The clinical use of photodynamic therapy (PDT) with rose bengal (RB) is emerging as an effective t reatment for a range of applications given its non-invasive and localised mode of delivery. In particular, rose bengal PDT has shown promising antifungal action in vitro. While focus has largely been on the physical and chemical impacts of PDT on the cell, an understanding of the role of genetics underpinning the cellular response is still limited. We have, therefore, reported a screen of the entire non-essential gene library of the model organism, Saccharomyces cerevisiae, using rose bengal PDT to ascertain the key genetic pathways affecting fungal tolerance to PDT. We also investigated the dosage of PDT required to eradicate Trichophyton rubrum spores, the main causative organism of onychomycosis infection. Following this, we conducted a pilot patient study of six patients (seven toenails) for the treatment of onychomycosis using rose bengal PDT (140 μM RB and ~763 J/cm2 green light), where the clinical treatment protocol was developed on the basis of the in vitro outcomes. The key biochemical pathways identified by the genetic screen as having altered tolerance to PDT included ergosterol biosynthesis, vacuolar acidification, and purine/S-adenosyl-L-methionine biosynthesis. The subsequent pilot patient study saw the complete cure of onychomycosis for all patients within three to five treatment sessions in the absence of pain or other local side effects. The outcome of the genetic screen for tolerance may thus inform the development of efficient clinical treatments using rose bengal PDT.
Peripheral nerve injuries are difficult to treat because axon regeneration is limited and functional recovery is often unsatisfactory in patients. Brief electrical stimulation of injured nerves is emerging as a new promising therapy that can relieve pain or induce better axon regeneration and functional recovery than untreated nerves. In this study, we report an innovative wireless and biocompatible stimulator that is also a scaffold for injured nerves when an autograft is applied to bridge a gap in rat sciatic nerves. We have named this device “graft-antenna” to highlight the double functionality of the implant. The scaffold is made of chitosan and incorporates a gold loop antenna (diameter ~1.3 mm, thickness ~70 nm) powered wirelessly by a transcranial magnetic stimulator (TMS). The device is bonded to tissue non-invasively and without sutures, exploiting the photo-adhesion properties of the chitosan scaffold. The stimulator did not migrate after implantation on healthy sciatic nerves in rats and was able to trigger a steady compound muscle action potential for 12 weeks (CMAP ~1.3 mV). No CMAP was elicited by the TMS when the wireless stimulator was not implanted on nerves. Axon regeneration was facilitated in sciatic nerves that were grafted with the graft-antenna and stimulated for 1 hour, once a week (magnetic field magnitude~0.72 T, pulse duration ~350 μs, repetition rate=1 pulse/sec). Eight weeks post-operatively, myelinated axon count, CMAP and nerve conduction velocity were statistically higher in the graft-antenna group (n=5) than in nerves grafted with the chitosan scaffold without antenna.
Background. Laser tissue repair usually relies on haemoderivate solders, based on serum albumin. These solders have intrinsic limitations that impair their widespread use, such as limited repair strength, high solubility, brittleness and viral transmission. Furthermore, the solder activation temperature (65-70 °C) can induce significant damage to tissue. In this study, a new laser-activated biomaterial for tissue repair was developed and tested in vitro and in vivo to overcome some of the shortcomings of traditional solders.
Materials and Methods. Flexible and insoluble strips of chitosan adhesive (surface area ~34 mm2, thickness ~20 μm) were developed and bonded on sheep intestine with a laser fluence and irradiance of 52 ± 2 J/cm2 and ~15 W/cm2 respectively. The temperature between tissue and adhesive was measured using small thermocouples. The strength of repaired tissue was tested by a calibrated tensiometer. The adhesive was also bonded in vivo to the sciatic nerve of rats to assess the thermal damage induced by the laser (fluence = 65 ± 11 J/cm2, irradiance = 15 W/cm2) four days post-operatively.
Results. Chitosan adhesives successfully repaired intestine tissue, achieving a repair strength of 0.50 ± 0.15 N (shear stress = 14.7 ± 4.7 KPa, n=30) at a temperature of 60-65 °C. The laser caused demyelination of axons at the operated site; nevertheless, the myelinated axons retained their normal morphology proximally and distally.
Background. Laser tissue soldering (LTS) is an alternative technique to suturing for tissue repair. One of the major drawbacks of LTS is the weak tensile strength of the solder welds when compared to sutures. In this study, the possibility was investigated for a low cytotoxic crosslinker, acting on amino groups, to enhance the bond strength of albumin solders. Materials and Methods. Solder strips were welded onto rectangular sections of sheep small intestine by a diode laser. The laser delivered in continuous mode mode a power of 170 ± 10 mW at λ=808 nm, through a multimode optical fiber (core size = 200 μm) 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 ± 1 mm, area = 12 ± 1.2 mm2). The solder incorporated 62% bovine serum albumin, 0.38% genipin, 0.25% indocyanin green dye (IG) and water. Tissue welding was also performed with a similar solder, which did not incorporate genipin, as a control group. The repaired tissue was tested for tensile strength by a calibrated tensiometer. Results. The tensile strength of the “genipin” solder was twice as high as the strength of the BSA solder (0.21 ± 0.04 N and 0.11 ± 0.04 N respectively; p~10-15 unpaired t-test, N=30). Discussion. 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.
