The overall goal of this protocol is to develop a biodegradable hemostatic glue for surgical application inspired by a ubiquitous compound in plants. This method can help us understand the water resistant adhesion mechanism of biometrics, such as tissue adhesives and sealant in biomedical application. The main advantage of this technique is that the process of making TAPE is extremely simple, scalable, and environmentally friendly.
First, prepare a Tannic Acid solution in a four milliliter glass vial on a magnetic stirrer. Add one milliliter of distilled water and one gram of Tannic Acid with stirring at 200 rpm. After an hour, the Tannic Acid will be completely dissolved in a brown solution.
Next, add one milliliter of distilled water to one gram of PEG powder and vortex them into a white slurry. Keep the slurry at 60 degrees for 10 minutes. It should become completely clear.
Then, return it to room temperature. Next, in a microcentrifuge tube, mix 329 microliters of PEG solution with 671 microliters of the Tannic Acid solution. Take the aliquat slowly as the solutions are both quite viscous.
Gently blend the solutions into a honey-like mixture with a narrow spatula. Spin the mixture at 12, 300 gs for three minutes in a fixed angle rotor. Then, carefully pipette off as much of the supernatant as possible, leaving the fully formed TAPE.
Store the TAPE at four to eight degrees Celsius for several weeks. To measure the adhesive strength of the TAPE, prepare a test tissue from a porcine biopsy skin punch. Remove all the fat and then prepare two six millimeter diameter test tissues.
Apply commercial cyanoacrylate glue to the outer side of each tissue and attach the rods or a similar object to be gripped by the test machine. Next, apply a drop of TAPE to one side of the tissue and spread it uniformly between the inner sides of the two tissues so they are attached. Then, attach and detach the tissues several times to make a homogeneous mix that fully covers the interface.
Next, attach the rods to the testing apparatus. Begin with applying a force of 20 Newtons for one minute. Next, have the rods pull apart at a rate of one millimeter per minute until the tissues are completely detached.
The data is given as a force-distance curve. To test adhesion strength in the presence of water, add 20 microliters of water to the detached area between the two tissues and immediately attach the wet surfaces with the TAPE. Repeat the test as before with no other changes.
First, cut an eight millimeter diameter cap off a microcentrifuge tube and weigh it. Next, fill the cap with 150 milligrams of TAPE and measure the total weight. Do not fill the cap beyond the lip, which will be needed as a barrier.
Add 50 milliliters of PBS buffer to the 75 square centimeter cell culture flask. Put the loaded cap into the flask and submerge the cap. Incubate the flask at 37 degrees Celsius on an orbital shaker set to 50 rpm, but not higher.
At the desired time, remove the cap and dry it with nitrogen gas. Then, weigh the cap. Next, replace the PBS in the flask and continue the incubation with gentle shaking until the next time point.
From the measurements, calculate the relative remaining weight. Begin with a fully anesthetized six-week old male mouse tested with a toe pinch. Be sure to apply vet ointment to the eyes prior to beginning surgery.
Now, expose the liver using a midline abdominal incision, and place filter paper of known mass under the liver to immobilize it. Next, prick the liver with an 18-gauge needle to induce bleeding. While absorbing the blood with sterile gauze, immediately put 100 microliters of TAPE on the site of the incision.
For positive controls, use cyanoacrylate or a similar glue and do nothing for negative controls. No further suturing is needed. After placing the adhesive, collect the blood from the damage site onto the paper.
Replace the paper every 30 second for two minutes. Measure the mass of the absorbed blood on the four filter papers. The animal should be euthanized.
Several ratios of TA and PEG were tested and the ideal combination to make TAPE was a two-to-one volume ratio. Such TAPE was applied between two porcine skins with a diameter of six millimeters then tested on a tensile machine as described. The force needed to detach two porcine skins was initially measured at 200 kilopascals and increased to 250 kilopascals with repeated cycles.
When the skin samples were moistened after each cycle of attachment/detachment, the tensile strength of the bond dropped to 90 kilopascals initially and decreased to 50 kilopascals after 20 cycles. The durability of TAPE was tested in small volumes diluted in PBS with shaking at physiological light conditions. Mass was retained for 21 days using the two-to-one ratio formulation.
A one-to-one ratio formulation of TAPE only lasted 13 days under these conditions. Next, a hemostatic test performed in vivo was conducted. The TAPE sealed liver was compared with commercial fibrin glue.
The TAPE sealed liver bled significantly less than the fibrin glue sealed liver. Once mastered, the preparation of TAPE can be done in five minutes if it is performed properly. While attempting this procedure, it is important to remember to keep the optimized ratio between Tannic Acid and PEG because it can dramatically affect the adhesion force of TAPE.
After its development, this technique paved the way for researchers in the field of biometrics to explore the nature inspired way to solve current problems in developing water resistant adhesive materials. Though this method can provide insight into hemostatic materials, it can also be applied to other systems such as tissue sealant for wound healing and implantable patch for drug delivery.
