximum of 105 plus or minus 10 N.cm-2 at 12.7 W.cm-2. When higher laser irradiances were used the tensile strength of the resulting bonds dropped. Histopathological analysis of the laser- soldered nerves, conducted immediately after surgery, showed the solder to have adhered well to the perineurial membrane, with minimal damage to the inner axons of the nerve. The maximum temperature reached at the solder surface and at the solder/nerve interface, measured using a non-contact fiber optic radiometer and thermocouple respectively, also rose steadily with laser irradiance. At 12.7 W.cm-2, the temperatures reached at the surface and at the interface were 85 plus or minus 4 and 68 plus or minus 4 degrees Celsius respectively. This study demonstrates the feasibility of the laser-solder repair technique for nerve anastomosis resulting in improved tensile strength. The welding temperature required to achieve optimal tensile strength has been identified.

Comparison of scaffold-enhanced albumin and n-butyl-cyanoacrylate adhesives for joining of tissue in a porcine model

An ex vivo study was conducted to compare the tensile strength of tissue samples repaired using three different techniques: (i) application of a scaffold-enhanced light-activated albumin protein solder, (ii) application of a scaffold-enhanced n-butyl-cyanoacrylate adhesive, and (iii) repair via conventional suture technique. Biodegradable polymer scaffolds of controlled porosity were fabricated with poly(L-lactic-co-glycolic acid) and salt particles using a solvent-casting and particulate-leaching technique. Group I porous scaffolds were doped with protein solder composed of 50%(w/v) bovine serum albumin solder and 0.5mg/ml indocyanine green dye mixed in deionized water, and activated with an 808-nm diode laser. Group II scaffolds were doped with n-butyl-cyanoacrylate, and required no light-activation. No stay sutures were required for Group I or II experiments. Group III repairs were performed using a single 4-0 suture. Thirteen organs were tested ranging from skin to liver to the small intestine, as well as the coronary, pulmonary, carotid, femoral and splenic arteries. Acute breaking strengths were measured and the data were analyzed by Student’s T-test. Using the protein solder of Group I, repairs formed on the ureter were most successful followed by small intestine, sciatic nerve, spleen, atrium, kidney, muscle, skin and ventricle. The strongest vascular repairs were achieved in the carotid artery and femoral artery. Overall, the tensile strength of Group III repairs performed via suture techniques were equivalent in magnitude to that of Group I repairs, however, a larger variance was observed in the suture repair group. Group II repairs utilizing the cyanoacrylate-doped scaffold all performed extremely well. Bonds formed using the Group II adhesive were approximately 30% stronger than Group I and III organ repairs and approximately 20% stronger than Group I and III vascular repairs. Application of the polymer scaffold assists in tissue alignment and reduces problems associated with adhesive runaway from the repair site. Scaffold-enhanced adhesives could possibly be used as a simple and effective method to join tissue together quickly and effectively in an emergency situation, or as a substitute to mechanical sutures or staples in many clinical applications.

Biodegradable polymer thin film for enhancement of laser-assisted incision closure with an indocyanine-green-doped liquid albumin solder

The purpose of this study was to determine if solid material reinforcement of a liquid albumin solder coagulum could improve the cohesive strength of the solder and thus the ultimate breaking strength of the incision repair in vitro. A 50%(w/v) bovine serum albumin solder with 0.5 mg/mL Indocyanine Green (ICG) dye was used to repair an incision in bovine aorta. The solder was coagulated with an 806 nm CW diode laser. A 50 micrometer thick poly(DL-lactic-co-glycolic acid) film was used to reinforce the solder (the controls had no reinforcement). Acute breaking strengths were measured and the data were analyzed by one-way ANOVA (P less than 0.05). Multiple comparisons of means were performed using the Newman- Keuls test. Observations of the failure modes indicated cohesive strength reinforcement of the test specimens versus the controls. At the higher laser powers used in this study (400 and 450 mW), the reinforced solder was consistently stronger than the controls. Reinforcement of liquid albumin solders in laser-assisted incision repair may have mechanical advantages in terms of acute breaking strength over conventional methods that do not reinforce the cohesive strength of the solder.

Solid protein solder-doped biodegradable polymer membranes for laser-assisted tissue repair

Solid protein solder-doped polymer membranes have been developed for laser-assisted tissue repair. Biodegradable polymer films of controlled porosity were fabricated with poly(L-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) using a solvent-casting and particulate-leaching technique. The films provided a porous scaffold that readily absorbed the traditional protein solder mix composed of bovine serum albumin (BSA) and indocyanine green (ICG) dye. In vitro investigations were conducted to assess the influence of various processing parameters on the strength of tissue repairs formed using the new membranes. These parameters included the PLGA copolymer and PLGA/PEG blend ratio, the salt particle size, the initial bovine serum albumin (BSA) weight fraction, and the laser irradiance used to denature the solder. Altering the PLGA copolymer ratio had little effect on repair strength, however, it influenced the membrane degradation rate. Repair strength increased with increased membrane pore size and BSA concentration. The addition of PEG during the film casting stage increased the flexibility of the membranes but not necessarily the repair strength. The repair strength increased with increasing irradiance from 12 W/cm2 to 15 W/cm2. The new solder-doped polymer membranes provide all of the benefits associated with solid protein solders including high repair strength and improved edge coaptation. In addition, the flexible and moldable nature of the new membranes offer the capability of tailoring the membranes to a wide range of tissue geometries, and consequently, improved clinical applicability of laser- assisted tissue repair.

Free-electron laser tissue-soldering in vitro with an albumin solder

The purpose of this study was to explore the feasibility of using a free-electron laser (FEL) to photothermally coagulate an albumin solder for laser-assisted incision closure. A 50%(w/v) bovine serum albumin solder was used to repair an incision in bovine aorta. The solder was coagulated by targeting absorption peaks in the solder infrared absorption spectrum using the FEL. Acute breaking strengths of repaired incisions were measured and the data analyzed by one-way ANOVA (P < 0.05). Multiple comparisons of means were performed using the Newman-Keuls test. The solder absorption spectrum from 2 - 10 microns was similar to water with an additional peak at 6.45 microns (amide II) due to the albumin. Preliminary results indicated that wavelengths at or very close to the absorption peaks were excessively absorbed, resulting in only the top surface of the solder being coagulated. Using wavelengths at points of weak absorption on the water absorption curve yielded better results.