In this study, a two layer (TL) solid solder was developed with a fixed thickness to minimize the difference in temperature across the solder (ΔT) and to weld at low temperature. Solder strips comprising two layers (65% albumin, 35% water) were welded onto rectangular sections of dog small intestine by a diode laser (λ = 808 nm). The laser delivered a power of 170 ± 10 mW through an optical fiber (spot size approximately 1 mm) for 100 seconds. A solder layer incorporated also a dye (carbon black, 0.25%) to absorb the laser radiation. A thermocouple and an infrared thermometer system recorded the temperatures at the tissue interface and at the external solder surface, during welding. The repaired tissue was tested for tensile strength by a calibrated tensiometer. The TL strips were able to minimize ΔT (12 ± 4°C) and control the temperature at tissue-interface. The strips fused on tissue at 55≤T≤62°C had higher tensile strength than the strips soldered at 51≤T<55°C (19.1 ± 6.6 versus 13.1 ± 6.4 gmf). The solid solder could efficiently weld at 60°C as it became insoluble and formed stable bonds with tissue. Fluid albumin solders, by contrast, requires temperatures ≥70°C for tissue repair, which cause more irreversible thermal damage.
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.
A new laser activated solder weld technique is described for the microsurgical repair of the inferior alveolar nerve in rats. The laser weld technique used an albumin based solder, containing indocyanine cardiogreen, plus an infrared diode laser. Seven animals had inferior alveolar nerve repairs performed using the laser weld technique and these were compared against corresponding unoperated controls plus three cases of nerve section without repair. Histochemical analysis was performed utilizing neuron counts and horseradish peroxidase tracer (HRP) uptake in the trigeminal ganglion following sacrifice and staining of frozen sections with cresyl violet and diaminobenzidene. The results of this analysis showed comparable mean neuron counts and mean HRP uptake by neurons for the unoperated control and laser weld groups with considerable reduction of mean values in cases of nerve section with no repair. Sections of the repaired inferior alveolar nerves, stained with Masson's trichrome, showed no adverse reactions by axons or epineurium to the coagulative repair with the solder and demonstrated regeneration of myelinated axons at the time of sacrifice. In summary a new technique of laser weld repair of the inferior alveolar nerve is described which, on initial analysis, appears to be a reliable alternative to traditional techniques.
A 100 micrometer core optical fiber-coupled 75 mW diode laser operating at a wavelength of 800 nm has been used in conjunction with a protein solder to stripe weld severed rat tibial nerves, reducing the long operating time required for microsurgical nerve repair. Welding is produced by selective laser denaturation of the protein based solder which contains the dye indocyanine green. Operating time for laser soldering was 10 plus or minus 5 min. (n equals 24) compared to 23 plus or minus 9 min (n equals 13) for microsuturing. The laser solder technique resulted in patent welds with a tensile strength of 15 plus or minus 5 g, while microsutured nerves had a tensile strength of 40 plus or minus 10 g. Histopathology of the laser soldered nerves, conducted immediately after surgery, displayed solder adhesion to the outer membrane with minimal damage to the inner axons of the nerves. An in vivo study, with a total of fifty-seven adult male wistar rats, compared laser solder repaired tibial nerves to conventional microsuture repair. Twenty-four laser soldered nerves and thirteen sutured nerves were characterized at three months and showed successful regeneration with average compound muscle action potentials (CMAP) of 2.4 plus or minus 0.7 mV and 2.7 plus or minus 0.8 mV respectively. Histopathology of the in vivo study, confirmed the comparable regeneration of axons in laser and suture operated nerves. A faster, less damaging and long lasting laser based anastomotic technique is presented.
A 100 micrometers core optical fiber-coupled 75 mW diode laser operating at a wavelength of 800 nm has been used in conjunction with a protein solder to stripe weld severed rat tibial nerves, reducing the long operating time required for microsurgical nerve repair. Welding is produced by selective laser denaturation of the albumin based solder which contains the dye indocyanine green. Operating time for laser soldering was 10 +/- 5 min. (n equals 20) compared to 23 +/- 9 min. (n equals 10) for microsuturing. The laser solder technique resulted in patent welds with a tensile strength of 15 +/- 5 g, while microsutured nerves had a tensile strength of 40 +/- 10 g. Histopathology of the laser soldered nerves, conducted immediately after surgery, displayed solder adhesion to the outer membrane with minimal damage to the inner axons of the nerves. An in vivo study is under way comparing laser solder repaired tibial nerves to conventional microsuture repair. At the time of submission 15 laser soldered nerves and 7 sutured nerves were characterized at 3 months and showed successful regeneration with compound muscle action potentials of 27 +/- 8 mV and 29 +/- 8 mW respectively. A faster, less damaging and long lasting laser based anastomotic technique is presented.
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