Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration

This protocol improves a basic concept of wet-spinning to develop a material from a single fiber strip into various strips that can be used for any preferred medical application. This technique is simple, inexpensive, and does not require the addition of synthetic polymers for customization of the natural polymers into a resource of protein for materials of interest. This technique can be extended to biomaterial development for killing cold therapy such as central or peripheral nervous system treatment, blood vessel replacement, or direct urinary tract reconstruction.

This method is intended for medical application including as a strategy for tissue neuralization or regenerative medicine. This technique can be easily implemented by beginners. Note that, after several molding repetitions, the quality of the solution should always be checked to maintain an optimal formation.

To form the wet spinning tube, first submerge a 24 gauge by three quarter inch peripheral venous catheter into freshly prepared gelatin solution for three seconds, followed by immersion in freshly prepared acetone solution for one minute. Repeat these steps 20 times. After the last acetone immersion, let the molded tube dry for five minutes at room temperature, and use a hemostat to carefully transfer the gelatin tube from the catheter into a glass Pasteur pipette with 2.5%polycaprolactone dichloromethane solution for an overnight under-hood incubation at room temperature.

The next morning, sterilize the gelatin for two hours under ultraviolet light before briefly submerging the tube in 75%ethanol. Then wash the tube two times with stem cell medium to remove the residual ethanol. To cultivate the gelatin tube with the cell culture, replace the supinate end of human adipose stem cells from a culture flask with one milliliter of 0.25%Trypsin-EDTA for a 3.5 minute incubation at 37 degrees Celsius and 5%carbon dioxide.

When the cells have detached, arrest the reaction with one milliliter of fetal bovine serum and transfer the cell suspension into a microcentrifuge tube. Collect the cells by centrifugation and resuspend the pellet in one milliliter of stem cell medium. Then place the gelatin tube in one well of a six-well plate containing three milliliters of stem cell medium.

After counting the cells, seed four times 10 to the fourth of human adipose stem cells into the tube for a two week incubation in the cell culture incubator. After confirming the appropriate level of sedation by lack of response to toe pinch in a female eight week old Sprague Dawley rat, shave the hair from the trapezius area and sterilize the exposed skin with povidone-iodine solution. Cover the rat with sterile surgical drapes to reduce the bacterial contamination of the surgical site and use surgical scissors to make a shallow, two centimeter long skin incision.

Place the gelatin tube directly into the layer between the fascia and the muscle and use a nylon suture to close the wound. Then sterilize the incision with povidone-iodine solution. Induce the rat via an intramuscular injection of ketoprofen and cefazolin and maintain the animal under aseptic conditions for seven days.

This user friendly wet spinning concept can be used to develop gelatin into fibers and tubes. Morphological observations of the gelatin tube by scanning electron microscopy reveals a not very smooth surface with a greater than 200 micrometer inner diameter and a thickness of approximately 20 micrometers. Staining of a human adipose stem cell culture gelatin tube for neural cell marker expression demonstrates a positive staining for the marker along with multiple detected nuclei suggesting that the cells can penetrate and adhere to the gelatin tube and differentiate into neural progenitor cells.

Implantation of the gelatin tube into the fascia layer confirms its safety and biocompatibility, as no indication of redness, swelling, or other inflammation symptoms are observed within the local tissue and the tube is surrounded by well-developed connective tissue. While attempting this procedure of two point one, ensuring immersion of the mold in the specific coating time in each solution are very important to building up a precise tube form. Following this procedure, our experiments use animal model of human disease can be performed to accurately investigate the outcome of the biomaterials for tissue regeneration strategy.

After its development, this technique paved the way for researchers in developing simple correlative nature polymers into any type of material for a decided medical application. Under normal use condition, the polycaprolactone dichloromethane solution is considered to present minimal environmental hazard, but don't forget to perform any steps with this chemical in an appropriate biosafety cabinet.