Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

The overall goal of this procedure is to fabricate a laser activated chitosan adhesive and illustrate how to apply it when repairing peripheral nerves. This is accomplished by first Solubilizing chitosan and rose Bengal powder in acetic acid solution. The second step of the procedure is to dry cast the solution to obtain a thin film.

The third step is to expose the rat nerve and dissect it. The final step is to place the film around the dissected nerve and irradiate it with a green laser to bond the film to the tissue. Ultimately, the strength with which rose film bonds to the tissue is measured using a calibrated tensiometer.

The laser adhesive is biocompatible and has the advantage of bonding directly to the tissue. This makes it very useful for repairing tissue that is deep and difficult to get to where sutures would be difficult. It would also have the advantage of repairing tissue where watertight closure is important, such as the dura or the bowel.

It is important to demonstrate visually the alto and adhesive technique because several steps are involved in the adhesive preparation and also because the surgical procedure requires particular care when the adhesive is applied on tissue. Most of the demonstration will be done by Matt Barton, who is a postgraduate student in my laboratory, and the surgical operation will be done by Professor Marcus Studley from Quare University. Begin by preparing a Kita sand solution in rose Bengal laced acetic acid.

A control solution can simply lack the rose Bengal set up the solution with a magnetic stir at room temperature. It will be ready in about two weeks or five days for controls without rose Bengal. During this time, the pH increases from roughly 2.6 to 3.9 as the chitosan dissolves.

Once prepared, centrifuge the solution for an hour and separate the solution from the impurity pellet. By gentle aspiration, do not disturb the pellet. Using a sterile syringe, inject the solution on a flat perspec slab.

Spread the solution evenly without making any air bubbles For a film with a thickness of roughly 13 microns, spread a milliliter of solution over 12 square centimeters. Cover the Perspex slab with an aluminum foil screen to avoid photo bleaching of the solution, but leave enough ventilation space for the film to dry out. Leave the solution to dry at room temperature and pressure.

Once the film has dried. Use a thin spatula to carefully pry it from the slab to test. If the adhesive is dry, cut a small sample and place it in a vial containing water.

When the film is insoluble and does not swell macroscopically, it is dry. This typically takes two weeks. Then measure the thickness of the film using a micrometer with clean, sharp scissors.

Cut the film into rectangular strips of the size required for your application. Store the pieces of rose adhesive or control adhesive between sterile glass slides wrapped in aluminum foil and at room temperature. Before using the film in an application, determine its optical attenuation.

This is accomplished by fixing a small piece of film in a quartz vete. Then in a dual beam spectrophotometer record the attenuated spectrum of the film between 400 and 800 nanometers. The measurements can be interpreted using beer's law to provide the attenuation length, which will be comparable to the thickness of the film.

For this protocol, calf intestine must have been harvested immediately after animal euthanasia and stored at minus 80 degrees Celsius. Defrost and hydrate the tissue by immersing it in deionized water for 10 minutes. Then bisect the tissue into two centimeter by one centimeter sections using a number 10 blade under an operating microscope at low power.

Keep the sections moist with drops of water to bond two sections. Place them end to end and wipe any excess water off the surface. Using cotton tips with micro forceps.

Place a strip of rose adhesive film across the incision on the ciero layer. Make certain the film is in full contact with the intestine. Prepare the DDE pumped solid state laser coupled to a multi-mode optical fiber.

Check that the laser parameters are set correctly. Now irradiate the adhesive. The beam spot size should be about five millimeters wide where it contacts the rows adhesive spot irradiate each portion of the adhesive for about five seconds.

Ultimately, the surface is scanned several times over about six minutes. Remove the irradiated sample. To assess the tissue bonding strength.

Clamp a sample to a calibrated single column tensiometer. Using mechanical grips. Move the grips at 22 millimeters per minute until the two two tissue stumps separate as a control.

Place a rose adhesive on the tissue. Do not irradiate the sample and measure the tissue's bonding strength. For this procedure, the rose adhesive films must be sterilized.

This can be done using 90%ethanol or with gamma radiation. Sedate a wistar rat with 4%gaseous isof fluorine and reduce the concentration to 2%when the animal is no longer responsive to a pain reflex. Test like a tow pinch, shave and sanitize the rat's thigh.

Position it for surgery under an operating microscope, make an oblique skin incision of about three centimeters in the dorsolateral part of the right thigh. Expose the tibial nerve with the muscle splitting approach through the gluteal muscles using straight micro scissors. Partially dissect and trim the adventitia of the tibial nerve.

Absorb any excess fluids with sterile gauze or cotton tips. Now cut the tibial nerve using micro forceps. Position a sterile rose adhesive strip underneath the tibial nerve.

Then approximate the nerve stumps end to end over the rose adhesive strip. It is important that the nerves be tension free. The strip will fully adhere to the nerve forming a collar which can assist with the rotation of the nerve.

During laser irradiation, proceed to activate the rose adhesive with the laser as previously described. Finally, close the muscles and skin with five three aught sutures and follow standard postoperative protocols for the rat's recovery. Following the outlined protocol.

The films obtained are bright rows in color, thin, and have a smooth surface. The films are also flexible and can be rolled into tubes of small diameter without causing tears or any other apparent damage. The rose adhesive has two absorption peaks at 530 and 562 nanometers.

The green laser is thus strongly absorbed by the adhesive and the corresponding attenuation length at 532 nanometers is roughly 12 microns. In contrast, Esan films without RB attenuate weekly with the laser. The rose adhesive bonds firmly to the intestine upon laser irradiation achieving a typical maximum load at failure of about one Newton.

The adhesive strength was estimated as the maximum load divided by the adhesive surface area. The non irradiated rose adhesive bonded significantly less to tissue. It is important to dry the keto and adhesive until it's completely insoluble in water.

It takes about two weeks. Also, whenever you make the adhesive, you need acids and acids should be handled in a fume hood. Remember also that lasers are involved in this procedure and they can be very dangerous, so you need to wear laser goggles all the time.